BENZOFURAN DERIVATIVES FOR THE TREATMENT OF HEPATITIS C

BACKGROUND OF THE INVENTION The disclosure generally relates to the novel compounds of formula I, including their salts, which have activity against hepatitis C virus (HCV) and are useful in treating those infected with HCV. The disclosure also relates to compositions and methods of using these compounds. Hepatitis C virus (HCV) is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma (Lauer, G. M.; Walker, B. D. N. Engl. J. Med. 2001, 345, 41-52).

HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5 '-untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encoded amino acid sequence throughout the HCV genome. At least six major genotypes have been characterized, and more than 50 subtypes have been described. The major genotypes of HCV differ in their distribution worldwide, and the clinical significance of the genetic heterogeneity of HCV remains elusive despite numerous studies of the possible effect of genotypes on pathogenesis and therapy.

The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is believed to be a metalloprotease and cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B- NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV. The HCV NS5B protein is described in "Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides (Bressanelli; S. et al, Journal of Virology 2002, 3482-3492; and Defrancesco and Rice, Clinics in Liver Disease 2003, 7, 211-242. Currently, the most effective HCV therapy employs a combination of alpha- interferon and ribavirin, leading to sustained efficacy in 40% of patients (Poynard, T. et al. Lancet 1998, 352, 1426-1432). Recent clinical results demonstrate that pegylated alpha-interferon is superior to unmodified alpha- interferon as monotherapy (Zeuzem, S. et al. N. Engl. J. Med. 2000, 343, 1666- 1672). However, even with experimental therapeutic regimens involving combinations of pegylated alpha-interferon and ribavirin, a substantial fraction of patients do not have a sustained reduction in viral load. Thus, there is a clear and important need to develop effective therapeutics for treatment of HCV infection. HCV-796, an HCV NS5B inhibitor, showed an ability to reduce HCV

RNA levels in patients. The viral RNA levels decreased transiently and then rebounded during dosing when treatment was with the compound as a single agent but levels dropped more robustly when combined with the standard of care which is a form of interferon and ribavirin. The development of this compound was suspended due to hepatic toxicity observed during exteneded dosing of the combination regimens. US patent 7,265,152 and the corresponding PCT patent application WO2004/041201 describe compounds of the HCV-796 class. Other compounds have been disclosed, see for example, WO2009/101022.

The invention provides technical advantages, for example, the compounds are novel and are effective against hepatitis C. Additionally, the compounds provide advantages for pharmaceutical uses, for example, with regard to one or more of their mechanism of action, binding, inhibition efficacy, target selectivity, solubility, safety profiles, or bioavailability.

DESCRIPTION OF THE INVENTION

The present invention relates to compounds having the Formula I, their pharmaceutical formulations, and use in treating hepatitis C.

One aspect of the invention is a compound of formula I

where:

R ¹ is phenyl or pyridinyl and is substituted with 0-3 substituents selected from the group consisting of halo, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, cycloalkoxy, hydroxyalkyloxy, and alkoxyalkyloxy, and wherein the phenyl or pyridinyl is also substituted with 1 CON(R ⁹ )(R ¹⁰ ) substituent; hydrogen, halo, alkyl, or alkoxy; R ³ is cyano, alkoxycarbonyl, (cycloalkyl)oxycarbonyl,

(alkylsulfonyl)aminocarbonyl, CON(R ¹³ )(R ¹⁴ ), (R ¹³ )(R ¹⁴ )NCONH, thiazolyl, tetrazolyl, triazolyl, or imidazolyl wherein the thiazolyl, tetrazolyl, triazolyl, or imidazolyl is substituted with 0-3 halo or alkyl substituents;

R ⁴ is phenyl that is independently substituted with 0-2 halo, alkyl, or alkoxy or is para substituted with X-Ar ¹ ;

R ⁵ and R ⁶ are independently hydrogen, nitro, halo, alkyl, alkoxy, N(R ⁷ )(R ⁸ ), or alkylsulfonyl; R ⁷ and R ⁸ are independently hydrogen, alkyl, cyanoalkyl, haloalkyl,

(cycloalkyl)alkyl, cycloalkyl, hydroxyalkyl, alkoxyalkyl, alkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylsulfonyl, alkylsulfonylalkyl,

S0 ₂ N(R ¹⁵ )(R ¹⁶ ), or benzyl where said benzyl is substituted with 0-3 substituents selected from halo, alkyl, haloalkyl, alkoxy, haloalkoxy, carboxy, and

alkoxycarbonyl; or N(R ⁷ )(R ⁸ ) taken together is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2 substituents selected from alkyl, hydroxyalkyl, or hydroxy;

R is hydrogen;

R ¹¹ and R ¹² taken together with the carbon to which they are attached is azetidinyl substituted with 0-3 alkyl substituents;

R is hydrogen or alkyl;

R is hydrogen or alkyl;

R ¹⁵ is hydrogen or alkyl; R is hydrogen or alkyl;

X is -O- or -NH-; Ar ^x is phenyl or para-halophenyl; and

Ar ² is phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyrazolyl, isoxazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, oxadiathiazolyl, triazolyl, or tetrazolyl, and is substituted with 0-3 substituents selected from halo, alkyl, or dialkylamino; or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a compound of formula I where where R ¹ is phenyl substituted with 0-3 substituents selected from the group consisting of halo and alkoxy, and is also substituted with 1 CON(R ⁹ )(R ¹⁰ ) substituent;

R ² is hydrogen or halo;

R ³ is CON(R ¹³ )(R ¹⁴ );

R ⁴ is phenyl that is para substituted with halo; R ⁵ is hydrogen;

R ⁶ is hydrogen, nitro, halo, alkyl, alkoxy, N(R ⁷ )(R ⁸ ), or alkylsulfonyl;

Ar ² is phenyl, pyridinyl, or pyrimidinyl, and is substituted with 0-3 substituents selected from halo or alkyl; or a pharmaceutically acceptable salt thereof. Another aspect of the invention is a compound of formula I wherein R ¹ is phenyl substituted with 2 substituents selected from the group consisting of halo, alkyl, and alkoxy, and is also substituted with 1 CON(R ⁹ )(R ¹⁰ ) substituent; R ² is halo; R ³ is CONHMe; R ⁴ is phenyl that is para substituted with halo; R ⁵ is hydrogen; R ⁶ is hydrogen; and Ar ² is pyrimidinyl; or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a compound of formula I where R ¹ is phenyl or pyridinyl wherein the phenyl or pyridinyl is substituted with 0-3 substituents selected from the group consisting of halo, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, cycloalkoxy, hydroxyalkyloxy, and alkoxyalkyloxy, and is also substituted with 1 CON(R ⁹ )(R ¹⁰ ) substituent; or a pharmaceutically acceptable salt thereof. Another aspect of the invention is a compound of formula I where R ² is hydrogen, halo, alkyl, or alkoxy.

Another aspect of the invention is a compound of formula I where R ² is hydrogen or halo.

Another aspect of the invention is a compound of formula I where R ³ is

CON(R ¹³ )(R ¹⁴ ).

Another aspect of the invention is a compound of formula I where R ³ is imidazolyl.

Another aspect of the invention is a compound of formula I where R ³ is imidazol- 2-yl.

Another aspect of the invention is a compound of formula I where R ⁴ is phenyl that is independently substituted with 0-2 halo, alkyl, or alkoxy substuituents or is para substituted with X-Ar ¹ . Another aspect of the invention is a compound of formula I where R ⁴ is phenyl that is substituted with 0-1 halo substituent.

Another aspect of the invention is a compound of formula I where R ⁵ and R ⁶ are independently hydrogen, nitro, halo, alkyl, alkoxy, N(R ⁷ )(R ⁸ ), or alkylsulfonyl.

Another aspect of the invention is a compound of formula I where R ⁵ is hydrogen and R ⁶ is hydrogen or N(R ⁷ )(R ⁸ ). Another aspect of the invention is a compound of formula I where R ⁷ and R ⁸ are independently hydrogen, alkyl, cyanoalkyl, haloalkyl, (cycloalkyl)alkyl, cycloalkyl, hydroxyalkyl, alkoxyalkyl, alkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylsulfonyl, alkylsulfonylalkyl, S0 ₂ N(R ¹³ )(R ¹⁴ ), or benzyl where said benzyl is substituted with 0-3 substituents selected from halo, alkyl, haloalkyl, alkoxy, haloalkoxy, carboxy, and alkoxycarbonyl.

