WO2019235919A1

WO2019235919A1 - Use of 6'-fluorq-neplanocin a derivatives against chikungunya virus

Info

Publication number: WO2019235919A1
Application number: PCT/NL2019/050328
Authority: WO
Inventors: J. Martijn VAN HEMERT; Kristina KOVACIKOVA; Eric J. Snijder; Ji-Seong Yun; Lak Shin Jeong; Dnyandev B. JARHAD; Gyudong Kim; Pramod Kumar SAHU; Hong-Rae Kim
Original assignee: Seoul National University; Future Medicine Co Ltd; Leids Universitair Medisch Centrum LUMC
Current assignee: Seoul National University; Future Medicine Co Ltd; Leids Universitair Medisch Centrum LUMC
Priority date: 2018-06-04
Filing date: 2019-06-04
Publication date: 2019-12-12
Anticipated expiration: 2020-12-04
Also published as: NL2021049B1

Abstract

The present invention relates to a compound of formula (I): wherein R is -H or-F, P is -H or -a phosphoramidate, n is r 2, X is -NH2, -NHR or -OH, Y is -H or -NH2 and Z is -CH or -N, or a pharmacologically acceptable salt, or a pharmacologically acceptable salt, solvate, tautomeric and/or polymorphic form of the following compound (I), for use against alphavirus infections, in particular against CHIKV infections.

Description

l

USE OF 6'-FLUORQ-NEPLANOCIN A DERIVATIVES AGAINST CHIKUNGUNYA VIRUS

Field of the Invention

The present invention relates to neplanocin A derivatives, particularly 6'-fluoro neplanocin A derivatives, for use against alphavirus infections, particularly chikungunya virus (CHIKV) infections.

Background ofthe Invention

Over the past 15 years, outbreaks of a number of emerging positive-stranded RNA viruses like the severe acute respiratory syndrome coronavirus (SARS-CoV) virus, Middle East respiratory syndrome coronavirus (MERS-CoV), chikungunya virus (CHIKV), and Zika virus (ZIKV) have seriously threatened human health and had a substantial socio economic impact. CHIKV is transmitted by mosquitos and causes severe arthritis that can persist for months. Currently, there are no effective chemotherapeutic agents or vaccines to prevent or cure CHIKV infections.

Chandra et al Journal of Medicinal Chemistry 2015 58, 5108-5120 and Kim et al

Bioorganic & Medicinal Chemistry Letters 2004 14, 2091-2093 have described the anti viral activity of certain neplanocin A derivatives. However, these compounds have not shown enhanced selectivity and/or activity against alphavirus infections, in particular chikungunya viruses.

Accordingly, an effective chemotherapeutic agent to prevent and/or cure CHIKV infections, as well as other alphaviruses, has continued to be sought.

Summary of the Invention

The present invention relates to a compound of formula (I):

wherein R is -H or -F, P is -H or -a phosphoramidate, n is 2, X is -NH₂, -NHR or -OH, Y is -H or -NH₂ and Z is -CH- or N, or a pharmacologically acceptable salt of the compound, for use against alphavirus infections, particularly CHIKV infections. Advantageously, P is -H in the compound of formula (I). Also advantageously, X is

-NH₂, Y is -H and Z is N in the compound. Also advantageously, R is -F in the compound.

More advantageously, the compound is of the formula (la):

wherein X is as above, advantageously -NH₂, and Y is as above, advantageously -H.

In another aspect, the present invention also relates to a pharmaceutical composition of the compound of formula (I) or of a pharmacologically acceptable salt or a pharmacologically acceptable salt solvate, tautomeric form and/or polymorphic form thereof for use in the prevention or treatment of alphavirus infections, particularly CHIKV infections.

In a further aspect, the present invention also relates to a method of therapeutic or prophylactic treatment of a subject against viral infection comprising administering a therapeutically effective amount of at least one compound according to the invention, to a subject in need of antiviral therapeutic or prophylactic treatment.

