Can We Make Small Molecules Lean? Optimization of a Highly

Publication Date (Web): March 21, 2017 ... We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target ...
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Can We Make Small Molecules Lean? Optimization of a Highly Lipophilic TarO Inhibitor Mihirbaran Mandal,*,† Zheng Tan,† Christina Madsen-Duggan,† Alexei V. Buevich,† John P. Caldwell,† Reynalda Dejesus,† Amy Flattery,⊥ Charles G. Garlisi,# Charles Gill,⊥ Sookhee Nicole Ha,† Ginny Ho,† Sandra Koseoglu,# Marc Labroli,† Kallol Basu,† Sang Ho Lee,‡ Lianzhu Liang,⊥ Jenny Liu,⊥ Todd Mayhood,# Debra McGuinness,# David G. McLaren,# Xiujuan Wen,# Emma Parmee,† Diane Rindgen,∥ Terry Roemer,‡ Payal Sheth,# Paul Tawa,# James Tata,† Christine Yang,† Shu-Wei Yang,† Li Xiao,† Hao Wang,‡ Christopher Tan,‡ Haifeng Tang,† Paul Walsh,§ Erika Walsh,§ Jin Wu,∥ and Jing Su*,† †

Global Chemistry, ‡Early Discovery Bacteriology, §Discovery Pharmaceutical Sciences, ∥Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, ⊥In Vivo Pharmacology, and #In Vitro Pharmacology, MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States S Supporting Information *

ABSTRACT: We describe our optimization efforts to improve the physicochemical properties, solubility, and offtarget profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure− activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

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INTRODUCTION After the discovery of penicillin by Alexander Fleming from Penicillium notatum in 1929,1 history witnessed tremendous efforts poured into antibacterial research, which led to the golden age of antibiotic discovery (1940s to 1960s) and revolutionized the clinical practice.2 Although several important classes of new drugs such as carbapenem,3 oxazolidinone,4 monobactam,5 and daptomycin6 were discovered during the 1970−1990 period, the next 25 years of antibacterial research has been, in general, disappointing in delivering novel antibiotics for various reasons.7 Additionally, antibiotic resistance,8 warned by Fleming in his Nobel Prize lecture in 1945, has been rising steadily, including the most recent astonishing reports on colistin resistance9 in animals as well as in humans. The emergence of bacterial resistance has been considered as one of the top threats to the global public health and has prompted governments and World Health Organization to take actions to address this potential global crisis.10 The resistance issue has been studied extensively and reviewed.8,11 Resistance mechanisms range from endogenous mechanisms (such as cognate on target mutations, reduced uptake by loss of porins, overexpression of β-lactamases, and efflux pumps) to exogenous mechanisms such as horizontal gene transfer.12 To overcome drug resistance by combining two © 2017 American Chemical Society

drugs with different mechanisms of action has been extensively applied for the treatment of Gram-negative infection, and the βlactam/β-lactamase combination therapy as exemplified by amoxicillin/clavulanate developed by the Beecham Research Laboratories to address the stability issue of amoxicillin represents a seminal achievement in this field.13 The most recent successful examples are the FDA approved ceftazidime/ avibactam14 and ceftolozane/tazobactam.15 Combination therapy other than β-lactam/β-lactamase therapies are also being investigated at clinical or preclinical stages.11a For example, cadazolid, a hybrid of fluoroquinolone and oxazolidinone in one molecule, is in phase III clinical trials and has received “qualified infectious disease product” (QIDP) and “fast track” development status from the FDA.16 In our own research on methicillin resistant Staphylococcus aureus (MRSA) resistance to β-lactam antibiotics, an antisensebased screening of 245 MRSA essential genes revealed that certain genes, such as TarL (an essential enzyme involved in wall teichoic acid (WTA) biosynthesis), when partially depleted, led to resensitization of MRSA to existing β-lactams.17 In accordance with the importance of WTA pathway in Received: January 24, 2017 Published: March 21, 2017 3851

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

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Figure 1. Physicochemical properties of hit 1 and its profile and the structure of imipenem (IPM), one of the putative antibiotics partner. sMITC95 is the synergistic minimum inhibitory threshold concentration to inhibit 95% growth of methicillin-resistant Staphylococcal aureus (MRSA) COL in the presence of 4 μg/mL of IPM. iMITC95 is the intrinsic minimum inhibitory threshold concentration to inhibit 95% growth of MRSA COL. LLE: lipophilic ligand efficiency.

Scheme 1. Synthesis of Analogs 1−5 with an Exocyclic Amide Linkagea

a Reagents and conditions: (a) i-PrMgCl, THF, −10 °C, 95%; (b) toluene, 2-propanol, Al(O-i-Pr)3, 50 °C, then KOH, H2O, rt, 91%; (c) 2(naphthalen-1-yl)acetic acid, 1,1′-carbonyldiimidazole, DBU, MeCN, rt, 12−56%.

resensitizing MRSA to β-lactams, Campbell et al. reported that the deletion of TarO (the first step in the WTA pathway) also resensitized MRSA to β-lactams.18 Our subsequent WTApathway-based whole cell screening resulted in the discovery of several inhibitors targeting the WTA transporter protein TarG.19 Resistant mutant selection in MRSA and MRSE and subsequent whole-genome sequence analysis indicated that in addition to nonsynonymous mutations that confer resistance due to the loss of binding of TarG inhibitors to its target, loss of function mutations mapping to TarB, TarD, TarI, as well as early stage genes TarO and TarA were identified.20 Loss of function mutations in TarO or TarA do not play a role in the viability of Staphylococci since WTA is a nonessential polymer.20 However, the absence of WTA greatly enhances the susceptibility of MRSA to β-lactam antibiotics in vitro as well as in vivo.21 Analogous to the β-lactam/β-lactamase combination therapy, as described above, finding an inhibitor that targets a nonessential target, yet is synergistic with β-lactam antibiotics, could be a viable approach to address drug resistance for Gram-positive MRSA infection.21 This synergistic characteristic of the WTA pathway with β-lactam antibiotics

inspired us to design a targeted phenotypic screening to identify nonbioactive agents that specifically inhibit the early steps in the WTA biosynthetic pathway, such as TarO or TarA.22 Thorough interrogation of our small molecule library resulted in the discovery of tarocin (1, Figure 1) and tarocin B (structure in the Supporting Information) as specific inhibitors of TarO in MRSA.21 Unlike tunicamycin, a known inhibitor of both TarO and MraY, which binds in the polar substrate-binding site in the intracellular domain, compound 1 and tarocin B were predicted to bind in the extracellularly exposed hydrophobic pocket, the putative binding domain of hydrophobic bactoprenol substrate.22 These hits possessed no intrinsic antibacterial activity (iMITC95 > 200 μM, minimal concentration to inhibit 95% of the growth as measured by OD600) but rescued the bactericidal activity of IPM (MIC = 32 μg/mL) to its clinical break point concentration (4 μg/mL) against IPM resistant MRSA COL at concentrations of 0.9 μM (0.4 μg/mL) and 1.7 μM (0.9 μg/ mL), respectively. Unfortunately, both compound 1 and tarocin B were highly lipophilic and would require significant optimization to identify a tool molecule for in vivo efficacy 3852

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

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Scheme 2. Synthesis of Urea Derivatives 6−11a

a

Reagents and conditions: (a) n-BuLi, THF, trichloromethyl carbonochloridate, −78 °C; (b) amine, Et3N, DCM, rt, 20−50%.

