Discovery of 3, 5-Diphenyl-4-methyl-1, 3-oxazolidin-2-ones as Novel

Article Cite This: J. Med. Chem. 2017, 60, 8963-8981

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Discovery of 3,5-Diphenyl-4-methyl-1,3-oxazolidin-2-ones as Novel, Potent, and Orally Available Δ‑5 Desaturase (D5D) Inhibitors Jun Fujimoto,* Rei Okamoto,† Naoyoshi Noguchi,‡ Ryoma Hara,‡ Shinichi Masada, Tetsuji Kawamoto,† Hiroki Nagase, Yumiko Okano Tamura, Mitsuaki Imanishi,† Shuichi Takagahara,§ Kazuki Kubo, Kimio Tohyama, Koichi Iida, Tomohiro Andou,† Ikuo Miyahisa, Junji Matsui,† Ryouta Hayashi,† Tsuyoshi Maekawa,‡ and Nobuyuki Matsunaga§ Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan S Supporting Information *

ABSTRACT: The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.

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INTRODUCTION Δ-5 desaturase (D5D) is a transmembrane protein mainly expressed in the liver and associated with polyunsaturated fatty acid (PUFA) biosynthesis (Figure 1).1−3 In this pathway, an essential ω-6 fatty acid, linoleic acid, is desaturated at the 6position by Δ-6 desaturase (D6D) to afford γ-linolenic acid, which is converted by elongase to dihomo-γ-linolenic acid (DGLA). D5D is involved in the production of arachidonic acid (AA) from DGLA by desaturation of the 5-postiion of DGLA. DGLA is known as a precursor of anti-inflammatory eicosanoids, such as prostaglandin E1 (PGE1) and 15hydroxytrienoic acid.4 In contrast, AA is a precursor of inflammatory unsaturated fatty acids, such as prostaglandins (PGs), leukotrienes (LTs), and thromboxanes (TXs). Therefore, D5D inhibitors are expected to drive anti-inflammatory properties through reduction of AA biosynthesis and simultaneous accumulation of DGLA. This concept is supported by a report regarding D5D-deficient mice that displayed increases in PGE1 and concurrent decreases in PGE2 levels compared to wild-type mice.5 So far, a few groups disclosed D5D inhibitors in the literature (Figure 2), but none have yet entered into clinical trials. In © 2017 American Chemical Society

Figure 1. Biosynthetic pathway of PUFA.

Received: August 16, 2017 Published: October 12, 2017 8963

DOI: 10.1021/acs.jmedchem.7b01210 J. Med. Chem. 2017, 60, 8963−8981

Journal of Medicinal Chemistry

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Figure 2. Reported D5D inhibitors (representative compounds).

1998, Obukowicz et al. reported 4-(3-methylbutoxy)aniline (CP-24879)6 with dual D5D/D6D inhibition and 2-amino-N(3-chlorophenyl)benzamide (CP-74006)7 with selective D5D inhibition, confirmed in rat liver microsomes and mastocytoma ABMC-7 cells. Starting from the structure of the latter, Lexicon Pharmaceuticals employed a cyclization approach, leading to the identification of the indazole compound shown in Figure 2 that displayed in vivo D5D inhibition in mouse liver.8 Takeda has also been interested in this target for its potential in antiinflammatory drug development and has disclosed fused and monocyclic pyrimidinones.9−11 Herein, we describe our efforts to discover and optimize a novel series of D5D inhibitors. Starting points were identified through a high-throughput screening (HTS) campaign utilizing a binding assay,12 which revealed a number of hit compounds. Among these, compounds 1−4 were highlighted due to their possessing moderate to good potencies and low molecular weights (Figure 3). Moreover, we noticed their structural similarities, which are characterized by aromatic rings at each end of the molecule and a spacer containing a carbonyl group present in the form of an amide or urea linkage. On the basis of these features, we constructed a pharmacophore model using Molecular Operating Environment (MOE)13 software as illustrated in Figure 4a and Figure 4b and hypothesized that a novel scaffold for D5D inhibitors could be discovered by the design of compounds matching this model. With this aim, two general structures, 1,3-oxazolidin-2-ones (5-Ph and 5-Bn in Figure 4c), were designed as compounds possessing two aromatic rings linked by a central 1,3-oxazolidin-2-one core. As shown in Figure 4d, both compounds fit well with the threepoint pharmacophore model, which encouraged us to embark

Figure 4. (a) A D5D inhibitor pharmacophore model constructed by common features of hit compounds. Nitrogens and oxygens are colored blue and red, respectively. The model includes (i) top and bottom aromatic rings (orange spheres) and (ii) a hydrogen-bond acceptor (cyan sphere). (b) Distances (Å) between each sphere. (c) Structures of the designed 1,3-oxazolidin-2-ones 5-Ph and 5-Bn. (d) Results of docking 5-Ph (light green) and 5-Bn (purple) to the pharmacophore model illustrated in (b).

on synthetic activity around 1,3-oxazolidin-2-one derivatives. In this article, our medicinal chemistry efforts on the discovery of a novel lead scaffold and further optimization to generate potent and orally available D5D inhibitors are discussed.

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CHEMISTRY The preparation of 3-benzyl-1,3-oxazolidin-2-one 5a was carried out following the procedure shown in Scheme 1. The oxazolidin-2-one compound 7 was formed by the reaction of 2amino-1-phenylethanol (6) and 1,1′-carbonyldiimidazole (CDI) in the presence of N,N-dimethyl-4-aminopyridine (DMAP). After methanesulfonylation of 8 using methanesulfonyl chloride (MsCl) in the presence of triethylamine (NEt3), the mesylated intermediate was reacted with 7 to afford the target compound 5a. Scheme 2 illustrates the synthesis of 5b−f. The Weinreb amide 10 was prepared from carboxylic acid 9, which was then treated with 4-fluorophenylmagnesium bromide to yield ketone 11. Compound 11 underwent reduction with sodium borohydride (NaBH4) followed by oxazolidin-2one ring formation by treatment with sodium hydride (NaH) to afford the racemate 13. The 3-benzyl-1,3-oxazolidin-2-one

Figure 3. D5D inhibitors identified by HTS. 8964



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Scheme 1. Synthesis of 3-Benzyl-1,3-oxazolidin-2-one 5aa

NaBH 4 and removal of the tert-butoxycarbonyl (Boc) protecting group under acidic conditions afforded the key 2aminoethanol intermediate 19 as a cis/trans mixture. According to the procedure shown in Scheme 2, we first prepared the oxazolidin-2-one intermediate from 19 and attempted a Buchwald−Hartwig reaction; however, the coupling reaction did not proceed, most likely due to steric hindrance of the 4methyl group of the oxazolidin-2-one. Thus, a Cu(I)-coupling reaction using 19 in 2-propanol was investigated according to a reported procedure,14 and this approach provided the desired coupling product 20. Formation of the 1,3-oxazolizin-2-one ring from 20 afforded the product as a mixture of diastereomers, which were separated into the each isomer 21 (trans) and 22 (cis) by silica gel column chromatography. Cyano derivatives 5i and 5j were prepared using zinc cyanide and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) from the precursors 21 and 22, respectively. The trans derivative 5i was subjected to chiral HPLC separation to yield enantiomers (4S,5S)-5i and (4R,5R)-5i. Scheme 5 shows the synthetic procedure for 4-gem-dimethyl1,3-oxazolidin-2-one derivative 5k. In a manner analogous to Scheme 4, the 2-aminoethanol intermediate 26 was obtained from carboxylic acid 23 in three steps. The following Cu(I)coupling reaction proceeded even with the sterically hindered gem-dimethyl substrate 26 to provide the coupling product 27, which was converted into target compound 5k via oxazolidin-2one ring formation and installment of a nitrile group using zinc cyanide and Pd(PPh3)4. Preparation of 5l−n was conducted using key intermediate 19 as the starting compound (Scheme 6). The Cu(I)-mediated coupling of diastereomixture 19 and the appropriate aryl iodides followed by 1,3-oxazolidin-2-one formation gave 30a and 30b as cis/trans mixtures, respectively. The bromo atom in

Reagents and conditions: (a) CDI, DMAP, THF, 80 °C, 46%; (b) (i) MsCl, NEt3, THF, 0 °C; (ii) 7, K2CO3, THF, 80 °C, then NaOH, THF, 0 °C, 2% in 2 steps.

a

compound 5b was obtained by the benzylation of 13 with 2,4dichlorobenzyl chloride. Intermediate 13 was also used for the preparation of 3,5-diphenyl-1,3-oxazolidin-2-ones 5c−f by a Buchwald−Hartwig coupling reaction. The enantiomers (S)-5f and (R)-5f were obtained by chiral high performance liquid chromatography (HPLC) separation of the racemic 5f. Compounds 5g and 5h were prepared as shown in Scheme 3. The ring opening reaction of oxirane 14 with the appropriate anilines gave aminoethanols 15a and 15b, which were converted to the corresponding target compounds 5g and 5h by 1,3-oxazolidin-2-one formation using CDI and DMAP. The 1,3-diphenyl-4-methyl-1,3-oxazolidin-2-one analogues 5i and 5j were obtained in six steps from the commercially available starting material 16 (Scheme 4). Compound 16 was converted to phenyl ketone 18 via Weinreb amide formation followed by Grignard reaction. Subsequent reduction with Scheme 2. Synthesis of 5b−fa

Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, EDCI, HOBt, NEt3, DMF, 0 °C to room temperature, 82%; (b) 4fluorophenylmagnesium bromide, THF, 0 °C to room temperature, 87%; (c) NaBH4, MeOH, 0 °C to room temperature, 98%; (d) NaH, THF, 0 °C to room temperature, 68%; (e) 2,4-dichlorobenzyl chloride, NaH, tetrabutylammonium iodide, THF, 0 °C to room temperature, 95%; (f) ArBr or ArI, Pd2(dba)3, Xantphos, NaOtBu, toluene, 120 °C, microwave irradiation, 12−47%; (g) chiral HPLC separation. a

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Scheme 3. Synthesis of 5g and 5ha

a

Reagents and conditions: (a) ArNH2, 2-propanol, 90 °C; (b) CDI, DBU, DMF, 80 °C, 3−4% in 2 steps.

Scheme 4. Synthesis of 5i and 5ja

a

Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, EDCI, HOBt, NEt3, DMF, room temperature, 81%; (b) 4fluorophenylmagnesium bromide, THF, 0 °C to room temperature, 76%; (c) NaBH4, THF, MeOH, 0 °C, then HCl/EtOAc, room temperature, 38%; (d) 1-bromo-3-iodobenzene, CuI, NaOH, 2-propanol, 90 °C, 72%; (e) CDI, DBU, THF, 80 °C, 37% (21) and 27% (22); (f) zinc cyanide, Pd(PPh3)4, DMF, 100 °C, 67% (5i) and 30% (5j); (g) chiral HPLC separation.