Another aspect of the invention is a compound of formula I where N(R ⁷ )(R ⁸ ) taken together is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2 substituents selected from alkyl, hydroxyalkyl, or hydroxyl.

R^R ¹²

Another aspect of the invention is a compound of formula I where R ⁹ is * ^Al"2 .

Another aspect of the invention is a compound of formula I where R ¹⁰ is hydrogen.

Another aspect of the invention is a compound of formula I where R ¹³ is hydrogen or alkyl.

Another aspect of the invention is a compound of formula I where R is hydrogen or alkyl. Another aspect of the invention is a compound of formula I where R ¹⁵ is hydrogen or alkyl.

Another aspect of the invention is a compound of formula I where R ¹⁶ is hydrogen or alkyl.

Another aspect of the invention is a compound of formula I where X is -O- or - NH-. Another aspect of the invention is a compound of formula I where X is -0-.

Another aspect of the invention is a compound of formula I where Ar ^x is phenyl or para-halophenyl. Another aspect of the invention is a compound of formula I where Ar ² is phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyrazolyl, isoxazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, oxadiathiazolyl, triazolyl, or tetrazolyl, and is substituted with 0-3 substituents selected from halo, alkyl, or dialkylamino; Another aspect of the invention is a compound of formula I where Ar ² is phenyl, pyridinyl, pyrazinyl, or pyrimidinyl; or a pharmaceutically acceptable salt thereof.

Any scope of any variable, including R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , X, Ar ¹ , or Ar ² can be used independently with the scope of any other instance of a variable.

Unless specified otherwise, these terms have the following meanings. "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 7 carbons.

"Hydroxy alkyl," "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. "Halo" includes all halogenated isomers from monohalo substituted to perhalo substituted in substituents defined with halo, for example, "Haloalkyl" and "haloalkoxy", "halophenyl", "halophenoxy." Ethylene means ethanediyl or -CH ₂ CH ₂ -; propylene means propanediyl or -CH ₂ CH ₂ CH ₂ -; butylene means butanediyl or -CH ₂ CH ₂ CH ₂ CH ₂ -; pentylene means pentanediyl or

-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -. "Aryl" means a monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms, or a bicyclic fused ring system wherein one or both of the rings is a phenyl group. Bicyclic fused ring systems consist of a phenyl group fused to a four- to six-membered aromatic or non- aromatic carbocyclic ring. Representative examples of aryl groups include, but are not limited to, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. "Heteroaryl" means a 5 to 7 membered monocyclic or 8 to 1 1 membered bicyclic aromatic ring system with 1 -5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 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. Substituents which are illustrated by chemical drawing to bond at variable positions on a multiple ring system (for example a bicyclic ring system) are intended to bond to the ring where they are drawn to append. For example, substituents R ¹ and R ² of formula IV are intended to bond to the benzene ring of formula IV and not to the thiophene ring.

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.

Some of the compounds of the invention possess asymmetric carbon atoms. 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.

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 ¹³ C and ¹⁴ C. 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.

Pharmaceutical Compositions and Methods of Treatment

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

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.

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.

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.

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.

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.

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

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.

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

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.

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

Another aspect of the invention is the method where the cyclosporin is cyclosporin A.

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.

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.

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.

"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.

"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.

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

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, losenges, and powders as well as liquid suspensions, syrups, elixers, 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. 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.

Liquid compositions are usually in dosage unit ranges. Generally, the liquid composition will be in a unit dosage range of 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.

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 1-100 mg/kg body weight daily. Generally, more compound is required orally and less parenterally. The specific dosing regime, however, will be determined by a physician using sound medical judgement.

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 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 regime, however, will be determined by a physician using sound medical judgement.

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

Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

Vertex

Merimepodib Pharmaceuticals

Antiviral IMPDH inhibitor

(VX-497) Inc., Cambridge,

MA

XTL

XTL-6865 B iopharmac eutical s

Antiviral monoclonal antibody

(XTL-002) Ltd., Rehovot,

Isreal

Vertex

Pharmaceuticals

Telaprevir

NS3 serine protease Inc., Cambridge, (VX-950, LY- Antiviral

inhibitor MA/ Eli Lilly and 570310)

Co. Inc., Indianapolis, IN

NS5B Replicase Wyeth /

HCV-796 Antiviral

Inhibitor Viropharma

NS5B Replicase

NM-283 Antiviral Idenix / Novartis

Inhibitor

NS5B Replicase Gene Labs /

GL-59728 Antiviral

Inhibitor Novartis

NS5B Replicase Gene Labs /

GL-60667 Antiviral

Inhibitor Novartis

NS5B Replicase

2'C MeA Antiviral Gilead

Inhibitor

NS5B Replicase

PSI 6130 Antiviral Roche

Inhibitor

NS5B Replicase

R1626 Antiviral Roche

Inhibitor

2'C Methyl NS5B Replicase

Antiviral Merck adenosine Inhibitor

Japan Tobacco

JTK-003 Antiviral RdRp inhibitor

Inc., Tokyo, Japan Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

ICN

Levovirin Antiviral ribavirin Pharmaceuticals,

Costa Mesa, CA

Schering-Plough

Ribavirin Antiviral ribavirin Corporation,

Kenilworth, NJ

Ribapharm Inc.,

Viramidine Antiviral Ribavirin Prodrug

Costa Mesa, CA

Ribozyme

Heptazyme Antiviral ribozyme Pharmaceuticals

Inc., Boulder, CO

Boehringer serine protease Ingelheim Pharma

BILN-2061 Antiviral

inhibitor KG, Ingelheim,

Germany serine protease

SCH 503034 Antiviral Schering Plough inhibitor

SciClone

Pharmaceuticals

Zadazim Immune modulator Immune modulator

Inc., San Mateo, CA

Maxim

Pharmaceuticals

Ceplene Immunomodulator immune modulator

Inc., San Diego, CA

F. Hoffmann-La

HCV IgG immuno¬

CellCept Immunosuppressant Roche LTD, Basel, suppressant

Switzerland

Nabi

HCV IgG immunoB iopharmac eutical s

Civacir Immunosuppressant

suppressant Inc., Boca Raton,

FL Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

Human Genome

Albuferon - a Interferon albumin IFN-a2b Sciences Inc.,

Rockville, MD

InterMune

IFN

Infergen A Interferon Pharmaceuticals alfacon-1

Inc., Brisbane, CA

Intarcia

Omega IFN Interferon IFN-co

Therapeutics

Transition

IFN-β and

Interferon IFN-β and EMZ701 Therapeutics Inc., EMZ701

Ontario, Canada

Serono, Geneva,

Rebif Interferon IFN^la

Switzerland

F. Hoffmann-La

Roferon A Interferon IFN-a2a Roche LTD, Basel,

Switzerland

Schering-Plough

Intron A Interferon IFN-a2b Corporation,

Kenilworth, NJ

RegeneRx Biopharma. Inc.,

Intron A and Bethesda, MD/

Interferon IFN-a2b/al-thymosin

Zadaxin SciClone

Pharmaceuticals Inc, San Mateo, CA

Schering-Plough

Rebetron Interferon IFN-a2b/ribavirin Corporation,

Kenilworth, NJ

InterMune Inc.,

Actimmune Interferon INF-γ

Brisbane, CA

Interferon-β Interferon Interferon^-la Serono

Viragen/

Multiferon Interferon Long lasting IFN

Valentis Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

Lympho-blastoid IFN- GlaxoSmithKline

Wellferon Interferon

ocn 1 pic, Uxbridge, UK

Viragen Inc.,

Omniferon Interferon natural IFN-a

Plantation, FL

F. Hoffmann-La

Pegasys Interferon PEGylated IFN-a2a Roche LTD, Basel,

Switzerland

Maxim

Pegasys and PEGylated IFN-a2a/ Pharmaceuticals

Interferon

Ceplene immune modulator Inc., San Diego,

CA

F. Hoffmann-La

Pegasys and PEGylated IFN-

Interferon Roche LTD, Basel, Ribavirin a2a/ribavirin

Switzerland

Schering-Plough

PEG-Intron Interferon PEGylated IFN-a2b Corporation,

Kenilworth, NJ

Schering-Plough

PEG-Intron / PEGylated IFN-

Interferon Corporation, Ribavirin a2b/ribavirin

Kenilworth, NJ

Indevus

Pharmaceuticals

IP-501 Liver protection antifibrotic

Inc., Lexington, MA

Idun

Pharmaceuticals

IDN-6556 Liver protection caspase inhibitor

Inc., San Diego, CA

InterMune

ITMN-191 (R- serine protease

Antiviral Pharmaceuticals 7227) inhibitor

Inc., Brisbane, CA

NS5B Replicase

GL-59728 Antiviral Genelabs

Inhibitor

ANA-971 Antiviral TLR-7 agonist Anadys Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

