Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor

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Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle Richard L. Mackman,*,† Victoria A. Steadman,‡ David K. Dean,‡ Petr Jansa,† Karine G. Poullennec,‡,∥ Todd Appleby,† Carol Austin,‡ Caroline A. Blakemore,‡,⊥ Ruby Cai,† Carina Cannizzaro,† Gregory Chin,† Jean-Yves C. Chiva,‡ Neil A. Dunbar,‡ Hans Fliri,§ Adrian J. Highton,‡ Hon Hui,† Mingzhe Ji,† Haolun Jin,† Kapil Karki,† Andrew J. Keats,‡ Linos Lazarides,‡ Yu-Jen Lee,† Albert Liclican,† Michael Mish,† Bernard Murray,† Simon B. Pettit,‡ Peter Pyun,† Michael Sangi,† Rex Santos,† Jonathan Sanvoisin,‡ Uli Schmitz,† Adam Schrier,† Dustin Siegel,† David Sperandio,† George Stepan,† Yang Tian,† Gregory M. Watt,‡,# Hai Yang,† and Brian E. Schultz† †

Gilead Sciences Inc., 333 Lakeside Drive, Foster City, California 94404, United States Selcia Ltd., Fyfield Business and Research Park, Fyfield Road, Ongar, Essex CM5 0GS, United Kingdom § Cypralis Ltd., Babraham Research Campus, Cambridge CB22 3AT, United Kingdom ‡

S Supporting Information *

ABSTRACT: Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition (Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

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INTRODUCTION Cyclophilins are a family of peptidyl-prolyl isomerases that are involved in a variety of biological processes.1,2 They are known to be mediators of the immunosuppressive effects of cyclosporin A (CsA), which acts by simultaneously binding to calcineurin and the cyclophilin (Cyp) isomerase active site.3,4 The modified CsA analogue, NIM-811, is an inhibitor of Cyp isomerase activity but does not bind calcineurin, making it a valuable tool for studying the non-immunosuppressive biology of Cyp isomerases.5,6 These studies have implicated Cyp’s in a variety of diseases ranging from asthma to viral replication.7−10 Indeed, several non-immunosuppressive cyclosporin analogues discovered after NIM-811, e.g., alisporivir and SCY-635, have been investigated in clinical trials as potential therapies for hepatitis C virus (HCV) infection.11,12 Alisporivir, © 2018 American Chemical Society

the furthest advanced, demonstrated an antiviral effect in HCV-infected patients, but was halted following toxicity observations when dosed in combination with ribavirin and pegylated interferon.13 The HCV NS5a protein was identified as the binding target of intracellular Cyp A, one member of the family of human cyclophilins, and although the exact mechanism of viral inhibition remains to be elucidated, it is presumed that interference of the Cyp A and HCV NS5a protein interaction prevents viral replication.14−18 Cyp inhibitors have also been demonstrated to reduce hepatitis B virus (HBV) infection through interaction with the hepatocyte sodium taurocholate cotransporting polypeptide (NTCP) Received: May 20, 2018 Published: August 3, 2018 9473

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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membered ring. These modifications improved binding affinity and anti-HCV replicon potency in addition to reducing human plasma protein binding. In the second phase, the C1 lactone and C13 lactam groups were switched along with changing from isoquinoline to quinoline, which introduced an intramolecular hydrogen bond. This resulted in significantly improved membrane permeability leading to high oral absorption. The final phase included modification of the C14 substituents to a spiro-1,3-dioxane that lowered log D, reduced oxidative metabolism, and also reduced pregnane X receptor (PXR) activation, which was a liability with respect to drug− drug interactions. Compound 3 has high binding affinity, a tractable synthesis, and high oral bioavailability in rat and dog. While 3 is a potential oral development candidate for antiHCV combination regimens, it also has utility in interrogating the role of Cyp inhibition in a variety of other preclinical disease models.

surface receptor that is required for entry of HBV particles into hepatocytes.19,20 Despite the broad therapeutic potential of Cyp inhibition, progress in the field has been hampered by the fact that potent Cyp inhibitors are derived from structurally complex natural products such as CsA and sanglifehrins.21−26 While semisynthesis strategies have been successfully utilized to improve upon these natural products, this approach fundamentally limits the structure−activity relationship (SAR) investigations necessary for improving oral pharmacokinetic (PK) properties. Synthetic small molecule inhibitors have been identified from screening campaigns, but, to the best of our knowledge, no highly potent, small molecule with oral properties has been reported to date.27,28 Our interest in Cyp inhibition stemmed from the potential to administer an oral Cyp A inhibitor in combination with other oral anti-HCV agents as part of a pan genotype, all oral, anti-HCV regimen. It was anticipated that inhibition of a host enzyme such as Cyp A would confer a high barrier to resistance and broad, pan-genotypic activity. We therefore initiated an effort to discover an orally bioavailable, small molecule Cyp A inhibitor that would have a low propensity for drug−drug interactions. Our first report29 detailed efforts based on the sanglifehrin A macrocycle, following encouraging reports of anti-HCV activity of a sanglifehrin derived semisynthetic analogue AAE931.30 This work culminated in the identification of leads 1 and 2 (Figure 1) that had potent Cyp inhibition and

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RESULTS AND DISCUSSION A time-resolved fluorescence resonance energy transfer (TRFRET) biochemical assay assessed binding affinity of the compounds toward Cyp A.29 The HCV replicon genotype 2a assay in huh-7 cells served as a cell-based functional assay (EC50) of Cyp inhibition. The free fraction (% free) in human plasma was determined using equilibrium dialysis between human plasma and buffer. The ratio of cell culture media (CCM) % free and human plasma % free was used to calculate the human plasma-adjusted (pa) EC50. For example, 2 is 59% free in CCM/buffer and 12% free in human plasma/buffer resulting in a 4.9-fold human plasma/CCM shift and a calculated paEC50 ≈ 1.6 μM. Exposures of 2 exceeding 1.6 μM would therefore be required to maintain Cyp inhibition above 50%, so significant improvements in the paEC50 potency of 2 were desirable. Oral absorption potential was assessed in vitro using log D and Caco-2 assays, while clearance was optimized using predictions from in vitro microsomal stability assays. In vivo correlations to the in vitro PK properties were determined in rat, dog, and cynomolgus monkey following intravenous and oral dosing. In the discovery of 2, the binding mode of the macrocycle to Cyp A was elucidated wherein the styrene group forms a πstacking interaction with arginine 55 (Figure 2A).29 This new interaction enabled the m-tyrosine to be replaced by alanine, thereby reducing molecular weight and hydrogen bond donor count without a loss in potency. Given the importance of the arginine 55 π-stacking interaction, our initial efforts to optimize 2 explored a variety of aromatic replacements of styrene, including biphenyl, phenol, and bicyclic heteroaryls.31−33 This culminated in the identification of isoquinolines 4−9 (Scheme 1). Analogues 4−9 were synthesized using methods similar to those reported earlier, employing ring closing metathesis (RCM) in the key macrocyclization step (Scheme 1).29 The valine terminus of tripeptide 10 was N-BOC deprotected and the amine coupled with unsaturated acid precursors (e.g., 14). The trichloroethyl (TCE) ester was then hydrolyzed and the acid was re-esterified with isoquinoline 17 to generate the required RCM precursors 12a−e. The acid 14 was prepared in three steps from Oppolzer’s sultam 13, using methods similar to those described previously for 2.29 The chiral (R)hydroxyalkyl isoquinoline, 17, was prepared in a Stille coupling, acid hydrolysis, and Noyori reduction sequence, that generated 16 stereoselectively, followed by a final Stille

Figure 1. Simplified sanglifehrin macrocycles that bind to Cyp A.

modest ant-HCV activity but poor pharmacokinetics. Here we describe the structure-guided optimization of these leads into a potent and orally bioavailable Cyp A inhibitor 3 (Figure 1). A pivotal part of our strategy was to maintain a tractable convergent synthesis that employed readily accessible building blocks. The key advances from 2 to 3 are described in three phases, beginning with the isoquinoline replacement of the styrene in 2 and a reduction in macrocycle ring size to a 219474

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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Figure 2. (A) Overlay of sanglifehrin A (white) with lead 2 (yellow) illustrating that the piperazic acid-alanine/m-tyrosine-valine backbone atoms are positioned in a similar pose. The sanglifehrin A m-tyrosine group forms a hydrogen bond with histidine 126, and the diene is perpendicular to arginine 55, while the styrene in 2 invokes a π-stacking interaction with arginine 55 and the m-tyrosine is replaced with alanine. (B) Overlay of 2 (yellow) and the 21-membered ring macrocycle 8 (cyan) reflecting the differences upon changing the π-stacking group and the macrocycle ring size. In the smaller macrocycle 8, the isoquinoline aryl ring is positioned over arginine 55 forming the π-stacking interaction, and the C13 to C16 carbon atoms in the spacer region are situated further away from the protein surface relative to C13 to C17 of 2. A similar distance from arginine 55 is maintained for the π-stacking interactions of both 2 and 8. (C) Overlay of isoquinoline 8 (cyan) and the corresponding quinoline 19 (purple) illustrating a close overlap of the macrocycle; the most significant difference is the orientation of the trans double bond. (D) Overlay of quinoline 19 (purple) and its C14 gem-dimethyl analogue 20 (orange) illustrating the positioning of the C13−C16 atoms closer to the protein surface and the C14 pro-R methyl group forming a lipophilic interaction with threonine 73. (E) Overlay of C1 lactone/C13 lactam 20 (orange), with C1 lactam/C13 lactone 34 (pink), illustrating that the respective switch of the C1 and C13 groups leads to a similar binding mode. (F) Overlay of C14 spiro-1,3-dioxane 3 (green) with C14 gem-dimethyl 34 (pink) illustrating a similar binding mode of the macrocycle and positioning of the C14 substituents.

vinylation. The RCM was conducted using Hoveyda-Grubbs second generation catalyst at a concentration of 3 mM to provide 4−7 in unoptimized yields ranging from 31 to 63%. The yield was reduced to 23% for the 21-membered ring analogue 8. In example 8, the RCM substrate 12e containing the trisubstituted pent-3-enoic acid was found to be superior to but-3-enoic acid (not shown), which cyclized under similar conditions but provided a mixture of cis and trans double bond isomers in 100-fold selectivity in the replicon EC50 relative to the MT4 cell line CC50 (Table 1, column 4), a cell line sensitive to the potential cytotoxic effects of compounds. Interestingly, the smaller macrocycle 8 also displayed a significantly improved microsomal predicted clearance (Table 1, column 7), presumably due to its lower log D or, alternatively, the shorter more compact C14−C17 spacer region contributing fewer metabolic soft spots relative to 5. Saturated analogue 9 was significantly weaker in binding affinity suggesting that the conformational rigidity of the macrocycle was a critical feature of potent binding. The X-ray structure of isoquinoline 8 complexed to Cyp A was solved and compared to styrene 2 (Figure 2B).29 The tripeptide backbone that forms the key hydrogen bond interactions with the Cyp protein remains in a comparable pose to that of 2. As expected, the isoquinoline heterocycle is positioned over arginine 55 resulting in a π-stacking interaction. The most notable difference was the positioning of the C13−C16 atoms between the isoquinoline and the C13 NH, where the removal of one methylene resulted in these atoms moving away from the protein surface. Armed with the structural information on 8 and its promising profile, efforts were focused on further evaluation of this novel 21-membered macrocycle. 9475

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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Scheme 1. Synthesis of Macrocycles 4-9 via RCMa

Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C; (b) HATU, i-Pr2NEt, CH3CN, 14 (93% over two steps), pent-4-enoic acid (62% over two steps), hex-5-enoic acid (82% over two steps), hept-6-enoic acid (75% over two steps); (c) Zn, NH4OAc, H2O, THF, 86−96%; (d) EDC, 4DMAP, CH2Cl2, 17, 42−89%; (e) Hoveyda-Grubbs II, PhMe, reflux, 3 mmol (2 mmol for 12d), 23−63%; (f) EDC, HOBT, CH3CN, (E)-pent-3enoic acid, 88% over two steps from 10; (g) LiOH, THF, H2O, 95−97%; (h) 10% Pd/C, THF, EtOAc, H2, 48%; (i) TMSOTf, Et3N, CH2Cl2, crotonaldehyde, 1 M TiCl4 in CH2Cl2, −78 °C, 89%; (j) proton sponge, Me3OBF4, CH2Cl2, quant.; (k) 2 M LiOH, THF, 60 °C, 59%; (l) PdCl2(PPh3)2, tri-n-butyl(1-ethoxyvinyl)tin, PhMe, 50 °C, quant.; (m) 2 M HCl, 1,4-dioxane, 89%; (n) dichloro(p-cymene) ruthenium(II) dimer, (1R, 2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine, THF, H2O, HCO2Na, 40 °C, 92%; (o) tri-n-butyl(vinyl)tin, PdCl2(PPh3)2, 1,4-dioxane, 150 °C, 50%. a

heterocycles to be cross coupled with the different C13−C16 unsaturated acids (Scheme 2), prior to coupling with the tripeptide and final macrocyclization described in Schemes 3 and 4. Dibromo intermediate 22 was converted to a monoketone, stereoselectively reduced to the (R)-hydroxyalkyl intermediate 23 using a Noyori reduction, and then coupled with but-3-enoic acid in a Heck reaction to provide 24. The 2chloroquinoline 25 was converted to the 2-iodo analogue in situ to promote a more efficient Stille coupling with tri-nbutyl(1-ethoxyvinyl)tin, and then the product was subsequently hydrolyzed to the monoketone. Noyori reduction of the ketone generated the (R)-hydroxyalkyl intermediate 26, which was then coupled with the respective unsaturated ester/ acid in a Heck reaction to provide 27 and 28 respectively. The gem dimethyl isoquinoline precursor 29 was formed in a Suzuki cross coupling between 2-chloroisoquinoline 16 and the gemdimethyl boronate ester 3131 followed by ester hydrolysis. The macrolactonization at C1 (Scheme 3, path a) began with NBOC removal in tripeptide 10 followed by HATU mediated coupling of the acid precursors 24, 28, or 29 to afford the intermediates 32a−c. The TCE ester, and hydroxyl protecting group present in 32b, were then removed to reveal the secoacid intermediates, which were cyclized by slow addition over 6−10 h to 2-methyl-6-nitrobenzoic acid anhydride (Shiina conditions36) at a maximal concentration of 3 mmol. The C14 gem-dimethyl analogues 20 and 21 were isolated in ∼50% yield

Oral dosing of 8 in dog and cynomolgus monkey revealed low oral bioavailability, likely due to its low log D and poor Caco-2 permeability. This was confirmed in an oral study in portal vein-cannulated dogs that established only 8% of a 5 mg/kg oral dose was being absorbed (data not shown). We sought to improve the permeability and oral properties of 8 through continued SAR exploration of the isoquinoline heterocycle and modifications to the C13−C16 region where modeling suggested that the C14 methylene would likely tolerate substitution. The immediate goal was to raise log D and improve permeability. Naphthyl and quinoline analogues, 18 and 19, respectively, were targeted, along with the C14 gemdimethyl analogues 20 and 21 (Table 2). The gem-dimethyl groups avoided the need for introducing chirality into this region, and it was thought they would not only raise log D but also improve permeability by shielding the polarity of the nearby C13 lactam group. We also explored the impact of switching the C1 lactone and C13 lactam groups in the context of the quinoline and isoquinoline heterocycles, analogues 34 and 35, respectively (Table 2). In the case of quinoline 34, this could potentially improve permeability through the introduction of an intramolecular hydrogen bond between the quinoline nitrogen and C1 lactam NH. Macrolactamization and macrolactonization approaches to the macrocycles were developed as alternative methods to the RCM.34,35 This strategy permitted the isoquinoline and related 9476

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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Table 1. Profile of Isoquinoline Analogues 4−9a

cpd #

TR-FRET Cyp A Kd (nM)

HCV 2a EC50 (nM)

SfAb CsAc 1 2 4 5 6 7 8 9

3.3 17 48 24 19 22 79 140 10 470

773 282 333 58 284 604 894 110 2777

MT4 CC50 (nM)

% free CCM/HP

plasma stab. t1/2 h/d/r (min)

MS pred. CL h/d (L/h/kg)

Caco-2 AB/BA (10−6 cm s−1)

log D pH 7.4

5608

14/0.5

0.77/−

29751 6490 >50000 >50000 >50000 36853 >50000

59/12 79/14 88/12

Sd/600/5 750/1047/5 Sd/ Sd/8

1.1/1.3 1.0/0.6 0.52/0.30

12/31 4.4/37 0.33/25

2.9 2.1 1.6

75/32

559/417/8

50000 >30000 >30000 >30000 26947

75/32 32/5 49/40 84/19 51/18 62/9.6 85/23

559/417/8 S/S/− 532/S/− 1023/S/15 S/S/− S/S/412 S/729/564

1500

10/10 1/5

MS pred. CL (L/h/kg) Hep pred. CL (L/h/kg) CL (L/h/kg) 1.40 0.24 0.68 0.79 0.80 0.42

Vss (L/kg)

t1/2 (h)

F%

3.9 10

90 100

24 25

100 55

0.68 0.44

0.65 0.36

2.5 3.9

1.35 0.16 0.30

0.42 0.08

12 2.3

a

Dosed in solution, formulation and dosing details are in the Supporting Information; Hep, hepatocytes; CL, clearance; Vss, steady state volume of distribution; t1/2, terminal half-life. bDogs used in this study were bile duct-cannulated in the IV arm and portal vein-cannulated in the oral arm.

Table 4. Profile of Quinoline Macrocycles 3, 47−51, and 53−54a

cpd #

TR-FRET Cyp A Kd (nM)

HCV 2a EC50 (nM)

MT4 CC50 (nM)

% free CCM/HP

MS pred. CL h/d (L/h/kg)

Caco-2 AB/BA (10−6 cm s−1)

PXR % act. 1.5/15 μMb

log D pH 7.4

34 3 47 48 49 50 51 53 54

9 5 5 4 5 11 23 8 11

98 98 47 60 87 59 355 80 320

>40000 >40000 >40000 >40000 37000 >40000 >40000 >40000 >40000

62/9.6 100/41 100/40 97/28 68/11 100/28 100/51 100/52 88/66

0.69/0.24 0.42/0.80 0.41/0.34 0.45/0.59 0.68/0.67 0.79/0.52 0.24/4fold above the paEC50 of 240 nM. Oral bioavailability was 100% in rat and 55% in dog, demonstrating good intestinal permeability properties. The superior plasma-adjusted antiHCV potency and optimal PK properties of 3 resulted in its selection as an oral candidate for in vivo efficacy studies in a variety of disease models. Moreover, the low projected clearance in humans together with the oral bioavailability data across multiple species supports the potential of 3 to also be used in the clinical setting as an oral inhibitor of Cyps.

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CONCLUSION

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EXPERIMENTAL SECTION

Cyp inhibition by oral small molecules holds significant promise for the interrogation of Cyps and their role in disease. The current report extends our previous findings on sanglifehrin based macrocyclic inhibitors resulting in the identification of the potent and orally bioavailable Cyp inhibitor 3. Compound 3 has Cyp A Kd = 5 nM, a high plasma free fraction leading to a calculated paEC50 = 240 nM, excellent oral bioavailability above 50% in two preclinical species, and minimal PXR activation potential. Critical advances during the structure driven optimization process were reducing the macrocycle size and combining a quinoline heterocycle with a C1 lactam and C13 lactone switch that dramatically improved permeability through formation of an intramolecular hydrogen bond. Late stage elaboration of the C14 substituent to a spirocyclic1,3-dioxane lowered log D, reduced PXR activation, and improved metabolic stability resulting in the identification of 3 as the best oral inhibitor. Importantly, 3 can be derived from three readily available precursors following a short four-step sequence. Synthesis developments have since been introduced beyond this report to generate 3 on a 100−1000 g scale to enable its use in a variety of preclinical disease models. These advances and results from the models will be reported in due course.

