NZ754996B2 - Nucleotide hemi-sulfate salt for the treatment of hepatitis c virus - Google Patents

Abstract

hemi-sulfate salt of the structure as depicted in the drawing below, to treat a host infected with hepatitis C, as well as pharmaceutical compositions and dosage forms, including solid dosage forms, thereof.

Description

WO 44640 NUCLEU’E‘lDE RENEE—SULFATE SALVE‘ F08: THE 'l‘ll/EENT {3F HEPA'HTES C VERUS CRGSSEREFERENCE TO RELA’lTEB Al’l’LlCATlQNS This application claims the benefit of provisional US, Application Nos. 62145143? tiled February 1, 2917; 62/469,912 filed March 10, 2917; 62/488,366 filed April 2i, 20M; and, 62/575,248 filed Qctober 20, ZOl’I. The entirety of these applications are incorporated by reference.

FEELI} 0F TEE INVENTION The present invention is the herni—sulfate salt of a selected nucleotide compound that has unexpected eutic properties to treat a host infected with hepatitis C, as well as pharmaceutical compositions and dosage forms thereof.

BACKGRGE Ni) 0F Til E ENVENTEGN l-iepatitis C (HCV) is an RNA single-stranded Virus and member ofthe Hepacivirns genus. it is estimated that 75% of all cases of liver disease are caused by l-iCV, HCV infection can lead to cirrhosis and, liver cancer, and iflett to progress, liver e that may require a liver transplant.

Approximately 7 1 million people worldwide are living with chronic l-ECV infections and approximately 399,000 people die each year from HCV, mostly from cirrhosis and hepatocellular carcinoma.

RNA polymerase is a key target for drug development againstRNA single stranded Viruses.

The HCV non—structural protein NSSB RNA—dependent RNA polymerase is a. key enzyme responsible for initiating and catalyzing Viral RNA synthesis. There are two maj or subclasses of NSSB inhibitors: nucleoside analogs and non—nucleoside tors (l‘fl‘lls). Nucleoside s are anaholized to acti ye tri phosphates that act as alternative substrates for the polymerase and non— nncleoside inhibitors (Nle) hind to allosteric regions on the protein. Nucleoside or nucleotide inhibitors mimic natural polymerase substrates and act as chain terminators. They t the initiation of RNA ription and elongation of a nascent RNA chain.

In addition to targeting RNA polymerase, other RNA Viral ns may also be targeted in combination therapies. For example, HCV proteins that are additional targets for therapeutic approaches are 'NS3/4A (a, serine protease) and NSSA (a non—stmeturai protein that is an essentiai component ofHCV replicase and exerts a range of effects on cellular pathways).

U1 in December 2013, the first ntieleoside NSSB poiynierase inhibitor uvir (SO\/’aldi®, Gilead Sciences) was approved. Sevaidi® is a uridine phosphoraniidate prodrug that is taken up by eytes and widergoes intraeeiiuiar tion to afford the active metabolite, Z’—deoxy-2’~ riwfiuoro—ii—C—rnethyluridine- 5 ’ —triphosphate. :0NH 000 ix Homi§~o~i§~o~i§~o N e on on on XL .: ‘L- 2’ mDeoxy—Z’met—fluoro—Bmcmmethyiuridine—S’—triphosphate Sovaidi® is the first drug that has demonstrated safety and, efficacy to treat certain types of i—iCV infection without the need for co-adrninistration of interferon Sovaidi® is the third drug with breakthrough therapy designation to receive FDA approvai. in 2014, the US) FDA approved i—iarvoni® (iedispasvin a. NSSA inhibitor? and sofosbnvir) to treat chronic hepatitis C virus Genotype 1 ion; Harvoni® is the first combination piii approved to treat chronic HCV Genotype 1 infection. it is aiso the first approved n that does not require administration with interferon or rihavirin. in on, the FDA approved simeprevir (GlysioTM) in combination with sofoshuvir (Sovaldig/‘l as a onceudaily, all oral, interferon and ribavirin-ttee treatment for adults with Genotype l HCV ion The US. FDA also approved Athie’s A PakTM in 2014, a inulti—pill pack containing dasa‘ouvir (a nonmnucleoside NSSB polymerase inhibitor), omhitasvir (a NSSA U1 inhibitor), previr {a NS3/4A inhibitor), and ritonavir. The VilllfiKlRA. PaklM can he used with or without the ribayirin to treat Genotype l HCV infected patients including patients with compensated sis. VIEKIRA PakTM does not require interferon, co—therapy, in Jul},7 2015, the US. FDA approved 'I‘eclmivieTM and DalrlinzaTM for the treatment of HCV genotype 4 and HCV Genotype 3 , respectively, l,"<—:elnni\,/ie..TM (Omhitasvir/paritaprevir/ritona‘v’ir) was approved for use in combination with rihavirin for the treatment of HCV genotype 4l- in patients Without scarring and cirrhosis and is the first option for HCV~4 infected patients who do not require co~adrninistrati on with interferon. Dairlnizam was approved for use with Sovaldi® to treat HCV genotype 3 infections. DaklinzaTM is the first drug that has demonstrated safety and efficacy in treating 'HCV (El-enotype 3 withoizit the need for co- stration of interferon or rihavirin. in October 20l 5, the US. FDA warned that l-ICV treatments Viekira Fair, and Technivie can cause serious liver injury primarily in patients with underlying advanced liver disease and required that additional information about safety he added to the label.

Other current approved ies for HCV include interferon alpha—2h or pegylated interferon 2b trongl), which can be administered with ribavirin (Rebetolqi), NS3/4A telaprevir (Inch/ekg’, Vertex and Johnson 8; Johnson), hoceprevir (Victrelism, Merck}, simeprevir (OlysioTM, Johnson & Johnson), paritaprevir (Abeie), Ombitasvir (Abeie), the NNI Dasabuvir {A8133} and Merck’ s ZepatierTM {a single—tablet combination of the two drugs grazoprevir and el'basvir).

Additional NSSB polymerase inhibitors are currently under development Merck is developing the uridine tide prodrug MK—3682 rly idenix IDX21437‘) and the drug is tly in Phase II combination trials.

United States patents and W0 applications that descri he nucleoside polymerase tors for the treatment of Flaviviridae, including HOV, e those filed by Idenix Pharmaceuticals (6,812,219; 6,914,054; 7,705,493, 7,138,376; 7,148,206; 7,157,441, 7,163,929; 766, 7,192,936; 7,365,057, 7,384,924, 7 456,i5 LI] , 7,547,704, 7 582,618; 7 608,597; 7 7 f' It i? 608,600; 7,625,875, 7,635,689, 7,662,798, 7,824,851, 7,902,202, 7,932,240, 7,951,789, 8,193,372, 8,299,038; 8,343,937; 8,362,068, 8,507,460, 8,637,475; 8,674,085; 8,680,071, 8,691,788, 8,742,101, 8,951,985, 9,109,001; 9,243,025, 1182016/0002281; 1182013.:"0064794, 1117’0/2015/095305; WO/2015/081133, WO/2015/061683; WO/ZQ13/177219; WO/’2013/039920; U1 WOx’2014I/I37930; 4/052638; WO/2012/154321); Merck (6,777,395; 7,105,499; 7,125,855, 7,202,224, 7,323,449, 7,339,054, 7,534,767, 7,632,821, 815, 8,071,568, 8,148,349; 8,470,834; 8,481,712; 8,541,434; 8,697,694; 8,715,638, 9,061,041.; 9,156,872 and, WO/2013/009737), Emory University (6,348,587; 6,911,424; 7,307,065, 7,495,006; 7,662,938, 7,772,208; 8,114,994; 8,168,583; 8,609,627; US 2014/0212382; and W02014/1244430); Gilead Sciences/ Pharmasset 1116. (7,842,672, 013; 8,008,264; 8,012,941; 8,012,942; 8,318,682, 8,324,179, 8,415,308, 451, 8,563,530, 8,841,275, 8,853,171, 785, 8,877,733, 159; 8,906,880; 321; 8,957,045; 8,957,046, 9,045,520; 9,085,573; 9,090,642, and 9,139,604) and (6,908,924; 522; 770; 7,211,570; 7,429,572; 7,601,820, 7,638,502, 7,718,790; 7,772,208; 1113242315; 7,919,247; 7,964,580; 8,093,380, 8,114,997; 8,173,621; 8,334,270, 8,415,’22,Lay 8,481,713; 8,492,539; 8,551,973, 8,580,765, 076, 8,629,263, 8,633,309; 8,642,756, 8,716,262; 8,716,263, 8,735,345; 8,735,372; 8,735,569; 8,759,510 and 8,765,710); Hoffman he (6,660,721), Roche (6,784,166, 7,608,599, 7,608,601 and 8,071,567); A1108 B10Ph31111a 1118, (8,895,723; 8,877,731; 8,871,737, 8,846,896, 8,772,474, 8,980,865, 9,012,427; US 105341; US 011497, US 2010/0249068; US2012/0070411; W0 20157054465, WC) 20147209979; WO 00505, Emma Pharmaceuticals (US 119, 8,846,638; 9,085,599; WO 2013/044030; WO 2012/125900), 13101.21, (7,268,119; 7,285,658; 7,713,941; 8,119,607; 8,415,309; 8,501,699 and 8,802,840), Biocryst PharmaceuticaEs (7,388,002; 7,429,571; 7,514,410; 434, 7,994,139, 8,133,870; 8,163,703; 8,242,085 211161 8,440,813), A1121 C1‘121’1‘1, LLC (8,889,701 and WO 201 57053662), 111111111168: (8,759,318 and 9170/2012/092484), 181188611 Precincts (8,399,429; 8,431,588, 8,481,510, 8,552,021, 8,933,052, 9,006,29 and 428) the University 61‘ Georgia Faundatim (6,348,587; 065, 7,662,938; 8,168,583; 8,673,926, 8,816,074; 8,921,384 and 8,946,244), RPS P11811118, LLC (8,895,531; 8,859,595; 8,815,829; 627; 7,560,550; US 2014/0066395; US 2014/0235566; US 201070279969, 121"13/2010/091386 and W9 2012/15881 1) University Cflflfige Cardiff Consultants Limited (WO/2014/076490, WO 2010/081082; "70/2008/062206), ion Pharmaceuticals, inc. (WO/ZOl-‘i/l69278 and Cocr‘ystal Phanna, lnc. (US 9,173,893): Katholieke Universiteit Leuven (WO 20] 5/1589l3), Catabasis (WO ZOl 3/090420) and the Regents ofthe University of Minnesota {W0 "2006/00463 7).

Area Pharmaceuticals, inc. has sed B~D—2’—deoxy—2‘nd—tliioro—2'—B—C-suhstituted—Z— U1 inoditied—Nfi—(mono— and thyl) purine nucleotides for the treatment of l-lCV in US Patent No. 9,828,410 and PCT Application No. WU 20l6/l4-49l8. Atea has also disclosed ndeoxym 2‘~suhstitnted—il'—suhstituted~2—N6~suhstituted~6~arninopurine nucleotides for the treatment of paramyxovirus and orthomyxovirus infections in US EMS/0009836 and WE) 2018/009623.

There remains a strong medical need to develop anti v'lrlCV therapies that are safe effective and olerated. The need is uated by the expectation of drug ance. More potent direct—acting antivirals could significantly shorten treatment duration and improve compliance and SVR (sustained viral response) rates for patients infected with all HOV genotypes. it is therefore an object of the present invention to provide compounds, pharmaceutical compositions, methods, and dosage forms to treat and/or prevent infections of HEW.

SUMMARY GK? THE :ENVENTIGN it has been surprisingly discovered that the hemisulfate salt of nd 1, which is provided below as Compound 2, exhibits unexpected advantageous therapeutic properties? including enhanced hioavailability and target organ selectivity, over its free base (Compound 1).

These unexpected advantages could not have been predicted in e. Compound 2 is thus a therapeutically superior composition of matter to administer in an eti‘ective amount to a host in need thereof, typically a human, for the treatment of hepatitis C. Compound 2 is referred to as the helm—sulfate salt of isopropyl((S)-(((2R,3R,4R,SR)~S~(2—amino—6—(nrethylarnino}9H—purin—9~yl)~ ro—3whydroxyuzi-methyltetrahydrofnran—2—yl)rnethoxyixiphenoxy)phosphoryl)—L~alaninate. nd i. is disclosed in US. PatentNo. 9328,4l0.

HN,CH3 N \ CH3 0 (3 l i Orn’ii‘ofi; N H36 ""2 H o .

CH3 0 ., e: Ht)" ’F Compound 1 HN/Ci'iffl N \N H3C: (Kn/\fi ,3?EC CH3 ------ CH3 0 a 0.5 H2304 HO 9F Compoundz nd 2, as Compound 1, is converted to its corresponding triphosphate nucleotide (Compound L6) in the cell, wlrtich is the active metabolite and inhibitor ot‘RNA polymerase (see Scheme 1 below). Since Compound 1—6 is produced in the cell and does not leave the cell, it is not measurable in the plasma, ever, the 510161" metabolite Compound 1—7 (see Scheme l) is exported from the celi, and therefore is measurable in plasma and acts as a surrogate of the concentration oi‘intracellular active lite Compound 143.

It has been discovered that the plasma concentration in two of surrogate Compound 1-7, and thus intracellular Compound 14%, is substantially higher when Compound 2 is administered, in viva than when Compound I is administered in viva. in a head—to-head comparison of dogs dosed with Compound 1 and Compound 2 (Example 19, Table 28), dosing with Compound 2 achieved an AUCgemg ofthe ultimate guanine 5’~OH side metabolite (l 37) that is twice as high as the AUC following nd 1 dosing. .lt is cted that a non—covaient salt has such an effect on plasma concentration of the parent drug (Cornpound l) Additionally, Compound 2 selectively partitions in vivo to the liver over the heart (Example l9, Tahle 29), which is beneficial since the liver is the diseased organ in hosts infected with HCV.

Dogs were dosed with Compound 1 or Compound 2 and the concentration of the active triphosphate (land) in the liver and heart was measured. The liver to heart ratio of the active U1 triphosphate concentration was higher after dosing with Compound 2 compared to Compound 1 as shown in Table 29. Specifically, the liver/heart ioning ratio for Compound 2 is 20 compared to a liver/heart partitioning ratio of 3.1 for Compound .1. This data indicates, ctedly, that the stration of nd 2 results in the preferential distribution of the active guanine triphosphate (Compound lwd) in the liver over the heart when compared to Compound l, which reduces potential off—target effects. It was unexpected that administration of Compound 2 would significantly reduce undesired offmtarget partitioning. This allows for the stration of Compound 2 at a higher dose than Compound 1, if desired by the healthcare practitioner. in addition, liver and heart tissue levels of the active guanine triphosphate tive of nd 2 (metabolite 1-6) were measured after oral doses of Compound 2 in rats and monkeys (Example 20), High levels of the active e triphosphate {he} were measured in the liver of all species tested. lmportantly, unquantitiahle levels of the e triphosphate (fl-=6) were measured in monkey hearts, and this is tive of specific formation of the active triphosphate. it was thus discovered that compared to Compound 1 dosing, Compound 2 dosing improves guanine triphosphate (1—6) distribution.

When administered to healthy and hepatitis C infected patients, Compound 2 was well tolerated after a single oral dose and Cit—tax, Tmax and AUCtoi pharmacokinetic parameters were comparable in both groups (Tables 34 and 35). As described in liixample 2.4, a single dose of Compound 2 in HCV—infeeted patients resulted in a significant antiviral activity. Plasma exposure of metabolite la"? was mostly dose—proportional over the studied range.

Individual pharniacolrinetie/pharmacodynamic analyses of patients dosed with Compound 2 showed that the viral response correlated with plasma re of metabolite L" of Compound 2 (Example 24, lillGS. 23A—23l"), indicating that profound vial responses are achievable with robust doses of Compound 2.

Zliixample 24 confirms that, as non—limiting embodiments, single oral doses of 300 mg, 400 mg, and 600 mg result in significant antiviral activity in humans. The C24 trough plasma concentration of metabolite in? following a 600 mg dose of Compound 2 doubled from the C24 trough plasma concentration of metabolite 1-7 following a 3th mg dose of Compound 2.

Flt}. 24 and lilxarnple 25 highlight the strilting ion provided lay Compound 2 for the treatment of hepatitis C. As shown in , the steady—state trough plasma levels (C2455) of U1 lite 1—7 following Compound 2 dosing in humans (600 mg Ql) {550 mg free base equivalent) and 450 mg QB (4-00 mg free base equivalent)) was predicted and compared to the lEC95 of Compound l. in vitro across a range of HCV al isolates to determine if the steady state plasma concentration is consistently higher than the £095, which would result in high efficacy against le clinical isolates in viva. The EC95 for Compound 1 is the same as the EC95 of Compound 2. For Compound 2 to be effective, the —state trough plasma level of lite in? should exceed the E025 As shown in , the liCss of Compound 2 against all tesed clinical es ranged from approximately l8 nM to 24 nM.

As shown in li‘lG. 24, Compound 2 at a dose of450 mg Q1) (400 mg free base equivalent) in humans provides a predicted steady state trough plasma concentration (€24,537; of approximately 40 ... Compound 2 at a dose ofoOO mg QD (550 mg free 1ease equivalent} in humans provides a predicted steady state trough plasma concentration ((32455) of approximately 50 ng/mL.

Therefore, the predicted steady state plasma concentration of surrogate metabolite 137 is almost douhle the 3505 t all tested clinical isolates (even the hard to treat G'l'3a), which indicates superior performance. in contrast, the M395 of the rd of care nucleotide sofoshuvir (Sovaldi) ranges from 50 nM to 265 nM across all tested HCV clinical isolates, with an EC95 less than the predicted steady state concentration at the commercial dosage of 400 mg for only two isolates, GIT/la and (Elf/1h.

The 3395 for the cial dosage of 400 mg of sofoshuvir is greater than the predicted steady state concentration for other clinical isolates, fill a, 0le, GT3a, GT4a, and Gléld.

The data comparing the efficacy and pharmacokinetic steady state parameters in 'li‘lG. 24 clearly demonstrates the unexpected therapeutic importance of Compound 2 for the treatment of hepatitis C. in fact, the predicted steady—state 3) plasma level after administration of Compound, 2 is predicted to he at least 2—fold higher than the ECss for all genotypes tested, and is 3 to 5—fold more potent against GT2. This data indicates that Compound 2 has potent pan— genotypic antiviral activity in humans. As shown in , the ECss of sofoshuvir against GTl, G’l‘3, and GT4 is greater than it‘ll) ng/mL. Thus surprisingly, nd 2 is active t HCV at a dosage form that delivers a lower steady-state trough concentration (40-50 ng/ml...) than the steady—state tough concentration (approximately 100 ng/mL) achieved by the equivalent dosage form of sofosbuvir.

U1 in one embodiment, therefore, the invention includes a dosage form of Compound 2 that provides a metabolite in"? steady—state plasma trough concentration (€2,455) between approximately —75 rig/mil: for example, 20—60 ng/rniL, 25—50 ng/rnL 40'60 rig/rials, or even 4060 lla This is unexpected in light of the fact that the steady state concentration of the equivalent metabolite of sot‘osbuvir is approximately 100 rig/ml." Additionally, it has been discovered that Compound 2 is an unusually stable, highly soluble, non-hygroscopic salt with activity against HCV. This is surprising because a number of salts of Compound 1 other than the hemi~sulfate salt (Compound 2), including the monossulfate salt und 3), are not physically stable, but instead deliquesce or become gummy solids {Example 4), and thus are not suitable for stable solid pharmaceutical dosage forms. singly? While Compound '2 does not become gummy, it is up to 43 times more soluble in water ed to Compound Ti, and is over 6 times more e than Compound 1 under simulated gastric fluid (SGF) ions (Example 15).

As discussed in Example l6, Compound 2 remains a white solid with an IR that corresponds to the reference standard for 6 months under accelerated stability ions (40 00/759?) RH). Compound 2 is stable for 9 months at ambient conditions (25 °C/60% RH) and refrigerator conditions (5 0C).

Solid dosage forms (50 mg and 10% mg tablets) of Compound 2 are also chemically stable under accelerated (40 0(775% RH) and refrigeration conditions (5 0C) for ‘6 months (:lEixample 26) nd 2 is stable under ambient conditions (:25. 006096 RH) in a solid dosage form for at least 9 months.