The biological activities of representative screening hits and an initial set of compounds 5a, 5b, 5g, and 5h are illustrated in Table 1. Screening hits 1 and 2 showed moderate to good D5D binding activity sufficient for us to view them as reasonable starting points for drug discovery (IC50 of 39 and 360 nM, respectively), and further enhancement of potency was sought from a novel scaffold based on a 1,3-oxazolidin-2-one framework (5-Ph and 5-Bn in Figure 4c). In order to quickly assess the merit of our design concept, 3-benzyl-1,3-oxazolidin2-one derivative 5a was first designed by installment of the 2,4dichloro-5-hydroxybenzyl moiety present in hit 1 into the 5-Bn scaffold. As expected, 5a displayed 3-fold more potent D5D binding activity as compared to 1. Due to potential toxicity concerns, the phenolic group15,16 of 5a was removed in the design of 5b, which showed somewhat moderated binding affinity while cellular activity was enhanced, possibly due to improved cellular permeability. Encouraged by these promising results, our attention was then directed to another 1,3oxazolidin-2-one series, the 5-Ph derivatives, which also fit

30a was converted to a cyano group by Pd(0)-catalyzed cyanation and following silica gel column chromatography separation provided the trans 5l. In the case where chloro derivative 30b was used, the cyanation reaction produced a mixture of both mono- and dicyano compounds, and their trans isomers 5m and 5n were isolated by silica gel column chromatography. The resulting dicyano compound 5n was further separated by chiral HPLC to yield (4S,5S)-5n. In addition, introduction of a cyano group from 30b and chiral HPLC purification of the resulting diastereomeric mixture afforded the enantiomer (4S,5S)-5m.

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RESULTS AND DISCUSSION

The compounds synthesized were evaluated for D5D binding activity in rat liver microsomes using [3H]T-336436612 as a ligand of D5D. D5D inhibitory activities in HepG2 cells were also evaluated by measuring the production of [14C]AA from [14C]DGLA. The in vitro activities are indicated as IC50 values. 8966



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Scheme 5. Synthesis of 5ka

a

Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, EDCI, HOBt, NEt3, DMF, room temperature, 57%; (b) 4fluorophenylmagnesium bromide, THF, 0 °C to room temperature; (c) NaBH4, THF, MeOH, 0 °C, then HCl/EtOAc, 0 °C to room temperature, 28% in 2 steps; (d) 1-bromo-3-iodobenzene, CuI, NaOH, 2-propanol, 90 °C; (e) CDI, DBU, THF, room temperature to 80 °C, 56% in 2 steps; (f) zinc cyanide, Pd(PPh3)4, DMF, 100 °C, 76%.

Scheme 6. Synthesis of 5l−na

a Reagents and conditions: (a) ArI, CuI, NaOH, 2-propanol, room temperature to 100 °C; (b) CDI, DBU, THF, 80 °C, 32% in 2 steps (30a) and 28% in 2 steps (30b); (c) (i) zinc cyanide, Pd(PPh3)4, DMF, room temperature, 100 °C; (ii) chiral HPLC separation, 16% in 2 steps. (d) For 5l: 30a, zinc cyanide, Pd(PPh3)4, DMF, room temperature to 100 °C, 49%. For 5m and 5n: 30b, zinc cyanide, Pd(PPh3)4, DMF, room temperature to 100 °C, 72% (5m) and 13% (5n). (e) Chiral HPLC separation.

provide suitable pharmacokinetic (PK) parameters for use in oral administration. The novel 3,5-diphenyl-1,3-oxazolidin-2-one 5h showed potent activity; however, it also demonstrated low metabolic stability in human and mouse liver microsomes (201 and 234 mL min−1 mg−1, respectively), an important issue in our quest to discover orally active agents. In order to address this matter in a rational way, metabolite analyses of 5h in human and mouse hepatocytes were conducted, which estimated phenol M-1, O-sulfate conjugate M-2, and O-glucuronate conjugates (M-3 and M-4) as the apparent metabolites of 5h (Figure 5). The analysis was also conducted in vivo, where five metabolites except M-3 (Figure 5) were detected in liver after oral treatment of 5h as dietary admixture to mice. Both cases suggested that the methoxy group and the 4-methylene of the 1,3-oxazolin-2-one ring on the test compound predominantly

well with our pharmacophore model (Figure 4d). We took notice of the 2,5-dimethoxyanilino moiety of hit compound 2 and introduced this fragment into the 5-Ph scaffold, resulting in the synthesis of 3-phenyl-1,3-oxazolidin-2-one derivative 5g. As we had hoped, both the binding and cellular activities of 5g were high (IC50 of 13 and 18 nM, respectively), and further modification showed that removal of the 2-methoxy substituent (5h) resulted in even more robust D5D inhibitory activity, with single-digit nM IC50 values in both the binding and cellular assays. In addition to the substantial enhancement in potency, 5g and 5h have an advantage in terms of lower lipophilicity, resulting in higher ligand lipophilic efficiency (LLE) values than the 5-Bn analogues (5a and 5b) and other hit compounds. Therefore, 3,5-diphenyl-1,3-oxazolidin-2-one 5h was selected as a lead compound and proceeded to the next stage of study, in which we sought to further increase biological potency and 8967



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Table 1. Biological Activities and Lipophilicities of Hit Compounds and 1,3-Oxazolidin-2-ones

D5D, IC50 (nM) compd

bindinga

cellb

cLogPd

LLEe

1 2 5a 5b 5g 5h

39 360 12 79 13 8.5

NTc NTc 1100 150 18 4.1

4.1 4.4 4.2 4.9 2.9 2.8

3.0 2.0 3.7 2.2 5.0 5.3

a

Inhibition of radiolabeled ligand binding to D5D in rat liver microsomes. bD5D inhibition in HepG2 cells measured by the production of [14C]AA from [14C]DGLA. cNot tested. dlog P value calculated by Daylight program. eLigand lipophilic efficiency LLE = pIC50(binding) − cLogP.

Figure 5. Structures of metabolites of 5h found in human or mouse hepatocytes (in vitro) and mice livers (in vivo) based on LC/MS/MS analysis, and quantification of each metabolite. aFormation ratio (%) = [(UV peak area of metabolite after incubation)/(UV peak area of parent compound before incubation)] × 100. Metabolites were quantified assuming that the molecular extinction coefficient is equal to that of 5h. bNot detected. c Concentrations in mice livers after oral dosing of 5h as dietary admixture (488 mg kg−1 day−1). Metabolites were quantified assuming that the molecular extinction coefficient is equal to that of 5h.

suffered from phase I oxidative metabolism and subsequent phase II conjugation reaction. Therefore, modification of these sites was explored with the goal of improving metabolic stability, which is summarized in Table 2. First, removal of the metabolically labile methoxy group (5c) was examined, which resulted in significantly reduced activity and showed the

importance of retaining a substituent at this position for substantial potency. Hence replacement of the methoxy with less metabolically susceptible substituent, such as fluoro or chloro atom, was next conducted to provide compounds 5d and 5e, which resulted in partially recovered potency but insufficient improvement in microsomal stability. Aiming 8968



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Table 2. Biological Activities and Metabolic Stabilities of 3,5-Diphenyl-1,3-oxazolidin-2-one Derivatives

a Inhibition of radioligand binding to D5D in rat liver microsomes. bD5D inhibition in HepG2 cells measured by production of [14C]AA from [14C]DGLA. cNot tested. dMetabolic stability in human or mouse hepatic microsomes. Values equal to or less than zero are indicated as “0” in this table.

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further improvement of metabolic stability, we chosed a substituent that was not only less metabolically labile but also polar, which resulted in the cyano derivative 5f showing the excellent metabolic stability in human microsomes with moderate binding affinity. Comparing the stereoisomers of 5f revealed that the S-enantiomer ((S)-5f) was the eutomer, showing relatively high D5D binding affinity with an IC50 value of 49 nM, in clear contrast to the significantly less active Ranalogue ((R)-5f). This suggested that the configuration of the 5-phenyl moiety might be crucial for gaining effective interaction with the enzyme. Encouraged by the improved metabolic stability of 5f, we attempted to block another metabolically labile position, the 4-methylene of the 1,3oxazolidin-2-one core. Initially, the 4-gem-dimethyl derivative 5k was prepared for studying substituent effects on metabolism, which showed expectedly high metabolic stability in both species supporting the validity of our drug design strategy. Unfortunately, D5D binding affinity of 5k was more than 5 times weaker than the corresponding nonmethylated compound 5f. We postulated that the drop-off in the binding affinity was due to steric hindrance at the 4,4-dimethylmethylene group, which could substantially alter the conformational rigidity of the 5-phenyl moiety that was deemed important for D5D binding. In order to test this assumption, the energetic effects of altering the dihedral angle between the central 1,3-oxazolidin-2-one and the 5-phenyl group were examined computationally using MOE. Two simplified compounds shown in Figure 6a and Figure 6b were built as (S)-enantiomers, and the variation in potential energy vs dihedral angle was calculated for each compound. The resulting calculations for the compound in Figure 6a, which has no substituents at the 4-position, showed an energy minimum for a dihedral angle between 90° and 135° (potential energy of ≤0.5 kcal/mol). On the other hand, the compound in Figure 6b, which is a model of the gem-dimethyl analogue, displayed an energy minimum with a narrower range of dihedral angles (95− 115°). We concluded that this difference in conformational rigidity is likely to result in a greater energy gap between the stable conformer in solution and the bound, bioactive conformation for gem-dimethyl compound 5k, leading to a decrease in potency. Although conclusive information regarding the bioactive conformation of our D5D inhibitor was not available due to the difficulty in obtaining crystal structures of D5D to date, we hypothesized that a substituent could be installed into the 4-position in a manner that would give rise to a conformation similar to model in Figure 6a, thus retaining potent D5D binding affinity. Hence 4-monomethyl compounds of Figure 6c and Figure 6d were examined by dihedral angle calculations for the models of trans and cis derivatives, respectively. As a result, the trans model in Figure 6c showed a similar dihedral angle plot as in Figure 6a, while cis compound of Figure 6d provided results similar to the less-active gemdimethyl compound of Figure 6b. This encouraged us to test the trans diastereomer with the expectation of retaining high binding affinity for D5D. The trans derivative 5i was first prepared as a racemate, which showed more potent binding activity (IC50 = 13 nM) than 5f. Moreover, the active enantiomer of 5i ((4S,5S)-5i) displayed higher potencies in both binding and cellular assays, reaching IC50 values of less than 10 nM, which are more than 6-fold more potent than the corresponding derivative (S)-5f. The high potency of (4S,5S)5i is possibly explained by our prediction that the (4S)-methyl group could minimize the conformational strain/energy gap

Figure 6. Dihedral angle plots for the model compounds (a−d) calculated by MOE. The molecules were built and optimized in the (5S)-configuration, followed by potential energy sampling around the dihedral angles (designated as bold lines) in vacuum. The 10 kcal/mol higher potential energy angles from the most stable one were plotted at 10 kcal/mol. In each step, Amber10:EHT force field and partial charges were used with the distant-dependent dielectric constant (r). Green lines indicate the dihedral angles of the lowest energy conformers.

between the low-energy and bioactive conformations (as discussed in Figure 6) and by additional lipophilic interaction between this methyl group and the protein. In contrast, the binding affinity of cis derivative 5j decreased relative to 5f, which is consistent with the calculations for model compounds of Figure 6b and Figure 6c. In terms of metabolic stability, (4S,5S)-5i displayed excellent microsomal stability (0 mL min−1 mg−1 in human and 24 mL min−1 mg−1 in mouse), which we reasoned is due to steric blockage of this position from metabolic enzymes. The high metabolic stability of compound (4S,5S)-5i was reflected in its excellent bioavailability, as found in a mouse cassette dosing study (F = 87.2%). Encouraged by the promising PK profile, in vivo D5D inhibition of (4S,5S)-5i was evaluated. The test compound was administered orally to C57BL/6J mice for 4 days, and AA and DGLA content of liver samples was measured. The decrease rate of AA/DGLA values relative to that of the control group as 100% was used as an index for assessing in vivo D5D inhibition of the test compound. In the course of previous studies of other chemotypes, we have learned that the maximum level of in vivo D5D inhibition is projected to be about 70% of the in vivo D5D inhibition index after 4 days of treatment.17 Hence, we set our goal to achieve the same level of in vivo efficacy with our novel 3,5-diphenyl-1,3-oxazolidin-2-one derivatives. However, 8970



