HK40082027B - Antiviral compounds - Google Patents

Description

antiviral compounds

Cross-references to related applications

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/978,199, filed on February 18, 2020, entitled “ANTIVIRAL COMPOUNDS,” the entire contents of which are incorporated herein by reference.

sequence list

This application includes a sequence list, which is submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy was created on February 17, 2021, and is named 1225-WO-PCT_SL.txt, with a size of 800 bytes.

Background Technology

Viruses belonging to the family Flaviviridae comprise at least three distinguishable genera: pestivirus, flavivirus, and hepacivirus (Calisher et al., J. Gen. Virol., 1993, 70, 37-43). While pestiviruses cause many economically important animal diseases, such as bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, swine cholera), and borderline sheep disease (BDV), their importance in human diseases has not been fully characterized (Moennig, V. et al., Adv. Vir. Res. 1992, 48, 53-98). Flaviviruses are the cause of important human diseases such as dengue fever and yellow fever, while hepaciviruses cause hepatitis C virus infection in humans. Other important viral infections caused by Flaviviridae include West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, Junjin virus, Murray Valley encephalitis, St. Louis encephalitis, Omsk hemorrhagic fever virus, and Zika virus. In summary, Flaviviridae infections cause significant mortality, morbidity, and economic losses worldwide. Therefore, there is a need to develop effective treatments for Flaviviridae viral infections.

Pneumoviridae viruses are negative-sense single-stranded RNA viruses that cause many prevalent human and animal diseases. Pneumoviridae viruses include human respiratory syncytial virus (HRSV) and human metapneumovirus. Almost all children will be infected with HRSV before the age of two. HRSV is a leading cause of lower respiratory tract infections in infancy and childhood, with 0.5% to 2% of infections requiring hospitalization.

Currently, there is no vaccine to prevent HRSV infection. The monoclonal antibody palizumab can be used for immunization, but its use is limited to high-risk infants, such as premature infants or those with congenital heart or lung diseases, and its general cost is often prohibitive. Additionally, the nucleoside analog ribavirin has been approved as the only antiviral agent for treating HRSV infection, but its efficacy is limited. Therefore, antiviral treatments targeting pulmonary viruses are needed.

Examples of pyrrolo[2,3-d]pyrimidine compounds that can be used to treat viral infections are found in U.S. 2012/0009147A1 (Cho et al.), U.S. 2012/0020921 A1 (Cho et al.), WO 2008/089105 A2 (Babu et al.), WO 2008/141079 A1 (Babu et al.), WO 2009/132135 A1 (Butler et al.), WO 2010/002877 A2 (Francom), and WO 2011/035231 A1. As described in (Cho et al.), WO 2011/035250 A1 (Butler et al.), WO 2011/150288 A1 (Cho et al.), WO 2012/012465 (Cho et al.), WO 2012/012776 A1 (Mackman et al.), WO 2012/037038 (Clarke et al.), WO 2012/087596 A1 (Delaney et al.) and WO 2012/142075 A1 (Girijavallabhan et al.).

Therefore, there is a need for compositions and methods that are effective and have acceptable toxicity characteristics for treating infections caused by pulmonary viral diseases (such as HRSV infection), flaviviridae infections (including dengue fever), and EBOV infection. This disclosure addresses these and other needs.

Summary of the Invention

In one embodiment, this disclosure provides a compound of formula (I):

Or its pharmaceutically acceptable salt, wherein:

The base is

^R1 and ^R2 are each independently H or -C(O) ^R1A , wherein ^R1A is a _C1-6 alkyl group;

^R3 is -N(H) ^R3A ;

^R3A is H, _-CH2OP (O)(OH) ₂ , or -C(O) ^R3D , where

R ^3D is a C _6-12 aryl or C _1-6 alkyl group optionally substituted with a C _3-6 cycloalkyl group;

R ^4A is O;

R ^4B and R ^4C are independently:

(A)-OH;

(B)–OR ^4B1 , where R ^4B1 is C _6-12 aryl;

(C) where

The subscript m is 0, 1, 2, 3, 4, or 5; and

Each R ^4D is independently a C _1-6 alkyl group;

(D) where

^R4E1 and ^R4E2 are each independently H or _C1-6 alkyl;

^R4F1 and ^R4F2 together form an oxo group;

R ^4G is a C _1-8 alkyl, C _3-8 cycloalkyl, or a 3- to 8-membered heterocyclic group having 1 to 3 heteroatoms selected from N, O, and S, optionally substituted with 1 to 3 R ^{4G1 .}

Each R ^4G1 is independently -OH, C _1-6 alkoxy, -(CH ₂ OCH ₂ ) _1-5 -CH ₃ , C _1-3 haloalkyl, or optionally C _3-8 cycloalkyl substituted with 1 to 3 R ^4G9 ;

Each R ^4G3 and R ^4G9 is independently a _C1-6 alkyl group; or

(E)-(OP(O)(OH)) _1-2 -OH; and

^R5A and ^R5B are each independently _C1-6 alkyl groups substituted with -OP(O)(OH) ₂ .

In another embodiment, this disclosure provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is:

In another embodiment, this disclosure provides a pharmaceutical formulation comprising a pharmaceutically effective amount of the compound of this disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In another embodiment, this disclosure provides a method for treating a person in need of a pulmonary viral infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating a person in need of a microribonucleovir infection, the method comprising administering to the person a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating a person in need of a flaviviridae virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for treating a filoviridae virus infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for preparing a medicament for treating pulmonary viral infections in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for preparing a medicament for treating microribonucleoviridae virus infection in persons in need of it, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for preparing a medicament for treating flaviviridae virus infections in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides a method for preparing a medicament for treating filoviridae virus infections in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating human pulmonary viral infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating human microribonucleoviridae virus infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating human flaviviridae virus infections.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating human filoviridae virus infections.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of pulmonary viral infections in persons in need.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of microribonucleoviridae virus infections in persons in need of such treatment.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of flaviviridae virus infections in persons in need.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of filoviridae virus infections in persons in need.

In another embodiment, this disclosure provides a method for treating or preventing exacerbations of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides a method for preparing a medicament for treating or preventing exacerbations of respiratory symptoms caused by viral infection in persons in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating or preventing exacerbations of a respiratory condition in a person caused by a viral infection, wherein the respiratory condition is chronic obstructive pulmonary disease.

In another embodiment, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbations of respiratory conditions caused by viral infection in persons in need, wherein the respiratory condition is chronic obstructive pulmonary disease.

Detailed Implementation

I. Overview

This disclosure provides 2',3'-dihydroxy-4'-fluoromethyl nucleoside and related compounds for treating viral infections such as Ebola, Zika, West Nile, yellow fever, dengue fever, HBV, HCV, RSV, etc.

II. Definition

"Alkyl" is a straight-chain or branched saturated monovalent hydrocarbon. For example, an alkyl group may have 1 to 10 carbon atoms (i.e., _C1-10 alkyl), 1 to 8 carbon atoms (i.e., _C1-8 alkyl), 1 to 6 carbon atoms (i.e., _C1-6 alkyl), or 1 to 4 carbon atoms (i.e., _C1-4 alkyl). Examples of alkyl groups include, but are not limited to _, methyl ₍ Me, _-CH3 ), ethyl (Et, _-CH2CH3 ), 1-propyl (n-Pr, n-propyl, _-CH2CH2CH3 ), 2-propyl (i-Pr, _isopropyl , -CH( _CH3 ) ₂ ), 1-butyl (n-Bu, n _- butyl, _{-CH2CH2CH2CH3 )} , 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH( _CH3 ) _{2 )} , ₂ -butyl (s-Bu, sec-butyl, -CH( _CH3 ) _2CH3 ), 2- _methyl -2-propyl (t-Bu, tert-butyl, -C( _CH3 ) ₃ ), 1 _- pentyl ( _{n -} pentyl, _{-CH2CH2CH2CH2CH3} ), ₂ -pentyl (-CH( _{CH3 ) 2CH2CH3 )} ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ ) ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ ) ), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH _{3 )} ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ ) ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ ) and octyl (-(CH ₂ ) ₇ CH ₃ ).

"Alkoxy" refers to an alkyl group having an oxygen atom attached to the alkyl group at the attachment point: alkyl-O-. Like alkyl groups, alkoxy groups can have any suitable number of carbon atoms, such as _C1-6 . Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups can be further substituted by the various substituents described herein. Alkoxy groups can be substituted or unsubstituted.

"Hydroxy group" refers to –OH.

As used in this article, “halogen” or “halogen” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

As used herein, “haloalkyl” means an alkyl group in which one or more hydrogen atoms of the alkyl group are independently replaced by halogen substituents, which may be the same or different. For example, a _C1-4 haloalkyl group is a _C1-4 alkyl group in which one or more hydrogen atoms of the _C1-4 alkyl group have been replaced by halogen substituents. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, fluorochloromethyl, difluoromethyl, difluorochloromethyl, trifluoromethyl, 1,1,1-trifluoroethyl, and pentafluoroethyl.

“Cycloalkyl” refers to a single saturated or partially unsaturated full carbon ring having 3 to 20 cyclic carbon atoms (i.e., _C3-20 cycloalkyl) (e.g., 3 to 12 cyclic atoms, such as 3 to 10 cyclic atoms, or 3 to 8 cyclic atoms, or 3 to 6 cyclic atoms, or 3 to 5 cyclic atoms, or 3 to 4 cyclic atoms). The term “cycloalkyl” also includes polyfused, saturated, and partially unsaturated full carbon ring systems (e.g., ring systems containing 2, 3, or 4 carbon rings). Thus, cycloalkyl includes polycyclic carbon rings, such as bicyclic carbon rings (e.g., bicyclic carbon rings having about 6 to 12 cyclic carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane) and polycyclic carbon rings (e.g., tricyclic and tetracyclic carbon rings having up to about 20 cyclic carbon atoms). When valence requirements permit, the rings of polyfused ring systems can be interconnected by fused bonds, spiral bonds, or bridging bonds. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl.

As used herein, “heterocyclic group” or “heterocyclic alkyl group” refers to a single saturated or partially unsaturated non-aromatic ring or non-aromatic polycyclic system having at least one heteroatom (i.e., at least one cyclic heteroatom selected from oxygen, nitrogen, and sulfur) in the ring. Unless otherwise specified, heterocyclic groups have 3 to about 20 cyclic atoms, for example 3 to 12 cyclic atoms, such as 3 to 10 cyclic atoms, or 3 to 8 cyclic atoms, or 3 to 6 cyclic atoms, or 3 to 5 cyclic atoms, or 4 to 6 cyclic atoms, or 4 to 5 cyclic atoms. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3, 4, 5, 6, or 7-membered ring) having about 1 to 6 cyclic carbon atoms and about 1 to 3 cyclic heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. When valence requirements permit, the rings of a polyfused ring (e.g., a bicyclic heterocyclic group) system can be connected to each other by fusion, spirocyclic, and bridging. Heterocyclic compounds include, but are not limited to, aziridine, imidazoline, morpholine, ethylene oxide (epoxide), oxacyclobutane, thioheterobutane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidone, tetrahydrofuran, tetrahydrothiophene, dihydropyridine, tetrahydropyridine, quinine ring, 2-oxa-6-azaspiro[3.3]hept-6-yl, 6-oxa-1-azaspiro[3.3]hept-1-yl, 2-Thia-6-azaspiro[3.3]hept-6-yl, 2,6-diazaspiro[3.3]hept-2-yl, 2-azabicyclo[3.1.0]hex-2-yl, 3-azabicyclo[3.1.0]hexyl, 2-azabicyclo[2.1.1]hexyl, 2-azabicyclo[2.2.1]hept-2-yl, 4-azaspiro[2.4]heptyl, 5-azaspiro[2.4]heptyl, etc.

As used herein, “aryl” refers to a single all-carbon aromatic ring or a polyfused all-carbon ring system, wherein at least one ring is aromatic. For example, in some embodiments, the aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Aryl groups include phenyl radicals. Aryl groups also include polyfused ring systems having about 9 to 20 carbon atoms (e.g., ring systems comprising 2, 3, or 4 rings), wherein at least one ring is aromatic, and wherein the other rings may be aromatic or non-aromatic (i.e., carbon rings). Such polyfused ring systems are optionally substituted by one or more (e.g., 1, 2, or 3) oxo groups on any carbon ring portion of the polyfused ring system. When valence requirements permit, the rings of a polyfused ring system may be interconnected by fused bonds, spiral bonds, or bridging bonds. It should also be understood that when referring to a range of atomically defined aryl groups (e.g., 6-10-membered aryl groups), the atomic range refers to the total number of ring atoms in that aryl group. For example, a 6-membered aryl group will include a phenyl group, and a 10-membered aryl group will include a naphthyl group and a 1,2,3,4-tetrahydronaphthyl group. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthraceneyl, etc.

As used herein, "heteroaryl" refers to a single aromatic ring having at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen, and sulfur; "heteroaryl" also includes polyfused ring systems having at least one such aromatic ring, which will be further described below. Thus, "heteroaryl" includes a single aromatic ring having about one to six carbon atoms and about one to four heteroatoms selected from oxygen, nitrogen, and sulfur. Sulfur and nitrogen atoms may also be present in oxidized forms, provided that the ring is aromatic. Exemplary heteroaryl ring systems include, but are not limited to, pyridinyl, pyrimidinyl, oxazolyl, or furanyl. "Heteroaryl" also includes polyfused ring systems (e.g., ring systems comprising 2, 3, or 4 rings), wherein the heteroaryl group as defined above is fused with one or more rings selected from heteroaryl (to form, for example, 1,8-naphthidyl), heterocyclic (to form, for example, 1,2,3,4-tetrahydro-1,8-naphthidyl), carbocyclic (to form, for example, 5,6,7,8-tetrahydroquinolinyl), and aryl (to form, for example, indazole) to form a polyfused ring system. Thus, a heteroaryl (single aromatic ring or polyfused ring system) has about 1-20 carbon atoms and about 1-6 heteroatoms within the heteroaryl ring. Such polyfused ring systems may optionally be substituted with one or more (e.g., 1, 2, 3, or 4) oxo groups on the carbocyclic or heterocyclic portion of the fused ring. When valence requirements permit, the rings of a polyfused ring system can be interconnected by fused bonds, spiro bonds, or bridging bonds. It should be understood that the rings of this polyfused ring system can be connected relative to each other in any order. It should be understood that the attachment points of the heteroaryl group or the polyfused ring system can be located on any suitable atom of the heteroaryl group or the polyfused ring system, including carbon atoms and heteroatoms (e.g., nitrogen). It should also be understood that when referring to a range of atomic-membered heteroaryl groups (e.g., 5- to 10-membered heteroaryl groups), the atomic range is the total number of ring atoms of the heteroaryl group and includes carbon atoms and heteroatoms. For example, a 5-membered heteroaryl group would include a thiazolyl group, and a 10-membered heteroaryl group would include a quinolinyl group. Exemplary heteroaryl groups include, but are not limited to, pyridyl, pyrroloyl, pyrazinyl, pyrimidinyl, pyrazinyl, thiophene, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzooxazolyl, inzolyl, quinoxolinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzoimidazolyl, thiophene, pyrrolo[2,3-b]pyridyl, quinazolinyl-4(3H)-one, and triazolyl.

"Compounds of this disclosure" includes compounds disclosed herein, such as compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In), including compounds of the embodiments.

"Pharmaceutically effective amount" means the amount of the compound of this disclosure that provides the desired therapeutic or pharmaceutical outcome in a formulation or combination thereof.

"Pharmaceutical acceptable excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for acceptable use in humans or livestock.

As used herein, “treatment” or “treat” or “treating” refers to a method for achieving a beneficial or desired outcome. For the purposes of this disclosure, beneficial or desired outcomes include, but are not limited to, the reduction of symptoms and/or the attenuation of the severity of symptoms and/or the prevention of the worsening of symptoms associated with a disease or condition. In one embodiment, “treatment” or “treating” includes one or more of the following: a) suppressing a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition, and/or attenuating the severity of a disease or condition); b) slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, delaying the worsening or progression of a disease or condition); and c) alleviating a disease or condition, such as causing the disappearance of clinical symptoms, improving disease status, delaying disease progression, improving quality of life, and/or prolonging survival.

"Prevention" refers to preventing or delaying the progression of clinical disease in patients with viral infections.

As used herein, “therapeutic effective amount” or “effective amount” means an amount that can effectively elicit the desired biological or medical response, including amounts of compounds sufficient to affect the disease when administered to a subject to treat the disease. Effective amounts will vary depending on the compound being treated, the disease and its severity, and age, weight, etc. Effective amounts may include a range of amounts. As understood in the art, an effective amount may be one or more doses; that is, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. Effective amounts may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered at an effective amount if, in combination with one or more other agents, a desired or beneficial outcome can be achieved or realized. The appropriate dose of any co-administered compounds may optionally be reduced due to the combined effects of the compounds (e.g., additive or synergistic effects).

As used herein, “co-administration” means administering a unit dose of the compound disclosed herein before or after administering a unit dose of one or more additional therapeutic agents, for example, administering the compound disclosed herein within seconds, minutes, or hours after administering one or more additional therapeutic agents. For example, in some embodiments, a unit dose of the compound disclosed herein is administered first, followed by a unit dose of one or more additional therapeutic agents within seconds or minutes. Alternatively, in other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by a unit dose of the compound disclosed herein within seconds or minutes. In some embodiments, a unit dose of the compound disclosed herein is administered first, followed by a unit dose of one or more additional therapeutic agents after several hours (e.g., 1-12 hours). In other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by a unit dose of the compound disclosed herein after several hours (e.g., 1-12 hours). Co-administration of the compound disclosed herein with one or more additional therapeutic agents generally means administering the compound disclosed herein and one or more additional therapeutic agents simultaneously or sequentially, such that a therapeutically effective amount of each agent is present in the patient.

Pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, and prodrugs of the compounds described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" means compounds, salts, compositions, dosage forms, and other substances that can be used to prepare pharmaceutical compositions suitable for veterinary or human use.

The compounds described herein can be prepared and/or formulated as pharmaceutically acceptable salts, or, where appropriate, as free bases. A "pharmaceutically acceptable salt" is a non-toxic salt of a compound in its free base form, possessing the desired pharmacological activity of a free base. These salts can be derived from inorganic acids, organic acids, or bases. For example, compounds containing basic nitrogen can be prepared as pharmaceutically acceptable salts by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propargylates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, and butyrates. Alkyne-1,4-diacidate, hexyne-1,6-diacidate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methanesulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, and mandelate. A list of other suitable pharmaceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

Examples of pharmaceutically acceptable salts of ^{the compounds disclosed herein also include salts derived from suitable bases such as alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., magnesium), ammonium, and NX 4+} ₍ where X is a C ₁ -C ₄ alkyl group). Base addition salts, such as sodium or potassium salts, are also included.

Also provided are compounds described herein, or pharmaceutically acceptable salts, isomers, or mixtures thereof, wherein one to n hydrogen atoms attached to a carbon atom may be replaced by a deuterium atom or D, where n is the number of hydrogen atoms in the molecule. As is known in the art, a deuterium atom is a non-radioactive isotope of a hydrogen atom. Such compounds can increase resistance to metabolism and are therefore used to increase the half-life of compounds described herein, or pharmaceutically acceptable salts, isomers, or mixtures thereof, when administered to mammals. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized, for example, by using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^15O , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , ^123I , and ^125I , respectively. Substitution with positron-emitting isotopes such as ^11C , ^18F , ^15O , and ^13N can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotope-labeled compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) can generally be prepared by means of a method similar to that described in the examples below, using an appropriate isotope-labeled reagent instead of the previously used unlabeled reagent.

The compounds of the embodiments disclosed herein, or their pharmaceutically acceptable salts, may include one or more asymmetric centers, and thus may produce enantiomers, diastereomers, and other stereoisomers that may be defined by absolute stereochemistry as (R)- or (S)- or for amino acids as (D)- or (L)-. This disclosure is intended to include all such possible isomers as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for preparing/separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain an alkene double bond or other geometrically asymmetric centers, and unless otherwise stated, the compounds are intended to include E and Z geometric isomers. Similarly, all tautomers are also intended to be included. When a compound is represented in its chiral form, it should be understood that the embodiments cover, but are not limited to, specific diastereomeric or enantiomerically enriched forms. When chirality is not specified but is present, it should be understood that the embodiments relate to specific diastereomeric or enantiomerically enriched forms; or racemic or non-racemic mixtures of such compounds. As used herein, a “non-racemic mixture” is a mixture of stereoisomers in a ratio not equal to 1:1.

A "racemate" is a mixture of enantiomers. The mixture may contain equal or unequal amounts of each enantiomer.

"Stereoisomers" are compounds with different chiralities of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. If a compound has one or more asymmetric centers or contains asymmetrically substituted double bonds, it may exist in stereoisomeric form and thus may be produced as individual stereoisomers or as mixtures. Unless otherwise specified, this specification is intended to include individual stereoisomers as well as mixtures. Methods for determining stereochemistry and isolating stereoisomers are well known in the art (see, for example, Chapter 4 of Advanced Organic Chemistry, 4th Edition, J. March, John Wiley and Sons, New York, 1992).

"Tautomers" refer to alternative forms of compounds with different proton positions, such as enol-ketone and imine-enamine tautomers, or tautomers containing heteroaryl groups attached to ring atoms of ring-NH- and ring=N-, such as pyrazole, imidazole, benzimidazole, triazole and tetraazole.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Dashes at the beginning or end of chemical groups are for convenience; chemical groups may be depicted without one or more dashes without losing their ordinary meaning. Wavy lines drawn through lines in the structure indicate the attachment point of a group. Dashed lines indicate optional bonds. Unless chemically or structurally required, the order in which chemical groups are written or their attachment point to the rest of the molecule does not indicate or imply directionality. For example, the group “ _-SO₂CH₂- ” is equivalent to “ _-CH₂SO₂- ”, and both can be attached in either direction. Similarly, an “arylalkyl” group may, for _{example ,} be attached to the rest of the molecule at the aryl or alkyl portion of the group. Prefixes such as “ _Cuv ” or ( _Cu - _Cv ) indicate that the following group has u to v carbon atoms. For example, “C₁ _- 6alkyl” and “ _C₁ - _C₆alkyl ” both indicate that the alkyl group has 1 to 6 carbon atoms.

As used herein, "solvent" refers to the result of the interaction between the solvent and the compound. Solvents of salts of the compounds described herein are also provided. Hydrates of the compounds described herein are also provided.

As used in this article, "prodrug" refers to a drug derivative that is converted into an active drug through some chemical or enzymatic pathways after being administered to the human body.

III. Compounds

This disclosure describes compounds of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), and (In).

In some embodiments, this disclosure provides compounds of formula (I):

Or its pharmaceutically acceptable salt, wherein:

The base is

^R1 and ^R2 are each independently H or -C(O) ^R1A , wherein ^R1A is a _C1-6 alkyl group;

^R3 is -N(H) ^R3A ;

^R3A is H, _-CH2OP (O)(OH) ₂ , or -C(O) ^R3D , where

R ^3D is a C _6-12 aryl or C _1-6 alkyl group optionally substituted with a C _3-6 cycloalkyl group;

R ^4A is O;

R ^4B and R ^4C are independently:

(A)-OH;

(B)–OR ^4B1 , where R ^4B1 is C _6-12 aryl;

(C) where

The subscript m is 0, 1, 2, 3, 4, or 5; and

Each R ^4D is independently a C _1-6 alkyl group;

(D) where

^R4E1 and ^R4E2 are each independently H or _C1-6 alkyl;

^R4F1 and ^R4F2 together form an oxo group;

R ^4G is a C _1-8 alkyl, C _3-8 cycloalkyl, or a 3- to 8-membered heterocyclic group having 1 to 3 heteroatoms selected from N, O, and S, optionally substituted with 1 to 3 R ^{4G1 .}

Each R ^4G1 is independently -OH, C _1-6 alkoxy, -(CH ₂ OCH ₂ ) _1-5 -CH ₃ , C _1-3 haloalkyl, or optionally C _3-8 cycloalkyl substituted with 1 to 3 R ^4G9 ;

Each R ^4G3 and R ^4G9 is independently a _C1-6 alkyl group; or

(E)-(OP(O)(OH)) _1-2 -OH; and

^R5A and ^R5B are each independently _C1-6 alkyl groups substituted with -OP(O)(OH) ₂ .

In some embodiments, the compound may be represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the base is

In some embodiments, the compound may be represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the base is

In some embodiments, the compound may be represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the base is

In some embodiments, the compound may be represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the base is

In some embodiments, the compound may be represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the base is

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R1 and ^R2 are each independently H or -C(O) ^R1A , wherein ^R1A is a _C1-6 alkyl group. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R1 and ^R2 are each H. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R1 and ^R2 are each -C(O) ^R1A , and ^R1A is a _C1-6 alkyl group. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R1 and ^R2 are each -C(O) ^R1A ; and ^R1A is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R1 and ^R2 are each -C(O) ^R1A ; and ^R1A is methyl, ethyl, or isopropyl.

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ia):

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R3 is -N(H) ^R3A ; ^R3A is H, _-CH2OP (O)(OH) ₂ , or -C(O) ^R3D , wherein ^R3D is a _C6-12 aryl or _C1-6 alkyl group optionally substituted with a _C3-6 cycloalkyl group. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R3A is H. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R3A is _-CH2OP (O)(OH) ₂ . In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R3A is -C(O) ^R3D , and ^R3D is a _C6-12 aryl or _C1-6 alkyl group optionally substituted with a _C3-6 cycloalkyl group. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R3A is -C(O) ^R3D , and ^R3D is a phenyl group or a _C1-3 alkyl group optionally substituted with a _C3-6 cycloalkyl group.

In some embodiments, the compound may be represented by formula (Ia) or a pharmaceutically acceptable salt thereof, wherein ^R3 is -N(H) ^R3A ; ^R3A is H or -C(O) ^R3D ; and ^R3D is phenyl or optionally a _C1-3 alkyl group substituted with a _C3-6 cycloalkyl group. In some embodiments, the compound may be represented by formula (Ia) or a pharmaceutically acceptable salt thereof, wherein ^R3 is _–NH2 .

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), or (Id) or a pharmaceutically acceptable salt thereof, wherein ^R4B and ^R4C are each independently:

(A)-OH;

(B)–OR ^4B1 , where R ^4B1 is C _6-12 aryl;

(C) where

The subscript m is 0, 1, 2, 3, 4, or 5; and

Each R ^4D is independently a C _1-6 alkyl group;

(D) where

^R4E1 and ^R4E2 are each independently H or _C1-6 alkyl;

^R4F1 and ^R4F2 together form an oxo group;

R ^4G is a C _1-8 alkyl, C _3-8 cycloalkyl, or a 3- to 8-membered heterocyclic group having 1 to 3 heteroatoms selected from N, O, and S, optionally substituted with 1 to 3 R ^{4G1 .}

Each R ^4G1 is independently -OH, C _1-6 alkoxy, -(CH ₂ OCH ₂ ) _1-5 -CH ₃ , C _1-3 haloalkyl, or optionally C _3-8 cycloalkyl substituted with 1 to 3 R ^4G9 ;

Each R ^4G3 and R ^4G9 is independently a _C1-6 alkyl group; or

(E)-(OP(O)(OH)) _1-2 -OH.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), or (Id) or a pharmaceutically acceptable salt thereof, wherein ^R4B and ^R4C are each independently:

(C) where

The subscript m is 0, 1, 2, 3, 4, or 5; and

Each R ^4D is independently a C _1-6 alkyl group;

(D) where

^R4E1 and ^R4E2 are each independently H or _C1-6 alkyl;

^R4F1 and ^R4F2 together form an oxo group;

R ^4G is a C _1-8 alkyl, C _3-8 cycloalkyl, or a 3- to 8-membered heterocyclic group having 1 to 3 heteroatoms selected from N, O, and S, optionally substituted with 1 to 3 R ^{4G1 .}

Each R ^4G1 is independently -OH, C _1-6 alkoxy, -(CH ₂ OCH ₂ ) _1-5 -CH ₃ , C _1-3 haloalkyl, or optionally C _3-8 cycloalkyl substituted with 1 to 3 R ^4G9 ;

Each R ^4G3 and R ^4G9 is independently a _C1-6 alkyl group; or

(E)-(OP(O)(OH)) _1-2 -OH.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), or (Id) or a pharmaceutically acceptable salt thereof, wherein ^R4B and ^R4C are each independently:

(A)-OH;

(B)–OR ^4B1 , where R ^4B1 is a naphthyl group;

(C) where

The subscript m is 0 or 1; and

R ^4D is a _C1-6 alkyl group;

(D) where

^R4E1 is a _C1-3 alkyl group;

R ^4E2 is H;

^R4F1 and ^R4F2 together form an oxo group;

R ^4G is a _C1-8 alkyl, _C4-6 cycloalkyl, or optionally substituted with one R ^4G1.

A 4- to 6-membered heterocyclic group with one heteroatom selected from N and O, substituted with R ^4G3 ;

Each R ^4G1 is independently -OH, C _1-4 alkoxy, -(CH ₂ OCH ₂ ) _1-2 -CH ₃ , C _1-3 haloalkyl, or optionally a C _3-6 cycloalkyl substituted with one R ^4G9 ;

Each R ^4G3 and R ^4G9 is independently a _C1-3 alkyl group; or

(E)-(OP(O)(OH)) _1-2 -OH.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (Ih), or (Ik) or a pharmaceutically acceptable salt thereof, wherein R ^4B is:

(B)–OR ^4B1 , where R ^4B1 is a naphthyl group; or

(C) where

The subscript m is 0 or 1; and

Each R ^4D is independently tert-butyl; or

(E)-(OP(O)(OH)) _1-2 -OH.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), or (If) or a pharmaceutically acceptable salt thereof, wherein R ^4C is:

(A)-OH; or

(D) where

R ^4E1 is methyl;

R ^4E2 is H;

^R4F1 and ^R4F2 together form an oxo group; and

R ^4G is

Each of the following can be optionally substituted with OH, methoxy, ethoxy, propoxy, butoxy, _CF3 , Me( _CH2OCH2 ) _2- , cyclopropyl or 1-methylcyclopropyl, and the following substituted compounds: methyl, ethyl, n-propyl, isopropyl, n _- butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-

Dimethyl-butyl, 2-ethyl-butyl, or 2-n-propyl-pentyl

Cyclobutyl, cyclopentyl, or cyclohexyl

Each of the pyrrolidinyl, oxecyclobutyl, or tetrahydropyranyl groups may be optionally substituted with methyl, ethyl, n-propyl, or isopropyl groups.

In some embodiments, the compound may be represented by formula (I), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R5A and ^R5B are each independently a _C1-6 alkyl group substituted with -OP(O)(OH) ₂ . In some embodiments, the compound may be represented by formula (I), (Ib), or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R5A and ^R5B are each independently a _C1-3 alkyl group substituted with -OP(O)(OH) ₂ . In some embodiments, the compound may be represented by formula (I) or (Ic) or a pharmaceutically acceptable salt thereof, wherein ^R5B is _-CH2OP (O)(OH) ₂ . In some embodiments, the compound may be represented by formula (I) or (Ib) or a pharmaceutically acceptable salt thereof, wherein ^R5A is _-CH2OP (O)(OH) ₂ .

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ib):

Where ^R3A is H; and ^R5A is _-CH2OP (O)(OH) ₂ .

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ic):

Where ^R3A is H; and ^R5B is _-CH2OP (O)(OH) ₂ .

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Id):

            

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ie):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by the formula (If):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by the formula (Ig):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ih):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ij):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by formula (Ik):

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by the formula (Im):

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), or (If) or a pharmaceutically acceptable salt thereof, wherein R ^4C is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), or (If) or a pharmaceutically acceptable salt thereof, wherein R ^4C is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), or (If) or a pharmaceutically acceptable salt thereof, wherein R ^4C is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), ⁽ Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is a _C1-8 alkyl, _C3-8 cycloalkyl, or a 3- to 8-membered heterocyclic group having 1 to 3 heteroatoms selected from N, O, and S, optionally substituted with 1 to 3 ^R4G3 ; each ^R4G1 is independently -OH, _C1-6 alkoxy, -( _CH2OCH2 ) _1-5 - _CH3 , _C1-3 haloalkyl, or _{a C3-8} cycloalkyl optionally substituted with 1 to 3 ^R4G9 ; and each ^R4G3 and ^R4G9 is independently _C1-6 alkyl.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein R ^4G is

Each of the following can be optionally substituted with OH, methoxy, ethoxy, propoxy, butoxy, _CF3 , Me( _CH2OCH2 ) _2- , cyclopropyl, or 1-methylcyclopropyl, and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec _- butyl, tert-butyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, or 2-n-propyl-pentyl.

Cyclobutyl, cyclopentyl, or cyclohexyl

Each of the pyrrolidinyl, oxecyclobutyl, or tetrahydropyranyl groups may be optionally substituted with methyl, ethyl, n-propyl, or isopropyl groups.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein R ^4G is

The methyl group may be optionally substituted with Me( _CH₂OCH₂ ) _₂- , cyclopropyl _, or 1-methylcyclopropyl.

Ethyl groups optionally substituted with butoxy groups,

The n-propyl group may be optionally substituted with a methoxy group.

Isopropyl, n-butyl,

The isobutyl group may be optionally substituted with OH, methoxy, or _CF3 .

n-Pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, 2-n-propyl-pentyl

Cyclobutyl, cyclohexyl

N-methylpyrrolidinyl, oxetyl butyl or tetrahydropyranyl.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein R ^4G is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, 2-n-propyl-pentyl, cyclobutyl, cyclohexyl, N-methyl-pyrrolidinyl, oxetyl, or tetrahydropyranyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein R ^4G is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, or 2-n-propyl-pentyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is isopropyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, or 2-ethyl-butyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is isopropyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is n-hexane. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is 2,2-dimethyl-butyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein ^R4G is 3,3-dimethyl-butyl. In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein R ^4G is 2-ethyl-butyl.

In some embodiments, the compound may be represented by formula (Ia) or a pharmaceutically acceptable salt thereof, wherein

^R1 and ^R2 are both H or -C(O) ^R1A , where ^R1A is methyl, ethyl or isopropyl;

^R3 is _–NH2 ;

R ^4B is:

-OPh; and

R ^4C is:

in

R ^4G is

Each of the following can be optionally substituted with OH, methoxy, ethoxy, propoxy, butoxy, _CF3 , Me( _CH2OCH2 ) _2- , cyclopropyl, or 1-methylcyclopropyl, and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec _- butyl, tert-butyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, or 2-n-propyl-pentyl.

Cyclobutyl, cyclopentyl, or cyclohexyl

Each of the pyrrolidinyl, oxecyclobutyl, or tetrahydropyranyl groups may be optionally substituted with methyl, ethyl, n-propyl, or isopropyl groups.

In some embodiments, the compounds of this disclosure or pharmaceutically acceptable salts thereof are represented by the formula (In):

in

^R1 and ^R2 are both H or -C(O) ^R1A , where ^R1A is methyl, ethyl, or isopropyl; and

R ^4G is

Each of the following can be optionally substituted with OH, methoxy, ethoxy, propoxy, butoxy, _CF3 , Me( _CH2OCH2 ) _2- , cyclopropyl, or 1-methylcyclopropyl, and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec _- butyl, tert-butyl, n-pentyl, neopentyl, n-hexane, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 2-ethyl-butyl, or 2-n-propyl-pentyl.

Cyclobutyl, cyclopentyl, or cyclohexyl

Each of the pyrrolidinyl, oxecyclobutyl, or tetrahydropyranyl groups may be optionally substituted with methyl, ethyl, n-propyl, or isopropyl groups.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the compounds in Tables 1A, 1B, 1C, and 1D.

Table 1A.

Table 1B.

Table 1C.

Table 1D.

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, the compound may be represented by formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, wherein the compound is:

In some embodiments, this disclosure provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is:

In vivo metabolites of the compounds described herein also fall within the scope of this document, provided that such products are novel and not readily apparent relative to existing technology. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc., of the applied compound, primarily due to enzymatic processes. Therefore, this includes methods that involve exposing the compound to a mammal for a sufficient period of time to produce its metabolites, thus generating novel and non-obvious compounds. Such products are typically identified by the following process: preparing a radiolabeled (e.g., ^14C or ^3H ) compound, administering it parenterally at a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal (such as a rat, mouse, guinea pig, monkey) or human, allowing sufficient time for metabolism (typically about 30 seconds to 30 hours), and separating its metabolites from urine, blood, or other biological samples. These products are readily separated because they are labeled (other products are separated using antibodies capable of binding to epitopes that survive in the metabolites). The metabolite structure is determined in a conventional manner (e.g., by MS or NMR analysis). Typically, the analysis of metabolites is performed in the same manner as routine drug metabolism studies well known to those skilled in the art. Transformation products can be used for the diagnostic determination of the therapeutic dose of compounds, provided they are not found in vivo in another way, even if they do not have HSV antiviral activity themselves.