Scheme 3. Synthesis of Analog 14a

a Reagents and conditions: (a) 30% HBr in acetic acid, 100 °C, 70%; (b) prop-2-en-1-ol, NaH, THF, 0 °C, 81%; (c) n-BuLi, THF, −78 °C, 53%; (d) toluene, 2-propanol, Al(O-i-Pr)3, 50 °C, 100%; (e) KHMDS, THF, −20 °C, 47%; (f) (Boc)2O, DMAP (cat.), rt, 98%; (g) Pd(PPh3)4, 1,3dimethylpyrimidine-2,4,6(1H,3H,5H)-trione, DCM, rt, 97%; (h) CSF, DMF, iodomethane, rt, 50%; (i) CF3CO2H, DCM, rt; (j) n-BuLi, THF, trichloromethyl carbonochloridate, −78 °C; (k) (2,6-naphthyridin-4-yl)methanamine, Et3N, DCM, rt, 18%.

study. Medicinal chemistry efforts were directed to improve the LLE with concomitant improvement in physicochemical properties while maintaining the synergistic antibacterial effect observed when combined with IPM at its breakpoint concentration. We have recently communicated our efforts on the progression of tarocin B toward lead like molecules.23 Herein, we describe our optimization efforts to improve the physicochemical properties and aqueous solubility of 1 utilizing a “hydrogen-bond hypothesis”24 that resulted in a series of molecules with improved LLE, and a tool compound 9 that demonstrated in vivo efficacy in a mouse infection model. Subsequent medicinal chemistry efforts led to a more polar compound 26 (clogP = 1.9). This example of turning a highly lipophilic compound to an antibiotic-like lead by lowering clogP by 5 log units demonstrated the power of the hypothesis

driven approach. Details of this medicinal chemistry effort and the in vivo results associated with 9 will be the central focus in this report.

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CHEMISTRY The synthesis of 1, 2, and analogs 3−5 with additional methylene spacers between the exocyclic carbonyl and the naphthalene moiety is described in Scheme 1. Condensation of the key intermediate 30, prepared following a known protocol,25 with 2-(naphthalen-1-yl)acetic acid using 1,1′carbonyldiimidazole as the activating agent afforded 1. Analogs 2−5 were prepared accordingly from the corresponding acetic acid derivatives. The synthesis of urea derivatives 6−11 from the corresponding oxazolidin-2-ones (30−33) is shown in Scheme 2. As a representative example, treatment of oxazolidinone 30 with n3853

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Scheme 4. Synthesis of Analogs 18−22a

Reagents and conditions: (a) HNMe(OMe)·HCl, NMM, EDCI, DCM, −5 °C, 95%; (b) TBDMSCl, imidazole, DCM, rt, 95%; (c) i-PrMgCl, THF, −5 °C, 93%; (d) toluene, 2-propanol, Al(O-i-Pr)3, 50 °C, 15%; (e) KOH, H2O, rt, 87%; (f) n-BuLi, THF, trichloromethyl carbonochloridate, −78 °C; (g) amine, Et3N, DCM, rt, 20−50%; (h) AcCl, MeOH, 71−74%; (i) RuCl3.H2O, NaIO4, MeCN, EtOAc, H2O, 6%. a

Scheme 5. Synthesis of Analogs 23 and 24a

a

Reagents and conditions: (a) HNMe(OMe)·HCl, HOBt, EDCI, DIEA, THF, 84%; (b) 3-bromo-5-fluorobenzotrifluoride, i-PrMgCl, 87%; (c) toluene, 2-propanol, Al(O-i-Pr)3, 50 °C, then KOH, 78%; (d) n-BuLi, THF, trichloromethyl carbonochloridate, −78 °C; (e) isoquinolin-4ylmethanamine, Et3N, DCM, rt, 37%; (f) pentamethylbenzene, BCl3, DCM, 46%; (g) RuCl3·H2O, NaIO4, MeCN, EtOAc, H2O, 24%.

BuLi at −78 °C followed by quenching with trichloromethyl carbonochloridate generated the corresponding oxazolidinone3-carbonyl chloride, which upon treatment with isoquinolin-4ylmethanamine afforded 6 in 20% yield. The other analogs 7− 11 were prepared using the similar protocol from the corresponding oxazolidinones (31−33) and the appropriate amines. While this strategy proved general for most analogs, alkoxy substituted derivatives such as 14 required the development of a slightly different synthetic strategy as illustrated in Scheme 3. Bis-bromide 35, prepared readily from commercially available 2,6-dichloro-4-(trifluoromethyl)pyridine (34), was converted to monoalkoxy derivative 36 in 70% yield. Metal−halogen exchange followed by treatment with Weinreb amide 28 afforded a ketone, which upon stereocontrolled reduction employing Meerwein−Ponndorf−Verley

conditions led to the formation of alcohol 37 in 53% yield. Oxazolidinone formation employing KHMDS followed by several functional group manipulations afforded the key intermediate 42 in 22% yield. Installation of urea fragment employing similar protocol shown in Scheme 2 produced the desired analog 14 in 18% yield. Urea derivatives 18−22 possessing a hydroxymethyl or a carboxyl group at the C-4 position (Scheme 4, generalized structure, R1 = hydroxymethyl or carboxyl group) were synthesized from the commercially available N-((benzyloxy)carbonyl)-L-serine 43 following the steps shown in Scheme 4. Ruthenium(III) chloride mediated oxidation of alcohol functionality of 21 afforded carboxylic acid derivative 22. By utilization of O-benzyl-N-(tert-butoxycarbonyl)-L-homoserine 50, one carbon homologation was achieved (Scheme 5) for 3854

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Table 1. Initial SAR Investigation with a Linker Expansion Strategya

MITC95: minimum concentration to inhibit 95% growth of MRSA COL in the presence of 4 μg/mL of IPM. iMITC95: minimum concentration to inhibit 95% growth of MRSA COL. LLE: lipophilic ligand efficiency.

as

After the confirmation that 1 was indeed the superior isomer among its enantiomeric/diastereomeric pairs,27 chemistry efforts were initiated to lower the clogP to align with our objective to administer the TarO inhibitor via intravenous route in combination with the antibiotics partner. Additionally, analog 1 displayed poor pharmacokinetic properties. Thus, to improve the PK profile and to lower clogP, our SAR studies were pursued by replacing the naphthalene ring with polar heteroaromatics as well as by substituting the bis-trifluoromethylphenyl with less lipophilic aromatic moieties. Unfortunately, these efforts resulted in compounds with significant loss of potencies. Only the isoquinoline moiety was demonstrated to be weakly tolerated with 15-fold drop in TarO IC50 relative to analog 1. To overcome this steep SAR issue, several approaches were undertaken, including a linker insertion and a linker expansion strategy (Table 1). From these efforts, analog 3 with a two-methylene spacer between the exocyclic amide carbonyl and naphthalene emerged with 19-fold improvement in TarO IC50, and further lengthening the linker resulted in a decrease in potency (analog 4). With this optimized linker, the SAR at both the naphthalene and bis-trifluoromethylphenyl regions was revisited. Soon it was discovered that either the insertion of a polar atom into the naphthalene region or the replacement of the highly lipophilic bis-trifluoromethylphenyl with less hydrophobic aromatic moieties was tolerated in terms of TarO potency, but concurrent reduction in polarity at the both sides led to loss in potency. From this exercise, analog 5 with an isoquinoline moiety replacing the naphthalene was

the preparation of analogs 23 and 24 following the similar synthetic sequence described in Scheme 4. The analogs 25 and 26 were prepared from 2-bromo-6-methoxy-4(trifluoromethyl)pyridine following the sequences of reaction described in Scheme 3, and details of the synthesis were incorporated into Supporting Information.