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Table 3. Biological Activities and PK Profiles of 5-Substituted Derivatives

D5D

in vivo

IC50 (nM)

D5D indexc

a

b

compd

configuration

R

binding

cell

(4S,5S)-5i 5l (4S,5S)-5m (4S,5S)-5n

4S,5S racemate 4S,5S 4S,5S

H F Cl CN

5.3 4.0 95%). 3-(2,4-Dichloro-5-hydroxybenzyl)-5-phenyl-1,3-oxazolidin2-one (5a). To a solution of 2,4-dichloro-5-(hydroxymethyl)phenol (8, 5.0 g, 25.9 mmol) in THF (200 mL) and NEt3 (10.8 mL, 77.7 mmol) was added MsCl (4.0 mL, 51.8 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc (×2). The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. This product (6.5 g) and K2CO3 (7.7 g, 55.5 mmol) were added to a solution of 7 (3.0 g, 18.5 mmol) in THF (160 mL) at room temperature. The mixture was stirred at 80 °C overnight. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc (×2). The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 20−70% EtOAc/hexane). The obtained product (5.7 g) was dissolved with THF (30 mL). To the mixture was added 1 N aq NaOH (41.0 mL, 41.0 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min. The mixture was neutralized with 1 N aq HCl at 0 °C and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 20−70% EtOAc/hexane) to yield 5a (0.20 g, 2%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 3.32−3.39 (m, 1H), 3.90 (t, J = 8.9 Hz, 1H), 4.27−4.54 (m, 2H), 5.59 (t, J = 8.1 Hz, 1H), 7.02 (s, 1H), 7.31−7.45 (m, 5H), 7.50 (s, 1H), 10.58 (br s, 1H). Anal. Calcd for C16H13Cl2NO3: C, 56.82; H, 3.87; N, 4.14. Found: C, 56.50; H, 4.09; N, 3.89. MS (ESI) m/z 338.0 [M + H]+. HPLC purity 100%. 3-(2,4-Dichlorobenzyl)-5-(4-fluorophenyl)-1,3-oxazolidin-2one (5b). To a solution of 13 (320 mg, 1.8 mmol) in THF (25 mL) was added NaH (60% oil dispersion, 92 mg, 2.3 mmol) at 0 °C. After being stirred at room temperature for 5 min, 2,4-dichlorobenzyl chloride (0.37 mL, 2.7 mmol) and tetrabutylammonium iodide (65 mg, 0.18 mmol) were added to the reaction mixture. The mixture was stirred at room temperature overnight. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc (×3). The organic layer was separated, washed with 1 N aq HCl and brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 5−50% EtOAc/hexane) to yield 5b (570 mg, 95%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 3.34 (dd, J = 8.7, 7.5 Hz, 1H), 3.69−3.93 (m, 1H), 4.34−4.74 (m, 2H), 5.47 (t, J = 8.1 Hz, 1H), 6.89−7.16 (m, 2H), 7.18−7.50 (m, 5H). Anal. Calcd for C16H12Cl2FNO2: C, 56.49; H, 3.56; N, 4.12. Found: C, 56.44; H, 3.69; N, 4.13. MS (ESI) m/z 340.0 [M + H]+. HPLC purity 99.2%. 5-(4-Fluorophenyl)-3-phenyl-1,3-oxazolidin-2-one (5c). To a solution of 13 (91 mg, 0.50 mmol), bromobenzene (118 mg, 0.75 mmol), Xantphos (15 mg, 0.03 mmol), and NaOtBu (72 mg, 0.75 mmol) in toluene (2 mL) was added Pd2(dba)3 (12 mg, 0.01 mmol) at room temperature. The mixture was stirred at 120 °C for 1 h under microwave irradiation. The mixture was filtered through Celite, and the filtrate was treated with water at room temperature and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) followed by trituration with EtOAc/heptane to yield 5c (16 mg, 12%) as an off-white solid. 1H NMR (300 MHz, DMSOd6): δ 4.02 (dd, J = 9.3, 7.8 Hz, 1H), 4.46 (t, J = 9.0 Hz, 1H), 5.77 (t, J = 8.2 Hz, 1H), 7.11−7.18 (m, 1H), 7.25−7.34 (m, 2H), 7.36−7.45 (m, 2H), 7.55−7.64 (m, 4H). Anal. Calcd for C15H12FNO2: C, 70.03; H,

4.70; N, 5.44. Found: C, 69.94; H, 4.81; N, 5.43. MS (ESI) m/z 258.1 [M + H]+. 3-(3-Fluorophenyl)-5-(4-fluorophenyl)-1,3-oxazolidin-2-one (5d). To a solution of 13 (150 mg, 0.83 mmol), 1-bromo-3fluorobenzene (217 mg, 1.2 mmol), Xantphos (24.0 mg, 0.04 mmol), and NaOtBu (111 mg, 1.2 mmol) in toluene (2 mL) was added Pd2(dba)3 (19.0 mg, 0.02 mmol). The mixture was stirred at 120 °C for 1 h under microwave irradiation. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 5−30% EtOAc/ hexane) to yield 5d (108 mg, 47%) as an off-white solid. 1H NMR (300 MHz, CDCl3): δ 3.92 (dd, J = 9.0, 7.6 Hz, 1H), 4.37 (t, J = 8.8 Hz, 1H), 5.64 (t, J = 8.1 Hz, 1H), 6.86 (tdd, J = 8.2, 2.5, 1.0 Hz, 1H), 7.14 (t, J = 8.6 Hz, 2H), 7.22−7.48 (m, 5H). Anal. Calcd for C15H11F2NO2: C, 65.45; H, 4.03; N, 5.09. Found: C, 65.43; H, 4.14; N, 5.07. MS (ESI) m/z 276.0 [M + H]+. HPLC purity 98.8%. 3-(3-Chlorophenyl)-5-(4-fluorophenyl)-1,3-oxazolidin-2-one (5e). To a suspension of 13 (150 mg, 0.83 mmol), 1-chloro-3iodobenzene (218 mg, 0.91 mmol), Xantphos (24 mg, 0.04 mmol), and NaOtBu (111 mg, 1.2 mmol) in toluene (2 mL) was added Pd2(dba)3 (19 mg, 0.02 mmol). The resulting mixture was stirred at 120 °C for 1 h under microwave condition. The reaction mixture was partitioned between EtOAc (20 mL) and water (20 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, and concentrated to dryness under reduced pressure. The crude product was purified by preparative TLC (eluent, petroleum ether/EtOAc = 4/ 1). The crude product was treated with methyl tert-butyl ether (10 mL), and then the mixture was concentrated to dryness. The solid was washed petroleum ether (10 mL × 3) and then dried over high vacuum to afford 5e (73 mg, 30%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 3.92 (dd, J = 9.2, 8.0 Hz, 1H), 4.36 (t, J = 8.8 Hz, 1H), 5.64 (t, J = 8.0 Hz, 1H), 7.07−7.20 (m, 3H), 7.31 (t, J = 8.0 Hz, 1H), 7.36−7.45 (m, 2H), 7.46−7.51 (m, 1H), 7.58 (t, J = 2.0 Hz, 1H). Anal. Calcd for C15H11NO2ClF·0.6H2O: C, 59.55; H, 4.06; N, 4.63. Found: C, 59.33; H, 3.80; N, 4.56. MS (ESI) m/z 291.9 [M + H]+. HPLC purity 99.0%. 3-[5-(4-Fluorophenyl)-2-oxo-1,3-oxazolidin-3-yl]benzonitrile (5f). Compound 5f was prepared in a manner similar to that described for 5b. Yield: 113 mg, 21%, orange solid. 1H NMR (300 MHz, CDCl3): δ 3.95 (dd, J = 8.9, 7.6 Hz, 1H), 4.40 (t, J = 8.8 Hz, 1H), 5.68 (t, J = 8.1 Hz, 1H), 7.05−7.21 (m, 2H), 7.37−7.55 (m, 4H), 7.73− 7.93 (m, 2H). Anal. Calcd for C16H11FN2O2: C, 68.08; H, 3.93; N, 9.92. Found: C, 68.28; H, 4.08; N, 9.54. MS (ESI) m/z 283.0 [M + H]+. HPLC purity 99.5%. 3-[(5R)-5-(4-Fluorophenyl)-2-oxo-1,3-oxazolidin-3-yl]benzonitrile ((R)-5f). Compound 5f (1.8 g, 6.5 mmol) was separated by a YMCK-Prep system (column, CHIRALPAK AS (CC001), 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 60 mL/min, hexane/EtOH = 400/600 (v/v) at 30 °C. The second eluting enantiomer was collected to yield (R)-5f (870 mg, 48%) as a white solid. Liquid chromatography analysis was performed on a Hitachi system, equipped with a CHIRALPAK AS (MB014) (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 0.5 mL/min, hexane/EtOH = 400/600 (v/v) at 30 °C. Retention time: 25.80 min (99.4% ee). 1H NMR (300 MHz, CDCl3): δ 3.95 (dd, J = 8.9, 7.6 Hz, 1H), 4.40 (t, J = 8.8 Hz, 1H), 5.68 (t, J = 8.1 Hz, 1H), 7.03−7.21 (m, 2H), 7.35−7.56 (m, 4H), 7.80−7.94 (m, 2H). Anal. Calcd for C16H11FN2O2: C, 68.08; H, 3.93; N, 9.92. Found: C, 68.20; H, 4.20; N, 9.72. MS (ESI) m/z 283.0 [M + H]+. HPLC purity 100%. 3-[(5S)-5-(4-Fluorophenyl)-2-oxo-1,3-oxazolidin-3-yl]benzonitrile ((S)-5f). Compound 5f (1.8 g, 6.5 mmol) was separated by a YMCK-Prep system (column, CHIRALPAK AS (CC001), 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 60 mL/min, hexane/EtOH = 400/600 (v/v) at 30 °C. The first eluting enantiomer was collected to afford (S)-5f (900 mg, 49%) as a white solid. Liquid chromatography analysis was performed on a Hitachi system, equipped with a CHIRALPAK AS (MB014) (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 0.5 mL/min, hexane/EtOH = 400/600 (v/v) at 30 °C. Retention time: 13.31 min (>99.9% ee). 1H NMR (300 MHz, CDCl3): δ 3.95 (dd, J = 8.9, 7.6 8974