Short Description of the Figures

Fig. 1. shows a picture of CHIKV positive-stranded genomic (gRNA) and subgenomic RNA (sgRNA) and negative strand (-RNA) levels in CHIKV-infected Vero E6 cells at 15 hpi, as detected by in gel hybridization with specific radioactive probes. Herein, 18S ribosomal RNA was used as loading control and treatment with 20 uM of 6-azauridine, a known inhibitor of CHIKV replication as described by Scholte FE, Tas A, Martina BE, Cordioli P, Narayanan K, Makino S, Snijder EJ, van Hemert MJ. 2013. "Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates. PLoS ONE 8:e71047" was included as positive control. Mock- infected cells were included as negative control.

Fig.2. shows infectious progeny titers at 20 hpi of CHIKV-infected Vero E6 cells that were left untreated (UTR) or were treated with various concentrations of COMPOUND A. CHIKV titers were determined by plaque assay as described by Scholte et al, 2013 (1). Fig. 3. Shows a time-of-addition assay. CHIKV-infected Vero E6 cells were treated with IOmM of COMPOUND A starting 12 h before infection (-12) until 14 h p.i. Untreated (UTR) cells were included as controls.

A) Infectious progeny titers at 16 hpi were determined by plaque assay.

B) Extracellular CHIKV RNA copy numbers were determined by RT-qPCR as described by Scholte FE, Tas A, Albulescu 1C, Zusinaite E, Merits A, Snijder EJ, and van Hemert MJ. 2015. Stress Granule Components G3BP1 and G3BP2 Play a Proviral Role Early in Chikungunya Virus Replication. Journal of virology 89: 4457-4469.

Detailed Description of the Invention

The embodiments of this invention are not limited to particular compounds, methods of preparation and/or treatment, which can vary and are understood by skilled artisans. It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an" and "the" can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range format.

It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, the term "alkyl" or "alkyl groups" refers to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert- butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). Unless otherwise specified, the term "alkyl" includes both "unsubstituted alkyls" and

"substituted alkyls."

As used herein, the term "substituted alkyls" refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups. In some embodiments, substituted alkyls can include a heterocyclic group. As used herein, the term "heterocyclic group" includes closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur or oxygen.

Heterocyclic groups can be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane (episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane, dithietane, dithietine, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan. The term "substituted" as referred to herein refers to a substitution at a carbon (or nitrogen) position mentioned, with the referenced group, which can include hydroxyl, carboxyl, cyano, nitro, halogen(s), thiol, one or more alkyl groups,

preferablyone or more C₁-C₆ alky groups, alkoxyl group, preferably C₁-C₆ alkyl or aryl, including phenyl alkoxyl groups, esters, including alkylene esters, thioethers, thioester, nitro or amines, alkanol, alkanoic acids or the like.

The term "weight percent," "wt-%," "percent by weight," "% by weight," and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, "percent," "%," and the like are intended to be synonymous with "weight percent," "wt-%," etc.

The compounds, compositions, and methods of the present invention can comprise, consist essentially of, or consist of the components and steps disclosed herein as well as other components and steps described herein. As used herein, "consisting essentially of" means that the compounds, compositions, and methods can include additional components and steps, but only if the same do not materially alter the basic and novel characteristics of the claimed compounds, compositions, and methods. In accordance with this invention, a compound of formula (I):

wherein R is -H or-F, P is -H or -a phosphoramidate, n is 2, X is -NH₂, -NHR or -OH, Y is -H or -NH₂ and Z is -CH or -N, or a pharmacologically acceptable salt of the compound (I) can be used to treat or prevent an alphavirus infection, particularly a CHIKV infection. The phosphoramidate can be substituted and preferably comprises one or more substituted alkyl substituents.

In a preferred embodiment, the compound of formula (l)as follows: P represents - H; and/or X is -NH₂, Y is -H and Z is -N; and/or R is -F. More preferably, the compound is of the formula (la):

wherein X is -NH₂, -NHR or -OH, preferably -NH₂, and Y is -H or -NH₂, preferably -H.