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RESULTS AND DISCUSSION TarO antagonism of all the final compounds was evaluated by the liquid chromatography−mass spectrometry (LC−MS) based assay using membrane bound TarO enzyme. Intrinsic antibacterial activity of all compounds was assessed using MRSA COL as the primary strain and reported as iMITC95, intrinsic minimum inhibitory threshold concentration to inhibit 95% growth based on OD600 reading. Their ability to potentiate β-lactam based antibiotics was measured in the presence of 4 μg/mL of IPM and reported here as sMITC95, synergistic minimum inhibitory threshold concentration to inhibit 95% growth of methicillin-resistant Staphylococcal aureus (MRSA) COL. The compounds were also evaluated in secondary assays such as the late stage targocil suppression assay (the ability of such compounds to reverse the TarG inhibition) and the phage K protection assay (recognition of the TarO enzyme by phage K to lyse cells).22 The toxicity of the compounds was routinely profiled using the Click-iT EdU assay.26 The results from the secondary assays and in vitro toxicity data were reported in the Supporting Information. 3855

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identified with an sMITC95 of 23 nM and was considered as a potential tool compound for an in vivo study. Unfortunately, analog 5 had poor pharmacokinetic properties with AUC0−24h of 1.7 μM·h at 100 mg/kg [formulated with Imwitor 742/ Tween 80 (50:50)] oral dose in mice. In addition, adequate solubility under variety of formulation conditions was not achieved for either subcutaneous or intravenous dosing despite the improvement in LLE (1.64 relative to −0.38 for analog 1). Thus, analog 5 was deemed unsuitable as a tool compound for in vivo efficacy studies. To expand SAR while improving LLE, previous SAR was carefully analyzed, and three key findings were noted: (a) substitution of oxygen (designated as O1) of the oxazolidinone 5 with “NH” (O1 → NH, X = CH2) resulted in complete loss of potency (sMITC95 > 200 μM); (b) replacement of O2 with “S” (O2 → S, X = CH2) and (c) removal of O3 (CO3 → CH2, X= CH2) led to a 150- and 4-fold drop in TarO enzymatic activity, respectively. On the basis of these observations, we speculated that O1, O2, and O3 could act as hydrogen bond acceptors in the binding site. To enhance the strength of the postulated hydrogen bonds by increasing the net negative charge on these oxygens, we proposed to incorporate nitrogen at 2 position of the linker by converting the exocyclic amide to urea (5 → 6) with minimum perturbation of the overall structure. Electrostatic potential (ESP) derived atomic charges showed that such modifications enhanced the net negative charge on oxygen O3 (Figure 2).28

significant drop in potency (9 vs 11) relative to incorporation of additional nitrogen into the isoquinoline moiety (9 vs 10). From these SAR efforts, analog 9 was quickly selected as a candidate for in vivo studies based on the consideration of its in vitro potency (COL sMITC95 = 250 nM), LLE, and cell toxicity (Table 1S in Supporting Information) in conjunction with its favorable preliminary pharmacokinetic profile (vide infra) while we continued medicinal chemistry efforts to further improve physicochemical properties, such as LLE and solubility. Seeking an opportunity to replace the 3,5-bis(trifluoromethyl)phenyl with heteroaromatics, we recognized that while analog 11 displayed IC50 of 5.3 μM, it possessed an LLE of 2.05 and a reasonably low clogP of 3.2. Therefore, to improve potency without deteriorating clogP, selective modification of the 4-trifluoropyridine of 11 was undertaken based on the previous SAR finding that the introduction of a methoxy group at the 6-position of the pyridine of analog 12 resulted in significant improvement of potency (Figure 3). Taking these observations into account, 6-methoxy-4(trifluoromethyl)pyridine was designed to restore the potency, and analog 14 with clogP of 3.2 was prepared and shown to have a TarO IC50 of 260 nM with a significant improved LLE of 3.47 (Table 2) with phage K IC50 and targocil reversal IC50 of 600 and 200 nM, respectively (Table 1S in Supporting Information). In addition, analog 14 has a solubility of 32 μM in PBS buffer. While this improvement in solubility relative to that of lead 1 (32 μM for 14 vs 95% purity by either silica gel chromatography or using reverse phase C18 column. 400 and 500 MHz NMR spectrometers were used to collect NMR spectra, and all the peaks were reported in ppm relative to the residual solvent peak, which served as the calibrant. The number of protons and coupling constants were reported parenthetically after each peak. Ultrahigh performance liquid chromatography equipped with high-resolution tandem mass spectrometry (UHPLC−HRMS/ MS) and C18 reverse phase column with the gradient of 5−95% MeCN in water with 0.05% TFA as the mobile phase was used to obtain the mass spectra and to assess the purity of the samples. Electrostatic potential derived atomic charges were calculated at the B3LYP/6-31+G(d,p) level of theory. Conformational analysis of 3 was done at the B3LYP/6-31+G(d,p) level of theory with inclusion of polarizable continuum model (PCM) for water (scrf = (solvent = water)). All calculations were carried out with the Gaussian 09 software package.32 The chemical structures of all compounds in the manuscript were compared to the list of reported PAINS structures, and no match was found. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-4-methyl-3-(2(naphthalen-1-yl)acetyl)oxazolidin-2-one (1). To a mixture of 27 (10.0 g, 34.0 mmol) and 28 (10.8 g, 40.0 mmol) in THF (100 mL) was added dropwise a solution of i-PrMgCl (85 mL, 1 M in THF, 85.0 mmol) at −10 °C. After the addition, the reaction mixture was warmed to rt and stirred for 12 h. Then the reaction mixture was added slowly into a solution of 1 N HCl (190 mL) at 0 °C. The resulting solution was extracted with EtOAc (150 mL × 3), and the combined EtOAc layers were dried with MgSO4, filtered, and concentrated to afford 29, which was dissolved in a mixture of i-PrOH (27 mL) and toluene (60 mL) and added Al(O-i-Pr)3 (6.2 g, 34.0 mmol). The resulting mixture was heated at 50 °C for 12 h and then cooled to rt. To this solution was added a solution of KOH in water (3.9 g of KOH in 4 mL of H2O), and the resulting mixture was stirred at 20 °C for 3 h, then quenched with brine (100 mL) and extracted with EtOAc (100 mL × 2). The EtOAc layers were combined and washed with brine (100 mL

on average 2- to 4- fold higher than that in the combination treatment group. This change in pharmacokinetic profile of IPM in the presence of 9 was not clearly understood. The synergistic pharmacological effect observed with IPM in the combination group provided strong support that the alternation of early stage WTA biosynthetic pathway is a viable alternative for the potentiation of β-lactam based antibiotics.

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CONCLUSIONS Continual discovery of novel antibiotics is vital for the survival of humankind in our battle against bacteria. Recent decades have witnessed both the rise in power due to their resistance to many once effective antibiotics as well as the decline in discovery of novel antibiotics. If these trends continue, experts predict that by 2050, 10 million deaths will occur annually from bacterial infections.31 In our continuous efforts to discover novel antibacterial mechanisms, we performed a phenotypic screening to identify WTA early stage inhibitors to intervene WTA polymer biosynthesis for revitalization of the important class of β-lactam based antibiotics. These efforts identified analogs with “undruglike” properties (for example, lead 1, clogP = 7.1 with unmeasurable solubility in PBS buffer) while invigorating our continued debate on whether the compound collection should be enriched with only “druglike” molecules. Clearly, in this case, identification of 1 would have been missed had we decided to screen a collection of molecules with only “druglike” properties. Furthermore, our optimization of the lead 1 to improve its physicochemical properties for in vivo study proved to be very challenging. Conventional wisdom suggested that it was much easier for medicinal chemists to add lipophilic moieties to a lead compound to achieve optimized potency, and 3860