Scheme 1 provides the metabolic pathway of Compound 1 and Compound 2, which involves the initial de-esteril‘ication of the phosphorantiidate (metabolite hi.) to form metabolite ft.— 2~ Metabolite l~2 is then converted to the 'NG-methyl—2,6—diaminopurine—S"—monophosphate derivative (metabolite 1—3); which is in tum lized to the free 5’—hydroxyl~N6—methylu2,6~ diaminopurine nucleoside (metabolite 1—8) and (:(IZR,3R,4R,5R)-5—(2—amino—o—oxo—l,o~dihydro— 9H—purin—9-yl)—lluoro—3—hydroxy—éi—methyltetrahydrot’uran—Z—ylhnethyl dihydrogen phosphate as the 5’—menephospha€e (metabolite 1—4). Metabeiite 1-4 is anabeiized to the correspending diphosphate (metabolite i—S) and then the active triphosphate derivative (metabolite 1-6). The 5’— tfiflmghfleemrbeihmmrnmmbmhaEHBgmmmm‘lemm0@{QR3RARfiR}$flmmm¢ hydroxy—S{hydroxymethyD—3 —methy1tetrahydrofuran—Z-yl} i ,9-dihy'er—{éH—purinné—one (1—7).

U1 Metabolite 15.7 i3 able in plasma and i3 ore a surrogate for the active triphesphate (i— 6), which is not measurable in plasma.

Scheme 1 \ HO F Cempaund 1 1-2 \NH \"NH O 3"! \ N N \ N HO"$"O < I A <1 ' A 0 N -"" HOW :0: :N . N NH? N NHn OH ‘ H5 "’5: H5 ’1:- 1—3 i=8 H H E! (ENH {INH HO"$"O"?"O"HID"O O N W N MHZ HO O N N MHZ OH OH OH In one embodiment, the invention is Compound 2 and its use to treat hepatitis C (HCV) in a host in need thereof, optionally in a pharmaceutically acceptable carrier. In one aspect, Compound 2 is used as an amorphous solid. in another aspect, Compound 2; is used as a crystalline solid.

U1 The present invention further includes an exemplary oil-limiting process for the preparation of nd 2 that includes (i) a first step of dissolving Compound it in. an organic solvent, for example, acetone, ethyl acetate, methanol, acetonitrile, or ether, or the like, in a flash or container, (ii) charging a second flash or ner with a second organic solvent, which may he the same as or different from the organic t in step (i), optionally cooling the second t to O—ll'l degrees C, and adding dropwise H2804 to the second organic solvent to create a l?le()4/organic solvent e; and wherein the solvent for example may he methanol; (iii) adding dropwise the il—leO/imolvent mixture at a molar ratio of OS/l .0 from step (ii) to the solution of Compound 1 of step (i) at ambient or slightly increased or decreased ature (for example 23—35 degrees C); (iv) stirring the reaction of step (iii) until precipitate of Compound 2 is formed, for example at ambient or slightly increased or decreased temperature; (v) optionally filtering the resulting precipitate from step (iv) and washing with an organic solvent; and (vi) optionally drying the resulting Compound 2 in a vacuum, optionally at elevated a temperature, for example, 55, 56, 5‘7, 58, 59, or 60 °C. in one embodiment, the organic solvent in step (i) is yl~2—pentanone. in one embodiment, the c t in step ti) is ethyl isopropyl ketone, In one embodiment, the organic solvent in step (i) is methyl nate. In one embodiment, the organic solvent in step (i) is ethyl hutyrate.

Despite the volume of antiviral nucleoside literature and patent filings, Compound 2 has not been specifically disclosed. Accordingly, the present invention es Compound 2, or a pharmaceutically acceptable composition or dosage form thereof, as bed herein.

Compounds, methods, dosage forms, and compositions are provided for the ent of a host infected with a HCV virus via administration of an effective amount of Compound 2. in certain embodiments, Compound 2 is administered at a dose of at least about 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or men mg, In certain embodiments, Compound 2 is administered for up to l2 weeks, for up to 10 weeks, for up to 8 weeks, for up to 6 weeks, or for up to 4- weeks. In alternative embodiments, Compound 2 is U1 administered for at least 4 weeks, for at least 6 weeks, for at least 8 weeks, for at least to weeks, or for at least l2 weeks. in certain embodiments, Compound 2 is administered at least once a day or every other day. In. n. ments, Compound 2 is administered in a dosage form that achieves a steady—state trough plasma level (C2453) of metabolite 1—7 n approximately 15" 75 ng/rnL. in one ment, Compound 2 is administered in a dosage form that achieves a steady—state trough plasma level (C2455) of metabolite 1-7 between approximately 20—60 ng/mL. In certain embodiments, Compound '2 is administered in a dosage form that achieves an AUC of metabolite 1~7 between approximately l,200 ng’l‘h/mL and 3,000 ngt‘h/‘mls, In one embodiment, Compound 2 is administered in a dosage form that achieves an AUC of metabolite 1—7 between approximately LSOO and 2, l 00 ng’i‘h/inL.

The compounds, compositions, and dosage forms can also be used to treat related ions such as anti~HCV antibody positive and antigen positive conditions, viral—based chronic liver inflammation, liver cancer ing from advanced hepatitis C (hepatocellular carcinoma (ECG), cirrhosis, chronic or acute hepatitis C, fulrninant hepatitis C, chronic persistent tis C and anti—HCV-based fatigue. The compound or formulations that include the compounds can also be used prophylacticalljvr to prevent or ct the progression of clinical illness in individuals who are anti—HCV antibody— or antigen-positive or who have been exposed to hepatitis C.

The present invention thus includes the following features: (a) Compound 2 as hed herein, (b) gs of Compound 2 (c) Use of Compound 2 in the m anufacture of a ment for treatment ot‘a hepatitis C virus infection; (d) Compound. 2 for use to treat hepatitis C, optionally in a pharmaceutically acceptable carrier, (e) A method for manufacturing a medicament intended for the therapeutic use for treating a hepatitis C virus infection, terized in that Compound 2, or a pharmaceutically acceptable salt, as described herein is used in the manufacture; WO 44640 (e) A pharmaceutical formulation comprising an effective host—treating amount of Compound 2 with a. phannaeeutically acceptable. carrier or diluent; (l’) Processes for the preparation of therapeutic products that n an effective amount of Compound 2; U1 (g) Solid dosage forms, including those that provide an advantageous pharmaeoklnetic profile; and (h) ses for the manufacture of Compound 2, as described herein BRIEF DESCRI?T10N OF THE FEGURES FlG. 1A is an overlay of XRPD diffractograms of samples l—l (amorphous Compound l), M alline Compound 1), and l—3 (amorphous Compound 2) prior to ity studies for characterization purposes as bed in Example 2 and Example 5. The x-axis is 2Theta measured in degrees and the y—axis is intensity measured in counts.

FIG. lB is the l—ll’LC chromatograph of amorphous Compound l (sample 1—1) to determine purity as described in Example 2. The purity of the sample was 98.70/25, 'l‘he X-axis is time measured in minutes and the yards is ity measured in counts is the HPLC elu'omatograph of crystalline Compound 1 (sample l—Z) to determine purity as described in Example 2. The purity of the sample was 99.1l%. The x-axis is time measured in minutes and the y—axis is intensity measured in counts.

FlG. 2B is a DSC and TGA graph of crystalline Compound 1 e l—2) prior to any stability studies for characterization purposes as described in Example 2. The x—axi s is temperature measured in °C, the left y—axis heat flow measured in (W/g), and the right y—axis is weight measured in percent.

FIG 3 is an X—ray llography image of Compound 1 showing the absolute stereochemistry as described in Example 2, is an overlay of XRPD diffractograms of samples l—l (amorphous Compound 1), l. -2 (crystalline Compound 1), and 1—3 (amorphous Compound 2) after storing at 25 °C and 60% ve humidity for l 4 days as described in e 2. The x—axis is ZTheta measured in s and the y—axis is intensity measured in counts. lilG. 48 is an overlay of XRPD diffractegrams of samples l—4, 1—5, l—6, l—7, and l—9 after storing at 25 0C and 60% relative humidity for 7 days as described in Example 4. The x—axis is U1 Zl‘heta measured in degrees and the y—axis is intensity ed in counts. is arr overlay 0f XRPD d,itlraetograihs of samples l—4, l—d, l—7, and l—9 after g at 25 °C and 609/5 relative hurtsidity for 14 days as described in Example 4. The X~axis is ta measured in degrees and the y—axis is intensity ed in eeurits.

FIG. SE is the XRPD pattern, of amorphous Compound 2 (sample l—3) as described in Example 5, The x—axis is ZTheta measured is degrees and the y—axis is intensity measured in counts. is the lilPLC chrematograph of amorphous Compound 2 (sample l—3) to determine purity as described in Example 5. The purity of the sample was 99.6%. The x—axis is time measured in s and the y—axis is intensity ed in s.

PK}. 68 is a DSC and TGA graph for amorphous Compound 2 (sample l—3) prior to any stability studies for eharaeteriaation purposes as described in, Example 5. The x—axis is temperature measured in 0C, the left y-axis heat flow measured in (‘v 7g), and the right y—axis is weight measured in percent. is an everlay of XRPD diffractograms of lline samples (samples 2—2, 2—6, and 2—7) and peorly crystalline samples (samples 2—3, 2—4, 2—5, and 2—8) identified from the erystallizations of Compound 2 (Example 6). The x—axis is Z'I‘heta measured in degrees and the y—axis intensity measured in counts.

PEG. "ill is an overlay ot‘XRPD etograiris 0f amorphous samples (samples 2—9, 2—l O, and 2—l 1) identified frem the erystallizatiens of Compound 2 (Example 6:). The x—axis is Z'l‘heta measured in degrees and the y—axis iriterisity rrieasured in eeunts. is an overlay of XRPD diffraetograms of samples (samples 2—2, 2—3, 2—4, 2—5, 2— 6, 2—7 and 2—8) after 6 days storage at 25 °C and 609-4; relative humidity (Example 6). The x—axis is Z'I‘heta measured in degrees and the y—axis intensity measured in counts. is a DSC and TGA graph for sample 2—12 (Example 6). The x—axis is temperature measured in "C, the left y—axis heat flow measured in (V "/g), and the right y—axis is weight measured in percent. Experimental procedures for DSC and 'l'GA collection are given in Example 2. is a DSC and 'l‘ti‘rA graph for sample 2—3 (liixample 6:). The X—axis is temperature measured in 0C, the left y—axis heat flow measured in (WI/g), and the right y—axis is weight U1 measured in percent. .lEixperime-ntal procedures for DSC and TGA collection are given in FIG. QB is a DSC and TGA graph. for sample 2~4 (Example 6). The xmaxis is temperature measured in "C, the left y—axis heat flow measured in (W/g), and the right y—axis is weight measured in percent. Experimental procedures for DSC and TGA collection are given in Example 2 A is a DSC and TGA graph for sample 2—5 (Example 6). The x—axis is temperature measured in "C, the left y~axis heat llow measured in (\ 715g), and the right. y~axis is weight measured in percent. Experimental procedures for DSC and TGA collection are given in Example 2..

FlG. 103 is a DSC and TGA graph for sample 2-6 le 6). The x—axis is ature measured in 0C, the left y—axis heat flow measured in (W/g), and the right. y—axis is weight measured in t. Experimental procedures for DSC and TGA collection are given in Example 2.

A is a DSC and TGA graph for sample 2—7 (Example 6). The x—axis is temperature measured in °C, the left y—axis heat flow measured in (W/g), and the right ynaxis is weight measured in percent. Experimental procedures for DSC and A collection are given in Example 2.

PEG. l 18 is a DSC and TGA graph for sample 2—8 (lilxample 6). The x—axis is temperature measured in "C, the left ywaxis heat flow measured in (W/g), and the right y—axis is weight measured in percent. Experimental procedures for DSC and TGA collection are given in Example FIG. lZA is the XRPD pattern of amorphous Compound 4 (sample 3—12) as discussed in Example 7. The x-axis is Z’l‘lreta measured in degrees and the yeaxis is intensity measured in counts. No crystallization of a malonate salt was observed regardless of the solvent used. lilG. lZB is an overlay of XRPD ractograms of amorphous s es 3—6, 3— l0, 3-11, and 3-12) identified from the attempted crystallization of compound 1 with malonate salt (Example 7). The xuaxis is Z'l‘heta measured in degrees and the yuaxis is intensity measured in counts.

Flt}. l3A is the l-[PLC chromatogram of sample 3—l2 from the ted crystallizations ofcompound l with malonate salt as described in Example 7. The sample was 99.2% pure. The xn U1 axis is time measured in minutes and the y—aXis is intensity measured in mAu.

FlG. l3B is an y of XRPD diffractograms of solid samples obtained from the crystallization using LAG (samples 443, 4-12, 44.3, 4-3? and 4~l) compared to nd 1 (sample 1—2) as described in Example 8. All the XRDP match the patterns of the crystalline acid counter ion with no additional peaks, The x-axis is ZTheta measured in degrees and the y—axis is intensity measured in counts.

FlG. MA is an overlay ol‘ml) diffractograms of samples obtained from utilizing ethyl acetate as a cwstallization t (samples 613" 642, 6-11, 64.0, 6-8, 6—7, 646, 6—5, 64, and 6- 2) compared to crystalline Compound 1 (sample id?) as bed, in Example 10. The MD patterns were generally found to match the Compound 1 pattern with the exception of samples 6- 2, 6-4, and 6—5 that exhibit slight differences. The x—axis is 2'l'heta measured in degrees and the y- axis is intensity measured in counts) Fig. MB is an overlay oleWD diffraetogram of sample S-l following a second dissolution in MEK and the addition of the antisolvent cyclohexane and pamioc acid as described in Example 9. Sample 5"}, crystallized in pamioc acid, was a solid following maturation, but the XRPD pattern matched the pattern of paniioc acid.

Flt}. lSA is an overlay ot‘XRPD ractograms of s obtained from utilizing ethyl acetate as a crystallization solvent (samples 6—5, 6—4, and 6—2) compared to crystalline Compound 1 (sample l-Z) as described in Example 10. The XRPD ns were generally found to match the nd 1 pattern with the exception of samples 6—2, 644, and 6—5 that exhibit slight differences. 'l‘he X—axis is Z'l‘heta measured in degrees and the yuaxis is intensity measured in counts and laheled with the acid used in crystallization. lilG. 1le is the XRPD pattern for Compound 2 as described in Example l4. The x—axis is ZTheta measured in degrees and the yards is ity measured in counts.

U1 EEG. l6A is a graph of the active ’1‘}? olite 1—6) trati on levels in the livers and hearts of rats, dogs, and monkeys (Example 18). The x—axis is the dosage measured in mg/kg for each species and the y—axis is the active TIP concentration measured in rig/g.

B is a graph of the active 1‘? (metabolite lad) concentration levels in the liver and heart of dogs (n====2) measured 4 hours alter a single oral dose of Compound 1 or Compound 2 (Example l 9). The x—axis is the dosage of each compound ed in mg/kg and the y—axis is the active TP tration measured in rig/g. is the plasma profile of Compound 1. and metabolite 1-7 in rats given a single 500 mg/kg oral dose of Compound 2 (Example 20) ed 72 hours post—dose. The x—axis is time measured in hours and the y—axis is plasma tration measured in ng/m'L.

FlG. 18 is the plasma profile of Compound 1 and metabolite l-x’7 in monkeys given single oral doses of '30 mg, l 00 mg? or 300 mg of Compound 2 (Example 20) measured 72 hours post~ dose. The roads is time measured in hours and the y-axis is plasma concentration measured in rig/ml...

FIG 19 is a graph of EC95 measured in nM of sofos‘ouvir and Compound 1 against HCV clinical es. EC95 values for Compound 1 are 7—33 times lower than sofosbuvir (Example '22".

The x-axis is labeled with the genotype and the y—axis is ECas measured in my}. is a graph of ECso measured in old of sofoshuvir and Compound 1 against laboratory strains ot‘l—lCV {Ilenotypes la, lb, 2a, 3a, 4a, and 5a. (filonrpouncl 'l is approximately 6* ll times more potent than sofosbuvir in Genotypes "1-5 (Example 22). The X—axis is labeled with the genotype and the y-axis is ECso measured in nM. is a graph of the mean plasma concentrationwtime e of Compound 1 following tl:1e administration ot‘a single dose of Compound 2 in all cohorts of Part B of the study as described in Example 24. Compound l was quickly ahsorhed and rapidly lized within approximately 8 hours in all cohorts from Part B. The xuaxis is the time measured in hours and the y—axis is the geometric mean plasma concentration measured in ng/mL.

Flt}. 22 is a graph of the mean plasma concentration—time profile of metabolite l~7 following the administration of a single dose of nd 2 in all cohorts of Part B of the study U1 as described in Example 24. Metabolite l.-’7 exhibited ned plasm a concentration in all cohorts from Part B. The x—axis is the time measured in hours and the yards is the geometric mean plasma concentration measured in rig/ml.,.

A is an individual cokinetic/pharmacodynamic analysis of a subject enrolled in the lb cohort as described in Example 24. The graph shows plasma metabolite 137 exposure and HC V RNA reduction levels. The dashed line represents the minimum concentration of metabolite 1J7 required to sustain a viral response greater than the E6395 value against Gle.

The seals is time measured in hours. The left y-axis is metabolite 1—7 plasma concentration measured in ngx’mL and the right y-axis is the HCV RNA reduction measured in logic lU/mL.

Fl'G. 23B is an individual pharmacokinetic/phannacodynamic analysis of a subject enrolled in the lb cohort as bed in Example 24. The graph shows plasma lite l—7 exposure and HCV RNA ion levels. The dashed line represents the minimum concentration of metabolite 1—7 required to sustain a viral response greater than the ECss value against (Till).

The x~axis is time measured in hours. The left y—axis is metabolite lw7 plasma. concentration measured in ng/mL and the right yuaxis is the HCV RNA reduction measured in logrn Iii/ml; C is an individual pharmacokinetic,"pharmacodynamic analysis of a subject enrolled in the lb cohort as described in e 24. The graph shows plasma metabolite l~7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite la»? required to sustain a viral response greater than the M195 value against GTlh.

The X—axis is time ed in hours. The left y—axis is metabolite 1-7 plasma concentration measured in ng/ml, and the right y—axis is the HCV RNA ion measured in logic lU/mL.

B is an individual pharmacokinetic/pharmacodynamic analysis of a subject enrolled in the 3b cohort as described. in e 24. Each graph shows plasma metabolite 1—7 exposure and lit:V RNA ion levels. The dashed line represents the minimum concentration of metabolite l~7 required to sustain a viral response r than the E6395 value against GTlh.

The X—axis is time ed in hours. The left y—aXis is metabolite 1—7.7 plasma concentration to easured in rig/ml. and the right y—axis is the HCV RNA reduction ed in logic ,. lilG. 23 ii is an individual pharmacolrinetic/pharmacodynamic analysis of a subi ect enrolled in the 3b cohort as described in Example 24. Each graph shows plasma metabolite 1-7 exposure U1 and l-lCV iltNA reduction levels. The dashed line represents the minimum concentration of rnetaholite 1—7 required to sustain a viral se greater than the E025 value against GTll). The x—axis is time measured in hours. The left y—axis is metabolite l~7 plasma concentration measured in ng/mL and the right y—axis is the HCV RNA ion measured in login ill/ml; FlG. 23F is an individual pharmacokinetic/pharmacodynamic analysis ofa subject enrolled in the 3h cohort as described in e 24. Each graph shows plasma metabolite 1—7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolite l~7 required to sustain a viral se greater than the ECss value against (Il-Tlh. The Xmaxis is time measured in hours. The left ymaxis is lite l~’7 plasma concentration measured in ng/mL and the right y-axis is the l—lCV RNA reduction measured in lOglO Iii/ml...

FlG. 24 is a graph of the £095 values of Compound 1 and sofosbuvir t al isolates of GTl? GT2, GT3; and GT4 l-lCV—int‘ected patients The dashed ntal line (mmmmm) represents the steady-state trough concentration (€24,ss) of sofoshuvir nucleoside following a dose of 400 mg QT) of sofoshuvir. The full horizontal line ( ) represents the steady~state trough concentration (C2455) of metabolite i=7 following 600 mg of Compound 2 (equivalent to 550 mg of Compound 1). The dotted horizontal line (—————————) represents the steady—state trough concentration ((124,53) of metabolite l—7 following 450 mg of Compound 2 (equivalent to 400 mg of Compound 1). As discussed in Example 25, the predicted steadynstate trough plasma level ) of metabolite la"? following 600 mg and 450 mg of Compound 2 exceeds the in viii/'0 lles of Compound 1 against all tested clinical isolates. The steady state trough plasma level (C2455) of sofoshuvir only exceeds the EC95 at GT2 clinical isolates. The x—axis is labeled with the clinical isolates and the table under the wards lists the ECas values for Compound l and sofosbuvir The y~aXis is the l3C95 against the al es measured in rig/mid. EC95 is expressed as nucleoside equivalent Sofosbuvir and Compound 2 were stered daily (QED).