All research compounds were synthesized at Gilead Sciences Inc. (Foster City, CA, USA) or Selcia Ltd. (Ongar, UK). Commercially available solvents and reagents were used as received without further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300, 400, or 500 MHz NMR or a Varian Mercury Plus 400 MHz NMR. Chemical shifts are reported in ppm (δ) using the residual solvent line as internal standard (CDCl3: δ 7.26, CD3OD: δ 3.31, CD3CN: δ 1.94 DMSO-d6: δ 2.50). Splitting patterns

Figure 3. Macrocycle 3 synthesis schematic. 9483

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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1.02 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 580 [M + H], Tr = 4.36 min. (53S,2R,7S,10S,E)-10-Isopropyl-2,7-dimethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclohexadecaphan-15-ene-4,6,9,12-tetraone (5). A solution of 12b (180 mg, 0.32 mmol) in toluene (100 mL) was stirred at rt under N2. Hoveyda-Grubbs second generation catalyst (20 mg, 0.032 mmol) was added, and the mixture was heated at reflux under N2 for 2 h. Additional Hoveyda-Grubbs second generation catalyst (10 mg, 0.016 mmol) was added, and the reaction mixture was heated at reflux for 1 h. The reaction mixture was cooled to rt and filtered. The majority of the filtrate was concentrated in vacuo, silica gel was added, and the mixture was further concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with EtOAc/acetone (1:0 to 5:1). The residue was triturated with Et2O and EtOAc, and the resulting solid was collected and dried to afford the title compound (66 mg, 39%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.13 (s, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.76 (s, 1H), 7.67 (s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 6.80−6.65 (m, 2H), 6.42 (d, J = 8.5 Hz, 1H), 6.31 (d, J = 9.6 Hz, 1H), 6.09 (q, J = 6.6 Hz, 1H), 5.65−5.55 (m, 1H), 4.55 (br d, 1H), 4.44−4.38 (m, 1H), 3.64−3.60 (m, 2H), 2.90−1.75 (m, 10H), 1.67 (d, J = 6.5 Hz, 3H), 1.54 (d, J = 7.1 Hz, 3H), 1.00 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). LC/ MS Method A (m/z) 536 [M + H], Tr = 1.33 min. HRMS C29H37N5O5 calculated 535.2795, found 535.2806. (53S,2R,7S,10S,E)-10-Isopropyl-2,7-dimethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacycloheptadecaphan-16-ene-4,6,9,12tetraone (6). 12c (209 mg, 0.362 mmol) was subjected to the procedure for 5 at a concentration of 3 mmol to afford the title compound (125 mg, 63%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 9.00 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 6.64 (m, 1H), 6.29 (d, J = 15.4 Hz, 1H), 5.97 (q, J = 5.9 Hz, 1H), 5.53 (q, J = 7.0 Hz, 1H), 4.37−4.18 (m, 2H), 3.65 (d, J = 9.8 Hz, 1H), 2.65 (t, J = 12.3 Hz, 1H), 2.36− 2.18 (m, 4H), 1.98 (m, 1H), 1.94−1.73 (m, 3H), 1.71−1.57 (m, 3H), 1.55 (d, J = 5.6 Hz, 6H), 0.83 (d, J = 6.6 Hz, 3H), 0.78 (d, J = 6.6 Hz, 3H). LC/MS Method C (m/z) 550.2 [M + H], Tr = 2.55 min. HRMS C30H39N5O5 calculated 549.2951, found 549.2972. (53S,2R,7S,10S,E)-10-Isopropyl-2,7-dimethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclooctadecaphan-17-ene-4,6,9,12-tetraone (7). 12d (255 mg, 0.431 mmol) was subjected to the procedure for 5 at a concentration of 2 mmol to afford the title compound (76 mg, 31%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 9.01 (s, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.57 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 6.55 (m, 1H), 5.95 (m, 1H), 5.43 (m, 1H), 5.23 (m, 3H), 4.32 (m, 1H), 4.20 (m, 1H), 3.67 (m, 1H), 2.69 (m, 1H), 2.30 (m, 2H), 2.13 (m, 2H), 1.96 (m, 3H), 1.83 (m, 1H), 1.64 (m, 3H), 1.56 (d, J = 6.7 Hz, 3H), 1.44 (d, J = 7.4 Hz, 3H), 0.92−0.72 (m, 6H). LC/MS Method C (m/z) 564.4 [M + H], Tr = 2.60 min. HRMS C31H41N5O5 calculated 563.3108, found 563.3121. (53S,2R,7S,10S,E)-10-Isopropyl-2,7-dimethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12tetraone (8). 12e (1.13 g, 2 mmol) was subjected to the general RCM method for 5 to afford the title compound (245 mg, 23%) containing ∼10% cis isomer. 1H NMR (300 MHz, CDCl3) δ 9.13 (s, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.72 (s, 1H), 7.55 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 6.72 (d, J = 16.3 Hz, 1H), 6.53−6.38 (m, 3H), 6.04 (q, J = 6.7 Hz, 1H), 5.76−5.65 (m, 1H), 4.57−4.53 (m, 1H), 4.29−4.23 (m, 1H), 3.77−3.63 (m, 2H), 3.40−3.20 (m, 2H), 2.70−1.75 (m, 6H), 1.71 (d, J = 6.9 Hz, 3H), 1.53 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H). LC/MS Method A (m/z) 522.0 [M + H], Tr = 1.40 min. HRMS C28H35N5O5 calculated 521.2638, found 521.2651. (53S,2R,7S,10S)-10-Isopropyl-2,7-dimethyl-51,52,53,54,55,56hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclopentadecaphane-4,6,9,12-tetraone (9). A mixture of 8 (16 mg, 0.03 mmol) in EtOAc (5 mL) and THF (2 mL) was treated with 10% palladium on carbon (15 mg), and the mixture was

are designated as s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; br, broad; ABq, AB quartet; app, apparent. Compound 3 13 C NMR assignment can be found in Supporting Information. All isolated characterized final compounds for assay were determined to have a purity of >95% at 254 nM by HPLC (see Supporting Information). High resolution mass spectrometry (HRMS) and low resolution (unit mass) liquid chromatography/mass spectrometry (LC/MS) details are provided in the Supporting Information. Retention times are denoted as Tr. Preparative normal phase silica gel chromatography was carried out manually or using a Teledyne ISCO CombiFlash Companion instrument with silica gel cartridges.. All in vivo studies were performed in accordance with local IACUC guidelines. (2R,3S,7S,10S,E)-10-Isopropyl-2,7-dimethylspiro[11-oxa3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanene-13,5′-[1,3]dioxan]-14-ene-4,6,9,12-tetraone (3). 73 (398.4 mg, 0.745 mmol), 68a (117.9 mg, 0.745 mmol), tri(otolyl)phosphine (45.3 mg, 0.149 mmol), and Et3N (0.32 mL, 2.235 mmol) were mixed in 1,4-dioxane (15 mL) and degassed with N2 for 5 min. The mixture was heated to 50 °C and treated with tris(dibenzylideneacetone)dipalladium(0) (68.2 mg, 0.074 mmol). The mixture was stirred at 100 °C for 40 min, cooled to rt, and filtered through a pad of Celite, and the Celite pad was rinsed with EtOAc. The mixture was concentrated in vacuo to afford the seco-acid that was used without further purification. LC/MS Method A (m/z) 612.2 [M + H], Tr = 1.70 min. A solution of the seco-acid (0.745 mmol) in anhydrous CH2Cl2 (18 mL) was added via syringe pump over 3 h to a solution of 2-methyl-6-nitrobenzoic anhydride (385.0 mg, 1.12 mmol), 4-DMAP (273.0 mg, 2.24 mmol) in anhydrous CH2Cl2 (230 mL) containing 4 Å molecular sieves. The reaction mixture was stirred at rt for 40 min, filtered, and partially concentrated in vacuo. The organic layer was washed with pH 4 citrate buffer (100 mL), a saturated solution of sodium bicarbonate (100 mL), and filtered. The mixture was concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with isohexanes/ acetone (1:0 to 1:1) to provide the title compound (134 mg, 30%). 1 H NMR (400 MHz, CD3OD) δ 8.26 (d, J = 8.5 Hz, 1H, Ar−H4), 7.85 (d, J = 8.5 Hz, 1H, Ar−H5), 7.74 (s, 1H, Ar−H8), 7.69 (dd, J = 8.5, 1.6 Hz, 1H, Ar−H6), 7.48 (d, J = 8.5 Hz, 1H, Ar−H3), 6.63 (d, J = 16.6 Hz, 1H, Ar−CHC), 6.15 (d, J = 16.6 Hz, 1H, Ar−CCH), 5.82 (q, J = 7.2 Hz, 1H, C(O)−CHNHCO), 5.36 (d, J = 9.2 Hz, 1H, C(O)−CH−O), 5.09−5.06 (m, 1H, Ar−CHN), 4.99 (d, J = 6.1 Hz, 1H, OCH2aO), 4.81 (d, J = 6.1 Hz, 1H, OCH2bO), 4.68 (dd, J = 11.3, 2.3 Hz, 1H, CH2aO), 4.51 (dd, J = 11.2, 2.3 Hz, 1H, CH2aO), 4.47−4.39 (m, 1H, CH2aN-NH), 3.88 (dd, J = 28.4, 11.3 Hz, 2H, CH2bO x 2), 3.69−3.58 (m, 1H, CHNH-N), 2.71 (td, J = 12.9, 3.2 Hz, 1H, CH2bN-NH), 2.33−2.11 (m, 2H, CH2aCHNH and CHCH− O), 2.01−1.88 (m, 1H, CH2aCH2N), 1.74−1.48 (m, 8H, CH3CHNH x 2, CH2bCH2N, CH2bCHNH), 1.11 (d, J = 6.8 Hz, 3H, CH3CHCH3), 1.06 (d, J = 6.6 Hz, 3H, CH3CHCH3). 13C NMR (126 MHz, CD3OD) δ 173.65, 171.29, 170.05, 169.60, 160.35, 146.74, 137.90, 136.57, 133.44, 128.74, 127.64, 126.94, 126.02, 122.75, 119.10, 93.46, 78.10, 70.99, 70.42, 60.73, 50.83, 49.68, 46.26, 41.35, 30.30, 25.68, 23.12, 20.64, 17.35, 17.11, 16.34. LC/MS Method A (m/z) 594.1 [M + H], Tr = 2.60 min. HRMS C31H39N5O7 calculated 593.2849, found 593.2866. (53S,2R,7S,10S,13R,14R,E)-10-Isopropyl-14-methoxy-2,7,13trimethyl-51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)isoquinolina-5(3,1)-pyridazinacyclohexadecaphan-15-ene4,6,9,12-tetraone (4). 12a (54 mg, 0.087 mmol) was subjected to the procedure for 5 at 3 mmol to afford the title compound (18 mg, 36%) as a white solid. 1H NMR (300 MHz, CD3CN) δ 9.20 (s, 1H), 8.15 (br s, 1H), 8.02 (d, J = 8.5 Hz, 1H), 8.00 (br s, 1H), 7.52 (dd, J = 8.5, 1.6 Hz, 1H), 7.33 (d, J = 9.2 Hz, 1H), 7.20 (dd, J = 16.3, 8.5 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 16.3 Hz, 1H), 6.11 (q, J = 6.5 Hz, 1H), 5.80−5.69 (m, 1H), 4.41 (br d, 1H), 4.33−4.26 (m, 2H), 3.99 (dd, J = 8.2, 2.9 Hz, 1H), 3.73−3.64 (m, 1H), 3.41 (s, 3H), 2.75−2.65 (m, 1H), 2.55−2.47 (m, 1H), 2.15−1.65 (m, 5H), 1.61 (d, J = 6.7 Hz, 3H), 1.47 (d, J = 7.1 Hz, 3H), 1.35 (d, J = 7.4 Hz, 3H), 9484

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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stirred under H2 for 3 h. The reaction mixture was degassed and filtered through filter aid, and the filter pad was washed with EtOAc. The filtrate was concentrated in vacuo, and the residue was triturated with Et2O (2 × 2 mL). The solid was dried to afford the title compound (7.6 mg, 48%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 9.17 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.80 (s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.34 (s, 1H), 6.33 (d, J = 7.8 Hz, 1H), 6.11−6.05 (m, 2H), 5.92−5.83 (m, 1H), 4.49−4.44 (m, 1H), 4.37−4.32 (m, 1H), 3.90 (d, J = 11.8 Hz, 1H), 3.75−3.68 (m, 1H), 3.10−2.70 (m, 3H), 2.40−1.60 (m, 9H), 1.68 (d, J = 6.5 Hz, 3H), 1.50 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 524.3 [M + H], Tr = 0.59 min. HRMS C28H37N5O5 calculated 523.2795, found 523.2810. 2,2,2-Trichloroethyl (S)-1-(((2R,3R)-3-Methoxy-2-methylpent-4-enoyl)-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (11a). A solution of 1029 (2.17 g, 4.09 mmol) in anhydrous CH2Cl2 (50 mL) was cooled to 0 °C, treated with TMSOTf (1.2 mL, 6.81 mmol), and stirred for 30 min. N,N-Diisopropylethylamine (2.4 mL, 13.63 mmol) was then added, and the mixture was concentrated in vacuo. The residue was treated with CH3CN (50 mL), 14 (539.0 mg, 3.407 mmol), and N,N-diisopropylethylamine (2.4 mL, 13.63 mmol). The reaction mixture was cooled to 0 °C and 2-(1H-7azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (1.814 g, 4.770 mmol) was added. The mixture was stirred at rt for 16 h, quenched with 1 M HCl (100 mL), and extracted with EtOAc (2 × 100 mL). The organic extracts were combined, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (1:0 to 1:4) to afford the title compound (2.19 g, 93%) as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ 6.76 (d, J = 7.3 Hz, 1H), 5.72 (dq, J = 15.1, 6.7 Hz, 1H), 5.57 (d, J = 8.5 Hz, 1H), 5.38−5.19 (m, 2H), 4.96 (d, J = 11.8 Hz, 1H), 4.72 (d, J = 11.8 Hz, 1H), 4.44−4.30 (m, 2H), 3.85 (d, J = 11.2 Hz, 1H), 3.70 (td, J = 10.3, 3.4 Hz, 1H), 3.56 (app t, J = 8.5 Hz, 1H), 3.24 (s, 3H), 2.95− 2.84 (m, 1H), 2.34 (app p, J = 7.6 Hz, 1H), 2.25−2.09 (m, 2H), 2.00−1.86 (m, 1H), 1.82−1.62 (m, 2H), 1.76 (d, J = 6.4 Hz, 3H), 1.32 (d, J = 6.9 Hz, 3H), 1.08 (d, J = 7.1 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). LC/MS Method A1 (m/z) 571.1 [M + H], Tr = 5.00 min. 2,2,2-Trichloroethyl (S)-1-(Pent-4-enoyl-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (11b). 10 (1.06 g, 2 mmol) was combined with pent-4-enoic acid (220 mg, 2.2 mmol) as described for the preparation of 11a. The crude 11b was subjected to silica gel chromatography eluting with a gradient of isohexanes/ EtOAc (3:1 to 1:3) to afford the title compound (634 mg, 62%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 6.66 (d, J = 7.6 Hz, 1H), 6.18 (d, J = 8.7 Hz, 1H), 5.92−5.78 (m, 1H), 5.36−5.26 (m, 1H), 5.10 (dd, J = 17.2, 1.6 Hz, 1H), 5.06 (dd, J = 10.3, 1.6 Hz, 1H), 4.97 (d, J = 12.0 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.48−4.30 (m, 2H), 3.88−3.66 (m, 3H), 3.00−2.88 (m, 1H), 2.50−1.60 (m, 8H), 1.32 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 513/515 [M + H], Tr = 4.20 min. 2,2,2-Trichloroethyl (S)-1-(hex-5-enoyl- L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (11c). 10 (1.06 g, 2 mmol) was combined with hex-5-enoic acid (251 mg, 2.2 mmol) as described for the preparation of 11a. The crude 11c was subjected to silica gel chromatography eluting with 0 to 40% EtOAc/MeOH (4:1 mixture) in isohexanes to afford the title compound (864 mg, 82%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.84 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 7.1 Hz, 1H), 5.72 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 5.28 (m, 1H), 4.97−4.84 (m, 2H), 4.91 (d, J = 12.1 Hz, 1H), 4.71 (d, J = 12.1 Hz, 1H), 4.14−4.07 (m, 1H), 3.75−3.22 (m, 3H), 2.22−2.15 (m, 2H), 1.98 (m, 4H), 1.78 (m, 2H), 1.66−1.56 (m, 3H), 1.20 (d, J = 7.0 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H). LC/MS Method C (m/z) 527.4/529.2 [M + H], Tr = 3.56 min. 2,2,2-Trichloroethyl (S)-1-(Hept-6-enoyl-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (11d). 10 (1.06 g, 2 mmol) was combined with hept-6-enoic acid (220 mg, 2.2 mmol) as described for the preparation of 11a. The crude 11d was subjected to silica gel chromatography eluting with isohexanes/(EtOAc/MeOH

4:1 mixture) (1:0 to 5:2) to afford the title compound (817 mg, 75%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 8.05−7.87 (m, 1H), 5.80 (td, J = 17.0, 6.8 Hz, 1H), 5.36 (m, 1H), 5.01 (d, J = 12.1 Hz, 1H), 5.03−4.89 (m, 2H), 4.81 (d, J = 12.1 Hz, 1H), 4.20 (m, 1H), 3.79−3.38 (m, 3H), 2.27 (m, 2H), 2.07 (m, 4H), 1.87 (m, 2H), 1.74−1.66 (m, 1H), 1.62 (m, 2H), 1.48−1.37 (m, 2H), 1.29 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H). LC/ MS Method C (m/z) 541.3/543.3 [M + H], Tr = 3.75 min. 2,2,2-Trichloroethyl (S)-1-(((E)-Pent-3-enoyl)- L -valyl- L alanyl)hexahydropyridazine-3-carboxylate (11e). Compound 10 (10.6 g, 20 mmol) in CH2Cl2 (300 mL) was stirred at 0 °C under N2 and treated dropwise with TMSOTf (6.66 g, 5.4 mL, 30 mmol). The reaction mixture was stirred at 0 °C for 1 h and then treated with cold saturated sodium hydrogen carbonate solution (200 mL). The mixture was stirred at 0 °C for 15 min, the organic layer separated, washed with saturated sodium hydrogen carbonate solution (100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue in CH3CN (240 mL) was stirred at 0 °C under N2 and treated with (E)-pent-3-enoic acid (2.20 g, 2.2 mL, 22 mmol), 1-hydroxybenzotriazole hydrate (3.82 g, 20 mmol, wetted with not less than 20 wt % water), and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (5.38 g, 28 mmol). The mixture was stirred at 0 °C for 15 min, then at rt for 20 h, and then concentrated in vacuo. The residue was partitioned between EtOAc (100 mL) and water (100 mL), and the organic layer was separated. The aqueous layer was washed with EtOAc (100 mL), and the combined organic extracts washed with water (100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (3:1 to 0:1) to afford the title compound (9.0 g, 88%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 6.69 (d, J = 7.6 Hz, 1H), 6.28 (d, J = 8.7 Hz, 1H), 5.73−5.51 (m, 2H), 5.36−5.27 (m, 1H), 4.96 (d, J = 11.8 Hz, 1H), 4.72 (d, J = 11.8 Hz, 1H), 4.40−4.27 (m, 2H), 3.88 (d, J = 11.2 Hz, 1H), 3.74−3.67 (m, 1H), 2.99 (d, J = 6.7 Hz, 2H), 2.95−2.88 (m, 1H), 2.75−1.75 (m, 5H), 1.74 (d, J = 5.4 Hz, 3H), 1.32 (d, J = 6.6 Hz, 3H), 0.93 (app t, J = 6.6 Hz, 6H). LC/ MS Method A (m/z) 513.1/515.1 [M + H], Tr = 2.59 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethyl (S)-1-(((2R,3R)-3-Methoxy-2-methylpent-4-enoyl)-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (12a). 11a (763.4 mg, 1.34 mmol) in THF (25 mL) was treated with zinc powder (1.92 g, 29.4 mmol) and a solution of ammonium acetate (1.54 g, 20 mmol) in water (5 mL). The mixture was stirred for 16 h, filtered through Celite, and then treated with 2 M HCl. The mixture was extracted with EtOAc (2 × 50 mL), and the combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was azeotroped with toluene to afford (S)-1-(((2R,3R)-3-methoxy-2methylpent-4-enoyl)-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylic acid (566 mg, 96%) as a light orange solid. 1H NMR (300 MHz, DMSO-d6) δ 7.74 (d, J = 9.1 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 5.67 (dq, J = 15.4, 6.0 Hz, 1H), 5.25−5.09 (m, 2H), 5.04 (d, J = 10.5 Hz, 1H), 4.16 (dd, J = 8.7, 6.2 Hz, 1H), 4.00−3.90 (m, 1H), 3.06 (s, 3H), 3.00−2.86 (m, 1H), 2.10−1.97 (m, 1H), 1.95−1.85 (m, 1H), 1.71 (d, J = 6.5 Hz, 4H), 1.61−1.46 (m, 2H), 1.14 (d, J = 6.9 Hz, 3H), 0.89− 0.79 (m, 9H). (S)-1-(((2R,3R)-3-methoxy-2-methylpent-4-enoyl)-Lvalyl-L-alanyl)hexahydropyridazine-3-carboxylic acid (180 mg, 0.40 mmol) and 17 (111 mg, 0.56 mmol) were added to CH2Cl2 (10 mL). The mixture was stirred at rt under N2 and treated with N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (108 mg, 0.56 mmol) and 4-DMAP (25 mg, 0.2 mmol). The mixture was stirred for 20 h, diluted with CH2Cl2, washed with saturated sodium hydrogen carbonate solution, water, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography using a gradient of isohexanes/EtOAc (1:1 to 0:1) to afford the title compound (103 mg, 42%) as a clear viscous oil. 1 H NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.76 (br s, 1H), 7.63 (br s, 1H), 7.54 (dd, J = 8.5, 1.3 Hz, 1H), 6.97 (dd, J = 17.2, 10.7 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 6.51 (d, J = 8.7 Hz, 1H), 6.41 (d, J = 17.2 Hz, 1H), 6.11 (q, J = 6.7 Hz, 1H), 5.72− 5.65 (m, 1H), 5.52 (d, J = 10.7 Hz, 1H), 5.34−5.20 (m, 2H), 4.43 (br 9485

