Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of

Article pubs.acs.org/jmc

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing Li-Qiang Sun,*,† Eric Mull,† Barbara Zheng,† Stanley D’Andrea,† Qian Zhao,† Alan Xiangdong Wang,† Ny Sin,† Brian L. Venables,† Sing-Yuen Sit,† Yan Chen,† Jie Chen,† Anthony Cocuzza,† Donna M. Bilder,† Arvind Mathur,⧫ Richard Rampulla,⧫ Bang-Chi Chen,⧫ Theerthagiri Palani,△ Sivakumar Ganesan,△ Pirama Nayagam Arunachalam,△ Paul Falk,‡ Steven Levine,‡ Chaoqun Chen,‡ Jacques Friborg,‡ Fei Yu,‡ Dennis Hernandez,‡ Amy K. Sheaffer,‡ Jay O. Knipe,∥ Yong-Hae Han,∥ Richard Schartman,∥ Maria Donoso,∥ Kathy Mosure,∥ Michael W. Sinz,∥ Tatyana Zvyaga,# Ramkumar Rajamani,§ Kevin Kish,● Jeffrey Tredup,● Herbert E. Klei,● Qi Gao,○ Alicia Ng,○ Luciano Mueller,∇ Dennis M. Grasela,⊥ Stephen Adams,▲ James Loy,∥ Paul C. Levesque,▲ Huabin Sun,▲ Hong Shi,▲ Lucy Sun,▲ William Warner,▲ Danshi Li,▲ Jialong Zhu,▲ Ying-Kai Wang,◊ Hua Fang,◊ Mark I. Cockett,‡ Nicholas A. Meanwell,† Fiona McPhee,‡ and Paul M. Scola† †

Department of Discovery Chemistry, ‡Department of Virology Discovery Biology, §Department of Computer-Assisted Drug Design, ∥ Department of Pharmaceutical Candidate Optimization, #Department of Lead Discovery and Optimization, and ◊Department of Lead Evaluation, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ⊥ Department of Clinical Development Infectious Disease, ∇Department of Mechanistic Biochemistry, and ●Department of Protein Science and Structure, Bristol-Myers Squibb Research and Development, P.O. Box 5400 Princeton, New Jersey 08543, United States ○ Department of Pharmaceutical Development, Bristol-Myers Squibb Research and Development, 1 Squibb Drive, New Brunswick, New Jersey 08903, United States ⧫ Department of Discovery Synthesis, Bristol-Myers Squibb Research and Development, Route 206 and Provinceline Road, Princeton, New Jersey 08543, United States △ Biocon Bristol-Myers Squibb R&D Center, Biocon Park, Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India ▲ Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States S Supporting Information *

ABSTRACT: The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure−activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

Received: June 1, 2016

© XXXX American Chemical Society

A

DOI: 10.1021/acs.jmedchem.6b00821 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

■

Article

INTRODUCTION Hepatitis C virus (HCV) is a chronic viral infection which afflicts approximately 200 million people worldwide.1−5 In the United States (US) alone, some 4 million people are believed to be infected, with 25 000 to 40 000 new cases reported annually. HCV is one of the most common causes of liver disease and has emerged as a leading cause of cirrhosis, hepatocellular carcinoma, and liver transplants. Moreover, the annual mortality attributed to HCV in the US has recently been shown to surpass the combined number of deaths associated with 60 notifiable infections.6−8 HCV has a single-stranded, positive sense RNA genome that encodes for three structural proteins (C, E1, and E2), a small viroporin designated as p7, and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS3 protease is comprised of the 181-residue amino terminus of the full-length protease-helicase that is optimally activated by forming a complex with its cofactor, NS4A. The NS3/4A enzyme complex processes the nonstructural elements of the HCV polyprotein to yield the mature viral replication proteins and is an essential element of the viral lifecycle. This virally encoded enzyme is an established target for drug discovery efforts, and a number of inhibitors of this protease have demonstrated clinical efficacy of which a subset have been approved by the U.S. Food and Drug Administration.9 Recently we reported the discovery of BMS-605339 (1), a potent inhibitor of HCV NS3 protease that was advanced into early clinical development.10 The antiviral activity of this compound was favorable: 1 exhibited a dose-dependent effect on viremia with a mean 1.8 log10 IU/mL reduction in viral load observed in HCV genotype-1-infected subjects measured 12 h after a single 120 mg oral dose. Despite this significant antiviral response, the clinical development of 1 was halted due to a cardiac liability which manifested as mild bradycardia, PR interval prolongation, and junctional escape rhythms, all of which were asymptomatic. In response to these findings, efforts were focused on the discovery of a protease inhibitor that presented preclinical properties similar to 1 but with an optimized cardiovascular (CV) profile. This objective proved to be challenging since the profile of 1 in preclinical species did not indicate the potential for CV findings at therapeutically relevant exposures. For example, 1 induced mild bradycardia in the dog at plasma levels of 12 μM; however, in human subjects similar findings were reported at drug plasma levels as low as 150 nM. This observation suggested that humans were more sensitive to the CV effects of 1 than the preclinical toxicology species. After some experimentation, however, an ex vivo toxicology model, specifically the Langendorff isolated rabbit heart preparation, was employed that enabled a more detailed assessment of the CV effects of 1.11,12 For example, perfusing a buffered solution of 1 directly onto an isolated rabbit heart preparation induced bradycardia in a doseand time-dependent manner with a no-observed-effect level (NOEL) observed at concentrations of ∼100 nM of 1. Thus, the CV effects of 1 in humans were reproduced in the isolated heart but at protein-free concentrations of >100 nM. With this finding, the Langendorff rabbit heart model was integrated into the program screening tier with the objective of identifying a compound that did not induce CV changes in this ex vivo model. This modification to the program screening tier was critical as it enabled the discovery of asunaprevir (2).12 Preclinically, 2 demonstrated an improved virology profile when compared to 1, with enhanced liver exposure observed after PO dosing to rat, dog, and monkey. The higher liver-to-plasma ratios and improved potency translated to the clinic with 2 providing a similar antiviral

response to 1 but at lower plasma levels. Importantly, the CV profile of 2 in humans was free of any of the findings associated with 1, which provided confidence in the predictive value of the Langendorff isolated heart model for this chemical series. The early clinical data obtained with 2 warranted full development of this compound in combination with daclatasvir (BMS-790052, 3), a NS5A replication complex inhibitor; this antiviral combination was approved for the treatment of individuals with chronic genotype-1b hepatitis C infection in Japan in July of 2014, becoming the first alloral, interferon- and ribavirin-free regimen to be marketed for this indication.12−14 In addition, 2 is currently being evaluated clinically in combination with 3 and the non-nucleoside NS5B polymerase inhibitor beclabuvir (BMS-791325, 4). This triple antiviral cocktail has proven effective for the treatment of individuals infected with both genotype-1a and genotype-1b infections, with the former the more challenging patient population to treat with direct-acting antiviral agents (DAAs).15

Following the discovery of 2, a compound was sought that recapitulated the virological and CV profile of 2 but which had an improved PK profile across species that would predict for a lower efficacious dose in humans infected with HCV genotype-1. B



Article

predictions were derived from drug pharmacokinetics in plasma, a key objective in a follow-up to 2 was the identification of a compound that demonstrated high concentrations of drug in the liver after oral dosing while maintaining a PK profile in plasma that was predictive of once-daily dosing.

A compound with this profile was viewed as a mitigation strategy should long-term development issues emerge with 2. Notably, a lower dose would ultimately facilitate the development of a once-daily, single tablet formulation combining a protease inhibitor with inhibitors of NS5A and NS5B for the treatment of genotype-1 infections. Herein, our efforts toward the discovery of a third protease inhibitor with the potential for a low QD dose in humans are described. In contemplating compounds that might provide an advantage over 2, the relatively high clearance of this compound in rat provided an early point of focus. Interestingly, the high clearance observed with 2 and close analogues thereof correlated with correspondingly high liver levels of drug. Given that the primary site of replication of HCV is in hepatocytes, high liver exposure was seen as an important property to maintain in a third generation compound. However, as human dose

■

RESULTS AND DISCUSSION Initial efforts were focused on exploration of structure−activity relationships (SARs) at the P1 subsite (see 1 for the designation of subsites). In earlier studies, P1 analogues had been identified with lower clearance, although other properties had been compromised. More specifically, in the previously disclosed effort that led to the discovery of 2, the effect of saturating the P1 vinyl moiety had been evaluated, and 5 was found to be slightly less active in the biochemical and replicon assays (Table 1).9h,i,16,17

Table 1. Antiviral, Rat PK, and CV Profiles of Compounds 1−8

a

GT-1a = genotype-1a; HCV NS3 enzyme inhibitory activity was assessed according to the conditions described in ref 16. bGT-1b = genotype-1b; HCV replicon inhibitory activity was assessed in the presence of 10% fetal bovine serum (FBS) according to the conditions described in ref 17. c IV/PO dosing levels: 5/15 mg/kg, n = 3. Vehicle: PEG-400/ethanol (90/10, v/v). dOral bioavailability. ePharmacokinetic area under curve. f Pharmacokinetic clearance. gPlasma half-life. hCompounds were dosed at 10 μM for 20 min. i10 μM for 60 min. jHR = heart rate. kSNRT = sinoatrial node recovery time. % Changes in HR and SNRT were meant to be the maximal changes over the course of the experiments. When vehicle only was dosed for 20 min, changes in HR and SNRT were −3% and 4%, n = 3, respectively. C



Article

microsomes (HLM) (t1/2 = 83 min) was significantly improved over 7 (t1/2 = 5 min) and approximated that of 6. The favorable in vitro properties found with 8 prompted further evaluation of in vivo PK and in vitro CV profiling studies. The PK profile of 8 in the rat represented some improvement compared to 5. For example, while oral bioavailability and liver levels at 24 h postdose were similar for these two analogues, the clearance of 8 was 5 mL/min/kg, ∼3-fold lower than that observed for 5. The improved clearance rate for 8 likely contributed to the ∼2.5-fold increase in plasma exposure observed for this compound when compared to 5. Upon perfusion in the isolated rabbit heart model, 8 had a significant effect on sinus node recovery time (SNRT) but minimal effects on heart rate (HR) over the 20 min evaluation period at a bath concentration of 10 μM. While the experimental results with 8 in this model represented an improvement over 5, the data nonetheless suggested that this compound posed a greater risk with respect to potential CV issues compared to the clinical lead 2, and hence further optimization would be required. In a more detailed comparison of the profiles of 8 and the 2, the former demonstrated a 6-fold reduction in intrinsic enzyme inhibitory activity and 3-fold weaker activity in the replicon than the latter. However, 8 demonstrated a significant advantage with respect to PK in rat with clearance 8-fold less than 2, and a plasma AUC over 24 h that was ∼20-fold greater. Moreover, the oral bioavailability of 8 was substantially improved compared to 2 (44% versus 12%), and the liver levels for 8 measured at 24 h postdose were more than twice that observed for 2. Hence, moving forward, efforts were directed toward further optimization of the bi-cyclopropyl series with the objective of improving replicon inhibitory activity and attenuating the unfavorable aspects of the CV profile of 8. To address these objectives, SARs at the P4 subsite of 8 were explored. Replacement of the carbamate tert-butyl moiety at P4 by an isopropyl group, compound 9, resulted in a 2−3-fold loss of potency in the enzyme and replicon assays (Table 2). However, replacement of the tert-butyl cap of 8 with a cyclobutyl moiety (10) maintained both enzyme and cellular activities. Collectively, these findings were suggestive that a threshold of lipophilic bulk was required at the P4 position for optimal activity, and, building upon this hypothesis, the P4 cyclopentyl series was explored. Interestingly, while the parent compound 11 was similar in activity to the parent P4 Boc analogue 8, the addition of a simple methyl group to the cyclopentyl ring, as in 12, improved activity 3−4-fold in the biochemical (IC50 = 5 nM) and replicon (EC50 = 3.7 nM) assays. Unfortunately, the enhanced antiviral activity observed with 12 was associated with a significant loss in liver microsomal stability, which prompted the preparation of derivatives of 11 designed to block potential metabolic soft spots on the cyclopentyl moiety. To this end, the cyclopropanated analogue 13 was prepared, which, compared to progenitor 8, resulted in a significantly longer in vitro half-life in HLM, t1/2 = 118 min, while also providing a noticeable improvement in both GT-1a intrinsic activity (6-fold) and GT-1b cell-based activity (2-fold). However, additional profiling revealed that 13 was an inhibitor of cytochrome P450 3A4 (CYP 3A4) with an EC50 value of 2 μM. While this level of inhibition is considered to be moderate, high and prolonged liver exposure of the compound was anticipated in HCV-infected subjects, presenting a risk for drug−drug interactions in combination therapy that precluded further evaluation of this compound. Nevertheless, the discovery of 13 was viewed as a significant advancement since it demonstrated that the targeted antiviral activity combined with enhanced microsomal stability could be achieved in the P1 bi-cyclopropyl series by optimizing the P4 functionality.

However, the PK profile of this compound in the rat was improved compared to 2 since the clearance of 5 was determined to be 18 mL/min/kg, 2-fold lower than that observed for 2.9h,i The improved clearance rate for 5 likely contributed to the ∼3-fold increase in half-life and ∼7-fold increase in plasma exposure observed for this compound compared to 2. Liver levels of 5 determined 24 h after dosing were 40 μM, approximately 3-fold higher than that recorded for 2, and the oral bioavailability was 43%, a more than 3-fold improvement when compared with 2. The improvement in PK properties for 5 compared to 2 was compelling given the relatively small structural change that distinguishes this matched pair. As previously described,12a,18 in vivo metabolism studies had provided evidence that the dominant clearance pathway for 2 was uptake of the parent compound into the liver. Data from subsequent in vitro studies were suggestive that the liver uptake process was modulated by transporters, thereby providing an explanation for the relatively low plasma levels and high liver levels observed in vivo for 2. Hence, in contemplating the differences in PK profiles between 2 and 5, (P1 vinyl vs ethyl) the simple change in hybridization of the P1 substituent in these compounds was thought to have impacted the transport of compound from plasma into the liver.9h,12a The observed increase in bioavailability of 5 compared to 2 is consistent with this hypothesis. It should be noted that compound 5 had been dropped from contention as a clinical candidate based on the results from the cardiovascular screen, which implicated this compound as presenting a substantial risk for cardiac issues. Although compound 5 was limited in its progression because of the CV liability, it nonetheless served as a starting point for additional analogue synthesis. Optimizing SARs at the P1 position proved to be challenging as seemingly small modifications at this site led to a substantial erosion in potency. This was due, in part, to the relatively small invagination that structurally defines the S1 subsite. For example, replacing the P1 ethyl moiety of 5 with an isopropyl group (6) led to a significant reduction in biological activity (Table 1). In contrast, the corresponding unsaturated isomer of 6, the isopropenyl derivative 7, was potent in both the biochemical assay and the replicon screen. The matched pair comparison between 6 and 7 illustrates the significance of a change in the degree of saturation and therein carbon hybridization on biological activity. Unfortunately, the gain in activity observed with 7 was offset by a substantial loss in liver microsomal stability. While detailed metabolic identification studies were not conducted on 7, we hypothesized that the methyl moiety at P1 may be subject to P450-mediated metabolism. In considering an immediate direction with respect to further analogue efforts, a model of both the active P1 isopropenyl compound 7 and the corresponding isopropyl analogue 6 was considered (Figure 1). A comparison of these analogues in the active site suggested that the loss in activity for 6 was driven by a lack of fit to the curvature of the S1 pocket as defined by residues L135, K136, S138, and F154 (Figure 1b). This unfavorable interaction was circumvented in the case of 7 by the presence of the olefin moiety at P1, which restricted the three carbon atoms of the vinyl methyl functionality to a single plane (Figure 1c). On the basis of these observations, the construction of a cyclopropyl moiety as the substituent attached to the P1 cyclopropyl ring as in 8 was considered. Modeling of 8 bound to the enzyme supported this hypothesis as the cyclopropyl ring docked well in the S1 pocket (Figure 1d). In the event, the activity of the bi-cyclopropyl analogue 8 in the biochemical and replicon assay was similar to 7. Moreover, the in vitro metabolic profile of 8 in human liver D



Article

Figure 1. (a) Model of 2 bound to the active site of HCV NS3 protease. (b−d) Models of the P1 element of analogues 6, 7, and 8 bound to the S1 subpocket of HCV NS3 protease.