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time: 10.92 min (99.9% ee). 1H NMR (300 MHz, CDCl3): δ 1.47 (d, J = 6.1 Hz, 3H), 4.31 (t, J = 6.3 Hz, 1H), 5.14 (d, J = 6.5 Hz, 1H), 7.05−7.22 (m, 2H), 7.36−7.44 (m, 2H), 7.46−7.57 (m, 2H), 7.64− 7.75 (m, 2H). Anal. Calcd for C17H13FN2O2: C, 68.91; H, 4.42; N, 9.45. Found: C, 68.53; H, 4.54; N, 9.42. MS (ESI) m/z 297.0 [M + H]+. HPLC purity 100%. 3-[(4S,5S)-5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzonitrile ((4S,5S)-5i). Compound 5i (180 mg, 0.61 mmol) was separated by a YMCK-Prep system (column, CHIRALPAK AD (AK001), 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 80 mL/min, hexane/EtOH = 800/200 (v/v) at 30 °C. The first eluting enantiomer was collected to yield (4S,5S)-5i (91 mg, quant) as a white solid. Liquid chromatography analysis was performed on a HITACHI system, equipped with a CHIRALPAK AD (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 0.5 mL/min, hexane/EtOH = 300/700 (v/v) at 30 °C. Retention time: 9.19 min (99.9% ee). 1H NMR (300 MHz, CDCl3): δ 1.47 (d, J = 6.1 Hz, 3H), 4.31 (t, J = 6.3 Hz, 1H), 5.14 (d, J = 6.5 Hz, 1H), 7.05−7.22 (m, 2H), 7.36−7.44 (m, 2H), 7.46−7.57 (m, 2H), 7.64− 7.75 (m, 2H). MS (ESI) m/z 297.0 [M + H]+. HPLC purity 99.9%. cis-3-[5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3yl]benzonitrile (5j). Compound 5j was prepared in a manner similar to that described for 5i. Yield: 64 mg, 30%, white solid. 1H NMR (300 MHz, CDCl3): δ 0.86 (br s, 3H), 4.66−4.82 (m, 1H), 5.80 (d, J = 7.6 Hz, 1H), 6.95−7.22 (m, 2H), 7.28−7.40 (m, 2H), 7.40−7.58 (m, 2H), 7.80−7.94 (m, 2H). Anal. Calcd for C17H13FN2O2: C, 68.91; H, 4.42; N, 9.45. Found: C, 68.66; H, 4.47; N, 9.45. MS (ESI) m/z 297.3 [M + H]+. HPLC purity 99.0%. 3-[5-(4-Fluorophenyl)-4,4-dimethyl-2-oxo-1,3-oxazolidin-3yl]benzonitrile (5k). To a solution of 28 (426 mg, 1.2 mmol) and zinc cyanide (275 mg, 2.3 mmol) in DMF (10 mL) was added Pd(PPh3)4 (203 mg, 0.18 mmol) at room temperature. The mixture was stirred at 100 °C under N2 for 6 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0%−30% EtOAc/hexane) followed by crystallization form IPE/hexane to yield 5k (277 mg, 76%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 0.84 (s, 3H), 1.49 (s, 3H), 5.38 (s, 1H), 7.09− 7.21 (m, 2H), 7.32−7.47 (m, 2H), 7.51−7.65 (m, 3H), 7.65−7.73 (m, 1H). Anal. Calcd for C18H15FN2O2: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.53; H, 5.03; N, 9.02. MS (ESI) m/z 311.3 [M + H]+. HPLC purity 100%. trans-3-Fluoro-5-[5-(4-fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzonitrile (5l). To a solution of 30a (1.4 g, 3.8 mmol) and zinc cyanide (0.57 g, 4.9 mmol) in DMF (20 mL) was added Pd(PPh3)4 (0.87 g, 0.8 mmol) at room temperature. The mixture was stirred at 100 °C under Ar for 10 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−20% EtOAc/hexane) followed by crystallization from IPE to yield 5l (0.57 g, 49%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 1.51 (d, J = 6.1 Hz, 3H), 4.30 (t, J = 6.1 Hz, 1H), 5.15 (d, J = 5.9 Hz, 1H), 7.08−7.22 (m, 3H), 7.34−7.45 (m, 2H), 7.48 (s, 1H), 7.60 (dt, J = 10.3, 2.2 Hz, 1H). Anal. Calcd for C17H12F2N2O2: C, 64.97; H, 3.85; N, 8.90. Found: C, 64.91; H, 4.01; N, 8.83. MS (ESI) m/z 314.9 [M + H]+. HPLC purity 99.3%. trans-3-Chloro-5-[5-(4-fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzonitrile (5m). The compound 5m was prepared from the corresponding bromo derivatives 30b by a method similar to that described for 5l. Yield: 169 mg, 72%, white solid. 1H NMR (300 MHz, CDCl3): δ 1.50 (d, J = 6.1 Hz, 3H), 4.30 (t, J = 6.1 Hz, 1H), 5.15 (d, J = 6.1 Hz, 1H), 7.15 (t, J = 8.5 Hz, 2H), 7.35−7.47 (m, 3H), 7.63 (s, 1H), 7.75 (t, J = 1.8 Hz, 1H). Anal. Calcd for C17H12ClFN2O2: C, 61.73; H, 3.66; N, 8.47. Found: C, 61.70; H, 3.89; N, 8.36. MS (ESI) m/z 330.8 [M + H]+. HPLC purity 100%. 3-(4S,5S)-Chloro-5-[5-(4-fluorophenyl)-4-methyl-2-oxo-1,3oxazolidin-3-yl]benzonitrile (4S,5S)-5m). To a solution of 30b

Hz, 1H), 4.40 (t, J = 8.8 Hz, 1H), 5.68 (t, J = 8.1 Hz, 1H), 6.99−7.21 (m, 2H), 7.35−7.58 (m, 4H), 7.79−7.95 (m, 2H). Anal. Calcd for C16H11FN2O2: C, 68.08; H, 3.93; N, 9.92. Found: C, 68.05; H, 4.04; N, 9.82. MS (ESI) m/z 283.0 [M + H]+. HPLC purity 99.8%. 3-(2,5-Dimethoxyphenyl)-5-(4-fluorophenyl)-1,3-oxazolidin2-one (5g). To a solution of 2-(4-fluorophenyl)oxirane (14, 1.0 g, 7.2 mmol) in propan-2-ol (20 mL) was added 2,5-dimethoxyaniline (1.1 g, 7.2 mmol) at room temperature. The mixture was stirred at 90 °C for 16 h. The mixture was treated with water at room temperature and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−50% EtOAc/hexane) to yield crude 15a (670 mg) as a yellow oil. The product was dissolved in DMF (20 mL). To the solution were added DBU (0.69 mL, 4.6 mmol) and CDI (746 mg, 4.6 mmol) at room temperature. The mixture was stirred at 80 °C for 3 h. The mixture was treated with water at room temperature and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0%−50% EtOAc/hexane) and preparative HPLC (L-column 2 ODS, eluted with H2O in acetonitrile containing 0.1% TFA). The desired fraction was neutralized with sat. aq NaHCO3 and extracted with EtOAc. The organic layer was separated, dried over MgSO4, and concentrated under vacuum. The residue was triturated with EtOAc/IPE/heptane to yield 5g (70 mg, 3.1% in 2 steps) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 3.72 (s, 3H), 3.77−3.85 (m, 4H), 4.27 (t, J = 8.7 Hz, 1H), 5.63−5.82 (m, 1H), 6.83−6.97 (m, 1H), 6.99−7.12 (m, 2H), 7.26−7.36 (m, 2H), 7.53−7.67 (m, 2H). Anal. Calcd for C17H16FNO4· 0.5H2O: C, 62.57; H, 5.25; N, 4.29. Found: C, 62.82; H, 5.07; N, 4.26. MS (ESI) m/z 318.1 [M + H]+. HPLC purity 100%. 5-(4-Fluorophenyl)-3-(3-methoxyphenyl)-1,3-oxazolidin-2one (5h). To a solution of 15b (3.6 g, 13.7 mmol) and DBU (3.1 mL, 20.5 mmol) in DMF (50 mL) was added CDI (3.3 g, 20.5 mmol), and the mixture was stirred at 80 °C for 16 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) to yield 5h (0.88 g, 22%) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 3.83 (s, 3H), 3.92 (dd, J = 9.1, 7.6 Hz, 1H), 4.36 (t, J = 8.8 Hz, 1H), 5.61 (t, J = 8.1 Hz, 1H), 6.66−6.77 (m, 1H), 6.97−7.10 (m, 1H), 7.10−7.18 (m, 2H), 7.23− 7.32 (m, 2H), 7.34−7.48 (m, 2H). Anal. Calcd for C16H14FNO3: C, 66.89; H, 4.91; N, 4.88. Found: C, 66.76; H, 5.04; N, 4.82. MS (ESI) m/z 288.0 [M + H]+. HPLC purity 100%. trans-3-[5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin3-yl]benzonitrile (5i). To a solution of 21 (354 mg, 1.0 mmol), zinc cyanide (178 mg, 1.5 mmol) in DMF (10 mL) was added Pd(Ph3P)4 (117 mg, 0.10 mmol) at room temperature, and the mixture was stirred at 100 °C under N2 for 11 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) followed by crystallization from Et2O/hexane to yield 5i (201 mg, 67%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 1.46 (d, J = 6.1 Hz, 3H), 4.31 (t, J = 6.3 Hz, 1H), 5.14 (d, J = 6.5 Hz, 1H), 7.07−7.21 (m, 2H), 7.35−7.46 (m, 2H), 7.46−7.57 (m, 2H), 7.66−7.75 (m, 2H). Anal. Calcd for C17H13FN2O2·0.1H2O: C, 68.50; H, 4.46; N, 9.40. Found: C, 68.23; H, 4.65; N, 9.30. MS (ESI) m/z 297.3 [M + H]+. HPLC purity 99.8%. 3-[(4R,5R)-5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzonitrile ((4R,5R)-5i). Compound 3i (180 mg, 0.61 mmol) was separated by a YMCK-Prep system (column, CHIRALPAK AD (AK001), 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 80 mL/min, hexane/EtOH = 800/200 (v/v) at 30 °C. The second eluting enantiomer was collected to yield (4R,5R)-5i (89 mg, quant) as a white solid. Liquid chromatography analysis was performed on a HITACHI system, equipped with a CHIRALPAK AD (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 0.5 mL/min, hexane/EtOH = 300/700 (v/v) at 30 °C. Retention 8975