serine protease

Boceprevir Antiviral Schering Plough inhibitor

serine protease Tibotec BVBA,

TMS-435 Antiviral

inhibitor Mechelen, Belgium

Boehringer serine protease Ingelheim Pharma

BI-201335 Antiviral

inhibitor KG, Ingelheim,

Germany serine protease

MK-7009 Antiviral Merck

inhibitor

PF-00868554 Antiviral replicase inhibitor Pfizer

Anadys

Non-Nucleoside

Pharmaceuticals,

ANA598 Antiviral NS5B Polymerase

Inc., San Diego, Inhibitor

CA, USA

Idenix

Non-Nucleoside Pharmaceuticals,

IDX375 Antiviral

Replicase Inhibitor Cambridge, MA,

USA

Boehringer

NS5B Polymerase Ingelheim Canada

BILB 1941 Antiviral

Inhibitor Ltd R&D, Laval,

QC, Canada

Nucleoside Pharmasset,

PSI-7851 Antiviral

Polymerase Inhibitor Princeton, NJ, USA

Nucleotide NS5B Pharmasset,

PSI-7977 Antiviral

Polymerase Inhibitor Princeton, NJ, USA

Antiviral NS5B Polymerase

VCH-759 ViroChem Pharma

Inhibitor

Antiviral NS5B Polymerase

VCH-916 ViroChem Pharma

Inhibitor

Antiviral NS5B Polymerase

GS-9190 Gilead

Inhibitor Type of Inhibitor or

Brand Name Physiological Class Source Company

Target

Peg-interferon Antiviral ZymoGenetics/Bris

Interferon

lamda tol-Myers Squibb

Synthetic Methods

The compounds may be made by methods known in the art including those described below. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using commercially 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 and are not to be confused with variables used in the claims or in other sections of the specification. Abbreviations used within the schemes generally follow conventions used in the art.

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; "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 ₂ 0" for diethyl ether; "DMAP" for 4- dimethylaminopyridine; "DCE" for 1 ,2-dichloroethane; "ACN" for acetonitrile; "DME" for 1,2-dimethoxy ethane; "HOBt" for 1-hydroxybenzotriazole hydrate; "DIEA" for diisopropylethylamine.

Abbreviations as used herein, are defined as follows: "1 x" for once, "2 x" for twice, "3 x" 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, "μΕ" 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, "cone." 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 "tic" for thin layer chromatography, "NMR" for nuclear magnetic resonance spectroscopy, "!H" for proton, "δ" for delta, "s" for singlet, "d" for doublet, "t" for triplet, "q" for quartet, "m" for multiplet, "br" for broad, "Hz" for hertz, and "α", "β", "R", "S", "E", and "Z" are stereochemical designations familiar to one skilled in the art.

A route to prepare compounds of formula 1 is shown in Schemes 1.

Scheme 1.

Preparation of tert-butyl 3-oxoazetidine-l-carboxylate

A mixture of oxalyl chloride (2.78 ml, 31.8 mmol) in (¾(¾ (80 ml) in a 500 ml round-bottomed flask was cooled to -78°C, and DMSO (4.51 ml, 63.5 mmol) was added dropwise to the mixture over 15 min. The reaction mixture was then stirred at the same temperature for 15 min. A solution of tert-butyl 3- hydroxyazetidine-l-carboxylate (5 g, 28.9 mmol) in CH2CI2 (50 ml) followed by a solution triethylamine (16.09 ml, 1 15 mmol) in CH2CI2 (70 ml) were added dropwise to the reaction mixture. The reaction mixture was warmed to room temperature, and then stirred overnight. The reaction mixture was washed with brine, and the aqueous layer back extracted with CH2CI2 (200 ml). The combined organic layers were washed with water, dried over Na ₂ S0 ₄ , filtered and concentrated in vacuum to provide a crude mixture. The crude product was then purified by column chromatography (15 % EtOAc in Hexane) to afford tert-butyl 3-oxoazetidine-l-carboxylate Yield: 4 g (81 %). ^X H NMR (400 MHz, CDC1 ₃ ): δ 4.69 (s, 4H), 1.49 (s, 9H). Preparation of tert-butyl 3 -(tert-butylsulfinylimino)azetidine- 1 -carboxylate

A mixture of tert-butyl 3-oxoazetidine-l-carboxylate (4 g, 23.37 mmol), 2-methylpropane-2-sulfinamide (3.12 g, 25.7 mmol) and tetraethoxytitanium (10.66 g, 46.7 mmol) in THF (40 ml) in a 20 ml sealed tube was stirred at 70°C overnight. The reaction mixture was cooled to room temperature, quenched with water and filtered through a bed of Celite. The solid material was washed with ethyl acetate. The organic layer was separated, dried over a2S04, filtered and concentrated to obtain a crude mixture. The crude product was then purified by column chromatography (20 % EtOAc in Hexane) to afford tert-butyl 3-(tert- butylsulfinylimino)azetidine-l -carboxylate Yield: 1.5 g (24 %). ^X H NMR (400 MHz, CDCI ₃ ): δ 5.15-4.94 (m, 1H), 4.75 (m, 1H), 4.68 (s, 2H), 1.45 (s, 9H), 1.27 (s, 9H). Preparation of tert-butyl 3 -cyano-3 -(1,1 -dimethylethylsulfinamido)azetidine- 1 - carboxylate

To a mixture of tert-butyl 3-(ter?-butylsulfinylimino)azetidine-l- carboxylate (1.5 g, 5.47 mmol) and Ti(OEt) ₄ (2.494 g, 10.93 mmol) in CH ₂ C1 ₂ (10 ml) under a nitrogen atmosphere in a 50 ml round-bottomed flask was added TMS-CN (1.83 ml, 13.67 mmol). The reaction mixture was stirred overnight and then quenched with water. The solid formed was filtered through a bed of Celite and washed with ethyl acetate. The organic layer was separated, dried over a2S04, filtered, and concentrated to give a crude mixture. The crude product was taken for next step without further purification. Yield: 1.5 g (91 %). ^l H NMR (400 MHz, CDC1 ₃ ): δ 4.49-4.35 (m, 2H), 4.14-4.4.11 (m, 2H), 1.45 (s, 9H), 1.27 (s, 9H). LCMS: (ES+) m/z observed = 202.2 (M+H) ⁺ of N-(3-cyanoazetidin-3- yl)-2-methylpropane-2-sulfinamide. Column-Ascentis Express C18 (5 X 4.6mm- 5μιη).

Mobile phase A : 2% MeCN -98% H ₂ O-10mM NH ₄ COOH

Mobile phase B : 98% MeCN - 2% H ₂ O-10mM NH ₄ COOH

Flow : lml/Min

Time %A %B

0.0 100.0 0

1.5 0.0 100.0

3.2 0.0 100.0

RetentionTime min: 1.8, wavelength: 220nm Synthesis of tert-butyl 3-carbamimidoyl-3-(l, l- dimethylethylsulfinamido)azetidine- 1 -carboxylate

A mixture of (R)-2-acetamido-3-mercaptopropanoic acid (N- acetylcycteine, 0.812 g, 4.98 mmol), tert-butyl 3-cyano-3-(l,l- dimethylethylsulfinamido)azetidine-l -carboxylate (1.5 g, 4.98 mmol), and ammonium acetate (1.918 g, 24.88 mmol) in MeOH (5 ml) in a 15 ml sealed tube was heated at 70 °C overnight. Analysis of the reaction mixture by LCMS showed 38% desired product with other impurities but no starting material present. The reaction mixture was used for the next step without workup.

