Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa® John O. Linka, , James G. Taylora, Alejandra Trejo-Martina, Darryl Katoa, Ashley A. Katanaa,f, Evan S. Krygowskia,f, Zheng-Yu Yanga, Sheila Zipfela, Jeromy J. Cottella, Elizabeth M. Bacona, Chinh V. Trana,f, Cheng Y. Yangb,f, Yujin Wangb,f, Kelly W. Wangb, Guangyu Zhaob, Guofeng Chengc, Yang Tianc, Ruoyu Gongc, Yu-Jen Leec, Mei Yuc, Eric Gormand, Erik Mogaliand,f, Jason K. Perrye ⁎

a

Medicinal Chemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, United States Drug Metabolism, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, United States c Biology, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, United States d Formulation and Process Development, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, United States e Structural Chemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, United States b

ARTICLE INFO

ABSTRACT

Keywords: HCV Hepatitis C Virus Non-structural protein 5A NS5A Sustained viral response SVR Direct-acting-antiviral DAA Rule-of-five Single-tablet regimen Velpatasvir GS-5816 Epclusa® Sofosbuvir

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1–6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1–6 HCV patients who have previously failed another treatment regimen.

Chronic hepatitis C virus (HCV) infection afflicts an estimated 71 million individuals worldwide and leads to significant morbidity and mortality, including liver cirrhosis and hepatocellular carcinoma.1 In 2013 US deaths related to HCV infection exceeded all other Center for Disease Control notifiable infections combined (including for example human immunodeficiency virus [HIV], tuberculosis and influenza). Infectious liver disease (from HCV and hepatitis B infection) has risen to the seventh leading cause of death worldwide and has surpassed other epidemic infectious disease deaths such as tuberculosis, HIV and malaria.2 Prior to 2013 HCV treatment therapy included ribavirin and some form of interferon (IFN) administered in complex, poorly tolerated regimens with cure-rates up to 56% in clinical trial settings.

Importantly, real-world cure-rates (post-approval and outside of clinical trial settings) with IFN and ribavirin are significantly lower at 3–10%.3 The longstanding high unmet need in HCV therapy is the basis for the rapid uptake of HCV direct-acting antivirals (DAAs). In succession the DAAs telaprevir (2011), sofosbuvir (2013), and the combination drug ledipasvir/sofosbuvir (LDV/SOF, 2015) each became the largest drug launches in history.4 Starting with the approval of the first sofosbuvirbased regimen5, the treatment and cure of chronic HCV infection has undergone a remarkable revolution. The standard-of-care has progressed from complex, side-effect ridden, and poorly tolerated IFNbased regimens to safe, simple, single tablet regimens with cure-rates at or above 95%.6 Key components in sofosbuvir-based DAA regimens are

Corresponding author. E-mail address: [email protected] (J.O. Link). f Former Gilead Employees. ⁎

https://doi.org/10.1016/j.bmcl.2019.04.027 Received 24 November 2018; Received in revised form 13 April 2019; Accepted 16 April 2019 0960-894X/ © 2019 Published by Elsevier Ltd.

Please cite this article as: John O. Link, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.04.027

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

non-structural protein 5A (NS5A) inhibitors.7 The NS5A inhibitor ledipasvir (1), combined with sofosbuvir was the first approved singletablet regimen (STR) for the cure of HCV infection (brand name Harvoni®).6 The importance of STRs for improved patient compliance and the associated avoidance of drug resistance is well established in HIV antiretroviral therapy;8 we targeted STRs for the treatment of HCV envisioning parallel advantages. In phase 3 clinical trials, LDV/SOF affords high cure rates (94–97%) in as little as 8 weeks of treatment for genotype 1 (GT1) HCV infected patients, and importantly demonstrates comparable effectiveness in real-world studies.9 During our discovery program for ledipasvir, the greatest unmet need was for the cure of GT1 infected patients, as GT1 represented the largest percentage of HCV infected patients worldwide, and the standard-of-care IFN/ribavirin regimens produced the lowest cure-rate for GT1 patients among the six main HCV genotypes. Our work focusing on optimizing GT1 replicon potency and pharmacokinetics in the discovery of ledipasvir has been previously described.10,6b After discovering ledipasvir, we directed our efforts to the discovery of a pan-genotypic agent that would produce high potency against the major HCV genotypes 1–6 and provide a safe, simple and effective treatment for the broadest range of HCV infected patients in a pan-genotypic STR. The following requirements for a pangenotypic STR guided our discovery efforts: once-daily oral dosing; sufficiently high potency across the major HCV genotypes and good pharmacokinetic properties (good bioavailability, high metabolic stability, long half-life) to afford a low-dose; appropriate drug properties and drug-drug interaction profile to allow for co-formulation with other HCV DAAs. Our discovery of the high resistance barrier, pan-genotypic, oncedaily oral NS5A inhibitor velpatasvir (39) is described herein. Velpatasvir is a component in the only approved pan-genotypic STRs. Velpatasvir, combined with sofosbuvir as Epclusa® is the STR for the treatment of naïve cirhhotic or non-cirrhotic HCV GT1-6 patients,11 and velpatasvir combined with sofosbuvir and voxilaprevir12a (HCV nonstructural protein 3 protease inhibitor) as Vosevi® is the pan-genotypic STR that is approved for treatment experienced GT1-6 patients.12b Velpatasvir possesses potency improvements as great as 33,000 fold (eg GT2b, Table 1) and an improved resistance barrier (vide infra) relative to LDV.13 The high genetic diversity of HCV is the basis for its chronicity,14 and presents significant challenges to the discovery of efficacious drugs. HCV is comprised of six main genotypes and 86 known subtypes.15 Further, within these subtypes there are multiple resistance associated substitutions (RAS, plural RASs) in the HCV genome measurable at baseline or appearing during or after treatment. At an early point in our efforts to discover VEL, a prevalent RAS in GT2 patient isolates was described that exhibited reduced susceptibility to first generation NS5A