Formulations and methods for determining the stability of compounds in alternative gastrointestinal secretions are known. Compounds are defined herein as stable in the gastrointestinal tract, wherein less than about 50 mol% of the protecting group is deprotected in alternative intestinal or gastric juices after incubation at 37°C for 1 hour. The fact that compounds are stable in the gastrointestinal tract does not mean they cannot be hydrolyzed in vivo. Prodrugs are generally stable in the digestive system but can be hydrolyzed substantially to the parent drug in the digestive lumen, liver, lungs, or other metabolic organs, or generally intracellularly. As used herein, a prodrug is understood to be a compound chemically engineered to efficiently release the parent drug after overcoming the biological barriers of oral delivery.

IV. Pharmaceutical Preparations

In some embodiments, this disclosure provides a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of the disclosed formula or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. This document also provides a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), and (In) thereof, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

The compounds described herein are formulated with conventional carriers and excipients. Tablets will contain excipients, flow aids, fillers, binders, etc. Aqueous formulations are prepared aseptically and will generally be isotonic when intended for delivery by non-oral administration. All formulations will optionally contain excipients, such as those described in the "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, etc. The pH range of the formulations is from about 3 to about 11, for example, from about 7 to 10.

While active ingredients can be administered alone, it may be preferred to provide them as pharmaceutical formulations. Formulations for veterinary and human use contain at least one active ingredient as defined above, along with one or more acceptable carriers and optional other therapeutic ingredients, particularly those additional therapeutic ingredients discussed herein. The carrier must be “acceptable,” meaning it is compatible with the other components of the formulation and physiologically harmless to its recipient.

Formulations include those suitable for the aforementioned routes of administration. Formulations can conveniently exist in unit dosage forms and can be prepared by any suitable method. Techniques and formulations are commonly found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods involve the step of associating the active ingredient with a carrier constituting one or more auxiliary ingredients. Generally, formulations are prepared by uniformly and tightly associating the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may exist as discrete units such as capsules, granules, or tablets, each containing a predetermined amount of the active ingredient; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions. The active ingredient may also be administered as pills, granules, or pastes.

Tablets are made by compression or molding, optionally using one or more excipients. Compressed tablets are prepared by compressing an active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets are prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored, and optionally formulated to provide a slow or controlled release of the active ingredient therefrom.

For infections of the eyes or other external tissues (e.g., the mouth and skin), the formulation is preferably applied as a topical ointment or cream containing active ingredients in amounts, for example, from 0.075% w/w to 20% w/w (including active ingredients in increments of 0.1% w/w, such as 0.6% w/w, 0.7% w/w, etc.), preferably from 0.2% w/w to 15% w/w, and most preferably from 0.5% w/w to 10% w/w. When formulated as an ointment, the active ingredients may be used with a paraffin base or a water-miscible ointment base. Alternatively, the active ingredients may be formulated as a cream with an oil-in-water emulsion base.

If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w polyols, i.e., alcohols having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG400), and mixtures thereof. Topical formulations may ideally include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

The oil phase of an emulsion can be composed of known components in a known manner. While this phase may consist only of emulsifiers (or simply emulsifiers), it ideally includes at least one emulsifier with fats or oils, or with a mixture of both. Preferably, hydrophilic emulsifiers are included together with lipophilic emulsifiers that act as stabilizers. It is also preferable to include both oils and fats. Emulsifiers, with or without stabilizers, together constitute a so-called emulsified wax, and the wax, together with the oils and fats, constitutes a so-called emulsified ointment matrix, which forms the oily dispersed phase of the ointment formulation.

Emulsifiers and emulsion stabilizers suitable for formulations include 60, 80, cetearyl alcohol, benzyl alcohol, myristicin, glyceryl monostearate, and sodium lauryl sulfate.

The appropriate oil or fat is selected for the formulation based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining, and washable product with a suitable consistency to prevent leakage from tubes or other containers. Straight-chain or branched monoalkyl or dialkyl esters can be used, such as diisohexyl adipate, isohexadecanoyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or mixtures of branched esters known as Crodamol CAP, the last three being preferred esters. These esters can be used alone or in combination, depending on the desired properties. Alternatively, high-melting-point lipids, such as white soft paraffin and/or liquid paraffin or other mineral oils, can be used.

The pharmaceutical formulations described herein comprise a combination with one or more pharmaceutically acceptable carriers or excipients and optional other therapeutic agents. Pharmaceutical formulations containing an active ingredient can be in any form suitable for the intended method of administration. When intended for oral use, they may be prepared, for example, tablets, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, solutions, syrups, or elixirs. Compositions intended for oral use may be prepared according to any method used to manufacture pharmaceutical compositions, and such compositions may contain one or more pharmaceutical agents, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Tablets containing an active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for manufacturing tablets are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known techniques, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract, thereby providing sustained action over a longer period. For example, delaying materials such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with waxes.

Formulations intended for oral use may also be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (such as calcium phosphate or kaolin) or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium (such as peanut oil, liquid paraffin, or olive oil).

Aqueous suspensions contain active substances mixed with excipients suitable for manufacturing aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; and dispersing or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of olefinic oxygen and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecanethoxycetyl alcohol), and condensation products of ethylene oxide and esters derived from fatty acids and hexyl anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethylparaben or n-propylparaben, one or more colorants, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

Oily suspensions can be prepared by suspending the active ingredients in vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil) or mineral oils (such as liquid paraffin). Oral suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (such as those mentioned above) and flavoring agents may be added to provide palatable oral formulations. These compositions may be preserved by adding antioxidants (such as ascorbic acid).

Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide an active ingredient that can be mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be vegetable oils (such as olive oil or peanut oil), mineral oils (such as liquid paraffin), or mixtures thereof. Suitable emulsifiers include naturally occurring gums, such as gum arabic and tragacanth; naturally occurring phospholipids, such as soybean lecithin; esters or metaesters derived from fatty acids and hexitan anhydrides, such as sorbitan monooleate; and condensation products of these metaesters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavoring agents. Syrups and elixirs may be formulated with sweeteners such as glycerin, sorbitol, or sucrose. Such formulations may also contain modifiers, preservatives, flavoring agents, or coloring agents.

The pharmaceutical composition may be in the form of a sterile injectable or intravenous preparation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents already mentioned above, according to known techniques. The sterile injectable or intravenous preparation may also be a sterile injectable solution or suspension in a non-toxic, parenteral-acceptable diluent or solvent (such as a solution in 1,3-butanediol), or prepared as a lyophilized powder. Acceptable solvents and media are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid may also be used in the preparation of the injection.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. For example, a sustained-release formulation intended for oral administration to humans may contain approximately 1 mg to 1000 mg of the active material, compounded with an appropriate and convenient amount of carrier material, which may vary between approximately 5% to approximately 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable dosages. For example, an aqueous solution intended for intravenous infusion may contain approximately 3 to 500 μg of the active ingredient per milliliter to allow for the infusion of an appropriate volume at a rate of approximately 30 mL/hr.

Formulations suitable for topical application to the eyes also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly in an aqueous solution of the active ingredient. The active ingredient is preferably present in such formulations at a concentration of 0.5% to 20%, advantageously 0.5% to 10%, and particularly about 1.5% w/w.

Preparations suitable for topical application in the oral cavity include lozenges containing flavoring active ingredients, typically sucrose and gum arabic or tragacanth; tablets containing inert active ingredients, such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes containing the active ingredients in a suitable liquid carrier.

Formulations for rectal administration may be provided as suppositories with a suitable matrix, including, for example, cocoa butter or salicylates.

Formulations suitable for intrapulmonary or nasal administration have particle sizes ranging from, for example, 0.1 micrometers to 500 micrometers (such as 0.5 micrometers, 1 micrometer, 30 micrometers, 35 micrometers, etc.) and are administered by rapid inhalation through the nasal passage or by oral inhalation to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered together with other therapeutic agents, such as compounds used to date for the treatment or prevention of pulmonary viral infections, as described below.

Another embodiment provides a novel, effective, safe, non-irritating, and physiologically compatible inhalable composition comprising a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof, suitable for treating pulmonary viral infections and potentially associated bronchiolitis. Preferred pharmaceutically acceptable salts are inorganic acid salts, including hydrochlorides, hydrobromides, sulfates, or phosphates, as they may cause less lung irritation. Preferably, the inhalable formulation is delivered into the bronchial space in the form of an aerosol comprising particles with a median mass aerodynamic diameter (MMAD) between about 1 μm and 5 μm. Preferably, compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) are formulated for aerosol delivery using a nebulizer, a pressurized metered-dose inhaler (pMDI), or a dry powder inhaler (DPI).

Non-limiting examples of nebulizers include atomizing nebulizers, jet nebulizers, ultrasonic nebulizers, pressurized nebulizers, vibrating perforated plate nebulizers, or equivalent nebulizers, including those utilizing adaptive aerosol delivery technologies (Denyer, J. Aerosol Medicine Pulmonary Drug Delivery 2010, 23 Supplement 1, S1-S10). Jet nebulizers use air pressure to break down a liquid solution into aerosol droplets. Ultrasonic nebulizers operate by using piezoelectric crystals to shear liquid into smaller aerosol droplets. Pressurized nebulizer systems force a solution under pressure through a small orifice to generate aerosol droplets. Vibrating perforated plate devices utilize rapid vibration to shear a liquid stream into appropriate droplet sizes.

In a preferred embodiment, a nebulizer capable of atomizing a formulation of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) into particles with the desired MMAD is used to deliver the atomized formulation into the bronchial space in the form of an aerosol containing particles with an MMAD primarily between about 1 μm and 5 μm. For optimal therapeutic effect and to avoid upper respiratory tract and systemic side effects, most atomized particles should not have an MMAD greater than about 5 μm. If the aerosol contains a large number of particles with an MMAD greater than 5 μm, these particles will deposit in the upper airway, reducing the amount of drug delivered to sites of inflammation and bronchoconstriction in the lower respiratory tract. If the aerosol has an MMAD less than about 1 μm, these particles tend to remain suspended in the inhaled air and are subsequently exhaled.

When formulated and delivered according to the methods described herein, the aerosol formulation for nebulization delivers a therapeutically effective dose of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) to the site of pulmonary viral infection. The amount of drug administered must be adjusted to reflect the delivery efficiency of a therapeutically effective dose of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In). In a preferred embodiment, the combination of the aqueous aerosol formulation with a nebulizer, jet nebulizer, pressurized nebulizer, vibrating perforated plate nebulizer, or ultrasonic nebulizer allows (depending on the nebulizer) delivery of at least 20% to about 90%, typically about 70%, of the applied dose of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) into the airway. In a preferred embodiment, at least about 30% to about 50% of the active compound is delivered. More preferably, about 70% to about 90% of the active compound is delivered.

In another embodiment, formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In), or a pharmaceutically acceptable salt thereof, are delivered as a dry, inhalable powder. The compound is administered intrabronchially as a dry powder formulation to efficiently deliver fine particles of the compound into the bronchial space using a dry powder or metered-dose inhaler. For delivery via DPI, compounds of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) are processed into particles with MMAD primarily between about 1 μm and about 5 μm by milling spray drying, critical fluid processing, or precipitation from solution. Media milling, jet milling, and spray drying apparatus and procedures capable of producing particle sizes with MMAD between about 1 μm and about 5 μm are well known in the art. In one embodiment, the excipient is added to a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) before processing into particles of the desired size. In another embodiment, the excipient is blended with particles of the desired size to facilitate the dispersion of the drug particles, for example by using lactose as an excipient.

Particle size determination is performed using devices well known in the art. For example, a multi-stage Anderson cascade impactor or other suitable methods (such as those specifically cited in Chapter 601 of the United States Pharmacopeia) are used as devices for characterizing the dosage and aerosols within dry powder inhalers.

In another preferred embodiment, a device such as a dry powder inhaler or other dry powder dispersing device is used to deliver compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) in dry powder form. Non-limiting examples of dry powder inhalers and devices include those disclosed in US 5,458,135; US 5,740,794; US 5775320; US 5,785,049; US 3,906,950; US 4,013,075; US 4,069,819; US 4,995,385; US 5,522,385; US 4,668,218; US 4,667,668; US 4,805,811; and US 5,388,572. There are two main designs for dry powder inhalers. One design is a metering device in which a reservoir for the drug is placed within the device, and the patient adds a dose of the drug to the inhalation chamber. A second design is a factory-metered device, where each individual dose is manufactured in a separate container. Both systems rely on formulating the drug into small particles with an MMAD of 1 μm to approximately 5 μm, and typically involve co-formulation with larger excipient particles (such as, but not limited to, lactose). The drug powder is placed in the inhalation chamber (either metered by the device or by breaking down a factory-metered dose), and the patient's inhalation airflow accelerates the powder out of the device and into the mouth. The non-laminar properties of the powder path cause the excipient-drug aggregates to break down, and the mass of the larger excipient particles causes them to impact the back of the throat, while the smaller drug particles deposit deep in the lungs. In a preferred embodiment, a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof is delivered in dry powder form using any type of dry powder inhaler as described herein, wherein the MMAD of the dry powder, which does not contain any excipients, is primarily in the range of 1 μm to about 5 μm.

In another embodiment, a metered-dose inhaler is used to deliver compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) in dry powder form. Non-limiting examples of metered-dose inhalers and devices include those disclosed in US 5,261,538; US 5,544,647; US 5,622,163; US 4,955,371; US 3,565,070; US 3,361,306; and US 6,116,234. In a preferred embodiment, a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) or a pharmaceutically acceptable salt thereof is delivered in dry powder form using a metered-dose inhaler, wherein the MMAD of the dry powder, which does not contain any excipients, is primarily in the range of about 1-5 μm.

Preparations suitable for vaginal application may be provided in the form of pessaries, tampons, creams, gels, pastes, foams or sprays, and contain, in addition to the active ingredient, a suitable carrier known in the art.

Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes to make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

The formulation is present in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions, requiring only immediate addition of a sterile liquid carrier, such as water for injection, before use. Temporary injectable solutions and suspensions are prepared from sterile powders, granules, and tablets of the aforementioned types. Preferred single-dose formulations are those containing a daily dose or a sub-daily dose of the active ingredient as described above, or a suitable portion thereof.

It should be understood that, in addition to the ingredients specifically mentioned above, these formulations may include other reagents conventional in the field related to the type of formulation discussed, such as flavoring agents, those suitable for oral administration.

Further, veterinary compositions comprising at least one active ingredient as defined above and its veterinary carrier are provided.

Veterinary drug carriers are materials that can be used to administer compositions and can be solid, liquid, or gaseous materials. They are otherwise inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary drug compositions can be administered orally, parenterally, or via any other desired route.

The compounds described herein are intended to provide controlled-release pharmaceutical formulations (“controlled-release formulations”) containing one or more compounds as active ingredients, wherein the release of the active ingredient is controlled and modulated to allow administration at a lower frequency or to improve the pharmacokinetic or toxicological characteristics of a given active ingredient.

The effective dose of the active ingredient depends at least on the nature of the condition being treated, its toxicity, whether the compound is used prophylactically (at a lower dose) or against an active viral infection, the delivery method, and the pharmaceutical formulation, and will be determined by clinicians using routine dose-escalation studies. The dose is expected to be from about 0.0001 to about 100 mg/kg body weight per day; typically, from about 0.01 to about 10 mg/kg body weight per day; more typically, from about 0.01 to about 5 mg/kg body weight per day; and most typically, from about 0.05 to about 0.5 mg/kg body weight per day. For example, the candidate daily dose range for an adult weighing approximately 70 kg would be from 1 mg to 1000 mg, preferably from 5 mg to 500 mg, and could be in single or multiple doses.

V. Route of application

One or more of the compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) (referred to herein as the active ingredient) may be administered via any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, local (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It should be understood that preferred routes may vary depending on, for example, the condition of the recipient. An advantage of the compounds herein is that they are orally bioavailable and can be administered orally.

The compounds disclosed herein (also referred to herein as the active ingredients) may be administered via any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It should be understood that preferred routes may vary depending on, for example, the recipient's condition. An advantage of some of the compounds disclosed herein is that they are orally bioavailable and can be administered orally.

The compounds disclosed herein can be administered to an individual for the required period of time or duration according to an effective dosing regimen, such as at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months or longer. In one variant, the compounds are administered on a daily or intermittent schedule throughout the individual's life.

The dosage or frequency of administration of the compounds disclosed herein may be adjusted during treatment based on the judgment of the physician administering the medication.

The compound can be applied in an effective amount to an individual (e.g., a human). In some embodiments, the compound is administered once daily.

The compound may be administered by any available route and means, such as by oral or parenteral (e.g., intravenous) administration. Therapeutic amounts of the compound may include from about 0.00001 mg/kg body weight/day to about 10 mg/kg body weight/day, such as from about 0.0001 mg/kg body weight/day to about 10 mg/kg body weight/day, or such as from about 0.001 mg/kg body weight/day to about 1 mg/kg body weight/day, or such as from about 0.01 mg/kg body weight/day to about 1 mg/kg body weight/day, or such as from about 0.05 mg/kg body weight/day to about 0.5 mg/kg body weight/day, or such as from about 0.3 mg to about 30 mg/day, or such as from about 30 mg to about 300 mg/day.

The compounds disclosed herein may be combined with one or more additional therapeutic agents at any dose of the disclosed compounds (e.g., 1 mg to 1000 mg of the compound). Therapeuticly effective doses may include about 1 mg/dose to about 1000 mg/dose, such as about 50 mg/dose to about 500 mg/dose, or such as about 100 mg/dose to about 400 mg/dose, or such as about 150 mg/dose to about 350 mg/dose, or such as about 200 mg/dose to about 300 mg/dose. Other therapeutically effective doses of the compounds disclosed herein are about 100 mg/dose, about 125 mg/dose, about 150 mg/dose, about 175 mg/dose, about 200 mg/dose, about 225 mg/dose, about 250 mg/dose, about 275 mg/dose, about 300 mg/dose, about 325 mg/dose, about 350 mg/dose, about 375 mg/dose, about 400 mg/dose, about 425 mg/dose, about 450 mg/dose, about 475 mg/dose, or about 500 mg/dose. Other therapeutically effective amounts of the compounds disclosed herein are about 100 mg/dose, or about 125 mg/dose, about 150 mg/dose, about 175 mg/dose, about 200 mg/dose, about 225 mg/dose, about 250 mg/dose, about 275 mg/dose, about 300 mg/dose, about 350 mg/dose, about 400 mg/dose, about 450 mg/dose, or about 500 mg/dose. A single dose may be administered hourly, daily, or weekly. For example, a single dose may be administered every 1, 2, 3, 4, 6, 8, 12, or 16 hours, or every 24 hours. A single dose may also be administered every 1, 2, 3, 4, 5, or 6 days, or every 7 days. A single dose may also be administered every 1, 2, or 3 weeks, or every 4 weeks. In some embodiments, a single dose may be administered weekly. A single dose may also be administered monthly.

Other therapeutically effective amounts of the compounds disclosed herein are about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 mg/dose.

The frequency of dosage of the disclosed compound will be determined by the individual patient's needs and may be, for example, once daily or twice daily or more. Administration of the compound will continue as long as treatment of the viral infection is required. For example, the compound may be administered to a person infected with the virus for a period of 20 to 180 days, or for a period of, for example, 20 to 90 days, or for example, 30 to 60 days.

Administration may be intermittent, with the patient receiving a daily dose of the compound for periods of several days or more, followed by periods of several days or more without receiving a daily dose of the compound. For example, the patient may receive a dose of the compound every other day or three times a week. Again, by way of example, the patient may receive a daily dose of the compound for periods of 1 to 14 days, followed by periods of 7 to 21 days without receiving a dose of the compound, followed by periods of a daily dose of the compound again in subsequent periods (e.g., 1 to 14 days). The alternating periods of administration followed by non-administration of the compound may be repeated as needed to treat the patient's clinical needs.

In one embodiment, pharmaceutical compositions are provided comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in combination with one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents, and a pharmaceutically acceptable excipient.

In one embodiment, kits are provided comprising compounds of the present disclosure or pharmaceutically acceptable salts thereof in combination with one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents.

In some embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with one, two, three, four, or more additional therapeutic agents. In some embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with two additional therapeutic agents. In other embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with three additional therapeutic agents. In still other embodiments, the compound of this disclosure or a pharmaceutically acceptable salt thereof is combined with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents, and/or they may be selected from different classes of therapeutic agents.

In some embodiments, when the compounds of this disclosure are combined with one or more additional therapeutic agents as described above, the components of the composition are administered simultaneously or sequentially. When administered sequentially, the combination may be administered in two or more doses.

In some embodiments, the compounds of this disclosure are combined with one or more additional therapeutic agents in a single dosage form for simultaneous administration to a patient, such as as a solid dosage form for oral administration.

In some embodiments, the compounds of this disclosure are administered in combination with one or more additional therapeutic agents.

To prolong the effects of the disclosed compounds, it is generally necessary to slow down the absorption of the compounds from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The absorption rate of the compound depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of the compound in the form of a parenteral administration can be achieved by dissolving or suspending the compound in an oil medium. Injectable reservoir formulations are prepared by forming a microcapsule matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolic acid. The rate of compound release can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Injectable reservoir formulations are also prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

VI. Combination Therapy

The compounds and compositions of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) provided herein are also used in combination with other active therapeutic agents for the treatment of viral infections, such as those of the Pneumoviridae, Picornaviridae, Flaviviridae, or Filoviridae families.

Combination therapy for treating pulmonary virology

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of pulmonary viral infections, preferably, the other active therapeutic agents are active against pulmonary viral infections, particularly respiratory syncytial virus (RSV) and/or metapneumovirus (MRV) infections. Non-limiting examples of these other active therapeutic agents active against RSV are ribavirin, palilizumab, motavizumab, RSV-IGIVMEDI-557, A-60444 (also known as RSV604), MDT-637, BMS-433771, ALN-RSV0, ALX-0171, and mixtures thereof. Other non-limiting examples of other active therapeutic agents that are active against respiratory syncytial virus (RSV) infection include RSV protein F inhibitors such as AK-0529; RV-521, ALX-0171, JNJ-53718678, BTA-585, and presatovir; RNA polymerase inhibitors such as luteicitabine and ALS-8112; anti-RSV G protein antibodies such as anti-G protein mAbs; and viral replication inhibitors such as nitrozonide.

In some implementations, other active therapeutic agents may be vaccines for the treatment or prevention of RSV, including but not limited to MVA-BN RSV, RSV-F, MEDI-8897, JNJ-64400141, DPX-RSV, SynGEM, GSK-3389245A, GSK-300389-1A, RSV-MEDIδM2-2 vaccine, VRC-RSVRGP084-00VP, Ad35-RSV-FA2, Ad26-RSV-FA2, and RSV fusion glycoprotein subunit vaccines.

Non-limiting examples of other active therapeutic agents that are active against metapneumovirus infection include sialidase modulators, such as DAS-181; RNA polymerase inhibitors, such as ALS-8112; and antibodies for the treatment of metapneumovirus infection, such as EV-046113.

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent metapneumovirus infection, including but not limited to mRNA-1653 and rHMPV-Pa vaccines.

Combination therapy for treating Picornaviridae

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of microviral infections, preferably, the other active therapeutic agents are active against microviral infections, particularly enterovirus infections. Non-limiting examples of these other active therapeutic agents are capsid-binding inhibitors, such as pracinamide, BTA-798 (vapendavir), and other compounds disclosed by Wu et al. (US 7,078,403) and Watson (US 7,166,604); fusion sialidase proteins, such as DAS-181; capsid protein VP1 inhibitors, such as VVX-003 and AZN-001; viral protease inhibitors, such as CW-33; phosphatidylinositol 4-kinase β inhibitors, such as GSK-480 and GSK-533; and anti-EV71 antibodies.

In some implementations, other active therapeutic agents may be vaccines for the treatment or prevention of infection with microribonucleoviridae viruses, including but not limited to EV71 vaccines, TAK-021, and vaccines based on the EV-D68 adenovirus vector.

Combination therapy for respiratory infections

Many infections caused by viruses of the Pneumoviridae and Picornaviridae families are respiratory infections. Therefore, adjunctive active therapeutic agents for the treatment of respiratory symptoms and post-infectious sequelae can be used in combination with the compounds provided herein. The adjunctive agents are preferably administered orally or by direct inhalation. Other preferred adjunctive therapeutic agents, for example, for the treatment of viral respiratory infections in combination with the compounds provided herein include, but are not limited to, bronchodilators and corticosteroids.

Glucocorticoids

Glucocorticoids were first introduced as a treatment for asthma in 1950 (Carryer, Journal of Allergy, 21, 282-287, 1950). Although their mechanism of action is not fully understood, they remain the most potent and consistently effective treatment for the disease (Morris, J. Allergy Clin. Immunol., 75(1Pt)1-13, 1985). Unfortunately, oral glucocorticoid therapy is associated with serious adverse side effects, such as truncal obesity, hypertension, glaucoma, impaired glucose tolerance, accelerated cataract formation, bone mineral loss, and psychological effects. All these side effects limit their use as long-term treatment agents (Goodman and Gilman, 10th ed., 2001). A solution to address systemic side effects is to deliver steroid medications directly to the site of inflammation. Inhaled corticosteroids (ICS) have been developed to alleviate the severe side effects of oral steroids. Non-limiting examples of corticosteroids that may be used in combination with the compounds provided herein are dexamethasone, dexamethasone sodium phosphate, flumethonone, flumethonone acetate, clotipreno, clotipreno ecaponicate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinolone, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone acetonide, flunisolone, flucodone-21-butyrate, flumethasone, flumethasone valerate, budesonide, halobetasol propionate, mometasone furoate, fluticasone, AZD-7594, cyclosporine; or pharmaceutically acceptable salts thereof.

anti-inflammatory agents

Other anti-inflammatory agents that act through an anti-inflammatory cascade mechanism can also be used as adjunctive therapies in combination with the compounds presented herein for the treatment of viral respiratory infections. The application of “anti-inflammatory signal transduction modulators” (referred to herein as AISTMs) such as phosphodiesterase inhibitors (e.g., PDE-4, PDE-5, or PDE-7 specific), transcription factor inhibitors (e.g., blocking NFκB via IKK inhibition), or kinase inhibitors (e.g., blocking p38 MAP, JNK, PI3K, EGFR, or Syk) is a logical approach to interrupting inflammation because these small molecules target a limited number of common intracellular pathways (which are key points of anti-inflammatory therapeutic interventions) (see the review by P.J. Barnes, 2006). These non-restrictive adjunctive therapeutic agents include: 5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl)-amide (P38 Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluoromethoxy-benzamide (PDE-4 inhibitor Roflumilast); 4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenyl-ethyl]pyridine (PDE-4 inhibitor CDP- 840); N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8-[(methanesulfonyl)amino]-1-dibenzofuran carboxamide (PDE-4 inhibitor Oglemilast); N-(3,5-dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide (PDE-4 inhibitor AWD12-281); 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl) 4-Amide (PDE-4 inhibitor Sch 351591); 4-[5-(4-fluorophenyl)-2-(4-methanesulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine (P38 inhibitor SB-203850); 4-[4-(4-fluorophenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethylamino- Ethyl ester (2-diethyl-ethyl ester prodrug of Cilomilast (PDE-4 inhibitor); (3-chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (Gefitinib, EGFR inhibitor); and 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (Imatinib, EGFR inhibitor).

β2-adrenergic receptor agonists and bronchodilators

Combinations of inhaled β2-adrenergic receptor agonists and bronchodilators such as formoterol, salbutamol, or salmeterol with the compounds provided herein are also suitable, but not limiting, combinations for the treatment of respiratory viral infections.

Inhaled β2-adrenergic receptor agonist bronchodilators such as formoterol or salmeterol, in combination with ICS, are also used to treat bronchoconstriction and inflammation (respectively, and). Combinations of these ICS and β2-adrenergic receptor agonists, as well as combinations of the compounds described herein, are also suitable, but not limiting, combinations for the treatment of respiratory viral infections.

Other examples of β2-adrenergic receptor agonists are bedoradine, vilanterol, indacaterol, olodaterol, tuloterrol, formoterol, abiraterrol, salbutamol, aforterol, levosalbutamol, fenoterol, and TD-5471.

Anticholinergic agents

Anticholinergic agents have potential use in the treatment or prevention of bronchoconstriction and can therefore be used as adjunctive therapeutic agents in combination with the compounds provided herein for the treatment of viral respiratory infections. These anticholinergic agents include, but are not limited to, antagonists of muscarinic receptors (especially the M3 subtype), which have shown therapeutic efficacy in controlling cholinergic tension in COPD in humans (Witek, 1999); 1-{4-hydroxy-1-[3,3,3-tris-(4-fluoro-phenyl)-propionyl]-pyrrolidine-2-carbonyl}-pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl)-amide; 3-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-8-isopropyl-8-methyl-8-aza-onium-bicyclo[3.2.1]octane (ipratropium-N,N-diethylglycinate); 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo[2.2] [2.2.2] Oct-3-yl ester (Solifenacin); 2-hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid 1-aza-bicyclo[2.2.2] Oct-3-yl ester (Revatropate); 2-{1-[2-(2,3-dihydro-benzofuran-5-yl)-ethyl]-pyrrolidine-3-yl}-2,2-diphenyl-acetamide (Darifenacin); 4-azacycloheptane-1-yl-2,2-diphenyl-butyramide (Buzepide); 7-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9-aza-tricyclo[3 .3.1.02,4]nonane (oxytropin-N,N-diethylglycine ester); 7-[2-(2-diethylamino-acetoxy)-2,2-di-thiophene-2-yl-acetoxy]-9,9-dimethyl-3-oxa-9-aza-onium-tricyclo[3.3.1.02,4]nonane (tiotropium-N,N-diethylglycine ester); dimethylamino-acetic acid 2-(3-diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester (tolterodine-N,N-dimethylglycine ester); 3-[4,4-bis-(4-fluoro-phenyl)-2-oxo-imidazolinidin-1-yl]-1-methyl-1-(2-oxo-2-pyridin-2-yl-ethyl)-pyrrolidineonium; 1-[1-(3-fluoro-benzyl)- Piperidin-4-yl]-4,4-bis-(4-fluoro-phenyl)-imidazolinidin-2-one; 1-cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1-phenyl-prop-2-yn-1-ol; 3-[2-(2-diethylamino-acetoxy)-2,2-di-thiophene-2-yl-acetoxy]-1-(3-phenoxy-propyl)-1-aza-bicyclo[2.2.2]octane (aldecanedin-N,N-diethylglycine ester); or (2-diethylamino-acetoxy)-di-thiophene-2-yl-acetic acid 1-methyl-1-(2-phenoxy-ethyl)-piperidin-4-yl ester; refenapyridine, glycopyrronium bromide, urodimethyl bromide, tiotropium bromide, adecanedinium bromide, benzylquinoline bromide.

Mucus Dissolving Agent

The compounds and compositions described herein can also be combined with mucolytics to treat symptoms of infections and respiratory tract infections. A non-limiting example of a mucolytic is ambroxol. Similarly, compounds of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) can be combined with expectorants to treat symptoms of infections and respiratory tract infections. A non-limiting example of an expectorant is guaiacol.

Nebulized hypertonic saline is used to improve immediate and long-term airway clearance in patients with lung disease (Kuzik, J. Pediatrics 2007, 266). Therefore, the compounds described herein can also be combined with nebulized hypertonic saline, particularly when a pulmonary viral infection is complicated by bronchiolitis. Combinations of compounds of formula (I) or (II) with hypertonic saline may also include any of the aforementioned additional agents. In one embodiment, approximately 3% hypertonic saline is used.

Combination therapy for the treatment of COPD

The compounds and compositions described herein can also be used in combination with other active therapeutic agents. Other active therapeutic agents for the treatment of COPD respiratory exacerbations include other anti-COPD active agents. Non-limiting examples of these other active therapeutic agents include anti-IL5 antibodies, such as benazolizumab and mepolizumab; dipeptidyl peptidase I (DPP1) inhibitors, such as AZD-7986 (INS-1007); DNA gyrase inhibitors/topoisomerase IV inhibitors, such as ciprofloxacin hydrochloride; MDR-associated protein 4/phosphodiesterase (PDE) 3 and 4 inhibitors, such as RPL-554; CFTR stimulants, such as ivacaftor and QBW-251; and MMP-9. MMP-12 inhibitors, such as RBx-10017609; adenosine A1 receptor antagonists, such as PBF-680; GATA 3 transcription factor inhibitors, such as SB-010; muscarinic receptor modulators/nicotinic acetylcholine receptor agonists, such as ASM-024; MARCKS protein inhibitors, such as BIO-11006; kit tyrosine kinase/PDGF inhibitors, such as masitinib; phosphodiesterase (PDE) 4 inhibitors, such as roflumilast and CHF-6001; phosphoinositol-3 kinase δ inhibitors, such as nanolixetine. miralisib; 5-lipoxygenase inhibitors, such as TA-270; muscarinic receptor antagonists/β2-adrenergic receptor agonists, such as bataflon succinate, AZD-887, and ipratropium bromide; TRN-157; elastase inhibitors, such as erdosteine; metalloproteinase-12 inhibitors, such as FP-025; interleukin-18 ligand inhibitors, such as tadekinigα; skeletal muscle troponin activators, such as CK-2127107; p38 MAP kinase inhibitors, such as acumapimod. ; IL-17 receptor modulators, such as CNTO-6785; CXCR2 chemokine antagonists, such as danirixin; leukocyte elastase inhibitors, such as POL-6014; epoxide hydrolase inhibitors, such as GSK-2256294; HNE inhibitors, such as CHF-6333; VIP agonists, such as avitadil; phosphoinositol-3 kinase δ/γ inhibitors, such as RV-1729; complement C3 inhibitors, such as APL-1; and G protein-coupled receptor-44 antagonists, such as AM-211.

Other non-limiting examples of active therapeutic agents include budesonide, adipocell, nitric oxide, PUR-1800, YLP-001, LT-4001, azithromycin, gamunex, QBKPN, sodium pyruvate, MUL-1867, mannitol, MV-130, MEDI-3506, BI-443651, VR-096, OPK-0018, TEV-48107, doxophylline, TEV-46017, OligoG-COPD-5/20, ZP-051, and lysine acetylsalicylate.

In some implementations, other active therapeutic agents may be vaccines that are active against COPD, including but not limited to MV-130 and GSK-2838497A.

Combination therapy for the treatment of dengue fever

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of flaviviridae virus infections, preferably, the other active therapeutic agents are active against flaviviridae virus infections, particularly dengue fever. Non-limiting examples of these other active therapeutic agents are host cytokine modulators, such as GBV-006; fenivel-Amine ABX-220, BRM-211; α-glucosidase 1 inhibitors, such as celgosivir; platelet-activating factor receptor (PAFR) antagonists, such as modipafant; cadherin-5/factor Ia modulators, such as FX-06; NS4B inhibitors, such as JNJ-8359; viral RNA splicing modulators, such as ABX-202; NS5 polymerase inhibitors; NS3 protease inhibitors; and TLR modulators.

In some implementations, other active therapeutic agents may be vaccines used to treat or prevent dengue fever, including but not limited to TetraVax-DV, DPIV-001, TAK-003, live attenuated dengue vaccine, quadrivalent dengue vaccine, quadrivalent DNA vaccine, and rDEN2δ30-7169DENV-1PIV.

Combination therapy for the treatment of Ebola

The compounds and compositions provided herein can also be used in combination with other active therapeutic agents. For the treatment of filoviridae virus infections, preferably, the other active therapeutic agents are active against filoviridae virus infections, particularly Marburg virus, Ebola virus, and Cueva virus infections. Non-limiting examples of these other active therapeutic agents are: ribavirin, palizumab, mavitizumab, RSV-IGIVMEDI-557, A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM-100201, BCX4430 ((2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), TKM-Ebola, T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (1-N,7-N-bis[3-( [dimethylamino]propyl]-3,9-dimethylquinoline[8,7-h]quinolone-1,7-diamine), rNAPc2, OS-2966, brincidofovir, remdesivir; RNA polymerase inhibitors, such as galidesivir, favipiravir (also known as T-705 or Avigan), JK-05; host cytokine modulators, such as GMV-006; cadherin-5/factor Ia modulators, such as FX-06; and antibodies used to treat Ebola, such as REGN-3470-3471-3479 and ZMapp.

Other non-restricted active therapeutic agents against Ebola include alpha-glucosidase 1 inhibitors, cathepsin B inhibitors, CD29 antagonists, dendritic ICAM-3 grasping integrin 1 inhibitors, estrogen receptor antagonists, factor VII antagonists, HLA class II antigen modulators, host cytokine modulators, interferon alpha ligands, neutral alpha-glucosidase AB inhibitors, Niemann-Pick C1 protein inhibitors, nucleoprotein inhibitors, polymerase cofactor VP35 inhibitors, serine protease inhibitors, tissue factor inhibitors, TLR-3 agonists, viral envelope glycoprotein inhibitors, and Ebola virus entry inhibitors (NPC1 inhibitors).