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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d, 1H), 4.33 (dd, J = 8.7, 5.6 Hz, 1H), 3.85−3.52 (m, 3H), 3.24 (s, 3H), 2.85−2.75 (m, 2H), 2.40−1.75 (m, 5H), 1.75 (d, J = 6.2 Hz, 3H), 1.67 (d, J = 6.5 Hz, 3H), 1.31 (d, J = 6.7 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H). LC/ MS Method A (m/z) 622 [M + H], Tr = 5.15 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethyl (S)-1-(Pent-4-enoyl-Lvalyl-L-alanyl)hexahydropyridazine-3-carboxylate (12b). 11b (616 mg, 1.2 mmol) was converted to 12b according to the procedure reported for compound 12a to afford the title compound (180 mg, 58%) as a foam. 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.76 (br s, 1H), 7.62 (br s, 1H), 7.54 (dd, J = 8.5, 1.6 Hz, 1H), 6.97 (dd, J = 17.2, 10.7 Hz, 1H), 6.63 (d, J = 7.6 Hz, 1H), 6.41 (dd, J = 17.2, 1.6 Hz, 1H), 6.15−6.08 (m, 2H), 5.90−5.78 (m, 1H), 5.53 (dd, J = 10.7, 1.4 Hz, 1H), 5.35−5.23 (m, 1H), 5.09 (dd, J = 17.2, 1.6 Hz, 1H), 5.02 (dd, J = 10.2, 1.6 Hz, 1H), 4.42 (br d, 1H), 4.31 (dd, J = 8.7, 6.0 Hz, 1H), 3.82−3.76 (m, 1H), 3.62−3.55 (m, 1H), 2.83−2.77 (m, 1H), 2.50−1.90 (m, 9H), 1.67 (d, J = 6.5 Hz, 3H), 1.31 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 564 [M + H], Tr = 2.04 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethyl (S)-1-(Hex-5-enoyl-Lvalyl-L-alanyl)hexahydropyridazine-3-carboxylate (12c). A solution of 11c (830 mg, 1.57 mmol) in THF (40 mL) was treated with water (10 mL) and lithium hydroxide hydrate (57 mg, 2.38 mmol). The mixture was stirred for 2 h and then filtered through a 5 cm layer of DOWEX D50 × 8 resin in H+ cycle (resin first washed with water). The resin was washed with additional water (50 mL), the filtrates collected, concentrated in vacuo, azeotroped with toluene (2 × 10 mL), and dried in vacuo to afford (S)-1-(hex-5-enoyl-L-valyl-Lalanyl)hexahydropyridazine-3-carboxylic acid (590 mg, 95%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.84 (s, 2H), 5.72 (m, 1H), 5.24 (m, 1H), 4.93 (d, J = 16.9 Hz, 1H), 4.87 (d, J = 10.4 Hz, 1H), 4.10 (m, 1H), 3.98 (m, 1H), 3.43 (m, 1H), 2.96 (m, 1H), 2.19 (m, 2H), 1.98 (m, 4H), 1.74 (s, 1H), 1.67−1.56 (m, 4H), 1.20 (d, J = 5.9 Hz, 3H), 0.91−0.81 (m, 6H). LC/MS Method C (m/z) 397.3 [M + H], Tr = 2.43 min. The ester formation was performed as described for compound 12a using (S)-1-(hex-5-enoyl-L -valyl-L -alanyl)hexahydropyridazine-3-carboxylic acid (238 mg, 0.6 mmol) and 17 (120 mg, 0.6 mmol) to afford the title compound (307 mg, 89%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 9.17 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.92 (s, 1H), 7.86 (s, 1H), 7.81 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.15−6.87 (m, 1H), 6.31 (d, J = 17.9 Hz, 1H), 6.09 (m, 1H), 5.78 (m, 1H), 5.50 (d, J = 10.6 Hz, 1H), 5.36 (m, 1H), 5.00 (d, J = 17.3 Hz, 1H), 4.94 (d, J = 10.1 Hz, 1H), 4.18 (m, 1H), 3.89 (m, 1H), 3.72 (m, 1H), 2.25 (m, 2H), 2.05 (m, 4H), 1.81 (m, 3H), 1.66 (m, 6H), 1.31 (d, J = 6.3 Hz, 3H), 0.90 (m, 6H). LC/MS Method C (m/z) 578.3 [M + H], Tr = 3.23 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethyl (S)-1-(Hept-6-enoyl-Lvalyl-L-alanyl)hexahydropyridazine-3-carboxylate (12d). 11d (790 mg, 1.46 mmol) was converted to 12d according to the procedure reported for compound 12c to afford the title compound (316 mg, 79%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 9.06 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.71 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 6.87 (dd, J = 17.4, 10.9 Hz, 1H), 6.21 (d, J = 17.5 Hz, 1H), 5.99 (q, J = 6.3 Hz, 1H), 5.67 (m, 1H), 5.40 (d, J = 10.8 Hz, 1H), 5.26 (m, 1H), 4.87 (d, J = 17.1 Hz, 1H), 4.80 (d, J = 9.6 Hz, 1H), 4.08 (d, J = 7.0 Hz, 1H), 3.80 (s, 1H), 3.61 (m, 1H), 2.15 (m, 2H), 1.94 (m, 4H), 1.78−1.66 (m, 2H), 1.63−1.44 (m, 3H), 1.53 (d, J = 6.6 Hz, 3H), 1.29 (m, 3H), 1.21 (d, J = 6.9 Hz, 3H), 0.81 (m, 6H). LC/MS Method C (m/z) 592.2 [M + H], Tr = 3.41 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethyl (S)-1-(((E)-Pent-3-enoyl)L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (12e). 11e (9.0 g, 17.5 mmol) was converted to 12e according to the procedure reported for compound 12a to afford the title compound (3.91 g, 51%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.76 (br s, 1H), 7.62 (s, 1H), 7.53 (dd, J = 8.5, 1.6 Hz, 1H), 6.98 (dd, J = 17.4, 10.7 Hz, 1H), 6.64 (d, J = 7.4 Hz, 1H), 6.41 (dd, J = 17.2, 1.3 Hz, 1H), 6.22 (d, J = 8.5 Hz, 1H), 6.11 (q, J = 6.7 Hz, 1H), 5.71−5.57 (m, 2H), 5.53 (dd, J = 10.7, 1.3 Hz, 1H), 5.33−5.26 (m, 1H), 4.46−4.40 (m, 1H), 4.28 (dd, J = 8.7, 6.0 Hz, 1H), 3.79 (d, J = 11.6 Hz, 1H), 3.62−3.55 (m, 1H), 2.98 (d, J =

6.7 Hz, 2H), 2.85−2.78 (m, 1H), 2.16−1.86 (m, 5H), 1.74 (dd, J = 6.0, 0.9 Hz, 3H), 1.67 (d, J = 6.7 Hz, 3H), 1.31 (d, J = 6.7 Hz, 3H), 0.92 (app t, J = 6.6 Hz, 6H). LC/MS Method A (m/z) 564.3 [M + H], Tr = 2.06 min. (2R,3R,E)-3-Methoxy-2-methylhex-4-enoic Acid (14). A solution of 1329 (6.0 g, 22.1 mmol) in anhydrous CH2Cl2 (24 mL) was treated with TMSOTf (5.0 mL, 22.1 mmol), followed by Et3N (3.54 mL, 25.4 mmol). The reaction mixture was stirred under N2 at rt for 15 h. The dark solution was concentrated in vacuo, and the residue was dissolved in anhydrous CH2Cl2 (22 mL). The mixture was treated dropwise with a solution of crotonaldehyde (3.66 mL, 44.2 mmol) and 1 M titanium tetrachloride in CH2Cl2 (44.2 mL, 44.2 mmol), at −78 °C under N2. The reaction mixture was stirred at −78 °C for 1 h, treated with ammonium chloride solution (30 mL), and then allowed to warm to rt. The aqueous layer was extracted with CH2Cl2 (2 × 25 mL), and the combined organics were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/ EtOAc (4:1) to afford (2R,3R,E)-1-((3aR,6S,7aS)-8,8-dimethyl-2,2dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3hydroxy-2-methylhex-4-en-1-one (6.7 g, 89%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 5.82−5.67 (m, 1H), 5.57−5.46 (m, 1H), 3.94−3.87 (m, 1H), 3.58−3.42 (m, 2H), 3.29−3.18 (m, 1H), 2.47 (d, J = 8.5 Hz, 1H), 2.17−2.04 (m, 3H), 2.00−1.84 (m, 3H), 1.69 (d, J = 6.5 Hz, 3H), 1.46−1.32 (m, 2H), 1.24−1.17 (m, 6H), 0.98 (s, 3H). LC/MS Method A1 (m/z) 364.08 [M + Na], Tr = 3.47 min. A solution of (2R,3R,E)-1-((3aR,6S,7aS)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-hydroxy-2methylhex-4-en-1-one (4.15 g, 12.1 mmol) in anhydrous CH2Cl2 (80 mL) was treated with 1,8-bis(dimethylamino)naphthalene (7.78 g, 36.3 mmol) followed by trimethyloxonium tetrafluoroborate (3.6 g, 24.2 mmol). The reaction mixture was stirred at rt for 3 h, treated with MeOH (3 mL), stirred for another 5 min, and then 2 M HCl (200 mL) and EtOAc (250 mL) were added. The mixture was filtered, the organic layer separated, and the aqueous layer extracted with EtOAc (2 × 100 mL). The combined organic extracts were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo to afford (2R,3R,E)-1-((3aR,6S,7aS)-8,8dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol1(4H)-yl)-3-methoxy-2-methylhex-4-en-1-one (4.80 g, 100%) as a pale brown solid. 1H NMR (300 MHz, CDCl3) δ 5.79−5.65 (m, 1H), 5.34−5.22 (m, 1H), 3.90 (t, J = 6.2 Hz, 1H), 3.66 (t, J = 9.0 Hz, 1H), 3.56−3.38 (m, 2H), 3.32−3.19 (m, 1H), 3.17 (s, 3H), 2.10−2.05 (m, 3H), 2.00−1.85 (m, 3H), 1.74 (dd, J = 6.5, 1.6 Hz, 2H), 1.45−1.31 (m, 2H), 1.24−1.18 (m, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.98 (s, 3H). LC/MS Method A1 (m/z) 378.14 [M + Na], Tr = 3.76 min. A 2 M solution of lithium hydroxide (50 mL, 100 mmol) was added to a stirred solution of (2R,3R,E)-1-((3aR,6S,7aS)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-methoxy-2-methylhex-4-en-1-one (4.80 g, 12.1 mmol) in THF (130 mL). The reaction mixture was heated at 60 °C for 15 h. The reaction mixture was cooled to rt, partially concentrated in vacuo, and treated with 2 M HCl (150 mL). The mixture was extracted with EtOAc (3 × 50 mL), and the combined extracts dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/Et2O (1:1) to afford the title compound (1.132 g, 59%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 11.15 (br s, 1H), 5.82−5.67 (m, 1H), 5.30−5.18 (m, 1H), 3.65 (t, J = 8.8 Hz, 1H), 3.26 (s, 3H), 2.63−2.51 (m, 1H), 1.77 (dd, J = 6.5, 1.6 Hz, 3H), 1.10 (d, J = 7.1 Hz, 3H). (R)-1-(3-Chloroisoquinolin-6-yl)ethan-1-ol (16). A solution of 15 (972 mg, 4 mmol) and tri-n-butyl(1-ethoxyvinyl)tin (2.5 g, 2.5 mL, 7 mmol) in toluene (12 mL) was degassed with N2 for 30 min. Bis(triphenylphosphine)palladium(II) dichloride (280 mg, 0.4 mmol) was added, and the reaction mixture was heated at 50 °C for 18 h. The reaction mixture was cooled to rt and concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (19:1 to 10:1) to afford 3-chloro-6-(1ethoxyvinyl)isoquinoline (964 mg, ∼ 100%). 1H NMR (300 MHz, CDCl3) δ 8.94 (s, 1H), 8.04 (s, 1H), 7.97−7.85 (m, 2H), 7.76 (s, 9486

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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solution was heated at 50 °C. The seco-acid (20 mg, 0.037 mmol) in N,N-dimethylformamide (1 mL) was added over 10 h by syringe pump, washing the syringe pump with additional N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 50 °C for an additional 2 h and concentrated in vacuo, and the residue was subjected to preparative HPLC (0 to 100% CH3CN/water) to afford the title compound (4 mg, 22%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.84−7.74 (m, 3H), 7.56 (dd, J = 8.5, 1.7 Hz, 1H), 7.47−7.43 (m, 1H), 7.35 (dd, J = 8.5, 1.9 Hz, 1H), 6.52 (d, J = 16.3 Hz, 1H), 6.31 (ddd, J = 16.2, 6.1, 4.8 Hz, 1H), 6.05 (q, J = 6.6 Hz, 1H), 5.58 (q, J = 7.2 Hz, 1H), 4.48−4.38 (m, 1H), 4.31 (d, J = 10.0 Hz, 1H), 3.80 (dd, J = 11.3, 2.8 Hz, 1H), 3.37 (dd, J = 6.2, 1.7 Hz, 1H), 2.99 (ddd, J = 15.1, 4.7, 2.0 Hz, 1H), 2.76 (td, J = 12.8, 3.2 Hz, 1H), 2.05−1.70 (m, 5H), 1.69 (d, J = 1.7 Hz, 3H), 1.67 (d, J = 2.3 Hz, 3H), 1.03 (dd, J = 13.8, 6.7 Hz, 6H). LC/MS Method C (m/z) 521.1 [M − H], Tr = 3.18 min. HRMS C29H36N4O5 calculated 520.2686, found 520.2694. (53S,2R,7S,10S,E)-10-Isopropyl-2,7-dimethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(2,7)-quinolina5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12-tetraone (19). A solution of 33 (342 mg, 0.492 mmol) in CH2Cl2 (2.5 mL) was cooled to 0 °C and treated with TMSOTf (0.18 mL, 0.96 mmol). The mixture was stirred at 0 °C for 2.75 h, and additional TMSOTf (0.073 mL, 0.45 mmol) was then added. The mixture was stirred for another 80 min, treated with N,N-diisopropylethylamine (0.6 mL, 3.44 mmol), and stirred an additional 10 min. The mixture was concentrated in vacuo to afford the seco-acid (E)-4-(2-((R)-1(((S)-1-(L-valyl-L-alanyl)hexahydropyridazine-3-carbonyl)oxy)ethyl)quinolin-7-yl)but-3-enoic acid (266 mg, 0.45 mmol). The seco-acid was dissolved in CH3CN (50 mL) at 0 °C under N2 and treated with N,N-diisopropylethylamine (0.343 mL, 1.971 mmol) and 2-(1H-7azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (262 mg, 0.70 mmol). The mixture was stirred at 0 °C and allowed to warm to rt over 16 h. The mixture was treated with 2 M HCl (20 mL), and the aqueous layer was extracted with CH2Cl2/MeOH (9:1) (2 × 100 mL). The combined organic extracts were washed with saturated sodium hydrogen carbonate solution (2 × 200 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give a crude residue. The residue was subjected to silica gel chromatography eluting with gradient of EtOAc/acetone (97:3 to 94:6) to afford a solid, which was then triturated with Et2O to afford the title compound (25.4 mg, 10%) as a pale yellow solid. 1H NMR (300 MHz, CD3OD) δ 8.21 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.75 (dd, J = 8.7, 1.3 Hz, 1H), 7.65 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.55 (dt, J = 16.7, 4.7 Hz, 1H), 6.41 (d, J = 16.7 Hz, 1H), 5.94 (q, J = 6.9 Hz, 1H), 5.72 (q, J = 7.1 Hz, 1H), 4.43 (br d, J = 11.2 Hz, 1H), 4.23 (d, J = 10.5 Hz, 1H), 3.88− 3.79 (m, 1H), 3.40−3.35 (m, 1H), 3.07−2.97 (m, 1H), 2.75 (dd, J = 12.9, 2.9 Hz, 1H), 2.08−1.85 (m, 4H), 1.74 (d, J = 6.9 Hz, 3H), 1.72−1.66 (m, 1H), 1.64 (d, J = 7.1 Hz, 3H), 1.01 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.48 Hz, 3H). LC/MS Method A (m/z) 522.2 [M + H], Tr = 1.90 min. HRMS C28H38N5O5 calculated 520.2638, found 521.2649. (5 3 S,2R,7S,10S,E)-10-Isopropyl-2,7,13,13-tetramethyl1 2 3 4 5 6 5 ,5 ,5 ,5 ,5 ,5 -hexahydro-3-oxa-8,11-diaza-1(2,7)-quinolina5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12-tetraone (20). 32b (1.11 g, 1.50 mmol) was hydrolyzed to the seco-acid according to the method for 18 except using 4 equiv of LiOH. The seco-acid was cyclized according to the method for 18 with addition by syringe pump over 6 h reaching a concentration of 3 mmol. The crude residue was subjected to silica gel chromatography eluting with isohexanes, then isohexanes/acetone (4:1), then isohexanes/acetone (7:3), and finally isohexanes/acetone (3:2). The product was treated with brine (200 mL) and then extracted with EtOAc (2 × 100 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to a second silica gel chromatography eluting with isohexanes/acetone (95:5), then isohexanes/acetone (93:7), then isohexanes/acetone (88:12) (3 L), and finally, isohexanes/acetone (82:16). Two purest fractions were combined, concentrated in vacuo, and triturated twice with Et2O to

1H), 4.90 (d, J = 2.8 Hz, 1H), 4.45 (d, J = 2.8 Hz, 1H), 4.01 (q, J = 7.0 Hz, 2H), 1.51 (t, J = 7.0 Hz, 3H). LC/MS Method A (m/z) 234/ 236 [M + H], Tr = 5.40 min. A solution of 3-chloro-6-(1ethoxyvinyl)isoquinoline (934 mg, 4 mmol) in 1,4-dioxane (10 mL) and 2 M HCl (5 mL) was stirred at rt for 30 min. The mixture was concentrated in vacuo, and the residue was partitioned between EtOAc (30 mL) and water (30 mL). The organic layer was separated, and the aqueous phase was extracted with EtOAc (30 mL). The organic extracts were combined, washed with water, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (9:1 to 4:1) to afford 1-(3-chloroisoquinolin-6yl)ethan-1-one (732 mg, 89%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.17 (s, 1H), 8.37 (br s, H), 8.15 (dd, J = 8.5, 1.4 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.88 (s, 1H), 2.77 (s, 3H). LC/MS Method A1 (m/z) 206/208 [M + H], Tr = 4.40 min. Dichloro(pcymene)ruthenium(II) dimer (3 mg, 0.005 mmol) and (1R,2R)(−)-N-p-tosyl-1,2-diphenylethylenediamine (4.4 mg, 0.012 mmol) were suspended in N2 degassed water (2 mL), and the mixture was then further degassed with N2 for 15 min. The mixture was stirred at 70 °C under N2 for 90 min. The yellow solution was cooled to rt and 1-(3-chloroisoquinolin-6-yl)ethan-1-one (206 mg, 1 mmol), sodium formate (340 mg, 5 mmol), and degassed THF (1 mL) were added. The reaction mixture was degassed with N2 for 5 min and then vigorously stirred at 40 °C for 2.5 h. The reaction mixture was cooled to rt and EtOAc (30 mL) was added. The organic layer was separated, washed with water, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (4:1 to 2:1) to afford the title compound (193 mg, 92%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.04 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.77 (br s, 1H), 7.72 (s, 1H), 7.63 (dd, J = 8.6, 1.6 Hz, 1H), 5.16− 5.08 (m, 1H), 2.14 (d, J = 3.1 Hz, 1H), 1.60 (d, J = 5.9 Hz, 3H). LC/ MS Method A1 (m/z) 208/210 [M + H], Tr = 4.25 min. (R)-1-(3-Vinylisoquinolin-6-yl)ethan-1-ol (17). 1,4-Dioxane (5 mL) was degassed with N2, and 16 (208 mg, 1 mmol), tri-nbutyl(vinyl)tin (951 mg, 0.9 mL, 3 mmol), and bis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol) were added. The reaction mixture was heated at 150 °C in a microwave reactor for 1 h. Additional tri-n-butyl(vinyl)tin (0.3 mL, 1 mmol) and bis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol) were added, and the reaction mixture was heated at 150 °C in a microwave reactor for 1 h. The mixture was cooled to rt and filtered, and the filter pad washed with EtOAc. The filtrate was concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (4:1 to 2:1) followed by a second silica gel chromatography eluting with isohexanes/EtOAc (3:1) to afford the title compound (100 mg, 50%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.19 (s, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.78 (br s, 1H), 7.58 (dd, J = 8.5, 1.6 Hz, 1H), 7.57 (s, 1H), 6.95 (dd, J = 17.2, 10.7 Hz, 1H), 6.40 (dd, J = 17.2, 1.6 Hz, 1H), 5.52 (dd, J = 10.6, 1.6 Hz, 1H), 5.10 (q, J = 6.5 Hz, 1H), 2.11 (br s, 1H), 1.60 (d, J = 6.5 Hz, 3H). LC/MS Method A (m/z) 200 [M + H], Tr = 1.50 min. (13S,4R,11S,14S,E)-11-Isopropyl-4,14-dimethyl11,12,13,14,15,16-hexahydro-3-oxa-10,13-diaza-1(3,1)-pyridazina-5(2,7)-naphthalenacyclopentadecaphan-6-ene-2,9,12,15tetraone (18). 32a (55 mg, 0.08 mmol) was dissolved in a mixture of THF (2 mL), MeOH (1 mL), and water (1 mL). The solution was treated with lithium hydroxide hydrate (4 mg, 0.16 mmol), and the mixture was stirred at rt for 1 h. CH2Cl2 (10 mL) and water (10 mL) were added to the reaction mixture followed by 1 M HCl until pH ≈ 2. The organic layer was separated, and the aqueous layer was back extracted with CH2Cl2 (2 × 10 mL). The combined organic extracts were concentrated in vacuo to afford the seco-acid 2,2,2-trichloroethyl (S)-1-(((E)-4-(7-((R)-1-hydroxyethyl)naphthalen-2-yl)but-3-enoyl)L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (37 mg, 84%) as a white solid. LC/MS Method C (m/z) 537.2 [M − H]. 2-Methyl-6nitrobenzoic anhydride (19 mg, 0.056 mmol) and 4-DMAP (23 mg, 0.185 mmol) were dissolved in 1,2-dichloroethane (18 mL), and the 9487

