WO2012048421A1

WO2012048421A1 - Hepatitis c inhibitor compounds

Info

Publication number: WO2012048421A1
Application number: PCT/CA2011/050636
Authority: WO
Inventors: Carl Thibeault; Paul J. Edwards; Cyrille Kuhn; Benoît MOREAU; Maude Poirier; Simon Surprenant
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 2010-10-14
Filing date: 2011-10-11
Publication date: 2012-04-19
Anticipated expiration: 2013-04-14

Abstract

Compounds of Formula (I) and (II) wherein R¹, R², R³, R⁶, A and A' are defined herein. The compounds are useful as inhibitors of the function of NS5A protein encoded by HCV for the treatment of hepatitis C viral infection.

Description

HEPATITIS C INHIBITOR COMPOUNDS

RELATED APPLICATIONS

This application claims benefit of U.S. Serial No. 61/393256, filed October 14, 2010, and U.S. Serial No. 61/449939 filed March 7, 2011 , both of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compounds, and their use as inhibitors of the function of NS5A protein encoded by HCV, pharmaceutical compositions containing such compounds and methods for using these compounds in the treatment of HCV infection.

BACKGROUND OF THE INVENTION

It is estimated that at least 170 million persons worldwide are infected with the hepatitis C virus (HCV). Acute HCV infection progresses to chronic infection in a high number of cases, and, in some infected individuals, chronic infection leads to serious liver diseases such as cirrhosis and hepatocellular carcinoma.

HCV replicates to very high levels and the HCV polymerase is error-prone resulting in a wide variety of new sequence variants (Science 1998, 282, 103-107). Some new sequence variants confer resistance to drug candidates currently undergoing clinical trials. The emergence of such resistance mutations is one cause of treatment failure in HCV antiviral trials (New England Journal of Medicine 2009, 360, 1827-1838, New England Journal of Medicine 2009, 360, 1839-1850, New England Journal of Medicine 2010, 362, 1292-1303, and The Lancet 2010, 376, 705-716).

Activity of HCV inhibitors, including NS5A inhibitors, is most effectively measured in vitro using the subgenomic replicon system, in which inhibition of the physiologically relevant HCV replication complex can be directly measured (Journal of Viral Hepatitis, 2007, 14 (Suppl. 1) 64-67). Inhibition in this system has translated into clinical efficacy as shown for many clinical candidates including protease inhibitors (Antimicrobial Agents and

Chemotherapy 2009, 53(4) 1377-85, Antimicrobial Agents and Chemotherapy 2008, 52(12) 4432-41 , Antimicrobial Agents and Chemotherapy 2010, 54(1) 305-31 1 , Antimicrobial Agents and Chemotherapy 2010 published online 7 Sep 2010 doi: 10.1 128/AAC.00787- 10) and also for an NS5A inhibitor (Nature 2010, 465, 96-100). Furthermore, it is possible to select for resistance using the replicon system, and there is good correlation between mutations selected in the replicon and those observed in HCV patients participating in clinical trials (Journal of Viral Hepatitis 2009, 16, 377-387, Antimicrobial Agents for Chemotherapy 2008, 52, 1 101-1 1 10, New England Journal of Medicine 2009, 360, 1827- 1838, Nature 2010, 465, 96-100).

The impact of emergence of any one resistance mutant on the outcome of therapy is determined not only by the effect of the particular resistance mutant on drug potency, but also by the fitness of the resulting virus variant. A resistance mutation which results in a virus with poor fitness will be more difficult to select under drug pressure, even if it results in a large decrease in potency for the drug. As mentioned above, resistance to HCV drugs is typically studied preclinically using the subgenomic replicon system. Techniques have been developed to evaluate the fitness or replication capacity of replicons bearing different resistance mutations, and thus the probable importance of these mutations in the clinic (Antimicrobial Agents and Chemotherapy 2008, 52, 1101-1 1 10). Because emergence of relatively fit resistance mutations can lead to treatment failure, there is a need for new inhibitors against each viral target which have improved activity against the resistant variants which emerge on treatment with each class of inhibitor. Potency of the compounds of the invention has been evaluated against genotype 1 a resistant mutants formed by a substitution at amino acid 30 of HCV NS5A Gin to Glu (Q30E) or a substitution at amino acid 31 of HCV NS5A Leu to Val (L31 V). Q30E and L31 V, are observed in in vitro experiments and are used herein as representative of clinically relevant resistant mutants (Antimicrobial Agents and Chemotherapy 2010, 54, 3641-3650; Nature 2010, 465, 96-100; Journal of Virology 2010, 84, 482-491 ; Richard Colonno, "Discovery and characterization of PPI-461 , a potent and selective HCV NS5A inhibitor with broad-spectrum coverage of all HCV genotypes", 3rd Annual HCV Drug Discovery Conference, April 2010, San Diego, CA).

WO 2008/021936, WO 2008/102318, WO 2008/021928, WO 2008/144380, WO

2008/021927, WO 2009/020825, WO 2009/020828, WO 2009/102325, WO 2009/102568, WO 2009/102633 WO 2009/102694, US 2010-0249190, WO 2010/017401 , WO 2010/096302, WO 2010/062821 , WO 2010/065674, WO 2010/065668, WO 2010/065681 , WO 2010/096777, WO 2010/1 1 1673, WO 2010/1 1 1534, WO 2010/091413, WO

2010/099527, WO 2010/096462, WO 2010/1 1 1483 disclose NS5A inhibitors that are described as being useful to treat HCV infection.

SUMMARY OF THE INVENTION

The present invention provides a novel series of compounds having inhibitory activity against HCV replication. The compounds of the invention may be used to inhibit the function of the NS5A protein encoded by HCV and may be used to reduce HCV replication. Compounds of the invention also maintain potent activity against clinically relevant resistance mutations as represented by genotype 1 a Q30E and L31V resistance mutations.

Further objects of this invention arise for one skilled in the art from the following description and the examples.

The invention provides a

wherein:

ring A and A' are each independently optionally substituted one time with halo;

R¹ is halo and R² is hydrogen; or

R¹ is hydrogen and R² is halo;

3 is selected from the group consisting of:

R⁴ is selected from (C _₆)alkyl optionally substituted one time with -0-(C-|.₆)alkyl or

-0-(Ci_₆)haloalkyl;

R⁵ is either absent or selected from the group consisting of -0-(C-|.₆)alkyl and

-S-(C_{1 -6})alkyl;

R⁷ is selected from (C ₆)alkyl optionally substituted one time with -0-(C-|.₆)alkyl

-O-CC^haloalkyl;

R⁸ is either absent or selected from the group consisting of

and

-S-(C_1-6)alkyl.

The invention provid

(II) wherein:

ring A and A' are each independently optionally substituted one time with halo;

R⁴ is selected from (C-|.₆)alkyl optionally substituted one time with -0-(C-|.₆)alkyl or -0-(C-,_

₆)haloalkyl;

R⁵ is -S-(C_1-6)alkyl;

6 is selected from the group consisting of:

R⁷ is selected from (Ci_₆)alkyl optionally substituted one time with -0-(Ci_₆)alkyl or

-0-(C-,.₆)haloalkyl;

R⁸ is either absent or selected from the group consisting of -0-(C-|.₆)alkyl and-S-(C-|.₆)alkyl. Another aspect of this invention provides a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, as a medicament.

Included within the scope of this invention is a pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, in admixture with at least one pharmaceutically acceptable carrier medium or auxiliary agent.

According to a further aspect of this invention the pharmaceutical composition according to this invention further comprises a therapeutically effective amount of at least one other antiviral agent.

The invention also provides the use of a pharmaceutical composition as described hereinabove for the treatment of a hepatitis C viral infection in a human being having or at risk of having the infection.

Another important aspect of the invention involves a method of treating or preventing a hepatitis C viral infection in a human being by administering to the human being an anti- hepatitis C virally effective amount of a compound of Formula (I) or (II), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, alone or in combination with at least one other antiviral agent, administered together or separately.

Also within the scope of this invention is the use of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, as described herein, for the manufacture of a medicament for the treatment or prevention of hepatitis C viral infection in a human being.

An additional aspect of this invention refers to an article of manufacture comprising a composition effective to treat a hepatitis C viral infection; and packaging material comprising a label which indicates that the composition can be used to treat infection by the hepatitis C virus; wherein the composition comprises a compound of Formula (I) or (II), according to this invention or a pharmaceutically acceptable salt thereof in admixture with at least one pharmaceutically acceptable carrier medium or auxiliary agent. Still another aspect of this invention relates to a method of inhibiting the replication of hepatitis C virus comprising exposing the virus to an effective amount of the compound of Formula (I) or (II), or a salt thereof, under conditions where replication of hepatitis C virus is inhibited.

Further included in the scope of the invention is the use of a compound of Formula (I) or (II), or a salt thereof, to inhibit the replication of hepatitis C virus.

In another aspect the invention provides novel intermediates useful in the production of compounds of Formula (I) or (II). In a preferred embodiment the invention provides one or more intermediates selected from 7a1 , 7b1 , 7e1 , 7h6, 7i2, 7j2, 8e1 , 8f1 , 9a1 , 9c1 , 9e1 , 9f1 , 9g1, 9h1, 9i1, 9j1, 911, 12a1, 12b1, 13c1, 14a1, 14a2, 15a1, 18a2, 18b1, 18c1, 18d1, 18e1, 19a1, 19a2, 20a1, 20a2 and 20a3.

In another aspect the invention provides novel intermediates useful in the production of compounds of Formula (I) or (II). In a preferred embodiment the invention provides one or more intermediates selected from 4a9, 7a1 , 7b1 , 7e1 , 7h6, 7g2, 7i2, 7j2, 8e1 , 8f1 , 9a1 , 9c1, 9e1, 9f1, 9g1, 9h1, 9i1, 9j1, 911, 9m1, 9n1, 10c1, 12a1, 12b1, 13c1, 14a1, 14a2, 15a1, 18a2, 18b1, 18c1, 18d1, 18e1, 19a1, 19a2, 20a1, 20a2 and 20a3, 21a1, 21a2, 21a3, 21a4 and 21a5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS DEFINITIONS

Terms not specifically defined herein should be given the meanings that would be given to them by one of skill in the art in light of the disclosure and the context. As used in the specification, however, unless specified to the contrary, the following terms have the meaning indicated and the following conventions are adhered to.

In the groups, radicals, or moieties defined below, the number of carbon atoms is often specified preceding the group, for example, C-|.₆-alkyl means an alkyl group or radical having 1 to 6 carbon atoms. In general, for groups comprising two or more subgroups, the first named subgroup is the radical attachment point, for example, the substituent "-C-i_₃- alkyl-aryl" means an aryl group which is bound to a -C-|.₃-alkyl group, wherein the -C_1-3- alkyl-group is bound to the core. In the previous example of

substituents may be attached to either the Ci_₃-alkyl or aryl portion thereof or both, unless specified otherwise.

In case a compound of the present invention or an intermediate used in the synthesis of a compound of the present invention is depicted in the form of a chemical name and as a formula, the formula shall prevail in case of any discrepancy between the name and formula.

The term "Ci__n-alkyl", wherein n is an integer from 2 to n, either alone or in combination with another radical denotes an acyclic, saturated, branched or linear hydrocarbon radical with 1 to n C atoms. For example the term C-|.₄-alkyl embraces the radicals H₃C-, H₃C-CH₂-, H₃C-CH₂-CH₂-, H₃C-CH(CH₃)-, H₃C-CH₂-CH₂-CH₂-, H₃C-CH₂"CH(CH₃)-, H₃C-CH(CH₃)- CH2-, (H₃C)₃C- and H₃C-C(CH₃)₂-.

