Potent Nonimmunosuppressive Cyclophilin Inhibitors With Improved

Article pubs.acs.org/jmc

Potent Nonimmunosuppressive Cyclophilin Inhibitors With Improved Pharmaceutical Properties and Decreased Transporter Inhibition Jiping Fu, Meiliana Tjandra, Christopher Becker, Dallas Bednarczyk, Michael Capparelli, Robert Elling, Imad Hanna, Roger Fujimoto, Markus Furegati, Subramanian Karur, Theresa Kasprzyk, Mark Knapp, Kwan Leung, Xiaolin Li, Peichao Lu, Wosenu Mergo, Charlotte Miault, Simon Ng, David Parker, Yunshan Peng, Silvio Roggo, Alexey Rivkin, Robert L. Simmons, Michael Wang, Brigitte Wiedmann, Andrew H. Weiss, Linda Xiao, Lili Xie, Wenjian Xu, Aregahegn Yifru, Shengtian Yang, Bo Zhou, and Zachary K. Sweeney* Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, California 94608, United States S Supporting Information *

ABSTRACT: Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.

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INTRODUCTION Cyclosporin A (CsA) (1, Figure 1) is a cyclic peptide originally isolated from broths of the fungus Tolypocladium inflatum. Seven of the 11 amino acids in CsA are N-methylated, and the structure contains a unique (4R)-4-((E)-2-butenyl)-4,N-dimethylthreonine residue.1 CsA binds strongly to a subset of peptidyl-prolyl isomerase proteins commonly known as cyclophilins. These proteins are thought to ensure proper folding and stabilization of other proteins through their isomerase and chaperone functionalities.2 In vivo, CsA functions as a noncytotoxic immunosuppressant by reversibly inhibiting cytokine secretion from stimulated Tcells.3 This activity, which led to approval of CsA for prevention of organ rejection in transplant patients, is a result of inhibition of calcineurin phosphatase activity by the CsA/cyclophilin A complex.4 Extensive chemical modification of the cyclosporin scaffold led to the discovery of cyclosporin analogues that bind strongly to cyclophilins but are significantly less immunosuppressive than CsA.5−7 This class of cyclosporins is known collectively as “nonimmunosuppressive cyclophilin inhibitors” and is exemplified by NIM811 (2), alisporivir (3), and SCY635 (4) (Figure 1).8 Nonimmunosuppressive cyclophilin inhibitors have potential utility in the treatment of a variety of medical conditions. Compound 2, for example, has been reported to reduce fibrosis, © XXXX American Chemical Society

asthma, cardiac reperfusion injury, and traumatic brain injury in rodent disease models.9 In vitro studies have revealed that cyclophilin inhibitors may also have activity against infectious agents, including human immunodeficiency virus (HIV), influenza virus, and human papilloma virus.10 A number of nonimmunosuppressive cyclophilin inhibitors are also potent inhibitors of hepatitis C (HCV) replication in vitro. These compounds appear to prevent appropriate viral RNA production by interfering with the interaction of cyclophilin A with the viral NS5A protein.10 Clinical trials with alisporivir and 4 have demonstrated that administration of these compounds results in a significant decrease in viral RNA in the plasma of infected patients.11,12 Importantly, as hosttargeted agents, cyclophilin inhibitors maintain activity against all major HCV genotypic variants. These drugs also exhibit a much higher barrier to resistance development than most direct-acting antiviral agents and have the potential to serve as an important component of combination therapy for the treatment of HCV infected patients.13 Alisporivir administration is associated with an increase in circulating levels of conjugated and unconjugated bilirubin in a subset of patients.14 Bilirubin, which is produced in the body by Received: July 3, 2014

A

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Figure 1. Structures of cyclosporin A (1), NIM811 (2), alisporivir (3), and SCY-635 (4). The cyclosporins contain different groups at P3 and P4.

heme degradation, is normally transported from the plasma into the liver by OATP1B1 and OATP1B3. In the liver, bilirubin is glucuronidated by UGT1A1, and the glucuronide conjugate is actively transported into bile by MRP2.15,16 The hyperbilirubinemia observed in some patients taking alisporivir has been attributed to inhibition of OATP1B1, OATP1B3, and MRP2.8,11 OATP inhibition has also been shown to influence the pharmacokinetics of coadministered medications.17,18 Other cyclosporins are also known to be promiscuous inhibitors of transporter activity; CsA, for example, blocks OATP1B1 activity in vitro.19 Valspodar, a CsA derivative modified to decrease binding to both cyclophilin and calcineurin, was studied clinically as an inhibitor of MDR1 activity.20 Recent analyses have led to the conclusion that large, hydrophobic peptides often reduce OATP activity.18 Several dozen agents have been tested in the clinic against HCV infection. However, most of these drugs are highly susceptible to resistance development and are effective for the treatment of only a few HCV variants.8,21 We therefore sought to identify a next-generation cyclophilin inhibitor with excellent anti-HCV potency, decreased transporter inhibition, and pharmaceutical and pharmacokinetic properties consistent with possible coadministration of other drugs. Our medicinal chemistry strategy was defined by structure−activity relationships obtained by screening our library of cyclosporins and from published data (Figure 2). An extensive survey of cyclosporins containing P3 groups has revealed that small, nonbranched groups such as CH3 or CH2OH increase cyclophilin binding (see Figure 1 for numbering). Structural studies indicate that this substitution improves affinity by stabilizing the conformation of the bound cyclosporin.22,23 Interestingly, although groups introduced at this position are remote from the cyclosporin/cyclophilin binding interface, branching at the β-position of the P4 amino acid has also been demonstrated to increase binding to cyclophilins in vitro.24 To

Figure 2. Medicinal chemistry strategy to preserve cyclophilin inhibition while lessening transporter interactions and improving pharmaceutical properties.

maintain potency comparable to alisporivir, our efforts focused on the preparation of analogues with D-alanine at P3 and valine or leucine-type branched motifs at P4. At the outset of our program, there were few data that could be used to guide a rational approach to address the undesired transporter inhibition observed for alisporivir and cyclosporin A. Although we were interested in lessening both OATP1B1 and MRP2 inhibition, most published data focused on the interactions of MDR1 with cyclosporin analogues. These studies indicated that decreasing the lipophilicity of the P4 residue (i.e., by hydroxylation of the leucine side-chain) could significantly lessen MDR1 inhibition by cyclosporin A.25 Experiments with photolabeled cyclosporin analogues and MDR-overexpressing cells have suggested that cyclosporins interact with MDR1 primarily via the calcineurin binding domain (P4−P8).26 Furthermore, it has been reported that 4, which contains a hydrophilic γ-hydroxyleucine P4, is a less potent inhibitor of MDR1 and MRP2 than cyclosporin A.27,28 With the expectation that compounds containing a hydrophilic P4 group might similarly be less potent than alisporivir as an B

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Scheme 2. Preparation of Ether-Containing Dipeptide 12a

OATP1B1 inhibitor, we chose to focus on hydrophilic P4 modification in the synthesis of new cyclosporin analogues. In this article, we report the cyclophilin binding properties and HCV replicon inhibition potencies of a variety of novel nonimmunosuppressive cyclosporins. Several compounds were determined to be less potent transporter inhibitors than alisporivir and show comparable cyclophilin binding potency and cellular antiviral efficacy.

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CHEMISTRY Substituted dipeptides required for synthesis of our cyclophilin inhibitors were prepared according to methods outlined in Scheme 1. A stereoselective, proline-catalyzed aldol reaction Scheme 1. Selective Synthesis of Dipeptide Intermediate 8a a

Reagents and conditions. (a) bromoacetic acid, NaI, NaH, THF, 0 °C; (b) trimethylsilyldiazomethane, CH3OH, 0 °C; (c) NaBH4, CH3OH, 0 °C; (d) TBDPSCl, imidazole, DCM, rt; (e) HCl in dioxane, 0 °C; (f) (R)-(N-benzyloxycarbonyl-N-methylaminopropanoic acid, HATU, DIPEA, DCM, rt; (g) NaIO4, RuCl3, heptane, EtOAc, H2O, rt.

Scheme 3. Synthesis of Nitrile Dipeptide 16a

a Reagents and conditions. (a) 1-propanal, (2S,4R)-4-(tert-butyldiphenylsilyloxy)-pyrrolidine-2-carboxylic acid, acetonitrile, 4 °C; (b) NaBH4, MeOH, 0 °C; (c) TBDPSCl, imidazole, DMF, rt; (d) CH3I, NaH, THF, 0 °C; (e) TFA, DCM, 0 °C; (f) N-protected (R)-2(methylamino)propanoic acid, HATU, DIPEA, DCM, rt; (g) (i) NaIO4, RuCl3, acetonitrile, H2O, CCl4, rt, (ii) NaHCO3, H2O2, EtOAc, MeOH, water.

between imine 5 and propionaldehyde provided, after reduction and repeated crystallization, the isomerically pure protected amine 6.29 O-Silylation and N-methylation then gave intermediate 7. Finally, removal of the Boc group, coupling with Fmoc-protected D-alanine, and selective furan oxidative cleavage led to dipeptide 8. Dipeptides containing an ether linkage were constructed as shown in Scheme 2. Treatment of the primary alcohol 9 with bromoacetic acid and sodium hydride gave an α-oxo ester that could be reduced and protected to provide 11. Acid 12 was obtained by removal of the Boc group, followed by coupling and oxidation. The synthesis of nitrile-containing cyclosporin 25 (Table 1) utilized intermediate 13, which was obtained from 8 as shown in Scheme 3. Mesylation of the alcohol and displacement with sodium cyanide gave dipeptide 15, which could be converted to acid 16. Alkylation of the intermediate alcohol 6 with methyl iodide produced an ether intermediate used for the synthesis of cyclosporin 26. Aldehyde 17 was prepared in high yield by oxidation of 9 under Swern conditions. Wittig transformations provided key intermediates for the synthesis of the homologated derivative 34 (Scheme 4). For example, treatment of 17 with (methoxymethyl)triphenylphosphonium chloride and sodium

a

Reagents and conditions. (a) methanesulfonyl chloride, TEA, DCM, 0 °C; (b) NaCN, DMF, 70 °C; (c) NaIO4, RuCl3, acetonitrile, H2O, CCl4, rt.