inhibitors. It has been noted that first generation NS5A inhibitors can have significantly less GT2 replicon activity when the wild-type (WT) leucine at position 31 of the NS5A protein is substituted by methionine (L31M).16 Notably, reduced susceptibility has been seen clinically in monotherapy where an NS5A inhibitor produced limited (0.3–0.5 log10) viral load reduction (VLR) in GT2 patients with L31M virus and > 3 log10 VLR for patients with L31 virus.17 The concern with the L31M RAS is significant since its prevalence is > 50% among deposited GT2a and GT2b sequences. Early reports of NS5A inhibitor potency for GT2a were based on a replicon derived from the JFH1 clone which bears the more readily inhibited L31 and therefore did not reflect the weaker potency observed for L31M GT2 virus.18 A major focus in our early work toward VEL was the potency optimization against GT2 replicons bearing L31M. As our work progressed, our definition of a “pan-genotypic” inhibitor evolved, and drove changes in our screening paradigm and in the structures of the inhibitors we designed and synthesized. We initiated the program with GT1a/b, 2a replicons in place and soon added GT3a and 4a. We then added further subtypes of these four genotypes (eg GT2b), RASs (eg GT2a L31M), and then expanded to genotypes 5 and 6 with GT5a, 6a, 6e. We also assessed numerous RASs where resistance to first-generation NS5A inhibitors was known within these genotypes/subtypes, and added prevalent RASs based on our analysis of published NS5A sequences and sequences from patients in our own earlier clinical trials.13n Interestingly we found that as we gained broader activity in GT1-4 subtypes, some of those same inhibitors showed improved activity against GT1-4 RASs and then against other genotypes (ie GT5, 6), subtypes or RASs. Our inhibitors evolved as our screening paradigm evolved. Initially we sought to find a potent GT1 NS5A inhibitor that also possessed potent inhibition against the more difficult to inhibit GT2 NS5A L31M variants. We had multiple GT2 replicons with L31M. As a surrogate for GT2 viruses bearing L31M, we utilized site-directed mutagenesis to generate the JFH1 NS5A L31M construct (second GT2 entry in Table 2). Additionally, the GT2a J6 strain has L31M in its sequence (third GT2 entry in Table 2), as does the viral strain we used for GT2b (fourth GT2 entry in Table 2). The potency difference among these three L31M viral strains is based on underlying sequence differences in the rest of the NS5A gene. Where we have data for GT2a J6 available we consider it the most relevant GT2a 31 M data as it is a patient isolate derived strain (rather than an engineered strain such as JFH1 L31M), and it is typically the most difficult to inhibit. Throughout this text all inhibitor potency EC50 values (effective concentration to inhibit viral replication by 50%) are replicon cell-based assays. In our earlier studies we found that fused ring systems, such as the tricyclic difluorofluorene within the core of ledipasvir,10a could afford enhanced potency in the GT1 replicon. We define the core herein as the

Table 1 Replicon cellular potency of ledipasvir and velpatasvir (GT1-6 replicons and subtypes).

39 GS-5816, velpatasvir (VEL)

1 GS-5885, ledipasvir (LDV)

GT EC50 (pM)

LDV VEL

1a13a

1b13b

2a JFH113c

2a J613d

2b13f

3a13g

4a13i

5a13k

6a13l

6e13m

31 14

4 16

21,000 8

249,000 16

530,000 6

168,000 4

390 9

150 54

1,100 6

264,000 130

2

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 2 Cores with fused ring systems provide high potency: replicon potency data and human liver microsomal metabolic stability values.

2

2–10

Cpd

a

4

5

6

7

8

9

10

GT EC50 (pM)

Pred CL (L/h/Kg)

13a

1b

92 528 5990 220 156 65 63 87 29

5 18 10 13 21 28 22 21 22

1a 2 3 4 5 6 7 8 9 10a

3

13b

2a

13c

20 223 1470 73 131 16 14 19 7

JFH1 (L31)

13e

13d

2a JFH1-L31M

2a J6

6,790 – – – 13,300 – – 9,850 5,900

– – – – – 23,700 19,700 – 8810

(M31)

2b

13f

(M31)

– – – – – – 10,200 – 1,770

HLM < 0.16 < 0.16 < 0.16 0.43 0.19 0.39 0.29 – 0.18

Additional replicon EC50 values for compound 10, GT(EC50 value): 3a13h (55 pM); 4a13j (29 pM); 5a13k (56 pM); 6a13l (16 pM); 6e13m (1160 pM).

central structure spanning between (modified) pyrrolidines. As we worked to discover a pan-genotypic inhibitor, we found that differing core structures produced unique profiles against genotypes/subtypes/ RASs. We hypothesized that a highly developed core could provide an important foundation for gaining broad activity against diverse NS5A proteins. In this endeavor we synthesized and tested over 100 different core systems within fully elaborated inhibitors.10,19,20 Many of the important cores we discovered incorporated fused ring systems. Table 2 provides GT1 and GT2 replicon potency and human predicted clearance (Pred CL) from human liver microsomes (HLM) for a number of novel inhibitors possessing four to five fused rings in cores with varying topologies, levels of unsaturation, and heteroatom count. Triphenylene 2 has good overall potency with a significant improvement over ledipasvir 1 for GT2a JFH1 but a three-fold loss in GT1a potency. Replacing two triphenylene methines with nitrogens provided a loss in GT1a potency to 530 pM for dibenzoquinoxaline inhibitor 3. Adding another fused aromatic ring in pentacyclic inhibitor 4 resulted in even greater losses in GT1a potency to 5990 pM. From inhibitors 2–4 the rank order in GT1a potency followed the potency rank order in the WT L31 GT2a JFH1 strain. We opted to change the topology of the tetracyclic ring system. The cyclopentaphenanthrenone 5 showed a decrease in metabolic stability and a potency of 220 pM against GT1a. We further changed the tetracycle topology in entries 6–10. Tetrahydropyrene-based inhibitor 6 regained GT1a potency to 156 pM, and had a GT2a JFH1 L31M potency of 13,300 pM. Changing a peripheral

ethylene to a methylenoxy in 7, and the dehydro version in 8 afforded further potency improvements in GT1a (65 and 63 pM respectively) and were 23,700 and 19,700 pM respectively versus the difficult to inhibit GT2a J6 strain. Replacing the ethylene group in 8 with another methylenoxy (9) slightly weakened the GT1a potency (87 pm). But reversing the orientation of the methylenoxy from 9 produced inhibitor 10, the most potent inhibitor versus GT1a and GT2 (all four strains) in Table 2. Importantly, tetracyclic benzopyrano-benzopyran inhibitor 10 represented a compound that relative to ledipasvir was equipotent against GT1a, and improved in GT2-6 replicons, notably GT2a J6 (28fold), GT2b (300-fold) and GT3a (4800-fold). Despite the potency improvements of 10 over ledipasvir, human liver microsomal stability – a key pharmacokinetic parameter for once-daily dosing – is a shortcoming for 10 and its related structures in Table 2. The HLM Pred CL for compound 10 is 0.18 L/h/Kg, while ledipasvir does not show measurable instability in the same assay (< 0.16 L/h/kg, lower limit of assay). For reference, a more sensitive assay for metabolic stability with 3Hlabled ledipasvir in human hepatocytes results in its predicted clearance of 0.012 L/h/kg.10a While a good initial direction for potency, benzopyrano-benzopyran 10 required further improvement in its oxidative metabolic stability in order to meet our goal of once-daily dosing. Our study of core structures required a significant chemistry resource commitment, as many of these complex ring systems were unprecedented, and required novel multi-step syntheses as exemplified in Scheme 1 for fused tetracyclic core inhibitor 7.19,20 3

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Scheme 1. Synthesis of 7a. (i) N-bromosuccinimide, DMF (ii) BnBr, K2CO3 , DMF b. Pd(PPh3)4, CuI, Et3N, 80 °C. c. (i) H2, Pd/C, 60 PSI (ii) Tf2O, Py, CH2Cl2 d. Pd (OAc)2, PPh3, Cy2NMe, DMF, 110 °C e. LAH, THF f. BBr3 g. Tf2O, Py, CH2Cl2 h. (i) KOAc, Pd(dppf)Cl2, (Bpin)2, dioxane, 110 °C (ii) 7j, Pd(PPh3)4, K2CO3, DMSO 100 °C i. KOAc Pd(dppf)Cl2, X-Phos, (Bpin)2, dioxane, 110 °C 7j, Pd(PPh3)4, K2CO3, DMSO, 100 °C j. (i) HCI, dioxane, CH2Cl2 (ii) methoxycarbonyl-valine, HATU, iPr2NEt.