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hexane) to yield 7 (3.9 g, 46%) as a colorless oil. 1H NMR (300 MHz, DMSO-d6): δ 3.32−3.38 (m, 1H), 3.72−4.02 (m, 1H), 5.59 (dd, J = 8.3, 7.6 Hz, 1H), 7.18−7.49 (m, 5H), 7.67 (br s, 1H). MS (ESI) m/z 164.0 [M + H]+. tert-Butyl {2-[Methoxy(methyl)amino]-2-oxoethyl}carbamate (10). To a solution of 2-((tert-butoxycarbonyl)amino)acetic acid (9, 25.5 g, 145.6 mmol) in DMF (250 mL) were added HOBt (19.7 g, 145.6 mmol), NEt3 (60.9 mL, 436.7 mmol), N,Odimethylhydroxylamine hydrochloride (21.3 g, 218.3 mmol), and EDCI hydrochloride (36.3 g, 189.2 mmol) at 0 °C. The mixture was stirred at room temperature over the weekend. The mixture was quenched with water at 0 °C and extracted with EtOAc (×2). The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was washed with IPE/hexane to yield 10 (26.1 g, 82%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.38 (s, 9H), 3.08 (s, 3H), 3.67 (s, 3H), 3.82 (d, J = 6.1 Hz, 2H), 6.72−7.19 (m, 1H). tert-Butyl [2-(4-Fluorophenyl)-2-oxoethyl]carbamate (11). To a solution of 10 (6.0 g, 27.5 mmol) in THF (100 mL) was added 4-fluorophenylmagnesium bromide (90 mL, 180.0 mmol) at 0 °C. The mixture was stirred at room temperature overnight. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc. The organic layer was separated, washed with 1 N aq HCl and brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 5−50% EtOAc/hexane) to afford a yellow solid. The solid was washed with IPE to yield 11 (6.1 g, 87%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.39 (s, 9H), 4.42 (d, J = 6.1 Hz, 2H), 7.08 (t, J = 5.7 Hz, 1H), 7.24−7.55 (m, 2H), 7.79−8.31 (m, 2H). tert-Butyl [2-(4-Fluorophenyl)-2-hydroxyethyl]carbamate (12). To a solution of 11 (3.5 g, 13.7 mmol) in MeOH (100 mL) was added NaBH4 (1.6 g, 41.1 mmol) at 0 °C. The mixture was stirred at room temperature for 1 h. The mixture was quenched with sat. aq NH4Cl at 0 °C, concentrated under vacuum, and extracted with EtOAc (×3). The organic layer was separated, dried over MgSO4, and concentrated under vacuum. The residue was washed with IPE to yield 12 (3.4 g, 98%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.34 (s, 9H), 2.86−3.20 (m, 2H), 4.31−4.89 (m, J = 6.1 Hz, 1H), 5.38 (d, J = 4.5 Hz, 1H), 6.71 (br s, 1H), 6.95−7.24 (m, 2H), 7.33 (dd, J = 8.3, 6.1 Hz, 2H). MS (ESI) m/z 254.1 [M − H]−. 5-(4-Fluorophenyl)-1,3-oxazolidin-2-one (13). To a solution of 12 (1.5 g, 6.0 mmol) in THF (50 mL) and DMF (50 mL) was added NaH (60% oil dispersion, 0.31 g, 7.8 mmol) at 0 °C. The mixture was stirred at room temperature for 2 h. The mixture was stirred at 60 °C for 1 h. The mixture was stirred at 80 °C for 1 h. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc (×3). The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 20−100% EtOAc/ hexane) to yield 13 (0.74 g, 68%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 3.32−3.37 (m, 1H), 3.86 (t, J = 8.7 Hz, 1H), 5.60 (t, J = 7.9 Hz, 1H), 7.11−7.37 (m, 2H), 7.37−7.52 (m, 2H), 7.69 (br s, 1H). 1-(4-Fluorophenyl)-2-[(3-methoxyphenyl)amino]ethanol (15b). To a solution of 2-(4-fluorophenyl)oxirane (14, 1.0 g, 7.2 mmol) in 2-propanol (10 mL) was added 3-methoxyaniline (0.80 g, 6.5 mmol), and the mixture was stirred at 90 °C for 5 h. The mixture was concentrated under vacuum and the residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/ hexane) to yield 15b (0.30 g, 16%). 1H NMR (300 MHz, CDCl3): δ 2.37 (d, J = 2.4 Hz, 1H), 3.19−3.32 (m, 1H), 3.32−3.47 (m, 1H), 3.76 (br s, 3H), 4.06 (1H, br s), 4.92 (d, J = 6.5 Hz, 1H), 6.13−6.25 (m, 1H), 6.25−6.38 (m, 2H), 7.00−7.16 (m, 3H), 7.35−7.47 (m, 2H). MS (ESI) m/z 262.1 [M + H]+. N2-(tert-Butoxycarbonyl)-N-methoxy-N-methylalaninamide (17). To a solution of 2-((tert-butoxycarbonyl)amino)propanoic acid (10.0 g, 52.9 mmol) in DMF (250 mL) were added HOBt (8.6 g, 63.4 mmol), NEt3 (22.1 mL, 158.6 mmol), N,O-dimethylhydroxylamine hydrochloride (7.7 g, 79.3 mmol), and EDCI hydrochloride (20.3 g, 105.7 mmol) at room temperature. The mixture was stirred at room

(1.3 g, 3.3 mmol), zinc cyanide (0.50 g, 4.3 mmol) in DMF (20 mL) was added Pd(PPh3)4 (0.76 g, 0.66 mmol) at room temperature. The mixture was stirred at 100 °C under Ar for 11 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0%−20% EtOAc/hexane) to yield a diastereomixture of 3-chloro-5-(5(4-fluorophenyl)-4-methyl- 2-oxooxazolidin-3-yl)benzonitrile (0.66 g). This product was separated by a YMCK-Prep system (column, CHIRALPAK IC, 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 85 mL/min, hexane/EtOH = 800/200 (v/v) at 30 °C. The third and fourth eluting enantiomers were combined and further separated by a YMCK-Prep system (column, CHIRALPAK IC, 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 85 mL/ min, hexane/EtOH = 700/300 (v/v) at 30 °C. The second eluting enantiomer was collected to yield (4S,5S)-5m (189 mg, 16%) as a white solid. Liquid chromatography analysis was performed on a HITACHI system, equipped with a CHIRALPAK IC (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 1.0 mL/min, hexane/EtOH = 900/100 (v/v) at 30 °C. Retention time: 27.33 min (99.9% ee). 1H NMR (300 MHz, CDCl3): δ 1.50 (d, J = 6.1 Hz, 3H), 4.30 (quintet, J = 6.1 Hz, 1H), 5.15 (d, J = 6.2 Hz, 1H), 7.11−7.20 (m, 2H), 7.35−7.43 (m, 2H), 7.43−7.46 (m, 1H), 7.63 (dd, J = 2.0, 1.4 Hz, 1H), 7.75 (t, J = 2.0 Hz, 1H). Anal. Calcd for C17H12N2O2ClF: C, 61.73; H, 3.66; N, 8.47. Found: C, 61.70; H, 3.74; N, 8.43. MS (ESI) m/z 330.8 [M + H]+. HPLC purity 100%. trans-5-[5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin3-yl]benzene-1,3-dicarbonitrile (5n). To a solution of 30b (1.3 g, 3.3 mmol), zinc cyanide (0.50 g, 4.3 mmol) in DMF (20 mL) was added Pd(PPh3)4 (0.76 g, 0.66 mmol) at room temperature. The mixture was stirred at 100 °C under Ar for 11 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−20% EtOAc/hexane) to yield 5n (0.14 g, 13%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 1.50−1.54 (m, 3H), 4.34 (quintet, J = 6.1 Hz, 1H), 5.19 (d, J = 6.0 Hz, 1H), 7.12−7.20 (m, 2H), 7.35−7.43 (m, 2H), 7.72 (t, J = 1.4 Hz, 1H), 8.00 (d, J = 1.4 Hz, 2H). MS (ESI) m/z 321.7 [M + H]+. HPLC purity 100%. 5-[(4S,5S)-5-(4-Fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile ((4S,5S)-5n). To a solution of 39 (3.6 g, 8.4 mmol) and zinc cyanide (2.5 g, 21.0 mmol) in DMF (75 mL) was added Pd(PPh3)4 (2.9 g, 2.5 mmol) at room temperature. The mixture was stirred at 100 °C under Ar for 17 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 5%−100% EtOAc/hexane) followed by recrystallization from EtOAc (55 mL) to yield (4S,5S)-5n (1.2 g, 43%) as a white solid. Liquid chromatography analysis was performed on a Hitachi system, equipped with a CHIRALPAK AD-H (PE164) (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 0.5 mL/min, hexane/ EtOH = 600/400 (v/v) at 30 °C. Retention time: 13.10 min (>99.9% ee). 1H NMR (300 MHz, CDCl3): δ 1.53 (d, J = 6.1 Hz, 3H), 4.34 (quintet, J = 6.1 Hz, 1H), 5.19 (d, J = 6.0 Hz, 1H), 7.08−7.22 (m, 2H), 7.34−7.45 (m, 2H), 7.72 (t, J = 1.3 Hz, 1H), 8.00 (d, J = 1.3 Hz, 2H). Anal. Calcd for C18H12FN3O2: C, 67.29; H, 3.76; N, 13.08. Found: C, 67.22; H, 3.80; N, 12.98. MS (ESI) m/z 322.0 [M + H]+. HPLC purity 99.9%. 5-Phenyl-1,3-oxazolidin-2-one (7). To a solution of 2-amino-1phenylthanol (6, 7.1 g, 51.5 mmol) and DMAP (6.3 g, 51.5 mmol) in THF (100 mL) was added CDI (10.0 g, 61.8 mmol) at room temperature. The mixture was stirred at 80 °C for 4 h. The mixture was neutralized with 1 N aq HCl at 0 °C and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 10%−70% EtOAc/ 8976



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temperature over the weekend. The mixture was quenched with water at 0 °C and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was recrystallized from IPE to yield 17 (9.9 g, 81%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.14 (d, J = 7.2 Hz, 3H), 1.36 (s, 9H), 3.09 (s, 3H), 3.72 (s, 3H), 4.12−4.62 (m, J = 7.4, 7.4 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H). tert-Butyl [1-(4-Fluorophenyl)-1-oxopropan-2-yl]carbamate (18). To a solution of 17 (10.0 g, 43.1 mmol) in THF (100 mL) was added 4-fluorophenylmagnesium bromide (90 mL, 180.0 mmol) at 0 °C. The mixture was stirred at room temperature overnight. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc. The organic layer was separated, washed with 1 N aq HCl and brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 5−50% EtOAc/hexane) to yield a yellow solid. The solid was washed with IPE to yield 18 (8.8 g, 76%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.13−1.39 (m, 12H), 4.59−5.19 (m, J = 7.2, 7.2 Hz, 1H), 7.16−7.53 (m, 3H), 7.90−8.26 (m, 2H). 2-Amino-1-(4-fluorophenyl)propan-1-ol (19). To a solution of 18 (5.7 g, 21.3 mmol) in THF (50 mL) and MeOH (170 mL) was added NaBH4 (2.4 g, 63.8 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min. The mixture was quenched with sat. aq NH4Cl at 0 °C, concentrated under vacuum, and extracted with EtOAc (×3). The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 10−30% EtOAc/hexane) to yield a white solid. The solid was washed with hexane to yield the crude product (3.2 g). This product was treated with 4 N HCl/EtOAc solution (44.1 mL, 176.6 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with MeOH and concentrated under vacuum. The residue was washed with IPE to yield a white solid. The solid was diluted with EtOAc, neutralized with sat. aq NaHCO3 at room temperature, and extracted with EtOAc (×3). The organic layer was separated, dried over MgSO4, and concentrated under vacuum to yield 19 (1.4 g, 38%). 1H NMR (300 MHz, CDCl3): δ 0.93 (d, J = 6.5 Hz, 3H), 1.94 (br s, 3H), 3.04−3.33 (m, 1H), 4.51 (d, J = 4.6 Hz, 1H), 6.82−7.10 (m, 2H), 7.14−7.42 (m, 2H). MS (ESI) m/z 170.3 [M + H]+. 2-[(3-Bromophenyl)amino]-1-(4-fluorophenyl)propan-1-ol (20). To a solution of 19 (1.0 g, 5.9 mmol), sodium hydroxide (0.47 g, 11.8 mmol), and 1-bromo-3-iodobenzene (2.0 g, 7.1 mmol) in 2propanol (20 mL) was added CuI (0.03 g, 0.15 mmol) at room temperature, and the mixture was stirred at 90 °C under N2 for 16 h. The mixture was concentrated under vacuum, and the residue was treated with water and brine and extracted with EtOAc. The organic layer was separated, washed with brine and 0.1 N NaOH, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−25% EtOAc/ hexane) to yield 20 (1.4 g, 72%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 0.93−1.10 (m, 3H), 2.17−2.82 (m, 1H), 3.53−3.90 (m, 2H), 4.49−4.98 (m, 1H), 6.79−6.89 (m, 2H), 6.98−7.11 (m, 4H), 7.31−7.41 (m, 2H). MS (ESI) m/z 324.1 [M + H]+. trans-3-(3-Bromophenyl)-5-(4-fluorophenyl)-4-methyl-1,3oxazolidin-2-one (21) and cis-3-(3-Bromophenyl)-5-(4-fluorophenyl)-4-methyl-1,3-oxazolidin-2-one (22). To a solution of 20 (1.4 g, 4.3 mmol) in THF (30 mL) were added DBU (0.96 mL, 6.4 mmol) and CDI (1.0 g, 6.4 mmol) at room temperature. The mixture was stirred at 80 °C for 2 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−50% EtOAc/hexane) and preparative HPLC (L-column 2 ODS, eluted with H2O in acetonitrile containing 0.1% TFA). The desired fraction was neutralized with sat. aq NaHCO3 and extracted with EtOAc. The organic layer was separated, dried over MgSO4, and concentrated under vacuum to yield 21 (0.55 g, 37%) and 22 (0.40 g, 27%) as white solids. 21: 1H NMR (300 MHz, CDCl3) δ 1.44 (d, J = 6.2 Hz, 3H), 4.08−4.35 (m, 1H), 5.10 (d, J = 6.6 Hz, 1H), 7.09−7.18 (m, 2H), 7.22−7.30 (m, 1H), 7.32−7.45 (m, 4H), 7.56 (t, J = 1.9 Hz,