The compounds of formula (I) can all be made in a well-known manner as described, for example in US patent 9,120,760. In this regard, 2,2-Dimethyl-propionic acid 2-[(2R,3S,4S)-4-(6-chloro-purine-9-yl)-5-fluoro-2, 3-(0-(0-lsopropylidenedioxy))- l(5)-en]-ethyl ester (Compound A) can be made by reacting 2,2-Dimethyl-propionic acid2-((3S,4R,5S)-2-fluoro-4,5-(0-isopropylidenedioxy)-3-ol-cyclopent-l(2)-en]-ethyl ester (Compound B) with 6-chloro-purine ( 2,2-Dimethyl-propionic acid 2-[(2R,3S,4S)-4- ( 6-chloro-purine-9-yl)-5-fluoro-2, 3-dihydroxy cyclopent-l(5)-en]-ethyl ester (Compound C) can then be made by reacting Compound A with an acid; and the neplanocin A derivatives of formula (I) can then be made by reacting 2,2-Dimethyl-propionic acid 2- [(2R,3S,4S)-4-(6-chloro-purine-9-yl)-5-fluoro-2, 3-dihydroxy cyclopent-l(5)-en]-ethyl ester (Compound C) with NH₃ or NH₂(CH₃) and then treating with NaOMe to remove the protecting group.

In addition, the present invention provides a pharmaceutical composition of the compound of formula (I) or of a pharmacologically acceptable salt thereof for prevention or treatment of alphavirus infections, particularly CHIKV infections. The pharmaceutical composition can further comprise a pharmacologically or pharmaceutically acceptable carrier.

As used herein, the term "patient" preferably means any recipient of a compound of formula (I) or a pharmacologically acceptable salt or a pharmaceutical composition thereof.

As used herein, the terms "subject" and "patient" may be used interchangeably herein.

The terms "subject" and "subjects" may refer to an animal, such as a mammal including a non-primate, e.g., a cow, pig, horse, cat, dog, rat, and mouse, and a primate {e.g., a monkey such as a cynomolgous monkey, a chimpanzee and a human, and for example, a human. In certain embodiments, the subject is refractory or non-responsive to current treatments for CHIKV infection. In another embodiment, the subject is a farm animal, e.g., a horse, a cow, a pig, etc., or a pet, e.g., a dog or a cat. In certain

embodiments, the subject is a human.

The term "host," as used herein, refers to any unicellular or multicellular organism in which an alphavirus, such as a Chikungunya virus, can replicate, including cell lines and animals, and in certain embodiments, a human. Alternatively, the host can be carrying a part of a viral genome, such as a Chikungunya viral genome, whose replication or function can be altered by the compounds of the present invention. The term "host" specifically includes infected cells, cells transfected with all or part of a viral genome, such as a Chikungunya viral genome, and animals, in particular, primates, including chimpanzees and humans. In most animal applications of the present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly anticipated by the present invention such as for chimpanzees.

As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) which can be used in the treatment or prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" includes a compound provided herein. In certain embodiments, a therapeutic agent is an agent which is known to be useful for or has been or is currently being used for the treatment or prevention of a disorder or one or more symptoms thereof.

"Therapeutically effective amount" refers to an amount of a compound or composition that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" can vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

"Treating" or "treatment" of any disease or disorder refers, in certain

embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, "treating" or "treatment" includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, "treating" or "treatment" includes modulating the disease or disorder, either physically, e.g., stabilization of a discernible symptom, or physiologically, such as stabilization of a physical parameter, or both. In yet another embodiment, "treating" or "treatment" includes delaying the onset of the disease or disorder.

As used herein, the terms "prophylactic agent" is used refer to any agent(s) which can be used in the prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term "prophylactic agent" includes a compound provided herein.