Follow-up SAR studies continued to focus on P4 in an effort to identify a replacement group that captured the potency and stability observed with 13 but was free of the CYP 450 inhibition observed with this analogue. The observation noted above wherein lipophilicity at P4 was critical in securing optimal cellbased activity encouraged the consideration of fluorinated P4 moieties. The enhanced metabolic stability often observed with fluorination was considered as a favorable attribute.19 This effort was initiated by replacing the P4 tert-butyl group in 8, with a trifluoro tert-butyl group to afford 14. This analogue demonstrated similar activity in the enzyme inhibitory assay as 8 but was 9-fold more potent in the replicon. Compound 14 also exhibited an increase in liver microsomal stability when compared to 8. The in vivo profile of 14 in the rat was quite similar to 8, highlighted by good oral bioavailability and low clearance. However, the CV profile of 14 in the isolated heart model was less favorable than that found for 8, with this P4 halogenated moiety associated with a more significant change in HR and SNRT. Nonetheless, 14 was considered to be an

important lead since the in vitro and in vivo (rat PK) profiles were favorable, and additional SAR studies were focused on further refining this P4 trifluoromethyl cap. For example, removal of a single fluorine atom from the P4 group of 14 provided the difluoro tert-butyl analogue 15 which was 30-fold less potent in the replicon. The loss in whole cell activity with 15 was attributed to the additional polarity associated with the difluoromethyl moiety compared to the symmetrically functionalized trifluoromethyl group of 14.19e A similar outcome resulted when the diastereotopic methyl groups in the P4 cap of 14 were removed to give 16, with replicon potency diminished by 25-fold (EC50 = 48 nM) compared to 14. As was observed with 15, the reduced whole cell activity associated with 16 was considered to be a consequence of an increase in polarity, realized in this case by a reduction in lipophilicity at the P4 position. Interestingly, the loss of replicon activity observed with 15 and 16 was not associated with a reduction in intrinsic activity as measured in the enzyme inhibition assay. This observation prompted further exploration of SAR around the P4 cap of 14; specifically, the removal of a E



Article

Table 2. GT-1a/1b Inhibitory Activity, t1/2 in HLM, Rat PK, and CV Profiles of P4-Modifed Analogues 8−18

a IV/PO dosing levels: 5/15 mg/kg, n = 3. Vehicle: PEG-400/ethanol (90/10, v/v). bCompounds were dosed at 10 μM for 20 min. c10 μM for 60 min. % Changes in HR and SNRT were meant to be the maximal changes over the course of the experiments. dMeasurement not obtainable due to inability to stimulate hearts. When vehicle only was dosed for 20 min, % change in HR and SNRT were −3 and 4, n = 3, respectively. eHCV NS3 enzyme inhibition was assessed according to the conditions described in ref 16. fHCV replicon inhibition was assessed in the presence of 10% fetal bovine serum (FBS) according to the conditions described in ref 17.

the activity of 18 can be further appreciated by comparing the profile of this compound with that of 9, a nonhalogenated P4 isopropyl analogue. Specifically, replacement of the P4, pro-R methyl of 9 with a CF3, provided 18, and therein a 10-fold increase in activity in the biochemical assay and a corresponding 13-fold increase in the replicon assay. In contrast, replacement of the pro-S methyl of 9 with a CF3 group provides 17 as a compound with similar activity to the progenitor. Hence, the stereoselective introduction of a CF3 group to the P4 subsite resulted in the identification of a compound with potent antiviral activity and excellent microsomal activity. Compound 18 was profiled in more detail and exhibited moderate clearance (11.5 mL/min/kg) and a long half-life of

single methyl group, examined in the context of 17 and 18 which were prepared as P4 stereoisomers epimeric at the CF3-bearing carbon. These compounds proved to be significantly different from each other in their activity in both biochemical and whole cell assays. For example, 18, which bears the (R)-configuration at P4, was found to be 6-fold more potent in the biochemical assay and 30-fold more potent in the cell-based assay than its isomer 17 which bears the P4 (S)-configuration. Molecular modeling studies of 17 and 18 suggested that the (R)-configured P4 cap of 18 can adopt a preferred binding mode in the S4 pocket that allows optimal van der Waals contact with the enzyme surface, while this orientation is not accessible to 17 (Figure 2). The significance of fluorination to F



Article

Hence, at this point, SAR efforts migrated to the P3 position of 18 in an effort to further optimize the CV properties of this compound while maintaining the favorable potency and PK attributes. The final element examined in order to improve the in vivo CV profile of 18 focused on modification of the P3 substituent, and the series of analogues probing alkyl substituents at this position are summarized in Table 4. These data demonstrate that the P3 moiety is an important modulator of potency, PK, and CV. For example, replacement of the tert-butyl-glycine of 18 with valine (23) had a negligible impact on inhibitory activity; however, the PK profile of 23 was significantly compromised when compared to 18. Moreover, similarly close analogues such as the cyclopropyl derivative 24, the isoleucine-based 25, the cyclobutyl analogue 26, and the 3-pentyl homologue 27 each resulted in a reduction of antiviral potency. However, the cyclopentyl and cyclohexyl derivatives 28 and 29 proved to be potent antiviral agents and demonstrated PK properties in the rat that were similar to 18. Compounds 28 and 29 were investigated in the rabbit isolated heart model, and both analogues exerted a minimal effect on HR and SNRT during the 20 min perfusion period at a bath concentration of 10 μM (Table 4). When the perfusion time was extended to 60 min in a follow-up study, again only mild effects on HR and SNRT were observed. These findings were similar to those observed with both vehicle and 2, while the concentration of 28 and 29 in atrial tissues, measured at the end of the experiment, was significantly higher (28 = 225 μM, 29 = 204 μM) relative to 2 (61 μM). The CV findings described herein, in conjunction with those described as part of the identification of 2, add further to the suggestion that no single structural feature is responsible for the CV signal but is rather an effect based on the overall composition of the molecule. Compounds 28 and 29 were subsequently evaluated in the dog and cynomolgus monkey where both compounds demonstrated good PK profiles, with lower clearance, enhanced bioavailability, significantly improved plasma exposure, and longer half-lives relative to 2 and 18. As a point of differentiation, after PO dosing to the cynomolgus monkey at 10 mg/kg, 28 showed signs of gastrointestinal (GI) irritation (mild vomiting and diarrhea), while no signs of GI tract irritation were noted for 29. While this observation was anecdotal in nature since it was not part of a formal toxicology study, it nonetheless shifted the focus toward 29, and this compound was further profiled, including a more extensive in vitro assessment of properties and potential liabilities as well as toxicology studies in both the rat and dog. The spectrum of genotype inhibition associated with 29 was assessed by determining IC50 values toward HCV NS3 protease complexes representative of six of the major genotypes of HCV, and these data are summarized in Table 6.12b In these assays, the profile of 29 was comparable to 2, as both compounds demonstrated single digit nanomolar activity against genotypes 1, 4, 5, and 6 with a loss in potency observed against genotypes 2 and 3. The activity of 29 and 2 as inhibitors of HCV RNA replication was measured using replicons representing genotypes 1a and 1b, as well as the 2a JFH-1 strain. In addition, the effects of 29 toward hybrid replicons encoding the NS3 protease domain representing genotypes 2b, 3a, and 4a were also determined.12b As shown in Table 7, the replicon profile of compound 29 across genotypes was similar to that for 2 and a recapitulation of the enzymatic activity of these compounds. The activity of analogues 2 and 29 against resistant mutants were similar in nature, with a significant loss in activity observed toward specific viruses, data summarized in Table 8.20 The similar virology profile of compounds 2 and 29 was not surprising since these analogues

Figure 2. (a) Model of the (S)-configuration CF3 group of 17 bound to the S4 groove. (b) Model of the (R)-configuration CF3 group of 18 bound to the S4 groove.

10.3 h after IV dosing to rats at a dose of 5 mg/kg. Following oral administration at a dose of 15 mg/kg, 18 showed significantly higher liver and plasma exposure relative to 8, with a liver concentration of 61.6 μM measured at 24 h postdose and a plasma AUC of 13.8 μM·h. Adding further to the interest in 18, the PK profile in the dog was similarly improved over 2, while cynomolgus monkey PK was comparable. When 18 was perfused to isolated rabbit hearts over a 20 min period at a concentration of 10 μM, only a mild decrease in HR and a mild increase in SNRT was noted. In a follow-up in vivo study evaluating the effect of 18 on the cardiac electrophysiology of anesthetized rabbits, no significant decrease in HR was observed. It should be noted, however, that both the QRS and HV (the interval between the His wave and the ventricular wave) intervals were prolonged in one out of three rabbits, changes that diminished slowly after completing the infusion. This finding was viewed as being consistent with the mild-to-moderate sodium channel inhibition observed with 18 in a patch-clamp assay (72% inhibition at 10 μM (1 Hz); 76% inhibition at 10 μM (4 Hz)), and progression of 18 was halted. Nonetheless, compound 18 represented a milestone compound as the targeted properties had been achieved with respect to potency, in vitro metabolic stability, and in vivo PK in both the rat and dog. Interestingly, the targeted properties in 18 had been captured by the introduction of relatively small structural changes at P1 and P4, and with these elements established, the next phase of the study focused on consideration of combining structural modifications at P2* and P3. Previous SAR studies at P2* had demonstrated that small structural changes to this element of the pharmacophore could exert a substantial impact on both the PK and CV properties.12a Consequently, the structural modifications that were considered for synthesis and evaluation at this position were limited in scope. Replacement of the C4-methoxy, C7-chloro isoquinoline of 18 with the C-3, C-6 dimethoxy analogue resulted in 19, a compound with enhanced potency but with poor exposure after oral dosing to rats. Additionally, when 19 was evaluated in the rabbit isolated heart model, a decrease in HR was noted, to the extent that additional parameters could not be measured. Even small structural perturbations to the P2* element of 18 proved to be unfavorable. For example, moving the C-7 chloro of 18 to either the C-5 or C-6 position of the isoquinoline resulted in compounds 20 and 21, both of which retained potent antiviral activity but demonstrated a substantial increase in clearance after IV dosing to rats. In the case of 20, reinstalling the C-7 chloro group did not favorably modulate PK since the clearance of 22 remained high in the rat (40 mL/min/kg). These findings recapitulated the previously noted SAR trends with respect to PK and CV effects, and illustrated the importance of maintaining the P2* functionality in 18 (and 2) in efforts to further optimize this chemical series. G



Article

Table 3. GT-1a/1b Inhibitory Activity, t1/2 in HLM, Rat PK, and CV Profile of P2* Analogues 19−22

IV/PO dosing levels: 5/15 mg/kg, n = 3. Vehicle: PEG-400/ethanol (90/10, v/v). bCompounds were dosed at 10 μM for 20 min. % Changes in HR and SNRT were meant to be the maximal changes over the course of the experiments. cMeasurement not obtainable due to inability to stimulate hearts. When vehicle only was dosed for 20 min, % change in HR and SNRT were −3 and 4, n = 3, respectively. dHCV NS3 enzyme inhibition was assessed according to the conditions described in ref 16. eHCV replicon inhibition was assessed in the presence of 10% fetal bovine serum (FBS) according to the conditions described in ref 17. a

revealed benign effects similar to those measured with 2: no effect on either HR, SNRT, QRS, or PR interval was noted, while mild prolongation of QTcf, but not QTcv, was observed at the end of the dosing period. The plasma concentration of 29 in this experiment (253 μM) matched that found with 2 (240 μM) at the same dose and was 10-fold higher than the concentration at which 1 induced bradycardia in this model. Compound 29 was also evaluated in a single-dose, cardiovascular telemetry study in dogs at 30 and 100 mg/kg, which resulted in no significant findings with respect to HR or related CV parameters. Collectively these results classified 29 as a compound with minimal CV risk. Compound 29 was advanced through human CYP 450 inhibition assessment with no significant liability identified. In addition, the cytotoxic effects of 29 toward HepG2, HuH-7, HEK 293T, HeLa, and MRC5 cell lines was found to be low, with CC50 values ranging from 8 to 14 μM. Compound 29 was negative in an AMES assay with and without S9 activation. This favorable profile for 29 led to the initiation of toxicology studies in the rat and the dog. A single dose exploratory toxicokinetic study conducted with 29 in rats at doses of 30, 100, and 300 mg/kg surfaced no significant issues. Similarly, a single dose toxicokinetic and tolerability study in

share a common silhouette when bound to the enzyme. More specifically, 29 is 106-fold less potent toward GT-1b NS3 incorporating an A156V change, 32-fold less active toward D168E GT-1b NS3 protease, 1900-fold less potent toward GT-1b NS3 with a D168V mutation and 65-fold less active toward a GT-1a NS3 enzyme incorporating R155K. It should be noted that activity against GT 2b and 3a, as well as the resistant variants noted, was ultimately addressed by employing a macrocyclization strategy, and these findings will be communicated in due course.21 Compound 29 was shown to be specific for the HCV NS3 serine protease, with no significant inhibitory activity measured against the closely related GBV-B NS3 protease (IC50 > 25 μM) and human leukocyte elastase (IC50 = 21 μM) enzymes or bovine viral diarrhea virus (BVDV) and HIV-1 in cell culture. Further in vitro profiling included evaluation of 29 in a hERG patch-clamp assay which revealed an IC50 of 12.5 μM, while in a sodium channel patchclamp assay 46.4% inhibition was observed at a concentration of 10 μM (4 Hz). The result for 29 in the sodium channel assay represented an improvement over that for 18, a more favorable outcome that was recapitulated in vivo. Specifically, evaluation of 29 in anesthetized rabbits given as a 30 mg/kg IV infusion H



Article

Table 4. GT-1a/1b Inhibitory Activity, t1/2 in HLM, and CV Profile of P3-Modified Derivatives 23−29d

10 μM for 60 min. % Changes in HR and SNRT were meant to be the maximal changes over the course of the experiments. When vehicle only was dosed for 20 min, the % change in HR and SNRT was −3 and 4, n = 3, respectively. bHCV NS3 enzyme inhibition was assessed according to the conditions described in ref 16. cHCV replicon inhibition was assessed in the presence of 10% fetal bovine serum (FBS) according to the conditions described in ref 17. dAstertisk (*): Compounds were dosed at 10 μM for 20 min. a

Table 5. Pharmacokinetic Properties of Compounds 2, 8, 18, 19, 20, 28, and 29 in the Rat, Dog, and Cynomolgus Monkeya Compd Rat t1/2 (h)b Cl (mL/min/kg)c F (%)d AUC (μM·h)e PO liver @ 24 h (μM) Dog t1/2 (h) Cl (mL/min/kg) F (%) AUC (μM·h) cynomolgus monkey t1/2 (h) Cl (mL/min/kg) F (%) AUC (μM·h)

2

8

18

19

20

28

29

4.2 38.4 14 1.26 15.2

10.0 5.2 44.2 19.6 35.7

10.3 11.5 58 13.8 61.6

3.0 14.0 3.5 0.794 19.2

10.9 40.7 121.8 8.6 49.3

5.8 12.4 66.0 15.0 11.5

8.6 12.5 86.4 21.1 51.0

5.6 6.2 95 9.5

5.1 4.3 57.5 8.3

2.9 15.8 21.9 2.9

6.1 9.4 15.4 4.4

1 18.7 61 2.20 1 19.5 18 0.65

6.7 8.5 157 12.7 1.2 11.4 12.9 0.772

a

Respective IV/PO dosing levels: rat (5/15 mg/kg, n = 3), dog (1/3 mg/kg, n = 3), monkey (1/3 mg/kg for 1 and 17; 3/10 mg/kg for 31 and 32, n = 3). Vehicle: PEG-400/ethanol (90/10, v/v) for rat-IV/PO and PEG-400/water (85:15, v/v) for dog-IV/PO and monkey IV/PO. bPlasma half-life. cPharmacokinetic clearance. dOral bioavailability. ePharmacokinetic area under curve. I



■

Table 6. Inhibitory Activities of Compounds 2 and 29 against Recombinant Full-Length HCV NS3/4A Protease Complexes Representing Different HCV Genotypesa

a

2

29

1a (H77) 1b (J4L6S) 2a (HC-J6) 2b (HC-J8) 3a (S52) 4a (ED43) 5a (SA13) 6a (HK-6A)

0.6 0.4 26 100 488 2.1 3.4 1.3

1.5 1.6 38 220 1402 6.0 4.1 3.4

Data are the mean values from at least three independent experiments.

