Ammonium derivatives for the treatment of hepatitis C

Abstract

Compounds of Formula I, including pharmaceutically acceptable salts thereof, are set forth, in addition to compositions and methods of using these compounds. The compounds have activity against hepatitis C virus (HCV) and may be useful in treating those infected with HCV. ##STR00001##

Claims

1. A compound, including a pharmaceutically acceptable salt thereof, which is selected from the group of ##STR00044## ##STR00045## ##STR00046##

2. A compound, including a pharmaceutically acceptable salt thereof, which is selected from the group of ##STR00047## ##STR00048##

3. A composition comprising a compound of claim 1, and a pharmaceutically acceptable carrier.

4. A composition comprising a compound of claim 2, and a pharmaceutically acceptable carrier.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) Unless otherwise specifically set forth elsewhere in the application, these terms shall have the following meanings. H refers to hydrogen, including its isotopes, such as deuterium. Halo means fluoro, chloro, bromo, or iodo. Alkyl means a straight or branched alkyl group composed of 1 to 6 carbons. Alkenyl means a straight or branched alkyl group composed of 2 to 6 carbons with at least one double bond. Cycloalkyl means a monocyclic ring system composed of 3 to 8 carbons. Alkylene means a straight or branched divalent alkyl group. Alkenylene means a straight or branched divalent alkyl group with at least one double bond. Cycloalkylene means a divalent cycloalkane moiety composed of 3 to 7 carbons and includes gem-divalency (for example 1,1-cyclopropanediyl) as well as non-gem-divalency (for example, 1,4-cyclohexanediyl). Alkylidinyl means a divalent alkene substituent where the divalency occurs on the same carbon of the alkene. Hydroxyalkyl, alkoxy and other terms with a substituted alkyl moiety include straight and branched isomers composed of 1 to 6 carbon atoms for the alkyl moiety. Haloalkyl and haloalkoxy include all halogenated isomers from monohalo substituted alkyl to perhalo substituted alkyl. Aryl includes carbocyclic and heterocyclic aromatic substituents. Phenylene is a divalent benzene ring. 1,4-Phenylene means 1,4-benzenediyl with respect to regiochemistry for the divalent moiety. Parenthetic and multiparenthetic terms are intended to clarify bonding relationships to those skilled in the art. For example, a term such as ((R)alkyl) means an alkyl substituent further substituted with the substituent R.

(2) The substituents described above may be attached at any suitable point of attachment unless otherwise specified. However, it is understood that the compounds encompassed by the present invention are those that are chemically stable as understood by those skilled in the art. Additionally, the compounds encompassed by the present disclosure are those that are suitably stable for use as a pharmaceutical agent.

(3) The invention includes all pharmaceutically acceptable salt forms of the compounds. Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents. Some anionic salt forms include acetate, acistrate, besylate, bromide, camsylate, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

(4) Some of the compounds of the invention possess asymmetric carbon atoms (see, for example, the structures below). The invention includes all stereoisomeric forms, including enantiomers and diastereomers as well as mixtures of stereoisomers such as racemates. Some stereoisomers can be made using methods known in the art. Stereoisomeric mixtures of the compounds and related intermediates can be separated into individual isomers according to methods commonly known in the art. The use of wedges or hashes in the depictions of molecular structures in the following schemes and tables is intended only to indicate relative stereochemistry, and should not be interpreted as implying absolute stereochemical assignments.

(5) The invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include .sup.13C and .sup.14C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds may have a variety of potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to favorably modify biological, pharmacological, or pharmacokinetic properties.

(6) As set forth above, the invention is directed to compounds of Formula I, including pharmaceutically acceptable salts thereof:

(7) ##STR00003##
wherein a, b and c are nitrogen; or a and b are nitrogen, while c is CH; or b and c are nitrogen, while a is CH; or a and c are nitrogen, while b is CH; R.sup.1 is selected from alkyl, hydroxyalkyl, alkoxyalkyl, haloalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, halocycloalkyl, cycloalkenyl, indanyl, alkylcarbonyl, and benzyl, wherein the benzyl moiety is substituted with 0-3 substituents selected from halo, alkyl, cycloalkyl, alkenyl, alkynyl, hydroxyl, cyano, haloalkyl, alkoxy, and haloalkoxy; R.sup.2 is selected from alkyl, cycloalkyl, ((Ar.sup.1)alkyl, (Ar.sup.1)cycloalkyl, ((Ar.sup.1)cycloalkyl)alkyl, ((Ar.sup.1)alkyl)cycloalkyl, and (((Ar.sup.1)alkyl)cycloalkyl)alkyl; Ar.sup.1 is phenyl substituted with 0-3 substituents selected from halo, hydroxy, cyano, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, alkoxy, and haloalkoxy; R.sup.3 is hydrogen, alkyl or cycloalkyl; R.sup.4 is selected from the group of hydrogen, halogen, alkyl, alkoxyl, alkylamino and alkylthio; or R.sup.2 and R.sup.4 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a ring; X is selected from the group of O, NR.sup.11, CONR.sup.11, NR.sup.11NR.sup.12 and CONR.sup.11NR.sup.12; R.sup.11 is selected from the group of hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, haloalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, halocycloalkyl, cycloalkenyl, indanyl, alkylcarbonyl, and benzyl, wherein the benzyl moiety is substituted with 0-3 substituents selected from halo, alkyl, haloalkyl, alkoxy, and haloalkoxy; R.sup.12 is selected from the group of hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, haloalkyl, cycloalkyl, hydroxycycloalkyl, alkoxycycloalkyl, halocycloalkyl, cycloalkenyl, indanyl, alkylcarbonyl, and benzyl, wherein the benzyl moiety is substituted with 0-3 substituents selected from halo, alkyl, haloalkyl, alkoxy, and haloalkoxy; R.sup.5 is selected from the group of alkyl, cycloalkyl, (cycloalkyl)alkyl, (alkyl)cycloalkyl, ((alkyl))cycloalkyl)alkyl, bridged bicycloalkyl, fused bicycloalkyl, and spiro bicycloalkyl, and is substituted with 0-4 substituents selected from the group of halo, alkyl, cycloalkyl, hydroxyalkyl, alkoxyalkyl, hydroxy, alkoxy, benzyloxy, CO.sub.2R.sup.13, CON(R.sup.14)(R.sup.15), N(R.sup.14)(R.sup.15), tetrahydrofuranyl, tetrahydropyranyl, and Ar.sup.2; or R.sup.5 is hydrogen, N-alkoxycarbonylpiperidinyl, piperidinonyl, or Ar.sup.3; R.sup.13 is selected from the group of hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, ((hydroxyalkyl)alkoxy)alkoxy, and ((alkoxy)alkoxy)alkoxy; R.sup.14 is selected from the group of hydrogen, alkyl, cycloalkyl, alkylcarbonyl, and alkoxycarbonyl; R.sup.15 is hydrogen or alkyl; or R.sup.14 and R.sup.15 taken together with the nitrogen to which they are attached is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, and is substituted with 0-2 substituents selected from alkyl, alkylcarbonyl, and alkoxycarbonyl;

(8) Ar.sup.2 is selected from the group of phenyl, indanyl, tetrahydronaphthyl, isochromanyl, benzodioxolyl, pyridinyl, pyrazolyl, imidazolyl, and triazolyl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, alkyenyl, haloalkyl, alkoxy, haloalkoxy, N(R.sup.16)(R.sup.17), and alkylCO; R.sup.16 is selected from the group of hydrogen, alkyl, cycloalkyl, alkylcarbonyl, and alkoxycarbonyl; R.sup.17 is hydrogen or alkyl; or R.sup.16 and R.sup.17 taken together with the nitrogen to which they are attached is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, and is substituted with 0-2 substituents selected from alkyl, alkylcarbonyl, and alkoxycarbonyl; Ar.sup.3 is selected from the group of phenyl, indanyl, fluorenyl, biphenyl, terphenyl, pyridinyl, pyrazolyl, isoxazolyl, imidazolyl, thiazolyl, triazolyl, benzoxazolyl, indolinyl, and dibenzofuranyl, and is substituted with 0-3 substituents selected from the group of cyano, halo, alkyl, alkenyl, haloalkyl, cycloalkyl, (CO.sub.2R.sup.13)alkyl, (CO.sub.2R.sup.13)alkenyl, (CON(R.sup.16)(R.sup.17))alkyl, phenyl, hydroxyl, alkoxy, haloalkoxy, alkylcarbonyl, CO.sub.2R.sup.13, CON(R.sup.16)(R.sup.17), and PhCONHSO.sub.2; or Ar.sup.3 is phenyl substituted with 1 substituent selected from benzyl, tetrazolyloxy, thiazolyl, phenylpyrazolyl, methyloxadiazolyl, thiadiazolyl, triazolyl, methyltriazolyl, tetrazolyl, pyridinyl, and dimethoxypyrimdinyl; and R.sup.6 is alkyl or cycloalkyl; R.sup.7 is alkyl or cycloalkyl; R.sup.8 is alkyl or cycloalkyl; or R.sup.7 and R.sup.8 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a ring; or R.sup.6, R.sup.7 and R.sup.8 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a bicyclic ring; or R.sup.5 and R.sup.6 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a ring; or R.sup.5, R.sup.6 and R.sup.7 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a bicyclic or tricyclic ring;

(9) or R.sup.5, R.sup.6, R.sup.7 and R.sup.8 can be connected by a carbon, oxygen, nitrogen or sulfur atom to form a tricyclic or tetracyclic ring.

(10) More preferred compounds, including pharmaceutically acceptable salts thereof, are selected from the group of

(11) ##STR00004## ##STR00005## ##STR00006##

(12) In addition, other preferred compounds, including pharmaceutically acceptable salts thereof, are selected from the group of

(13) ##STR00007## ##STR00008## ##STR00009##

Pharmaceutical Compositions and Methods of Treatment

(14) The compounds demonstrate activity against HCV NS5B and can be useful in treating HCV and HCV infection. Therefore, another aspect of the invention is a composition comprising a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

(15) Another aspect of the invention is a composition further comprising a compound having anti-HCV activity.