The term "prophylactic agent" for example may be an agent useful for preventing or impeding the onset, development, progression and/or severity of a disorder.

As used herein, the phrase "prophylactically effective amount" refers to the amount of a therapy, e.g., prophylactic agent, which is sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with a disorder, or to enhance or improve the prophylactic effect(s) of another therapy, e.g., another prophylactic agent.

Also as used herein, the term "phosphoramidate" preferably means a conventional phosphoramidate group as described, for example, in US patent 7964580B1, more preferably a group of the formula:

Also as used herein, the term "alphavirus" preferably means any virus of the group IV Togaviridae family of viruses, such as a chikungunya virus, further referred to herein as CHIKV and the Semliki Forest virus (SFV), particularly CHIKV.

Also as used herein, the term "compound of formula (I)" preferably includes crystalline and amorphous forms for the compound. In crystalline form, the compound can be randomly hydrated or solvated and can contain stoichiometric amounts of water, as well as lesser amounts of water.

Also as used herein, the term "pharmacologically acceptable salt" preferably means a salt of a compound of formula (I), prepared by using conventional procedures known to persons skilled in the art. Specifically, pharmacologically acceptable salts include, but the term is not limited to a salt derived from a pharmacologically and/or physiologically acceptable, organic or inorganic acid or base. The salt can be derived from a suitable acid, such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. The salt can also be derived from a suitable base, such as a hydroxide of an alkali metal, such as sodium or potassium, or an alkali earth metal, such as magnesium.

The term "pharmaceutically acceptable salt" herein refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric,

methanesulfonic, ethanesulfonic, 1 ,2-ethane-disulfonic, 2- hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4- toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic,

glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminium ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, N- methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,

tetramethylammonium hydroxide, and the like.

Pharmaceutically acceptable salts further include, by way of example only and without limitation, sodium, potassium, calcium, magnesium, ammonium,

tetraalkylammonium and the like, and when the compound contains a basic

functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4- hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),

ethanesulfonate, 1 ,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4- toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-l- carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butyl acetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate,

cyclohexylsulfamate, quinate, muconate and the like.

As used herein, the term ECso refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

As used herein, the term CC50 refers to the cytotoxic concentration to cause death to 50% of viable cells in the host. These values are measured through dilution assays of compounds added to virus infected host cells.

In order to use the compound of formula (I) for the treatment or prevention of alphavirus infections, particularly CHIKV infections, the compound can be formulated as a pharmaceutical composition using well-known methods to provide rapid, continued, or delayed release of the compound after its administration to a patient. The use of a pharmaceutical composition of the compound of formula (l)_can involve enteral and/or parenteral (e.g., intravenous or subcutaneous or intramuscular) treatment of a human patient. The dosage administered will generally depend on the condition of the patient, as well as the patient's weight and the progression of the alphavirus infection.

In preparing a dosage form of a pharmaceutical composition of the compound of formula (I) for use in treating or preventing alphavirus infections, it is preferable to mix or dilute the compound with a carrier or seal it in a carrier in a container. The compound can be formulated in a conventional manner for oral administration in the form of a powder, granule, tablet, capsule, solution, suspension, emulsion, syrup or aerosol and for external administration in the form of a suppository or sterile injection. Specifically, it is possible to prepare the compound for administration using a conventional diluent, inert filler, weighting agent, bonding agent, wetting agent, disintegrating agent and/or or surfactant agent. For oral administration, a tablet, pill, powder, granule, and capsule, can be formulated with one or more diluting agents, such as starch, calcium carbonate, sucrose, lactose and/or or gelatine, as well as a lubricant, such as magnesium stearate or talc, and a diluting agent, such as a wetting agent, a sweetening agent, a flavouring agent, or a preserving agent.

Dietetic water or liquid paraffin can be used for oral administration of the compound in liquid form. For external, i.e, non-oral, administration, possible

formulations of the compound can include sterile water or a non-aqueous solvent, a suspending agent, an emulsifier, a lyophilization agent and/or a suppository base.