The term "halo" as used herein is intended to mean a halogen substituent selected from fluoro, chloro or bromo.

Many of the terms given above may be used repeatedly in the definition of a formula or group and in each case have one of the meanings given above, independently of one another.

Unless specifically indicated, throughout the specification and the appended claims, a given chemical formula or name shall encompass tautomers and all stereo, optical and geometrical isomers (e.g. enantiomers, diastereomers, E/Z isomers, atropisomers) and racemates thereof as well as mixtures in different proportions of the separate enantiomers, mixtures of diastereomers, or mixtures of any of the foregoing forms where such isomers and enantiomers exist, as well as salts, including pharmaceutically acceptable salts thereof and solvates thereof such as for instance hydrates including solvates of the free compounds or solvates of a salt of the compound.

One skilled in the art would know how to separate, enrich, or selectively prepare the enantiomers of the compounds of the present invention. Preparation of pure stereoisomers, e.g. enantiomers and diastereomers, or mixtures of desired enantiomeric excess (ee) or enantiomeric purity, are accomplished by one or more of the many methods of (a) separation or resolution of enantiomers, or (b) enantioselective synthesis known to those of skill in the art, or a combination thereof. These resolution methods generally rely on chiral recognition and include but not limited to chromatography using chiral stationary phases, enantioselective host-guest complexation, resolution or synthesis using chiral auxiliaries, enantioselective synthesis, enzymatic and nonenzymatic kinetic resolution, or spontaneous enantioselective crystallization. Such methods are disclosed generally in Chiral Separation Techniques: A Practical Approach (2nd Ed.), G. Subramanian (ed.), Wiley-VCH, 2000; T.E. Beesley and R.P.W. Scott, Chiral Chromatography, John Wiley & Sons, 1999; and Satinder Ahuja, Chiral Separations by Chromatography, Am. Chem. Soc, 2000. Furthermore, there are equally well-known methods for the quantitation of enantiomeric excess or purity, including but not limited to GC, HPLC, CE, or NMR, and assignment of absolute configuration and conformation, including but not limited to CD, ORD, X-ray

crystallography, or NMR.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.

Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. For example, such salts include acetates, ascorbates, benzenesulfonates, benzoates, besylates, bicarbonates, bitartrates, bromides/hydrobromides, Ca-edetates/edetates, camsylates, carbonates,

chlorides/hydrochlorides, citrates, edisylates, ethane disulfonates, estolates esylates, fumarates, gluceptates, gluconates, glutamates, glycolates, glycollylarsnilates,

hexylresorcinates, hydrabamines, hydroxy ma leates, hydroxynaphthoates, iodides, isothionates, lactates, lactobionates, malates, maleates, mandelates, methanesulfonates, mesylates, methylbromides, methylnitrates, methylsulfates, mucates, napsylates, nitrates, oxalates, pamoates, pantothenates, phenylacetates, phosphates/diphosphates, polygalacturonates, propionates, salicylates, stearates subacetates, succinates, sulfamides, sulfates, tannates, tartrates, teoclates, toluenesulfonates, triethiodides, ammonium, benzathines, chloroprocaines, cholines, diethanolamines, ethylenediamines, meglumines and procaines. Further pharmaceutically acceptable salts can be formed with cations from metals like aluminium, calcium, lithium, magnesium, potassium, sodium, zinc and the like, (also see Pharmaceutical salts, Birge, S.M. et al., J. Pharm. Sci., (1977), 66, 1-19).

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.

Salts of other acids than those mentioned above which for example are useful for purifying or isolating the compounds of the present invention (e.g. trifluoro acetate salts) also comprise a part of the invention.

The term "other anti-HCV agent" as used herein means those agents that are effective for diminishing or preventing the progression of hepatitis C related symptoms of disease. Such agents can be selected from: immunomodulatory agents, inhibitors of HCV NS3 protease, inhibitors of the function of NS5A protein encoded by HCV, inhibitors of HCV polymerase or inhibitors of another target in the HCV life cycle. Examples of anti-HCV agents include, a- (alpha), β- (beta), δ- (delta), γ- (gamma), ω- (omega) or x- (tau) interferon, pegylated a- interferon, ribavirin, amantadine, taribavirin (Viramidine), Nitazoxannide, ABT-267 and BMS-791325.

The term "immunomodulatory agent" as used herein includes those agents (compounds or biologicals) that are effective to enhance or potentiate the immune system response in a human being. Immunomodulatory agents include, but are not limited to, inosine

monophosphate dehydrogenase inhibitors, class I interferons, class II interferons, consensus interferons, asialo-interferons, pegylated interferons and conjugated interferons, including but not limited to interferons conjugated with other proteins including but not limited to human albumin. Class I interferons are a group of interferons that all bind to receptor type I, including both naturally and synthetically produced class I interferons, while class II interferons all bind to receptor type II. Examples of class I interferons include, but are not limited to, α-, β-, δ-, ω-, and τ-interferons, while examples of class II interferons include, but are not limited to, γ-interferons.

The term "inhibitor of HCV NS5A" as used herein means an agent (compound or biological) that is effective to inhibit the function of HCV NS5A in a human being. Inhibitors of HCV NS5A include, for example, BMS-824393, BMS-790052, ITMN-10050, ITMN-9916, EDP- 239, AZD7295, PPI-1301 and PPI-461.

The term "inhibitor of HCV NS3 protease" as used herein means an agent (compound or biological) that is effective to inhibit the function of HCV NS3 protease in a human being. Inhibitors of HCV NS3 protease include, for example, the candidates ABT-450, ACH-1625, BMS-650032, PHX1766, VX-813, telaprevir (VX-950), AVL-181 , boceprevir (SCH-503034), narlaprevir (SCH-900518), danoprevir (ITMN-191), TMC 435350, vaniprevir (MK7009), MK 5172, Bl 201335, IDX320, GS 9256 and VX-985.

The term "inhibitor of HCV polymerase" as used herein means an agent (compound or biological) that is effective to inhibit the function of an HCV polymerase in a human being. This includes, for example, inhibitors of HCV NS5B polymerase. Inhibitors of HCV polymerase include, for example, the candidates RG-7128, tegobuvir (GS9190), IDX184, IDX375, PSI-7977, MK-3281 , filibuvir (PF868554), VX-222, VCH-759, ANA598, JTK-853, INX189, PSI-938, RG-7348, ABT-333, ABT-072 and Bl 207127.

The term "inhibitor of another target in the HCV life cycle" as used herein means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HCV in a human being other than by inhibiting the function of the HCV NS3 protease. This includes agents that interfere with either host or HCV viral targets necessary for the HCV life cycle or agents which specifically inhibit in HCV cell culture assays through an undefined or incompletely defined mechanism. Inhibitors of another target in the HCV life cycle include, for example, agents that inhibit viral targets such as Core, E1 , E2, p7, NS2/3 protease, NS3 helicase, NS4A, NS5B polymerase, NS5A and internal ribosome entry site (IRES), or host targets such as cyclophilin B, phosphatidylinositol 4-kinase Ilia, CD81 , SR- B1 , Claudin 1 , VAP-A, VAP-B. Specific examples of inhibitors of another target in the HCV life cycle include SCY-635, ITX5061 , NOV-205, BIT-225, NA808, MK-1220, PF-4878691 , MX-3253, GS 9450, ISIS-14803, NIM-81 1 , and DEBIO-025.

The term "HIV inhibitor" as used herein means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HIV in a human being. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HIV in a human being. HIV inhibitors include, for example, nucleoside inhibitors, non-nucleoside inhibitors, protease inhibitors, fusion inhibitors and integrase inhibitors.

The term "HAV inhibitor" as used herein means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HAV in a human being. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HAV in a human being. HAV inhibitors include Hepatitis A vaccines, for example, Havrix^® (GlaxoSmithKline), VAQTA^® (Merck) and Avaxim^® (Aventis Pasteur).

The term "HBV inhibitor" as used herein means an agent (compound or biological) that is effective to inhibit the formation and/or replication of HBV in a human being. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of HBV in a human being. HBV inhibitors include, for example, agents that inhibit HBV viral DNA polymerase or HBV vaccines. Specific examples of HBV inhibitors include Lamivudine (Epivir-HBV^®), Adefovir Dipivoxil, Entecavir, FTC (Coviracil^®), DAPD (DXG), L-FMAU (Clevudine^®), AM365 (Amrad), Ldt (Telbivudine), monoval-LdC (Valtorcitabine), ACH-126,443 (L-Fd4C) (Achillion), MCC478 (Eli Lilly), Racivir (RCV), Fluoro-L and D nucleosides, Robustaflavone, ICN 2001-3 (ICN), Bam 205 (Novelos), XTL- 001 (XTL), Imino-Sugars (Nonyl-DNJ) (Synergy), HepBzyme; and immunomodulator products such as: interferon alpha 2b, HE2000 (Hollis-Eden), Theradigm (Epimmune), EHT899 (Enzo Biochem), Thymosin alpha-1 (Zadaxin^®), HBV DNA vaccine (PowderJect), HBV DNA vaccine (Jefferon Center), HBV antigen (OraGen), BayHep B^® (Bayer), Nabi- HB^® (Nabi) and Anti-hepatitis B (Cangene); and HBV vaccine products such as the following: Engerix B, Recombivax HB, GenHevac B, Hepacare, Bio-Hep B, TwinRix, Comvax, Hexavac. The term "treatment" as used herein is intended to mean the administration of a compound or composition according to the present invention to alleviate or eliminate symptoms of the hepatitis C disease and/or to reduce viral load in a patient. The term "treatment" also encompasses the administration of a compound or composition according to the present invention post-exposure of the individual to the virus but before the appearance of symptoms of the disease, and/or prior to the detection of the virus in the blood, to prevent the appearance of symptoms of the disease and/or to prevent the virus from reaching detectible levels in the blood.

The term "prevention" as used herein means the administration of a compound or composition according to the present invention post-exposure of the individual to the virus but before the appearance of symptoms of the disease, and/or prior to the detection of the virus in the blood, to prevent the appearance of symptoms of the disease.

The term "therapeutically effective amount" means an amount of a compound according to the invention, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a compound according to the invention which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the invention, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

PREFERRED EMBODIMENTS

In the following preferred embodiments, groups and substituents of the compounds of Formula (I) according to this invention are described in detail.

Any and each of the definitions below may be combined with each other.

Ring A and A'

Ring A and A'-A: Ring A and A' are each independently optionally substituted one time with halo.

Ring A and A'-B: Ring A and A' are each independently optionally substituted one time with F.

Ring A and A'-C: Ring A and A' are each unsusbtituted. R¹/R²

R¹/R²-A: R¹ is halo and R² is hydrogen; or R¹ is hydrogen and R² is halo.

R¹/R²-B: R¹ is F or CI and R² is hydrogen; or R¹ is hydrogen and R² is F or CI. R¹/R²-C: R¹ is CI and R² is hydrogen; or R¹ is hydrogen and R² is CI.

R¹/R²-D: R¹ is F and R² is hydrogen; or R¹ is hydrogen and R² is F.

E

R³-A: ³ is selected from the group consisting of:

R³-B:

R⁴-A: R⁴ is selected from (C-_!^alkyl optionally substituted one time with

-0-(Ci_-6)alkyl or -0-(Ci_-6) aloalkyl.