(16) Another aspect of the invention is a composition where the compound having anti-HCV activity is an interferon or a ribavirin. Another aspect of the invention is where the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, interferon lambda, and lymphoblastoid interferon tau.

(17) Another aspect of the invention is a composition where the compound having anti-HCV activity is a cyclosporin. Another aspect of the invention is where the cyclosporin is cyclosporin A.

(18) Another aspect of the invention is a composition where the compound having anti-HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

(19) Another aspect of the invention is a composition where the compound having anti-HCV activity is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.

(20) Another aspect of the invention is a composition comprising a compound, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, an interferon and ribavirin.

(21) Another aspect of the invention is a method of inhibiting the function of the HCV replicon comprising contacting the HCV replicon with a compound or a pharmaceutically acceptable salt thereof.

(22) Another aspect of the invention is a method of inhibiting the function of the HCV NS5B protein comprising contacting the HCV NSSB protein with a compound or a pharmaceutically acceptable salt thereof.

(23) Another aspect of the invention is a method of treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof. In another embodiment the compound is effective to inhibit the function of the HCV replicon. In another embodiment the compound is effective to inhibit the function of the HCV NSSB protein.

(24) Another aspect of the invention is a method of treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, in conjunction with (prior to, after, or concurrently) another compound having anti-HCV activity.

(25) Another aspect of the invention is the method where the other compound having anti-HCV activity is an interferon or a ribavirin.

(26) Another aspect of the invention is the method where the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, interferon lambda, and lymphoblastoid interferon tau.

(27) Another aspect of the invention is the method where the other compound having anti-HCV activity is a cyclosporin.

(28) Another aspect of the invention is the method where the cyclosporin is cyclosporin A.

(29) Another aspect of the invention is the method where the other compound having anti-HCV activity is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

(30) Another aspect of the invention is the method where the other compound having anti-HCV activity is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.

(31) Another aspect of the invention is the method where the other compound having anti-HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS5B protein.

(32) Therapeutically effective means the amount of agent required to provide a meaningful patient benefit as understood by practitioners in the field of hepatitis and HCV infection.

(33) Patient means a person infected with the HCV virus and suitable for therapy as understood by practitioners in the field of hepatitis and HCV infection.

(34) Treatment, therapy, regimen, HCV infection, and related terms are used as understood by practitioners in the field of hepatitis and HCV infection.

(35) The compounds of this invention are generally given as pharmaceutical compositions comprised of a therapeutically effective amount of a compound or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier and may contain conventional excipients. Pharmaceutically acceptable carriers are those conventionally known carriers having acceptable safety profiles. Compositions encompass all common solid and liquid forms including for example capsules, tablets, lozenges, and powders as well as liquid suspensions, syrups, elixirs, and solutions. Compositions are made using common formulation techniques, and conventional excipients (such as binding and wetting agents) and vehicles (such as water and alcohols) are generally used for compositions. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985.

(36) Solid compositions are normally formulated in dosage units and compositions providing from about 1 to 1000 mg of the active ingredient per dose are preferred. Some examples of dosages are 1 mg, 10 mg, 100 mg, 250 mg, 500 mg, and 1000 mg. Generally, other agents will be present in a unit range similar to agents of that class used clinically. Typically, this is 0.25-1000 mg/unit.

(37) Liquid compositions are usually in dosage unit ranges. Generally, the liquid composition will be in a unit dosage range of about 1-100 mg/mL. Some examples of dosages are 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL. Generally, other agents will be present in a unit range similar to agents of that class used clinically. Typically, this is 1-100 mg/mL.

(38) The invention encompasses all conventional modes of administration; oral and parenteral methods are preferred. Generally, the dosing regimen will be similar to other agents used clinically. Typically, the daily dose will be about 1-100 mg/kg body weight daily. Generally, more compound is required orally and less parenterally. The specific dosing regimen, however, will be determined by a physician using sound medical judgment.

(39) The invention also encompasses methods where the compound is given in combination therapy. That is, the compound can be used in conjunction with, but separately from, other agents useful in treating hepatitis and HCV infection. In these combination methods, the compound will generally be given in a daily dose of about 1-100 mg/kg body weight daily in conjunction with other agents. The other agents generally will be given in the amounts used therapeutically. The specific dosing regimen, however, will be determined by a physician using sound medical judgment.

(40) Some examples of compounds suitable for compositions and methods are listed in Table 1.