Table 3 Potency and metabolic stability derived from terminal amino acid modifications in benzypyrano-benzopyran series.

B

A 11 20

Cpd

GT EC50 (pM)

Pred CL (L/h/Kg)

1a13a

1b13b

2a13c JFH1 (L31)

2a J613d (M31)

2a JFH1-L31M13e

2b13f (M31)

HLM

11

31

19

9

–

12,100

1,850

0.18

12

53

20

20

–

31,300

–

0.21

13

55

22

22

–

> 44,000

–

–

14

380

800

74

330

–

600

–

15

93

48

7

240

145

22

0.19

16

185

172

14

1430

1,330

–

0.23

17

87

79

10

–

3,670

–

0.21

18

50

31

4

1820

600

76

0.24

19

750

580

38

1800

–

1,210

0.26

20

320

315

22

280

320

57

–

A

B

4

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

inhibitors show acceptable rat and cyno Fa%. Unfortunately the bis-DPhGly compound 15 has a nearly unmeasurable Fa% in rat and cyno at 2% each. Compound 15 has an Fa% of 34 in dog, exemplifying our concerns regarding the translatability from dog Fa to human, vide supra. (Note that dog Fa% is also substantially higher than rat and cyno for compounds 9 and 10). As with compound 15 we found that as we further elaborated compounds to improve pan-genotypic potency, maintaining sufficient permeability became correspondingly challenging to attain (vide infra). The value of D-PhGly in attaining pan-genotypic potency was apparent, although the permeability liability of using it on both sides of the molecule appeared daunting. We therefore investigated amino-acid changes in unsymmetric inhibitors with one amino-acid held constant as valine. Potency improvements can be seen in the unsymmetric series in the progression from 4-THP-Gly (16) to methylthreonine (17), to DPhGly (18) where the latter has excellent potency of 50 pM against GT1a and ∼10 fold improved GT2a JFH1 L31M over the valine inhibitor 10. The 3-pyridyl glycine analog 19 (mixture of epimers) showed erosion of its profile (beyond what would be expected for an ∼1:1 epimeric mixture) in GT1a, 1b and 2b, dissuading us from phenylglycine modification as an avenue for modulation of the PK properties. Finally, the unsymmetric D-PhGly/4-THP-Gly inhibitor 20 had an unacceptable level of GT1 potency loss, being more similar to the symmetric 4-THP-Gly inhibitor 14 than the symmetric D-PhGly inhibitor 15 in this regard. D-PhGly represented an important terminal amino-acid for further study. Nonetheless, the benzopyrano-benzopyran series still did not meet our criteria for progression. The most promising compound in Table 3 from the perspective of pan-genotypic activity is symmetric D-PhGly inhibitor 15, but it shows almost no fraction absorbed in rat and cyno. Further, compounds in this series have HLM metabolic instability that would be incompatible with oncedaily dosing, tightly ranging from 0.18 to 0.26 L/h/Kg (Table 3). We decided to investigate even more complex polycyclic cores with the goal of improving upon the pan-genotypic potency and HLM stability of the benzopyrano-benzopyran series. One direction we undertook was the design and synthesis of a pair of benzopyrano-naphthimidazoles (Table 5). These unprecedented structures have contiguous fused pentacyclic ring systems comprising one side of the core. In this ring system there is only one rotatable bond remaining in the core (where the benzopyrano-naphthimidazole connects to the imidazole). Interestingly, in the linear contiguous fused ring system we envisioned, there are two possible positions for the methylenoxy group – either on the “same side” as the central ring of the naphthimidazole portion, as in benzopyrano-naphthimidazole 21, or the opposing side of this ring as in pentacycle 22. During the intensive synthetic design, synthesis, and purification process, pentacycle 21 was synthesized and tested before pentacycle 22. The data resulting from pentacycle 21 was a discouraging setback, with potency losses against all GT2 variants relative to the earlier benzypyrano-benzopyran 10. Despite the dispiriting results with pentacycle 21, we continued with the challenging synthesis and

Table 4 Fraction absorbed (Fa%) for benzopyrano-benzopyran series; bis-D-PhGly inhibitor 15 has low Fa% in rat and cyno. Cpd

9 10 15

Fraction absorbed (Fa%) Rat

Dog

Cyno

18 23 2

100 70 34

– 15 2

We next investigated varying the sidechains of the amino-acid termini of the optimized core inhibitor 10 to assess their effect on broad genotype potency as shown in Table 3. Moving from valine (10) to isoleucine (11) had minimal impact on the profile. Incorporation of cyclobutyl or cyclopentyl glycine provided little change in GT1b or GT2a JFH1 activity, but showed losses in GT1a, and GT2a JFH1 L31M potency (compounds 12 and 13). Interestingly, the 4-tetrahydropyranyl glycine (4-THP-Gly) inhibitor 14 possessed similar activity over a range of genotypes and subtypes. Although the GT1a activity of 14 shows a 12-fold loss in potency relative to valine inhibitor 10, potencies of ≤800 pM are maintained in eight out of nine replicons tested, including a potency of 330 pM against the challenging GT2a J6. The symmetric Dphenylglycine (D-PhGly) inhibitor 15 displays an even better profile with five replicons all less than the GT2a J6 value of 240 pM, and GT1a at 93 pM. At this point we were interested in the pharmacokinetic properties of the important inhibitor 15. We have found that these high logD, high MW, low buffer solubility compounds appear to adhere to the surface in the Caco2 in vitro assay apparatus (where the surface area in the measurement system is high). Ledipasvir, velpatasvir and other compounds detailed herein do not have sufficient cell-free permeability (ability to pass from loading to recovery chamber in the absence of Caco2 cells) in a Caco2 well system to provide reliable permeability readouts. Nonetheless LDV and VEL perform quite well in vivo both in preclinical species and humans, and are safe and effective drugs with desirable pharmacokinetic properties.21 To gauge the permeability of such compounds, we rely on percent fraction absorbed (Fa%) of the inhibitors after oral dosing of a lowdose non-precipitating solution formulation in preclinical species. The use of a low-dose with a non-precipitating solution formulation is to remove solubility differences among compounds. The Fa% calculation removes the liver metabolism component from bioavailability (F%).22a Further, we found that the Fa% measured in dog is consistently higher than other species. The dog intestinal system is known to have “loose junctions” and consequently we de-emphasized dog Fa in our analysis with the concern that the dog Fa represented a species-specific permeability not representative of human.22b Thus during these studies we relied on rat as a screening species, and then cynomolgous monkey (cyno) for confirmation of important compounds. Table 4 shows Fa% results for benzopyrano-benzopyrans 9, 10 and 15. The bis-valine Table 5 Potency and metabolic stability of benzopyran-naphthimidazoles. Cpd

GT EC50 (pM) 13a

1a

1b

21

41

22

16

13b

13c

13d

13f

13h

Fraction Abs. (Fa%)

4a

Pred CL (L/h/ Kg) HLM

Rat

Dog

13j

2a JFH1 (L31)

2a J6 (M31)

2b (M31)

3a

17

16

20,900

32,300

12

130

0.22

31

54

17

4

1,160

162

4

8

< 0.16

15

50

5

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 6 Pyrrolidine modifications in the benzopyran-naphthimidazole series.

