A Unified Approach to Synthesizing Four Linezolid Metabolites That May Cause Thrombocytopenia

Abstract

Background/Objectives: Linezolid is a first-in-class oxazolidinone antibiotic that exhibits activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. However, its clinical use is often restricted because of hematological toxicities, particularly thrombocytopenia, in patients with renal impairment. That side effect is thought to result from the systemic accumulation of pharmacologically inactive metabolites generated by oxidative degradation and ring-opening of the morpholine, but the details remain unclear. In this study, we established a novel synthetic route for four linezolid metabolites (PNU-142618, 142300, 142586 and 173558). Methods: The four major metabolites, which are secondary or tertiary amines, were synthesized using the aniline derivatives protected with a 2-nitrobenzensulfonyl (Ns) group. Results: Application of this Ns strategy enabled selective N-alkylation, enabling efficient synthesis of the target metabolites. The desired metabolites containing a carboxylic acid group were obtained as their sodium salts. This is the first report on the synthesis of PNU-142618 and 173558. Conclusions: The established synthetic pathway provides access to four linezolid metabolites. The results facilitated the provision of compounds necessary for comprehensive pharmacokinetic and toxicological studies.

1. Introduction

Linezolid is an oxazolidinone antibiotic that exhibits broad-spectrum activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci [1]. It has been approved to treat various infections caused by multidrug-resistant organisms and has demonstrated superior efficacy to other antibiotics, such as quinupristin–dalfopristin. The antibacterial mechanism of action involves selective binding to the 50S ribosomal subunit, which inhibits the formation of the 70S initiation complex and inhibits bacterial protein synthesis.

Despite its clinical effectiveness, linezolid treatment is associated with the incidence of hematological adverse events, particularly thrombocytopenia, which prevents its long-term use. These toxicities are more pronounced in patients with renal impairment, which has prompted the re-evaluation of dosing strategies based on renal function [2]. Linezolid-induced thrombocytopenia may be caused by the accumulation of its major inactive metabolites, PNU-142300 (1) and PNU-142586 (2) [3,4,5,6]. These are generated by oxidative cleavage of the morpholine ring on linezolid through routes a and b, respectively, and are primarily eliminated through the kidneys. And these metabolites are known to be P450-independent [7,8]. Furthermore, metabolites such as PNU-142618 (3) and PNU-173558 (4) may also form from PNU-142300 (1) and/or PNU-142586 (2), although the mechanism of their metabolic pathways remains unclear, and toxicological evaluations have not yet been conducted (Figure 1, see Supplementary Materials) [9].

It was also suggested that PNU-142586 (2) may cause myelosuppression by exerting a concentration-dependent cytotoxic effect on megakaryocytic cells. In addition, the transport of this metabolite via human organic anion transporter 3 (hOAT3) and its inhibition by other drugs, such as proton pump inhibitors, may further exacerbate its accumulation [10]. The complexity of linezolid metabolism and interindividual variability in renal clearance highlight the need for methods to precisely measure and evaluate parent drug and metabolite concentrations in the plasma and urine [11,12]. So, advanced analytical techniques such as UPLC–MS/MS have been established to facilitate these pharmacokinetic analyses in healthy individuals and individuals with impairment in hepatic/renal function [6,13].

Although the primary metabolic pathways of linezolid have been partially elucidated, the importance of pharmacokinetics for linezolid metabolites and their toxicology remains unclear [14,15]. One of the reasons for this limitation is the difficulty in acquiring the metabolites as standard materials for the studies. The synthesis of linezolid metabolites is necessary for elucidating the mechanisms underlying linezolid-associated adverse effects. Therefore, synthetic strategies to prepare these metabolites are important, but to date, only one paper has been published on the chemical synthesis of linezolid metabolites PNU-142300 (overall yield 23%) and PNU-142586 (overall yield 7%) by Hanaya and Sugai et al. [16]. In this study, we adopted the 2-nitrobenzenesulfonyl (Ns)-strategy developed by Kan and Fukuyama [17,18], which enables efficient protection, selective N-alkylation, and mild deprotection of amines, thereby enabling the introduction of selective alkyl chains onto aniline derivatives.

2. Results and Discussion

2.1. Retrosynthetic Analysis

Since the desired linezolid metabolites are aromatic secondary or tertiary amine derivatives, we incorporated the Ns group into the designed synthetic route, which is useful as a protective group for amines, and adopted compound 5 as a common intermediate [19]. Compound 5 can be prepared from carbamate obtained by Curtius rearrangement reaction. And the ^tBu esters used in Curtius rearrangement reaction were also obtained from 2-fluoro-4-nitrobenzoic acid as starting material (Figure 2).

2.2. Preparation of the Common Intermediate 5

2-Fluoro-4-nitrobenzoic acid was treated with tert-butoxycarbonyl (Boc) anhydride to obtain the corresponding tert-butyl ester 6. Next, catalytic hydrogenation of 6 afforded the aniline derivative 7, whose primary amine group was then treated with benzyl chloroformate (Cbz-Cl) to give the carbamate 8. The oxazolidinone derivative 9 was yielded with 8 and a chiral glycidyl ester under n-butyllithium conditions based on Manninen and Brickner’s procedure [1,19]. Resulting primary alcohol on 9 was substituted with azide group via mesylate to give 10. tert-Butyl ester derivative 10 was treated with trifluoroacetic acid (TFA) to afford carboxylic acid without further purification, followed by Curtius rearrangement reaction to obtain aniline derivative 11. Using catalytic hydrogenation for azide group on 11 afforded the corresponding primary amine 12, followed by acetylation with acetic anhydride (Ac₂O) to yield 13. Then, 13 was treated with TFA to eliminate the Boc group, yielding the aniline derivative 14. Finally, treatment of 14 with 4-nitrobenzenesulfonyl chloride in pyridine gave the desired common intermediate 5 (overall yield: 25%, 12 steps) (Scheme 1).