Table 7. Cell Culture Potency of Compounds 2 and 29 in HCV Repliconsa EC50 (nM) genotype (strain)

2

29

1a (H77) 1b (Con1) 2a (JFH1) 2b (HC-J8)b 3a (S52)b 4a (ED43)b

4 3 217 621 1100 1.7

4.5 4.5 136 562 1650 2.8

a

Data are the mean values from at least three independent experiments. bHybrid replicons were used to assess the NS3 protease activity representing specified genotypes. For the genotype 4a NS3 construct, the replicon backbone was genotype 1b while for the genotype 2b and 3a NS3 constructs, the replicon backbone was genotype 2a.

Table 8. Fold Loss in Potency of Compounds 2 and 29 against Known Resistance Substitutions fold resistancea replicon

2

29

GT-1a NS3-R155K GT-1b NS3-A156V GT-1b NS3-D168E GT-1b NS3-D168V

21 20 78 280

65 106 32 1900

CHEMISTRY

The synthesis of the P1 bi-cyclopropane acylsulfonamide tripeptide 29 as a representative example is outlined in Scheme 3, and this route was used to provide general access to all of the analogues described herein. In the case of 29, the synthesis started from 30, as depicted in Scheme 1, to which the introduction of the cyclopropane motif was achieved via a directed cyclopropanation using Pd(OAc)2 as the catalyst in the presence of an excess of diazomethane, a protocol that has been shown to efficiently cyclopropanate a wide variety of olefins.23 However, the reaction failed to proceed to completion (∼50% conversion based on liquid chromatography/mass spectrometry (LC/MS)), and the product 31 was inseparable from the starting material 30 by silica gel chromatography. While the screening of modified reaction conditions did not improve the conversion of 30 to 31, we were pleased to find that full conversion was achieved in 78% yield after the product mixture was resubjected to the reaction conditions for 2−3 cycles. The next series of reactions involved converting the ester functionality of 31 to the corresponding P1−P1′ acylsulfonamide 33. In the event, ester 31 was hydrolyzed with aqueous NaOH to afford the corresponding carboxylic acid 32, which, in turn, was converted into acylsulfonamide 33 using HATU and cyclopropanesulfonamide24 in the presence of DBU as the base. Removal of the Boc protecting group from 33 followed by HATU-mediated coupling with the previously described proline derivative 3512a afforded the dipeptide acylsulfonamide 36 in 93% yield over the two steps. In view of the difficulty of completing the cyclopropanation reaction and the challenging purification of 31 from 30, an alternative approach to 36 was developed in which the cyclopropane moiety was installed after the introduction of the functionalized proline derivative 35, as summarized in Scheme 2. HATU-mediated coupling of 35 with amine salt 37, prepared from 30 by deprotection of the Boc group, provided vinyl ester 38. In this example, the Pd-catalyzed cyclopropanation of 38 with diazomethane generated in situ from N-methyl-N-nitrosourea−NaOH in Et2O proceeded almost to completion in good yield (88%), and 39 was easily purified by column chromatography. Ester 39 was hydrolyzed with aqueous NaOH to yield the corresponding carboxylic acid 40, which, in turn, was transformed into acylsulfonamide 36 using HATU and cyclopropanesulfonamide in the presence of DBU. This alternative approach offered a more convenient procedure to synthesize 36 and proceeded in a higher overall yield. Conversion of 36 to the final compound 29 was then accomplished as outlined in Scheme 3. Removal of the Boc protecting group from 36 using HCl in dioxane was followed by HATUmediated coupling with the commercially available (S)-2-(tertbutoxycarbonylamino)-2-cyclohexylacetic acid to produce the tripeptide 42. Compound 42 was deprotected to provide the amine salt 43, which was then coupled with (R)-pyridin-2-yl 1,1,1-trifluoropropan-2-yl carbonate 44, prepared from commercially available (R)-1,1,1-trifluoropropan-2-ol,25 to provide the final compound 29 in 98% yield over the two steps. The absolute stereochemistry of 29 was determined by single crystal X-ray diffraction analysis, and the structure is presented in Figure 3.26

IC50 (nM) genotype (strain)

Article

a

Fold resistance represents the compound EC50 value against a replicon harboring an NS3 substitution divided by the compound EC50 value against wild-type replicon (GT-1a for GT-1a NS3 substitutions and GT-1b for GT-1b NS3 substitutions.

dogs that evaluated 29 at doses of 20, 60, and 200 mg/kg provided a similarly favorable outcome, with no significant issues identified. Compound 29 emerged from this preclinical assessment as a compound that met the criteria that had been set for a NS3 protease inhibitor clinical candidate. While both the potency of 29 and the CV profile were similar to that observed with 2, the PK properties were significantly improved across species, with lower clearance and higher oral bioavailability. The improved pharmacokinetic parameters coupled with low nanomolar whole cell antiviral activity associated with 29 resulted in a human dose prediction of 6 mg once daily to cover GT-1a and GT-1b viruses, which met our primary objective and triggered the onset of early development activities with this compound.22 The subtle structural changes that distinguish 29 (BMS-890068) from 2, that is, modifications to P1, P4 and ultimately P3, underscore the significance of relatively small structural changes on the key properties of potency, PK, and CV profiles of analogues in this chemical series.

■

CONCLUSION We have described efforts to optimize a series of HCV NS3 protease inhibitors which culminated in the discovery of 29, a compound which preserves the favorable properties of clinical compound 2 while demonstrating improved PK parameters predictive of once daily oral dosing in humans. The optimization J



Article

Scheme 1. Synthesis of P1 Bi-cyclopropane-Based Dipeptide 36a

a Reagents and conditions: (a) CH2N2, Pd(OAc)2, ether, 0 °C to RT, 78%; (b) NaOH, MeOH, H2O, reflux, 98%; (c) CDI, cyclopropanesulfonamide, DBU, THF, reflux to RT, 88%; (d) 4 M HCl, dioxane, RT, quantitative yield; (e) HATU, i-Pr2NEt, DCM, RT, 93%.

Scheme 2. An Alternative Route for Synthesis of P1 Bi-cyclopropanedipeptide 36a

a

Reagents and conditions: (a) (1R,2S)-ethyl 1-amino-2-vinylcyclopropanecarboxylate (HCl salt, 37), HATU, i-Pr2NEt, DCM, RT, 88%; (b) CH2N2, Pd(OAc)2, ether, 0 °C to RT, 88%; (c) NaOH, MeOH, H2O, reflux, quantitative yield; (d) CDI, cyclopropanesulfonamide, DBU, THF, reflux to RT, 94%.

campaign began first with replacement of the P1 terminal alkene with a cyclopropane moiety, represented by 8. This change significantly improved the PK profile in rat and likewise improved the metabolic stability in HLM but was accompanied by a loss in potency. Optimization of the P4 substituent restored potency in both the isolated enzyme and replicon assays while maintaining all an optimal PK and microsomal stability profile, represented by compound 18. In an attempt to mitigate an unfavorable CV signal observed with this molecule, replacements for P2* were surveyed. While analogues with enhanced potency were identified, such tuning of the P2* region failed to fully resolve the CV liability. Finally, structural modifications to the P3 region afforded 29, a compound that demonstrated a favorable balance of potency, metabolic stability, PK parameters, and CV safety. This compound was moved to the development stage and was positioned as a backup clinical candidate to 2 as it offered

a projected benefit of once daily dosing based on preclinical PK parameters.

■

EXPERIMENTAL SECTION

All reagents were purchased from commercial suppliers and used without purification unless otherwise noted. All anhydrous reactions were performed under a N2 atmosphere using SureSeal solvents from Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AM FT 300 MHz spectrometer, a Bruker AvanceIII 500 MHz spectrometer, or a Bruker Ultrashield 400 MHz spectrometer, each equipped with a 5 mm TXI cryoprobe. All spectra were determined in the solvents indicated, and residual protio-solvent was used as internal standard for chemical shift assignments. Interproton coupling constants are reported in Hertz (Hz). Multiplicity patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd, doublet of doublets; dt, doublet of triplets; dq, doublet of quartets. Most spectra were analyzed using the ACDLABS K



Article

Scheme 3. Synthesis of P1 Bi-cyclopropane-Based Tripeptide 29a

a Reagents and conditions: (a) 4 M HCl, dioxane, RT, quantitative yield; (b) HATU, i-Pr2NEt, (S)-2-(tert-butoxycarbonylamino)-2-cyclohexylacetic acid, DCM, RT, 92%; (c) 4 M HCl, dioxane, RT, quantitative yield; (d) 44, i-Pr2NEt, DCM, RT, 98%.

(30, 2.4 g, 9.40 mmol) and diacetoxypalladium (120 mg, 0.535 mmol) was added 0.8 M diazomethane in ether (40 mL, 32.0 mmol) dropwise at 0 °C. The formed black suspension was stirred at 0 °C for 2 h, warmed to RT, and stirred for 2 h. The reaction was incomplete as indicated by LC/MS, and the mixture contained 50% starting material. After filtration through a Celite plug and washing with 50% ether in THF, the filtrate was concentrated to give 2.45 g of a residue that was resubjected to the cyclopropanation reaction conditions described above until no starting material was detected by LC/MS (the reaction was typically repeated 2−3 times). The resulting residue was purified by silica gel chromatography (20% EtOAc in hexanes) to provide 31 (2.08 g, 78%) as a solid that was used in the next step without further purification. 1H NMR (400 MHz, MeOH-d4) δ 4.29−4.06 (m, 2H), 1.59 (dd, J = 7.8, 5.0 Hz, 1H), 1.44 (s, 9H), 1.30−1.23 (m, 3H), 1.20− 1.13 (m, 1H), 1.11−1.01 (m, 1H), 0.81 (qt, J = 7.9, 4.9 Hz, 1H), 0.60− 0.52 (m, 1H), 0.48−0.41 (m, 1H), 0.37−0.26 (m, 2H); LC/MS (Method A): tR = 2.67 min; LC/MS (ESI) m/z calcd for C14H23NO4: 269.16; found: 292.26 (M + Na)+. (1S,2R)-2-((tert-Butoxycarbonyl)amino)-[1,1′-bi(cyclopropane)]-2-carboxylic acid (32). To a solution of 31 (36 g, 149 mmol) in MeOH (400 mL) and H2O (200 mL) was added NaOH (12.18 g, 304 mmol). After heating at reflux for 1 h, the mixture was concentrated, neutralized with 1 N HCl, and extracted with EtOAc. The combined extracts were washed with brine, dried over MgSO4, and concentrated to give crude 32 (36 g, 98%), which was used in the next step without further purification. 1H NMR (400 MHz, MeOH-d4) δ 1.60−1.47 (m, 1H), 1.41 (s, 9H), 1.18−1.10 (m, 1H), 1.00 (q, J = 8.4 Hz, 1H), 0.85 (qt, J = 8.1, 4.8 Hz, 1H), 0.56−0.41 (m, 2H), 0.36− 0.23 (m, 2H); LC/MS (Method P): tR = 1.95 min; LC/MS (ESI) m/z calcd for C12H19NO4: 241.13; found: 264.27 (M + Na)+. tert-Butyl ((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′bi(cyclopropan)]-2-yl)carbamate (33). A solution of 32 (40 g, 166 mmol) and CDI (40.3 g, 249 mmol) in THF (414 mL) was heated at reflux for 2 h. After cooling to RT, cyclopropanesulfonamide (32.1 g, 265 mmol) and DBU (40.0 mL, 265 mmol) were added to the solution which was stirred overnight. After concentration, the residue was extracted with EtOAc, the extracts were washed with 1 N HCl and brine, and dried over MgSO4. The solution was filtered and concentrated, and

Figure 3. Single crystal X-ray structure of 29. The molecular structure of 29 free acid determined by X-ray crystallography from a monohydrate crystal. Water molecules are excluded for clarity. SpecManager 12.0 program software. Liquid chromatography/mass spectra (LC/MS) were performed on a Shimadzu LC instrument coupled to a Water Micromass ZQ instrument, and the LC/MS conditions are compiled in Table 9. Purities of the final compounds were determined by HPLC and were greater than 95%. HPLC conditions to assess purity are compiled in Table 10. (1S,2R)-Ethyl 2-((tert-butoxycarbonyl)amino)-[1,1′-bi(cyclopropane)]-2-carboxylate (31). To a solution of (1R,2S)ethyl 1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate L



Article

Table 9. LC/MS Conditions Used in the Characterization of All Compounds Found in the Experimental Section LC/MS conditiona b

Method A Method B Method Cb Method D Method E Method F Method G Method H Method I Method J Method Kb Method L Method M Method Nb Method O Method P

Colc

GTd

FRe

Sol Af

Sol Bg

1 1 4 3 1 2 3 4 4 3 4 2 1 3 4 3

3 3 3 2 3 3 4 4 3 3 3 2 3 3 4 3

4 5 5 5 4 1 4 4 4 4 5 1 4 5 4 5

A1 A1 A1 A1 A1 A1 A1 A2 A2 A1 A1 A1 A1 A1 A1 A1

B1 B1 B1 B1 B1 B1 B1 B2 B2 B1 B1 B1 B1 B1 B1 B1

Table 10. HPLC Purity Conditions Used in the Characterization of All Compounds Found in the Experimental Section

a

Gradient = 0%B to 100%B. bGradient = 30%B to 100%B; wavelength (λ) = 220 nm; injection volume = 5 μL unless otherwise stated. cCol = Column where column 1 = Phenomenex-Luna C18 (4.6 × 30 mm, S10); column 2 = Phenomenex-Luna C18 (2.0 × 30 mm, 3 μm); column 3 = Phenomenex-Luna C18 (3.0 × 50 mm, S10); Column 4 = Phenomenex-Luna (4.6 × 50 mm, S10). dGT = gradient time (min). e FR = flow rate (mL/min). fSol A = Solvent A where solvent A1 = 0.1% TFA/10% MeOH/90% H2O; A2 = 0.1% TFA/10% MeCN/90% H2O. gSol B = Solvent B where solvent B1 = 0.1% TFA/90% MeOH/ 10% H2O; B2 = 0.1% TFA/90% MeCN/10% H2O.