FIG. '25 is a flow diagram showing the manufacturing process of 50 mg and 100 mg tablets of Compound 2 as described in Example 26. in step l, microerystalline cellulose, Compound 2, lactose monohydratej and, croscarmellose sodium are filtered through a 600 uM screen. In step 2, the contents from step l are loaded into a V—blender and mixed for 5 minutes at 25 rpm. in step 3, ium stearate is filtered through a 600 old screen. in step 4, magnesium stearate is loaded into the V-hlender containing the contents from step 2 (microcrystalline cellulose, Compound 2, lactose monohydrate, and eroscarmellose sodium) and mixed for ‘2 minutes at 25 rpm. The U1 common blend is then divided for the production of 50 mg tablets and lOO mg tablets. To produce 50 mg tablets, the blend from step 4 is compressed with 6 mm round standard concave tooling. To produce 100 mg s, the blend from step 4 is compressed with 8 mm round rd concave tooling. The tablets are then packaged into HDPE bottles induction—sealed with PP caps with desiccant, HS. 26 is the hemi—sulfate salt that ts advantageous pharmacological properties over its corresponding free base for the treatment of an HCV Virus.

DE'E‘AELED DESCREP'E‘KGN 0F THE l‘lflN The invention sed herein is a compound, , ition, and solid dosage form for the treatment of infections in or exposure to humans and other host animals of the HCV Virus that includes the administration of an reflective amount of the hood—sulfate salt of isopropyl({S}~ (((2R,3R,4R,SRl—S—(2~amino-6(methylamino)H9H-pnrin9—yl)—4-fluoro-S ~hVdroxywll- niethyltetrahydrol‘hran~2—yl)methoxyXphenoxflphosphoryl)«L—alaninate (Compound 2) as described herein, optionally in a pharmaceutically acceptable carrier. in one embodiment, Compound 2 is an amorphous solid. ln yet another ment, Compound 2 is a crystalline solid. 0{)CmH:%W 39 ()5 H2304 Compound 2 The compound, compositions, and dosage forms can also be used to treat conditions related to or occurring as a result of an HCV viral exposure. For example, the active compound can he used to treat HCV antibody positive— and HCV antigen—positive conditions, viral—based cln‘onic liver inflammation, liver cancer resulting from advanced hepatitis C (eg, hepatocellular oma), cirrhosis, acute hepatitis C, fuiminant hepatitis C, chronic persistent hepatitis C, and anti—i-iCV—based fatigue.

The active compounds and compositions can also be used to treat the range ot‘ HCV genotypes. At least six distinct genotypes of HCV, each of which have multiple subtypes, have U1 been identified globally. Genotypes 1—3 are ent woridwide, and Genotypes 4, 5, and 6 are more limited geographically. pe 4 is common in the Middle East and Africa. Genotype 5 is mostly found in South Attica. Genotype 6 predominately exists in ast Asia, Although the most common genotype in the United States is Genotype i, defining the genotype and subtype can assist in treatment type and duration, For example, different genotypes respond 'entiy to different medications and optimai treatment times vary depending on the genotype infection, Within genotypes, subtypes, such as Genotype la and Genotype lb, d differently to treatment as weii. infection with one type of genotype does not preclude a later infection with a different genotype.

As described in Example 22, Compound 2 is active against the range of HCV genotypes, ing Genotypes l—S. in one embodiment, Compound 2 is used to treat HC V Genotype l, HCV Genotype 2, HCV Genotype 3, HCV Genotype 4, HCV Genotype 5, or HCV Genotype 6. in one embodiment, Compound ‘2 is used to treat HCV Genotype la. in one embodiment, Compound 2 is used to treat HCV Genotype lb. in one embodiment, nd 2 is used to treat ithV Genotype 2a. in one embodiment, Compound 2 is used to treat HCV Genotype 2b. in one embodiment, Compound 2 is used to treat HCV Genotype 3a. In one embodiment, Compound 2 is used to treat HCV Genotype 4a. in one embodiment, Compound 2 is used to treat i—lCV Genotype in one embodiment, Compound 1 or Compound 2 is used to treat i—lCV Genotype 5a, in one embodiment, Compound 1 or nd 2 is used to treat HCV Genotype 6a. in one embodiment, Compound 1 or Compound 2 is used to treat HCV Genotype 6b, 6c, 6d, 6e, 61'", 6g, 6h, 6i, 6}, 6k, oi, om, on, do, 6p, oq, 6r, 63, fit, or 6u.

As discussed in e 25 and shown in , the ted ~state trough concentration (CZ/11,55) of metaboiite in»? ing a dose of 450 mg (400 mg free base) and a dose of 690 mg (550 mg free base) of Compound 2 is approximately 4-0 ng/mL to 59 ng/mL. This (324,55 levei exceeded the lEiCss of Compound 1 at i—lCV Genotypes 1a, lb, 2a, 2b, 3a, 4a, and 4d. This data confirms that Compound 2 has potent-pan genotypie activity. This is surprising beeause Compound 2 achieves a smaller steadyustate trough concentration (C2455) than the steady—state trough concentration (C2455) of the nucleoside metabolite of sot‘oshuvir following equivalent sofoshuvir dosing. The steady—state trough concentration ((32453) of the corresponding nucleoside lite of sofosbuvir is approximately lOG ng/mL, but this level only s the E0): of U1 sofoshuvir against G’l'Z clinical isolates (37le 24). Compound 2 is more potent than sofosbuvir against GTl, GT2, GT3, and GT4, and therefore allows a dosage form that delivers a smaller steady—state trough concentration of its metabolite which is nonetheless efficacious t all tested pes of HCV. In one embodiment, a dosage form of Compound 2 is delivered that achieves a metabolite lw’?’ steady—state trough concentration (C2455) between approximately l5—75 ng/mL. In one embodiment, a dosage form of Compound 2 is delivered that achieves a metabolite In? steady—state trough concentration (C2455) between approximately 20—60 ng/niL, 20-50 ng/niL, or 2040 rig/nth.

In one embodiment, the compound, formulations, or solid dosage forms that include the compound can also be used lactically to prevent or retard the progression of al illness in individuals who are HCV antibody— or IICV antigen—positive or who have been exposed to tis C.

In particular, it has been discovered that nd 2 is active against HCV and exhibits superior drug—like and cological properties compared to its free base (Compound I), Surprisingly, Compound '2 is more bioavailahle and achieves a higher AUC than Compound 1 (_Example 19) and Compound 2 is more selective for the target organ, the liver, than Compound 1 (Example 19).

Compound 2 is also advantageous over Compound 1 in terms of solubility and chemical stability, This is surprising because the mono—sulfate salt of isopropylflfl—(«ZK3R,4R,5R)—5—(2~ amino-o-(methylamino)—9Hupurin—9—yl)—4—fluoro~3 —hydroxv—él—methyltetrahydrofuran—Z— vi)rnethoxyXphenoxy)phosphoryl)~L—alaninate und 3) is unstable and exhibits the appearance of a sticky gum, while Compound 2, the hemi—sulfate salt, is a stable white solid. The ulfate salt, both as a solid and in a solid dosage form, is very stable over 9 months and is not hydroscopic. ’7 "I 145.: CH3 0 a \ HO" 3F H2S04 Compoundil Despite the volume of antiviral nucleoside literature and patent filings, Compound 2 has not been cally disclosed.

Compound 2 has S~stereochemistry at the phosphorus atom which has been confirmed with X—ray crystallography ( e 2). in alternative embodiments, nd 2 can he used in the form of any desired ratio of phosphorus R— and S-enantiomers, including up to pure enantiomers. in some embodiments, nd 2 is used in a form that is at least 90% free of the opposite enantiom er, and can he at least, 98%, 99%, or even 100% free of the opposite enantiomer Unless described otherwise, an enantiomerically enriched Compound 2 is at least 90% free of the opposite enantiomer In addition, in an alternative embodiment, the amino acid of the phosphoramidate can he in the Du or Lwconfiguration, or a mixture thereof, including a racemio mixture.

Unless otherwise specified, the compounds described herein are provided in the (id)— configurarion. In an alternative embodiment, the nds can be provided in a B—L— configuration. Likewise, any substituent group that exhibits chirality can he provided in racemic, omeric, diastereomeric form, or any mixture thereof. "Where a phosphoramidate exhibits ehirality, it can he ed as an R or S chiral phosphorus derivative or a mixture ti’iereof, including a racemic mixture. All of the combinations of these stereo configurations are alternative embodiments in the invention described herein, In another embodiment, at least one of the hydrogens of Compound 2 (the nucleotide or the herni—suli‘ate salt) can be replaced with deuterium.

These alternative configurations include, but are not limited to, fix) on CH3 0 HWCHQ W59 0 NAN/7km wg 2 CH 0 ~" ‘2 3 \. HQ‘ F 6 0.5 H2804 CH 0 <’ El 1 _ H 0 NAN/0 NH "W0 Maw/"i m3 2 H E) ------ CHM) c ., Q\r’ H? "F :9 0.5H2504 CH3? 0 Mac/k ‘ N NH: Hot, 0 ,R 2 " Y m/LN \0 H o .

CH3 0 Synthesis of Compound 2 The present invention further includes a nonnlirniting illustrative process for the preparation of Compound 2 that includes (i) a first step of ving Compound 1 in an organic solvent, for example, acetone, ethyl acetate, methanol, acetonitrile, or ether, or the like, in, a flash or container; (ii) charging a second flask or container with a second organic solvent, which may be the same as or different from the organic solvent in step (i), optionally cooling the second solvent to 0-10 s C, and adding dropwise Zl-leCm to the second organic 29 t to create a organic solvent mixture; and wherein the solvent for example may be methanol; (iii) adding dropwise the HzSOi/solvent mixture at a molar ratio of 0.5/ l .0 from step {ii} to the solution of (fiionipound l of step (i) at ambient or slightly increased or decreased ature (for e 23—35 degrees C); (iv) stirring the reaction of step (iii) until precipitate of Compound 2 is , for example at ambient or slightly increased or decreased temperature; (v) optionally filtering the ing precipitate from step (iv) and washing with an c solvent; and (vi) optionally drying the resulting Compound 2 in a vacuum, optionally at elevated a temperature, for example, 55, 56, 5‘7, 58, 59, or 60 0C.

U1 in certain embodiments, step (i) above is carried out in acetone, Ili'urther, the second organic solvent in step (ii) may be for example methanol and the mixture of organic solvents in step (v) is methanol/acetone. in one ment, Compound 1 is dissolved in ethyl acetate in step (i). in one embodiment, Compound l is dissolved in tetrahydrofuran in step (i). In one embodiment, Compound 1 is dissolved in itrile in step (i). in an additional embodiment, Compound 1 is dissolved in dimethylt‘ormamide in step (i).

Zlin one embodiment, the second c solvent in step (ii) is ethanol. In one embodiment, the second organic solvent in step (ii) is isopropanol, In one embodiment, the second organic solvent in step (ii) is mhutanol. in one embodiment, a mixture of ts are used for washing in step (v), for example, ethanol/acetone, In one embodiment, the mixture of solvent for washing in step (v) is isopropanolfacetone. in one embodiment, the mixture of t for washing in step (v) is n— butanol/acetone. in one embodiment, the mixture of t for washing in step (v) is l/ethyl acetate. in one embodiment, the mixture of solvent for washing in step (v) is isopropanol/ethyl acetate. in one ment, the mixture of solvent for washing in step (v) is n~butanol/ethyl acetate, in one embodiment, the mixture of t for washing in step (v) is ethanol/ tetrahydrofuran. In one embodiment, the e of solvent for washing in step (v) is isopropanol/ tetrahydrofuran. in one embodiment, the mixture of solvent for washing in step (v) is n—butanol/ tetrahydrofuran. in one embodiment, the mixture of solvent for washing in step (v) is ethanol/ acetonitrile. in one embodiment, the mixture of solvent for g in step (v) is isopropanol/ aeetoniti‘ile. In one embodiment, the mixture of solvent for washing in step (v) is nubutanol/ aeetonitrile. In one embodiment, the mixture of solvent for washing in step (v) is ethanol/ dimethylformamide. in one embodiment, the mixture of solvent for washing in step (v) is panol/ dimethylformamide. In one embodiment, the mixture of solvent for washing in step (v) is n—butanol/ dimethylformamide lit Metabolism of Esopropyl({S}(({2R,§R,4R,5R)~5~(2uamino~6—(methylaminolullfiégtiirinw 9—yl}—4-llu0ro—S—hydrosy—4—methyltetmhytlrofuran~2~yl)methoxy)(phennxymhosphoryl)—L— alanlnate und 2) The metabolism of Compound 1 and Compound 2 involves the production of a 5’" U1 mono-phosphate and the subsequent ism of the 'Nfi-methyl—2,6—cliaminopurine base (L3) to generate ((21%,3Rfl-R, 5R)—5 —{2mamino—6-oxon l ,6—dihydro—9H—purin—9~yl)m4—fluoro—3—hydroxy—4~ methyltetrahydrofuran—Z~yl)methyl dillydtogen phosphate (1»4) as the 5’~monophosphate. The monophosphate is then further anaholized to the active triphosphate species: the 5’—triphosphate (L6). The S’—tiipliospliate can be further metabolized to generate 2—amino—9—((2R,3R,4R,SIG—3— fluoro—él—hydroxy—S—(hydroxymethyl)—3 —niethyltetrahyd1‘ofuran—2-yl)- 1 ,9-dihydro—6H-purin—6—one (137). Alternatively, 5’—monophophate M can be metabolized to generate the purine base L8. The metabolic pathway for pyl((S’)—(((2R,3R,4R,5R)-5*(2~amino~6~(methylamino)-9H-pm'in-9— yl)—4~flooro—3 —hydroxy4—methyltetrahydroforanullmyl)methoxy)(phenoxflphosphoryl)mL— a‘te is illustrated in Scheme 1 (shown above). ill. Additional Salts of Compound 1 In alternative embodiments, the present invention provides nd 1 as an oxalate salt (Compound 4) or an HCl salt (Compound 5), Hot/CA3l- . HNLHJ» / E The terms "coadminister‘l and "coadministration" or ation therapy are used to describe the administration ofCompound 2 according to the present ion in combination with at least one other active agent, for example where appropriate at least one additional anti-I-iCV agent. The timing of the coadministration is best ined by the medical specialist treating the patient. it is sometimes preferred that the agents be administered at the same time. Alternatively, the drugs selected for combination therapy may be administered at different times to the patient, Of , when more than one viral or other infection or other condition is t, the present compounds may be combined with other agents to treat that other infection or condition as The term "host", as used herein, refers to a unicellular or multicellular organism in which a HCV virus can replicate, including cell lines and animals, and typically a human. The term host specifical ly refers to ed cells, cell s transfected with all or part of a HCV genome, and s, in particular, primates (_including chimpanzees) and humans. in most animal applications of the present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly pated by the present invention (such as chimpanzees). The host can be for example, bovine, equine, avian, canine, feline, etc, Isotopic Substitution The present invention includes compounds and the use ofcornpound 2 with desired isotopic substitutions of atoms at amounts above the natural abundance of the isotope, ie, enriched. isotopes are atoms having the same atomic number but different mass numbers, ie, the same number of s but a different number of ns. By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H) may be used anywhere in described structures Alternatively or in addition, isotopes of carbon, eg 13C and "C, may be used. A preferred isotopic substitution is deuterium for hydrogen at one or more locations on the le to improve the performance of the drug The deuterium can be bound in U1 a location ot‘bond breakage during metabolism (an u—deuteriurn kinetic isotope effect) or next to or near the site ofbond breakage (a tindeuterium kinetic e effect). Achillion Pharmaceuticals, Inc. (WO/‘ZOl4/169278 and WO/2014/169280) describes deuteration of tides to improve their pharmacolrinetic or pharmacodynamic, including at the 5—positi on of the molecule.

Substitution with isotopes such as ium can ati‘ord certain therapeutic ages resulting from greater metabolic stability, such as, for example, increased in Vivo half—life or reduced dosage requirements. Substitution of deuterium for hydrogen at a site of metabolic break down can reduce the rate of or eliminate the metabolism at that bond, At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including protium (ll-l), deuterium (2H) and tritium (3H). Thus, reference herein to a compound encompasses all ial isotopic forms unless the context clearly dictates otherwise.

The term "isotopically—labeled" analog refers to an analog that is a "deuterated analog", a "BC-labeled analog," or a "deuterated/l3C-labeled analog.ll The term "deuterated analog" means a compound described in, y a tope, i.e., hydrogen/protium (ll-i), is substituted by a Hnisotope, i.e., deuterium (2H). Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. in certain embodiments, the isotope is 90, 95 or 99% or more ed in an isotope at any location of interest. in some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location" Unless indicated to the contrary, the deuteration is at least 80% at the selected location.

Deuteration of the nucleoside can occur at any replaceable en that es the desired results.

V. Methods of Treatment or E’rophylaxis ent, as used herein, refers to the administration of Compound 2 to a host, for example a human that is or may become infected with a HCV virus.

The term ylactic" or preventative, when used, refers to the administration of Compound 2 to prevent or reduce the likelihood of an occurrence ofthe viral disorder. The present invention includes both treatment and prophylactic or preventative therapies. in one embodiment, Compound 2 is administered to a host who has been exposed to and thus is at risk ot‘int‘ection by a tis C virus infection.

The ion is directed to a method of treatment or laxis of a hepatitis C virus, U1 including drug resistant and iug resistant forms of l-lCV and related disease states, conditions, or complications of an HCV infection, including cirrhosis and d hepatotoxicities, as well as other conditions that are secondary to a lElCV infection, such as weakness, loss of appetite, weight loss, breast enlargement (especially in men), rash (especially on the palms), difficulty with clotting of blood, spider—like blood vessels on the shin, confusion, coma (encephalopathy), buildup of fluid in the abdominal cavity (ascites), esophageal varices, portal hypertension, kidney failure, enlarged spleen, decrease in blood cells, anemia, thrombocytopenia, jaundice, and cellular cancer, among others. The method comprises administering to a host in need thereof, typically a human, with an effective amount of Compound ‘2 as described herein, optionally in combination with at least one additional bioactive agent, for example, an additional anti-HOV agent, r in ation with a pharniaceutically acceptable carrier additive and/or excipient.

In yet another aspect, the present invention is a method for prevention or prophylaxis of an HCV infection or a disease state or related or follow~on disease state, condition or complication of an HCV infection, including cirrhosis and related toxicities, weakness, loss of appetite, weight loss, breast enlargement (especially in men), rash (especially on the palms), difficulty with clotting of blood, spider~like blood vessels on the skin, confusion, coma (encephalopathy), buildup of fluid in the abdominal cavity (ascites), esophageal varices, portal hypertension, kidney failure, enlarged spleen, decrease in blood cells, , thrombocytopenia, jaundice, and hepatocellular ) , among others, said method comprising administering to a patient at risk with an effective amount Compound 2 as described above in, combination with a pharmaceutically acceptable carrier, additive, or excipient, optionally in combination with another antiuHCV agent. in. r embodiment, the active compounds of the invention can be administered to a patient after a hepatitis—related liver transplantation to t the new organ.

In an alternative embodiment, Compound. 2 is provided as the hemisult‘ate salt of a phosphoramidate of Compound 1 other than the specific phosphoramidate described in the compound illustration. A wide range ot‘phosphoramidates are known to those skilled in the art that U) C) e various esters and phosphouesters, any combination of which can be used to provide an active compound as described herein in the form of a heini sulfate salt.