2.3. Synthesis of PNU-142300 (1)

The Ns-protected intermediate 5 was treated with (2-bromoethoxy)-tert-butyldimethylsilane in N,N-dimethylformamide (DMF) in the presence of potassium carbonate, which yielded compound 15. Deprotection of the tert-butyldimethylsilyl group with tetrabutylammonium fluoride (TBAF) yielded the primary alcohol derivative 16. O-Alkylation of 16 was carried out under biphasic conditions using tetrabutylammonium hydrogen sulfate as a phase-transfer catalyst according to the procedure of Sugai et al. [16] to obtain 17. In this O-alkylation reaction, the reaction proceeded as intended and the desired compound 17 was obtained with 77% (conversion yield), but the reaction was incomplete (recovered 16 with 43%). Finally, Ns group of 17 was removed with thiophenol, followed by deprotection of the tert-butyl group in 17 using TFA, and neutralization with 1 M sodium hydroxide solution to yield a sodium salt of PNU-142300 (1) (overall yield: 11%, 17 steps) (Scheme 2).

2.4. Synthesis of PNU-142586 (2)

A common intermediate 5 was alkylated with a benzyl 2-bromoethyl ether to give 18, followed by the removal of the Ns group using thiophenol to afford secondary amine 19. The N-alkylation reaction of 19 treated with ethyl bromoacetate in the presence of potassium carbonate to obtain 20 required high temperatures and extended reaction time to improve yield. Then, the benzyl group on 20 was removed by hydrogenation over Pd/C to afford primary alcohol 21. Then, 21 in THF was treated with aqueous sodium hydroxide to hydrolyze the ester, which afforded 2 with precipitation as sodium salt (overall yield: 12%, 17 steps) (Scheme 3).

2.5. Synthesis of PNU-142618 (3)

PNU-142618 (3) was obtained by debenzylation with Pd/C and H₂ of 19 (overall yield: 22%, 15 steps) (Scheme 4).

2.6. Synthesis of PNU-173558 (4)

Analogous to compound 2, PNU-173558 (4) was also designed for preparation as the sodium salt. Specifically, compound 4 was synthesized by N-alkylation of the common intermediate 5 with ethyl bromoacetate and potassium carbonate to afford 22, followed by deprotection of the Ns group using thiophenol, and ester hydrolysis with 1 M NaOH (overall yield: 23%, 15 steps) (Scheme 5).

3. Materials and Methods

3.1. General Remarks

All reagents and solvents were of the highest commercial grade and used without further purification. ¹H-NMR spectra were measured at 600 MHz on a JEOL JNM-ECX600 spectrometer (JOEL, Tokyo, Japan). Chemical shifts are reported relative to internal standards (tetramethylsilane, δH 0.00, CDCl₃, δH 7.26, CD₃OD, δH 3.31). Data are presented as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), coupling constant, and integration. ¹³C-NMR spectra were measured at 151 MHz using a JEOL JNM-ECX600 spectrometer. The following internal references were used: tetramethylsilane: δ 0.00, CDCl₃ δ 77.0, CD₃OD, δ 49.0). High-resolution mass spectra were recorded using a Waters Xevo G2-S QTOF instrument (Waters, Milford, MA, USA). Silica gel column chromatography was done using Silica Gel 60N (spherical and neutral, particle size 40 to 100 µm, Kanto Chemical Co., Tokyo, Japan). Analytical thin-layer chromatography was conducted using Merck silica gel 60 F₂₅₄ plates with visualization by ultraviolet light or spraying with Ninhydrin–ethanol test solution spray on a hot plate. Preparative thin-layer chromatography was performed using Merck silica gel 60 F₂₅₄ plates with visualization by ultraviolet light. The high-performance liquid chromatography system (HPLC) consisted of a PU-1580 (JASCO, Tokyo, Japan), SSC-5410 (Senshu Scientific Co., Ltd., Tokyo, Japan), and column (SG80, 5 μm, 20 mm I.D. × 250 mm). Optical rotations were measured on a JASCO P-1030 digital polarimeter at the sodium D line (589 nm). Melting points were measured on an ATM-01 (AS ONE Co., Osaka, Japan).

3.2. Synthesis

3.2.1. tert-Butyl 2-fluoro-4-nitrobenzoate (6) [20]

To a solution of 2-fluoro-4-nitrobenzoic acid (6.0 g, 32 mmol) in dichloromethane (DCM) (60 mL), triethylamine (TEA) (13 mL, 93 mmol), dimethylaminopyridine (DMAP) (1.2 g, 9.8 mmol), and di-tert-butyl dicarbonate (Boc₂O) (11 mL, 49 mmol) were added. After stirring for 2 h at room temperature, the residue was treated with ethyl acetate (EtOAc) and 5% citric acid solution. The organic layer was washed with brine and dried over anhydrous Na₂SO₄. The filtrate was concentrated under reduced pressure to obtain a colorless solid 6 (6.6 g, 83%). M.P. 81–82 °C. ¹H-NMR (600 MHz, CDCl₃) δ 8.07–8.03 (m, 2H), 7.99–7.97 (m, 1H), 1.62 (s, 9H).

3.2.2. tert-Butyl 4-amino-2-fluorobenzoate (7) [20]

To a solution of 6 (1.1 g, 5.0 mmol) in methanol (MeOH) (30 mL), Pd/C (200 mg) was added. After stirring for 2 h under a H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and a colorless solid 7 was obtained (1.0 g, quant.). M.P. 94–96 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.68 (t, J = 8.6 Hz), 6.39 (dd, J = 2.4, 8.6 Hz), 6.31 (dd, J = 2.0, 12.7 Hz), 4.13 (bs, 2H), and 1.57 (s, 9H). HRMS (ESI): m/z calculated for C₁₁H₁₄FNNaO₂: 234.0906, found [M + Na]⁺ 234.0914.