LCMS: (ES+) m/z = 319.2 (M+H) ⁺

Column-Ascentis Express CI 8 (5 X 4.6ηηη-5μιη)

Mphase A : 2% MeC -98% H ₂ O-10mM H ₄ COOH

Mphase B : 98% MeCN - 2% H ₂ O-10mM NH ₄ COOH

Flow : lml/Min

Time %A %B

0.0 100.0 0

1.5 0.0 100.0

3.2 0.0 100.0

RT min: 1.59, wavelength: 220nm

Preparation of tert-butyl-3 -(1,1 -dimethylethylsulfinamido)-3 -(pyrimidin-2- yl)azetidine- 1 -carboxylate

To the above reaction mixture of tert-butyl 3-carbamimidoyl-3-(l,l- dimethylethylsulfinamido)azetidine-l -carboxylate (assumed 4.98 mmol) in methanol (5 mL) was added sodium methoxide (0.509 g, 9.42 mmol) and (E)-3- (dimethylamino)acrylaldehyde (0.560 g, 5.65 mmol), and the resulting solution was heated at 70°C overnight. The reaction mixture was then concentrated and the residue dissolved in ethyl acetate. The mixture was washed with water followed by saturated brine solution, and then dried over Na ₂ S0 ₄ and

concentrated. The crude product was then purified by column chromatography (50 % EtOAc in Hexane) to afford tert-butyl 3-(l,l-dimethylethylsulfinamido)-3- (pyrimidin-2-yl)azetidine-l -carboxylate Yield: 200 mg (overall 12 %). ^X H NMR (400 MHz, CDC1 ₃ ): δ 8.78 (d, J= 4.8 Hz, 2H), 7.24 (m, 1H), 4.63-4.48 (m, 2H), 4.23 (d, J = 9.2 Hz, 2H), 1.47 (s, 9H), 1.29 (s, 9H). LCMS: (ES+) m/z observed = 255.2 (M+H) ⁺ of 2-methyl-N-(3-(pyrimidin-2-yl)azetidin-3-yl)propane-2- sulfinamide.

Column-Ascentis Express CI 8 (5 X 4.6ιηιη-5μιη)

Mphase A : 2% MeC -98% H ₂ O-10mM H ₄ COOH

Mphase B : 98% MeCN - 2% H ₂ O-10mM NH ₄ COOH

Flow : lml/Min

Time %A %B

0.0 100.0 0

1.5 0.0 100.0

3.2 0.0 100.0

RT min: 1.69, wavelength: 220nm Preparation of tert-butyl 3 -amino-3 -(pyrimidin-2-yl)azetidine- 1 -carboxylate hydrochloride

tert-Butyl 3 -( 1 , 1 -dimethylethylsulfinamido)-3 -(pyrimidin-2-yl)azetidine- 1-carboxylate (40 mg, 0.1 13 mmol) in MeOH (0.5 ml) in a 10 ml round-bottomed flask was cooled to 0°C. A solution of HCl in ether (0.141 ml, 0.564 mmol, 4 ) was added. The mixture was then stirred at 0°C for 15min and then concentrated at room temperature. The residue was triturated with hexane and diethyl ether. The resulting solid was allowed to settle and supernatant liquid was decanted. The solid obtained was dried under vacuo to afford tert-butyl 3 -amino-3 - (pyrimidin-2-yl)azetidine- 1-carboxylate hydrochloride Yield: 28mg (87 %). ^X H NMR (400 MHz, DMSO-d ₆ ): δ 9.08 (bs, 2H), 9.0 (d, J= 4.8 Hz, 2H), 7.63 (t, J = 4.8 Hz, 1H), 4.23-4.21 (m, 4H), 1.43 (s, 9H). LCMS: (ES+) m/z observed = 151.2 (M+H) ⁺ of 3-(pyrimidin-2-yl)azetidin-3-amine.

Column-Ascentis Express CI 8 (5 X 4.6ηΐΓη-5μιη)

Mphase A : 2% MeC -98% H ₂ O-10mM H ₄ COOH

Mphase B : 98% MeCN - 2% H ₂ O-10mM NH ₄ COOH

Flow : lml/Min

Time %A %B

0.0 100.0 0

1.5 0.0 100.0

3.2 0.0 100.0

RT min: 1.49, wavelength: 220nm Preparation of ethyl 4-fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran-3- carboxylate

To a mixture of ethyl 2-(4-fluorophenyl)-5-hydroxybenzofuran-3- carboxylate (500 mg, 1.665 mmol) in acetonitrile (10 mL) at r.t. under 2 was added l-(chloromethyl)-4-fluoro-l,4-diazoniabicyclo[2.2.2]octane

tetrafluoroborate (708 mg, 1.998 mmol). The mixture was stirred at r.t. (the mixture turned bright yellow in color) for 20 hours. The mixture was evaporated. The residue was added with 10 ml H ₂ 0. The aqueous decanted, and the residue further washed with 2 x 5 ml H2O. The mixture was dissolved in MeOH (about 10 ml), and the insoluble filtered. The filtrate was purified by Shimadzu-VP preparative reverse phase HPLC using the separation method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10% H ₂ O-0.1% TFA, Start %B = 60, Final %B = 100, Gradient time = 10 min, Stop time = 12 min, Flow Rate = 25 mL/min, Column: Waters-Sunfire 19 x 100 mm S5, Fraction Collection: 6.44 - 7.24 min. (UV detection at 220 nm). The desired fractions were combined and evaporated to give a yellow solid. The yellow solid was further purified by Biotage Horizon flash chromatography (0 to 70%

EtOAc/Hexane, 3 x 80 g silica gel column) to give a light yellow solid (108.9 mg). ^X H NMR (500 MHz, CD ₃ OD) δ 7.95 (m, 2H), 7.26 (t overlapping with dd, 2H), 7.25 (dd, 1H), 7.03 (t, J= 8.39, 1H), 4.39 (q, J= 7.17, 2H), 1.36 (t, J= 7.17, 3H). ¹⁹ F NMR (470.45 MHz, CD ₃ OD) δ -112.36, -142.29 (The ¹⁹ F chemical shift was referenced to CFCI ₃ at 0.0 ppm). The position of the F atom at C4 was confirmed by through bond correlation between H6 and H7, ^l H- ^u C

HMBC and F-C4 coupling in ¹³ C NMR (125.75 MHz, CD ₃ OD) (δ 144.8 ppm, d, J= 247, C4). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 319.14, HPLC R _t = 1.718 min. The minor fractions collected at about 7.69 - 8.20 min. was confirmed by through bond correlation, ^l H- ^u C

HMBC and F-C6 coupling in ¹³ C NMR (125.75 MHz, CD ₃ OD) (δ 152.5 ppm, d, J= 242 Hz, C6) to be the isomer of the F-atom at C6 (C4 : C6 about 3: 1 based on preparative HPLC %area of the UV trace); ¾ NMR (500 MHz, CD ₃ OD) δ 8.04 (dd, J= 8.55, 5.49, 2H), 7.59 (d, J= 8.85, 1H), 7.38 (d, J= 10.07, 1H), 7.25 (t, J = 8.70, 2H), 4.40 (q, J= 7.17, 2H), 1.41 (t, J= 7.17, 3H). ¹⁹ F NMR (470.45 MHz, CD ₃ OD) δ -112.29, -138.52. LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90%

MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 319.14, HPLC R _t = 1.798 min.

(Alternatively, the two isomers were separated after the ester hydrolysis by

Shimadzu-VP preparative reverse phase HPLC using the same method as above but with Start %B = 40).

