Article pubs.acs.org/jmc
Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily Oral NS5A Inhibitor for the Treatment of Hepatitis C Virus Infection John O. Link,*,† James G. Taylor,† Lianhong Xu,† Michael Mitchell,† Hongyan Guo,† Hongtao Liu,† Darryl Kato,† Thorsten Kirschberg,† Jianyu Sun,† Neil Squires,† Jay Parrish,† Terry Keller,† Zheng-Yu Yang,† Chris Yang,‡ Mike Matles,‡ Yujin Wang,‡ Kelly Wang,‡ Guofeng Cheng,§ Yang Tian,§ Erik Mogalian,± Elsa Mondou,∥ Melanie Cornpropst,∥ Jason Perry,⊥ and Manoj C. Desai† †
Medicinal Chemistry, ‡Drug Metabolism, §Biology, ±Formulation and Process Development, ∥Clinical Research, and ⊥Structural Chemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States S Supporting Information *
ABSTRACT: A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an extended plasma half-life of 37−45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. 39 has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.
■
INTRODUCTION Hepatitis C virus (HCV) infection is a significant public health concern, with approximately 170 million infected individuals worldwide, and is the leading cause of liver transplant and hepatocellular carcinoma.1 HCV is the most common chronic blood-borne pathogen in the U.S., and the Center for Disease Control and the U.S. Preventative Services Task Force are aligned in recommending that all baby-boomers (individuals born between 1945 and 1965) undergo testing for HCV infection.2 Until recently, the standard of care for treatment of genotype 1 (GT1) infection (60% of total infections worldwide among the seven known genotypes, with both GT1a and GT1b as major subtypes)3 consisted of weekly pegylated interferon (PEG) injections and twice-daily oral ribavirin (RBV) for 24 or 48 weeks (duration based on response-guided therapy). PEG/ RBV treatments achieve up to 54−63% sustained virologic response (SVR) in GT1 patients,4 but treatment is accompanied by considerable toxicity including flu-like symptoms, depression, and anemia.5 Triple therapy containing PEG/RBV combined with three-times-daily dosing of the recently approved directacting antiviral (DAA) protease inhibitor telaprevir or boceprevir has improved the GT1 HCV SVR rates to 66−79% for treatment naı̈ve patients but with increased toxicities including rash (telaprevir) or grade 3 or 4 anemia.6 Prior null responders (patients who attained less than a 1 log viral load reduction on PEG/RBV) are not indicated for retreatment with PEG/RBV because they achieve SVR rates 600-fold potency enhancement, whereas the difference between phenyl and naphthyl linkers in the bis-benzimidazole series is 44 nM means that no inhibition was observed at this top well concentration.
more centrally positioned alkyne in 27 is improved 6-fold in potency. As in Table 1, introduction of alkynes in the linker affords lower potency relative to inhibitors with aromatic elements. Notably, the biphenyl in inhibitor 29 provides the highest level of potency among the nonfused central connectors in Tables 1 or 2. The high potency of biphenyl 29, along with the theme throughout Tables 1 and 2 that fused central ring systems consistently provide high potency (compounds 20, 21, and 25), prompted us to study constraint of the biphenyl to form tricyclic fused-ring systems. Initial SAR of central fused tricycles was studied in a symmetric bis-imidazole series to obviate tricycle desymmetrization and thereby simplify compound synthesis. Fluorene ring-linked inhibitor 30 (Table 3) suffered a mild loss in potency relative to biaryl 6 and posed stability concerns because the fluorene ring system readily underwent autoxidation upon standing. The F
dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Table 2. In Vitro Activitya
not well-tolerated. We postulated that a smaller, lipophilic blocking group such as a difluoromethylene group might provide an optimal connector. We decided to introduce this important modification directly in the imidazole/benzimidazole series of interest because of the synthetic challenge posed by introduction of the difluoromethylene group. Use of the difluoromethylene connector produced the most potent inhibitor in the optimized unsymmetric series, difluorofluorene 33 (EC50 = 40 pM). We measured the pharmacokinetics of 29 and 33 in rats and dogs (Table 4) and found that both inhibitors showed similar good half-lives in plasma, low systemic clearance (CL), and moderate volumes of distribution (Vss) that are greater than total body water volume. Unexpectedly, the modest 11% oral bioavailability of biphenyl inhibitor 29 was improved to over 35% in difluoroflourene 33. Incorporation of difluorofluorene in compound 33 therefore provided combined improvements in potency and bioavailability. Next, we assessed terminal heterocycle modifications in the symmetric bis-imidazole system. Piperidine 34 (Table 5) was less potent than the [2.2.1]azabicyclic inhibitor 35. Importantly, we found that 35 possessed a long half-life of 5.3 h in dog (Table 4). Table 5. In Vitro Activitya
a
See the Experimental Section for detailed assay protocols. bFor standard deviations, see the Experimental Section. cA value of >44 nM means that no inhibition was observed at this top well concentration.