3.2.3. tert-Butyl 4-(((benzyloxy)carbonyl)amino)-2-fluorobenzoate (8) [21]

To a solution of 7 (3.8 g, 18 mmol) in pyridine (Py) (35 mL), benzyl chloroformate (13 g, 73 mmol) was added dropwise at 0 °C. The mixture was allowed to warm to room temperature. After stirring overnight at room temperature, the mixture was diluted with EtOAc. The residue was treated with EtOAc and 5% hydrochloric acid. The organic layer was washed with brine and dried over anhydrous Na₂SO₄. The filtrate was concentrated under reduced pressure. The resulting residue was separated by column chromatography (C.C.) (hexane/EtOAc = 6:1) to yield 8 as a white solid (6.2 g, 99%). M.P. 115–120 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.80 (t, J = 8.1 Hz, 1H), 7.41–7.35 (m, 6H), 7.04–6.97 (m, 2H), 5.21 (s, 2H), 1.58 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 163.2 (d, J = 4.3 Hz), 162.8 (d, J = 258.7 Hz), 152.7, 143.0 (d, J = 11.6 Hz), 135.5, 132.8, 128.7, 128.6, 128.4, 114.9 (d, J = 10.1 Hz), 113.0, 106.3 (d, J = 28.9 Hz), and 81.6, 67.5, 28.2. HRMS (ESI): m/z calculated for C₁₉H₂₀FNNaO₄: 368.1274, found [M + Na]⁺ 368.1264.

3.2.4. tert-Butyl (R)-2-fluoro-4-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)benzoate (9) [21]

To a solution of 8 (2.8 g, 8.0 mmol) in THF (80 mL), n-butyllithium in hexane (6 mL, 9.6 mmol) was added dropwise at −78 °C. After stirring for 1 h at −78 °C, (R)-glycidyl butyrate (1.2 mL, 8.8 mmol) was added, and the mixture was allowed to warm to room temperature. After stirring overnight at room temperature, saturated NH₄Cl was added, and the organic materials were extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by C.C. (hexane/EtOAc = 1:1) to yield 9 as a white solid (1.8 g, 71%). M.P. 143–145 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.87 (t, J = 8.4 Hz, 1H), 7.47 (dd, J = 13.1, 2.4 Hz, 1H), 7.26 (dd, J = 8.9, 2.4 Hz, 1H), 4.79–4.77 (m, 1H), 4.13–4.00 (m, 3H), 3.77 (d, J = 12.4 Hz, 1H), 1.59 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 163.0 (d, J = 4.3 Hz), 162.3 (d, J = 258.6 Hz), 154.3, 142.8 (d, J = 11.5 Hz), 132.8, 115.3 (d, J = 8.7 Hz), 112.4 (d, J = 2.9 Hz), 106.0 (d, J = 28.9 Hz), and 81.9, 62.5, 46.0, 28.2. HRMS (ESI): m/z calculated for C₁₅H₁₈FNNaO₅: 334.1067, found [M + Na]⁺ 334.1076. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −118.5° (c 0.10, CHCl₃).

3.2.5. tert-Butyl (R)-4-(5-(azidomethyl)-2-oxooxazolidin-3-yl)-2-fluorobenzoate (10) [21]

To a solution of 9 (2.5 g, 8.0 mmol) in DCM (80 mL), TEA (7 mL) and methanesulphonyl chloride (1.9 mL, 24 mmol) were added dropwise at 0 °C, and the mixture was allowed to warm to room temperature. After stirring for 1 h at room temperature, the solvent was removed by vacuum drying. The reaction mixture was diluted with water, and the organic material was extracted using EtOAc. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue (3.1 g, 8.0 mmol) was solved in DMF (35 mL), sodium azide (0.9 g, 15 mmol) was added. After stirring overnight at 60 °C, the mixture was diluted with hexane/EtOAc = 1:1. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and the filtrate was concentrated under reduced pressure. The resulting residue was separated by C.C. (hexane/EtOAc = 6:1) to yield 10 as a white solid (2.2 g, 82%). M.P. 93–95 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.87 (t, J = 8.4 Hz, 1H), 7.47 (dd, J = 13.1, 2.1 Hz, 1H), 7.26 (dd, J = 8.8, 2.2 Hz, 1H), 4.87–4.83 (m, 1H), 4.14–4.09 (m, 1H), 3.88 (dd, J = 8.9, 6.2 Hz, 1H), 3.76 (dd, J = 13.4, 4.1 Hz, 1H), 3.62 (dd, J = 13.4, 4.5 Hz, 1H), 1.59 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 162.9 (d, J = 2.9 Hz), 162.4 (d, J = 258.7 Hz), 153.5, 142.7 (d, J = 11.5 Hz), 132.8, 115.5 (d, J = 8.6 Hz), 112.4 (d, J = 2.9 Hz), 106.1 (d, J = 28.9 Hz), and 81.9, 70.9, 52.9, 47.0, 28.2. HRMS (ESI): m/z calculated for C₁₅H₁₇FN₄NaO₄: 359.1132, found [M + Na]⁺ 359.1133. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −320.5° (c 0.20, CHCl₃).

3.2.6. tert-Butyl (R)-(4-(5-(azidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)carbamate (11) [22]

To a solution of 10 (672 mg, 2.0 mmol) in DCM (4 mL) was added TFA (4 mL, 52 mmol) dropwise at 0 °C, and the mixture was allowed to warm to room temperature. After stirring overnight at room temperature, the solvent was removed in vacuo. To a solution of the resulting residue (565 mg, 2.0 mmol) in THF (7.5 mL), tert-butyl alcohol (t-BuOH) (950 μL, 10 mmol), TEA (360 μL, 2.6 mmol), and diphenylphosphoryl azide (DPPA) (560 μL, 2.6 mmol) were added. After stirring for 2 h at room temperature and overnight at 70 °C, the solvent was concentrated under reduced pressure, and the mixture was diluted with EtOAc. Saturated NaHCO₃ was added to the mixture, and the organic materials were extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by C.C. (hexane/EtOAc = 1:1) to yield 11 as a white solid (390 mg, 56%). M.P. 85–88 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.66–7.64 (m, 1H), 6.98 (d, J = 8.6 Hz, 1H), 6.67 (s, 1H), 4.79 (qd, J = 4.5, 1.7 Hz, 1H), 4.06 (t, J = 8.9 Hz, 1H), 3.83 (dd, J = 8.9, 6.2 Hz, 1H), 3.71 (dd, J = 13.4, 4.5 Hz, 1H), 3.59 (dd, J = 13.2, 4.6 Hz, 1H), 1.53 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 153.8, 152.4, 152.0 (d, J = 53.5 Hz), 133.1 (d, J = 10.1 Hz), 123.2 (d, J = 10.1 Hz), 113.3 (d, J = 2.8 Hz), 106.1 (d, J = 24.6 Hz),and 81.1, 70.6, 53.0, 47.4, 28.3. HRMS (ESI): m/z calculated for C₁₅H₁₈FN₅NaO₄: 374.1241, found [M + Na]⁺ 374.1241. [${\alpha ]}_{\mathrm{D}}^{20}$ = −178.0° (c 0.20, CHCl₃).

