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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 Paul M. Scola,*,† Alan Xiangdong Wang,† Andrew C. Good,§ Li-Qiang Sun,† Keith D. Combrink,† Jeffrey A. Campbell,† Jie Chen,† Yong Tu,† Ny Sin,† Brian L. Venables,† Sing-Yuen Sit,† Yan Chen,† Anthony Cocuzza,† Donna M. Bilder,† Stanley D’Andrea,† Barbara Zheng,† Piyasena Hewawasam,† Min Ding,† Jan Thuring,† Jianqing Li,× Dennis Hernandez,‡ Fei Yu,‡ Paul Falk,‡ Guangzhi Zhai,‡ Amy K. Sheaffer,‡ Chaoqun Chen,‡ Min S. Lee,‡ Diana Barry,‡ Jay O. Knipe,∥ Wenying Li,∥ Yong-Hae Han,∥ Susan Jenkins,∥ Christoph Gesenberg,∥ Qi Gao,∞ Michael W. Sinz,∥ Kenneth S. Santone,∥ Tatyana Zvyaga,# Ramkumar Rajamani,§ Herbert E. Klei,§ Richard J. Colonno,‡ Dennis M. Grasela,⊥ Eric Hughes,⊥ Caly Chien,⊥ Stephen Adams,∥ Paul C. Levesque,∥ Danshi Li,∥ Jialong Zhu,∥ Nicholas A. Meanwell,† and Fiona McPhee‡ †

Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ‡ Department of Virology Discovery Biology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States § Department of Computer-Assisted Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ∥ Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ⊥ Department of Early Clinical and Translational Research, Discovery MedicineVirology, Bristol-Myers Squibb Research and Development, Hopewell, New Jersey 08543, United States # Department of Lead Discovery and Optimization, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, 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 Chemical Synthesis, Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States S Supporting Information *

ABSTRACT: The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1′ site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

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INTRODUCTION

Hepatitis C virus (HCV) is a global pandemic that afflicts approximately 3% of the world’s population.1−4 The insidious nature of this disease is underscored by its benign presentation © 2014 American Chemical Society

Special Issue: HCV Therapies Received: November 27, 2013 Published: February 20, 2014 1708

dx.doi.org/10.1021/jm401840s | J. Med. Chem. 2014, 57, 1708−1729

Journal of Medicinal Chemistry

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Figure 1. Details of the binding interactions of peptide inhibitor 1 bound to the NS3/4A complex.

Figure 2. Surface of NS3/4A enzyme illustrating the S1 and S1′ subsites. Close-up of 1 bound to NS3/4A.

In addition, treatment with boceprevir therapy is further complicated by a 4-week lead-in with PEG interferon-α and ribavirin, while telaprevir has recently been issued a black box warning by the FDA in response to reports of toxic epidermal necrolysis in patients receiving the drug.16−18 Hence, a need exists for the identification of effective HCV inhibitors that are amenable to less frequent dosing schedules while also demonstrating improved efficacy and safety profiles that can be combined with mechanistically orthogonal agents.19 In this article, we describe the design concept behind the discovery of acylsulfonamidebased tripeptidic inhibitors of the viral NS3/4A protease and the prosecution of this chemical series that culminated in the

during the acute phase and the paucity of specific symptoms throughout the early stages. In its advanced stages, however, HCV is debilitating, as a significant number of patients suffer from advanced cirrhosis and ultimately hepatocellular carcinoma.5,6 Hepatitis C virus exists as six major genotypes (GT-1−6), each of which can be found as distinct clusters in regions throughout the world.7,8 Upon treatment, individuals infected with hepatitis C can potentially be cleared of the virus, but the drug regimen employed and viral response rates are dependent on genotype. Immune modulation was the initial cornerstone of therapy with pegylated (PEG) interferon-α combined with ribavirin administered to accomplish this goal.9 Patients infected with GT-2 or GT-3 respond best to treatment with this regimen, with sustained viral responses (SVRs) of 70−80% measured at 24 weeks after treatment. In contrast, the SVR rates for GT-1 patients treated with PEG interferon-α and ribavirin are less than 50%. Hence, in the development of direct acting antivirals, the GT-1 virus has been a primary focus of efforts due to the more acute medical need. As a consequence of those efforts, treatment for individuals infected with HCV GT-1 has evolved to depend on the use of PEG interferon-α and ribavirin9−12 in conjunction with one of the two recently approved HCV NS3 protease inhibitors, boceprevir or telaprevir.13,14 Supplementing the former standard of care regimen with either of these small molecule, direct-acting antiviral agents has resulted in an increase in SVR rates.15 However, both of these protease inhibitors require thrice daily (t.i.d.) dosing, a regimen generally associated with compliance issues, particularly against the backdrop of the side effect profile of the combination of PEG interferon-α and ribavirin, which in turn may facilitate the emergence of drug resistant virus.

advancement of a compound into phase I clinical trials for the treatment of HCV-infected patients.

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RESULTS AND DISCUSSION Our efforts in this area began with analysis of the hexapeptide 1, reported as a potent inhibitor of the NS3/4A protease complex.20 As described, when bound to the active site of the enzyme, this peptide engaged in a series of nonbonding interactions with the 1709



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enzyme that extended along the nonprime binding groove (Figure 1). Of particular interest were interactions involving the C-terminus carboxylic acid group of 1 which engages in a salt bridge interaction with the catalytic histidine while also functioning as an acceptor of a hydrogen bond from the backbone of glycine 139 (Figure 1). Single point mutagenesis studies and structure−activity relationship determinations indicated that these nonbonding interactions between the P1 acid of peptide 1 and the NS3/4A enzyme complex were essential for enzyme inhibition.20 In the evaluation of strategies to further the binding capacity of peptidic inhibitors such as 1, the concept of improving potency via interactions within the S1′ subsite of the NS3/4A enzyme was considered.21 As illustrated in Figure 2, the S1′ site is a shallow pocket along the surface of the peptide that is contiguous with the oxyanion hole. In an effort to enhance the binding interaction of these peptidic inhibitors of the NS3/4A enzyme, a ligand design strategy was pursued in which the essential interactions between the carboxylic acid group of 1 and the catalytic site were maintained while projecting functionality into the S1′ site. On the basis of modeling considerations, acylsulfonamides were proposed as a structural motif that could maintain the electrostatic interactions of the parent carboxylic acid while offering the potential to extend structural elements into the prime side of the enzyme and engage in favorable nonbonding interactions. The disclosure of tripeptidic carboxylic acids represented by 2 (Table 1) as potent inhibitors of HCV NS3/4A led to the selection of this chemotype as a suitable vehicle with which to explore the concept.22 The tripeptidic methylacylsulfonamide 3 presented in Table 1 was the first compound prepared to explore the design concept and proved to be equipotent in a GT-1a enzyme inhibition assay with the carboxylic acid prototype 2.23 This result was meaningful, since it indicated that the methylsulfonamide moiety of 3 bound effectively in the catalytic site of the enzyme and thus served as a functional replacement for the terminal carboxylic acid. Guided by the model, the prime side SAR was further explored by introducing structural features designed to more effectively complement the uniquely defined S1′ subsite. Most immediately, the cyclopropylacylsulfonamide 6 was evaluated and this compound inhibited HCV NS3/4A activity with an IC50 of 1 nM. The potency of 6 represents an approximately 50-fold enhancement in intrinsic activity compared to carboxylic acid 2 and a 30-fold increase in activity compared to the prototypical acylsulfonamide 3. Close analogues of cyclopropylsulfonamide 6 were significantly less active, with the ethylacylsulfonamide 4 showing 6-fold reduced intrinsic activity (IC50 = 6 nM) when compared to 6, while the isopropylacylsulfonamide 5, which adds just two hydrogen atoms to 6, is 19-fold weaker, IC50 = 19 nM. The rationale for the enhanced potency associated with cyclopropylsulfonamide 6 compared to carboxylic acid 2 was evident from a modeled structure of 6 bound to the NS3/4A enzyme, depicted in Figure 3a. In general, the spatial orientation of the acylsulfonamide group of 6 with respect to the catalytic site and S1′ pocket of the enzyme suggests that productive nonbonding interactions in these regions are responsible for the observed gain in potency. Specifically, the nitrogen of the acylsulfonamide group participates in a salt bridge with the catalytic histidine of NS3 protease, an observation consistent with the relative pKa of this functionality,24−26 while the carbonyl moiety accepts a hydrogen bond from glycine 137 (Figure 3b). The presence of charge on the amide nitrogen rather than oxygen is supported by calculations of the electrostatic potential for the acylsulfonamide group as well as crystal structure data reported for

Table 1. HCV Inhibitory Profile of the Tripeptide Carboxylic Acid 2 and Acylsulfonamide Homologues

acylsulfonamides.27 Most interestingly, the electron withdrawing sulfonyl group of 6 has multiple functions, since it acidifies the amide N−H while presenting hydrogen-bond-accepting capacity to the oxyanion whole via its diastereotopic oxygen atoms, each of which engages a backbone NH, one to Gly137 and the other to Ser139 (Figure 3b). Moreover, the bridging sulfonyl moiety positions the terminal cyclopropyl ring in the S1′ pocket where it engages in favorable hydrophobic interactions with the enzyme subsite. This specific aliphatic binding element was critical to activity while also sensitive to small structural modifications, since simple ring-opening to 5 or expansion to a cyclobutane (7), cyclopentane (8), or cyclohexane (9) resulted in a marked reduction in inhibitory activity although the phenyl analogue 10, the unsaturated homologue of 9, is 18-fold more potent than 9 but still markedly less potent than 6. These prime side SARs were 1710



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Figure 3. (a) Model of acylsulfonamide 6 bound to the NS3/4A protease enzyme. (b) Key interactions between the acylsulfonamide moiety and the NS3 protein. (c) Cyclopropyl moiety bound to the S1′ pocket.

of 55 mL min−1 kg−1 and a half-life of 1 h. A similar PK profile was observed for 6 in the dog (3 mg/kg dose) with exposure of 2.0 μM·h, oral bioavailability of 20%, clearance of 16 mL min−1 kg−1, and a half-life of 0.6 h. Since the overall PK profile of 6 reflected poor oral exposure, high clearance, and a short half-life, a critical objective in the identification of a first-generation protease inhibitor in the acylsulfonamide series became the optimization of these parameters. Two additional observations with respect to compound 6 proved to be instrumental in shaping the prosecution of this program. First, the metabolic stability of this compound in rat and dog liver microsomes was high, t1/2 of 162 and 170 min, respectively. This level of microsomal stability is predictive of low oxidative clearance in these species. Second, after oral dosing to rats, the concentration of compound 6 was greater in the liver than in plasma, and this hepatotropic distribution was maintained at each of the time points measured. For example, at the 4 h time point, the liver concentration of 6 was 5.8 μM and the plasma level was 0.58 μM, while at 8 h, the liver level was 0.6 μM and the plasma level was 0.004 μM. Collectively, these findings were suggestive that the in vivo clearance for 6 in both the rat and dog was not exclusively driven by metabolic events and raised the prospect that transporters may be playing a role in compound uptake into the liver of both of these species.32,33 The elevated and sustained liver levels observed for 6 were considered a favorable attribute in the context of a potential treatment for HCV, since the replication cycle of this virus is believed to occur predominantly in the liver.34 Hence, in the optimization of compound 6, the potential relevance of identifying a compound that possessed a hepatotropic distribution profile was recognized. To this end, an in vivo assay was developed in which compounds were dosed intraduodenally to surgically prepared rats and plasma levels

rationalized using a plot of the van der Waals surface of the protein which highlights the shallow cavity within the S1′ site that is defined by Gln41, Phe43, Val55, and Gly58, with Phe43 forming the base (Figure 3c). An assessment of the optimized geometries of the sulfonamide caps in analogues 3−9 suggested that the cyclopropyl moiety exhibited a shape most complementary with the S1′ pocket, thus maximizing van der Waals surface contact and explaining the potency of 6. Though most of the S1′ site is static, based on modeling efforts, the mobility of Gln41 allows for accommodation of larger groups such as phenyl substitution (10), albeit being a suboptimal fit in the shallow cavity. The tripeptidic acylsulfonamide 6 and carboxylic acid 2 were evaluated in a cell-based HCV replicon assay28 derived from HCV GT-1b wherein 6 with EC50 = 4 nM was 120-fold more potent than 2, EC50 = 550 nM. Importantly, the enhanced cellbased antiviral activity of acylsulfonamide 6 compared to carboxylic acid 2 was consistent with the comparative potency of these inhibitors in the biochemical screen. From a drug design perspective, the acylsulfonamide functionality provides enhanced potency both in biochemical and in cell-based assays without resort to the use of either conformational restraint tactics29 (e.g., macrocyclization) or mechanism-based motifs30 and this structural feature has been widely adopted in NS3 protease inhibitor design.31 The low nanomolar potency of 6 in the cell-based assay met our target criteria for antiviral activity in cell culture, and efforts turned to optimizing the pharmacokinetics (PK) properties of this series. The PK profile of 6 was determined in both rat and dog. When dosed orally to rats at 20 mg/kg, 6 demonstrated an exposure of 0.73 μM·h and an oral bioavailability of 9%. Intravenous adminstration of 6 at 5 mg/kg resulted in clearance 1711



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Table 2. Virology and PK Parameters for Acylsulfonamide Derivativesa

a

The asterisk (∗) denotes that the compound was dosed as a mixture of diastereoisomers epimeric at the C2 position of the P1 cyclopropane ring.