Preparation of 4-fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran-3-carboxylic acid

To a mixture of ethyl 4-fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran- 3-carboxylate (108.9 mg, 0.342 mmol) in a mixture of MeOH (2 mL)/THF (2 mL) at r.t. under N ₂ was added sodium hydroxide (1.0 mL, 1.0 mmol) (1 M aq.). The mixture was stirred at 100 °C for 1.5 hours. The mixture was cooled to r.t., added with 1.5 ml IN HC1, and then added 10 ml H2O. The white precipitates were filtered and washed with 3 x 2 ml H ₂ 0 and dried (73 mg). *H NMR (500 MHz, CD ₃ OD) δ 7.98 (m, 2H), 7.25 (t overlapping with dd, 2H), 7.24 (dd, 1H), 7.02 (t, J= 8.39, 1H). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 291.01, HPLC R _t = 1.478 min.

Preparation of 4-fluoro-2-(4-fluorophenyl)-5-hydroxy-N-methylbenzofuran-3- carboxamide

To a mixture of 4-fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran-3- carboxylic acid (73 mg, 0.252 mmol), methylamine, HCl (25.5 mg, 0.377 mmol), HOBT hydrate (65.5 mg, 0.428 mmol) and EDC hydrochloride (87 mg, 0.453 mmol) at r.t. under 2 was added N,N-diisopropylethylamine (0.220 mL, 1.258 mmol). The mixture was stirred at r.t. for 16 hours. After concentration, the mixture was added with 5 ml IN HCl, and then 14 ml H2O. The white solid was filtered and washed with 3 x 5 ml H ₂ 0 and dried (64 mg). ^X H NMR (500 MHz, CD ₃ OD) δ 7.89 (dd, J= 8.09, 5.34, 2H), 7.25 (t overlapping with dd, 2H), 7.23 (dd, 1H), 6.99 (t, J= 8.55, 1H), 2.96 (s, 3H). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 304.06, HPLC R _t = 1.262 min. Preparation of 4-fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofuran-5- yl trifluoromethanesulfonate

To a white suspension of 4-fluoro-2-(4-fluorophenyl)-5-hydroxy-N- methylbenzofuran-3-carboxamide (64 mg, 0.211 mmol) in CH2CI2 (2 mL) at r.t. under 2 was added triethylamine (0.059 mL, 0.422 mmol). The mixture was cooled to 0 °C, and then added 1, 1, 1-trifluoro-N-phenyl-N- (trifluoromethylsulfonyl)methanesulfonamide (1 13 mg, 0.317 mmol). The mixture was then stirred at r.t. (the white suspension turned into a light yellow solution after stirring for about 10 min) for 2 hours 35 min. The mixture was left standing at r.t. overnight, and then evaporated. The residue was cooled in an ice- water bath, added with 2 ml H ₂ 0. The solids were filtered and washed with 3 x 2 ml H ₂ 0, and dried (94 mg). ¾ NMR (500 MHz, CD ₃ OD) δ 7.95 (m, 2H), 7.59 (dd, J= 9.00, 1.00, 1H), 7.50 (dd, J= 9.00, 7.50, 1H), 7.30 (t, J= 8.55, 2H), 2.99 (s, 3H). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S 10; (ES+) m/z (M+H) ⁺ = 436.04, HPLC R _t = 1.678 min.

Preparation of methyl 5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-2-methoxy-4-methylbenzoate

A mixture of the above prepared 4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl trifluoromethanesulfonate (assumed 0.211 mmol), methyl 2-methoxy-4-methyl-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan -2- yl)benzoate (0.078 g, 0.253 mmol), (Ph ₃ P) ₄ Pd (0.024 g, 0.021 mmol) and cesium carbonate (0.103 g, 0.317 mmol) in a mixture of H ₂ 0 (0.2 mL)/l,4-dioxane (1 mL) was stirred at 95 °C for 2 hours 30 min. The mixture was left standing at r.t. overnight. The mixture was diluted with 3.5 ml 1,4-dioxane, filtered through a Whatman PVDF 0.45 um disk (with 3 x 1 ml washing). The filtrate was concentrated. The mixture was added with 3.5 ml IN HC1, and then 6 ml H ₂ 0 (yellow solid deposited on the wall of the flask). The aqueous was decanted, and the residue washed with 3 x 2 ml H ₂ 0 and dried. LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column:

Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 466.27, HPLC R _t = 1.708 min.

Preparation of 5-(4-fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofuran -5- yl)-2-methoxy-4-methylbenzoic acid

To the above prepared methyl 5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-2-methoxy-4-methylbenzoate (assumed

0.211 mmol) in a mixture MeOH (2 mL)/THF (2 mL) at r.t. under N ₂ was added sodium hydroxide (0.84 mL, 0.84 mmol). The mixture was stirred at r.t. for 24 hours. The mixture was added with 2 ml IN HC1, and concentrated until off white solids formed. The mixture was added with 5 ml H ₂ 0, the solids filtered and washed with 3 x 2 ml H ₂ 0 and dried (75.1 mg). ^X H NMR (500 MHz, CD ₃ OD) δ 7.95 (m, 2H), 7.73 (s, 1H), 7.51 (d, J= 8.24, 1H), 7.30-7.25 (t ovelapping with m, 3H), 7.13 (s, 1H), 3.99 (s, 3H), 2.96 (s, 3H), 2.28 (s, 3H). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 452.23, HPLC R _t = 1.582.

The following intermediates were prepared in a similar manner as described.

Methyl 3 -fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3 -(methylcarbamoyl)benzofuran-

5-yl)-4-methylbenzoate

LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S 10; (ES+) m/z (M+H) ⁺ = 454.08, HPLC R _t = 1.838 min.

3-Fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3-(methylcarbamoy l)benzofuran-5-yl)-4- methylbenzoic acid

^X H NMR (500 MHz, CD ₃ OD) δ 7.96 (m, 2H), 7.76 (s, 1H), 7.75-7.73 (d, 1H), 7.56 (d, J= 8.24, 1H), 7.33-7.28 (t ovelapping with m, 3H), 2.96 (s, 3H), 2.20 (s, 3H). LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S10; (ES+) m/z (M+H) ⁺ = 440.09, HPLC R _t = 1.720. The following acid intermediate was prepared by either one of the methods shown below in a similar manner as described.

3-(4-Fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofu ran-5-yl)-4- methylbenzoic acid

LC/MS were performed by using Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. HPLC method: Solvent A = 10% MeOH-90% H ₂ O-0.1% TFA, Solvent B = 90% MeOH-10%H ₂ O-0.1% TFA, Start %B = 0, Final %B = 100, Gradient time = 2 min, Stop time = 3 min, Flow Rate = 5 ml/min, Column: Phenomenex-Luna, 3.0 x 50 mm, S 10; (ES+) m/z (M+H) ⁺ = 422.19, HPLC R _t = 1.653 min.

Synthesis of tert-butyl 3-(3-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3-(pyri midin-2- yl)azetidine- 1 -carboxylate

To a mixture of 3-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzoic acid (60 mg, 0.142 mmol) and tert-butyl 3 -amino-3-(pyrimidin-2-yl)azetidine-l -carboxylate hydrochloride (40.8 mg, 0.142 mmol) in DMF (1 ml) was added PyBOP (74.1 mg, 0.142 mmol) and triethylamine (0.020 ml, 0.142 mmol). The reaction mixture was stirred overnight, and then diluted with water and extracted with EtOAc. The organic layer was further washed with water, dried over sodium sulphate and

concentrated. The crude product was then purified by column chromatography (2% methanol in chloroform) to afford tert-butyl 3-(3-(4-fluoro-2-(4- fluorophenyl)-3 -(methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3 -

(pyrimidin-2-yl)azetidine-l -carboxylate Yield: 55 mg (59 %). LCMS: (ES+) m/z observed = 554.2 (M+H) ⁺ of 4-fluoro-2-(4-fluorophenyl)-N-methyl-5-(2-methyl- 5-(3-(pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)benzofura n-3-carboxamide. Column-ZORBAX SB C18 (50 X 4.6ηιηι-5μιη)

Mphase A : 10% MeOH -90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH -10% H ₂ O-0.1% TFA