3.2.7. tert-Butyl (S)-(4-(5-(aminomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)carbamate (12) [22]

To a solution of 11 (754 mg, 2.1 mmol) in methanol (MeOH) (50 mL), Pd/C (301 mg) was added. After stirring for 1 h under a H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure to obtain a colorless solid 12 (704 mg, quant.). M.P. 95–100 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.68 (d, J = 13.4 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.68 (bs, 1H), 4.68–4.66 (m, 1H), 4.02 (t, J = 8.8 Hz, 1H), 3.83 (t, J = 7.6 Hz, 1H), 3.11 (dd, J = 13.6, 4.0 Hz, 1H), 2.98 (dd, J = 13.6, 5.7 Hz, 1H), 1.53 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 154.5, 152.4, 152.0 (d, J = 243.0 Hz), 133.5 (d, J = 10.1 Hz), 129.7 (d, J = 24.6 Hz), 122.9 (d, J = 10.1 Hz), 113.2, 105.9 (d, J = 24.6 Hz), and 81.1, 73.8, 47.6, 44.9, 28.3. HRMS (ESI): m/z calculated for C₁₅H₂₀FN₃NaO₄: 348.1336, found [M + Na]⁺ 348.1329. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −119.0° (c 0.20, CHCl₃).

3.2.8. tert-Butyl (S)-(4-(5-(acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)carbamate (13)

To a solution of 12 (978 mg, 3.0 mmol) in DCM (7 mL), TEA and Ac₂O (473 μL, 4.3 mmol) were added dropwise at 0 °C, and the mixture was allowed to warm to room temperature. After stirring for 0.5 h at room temperature, the solvent was removed in vacuo, and a colorless solid 13 was obtained (814 mg, quant.). M.P. 105–108 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.90–8.17 (bs, 1H), 7.55 (dd, J = 13.1, 2.4 Hz, 1H), 6.99 (dd, J = 8.9, 1.4 Hz, 1H), 6.69 (bs, 1H), 6.59 (t, J = 6.2 Hz, 1H), 4.80–4.76 (m, 1H), 4.02 (t, J = 8.9 Hz, 1H), 3.76 (dd, J = 9.1, 6.7 Hz, 1H), 3.68–3.63 (m, 2H), 2.03 (s, 3H), 1.53 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ171.4, 154.4, 152.5, 152.0 (d, J = 242.8 Hz), 133.1 (d, J = 10.5 Hz), 123.2 (d, J = 11.5 Hz), 113.5 (d, J = 2.8 Hz), 106.0 (d, J = 2.6 Hz), and 81.2, 72.0, 47.5, 41.9, 28.3, 23.0. HRMS (ESI): m/z calculated for C₁₇H₂₂FN₃NaO₅: 390.1441, found [M + Na]⁺ 390.1432. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −102.5° (c 0.20, CHCl₃).

3.2.9. (S)-N-((3-(4-Amino-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (14)

To a solution of 13 (332 mg, 0.9 mmol) in DCM (1 mL), TFA (1 mL) was added. After stirring for 1 h at room temperature, the solvent was removed in vacuo, and a colorless solid 14 was obtained (215 mg, 90%). M.P. 92–95 °C. ¹H-NMR (600 MHz, CD₃OD) δ 7.58 (dd, J = 12.7, 2.4 Hz, 1H), 7.49 (t, J = 8.6 Hz, 1H), 7.23–7.21 (m, 1H), 4.73–4.69 (m, 1H), 4.06 (t, J = 8.9 Hz, 1H), 3.73 (dd, J = 9.1, 6.4 Hz, 1H), 3.47–3.46 (m, 2H), 1.86 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ 172.8, 156.7, 156.1 (d, J = 37.5 Hz), 155.0 (d, J = 13.0 Hz), 138.5 (d, J = 10.1 Hz), 126.6, 118.3 (d, J = 13.0 Hz), 113.4 (d, J = 2.9 Hz), 106.0 (d, J = 24.6 Hz), 72.2, 41.8, 21.1.

3.2.10. (S)-N-((3-(3-Fluoro-4-((2-nitrophenyl)sulfonamido)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (5)

To a solution of 14 (59 mg, 0.2 mmol) in pyridine (Py) (1.5 mL), 2-nitrobenzenesulfonyl (Ns) chloride was added. After stirring for 1.5 h at room temperature, the solvent was concentrated under reduced pressure, and the mixture was diluted with CHCl₃. Saturated NH₄Cl was added to the mixture, and the organic materials were extracted with CHCl₃. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by C.C. (CHCl₃/MeOH = 40:1) to yield 5 as a yellow solid (91 mg, 92%). M.P. 98–100 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.92 (dd, J = 7.9, 1.0 Hz, 1H), 7.81 (dd, J = 7.9, 1.4 Hz, 1H), 7.75 (td, J = 7.7, 1.4 Hz, 1H), 7.63 (td, J = 7.7, 1.1 Hz, 1H), 7.54 (t, J = 8.6 Hz, 1H), 7.46 (dd, J = 12.4, 2.4 Hz, 1H), 7.10 (dt, J = 8.9, 1.4 Hz, 1H), 6.42 (t, J = 6.2 Hz, 1H), 4.80–4.78 (m, 1H), 4.03 (t, J = 9.1 Hz, 1H), 3.77 (dd, J = 9.1, 6.7 Hz, 1H), 3.68–3.63 (m, 2H), 2.01 (s, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ171.4, 155.8 (d, J = 247.1 Hz), 154.2, 147.9, 137.7 (d, J = 10.1 Hz), 134.3, 132.8, 132.6, 131.2, 127.5, 125.7, 119.0 (d, J = 14.5 Hz), 113.6, 113.6, 106.1 (d, J = 26.0 Hz), and 72.1, 47.4, 41.8, 23.1. HRMS (ESI): m/z calculated for C₁₈H₁₇FN₄NaO₇S: 475.0700, found [M + Na]⁺ 475.0705. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −70.5° (c 0.20, CHCl₃).