to binding. Moreover, each of the side chains as well as the P4 moiety interfaced with the enzyme through van der Waals contact and effective desolvation of enclosed pockets. Since we sought to maintain the key H-bond donor−acceptor interactions between ligand and enzyme, attention was focused on strategically reducing the size of the amino acid side chains of 6. Truncation of the P1 and P3 amino acid side chains of 6, as well as P4, was examined with the result that each analogue, compounds 11−13 (Table 2), led to a significant loss in activity in both the biochemical and cell-based assays. In addition, while the P4 methyl carbamate derivative 14 was active in both the biochemical assay and the replicon assay, it performed poorly in the 4 h rat screen compared to the prototype 6. Efforts then turned to the optimization of the heterocycle attached to the P2

were monitored over 4 h, while liver levels were captured at the 4 h time point at the end of the experiment. This route of administration was selected in order to circumvent potential absorption issues associated with the oral dosing of compounds exhibiting low solubility in vehicle. Additionally, the throughput in this abbreviated assay was greater than that for more complete rat PK studies, which facilitated the rapid evaluation of this series of compounds. In optimizing a given chemical series, the reduction of molecular weight is one tactic by which pharmacokinetics properties may be improved, and this approach was examined in the context of compound 6.35 The model structure of compound 6 with the NS3/4A enzyme suggsted that each of the H-bond donors and acceptors along the tripeptide backbone of 6 was complemented by the enzyme and appeared to contribute 1712



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Figure 4. (A−H) Models of compounds 19, 22, 41, 24, and 31 bound to the active site of HCV NS3/4A protease.

develop SAR. For example, on the basis of the model presented in Figure 4 A, the P2* (Table 2) heterocycle of 19 largely overlaid the aliphatic region of the side chain of Arg155. Hence, it was anticipated that the judicious addition of small lipophilic substituents to the core of this ring system might be used to productively modulate potency, as had been described in related ring systems.36 Additionally, a model of 19 bound to the enzyme demonstrates that the charged guanidine functionality at the terminus of Arg155 is proximal to the fused phenyl ring of the isoquinoline in 19, and this observation raised the possibility of engineering and refining favorable electronic interactions between the inhibitor and enzyme. Moreover, the isoquinoline ring system offered the unique potential to explore substitution at the C4 position as a means of influencing potency. Finally, the observation that the exposure of isoquinoline 19 in the rat screen was substantially greater than that for the closely related quinoline analogue 15 suggested that modulating the polarity of the isoquinoline ring system through substituent effects might be a useful tool to further optimize PK properties. The SAR study that is summarized in Table 3 revealed that substitution at each position of the isoquinoline ring provided unique properties, driven by an interplay of steric and electronic effects between the substituent, the heterocycle, and the enzyme surface. In some examples, the SAR was relatively straightforward and readily interpretable. For example, a simple methyl group at C-4 (22) or C-6 (33) provided a several-fold increase in intrinsic activity (IC50) compared to the unsubstituted isoquinoline 19, likely driven by van der Waals contact between the methyl substituent and the enzyme surface (Figure 4C). Similarly, the reduction in potency observed for the analogue substituted at C-8 with a simple methyl group (41) could readily be understood as the result of a steric clash between this substituent and alanine 156 of the enzyme (Figure 4D). While the use of small isoquinoline substituents was emphasized, larger groups were also explored to a limited extent in order to establish the boundaries of SARs. A simple phenyl substituent at C-3 (20) or a morpholino group at C-6 (29) led to a modest increase in activity, whereas large substituents at C-5 were poorly tolerated (e.g., 24), attributed to a steric clash with the EF loop of the enzyme (Figure 4E).

proline moiety in 6. For example, replacing the phenyl moiety of 6 with a hydrogen atom provided quinoline 15 which maintained an acceptable level of potency. However, 15 performed poorly in the rat PK screen with exposures in both plasma and liver significantly lower than that observed for the parent compound 6. A further simplification of the quinoline motif found in 15 to the corresponding pyridyl analogue 16 resulted in a significant loss in inhibitory activity in both the enzyme and cell-based assays. Activity was restored by the addition of a phenyl ring to the 2-position of the pyridyl moiety to give 17; however, cellbased activity for this compound remained modest, and exposure in the rat PK screen was poor. These data points returned attention to fused bicyclic ring systems as vehicles for further optimization. The C-2-linked quinoline 18 performed markedly better in the rat PK screen than the 7-methoxy, 4-substituted quinoline 15, although the antiviral activity of 18 was 10-fold inferior to 15. Interestingly, the C-2-linked isoquinoline 19, which hybridized structural aspects derived from 15 and 18, demonstrated an improvement in intrinsic activity compared to 18 while maintaining good exposure in the rat PK screen. To confirm these observations in the rat PK screen, isoquinoline 19 was dosed to rats in a more thorough study with the results demonstrating a significant improvement in the in vivo profile of this compound compared to quinoline derivative 6. Specifically, dosing of a solution of 19 to rats at 15 mg/kg revealed an oral exposure of 2.6 μM·h, with F = 20%, which compared favorably to the 0.577 μM·h value and oral bioavailability of 9% previously noted for 6. Moreover, clearance for 19 was moderate at 19 mL mg−1 kg−1, an improvement over the rate of 55 mL min−1 kg−1 found with 6. Finally, the half-life of 2.2 h measured for 19 was more than twice that of 6. Thus, by reduction of the molecular size of the heterocycle found in 6 and replacement of the quinoline ring system with an isoquinoline moiety, the PK profile of 19 represented a substantial improvement. This data set suggested that further exploration of SARs in the isoquinoline series was warranted with the objective of improving the antiviral EC50 toward genotypes 1a and 1b viruses to less than 20 nM while further optimizing the pharmacokinetic properties in rat. In several respects, the unsubstituted isoquinoline 19 proved to be a particularly useful template with which to further 1713



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Table 3. Profiling Data for Isoquinoline-Based HCV NS3 Inhibitorsa

a

The asterisk (∗) indicates that this compound was dosed as a mixture of diastereoisomers epimeric at the C2 position of the P1 cyclopropane ring.

Interestingly, the relative conformation between the heterocycle ring system and the ring substituent was also an important contributing factor to potency. For example, the C-6 pyrrole analogue 30 demonstrated good activity in the enzyme inhibition assay, while the C-6 dimethylamide derivative 31 proved to be significantly less active. A model of 30 bound to the protease suggests that the C6 pyrrole substituent can readily assume a coplanar arrangement with the aromatic ring system. In contrast,

the C-6 amide group of 31 and the isoquinoline ring adopt an orthogonal orientation (Figure 4G), a conformation driven by A1,3 strain between the amide group and the isoquinoline ring.37 The significance of a coplanar orientation between the C-6 substituent and the isoquinoline ring was also apparent when comparing the relative activity of the methyl analogue 33 and the corresponding tert-butyl analogue 34. The reason for the loss in activity associated with an orthogonal orientation of the amide in 1714



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Figure 5. (a) X-ray cocrystallographic structure of 35 bound to NS3 Protease. (b) Electrostatic potential maps for analogues 35, 32, 25, and 36.

31 or the bulky tert-butyl group in 34 is not fully understood. However, on the basis of modeling studies, it appears that when bound to the enzyme, these C-6 groups alter the relative conformation of the C-1 alkoxyproline moiety with respect to the isoquinoline ring, more specifically, by reducing the planarity defined by the C-4 proline O−C-1 carbon−N2 nitrogen bond of the isoquinoline ring. Hence, the binding interface between the P2* of 31 (or 34) and the enzyme is compromised with this compound compared to 19 (Figure 4H). The role of electronic effects was also examined in each of the fused aromatic rings of the P2* heterocycle. Interestingly, these effects proved to be minimal at C-4, since both electron withdrawing and electron donating groups engendered good activity in the enzyme inhibition assay, as exemplified by the comparison between chloro derivative 21 and the methoxy analogue 23. However, a significant electronic effect was noted for substituents at C-6 of the isoquinoline ring. For example, the C-6 methoxy derivative 35 demonstrated excellent activity in the biochemical screen, whereas the corresponding C-6 chloro analogue 32 exhibited more modest activity in this assay. It was proposed that the differing impact of these substituents on intrinsic potency

may be a consequence of their opposing influence on the electronic surface of the heterocyclic ring. A cocrystal structure of 35 with the NS3/4A enzyme (Figure 5a) was obtained that confirmed the proposed acylsulfonamide conformation of 6 with respect to backbone orientation and interactions with the enzyme as described. Further analysis of the cocrystal structure of 35 illustrates the position of the isoquinoline ring with respect to the protein and highlights potential binding interactions between them. As previously noted, the isoquinoline of 35 is positioned over the side chain of Arg155, with the terminus of this amino acid in a region proximal to the fused phenyl ring of the isoquinoline core. The C-6 methoxy group of 35 adopts a coplanar arrangement with respect to the aromatic ring and the terminal methyl group projects in the direction of the EF loop.38 This binding conformation of the methoxy group potentiates its electron releasing effect which increases electron density of the isoquinoline ring and, since the diffused negative surface charge is proximal to the positively charged terminus of Arg155, may result in increased binding affinity for this compound compared to the des-methoxy analogue 19. On the other hand, the Cl substituent in 32 withdraws electron density from the ring, thus decreasing 1715



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observed for isoquinoline analogues incorporating substituents with a greater lipophilic character. For example, the C-3 phenyl derivative 20 and C-6 tert-butyl analogue 34 exhibited CC50 values of less than 20 μM, while the CC50 values for the chloro analogues 21, 25, and 32 were less than 46 μM. The performance of isoquinoline derivatives in the rat screen was also influenced by the nature and site of attachment of the substituent to the heterocycle core. Most notably, the C-5substituted isoquinolines 26 and 27 demonstrated higher plasma levels but lower liver levels in rats following oral administration when compared to the corresponding C-4 isomers 22 and 23, respectively. Hence, on the basis of these limited rat screen data, it appeared that isoquinolines with substituents in the nitrogencontaining ring (22, 23) presented in vivo profiles distinct from those analogues bearing substituents in the adjacent all-carbon ring. The rat screen result for the C-6 methoxy analogue 35 was consistent with this observation, since both plasma and liver levels were elevated compared to the prototype 19. The significantly higher exposure observed with 35 in the rat screen and in subsequent full PK studies when compared to either the C-5 isomer 27 or the unsubstituted isoquinoline 19 provided a striking example of the impact of small structural changes on the in vivo performance of molecules in this series. In considering the profiling results on the compounds compiled in Table 3, the C-4 and C-6 methoxy isoquinoline analogues 23 and 35, respectively, emerged as optimal with respect to activity in the cell-based assay, in vitro cytotoxicity profile, and exposure in the coarse rat screen, and hence, these compounds were evaluated more extensively. Although the oral bioavailability of 23 after dosing to rats as a solution in PEG-400/ethanol (90/10, v/v) was modest, the compound was not detectable in plasma at the 24 h time point. Interestingly, the liver levels of 23 measured 24 h after po dosing were high (4.2 μM). After iv dosing, the clearance of 23 was high and the 24 h liver concentration was similarly high at 8.0 μM. Thus, after po or iv dosing, 23 was cleared rapidly from plasma and partitioned into the liver, resulting in low plasma exposure and high C24 liver levels. The high clearance observed for 23 coupled with the low plasma exposure after po dosing was not consistent with the targeted PK profile, and this compound was not progressed further. In contrast, the PK profile of 35, a regiosiomer of compound 23, was more favorable. The oral bioavailability of 35 after solution dosing was 18%, with plasma exposure of this compound relatively high at 14 μM·h. At the 24 h time point, the concentration of 35 in plasma was 8 nM, while the liver concentration was elevated relative to plasma at 730 nM. After iv dosing, the clearance of 35 from plasma was low at 4.4 mL min−1 kg−1, resulting in a substantial half-life of ∼4 h. The sustained plasma levels of 35 over 24 h after po dosing, coupled with the high EC50 multiples of this compound in the liver at the 24 h time point, focused attention on this compound as a program lead. Given the structural similarity of analogues 35 and 23, the significant difference in clearance rates in rat for these compounds warranted further consideration. Interestingly, despite this differential in clearance, the in vitro stability of 35 and 23 in a rat liver microsomal assay was quite similar, with each having a t1/2 of greater than 60 min. Moreover, while the liver-to-plasma ratios for each of these compounds 24 h after dosing was high, the compound with the higher clearance, 23, also demonstrated a higher liver-to-plasma ratio. These results were suggestive of transporters playing a role in governing the in vivo disposition of these compounds, as noted for 6. Additionally, the profile of the regioisomeric analogues 23 and 35 provide yet a further

the potential for a favorable cation−π interaction between the isoquinoline ring system and the guanidine terminus of Arg155.39−41 This rationale for the difference in potency between 32 and 35 was further confirmed by a detailed analysis of the electrostatic potential (ESP) generated by the functionalized heterocycles within these analogues in the presence of protein, which was represented as a field of partial changes. Plots of the field-induced electrostatic potential for the functionalized isoquinolines present in compounds 32 and 35 (Figure 5b) provides a pictorial representation of these presumed electronic effects. As shown, the C-6 methoxy group of 35 introduces a build-up of negative potential within the isoquinoline core while the C-6 Cl substituent of 32 depletes electron density within this ring system, with partial negative charge localized on this atom. This electronic effect proved to be specific for position 6 of the isoquinoline ring, as the intrinsic enzyme inhibitory activities of the C-5 chloro and C-5 methoxy analogues 25 and 27, respectively, are similar. This observation can be rationalized in a similar fashion based on ESP plots where the reduced effectiveness of the C-5 chloro atom of 25 to withdraw electron density from the isoquinoline ring system is evident. Interestingly, while the C-6 chloro derivative 25 was less active than the corresponding methoxy analogue 35, an electron withdrawing cyano group at C-6 (36) was equipotent to 35. The apparent discrepancy between the activity of 25 and 36 can be reconciled based on the distinct mode of interaction of 36 with the enzyme. Specifically, an analysis of the field-induced electrostatic potential of 36 (Figure 5b) indicates that a large charge density of negative potential is localized at the terminus of the cyano substituent. This is of relevance since in the bound conformation this group is proximal to the positively charged center of Arg155, which results in a dominant and favorable local interaction. This analysis is consistent with reported crystal structures of structurally differentiated cyano compounds in which the cyano moiety is docked over the charged terminus of an arginine.42 The potential electronic interaction between the terminus of the cyano group in 36 and Arg155 complements the interactions of 35 and Arg155 in which a cation−π interaction is induced by the methoxy group of 35. Hence, there appear to exist two distinct modes by which C-6 substituents attached to the isoquinoline ring system can induce a favorable electronic interaction with Arg155 of the enzyme. The observation that substituents at the C-6 position of the isoquinoline ring system could influence binding by potentially modifying electron density in the ring was supported by the activity of the C-6 hydroxy analogue 37, which was equipotent to 35 in the enzyme inhibition assay. However, the IC50 results for the C-5 and C-6 fluoro analogues 28 and 38, respectively, were confounding because both compounds demonstrated a similar reduction in potency in contrast to the corresponding chloro analogues 25 and 32. This suggested that further evaluation of the underlying electronic factors governing potency was required, yet such a study was beyond the scope of this immediate work. Nevertheless, the data presented in Table 3 demonstrated that small substituents could be used to modulate enzyme inhibitory potency (IC50) within the isoquinoline series, and this effect extended to the cell-based replicon assay. However, while the activity of analogues in the replicon assay generally tracked with intrinsic enzyme inhibitory activity (IC50), a precipitous drop-off in antiviral activity was observed with analogues bearing polar substituents, as in 36 and 37.43 In addition, in the in vitro cytotoxicity assay, SAR emerged in which lower CC50 values were 1716