In some implementations, other active therapeutic agents may be vaccines for the treatment or prevention of Ebola, including but not limited to VRC-EBOADC076-00-VP, adenovirus-based Ebola vaccines, rVSV-EBOV, rVSVN4CT1-EBOVGP, MVA-BN Filo+Ad26-ZEBOV regimen, INO-4212, VRC-EBODNA023-00-VP, VRC-EBOADC069-00-VP, GamEvac-combi vaccine, SRC VB vector, HPIV3/EboGP vaccine, MVA-EBOZ, recombinant Ebola glycoprotein vaccine, Vaxart adenovirus vector 5-based Ebola vaccine, FiloVax vaccine, GOVX-E301, and GOVX-E302.

The compounds and compositions provided herein can also be used in combination with aminophosphate morpholino oligomers (PMOs), which are synthetic antisense oligonucleotide analogs designed to interfere with translation by forming base-pair duplexes with specific RNA sequences. Examples of PMOs include, but are not limited to, AVI-7287, AVI-7288, AVI-7537, AVI-7539, AVI-6002, and AVI-6003.

The compounds and compositions provided herein are also intended for use with general care provided to patients with infections caused by filoviridae viruses, including parenteral fluids (including glucose saline and Ringer's lactate) and nutrients, antibiotics (including metronidazole and cephalosporin antibiotics such as ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever and pain medications, antiemetics (such as metoclopramide) and/or antidiarrheals, vitamin and mineral supplements (including vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medications, and medications for other common illnesses in the patient population, such as antimalarial agents (including artemether and artemisinin-benfluridine combination therapy), typhoid vaccines (including quinolone antibiotics such as ciprofloxacin, macrolide antibiotics such as azithromycin, cephalosporin antibiotics such as ceftriaxone, or aminopenicillins such as ampicillin) or Shigella vaccines.

VII. Methods for treating viral infections

This disclosure provides methods for treating a variety of diseases, such as respiratory syncytial virus (RSV), Ebola, Zika, West Nile, dengue fever, HCV, and HBV, using compounds of the disclosed formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In), using compounds of the ... or (In), using compounds of the formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Im), or (In), using compounds of the formulas (I), (Ig), (Ih), (Ij), (Im), or (In), using compounds of the formulas (I), (Ig), (Ih), and (In), using compounds of the formulas (Ig), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ig), (Ih), and (HBV).

Paramyxoviridae

In some embodiments, this disclosure provides a method for treating paramyxoviridae infections, the method comprising administering to an individual (e.g., a human) infected with a paramyxoviridae virus a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Paramyxoviridae viruses include, but are not limited to, Nipah virus and parainfluenza virus.

Pulmonary Virology

In some embodiments, this disclosure provides a method of treating a person in need of a pulmonologic virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Pulmonologic viruses include, but are not limited to, respiratory syncytial virus (RSV) and human metapneumovirus (HMV). In some embodiments, the pulmonologic virus infection is an RSV infection. In some embodiments, the pulmonologic virus infection is a HMV infection.

In some embodiments, this disclosure provides a method for preparing a medicament for treating pulmonary viral infections in persons in need, characterized by the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary viral infections in persons. In some embodiments, the pulmonary viral infection is respiratory syncytial virus infection. In some embodiments, the pulmonary viral infection is human metapneumovirus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of pulmonary viral infections in persons in need. In some embodiments, the pulmonary viral infection is respiratory syncytial virus infection. In some embodiments, the pulmonary viral infection is human metapneumovirus infection.

In some embodiments, this disclosure provides a method for treating RSV infection, the method comprising administering to an individual (e.g., a person) infected with respiratory syncytial virus a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Typically, individuals suffer from chronic respiratory syncytial virus infection, but this disclosure is applicable to individuals with acute RSV infection.

In some embodiments, a method for inhibiting RSV replication is provided, which includes administering to an individual (e.g., a human) a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

In some embodiments, this disclosure provides a method for reducing viral load associated with RSV infection, wherein the method comprises administering to an individual (e.g., a person) infected with RSV a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt thereof, wherein the therapeutically effective amount is sufficient to reduce the individual's RSV viral load.

As described more fully herein, the compounds of this disclosure may be administered together with one or more adjunctive therapeutic agents to an individual (e.g., a human) infected with RSV. The adjunctive therapeutic agent may be administered simultaneously with, before, or after administration of, the compounds of this disclosure to the infected individual (e.g., a human).

In some embodiments, compounds of the present disclosure or pharmaceutically acceptable salts thereof are provided for the treatment or prevention of RSV infection. In some embodiments, compounds of the present disclosure (e.g., compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In)) or pharmaceutically acceptable salts thereof are provided for the preparation of medicaments for the treatment or prevention of RSV infection.

As described more fully herein, the compounds of this disclosure can be administered together with one or more additional therapeutic agents to an individual (e.g., a human) infected with RSV. Furthermore, in some embodiments, when used for the treatment or prevention of RSV, the compounds of this disclosure can be administered together with one or more (e.g., one, two, three, four, or more) additional therapeutic agents selected from the group consisting of: RSV combination drugs, RSV vaccines, RSV DNA polymerase inhibitors, immunomodulators, Toll-like receptor (TLR) modulators, interferon α receptor ligands, hyaluronidase inhibitors, respiratory syncytial antigen inhibitors, cytotoxic T-lymphocyte-associated protein 4 (ipi4) inhibitors, cyclophilic protein inhibitors, RSV viral entry inhibitors, antisense oligonucleotides targeting viral mRNA, short interfering RNA (siRNA), and ddR. NAi endonuclease modulators, ribonucleotide reductase inhibitors, RSV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, farnesol X receptor agonists, RSV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein modulators, retinoic acid-induced gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2,3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin α-1, Bruton's tyrosine kinase (BTK) inhibitors, KDM inhibitors, RSV replication inhibitors, arginase inhibitors, and other RSV drugs.

Microribonucleoviridae

In some embodiments, this disclosure provides a method for treating a person in need of a picornaviridae virus infection, the method comprising administering to the person a therapeutically effective amount of the disclosed compound or a pharmaceutically acceptable salt thereof. Picornaviridae viruses are a heterogeneous group of enteroviruses that cause infections including herpetic pharyngitis, aseptic meningitis, common cold-like syndrome (human rhinovirus infection), nonparalytic poliomyelitis-like syndrome, epidemic pleuropneumonia (an acute, febrile, infectious disease that typically occurs during epidemics), hand-foot-mouth disease, pancreatitis in children and adults, and severe myocarditis. In some embodiments, the picornaviridae virus infection is a human rhinovirus infection.

In some embodiments, this disclosure provides a method for preparing a medicament for treating microviral infections in persons in need, characterized by the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides the use of a compound of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating microviral infections in persons. In some embodiments, the microviral infection is a human rhinovirus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of microRNAviridae virus infections in persons in need. In some embodiments, the microRNAviridae virus infection is a human rhinovirus infection.

Flaviviridae

In some embodiments, this disclosure provides a method of treating a person in need of a flaviviridae virus infection, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Representative flaviviridae viruses include, but are not limited to, dengue fever, yellow fever, West Nile virus, Zika virus, Japanese encephalitis virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). In some embodiments, the flaviviridae virus infection is dengue virus infection. In some embodiments, the flaviviridae virus infection is yellow fever virus infection. In some embodiments, the flaviviridae virus infection is West Nile virus infection. In some embodiments, the flaviviridae virus infection is Zika virus infection. In some embodiments, the flaviviridae virus infection is Japanese encephalitis virus infection. In some embodiments, the flaviviridae virus infection is hepatitis C virus infection. In some embodiments, the flaviviridae virus infection is hepatitis B virus infection.

In some embodiments, this disclosure provides a method for preparing a medicament for treating flaviviride virus infections in persons in need, characterized by the use of compounds of this disclosure or pharmaceutically acceptable salts thereof. In some embodiments, this disclosure provides the use of compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating flaviviride virus infections in persons. In some embodiments, the flaviviride virus infection is dengue virus infection. In some embodiments, the flaviviride virus infection is yellow fever virus infection. In some embodiments, the flaviviride virus infection is West Nile virus infection. In some embodiments, the flaviviride virus infection is Zika virus infection. In some embodiments, the flaviviride virus infection is hepatitis C virus infection. In some embodiments, the flaviviride virus infection is hepatitis B virus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of flaviviridae virus infection in persons in need. In some embodiments, the flaviviridae virus infection is dengue virus infection. In some embodiments, the flaviviridae virus infection is yellow fever virus infection. In some embodiments, the flaviviridae virus infection is West Nile virus infection. In some embodiments, the flaviviridae virus infection is Zika virus infection. In some embodiments, the flaviviridae virus infection is hepatitis C virus infection. In some embodiments, the flaviviridae virus infection is hepatitis B virus infection.

Filoviridae

In some embodiments, this disclosure provides a method of treating a filoviridae virus infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof. Representative filoviridae viruses include, but are not limited to, Ebola and Marburg. In some embodiments, the filoviridae virus infection is Ebola virus infection.

In some embodiments, this disclosure provides a method for preparing a medicament for treating filoviridae virus infections in persons in need, characterized by the use of compounds of this disclosure or pharmaceutically acceptable salts thereof. In some embodiments, this disclosure provides the use of compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating filoviridae virus infections in persons. In some embodiments, the filoviridae virus infection is Ebola virus infection.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment of filoviridae virus infections in persons in need. In some embodiments, the filoviridae virus infection is Ebola virus infection.

VIII. Methods for treating or preventing worsening of respiratory symptoms caused by viral infection

The compounds disclosed herein can also be used to treat or prevent the worsening of respiratory symptoms caused by viral infections in persons in need. Compounds of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Im), or (In) can also be used to treat or prevent the worsening of respiratory symptoms caused by viral infections in persons in need.

In some embodiments, this disclosure provides a method for treating or preventing exacerbations of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for treating or preventing exacerbations of respiratory symptoms caused by a viral infection in a person in need, the method comprising administering to the person a therapeutically effective amount of a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for preparing a medicament for treating or preventing exacerbations of respiratory symptoms caused by a viral infection in a person in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptoms are chronic obstructive pulmonary disease (COPD). In some embodiments, the viral infection is caused by respiratory syncytial virus (RSV), rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides a method for preparing a medicament for treating or preventing exacerbations of respiratory symptoms caused by a viral infection in a person in need, characterized by using a compound of this disclosure or a pharmaceutically acceptable salt thereof, wherein the respiratory symptom is asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides the use of the compounds of this disclosure or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating or preventing exacerbations of a respiratory condition in humans caused by a viral infection, wherein the respiratory condition is chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides the use of the disclosed compounds or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating or preventing exacerbations of a respiratory condition in humans caused by a viral infection, wherein the respiratory condition is asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbations of respiratory conditions caused by viral infections in persons in need, wherein the respiratory condition is chronic obstructive pulmonary disease. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, or metapneumovirus.

In some embodiments, this disclosure provides compounds of the present disclosure or pharmaceutically acceptable salts thereof for the treatment or prevention of exacerbations of respiratory symptoms caused by viral infection in persons in need, wherein the respiratory symptoms are asthma. In some embodiments, the viral infection is caused by respiratory syncytial virus, rhinovirus, enterovirus, or metapneumovirus.

IX. Implementation Examples

Abbreviations: Certain abbreviations and acronyms are used to describe experimental details. While most of these are understandable to those skilled in the art, Table 1 contains a list of abbreviations and acronyms that have many of these.

Table 2: List of abbreviations and acronyms .

Compound structures using the symbol “P ^* ” refer to isolated (R)- or (S) isomers where the specific stereochemistry at that position is not specified.

A. Intermediate

Intermediate 1.((3aS,4S,6S,6aS)-6-(4-((tert-butoxycarbonyl)amino)pyrrolo[2,1-f][1,2, 4] Triazine-7-yl)-2,2-dimethyl-4-((((trifluoromethyl)sulfonyl)oxy)methyl)tetrahydrofurano[3,4-d][1, 3] Dioxacyclopenten-4-yl)methyl acetate

To a solution of (7-((3aS,4S,6aS)-6,6-bis(hydroxymethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate tert-butyl (WO2015069939; 100 mg, 0.229 mmol) and Novozyme-435 (50 mg, 50% w/w) in anhydrous tetrahydrofuran (1 mL), vinyl acetate (0.03 mL, 0.321 mmol) was added, and the reaction mixture was stirred at 45 °C for 7 hours. The reaction mixture was filtered, and the enzyme was washed repeatedly with THF. The combined filtrates were concentrated, and the resulting residue was purified by silica gel chromatography in 30–100% ethyl acetate/hexane to give the compound. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.46(s,1H),8.21(s,1H),7.21(s,1H),6.88(d,J＝4.6Hz,1H),5.39(d,J＝4.9Hz,1H),5.17(dd,J＝6.1,4.9Hz,1 H), 4.78 (d, J = 6.1Hz, 1H), 4.03 (q, J = 11.2Hz, 2H), 3.63 (s, 2H), 1.96 (s, 3H), 1.50 (d, J = 3.0Hz, 12H), 1.29 (s, 3H). LCMS: MS m/z = 478.92 [M+1]; t _R = 0.96 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

Intermediate 2.((3aS,4S,6S,6aS)-6-(4-((tert-butoxycarbonyl)amino)pyrrolo[2,1-f][1,2, 4] Triazine-7-yl)-2,2-dimethyl-4-((((trifluoromethyl)sulfonyl)oxy)methyl)tetrahydrofurano[3,4-d][1, 3] Dioxacyclopenten-4-yl)methyl acetate

Intermediate 1 (350 mg, 0.731 mmol) and a solution of pyridine (0.300 mL, 3.66 mmol) in dichloromethane (3.67 mL) were added to a solution of trifluoromethanesulfonic anhydride in dichloromethane (1 M, 1.46 mL, 1.46 mmol) at 0 °C. After 20 minutes, the reaction mixture was diluted with water (5 mL), and the resulting mixture was extracted with dichloromethane (2 × 5 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude compound was used directly in the next step. LCMS: MS m/z = 610.79 [M+1], t _R = 1.51 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

Intermediate 3.((3aS,4R,6S,6aS)-6-(4-((tert-butoxycarbonyl)amino)pyrrolo[2,1-f][1,2, [4]triazine-7-yl)-4-(fluoromethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-yl)ethyl Methyl ester

A solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M, 2.87 mL, 2.87 mmol) was added to a solution of crude intermediate 2 (350 mg, 0.573 mmol) in tetrahydrofuran (3 mL) at room temperature. After 4 hours, the reaction mixture was diluted with ethyl acetate (5 mL) and washed with water (2 × 5 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (eluting with a hexane solution of 0–100% ethyl acetate) to give the compound. ¹ H NMR (400MHz, methanol-d ₄ ) δ8.12 (br s,1H),7.18(d,J＝4.7Hz,1H),6.89(d,J＝4.7Hz,1H),5.55(d,J＝4.4Hz,1H),5.29(dd,J＝6.5,4.5Hz,1H) ,4.96(d,J＝6.4Hz,1H),4.77–4.68(m,1H),4.65–4.56(m,1H),4.24–4.13(m,2H),2.02(s,3H),1.58(br s,12H),1.37(s,3H). LCMS: MS m/z = 480.97 [M+1], t _R = 1.39 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 3.04 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min.

Intermediate 4.((3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-4- (fluoromethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-yl)methanol

The solution of intermediate 3 (189 mg, 0.393 mmol) in water (0.6 mL) and dioxane (2.4 mL) was heated to 100 °C. After 4 hours, the resulting mixture was concentrated under reduced pressure. The crude residue was dissolved in methanol (2 mL), and 1 N potassium carbonate solution (1 mL) was added to the mixture at room temperature. After 1.25 hours, the reaction mixture was concentrated under reduced pressure, and the crude residue was purified by silica gel chromatography (eluting with a hexane solution of 0-100% ethyl acetate) to give the compound. ¹ H NMR (400MHz, methanol-d ₄ )δ7.79(s,1H),6.86(d,J＝4.5Hz,1H),6.74(d,J＝4.5Hz,1H),5.36(d,J＝5.7Hz,1H),5.24(t,J＝5.9Hz,1H),4.99(d,J＝6.3Hz,1H),4.71(dd, J＝31.9,10.0Hz,1H),4.59(dd,J＝33.4,10.0Hz,1H),3.73(dd,J＝11.5,1.6Hz,1H),3.64(dd,J＝11.5,2.2Hz,1H),1.58(s,3H),1.35(s,3H). LCMS: MS m/z = 339.24 [M+1], t _R = 1.02 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 1.94 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min.

Intermediate 5.(2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl) 2-(hydroxymethyl)tetrahydrofuran-3,4-diol

A concentrated aqueous hydrochloric acid solution (12N, 0.172 mL, 2.07 mmol) was added to a solution of intermediate 4 (50 mg, 0.148 mmol) in acetonitrile (1 mL) at room temperature. After 45 minutes, the resulting mixture was neutralized with an aqueous sodium hydroxide solution of 2N and purified by preparative HPLC (Gemeni C18 5 μM 100 x 30 mm column, 5-100% acetonitrile/water gradient) to give the compound. ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.78 (s, ¹H), 6.87 (d, J = 4.5 Hz, ¹H), 6.75 (d, J = 4.5 Hz, ¹H), 5.21 (d, J = 8.9 Hz, ¹H), 4.80–4.74 (m, 1.5H), 4.65 (dd, J = 9.9, 6.1 Hz, ¹H), 4.52 (d, J = 9.9 Hz, 0.5H), 4.32 (d, J = 5.4 Hz, ¹H), 3.71 (t, J = 1.9 Hz, 2H). ^¹⁹F NMR (376 MHz, methanol- _d⁴ ) δ -237.67 (t, J = 47.7 Hz). LCMS: MS m/z = 299.20 [M+1], t _R = 0.48 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _{_R} = 1.10 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 2.05 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Intermediate 6.((S)-(((3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- (2-(fluoromethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxane-4-yl)methoxy)(phenoxy) 2-Ethylbutyl phosphoyl-L-alanine

Tetrahydrofuran (18.75 mL) was added to a mixture of intermediate 4 (1.5 g, 4.433 mmol), intermediate B1 (J.Med.Chem.2017, 60(5), pp. 1648-1661; 2.197 g, 4.877 mmol), and magnesium chloride (0.633 g, 6.65 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (1.931 mL, 11.08 mmol). After 2 hours at 50 °C, the reaction mixture was diluted with ethyl acetate (400 mL), and the resulting mixture was washed with water (2 × 100 mL) and brine (200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was subjected to silica gel chromatography, eluting with a solution of 0-20% methanol in dichloromethane to give the intermediate. LCMS: MS m/z = 650.28 [M+1], t _R = 1.74 min; LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μC18 100A, 50x3.0 mm; solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; gradient: 0 min-1.6 min 2-100% acetonitrile, 1.6 min-1.8 min 100% acetonitrile, 1.80 min-1.90 min 100%-2% acetonitrile, 1.90 min-2.20 min 2% acetonitrile, 1100 μl/min.

B. Compounds

Example 1. ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester

Acetonitrile (0.4 mL) was added to a mixture of intermediate 4 (27 mg, 0.080 mmol), intermediate B1 (J.Med.Chem.2017, 60(5), pp. 1648-1661; 36 mg, 0.080 mmol), and magnesium chloride (8 mg, 0.08 mmol) at room temperature. The mixture was heated to 50 °C for 10 minutes, and N,N-diisopropylethylamine (0.035 mL, 0.20 mmol) was added. After 5 hours, the reaction mixture was cooled to room temperature, and concentrated hydrochloric acid aqueous solution (0.093 mL) was added dropwise. After 30 minutes, the reaction mixture was diluted with ethyl acetate (20 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was subjected to silica gel chromatography and eluted with a hexane solution of 0-100% ethyl acetate to obtain the product, which was determined to be a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, methanol-d ₄ )δ7.79(s,1H),7.40–7.31(m,2H),7.26–7.14(m,3H),6.85(d,J＝4.5Hz,1H ),6.73(d,J＝4.6Hz,1H),5.36(d,J＝8.3Hz,1H),4.80–4.70(m,1H),4.70–4. 58(m,2H),4.33(d,J＝5.2Hz,1H),4.21(dd,J＝5.7,1.7Hz,2H),4.07–3.87( m,3H),1.48(appp,J＝6.2Hz,1H),1.39–1.27(m,7H),0.86(t,J＝7.5Hz,6H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.79 (t, J = 47.7 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.53. LCMS: MS m/z = 610.32 [M+1], t _R = 1.22 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _{_R} = 3.03 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.11 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 2. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro Sodium methyl-3,4-dihydroxytetrahydrofuran-2-yl)methyltriphosphate

At 0 °C, _NaHCO₃ (10 mg, 0.12 mmol) and POCl₃ ₍ 0.03 mL, 0.33 mmol) were added to a solution of intermediate 5 (10 mg, 0.034 mmol) in PO₄(OMe) _₃ (0.5 mL). The reaction mixture was stirred at 0 °C for 4 hours, during which time a solution of tributylammonium pyrophosphate (250 mg, 0.46 mmol) in ACN (0.5 mL) was added, followed by the addition of tributylamine (0.14 mL, 0.59 mmol). The reaction mixture was stirred at 0 °C and monitored by ion-exchange HPLC. After 1 hour, the reaction was quenched with triethylammonium bicarbonate buffer (1 M, 8 mL). The reaction mixture was stirred at room temperature for 0.5 hours, then concentrated and co-evaporated twice with water. The residue was dissolved in water (2 mL) and _NaHCO₃ (400 mg) was added. The resulting mixture was concentrated. The residue was dissolved in water (approximately 2 mL) and loaded onto a C-18 column, eluted with water. The fractions containing the product were combined, acidified with HCl (1N, 150 μL), and concentrated to approximately 4 mL. The product was loaded onto an ion exchange column and eluted successively with water and 10–40% triethylammonium bicarbonate buffer (1M) _-H₂O . The fractions containing the product were combined and concentrated. The residue was dissolved in water (1 mL) and NaOH (1N, 0.1 mL) was added. The resulting mixture was concentrated to approximately 0.2 mL and purified using a C-18 column, eluting with water. The fractions containing the product were combined and concentrated to obtain the product. ¹ H NMR (400MHz, water-d ₂ )δ7.88(s,1H),7.06(d,J＝4.7Hz,1H),6.96(d,J＝4.7Hz,1H),5.46(d,J＝9.2Hz,1H),4.94–4.84(m,2H) ,4.82–4.75(m,1H),4.72(d,J＝5.3Hz,1H),4.13(dd,J＝10.8,5.8Hz,1H),3.96(dd,J＝10.6,5.1Hz,1H). ³¹ P NMR (162MHz, water-d ₂ ) δ -5.74 (d,J=19.7Hz), -11.10 (d,J=19.4Hz), -21.68 (t,J=19.7Hz). LCMS: MS m/z = 538.88 [M+1], t _R = 0.45 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min. Ion exchange HPLC: t _R = 10.303 min; Ion exchange 0-80 Milli-Q water/0.5 M TEAB, 14 min gradient; 1 mL/min.

Example 3. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine neopentyl ester

(tert-butoxycarbonyl)-L-alanine neopentyl ester. (tert-butoxycarbonyl)-L-alanine (20.61 g, 0.109 mol) was dissolved in acetonitrile (100 mL), and 2,2-dimethylprop-1-ol (8 g, 0.091 mol) was added, followed by EDCI (18.32 g, 0.118 mol) and DMAP (16.63 g, 0.136 mol). The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was achieved by silica gel chromatography with 0–25% ethyl acetate/hexane to give intermediate D1. ¹ H NMR (400MHz, DMSO-d6) δ7.27(d,J＝7.5Hz,1H),4.00(p,J＝7.4Hz,1H),3.79(d,J＝10 .5Hz,1H),3.64(d,J＝10.5Hz,1H),1.35(s,9H),1.23(d,J＝7.4Hz,3H),0.87(s,9H).

(S)-1-(neopentoxy)-1-oxopropyl-2-ammonium chloride. Neopentyl (tert-butoxycarbonyl)-L-alanine ester (17.45 g, 0.067 mol) was dissolved in anhydrous dichloromethane (175 mL) and a dioxane solution of 4N HCl (84.11 mL, 0.336 mol). The reaction was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The resulting residue was placed under high vacuum overnight to give intermediate D2, which was used as is in the next step without purification. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.60 (s, 3H), 4.08 (q, J = 7.2Hz, 1H), 3.90 (d, J = 10.4Hz, 1H), 3.78 (d, J = 10.4Hz, 1H), 1.43 (d, J = 7.2Hz, 3H), 0.90 (s, 9H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine neopentyl ester. Triethylamine (20.78 mL, 147.84 mmol) was added to a solution of intermediate D2 (13.15 g, 67.2 mmol) and phenyl dichlorophosphate (10 mL, 67.2 mmol) in anhydrous dichloromethane (227 mL) under an argon atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (9.348 g, 67.2 mmol) and triethylamine (10.39 mL, 73.92 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80 g _SiO₂ Combiflash HP Gold column, 100% dichloromethane, followed by 0-45% ethyl acetate/hexane) to obtain the desired compound as a diastereomeric mixture (19 g, 64.79%, diastereomeric mixture). The obtained compound was dried under high vacuum to partially solidify. Diisopropyl ether was added to the partially solidified material and sonicated to obtain a fine solid. The solid was separated by filtration. A second round of sonication with diisopropyl ether and filtration yielded intermediate D3. Intermediate D3 was identified as a single diastereomeric compound by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 8.31–8.23 (m, 2H), 7.52–7.43 (m, 2H), 7.38 (dd, J = 8.6, 7.2 Hz, 2H), 7.28–7.18 (m, 3H), 4.09 (dq, J = 9.8, 7.2 Hz, 1H), 3.83–3.72 (m, 2H), 1.34 (dd, J = 7.2, 1.2 Hz, 3H), 0.91 (s, 9H). ^³¹P NMR (162 MHz, methanol- _d⁴ ) δ -1.32 (s). LCMS: MS m/z = 436.85 [M+1]; t _R = 1.67 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine neopentyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (0.015 g, 0.044 mmol), intermediate D3 (0.021 g, 0.049 mmol), and magnesium chloride (0.006 g, 0.067 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.019 mL, 0.111 mmol). The resulting mixture was stirred at 50 °C for 1.5 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was dissolved in anhydrous acetonitrile (0.5 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (0.088 mL, 1.058 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with 3N sodium hydroxide aqueous solution. The resulting mixture was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 15%–85% acetonitrile/water gradient, 30 min run) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, methanol-d ₄ )δ7.78(s,1H),7.34(dd,J＝8.6,7.1Hz,2H),7.27–7.13(m,3H),6.84(d,J＝4 .5Hz,1H),6.73(d,J＝4.5Hz,1H),5.35(d,J＝8.4Hz,1H),4.81–4.55(m,3H), 4.33(d,J＝5.2Hz,1H),4.24–4.17(m,2H),4.02–3.86(m,1H),3.83(d,J＝10. 5Hz, 1H), 3.73 (d, J = 10.5Hz, 1H), 1.33 (dd, J = 7.1, 1.0Hz, 3H), 0.91 (s, 9H). ³¹ PNMR (162MHz, methanol- _d4 ) δ 3.54. LCMS: MS m/z = 596.10 [M+1]; t _R = 1.16 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18100A, 50x3.0mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 4.848 min; HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 4. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 3,3-dimethylbutane ester

(tert-Butoxycarbonyl)-L-alanine 3,3-dimethylbutyl ester. (tert-Butoxycarbonyl)-L-alanine (22.38 g, 0.118 mol) was dissolved in acetonitrile (100 mL), and 3,3-dimethylbut-1-ol (10.07 g, 0.099 mol) was added in a single addition, followed by EDCI (19.89 g, 0.128 mol) and DMAP (18.06 g, 0.148 mol). The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was achieved by silica gel chromatography with 0–25% ethyl acetate/hexane to give intermediate E1. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.22 (d, J = 7.4 Hz, 1H), 4.13-3.82 (m, 3H), 1.47 (t, J = 7.2 Hz, 2H), 1.35 (s, 9H), 1.19 (d, J = 7.3 Hz, 3H), 0.88 (s, 9H).

(S)-1-(3,3-dimethylbutoxy)-1-oxopropyl-2-ammonium chloride. Intermediate E1 (20.9 g, 0.076 mol) was dissolved in anhydrous dichloromethane (200 mL) and a dioxane solution of 4N HCl (95.57 mL, 0.382 mol). The reaction was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The resulting residue was placed under high vacuum overnight, and intermediate E2 was used as is in the next step without purification. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.67 (s, 3H), 4.32–4.07 (m, 2H), 3.97 (d, J = 7.2 Hz, 1H), 1.52 (t, J = 7.3 Hz, 2H), 1.39 (d, J = 7.1 Hz, 3H), 0.89 (s, 9H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 3,3-dimethylbutyl ester. Triethylamine (23.5 mL, 167.1 mmol) was added to a solution of intermediate E2 (15.93 g, 75.96 mmol) and phenyl dichlorophosphate (11.3 mL, 75.96 mmol) in anhydrous dichloromethane (300 mL) under an argon atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (10.57 g, 75.96 mmol) and triethylamine (11.74 mL, 83.56 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80 g _SiO₂ Combiflash HP Gold column, 100% dichloromethane, followed by 0-35% ethyl acetate/hexane) to give intermediate E3, a diastereomeric mixture. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.28(d,J＝9.1Hz,2H),7.53–7.34(m,4H),7.30–7.16(m,3H),6.66(td,J＝13.2,1 0.0Hz,1H),4.06–3.88(m,3H),1.40–1.29(m,2H),1.24–1.11(m,3H),0.83(s,9H). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ -1.26, -1.57. LCMS: MS m/z = 450.96 [M+1]; t _R = 1.71 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 3,3-dimethylbutyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (0.015 g, 0.044 mmol), intermediate E3 (0.022 g, 0.049 mmol), and magnesium chloride (0.006 g, 0.067 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.019 mL, 0.111 mmol). The resulting mixture was stirred at 50 °C for 1.5 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was dissolved in anhydrous acetonitrile (0.5 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (0.088 mL, 1.058 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with 3N sodium hydroxide aqueous solution. The resulting mixture was purified by preparative HPLC (Phenominex Synergi4u Hydro-RR 150x30 mm column, 15%–85% acetonitrile/water gradient, 30 min run) to give the product. ^¹H NMR (400 MHz, methanol- _d4) )δ7.78(d,J＝1.8Hz,1H),7.39–7.28(m,2H),7.28–7.12(m,3H),6.85(dd,J＝5.7, 4.5Hz,1H),6.75(dd,J＝8.7,4.5Hz,1H),5.37(dd,J＝8.2,6.2Hz,1H),4.83–4.53( m,3H),4.36(dd,J＝18.1,5.2Hz,1H),4.30–4.04(m,4H),3.96–3.81(m,1H),1.51( td, J＝7.5, 2.2Hz, 2H), 1.27 (ddd, J＝19.5, 7.1, 1.1Hz, 3H), 0.90 (d, J＝1.9Hz, 9H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.7, 3.51. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.40–-238.89 (m). LCMS: MS m/z = 610.05 [M+1]; t _R = 1.20 min (minor isomer), 1.22 min (major isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 5.024 min (minor isomer), 5.1 min (major isomer); HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B gradient at 1.5 mL/min for 8.5 min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by chiral preparative HPLC (Chiralpak IA 5 μm, 21 x 250 mm; 100% ethanol).

Example 5. First eluted diastereomer of Example 4: ^¹H NMR (400MHz, methanol- _d4) )δ7.78(s,1H),7.32(t,J＝7.9Hz,2H),7.23–7.13(m,3H),6.86(d,J＝4.6Hz,1H ),6.75(d,J＝4.5Hz,1H),5.37(d,J＝8.1Hz,1H),4.83–4.71(m,1H),4.71–4.59( m,2H),4.38(d,J＝5.3Hz,1H),4.26(dd,J＝5.1,1.7Hz,2H),4.16–4.07(m,2H),3 .93–3.74(m,1H),1.56–1.47(m,2H),1.24(dd,J=7.2,1.2Hz,3H),0.90(s,9H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.73. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.61 (t, J = 47.7 Hz). LCMS: MS m/z = 610.13 [M+1]; t _R = 1.20 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μXB-C18100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 4.996 min; HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 6. Second eluted diastereomer of Example 4: ^¹H NMR (400MHz, methanol- _d⁴ ) δ 7.79 (s, 1H), 7.35 (dd, J = 8.6, 7.1Hz, 2H), 7.27–7.14 (m, 3H), 6.84 (d, J = 4.6Hz, 1H), 6.73 (d, J = 4.6Hz, 1H), 5.36 (d, J = 8.3Hz, 1H), 4.82–4.56 (m, 3H), 4.33 (d, J = 5.2Hz, 1H), 4.24–4.04 (m, 4H), 3.94–3.80 (m, 1H), 1.50 (t, J = 7.4Hz, 2H), 1.29 (dd, J = 7.1, 1.0Hz, 3H), 0.90 (s, 9H). ³¹ PNMR (162 MHz, methanol- _d⁴ ) δ 3.51. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.84 (t, J = 47.7 Hz). LCMS: MS m/z = 610.14 [M+1]; t _R = 1.21 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μXB-C18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 5.079 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 7. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2,2-dimethylbutane ester

(tert-Butoxycarbonyl)-L-alanine 2,2-dimethylbutyl ester. (tert-Butoxycarbonyl)-L-alanine (11.11 g, 0.059 mol) was dissolved in acetonitrile (60 mL), and 2,2-dimethylbut-1-ol (5.0 g, 0.049 mol) was added, followed by EDCI (9.876 g, 0.064 mol) and DMAP (8.967 g, 0.073 mol). The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was performed using silica gel chromatography with 0–20% ethyl acetate/hexane to give intermediate H1. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.26(d,J＝7.5Hz,1H),3.99(p,J＝7.4Hz,1H),3.81(d,J＝10.6Hz,1H),3.66(d,J＝10.6Hz,1H),1.35(s,9H),1.23(m,5H),0.84–0.72(m,9H).

(S)-1-(2,2-Dimethylbutoxy)-1-oxopropyl-2-ammonium chloride. Intermediate H1 (10.34 g, 0.038 mol) was dissolved in anhydrous dichloromethane (100 mL) and a dioxane solution of 4N HCl (47.28 mL, 0.189 mol). The reaction was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The residue was placed under high vacuum overnight, and intermediate H2 was used as is in the next step without purification. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.65(s,3H),4.05(q,J＝7.2Hz,1H),3.91(d,J＝10.6Hz,1H),3.79(d,J＝10 .6Hz, 1H), 1.42 (d, J = 7.2Hz, 3H), 1.26 (q, J = 7.6Hz, 2H), 0.87–0.73 (m, 9H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 2,2-dimethylbutyl ester. Triethylamine (11.64 mL, 82.87 mmol) was added to a solution of intermediate H2 (7.9 g, 37.67 mmol) and phenyl dichlorophosphate (5.605 mL, 37.67 mmol) in anhydrous dichloromethane (150 mL) under an argon atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (5.24 g, 37.67 mmol) and triethylamine (5.82 mL, 41.43 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80 g _SiO₂ Combiflash HP Gold column, 100% dichloromethane, followed by 0-35% ethyl acetate/hexane) to give intermediate H3. ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 8.32–8.23 (m, 2H), 7.52–7.34 (m, 4H), 7.31–7.18 (m, 3H), 4.15–4.02 (m, 1H), 3.86–3.74 (m, 2H), 1.39–1.28 (m, 3H), 1.32–1.19 (m, 2H), 0.89–0.76 (m, 9H). ^³¹P NMR (162 MHz, methanol- _d⁴ ) δ -1.35, -1.57. LCMS: MS m/z = 450.94 [M+1]; t _R = 1.71 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2,2-dimethylbutyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (0.015 g, 0.044 mmol), intermediate H3 (0.022 g, 0.049 mmol), and magnesium chloride (0.006 g, 0.067 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.019 mL, 0.111 mmol). The resulting mixture was stirred at 50 °C for 1.5 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was dissolved in anhydrous acetonitrile (0.5 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (0.088 mL, 1.058 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with an aqueous solution of 3N sodium hydroxide. The resulting mixture was purified by preparative HPLC (Phenominex Synergi4u Hydro-RR 150 x 30 mm column, 15%–85% acetonitrile/water gradient, 30-minute run) to obtain the product. ¹ HNMR (400MHz, methanol-d ₄ )δ7.78(d,J＝1.4Hz,1H),7.37–7.27(m,2H),7.27–7.12(m,3H),6.85(dd,J＝5.8,4.5Hz,1H),6.74(dd,J＝9.9,4.5Hz,1H),5.37(dd,J＝8.2,6.7Hz,1 H),4.83–4.53(m,3H),4.36(dd,J＝16.6,5.3Hz,1H),4.24(ddd,J＝21.2,5 .7,1.8Hz,2H),4.01–3.72(m,3H),1.36–1.24(m,5H),0.90–0.77(m,9H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.72, 3.54. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.38–-238.82 (m). LCMS: MS m/z = 610.05 [M+1]; t _R = 1.32 min (minor isomer), 1.33 min (major isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 5.021 min (minor isomer), 5.093 min (major isomer); HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B gradient at 1.5 mL/min for 8.5 min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by chiral preparative HPLC (Chiralpak IA 5 μm, 21 x 250 mm; 100% ethanol).