Vl. Pharmaceutical Compositions and Dosage Forms U1 in an aspect of the invention, pharmaceutical compositions according to the present invention comprise an anti~HCV virus effective amount of nd 2 as described herein, ally in combination with a pharrnaceutically acceptable r, additive, or excipient, further optionally in combination or alternation with at least one other active compound. in one embodiment, the invention includes a. solid dosage form of Compound 2 in a pliarrnaceutically acceptable carri er: in an aspect of the ion, pharmaceutical compositions according to the present invention comprise an anti-l-ICV effective amount of Compound 2 bed herein, optionally in combination with a pharmaceutically able carrier, additive, or eacipient, further optionally in combination with at least one other antiviral agent, such as an -ECV agent, The invention includes pharmaceutical compositions that include an effective amount to treat a. hepatitis C virus infection of Compound 2 of the present ion or prodrug, in a ceutically able carrier or excipient. in an alternative embodiment, the invention includes pharmaceutical compositions that include an effective amount to prevent a. hepatitis C virus infection of Compound 2 of the present invention or g, in a pharmaceuticaliy able carrier or excipient. ()ne of ordinary skill in the art will recognize that a therapeutically effective amount will vary with the infection or condition to be treated, its severity, the treatment regimen to he employed, the pharmacolrinetic of the agent used, as well as the patient or subject (animal or human) to he d, and such therapeutic amount can be determined by the attending physician or specialist.

Compound 2 according to the present invention can he formulated in a mixture with a pharmaceutically acceptable carrier. in general, it is preferable to administer the pharmaceutical composition in orally—adniinistrable form, an in particular, a solid dosage form such as a pill or tablet. Certain formulations may be administered via a parenteral, enous, intramuscular, topical, transdermal, huccal, subcutaneous, suppository, or other route, including intranasal spray. intravenous and intramuscular formulations are often administered in sterile saline. One of U) i... ordinary skill in the art may modify the formulations to render them more soluhle in water or another vehicle, for example, this can be easily accomplished by minor modifications (salt formulation, esterification, etc) that are well within the ordinary sltill in the art. it is also well within the routineers’ skill to modify the route of stration and dosage regimen of Compound U1 2 in order to manage the pharmacokinetic of the present compounds for maximum heneti cial effect in patients, as described in more detail herein.

In certain phannaceutical dosage forms, the prodrug form of the compounds, especially including acylated (acetylated or other), and ether (alkyl and related) derivatives, phosphate esters, osphoramidates, phosphoramidates, and venous salt forms of the present compounds, may he used to achieve the desired effect. One of ordinary skill in the art will ize how to readily modify the present compounds to prodrug forms to facilitate delivery of active compounds to a ed site within the host organism or patient. The person of ordinary skill in the art also will take advantage of favorable pharmacokinetic parameters of the prodrug forms, where applicable, in delivering the present nds to a targeted site within the host organism or patient to maximize the intended effect of the compound.

The amount of Compound 2 included within the tl‘ierapeutically active formulation according to the present invention is an effective amount to achieve the desired outcome according to the present invention, for example, for treating the HCV infection, reducing the likelihood of a HCV infection or the inhibition, reduction, and/or abolition of HCV or its secondary effects, including disease states, conditions, and/or complications which occur secondary to HCV. in general, a eutically ive amount of the present compound in a ceutical dosage form may range from about 0.001 mg/kg to about 100 rug/kg per day or more, more often, slightly less than about 0, l mg/kg to more than about. 25 ntg/kg per day of the patient or considerably more, ing upon the compound used, the condition or ion treated and the route of administration. Compound 2 is often administered in amounts ranging from about 0.l mg/kg to ahout 15 mg/kg per day of the patient, ing upon the pharrnacolrinetic of the agent in the patient. This dosage range generally produces tive blood level corticentrations of active compound which may range from about 0.001 to about lOO, about 005 to about 1th microgram s/cc of blood in the t. ()ften, to treat, prevent or delay the onset of these infections and/or to reduce the likelihood of an HCV virus infection, or a secondary disease state, ion or complication of HCV, D») Em.) Compound '2 will be stered in a solid dosage form in an amount ranging from about 250 micrograms up to about 800 milligrams or more at least once a day, for example, at least about 5, l0, 20, 25, 50, 75, l00, l50, 200, 250, 300, 350, 400, 450, see, 550, 600, 650, 700, 750, or 800 milligrams or more, once, twice, three, or up to four times a day according to the direction of the U1 heal tli care provider. Compound 2 often administered orally, but may be administered parenteral l y, topically, or in suppository form, as well as intranasally, as a nasal spray or as otherwise described herein, More generally, Compound ,2 can be administered in a tablet, capsule, injection, enous formulation, suspension, liquid, on, implant, particle, sphere, cream, ointment, suppository, inhalable form, transdermal form, buccal, sublingual, topical, gel, mucosal, and the like.

When a dosage form herein refers to a milligram weight dose, it refers to the amount of Compound 2 Ge, the weight of the bemi~sulfate salt) unless otherwise ied to the contrary. ln n embodiments, the pharmaceutical composition is in a dosage form that contains from about i mg to about 2000 mg, from about 10 mg to about l000 mg, from about l00 mg to about 800 mg, from about 2.00 mg to about 600 mg, from about 300 mg to about 500 mg, or from about 400 mg to about 450 mg of nd 2 in a unit dosage form. In certain embodiments, the ceutical composition is in a dosage form, for example in a solid dosage form, that contains up to about l0, about 50, about l00, about 125, about 150, about l75, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, or about 1000 mg or more of Compound 2 in a unit dosage form. in one embodiment, Compound 2 is administered in a dosage form that delivers at least about 300 mg. in one embodiment, Compound 2 is administered in a dosage form that delivers at least about 400 mg, in one embodiment, Compound 2 is administered in a dosage form that delivers at least about 500 mg. in one ment, Compound 2 is administered in a dosage form that delivers at least about 600 mg. in one embodiment, Compound. 2 is administered in. a dosage form that delivers at least about 700 mg. in one embodiment, Compound 2 is administered in a dosage form that delivers at least about 800 mg. In certain embodiments, Compound 2 is administered at least once a day for up to l2 weeks, in n embodiments, Compound 2 is administered at least once a day for up to l0 weeks. In certain embodiments, Compound 2 is U) U) WO 44640 administered at least once a day for up to 8 weeks. in certain embodiments, Compound 2 is administered at least once a day for up to 6 weeks, in certain embodiments, nd 2 is administered at least once a day for up to 4 weeks, in certain embodiments, Compound 2 is administered at least once a day for at least 4 weeks. in certain embodiments, Compound 2 is U1 administered at least once a day for at least 6 weeks, in certain ments, Compound 2 is administered at least once a day for at least 8 weeks. In certain embodiments, Compound 2 is administered at least once a day for at least 10 weeks, in certain ments, Compound 2 is administered at least once a day for at least 12 weeks. in certain embodiments, Compound 2 is administered at least every other day for up to l2 weeks, up to if) weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In certain embodiments, Compound 2 is administered at least every other day for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least it) weeks, or at least l2 weeks.

In one embodiment, at least about 600 mg of Compound 2 is administered at least once a day for up to 6 weeks. in one embodiment, at least about 500 mg of nd 2 is administered at least once a day for up to 6 weeks. in one embodiment, at least about 400 mg oi" Compound 2 is administered at least once a day for up to 6 weeks, In one embodiment, at least 300 mg of Compound 2 is administered at least once a day for up to 6 weeks, In one embodiment, at least 200 mg of Compound 2 is administered at least once a day for up to 6 weeks. in one embodiment, at least llll‘) mg of Compound 2 is administered at least once a day for up to 6 weeks.

Metabolite 1-6 is the active sphate of Compound 2, but metabolite 1—6 is not measurable in . A surrogate for metabolite L6 is metabolite L7. Metabolite 1—7 is a nucleoside lite measurable in plasma and is therefore an tion of the intracellular concentrations of metabolite L6. For maximum HCV antiviral activity, a dosage form of Compound 2 must achieve a metabolite in? steady—state trough concentration (til/24,55) that exceeds the ECss value of Compound 2. As shown in , the E0): of Compound 1 t clinical isolates of GT1, GT2, (3T3, and GT4 is less than 25 ngme (Compound l ECss and Compound 2 ECss values are the same). In one embodiment, a dosage form of Compound 2 is delivered that achieves a steady~state trough concentration (Cate) of metabolite 1—7 that is between approximately if? to 75 ng/ml... in one embodiment, a dosage form of Compound 2 is delivered that achieves a steady~state trough tration (C24,ss) of metabolite 1—7 that is between approximately 20 to 60 ng/mL. In one embodiment, a dosage form of Compound 2 is delivered that achieves a steady—state trough concentration (€24,5s) of metabolite 1~7 that is between approximately 30 to 60 ng/mL. in one embodiment, a dosage form of Compound 2 is delivered that ael'iieves a steady-state trough concentration (Cares) of metabolite 1~7 that is between approximately 20 to 50 ng/mL. in one embodiment, a dosage form of Compound 2 is delivered that achieves a steady—state trough concentration (C2455) of lite 1~7 that is between U1 approximately 30 to 50 rig/nil... In one embodiment, a dosage form of Compound 2 is delivered that achieves a steady—state trough concentration (C24,ss) of metabolite 1—7 that is between approximately 20 to 45 ng/mL. In one embodiment, a dosage form of nd 2 is delivered that achieves a steady—state trough concentration (Carts) of metabolite 1-7 that is between approximately 20 to 30 n,g;’n:il_,. In one embodiment, a dosage form of Compound 2 is delivered that achieves a steady—state trough concentration (€24,533) of metabolite 1—7 that is n approximately 20 to 35 ng/mL. In one embodiment, a dosage form of Compound 2- is delivered, that es a steady-state trough concentration ((jZ/i,ss) of lite 1~7 that is between approximately 20 to 25 ng/mL. Approximate dosage forms are jg l0% of the steadywstate trough concentration. in one embodiment, Compound 2 is dosed at an amount that achieves a metabolite 1—7 AUC (_area under the curve) of between approximately 1,200 and 3,000 ng/mL, in one embodiment, Compound 2 is dosed at an amount that achieves a metabolite 1-7 AUC‘ of between imately l,500 and 3,000 rig/ml." in one embodiment, Compound 2 is dosed at an amount that achieves a metabolite 1—7 AUC of between approximately 1,800 and 3,000 ng/mL In one embodiment, Compound ‘2 is dosed at an amount that achieves a metabolite 1—7 AUC of between approximately 2,100 and 3,000 ng/"ml_,. in a preferred embodiment, Compound 2 is dosed at amount that achieves a metabolite 1—7 AUC of imately 2,200 ng*h/mL. Approximate dosage forms are j; 10% ot‘ the AUC.

In the case of the co—administration of nd 2 in ation with another CV compound as otherwise described herein, the amount of nd 2 according to the present ion to be administered in ranges from about 0.0l rug/kg of the patient to about 800 rug/kg or more of the patient or considerably more, depending upon the second agent to be co— administered and its potency against the virus, the condition of the patient and severity of the disease or infection to be treated and the route of administration. The other anti—HCV agent may for example be administered in amounts ranging from about 0.0l ring/kg to about 800 mg/lrg.

Examples of dosage s of the second active agent are s ranging from about 250 U) k)! micrograms up to about 750 mg or more at least once a day, for example, at least about 5, 10, 20, '25, 50, 75, it’ll"), lSO, 200, 250, 300, 350, 400, 450, 500, 600, 700, or 800 milligrams or more, up to four times a day. In certain red embodiments, Compound 2 may he often administered in an amount ranging from about 0.5 mg/kg to about 50 mgx’kg or more (usually up to about lOQ U1 ing/kg), generally ing upon the pharmacokinetic of the two agents in the patient. These dosage ranges generally produce effective blood level concentrations of active compound in the patient.

For purposes of the t invention, a prophylactically or preventive effective amount of the compositions according to the present invention falls within the same concentration range as set forth above for therapeutically effective amount and is usually the same as a therapeutically effective amount.

Administration of Compound 2 may range from continuous (intravenous drip) to several oral or intranasal administrations per day (for example, QlD.) or transdermal administration and may include oral, topical, parenteral, uscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), huccal, and suppository administration, among other routes of administration. Enteric coated oral tablets may also he used to enhance bioavailahility of the compounds for an oral route of administration. The most ive dosage form will depend upon the hioavailahility/pharrnacolrinetic of the particular agent chosen as well as the severity of disease in the patient. Oral dosage forms are particularly red, because of ease of administration and prospective favorable patient compliance.

To prepare the pharmaceutical compositions ing to the present invention, a eutically effective amount of Compound 2 according to the present invention is often intimately d with a ceutically acceptahle r according to conventional ceutical nding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, erg, oral or parenteral. in preparing pharmaceutical compositions in oral dosage form, any ofthe usual pharmaceutical media may he used. Thus, for liquid oral preparations such as sions, elixirs, and solutions, suitable carriers and additives including water, s, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may he used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, manifold, lactose, and related. carriers, diluents, U) 0‘. granulating agents, lubricants, hinders, disintegrating agents, and the like may he used. if desired, the tablets or capsules may he enteric—coated or sustained release by standard techniques. The use of these dosage forms may significantly enhance the hioavailability of the nds in the patient.

U1 For parenteral formulations, the r will usually se sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, also may be included. Of , where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. lnjectable suspensions may also he prepared, in which case appropriate liquid carriers, suspending agents, and the like may be employed.

Liposomal suspensions (including liposornes targeted to Viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable rs. This may be appropriate for the delivery of free nucleosides, acyl/alkyl nucleosides or phosphate ester pro~drug forms of the side compounds according to the present invention. in typical embodiments according to the present invention, Compound 2 and the compositions described are used to treat, prevent or delay a HCV infection or a secondary disease state, condition or complication ofHCV.

Vii. Combination and Alternation Therapy it is well recognized that druguresistant variants of s can emerge after prolonged treatment with an antiviral agent. Drug resistance sometimes occurs by mutation of a gene that encodes for an enzyme used in viral replication, The eff; cacy ot‘a drug against an l-ECV infection, can be prolonged, augmented, or restored by stering the compound in combination or ation with another, and perhaps even two or three other, antiviral compounds that induce a different mutation or act through a different pathway, from that of the principle drug.

Alternatively, the pharm acoltinetic, bio distribution, hall'llife, or other parameter of the drug can be altered by such combination y (which may include ation y if considered concerted). Since the disclosed Compound 2 is an. NSSB polymerase inhibitor, it may be useful to administer the compound to a host in combination with, for example a (l) Protease inhibitor, such as an NS3/Al-A se tor, {2) NSSA inhibi tor, (3) Another NSSB polymerase inhibitor, (2») \3 (4») NSSB bstrate inhibitor; (5) Interferon sulfa—2a., which may he pegylated or nil'ieiwise modified, and/or rihavii‘in; (6) Non—substratembascd inhibitor; U1 (7) Hehcase inhibitor; (8) Antisense oligodeoxynncleotido (SmODN); (9) Aptainer; (l0) N’ucleasewresistant i‘ibozynie; ( l l) iRNAfl including microRNA, and SiRNA; (l2) Antibody, partial antibody or domain dy to the Virus, or (l3) Viral antigen or partiai antigon that induces a host antibody se.

Non limiting examples of -EZCV agents that can be administered in combination with Compound 2 ofthe invention, alone or with le drugs from this lists, are (i) R) protsase inhibitors such as telaprevir (lncivohQ ), boceprevir (Vietrelisfi‘f‘fi sinieprevir om), paritaprevir {ART-450), glecaprevit (ABT-493), ritonavir (Nowii‘), ACH— 26843 AZD~7295, BMS—79l3‘25, danopi‘evir, Filibuvir, 638—9256, GSoMSlj MK—5172, Setiobuvir, Sovaprevii, ’I‘ego’buvir, VX~135, VX—ZZZ, and, ALS-ZZO; (ii) NSSA inhibitor snoh as ACH~2928, , BOX-719, daolatasvii', ledispasvin velpatasvir (Epclusa), elhasvir (h/iKn8742), grazoprevii‘ (MKw5172), and Onibitasvir (@3267); (iii) NSSB inhibitors such as AZDJZQS, Clomizoie, dasahiwir (lifixviera), i’I‘X—SOol, PP}— 461, I’M—688, sofosbuvir (Sovaldi®), hfi£~3682, and mericitahine; (iv) NSSB inhihitors such as Ali‘u’iT—Z‘SZSS, and MBX~700g (V) Antibody such as (ES—6624; (vi) Combination drugs such as l—larvoni (lodipasVir/sofosbuvir); a Pah (omhitasvin’pantapievir/ritonavin’dasahnvir); 'Viekii‘ax (ombitasvir/paiitapi'evir/ritonavii‘); G/l.) (paritaprevir and glecapi'evir); Toohnivie (onihitasviiz/ pantapi‘e'vir/iitonavii‘) and usa huvir/volpatasvir) and Zepatiei (ei‘oasvir and grazoprevir).

U) C133 if Compound 2 is administered to treat advanced hepatitis C virus leading to liver cancer or cirrhosis, in one embodiment, the compound can be administered in combination or ation with another drug that is typically used to treat hepatocelluiar carcinoma (ii-{CC}, for e, as described by Andrew Zhu in "New Agents on the Horizon in Hepatocellular Carcinoma" U1 'l‘herapeutic Advances in Medical Oncology, V 5(1), y 20l3, All—50¢ Exarnpl es oi." suitable nds for combination y where the host has or is at risk of HCC include anti~ angiogenie agents? snnitinib, brivaniln linifanih, rarnucironiah, hevacizumab, cediranib, pazopanih, Tsar—es, lenvatinih, antibodies against EGFR, in’l‘or inhibitors, MEK inhibitors, and histone decetylace tors.

EXAl‘l/EPLES General s 1H, 19F and "P NMR spectra were recorded, on a 4-00 MHZ Fourier orm Blocker spectrometer. a were obtained DMSO—ds unless stated otherwise. The spin multiplicities are indicated by the symbols 3 (singlet), d et): t (triplet), in (multiplet) and, hr (broad).

Coupling constants (J) are reported in Hz. The reactions were generally carried out under a dry en atmosphere using Sigma—Aldrich anhydrous solvents. All common chemicals were purchased from commercial sources.

The following abbreviations are used in the Examples: AUC: Area under the Curve C24: Concentration of the drug in plasma at 24- hours (324,55: Concentration at 2.4 hours after dosing at steady state Cmax: Maximum concentration of the drug achieved in plasma DCM: Dichloromethane EtOAc: Ethyl acetate EtOI-l: Ethanol l—lPLC: High pressure liquid chromatography NaOH: Sodium hydroxide NazStZlii: Sodium sulphate (anhydrous) MeCN: Acetonitrile MeNHz: Methylamine MeOE-I: Methanol NazSiilxi: Sodium sulfate.

NaHCQs: Sedium bicarbonate U1 NE'irlClI Ammi‘mium chlcrlde NH4OH: Ammcnium ide PE: Petroleum. ether Phfl’: 'l'riphenylphosphine RH: relative humidity Silica gel (230 to 490 mesh, Sorbent) t—BuMgCl: l—Butyl magnesium chloride "Pam: Time at which Cmax is achieved THF: Tetrahydmfumn (THF), anhydmus 'I'P: phi'mphate.‘ Example 1. Synthesis (if Con'apound 1 Scheme 2 Ci t—BuMgCi/THF {N \N ————————»» CHso I Step2 ' NAN HOCY H2 r ’TOOHQfix:tie/NiéficnsN Compoundt Step 1: Synthesis of ,4R,5R)—5—(2—An1ino—é-(methylamino)—§H—purin—9—yl)—4—flnoro—2— (hydroxymethyl)—4~methyltetrahydrefuran~3~ol {2%) A 50 L flask was charged with methanol (30 L) and stirred at 10 j; 5 0C. NH2CH3 (3.95 Kg) was slowly ated into the reactor at 10 _l; 5. °C Compound 2~1 (3.77 kg) was added in, batches at 20 j; 5 "C and stirred for 1 hour to obtain a clear solution. The reaction was stirred for an additional 6 — 8 hours, at which point HPLC indicated that the intermediate was less than 91% of the solution. The reactor was charged with solid Na0l-E (254 g), stirred for 30 minutes and concentrated at 50 __+_ 5 DC (vacuum degree: —0.095)‘ The resulting e was charged with EtOH (40 L) and re~slurried for l hour at 60 0C. The mixture was then filtered through celite and the filter cake was re—slurried with EtOH (15 L) for 1 hour at 60 0C. The filtrate was filtered once more, combined with the filtrate from the previous filtration, and then concentrated at 50 j; 5 °C {vacuum degree: 43.095). A large amount of solid was itated. EtOAc (6 L) was added to the solid residue and the mixture was concentrated at 50 j; 5 0C (vacuum degree: -0,095). DCM was then added to the e and the mixture was re—slurried at reflux for l hour, cooled to room ature, filtered, and dried at 50 i 5 CC in a vacuum oven to afford, compound, 2—2 as an off" white solid (l89 Kg, 95.3%, purity ol’99.2%).