Flow : lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

RT min: 2.1, wavelength: 220nm

Synthesis of 4-fluoro-2-(4-fluorophenyl)-N-methyl-5-(2-methyl-5-(3-(pyrim idin- 2-yl)azetidin-3-ylcarbamoyl)phenyl)benzofuran-3-carboxamide

To a mixture of tert-butyl 3-(3-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3-(pyri midin-2- yl)azetidine- 1 -carboxylate (55 mg, 0.084 mmol) in MeOH (0.5ml) in a 10 ml round-bottomed flask was added a solution of HC1 in ether (0.021 ml, 0.084 mmol, 4 N). The mixture was stirred at room temperature for 30 min. After which time, no starting material was observed by LCMS. The reaction mixture was concentrated, and the crude residue was diluted with water and washed with ethyl acetate (10 ml). The aq. layer was basified with solid NaHC03, and back extracted with ethyl acetate (3 x 10 ml). The combined the organic layers were washed with brine solution (3 x 15 ml), dried with Na ₂ S0 ₄ , filtered and concentrated. The product obtained was re-crystallized from ethyl acetate ether. Yield : 42 mg (90 %). ^X H NMR (400 MHz, DMSO-d ₆ ): δ 9.38 (s, 1H), 8.80 (d, J = 4.8 Hz, 2H), 8.71 (broad d, J= 4.4 Hz, 1H), 7.96 - 7.86 (m, 4H), 7.66 (d, J = 8.4 Hz, 2H), 7.48 - 7.35 (m, 5H), 4.10 (d, J= 8.8 Hz, 2H), 3.84 (d, J= 8.4 Hz, 2H), 2.80 (d, J= 4.8 Hz, 3H), 2.23 (s, 3H). ¹⁹ FNMR (376.47 MHz, DMSO-d ₆ ): δ -110.43, -121.44. LCMS: (ES+) m/z = 554.2 (M+H) ⁺ Column-ZORBAX SB C18 (50 X 4.6ηηη-5μιη) Mphase A : 10% MeOH -90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH -10% H ₂ O-0.1% TFA

Flow lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

RT min: 1.69, wavelength: 220nm

HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron

Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 6.90

Wavelength: 220 nm, RT min: 6.90

HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 8.13

Wavelength: 220 nm, RT min: 8.13 Synthesis 4-fluoro-2-(4-fluorophenyl)-N-methyl-5-(2-methyl-5-(l-methyl -3- (pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)benzofuran-3-c arboxamide

To a colorless suspension of 4-fluoro-2-(4-fluorophenyl)-N-methyl-5-(2- methyl-5-(3-(pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)be nzofuran-3- carboxamide (25 mg, 0.045 mmol) and paraformaldehyde (2.034 mg, 0.068 mmol) in MeOH (1 ml) in a 5 ml round-bottomed flask was added sodium cyanoborohydride (4.26 mg, 0.068 mmol). The reaction mixture was stirred at room temperature for 1 hour and concentrated to remove methanol. The crude residue was diluted with ethyl acetate (10 ml), and washed with sat. NH4CI solution. The organic solution was dried over Na ₂ S04, filtered and concentrated. The product was purified by Prep-HPLC and obtained as TFA salt. Yield 19 mg (74%). ¾ NMR (400 MHz, CD ₃ OD at 55°C): δ 8.92 (s, 1H), 7.97 - 7.92 (m, 3H), 7.86 (d, J= 1.6 Hz, 1H), 7.54 - 7.50 (m, 3H), 7.32 - 7.25 (m, 3H), 5.1-4.8 (broad m, 4 H), 3.21 (s, 3H), 2.96 (s, 3H), 2.31 (s, 3H). ¹⁹ F NMR (376.57 MHz, CD ₃ OD): δ -77.03 (TFA), -112.14, -122.86. LCMS: (ES+) m/z = 568.2 (M+H) ⁺ Column-ZORBAX SB C18 (50 X 4.6ιηιη-5μιη)

Mphase A : 10% MeOH -90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH -10% H ₂ O-0.1% TFA

Flow : lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

RT min: 1.7, wavelength: 220nm

HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron

Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5) Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 7.06

Wavelength: 220 nm, RT min: 7.06 HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron

Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 9.10

Wavelength: 220 nm, RT min: 9.10

PREPARATIVE HPLC METHOD

Column : Sunfire C18 (19χ150χ5μ)

Mobile Phase: 0.05% TFA (A), MeCN (B)

Gradient: Time Flow B%

0 18ml/min 20

12 18ml/min 100

RT: 5.1min Synthesis of tert-butyl 3-(5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-2-methoxy-4-methylbenzamid o)-3- (pyrimidin-2-yl)azetidine- 1 -carboxylate

To a mixture of 5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-2-methoxy-4-methylbenzoic acid (50 mg, 0.111 mmol) and tert-butyl 3 -amino-3-(pyrimidin-2-yl)azetidine-l -carboxylate hydrochloride (31.8 mg, 0.111 mmol) in DMF (1 ml) in a 25 ml round-bottomed flask was added PyBOP (69.2 mg, 0.133 mmol) and then triethylamine (0.046 ml, 0.332 mmol). The reaction mixture was stirred overnight, and then diluted with water and extracted with EtOAc. The organic layer was further washed with water, dried over sodium sulphate and concentrated. The crude product was then purified by column chromatography (2% methanol in chloroform) to afford tert- butyl 3-(5-(4-fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofu ran-5-yl)-2- methoxy-4-methylbenzamido)-3-(pyrimidin-2-yl)azetidine- 1 -carboxylate. Yield: 40 mg (53 %). LCMS: (ES+) m/z observed = 584.2 (M+H) ⁺ of 4-fluoro-2-(4- fluorophenyl)-5-(4-methoxy-2-methyl-5-(3-(pyrimidin-2-yl)aze tidin-3- ylcarbamoyl)phenyl)-N-methylbenzofuran-3 -carboxamide

Column-PUROSPHER@ star RP-18 (55 X 4 ιηιη-3μιη)

Buffer : 20 Mm NH ₄ OAC in water

Mobile Phase A : Buffer : MeCN (90: 10)

Mobile Phase B : MeCN : Buffer (90: 10)

Flow : 2.5ml/Min

Time %A %B

0.0 100.0 0.00

2 00.0 100.0

2.5 00.0 100.0

3 100.0 00.0

RT min: 2.7, wavelength: 220nm Synthesis 4-fluoro-2-(4-fluorophenyl)-5-(4-methoxy-2-methyl-5-(3-(pyri midin-2- yl)azetidin-3-ylcarbamoyl)phenyl)-N-methylbenzofuran-3-carbo xamide

To a clear solution of tert-butyl 3-(5-(4-fluoro-2-(4-fluorophenyl)-3-

(methylcarbamoyl)benzofuran-5-yl)-2-methoxy-4-methylbenza mido)-3- (pyrimidin-2-yl)azetidine-l-carboxylate (40 mg, 0.059 mmol) MeOH (0.5 ml) in a 10 mL round-bottomed flask was added a solution of HQ in ether (0.146 ml, 0.585 mmol, 4 N). The mixture was stirred at room temperature for 30 min. After which time, no starting material was observed by LCMS analysis. The reaction mixture was concentrated, and the residue diluted with water and extracted with ethyl acetate (10 ml). The aqueous layer was basified with solid NaHC03 and extracted with ethyl acetate (3 x 10 ml). The combined the organic extracts were washed with brine solution (3 x 15 ml), dried over Na ₂ S0 ₄ , filtered and concentrated. The product was purified by Prep-HPLC and obtained as TFA salt. Yield : 18 mg (53 %).