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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afford the title compound ∼241 mg. The less pure fractions were subjected to preparative reverse phase HPLC, and the isolated product combined to afford the title compound (422 mg, 51%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 8.5 Hz, 1H), 7.81−7.71 (m, 2H), 7.61 (s, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 9.6 Hz, 1H), 6.46 (d, J = 16.5 Hz, 1H), 6.25 (d, J = 16.5 Hz, 1H), 5.89 (q, J = 6.8 Hz, 1H), 5.72 (qd, J = 7.2, 4.8 Hz, 1H), 4.37 (d, J = 12.1 Hz, 1H), 4.29 (t, J = 10.0 Hz, 1H), 3.83−3.73 (m, 1H), 2.72 (td, J = 12.6, 3.5 Hz, 1H), 1.97 (m, 2H), 1.88 (m, 1H), 1.71 (d, J = 6.9 Hz, 3H), 1.67 (m, 2H), 1.59 (d, J = 7.2 Hz, 3H), 1.48 (s, 3H), 1.34 (s, 3H), 0.94 (t, J = 6.3 Hz, 6H). LC/MS Method A (m/z) 550.1 [M + H], Tr = 2.24 min. HRMS C30H39N5O5 calculated 549.2951, found 549.2968. (5 3 S,2R,7S,10S,E)-10-Isopropyl-2,7,13,13-tetramethyl51,52,53,54,55,56-hexahydro-3-oxa-8,11-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12tetraone (21). 32c (1.50 g, 2.15 mmol) was hydrolyzed to the secoacid according to the method for 18 except using 4 equiv of LiOH. The seco-acid was cyclized according to the method for 18 with addition by syringe pump over 6 h reaching a concentration of 3 mmol. The residue was subjected to silica gel chromatography eluting with 35 to 65% acetone in isohexanes to afford the title compound (445 mg). The impure fractions were subjected to recrystallization from acetone/isohexanes to afford additional material and a combined yield of title compound (619 mg, 52%). 1H NMR (400 MHz, CD3OD) δ 9.13 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.89 (s, 1H), 7.55 (dd, J = 8.5, 1.6 Hz, 1H), 7.53 (s, 1H), 6.73−6.34 (m, 2H), 6.03 (q, J = 6.6 Hz, 1H), 5.63 (q, J = 7.2 Hz, 1H), 4.67 (d, J = 12.2 Hz, 1H), 4.47−4.21 (m, 2H), 3.81−3.73 (m, 1H), 2.76−2.64 (m, 1H), 2.08− 1.81 (m, 3H), 1.80−1.58 (m, 8H), 1.51 (s, 3H), 1.35 (s, 3H), 0.98 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). LC/MS Method B1 (m/z) 550.2 [M + H], Tr = 2.74 min. HRMS C30H39N5O5 calculated 549.2951, found 549.2964. (R)-1-(7-Bromonaphthalen-2-yl)ethan-1-ol (23). 2,7-Dibromonaphthalene 22 (1 g, 3.50 mmol) in anhydrous THF (18 mL), at −78 °C under N2, was treated dropwise with a solution of nbutyllithium (2.5 M in hexanes, 1.5 mL, 3.67 mmol). The mixture was stirred at −78 °C for 20 min, and then N-methoxy-N-methylacetamide (409 μL, 3.85 mmol) was added. The mixture was stirred for 15 min, warmed to rt, and stirred an additional 30 min. The reaction was quenched with 2 M HCl and extracted with CH2Cl2 (2 × 50 mL). The combined organic extracts were dried through a hydrophobic frit and concentrated in vacuo. The residue was subjected to silica flash chromatography eluting with isohexanes/EtOAc (7:1) to afford 1-(7bromonaphthalen-2-yl)ethan-1-one (650 mg, 75%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 8.15 (s, 1H), 8.06 (dd, J = 8.7, 1.6 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.69 (dd, J = 8.7, 1.8 Hz, 1H), 2.74 (s, 3H). Dichloro (pcymene) ruthenium(II) dimer (8 mg, 0.013 mmol) in water (5 mL) was treated with (1R, 2R)-(−)-N-p-tosyl-1, 2-diphenylethylenediamine (11.5 mg, 0.031 mmol). The mixture was degassed for 15 min and then heated at 70 °C for 1.5 h. The reaction was cooled and treated with a solution of 1-(7-bromonaphthalen-2-yl)ethan-1-one (650 mg, 2.61 mmol) in degassed anhydrous THF (2 mL) followed by sodium formate (874 mg, 13.1 mmol). The mixture was heated at 40 °C for 3 h, cooled to rt, and extracted with CH2Cl2 (2 × 50 mL). The combined organic extracts were dried through a hydrophobic frit and concentrated in vacuo. The product was subjected to silica flash chromatography eluting with isohexanes/EtOAc (4:1) to afford the title compound (450 mg, 69%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 1.8 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.77−7.68 (m, 2H), 7.59−7.51 (m, 2H), 5.09 (qd, J = 6.5 Hz, 3.6 Hz, 1H), 1.90 (d, J = 3.6 Hz, 1H), 1.60 (d, J = 6.5 Hz, 3H). (R,E)-4-(7-(1-Hydroxyethyl)naphthalen-2-yl)but-3-enoic Acid (24). 23 (42 mg, 0.17 mmol) was dissolved in CH3CN (2 mL) and treated with but-3-enoic acid (35 mg, 0.41 mmol), palladium(II) acetate (4 mg, 0.017 mmol), tri-(o-tolyl)phosphine (10 mg, 0.034 mmol), and Et3N (0.12 mL). The mixture was heated at 120 °C in the microwave reactor for 15 min. The reaction mixture was then filtered and concentrated in vacuo, and the residue was subjected to

silica gel chromatography eluting with MeOH/CH2Cl2 (1:3) to afford the title compound (35 mg, 81%) as a yellow solid. LC/MS Method C (m/z) 255.2 [M − H], Tr = 1.80 min. (R)-1-(7-Bromoquinolin-2-yl)ethan-1-ol (26). A slurry of 25 (8.10 g, 33.4 mmol) and sodium iodide (50.0 g, 334 mmol) in CH3CN (27 mL) was treated with acetyl chloride (3.56 mL, 50.0 mmol). The flask was sealed and heated at 80 °C for 3 h. The cooled mixture was treated sequentially with 10% w/w aqueous potassium carbonate solution (80 mL), 5% w/w aqueous sodium sulfite solution (80 mL) and saturated aqueous sodium thiosulfate solution (80 mL). The mixture was extracted with CH2Cl2 (2 × 250 mL), and the combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo to provide crude 7-bromo-2-iodoquinoline. The crude 7-bromo-2-iodoquinoline was treated with tri-n-butyl(1ethoxyvinyl)tin (13.6 mL, 40.1 mmol), 1,4-dioxane (67 mL), and bis(triphenylphosphine)palladium(II) dichloride (2.37 g, 3.34 mmol), and the reaction mixture was heated at 100 °C for 5 h. The mixture was treated with 2 M HCl (67 mL), stirred for 1 h, and the solids were removed and washed with EtOAc. The filtrate was concentrated in vacuo to remove organics, and then the aqueous solution was extracted with EtOAc (3 × 250 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of 0 to 6% EtOAc in isohexanes, and first doping the silica gel with 10% w/w potassium carbonate, to afford 1-(7-bromoquinolin-2yl)ethan-1-one 40 (5.5 g, 66%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.42 (s, 1H), 8.21 (ABq, ΔδAB = 0.11, JAB = 8.5 Hz, 2H), 7.80−7.72 (m, 2H), 2.86 (s, 3H). LC/MS Method A (m/z) 250.1/ 252.1 [M + H], Tr = 2.90 min. Dichloro (p-cymene) ruthenium(II) dimer (61 mg, 0.100 mmol) and (1R,2R)-(−)-N-p-tosyl-1,2-diphenylethylenediamine (88 mg, 0.012 mmol) were suspended in degassed water (40 mL), and the mixture was degassed with N2 for 5 min. The mixture was stirred at 70 °C for 90 min. The turbid orange mixture was allowed to cool to rt, and then 1-(7-bromoquinolin-2-yl)ethan-1-one (5.00 g, 20 mmol) in degassed THF (40 mL) was added followed by sodium formate (6.8 g, 100 mmol). The reaction mixture was degassed with N2 for 5 min and then vigorously stirred at 40 °C for 4 h. The mixture was diluted with EtOAc (100 mL) and water (100 mL), and the organic layer was separated, washed with water (100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of 0− 30% EtOAc in isohexanes to afford the title compound (4.96 g, 98%) as an off-white solid. 1H NMR (300 MHz, CDCl3) δ 8.28 (d, J = 1.6 Hz, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.64 (dd, J = 8.7, 1.6 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 5.07−5.04 (m, 1H), 4.85 (d, J = 4.5 Hz, 1H), 1.59 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 252.1/254.1 [M + H], Tr = 1.74 min. tert-Butyl (R,E)-4-(7-(1-hydroxyethyl)naphthalen-2-yl)but-3enoate (27). A solution of 26 (295 mg, 1.17 mmol) in anhydrous CH3CN (12 mL) was treated with but-3-enoic acid tert-butyl ester (0.44 mL, 2.75 mmol), bis(tritert-butylphosphine)palladium(0) (25 mg, 0.049 mmol), and N,N-dicyclohexylmethylamine (0.39 mL, 1.83 mmol). The mixture was heated at reflux for 2 h and concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (95:5) to afford the title compound (348 mg, 95%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.11 (d, J = 8.5 Hz, 1H), 7.97 (app s, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.67 (dd, J = 8.5, 1.6 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 6.69 (d, J = 16.1 Hz, 1H), 6.60−6.47 (m, 1H), 5.10−4.96 (m, 2H), 3.26 (dd, J = 1.1, 6.9 Hz, 2H), 1.59 (d, J = 6.3 Hz, 3H), 1.51 (s, 9H). LC/MS Method A (m/z) 314.2 [M + H], Tr = 1.91 min. (R,E)-4-(2-(1-Acetoxyethyl)quinolin-7-yl)-2,2-dimethylbut3-enoic Acid (28). A solution of 26 (1.00 g, 3.97 mmol) and Et3N (1.65 mL, 11.9 mmol) in anhydrous CH2Cl2 at 0 °C was treated with acetic anhydride (0.75 mL, 7.93 mmol) and 4-(dimethylamino)pyridine (24 mg, 0.197 mmol). The reaction mixture was allowed to warm to rt and stirred for 1.5 h. Water (100 mL) was added, the organic layer was separated, and the aqueous layer was back extracted with CH2Cl2 (2 × 100 mL). The combined organic extracts were 9488

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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CD3OD) δ 7.88−7.75 (m, 4H), 7.56 (dd, J = 8.8, 1.6 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 6.71 (d, J = 16.4 Hz, 1H), 6.51−6.42 (m, 1H), 5.38 (q, J = 7.6 Hz, 1H), 4.98−4.93 (m, 2H), 4.78 (d, J = 12.8 Hz, 1H), 4.25 (d, J = 10.4 Hz, 1H), 3.78−3.75 (m, 2H), 3.24 (d, J = 10.2 Hz, 2H), 2.10−2.02 (m, 2H), 1.84−1.81 (m, 2H), 1.65−1.62 (m, 1H), 1.52 (d, J = 7.2 Hz, 3H), 1.30−1.24 (m, 3H), 0.98−0.92 (m, 6H). LC/MS Method C (m/z) 668.9 [M + H], Tr = 2.17 min. 2,2,2-Trichloroethyl (S)-1-(((E)-4-(2-((R)-1-Acetoxyethyl)quinolin-7-yl)-2,2-dimethylbut-3-enoyl)- L -valyl- L -alanyl)hexahydropyridazine-3-carboxylate (32b). A solution of 10 (1.29 g, 2.42 mmol) in CH2Cl2 (12 mL) was cooled in an ice−water bath under N2. TMSOTf (0.715 mL, 4.84 mmol) was added dropwise, and the mixture stirred for 1 h. N,N-Diisopropylethylamine (1.69 mL, 9.68 mmol) was added, and the reaction mixture was concentrated in vacuo to afford (S)-1-[(S)-2-((S)-2-amino-3-methylbutyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylic acid 2,2,2-trichloro-ethyl ester as a white solid. A solution of 28 (792 mg, 2.42 mmol) in N,N-dimethylformamide (10 mL) at 0 °C was treated with N,N-diisopropylethylamine (2.11 mL, 12.1 mmol) and 2(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (1.01 g, 2.66 mmol). The mixture was stirred at rt for 15 min and then cooled to 0 °C. (S)-1-[(S)-2-((S)-2amino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3carboxylic acid 2,2,2-trichloro-ethyl ester dissolved in N,N-dimethylformamide (10.7 mL) was added, and the stirred reaction mixture was then allowed to warm to rt. The mixture was stirred for 1 h and then diluted with EtOAc (100 mL) and water (100 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (100 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/acetone (95:5) followed by isohexanes/acetone (9:1), followed by isohexanes/acetone (85:15), and finally, isohexanes/acetone (3:1) to afford the title compound (1.11 g, 62%) as a colorless solid. 1H NMR (300 MHz, CD3OD) δ 8.21 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.75 (dd, J = 1.3, 8.7 Hz, 1H), 7.65 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.55 (dt, J = 4.7, 16.7 Hz, 1H), 6.41 (d, J = 16.7 Hz, 1H), 5.94 (q, J = 6.9 Hz, 1H), 5.72 (q, J = 7.1 Hz, 1H), 4.43 (br d, J = 11.2 Hz, 1H), 4.23 (d, J = 10.5 Hz, 1H), 3.88−3.79 (m, 1H), 3.40−3.35 (m, 1H), 3.07−2.97 (m, 1H), 2.75 (dd, J = 2.9, 12.9 Hz, 1H), 2.08−1.85 (m, 4H), 1.74 (d, J = 6.9 Hz, 3H), 1.72−1.66 (m, 1H), 1.64 (d, J = 7.1 Hz, 3H), 1.01 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.48 Hz, 3H). LC/MS Method A (m/z) 522.2 [M + H], Tr = 1.90 min. 2,2,2-Trichloroethyl (S)-1-(((E)-4-(6-((R)-1-Hydroxyethyl)isoquinolin-3-yl)-2,2-dimethylbut-3-enoyl)-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (32c). 29 (1.05 g, 2.84 mmol) and 10 (1.61 g, 3.03 mmol) were combined as described in 32b. The crude residue was subjected to silica gel chromatography eluting with 35−60% acetone in iso-hexanes to afford the title compound (1.51 g, 71% over two steps from 10) as a colorless oil. 1H NMR (400 MHz, CDCl3) 9.14 (s, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.61−7.48 (m, 2H), 7.07 (d, J = 15.9 Hz, 1H), 6.77 (d, J = 15.9 Hz, 1H), 6.66 (d, J = 7.7 Hz, 1H), 6.39 (d, J = 8.4 Hz, 1H), 5.27 (m, 1H), 5.07 (q, J = 6.4 Hz, 1H), 4.91 (d, J = 11.9 Hz, 1H), 4.67 (d, J = 11.9 Hz, 1H), 4.31 (d, J = 12.2 Hz, 1H), 4.25 (dd, J = 8.4, 6.1 Hz, 1H), 3.84 (d, J = 11.2 Hz, 1H), 3.70−3.62 (m, 1H), 2.88−2.80 (m, 1H), 2.20−2.04 (m, 3H), 1.97−1.84 (m, 1H), 1.77−1.62 (m, 2H), 1.57 (d, J = 6.5 Hz, 3H), 1.48 (br s, 6H), 1.28 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 7.5 Hz, 3H). LC/MS Method B1 (m/z) 697.8 [M + H], Tr = 3.08 min. (R)-1-(7-((E)-4-(tert-Butoxy)-4-oxobut-1-en-1-yl)quinolin-2yl)ethyl (S)-1-((tert-Butoxycarbonyl)- L -valyl- L -alanyl)hexahydropyridazine-3-carboxylate (33). A solution of compound 10 (804 mg, 1.51 mmol) in THF (38 mL) was treated with zinc powder (2.18 g, 33.3 mmol) followed by a solution of ammonium acetate (1.75 g, 22.7 mmol) in water (9.4 mL). The reaction mixture was stirred at room temperature for 72 h. The reaction mixture was filtered through a Hyflo-supercel, washing with EtOAc and saturated aqueous potassium hydrogen sulfate. The

washed with brine (100 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes followed by isohexanes/ EtOAc (95:5) and finally isohexanes/EtOAc (9:1) to afford (R)-1-(7bromoquinolin-2-yl)ethyl acetate (1.16 g, 99%) as a colorless oil. The oil was dissolved in anhydrous CH3CN (20 mL) and treated with palladium(II) acetate (89 mg, 0.395 mmol), 2,2-dimethyl-but-3-enoic acid (496 mg, 4.35 mmol), tri(o-tolyl)phosphine (241 mg, 0.790 mmol), and Et3N (1.09 mL, 7.90 mmol). The mixture was heated in the microwave at 100 °C for 20 min and then concentrated in vacuo. Water (200 mL) was added and the mixture was extracted with EtOAc (2 × 50 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/ EtOAc (95:5) followed by isohexanes/EtOAc (9:1) and finally isohexanes/EtOAc (3:1) to afford the title compound (792 mg, 61%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.31 (d, J = 8.5 Hz, 1H), 7.94 (s, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.77 (dd, J = 8.5, 1.3 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 6.74 (ABq, ΔδAB = 0.02, JAB = 16.5 Hz, 2H), 5.98 (q, J = 6.8 Hz, 1H), 2.16 (s, 3H), 1.65 (d, J = 6.8 Hz, 3H), 1.47 (s, 6H). LC/MS Method A (m/z) 328.1 [M + H], Tr = 2.11 min. (R,E)-4-(6-(1-Hydroxyethyl)isoquinolin-3-yl)-2,2-dimethylbut-3-enoic Acid (29). 16 (880 mg, 4.23 mmol), 31 (1.24 g, 4.88 mmol), bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine]palladium(II) chloride (173 mg, 0.21 mmol), and potassium phosphate tribasic (2.64 g, 12.4 mmol) were dissolved in cyclopentyl methyl ether (11.9 mL) and water (5.1 mL). The biphasic mixture was vigorously stirred at 90 °C for 3.5 h then cooled to rt and diluted with EtOAc (50 mL) and water (40 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (2 × 50 mL). The combined organic phases were dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 25−60% EtOAc in iso-hexanes to afford methyl (R,E)-4-(6-(1-hydroxyethyl)isoquinolin-3-yl)-2,2-dimethylbut-3-enoate (1.07 g, 85%) as a yellow oil. A solution of the methyl (R,E)-4-(6-(1-hydroxyethyl)isoquinolin-3-yl)-2,2-dimethylbut-3enoate (1.05 g, 3.51 mmol) in THF (8 mL), MeOH (4 mL), and water (4 mL) was treated with lithium hydroxide hydrate (297 mg, 7.08 mmol). The mixture was stirred for 6 h and then quenched with 1 M HCl (7.2 mL, 7.2 mmol). The mixture was concentrated and then azeotroped with anhydrous MeOH (50 mL) followed by toluene (50 mL) to afford the title compound (1.3 g, 100%) as a yellow solid that was used without further purification. LC/MS Method A (m/z) 286.1 [M + H], Tr = 1.97 min. Methyl (E)-2,2-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-3-enoate (31). Bis(cyclopentadienyl)zirconium chloride hydride (290 mg, 1.1 mmol) was cooled in an ice water bath under argon. A solution of 30 (1.4 g, 11.1 mmol) and pinacolborane (2.4 mL, 16.5 mmol) in CH2Cl2 (3 mL) was added by cannula, washing the cannula and flask with an additional portion of CH2Cl2 (2 mL). The mixture was removed from the cold bath and stirred for 72 h at rt. The mixture was then diluted with EtOAc (50 mL), quenched with dropwise water (5 mL), and then further diluted with water (50 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (30 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 5−15% EtOAc in hexanes to afford the title compound (1.6 g, 57%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 6.73 (d, J = 18.5 Hz, 1H), 5.50 (d, J = 18.5 Hz, 1H), 3.67 (s, 3H), 1.31 (s, 6H), 1.27 (s, 12H). LC/MS Method B1 (m/z) 255.2 [M + H], Tr = 4.03 min. 2,2,2-Trichloroethyl (S)-1-(((E)-4-(7-((R)-1-Hydroxyethyl)naphthalen-2-yl)but-3-enoyl)-L-valyl-L-alanyl)hexahydropyridazine-3-carboxylate (32a). 10 (95 mg, 0.18 mmol) was deprotected as described in the synthesis of 32b and then combined with 24 (40 mg, 0.08 mmol) as described in the synthesis of 32b. The crude residue was subjected to silica gel chromatography eluting with MeOH/CH2Cl2 (1:10) to afford the title compound (68 mg, 65%) as a yellow solid. 1H NMR (400 MHz, 9489