R⁴-B: R⁴ is selected from (C^alkyl optionally substituted one time with

-0-(C_1-3)alkyl.

R⁴-C: R⁴ of:

R⁵-A: R⁵ is either absent or selected from the group consisting of -0-(C-|.₆)alkyl and -S-(C_1-6)alkyl.

R⁵-B: R⁵ is either absent or selected from the group consisting of -0-(C _-3)alkyl and -S-(C_1-3)alkyl.

R⁵-C: R⁵ is either absent or selected from the group consisting of -0-(C-,)alkyl and -S-(d)alkyl.

R⁶-A: R⁶ is selected from the group consisting of:

R⁶-B: R is selected from the group consisting of:

R⁷-A: R⁷ is selected from (C _₆)alkyl optionally substituted one time with

-0-(Ci_₆)alkyl or -0-(Ci_₆)haloalkyl.

R⁷-B: R⁷ is selected from (C-|.₄)alkyl optionally substituted one time with

-0-(C_1-3)alkyl.

R⁷-C: R⁷

R -A: R is either absent or selected from the group consisting of -0-(Ci_₆)alkyl and -S-(C_{1 -6})alkyl.

R⁸-B: R⁸ is either absent or selected from the group consisting of -0-(Ci_₃)alkyl and -S-(C_{1 -3})alkyl.

R⁸-C: R⁸ is either absent or selected from the group consisting of -0-(Ci)alkyl and -S-(d)alkyl.

The following table represents further embodiments E- 1 to E- 10 of the compounds of Formula (I):

Ring A & A' R /R² R³ R⁴ R⁵ R⁶ R⁷ R⁸

E-2 Ring A&A'-A R¹/R -C R³-A R⁴-A R^b-A R^e-A R'-A R⁸-A

E-3 Ring A&A'-A R¹/R -C R³-B R⁴-B R^b-B R^e-B R'-B R^a-B

E-4 Ring A&A'-B R¹/R -B R³-A R⁴-B R^b-B R^e-A R'-B R^a-B

E-5 Ring A&A'-B R¹/R -C R³-B R⁴-B R^b-B R^e-A R'-B R^a-B

E-6 Ring A&A'-C R¹/R -C R³-C R⁴-C - R^e-C R -C -

E-7 Ring A&A'-C R /R²-B R³-C R⁴-C - R⁶-C R⁷-C -

E-8 Ring A&A'-C R¹/R -B R³-B R⁴-C R^b-C R^e-B R'-C R^a-C

E-9 Ring A&A'-C R¹/R -B R³-A R⁴-A R^b-A R^e-A R'-A R^a-A

E-10 Ring A&A'-C R /R²-B R³-C R⁴-A - R⁶-B R⁷-A R⁸-A

E-16 Ring A&A'-C R¹/R -D R³-C R⁴-C - R^e-C R -C -

In the following preferred embodiments, groups and substituents of the compounds of Formula (II) according to this invention are described in detail.

Any and each of the definitions below may be combined with each other.

Ring A and A'

Ring A and A'-C: Ring A and A' are each unsusbtituted.

R⁴-A: R⁴ is selected from (C _₆)alkyl optionally substituted one time with

-0-(Ci_₆)alkyl or -0-(Ci_₆)haloalkyl.

R⁴-B: R⁴ is selected from (C-|.₄)alkyl optionally substituted one time with -0-(C₁_₃)alkyl.

R⁵-A: R⁵ is -S-(C_1-6)alkyl.

R⁵-B: R⁵ is -S-(C₁_₃)alkyl.

R⁵-C: R⁵ is -S-(d)alkyl.

R⁶-A: R⁶ is selected from the group consisting of:

6-B: R⁶ is selected from the group consisting of:

R⁷-A: R⁷ is selected from (C^alkyl optionally substituted one time with -0-(Ci_-6)alkyl or -0-(Ci_-6)haloalkyl. R⁷-B: R⁷ is selected from (Ci_₄)alkyl optionally substituted one time with

-0-(C_1-3)alkyl.

R⁷-C: of:

R -A: R is either absent or selected from the group consisting of -0-(Ci_₆)alkyl and

-S-(C_{1 -6})alkyl.

R⁸-B: R⁸ is either absent or -S-(Ci_₃)alkyl.

R⁸-C: R⁸ is either absent or -S-(Ci)alkyl.

The following table represents further embodiments E- 1 1 to E- 15 of the compounds of Formula (I I):

Examples of most preferred compounds according to this invention are compounds 1001 , 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 101 1 , 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1021 , 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031 , 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041 , 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051 , 1052, 1053, 1054, 1055 and 1056.

Examples of most preferred compounds according to this invention are compounds 1001 , 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 101 1 , 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1020, 1021 , 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031 , 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041 , 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051 , 1052, 1053 and 1054. Examples of most preferred compounds according to this invention are compounds 1001 , 1002, 1003, 1004, 1005, 1006, 1008, 1009, 1010, 101 1 , 1012, 1013, 1014, 1015, 1016, 1020, 1021 , 1022, 1023, 1024, 1025, 1026, 1027, 1029, 1030, 1031 , 1032, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041 , 1042, 1043, 1044, 1046, 1047, 1048, 1049, 1053, 1054.

Examples of most preferred compounds according to this invention are compounds 1017, 1024, 1036, 1037, 1038, 1039, 1045 and 1047.

Examples of most preferred compounds according to this invention are compounds 1024, 1036, 1037, 1038, 1039 and 1047.

Examples of most preferred compounds according to this invention are compounds 1006, 1007, 101 1 , 1020, 1024, 1034, 1035, 1037, 1045 and 1051.

Examples of most preferred compounds according to this invention are compounds 1006, 101 1 , 1020, 1024, 1035 and 1037.

Examples of most preferred compounds according to this invention are compounds 1006, 101 1 , 1020, 1024, 1035, 1037 and 1045.

An example of a most preferred compound according to this invention is compound 1006. An example of a most preferred compound according to this invention is compound 101 1. An example of a most preferred compound according to this invention is compound 1020. An example of a most preferred compound according to this invention is compound 1024. An example of a most preferred compound according to this invention is compound 1035. An example of a most preferred compound according to this invention is compound 1037. An example of a most preferred compound according to this invention is compound 1045. An example of a most preferred compound according to this invention is compound 1056. PHARMACEUTICAL COMPOSITION

Suitable preparations for administering the compounds of Formula (I) and (II) will be apparent to those with ordinary skill in the art and include for example tablets, pills, capsules, suppositories, lozenges, troches, solutions, syrups, elixirs, sachets, injectables, inhalatives and powders etc. The content of the pharmaceutically active compound(s) should be in the range from 0.05 to 90 wt.-%, preferably 0.1 to 50 wt.-% of the composition as a whole.

Suitable tablets may be obtained, for example, by mixing one or more compounds according to Formula (I) or (II) with known excipients, for example inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and/or lubricants . The tablets may also consist of several layers.

According to an alternate embodiment, the pharmaceutical composition of this invention may additionally comprise at least one other anti-HCV agent.

As discussed above, combination therapy is contemplated wherein a compound of the invention, or a pharmaceutically acceptable salt thereof, is co-administered with at least one additional agent selected from: an antiviral agent, an immunomodulatory agent, an inhibitor of HCV NS3 protease, an inhibitor of HCV polymerase, an inhibitor of another target in the HCV life cycle, an HIV inhibitor, an HAV inhibitor and an HBV inhibitor. These additional agents may be combined with the compounds of this invention to create a single pharmaceutical dosage form. Alternatively these additional agents may be separately administered to the patient as part of a multiple dosage form, for example, using a kit. Such additional agents may be administered to the patient prior to, concurrently with, or following the administration of a compound of the invention, or a pharmaceutically acceptable salt thereof. The dose range of the compounds of the invention applicable per day is usually from 0.01 to 100 mg/kg of body weight, preferably from 0.1 to 50 mg/kg of body weight. Each dosage unit may conveniently contain from 5% to 95% active compound (w/w). Preferably such preparations contain from 20% to 80% active compound.

The actual pharmaceutically effective amount or therapeutic dosage will of course depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease. In any case the combination will be administered at dosages and in a manner which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.

When the composition of this invention comprises a combination of a compound of the invention and one or more additional therapeutic or prophylactic agent, both the compound and the additional agent should be present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.

EXAMPLES

Other features and advantages of the present invention will become apparent from the following more detailed Examples which illustrate, by way of example, the principles of the invention. As is well known to a person skilled in the art, reactions are performed in an inert atmosphere (including but not limited to nitrogen or argon) where necessary to protect reaction components from air or moisture. Temperatures are given in degrees Celsius (°C). Solution percentages and ratios express a volume to volume relationship, unless stated otherwise. Flash chromatography is performed on Teledyne Isco CombiFlash® Rf system using RediSep® Normal-phase Silica Flash Columns or RediSep Rf Gold® Normal-Phase

Silica Columns or SiliaSep Universal Closed-Top Flash Cartridges or InnoFlash Silica Flash Column.

All of the compounds of the invention are synthesized analogously to the specific Examples described below. Retention times (t_R) for each compound are measured using the standard analytical HPLC or UPLC conditions described below. As is well known to one skilled in the art, retention time values are sensitive to the specific measurement conditions.

Therefore, even if identical conditions of solvent, flow rate, linear gradient, and the like are used, the retention time values may vary when measured, for example, on different HPLC or UPLC instruments. Even when measured on the same instrument, the values may vary when measured, for example, using different individual HPLC or UPLC columns, or, when measured on the same instrument and the same individual column, the values may vary, for example, between individual measurements taken on different occasions. A person skilled in the art will recognize that obvious modifications to the synthetic methods, including the amount of time indicated to perform the various steps, may be required to generate each of the specific compounds listed in the Examples section.

Preparative RP-HPLC is performed under standard conditions using one of the following specific measuring conditions:

Compounds are purified by preparative RP-HPLC under standard conditions using a Waters SunFire Prep OBD™ C18 column (5μηι, 19 x 50 mm) eluting with a linear methanol : water gradient containing 10 mM Ammonium Formate (pH 3.8) over 10 minutes at 30 ml/min. Fractions containing the desired product are pooled and lyophilized.

Compounds are purified by preparative RP-HPLC under standard conditions using a Waters XBridge Prep OBD™ C18 (5μηι, 19 x 50 mm) eluting with a linear methanol : water gradient containing 10 mM Ammonium Bicarbonate (pH 10) over 10 minutes at 30 ml/min. Fractions containing the desired product are pooled and lyophilized.

Compounds are purified by preparative RP-HPLC under standard conditions using a Waters SunFire Prep OBD™ C18 column (5μηι, 19 x 50 mm) eluting with a linear acetonitrile : water gradient containing 0.06 % TFA (v/v) 10 minutes at 30 ml/min. Fractions containing the desired product are pooled and lyophilized.

Analytical UPLC is performed under standard conditions using one of the following specific measuring conditions:

Analytical UPLC is carried out under standard conditions using a Waters ACQUITY UPLC^® HSS T3 column (1 .8μηι, 2.1 x 50 mm) eluting with a segmented linear MeCN gradient containing 0.06%TFA (v/v) over 2.6 min at 0.9 ml/min. Analytical UPLC is also carried out under standard conditions using a Waters ACQUITY UPLC^® BEH C18 column (1.8μηι, 2.1 x 30 mm) eluting with a linear methanol gradient containing 10 mM Ammonium Bicarbonate (pH 10) over 2.2 min at 0.75 ml/min or a Waters ACQUITY UPLC^® HSS C18 column (1.8μηι, 2.1 x 30 mm) eluting with a linear MeOH gradient containing 10 mM Ammonium Formate (pH 3.8) over 2.3 min at 0.8 ml/min.