HPLC conditiona

Colg

GTh

FRi

Sol Aj

Sol Bk

Method A Method B Method C Method D Method E Method F Method Gb Method Hb Method Ic Method Jb Method Kd Method Ld Method Md Method Ne Method Oe Method Pf Method Q Method R Method Sb Method Tb Method Ub Method Vb Method Wb

1 2 1 2 3 4 5 6 1 1 7 1 7 1 7 7 6 8 5 6 1 2 1

15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 25

1 1 0.5 0.5 0.5 0.5 1 1 1 1 1 1 1.5 1 1.5 1.5 0.5 0.5 0.5 0.5 1 1 1

A1 A1 A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 A2 A2 A2 A2 A3 A3 A1 A1 A1 A1 A2

B1 B1 B1 B1 B1 B1 B1 B1 B2 B2 B2 B2 B2 B2 B2 B2 B3 B3 B1 B1 B1 B1 B2

a Gradient = 10%B to 100%B. bGradient = 40%B to 100%B. cGradient = 60%B to 100%B. dGradient = 70%B to 100%B. eGradient = 80%B to 100%B. fGradient = 30%B to 100%B; wavelength (λ) = 220 nm; injection volume = 5 μL unless otherwise stated. gCol = Column where column 1 = Waters Sunfire C18 (4.6 × 150 mm, 3.5 μm); column 2 = Xbridge Phenyl (4.6 × 150 mm, 3.5 μm); column 3 = XSELECT SCH C18 (3.0 × 150 mm, 3.5 μm); column 4 = Zorbax Bonus-RP (3.0 × 150 mm, 3.5 μm); Column 5 = Sunfire C18 (3.0 × 150 mm, 3.5 μm); Column 6 = Xbridge Phenyl (3.0 × 150 mm, 3.5 μm); Column 7 = Phenomenex (4.6 × 150 mm, 5 μm C-18); Column 8 = Xbridge C18 (3.0 × 150 mm, 3.5 μm). hGT = gradient time (min). iFR = flow rate (mL/min). jSol A = Solvent A where solvent A1 = 0.1% TFA/5% ACN/95% H2O; solvent A2 = 10% MeOH/90% H2O/0.2% H3PO4; solvent A3 = 10 mM NH4HCO3/5% MeOH/95% H20. kSolvent B = Solvent B where solvent B1 = 0.1% TFA/95% ACN/5% H2O; solvent B2 = 90% MeOH/10% H2O/ 0.2% H3PO4; solvent B3 = 10 mM NH4HCO3/95% MeOH/5% H20.

the residual solid crystallized from EtOAc (300 mL) to yield 33 (48 g) as a white solid. The mother liquor was concentrated, and the residue was crystallized from ethyl acetate (20 mL) to yield an additional 2 g of 33, amounting to a combined yield of 88%. 1H NMR (400 MHz, MeOH-d4) δ 2.96 (br. s., 1H), 1.68 (dd, J = 6.4, 4.4 Hz, 1H), 1.43 (s, 9H), 1.32−0.97 (m, 6H), 0.72−0.59 (m, 1H), 0.56−0.42 (m, 2H), 0.33−0.19 (m, 2H); LCMS (Method P): tR = 1.95 min; LC/MS (ESI) m/z calcd for C15H24N2O5S: 344.14; found: 367.30 (M + Na)+. (1 S,2R )-2-Amino-N-(cyclopropylsulfonyl)-[1,1′ -b i(cyclopropane)]-2-carboxamide, HCl salt (34). A solution of 33 (48 g, 139 mmol) and 4 M HCl in dioxane (139 mL, 557 mmol) was stirred for 4 h. Concentration gave 34 (39.1 g, quantitative yield) as a slightly hygroscopic white solid. 1H NMR (400 MHz, MeOH-d4) δ 3.06 (tt, J = 8.0, 4.8 Hz, 1H), 2.04−1.89 (m, 1H), 1.57−1.41 (m, 2H), 1.34− 1.06 (m, 4H), 0.88−0.75 (m, 1H), 0.71−0.56 (m, 2H), 0.49−0.27 (m, 2H); LCMS (Method A): tR = 0.18 min; LCMS (ESI) m/z calcd for C10H16N2O3S: 244.08; found: 267.10 (M + Na)+ (2S,4R)-tert-Butyl 4-((7-chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidine-1-carboxylate (36). HATU (21.58 g, 56.8 mmol) was added to a solution of 35 (20 g, 47.3 mmol), 34 (14.61 g, 52.0 mmol), and i-Pr2NEt (41.3 mL, 236 mmol) in CH2Cl2 (200 mL), and the mixture was stirred for 4 h. After concentration, the residue was purified by silica gel chromatography eluting with 20% acetone in hexanes to afford 36 (33 g, 93%) as a solid. 1H NMR (400 MHz, MeOH-d4) δ 8.15−8.05 (m, 2H), 7.72 (dd, J = 8.8, 2.0 Hz, 1H), 7.58 (s, 1H), 5.73 (br. s., 1H), 4.40 (dd, J = 9.5, 7.0 Hz, 1H), 4.02 (s, 3H), 3.92−3.77 (m, 2H), 3.06−2.95 (m, 1H), 2.61− 2.48 (m, 1H), 2.35−2.22 (m, 1H), 1.80 (dd, J = 8.0, 5.0 Hz, 1H), 1.48 (s, 9H), 1.35−0.97 (m, 6H), 0.94−0.84 (m, 1H), 0.66−0.49 (m, 2H), 0.36 (d, J = 4.0 Hz, 2H); LC/MS (Method A): tR = 2.75 min; LC/MS (ESI) m/z calcd for C30H37ClN4O8S: 648.20; found: 649.25 (M + H)+. (2S,4R)-tert-Butyl 4-((7-chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1R,2S)-1-(ethoxycarbonyl)-2-vinylcyclopropyl)carbamoyl)pyrrolidine-1-carboxylate (38). HATU (11.87 g, 31.2 mmol) was added to a solution of 35 (11 g, 26.0 mmol), (1R,2S)-ethyl 1-amino-2-vinylcyclopropanecarboxylate, HCl salt (37)

(4.99 g, 26.0 mmol) and i-Pr2NEt (22.72 mL, 130 mmol) in CH2Cl2 (200 mL), and the mixture was stirred for 4 h. After concentration, the residue was purified by silica gel chromatography eluting with 20% acetone in hexanes to give 38 (13 g, 88%) as a solid. 1H NMR (400 MHz, MeOH-d4) δ 8.05 (d, J = 9.1 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.66 (dd, J = 8.9, 2.1 Hz, 1H), 7.52 (s, 1H), 5.83−5.70 (m, 1H), 5.69−5.63 (m, 1H), 5.36−5.24 (m, 1H), 5.14−5.05 (m, 1H), 4.46−4.36 (m, 1H), 4.19−4.06 (m, 2H), 3.98 (s, 3H), 3.86 (d, J = 2.5 Hz, 2H), 2.70−2.56 (m, 1H), 2.39 (ddd, J = 13.6, 8.4, 4.9 Hz, 1H), 2.20 (q, J = 8.7 Hz, 1H), 1.83−1.66 (m, 1H), 1.46−1.38 (m, 10H), 1.23 (t, J = 7.2 Hz, 3H); LCMS (Method A): tR = 2.79 min; LC/MS (ESI) m/z calcd for C28H34ClN3O7: 559.20; found: 560.24 (M + H)+. (2S,4R)-tert-Butyl 4-((7-chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2-(ethoxycarbonyl)-[1,1′-bi(cyclopropan)]-2yl)carbamoyl)pyrrolidine-1-carboxylate (39). A solution of diazomethane in Et2O (49.1 mL, 0.8 M, 39.3 mmol) was added dropwise to a solution of 38 (2.2 g, 3.93 mmol) and diacetoxypalladium (0.220 g, 0.982 mmol) in THF (19.64 mL) maintained at 0 °C. The formed black suspension was stirred at 0 °C for 2 h, warmed to RT, and stirred overnight. After filtration through a Celite plug washed with 50% ether in THF, the filtrate was concentrated to give 2.5 g of a residue that was M



Article

that was used in the next step without further purification. 1H NMR (400 MHz, MeOH-d4) δ 8.14−8.05 (m, 2H), 7.72 (dd, J = 8.9, 2.1 Hz, 1H), 7.56 (s, 1H), 5.86 (br. s., 1H), 4.64 (dd, J = 10.4, 6.9 Hz, 1H), 4.23−4.15 (m, 1H), 4.14−4.05 (m, 2H), 3.99 (s, 3H), 3.05−2.93 (m, 1H), 2.69−2.54 (m, 1H), 2.33 (ddd, J = 14.1, 10.2, 4.4 Hz, 1H), 2.01− 1.65 (m, 7H), 1.40−1.04 (m, 11H), 0.89−0.77 (m, 1H), 0.64−0.45 (m, 2H), 0.38−0.24 (m, 2H); LCMS (Method A): tR = 1.89 min; LC/MS (ESI) m/z calcd for C33H42ClN5O7S: 687.24; found: 688.25 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl ((S)-2-((2S,4R)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)carbamate (29). A solution of 43 (3.476 g, 4.33 mmol), i-Pr2NEt, (3.78 mL, 21.66 mmol), and (R)-pyridin-2-yl 1,1,1-trifluoropropan-2-yl carbonate (1.223 g, 5.20 mmol) (44) in CH2Cl2 (50 mL) was stirred overnight. After concentration, the residue was purified by silica gel chromatography eluting with 25−33% acetone in hexanes to give 29 (3.50 g, 98%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ ppm 10.37 (s, 1 H), 8.89 (br. s., 1H), 8.09 (d, J = 1.94 Hz, 1H), 8.07 (d, J = 9.16 Hz, 1H)), 7.95 (d, J = 5.55 Hz, 1H), 7.79 (dd, J = 9.02, 2.08 Hz, 1H), 7.66 (s, 1H), 5.72 (br. s., 1H), 4.78 (ddd, J = 13.46, 6.66, 6.52 Hz, 1H), 4.36−4.47 (m, 2H), 3.97 (s, 3H), 3.82−3.93 (m, 2H), 2.90−3.00 (m, 1H), 2.45 (dd, J = 13.59, 6.66 Hz, 1H), 2.15−2.26 (m, 1H), 1.82 (d, J = 11.65 Hz, 1 H), 1.73 (t, J = 12.21 Hz, 2H), 1.50−1.68 (m, 4H), 1.17 (d, J = 6.38 Hz, 3H), 1.07−1.15 (m, 5H), 0.82−1.03 (m, 6H), 0.64−0.78 (m, 1H), 0.38−0.57 (m, 2H), 0.16−0.29 (m, 2H); HPLC purity (retention time): 100% (13.90 min, method U); 100% (11.30 min, method V); LCMS (Method A): tR = 3.06 min; LC/MS (ESI) m/z calcd for C37H45ClF3N5O9S: 828.29; found: 850.46 (M + Na)+. tert-Butyl (S)-1-((2S,4R)-4-(7-chloro-4-methoxyisoquinolin-1yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-ylcarbamate (8). 1H NMR (400 MHz, MeOH-d4) δ 8.13−8.02 (m, 2H), 7.69 (d, J = 8.3 Hz, 1H), 7.58 (s, 1H), 5.81 (br. s., 1H), 4.62−4.50 (m, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.20 (d, J = 9.5 Hz, 1H), 4.09−3.95 (m, 4H), 3.04−2.94 (m, 1H), 2.58 (dd, J = 13.7, 6.9 Hz, 1H), 2.35−2.21 (m, 1H), 1.78 (dd, J = 8.0, 5.3 Hz, 1H), 1.34−0.97 (m, 24H), 0.91−0.80 (m, 1H), 0.64−0.47 (m, 2H), 0.38−0.26 (m, 2H); HPLC purity (retention time): 96.7% (16.3 min, method P); 96.3% (17.7 min, method J); LCMS (Method J): tR = 3.39 min; LC/MS (ESI) m/z calcd for C36H48ClN5O9S: 761.29; found: 762.65 (M + H)+. Isopropyl ((S)-1-((2S,4R)-4-((7-Chloro-4-methoxyisoquinolin1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl1-oxobutan-2-yl)carbamate (9). 1H NMR (400 MHz, MeOH-d4) δ 8.15−8.06 (m, 2H), 7.70 (dd, J = 8.9, 1.9 Hz, 1H), 7.58 (s, 1H), 5.81 (br. s., 1H), 4.59−4.42 (m, 2H), 4.29−4.22 (m, 1H), 4.09−3.99 (m, 4H), 3.04−2.95 (m, 1H), 2.59 (dd, J = 13.8, 7.0 Hz, 1H), 2.34−2.22 (m, 1H), 1.78 (dd, J = 8.0, 5.3 Hz, 1H), 1.38−0.81 (m, 23H), 0.64−0.48 (m, 2H), 0.33 (d, J = 4.5 Hz, 2H); HPLC purity (retention time): 99.4% (13.67 min, method R); 100% (13.01 min, method Q); LCMS (Method L): tR = 2.43 min; LC/MS (ESI) m/z calcd for C35H46ClN5O9S: 747.27; found: 748.34 (M + H)+. Cyclobutyl (S)-1-((2S,4R)-4-(7-chloro-4-methoxyisoquinolin1-yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)-bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-ylcarbamate (10). 1H NMR (400 MHz, MeOH-d4) δ 9.01 (s, 1H), 8.16−8.07 (m, 2H), 7.72 (dd, J = 8.9, 1.9 Hz, 1H), 7.59 (s, 1H), 5.82 (br. s., 1H), 4.61−4.50 (m, 2H), 4.43 (d, J = 11.5 Hz, 1H), 4.24 (s, 1H), 4.10−4.01 (m, 4H), 3.06−2.95 (m, 1H), 2.60 (dd, J = 13.8, 6.8 Hz, 1H), 2.35−2.23 (m, 1H), 2.14 (br. s., 1H), 2.09−1.98 (m, 1H), 1.98−1.90 (m, 1H), 1.90−1.82 (m, 1H), 1.79 (dd, J = 8.0, 5.3 Hz, 1H), 1.74−1.63 (m, 1H), 1.55−1.43 (m, 1H), 1.32−1.22 (m, 3H), 1.18−1.09 (m, 3H), 1.06 (s, 9H), 0.92−0.79 (m, 1H), 0.65−0.49 (m, 2H), 0.34 (d, J = 3.8 Hz, 2H); HPLC purity (retention time): 97.9% (14.3 min, method K); 97.4% (14.3 min, method L); LCMS (Method J): tR = 2.86 min; LC/MS (ESI) m/z calcd for C36H46ClN5O9S: 759.27; found: 760.41 (M + H)+. Cyclopentyl ((S)-1-((2S,4R)-4-((7-Chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (11). 1H NMR (400 MHz,

