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Article Cite This: J. Med. Chem. 2018, 61, 5138−5153

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Identification of Highly Potent Human Immunodeficiency Virus Type‑1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine

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Koushi Hidaka,† Tooru Kimura,‡ Rajesh Sankaranarayanan,‡ Jun Wang,‡ Keith F. McDaniel,§ Dale J. Kempf,§ Masanori Kameoka,∥ Motoyasu Adachi,⊥ Ryota Kuroki,#,Δ Jeffrey-Tri Nguyen,‡ Yoshio Hayashi,∇ and Yoshiaki Kiso*,○ †

Laboratory of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe 650-8586, Japan Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan § Global Pharmaceutical Research and Development, AbbVie, North Chicago, Illinois 60064, United States ∥ Department of International Health, Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan ⊥ Quantum Beam Science Drectorate, National Institutes for Quantum and Radiological Science and Technology, Tokai, Ibaraki 319-1106, Japan # Quantum Beam Science Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan ∇ Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan ○ Laboratory of Peptide Sciences, Nagahama Institute of Bio-Science and Technology, Nagahama 526-0829, Japan ‡

S Supporting Information *

ABSTRACT: The emergence of drug-resistant HIV from a widespread antiviral chemotherapy targeting HIV protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors. We synthesized a series of allophenylnorstatine-based peptidomimetics with various P3, P2, and P2́ moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild type HIV-1 protease and the virus, leading to a highly potent compound 21f (KNI-1657) against lopinavir/ritonavir- or darunavir-resistant strains. Co-crystal structures of 21f and the wild-type protease revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy to design allophenylnorstatine-based peptidomimetics combined with Thfg residue would be promising for generating candidates to overcome multidrug resistance.

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been, accordingly, recognized as a controllable chronic illness.4 Despite the therapeutic success, drug resistance and side effects still remain a great concern in long-term treatment. Resistance to HIV protease inhibitors is one of the problems because of the market for generic drugs.5 The situation is complicated by the existence of other HIV-1 subtypes and circular recombinant

INTRODUCTION In the past decades, antiretroviral therapy for human immunodeficiency virus type-1 (HIV-1) infection using nucleoside and non-nucleoside reverse transcriptase inhibitors, and protease inhibitors has advanced to become convenient for patients because of the once-daily dosing regimen and reduced numbers of orally administered tablets and capsules.1 Recent approvals of entry and integrase inhibitors provide new options for patients struggling with drug resistance.2,3 HIV/AIDS has © 2018 American Chemical Society

Received: November 30, 2017 Published: May 31, 2018 5138

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

Figure 1. Allophenylnorstatine-containing HIV protease inhibitors 1 and 2.

forms because of the altered susceptibility to drugs.6,7 Therefore, the development of alternative HIV protease inhibitors is still a challenge for researchers.8 Progress on recently approved HIV protease inhibitors led to drugs that possess a high genetic barrier against the virus. Lopinavir, as an example, requires more than six mutations to lose ten times its antiviral activity in vitro.9 Another example, darunavir, is now the first option for therapeutic regimens and would take more than one hundred days to induce drug resistance.10 Nevertheless, a research group of Abbott Laboratories has generated a lopinavir/ritonavir resistant HIV strain, A17.11 The mutant clone displayed approximately 50fold resistance to lopinavir. Although the mutation sites were identified in the protease coding region, a detailed study on the decreasing activity was not reported. Koh et al. reported high resistance to darunavir using mixed clones from four patients with therapeutic failure.12 A combination of the four mutations surprisingly attenuated the activity approximately 100-fold. Overcoming these resistances is the target of developing nextgeneration inhibitors. Allophenylnorstatine [Apns: (2S,3S)-3-amino-2-hydroxy-4phenylbutyric acid] is an unnatural α-hydroxy-β-amino acid residue that contains a hydroxymethylcarbonyl (HMC) group as a transition-state isostere to act as a P1 residue in peptidic HIV protease inhibitors.13−15 The ideal binding interactions of HMC isostere with two catalytic Asp residues of HIV-1 protease have been disclosed by neutron crystallography16 in addition to former X-ray crystallography17 and NMR analyses.18 Previously developed Apns-based compounds 1 and 2, known as KNI-272 and KNI-764, respectively, are highly potent against the protease and the virus, and both were tested orally in clinical studies (Figure 1).19,20 Modifications of these inhibitors were extensively studied for improving antiviral activity against drug resistant strains, water-solubility, and pharmacokinetic profile.21−24 Their effective potency against resistances was unfortunately limited. To overcome the multidrug-resistant mutations, we planned a series of Apnsbased tripeptidic compounds with a combination of residues and terminal caps that were selected from previous experiments exhibiting high protease inhibitory potency. Especially, unnatural P2 residue tetrahydrofuranylglycine (Thfg), originally designed by Ghosh et al.25 and further exploited in urethanes in amprenavir26 and darunavir,27 is newly applied to the Apnsbased derivatives, leading to compounds with highly potent anti-HIV activity, even against lopinavir/r- or darunavirresistant strains.

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LIGAND SELECTIONS FOR COMBINATIONAL MODIFICATIONS The general structure of the HIV protease inhibitor synthesized in this study is depicted in Figure 2. Our combinational

Figure 2. Incorporation of Thfg at the P2 residue and the corresponding binding subsites.

modifications were intended to be structurally focused on yielding high potency against the protease. First, Apns was chosen as the P1 residue from a previous comparison with phenylnorstatine.15 (R)-5,5-Dimethylthiazolidine-4-carboxylic acid (Dmt) was also selected as the P1́ residue from the reported proline-based modifications.19 Three residues were selected to optimize the P2 residue. Asn was known as the most effective for protease inhibition among many natural and nonnatural amino acid residues. Val was also selected with a little less potency than Asn. In addition to the above branched residues, Thfg, which is a hybrid structure of Asn and Val, was applied. The P3 cap was searched among our results from previous structure−activity relationship studies.24 Three types of phenoxyacetyl moieties, including 5-isoquinolinyloxyacetyl in compound 1 and the oxygen-mimicking chromonylcarbonyl and bezofurancarbonyl groups, were chosen. The seven P2́ amide residues were selected to include β-methallylamide from a previous study on highly potent dipeptidic inhibitors such as KNI-1689.22

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CHEMISTRY The preparation of Thfg was performed as reported28 (Scheme 1). (R)-3-Hydroxytetrahydrofuran (3) was converted to the substituted malonate 4, and then decarboxylated with cuprous oxide to generate tetrahydrofuranylacetic acid 5. The acid was coupled to (S)-Evans auxiliary and then subjected to stereoselective and electrophilic azidation using trisyl azide to afford (2S,3′R)-azidotetrahydrofuranylacetate 7. The intermediate was subjected to reduction using SnCl2 in ethanol and in situ Boc protection to give a mixture of 8a and 8b in 45 and 30% yields, 5139

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

Scheme 1a

Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, −10 °C, 2 h; (b) NaH, CH2(CO2Et)2, DMF, reflux, 20 h; (c) 1 M NaOH aq., 40 °C, 72 h then H3O+; (d) Cu2O, CH3CN, 80 °C, 12 h; (e) Me3COCl, Et3N, THF, −78 °C then N-lithio-(S)-(−)-4-benzyloxazolidine, 2 h; (f) KN(TMS)2, THF, −78 °C, 30 min then trisyl azide, −78 °C, 2 min then AcOH, 35 °C, 1 h; (g) SnCl2, EtOH, 0 °C, 3.5 h; (h) (Boc)2O, NaHCO3 aq., dioxane, rt, 19 h; (i) LiOH, THF−H2O, rt, 2.5 h. a

then removal of the Boc group and condensations with six types of P3 carboxylic acids afforded compounds 21a−f. Similarly, compounds 24a−f were synthesized from P2′intermediates except for the last BOP coupling procedure with P3 3-(dimethylamino)phenoxyacetic acid as shown in Scheme 4. Scheme 5 describes the P2 derivative synthesis using Boc-Asn-OH and Boc-Val-OH to couple with the amine generated from 19. The subsequent deprotection and BOP coupling with benzofurancarboxylic acid derivatives gave the compounds 27a−d.

respectively. Both were hydrolyzed to give (2S,3′R)-Boc-ThfgOH (9). In the case of (2S,3′S)-isomer, (S)-3-hydroxytetrahydrofuran was used as the starting material and transformed in a manner similar to the (R)-form, and the intermediate azide 11 with Evans auxiliary attached was hydrolyzed using LiOH to yield (2S,3′S)-2-azidotetrahydrofuranylacetic acid 12. P3 phenoxyacetic acid analogues were synthesized from the phenol derivatives as described before.24 Scheme 2 illustrates the synthesis of Thfg-containing inhibitors. Compounds 15a and 15b were obtained from a previously reported intermediate, Boc-protected dipeptide carboxamides 13.20 The deprotection of the Boc group using HCl-dioxane gave the amine component to react with 9 using EDC−HOBt coupling reagents, and the subsequent deprotection and BOP coupling with quinolinecarboxylic acid derivatives afforded the desired compounds. The (2S,3′S)-Thfg isomer was incorporated as an azido form to give the intermediate 16. The reduction to amine and BOP coupling with the P3 carboxylic acids gave compounds 17a and 17b in good yields. The intermediate 14 was deprotected and used to combine 5isoquinolinyloxyacetic acid to give compound 18. Preparation of a series of P3 modifications was started from P2′-dimethylbenzyl intermediate 19 (Scheme 3). The Boc group was removed to couple with (2S,3′R)-Boc-Thfg-OH, and