Mass spectral analyses are recorded using electrospray mass spectrometry.

Abbreviations or symbols used herein include:

Ac: acetyl; AcOH: acetic acid; BEH: ethylene bridged hybrid; Bn: benzyl (phenylmethyl); BOC or Boc: fe/f-butyloxycarbonyl; Bu: butyl; DCM: dichloromethane; DIPEA: N,N- diisopropylethylamine; DME: dimethoxyethane; DMF: W,W-dimethylformamide; EC₅₀: 50% effective concentration; EDCI:1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Et: ethyl; Et₃N: triethylamine; Et₂0: diethyl ether; EtOAc: ethyl acetate; EtOH: ethanol; Hex:

hexanes; HATU: N,N,N',N'-tetramethyl-0-(7-azabenzotriazol-1-yl)uronium

hexafluorophosphate; HOAt: 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; HSS: high strength silica; 'Pr or i-Pr: 1-methylethyl (/so- propyl); LC-MS: liquid chromatography-mass spectrometry; m/z: mass-to-charge ratio; [M+H]⁺: protonated molecular ion; Me: methyl; MeCN: acetonitrile; MeOH: methanol; MS: mass spectrometry; NMP: N-methyl-2-pyrrolidone; OBD: optimum bed density; Pd(dppf)CI₂: 1 ,1 '- bis(diphenylphosphino)-ferrocenedichloropalladium(ll); Ph: phenyl; Pr: propyl; Prep LCMS: preparative liquid chromatography-mass spectrometry; RBF: round bottom flask; RP- HPLC: reversed-phase high pressure liquid chromatography; RT: room temperature (approximately 18°C to 25°C); ferf-butyl or t-butyl: 1 ,1-dimethylethyl; TBTU: O- (benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; TFA: trifluoroacetic acid; THF: tetrahydrofuran; t_R: retention time; UPLC: ultra performance liquid chromatography; X-PHOS: 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl.

Example 1

Preparation of intermediate 1a4

1a4

Step 1 :

To a solution of 4-bromo-3-fluorobenzoic acid 1a1 (Oakwood) (10.7 g, 48.8 mmol), N, 0- dimethylhydroxylamine hydrochloride ( 4.76 g, 48.8 mmol), EDCI (14.0 g, 73.2 mmol) and HOAt (4.92 g, 9.4 mmol) in DMF (79 mL) is added DIPEA (25.5 mL, 146.4 mmol). After stirring at RT for 24 h, the reaction mixture is concentrated to 50 mL and 300 mL of toluene is added. The layers are separated; the organic layer is washed with water (100 mL), 0.1 N HCI (100 mL) and 1 N NaOH (100 mL) dried with MgS0₄, filtered and concentrated to afford 1a2.

Step 2:

To a solution of 1a2 (12 g, 45.9 mmol) in THF (450 mL) at 0°C is added a

methylmagnesium chloride solution in THF (3M, 19.1 mL, 57.4 mmol). The reaction mixture is stirred for 80 min and then 1 N HCI (75 mL) is added. Stirring is continued and EtOAc is added. The layers are separated and the aqueous layer is extracted with EtOAc (500 mL). The combined organic layers are washed with brine, dried with MgS0₄, filtered and concentrated to afford 1a3.

Step 3:

To a stirred solution of 1a3 (9.8 g, 45.3 mmol) in THF (90 mL) is added portionwise phenyltrimethylammonium tribromide (17.0 g, 45.3 mmol). The reaction mixture is stirred for 1 h at RT. Water is added, followed by EtOAc and the layers are separated. The organic layer is washed with water, brine, dried over MgS0₄, filtered and concentrated. The product is purified by flash chromatography using 100% hexanes to afford 1a4.

Example 2

Preparation of intermediate 2a2

2a1 2a2

2a1 (6 g, 26 mmol) (Novabiochem) in THF (95 mL) is treated with NaH (60% in oil, 2.1 g, 86.5 mmol). After 45 min, iodomethane (3.2 mL, 52 mmol) is added and the reaction mixture is stirred for 3 h at reflux, then 16 h at RT. The reaction mixture is concentrated in vacuo, diluted with DCM and washed with water. The aqueous layer is acidified with 1 N HCI and extracted with DCM. The combined organics layers are dried over Na₂S0₄, filtered and concentrated in vacuo to afford 2a2.

Example 3

Preparation of intermediate 3a5

3a5

Step 1 :

3a1 (12.5 g, 51 mmol) (Aldrich) is dissolved in DCM (300 mL) and to this is added brosyl chloride 3a2 (26 g, 102 mmol) followed by addition of Et₃N (28.4 mL, 204 mmol) and N,N- dimethylaminopyridine (623 mg, 5 mmol). The reaction mixture is stirred at reflux for 16 h. The resulting mixture is concentrated in vacuo and the product is placed in EtOAc (500 mL). The organic layer is washed with water (80 mL), saturated aqueous solution of NaHC0₃ (80 mL), water (80 mL), brine (80 mL), dried over MgS0₄, filtered and concentrated in vacuo. The crude is purified by flash chromatography (90:10

hexanes: EtO Ac to 75:25 hexanes: EtO Ac) to afford 3a3 (t_R = 1.9 min, (M+H)⁺ 364.0; 366.0).

Step 2:

3a3 (7.8 g, 16.8 mmol) is dissolved in DMF (52 mL), potassium thioacetate (2.5 g, 21.8 mmol) is added and the reaction mixture is heated to 65°C for 2 h and stirred at RT for 16 h. The resulting reaction mixture is diluted with EtOAc (200 mL), washed with brine (200 mL) containing 1 N HCI to adjust the pH of the aqueous layer to approximately 4. The organic layer is washed with brine (200 mL), dried over Na₂S0₄, filtered and concentrated in vacuo. The crude mixture is purified by flash chromatography (90:10 hexanes: EtOAc to 75:25 hexanes: EtOAc) to afford 3a4 (t_R = 1.7 min, (M+H)⁺ 304.2).

Step 3:

3a4 (4.59 g, 8.1 mmol) is dissolved in MeOH (79 mL), 1 N NaOH solution (16.3 mL, 16.3 mmol) is added followed by dimethyl sulfate (1.65 mL, 17.4 mmol). After 60 min, 1 N NaOH solution (33.3 mL, 33.3 mmol) is added and the reaction mixture is stirred at RT for 3 h. The volatiles are removed under reduced pressure, and the residue is partioned between 1 N HCI and EtOAc. The organic layer is washed with brine, dried over Na₂S0₄, filtered and concentrated in vacuo to afford 3a5 (t_R = 1.39 min, (M-H)⁺ 260.1).

Example 4

Step 1 :

To a stirred cooled (10°C) suspension of (S)-pyroglutamic acid 4a1 (Aldrich) (50.1 g, 0.388 mol) in MeCN (200 mL), DIPEA (49.1 g, 0.380 mol) is added dropwise. To this solution, iodoethane (94.8 g, 0.608 mol) is added. The reaction mixture is stirred for 3 days at RT and concentrated under reduced pressure. The residue is treated with EtOAc (500 mL) and the resultant mixture is filtered. Removal of the solvent from the filtrate under reduced pressure provides 4a2.

Step 2:

To a stirred cooled (10°C) solution of ester 4a2 (48.5 g, 0.309 mol) in EtOH (400 mL), sodium borohydride (1 1 .7 g, 0.31 mol) is added portionwise at ~10°C to 12°C. The reaction mixture is stirred for 17 h at RT, cooled to 5°C and quenched with 12 N HCI (27 mL) until the pH is approximately 3. After 3 h of stirring at RT, the resultant mixture is neutralized with solid NaHC0₃ (20 g). The precipitate is filtered off and the filtrate is concentrated under reduced pressure. The residue is dissolved in THF (400 mL) and dried over MgS0₄. Filtration and removal of the solvent from the filtrate under reduced pressure affords crude 4a 3

Step 3:

To a stirred cooled (0°C) solution of alcohol 4a3 (39.4 g, ca. 0.307 mol) and 1 ,3-imidazole (20.9 g, 0.307 mol) in anhydrous DMF (300 mL), te/f-butyldiphenyl chlorosilane (84.4 g, 0.307 mol) is added dropwise. The reaction solution is stirred for 2 days at RT and concentrated under reduced pressure. The residue is partitioned between with EtOAc (500 mL) and water (200 mL). The organic phase is separated, washed with water (100 mL), saturated aqueous NaH₂P0₄ (2 ^χ 100 mL), saturated aqueous sodium bicarbonate (200 mL), brine (200 mL) and dried over MgS0₄. Filtration and removal of the solvent from the filtrate under reduced pressure provides crude 4a4.

Step 4:

To a stirred solution of 4a4 (106.0 g, ca. 0.265 mol) and 4-(dimethylamino)pyridine (6.47 g, 0.053 mol) in anhydrous MeCN (200 mL), di-te/f-butyl dicarbonate (69.4 g, 0.318 mol) is added portionwise at RT. The reaction solution is stirred at RT for 8 h and concentrated under reduced pressure. The residue is dissolved in EtOAc (700 mL), washed with saturated aqueous NaH₂P0₄ (3 ^χ 200 mL), brine (300 mL) and dried over MgS0₄. Filtration and removal of the solvent from the filtrate under reduced pressure provides crude 4a5. The crude product is suspended in hexanes (400 mL). The precipitate is filtered, washed with hexanes (3x300 mL) and dried in vacuo to provide 4a5.

Step 5:

To a stirred cooled (-55°C) solution of 4a5 (102.4 g, 0.226 mol) in anhydrous toluene (370 mL), a 1 M solution of lithium triethylborohydride in THF (235 mL, 0.235 mol) is added dropwise. The reaction solution is stirred for 50 min at ~-50°C to -47°C, then DIPEA (123.7 g, 0.957 mol) is added dropwise followed by addition of 4-(dimethylamino)pyridine (0.345 g, 0.0028 mol). To the resultant solution, trifluoroacetic anhydride (54.5 g, 0.26 mol) is added dropwise at ~-52°C to -50°C. The reaction solution is stirred for 10 min at the same temperature and is then warmed to RT. After 16 h of stirring, the reaction solution is diluted with toluene (80 mL) and cooled to -20°C. Water (500 mL) is added dropwise at ~-20°C to 0°C. The organic phase is separated, washed with water (2 ^χ 500 mL), saturated aqueous sodium bicarbonate (500 mL) and dried over MgS0₄. The reaction mixture is filtered, concentrated under reduced pressure and subjected to flash chromatography (5%

EtOAc: hexanes) to afford 4a6.

Step 6:

To a stirred cooled (-35°C) solution of 4a6 (42.3 g, 0.0967 mol) in anhydrous toluene (307 mL), a 1.1 M solution of diethylzinc in toluene (200 mL, 0.22 mol) is added dropwise followed by addition of chloroiodomethane (76.3 g, 0.433 mol) at the same temperature. The reaction solution is stirred for 20 h at ~-24°C to -21 °C and quenched by addition of 3.8% aqueous sodium bicarbonate (200 mL) at ~-20°C to

-10°C. The resultant mixture is filtered through a pad of Celite®. The organic phase is separated, dried over MgS0₄ and filtered. The solvent is removed from the filtrate under reduced pressure and the residue is subjected to flash chromatography (2-10%

EtOAc: hexanes) to afford 4a7.