3.2.11. (S)-N-((3-(4-((N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-2-nitrophenyl)sulfonamido)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (15)

To a solution of 5 (113 mg, 0.25 mmol) in DMF (0.3 mL), (2-bromoethoxy)-tert-butyldimethylsilane (107 μL, 0.5 mmol) and K₂CO₃ (52 mg, 0.75 mmol) were added. After stirring for 2 h at room temperature and 48 h at 50 °C, the reaction mixture was diluted with water, and the organic material was extracted with hexane/EtOAc (1:1). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by C.C. (CHCl₃/MeOH = 40:1) to yield 15 as a yellow solid (137 mg, 86%). M.P. 88–90 °C. ¹H-NMR (600 MHz, CDCl₃) δ 6.80–6.77 (m, 2H), 6.70 (dd, J = 7.7, 0.9 Hz, 1H), 6.67–6.60 (m, 2H), 6.53 (t, J = 8.8 Hz, 1H), 6.22 (dd, J = 8.6, 2.1 Hz, 1H), 5.28 (t, J = 6.2 Hz, 1H), 3.90–3.88 (m, 1H), 3.15 (t, J = 9.1 Hz, 1H), 2.94–2.87 (m, 3H), 2.82–2.78 (m, 3H), 2.74–2.71 (m, 1H), 1.13 (s, 3H), -0.08 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 171.1,159.7 (d, J = 251.4 Hz), 154.0, 148.0, 139.7 (d, J = 10.2 Hz), 134.1, 133.7, 132.4, 131.3, 131.0, 124.0, 121.0 (d, J = 13.0 Hz), 113.1, 106.5 (d, J = 26.0 Hz), and 72.0, 61.1, 53.4, 47.4, 41.9, 25.8, 23.1, 18.1, -5.5. HRMS (ESI): m/z calculated for C₂₆H₃₅FN₄NaO₈SSi: 633.1827, found [M + Na]⁺ 633.1805. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −87.5° (c 0.20, CHCl₃).

3.2.12. (S)-N-((3-(3-Fluoro-4-((N-(2-hydroxyethyl)-2-nitrophenyl)sulfonamido)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (16)

To a solution of 15 (137 mg, 0.23 mmol) in THF (0.5 mL), Tetrabutylammonium fluoride (TBAF) (350 μL, 0.35 mmol) was added dropwise at 0 °C, and the mixture was allowed to warm to room temperature. After stirring for 2 h at room temperature, the solvent was removed in vacuo. The reaction mixture was diluted with water, and the organic material was extracted with EtOAc. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by C.C. (CHCl₃/MeOH = 10:1) to yield 16 as a yellow solid (99 mg, 85%). M.P. 102–104 °C. ¹H-NMR (600 MHz, CD₃OD) δ 7.80 (ddd, J = 8.3, 7.0, 1.1 Hz, 1H), 7.74 (dd, J = 7.9, 1.4 Hz, 1H), 7.70 (dd, J = 7.9, 1.4 Hz, 1H), 7.66 (td, J = 7.6, 1.1 Hz, 1H), 7.56 (dd, J = 13.1, 2.4 Hz, 1H), 7.43 (t, J = 8.6 Hz, 1H), 7.31–7.29 (m, 1H), 4.81–4.80 (m, 1H), 4.15 (t, J = 8.9 Hz, 1H), 3.82 (dd, J = 9.3, 6.2 Hz, 3H), 3.59–3.56 (m, 4H), 1.96 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ 174.1, 161.2 (d, J = 250.0 Hz), 156.2, 149.6, 142.1 (d, J = 11.5 Hz), 135.6, 135.0, 133.0, 132.6, 132.2, 125.3, 121.6 (d, J = 13.0 Hz), 114.8, 107.5 (d, J = 27.4 Hz), and 73.6, 60.8, 54.5, 49.9, 49.5, 49.3, 49.2, 49.0, 48.9, 48.8, 48.6, 43.1, 22.5. HRMS (ESI): m/z calculated for C₂₀H₂₂FN₄O₈S: 497.1142, found [M + H]⁺ 497.1154. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −64.5° (c 0.20, CH₃OH).

3.2.13. tert-Butyl (S)-2-(2-((N-(4-(5-(acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-2-nitrophenyl)sulfonamido)ethoxy)acetate (17)

To a solution of 16 (30 mg, 0.06 mmol) in DCM (1 mL), tert-butyl bromoacetate (13 μL, 0.09 mmol), tetrabutylammonium hydrogen sulfate (20 mg, 0.06 mmol), and 1 M NaOH (90 μL, 0.09mmol) were added. After stirring vigorously for 3 h at room temperature, 1 M NaOH (90 μL, 0.09 mmol) was added to the reaction mixture. After stirring vigorously for 1 h at room temperature, the reaction mixture was diluted with water, and the organic material was extracted with DCM. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by HPLC (CHCl₃/MeOH = 40:1) to yield 17 as a yellow solid (16 mg, 75% based on conversion yield, 43% recovered 16). ¹H-NMR (600 MHz, CDCl₃) δ 7.73–7.68 (m, 2H), 7.61 (dd, J = 7.9, 1.4 Hz, 1H), 7.58–7.52 (m, 2H), 7.47 (t, J = 8.6 Hz, 1H), 7.13 (dd, J = 8.9, 2.4 Hz, 1H), 6.17 (t, J = 6.0 Hz, 1H), 4.79 (q, J = 3.0 Hz, 1H), 4.06 (t, J = 8.9 Hz, 1H), 3.92 (s, 2H), 3.78 (dd, J = 9.3, 6.9 Hz, 1H), 3.73–3.66 (m, 3H), 3.64–3.60 (m, 1H), 2.04 (s, 3H), 1.46 (s, 9H), ¹³C-NMR (151 MHz, CDCl₃) δ 172.4, 160.8 (d, J = 248.7 Hz), 151.0 (d, J = 11.6 Hz), 147.9, 137.8, 133.7, 133.1, 132.3, 131.5, 131.4, 123.8, 123.2, 112.1 (d, J = 11.5 Hz), 109.9, 108.9, 99.5 (d, J = 24.6 Hz), and 69.1, 59.9, 59.0, 54.0, 46.5, 43.7, 29.7, 24.0, 23.1, 19.7, 13.7. HRMS (ESI): m/z calculated for C₂₆H₃₁FN₄NaO₁₀S: 633.1643, found [M + Na]⁺ 633.1651. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −77.0° (c 0.20, CHCl₃).