Anaiytie Method for Compound 2%: The purity of compound as (15 mg) was obtained, using an Agilent l 100 HPLC system with a Agilent Poroshell 120 EEC-Cid 46* 150mm 4—h/licron column with the following conditions: 1 mL/min flow rate, read at 254 nm, 30 "C column temperature, l5 uL ion volume, and a 31 minute run time, The sample was dissolved in acetonitrile water (20:80) (v/v) The gradient method is shown below.

A% ll05 TFAin water) % [Acetonitrile Step 2: Synthesis of isopropyl{(5}{{(2R93R14R5Ry5-(2uAminowddmerhylarninn)~9}{~purim 9—yl}fluoro—3—hydrosymethyltetrahydrofuran—Z-yl)methoxy)(phenoxy)phosphorylHZ— alaninate (Compound 1) Compound 2—2 and compound 2—3 (isopropyl ((perfluorophenoxyXphenoxy}phosphoryl)~ L~alaninate) were dissolved in Till? (l L) and d under en. The suspension was then cooled to a temperature helowS~5"C and a l.7 M solution of t—BuMgCl solution (384 mL) was slowly added over l5 hours while a temperature of S—lG °C was maintained. A solution ofNHziCl {2 l...) and water (8 L) was added to the suspension at room temperature followed by DCM. The mixture was 3tirred for 5 minutes before a 5% aqueous solution of K2C03 (10 L) was added and the mixture was stirred for 5 additional s before filtering through diatomite (500 g). The diatomite was washed with DCM and the filtrate was separated. The organic. phase was washed with a 5%oaqueous KzCO3 on (l O L x 2), brine (l O L x 3), and dried overNa2SO4 (500 g) tor approximately l hour. ile this entire process was repeated?times in parallel and the 8 batches were combined. The organic. phases were filtered and trated at 45 i 5 °C (vacuum degree of 0.09 llea). EtQAC was added and the e was d for l hour at 60 CC and then at room temperature for 18 hours. The mixture was then filtered. and washed, with EtOAc (2 L) to afford crude Compound l" The crude material was dissolved in DCM (12 L), heptane (l 8 L) was added at ill-20 CC, and the mixture was allowed to stir for 30 minutes at this temperature. The mixture was illtered washed with heptane(5 L), and dried at 50;5’C to afford pure Compound r (1650 g 60%;).

Analytic Method for Compound l: The purity of Compound 1 (25 rng) was obtained usin0 an Agilent 1100 HPLC system with a Waters Xlerra Phenyl Stun 0mm column with the following conditions: 1 mL/min flow rate, read at 254 nm, 30 C column temperature 15 LLL injection volume, and a 25 minute run time. The sample. was dissolved in aeetonitrile — water {50:50) {v/V). The gradient method is shown below.

Time (min A99 0.1% 14151394 in water/ wAcetonitrile Example 2. Characterization of Amorphous and Crystalline Compound 1 Amorphous Compound 1 and crystalline Compound 1 were initially analyzed by XRPD, 1lell‘IMR, and. HPLC. The XRPD patterns for both compounds are shown in FIG lA and the HPLC traces to ine purity are shown in FIGS. 1}} and 2A, respectively. Table l is a list of peaks from the MD of lline Compound 1 and Table 2 is a list of relative ion times (RTT) from the HPLC traces, Amorphous Compound 1 was 98.63% pure and crystalline Compound l was 99.11% pure. Both nds were a white solid. is the "l‘GA and DSC graphs of crystalline Compound 1. For crystalline Compound 1, an endotherrn was observed at 886 °C and ll} there was a 7.8% mass loss from 80 110 "C.

A sample of Compound, 1 was recrystallized from EtQAe/hexane and drawn with ORTEP.

The absolute structure of Compound 1 was contl rrned by the recrystallization of a single crystal. is the ORTEP drawing of Compound 1. Crystal data and measurement data are shown in "llahle 3: The absolute stereochemistry ofCompound l based on the X—ray erystailography is shown below: H:N a rf"N €3qu * A : Q N N’EOALXCHit N Has N"? ° 0Vii o " . ., DSC data were collected on a TA instruments Q2000 equipped with a 50 position auto— sampler The calibrati on for tl’iermai capacity was carried out using re and the calibration for energy and temperature was carried out using certified indium. 'l'ypically approximately 3 mg of each sample, in a pin—holed aluminum pan, was heated at 10 CC/min from 25 C'C to 200 0C. A purge of dry nitrogen at 50 ml/min was maintained over the sample. The ment control software was Advantage for Q Series VZ.8.0.394 and l Advantage v5.5.3 and the data were analyzed using Universal Analysis v4.5A. l6A data were eelleeted (111 a TA Instruments QSOO TGA, equipped with a l6 pesitien aute— sampler. The, instrument was temperature calibrated using certified Alumel and Nickel. ’l‘ypieallyfi 210 mg of each sample was loaded 01110 a p16:Eared aluminum DSC pan and heated at ll} CC/min frnm ambient temperature to 351’) C‘C. A nitrogen purge at 60 ml/min was maintained U1 over the sample. The instrument central sowaare was Advantage for Q Series 17.25.0256 and l Advantage v5.5.3 and the data were analyzed using Universal Analysis V4.5.

Amorphens Campennd ‘l ): 1H NMR. (400 MHZ, DMiSO—ds) 6 ppm llll ~ 1.15 (111, 9 H), 1.2l (11, 1227.20 Hz, 3 H), 2.75 — 3.08 (111, 3 H) 3.71 — 3.87 (m, l H), 4.02 - 4.l3 (m, l H), 4.22 — 4,53 (1n, 3 H.)7 , lH)f1.69 2 5.86 (111 l H) 6.04 (hi 1’1,J=19.33 Hz, I—‘l H), 712— 7.27(111 3. H) 7 7 7.27 - 7.441111, 3 1-1:), 7.81 (s, 1 1:1) Crystalline C11111pe11111l 1 (1—2): JH NMR (400 MHZ, s) 5 ppm 0.97 — HE) (111, 16 H), 'E .21 (d, J=7fi7 Hz, 3 H) 2.87 (br s, 3 H), 3.08 (s, ‘2; H), 3.79 (br d, J227O7 Biz, 1 H), 4.08 (br El, J227.58 Hz, l H), 4.17 — 4.55 (m, 3 H) 4.81 (quill, J=6.25 Hz, 1 H), 5.78 (b1 5, l H), 5.9l - 6.15 (111, 4 H), 7‘10 - 726(111, '3 H), 7.26 — 1, 3 H), 7.81 (s, 1H) 'l‘ahle 1. Peak list fer crystalline Campanild l WO 44640 2807 200 WO 44640 Tabia 2. Relative Retention Times fmm HPLC chmmatographs 9f Amarphous Campaund I and; Crystalline Compound 1 048 03:) ; {>48 Oil ' 004 0:7 Tabfie 3. (:rystal and Data M'aasurement 0f ijgcund1 Bond Precisien ;;'f5f'i'"87§i'{ii§m:15=226422(9) "1::'E29497Z'5'}""""""""". 2113555390 beta: 1 13 . 184(4) gamma==90 Temperature 150K Calculated ............................................................................................................................................................................................................................................

Volume 3745(2" :- .57" SpaCLGmup "221"" ..................................................................................................................................................................................................................................................

Hal} Group P 2yb Moiety Farmuia C24 H34 F N7 07 P 2({324 H34 F N7 ()7 P) Sum Farmula (:24 H34 F N7 07 P C48 H68 F2 N14 014 P2.

Mir 58255 316510 D51, g cm"1 151 E4 1334 Z 4 Mu (mm’l) 1.139 1139 F000 1228.0 F000’ E23321 by 1:, 1mm; 12,3425 ................................................................................................................................................................................................................................................

Tmm, rm 0.790; 0.815 CorrCenonN/lethod #ReportedTeritqu=0808Tma=l00 """"""""""""""""KBEEOHMULitsr/w mm"mliéiQEEliilllEiEilEEé"""""""" """"""""""""""""""""""""""""""""""""""""iii/7575377"""""""""""""""""""""""""""""""""""""""" Theta (max) @8244 R tions) 0.2091 (: 7995) WRZ (retl eetions) 0.5338 ( 8259) S 2. 875 Npar 7 l 6 This initial characterization was followed by storage at 25 0C 60% relative humidity (RH) for 14 days with analysis by HPLC and XRPD after 7 and l4 days is the XRPD after l4 days at 25 "(I /‘ 60% (Ml). Amorphous Compound 1 {sample l-l) remained poorly crystalline, whereas crystalline Compound 1 (sample an) retained its emstallinity, but both nds were stable after l4 days at 25 "C 60% (RE-l).

Example 3. Formation of Oxalate Salt €ompnnnd 4 Initially, the oxalate salt of Compound 1, Compound 4, was formed by mixing the oxalic salt with solvent, (5 vol, 100 all) and allowing any solution to evaporate at room temperature. Any suspension was matured (room temperature 50 °C) for 3 hours and eiystallinlty was accessed.

HN ’J 0' -' HO"; F ‘ file nd4 ‘ Table 4 shows the different solvents used in the tion of Compound 4. All solvents except for two (cyclohexane and n~heptanel afforded crystalline products. Despite the hi gh crystallinity and solubility of Compound 4, oxalate salts are not acceptable for clinical development clue to the potential formation ofkidney stones and other salts of compound l were explored.

Table 4. ion of Gxalate Compound 4 Solvent. Glasers'atiou post send Qbservalion after addition at room . , g a maturation/evaporation ________________________________________________________________________________itanaemtm ' : tOH 8011mm (WA Emmi _________________________________E EPA Schism 0’ rm! ______________________________ Acetone Solution GXA-n Form 1 MEK Solution GXA m Form 1 Eli) Ac. Sus g ension GXA — Form 1 iPrGAc Sus ension GXA— Form 1 "fl-l]: Solution ()XA — Form 1 'lToluene on GXA — Form 1 MeCN Solution ()XA — Form l WHO/water ______________________________Sstatics_____________________________ _______________l.l§a_:__tism__l______________________ IEME EUSPBWOHOMEWM Cvcloliexane Sus c ension Amer aheus n—Hetane Susenslon Amorhous Example 4. Salt Compounds of Amorphous Compound l Since the oxalate salt compound 4 (Example 3) could not be carried foiward in clinical trials clue to its potential to form kidney , ous salts of Compound 1 were formed with the counter ions listed in Table 5. Compound 1 was dissolved in t~butanol (20 vol, 6; ml) and the solution was treated with the acid counter—ions (1 equivalent for each sample except sample l_~9 which had 0.5 equivalent of sulfate). The samples were then frozen with the t removed by lyoplillizati on. The residual solid in samples L4,, l—S, l—6fl l—7, l—8, and 1—9 was initially analyzed by XRPD and HFLC.

T111311: 5. Amerphous $1111 farmmian (1111111114 Sampie Sampie details Stock solution ()hservation NMR 11) (11:11:11.1: 11C] (1 1) T1113 1M. White 5011(1 3 fewer pm‘mns ~03 ea l—BuGH 1—5 11c (1: 1) THF 1M White 301111 3 fewer s .................................................................................................................................................................................................................................... 1—6 Fumaric (1:1) MeOH:THF Glassy $0111.05 eq fumaric acid (1: 1) 0 5M 0.84 er i—BuGH 86112011: THF White 501111 €C£b6112010 1113111 ~ 1.1 sq 51113611111: acid 81101311110 M13011 Sticky wh1te 0.37 eq t—BuOH : : . 1M 3011(1 81215113: 3 fewer prawns THF White 301111, ~03 aq ("1311011 acid: APP) 11-1NMR spectrum were taken 111172111 samples.

Sampie 144,118 (1:1) sail: JH NMR (400 MHZ, 1911418104511) 5 ppm 0.93 — 1.39 (m, 16 H), 2.97 (br s, 2 H), 3.70 - 3.38011, 1 H), 4.100315, 11:1),418 - 4.49(m,31-1),4.70 — 4.881311, 1 1:1),571 — 5.94011, 1 H), 6.07 (131" {1, 7:19.07 Hz, 2 H), 7.14 — 7.27 (111, 3 H), 7.29 - 7.44 (111, 2 H)7 7.83 — 8.19011, 1H) Sampie 1-5, 1111111: (121) $311: 1H NMR (400 ME-iz, £311'{Sf()-d6) 5 ppm 0.97 - 1.38 (m, 15 11)., 2.96 (br s, 2 H), 4.06 ~ 4.18 (m, 1 H), 4.19 _ 4.49 (111, 3 H), 4.66 — 4.91 (m, 1 H), 5.70 w 595(111, '1 H) 5.96 — 6.16011, 2 H), 7.10 — 7.27(m,31—1),7.30 — 7.43(1n,,21¥1),7.88 — 8.19011, 1 1-1) Sampie 1—6., Fumaric (1:1) 51111: 1H NMR (400 MHZ, O—de) 5 ppm 0.95 _ 1.31 (m, 21 H), 2.87 (br s, 3 H), 3.79 {111 d, J=7.20 Hz, 1 H), 4.01 — 4.13011, '1 H), 4.16 ~ 4.23 (111, 111), 4.16 — 4.1241111, 1H), 4.20 (s, 1 H), 4.18 - 4.23 (m, 1 H), 4.24 - 4.52 (m, 1 H), 4.24 ~ 4.52. (m, 1 H), 4.24 ~ 449(111, 1H), 4.72 — 4.881111, '1 H), 5.68 — 5.86011, 1H), 6.04 (111‘ d, J=1933 Hz, 4 H), 6.63 WO 44640 (s, 1 H), 6.61 _ 666(111, 1 H), 7.12 _ 727(111, 5 H), 7.27 _ 7.45 (m, 5 H), 7.81 (s, 1H), 151411113, Sampie 147, Henzoie (1:1) sait: U1 JH NMR (400 MHZ, DMSO-ds) 5 ppm 0.96 — 1.30 (m, 15 1-1.), 2.87 (bf s, 3 H), 379(111 d, .J=7.07 Hz, 1 H), 407(1):" s, 1 H), 420(1), 1 H), 4.25 — 4.512(111, 3 H).4.81(s, 1 H), 5.71 , 5.85 (m, 1 H), 15.04 (by (:1, $319.33 Hz, 4 H), 7.08 ~ 7.27 (m, 3 H), 7.27 ~ 7.43 (m, 3 H), 7.45 ~ 7.57 (m, 2 H), 7.63 (s, 1 H), 7.81 (s, 1 H), 7.95 (dd, J=8.27, 1.33 Hz, 2 H), 1298(‘015, 1 H) Sampie 1—8., Sammie (1:1) 15211: 1H NMR (400 M1912, -ds) 5 ppm 0.98 — 1.28 (m, 15 H), 2.42 (s, S H), 2.87 (111 s, 3 H), 3.57 — 3.62 (m, 1 H), 3.70 m 3.86 (m, 1 H), 4.02 — 4.14 (m, 1 H), 4.20 (s, 1 H), 4.24 - 4,51 (m, 3 H), 4.70 - 4.88 (m, 1 H), 5.69 — 586(1)), 1 H), 6.04 (111 d,J===19.331-12,41—1), 7.12 — 7.27 (m, 3 H), 7.27 — 7.441111, 3 H), 7.81 (s, 1 H), 11.95 — 12581111,.2 H) Sampie 1-9, Suifurie (11.531) sait: 1H NMR (400 MHZ, 121450—216) 1 ppm 1.02 . 1.31 (m, 15 H), 2.94 (1.11- s, 3 H), 3.79 (111 d, J=7.2e Hz, 2 H), 4.09 (111- s, 1 H), 4.22 — 4.48 (m, 3 H), 4.72 — 4.90 (m, 1 H), 5.71 - 11, 1H), 6.07 (131 11,]:11'107 112,2 H), 7.12 - 7.28 (m, 3 H), 7.31 _ 7.441111, 2 H), 7.75 4 8.191111, 1 H).

The samples were then subjected to storage at 17.5 °C .I' 60% reiative humidity (RH) for 14 days with is by HPLC and XRPD after 7 (1:11:33 413) and 14 days (1711:]. 5A). AH prepared salts remained amorpheus and the obsewations are shewn in ’l‘ab1e 6. The memo suifate (samp1e 1—5) and suceinate salts (sa111p1e 1—8) were found ‘10 be physicafly 1111111111111) and dehquesced or became a gum during the course of the study. Both the fumarate (sample 1—6) and benzeate salts (sample 1-7) were found. to be giassy solids. The HC}. 3211: (samp1e 1—4) was found to retain its physical appearance. Surprisingiy, the hemiwsulfate $1111. (sampie 1-9) 21110 retained its physicaE appearance as a white se1id in contrast to meno~sulfate compound e 1—5), which was a sticky gum. Results are ShOWE‘a in Table 6. The mane HC1 salt (samp1e 1—4) and the he1ni~sulfate sa11. e 1—9) were found ’10 be physieafly and chemically stable after 2 weeks sterage at 25 C'C / 60% ve humidity (RH). Although both salts were stable over the two weeks, the hemi—sulfate salt was or to the H0 salt because the l-lCl salt was hygroscopic, rendering it less useful compared to the hemi~sulfate salt for long—term storage or use Table 6. ity of samples after 7 and 14 days at 25 0C / 66% RH Sample Time exposed to 25 0C /' 66% RH (days) HPLC Gbservation Gbservation HPLC Observation l-l 98.6 White solid White solid 1-2 99.1 White solid Vv'lllle solid to 99.7 White solid White solid White solid thitewhdhhitewhd .____________l:5?______________________98...?_________l____llif_lti_ts_sstis_____________________________________Ti l J3 98.4 White solid Sticky white — Sticky gum solid 1—6 98.7 Glassy solid 98.6 Clear glassy White glassy solid solid 98.8 White solid l-7 , . Clear glassy . .. Clear glassy . 98.7 Stiekv white i'g "’ esced Deliquesced solid, a . i , / strokv orl 98.7 White solid 98.1 White solid White solid Example 5 (:Tharacterizatlon of Amorphous (Tonipound 2 Amorphous Compound, 2 was initially analyzed by XRPD, iHNl‘t/lR, DSC, TGA, and, li-ll’LC. The XRPD pattern for amorphous (Ilonipound 2 overlaid with ous Compound 1 and crystalline Compound 1 is shown in FIG. lA and the XRPD pattern of amorphous Compound 2 alone is shown in 3. Table 7 is a peak list from, the XRPD pattern shown in 3. The HPLC trace to determine purity is shown in FIG 6A. ’l‘ahle S is a list of relative retention times (RTT) from the l-lPl_,C trace shown in . Amorphous Compound 2 was 9.968% pure. FIG 68 is a 'l‘GA and DSC graph of amorphous nd 2. Experimental details for the TGA and DSC experiments are given in Example 2.

Table 7. Peak list for Amorphous Compound 2 Amwphnns Cnmponnd 2: 11-1 NMR (400 Ml-lz, ,DilctSO-ds) 5 ppm 0.93 — 1.29 (in, 13 1-1), 2.94 (br s, 3 1-1), 3.79 (td, J=10.04, 7.07 Hz, 2 H), 4.05 - 4.19 (in, 1 H) 4.19 — 4.50 (m, 3 H), 4.81(quin,J=6.25 Hz,1H),5.71— (111,1H), 5.9"] ~ 6.16 (tn, 2 H), 7.14 — 7128011, 3 H), 7.31 - 7.44 (m, 2 H), 7.82 - 8.09 (in, 1 ll) Example 6. Crystallization of Aniei'plieiis Cempnimd 2 Since the sulfate salt was found to remain as a solid after the 14 day ity study as shown in Table 6, preliminary tests studying crystallization cenditions using 1 1 different selvents was conducted. Arnerphous Cornpennd 2 was suspended in 5 volumes of solvent at 25 "C (sample 2—1, 2—2, 2-3, 24, 2—5, 2-6, 2-7, 2w8, 2-9, 240, and 2~1 1). To those s that were not free flowing (2—1, 242, 26, 2—1-1, 2—5, 2—6, 2—7, 2—8, and 2—10), an additional 5 volumes of solvent was added. The samples were then matured at 25 50 "(I {1 O{ii/min between temperatures and ll hour at each temperature) for 6 days except for sample 2n1, which was observed to be a clear solution after 1 day and was allowed tn evaporate under t conditions. The results are shown in Table 9. Crystalline patterns resulted from crystallization with isobatanel (sample 2~l), e (sample 2~2), EtOAe (sample 2—6), and iPrOAe (sample 2w?) 'l'we poorly crystalline samples were also identified from crystallization with MEK (sample 2—4) and MIBK (sample 26). The XRPD patterns are shown in F16. 7A.