Step 7:

To a stirred solution of 4a7 (59.2 g, 0.201 mol) in anhydrous THF (560 mL), a 1 M solution of tetrabutylammonium fluoride in THF (144 mL, 0.144 mol) is added dropwise at RT. After 6 h of stirring, the reaction solution is quenched with saturated aqueous solution of NH₄CI (95 mL) and the reaction mixture is concentrated under reduced pressure. The residue is treated with DCM (400 mL), saturated aqueous solution of NH₄CI (250 mL) and water (250 mL). The organic phase is separated and the aqueous phase is extracted with DCM (3 ^χ 100 mL). The combined organic extracts are dried over MgS0₄ and filtered. The solvent is removed from the filtrate under reduced pressure and the residue is subjected to flash chromatography (5-30% EtOAc:hexanes] to afford 4a8.

Step 8:

To a stirred solution of 4a8 (26.6 g, 0.125 mol) in a mixture of MeCN (250 mL) and CCI₄ (250 mL), water (375 mL) and sodium periodate (109.4 g, 0.51 1 mol) are added at RT. The resultant mixture is stirred for 0.5 h and ruthenium trichloride (0.57 g, 0.0027 mol) is added in one portion. The reaction mixture is stirred for 2 h maintaining the temperature from 18°C to 24°C by periodical cooling. The resulting precipitate is filtered off and washed with DCM (3 x 100 mL). The organic phase is separated and the aqueous phase is extracted with DCM (3 x 100 mL). The combined organic extracts are dried over MgS0₄ and filtered. Removal of the solvent from the filtrate under reduced pressure provides a residue that is dissolved in diethyl ether (1 L). The resultant solution is filtered. The solvent is removed from the filtrate under reduced pressure and the residue is recrystallized two times with EtOAc to afford 4a9 (t_R = 3.06 min; (M-H)⁺: 225.9).

Example 5

5a1 5a2 5a3

5a4 5a5 Step 1 :

L-Serine 5a1 (Sigma) (8 g, 142 mmol) is dissolved in water / THF (8 mL / 375 mL), potassium carbonate (99 g, 714 mmol) and benzyl bromide 5a2 (59 mL, 500 mmol) are added. The mixture is heated at 55°C for 24 h, then cooled, filtered and washed with EtOAc. Purification by flash chromatography (5-30% EtOAc:hexanes) affords 5a3 (t_R = 2.19 min, (M+H)⁺ 377.3).

Step 2:

To 5a3 (25 g, 67 mmol) and sodium sulfate (2.27 g, 16 mmol) in MeCN (225 mL) at 40°C is added difluoro(fluorosulfonyl)acetic acid (1 1.9 g, 67 mmol) dropwise for 90 min. The reaction mixture is concentrated in vacuo and the residue is purified by flash

chromatography eluting 0-10-20% EtOAc:hexanes. A second purification column is performed (0-5% EtOAc:hexanes, RediSep Rf Gold® Normal-Phase Silica Columns) to afford 5a4 (t_R = 2.3 min, (M+H)⁺ 426.3).

Step 3:

5a4 (1.3 g, 3 mmol) is stirred with Pd(OH)₂ (10 % ,100 mg) in MeOH (10 mL). Air is removed under vacuum and a hydrogen atmosphere (1 atm; balloon) is made. After three cycles, the reaction mixture is stirred for 24 h then filtered and concentrated in vacuo to afford 5a5.

Example 6

6a1 6a2

To a solution of L-alanine 6a1 (Aldrich) (8 g, 168 mmol) in 1 N NaOH (168 mL) is added Na₂C0₃ (8.9 g, 84 mmol). The solution is cooled to 0°C and methyl chloroformate (20.5 mL, 172 mmol) is added dropwise. The reaction mixture is stirred at RT for 3.5 h. The solution is washed with Et₂0 and acidified to a pH of approximately 1 with 1 N HCI. The aqueous layer is extracted with DCM and the organic layer is washed with brine, dried over Na₂S0₄ and concentrated to afford 6a2. The following intermediates are prepared analogously to the procedure described in

Example 6 using the appropriate amino acid derivative.

Example 7

Preparation of intermediate 7a2

3a5 7a1 7a2

3a5 (0.67 g, 2.6 mmol) is dissolved in MeCN (12.6 mL). 7a1 (Matrix) (0.65 g, 2.3 mmol) and DIPEA (0.41 mL, 2.3 mmol) are added and the reaction is stirred at RT for 16 h. The MeCN is evaporated and toluene (17 mL) is added. The resulting solids are removed and NH₄OAc (1.8 g, 23.4 mmol) is added to the toluene solution. The solution is heated to 100°C overnight and concentrated in vacuo. The organic layer is washed with water, brine, dried over Na₂S0₄, filtered, concentrated in vacuo and purified by flash chromatography (30-70 % EtOAc:hexanes) to afford 7a2 (t_R = 1.94 min, (M+H)⁺ 438.1 ; 440.1).

The following intermediates are prepared analogously to the procedure described in

Example 7 starting from the appropriate dibromoketone derivative and the appropriate protected amino acid.

Example 7h

Preparation of intermediate 7h6

7h5 7a1 7h6 7h7

Step 1 :

7h1 (16 mL, 178 mmol) is placed into a mixture of hexanes (80 mL) and toluene (80 mL) and cooled to 0°C. Trimethylsilyl diazomethane 7h2 (2M in hexanes, 90 mL, 180 mmol) is added slowly and the reaction mixture is stirred at RT for 16h. The reaction mixture is concentrated in vacuo keeping the heating bath at 50°C to afford 7h3 in toluene.

Step 2:

7h3 (8 ml, 53 mmol), /V-methylpyrrolidinone (179 mL), 6b2 (1 1.2 g, 63.9 mmol), TBTU (20.5 g, 63.9 mmol) and 2,4,6-collidine (21 mL, 160 mmol) are charged in a 500 mL flask. The reaction mixture is stirred at RT for 16 h. 0.5 equivalent of 6b2 (4.7 g, 26.8 mmol), TBTU (8.5 g, 26.8 mmol) and 2,4,6-collidine (3.5 mL, 26.8 mmol) are added and the reaction mixture is stirred for 16h. The reaction mixture is poured into a saturated aqueous solution of NaHC0₃ (800 mL) and EtOAc is added. The layers are separated and the organic layer is washed with a saturated aqueous solution of NaHC0₃, water, dried over Na₂S0₄, filtered and concentrated in vacuo. The product is purified by flash

chromatography (100% hexanes to 20% MeOH / EtOAc) to provide 7h4 (t_R = 1.1 min, (M+H)⁺ 286.2).

Step 3:

To 7h4 (3.5 g, 12 mmol) in THF (26 mL) and water (35 mL) is added lithium hydroxide monohydrate (1.5 g, 36.5 mmol). The reaction mixture is stirred at RT for 10 min and acidified to approximately pH=4 using 10% HCI aqueous solution. The aqueous layer is extracted with EtOAc (3x) and the combined organic layers are dried over MgS0₄, filtered and concentrated in vacuo to afford 7h5 (t_R = 0.9 min, (M+H)⁺ 272.2).

Step 4:

7h5 (2.9 g, 10.6 mmol) is dissolved in MeCN (48 mL). 7a1 (3.2 g, 11.6 mmol) and DIPEA (1.8 mL, 10.6 mmol) are added and the reaction is stirred at RT for 1 h. The MeCN is evaporated and toluene (100 mL) is added. The resulting solids are removed and NH₄OAc (8 g, 105.8 mmol) is added to the toluene solution. The solution is heated to 90°C overnight and concentrated in vacuo. The reaction mixture is diluted with EtOAc, washed with water, brine, dried over Na₂S0₄, filtered and concentrated in vacuo. The residue is purified by flash chromatography (0-100% EtOAc in hexanes) and then a second purification column (60% EtOAc in hexanes, RediSep Rf Gold® Normal-Phase Silica Column) is performed to isolate the diastereoisomers 7h6 and 7h7 (7h6: t_R = 1.9 min, (M+H)+ 448.3; 450.2; 7h7: t_R = 1.7 min, (M+H)+ 448.3; 450.2).

Example 7i

7g1 7i1 _7i2

Step 1 :

7g1 (2.6 g, 7.4 mmol) is dissolved in THF (50 mL) and bromine (0.42 mL, 8.1 mmol) is added dropwise. After 1 h at RT, the reaction is neutralized with a saturated aqueous solution of Na₂S₂0₃, then diluted with EtOAc and saturated aqueous solution of NaHC0₃. The layers are separated and the organic layer is washed with water and brine, dried over MgS0₄, filtered and concentrated in vacuo. Purification by flash chromatography (10-50 % EtOAc:hexanes) gives 7i1 (t_R = 2.09 min, (M+H)⁺ 426.1 ;428.1 ;429.9).

Step 2:

An oven dried flask is charged with 7i1 (1 g, 2.3 mmol) and THF (7.5 mL) is added. The reaction mixture is cooled to -78°C and then treated with a solution of te/f-butyllithium (1.7 M in pentane, 5.7 mL, 9.6 mmol). The reaction mixture is stirred for 5 min. A solution of N- fluorobenzenesulfonimide (961 mg, 3.1 mmol) in THF (2.5 mL) is added and the reaction mixture is stirred at -78°C for 30 min. Another portion of /V-fluorobenzenesulfonimide (324 mg, 1.0 mmol) in THF (1 mL) is added and the mixture is stirred for 8 min. The reaction is neutralized with a saturated aqueous solution of NH₄CI and the mixture is allowed to warm to RT. The mixture is extracted with EtOAc and the organic layer is washed with water, brine, dried over MgS0₄, filtered and concentrated. Purification by flash chromatography (20-40% EtOAc: hexanes, RediSep Rf Gold® Normal-Phase Silica Columns) affords 7i2.

Example 7j

7g2 7j1 _7j2

Step 1 :

7g2 (1.47 g, 4.1 mmol) is dissolved in THF (30 mL). Bromine (0.23 mL, 4.5 mmol) is added dropwise. After 2 h at 23°C, the reaction is neutralized with a saturated aqueous solution of Na₂S₂0₃, diluted with EtOAc and a saturated aqueous solution of NaHC0₃. The layers are separated and the organic layer is washed with water and brine, dried over MgS0₄, filtered and concentrated in vacuo. Purification by flash chromatography (10-50% EtOAc in hexanes) gives 7j1 (t_R = 2.00 min, (M+H)+ 438.0;440.0).

Step 2:

An oven dried flask is charged with 7j1 (1 g, 2.3 mmol) and THF (7.5 mL) is added. The mixture is cooled to -78°C and then treated with a solution of methyllithium (1.6 M in diethyl ether, 1.6 mL, 2.5 mmol). After 10 min, a solution of fe/f-butyllithium (1.7 M in pentane, 2.95 mL, 5.0 mmol) is added. The mixture is stirred for 10 min, then a solution of N- fluorobenzenesulfonimide (1.22 g, 1.7 mmol) in THF (3.2 mL) is added and the mixture is stirred at -78°C for 15 min. The reaction is quenched with a saturated aqueous solution of NH₄CI and the mixture is allowed to reach 23°C. The mixture is extracted with EtOAc and the organic layer is washed with water, brine, dried over MgS0₄, filtered and concentrated. Purification by flash chromatography (20-40% EtOAc in hexanes) followed by purification by preparative HPLC (CH₃CN / water containing 0.6 % TFA) affords 7j2 (t_R = 2.08 min, (M- H)⁺ 376.0;378.0).