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RESULTS AND DISCUSSION To test the matching of Thfg residue with Apns-based peptidomimetics, we first combined a P3−P2 structure based on Merck’s compounds,25 quinolinecarbonyl-Thfg, with ApnsDmt-(2-methyl)benzylamide of 2. Compound 15a with P2 (2S,3′R)-Thfg exhibited highly potent HIV-1 protease inhibitory activity, 96% inhibition at 1 nM inhibitor concentration (Table 1). As expected from the results in Merck’s report, compound 17a with P2 (2S,3′S)-Thfg was less potent than compound 15a. The EC50 value of compound 15a was less than 3 nM. The anti-HIV activity of 17a was weak (26 nM), in agreement with its protease inhibition. The activities of these Thfg-containing derivatives were relatively potent compared 5140

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

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Scheme 2a

a Reagents and conditions: (a) 4 M-HCl/dioxane, anisole, rt, 15−60 min; (b) 9, EDC·HCl, HOBt, Et3N, DMF, rt, 14 h; (c) 2-quinolinecarboxylic acid or 2-quinoxalinecarboxylic acid, BOP, Et3N, DMF, rt, 14−19 h, then RP-HPLC purification; (d) 12, EDC·HCl, HOBt, Et3N, DMF, rt, 13 h, then RP-HPLC purification; (e) 5% Pd(OH)2, EtOH-THF, rt, 10 h; (f) 7-isoquinolinyloxyacetic acid, BOP, HOBt, Et3N, DMF, rt, 14 h, then RPHPLC purification.

Scheme 3a

a Reagents and conditions: (a) 4 N HCl/dioxane, anisole, rt, 15−30 min; (b) 9, EDC·HCl, HOBt, Et3N, DMF, rt, 21 h; (d) carboxylic acid derivatives, BOP, Et3N, DMF, rt, 12−40 h, then RP-HPLC purification.

with the parent compounds 1 and 2 (176 and 60 nM, respectively). Replacement of P3 quinolinecarbonyl with quinoxalinecarbonyl resulted in a slight increase in protease inhibition. An improvement was also observed in the anti-HIV activity under 50% human serum. Additional aromatic nitrogen may increase the basicity affecting the cellular membrane permeability. These successful results on the combination with Thfg urged us to optimize the P3 position. Selection of the 5-isoquinolinyloxyacetyl moiety was derived from inhibitor 1. The resultant compound 18 exhibited sufficient HIV-1 protease inhibition and anti-HIV activity

with EC50 of 21 nM (Table 2). However, the activity under 50% human serum was dramatically attenuated to 4.35 μM. We shifted to use the P2́ structure with an additional o-methyl group, that is 2,6-dimethylbenzylamide, which is the most favorable P2́ for enzyme inhibition among the previously reported dipeptidic inhibitors.22 Compound 21a exhibited 98% enzyme inhibition and 60 nM anti-HIV activity. Interestingly, the addition of serum slightly affected the activity with a serum binding effect of 2.4. Replacement with 3-(phenylamino)phenoxyacetyl (21b) increased the anti-HIV activity up to 5 nM. A minor variant with the dimethylamino moiety in 5141

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

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Scheme 4a

a Reagents and conditions: (a) 4 N HCl/dioxane, anisole, rt, 15−60 min for a, b, c, f or 2−5 min for d, e; (b) 9, EDC·HCl, HOBt, Et3N, DMF, rt, 13−21 h; (d) (3-dimethylamino)phenoxyacetic acid, BOP, Et3N, DMF, rt, 13−23 h, then RP-HPLC purification.

Scheme 5a

a

Reagents and conditions: (a) 4 N HCl/dioxane, anisole, rt, 30 min; (b) Boc-Val-OH or Boc-Asn-OH, EDC·HCl, HOBt, Et3N, DMF, rt, 14−15 h; (d) benzofuran-2-carboxylic acid or 7-methoxybenzofuran-2-carboxylic acid, BOP, Et3N, DMF, rt, 14−15 h, and prep. HPLC.

Table 1. HIV Protease Inhibitory and Anti-HIV Activity of P2-Thfg Derivativesa

Anti-HIV-1pNL4−3, EC50 (nM) Compound

X

Configuration of Thfg

HIV-1 PR % inhibition at 1 nM

0% HS

50% HS

SBE

15a (KNI-1640) 15b (KNI-1639) 17a (KNI-1603) 17b (KNI-1609) 1 2

CH N CH N

(2S,3′R) (2S,3′R) (2S,3′S) (2S,3′S)

96 97 69 86 14 48

137 21 23 11 16 2

a

HS, human serum; SBE, serum binding effect, EC50 with 50% HS/EC50 0% HS. % inhibition values were obtained by single determination. EC50 values were averaged from multiple determinations (n = 3).31 bIIIB strain.

compound 21c improved anti-HIV inhibition (95%. The purity of compound 27a was determined to be 90.2%. Analytical HPLC was performed using a

Figure 4. X-ray crystal structures of 21f bound to wild-type (PDB ID, 5YOK) and lopinavir-resistant (PDB ID, 5YOJ) proteases. (a) Hydrogen bond interactions in the active sites of wild-type HIV-1 protease. (b) Superimposition of cocrystals complexed with WTm5 (green) and A17m5 (red). Side chains of the mutated residues in the S2/S2′ pockets were presented with molecular surfaces. Figures were generated using PyMOL (Schrödinger LLC, OR, SA). 5146

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

C18 reversed-phase column (4.6 × 150 mm; YMC Pack ODS AM302) with binary solvent systems: (A) linear gradient of CH3CN 40−100% in 0.1% aqueous TFA in 15 min, (B) linear gradient of CH3CN 20−80% in 0.1% aqueous TFA in 30 min at a flow rate of 0.9 mL/min, detected at 230 nm. Preparative HPLC was carried out on a C18 reversed-phase column (20 × 250 mm; YMC Pack ODS SH343− 5) with a binary solvent system: a linear gradient of CH3CN in 0.1% aqueous TFA with a flow rate of 5.0 mL/min and detection at 230 nm. 1 H NMR spectra were recorded on a JNM-AL 300 (JEOL, Ltd.), UNITY INOVA 400NB (Varian, Inc.), or DPX400 (Bruker, Ltd.) spectrometer with TMS as an internal standard. Mass spectra with electrospray ionization using 50% aqueous methanol as mobile phase were recorded on a micrOTOF-Q II spectrometer (Bruker, Ltd.). (S)-2-tert-Butoxycarbonylamino-2-((R)-tetrahydrofuran-3yl)acetic Acid (9). 1H NMR (400 MHz, DMSO-d6): δ (ppm): 12.56 (br s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 3.78 (t, J = 8.8 Hz, 1H), 3.74− 3.69 (m, 2H), 3.61 (dd, J = 15.8, 7.3 Hz, 1H), 3.39 (dd, J = 8.4, 7.0 Hz, 1H), 2.45−2.44 (m, 1H), 1.91−1.85 (m, 1H), 1.70−1.63 (m, 1H), 1.38 (s, 9H). TOF-MS (ESI) m/z: calcd for C11H19NO5Na [M + Na]+ 268.1155; found 268.1140. (S)-2-Azido-2-((S)-tetrahydrofuran-3-yl)acetic Acid (12). 1H NMR (300 MHz, CDCl3) δ (ppm): 3.99−3.92 (m, 2H), 3.91 (d, J = 7.5 Hz, 1H), 3.80 (dd, J = 16.0, 7.5 Hz, 1H), 3.72 (dd, J = 9.2, 6.6 Hz, 1H), 2.74 (dt, J = 14.9, 7.5 Hz, 1H), 2.18−2.07 (m, 1H), 1.98−1.86 (m, 1H). TOF-MS (ESI) m/z: calcd for C6H8N3O3 [M − H]− 170.0571; found 170.0567. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (14). Compound 14 was prepared from BocApns-Dmt-NH(2-methylbenzyl) 1320 in a similar manner to that described for compound 20; yield 65%. 1H NMR (400 MHz, DMSOd6) δ (ppm): 8.38 (br s, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.32−7.30 (m, 1H), 7.28 (d, J = 7.3 Hz, 3H), 7.20−7.07 (m, 6H), 6.93 (d, J = 9.2 Hz, 1H), 5.26 (d, J = 7.0 Hz, 1H), 5.01 (d, J = 9.0 Hz, 1H), 4.93 (d, J = 9.2 Hz, 1H), 4.50 (s, 1H), 4.43 (d, J = 6.2 Hz, 1H), 4.40−4.34 (m, 2H), 4.27−4.2 (br s, 1H), 4.17 (dd, J = 14.8, 4.6 Hz, 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.65−3.59 (m, 1H), 3.56 (t, J = 8.1 Hz, 1H), 3.46 (dd, J =15.4, 7.5 Hz, 1H), 3.27 (t, J = 7.0 Hz, 1H), 2.73 (br d, J = 11.7 Hz, 1H), 2.63 (br d, J = 13.6 Hz, 1H), 2.27 (s, 3H), 2.28−2.25 (m, 1H), 1.50 (s, 3H), 1.44 (m, 2H), 1.37 (s, 9H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C35H49N4O7S [M + H]+ 669.3316; found 669.3288. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoline-2carbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (15a, KNI-1640). A mixture of intermediate 14 (84.9 mg, 0.13 mmol), anisole (27.6 μL, 0.25 mmol), and 4 M HCl in dioxane (1.27 mL) was stirred for 60 min at rt. After removal of the solvent in vacuo, the residue was precipitated from ether to give the hydrochloride salt. To a solution of the salt in DMF (2 mL) were added triethylamine (35.4 μL, 0.25 mmol), quinoline-2-carboxylic acid (24.2 mg, 0.14 mmol), and BOP (67.2 mg, 0.15 mmol) in an ice bath, and the mixture was stirred overnight at room temperature. After removal of the solvent in vacuo, the residue was added into EtOAc, washed sequentially with 5% NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo to give a product as a solid, 78 mg. Purification of the product by preparative HPLC gave compound 15a as a white powder; yield 85%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.78 (d, J = 9.3 Hz, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.18−8.15 (m, 2H), 8.10 (dd, J = 8.4, 1.1 Hz, 1H), 7.88 (dt, J = 7.6, 1.5 Hz, 1H), 7.74 (dt, J = 7.4, 1.1 Hz, 1H), 7.31−7.29 (m, 1H), 7.27 (d, J = 7.1 Hz, 2H), 7.17−7.06 (m, 6H), 6.96 (t, J = 7.5 Hz, 1H), 5.34 (d, J = 7.1 Hz, 1H), 5.06 (d, J = 9.2 Hz, 1H), 4.98 (d, J = 9.0 Hz, 1H), 4.53 (t, J = 8.8 Hz, 1H), 4.51 (s, 1H), 4.46−4.38 (m, 2H), 4.3−4.23 (m, 1H), 4.18 (dd, J = 15.0, 4.8 Hz, 1H), 3.71 (dt, J = 7.9, 5.1 Hz, 1H), 3.64 (dd, J = 8.8, 7.3 Hz, 1H), 3.54 (dd, J = 15.8, 7.7, Hz, 1H), 3.45 (dd, J = 8.8, 6.4 Hz, 1H), 2.75 (dd, J = 13.6, 2.2 Hz, 1H), 2.67−2.61 (m, 1H), 2.26 (s, 3H), 1.66− 1.59 (m, 2H), 1.51 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C40H46N5O6S [M + H]+ 724.3163; found 724.3161.