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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was concentrated in vacuo, and then dissolved in N,N-dimethylformamide (5 mL). The mixture was added into an argon purged flask containing 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (276 mg, 0.73 mmol), N,Ndiisopropylethylamine (312 mg, 2.42 mmol), and CH2Cl2 (200 mL). The reaction mixture was repurged with argon and then stirred at rt for 2 h. The mixture was washed with water (100 mL), brine (100 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 0− 40% EtOAc/MeOH (4/1) in isohexanes to afford the title compound (4 mg, 3%) as a white solid. 1H NMR (400 MHz, CD3CN) δ 9.12 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.28 (dd, J = 18.5, 6.4 Hz, 2H), 7.03 (d, J = 15.9 Hz, 1H), 6.81 (d, J = 15.9 Hz, 1H), 5.65 (d, J = 3.5 Hz, 1H), 4.95 (dq, J = 13.2, 6.8 Hz, 2H), 4.29 (d, J = 11.7 Hz, 1H), 3.24 (s, 1H), 2.92 (d, J = 12.9 Hz, 1H), 2.42−2.23 (m, 1H), 1.70 (d, J = 16.1 Hz, 2H), 1.59 (s, 3H), 1.52 (d, J = 6.1 Hz, 2H), 1.49−1.39 (m, 9H), 1.01 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H). LC/MS Method C (m/z) 550.4 [M + H], Tr = 1.81 min. HRMS C30H39N5O5 calculated 549.2951, found 549.2963. (R)-N-((R)-1-(3-Chloroisoquinolin-6-yl)ethyl)-2-methylpropane-2-sulfinamide (37). A solution of 1-(3-chloroisoquinolin-6yl)ethan-1-one (see compound 16) (1.72 g, 8.3 mmol) in THF (40 mL) was stirred under N2. Titanium(IV) ethoxide (3.8 g, 3.45 mL, 16.6 mmol, tech. grade) was added followed by (R)-(+)-2methylpropanesulfinimide (1.11 g, 9.2 mmol), and the reaction mixture was stirred at 60 °C under N2 for 18 h. Additional (R)-(+)-2methylpropanesulfinimide (190 mg, 1.67 mmol) was added, and the reaction mixture was stirred at 65 °C for a further 2 h. The reaction mixture was cooled to room temperature, treated with EtOAc and brine, and the suspension was filtered through Celite, washing the filter pad with EtOAc. The EtOAc layer was separated, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc 7:3 to 3:7 to afford the intermediate sulfinimide (2.2 g, 86%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 9.13 (s, 1H), 8.19 (s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.84 (s, 1H), 2.92 (s, 3H), 1.39 (s, 9H). LC/MS Method B (m/z) 309.1/311.1 [M + H], Tr = 2.31 min. A mixture of (1S, 2R)-(−)-cis-1-amino-2-indanol (60 mg, 0.4 mmol), dichloro (pcymene) ruthenium(II) dimer (122 mg, 0.2 mmol), and powdered 4 Å molecular sieves (2 g) was suspended in anhydrous 2-propanol (9 mL) and stirred under N2. The suspension was heated at 90 °C for 20 min, cooled to 40 °C, and treated with a solution of (R)-2methylpropane-2-sulfinic acid [1-(3-chloro-isoquinolin-6-yl)-eth-(E)ylidene]-amide (1.23 g, 4 mmol) in 2-propanol (28 mL) followed by a solution of potassium tert-butoxide (122 mg, 1.1 mmol) in 2-propanol (10 mL). The reaction mixture was stirred at 40 °C for 2 h and then allowed to cool. The mixture was poured directly onto a silica gel cartridge and eluted with EtOAc to afford the title compound (1.19 g, 96%) as a brown gum. 1H NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.74 (br s, 2H), 7.63 (dd, J = 8.5, 1.3 Hz, 1H), 4.71−4.79 (m, 1H), 3.53 (d, J = 3.4 Hz, 1H), 1.63 (app t, J = 6.7 Hz, 3H), 1.28 (s, 9H). LC/MS Method B (m/z) 311.0/313.0 [M + H], Tr = 2.19 min. tert-Butyl (R)-(1-(3-chloroisoquinolin-6-yl)ethyl)carbamate (38). A solution of 37 (932 mg, 3 mmol) in MeOH (9 mL) was stirred at rt under N2. 4 M HCl in 1,4-dioxane (3 mL, 12 mmol) was added, and the reaction mixture stirred at rt for 30 min. The mixture was concentrated in vacuo, and the residue was triturated with Et2O to form an orange solid that was collected, washed with Et2O, and dried to afford (R)-1-(3-chloroisoquinolin-6-yl)ethan-1-amine hydrochloride salt (777 mg, quant). 1H NMR (300 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.80−8.65 (br, 3H), 8.26 (d, J = 8.5, 1H), 8.05 (br, 1H), 8.04 (br, 1H), 7.90 (dd, J = 8.5, 1.4 Hz, 1H), 4.67−4.58 (m, 1H), 1.61 (d, J = 6.7 Hz, 3H). LC/MS Method C (m/z) 207/209 [M + H], Tr = 0.72 min. A mixture of the hydrochloride salt (730 mg, 3 mmol) and Et3N (909 mg, 1.25 mL, 9 mmol) in CH2Cl2 (25 mL), was stirred at rt until a solution formed. A solution of di-tert-butyl dicarbonate (981 mg, 4.5 mmol) in CH2Cl2 (5 mL) was then added, and the reaction

mixture was treated with 1 M HCl (3 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (2 × 100 mL). The organic layers were combined, washed with brine, filtered, and concentrated to give a colorless gum. The residue was azeotroped with in vacuo toluene (3 × 200 mL) to afford (S)-1-[(S)-2-((S)-2-tertbutoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydropyridazine-3-carboxylic acid (605 mg, quantitative) as a white solid. LC/MS Method A (m/z) 401.2 [M + H], Tr = 1.78 min. Compound 27 (363 mg, 1.16 mmol) and (S)-1-[(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine3-carboxylic acid (505 mg, 1.26 mmol) were dissolved in CH2Cl2 (20 mL) and treated with N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (311 mg, 1.62 mmol) and 4-DMAP (142 mg, 1.16 mmol). The mixture was stirred at rt for 16 h and then treated with saturated aqueous ammonium chloride solution (100 mL). The mixture was extracted with CH2Cl2 (3 × 50 mL), and the organic extracts were washed with brine (50 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (4:1) followed by isohexanes/EtOAc (7:3), then isohexanes/EtOAc (3:2) to afford the title compound (357 mg, 44%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 8.5 Hz, 1H), 7.97 (s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 1.6, 8.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.77− 6.63 (m, 2H), 6.59−6.46 (m, 1H), 6.13 (q, J = 6.7 Hz, 1H), 5.35− 5.23 (m, 1H), 5.12 (br d, J = 8.3 Hz, 1H), 4.46 (br d, J = 12.9 Hz, 1H), 4.01−3.91 (m, 1H), 3.83 (d, J = 11.6 Hz, 1H), 3.68−3.57 (m,1H), 3.26 (d, J = 6.9 Hz, 2H), 2.72 (m, 1H), 2.23−2.02 (m, 3H), 1.99−1.86 (m, 1H), 1.73 (d, J = 6.9 Hz, 3H), 1.70−1.65 (m, 1H), 1.51 (s, 9H), 1.46 (s, 9H), 1.33 (d, J = 6.9 Hz, 3H), 1.00−0.86 (m, 6H). LC/MS Method A (m/z) 696.4 [M + H], Tr = 3.29 min. (5 3 S,2R,7S,10S,E)-10-Isopropyl-2,7,13,13-tetramethyl51,52,53,54,55,56-hexahydro-11-oxa-3,8-diaza-1(2,7)-quinolina5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12-tetraone (34). 45 (117 mg, 0.201 mmol) was hydrolyzed according to the method for 18 over 20 h followed by preparative HPLC eluting with a gradient of 20 to 100% CH3CN in water to afford the seco-acid (60 mg, 53%). LC/MS Method A (m/z) 568.3 [M + H], Tr = 1.60 min. A slurry of 2-methyl-6-nitrobenzoic anhydride (177 mg, 0.500 mmol), 4-DMAP (92 mg, 0.750 mmol), and powdered 4 Å molecular sieves (1 g) in 1,2-dichloroethane (33 mL) was stirred under N2 at 50 °C. The seco-acid (57 mg, 0.100 mmol) was dissolved in N,Ndimethylformamide (5 mL), and the solution was added to the slurry over 5 h via syringe pump. The acid containing flask was washed with further N,N-dimethylformamide (1 mL), and the washings were transferred to the reaction vessel over 30 min. The reaction mixture was stirred at 50 °C for 1 h and then concentrated in vacuo. The residue was subjected to preparative HPLC eluting with a gradient of 5−100% CH3CN in water to afford the title compound (24.5 mg, 45%) as a white solid.1H NMR (400 MHz, CD3OD) δ 8.22 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.58 (dd, J = 8.5, 1.7 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 6.51 (d, J = 16.3 Hz, 1H), 6.33 (d, J = 16.3 Hz, 1H), 5.77 (q, J = 7.2 Hz, 1H), 5.20 (d, J = 9.0 Hz, 1H), 5.09−5.01 (m, 1H), 4.40 (d, J = 12.1 Hz, 1H), 3.65−3.51 (m, 1H), 2.67 (td, J = 12.9, 3.2 Hz, 1H), 2.25 (d, J = 13.7 Hz, 1H), 2.20−2.06 (m, 1H), 1.92 (d, J = 13.6 Hz, 1H), 1.66 (dt, J = 12.9, 4.0 Hz, 1H), 1.57 (dd, J = 18.2, 7.0 Hz, 5H), 1.50 (s, 3H), 1.39 (s, 3H), 1.25 (d, J = 9.9 Hz, 1H), 1.23−1.11 (m, 1H), 1.03 (d, J = 6.8 Hz, 2H), 0.97 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 550.2 [M + H], Tr = 2.05 min. HRMS C30H39N5O5 calculated 549.2951, found 549.2962. (5 3 S,2R,7S,10S,E)-10-Isopropyl-2,7,13,13-tetramethyl1 2 3 4 5 6 5 ,5 ,5 ,5 ,5 ,5 -hexahydro-11-oxa-3,8-diaza-1(6,3)-isoquinolina-5(3,1)-pyridazinacyclopentadecaphan-14-ene-4,6,9,12tetraone (35). 46 (390 mg, 0.48 mmol) in THF (20 mL) was treated with a solution of lithium hydroxide (13 mg, 0.53 mmol) in water (10 mL), and the mixture was stirred at rt for 2 h. 1 M HCl was added (0.55 mL) and the reaction mixture was concentrated in vacuo. The residue was partitioned between CH2Cl2 (50 mL) and water (50 mL), and the organic layer was separated, concentrated in vacuo, and dried under high vacuum. The residue was treated with 4 M HCl in 1,4-dioxane (10 mL, 40 mmol) and stirred at rt for 4 h. The mixture 9490

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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mixture was stirred at rt for 20 h. The mixture was washed with water (30 mL), brine (30 mL), dried, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 10−30% EtOAc in isohexanes to afford the title compound (714 mg, 79% over two steps) as a white solid. 1H NMR (300 MHz, CDCl3) δ 9.05 (s, 1H), 7.96 (d, J = 8.5, 1H), 7.71 (br, 1H), 7.67 (br, 1H), 7.57 (dd, J = 8.5, 1.4 Hz, 1H), 5.00−4.90 (br, 2H), 1.53 (d, J = 6.5 Hz, 3H), 1.44 (s, 9H). LC/MS Method C (m/z) 307/309 [M + H], Tr = 2.67 min. (R,E)-4-(6-(1-((tert-Butoxycarbonyl)amino)ethyl)isoquinolin3-yl)-2,2-dimethylbut-3-enoic Acid (39). 38 (200 mg, 0.65 mmol) was subjected to the same coupling procedure with 31 as described for compound 29. The crude product was dissolved in THF (9.5 mL, 0.1 M) and then treated with 0.1 M NaOH (9.5 mL, 1.03 mmol) followed by heating the mixture at 40 °C for 3 h. The mixture was cooled to rt, treated with 1 M HCl (3.6 mL) until pH ≈ 4, and then concentrated in vacuo to afford the crude acid (191 mg, 76% over two steps from 38) as a yellow solid that was used without further purification. LC/MS Method C (m/z) 385.1 [M + H]. (R)-N-((R)-1-(7-Bromoquinolin-2-yl)ethyl)-2-methylpropane-2-sulfinamide (41). A solution of 1-(7-bromoquinolin-2yl)ethan-1-one 40 (1.42 g, 5.68 mmol, see preparation of 26) in THF (28 mL) was treated with titanium(IV) ethoxide (2.6 g, 2.35 mL, 11.4 mmol) followed by (R)-(+)-2-methylpropanesulfinimide (825 mg, 6.82 mmol). The reaction mixture was stirred at 60 °C for 6 h. Brine was added followed by EtOAc (25 mL), and the suspension was filtered through Celite, washing the filter pad with EtOAc. The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (9:1 to 3:1) to afford the intermediate sulfinimide (448 mg, 22%) as an orange solid. 1H NMR (300 MHz, CDCl3) δ 8.37 (s, 1H), 8.24 (d, J = 8.6 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.71 (m, 2H), 2.99 (s, 3H), 1.38 (s, 9H). LC/MS Method A (m/z) 352.9/354.9 [M + H], Tr = 3.14 min. A mixture of (1S, 2R)-(−)-cis-1-amino-2-indanol (19 mg, 0.13 mmol), [Ru(p-cymene)Cl2]2 (39 mg, 0.064 mmol) and powdered 4 Å molecular sieves (0.7 g) was suspended in anhydrous 2-propanol (3 mL) and stirred under N2. The suspension was heated at 90 °C for 30 min. The reaction mixture was cooled to 40 °C, and a solution of the intermediate sulfinimide (448 mg, 1.27 mmol) in 2propanol (9 mL) was added followed by a solution of potassium tertbutoxide (36 mg, 0.32 mmol) in 2-propanol (3 mL). The reaction mixture was stirred for 2 h at 40 °C and then allowed to cool before filtration through a silica gel plug eluting with EtOAc. The eluant was concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with a gradient of iso-hexanes/EtOAc (1:1 to 0:1) to afford the title compound (287 mg, 64%) as a brown gum. 1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 8.5, 1.2 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 5.42 (br d, J = 4.2 Hz, 1H), 4.80 (m, 1H), 1.60 (d, J = 6.7 Hz, 3H), 1.33 (s, 9H). LC/MS Method A (m/z) 354.9/356.8 [M + H], Tr = 2.49 min. Methyl (R,E)-4-(2-(1-Aminoethyl)quinolin-7-yl)-2,2-dimethylbut-3-enoate (42). 41 (207 mg, 0.583 mmol), 31 (170 mg, 0.670 mmol), bis[(dicyclohexyl)(4-dimethylaminophenyl)phosphine] palladium(II) chloride (24 mg, 0.029 mmol), and potassium phosphate tribasic (371 mg, 1.75 mmol) were suspended in cyclopentylmethyl ether (3 mL) and water (1.5 mL). The mixture was stirred and heated at 80 °C under N2 for 5 h. The reaction mixture was allowed to cool and diluted with EtOAc (20 mL) and water (20 mL). The aqueous layer was separated and washed with further EtOAc (20 mL), and the combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of EtOAc in isohexanes (1:1 to 3:1) to afford methyl (E)-4-(2-((R)-1(((R)-tert-butylsulfinyl)amino)ethyl)quinolin-7-yl)-2,2-dimethylbut3-enoate as a yellow oil (201 mg, 86%). 1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 8.5, 1.8 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 6.63 (s, 2H), 5.48 (d, J = 4.0 Hz, 1H), 4.85−4.74 (m, 1H), 3.74 (s, 3H), 1.59 (d, J = 6.7

Hz, 3H), 1.48 (s, 6H), 1.33 (s, 9H). LC/MS Method A (m/z) 403.2 [M + H], Tr = 2.56 min. Methyl (E)-4-(2-((R)-1-(((R)-tertbutylsulfinyl)amino)ethyl)quinolin-7-yl)-2,2-dimethylbut-3-enoate (193 mg, 0.48 mmol) in MeOH (20 mL) was treated with 4 M HCl in 1,4-dioxane (10 mL). The mixture was stirred at rt for 2 h, concentrated in vacuo, and azeatroped with toluene (3 × 15 mL) to afford the title compound (166 mg, quant.) as a yellow solid that was used without purification. LC/MS Method A (m/z) 299.1 [M + H], Tr = 1.35 min. 2,2,2-Trichloroethyl (S)-1-(((S)-2-hydroxy-3-methylbutanoyl)-L-alanyl)hexahydropyridazine-3-carboxylate (44). 2,2,2-Trichloroethyl (S)-1-((tert-butoxycarbonyl)-L-alanyl)hexahydropyridazine-3-carboxylate 4329 (1.5 g, 3.5 mmol) in anhydrous CH2Cl2 (30 mL) was cooled to 0 °C and treated with TMSOTf (941 μL, 5.2 mmol). The reaction mixture was stirred at 0 °C for 1 h, N,N-diisopropylethylamine (2.2 mL, 13.9 mmol) was added, and the mixture was then concentrated in vacuo to afford crude 2,2,2-trichloroethyl (S)-1-(L-alanyl)hexahydropyridazine-3-carboxylate. The residue was dissolved in anhydrous CH3CN (20 mL), cooled to 0 °C and treated with (S)-2-hydroxy-3-methylbutanoic acid (413 mg, 3.5 mmol), hydroxybenzotriazole monohydrate (796 mg, 5.2 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (1.02 g, 5.2 mmol), and N,N-diisopropylethylamine (1.7 mL, 10.4 mmol). The reaction mixture was stirred at 0 °C for 90 min, warmed to rt, and then stirred another 72 h. The mixture was concentrated in vacuo, and the residue was treated with EtOAc (30 mL) washed with saturated ammonium chloride (50 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexane/acetone (2:1) to afford the title compound (1.1 g, 73% over two steps) as a white solid. 1H NMR (300 MHz, CDCl3) δ 7.09 (d, J = 7.1 Hz, 1H), 5.46−5.34 (m, 1H), 4.97 (d, J = 12.0 Hz, 1H), 4.73 (d, J = 12 Hz, 1H), 4.00−3.95 (m, 1H), 4.43−4.31 (m, 1H), 3.86 (d, J = 11.2 Hz, 1H), 3.78−3.67 (m, 1H), 3.02−2.85 (m, 1H), 2.67 (d, J = 5.6 Hz, 1H), 2.26−2.08 (m, 2H), 2.02−1.90 (m, 1H), 1.84−1.66 (m, 2H), 1.36 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H). LC/MS Method A (m/z) 434.63 [M + H], Tr = 2.26 min. Methyl (E)-4-(2-((R)-1-((S)-1-(((S)-2-Hydroxy-3-methylbutanoyl)-L-alanyl)hexahydropyridazine-3-carboxamido)ethyl)quinolin-7-yl)-2,2-dimethylbut-3-enoate (45). 44 (502 mg, 1.16 mmol) was suspended in THF (15 mL) and water (3 mL) and treated with lithium hydroxide monohydrate (69 mg, 1.65 mmol). The reaction mixture was stirred for 45 min, and then 2 M HCl (0.75 mL) was added. The reaction mixture was stirred for 5 min and concentrated in vacuo to afford the crude (S)-1-(((S)-2-hydroxy-3methylbutanoyl)-L-alanyl)hexahydropyridazine-3-carboxylic acid (350 mg, quant.) that was used without further purification. 42 (166 mg, 0.48 mmol), (S)-1-[(S)-2-((S)-2-hydroxy-3-methyl-butyrylamino)propionyl]-hexahydro-pyridazine-3-carboxylic acid (190 mg, 0.48 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (256 mg, 0.67 mmol), and powdered 4 Å molecular sieves were suspended in CH3CN (9.6 mL) under N2. The mixture was treated with N,N-diisopropylethylamine (186 mg, 250 μL, 1.44 mmol) and stirred for 20 h. The mixture was filtered through a phase separator, and the filtrate was concentrated in vacuo. The residue was dissolved in CH2Cl2 (50 mL) and washed with saturated sodium bicarbonate solution (30 mL), water (30 mL), saturated aq. ammonium chloride (30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of EtOAc in isohexanes (1:1 to 1:0) to afford the title compound as a yellow gum (77 mg, 28% over two steps from 44). 1H NMR (300 MHz, CDCl3) δ 8.18−8.07 (m, 3H), 7.74 (d, J = 8.5 Hz, 1H), 7.67 (dd, J = 8.7, 1.3 Hz, 1H), 7.25 (m, 1H), 6.76 (d, J = 16.3 Hz, 1H), 6.64 (m, 1H), 5.42 (m, 1H), 5.24 (m, 1H), 4.55 (br d, J = 13.2 Hz, 1H), 3.97 (br s, 1H), 3.80 (d, J = 11.8 Hz, 1H), 3.73 (s, 3H), 3.46 (m, 1H), 2.87 (br s, 1H), 2.70 (m, 1H), 2.27 (m, 1H), 2.11 (m, 1H), 1.71−1.58 (m, 3H), 1.57 (d, J = 6.7 Hz, 3H), 1.48 (s, 6H), 1.42 (d, J = 6.9 Hz, 3H), 1.00 (d, J = 6.9 Hz, 9491