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The comparative profiles of acylsulfonamide 35 and carboxylic acid 50 further underscore the significant advantage offered by the acylsulfonamide moiety within this chemical series in terms of both potency and PK. From this survey of analogues in the tripeptidic acylsulfonamide chemotype, 35 (BMS-605339) emerged as the most promising HCV inhibitor. The antiviral activity of 35 in the cellbased replicon assay was 8.3 nM against GT-1a and 2.8 nM versus GT-1b, while the in vitro cytotoxicity profile of 33 in a hepatotoma-derived cell line was also favorable (CC50 = 167 μM), providing a significant in vitro therapeutic index. The selectivity of 35 for the HCV NS3 protease was examined by evaluating its effects against a range of serine and cysteine proteases in enzyme assays. This compound displayed >1000-fold selectivity for the HCV protease versus cellular proteases including the most homologous protease, GBV-B NS3. The anti-HCV specificity of 35 was examined in different cell culture systems. This compound was inactive against bovine viral diarrhea virus (BVDV), canine parainfluenza virus, and HIV. In addition, the PK properties of 35 in the rat represented a marked improvement over the early isoquinoline lead 19, as 35 demonstrated greater plasma and liver exposure following oral adminstration, lower clearance, and a longer half-life (Table 3). Given this favorable profile, 35 was progressed into PK studies in higher species. The oral bioavailability of 35 in the dog was 51%, with moderate to high clearance and a relatively short plasma half-life. Pharmacokinetic studies in monkeys indicated that the 35 was poorly absorbed (oral bioavailability less than 1%), with moderate clearance and a short half-life. Consistent with this observation, 35 was rapidly metabolized in monkey liver microsomes in vitro, suggesting that the poor bioavailability of 35 after oral dosing to monkeys was a consequence of a significant firstpass effect. In contrast, 35 proved to be more stable in rat, dog, and human liver microsomes. In an effort to gain insight into the hepatic exposure of 35 in different species following oral administration, additional studies were conducted in both the rat and dog. After dosing to rats at 15 mg/kg, 35 demonstrated liver levels that were higher than plasma. For example, rat liver levels for 35 24 h after dosing were 0.733 μM while plasma levels were measured as 0.005 μM. A similar hepatotropic distribution of 35 was observed in dogs 24 h after dosing at 14 mg/kg, where liver levels were measured at 384 nM and plasma levels at 6 nM. The liver-to-plasma ratios of 35 24 h after oral dosing to rat and dog were 136 and 75, respectively. The high liver-to-plasma ratio observed in multiple species again suggested the possibility of transporters playing a role in the uptake of compounds into the liver. High and sustained concentrations of compound in the liver were considered a favorable distribution profile in the context of treating hepatitis C virus infection, and further studies directed at developing an understanding of the hepatotropic distribution were conducted. In general, studies on the cellular uptake of 35 into hepatocytes established the role of an energy-dependent process and the Km values for this cellular uptake process were similar across species (1.4−5.5 μM), suggestive of similar kinetics.44 On the basis of these preliminary findings, the elevated liver levels of 35 after oral dosing in both rat and dog appeared to be mediated, at least in part, by an active transport process. In addition, by extrapolation of the available in vitro and in vivo data, the human liver disposition of 35 was predicted to be high relative to plasma. Thus, while the PK profile of 35 was different among each of the three preclinical species in which it was evaluated, the sustained

illustration of the significant impact of relatively small structural changes within a large molecule on the PK properties of compounds in this series. In an effort to further optimize the properties of the 6-methoxyisoquinoline 35, select structural changes were made to the P2* region and subsequently on the peptidic scaffold of this compound. For example, replacing the methoxy group at the C6 position of the isoquinoline ring with an ethoxy or amine substituent resulted in analogues 39 and 29, respectively, which exhibited potency similar to that of 35; however, both compounds were observed to have low exposure in the rat PK screen compared to the prototype. As previously noted, the O-demethylated analogue of 35, compound 37, was suitably potent in the enzyme inhibition assay; however, the antiviral activity of 37 in the replicon was poor and well below the targeted potency. Thus, a methoxy group at the C6 position of the isoquinoline ring system was optimal with respect to potency and PK. The potency of 35 was improved 6-fold by the addition of a second methoxy group to the C-4 position of the isoquinoline ring system to give 42; however, the oral exposure of this compound was poor. Additionally, the in vivo clearance of 42 was relatively high compared to 35, and for these reasons this compound was not considered for further study. Nevertheless, the potency observed with 42 prompted the synthesis of heterocycles in which the electronic properties of the nitrogencontaining ring system in 35 were modified. The enzyme inhibitory activity of the C-3 linked isoquinoline 43 and the quinazoline analogue 44 was comparable to 35, while the antiviral activity in the cell-based replicon for both compounds was lower. However, the plasma exposure of both isoquinoline 43 and quinazoline 44 in the rat screen was significantly less than that observed for 35. Consistent with this observation are the previously noted results for quinoline analogue 15 which was also inferior to isoquinoline 35. The observed discrepancy in rat PK performance between the lead isoquinoline 35 and the isomeric isoquinoline 43 and quinoline 15 is noteworthy. Moreover, the observation that isoquinolines performed better than the corresponding quinoline in the rat screen has proved to be a general phenomenon.31r For example, the C-3 phenylisoquinoline 45 had exposure in both liver and plasma that was more than 10-fold greater than that observed for the corresponding quinoline 6. The superior performance of isoquinolines in the rat screen compared to structurally similar quinolines once again underscores the impact of small structural changes on the PK profile of compounds in this tripeptide acylsulfonamide series. Additional structural changes were also probed at the P1, P2, and P3 subsites of 35, but in each case the resulting analogue had inferior properties. Saturation of the olefin at P1 provided 47, which retained antiviral activity but demonstrated poor oral exposure in rats. A similar result was found with 48 in which the P4 tert-butyl group is replaced with a tetrahydrofuranyl moiety. Interestingly, the P3 valine derivative 49 demonstrated a similar PK profile to 35; however, the cell-based replicon inhibitory activity was ∼3-fold less than that observed for 35. To provide an additional point of reference of the impact of the acylsulfonamide group with respect to both the potency and PK properties of 35, the carboxylic acid analogue 50 was prepared. As shown in Table 4, the NS3/4A inhibitory activity and antiviral properties of 50 were considerably poorer, with an IC50 of 247 nM and an EC50 of 2000 nM, values that are approximately 120-fold less than those observed for acylsulfonamide 35. The performance of 50 in the rat screen was also poor, with compound not detectable in either the plasma or the liver after intraduodenal dosing. 1717



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Table 4. Profiling Data for Analogues of 35

and elevated liver levels observed after po dosing to the rat and dog in conjunction with the active uptake process observed in vitro across species provided the confidence to progress 35 into advanced profiling and toxicology studies. Compound 35 was extensively profiled in vitro in an effort to define off-target liabilities and assess the potential risk of such findings in a clinical setting. This compound was found to be a P-glycoprotein (P-gp) substrate and a mild inhibitor of CYP 3A4 (IC50 ≈ 2.0 μM). However, the inhibitory effect was not timedependent and there was no evidence implicating 35 as an inducer of CYP 3A4 expression. The in vitro toxicology profile of

35 was also favorable, as there was no evidence of mutagenicity in either the Ames or in vitro micronucleus assay. The in vivo toxicology assessment of 35 was also supportive of the clinical development of this compound based on Cmax and AUC margins for a projected human clinical dose, including a reduction in heart rate observed in dogs. Compound 35 was advanced into phase 1 clinical trials where the initial study examined administration of the compound to healthy volunteers at doses of 10, 30, 60, and 120 mg. Plasma drug levels increased with each incremental dose with the average Cmax observed at ∼1.5 h, indicating relatively rapid absorption of 1718



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appeared to be significantly more sensitive than anticipated from initial preclinical evaluations. The results of these studies will be described in a forthcoming publication. Acylsulfonamide tripeptide 35 was synthesized by the coupling of three constituent fragments (Schemes 1 and 2), and this general approach was used to provide access to the analogues described herein.45 The synthesis of intermediate 54 began with the construction of the isoquinoline heterocycle 53, which was available by a scalable, two-step sequence starting with 3-methoxycinnamic acid (51). This commercially available acid was subjected to a Curtius rearrangement followed by electrocyclization to afford the isoquinolone ring system in 52.46 Intermediate 52 was converted to 1-chloro-6-methoxyisoquinoline (53) under standard conditions, and this activated aromatic derivative was coupled with N-protected 4-hydroxyproline under basic conditions to provide the P2 element 54. The P1−P1′ moiety 58 was synthesized by coupling the Boc-protected vinylcyclopropylamino acid 5547 with cyclopropylsulfonamide (56),48 followed by CF3CO2H-induced unmasking of the amine. The amine 58 was coupled efficiently at room temperature with the proline derivative 54 using HATU as the coupling reagent to provide the dipeptide acylsulfonamide 59. The Boc group in 59 was removed under acidic conditions which directly provided the HCl salt of amine 60, a compound that cleanly coupled to commercially available tert-butylglycine (61) to provide 35 in excellent yield. Compound 35 crystallized from ethanol as the free acid which in the solid state revealed an intramolecular hydrogen bond between the sulfonamide NH and the carbonyl of the P3 tert-butylglycine moiety (Figure 7).49

drug. The terminal half-life was 4−8 h, and compound was detectable in plasma 24 h after administration across the dose range. Compound 35 was administered to human subjects infected with genotype 1 hepatitis C virus at doses of 10, 60, and 120 mg. The antiviral response was dose-dependent with a mean 1.8 log10 reduction in viral load observed 12 h after a single 120 mg dose of 35 (Figure 6). While the antiviral response of 35

Figure 6. Mean reduction in HCV plasma level following single oral doses of 35.

in human subjects was favorable, clinically important electrocardiographic changes were noted in one healthy volunteer and one HCV-infected subject following the administration of a 120 mg dose of drug. Note that while these transient electrocardiographic findings, which are manifested in mild bradycardia, PR-interval prolongation, and junctional rhythm disturbances, were asymptomatic in their presentation, the potential risk associated with the administration of this drug to the HCV-infected population was nonetheless deemed significant. These findings caused the cessation of clinical trials on 35 and prompted a detailed investigation of this cardiac finding, since humans

■

CONCLUSION The discovery of 35, a potent tripeptidic inhibitor of HCV NS3 protease, has been described along with the preclinical profiling that supported advancement of this compound into clinical trials. This compound incorporates a cyclopropylacylsulfonamide P1′ moiety as a key structural motif that is associated with markedly enhanced potency compared to the carboxylic acid prototype in a series of tripeptide inhibitors. In the development of the

Scheme 1. Preparation of P2−P2* and P1−P1′a

Reagents and conditions: (a) EtOC(O)Cl, TEA, acetone, 0 °C; (b) NaN3, rt; (c) Ph2CH2, Bu3N, 210 °C, 49% overall for three steps; (d) POCl3, reflux, 88%; (e) N-Boc-3-(R)-hydroxy-L-proline, t-BuOK, DMSO, 10 °C to rt, 100%; (f) CDI, cyclopropanesulfonamide (56), reflux to rt, 100%; (g) TFA, CH2Cl2, then HCl, ether, 98%.

a

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Scheme 2. Coupling of Fragments for the Synthesis of 35a

a Reagents and conditions: (a) HATU, Hunig’s base (i-Pr2NEt), 58, DCM, 0 °C to rt, 64%; (b) TFA, CH2Cl2, then HCl, ether, 100%; (c) HATU, Hunig’s base, N-Boc-tert-butyl-L-glycine (61), DCM, 0 °C to rt, 78%.

triplets; dq, doublet of quartets. Most spectra were analyzed using the ACDLABS SpecManager 12.0 program software. Note that at ambient temperature in DMSO-d6, most of the final products exist predominantly as a mixture of rotamers. All compounds were determined to be at a purity level of greater than 95% as detemined by liquid chromatography methodologies. Liquid chromatography/mass spectrometry (LCMS) was performed on a Shimadzu LC instrument coupled to a Water Micromass ZQ instrument, and the LCMS conditions are shown in Table 5.