1H). Anal. Calcd for C16H13BrFNO2: C, 54.88; H, 3.74; N, 4.00. Found: C, 54.85; H, 3.55; N, 3.87. MS (ESI) m/z 351.3 [M + H]+. 22: 1 H NMR (300 MHz, CDCl3) δ 0.86 (d, J = 6.5 Hz, 3H), 4.51−4.78 (m, 1H), 5.77 (d, J = 7.7 Hz, 1H), 7.07−7.18 (m, 2H), 7.22−7.41 (m, 4H), 7.44−7.53 (m, 1H), 7.72 (t, J = 1.9 Hz, 1H). Anal. Calcd for C16H13BrFNO2: C, 54.88; H, 3.74; N, 4.00. Found: C, 54.75; H, 3.62; N, 3.86. MS (ESI) m/z 351.2 [M + H]+. N2-(tert-Butoxycarbonyl)-N-methoxy-N,2-dimethylalaninamide (24). To a solution of 2-((tert-butoxycarbonyl)amino)-2methylpropanoic acid (23, 18.0 g, 88.6 mmol), N,O-dimethylhydroxylamine hydrochloride (13.0 g, 132.9 mmol), NEt3 (37.0 mL, 265.7 mmol), and HOBt monohydrate (16.3 g, 106.3 mmol) in DMF (50 mL) was added EDCI (20.6 g, 132.9 mmol) at room temperature. The mixture was stirred at room temperature for 16 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum to yield 24 (12.5 g, 57%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 1.44 (s, 9H), 1.55 (s, 6H), 3.21 (s, 3H), 3.68 (s, 3H). 2-Amino-1-(4-fluorophenyl)-2-methylpropan-1-ol Hydrochloride (26). To a solution of 24 (6.0 g, 24.4 mmol) in THF (100 mL) was added dropwise 4-fluorophenylmagnesium bromide (48.7 mL, 97.4 mmol) at 0 °C. The mixture was stirred at room temperature for 16 h. The mixture was quenched with sat. aq NH4Cl at 0 °C and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−10% EtOAc/hexane) to yield the crude 25 (4.39 g). The product was dissolved in MeOH (40 mL) and THF (40 mL). To the mixture was added NaBH4 (1.77 g, 46.8 mmol) at 0 °C, and the mixture was stirred at 0 °C for 1 h. The mixture was quenched with water at 0 °C and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) followed by crystallization from IPE/hexane. This product (2.0 g) was added to a solution of 4 N HCl/EtOAc solution (17.8 mL, 71.3 mmol) at 0 °C. The mixture was stirred at room temperature for 1 h. The mixture was diluted by toluene and concentrated under vacuum. The resulting solid was washed by IPE to yield 26 (1.5 g, 28%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 1.09 (d, J = 14.5 Hz, 6H), 4.63 (br s, 1H), 6.31 (d, J = 3.8 Hz, 1H), 7.04−7.29 (m, 2H), 7.36−7.52 (m, 2H), 7.96 (br s, 3H). MS (ESI) m/z 184.1 [M + H]+. 3-(3-Bromophenyl)-5-(4-fluorophenyl)-4,4-dimethyl-1,3-oxazolidin-2-one (28). To a solution of 26 (500 mg, 2.3 mmol), 1bromo-3-iodobenzene (773 mg, 2.7 mmol), and NaOH (273 mg, 6.8 mmol) in 2-propanol (10 mL) was added CuI (11 mg, 0.06 mmol) at room temperature. The mixture was stirred at 90 °C under N2 for 16 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) to yield the crude 27 (650 mg). This product was dissolved in THF (20 mL), and to the mixture were added DBU (0.58 mL, 3.8 mmol) and CDI (623 mg, 3.8 mmol) at room temperature. The mixture was stirred at 80 °C for 2 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) followed by crystallization from IPE to yield 28 (462 mg, 56%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 0.82 (s, 3H), 1.46 (s, 3H), 5.35 (s, 1H), 7.13 (t, J = 8.6 Hz, 2H), 7.19−7.25 (m, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.36−7.46 (m, 3H), 7.52 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 365.2 [M + H]+. 3-(3-Bromo-5-fluorophenyl)-5-(4-fluorophenyl)-4-methyl1,3-oxazolidin-2-one (30a). To a solution of 19 (2.0 g, 11.8 mmol), 1-bromo-3-fluoro-5-iodobenzene (3.6 g, 11.8 mmol), and NaOH (0.95 g, 23.6 mmol) in 2-propanol (40 mL) was added CuI (0.056 g, 0.30 mmol) at room temperature. The mixture was stirred at 100 °C under 8977



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Ar for 16 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO 4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−20% EtOAc/hexane) to yield the crude 29a (2.1 g) as a dark yellow oil. To a solution of the product in THF (50 mL) were added CDI (1.5 g, 9.0 mmol) and DBU (1.4 mL, 9.0 mmol), and the mixture was stirred at 80 °C for 2 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−15% EtOAc/hexane) to yield 30a (1.4 g, 32% in 2 steps) as a pale yellow oil. 1H NMR (300 MHz, CDCl3): δ 1.49 (s, 3H), 4.25 (t, J = 6.2 Hz, 1H), 5.11 (d, J = 6.1 Hz, 1H), 7.05−7.18 (m, 3H), 7.23 (t, J = 2.2 Hz, 1H), 7.31−7.42 (m, 3H). MS (ESI) m/z 368.2 [M + H]+. 3-(3-Bromo-5-chlorophenyl)-5-(4-fluorophenyl)-4-methyl1,3-oxazolidin-2-one (30b). To a solution of 19 (2.0 g, 11.8 mmol), 1-bromo-3-chloro-5-iodobenzene (3.8 g, 11.8 mmol), and NaOH (0.95 g, 23.6 mmol) in 2-propanol (40 mL) was added CuI (0.056 g, 0.30 mmol) at room temperature. The mixture was stirred at 90 °C under Ar for 16 h. CuI (0.056 g, 0.30 mmol) and 1-bromo-3-chloro-5iodobenzene (1.0 g, 3.1 mmol) were added to the mixture, and the mixture was stirred at 90 °C under Ar for 8 h. The mixture was filtered through Celite, and the filtrate was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−15% EtOAc/hexane) to yield crude 29b. The product was dissolved in THF (40 mL). To the solution were added CDI (1.9 g, 11.8 mmol) and DBU (1.8 mL, 11.8 mmol) at room temperature. The mixture was stirred at 80 °C for 3 h. The mixture was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−15% EtOAc/hexane) to yield 30b (1.3 g, 28% in 2 steps) as a pale yellow solid. MS (ESI) m/z 384.2 [M + H]+. 2-Bromo-1-(4-fluorophenyl)propan-1-one (32). To a solution of 1-(4-fluorophenyl)propan-1-one (31, 25.2 g, 165.6 mmol) in Et2O (300 mL) was added bromine (9.3 mL, 182.2 mmol) at 0 °C. The mixture was stirred at room temperature for 2 h. The mixture was poured into sat. aq NaHCO3 and sat. aq Na2S2O3, and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 100% hexane) to yield 32 (36.2 g, 95%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 1.91 (d, J = 6.6 Hz, 3H), 5.24 (q, J = 6.6 Hz, 1H), 7.12−7.21 (m, 2H), 8.03−8.10 (m, 2H). 2-(Dibenzylamino)-1-(4-fluorophenyl)propan-1-one (33). A mixture of 32 (36.2 g, 156.6 mmol), dibenzylamine (31.6 mL, 164.5 mmol), Na2CO3 (15.8 g, 188.0 mmol), and CH3CN (300 mL) was stirred at 60 °C overnight. The precipitate was removed by filtration. The filtrate was concentrated under vacuum. The residue was poured into 1 N aq HCl and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−5% EtOAc/hexane) to yield 32 (48.7 g, 89%) as a yellow oil. 1H NMR (300 MHz, DMSO-d6): δ 1.25 (d, J = 6.6 Hz, 3H), 3.53 (s, 4H), 4.30 (q, J = 6.6 Hz, 1H), 7.11−7.17 (m, 4H), 7.18− 7.33 (m, 8H), 7.58−7.67 (m, 2H). MS (ESI) m/z 348.2 [M + H]+. (1RS,2RS)-2-(Dibenzylamino)-1-(4-fluorophenyl)propan-1-ol (34). A mixture of 33 (48.7 g, 140.1 mmol), LAH (5.3 g, 140.1 mmol), and Et2O (500 mL) was stirred at room temperature for 1 h. The mixture was quenched with sat. aq Na2SO4. The mixture was filtered and the filtrate was concentrated under vacuum to yield 34 (39.9 g, 81%). 1H NMR (300 MHz, DMSO-d6): δ 0.84 (d, J = 6.7 Hz, 3H), 2.60−2.71 (m, 1H), 3.44 (d, J = 13.7 Hz, 2H), 3.88 (d, J = 13.7 Hz, 2H), 4.54 (dd, J = 8.3, 1.8 Hz, 1H), 4.99 (d, J = 1.8 Hz, 1H), 7.04− 7.36 (m, 14H). MS (ESI) m/z 350.2 [M + H]+.

(1RS,2RS)-2-Amino-1-(4-fluorophenyl)propan-1-ol (35). A mixture of 34 (29.9 g, 85.5 mmol) and 10% palladium on carbon (3 g) in MeOH (400 mL) was hydrogenated under balloon pressure at room temperature overnight. The catalyst was removed by filtration, and the filtrate was concentrated under vacuum. The residue was treated with hexane and the precipitate was collected by filtration, washed with hexane, and dried under vacuum to yield 35 (13.5 g, 93%) as a off-white solid. 1H NMR (300 MHz, DMSO-d6): δ 0.75 (d, J = 6.5 Hz, 3H), 1.53 (br s, 2H), 2.71−2.82 (m, 1H), 4.12 (d, J = 6.7 Hz, 1H), 5.29 (br s, 1H), 7.07−7.16 (m, 2H), 7.28−7.36 (m, 2H). tert-Butyl [(1S,2S)-1-(4-Fluorophenyl)-1-hydroxypropan-2yl]carbamate (36). To a solution of 35 (7.6 g, 44.6 mmol) and NEt3 (9.3 mL, 66.9 mmol) in THF (90 mL) was added Boc2O (12.3 mL, 53.6 mmol) at 0 °C. The mixture was stirred at room temperature overnight. The mixture was poured into sat. aq NH4Cl and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was suspended in hexane. The precipitate was collected by filtration, washed with hexane, and dried under vacuum to yield tert-butyl [(1RS,2RS)-1-(4-fluorophenyl)-1-hydroxypropan-2-yl]carbamate (11.0 g, 91%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 0.90 (d, J = 6.7 Hz, 3H), 1.33 (s, 9H), 3.57−3.73 (m, 1H), 4.52 (dd, J = 5.0 Hz, 1H), 5.32 (d, J = 5.0 Hz, 1H), 6.30 (d, J = 7.9 Hz, 1H), 7.06−7.17 (m, 2H), 7.24−7.33 (m, 2H). This product was separated by a YMCKPrep system (column, CHIRALPAK AD, 50 mm i.d. × 500 mm L, Daicel, Japan), eluting with a flow rate of 80 mL/min, hexane/IPA = 850/150 (v/v) at 30 °C. The first eluting enantiomers were collected to yield 36 (5.9 g). Liquid chromatography analysis was performed on a Hitachi system, equipped with a CHIRALPAK IC (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 1.0 mL/min, hexane/IPA = 850/150 (v/v) at 30 °C. Retention time: 5.34 min (>99.9% ee). (1S,2S)-2-Amino-1-(4-fluorophenyl)propan-1-ol (37). A mixture of 44 (15.2 g, 78.1 mmol) in 4 M aq LiOH solution (234 mL, 937 mmol) was stirred at 90 °C for 1.5 h. After the reaction has cooled, the mixture was acidified with 6 N aq HCl (182 mL, 1093 mmol). The solution was washed with methyl tert-butyl ether. The aqueous layer was basified with 4 M aq LiOH (42.9 mL, 172 mmol) with ice-cooling. The precipitate was collected by filtration, washed with cold water, and dried under vacuum to yield 37 (9.9 g, 75%) as a colorless solid. Liquid chromatography analysis was performed on a Hitachi system, equipped with a CHIRALCEL OD-H (4.6 mm i.d. × 250 mm L, Daicel, Japan), eluting with a flow rate of 1.0 mL/min, hexane/EtOH/ diethylamine = 900/100/1 (v/v/v) at 30 °C. Retention time: 6.97 min (>99.9% ee). 1H NMR (300 MHz, DMSO-d6): δ 0.75 (d, J = 6.4 Hz, 3H), 1.44 (br s, 2H), 2.76 (quintet, J = 6.5 Hz, 1H), 4.12 (d, J = 6.8 Hz, 1H), 5.29 (br s, 1H), 7.07−7.16 (m, 2H), 7.28−7.36 (m, 2H). (1S,2S)-2-Amino-1-(4-fluorophenyl)propan-1-ol Hydrochloride (37-HCl). To a solution of 36 (5.9 g, 21.9 mmol) in EtOAc (60 mL)/MeOH (30 mL) was added 4 N HCl/EtOAc solution (43.7 mL, 175.0 mmol) at 0 °C. The mixture was stirred at room temperature for 5 h. The mixture was concentrated under vacuum. The residue was suspended in IPE. The precipitate was collected by filtration, washed with IPE, and dried under vacuum to yield 37-HCl (4.2 g, 93%) as a white solid. 1H NMR (300 MHz, DMSO-d6): δ 0.96 (d, J = 6.6 Hz, 3H), 3.16−3.30 (m, 1H), 4.48 (d, J = 8.5 Hz, 1H), 6.22 (br s, 1H), 7.16−7.26 (m, 2H), 7.37−7.46 (m, 2H), 8.04 (br s, 3H). (1S,2S)-2-[(3,5-Dibromophenyl)amino]-1-(4-fluorophenyl)propan-1-ol (38). To a solution of 37-HCl (4.0 g, 19.3 mmol), 1,3dibromo-5-iodobenzene (7.0 g, 19.3 mmol), and NaOH (2.3 g, 57.9 mmol) in 2-propanol (20 mL) was added CuI (0.092 g, 0.48 mmol) at room temperature. The mixture was stirred at 90 °C under Ar for 20 h. The mixture was filtered through Celite, and the filtrate was concentrated under vacuum. The residue was treated with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) to yield 38 (3.8 g, 49%) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 1.09 (d, J = 6.4 Hz, 3H), 2.49 (d, J = 2.4 Hz, 1H), 3.46−3.68 (m, 1H), 3.77 (d, J = 8.6 Hz, 1H), 4.61 (d, J = 4.2 8978



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MgCl2, 0.005% Tween 20, and 1 mM GSH). [3H]T-3364366 was added to a final concentration of 3 nM, and the mixture was incubated for 150 min. For bound/free separation, the membrane fraction was trapped on a GF/C glass filter using a cell harvester. The GF/C glass filter was washed 5 times with 300 μL of ice-cooled assay buffer (without 0.005% Tween 20) to separate the ligand bound to D5D. 50 μL of MicroScint-O (PerkinElmer) was added, and the radioactivity on the filter was measured by TopCount (PerkinElmer). To evaluate nonspecific binding, 10 μM nonlabeled T-3364366 was used. IC50 values of test compounds were determined by fitting the following three-parameter logistic equation to the data: % control = bottom + (top − bottom)/(1 + 10(log[I]−logIC50)). HepG2 Cellular Assay. HepG2 was plated on a 96-well plate at 1 × 105 cells/well and cultured overnight in DMEM (Dulbecco’s modified Eagle medium) containing 10% FBS (fetal bovine serum). After washing (200 μL × 2) with PBS (phosphate buffered saline), 40 μL of a reaction medium (DMEM, penicillin/streptomycin, 0.3% BSA) containing the test compound was added, and the mixture was preincubated in a CO2 incubator at 37 °C for 30 min. The reaction was started by adding a reaction medium (20 μL) containing 0.1 μCi [14C]eicosatrienoic acid, and the mixture was incubated in a CO2 incubator at 37 °C. Incubation was performed for 3 h, the cells were washed twice with 200 μL of PBS, detached with 50 μL of trypsin− EDTA, and placed on a 96-deep-well block. Furthermore, 20 μL of 2.5 N NaOH was added, and the plate was sealed and incubated overnight at 55 °C to allow saponification. The fatty acid extraction included acidifying with 10 μL of formic acid, adding chloroform/MeOH (1:4), stirring, and further adding 200 μL of pure water to separate into two layers. The operation after development with TLC followed the method of detection system using TLC in a system using rat liver microsomes. A D5D inhibitory rate (%) of 1 μM test compound was determined. D5D Inhibitory Action in Vivo. A suspension of the test compound (3 mg/kg) in 0.5% methylcellulose was administered by gavage every morning at 10 mL/kg for 4 days to 7- to 9-week-old male normal mice (C57BL/6J, CLEA Japan, Inc.) acclimated with a normal chow feed (CE-2, CLEA Japan, Inc.) in individual cages. In the next morning of the final administration, the mice were anesthetized, and the liver was isolated. Total lipid was extracted from about 40 mg of a liver sample with hexanepropanol solution, and a phospholipid fraction was extracted using a solid phase extraction column (Sep-Pak Vac NH2, Waters) and labeled with an esterified long-chain and shortchain fatty acid labeling reagent (YMC). The arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) content of the sample after labeling were measured by high performance liquid chromatography (Agilent 1200, Agilent Technologies). AA content was divided by the content of DGLA to obtain an AA/DGLA ratio. The decrease rate of the test compound administration group was calculated based on the AA/DGLA ratio of the solvent administration group as 100%, which was used as an index of the liver D5D inhibitory action in vivo. D5D Inhibitory Action in Atherosclerosis Model. Thirteenweek-old apoE KO mice were fed Western diet (Research Diets, D12079B) for 2 weeks. One day after the diet feeding start, a suspension of a test compound (0.3−3 mg/kg) was administered by gavage every morning at 10 mL/kg to the mice with Western diet feeding. In the next morning of the final administration, the mice were anesthetized and the livers isolated. The procedure for measuring the liver AA/DGLA ratio is described above. X-ray Structure Analysis. Crystal data for (4S,5S)-5n: C18H12FN3O2, MW = 321.31; crystal size, 0.23 mm × 0.19 mm × 0.19 mm; colorless, block; monoclinic, space group P21, a = 9.19260(17) Å, b = 8.44710(15) Å, c = 9.83821(18) Å, α = γ = 90°, β = 92.104(7)°, V = 763.43(2) Å3, Z = 2, Dx = 1.398 g/cm3, T = 100 K, μ = 0.853 mm−1, λ = 1.54187 Å, R1 = 0.025, wR2 = 0.063, Flack parameter21 = −0.02(2). All measurements were made on a Rigaku R-AXIS RAPID-191R diffractometer using graphite monochromated Cu Kα radiation. The structure was solved by direct methods with SIR200822 and was refined using full-matrix least-squares on F2 with SHELXL-2014/7.23

Hz, 1H), 6.67 (d, J = 1.5 Hz, 2H), 6.95 (d, J = 1.6 Hz, 1H), 7.00−7.11 (m, 2H), 7.30−7.38 (m, 2H). MS (ESI) m/z 402.1 [M + H]+. (4S,5S)-3-(3,5-Dibromophenyl)-5-(4-fluorophenyl)-4-methyl1,3-oxazolidin-2-one (39). To a solution of 38 (3.8 g, 9.5 mmol) and CDI (2.3 g, 14.3 mmol) in DMF (50 mL) was added DBU (2.1 mL, 14.3 mmol) at room temperature. The mixture was stirred at 100 °C for 1.5 h. The mixture was treated with water and the resulting solid was collected by filtration and washed with water and hexane to yield 39 (1.8 g, 45%). The filtrate was extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0−30% EtOAc/hexane) to yield 39 (1.8 g, 44%) as a brown oil. 1H NMR (300 MHz, CDCl3): δ 1.38−1.50 (m, 3H), 4.25 (quintet, J = 6.2 Hz, 1H), 5.11 (d, J = 6.4 Hz, 1H), 7.09−7.20 (m, 2H), 7.35−7.43 (m, 2H), 7.48−7.52 (m, 1H), 7.54 (d, J = 1.6 Hz, 2H). MS (ESI) m/z 430.1 [M + H]+. N2-(tert-Butoxycarbonyl)-N-methoxy-N-methyl-L-alaninamide (41). To a suspension of Boc-L-alanine (40, 25.4 g, 134.1 mmol), N,O-dimethylhydroxylamine hydrochloride (15.7 g, 160.9 mmol), NEt3 (56.1 mL, 402.3 mmol), and HOBt hydrate (24.6 g, 160.9 mmol) in DMF (200 mL) was added EDCI hydrochloride (30.8 g, 160.9 mmol) in one portion with ice-cooling. The mixture was stirred at room temperature for 16 h. The mixture was poured into cold water (1 L) and stirred for 20 min with ice-cooling. The precipitate was collected by filtration, washed with water (500 mL) and isopropyl ether (200 mL), and dried under vacuum at 50 °C to yield 41 (27.3 g, 88%) as a colorless solid. 1H NMR (300 MHz, CDCl3): δ 1.31 (d, J = 6.8 Hz, 3H), 1.44 (s, 9H), 3.21 (s, 3H), 3.77 (s, 3H), 4.63−4.74 (m, 1H), 5.25 (d, J = 7.9 Hz, 1H). tert-Butyl [(2S)-1-(4-Fluorophenyl)-1-oxopropan-2-yl]carbamate (42). To an ice-cold suspension of 41 (27.3 g, 117.4 mmol) in THF (293 mL) was added dropwise a solution of 0.9 M 4fluorophenylmagnesium bromide in THF (326 mL, 293.4 mmol). The resulting mixture was stirred at room temperature for 20 h. The reaction was quenched by addition of sat. aq NH4Cl and stirred for 10 min. The resulting solution was extracted with EtOAc, and the extract was washed brine. After removal of the solvent, the residue was purified by column chromatography (silica gel, eluted with 0−10% EtOAc/hexane) to yield 42 (34.0 g, quant) as a colorless solid. 1H NMR (300 MHz, CDCl3): δ 1.40 (d, J = 7.2 Hz, 3H), 1.46 (s, 9H), 5.25 (quintet, J = 7.3 Hz, 1H), 5.53 (d, J = 7.2 Hz, 1H), 7.12−7.20 (m, 2H), 7.97−8.05 (m, 2H). tert-Butyl [(1R,2S)-1-(4-Fluorophenyl)-1-hydroxypropan-2yl]carbamate (43). A mixture of 42 (29.0 g, 108.5 mmol), 2propanol (92 mL, 1193.4 mmol), and Al(OiPr)3 (4.4 g, 21.7 mmol) in toluene (141 mL) was stirred at 50 °C for 18 h under Ar. The mixture was quenched with sat. aq NH4Cl and extracted with EtOAc (×2). The combined extracts were washed with brine and dried over MgSO4. The solvent was removed under vacuum and the residue was recrystallized from EtOAc (57 mL) and hexane (459 mL) to yield 43 (19.4 g, 67%) as a colorless solid. 1H NMR (300 MHz, CDCl3): δ 0.98 (d, J = 6.8 Hz, 3H), 1.46 (s, 9H), 3.36 (br s, 1H), 3.98 (br s, 1H), 4.56 (d, J = 4.9 Hz, 1H), 4.83 (t, J = 3.6 Hz, 1H), 6.99−7.07 (m, 2H), 7.27−7.34 (m, 2H). (4S,5S)-5-(4-Fluorophenyl)-4-methyl-1,3-oxazolidin-2-one (44). A mixture of 43 (24.1 g, 89.31 mmol), NEt3 (13.7 mL, 98.3 mmol), and MsCl (7.6 mL, 98.3 mmol) in THF (298 mL) was stirred at 50 °C for 4 h. NEt3 (13.7 mL, 98.3 mmol) and MsCl (7.6 mL, 98.3 mmol) were added, and the mixture was stirred at 50 °C for 4 h. The mixture was quenched with NaHCO3 solution and extracted with EtOAc. The extract was washed with brine and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, eluted with 30−50% EtOAc/hexane) to yield 44 (15.2 g, 87%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 1.38 (d, J = 6.4 Hz, 3H), 3.82 (quintet, J = 6.3 Hz, 1H), 5.02 (d, J = 7.2 Hz, 1H), 6.06 (br s, 1H), 7.06−7.14 (m, 2H), 7.32−7.40 (m, 2H). D5D Binding Assay. All tests were performed at room temperature at 200 μL/well. A rat liver microsome fraction (30 μg/ well) was preincubated with a test compound for 15 min in an assay buffer (10 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM NaF, 3 mM 8979