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× 4), dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−100% EtOAc in hexane) to provide 30 (9.6 g, 91% yield). To a solution of 1,1′-carbonyldiimidazole (36.0 mg, 0.22 mmol) in acetonitrile (1 mL) was added 2-(naphthalen-1-yl)acetic acid (41.0 g, 0.22 mmol), and the reaction mixture was stirred at rt for 1 h. To this resulting mixture was added 30 (50.0 mg, 0.16 mmol) followed by DBU (34.0 mg, 0.22 mmol), and the reaction mixture was stirred at rt for 12 h. The resulting mixture was concentrated, and the crude product was purified by reverse phase HPLC to provide 1 (70.0 mg, 91% yield). 1H NMR (500 MHz, CDCl3) δ 7.97 (s, 1H), 7.95−7.90 (m, 2H), 7.87 (dd, J = 7.3, 2.3 Hz, 1H), 7.84 (s, 2H), 7.56 (m, 2H), 7.51−7.45 (m, 2H), 5.84 (d, J = 7.3 Hz, 1H), 4.94 (p, J = 6.7 Hz, 1H), 4.83 (d, J = 17.2 Hz, 1H), 4.77 (d, J = 17.2 Hz, 1H), 0.97 (d, J = 6.6 Hz, 3H). m/z: 482.2. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(isoquinolin4-yl)acetyl)-4-methyloxazolidin-2-one (2). Compound 2 was prepared from 2-(isoquinolin-4-yl)acetic acid and intermediate 30 following the acylation protocol used to prepare 1 in 40% yield. 1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H), 8.66 (s, 1H), 8.35 (d, J = 8.1 Hz, 1H), 8.16 (m, 2H), 7.97 (m, 2H), 7.85 (m, 2H), 5.97 (d, J = 7.2 Hz, 1H), 5.03−4.96 (m, 1H), 4.96−4.84 (m, 2H), 0.97 (d, J = 6.4 Hz, 3H). m/z: 482.9. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-4-methyl-3-(3(naphthalen-1-yl)propanoyl)oxazolidin-2-one (3). Compound 3 was prepared from 3-(naphthalen-1-yl)propanoic acid and intermediate 30 following the acylation protocol used to prepare 1 in 17% yield. 1 H NMR (500 MHz, CDCl3) δ 8.13 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.84−7.77 (m, 3H), 7.59 (dd, J = 8.4, 6.9, 1H), 7.54 (dd, J = 7.4, 6.7, 1H), 7.48−7.42 (m, 2H), 5.68 (d, J = 7.4 Hz, 1H), 4.96−4.85 (m, 1H), 3.57−3.39 (m, 4H), 0.95 (d, J = 6.7, 3H). m/z: 495.9. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-4-methyl-3-(4(naphthalen-1-yl)butanoyl)oxazolidin-2-one (4). Compound 4 was prepared from 4-(naphthalen-1-yl)butanoic acid and intermediate 30 following the acylation protocol used to prepare 1 in 56% yield. 1H NMR (400 MHz, CD3OD) δ 8.16 (d, J = 8.4 Hz, 1H), 8.03 (bs, 3H), 7.84 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.54−7.42 (m, 2H), 7.41−7.31 (m, 2H), 5.92 (d, J = 7.5 Hz, 1H), 4.94 (p, J = 6.8 Hz, 1H), 3.16 (t, J = 7.8 Hz, 2H), 3.12−2.96 (m, 2H), 2.09 (p, J = 7.4 Hz, 2H), 0.84 (d, J = 6.6 Hz, 3H). m/z: 510.1. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(isoquinolin4-yl)propanoyl)-4-methyloxazolidin-2-one (5). Compound 5 was prepared from 3-(isoquinolin-4-yl)propanoic acid and intermediate 30 following the acylation protocol used to prepare 1 in 12% yield. 1H NMR (400 MHz, CD3OD) δ 9.11 (s, 1H), 8.37 (s, 1H), 8.20 (dd, J = 8.6, 1.1 Hz, 1H), 8.12 (dq, J = 8.2, 1.2 Hz, 1H), 8.02 (s, 3H), 7.87 (ddt, J = 8.4, 6.8, 1.3 Hz, 1H), 7.71 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 5.93 (dd, J = 7.9, 3.2 Hz, 1H), 5.02−4.91 (m, 1H), 3.54−3.32 (m, 4H), 0.84 (d, J = 6.6 Hz, 3H). m/z: 497.2. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-N-(isoquinolin-4ylmethyl)-4-methyl-2-oxooxazolidine-3-carboxamide (6). To a solution of 30 (8.0 g, 26.1 mmol) in THF (50 mL) was added slowly a solution of n-BuLi (10.4 mL, 2.5 M in hexane, 26.1 mmol) at −78 °C, and the reaction mixture was stirred at −78 °C for 20 min. To this resulting solution was added rapidly trichloromethyl carbonochloridate (3.2 mL, 26.1 mmol), and the mixture was stirred at −78 °C for an additional 30 min before slowly warming to rt. The reaction mixture was carefully evaporated to obtain the 3-carbonyl chloride of 30, which was used directly in the next step without further purification. To a solution of this crude 3-carbonyl chloride of 30 (64.0 mg, 0.17 mmol) in DCM (1 mL) was added a mixture of isoquinolin-4ylmethanamine (93.0 mg, 0.34 mmol) and triethylamine (0.19 mL, 1.38 mmol) in DCM (1 mL) at 0 °C, and the resulting mixture was slowly warmed to rt and stirred for 12 h. The reaction mixture was quenched with water, extracted with DCM, dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (5−50% EtOAc in hexane) to obtain analog 6 (33.0 g, 20% yield). 1H NMR (500 MHz, CDCl3) δ 9.27 (s, 1H), 8.56 (s, 1H), 8.20 (t, J = 5.7 Hz, 1H), 8.11−8.04 (m, 2H), 7.96−7.93 (m, 1H),