(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoxaline-2carbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (15b, KNI-1639). Compound 15b was prepared from compound 14 using quinoxaline-2-carboxylic acid in a manner similar to that described for compound 15a; yield 61%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 9.48 (s, 1H), 8.77 (d, J = 9.1 Hz, 1H), 8.45−8.35 (m, 2H), 8.27−8.20 (m, 2H), 8.04−7.97 (m, 2H), 7.33−7.25 (m, 3H), 7.13−7.05 (m, 6H), 6.98−6.92 (m, 1H), 5.34 (d, J = 7.2 Hz, 1H), 5.06 (d, J = 8.9 Hz, 1H), 4.98 (d, J = 9.2 Hz, 1H), 4.56−4.49 (m, 2H), 4.47−4.38 (m, 2H), 4.31−4.25 (m, 1H), 4.18 (dd, J = 15.0, 4.77 Hz, 1H), 3.75−3.69 (m, 1H), 3.65 (dd, J = 8.7, 7.2 Hz, 1H), 3.54 (q, J = 7.6 Hz, 1H), 3.46−3.42 (m, 1H), 2.79−2.73 (m, 1H), 2.69−2.61 (m, 2H), 2.26 (s, 3H), 1.65−1.58 (m, 2H), 1.51 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C39H45N6O6S [M + H]+ 725.3116; found 725.3091. Anal. Calcd for C39H44N6O6S·1CF3COOH·0.4H2O: C, 58.2; H, 5.46; N, 9.93. Found: C, 58.26; H, 5.56; N, 9.81. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-azido-2-((S)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]5,5-dimethyl-1,3-thiazolidine-4-carboxamide (16). Compound 16 was prepared from 13 in a similar manner to that described for compound 20; yield quantitative. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.50 (d, J = 8.6 Hz, 1H), 8.41 (t, J = 5.4 Hz, 1H), 7.33−7.24 (m, 3H), 7.23−7.17 (m, 2H), 7.17−7.10 (m, 4H), 5.41 (d, J = 7.1 Hz, 1H), 5.05−4.93 (m, 2H), 4.51 (s, 1H), 4.45−4.37 (m, 2H), 4.27−4.16 (m, 2H), 3.73−3.70 (m, 1 H), 3.69−3.65 (m, 1H), 3.62−3.57 (m, 2H), 3.44−3.40 (m, 1H), 2.77−2.71 (m, 1H), 2.66−2.59 (m, 1H), 2.27 (s, 3H), 1.95−1.87 (m, 1H), 1.73 (s, 9H), 1.69−1.55 (m, 2H), 1.50 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C30H39N6O5S [M + H]+ 595.2697; found 595.2672. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoline-2carbonyl)amino-2-((S)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (17a, KNI-1603). A mixture of compound 16 (680 mg, 1.14 mmol) and 5% Pd(OH)2 (50 mg) in ethanol−THF (1:1, 40 mL) was stirred under hydrogen gas for 10 h at rt. After removal of the solvent in vacuo, the residue was purified by silica gel column chromatography to give the amino intermediate. To a solution of the intermediate in DMF were added triethylamine, quinoline-2-carboxylic acid, and BOP in an ice bath, and the mixture was stirred overnight at room temperature. After removal of the solvent in vacuo, the residue was added into EtOAc, washed sequentially with 5% NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo to give a product as a solid. Purification of the product by preparative HPLC gave compound 17a as a white powder; yield 83%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.80 (d, J = 9.2 Hz, 1H), 8.60 (d, J = 8.6 Hz, 1H), 8.43−8.36 (m, 2H), 8.18−8.15 (m, 2H), 8.10 (dd, J = 8.1, 0.7 Hz, 1H), 7.88 (dt, J = 7.9, 1.1 Hz, 1H), 7.74 (t, J = 7.1 Hz, 1H), 7.31− 7.29 (m, 1H), 7.25 (d, J = 7.3 Hz, 2H), 7.15−7.08 (m, 4H), 7.06 (d, J = 7.5 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 5.39 (d, J = 7.6 Hz, 1H), 5.05 (d, J = 9.0 Hz, 1H), 4.98 (d, J = 9.2 Hz, 1H), 4.54 (d, J = 8.8 Hz, 1H), 4.51 (s, 1H), 4.46−4.38 (m, 2H), 4.29−4.23 (m, 1H), 4.17 (dd, J = 15.0, 4.8 Hz, 1H), 3.70 (dt, J = 8.1, 4.9 Hz, 1H), 3.57 (dd, J = 15.4, 7.5 Hz, 1H), 3.50 (t, J = 7.5 Hz, 1H), 3.43−3.42 (m, 1H), 2.76−2.61 (m, 3H), 2.26 (s, 3H), 1.90−1.83 (m, 1H), 1.67−1.59 (m, 1H), 1.51 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C40H46N5O6S [M + H]+ 724.3163; found 724.3147; Anal. Calcd for C40H45N5O6S· 0.1H2O: C, 66.20; H, 6.28; N, 9.65. Found: C, 66.22; H, 6.19; N, 9.47. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(quinoxaline-2carbonyl)amino-2-((S)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (17b, KNI-1609). Compound 17b was prepared from compound 16 in a manner similar to that described for compound 17a; yield 86%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.48 (s, 1H), 8.80 (d, J = 9.0 Hz, 1H), 8.40−8.38 (m, 2H), 8.26−8.2 (m, 2H), 7.31−7.29 (m, 1H), 7.25 (d, J = 7.0 Hz, 2H), 7.12−7.07 (m, 3H), 7.05 (d, J = 7.7 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 5.4 (d, J = 7.3 Hz, 1H), 5.05 (d, J = 9.0 Hz, 1H), 4.98 (d, J = 9.3 Hz, 1H), 4.52 (t, J = 9.0 Hz, 1H), 4.51 (s, 1H), 4.45−4.38 (m, 2H), 4.28−4.23 (m, 1H), 4.18 (dd, J = 14.8, 5.0 Hz, 1H), 3.70 (dt, J = 8.1, 5.0 Hz, 1H), 3.57 (dd, J = 15.6, 7.5 Hz, 1H), 3.52−3.32 (m, 2H, overlapped with water), 2.77−2.63 5147