3.2.14. (S)-2-(2-((4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)amino)ethoxy)acetic acid (1)

To a solution of 17 (30 mg, 0.06 mmol) in DMF (300 μL), K₂CO₃ (30 mg, 0.22 mmol) and thiophenol (17 μL, 0.17 mmol) were added. After stirring for 6 h at room temperature, the reaction mixture was diluted with water, and the organic material was extracted with DCM. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by HPLC (CHCl₃/MeOH = 40:1) to yield a colorless solid (16 mg, 86%). To a solution of the solid (15 mg, 0.04 mmol) in DCM (440 μL), TFA was added. After stirring for 1 h at room temperature and concentrating under reduced pressure, a colorless solid was obtained. To a solution of the solid in water, 1 M NaOH (60 μL) was added dropwise. The crude solution was purified using Sep Pak C18 (H₂O-MeOH) to yield PNU-142300 (1) as a sodium salt (19 mg, 94%). M.P. 101–103 °C. ¹H-NMR (600 MHz, CD₃OD) δ 7.34 (dd, J = 13.4, 2.4 Hz, 1H), 7.03 (dt, J = 8.8, 1.3 Hz, 1H), 6.79 (t, J = 9.1 Hz, 1H), 4.75 (dd, J = 8.9, 6.2 Hz, 1H), 4.08 (t, J = 8.9 Hz, 1H), 3.89 (s, 2H), 3.75 (dd, J = 9.1, 6.4 Hz, 1H), 3.69 (t, J = 5.5 Hz, 2H), 3.54 (d, J = 4.8 Hz, 2H), 3.33 (t, J = 5.3 Hz, 2H), 1.97 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ 178.1, 174.1, 157.1, 153.4 (d, J = 238.4 Hz), 135.6 (d, J = 11.5 Hz), 129.0, 116.9, 113.5 (d, J = 4.3 Hz), and 108.5, 108.4, 73.4, 71.7, 70.3, 49.8, 44.5, 43.2, 22.5. HRMS (ESI): m/z calculated for C₁₆H₂₀FN₃NaO₆: 392.1234, found [M + H]⁺ 392.1219. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −63.0° (c 0.20, CH₃OH).

3.2.15. (S)-N-((3-(4-((N-(2-(Benzyloxy)ethyl)-2-nitrophenyl)sulfonamido)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (18)

To a solution of 5 (45 mg, 0.1 mmol) in DMF (0.5 mL), K₂CO₃ (21 mg, 0.15 mmol) and benzyl 2-bromoethyl ether (32 μL, 0.2 mmol) were added. After stirring overnight at 50 °C, the reaction mixture was diluted with water, and the organic material was extracted with DCM. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by C.C. (CHCl₃/MeOH = 30:1) to yield 18 as a colorless solid (137 mg, 93%). ¹H-NMR (600 MHz, CDCl₃) δ 7.72 (dd, J = 8.1, 1.2 Hz, 1H), 7.65 (td, J = 7.7, 1.4 Hz, 1H), 7.58 (dd, J = 8.1, 1.2 Hz, 1H), 7.51–7.48 (m, 2H), 7.35–7.25 (m, 4H), 7.22 (d, J = 6.5 Hz, 2H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.42 (t, J = 6.2 Hz, 1H), 4.79–4.75 (m, 1H), 4.43 (s, 2H), 4.02–3.96 (m, 2H), 3.75 (dd, J = 9.1, 6.7 Hz, 1H), 3.67 (ddd, J = 14.6, 6.0, 3.6 Hz, 1H), 3.62–3.56 (m, 3H), 2.01 (s, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ 171.3, 159.8 (d, J = 252.9 Hz), 154.0, 148.0, 139.8 (d, J = 10.1 Hz), 137.8, 133.8 (d, J = 8.6 Hz), 132.3, 131.4, 131.1, 128.3, 127.7, 127.7, 123.9, 120.5 (d, J = 11.5 Hz), 113.3, 106.4 (d, J = 26.0 Hz), and 72.8, 72.0, 67.9, 50.9, 47.4, 41.8, 23.1. HRMS (ESI): m/z calculated for C₂₇H₂₇FN₄NaO₈S: 609.1431, found [M + Na]⁺ 609.1418. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −85.5° (c 0.20, CHCl₃).

3.2.16. (S)-N-((3-(4-((2-(Benzyloxy)ethyl)amino)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (19)

To a solution of 18 (79 mg, 0.14 mmol) in DMF (0.5 mL), K₂CO₃ (75 mg, 0.54 mmol), and thiophenol (41 μL, 0.4 mmol) were added. After stirring for 6 h at room temperature, the reaction mixture was diluted with water, and the organic material was extracted with DCM. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by HPLC (CHCl₃/MeOH = 40:1) to yield a colorless solid 19 (15.6 mg, 86%). M.P. 80–83 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.37–7.30 (m, 6H), 6.97 (dq, J = 8.7, 1.2 Hz, 1H), 6.66 (t, J = 9.1 Hz, 1H), 6.23 (bs, 1H), 4.74 (t, J = 3.1 Hz, 1H), 4.56 (s, 2H), 4.19–4.31 (1H), 3.99 (t, J = 9.1 Hz, 1H), 3.72–3.68 (m, 4H), 3.61–3.57 (m, 1H), 3.33 (t, J = 4.6 Hz, 2H), 2.02 (s, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ13C-NMR (151 MHz, CHLOROFORM-D) δ 171.1, 154.6, 151.3 (d, J = 240.0 Hz), 137.9, 133.9 (d, J = 11.5 Hz), 128.5, 127.8, 127.8, 114.9, 112.2 (d, J = 4.4 Hz), 107.0 (d, J = 23.1 Hz), and 73.2, 71.8, 68.3, 48.0, 43.5, 42.0, 23.1. HRMS (ESI): m/z calculated for C₂₁H₂₄FN₃NaO₄: 424.1649, found [M + Na]⁺ 424.1671. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −89.5° (c 0.20, CHCl₃).