Example 8

7c1 8a 1

7c1 (9.5 g, 24 mmol), bis(pinacolato)diboron (12.9 g, 51 mmol), potassium acetate (6.2 g, 63 mmol) and Pd[PPh₃]₄ (1.1 g, 0.97 mmol) are placed in 1 ,4-dioxane (200 mL). The reaction mixture is degassed with argon for 30 min and heated at 80°C for 16 h. The reaction mixture is cooled to RT, filtered and partitioned between DCM and water / NaHC0₃. The layers are separated and the aqueous phase is extracted with DCM. The combined organic layers are washed with water, brine, dried over Na₂S0₄ and

concentrated in vacuo. The product is purified by flash chromatography (10%-30%

EtOAc:petroleum ether) to afford 8a1 ((M+H)⁺ 440.2).

Example 8 starting from the appropriate halogeno derivative.

Example 9

Preparation of intermediate 9a 1

7a2 (884 mg, 2 mmol), 8g1 (975 mg, 2 mmol), 2M aqueous solution of sodium carbonate (6.7 ml_, 20.3 mmol) and Pd(dppf)CI₂ ^*CH₂Cl₂ (74 mg, 0.10 mmol) are placed in DME (10 ml_). The mixture is degassed with argon for 10 min and heated at 90°C for 8 h. The reaction mixture is cooled to RT, filtered over Celite® and washed with DCM. The filtrate is passed through a phase separator (rinsed with DCM) and concentrated in vacuo. The product is purified by flash chromatography (50%-100% THF:hexanes) to afford 9a1 (t_R = 2.07 min, (M+H)⁺ 715.6).

Example 9 starting from the appropriate halogeno derivative and boronic acid or boronic ester derivative.

Preparation of intermediate 911

7i2 (69.5 mg, 0.19 mmol), 8a1 (83.5 mg, 0.19 mmol), sodium carbonate (80.5 mg, 0.76 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (7.8 mg, 0.02 mmol), tris(dibenzylideneacetone)dipalladium (5.2 mg, 0.01 mmol) are placed in a mixture of 2- methyltetrahydrofuran (0.8 mL) and water (0.2 ml_). The reaction mixture is degassed with argon for 2 min and heated at 100°C for 7 h. The reaction mixture is cooled to RT, DCM is added, filtered over Celite® and washed with DCM. The filtrate is passed through a phase separator (rinsed with DCM) and concentrated in vacuo. The product is purified by flash chromatography (50%- 100% THF:hexanes) to afford 911 (t_R = 2.06 min, (M+H)⁺ 643.9).

7j2 8c1 9m1

7j2 (146 mg, 0.39 mmol), 8c1 (192 mg, 0.43 mmol) and 0.5 M solution of K₃P0₄ in water (1 .55 mL, 0.77 mmol) are placed in 2-methyltetrahydrofuran (0.8 mL). The mixture is degassed with argon for 10 min and X-PHOS precatalyst (J. Am. Chem. Soc, 2010, 132 (40), pp 14073-14075) (30 mg, 0.04 mmol) is added. The mixture is heated at 60°C for 2 h The reaction mixture is cooled to 23°C, EtOAc and water are added. The layers are separated and the organic layer is dried over MgS0₄, filtered and concentrated in vacuo. The product is purified by flash chromatography (0%-100% EtOAc in hexanes) to afford 9m1 (t_R = 1 .30 min, (M+H)+ 667.2).

The following intermediate is prepared analogously to the procedure described in Example 911 starting from the appropriate halogeno derivative and boronic acid or boronic ester derivative.

Example 10

Preparation of intermediate 10a1

7c1 10a1

7c1 (5 g, 12.8 mmol) is dissolved in 4M HCI solution in 1 ,4-dioxane (32 mL, 127 mmol) and the reaction mixture is stirred at RT until completion. The solvent is evaporated and the residue is dried under high vacuum to afford 10a1 (t_R = 1 .35 min, (M+H)⁺ 292.1 ;294.1).

The following intermediate is prepared analogously to the procedure described in Example 10 starting from the appropriate fe/f-butyl carbamate derivative.

Example 11

Preparation of intermediate 11a2

10a1 11a1 11a2

Step 1 :

10a1 (4.2 g, 12.8 mmol) is placed in water (10 mL) and 1 N NaOH (50 mL, 50 mmol) is added. The reaction mixture is sonicated for 3 min and is stirred for 3 h. The residue is filtered and dried under high vacuum to afford 11a1 (t_R = 1.35 min, (M+H)⁺ 292.1 ; 294.1).

Step 2:

11a1 (3.7 g, 12.8 mmol) is suspended in DCM (35 mL) and treated with DIPEA (2.6 mL, 14.8 mmol). 2-(trimethylsilyl)ethoxycarbonyl-0-succinimide (3.5 g, 20.6 mmol) is added and the mixture is stirred at RT for 2.5 h. The reaction mixture is diluted with DCM and neutralized with water. The layers are separated and the aqueous layer is extracted with DCM. The organic layers are combined, dried over MgS0₄, filtered and concentrated. Purification by flash chromatography (0-50% EtOAc:hexanes) affords 11a2.

Example 12

9a1 12a1

9a1 (1.12 g, 1.6 mmol) is dissolved in THF (8 mL) and a solution of tetrabutylammonium fluoride (1 M in THF, 4.7 mL, 4.7 mmol) is added. The solution is stirred for 16 h at RT, then diluted with DCM, washed with water, brine, dried over MgS0₄, filtered and concentrated. Water is added to the crude compound followed by diethyl ether and hexanes. The resulting mixture is stirred for 16 h at RT, then filtered, washed with water and dried under high vacuum to afford 12a1 (t_R = 1.87 min, (M+H)⁺ 571.5).

The following intermediate is prepared analogously to the procedure described in Example 12 starting from the appropriate 2-(trimethylsilyl)ethoxycarbonyl carbamate derivative.

Example 13

Preparation of intermediate 13a1

10a1 (35 g, 96 mmol), 6b2 (16.8 g, 96 mmol) and HATU (40 g, 105 mmol) are charged in a 1 L round bottom flask. DMF (350 mL) is added followed by DIPEA (66 ml_, 383 mmol) and the reaction mixture is stirred at RT for 2 h. To the reaction mixture is added water followed by EtOAc. The layers are separated and the aqueous layer is extracted with EtOAc. The organic layers are combined, washed with water, brine, dried over Na₂S0₄, filtered and concentrated. Purification by flash chromatography (0-40% EtOAc: hexanes) affords 13a1.

The following intermediates and compound 1056 are prepared analogously to the procedure described in Example 13 starting from the appropriate amine derivative and the appropriate carboxylic acid derivative.

Example 14

Step 1 :

12a1 (195 mg, 0.34 mmol), /V-methylpyrrolidinone (2 mL), 6b2 (1.5 M in NMP, 0.342 mL, 0.51 mmol), HATU (195 mg, 0.51 mmol) and 2,4,6-collidine (0.205 mL, 1.0 mmol) are charged in a 20 mL vial. The reaction mixture is stirred at RT for 16 h, then poured into water and EtOAc. The layers are separated and the organic layer is washed with water, brine, dried over MsS0₄, filtered and concentrated in vacuo to afford 14a1 (t_R = 1.92 min, (M+H)⁺ 728.5).

Step 2:

14a1 (247 mg, 0.34 mmol) is dissolved in 1 ,4-dioxane (2 mL) and a 4 M HCI solution in 1 ,4- dioxane (8 mL, 32 mmol). The reaction mixture is stirred at RT for 16 h, then the reaction mixture is concentrated in vacuo to afford 14a2 (t_R = 1.79 min, (M+H)⁺ 628.4).

Step 3:

14a2 (60.6 mg, 0.09 mmol), /V-methylpyrrolidinone (1.0 mL), 6b2 (1.5 M in NMP, 0.086 mL, 0.20 mmol), HATU (0.5 M in NMP, 0.257 mL, 0.20 mmol) and 2,4,6-collidine (0.084 mL, 0.66 mmol) are charged in a 8 mL vial. The reaction mixture is stirred at RT for 90 min, and the product is purified by preparative RP-HPLC. The fractions are concentrated and lyophilized to afford 1001.

Compounds 1002-1016 are prepared analogously to the procedure described in Example 14 starting from the appropriate amine derivative and the appropriate carboxylic acid derivatives in sequence.

Example 15

P

Step 1 :

9e1 (126 mg, 0.12 mmol) is dissolved in a 4 M HCI solution in 1 ,4-dioxane (1.21 mL, 4.9 mmol) and the reaction mixture is stirred at RT for 16 h. The solvent is evaporated to afford 15a1.

Step 2:

15a1 (69 mg, 0.12 mmol), /V-methylpyrrolidinone (0.9 mL), 6b2 (63.7 mg, 0.36 mmol), HATU (208 mg, 0.36 mmol) and 2,4,6-collidine (0.145 mL, 1.46 mmol) are charged in a 8 mL vial. The reaction mixture is stirred at RT for 2 h, and the product is purified by preparative RP-HPLC. The fractions are concentrated and lyophilized to afford 1017.

The following intermediate 15b1 and compounds 1018, 1020, 1021 and 1055 are prepared analogously to the procedure described in Example 15 starting from the appropriate intermediates.

Example 16

Preparation of compound 1022

9d1 (40 mg, 0.052 mmol) is dissolved in DMF (0.5 mL), and NBS (10.3 mg, 0.058 mmol) is added and solution is stirred at RT for 20 h. The resulting product is purified by preparative RP-HPLC, and the fractions are concentrated and lyophilized to afford 1022.

Example 17

9d1 (200 mg, 0.37 mmol) is dissolved in THF (2 mL) and MeCN (1 mL), and N- chlorosuccinimide (79.5 mg, 0.60 mmol) is added and solution is stirred at 50°C for

2 h. The resulting product is separated and purified by preparative RP-HPLC to afford 1023 following concentration and lyophilization of the combined fractions.

Compounds 1024 and 1025 are prepared analogously to the procedure described in

Example 17 starting from the appropriate intermediate.

Example 18

15t>1 18a1 18a2

Aryl bromide 15b1 (322 mg, 0.69 mmol) and 4-acetylphenylboronic acid 18a1 (Aldrich) (1 19 mg, 0.73 mmol) are charged in a flask to which is added DME (3.5 mL) and a 2M Na₂C0₃ aqueous solution (1 mL, 2 mmol). The flask is purged for 10 min by bubbling argon into the reacton mixture. Pd(dppf)CI₂ ^*CH₂Cl₂ (25 mg, 0.030 mmol) is added and the reaction mixture is heated at 90°C for 16h. The reaction mixture is cooled to RT and partitioned between DCM and water. The layers are separated and the aqueous phase is extracted twice with DCM. The combined organic layers are washed with brine, dried over MgS0₄ and concentrated. The product is purified by flash chromatography (0-10% methanol:DCM) to afford 18a2 (t_R = 1.86 min, (M+H)⁺ 507.3).

Example 18 starting from the appropriate bromo derivative and the appropriate boronic acid derivative.