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

1,3-thiazolidine-4-carboxamide (21b, KNI-1650). Compound 21b was prepared from compound 20 in a manner similar to that described for compound 15a; yield 57%. 1H NMR (400 MHz, DMSOd6) δ (ppm): 8.24 (d, J = 8.6 Hz, 1H), 8.20 (br s, 1H), 8.11 (dd, J = 5.6, 3.7 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.23−7.22 (m, 3H), 7.20 (d, J = 7.7 Hz, 1H), 7.01−7.05 (m, 3H), 7.01 (d, J = 7.5 Hz, 2H), 6.84 (t, J = 7.0 Hz, 1H), 6.67 (dd, J = 8.1, 1.8 Hz, 1H), 6.63 (t, J = 2.2 Hz, 1H), 6.36 (dd, J = 8.2, 2.4 Hz, 1H), 5.05 (d, J = 9.0 Hz, 1H), 4.91 (d, J = 9.0 Hz, 1H), 4.53 (s, 1H), 4.50 (d, J = 14.8 Hz, 1H), 4.46−4.40 (m, 1H), 4.41 (d, J = 15.0 Hz, 1H), 4.40 (d, J = 2.6 Hz, 1H), 4.27 (t, J = 8.6 Hz, 1H), 4.21−4.15 (m, 1H), 4.17 (dd, J = 13.9, 3.3 Hz, 1H), 3.60 (dd, J = 13.4, 8.4 Hz, 1H), 3.50−3.42 (m, 2H, overlapped with water), 3.25−3.21 (m, 1H), 2.71−2.61 (m, 2H), 2.41−2.36 (m, 1H), 2.30 (s, 6H), 1.51− 1.45 (m, 1H), 1.45 (s, 3H), 1.40−1.29 (m, 1H), 1.35 (s, 3H). TOFMS (ESI) m/z: calcd for C45H54N5O7S [M + H]+ 808.3738; found 808.3704. Anal. Calcd for C45H53N5O7S·0.1CF3COOH: C, 66.25; H, 6.53; N, 8.55. Found: C, 66.21; H, 6.66; N, 8.41. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl1,3-thiazolidine-4-carboxamide (21c, KNI-1652). Compound 21c was prepared from compound 20 in a manner similar to that described for compound 15a; yield 45%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.26 (d, J = 8.0 Hz, 1H), 8.13−8.09 (m, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.33 (d, J = 7.0 Hz, 2H), 7.21 (t, J = 7.3 Hz, 2H), 7.12−7.06 (m, 3H), 7.01 (d, J = 7.5 Hz, 2H), 6.37 (d, J = 7.6 Hz, 1H), 6.27 (s, 1H), 6.22 (d, J = 8.0 Hz, 1H), 5.06 (d, J = 8.7 Hz, 1H), 4.92 (d, J = 9.0 Hz, 1H), 4.54 (s, 1H), 4.50 (d, J = 15.2 Hz, 1H), 4.43 (m, 1H), 4.41 (d, J = 15.2 Hz, 1H), 4.408 (d, J = 2.0 Hz, 1H), 4.03 (t, J = 8.8 Hz, 1H), 4.21−4.17 (m, 2H), 3.64−3.59 (m, 1H), 3.52−3.46 (m, 2H), 3.28−3.22 (m, 1H), 2.87 (s, 6H), 2.74−2.58 (m, 2H), 2.30 (s, 6H), 2.02−1.95 (m, 1H), 1.55−1.49 (m, 2H), 1.46 (s, 3H), 1.35 (s, 3H). TOF-MS (ESI) m/z: calcd for C41H54N5O7S [M + H]+ 760.3738; found 760.3723. Anal. Calcd for C41H53N5O7S·0.2CF3COOH: C, 63.52; H, 6.85; N, 8.95. Found: C, 63.46; H, 6.94; N, 9.01. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(chromon2-carbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (21d, KNI-1654). Compound 21d was prepared from compound 20 in a manner similar to that described for compound 15a; yield 42%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.88 (d, J = 8.6 Hz, 1H), 8.33 (d, J = 8.2 Hz, 1H), 8.10 (br. s, 1H), 8.06 (dd, J = 7.9, 1.7 Hz, 1H), 7.90 (dt, J = 7.9, 1.8 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.55 (dt, J = 7.6, 0.9 Hz, 1H), 7.33 (d, J = 7.1 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 7.07−7.03 (m, 2H), 7.00 (d, J = 7.3 Hz, 2H), 6.84 (s, 1H), 5.3 (d, J = 7.1 Hz, 1H), 5.06 (d, J = 9.0 Hz, 1H), 4.94 (d, J = 9.0 Hz, 1H), 4.53 (s, 1H), 4.47−4.43 (m, 2H), 4.40 (t, J = 9.0 Hz, 1H), 4.23−4.2 (m, 1H), 4.17 (dd, J = 13.9, 3.3 Hz, 1H), 3.75 (dt, J = 8.1, 5.1 Hz, 1H), 3.69 (dd, J = 8.4, 7.0 Hz, 1H), 3.59 (dt, J = 15.1, 7.5 Hz, 1H), 3.40 (dd, J = 8.8, 6.4 Hz, 1H), 2.75 (dt, J = 13.7, 2.4 Hz, 1H), 2.69−2.59 (m, 2H), 2.30 (s, 6H), 1.72−1.69 (m, 1H), 1.68−1.62 (m, 1H), 1.45 (s, 3H), 1.34 (s, 3H). TOF-MS (ESI) m/z: calcd for C41H47N4O8S [M + H]+ 755.3109; found 755.3094. Anal. Calcd for C41H46N4O8S·0.75H2O: C, 64.09; H, 6.23; N, 7.29. Found: C, 64.06; H, 6.13; N, 7.27. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (21e, KNI-1666). Compound 21e was prepared from compound 20 in a manner similar to that described for compound 15a; yield 39%; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.55 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.15−8.08 (m, 1H), 7.79 (d, J = 7.0 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 7.49 (t, J = 8.2 Hz, 1H), 7.38−7.34 (m, 3H), 7.15 (d, J = 7.3 Hz, 2H), 7.06 (dd, J = 14.8, 6.8 Hz, 2H), 7 (d, J = 7.5 Hz, 2H), 5.05 (d, J = 9.0 Hz, 1H), 4.93 (d, J = 9.3 Hz, 1H), 4.53 (s, 1H), 4.47−4.36 (m, 3H), 4.25−4.14 (m, 2H), 3.72−3.63 (m, 2H), 3.52 (dd, J = 15.2, 10.4 Hz, 1H), 3.46−3.31 (m, 1H, overlapped with water), 2.74−2.59 (m, 2H), 2.49−2.45 (m, 1H), 2.30 (s, 6H), 1.54−1.47 (m, 2H), 1.45 (s, 3H), 1.35 (s, 3H), 1.31−1.20 (m, 1H). TOF-MS (ESI) m/z: calcd for