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Diseases: Molecular Mechanisms and Perspectives in Therapeutics; Springer: Cham, Switzerland, 2014; pp 61−81, DOI: 10.1007/978-3319-07320-0_7. (2) Nakamura, M. T.; Nara, T. Y. Structure, function and dietary regulation of delta6, delta5, delta9 desaturases. Annu. Rev. Nutr. 2004, 24, 345−376. (3) Cho, H. P.; Nakamura, M.; Clarke, S. D. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase. J. Biol. Chem. 1999, 274, 37335−37339. (4) Wang, X.; Lin, H.; Gu, Y. Multiple roles of dihomogammalinolenic acid against proliferation diseases. Lipids Health Dis. 2012, 11, 25. (5) Fan, Y. Y.; Monk, J. M.; Hou, T. Y.; Callway, E.; Vincent, L.; Weeks, B.; Yang, P.; Chapkin, R. S. Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model. J. Lipid Res. 2012, 53, 1287−1295. (6) Obukowicz, M. G.; Raz, A.; Pyla, P. D.; Rico, J. G.; Wendling, J. M.; Needleman, P. Identification and characterization of a novel Δ6/ Δ5 fatty acid desaturase inhibitor as a potential anti-inflammatory agent. Biochem. Pharmacol. 1998, 55, 1045−1058. (7) Obukowicz, M. G.; Welsch, D. J.; Salsgiver, W. J.; Martin-Berger, C. L.; Chinn, K. S.; Duffin, K. L.; Raz, A.; Needleman, P. Novel, selective Δ6 or Δ5 fatty acid desaturase inhibitors as anti-inflammatory agents in mice. J. Pharmacol. Exp. Ther. 1998, 287, 157−166. (8) Baugh, S. D. P.; Pabba, P. K.; Barbosa, J.; Coulter, E.; Desai, U.; Gay, J. P.; Gopinathan, S.; Han, Q.; Hari, R.; Kimball, S. D.; Nguyen, H. V.; Ni, C.; Powell, D. R.; Smith, A.; Terranova, K. M.; Wilson, A.; Yu, X.; Lombardo, V. K. Design, synthesis, and in vivo activity of novel inhibitors of delta-5 desaturase for the treatment of metabolic syndrome. Bioorg. Med. Chem. Lett. 2015, 25, 3836−3839. (9) (a) Suzuki, H.; Fujimoto, T.; Yamamoto, T. Fused ring compound and use thereof. US2010190747, 2010. (b) Yashiro, H.; Takagahara, S.; Tamura, Y. O.; Miyahisa, I.; Matsui, J.; Suzuki, H.; Ikeda, S.; Watanabe, M. A novel selective inhibitor of delta-5 desaturase lowers insulin resistance and reduces body weight in dietinduced obese C57BL/6J mice. PLoS One 2016, 11, e0166198. (10) Matsunaga, N.; Suzuki, H.; Asano, K.; Tokuhara, H.; Yamamoto, K.; Okamoto, R. Fused Heterocyclic Compound and Application Thereof. WO2012011591, 2012. (11) Matsunaga, N.; Igawa, H.; Suzuki, H.; Okamoto, R.; Furukawa, H.; Murayama, K. Heterocyclic Compound and Application Thereof. WO2012011592, 2012. (12) Miyahisa, I.; Suzuki, H.; Mizukami, A.; Tanaka, Y.; Ono, M.; Hixon, M. S.; Matsui, J. T-3364366 targets the desaturase domain of delta-5 desaturase with nanomolar potency and a multihour residence time. ACS Med. Chem. Lett. 2016, 7, 868−872. (13) Molecular Operating Environment (MOE), version 2013.08; Chemical Computing Group Inc. (1010 Sherbooke St. West, Suite No. 910, Montreal, QC, Canada, H3A 2R7), 2016. (14) Job, G. E.; Buchwald, S. L. Copper-catalyzed arylation of βamino alcohols. Org. Lett. 2002, 4, 3703−3706. (15) Vearrier, D.; Jacobs, D.; Greenberg, M. I. Phenol toxicity following cutaneous exposure to creolin®: a case report. J. Med. Toxicol. 2015, 11, 227−231. (16) Kavlock, R. J. Structure-activity relationships in the developmental toxicity of substituted phenols: in vivo effects. Teratology 1990, 41, 43−59. (17) Manuscript in preparation. (18) De Graauw, C. F.; Peters, J. A.; Van Bekkum, H.; Huskens, J. Meerwein-Ponndorf-Verley reductions and oppenauer oxidations: an integrated approach. Synthesis 1994, 1994 (10), 1007−1017. (19) Poelert, M. A.; Hof, R. P.; Peper, N. C. M. W.; Kellogg, R. M. Enantiomerically pure β-amino sulfides and β-amino thiols from ephedrine. Heterocycles 1994, 37, 461−475. (20) (a) Benedetti, F.; Norbedo, S. Facile inversion of configuration of N-Boc-β-aminoalcohols via SN2 cyclization to oxazolidinones. Tetrahedron Lett. 2000, 41, 10071−10074. (b) Bertau, M.; Bürli, M.; Hungerbühler, E.; Wagner, P. A novel highly stereoselective synthesis of chiral 5- and 4,5-substituted 2-oxazolidinones. Tetrahedron:

All non-H atoms were refined with anisotropic displacement parameters. CCDC 1519191 for compound (4S,5S)-5n contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/Community/ Requestastructure/Pages/DataRequest.aspx?.

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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.7b01210. Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-466-32-1086. Fax: +81-466-29-4448. E-mail: jun. [email protected]. ORCID

Jun Fujimoto: 0000-0002-8857-2059 Present Addresses †

R.O., T.K., M.I., T.A., J.M., and R.H.: Axcelead Drug Discovery Partners, Inc., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, Japan. ‡ N.N., R.H., and T.M.: SCOHIA PHARMA, Inc., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, Japan. § S.T., N.M.: Cardurion Pharmaceuticals KK, 26-1, MuraokaHigashi 2-chome, Fujisawa, Kanagawa, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kazuko Yonemori, Akihiro Yokota, and Yumi Imai for their support in computational chemistry and Mitsuyoshi Nishitani for his assistance in crystallographic data deposition to the Cambridge Crystallographic Data Centre. We express our gratitude to Shinobu Sasaki and Mika Inoue for their support in synthetic activities and to Katsuhiko Miwa for his assistance in separations of stereoisomers using a chromatography technique. We also express our appreciation to Yoshinori Kawamura for supporting dog ECG study. We thank Douglas R. Cary for his valuable advice in preparing the manuscript.

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ABBREVIATIONS USED IPE, isopropyl ether; THF, tetrahydrofuran; EtOAc, ethyl acetate; Et2O, diethyl ether; MsCl, methanesulfonyl chloride; NEt3, triethylamine; CDI, 1,1′-carbonyldiimidazole; DMAP, 4dimethylaminopyridine; DBU, 1,8-diazabicyclo[5.4.0]undec-7ene; NaBH4, sodium borohydride; NaH, sodium hydride; NaOtBu, sodium tert-butoxide; LAH, lithium aluminum hydride; Pd(PPh3)4, tetrakis(triphenylphosphine)platinum(0); Xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; CuI, copper(I) iodide; EDCI, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole; Al(OiPr)3, aluminum isopropoxide

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REFERENCES

(1) Tosi, F.; Sartori, F.; Guarini, P.; Olivieri, O.; Martinelli, N. Delta5 and Delta-6 Desaturases: Crucial Enzymes in Polyunsaturated Fatty Acid-Related Pathways with Pleiotropic Influences in Health and Disease. In Oxidative Stress and Inflammation in Non-Communicable 8980



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Asymmetry 2001, 12, 2103−2107. (c) Tanaka, N.; Tamai, T.; Mukaiyama, H.; Hirabayashi, A.; Muranaka, H.; Ishikawa, T.; Kobayashi, J.; Akahane, S.; Akahane, M. Relationship between stereochemistry and the b3-adrenoceptor agonistic activity of 4′hydroxynorephedrine derivative as an agent for treatment of frequent urination and urinary incontinence. J. Med. Chem. 2003, 46, 105−112. (d) Groeper, J. A.; Hitchcock, S. R.; Ferrence, G. M. A scalable and expedient method of preparing diastereomerically and enantiomerically enriched pseudonorephedrine from norephedrine. Tetrahedron: Asymmetry 2006, 17, 2884−2889. (21) Flack, H. D. On enantiomorph-polarity estimation. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, 39, 876−881. (22) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Siliqi, D.; Spagna, R. IL MILIONE: a suite of computer programs for crystal structure solution of proteins. J. Appl. Crystallogr. 2007, 40, 609−613. (23) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112−122.

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Discovery of 3, 5-Diphenyl-4-methyl-1, 3-oxazolidin-2-ones as Novel

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