3.2.17. Ethyl (S)-N-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-N-(2-(benzyloxy)ethyl)glycinate (20)

To a solution of 19 (20 mg, 0.05 mmol) in acetonitrile (MeCN) (0.5 mL), K₂CO₃ (11 mg, 0.08 mmol) and ethyl bromoacetate (16 μL, 0.1 mmol) were added. After stirring for 2 days at 90 °C, the reaction mixture was diluted with water, and the organic material was extracted with DCM. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by HPLC (CHCl₃/MeOH = 40:1) to yield 20 a colorless solid (16 mg, 68%). ¹H-NMR (600 MHz, CDCl₃) δ 7.42–7.22 (m, 6H), 7.03–6.99 (m, 1H), 6.93 (t, J = 9.3 Hz, 1H), 6.44–6.38 (m, 1H), 4.76–4.69 (m, 1H), 4.49 (s, 2H), 4.16–4.07 (m, 3H), 3.99 (t, J = 9.1 Hz, 1H), 3.75–3.66 (m, 4H), 3.61–3.54 (m, 3H), 3.48 (s, 2H), 2.06–1.96 (m, 3H), 1.22 (t, J = 7.2 Hz, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ 171.2, 171.1, 154.4, 154.3 (d, J = 244.2 Hz), 138.2, 134.3 (d, J = 8.6 Hz), 131.4 (d, J = 10.1 Hz), 128.5, 128.4, 127.8, 127.6, 127.5, 120.1 (d, J = 4,2 Hz), 114.0, 107.8 (d, J = 26.0 Hz), 73.2, 71.9, 69.1, 60.7, 54.4 (d, J = 4.4 Hz), 52.5, 50.8, 47.7, 41.9, 23.1, 14.2. HRMS (ESI): m/z calculated for C₂₅H₃₀FN₃NaO₆: 510.2016, found [M + Na]⁺ 510.2015. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −86.0° (c 0.20, CHCl₃).

3.2.18. Ethyl (S)-N-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-N-(2-hydroxyethyl)glycinate (21)

To a solution of 20 (16 mg, 0.04 mmol) in THF (1 mL), Pd/C (20 mg) was added. After stirring for 24 h under a H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and a colorless solid 21 was obtained (20 mg, quant.). M.P. 75–78 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.40–7.37 (m, 1H), 7.02–7.00 (m, 2H), 6.77–6.77 (m, 1H), 4.76–4.75 (m, 1H), 4.20 (q, J = 7.1 Hz, 1H), 4.01–3.97 (m, 3H), 3.75–3.72 (m, 2H), 3.67–3.61 (m, 4H), 3.47 (t, J = 4.6 Hz, 2H), 2.01 (s, 3H), 1.27 (q, J = 7.1 Hz, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ 172.6, 171.4, 155.0 (d, J = 244.2 Hz), 154.5, 133.6 (d, J = 10.1 Hz), 132.3 (d, J = 10.1 Hz), 120.9 (d, J = 2.9 Hz), 114.0, 107.8 (d, J = 26.0 Hz), and 72.0, 61.3, 59.5, 55.5, 55.5, 47.6, 41.9, 23.0, 14.2. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −55.0° (c 0.20, CHCl₃).

3.2.19. (S)-N-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-N-(2-hydroxyethyl)glycine (2)

To a solution of 21 (40 mg, 0.01 mmol) in THF (1 mL), 1 M NaOH (300 μL) was added. After stirring for 2 h at room temperature, the supernatant was removed to obtain a colorless solid. A solution of the solid in water was purified by Sep Pak C18 (water, MeOH) to yield PNU-142586 (2) as a sodium salt (38 mg, 80%). M.P. 80–82 °C.¹H-NMR (600 MHz, CD₃OD) δ 7.37 (dd, J = 15.8, 2.7 Hz, 1H), 7.06–7.04 (m, 1H), 6.93 (t, J = 9.5 Hz, 1H), 4.75 (dd, J = 8.8, 6.4 Hz, 1H), 4.09 (t, J = 9.1 Hz, 1H), 3.79 (d, J = 1.4 Hz, 2H), 3.75 (dd, J = 9.3, 6.5 Hz, 1H), 3.66 (t, J = 5.2 Hz, 2H), 3.54–3.50 (m, 4H), 1.96 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ180.2, 174.1, 156.9, 154.8 (d, J = 242.7 Hz), 136.1, 131.7 (d, J = 10.1 Hz), and 120.1, 115.9, 109.2 (d, J = 27.5 Hz), 73.4, 60.9, 59.1, 56.9, 43.2, 22.5. HRMS (ESI): m/z calculated for C₁₆H₂₀FN₃NaO₆: 392.1234, found [M + H]⁺ 392.1252. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −52.0° (c 0.20, CH₃OH).

3.2.20. (S)-N-((3-(3-Fluoro-4-((2-hydroxyethyl)amino)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (3)

To a solution of 19 (15 mg, 0.04 mmol) in THF (1 mL), Pd/C (13 mg) was added. After stirring overnight under a H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and a colorless solid 3 was obtained (9 mg, 86%). M.P. 98–100 °C. ¹H-NMR (600 MHz, CD₃OD) δ 7.35 (dd, J = 13.6, 2.6 Hz, 1H), 7.03 (dq, J = 8.8, 1.2 Hz, 1H), 6.78 (t, J = 9.3 Hz, 1H), 4.76–4.74 (m, 1H), 4.07 (t, J = 8.9 Hz, 1H), 3.76–3.72 (m, 3H), 3.54 (d, J = 4.8 Hz, 2H), 3.26 (t, J = 5.7 Hz, 2H), 1.96 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ 174.1, 157.1, 152.5 (d, J = 238.4 Hz), 135.5 (d, J = 13.0 Hz), 129.0 (d, J = 10.1 Hz), 116.9, 113.3 (d, J = 4.4 Hz), 108.4 (d, J = 24.6 Hz), and 73.4, 61.4, 49.7, 49.4, 49.3, 49.1, 49.0, 48.9, 48.7, 48.6, 46.7, 43.2, 30.9, 22.4. HRMS (ESI): m/z calculated for C₁₄H₁₈FN₃NaO₄: 334.1179, found [M + Na]⁺ 334.1263. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −48.5° (c 0.20, CH₃OH).