purified by silica gel chromatography eluting with 10−20% hexanes in acetone to provide 39 (2.0 g, 88%) as a solid. 1H NMR (400 MHz, MeOH-d4) δ 8.05 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.66 (dd, J = 8.9, 2.1 Hz, 1H), 7.52 (s, 1H), 5.71−5.60 (m, 1H), 4.43−4.33 (m, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 3.88−3.80 (m, 2H), 2.68− 2.54 (m, 1H), 2.37 (ddd, J = 13.4, 8.5, 4.8 Hz, 1H), 1.70−1.53 (m, 1H), 1.40 (s, 9H), 1.30−1.06 (m, 5H), 0.92−0.80 (m, 1H), 0.64−0.51 (m, 1H), 0.51−0.36 (m, 2H), 0.35−0.26 (m, 1H); LCMS (Method A): tR = 2.79 min; LC/MS (ESI) m/z calcd for C29H36ClN3O7: 574.06; found: 574.27 (M + H)+. (1S,2R)-2-((2S,4R)-1-(tert-Butoxycarbonyl)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)pyrrolidine-2-carboxamido)[1,1′-bi(cyclopropane)]-2-carboxylic acid (40). A mixture of 39 (2.4 g, 4.18 mmol), NaOH (0.669 g, 16.72 mmol), MeOH (10 mL), and H2O (5 mL) was heated at reflux for 1 h. After concentration, the residue was neutralized with 1 N HCl, extracted with EtOAc, washed with brine, dried over MgSO4, and concentrated to give crude 40 (2.283 g, quantitative yield), which was used in the next step without further purification. 1H NMR (500 MHz, MeOH-d4) δ 8.15−8.00 (m, 1H), 7.70 (br. s., 1H), 7.57 (br. s., 1H), 5.68 (br. s., 1H), 4.42 (br. s., 1H), 4.17−4.08 (m, 1H), 4.02 (br. s., 3H), 3.88 (br. s., 2H), 2.65 (br. s., 1H), 2.40 (br. s., 1H), 1.70−1.56 (m, 1H), 1.45 (br. s., 9H), 1.33−1.17 (m, 2H), 1.08 (d, J = 7.9 Hz, 1H), 0.96 (br. s., 1H), 0.67−0.42 (m, 3H), 0.38−0.26 (m, 1H); LCMS (Method A): tR = 2.64 min; LC/MS (ESI) m/z calcd for C27H32ClN3O7: 545.13; found: 546.15 (M + H)+. (2S,4R)-tert-Butyl 4-((7-chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidine-1-carboxylate (36). A solution of 40 (16.5 g, 30.2 mmol) and CDI (7.35 g, 45.3 mmol) in THF (100 mL) was heated at reflux for 2 h. After cooling to RT, cyclopropanesulfonamide (5.86 g, 48.4 mmol) and DBU (7.29 mL, 48.4 mmol) were added to the mixture. The resulting solution was stirred overnight and concentrated, and the residue was acidified with 1 N HCl. The mixture was extracted with EtOAc, and the combined extracts were dried over MgSO4, concentrated, and purified by silica gel chromatography eluting with 20% acetone in hexanes to yield 36 (18.6 g, 94%) as a solid. (2S,4R)-4-((7-Chloro-4-methoxyisoquinolin-1-yl)oxy)-N((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)pyrrolidine-2-carboxamide HCl salt (41). A solution of 36 (2.6 g, 4.01 mmol) and 4 M HCl in dioxane (8 mL, 32.0 mmol) was stirred for 4 h. Concentration in vacuo gave 41 (2.348 g, quantitative yield) as the HCl salt which was used in the next step without further purification. 1H NMR (500 MHz, MeOH-d4) δ 8.29 (s, 1H), 8.15 (d, J = 8.9 Hz, 1H), 7.79−7.72 (m, 1H), 7.59 (s, 1H), 5.90 (br. s., 1H), 4.73−4.65 (m, 1H), 4.03 (s, 3H), 3.90−3.74 (m, 2H), 3.09− 2.98 (m, 1H), 2.90 (dd, J = 14.2, 7.5 Hz, 1H), 2.43−2.32 (m, 1H), 1.88− 1.79 (m, 1H), 1.37−1.03 (m, 6H), 0.84−0.73 (m, 1H), 0.64−0.49 (m, 2H), 0.41−0.29 (m, 2H); LCMS (Method A): tR = 1.70 min; LC/MS (ESI) m/z calcd for C25H29ClN4O6S: 548.15; found: 549.04 (M + H)+. tert-Butyl ((S)-2-((2S,4R)-4-((7-chloro-4-methoxyisoquinolin1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl2-oxoethyl)carbamate (42). HATU (2.84 g, 7.48 mmol) was added to a solution of 41 (3.1 g, 4.98 mmol), (S)-2-(tert-butoxycarbonylamino)2-cyclohexylacetic acid (1.411 g, 5.48 mmol) and i-Pr2NEt (4.35 mL, 24.92 mmol) in CH2Cl2 (10 mL), and the mixture was stirred for 4 h. After concentration, the residue was purified by silica gel chromatography eluting with 30% acetone in hexanes to afford 42 (3.6 g, 92%) as a solid. 1 H NMR (400 MHz, MeOH-d4) δ 8.16−8.04 (m, 2H), 7.69 (dd, J = 8.8, 2.0 Hz, 1H), 7.58 (s, 1H), 5.81 (br. s., 1H), 4.63−4.47 (m, 2H), 4.09−3.96 (m, 5H), 3.05−2.94 (m, 1H), 2.56 (dd, J = 13.7, 6.9 Hz, 1H), 2.37−2.25 (m, 1H), 1.87−1.62 (m, 7H), 1.37−0.82 (m, 22H), 0.65−0.46 (m, 2H), 0.40−0.25 (m, 2H); LCMS (Method A): tR = 2.97 min; LC/MS (ESI) m/z calcd for C38H50ClN5O9S: 787.30; found: 788.30 (M + H)+. (2S,4R)-1-((S)-2-Amino-2-cyclohexylacetyl)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)-N-((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)pyrrolidine-2-carboxamide HCl salt (43). A solution of 42 (24 g, 30.4 mmol) and 4 M HCl in dioxane (122 mL) was stirred for 4 h. Concentration provided 43 (22.03 g, quantitative yield) as the HCl salt N



Article

MeOH-d4) δ 8.15−8.05 (m, 2H), 7.70 (d, J = 8.8 Hz, 1H), 7.58 (s, 1H), 5.81 (br. s., 1H), 4.67 (br. s., 1H), 4.61−4.51 (m, 1H), 4.43 (d, J = 12.0 Hz, 1H), 4.25 (s, 1H), 4.09−3.99 (m, 4H), 3.05−2.92 (m, 1H), 2.59 (dd, J = 13.4, 6.9 Hz, 1H), 2.35−2.20 (m, 1H), 1.82−0.78 (m, 25H), 0.65−0.48 (m, 2H), 0.33 (d, J = 4.5 Hz, 2H); HPLC purity (retention time): 98.9% (10.5 min, method O); 98.8% (13.4 min, method F); LCMS (Method H): tR = 3.28 min; LC/MS (ESI) m/z calcd for C37H48ClN5O9S: 773.29; found: 774.03 (M + H)+. 1-Methylcyclopentyl ((S)-1-((2S,4R)-4-((7-chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2-((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (12). 1H NMR (400 MHz, MeOH-d4) δ 8.12 (d, J = 8.8 Hz, 1H), 8.07 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.60 (s, 1H), 5.81 (br. s., 1H), 4.63−4.42 (m, 2H), 4.25−4.19 (m, 1H), 4.11−3.99 (m, 4H), 3.02−2.96 (m, 1 H); 2.59 (dd, J = 13.7, 6.9 Hz, 1H), 2.39−2.23 (m, 1H), 1.89 (d, J = 9.0 Hz, 1H), 1.83−0.98 (m, 25H), 0.94−0.83 (m, 2H), 0.65−0.50 (m, 2H), 0.34 (d, J = 2.5 Hz, 2H); HPLC purity (retention time): 99.2% (11.8 min, method O); 100% (13.8 min, method F); LC/MS (Method H): tR = 3.40 min; LCMS (ESI) m/z calcd for C38H50ClN5O9S: 787.30; found: 809.90 (M + Na)+. (1R,3r,5S)-Bicyclo[3.1.0]hexan-3-yl ((S)-1-((2S,4R)-4-((7chloro-4-methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (13). 1H NMR (400 MHz, MeOH-d4) δ 8.02−8.14 (m, 2H), 7.67 (dd, J = 8.8, 2.0 Hz, 1H), 7.54 (s, 1H), 6.87 (d, J = 9.3 Hz, 1H), 5.76 (br. s., 1H), 4.68 (t, J = 6.9 Hz, 1H), 4.52 (dd, J = 10.4, 6.9 Hz, 1H), 4.37 (d, J = 11.8 Hz, 1H), 4.21 (d, J = 9.6 Hz, 1H), 3.93−4.11 (m, 5H), 2.93−3.01 (m, 1H), 2.56 (dd, J = 13.7, 6.9 Hz, 1H), 2.19−2.31 (m, 1H), 1.92−2.04 (m, 1H), 1.71−1.88 (m, 2H), 1.66 (d, J = 14.6 Hz, 1H), 1.40 (d, J = 14.6 Hz, 1H), 1.03−1.33 (m, 10H), 0.91−1.03 (m, 10H), 0.83 (ddt, J = 12.7, 8.2, 4.0 Hz, 2H); HPLC purity (retention time): 99% (13.89 min, method S); 97% (10.34 min, method T); LC/MS (Method J): t R = 3.01 min; LC/MS (ESI) m/z calcd for C38H48ClN5O9S: 786.33; found: 786.16 (M + H)+. 1,1,1-Trifluoro-2-methylpropan-2-yl-(S)-1-((2S,4R)-4-(7chloro-4-methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)-bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (14). 1 H NMR (400 MHz, MeOH-d4) δ 8.14−8.03 (m, 2H), 7.70 (dd, J = 9.0, 2.0 Hz, 1H), 7.59 (s, 1H), 5.80 (br. s., 1H), 4.58 (dd, J = 10.5, 6.8 Hz, 1H), 4.44 (d, J = 11.8 Hz, 1H), 4.17 (s, 1H), 4.09−3.96 (m, 4H), 3.04− 2.94 (m, 1H), 2.59 (dd, J = 13.8, 6.8 Hz, 1H), 2.34−2.21 (m, 1H), 1.78 (dd, J = 8.2, 5.4 Hz, 1H), 1.43−0.99 (m, 21H), 0.92−0.79 (m, 1H), 0.57 (dd, J = 18.4, 8.2 Hz, 2H), 0.33 (d, J = 4.5 Hz, 2H); HPLC purity (retention time): 95% (15.6 min, method E); 97.1% (13.6 min, method F); LC/MS (Method O): tR = 4.23 min; LCMS (ESI) m/z calcd for C36H45ClF3N5O9S: 815.25; found: 816.3 (M + H)+. 1,1-Difluoro-2-methylpropan-2-yl-(S)-1-((2S,4R)-4-(7-chloro4-methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)-bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (15). 1H NMR (400 MHz, MeOH-d4) δ 8.12−8.03 (m, 2H), 7.69 (dd, J = 8.9, 2.1 Hz, 1H), 7.58 (s, 1H), 5.77 (br. s., 1H), 4.58 (dd, J = 10.5, 6.8 Hz, 1H), 4.47 (d, J = 11.5 Hz, 1H), 4.15 (d, J = 8.8 Hz, 1H), 4.06−3.98 (m, 4H), 3.04−2.94 (m, 1H), 2.64−2.55 (m, 1H), 2.33−2.22 (m, 1H), 1.78 (dd, J = 8.3, 5.3 Hz, 1H), 1.33−1.22 (m, 3H), 1.20−0.99 (m, 19H), 0.90−0.80 (m, 1H), 0.64−0.48 (m, 2H), 0.35−0.30 (m, 2H); HPLC purity (retention time): 96.3% (14.9 min, method N); 98.4% (13.2 min, method F); LCMS (Method J): t R = 2.90 min; LC/MS (ESI) m/z calcd for C36H46ClF2N5O9S: 797.26; found: 798.37 (M + H)+. 2,2,2-Trifluoroethyl-(S)-1-((2S,4R)-4-(7-chloro-4-methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)-bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (16). 1H NMR (400 MHz, MeOHd4) δ 8.16−8.02 (m, 2H), 7.68 (dd, J = 8.9, 2.1 Hz, 1H), 7.55 (s, 1H), 5.79−5.74 (m, 1H), 5.47 (s, 1H), 4.51 (dd, J = 10.6, 7.1 Hz, 1H), 4.44− 4.30 (m, 2H), 4.24 (s, 1H), 4.23−4.13 (m, 1H), 4.05−3.97 (m, 4H), 3.01−2.90 (m, 1H), 2.58 (dd, J = 13.7, 6.9 Hz, 1H), 2.23 (ddd, J = 13.9, 10.3, 4.3 Hz, 1H), 1.75 (dd, J = 8.2, 5.4 Hz, 1H), 1.31−1.18 (m, 3H),