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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3H), 0.87 (d, J = 6.9 Hz, 3H). LC/MS Method A (m/z) 582.2 [M + H], Tr = 1.91 min. 2,2,2-Trichloroethyl (S)-1-(((S)-2-(((E)-4-(6-((R)-1-((tertbutoxycarbonyl)amino)ethyl)isoquinolin-3-yl)-2,2-dimethylbut-3-enoyl)oxy)-3-methylbutanoyl)-L-alanyl)hexahydropyridazine-3-carboxylate (46). 2-Methyl-6-nitrobenzoic anhydride (344 mg, 1 mmol), 4-DMAP (128 mg, 1.05 mmol), and 39 (191 mg, 0.50 mmol) were stirred in anhydrous CH2Cl2 (20 mL). The mixture was treated with N,N-diisopropylethylamine (0.26 mL, 1.50 mmol) and then stirred at rt for 10 min. 44 (331 mg, 0.75 mmol) in anhydrous CH2Cl2 (10 mL) was added dropwise, and the mixture was stirred at rt for 12 h. The mixture was washed with water (20 mL) and brine (10 mL). The aqueous phase was back extracted with CH2Cl2 (20 mL), and the combined organic extracts were washed with brine (20 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 0−40% EtOAc/MeOH (4:1 mixture) in isohexanes to afford the title compound (391 mg, 97%) as a white solid. LC/MS Method C (m/z) 798.1 [M + H], Tr = 2.82 min. (2′R,3′S,7′S,10′S,E)-4-Hydroxy-10′-isopropyl-2′,7′dimethylspiro[cyclohexane-1,13′-11-oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanen]-14′-ene4′,6′,9′,12′-tetraone (47 Major and 48 Minor). A mixture of 62a (90 mg, 0.093 mmol) in THF (1 mL), MeOH (1 mL), and water (1 mL) was treated with K2CO3 (89 mg, 0.644 mmol), and the mixture was stirred at 0 °C for 2.5 h. The mixture was stored for 64 h in a freezer, stirred at rt for 3 h, and then at 30 °C for 1.5 h. The mixture was diluted with brine and extracted with EtOAc (2 × 50 mL). The aqueous fractions were back extracted with EtOAc (50 mL), and the combined organic extracts dried over MgSO 4, filtered, and concentrated in vacuo. The residue was dissolved in 4 M HCl in 1,4-dioxane (3 mL) and stirred at rt for 45 min. The mixture was concentrated and dried in vacuo for 2 h to obtain the crude seco-acid. The crude seco-acid (0.093 mmol) was treated with 5 equiv of HATU and 8 equiv of N,N-diisopropylethylamine according to the procedure for compound 35 in CH2Cl2 (100 mL). The mixture was concentrated in vacuo, and the residue was diluted with EtOAc (50 mL). The solution was washed with 5% LiCl solution (2 × 50 mL), 0.1 M HCl (50 mL), and water (50 mL). The aqueous fractions were back extracted with EtOAc (50 mL), and the combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to preparative HPLC using a 21.2 × 250 mm 10 μm C18 Phenomenex Gemini semipreparative column and CH3CN/water (containing 0.1% TFA modifier) as mobile phase at a flow rate of 20 mL/min with a 50 min gradient consisting of 0−5 min: 20% CH3CN; 5−48 min: 20% CH3CN to 80% CH3CN; 48−50 min: 80% CH3CN to obtain the product fractions. Each combined product fraction was concentrated in vacuo to remove CH3CN, neutralized with NaHCO3 solution, diluted with water, and then extracted with EtOAc (2 × 50 mL). The organic extracts were then concentrated to afford the title compound (major isomer 47, 4.6 mg, 8%; and minor isomer 48, 1.1 mg, 2%). Compound 47: 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 1.5 Hz, 1H), 7.62 (dd, J = 8.5, 1.6 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 6.61 (d, J = 16.4 Hz, 1H), 6.29 (d, J = 16.4 Hz, 1H), 5.79 (q, J = 7.2 Hz, 1H), 5.23 (d, J = 8.6 Hz, 1H), 5.06 (q, J = 6.6 Hz, 1H), 4.41 (dd, J = 13.2, 4.3 Hz, 1H), 3.88 (s, 1H), 3.57 (dd, J = 11.8, 2.9 Hz, 1H), 2.68 (td, J = 13.0, 3.3 Hz, 1H), 2.25 (d, J = 12.5 Hz, 1H), 2.16 (d, J = 12.8 Hz, 3H), 2.00−1.88 (m, 3H), 1.84−1.63 (m, 5H), 1.60 (d, J = 7.3 Hz, 3H), 1.57 (d, J = 6.7 Hz, 3H), 1.61−1.47 (m, 1H), 1.28 (s, 1H), 1.05 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.7 Hz, 3H). LC/ MS Method B (m/z) 606.3 [M + H], Tr = 2.03 min. HRMS C33H43N5O6 calculated 605.3213, found 605.3227. Compound 48: 1H NMR (400 MHz, CD3OD) δ 8.22 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.76 (s, 1H), 7.59 (dd, J = 8.5, 1.7 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 16.4 Hz, 1H), 6.24 (d, J = 16.4 Hz, 1H), 5.85−5.74 (m, 1H), 5.28 (d, J = 8.6 Hz, 1H), 5.06 (dd, J = 7.2, 4.8 Hz, 1H), 4.46−4.36 (m, 1H), 3.68−3.61 (m, 1H), 3.57 (dd, J = 11.7, 3.0 Hz, 1H), 2.68 (td, J = 13.0, 3.3 Hz, 1H), 2.57 (dd, J = 14.1, 3.0 Hz, 1H), 2.33 (dd, J = 13.0, 3.3 Hz, 1H), 2.24 (d, J

= 13.7 Hz, 1H), 2.18 (dt, J = 8.1, 6.4 Hz, 1H), 2.00−1.86 (m, 3H), 1.76−1.62 (m, 2H), 1.61 (d, J = 7.3 Hz, 3H), 1.57 (d, J = 6.8 Hz, 3H), 1.55−1.26 (m, 5H), 1.07 (d, J = 6.8 Hz, 3H), 1.04 d, J = 6.6 Hz, 3H). LC/MS Method B (m/z) 606.3 [M + H], Tr = 2.02 min. HRMS C33H43N5O6 calculated 605.3213, found 605.3226. (1r,2′R,3′S,4S,7′S,10′S,E)-10′-Isopropyl-4-methoxy-2′,7′dimethylspiro[cyclohexane-1,13′-11-oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanen]-14′-ene4′,6′,9′,12′-tetraone (49). 62b (0.32 mmol) was cyclized according to the procedure for the preparation of 35 from 46 in CH2Cl2 (400 mL) and then subjected to HPLC purification (21.2 × 250 mm 10 μm C18 Phenomenex Gemini semipreparative column eluting with CH3CN/water (containing 0.1% TFA modifier) at a flow rate of 20 mL/min; 50 min gradient consisting of 0−5 min, 20% CH3CN; 5−48 min, 20% to 80% CH3CN gradient, 48−50 min, 80% CH3CN) to afford 49 (40 mg, 20%). 1H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.79 (s, 1H), 7.63 (dd, J = 8.4, 1.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 6.59 (d, J = 16.4 Hz, 1H), 6.29 (d, J = 16.4 Hz, 1H), 5.82 (q, J = 7.2 Hz, 1H), 5.27 (d, J = 8.6 Hz, 1H), 5.09 (q, J = 6.7 Hz, 1H), 4.44 (dd, J = 13.3, 3.6 Hz, 1H), 3.68 (s, 1H), 3.60 (dd, J = 11.7, 2.8 Hz, 1H), 3.48 (s, 1H), 3.37 (s, 3H), 2.70 (td, J = 12.9, 3.1 Hz, 1H), 2.32−2.11 (m, 3H), 2.11−1.83 (m, 6H), 1.71 (dd, J = 15.4, 11.1 Hz, 2H), 1.65−1.49 (m, 8H), 1.06 (dd, J = 19.3, 6.7 Hz, 6H). LC/MS Method B (m/z) 620.6 [M + H]. HRMS C34H45N5O6 calculated 619.3370, found 619.3383. (2′R,3′S,7′S,10′S,E)-10′-Isopropyl-2′,7′-dimethyl-2,3,5,6tetrahydrospiro[pyran-4,13′-11-oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanen]-14′-ene4′,6′,9′,12′-tetraone (50). A solution of 62c (0.70 g, 0.83 mmol) in THF (10 mL) was treated with 1 N KOH in water (0.83 mL). The reaction mixture was vigorously stirred at rt for 20 min and then acidified by adding 1 M HCl in water (0.83 mL). The mixture was concentrated in vacuo, and the residue was dissolved in 4 M HCl in 1,4-dioxane (2 mL). The mixture was stirred for 1 h, concentrated in vacuo, and azeotroped with toluene (3 × 5 mL) to afford the crude seco-acid. The seco-acid (0.83 mmol) was cyclized with HATU according to the procedure for compound 35 in CH2Cl2 (800 mL) except including a catalytic amount of 4-DMAP. The mixture was concentrated and the residue subjected to preparative HPLC to obtain the title product (0.22 g, 45%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.78 (s, 1H), 7.63 (dd, J = 8.4, 1.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 6.60 (d, J = 16.3 Hz, 1H), 6.29 (d, J = 16.3 Hz, 1H), 5.80 (q, J = 7.2 Hz, 1H), 5.29 (d, J = 8.6 Hz, 1H), 5.08 (d, J = 5.3 Hz, 1H), 5.05 (d, J = 6.7 Hz, 1H), 4.41 (dd, J = 13.1, 3.4 Hz, 1H), 3.93 (tt, J = 11.9, 3.7 Hz, 2H), 3.65 (s, 2H), 3.63−3.45 (m, 3H), 2.68 (td, J = 12.9, 3.3 Hz, 1H), 2.40 (d, J = 13.7 Hz, 1H), 2.32−2.10 (m, 3H), 2.00−1.89 (m, 2H), 1.85 (ddd, J = 13.8, 10.8, 4.3 Hz, 1H), 1.67 (dt, J = 13.0, 4.2 Hz, 1H), 1.60 (d, J = 7.2 Hz, 3H), 1.57 (d, J = 6.7 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H). LC/MS Method B (m/z) 592.3 [M + H], Tr = 2.15 min. HRMS C32H41N5O6 calculated 591.3057, found 591.3069. (2′R,3′S,7′S,10′S,E)-10′-Isopropyl-2′,7′-dimethyl-2,3,5,6tetrahydrospiro[thiopyran-4,13′-11-oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanen]-14′-ene4′,6′,9′,12′-tetraone 1,1-Dioxide (51). 73 (187 mg, 0.35 mmol) and 65 (79 mg, 0.38 mmol) were combined using the same method as 3, except heating at 80 °C for 30 min to form the seco-acid. LC/MS Method A (m/z) 658.2 [M + H], Tr = 1.57 min. The seco-acid was cyclized in the same manner as 3 except by dropwise addition over 4 h, followed by stirring of the mixture for 4 h. The residue was subjected to silica gel chromatography eluting with isohexanes/ EtOAc/MeOH (1:1:0 to 0:1:0 to 0:9:1) followed by reverse phase preparative HPLC eluting with a gradient of CH3CN (containing 0.1% trifluoroacetic acid)/water (containing 0.1% trifluoroacetic acid) from 9:1 to 1:1. The resultant product was partitioned between CH2Cl2 (20 mL) and saturated sodium hydrogen carbonate solution (20 mL), separated, and the organic layer washed with brine (30 mL). The organic solution was filtered through a hydrophobic frit and then concentrated in vacuo to afford the title compound (14 mg, 7% over two steps) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.28 (d, J 9492

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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= 8.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.85 (br s, 1H), 7.68 (dd, J = 8.5, 1.6 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 6.73 (d, J = 16.3 Hz, 1H), 6.36 (d, J = 16.3 Hz, 1H), 5.85 (q, J = 7.1 Hz, 1H), 5.33 (d, J = 8.3 Hz, 1H), 5.09 (q, J = 6.7 Hz, 1H), 4.46−4.40 (m, 1H), 3.65−3.55 (m, 1H), 2.25−2.17 (m, 11H), 1.97−1.92 (m, 1H), 1.71−1.63 (m, 2H), 1.62 (d, J = 7.1 Hz, 3H), 1.59 (d, J = 6.7 Hz, 3H), 1.09 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 640.2 [M + H], Tr = 2.09 min. HRMS C32H41N5O7S calculated 639.2727, found 639.2739. (2R,3S,7S,10S,E)-10-Isopropyl-2,2′,2′,7-tetramethylspiro[11oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanene-13,5′-[1,3]dioxan]-14-ene-4,6,9,12-tetraone (52). 73 (228 mg, 0.426 mmol) and 68b (79 mg, 0.426 mmol) were combined using the same method as 3, except heating at 60 °C for 1 h to form the seco-acid. The seco-acid was cyclized in the same manner as 3 except by dropwise addition over 4 h. The crude product was subjected to reverse phase preparative HPLC eluting with water/ CH3CN 3:2 to afford the title compound (53 mg, 20% over two steps) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.24 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.74−7.66 (m, 2H), 7.46 (d, J = 8.7 Hz, 1H), 6.64 (d, J = 16.7 Hz, 1H), 6.17 (d, J = 16.7 Hz, 1H), 5.87−5.75 (m, 1H), 5.38 (d, J = 9.1 Hz, 1H), 5.12−5.01 (m, 1H), 4.51−4.39 (m, 2H), 4.37−4.29 (m, 1H), 4.16−4.03 (m, 2H), 3.64− 3.56 (m, 1H), 2.80−2.65 (m, 1H), 2.29−2.14 (m, 2H), 2.00−1.90 (m, 1H), 1.64 (d, J = 7.4 Hz, 3H), 1.60 (d, J = 6.7 Hz, 3H), 1.77− 1.58 (m, 2H), 1.55 (s, 3H), 1.35 (s, 3H), 1.11 (d, J = 6.7 Hz, 3H), 1.06 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 622.3 [M + H], Tr = 2.38 min. HRMS C33H43N5O7 calculated 621.3162, found 621.3176. (2R,3S,7S,10S,E)-10-Isopropyl-2,7-dimethylspiro[11-oxa3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphanene-13,6′-[1,4]dioxepan]-14-ene-4,6,9,12-tetraone (53). 73 (68 mg, 0.128 mmol) and 71 (79 mg, 0.426 mmol) were combined using the same method as 3, except heating at reflux for 40 min to form the seco-acid. LC/MS Method A (m/z) 626.2 [M + H], Tr = 1.67 min. The seco-acid was cyclized in the same manner as 3 except by dropwise addition over 2.5 h. The crude residue was subjected to reverse phase preparative HPLC eluting with a gradient of CH3CN/water containing 0.1% formic acid (1:9 to 7:3). The isolated residue was then triturated with Et2O, and collected to provide the title compound (6.5 mg, 8% over two steps) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.25 (d, J = 8.5 Hz, 1H), 8.19 (br s, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.73−7.68 (m, 2H), 7.46 (d, J = 8.5 Hz, 1H), 6.49 (d, J = 16.5 Hz, 1H), 6.30 (d, J = 16.5 Hz, 1H), 5.86−5.76 (m, 1H), 5.35 (d, J = 9.2 Hz, 1H), 5.07 (q, J = 6.7 Hz, 1H), 4.53 (d, J = 12.7 Hz, 1H), 4.46−4.38 (m, 1H), 4.38 (d, J = 12.7 Hz, 1H), 4.13 (d, J = 12.5 Hz, 1H), 3.91−3.76 (m, 5H), 3.65−3.57 (m, 1H), 2.75−2.63 (m, 1H), 2.28−2.10 (m, 2H), 2.00−1.90 (m, 1H), 1.66 (d, J = 7.4 Hz, 3H), 1.60 (d, J = 6.7 Hz, 3H), 1.73−1.53 (m, 2H), 1.08 (d, J = 6.7 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 608.2 [M + H], Tr = 2.12 min. HRMS C32H41N5O7 calculated 607.3006, found 607.3014. (53S,2R,7S,10S,E)-13,13-Bis(hydroxymethyl)-10-isopropyl2,7-dimethyl-51,52,53,54,55,56-hexahydro-11-oxa-3,8-diaza-1(2,7)-quinolina-5(3,1)-pyridazinacyclopentadecaphan-14ene-4,6,9,12-tetraone (54). A solution of compound 52 (20 mg, 0.032 mmol) in MeOH (1 mL) was treated with 1 M HCl (3 mL) and then stirred for 1 h at rt. The mixture was neutralized by addition of saturated sodium bicarbonate solution followed by NaCl to saturate the solution. The aqueous layer was extracted with CH2Cl2, the organic extracts filtered through a hydrophobic frit, and then concentrated in vacuo to afford the title compound (14 mg, 75%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.24 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.74−7.65 (m, 2H), 7.45 (d, J = 8.5 Hz, 1H), 6.63 (d, J = 16.7 Hz, 1H), 6.31 (d, J = 16.7 Hz, 1H), 5.81 (q, J = 7.3 Hz, 1H), 5.32 (d, J = 9.1 Hz, 1H), 5.06 (q, J = 6.7 Hz, 1H), 4.46− 4.37 (m, 1H), 4.16−3.93 (m, 4H), 3.64−3.56 (m, 1H), 2.77−2.64 (m, 1H), 2.28−2.08 (m, 2H), 1.96−1.84 (m, 1H), 1.65 (d, J = 7.4 Hz, 3H), 1.60 (d, J = 6.9 Hz, 3H), 1.77−1.49 (m, 2H), 1.04 (d, J = 6.7 Hz, 3H), 1.01 (d, J = 6.7 Hz, 3H). LC/MS Method A (m/z) 582.5

[M + H], Tr = 1.60 min. HRMS C30H39N5O7 calculated 581.2849, found 581.2858. tert-Butyl (R)-(1-(7-Bromoquinolin-2-yl)ethyl)carbamate (55). 41 (533 mg, 1.5 mmol) was dissolved in CH2Cl2 (23 mL) and 4 M HCl in 1,4-dioxane (7.5 mL). The mixture was stirred for 2 h and then concentrated in vacuo. The residue was passed though an SCX cation exchange cartridge eluting with MeOH and then methanolic ammonia to afford the crude (R)-1-(7-bromoquinolin-2yl)ethan-1-amine 72. A solution of 72 (3.5 g, 12.2 mmol) in anhydrous CH2Cl2 at 0 °C under an atmosphere of N2 was treated with Et3N (5.1 mL, 36.5 mmol), 4-DMAP (297 mg, 2.4 mmol), and di-tert-butyl dicarbonate (4.0 g, 18.3 mmol). The reaction mixture was stirred at rt for 2 h. The reaction mixture was cooled to 0 °C and quenched with 1 M HCl. The organic layer was separated, washed with brine, dried through a hydrophobic frit, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc 6:1 to afford the title compound (3 g, 70%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 8.29 (s, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 8.7, 1.8 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.20−6.01 (m, 1H), 5.10−4.93 (m, 1H), 1.55 (d, J = 6.9 Hz, 3H), 1.49 (s, 9H). LC/MS Method A (m/z) 353.0 [M + H], Tr = 3.02 min. Ethyl (R,E)-1-(2-(2-(1-((tert-butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-oxocyclohexane-1-carboxylate (56a). A mixture of 55 (556 mg, 1.58 mmol), 58 (559 mg, 2.85 mmol, obtained from Small Molecules, Inc., Hoboken, USA), Pd(OAc)2 (70 mg, 0.31 mmol), and (o-Tol)3P (100 mg, 0.33 mmol) in 1,4-dioxane (5 mL) was treated with Et3N (0.72 mL, 5.17 mmol), and the mixture was heated at 100 °C for 30 min in a microwave reactor. The mixture was concentrated and diluted with water (50 mL) and EtOAc (50 mL). The mixture was filtered through Celite pad, and the organic layer was separated. The aqueous fraction was extracted with EtOAc (50 mL), and the combined organic fractions were washed with water (50 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with hexanes/EtOAc to obtain the title product (479 mg, 65%). 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 8.3 Hz, 1H), 8.01 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 6.74 (d, J = 16.1 Hz, 1H), 6.47 (d, J = 16.3 Hz, 1H), 6.16 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 2.76−2.56 (m, 2H), 2.57−2.38 (m, 4H), 2.18−2.00 (m, 2H), 1.47 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H). LC/MS Method B (m/z) 467.0 [M + H], Tr = 2.33 min. Methyl (1r,4R)-1-((E)-2-(2-((R)-1-((tert-Butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-methoxycyclohexane-1carboxylate (56b). 59 (223 mg, 1.05 mmol) and 55 (369 mg, 1.05 mmol) were combined as described in the preparation of 56a to afford the title compound (424 mg, 89%). LC/MS Method B (m/z) 455.1 [M + H]. (R,E)-4-(2-(2-(1-((tert-Butoxycarbonyl)amino)ethyl)quinolin7-yl)vinyl)tetrahydro-2H-pyran-4-carboxylic acid (56c). 61 (0.726 g, 4.27 mmol) and 55 (1.5 g, 4.27 mmol) were combined as described in the preparation of 56a to afford the title compound (1.1 g, 59%). LC/MS Method B (m/z) 441.2 [M + H], Tr = 2.21 min. 1-((E)-2-(2-((R)-1-((tert-Butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylic Acid (57). A solution of 56a (237 mg, 0.51 mmol) in THF (5 mL) was stirred at −60 °C and treated with 1 M K-selectride in THF (0.61 mL). The reaction mixture was slowly warmed to 0 °C over 30 min, diluted with EtOAc (50 mL), and washed with water (2 × 50 mL). The aqueous fractions were back extracted with EtOAc (50 mL), and the combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with hexanes/EtOAc to provide ethyl 1-((E)-2-(2-((R)-1-((tertbutoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-hydroxycyclohexane-1-carboxylate (192 mg, 81%) as a ∼4:1 isomer mixture. LC/MS Method B (m/z) 469.0 [M + H], Tr = 2.20 min. A mixture of ethyl 1((E)-2-(2-((R)-1-((tert-butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-hydroxycyclohexane-1-carboxylate (192 mg, 0.41 mmol) and LiOH (87.7 mg, 2.09 mmol) in THF (2 mL), MeOH (2 mL), and 9493

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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water (2 mL) was stirred at 50 °C for 3 h. The mixture was concentrated to ∼1/3 volume, diluted with water (20 mL), neutralized with 1 M HCl (2.2 mL), and then extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with water (50 mL), dried over MgSO4, filtered, and concentrated in vacuo to provide crude 1-((E)-2-(2-((R)-1-((tert-butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-hydroxycyclohexane-1-carboxylic acid. A solution of the crude 1-((E)-2-(2-((R)-1-((tertbutoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-hydroxycyclohexane-1-carboxylic acid, imidazole (115 mg, 1.69 mmol), and TBSCl (79 mg, 0.52 mmol) in DMF was stirred at 35 °C bath for 1.5 h. The mixture was treated with additional imidazole (102 mg, 1.50 mmol), and TBSCl (77 mg, 0.51 mmol) and stirred at 35 °C for 17 h. The mixture was concentrated to 1/3 volume in vacuo and then diluted with EtOAc (50 mL). The solution was washed with 5% LiCl solution (2 × 50 mL). The aqueous fractions were back extracted with EtOAc (50 mL), and the combined organic fractions dried over MgSO4, filtered, and concentrated in vacuo. The residue was was dissolved in THF (2 mL), MeOH (2 mL), and water (2 mL), and stirred with K2CO3 (226 mg, 1.64 mmol) at rt for 30 min. The solution was concentrated to ∼1/2 volume, diluted with water (20 mL), and acidified with 1 M HCl (∼3.5 mL). The solution was extracted with EtOAc (2 × 50 mL). The extracts were washed with water (50 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with hexanes/ EtOAc to afford the title compound (193 mg, 85%) as an apparent ∼4:1 isomer mixture. LC/MS Method B (m/z) 555.1 [M + H], Tr = 2.60 min. (1r,4r)-4-Methoxy-1-vinylcyclohexane-1-carboxylic Acid (59). 58 (3.0 g, 15.29 mmol) in MeOH (80 mL) was treated dropwise with NaBH4 (697 mg, 18.35 mmol) in MeOH (80 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min, and then at rt for 48 h before quenching with saturated NH4Cl solution. The mixture was extracted with EtOAc (100 mL), washed with brine (50 mL) dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with 30% EtOAC in isohexanes to isolate methyl (1r,4r)-4-hydroxy-1-vinylcyclohexane-1carboxylate (425 mg, 16%). 1H NMR (400 MHz, CDCl3) δ 5.84− 5.71 (m, 1H), 5.23−5.11 (m, 2H), 4.11 (q, J = 7.5 Hz, 2H), 3.79 (tq, J = 6.8, 3.6 Hz, 1H), 1.90 (ddd, J = 11.7, 8.2, 4.2 Hz, 4H), 1.71 (ddt, J = 11.9, 7.8, 3.7 Hz, 2H), 1.61−1.50 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H). Combined cis and cis/trans mixtures (1.3 g, 43%) were also obtained. The methyl (1r,4r)-4-hydroxy-1-vinylcyclohexane-1-carboxylate (425 mg, 2.13 mmol) was dissolved in CH2Cl2 (10 mL) and treated with CH3OBF4 (1.66 g, 10.7 mmol) and 1,8-bis(dimethylamino)naphthalene (2.3 g, 10.7 mmol). The mixture was stirred at rt for 24 h and then filtered through Celite. The filtrate was washed with saturated NaHCO3 (30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with EtOAC/hexane to afford methyl (1r,4r)-4-methoxy-1-vinylcyclohexane-1-carboxylate (223 mg, 49%). 1 H NMR (400 MHz, CDCl3) δ 5.87−5.68 (m, 1H), 5.20−4.99 (m, 2H), 4.23−3.95 (m, 2H), 3.36−3.19 (m, 4H), 1.87 (pd, J = 13.5, 5.6 Hz, 4H), 1.75−1.54 (m, 4H), 1.29−1.14 (m, 3H). Methyl (1r,4r)-4methoxy-1-vinylcyclohexane-1-carboxylate (223 mg, 1.05 mmol) and 1 M aq. LiOH (2 mL) in THF (4 mL) and MeOH (0.5 mL) was stirred at room temperature for 1 h. The reaction mixture was acidified with 1 M HCl, and extracted with EtOAc (10 mL) followed by 20% iPrOH in CHCl3 (10 mL), twice. The extracts were dried over MgSO4, filtered, and concentrated under reduced pressure to afford the title compound (202 mg, quant.). LCMS Method B (m/z) 185.11 [M + H]. Methyl 4-vinyltetrahydro-2H-pyran-4-carboxylate (61). Methyl tetrahydro-2H-pyran-4-carboxylate 60 was reacted according to the method of Bercot, E. A. et al.46 to afford the title compound 61 (1.6 g, 45%). 2,2,2-Trichloroethyl (3S)-1-(((2S)-2-((1-((E)-2-(2-((R)-1-((tertButoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-((tertbutyldimethylsilyl)oxy)cyclohexane-1-carbonyl)oxy)-3-methylbutanoyl)- L -alanyl)hexahydropyridazine-3-carboxylate

(62a). A mixture of 57 (131 mg, 0.236 mmol), 44 (125 mg, 0.289 mmol), 2-methyl-6-nitrobenzoic anhydride (98 mg, 0.285 mmol), and 4-DMAP (72 mg, 0.589 mmol) was dissolved in CH2Cl2 (3 mL), and the mixture was stirred for 3 min. N,N-Diisopropylethylamine (0.1 mL, 0.574 mmol) was added, and the resulting mixture was stirred at rt for 17 h. The mixture was diluted with EtOAc (50 mL) and washed with water (2 × 50 mL). The aqueous fractions were back extracted with EtOAc (50 mL), and the combined organic fractions dried over MgSO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with hexanes/EtOAc to obtain the title compound (90 mg, 39%) as a ∼4:1 mixture. LC/MS Method B (m/z) 968.2 [M + H], Tr = 3.04 min. 2,2,2-Trichloroethyl (S)-1-(((S)-2-(((1r,4S)-1-((E)-2-(2-((R)-1((tert-Butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)-4-methoxycyclohexane-1-carbonyl)oxy)-3-methylbutanoyl)- L alanyl)hexahydropyridazine-3-carboxylate (62b). 56b (243 mg, 0.53 mmol) and 44 were combined following the same procedure as the preparation of 62a to afford the title compound (287 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.74−7.54 (m, 2H), 7.26 (d, J = 11.4 Hz, 2H), 7.00 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 16.3 Hz, 1H), 6.42 (d, J = 16.2 Hz, 1H), 6.17 (s, 1H), 5.25−5.14 (m, 1H), 5.08 (d, J = 3.7 Hz, 1H), 4.94 (d, J = 9.2 Hz, 1H), 4.89 (d, J = 12.0 Hz, 1H), 4.66 (d, J = 11.9 Hz, 1H), 4.23 (d, J = 13.6 Hz, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.66 (m, 2H), 3.34 (d, J = 8.9 Hz, 1H), 2.77 (s, 1H), 2.30 (td, J = 7.0, 3.9 Hz, 1H), 2.14 (d, J = 18.3 Hz, 5H), 2.02 (s, 3H), 1.89−1.56 (m, 7H), 1.50 (d, J = 6.8 Hz, 3H), 1.44 (s, 9H), 1.28−1.15 (m, 6H), 0.91 (d, J = 6.9 Hz, 6H). LC/ MS Method B (m/z) 870.3 [M + H]. 2,2,2-Trichloroethyl (S)-1-(((S)-2-((4-((E)-2-(2-((R)-1-((tertButoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)tetrahydro2H-pyran-4-carbonyl)oxy)-3-methylbutanoyl)- L -alanyl)hexahydropyridazine-3-carboxylate (62c). A solution of 56c (1.0 g, 2.27 mmol) in THF (10 mL) and MeOH (5 mL) was treated with 1 N KOH in water (11.4 mL). The reaction mixture was stirred at 50 °C for 2 h and then acidified by adding 1 M HCl in water (11.4 mL). The mixture was extracted with EtOAc (2 × 100 mL), dried over Na2SO4, and concentrated in vacuo to afford (R,E)-4-(2-(2-(1((tert-butoxycarbonyl)amino)ethyl)quinolin-7-yl)vinyl)tetrahydro2H-pyran-4-carboxylic acid that was used without further purification. The (R,E)-4-(2-(2-(1-((tert-butoxycarbonyl)amino)ethyl)quinolin-7yl)vinyl)tetrahydro-2H-pyran-4-carboxylic acid (0.328 g, 0.77 mmol), 44 (0.398, 0.92 mmol), 4-DMAP (0.225 g, 1.85 mmol), N,Ndiisopropylethylamine (0.239 g, 1.85 mmol), and 2-methyl-6nitrobenzoic anhydride (0.632 g, 1.84 mmol) were dissolved in CH2Cl2 (10 mL). The reaction mixture was stirred at rt for 16 h and then concentrated in vacuo. The residue was diluted with EtOAc (100 mL), washed with saturated NH4Cl (100 mL), dried over Na2SO4, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (1:0 to 0:1) to afford the title compound (0.55 g, 85%). LC/MS Method B (m/z) 841.2 [M + H], Tr = 2.56 min. Methyl 4-(1-hydroxyethyl)tetrahydro-2H-thiopyran-4-carboxylate 1,1-Dioxide (64). A solution of N,N-diisopropylamine (1.51 g, 2.1 mL, 15 mmol) in anhydrous THF (10 mL) was stirred at −78 °C under N2. n-Butyl lithium (6 mL, 15 mmol, 2.5 M solution in hexane) was added dropwise, and the reaction mixture stirred at −78 °C for 30 min. A solution of 63 (1.60 g, 10 mmol) in THF (4 mL) was added, and the reaction mixture was stirred at −78 °C for 20 min. Acetaldehyde (1.32 g, 1.7 mL, 30 mmol) was then added in one portion, the cooling bath was removed, and the mixture was stirred at rt for 1 h. The reaction mixture was cooled to 0 °C, and ice-cold 2 M HCl was added to adjust to pH 2. Sodium chloride was added to saturate the aqueous phase, and the mixture was extracted with Et2O (3 × 50 mL). The organic extracts were combined and washed with brine (100 mL), filtered through a hydrophobic frit, and then concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with pentane/Et2O (3:1 to 1:3) to afford methyl 4-(1-hydroxyethyl)tetrahydro-2H-thiopyran-4-carboxylate (1.77 g, 87%) as a yellow oil. 1H NMR (300 MHz, DMSO-d6) δ 4.85 (d, J = 5.8 Hz, 1H), 3.63 (s, 3H), 3.58−3.50 (m, 1H), 2.57−2.43 9494

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

Journal of Medicinal Chemistry

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and then heated at 50 °C for 17 h. The mixture was cooled to rt and extracted with EtOAc (2 × 200 mL), and the combined organic extracts were washed with water (300 mL), dried over MgSO4, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with petroleum ether/EtOAc (15:1 to 9:1) to afford the title compound 67a (33 g, 14% over two steps) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.82 (d, J = 6.0 Hz, 1H), 4.77 (d, J = 6.0 Hz, 1H), 4.35 (d, J = 12.0 Hz, 2H), 4.25−4.19 (m, 4H), 2.31 (s, 3H), 1.25 (m, 3H). Ethyl 5-Acetyl-2,2-dimethyl-1,3-dioxane-5-carboxylate (67b). Ethyl 2,2-bis(hydroxymethyl)-3-oxobutanoate (for preparation see 67a) (7.6 g, 40 mmol), acetone (30 mL, 400 mmol), 2,2dimethoxypropane (41.6 g, 50 mL, 400 mmol), and 4-toluenesulfonic acid hydrate (152 mg, 0.8 mmol) were mixed together and stirred at rt for 18 h. The mixture was concentrated in vacuo to a volume of ∼20 mL and treated with saturated sodium hydrogen carbonate solution (50 mL). The mixture was extracted with EtOAc (3 × 70 mL), and the combined organic extracts were washed with water (100 mL), brine (100 mL), filtered through a hydrophobic frit, and then concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (9:1 to 4:1) to afford the title compound (8.5 g, 31% over two steps) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 4.30 (ABq, ΔδAB = 0.04, JAB = 11.9 Hz, 4H), 4.23 (q, J = 7.1 Hz, 2H), 2.33 (s, 3H), 1.40 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). 5-Vinyl-1,3-dioxane-5-carboxylic Acid (68a). 67a (22.3 g, 0.11 mol) was dissolved in EtOH (100 mL) and treated portionwise with sodium borohydride (2.08 g, 0.055 mol) at 0 °C. The mixture was stirred at 0 °C for 1 h and then treated with acetone. The solution was extracted with EtOAc (3 × 600 mL), and the combined organic extracts were concentrated in vacuo to afford ethyl 5-(1hydroxyethyl)-1,3-dioxane-5-carboxylate (18.9 g, 84%) as a colorless oil. A mixture of ethyl 5-(1-hydroxyethyl)-1,3-dioxane-5-carboxylate (16.8 g, 83 mmol) in anhydrous pyridine (40 mL) at 0 °C was treated with 4-toluenesulfonyl chloride (19.1 g, 99 mmol). The mixture was stirred at rt for 16 h, and then water (40 mL) was added. The mixture was extracted with EtOAc (2 × 400 mL), and the extracts were washed with brine (3 × 200 mL) and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with petroleum ether/EtOAc (25:1 to 5:1) to provide ethyl 5-(1(tosyloxy)ethyl)-1,3-dioxane-5-carboxylate (22.7 g, 76%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 5.03−4.98 (m, 1H), 4.20−4.17 (m, 2H), 4.08 (d, J = 12.0 Hz, 1H), 4.02 (d, J = 11.6 Hz, 1H), 3.87 (d, J = 11.6 Hz, 1H), 3.80 (d, J = 11.6 Hz, 1H), 2.45 (s, 3H), 1.34 (d, J = 6.8 Hz, 3H), 1.26 (m, 3H). Ethyl 5-(1-(tosyloxy)ethyl)-1,3-dioxane-5-carboxylate (20.7 g, 58 mmol) in DBU (25 mL) was heated at 140 °C for 3 h and then cooled to rt. The mixture was poured into Et2O (300 mL), washed with brine (3 × 100 mL), dried over Na2SO4, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with petroleum ether/Et2O (30:1 to 20:1) to provide an oil which was then distilled in vacuo (45 to 49 °C/0.3 mbar) to afford ethyl 5-vinyl1,3-dioxane-5-carboxylate (3.6 g, 33%) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.65 (m, 1H), 5.27 (m, 1H), 5.24 (m, 1H), 4.95 (d, J = 6.0 Hz, 1H), 4.71 (d, J = 8.0 Hz, 1H), 4.44 (t, J = 11.2 Hz, 2H), 4.25 (t, J = 7.2 Hz, 2H), 3.70 (d, J = 11.6 Hz, 2H), 1.29 (t, J = 11.0 Hz, 3H). Ethyl 5-vinyl-1,3-dioxane-5-carboxylate (397.7 mg, 2.136 mmol) in THF/MeOH/water (15 mL, 2:2:1) was treated with lithium hydroxide monohydrate (269 mg, 6.408 mmol). The mixture was stirred at rt for 1.5 h, concentrated in vacuo, cooled to 0 °C, and quenched with 1 M HCl. The aqueous layer was saturated with sodium chloride and then extracted with CH2Cl2 (3 × 100 mL). The combined organic extracts were filtered through a phase separator and concentrated in vacuo to provide the title compound (320.3 mg, 95%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 5.69 (dd, J = 17.6, 10.9 Hz, 1H), 5.27−5.39 (m, 2H), 5.02 (d, J = 6.0 Hz, 1H), 4.73 (d, J = 6.2 Hz, 1H), 4.50 (d, J = 11.4 Hz, 2H), 3.72 (d, J = 11.6 Hz, 2H). 2,2-Dimethyl-5-vinyl-1,3-dioxane-5-carboxylic Acid (68b). N,N-Diisopropylamine (1.51 g, 2.1 mL, 15 mmol) in anhydrous THF (30 mL) was stirred and cooled to −78 °C under N2. n-Butyl