B A A

a

B

Cpd

23 30

GT EC50 (pM)

Pred CL (L/h/Kg)

Fraction Absorbed (Fa%)

1a13a

1b13b

2a13c JFH1 (M31)

2a J613d (M31)

2b13f

3a13h

4a13j

HLM

Rat

Dog

Cyno

23a

11

18

19

234

–

7713g

2013i

–

–

–

–

24

11

18

7

443

132

18

11

< 0.16

10

–

–

25

15

18

7

378

265

48

20

< 0.16

–

–

–

26

21

26

8

280

135

24

14

< 0.16

16

56

–

27

24

34

10

61

180

206

36

< 0.16

10

–

–

28

14

19

9

106

166

58

22

< 0.16

11

–

–

29

11

17

12

67

157

61

15

< 0.16

13

–

64

30

8

12

14

280

63

20

8

< 0.16

27

100

53

For compound 23, the sidechain of the valine on the methylpyrrolidine side is perdeuterated.

isolation of 22. Remarkably, there is striking divergence in potency between these isomeric pentacycles. Pentacycle 22 contributes the greatest potency across genotypes among the 100 + cores that we have synthesized. Compound 22 is more potent than 10 or 21 against all GT14 replicons we tested. Compound 22 is more potent than 21 by 200 fold in GT2b, 18 fold in GT2a J6 and 16 fold in GT4a potency. The basis for this divergence in potency between 21 and 22 remains difficult for us to rationalize. There is an additional important attribute of the benzopyrano-naphthimidazole based inhibitor 22; it does not have measurable oxidative metabolism under the conditions employed in the HLM

assay. This further differentiates 22 from the benzopyrano-naphthimidazole based inhibitor 21 (which has a Pred CL of 0.22 L/h/kg) and the tetracyclic benzopyrano-benzopyran series inhibitors in Table 3. It should be noted that the benzopyrano-naphthimidazole ring system is unprecedented within drug-space. According to a recent summary of rings in drugs, new ring systems are relatively rare, with six new ring systems entering drug-space on average each year.23 The high potency contributed by the benzopyrano-naphthimidazole, along with its contribution to improved metabolic stability, has proven transformative for our program.

Table 7 Compound 26 has improved GT1 resistance profile. GT1a EC50 (nM)

LDV Compound 26

GT1b EC50 (nM)

WT

M28T

Q30H

Q30R

L31M

Y93C

Q30E

Y93H

WT

Y93H

0.031 0.037

1.9 0.19

5.7 0.15

19.6 0.22

17 0.37

49.6 0.22

169 10.4

52.0 4.7

0.004 0.026

7.2 0.086

All RAS are transiently transfected GT1a or GT1b subgenomic HCV replicons. 6

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 8 D-PhGly improves overall potency profile.

B

A Cpd A

31, 32

GT EC50 (pM)

Pred CL (L/h/Kg)

Fraction absorbed (Fa%)

1a13a

1b13b

2a13c JFH1

2a J613d (M31)

2b13f (M31)

3a13h

4a13j

HLM

Rat

Dog

Cyno

31

22

21

4

134

12

5

8

< 0.16

13

56

16

32

35

33

6

73

16

6

11

< 0.16

–

–

–

B

In our previous work, modification of the pyrrolidine structure provided improvements in pharmacokinetic properties and potency.10a Here we pursued modifications of the pyrrolidines in the benzopyranonaphthimidazole series as shown in Table 6. The 5-methyl pyrrolidine 23 improved the GT2a J6 potency to 234 pM. The 4-methyl pyrrolidine 24 showed a lesser improvement of the GT2a J6 potency relative to 23 at 443 pM, but was superior in GT3a potency at 18 pM versus 77 pM. The 4-ethoxy inhibitor 25 was intermediate in potency for both the GT2a J6 and GT3a potency (378 and 48 pM respectively) relative to methyl pyrrolidines 23 and 24. The 4-methoxymethyl pyrrolidine in inhibitor 26 provides a good balance of GT2a J6 and GT3a potency at 280 and 24 pM respectively. Further, the methoxymethyl inhibitor 26 has a somewhat improved Fa% in rat over the 4-methyl inhibitor 24, and Fa% comparable to the unsubstituted pyrrolidine. It is notable that metabolic stability under the conditions of the HLM assay is maintained across the series of benzopyrano-naphthimidazole inhibitors tested in Table 6. The methoxymethyl pyrrolidine/pentacyclic core inhibitor 26 has low picomolar inhibition across a range of GT1-4 replicons, Fa of 16% in rat, and good HLM stability. We assessed the potency of compound 26 against a range of clinically relevant GT1a and 1b RASs as shown in Table 7, and the inhibitor demonstrates improvements in its resistance profile over LDV. Inhibitor 26 shows no significant decrease in potency relative to WT for GT1a M28T, Q30H, L31M, Q30R and Y93C while LDV shows increasing losses in potency across this RAS series. Inhibitor 26 has low nanomolar potency against the challenging GT1a Y93H RAS, again superior to LDV, and minimal loss in activity against GT1b Y93H relative to WT. We pursued further optimization through modification of the second pyrrolidine of 26 as shown in Table 6. Adding a 4-methoxymethyl (27), a 4-methyl (28) or a 5-methyl (29) to this pyrrolidine all produced improvements in GT2a J6 potency over compound 26. Compounds 27 and 29 had the best GT2a J6 potencies we had yet seen at 61 and 67 pM respectively. Compound 29 additionally had strong potency against GT1a (11 pM) and GT1b (17 pM) and GT2b (157 pM). Compound 29 and 26 have comparable Fa% in rat with 29 showing good Fa% in cyno. Thus, the 4-methoxymethyl pyrrolidine and the 5methyl pyrrolidine emerged as important groups, and worked well when paired (29). Since the 4-methoxymethyl pyrrolidine had been tested on both sides of the molecule (27), the inhibitor 30 with 5-methyl pyrrolidine on both sides was also assessed. Compound 30 had inferior GT2a J6 potency to pyrrolidines 27 and 29, but showed superior GT2b potency and improved Fa% in the rat. Additionally, compounds 28, 29 and 30 require improvement in GT6e potency, with EC50 values of 864, 857 and 2080 picomolar respectively. As shown in Table 3, incorporation of D-PhGly improved potency