Example 8. First eluted diastereomer of Example 7: ^¹H NMR (400MHz, methanol- _d⁴ ) δ 7.78 (s, 1H), 7.32 (t, J = 7.8Hz, 2H), 7.19 (m, 3H), 6.86 (d, J = 4.5Hz, 1H), 6.75 (d, J = 4.5Hz, 1H), 5.37 (d, J = 8.2Hz, 1H), 4.81–4.56 (m, 3H), 4.37 (d, J = 5.3Hz, 1H), 4.26 (d, J = 4.9Hz, 2H), 3.99–3.66 (m, 3H), 1.36–1.19 (m, 5H), 0.87 (s, 6H), 0.82 (t, J = 7.6Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.72. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.59 (t, J = 47.7 Hz). LCMS: MS m/z = 610.11 [M+1]; t _R = 1.21 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μXB-C18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 5.007 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 9. Second eluted diastereomer of Example 7: ^¹H NMR (400MHz, methanol- _d4) )δ7.79(s,1H),7.34(t,J＝7.9Hz,2H),7.27–7.14(m,3H),6.84(d,J＝4.5Hz, 1H),6.73(d,J＝4.5Hz,1H),5.35(d,J＝8.3Hz,1H),4.81–4.55(m,3H),4.33(d ,J＝5.2Hz,1H),4.21(d,J＝5.8Hz,2H),3.94(dd,J＝9.9,7.1Hz,1H),3.85(d,J ＝10.7Hz,1H),3.76(d,J＝10.6Hz,1H),1.36–1.24(m,5H),0.89–0.77(m,9H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.54. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.76 (t, J = 47.7 Hz). LC-MS: MS m/z = 610.14 [M+1]; t _R = 1.22 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μX B-C18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 5.085 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 10. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine methyl ester

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine methyl ester. L-alanine methyl ester hydrochloride (14 g, 100 mmol) was mixed with 50 mL of anhydrous DCM and stirred in an ice bath under atmospheric nitrogen. Phenyl dichlorophosphate (16.4 mL, 110 mmol) was added dropwise to the reaction mixture, and the reaction mixture was stirred for 30 min. Triethylamine (29.4 mL, 210 mmol) was mixed with 20 mL of anhydrous DCM and added dropwise to the reaction mixture. The reaction mixture was stirred for 1 h. Pentafluorophenol (18.4 g, 100 mmol) was added in a single addition. Triethylamine (14.7 mL, 105 mmol) was mixed with 30 mL of anhydrous DCM and added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with DCM (50 mL) and washed with water (5 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to give a solid. Isopropyl ether (130 mL) was added to the solid. Large solid particles were crushed, then sonicated for 20 minutes, and the mixture was stirred for 24 hours. The solid was collected and washed with a small amount of isopropyl ether (30 mL), and dried under high vacuum to obtain intermediate K1. Intermediate K1 was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, chloroform-d) δ 7.40–7.32 (m, 2H), 7.28–7.19 (m, 3H), 4.20 (m, 1H), 3.96–3.85 (m, 1H), 3.74 (s, 3H), 1.47 (d, J = 7.1 Hz, 3H). ^³¹P NMR (162 MHz, chloroform-d) δ -1.62. ¹⁹ F NMR (376 MHz, chloroform-d) δ -153.82 (dd, J = 18.5, 2.7 Hz), -159.99 (td, J = 21.8, 3.8 Hz), -162.65 (dd, J = 22.2, 17.6 Hz). LCMS: MS m/z = 425.9 [M+1], 423.9 [M-1], t _R = 1.68 min; LC system: Thermo Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6 μC18 100A, 50 x 3 mm; solvent: A: water containing 0.1% acetic acid, B: acetonitrile containing 0.1% acetic acid; gradient: 0 min - 0.3 min 5% B, 0.3 min - 1.5 min 5 - 100% B, 1.5 min - 2 min 100% B, 2 min - 2.2 min 100 - 5% B, 2 mL/min. HPLC: t _R = 3.76 min; HPLC system: Agilent 1100 series; column: Phenomenex Gemini 5μC18 110A, 50 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 2 mL/min within 5 min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine methyl ester. Intermediate 4 (56 mg, 0.165 mmol) and intermediate K1 (74 mg, 0.174 mmol) were mixed and dissolved in 5 mL of anhydrous THF. Magnesium chloride (47 mg, 0.495 mmol) was added in a single addition. DIPEA (72 μL, 0.414 mmol) was added, and the reaction was stirred at 45 °C for 20 h. More intermediate K1 (74 mg, 0.174 mmol) was added, and the reaction was stirred at 45 °C for 8 h. The reaction was then stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (10 mL) and washed with water (3 × 5 mL), followed by washing with brine (5 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (4 g _SiO2 Combiflash HP Gold column, 0-100% ethyl acetate/hexane). The fractions were combined and concentrated under reduced pressure. The residue was dissolved in ACN (5 mL) and stirred in an ice bath. A concentrated aqueous hydrochloric acid solution (250 μL) was added dropwise. The reaction mixture was stirred in an ice bath for 1 hour. The ice bath was removed, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with EtOAc (15 mL), and a saturated aqueous sodium bicarbonate solution (20 mL) was added. The mixture was stirred for 10 minutes. The organic extract was collected and washed with brine (10 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by C18 column chromatography (Phenomenex Gemini column, 5-95% ACN/water). The fractions were combined and freeze-dried to obtain the product. NMR spectroscopy confirmed that the product was a single diastereomer. ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.79 (s, ¹H), 7.38–7.30 (m, 2H), 7.26–7.15 (m, 3H), 6.85 (d, J = 4.5 Hz, 1H), 6.73 (d, J = 4.5 Hz, 1H), 5.36 (d, J = 8.4 Hz, 1H), 4.81–4.57 (m, 3H), 4.34 (d, J = 5.2 Hz, 1H), 4.20 (dt, J = 5.7, 1.9 Hz, 2H), 3.91 (dq, J = 10.0, 7.1 Hz, 1H), 3.65 (s, 3H), 1.29 (dd, J = 7.1, 1.1 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.49. LCMS: MS m/z = 540.0 [M+1], 538.2 [M-1], t _R = 1.14 min; LC system: Thermo Dionex Ultimate 3000U HPLC; column: Phenomenex Kinetex 2.6 μC18 100A, 50 x 3 mm; solvent: A: water containing 0.1% acetic acid, B: acetonitrile containing 0.1% acetic acid; gradient: 0 min–0.3 min 5% B, 0.3 min–1.5 min 5–100% B, 1.5 min–2 min 100% B, 2 min–2.2 min 100–5% B, 2 mL/min. HPLC: t _{_R} = 2.23 min; HPLC system: Agilent 1100 series; column: Phenomenex Gemini 5μC18 110A, 50 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 2 mL/min for 5 min. HPLC: t_{_R} = 3.770 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 11. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((((S)-1-methoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran 3,4-dimethylbis(2-methylpropionate)

Example 10 (13 mg, 0.024 mmol) was dissolved in 2 mL of anhydrous THF and stirred at room temperature. Isobutyric anhydride (8 μL, 0.048 mmol) and DMAP (0.3 mg, 0.0024 mmol) were added, and the reaction was stirred for 30 minutes. More isobutyric anhydride (4 μL, 0.024 mmol) was added, and the reaction was stirred for 30 minutes. The reaction was then diluted with EtOAc (10 mL), washed with saturated sodium bicarbonate aqueous solution (2 × 5 mL), and then washed with brine (5 mL). The organic extract was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (4 g _SiO2 Combiflash HP Gold column, 0-10% methanol/DCM). The fractions were combined and concentrated under reduced pressure. The residue was dissolved in ACN, diluted with water, and lyophilized to give the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, methanol- _d4) )δ7.79(s,1H),7.38–7.13(m,5H),6.79(d,J＝4.5Hz,1H),6.61(d,J＝4.6Hz,1H),5.88–5 .77(m,2H),5.60(d,J＝7.8Hz,1H),4.79–4.52(m,2H),4.39–4.21(m,2H),3.96(dq,J＝10. 2, 7.1 Hz, 1H), 3.66 (s, 3H), 2.68 (p, J = 7.0 Hz, 1H), 2.46 (p, J = 7.0 Hz, 1H), 1.31 (dd, J = 7.1, 1.0 Hz, 3H), 1.22 (dd, J = 7.0, 1.1 Hz, 6H), 1.07 (d, J = 7.0 Hz, 3H), 1.03 (d, J = 7.0 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.38. LCMS: MS m/z = 680.2 [M+1], 678.3 [M-1], t _R = 1.60 min; LC system: Thermo Dionex Ultimate 3000U HPLC; Column: Phenomenex Kinetex 2.6μC18 100A, 50x3mm; Solvent: A: water containing 0.1% acetic acid, B: acetonitrile containing 0.1% acetic acid; Gradient: 0 min-0.3 min 5% B, 0.3 min-1.5 min 5-100% B, 1.5 min-2 min 100% B, 2 min-2.2 min 100-5% B, 2 mL/min. HPLC: t _{_R} = 3.17 min; HPLC system: Agilent 1100 series; column: Phenomenex Gemini 5μC18 110A, 50 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 2 mL/min for 5 min. HPLC: t_{_R} = 5.460 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 12. ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester

Tetrahydrofuran (1 mL) was added to a mixture of intermediate 4 (200 mg, 0.591 mmol), intermediate M1 (J. Org. Chem. 2011, 76(20), pp. 8311-8319; 322 mg, 0.709 mmol), and magnesium chloride (84 mg, 0.887 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.257 mL, 1.478 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (7 mL), and concentrated hydrochloric acid aqueous solution (0.493 mL) was added dropwise at 0 °C. After 2 hours at 0°C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0°C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by a gradient of 10-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.71(bs,2H),7.43–7.30(m,2H),7.25–7.15(m,3H),6.84(d,J＝4.5Hz,1H), 6.68(d,J＝4.5Hz,1H),6.04(dd,J＝13.2,10.1Hz,1H),5.29–5.22(m,2H),5.14(d,J＝7.3Hz, 1H),4.86(hept,J＝6.2Hz,1H),4.70–4.58(m,1H),4.57–4.44(m,2H),4.20(t,J＝4.8Hz,1H) ,4.05–3.91(m,2H),3.85–3.68(m,1H),1.21(d,J＝7.0Hz,3H),1.15(dd,J＝6.3,2.8Hz,6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.69 (t, J=48.1Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.51. LCMS: MS m/z = 568.15 [M+1], t _R = 1.19 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 2.55 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.32 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 13. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine ethyl ester

Tetrahydrofuran (4 mL) was added to a mixture of intermediate 4 (200 mg, 0.591 mmol), intermediate N1 (WO2012075140; 415 mg, 0.946 mmol), and magnesium chloride (84 mg, 0.887 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.257 mL, 1.478 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (3 mL), and concentrated hydrochloric acid aqueous solution (0.514 mL) was added dropwise at 0 °C. After 2 hours at 0°C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0°C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by a gradient of 10-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.71(bs,2H),7.45–7.33(m,2H),7.31–7.09(m,3H),6.84(d,J ＝4.5Hz,1H),6.68(d,J＝4.5Hz,1H),6.08(dd,J＝13.3,10.1Hz,1H),5.32–5.22 (m,2H),5.14(d,J＝7.3Hz,1H),4.70–4.44(m,3H),4.19(t,J＝4.8Hz,1H),4.11 –3.94(m,4H),3.88–3.74(m,1H),1.22(d,J＝6.7Hz,3H),1.15(t,J＝7.1Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.69 (t, J=48.0Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.46. LCMS: MS m/z = 554.11 [M+1], t _R = 1.13 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 2.42 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.05 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 14. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester

(S)-1-(cyclohexyloxy)-1-oxopropyl-2-ammonium chloride. Trimethylsilyl chloride (76.56 mL, 695.9 mmol) was added to a mixture of L-alanine (20.0 g, 224.48 mmol) and cyclohexanol (213.6 g, 2132.6 mmol). The reaction was stirred overnight at 80 °C. The reaction was concentrated, and the resulting residue was co-evaporated with 2 × 100 mL of toluene, followed by co-evaporation with 500 mL of hexane. The resulting residue was dried under high vacuum for 15 min, with hexane added slowly while stirring. The mixture was stirred at room temperature for 30 min, and the solid was separated by filtration, washed with hexane, and dried under high vacuum overnight to give intermediate O1. ¹ H NMR (400MHz, DMSO-d6) δ8.53 (d, J = 17.7Hz, 3H), 4.77 (tt, J = 8.4, 3.7Hz, 1H), 3.99 (t, J = 6.9Hz, 1H), 1.88-1.59 (m, 4H), 1.54-1.12 (m, 8H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester. Triethylamine (35 mL, 251.33 mmol) was added to a solution of intermediate O1 (23.2 g, 111.7 mmol) and phenyl dichlorophosphate (16.2 mL, 108.91 mmol) in anhydrous dichloromethane (400 mL) at 0 °C under an argon atmosphere. The resulting mixture was stirred at room temperature for 1.5 h. Then, 4-nitrophenol (14.53 g, 104.44 mmol) and triethylamine (18 mL, 125.66 mmol) were added at 0 °C. The reaction mixture was stirred at room temperature for 1 h, diluted with _Et₂O , and the solid was filtered off. The crude product was concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate and washed with a saturated aqueous sodium carbonate solution and brine. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The crude material was purified by silica gel chromatography (330 g _SiO₂ Combiflash HP Gold column, 0-10% methanol/dichloromethane) to obtain the desired compound as a diastereomeric mixture (41.3 g, 83%, diastereomeric mixture). The obtained material was dried overnight under high vacuum, resulting in solidification. Diisopropyl ether (225 mL) was added to the solidified material and the mixture was thoroughly sonicated to obtain a fine solid. The solid was separated by filtration to obtain intermediate O₂, which was identified as a single isomer by ^1H NMR and ^31P NMR. ^¹H NMR (400MHz, methanol- _d⁴ ) δ 8.32–8.23 (m, 2H), 7.52–7.40 (m, 2H), 7.38 (dd, J = 8.6, 7.2Hz, 2H), 7.29–7.17 (m, 3H), 4.68 (dp, J = 8.7, 3.8Hz, 1H), 4.02 (dq, J = 9.8, 7.1Hz, 1H), 1.78–1.64 (m, 3H), 1.57–1.46 (m, 1H), 1.44–1.22 (m, 9H). ³¹ P NMR (162 MHz, methanol-d4) δ -1.32 (s) LC-MS: MS m/z = 448.86 [M+1]; t _R = 1.3 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μX B-C18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2 - 100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclohexyl ester. Tetrahydrofuran (2 mL) was added to a mixture of intermediate 4 (200 mg, 0.591 mmol), intermediate O2 (318 mg, 0.709 mmol), and magnesium chloride (84 mg, 0.887 mmol) at room temperature. The mixture was heated to 50 °C for 10 minutes, and N,N-diisopropylethylamine (0.257 mL, 1.478 mmol) was added. After stirring at 50 °C for 3 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (7 mL), and concentrated hydrochloric acid aqueous solution (0.493 mL) was added dropwise at 0 °C. After 2 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.71(s,2H),7.43–7.30(m,2H),7.27–7.11(m,3H),6.85(d,J＝4 .4Hz,1H),6.69(d,J＝4.5Hz,1H),6.17–5.94(m,1H),5.36–5.19(m,2H),5.14(d d,J＝7.3,1.0Hz,1H),4.70–4.59(m,2H),4.58–4.45(m,2H),4.21(t,J＝4.8Hz,1 H),4.05–3.91(m,2H),3.85–3.70(m,1H),1.76–1.55(m,4H),1.50–1.12(m,9H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.73 (t, J=48.1Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.52. LCMS: MS m/z = 608.19 [M+1], t _R = 1.31 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 2.89 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.90 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 15. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-(cyclohexyloxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl) Tetrahydrofuran-3,4-dimethyldiacetic acid ester

N,N'-diisopropylcarbodiimide (42 mg, 0.33 mmol) and acetic acid (20 mg, 0.33 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. Example 14 (40 mg, 0.07 mmol) and 4-dimethylaminopyridine (8 mg, 0.07 mmol) were added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, chloroform-d) δ 7.92 (s, 1H), 7.32–7.25 (m, 3H), 7.25–7.20 (m, 2H), 7.17–7.10 (m, 1H), 6.62 (d, J = 4.6Hz, 1H), 6.51 (d, J = 4.6Hz, 1H), 5.86 (dd, J = 7.5, 5.4Hz, 1H), 5.80 (d, J = 5.4Hz, 1H), 5.64 (d, J = 7.5Hz, 1H), 4.75 ( tt, J＝8.8,3.9Hz,1H), 4.71–4.63(m,1H), 4.60–4.52(m,1H), 4.33(ddd, J＝10.8,5.7,1.9Hz,1H), 4.25(ddd, J＝10.8,5.8,2.2Hz,1H), 4.08–3.97(m,2H), 2.12(s,3H), 2.00(s,3H), 1.96–1.62(m,4H), 1.57–1.17(m,7H). ^19F NMR (376MHz, chloroform-d) δ -234.25(t, J＝46.8Hz). ^31P NMR (162MHz, chloroform-d) δ 2.69. LCMS: MS m/z = 692.34 [M+1], t _R = 1.51 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _R = 3.30 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min.

Example 16. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-(cyclohexyloxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl) Tetrahydrofuran-3,4-dimethyldipropionate

Propionic anhydride (17 mg, 0.13 mmol) and Example 14 (40 mg, 0.07 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (8 mg, 0.07 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR(400MHz, chloroform-d)δ7.91(s,1H),7.32–7.25(m,3H),7.24–7.20(m,2H),7 .16–7.11(m,1H),6.60(d,J＝4.6Hz,1H),6.50(d,J＝4.6Hz,1H),5.98–5.9 1(m,2H),5.88(dd,J＝7.3,5.4Hz,1H),5.83(d,J＝5.4Hz,1H),5.63(d,J＝7 .3Hz,1H),4.79–4.71(m,1H),4.67(d,J＝4.0Hz,1H),4.56(d,J＝5.0Hz,1H) ,4.33(ddd,J＝10.8,5.6,1.8Hz,1H),4.25(ddd,J＝10.8,5.8,2.2Hz,1H), 4.11–4.06(m,1H),4.05–3.97(m,1H),2.39(q,J＝7.6Hz,2H),2.26(qd,J＝ 7.6,1.9Hz,2H),1.98(d,J＝14.2Hz,1H),1.83–1.75(m,2H),1.68(t,J＝8. 1Hz, 2H), 1.55–1.28 (m, 6H), 1.17 (t, J = 7.6Hz, 3H), 1.07 (t, J = 7.6Hz, 3H). ¹⁹ F NMR (376 MHz, chloroform-d) δ -234.18 (t, J = 46.9 Hz). ³¹ P NMR (162 MHz, chloroform-d) δ 2.73. LCMS: MS m/z = 720.60 [M+1], t _R = 1.63 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _R = 3.52 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min.

Example 17. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine hexyl ester

L-Alanine Hexyl Ester Hydrochloride. L-Alanine (4.45 g, 50 mmol) was mixed with 1-hexanol (30 mL). TMS-Cl (19.1 mL, 150 mmol) was added dropwise, and the reaction was stirred at room temperature for 16 hours. More 1-hexanol (10 mL) and TMS-Cl (5 mL) were added. The reaction mixture was heated to 80 °C and stirred for 20 hours. The reaction mixture was concentrated under reduced pressure. The resulting oil was dried under high vacuum and allowed to solidify slowly to give intermediate R1, the hydrochloride. ^¹H NMR (400 MHz, chloroform-d) δ 8.77 (s, 3H), 4.20 (m, 3H), 1.71 (m, 5H), 1.47–1.19 (m, 6H), 1.01–0.78 (m, 3H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine hexyl ester. Phenyl dichlorophosphate (3.7 mL, 25 mmol) was dissolved in anhydrous DCM (50 mL) and stirred in an ice bath under atmospheric nitrogen. Intermediate R1 (5.2 g, 25 mmol) was added in one step. The reaction mixture was stirred for 30 min. Triethylamine (8.4 mL, 60 mmol) was added dropwise, followed by stirring for 60 min. p-Nitrophenol (3.1 g, 22.5 mmol) and triethylamine (4.2 mL, 30 mmol) were added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with DCM (100 mL) and washed with water (3 × 20 mL). The organic matter was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (120 g _SiO2 Combiflash HP Gold column, 0-20% ethyl acetate/hexane). The fractions were combined and concentrated under reduced pressure to give intermediate R2 (a mixture of diastereomers). ^¹H NMR (400 MHz, chloroform-d) δ 8.25 (d, J = 9.1 Hz, 2H), 7.51–7.32 (m, 4H), 7.32–7.15 (m, 3H), 4.14 (m, 3H), 3.93 (m, 1H), 1.62 (m, 2H), 1.44 (m, 3H), 1.39–1.20 (m, 6H), 0.99–0.82 (m, 3H). ^³¹P NMR (162 MHz, chloroform-d) δ -3.03 (s), -3.08 (s).

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine hexyl ester. Tetrahydrofuran (1 mL) was added to a mixture of intermediate 4 (100 mg, 0.296 mmol), intermediate R2 (173 mg, 0.384 mmol), and magnesium chloride (42 mg, 0.443 mmol) at room temperature. The mixture was heated to 50 °C for 10 minutes, and N,N-diisopropylethylamine (0.129 mL, 0.739 mmol) was added. After stirring at 50 °C for 3 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (7 mL), and concentrated hydrochloric acid aqueous solution (0.246 mL) was added dropwise at 0 °C. After 2 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to give the product (diasteremeric mixture). ^¹H NMR (400 MHz, DMSO- _d6) )δ7.83(s,0.24H),7.83(s,0.76H),7.72(bs,2H),7.41–7.33(m,2H),7.26–7.14(m ,3H),6.88–6.82(m,1H),6.70(d,J＝4.5Hz,0.26H),6.68(d,J＝4.5Hz,0.74H),6.13– 5.99(m,1H),5.37–5.06(m,3H),4.74–4.44(m,3H),4.25–4.11(m,1H),4.06–3.93( m,4H),3.90–3.76(m,1H),1.58–1.44(m,2H),1.34–1.11(m,9H),0.87–0.74(m,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -236.47 (t, J = 48.1 Hz), -236.76 (t, J = 48.1 Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 3.55, 3.47. LCMS: MS m/z = 610.16 [M+1], t _R = 1.35 min (minor isomer) and 1.37 min (major isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 3.02 min (minor isomer) and 3.06 min (major isomer); HPLC system: Agilent 1100 series; column: Gemini 5μC18110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t _R = 5.16 min (minor isomer) and 5.23 min (major isomer); HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B gradient at 1.5 mL/min for 8.5 min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (Chiralpak AD-H 5 μm, 21 x 250 mm; 30% methanol) to obtain Examples 18 and 19.

Example 18. ((R)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine hexyl ester

First eluted diastereomer of Example 17: ^1H NMR (400MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.36–7.28(m,2H),7.23–7.09(m,3H),6.88(d,J＝4.6Hz,1H), 6.69(d,J＝4.5Hz,1H),5.99(dd,J＝13.0,10.1Hz,1H),5.22(d,J＝8.6Hz,1H),4 .69–4.57(m,1H),4.54–4.42(m,2H),4.17(d,J＝5.2Hz,1H),4.03–3.90(m,4H) ,3.84–3.69(m,1H),1.53–1.40(m,2H),1.30–1.05(m,9H),0.82–0.73(m,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.46 (t, J=48.1Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.54. LCMS: MS m/z = 610.16 [M+1], t _R = 1.35 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 3.02 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.16 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 19. ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine hexyl ester

Second eluted diastereomer of Example 17: ^¹H NMR (400MHz, DMSO- _d₆ ) δ 7.83 (s, 1H), 7.73 (bs, 2H), 7.45–7.27 (m, 2H), 7.29–7.10 (m, 3H), 6.86 (d, J = 4.5Hz, 1H), 6.68 (d, J = 4.5Hz, 1H), 6.06 (dd, J = 13.2, 10.1Hz, 1H), 5.28–5.19 (m, 2H), 5.1 8–5.09(m,1H),4.70–4.57(m,1H),4.58–4.45(m,2H),4.23–4.17(m,1H),4.05–3.95 (m,4H),3.88–3.79(m,1H),1.57–1.48(m,2H),1.30–1.18(m,9H),0.85–0.80(m,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.75 (t, J=48.1Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.47. LCMS: MS m/z = 610.16 [M+1], t _R = 1.37 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 3.06 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.23 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 20. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((S)-(((S)-1-isopropoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) Tetrahydrofuran-3,4-dimethyldiacetic acid ester

N,N'-diisopropylcarbodiimide (56 mg, 0.44 mmol) and acetic acid (26 mg, 0.44 mmol) were dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. Example 12 (50 mg, 0.09 mmol) and 4-dimethylaminopyridine (11 mg, 0.09 mmol) were added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10–100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ HNMR (400MHz, DMSO-d ₆ )δ7.86(s,1H),7.80(bs,2H),7.41–7.32(m,2H),7.28–7.11(m,3H),6.83(d,J＝4.5Hz,1H),6.66(d, J＝4.5Hz,1H),6.13(dd,J＝13.2,10.1Hz,1H),5.75(dd,J＝8.7,5.5Hz,1H),5.67(d,J＝5.4Hz,1H),5.5 3(d,J＝8.7Hz,1H),4.86(hept,J＝6.2Hz,1H),4.73–4.61(m,1H),4.61–4.49(m,1H),4.21–4.07(m,2 H), 3.89–3.71 (m, 1H), 2.13 (s, 3H), 1.93 (s, 3H), 1.21 (d, J = 7.0Hz, 3H), 1.15 (dd, J = 6.2, 2.8Hz, 6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-234.62 (t, J=46.9Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.49. LCMS: MS m/z = 652.41 [M+1], t _R = 1.38 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 2.98 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.03 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 21. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-ethoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl)tetrahydro Furan-3,4-dimethyldiacetate

N,N'-diisopropylcarbodiimide (57 mg, 0.45 mmol) and acetic acid (27 mg, 0.45 mmol) were dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. Example 13 (50 mg, 0.09 mmol) and 4-dimethylaminopyridine (11 mg, 0.09 mmol) were added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ HNMR (400MHz, DMSO-d ₆ )δ7.86(s,1H),7.80(bs,2H),7.40–7.33(m,2H),7.25–7.16(m,3H),6.83(d,J＝4.5Hz,1H), 6.66(d,J＝4.5Hz,1H),6.16(dd,J＝13.3,10.1Hz,1H),5.75(dd,J＝8.7,5.5Hz,1H),5.67(d,J ＝5.4Hz,1H),5.53(d,J＝8.7Hz,1H),4.74–4.44(m,2H),4.21–4.09(m,2H),4.05–3.97(m,2H) ,3.91–3.75(m,1H),2.13(s,3H),1.93(s,3H),1.22(d,J＝7.0Hz,3H),1.14(t,J＝7.1Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-234.59 (t, J=46.8Hz). ³¹ PNMR (162MHz, DMSO-d ₆ ) δ3.44. LCMS: MS m/z = 638.26 [M+1], t _R = 1.32 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 2.84 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.78 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 22. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((S)-(((S)-1-isopropoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) Tetrahydrofuran-3,4-dimethyldipropionate

Example 12 (50 mg, 0.09 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere. Propionic anhydride (24 mg, 0.19 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) were added, and the reaction mixture was stirred at room temperature. After 30 minutes, the reaction mixture was diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO- _d6) )δ7.85(s,1H),7.80(bs,2H),7.41–7.32(m,2H),7.28–7.13(m,3H),6.83(d,J＝4.5Hz,1H),6.66(d,J＝4.5Hz,1H) ,6.13(dd,J＝13.2,10.1Hz,1H),5.78(dd,J＝8.5,5.5Hz,1H),5.69(d,J＝5.5Hz,1H),5.53(d,J＝8.5Hz,1H),4.96– 4.76(m,1H),4.73–4.62(m,1H),4.62–4.47(m,1H),4.26–4.05(m,2H),3.91–3.68(m,1H),2.46–2.37(m,2H),2.3 1–2.00(m,2H),1.21(d,J＝7.1Hz,3H),1.14(dd,J＝6.2,2.4Hz,6H),1.07(t,J＝7.5Hz,3H),0.93(t,J＝7.5Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-234.55 (t, J=46.8Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.50. LCMS: MS m/z = 680.51 [M+1], t _R = 1.48 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 3.23 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.50 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 23. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((S)-(((S)-1-isopropoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) Tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Example 12 (50 mg, 0.09 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere. Isobutyric anhydride (29 mg, 0.19 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) were added, and the reaction mixture was stirred at room temperature. After 4 hours, the reaction mixture was diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO- _d6) )δ7.85(s,1H),7.78(bs,2H),7.41–7.31(m,2H),7.26–7.13(m,3H),6.82(d,J＝4.5Hz,1H),6.64(d,J＝4.5Hz,1 H),6.13(dd,J＝13.2,10.0Hz,1H),5.75(dd,J＝8.3,5.4Hz,1H),5.69(d,J＝5.5Hz,1H),5.53(d,J＝8.3Hz,1H),4 .86(hept,J＝6.3Hz,1H),4.74–4.63(m,1H),4.63–4.51(m,1H),4.23–4.06(m,2H),3.90–3.70(m,1H),2.73–2. 58(m,1H),2.41(hept,J＝7.0Hz,1H),1.21(d,J＝7.1Hz,3H),1.17–1.10(m,12H),0.95(dd,J＝17.5,7.0Hz,6H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ-234.04 (t, J=46.7Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.53. LCMS: MS m/z = 708.42 [M+1], t _R = 1.61 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 3.46 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.92 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 24. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-ethoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl)tetrahydro Furan-3,4-dimethylbis(2-methylpropionate)

Example 13 (50 mg, 0.09 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere. Isobutyric anhydride (30 mg, 0.19 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) were added, and the reaction mixture was stirred at room temperature. After 4 hours, the reaction mixture was diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO- _d6) )δ7.85(s,1H),7.79(bs,2H),7.40–7.32(m,2H),7.26–7.14(m,3H),6.82(d,J＝4.5Hz,1H),6.64(d,J＝4.5Hz, 1H),6.16(dd,J＝13.3,10.1Hz,1H),5.75(dd,J＝8.4,5.5Hz,1H),5.69(d,J＝5.5Hz,1H),5.53(d,J＝8.3Hz,1H), 4.77–4.65(m,1H),4.62–4.51(m,1H),4.21–4.12(m,2H),4.10–3.96(m,2H),3.95–3.73(m,1H),2.65(hept,J＝ 7.0Hz,1H),2.41(hept,J＝7.0Hz,1H),1.22(d,J＝7.0Hz,3H),1.18–1.03(m,9H),0.96(dd,J＝17.6,7.0Hz,6H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ-234.01 (t, J=46.7Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.49. LCMS: MS m/z = 694.42 [M+1], t _R = 1.55 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 3.34 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.70 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 25. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-ethoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl)tetrahydro Furan-3,4-dimethyldipropionate

Example 13 (50 mg, 0.09 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 mL) under argon atmosphere. Propionic anhydride (25 mg, 0.19 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) were added, and the reaction mixture was stirred at room temperature. After 30 minutes, the reaction mixture was diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO- _d6) )δ7.85(s,1H),7.79(bs,2H),7.41–7.31(m,2H),7.27–7.14(m,3H),6.83(d,J＝4.5Hz,1H),6.66(d,J＝4.5Hz,1 H),6.16(dd,J＝13.3,10.1Hz,1H),5.78(dd,J＝8.6,5.5Hz,1H),5.69(d,J＝5.5Hz,1H),5.53(d,J＝8.5Hz,1H),4. 73–4.62(m,1H),4.61–4.49(m,1H),4.21–4.11(m,2H),4.09–3.98(m,2H),3.91–3.74(m,1H),2.46–2.37(m,2H) ,2.29–2.12(m,2H),1.22(d,J＝7.0Hz,3H),1.14(t,J＝7.1Hz,3H),1.07(t,J＝7.5Hz,3H),0.93(t,J＝7.5Hz,3H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ-234.52 (t, J=46.8Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.45. LCMS: MS m/z = 666.35 [M+1], t _R = 1.43 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 3.11 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 5.27 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 26. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-hydroxy-2-methyl propyl ester

(S)-1-(2-(benzyloxy)-2-methylpropoxy)-1-oxopropan-2-ammonium chloride. DMAP (2.04 g, 8.32 mmol) was added to a mixture of Boc-L-alanine (1.26 g, 6.66 mmol), 2-benzyloxy-2-methylpropanol (1.0 g, 5.55 mmol), and EDCI (1.12 g, 7.21 mmol) in acetonitrile (20 mL). The mixture was then stirred at room temperature for 2 hours, diluted with EtOAc, washed with brine, dried over sodium sulfate, and concentrated under vacuum. The resulting residue was purified by silica gel chromatography (EtOAc 0 to 60% hexane solution) to give Boc-L-alanine propyl ester, which was dissolved in DCM (10 mL) and a dioxane solution of 4N HCl (5.5 mL, 22.19 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 2 hours, concentrated under vacuum, dissolved in ACN (10 mL), and lyophilized overnight to obtain intermediate AA1, which was used in the next reaction. ^¹H NMR (400 MHz, chloroform-d) δ 8.82 (s, 3H), 7.42–7.07 (m, 5H), 4.44 (s, 2H), 4.24 (m, 2H), 4.08 (d, J = 11.2 Hz, 1H), 1.70 (d, J = 7.0 Hz, 3H), 1.28 (d, J = 2.4 Hz, 6H). LCMS m/z = 251.97 [free base M+1], t _R = 0.85 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 2-(benzyloxy)-2-methylpropyl ester. Phenyl dichlorophosphate (0.43 mL, 2.89 mmol) was added in a single dose to a solution of intermediate AA1 (832 mg, 2.89 mmol) in DCM (20 mL) at -78 °C, followed by dropwise addition of triethylamine (0.80 mL, 5.76 mmol) over 5 minutes at -78 °C. After removing the dry ice bath, the resulting mixture was stirred for 30 minutes and cooled to -78 °C, followed by a single dose of p-nitrophenol (402 mg, 2.89 mmol), and then triethylamine (0.40 mL, 2.89 mmol) over 5 minutes at -78 °C. After removing the dry ice bath, the resulting mixture was stirred for 50 minutes, then diluted with DCM, washed with brine, concentrated under vacuum, and the residue was purified by silica gel column chromatography (EtOAc 0 to 60% hexane solution) to give intermediate AA2 (a mixture of diastereomers). ^¹H NMR (400 MHz, chloroform-d) δ 8.25–8.12 (m, 2H), 7.41–7.14 (m, 12H), 4.45 (m, 2H), 4.31–4.12 (m, 2H), 4.07 (m, 1H), 3.89 (m, 1H), 1.41 (m, 3H), 1.27 (m, 6H). ^³¹P NMR (162 MHz, chloroform-d) δ -3.10, -3.18. LCMS m/z = 528.78 [M+1], t _R = 1.70 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-(benzyloxy)-2-methylpropyl ester. N,N-dimethylformamide (2 mL) was added to a mixture of intermediate 5 (0.04 g, 0.134 mmol), intermediate AA2 (0.081 g, 0.153 mmol), and magnesium chloride (0.064 g, 0.671 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.07 mL, 0.402 mmol). The resulting mixture was stirred at 50 °C for 3 hours. The reaction mixture was then purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30mm column, 0-100% acetonitrile/water) to obtain intermediate AA3. LCMS: MS m/z = 688.18 [M+1], t _R = 1.20 min (major isomer) and 1.22 min (minor isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-hydroxy-2-methylpropyl ester. 10% Pd/C (11 mg, 0.01 mmol) was added to a solution of intermediate AA3 (14 mg, 0.02 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature under _H₂ atmosphere for 15 hours and filtered. The filtrate was concentrated under vacuum, dissolved in ACN, and purified by preparative HPLC (Phenominex Gemini-NX 10u C18 250x30 mm column, ACN 10% to 100% aqueous solution) to give the product as a diastereomeric mixture. ¹ H NMR (400MHz, methanol-d4) δ7.79(s,1H),7.39–7.27(m,2H),7.28–7.13(m,3H),6.85(dd,J＝6.9, 4.5Hz,1H),6.74(dd,J＝11.1,4.5Hz,1H),5.36(dd,J＝8.3,5.9Hz,1H),4.83–4.68(m,1H), 4.73–4.64 (m, 1H), 4.69–4.56 (m, 1H), 4.36 (dd, J = 16.6, 5.2 Hz, 1H), 4.30–4.15 (m, 2H), 4.05–3.92 (m, 1H), 3.96–3.86 (m, 2H), 1.32 (ddd, J = 19.2, 7.2, 1.1 Hz, 3H), 1.18–1.19 (m, 6H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.78, 3.54 ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) -238.51–-238.97 (m). LCMS: MS m/z = 598.05 [M+1], t _R = 0.94 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 3.697 min (major isomer), 3.734 min (minor isomer); HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B gradient at 1.5 mL/min for 8.5 min.