(m, 4H), 2.28−2.12 (m, 2H), 1.73−1.46 (m, 2H), 0.96 (d, J = 6.5 Hz, 3H). LC/MS Method A (m/z) 227.1 [M + Na], Tr = 1.53 min. A solution of methyl 4-(1-hydroxyethyl)tetrahydro-2H-thiopyran-4carboxylate (408 mg, 2 mmol) in CH2Cl2 (10 mL) was stirred at 0 °C and treated with meta-chloroperbenzoic acid (70% pure containing 20% water and 10% meta-chlorobenzoic acid, 830 mg, 4.8 mmol). The mixture was stirred at 0 °C for 3 h, and then additional metachloroperbenzoic acid (830 mg, 4.8 mmol) was added. The mixture was stirred at 0 °C for 3 h, and then sodium thiosulfate solution and saturated sodium hydrogen carbonate solution were added. The mixture was extracted with CH2Cl2 (3 × 50 mL), and the combined organic extracts were washed with brine (50 mL), filtered through a hydrophobic frit, and then concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/EtOAc (1:1 to 0:1) to afford the title compound (281 mg, 60%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 5.12 (d, J = 5.8 Hz, 1H), 3.71−3.64 (m, 4H), 3.11−2.92 (m, 4H), 2.30−1.90 (m, 4H), 0.99 (d, J = 6.5 Hz, 3H). LC/MS Method A (m/z) 237.1 [M + H], Tr = 0.76 min. 4-Vinyltetrahydro-2H-thiopyran-4-carboxylic Acid 1,1-Dioxide (65). A solution of 64 (281 mg, 1.2 mmol) in CH2Cl2 (20 mL) was stirred at 0 °C under N2. Pyridine (284 mg, 0.3 mL, 3.6 mmol) was added followed by dropwise addition of trifluoromethanesulfonic anhydride (677 mg, 0.4 mL, 2.4 mmol). The reaction mixture was stirred at 0 °C for 30 min, and then saturated sodium hydrogen carbonate solution was added. The organic layer was separated, washed with brine (10 mL), filtered through a hydrophobic frit, and then concentrated in vacuo to a volume of ∼5 mL. 1,8Diazabicycloundec-7-ene (0.6 mL, 3.6 mmol) was added, and the reaction mixture was stirred at rt for 45 min. The reaction mixture was cooled to 0 °C and adjusted to pH 2 with ice-cold 2 M HCl. The mixture was extracted with CH2Cl2 (3 × 30 mL), and the combined organic extracts were washed with water (30 mL), brine (30 mL), filtered through a hydrophobic frit, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (4:1 to 1:1) to afford methyl 4-vinyltetrahydro2H-thiopyran-4-carboxylate 1,1-dioxide (182 mg, 70% over two steps) as a white solid. 1H NMR (300 MHz, CDCl3) δ 5.82 (dd, J = 17.6, 10.7 Hz, 1H), 5.31 (d, J = 10.7 Hz, 1H), 5.23 (d, J = 17.6 Hz, 1H), 3.13−3.05 (m, 4H), 3.79 (s, 3H), 2.66−2.59 (m, 2H), 2.34−2.25 (m, 2H). LC/MS Method A (m/z) 219.1 [M + H], Tr = 1.33 min. The methyl 4-vinyltetrahydro-2H-thiopyran-4-carboxylate 1,1-dioxide (174 mg, 0.8 mmol) in THF (4 mL) and MeOH (0.5 mL) was stirred at rt under N2 and treated with a solution of lithium hydroxide monohydrate (134 mg, 3.2 mmol) in water (1 mL). The reaction mixture was stirred at rt for 1 h, cooled to 0 °C, and adjusted to pH 2 with 2 M HCl. Sodium chloride was added, and the mixture was extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with brine (50 mL), filtered through a hydrophobic frit, and then concentrated in vacuo to afford the title compound (163 mg, 100%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.1−12.9 (br s, 1H), 5.87 (dd, J = 17.6, 10.7 Hz, 1H), 5.25 (d, J = 10.7 Hz, 1H), 5.23 (d, J = 17.6 Hz, 1H), 3.13−2.96 (m, 4H), 2.42−2.33 (m, 2H), 2.12−2.03 (m, 2H). LC/MS Method A (m/z) 227.1 [M + Na], Tr = 0.96 min. 5-Vinyl-1,3-dioxane-5-carboxylic Acid (67a). A mixture of 66 (150 g, 1.2 mol) in formaldehyde (210 mL, 2.8 mol, 40% aq.) and 1,4-dioxane (550 mL) was treated dropwise with Et3N (1.0 M in THF, 57 mL, 0.06 mol) at 0 °C. The mixture was heated to 35−40 °C, stirred for 1 h and then diluted with water (400 mL). The solution was extracted with toluene (3 × 600 mL), and the aqueous phase was concentrated in vacuo at 35 °C to ∼1/4 of the initial volume. The aqueous solution was extracted with EtOAc (3 × 900 mL), and the organic extracts were dried over Na2SO4 and concentrated in vacuo to afford ethyl 2,2-bis(hydroxymethyl)-3oxobutanoate (75 g, 0.39 mol). 1H NMR (300 MHz, DMSO-d6) δ 4.86 (t, J = 5.3 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.87 (d, J = 5.3 Hz, 4H), 2.07 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H). Ethyl 2,2bis(hydroxymethyl)-3-oxobutanoate (75 g, 0.39 mol) was mixed with 40% formaldehyde (360 mL) in concentrated HCl (360 mL) 9495

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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3.81 (m, 4H). LC/MS Method A (m/z) 182.1 [M + Na], Tr = 1.04 min. 6-Vinyl-1,4-dioxepane-6-carboxylic Acid (71). Toluene (13 mL) and water (3.25 mL) were deoxygenated by bubbling N2 through for 20 min and then treated with 70 (512 mg, 3.22 mmol), 2,2′bis(diphenylphosphino)-1,1′-binaphthalene (218 mg, 0.35 mmol), and potassium vinyltrifluoroborate (1.73 g, 12.9 mmol). The mixture was deoxygenated with N2 for 5 min and then treated with bis(1,5cyclooctadiene)rhodium(I) tetrafluoroborate (131 mg, 0.32 mmol). The reaction mixture was stirred and heated at reflux under N2 for 4.5 h. The mixture was partitioned between Et2O (30 mL) and water (30 mL). The aqueous layer was separated and extracted with Et2O (2 × 30 mL), and the combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with a gradient of isohexanes/Et2O (3:1 to 13:7) to afford 6-(nitromethyl)-6-vinyl-1,4-dioxepane (155 mg, 26%) as a pale brown oil. 1H NMR (300 MHz, CDCl3) δ 5.79 (dd, J = 17.8, 11.2 Hz, 1H), 5.30 (d, J = 11.2 Hz, 1H), 5.22 (d, J = 17.9 Hz, 1H), 4.64 (s, 2H), 3.93−3.77 (m, 8H). LC/MS Method A (m/z) 210.1 [M + Na], Tr = 1.51 min. 6-(Nitromethyl)-6-vinyl-1,4dioxepane (214 mg, 1.14 mmol) in N,N-dimethylformamide (5 mL) under N2 was treated with acetic acid (684 mg, 652 μL, 11.4 mmol) and sodium nitrite (237 mg, 3.43 mmol). The mixture was heated at 40 °C for 23 h, cooled, and adjusted to pH 1 with 2 M HCl. The aqueous layer was extracted with Et2O (3 × 20 mL), and the combined organic extracts were washed with water (20 mL), dried over MgSO4, filtered, concentrated in vacuo. The residue was azeotroped with toluene to afford a yellow gum. The original aqueous layer was saturated with NaCl and extracted with Et2O (3 × 20 mL), and the combined organic extracts were washed with water, dried over MgSO4, filtered, and concentrated in vacuo. The residue was azeoptroped with toluene and then combined with the first extract to afford the title compound (101 mg, 51%).1H NMR (300 MHz, CDCl3) δ 5.79 (dd, J = 17.4, 10.9 Hz, 1H), 5.27 (d, J = 10.9 Hz, 1H), 5.26 (d, J = 17.8 Hz, 1H), 4.35 (d, J = 12.7 Hz, 2H), 3.88 (d, J = 12.5 Hz, 2H), 3.93−3.76 (m, 4H). LC/MS Method A (m/z) 195.2 [M + Na], Tr = 0.96 min. (R)-1-(7-Bromo-quinolin-2-yl)-ethylamine Hydrochloride (72). Prepared as described in the synthesis of 55. (S)-N-((R)-1-(7-Bromoquinolin-2-yl)ethyl)-1-(((S)-2-hydroxy3-methylbutanoyl)-L-alanyl)hexahydropyridazine-3-carboxamide (73). 44 (400 mg, 0.92 mmol) in THF (20 mL) was treated with zinc dust (1.3 g, 20.3 mmol) and ammonium acetate (1.1 g, 13.9 mmol) in water (7 mL). The mixture was stirred at rt for 16 h, the zinc residues were filtered off, and the volatiles were removed in vacuo. The residue was partitioned between EtOAc and saturated potassium hydrogen sulfate solution, the aqueous layer was extracted twice with EtOAc (50 mL), and the combined organic extracts were concentrated in vacuo. The residual acetic acid was azeotroped off with toluene (3 × 10 mL) to afford crude (S)-1-[(S)-2-((S)-2hydroxy-3-methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3carboxylic acid (280 mg, 99%). (S)-1-[(S)-2-((S)-2-Hydroxy-3methyl-butyrylamino)-propionyl]-hexahydro-pyridazine-3-carboxylic acid (140 mg, 0.46 mmol) was combined with 72 (194 mg, 0.60 mmol) and N,N-diisopropylethylamine (0.400 mL, 2.3 mmol) in anhydrous CH3CN (10 mL). The mixture was then treated with 2(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (227 mg, 0.60 mmol) and stirred at rt for 3 h. The mixure was concentrated in vacuo, the residue dissolved in EtOAc and subsequently washed with saturated ammonium chloride solution and sodium bicarbonate solution. The organic layer was concentrated in vacuo, and the residue was subjected to silica gel chromatography eluting with isohexanes/acetone (1:0 to 1:1) to afford the title compound (136 mg, 55%) as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.33 (d, J = 8.7 Hz, 1H), 8.26 (d, J = 1.6 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.69 (dd, J = 8.7, 1.8 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 5.40−5.31 (m, 1H), 5.28−5.18 (m, 1H), 4.34−4.24 (m, 1H), 3.81 (d, J = 3.4 Hz, 1H), 3.62−3.55 (m, 1H), 3.00−2.87 (m, 1H), 2.12−2.02 (m, 2H), 1.96−1.87 (m, 1H), 1.75−1.65 (m, 3H), 1.60 (d, J = 6.9 Hz, 3H), 1.38 (d, J = 6.9 Hz, 3H), 0.98 (d, J = 6.9 Hz,

lithium (2.5 M in hexanes, 5.6 mL, 14 mmol) was added dropwise, and the mixture was stirred at −78 °C for 30 min. 67b (2.3 g, 10 mmol) in THF (5 mL) was added to the reaction mixture, and the mixture was stirred at −78 °C for 15 min. N-Phenyl-(bistrifluoromethanesulfonamide) (3.93 g, 11 mmol) in THF (30 mL) was added, and the mixture was stirred at −78 °C for 15 min. The mixture was allowed to warm to rt and stirred for 30 min. The mixture was concentrated in vacuo, and Et2O (50 mL) was added. The mixture was cooled to 5 °C and washed with cold 1 M sodium hydroxide solution (3 × 30 mL) and brine (30 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue in N,N-dimethylformamide (20 mL) was treated with tri-nbutylamine (5.55 g, 7 mL, 30 mmol) and stirred at rt under N2. Bis(triphenylphosphine) palladium(II) dichloride (350 mg, 0.5 mmol) and formic acid (920 mg, 0.77 mL, 20 mmol) were added, and the reaction mixture was heated at 60 °C for 90 min. The reaction mixture was cooled to room temperature and EtOAc (100 mL) and water (100 mL) were added. The organic layer was separated, washed with water (5 × 30 mL), brine (50 mL), filtered through a hydrophobic frit, and then concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with isohexanes/EtOAc (9:1) followed by silica gel chromatography eluting with isohexanes/ EtOAc (19:1 to 9:1) to afford ethyl 2,2-dimethyl-5-vinyl-1,3-dioxane5-carboxylate (1.07 g, 50%) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 5.24 (dd, J = 17.4, 10.9 Hz, 1H), 5.28 (d, J = 10.9 Hz, 1H), 5.24 (d, J = 17.4 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.10 (ABq, ΔδAB = 0.44, JAB = 11.9 Hz, 4H), 1.46 (s, 3H), 1.41 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). LC/MS Method A (m/z) 237.0 [M + Na], Tr = 2.14 min. A solution of 2,2-dimethyl-5-vinyl-[1,3]dioxane-5-carboxylic acid ethyl ester (150 mg, 0.7 mmol) in THF (3 mL) was stirred at 5 °C under N2. A solution of lithium hydroxide monohydrate (59 mg, 1.4 mmol) in water (1 mL) was added, and the reaction mixture was stirred at 5 °C for 30 min and then at room temperature for 5 h. MeOH (0.5 mL) was added to give a clear solution, and the reaction mixture was stirred at room temperature for 22 h. The mixture was concentrated in vacuo. Water (2 mL) was added to the residue, and the solution was acidified to pH 2 with 2 M HCl. Brine was added and the mixture was extracted with EtOAc. The organic extracts were combined and washed with brine. The organic extract was separated, washed with water (×5), and brine. The organic solution was filtered through a hydrophobic frit, and the filtrate was concentrated in vacuo to afford the title compound (117 mg, 90%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 5.76 (dd, J = 17.8, 10.7 Hz, 1H), 5.35 (d, J = 10.7 Hz, 1H), 5.30 (d, J = 17.8 Hz, 1H), 4.32 (d, J = 12.0 Hz, 2H), 3.91 (d, J = 12.0 Hz, 2H), 1.49 (s, 3H), 1.44 (s, 3H). LC/MS Method A (m/z) 185.1 [M − H], Tr = 1.26 min. 6-(Nitromethylene)-1,4-dioxepane (70). MgSO4 (2.05 g, 17.0 mmol) in nitromethane (4 mL) was vigorously stirred under N2 and treated with a solution of 6947 (899 mg, 7.74 mmol) in nitromethane (2 mL). The mixture was stirred at rt for 5 min, and a solution of 2,8,9-triisopropyl-2,5,8,9-tetraaza-1-phosphabicyclo[3,3,3]undecane (231 mg, 250 μL, 0.77 mmol) in nitromethane (2 mL) was then added. The mixture was heated at 40 °C for 19 h, cooled, and directly subjected to silica gel chromatography eluting with pentane/Et2O (1:1 to 1:3) to afford 6-(nitromethyl)-1,4-dioxepan-6-ol (798 mg, 58%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 4.58 (s, 2H), 3.85 (s, 4H), 3.93−3.77 (m, 4H), 3.27 (s, 1H). 6-(Nitromethyl)-1,4dioxepan-6-ol (0.74 g, 4.18 mmol) and Et3N (1.86 g, 2.56 mL, 18.4 mmol) in CH2Cl2 (20 mL), under N2, were cooled to −78 °C. Methanesulfonyl chloride (1.43 g, 970 μL, 12.5 mmol) was added dropwise, and the mixture was stirred for 30 min. The mixture was quenched with saturated ammonium chloride solution, and the organic layer was separated. The aqueous layer was back extracted with CH2Cl2 (20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography eluting with pentane/Et2O (3:1 to 1:1) to afford the title compound (512 mg, 77%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.02 (t, J = 2.5 Hz, 1H), 5.04 (d, J = 2.5 Hz, 2H), 4.36 (d, J = 0.5 Hz, 2H), 3.90− 9496

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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3H), 0.83 (d, J = 6.9 Hz, 3H). LC/MS Method A (m/z) 534.1/536.1 [M + H], Tr = 2.27 min. Cyclophilin A TR-FRET Competitive Binding Assay. Cyclopilin A protein isolation and binding assay procedures were described previously.29 For sanglifehrin A data reported in Table 1, a modified version of the previously described assay was used as described in Supporting Information. Cell Based HCV Replicon Genotype 2a, MT4 Cell Viability Assays, in Vitro and in Vivo Pharmacokinetic Assessments and Crystallography Methods. See Supporting Information. All in vivo studies were performed in accordance with local IACUC guidelines.

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B virus; NTCP, sodium taurocholate cotransporting polypeptide; SAR, structure−activity relationships; EC50, effective concentration for 50% inhibition; PXR, pregnane X receptor; TR-FRET, time-resolved fluorescence resonance energy transfer; CCM, cell culture media; RCM, ring closing metathesis; BOC, tert-butyloxycarbonyl; TCE, trichloroethyl; CC50, concentration for 50% cell viability loss; HATU, O-(7azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide; DMAP, 4-(N,N-dimethylamino)pyridine; hERG, potassium channel of the human ether-à-go-go related gene; IACUC, International Animal Care and Use Committee; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, Tris(2-carboxyethyl)phosphine; SfA, sanglifehrin A

ASSOCIATED CONTENT

* Supporting Information S

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00802. HCV replicon assay methods in huh-7 cells, MT4 viability and TR-FRET assay method for sanglifehrin A; organic compound liquid chromatography mass spectrometry methods; pharmacokinetic in vitro and in vivo methods; X-ray crystallography; 1H NMR spectra for assayed compounds, carbon assignment for compound 3, intramolecular hydrogen bond NMR data, and HPLC spectra (PDF) Molecular formula strings for 3−9, 18−21, 34−35, 47− 51, and 53−54 (CSV)

(1) Lang, K.; Schmid, F. X.; Fischer, G. Catalysis of protein folding by prolyl isomerase. Nature 1987, 329, 268−270. (2) Fischer, G.; Wittmann-Liebold, B.; Lang, K.; Kiefhaber, T.; Schmid, F. X. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 1989, 337, 476−478. (3) Handschumacher, R. E.; Harding, M. W.; Rice, J.; Drugge, R. J.; Speicher, D. W. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 1984, 226, 544−547. (4) Liu, J.; Farmer, J. D.; Lane, W. S.; Friedman, J.; Weissman, I.; Schreiber, S. L. Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes. Cell 1991, 66, 807−815. (5) Rosenwirth, B.; Billich, A.; Datema, R.; Donatsch, P.; Hammerschmid, F.; Harrison, R.; Hiestand, P.; Jaksche, H.; Mayer, P.; Peichl, P. Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog. Antimicrob. Agents Chemother. 1994, 38, 1763−1772. (6) Traber, R.; Kobel, H.; Loosli, H.-R.; Senn, H.; Rosenwirth, B.; Lawen, A. [MeIle4]Cyclosporin, a novel natural cyclosporin with antiHIV activity: structural elucidation, biosynthesis and biological properties. Antiviral Chem. Chemother. 1994, 5, 331−339. (7) Nigro, P.; Pompilio, G.; Capogrossi, M. C. Cyclophilin A: a key player for human disease. Cell Death Dis. 2013, 4, e888. (8) Naoumov, N. V. Cyclophilin inhibition as potential therapy for liver diseases. J. Hepatol. 2014, 61, 1166−1174. (9) Elrod, J. W.; Molkentin, J. D. Physiologic functions of cyclophilin D and the mitochondrial permeability transition pore. Circ. J. 2013, 77, 1111−1122. (10) Hopkins, S.; Gallay, P. A. The role of immunophilins in viral infection. Biochim. Biophys. Acta, Gen. Subj. 2015, 1850, 2103−2110. (11) Flisiak, R.; Horban, A.; Gallay, P.; Bobardt, M.; Selvarajah, S.; Wiercinska-Drapalo, A.; Siwak, E.; Cielniak, I.; Higersberger, J.; Kierkus, J.; Aeschlimann, C.; Grosgurin, P.; Nicolas-Ḿ etral, V.; Dumont, J.-M.; Porchet, H.; Crabbe, R.; Scalfaro, P. The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus. Hepatology 2008, 47, 817−826. (12) Hopkins, S.; DiMassimo, B.; Rusnak, P.; Heuman, D.; Lalezari, J.; Sluder, A.; Scorneaux, B.; Mosier, S.; Kowalczyk, P.; Ribeill, Y.; Baugh, J.; Gallay, P. The cyclophilin inhibitor SCY-635 suppresses viral replication and induces endogenous interferons in patients with chronic HCV genotype 1 infection. J. Hepatol. 2012, 57, 47−54. (13) Keller, D. M. Pancreatitis Events Halt Development of Alisporivir for HCV. Medscape, Apr 19, 2012. (14) Puyang, X.; Poulin, D. L.; Mathy, J. E.; Anderson, L. J.; Ma, S.; Fang, Z.; Zhu, S.; Lin, K.; Fujimoto, R.; Compton, T.; Wiedmann, B. Mechanism of resistance of hepatitis C virus replicons to structurally distinct cyclophilin inhibitors. Antimicrob. Agents Chemother. 2010, 54, 1981−1987. (15) Yang, F.; Robotham, J. M.; Grise, H.; Frausto, S.; Madan, V.; Zayas, M.; Bartenschlager, R.; Robinson, M.; Greenstein, A. E.; Nag, A.; Logan, T. M.; Bienkiewicz, E.; Tang, H. A major determinant of

Accession Codes

Cocrystal structures of Cyp A and 3, 8, 19, 20, 34. Authors will release the atomic coordinates upon article publication.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 650 522 5258. ORCID

Richard L. Mackman: 0000-0001-8861-7205 Victoria A. Steadman: 0000-0003-1583-0202 Brian E. Schultz: 0000-0001-9757-2547 Present Addresses ∥

(K.G.P.) UCB, Avenue de l’Industrie, 1420 Braine-l’Alleud, Belgium. # (G.M.W.) Carbosynth, 8 and 9 Old Station Business Park, Compton, Berkshire, RG20 6NE, United Kingdom. ⊥ (C.A.B.) Pfizer, 280 Shennecossett Rd, Groton, CT 06340, USA. Notes

The authors declare the following competing financial interest(s): The authors are current or former employees of Selcia, Ltd., Cypralis, Ltd., or Gilead Sciences, and may own company stock.

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ACKNOWLEDGMENTS We express our thanks to the following individuals: Kathy Brendza and Loredana Serafini for HRMS analysis, Ke-Yu Wang and Ian Marshall (Selcia) for NMR assistance. This work was funded by Gilead Sciences Inc. and Selcia Ltd.

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ABBREVIATIONS USED Cyp, cyclophilin; HCV, hepatitis C virus; PK, pharmacokinetic; pa, plasma-adjusted; CsA, cyclosporin A; HBV, hepatitis 9497

DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499

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Journal of Medicinal Chemistry

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(44) Bahar, F. G.; Ohura, K.; Ogihara, T.; Imai, T. Species differences of esterase expression and hydrolase activity in plasma. J. Pharm. Sci. 2012, 101, 3979−3988. (45) Ma, X.; Idle, J. R.; Gonzalez, F. J. The pregnane X receptor: From bench to bedside. Expert Opin. Drug Metab. Toxicol. 2008, 4, 895−908. (46) Bercot, E. A.; Caille, S.; Bostick, T. M.; Ranganathan, K.; Jensen, R.; Faul, M. M. Diastereoselective palladium-catalyzed αarylation of 4-substituted cyclohexyl esters. Org. Lett. 2008, 10, 5251− 5254. (47) Dreyer, A.; Doods, H.; Gerlach, K.; Gottschling, D.; Heimann, A.; Mueller, S. G.; Rudolf, K.; Schaenzle, G. New Compounds. International Patent WO 2010139717A1, 2010.

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DOI: 10.1021/acs.jmedchem.8b00802 J. Med. Chem. 2018, 61, 9473−9499