¾ NMR (400 MHz, DMSO-d ₆ ): δ 10.06 (s, 1H), 9.21 (b s, 2H), 9.03 (d, J= 4.8 Hz, 2H), 8.70 (d, J= 4.8 Hz, 1H), 7.93 (m, 2H), 7.88 (s, 1H), 7.65-7.62 (m, 2H), 7.43 (t, J= 8.8 Hz, 2H), 7.33 (m, 1H), 7.30 (s, 1H), 4.71 (m, 2H), 4.51 (m, 2H), 4.13 (s, 3H), 2.80 (d, J= 4.8 Hz, 3 H), 2.27 (s, 3 H). ¹⁹ F NMR (376.47 MHz, DMSO-d ₆ ): δ -73.55 (TFA), -110.39, -121.65. LCMS: (ES+) m/z observed = 584.0 (M+H) ⁺

Column-Ascentis Express CI 8 (5 X 4.6ιηιη-5μιη)

Mphase A : 2% MeC -98% H ₂ O-10mM H ₄ COOH

Mphase B : 98% MeCN - 2% H ₂ 0- lOmM NH ₄ COOH

Flow : lml/Min

Time %A %B

0.0 100.0 0

1.5 0.0 100.0

3.2 0.0 100.0

RT min: 1.76, wavelength: 220nm HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 7.31

Wavelength: 220 nm, RT min: 7.31

HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 8.64

Wavelength: 220 nm, RT min: 8.64 PREPARATIVE HPLC METHOD

Column : XBridege phenyl (19x250x5 μ)

Mobile Phase: 0.05% TFA (A), MeCN (B)

Gradient: Time Flow B%

0 15ml/min 20 10 15ml/min 60

RT: 9.50min Synthesis 4-fluoro-2-(4-fluorophenyl)-5-(4-methoxy-2-methyl-5-(l-methy l-3- (pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)-N-methylbenzo furan-3- carboxamide

To a colorless suspension of 4-fluoro-2-(4-fluorophenyl)-5-(4-methoxy-2- methyl-5-(3-(pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)-N - methylbenzofuran-3-carboxamide (30 mg, 0.051 mmol) and paraformaldehyde (2.315 mg, 0.077 mmol) in MeOH (1 ml) in a 5 mL round-bottomed flask was added sodium cyanoborohydride (4.85 mg, 0.077 mmol) The reaction mixture was stirred at room temperature for 1 h. and concentrated to remove methanol. The residue was diluted with ethyl acetate (10 ml), washed with sat. NH4CI solution. The organic solution was dried over Na ₂ S0 ₄ , filtered and concentrated. The product was purified by Prep-HPLC and obtained as a TFA salt. Yield 10 mg (33%). ¾ NMR (400 MHz, CD ₃ OD at 55°C): δ 9.04 (d, J= 4.8 Hz, 2H), 8.08 (b s, 1H), 7.95 (m, 2H), 7.59 (t, J= 4.8 Hz, 1H), 7.52 (d, J= 8.8 Hz, 1H), 7.29-7.23 (m, 4H), 5.20 (d, J= 11.6 Hz, 2H), 4.71 (d, J= 10.8 Hz, 2H), 4.20 (s, 3 H), 3.26 (s, 3 H), 2.96 (s, 3 H), 2.32 (s, 3 H). ¹⁹ F NMR (376.57 MHz, CD ₃ OD): δ -77.01 (TFA), -1 12.22, -122.88. LCMS: (ES+) m/z observed = 599.2 (M+2H) ⁺ Column-ZORBAX SB C18 (50 X 4.6ιηιη-5μιη)

Mphase A : 10% MeOH -90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH -10% H ₂ O-0.1% TFA

Flow : lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

RT min: 1.78, wavelength: 220nm HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron

Buffer : 0.05% TFA in water pH 2.5 Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 7.42

Wavelength: 220 nm, RT min: 7.42

HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron

Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 9.86

Wavelength: 220 nm, RT min: 9.86

PREPARATIVE HPLC METHOD

Column : Sunfire C18 (19χ150χ5μ)

Mobile Phase: 0.05% TFA (A), MeCN (B)

Gradient: Time Flow B%

0 18ml/min 20

12 18ml/min 45

RT: 9.9min Synthesis of tert-butyl 3-(3-fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3-(pyri midin-2- yl)azetidine- 1 -carboxylate

To a mixture of 3-fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzoic acid (50 mg, 0.114 mmol) and tert-butyl 3 -amino-3-(pyrimidin-2-yl)azetidine-l -carboxylate hydrochloride (35.9 mg, 0.125 mmol) in DMF (1 ml) in a 25 ml round-bottomed flask was added PyBop (77 mg, 0.148 mmol) and then triethylamine (0.032 ml, 0.228 mmol). The reaction mixture was stirred overnight, and then diluted with water and extracted with EtOAc. The organic layer was further washed with water, dried over sodium sulphate and concentrated. The crude product was then purified by column chromatography (2% methanol in chloroform) to afford tert- butyl 3-(3-fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3-(pyri midin-2- yl)azetidine-l -carboxylate. Yield: 40 mg (52.4 %). LCMS: (ES+) m/z observed = 572.2 (M+H) ⁺ of 4-fluoro-5-(3-fluoro-2-methyl-5-(3-(pyrimidin-2-yl)azetidin- 3- ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3- carboxamide Column-PUROSPHER@ star RP- 18 (55 X 4 ιηιη-3μιη).

Buffer : 20 mM NH ₄ OAC in water

Mobile Phase A : Buffer : MeCN (90: 10)

Mobile Phase B : MeCN : Buffer (90: 10)

Flow : 2.5ml/Min

Time %A %B

0.0 100.0 0.00

2 00.0 100.0

2.5 00.0 100.0

3 100.0 00.0

RT min: 2.1, wavelength: 220nm Synthesis 4-fluoro-5-(3-fluoro-2-methyl-5-(3-(pyrimidin-2-yl)azetidin- 3- ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3- carboxamide

To a solution of tert-butyl 3-(3-fluoro-5-(4-fluoro-2-(4-fluorophenyl)-3- (methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)-3-(pyri midin-2- yl)azetidine- 1 -carboxylate (40 mg, 0.060 mmol) in methanol (0.5 ml) in a 10 ml round-bottomed flask was added a solution of HQ in ether (0.199 ml, 0.596 mmol, 3 N). The reaction mixture was stirred at room temperature for 30 min. After which time, no starting material was observed by LCMS analysis. The reaction mixture was concentrated, diluted with water and extracted with ethyl acetate (10 ml). The aqueous layer was basified with solid NaHCC and extracted with ethyl acetate (3 x 10 ml). The combined organic extracts were washed with brine solution (3 x 15 ml), dried over Na ₂ S0 ₄ , filtered and concentrated. The product was purified by Prep-HPLC and obtained as TFA salt. Yield : 20 mg (59 %). 'H NMR (400 MHz, DMSO-d ₆ ): δ 9.81 (s, 1H), 9.20 (b s, 2H), 8.91 (d, J= 4.8 Hz, 2H), 8.72 (d, J= 4.8 Hz, 1H), 7.94 (m, 2H), 7.78 (d, 1H), 7.77 (s, 1H), 7.71 (d, J= 8.8 Hz, 1H), 7.53 (t, J= 4.8 Hz, 1H), 7.46-7.38 (m, 3H), 4.62 (m, 2H), 4.43 (m, 2H), 2.81 (d, J= 4.8 Hz, 3 H), 2.16 (s, 3 H). ¹⁹ F NMR (376.57 MHz, DMSO-d ₆ ): δ -73.64 (TFA), -110.21, -114.41, -121.40. LCMS: (ES+) m/z = 572.2 (M+H) ⁺

Column-ZORBAX SB C18 (50 X 4.6ιηιη-5μιη)

Mphase A : 10% MeOH -90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH -10% H ₂ O-0.1% TFA

Flow : lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

PvT min: 1.75, wavelength: 220nm HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 7.14

Wavelength: 220 nm, RT min: 7.14

HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN :Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 8.39

Wavelength: 220 nm, RT min: 8.39 PREPARATIVE HPLC METHOD

Column : XBridge phenyl (19χ250χ5μ)

Mobile Phase: 0.05% TFA (A), MeCN (B)

Gradient: Time Flow B%

0 15ml/min 20 10 15ml/min 60

RT: 9.00min Synthesis 4-fluoro-5-(3-fluoro-2-methyl-5-(l-methyl-3-( yrimidin-2-yl)azetidin- 3-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran- 3-carboxamide