Example 19

Preparation of compound 1026

Step 1 :

18c1 (500 mg, 1.0 mmol) is dissolved in THF (7 mL) and bromine (105 μΙ_, 2.1 mmol) is added. The solution is stirred at RT for 1 h. A saturated aqueous solution of NaHC0₃ is added followed by EtOAc. The layers are separated and the aqueous layer is extracted with EtOAc (3x). The organic layers are combined, washed with water, brine, dried over Na₂S0₄ and concentrated to afford 19a1 (t_R = 2.06 min, (M+H)⁺ 645; 647; 649). Step 2:

3a5 (66 mg, 0.25 mmol) is dissolved in MeCN (1 mL). 19a1 (1 16 mg, 0.18 mmol) and DIPEA (0.044 mL, 0.25 mmol) are added and the reaction mixture is stirred at RT for 4 h. The MeCN is evaporated, the mixture is placed into toluene (1.34 mL) and the salts are removed by filtration. NH₄OAc (208 mg, 1.8 mmol) is added and the solution is heated to 100°C for 16h. The reaction mixture is cooled to RT, concentrated in vacuo, diluted with EtOAc and washed with water and brine. The organic layer is dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography (0-10 % MeOH:DCM) affords 19a2 (t_R = 2.04 min, (M+H)⁺ 806.5; 808.5).

Step 3:

19a2 (72 mg, 0.09 mmol) is dissolved in 4M HCI solution in 1 ,4-dioxane (0.45 mL, 1.8 mmol). The reaction mixture is stirred at RT for 16 h, and the solvent is evaporated. The crude product, /V-methylpyrrolidinone (1.0 mL), 6b2 (1.5 M in NMP, 90 μΐ, 0.20 mmol), HATU (26 mg, 0.07 mmol) and 2,4,6-collidine (30 μΐ, 0.22 mmol) are charged in a 8 mL vial. The reaction mixture is stirred at RT for 2 h. The resulting product is purified by preparative RP-HPLC and the fractions are concentrated and lyophilized to afford 1026 (t_R = 1.99 min, (M+H)⁺ 863.6; 865.6).

Compound 1027 is prepared analogously to the procedure described in Example 19 starting from the appropriate ketone derivative and the appropriate carboxylic acid derivative.

Example 20

Preparation of compound 1028

Step 1 :

18c1 (68 mg, 1.3 mmol) is dissolved in a mixture of MeCN (5 mL) and THF (1.7 mL) and N- chlorosuccinimide (246 mg, 1.8 mmol) is added. The resulting solution is stirred at 50°C for 6 h, and concentrated in vacuo. Purification by flash chromatography (0-10% MeOH:DCM) affords 20a1 (t_R = 2.01 min, (M+H)⁺ 523.4; 525.4).

Step 2:

20a1 (693 mg, 1.3 mmol) is dissolved in THF (9 mL) and bromine (65 μί, 1.3 mmol) is added. The solution is stirred at RT for 24 h. A saturated aqueous solution of NaHC0₃ is added followed by EtOAc. The layers are separated and the aqueous layer is extracted with EtOAc (3x). The organic layers are combined, washed with water, brine, dried over Na₂S0₄ and concentrated in vacuo. Purification by flash chromatography (25-100% THF:hexanes) affords 20a2 (t_R = 2.06 min, (M+H)⁺ 601.3; 603.3).

Step 3: 20a2 (210 mg, 0.49 mmol) is dissolved in MeCN (2.2 mL). 3a5 (127 mg, 0.49 mmol) and DIPEA (0.085 mL, 0.49 mmol) are added and the reaction mixture is stirred at RT for 2 h. The MeCN is evaporated, then the mixture is placed into toluene (4.6 mL) and the salts are removed by filtration. NH₄OAc (750 mg, 9.7 mmol) is added and the solution is heated to 100°C for 16h. The reaction mixture is cooled to RT, concentrated in vacuo, diluted with EtOAc and washed with water and brine. The organic layer is dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography (0-100 %

EtOAc:hexanes) affords 20a3.

Step 4:

20a3 (373 mg, 0.49 mmol) is dissolved in 4M HCI solution in 1 ,4-dioxane (2.4 mL, 9.7 mmol) and the reaction mixture is stirred at RT for 16 h. The solvent is evaporated, and the crude product, /V-methylpyrrolidinone (1.0 mL), 6b2 (128 mg, 0.73 mmol), HATU (280 mg, 0.73 mmol) and 2,4,6-collidine (290 μί, 2.9 mmol) are charged in a 8 mL vial. The reaction mixture is stirred at RT for 5 h. Purification by flash chromatography (50% EtOAc in hexanes to 20% MeOH in EtOAc) followed by a second purification by preparative RP- HPLC, concentrating and lyophilizing the fractions affords 1028.

Compounds 1029-1054 are prepared analogously to the procedure described in Example 20 starting from the appropriate ketone derivative and the appropriate carboxylic acid derivative.

Example 21

Preparation of compound 1024

Step 1 :

4a9 (30.0 g, 132 mmol) and 21a1 (42.9 g, 132 mmol) are added to a 1 L RBF and the mixture is purged with argon. Anhydrous acetonitrile (300 mL) and diisopropylethylamine (57.5 mL, 330 mmol) are added. Stirring is initiated and the mixture is then agitated at RT until complete conversion (>99 A%, 220 nm with respect to 21a1). The mixture is concentrated via reduced pressure. Toluene (150 mL) is added to the mixture. The mixture is reduced to ~100 mL and is filtered through a filter frit into a 1 L RBF containing ammonium acetate (102 g, 1.32 mol). The original 1 L RBF is rinsed with 2x100 mL of toluene and the washes are passed through the solids within the filter frit. Stirring is initiated and the mixture is purged with argon. The mixture is heated to 98-102 °C and agitated for 6 h. The mixture is cooled to RT until complete conversion (>99 A%, 220 nm). The mixture is washed with water. The mixture is again concentrated to via reduced pressure. 1 ,4-dioxane (100 mL) is added and the mixture is reduced. 1 ,4-dioxane (150 mL) is added and the mixture is stirred. The mixture is purged with argon, heated to 45-52 °C and agitated until a homogeneous solution is obtained. 264 mL (1.06 mol) of a 4 N HCI solution in 1 ,4-dioxane is added drop-wise. The mixture is agitated at 45-52 °C until complete conversion (>99 A, 220 nm). The mixture is cooled to 10-12 °C and agitated at 10-12°C for 30 min; the solids are collected by filtration. The filtrate is charged back to the RBF and cooled to 10-12 °C. The filtrate is passed through the filter pad, and the resulting solids are washed with 1 ,4-dioxane (50 ml.) pre-cooled to 1 1-13 °C. The solids are suction dried for 1 h and dried in a vacuum oven at 50°C under house vacuum with a nitrogen bleed for 15 h. 21a2 (52.4 g) is recovered.

Step 2:

21a2 (10.0 g, 23.6 mmol), 6a2 (4.54 g, 25.9 mmol), TBTU (8.23 g, 25.9 mmol) are added to a 250 mL 2 neck RBF, and the mixture is purged with argon. /V,/V-dimethylformamide (75 mL) and diisopropylethylamine (20.5 mL, 118 mmol) is added to the mixture. The mixture is stirred and agitated at RT until complete conversion (>98 A% conversion, 220 nm with respect to 21a2). The mixture is diluted with ethyl acetate (120 mL) and transferred to a separatory funnel. The organic layer is washed with water (1x200 mL and then 1x150 mL). The combined water washes are extracted with dichloromethane (50mL). The organic layers are combined and concentrated via reduced pressure. The residue is subjected to flash silica chromatography (30-60 % EtOAc/Hexs) to produce 21a3 (12.6 g).

Step 3:

21a3 (12.2 g, 22.7 mmol) and /V-chlorosuccinimide (3.1 1 g, 23.3 mmol) are added to a 250 mL 2-3 neck RBF and purged with argon. Anhydrous acetonitrile (53 mL) is added to the mixture. The mixture is stirred and the temperature is adjusted to 72-77°C. The mixture is agitated at 72-77 °C for 7 h and cooled to RT until complete conversion (>98 A% conversion, 220 nm). The temperature is adjusted to -5 to 0 °C and the mixture is agitated for 30 min. The solids are collected by filtration. The filtrate is added back to the RBF and the temperature of the filtrate is adjusted to -5 to 0 °C. The solution is then passed through the filter medium. The solids are then washed with precooled (-5 to 0 °C) acetonitrile (20 mL). The solids are suction dried for 1 h and then dried in a vacuum oven (50 °C at house vacuum with a nitrogen bleed) for 10-15 h to afford 21a4 (10.2 g).

Step 4:

21a3 (12.6 g, 20.1 mmol), bis(pinacolato)diboron (6.31 g, 24.9 mmol), Pd(dppf)CI₂ complex with dichloromethane (733 mg, 1.00 mmol) and potassium acetate (8.86 g, 90.3 mmol) is added to a RBF (250 mL), and purged with argon. /V,/V-dimethylformamide (100 mL) is added to the mixture. The mixture is stirred, evacuated (house vacuum) and then purged with argon (3X). The temperature is then adjusted to 90°C and the mixture is agitated until complete conversion (>98 A% conversion, 220 nm). The mixture temperature is then adjusted to 18-22 °C. The mixture is transferred to a separatory funnel and diluted with isopropyl acetate (200 mL). The resulting solution is washed with water (200mL and then 50 mL). The organic layer is dried with anhydrous sodium sulfate, filtered and then concentrated via reduced pressure. The resulting material is subjected to flash silica chromatography (short column, 40-60% EtOAc/hexs) to produce 21a5 (7.80 g).

Step 5:

21a4 (7.20 g, 13.1 mmol), 21a5 (7.54 g, 13.8 mmol), Pd(dppf)CI₂ complex with dichloromethane (480 mg, 0.59 mmol), sodium carbonate (6.96 g, 65.7 mmol) is added to a RBF (500 mL), and the mixture is purged with argon. 1 ,2-dimethoxyethane (75 mL) and water (25 mL) are added to the mixture. The mixture is stirred, evacuated and purged with argon (3X). The temperature is adjusted to 72-77°C, and the mixture is agitated until complete conversion (>98 A% conversion, 220 nm, with respect to 21a4). The temperature is adjusted to 18-22 °C and the mixture is diluted with isopropyl acetate (150 mL) and water (150 mL). The resulting bi-phasic solution is mixed for 10 min, and the resulting solution is passed through a short celite plug. The aqueous phase is cut and the resulting organic layer is washed with water (150 mL) and then concentrated via reduced pressure. The residue is subjected to flash silica chromatography (EtOAc) and further dried at 9-15 mbar at 55 °C to afford 1024 (9.81 g).

Example 22

HCV replicon RNA replication assay

HCV replicons:

Two subgenomic replicons designated HCVPVI a and HCVPVI b are generated based on the wildtype sequence for genotype 1 a, strain H77 (GenBank accession no. AF009606) and the wildtype sequence CON-1 genotype b (GenBank accession number AJ238799), see Science 1999, 285: 1 10-1 13. HCV genotype 1 a subgenomic fragment NS2-NS3- NS4A-NS4B-NS5A-NS5B is drawn from the reference nucleic acid encoding residues 81 1 to 301 1 of AF009606, HCV genotype 1 b subgenomic fragment NS2-NS3-NS4A-NS4B- NS5A-NS5B is drawn from the reference nucleic acid encoding residues 81 1 to 3010 of CON-1 (GenBank accession number AJ238799). Both subgenomic replicons contain a hybrid HCV-poliovirus (PV) 5'UTR, a modified luciferase reporter gene expressed as a luciferase-FMDV2A-neomycin phosphotransferase gene fusion and a NS2-NS5B subgenomic fragment with its 3'UTR. The replication of both HCV NS2-NS5B subgenomic replicons is enhanced by cell-culture adaptive mutations in the NS3 and the NS4B coding regions for the genotype 1 a replicon and in the NS3, NS4A and NS5A coding regions for the genotype 1 b.