(41) TABLE-US-00001 TABLE 1 Type of Physiological Inhibitor Brand Name Class or Target Source Company NIM811 Cyclophilin Novartis Zadaxin Inhibitor Sciclone Suvus Immuno- Bioenvision Actilon modulator Coley (CPG10101) Methylene blue TLR9 agonist Batabulin (T67) Anticancer -tubulin Tularik Inc., South inhibitor San Francisco, CA ISIS 14803 Antiviral antisense ISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., New York, NY Summetrel Antiviral antiviral Endo Pharmaceuticals Holdings Inc., Chadds Ford, PA GS 9132 (ACH- Antiviral HCV Achillion/Gilead 806) Inhibitor Pyrazolopyrimidine Antiviral HCV Arrow Therapeutics compounds and Inhibitors Ltd. salts From WO- 2005047288 May 26, 2005 Levovirin Antiviral IMPDH Ribapharm Inc., inhibitor Costa Mesa, CA Merimepodib Antiviral IMPDH Vertex (VX-497) inhibitor Pharmaceuticals Inc., Cambridge, MA XTL-6865 (XTL- Antiviral monoclonal XTL 002) antibody Biopharmaceuticals Ltd., Rehovot, Isreal Telaprevir Antiviral NS3 serine Vertex (VX-950, LY- protease Pharmaceuticals 570310) inhibitor Inc., Cambridge, MA/Eli Lilly and Co. Inc., Indianapolis, IN HCV-796 Antiviral NS5B Wyeth/Viropharma Replicase Inhibitor NM-283 Antiviral NS5B Idenix/Novartis Replicase Inhibitor GL 59728 Antiviral NS5B Gene Labs/ Replicase Novartis Inhibitor GL-60667 Antiviral NS5B Gene Labs/ Replicase Novartis Inhibitor 2C MeA Antiviral NS5B Gilead Replicase Inhibitor PSI 6130 Antiviral NS5B Roche Replicase Inhibitor R1626 Antiviral NS5B Roche Replicase Inhibitor 2C Methyl Antiviral NS5B Merck Replicase adenosine Inhibitor JTK-003 Antiviral RdRp Japan Tobacco Inc., inhibitor Tokyo, Japan Levovirin Antiviral ribavirin ICN Pharmaceuticals, Costa Mesa, CA Ribavirin Antiviral ribavirin Schering-Plough Corporation, Kenilworth, NJ Viramidine Antiviral Ribavirin Ribapharm Inc., Prodrug Costa Mesa, CA Heptazyme Antiviral ribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO BILN-2061 Antiviral serine Boehringer protease Ingelheim Pharma inhibitor KG, Ingelheim, Germany SCH 503034 Antiviral serine Schering Plough protease inhibitor Zadazim Immune Immune SciClone modulator modulator Pharmaceuticals Inc., San Mateo, CA Ceplene Immuno- immune Maxim modulator modulator Pharmaceuticals Inc., San Diego, CA CellCept Immuno- HCV IgG F. Hoffmann-La suppressant immuno- Roche LTD, Basel, suppressant Switzerland Civacir Immuno- HCV IgG Nabi suppressant immuno- Biopharmaceuticals suppressant Inc., Boca Raton, FL Albuferon- Interferon albumin Human Genome IFN-2b Sciences Inc., Rockville, MD Infergen A Interferon IFN InterMune alfacon-1 Pharmaceuticals Inc., Brisbane, CA Omega IFN Interferon IFN- Intarcia Therapeutics IFN- and EMZ701 Interferon IFN- and Transition EMZ701 Therapeutics Inc., Ontario, Canada Rebif Interferon IFN-1a Serono, Geneva, Switzerland Roferon A Interferon IFN-2a F. Hoffmann-La Roche LTD, Basel, Switzerland Intron A Interferon IFN-2b Schering-Plough Corporation, Kenilworth, NJ Intron A and Interferon IFN-2b/ RegeneRx Zadaxin 1- Biopharma. Inc., thymosm Bethesda, MD/ SciClone Pharmaceuticals Inc, San Mateo, CA Rebetron Interferon IFN-2b/ Schering-Plough ribavirin Corporation, Kenilworth, NJ Actimmune Interferon INF- InterMune Inc., Brisbane, CA Interferon- Interferon Interferon- Serono -1a Multiferon Interferon Long Viragen/ lasting Valentis IFN Wellferon Interferon Lympho- GlaxoSmithKline blastoid plc, Uxbridge, UK IFN-n1 Omniferon Interferon natural Viragen Inc., IFN- Plantation, FL Pegasys Interferon PEGylated F. Hoffmann-La IFN-2a Roche LTD, Basel, Switzerland Pegasys and Interferon PEGylated Maxim Ceplene IFN-2a/ Pharmaceuticals immune Inc., San Diego, CA modulator Pegasys and Interferon PEGylated F. Hoffmann La Ribavirin IFN-2a/ Roche LTD, Basel, ribavirin Switzerland PEG-Intron Interferon PEGylated Schering-Plough IFN-2b Corporation, Kenilworth, NJ PEG-Intron/ Interferon PEGylated Scheringng-Plough Ribavirin IFN-2b/ Corporation, ribavirin Kenilworth, NJ IP-501 Liver antifibrotic Indevus protection Pharmaceuticals Inc., Lexington, MA IDN-6556 Liver caspase Idun protection inhibitor Pharmaceuticals Inc., San Diego, CA ITMN-191 Antiviral serine InterMune (R-7227) protease Pharmaceuticals inhibitor Inc., Brisbane, CA GL-59728 Antiviral NS5B Genelabs Replicase Inhibitor ANA-971 Antiviral TLR-7 Anadys agonist Boceprevir Antiviral serine Schering Plough protease inhibitor TMS-435 Antiviral serine Tibotec BVBA, protease Mechelen, Belgium inhibitor BI 201335 Antiviral serine Boehringer protease Ingelheim Pharma inhibitor KG, Ingelheim, Germany MK-7009 Antiviral serine Merck protease inhibitor PF-00868554 Antiviral replicase Pfizer inhibitor ANA598 Antiviral Non- Anadys Nucleoside Pharmaceuticals, NS5B Inc., San Diego, CA, Polymerase USA Inhibitor IDX375 Antiviral Non- Idenix Nucleoside Pharmaceuticals, Replicase Cambridge, MA, Inhibitor USA BILB 1941 Antiviral NS5B Boehringer Polymerase Ingelheim Canada Inhibitor Ltd R&D, Laval, QC, Canada PSI-7851 Antiviral Nucleoside Pharmasset, Polymerase Princeton, NJ, USA Inhibitor PSI-7977 Antiviral Nucleotide Pharmasset, NS5B Princeton, NJ, USA Polymerase Inhibitor VCH-759 Antiviral NS5B ViroChem Pharma Polymerase Inhibitor VCH-916 Antiviral NS5B ViroChem Pharma Polymerase Inhibitor GS 9190 Antiviral NS5B Gilead Polymerase Inhibitor Peg-interferon Antiviral Interferon ZymoGenetics/ lamda Bristol-Myers Squibb