(m, 3H), 2.25 (s, 3H), 1.91−1.86 (m, 1H), 1.67−1.58 (m, 1H), 1.50 (s, 3H), 1.35 (s, 3H). TOF-MS (ESI) m/z: calcd for C39H45N6O6S [M + H]+ 725.3116; found 725.3099. Anal. Calcd for C39H44N6O6S: C, 64.62; H, 6.12; N, 11.59. Found: C, 64.33; H, 6.14; N, 11.30. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((S)-2-(isoquinolin-5yloxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (18, KNI-1641). Compound 18 was prepared from compound 14 in a manner similar to that described for compound 15a; yield 66%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.29 (s, 1H), 8.54 (d, J = 5.9 Hz, 1H), 8.38 (t, J = 6.0 Hz, 1H), 8.20 (d, J = 8.8 Hz, 2H), 8.02 (dt, J = 5.9, 0.9 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.31−7.29 (m, 1H), 7.26 (d, J = 7.7 Hz, 2H), 7.17 (t, J = 7.5 Hz, 3H), 7.15−7.09 (m, 4H), 5.4 (d, J = 7.1 Hz, 1H), 5.03 (d, J = 9.0 Hz, 1H), 4.96 (d, J = 9.2 Hz, 1H), 4.80 (d, J = 14.7 Hz, 1H), 4.72 (d, J = 14.7 Hz, 1H), 4.49 (s, 1H), 4.43−4.37 (m, 2H), 4.32 (t, J = 8.4 Hz, 1H), 4.26−4.21 (m, 1H), 4.17 (dd, J = 14.8, 4.6 Hz, 1H), 3.66 (dt, J = 8.1, 4.8 Hz, 1H), 3.57−3.48 (m, 2H), 3.40−3.30 (m, 1H, overlapped with water), 2.77 (dd, J = 14.1, 2.8 Hz, 1H), 2.69−2.63 (m, 2H), 2.26 (s, 3H), 1.68−1.61 (m, 1H), 1.59−1.52 (m, 1H), 1.50 (s, 3H), 1.34 (s, 3H). TOF-MS (ESI) m/z: calcd for C41H48N5O7S [M + H]+ 754.3269; found 754.3244. Anal. Calcd for C41H47N5O7S· 1.5H2O: C, 63.06; H, 6.45; N, 8.97. Found: C, 63.02; H, 6.21; N, 8.85. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (20). A mixture of Boc-Apns-Dmt-NH(2,6dimethylbenzyl) 1922 (789 mg, 1.42 mmol), anisole (307 μL, 2.84 mmol), and 4 M HCl in dioxane (4.6 mL) was stirred for 30 min at rt. After removal of the solvent in vacuo, the residue was precipitated from ether to give a white solid. To a solution of the hydrochloride salt in DMF (15 mL) were added triethylamine (198 μL, 1.29 mmol), HOBt·H2O (198 mg, 1.29 mmol), Boc-(2S,3R)-Tfg−OH 9 (316 mg, 1.29 mmol), and EDC·HCl (247 mg, 1.29 mmol) in an ice bath, and the mixture was stirred overnight at room temperature. After removal of the solvent in vacuo, the residue was added into EtOAc, washed sequentially with 10% citric acid, 5% NaHCO3, and brine, dried over MgSO4, and concentrated in vacuo to give the titled compound as an amorphous solid; quantitative yield. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.15−8.07 (m, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 7.2 Hz, 2H), 7.25−7.19 (m, 2H), 7.17−7.13 (m, 1H), 7.10−7.05 (m, 1H), 7.03−6.99 (m, 2H), 6.92 (d, J = 9.2 Hz, 1H), 5.15 (d, J = 7.5 Hz, 1H), 5.03 (d, J = 9.1 Hz, 1H), 4.90 (d, J = 8.9 Hz, 1H), 4.54 (s, 1H), 4.47 (dd, J = 13.9, 6.1 Hz, 1H), 4.37 (dd, J = 7.3, 2.0 Hz, 1H), 3.78 (t, J = 9.2 Hz, 1H), 4.24−4.13 (m, 2H), 3.62 (td, J = 8.0, 4.8 Hz, 1H), 3.55 (t, J = 7.9 Hz, 1H), 3.46 (q, J = 7.7 Hz, 1H), 3.28−3.24 (m, 1H), 2.73−2.61 (m, 2H), 2.31 (s, 6H), 2.24 (dd, J = 16.3, 7.9 Hz, 1H), 1.46 (s, 3H), 1.41−1.24 (m, 14H). TOF-MS (ESI) m/z: calcd for C36H51N4O7S [M + H]+ 683.3473; found 683.3440. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(isoquinolin-5yloxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (21a, KNI-1648). Compound 21a was prepared from compound 20 in a manner similar to that described for compound 15a; yield 76%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.56 (s, 1H), 8.62 (d, J = 6.0 Hz, 1H), 8.29 (br s, 1H), 8.28 (d, J = 6.8 Hz, 1H), 8.24 (d, J = 8.8 Hz, 1H), 8.12 (dd, J = 5.9, 3.7 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 7.1 Hz, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.21 (t, J = 7.3 Hz, 2H), 7.15−7.05 (m, 2H), 7.01 (d, J = 7.3 Hz, 2H), 5.07 (d, J = 8.8 Hz, 1H), 4.92 (d, J = 8.8 Hz, 1H), 4.85 (d, J = 14.7 Hz, 1H), 4.78 (d, J = 14.7 Hz, 1H), 4.53 (s, 1H), 4.47−4.42 (m, 2H), 4.35 (t, J = 8.4 Hz, 1H), 4.22−4.17 (m, 1H), 4.17 (dd, J = 13.7, 4.2 Hz, 1H), 3.67 (dt, J = 8.4, 5.0 Hz, 1H), 3.58−3.32 (m, 3H, overlapped with water), 2.75−2.60 (m, 3H), 2.30 (s, 6H), 1.70−1.64 (m, 1H), 1.62−1.55 (m, 1H), 1.45 (s, 3H), 1.35 (s, 3H). TOF-MS (ESI) m/z: calcd for C42H50N5O7S [M + H]+ 768.3425; found 768.3383. Anal. Calcd for C42H49N5O7S·0.75H2O: C, 64.55; H, 6.51; N, 8.96. Found: C, 64.57; H, 6.56; N, 8.94. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3(phenylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl5148

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

C40H47N4O7S [M + H]+ 727.3160; found 727.3143. Anal. Calcd for C40H46N4O7S·0.6H2O: C, 65.13; H, 6.45; N, 7.59. Found: C, 65.03; H, 6.43; N, 7.75. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(7-methoxybenzofuran-2-carbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3thiazolidine-4-carboxamide (21f, KNI-1657). Compound 21f was prepared from compound 20 in a manner similar to that described for compound 15a; yield 57%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.39 (d, J = 9.5 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.06 (br s, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.28 (t, J = 7.9 Hz, 3H), 7.22 (t, J = 7.9 Hz, 1H), 7.1 (t, J = 7.5 Hz, 2H), 7.05−6.95 (m, 5H), 5.00 (d, J = 9.0 Hz, 1H), 4.89 (d, J = 8.6 Hz, 1H), 4.49 (s, 1H), 4.43−4.32 (m, 3H), 4.18−4.1 (m, 2H), 3.93 (s, 3H), 3.67−3.64 (m, 2H), 3.51−3.48 (m, 2H, overlapped with water), 2.70−2.56 (m, 3H), 2.25 (s, 6H), 1.48−1.44 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ (ppm): 170.12, 169.92, 167.72, 157.57, 148.49, 145.26, 143.62, 139.21, 137.29, 134.45, 129.44, 128.62, 127.89, 127.79, 127.26, 125.77, 124.59, 114.45, 110.64, 108.76, 72.51, 72.28, 71.64, 70.88, 69.82, 67.19, 63.08, 60.25, 55.72, 55.26, 53.96, 53.12, 51.14, 47.68, 41.34, 37.23, 33.11, 30.32, 28.38, 24.57, 19.41. TOF-MS (ESI) m/z: calcd for C41H49N4O8S [M + H]+ 757.3266; found 757.3248. Anal. Calcd for C40H51N5O6S·0.6H2O: C, 64.14; H, 6.46; N, 7.30. Found: C, 64.07; H, 6.43; N, 7.38. (R)-N-[(S)-Indan-1-yl]-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23a). Compound 23a was prepared from 22a22 in a manner similar to that described for compound 20; yield 82%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.36 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.28−7.25 (m, 3H), 7.23−7.21 (m, 2H), 7.17 (d, J = 7.3 Hz, 2H), 7.14−7.12 (m, 2H), 6.92 (d, J = 8.8 Hz, 1H), 5.31 (dd, J = 15.4, 7.7 Hz, 1H), 5.19 (d, J = 7.3 Hz, 1H), 5.01 (d, J = 9.2 Hz, 1H), 4.95 (d, J = 9.2 Hz, 1H), 4.46 (s, 1H), 4.39 (dd, J = 7.3, 2.2 Hz, 1H), 4.25 (br. s., 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.62 (dt, J = 5.8 Hz, 1H), 3.55 (t, J = 7.7 Hz, 1H), 3.46 (dd, J = 15.4, 7.7 Hz, 1H), 3.31−3.24 (m, 1H), 2.94 (ddd, J = 15.9, 8.6, 3.2 Hz, 1H), 2.84− 2.78 (m, 2H), 2.72−2.68 (m, 2H), 2.42−2.34 (m, 1H), 2.27−2.22 (m, 1H), 1.91−1.80 (m, 1H), 1.51 (s, 3H), 1.46 (s, 3H), 1.37 (s, 9H), 1.36−1.27 (m, 2H). TOF-MS (ESI) m/z: calcd for C36H49N4O7S [M + H]+ 681.3316; found 681.3275. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23b). Compound 23b was prepared from 22b22 in a manner similar to that described for compound 20; quantitative yield. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.10 (d, J = 8.80 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 7.2 Hz, 2H), 7.27 (d, J = 7.3 Hz, 1H), 7.25−7.05 (m, 7H), 6.92 (d, J = 9.1 Hz, 1H), 5.28 (dd, J = 8.8, 5.1 Hz, 1H), 5.22−5.14 (m, 1H), 5.10−4.99 (m, 2H), 4.95 (d, J = 9.1 Hz, 1H), 4.75 (s, 1H), 4.48−4.38 (m, 2H), 4.27−4.19 (m, 1H), 3.77 (t, J = 9.2 Hz, 1H), 3.64−3.58 (m, 1H), 3.54 (t, J = 7.8 Hz, 1H), 3.47−3.42 (m, 1H), 3.28−3.22 (m, 1H), 3.08−3.02 (m, 1H), 2.86−2.75 (m, 2H), 2.68−2.62 (m, 2H), 2.22 (dt, J = 16.1, 8.0 Hz, 1H), 1.56 (s, 3H), 1.48 (s, 3H), 1.37 (s, 9H), 1.31−1.21 (m, 2H). TOF-MS (ESI) m/z: calcd for C36H49N4O8S [M + H]+ 697.3266; found 697.3209. (R)-N-Cyclopentyl-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23c). Compound 23c was prepared from 22c22 in a manner similar to that described for compound 20; yield 92%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.92 (d, J = 7.1 Hz, 2H), 7.31 (d, J = 7.3 Hz, 2H), 7.20 (d, J = 7.6 Hz, 2H), 7.15−7.13 (m, 1H), 6.91 (d, J = 9.2 Hz, 1H), 5.14 (d, J = 7.3 Hz, 1H), 4.98 (d, J = 9.0 Hz, 1H), 4.92 (d, J = 9.0 Hz, 1H), 4.42 (s, 1H), 4.38 (dd, J = 7.5, 2.3 Hz, 1H), 4.26−4.20 (br. s., 1H), 4.05−3.97 (m, 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.61 (dt, J = 7.9, 4.8 Hz, 1H), 3.54 (t, J = 7.7 Hz, 1H), 3.42 (dd, J = 15.4, 7.5 Hz, 1H), 3.29−3.23 (m, 1H), 2.69−2.65 (m, 1H), 2.62−2.58 (m, 1H), 2.26− 2.18 (m, 1H), 1.80−1.72 (m, 2H), 1.65−1.60 (s, 2H), 1.49−1.37 (s,