1.14−1.01 (m, 11H), 0.86−0.75 (m, 1H), 0.61−0.46 (m, 2H), 0.29 (q, J = 4.6 Hz, 2H); HPLC purity (retention time): 98.7% (14.57 min, method E); 100% (12.83 min, method F); LCMS (Method E): tR = 3.04 min; LC/MS (ESI) m/z calcd for C34H41ClF3N5O9S: 787.23; found: 788.30 (M + H)+. (S)-1,1,1-Trifluoropropan-2-yl-(S)-1-((2S,4R)-4-(7-chloro-4methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2-(cyclopropylsulfonylcarbamoyl)-bi(cyclopropan)-2-ylcarbamoyl)pyrrolidin-1yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (17). 1H NMR (400 MHz, MeOH-d4) δ 8.13−8.08 (m, 2H), 7.70 (dd, J = 8.9, 2.1 Hz, 1H), 7.58 (s, 1H), 5.79 (br. s., 1H), 4.99−4.90 (m, 1H), 4.55 (dd, J = 10.3, 7.0 Hz, 1H), 4.44 (d, J = 11.8 Hz, 1H), 4.29−4.23 (m, 1H), 4.07− 4.00 (m, 4H), 2.99 (tt, J = 8.0, 4.8 Hz, 1H), 2.61 (dd, J = 13.7, 6.9 Hz, 1H), 2.27 (ddd, J = 14.0, 10.4, 4.3 Hz, 1H), 1.78 (dd, J = 8.0, 5.3 Hz, 1H), 1.33−1.22 (m, 4H), 1.18−1.03 (m, 14H), 0.87−0.79 (m, 1H), 0.63− 0.50 (m, 2H), 0.35−0.28 (m, 2H); HPLC purity (retention time): 95.7% (14.1 min, method R); 95.5% (13.5 min, method Q); LCMS (Method E): tR = 3.05 min; LC/MS (ESI) m/z calcd C35H43ClF3N5O9S: 801.24; found: 802.14 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-(S)-1-((2S,4R)-4-(7-chloro-4methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2(cyclopropylsulfonylcarbamoyl)bi(cyclopropan)-2ylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (18). 1H NMR (400 MHz, MeOH-d4) δ 8.17−8.06 (m, 2H), 7.70 (dd, J = 9.0, 2.0 Hz, 1H), 7.58 (s, 1H), 5.81 (br. s., 1H), 4.56 (dd, J = 10.4, 6.9 Hz, 1H), 4.46 (d, J = 11.8 Hz, 1H), 4.27 (s, 1H), 4.09−3.98 (m, 4H), 3.06−2.94 (m, 1H), 2.61 (dd, J = 13.8, 7.0 Hz, 1H), 2.35−2.22 (m, 1H), 1.79 (dd, J = 8.0, 5.3 Hz, 1H), 1.35−1.21 (m, 7H), 1.18−1.04 (m, 11H), 0.90−0.73 (m, 1H), 0.65−0.48 (m, 2H), 0.33 (d, J = 4.5 Hz, 2H); HPLC purity (retention time): 97.9% (9.6 min, method O); 96.9% (12.4 min, method N); LC/MS (Method J): tR = 2.92 min; LCMS (ESI) m/z calcd for C35H43ClF3N5O9S: 801.24; found: 802.19 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-(S)-1-((2S,4R)-2-((1S,2R)-2(cyclopropylsulfonylcarbamoyl)bi(cyclopropan)-2-ylcarbamoyl)-4-(3,6-dimethoxyisoquinolin-1-yloxy)pyrrolidin-1-yl)-3,3dimethyl-1-oxobutan-2-ylcarbamate (19). 1H NMR (400 MHz, MeOH-d4) δ 7.88 (d, J = 9.1 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 6.81 (dd, J = 9.2, 2.4 Hz, 1H), 6.53 (s, 1H), 5.83−5.78 (m, 1H), 4.93−4.87 (m, 1H), 4.48 (dd, J = 10.3, 7.1 Hz, 1H), 4.41 (d, J = 11.3 Hz, 1H), 4.30 (s, 1H), 4.04 (dd, J = 11.7, 3.7 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.01− 2.92 (m, 1H), 2.57 (dd, J = 13.8, 7.1 Hz, 1H), 2.24 (ddd, J = 13.8, 10.1, 4.3 Hz, 1H), 1.75 (dd, J = 8.1, 5.3 Hz, 1H), 1.30−1.18 (m, 6H), 1.14− 0.99 (m, 12H), 0.88−0.75 (m, 1H), 0.62−0.45 (m, 2H), 0.35−0.22 (m, 2H); HPLC purity (retention time): 100% (13.89 min, method A); 100% (12.72 min, method B); LCMS (Method F): tR = 3.11 min; LC/MS (ESI) m/z calcd for C36H46F3N5O10S: 797.29; found: 798.46 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl ((S)-1-((2S,4R)-4-((5-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (20). 1H NMR (400 MHz, MeOH-d4) δ 8.10 (dd, J = 8.3, 1.3 Hz, 1H), 7.73 (dd, J = 7.6, 1.3 Hz, 1H), 7.64 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 5.79−5.75 (m, 1H), 4.73 (quin, J = 6.7 Hz, 1H), 4.50 (dd, J = 10.4, 7.2 Hz, 1H), 4.41 (d, J = 11.3 Hz, 1H), 4.24 (s, 1H), 4.01 (dd, J = 11.7, 3.7 Hz, 1H), 3.93 (s, 3H), 3.02−2.92 (m, 1H), 2.56 (dd, J = 13.7, 7.2 Hz, 1H), 2.24 (ddd, J = 13.9, 10.3, 4.3 Hz, 1H), 1.75 (dd, J = 8.1, 5.3 Hz, 1H), 1.30−1.19 (m, 6H), 1.14−1.05 (m, 3H), 1.02 (s, 9H), 0.88−0.75 (m, 1H), 0.62−0.45 (m, 2H), 0.29 (q, J = 4.8 Hz, 2H); HPLC purity (retention time): 100% (14.76 min, method E); 100% (12.99 min, method F); LCMS (Method D): tR = 2.28 min; LC/MS (ESI) m/z calcd for C35H43ClF3N5O9S: 801.24; found: 802.22 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl ((S)-1-((2S,4R)-4-((6-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (21). 1H NMR (400 MHz, MeOH-d4) δ 8.19−7.97 (m, 2H), 7.57 (s, 1H), 7.47 (dd, J = 8.8, 2.3 Hz, 1H), 5.77 (br. s., 1H), 4.82− 4.72 (m, 1H), 4.50 (dd, J = 10.2, 6.9 Hz, 1H), 4.39 (d, J = 12.1 Hz, 1H), 4.24 (s, 1H), 4.08−3.96 (m, 4H), 3.02−2.90 (m, 1H), 2.56 (dd, J = 13.5, 7.2 Hz, 1H), 2.23 (ddd, J = 14.0, 10.2, 4.3 Hz, 1H), 1.75 (dd, J = 8.1, 5.3 Hz, 1H), 1.34−1.18 (m, 6H), 1.15−0.97 (m, 12H), 0.88−0.75 O



Article

(m, 1H), 0.53 (dd, J = 18.5, 8.4 Hz, 2H), 0.29 (d, J = 4.8 Hz, 2H); HPLC purity (retention time): 100% (14.04 min, method S); 100% (10.32 min, method T); LC/MS (Method A): tR = 2.97 min; LC/MS (ESI) m/z calcd for C35H43ClF3N5O9S: 801.24; found: 802.38 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl ((S)-1-((2S,4R)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)-4-((5,7-dichloro-4-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (22). 1 H NMR (400 MHz, MeOH-d4) δ 9.01 (b, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.71 (s, 1H), 5.80 (br. s., 1H), 4.56 (dd, J = 10.3, 7.0 Hz, 1H), 4.48 (d, J = 11.8 Hz, 1H), 4.22 (s, 1H), 4.03 (dd, J = 11.8, 3.5 Hz, 1H), 3.97 (s, 3H), 3.04−2.96 (m, 1H), 2.61 (dd, J = 13.4, 6.9 Hz, 1H), 2.29 (ddd, J = 13.9, 10.3, 4.1 Hz, 1H), 1.79 (dd, J = 7.9, 5.4 Hz, 1H), 1.38−1.22 (m, 5H), 1.18−1.00 (m, 11H), 0.93−0.77 (m, 1H), 0.66− 0.49 (m, 2H), 0.38−0.27 (m, 2H); HPLC purity (retention time): 97.2% (17.8 min, method I); 97.5% (27.4 min, method W); LC/MS (Method C): tR = 2.93 min; LCMS (ESI) m/z calcd for C35H42Cl2F3N5O9S: 835.20; found: 836.10 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-((S)-2-((2S,4R)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclopropyl-2-oxoethyl)carbamate (23). 1H NMR (400 MHz, MeOH-d4) δ 8.14 (d, J = 1.8 Hz, 1H), 8.04−8.11 (m, 1H), 7.67 (dd, J = 8.9, 2.1 Hz, 1H), 7.53 (s, 1H), 5.77 (br. s., 1H), 4.78−4.83 (m, 1H), 4.53 (dd, J = 10.3, 7.1 Hz, 1H), 4.40 (d, J = 11.3 Hz, 1H), 3.93−4.04 (m, 4H), 3.73 (d, J = 9.1 Hz, 1H), 3.31−3.35 (m, 4H), 3.18−3.27 (m, 2H), 3.11 (dt, J = 3.3, 1.6 Hz, 1H), 2.94−3.03 (m, 1H), 2.55 (dd, J = 13.7, 6.9 Hz, 1H), 2.29 (ddd, J = 14.0, 10.2, 4.3 Hz, 1H), 1.78 (dd, J = 7.7, 4.9 Hz, 1H), 1.02−1.32 (m, 12H); HPLC purity (retention time): 100% (12.07 min, method U); 100% (10.10 min, method V); LCMS (Method N): tR = 2.79 min; LC/MS (ESI) m/z calcd for C34H39ClF3N5O9S: 786.21; found: 786.20 (M + H)+. (2R)-1,1,1-Trifluoropropan-2-yl-N-[(2S)-1-[(2S,4R)-4-[(7chloro-4-methoxyisoquinolin-1-yl)oxy]-2-{[(1R,2S)-1[(cyclopropanesulfonyl)carbamoyl]-2-cyclopropylcyclopropyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate (24). 1H NMR (400 MHz, MeOH-d4) δ 8.15−8.04 (m, 2H), 7.68 (d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 5.80 (br. s., 1H), 4.78−4.71 (m, 1H), 4.60−4.51 (m, 2H), 4.05−3.97 (m, 5H), 3.04−2.94 (m, 1H), 2.58 (dd, J = 13.9, 6.9 Hz, 1H), 2.31 (ddd, J = 14.0, 10.4, 4.3 Hz, 1H), 2.16−2.05 (m, 1H), 1.80 (dd, J = 8.0, 5.0 Hz, 1H), 1.33−1.27 (m, 2H), 1.26−1.20 (m, 4H), 1.16−1.08 (m, 3H), 0.99 (t, J = 6.7 Hz, 6H), 0.92− 0.82 (m, 1H), 0.64−0.56 (m, 1H), 0.56−0.48 (m, 1H), 0.39−0.28 (m, 2H); HPLC purity (retention time): 98.4% (14.2 min, method K); 98.2% (14.2 min, method L); LC/MS (Method J): tR = 2.84 min; LCMS (ESI) m/z calcd for C34H41ClF3N5O9S: 787.23; found: 788.15 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-(2S,3R)-1-((2S,4R)-4-(7-chloro4-methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2(cyclopropylsulfonylcarbamoyl)bi(cyclopropan)-2ylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-oxopentan-2-ylcarbamate (25). 1H NMR (400 MHz, MeOH-d4) δ 8.13−8.07 (m, 2H), 7.68 (dd, J = 8.9, 2.1 Hz, 1H), 7.57 (s, 1H), 5.80 (t, J = 3.1 Hz, 1H), 4.76−4.67 (m, 1H), 4.60−4.52 (m, 2H), 4.10−4.04 (m, 1H), 4.04−3.97 (m, 4H), 3.04−2.96 (m, 1H), 2.59 (dd, J = 13.6, 6.8 Hz, 1H), 2.31 (ddd, J = 13.9, 10.3, 4.1 Hz, 1H), 1.91 (dt, J = 9.3, 6.6 Hz, 1H), 1.80 (dd, J = 8.0, 5.3 Hz, 1H), 1.68−1.54 (m, 1H), 1.36−1.06 (m, 10H), 0.99−0.80 (m, 7H), 0.64−0.48 (m, 2H), 0.38−0.27 (m, 2H); HPLC purity (retention time): 95.0% (15.2 min, method L); 97.4% (13.5 min, method Q); LCMS (Method J): tR = 2.96 min; LC/MS (ESI) m/z calcd for C35H43ClF3N5O9S: 801.24; found: 802.30 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-(S)-2-((2S,4R)-4-(7-chloro-4methoxyisoquinolin-1-yloxy)-2-((1S,2R)-2(cyclopropylsulfonylcarbamoyl)bi(cyclopropan)-2ylcarbamoyl)pyrrolidin-1-yl)-1-cyclobutyl-2-oxoethylcarbamate (26). 1H NMR (400 MHz, MeOH-d4) δ 8.18−7.94 (m, 2H), 7.68 (dd, J = 8.9, 2.1 Hz, 1H), 7.57 (s, 1H), 5.86−5.75 (m, 1H), 4.60−4.47 (m, 2H), 4.32 (d, J = 9.3 Hz, 1H), 4.11−3.96 (m, 4H), 2.84−2.72 (m, 1H), 2.58 (dd, J = 13.7, 6.9 Hz, 1H), 2.30 (ddd, J = 13.9, 10.2, 4.3 Hz, 1H), 2.13−1.99 (m, 2H), 1.94−1.75 (m, 5H), 1.45−1.06 (m, 11H), 0.93−0.79 (m, 1H), 0.65−0.48 (m, 2H), 0.39−0.26 (m, 2H); HPLC purity (retention time): 95.5% (14.7 min, method N); 95.2% (15.8 min,

method E); LCMS (Method J): tR = 2.93 min; LC/MS (ESI) m/z calcd for C35H41ClF3N5O9S: 799.22; found: 800.38 (M + H)+. (R)-1,1,1-Trifluoropropan-2-yl-((S)-1-((2S,4R)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-3-ethyl-1-oxopentan-2-yl)carbamate (27). 1H NMR (MeOH-d4) δ 8.03−8.11 (m, 2H), 7.66 (dd, J = 8.9, 2.1 Hz, 1H), 7.53−7.57 (m, 1H), 5.77 (br. s., 1H), 4.63−4.71 (m, 1H), 4.49−4.58 (m, 2H), 4.25 (d, J = 9.3 Hz, 1H), 3.96−4.02 (m, 4H), 2.93−3.00 (m, 1H), 2.56 (dd, J = 13.8, 6.8 Hz, 1H), 2.24−2.33 (m, 1H), 1.74−1.86 (m, 2H), 1.54−1.02 (m, 16H), 1.00−1.15 (m, 8H); HPLC purity (retention time): 99% (14.30 min, method S); 99% (10.53 min, method T); LC/MS (Method A): tR = 2.93 min; LC/MS (ESI) m/z calcd for C36H45ClF3N5O9S: 816.28; found: 838.31 (M + Na)+. (R)-1,1,1-Trifluoropropan-2-yl-((S)-2-((2S,4R)-4-((7-chloro-4methoxyisoquinolin-1-yl)oxy)-2-(((1S,2R)-2((cyclopropylsulfonyl)carbamoyl)-[1,1′-bi(cyclopropan)]-2-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclopentyl-2-oxoethyl)carbamate (28). 1H NMR (400 MHz, MeOH-d4) δ 8.02−8.17 (m, 2H), 7.66 (dd, J = 8.9, 2.1 Hz, 1H), 7.54 (s, 1H), 5.73−5.80 (m, 1H), 4.47−4.73 (m, 3H), 3.90−4.17 (m, 5H), 2.93−3.02 (m, 1H), 2.56 (dd, J = 13.7, 6.9 Hz, 1H), 2.21−2.37 (m, 2H), 1.69−1.87 (m, 3H), 1.45− 1.69 (m, 5H), 1.35 (br. s., 1H), 1.02−1.31 (m, 14H); HPLC purity (retention time): 98% (13.39 min, method U); 98% (11.02 min, method V); LC/MS (Method A): tR = 2.95 min; LCMS (ESI) m/z calcd for C36H43ClF3N5O9S: 814.27; found: 814.53 (M + H)+. Isolated Perfused Rabbit Heart. Hearts were excised from anesthetized rabbits and perfused in a retrograde manner through the aorta at a constant pressure and temperature (37 ± 0.2 °C) with oxygenated Krebs-Henseleit bicarbonate buffer. A bipolar ECG was recorded via electrodes connected to an ECG amplifier, and a pair of recording electrodes were placed on the epicardium of the left atrium and pair of stimulating electrodes was placed on the epicardium of the right atrium for atrial stimulation. Flow of the perfusate through the coronary arteries was monitored and recorded via an extracorporeal flow probe. The atrial electrogram, ECG and coronary flow were continuously monitored and recorded via a Po-Neh-Mah physiology platform. Data were expressed as mean ± SD and were compared using student t test. The primary end point of the studies was SA node function; the secondary end points were cardiac conduction (A-V conduction, QRS duration), QT interval, and coronary flow. SA node function was evaluated by measuring SA node recovery time (SNRT) and sinus rate (HR). SNRT was defined as the longest A1−A2 cycle length after atrial pacing. In some experiments, test agents were perfused at different concentrations in ascending order in each heart. Each concentration was perfused for 10 min without washout between concentrations. All parameters were measured at baseline and after 10 min perfusion at each concentration. Data were averaged from 1 min recording at baseline and after 10 min perfusion at each dose. To study the effects of a single concentration of test agent over a longer time period, rabbit hearts were perfused for 1 h with test agent and data were analyzed every 10 min throughout the perfusion period. Anesthetized Rabbit Electrophysiology. Rabbits were anesthetized with propofol-fentanyl and a decapolar electrode catheter was inserted into the heart with the distal electrode placed in the right ventricle (RV) and the proximal electrode located in the right atrium (RA). The femoral artery was cannulated for measuring blood pressure and to take blood samples. The primary end point was SA node function reflected by SA node recovery time (SNRT) and sinus rate (HR). SNRT is defined as the longest A1-A2 cycle length after atrial pacing. A1 represents the last beat of an atrial pacing train, and A2 the first spontaneous sinus beat after pacing. The secondary end points were cardiac conduction (A-V conduction, QRS duration) and QT interval. The atrial and His-bundle electrograms were continuously monitored and recorded via intracardiac leads that were connected to Prucka CardioLab 7000. Body surface ECG (lead II and III) and arterial blood pressure (BP) were continuously monitored and recorded at 500 Hz via Po-Neh-Mah at a logging rate of 10 s. Compound 29 was intravenously infused at 30 mg/kg, via an infusion pump over 10 min. Blood samples were taken at 5 min during the infusion and immediately at the end of the P