Synthetic Methods

(42) The compounds may be made by methods available in the art, as well as those described below and including variations within the skill of the art. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using readily available materials. The variables (e.g. numbered R substituents) used to describe the synthesis of the compounds are intended only to illustrate how to make the compounds and are not to be confused with variables used in the claims or in other sections of the specification. The following methods are for illustrative purposes and are not intended to limit the scope of the invention.

(43) Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows: NaHMDS for sodium bis(trimethylsilyl)amide; DMF for N,N-dimethylformamide; MeOH for methanol; NBS for N-bromosuccinimide; Ar for aryl; TFA for trifluoroacetic acid; LAH for lithium aluminum hydride; BOC, DMSO for dimethylsulfoxide; h for hours; rt for room temperature or retention time (context will dictate); min for minutes; EtOAc for ethyl acetate; THF for tetrahydrofuran; EDTA for ethylenediaminetetraacetic acid; Et.sub.2O for diethyl ether; DMAP for 4-dimethylaminopyridine; DCE for 1,2-dichloroethane; ACN for acetonitrile; DME for 1,2-dimethoxyethane; HOBt for 1-hydroxybenzotriazole hydrate; DIEA for diisopropylethylamine, Nf for CF.sub.3(CF.sub.2).sub.3SO.sub.2; and TMOF for trimethylorthoformate.

(44) Abbreviations are defined as follows: 1 for once, 2 for twice, 3 for thrice, C. for degrees Celsius, eq for equivalent or equivalents, g for gram or grams, mg for milligram or milligrams, L for liter or liters, mL for milliliter or milliliters, L for microliter or microliters, N for normal, M for molar, mmol for millimole or millimoles, min for minute or minutes, h for hour or hours, rt for room temperature, RT for retention time, atm for atmosphere, psi for pounds per square inch, conc. for concentrate, sat or sat'd for saturated, MW for molecular weight, mp for melting point, ee for enantiomeric excess, MS or Mass Spec for mass spectrometry, ESI for electrospray ionization mass spectroscopy, HR for high resolution, HRMS for high resolution mass spectrometry, LCMS for liquid chromatography mass spectrometry, HPLC for high pressure liquid chromatography, RP HPLC for reverse phase HPLC, TLC or tlc for thin layer chromatography, NMR for nuclear magnetic resonance spectroscopy, .sup.1H for proton, for delta, s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad, Hz for hertz, and , , R, S, E, and Z are stereochemical designations familiar to one skilled in the art.

CHEMISTRY EXPERIMENTAL

(45) LC/MS Method (i.e., Compound Identification)

(46) All Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10AS or LC-20AS liquid chromotograph using a SPD-10AV or SPD-20A UV-Vis detector and Mass Spectrometry (MS) data were determined with a Micromass Platform for LC in electrospray mode.

(47) HPLC Method (i.e., Compound Isolation)

(48) Compounds purified by preparative HPLC were diluted in methanol (1.2 mL) and purified using a Shimadzu LC-8A or LC-10A or Dionex APS-3000 or Waters Acquity automated preparative HPLC system.

(49) Syntheses of Intermediates:

Preparation of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid

(50) ##STR00010##

(51) Step 1: To a solution of 2,4,6-trichloro-1,3,5-triazine (15 g) in THF (300 mL) was added 2,2,2-trifluoroethanol (8.14 g) and Hunig's Base (15.63 mL). The resulting mixture was stirred for 16 hours. After removal of most THF and precipitate through a plug washing with THF, the filtrate was concentrate to give a crude that will be used as it is.

(52) Step 2: To a solution of the product in Step 1 above (10 g) in THF (100 mL) was added tert-butyl 4-aminobenzoate (7.79 g) and Hunig's Base (7.04 mL). The resulting mixture was stirred for 16 h. The precipitate was filtered and washed with Et.sub.2O, dried, then washed with water and dried to give 10.6 g of tert-butyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoate as a solid.