Table 5. LCMS Conditions Used in the Characterization of All Compounds Found in the Experimental Section

Figure 7. Single crystal X-ray structure of 35.

acylsulfonamide chemotype, the utility of P2* as a vehicle to modulate both potency and PK properties was realized. Notably, subtle structural changes were found to exert a significant impact on both potency and PK parameters with the C-6 methoxyisoquinoline present in 35 found to be optimal. The compound exhibited variable oral bioavailability between rat and dog, and yet high concentrations of drug was observed in the liver after oral dosing to both species. The antiviral activity of 35 following administration of a single dose to HCV genotype 1 infected patients was significant, with a mean 1.8 log10 reduction in viral load measured at 12 h after a single 120 mg dose. However, clinical trials of 35 were terminated because of a cardiovascular liability assessed in more than one subject at the 120 mg dose. Although exposure margins in preclinical toxicology studies with 35 were sufficient to advance this compound into clinical trials, humans proved to be significantly more sensitive to the cardiovascular effects of 35 than the dog. These findings were fully integrated into the program which subsequently led to the discovery of asunaprevir (BMS-650032), a compound currently in advanced clinical trials for the treatment of HCV infection.50,51

■

LCMS conditiona

Colb

GTc

FRd

Sol Ae

Sol Bf

method A method B method C method D method E method F method G method H

3 4 2 1 5 3 6 2

2 3 2 3 2 3 4 3

5 4 1 0.8 5 5 4 0.8

A1 A1 A1 A1 A1 A1 A1 A1

B1 B1 B1 B1 B1 B1 B1 B1

a Gradient = 0% B to 100% B; wavelengh (λ) = 220 nm; injection volume = 5 μL unless otherwise stated. bCol = column, where column 1 is Phenomenex-Luna C18 (2.0 mm × 50 mm, 3 μm); column 2 is Phenomenex-Luna C18 (2.0 mm × 30 mm, 3 μm); column 3 is XTERRA C18 (3.0 mm × 50 mm, S7); column 4 is PhenomenexLuna C18 (3.0 mm × 50 mm, 5 μm); column 5 is YMC ODS C18 S7 (3.0 mm × 50 mm); column 6 is XTERRA C18 (4.6 mm × 50 mm, S7). cGT = gradient time (min). dFR = flow rate (mL/min). eSol A = solvent A, where solvent A1 is 0.1% TFA/10% MeOH/90% H2O. fSol B = solvent B, where solvent B1 is 0.1% TFA/90% MeOH/10% H2O.

Preparation of 6-Methoxy-2H-isoquinolin-1-one (52). To a stirred solution of 3-methoxycinnamic acid 51 (200 g, 1.12 mol) and Et3N (227 g, 2.25 mol) in acetone (1.5 L) maintained at 0 °C was added ethyl chloroformate (188 g, 1.68 mol) dropwise over 45 min. After the mixture was stirred at this temperature for 1 h, aqueous NaN3 (118 g, 1.81 mol) in H2O (181 mL) was added over 10 min, and the reaction mixture was stirred for 16 h at ambient temperature. H2O (1.8 L) was added to the reaction mixture, and the volatile components were removed in vacuo. The resulting slurry was extracted 3 times with toluene (the following volumes of toluene were employed: 800, 500, and 300 mL), and the combined organic layers were dried over MgSO4 and filtered. The filtrate was concentrated in vacuo to a volume of approximately 1.2 L (to remove residual acetone) and diluted to 1.5 L with toluene. This solution containing the acylazide intermediate was added dropwise over 2.5 h to a mixture of diphenylmethane (900 mL) and tributylamine (535 mL) maintained at 190 °C. In the course of this addition, toluene was removed from the reaction mixture by distillation. After the addition of the intermediate acylazide was complete, the

EXPERIMENTAL SECTION

All reagents were purchased from commercial suppliers and used without purification unless otherwise noted. All anhydrous reactions were performed under a nitrogen 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. 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 1720



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temperature of the reaction mixture was raised to 210 °C. After 2 h, the reaction mixture was cooled to room temperature and the precipitated product collected by filtration. The filter cake was washed several times with hexanes and allowed to dry in air to give 52 as a light yellow solid (96.6 g, 49%). 1H NMR (400 MHz, DMSO-d6) δ 11.07 (br s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.20−7.10 (m, 2H), 7.05 (dd, J = 8.8, 2.5 Hz, 1H), 6.48 (d, J = 7.0 Hz, 1H), 3.87 (s, 3H). LCMS (method A): tR = 0.82 min. LCMS (ESI) m/z calcd for C10H9NO2: 175.06. Found: 175.87 (M + H)+. Preparation of 1-Chloro-6-methoxyisoquinoline (53). A mixture of 52 (93.5 g, 0.534 mol) in POCl3 (900 mL) was heated at a gentle reflux for 3 h and then concentrated in vacuo. Cold H2O (500 mL) and EtOAc (500 mL) were added to the crude reaction mixture. The resulting bilayer was chilled in an ice bath and, with rapid stirring, brought to a pH of 7 by the slow addition of solid NaHCO3. The organic layer was removed and the remaining aqueous phase extracted with EtOAc (4 × 350 mL). The combined organic extracts were washed sequentially with saturated aqueous NaHCO3 (2 × 250 mL) and brine (200 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo to provide a yellow-brown solid. The residue was purified by flash chromatography over silica gel using CH2Cl2 as eluent, and the mixed fractions were combined and rechromatographed with a mixture of hexanes and EtOAc (7:1) to afford 53 (91.3 g, 88.3%) as a white solid. 1 H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 9.5 Hz, 1H), 8.10 (d, J = 6.0 Hz, 1H), 7.69 (d, J = 5.5 Hz, 1H), 7.38−7.34 (m, 2H), 3.98 (s, 3H). LCMS (method A): tR = 1.42 min. LCMS (ESI) m/z calcd for C10H8ClNO: 193.03. Found: 193.91 (M + H)+. Preparation of N-Boc-3-(R)-(6-methoxy-1-oxo-isoquinoline)L-proline (54). N-Boc-3-(R)-hydroxy-L-proline (25.0 g, 0.108 mol) was added in small portions to a stirred suspension of potassium tertbutoxide (28.1 g, 0.238 mol) in DMSO (432 mL) in a 1 L three-neck flask equipped with an overhead stirrer. The resulting heterogeneous reaction mixture was stirred at room temperature for 2 h before addition of 53 (21.6 g, 0.111 mol) as a solid in small portions. The reaction mixture was stirred at ambient temperature for 12 h, quenched with H2O (100 mL), and extracted with EtOAc (2 × 200 mL). The aqueous and DMSO mixture was treated with 10% aqueous citric acid (200 mL) and extracted with EtOAc (4 × 500 mL). The combined organic layers were washed sequentially with H2O and brine, dried over MgSO4, and concentrated in vacuo to provide 54 (46g, quantitative yield) as a yellow solid which was used without further purification. 1H NMR (400 MHz, CD3OD) δ 8.07 (d, J = 8.5 Hz, 1H), 7.88−7.87 (m, 1H), 7.25−7.24 (m, 1H), 7.18−7.16 (m, 2H), 5.73 (b, 1H), 4.52−4.44 (m, 1H), 3.92 (s, 3H), 3.87−3.80 (m, 2H), 2.72−2.66 (m, 1H), 2.43−2.38 (m, 1H), 1.42 and 1.44 (s, s, rotamers, 9H). LCMS (method A): tR = 1.62 min. LCMS (ESI) m/z calcd for C20H24N2O6: 388.16. Found: 388.94 (M + H)+. Preparation of 2-[[[(1R,2S)-1-[[(Cyclopropylsulfonyl)amino]carbonyl]-2-ethenylcyclopropyl]amino]carbonyl]-4-[(6-methoxy-1-isoquinolinyl)oxy]-(2S,4R)-1-pyrrolidinecarboxylic Acid, 1,1-Dimethylethyl Ester (59). DIPEA (38.3 g, 342 mmol) was added to a stirred mixture of 54 (33.22 g, 85.5 mmol), HATU (48.75 g, 128 mmol), and (1-(R)-amino-2-(S)-vinylcyclopropanecarbonyl)amide HCl salt (58) (25.02 g, 94.1 mmol) in CH2Cl2 (1 L) at 0 °C. After the mixture was stirred at ambient temperature for 12 h, the solvent was removed in vacuo and the residue stirred with EtOAc (1 L) and filtered. The filtrate was washed twice with ice cold 5% citric acid (aq) and brine, dried over MgSO4, and filtered. The filtrate was evaporated in vacuo and residue recrystallized from MeOH (210 mL) to yield 59 (26.5 g) of the desired product as a white cube. The mother liquor was evaporated in vacuo and the residue purified through a plug of silica gel eluted with hexane−EtOAc (2:1) to yield an additional 10.5 g of 59 as a foam. Recrystallization from MeOH (50 mL) yielded an additional 6.5 g of 59 to afford a combined yield of 64.3%. 1H NMR (400 MHz, CD3OD) δ 8.07 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 6.0 Hz, 1H), 7.26 (d, J = 6.0 Hz, 1H), 7.20−7.17 (m, 2H), 5.81−5.72 (m, 2H), 5.32 (d, J = 18.0 Hz, 1H), 5.13 (d, J = 10.5 Hz, 1H), 4.42−4.40 (m, 1H), 3.93 (s, 3H), 3.89−3.86 (m, 1H), 3.80 (d, J = 12.5 Hz, 1H), 2.98−2.94 (m, 1H), 2.57−2.53 (m, 1H), 2.30−2.24 (m, 2H), 1.88 (dd, J = 8.09, 5.34 Hz, 1H), 1.45−1.42 (m, 10H), 1.27−1.24 (m, 1H), 1.20−1.16 (m, 1H), 1.08−1.05 (m, 2H).

LCMS (method A): tR = 1.74 min. LCMS (ESI) m/z calcd for C29H36N4O8S: 600.23. Found: 601.00 (M + H)+. Preparation of 2-[[[(1R,2S)-1-[[(Cyclopropylsulfonyl)amino]carbonyl]-2-ethenylcyclopropyl]amino]carbonyl]-4-[(6-methoxy-1-isoquinolinyl)oxy]-(2S,4R)-1-pyrrolidine HCl Salt (60). To an ice cold solution of 59 (32.83 g, 54.7 mmol) in CH2Cl2 (100 mL) was added TFA (100 mL), and the solution was allowed to warm to ambient temperature and stirred for 2 h. The solvent was removed in vacuo, the residue triturated with 1 M HCl in Et2O, filtered, and the solid washed with Et2O. The solid was stirred in 1 M HCl in Et2O (200 mL) for 12 h, filtered, and washed with Et2O to give 60 as a slightly hygroscopic white solid (32.2 g). 1H NMR (400 MHz, CD3OD) δ 9.14 (b, 1H), 8.34 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 6.5 Hz, 1H), 7.47 (d, J = 6.0 Hz, 1H), 7.34−7.30 (m, 2H), 5.97 (b, 1H), 5.62−5.69 (m, 1H), 5.33 (d, J = 18.0 Hz, 1H), 5.16 (d, J = 10.5 Hz, 1H), 4.42−4.40 (m, 1H), 3.98 (s, 3H), 3.88 (d, J = 12.5 Hz, 2H), 2.95−2.95 (m, 1H), 2.48−2.42 (m, 1H), 2.37−2.32 (m, 1H), 1.97−1.95 (m, 1H), 1.40−1.37 (m, 1H), 1.31−1.26 (m, 1H), 1.20−1.03 (m, 4H). LCMS (method A): tR = 1.12 min. LCMS (ESI) m/z calcd for C24H28N4O6S: 500.17. Found: 500.93 (M + H)+. Preparation of tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (35). DIPEA (32.0 g, 286 mmol) was added dropwise to a suspension of 60 (32.1 g, 56.02 mmol) in CH2Cl2 (500 mL), and the mixture was stirred at 0 °C. After 30 min, HATU (31.9 g, 84.0 mmol) and N-Boc-tert-butylL-glycine (14.2 g, 61.62 mmol) were added as solids in one portion to the mixture which had turned into a slightly brownish solution. The mixture was warmed to ambient temperature, stirred for 4 h and the solvent removed in vacuo. The residue was stirred with EtOAc (1 L), filtered and the filtrate washed with ice cold 5% aqueous citric acid (twice, keeping the aqueous phase acidic) and brine, respectively. The organic layer was dried over MgSO4, filtered and the filtrate evaporated in vacuo. The residue was purified through a plug of silica gel, eluting with hexane−EtOAc (1.5:1) to yield 39.0 g of a foam that was recrystallized from IPA (1 L) to furnish 35 (33.5 g, 77.5%) as white flakes with purity determined as 97.9% by HPLC. The following spectral data were obtained from a sample recrystallized from MeOH−H2O: 1H NMR (400 MHz, CD3OD) δ 8.09 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.18 (s, 1H), 7.10−7.08 (m, 1H), 6.60 (d, J = 10.0 Hz, 1H), 5.81 (b, 1H), 5.74−5.69 (m, 1H), 5.28 (d, J = 18.0 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.51−4.49 (m, 1H), 4.24−4.21 (m, 1H), 4.06−4.02 (m, 1H), 3.92 (s, 3H), 2.97−2.91 (m, 1H), 2.61−2.55 (m, 1H), 2.30−2.20 (m, 2H), 1.88−1.85 (m, 1H), 1.45−1.40 (m, 1H), 1.27 (s, 9H), 1.25−1.23 (m, 1H), 1.08−1.00 (m, 12H). LCMS (method A): tR = 1.75 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 714.11 (M + H)+; Anal. Calcd for C35H47N5O9S·0.5H2O: C, 58.16; H, 6.69; N, 9.69. Found: C, 58.01; H, 6.46; N, 9.55. tert-Butyl ((2S)-1-((2S,4R)-4-((7-Methoxy-2-phenylquinolin-4yl)oxy)-2-((1-((methylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (3). 1H NMR (500 MHz, CD3OD) δ 8.36 (d, J = 9.2 Hz, 1H), 8.15−8.06 (m, J = 7.6 Hz, 2H), 7.84−7.72 (m, 3H), 7.68 (br s, 1H), 7.57 (br s, 1H), 7.43 (d, J = 7.0 Hz, 1H), 5.88 (br s, 1H), 5.84−5.72 (m, 1H), 5.33 (d, J = 17.1 Hz, 1H), 5.17 (d, J = 10.1 Hz, 1H), 4.73−4.56 (m, 2H), 4.27−4.14 (m, 2H), 4.13−3.99 (m, 3H), 3.23 (br s, 3H), 2.85− 2.75 (m, 1H), 2.53−2.39 (m, 1H), 2.34−2.19 (m, 1H), 1.93 (dd, J = 7.9, 5.5 Hz, 1H), 1.53−1.42 (m, 1H), 1.22 (s, 9H), 1.06 (br s, 9H). LCMS (method D): tR = 2.71 min. LCMS (ESI) m/z calcd for C39H49N5O9S: 763.33. Found: 764.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Ethylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (4). lH NMR: (500 MHz, CD3OD) δ 8.10− 8.03 (m, 3H), 7.47−7.54 (m, 3H), 7.37 (s, 1H), 7.24 (s, 1H), 7.06 (d, J = 9 Hz, 5.89−5.75 (m, 1H), 5.53 (m, 1H), 5.23 (d, J = 17 Hz, 1H), 5.05 (d, J = 10 Hz, 1H), 4.51−4.57 (m, 2H), 4.25 (m, 1H), 4.02−4.14 (m, 1H), 3.93 (s, 3H), 3.03−3.30 (m, 2H), 2.66−2.77 (m, 1H), 2.41 (m, 1H), 2.09−2.18 (m, 1H), 1.83 (m, 1H), 1.26 (s, 9H), 1.24−1.38 (m,3H), 1.03 1721