19H), 1.34−1.25 (m, 2H). TOF-MS (ESI) m/z: calcd for C32H49N4O7S [M + H]+ 633.3316; found 633.3289. (R)-N-Allyl-3-[(2S,3S)-3-((S)-2-(tert-butoxycarbonyl)amino-2((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23d). Compound 23d was prepared from 22d22 in a manner similar to that described for compound 20; yield 94%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.16 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1), 7.29 (d, J = 7.2 Hz, 2H), 7.15−7.11 (m, 1H), 7.22−7.17 (m, 2 H), 6.94 (d, J = 8.8 Hz, 1H), 5.28−5.17 (m, 2H), 5.83−5.73 (m, 1H), 5.03 (dq, J = 10.3, 1.67 Hz, 1H), 4.42 (s, 1H) 5.04−4.92 (m, 3H), 4.36 (dd, J = 7.2, 3.42 Hz, 1H), 4.24 (t, J = 9.4 Hz, 1H), 3.65−3.53 (m, 2H) 3.78−3.69 (m, 3H), 3.28−3.23 (m, 1H) 3.49−3.42 (m, 1H), 2.73− 2.63 (m, 2H), 2.24 (dd, J = 15.9, 7.6 Hz, 1H), 1.51 (s, 3H), 1.41−1.33 (m, 12H), 1.30−1.24 (m, 2H). TOF-MS (ESI) m/z: calcd for C30H45N4O7S [M + H]+ 605.3003; found 605.2969. (R)-N-(2-Methylallyl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23e). Compound 23e was prepared from 22e22 in a manner similar to that described for compound 20; yield 99%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.17 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.19 (t, J = 7.3 Hz, 2H), 7.16−7.12 (m, 1H), 6.94 (d, J = 9.1 Hz, 1H), 5.25 (d, J = 7.5 Hz, 1H), 5.02−4.96 (m, 1H), 4.96−4.88 (m, 2H), 4.74 (s, 1H), 4.45 (s, 1H), 4.36 (dd, J = 7.3, 3.2 Hz, 1H), 4.23 (t, J = 9.4 Hz, 1H), 3.77− 3.72 (m, 2H), 3.64−3.53 (m, 3H), 3.46 (q, J = 7.7 Hz, 1H), 3.28−3.23 (m, 1H), 2.73−2.62 (m, 2H), 2.24 (dd, J = 16.3, 7.7 Hz, 1H), 1.51 (s, 3H) 1.66 (s, 3H), 1.41−1.23 (m, 14H). TOF-MS (ESI) m/z: calcd for C31H47N4O7S [M + H]+ 619.3160; found 619.3148. (R)-N-(cis-4-Hydroxy-2-buten-1-yl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23f). Compound 23f was prepared from 22f22 in a manner similar to that described for compound 20; yield 85%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.14 (br. s., 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 7.3 Hz, 2H), 7.24−7.08 (m, 5H), 6.93 (d, J = 9.1 Hz, 1H), 5.57−5.50 (m, 1H), 5.37−5.29 (m, 1H), 5.23 (d, J = 7.3 Hz, 1H), 4.99−4.89 (m, 2H), 4.66 (t, J = 5.3 Hz, 1H), 4.40−4.32 (m, 2H), 4.23 (br. s., 1H), 4.03 (t, J = 5.8 Hz, 2H), 3.80−3.70 (m, 3H), 3.65−3.52 (m, 2H), 3.49−3.43 (m, 1H), 3.29−3.23 (m, 1H), 2.73−2.65 (m, 2H), 2.28−2.19 (m, 1H), 1.53−1.47 (m, 3H), 1.42− 1.26 (m, 14H). TOF-MS (ESI) m/z: calcd for C31H47N4O8S [M + H]+ 635.3109; found 635.3078. (R)-N-[(S)-Indan-1-yl]-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24a, KNI-1665). Compound 24a was prepared from compound 23a in a manner similar to that described for compound 15a; yield 40%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.36 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 7.1 Hz, 2H), 7.23−7.15 (m, 6H), 7.13−7.08 (m, 2H), 6.43 (d, J = 7.0 Hz, 1H), 6.35 (br s, 1H), 6.29 (d, J = 7.5 Hz, 1H), 5.30 (dd, J = 15.6, 7.9 Hz, 1H), 5.03 (d, J = 9.2 Hz, 1H), 4.97 (d, J = 9.2 Hz, 1H), 4.51 (d, J = 14.8 Hz, 1H), 4.46 (s, 1H), 4.43 (d, J = 14.8 Hz, 1H), 4.42 (d, J = 3.1 Hz, 1H), 4.28 (t, J = 8.6 Hz, 1H), 4.25− 4.19 (m, 1H), 3.73−3.45 (m, 4H, overlapped with water), 3.25 (dd, J = 8.6, 7.0 Hz, 1H), 2.96−2.91 (m, 1H), 2.89 (s, 6H), 2.83−2.58 (m, 3H), 2.42−2.35 (m, 2H), 1.86−1.81 (m, 1H), 1.56−1.47 (m, 2H), 1.50 (s, 3H), 1.45 (s, 3H). TOF-MS (ESI) m/z: calcd for C41H52N5O7S [M + H]+ 758.3582; found 758.3556. Anal. Calcd for C41H51N5O7S·1CF3COOH: C, 59.23; H, 6.01; N, 8.03. Found: C, 59.38; H, 6.16; N, 8.33. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-((S)-2-(3(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl1,3-thiazolidine-4-carboxamide (24b, KNI-1749). Compound 24b was prepared from compound 23b in a manner similar to that described for compound 15a; yield 31%. 1H NMR (400 MHz, DMSOd6) δ (ppm): 8.26 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 7.3 Hz, 2H), 7.26 (d, J = 7.0 Hz, 1H), 5149

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

Article

7.22−7.14 (m, 4H), 7.11−7.05 (m, 3H), 6.37 (d, J = 8.8 Hz, 1H), 6.28 (br s, 1H), 6.23 (d, J = 7.9 Hz, 1H), 5.27 (dd, J = 9.0, 5.5 Hz, 1H), 5.04 (d, J = 9.2 Hz, 1H), 4.95 (d, J = 9.0 Hz, 1H), 4.74 (s, 1H), 4.49 (d, J = 15.0 Hz, 1H), 4.46 (d, J = 2.8 Hz, 1H), 4.42 (d, J = 14.7 Hz, 1H), 4.41 (m, 1H), 4.27 (t, J = 8.8 Hz, 1H), 4.20−4.19 (m, 1H), 3.61 (dd, J = 13.0, 7.7 Hz, 1H), 3.50−3.29 (m, 2H, overlapped with water), 3.24−3.20 (m, 1H), 3.04 (dd, J = 15.8, 4.6 Hz, 1H), 2.87 (s, 6H), 2.83−2.79 (m, 3H), 2.70−2.63 (m, 1H), 2.43−2.33 (m,1H), 1.55 (s, 3H), 1.54−1.49 (m, 2H), 1.48 (s, 3H). TOF-MS (ESI) m/z: calcd for C41H52N5O8S [M + H]+ 774.3531; found 774.3512. (R)-N-Cyclopentyl-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24c, KNI-1729). Compound 24c was prepared from compound 23c in a manner similar to that described for compound 15a; yield 66%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.22 (d, J = 8.4 Hz, 1H), 7.94−7.91 (m, 2H), 7.30 (d, J = 7.0 Hz, 2H), 7.20 (t, J = 7.5 Hz, 2H), 7.15−7.08 (m, 2H), 6.43 (br. d, J = 7.3 Hz, 1H), 6.34 (br s, 1H), 6.28 (d, J = 7.9 Hz, 1H), 5.00 (d, J = 9.0 Hz, 1H), 4.94 (d, J = 9.2 Hz, 1H), 4.31 (d, J = 15 Hz, 1H), 4.43 (d, J = 13.2 Hz, 1H), 4.428 (s, 1H), 4.41 (m, 1H), 4.29 (t, J = 8.8 Hz, 1H), 4.23−4.19 (br. s, 1H), 4.00 (dt, J = 12.1, 5.1 Hz, 1H), 3.62 (dt, J = 8.1, 5.3 Hz, 1H), 3.52−3.46 (m, 2H, overlapped with water), 3.24 (dd J = 8.8, 7.0 Hz, 1H), 2.89 (s, 6H), 2.70 (dd, J = 13.7, 2.4 Hz, 1H), 2.63 (dd, J = 13.9, 2.9 Hz, 1H), 2.44−2.37 (m,1H), 1.78−1.74 (m, 2H), 1.64−1.59 (m, 2H), 1.56−1.38 (m, 6H), 1.49 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C37H52N5O7S [M + H]+ 710.3582; found 710.3553. (R)-N-Allyl-3-[(2S,3S)-3-((S)-2-(3-(dimethylam ino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24d, KNI-1742). Compound 24d was prepared from compound 23d in a manner similar to that described for compound 15a; yield 56%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.22−8.09 (m, 2H), 7.93 (d, J = 8.9 Hz, 1H), 7.32−7.23 (m, 2H), 7.22−7.16 (m, 2H), 7.15−7.06 (m, 2H), 6.39 (d, J = 8.3 Hz, 1H), 6.30 (br. s., 1H), 6.25 (d, J = 8.1 Hz, 1H), 5.82−5.72 (m, 1H), 5.25−5.19 (m, 1H), 5.06−4.92 (m, 3H), 4.53−4.48 (m, 1H), 4.46− 4.35 (m, 3H), 4.29−4.19 (m, 2H), 3.78−3.58 (m, 4H), 3.52−3.47 (m, 2H), 3.27−3.23 (m, 1H), 2.88 (s, 6 H), 2.76−2.70 (m, 1H), 2.68−2.63 (m, 1H), 2.43−2.37 (m, 1H), 1.58−1.44 (m, 5H), 1.36 (s, 3H). TOFMS (ESI) m/z: calcd for C35H48N5O7S [M + H]+ 682.3269; found 682.3234. (R)-N-(2-Methylallyl)-3-[(2S,3S)-3-((S)-2-(3-(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran-3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24e, KNI-1728). Compound 24e was prepared from compound 23e in a manner similar to that described for compound 15a; yield 37%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.18−8.16 (m, 2H), 7.95 (d, J = 8.8 Hz, 1H), 7.27 (d, J = 7.1 Hz, 2H), 7.19 (t, J = 7.3 Hz, 2H), 7.15−7.08 (m, 2H), 6.42 (d, J = 7.9 Hz, 1H), 6.3 (s, 1H), 6.27 (d, J = 8.1 Hz, 1H), 5.01 (d, J = 9.2 Hz, 1H), 4.94 (d, J = 9.3 Hz, 1H), 4.90 (s, 1H), 4.74 (s, 1H), 4.51 (d, J = 14.8 Hz, 1H), 4.44 (s, 1H), 4.43 (d, J = 14.7 Hz, 1H), 4.38 (d, J = 3.7 Hz, 1H), 4.27 (t, J = 8.8 Hz, 1H), 4.23−4.18 (m, 1H), 3.74 (dd, J = 15.4, 6.2 Hz, 1H), 3.64−3.57 (m, 2H), 3.24 (dd, J = 7, 8.6 Hz, 1H), 2.88 (s, 6H), 2.74−2.60 (m, 2H), 2.43−2.37 (m, 1H), 1.65 (s, 3H), 1.56−1.45 (m, 2H), 1.51 (s, 3H), 1.36 (s, 3H). TOF-MS (ESI) m/z: calcd for C36H50N5O7S [M + H]+ 696.3425; found 696.3409. (R)-N-(cis-4-Hydroxy-2-buten-1-yl)-3-[(2S,3S)-3-((S)-2-(3(dimethylamino)phenoxyacetyl)amino-2-((R)-tetrahydrofuran3-yl)acetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl1,3-thiazolidine-4-carboxamide (24f, KNI-1653). Compound 24f was prepared from compound 23f in a manner similar to that described for compound 15a; yield 24%. 1H NMR (400 MHz, DMSOd6) δ (ppm): 8.22−8.08 (m, 2H), 7.93 (d, J = 8.93 Hz, 1H), 7.32−7.24 (m, 2H), 7.24−7.17 (m, 2H), 7.14−7.07 (m, 2H), 6.40 (d, J = 8.2 Hz, 1H), 6.30 (br. s., 1H), 6.25 (d, J = 7.7 Hz, 1H), 5.58−5.50 (m, 1H), 5.36−5.29 (m, 1H), 4.96 (q, J = 9.2 Hz, 2H), 4.50 (d, J = 14.9 Hz, 1H), 4.43 (d, J = 14.8 Hz, 1H), 4.40−4.34 (m, 2H), 4.29−4.18 (m, 2H), 4.05−4.00 (m, 2H), 3.81−3.69 (m, 4H), 3.62 (d, J = 4.9 Hz,