(53) TABLE-US-00002 tert-butyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy)- 1,3,5-triazin-2-ylamino)benzoate MS (M + H).sup.+ Calcd. 405.1 MS (M + H).sup.+ Observ. 405.0 LC Condition Solvent A 100% Water-0.1% TFA Solvent B 100% ACN-0.1% TFA Start % B 2 Final % B 98 Gradient Time 1.6 min Stop Time 1.8 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair ACN-H.sub.2O-0.1% TFA Column Aquity UPLC BEH C18 1.7 um

(54) Step 3: To a slurry of tert-butyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoate (3.6 g) and 1-(4-chlorophenyl)cyclopropanamine (1.49 g) in THF (50 mL) was stirred for 5 hours at 80 C. The precipitate was filtrated through a plug washing with THF to give a crude product that was purified by Biotage eluting with 4/1-hexane/ethyl acetate to give 1.8 g of tert-butyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoate as a solid.

(55) TABLE-US-00003 tert-butyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6- (2,2,2-trifluoroethoxy)-1,3 5-triazin-2-ylamino)benzoate MS (M + H).sup.+ Calcd. 536.2 MS (M + H).sup.+ Observ. 536.0 LC Condition Solvent A 100% Water-0.1% TFA Solvent B 100% ACN-0.1% TFA Start % B 2 Final % B 98 Gradient Time 1.6 min Stop Time 1.8 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair ACN-H.sub.2O-0.1% TFA Column Aquity UPLC BEH C18 1.7 um

(56) Step 4: A solution of above tert-butyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoate (4 g) and HCl in dioxane (7.46 ml, 4M) was stirred for 4 hours. Concentration gave 3.58 g of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid as a solid.

(57) TABLE-US-00004 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2- trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid MS (M + H).sup.+ Calcd. 480.1 MS (M + H).sup.+ Observ. 480.1 LC Condition Solvent A 100% Water-0.1% TFA Solvent B 100% ACN-0.1% TFA Start % B 2 Final % B 98 Gradient Time 1.6 min Stop Time 1.8 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair ACN-H.sub.2O-0.1% TFA Column Aquity UPLC BEH C18 1.7 um

Preparation of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoyl chloride

(58) ##STR00011##

(59) A mixture of sulfuryl dichloride (1.68 g) and 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid (0.40 g) in CH.sub.2Cl.sub.2 (4 mL) in sealed tube was heated at 106 C. for 16 hours. All the solvents were removed under vacuum to give crude 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoyl chloride (0.42 g) as yellow solid, which was used in the further reactions without purification.

Preparation of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoyl chloride

(60) ##STR00012##

(61) Step 1: iPr.sub.2NEt (2.65 g) was added into the solution of 2,4,6-trichloro-1,3,5-triazine (3.78 g, 20.48 mmol) and 2,2,2-trifluoroethanol-D2 (2 g) in THF (150 mL). The reaction was stirred at room temperature for 16 hours.

(62) Step 2: iPr.sub.2NEt (2.65 g) and methyl 4-aminobenzoate (3.10 g) were added into the solution in step 1 and the reaction was stirred at room temperature for 16 hours. Solvents were removed under vacuum to give a residue, to which 20 ml of water and 100 ml of EtOAc were added. The resulted mixture was stirred t room temperature for 16 hours. The solid was isolated by filtration and dried under vacuum to give methyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoate (7 g).

(63) TABLE-US-00005 methyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy-D2)- 1,3,5-triazin-2-ylamino)benzoate MS (M + H).sup.+ Calcd. 365.1 MS (M + H).sup.+ Observ. 565.1 Retention Time 1.99 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 5 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 4.6 50 mm S10

(64) Step 3: iPr.sub.2NEt (0.35 g) was added into the solution of methyl 4-(4-chloro-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoate (1 g) and 1-(4-chlorophenyl)cyclopropanamine (0.46 g) in THF (15 mL). The reaction was stirred at 70 C. for 16 hours. Solvents were removed under vacuum to give a residue, to which 100 ml of EtOAc were added. The organic phase was washed with water (220 mL) and brine (15 mL), dried over MgSO.sub.4 and concentrated under vacuum to give methyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoate (1.30 g) which was used in the further reaction without purification.

(65) TABLE-US-00006 methyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2- trifluoroethoxy-D2)-1,3 5-triazin-2-ylamino)benzoate MS (M + H).sup.+ Calcd. 496.1 MS (M + H).sup.+ Observ. 496.0 Retention Time 2.18 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 5 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 4.6 50 mm S10

(66) Step 4: The mixture of K.sub.2CO.sub.3 (0.84 g) and methyl 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoate (0.75 g) in acetone (10 mL) and water (10 mL) in sealed tube was heated at 80 C. for 16 hours. After cooling, the mixture was added with 1N HCl to adjust pH to 2 when white solid formed. This white solid was isolated via filtration and dried to give 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoic acid (0.75g) which was used in the next step with purification.