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(s, 9H). LCMS (method D): tR = 2.76 min. LCMS (ESI) m/z calcd for C40H51N5O9S: 777.34. Found: 778.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Isopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5). 1H NMR (500 MHz, CD3OD) δ 8.37 (d, J = 9.3 Hz, 1H), 8.13−8.07 (m, 2H), 7.84−7.73 (m, 3H), 7.68 (s, 1H), 7.58−7.52 (m, 1H), 7.42 (dd, J = 9.3, 2.3 Hz, 1H), 5.86 (br s, 1H), 5.74 (dt, J = 17.1, 9.6 Hz, 1H), 5.33 (d, J = 17.1 Hz, 1H), 5.17 (dd, J = 10.4, 1.5 Hz, 1H), 4.71 (d, J = 12.7 Hz, 1H), 4.64 (dd, J = 10.4, 7.0 Hz, 1H), 4.19 (s, 1H), 4.18−4.12 (m, 1H), 4.08 (s, 3H), 3.87−3.75 (m, J = 13.8, 6.8, 6.8 Hz, 1H), 2.79 (dd, J = 14.0, 6.8 Hz, 1H), 2.50−2.40 (m, 1H), 2.28 (q, J = 8.9 Hz, 1H), 1.92 (dd, J = 8.1, 5.5 Hz, 1H), 1.44 (dd, J = 9.5, 5.5 Hz, 1H), 1.37 (d, J = 7.0 Hz, 6H), 1.21 (s, 9H), 1.06 (s, 9H). LCMS (method D): tR = 2.84 min. LCMS (ESI) m/z calcd for C41H53N5O9S: 791.36. Found: 792.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (6). lH NMR (300 MHz, CD3OD) δ 8.07− 8.04 (m, 3H) 7.55−7.47 (m, 3H), 7.37 (d, J = 2 Hz, 1H), 7.23 (s, 1H), 7.07−7.05 (m, 1H), 5.84−5.77 (m, 1H), 5.53 (m, 1H), 5.25 (d, J = 17.1 Hz, 1H), 5.07 (d, J = 10.1 Hz, 1H), 4.55−4.50 (m, 2H), 4.25 (s, 1H), 4.10−4.08 (m, 1H), 3.93 (s, 3H), 2.84 (bs, 1H), 2.60−2.70 (m, 1H), 2.35−2.40 (m, 1H), 2.15−2.20 (m, 1H), 1.85−1.83), (m, 1H), 1.41−1.38 (m, 1H), 1.27 (s, 9H), 1.17 (s, 2H), 1.03 (s, 9H), 1.10−0.80 (m, 2H). LCMS (method D): tR = 2.80 min. LCMS (ESI) m/z calcd for C41H51N5O9S: 789.34. Found: 790.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclobutylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (7). 1H NMR (400 MHz, CD3OD) δ 8.11− 8.00 (m, 3H), 7.58−7.45 (m, 3H), 7.39 (d, J = 2.3 Hz, 1H), 7.25 (s, 1H), 7.07 (dd, J = 9.2, 2.1 Hz, 1H), 5.77−5.62 (m, 1H), 5.56 (br s, 1H), 5.26 (d, J = 17.1 Hz, 1H), 5.10 (d, J = 10.3 Hz, 1H), 4.62−4.46 (m, 2H), 4.38−4.23 (m, 2H), 4.14−4.06 (m, 1H), 3.94 (s, 3H), 2.66 (dd, J = 13.8, 6.8 Hz, 1H), 2.55−2.42 (m, 2H), 2.37−2.12 (m, 4H), 2.07−1.90 (m, 2H), 1.83 (dd, J = 8.1, 5.3 Hz, 1H), 1.47−1.34 (m, 1H), 1.31−1.21 (m, 9H), 1.08−0.93 (m, 10H). LCMS (method B): tR = 2.38 min. LCMS (ESI) m/z calcd for C42H53N5O9S: 803.36. Found: 804.29 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopentylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (8). 1H NMR (300 MHz, CDCl3) δ 7.59−7.46 (m, 3H), 7.05−6.86 (m, 3H), 6.85 (d, J = 2 0.2 Hz, 1H), 6.67 (s, 1H), 6.53 (dd, J = 9.1, 2.2 Hz, 1H), 5.36−5.14 (m, 1H), 5.00 (br s, 1H), 4.80− 4.62 (m, 1H), 4.56 (d, J = 10.2 Hz, 1H), 4.03−3.92 (m, 2H), 3.80−3.66 (m, 1H), 3.64−3.52 (m, 1H), 3.41 (s, 3H), 2.22−2.05 (m, J = 13.2, 7.0 Hz, 1H), 1.95−1.74 (m, 1H), 1.74−1.57 (m, 1H), 1.53−1.31 (m, 6H), 1.29−1.04 (m, 6H), 0.77 (s, 8H), 0.51 (s, 9H). LCMS (method D): tR = 2.93 min. LCMS (ESI) m/z calcd for C43H55N5O9S: 817.37. Found: 818.30 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclohexylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (9). lH NMR (400 MHz, CD3OD) δ 7.99− 8.07 (m. 3H), 7.47−7.55 (ml 3H), 7.37 (d, J = 2.2 Hz), 7.18 (s, 1H), 7.05 (dd, J = 9, 2 Hz, 1H), 5.69−5.84 (m, 1H), 5.51 (m, H), 5.21−5.28 (m, 1H), 5.05−5.09 (m, 1H), 4.47−4.56 (m, 2H), 4.24−4.28 (m, 1H), 4.04−4.13 (m, 1H), 3.94 (s, 3H), 3.41 (m, 1H), 2.63−2.69 (m, 1H), 2.30−2.49 (m, 1H), 2.04−2.23 (m, 3H), 1.73−1.92 (m, 3H), 1.59−1.73 (m, 1H), 1.29 (s, 9H), 1.14−1.55 (m, 6H), 1.03 (s, 9H). LCMS (method D): tR = 3.01 min. LCMS (ESI) m/z calcd for C44H57N5O9S: 831.39. Found: 832.30 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-4-((7-Methoxy-2-phenylquinolin-4yl)oxy)-2-(((1R,2S)-1-((phenylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (10). lH NMR (500 MHz, CD3OD) δ 8.10 (d, J = 9 Hz, 1H), 8.05, 8.04 (2s, 2H), 7.96−7.97 (m, 2H), 7.61− 7.65 (m, 1H), 7.49−7.58 (m, 5H), 7.40 (d, J = 2 Hz, 1H), 7.27 (s, 1H), 7.10 (dd, J = 9, 2 Hz, 1H), 5.57 (m, 1H), 5.37−5.46 (m, 1H), 5.15 (d, J = 17 Hz, 1H), 4.91 (d, J = 10 Hz, 1H), 4.52−4.57 (m, 2H), 4.25−4.29

(m, 1H), 4.11 (dd, J = 12, 2.6 Hz, 1H), 3.95 (s, 3H), 2.64−2.72 (m, 1H), 2.30−2.39 (m, 1H), 2.11−2.16 (m, 1H), 1.71 (dd, J = 8, 5.5 Hz, 1H), 1.31−1.34 (m, 1H), 1.28 (s, 9H), 1.06 (s, 9H). LCMS (method D): tR = 2.881 min. LCMS (ESI) m/z calcd for C44H51N5O9S: 825.34. Found: 826.30 (M + H)+. tert-Butyl (1-((2S,4R)-2-((1-((Cyclopropylsulfonyl)carbamoyl)cyclopropyl)carbamoyl)-4-((7-methoxy-2-phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (11). lH NMR (300 MHz, CD3OD) δ 8.07−8.03 (m, 3H), 7.47−7.57 (m, 3H), 7.38 (d, J = 2.2 Hz, 1H), 7.38−7.36 (m, 1H), 7.24 (s, 1H), 6.67 (d, J = 9.5 Hz, NH), 5.56 (s, 1H), 4.53−4.48 (m, 1H), 4.26 (d, J = 9.2 Hz, 1H), 4.07−4.13 (m, 1H), 3.94 (s, 3H), 3.01−2.92 (m, 1H), 2.65 (dd, J = 13.7, 6.8 Hz, 1H), 2.37−2.27 (m, 1H), 1.65−1.52 (m, 11H), 1.44−1.52 (m, 1H), 1.26 (s, 9H), 1.03 (s, 9H). LCMS (method D): tR = 2.71 min. LCMS (ESI) m/z calcd for C39H49N5O9S: 763.33. Found: 764.25 (M + H)+. tert-Butyl (2-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-2-oxoethyl)carbamate (12). 1H NMR (400 MHz, CD3OD) δ 7.99−8.12 (m, 3H), 7.59−7.46 (m, 3H), 7.40 (d, J = 2.52 Hz, 1H), 7.28 (s, 1H), 7.15 (dd, J = 2.52, 9.06 Hz, 1H), 5.80−5.60 (m, 2H), 5.30 (dd, J = 1.51, 17.12 Hz, 1H), 5.15−5.06 (m, 1H), 4.55 (dd, J = 7.43, 9.44 Hz, 1H), 4.15−3.98 (m, 3H), 3.95 (s, 3H), 3.89−3.80 (m, 1H), 3.00−2.89 (m, 1H), 2.65 (dd, J = 7.18, 13.98 Hz, 1H), 2.40 (ddd, J = 4.15, 9.82, 13.98 Hz, 1H), 2.24 (q, J = 8.81 Hz, 1H), 1.87 (dd, J = 5.29, 8.06 Hz, 1H), 1.50 (s, 1H), 1.44−0.98 (m, 13H). LCMS (method B): tR = 2.03 min. LCMS (ESI) m/z calcd for C37H43N5O9S: 733.28. Found: 734.28 (M + H)+. (2S,4R)-1-(2-Amino-3,3-dimethylbutanoyl)-N-((1R,2R)-1((cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)-4-((7methoxy-2-phenylquinolin-4-yl)oxy)pyrrolidine-2-carboxamide (13). lH NMR (500 MHz, CD3OD) δ 8.48 (m, 1H), 8.15 (m, 2H), 7.74 (m, 3H), 7.63 (s, 2H), 7.47 (m, 1H), 5.90 (m, 1H), 5.76−5.65 (m, 1H), 5.32 (d, J = 17 Hz, 1H), 5.13 (d, J =10 Hz, 1H), 4.76 (m, 1H), 4.56 (m, 1H), 4.19 (m, 2H), 4.06 (s, 3H), 2.95 (s, 1H), 2.84 (m, 1H), 2.43 (m, 1H), 2.33 (m, 1H), 1.89 (m, 1H), 1.42 (m, 1H), 1.17 (s, 9H), 1.04−1.33 (m, 4H). LCMS (method D): tR = 2.24 min. LCMS (ESI) m/ z calcd for C36H43N5O7S: 689.29. Found: 690.25 (M + H)+. Methyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxy-2phenylquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (14). 1H NMR (400 MHz, CD3OD) δ 8.36 (d, J = 9.5 Hz, 1H), 8.09 (dd, J = 6.9, 1.7 Hz, 1H), 7.79−7.72 (m, 3H), 7.65 (s, 1H), 7.54 (d, J = 2.1 Hz, 1H), 7.43 (dd, J = 9.3, 2.3 Hz, 1H), 5.86 (br s, 1H), 5.77−5.70 (m, 1H), 5.30 (dd, J = 17.1, 1.2 Hz, 1H), 5.13 (dd, J = 10.4, 1.5 Hz, 1H), 4.64−4.58 (m, 2H), 4.23 (s, 1H), 4.14 (dd, J = 12.2, 3.1 Hz, 1H), 4.06 (s, 3H), 3.38 (s, 3H), 2.97−2.92 (m, 1H), 2.77 (dd, J = 13.9, 6.9 Hz, 1H), 2.46−2.40 (m, 1H), 2.24 (q, J = 8.7 Hz, 1H), 1.90 (q, J = 5.7 Hz, 1H), 1.44 (q, J = 5.2 Hz, 1H), 1.22−1.26 (m, 2H), 1.11−1.07 (m, 2H), 1.05 (s, 9H). LCMS (method E): tR = 1.47 min. LCMS (ESI) m/z calcd for C38H45N5O9S: 747.29. Found: 748.45 (M + H)+. tert-Butyl ((2S)-1-((2S,4R)-2-((1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxyquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (15). 1H NMR (300 MHz, CD3OD) δ 8.62 (d, J = 5.1 Hz, 1H), 8.18−8.07 (m, 1H), 7.28 (s, 1H), 7.11 (d, J = 9.1 Hz, 1H), 6.99− 6.88 (m, 1H), 6.08−5.75 (m, 1H), 5.48−5.39 (m, 1H), 5.30−5.15 (m, J = 17.2 Hz, 1H), 5.08−4.92 (m, 1H), 4.68−4.40 (m, 2H), 4.28−4.21 (m, 1H), 4.18−4.06 (m, 1H), 3.97−3.89 (m, 3H), 2.84−2.64 (m, 2H), 2.60−2.44 (m, 1H), 2.20−2.05 (m, 1H), 1.95−1.74 (m, 1H), 1.33−1.21 (m, 9H), 1.06 (s, 9H), 1.51−0.82 (m, 5H). LCMS (method D): tR = 2.66 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 714.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-(pyridin-4yloxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (16). 1H NMR (400 MHz, CD3OD) δ 8.35 (d, J = 5.54 Hz, 2H), 6.99 (d, J = 5.79 Hz, 2H), 5.75 (ddd, J = 9.06, 10.26, 17.19 Hz, 1H), 5.33−5.24 (m, 2H), 5.10 (dd, J = 1.64, 10.20 Hz, 1H), 4.39 (dd, J = 6.92, 10.45 Hz, 1H), 4.28−4.17 (m, 2H), 4.06−3.98 (m, 1H), 2.92 (tt, J = 4.78, 8.06 Hz, 1H), 2.46 (dd, J = 7.18, 13.47 Hz, 1H), 2.29−2.16 (m, 2H), 1.85 (dd, J = 5.41, 8.18 Hz, 1H), 1.46−1.38 (m, 1H), 1.36−1.17 1722