Example 27. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((((S)-1-(2-hydroxy-2-methylpropoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy 3,4-dimethylbis(2-methylpropionate)tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

((((3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-(fluoromethyl)-2,2-dimethyltetrahydrofuranolo[3,4-d][1,3]dioxacyclopenten-4-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-(benzyloxy)-2-methylpropyl ester. Tetrahydrofuran (2.5 mL) was added to a mixture of intermediate 4 (0.2 g, 0.591 mmol), intermediate AA2 (0.375 g, 0.709 mmol), and magnesium chloride (0.112 g, 1.182 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.258 mL, 1.478 mmol). The resulting mixture was stirred at 50 °C for 2 hours. The reaction mixture was then diluted with EtOAc, washed with brine, dried over sodium sulfate, and concentrated under vacuum. The resulting residue was purified by silica gel chromatography (EtOAc 0 to 100% hexane solution) to give intermediate BB1 (a diastereomeric mixture). LCMS: MS m/z = 728.18 [M+1], t _R = 1.39 min (minor isomer) and 1.42 min (major isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-(benzyloxy)-2-methylpropyl ester. Concentrated hydrochloric acid (0.35 mL, 9.6 mmol) was added dropwise to an ice-cold solution of intermediate BB1 (0.35 g, 0.481 mmol) in acetonitrile (14 mL). The reaction mixture was stirred at 0 °C for 4 hours. The reaction mixture was then diluted with ice and neutralized with saturated sodium bicarbonate solution. The reaction mixture was extracted with ethyl acetate, and the organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The residue obtained was purified by silica gel chromatography (0-20% methanol/dichloromethane) to give intermediate BB2 (a mixture of diastereomers). ¹ H NMR (400MHz, methanol-d ₄ )δ7.78(d,J＝1.1Hz,1H),7.38–7.12(m,10H),6.88–6.81(m,1H),6.74(dd,J＝8.3,4.5Hz,1H),5.36(dd,J＝8.3,6.6Hz,1H),4.82–4.67(m,1H),4.7 1–4.63(m,1H),4.67–4.55(m,1H),4.45(d,J＝4.8Hz,2H),4.34(dd,J＝16 .4,5.2Hz,1H),4.29–4.12(m,3H),4.10–3.90(m,2H),1.35–1.21(m,9H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.39–-238.76 (m). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.69, 3.49. LCMS: MS m/z = 688.10 [M+1]; t _R = 1.20 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

(2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-((((((S)-1-(2-(benzyloxy)-2-methylpropoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)-oxy)methyl)-2-(fluoromethyl)tetrahydrofuran-3,4-dimethylbis(2-methylpropionate). Isobutyric anhydride (0.072 mL, 0.436 mmol) was added to a solution of intermediate BB2 (0.05 g, 0.073 mmol) in THF (2 mL) at room temperature, followed by the addition of DMAP (1.3 mg, 0.011 mmol). The resulting mixture was stirred for 30 minutes and monitored by LCMS. The reaction mixture was quenched with methanol and concentrated. The obtained residue was purified by silica gel chromatography (0-15% methanol/dichloromethane) to give intermediate BB3 (a mixture of diastereomers). ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.79 (d, J = 4.1 Hz, 1H), 7.36–7.12 (m, 10H), 6.87–6.73 (m, 1H), 6.60 (d, J = 4.6 Hz, 1H), 5.87–5.76 (m, 2H), 5.59 (dd, J = 7.8, 4.4 Hz, 1H), 4.76–4.63 (m, 1H), 4.65–4.51 (m, 1H), 4.46 (d, J = 1.4 Hz, 2H), 4.35 (dt, J = 1.4 Hz, 2H), 4.35 (dt, J = 1.4 Hz, 2H). 9.1, 2.8 Hz, 1H), 4.26 (ddd, J = 10.7, 5.4, 2.1 Hz, 1H), 4.18 (dd, J = 11.5, 2.7 Hz, 1H), 4.13–3.93 (m, 2H), 2.73–2.59 (m, 1H), 2.53–2.39 (m, 1H), 1.34 (dd, J = 7.1, 1.1 Hz, 2H), 1.31–1.18 (m, 13H), 1.01–1.09 (m, 6H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.36 (t, J = 47.0 Hz), -235.91 (t, J = 47.0 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.45, 3.33. LCMS: MS m/z = 828.21 [M+1]; t _R = 1.60 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

(2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-2-((((((S)-1-(2-hydroxy-2-methylpropoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3,4-dimethylbis(2-methylpropionate). 10% Pd/C (26 mg, 0.024 mmol) was added to a solution of intermediate BB3 (40 mg, 0.048 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature under _H₂ for 15 hours and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel chromatography (0-15% methanol/dichloromethane) to give the product (diasteremeric mixture). ^¹H NMR (400 MHz, methanol- _d⁻¹) )δ7.79(d,J＝4.2Hz,1H),7.35–7.29(m,2H),7.27–7.15(m,3H),6.79(d,J＝4 .6Hz,1H),6.62(d,J＝4.6Hz,1H),5.86–5.76(m,2H),5.60–5.56(m,1H),4.76 –4.53(m,2H),4.37–4.21(m,2H),4.07–3.88(m,3H),2.67(p,J＝6.9Hz,1H),2 .53–2.38(m,1H),1.40–1.25(m,3H),1.25–1.16(m,12H),1.01–1.09(m,6H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.56 (t, J = 46.9 Hz), -235.96 (t, J = 46.9 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.50, 3.45. LCMS: MS m/z = 738.17 [M+1]; t _R = 1.29 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 5.31 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC Chiralpak AD-H 5 μm, 21 x 250 mm; 30% isopropanol).

Example 28. First eluted diastereomer of Example 27: ^¹H NMR (400MHz, methanol- _d⁴ ) δ 7.80 (s, 1H), 7.36–7.27 (m, 2H), 7.25–7.12 (m, 3H), 6.85 (d, J = 4.6Hz, 1H), 6.76 (d, J = 4.6Hz, 1H), 5.92 (dd, J = 7.6, 5.6Hz, 1H), 5.84 (d, J = 5.6Hz, 1H), 5.59 (d, J = 7.5Hz, 1H), 4. 72 (s, 1H), 4.61 (s, 1H), 4.34 (d, J = 4.5 Hz, 2H), 4.04–3.87 (m, 3H), 2.73–2.58 (m, 1H), 2.48 (p, J = 7.0 Hz, 1H), 1.30 (dd, J = 7.2, 1.2 Hz, 3H), 1.25–1.16 (m, 12H), 1.03–1.10 m, (6H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.93 (t, J = 47.0 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.49. LCMS: MS m/z = 738.16 [M+1]; t _R = 1.31 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 5.282 min; HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 29. Second eluted diastereomer of Example 27: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.79 (s, 1H), 7.37–7.28 (m, 2H), 7.25 (dt, J = 8.7, 1.2 Hz, 2H), 7.18–7.20 (m, 1H), 6.79 (d, J = 4.5 Hz, 1H), 6.62 (d, J = 4.6 Hz, 1H), 5.88–5.77 (m, 2H), 5.59 (d, J = 7.8 Hz, 1H), 4.78–4.65 (m, 1H), 4.66–4.53 (m, 1H), 4.34 (ddd, J = 8.7, 1.2 Hz, 2H) 10.8, 4.8, 1.8 Hz, 1H), 4.27 (ddd, J = 10.7, 5.6, 2.1 Hz, 1H), 4.09–3.92 (m, 2H), 3.91 (d, J = 10.9 Hz, 1H), 2.67 (hept, J = 7.0 Hz, 1H), 2.46 (hept, J = 7.1 Hz, 1H), 1.37 (dd, J = 7.1, 1.0 Hz, 3H), 1.26–1.10 (m, 12H), 1.02–1.08 (m, 6H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.54 (t, J = 46.8 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.44. LCMS: MS m/z = 738.17 [M+1]; t _R = 1.30 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 5.277 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 30. (S)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-methoxy propyl ester

(S)-(tert-butoxycarbonyl)-L-alanine 2-methoxypropyl ester. (tert-butoxycarbonyl)-L-alanine (2.519 g, 0.013 mol) was dissolved in acetonitrile (12 mL), and (S)-2-methoxyprop-1-ol (1 g, 0.011 mol) was added in a single addition, followed by EDCI (2.239 g, 0.014 mol) and DMAP (2.033 g, 0.017 mol). The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was achieved by silica gel chromatography with 0–30% ethyl acetate/hexane to give intermediate EE1. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.31(d,J＝7.4Hz,1H),4.10–3.92(m,3H),3.50(td,J＝6.3,4.2Hz,1H),3.26(s,3H),1.38(s,9H),1.25(d,J＝7.4Hz,3H),1.07(d,J＝6.4Hz,3H).

(S)-2-Methoxypropyl-L-alanine ester hydrochloride. Intermediate EE1 (2.165 g, 0.008 mol) was dissolved in anhydrous dichloromethane (22 mL) and a dioxane solution of 4N HCl (10.36 mL, 0.041 mol). The reaction was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The residue was placed under high vacuum overnight, and intermediate EE2 was used as is for the next step without purification. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.64 (s, 3H), 4.17–3.99 (m, 3H), 3.53 (m, 1H), 3.24 (s, 3H), 1.41 (d, J = 7.2 Hz, 3H), 1.07 (d, J = 6.4 Hz, 3H).

(S)-((4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxypropyl ester. Triethylamine (2.34 mL, 16.66 mmol) was added to a solution of intermediate EE2 (1.5 g, 7.589 mmol) and phenyl dichlorophosphate (1.129 mL, 7.589 mmol) in anhydrous dichloromethane (26 mL) at 0 °C under an argon atmosphere. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (1.056 g, 7.589 mmol) and triethylamine (1.17 mL, 8.33 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80 g _SiO₂ Combiflash HP Gold column, 100% dichloromethane, followed by 0-75% ethyl acetate/hexane) to give intermediate EE3 (a diastereomeric mixture). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.32–8.23(m,2H),7.53–7.44(m,1H),7.49–7.34(m,3H),7.30–7.16(m,3H),6.70(ddd,J＝13.6,10.0,7.9Hz,1 H),4.08–3.89(m,3H),3.48–3.36(m,1H),3.19(d,J＝1.0Hz,3H),1.26–1.11(m,3H),1.01(dd,J＝6.4,1.5Hz,3H). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ -1.26, -1.47. LCMS: MS m/z = 438.99 [M+1]; t _R = 1.43 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

(S)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxypropyl ester. Tetrahydrofuran (1.5 mL) was added to a mixture of intermediate 4 (0.15 g, 0.443 mmol), intermediate EE3 (0.214 g, 0.488 mmol), and magnesium chloride (0.063 g, 0.665 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.193 mL, 1.108 mmol). The resulting mixture was stirred at 50 °C for 1.5 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 15%–85% acetonitrile/water). The purified fractions were combined and concentrated under reduced pressure. The resulting residue was dissolved in anhydrous acetonitrile (3 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (0.2 mL, 2.4 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and diluted with ice, then neutralized with an aqueous sodium bicarbonate solution. The resulting mixture was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 15%–85% acetonitrile/water gradient, 30 min run) to give the product (diasteremeric mixture). ^¹H NMR (400 MHz, DMSO- _d6) was used. )δ7.81(d,J＝2.4Hz,1H),7.67(s,2H),7.35(dt,J＝8.5,6.9Hz,2H),7.24–7.11(m,3H),6.83(dd,J＝5.6,4.5Hz ,1H),6.67(dd,J＝8.0,4.5Hz,1H),6.05(td,J＝13.9,10.1Hz,1H),5.30–5.18(m,2H),5.10(dd,J＝7.3,2.6Hz,1 H),4.67–4.55(m,1H),4.50(dd,J＝14.2,7.0Hz,2H),4.18(q,J＝5.2Hz,1H),3.96(m,4H),3.83(dd,J＝10.5,7. 0Hz, 1H), 3.49–3.40 (m, 1H), 3.20 (d, J = 2.0Hz, 3H), 1.20 (dd, J = 15.3, 7.1Hz, 3H), 1.02 (dd, J = 6.3, 3.8Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.33–-236.84(m). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 3.57, 3.44. LCMS: MSm/z = 598.03 [M+1]; t _R = 1.00 min (minor isomer) and 1.10 min (major isomer); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 3.95 min (minor isomer), 4.018 min (major isomer); HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B gradient at 1.5 mL/min for 8.5 min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC Chiralpak AD-H 5 μm, 21 x 250 mm; 30% isopropanol).

Example 31. First eluted diastereomer of Example 30: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.78 (s, ¹H), 7.33 (dd, J = 8.5, 7.3 Hz, 2H), 7.24–7.13 (m, 3H), 6.86 (d, J = 4.6 Hz, 1H), 6.75 (d, J = 4.5 Hz, 1H), 5.37 (d, J = 8.2 Hz, 1H), 4.82–4.71 (m, 1H), 4.71–4.59 (m, 2H), 4 0.38 (d, J = 5.3 Hz, 1H), 4.26 (dd, J = 5.2, 1.7 Hz, 2H), 4.07–3.99 (m, 2H), 3.98–3.85 (m, 1H), 3.58–3.49 (m, 1H), 3.32 (s, 3H), 1.26 (dd, J = 7.2, 1.2 Hz, 3H), 1.11 (d, J = 6.4 Hz, 3H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.62 (t, J = 47.8 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.69. LCMS: MS m/z = 597.94 [M+1]; t _R = 1.11 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 3.939 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 32. Second eluted diastereomer of Example 30: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.79 (s, ¹H), 7.39–7.29 (m, 2H), 7.27–7.14 (m, 3H), 6.85 (d, J = 4.5 Hz, ¹H), 6.73 (d, J = 4.6 Hz, ¹H), 5.36 (d, J = 8.3 Hz, ¹H), 4.82–4.55 (m, 3H), 4.33 (d, J = 5.2 Hz, ¹H). 4.21 (dt, J = 5.8, 1.8 Hz, 2H), 4.04 (d, J = 5.0 Hz, 2H), 3.93 (dq, J = 9.8, 7.2 Hz, 1H), 3.59–3.48 (m, 1H), 3.31 (s, 3H), 1.31 (dd, J = 7.1, 1.0 Hz, 3H), 1.10 (d, J = 6.4 Hz, 3H). ^19F NMR (376 MHz, methanol- _d⁴ ) δ -238.78 (t, J = 47.8 Hz). ^31P NMR (162 MHz, methanol- _d⁴ ) δ 3.44. LCMS: MS m/z = 597.97 [M+1]; t _R = 1.12 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2 - 100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 4.005 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 33. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((((S)-1-methoxy-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran 3,4-dimethyldipropionate

At room temperature, propionic anhydride (0.041 mL, 0.317 mmol) was added to a 2 mL solution of THF (0.057 g, 0.106 mmol) from Example 10, followed by the addition of DMAP (2.58 mg, 0.021 mmol). The resulting mixture was stirred for 10 minutes and monitored by LCMS. The reaction mixture was quenched with methanol and diluted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium bicarbonate solution, separated, dried over sodium sulfate, filtered, and concentrated. The residue obtained was purified by silica gel chromatography (0-15% methanol/dichloromethane) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.79(s,1H),7.38–7.29(m,2H),7.33–7.21(m,2H),7.23–7.14(m,1H),6.79(d,J＝4.5Hz,1H),6.62(d,J ＝4.6Hz,1H),5.91–5.78(m,2H),5.58(d,J＝7.9Hz,1H),4.76–4.64(m,1H),4.58(q,J＝10.0Hz,1H),4.37–4 .28 (m, 1H), 4.25 (ddd, J = 10.5, 5.5, 2.1 Hz, 1H), 4.02–3.89 (m, 1H), 3.66 (s, 3H), 2.45 (qd, J = 7.5, 1.0 Hz, 2H), 2.26 (qd, J = 7.6, 3.2 Hz, 2H), 1.31 (dd, J = 7.1, 1.0 Hz, 3H), 1.16 (t, J = 7.6 Hz, 3H), 1.04 (t, J = 7.5 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.37. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -236.42 (t, J = 46.6 Hz). LCMS: MS m/z = 652.09 [M+1]; t _R = 1.23 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 4.994 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 34. ((S)-(((2R,3S,4R,5S)-2-(fluoromethyl)-3,4-dihydroxy-5-(4-imino-3- ((phosphonoyloxy)methyl)-3,4-dihydropyrrolo[2,1-f][1,2,4]triazin-7-yl)tetrahydrofuran-2-yl)methoxy (phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester

((S)-((((2R,3S,4R,5S)-5(3-(((((benzyloxy)(hydroxy)phosphoryl)oxy)methyl)-4-imino-3,4-dihydropyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester. Dibenzylchloromethyl phosphate and sodium iodide were added to a solution of Example 1 (0.125 g, 0.205 mmol) in DMF (2.25 mL). The reaction mixture was stirred overnight at room temperature, and the resulting reaction mixture was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 0-100% acetonitrile/water) to give intermediate II1. ^1H NMR (400 MHz, DMSO- _d6) )δ12.49(s,1H),10.31(d,J＝10.5Hz,1H),8.42(s,1H),7.45(dd,J＝4.8,2.3Hz,1H),7.36(t,J＝7.9Hz,2H),7.26(d,J＝4.3Hz,4H) ,7.20(dt,J＝11.9,6.6Hz,4H),6.91(d,J＝4.7Hz,1H),6.07(dd,J＝13.0,10.1Hz,1H),5.64(d,J＝10.8Hz,2H),5.39(d,J＝4.7Hz,1 H),5.29(s,1H),5.20(d,J＝8.7Hz,1H),4.76(d,J＝6.9Hz,2H),4.68–4.58(m,1H),4.59–4.48(m,1H),4.40(s,1H),4.19(t,J＝3.8 Hz,1H),3.98(ddd,J＝19.0,10.9,5.6Hz,3H),3.93–3.79(m,2H),1.43(h,J＝6.3Hz,1H),1.32–1.20(m,7H),0.80(t,J＝7.4Hz,6H). ³¹ P NMR (162MHz, DMSO- _d₆ ) δ 3.59, 0.13. ¹⁹ F NMR (376MHz, DMSO- _d₆ ) δ -235.99 (t, J = 47.6Hz). LCMS: MS m/z = 810.23 [M+1]; t _R = 1.30 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2 - 100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min.

((S)-(((2R,3S,4R,5S)-2-(fluoromethyl)-3,4-dihydroxy-5-(4-imino-3-((phosphonooxy)methyl)-3,4-dihydropyrrolo[2,1-f][1,2,4]triazine-7-yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester. 10% Pd/C (6.17 mg, 0.058 mmol) was added to a solution of intermediate II1 (47 mg, 0.058 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature under _H₂ atmosphere for 1.5 hours and filtered. The filtrate was concentrated, and the resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30mm column, 0-100% acetonitrile/water containing TFA modifier) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400MHz, DMSO- _d⁶ ) δ 10.33 (s, ¹H), 8.42 (s, ¹H), 7.48 (d, J = 4.8Hz, ¹H), 7.40–7.30 (m, 2H), 7.24–7.13 (m, 3H), 6.90 (d, J = 4.7Hz, ¹H), 6.07 (dd, J = 13.1, 10.1Hz, ¹H), 5.67 (d, J = 11.5Hz, 2H), 5.41 (s, ¹H), 5.19 (d, J) ＝8.7Hz,1H),4.63(q,J＝10.2Hz,1H),4.57–4.44(m,1H),4.39(dd,J＝8.8,4.9Hz,1H),4.18(d,J＝ 4.9Hz, 1H), 4.06–3.77 (m, 5H), 1.42 (h, J = 6.2Hz, 1H), 1.33–1.19 (m, 7H), 0.80 (t, J = 7.5Hz, 6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -74.19 (s), -236.54 (t, J=47.9Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 3.59, 0.12. LCMS: MS m/z = 720.16 [M+1]; t _R = 1.21 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 4.854 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 35. (R)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-1-methyl pyrrolidine-3-yl ester

(R)-(tert-butoxycarbonyl)-L-alanine 1-methylpyrrolidine-3-yl ester. Triphenylphosphine (3.63 g, 0.014 mol) was added in a single addition to a solution of (S)-1-methylpyrrolidine-3-ol (1.0 g, 0.01 mol) and (tert-butoxycarbonyl)-L-alanine (2.058 g, 0.011 mol) in THF (20 mL). Diisopropyl azodicarbonate (2.53 mL, 0.013 mol) was added to the resulting reaction mixture, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with reaction EtOAc and washed with saturated sodium bicarbonate aqueous solution, followed by washing with 5% citric acid aqueous solution. The citric acid extract was washed with EtOAc (2×). The acid extract was alkalized with 2N NaOH aqueous solution to obtain a pH of 9, and extracted with EtOAc (2×). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate JJ1. ^1H NMR (400MHz, chloroform-d) δ 5.29–5.18 (m, 1H), 5.02 (s, 1H), 4.28 (t, J = 7.6 Hz, 1H), 2.80 (dd, J = 9.6, 5.4 Hz, 1H), 2.69 (d, J = 3.6 Hz, 2H), 2.42–2.19 (m, 5H), 1.89–1.72 (m, 1H), 1.43 (s, 9H), 1.36 (d, J = 7.2 Hz, 3H).

(R)-1-methylpyrrolidone-3-yl-L-alanine ester dihydrochloride. Intermediate JJ1 (2.27 g, 0.008 mol) was dissolved in anhydrous dichloromethane (20 mL), and a dioxane solution of 4N HCl (14.59 mL, 0.058 mol) was added. The mixture was stirred at ambient temperature for 4 hours. The solution was concentrated under reduced pressure and co-evaporated with dichloromethane. The mixture was then placed under high vacuum overnight, and intermediate JJ2 was used as is for the next step. ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),8.81(s,3H),5.38(s,1H),4.06(q,J＝7.1Hz,1H),3.44(d,J＝78.6Hz,5H),2.84(s,3H),2.12(s,1H),1.43(d,J＝7.1Hz,3H).

(R)-((4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 1-methylpyrrolidine-3-yl ester. Triethylamine (1.848 mL, 13.155 mmol) was added to a solution of intermediate JJ2 (1.5 g, 4.079 mmol) and phenyl dichlorophosphate (0.607 mL, 4.079 mmol) in anhydrous dichloromethane (19 mL) at 0 °C under an argon atmosphere. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (0.567 g, 4.079 mmol) and triethylamine (0.616 mL, 4.385 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80 g _SiO₂ Combiflash HP Gold column, 0-10% methanol/dichloromethane) to give intermediate JJ3 (a diastereomeric mixture). ¹ H NMR (400MHz, chloroform-d) δ8.26–8.18(m,2H),7.44–7.30(m,4H),7.26–7.15(m,3H),5.20(d,J＝8.5Hz,1H),4.24–3.89(m,1H),3.80(dd,J＝11.5,8. 8Hz,1H),2.97–2.80(m,1H),2.75(d,J＝21.6Hz,1H),2.68–2.58(m,1H),2.39–2.18(m,5H),1.78(dd,J＝14.6,7.8Hz,1H),1.45–1.34(m,3H). LCMS: MS m/z = 450.23 [M+1]; t _R = 1.07 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2 - 100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min.

(R)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 1-methylpyrrolidine-3-yl ester. Tetrahydrofuran (3 mL) was added to a mixture of intermediate 4 (0.1 g, 0.296 mmol), intermediate JJ3 (0.5 g, 1.113 mmol), and magnesium chloride (0.141 g, 1.478 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.129 mL, 0.739 mmol). The resulting mixture was stirred at 50 °C for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography using 0-10% methanol/dichloromethane. The obtained pure fractions were combined and concentrated. The obtained residue was dissolved in anhydrous acetonitrile (3 mL) and cooled in an ice bath, then concentrated hydrochloric acid (0.257 mL, 3.086 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and concentrated. The obtained residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 0-100% acetonitrile/water with TFA modifier). The pure fractions were combined and concentrated under reduced pressure. The obtained residue was diluted with water and neutralized with saturated sodium bicarbonate aqueous solution. The aqueous layer was extracted with ethyl acetate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in acetonitrile and water and lyophilized to give the product (a mixture of diastereomers). ^¹H NMR (400 MHz, methanol- _d4) )δ7.79(d,J＝0.7Hz,1H),7.40–7.29(m,2H),7.28–7.15(m,3H),6.86(dd,J＝6.7,4.5Hz,1H),6.75(dd ,J＝13.6,4.6Hz,1H),5.41–5.32(m,1H),5.14(ddt,J＝8.1,5.4,2.6Hz,1H),4.82–4.55(m,3H),4.36(d d, J = 14.7, 5.3 Hz, 1H), 4.28–4.18 (m, 2H), 3.97–3.79 (m, 1H), 2.87–2.76 (m, 1H), 2.76–2.59 (m, 2H), 2.34 (s, 3H), 2.40–2.19 (m, 2H), 1.79 (ddt, J = 14.5, 7.6, 3.9 Hz, 1H), 1.28 (ddd, J = 17.1, 7.1, 1.1 Hz, 3H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.41–-238.79 (m). ³¹ PNMR (162 MHz, methanol- _d⁴ ) δ 3.72, 3.44. LCMS: MS m/z = 609.20 [M+1]; t _R = 0.80 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min; HPLC: t _R = 3.113 min (major isomer) and 3.168 min (minor isomer); HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6 μL 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 36. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((((S)-1-(((R)-1-methylpyrrolid-3-yl)oxy)-1-oxopropyl-2-yl)amino)(phenoxy) Phosphoryl)oxy)methyl)tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Isobutyric anhydride (0.074 mL, 0.444 mmol) was added to a solution of Example 35 (0.045 g, 0.074 mmol) in THF (2 mL) at room temperature, followed by the addition of DMAP (1.3 mg, 0.011 mmol). The resulting mixture was stirred for 30 min and monitored by LCMS. The reaction mixture was quenched with methanol and concentrated. The resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150 x 30 mm column, 0-100% acetonitrile/water containing TFA modifier) to give the product (diasteremeric mixture). ^¹H NMR (400 MHz, methanol- _d4) was used to further purify the product. )δ7.80(d,J＝3.4Hz,1H),7.33(dd,J＝8.4,7.4Hz,2H),7.28–7.15(m,3H),6.87–6.58(m,2H),5.96–5 .71(m,2H),5.59(dd,J＝7.9,2.7Hz,1H),5.20(d,J＝5.2Hz,1H),4.77–4.49(m,2H),4.40–4.22(m,2H) ,4.02–3.83(m,1H),2.94–2.62(m,3H),2.54–2.35(m,5H),2.25(dq,J＝22.6,7.4Hz,2H),1.89–1.72(m,1H),1.32(dd,J＝7.1,1.1Hz,3H),1.22(dd,J＝7.0,1.1Hz,6H),1.06(ddd,J＝18.6,7.0,4.7Hz,6H). ¹⁹ F NMR (376MHz, methanol- _d⁴ ) δ -235.33(t,J＝46.8Hz), -235.86(t,J＝46.7Hz). ³¹ PNMR (162MHz, methanol- _d⁴ ) δ 3.38. LCMS: MS m/z = 749.24 [M+1]; t _R = 1.13 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC Chiralpak IA 5 μm, 25 x 250 mm; 25% isopropanol).

Example 37. First eluted diastereomer of Example 36: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.81 (s, 1H), 7.38–7.29 (m, 2H), 7.27–7.14 (m, 3H), 6.84 (d, J = 4.5 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 5.96–5.79 (m, 2H), 5.59 (d, J = 7.8 Hz, 1H), 5.22 (dq, J = 6.8, 3.6 Hz, 1H), 4.73 (s, 1H), 4.61 (s, 1H), 4.42–4.26 ( m, 2H), 3.99–3.85(m, 1H), 3.14–2.92(m, 3H), 2.68(dt, J = 14.0, 7.0 Hz, 2H), 2.58–2.42(m, 4H), 2.32(dq, J = 14.7, 7.4 Hz, 1H), 2.04–1.83(m, 1H), 1.25(ddd, J = 20.9, 7.1, 1.1 Hz, 9H), 1.06(dd, J = 17.9, 7.0 Hz, 6H). ^19F NMR (376 MHz, methanol- _d⁴ ) δ -235.85(t, J = 46.7 Hz). ³¹ P NMR (162MHz, methanol- _d4 ) δ 3.43 LC-MS: MS m/z = 749.24 [M+1]; t _R = 1.16 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min; HPLC: t _R = 4.614 min; HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5 min.

Example 38. Second eluted diastereomer of Example 36: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.80 (s, ¹H), 7.38–7.28 (m, 2H), 7.26–7.16 (m, 3H), 6.80 (d, J = 4.6 Hz, ¹H), 6.63 (d, J = 4.6 Hz, 1H), 5.89–5.75 (m, 2H), 5.59 (d, J = 7.9 Hz, 1H), 5.19 (ddt, J = 8.0, 5.4, 2.7 Hz, 1H), 4.79–4.51 (m, 2H), 4.42–4.19 (m, 2H) ), 3.95(dq, J＝9.8, 7.1Hz, 1H), 2.96–2.60(m, 4H), 2.53–2.37(m, 5H), 2.32–2.17(m, 1H), 1.82(dtd, J＝14.3, 7.2, 2.6Hz, 1H), 1.33(dd, J＝7.1, 1.1Hz, 3H), 1.22(dd, J＝7.0, 1.2Hz, 6H), 1.05(dd, J＝18.3, 7.0Hz, 6H). ¹⁹ F NMR (376MHz, methanol- _d⁴ ) δ -235.32(t, J＝46.8Hz). ³¹ P NMR (162MHz, methanol- _d⁴ ) δ 3.37. LCMS: MS m/z = 749.23 [M+1]; t _R = 1.13 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 4.553 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 39. (R)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-methoxy propyl ester

(R)-((2-methoxypropoxy)methyl)benzene. Sodium hydride (0.866 g, 0.036 mol) was added in portions to an ice-cold solution of (R)-(-)-1-benzyloxy-2-propanol (3.0 g, 0.018 mol) in 20 mL of THF. The reaction mixture was stirred at 0 °C for 30 min, followed by the addition of methyl iodine (2.247 mL, 0.036 mol). The reaction mixture was stirred at 0 °C for 1 h and diluted with ice-cold water and ethyl acetate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using 0–40% ethyl acetate/hexane to give intermediate NN1. ^1H NMR (400MHz, chloroform-d) δ 7.37 (d, J = 4.3Hz, 4H), 7.31 (dd, J = 10.5, 6.1Hz, 1H), 4.66–4.53 (m, 2H), 3.64–3.38 (m, 3H), 3.42 (s, 3H), 1.18 (d, J = 6.3Hz, 3H).

(R)-(tert-butoxycarbonyl)-L-alanine 2-methoxypropyl ester. 10% Pd/C (0.3 g, 0.003 mol) was added to a degassed solution of intermediate NN1 (2.0 g, 0.011 mol) in THF (20 mL), and the reaction mixture was stirred overnight under hydrogen. The reaction mixture was filtered, and (tert-butoxycarbonyl)-L-alanine (2.519 g, 0.013 mol), acetonitrile (10 mL), EDCI (2.5 g, 0.016 mol), and DMAP (2.169 g, 0.018 mol) were added to the filtrate in a single addition. The reaction mixture was stirred overnight at room temperature and diluted with ethyl acetate and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The intermediate NN2 was purified by silica gel chromatography with 0–30% ethyl acetate/hexane. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.26(d,J＝7.4Hz,1H),4.10–3.93(m,2H),3.91(dd,J＝11.4,5.7Hz,1H),3.46(td,J =6.2,4.2Hz,1H),3.23(s,3H),1.35(s,9H),1.25–1.10(m,3H),1.05(d,J＝6.4Hz,3H).

(R)-2-Methoxypropyl-L-alanine ester hydrochloride. Intermediate NN2 (2.5 g, 0.010 mol) was dissolved in anhydrous dichloromethane (25 mL) and a dioxane solution of 4N HCl (11.96 mL, 0.048 mol). The resulting solution was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The resulting residue was placed under high vacuum overnight, and intermediate NN3 was used as is in the next step. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.63(s,3H),4.19(dd,J＝11.4,3.8Hz,1H),4.10–3.96(m,2H),3.59–3.46(m,1H),3.24(s,3H),1.41(d,J＝7.2Hz,3H),1.08(d,J＝6.4Hz,3H).

(R)-((4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxypropyl ester. Triethylamine (3.05 mL, 21.71 mmol) was added to a solution of intermediate NN3 (1.95 g, 9.865 mmol) and phenyl dichlorophosphate (1.468 mL, 9.865 mmol) in anhydrous dichloromethane (34 mL) at 0 °C under an argon atmosphere. The resulting mixture was stirred at 0 °C for 1 hour. Then, 4-nitrophenol (1.372 g, 9.865 mmol) and triethylamine (1.525 mL, 10.85 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography (80g _SiO2 Combiflash HP Gold column, 100% dichloromethane, followed by 0-100% ethyl acetate/hexane) to give intermediate NN4 (a mixture of diastereomers). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.33–8.23(m,2H),7.54–7.44(m,1H),7.49–7.33(m,3H),7.23(m,3H),6.70(ddd,J＝13.7,9.9,8.3Hz,1H),4.09–3.94(m,2H),3.92( ddd,J＝11.4,5.7,1.4Hz,1H),3.48–3.36(m,1H),3.20(d,J＝1.5Hz,3H),1.23(ddd,J＝7.2,4.0,1.2Hz,3H),1.01(dd,J＝6.4,1.1Hz,3H). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ -1.26, -1.48. LCMS: MS m/z = 439.00 [M+1]; t _R = 1.43 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min.

(R)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxypropyl ester. Tetrahydrofuran (1.5 mL) was added to a mixture of intermediate 4 (0.15 g, 0.443 mmol), intermediate NN4 (0.214 g, 0.488 mmol), and magnesium chloride (0.063 g, 0.665 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.193 mL, 1.108 mmol). The resulting mixture was stirred at 50 °C for 1.5 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 0-100% acetonitrile/water). The purified fractions were combined and concentrated under reduced pressure. The resulting residue was dissolved in anhydrous acetonitrile (3 mL) and cooled in an ice bath, followed by the dropwise addition of concentrated hydrochloric acid (0.2 mL, 2.4 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and diluted with ice, then neutralized with an aqueous sodium bicarbonate solution. The resulting mixture was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30 mm column, 0-100% acetonitrile/water) to give the product (diasteremeric mixture). ^¹H NMR (400 MHz, methanol- _d4) )δ7.78(d,J＝1.1Hz,1H),7.33(q,J＝7.8Hz,2H),7.27–7.13(m,3H),6.85(dd,J＝6.2,4.6Hz,1H),6 .74(dd,J＝10.6,4.6Hz,1H),5.36(dd,J＝8.3,6.1Hz,1H),4.82–4.55(m,3H),4.35(dd,J＝17.4,5.3 Hz, 1H), 4.29–4.15(m, 2H), 4.10(ddd, J＝11.5, 4.0, 2.6Hz, 1H), 4.06–3.86(m, 2H), 3.53(ddt, J＝9.8, 6.3, 3.5Hz, 1H), 3.31(s, 3H), 1.29(ddd, J＝19.7, 7.2, 1.1Hz, 3H), 1.11(dd, J＝6.4, 2.3Hz, 3H). ¹⁹ F NMR (376MHz, methanol- _d⁴ ) δ -238.52–-238.94(m). ³¹ P NMR (162MHz, methanol- _d⁴ ) δ 3.70, 3.41. LCMS: MS m/z = 598.04 [M+1]; t _R = 0.99 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min; HPLC: t _R = 3.947 min (minor isomer), 4.011 min (major isomer); HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6 μL 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC Chiralpak AD-H 5 μm, 21 x 250 mm; 30% isopropanol).