Vegetable oils such as propylene glycol, polyethylene glycol, or olive oil, or an ester such as ethyl oleate, can be used as the non-aqueous solvent. Witepsol, macrogol, Twin 61, cacao butter, laurinum, or glycerogelatin can be used as the suppository base.

The present invention preferably also relates to a method of therapeutic or prophylactic treatment of a subject against viral infection comprising administering a therapeutically effective amount of at least one compound of the invention to a subject in need of antiviral therapeutic or prophylactic treatment. Preferably, the administering is by ingestion, injection, infusion, or other bodily administration. Preferably the virus is an alphavirus of the group IV Togaviridae family of viruses. Preferably the alphavirus is a Chikungunya virus.

In one aspect, the compounds provided herein may advantageously be provided or administered in combination with a second therapeutic agent, such as one useful for the treatment or prevention of alphavirus infections. Exemplary second therapeutic agents are provided in detail elsewhere herein.

In another aspect, provided herein are pharmaceutical compositions, single unit dosage forms, and kits suitable for use in treating or preventing disorders such as alphavirus of the group IV Togaviridae family of viruses' infections, in particular wherein the alphavirus infection is a Chikungunya virus infection. The pharmaceutical

composition preferably may comprise a therapeutically or prophylactically effective amount of a compound provided herein, e.g., of Formula I or la, and preferably, a therapeutically or prophylactically effective amount of a second therapeutic agent such as one useful for the treatment or prevention of Chikungunya infections.

Preferably the amount of compound I administered is an antiviral effective amount sufficient to improve, inhibit, prevent or ameliorate the viral infection. Preferably the antiviral effective amount is an amount that prevents the occurrence or one or more symptoms of the infection or reduces the severity of, or the length of time during which the subject suffers from, one or more symptoms of the infection by at least 50%.

Preferably, the present invention also relates to a pharmaceutical composition for treating a subject with a viral infection or in need of prophylactic antiviral treatment comprising: an antiviral sufficient amount of a compound of the invention, to produce antiviral effects. Preferably the compound is formulated into tablets, gel capsules, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a diluent, adjuvant, excipient, vehicle or combinations thereof. Preferably the antiviral amount of the compound is an amount sufficient to improve, inhibit, prevent or ameliorate the viral infection.

Preferably the antiviral effective amount is an amount that prevents the occurrence or one or more symptoms of the infection or reduces the severity of, or the length of time during which the subject suffers from, one or more symptoms of the infection by at least 50%. Preferably the antiviral effective amount is an amount that reduces viral replication or kills viral cells by at least 50%. Preferably, the present invention also relates to a method for preparing a compound, according to the invention, comprising providing a precursor compound comprising one or more protective groups, and removing the protecting groups.

The following, non-limiting examples illustrate the effectiveness of the compounds according to the invention.

Example 1: Effectiveness of Compound A

Compound A was provided, which is a compound according to formula (I), wherein

R is F, P is -H, n is 2, X is -NH₂, Y is -H, and Z is N.

Cells and viruses

VeroE6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM;

Lonza), supplemented with 8% fetal calf serum (FCS; PAA), 2 mM L-glutamine, 100 ILJ/ml of penicillin and 100 pg/ml of streptomycin and were grown at 37°C in a humidified incubator with 5% CO2. Infections were performed in Eagle's minimal essential medium (EMEM;

Lonza) with 25 mM HEPES (Lonza) supplemented with 2% FCS, L-glutamine, and antibiotics. Infectious clone-derived chikungunya virus (CH1KV-LS3) was synthesized as described by Scholte et a I, 2013 (1). Semliki Forest virus (SFV) strain SFV4 was used, and SINV strain HR LP was used. Compound A was dissolved in DMSO (10 mM stocks). All work with infectious CHIKV was performed inside biosafety cabinets in biosafety level 3 facilities at Leiden University Medical Center.