Article

(m, 11H), 1.09−0.94 (m, 11H). LCMS (method B): tR = 1.96 min. LCMS (ESI) m/z calcd for C30H43N5O8S: 633.28. Found: 634.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((2-phenylpyridin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (17). 1H NMR (400 MHz, CD3OD) δ 8.43 (d, J = 5.8 Hz, 1H), 7.87 (dd, J = 8.1, 1.5 Hz, 2H), 7.55−7.28 (m, 4H), 6.97 (dd, J = 5.8, 2.0 Hz, 1H), 6.68 (d, J = 9.6 Hz, 1H), 5.75 (ddd, J = 17.2, 10.3, 8.9 Hz, 1H), 5.36 (br s, 1H), 5.29 (dd, J = 17.1, 1.3 Hz, 1H), 5.14−5.08 (m, 1H), 4.43 (dd, J = 10.4, 6.9 Hz, 1H), 4.33−4.18 (m, 2H), 4.09−3.99 (m, 1H), 2.93 (tt, J = 8.1, 4.8 Hz, 1H), 2.50 (dd, J = 13.5, 6.7 Hz, 1H), 2.31−2.15 (m, 2H), 1.86 (dd, J = 8.2, 5.4 Hz, 1H), 1.48−1.37 (m, 1H), 1.32−0.96 (m, 22H). LCMS (method B): tR = 2.14 min. LCMS (ESI) m/z calcd for C36H47N5O8S: 709.31. Found: 710.31 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-(quinolin-2yloxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (18). 1H NMR (CD3OD) δ 9.18 (d, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.82−7.78 (m, 2H), 7.64 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 6.92−6.90 (m, 1H), 5.92 (b, 1H), 5.78−5.73 (m, 1H), 5.32 (d, J = 17.0 Hz, 1H), 5.13 (d, J = 10 Hz, 1H), 4.51−4.47 (m, 1H), 4.15− 4.09 (m, 1H), 4.14−4.10 (m, 1H), 2.98−2.89 (m, 1H), 2.57−2.52 (m, 1H), 2.30−2.23 (m, 2H), 1.91−1.87 (m, 1H), 1.47−1.44 (m, 1H), 1.26−1.17 (m, 10H), 1.08−1.01 (m, 12H). LCMS (method A): tR = 1.75 min. LCMS (ESI) m/z calcd for C34H45N5O8S: 683.30. Found: 684.07 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-(isoquinolin-1yloxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (19). 1H NMR (CD3OD) δ 9.18 (b, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H), 7.32 (d, J = 6 Hz, 1H), 5.88 (b, 1H), 5.82−5.70 (m, 1H), 5.27 (d, J = 16.8 Hz, 1H), 5.12 (d, J = 10.2 Hz, 1H), 4.57−4.45 (m, 2H), 4.26 (b, 1H), 4.11−4.06 (m, 1H), 2.99−2.92 (m, 1H), 2.67−2.60 (m, 1H), 2.34−2.22 (m, 2H), 1.90−1.86 (m, 1H), 1.46−1.42 (m, 1H), 1.27−1.15 (m, 10H), 1.09−1.00 (m, 12H). LCMS (method A): tR = 1.80 min. LCMS (ESI) m/z calcd for C34H45N5O8S: 683.30. Found: 684.07 (M + H)+. Compound 19a. 1H NMR (400 MHz, methanol-d4) δ 8.22 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 5.8 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.76−7.68 (m, 1H), 7.58−7.51 (m, 1H), 7.34 (d, J = 5.8 Hz, 1H), 6.71−6.57 (m, 1H), 5.90 (br s, 1H), 5.78 (dt, J = 17.3, 8.7 Hz, 1H), 5.39−5.27 (m, 1H), 5.18−5.10 (m, 1H), 4.65−4.45 (m, 2H), 4.27 (t, J = 9.2 Hz, 1H), 4.10 (d, J = 11.3 Hz, 1H), 2.97 (dq, J = 12.4, 4.0 Hz, 1H), 2.73−2.60 (m, 1H), 2.39−2.23 (m, 2H), 1.93−1.78 (m, 1H), 1.49−1.39 (m, 1H), 1.34−0.98 (m, 21H). LCMS (method H): tR = 3.21 min. LCMS (ESI) m/z calcd for C34H45N5O8S: 683.30. Found: 684.5 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((3-phenylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (20). 1H NMR (CD3OD) δ 8.20−8.16 (m, 3H), 7.89−7.85 (m, 2H), 7.70 (t, J = 7.5 Hz, 1H), 7.49−7.46 (m, 3H), 7.40−7.37 (m, 1H), 6.10 (b, 1H), 5.79−5.72 (m, 1H), 5.29 (d, J = 18.0 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.58−4.55 (m, 1H), 4.48−4.46 (m, 1H), 4.30−4.18 (m, 2H), 2.97−2.93 (m, 1H), 2.75−2.70 (m, 1H), 2.41−2.36 (m, 1H), 2.26−2.21 (m, 1H), 1.90−1.87 (m, 1H), 1.46−1.43 (m, 1H), 1.30−1.26 (m, 10H), 1.09−0.92 (m, 12H). LCMS (method F): tR = 2.08 min. LCMS (ESI) m/z calcd for C40H49N5O8S: 759.33. Found: 760.11 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-4-((4-Chloroisoquinolin-1-yl)oxy)-2(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (21). 1H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 8.31 Hz, 1H), 8.11 (d, J = 8.56 Hz, 1H), 8.06 (s, 1H), 7.86 (t, J = 7.70 Hz, 1H), 7.63 (t, J = 7.58 Hz, 1H), 6.60 (d, J = 8.80 Hz, 1H), 5.85 (s, 1H), 5.81−5.70 (m, 1H), 5.29 (d, J = 17.12 Hz, 1H), 5.12 (d, J = 10.76 Hz, 1H), 4.59−4.46 (m, 2H), 4.22 (d, J = 9.29 Hz, 1H), 4.06 (dd, J = 11.49, 2.45 Hz, 1H), 2.98−2.89 (m, 1H), 2.63 (dd, J = 13.82, 6.97 Hz, 1H), 2.36−2.18 (m, 2H), 1.88 (dd, J = 7.83, 5.62 Hz, 1H), 1.44 (dd, J = 9.41, 5.26 Hz, 1H), 1.28−1.23 (m, 1H), 1.20 (s, 9H), 1.11−1.05

(m, 3H), 1.03 (s, 9H). LCMS (method C): tR = 2.49 min. LCMS (ESI) m/z calcd for C34H44ClN5O8S: 717.26. Found: 718.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((4-methylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (22). 1H NMR (400 MHz, CD3OD) δ 8.22 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.80 (s, 1H), 7.76 (t, J = 8.1 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 6.63 (d, J = 8.6 Hz, 1H), 5.77 (m, 2H), 5.29 (d, J = 17.1 Hz, 1H), 5.12 (d, J = 10.0 Hz, 1H), 4.53 (dd, J = 10.3, 6.6 Hz, 1H), 4.45 (d, J = 11.3 Hz, 1H), 4.25 (m, 1H), 4.06 (dd, J = 12.0, 3.4 Hz, 1H), 2.94 (m, 1H), 2.62 (dd, J = 13.7, 7.1 Hz, 1H), 2.49 (s, 3H), 2.26 (m, 2H), 1.88 (dd, J = 8.1, 5.4 Hz, 1H), 1.44 (dd, J = 9.5, 5.1 Hz, 1H), 1.26 (m, 10H),1.04 (m, 12H). LCMS (method A): tR = 1.82 min. LCMS (ESI) m/z calcd for C35H47N5O8S: 697.31. Found: 720.37 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((4-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (23). 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.87 (br s, 1H), 8.17 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.69−7.55 (m, 2H), 6.62 (d, J = 8.3 Hz, 1H), 5.73 (br s, 1H), 5.70−5.55 (m, 1H), 5.24 (d, J = 16.8 Hz, 1H), 5.16−5.05 (m, 1H), 4.49−4.31 (m, 2H), 4.08 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.97−3.82 (m, 1H), 2.99−2.87 (m, 1H), 2.48−2.70 (m, 1H), 2.24−2.10 (m, 2H), 1.72 (dd, J = 7.9, 5.4 Hz, 1H), 1.43−1.32 (m, 1H), 1.29−0.93 (m, 22H). LCMS (method H): tR = 3.22 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 736.29 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((5-morpholinoisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (24). 1H NMR (500 MHz, DMSO-d6) δ 10.41 (br s, 1H), 8.87 (br s, 1H), 8.02 (d, J = 6.1 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 5.8 Hz, 1H), 7.50−7.43 (m, 1H), 7.36 (d, J = 7.3 Hz, 1H), 6.61 (d, J = 7.9 Hz, 1H), 5.78 (br s, 1H), 5.67−5.56 (m, 1H), 5.23 (d, J = 17.2 Hz, 1H), 5.10 (d, J = 11.6 Hz, 1H), 4.48−4.35 (m, 2H), 4.07 (d, J = 8.4 Hz, 1H), 3.95 (d, J = 8.9 Hz, 1H), 3.86 (t, J = 4.4 Hz, 4H), 3.06−2.90 (m, 6H), 2.24−2.11 (m, 2H), 1.71 (dd, J = 7.9, 5.3 Hz, 1H), 1.43−0.92 (m, 23H). LCMS (method D): tR = 3.25 min. LCMS (ESI) m/z calcd for C38H52N6O9S: 768.35. Found: 769.52 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-4-((5-Chloroisoquinolin-1-yl)oxy)-2(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)scarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (25). 1H NMR (400 MHz, CD3OD) δ 8.13 (d, J = 8.3 Hz, 1H), 8.05 (d, J = 6.1 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 6.1 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 6.52 (d, J = 9.3 Hz, 1H), 5.82 (s, 1H), 5.76−5.64 (m, 1H), 5.24 (d, J = 16.9 Hz, 1H), 5.06 (d, J = 10.3 Hz, 1H), 4.52−4.41 (m, 2H), 4.16 (d, J = 9.3 Hz, 1H), 4.01 (dd, J = 11.9, 2.8 Hz, 1H), 2.92−2.84 (m, 1H), 2.58 (dd, J = 13.8, 7.0 Hz, 1H), 2.30−2.12 (m, 2H), 1.82 (dd, J = 7.9, 5.5 Hz, 1H), 1.38 (dd, J = 9.4, 5.3 Hz, 1H), 1.24−1.16 (m, 1H), 1.15 (s, 9H), 1.05−0.99 (m, 3H), 0.97 (s, 9H). LCMS (method C): tR = 2.46 min. LCMS (ESI) m/z calcd for C34H44ClN5O8S: 717.26. Found: 718.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((5-methylsisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (26). 1H NMR (400 MHz, CD3OD) δ 8.06 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 6.1 Hz, 1H), 7.53 (d, J = 7.1 Hz, 1H), 7.44 (d, J = 5.9 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 6.62 (d, J = 9.3 Hz, 1H), 5.86 (s, 1H), 5.81−5.70 (m, 1H), 5.29 (d, J = 16.9 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.53 (dd, J = 10.3, 7.1 Hz, 1H), 4.46 (d, J = 11.5 Hz, 1H), 4.25 (d, J = 9.5 Hz, 1H), 4.07 (dd, J = 11.9, 3.3 Hz, 1H), 2.98−2.90 (m, 1H), 2.66−2.58 (m, 4H), 2.34−2.18 (m, 2H), 1.87 (dd, J = 8.1, 5.4 Hz, 1H), 1.47−1.40 (m, 1H), 1.30−1.17 (m, 1H), 1.23 (s, 9H), 1.10−0.96 (m, 3H), 1.03 (s, 9H). LCMS (method C): tR = 2.41 min. LCMS (ESI) m/z calcd for C35H47N5O8S: 697.31. Found: 698.30 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((5-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (27). 1H NMR (400 MHz, CD3OD) δ 7.89 (d, J = 5.9 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 5.9 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 9.5 Hz, 1H), 5.79 (s, 1H), 5.81−5.70 (m, 1H), 5.23 (d, J = 16.9 Hz, 1H), 5.06 (d, J = 10.0 Hz, 1H), 4.47 (dd, J = 9.7, 7.0 Hz, 1H), 4.39 (d, J = 12.2 Hz, 1H), 4.20 (d, 1723