Example 40. First eluted diastereomer of Example 39: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.78 (s, 1H), 7.37–7.28 (m, 2H), 7.24–7.13 (m, 3H), 6.86 (d, J = 4.5 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 5.37 (d, J = 8.2 Hz, 1H), 4.83–4.71 (m, 1H), 4.71–4.59 (m, 2H), 4.37 (d, J = 5.2 Hz, 1H), ...δ 7.37 (d, J = 8.2 Hz, 1H), δ 7.37 (s, J = 8.2 Hz, 1H), δ 7.37 (s, J = 8.2 Hz, 1H), δ 7.37 (s, J = 8.2 Hz, 1H), δ 7.37 (s, J = 8.2 Hz 4.26 (dd, J = 5.0, 1.7 Hz, 2H), 4.09 (dd, J = 11.5, 4.0 Hz, 1H), 4.06–3.86 (m, 2H), 3.60–3.48 (m, 1H), 3.31 (s, 3H), 1.26 (dd, J = 7.2, 1.2 Hz, 3H), 1.12 (d, J = 6.4 Hz, 3H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.68 (t, J = 47.9 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.71. LCMS: MS m/z = 598.08 [M+1]; t _R = 0.96 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μl/min. HPLC: t _R = 3.938 min; HPLC system: Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 41. Second eluted diastereomer of Example 39: ^¹H NMR (400MHz, methanol- _d4) )δ7.78(s,1H),7.39–7.29(m,2H),7.27–7.14(m,3H),6.84(d,J＝4.5Hz,1H),6.7 3(d,J＝4.5Hz,1H),5.36(d,J＝8.4Hz,1H),4.82–4.56(m,3H),4.33(d,J＝5.2Hz,1 H),4.21(tt,J＝6.0,3.6Hz,2H),4.10(dd,J＝11.5,3.9Hz,1H),4.03–3.88(m,2H) ,3.53(pd,J＝6.3,4.0Hz,1H), 1.31(dd,J＝7.2,1.0Hz,3H), 1.11(d,J＝6.4Hz,3H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.81 (t, J = 47.6 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.42 LC MS: MS m/z = 598.05 [M+1]; t _R = 0.99 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μXB-C18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2 - 100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 4.004 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 42. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (Fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-butoxyethyl ester

(tert-butoxycarbonyl)-L-alanine 2-butoxyethyl ester. N-methylmorpholine (16.75 mL, 152 mmol), 4-(dimethylamino)pyridine (0.12 g, 1 mmol), and tripropylphosphonic anhydride (36.27 mL, 61 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (10.57 g, 56 mmol) and 2-butoxyethyl-1-ol (6.00 g, 51 mmol) in anhydrous dichloromethane (100 mL) at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with water (50 mL), twice with 10% citric acid solution (2 × 40 mL), twice with saturated sodium bicarbonate aqueous solution (2 × 40 mL), and once with brine (50 mL). After drying with sodium sulfate, the mixture was filtered through a 3 cm silica gel filter, which was then washed with additional dichloromethane. The combined organic compounds were concentrated under reduced pressure, co-distilled with dichloromethane, and dried under high vacuum overnight to give intermediate QQ1. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.27 (d, J = 7.4 Hz, 1H), 4.26–4.14 (m, 1H), 4.14–4.06 (m, 1H), 4.05–3.93 (m, 1H), 3.58–3.49 (m, 2H), 3.39 (t, J = 6.5 Hz, 2H), 1.50–1.42 (m, 2H), 1.38 (s, 9H), 1.35–1.27 (m, 2H), 1.23 (d, J = 7.4 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine 2-butoxyethyl ester. Intermediate QQ1 (14.2 g, 49.07 mmol) was dissolved in 50 mL of 4 M HCl in 1,4-dioxane. The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and co-distilled with toluene to obtain a pure solid, which was dried under high vacuum for 1 hour. The solid was suspended in dichloromethane (200 mL), and phenyl dichlorophosphate (8.03 mL, 53.98 mmol) and triethylamine (14.96 mL, 107.96 mmol) were added sequentially at -78 °C, and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 °C, and pentafluorophenol (9.03 g, 49.07 mmol) and triethylamine (8.84 mL, 63.79 mmol) were added sequentially, followed by warming the mixture to room temperature. After 3 hours, the reaction mixture was cooled to 0°C and the solid was filtered off. The filtrate was washed with saturated ammonium chloride aqueous solution (100 mL), water (100 mL), and brine (50 mL). The organic matter was dried over sodium sulfate and filtered through a 3 cm silica gel layer, which was washed with a 1:5 mixture of ethyl acetate and dichloromethane (100 mL). The combined organic matter was concentrated under reduced pressure to give 22.5 g of crude solid product, which, based on NMR, was a mixture of two phosphorus isomers. The solid was dissolved in a boiling mixture of diisopropyl ether (6 mL) and hexane (200 mL), and the mixture was stirred vigorously overnight at room temperature. The solid product was separated by filtration and washed with hexane (3 × 40 mL) to give intermediate QQ2. Intermediate QQ2 was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.35(m,2H),7.31–7.16(m,3H),6.93(dd,J＝14.2,9.9Hz,1H),4.23–4.08(m,2H),4.07–3.90(m,1H) ,3.52(t,J＝4.8Hz,2H),3.35(t,J＝6.5Hz,2H),1.46–1.37(m,2H),1.36–1.18(m,5H),0.84(t,J＝7.4Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.25 (d, J = 21.6 Hz, 2F), -160.87 (td, J = 23.3, 3.2 Hz, 1F), -163.70 (td, J = 24.0, 4.2 Hz, 2F). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.42. LCMS: MS m/z = 511.95 [M+1], t _R = 1.70 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-butoxyethyl ester. Tetrahydrofuran (0.7 mL) was added to a mixture of intermediate 4 (100 mg, 0.296 mmol), intermediate QQ2 (196 mg, 0.384 mmol), and magnesium chloride (42 mg, 0.443 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.129 mL, 0.739 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (5 mL), and concentrated hydrochloric acid aqueous solution (0.246 mL) was added dropwise at 0 °C. After 4 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ8.10–7.75(m,3H),7.43–7.33(m,2H),7.27–7.15(m,3H),6.91(d,J＝4.4Hz,1H),6.71(d,J＝ 4.5Hz,1H),6.11(dd,J＝13.3,10.1Hz,1H),5.43–5.10(m,3H),4.70–4.57(m,1H),4.58–4.44( m,2H),4.23–4.12(m,2H),4.13–4.02(m,1H),3.99(d,J＝5.3Hz,2H),3.93–3.78(m,1H),3.55– 3.49(m,2H),3.35–3.33(m,2H),1.47–1.37(m,2H),1.34–1.17(m,5H),0.84(t,J＝7.4Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.74 (t, J=47.9Hz). ³¹ P NMR (162MHz, DMSO-d6) δ 3.44. LCMS: MS m/z = 626.03 [M+1], t _R = 1.26 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 2.78 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.68 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 43. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-methoxy-2-methyl propyl ester

(tert-butoxycarbonyl)-L-alanine 2-methoxy-2-methylpropyl ester. Under an argon atmosphere at 0 °C, N-methylmorpholine (6.33 mL, 58 mmol), 4-(dimethylamino)pyridine (0.05 g, 0.4 mmol), and tripropylphosphonic anhydride (13.72 mL, 23 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (4.00 g, 21 mmol) and 2-methoxy-2-methylprop-1-ol (2.00 g, 19 mmol) in anhydrous dichloromethane (50 mL). The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with water (30 mL), twice with 10% citric acid solution (2 × 20 mL), twice with saturated sodium bicarbonate aqueous solution (2 × 20 mL), and once with brine (20 mL). After drying with sodium sulfate, the mixture was filtered through a 3 cm silica gel layer, which was then washed with a 3:1 mixture of dichloromethane and ethyl acetate. The combined organic compounds were concentrated under reduced pressure, co-distilled with dichloromethane, and dried overnight under high vacuum to give intermediate RR1. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.30 (d, J = 7.4 Hz, 1H), 4.10–3.77 (m, 3H), 3.11 (s, 3H), 1.37 (s, 9H), 1.24 (d, J = 7.4 Hz, 3H), 1.10 (s, 6H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxy-2-methylpropyl ester. Intermediate RR1 (5.1 g, 18.52 mmol) was dissolved in 15 mL of 4 M HCl in 1,4-dioxane, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and co-distilled with toluene to obtain a pure solid, which was dried under high vacuum for 1 hour. The solid was suspended in dichloromethane (100 mL), and phenyl dichlorophosphate (3.03 mL, 20.37 mmol) and triethylamine (5.65 mL, 40.75 mmol) were added sequentially at -78 °C, and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 °C, and pentafluorophenol (3.41 g, 18.52 mmol) and triethylamine (3.59 mL, 25.93 mmol) were added sequentially, followed by warming the mixture to room temperature. After 3 hours, the reaction mixture was cooled to 0°C and the solid was filtered off. The filtrate was washed with saturated ammonium chloride aqueous solution (100 mL), water (100 mL), and brine (50 mL). The organic matter was dried over sodium sulfate and filtered through a 3 cm silica gel layer, which was washed with a 1:3 mixture of ethyl acetate and dichloromethane (100 mL). The combined organic matter was concentrated under reduced pressure to give 6.23 g of crude product. Based on NMR, the crude solid product was a mixture of two phosphorus isomers. The solid was dissolved in boiling diisopropyl ether (50 mL), and the mixture was stirred vigorously overnight at room temperature. The solid product was filtered off and washed with cold diisopropyl ether (2 × 10 mL) and hexane (3 × 20 mL) to give intermediate RR2. Intermediate RR2 was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.46–7.36(m,2H),7.29–7.16(m,3H),6.92(dd,J＝14.2,9.9Hz,1H),4.12–3.86(m,3H),3.09(s,3H),1.31(d,J＝7.1Hz,3H),1.09(s,6H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ -154.22 (d, J = 21.4Hz, 2F), -160.89 (td, J = 23.4, 3.2Hz, 1F), -163.69 (td, J = 23.4, 4.0Hz, 2F). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.43. LCMS: MS m/z = 497.86 [M+1], t _R = 1.65 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-methoxy-2-methylpropyl ester. Tetrahydrofuran (0.7 mL) was added to a mixture of intermediate 4 (100 mg, 0.296 mmol), intermediate RR2 (191 mg, 0.384 mmol), and magnesium chloride (42 mg, 0.443 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.129 mL, 0.739 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (5 mL), and concentrated hydrochloric acid aqueous solution (0.246 mL) was added dropwise at 0 °C. After 4 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.70(bs,2H),7.44–7.30(m,2H),7.26–7.14(m,3H),6.84(d,J ＝4.4Hz,1H),6.68(d,J＝4.4Hz,1H),6.10(dd,J＝13.1,10.1Hz,1H),5.29–5.21 (m,2H),5.13(d,J＝7.3Hz,1H),4.72–4.43(m,3H),4.25–4.15(m,1H),4.03–3. 95(m,3H),3.95–3.82(m,2H),3.08(s,3H),1.26(d,J=7.2Hz,3H),1.09(s,6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.71 (t, J=48.1Hz). ³¹ PNMR (162MHz, DMSO-d ₆ ) δ3.47. LCMS: MS m/z = 612.03 [M+1], t _R = 1.16 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _R = 2.47 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min.

Example 44. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(naphthal-1-yloxy)phosphoyl)-L-alanine ethyl ester

((naphthyl-1-yloxy)(perfluorophenoxy)phosphoryl)-L-alanine ethyl ester. Naphthyl-1-ol (2.82 g, 19.57 mmol) was added to a solution of phosphoric acid trichloro(3.00 g, 19.57 mmol) in anhydrous tetrahydrofuran (50 mL) at -78 °C under argon atmosphere. Triethylamine (6.00 mL, 43.05 mmol) was added dropwise. After 15 minutes, the reaction was heated to 0 °C. After 30 minutes, the reaction was cooled again to -78 °C, and L-alanine ethyl ester hydrochloride (3.01 g, 19.57 mmol) was added, followed by triethylamine (2.73 mL, 19.57 mmol). The reaction mixture was heated to room temperature and stirred for 3 hours. The reaction was cooled to 0 °C, and pentafluorophenol (3.60 g, 19.57 mmol) was added, followed by triethylamine (3.00 mL, 21.52 mmol). The reaction mixture was heated to room temperature and stirred for 2 hours. It was diluted with ethyl acetate (100 mL) and washed with saturated ammonium chloride aqueous solution (100 mL), water (100 mL), and brine (100 mL). The organic matter was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography using a gradient of 0 to 35% ethyl acetate in hexane to give 4.9 g of crude product, a mixture of diastereomers. The solid was dissolved in boiling diisopropyl ether (70 mL), and the mixture was stirred vigorously overnight at room temperature. The solid product was filtered off and washed with cold diisopropyl ether (2 × 10 mL) and hexane (3 × 20 mL) to give intermediate SS1. NMR spectroscopy confirmed that intermediate SS1 was a single diastereomer. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.14–8.08(m,1H),8.03–7.98(m,1H),7.82(d,J＝7.7Hz,1H),7.67–7.44(m,4H),7.11( dd,J＝14.3,9.9Hz,1H),4.15–3.92(m,3H),1.33(d,J＝7.1Hz,3H),1.09(t,J＝7.1Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.19 (d, J = 21.6Hz, 2F), -160.70 (t, J = 23.4Hz, 1F), -163.61 (t, J = 21.9Hz, 2F). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.68. LCMS: MS m/z = 489.95 [M+1], t _R = 1.76 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(naphthyl-1-yloxy)phosphoryl)-L-alanine ethyl ester. Tetrahydrofuran (0.4 mL) was added to a mixture of intermediate 4 (50 mg, 0.148 mmol), intermediate SS1 (94 mg, 0.192 mmol), and magnesium chloride (21 mg, 0.222 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.064 mL, 0.369 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (5 mL), and concentrated hydrochloric acid aqueous solution (0.123 mL) was added dropwise at 0 °C. After 4 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ8.19–8.10(m,1H),8.02–7.93(m,1H),7.87(s,1H),7.80–7.72(m,1H),7.62–7.5 5(m,2H),7.52–7.43(m,2H),6.91(d,J＝4.4Hz,1H),6.67(d,J＝4.5Hz,1H),6.26(dd, J＝13.0,10.1Hz,1H),5.38–5.11(m,3H),4.74–4.46(m,3H),4.22(d,J＝4.9Hz,1H), 4.13–3.99(m,4H),3.95–3.86(m,1H),1.22(d,J＝7.1Hz,3H),1.12(t,J＝7.1Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.51 (t, J=47.7Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 3.98. LCMS: MS m/z = 604.02 [M+1], t _R = 1.21 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min. HPLC: t _{_R} = 2.69 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.51 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 45. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-(2-ethoxyethyl) oxyethyl ester

(tert-butoxycarbonyl)-L-alanine 2-(2-ethoxyethoxy)ethyl ester. N-methylmorpholine (19.67 mL, 179 mmol), 4-(dimethylamino)pyridine (0.15 g, 1.2 mmol), and tripropylphosphonic anhydride (42.6 mL, 72 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (12.41 g, 66 mmol) and 2-(2-ethoxyethoxy)ethanol-1-ol (8.00 g, 60 mmol) in anhydrous dichloromethane (100 mL) at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with water (50 mL), twice with 10% citric acid solution (2 × 40 mL), twice with saturated sodium bicarbonate aqueous solution (2 × 40 mL), and once with brine (50 mL). It was dried over sodium sulfate, filtered through a 3 cm silica gel layer, and the silica gel layer was washed with dichloromethane. The combined organic compounds were concentrated under reduced pressure, co-distilled with dichloromethane, and dried overnight under high vacuum to give intermediate TT1. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.27(d,J＝7.4Hz,1H),4.23–4.14(m,1H),4.14–4.06(m,1H),4.05–3.94(m,1H),3.64–3.56(m,2H ), 3.55–3.49 (m, 2H), 3.49–3.39 (m, 4H), 1.38 (s, 9H), 1.23 (d, J = 7.4Hz, 3H), 1.09 (t, J = 7.0Hz, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine 2-(2-ethoxyethoxy)ethyl ester. Intermediate TT1 (18.3 g, 59.93 mmol) was dissolved in 50 mL of 4 M HCl in 1,4-dioxane. The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and co-distilled with toluene to give a crude product, which was dried under high vacuum for 1 hour. The resulting solid was suspended in dichloromethane (100 mL), and phenyl dichlorophosphate (9.81 mL, 65.92 mmol) and triethylamine (18.28 mL, 131.84 mmol) were added sequentially at -78 °C, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 °C, and pentafluorophenol (11.03 g, 59.93 mmol) and triethylamine (10.80 mL, 78.05 mmol) were added sequentially, followed by warming the mixture to room temperature. After 3 hours, the reaction mixture was cooled to 0°C and the solid was filtered off. The filtrate was washed with saturated ammonium chloride aqueous solution (100 mL), water (100 mL), and brine (50 mL). The organic matter was dried over sodium sulfate and filtered through a 3 cm silica gel layer, which was washed with a 1:1 mixture of ethyl acetate and dichloromethane (100 mL). The combined organic matter was concentrated under reduced pressure to give 21.7 g of crude product, a mixture of diastereomers. The solid was dissolved in a minimal amount of boiling diisopropyl ether, and the mixture was stirred vigorously overnight at room temperature. The solid product was filtered off and washed with cold diisopropyl ether (2 × 20 mL) and hexane (3 × 40 mL) to give intermediate TT2. Intermediate TT2 was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.36(m,2H),7.30–7.20(m,3H),6.92(dd,J＝14.2,9.9Hz,1H),4.21–4.08(m,2H),4.07–3.92(m,1 H), 3.62–3.56 (m, 2H), 3.53–3.47 (m, 2H), 3.45–3.36 (m, 4H), 1.29 (d, J = 7.1Hz, 3H), 1.07 (t, J = 7.0Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.24 (d, J = 21.5Hz, 2F), -160.86 (t, J = 23.1Hz, 1F), -163.68 (t, J = 21.7Hz, 2F). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.40. LCMS: MS m/z = 528.06 [M+1], t _R = 1.64 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-(2-ethoxyethoxy)ethyl ester. Tetrahydrofuran (0.7 mL) was added to a mixture of intermediate 4 (100 mg, 0.295 mmol), intermediate TT2 (203 mg, 0.384 mmol), and magnesium chloride (42 mg, 0.443 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.129 mL, 0.739 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (5 mL), and concentrated hydrochloric acid aqueous solution (0.246 mL) was added dropwise at 0 °C. After 4 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ7.84(s,1H),7.77(bs,2H),7.42–7.34(m,2H),7.28–7.13(m,3H),6.86(d,J＝4.5Hz,1H),6.69(d,J ＝4.5Hz,1H),6.11(dd,J＝13.3,10.1Hz,1H),5.37–5.22(m,2H),5.20–5.10(m,1H),4.64(q,J＝10.2Hz, 1H),4.57–4.43(m,2H),4.24–4.14(m,2H),4.12–4.05(m,1H),4.03–3.97(m,2H),3.92–3.79(m,1H),3 .59–3.54(m,2H),3.51–3.45(m,2H),3.43–3.38(m,4H),1.24(d,J=7.1Hz,3H),1.07(t,J=7.0Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.74 (t, J=48.1Hz). ³¹ PNMR (162MHz, DMSO-d ₆ ) δ3.43. LCMS: MS m/z = 642.08 [M+1], t _R = 1.13 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 2.43 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.05 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 46. ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine butyl ester

(tert-Butoxycarbonyl)-L-alanine butyl ester. N-methylmorpholine (22.25 mL, 202 mmol), 4-(dimethylamino)pyridine (0.17 g, 1.4 mmol), and tripropylphosphonic anhydride (48.19 mL, 81 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (14.04 g, 74 mmol) and but-1-ol (5.00 g, 67 mmol) in anhydrous dichloromethane (100 mL) at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with water (50 mL), twice with 10% citric acid solution (2 × 40 mL), twice with saturated sodium bicarbonate aqueous solution (2 × 40 mL), and once with brine (50 mL). After drying with sodium sulfate, the mixture was filtered through a 3 cm silica gel filter, which was then washed with additional dichloromethane. The combined organic compounds were concentrated under reduced pressure, co-distilled with dichloromethane, and dried overnight under high vacuum to give intermediate UU1. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.27 (d, J = 7.4 Hz, 1H), 4.19–3.89 (m, 3H), 1.60–1.48 (m, 2H), 1.42–1.28 (m, 11H), 1.22 (d, J = 7.4 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine butyl ester. Intermediate UU1 (6.75 g, 27.5 mmol) was dissolved in 30 mL of 4 M HCl in 1,4-dioxane. The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and co-distilled with toluene to obtain a solid. This pure solid was dried under high vacuum for 1 hour. The solid was suspended in dichloromethane (30 mL), and phenyl dichlorophosphate (4.51 mL, 30.3 mmol) and triethylamine (8.39 mL, 60.6 mmol) were added sequentially at -78 °C. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 °C, and then pentafluorophenol (5.07 g, 27.5 mmol) and triethylamine (4.20 mL, 30.0 mmol) were added sequentially. The resulting mixture was then heated to room temperature. Three hours later, the reaction mixture was cooled to 0°C and the solid was filtered off. The filtrate was washed with saturated ammonium chloride aqueous solution (50 mL), water (50 mL), and brine (20 mL). The organic matter was dried over sodium sulfate and filtered through a 3 cm silica gel layer, which was then washed with dichloromethane (50 mL). The combined organic matter was concentrated under reduced pressure to give 12.2 g of crude product, a mixture of diastereomers. The solid was dissolved in boiling hexane (120 mL), and the mixture was stirred vigorously overnight at room temperature. The solid product was filtered off and washed with hexane (3 × 30 mL) to give intermediate UU2. NMR spectroscopy confirmed that intermediate UU2 was a single diastereomer. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.36(m,2H),7.30–7.17(m,3H),6.89(dd,J＝14.2,9.9Hz,1H),4.12 –3.91(m,3H),1.59–1.45(m,2H),1.38–1.19(m,5H),0.85(t,J＝7.3Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.24 (d, J = 20.7Hz, 2F), -160.88 (t, J = 23.1Hz, 1F), -163.70 (t, J = 21.6Hz, 2F). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.48. LCMS: MS m/z = 467.92 [M+1], t _R = 1.86 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% acetonitrile, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine butyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (70 mg, 0.207 mmol), intermediate UU2 (126 mg, 0.269 mmol), and magnesium chloride (30 mg, 0.310 mmol) at room temperature. The mixture was heated to 40 °C for 10 minutes, and N,N-diisopropylethylamine (0.090 mL, 0.517 mmol) was added. After stirring at 40 °C for 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (20 mL), and the resulting mixture was washed with water (20 mL) and brine (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was dissolved in acetonitrile (2 mL), and concentrated hydrochloric acid aqueous solution (0.172 mL) was added dropwise at 0 °C. After 4 hours at 0 °C, the reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL) at 0 °C, and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100x30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, DMSO-d6 ₎ )δ7.83(s,1H),7.73(bs,2H),7.43–7.32(m,2H),7.25–7.13(m,3H),6.85(d,J＝4.5Hz ,1H),6.69(d,J＝4.5Hz,1H),6.08(dd,J＝13.2,10.2Hz,1H),5.34–5.21(m,2H),5.20– 5.07(m,1H),4.71–4.58(m,1H),4.57–4.41(m,2H),4.29–4.15(m,1H),4.10–3.94(m, 4H), 3.93–3.74 (m, 1H), 1.60–1.42 (m, 2H), 1.36–1.13 (m, 5H), 0.84 (t, J = 7.4Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-236.73 (t, J=47.8Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ3.49. LCMS: MS m/z = 582.02 [M+1], t _R = 1.22 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-0.2 min 2% acetonitrile, 0.2 min-1.5 min 2-100% acetonitrile, 1.5 min-2.2 min 100% acetonitrile, 2.2 min-2.4 min 100%-2% acetonitrile, 2.4 min-2.5 min 2% acetonitrile, 2 μl/min. HPLC: t _{_R} = 2.75 min; HPLC system: Agilent 1100 series; column: Gemini 5μC18 110A, 50 x 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL/min. HPLC: t_{_R} = 4.60 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 8.5 min gradient 2-98% B, 1.5 mL/min.

Example 47. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(4-(tert-butyl)phenoxy)phosphoyl)-L-alanine ethyl ester

((4-(tert-butyl)phenoxy)(4-nitrophenoxy)phosphoryl)-L-alanine ethyl ester. L-alanine ethyl ester (1.00 g, 6.52 mmol) was added to a solution of phosphorus oxychloride (V) (0.61 mL, 6.52 mmol) in dichloromethane (20 mL) at -78 °C under an argon atmosphere. Triethylamine (2.00 mL, 14.35 mmol) was slowly added dropwise. After 15 minutes, the reaction was heated to 0 °C. After 30 minutes, the reaction was cooled to -78 °C and 4-tert-butylphenol (0.98 g, 6.52 mmol) was added. Triethylamine (0.91 mL, 6.52 mmol) was added. The reaction was heated to room temperature and stirred for 3 hours. The reaction was cooled to 0 °C. 4-Nitrophenol (0.91 g, 6.52 mmol) was added, followed by the dropwise addition of triethylamine (0.91 mL, 6.52 mmol). The reaction mixture was heated to room temperature and stirred for 2 hours. The reactants were diluted with ethyl acetate and washed with ammonium chloride, water, and brine. The organic matter was dried over sodium sulfate, filtered, and concentrated. Intermediate VV1 (a mixture of diastereomers) was separated as an oil by silica gel chromatography (using a gradient of 0-50% ethyl acetate in hexane). ^¹H NMR (400 MHz, DMSO- _d₆₆ ) δ 8.31 (d, J = 8.8 Hz, 2H), 7.57–7.36 (m, 4H), 7.24–7.09 (m, 2H), 6.76–6.57 (m, 1H), 4.12–3.77 (m, 3H), 1.30–1.21 (m, 12H), 1.11 (t, J = 7.1 Hz, 3H). ^³¹P NMR (162 MHz, DMSO- _d₆₆ ) δ -1.16, -1.24. LCMS: MS m/z = 451.00 [M+1], t _R = 1.84 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(4-(tert-butyl)phenoxy)phosphoryl)-L-alanine ethyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate VV1 (71 mg, 0.16 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.6 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium carbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, gradient of 5-100% acetonitrile aqueous solution) to give Example VV (diasteremeric mixture). LCMS: MS m/z = 609.96 [M+1], t _R = 1.42 min (minor), 1.44 min (major); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 μXB-C18 100A, 50 x 4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min - 2.0 min 2 - 100% acetonitrile, 2.0 min - 3.05 min 100% acetonitrile, 3.05 min - 3.2 min 100% - 2% acetonitrile, 3.2 min - 3.5 min 2% ACN, 2 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC chiralpack AD-H 5 μm, 250 × 21 mm; methanol 25%).

Example 48. First eluted diastereomer of Example 47: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 8.00 (s, 1H), 7.40–7.33 (m, 3H), 7.16–7.09 (m, 2H), 6.99 (d, J = 4.8 Hz, 1H), 5.40 (d, J = 8.1 Hz, 1H), 4.82–4.72 (m, 1H), 4.70–4.57 (m, 2H), 4.39 (d, J = 5.2 Hz, 1H), 4.34–4.20 (m, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.96–3.83 (m, 1H), 1.33–1.26 (m, 12H), 1.22 (t, J = 7.1 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.93. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.56 (t, J = 47.8 Hz). LCMS: MS m/z = 609.96 [M+1], t _R = 1.42 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min - 2.0 min 2-100% acetonitrile, 2.0 min - 3.05 min 100% acetonitrile, 3.05 min - 3.2 min 100% - 2% acetonitrile, 3.2 min - 3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.91 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 49. Second eluted diastereomer of Example 47: ^¹H NMR (400MHz, methanol- _d4) )δ7.99(s,1H),7.43–7.35(m,2H),7.35–7.30(m,1H),7.20–7.13(m,2H),6.96(d,J＝4.7Hz,1H),5.39(d,J＝8.4Hz,1H),4.82–4.69(m,1H),4. 69–4.53(m,2H),4.35(d,J＝5.2Hz,1H),4.28–4.15(m,2H),4.15–4.07(m,2H),3.99–3.84(m,1H),1.35–1.26(m,12H),1.22(t,J＝7.1Hz,3H). ³¹ PNMR (162MHz, methanol-d ₄ ) δ 3.78. ^19F NMR (376MHz, methanol- _d⁴ ) δ -238.79 (t, J = 47.8Hz). LCMS: MS m/z = 609.96 [M+1], t _R = 1.45 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.94 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 50. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine(1-methylcyclopropane) Methyl ester

(tert-butoxycarbonyl)-L-alanine (1-methylcyclopropyl) methyl ester. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.22 g, 6.36 mmol) was added to a solution of (tert-butoxycarbonyl)-L-alanine (1.00 g, 5.29 mmol) in 10 mL of acetonitrile under an argon atmosphere. After 15 minutes, 4-(dimethylamino)-pyridine (0.71 g, 5.81 mmol) was added, followed by (1-methylcyclopropyl)methanol (0.51 mL, 5.29 mmol). The reaction was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with 5% citric acid aqueous solution (2 × 10 mL). The organic compound was washed with saturated sodium bicarbonate aqueous solution (10 mL), water (5 mL), and then brine (10 mL). The organic compound was dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (using a gradient of 0–10% ethyl acetate in hexane) to give intermediate YY1. ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 7.26 (d, J = 7.4 Hz, 1H), 4.09–3.69 (m, 3H), 1.38 (s, 9H), 1.25 (d, J = 7.5 Hz, 3H), 1.06 (s, 3H), 0.56–0.39 (m, 2H), 0.38–0.24 (m, 2H).

L-Alanine (1-methylcyclopropyl) methyl ester hydrochloride. A solution of 4M hydrogen chloride in 1,4-dioxane (3.7 mL, 14.80 mmol) was added to a solution of intermediate YY1 (1.85 g, 7.19 mmol) in dichloromethane (10 mL) at 0 °C. After 1 hour, the reaction was concentrated. The residue was dissolved in dichloromethane (10 mL) and concentrated. The previous step was repeated. Intermediate YY2 was used in the next reaction without further purification. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.41(s,3H),4.13(q,J＝7.2Hz,1H),4.07–3.91(m,2H),1.43(d,J＝7.2Hz,3H),1.10(s,3H),0.56–0.47(m,2H),0.39–0.33(m,2H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine (1-methylcyclopropyl) methyl ester. Phenyl dichlorophosphate (0.82 mL, 5.45 mmol) and triethylamine (1.58 mL, 11.36 mmol) were successively added to a suspension of intermediate YY2 (880 mg, 4.54 mmol) in dichloromethane (15 mL) at 0 °C. After 1 hour, 4-nitrophenol (0.63 g, 4.54 mmol) and triethylamine (0.79 mL, 5.8 mmol) were then successively added at 0 °C, and the resulting mixture was warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (eluting with hexane solution of 0-100% ethyl acetate) to give intermediate YY3 (a mixture of diastereomers). ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 8.34–8.27 (m, 2H), 7.55–7.37 (m, 4H), 7.31–7.17 (m, 3H), 6.78–6.65 (m, 1H), 4.13–3.97 (m, 1H), 3.89–3.76 (m, 2H), 1.31–1.21 (m, 3H), 1.02 (d, J = 1.5 Hz, 3H), 0.49–0.37 (m, 2H), 0.35–0.23 (m, 2H). ^³¹P NMR (162 MHz, DMSO- _d₆ ) δ -1.25, -1.44. LCMS: MS m/z = 433.02 [M⁻¹], t _R = 1.75 min (minor), 1.77 min (major); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6 μXB-C18 100A, 50 x 4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min–2.0 min 2–100% acetonitrile, 2.0 min–3.05 min 100% acetonitrile, 3.05 min–3.2 min 100%–2% acetonitrile, 3.2 min–3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine (1-methylcyclopropyl)methyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate YY3 (67 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.60 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to give the product (diasteremeric mixture). LCMS: MS m/z = 593.95 [M+1], t _R = 1.33 min (minor), 1.35 min (major); LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by chiral preparative HPLC (Chiralpak IA 5 μm, 250 × 22 mm; 100% ethanol).

Example 51. First eluted diastereomer of Example 50: ^¹H NMR (400MHz, methanol- _d4) )δ7.87(s,1H),7.36–7.29(m,2H),7.25–7.15(m,3H),7.07(d,J＝4.6Hz,1 H),6.86(d,J＝4.6Hz,1H),5.38(d,J＝8.2Hz,1H),4.81–4.73(m,1H),4.70 –4.59(m,2H),4.38(d,J＝5.3Hz,1H),4.33–4.23(m,2H),4.00–3.86(m,3H ),1.34–1.28(m,3H),1.10(s,3H),0.51–0.47(m,2H),0.38–0.33(m,2H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.75. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.49 (t, J = 48.0 Hz). LCMS: MS m/z = 594.00 [M+1], t _R = 1.33 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.70 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 52. Second eluted diastereomer of Example 50: ^¹H NMR (400MHz, methanol- _d4) )δ7.99(s,1H),7.42–7.31(m,3H),7.29–7.15(m,3H),6.93(d,J＝4.7Hz,1 H),5.38(d,J＝8.4Hz,1H),4.80–4.69(m,1H),4.69–4.53(m,2H),4.33(d, J＝5.2Hz,1H),4.29–4.15(m,2H),4.03–3.90(m,2H),3.85(d,J＝11.1Hz,1 H),1.38–1.32(m,3H),1.10(s,3H),0.52–0.43(m,2H),0.39–0.32(m,2H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.60. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.78 (t, J = 47.7 Hz). LCMS: MS m/z = 594.00 [M+1], t _R = 1.35 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.76 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 53. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 3,3,3-trifluoro-2, 2-Dimethylpropyl ester

(tert-butoxycarbonyl)-L-alanine 3,3,3-trifluoro-2,2-dimethylpropyl ester. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.43 g, 12.69 mmol) was added to a solution of (tert-butoxycarbonyl)-L-alanine (2.00 g, 10.57 mmol) in 10 mL of acetonitrile under an argon atmosphere. After 15 minutes, 4-(dimethylamino)-pyridine (1.42 g, 11.62 mmol) was added, followed by 3,3,3-trifluoro-2,2-dimethylprop-1-ol (1.69 mL, 10.55 mmol). The reaction was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with 5% citric acid aqueous solution (2 × 10 mL). The organic matter was washed with saturated sodium bicarbonate aqueous solution (10 mL), water (5 mL), and then brine (10 mL). The organic matter was dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (using a gradient of 0-10% ethyl acetate in hexane) to give intermediate BBB1. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.32 (d, J = 7.3 Hz, 1H), 4.15 (d, J = 11.6 Hz, 1H), 4.08–3.95 (m, 2H), 1.37 (s, 9H), 1.25 (d, J = 7.3 Hz, 3H), 1.13–1.09 (m, 6H).

((4-Nitrophenoxy)(phenoxy)phosphoryl)-L-alanine 3,3,3-trifluoro-2,2-dimethylpropyl ester. A solution of 1,4-dioxane in 4M hydrogen chloride (10 mL, 40.00 mmol) was added to a solution of intermediate BBB1 (1.88 g, 6.00 mmol) in dichloromethane (5 mL). After 1 hour, the reaction was concentrated. The residue was dissolved in dichloromethane (15 mL) and cooled to 0 °C. Phenyl dichlorophosphate (1.07 mL, 7.21 mmol) and triethylamine (1.83 mL, 13.22 mmol) were added sequentially. After 1 hour, 4-nitrophenol (0.836 g, 6.00 mmol) and triethylamine (0.92 mL, 7.00 mmol) were then added sequentially at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (eluting with hexane solution of 0-100% ethyl acetate) to give intermediate BBB2 (a mixture of diastereomers). ^¹H NMR (400 MHz, DMSO- _d₆₆ ) δ 8.30 (d, J = 8.9 Hz, 2H), 7.56–7.36 (m, 4H), 7.35–7.16 (m, 3H), 6.85–6.64 (m, 1H), 4.18–3.94 (m, 3H), 1.30–1.23 (m, 3H), 1.10 (s, 6H). ^³¹P NMR (162 MHz, DMSO- _d₆₆ ) δ -1.29, -1.46. ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -76.19, -76.19.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 3,3,3-trifluoro-2,2-dimethylpropyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), BBB2 (73 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.60 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100 acetonitrile aqueous solution) to give the product. LCMS: MS m/z = 649.97 [M+1], t _R = 1.38 min (minor), 1.40 min (major); LC system: Thermo Accela 1250 UHPLCz; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 μXB-C18 100A, 50 x 4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min - 2.0 min 2 - 100% acetonitrile, 2.0 min - 3.05 min 100% acetonitrile, 3.05 min - 3.2 min 100% - 2% acetonitrile, 3.2 min - 3.5 min 2% ACN, 2 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other by a chiral preparative SFC (SFC chiralpack AD-H 5 μm, 250 × 21 mm; isopropanol 30%).

Example 54. First eluted diastereomer of Example 53: ^¹H NMR (400MHz, methanol- _d4) )δ7.84(s,1H),7.38–7.30(m,2H),7.24–7.15(m,3H),7.00(d,J＝4.6Hz,1H) ,6.82(d,J＝4.6Hz,1H),5.38(d,J＝8.2Hz,1H),4.82–4.73(m,1H),4.70–4.6 2(m,2H),4.38(d,J＝5.3Hz,1H),4.31–4.24(m,2H),4.17(d,J＝11.5Hz,1H), 4.09–4.03(m,1H),4.01–3.93(m,1H),1.37–1.26(m,3H),1.19–1.12(m,6H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.69. ¹ H decoupled ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -78.76 (s), -238.55 (t, J = 47.8 Hz). LCMS: MS m/z = 649.97 [M+1], t _R = 1.38 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.88 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 55. Second eluted diastereomer of Example 53: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.89 (s, 1H), 7.40–7.32 (m, 2H), 7.28–7.17 (m, 3H), 7.10 (d, J = 4.6 Hz, 1H), 6.83 (d, J = 4.6 Hz, 1H), 5.38 (d, J = 8.3 Hz, 1H), 4.81–4.70 (m, 1H), 4.70–4.57 (m, 2H), 4.33 (d, J = 5.2 Hz, 1H), 4.29–4.14 (m, 3H), 4.07–3.91 (m, 2H), 1.34 (dd, J = 7.2, 1.0 Hz, 3H), 1.17–1.11 (m, 6H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.50. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -78.77 (s), -238.67 (t, J = 47.8 Hz). LCMS: MS m/z = 649.97 [Mz+1], t _R = 1.40 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.89 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 56. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine propyl ester

L-alanine propyl ester hydrochloride. Trimethylchlorosilane (30 mL, 272.68 mmol) was added to a mixture of L-alanine (20.00 g, 224.48 mmol) and 1-propanol (200 mL). The resulting mixture was stirred in a sealed container at 70 °C for 15 hours. The reaction mixture was concentrated under reduced pressure. The solid was crushed, dissolved in a mixture of ethyl acetate/hexane (100 mL, 50:50) and filtered. The filter cake was washed with ethyl acetate/hexane (20 mL, 50:50) and dried under high vacuum to give intermediate EEE1. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.63 (s, 3H), 4.20–3.95 (m, 3H), 1.69–1.54 (m, 2H), 1.41 (d, J = 7.2 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine propyl ester. Phenyl dichlorophosphate (4.88 mL, 32.81 mmol) and triethylamine (9.12 mL, 65.62 mmol) were successively added to a suspension of EEE1 (5.00 g, 29.83 mmol) in dichloromethane (50 mL) at 0 °C. After 1 hour, pentafluorophenol (5.49 g, 29.83 mmol) and triethylamine (4.55 mL, 32.81 mmol) were successively added at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate solution (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (using a gradient of 0-30% ethyl acetate in hexane) to give intermediate EEE2 (a diastereomeric mixture). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.46–7.38(m,2H),7.30–7.18(m,3H),6.89(dd,J＝14.1,9.9Hz,1H),4.03– 3.92(m,3H),1.62–1.48(m,2H),1.29(d,J＝6.8Hz,3H),0.85(t,J＝7.4Hz,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.11–-154.32(m), -160.60–-161.00(m), -163.56–-163.91(m). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.48. LCMS: MS m/z = 453.9 [M+1], t _R = 1.81 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine propyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate EEE2 (63 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.60 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to give Example EEE (diastereomer mixture). LCMS: MS m/z = 567.94 [M+1], t _R = 1.24 min (minor), 1.26 min (major); LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other using a chiral preparative SFC (SFC chiralpack AD-H 5 μm, 250 × 21 mm; ethanol 35%).