7.86−7.78 (m, 3H), 7.70 (ddd, J = 8.0, 6.9, 1.1 Hz, 1H), 5.87−5.81 (m, 1H), 5.05−4.90 (m, 3H), 1.01 (d, J = 6.6 Hz, 3H). m/z: 498.4. (4S,5R)-N-((1,6-Naphthyridin-8-yl)methyl)-5-(3,5-bis(trifluoromethyl)phenyl)-4-methyl-2-oxooxazolidine-3-carboxamide (7). Compound 7 was prepared from (1,6-naphthyridin-8yl)methanamine and intermediate 30 according to the procedure for 6 in 59% yield. 1H NMR (500 MHz, CDCl3) δ 9.26 (s, 1H), 9.18 (dd, J = 4.3, 1.8 Hz, 1H), 8.83 (t, J = 6.0 Hz, 1H), 8.78 (s, 1H), 8.33 (dd, J = 8.3, 1.8 Hz, 1H), 7.90 (s, 1H), 7.79 (d, J = 1.5 Hz, 2H), 7.60 (dd, J = 8.3, 4.3 Hz, 1H), 5.79 (d, J = 7.6 Hz, 1H), 5.07 (d, J = 6.0 Hz, 2H), 4.90 (dt, J = 7.6, 6.5 Hz, 1H), 0.94 (d, J = 6.6 Hz, 3H). m/z: 499.7. (4S,5R)-5-(3-Fluoro-5-(trifluoromethyl)phenyl)-N-(isoquinolin-4-ylmethyl)-4-methyl-2-oxooxazolidine-3-carboxamide (8). Compound 8 was prepared from isoquinolin-4-ylmethanamine and intermediate 31 according to the procedure for 6 in 2% yield. 1H NMR (500 MHz, CDCl3) δ 9.30 (s, 1H), 8.56 (s, 1H), 8.23 (t, J = 5.7 Hz, 1H), 8.10 (m, 2H), 7.86 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.73 (ddd, J = 8.2, 6.9, 1.0 Hz, 1H), 7.43−7.35 (m, 2H), 7.29 (m, 1H), 5.81−5.74 (m, 1H), 5.03 (dd, J = 15.0, 5.9 Hz, 1H), 4.96 (dd, J = 15.0, 5.6 Hz, 1H), 4.90 (dq, J = 7.7, 6.6 Hz, 1H), 1.03 (d, J = 6.6 Hz, 3H). m/z: 448.1. (4S,5R)-N-(Isoquinolin-4-ylmethyl)-4-methyl-2-oxo-5-(3(trifluoromethyl)phenyl)oxazolidine-3-carboxamide (9). Compound 9 was prepared from isoquinolin-4-ylmethanamine and intermediate 32 according to the procedure for 6 in 33% yield. 1H NMR (500 MHz, CDCl3) δ 9.26 (s, 1H), 8.55 (s, 1H), 8.24 (t, J = 5.7 Hz, 1H), 8.08 (dt, J = 8.5, 0.9 Hz, 1H), 8.05 (dt, J = 8.2, 1.0 Hz, 1H), 7.81 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.68 (m, 2H), 7.61−7.55 (m, 2H), 7.50 (d, J = 8.1 Hz, 1H), 5.78 (d, J = 7.7 Hz, 1H), 4.99 (dd, J = 14.9, 5.8 Hz, 1H), 4.94 (dd, J = 14.9, 5.6 Hz, 1H), 4.88 (dq, J = 7.7, 6.5 Hz, 1H), 0.99 (d, J = 6.5 Hz, 3H). m/z: 430.2. (4S,5R)-N-((1,6-Naphthyridin-8-yl)methyl)-4-methyl-2-oxo5-(3-(trifluoromethyl)phenyl)oxazolidine-3-carboxamide (10). Compound 10 was prepared from (1,6-naphthyridin-8-yl)methanamine and intermediate 32 according to the procedure for 6 in 31% yield. 1H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H), 9.17 (dd, J = 4.2, 1.9 Hz, 1H), 8.84 (t, J = 6.0 Hz, 1H), 8.79 (s, 1H), 8.32 (dd, J = 8.3, 1.8 Hz, 1H), 7.65−7.62 (m, 1H), 7.61−7.52 (m, 3H), 7.48 (dd, J = 8.0, 1.5 Hz, 1H), 5.73 (d, J = 7.6 Hz, 1H), 5.07 (d, J = 6.0 Hz, 2H), 4.82 (dq, J = 7.7, 6.6 Hz, 1H), 0.92 (d, J = 6.6 Hz, 3H). m/z: 431.3. (4S,5S)-N-(Isoquinolin-4-ylmethyl)-4-methyl-2-oxo-5-(4(trifluoromethyl)pyridin-2-yl)oxazolidine-3-carboxamide (11). Compound 11 was prepared from isoquinolin-4-ylmethanamine and intermediate 33 according to the procedure for 6 in 12% yield. 1H NMR (400 MHz, CD3OD) δ 9.49 (s, 1H), 8.86 (s, 1H), 8.69 (s, 1H), 8.51 (s, 1H), 8.38 (m, J = 11.0 Hz, 2H), 8.11 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.70 (m,, 1H), 5.91 (d, J = 7.8 Hz, 1H), 5.04 (d, J = 5.6 Hz, 2H), 4.07 (q, J = 7.4 Hz, 1H), 0.89 (d, J = 7.4 Hz, 3H). m/ z: 431.5. (4S,5S)-N-((1,6-Naphthyridin-8-yl)methyl)-5-(6-methoxy-4(trifluoromethyl)pyridin-2-yl)-4-methyl-2-oxooxazolidine-3carboxamide (14). A mixture of 2,6-dichloro-4-(trifluoromethyl)pyridine 34 (5.0 g, 23.2 mmol) and 30% HBr in acetic acid (4.2 mL, 23.0 mmol) was heated at 100 °C for 24 h in a sealed tube. The reaction mixture was cooled to 0 °C and neutralized carefully with an aqueous solution of 6 N NaOH. The resulting mixture was extracted with diethyl ether, and the ether layer was dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 35 (5.0 g, 70% yield). To a solution of prop-2-en-1-ol (3.6 mL, 52.5 mmol) in THF (40 mL) was added NaH (2.1 g, 52.5 mmol) in several portions at 0 °C. The resulting solution was slowly warmed to rt and stirred for 10 min. Then the reaction mixture was cooled to 0 °C, and to it was added a solution of 2,6-dibromo-4-(trifluoromethyl)pyridine 35 (16.0 g, 52.5 mmol) in THF (50 mL). The resulting mixture was slowly warmed to rt and stirred for 30 min. The reaction mixture was diluted with an aqueous solution of saturated NH4Cl and extracted with EtOAc. The EtOAc layer was dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 36 (12.0 g, 81% yield). 3861

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

Journal of Medicinal Chemistry

Article

To a solution of n-BuLi (18 mL, 2.5 M in hexane, 45.1 mmol) in THF (25 mL) was added a solution of 36 (12.7 g, 45.1 mmol) in THF (20 mL) at −78 °C. The resulting solution was stirred at −78 °C for 20 min, and to it was added a solution of 28 (6.0 g, 22.5 mmol) in THF (20 mL). The reaction mixture was slowly warmed to 0 °C over 1 h. The resulting mixture was quenched with an aqueous solution of saturated NH4Cl and extracted with EtOAc. The EtOAc layer was dried with MgSO4, filtered, concentrated, and the crude product was purified using silica gel chromatography (0−100% EtOAc in hexane) to provide benzyl (S)-(1-(6-(allyloxy)-4-(trifluoromethyl)pyridin-2yl)-1-oxopropan-2-yl)carbamate (7.0 g, 53% yield). m/z: 409.25. To a solution of this (S)-benzyl (1-(6-(allyloxy)-4(trifluoromethyl)pyridin-2-yl)-1-oxopropan-2-yl)carbamate (5.0 g, 12.2 mmol) in a mixture of toluene (10 mL) and 2-propanol (5 mL) was added aluminum isopropoxide (0.5 g, 2.4 mmol) at rt, and the resulting solution was heated at 50 °C for 4 h. The reaction mixture was cooled to rt, diluted with brine, extracted with EtOAc, and the EtOAc layer was dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 37 (5.0 g, 100% yield). m/z: 411.2. To a solution of 37 (0.9 g, 2.2 mmol) in THF (3 mL) was added potassium bis(trimethylsilyl)amide (4.8 mL, 4.8 mmol) at −20 °C. The resulting mixture was stirred at −20 °C for 10 min before slowly warming to 0 °C. An aqueous solution of saturated NH4Cl was added, and the resulting mixture was extracted with EtOAc. The EtOAc layer was washed with a solution of saturated NaHCO3 followed by brine, dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 38 (0.32 g, 47% yield). m/z: 303.1. The mixture of 38 (0.3 g, 1.0 mmol), di-tert-butyl dicarbonate (0.46 mL, 2.0 mmol) and DMAP (24.0 mg, 0.2 mmol) was stirred in a sealed tube for 12 h at rt. Then the reaction mixture was directly loaded into a column and purified by silica gel chromatography (0− 10% EtOAc in hexane) to provide 39 (0.4 g, 98% yield). m/z: 805.4. To a solution of 39 (0.4 g, 1.0 mmol) in DCM (2 mL) was added Pd(Ph3)4 (0.11 g, 0.1 mmol) followed by 1,3-dimethylpyrimidine2,4,6-(1H,3H,5H)-trione (0.46 g, 3.0 mmol). The resulting mixture was cooled to −78 °C and degassed and backfilled with nitrogen. Then the reaction mixture was warmed to rt and stirred for 12 h. The resulting mixture was diluted with EtOAc and washed with a solution of saturated NaHCO3. The EtOAc layer was separated, dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 40 (0.35 g, 97% yield). m/z: 725.4. To a solution of 40 (0.20 g, 0.55 mmol) in DMF (2 mL) were added CsF (0.25 g, 1.66 mmol) and iodomethane (0.1 mL, 1.66 mmol) at rt, and the resulting solution was stirred for 12 h. The reaction mixture was quenched with a solution of saturated NH4Cl and extracted with EtOAc. The EtOAc layer was washed with brine, dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−10% EtOAc in hexane) to provide 41 (0.10 g, 50% yield). To a solution of 41 (0.53 g, 1.42 mmol) in DCM (5 mL) was added trifluoroacetic acid (0.5 mL, 7.12 mmol) at rt, and the mixture was stirred for 12 h. The reaction mixture was quenched with a solution of saturated NaHCO3 (5 mL), extracted with DCM, dried over MgSO4, filtered, and concentrated to provide 42 (0.38 g), which was taken directly to the next step without further purification. To a solution of 42 (0.38 g, 1.38 mmol) in THF (5 mL) was added n-BuLi (0.83 mL, 2.5 M in hexane, 2.07 mmol) at 0 °C, and the mixture was stirred for 30 min. The reaction mixture was cooled to −78 °C, and to it was added rapidly trichloromethyl carbonochloridate (0.2 mL, 2.00 mmol). The resulting mixture was stirred for 30 min at −78 °C before warming to rt. The reaction mixture was evaporated to dryness, and the resulting crude 3-carbonyl chloride of 41 was taken directly to the next step without further purification. A portion of 3carbonyl chloride of 41 (0.16 g, 0.46 mmol) was added to a mixture of (1,6-naphthyridin-8-yl)methanamine·3HCl (0.24 g, 0.92 mmol) and triethylamine (0.5 mL, 3.69 mmol) in DCM (2 mL) at 0 °C, and the resulting mixture was warmed to rt and stirred for 4 h. The reaction