Table 9. Crystallization: Conditions of Coniponnd 2 Sample Gbservation Gliservation Qbsewation after 5 after 10 after 1 day volumes s maturation Solid not Free flowing Solution, free flowing snsnension evaporated at RT yielding a lsohutanol Solid not Free flowing Suspension Crystalline free ‘l‘lowin0 l: Jension Pattern '2 Acetone Solid not Free ilowing '_.uspension Crystalline free flowin sus ension Pattern 3 Solid — not Free llowing Suspension Poorly free flowing suspension crystalline Pattern 4 M ll}K Solid not Free g Suspension Poorly free g sion crystalline — ___llsttsrs_fl________ EtOAo Solid—not Free flowing : Suspension Crystalline — l free llowino sus ension Pattern l iPrOAe Solid not Free flowing Suspension lline free flowin susension Pattern l Solid — not Free flowing Suspension Poorly free flowing; sus ension c 'stalline TEE/[E Free flowing Suspension Amorphous 'l‘oluene Solid not Free flowing Suspension ous free flowin sus en si on l-leptane Free flowing Suspension Amorphous snsaension - The seven samples (Samples 2—2, 293, 2—4, 2—5, Zwo, 2—7 and 243) were analyzed by DSC, TGA’ 1H—NMR and 1C (Table 10, : FIG. BB, ,, FlG, 9B, FIG. WA? FIG, lO’B, FIG. ll A, and FIG. ll B) as well as by XRPD following 6 days storage at 25. °C 60% relative humidity (RH) (all samples remained crystalline / poorly lline lollowing stability) All samples retained roughly half an equivalent of sulfate, but contained a, relatively large amount of residual t. An overlay of the X—ray diffraotograrns of amorphous samples 1L9, 240, and Z-ll is shown in HQ 73, Tame 111. Characterizatian (31‘ crystailine Campound 2 samples 1 3C Sampfle Soivem "H NMR (carrected 3}) for ’E‘GA) ; 3395 2-2 Isobutanol Ends 1138 °C t— ‘ t} isobutanol 1 40 CC ,. 713% 1 13111010 30—95 0C '7_’ Acciom .

I ».‘ a i ambient,, .n 0.5 mg "magma3. a" I) H 3 ’_ ( Endo 100445 0C 111-0 0C Broad cmnplex 8.5 % 2-4 endo 304 15 0C ambient, ~ 0.8 sq MEK 1 Endo 115—145 "C 140 0C . 5.2% Bread cndo 30-105 0C. 2—5 ambmnt _. , ' $160 114 7 0C‘ 110 0C 1 2.00/13 2~6 Sharp endo 1 136 OC ambient— 0.9 sq EtOAc 1 190°C 2—7 iPrOAc Endo 30—90 0C ambient— 9.8 eq iPrOAC 1 90°C ; Endo304000c 4296 2—8 " r 211310 1, 15.6 anfijignt— 130 9C JHNMR spectmm were taken for 2111 sampies and 1isted be10w.

Sampie 2-2: 1&1 NMR (400 M112, ATM/£8046) 5 ppm 0.83 {(1, 37516.69 Hz, 7 H), 0.99 - 1.26 (m, 14 H), 1.61 (ab; J=13.26, 6.63 Hz, 1 H), 3.73 — 3.87 (m, 2 H), 4.03 - 4.18 (m, 1 H), 4.18 — 4.51, (m, 4 H), 4.66 — 4.92; (m, 1 H), 4.70 ~ 4.90 (m, 1, H), 4.72 - 4.88 (m, I H), .81 (br s, 1 H), 5,93 - 6.11 (m, 2 H), 7110 — 7,26 (m, 3 H), 7.14 - 7.26 (m, 1 3H), 7.30 - 7.41 £311,211), 7.94 (br s 1 H) Sampie 2-3: 1H NMR {400 MHz, Dfi/fSO—da) 5 ppm 100 ~ 1.26 (m, 13 H), 209 (s 3 7 33) 3.74 — 3.87 (m, 17. H), 4.11) (hr d, J=7.70 Hz, 1 H), 4.22 ~ 4.50 (m, 3 H), 4.81(quin,J=6.28 Hz, 1 H), 5.71 _ 1, 1 H), 5.96 _ 6.15 (111,2 H), 7.12 — 7.261111, 3 11), 7.31 — 7.41 (111, 21-1), 7.79 — 3.07 (m, 1 1-1) Sampie 214: U1 JH NMR (400 MHZ, DM’S‘O—a’e) 5 ppm (1.91 (t, J11733 Hz, 3 H), 1.01 — 1.213 (m, 13 H), 2.08 (s, 2 H), 3.72 1 3.89 (m, 2 H), 4.10 (hr :1, 11:13.08 Hz, 1 H), 4.23 1 4.47 (m, 3 H), 4.8.1 11, J===6.25 1-12., 1. H), 5.69 - 5.890111, 1 H), 5.94 — 6.13 (111, 21-1), 7.14 1 7.25 (m, 3 H), 7.32 — 7.41(m,2H),7.79 _ 8.11611, 1H) Samyie 2—5: 1H NMR (400 MHz, DA/[fs’O—ds) 5 ppm 0.86 (d, J=6.69 Hz, 1 H), 0.98 - 1.33 (m, 13 H), 2.02 - 2.091161, 1 1-1), 4.03 — 4.17 (m, 1 191),4.22 - 4.50 (m, 3 11:), 4.81 (1111111,.1’1625 iii-12., 1. 131), 5.81 {131 s, 1 H), 5.93 — 6.15 (m, 2 H), 7.11 - 7.27 (111, 3 H), 7.31 — 7.41 (m, 2 H), 7.77 - 8.21011, 1 H) Samyie 2~6: 1H. NMR (400 MHz, [9.11430411) :3 ppm 0.98 - 1.28 (m, 15 .H), 2.00 (s, 3 H), 3.99 — 4.14 (m, 3 H), 4.21 — 4.49 (m, 3 H), 4.81 (1111111, J=1622 Hz, 1 H), 5.82 {br s, 1 H), .93 — 6.14 (m, 2 H), 7.11 _ 7.26 (m, 3 H), 7.29 _ 7.42 (m, 2 H), 7.79 ~ 8.171311, 1 H) Sammie 2-7: 1H NMR (400 MHZ, [DA/[301076) 5 ppm 092 ~ 1.28 (m, 17 H), 1.97 (s, 2 H), 4.04 1 4.16 (m, 1 H), 4.20 - 4.51 (m, 3 H), 4.71 — 4.93 (m, 2 H), 5.82 (131 S, 1 H), .95 — 6.14 (m, 2 H), 7.11 - 7.28011, 3H), 7.31 .. 7.43 (111,2 1-1), 7.75 ~ 8.21(1n, 1H) Sampie 2-8: 1H NMR (400 1141-12, 112145016116) 5 ppm 081. ~ 1.11 (m, 13 H), 1.19 (s, .1 H), 1.53 — 1.66 (m, 1 H), 3.87 - 4.111 (m, 1 H), 4.06 — 4.32 (m, 3 H), 4,64 (quin, 7116.25 Hz, 1 H), 5.55 — 5.75 (m, 1 H), 5.77 — 5.97 (m, 2 H), 6.94 - 7.10 (m, 3 H), 7.13 — 7.26 (m, '2 H), 7.66 - 7.96 (m, 1 H) e 7. ill‘aiiure to Crystallize Amorphous ll’lalonate Salt (Compound 4) As shown in Example 3, a crystalline oxalate salt was identified when determining appropriate salts for Compound l, but oxalate salt Compound 4 could not be carried forward in. clinical trials due to its ial for causing kidney . Therefore, crystallization of the chemically related malonate salt {Compound 5) was attempted using the same ll solvents as for the hemi—sulfate salt. Compound 1 (l2 X 50 mg, samples 3~l, 3—2, 3—3, 34, 3—5, 36, 3~7, 3—8, 3_ 9, 3—10, 3-11, and 3—l2) was dissolved in z—hutanol (20 vol) and the solutions were then treated with l equivalence ol‘a rnalonie acid stock solution (l M in "ill-1F). The samples were then frozen with the t removed by lyophilisation. To samples 3nl, 3-12, 36, 34-, 3—5, 3M6, 35.7, $8, 3" 9., 3—10, and 3~l 1. relevant solvent (5 volumes) was added at room temperature. Any resulting solutions were allowed to evaporate under ambient conditions, while gums or solids were matured at 25 — 50 0C (l °C/min between atures and 4 hour at each temperature) for 5 days. The solids were analyzed by XRPD (3), but all s were found to either form a gum or were amorphous (FlG. lZB). Results are sl'iown in Table l l. The one solid (amorphous) sample {342) was analyzed by J'H—NMR and HPLC, and was found to contain around l equivalence of malonio acid (peaks overlap) as well as 0.6 eq. r—BuOE-l. The nd was 99.2% pure (A). FlG. 12A is an XRDP of sample 3u’l‘2 and FIG. BA is the HPLC ehrotnatograph of sample 3— l2, Sample 342: 1H NMR {400 MHz, [)1l/fSt'3-d6) 5 ppm oat - ll] (m. is it), 1.19 (is, 1 H). 1.53 _ 1.66 (m, 1 £113.87 - 4.01 (m, 114114.96 — 4.32 (m, 3 H), 4.64 (quin, J=6.25 Hz, 1H), 5.55 - 5.75. (m, 1 n). 5.77 . 5.97 (m? 2 H), 6.94 - 7.10 (m, 3 it), 7.i3 — 7.26 (m. '2 H), 7.66~7.96(m, 1H) Table ll. Crystallization Conditions of Amorphous te Salt Composed 4 Sample ID Solvent thervation Ghservation after 5 after 5 s days maturation / eva iteration lill’A Clear solutionl‘ Clear Quin - Acetone ear solution* lviEK Clear solution’l Clear Gum Solution 8:, Clear gum Clear solution* Cleargumé’éenstalAmorphous like a) wearance ....................................................................................................................................................................................................................................

Clear gum sus enslon Toluene White gum / White gum Amorphous solid Heptane White solid - static) : {White solid — y white solid — Amorphous *Evaporated at room temperature Example 8. Failure of Adequate Salt Formation using Liquid Assisted Grinding (Lao) A liquid assisted grinding (LAG) snidy to determine appropriate salts other than hemi— sulfate was performed using the £4 acidic counter ions in Table l2.

Table 12. Counter—ion stock ons used in LAG Crystallization (ountermn seaward Fem": Malonic DuGiucumnic mdellc ..................l.

D—Gluconic lecolie L~Lactie Gleic corbic Adi ie ’l‘HF (heat) .........................................................................................................................

Stearic Psimim _________________________________________________________________________________ Methanesulfonic Compound 1 (30 mg) was placed in HPLC Vials with two 3 mm ball bearings. The als were wett‘ed with solvent, (15 ill ethanol, sample 44, 4—2, 4—3, 4—4, 4'5, 4—6., 4'7, 4~83 4~9, 4-10, AH 17 4-12, 44 3, and 1-H 4) and 1 equivalence of the acid counter-ion was added The samples were then ground fer 2 hours at 650 rpm using a Fritsch milling system with an Automaxien adapter. "Most of the samples after grinding were found to be clear gums and were not ed further ('l'ahle 13), Those that were ehser‘ved to contain sclid were analyzed by XRPD and, in all cases, the patterns ebtained were found it) match these of the crystalline acid U1 counter ion with no additional peaks (Fifi. BB).

Table 13. ()hservatiens and XRPE} s from LAG at" (Innrpeunds l Malomc _ __§lssr__ssst__ DmGlueurenic "militias gum/s01iiimmm D—Glucurenic acid & anter hoes hal 0 DL—Mahdelie Clear gum ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________ 5136111601116 ("lawsuits ____________it 46 (31300310__________________________________________________tilestssst_________________________________________________________________________________________ ‘ Liars-tic Clear urn — 4—9 ,seorhic White gum/selid L—Ascerbic acid 81. 440 Adi ic Clear run] — 4m Stearic White gum/solid Stearic acid & ___________________________________________________________________________________________________________________________________________________________________________________________a.testshssshsts_________ ‘ 4—1;{4 Palmitie White gum/solid Palmitic acid 8: amer 'hous halo Methanesuifcnic cream — Example 9. Failure tn Obtain Adequate Salt Fermatien using Methyl Ethyl .Ketane (It/EEK) Methyl ethyl ltetene (MEK) was next utilized as a solvent to study riate salts other than the hemi—sulfate salt. Using the 14 acidic. counter ions in Table l 2., the study was performed by disselving Ccmpeund l {50 mg) in h/EEK (20 vol) at room temperature. The solutiens were treated with l equivalence ofthe selected counter—lens ('l‘able l2). The s were then ccoled down it) 5 °C at Oi OC/rnin and stirred at this ature evernight. All samples were allowed to evaporate under ambient conditions and any selids observed were analyzed by XRPD. This evaporation mainly ed gums, with the exception of the samples with steric acid (sample 442) and ie acid (sample 5—l3), which ati‘crded glassy sclvents. These selids were amorphous by XRPD, but ne crystalline farms ef the salt were ebtalned. Results are sliewn in Table l4. (F16. 13A,).

Table 14. Resalts frem ving Compeund l. in l‘w’lEK (2G velumes) Sample Placid Solvent Qbservatien Gbsei‘yafien allen ll) for aeltl at npen acid upen eoellng 11pm: l M additlun ' evaleralleit Y ll ' . Pamela DMSQ Yellow wing; Y ellOW gum a): x ....6 solution ~2 Malomc Solution Selution 3 D—Glueuronle Solution Selution —5 D—Glueeme lHE " ’ 'l‘urbid Clear gum _‘ ‘. r. selu'tlen 6 666666 6 -7 semen 666666666 "J: i 8 SOlUtlQl’l Selution — l 0 Adlple lllF Soluti en Selutlon Clear gum —11 66666666 6666666 -12 Stearle llll? on Tu1l1id ' lefiiglassy solution so 1 Clear glassy S -l 3 Palm i ti 1: "El-ll" on Solution selid" elution Clear gum —_l__4_ Methanesulfenic lHE Solutien Stock solution prepared prim to all-id on *Samples were ed by XRPD and gave amorphous patterns plus peaks from the acid counter ion Since all samples were amorphous, all samples were redissolved in MEK (5 vel) and eyeloliexane was added (20 vol antiselvent) at room temperature followed by l hear of stirring at 25 OC. The samples were then matured between 50 — 5 0C (l oC/min between temperatures, 4 lmurs at each temperature) for 2. days befrire the cycle was changed to 50 — 25 "C for a further 4 days. The s were observed by eye following maturation. Results are shown in Table 15.

Following the maturation, all s except 5~l (with particle aeid) were feund to be gums.

Sample S-l, a yellow solid, was analyzed by MD, and the pattern was found, to match the known form of pamoic acid (FIG. MB), and therefore no crystalline forms of the salt were Table ES. s from redissolving Compound E in EVEEK {5 volumes} and antisolvent Sample ID Immediate Observation Gbseryation Ghservation ____________________________________ __________t2.121251111311111_______________s.Earthstitans...___a:f_ts1:_§_9__111_§1111ts§____l___stts1:__Ejttatsr_s_ttss___ Precipitate Gum Gum Yellow _ 1 sus ensi011* ‘2 itate &5 itate/Uum $8 Preeipltate LightGsuspension 5_9 Precipitate ,10 Preeipitate 51 Preeipitate Sal x, Preeipitate Light suspension Gum Gurn z,- 1 ' ' 543 Preeipitate Light suspension (IE-trot Gum rtate Gum Gurn (3:11:11 ple ed by XRPD with pattern niatelnng 111101111 form of pammc acid (110 addnional pealrs Example 101 Failure to Obtain Adequate Salt Formation using Ethyl Aeetate Ethyl e was next utilized to study appropriate salts other than herni—sulfate salt.

Utilizing the l4 acidic: counter ions in Table l2, the study was performed by dissolving Compound 1 (50 mg) in ethyl acetate (21’) vol) at 5% oC. The solutions were treated with l equivalent of the selected counter—ions (Table E2). The samples were then cooled down to 5 0C at OE "(Z/min and stirred at this temperature for 4 days. The solutions were allowed to ate under ambient conditions while any solids were analyzed by XRPD. The results from the erystalllzations using ethyl acetate are in Table to. En contrast to Example 8 where MEEEK was the solvent, the majority of samples were observed to be suspensions following cooling of the acideornponnd mixture (those that were solutions were allowed to evaporate under ambient conditions). However, the XRPD diffractograms were generally found to match crystalline Compound 1. Sampies 6—2, 644, and; 645 have some slight differences (FlG. 'lllA and A).

No crystalline forms ofthe salt were obtained.

Table 16. Results from dissolving Compound 1 in EtGAe (20 volumes) thervation for upon acid amen acid at addition Evaporation 1 M ; Pamoic DMSO Yellow Yellow on solution* Melanie 'l‘HE‘ on White Slight l suspension differenees to freebase D-Gluouronio Water Solution Dl_.r—lVlandelie TH}? Solu ti on White l suspension differences a to fi'eebase D—(Elueoni c "fl-ll? White Possi bl e Slight l ‘ precipitate white gum differences to fi'eebase Solution White sus wensi on Solution sus ension Solution Freebase susension LnAsoorbie Solution on" White solid E ‘ on side yellow gum — amen Jhous Adipie Solution White Freebase- Capi‘oie V " Solution White Freebase sue ension Stearie ‘- 5‘ on susension -- susension Methanesulfonie THE White -- precipitate clear gum" Example 11. Chemical Purity Determinatieh by t-EPLC Purity analysis in Example 2 and Example 4 was performed on an Agilent HPl 100 series system equipped with a diode array detecter and using Chem Statieri software VB0403 using the methed shewn in Table 17.

Table 17. HPLC method fer chemical parity inations Parameter Vahte Type of method Reverse phase with gradient elation Sample Preparation 0.5 mg/ml in aeetenitrile : water l:l Celumh Supelce Ascentis Express (318, lOO x 4.6 mm, 2.7 [am umn ature ("(1) 25 Injectien ( m1) 5 Wavelength, Bandwidth (hm) 255, 90 Flow Rate (ml/min) 2 Phase A 0. l% TFA in water Phase B 0.085% TFA in acetonitrile Time (min) % Phase A % Phase B __________________________________________________________________________ 0 9g Timetable Example 12. XwRay Powder Diffraction (X31313) Techniques The XRPD patterns in Eiixamples ’2', 3,, 4, 5, 6,, 7, 8, and 9 were ted on a PANalytieal Empyrean dit‘fraetonieter using Cu Kg radiation (45 kV, 40 111A) in transmission geometry. A 0.5'3 slit, 4 mm mask and 0.4 rad Seller slits with a focusing mirror were used en the incident beam. A el3D detector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Seller slits. The instrument is pet‘fermahce d using silicon pewder on a weekly basis. The sef‘tware used fer data celleetieh was X’Pert Data Celleeter V. 5.3 and the data were analyzed and, presented using Ditt‘rae Pius EVA v. H.000 or High score Plus v. 4.5. Samples were prepared and analyzed in either a metal or Millipere 96 well—plate in transmission made. X—ray transparent film was used between the metal sheets on the metal late and pewders (approximately 1—2. mg) were used as received. The Millipere plate was used to isolate and analyze sclids from suspensions by adding a small amount of suspension directly to the plate before filtration under a light vacuum.

The scan mode fer the metal plate used the gonio scan axis. whereas a 29 scan was utilized for the Millipore plate. A performance check was d out using silicon powder (metal well— U1 plate), The details of the data collection were an angular range of 2.5 to 32.0" 28, a step size of 0.01303 .49, and a total collection time of 2.07 s.

Samples were also ted on a Bruker D8 ditl‘ractoineter using Cu Kill radiation (40 kV, 40 nut), 63 - 28 gonionieter, and divergence of V4 and receiving slits, a Ge monoclironiator and a larynxeye detector. The instrument is performance d using a certified Corundurn rd (NlS’l‘ 1976). The software used for data collection was DiffracPlus XRD Commander v2.6.1 and the data were analyzed and presented using Diffrac Pius EVA V15‘0.0.0.

Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was gently packed into a cavity cut into polished, zero—background (510) silicon wafer. The sample was rotated in its own plane during is. The details of the data collection were an angular range of 2 to 42° 26, a step size cf {11.05" 29, and collection time of 0.5 s/step Examnle 13., Synthesis of Amorphous Cempeund 2 ,ca-in "CH3 HN " in n.‘ \ "Kr/N. < N l CHt o a! 9H3 (3 a /.

O N" 0'5H2504 NAN/«NH: Ht’r‘flnwaomcu. r N’A‘NH— ’- —* H—C o ii "r’ r lac/"(fies ° "W904 cat a i O as" "s CH3 0 —=~ Ho" r \ / \ / Compeund 1 Compound 2 A 250 ml; flask was charged with MeOH (l 5 'l niL) and the solution was coeled tn 0-5 0C.

A concentrated on of H2304 was added dropwise over 10 minutes. A separate flask, was charged with Compound 1 (151 g) and e (910 mic), and the HzSQ34/l‘vlefjifi solution was added dropwise at 25-30 0C over 2.5 hours. A large amount of solid was precipitated After the solution was stirred for 12—15 hours at 25-30 "C, the mixture was filtered, washed with MeOH/acetone (25 mL/150 mL), and dried at 55—60 CC in vacuum to afford Compound 2 (121 g, 74%).

Analytic Method fer Compeund 2: The purity of Compowtid 2 was obtained. using an Agilent llfiO HPLC system with a Waters X'I'erra Phenyi Slim 4.6*250mm column with the following ions: l mL/min flow rate, read at 254 nm, 30 0C column temperature, 10 til; injection volume, and a 30 minute run time. The sample was dissolved in ACNzwa‘ter (90K), V/V). The Gradient method for separation is shown below. Rfiniin) of Compound 2 was approximately 12.0 minutes. ___:tia2____:<___r:i£22.......l..9.;.tfcéaflaffiiéafiliaa_£::§2f’:§.W..:t:§§§2§ialli£l§_2?f2-__- o 90 i0 a/iia; (400 Mia-ix, DMSO-dg): a 8.41 (hi; 1H), 797 (s, 1H), 7.36 (t, J==== 8.0 Hz, 2H), 7.22 (d, J: 3.0 Hz, 2H ), ’7.l7 (i, J: 8.0 Hz, iii), 6.73 (s, 2H), 6.07 (d, J: 3.0 Hz, 1H), 6.00 (dd, J = 120, 8,0 Hz, 1H), 5.81631; 1H), .73 (m, 1H), .28 (m, 3H), 4. 10 (ft. J 8.0 Hz, 2H), 3.35—3.74 (m, lH), 2.95 (s, 3H), 1.21 (3, J: 4.0 Hz, 3H). "5-1.10 (m, 9H).

Example 14. terization of Compound 2 Compound 2 was further characterized by eye, ‘HNMR, "CNMR, "FNMR, MS, HPLC, and XRPD (FIG. lSB). Residual solvent was measured by GC. Water content. was measured by Karl Fischer Titration, and the water content was only 0.70%. Data is sworn arized in Table 18.

Table 18‘ Summary of Additional Characterization Data ef Compound 2 Result Appearance White Solid _______________________________________:______....._____________.....+____________....______________....._____________....._____________....._____________.....______________....._____________....._____________....._____________.....__________ lHT‘Jl‘i/ER. peaks are listed in Example 4 MSGiSl-l-ve) [M-t-E-{l'i' 582.3 conforms to structure l-lPl_.C 99. 8% by AUC at 254 nm (average of two preparations) iteeduaisiiltembyot Methanol57Elfin" Acetone — 752 ppm Diohloromethane 50 ppm Ethyl Acetate — l76 ppm .............................................................................................................................................................................................................................................

Water Content Example 15. Solubility of Compound 1 and Compound 2 Compound I and Compound 2 were both tested for solubility in biorelevant test medias, including simulated gastric- i‘luid (SGF), fastedvstate simulated gastric fluid (FaSSlF), and fed—state gastric fluid {FeSSlF} Results for Compound 1 are shown in ’I'ahle l 9 and results for Compound 2 are shown in Table 20, Samples were d at room, temperature (20 — 25 CC). Compound 2 was more than d more soluble than nd 1 in water at 2 hours and more than 25ufold more soluble at 24 hours. In 8GP conditions, Compound 2 had a solubility of 842 mg/niL at 24 hours compared to the solubility of l5.6 lug/ml. of Compound 1 at the same time point: Compound 2 was also more soluble at 2 hours in the SGF conditions than Compound 1; and soluble enough to allow for testing even after 48 hours while testing at 48 hours was not done with Compound 1.

Table 19. Compound l solubility testing s Test Media Solubility (in tug/ml.) ance Descriptive """""""""""""""""""""""""""""""Eéiiiiiiiilimm mm Cleai Solution Spaiinvly with gum at the Solubl e bottom FaSSlF Tuihid Slightly Soluble li‘eSSlF *Sample ed to be clear yet a solubility of only l5 mg/mL was achieved Upon furtherinvestigation it \T.as noted, that a gummy film formed on the stir bar. The compound l active pharmaceutical ingredient fomied a gummy hall in diluent (90% water/1094) acetonitrile) during standard preparation winch ed a long senication time to dissolve completely.

Tahie 29. Command 2 sainhiiity tearing reanits Test Media Sninhiiity {in mg/mL salt base) Appearanee ptive """"" """ mm lhnmslefihnnredghnnr‘; Water 65.3 68.0 N/A Soluble 8GP 89.0 34 2 81 3 Turhid Soluble """"" """""""" ""'2""(3""""""""+""""""N17K"""""""""""""""""""""""""""""""""""""effing}"""""" FaSSIF 19 Soluble FeSSIF 3.3 3.4 N/A Turbid, Slightly : : : Soluble e 16. Chemicai Stability 0f Cmnpennd 2 Compennd 2 was tested for chen‘aicai stability at 25 and 40 0C over a 6 month time period by inenitnring erganie purity, water content, 1kWh/1R, DSC, and Ranien IR. The container closure system. for the study was a combination m edieinai valve bag with a phannaeeuticai laminated film over the pouch and desiccant silica gel between the two layers. und 2 (1 g) was measured into each eentainer. Bags were then stored at 25°C./60%RH (reiative humidity) and 40"C/75%RH (reiative humidity). Organic purity, water content, 1HNMR, DSC and Raman were measured at Time 0, Month 1, Month 2, Month 3 and Month 6.

The purity of Compound 2 was obtained using a Shimadzu LC—ZOAD System with a Waters XTerra Phenyi, 5 nm, 4.6x250n1m celumn with the feiiowing cenditiens: 1 mL/min flow rate, read, at 254 nm, 35 "(2 column temperature, and 10 giL injection volume. The Sampie was ved in aeetoniti‘iie water (90: 10) (:V/V). The gradient methed is shown below.

Innetmmir‘rnf‘nIii/(water) 0 90 I O The water content of Compound 2 (250 mg) was ined by a water titration apparatus using the Kari Fischer titration method.

Resuite are shown in Table 21 and Tahie 22. When Compound 2 was stored for 6 months at 25 and 4-0 0C, the rate of degradation was minimal. At 3 months, Compound, 2 was 99.75% U1 percent pure at the 25 "C conditions and 99.58% pure at the 40 "C conditions. At 6 months, Compound 2 was stiii 99.74% pure at the 25 0C conditions and 99.30% pure at the 40 DC conditions. At '25 °C. the percent of degradation. product increased from 0.03% at Day 0 to 0.08% after 6 months. At 40 "C, the percent of degradation product increased from 0.03% to 039%. Over the course ot‘o months, the percent of water sed approximately 0.6% at 25 "C and increased approximately 0.7% at 40 "’C.

Characterization by EHNMR, Ranian, and DSC of Compound 2 at i, 2, 3, and 6 months was the same as the characterization of (filompound 2 on day 0 at both temperature conditions {Table 22), highlighting the iong—term stabiiity of Compound 2.

Tabie 21. Compound 2 rate of ation over ti months at 25 and 40 0C Pereent of Maximum Percent Percent ation 1mpurity ‘Water Purity Product Percent 49 0C .........................................................................................................

Month 1 E ‘ Menth 3 1.9 399.58 0.20 Menth 6 1.9 ‘ Tabie 22. Characterization of Compound 2 during degradation study Time, iE-ENMR Ragnar: Tested Day 0 initial Test Initial Test initial Test with I Ehe same as 118 same as Day 0 QC Month 2 {he same aeBay 0 line same as Day 0 {he same as Dag/O """"% """ MonthThcsmnedsDayTheameasDayO hesamaDm ‘ "‘ffiésameasnavn "'3‘E€"§§%§i'é"§§'3i55§"iim mi:i'i'éggfii'éfl'é;5575""- Menthé 4‘9 0C Month 2 The same as Day 0 The same as Day 0 The same as Day 0 Month '3 The same as Day 0 The same as Day 0 The same as Day 0 Menth 6 The same as Day 0 The same as Day 0 The same as Day 0 Additiena} al stebiiity studies of Cempound 2 were measured to determine the impurity and water levels Three cenditiens were tested: aeeeierated ity (40 i 20C 75. j: % RH) over a 6~month time periodfl ambient stability ('25 i- 2°C / 60 j; 5%Ri-1) over a 9-month period? and stabiiiiy under refrigerator conditie-ns (5 i 3°C) over a 9~rnenth nme permd The results for accelerated stability, ambient stability, and refrigerator conditions are shown in Table 23, Table 24, and Table 25: respectively. Based on the results of these studies, Compound 2 is very chemically stable. lo the accelerated stability study (’l‘able 23‘), at each tirne point (l 5‘ month, 3" month, and 6m month) where Compound 2 was measured, the appearance of Compound 2 was always a white solid and the lR matched the reference rd. After six months, the total related nce 1 impurities was only 008% and there was no detection of related substance 2 and isomers.

Table 23. Accelerated ity (4% j; 2°C / ‘75 1; Ell) «of Compound 2 ’l‘estino time s items Specification a 3""munth 6*" month White or off' White White White White A carancc‘ ' pp White solid solid solid solid solid correspond correspond correspond correspond with I ' with with with lR reference reference reference reference standard . standard ndsmddrd "dag/10% 45% 0.36% 0.41% lrn uritV A ' 530.15% impurity B <0. 1504') Impurity F ‘01" m< gox Related _ ,, Substance lmpunty H \0. 1 5 _. Any‘other" l ’0100/ srnnlc inrourrt' a 'lbtal . 0 (1° . . l "2/00’ 0.02% 0.02% 0.03% lnt unties Related Substance ty G <0 15% lntpuntjz C :0. l5/ _______________________________+ lscmer .lrn )uritv D SO 150/6: NDV lrn uri ‘E 50.15% -- ~ - a .

Assav 93n%~i02.0°/t 980 , 996% 99.5% TAMC a SlOOchu/ My?3:12":hi 1 Mold and Yeast filtlllct‘u/O ' ' ' e EColi Not Detected ND: Not Detected In the ambient stability study where the appearance, lR, water and ty levels were ed for nine months, the appearance of Compound 2 was always a white solid and the 1R always corresponded with the reference Sample. The results (Table 24) highlight new chemical l y stable Cnrnpound 2 is. After 9 months, the percentage of water in the sample was only 0.20% U1 and the total related substance 1 impurities was enly 0.02%. Similarly to the accelerated stability studies, related substance 2 and any isomers ef Centipennd 2 were not detected.

Table 24. Ambient stability (‘25 i 2°C / 66 j; 5% RH) 0f Campound ‘2 ---------------------------------------------------------------------------------------------1—---------------------------------------------------------------------------------------------------------------------------------------------------- I Testin- time eint 1'th rcatmn 1 ll month 3rd mnnth 631‘ innnth 9th nienth E ; mnnth.

White or of?~ White White White .7 . ite Appearance : = white solid. . sehd. . White solid .

E solid solid. solid cerrespend cenespend correspond pond 3 correspond a _. ~. g g With. w1th With. ._ .- , w ith iii a With nce_ g ,r t t g . g reference reterenee reference reference standard E ' ' standard standard standard standard Water 52.0% 0.45% 0.46% impurity i I, :‘o/ i ND. ND MO. 1-, UN?)4‘ Q 0/ (3 "Jill /o0/ ',T Related F ,. 0 .

S ' :~ ~ r i .r t . . , , Any other single f 001% 030% 530.10% 0.03% 0.02% impurity : Will. 001% 530.2% 0.02% ___________________________impunitet.

Related Substance pg:ritvJ 5301594) ' Ni). V '. t I . ND. 2 E Imp?" germ ND ________+_______________________________________________________________________________________________________________________________________________ isomer ""13"" l 50.15% ND. 1mg?" S0.l5% ND, 93013902".. o/N — i As. E 98.8% TAMC :{lt‘iflflcfw'rr The water cement after 9 menths was 0.32% and the total impurities of d sub stance l were enly 0.01% Ofthe sampie. Compound 2 is very chemically stable under refrigerator eerrditions.

Tabie 25. Stability under refrigerator conditions (5 j: 3°C) of Compound 2 Item 3pccificafim; Omenth-.

Fesungflmeflmm * menth a '3" menth th m9th menth White ereffu White , White Wlme Off—white A"39‘3""H white solid solid. solid solid solid ecrrespend Ecorrespond cerrespcncl correspond cerres endp With. wrth. wrth. ._ t , With ER wrth reference. .‘ / 1 eterence. _ reiererrce7» reference. . .reference. standard standard standard standard standard Water ’ 0,32% 042% 032% Impurity Related Any other 0', : single __f ' ’ /° r 0 010/ im uritv ' 0.01% Related Substance 980%~E 020 988% 101 1% 1002/ 986% 101.4149 TAMC <1000ciu/g__— ial Vicki and Testing Yeast ND; Net Detected Emrriple 17‘ Plasma levels (if lites l‘ellewing single eral deses 0f Cempound 2 A single oral dose of Compound 2 was administered to rats, dogs, and monkeys, and the plasma. levels of certain rrietabolites shown in. Scheme .1 were ed.

The conversion of Compound 2 to nd 1 and metabolite 1—7 are shown in Table 26 and the results for metabolite 1-8 and metabolite l«2 are shown in. Table 27. In rats, low levels of Cempeurid 1 exposure were Observed, but high levels of metabolite 1-7, the nucleoside metabolite of the active triphosphate olite 1—6)? were observed. in monkeys, roughly dose-proportional exposures of Compound 1 were measured. in dogs, suprepropertional Compound l exposures, indicative of first—pass metabolic clearance in the liver, were ed. Throughout the study, significantiy more vomiting in dogs (5/5 in high dose group) than in monkeys (US in high dose greup) was observed.

Table 26. Plasma levels (if Compemrd l anal lite 1—7 after single oral doses el’ Communal 2 {Iiempeiirlrl l Metabelite l—7 Dose* Species (mg/kg) AElCO-last (hr’Frig/ml") ltlorrkeyb 3 males per dose per species; *dose formulations: "‘05% ("ix/1010.5" Tween 80 in water; Bpowder in capsules Tebie 2’7. i’iasma ievets nt" metabotites "Ht and tui’; after singie oral dose of Compound 2 Dose" Speble‘s. 1 ) ,ALICOnlast I AVUCO-iast (hr* n) ' ’ ' ‘ ; (hr* ng/mL) Reta 506‘; Dog" 100 5690 Monkeyh 100 "2:3 00'" 3 males pet dose pet species; *dose fomitiiations: 305 CMC 0504Tween 80in water;bpowderin capsules Examine 18. Tissne Exposure of Active Triphosphaie foiiowing Comnound 2 Orai Dose Heart and liver tissue ieveis of the active triphosphate (T?) of Compound 2 (metabolite U": tun) were measured 4 hours after otai doses of Compound 2. Sampies of iiver and heart were obtained 21:4 hours after a single dose ot‘Componnd 2, flashmfrozen, nized and analyzed by {ALMS/MS for intraceiiniar ieveis ot‘ the active TP. Tissue levels were measured in rats, dogs; and monkeys as shown in FIG. toAi High levels of the aetive 1"? were measured in the iiver of all species tested. Reiativeiy tow ieveis ot‘ the active TP were measured in the hearts of dogs due to saturation of first—pass hepatic metabolism, and unqnantifiahle levels of '1‘? were measured in rat and monkey hearts, indicative of fiver—specific formation ot‘the active TF3 While not shown, compared to Compound 1 dosing, nd 2 dosing ed TP distribution.

Exampte 19., Pharmacological Comparison of Commund 1 and Compound 2 in Dogs A head—to—head ison of dogs dosed with Compound 1 and Compound 2 was conducted. The study measured piasm a Eeveis ot‘Componnd l and metabolite 1:; (from Scheme 1) out to 4 hours after dosing with Compound 1 (25 mgx’kg) and Compound 2 (:30 mgfkg) (Table 28), and the AUCmahr) of metabolite 1—7 was twice as great with Compound 2 compared to Compound 'E. Dose—normaiized exposures to Compound 1 and metaboiite L7 are shown in Tabie 28. Values for AUCtounm for Compound 1, metabolite 1—7, and the sum of Compound 1 U1 lite 1-3.7 were greater after dosing with Compound 2.

Tabie 23. ison of Plasma Levels foliowing dosing wfith (ionipound i and Compound 2 {'"m"''''"""""""''''"""""""""""""""?""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" Dosed Compound Mean Bose—normalized AUanaana (u'Mi*tnr} for: Compound 1 Metabolite 1—37 Compound 1 + Metabolite 1»? Compound} (25 02 2'} Ina/kg) Compound 2 (30 mgfkg) aAUC(o.4m;.VahI€S nonnahzed to a dose of 25 mglkg heart ratio triphosphate concentrations indicate that dosing with Compound 2, as compared. to Compound 1.. increases the selective delivety of the triphosphate to the liver, as shown in Tahie 29. The AUCm—ofi) of the active guanine metabolite {1—6) after stration of Compound 1 measured in the heart was 174 , while the AUCmam) of the active guanine hte (1—6) after administration of Compound 2 measured in the heart was 23 gen/WM. The liver/heart ratio for Compound 2 was 20 compared to a fiver/heart ratio of 3.1 for Compound 1.

Table 29. Comparison of Liver and l-leart Exposure following dosing with Compound 1 and Compound 2 ,................................................................................................................................................................................................................................................

Dos-ed Compound Mean Dosemnormalized AUCgsama (pit/Fin) for: Compound 2 565 28b 20 ............................................................................................................................................................................................................................................... aAetive TP concentrations (3—6; Scheme 1) normalized to a dose 01°25 reg/kg lExtrapolated below the lower limit ofquantitation of the calibration curve The effect of increased selectivity for the liver over the heart when Compound 2 was administered compared to Compound l is also shown in FIG. l 63 The heart and liver tissue levels of the active triphosphate following a dosage of Compound 2 ('30 mg/kg) were ed, to the tissue levels oi." the active triphosphate following a dosage of nd 1 (25 ring/kg).

The concentration of the active TP was higher in the liver than the heart for both Compound 1 and Compound 2, but the active '1‘? was more selective for the liver over the heart when nd 2 was closed compared to Compound 1. e 23. Plasma Profiles of Compound 2 Metabolites in Rats and Monkeys Male Spragne-Dawley rats and eynomolgus monkeys (’3 s per dose group) were given single oral doses of Compound 2. Aliquots of plasma prepared from blood samples treated with Diehlorvos were analyzed by LCmMS/TvlS for concentrations of Compound l and metabolite 1—7 (the nucleoside metabolite of the active triphosphate of Compound 2 shown in Scheme l), and pharmacokinetio parameters were determined using WinNonlin. The results for a single 500 mg/kg dose in rats is shown in and the results for a single 30, lOO, or 300 mgslrg dose in monkeys is shown in , The results are also summarized in Table 30.

High plasma levels of metabolite 1—7, the nneleoside metabolite of the active sphate (TP) of Compound 2, are indicative of formation of high levels of the TR even in rats where very low plasma levels nt no eleotide prodrug are ob served due to the short l'taltllit"e ofCompound l in rat blood (<2 min). Persistent plasma levels of metabolite in"? reflect the long ife of the in monkeys? plasma exposures (AUC) of Compound 1 were roughly dose—proportional, While metabolite l~7 exposures were somewhat less than proportional, although AUC values U1 for both parent drug and the nucleoside metabolite of the active ’1? continue to se up to the highest dose tested (300 trig/kg).