Example 57. First eluted diastereomer of Example 56: ^¹H NMR (400MHz, methanol- _d4) )δ7.96(s,1H),7.36–7.28(m,2H),7.25(d,J＝4.7Hz,1H),7.22–7.14(m,3H) ,6.94(d,J＝4.7Hz,1H),5.39(d,J＝8.2Hz,1H),4.81–4.73(m,1H),4.70–4.6 0(m,2H),4.38(d,J＝5.2Hz,1H),4.33–4.21(m,2H),4.11–3.99(m,2H),3.97 –3.87(m,1H),1.70–1.57(m,2H),1.33–1.27(m,3H),0.93(t,J=7.4Hz,3H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.59. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -236.94 (t, J = 47.7 Hz). LCMS: MS m/z = 567.94 [M], t _R = 1.24 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min. HPLC: t _R = 2.53 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 58. Second eluted diastereomer of Example 56: ^¹H NMR (400MHz, methanol- _d4) )δ7.90(s,1H),7.41–7.33(m,2H),7.28–7.17(m,3H),7.12(d,J＝4.6Hz,1H) ,6.85(d,J＝4.7Hz,1H),5.38(d,J＝8.4Hz,1H),4.80–4.70(m,1H),4.70–4.5 7(m,2H),4.34(d,J＝5.2Hz,1H),4.27–4.17(m,2H),4.11–3.98(m,2H),3.97 –3.85(m,1H),1.71–1.55(m,2H),1.36–1.29(m,3H),0.92(t,J＝7.4Hz,3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.59. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -237.39 (t, J = 47.7 Hz). LCMS: MS m/z = 567.94 [M+1], t _R = 1.26 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min. HPLC: t _R = 2.57 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 59. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine pentyl ester

(tert-Butoxycarbonyl)-L-alanine pentyl ester. 4-Methylmorpholine (7.93 mL, 72.15 mmol), 4-(dimethylamino)pyridine (59 mg, 0.48 mmol), and tripropylphosphonic anhydride (17.18 mL, 28.86 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (5.00 g, 26 mmol) and 1-pentanol (2.12 g, 24.05 mmol) in 100 mL of anhydrous dichloromethane at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with 2 × 10% citric acid aqueous solution (20 mL), twice with saturated sodium bicarbonate aqueous solution (20 mL), and once with brine (50 mL). The combined organic matter was dried over sodium sulfate, filtered through a 3 cm silica gel filter, and the silica gel was washed with additional dichloromethane (200 mL). The organic compound was concentrated under reduced pressure, co-distilled with DCM, and dried overnight under high vacuum. No further purification was performed; the intermediate HHH1 was used in the next step. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.26 (d, J = 7.4 Hz, 1H), 4.25–3.80 (m, 3H), 1.65–1.48 (m, 2H), 1.41–1.18 (m, 16H), 1.00–0.74 (m, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine pentyl ester. Intermediate HHH1 (6.11 g, 22.3 mmol) was dissolved in dichloromethane (20 mL) and cooled to 0 °C. Hydrogen chloride (30.00 mL, 120.00 mmol, 4 N dioxane solution) was added. After 2 hours, the reaction was concentrated under reduced pressure. The residue was dissolved in dichloromethane (30 mL) and cooled to 0 °C. Phenyl dichlorophosphate (3.65 mL, 24.53 mmol) and triethylamine (6.80 mL, 49.06 mmol) were added sequentially. After 1 hour, pentafluorophenol (4.0 g, 22.30 mmol) and triethylamine (3.40 mL, 24.53 mmol) were then added sequentially at 0 °C, and the resulting mixture was warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and filtered through a 3 cm silica gel filter. The pad was washed with additional dichloromethane (200 mL). The organic matter was concentrated under reduced pressure. The residue was dissolved in hot hexane (30 mL) and then diluted with hexane (120 mL). The reaction was stirred for 2 hours, and intermediate HHH2 was separated by filtration. NMR spectroscopy confirmed that intermediate HHH2 was a single diastereomer. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.46–7.37(m,2H),7.30–7.19(m,3H),6.90(dd,J＝14.3,9.9Hz,1H),4. 07–3.93(m,3H),1.58–1.48(m,2H),1.33–1.20(m,7H),0.88–0.77(m,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.24 (d, J = 21.5Hz), -160.86 (t, J = 23.1Hz), -163.69 (dd, J = 24.2, 20.4Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.47. LCMS: MS m/z = 481.8 [M+1], t _R = 1.91 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine pentyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate HHH2 (74 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.60 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100 acetonitrile aqueous solution) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, methanol-d ₄ )δ7.80(s,1H),7.39–7.31(m,2H),7.29–7.15(m,3H),6.87(d,J＝4.6Hz,1H),6.75(d,J＝4.5Hz,1H),5.36(d,J＝8.3Hz,1H),4.81–4.57(m,3H), 4.34(d,J＝5.2Hz,1H),4.21(d,J＝5.3Hz,2H),4.14–4.00(m,2H),3.95– 3.86(m,1H),1.66–1.52(m,2H),1.36–1.25(m,7H),0.93–0.85(m,3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.59. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.85 (t, J = 47.7 Hz). LCMS: MS m/z = 595.97 [M+1], t _R = 1.43 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.87 min; Agilent 1290II; Column: Phenomenex Kinetex. C18, 2.6u110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 60. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- ((((S)-(((S)-1-(2-ethylbutoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2- (Fluoromethyl)tetrahydrofuran-3,4-dimethyldiacetate

Acetic anhydride (11 mg, 0.11 mmol) and Example 1 (33 mg, 0.05 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (0.7 mg, 0.005 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.82(s,1H),7.38–7.31(m,2H),7.28–7.23(m,2H),7.22–7.17(m,1H),6.86(d,J＝4.6Hz, 1H),6.66(d,J＝4.6Hz,1H),5.89–5.82(m,1H),5.80(d,J＝5.5Hz,1H),5.59(d,J＝7.9Hz,1H), 4.76–4.68 (m, 1H), 4.65–4.54 (m, 1H), 4.36–4.30 (m, 1H), 4.30–4.24 (m, 1H), 4.10–3.91 (m, 3H), 2.14 (s, 3H), 1.98 (s, 3H), 1.57–1.43 (m, 1H), 1.41–1.28 (m, 7H), 0.88 (t, J = 7.5 Hz, 6H). ³¹ PNMR (162 MHz, methanol- _d⁴ ) δ 3.40. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -236.51 (t, J = 46.9 Hz). LCMS: MS m/z = 694.1 [M+1], t _R = 1.67 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2μL/min. HPLC: t _R = 3.32 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6μm. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 61. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- ((((S)-(((S)-1-(2-ethylbutoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2- (fluoromethyl)tetrahydrofuran-3,4-dimethyldipropionate

Propionic anhydride (14 mg, 0.11 mmol) and Example 1 (33 mg, 0.05 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (0.7 mg, 0.005 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.83(s,1H),7.41–7.29(m,2H),7.29–7.23(m,2H),7.23–7.16(m,1H),6.89(d,J＝4.6Hz,1H),6.67(d, J＝4.6Hz,1H),5.92–5.79(m,2H),5.59(d,J＝7.7Hz,1H),4.76–4.67(m,1H),4.65–4.54(m,1H),4.37–4.31 (m, 1H), 4.29–4.24(m, 1H), 4.09–4.04(m, 1H), 4.03–3.94(m, 2H), 2.49–2.42(m, 2H), 2.31–2.23(m, 2H), 1.54–1.46(m, 1H), 1.40–1.31(m, 7H), 1.17(t, J = 7.6 Hz, 3H), 1.05(t, J = 7.5 Hz, 3H), 0.88(t, J = 7.5 Hz, 6H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.41. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -236.30(t, J = 46.9 Hz). LCMS: MS m/z = 722.0 [M+1], t _R = 1.78 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 3.54 min; Agilent 1290II; Column: Phenomenex Kinetex. C18, 2.6u110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 62. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- ((((S)-(((S)-1-(2-ethylbutoxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2- (Fluoromethyl)tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Isobutyric anhydride (18 mg, 0.11 mmol) and Example 1 (34 mg, 0.06 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (0.7 mg, 0.006 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.88(s,1H),7.37–7.28(m,2H),7.28–7.21(m,2H),7.21–7.16(m,1H),6.99(d,J＝4.6Hz,1H),6.69(d,J＝4.6H z,1H),5.88–5.75(m,2H),5.66–5.53(m,1H),4.78–4.67(m,1H),4.63–4.54(m,1H),4.40–4.31(m,1H),4.31–4.2 3(m, 1H), 4.10–4.02(m, 1H), 4.02–3.89(m, 2H), 2.68(hep, J = 7.0 Hz, 1H), 2.47(hep, J = 7.0 Hz, 1H), 1.58–1.43(m, 1H), 1.39–1.30(m, 7H), 1.25–1.18(m, 6H), 1.08(d, J = 7.0 Hz, 3H), 1.03(d, J = 7.0 Hz, 3H), 0.87(t, J = 7.5 Hz, 6H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.44. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.60(t, J = 46.8 Hz). LCMS: MS m/z = 750.1 [M+1], t _R = 1.88 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2μL/min. HPLC: t _R = 3.73 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6μm. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 63. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (((((S)-1-(cyclohexyloxy)-1-oxopropyl-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)-2-(fluoromethyl) Tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Propionic anhydride (17 mg, 0.13 mmol) and Example 14 (40 mg, 0.07 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (8 mg, 0.07 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.83(s,1H),7.37–7.30(m,2H),7.29–7.23(m,2H),7.23–7.16(m,1H),6.87(d,J＝4.6Hz,1H),6.65(d,J＝4.6 Hz,1H),5.88–5.79(m,2H),5.59(d,J＝7.6Hz,1H),4.79–4.66(m,2H),4.66–4.55(m,1H),4.39–4.32(m,1H),4.3 1–4.25 (m, 1H), 3.98–3.88 (m, 1H), 2.68 (hep, J = 7.0, 1H), 2.47 (hep, J = 7.0, 1H), 1.85–1.76 (m, 2H), 1.75–1.67 (m, 2H), 1.58–1.49 (m, 1H), 1.47–1.28 (m, 8H), 1.24–1.21 (m, 6H), 1.08 (d, J = 7.0 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.45. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.50 (t, J = 46.9 Hz). LCMS: MS m/z = 748.1 [M+1], t _R = 1.85 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 3.64 min; Agilent 1290II; Column: Phenomenex Kinetex. C18, 2.6u110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 64. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine tetrahydro-2H-pyran- 4-yl ester

(S)-2-Aminopropionic acid tetrahydro-2H-pyran-4-yl ester hydrochloride. TMSCl (2 mL) was added to a mixture of L-alanine (500 mg, 5.61 mmol) and tetrahydro-2H-pyran-4-ol (5 g, 49.0 mmol). The resulting mixture was stirred at 70 °C for 15 hours and concentrated under vacuum. The resulting solid was extracted with a 5% hexane solution of EtOAc, filtered, and washed several times with the same solution. The resulting solid was dried under high vacuum for 15 hours to give intermediate MMM1. Due to the hygroscopic nature of the solid, it was used as is in subsequent reactions.

(2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate tetrahydro-2H-pyran-4-yl ester. Intermediate MMM1 (1.33 g, 6.34 mmol) was dissolved in methylene chloride (15 mL), cooled to -78 °C, and phenyl dichlorophosphate (1.137 mL, 7.61 mmol) was rapidly added. Triethylamine (2.2 mL, 15.2 mmol) was added over 30 minutes at -78 °C, and the resulting mixture was stirred at -78 °C for 30 minutes. Then, 4-nitrophenol (882 mg, 6.34 mmol) was added in a single batch, followed by triethylamine (1.1 mL, 7.61 mmol) over 30 minutes at -78 °C. The mixture was stirred at -78 °C for 30 minutes, washed with water (2×) and brine, and dried over sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (EtOAc 0 to 70% hexane solution) to give intermediate MMM2 (a mixture of diastereomers). ^¹H NMR (400 MHz, chloroform-d) δ 8.22 (m, 2H), 7.49–7.06 (m, 7H), 4.95 (m, 1H), 4.14 (m, 1H), 4.07–3.80 (m, 3H), 3.52 (m, 2H), 1.95–1.81 (m, 2H), 1.64 (m, 2H), 1.42 (m, 3H). ^³¹P NMR (162 MHz, chloroform-d) δ -3.09, -3.13.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine tetrahydro-2H-pyran-4-yl ester. Tetrahydrofuran (5 mL) was added to a mixture of intermediate 4 (250 mg, 0.74 mmol), intermediate MMM2 (433 mg, 0.96 mmol), and magnesium chloride (106 mg, 1.11 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (20 mL). The organic matter was washed with water (10 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid (0.6 mL, 7.39 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (5 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (20 mL), and the resulting mixture was washed with saturated sodium bicarbonate solution (10 mL) and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to give the product (diasteremeric mixture). ^¹H NMR (400MHz, methanol- _d⁴ ) δ 7.86–7.82 (m, 1H), 7.40–7.30 (m, 2H), 7.28–7.15 (m, 3H), 6.99–6.93 (m, 1H), 6.83–6.77 (m, 1H), 5.40–5.35 (m, 1H), 4.95–4.88 (m, 1H), 4.81–4.58 (m, 3H), 4.40–4.31 (m, 1H), 4.29–4.18 (m, 2H), 3.97–3.78 (m, 3H), 3.56–3.46 (m, 2H), 1.92–1.82 (m, 2H), 1.66–1.55 (m, 2H), 1.34–1.25 (m, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.75, 3.56. ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.53, -238.74. LCMS: MS m/z = 610.01 [M+1], t _R = 1.16 min (minor), 1.18 min (major); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 2.32 min (minor), 2.37 (major); Agilent 1290II; Column: Phenomenex Kinetex. C18, 2.6u 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5min gradient of 2-98% B, 1.5mL/min.

Example 65. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((((S)-1-oxo-1-((tetrahydro-2H-pyran-4-yl)oxy)propyl-2-yl)amino)(phenoxy)phospho Tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Isobutyric anhydride (26 mg, 0.16 mmol) and Example 64 (50 mg, 0.08 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (8 mg, 0.07 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10–100% acetonitrile aqueous solution to give the product (diasteremeric mixture). LCMS: MS m/z = 750.1 [M+1], t _R = 1.62 min (major), 1.65 (minor); LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

Separation of Sp and Rp diastereomers. The diastereomers were separated from each other using a chiral preparative SFC (SFC chiralpack 1A 5 μm, 250 × 21 mm; 30% isopropanol).

Example 66. First eluted diastereomer of Example 65: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.81 (s, 1H), 7.39–7.30 (m, 2H), 7.26–7.16 (m, 3H), 6.85 (d, J = 4.4 Hz, 1H), 6.77 (d, J = 4.6 Hz, 1H), 5.96–5.90 (m, 1H), 5.85 (d, J = 5.6 Hz, 1H), 5.60 (d, J = 7.7 Hz, 1H), 4.95–4.89 (m, 2H), 4.73 (s, 1H), 4.62 (s, 1H) ,1H), 4.38–4.29(m,2H), 3.96–3.80(m,2H), 3.56–3.46(m,2H), 2.73–2.64(m,1H), 2.53–2.44(m,1H), 1.91–1.83(m,2H), 1.67–1.56(m,2H), 1.35–1.20(m,9H), 1.09(d,J＝7.0Hz,3H), 1.05(d,J＝7.0Hz,3H). ³¹ P NMR (162MHz, methanol- _d4 ) δ3.50. ^1H decoupled ¹⁹ F NMR (376MHz, methanol- _d4 ) δ-235.96. LCMS: MS m/z = 750.08 [M+1], t _R = 1.62 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 3.23 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6 μL. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 67. Second eluted diastereomer of Example 65: ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.80 (s, ¹H), 7.38–7.30 (m, 2H), 7.29–7.23 (m, 2H), 7.22–7.17 (m, ¹H), 6.80 (d, J = 4.5 Hz, ¹H), 6.61 (d, J = 4.5 Hz, 1H), 5.87–5.79 (m, 2H), 5.60 (d, J = 7.8 Hz, 1H), 4.99–4.91 (m, 1H), 4.78–4.66 (m, 1H), 4.66–4.54 (m, 1H), 4.40–4.32 (m, 1H), 4 .32–4.25(m,1H), 4.02–3.92(m,1H), 3.90–3.82(m,2H), 3.57–3.46(m,2H), 2.74–2.64(m,1H), 2.51–2.42(m,1H), 1.95–1.83(m,2H), 1.69–1.56(m,2H), 1.37–1.28(m,3H), 1.23(d,J＝7.0Hz,6H), 1.08(d,J＝7.0Hz,3H), 1.03(d,J＝7.0Hz,3H). ³¹ P NMR (162MHz, methanol- _d⁴ ) δ 3.40. ^1H decoupled ¹⁹ F NMR (376MHz, methanol- _d⁴ ) δ -235.32. LCMS: MS m/z = 750.07 [M+1], t _R = 1.65 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2μL/min. HPLC: t _R = 3.24 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6μm. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 68. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (Fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-propylpentyl ester

(tert-Butoxycarbonyl)-L-alanine 2-propylpentyl ester. 4-Methylmorpholine (7.92 mL, 72.07 mmol), 4-(dimethylamino)pyridine (59 mg, 0.48 mmol), and tripropylphosphonic anhydride (17.16 mL, 28.83 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (5.00 g, 26.42 mmol) and 2-propylpentyl-1-ol (3.13 g, 24.02 mmol) in 100 mL of anhydrous dichloromethane at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with 2 × 10% citric acid aqueous solution (20 mL), twice with saturated sodium bicarbonate aqueous solution (20 mL), and once with brine (50 mL). The combined organic compounds were dried over sodium sulfate, filtered through a 3 cm silica gel layer, and washed with 200 mL of dichloromethane. The organic compounds were concentrated under reduced pressure, co-distilled with DCM, and dried under high vacuum overnight. No further purification was performed; the intermediate QQQ1 was used in the next step. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.27 (d, J = 7.5 Hz, 1H), 4.06–3.86 (m, 3H), 1.67–1.56 (m, 1H), 1.43–1.10 (m, 20H), 0.93–0.79 (m, 6H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine 2-propylpentyl ester. A 10 mL (40.00 mmol) solution of 4 M hydrogen chloride in 1,4-dioxane was added to a solution of intermediate QQQ1 (5.70 g, 18.93 mmol) in dichloromethane (5 mL). After 1 hour, the reaction was concentrated. The residue was dissolved in dichloromethane (15 mL) and cooled to 0 °C. Phenyl dichlorophosphate (3.10 mL, 20.82 mmol) and triethylamine (5.77 mL, 41.64 mmol) were added sequentially. After 1 hour, pentafluorophenol (3.48 g, 18.93 mmol) and triethylamine (2.89 mL, 20.82 mmol) were then added sequentially at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and filtered through a 3 cm silica gel filter. The silica gel cake was washed with another 100 mL of dichloromethane. The organic matter was concentrated. The residue was dissolved in 30 mL of hot hexane and then diluted with 120 mL of hexane. The reaction was stirred for 5 hours and then filtered to separate intermediate QQQ2. Intermediate QQQ2 was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.44–7.37(m,2H),7.48–7.36(m,1H),6.94–6.85(m,1H),4.06–3.90(m,3H),1.66–1.55(m,1H),1.32–1.18(m,11H),0.85–0.79(m,6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.23 (d, J = 22.6Hz), -160.88 (t, J = 23.5Hz), -163.71 (t, J = 22.0Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.44. LCMS: MS m/z = 523.78 [M+1], t _R = 2.04 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-propylpentyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate QQQ2 (80 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ HNMR (400MHz, methanol-d ₄ )δ7.81(s,1H),7.39–7.31(m,2H),7.28–7.16(m,3H),6.88(d,J＝4.5Hz,1H),6.75(d,J＝4.5Hz,1H),5.36(d,J＝8.3Hz,1H),4.81–4.57(m,3H) ,4.34(d,J＝5.2Hz,1H),4.26–4.17(m,2H),4.07–4.01(m,1H),3.98–3 .88(m,2H),1.69–1.61(m,1H),1.38–1.20(m,11H),0.94–0.83(m,6H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.55. ¹ H decoupled ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.83. LC-MS: MS m/z = 638.03 [M+1], t _R = 1.58 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 μX B-C18 100A, 50 x 4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min - 2.0 min 2-100% acetonitrile, 2.0 min - 3.05 min 100% acetonitrile, 3.05 min - 3.2 min 100% - 2% acetonitrile, 3.2 min - 3.5 min 2% ACN, 2 μL/min. HPLC: t _R = 3.29 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

Example 69. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-valine ethyl ester

L-valine ethyl ester hydrochloride. Trimethylchlorosilane (4.58 mL, 36 mmol) was added to a solution of L-valine (5.0 g, 43 mmol) in ethanol (20 mL). The reaction was heated at 70 °C for 18 h. The reaction was concentrated under reduced pressure. Without further purification, intermediate RRR1 was used in the next step. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.58 (s, 3H), 4.31–4.14 (m, 2H), 3.87–3.80 (m, 1H), 2.24–2.13 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H), 0.99 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-valine ethyl ester. Intermediate RRR1 (7.40 g, 40.74 mmol) was dissolved in dichloromethane (15 mL) and cooled to 0 °C. Phenyl dichlorophosphate (6.67 mL, 44.81 mmol) and triethylamine (12.42 mL, 89.62 mmol) were added sequentially. After 1 hour, pentafluorophenol (7.50 g, 40.74 mmol) and triethylamine (6.21 mL, 44.81 mmol) were added sequentially at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and filtered through a 3 cm silica gel filter. The silica gel cake was washed with additional dichloromethane (100 mL). The organic matter was concentrated. The residue was dissolved in 30 mL of hot hexane and then diluted with 120 mL of hexane. The reaction was stirred for 5 hours and then filtered to separate intermediate RRR2. Intermediate RRR2 was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.48–7.34 (m, 2H), 7.28–7.18 (m, 3H), 6.81–6.71 (m, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.69–3.58 (m, 1H), 2.01–1.88 (m, 1H), 1.16 (t, J = 7.1 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.30 (d, J = 21.4Hz), -161.10 (t, J = 23.3Hz), -163.77 (dd, J = 24.0, 20.1Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 1.95. LCMS: MS m/z = 467.87 [M+1], t _R = 1.85 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-valine ethyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate RRR2 (72 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^1H NMR (400 MHz, methanol- _d4) )δ7.84(s,1H),7.40–7.31(m,2H),7.27–7.15(m,3H),6.97(d,J＝4.5Hz,1H),6 .79(d,J＝4.5Hz,1H),5.36(d,J＝8.4Hz,1H),4.81–4.71(m,1H),4.70–4.59(m,2 H),4.35(d,J＝5.1Hz,1H),4.26–4.20(m,2H),4.16–4.05(m,2H),3.64(dd,J＝9. 9, 6.2Hz, 1H), 2.05–1.95 (m, 1H), 1.21 (t, J = 7.2Hz, 3H), 0.93 (t, J = 7.7Hz, 6H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 4.41. ¹ H decoupled ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.95. LCMS: MS m/z = 581.94 [M+1], t _R = 1.30 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2μl/min. HPLC: t _R = 2.64 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6μm. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 70. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine isobutyl ester

(tert-butoxycarbonyl)-L-alanine isobutyl ester. 4-methylmorpholine (1.74 mL, 15.86 mmol), 4-(dimethylamino)pyridine (13 mg, 0.11 mmol), and tripropylphosphonic anhydride (3.78 mL, 6.34 mmol, 50% ethyl acetate solution) were added to a stirred solution of (tert-butoxycarbonyl)-L-alanine (1.00 g, 5.29 mmol) and 1-methylprop-1-ol (0.60 mL, 6.34 mmol) in 10 mL of anhydrous dichloromethane at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was washed with 2 × 10% citric acid aqueous solution (20 mL), twice with saturated sodium bicarbonate aqueous solution (20 mL), and once with brine (50 mL). The combined organic matter was dried over sodium sulfate, filtered through a 3 cm silica gel filter, and the silica gel was washed with additional dichloromethane (200 mL). The organic compound was concentrated under reduced pressure, co-distilled with DCM, and dried overnight under high vacuum. No further purification was performed; the intermediate SSS1 was used in the next step. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.26 (d, J = 7.4 Hz, 1H), 4.08–3.94 (m, 1H), 3.93–3.72 (m, 2H), 1.93–1.78 (m, 1H), 1.44–1.29 (m, 9H), 1.24 (d, J = 7.4 Hz, 3H), 0.88 (d, J = 6.7 Hz, 6H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine isobutyl ester. A 10 mL (40.00 mmol) solution of 4 M hydrogen chloride in 1,4-dioxane was added to a solution of intermediate SSS1 (12.15 g, 49.54 mmol) in dichloromethane (5 mL). After 1 hour, the reaction was concentrated. The residue was dissolved in dichloromethane (15 mL) and cooled to 0 °C. Phenyl dichlorophosphate (8.11 mL, 54.50 mmol) and triethylamine (15.11 mL, 108.99 mmol) were added sequentially. After 1 hour, pentafluorophenol (9.12 g, 49.54 mmol) and triethylamine (7.55 mL, 54.50 mmol) were then added sequentially at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with 50 mL of dichloromethane, washed with 50 mL of saturated sodium bicarbonate solution and 50 mL of brine, dried over anhydrous sodium sulfate, and filtered through a 3 cm silica gel filter. The silica gel cake was washed with another 100 mL of dichloromethane. The organic matter was concentrated. The residue was dissolved in 30 mL of hot hexane and then diluted with 120 mL of hexane. The reaction was stirred for 5 hours, and then intermediate SSS2 was separated by filtration. Intermediate SSS2 was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.45–7.38(m,2H),7.23(dd,J＝11.2,7.8Hz,3H),6.91(dd,J＝14.2,10.0Hz,1H),4.07–3.94( m,1H),3.81(d,J＝6.6Hz,2H),1.89–1.79(m,1H),1.29(d,J＝7.1Hz,3H),0.86(d,J＝6.7Hz,6H). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.49. ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ -154.25 (d, J = 21.3Hz), -160.86 (t, J = 23.3Hz), -163.55--163.79 (m). LCMS: MS m/z = 467.8 [M+1], t _R = 1.88 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine isobutyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (40 mg, 0.12 mmol), intermediate SSS2 (72 mg, 0.15 mmol), and magnesium chloride (17 mg, 0.18 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (52 μL, 0.30 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100% acetonitrile aqueous solution) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ¹ HNMR (400MHz, methanol-d ₄ )δ7.84(s,1H),7.39–7.32(m,2H),7.27–7.17(m,3H),6.96(d,J＝4.6Hz,1H),6.79(d,J＝4.6Hz,1H),5.37(d,J＝8.3Hz,1H),4.81–4.70(m,1H),4 .70–4.58(m,2H),4.34(d,J＝5.1Hz,1H),4.25–4.17(m,2H),3.98–3.80( m,3H),1.95–1.85(m,1H),1.32(d,J＝7.1Hz,3H),0.91(d,J＝6.7Hz,6H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.57. ¹ H decoupled ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.78. LCMS: MS m/z = 581.97 [M+1], t _R = 1.35 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2μL/min. HPLC: t _R = 2.73 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6μm. 110A, 100x4.6mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 2-98% B, 1.5mL/min for 8.5min.

Example 71. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclobutyl ester

(tert-Butoxycarbonyl)-L-alanine cyclobutyl ester. (tert-Butoxycarbonyl)-L-alanine (9.447 g, 0.05 mol) was dissolved in acetonitrile (36 mL), and cyclobutanol (3 g, 0.042 mol) was added in a single addition, followed by EDCI (8.396 g, 0.054 mol) and DMAP (7.624 g, 0.062 mol). The reaction was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane and water. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. Purification was achieved by silica gel chromatography with 0–30% ethyl acetate/hexane to give intermediate TT1. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.21(d,J＝7.3Hz,1H),4.87(p,J＝7.4Hz,1H),3.91(h,J＝7.3Hz,1H),2.20–2.28(m,2H) ,2.10-1.85(m,2H),1.78-1.65(m,1H),1.58(m,1H),1.36(s,9H),1.19(d,J＝7.4Hz,3H).

L-Alanine cyclobutyl ester hydrochloride. Intermediate TTT1 (8 g, 0.033 mol) was dissolved in anhydrous dichloromethane (88 mL) and a dioxane solution of 4N HCl (41.1 mL, 0.164 mol). The reaction was stirred at ambient temperature for 4 hours. The reaction was concentrated under reduced pressure and co-evaporated with dichloromethane. The residue was placed under high vacuum overnight, and intermediate TTT2 was used as is in the next step without purification. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.65(s,3H),5.03–4.90(m,1H),3.97(q,J＝7.2Hz,1H),2.34–2.21(m,2H) ,2.04(m,2H),1.82–1.68(m,1H),1.68–1.51(m,1H),1.39(d,J=7.2Hz,3H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine cyclobutyl ester. Triethylamine (9.76 mL, 69.44 mmol) was added to a solution of intermediate TTT2 (5.67 g, 31.56 mmol) and phenyl dichlorophosphate (4.696 mL, 31.56 mmol) in anhydrous dichloromethane (100 mL) under an argon atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour. Then, pentafluorophenol (5.81 g, 31.56 mmol) and triethylamine (4.88 mL, 34.72 mmol) were added. After stirring at 0 °C for 1 hour, the reaction mixture was diluted with _Et₂O and the solid was filtered off. The crude product was concentrated under reduced pressure and purified by silica gel chromatography using a gradient eluent of 0-100% ethyl acetate/hexane to give the desired compound as a diastereomeric mixture. The obtained compound was dried under high vacuum to allow solidification. Diisopropyl ether was added to the solidified material and sonicated to obtain a fine solid. The solid was separated by filtration. A second round of sonication with diisopropyl ether and filtration yielded intermediate TTT3. NMR spectroscopy confirmed that intermediate TTT3 was a single diastereomer. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.40(dd,J＝8.8,7.0Hz,2H),7.27–7.16(m,3H),6.85(dd,J＝14.1,9.9Hz,1H),4.91–4.78(m,1H),4.00–3.8 2(m,1H),2.28–2.15(m,2H),1.99–1.84(m,2H),1.76–1.63(m,1H),1.56(m,1H),1.25(dd,J＝7.1,1.2Hz,3H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ -154.11–-154.31 (m), -160.88 (t, J = 23.3Hz), -163.67 (t, J = 23.6Hz). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 0.45. LCMS: MS m/z = 465.94 [M+1]; t _R = 1.67 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclobutyl ester. Tetrahydrofuran (1.5 mL) was added to a mixture of intermediate 4 (0.1 g, 0.296 mmol), intermediate TTT3 (0.151 g, 0.325 mmol), and magnesium chloride (0.042 g, 0.0443 mmol) at room temperature, followed by the addition of N,N-diisopropylethylamine (0.129 mL, 0.739 mmol). The resulting mixture was stirred at 50 °C for 1.5 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified using preparative HPLC (Phenominex Synergi 4u Hydro-RR 150x30mm column, 15%–85% acetonitrile/water). Fractions were combined and concentrated under reduced pressure. The obtained residue was dissolved in anhydrous acetonitrile (3 mL) and cooled in an ice bath, followed by dropwise addition of concentrated hydrochloric acid (0.133 mL, 1.6 mmol). The reaction mixture was stirred at room temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with a saturated aqueous sodium bicarbonate solution. The separated solids were separated by filtration, washed with water, and purified by silica gel chromatography with 0–20% methanol/dichloromethane to give the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.78(s,1H),7.39–7.30(m,2H),7.27–7.14(m,3H),6.84(d,J＝4.5Hz,1H),6 .73(d,J＝4.5Hz,1H),5.36(d,J＝8.3Hz,1H),4.99–4.86(m,1H),4.82–4.56(m,3 H),4.34(d,J＝5.2Hz,1H),4.24–4.15(m,2H),3.87(m,1H),2.35–2.22(m,2H),2 .02(m,2H),1.82–1.68(m,1H),1.69–1.53(m,1H),1.28(dd,J=7.1,1.0Hz,3H). ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -238.78 (dd, J = 48.6, 46.9 Hz). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.52. LCMS: MS m/z = 580.05 [M+1]; t _R = 1.08 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.8 min 2-100% acetonitrile, 1.8 min-1.85 min 100%-2% acetonitrile, 1.85 min-2.00 min 2% ACN, 1800 μL/min. HPLC: t _R = 4.415 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

Example 72. ((S)-(((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- 2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine oxocyclic ring Butane-3-yl ester

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine oxetane-3-yl ester. Phenyl dichlorophosphate (2.01 g, 9.55 mmol) was added in a single addition to a solution of L-alanine oxetane-3-yl ester (1.98 g, 9.55 mmol) in DCM (50 ml). The resulting mixture was cooled to 0 °C, and triethylamine (0.97 g, 9.55 mmol) was added dropwise. After removing the ice bath, the resulting mixture was stirred for 30 minutes and cooled to 0 °C, and 2,3,4,5,6-pentafluorophenol (1.76 g, 9.55 mmol) was added in a single addition, followed by the dropwise addition of triethylamine (0.97 g, 9.55 mmol). After removing the ice bath, the resulting mixture was stirred for 30 minutes, diluted with EtOAc, and washed with water and brine. The organic solvent was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (eluting with a hexane solution of 0-100% ethyl acetate) to give the intermediate. ^¹H NMR (400 MHz, chloroform-d) δ 7.38–7.34 (m, 2H), 7.28–7.19 (m, 3H), 5.49–5.43 (m, 1H), 4.91–4.85 (m, 2H), 4.63–4.56 (m, 2H), 4.27–4.22 (m, 1H), 4.06–4.02 (m, 1H), 1.51–1.48 (m, 3H). ^³¹P NMR (162 MHz, chloroform-d) δ 0.38, 0.22. LCMS: MS m/z = 468.01 [M+1], t _R = 1.63 min; LC system: Thermo Accela 1250U HPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min. HPLC: t _R = 3.15 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for 8.5 min.