mixture was concentrated, and the residue was purified by silica gel chromatography (0−100% EtOAc in hexane) to afford 14 (0.04 g, 19% yield). 1H NMR (500 MHz, CDCl3) δ 9.28 (s, 1H), 9.20 (dd, J = 4.3, 1.8 Hz, 1H), 8.84 (t, J = 6.0 Hz, 1H), 8.80 (s, 1H), 8.35 (dd, J = 8.3, 1.8 Hz, 1H), 7.62 (dd, J = 8.3, 4.3 Hz, 1H), 7.27−7.24 (m, 1H), 6.95 (dd, J = 1.4, 0.8 Hz, 1H), 5.61 (dt, J = 7.9, 0.8 Hz, 1H), 5.09 (d, J = 6.0 Hz, 2H), 4.96 (dq, J = 7.8, 6.5 Hz, 1H), 3.90 (s, 3H), 1.00 (d, J = 6.6 Hz, 3H). m/z: 462.1. (4S,5R)-5-(3,5-Bis(trifluoromethyl)phenyl)-4-(hydroxymethyl)-N-(isoquinolin-4-ylmethyl)-2-oxooxazolidine-3-carboxamide (18). To a solution of 43 (170 g, 800 mmol) in DCM (1.8 L) was added N,O-dimethylhydroxylamine hydrochloride (116 g, 900 mmol), and the resulting mixture was stirred at −15 °C for 5 min. Then N-methylmorpholine (220 mL, 900 mmol) and EDCI (160 g, 900 mmol) were added in portions while keeping the temperature below −5 °C. After the addition, the resulting mixture was stirred at −5 °C for 3 h and then was quenched with ice-cooled 1 N HCl (800 mL). The organic phase was separated, washed with 1 N HCl (300 mL × 2) followed by saturated NaHCO3, dried with MgSO4, filtered, and concentrated to afford 44 (190 g, 95% yield). 1H NMR (CDCl3, 400 MHz) δ 7.36−7.31 (m, 5H), 5.91 (s, 1H), 5.11 (d, J = 2.0 Hz, 2H), 4.87 (s, 1H), 3.85 (s, 2H), 3.77 (s, 3H), 3.22 (s, 3H). m/z: 282.9. To a solution of 44 (20.0 g, 72 mmol) in DCM (400 mL) were added imidazole (12.0 g, 176 mmol) and TBDMSCl (16.0 g, 106 mmol), and the resulting mixture was stirred at rt for 2 h. The reaction mixture was quenched with saturated NH4Cl and extracted with DCM. The DCM layer was washed with water followed by brine, dried with MgSO4, concentrated, and the crude product was purified by silica gel chromatography (7% ethyl acetate in hexanes) to afford 45 (23.0 g, 95% yield). 1H NMR (CDCl3, 400 MHz) δ 7.36−7.29 (m, 5H), 5.63 (s, 1H), 5.14−5.06 (m, 2H), 4.81 (s, 1H), 3.90−3.80 (m, 2H), 3.73 (s, 3H), 3.21 (s, 3H), 0.83(s, 9H), 0.01 (s, 6H). m/z: 396.9. To a mixture of 45 (23.0 g, 58 mmol) and 1-bromo-3,5-bistrifluoromethylbenzene (27.0 g, 70 mmol) in anhydrous THF (200 mL) was added i-PrMgCl (73 mL, 2 M in THF, 146 mmol) at −5 °C. After the addition, the resulting mixture was warmed to rt and stirred for 12 h. The reaction mixture was neutralized using aqueous solution of 3 N HCl and extracted with EtOAc. The EtOAc layer was washed with water, dried with MgSO4, filtered, evaporated, and the crude product was purified by silica gel chromatography (10% EtOAc in petroleum ether) to afford 46 (30.0 g, 93% yield). 1H NMR (CDCl3, 400 MHz) δ 8.37 (s, 2H), 8.08 (s, 1H), 7.37−7.34 (m, 5H), 5.84 (d, J = 4.0 Hz, 1H), 5.39−5.37 (m, 1H), 5.13 (s, 2H), 4.02−3.84 (m, 2H), 0.70 (s, 9H), −0.10 (d, J = 5.6 Hz, 6H). m/z: 549.9. A mixture of 46 (10.0 g, 18 mmol), Al(O-i-PrO)3 (3.9 g, 18 mmol) and i-PrOH (15.6 mL, 198 mmol) in anhydrous toluene (200 mL) was heated at 60 °C for 12 h. The reaction mixture was then cooled to rt and washed with water. The organic layer was dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (15% EtOAc in petroleum ether) to afford 47 (1.5 g, 15% yield). 1H NMR (CDCl3, 400 MHz) δ 7.89−7.79 (m, 3H), 7.40− 7.31 (m, 5H), 5.60 (s, 1H), 5.15−5.03 (m, 2H), 4.45−4.35 (m, 1H), 3.95 (s, 1H), 3.69−3.61 (m, 2H), 0.91 (s, 9H), 0.04 (d, J = 5.6 Hz, 6H). m/z: 551.9. A mixture of 47 (20.0 g, 36 mmol) and KOH (20 mL of 48% aqueous solution) in toluene (200 mL) was stirred at rt for 12 h. The reaction mixture was diluted with EtOAc, washed with water followed by brine. The EtOAc layer was dried with MgSO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (15% EtOAc in petroleum ether) to afford 48 (12.0 g, 87% yield). 1H NMR (CDCl3, 400 MHz) δ 7.88 (s, 1H), 7.84 (s, 2H), 5.86 (s, 1H), 5.77 (s, 1H), 4.17 (t, J = 4.2 Hz, 1H), 3.29−3.21 (m, 2H), 0.769 (s, 9H), −0.11 (d, J = 5.6 Hz, 6H). m/z: 443.9. To a solution of 48 (0.20 g, 0.96 mmol) in THF (5 mL) was added n-BuLi (0.39 mL, 2.5 M in hexane, 0.96 mmol) at 0 °C, and the resulting mixture was stirred for 20 min. The reaction mixture was cooled to −78 °C, and to it was added rapidly trichloromethyl chloroformate (0.23 mL, 0.96 mmol). The resulting mixture was stirred for 30 min before warming up to rt. The reaction mixture was 3862