2H), 3.27−3.23 (m, 1H), 2.88 (s, 6 H), 2.76−2.71 (m, 1H), 2.68−2.60 (m, 2H), 2.40 (dd, J = 15.5, 7.7 Hz, 1H), 1.58−1.42 (m, 5H), 1.35 (s, 3H). TOF-MS (ESI) m/z: calcd for C36H50N5O8S [M + H]+ 712.3375; found 712.3343. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-3-carbamoylpropionyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (25). Compound 25 was prepared from 1922 using BocAsn-OH in a manner similar to that described for compound 20; yield 94%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.09 (br. s., 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 7.7 Hz, 2H), 7.26−7.06 (m, 4H), 7.03−7.00 (m, 2H), 6.87 (br. s., 2H), 6.80 (d, J = 8.3 Hz, 1H), 5.22 (d, J = 7.2 Hz, 1H), 4.95 (d, J = 8.9 Hz, 1H), 4.87 (d, J = 9.1 Hz, 1H), 4.50 (s, 1 H), 4.45 (dd, J = 13.9, 5.9 Hz, 1H), 4.36 (dd, J = 7.2, 2.8 Hz, 1H), 4.22−4.11 (m, 3H), 2.72−2.61 (m, 2H), 2.31 (s, 6H), 2.29−2.20 (m, 2H), 1.45 (s, 3H), 1.41−1.32 (m, 12H). TOF-MS (ESI) m/z: calcd for C34H48N5O7S [M + H]+ 670.3269; found 670.3244. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(tertbutoxycarbonyl)amino-3-methylbutanoyl)amino-2-hydroxy4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (26). Compound 26 was prepared from 1922 using Boc-Val-OH in a manner similar to that described for compound 20; yield 77%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.12 (dd, J = 5.7, 3.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.2 Hz, 2H), 7.23−7.18 (m, 2H), 7.16−7.05 (m, 2H), 7.04−6.99 (m, 2H), 6.65 (d, J = 9.7 Hz, 1H), 5.14−5.03 (m, 2H), 4.89 (d, J = 9.1 Hz, 1H), 4.54 (s, 1H), 4.48 (dd, J = 13.9, 6.2 Hz, 1H), 4.38 (dd, J = 7.4, 1.9 Hz, 1H), 4.23−4.13 (m, 2H), 3.70−3.65 (m, 1H), 2.71−2.60 (m, 2H), 2.31 (s, 6H), 1.78−1.68 (m, 1H), 1.46 (s, 3H), 1.41−1.32 (m, 12H), 0.67 (d, J = 6.7 Hz, 3H), 0.56 (d, J = 6.6 Hz, 3H). TOF-MS (ESI) m/z: calcd for C35H51N4O6S [M + H]+ 655.3524; found 655.3492. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-3-carbamoylpropionyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (27a, KNI-1539). Compound 27a was prepared from compound 25 in a manner similar to that described for compound 15a; yield 33%; purity 90.2%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.60 (d, J = 8.2 Hz, 1H), 8.11 (dd, J = 5.8, 3.7 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83−7.78 (m, 1H), 7.69 (dd, J = 8.4, 0.8 Hz, 1H), 7.58 (s, 1H), 7.51−7.46 (m, 1 H), 7.38−7.28 (m, 4H), 7.09−7.03 (m, 3H), 7.02−6.96 (m, 2H), 6.90 (br. s., 2H), 5.74 (br. s., 1H), 5.01 (d, J = 8.9 Hz, 1H), 4.89 (d, J = 9.1 Hz, 1H), 4.80−4.73 (m, 1H), 4.52 (s, 1H), 4.50−4.40 (m, 2H), 4.17 (dd, J = 13.9, 3.4 Hz, 2H), 2.70−2.62 (m, 2H), 2.56−2.53 (m, 2H), 2.30 (s, 6H), 1.45 (s, 3H), 1.34 (s, 3H). TOF-MS (ESI) m/z: calcd for C38H44N5O7S [M + H]+ 714.2956; found 714.2951. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(3-(benzofuran-2-carbonyl)amino-3-methylbutanoyl)amino-2-hydroxy-4phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (27b, KNI-1540). Compound 27b was prepared from compound 25 in a manner similar to that described for compound 15a; yield 46%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.26 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 9.3 Hz, 1H), 8.12 (dd, J = 3.5, 6.0 Hz, 1H), 7.78 (td, J = 7.9, 1.3 Hz, 1H), 7.70 (dd, J = 8.4, 0.7 Hz, 1H), 7.62 (d, J = 0.9 Hz, 1H), 7.48 (dt, J = 7.3, 1.3 Hz, 1H), 7.37−7.32 (m, 3H), 7.16 (t, J = 7.5 Hz, 2H),7.09−7.05 (m, 2H), 7.01 (d, J = 7.7 Hz, 2H), 5.20 (br s, 1H), 5.09 (d, J = 8.8 Hz, 1H), 4.93 (d, J = 9.0 Hz, 1H), 4.54 (s, 1H), 4.47 (dd, J = 14.1, 6.2 Hz, 1H), 4.42 (br s, 1H), 4.28−4.14 (m, 3H), 2.74− 2.60 (m, 2H), 2.3 (s, 6H), 2.02−1.97 (m, 1H), 1.45 (s, 3H), 1.36 (s, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.68 (d, J = 6.8 Hz, 3H). TOF-MS (ESI) m/z: calcd for C39H47N4O6S [M + H]+ 699.3211; found 699.3201. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(7-methoxybenzofuran-2-carbonyl)amino-3-carbamoylpropionyl)amino2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4carboxamide (27c, KNI-1565). Compound 27c was prepared from compound 26 in a manner similar to that described for compound 15a; yield 29%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.54 (d, J = 8.2 Hz, 1H), 8.10 (dd, J = 5.8, 3.6 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.57−7.53 (m, 1H), 7.36−7.24 (m, 5H), 7.12−7.04 (m, 4H), 7.01− 6.97 (m, 2H), 6.90 (br. s., 1H), 5.75 (br. s., 1H), 5.00 (d, J = 8.9 Hz, 5150