3.2.21. Ethyl (S)-N-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)-N-((4-nitrophenyl)sulfonyl)glycinate (22)

To a solution of 5 (100 mg, 0.22 mmol) in DMF (0.2 mL), K₂CO₃ (45 mg, 0.33 mmol), and ethyl bromoacetate (49 μL) were added. After stirring overnight at 50 °C, the reaction mixture was diluted with water, and the organic material was extracted with hexane/EtOAc (1:1). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by C.C. (CHCl₃/MeOH = 20:1) to yield 22 as a colorless solid (112 mg, 95%). M.P. 78–80 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.73–7.66 (m, 4H), 7.60–7.53 (m, 2H), 7.12 (dd, J = 8.6, 2.1 Hz, 1H), 6.37 (s, 1H), 4.80–4.79 (m, 1H), 4.52 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 4.06 (t, J = 8.9 Hz, 1H), 3.80 (dd, J = 9.1, 6.7 Hz, 1H), 3.68–3.61 (m, 2H), 2.02 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ 171.3, 168.7, 159.4 (d, J = 251.4 Hz), 154.0, 147.8, 140.2 (d, J = 10.1 Hz), 134.4, 134.1, 132.4, 131.7, 131.3, 124.3, 120.8 (d, J = 13.0 Hz), 113.3, 106.4 (d, J = 26.0 Hz), and 72.0, 61.7, 52.5, 47.4, 41.9, 23.1, 14.1. HRMS (ESI): m/z calculated for C₂₂H₂₃FN₄NaO₉S: 561.1067, found [M + Na]⁺ 561.1073. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −138.0° (c 0.20, CHCl₃).

3.2.22. Ethyl (S)-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)glycinate (23)

To a solution of 22 (81 mg, 0.15 mmol) in DMF (0.2 mL), K₂CO₃ (83 mg, 0.6 mmol) and thiophenol (46 μL, 0.45 mmol) were added. After stirring overnight at room temperature, the reaction mixture was diluted with water, and the organic material was extracted with hexane/EtOAc (1:1). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was separated by HPLC (CHCl₃/MeOH = 1:1) to yield 23 a colorless solid (50 mg, 95%). M.P. 93–95 °C. ¹H-NMR (600 MHz, CDCl₃) δ 7.36 (dd, J = 13.1, 2.4 Hz, 1H), 6.96 (dq, J = 8.7, 1.2 Hz, 1H), 6.88 (t, J = 6.0 Hz, 1H), 6.54 (t, J = 8.9 Hz, 1H), 4.76–4.73 (m, 1H), 4.24 (q, J = 7.1 Hz, 2H), 3.98 (t, J = 8.9 Hz, 1H), 3.92 (s, 2H), 3.73–3.69 (m, 2H), 3.65–3.58 (m, 2H), 2.01 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H), ¹³C-NMR (151 MHz, CDCl₃) δ 171.5, 170.7, 154.8, 151.1 (d, J = 239.8 Hz), 132.7 (d, J = 11.6 Hz), 128.5 (d, J = 8.6 Hz), 114.9 (d, J = 2.9 Hz), 112.1 (d, J = 4.4 Hz), 107.1 (d, J = 23.2 Hz), and 72.0, 61.5, 48.0, 45.5, 41.9, 23.0, 14.2. HRMS (ESI): m/z calculated for C₁₆H₂₀FN₃NaO₅: 376.1285, found [M + Na]⁺ 376.1277. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −36.0° (c 0.20, CHCl₃).

3.2.23. (S)-(4-(5-(Acetamidomethyl)-2-oxooxazolidin-3-yl)-2-fluorophenyl)glycine (4)

To a solution of 23 (18 mg, 0.05 mmol) in THF (1 mL), 1M NaOH (79 μL) was added. After stirring for 2 h at room temperature, MeOH was added, and the reaction mixture was subjected to PTLC (CHCl₃/MeOH = 1:1) to yield PNU-173558 (4) as a sodium salt (15 mg, 95%). M.P. 118–120 °C. ¹H-NMR (600 MHz, CD₃OD) δ 7.36 (dd, J = 13.4, 2.4 Hz, 1H), 7.01 (dt, J = 8.7, 1.3 Hz, 1H), 6.64 (t, J = 9.3 Hz, 1H), 4.76–4.74 (m, 1H), 4.08 (t, J = 9.1 Hz, 1H), 3.75 (dd, J = 9.1, 6.4 Hz, 1H), 3.65 (s, 2H), 3.54 (d, J = 5.2 Hz, 2H), 1.97 (s, 3H), ¹³C-NMR (151 MHz, CD₃OD) δ 177.6, 174.0, 170.4, 157.1, 153.3 (d, J = 236.9 Hz), 135.5 (d, J = 13.0 Hz), 128.7 (d, J = 10.1 Hz), 116.9, 113.1 (d, J = 4.4 Hz), 108.3 (d, J = 24.4 Hz), and 73.4, 49.8, 48.4, 43.2, 30.8, 22.4. HRMS (ESI): m/z calculated for C₁₄H₁₆FN₃NaO₅: 348.0972, found [M + H]⁺ 348.0986. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −69.5° (c 0.20, CH₃OH).

4. Conclusions

In conclusion, we presented a novel synthesis route for linezolid metabolites using the Ns strategy. Four linezolid metabolites were synthesized from a common intermediate (5), which is Ns-protected aniline derivative obtained by Curtius rearrangement reaction of oxazolidinone derivatives. The protection of aromatic amines by the Ns group was effective in preparing the desired secondary and tertiary amine derivatives. This is the first report on the synthesis of PNU-142618 and PNU-173558. The linezolid metabolites obtained in this study are currently being used in clinical analysis of the pharmacokinetics of these metabolites, and the results will be reported in due course. The results in this study not only contribute to the pharmacokinetic and toxicological studies of linezolid metabolites but also suggest that the morpholine ring of linezolid can be replaced with various other substituents, and it is expected that novel linezolid derivatives with antimicrobial activity will be developed.