Article

1H), 5.10 (d, J = 11.5 Hz, 1H), 4.59−4.47 (m, 1H), 4.43 (d, J = 11.7 Hz, 1H), 4.23 (s, 1H), 4.05 (dd, J = 12.0, 3.4 Hz, 1H), 3.08−2.84 (m, 1H), 2.61 (dd, J = 13.9, 6.6 Hz, 1H), 2.49 (s, 3H), 2.35−2.11 (m, 2H), 1.86 (dd, J = 8.1, 5.6 Hz, 1H), 1.52−1.33 (m, 1H), 1.29−1.17 (m, 9H), 1.14− 1.03 (m, 2H), 1.03−0.92 (m, 11H). LCMS (method A): tR = 2.68 min. LCMS (ESI) m/z calcd for C35H47N5O8S: 697.31. Found: 720.37 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-4-((6-(tert-Butyl)isoquinolin-1-yl)oxy)-2-(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (34). 1H NMR (400 MHz, CD3OD) δ 8.28−8.07 (m, 1H), 8.04−7.87 (m, 1H), 7.78 (s, 1H), 7.72−7.60 (m, 1H), 7.38−7.22 (m, 1H), 6.00−5.86 (m, 1H), 5.86−5.71 (m, 1H), 5.33 (d, J = 17.3 Hz, 1H), 5.15 (d, J = 11.0 Hz, 1H), 4.64 (s, 1H), 4.56 (d, J = 10.0 Hz, 1H), 4.48 (d, J = 11.3 Hz, 1H), 4.28 (d, J = 9.3 Hz, 1H), 4.17− 3.98 (m, 1H), 3.06−2.90 (m, 1H), 2.72−2.59 (m, 1H), 2.47−2.16 (m, 2H), 1.91 (dd, J = 8.3, 5.5 Hz, 1H), 1.54−1.38 (m, 9H), 1.35−1.18 (m, 11H), 1.12−0.96 (m, 11H). LCMS (method A): tR = 2.35 min. LCMS (ESI) m/z calcd for C38H53N5O8S: 739.36. Found: 740.35 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-4-((6-Cyanoisoquinolin-1-yl)oxy)-2(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (36). 1H NMR (500 MHz, CD3OD) δ 8.42−8.24 (m, 2H), 8.14 (d, J = 5.8 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 5.5 Hz, 1H), 5.91 (br s, 1H), 5.82−5.65 (m, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.13 (d, J = 10.4 Hz, 1H), 4.64−4.51 (m, 1H), 4.47 (br s, 1H), 4.29−4.00 (m, 2H), 3.04−2.83 (m, 1H), 2.64 (dd, J = 13.7, 6.7 Hz, 1H), 2.42−2.15 (m, 2H), 1.89 (dd, J = 8.2, 5.5 Hz, 1H), 1.45 (s, 2H), 1.37−1.13 (m, 10H), 0.90 (d, J = 3.7 Hz, 10H). LCMS (ESI) m/z calcd for C35H44N6O8S: 708.29. Found: 709.40 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-hydroxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (37). 1H NMR (400 MHz, CD3OD) δ 8.03 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 6.0 Hz, 1H), 7.09 (d, J = 6.0 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.99 (s, 1H), 5.79−5.70 (m, 2H), 5.29 (d, J = 17.4 Hz, 1H), 5.10 (d, J = 11.2 Hz, 1H), 4.52−4.36 (m, 2H), 4.24 (s, 1H), 4.08− 4.02 (m, 1H), 2.92−2.88 (m, 1H), 2.62−2.58 (m, 1H), 2.26−2.18 (m, 2H), 1.94−1.84 (m, 1H), 1.48−1.38 (m, 1H), 1.32−0.99 (m, 23H). LCMS (method A): tR = 1.52 min. LCMS (ESI) m/z calcd for C34H45N5O9S: 699.29. Found: 700.18 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-fluoroisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (38). 1H NMR (400 MHz, CD3OD) δ 8.24 (dd, J = 9.0, 5.9 Hz, 1H), 7.96 (d, J = 6.1 Hz, 1H), 7.88 (s, 1H), 7.47 (dd, J = 9.7, 2.1 Hz, 1H), 7.32−7.23 (m, 2H), 6.59 (d, J = 9.0 Hz, 1H), 5.85 (br s, 1H), 5.74 (dt, J = 17.1, 9.6 Hz, 1H), 5.28 (d, J = 16.9 Hz, 1H), 5.10 (dd, J = 10.4, 1.3 Hz, 1H), 4.52 (dd, J = 10.1, 7.2 Hz, 1H), 4.44 (d, J = 11.7 Hz, 1H), 4.22 (d, J = 9.5 Hz, 1H), 4.05 (dd, J = 11.9, 3.3 Hz, 1H), 2.97−2.89 (m, 1H), 2.60 (dd, J = 13.8, 7.0 Hz, 1H), 2.32−2.17 (m, 2H), 1.86 (dd, J = 8.2, 5.5 Hz, 1H), 1.42 (dd, J = 9.3, 5.4 Hz, 1H), 1.22 (s, 8H), 1.08− 1.03 (m, 2H), 1.01 (s, 9H). LCMS (method G): tR = 3.81 min. LCMS (ESI) m/z calcd for C34H44FN5O8S: 701.29. Found: 702.28 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-ethoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (39). 1H NMR (500 MHz, CD3OD) δ 8.08 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 5.8 Hz, 1H), 7.22 (d, J = 5.8 Hz, 1H), 7.15 (s, 1H), 7.10−7.07 (m, 1H), 5.83 (s, 1H), 5.78−5.72 (m, 1H), 5.12 (d, J = 17.4 Hz, 1H), 4.54−4.49 (m, 1H), 4.42 (d, J = 11.3 Hz, 1H), 4.17 (q, J = 7.0 Hz, 2H), 4.09−4.03 (m, 1H), 2.97−2.91 (m, 1H), 2.63−2.57 (m, 1H), 2.32−2.19 (m, 2H), 1.90−1.85 (m, 1H), 1.46−1.42 (m, 1H), 1.31−1.24 (m, 10H), 1.09−0.98 (m, 15H). LCMS (ESI) m/z calcd for C36H49N5O9S: 727.33. Found: 728.38 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (40). 1H NMR (400 MHz, CD3OD)) δ 7.93 (s, 1H), 7.89 (d, J = 5.9 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 5.9 Hz, 1H), 5.87 (s, 1H), 5.82−5.70 (m, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.12 (dd, J = 10.3, 1.5 Hz, 1H), 4.57 (dd, J = 10.0,

J = 9.1 Hz, 1H), 4.01 (dd, J = 11.9, 3.3 Hz, 1H), 3.93 (s, 3H), 2.98−2.90 (m, 1H), 2.56 (dd, J = 13.6, 6.7 Hz, 1H), 2.34−2.18 (m, 2H), 1.82 (dd, J = 8.1, 5.6 Hz, 1H), 1.47−1.40 (m, 1H), 1.30−1.19 (m, 1H), 1.25 (s, 9H), 1.10−0.99 (m, 3H), 1.03 (s, 9H). LCMS (method C): tR = 2.37 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 714.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((5-fluoroisoquinolin-1-yl)oxy)spyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (28). 1H NMR (400 MHz, CD3OD) δ 8.06 (d, J = 6.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.47 (m, 3H), 6.59 (d, J = 9.1 Hz, 1H), 5.89 (s, 1H), 5.75 (m, 1H), 5.29 (d, J = 17.4 Hz, 1H), 5.12 (dd, J = 10.3, 1.5 Hz, 1H), 4.52 (m, 2H), 4.23 (d, J = 9.3 Hz, 1H), 4.07 (dd, J = 11.9, 3.1 Hz, 1H), 2.94 (m, 1H), 2.63 (dd, J = 13.8, 7.0 Hz, 1H), 2.28 (m, 2H), 1.88 (dd, J = 8.1, 5.4 Hz, 1H), 1.44 (dd, J = 9.5, 5.4 Hz, 1H), 1.25 (m, 10H), 1.05 (m, 12H). LCMS (method D): tR = 3.28 min. LCMS (ESI) m/z calcd for C34H44FN5O8S: 701.29. Found: 724.02 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-morpholinoisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (29). 1H NMR (400 MHz, CD3OD) δ 8.06 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 6.0 Hz, 1H), 7.40−7.03 (m, 3H), 6.63 (d, J = 9.3 Hz, 1H), 5.95−5.70 (m, 3H), 5.33 (d, J = 17.0 Hz, 2H), 5.15 (d, J = 10.7 Hz, 1H), 4.68−4.50 (m, 3H), 4.43 (d, J = 11.7 Hz, 1H), 4.29 (d, J = 9.1 Hz, 1H), 4.18−4.01 (m, 1H), 3.97 (s, 3H), 3.05−2.90 (m, 1H), 2.61−2.55 (m, 1H), 2.45−2.22 (m, 2H), 1.91−1.87 (m, 1H), 1.58−1.42 (m, 1H), 1.39 (s, 9H), 1.15−0.94 (m, 13H). LCMS (method A): tR = 1.70 min. LCMS (ESI) m/z calcd for C38H52N6O9S: 768.35. Found: 769.14 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-4-((6-(1H-Pyrazol-1-yl)isoquinolin-1yl)oxy)-2-(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (30). 1H NMR (400 MHz, CD3OD) δ 8.42 (d, J = 4.5 Hz, 1H), 8.31 (d, J = 15 Hz, 1H), 8.15 (s, 1H), 8.01 (d, J = 10 Hz, 1H), 7.80 (d, J = 2.5 Hz, 1H), 7.40 (d, J = 10 Hz, 1H), 6.61−6.59 (m, 1H), 5.89 (b, 1H), 5.83−5.70 (m, 1H), 5.29 (d, J = 18 Hz, 1H), 5.13 (d, J = 10.5 Hz, 1H), 4.60−4.46 (m, 2H), 4.24 (b, 1H), 4.11−4.07 (m, 1H), 2.98−2.92 (m, 1H), 2.68−2.61 (m, 1H), 2.29−2.22 (m, 2H), 1.91−1.87 (m, 1H), 1.47−1.43 (m, 1H), 1.27−1.23 (m, 10H), 1.10− 1.04 (m, 12H). LCMS (method F): tR = 1.77 min. LCMS (ESI) m/z calcd for C37H47N7O8S: 749.32. Found: 750.06 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-(dimethylcarbamoyl)isoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl1-oxobutan-2-yl)carbamate (31). 1H NMR (400 MHz, CD3OD) δ 8.27 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 5.9 Hz, 1H), 7.86 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.39 (m, 1H), 5.90 (br s, 1H), 5.67−5.45 (m, 1H), 5.37−5.22 (m, 1H), 5.20−5.03 (m, 1H), 4.68−4.53 (m, 1H), 4.50−4.36 (m, 1H), 4.34−4.17 (m, 1H), 4.14−3.98 (m, 1H), 3.19−3.07 (m, 3H), 3.03−2.89 (m, 4H), 2.65−2.54 (m, 2H), 2.52−2.40 (m, 1H), 2.35−2.14 (m, 1H), 2.00−1.74 (m, 1H), 1.25−1.18 (m, 11H), 1.02 (m, 11H). LCMS (method C): tR = 2.45 min. LCMS (ESI) m/z calcd for C37H50N6O9S: 754.34. Found: 777.43 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-4-((6-Chloroisoquinolin-1-yl)oxy)-2(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1oxobutan-2-yl)carbamate (32). 1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 9.0 Hz, 1H), 8.01 (d, J = 6.0 Hz, 1H), 7.86 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 6.0 Hz, 1H), 6.58 (d, J = 10.0 Hz, 1H), 5.88 (b, 1H), 5.80−5.72 (m, 1H), 5.29 (d, J = 18.0 Hz, 1H), 5.10 (d, J = 10.5 Hz, 1H), 4.56−4.53 (m, 1H), 4.47−4.45 (m, 1H), 4.23−4.21 (m, 1H), 4.08−4.06 (m, 1H), 2.97−2.92 (m, 1H), 2.64−2.60 (m, 1H), 2.31−2.22 (m, 2H), 1.90−1.87 (m, 1H), 1.46−1.43 (m, 1H), 1.26−1.20 (m, 10H), 1.11−0.99 (m, 12H). LCMS (method A): tR = 1.94 min. LCMS (ESI) m/z calcd for C34H44ClN5O8S: 717.26. Found: 718.25 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (33). 1H NMR (400 MHz, CD3OD) δ 8.07 (d, J = 8.6 Hz, 1H), 7.90 (s, 1H), 7.58 (s, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 5.9 Hz, 1H), 5.83 (br s, 1H), 5.79−5.60 (m, 1H), 5.28 (d, J = 17.1 Hz, 1724