Cytopathic effect (CPE) reduction assays

VeroE6 cells were seeded at a density of 5,000 cells/well (CHIKV) or 10,000 cells/well (SFV) in a 100 mI total volume in 96-well clusters. The following day, 2-fold dilutions of

COMPOUND A or control compounds were prepared in infection medium. After replacing medium with the respective dilutions of the compound, the cells were infected in quadruplicate with CHIKV-LS3 (MOI 0.005) or SFV (MOI 0.025). Viability assays were run in parallel. After 40 hours post-infection (hpi) for SFV or 96 hpi for CHIKV, an MTS colorimetric assay was started by adding 20pl/well of CellTiter 96^® AQu_eous One Solution Cell Proliferation Assay (MTS) reagent (Promega). The assay was stopped after 2-2.5 hours by fixing with 37% formaldehyde. The absorbance was measured at 495nm in a Berthold Mithras LB 940 plate reader and the values were expressed relative to uninfected (infection) or untreated

(viability) cells. The results represent an average of quadruplicate wells expressed as mean ±SD. EC50s were determined using Graphpad Prism.

RNA isolation, gel electrophoresis, and in-gel hybridization

CHIKV RNA isolation using acid phenol, denaturing gel electrophoresis, and detection by ingel hybridization with 32P-labeled oligonucleotides specific for positive or negative strand RNA were performed as described previously.

RT qPCR

CHIKV RNA copy numbers were determined by RT-qPCR as described by Scholte et al, 2015

(2).

Results

Compound A strongly inhibited CHIKV replication in CPE reduction assays with an average EC50 of 0.18 mM (see Table 1).

Table 1. ECso and CC₅₀ values of Compound A for different alphaviruses, determined by CPE reduction assays on Vero E6 cells. All values are in mM.

The CCso of the compound was over 250 mM, resulting in a very high selectivity index (SI) of >1300. SFV was less sensitive to the compound (EC50 of 5.2 mM; Table 1), while SINV was not inhibited by the compound at all.

CHIKV RNA levels at 15 hpi were analyzed in a single infectious cycle dose-response experiment with Vero E6 cells that were infected at an MOI of 1 and treated with various concentrations of Compound A. Compared to untreated cells (Fig 1., 0 mM), increasing concentrations led to a strong reduction in the levels of intracellular positive- and negative- stranded RNA levels CHIKV RNA (Fig. 1). The treatment of CHIKV-infected Vero E6 cells (MOI 1) with Compound A led to a dose dependent decrease in the production of infectious progeny (Fig. 2). Treatment of cells with 10 mM of the compound led to a 3-log reduction in the production of infectious virus.

Time-of-addition experiments with 10 mM Compound A demonstrated that the compound inhibited CHIKV replication when treatment was initiated as late as 6 hpi, suggesting it inhibits a post entry step of the CHIKV replication cycle (Fig. 3). In particular, pretreatment of cells with Compound A led to efficient inhibition of CHIKV, likely due to a loading effect or the build-up of S-adenosyl-homocysteine levels in the cell. It is important to note that Compound A led to a strong reduction in infectious progeny titers (Fig. 3A), while the effect on the total amount of CHIKV RNA that was released from the cell was less strong (Fig. 3B), as can be most clearly seen for the -12 h pretreated samples (Fig. 3). This increase in specific infectivity (i.e. the ratio between the quantity of genomic RNA (copies) and infectious units (PFU) per ml) suggests the compound interferes with the infectivity of the viral RNA. Selection of Compound A-resistant viruses, genotyping and reverse genetics studies indicated that 2 mutations (G230R and K299E) in nsPl are required to confer resistance to the compound, demonstrating that nsPl is the target of the compound. The compound likely interferes with the capping of viral RNA by nsPl, which is supported by the fact that Compound A treatment resulted in a strong reduction of infectious progeny titers (Fig 3A), while there was little effect on the total amount of viral RNA that was released from the infected cell (Fig. 3B).