relative to valine in GT2 L31M replicons in the benzopyrano-benzopyran based core series. This amino-acid was thus utilized with the benzopyrano-naphthimidazole scaffold (Table 8). The D-PhGly when proximal to the pentacycle (compound 31) improved the GT2a J6 potency > 8-fold over valine (134 pM) and ∼16-fold (73 pM) when proximal to the imidazole (compound 32). The improved GT2a J6 activity of 32 over 31 demonstrates a valuable aspect of the unsymmetric core. We have observed divergent SAR when modifying the pendant groups on differing sides of an unsymmetric core, and this property can prove advantageous.10a Also note here that compound 32 at 73 pM is 25-fold more potent against GT2a J6 than the analogous D-PhGly inhibitor 18 in the optimized tetracyclic benzopyrano-benzopyran series, again demonstrating the superiority of the pentacyclic benzopyranonaphthimidazole core series. Importantly, replacement of one valine by D-PhGly (compounds 21 and 31) results in little change to the fraction absorbed (15% versus 13% respectively) giving us encouragement in the strategy of utilizing a mixed valine/D-PhGly inhibitor to achieve pan-genotypic potency with acceptable Fa%. We next sought to understand the interplay of either 4- or 5-substitution of the pyrrolidines when combined with the D-PhGly terminus. This was assessed in the symmetric tetracyclic benzopyrano-benzopyran series with parallel modifications on each side of the inhibitor (Table 9). This study uncovered intriguing matched and mismatched pairings. Addition of the 5-methyl is a mismatch with the D-PhGly (33) relative to hydrogen in pyrrolidine 15 and imparts the following respective potency losses: GT1a (150 versus 93 pM), GT2a J6 (1835 versus 237 pM) and GT2b (94 versus 22 pM). In stark contrast, addition of the 4-methyl group is a match with the D-PhGly (compounds 34 versus 15) and imparts respective potency improvements in GT1a (31 versus 93 pM), GT1b (20 versus 48 pM), GT2a J6 (58 versus 237 pM), and GT4a (14 versus 24 pM). The difference in matched versus mismatched (34 versus 33) can be as great as 31-fold (GT2a J6). The GT1-4 potency profile of compound 34 is highly attractive (< 60 pM), but as previously seen when two D-PhGly termini are present the PK profile is untenable for progression with rat Fa% down at 3% (notably 2–3% for all three such compounds 15, 33 and 34). Our concern continued regarding the translatability of the higher dog Fa values (ie 30% for 34) to human when accompanied by low Fa in other species. The benefit of adding a 4-substituent to the pyrrolidine ring in combination with the D-PhGly was pursued in the pentacyclic benzopyrano-naphthimidazole series. Thus the highly optimized 4-position methoxymethyl pyrroldine was utilized with the D-PhGly terminus, and provided the best overall potency we had yet seen across seven GT1-4 subtypes as shown in Table 10 (inhibitor 35), ranging from 6 to 19 pM. Relative to inhibitor 32, the addition of the methoxymethyl improved 7

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 9 5-Methyl-pyrrolidine antagonizes, and 4-methyl-pyrrolidine synergizes with D-PhGly.

B

A

Cpd A

GT EC50 (pM) 13a

B

33, 34

13b

13c

1a

1b

33

150

20

8

1835

34

31

20

5

58

2a

JFH1

2a J6

13d

(M31)

2b

13f

(M31)

13h

13j

Pred CL (L/h/Kg)

Fraction Absorbed (Fa%)

HLM

Rat

Dog

3a

4a

94

4

15

< 0.16

3

43

26

6

13

< 0.16

2

30

Table 10 Pyrrolidine modification improves pan-genotypic potency and fraction absorbed resulting in velpatasvir, 39.

B

A Cpd A

35 41

GT EC50 (pM)

Pred CL (L/h/Kg)

Fraction absorbed (Fa%)

1a13a

1b13b

2a JFH113c

2a J613d(M31)

2b13f

3a13h

4a13i

HLM

Rat

Dog

Cyno

35

11

19

6

13

9

17

7

< 0.16

12

–

3

36

13

17

6

14

15

25

11

< 0.16

9

–

4

37

10

11

6

113

46

18

9

0.24

–

–

–

38

13

20

7

21

28

25

9

< 0.16

13

–

–

39

14

16

8

16

6

413g

9

< 0.16

36

29

37

40

8

5

4

53

15

20

< 0.16

4

–

–

41

33

30

18

45

27

50

< 0.16

4

–

–

B

8

63

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 11 Data from replacing the benzopyrano-naphthimidazole core of VEL with the benzopyrano-benzopyran core. O O

NH

O

O

H N

N

N

N N

N H

O

O

HN

O

42

O

GT EC50 (pM) 13a

13b

1a

1b

16

16

2a J6 480

13d

2b

13f

290

13h

13i

13k

13l

3a

4a

5a

6a

120

13

81

22

potency most dramatically for GT2a J6 (from 73 to 13 pM), with additional improvements in GT1a, 1b, 2b and 4a, and with only GT3a slightly losing some potency, from 6 to 17 pM. Further, compound 35 is stable at the lower limit of our routine HLM assay. Although sufficiently potent against GT1-4 for advancement, the next step was to improve permeability since inhibitor 35 has a low Fa% of 12 in rat, and 3 in cyno precluding it from selection for development. Further modifications to the pyrrolidine proximal to the benzopyrano-naphthimidazole were undertaken in order to improve the Fa% of potent GT1-4 inhibitor 35, with the intention of maintaining its favorable potency and metabolic stability. Addition of a 4-methyl group to the pyrrolidine of 35 afforded compound 36 which had little change in potency, and also left the Fa% largely unchanged in rat (9%) and cyno (4%). Fusion of a cyclopropyl group to the pyrrolidine can be thought of as an interpolation between the 4- and 5-methyl-pyrrolidine. Both the 4R, 5S (37) and the 4S, 5R (38) stereochemistry were tested. Potency was eroded for GT2a J6 and GT2b in compound 37, so its Fa% was not tested. Isomer 38 showed sufficient potency, but little improvement in rat Fa% (13%). The addition of a 5-methyl to the pyrrolidine in inhibitor 39 improved the GT3a potency four-fold relative to 35 with the other genotypes minimally changed. Importantly incorporation of the 5-methyl in 39 produced a dramatic improvement in the fraction absorbed with values of 36, 29 and 37% in rat, dog and cyno respectively. Further, what would be considered small structural changes from 39 such as replacing the 4-methoxymethyl group with methyl (compound 40) or moving the 5-methyl of 40 to the 4-position (compound 41) resulted in substantial losses in permeability (both have 4% Fa in rat). Thus the clear standout in Fa% is inhibitor 39 which is also among the most potent overall compounds in Table 10. This level of fraction absorbed is impressive for a compound of MW 883 amu bearing 16 heteroatoms, and with a structure comprised of nine rings,

O

Pred CL (L/h/Kg)

Fraction Abs. (Fa%)

6e

HLM

Rat

Monkey

110

0.20

29

26

13m

five of which are fused. Contrary to prevailing thought with “rulebreaker” compounds, it has been our experience that what might be considered a small structural change - such as the addition of a methyl group to a specific position of the pyrrolidine as seen for inhibitor 39 in Table 10 – can sometimes lead to dramatic effects on pharmacokinetic properties. To test the importance of the pentacyclic benzopyrano-naphthimidazole-based core in 39, we replaced it with the tetracyclic benzopyrano-benzopyran-based core to provide inhibitor 42 (Table 11). Although fused tetracycle 42 is the most potent inhibitor in the optimized benzopyrano-benzopyran series, it nonetheless possesses inferior replicon potency to fused pentacycle 39 in eight of out of nine GT1-6 subtypes; it is 34-fold less active against GT2a J6 at 480 pM, and 18-fold less active against GT2b at 290 pM, and 30-fold less active against GT3a (120 pM). Additionally 42 has inferior pharmacokinetic properties to 39; it has lower Fa% in rat and cyno (29 and 26% respectively) and is less stable in HLM with a Pred CL = 0.20 L/h/kg. Fig. 1 depicts additional compounds having a range of small structural changes relative to compound 39, which in each case have characteristics rendering them inferior. The dihydro version of 39 (compound 43) increases the human predicted CL by > 3-fold (0.19 L/h/kg, compared to 0.06 L/h/kg for 39, Table 12). The oxo-version (44) loses two- to twelve-fold in potency across genotypes (GTs 1a, 2a J6, 3a, 4a are respectively 31, 71, 156 and 28 pM). Finally, interchanging the ends of the molecule relative to the core (compound 45), results in lower Fa = 13% in cyno and underscores the importance of the unsymmetric linker in providing an element that allows for structural diversity that can be exploited for improved properties. Based on its exceptional overall profile, compound 39 was selected for development and is now known as velpatasvir (VEL). Compound 39 has excellent pan-genotypic potency with EC50 values across GT1-4 and