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7.1 Hz, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.23 (s, 1H), 4.09 (dd, J = 11.7, 3.2 Hz, 1H), 2.98−2.90 (m, 1H), 2.61 (dd, J = 13.8, 6.7 Hz, 1H), 2.50 (s, 3H), 2.36−2.18 (m, 2H), 1.88 (dd, J = 8.2, 5.5 Hz, 1H), 1.45 (dd, J = 9.4, 5.5 Hz, 1H), 1.29−1.21 (m, 1H), 1.18 (s, 9H), 1.10−1.05 (m, 2H), 1.05−0.99 (m, 1H), 1.02 (s, 9H). LCMS (method C): tR = 2.32 min. LCMS (ESI) m/z calcd for C35H45CN5O8S: 697.31. Found: 698.35 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((8-methylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2yl)carbamate (41). 1H NMR (500 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.94 (br s, 1H), 7.95 (d, J = 5.6 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.40−7.29 (m, 2H), 6.47 (d, J = 8.5 Hz, 1H), 5.89 (br s, 1H), 5.68−5.55 (m, 1H), 5.24 (d, J = 17.4 Hz, 1H), 5.11 (d, J = 10.7 Hz, 1H), 4.52−4.40 (m, 1H), 4.30 (d, J = 11.6 Hz, 1H), 4.06 (d, J = 8.7 Hz, 1H), 3.94 (d, J = 9.6 Hz, 1H), 2.93 (d, J = 4.7 Hz, 2H), 2.73 (s, 3H), 2.26−2.13 (m, 2H), 1.72 (dd, J = 7.7, 5.6 Hz, 1H), 1.41−1.32 (m, 2H), 1.28−0.91 (m, 21H). LCMS (method D): tR = 3.31 min. LCMS (ESI) m/z calcd for C35H47N5O8S: 697.31. Found: 720.45 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((4,6-dimethoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (42). 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.88 (br s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.61 (s, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 5.71 (br s, 1H), 5.69−5.55 (m, 1H), 5.25 (d, J = 17.1 Hz, 1H), 5.16−5.08 (m, 1H), 4.46−4.28 (m, 2H), 4.08 (d, J = 8.3 Hz, 1H), 4.01−3.86 (m, 7H), 2.99− 2.92 (m, 1H), 2.48−2.46 (m, 1H), 2.22−2.12 (m, 2H), 1.72 (dd, J = 7.9, 5.4 Hz, 1H), 1.41−1.34 (m, 1H), 1.31−0.92 (m, 22H); 1H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 9.05 Hz, 1H), 7.48 (s, 1H), 7.40 (d, J = 2.20 Hz, 1H), 7.12 (d, J = 9.05 Hz, 1H), 5.75 (m, 2H), 5.29 (d, J = 16.87 Hz, 1H), 5.12 (d, J = 10.52 Hz, 1H), 4.50 (m, 1H), 4.39 (d, J = 11.98 Hz, 1H), 4.24 (s, 1H), 4.04 (dd, J = 11.74, 2.93 Hz, 1H), 3.99 (s, 3H), 3.92 (s, 3H), 2.94 (m, 1H), 2.58 (dd, J = 13.57, 6.97 Hz, 1H), 2.24 (m, 2H), 1.87 (dd, J = 8.07, 5.62 Hz, 1H), 1.43 (m, 1H), 1.21 (m, 10H), 1.07 (m, 12H). LCMS (method H): tR = 3.22 min. LCMS (ESI) m/z calcd for C36H49N5O10S: 743.32. Found: 744.34 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxyisoquinolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (43). 1H NMR (400 MHz, CD3OD) δ 8.77 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.97 (s, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.30 (d, J = 9.3 Hz, 1H), 5.82−5.62 (m, 1H), 5.40 (br s, 1H), 5.27 (d, J = 17.4 Hz, 1H), 5.10 (d, J = 11.2 Hz, 1H), 4.55−4.38 (m, 2H), 3.93 (s, 3H), 2.98− 2.84 (m, 1H), 2.61 (dd, J = 13.6, 6.7 Hz, 1H), 2.34−2.13 (m, 2H), 1.85 (dd, J = 8.1, 5.6 Hz, 1H), 1.27 (s, 9H), 1.25−1.18 (m, 4H), 1.10−1.04 (m, 3H), 1.03−0.99 (m, 9H). LCMS (method C): tR = 1.42 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 714.20 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((7-methoxyquinazolin-4-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (44). 1H NMR (400 MHz, CD3OD) δ 8.71 (s, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.26−7.18 (m, 2H), 5.93 (br s, 1H), 5.84 (dt, J = 17.2, 9.7 Hz, 1H), 5.30−5.22 (m, 1H), 5.08 (dd, J = 10.4, 1.6 Hz, 1H), 4.66−4.53 (m, 2H), 4.21 (s, 1H), 4.16−4.08 (m, 1H), 3.98 (s, 3H), 2.95−2.85 (m, 1H), 2.75−2.66 (m, 1H), 2.45 (ddd, J = 14.1, 10.1, 4.4 Hz, 1H), 2.19−2.10 (m, 1H), 1.86 (dd, J = 7.8, 5.1 Hz, 1H), 1.47 (d, J = 5.4 Hz, 1H), 1.38 (dd, J = 9.4, 5.3 Hz, 1H), 1.24−1.12 (m, 9H), 1.09− 0.93 (m, 12H). LCMS (method D): tR = 3.03 min. LCMS (ESI) m/z calcd for C34H46N6O9S: 714.30. Found: 737.28 (M + Na)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methoxy-3phenylisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (45). 1H NMR (500 MHz, CD3OD) δ 8.23− 7.98 (m, 3H), 7.77 (br s, 1H), 7.56−7.33 (m, 3H), 7.26 (br s, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.05 (br s, 1H), 5.86−5.67 (m, 1H), 5.29 (d, J = 17.1 Hz, 1H), 5.12 (d, J = 10.1 Hz, 1H), 4.55 (d, J = 7.6 Hz, 1H), 4.42 (d, J = 10.7 Hz, 1H), 4.33−4.10 (m, 2H), 3.94 (s, 3H), 2.95 (dq, J = 8.4, 4.1 Hz, 1H), 2.68 (d, J = 6.4 Hz, 1H), 2.43−2.16 (m, 2H), 1.89 (d, J = 5.8 Hz, 1H), 1.45 (d, J = 4.9 Hz, 1H), 1.37−1.16 (m, 10H), 1.13−0.89

(m, 12H). LCMS (method F): tR = 2.18 min. LCMS (ESI) m/z calcd for C41H51N5O9S: 789.34. Found: 790.10 (M + H)+. tert-Butyl ((S)-1-((2S,4S)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (46). 1H NMR (400 MHz, CD3OD) δ 8.14 (d, J = 8.9 Hz, 1H), 7.88 (d, J = 6.1 Hz, 1H), 7.32−7.10 (m, 3H), 6.76 (d, J = 10.0 Hz, 1H), 6.58 (b, 1H), 5.76−5.69 (m, 1H), 5.28 (d, J = 8.0 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.51−4.49 (m, 1H), 4.24−4.21 (m, 1H), 4.06−4.02 (m, 1H), 3.92 (s, 3H), 2.97−2.91 (m, 1H), 2.61−2.55 (m, 1H), 2.30−2.20 (m, 2H), 1.88−1.85 (m, 1H), 1.45−1.40 (m, 1H), 1.27 (s, 9H), 1.25−1.23 (m, 1H), 1.08−1.00 (m, 12H). LCMS (method A): tR = 1.75 min. LCMS (ESI) m/z calcd for C35H47N5O9S: 713.31. Found: 714.11 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2R)-1-((Cyclopropylsulfonyl)carbamoyl)-2-ethylcyclopropyl)carbamoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan2-yl)carbamate (47). 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 6.0 Hz, 1H), 7.24 (d, J = 6.0 Hz, 1H), 7.18 (d, J = 1.5 Hz,1H), 7.10−7.08 (m, 1H), 6.59 (d, J = 10.0 Hz, 1H), 5.81 (b, 1H), 4.51−4.49 (m, 1H), 4.42−4.40 (m, 1H), 4.24−4.21 (m, 1H), 4.06−4.02 (m, 1H), 3.92 (s, 3H), 2.98−2.94 (m, 1H), 2.61−2.55 (m, 1H), 2.27−2.25 (m, 1H), 1.65−1.51 (m, 4H), 1.27 (s, 9H), 1.24−1.21 (m, 2H), 1.09−1.07 (m, 2H), 1.04 (s, 9H), 0.99−0.96 (m, 4H). LCMS (method A): tR = 1.70 min. LCMS (ESI) m/z calcd for C35H49N5O9S: 715.33. Found: 716.11 (M + H)+. (S)-Tetrahydrofuran-3-yl ((S)-1-((2S,4R)-2-(((1R,2S)-1((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (48). 1H NMR (CD3OD) δ 8.07 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.20 (s, 1H), 7.13 (d, J = 9.0 Hz, 1H), 7.06 (d, J = 10.0 Hz, 1H), 5.85 (b, 1H), 5.80−5.73 (m, 1H), 5.28 (d, J = 18.0 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.77−4.75 (m, 1H), 4.59−4.55 (m, 1H), 4.42−4.40 (m, 1H), 4.29−4.27 (m, 1H), 4.09−4.05 (m, 1H), 3.93 (s, 3H), 3.72−3.66 (m, 4H), 2.97−2.91 (m, 1H), 2.61−2.55 (m, 1H), 2.30−2.20 (m, 2H), 1.90−1.85 (m, 2H), 1.71−1.64 (m, 1H), 1.46−1.38 (m, 1H), 1.26−1.23 (m, 2H), 1.08−1.03 (m, 11H). LCMS (method A): tR = 1.52 min. LCMS (ESI) m/z calcd for C35H45N5O10S: 727.29. Found: 728.06 (M + H)+. tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((Cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2yl)carbamate (49). 1H NMR (CD3OD) δ 8.10 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.18 (s, 1H), 7.10 (d, J = 9.0 Hz, 1H), 5.86 (b, 1H), 5.81−5.74 (m, 1H), 5.32 (d, J = 18.1 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.55−4.47 (m, 1H), 4.06−4.03 (m, 2H), 3.92 (s, 3H), 2.97−2.94 (m, 1H), 2.61−2.57 (m, 1H), 2.35−2.22 (m, 2H), 1.89−1.88 (m, 1H), 1.43−1.40 (m, 1H), 1.25−1.21 (m, 10H), 1.09−1.07 (m, 3H), 0.98−0.94 (m, 6H). LCMS (method A): tR = 1.71 min. LCMS (ESI) m/z calcd for C34H45N5O9S: 699.29. Found: 700.07 (M + H)+. (1R,2S)-1-((2S,4R)-1-((S)-2-((tert-Butoxycarbonyl)amino)-3,3dimethylbutanoyl)-4-((6-methoxyisoquinolin-1-yl)oxy)pyrrolidine-2-carboxamido)-2-vinylcyclopropanecarboxylic Acid (50). 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 5.8 Hz, 1H), 7.12 (d, J = 5.5 Hz, 1H), 7.10−7.02 (m, 2H), 6.98 (s, 1H), 5.85−5.66 (m, 2H), 5.23 (d, J = 17.1 Hz, 1H), 5.08 (d, J = 10.4 Hz, 1H), 4.55 (t, J = 7.8 Hz, 1H), 4.40 (d, J = 11.3 Hz, 1H), 4.32 (d, J = 9.5 Hz, 1H), 4.01 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H), 2.61−2.48 (m, 2H), 2.11−2.03 (m, 1H), 1.93 (t, J = 6.4 Hz, 1H), 1.38−1.28 (m, 9H), 1.24− 1.19 (m, 1H), 1.00 (s, 9H). LCMS (method D): tR = 3.09 min. LCMS (ESI) m/z calcd for C32H42N4O8: 610.30. Found: 611.25 (M + H)+. Sample Analysis. Samples from in vitro and in vivo studies were analyzed by LC/MS/MS. Protein-containing samples (plasma, microsomal incubates) were treated with two volumes of ACN containing an internal standard. After the precipitated proteins were removed by centrifugation, the resulting supernatants were transferred to autosampler vials and 5 μL was injected onto an HPLC column for analysis. Tissue samples were weighed and homogenized in 2 volumes of a solution containing 20% Hanks buffered salt solution/80% ACN and 1725



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centrifuged. An aliquot of each tissue supernatant was removed and processed as described for plasma samples. The HPLC system consisted of two Shimadzu LC10AD pumps (Columbia, MD), a Shimadzu SILHTC autosampler, and a Hewlett-Packard series 1100 column compartment (Palo Alto, CA). The column was a YMC Pro C18 (2.0 mm × 50 mm, 3 μm particles), maintained at 60 °C. The mobile phase consisted of solvent A (10 mM ammonium formate and 0.1% formic acid in water) and solvent B (10 mM ammonium formate and 0.1% formic acid in methanol) at a flow rate of 0.3 mL/min. The initial mobile phase composition was 95% A/5% B which was changed to 15% A/85% B over 2 min and held at that composition for an additional 2 min. The mobile phase was then returned to initial conditions and the column reequilibrated. The total analysis time was 5 min. The HPLC was interfaced to a Micromass Quattro tandem mass spectrometer (Beverly, MA) equipped with an electrospray ionization source. Ions representing the (M + H)+ species for both BMS-605339 and the internal standard were selected in MS1 and collisionally dissociated with argon at a pressure of 2 × 10−3 Torr to form specific product ions which were subsequently monitored by MS2. The selected reaction monitoring transitions used were 714.7 → 439.1 for BMS605339 and 485.2 → 381.3 for the internal standard. The LLQ of BMS605339 was 5 nM in all samples, which measured concentrations of at least two-thirds of the quality control samples with 20% of nominal values. Microsomal Metabolic Stability. The metabolic stability of 35 was investigated in pooled liver microsomes from human, mouse, rat, dog, and monkey. The concentrations of CYP P450 protein in these preparations were 0.38, 0.52, 0.89, and 1.4 nmol/mg microsomal protein in human, rat, dog, and monkey, respectively. Incubation mixtures were prepared in triplicate for each species and consisted of BMS-605339 (3 μM) in potassium phosphate buffer (0.1 M, pH 7.4) at 37 °C, liver microsomes (protein concentration 0.9 mg/mL), magnesium chloride (0.033 mM), and a NADPH-regenerating system (NADPH, 0.43 mg/mL; glucose 6-phosphate, 0.52 mg/mL; glucose 6-phosphate dehydrogenase, 0.6 units/mL). The total concentration of ACN in the incubation mixtures was

Discovery and Early Clinical Evaluation of BMS ... - ACS Publications

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