This suggests that a large proportion of non-infectious particles was produced, which might contain uncapped genomes that will not be translated by the host cell and therefore cannot start a new round of replication. The fact that two mutations are required means there is a high genetic barrier to resistance.

Claims

2. wherein R is -H or-F, P is -H or -a phosphoramidate, n is 2_; X is -NH₂, -NHR or -OH, Y is -H or -NH₂ and Z is -CH or -N, or a pharmacologically acceptable salt, solvate, tautomeric and/or polymorphic form of the compound (I), for use against alphavirus infections. The compound of claim 1, wherein the alphavirus is an alphavirus of the group IV Togaviridae family of viruses.

3. The compound of claim 2, wherein the alphavirus is a Chikungunya (CHIKV)

virus.

4. The compound of any one of claims 1 to 3 wherein P is -H.

5. The compound of any one of claims lto4 2 wherein X is -NH₂, Y is -H and Z is - N.

6. The compound of any one of claims 1 to 5 wherein R is -F.

7. The compound of any one of claims 1 to 6 of formula (la):

8. The compound of claim 7 wherein X is -NH₂.

9. The compound of claim 7 or 8 wherein Y is -H.

10. A pharmaceutical composition of the compound of formula (I) or of a

pharmacologically acceptable salt thereof for use in the prevention or treatment of alphavirus infections.

11. The pharmaceutical composition according to claim 10 for use in the prevention or treatment of an infection by an alphavirus of the group IV Togaviridae family of viruses.

12. The pharmaceutical composition according to claim 11 for use in the prevention or Chikungunya virus infections.

13. A method of therapeutic or prophylactic treatment of a subject against viral infection comprising administering a therapeutically effective amount of at least one compound of any one of claims 1 to 9, or a pharmacologically acceptable salt solvate, tautomeric and/or polymorphic form thereof, to a subject in need of antiviral therapeutic or prophylactic treatment.

14. The method of claim 13, wherein the administering is by ingestion, injection, infusion, or other bodily administration.

15. The method according to any one of claim 13 or 14, wherein the virus is an

alphavirus.

16. The method according to claim 15, wherein the alphavirus is of the group IV Togaviridae family of viruses.

17. The method of claim 16, wherein the alphavirus is a Chikungunya virus.

18. The method of any one of claims 13 to 17, wherein the amount of compound (I) administered is an antiviral effective amount sufficient to improve, inhibit, prevent or ameliorate the viral infection.

19. The method of claim 18, wherein the antiviral effective amount is an amount that prevents the occurrence or one or more symptoms of the infection or reduces the severity of, or the length of time during which the subject suffers from, one or more symptoms of the infection by at least 50%.

20. A pharmaceutical composition for treating a subject with a viral infection or in need of prophylactic antiviral treatment comprising: an antiviral sufficient amount of a compound according to any one of claims 1 to 9 to produce antiviral effects.

21. The pharmaceutical composition of claim 20, wherein the compound is

formulated into tablets, gel capsules, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.

22. The pharmaceutical composition according to any one of claim 20 or 21, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a diluent, adjuvant, excipient, vehicle or combinations thereof.

23. The pharmaceutical composition according to any one of claim 20 to 22, wherein the antiviral amount of the compound is present an amount sufficient to improve, inhibit, prevent or ameliorate the viral infection.

24. The pharmaceutical composition according to any one of claim 20 to 23, wherein the antiviral effective amount is an amount that prevents the occurrence or one or more symptoms of the infection or reduces the severity of, or the length of time during which the subject suffers from, one or more symptoms of the infection by at least 50%.

25. The pharmaceutical composition according to any one of claim 20 to 24, wherein the antiviral effective amount is an amount that reduces viral replication or kills virally-infected cells by at least 50%.

26. A method for the preparation of a compound according to any one of claims 1 to 9, comprising providing a precursor compound comprising one or more protective groups, and removing the protecting groups.