Stable replicon cell lines are established as described, for example, in Science 1999, 285: 1 10-1 13. The amount of luciferase expressed by selected cells directly correlates with the level of HCV RNA replication, as measured by real-time PCR.

HCV replicon RNA replication assay: To generate cell lines harboring the replicon containing the NS3 substitutions, Huh-7 cells are electroporated with 1-10 of purified in vitro transcripts and stable cell lines are selected in the presence of G418 (0.25 mg /ml).

The stable HCV replicon cells are maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 0.25 mg/ml G418 (standard medium). During the assay, DMEM supplemented with 10% FBS, containing 0.5% DMSO and lacking neomycin are used as assay medium.

For the assay, the cell stocks are trypsinized and diluted in assay medium to distribute 70 μΙ (8,000 ells) in black 96-well plates. The plates are then incubated at 37° until compound addition. The test compound in 100% DMSO is first diluted in assay medium to a final DMSO concentration of 0.5%. Serial dilutions are prepared in assay medium to generate nine-concentration dose response curves. A fixed volume from each well of the compound dilution plate is transferred to a corresponding well of the cell culture plate. The cell culture plate is incubated at 37°C with 5% C0₂ for 72 hours. Following the 72h incubation period, the medium is aspirated from the 96-well assay plate and a volume of 50 μΙ of 1X Glo Lysis Buffer (Promega) is added to each well. The luciferase activity is determined using Bright- Glo luciferase substrate (Promega) according to the manufacturer's instructions and the luminescence is detected on a Packard Topcount instrument. The luminescence (CPS) in each well of the culture plate is a measure of the amount of HCV RNA replication in the presence of various concentrations of inhibitor. The % inhibition is calculated for each inhibitor concentration and used to determine the concentration that results in 50% inhibition of HCV replication (EC₅₀).

Selected compounds listed in Table 1 below are tested in the assay of Example 22. TABLE 1

EXAMPLE 23

Potency against HCV NS5A resistant variants Q30E and L31 V:

Production o†HCVNS5A replicons:

HCVPVI a is subgenomic replicon genotype 1 a (strain H77) used to generate replicons containing the Q30E and L31V resistant variants in the HCV NS5A gene. HCVPVI a contains a hybrid HCV-poliovirus (PV) 5'UTR, a modified luciferase reporter gene expressed as a luciferase-FMDV2A-neomycin phosphotransferase gene fusion and a NS2- NS5B subgenomic fragment with its 3'UTR. Replication of the HCV NS2-NS5B subgenomic replicon is enhanced by cell-culture adaptive mutations in the NS3 and NS4B coding regions and the amount of luciferase expressed by selected cells directly correlates with the level of HCV replication, as measured by real-time PCR . Stable replicon cell lines are established as taught by Lohman et al., 1999. Science 285: 1 10-113 and the desired amino acid substitutions are introduced by site-directed mutagenesis using Quick Change (Agilent Technologies Inc., Stratagene Products) according to the manufacturer's instructions. The stable replicon cell lines are generated by transfection of in vitro transcribed replicon RNA and selection of replicating replicon containing cells in the presence of Geneticin® G418 (Invitrogen).

SEQ ID NO: 1 is a nucleotide sequence representing the HCV genotype 1 a subgenomic fragment NS2-NS3-NS4A-NS4B-NS5A-NS5B with resistant variant Q30E in the NS5A portion. SEQ ID NO: 1 is 6609 bases wherein nucleotide bases 1-651 of SEQ ID NO:1 encode NS2, nucleotide bases 652-2544 encode NS3, nucleotide bases 2545-2706 encode NS4A, nucleotide bases 2707-3489 encode NS4B, nucleotide bases 3490-4833 encode NS5A, and nucleotide bases 4834-6606 encode NS5B. NS5A mutation Q30E is encoded by the codon of bases 3577-3579 of SEQ ID NO:1. SEQ ID NO: 2 is the corresponding polypeptide with adaptive mutations introduced in the NS3 and NS4B coding regions over the reference sequence. The reference sequence is GenBank accession number AF009606, residues 81 1 to 301 1 wherein residue 81 1 corresponds to residue 2 in SEQ ID NO: 2, residue 1 being methionine. The adaptive mutations are at residues 471 , 549, 622, 1000 and 1030 of SEQ ID NO: 2 and the HCV NS5A resistant mutation Q30E corresponds to residue 1 193 of SEQ ID NO: 2.

SEQ ID NO: 3 is a nucleotide sequence representing the HCV genotype 1 a subgenomic fragment NS2-NS3-NS4A-NS4B-NS5A-NS5B with resistant variant L31 V in the NS5A portion. SEQ ID NO: 3 is 6609 bases wherein nucleotide bases 1-651 of SEQ ID NO:3 encode NS2, nucleotide bases 652-2544 encode NS3, nucleotide bases 2545-2706 encode NS4A, nucleotide bases 2707-3489 encode NS4B, nucleotide bases 3490-4833 encode NS5A, and nucleotide bases 4834-6606 encoded NS5B. NS5A mutation L31V is encoded by the codon of bases 3580-3582 of SEQ ID NO: 3. SEQ ID NO: 4 is the corresponding polypeptide with adaptive mutations introduced in the NS3 and NS4B coding regions over the reference sequence. The reference sequence is GenBank accession number AF009606, residues 81 1 to 301 1 where 81 1 corresponds to residue 2 in SEQ ID NO:4, residue 1 being methionine. The adaptive mutation are at residues 471 , 549, 622, 1000 and 1030 of SEQ ID NO: 4 and the HCV NS5A resistant mutation L31V corresponds to residue 1 194 of SEQ ID NO: 4.

HCV replicon RNA replication assay: The stable HCV replicon cells are maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 0.25 mg/ml G418 (Standard Medium), while the replication assay uses DMEM supplemented with 10% FBS, containing 0.5% DMSO and lacking G418 (Assay Medium).

The cell stocks are trypsinized and diluted in Assay Medium; 70 μΙ (about 8,000 cells) are distributed in black 96-well plates. The plates are then incubated at 37° until compound addition. The test compound in 100% dimethyl sulfoxide (DMSO) is first diluted in Assay Medium to a final DMSO concentration of 0.5%. Serial dilutions are prepared in Assay Medium to generate nine concentrations dose response curves. A fixed volume from each well of the compound dilution plate is transferred to a corresponding well of the cell culture plate. The cell culture plate is incubated at 37°C with 5% C0₂ for 72 h. Following the 72 h incubation period, the medium is aspirated from the 96-well assay plate and a volume of 50 μΙ of 1X Glo Lysis Buffer (Promega) is added to each well. Luciferase activity is determined using Bright-Glo luciferase substrate (Promega) according to the manufacturer's instructions and the luminescence detected on a Packard Topcount instrument.

Luminescence (counts per second CPS) from each well of the culture plate is a measure of the amount of HCV RNA replication in the presence of various concentrations of inhibitor. The % inhibition is calculated for each inhibitor concentration and used to determine the concentration that results in 50% inhibition of HCV replication (EC₅₀) as a measure of potency against the NS5A genotype 1 a resistant variants by the non-linear regression routine Symyx® Assay Explorer 3.2 SP1.

Table 2 provides the EC₅₀ determined for the compounds of the invention against each of HCV NS5A genotype 1 a resistant variants Q30E and L31V when measured according to the assay, together with the EC₅₀ of two prior art compounds as determined according to the same assay.

TABLE 2

Each reference, including all patents, patent applications, and publications cited in the present application is incorporated herein by reference in its entirety, as if each of them is individually incorporated. Further, it would be appreciated that, in the above teaching of invention, the skilled in the art could make certain changes or modifications to the invention, and these equivalents would still be within the scope of the invention defined by the appended claims of the application.

Claims

1. A compound of F

wherein:

ring A and A' are each independently optionally substituted one time with halo;

R¹ is halo and R² is hydrogen; or

R¹ is hydrogen and R² is halo;

3 is selected from the group consisting of:

R⁴ is selected from (C^alkyl optionally substituted one time with -0-(C-|.₆)alkyl or -0-(Ci_₆)haloalkyl;

R⁵ is either absent or selected from the group consisting of -0-(Ci.₆)alkyl and

-S-(C_{1 -6})alkyl;

6 is selected from the group consisting of:

R⁷ is selected from (C^alkyl optionally substituted one time with -0-(C-|.₆)alkyl or -0-(Ci_₆)haloalkyl;

R⁸ is either absent or selected from the group consisting of -0-(C-|.₆)alkyl and -S-(C_{1 -6})alkyl.

2. The compound of Formula (I) according to claim 1 , wherein Ring A and A' are each unsusbtituted.

3. The compound of Formula (I) according to claim 1 or 2, wherein R¹ is F or CI and R² is hydrogen; or R¹ is hydrogen and R² is F or CI.

4. The compound of Formula (I) according to any one of claims 1 to 3, wherein R³

R⁴

; and

R⁵ is either absent or selected from the group consisting of -0-(Ci_₃)alkyl and -S-(Ci_₃)alkyl.

5. The compound of Formula (I) according to any one of claims 1 to 4, wherein R⁶

R⁷

; and

R is either absent or selected from the group consisting of -0-(C-|.₃)alkyl and -S-(Ci_₃)alkyl. 6. A compound of Formula (II) or salt thereof:

wherein:

ring A and A' are each independently optionally substituted one time with halo;

R⁴ is selected from (C _₆)alkyl optionally substituted one time with -0-(C-|.₆)alkyl or -0-(C-|.

₆)haloalkyl;

R⁵ is -S-(C_{1 -6})alkyl;

6 is selected from the group consisting of:

-0-(Ci_₆)haloalkyl;

R⁸ is either absent or selected from the group consisting of -0-(C-|.₆)alkyl and-S-(C-|.₆)alkyl.

7. The compound of Formula (II) according to claim 6, wherein Ring A and A' are each unsusbtituted.

8. The compound of Formula (II) according to claim 6 or 7, wherein R⁴ is selected from (C-|.₄)alkyl optionally substituted one time with -0-(C-i_₃)alkyl.

9. The compound of Formula (II) according to any one of claims 6 to 8, wherein R⁵ is - S-(C,)alkyl.

10. The compound of Formula (II) according to any one of claims 6 to 9, wherein R⁶ is selected from the group consisting of:

R⁷ is selected from (C-|.₄)alkyl optionally substituted one time with -0-(C-|.₃)alky; and R⁸ is either absent or -S-(Ci)alkyl.

11. The compound according to claim 1 or 6, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

12. A compound of Formula (I) according to claim 1 or a compound of Formula (II) according to claim 6, or a pharmaceutically acceptable salt thereof, as a medicament.

13. A pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of Formula (I) according to claim 1 or a compound of Formula (II) according to claim 6, or a pharmaceutically acceptable salt thereof, in admixture with at least one pharmaceutically acceptable carrier medium or auxiliary agent.

14. The pharmaceutical composition according to claim 13 further comprising a therapeutically effective amount of at least one other antiviral agent.

15. Use of a compound of Formula (I) according to claim 1 or a compound of Formula (II) according to claim 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of hepatitis C viral infection in a human being.