43

44

45 Fig. 1. Comparator structures to velpatasvir. 9

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 12 Velpatasvir in vitro and in vivo pharmacokinetic parameters. Dose (route)

Rat Dog Monkey Human

2 mg/Kg (IV) 0.25 mg/Kg (IV) 0.5 mg/Kg (IV) 100 mg (PO)

In vitro

In vivo

% Free plasma

Pred CL (L/hr/kg) LM

CL (L/hr/kg)

Vss (L/kg)

t½ (h)

Bioavailability (F%)

0.2 0.2 0.4 0.3

0.74 0.37 < 0.17 0.061

0.94 0.25 0.30 –

1.61 1.43 1.60 –

2.25 5.20 5.03 15.7

282 253 304 505

1. Data generated in human hepatocytes using 3H-VEL. 2. 2 mg/Kg PO. 3. 0.5 mg/Kg PO. 4. 1 mg/Kg PO. 5. Calculated from human oral CL/F and human predicted clearance from preclinical data, see text.

Scheme 2. Synthesis of velpatasvir. a. K2CO3, DMAc b. Pd2(dba)3, P(4-F-Ph)3, PivOH, K2CO3 c. CH2CHBF3K, Pd(OAc)2, S-Phos, n-PrOH d. NBS, THF/DMSO/H2O e. MnO2, CH2Cl2 f. 46, K2CO3, CH2Cl2 g. PyHBr3, CH2Cl2, MeOH h. 47, Cs2CO3, 2-Me-THF i. NH4OAc, toluene, 2-methoxyethanol j. (i) MnO2, CH2Cl2 (ii) HCl, dioxane, CH2Cl2 (iii) methoxycarbonyl-D-phenylglycine, COMU, i-Pr2NEt, DMF.

Scheme 3. Synthesis of Boc-methoxymethyl proline. a. (i) SOCl2, CH2Cl2 (ii) Boc2O, NaHCO3, CH2Cl2, H2O (iii) TosCl, Et3N, DMAP, CH2Cl2 b. NaCN, DMSO c. AcCl, MeOH d. Boc2O, NaHCO3, EtOAc, H2O e. NaOH, MeOH f. BH3·THF, 2-Me-THF g. NaOH, MTBE h. MeI, NaOtBu, THF.

notable paucity of new ring systems in chemical space, and the reuse of known systems, has been attributed to be due to the cost of synthesis in materials and time.25 The favorable results here demonstrate the benefit of pursuing such complex molecular structures. The synthesis of velpatasvir, and the novel pentacyclic benzopyrano-naphthimidazole ring structure embedded therein, is detailed in Scheme 2.20 The protected methoxymethyl proline 46 is synthesized as depicted

GT6a ranging from 6 to 16 pM, and GT5a at 75 pM, GT6e at 130 pM, and possesses uniquely good preclinical fraction absorbed and bioavailability. Additionally, VEL did not display cytotoxicity in replicon cells24 at the highest concentration tested (CC50 > 44,400 nM) affording a selectivity index (SI) relative to its GT1a potency of > 4,900,000 (SI = CC50/EC50). As previously noted, the pentacyclic ring-system in velpatasvir is unprecedented within drug-space. The 10

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

Table 13 LDV and VEL potency against clinically relevant GT1 resistance associated substitutions (RAS). GT1a EC50 (nM)

LDV VEL

GT1b EC50 (nM)

WT

M28T

Q30H

Q30R

L31M

Y93C

Q30E

Y93H

WT

Y93H

0.031 0.014

1.9 0.105

5.7 0.032

19.6 0.031

17 0.22

49.6 0.053

169 0.25

52.0 8.5

0.004 0.016

7.2 0.011

All RAS are transiently transfected GT1a or GT1b subgenomic HCV replicons. Table 14 LDV and VEL potency against GT2 and GT3 RAS. GT2a EC50 (nM)

LDV VEL

GT2b EC50 (nM) 1

GT3a EC50 (nM) 2

WT

K44R

N62V

N62S

WT (M31)

WT (M31)

R44K

P58S

WT

A30K

Y93H

209 0.009

164 0.005

222 0.01

123 0.003

865 0.004

211 0.007

943 0.011

292 0.01

> 44.8 0.013

> 88.8 0.21

> 88.8 3.2

GT2a WT is JFH1 Renilla luciferase (Rluc) subgenomic replicon. GT2a RAS are GT2a backbone which encode the full-length NS5A gene from various GT2a clinical isolates carrying the indicated RAS. GT2b WT are chimeric replicons based on GT2a JFH1 Rluc subgenomic replicon, encoding the full-length gene from GT2b 1MD2b-1 strain or 2AY232738. GT2b RAS were constructed similarly using GT2b clinical isolates carrying the indicated RAS. GT3a WT is GT3a S52 chimeric replicon based on GT1b Rluc subgenomic replicon backbone. GT3a RAS were produced by site-directed mutagenesis in the WT replicon. “ > ” indicates the highest concentration tested where 50% inhibition was not achieved. Table 15 Velpatasvir breaks most metric-based rules for bioavailability and “drug-likeness” (broken rules in red). Calculated values from ACD/ Labs software except for ClogP from ChemBioDraw14®, CambridgeSoft Corporation. # of H-bond acceptors is the sum of N and O’s as defined by Lipinski.31a Rule

Parameter

Limit value of rule

VEL

Lipinski Rule of 531a

Molecular Weight

≤ 500

883

Lipinski Rule of 5

CLogP

≤5

5.7

Lipinski Rule of 5

# of H-Bond Donors

≤5

4

Lipinski Rule of 5

# of H-Bond Acceptors

≤ 10

16

Veber31b

# of Rotatable Bonds

≤ 10

13

Veber

Polar Surface Area

< 140 Å2

193 Å2

# of Rings

≤ 5 is 95th percentile

9

# of Aromatic Rings

≤3

6

Rings in drugs23 Aromatic

Ring-Rule31c

in Scheme 3.19,20 Further characterization of VEL follows. In addition to the high potency across genotypes, we required an inhibitor possessing a halflife suitable for inclusion in an STR. Data on the favorable preclinical pharmacokinetic profile of VEL is detailed in Table 12. The predicted clearance from microsomes was low for the three preclinical species tested. The Pred CL is very similar to the in vivo CL for rat and dog, while being higher in vivo for cyno monkey. These data are consistent with the major route of CL being hepatic oxidative metabolism. This is important because VEL has a very low Pred CL based on human hepatic in vitro systems. In human liver microsomes VEL showed no measureable disappearance of parent during a 60 min incubation, leading to a Pred CL < 0.16 L/h/kg. Using 3H-39 allowed a lower detection limit along with quantification of metabolites. In cryopreserved human hepatocytes 3H-39 undergoes minimal metabolism and the Pred CL is exceedingly low at 0.06 L/h/kg (4.7% hepatic extraction, Eh%). VEL is highly protein-bound. The percent free drug in plasma measured by dialysis is similar across species ranging from 0.2 to 0.4% free for rat, dog and cyno; human is within this range at 0.3% free. The steady-state volume of distribution (Vss) is similar across the preclinical species and is larger than total body water ranging from 1.4 to 1.6 L/kg. With similar free-fractions across species, it would be expected that VEL would