Article

infusion; and then at 10, 20, 40, and 60 min after the end of the infusion. PEG400: ethanol (9:1) was used as the vehicle. Data were averaged from a 1 min recording interval immediately before blood sampling and are expressed as the mean ± SEM. Data were compared with vehicle control using student t test. In addition to blood samples, liver and heart (atrium and ventricle) samples were collected at the end of the observations for 29 concentration analyses. In Vivo Pharmacokinetic Studies. All animal studies were performed under the approval of the Bristol-Myers Squibb Animal Care and Use Committee and in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). Mouse PK Studies. The pharmacokinetic parameters of compound 29 were characterized in male FVB mice (Harlan Breeding Laboratories, Indianapolis, IN). Mice dosed orally were fasted overnight, while those intravenously dosed were not fasted. Two groups of mice (N = 9 per group, 20−25 g) received drug either as an intravenous (IV) bolus dose (2 mg/kg; vehicle of PEG-400/ethanol, 9:1) via the tail vein or by oral gavage (5 mg/kg; vehicle of PEG 400/ethanol, 9:1). Serum concentrations were measured at 0.05 (IV only), 0.25 (PO only), 0.5, 1, 3, 6, 8, and 24 h following dosing. Serum samples were stored at −20 °C until analysis by LC/MS/MS for 29. To evaluate tissue exposure, livers and brains were removed from the FVB mice at the terminal sampling points for each group (6, 8, and 24 h). The excised tissues were rinsed, blotted dry, weighed and stored at −20 °C until processed for analysis by LC/MS/MS for 29. Rat PK Studies. Male Sprague−Dawley rats (300−350 g, Hilltop Lab Animals, Inc., Scottsdale, PA) with single or dual indwelling cannulae implanted in the jugular vein were used in the pharmacokinetic studies of compounds 18, 28 and 29. Rats dosed orally were fasted overnight, while those intravenously dosed were not fasted. Drugs were administered to rats (N = 3 per group) as a 10 min infusion (5 mg/kg) via jugular vein cannula or orally by gavage (15 mg/kg). The vehicle used for dosing both IV and PO was PEG-400/ethanol (9:1). Serial blood samples were obtained from the jugular vein cannulae of all animals at 0 (predose) and at 0.17 (or 0.25 for oral), 0.5, 0.75, 1, 2, 4, 6, 8, 24 h postdose. These samples (∼0.3 mL) were collected into K3EDTAcontaining tubes and then centrifuged at 4 °C (1500−2000g) to obtain plasma, which was stored at −20 °C until analysis by LC/MS/MS for each analyte. To evaluate liver exposure, livers were removed at the terminal sampling time point for each dosing route. Sample preparation was the same as described below. Dog PK studies. The pharmacokinetics of compounds 18, 28, and 29 were evaluated in male beagle dogs (∼9−12 kg, Marshall Farms USA Inc., North Rose, NY) bearing vascular access ports. The studies were conducted in a crossover design (N = 2), with a two week washout period between the IV and PO doses. Dogs were fasted overnight prior to oral dosing, but not fasted prior to IV dosing and all dogs were fed 4 h after dosing. In the IV study, each compound was infused at 1 mg/kg over 5 min (solution in 85%PEG-400/15% water) into the cephalic vein. Serial blood samples were collected from a venous vascular access port at 0.08, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose. In the PO study, compounds were administered by oral gavage at 3 mg/kg (solution in 85% PEG-400/15% water). Serial blood samples were collected from the venous vascular access port at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose. Plasma was prepared from the blood samples and the concentrations of each compound in plasma samples were determined by LC/MS/MS. Cynomolgus Monkey PK Studies. The pharmacokinetics of compounds 18, 28, and 29 were conducted in three male cynomolgus monkeys bearing vascular access ports (3.1−5.7 kg, Charles River Biomedical Research Foundation, Houston, TX), with a two week washout period between doses. Monkeys were fasted overnight prior to oral dosing, but not prior to IV dosing and all animals were fed 4 h after dosing. For IV dosing, each compound was infused at 3 mg/kg over 5 min into the venous vascular port. Serial blood samples were collected via an arterial vascular access port at 0.08, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose. In the PO study, each compound was administered by oral gavage at 10 mg/kg. Both IV and PO dose solutions were prepared in 85 %PEG-400/15% water. Serial blood samples were collected via arterial vascular access port at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8,

and 24 h postdose. Plasma was prepared from the blood samples and the concentrations of each analyte were determined by LC/MS/MS. Data Analysis. The pharmacokinetic parameters of compounds 18, 28, and 29 were obtained by noncompartmental analysis of plasma concentration versus time data (KINETICA software, Version 2.4, InnaPhase Corporation, Philadelphia, PA). The peak concentration (Cmax) and time for Cmax (Tmax) were recorded directly from experimental observations. The area under the curve from time zero to the last sampling time (AUC(0−T)) and the area under the curve from time zero to infinity (AUC(INF)) were calculated using a combination of linear and log trapezoidal summations. The whole body plasma clearance (Cl), steady-state volume of distribution (Vss), apparent terminal t1/2, and mean residence time (MRT) were estimated following intravenous administration. The absolute oral bioavailability (F) was estimated as the ratio of dose-normalized AUC values following PO and IV doses.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00821. SMILES codes and GT-1a/1b inhibitory activity for compounds 6−29 (CSV)

■

AUTHOR INFORMATION

Corresponding Author

*Phone, 203-677-7460; fax, 203-677-7804; e-mail, LiQiang. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the following members of our analytical group for the roles that they played in enabling the work described in this paper: Xiaohu Huang, Jingfang Cutrone, Dieter Drexler, Edward S. Kozlowski, Christopher J. Poronsky, and Julia M. Nielson.

■

ABBREVIATIONS USED CDI, 1,1′-carbonyldiimidazole; CV, cardiovascular; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; GT, genotype; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-pyridinium 3-oxid hexafluorophosphate; PK, pharmacokinetic; RT, room temperature; SAR, structure−activity relationship; THF, tetrahydrofuran; US, United States; Vss, apparent volume of distribution at steady state

■

REFERENCES

(1) Kim, W. R. Global epidemiology and burden of hepatitis C. Microbes Infect. 2002, 4, 1219−1225. (2) WHO. Hepatitis C global prevalence (update). Weekly Epidemiol. Rec. 1999, 74, 425−427. (3) Hanafiah, K. M.; Groeger, J.; Flaxman, A. D.; Wiersma, S. T. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013, 57, 1333−1342. (4) Hajarazadeh, B.; Grebely, J.; Dore, G. J. Epidemiology and natural history of HCV infection. Nat. Rev. Gastroenterol. Hepatol. 2013, 10, 553−562. (5) Gravitz, L. Introduction: a smouldering public-health crisis. Nature 2011, 474, S2−4. (6) (a) Pawlotsky, J.-M. Pathophysiology of hepatitis C virus infection and related liver disease. Trends Microbiol. 2004, 12, 96−102. (b) Liang, T. J.; Heller, T. Pathogenesis of hepatitis C-associated hepatocellular carcinoma. Gastroenterology 2004, 127, S62−S71. (7) Brown, R. S. Hepatitis C and liver transplantation. Nature 2005, 436, 973−978.

Q



Article

(8) Ly, K. N.; Hughes, E. M.; Jiles, R. B.; Holmberg, S. D. Rising mortality associated with hepatitis C virus in the United States, 2003− 2013. Clin. Infect. Dis. 2016, 62, 1287−1288. (9) (a) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.; Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A. M.; Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace, L.; LaPlante, S. R.; Narjes, H.; Poupart, M. A.; Rancourt, J.; Sentjens, R. E., St; Sr George, R.; Simoneau, B.; Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C. L.; Llinàs-Brunet, M. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 2003, 426, 186−189. (b) Llinàs-Brunet, M.; Bailey, M. D.; Bolger, G.; Brochu, C.; Faucher, A.-M.; Ferland, J. M.; Garneau, M.; Ghiro, E.; Gorys, V.; Grand-Maître, C.; Halmos, T.; Lapeyre-Paquette, N.; Liard, F.; Poirier, M.; Rhéaume, M.; Tsantrizos, Y. S.; Lamarre, D. Structure-activity study on a novel series of macrocyclic inhibitors of the hepatitis C virus NS3 protease leading to the discovery of BILN 2061. J. Med. Chem. 2004, 47, 1605−1608. (c) Llinàs-Brunet, M.; Bailey, M.; Goudreau, D. N.; Bhardwaj, P. K.; Bordeleau, J.; Bös, M.; Bousquet, Y.; Cordingley, M. G.; Duan, J.; Forgione, P.; Garneau, M.; Ghiro, E.; Gorys, V.; Goulet, S.; Halmos, T.; Kawai, S. H.; Naud, J.; Poupart, M. A.; White, P. W. Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335). J. Med. Chem. 2010, 53, 6466−6476. (d) Raboisson, P.; de Kock, H.; Rosenquist, Å.; Nilsson, M.; Salvador-Oden, L.; Lin, T. I.; Roue, N.; Ivanov, V.; Wahling, H.; Wickstrom, K.; Hamelink, E.; Edlund, M.; Vrang, L.; Vendeville, S.; Van de Vreken, W.; McGowan, D.; Tahri, A.; Hu, L.; Boutton, C.; Lenz, O.; Delouvroy, F.; Pille, G.; Surleraux, D.; Wigerinck, P.; Samuelsson, B.; Simmen, K. Structureactivity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350. Bioorg. Med. Chem. Lett. 2008, 18, 4853−4858. (e) Cummings, M. D.; Lindberg, J.; Lin, T.-I.; de Kock, H.; Lenz, O.; Lilja, E.; Fellander, S.; Baraznenok, V.; Nystrom, S.; Nilsson, M.; Vrang, L.; Edlund, M.; Rosenquist, Å.; Samuelsson, B.; Raboisson, P.; Simmen, K. Induced-fit binding of the macrocyclic noncovalent inhibitor TMC435 to its HCV NS3/NS4A protease target. Angew. Chem., Int. Ed. 2010, 49, 1652−1655. (f) Rosenquist, Å.; Samuelsson, B.; Johansson, P. O.; Cummings, M. D.; Lenz, O.; Raboisson, P.; Simmen, K.; Vendeville, S.; de Kock, H.; Nilsson, M.; Horvath, A.; Kalmeijer, R.; de la Rosa, G.; BeumontMauviel, M. Discovery and development of simeprevir (TMC435), a HCV NS3/4a protease inhibitor. J. Med. Chem. 2014, 57, 1673−1693. (g) Liverton, N. J.; Holloway, M. K.; McCauley, J. A.; Rudd, M. T.; Butcher, J. W.; Carroll, S. S.; DiMuzio, J.; Fandozzi, C.; Gilbert, K. F.; Mao, S.-S.; McIntyre, C. J.; Nguyen, K. T.; Romano, J. J.; Stahlhut, M.; Wan, B.-L.; Olsen, D. B.; Vacca, J. P. Molecular modeling based approach to potent P2-P4 macrocyclic inhibitors of hepatitis C NS3/4A protease. J. Am. Chem. Soc. 2008, 130, 4607−4609. (h) McCauley, J. A.; McIntyre, C. J.; Rudd, M. T.; Nguyen, K. T.; Romano, J. J.; Butcher, J. W.; Gilbert, K. F.; Bush, K. J.; Holloway, M. K.; Swestock, J.; Wan, B.-L.; Carroll, S. S.; DiMuzio, J. M.; Graham, D. J.; Ludmerer, S. W.; Mao, S.-S.; Stahlhut, M. W.; Fandozzi, C. M.; Trainor, N.; Olsen, D. B.; Vacca, J. P.; Liverton, N. J. Discovery of vaniprevir (MK-7009), a macrocyclic hepatitis C virus NS3/4a protease inhibitor. J. Med. Chem. 2010, 53, 2443−2463. (i) Harper, S.; McCauley, J. A.; Rudd, M. T.; Ferrara, M.; DiFilippo, M.; Crescenzi, B.; Koch, U.; Petrocchi, A.; Holloway, M. K.; Butcher, J. W.; Romano, J. J.; Bush, K. J.; Gilbert, K. F.; McIntyre, C. J.; Nguyen, K. T.; Nizi, E.; Carroll, S. S.; Ludmerer, S. W.; Burlein, C.; DiMuzio, J. M.; Graham, D. J.; McHale, C. M.; Stahlhut, M. W.; Olsen, D. B.; Monteagudo, E.; Cianetti, S.; Giuliano, C.; Pucci, V.; Trainor, N.; Fandozzi, C. M.; Rowley, M.; Coleman, P. J.; Vacca, J. P.; Summa, V.; Liverton, N. J. Discovery of MK5172, a macrocyclic hepatitis C virus NS3/4a protease inhibitor. ACS Med. Chem. Lett. 2012, 3, 332−336. (j) Rudd, M. T.; Butcher, J. W.; Nguyen, K. T.; McIntyre, C. J.; Romano, J. J.; Gilbert, K. F.; Bush, K. J.; Liverton, N. J.; Holloway, M. K.; Harper, S.; Ferrara, M.; DiFilippo, M.; Summa, V.; Swestock, J.; Fritzen, J.; Carroll, S. S.; Burlein, C.; DiMuzio, J. M.; Gates, A.; Graham, D. J.; Huang, Q.; McClain, S.; McHale, C.; Stahlhut, M. W.; Black, S.; Chase, R.; Soriano, A.; Fandozzi, C. M.; Taylor, A.; Trainor, N.; Olsen, D. B.; Coleman, P. J.; Ludmerer, S. W.;