hexamethyldisilazide produced the vinyl ether 18. Hydrolysis, reduction, and protection then yielded the silyl ether 19. This intermediate could be used to generate dipeptide 20 using the same sequence of transformations as those employed for the synthesis of 8. Other cyclosporin derivatives formed from these precursors are shown in Scheme 5. Briefly, coupling of nonapeptide 2130 with dipeptide 8 generated the intermediate 22. Sequential deprotection of the amine and ester functionalities, followed by macrocyclization under dilute conditions, gave macrocycle 23. Selective modification of protected hydroxyl groups on the macrocyclic template generated additional series of functionalized cyclosporin derivatives. For example, as illustrated in Scheme 6, desilylation of the TBDPS-protected precursor 23, followed successively by oxidation, reductive amination, and hydrolysis, afforded the amine-functionalized cyclosporin derivatives 27−33. Similar approaches yielded cyclosporins 34 and 35 from 20 and 12, respectively. C

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Scheme 4. Preparation of Amino Acid 20a

interact strongly and reversibly with these cyclophilins. Antiviral activity, assessed for subgenomic GT1a, 1b, 2a, 3a, and 4a replicons coexpressing a luciferase reporter gene or for the GT2a JFH1 virus, was comparable to compound 3. For clarity, only the Kd for cyclophilin A binding and the EC50 values for the GT1b replicon are provided in Tables 1−3. There was a good correlation between the cyclophilin binding Kd and EC50 in the replicon system. The inhibition of OATP1B1 was routinely tested in CHO cells overexpressing OATP1B1 using 8-fluorescein-cAMP as the substrate.31 Initial profiling of compounds 25 and 26 revealed that hydrophilic functionality could be appended to the P4 valine of the alisporivir scaffold while maintaining binding affinity and cellular potency (Table 1). Remarkably, 26 was reproducibly found to be more soluble than alisporivir in our standard kinetic solubility assay (kinetic solubility 4 = 0.066 g/L, kinetic solubility 3 < 0.005 g/L). Inhibitor 26 was also a less potent inhibitor of OATP1B1 transporter activity than alisporivir. However, this analogue still did not meet our target OATP1B1 inhibition criteria (>5-fold reduction in cellular OATP1B1 inhibition relative to alisporivir). More substantial reductions in OATP1B1 inhibition could be observed with amine-containing analogues (Table 2). In this series, the extent of OATP1B1 inhibition was broadly correlated with basicity and hydrophilicity of the R group. For example, morpholine containing compound 27 (OATP1B1 IC50 = 4.3 μM) was a less potent inhibitor than the less basic compound 29 or the more hydrophobic bicyclic morpholine analogue 30. Hydrophilic piperazine 33 was a less potent OATP1B1 inhibitor than most other analogues in this series. No significant differences in the inherent cyclophilin binding affinity or the EC50 in the replicon cellular assay was observed for the compounds in Table 2. These results suggests that the difference in OATP inhibition observed for the cyclosporins is not likely to be caused by reduced cellular permeability or distribution properties. Utilizing 27 as a lead compound, the length of the alkyl chain connecting the morpholine group to the macrocycle was varied (Table 3). Compounds 34 and 35 were active in the replicon assay and were less potent inhibitors of OATP1B1 inhibition than alisporivir. However, screening studies indicated that 34 was a potential time-dependent inhibitor of CYP3A4, and this compound was not investigated further. Analogue 35 had a very promising in vitro and in vivo profile (in vivo data not shown), and this compound was selected as a potential alternative clinical candidate to inhibitor 33. To further characterize the cyclophilin interactions, a cocrystal structure of 33 in complex with rat cyclophilin D was obtained (Figure 3). As expected, the conformation of the

a

Reagents and conditions. (a) (methoxymethyl)triphenylphosphonium chloride, NaHMDS, THF, 0 °C; (b) PPTS, acetone, rt; (c) NaBH4, CH3OH, 0 °C; (d) TBDPSCl, imidazole, DMF, rt; (e) HCl in dioxane, 0 °C; (f) (R)-(N-benzyloxycarbonyl-Nmethylaminopropanoic acid, HATU, DIPEA, DCM, rt; (g) NaIO4, RuCl3, acetonitrile, H2O, CCl4, rt.

Table 1. Cyclosporin Analogues Containing Neutral P4 Residues

compd

R′

Kd (nM)a

EC50 (nM)b

OATP1B1 (μM)c

alisporivir (3) 25 26

H CN OCH3

2.2 1.3 1.7

46 28 36

0.8 1 2.8

a

Affinity to human cyclophilin A as determined by SPR. bInhibition of replication of a subgenomic HCV replicon coexpressing a luciferase reporter gene in Huh7.5 cells. cInhibition of OATP1B1 transporter activity in CHO cells using 8-fluorescein-cAMP as the substrate.

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RESULTS AND DISCUSSION The affinity of the new compounds for cyclophilins A, B, and F was tested in surface plasmon resonance experiments using biotinylated cyclophilin proteins. All inhibitors were found to Scheme 5. Preparation of Cyclosporin Intermediate 23a

Reagents and conditions. (a) 8, HATU, diisopropylethylamine, HOAt, DCM, rt; (b) Tris-N-(2-aminoethyl)amine; (c) 0.5 M NaOH 0 °C; (d) BOP, DMAP, DCM, rt.

a

D

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Scheme 6. Late-Stage R4 Modificationa

Reagents and conditions. (a) TBAF, THF, rt; (b) sulfur trioxide pyridine complex, DMSO, DIEA, DCM, −78 °C to rt; (c) acetic acid, morpholine, NaBH(OAc)3, DCE, rt; (d) 20% tetramethylammonium hydroxide in MeOH, THF, 0 °C. a

transporter inhibitor than alisporivir (Table 5).37 The combination of reduced inhibition of OATP1B1, OATP1B3, and MRP2 indicates that 33 is less likely to interfere with bilirubin transport than alisporivir. Similarly, the reduced inhibition could normalize the clearance of coadministered drugs that are substrates for these transporters. The higher concentrations required for significant inhibition of MDR1 by inhibitor 33 is consistent with earlier studies that explored the structure−activity relationships of the cyclosporins for inhibition of this widely expressed transporter.25 Pharmacokinetic studies of 33 were conducted in rat and dog (Table 6). Compounds that bind tightly to cyclophilins partition in a dose-dependent fashion into blood.27,38 This partitioning is thought to be driven by the high expression of cyclophilins in erythrocytes. At low compound plasma concentrations, cyclophilin binding drives distribution into blood cells. Higher plasma concentrations lead to saturation of binding to erythrocyte cyclophilins, and significant partitioning into plasma is observed. Both total blood exposure and total plasma exposure for 33 and 3 in rat and dog studies are described in Table 6. Following an oral dose of each compound to rats, blood and plasma exposures are higher for 33 than 3. Similar data was obtained for 33 in dog studies. The calculated bioavailabilities for 33 and 3 in rat, using blood and plasma compartments separately as the basis for comparison, were similar.

macrocyclic chain is nearly identical to the conformation of the cyclosporin A macrocycle in complex with human cyclophilin A (1CWA).32,33 The C−CH3 group of the D-Ala residue at P3 engages in surface-exposed, hydrophobic contacts with Thr73 of the cyclophilin protein. The small methyl group also is likely to reduce the conformational mobility of this region of the peptide macrocycle,34 while the hydroxymethylpiperazine functionality extends toward the P6 leucine side chain and disrupts the hydrophobic surface formed by the P1, P6, and P11 residues. This bulky P4 group is clearly much too large to be accommodated by the calcineurin pocket occupied by the P4-leucine of CsA in the ternary CsA/cyclophilin A/calcineurin complex7 The methoxyethylpiperazine unit extends across a significant portion of the calcineurin binding region of the cyclosporins (P4−P7). As previous work had suggested that this hydrophobic surface was responsible for transporter− cyclosporin interactions, the piperazine subunit of 33 could be expected to reduce the affinity of this cyclosporin for biological transporters.26 Compound 33 was further characterized in a series of antiviral and transporter assays. This cyclophilin inhibitor is significantly more soluble than alisporivir in aqueous media (solubility (H2O, pH 7 (33)) = 1.1 g/L, solubility (H2O, pH 7 (3)) < 0.005 g/L). The compound does not inhibit major CYP enzymes reversibly (IC50 > 40 μM) or inhibit CYP3A4 in a time-dependent fashion. Additional in vitro and in vivo data comparing compound 33 and 3 are shown in Tables 4−6. In cyclophilin binding assays and standard replicon assays, 33 and 3 have high affinity for cyclophilin A and improved replicon potency relative to 2 or 4. Consistent with the reduced lipophilicity (log D, pH 7.4 (33) = 3.9, log D pH 7.4 (3) > 6.0) and moderate basicity (measured pKa (3) = 7.3), the addition of human serum has less of an impact on the measured cellular potency of 33 than 3. Because the efficacy of HCV antiviral drugs is generally correlated with the ratio of minimum total plasma concentration and EC50 measured in the presence of human serum, the total trough plasma concentration required for antiviral response with 33 may be lower than that required for 3.35,36 Inhibitor 33 and 3 exhibit similar low cytotoxicity in several cell lines (i.e., CC50 in MT4 cells: 33 > 20 μM, 3 > 20 μM). As expected, 33 also does not significantly affect the function of immune cells in vitro and appears to be a less potent inhibitor of human peripheral blood mononuclear cell proliferation in vitro than 3 (PBMC EC50 33 = 13.2 ± 5.4 μM, PBMC EC50 3 = 0.5 ± 0.26 μM). Our design strategy was predicated on the hypothesis that the interaction between the cyclosporin and the target transporter is often driven by hydrophobic interactions involving the P4−P8 region. Broad transporter inhibition profiling revealed that 33 is consistently a less potent

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CONCLUSION

Nonimmunosuppressive cyclophilin inhibitors such as the modified cyclosporin molecules described in this report have considerable potential as medicines for the treatment of hepatitis C infection and other diseases. However, firstgeneration cyclosporins are potent inhibitors of biological transporters such as MRP2 and OATP1B1. Inhibition of these transporters can cause drug−drug interactions and interfere with the normal excretion of biomolecules. New synthetic methodology has enabled diversity-oriented synthesis of a variety of cyclosporin analogues containing hydrophilic P4 groups. Some of these compounds feature significantly improved physical properties and in vitro transporter inhibition profiles. As exemplified for inhibitor 33 (NIM258), the new cyclosporins have promising pharmacokinetic profiles in preclinical species, are not immunosuppressive in vitro, and have excellent potency in cellular antiviral assays. These molecules should be useful for studies investigating the utility of cyclophilin inhibitors for the treatment of infection and other diseases. E

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Table 2. Amine-Containing Inhibitors 6−12

Table 3. Modifications of the Linker Between Morpholine and Cyclosporin Moieties

compd alisporivir (3) 27 34 35

X

Kd (nM)a

EC50 (nM)b

OATP1B1 (μM)c

CH2 OCH2CH2

2.2 3.1 1.1 1.1

46 33 29 36

0.8 4.3 3.9 6.4

a

Affinity of compound to human cyclophilin A as determined by SPR. Inhibition of replication of a subgenomic HCV replicon coexpressing a luciferase reporter gene in Huh7.5 cells. cInhibition of OATP1B1 transporter activity in CHO cells using 8-fluorescein-cAMP as the substrate.

b

Figure 3. Surface representation of cyclosporin 33 bound to rat cyclophilin D (4TQT). Phenomenex Kinetix C18 column; 2.1 mm × 50 mm; 2.6 μ core size, column temperature = 50 °C. Eluents: water with 0.1% TFA and CH3CN with 0.1% TFA with a flow rate of 1.2 mL/min and a gradient of 2−88% CH3CN in 9.5 min. According to this method, the purity of all test compounds was greater than 95%. ESI-MS data were recorded using a SYNAPT G2 HDMS (Q-TOF mass spectrometer, Waters) with lockspray ESI source. The resolution of the MS system was approximately 15000. Leucine enkephalin was used as lock mass (internal standards) infused from the lockspray probe. The compound was introduced into the mass spectrometer by UPLC (Acquity, Waters) from the sample probe. The separation was performed on an Acquity UPLC BEH C18 1 mm × 50 mm column at 0.2 mL/min flow rate with the gradient from 5% to 95% (3 min) using water with 0.1% formic acid and acetonitrile with 0.1% formic acid. The mass accuracy of the system has been found to be 50

2.4 0.5 ± 0.26 4.7 13.2 ± 5.4

a

Affinity of compound to human cyclophilin as determined by SPR. bInhibition of replication of a subgenomic HCV replicon coexpressing a luciferase reporter gene in Huh7.5 cells. 40% HS values refer to the cellular EC50 obtained following addition human serum to the assay medium. c Inhibition of replication of MT-4 cells determined using CDCF as a probe substrate. dInhibition of incorporation of 5-bromo-2′-deoxyuridine in fresh PBMC cells stimulated with phytohemagglutinin. For experimental details, see the Supporting Information.

Table 5. In Vitro Transporter Inhibition Profile for Alisporivir (3) and 33 compd

OATP1B1 IC50 (μM)a

OATP1B3 IC50 (μM)a

MRP2 Ki (μM)b

BSEP IC50 (μM)c

BCRP IC50 (μM)d

MDR1 IC50 (μM)e

alisporivir (3) 33

0.18 1.2

0.22 0.88

18 140

0.11 1.6

2.5 >25

0.59 6.3

a

Inhibition of recombinant OATP1B1 and OATP1B3 transporter activity in HEK293 cells using [3H]estradiol-17β-glucuronide as the substrate. Inhibition of MRP2 activity determined using inside out vesicles prepared from insect Sf9 cells expressing hMRP2 and CDCF as the substrate. c Inhibition of BSEP activity determined using HEK293 cells and [3H]taurocholic acid as the substrate. dInhibition of BCRP activity determined using T8 cells and Bodipy FL-prazosin as the substrate. eInhibition of MDR1 activity determined using MDA T0.3 cells and rhodamine 123 as the substrate. b

Table 6. Comparison of Pharmacokinetic Parameters of 3 and 33 in Rat and Dog compd

rat PO AUC (μM·h)a blood/plasma

dog PO AUC (μM·h)b blood/plasma

estimated rat F%c blood/plasma

alisporivir (3) 33

22.1/2.3 58.4/5.8

2.19/0.4 22.1/0.9

20/46 15/49

a

25 mg/kg, 0−72 h, administered as a solution in MEPC. b1 mg/kg, 0−72 h, administered as a solution in MEPC. cRat IV, 2 mg/kg; dog IV, 1 mg/ kg administered as a solution in 75% PEG400 + 25% D5W. PO: oral administration. F%: fraction of administered drug that reached the systemic circulation (oral bioavailability). All animal studies were approved by the Novartis Emeryville IACUC. equiv) in dry tetrahydrofuran (30 mL) at 0 °C under nitrogen. After stirring at 0 °C for 15 min, methyl iodide (3.7 g, 16.0 mL, 26.0 mmol, 3.3 eqiuv) and DMF (3 mL) were added. The reaction mixture was then stirred at room temperature for 3 h. The reaction was quenched with satd aq ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated to give 7 (4.0 g, 100%). 1H NMR (400 MHz, CDCl3) 0.84 (m, 3H), 1.00 (s, 9H), 1.50 (s, 9H), 2.33 (m, 1H), 2.71 (m, 3H), 3.47 (m, 3H), 5.26 (m, 1H), 6.09 (m, 1H), 5.93 (s, 1H), 6.16 (m, 1H), 6.26 (m, 1H). HPLC purity: 96%. HPLC retention time: 1.13 min. (2S,3S)-2-((R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-N-methylpropanamido)-4-((tert-butyldiphenylsilyl)oxy)-3methylbutanoic Acid (8). Trifluoroacetic acid (10.0 mL, 130.0 mmol, 13.2 equiv) was added to a solution of 7 (5.0 g, 9.85 mmol, 1.0 equiv) in dichloromethane (20 mL) at 0 °C. After 1 h of stirring at 0 °C, the solution was concentrated. The residue was diluted with ethyl acetate and washed with saturated aqueous NaHCO3 and brine. The organic layer was dried over magnesium sulfate and concentrated to obtain 4.0 g (100% yield) (1S,2S)-3-((tert-butyldiphenylsilyl)oxy)-1-(furan-2-yl)N,2-dimethylpropan-1-amine, which was used in the next step without further purification. This material (4.0 g, 9.81 mmol) as a solution in DCM was added to a mixture of (R)-2-((9H-fluorenyl-methoxycarbonyl) methylamino)-propanoic acid (3.83 g, 11.8 mmol, 1.2 equiv), HATU (4.5 g, 11.8 mmol, 1.2 equiv), and DIPEA (3.8 g, 5.2 mL, 29.4 mmol, 3.0 equiv) in DCM (40 mL). After stirring at room temperature for 16 h, the solution was diluted with ethyl acetate and washed with 1.0 N hydrochloric acid and brine. The organic layer was dried over magnesium sulfate and concentrated. The crude material was then purified by silica gel column chromatography (heptanes/ethyl acetate) to give the desired furan (((R)-1-((1S,2S)-3-(tert-butyl-diphenylsilanyloxy)-1-furan-2-yl-2-methyl-propyl)-methyl-carbamoyl)-ethyl)methyl-carbamic acid-9H-fluoren-9-ylmethyl ester (2.0 g, 29% yield). MS m/z (M + 1) 715.2. Ruthenium(III) chloride (175 mg, 0.85 mmol, 0.3 equiv) was added to a well stirred suspension of sodium periodate (5.38 g, 25.2 mmol, 9.0 equiv) in acetonitrile/carbon tetrachloride/water (10 mL/6.67

acetate. The organic layer was washed with 1.0 N hydrochloric acid and brine, dried over sodium sulfate, and concentrated. The crude material was purified by silica gel column chromatography (heptanes/ ethyl acetate) to give (1S,2S)-(1-furan-2-yl-2-methyl-3-oxo-propyl)carbamic acid tert-butyl ester (29.0 g, 80% yield). 1H NMR (400 MHz, CDCl3) 1.11 (dm, 3H), 1.44 (s, 9H), 2.92 (m, 1H), 5.10 (m, 2H), 6.20 (s, 1H), 6.34 (m, 1H), 7.35 (m, 1H), 9.66 (m, 1H), 9.76 (s, 1H). Sodium borohydride (12.6 g, 332.0 mmol, 3.0 equiv) was slowly added to a solution of (1S,2S)-(1-furan-2-yl-2-methyl-3-oxo-propyl)carbamic acid tert-butyl ester (28 g, 111 mmol, 1.0 equiv) in MeOH (120 mL) at 0 °C. After stirring at 0 °C for 2 h, the reaction mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The crude material was recrystallized from hot heptane to provide 6 (12.2 g, 43% yield). 1H NMR (400 MHz, CDCl3) 0.74 (d, J = 6.6, 3H), 1.46 (s, 9H), 2.25 (m, 1H), 3.32 (m, 1H), 3.52 (m, 2H), 5.11 (m, 2H), 6.15 (m, 1H), 6.36 (m, 1H), 7.36 (m, 1H). HPLC purity: 95%. HPLC retention time: 2.04 min. tert-Butyl((1S,2S)-3-((tert-butyldiphenylsilyl)oxy)-1-(furan-2-yl)-2methylpropyl)(methyl)carbamate (7). tert-Butyl-chloro-diphenyl-silane (2.4 g, 8.6 mmol, 1.1 equiv) and imidazole (1.2 g, 17.2 mmol, 2.2 equiv) were added to a solution of 6 (2.0 g, 7.8 mmol, 1.0 equiv) in DMF (30 mL) were added. After stirring at room temperature for 18 h, the reaction was quenched by addition of 1.0 N hydrochloric acid solution and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated to give (1S,2S)-3-(tert-butyl-diphenyl-silanyloxy)-1furan-2-yl-2-methyl-propyl)-carbamic acid tert-butyl ester (3.9 g, 100%) as a crude product. 1H NMR (400 MHz, CDCl3) 0.85 (d, J = 7.04 3H), 1.08 (s, 9H), 1.43 (s, 9H), 2.30 (m, 1H), 3.41 (m, 1H), 3.54 (m, 1H), 4.89 (m, 1H), 5.93 (s, 1H), 6.17 (m, 1H), 6.32 (m, 1H), 7.31 (s, 1H), 7.4 (m, 6H), 7.65 (m, 4H). (1S,2S)-3-(tert-Butyl-diphenyl-silanyloxy)-1-furan-2-yl-2-methylpropyl)-carbamic acid tert-butyl ester (3.9 g, 7.9 mmol, 1.0 equiv) was added to a suspension of 60% sodium hydride (0.94 g, 23.5 mmol, 3.0 G

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butanoic Acid (12). HCl in dioxane (4M, 5 mL) was added to a solution of compound 11 (800.0 mg, 1.45 mmol) in DCM (1 mL) at 0 °C. After stirring at 10 °C for 45 min, the reaction mixture was concentrated in vacuo to give the deprotected intermediate (655.0 mg), which was used for the next step without further purification. NMe-Z-D-Ala-OH (688.0 mg, 2.90 mmol, 2.0 equiv), HATU (827.0 mg, 2.18 mmol, 1.5 equiv), and DIPEA (1.26 mL, 7.25 mmol, 5.0 equiv) were added to a solution of deprotected intermediate (655.0 mg, 1.45 mmol) in DCM (10 mL) at 0 °C. After stirring at RT for 12 h, the reaction mixture was concentrated in vacuo and purified by silica gel chromatography (EtOAc:heptane) to provide the coupled intermediate (300.0 mg, 31%, MS m/z (M + 1) 671.2). Sodium periodate (669.0 mg, 3.13 mmol, 7.0 equiv) followed by RuCl3 (75.0 mg, 0.29 mmol, 0.64 equiv) were added to a solution of the furan (300.0 mg, 0.45 mmol) in heptane/ethyl acetate/water (3/ 1/4, 40 mL) at RT. After stirring for 45 min, the reaction mixture was filtered through Celite. The filtrate was diluted with water and extracted with ethyl acetate. The combined organic layer was washed with saturated sodium bisulfite and brine, dried over sodium sulfate, and concentrated in vacuo to give compound 12 (291 mg, yield 100%). MS m/z (M + 1) 649.2. HPLC purity: 90%. (9H-Fluoren-9-yl)methyl ((R)-1-(((1S,2S)-1-(Furan-2-yl)-3-hydroxy-2-methylpropyl)(methyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (13). A plastic tube was charged with ((R)-1-(((1S,2S)-3(tert-butyl-diphenyl-silanyloxy)-1-furan-2-yl-2-methyl-propyl)-methylcarbamoyl)-ethyl)-methyl-carbamic acid-9H-fluoren-9-ylmethyl ester (500 mg) in THF (3.0 mL). Two equiv of tetrabutylammonium fluoride solution in THF were added, and the resulting solution was stirred at room temperature for 24 h. The solution was then diluted with EtOAc and made basic by addition of saturated NaHCO3 solution. The phases were separated, and the organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/heptane, 0−70%) to give 199 mg of 13 (yield 60%). MS m/z (M + 1) 477.3. HPLC purity: 80%. (2S,3S)-3-((R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-N-methylpropanamido)-3-(furan-2-yl)-2-methylpropyl Methanesulfonate (14). TEA (0.10 mL, 0.75 mmol, 2.0 equiv) and MsCl (0.044 mL, 0.56 mmol, 1.5 equiv) were added to a solution of 27 (180 mg, 0.38 mmol, 1.0 equiv) in DCM (3.0 mL) at 0 °C. After stirring at 0 °C for 1 h, the reaction was quenched by addition of EtOH (0.5 mL) and EtOAc. The resulting solution was washed with saturated aqueous NH4Cl solution, dried over Na2SO4, and concentrated to give 209 mg of 14 (yield 100%). MS m/z (M + 1) 555.2. HPLC purity: 80%. (9H-Fluoren-9-yl)methyl ((R)-1-(((1S,2R)-3-Cyano-1-(furan-2-yl)2-methylpropyl)(methyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (15). NaCN (47 mg, 0.97 mmol, 3,0 equiv) was added to a solution of 14 (180 mg, 0.325 mmol, 1.0 equiv) in DMF (1.0 mL), and the mixture was stirred at 70 °C for 2 h. The solution was filtered, and the filtrate was diluted with EtOAc. The solution was washed with saturated aqueous NaHCO3 solution and brine, dried over Na2SO4, and concentrated. The residue was dissolved in DCM (1.0 mL), and TEA (0.127 mL, 0.91 mmol, 3.0 equiv) and FmocCl (118 mg, 0.456 mmol, 1.5 equiv) were added. After stirring at RT for 1 h, the solution was concentrated and the residue was purified by silica gel column chromatography (EtOAc/heptane 0% to 70%) to give 15 (80 mg, 54%). MS m/z (M + 1) 486.3. HPLC purity: 90%. (2S,3R)-2-((R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-N-methylpropanamido)-4-cyano-3-methylbutanoic Acid (16). To a solution of NaIO4 (211 mg, 0.99 mmol, 6.0 equiv) in MeCN (0.6 mL), water (0.4 mL), CCl4 (0.6 mL), RuCl3 (10 mg, 0.049 mmol, 0.3 equiv) were added. After stirring at RT for 10 min, this mixture was added to a solution of 15 (80 mg, 0.165 mmol, 1.0 equiv) in MeCN (0.3 mL) and the resulting mixture was stirred at RT for 20 min. The mixture was diluted with EtOAc and quenched by addition of aq NaHSO3 solution. The phases were separated, and the organic layer was washed with brine, dried over Na2SO4, and concentrated to provide the desired product (50 mg, yield 66%). MS m/z (M + 1) 464.3. HPLC purity: 90%.

mL/10 mL). After 15 min, (((R)-1-((1S,2S)-3-(tert-butyl-diphenylsilanyloxy)-1-furan-2-yl-2-methyl-propyl)-methyl-carbamoyl)-ethyl)methyl-carbamic acid-9H-fluoren-9-ylmethyl ester (2.0 g, 2.80 mmol, 1.0 equiv) was added and stirring was continued for 5 min. The reaction mixture was then quenched with water (25 mL) and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bisulfite and then with brine, dried over magnesium sulfate, and concentrated to give 8 (1.94 g, 80% yield). MS m/z (M + 1) 693.4. Purity: 90%. ((1S,2S)-1-Furan-2-yl-3-hydroxy-2-methyl-propyl)-methyl-carbamic Acid tert-Butyl Ester (9). Tetrabutyl ammonium flouride (1.0 M in THF, 47.3 mL, 47.3 mmol, 1.2 equiv) was added to a solution of 7 (20 g, 39.4 mmol) in MeOH (197 mL) at RT. After stirring for 18 h, the reaction mixture was added water and EtOAc. The phases were separated, and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/heptane) to give (10.51 g, 99%). 1H NMR (400 MHz, CDCl3) 7.37 (s, 1H), 6.33 (s, 1H), 6.27 (br s, 1H), 5.41 (br s, 1H), 3.41 (br s, 2H), 3.05 (s, 1H), 2.67 (s, 3H), 2.37 (m, 1H), 1.49 (s, 9H), 1.01(m, 3H). HPLC purity: 95%. tert-Butyl ((1S,2S)-3-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)-1(furan-2-yl)-2-methylpropyl)(methyl)carbamate (11). Compound 9 (300.0 mg, 1.1 mmol), bromoacetic acid (464.0 mg, 3.4 mmol, 3.0 equiv), and sodium iodide (167.0 mg, 1.1 mmol) were added successively to a suspension of NaH (60%, 134.0 mg, 3.4 mmol, 3.0 equiv) in THF (2.2 mL) at 0 °C. After stirring for 7 h at RT, the reaction mixture was quenched with water at 0 °C, diluted with EtOAc, and acidified with aqueous 1.0 N HCl aq solution. The organic layer was separated, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give 2-((2S,3S)-3-((tert-butoxycarbonyl)(methyl)amino)-3-(furan-2-yl)-2-methylpropoxy)acetic acid (287.0 mg, MS m/z (M + Na) 350.0), which was used for the next step without further purification. Trimethylsilyldiazomethane (1.0 M in hexanes, 6 mL, 6.0 mmol, 2.0 equiv) was added dropwise to a solution of 2-((2S,3S)-3-((tertbutoxycarbonyl)(methyl)amino)-3-(furan-2-yl)-2-methylpropoxy)acetic acid (1.0 g, 3.1 mmol) in methanol (2 mL) at 0 °C. After stirring for 30 min at RT, acetic acid (0.179 mL, 3.1 mmol, 1.0 equiv) was added dropwise. The reaction mixture was concentrated in vacuo, and the residue was purified by silica gel chromatography (EtOAc:heptane) to give the corresponding methyl ester 10 (0.95 g, 89% yield). 1 H NMR (400 MHz, CDCl3) 7.30−7.40 (m, 1H), 6.32 (dd, J = 1.86, 3.13 Hz, 1H), 6.27 (br s) and 6.20 (br s, 1H), 5.21 (d, J = 11.15 Hz) and 5.03 (d, J = 10.22 Hz, 1H), 3.96−4.02 (m, 2H), 3.71 (s, 3H), 3.39−3.46 (m, 1H), 3.20 (dd, J = 6.92, 8.97 Hz, 1H), 2.69 (br s, 3H), 2.39−2.52 (m, 1H), 1.47 (br s, 9H), 1.08 (br s, 3H). MS m/z (M + Na) 364.3. Sodium borohydride (1.05 g, 27.8 mmol, 10.0 equiv) was added to a solution of this methyl ester (0.95 g, 2.78 mmol) in methanol (7 mL) at 0 °C. After stirring for 1 h, the reaction was quenched with 1.0 M sulfuric acid and extracted with DCM. The combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the alcohol (0.85 g, MS m/z (M + Na) 336.3), which was used for the next step without further purification. Imidazole (0.31 g, 4.55 mol, 1.5 equiv) and TBDPSCl (0.9 mL, 3.5 mmol, 1.5 equiv) were added to a solution of the alcohol (0.95 g, 3.03 mmol) in DCM (8 mL) at RT. After stirring for 2 h, the reaction mixture was diluted with DCM (20 mL) and washed with aqueous 1.0 M sulfuric acid. The combined organic layers were dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:heptane) to give 11 (1.6 g, 96%). 1H NMR (400 MHz, CDCl3) 7.64−7.75 (m, 5H), 7.30−7.46 (m, 6H), 6.28 (d, J = 1.86 Hz, 1H), 6.21 (br s) and 6.15 (br s, 1H), 5.15 (d, J = 10.37 Hz) and 4.98 (d, J = 10.56 Hz, 1H), 3.74 (t, J = 5.31 Hz, 2H), 3.44 (qt, J = 5.23, 10.91 Hz, 2H), 3.32 (br s, 1H), 3.12 (dd, 1H), 2.70 (br s, 3H), 2.43−2.36 (m, 1H), 1.46 (s, 9H), 1.03 (s, 3H), 1.07 (s, 9H). MS m/z (M + Na) 574.4. Purity: 95%. (2S,3S)-2-((R)-2-(((Benzyloxy)carbonyl)(methyl)amino)-N-methylpropanamido)-4-(2-((tert-butyldiphenylsilyl)oxy)ethoxy)-3-methylH

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tert-Butyl ((1S,2S)-1-(Furan-2-yl)-2-methyl-3-oxopropyl)(methyl)carbamate (17). TBAF (1.0 M in THF, 47.3 mL, 47.3 mmol, 1.2 equiv) was added to a solution of ((1S,2S)-3-(tert-butyl-diphenylsilanyloxy)-1-furan-2-yl-2-methyl-propyl)-methyl-carbamic acid tertbutyl ester (20 g, 39.4 mmol) in MeOH (197 mL) at RT. After stirring for 18 h, the reaction mixture was quenched by addition of water (30 mL). The aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/heptane) to give the intermediate alcohol (10.51 g, 99%). 1H NMR (400 MHz, CDCl3) 7.37 (s, 1H), 6.33 (s, 1H), 6.27 (br s, 1H), 5.41 (br s, 1H), 3.41 (br s, 2H), 3.05 (s, 1H), 2.67 (s, 3H), 2.37 (m, 1H), 1.49 (s, 9H), 1.01 (m, 3H). DIPEA (10.4 mL, 59.4 mmol, 4 equiv) was added to a solution of the alcohol (4.0 g, 14.85 mmol) in DCM (37 mL) and DMSO (37 mL) at 0 °C. Pyridine sulfur trioxide (7.1 g, 44.6 mmol, 3 equiv) was added, and the mixture was stirred for 30 min. The reaction mixture was quenched by addition of saturated aqueous ammonium chloride solution, diluted with EtOAc (200 mL), washed with brine (2 × 20 mL), and dried over Na2SO4. Filtration and concentration provided 17 (3.8 g, 96%). 1H NMR (400 MHz, CDCl3) 9.57 (br s, 1H), 7.36−7.39 (m, 1H), 6.30− 6.34 (m, 1H), 6.23 (br s, 1H), 5.55−5.69 (m, 1H), 2.96−3.17 (m, 1H), 2.65 (br s, 3H), 1.49 (br s, 9H), 1.11−1.16 (m, 3H). MS m/z (M + 1) 268.2. Purity: 80%. tert-Butyl ((1S,2R,E)-1-(Furan-2-yl)-4-methoxy-2-methylbut-3-en1-yl)carbamate (18). NaHMDS (48 mL, 3.38 mmol, 1 M in THF) was added to a solution of (methoxymethyl)triphenylphosphonium chloride (17.48 g, 56.9 mmol, 4 equiv) in THF (34 mL) at 0 °C. After 5 min, a solution of 17 in THF (13 mL) was added to the mixture, which was then stirred at 0 °C for 30 min, then overnight at RT. The reaction mixture was poured into Et2O and washed with saturated aqueous NH4Cl solution and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (EtOAc/heptane) to give 18 as a mixture of cis and trans isomer (3.55 g, 85%). 1H NMR (400 MHz, CDCl3) 7.31−7.37 (m, 1H), 6.11−6.33 (m, 3H), 5.74−5.79 (m), 5.07−5.17 (m), 4.81−4.94 (m), 4.46−4.58 (m, 1H), 4.13−4.23 (m), 3.58 (s), 3.39 (s, 3H), 3.16−3.28 (m), 2.66−2.82 (m, 4H), 2.59−2.64 (s). MS m/z (M + 1) 296.3. Purity: 95%. tert-Butyl ((1S,2R)-4-((tert-Butyldiphenylsilyl)oxy)-1-(furan-2-yl)2-methylbutyl)(methyl)carbamate (19). PPTS (4.53 g, 18.03 mmol, 1.5 equiv) was added to a solution of 18 (3.55 g, 12.02 mmol) in acetone. After stirring for 24 h, the reaction mixture was poured into saturated aqueous NaHCO3 solution and extracted with EtOAc. The phases were separated, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (EtOAc/heptane) to give the intermediate aldehyde (2.66 g, 79%). 1H NMR (400 MHz, CDCl3) 9.65 (s, 1H), 7.35 (s, 1H), 6.66−5.96 (br s, 2H), 5.25−4.78 (m, 1H), 2.80 (m, 1H), 2.72 (s, 3H), 2.72−2.59 (m, 2H), 1.47 (s, 9H), 0.74 (d, J = 6.65 Hz, 3 H). NaBH4 (1.8 g, 47.3 mmol, 5.0 equiv) was added to a solution of the aldehyde (2.66 g, 9.46 mmol) in methanol (38 mL) at 0 °C. After stirring for 1 h, the reaction mixture was diluted with EtOAc, washed with saturated aqueous NH4Cl and brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (EtOAc/heptane) to provide the corresponding alcohol (2 g, 75% yield). 1H NMR (400 MHz, CDCl3) 7.35 (s, 1H), 6.27 (br s, 2H), 5.13−4.87 (d, J = 9.78 Hz, 1H), 3.67 (m, 2H), 2.68 (s, 3H), 2.32 (m, 1H), 1.47 (s, 9H), 1.27 (m, 2H), 0.88 (d, J = 6.65 Hz, 3 H). Imidazole (988 mg, 14.52 mmol, 2.2 equiv) and TBDPSCl (2.18 g, 7.92 mmol, 1.2 equiv) were added to a solution of the alcohol (1.87 g, 6.60 mmol) in DMF (9.7 mL) at 0 °C. After stirring at RT for 12 h, the reaction mixture was diluted with EtOAc (20 mL) and washed sequentially with water (5 mL), aqueous 1 M HCl (5 mL), saturated aqueous NaHCO3 (5 mL), and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (EtOAc/heptane) to give 19 (3.55 g, yield 100%). 1H NMR (400 MHz, CDCl3) 7.66−7.73 (m, 2H), 7.33−7.45 (m, 10H), 6.28−6.31 (m, 1H), 6.25 (br s, 1H), 3.63−3.72 (m, 2H),

2.64 (s, 3H), 2.26−2.36 (m, 1H), 1.46 (s, 9H), 1.25−1.30 (m, 2H), 1.05 (s, 9H), 0.84−0.89 (m, 3H). MS m/z (M + 1) 522.3. HPLC purity: 80%. (2S,3R)-2-((R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-N-methylpropanamido)-5-((tert-butyldiphenylsilyl)oxy)-3methylpentanoic Acid (20). HCl in dioxane (4.0 M, 3.6 mL, 14.4 mmol, 15 equiv) was added to a solution of 19 in dioxane (4.8 mL) at 0 °C. After stirring for 6 h, the reaction mixture was diluted with DCM (30 mL) and washed with saturated aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated to give the deprotected intermediate (385 mg, 95%), which was used without further purification. DIPEA (2.3 mL, 4.57 mmol, 5.0 equiv) and HATU (694 mg, 1.8 mmol, 2.0 equiv) were added to a solution of (R)-2-(((benzyloxy)carbonyl)(methyl)amino)propanoic acid (433 mg, 1.8 mmol, 2.0 equiv) in DCM (9 mL). The mixture was stirred for 10 min, after which the deprotected intermediate (385 mg, 0.92 mmol) was added. After stirring at RT for 4 h, the reaction mixture was diluted with DCM (50 mL) and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (heptane/ acetone) to provide the coupled intermediate (242.0 mg, 41% yield). MS m/z (M + 1) 641.5. RuCl3 (26.6 mg, 0.13 mmol, 0.34 equiv) was added to a stirred mixture of NaIO4 (485.0 mg, 2.30 mmol, 6.0 equiv) in H2O/CCl4/CH3CN (3/2/3, 10.4 mL), and the mixture was stirred vigorously for 15 min. A solution of the furan (242.0 mg, 0.38 mmol) in CH3CN (3 mL) was added. After stirring for 15 min, the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layer was washed with saturated aqueous NaHSO3 solution, brine, dried over Na2SO4, and concentrated to give product 20 (242.0 mg), which was used for the next step without further purification. MS m/z (M + 1) 619.4. HPLC purity: 90%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3S)-4-((tert-butyldimethylsilyl)oxy)-3methyl-2-(methylamino)butanoic acid]-cyclosporin A (23). Intermediate 21 (2.0 g, 1.7 mmol, 1.0 equiv) was added to a suspension of 8 (1.43 g, 2.1 mmol, 1.0 equiv), HATU (784 mg, 2.1 mmol, 1.2 equiv), and DIPEA (666 mg, 0.90 mL, 5.16 mmol, 3 equiv) in DCM (10 mL). The resulting suspension was stirred at RT for 16 h and diluted with ethyl acetate and washed with 1.0 N hydrochloric acid and brine. The organic layer was dried over magnesium sulfate and concentrated. The crude material was then purified by silica gel column chromatography (heptane/acetone) to give the desired undecapeptide (3.2 g, 100% yield, MS m/z (M + 1) 1838.8). Tris(2-aminoethyl) amine (573 mg, 0.59 mL, 3.92 mmol, 4.0 equiv) was added to a solution of 22 (1.8 g, 0.98 mmol, 1 equiv) in DCM (12 mL) at RT, and the resulting mixture was stirred for 3 h. The solution was then diluted with ethyl acetate and washed with 1.0 N hydrochloric acid and finally with brine. The organic layer was dried over magnesium sulfate and concentrated to give the desired deprotected intermediate (327 mg, 100% yield). MS m/z (M + 1) 1617.0. Sodium hydroxide (0.5 M, 10.77 mL, 5.38 mmol, 6 equiv) was added to a solution of the amine (1.45 g, 0.88 mmol, 1 equiv) in THF (10 mL) at 0 °C, and the resulting solution was stirred at 0 °C 1 h. The solution was diluted with ethyl acetate and washed with 1.0 N hydrochloric acid and brine. The organic layer was dried over magnesium sulfate and concentrated to give the desired acid (1.35g, 100% yield). MS m/z (M + 1) 1518.0. A solution of DMAP (240 mg, 2.0 mmol, 2.0 equiv) and the unprotected undecapeptide (1.45 g, 0.98 mmol, 1.0 equiv) in DCM (50 mL) was added to a solution of BOP (523 mg, 1.2 mmol, 2.0 equiv) in DCM (50 mL) over 15 min at RT. After stirring at RT for 20 h, the solution was concentrated and the residue was dissolved in EtOAc. The solution was then washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (heptanes/acetone) to give product 23 (590 mg, 40% yield). MS m/z (M + 1) 1499.8. HPLC purity: 95%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3R)-4-cyano-3-methyl-2(methylamino)butanoic acid]-cyclosporin A (25). HATU (49 mg, 0.13 mmol, 1.2 equiv) was added to a solution of 16 (56 mg, 0.11 mmol, 1.0 equiv) in DCM (0.5 mL) at 0 °C. After stirring at 0 °C for 5 I

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min, compound 21 (113 mg, 0.097 mmol, 0.9 equiv) and NMM (33 mg, 0.32 mmol, 3.0 equiv) were added. The resulting solution was then stirred at 0 °C for 1 h and at RT for 18 h. The solution was diluted with EtOAc and washed with 1.0 N HCl solution, water, NaHCO 3 solution, and brine and dried over Na 2 SO 4 and concentrated. The residue was purified by silica gel column chromatography (acetone/heptane 0%−90%) to give the coupled intermediate (90 mg, yield 52%). MS m/z (M + Na) 1632.0. LiOH·H2O (14 mg, 0.34 mmol, 6.0 equiv) was added to a solution this ester (90 mg, 0.056 mmol, 1.0 equiv) in THF (1.0 mL)/water (0.5 mL). After stirring at 0 °C for 3 h, the reaction was acidified by addition of 1.0 N HCl solution to pH 6. The solution was extracted with EtOAc, washed with brine, dried over Na2SO4, and concentrated (70 mg, MS m/z (M + 1) 1287.8). This residue (70 mg, 0.054 mmol, 1.0 equiv) and DMAP (20 mg, 0.16 mmol, 3.0 equiv) in 20 mL of DCM were added to a solution of BOP (72 mg, 0.16 mmol, 3.0 equiv) in DCM (40 mL). After stirring at RT for 3 days, the solution was washed with 1.0 N HCl, saturated NaHCO3 solution, and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (acetone/heptane, 0−100%) to give (22 mg, 32%) of the acetylated intermediate (MS m/z (M + 1) 1269.9). A solution of Me4NOH (25% in MeOH, 63 mg, 0.17 mmol, 10 equiv) was added to a solution of the acetylated cyclosporin (22 mg, 0.017 mmol, 1.0 equiv) in MeOH (1.0 mL) at 0 °C. The solution was stirred at 0 °C for 1 h and at RT for 2 h. The solution was then diluted with EtOAc and acidified by addition of 10 N HCl solution until pH 6.0. The phases were separated, and the organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by reverse HPLC to give 25 (7 mg, overall yield 5%). HRMS m/z calcd for C63H111N12O12 [M + H]+ 1227.8444; found 1227.8428. HPLC purity: 96%. 3 - [ ( D ) - N ( C H 3 ) - A l a ] - 4 - [ (2 S , 3 S ) - 4 - m e th o x y - 3 - m e t h y l - 2 (methylamino)butanoic acid]-cyclosporin A (26). Compound 21 (265 mg, 0.23 mmol, 0.66 equiv) was added to a mixture of (2S,3S)-2((R)-2-((tert-butoxycarbonyl)(methyl)amino)-N-methylpropanamido)-4-methoxy-3-methylbutanoic acid (120 mg, 0.35 mmol, 1.0 equiv, see Supporting Information), HATU (158 mg, 0.42 mmol, 1.2 equiv), and DIPEA (134 mg, 0.18 mL, 1.04 mmol, 3 equiv) in dichloromethane (2 mL). After stirring at RT for 16 h, the reaction mixture was diluted with ethyl acetate and washed with 1.0 N hydrochloric acid and brine. The organic layer was dried over magnesium sulfate and concentrated. The coupled product was purified by silica gel column chromatography (heptanes/acetone) to give the desired undecapeptide (517 mg, 100% yield, MS m/z (M + 1) 1493.0). Sodium hydroxide solution (0.5 M, 3.75 mL, 1.88 mmol, 8 equiv) was added to a solution of the coupled product (350 mg, 0.24 mmol, 1.0 equiv) in THF (6 mL) at 0 °C, and the resulting solution was stirred at 0 °C for 1 h. The solution was then diluted with ethyl acetate, washed with 1.0 N hydrochloric acid and brine, dried over magnesium sulfate, and concentrated to give the desired acid (327 mg, 100% yield, MS m/z (M + 1) 1393.80). Trifluoroacetic acid (1.0 mL, 13.0 mmol, 121 equiv) was added to a solution of the acid (150 mg, 0.11 mmol, 1 equiv) in dichloromethane (2 mL) at 0 °C. After 1 h, the solvent was evaporated. The remaining oil was dissolved in ethyl acetate, washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, and concentrated to give the amino acid (139 mg, 100% yield, MS m/z (M + 1) 1293.8). The amino acid (40 mg, 0.03 mmol, 1.0 equiv) in dichloromethane (20 mL) was added to a solution of BOP (27.4 mg, 0.06 mmol, 2.0 equiv) and DMAP (7.56 mg, 0.06 mmol, 2 equiv) in dichloromethane (20 mL) over 15 min at RT. The resulting solution was stirred at for 20 h. The solution was concentrated, and the residue was dissolved in ethyl acetate, washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (heptanes/acetone) to give the target macrocycle (25 mg, 65% yield MS m/z (M + 1) 1275.80). A tetramethylammonium hydroxide solution (25% in MeOH, 71.5 mg, 0.196 mmol, 20 equiv) was added to a solution of the macrocycle (25 mg, 0.02 mmol, 1.0 equiv) in THF (2 mL) at 0 °C. After stirring

for 30 min, the solution was extracted with EtOAc. The organic layer was washed with saturated aqueous ammonium chloride and then with brine, dried with magnesium sulfate, and concentrated. The residue was purified by HPLC to provide 26 (12 mg, overall yield: 32%). HRMS m/z calcd for C63H114N11O13 [M + H]+ 1232.8598; found 1232.8596. HPLC purity: 98%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3R)-3-methyl-2-(methylamino)-4-morpholinobutanoic acid]-cyclosporin A (27). Compound 23 (2.7 g, 1.8 mmol, 1.0 equiv) was added to a solution of TBAF in THF (18.0 mL, 1.0 M in THF, 18.0 mmol, 10.0 equiv) at RT. After stirring at RT for 6 h, the solution was diluted with EtOAc and washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (acetone/heptane (0% to 100%) to provide the intermediate alcohol (1.7 g, 75%). MS (M + H) 1261.9. DIPEA (0.29 mL, 1.7 mmol, 3.0 equiv) and a solution of Py·SO3 (442 mg, 2.8 mmol, 5.0 eqiv) in DMSO (0.5 mL) were added to a solution of the alcohol (700 mg, 0.55 mmol) in DCM (1 mL) at 0 °C, and the resulting solution was stirred at 0 °C for 30 min. The reaction was quenched by addition of NH4Cl solution and then extracted with EtOAc. The combined organic layers were washed with brine, dried with MgSO4, and concentrated. The resulting aldehyde (MS m/z (M + 23) 1281.9) was used in the next step with no further purification. Morpholine (111 mg, 1.3 mmol, 2.0 equiv), acetic acid (38 mg, 0.64 mmol, 1.0 equiv), and Na2SO4 (50 mg) were added to a solution of the aldehyde (800 mg, 0.64 mmol, 1.0 equiv) in CH3CN (3.2 mL). After stirring at RT for 5 min, sodium triacetoxyborohydride (269 mg, 1.3 mmol, 2.0 equiv) was added and the resulting mixture was stirred at room temperature for 1 h. NH4Cl solution and EtOAc were added to the reaction mixture. The phases were separated, and the organic layer was washed with brine, dried over MgSO4, and concentrated to give the desired acetylated cyclosporin (846 mg, MS m/z (M + 1) 1331.0). Tetramethylammonium hydroxide (25% weight in MeOH, 2.3 g, 6.4 mmol, 10 equiv) was added to a solution of the acetylated intermediate (846 mg, 0.64 mmol) in MeOH (3.2 mL) at 0 °C. After stirring for 30 min at 0 °C, the reaction solution was diluted with EtOAc and washed with NH4Cl solution and brine, dried over MgSO4, and concentrated. The crude material was purified by reverse phase HPLC to give the desired product as a TFA salt. The resulting solution was basified with saturated NaHCO3 aqueous solution and extracted with ethyl acetate to obtain free 27 (430 mg, yield 53%). HRMS m/z calcd for C66H119N12O13 [M + H]+ 1287.9020; found 1287.9006. HPLC purity: 99%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3R)-3-methyl-2-(methylamino)-4-(1,4oxazepan-4-yl)butanoic acid]-cyclosporin A (28). Prepared according to the procedure employed for the synthesis of compound 27, substituting 1,4-oxazepane for morpholine in the reductive amination. Yield 9% (from 23). MS m/z (M + 1) 1302.9. HPLC purity 95%. 3-[(D)-N(CH 3)-Ala]-4-[(2S,3R)-4-(3,3-difluoropyrrolidin-1-yl)-3methyl-2-(methylamino)butanoic acid]-cyclosporin A (29). Prepared according to the procedure employed for the synthesis of compound 27, substituting 3,3-difluoropyrrolidine for morpholine in the reductive amination. Yield 6% (from 23). HRMS m/z calcd for C66H117N12O12F2 [M + H]+ 1307.8882; found 1307.8875. HPLC purity >95%. 3-[(D)-N(CH3)]-Ala-4-[(2S,3R)-4-(8-oxa-3-azabicyclo[3.2.1]octan3-yl)-3-methyl-2-(methylamino)butanoic acid]-cyclosporin A (30). Prepared according to the procedure employed for the synthesis of compound 27, substituting 8-oxa-3-azabicyclo[3.2.1]octane for morpholine in the reductive amination. Yield 12% (from 23). HRMS m/z calcd for C68H121N12O13 [M + H]+ 1313.9176; found 1313.9170. HPLC purity 98%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3R)-4-(4-cyanopiperidin-1-yl)-3-methyl2-(methylamino)butanoic acid]-cyclosporin A (31). Prepared according to the procedure employed for the synthesis of compound 27, substituting 4-cyanopiperidine for morpholine in the reductive amination. Yield 18% (from 23). HRMS m/z calcd for C68H120N13O12 [M + H]+ 1310.9179; found 1310.0189. HPLC purity 98%. 3-[(D)-N(CH3)-Ala-4-(2S,3R)]-4-((2-methoxyethyl)(methyl)amino)3-methyl-2-(methylamino)butanoic acid]-cyclosporin A (32). PreJ

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undecapeptide (516.0 mg, MS m/z (M + 1) 1531.9), which was used in the next step without further purification.. Cyclization, desilylation, reductive amination, and deacetylation were performed as described for the synthesis of compound 27 (overall yield 3% from 21). HRMS m/z calcd for C67H121N12O13 [M + H]+ 1301.9176; found 1301.9152. HPLC purity 97%. 3-[(D)-N(CH 3)-Ala]-4-[(2S,3S)-3-methyl-2-(methylamino)-4-(2morpholinoethoxy)butanoic acid]-cyclosporin A (35). Compound 12 and amine 21 were coupled using DIPEA, HATU, and HOAt as described above. Conversion of this intermediate to 35 was performed as described for compound 34 (overall yield 5% from 21). HRMS m/z calcd for C68H123N12O15 [M + H]+ 1379.8952; found 1379.8951. HPLC purity 99%. Inhibitor Binding to Cyclophilin Proteins. Binding of inhibitors to purified bacterially expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged monobiotinylated cyclophilin proteins were immobilized onto a Biotin CAPture chip (GE Healthcare). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophilins. Compounds at various concentrations were injected into the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasted 10 min postinjection. Data were analyzed using Biacore T200 evaluation software version 1.0, and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD. Inhibitory Effects in an HCV Replicon System. Susceptibility to compounds was analyzed in Huh7.5 cells containing genotype 1a or 1b subgenomic HCV replicons coexpressing a luciferase reporter gene. The original replicons, pFK389lucubineo_3_3′_ET and pH/SGNeo(L+I), were licensed from ReBlicon GmbH (Germany) and Apath LLC (Saint Louis, MO), respectively. The firefly reporter gene was replaced by renilla luciferase reporter gene using standard molecular biology techniques as follows. Generation of genotype 1b subgenomic replicon: The renilla firefly gene−neomycin phosphotransferase cassette was PCR-amplified from pF9 cytomegalovirus (CMV) hRluc-Neo Flexi(R) (Promega) using Accuprime super mix II (Invitrogen) and the primer sets AscI hRlucNeo Fwd and NotI hRluc Rev. These primers have the following sequences and introduce restriction sites for subsequent cloning: AscI hRlucNeo Fwd, 5′- GGG CGC GCC ATG GCT TCC AAG GTG TAC G-3′ (AscI site underlined) and NotI hRluc Rev, 5′-CGC GGC CGC TCA GAA GAA CTC GTC AAG −3′ (NotI site underlined). The amplification product was subcloned into pCR2.1-TOPO (Invitrogen). The resulting plasmid was digested with AscI and NotI, and the excised fragment (hRluc-Neo) was ligated with the Roche Quick Ligation Kit (Roche) into pFK1389lucubineo_3_3′_ET digested with the same enzymes. Generation of genotype 1a subgenomic replicon: pH/SG-Neo(L+I) was modified by introduction of the luciferase reporter gene, along with additional adaptive mutations, via replacement of the pH/SGNeo(L+I) NS3-NS5A coding region with the corresponding region of pH77-S to obtain pH/SG-lucubineo-H77S as described (Borawski et al.39). Subsequently, the firefly luciferase−neomycin phosphotransferase cassette was replaced by the renilla firefly−neomycin phosphotransferase cassette. To create a unique cloning site, the NotI restriction site in NS5B was removed using QuickChange II XL sitedirected mutagenesis kit (Agilent). The two site-directed mutagenesis primers have the following sequences and introduce a mutation to knock out the NotI site in NS5B for subsequent cloning: NotI KO Fwd 5′-CTC AAA CTC ACT CCA ATA GCT GCC GCT GGC CGG CTG GAC-3′ (G → T mutation underlined) and NotI KO Rev 5′GTC CAG CGG GCC AGC GGC AGC TAT TGG AGT GAG TTT GAG-′3 (C → A mutation underlined). The resulting vector, pH/SGlucubineo-H77S-NotIKO, was sequenced to confirm the correct sequence on the NS5B gene. The hRluc-Neo gene was amplified from pF9 cytomegalovirus (CMV) hRluc-Neo Flexi(R) (Promega) by PCR using Accuprime super mix II (Invitrogen) and primer sets AscI hRlucNeo Fwd and NotI hRluc Rev. These primers have the following

pared according to the procedure employed for the synthesis of compound 27, substituting 2-methoxy-N-methylethanamine for morpholine in the reductive amination. Yield 5% (from 23). MS m/ z (M + 1) 1289.9. HPLC purity 96%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3S)-4-((tert-butyldiphenylsilyl)oxy)-3methyl-2-(methylamino)butanoic acid]-cyclosporin A (33). TBAF (1.0 M in THF, 2.20 mL, 2.20 mmol, 6 equiv) was added to a solution of 23 (550 mg, 0.37 mmol, 1.0 equiv) in THF (2.0 mL), and the resulting solution was stirred at room temperature for 1 h. The reaction solution was then diluted with ethyl acetate, washed with brine, dried over magnesium sulfate, and concentrated. The crude material was purified by silica gel column chromatography (heptanes/ acetone) to give the desired alcohol (347 mg, 75% yield, MS m/z (M + 1) 1260.8). DMSO (198 mg, 0.18 mL, 2.54 mmol, 32 equiv) was added to a solution of oxalyl chloride (161 mg, 0.111 mL, 1.27 mmol, 16 equiv) in DCM (2 mL) at −78 °C, and the resulting solution was stirred at −78 °C for 10 min. The alcohol (100 mg, 0.08 mmol, 1 equiv) in DCM (1.0 mL) was added to the reaction mixture. After 10 min, TEA (281 mg, 0.387 mL, 2.78 mmol, 35 equiv) was added. The solution was stirred at −78 °C for 10 min and at RT for 30 min. The reaction was quenched by the addition of saturated aqueous NH4Cl solution. The mixture was extracted with ethyl acetate, washed with brine, dried with magnesium sulfate, and concentrated. The aldehyde (MS m/z (M + 1) 1258.9) was used in the next step with no further purification. 1-(2-methoxyethyl)piperazine (1.375 g, 9.53 mmol, 10 equiv) and acetic acid (0.546 mL, 9.53 mmol, 10 equiv) were added to a solution of the aldehyde (1.2 g, 0.953 mmol, 1.0 equiv) in dichloroethane (25 mL). After stirring at RT for 15 min, sodium triacetoxyborohydride (2.02 g, 9.53 mmol, 10.0 equiv) was added and the resulting mixture was stirred at RT for 3 h. The mixture was diluted with EtOAc and washed with aqueous NH4Cl solution and brine, dried over Na2SO4, and concentrated. The amine intermediate was used in the next step with no further purification. Tetramethylammonium hydroxide (25% weight in MeOH, 2.74 g, 9.01 mmol, 10 equiv) at 0 °C was added to a solution of the amine (1.25 g, 0.90 mmol, 1.0 equiv) in THF (15 mL). After stirring for 2 h at 0 °C, the reaction solution was diluted with ethyl acetate and washed with saturated aqueous NH4Cl and brine, dried over magnesium sulfate, and concentrated. The remaining material was purified by reverse phase HPLC. The collected fraction was dissolved in EtOAc and washed with satd aq K2CO3 solution to remove TFA to provide free 33 (580 mg, 48%). HRMS m/z calcd for C69H126N13O13 [M + H]+ 1344.9598; found 1344.9593. HPLC purity 99%. 3-[(D)-N(CH3)-Ala]-4-[(2S,3R)-3-methyl-2-(methylamino)-5-morpholinopentanoic acid]-cyclosporin A (34). DIPEA (191 mg, 258 μL, 1.48 mmol, 4 equiv), HATU (183 mg, 0.48 mmol, 1.3 equiv), and HOAt (65.3 mg, 0.48 mmol, 1.3 equiv) were added to a solution of 20 (240 mg, 0.39 mmol, 1.05 equiv) in DCM (3.7 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 10 min. Amine 21 (430 mg, 0.37 mmol) was added to the mixture. After stirring for 3 h at RT, the reaction mixture was diluted with DCM (20 mL) and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (acetone/heptane) to give the desired Cbz-protected undecapeptide (545 mg, 84% yield, MS m/z (M + Na) 1765.1). Triethylsilane (1.7 mL, 10.8 mmol, 35.0 equiv), triethylamine (0.366 mL, 2.63 mmol, 8.5 equiv), and palladium(II) acetate (69.3 mg, 0.31 mmol, 1.0 equiv) were added to a solution of the undecapeptide in DCM (3 mL). After stirring at RT for 1 h, the reaction mixture was quenched with saturated aqueous ammonium chloride solution and filtered. The filtrate was diluted with DCM and washed with saturated NaHCO3 solution. The combined organic layers were dried with Na2SO4, filtered, and concentrated to give the desired amine (504 mg, MS m/z (M + 1) 1631.2). The amine (504.0 mg, 0.31 mmol) in THF/water (1/1, 3.2 mL) at 0 °C was treated with LiOH·H2O (64.9 mg, 1.6 mmol, 5.0 equiv). After stirring at 0 °C for 1.5 h, the reaction mixture was diluted with DCM and washed with aqueous 1.0 N HCl solution and brine. The combined organic layer was dried with Na2SO4, filtered, and concentrated to give product the amino acid K

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sequences and introduce restriction sites for subsequent cloning: AscI hRlucNeo Fwd, 5′- GGG CGC GCC ATG GCT TCC AAG GTG TAC G-3′ (AscI site underlined), and NotI hRluc Rev, 5′-CGC GGC CGC TCA GAA GAA CTC GTC AAG −3′ (NotI site underlined). The amplification product was subcloned into pCR2.1-TOPO (Invitrogen), the resulting plasmid was digested with AscI and NotI, and the excised fragment (hRluc-Neo) was ligated with the Promega Rapid Ligation ligation kit (Promega) into pH/SG-lucubineo-H77SNotIKO digested with the same enzymes. Generation of stable replicon cell lines: 10 μg of in vitro-transcribed RNA were transfected into Huh7.5 cells by electroporation. Briefly, subconfluent cells were detached by trypsin treatment, collected by centrifugation, washed twice with ice-cold phosphate buffered saline (PBS), and resuspended at 107 cells/mL in Opti-MEM (Invitrogen). Replicon RNA was added to 400 μL of cell suspension in a Gene Pulser cuvette (0.4 cm gap). Cells were electroporated at 270 V and 960 μF (Bio-Rad Gene Pulser system; Bio-Rad, Hercules, CA). Pulsed cells were incubated at room temperature for 10 min after electroporation and then resuspended in 30 mL of Dulbecco’s Modified Eagle Medium (DMEM). Cells were plated into 100 mm diameter dishes for G418 selection. Colonies were pooled, expanded, and cryopreserved at early passage levels. Cells were grown in DMEM, 2 mM L-glutamine, 0.1 mM essential amino acids, 1× penicillin−streptomycin, 1 mM sodium pyruvate, 10% heat-inactivated FBS (Invitrogen), and 500 μg/mL geneticin (G418). Cells were routinely split 1:4 twice a week. The assay medium for both luciferase reporter HCV replicon and cytotoxicity assays contains phenol red-free DMEM and lacks G418. Sixteen half-log dilutions of compounds were stamped into 12 384well plates, allowing parallel EC50 determination in triplicate for genotype 1a and 1b replicon cells of cytotoxicity and protein binding via addition of 40% human serum to genotype 1b replicon cells. Luciferase activity of drug-treated cells was measured relative to DMSO-treated controls after 72 h incubation using Renilla-Glo luciferase assay (Promega). Cytotoxicity (CC50) was analyzed using Cell Titer-Glo (Promega). Anti-Proliferative or Immunosuppressive Activity. PBMCs of two human donors were obtained on the day of the experiment (AllCells, LLC), pelleted in their original 50 mL conical tubes at 1200 rpm for 10 min, and resuspended in 25 mL in X-VIVO 15 medium (Lonza). After adjusting to a concentration of 3.75 × 105 cells/mL, 80 μL aliquots were dispensed into 96-well assay plates coated with polyD-lysine. Compounds were diluted in 12-step half-logarithmic series in DMSO starting at 10 mM. This allowed selection of seven concentrations at the high or low end of the range depending on the expected IC50. Then 1.5 μL of aliquots of test compound were transferred to 30 μL of X-VIVO 15 medium, and 10 μL of this 10× dilution was added to the cells. To stimulate proliferation of T-cells, 10 μL of a 31.6 μg/mL PHA solution was added. Drug-free control wells (±PHA) containing the appropriate volume of medium served as background (low- and high-signal controls). Plates were incubated at 37 °C and 5% CO2 for 60 h. Twelve hours before stopping the assay, BrdU labeling solution was added to each well of the assay plates and they were further processed according to the manufacturer’s protocol (BrdU ELISA, Roche). Briefly, plates were centrifuged at 1200 rpm for 10 min and the medium was removed by gently flicking the plates and patting on paper towels. After drying at 60 °C for 1 h, each well received 200 μL of fix/denature solution and the plates were incubated for 30 min at room temperature. The solution was replaced with 100 μL/well of the Anti-BrdU-POD working solution, and the plates were incubated at room temperature for 90 min. After three washes with 200 μL/well of washing solution, 100 μL/well of substrate solution was added and the plates were incubated at room temperature for 10 min before the reaction was stopped with 25 μL/well of 1N H2SO4 with shaking for 1 min at 300 rpm. Absorbance was read at 450 nm using a PHERAstar FS plate reader (BMG). The compound effect was calculated as percent inhibition of compound-treated cells versus nontreated PHA-stimulated cells after

subtraction of the background signal. The IC50 of each compound was calculated using XLfit software and a four-parameter logistic curve fit. Transporter Interactions. OATP Assay. CHO-vector and CHOOATP1B1 cells were seeded at a density of 30000 cells/well of a 96well plate (Costar 3904). The cells were cultured in DMEM/F12 + 10% FBS + 2 mM Ala-Gln + 1% Pen-Strep for 2 days, and the medium was changed 1 day postseeding. Prior to the assay, the medium was aspirated, the cells were washed ∼3× with HBSS + 10 mM HEPES, and the solution was equilibrated for 15 min in HBSS + 10 mM HEPES. Solutions containing the inhibitor were prepared in HBSS + 10 mM HEPES (up to 1% DMSO). At time = 0, the wash solution was aspirated and the substrate (8-fluorescein-cAMP, 2.5 μM) or inhibitor (0−30 μM) solution was added to each well. After 10 min, the substrate or substrate/inhibitor solution was aspirated and the cells were washed 3× with HBSS + 10 mM HEPES. The amount of the transported probe substrate was determined by fluorescent photometry after lysis, and the IC50 was derived from the concentration− response curve. MRP2 Assay. Human MRP2 inhibition was tested using a BD Gentest transporter vesicle assay kit. Inside-out vesicles were prepared from insect Sf9 cells infected with baculovirus expressing hMRP2. The ATP-dependent uptake of the MRP2 probe substrate 5(6)-carboxy2′,7′-dichlorofluorescein (CDCF) was measured by the difference between uptake in the presence of ATP and in the presence of AMP. The percent remaining of ATP-dependent uptake activity was calculated as uptake in the presence of inhibitor divided by the uptake in the absence of inhibitor. The IC50 values were calculated as described by the vendor according to the equation IC50 = [(50% − Low % Inhibition)]/[ (High % Inhibition − Low % Inhibition)].

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ASSOCIATED CONTENT

S Supporting Information *

Assay details, synthetic procedures, and characterization data for compounds 27−35. Crystallography parameters for 4TQT. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Structural data for the cocrystal structure of 33 and rat cyclophilin D has been deposited with the RSC Protein Data Bank (code: 4TQT), http://www.pdb.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 510-879-9392. E-mail: zachary.sweeney@novartis. com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Editor and reviewers for helping us to improve the content of the manuscript during the review process. We acknowledge Kyoko Uehara for performing some of the SPR experiments.

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ABBREVIATIONS USED OATP1B1, organic anion transporting polypeptide 1B1; MRP2, multidrug resistance-associated protein 2; MDR1, multidrug resistance protein 1; CsA, cyclosporin A; TBDPS, tertbutyl diphenylsilyl; BrdU, bromodeoxyuridine; PHA, phytohemagglutinin; CYP, cytochrome p450; PBMC, peripheral mononuclear blood cells L

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