have a moderate human Vss, also similar to the preclinical species. The half-lives of VEL are 2.3, 5.2 and 5.0 h in rat, dog and cyno respectively. Taken together VEL has a strong preclinical pharmacokinetic profile supporting its potential for once-daily dosing, which is requisite for our goal of selecting a compound with potential for co-formulation in a STR. Table 13 depicts the excellent potency of VEL compared to LDV26 for a range of clinically relevant GT1 RASs.27 Against these GT1 RASs, the potency of VEL is 31–105 pM for M28T, Q30H, Q30R and Y93C, 220–250 pM for L31M and Q30E, and 6.7 nM against Y93H. In GT1b the Y93H RAS has similar susceptibility to that of WT virus. Table 14 depicts the potency of VEL compared to LDV26 against GT2 and GT3 RASs.27 For VEL, GT2a RAS activities range from hypersensitive to a 2-fold loss versus WT replicon for L31M (GT2a J6, Table 10), K44R, N62V and N62S. In GT2b VEL potency ranges from 4 to 11 pM for the L31M, R44K, and P58S replicons. In GT3 VEL has potencies of 210 pM against A30K and 3.2 nM against Y93H. In GT3a the presence of A30K prior to treatment has been associated with virologic failure for a first generation NS5A inhibitor in combination with sofosbuvir.28,18 Overall, the activity of VEL against a range of GT1-3 RASs is improved over first generation NS5A inhibitors.13n Gratifyingly VEL has good bioavailability and a pharmacokinetic 11

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

half-life ranging from 12 to 23 h in healthy human volunteers, which is consistent with the once-daily dosing.29 We calculate the human bioavailability for VEL to be 50% from its clinically used solid dosage form;30 this exceeds the bioavailability from non-precipitating solution formulations in rat, dog and cyno monkey (Table 12). VEL has good human bioavailability despite breaking the dominant “rules” for bioavailability in the medicinal chemistry literature (Table 15).31,23 We did not follow a “rules-based” approach (ie one guided by calculated metrics) in our discovery of velpatasvir, and instead our discovery program was driven by measureable data such as potency and in vitro and in vivo pharmacokinetic data. A rules-based approach to bioavailability would not have informed the successful path to VEL shown in Table 10; this is most evident when comparing VEL to its 4-methyl isomer 36. All rulebased metrics in Table 15 are identical for VEL and 36, and fail to account for the striking differences in Fa% between these two compounds. Indeed, our first calculations of most values in Table 15 were performed after discovery of velpatasvir for discussion purposes for publication. The 24 h trough concentrations of VEL in healthy volunteers are above its average protein-adjusted EC50s for GT1-6 at all doses tested (lowest total dose 5 mg).29 Accordingly, VEL demonstrates high antiviral activity in GT1-4 HCV infected patients treated once-daily for three days in monotherapy. GT1a, 1b and GT2 infected patients achieved mean maximum VLR > 4 log10, and GT3 and GT4 HCV infected patients achieved > 3 log10 VLR. For example, in GT1a patients, three once-daily 100 mg monotherapy doses of VEL produced a VLR of 4.1 log10. Viral load suppression persisted at > 3 log10 for three days post the last dose. Five days post the last 150 mg dose GT2 patients remained suppressed ∼4 log10 below baseline.29 In the ASTRAL Phase 3 clinical trials the STR of sofosbuvir 400 mg and VEL 100 mg for a treatment duration of 12 weeks achieved an overall sustained virologic response (SVR, cure) rate of 98% in noncirrhotic or compensated cirrhotic patients infected with GT1-6 virus. SVRs broken down by genotype are as follows: GT1, 99%; GT2, 99%; GT3, 95%; GT4, 100%; GT5, 97%; GT6 100% and SVR rates were not affected by the presence of pre-existing RASs. Patients with 35 known subtypes, 13 new or mixed subtypes, and a wide-range of pre-existing RASs were enrolled and are represented in these high cure rates – even a GT7 patient (one of the few documented cases worldwide) was enrolled and cured. Adverse event rates and severity for VEL were comparable to placebo.32 Recently real-world efficacy studies of SOF/VEL have demonstrated comparable SVR rates to the ASTRAL clinical trials.33 The ASTRAL trials and real-world results demonstrate that the preclinical design and testing paradigm described herein for velpatasvir has successfully translated to pan-genotypic clinical efficacy. Voxilaprevir (VOX, an NS3/4A protease inhibitor) is combined with sofosbuvir and velpatasvir in an STR and achieves an overall SVR of 97% for previously treated GT1-6 patients who failed to achieve SVR.12 Velpatasvir is a potent, high resistance barrier, once-daily pangenotypic NS5A inhibitor, and is a component of Epclusa® and Vosevi®, the only pan-genotypic STRs. With these safe, simple and effective STRs most patients can be cured of HCV infection regardless of genotype or treatment history.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.04.027. References 1. Blach S, et al. Lancet Gastroenterol Hepatol. 2017;2:161. 2. (a) Ly KN, Hughes EM, Jiles RB, Holmberg SD. Clin Infect Dis. 2016;62:1287–1288; (b) Stanaway JD, Flaxman AD, Naghavi M, et al. Lancet. 2016;388:1081. 3. (a) Strader DB, Seeff LB. Clinical Liver Disease. 2012;1:6; (b) Hoofnagle JH, Seeff LB. N Engl J Med. 2006;355:2444; (c) North CS, Hong BA, Adewuyi SA, et al. General Hosp Psychiatry. 2012;35:122. 4. (a) Weisman R. Boston Globe, Vertex to stop selling hepatitis C drug Incivek. https:// www.bostonglobe.com/business/2014/08/12/vertex-stop-selling-hepatitis-drugincivek/El0jtOpH9I1CaIgQpSUKWO/story.html (Last accessed June 19, 2018). (b) Palmer, E. FiercePharma, Vertex's Incivek unseats Celebrex as fastest drug launch ever. https://www.fiercepharma.com/sales-and-marketing/vertex-s-incivek-unseatscelebrex-as-fastest-drug-launch-ever (last accessed June 10, 2018) (c) Herper, M., Forbes August 17, 2014 issue; The Top Drug Launches of All Time. https://www. forbes.com/sites/matthewherper/2015/07/29/the-top-drug-launches-of-all-time/# 6fe13a386512 (Last accessed June 19, 2018). (d) EP Vantage, February 21st, 2018. The biggest drug launches – hep C dominates but Tecfidera stands out. http://www. epvantage.com/Universal/View.aspx?type=Story&id=766560&isEPVantage=yes (Last accessed June 21, 2018). 5. Sofia MJ. Med Chem Rev. 2015;50:397. 6. (a) https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/205834s001lbl. pdf (Last accessed June 10, 2018). (b) Sofia, M. J.; Link, J. O. in Comprehensive Medicinal Chemistry III, Chackalamannil, S.; Rotella, D.; Ward, S.; Eds. Elsevier Ltd.: Amsterdam, NL 2017; 558. 7. Macdonald A, Harris M. J Gen Virol. 2015;96:727–738. 8. Blanco JL, Montaner JS, Marconi VC, et al. AIDS. 2014;28:2531–2539. 9. (a) Kowdley KV, Gordon SC, Reddy KR, et al. N Engl J Med. 2014;370:1879–1888; (b) Backus LI, Belperio PS, Shahoumian TA, Loomis TP, Mole LA. Hepatology. 2016;64:405–414. 10. (a) Link JO, Taylor JG, Xu L, et al. J. Med. Chem. 2014;57:2033 (b) Guo H, Kato D, Kirschberg TA, et al. Patent Application. WO 2010/132601 A1, 2010. 11. https://www.gilead.com/~/media/files/pdfs/medicines/liver-disease/epclusa/ epclusa_pi.pdf (Last accessed June 24, 2018). 12. (a) Taylor James G, Zipfel S, Ramey K, et al. Bioorg Med Chem Lett. 2019. https:// doi.org/10.1016/j.bmcl.2019.03.037 (in press) (b) https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/209195s000lbl. pdf (Last accessed June 10, 2018). 13. (a) GT1a (strain H77). (b) GT1b Con-1. (c) GT2a JFH1. (d) GT2a J6. (e) GT2a JFH1 subgenomic transient replicon cells with L31M mutation. (f) GT2b MD2b-1 NS5A. (g) GT3a S52 transiently transfected subgenomic HCV replicon. (h) GT3a S52 NS5A transient chimeric replicon based on GT1b Renilla luciferase (Rluc) backbone. (i) GT4a ED43. (j) GT4a ED43 NS5A transient chimeric replicons based on GT1b Rluc backbone. (k) GT5a SA13 NS5A (9-184) transient chimeric replicons based on GT1b Rluc backbone. (l) GT6a HK6 stable subgenomic HCV replicon. (m) GT6e D88 NS5A (9-184) transient chimeric replicons based on GT1b Rluc backbone. In these replicons a-c, g and i are stable subgenomic replicon cells; d and f are NS5A transient chimeric replicons based on GT2a JFH1 Rluc backbone. (n) Cheng G, Yu M, Peng B, et al. J. Hepatol. 2013, 58(suppl):S484. http://www.natap.org/2013/EASL/EASL_ 34.htm (Last accessed June 10, 2018). 14. Torres-Puente M, Cuevas JM, Jimenez-Hernandez N, et al. J Viral Hepat. 2008;15:188. 15. https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/56/hcv-classification (last accessed October 14, 2018). 16. Scheel TK, Gottwein JM, Mikkelsen LS, Jensen TB, Bukh J. Gastroenterology. 2011;140:1032. 17. (a) Bilello JP, Lallos LB, McCarville JF, La Colla M, Serra I, Chapron JM, Pierra C, Sandring DN, Seifer M. Antimicrob. Agents Chemother. 2014;58:4431–4442 (b) http://www.natap.org/2013/HCV/013113_03.htm (Last accessed October 14, 2018). 18. Gao M, Nettles RE, Belema M, et al. Nature. 2010;465:96–100. 19. Bacon EM, Cottell JJ, Katana AA, et al. Patent Application WO 2012/068234 2012 A2. 20. Bacon EM, Cottell JJ, Katana AA, et al. Patent Application. WO 2013/075029, 2013. 21. Additionally, to confirm that the compounds herein provide valid data in microsomal or hepatocyte stability assays we assess for sufficient recovery of the test compound under the assay conditions (either non-radiolabeled, or tritiated compounds for advanced compounds such LDV or VEL). For protein-binding dialysis assays (such as plasma versus buffer or plasma versus cell-culture medium) we confirm that the system has achieved equilibrium by comparing results from testing addition of compound to the plasma well versus in a separate experiment addition of compound to the buffer or cell-culture medium well. We then confirm that the experimental