Table 3. In Vitro Activitya
compd
A
EC50 (1a, nM)b
EC50 (1b, nM)
30 31 32 33
CH2 CO CMe2 CF2
0.094 0.30 1.2 0.040
0.013 0.018 0.014 0.003
a
See the Experimental Section for detailed assay protocols. standard deviations, see the Experimental Section.
b
compd
EC50 (1a, nM)b
EC50 (1b, nM)
34 35
0.45 0.21
0.005 0.009
a
See the Experimental Section for detailed assay protocols. standard deviations, see the Experimental Section.
b
For
Attracted by the favorable pharmacokinetic properties that the [2.2.1]azabicyclic ring system imparted in 35, we sought to understand the SAR of the azabicyclic ring system in the context of an unsymmetric core. The more synthetically accessible imidazole-biphenyl-benzimidazole core was chosen for this study. We discovered matched and mismatched sets where the potency is markedly better when the [2.2.1]azabicyclic ring system is paired with the benzimidazole in 36 rather than with the imidazole in 37 (Table 6). We considered this differential
For
oxidation product, fluorenone 31, lost significant potency (EC50 = 300 pM). Blocking oxidation with gem-dimethyl (32) lost even more potency, giving some credence to the concept that significant out-of-plane steric bulk as part of the fused linker is Table 4. Rat and Dog Pharmacokineticsa,b compd
species
CL (L/h/kg)
Vss (L/kg)
t1/2 (hr)
MRT (hr)d
%F
29
rat dog rat dog rat dog rat dog
1.04 ± 0.17 0.78 ± 0.29 0.42 ± 0.04 0.53 ± 0.04 0.70 ± 0.02 0.05 ± 0.007 0.75 ± 0.04 0.40 ± 0.26
1.76 ± 0.17 2.31 ± 0.37 0.93 ± 0.04 1.96 ± 0.03 0.98 ± 0.08 0.31 ± 0.007 1.81 ± 0.26 2.03 ± 0.92
1.57 ± 0.19 2.30 ± 0.28 1.83 ± 0.22 2.63 ± 0.18 1.49 ± 0.02 5.29 ± 0.60 2.07 ± 0.19 4.01 ± 0.82
1.71 ± 0.16 3.00 ± 0.27 2.21 ± 0.21 3.69 ± 0.29 1.40 ± 0.07 6.60 ± 1.10 2.42 ± 0.22 5.47 ± 1.01
11.5 ± 8.7 n.d.c 36.7 ± 3.2 n.d.c 35.2 ± 10 n.d.c 26.3 ± 9.0 n.d.c
33 35 36
a
All parameters except for %F are from intravenous dosing. bSee the Experimental Section for detailed assay protocols. cn.d., not determined. dMean residence time. G
dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Table 6. In Vitro Activitya
compd
Y′
A
Y
EC50 (1a, nM)b
EC50 (1b, nM)
36 37 38
absent CH2 absent
absent absent CF2
CH2 absent CH2
0.16 0.66 0.056
0.006 0.021 0.004
a
See the Experimental Section for detailed assay protocols. standard deviations, see the Experimental Section.
b
For
SAR to be an important result; it further demonstrated to us that the unsymmetric core series opened opportunities for inhibitor optimization that would not be available if structures were limited to a symmetric cores. Unlike in the symmetric core system, the [2.2.1]azabicyclic ring system in the matched unsymmetric case did not demonstrate a loss in potency relative to the pyrrolidine (36 vs 29). Importantly, the extended pharmacokinetic half-life that the [2.2.1]azabicyclic ring system provided in symmetric core inhibitor 35 translated in unsymmetric core inhibitor 36; both the rat and dog plasma half-lives are improved in 36 over those of pyrrolidine analogue 29 having the same core (Table 4). To improve upon the potency of 36, biphenyl was replaced by difluorofluorene, affording inhibitor 38 (EC50 = 56 pM). 38 has a protein-adjusted EC50 of 784 pM, corresponding to a protein-binding shift of 14fold.36 During final optimization, replacement of the pyrrolidine in 38 with a spirocyclopropylpyrrolidine afforded 39 (ledipasvir, GS5885), the most potent inhibitor in the series (Figure 3).21 39 has GT1a and 1b EC50 values of 31 and 4 pM, respectively, and protein-adjusted EC50 values of 210 pM (GT1a) and 27 pM (GT1b). The protein-binding shift is 6.7-fold and is improved relative to pyrrolidine 38. Compound 39 is highly protein-bound both in human serum and in the cell-culture medium (containing 10% BSA) of the replicon assay (1.1% unbound fraction in cellculture medium). Accounting for the low unbound drug concentration in the replicon assay, the intrinsic EC50 of 39 is 310 fM for GT1a and 40 fM for GT1b. 39 is remarkable not only on the basis of its high replicon potency but also on the basis of its low clearance, good bioavailability, and long half-lives (4.7−10.3 h) in rat, dog, and monkey and low predicted clearance in human (0.012 L/h/kg) (Figure 3 and Table 7). In 14 day toxicology studies in rats and dogs with compound 39, there were no significant adverse findings. We selected 39 for clinical development, projecting that it would be highly efficacious in the treatment of HCV infection with a long half-life suitable for once-daily dosing. The pharmacokinetics of 39 were studied in fasted healthy volunteers in a placebo-matched double-blind phase 1 clinical trial at oral doses of 3, 10, 30, 60, and 100 mg (Figure 4 and Table 8) and were dose-proportional over the range tested. Long mean plasma half-lives of 37−45 h were observed. Twenty-four hour postdose drug concentrations were 12−470-fold over the GT1 protein-adjusted EC50 and remained over the GT1a proteinadjusted EC50 well past 24 h. These results were consistent with the potential for both once-daily dosing and low risk for
Figure 3. Structure of 39 (A), rat, dog, and cyno pharmacokinetic curves (B), potency and microsomal stability. EC50 values: GT1a = 31 pM, GT1b = 4 pM, intrinsic GT1a = 0.31 pM, intrinsic GT1b = 0.04 pM, protein-adjusted GT1a = 210 pM, protein-adjusted GT1b = 27 pM. Metabolic stability rat, dog, cyno and human microsomes:
