Discovery of Hepatitis C Virus NS3-4A Protease Inhibitors with

Article pubs.acs.org/jmc

Discovery of Hepatitis C Virus NS3-4A Protease Inhibitors with Improved Barrier to Resistance and Favorable Liver Distribution Benoît Moreau,*,† Jeff A. O’Meara,† Josée Bordeleau,† Michel Garneau,‡ Cedrickx Godbout,† Vida Gorys,† Mélissa Leblanc,† Elisia Villemure,† Peter W. White,‡ and Montse Llinàs-Brunet† †

Department of Medicinal Chemistry and ‡Department of Biological Sciences, Research and Development, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada S Supporting Information *

ABSTRACT: Given the emergence of resistance observed for the current clinical-stage hepatitis C virus (HCV) NS3 protease inhibitors, there is a need for new inhibitors with a higher barrier to resistance. We recently reported our rational approach to the discovery of macrocyclic acylsulfonamides as HCV protease inhibitors addressing potency against clinically relevant resistant variants. Using X-ray crystallography of HCV protease variant/ inhibitor complexes, we shed light on the complex structural mechanisms by which the D168V and R155K residue mutations confer resistance to NS3 protease inhibitors. Here, we disclose SAR investigation and ADME/PK optimization leading to the identification of inhibitors with significantly improved potency against the key resistant variants and with increased liver partitioning.

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INTRODUCTION

new HCV drugs with alternative mechanisms are approved, the standard therapy will become an IFN-α-sparing regimen comprising two or more DAA.12 In combination with other DAAs, next generation protease inhibitors with a higher barrier to resistance might significantly improve the response rate and/ or simplify treatment regimens. We recently reported crystal structures of HCV NS3 protease inhibitors bound to variants and compared the interactions with the 1/wild type complex.13 The analysis provided an understanding of the structural mechanism by which the variants confer resistance to inhibitors of HCV NS3 protease. When compound 1 is bound to wildtype NS3 protease, the Arg155 and Asp168 residues form a salt bridge that desolvates Arg155 and form a hydrophobic surface to which the quinoline moiety of the inhibitor can bind. In the mutated protease, the Arg155 and Asp168 salt bridge is disrupted, leading to the loss of the binding surface. This observation led to the design of compounds that are conformationally flexible to adapt to amino acid variation within the binding pocket and that predominantly capitalize on highly conserved residues. We reported the discovery of macrocyclic acylsulfonamides as HCV NS3 protease inhibitors addressing potency against clinically relevant resistant variants.13 Here, we disclose the SAR investigation and ADME/PK optimization leading to the identification of inhibitors with

The hepatitis C virus (HCV), identified in 1989,1 infects about 130−170 million individuals worldwide and is a major cause of chronic liver disease.2 The HCV NS3 protease, an essential protein required for the virus replication, was validated in 2002 as an attractive target in a clinical trial with inhibitor ciluprevir (BILN 2061).3 In 2011, the first generation of HCV direct acting antivirals (DAA), protease inhibitors boceprevir4 and telaprevir,5 which contain α-keto amide warheads, were approved by the FDA. These inhibitors must be administered in combination therapy with ribavirin (RBV) and injectable pegylated interferon-α (PEG IFN-α) in order to prevent the rapid emergence of resistance. In phase III trials, for treatment naive genotype 1 infected patients, these drugs combinations improved the SVR from 45% to approximately 67−75%.6,7 Second generation NS3 protease inhibitors, which have ionic interactions with the active site, are in advanced clinical trials. Among these, 1 (faldaprevir, BI 201335)8 is a C-terminal carboxylic acid developed by Boehringer Ingelheim and is currently completing phase III clinical trials. Common resistant variants for genotype 1 patients have been observed for 1 in the clinic. For genotype 1b, mutations encode changes at Asp168, most frequently to valine (D168V). For genotype 1a, the most common substitution occurs on the Arg155 residue to lysine (R155K), while substitutions of the Asp168 residue are also observed at higher doses.9 There is a significant overlap in the variants observed in the clinic for other second generation inhibitors. These substitutions are consistent with in vitro resistance studies performed in genotype 1a and 1b replicons.10,11 Given the well-known interferon side effects and the emergence of resistant variants, it is expected that when © XXXX American Chemical Society

Special Issue: HCV Therapies Received: January 25, 2013

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dx.doi.org/10.1021/jm400121t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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bound to wild-type,17 the modulation of the quinoline and the N-terminal capping group, which are in proximity to the mutation sites, should have the greatest impact on the resistance profile of the inhibitors. Therefore, we focused on the SAR of the quinoline moiety and the N-terminal capping group of compound 2 in order to assess the potential to improve on the potency versus the variant relicons. A strategy was designed for the synthesis of the inhibitors to allow modifications at these two sites. All inhibitors were synthesized from the previously reported macrocyclic brosylate 21. Inhibitors 2−20 were prepared as outlined in Scheme 1 (see also Supporting Information).18 Displacement of the brosylate with 2-alkoxy-4-hydroxyquinoline derivatives 22a−c in the presence of cesium carbonate followed by hydrolysis of the carboxylic acid and subsequent acylsulfonamide formation with 23a,b19 yielded compounds 2−4 and intermediate 24, respectively. After removal of the Boc protecting group under acidic conditions from the appropriate intermediate (2, 3, or 24), the resulting crude amine salt was reacted with the corresponding chloroformate to give carbamates 5 and 6 or with the corresponding acid under coupling conditions to afford amides 7−20. The synthesis of 2-alkoxy-4-hydroxyquinoline derivatives 22a−c was carried out as outlined in Scheme 2. Aniline 25 was treated with imidate 26, prepared from ethyl cyanoacetate and the required alcohol. The resulting intermediate 27 was carefully heated to produce 2-alkoxy-4hydroxyquinoline derivatives 22a−c. The impact of the 2-alkoxyquinoline substitution (compounds 2−4, Table 2) on potency against D168V is modest (within 2-fold), but a greater shift in potency against R155K is observed (entries 1−3). Small C-2 substitutions provided improved potency but resulted in a somewhat lower permeability and a considerably lower metabolic stability. In contrast, the size of the N-terminal carbamate (compounds 3, 5, and 6) plays a significant role on the potency against D168V in gt 1b, whereas no impact is observed on R155K gt 1a potency. Primary alkyl carbamates 6 provided improved potency compared to bulkier carbamates 3 and 5. In correlation with the binding mode of the inhibitor,13 the C-2 quinoline modifications have a more significant impact on the potency against the R155K variant, while N-terminal carbamate modifications mostly affect the potency against D168V, and the effect is more pronounced. Primary alkyl carbamates were then compared to amide groups (entries 6 and 7). The primary butyrylamide 7 provides similar potency to the corresponding carbamate but suffered from a decreased permeability. As was

significantly improved potency against the key resistant variants and with increased liver partitioning.

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RESULTS AND DISCUSSION Our first objective was to identify a HCV NS3 protease inhibitor class with enhanced potency against key resistant mutations relative to 1 (Table 1). The in-house collection of Table 1. Comparison of in Vitro Profiles of 1 and Compound 1

EC50 (nM) compd

WT gt 1b

WT gt 1a

D168V gt 1b

R155K gt 1a

HLM T1/2 (min)

Caco-2 AB (10−6 cm·s−1)

1 2

3.1 0.4

6.5

5700 150

5000 40

193 49

8.2 5.5

protease inhibitors was profiled against two distinct replicons containing the targeted resistance mutations: D168V in genotype 1b and R155K in genotype 1a. We selected inhibitors that possessed smaller, more compact carbamates and/or smaller P2 proline substituents relative to 1.13 This approach led to the identification of compound 1, a tripeptide macrocycle with an acylsulfonamide active site binding group and a simplified P2-alkoxyquinoline substituent. A C-terminal acylsulfonamide was originally shown to be a replacement for the carboxylic acid by the BMS group14 and the Uppsala group.15,16 Compound 2 exhibited a 125-fold improvement in potency for the R155K gt 1a variant compared to 1 and a 40-fold improvement for the D168V gt 1b variant. From a comparison of the in vitro profiles, 1 showed increased human liver microsomal stability and higher permeability. Thus, compound 2 represented an attractive lead structure but required improvements in both potency and ADME properties. Throughout lead optimization, a particular focus was placed on improving permeability and metabolic stability. On the basis of the structural information provided by the analysis of 1 Scheme 1. Synthesis of Compounds 2−20 and Intermediate 24a

Conditions: (a) Cs2CO3, 22a−c, NMP, 70 °C; (b) 1 M LiOH, THF, MeOH; (c) i-BuCOCl, Et3N, 23a or 23b, LiHMDS; (d) HCl/dioxanes; (e) corresponding alkyl chloroformate, Et3N, THF, or corresponding acid, TBTU or HATU, Et3N, DMF.

a

B



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Scheme 2. Synthesis of 2-Alkoxy-4-hydroxyquinoline Derivatives 23a−ca

a

Conditions: (a) R2OH, Et2O, HCl(g) 0 °C to rt; (b) R2OH, 40 °C, 4 h; (c) Ph2O, 230 °C, 8−10 min.

Table 2. Summary of Preliminary SAR at the C-2 Quinoline and N-Terminal Capping Group

Table 3. Optimization of Potency against D168V gt 1b and R155K gt 1a (Compounds 11−23)

the case with carbamates, the introduction of bulkier alkyl substitutions was in general not well tolerated and only amides with small groups were tolerated (results not shown). Among the best amides identified, compound 8 incorporating the 1H3-pyrazole stood out for its improved metabolic stability but lacked permeability. Throughout this exercise, addressing potency against the R155K gt 1a mutation proved to be more challenging than for D168V gt 1b. The 1H-3-pyrazole amide capping group was then combined with the bulkier isopropyloxy C-2 quinoline substitution in an effort to improve on permeability (Table 3, compound 9). In addtition to an improved metabolic stability, an interesting improvement in potency was observed for R155K gt 1a, providing the first inhibitor with single-digit nanomolar potency against both resistance variants. Compound 9 demonstrated desirable potency and metabolic stability, but permeability remained to be improved. A wide range of small heterocycles were then explored and were found to provide single-digit nanomolar potency against mutant D168V in gt 1b. We first identified the 5-methyl-2-thiophene amide 10, which provided 3-fold improvements in potency against the mutants and much higher permeability. The presence of a methyl group in a 1,3substitution pattern with regard to the amide carbonyl proved to be beneficial for potency. We then further screened small

five-membered ring heterocycles to identify the optimal heterocycle, regioisomer, and substitution pattern (compounds 11−16). The key methyl group could be attached to either carbon or nitrogen, but a 1,2-substitution pattern and larger heterocycles were on average not well tolerated (results not shown). The position of the heteroatom in the ring also played a crucial role for potency. Replacement of the thiophene moiety by the different thiazole regioisomers 11−13 while keeping the 1,3-methyl to amide relationship resulted in a 5- to 25-fold loss in potency against both resistance variants. However, the Nmethylation of pyrazole 9 to install the crucial 1,3-methyl amide substitution resulted in compound 14, which showed a superior potency profile. As with the thiazoles, a significant loss in potency was observed with the alternative pyrazole amide regioisomer 15. Overall, a strong impact of heteroatom C



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Table 4. Pharmacokinetic Profile of Compounds 17−20 in Rata oral 17 18 19 20 a

iv

RLM t1/2 (min)

Cmax (μM)

AUC (μM·h)

t1/2 (h)

Vss (L/kg)

CL (mL min−1 kg−1)

F (%)

[liver], 8 h (μM)

liver Kp

169 190 138 107

0.88 0.30 1.3 0.87

1.3 0.36 1.4 1.4

5.6 2.0 2.6 3.3

3.3 0.86 0.97 2.0

16 33 19 22

11 6.1 14 16

11 6.5 56 73

360 323 >1500 >1500

10 mg/kg po (1% MP, 0.3% Tween-80, and Methocel 0.5%); 4 mg/kg iv (70% PEG-400 and 30% water).

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CONCLUSIONS In summary, starting from compound 1, a macrocyclic acylsulfonamide, we designed inhibitors with improved potency against the key resistant mutations D168V gt 1b and R155K gt 1a. This series of inhibitors must have the 2-alkoxyquinoline at P2, the macrocyclic ring, and the acylsulfonamide to have adequate potency against the key resistant mutations. Acyclic inhibitors containing the same features are much less potent against these variants as are the corresponding macrocyclic carboxylic acids. The modulation of the capping group proved to be essential to achieve single-digit nanomolar potency against the resistance variants. During our optimization of the ADME properties, compounds with higher permeability were identified and selected for further profiling in rats. On the basis of their improved potency and overall profile, including high liver partition, compounds 19 and 20 were identified as suitable follow-ups for compound 1. Compound 19 was further profiled against other genotypes and resistance studies were performed. The X-ray structures of compound 19 in complex with variants were also studied.13 We believe that HCV protease inhibitors displaying superior potency against clinically relevant variants, such as compound 19, in combination with other direct-acting antivirals will lead to further improvements in therapy for HCV patients.

positioning on potency is observed. This can be partially rationalized from the crystal structure of compound 18 bound to the mutant proteases13 in which the C4-H of N-terminal methylpyrazole forms a CH···O interaction with the backbone oxygen of Ala-157. Heterocycles with acidic C4 hydrogen have the strongest interaction. The best heterocycles identified from this exercise were the 1N-methyl-3-pyrazole and the 2-methyl3-triazole, leading respectively to compounds 14 and 16. These two inhibitors with similar profiles met our desired potency and metabolic stability criteria, but permeability needed improvement. Our strategy to improve on permeability was to slightly increase lipophilicity on the N-alkyl substituent of the heterocyclic capping group or at the sulfonamide moiety. We observed that pyrazole capping groups with perfluorinated ethyl chains, as exemplified by compounds 17 and 18, provided a slight improvement in permeability while retaining similar potency profile. Interestingly, these modifications appreciably improved metabolic stability without changes in the CYP 450 inhibition profile. For the sulfonamide substitution, very steep SAR was found and only the cyclopropyl and α-methylcyclopropyl substitutions were tolerated for potency. The addition of an α-methyl substitution to the cyclopropylsulfonamide is well tolerated in terms of potency but resulted in a slightly decreased metabolic stability. However, it has a very beneficial effect on the permeability of these inhibitors. For the N-methyl pyrazole 19 and the N-methyltriazole 20, this resulted in a substantial gain in permeability that was coupled with a 3-fold gain in potency against both mutants. Overall, compounds matching the desired potency criteria were identified and could be divided into two groups. On one hand, fluorinated alkyl heterocyclic analogues 17 and 18 demonstrated improved metabolic stability but moderate permeability, whereas α-methylcyclopropylsulfonamide analogues 19 and 20 showed higher permeability but moderate metabolic stability. In order to differentiate between the selected compounds and further profile the best analogue, pharmacokinetic studies in rats were performed (Table 4). Compound 18 demonstrated higher clearance and lower bioavailability compared to the other three compounds. Compounds 17, 19, and 20 showed similar plasma exposure, clearance, and bioavailability. In an evaluation of the pharmacokinetics for this class of inhibitors, a particular focus was placed on the concentration of the inhibitor in the liver, the predominant site of viral replication.20 It was previously reported that clinical efficacy of HCV antivirals was better predicted using the liver-corrected inhibitory quotient (LCIQ, Kp*Cmin/EC50),21 Of the four inhibitors in Table 4, compounds 19 and 20 were found to have significantly greater Kp, and thus, these emerged as the preferred compounds from this lead optimization project.

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EXPERIMENTAL SECTION

General. All commercially obtained solvents and reagents were used as received without further purification. All reactions were carried out under an atmosphere of argon. Temperatures are given in °C. Solution percentages express a weight to volume relationship, and solution ratios express a volume to volume relationship, unless stated otherwise. NMR spectra were recorded on a Bruker Avance II (400 MHz for 1HNMR) spectrometer and were referenced to either DMSO-d6 (2.50 ppm) or CDCl3 (7.27 ppm). Mass spectra were obtained from a Waters Acquity SQD/TQD instrument using electrospray as ionization mode. Purification of crude material was performed either by flash column chromatography or by using a CombiFlash Rf Teledyne ISCO using RediSep silica or SilicaSep columns according to preprogrammed gradient and flow rate separation conditions in EtOAc/hexane or MeOH/DCM. The final compounds were purified either by column chromatography to yield the neutral compound or by preparative HPLC in a Waters 2767 sample manager with pump 2545, column fluidics organizer (SFO), PDA detector 2996 or mass detector 3100, and Mass Lynx 4.1 using either a Sunfire prep C18 OBD column, 19 mm × 50 mm, 5 μm and a linear gradient program of MeCN/water (0.1%TFA), or a XBridge prep C18 OBD column, 19 mm × 50 mm and a linear gradient program of water (10 nM ammonium bicarbonate, pH 10) and MeOH. Fractions were analyzed by analytical HPLC, and the pure fractions were combined, concentrated, frozen, and lyophilized to yield the desired compound as the trifluoroacetate salt in the case where TFA is used as buffer. The lyophilized TFA salt was dissolved in water, and either 2 or 10 M NaOH was added dropwise until a pH of 14 was maintained. The pH was readjusted to 7 with 2 M HCl and the neutral product extracted into EtOAc. The combined extracts were washed D



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the combined organics were washed with 3 × H2O and 1 × brine, dried over MgSO4, filtered, and concentrated in vacuo to give the corresponding acid (5.90 g, 100%) which was used without further purification. Step 3. The acid was dissolved in DCM (55 mL). Triethylamine (3.85 mL, 27.7 mmol) was added, and the solution was cooled to 0 °C in an ice bath. Isobutyl chloroformate (1.63 mL, 12.6 mmol) was added dropwise, and the mixture was stirred at 0 °C for 1 h and then allowed to warm slowly to room temperature and stirred overnight. The mixture was concentrated in vacuo. The residue was taken up in THF/Et2O (1:1, 100 mL), and the mixture was filtered through Celite to remove the salts. The mother liquor was concentrated to dryness to provide azalactone (5.67 g, 100%), which was used as such without further purification. Step 4. Sulfonamide 23a (922 mg, 7.61 mmol, 2.60 equiv) was dissolved in THF (25 mL) and cooled to −20 °C. LiHMDS (1.0 M solution in THF, 6.24 mL, 6.24 mmol, 2.14 equiv) was added all at once. The mixture was stirred at −20 °C for 5 min and then allowed to warm to room temperature for 20 min. The mixture was then recooled to −20 °C. The azalactone from above (1.97 g, 2.92 mmol) was dissolved in THF (20 mL) and added dropwise over 1 h to the sulfonamide anion solution. After the addition was completed, the mixture was allowed to warm to room temperature and the solution stirred overnight. Glacial HOAc (350 μL) was added, and the reaction mixture was concentrated to dryness. The material was purified by flash chromatography using 25−55% EtOAc/hexanes as the eluent. The pure fractions were combined and concentrated in vacuo to provide 1 as a white solid (1.75 g, 75%). 1H NMR (400 MHz, chloroform-d) δ 10.13 (s, 1H), 7.84 (d, J = 9.19 Hz, 1H), 6.91−7.00 (m, 1H), 6.61 (br s, 1H), 5.75 (q, J = 8.80 Hz, 1H), 5.60 (spt, J = 6.19 Hz, 1H), 5.25 (br s, 1H), 5.02 (t, J = 9.39 Hz, 2H), 4.51−4.69 (m, 2H), 4.27−4.39 (m, 1H), 3.97−4.04 (m, 1H), 3.94 (s, 3H), 2.88−2.96 (m, 1H), 2.54 (s, 6H), 2.27−2.36 (m, 1H), 1.78−2.00 (m, 3H), 1.31− 1.61 (m, 28H). FIA MS (electrospray): 798.5 (M + H)+. Intermediate 24. 24 was prepared analogously to compound 1 by performing the last step with sulfonamide 23b instead of sulfonamide 23a. Sulfonamide 23b (2.40 g, 25.1 mmol) was dissolved in THF (100 mL) and cooled to −20 °C. LiHMDS (1.0 M solution in THF, 21.8 mL, 21.8 mmol) was added all at once. The mixture was stirred at −20 °C for 5 min and then allowed to warm to room temperature for 20 min. The mixture was then recooled to −20 °C. The azalactone from above (5.67 g, 8.38 mmol) was dissolved in THF (40 mL) and added dropwise over 1 h to the sulfonamide anion solution. After the addition was completed, the mixture was allowed to warm to room temperature and the solution stirred overnight. Glacial HOAc (2.0 mL) was added, and the reaction mixture was concentrated to dryness. The material was purified by flash chromatography using 25−55% EtOAc/hexanes as the eluent. The pure fractions were combined and concentrated in vacuo to provide 23 as a white solid (6.11 g, 90%). 1H NMR (400 MHz, CDCl3) δ 10.03 (br s, 1H), 7.84 (d, J = 9.00 Hz, 1H), 6.91−7.02 (m, 1H), 6.75 (br s, 1H), 6.02 (s, 1H), 5.69−5.79 (m, 1H), 5.60 (spt, J = 6.23 Hz, 1H), 5.21−5.34 (m, 1H), 4.99−5.13 (m, 2H), 4.54−4.69 (m, 2H), 4.34 (t, J = 7.83 Hz, 1H), 3.97−4.05 (m, 1H), 3.94 (s, 3H), 2.56−2.76 (m, 2H), 2.25−2.35 (m, 1H), 2.12 (s, 3H), 2.07 (s, 3H), 1.75−2.00 (m, 4H), 1.25−1.68 (m, 27H). FIA MS (electrospray): 812.6 (M + H)+. Compound 6. Boc protected macrocyclic amine 2 (40 mg, 0.051 mmol) was charged in a vial with a 4 N solution of HCl in dioxane (3 mL). Solution was stirred at room temperature for 1 h, after which the solution was evaporated to dryness to provide the amine hydrochloride which was used as such. 1-propyl chloroformate (15 mg; 0.077 mmol, 1.2 equiv) was dissolved in THF (3.0 mL), TEA (28 μL; 0.20 mmol, 4.0 equiv) and the amine hydrochloride were added. The reaction was stirred at room temperature overnight. The resulting solution was filtered through a Millex filter and purified by preparative HPLC (X-Bridge column, 10 nM Ammonium Bicarbonate pH 10: MeOH). The pure fractions were combined, concentrated, frozen and lyophilized to provide compound 6 (24 mg; 61%). FIA MS. (electrospray) for C38H51N5O10S: 770.4 (M + H)+ . Reverse phase HPLC Homogeneity @ 220 nm (0.1% TFA; CH3CN: H20):

with water (2×) and brine (1×), dried over MgSO4, filtered, and evaporated to dryness. The obtained solid was dissolved in acetonitrile/water, frozen, and relyophilized to provide the neutral product. The neutral inhibitor HPLC purity was measured by using a Waters Alliance 2695 separation module with a Waters 2487 dual λ absorbance detector. A Sunfire C18, 4.6 mm × 30 mm, 3.5 μm column was used. Linear gradient at 220 and 254 nm was as follows: 5−100% B; solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/MeCN. All compounds tested in vitro were determined to be of >95% purity by HPLC. Cell-Based Replicon Assay. The bicistronic luciferase reporter replicon, encoding the Con1 genotype 1b or H77 1a NS2-NS5B coding region, as well as resistant variants of these, and the experimental procedures for measuring EC50 values have been described elsewhere.10 Briefly, compounds were incubated with cells for 72 h, and the relative levels of luciferase present were determined using the Bright-Glo luciferase substrate (Promega) on a Packard Topcount instrument, and EC50 values were determined by the nonlinear regression. In Vitro ADME Measurements. Compound stability in human and rat liver microsomes and permeability in Caco-2 cells were evaluated as previously described.22 Pharmacokinetics. Pharmacokinetic studies were performed as previously described,17 an oral vehicle consisting of 1% N-methyl-2pyrrolidone, 0.5% Methocel, and 0.3% Tween-80 and an iv vehicle consisting of 70% PEG-400/30%. Synthesis of 4-Hydroxy-7-methoxy-8-methyl-2-isopropoxyquinoline 22a. A solution of ethyl cyanoacetate (120 g, 1.06 mol) and isopropanol (70.1 g, 1.16 mol, 1.10 equiv) in anhydrous diethyl ether (1.00 L) was cooled to 0 °C. This solution was purged with HCl gas for 45 min, and then the reaction mixture was warmed to ambient temperature and stirred for 20 h. The solvent was removed in vacuo. The residue was triturated with hexanes, collected by filtration, and dried in vacuo to yield imidate hydrochloride (81 g, 34% yield). To this intermediate (81.0g, 386 mmol) were added 3-methoxy-2methylaniline (53.0 g, 386 mmol) and isopropanol (800 mL). This mixture was stirred at 40 °C for 3.5 h. The solvent was removed in vacuo, and the remaining residue was dissolved in EtOAc (1.5 L) and washed with brine (500 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 27 (R2 = isopropyl) (128 g) which was used in the next step without further purification. In a 1 L round-bottom flask, 27 (128 g) was dissolved in diphenyl ether (600 mL), and this mixture was quickly heated (heating mantle) to 230 °C. The temperature was kept between 230 and 245 °C for 8 min. The reaction mixture was then cooled to room temperature, passed through a pad of silica gel (∼1 kg), and washed with hexanes to remove the diphenyl ether. The column was then eluted with a 20−80% EtOAc in hexanes to afford 22a (40 g, 42% yield for two steps). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.07 (s, 1H), 7.84 (d, 1H, J = 9.2 Hz), 7.14 (d, 1H, J = 9.2 Hz), 6.04 (s, 1H), 5.44 (S, 1H, J = 6.3 Hz), 3.89 (s, 3H), 2.41 (s, 3H), 1.34 (d, 6H, J = 6.3 Hz). FIA MS (electrospray): 246.2 (M − H)−, 248.2 (M + H)+. Synthesis of Compound 2. Step 1. Brosylate 21 (for synthesis see Supporting Information) (10.0 g, 14.3 mmol) and hydroxyquinoline 22a (3.90 g, 15.8 mmol) were dissolved in NMP (150 mL). Cs2CO3 (9.33 g, 28.6 mmol) was added, and the mixture was heated to 70 °C for 8 h. The solution was cooled to room temperature and stirred an additional 8 h. The mixture was diluted with EtOAc and washed with 3 × H2O, 2 × NaHCO3 (sat.), 1 × 1.0 M NaOH, 2 × H2O, and 1 × brine. The organics were dried over MgSO4, filtered, and concentrated in vacuo. The material was purified by flash chromatography using 30−40% EtOAc/hexanes as the eluent. Step 2. The product containing fractions were combined and concentrated in vacuo to give a white solid (5.94 g, 59%). The solid (5.94 g, 8.38 mmol) was dissolved in THF/MeOH (2/1, 120 mL), and 1 M NaOH (67.0 mL, 67.0 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness and the residue taken up in EtOAc/H2O. The two phase mixture was acidified to pH ≈ 5 with 10% citric acid. The aqueous phase was extracted with 3 × EtOAc, and E



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98.5%, RT = 5.98 min. 1H NMR (400 MHz,DMSO-d6): δ = 11.06 (s, 1H), 8.80 (s, 1H), 7.88 (d, 1H, J = 9.0 Hz), 7.43 (d, 1H, J = 6.6 Hz), 7.08 (d, 1H, J = 9.0 Hz), 6.35 (s, 1H), 5.61 (m, 1H), 5.42 (s, 1H), 5.10 (t, 1H, J = 9.8 Hz), 4.50−4.40(m, 4H), 4.35 (m, 1H), 3.71 (t, 1H, J = 6.9 Hz), 2.92−2.87 (m, 1H), 2.68- 2.57 (m, 2H), 2.52−2.28 (m, 10H), 1.76−0.90 (m, 20H), 0.80 (t, 3H, J = 7.4 Hz). Compound 9. Boc protected macrocyclic amine 1 (100 mg, 0.136 mmol) was charged in a vial with a 4 N solution of HCl in dioxane (3.0 mL). The solution was stirred at room temperature for 1 h, after which the solution was evaporated to dryness to provide the amine hydrochloride which was used as such. 1H-Pyrazole-3-carboxylic acid (18.3 mg, 0.163 mmol, 1.20 equiv) was dissolved in DMF (2.0 mL). Triethylamine (75.9 μL, 0.545 mmol, 4.0 equiv) and HATU (62.1 mg, 0.163 mmol, 1.20 equiv) were added, and the mixture was stirred for 15 min. The Boc deprotected macrocyclic amine hydrochloride was dissolved in DMF (2.0 mL) and added to the acid solution. The mixture was stirred at room temperature overnight. The resulting solution was filtered through a Millex filter and purified by preparative HPLC (X-Bridge column, 10 nM ammonium bicarbonate, pH 10, MeOH). The pure fractions were combined, concentrated, frozen, and lyophilized to provide compound 9 as a white amorphous solid (107 mg, 99%). FIA MS (electrospray): 790.1 (M − H)−, 792.2 (M + H)+ . Reverse phase HPLC homogeneity at 220 nm (0.1% TFA, CH3CN/ H2O): 100%, tR = 5.247 min. 1H NMR (400 MHz, DMSO-d6): δ = 11.05 (s, 1H), 8.79 (s, 1H), 8.04 (bs, 1H), 7.82 (d, 1H, J = 9 Hz), 7.74 (d, 1H, J = 2.0 Hz), 7.07 (d, 1H, J = 9.0 Hz), 6.71 (s, 1H), 6.37 (s, 1H), 5.61 (dd, 1H, J = 8.3, 18.4 Hz), 5.49 (p, 1H, J = 5.8 Hz), 5.45 (bs, 1H), 5.14 (dd, 2H, J = 9, 10.2 Hz), 4.62−4.54 (m, 2H), 4.34 (dd, 1H, J = 7.0, 9.8 Hz), 4.02 (dd, 1H, J = 3.5, 11.3 Hz), 3.87 (s, 3H), 2.94−2.88 (m, 1H), 2.62- 2.56 (m, 2H), 2.43 (s, 3H), 2.38−2.31 (m, 2H), 2.01− 1.92 (m, 1H), 1.80−1.75 (m, 1H), 1.58−1.52 (m, 3H), 1.44−1.36 (m, 10H), 1.31−1.20 (m, 2H), 1.11−0.97 (m, 4H). Compound 19. Intermediate 24 (2.55 g, 3.14 mmol) was charged in a vial. Then a 4 N solution of HCl in dioxane (40.0 mL, 160 mmol) was added. The solution was stirred at room temperature for 2 h, after which a precipitate had formed. The solution was evaporated to dryness to give the amine hydrochloride. 1-Methyl-1H-pyrazole 4carboxylic acid (475 mg, 3.77 mmol, 1.20 equiv) was dissolved in DMF (10 mL). Then triethylamine (2.19 mL, 15.7 mmol, 5.0 equiv) was added followed by TBTU (1.43 g, 3.77 mmol, 1.20 equiv). The solution was stirred for 15 min, after which the amine hydrochloride was added in DMF (10 mL) and the solution was stirred at room temperature for 16 h. Work up was the following: Water (25 mL) was added. The organic layer was extracted with EtOAc (3 × 50 mL) and dried over MgSO4. The solvent was evaporated. The residue was purified on CombiFlash (25−70% EtOAc/hexane). The pure fractions were combined, concentrated, frozen, and lyophilized to provide compound 19 (1.90 g, 74%). FIA MS (electrospray): 820 (M + H)+. Reverse phase HPLC homogeneity at 220 nm (0.1% TFA, CH3CN/ H2O): 99%. 1H NMR (400 MHz,DMSO-d6): δ = 10.83 (s, 1H), 8.93 (s, 1H), 7.83 (d, 1H, J = 8.8 Hz), 7.79 (d, 1H, J = 7.0 Hz), 7.77 (d, 1H, J = 2.1 Hz), 7.08 (d, 1H, J = 9.0 Hz), 6.60 (d, 1H, J = 2.3 Hz), 6.37 (s, 1H), 5.66−5.58 (m, 1H), 5.62 (S, 1H J = 6.2 Hz), 5.47−5.44 (m, 1H), 5.07 (dd, 1H, J = 9.5, 9.2 Hz), 4.64−4.54 (m, 1H) 4.52 (d, 1H, J = 11.6 Hz), 4.40 (dd, 1H, J = 9.7, 7.1 Hz), 4.02 (dd, 1H, J = 11.8, 3.5 Hz), 3.89 (s, 3H), 3.88 (s, 3H), 2.66−2.57 (m, 1H), 2.51 (s, 3H), 2.43 (s, 3H), 2.38−2.29 (m, 2H), 2.02−1.90 (m, 1H), 1.88−1.77 (m, 1H), 1.58 (dd, 1H, J = 8.2, 5.1 Hz), 1.52 (dd, 1H, J = 9.3, 5.2 Hz), 1.45− 1.35 (m, 12H), 1.34−1.20 (m, 4H), 0.93−0.84 (m, 2H). Synthesis of 2-Methyl-2H-1,2,3-triazole-4-carboxylic Acid. Step 1. Methyl cyanoformate (1.00 g, 11.7 mmol) was charged in a flask and dissolved in THF (40.0 mL). Then a 0.6 M diazomethane solution in Et2O (58.8 mL, 35.3 mmol, 3.00 equiv) was added. This solution was stirred at room temperature for 16 h. Water (40 mL) and EtOAc (40 mL) were added, and then the layers were separated. The solvent was evaporated and purification was performed on Combiflash (20−100% hexane) to provide the 3,4-regioisomer (654 mg, 39%) and the desired 2,4-regioisomer (434 mg, 26%) as a clear yellow oil. 1H NMR (400 MHz,CDCl3): δ 8.05 (s, 1H), 4.28 (s, 3H) 3.96 (s, 3H). FIA MS (electrospray): 142.2 (M + H)+.

Step 2. 2-Methyl-2H-1,2,3-triazole-4-carboxylic acid methyl ester (263 mg, 1.86 mmol) was charged in a round-bottom flask. Then a mixture containing THF (15.0 mL), 1 M solution NaOH (9.30 mL, 9.30 mmol, 5.0 equiv), and MeOH (5.00 mL) was added. The solution was stirred at room temperature. After 4 h, 1 N HCl was added (10.0 mL) and the solvent was evaporated. EtOAc was added, and layers were separated. Solvent was evaporated. 2-Methyl-2H-1,2,3-triazole 4carboxylic acid was obtained as a white solid (215 mg, 91%). 1H NMR (400 MHz, DMSO-d6): δ 8.17 (s, 1H), 4.22 (s, 3H). FIA MS (electrospray): 128.0 (M + H)+. Compound 20. Boc protected amine 24 (90 mg, 0.12 mmol) was dissolved in DCM (4.0 mL), and a commercial solution of 4 N HCl in dioxane (4.0 mL, 4.0 mmol, 33 equiv) was added. This mixture was stirred at room temperature for 60 min and then concentrated under reduced pressure to afford a residue corresponding to the unprotected amine. The crude intermediate was dissolved in DMF (1.5 mL) along with TEA (81 μL, 0.58 mmol, 5.0 equiv), 2-methyl-2H-1,2,3-triazole 4carboxylic acid (18 mg, 0.14 mmol, 1.2 equiv), and TBTU (53 mg, 0.14 mmol, 1.2 equiv). The solution was stirred at room temperature for 16 h. The crude mixture was filtered with a Millex filter and purified directly by preparative HPLC. The appropriate fractions were combined, frozen, and lyophilized to give compound 20 as a white lyophilized solid (37 mg, 39%). 1H NMR (400 MHz,DMSO-d6): δ 10.80 (s, 1H), 8.92 (s, 1H), 8.07 (s, 1H), 8.35 (d, 1H, J = 6.7 Hz), 7.85 (d, 1H, J = 8.8 Hz), 7.10 (d, 1H, J = 8.8 Hz), 6.35 (s, 1H), 5.67−5.59 (m 1H), 5.53−5.43 (m, 2H), 5.10−5.03 (m, 1H), 4.62−4.52 (m, 2H), 4.45−4.39 (m, 1H), 4.20 (s, 3H), 4.06−3.98 (m, 1H), 3.89 (s, 3H), 2.69−2.66 (m, 1H), 2.44 (s, 3H), 2.40−2.31 (m, 2H), 2.06−1.94 (m, 1H), 1.89−1.76 (m, 1H), 1.62−1.49 (m, 2H), 1.47−1.35 (m, 14H), 1.34−1.20 (m, 5H), 0.94−0.83 (m, 2H). FIA MS (electrospray): 821.5 (M + H)+; 819.3 (M − H)−.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed synthesis of macrocycle 21, quinolines 22b,c, carbamates 2−6, and amides 7−20. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (450) 682-4641. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors express their gratitude to the biology, analytical, and structural research department members who contributed to the generation of the data. The authors also thank Drs. Richard Bethell and Michael Cordingley for their guidance and support during this work.

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ABBREVIATIONS USED ADME, administration, distribution, metabolism, and excretion; AUC, area under the curve; CYP, cytochrome P 450; DCM, dichloromethane; DAA, direct acting antiviral; DIPEA, diisopropylethylamine; DMAP, dimethylaminopyridine; DMSO, dimethylsulfoxide; CDCl3, deuterated chloroform; EtOAc, ethyl acetate; HCV, hepatitis C virus; HPLC, high pressure liquid chromatography; PEG, polyethylene glycol; PK, pharmacokinetic; LCIQ, liver-corrected inhibitory quotient; LiHMDS, lithium bis(trimethylsilyl)amide; MES, 2-(Nmorpholino)ethanesulfonic acid; SVR, sustained virological response; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TCEP, tris(2-carboxyethyl)F



Article

(13) O’meara, J.; Lemke, C. T.; Godbout, C.; Kukolj, G.; Lagacé, L.; Moreau, B.; Thibeault, D.; White, P. W.; Llinàs-Brunet, M. Molecular mechanism by which a potent hepatitis C virus NS3-NS4A protease inhibitor overcomes emergence of resistance. J. Biol. Chem. 2013, 288, 5673−5681. (14) Campbell, J. A.; Good, A. Hepatitis C Tripeptide Inhibitors. WO 2002/060926 A2, 2002. (15) Johansson, A.; Poliakov, A.; Akerblom, E.; Wiklund, K.; Lindeberg, G.; Winiwarter, S.; Danielson, U. H.; Samuelsson, B.; Hallberg, A. Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): a comparative study of different C-terminals. Bioorg. Med. Chem. 2003, 11, 2551−2568. (16) Ronn, R.; Sabnis, Y. A.; Gossas, T.; Akerblom, E.; Danielson, U. H.; Hallberg, A.; Johansson, A. Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS3. Bioorg. Med. Chem. 2006, 14, 544−559. (17) Lemke, C. T.; Goudreau, N.; Zhao, S.; Hucke, O.; Thibeault, D.; Llinas-Brunet, M.; White, P. W. Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335. J. Biol. Chem. 2011, 286, 11434−11443. (18) Yee, N. K.; Farina, V.; Houpis, I. N.; Haddad, N.; Frutos, R. P.; Gallou, F.; Wang, X. J.; Wei, X.; Simpson, R. D.; Feng, X.; Fuchs, V.; Xu, Y.; Tan, J.; Zhang, L.; Xu, J.; Smith-Keenan, L. L.; Vitous, J.; Ridges, M. D.; Spinelli, E. M.; Johnson, M.; Donsbach, K.; Nicola, T.; Brenner, M.; Winter, E.; Kreye, P.; Samstag, W. Efficient large-scale synthesis of BILN 2061, a potent HCV protease inhibitor, by a convergent approach based on ring-closing metathesis. J. Org. Chem. 2006, 71, 7133−7145. (19) Li, J.; Smith, D.; Wong, H. S.; Campbell, J. A.; Meanwell, N. A.; Scola, P. M. A facile synthesis of 1-substituted cyclopropylsulfonamides. Synlett 2006, 2006, 725−728. (20) Pal, S.; Shuhart, M. C.; Thomassen, L.; Emerson, S. S.; Su, T.; Feuerborn, N.; Kae, J.; Gretch, D. R. Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo. J. Virol. 2006, 80, 2280−2290. (21) Duan, J.; Bolger, G.; Garneau, M.; Amad, M.; Batonga, J.; Montpetit, H.; Otis, F.; Jutras, M.; Lapeyre, N.; Rheaume, M.; Kukolj, G.; White, P. W.; Bethell, R. C.; Cordingley, M. G. The liver partition coefficient-corrected inhibitory quotient and the pharmacokinetic− pharmacodynamic relationship of directly acting anti-hepatitis C virus agents in humans. Antimicrob. Agents Chemother. 2012, 56, 5381− 5386. (22) Duan, J.; Yong, C. L.; Garneau, M.; Amad, M.; Bolger, G.; De, M. J.; Montpetit, H.; Otis, F.; Jutras, M.; Rheaume, M.; White, P. W.; Llinas-Brunet, M.; Bethell, R. C.; Cordingley, M. G. Cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV genotype 1 NS3/4A protease inhibitor. Xenobiotica 2012, 42, 164−172.

phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tris, tris(hydroxymethyl)aminomethane

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Discovery of Hepatitis C Virus NS3-4A Protease Inhibitors with

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