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

Journal of Medicinal Chemistry

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1H), 4.89 (d, J = 8.9 Hz, 1H), 4.75 (td, J = 7.8, 5.8 Hz, 1H), 4.52 (s, 1 H), 4.48−4.41 (m, 2H), 4.20−4.12 (m, 2H), 3.98 (s, 3H), 2.72−2.62 (m, 2H), 2.57−2.53 (m, 2H), 2.30 (s, 6H), 1.45 (s, 3H), 1.34 (s, 3H). TOF-MS (ESI) m/z: calcd for C39H46N5O8S [M + H]+ 744.3062; found 744.3040. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-((S)-2-(7-methoxybenzofuran-2-carbonyl)amino-3-methylbutanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (27d, KNI-1566). Compound 27d was prepared from compound 26 in a manner similar to that described for compound 15a; yield 40%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.28 (d, J = 8.4 Hz, 1H), 8.12 (dd, J = 6, 3.5 Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H), 7.60 (s, 1H), 7.35 (d, J = 7.1 Hz, 2H), 7.31 (dd, J = 7.9, 1.1 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.16 (t, J = 7.7 Hz, 2H), 7.1−7.03 (m, 3H), 7.01 (d, J = 7.5 Hz, 2H), 5.19 (d, J = 7.1 Hz, 1H), 5.08 (d, J = 8.8 Hz, 1H), 4.93 (d, J = 9.2 Hz, 1H), 4.54 (s, 1H), 4.47 (dd, J = 14.1, 6.4 Hz, 1H), 4.42 (dd, J = 7.1, 2.7 Hz, 1H), 4.26 (t, J = 8.1 Hz, 1H), 4.24−4.20 (m, 1H), 4.17 (dd, J = 13.9, 3.3 Hz, 1H), 3.97 (s, 3H), 2.72 (dd, J = 13.9, 2.6 Hz, 1H), 2.64 (dd, J = 13.6, 2.6 Hz, 1H), 2.30 (s, 6H), 2.04− 1.97 (m, 1H), 1.45 (s, 3H), 1.36 (s, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.69 (d, J = 6.8 Hz, 3H). TOF-MS (ESI) m/z: calcd for C40H49N4O7S [M + H]+ 729.3316; found 729.3297. Anal. Calcd for C40H48N4O7S· 0.1H2O: C, 65.75; H, 6.65; N, 7.67. Found: C, 65.72; H, 6.71; N, 7.76. HIV Protease Inhibition. Recombinant HIV-1 protease was purchased from Bachem AG, Bubendorf, Switzerland. In the inhibition assay, 25 μL of 200 mM 2-(N-morpholino)ethanesulfonic acid (MES)−NaOH buffer (pH 5.5), containing 2 mM dithiothreitol, 2 mM EDTA-2Na, and 1 M NaCl, was mixed with 5 μL of the inhibitor (10 nM) dissolved in DMSO and 10 μL of HIV-1 protease (40 ng/ mL) in 50 mM AcOH (pH 5.0) containing 1 mM EDTA-2Na, 25 mM NaCl, 0.2% 2-mercaptoethanol, 0.2% Nonidet P-40, and 10% glycerol. The mixture was preincubated for 5 min at 37 °C, and the reaction was initiated by adding 10 μL of a 1.0 mM substrate solution (H-Lys-AlaArg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2). After incubation for 3 h at 37 °C, the reaction was terminated by the addition of 1 N HCl, and the N-terminal cleavage fragment (H-Lys-Ala-Arg-Val-Tyr-OH) was separated by reversed-phase HPLC on a C18 column (3.0 × 75 mm; YMC Pack ODS AS-3E7) with a linear gradient of CH3CN in 0.1% aqueous TFA at a flow rate of 1.0 mL/min, and its quantity was determined by monitoring fluorescence intensity (Ex, 275 nm; Em, 305 nm). Anti-HIV Activity. Anti-HIV activity of test compounds was determined based on inhibition of an HIV-induced cytopathic effect in MT-4 cells.31 Wild-type HIV-1 (pNL4-3) or LPV-resistant strain A1711 was inoculated to MT-4 cells in a 96-well plate. The resulting culture was treated with an equal volume of a 1% DMSO solution of each test compound with several concentrations and 10% fetal bovine serum, and was incubated for 5 days in a CO2 incubator at 37 °C, in triplicate. After treatment with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the optical density of the plate was measured, and percent cytopathic effect reduction was calculated, then EC50 values were estimated by fitting the data to a median-effect equation. Cytotoxicity (TD50) was determined by incubation in the absence of the virus. In addition, pNL4-3-based, wild-type, or DRV-resistant luciferase reporter lentiviral vector30 was generated by transfecting 293T cells with pCMV-ΔR8.91 or the derivatives containing V32I/L33F/I54M/ V82I or V32I/L33F/I54M/I84V mutations together with pLenti CMV Puro LUC (w168−1) (17477, Addgene, Cambridge, MA) and pHit/ G32 using FuGENE HD Transfection Regent (Promega, Madison, WI) in the presence or absence of HIV-1 protease inhibitor. Fortyeight hours later, 293T cells were infected with lentiviral vector. Twenty-four hours after infection, luciferase activity in infected cells was measured using a Steady-Glo Luciferase Assay System (Promega, Madison, WI) with an LB962 microplate luminometer (Berthold, Bad Wildbad, Germany). Drug susceptibility was evaluated as a reduction in luciferase activity in infected cells. The 50% inhibitory concentration (IC50) of protease inhibitors for suppressing the transduction of luciferase gene was calculated from the dose−response curve using a

standard function of GraphPad Prism 5 software (GraphPad Software, San Diego, CA). Preparation and X-ray Crystallographic Analysis of WTm5 and A17m5. The chemically synthesized DNA encoded the gene for the initial methionine and the 99 amino acid including five mutations of Q7K, L33I, L63I C67A, and C95A (WTm5) and the gene derived from A17 type HIV-1 protease11 with additional lopinavir/ritonavirresistant mutations of L10F, V32I, M46I, I47V, Q58E, and I84V (A17m5) were used for expression in E. coli. Preparation of WTm5 and A17m5 for crystallization was performed as reported.16 WTm5 and A17m5/21f complexes were prepared with a 2-fold molar excess of inhibitors to protease at 5.0 and 2.0 mg/mL protein concentrations, respectively. The both complexes were crystallized using hanging drop vapor diffusion method by mixing in equal volumes (2 μL each) of protease solution and crystallization buffer containing 126 mM phosphate buffer at pH 5.0, 63 mM sodium citrate. For crystallization of A17m5/21f complex, PEG4000 was used as a precipitant for crystallization at the final concentration of 20% (w/v). The obtained crystals were flash-frozen under a N2 gas cryo-stream (100 K), and subjected to X-ray at BL41XU beamline in SPring-8. Data for WTm5/ 21f and A17m5/21f crystals belong to the same space group of P21212 with similar cell constants, and the crystals were processed to the resolution of 0.85 and 1.50 Å resolution, respectively, by using HKL2000.33 The structures of WTm5/21f and A17m5/21f were refined to a crystallographic R-factor of 10.3% (free R-factor = 12.5%) using program SHELX-97,34 and R-factor of 17.1% (free R-factor = 18.8%) using program Refmac535 and PHENIX 1.9,36 respectively (see Supporting Information for X-ray diffraction data).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01709. HPLC analysis of target compounds; X-ray diffraction data collection and refinement statistics; Supplementary figure (PDF) Molecular formula strings (CSV) Accession Codes

The atomic coordinates for the crystal structures of WTm5 and A17m5 proteases with compound 21f can be assessed using PDB ID codes 5YOK and 5YOJ, respectively.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-749-64-8113. Fax: +81-749-76-8140. E-mail: y_ [email protected]. ORCID

Koushi Hidaka: 0000-0002-8956-6996 Keith F. McDaniel: 0000-0001-5704-3288 Yoshio Hayashi: 0000-0002-7010-6914 Yoshiaki Kiso: 0000-0002-7877-8221 Notes

The authors declare no competing financial interest. Δ Deceased on Aug 8, 2015.

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ACKNOWLEDGMENTS We are sincerely grateful to Ms. Y. Matsui, Ms. S. Shibakawa, and Mr. T. Hamada for the determination of HIV-1 protease inhibitory activity, and to Ms. Tatyana Dekhtyar for the determination of anti-HIV activity. This research was supported in parts by the Frontier Research Program and the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. 5151

DOI: 10.1021/acs.jmedchem.7b01709 J. Med. Chem. 2018, 61, 5138−5153

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The synchrotron radiation experiments were performed at the BL41-XU and BL38B1 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2011A1124 and 2014B1313).

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ABBREVIATIONS USED HIV, human immunodeficiency virus; AIDS, acquired immune deficiency syndrome; Apns, allophenylnorstatine; HMC, hydroxymethylcarbonyl; Dmt, (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid; Thfg, tetrahydrofuranylglycine; SAR, structure−activity relationships; Boc, tert-butyloxycarbonyl; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; EDC, 1-ethyl-3-(3,3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole

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