Oral administration of Compound. 2 in rats and monkeys produced high and dose~ dependent plasma exposures to lite 1—7 (the nucleoslde metabolite ofthe intracellular active triphosphate of Compound 2); metabolite 137 exposures continued to inerease up to the l'rlghest dose tested, reflecting ntial formation of the active '1‘? in these speeles.

Table 39. Plasma levels of Compounds l and in? after single oral dose of Compound 2 Compound 1 AUCu—aast (hrrug/mil) Metabolite ln7 Cum {ng/mL) — (hrllng/mlg) dose torrnul ations: a03/o CMC, 0.5% Tween 89 in water; '9 owdcr inE) ca )sulcsE Example 21. The Effect of the Active hesphate ei‘ Cemennnd l and Cnmpennd 2 en nndrial Integrity The relative efficiency of incorporation et‘the active triphesphate (Tl?) efCempeund 1 and, Compound 2, metabeiite 1-6 (Scheme '1), by human mitochondrial RNA polymerase was compared to the relative efficiency of the active TP of sofoebuvir and the active T? of INK—189.

Cempeund l and (Tenipeiind 2 are not likely to affect mitochondrial integrity since their active triphesphate is poorly ineomerated by human mitochondrial RNA polymerase with an ncy r to that of the sphate of sofesliuvir; the relative efficiency of incorperation 0f the triphesphate of lNXmlt‘éQ was up to 55—fnld greater. Results are shewn in Table 31. The incorporatien of these analogs by human mitochondrial RNA~dependent RNA polymerase (POLRM'I') were determined aceerding to Arneld et al. tivity 0f M’iteehendriai Tran seriptien and Resistance ef'RNA rase ii Dependent Nuclear Transcription to Antiviral Ribonucleetides PLOS Paiheg. el 003 ()3 O). , 2012, 8, ’l‘aliie 31. Kinetic: Parameters for Nucleetide Analegs Evaluated with Human Miteehondrial RNA Pelymernse Nucleotide Analog Kmfis") Kemp (31M) Reiative Efficiency" 2’~deexy~2’~F~2’~ 0.00034: 590 n -. . -250 l0 Kl0"6 yiUTP eeeees (active TP nf sefesbuvir) 2’—C—methylGTP 0,051 $0.00 (active "E? or m- Active TP ei' earn 7 -_:; Cornpeund l and 0.0092 Campeund 2 (metabolite 1—6) *Relative effi ClSl’lcy : (Knol/Kd,app>analog nucleotide/ 'Kd,app)nntural tide Example 22. Activity of Compound 1 against liepiicons Containing the NSSB Sequence A panel. ot‘replicons containing the NSSB sequences from various HCV genotypes derived from 6 laboratory reference strains (Gila, lh, 2a, 3a, 4a and 5a) (Fifi. l9) and from 8 HCV patient plasma samples (GT1 a, ih, 2a, 2b, 3a—l, 3a~2, 4a and 4d) (FIG 20) were used to determine the potency of Compound l and sofoshuvir.

Compound 1 was more potent than sofosbuvir t clinical and laboratory strains of iZ-lCV. Compound 1 showed potent pan—genotypic antiviral activity in vitro t wild—type clinical isolates with ECss < 80 nM, which is r—‘im to lr-imfold more potent than sofosbuvir. As shown in HQ 20, iiCss values for Compound .‘l were 7-33 times lower than sofoshuvir against clinical isolates of all HC‘V genotypes tested. ECso values for Compound 1 were 6~ll times lower than uvir against laboratory strains ot‘iiC‘V Genotypes l-S (FIG 19).

Example 23. Single ing Bose (SAD) Study of Compound 2 in Healthy Volunteers (Fart A) and GTLHCV infected ?atients (Part E} nd 2 was tested in a single ascending dose (SAD) study to measure its safety, tolerahility) and pharmacokinetic in y subjects (Part A). Part A was a randomized double blind, placebo—controlled SAD study. Healthy subjects in Part A, received a single dose of Compound 2 or placebo in the fasting state. Subjects were confined to the clinic from Day —l to Day 6.

Dosing in each cohort was red such that 2 subjects (1 activezl placebo) were evaluated for 48 hours after dosing before the remainder of the cohort was dosed. Each cohort received Compound 2 in ascending order. Dosing of sequential cohorts occurred based on review of available safety data (through Day 5) and plasma pharmacokinetic data (through 2-4 h) of the prior cohort.

Dose escalation proceeded ing satisfactory review of these data. As pharmacokinetic and safety data emerged from prior cohorts? doses evaluated in, Cohorts 3a~4a were adjusted by increments no more than 100 mg The total m dose evaluated in Part A did not exceed 800 mg. The dosing regimen for Part A is shown in Table 32. ’l‘abie 32. Dosing Regimen fer Cempnund 2 stratieti Part A at" Study Cehnrt ‘ -' ,: Cnmseuutiz Cemented l)* Health" : 50 (45‘) me x 1 day —liealthv ':1 160 (90' me x 1 day : 200 (180‘ ms x1 dav Healthv 2__ "499Q60)trig"X_ _l_ "(lag/___ *Cllmcal doses a1e expressed"111 terms bf Compound2"withthe apprexirnate Compound l base equivalentin parenthesis Healthy veluhteers in the Part A pertien 0f the study were male and female subjects between. the ages of 18 and 651 Active and placebo ents were pooled within each. Part A cehert to preserve the study blind.

Compound 2 was alse tested in a single ascending dose (SAD) study to measure its , tolerabiilty, pharmaeeklnetie, and antiviral activity in (:i’l‘l—l-lifii‘v’ infected patients (Part B). ts 111 Part B received a single dose ofCompound 2 in the fasting state. ts were confined, t0 the clinic from Day —1 to Day 6.

Part B was initiated after the safety (through Day 5) and plasma pharmaeekinetie (through 24 h) data review from Cohort 3a in Part A. Available safety data (through Day 5) and pharmacekinetic data (through 24 h) was reviewed fer the first cehert in Part B (Cehort lb) before enrolling subsequent Part B eelmrts, Subsequent Part B coherts were only clesetl fellewing review of available safety and pharmacokinetic data from the respective doses in Part A as well as available safety (through Day 5) from the prior Part B eoherts.

Dose escalatien up to 60% mg in E-l'CV-infected ts preceedecl ing satisfactory review of these data. The dosing regimen for Part B is shewn in Table ’33. 'l‘abie 33. Basing Regimen for Comeound 2 in Fart B of Study Cehert Peiulatieri N (active) Cam-cued 2 Cen'ieund l) G'l‘l 'liiltii‘il’élnfected G'l‘l l-iCVinfected .

GIHQI’infected 4bf!1H<‘\1ntected _____________ *Clinical doses are expressed,111 terms of Cempeund, 2, with the approxiritate eund l base equivalent in hesis.

Patients infected with HCV were treatment—naive, nen—eirrhetie Gill—infected subjects with a viral lead of}: 5 legio lU/mL.

Ne serious adverse events were reenrded and no ure tinuatinns were ed in, either Part A or Part B All adverse ett‘eets were mild to moderate in, intensity and n0 dose— related patterns, including laboratory parameters, vital signs, and ECGS were evident.

Example 24. Results at" the Single Ascending Dnse (SAD) Study at" {Iiempmrnri 2 Pharmaeekinetie of Compound 1 and nucleesitle metabelite 1-7 were measured fellowing the single dose of nd 2, The C24 trough plasma ntratiorts (C243) of metabolite i~7 in HCVuinfeetetl patients fellewing a 600 mg dose of Compennd 2 was 25.8 ng/mL, which is more than denble the plasma concentration rinse following a 300 mg close of Compound 24 Metabelite in? (shown in Scheme 1) can only be generated via dephespherylatien 0f the intracellular phesphate metabolite l4, metabolite 1-5, and metabolite 1-6, which is the active species. 'l‘herefere, metabolite 1—7 can be considered a surrogate of the active species, The pharmaeokinetie data far all ceherts is shnwn in Table 34 and, Table 35. Values are reperted as mean i SD, except tor Tim where median (range) is reported. Pharrnaeokinetic parameters were comparable in, healthy and feeted patients.

Table 34., Human i’harmaeekinetie ni‘ Cempnnnd l and Metal‘inlite 1—7 after Administration of a single dose of nd 2 in Healthy Voinnteers — 167::HO 656 it 255 4.4:) 1: lia/ Metabnlite 13.n- 509 l en 24—hr profile.

Table 35‘ Human l’liarniaeakinetie at" Campeund 1 and Metabelite 1+7 after Administration of Compound 2 in V Infected Fatients 5l54 60(40—60) 538: l"3* 8.4: 4.3 : Z7" Metabelite E60:’36.7 4.0(4.~.O40) 2l32:l20 116:12l 22.5:329l .........................................................................................................................................................................................................1 2i76+ ii6 —— 25.83;. 4.08 *Based en 24—hr' profile.

The mean plasma concentration-time profiles atCompound 1. and metabolite 1—7 were alse calculated for all eoherts 6f Part A and Part B of the study. is the mean plasma" concentration of Compeuml l ing a. single dose of Compound 2 and is the mean plasmamconcentration 6f metabelite i=7 fellewing, a single close 0f nd 2. AS shown in , Cempound l was quickly abserbed and rapidly/‘extensively i'uetaholized in all cohorts from Part B. As shown in , metabolite 1-7 was a majer metabelite and exhibited sustained plasma eeneentratiens. Plasma expesure of Cempeund l was lated while exposure of metabolite 1—7 was dose-proportional.

Fer the HCV—infected subj ects of Part B, measurements of HCV RNA tatien were performed e, during. and after administratien of Compound 2. Plasma HCV RNA determinations were performed through the use of a validated commercial assay. Baseline was defined as the iriean of Day ~l and Day 1 (pie—(lose). A single 300 mg dose of Compound 2 (equivalent to 270 mg of Compound 1:) resulted in significant antiviral activity in G’lTlh—HCV infected subjects. The mean maximum HCV RNA reduction 24 hours postudose following a single 300 mg dose was l.7 logic lU/mL and this compares to a ~12 loglO Ill/ml, reduction alter l day of 400 mg of sofoshuvir monotherapy in G'l'la l3lCV~infected subjects The mean maximum l-lCV RNA reduction 24 hours post—dose following a single lOO mg dose was 0.8 log in lU/mL The mean U1 maximum l-lCV RNA reduction was 2.2 logic lU/mla following a, single 400 mg dose Individual pharmacokinetic/pharmacodynamic analyses for the individual subjects from Part B of the study are shown in FIGS. 23A~23F Metabolite 1*? concentration. is plotted against HCV RNA. reduction, concentration, and as shown in FIGS. 23Au23F, plasma HCV RNA reduction correlates with plasma metabolite 137 exposure. Viral response is sustained with metabolite 1-7 plasma concentrations that are greater than the ECss value against (ill b. The correlation between plasma concentration and HCV RNA reduction levels indicates that a more profound response will be achievable with higher doses of Compound 2.

Example 25. Predicted Steady—State Trough s of lite lw’?’ exceed Compound Tl, EC95 Values against Clinical isolates of HCV G'l‘ 1-4 As shown in Flt}. 24,, the steady—state trough plasma levels (Cb/Les) of metabolite l~7 following Compound 2 dosing in humans (600 mg QD (559 mg free base lent) and 450 mg QD (400 mg free base equivalent» was ted and compared to the Files of Compound 1 m vz’tm across all tested clinical isolates to determine if the steady state plasma concentration is consistently higher than the ECas, which would result in high efficacy against any or all tested clinical isolates in vivo. The EC95 for Compound 'l is the same as the Elias of nd 2‘ For Compound 2 to be effective, the steady—state trough plasma level of metabolite 1-7 should exceed the EC 95.

As shown in FIG, 24, the ECas of Compound 2 against all tested clinical es ranged from approximately l8 to 24 anl As shown in FlG 24, nd 2 at a dose of 450 mg QB (400 mg free base equivalent) in humans of provides a ted steady state trough plasma concentration (C24,ss) of approximately 40 ng/mL. Compound 2 at a dose of 600 mg Q1) (550 mg free base equivalent) in humans of provides a ted steady state trough plasma concentration (C2455) of approximately Sling/mil.

Therefore, the predicted steady state plasma concentration of surrogate metabolite 1—7 is almost. double the ECss against all tested clinical isolates (even the hard to treat GT3a), which indicates superior performance, In contrast, the EC95 of the standard of care nucleotide sot‘osbnvir ranges from 50 to 265 U1 nlvl across all tested lilCV clinical isolates, with an 131C195 less than the predicted steady state concentration at the commercial dosage of 400 mg for only two es, GTZa and GTZh. The EC95 for the commercial dosage 01’4th ing of sofoshuvir is greater than the predicted steady state concentration for other clinical isolates, G’l‘la, G'l‘lh, GT3 a, G’l‘daa and G’l‘éld.

The Compound 2 450 mg steady state trough plasma concentration (C2455) was predicted using the 300 mg steady state trough plasma concentration (6324,55). The mean steady state trough plasma concentration (6324,55) at 300 mg was 26.4 ng/mL, and therefore the calculation was 26.4*450/’300==39.6 ng/mL.

The 6th mg steady state trough plasma tration ) was predicted using three approaches: 1) the (300 mg Day l (324 mean was 25.8 ngfmls and a 60% increase was assumed for reaching steady state. Therefore the calculation was 25.8*l.6=4l.3 ng/mL; 2) the 400 mg day l C24. mean was 22.5 ng/inL and a 60% increase was assumed for reaching steady state. Taking dose proportional PK. into t, the ation was 22.5*l.6*600/’4DO=54 ngz’mL; and 3) the 300 mg steady state trough plasma trati on (€24,85) was 26.4 rig/ml, and a proportional PK was assumed. Therefore the calculation was 26.4*2=52.8 ng/mL The 600 mg steady state trough plasma concentration (C2455) is the e of the 3 data points ( (4i .3+5£l-+52.8)/3 =49}. ng/mL).

There is generally about a 60% increase in C24 at steady state compared to (324 following a single dose.

The data comparing the et‘li cacy and pharmacoldnetic steady state parameters in FIG; 24 y demonstrates the unexpected therapeutic importance of Compound 2 for the treatment of hepatitis C. In fact, tl’ie predicted steady-state plasma level after administration of Compound 2 is ted to he at least 2—fold higher than the EC95 for all genotypes tested, and is 3— to 5—fold more potent against GT2. This data indicates that Compound 2 has potent pan—genotypic antiviral activity in humans As shown in FIG; 24, the M395 of soi‘oshuvir at GT1, GT3, and (Zl’l‘4 is greater than 190 ngr’mL Thus singly, Compound 2 is active against HCV at a dosage form that delivers a lower —state trough concentration (40—50 s) than the steady—state tough concentration (approximately lOO ng/mL) achieved by a similar dosage form of sot‘osbiwir.

Example 26? a’tien Bescriptlon and Manufacturing el" Cetnpound 2 A representative non—limiting batch fonnula for Compound 2 tablets (50 mg and HM") mg) is presented in Table 36, The tablets were produced from a common blend using a direct compression process as shown in . The active pharmaceutical ingredient (API) is adjusted U1 based on the as—ls assay, with the ment made in the percentage ot‘mlcrocrystalline cellulose.

The API and exeiplents crystalline ose, lactose monohydrate, and eroscarmellose sodium) were screened, placed into a V~blender (PK Blendmaster, 0.5L bowl) and mixed for 5 s at 25 rpm. Magnesium Stearate was then screened, added and the blend was mixed for an additional 2 minutes. The cemrnon blend was divided for use in producing 50 mg and 100 mg tablets. The lubricated blend was then compressed at a speed of l0 tablets/minutes using a single punch research tablet press (Korsch XPl) and a gravity powder . The 50 tablets were produced using round standard concave 6 mm tooling and 35 kN forces, The 100 mg tablets were produced using 8 mm round standard concave g and 3.9—4.2 kN forces.

Table 36. Formulation at" 5% mg and 160 mg Compound 2 ’l‘alrlets RawMaternl "aw/ag/aarchatgnmt 5t} mg Tablet 160 mg Tablet Mlcrocrystal line ose, USP/"NF, E? LactOScMonohxdiatc ' USP/NF, up, JEEP, JP Croscartnellose Sodium, 50 lat) 5.0 Magnesium Stearate, 1.0 3.6 ll) USP/NF, BF, El.) JP Total 100.0 200.0 Compound 2 was adjusted based on the as—ls assay, with the adjustment made in the percentage of microcr‘ystalline cellulose. nd 2 and excipients (microerystalllne cellulose, lactose monchydrate, and croscarrnellose sodium) were screened, placed into a V-blender (PK Blendrnaster, 0.5L bowl) and mixed for 5 minutes at 25 rpm. Magnesium stearate was then screened, added and the blend was mixed for an additional 2 minutes. The common blend was divided for use in producing 50 mg and 100 mg tablets The. lubricated blend was then compressed at a speed of l0 tablets/minutes using a single punch research tablet press (Korsoh Xll’l) and a gravity powder feeder. The 50 mg tablets were produced using round standard concave 6 mm U1 g and 3.5 kN . The 100 mg tablets were produced using 8 mm round standard concave tooling and 3.9—4.2 er forces. The specifications of the 50 mg and lOO mg tablets are shown in Table 37.

Table 37. Specifications of 50 mg and 189 mg Tablets of Compound 2 % mg Tablets 190 mg Tablets Average Weight (11:10) 200 _+_ it) mg Individual Weight 290 _+_ 20 mg Friability Nils/l"? 0.5% NMT 05% The 50 mg and 100 mg tablets produced as described above were subjected to 5 month stability studies under three conditions: 5°C (refrigeration), 2500/6096 Rl-l (ambient), and 4000/7596 RH (accelerated). Both the 50 mg and 100 mg tablets were chemically stable under all three ions tested.

Under refrigeration conditions (:5 "(3), both the 50 mg and l 00 mg tablets remained white solids that did not change in appearance from T--0 to T=-6 months. Tlrn'onghout the 6~month study, no impurities were reported that were greater than 0.05% for either the 50 mg tablets or the 100 mg tablets. The water content after 6 months was also less than 3.0 % w/w for both tablets. Similar results were reported when the tablets were subjected to t conditions (25 "(I/60% RH); no impurities that were greater than 00596 were reported hout the 6 months for both s and the water content did not exceed 3.0 % w/w at the 6~month mark. When the s were subjected to rated conditions (40 °C/75% RE), the appearance of the 50 mg and lOO mg s did not change from a white, round tablet One impurity was reported after 3 m onths, but the impurity was only 009%. A second impurity was reported after 6 months, but the total impurity percentage was only 0.21% for both the 50 mg and 100 mg tablets. Water content was 3. % w/w at 6 months for the 50 mg tablets and 3 .2 % w/w for the 100 mg tablets. in a separate study, the ity of 50 mg and l00 mg tablets of Compound 2 at ambient conditions (25 OC/oO‘l/{t RH) was measured. over 9 months. The appearance of the 50 mg and lOQ U1 mg tablet did not change from a white round tablet over the course of 9 months. impurities in the 50 mg tablet were less than O.l0% after 9 months and impurities in the 100 rng tablet were less than 005%. The water content of the 50 mg tablet and the 100 mg tablet after 9 months was only 2.7 % w/w and 2.6 % w/w, respectively.

This specification has been described with reference to embodiments of the ion However, one of ordinary skill in the art appreciates that various modifications and s can be made without ing from the scope of the invention as set forth in the claims below.

Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.

Claims (10)

The claims defining the invention are as follows:

1. A compound of the formula:

2. The compound of claim 1, wherein the compound is at least 90% free of the opposite phosphorus R-enantiomer.

3. The compound of claim 1, n the compound is at least 98% free of the te phosphorus R-enantiomer.

4. The compound of claim 1, n the compound is at least 99% free of the te phosphorus R-enantiomer.

5. A pharmaceutical composition comprising the compound of claim 1 in a pharmaceutically acceptable carrier.

6. A pharmaceutical composition comprising the compound of any one of claims 2-4 in a pharmaceutically acceptable carrier.

7. Use of a compound of claim 1, in the manufacture of a medicament for treating HCV in a human in need thereof.

8. Use of a compound of any one of claims 2-4, in the manufacture of a medicament for treating HCV in a human in need thereof.

9. A compound of the formula: n the compound is an amorphous solid.

10. A compound of the formula: wherein the compound is a crystalline solid. \\\ssss§\\\§\\\h\§c\\\\x§ \\s\§\\\\\\s\\\\\u z:\:\...‘..S.‘.\s.\x3....\..3.\\u}:.:\x:\\\.