At room temperature, tetrahydrofuran (1.0 mL) was added to a mixture of intermediate 4 (0.0345 g, 0.103 mmol), ((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine oxetane-3-yl ester (0.626 g, 0.134 mmol), and magnesium chloride (0.015 g, 0.155 mmol), followed by the addition of N,N-diisopropylethylamine (0.063 mL, 0.361 mmol). The resulting mixture was stirred at 50 °C for 1.5 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with saturated sodium chloride solution and ethyl acetate. The layers were separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified using preparative HPLC (Phenominex Synergi 4u Hydro-RR 150 x 30 mm column, 15%–85% acetonitrile/water). The fractions were combined and concentrated under reduced pressure. LCMS: MS m/z = 581.97 [M]; t _R = 1.12 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μXB-C18100A, 50x3.0mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 1.8 min 2-100% acetonitrile, 1.8 min - 1.85 min 100% - 2% acetonitrile, 1.85 min - 2.00 min 2% acetonitrile, 1800 μl/min. HPLC: t- _R = 3.37 min; Agilent Infinity 1290II; Column: Phenomenex Kinetex 2.8μC18 100A, 100x4.6 mm; Solvent: Acetonitrile containing 0.1% trifluoroacetic acid, water containing 0.1% trifluoroacetic acid; Gradient: 0-0.55 min 2% acetonitrile, 0.55-8.55 min 2-98% acetonitrile, 8.55-9.25 min 98% acetonitrile, 1500 μl/min

Example 73. (2R,3S,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoro methyl)-2-((((S)-(((S)-1-(oxecyclobutane-3-yloxy)-1-oxopropyl-2-yl)amino)(phenoxy)phospho Tetrahydrofuran-3,4-dimethylbis(2-methylpropionate)

Isobutyric anhydride (8.2 mg, 0.05 mmol) and Example 72 (15 mg, 0.03 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere, and the mixture was stirred at room temperature for 5 minutes. 4-Dimethylaminopyridine (0.3 mg, 0.003 mmol) was added, and the reaction mixture was stirred at room temperature. After 2 hours, methanol (0.5 mL) was added, and the mixture was stirred for 20 minutes. It was then diluted with ethyl acetate (10 mL), washed twice with saturated sodium bicarbonate aqueous solution (10 mL), and once with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) using a gradient of 10-100% acetonitrile aqueous solution to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ8.14(s,2H),7.37(t,J＝7.8Hz,1H),7.31–7.18(m,4H),6.98(d,J＝4.6Hz,2H),6.84(dd,J＝36.1,4.5Hz,1H), 5.45(d,J＝5.7Hz,2H),5.21(t,J＝6.1Hz,2H),4.97(d,J＝6.4Hz,2H),4.89–4.82(m,1H),4.71(d,J＝47.0Hz,1H), 4.62–4.54 (m, 1H), 4.37 (d, J = 4.7 Hz, 1H), 3.78 (d, J = 11.4 Hz, 2H), 3.67 (d, J = 11.4 Hz, 2H), 2.71 (p, J = 7.0 Hz, 1H), 2.52 (p, J = 7.0 Hz, 1H), 1.33–1.29 (m, 3H), 1.25 (d, J = 7.0 Hz, 6H), 1.12 (d, J = 7.0 Hz, 3H), 1.08 (d, J = 7.0 Hz, 3H). ³¹ P NMR (162 MHz, methanol- _d⁴ ) δ 3.50. Decoupled ¹⁹ F NMR (376 MHz, methanol- _d⁴ ) δ -235.96 (s). LCMS: MS m/z = 722.1 [M+1], t _R = 1.56 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6 mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min.

Example 74. ((S)-(((2R,3S,4R,5S)-5-(4-butyrylaminopyrrolo[2,1-f][1,2,4]triazine- 7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethyl Butyl ester

Butyryl chloride (0.026 mL, 0.254 mmol) was added dropwise to a solution of intermediate 6 (150 mg, 0.231 mmol) in anhydrous pyridine (1 mL) at room temperature under an argon atmosphere. After 30 minutes, the reaction mixture was purified by a gradient of 20-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) to give the compound. LCMS: MS m/z = 720.27 [M+1]; t _R = 1.76 min; LC system: Dionex Ultimate 3000U HPLC; Column: Phenomenex Kinetex 2.6μC18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min–0.2 min 40% acetonitrile, 0.2 min–1.55 min 40%–100% acetonitrile, 1.55 min–2.80 min 100% acetonitrile, 2.80–2.81 min 100%–40% acetonitrile, 1100 μl/min. The obtained compound was dissolved in acetonitrile (4 mL), and concentrated HCl (0.4 mL) was added dropwise at 0 °C. The reaction was stirred at ambient temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with an aqueous sodium bicarbonate solution. The resulting mixture was purified by a gradient of 20-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5μm C18 100x30mm column) to obtain the compound. ^¹H NMR (400MHz, DMSO-d6) δ 10.73 (s, 1H), 8.25 (s, 1H), 7.40–7.31 (m, 2H), 7.24–7.13 (m, 4H), 6.93–6.92 (m, 1H), 6.08–6.02 (m, 1H), 5.34–5.32 (m, 1H), 5.19 (s, 2H), 4.65–4.63 (m, 1H), 4.58 -4.45(m,2H),4.21-4.20(m,1H),4.03-3.92(m,3H),3.93-3.76(m,2H),2.69-2.65(m,2H),1. 65-1.60(m,2H),1.44-1.41(m,1H),1.32-1.18(m,7H),0.94-0.91(m,3H),0.79-0.77(m,6H). ³¹ P NMR (162MHz, DMSO-d6) δ 3.54. ¹⁹ F NMR (376MHz, DMSO-d6) δ-236.50 (t, J=47.9Hz). LCMS: MS m/z = 680.22 [M+1]; t _R = 1.37 min; LC system: Dionex Ultimate 3000 UHPLC; Column: Phenomenex Kinetex 2.6 μC18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min - 0.2 min 40% acetonitrile, 0.2 min - 1.55 min 40% - 100% acetonitrile, 1.55 min - 2.80 min 100% acetonitrile, 2.80 - 2.81 min 100% - 40% acetonitrile, 1100 μl/min.

Example 75. ((S)-(((2R,3S,4R,5S)-5-(4-benzoylaminopyrrolo[2,1-f][1,2,4]tri) (-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2- Ethyl butyl ester

Benzoyl chloride (0.018 mL, 0.154 mmol) was added dropwise to a solution of intermediate 6 (100 mg, 0.154 mmol) in anhydrous pyridine (1 mL) at room temperature under an argon atmosphere. After 30 minutes, the reaction mixture was purified by a gradient of 20-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) to obtain the compound. LCMS: MS m/z = 754.26 [M+1]; t _R = 1.88 min; LC system: Dionex Ultimate 3000 UHPLC; Column: Phenomenex Kinetex 2.6μC18 100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min–0.2 min 40% acetonitrile, 0.2 min–1.55 min 40%–100% acetonitrile, 1.55 min–2.80 min 100% acetonitrile, 2.80–2.81 min 100%–40% acetonitrile, 1100 μl/min. The obtained compound was dissolved in acetonitrile (3 mL), and concentrated HCl (0.3 mL) was added dropwise at 0 °C. The reaction was stirred at ambient temperature for 1 hour. After 1 hour, the reaction mixture was cooled in an ice bath and neutralized with an aqueous sodium bicarbonate solution. The resulting mixture was purified by a gradient of 20-100% acetonitrile aqueous solution using preparative HPLC (Phenomenex Gemini 5μm C18 100x30mm column) to give the compound. ^¹H NMR (400MHz, DMSO-d6) δ 13.15 (s, 0.3H), 11.21 (s, 0.7H), 8.33–7.99 (m, 3H), 7.62–7.48 (m, 3H), 7.38–7.34 (m, 2H), 7.23–6.97 (m, 5H), 6.09–6.03 (m, 1H), 5.33–5. 32(m,2H),5.23-5.21(m,1H),4.69-4.63(m,1H),4.57-4.49(m,2H),4.22-4.20(m ,1H),4.02-3.79(m,5H),1.46–1.40(m,1H),1.32-1.19(m,7H),0.79–0.77(m,6H). ^31P NMR (162MHz, DMSO-d6) δ3.55. LCMS: MS m/z = 714.27 [M+1]; t _R = 1.85 min; LC system: Dionex Ultimate 3000U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μC18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.6 min 2-100% acetonitrile, 1.6 min-1.8 min 100% acetonitrile, 1.80 min-1.90 min 100%-2% acetonitrile, 1.90 min-2.20 min 2% acetonitrile, 1100 μl/min.

Example 76. ((S)-(((2R,3S,4R,5S)-5-(4-(2-cyclohexylacetamyl)pyrrolo[2,1-f][1, [2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-propane 2-Ethylbutyl amino acid

Cyclohexylacetyl chloride (0.039 mL, 0.254 mmol) was added dropwise to a solution of intermediate 6 (150 mg, 0.231 mmol) in anhydrous pyridine (1 mL) at room temperature under an argon atmosphere. After 30 minutes, the reaction mixture was purified by a gradient of 20-100% acetonitrile in aqueous solution using preparative HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm column) to give the compound. LC-MS: MS m/z = 774.45 [M+1]; t _R = 1.98 min; LC system: Thermo Accela 1250 UHPLC; MS system: Dionex Ultimate 3000 UHPLC; Column: Phenomenex Kinetex 2.6 μC 18100A, 50 x 3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min–0.2 min 40% acetonitrile, 0.2 min–1.55 min 40%–100% acetonitrile, 1.55 min–2.80 min 100% acetonitrile, 2.80–2.81 min 100%–40% acetonitrile, 1100 μl/min. The obtained compound was dissolved in acetonitrile (4 mL), and concentrated HCl (0.4 mL) was added dropwise at 0 °C. The reaction was stirred at ambient temperature for 1 hour. One hour later, the reaction mixture was cooled in an ice bath and neutralized with an aqueous sodium bicarbonate solution. The resulting mixture was purified by preparative HPLC (Phenomenex Gemini 5μm C18 100x30mm column) using a gradient of 20-100% acetonitrile aqueous solution to give the compound. ^¹H NMR (400MHz, DMSO-d6 ₎ )δ10.71(s,1H),8.25(s,1H),7.37-7.34(m,2H),7.23-7.13(m,4H),6.94-6.9 3(m,1H),6.08-6.02(m,1H),5.34-5.30(m,2H),5.19(s,1H),4.68-4.48(m,3H ),4.20-4.19(m,1H),4.00-3.78(m,5H),2.55-2.53(m,2H),1.87-1.56(m,5H) ,1.45-1.38(m,1H),1.31-1.07(m,11H),1.04-0.92(m,2H),0.81-0.77(m,6H). ³¹ P NMR (162MHz, DMSO-d6) δ 3.54. ¹⁹ F NMR (376MHz, DMSO-d6) δ-236.47 (t, J=48.1Hz). LCMS: MS m/z = 734.20 [M+1]; t _R = 1.23 min; LC system: Dionex Ultimate 3000U HPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μC18 100A, 50x3.0 mm; Solvent: Acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; Gradient: 0 min-1.6 min 2-100% acetonitrile, 1.6 min-1.8 min 100% acetonitrile, 1.80 min-1.90 min 100%-2% acetonitrile, 1.90 min-2.20 min 2% acetonitrile, 1100 μl/min.

Example 77. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (Fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclopropylmethyl ester.

(tert-Butoxycarbonyl)-L-alanine cyclopropyl methyl ester. N-methylmorpholine (12.20 mL, 110.99 mmol), 4-(dimethylamino)pyridine (0.094 g, 0.74 mmol), and tripropylphosphonic anhydride (26.43 mL, 44.39 mmol, 50% ethyl acetate solution) were added to a stirred solution of boc-L-alanine (7.00 g, 37.00 mmol) and cyclopropane-methanol (3.20 g, 44.39 mmol) in 50 mL of anhydrous dichloromethane at 0 °C under argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours (conversion difficult to monitor by TLC or LCMS). The reaction mixture was washed with water (20 mL), 2 × 10% citric acid solution (20 mL), 2 × saturated _NaHCO3 aqueous solution (20 mL), and once with brine (20 mL). The organic compound was dried over _{Na₂SO₄ ,} filtered through a 3 cm silica gel layer, and the silica gel layer was washed with dichloromethane. The organic compound was concentrated under reduced pressure, co-distilled with dichloromethane, and dried under high vacuum overnight to obtain an intermediate. ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 7.25 (d, J = 7.3 Hz, 1H), 4.12–3.77 (m, 3H), 1.38 (s, 9H), 1.24 (d, J = 7.4 Hz, 3H), 1.12–0.99 (m, 1H), 0.57–0.42 (m, 2H), 0.31–0.19 (m, 2H).

((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine cyclopropyl methyl ester. (tert-butoxycarbonyl)-L-alanine cyclopropyl methyl ester (8.26 g, 33.96 mmol) was dissolved in dichloromethane (20 mL) and cooled to 0 °C. Hydrogen chloride (30.00 mL, 120.00 mmol, 4 N dioxane solution) was added. After 2 hours, the reaction was concentrated under reduced pressure. The residue was dissolved in dichloromethane (30 mL) and cooled to 0 °C. Phenyl dichlorophosphate (5.56 mL, 37.35 mmol) and triethylamine (10.36 mL, 74.70 mmol) were added successively. After 1 hour, pentafluorophenol (6.25 g, 33.96 mmol) and triethylamine (5.18 mL, 37.35 mmol) were added successively at 0 °C, and the resulting mixture was then warmed to room temperature. After 2.5 hours, the reaction mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and filtered through a 3 cm silica gel filter. The pad was washed with additional dichloromethane (200 mL). The organic matter was concentrated under reduced pressure. The residue was dissolved in warm tert-butyl methyl ether (100 mL) and then diluted with 100 mL of hexane. The reaction mixture was stirred for 2 hours, and the intermediate was separated by filtration. The intermediate was identified as a single diastereomer by NMR spectroscopy. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.38(m,2H),7.30–7.18(m,3H),6.98–6.82(m,1H),4.06–3.93(m,1H),3.87(d,J＝7 .2Hz,2H),1.30(d,J＝6.9Hz,3H),1.12–0.98(m,1H),0.53–0.43(m,2H),0.27–0.20(m,2H). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ -154.22 (d, J = 21.9Hz), -160.88 (t, J = 22.4Hz), -163.70 (t, J = 21.8Hz). ³¹ P NMR (162MHz, DMSO- _d₆ ) δ 0.47 (t, J = 12.5Hz). LCMS: MS m/z = 465.8 [M+1], t _R = 1.80 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(fluoromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine cyclopropyl methyl ester. Tetrahydrofuran (0.5 mL) was added to a mixture of intermediate 4 (10 mg, 0.03 mmol), ((perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine cyclopropyl methyl ester (15 mg, 0.03 mmol), and magnesium chloride (4 mg, 0.4 mmol) at room temperature. The mixture was stirred at room temperature for 20 minutes. N,N-diisopropylethylamine (15 μL, 0.09 mmol) was added. The reaction mixture was heated to 50 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (2 mL). The organic matter was washed with water (2 mL), dried over sodium sulfate, filtered, and concentrated. Hydrochloric acid aqueous solution (0.3 mL, 3.60 mmol, 12 M) was added dropwise to a solution of the residue in acetonitrile (2 mL) at 0 °C. After 1 hour, the reaction mixture was diluted with ethyl acetate (5 mL), and the resulting mixture was washed with saturated sodium bicarbonate aqueous solution (5 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by HPLC (Phenomenex Gemini 5 μm C18 100 x 30 mm, 5-100 acetonitrile aqueous solution) to obtain the product, which was identified as a single diastereomer by NMR spectroscopy. ^¹H NMR (400 MHz, methanol- _d4) )δ7.91(s,1H),7.40–7.33(m,2H),7.30–7.17(m,3H),7.14(d,J＝4.7Hz,1H) ,6.86(d,J＝4.7Hz,1H),5.37(d,J＝8.4Hz,1H),4.81–4.69(m,1H),4.68–4.5 7(m,2H),4.34(d,J＝5.2Hz,1H),4.29–4.16(m,2H),3.99–3.85(m,3H),1.32 (d,J＝7.1Hz,3H),1.17–1.04(m,1H),0.56–0.48(m,2H),0.29–0.22(m,2H). ³¹ P NMR (162 MHz, methanol- _d4 ) δ 3.53. ¹⁹ F NMR (376 MHz, methanol- _d4 ) δ -238.80 (t, J = 48.0 Hz). LCMS: MS m/z = 579.89 [M+1], t _R = 1.28 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18 100A, 50x4.6mm; Solvent: Acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% acetonitrile, 2.0 min-3.05 min 100% acetonitrile, 3.05 min-3.2 min 100%-2% acetonitrile, 3.2 min-3.5 min 2% ACN, 2 μl/min. HPLC: t _R = 2.62 min; Agilent 1290II; Column: Phenomenex Kinetex C18, 2.6u 110A, 100x4.6 mm; Solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; Gradient: 8.5 min gradient of 2-98% B, 1.5 mL/min.

C. Biological Examples

Example 78. Expression and purification of dengue NS5 protein

The gene encoding the full-length NS5 sequence of dengue serotype 2 (New Guinea C strain) was synthesized and cloned into the NheI/XhoI site of the pET28b vector to obtain the N-terminal HIS-tagged NS5. The sequenced plasmid was transformed into *E. coli* BL21(DE3), and the cells were grown in LB medium until the OD ₆₀₀ reached 0.5. Protein expression was induced by the addition of 0.5 mM IPTG, and the culture was incubated overnight at 16°C with continuous shaking at 250 rpm. After centrifugation, the cell pellet from 1 L of bacterial culture was resuspended in buffer A containing 50 mM HEPES (pH 7.6), 500 mM NaCl, 10% glycerol, 0.5% Triton X100, and 2 mM TCEP, supplemented with two tablets of a complete protease inhibitor mixture (Roche Diagnostics, Risch-Rotkreuz, Switzerland) without EDTA. Cells were lysed using a microfluidic apparatus and then centrifuged. The clarified supernatant was purified using a 5 mL Ni-NTA column (GE Healthcare) equilibrated in buffer B [25 mM HEPES 7.5, 500 mM NaCl, 10% Gly, 0.1% CHAPS, and 1 mM TCEP]. The column was washed with buffer B containing 40 mM imidazole, and NHIS-NS5 was eluted with a gradient of buffer B containing 500 mM imidazole. Fractions containing NHIS-NS5 were combined and further purified by size exclusion chromatography using a 120 mL Superdex 200 column (GE Healthcare) equilibrated in buffer C [25 mM HEPES 7.2, 200 mM NaCl, 10% glycerol, 0.1% CHAPS, and 2 mM DTT]. The quality and purity of the NHIS-NS5 protein were confirmed by mass spectrometry.

Alternatively, NHIS-NS5 purified by Ni-NTA was used to remove the N-terminal NHIS tag by adding thrombin protease and incubating overnight at 4°C with buffer B. The tag-detachable NS5 protein was separated from other substances on a second Ni-NTA column and further purified by size exclusion chromatography using a 120 mL Superdex 200 column (GE Healthcare) equilibrated in buffer C [25 mM HEPES 7.2, 200 mM NaCl, 10% glycerol, 0.1% CHAPS, 2 mM DTT]. The quality and purity of the NS5 protein were confirmed by mass spectrometry.

Example 79. DENV Pol IC50

In the DENV2-NS5 polymerase assay, a 244-nucleotide secondary unstructured heteropolymer RNA (sshRNA) with the sequence 5'-(UCAG)20(UCCAAG)14(UCAG)20-3' (SEQ ID NO:1) was used as a template, with 5'-CUG-3' as the primer. Six 2-fold dilutions of the compound, starting at 200 nM, and an inhibitor-free control were plated in 96-well plates. 100 nM DENV2 NS5 was pre-incubated for 5 minutes at room temperature in a reaction mixture containing 40 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM DTT, 0.2 units/μL RNasin PlusRNase inhibitor, 200 ng/μL sshRNA, 20 μM CUG, and ₂ mM MgCl2. The enzyme mixture was added to the compound dilution buffer, and the reaction was initiated by adding a mixture containing 20 μM of three natural NTPs plus _{2 μM} of an analogue (a competitive natural NTP with a base match of 1:100 α-33P-NTP). After 90 min at 30 °C, 5 μL of the reaction mixture was spotted onto DE81 anion exchange paper. The filter paper was washed three times with Na₂HPO₄ (125 mM, pH 9) for 5 min each time, rinsed with water and ethanol, then air-dried and exposed to a phosphorescent imager. The synthesized RNA was quantified using a Typhoon Trio imager and Image Quant TL software, and the reaction rate was calculated by linear regression using GraphPad Prism 5.0. The _IC₅₀ value was calculated in Prism by nonlinear regression analysis using the following dose-response (variable slope) equation (four-parameter logistic equation): Y = Bottom + (Top - Bottom) / (1 + 10^(( _LogIC₅₀ - X) * HillSlope)).

Example 80. Preparation of RSV RNP

The RSV ribonucleoprotein (RNP) complex was prepared according to a modified method from Mason et al. (1). HEp-2 cells were seeded at a density of 7.1 × ^10⁴ cells/ ^cm² in MEM + 10% fetal bovine serum (FBS) and allowed to adhere overnight at 37°C (5% _CO₂ ). After adhesion, the cells were infected with RSV A2 (MOI = 5) in 35 mL MEM + 2% FBS. Twenty hours after infection, the medium was replaced with MEM + 2% FBS supplemented with 2 μg/mL actinomycin D and the cells were restored to 37°C for one hour. The cells were then washed once with PBS and treated with 35 mL PBS + 250 μg/mL lysophosphatidylcholine for one minute, after which all liquid was aspirated. Cells were harvested by scraping them into 1.2 mL of buffer A [50 mM Tris acetate (pH 8.0), 100 mM potassium acetate, 1 mM DTT, and 2 μg/mL actinomycin D] and lysed by repeatedly passing them through an 18-gauge needle (10 times). The cell lysates were placed on ice for 10 minutes and then centrifuged at 2400 g for 10 minutes at 4 °C. The supernatant (S1) was removed, and the pellet (P1) was lysed in 600 μL of buffer B [10 mM Tris acetate (pH 8.0), 10 mM potassium acetate, and 1.5 mM _MgCl2 ] supplemented with 1% Triton X-100 by repeatedly passing it through an 18-gauge needle (10 times). The resuspended pellet was placed on ice for 10 minutes and then centrifuged at 2400 g for 10 minutes at 4 °C. Remove the supernatant (S2), and lyse the precipitate (P2) in 600 μL of buffer B supplemented with 0.5% deoxycholate and 0.1% Tween 40. Place the resuspended precipitate on ice for 10 minutes, then centrifuge at 2400 g for 10 minutes at 4 °C. Collect the supernatant fraction (S3) containing the enriched RSV RNP complex, and determine the protein concentration by UV absorbance at 280 nm. Store the aliquots of RSV RNP S3 fraction at -80 °C.

Example 81.RSV RNP determination

Transcription reactions were performed in 30 μL of reaction buffer [50 mM TRIS-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, _4.5 mM MgCl₂, 3 mM DTT, 2 mM ethylene glycol-bis(2-aminoethyl ether)-tetraacetic acid (EGTA), 50 μg/mL BSA, 2.5 URaNasin (Promega), ATP, GTP, UTP, CTP, and 1.5 μCi [α-32P]NTP (3000 Ci/mmol)] containing 25 μg of crude RSV RNP complex. Radiolabeled nucleotides were selected for the transcription assay to match the nucleotide analogs evaluated for inhibiting RSV RNP transcription. Cold competitive NTPs were added to a final concentration of half their Km (ATP = 20 μM, GTP = 12.5 μM, UTP = 6 μM, CTP = 2 μM). The remaining three nucleotides were added to a final concentration of 100 μM.

To determine whether the nucleotide analogues inhibited RSV RNP transcription, compounds were added in 5-fold increments using a 6-step serial dilution. After incubation at 30°C for 90 min, the RNP reaction was terminated with 350 μL of Qiagen RLT lysis buffer, and RNA was purified using the Qiagen RNeasy 96 kit. The purified RNA was denatured in RNA loading buffer (Sigma) at 65°C for 10 min and run on a 1.2% agarose/MOPS gel containing 2M formaldehyde. The agarose gel was dried and exposed to a Storm phosphorescence imaging screen, and developed using a Storm phosphorescence imaging system (GE Healthcare). The concentration of the compound that reduced the total radiolabeled transcript by 50% ( _IC50 ) was calculated by two repeated nonlinear regression analyses.

Example 82. DENV-2moDC EC50

Human monocyte-derived dendritic cells (moDCs) were derived from CD14+ monocytes (AllCells) cultured in human MoDC differentiation medium (Miltenyi Biotec) containing GM-CSF and IL-4. On day 7, moDCs were collected by mechanical disruption, washed, and resuspended in serum-free RPMI. The moDCs were infected in serum-free RPMI with Vero-derived dengue 2 New Guinea strain (NGC) at an MOI of 0.1 for two hours with gentle agitation at 37°C. The cells were washed and resuspended in RPMI (Gibco, supplemented with sodium pyruvate, NEAA, and penicillin-streptomycin) containing 10% serum. Cells were seeded in triplicate in 96-well plates, with compound dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. At 48 hours, the cells were washed with 1×PBS, and all supernatant was removed. Total RNA was extracted using RNEasy 96 plates (Qiagen), and first-strand cDNA was generated using XLT cDNA 5x Supermix (QuantaBio). This cDNA was used as a template in Taqman qPCR double-stranded reactions specific to DENV2 virus and GAPDH gene expression. _EC50 values were determined using Prism Graphpad software, and wells were normalized to positive and negative controls without compounds.

Example 83.moDC CC50

Human monocyte-derived dendritic cells (moDCs) were derived from CD14+ monocytes (AllCells) cultured in human MoDC differentiation medium (Miltenyi Biotec) containing GM-CSF and IL-4. On day 7, moDCs were harvested by mechanical disruption, washed, and cultured in triplicate at 1x10^5–5x10^4 cells/well in 96-well plates, with compound dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. After 48 hours, CellTiter Glo (Promega) was added and incubated at room temperature for 10 minutes, followed by readings on a photometer. % viability curves were calculated relative to wells without compound and cell-free controls. CC ₅₀ values were determined using Prism Graphpad software.

Example 84. DENV-2Huh-7 EC50

Huh7 (human hepatocellular carcinoma 7) cells were kept in complete DMEM medium containing 10% FCS. On the day of assay, cells were trypsinized (0.1% trypsin-EDTA), washed, and infected with dengue serotype 2 New Guinea C (NGC) in serum-free DMEM at an MOI of 0.1 for 2 hours with gentle agitation at 37°C. After 2 hours, cells were washed with serum-free medium and resuspended in DMEM (Gibco, supplemented with sodium pyruvate, NEAA, and penicillin-streptomycin) containing 10% FCS. Cells were seeded in triplicate in 96-well plates, and the compound was dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. At 48 hours, cells were washed with 1×PBS, and all supernatant was removed. Total RNA was extracted using RNEasy 96 plates (Qiagen), and first-strand cDNA was generated using XLT cDNA 5x Supermix (QuantaBio). This cDNA was used as a template in Taqman qPCR double-stranded reactions specific to DENV2 virus and GAPDH gene expression. _EC50 values were determined using Prism Graphpad software, and wells were normalized to positive and negative controls without compounds.

Example 85. Huh-7 CC50

Human hepatocellular carcinoma 7 (Huh7) cells were held in complete DMEM containing 10% FCS. On the day of assay, cells were trypsinized with 0.1% trypsin-EDTA, washed, and cultured in triplicate at 1–2 x 10^4 cells/well in 96-well plates, with the compound dispensed in fractionated doses (Hewlett-Packard D300 digital dispenser). All wells were normalized to 0.25% DMSO. After 48 hours, CellTiter Glo (Promega) was added and incubated at room temperature for 10 minutes, followed by readings on a spectrophotometer. % viability curves were calculated relative to wells without the compound and cell-free controls. CC ₅₀ values were determined using Prism Graphpad software.

Example 86. RSV HEp-2 EC50

Antiviral activity against RSV was determined in HEp-2 cells using an infectious cytopathic cell protection assay. In this assay, compounds that inhibit viral infection and/or replication exert a cytoprotective effect against virus-induced cell killing, which can be quantified using a cell viability assay. The technique used here is a novel adaptation of the method described in the published literature (Chapman et al., Antimicrob Agents Chemother. 2007, 51(9):3346-53).

HEp-2 cells were obtained from ATCC (Manassas, VI) and maintained in MEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were passaged twice weekly and maintained in the subconfluence phase. A commercial stock solution of RSV strain A2 (Advanced Biotechnologies, Columbia, MD) was titrated prior to compound assays to determine the appropriate dilution of the viral stock solution for producing the desired cytopathic effects in HEp-2 cells.

For the antiviral assay, HEp-2 cells were grown in large cell culture flasks until nearly confluent, but not completely confluent. The test compounds were pre-diluted in DMSO in 384-well compound dilution plates at a standardized dose of 8 or 40 samples per plate. Serial dilution increments of 3-fold were prepared for each test compound in the plates, and test samples were transferred to the 384-well cell culture assay plates at 100 nL per well using an acoustic transfer device (Echo, Labcyte). Each compound dilution was transferred to dry assay plates as a single sample or in quadruplicates, and these dry assay plates were stored until ready for assay. Positive and negative controls were arranged in vertical blocks (one column) opposite each other at both ends of the plate.

Subsequently, an infectious mixture was prepared by titrating a pre-determined appropriate dilution of the viral stock solution with cells at a density of 50,000/mL, and added to the test plates containing the compound at 20 μL/well using an automated system (uFlow, Biotek). Each plate included a negative control and a positive control (16 replicates each) to generate 0% and 100% viral inhibition standards, respectively. After RSV infection, the test plates were incubated in a 37°C cell culture incubator for 4 days. After incubation, the cell viability reagent CellTiterGlo (Promega, Madison, WI) was added to the test plates, the plates were briefly incubated, and luminescence readings were measured in all test plates (Envision, Perkin Elmer). The percentage of inhibition of RSV-induced cytopathic effects was determined by the level of residual cell viability. These values were calculated for each test concentration relative to the 0% and 100% inhibition controls, and the _EC50 value of each compound was determined by nonlinear regression as the concentration that inhibited RSV-induced cytopathic effects by 50%. Various potent anti-RSV tool compounds were used as positive controls for antiviral activity.

Example 87. HEp-2 CC50

The cytotoxicity of the test compounds was determined in uninfected HEp-2 cells in parallel with antiviral activity using a cell viability assay in a manner similar to that described previously for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008, 52(2):655-65). The same protocol used to determine antiviral activity was used to measure the cytotoxicity of the compounds, except that the cells were not infected with RSV. Instead, a mixture of uninfected cells of the same density was added at 20 μL/well to plates containing pre-diluted compounds (also 100 nL/sample). The assay plates were then incubated for 4 days, followed by cell viability testing with the addition of the same CellTiter Glo reagent and measurement of luminescent readings. Untreated cells and cells treated with 2 μM puromycin (Sigma, St. Louis, MO) served as 100% and 0% cell viability controls, respectively. The percentage of cell viability was calculated relative to the 0% and 100% controls for each test compound concentration, and the CC ₅₀ value was determined by nonlinear regression as the compound concentration that reduced cell viability by 50%.

Example 88. RSV NHBE EC50

Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Walkersville, MD, Cat#CC-2540) and cultured in bronchial epithelial growth medium (BEGM) (Lonza, Walkersville, MD, Cat#CC-3170). Cells were passaged 1-2 times per week to maintain <80% confluence. NHBE cells were discarded after 6 passages.

To perform the RSV A2 antiviral assay, NHBE cells were seeded in 96-well plates at a density of 7,500 cells/well from BEGM and allowed to adhere overnight at 37°C. After adhesion, 100 μL of cell culture medium was removed, and a 3-fold serially diluted compound was added using a Hewlett-Packard D300 digital dispenser. The final concentration of DMSO was normalized to 0.05%. Following the addition of the compound, NHBE cells were infected by adding 100 μL of RSV A2 at a titer of 1 × ^10⁴.⁵ tissue culture infection dose/mL from BEGM, and then incubated at 37°C for 4 days. NHBE cells were then equilibrated to 25°C, and cell viability was determined by removing 100 μL of culture medium and adding 100 μL of Cell-Titer Glo viability reagent. The mixture was incubated at 25°C for 10 minutes, and the luminescence signal was quantified on an Envision luminescent plate reader.

Table 3. Activity Data

Table 4. Activity Data

Comparative Example 1. ((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2- (chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester

((3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-(chloromethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-yl)methanol. CE1.1 (intermediate 14j from WO 2015/069939; 200 mg, 0.363 mmol) was dissolved in 5 mL of anhydrous pyridine. Trifluoromethanesulfonyl chloride (58 μL, 0.545 mmol) was added in one go and the mixture was stirred for 60 min. More trifluoromethanesulfonyl chloride (58 μL, 0.545 mmol) was added and the mixture was stirred for 30 min. Trifluoromethanesulfonyl chloride (100 μL) was added and the mixture was stirred for 30 min. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in 5 mL of anhydrous DMF, and lithium chloride (308 mg, 7.26 mmol) was added in one go. The reaction was stirred for 16 hours.

The reaction mixture was diluted with EtOAc (15 mL) and washed with brine (3 × 10 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (12 g _SiO2 Combiflash HP Gold column, 0-20% ethyl acetate/hexane). The fractions were combined and concentrated under reduced pressure. The residue was dissolved in dioxane (8 mL) and water (2 mL) and stirred at 120 °C for 2 hours. The reaction mixture was then concentrated under reduced pressure.

The residue was dissolved in THF (5 mL). Tetrabutylammonium fluoride trihydrate (97 mg, 0.307 mmol) was added, and the reaction was stirred for 1 hour. The reaction mixture was diluted with EtOAc (15 mL) and washed with brine (5 × 5 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (4 g _SiO2 Combiflash HP Gold column, 0-100% ethyl acetate/hexane). The fractions were combined and concentrated under reduced pressure to give intermediate CE1.2. ¹ H NMR (400MHz, chloroform-d) δ7.87(s,1H),6.65(d,J＝4.5Hz,1H),6.54(d,J＝4.5Hz,1H),6.12(bs,2H),5.33(dd,J＝6 .9,5.9Hz,1H),5.14(d,J＝6.9Hz,1H),5.05(d,J＝5.9Hz,1H),3.98–3.71(m,4H),1.63(s,3H),1.36(s,3H). LCMS: MS m/z = 355.3 [M+1], 353.4 [M-1], t _R = 1.04 min; LC system: Thermo Dionex Ultimate 3000U HPLC; column: Phenomenex Kinetex 2.6μC18100A, 50x3mm; solvent: A: water containing 0.1% acetic acid, B: acetonitrile containing 0.1% acetic acid; gradient: 0 min-0.3 min 5% B, 0.3 min-1.5 min 5-100% B, 1.5 min-2 min 100% B, 2 min-2.2 min 100-5% B, 2 mL/min. HPLC: t _R = 2.15 min; HPLC system: Agilent 1100 series; column: Phenomenex Gemini 5μC18 110A, 50 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B within 5 min, 2 mL/min.

((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine 2-ethylbutyl ester. CE1.2 (47 mg, 0.132 mmol) and CE1.3 (72 mg, 0.159 mmol) were mixed and dissolved in 2 mL of anhydrous THF. Magnesium chloride (38 mg, 0.396 mmol) was added in a single addition. DIPEA (57 μL, 0.33 mmol) was added, and the reaction was stirred at 40 °C for 16 hours. The reactants were diluted with EtOAc (15 mL) and washed with water (5 × 10 mL), followed by washing with brine (5 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dissolved in MeCN (5 mL) and stirred in an ice bath. Concentrated hydrochloric acid aqueous solution (250 μL) was added dropwise. The reaction was stirred in an ice bath for 2 hours. 1 M triethylammonium bicarbonate solution was added to the reaction, resulting in a pH of 8, and the reaction was then concentrated under reduced pressure. The crude residue was purified by preparative HPLC (5-100% MeCN/water) under neutral conditions. The fractions were combined and concentrated under reduced pressure. The residue was dissolved in MeCN and water and lyophilized to give the product. ^¹H NMR (400 MHz, methanol- _d⁴ ) δ 7.77 (m, ¹H), 7.39–7.09 (m, 5H), 6.84 (m, ¹H), 6.74 (m, ¹H), 5.36 (m, ¹H), 4.72 (m, ¹H), 4.41–4.23 (m, 3H), 4.07–3.84 (m, 5H), 1.53–1.40 (m, ¹H), 1.38–1.25 (m, 7H), 0.89–0.81 (m, 6H). ^³¹P NMR (162 MHz, methanol- _d⁴ ) δ 3.46, 3.59. LCMS: MS m/z = 626.2 [M+1], 624.6 [M-1], t _R = 1.30 min; LC system: Thermo Dionex Ultimate 3000U HPLC; Column: Phenomenex Kinetex 2.6μC18 100A, 50x3mm; Solvent: A: water containing 0.1% acetic acid, B: acetonitrile containing 0.1% acetic acid; Gradient: 0 min-0.3 min 5% B, 0.3 min-1.5 min 5-100% B, 1.5 min-2 min 100% B, 2 min-2.2 min 100-5% B, 2 mL/min. HPLC: t _{_R} = 3.14 min; HPLC system: Agilent 1100 series; column: Phenomenex Gemini 5μC18 110A, 50 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 2 mL/min for the first 5 min. HPLC: t_{_R} = 5.247, 5.327 min; HPLC system: Agilent 1290II; column: Phenomenex Kinetex C18, 2.6 μm 110A, 100 x 4.6 mm; solvent: A: water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA; gradient: 2-98% B, 1.5 mL/min for the first 8.5 min.

Comparative Example 2. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-2-(chloro) Methyl methyl (methyl)-3,4-dihydroxytetrahydrofuran-2-yl)tetrahydrotriphosphate

See compound TP13 (Example 33) in WO 2015/069939.

Although the foregoing invention has been described in considerable detail by way of illustration and examples for clarity of understanding, those skilled in the art will understand that certain variations and modifications may be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety as if it were incorporated individually. In the event of any conflict between this application and the references provided herein, this application shall prevail.

Claims (9)

1. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from the group consisting of: 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is: 3. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is: 4. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is: 5. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is: 6. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is: 7. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is: 8. A pharmaceutical preparation comprising a pharmaceutically effective amount of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. 9. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of a medicament for treating human flaviviridae virus infection, wherein the flaviviridae virus infection is dengue virus infection.