Acknowledgements The authors thank Kathy Brendza, Nina Soltero and Gregg Czerwieniec for HRMS determination, and Keyu Wang, and Kevin M. Page for NMR determination. The authors would also like to thank the patients and their families as well as the study site staff who participated in the clinical trials in support of velpatasvir, Epclusa® and Vosevi®.

12

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

J.O. Link, et al.

22.

23. 24. 25. 26. 27.

conditions employed afford a matching partition ratio within experimental error. We also assess time to reach equilibrium in these experiments. (a) The typically utilized “bioavailability, F%” includes percent fraction absorbed (Fa%), along with gut metabolism and hepatic clearance. Fa% represents the gut absorption component, through a calculation removing hepatic clearance and gut metabolism (we assume no gut metabolism as a simplification in our calculation). For more on Fa% see: Rowland, M., Tozer, T. N. Clinical Pharmacokinetics and Pharmacodynamics: Concepts and Applications 4th ed., 2011 Wolters Kluwer Health/Lippincott William & Wilkins Philadelphia. (b) He YL, Murby S, Warhurst G, et al. J Pharm Sci. 1998;87:626 Taylor RD, MacCoss M, Lawson AD. J Med Chem. 2014;57:5845. GT1a and 1b, stable subgenomic Renilla luciferase replicons derived from GT1a H77 and GT1b Con-1 strain. Lipkus AH, Yuan Q, Lucas KA, et al. J Org Chem. 2008;73:4443–4451. Cheng G, Tian Y, Doehle B, et al. Delaney W. 2016;60:1847–1853. (a) Cheng, G.; Tian, Y.; Yu, M., et al. 2013 GS-5816, a second-generation HCV NS5A inhibitor with potent antiviral activity, broad genotypic coverage, and a high resistance barrier. EASL 48th Annual Meet, Amsterdam, The Netherlands, 24 to 28

28. 29.

30. 31.

32. 33.

13

April 2013. http://www.natap.org/2013/EASL/EASL_34.htm (Last Accessed 10/12/ 18). (b) Lawitz EJ, Dvory-Sobo H, Doehle BP, Worht AS, McNally J, Brainard DM, Link JO, Miller MD, Mo H. Antimicrob Agents Chemother. 2016;60:5368–5378 Sulkowski MS, Gardiner DF, Rodriguez-Torres M, et al. N Engl J Med. 2014;370:211. (a) Lawitz E, Freilich B, Link J, et al. J Viral Hepat. 2015:1; (b) Based on its improved resistance barrier, the mean maximal GT1 VLR for VEL exceeds that of LDV in monotherapy, see: Lawitz EJ, Gruener D, Hill JM, et al. J. Hepatol. 2012;57:24. Mogalian E, German P, Kearney BP, et al. The human bioavailability was calculated using the CL estimated from preclinical studies herein, and CL/F from the following. Antimicrob Agents Chemother. 2017;61:1. (a) Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Adv Drug Delivery Rev. 1997;23:3; (b) Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. J Med Chem. 2002;45:2615; (c) Ritchie TJ, Macdonald SJ. Drug Discov Today. 2009;14:1011. Asselah T, Bourgeois S, Pianko S, et al. Liver Int. 2018;38:443–450. von Felden J, Vermehren J, Ingiliz P, et al. Aliment Pharmacol Ther. 2018;47:1288.