(67) TABLE-US-00007 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy- D2)-1,3,5-triazin-2-ylamino)benzoic acid MS (M + H).sup.+ Calcd. 482.1 MS (M + H).sup.+ Observ. 482.3 Retention Time 2.55 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair ACN:Water:Ammonium Actetate Column Phenomenex LUNA C18, 50 2, 3 u

(68) Step 5: A mixture of sulfuryl dichloride (0.42 g) and 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoic acid (0.31 g) in CH.sub.2Cl.sub.2 (2 mL) in sealed tube was heated at 90 C. for 16 hours. All the solvents were removed under vacuum to give crude 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoyl chloride (0.31g) as yellow solid, which was used in the further reactions without purification.

(69) Syntheses of Claim I:

Synthesis of Compound 1001, 2-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzamido)-N,N,N-trimethylethanaminium

(70) ##STR00013##

(71) iPr.sub.2NEt (26 mg) was added into the solution of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoyl chloride (50 mg) and 2-amino-N,N,N-trimethylethanaminium chloride hydrochloride (18 mg) in THF (4 mL). The reaction was stirred at room temperature for 16 hours. Solvents were removed under vacuum to give a residue which was purified by preparative HPLC to give 1001 (12 mg).

(72) TABLE-US-00008 1001 MS Calcd. 564.2 MS Observ. 564.3 Retention Time 2.86 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair ACN:Water:Ammonium Actetate Column Phenomenex LUNA C18, 50 2, 3 u

Synthesis of Compound 1002, 2-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzamido)-N,N,N-trimethylethanaminium

(73) ##STR00014##

(74) iPr.sub.2NEt (26 mg) was added into the solution of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy-D2)-1,3,5-triazin-2-ylamino)benzoyl chloride (50 mg) and 2-amino-N,N,N-trimethylethanaminium chloride hydrochloride (18 mg) in THF (4 mL). The reaction was stirred at room temperature for 16 hours. Solvents were removed under vacuum to give a residue which was purified by preparative HPLC to give 1002 (2.56 mg).

(75) TABLE-US-00009 1002 MS Calcd. 566.2 MS Observ. 566.1 Retention Time 1.74 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 5 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 4.6 50 mm S10

Synthesis of Compound 1003, 2-(2-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoyl)hydrazinyl)-N,N,N-trimethyl-2-oxoethanaminium

(76) ##STR00015##

(77) To a solution of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid (50 mg) and TBTU (33.5 mg) in DMF (2 mL) was added 2-hydrazinyl-N,N,N-trimethyl-2-oxoethanaminium hydrochloride (17.5 mg) and iPr.sub.2NEt (0.073 mL). After stirring at room temperature for 3 hours, the mixture was purified by preparative HPLC to give 1003 (30 mg).

(78) TABLE-US-00010 1003 MS Calcd. 593.2 MS Observ. 593.0 Retention Time 2.90 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 30 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 30 mm 3 um

Synthesis of Compound 1004, 3-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzamido)-N,N,N-trimethylpropan-1-aminium

(79) ##STR00016##

(80) To a solution of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid (50 mg) and TBTU (33.5 mg) in DMF (2 mL) was added 3-amino-N,N,N-trimethylpropan-1-aminium TFA salt (22.2 mg) and iPr.sub.2NEt (0.073 mL). After stirring at room temperature for 3 hours, the mixture was purified by preparative HPLC to give 1004 (31.5 mg).

(81) TABLE-US-00011 1004 MS Calcd. 578.2 MS Observ. 578.1 Retention Time 3.03 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 30 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 30 mm 3 um

Synthesis of Compound 1005, (S)-5-carboxy-5-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzamido)-N,N,N-trimethylpentan-1-aminium

(82) ##STR00017##

(83) To a solution of 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid (35 mg) and TBTU (25.8 mg) in DMF (2 mL) was added (S)-2-amino-6-(trimethylammonio)hexanoate hydrochloride (16.39 mg) and iPr.sub.2NEt (0.051 mL). After stirring at room temperature for 3 hours, the mixture was purified by preparative HPLC to give 1005 (17 mg).

(84) TABLE-US-00012 1005 MS Calcd. 650.2 MS Observ. 650.1 Retention Time 2.01 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 30 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 30 mm 3 um

Synthesis of Compound 1006, 3-(4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzamido)-N,N,N,2,2-pentamethylpropan-1-aminium

(85) ##STR00018##

(86) 1006 was prepared via the same synthetic procedure for Compound 1005, using 3-amino-N,N,N,2,2-pentamethylpropan-1-aminium as the coupling reagent.

(87) TABLE-US-00013 1006 MS Calcd. 606.3 MS Observ. 606.2 Retention Time 3.23 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 30 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

(88) Preparation of 2000, 3000 and 4000 have been described in detail in WO-2011/139513:

(89) ##STR00019##

(90) The following general procedure was applied to synthesize compounds of Claim I:

(91) ##STR00020##

(92) iPr.sub.2NEt or Et.sub.3N (1-20 eq.) was added into a solution of 2000 (1 eq.), amine (1-1.5 eq.) and TBTU (1-2 eq.) in DMF or THF at room temperature. The reaction was stirred for 16-72 hours at room temperature or increased temperature from 40 C. to 115 C., before quenched with saturated sodium bicarbonate aqueous solution. The aqueous layer was extracted with EtOAc. The combined organic phase was dried over Mg.sub.2SO.sub.4 and concentrated under vacuum to give a crude product, which was purified by preparative HPLC.