To a colorless suspension of 4-fluoro-5-(3-fluoro-2-methyl-5-(3- (pyrimidin-2-yl)azetidin-3-ylcarbamoyl)phenyl)-2-(4-fluoroph enyl)-N- methylbenzofuran-3-carboxamide (30 mg, 0.052 mmol) and paraformaldehyde (2.364 mg, 0.079 mmol) in MeOH (1 ml) in a 5 mL round-bottomed flask was added sodium cyanoborohydride (4.95 mg, 0.079 mmol). The reaction mixture was stirred at room temperature for 1 hr., and then concentrated to remove methanol and diluted with ethyl acetate (10ml). The organic layer was washed with sat. NH4CI solution, dried over a2S04, filtered and concentrated. The product was purified by Prep-HPLC and obtained as TFA salt. Yield: 10 mg (33%). ¾ NMR (400 MHz, CD ₃ OD at 55°C): δ 8.93 (d, J= 4.8 Hz, 2H), 7.9 - 7.94 (m, 2H), 7.73-7.72 (m, 2H), 7.56 (d, J= 8.4 Hz, 1H), 7.52 (t, J= 4.8 Hz, 1H), 7.35-7.26 (m, 3H), around 4.54 (m, 4H, obscured by solvent peak), 3.18 (s, 3 H), 2.96 (s, 3 H), 2.22 (s, 3 H). ¹⁹ F NMR (376.47 MHz, CD ₃ OD): δ -76.99 (TFA), -1 12.03, -1 16.00, -122.73. LCMS: (ES+) m/z observed = 587.2 (M+2H) ⁺ Column-ZORBAX SB C18 (50 X 4.6ιηιη-5μιη)

Mphase A : 10% MeOH-90% H ₂ O-0.1% TFA

Mphase B : 90% MeOH- 10% H ₂ O-0.1% TFA

Flow : lml/Min

Time %A %B

0.0 100.0 0

2 00.0 100.0

3 100.0 0.0

RT min: 1.73, wavelength: 220nm

HPLC Method: SUNFIRE C18 (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : MeCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 7.28

Wavelength: 220 nm, RT min: 7.28

HPLC Method: XBridge phenyl (4.6X150)mm, 3.5 micron Buffer : 0.05% TFA in water pH 2.5

Mobile Phase A : Buffer : MeCN (95:5)

Mobile Phase B : meCN : Buffer (95:5)

Flow: lml/min

Time B%

0 10

12 100

15 100

Wavelength: 254 nm, RT min: 9.43

Wavelength: 220 nm, RT min: 9.43

PREPARATIVE HPLC METHOD

Column : Sunfire C18 (19χ150χ5μ)

Mobile Phase: 0.05% TFA (A), MeCN (B)

Gradient: Time Flow B%

0 18ml/min 10

15 18ml/min 100 RT: 9.3min Biological Methods

HCVNS5B RdRp cloning, expression, and purification. The cDNA encoding the NS5B protein of HCV, genotype lb, was cloned into the pET21a expression vector. The protein was expressed with an 18 amino acid C-terminal truncation to enhance the solubility. The E. coli competent cell line BL21(DE3) was used for expression of the protein. Cultures were grown at 37 °C for ~ 4 hours until the cultures reached an optical density of 2.0 at 600 nm. The cultures were cooled to 20 °C and induced with 1 mM IPTG. Fresh ampicillin was added to a final concentration of 50 μg/mL and the cells were grown overnight at 20 °C.

Cell pellets (3L) were lysed for purification to yield 15-24 mgs of purified NS5B. The lysis buffer consisted of 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.5% triton X-100, 1 mM DTT, ImM EDTA, 20% glycerol, 0.5 mg/mL lysozyme, 10 mM MgCl ₂ , 15 ug/mL deoxyribonuclease I, and Complete TM protease inhibitor tablets (Roche). After addition of the lysis buffer, frozen cell pellets were resuspended using a tissue homogenizer. To reduce the viscosity of the sample, aliquots of the lysate were sonicated on ice using a microtip attached to a Branson sonicator. The sonicated lysate was centrifuged at 100,000 x g for 30 minutes at 4 °C and filtered through a 0.2 μιη filter unit (Corning).

The protein was purified using two sequential chromatography steps: Heparin sepharose CL-6B and polyU sepharose 4B. The chromatography buffers were identical to the lysis buffer but contained no lysozyme, deoxyribonuclease I, MgCl ₂ or protease inhibitor and the NaCl concentration of the buffer was adjusted according to the requirements for charging the protein onto the column. Each column was eluted with a NaCl gradient which varied in length from 5-50 column volumes depending on the column type. After the final chromatography step, the resulting purity of the enzyme is >90% based on SDS-PAGE analysis. The enzyme was aliquoted and stored at -80 °C.

HCVNS5B RdRp enzyme assay. An on-bead solid phase homogeneous assay was used in a 384-well format to assess NS5B inhibitors (WangY-K, Rigat K, Roberts S, and Gao M (2006) Anal Biochem, 359: 106-1 11). The biotinylated oligo dTi2 primer was captured on streptavidin-coupled imaging beads (GE, RPNQ0261) by mixing primer and beads in IX buffer and incubating at room temperature for three hours. Unbound primer was removed after centrifugation. The primer-bound beads were resuspended in 3X reaction mix (20 mM Hepes buffer, pH 7.5, dT primer coupled beads, poly A template, ³ H-UTP, and RNAse inhibitor (Promega 2515)). Compounds were serially diluted 1 :3 in DMSO and aliquoted into assay plates. Equal volumes (10 μί) of water, 3X reaction mix, and enzyme in 3X assay buffer (60 mM Hepes buffer, pH 7.5, 7.5 mM MgCl ₂ , 7.5 mM KCl, 3 mM DTT, 0.03 mg/mL BSA, 6 % glycerol) were added to the diluted compound on the assay plate. Final concentration of components in 384-well assay: 0.36 nM template, 15 nM primer, 0.29 μΜ ³ H-UTP (0.3 μϋϊ), 1.6 υ/μί RNAse inhibitor, 7 nM NS5B enzyme, 0.01 mg/mL BSA, 1 mM DTT, and 0.33 μg/μL beads, 20 mM Hepes buffer, pH 7.5, 2.5 mM MgCl ₂ , 2.5 mM KCl, and 0.1 % DMSO.

Reactions were allowed to proceed for 4 hours at 30° C and terminated by the addition of 50 mM EDTA (10 μΚ). After incubating for at least 15 minutes, plates were read on an Amersham LEADseeker multimodality imaging system. IC5 ₀ values for compounds were determined using ten different [I]. IC5 ₀ values were calculated from the inhibition using the four-parameter logistic formula y = A+((B-A)/(l+((C/x) ^A D))), where A and B denote minimal and maximal % inhibition, respectively, C is the IC5 ₀ , D is hill slope and x represents compound concentration.

Cell lines. The cell lines used to evaluate compounds consist of a human hepatocyte derived cell line (Huh-7) that constitutively expresses a genotype lb HCV replicon containing a Renilla luciferase reporter gene. These cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS, 100 U/mL penicillin/streptomycin and 1.0 mg/mL G418.

HCV replicon luciferase assay. To evaluate compound efficacy, titrated compounds were transferred to sterile 384-well tissue culture treated plates, and the plates were seeded with HCV replicon cells (50 μΐ _^ at a density of 2.4 x 10 ³ cells/well) in DMEM containing 4 % FBS (final DMSO concentration at 0.5 %). After 3 days incubation at 37°C, cells were analyzed for Renilla Luciferase activity using the EnduRen substrate (Promega cat #E6485) according to the manufacturer's directions. Briefly, the EnduRen substrate was diluted in DMEM and then added to the plates to a final concentration of 7.5 μΜ. The plates were incubated for at least 1 h at 37°C then read on a Viewlux Imager (PerkinElmer) using a luminescence program. The 50% effective concentration (EC ₅ 0) was calculated using using the four-parameter logistic formula noted above. To assess cytotoxicity of compounds, Cell Titer-Blue (Promega) was added to the EnduRen-containing plates and incubated for at least 4 hrs at 37°C. The fluorescence signal from each well was read using a Viewlux Imager. All CC ₅ 0 values were calculated using the four-parameter logistic formula.

Enzyme and replicon data for compound I is reported in Table 2.

Table 2.

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.