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

Journal of Medicinal Chemistry

Article

N,O-dimethylhydroxylamine hydrochloride (1.69 g, 17.38 mmol), DIPEA (6.17 mL, 35.3 mmol), and EDC (3.47 g, 18.10 mmol) at 0 °C. The resulting mixture was slowly warmed to rt and stirred for 12 h and then was diluted with EtOAc, washed with 1 N HCl followed by brine, dried with Na2SO4, filtered, and concentrated to yield 51 (4.20 g, 84%). m/z: 375.2. A mixture of 3-bromo-5-fluorobenzotrifluoride (4.40 g, 18.11 mmol) and 51 (4.48 g, 12.71 mmol) in THF (11.56 mL) was cooled to 0 °C, and to it was added i-PrMgCl (17.38 mL, 2 M in THF, 34.8 mmol). The resulting mixture was slowly warmed up to rt and stirred for 16 h. The reaction mixture was poured into 1 N HCl aqueous solution and extracted with EtOAc. The EtOAc layer was washed with brine, dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−30% EtOAc in hexane) to yield 52 (5.0 g, 87%). m/z: 456.2. To a mixture of 52 (5.01 g, 11 mmol) and 2-propanol (5.95 mL) in toluene (8.92 mL) was added Al(O-i-Pr)3 (0.88 g, 4.4 mmol), and the resulting mixture was heated at 50 °C for 14 h, and then cooled to rt. To this solution was added KOH (1.23 g, 22 mmol), and the resulting mixture was stirred for 8 h at rt. The reaction mixture was poured into 1 N HCl aqueous solution and extracted with EtOAc. The EtOAc layer was separated, dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−50% EtOAc in hexane) to give 53 (3.27 g, 78% yield). m/z: 384.6. To a solution of 53 (0.65 g, 1.70 mmol) in THF (10 mL) was added n-BuLi (0.717 mL, 2.5 M in hexane, 1.79 mmol) at −78 °C, and the resulting solution was stirred for 0.5 h, and then to it was added rapidly trichloromethyl chloroformate (0.20 mL, 1.70 mmol). The resulting mixture was slowly warmed to rt. The reaction mixture was concentrated and dissolved in DCM (15 mL). To this resulting mixture was added TEA (1.18 mL, 8.53 mmol) followed by isoquinolin-4-ylmethanamine hydrochloride (0.39 g, 1.70 mmol) at 0 °C, and the resulting mixture was warmed to rt and stirred for 16 h. The reaction mixture was poured into water, extracted with DCM, and the DCM layer was washed with brine, dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−70% EtOAc in hexane) to provide urea derivative of 53 (0.36 g, 37% yield). m/z: 568.3. To a portion of this urea derivative (64 mg, 0.11 mmol) in DCM (5 mL) was added pentamethylbenzene (84 mg, 0.56 mmol) followed by solution of boron trichloride in DCM (0.11 mL, 0.11 mmol) at −78 °C, and the reaction mixture was slowly warmed to 0 °C over 1 h. LC−MS showed the presence of starting material. The reaction mixture was cooled back to −78 °C, and to the mixture was added an additional amount of BCl3 (0.11 mL, 0.11 mmol), and the resulting solution was stirred for 1 h. The reaction mixture was diluted with EtOAc, washed with a solution of saturated NaHCO3 followed by with brine, dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−100% EtOAc in hexane) to afford 23 (25 mg, 46% yield). 1H NMR (500 MHz, CDCl3) δ 9.29 (s, 1H), 8.56 (s, 1H), 8.30 (t, J = 5.7 Hz, 1H), 8.09 (m, 2H), 7.85 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.71 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H), 7.43−7.37 (m, 2H), 7.27 (m, 1H), 5.87 (d, J = 7.2 Hz, 1H), 5.13−5.02 (m, 2H), 4.95 (dd, J = 14.9, 5.4 Hz, 1H), 3.73−3.37 (m, 2H), 1.34 (m, 2H). m/z: 478.7. 2-((4S,5R)-5-(3-Fluoro-5-(trifluoromethyl)phenyl)-3-((isoquinolin-4-ylmethyl)carbamoyl)-2-oxooxazolidin-4-yl)acetic Acid (24). To a solution of 23 (56 mg, 0.11 mmol) in a mixture of acetonitrile (0.5 mL), EtOAc (0.5 mL), and water (1 mL) were added sodium periodate (0.12 g, 0.58 mmol) and ruthenium(III) chloride hydrate (5 mg, 0.02 mmol), and the resulting mixture was stirred at rt for 1.5 h. Then the reaction mixture was diluted with EtOAc (10 mL) and washed with water (10 mL). The EtOAc layer was dried with Na2SO4, filtered, concentrated, and the crude product was purified by reverse phase HPLC to obtain 24 (15 mg, yield 24.3%). 1H NMR (500 MHz, CDCl3) δ 9.55 (s, 1H), 8.78 (dd, J = 7.4, 5.0 Hz, 1H), 8.53 (s, 1H), 8.39 (dd, J = 8.3, 1.1 Hz, 1H), 8.33−8.28 (m, 1H), 8.20 (ddd, J = 8.5, 6.9, 1.3 Hz, 1H), 8.01 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.41− 7.37 (m, 2H), 7.30 (m, 1H), 5.93 (d, J = 8.3 Hz, 1H), 5.38−5.27 (m, 1H), 5.10 (ddd, J = 8.6, 6.4, 2.9 Hz, 1H), 4.89 (dd, J = 16.3, 5.0 Hz,