(93) TABLE-US-00014 2001 Amine Used Product MS Calcd. 664.3 MS Observ. 664.2 Retention Time 4.27 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

(94) TABLE-US-00015 2002 Amine Used Product MS Calcd. 732.4 MS Observ. 732.5 Retention 3.89 min Time LC Condition Solvent A 5:95 methanol:water with 10 mM ammonium acetate Solvent B 95:5 methanol:water with 10 mM ammonium acetate Gradient: 0.5 min hold at 0% B, 0-100% B over 4 minutes, then a 0.5-minute hold at 100% B Flow Rate 0.5 mL/min Wavelength 220 Solvent Pair Water-Methanol-Ammonium Acetate Column Waters BEH C18, 2.0 50 mm, 1.7-m particles

(95) The following general procedure was applied to synthesize compounds of Claim I:

(96) ##STR00025##

(97) iPr.sub.2NEt or Et.sub.3N (1-20 eq.) was added into a solution of 3000 (1 eq.), amine (1-1.5 eq.) and TBTU (1-2 eq.) in DMF or THF at room temperature. The reaction was stirred for 16-72 hours at room temperature or increased temperature from 40 C. to 115 C., before quenched with saturated sodium bicarbonate aqueous solution. The aqueous layer was extracted with EtOAc. The combined organic phase was dried over Mg.sub.2SO.sub.4 and concentrated under vacuum to give a crude product, which was purified by preparative HPLC.

(98) TABLE-US-00016 3001 Amine Used Product MS Calcd. 618.3 MS Observ. 618.3 Retention 3.59 min Time LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

(99) TABLE-US-00017 3002 Amine Used Product MS Calcd. 632.2 MS Observ. 632.3 Retention Time 3.64 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

(100) TABLE-US-00018 3003 Amine Used 0 Product MS Calcd. 660.4 MS Observ. 660.2 Retention Time 3.87 min LC Condition Solvent A 90% Water-0% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

Preparation of 4001:

(101) ##STR00032##

(102) Step 1: Compound 4000 (20 mg) and sulfurous dichloride (63 mg) were mixed together. The reaction mixture was heated to 60 C. for 6 hours. Removal of solvents under vacuum provided crude Compound 4000-In which was used in the next step without purification.

(103) Step 2: iPr.sub.2NEt (9.14 mg) was added into a solution of 4000-In from Step 1 and 2-amino-N,N,N-trimethylethanaminium (4.7 mg) in DMF (1 mL) at room temperature. The reaction was stirred for 16 hours at room temperature. Compound 4001 was isolated by preparative HPLC.

(104) TABLE-US-00019 4001 MS Calcd. 650.3 MS Observ. 650.2 Retention Time 3.28 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10% Water-90% Methanol-0.1% TFA Start % B 0 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFA Column PHENOMENEX-LUNA 2.0 50 mm 3 um

Biological Methods

(105) Infection assays. HCV pseudoparticles, produced using standardized methodology (Bartosch, B., Dubuisson, J. and Cosset, F.-L. J. Exp. Med. 2003, 197:633-642) were made via a liposome-based transfection procedure of 293T cells with plasmids expressing the murine leukemia virus capsid and polymerase proteins, an MLV genome encoding the luciferase reporter gene, and envelope glycoproteins from either HCV or vesicular stomatitis virus (VSV). The genotype 1a HCV E1 and E2 envelope coding sequences were derived from the H77C isolate (GenBank accession number AF009606). Media containing pseudoparticles was collected 3 days following transfection, filtered, and stored at 20 C. as a viral stock. Infections were performed in 384-well plates by mixing pseudovirus with 110.sup.4 Huh7 cells/well in the presence or absence of test inhibitors, followed by incubation at 37 C. Luciferase activity, reflecting the degree of entry of the pseudoparticles into host cells, was measured 2 days after infection. The specificity of the compounds for inhibiting HCV was determined by evaluating inhibition of VSV pseudoparticle infection.

(106) Compounds and data analysis. Test compounds were serially diluted 3-fold in dimethyl sulfoxide (DMSO) to give a final concentration range in the assay of 50.0 M to 0.04 pM. Maximum activity (100% of control) and background were derived from control wells containing DMSO but no inhibitor or from uninfected wells, respectively. The individual signals in each of the compound test wells were then divided by the averaged control values after background subtraction and multiplied by 100% to determine percent activity. Assays were performed in duplicate and average EC.sub.50 values (reflecting the concentration at which 50% inhibition of virus replication was achieved) were calculated. Compound EC.sub.50 data is expressed as A=0.0110 nM; B=10-1000 nM. Representative data for compounds are reported in Table 2.

(107) TABLE-US-00020 TABLE 2 EC.sub.50 EC.sub.50 Compd# Structure (1a, nM) (1a, nM) 1001 A 1002 A 1003 64.090 B 1004 A 1005 A 1006 0.115 A 2001 A 3001 0 A 3002 B 3003 7.512 A 4001 A

(108) It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.