McCauley, J. A. P2-Quinazolinones and bis-macrocycles as new templates for next-generation hepatitis C virus NS3/4a protease inhibitors: discovery of MK-2748 and MK-6325. ChemMedChem 2015, 10, 727−735. (k) Neelamkavil, S. F.; Agrawal, S.; Bara, T.; Bennett, C.; Bhat, S.; Biswas, D.; Brockunier, L.; Buist, N.; Burnette, D.; Cartwright, M.; Chackalamannil, S.; Chase, R.; Chelliah, M.; Chen, A.; Clasby, M.; Colandrea, V. J.; Davies, I. W.; Eagen, K.; Guo, Z.; Han, Y.; Howe, J.; Jayne, C.; Josien, H.; Kargman, S.; Marcantonio, K.; Miao, S.; Miller, R.; Nolting, A.; Pinto, P.; Rajagopalan, M.; Ruck, R. T.; Shah, U.; Soriano, A.; Sperbeck, D.; Velazquez, F.; Wu, J.; Xia, Y.; Venkatraman, S. Discovery of MK-8831, A novel spiro-proline macrocycle as a pangenotypic HCV- NS3/4a protease inhibitor. ACS Med. Chem. Lett. 2016, 7, 111−116. (l) Jiang, Y.; Andrews, S. W.; Condroski, K. R.; Buckman, B.; Serebryany, V.; Wenglowsky, S.; Kennedy, A. L.; Madduru, M. R.; Wang, B.; Lyon, M.; Doherty, G. A.; Woodard, B. T.; Lemieux, C.; Geck Do, M.; Zhang, H.; Ballard, J.; Vigers, G.; Brandhuber, B. J.; Stengel, P.; Josey, J. A.; Beigelman, L.; Blatt, L.; Seiwert, S. D. Discovery of danoprevir (ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV) NS3/4A protease. J. Med. Chem. 2014, 57, 1753−1769. (m) Sheng, X. C.; Casarez, A.; Cai, R.; Clarke, M. O.; Chen, X.; Cho, A.; Delaney, W. E., IV; Doerffler, E.; Ji, M.; Mertzman, M.; Pakdaman, R.; Pyun, H.-J.; Rowe, T.; Wu, Q.; Xu, J.; Kim, C. U. Discovery of GS-9256: a novel phosphinic acid derived inhibitor of the hepatitis C virus NS3/4A protease with potent clinical activity. Bioorg. Med. Chem. Lett. 2012, 22, 1394−1396. (10) Scola, P. M.; Wang, A.; Good, A. C.; Sun, L.-Q.; Combrink, K. D.; Campbell, J. A.; Chen, J.; Tu, Y.; Sin, N.; Venables, B. L.; Sit, S. Y.; Chen, Y.; Cocuzza, A.; Bilder, D. M.; D’Andrea, S.; Zheng, B.; Hewawasam, P.; Ding, M.; Thuring, J.; Hernandez, D.; Yu, F.; Falk, P.; Zhai, G.; Sheaffer, A. K.; Chen, C.; Lee, M. S.; Barry, D.; Knipe, J. O.; Han, Y. H.; Jenkins, S.; Gesenberg, C.; Sinz, M. W.; Santone, K. S.; Zvyaga, T.; Rajamani, R.; Klei, H. E.; Colonno, R. J.; Grasela, D. M.; Hughes, E.; Chien, C.; Adams, S.; Levesque, P. C.; Li, D.; Zhu, J.; Meanwell, N. A.; McPhee, F. Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection. J. Med. Chem. 2014, 57, 1730−1755. (11) Langendorff, O. Untersuchungen am uberleberden saugerleizen. Pfluegers Arch. 1895, 61, 291−332. (12) (a) Scola, P. M.; Sun, L.-Q.; Wang, A. X.; Chen, J.; Sin, N.; Venables, B. L.; Sit, S.-Y.; Chen, Y.; Cocuzza, A.; Bilder, D. M.; D’Andrea, S. V.; Zheng, B.; Hewawasam, P.; Tu, Y.; Friborg, J.; Falk, P.; Hernandez, D.; Levine, S.; Chen, C.; Yu, F.; Sheaffer, A. K.; Zhai, G.; Barry, D.; Knipe, J. O.; Han, Y.-H.; Schartman, R.; Donoso, M.; Mosure, K.; Sinz, M. W.; Zvyaga, T.; Good, A. C.; Rajamani, R.; Kish, K.; Tredup, J.; Klei, H. E.; Gao, Q.; Mueller, L.; Colonno, R. J.; Grasela, D. M.; Adams, S. P.; Loy, J.; Levesque, P. C.; Sun, H.; Shi, H.; Sun, L.; Warner, W.; Li, D.; Zhu, J.; Meanwell, N. A.; McPhee, F. The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection. J. Med. Chem. 2014, 57, 1730−1752. (b) McPhee, F.; Sheaffer, A. K.; Friborg, J.; Hernandez, D.; Falk, P.; Zhai, G.; Levine, S.; Chaniewski, S.; Yu, F.; Barry, D.; Chen, C.; Lee, M. S.; Mosure, K.; Sun, L.-Q.; Sinz, M.; Meanwell, N. A.; Colonno, R. J.; Knipe, J.; Scola, P. Preclinical profile and characterization of the hepatitis C virus NS3 protease inhibitor asunaprevir (BMS-650032). Antimicrob. Agents Chemother. 2012, 56, 5387−5396. (13) (a) Chayama, K.; Takahashi, S.; Toyota, J.; Karino, Y.; Ikeda, K.; Ishikawa, H.; Watanabe, H.; McPhee, F.; Hughes, E.; Kumada, H. Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders. Hepatology 2012, 55, 742− 748. (b) Pasquinelli, C.; McPhee, F.; Eley, T.; Villegas, C.; Sandy, K.; Sheridan, P.; Persson, A.; Huang, S.-P.; Hernandez, D.; Sheaffer, A. K.; Scola, P.; Marbury, T.; Lawitz, E.; Goldwater, R.; Rodriguez-Torres, M.; DeMicco, M.; Wright, D.; Charlton, M.; Kraft, W. K.; Lopez-Talavera, J.C.; Grasela, D. M. Single- and multiple-ascending-dose studies of the NS3 protease inhibitor asunaprevir in subjects with or without chronic hepatitis C. Antimicrob. Agents Chemother. 2012, 56, 1838−1844. (c) Suzuki, Y.; Ikeda, K.; Suzuki, F.; Toyota, J.; Karino, Y.; Chayama, K.; R



Article

Kawakami, Y.; Ishikawa, H.; Watanabe, H.; Hu, W.; Eley, T.; McPhee, F.; Hughes, E.; Kumada, H. Dual oral therapy with daclatasvir and asunaprevir for patients with HCV genotype 1b infection and limited treatment options. J. Hepatol. 2013, 58, 655−662. (d) Lok, A. S.; Gardiner, D. F.; Hézode, C.; Lawitz, E. J.; Bourlière, M.; Everson, G. T.; Marcellin, P.; Rodriguez-Torres, M.; Pol, S.; Serfaty, L.; Eley, T.; Huang, S.-P.; Li, J.; Wind-Rotolo, M.; Yu, F.; McPhee, F.; Grasela, D. M.; Pasquinelli, C. Randomized trial of daclatasvir and asunaprevir with or without pegIFN/RBV for hepatitis C virus genotype 1 null responders. J. Hepatol. 2014, 60, 490−499. (e) Kumada, H.; Suzuki, Y.; Ikeda, K.; Toyota, J.; Karino, Y.; Chayama, K.; Kawakami, Y.; Ido, A.; Yamamoto, K.; Takaguchi, K.; Izumi, N.; Koike, K.; Takehara, T.; Kawada, N.; Sata, M.; Miyagoshi, H.; Eley, T.; McPhee, F.; Damokosh, A.; Ishikawa, H.; Hughes, E. Daclatasvir plus asunaprevir for chronic HCV genotype 1b infection. Hepatology 2014, 59, 2083−2091. (14) Poole, R. Daclatasvir + asunaprevir: first global approval. Drugs 2014, 74, 1559−1571. (15) (a) Everson, G. T.; Sims, K. D.; Rodriguez-Torres, M.; Hézode, C.; Lawitz, E.; Bourlière, M.; Loustaud-Ratti, V.; Rustgi, V.; Schwartz, H.; Tatum, H.; Marcellin, P.; Pol, S.; Thuluvath, P. J.; Eley, T.; Wang, X.; Huang, S.-P.; McPhee, F.; Wind-Rotolo, M.; Chung, E.; Pasquinelli, C.; Grasela, D. M.; Gardiner, D. F. Efficacy of an interferon- and ribavirinfree regimen of daclatasvir, asunaprevir, and BMS-791325 in treatmentnaive patients with HCV genotype 1 infection. Gastroenterology 2014, 146, 420−429. (b) Hassanein, T.; Sims, K. D.; Bennett, M.; Gitlin, N.; Lawitz, R.; Nguyen, T.; Webster, L.; Younossi, Z.; Schwartz, H.; Thuluvath, P. J.; Zhou, H.; Rege, B.; McPhee, F.; Zhou, N.; WindRotolo, M.; Chung, E.; Griffies, A.; Grasela, D. M.; Gardiner, D. F. A randomized trial of daclatasvir in combination with asunaprevir and beclabuvir in patients with chronic hepatitis C virus genotype 4 infection. J. Hepatol. 2015, 62, 1204−1206. (c) Toyota, J; Karino, Y.; Suzuki, F.; Ikeda, F.; Ido, A.; Tanaka, K.; Takaguchi, K.; Naganuma, A.; Tomita, E.; Chayama, K.; Fujiyama, S.; Inada, Y.; Yoshiji, H.; Watanabe, H.; Ishikawa, H.; Hu, W.; McPhee, F.; Linaberry, M.; Yin, P. D.; Swenson, E. S.; Kumada, H. Daclatasvir/asunaprevir/beclabuvir fixeddose combination in Japanese patients with HCV genotype 1 infection. J. Gastroenterol. 2015, ASAP, DOI 10.1007/s00535-016-1245-6. (16) Wang, X. A.; Sun, L.-Q.; Sit, S.-Y.; Sin, N.; Scola, P. M.; Hewawasam, P.; Good, A. C.; Chen, Y.; Campbell, J. A. Hepatitis C virus inhibitors. U.S. Patent 6,995,174, 2006. (17) (a) Pietschmann, T.; Bartenschlager, R. The hepatitis C virus replicon system and its application to molecular studies. Curr. Opin. Drug Discovery Dev. 2001, 4, 657−664. (b) Sheaffer, A. K.; Lee, M. S.; Hernandez, D.; Chaniewski, S.; Yu, F.; Falk, P.; Friborg, J.; Zhai, G.; McPhee, F. Development of a chimeric replicon system for phenotypic analysis of NS3 protease sequences from HCV clinical isolates. Antiviral Ther. 2011, 16, 705−718. (18) Mosure, K. W.; Knipe, J. O.; Browning, M.; Arora, V.; Shu, Y. Z.; Phillip, T.; Mcphee, F.; Scola, P.; Balakrishnan, A.; Soars, M. G.; Santone, K.; Sinz, M. Preclinical pharmacokinetics and in vitro metabolism of asunaprevir (BMS-650032), a potent hepatitis C virus NS3 protease inhibitor. J. Pharm. Sci. 2015, 104, 2813−2823. (19) (a) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 2015, 58, 8315−8359. (b) Diana, G. D.; Rudewicz, P.; Pevear, D. C.; Nitz, T. J.; Aldous, S. C.; Aldous, D. J.; Robinson, D. T.; Draper, T.; Dutko, F. J.; Aldi, C.; Gendron, G.; Oglesby, R. C.; Volkots, D. L.; Reurnan, M.; Bailey, T. R.; Czerniak, R.; Block, T.; Roland, R.; Oppermand, J. Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism. J. Med. Chem. 1995, 38, 1355−1371. (c) Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher, M. H.; He, H.; Hickey, G. J.; Kowalchick, J. E.; Leiting, B.; Lyons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.; Petrov, A.; Scapin, G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt, M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N. A.; Weber, A. E. (2R)-4-Oxo-4-[3(trifluoromethyl)-5,6-dihydro[1,2,4]-triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-amine: a potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J. Med. Chem. 2005, 48, 141−151. (d) Barnes-Seeman, D.; Jain, M.; Bell, L.;

Ferreira, S.; Cohen, S.; Chen, X. H.; Amin, J.; Snodgrass, B.; Hatsis, P. Metabolically stable tertbutyl replacement. ACS Med. Chem. Lett. 2013, 4, 514−516. (e) Huchet, Q. A.; Kuhn, B.; Wagner, B.; Kratochwil, N. A.; Fischer, H.; Kansy, M.; Zimmerli, D.; Carreira, E. M.; Muller, K. Fluorination patterning: a study of structural motifs that impact physicochemical properties of relevance to drug discovery. J. Med. Chem. 2015, 58, 9041−9060. (20) McPhee, F.; Friborg, J.; Levine, S.; Chen, C.; Falk, P.; Yu, F.; Hernandez, D.; Lee, M. S.; Chaniewski, S.; Sheaffer, A. K.; Pasquinelli, C. Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir. Antimicrob. Agents Chemother. 2012, 56, 3670−3681. (21) Scola, P. M.; Sun, L.-Q.; Gillis, E. P.; Bowsher, M. S.; Chen, J.; Wang, X. A.; Sit, S.-Y.; Chen, Y.; Zheng, Z.; D’Andrea, S.; Sin, N.; Venables, B.; Mull, E.; Chen, Q.; Kandhasamy, S.; Pulicharla, N.; Vishwakrishnan, S.; Reddy, S.; Trivedi, R.; Sinha, S.; Sivaprasad, S.; Rao, A.; Desai, S.; Ghosh, K.; Rajamani, R.; Friborg, J.; Levine, S.; Chen, C.; Falk, P.; Jenkins, S.; Kramer, M.; Haskel, R.; Johnson, K.; Loy, J.; Levesque, P.; Zhu, J.; Cockett, M.; Meanwell, N. A.; McPhee, F. Discovery of a second generation, pan genotype NS3/4A protease inhibitor (BMS-986144) for the treatment of hepatitis C. 250th ACS National Meeting & Exposition, Boston, MA, United States, August 16− 20, 2015; MEDI-309. (22) The human Vss and clearance values for compound 29 were determined via allometric scaling using species-invariant modeling of preclinical species. Absorption kinetics in humans were assumed to be the average determined from rat, dog and monkey following solution dosing. The efficacious dose was projected by translating the in vitro replicon potency to clinical viral load decline as stated in ref 18, and this resulted in a once daily dose projection of 6 mg for 29. (23) Paulissen, R.; Hubert, R. J.; Teyssie, Ph. Transition metal catalysed cyclopropanation of olefin. Tetrahedron Lett. 1972, 13, 1465− 1466. (24) Li, J.; Smith, D.; Wong, H. S.; Campbell, J. A.; Meanwell, N. A.; Scola, P. M. A facile synthesis of 1-substituted cyclopropylsulfonamides. Synlett 2006, 725−728. (25) Hiebert, S.; Rajamani, R.; Sun, L.-Q.; Mull, Eric; Gillis, E. P.; Bowsher, M. S.; Zhao, Q.; Meanwell, N. A.; Renduchintala, K. V.; Sarkunam, K.; Nagalakshmi, P.; Babu, P. V. K. S.; Scola, P. M. Preparation of macrocyclic peptides, especially proline-containing peptides, as inhibitors of hepatitis C virus replication. World Patent Application, WO 2012151195. (26) Full X-ray crystallographic data have been deposited with the Cambridge Crystallographic Data Center (CCDC reference number 1491198). Copies of the data can be obtained free of charge via the internet at http://www.ccdc.cam.ac.uk.

S


Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of

Recommend Documents