evaporated to dryness, and the crude product was taken directly to the next step without further purification. To a portion of this crude 3-carbonyl chloride of 48 (0.22.g, 0.45 mmol) in DCM (5 mL) was added isoquinolin-4-ylmethanamine (0.10 g, 0.68 mmol), triethylamine (0.23 mL, 1.80 mmol), and water (2 drops) at 0 °C, and the resulting mixture was slowly warmed to rt and stirred for 12 h. The reaction mixture was diluted with DCM (10 mL) and washed with water (10 mL). The DCM layer was dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−50% EtOAc in hexane) to provide the corresponding urea intermediate (0.16 g, 38% yield). To a solution of this urea intermediate (0.10 g, 0.16 mmol) in MeOH (5 mL) was added acetyl chloride (0.01 mL, 0.19 mmol), and the resulting mixture was stirred for 2 h at rt. The reaction mixture was diluted with DCM (10 mL) and washed with water (10 mL). The DCM layer was dried with Na2SO4, filtered, concentrated, and the crude product was purified by silica gel chromatography (0−50% EtOAc in hexane) to obtain 18 (29 mg, 36% yield). 1H NMR (400 MHz, CD3OD) δ 9.13 (s, 1H), 8.62 (t, J = 5.9 Hz, 1H), 8.44 (s, 1H), 8.17 (dq, J = 8.5, 0.9 Hz, 1H), 8.12−8.05 (m, 3H), 7.95 (tt, J = 1.8, 0.9 Hz, 1H), 7.83 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.68 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H), 5.95 (d, J = 8.1 Hz, 1H), 5.01−4.88 (m, 2H), 4.80 (ddd, J = 8.1, 3.2, 1.3 Hz, 1H), 3.84 (dd, J = 12.0, 3.2 Hz, 1H), 3.08 (dd, J = 12.0, 1.4 Hz, 1H). m/z: 514. (4S,5R)-N-((1,6-Naphthyridin-8-yl)methyl)-5-(3,5-bis(trifluoromethyl)phenyl)-4-(hydroxymethyl)-2-oxooxazolidine-3-carboxamide (19). Compound 19 was prepared from intermediate 48 using (1,6-naphthyridin-8-yl)methanamine according to the procedure for 18 in 18% yield. 1H NMR (500 MHz, CDCl3) δ 9.15 (dd, J = 4.3, 1.8 Hz, 1H), 9.05 (s, 1H), 8.95 (t, J = 6.1 Hz, 1H), 8.62 (s, 1H), 8.20 (dd, J = 8.3, 1.8 Hz, 1H), 7.95 (d, J = 1.5 Hz, 2H), 7.90 (s, 1H), 7.56 (dd, J = 8.3, 4.3 Hz, 1H), 5.83 (d, J = 8.1 Hz, 1H), 5.01−4.89 (m, 2H), 4.79 (dt, J = 8.2, 2.7 Hz, 1H), 3.93 (dd, J = 12.3, 3.0 Hz, 1H), 3.24 (dd, J = 12.4, 2.3 Hz, 1H). m/z: 514.9. (4S,5R)-5-(3-Fluoro-5-(trifluoromethyl)phenyl)-4-(hydroxymethyl)-N-(isoquinolin-4-ylmethyl)-2-oxooxazolidine-3-carboxamide (20). Compound 20 was prepared using 1-bromo-3fluoro-5-(trifluoromethyl)benzene according to the procedure for 18. 1 H NMR (400 MHz, CD3OD) δ 9.19 (s, 1H), 8.47 (s, 1H), 8.22 (dd, J = 8.5, 1.0 Hz, 1H), 8.18−8.12 (m, 1H), 7.88 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.73 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 7.63 (s, 1H), 7.54 (d, J = 9.3 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 5.89 (d, J = 8.0 Hz, 1H), 5.05−4.89 (m, 2H), 4.75 (ddd, J = 8.0, 3.3, 1.4 Hz, 1H), 3.83 (dd, J = 11.9, 3.3 Hz, 1H), 3.13 (dd, J = 12.0, 1.4 Hz, 1H). m/z: 464.2. (4S,5R)-4-(Hydroxymethyl)-N-(isoquinolin-4-ylmethyl)-2oxo-5-(3-(trifluoromethyl)phenyl)oxazolidine-3-carboxamide (21). Compound 21 was prepared using 1-bromo-3-(trifluoromethyl)benzene according to the procedure for 18. 1H NMR (500 MHz, CD3OD) δ 9.21 (s, 1H), 8.50 (s, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.90 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.83 (s, 1H), 7.78−7.73 (m, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 5.92 (d, J = 8.0 Hz, 1H), 5.04 (d, J = 15.3 Hz, 1H), 4.96 (d, J = 15.3 Hz, 1H), 4.76 (ddd, J = 8.1, 3.4, 1.4 Hz, 1H), 3.83 (dd, J = 11.8, 3.4 Hz, 1H), 3.15 (dd, J = 11.9, 1.5 Hz, 1H). m/z: 446.1. (4R,5R)-3-((Isoquinolin-4-ylmethyl)carbamoyl)-2-oxo-5-(3(trifluoromethyl)phenyl)oxazolidine-4-carboxylic Acid (22). To a solution of 21 (50 mg, 0.11 mmol) in a mixture of acetonitrile (0.15 mL), EtOAc (0.15 mL), and water (0.3 mL) were added sodium periodate (98 mg, 0.46 mmol) and ruthenium(III) chloride hydrate (1 mg, 0.004 mmol). The resulting mixture was stirred at rt for 12 h and was then diluted with water, extracted with EtOAc (3×), dried with Na2SO4, filtered, concentrated, and the crude product was purified by reverse phase HPLC to afford 22 (2.9 mg, 5.6% yield). 1H NMR (400 MHz, CD3OD) δ 9.21 (s, 1H), 8.58 (t, J = 5.8, 1H), 8.46 (s, 1H), 8.18 (m, 2H), 7.89 (t, J = 7.7 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.71−7.62 (m, 3H), 7.57 (t, J = 7.7 Hz, 1H), 6.01 (d, J = 8.9 Hz, 1H), 5.12 (d, J = 9.0 Hz, 1H), 4.97 (m, 2H). m/z: 460.2. (4S,5R)-5-(3-Fluoro-5-(trifluoromethyl)phenyl)-4-(2-hydroxyethyl)-N-(isoquinolin-4-ylmethyl)-2-oxooxazolidine-3-carboxamide (23). To a solution of 50 (4.48 g, 14.48 mmol) in THF (30 mL) was added 1-hydroxybenzotriazole hydrate (2.39 g, 15.64 mmol), 3863

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

Journal of Medicinal Chemistry

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1H), 2.96 (dd, J = 17.6, 6.4 Hz, 1H), 2.24 (dd, J = 17.6, 2.9 Hz, 1H). m/z: 492.2. (4S,5R)-5-(3-Fluoro-5-(trifluoromethyl)phenyl)-4-methyloxazolidin-2-one (31). Intermediate 31 was prepared from 1-bromo-3fluoro-5-(trifluoromethyl)benzene according to the procedure for 30. 1 H NMR (600 MHz, CDCl3) δ 7.35 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.58 (s, 1H), 5.82−5.62 (m, 1H), 4.27 (p, J = 6.9 Hz, 1H), 0.84 (d, J = 6.6 Hz, 3H). m/z: 264.1. (4S,5R)-4-Methyl-5-(3-(trifluoromethyl)phenyl)oxazolidin-2one (32). Intermediate 32 was prepared from 1-bromo-3(trifluoromethyl)benzene according 20 (to the procedure for 30). 1 H NMR (500 MHz, CDCl3) δ 7.67−7.64 (m, 1H), 7.61−7.52 (m, 3H), 6. s, 1H), 5.80 (d, J = 8.0 Hz, 1H), 4.29 (dq, J = 8.2, 6.6 Hz, 1H), 0.84 (d, J = 6.6 Hz, 3H). m/z: 246.1. (4S,5S)-4-Methyl-5-(4-(trifluoromethyl)pyridin-2-yl)oxazolidin-2-one (33). The intermediate 33 was prepared from 2bromo-4-(trifluoromethyl)pyridine according to the procedure for 30. 1 H NMR (400 MHz, CD3OD) δ 8.84 (d, J = 5.1 Hz, 1H), 7.71 (s, 1H), 7.69−7.65 (d, J = 5.1 Hz, 1H), 5.83 (d, J = 8.4 Hz, 1H), 4.39 (dq, J = 8.4, 6.5 Hz, 1H), 0.74 (d, J = 6.6, 3H). m/z: 247.01.

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REFERENCES

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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.7b00113. Synthetic procedures for compounds 12, 13, 15, 16, 17, 25, and 26, Table 1S containing the results from secondary confirmatory assays, such as suppression of TarG inhibition, and bacteriophage ØK (phage K) protection, as well as mammalian HeLa cell cytotoxicity data, and Table 2S containing data from the satellite PK study of 9 and IPM following the dosing regimen used in the in vivo efficacy study (PDF) Molecular formula strings and some data (CSV)

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Article

AUTHOR INFORMATION

Corresponding Authors

*M.M.: phone, +1 908-740-2369; e-mail, mihirbaran.mandal@ merck.com. *J.S.: phone, +1 908-740-3858; e-mail, [email protected]. ORCID

Mihirbaran Mandal: 0000-0002-7870-7271 Alexei V. Buevich: 0000-0002-5968-9151 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank to Melissa Lin for her help in compound characterization and Li-Kang Zhang and Junling Gao for their help in LC−MS analysis of the compounds. Kristine Devito and Elizabeth Smith are thanked for their efforts in assessing cytotoxicity of all the final compounds against mammalian cells.

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ABBREVIATIONS USED WTA, wall teichoic acid; LLE, lipophilic ligand efficiency; MITC95, minimum concentration necessary to inhibit 95% growth as determined by OD600 reading; CFU, colony forming unit; PBS, phosphate buffered saline; DIEA, diisopropylethylamine; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; DCM, dichloromethane; MRSA COL, methicillin-resistant Staphylococcal aureus COL 3864

DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.7b00113 J. Med. Chem. 2017, 60, 3851−3865