Synthesis of Linezolid Metabolites PNU-142300 and PNU-142586 toward the Exploration of Metabolite-Related Events

Abstract

Linezolid (1) is an oxazolidinone antibiotic that is partially metabolized in vivo via ring cleavage of its morpholine moiety to mainly form two metabolites, PNU-142300 (2) and PNU-142586 (3). It is supposed that accumulation of 2 and 3 in patients with renal insufficiency may cause thrombocytopenia, one of the adverse effects of linezolid. However, the poor availability of 2 and 3 has hindered further investigation of the clinical significance of the accumulation of these metabolites. In this paper, we synthesized metabolites 2 and 3 via a common synthetic intermediate, 4; this will encourage further exploration of events related to these metabolites and lead to improved clinical use of linezolid.

Linezolid (1) is a novel antimicrobial agent that possesses an oxazolidinone moiety; it exhibits a broad antibacterial spectrum against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE)^1–5) (Chart 1). Linezolid selectively binds to the 50s subunit of bacterial ribosomes to prevent the formation of the 70s ribosome, resulting in inhibition of bacterial protein synthesis.⁶⁾

The major adverse event associated with linezolid treatment is reversible myelosuppression, which may cause anemia and thrombocytopenia. Wu et al.,⁷⁾ Lin et al.,⁸⁾ and Matsumoto et al.⁹⁾ independently reported that linezolid-associated thrombocytopenia is induced more frequently in patients with renal insufficiency than in those with normal renal function. Brier et al. examined the pharmacokinetics of linezolid (1) and its major metabolites, PNU-142300 (2) and PNU-142586 (3).¹⁰⁾ It was found that clearance of linezolid (1) was not affected by the renal function of the patients and that 2 and 3 accumulated in the blood of patients with renal insufficiency. The toxicity of metabolites 2 and 3 has not been fully elucidated; it is suspected that accumulation of 2 and 3 leads to thrombocytopenia in patients with renal insufficiency. Unfortunately, the poor availability of 2 and 3 has hindered further investigation of the clinical significance of the accumulation of these metabolites. In addition, these metabolites are known to be P450-independent,^5,11) and P450 enzyme-mediated direct synthesis of 2 and 3 on a preparative scale is likely impossible. Herein, we synthesized the major metabolites 2 and 3 via a common synthetic intermediate, which may encourage the further exploration of metabolite-related events and improve the clinical use of linezolid.

Chart 1. Linezolid (1) and Its Major Metabolites, 2 and 3

Results and Discussion

For the convenient synthesis of metabolites 2 and 3, aminoalcohol derivative 4, bearing tert-butyldimethylsilyl (TBS) and benzyl (Bn) groups was designed as a common synthetic intermediate (Chart 2). We planned the selective removal of the protecting groups in 4 and subsequent alkylation with bromoacetate derivatives to form 2 and 3, respectively.

Chart 2. The Common Synthetic Intermediate 4 of 2 and 3

The synthesis of 4 was conducted by a procedure similar to the linezolid synthesis developed by Brickner et al.¹²⁾ Nucleophilic substitution of difluoride 5 with N-benzylethanolamine and subsequent protection of the alcohol with a TBS group gave 6 (Chart 3). In the nucleophilic substitution step, high temperature was required due to the higher bulkiness and lower reactivity of N-benzylethanolamine compared to morpholine in Brickner’s linezolid synthesis. Reduction of the nitro group with hydrogen in the presence of a palladium catalyst caused undesired debenzylation. Alternatively, complete selectivity in the reduction of the nitro group was achieved by using copper hydride.¹³⁾ The oxazolidinone ring was constructed from carbamate 8 and a glycidyl ester according to Manninen and Brickner’s procedure.¹⁴⁾ Finally, successive mesylation and azidation provided aminoalcohol derivative 4 in moderate yield.

Chart 3. Synthesis of Common Synthetic Intermediate 4

(a) N-Benzylethanolamine, N,N-diisopropylethylamine, ⁿBuOH, reflux, 81%. (b) TBSCl, imidazole, CH₂Cl₂, r.t., 94%. (c) Cu(acac)₂, NaBH₄, THF, EtOH, r.t., 95%. (d) CbzCl, NaHCO₃, THF, quant. (e) (R)-Glycidyl butyrate, ⁿBuLi, THF, −78°C to r.t., 71%. (f) MsCl, Et₃N, DMAP, CH₂Cl₂, r.t. (g) NaN₃, DMF, 50°C, 75% for 2 steps.

For PNU-142300 (2), O-alkylation was attempted after removal of the TBS group in 4. Using sodium hydride (NaH) as a base and benzyl bromoacetate as the alkylating agent in N,N-dimethylformamide (DMF) and tetrahydrofuran (THF), O-acylation mainly occurred, and a negligible amount of the desired O-alkylated product 10 was obtained. When a bulkier alkylating agent, tert-butyl bromoacetate, was applied to suppress O-acylation, the reaction was sluggish, and the desired product 10 was obtained in less than 10% yield (Chart 4). Alternatively, O-alkylation in the presence of excess tert-butyl bromoacetate under biphasic conditions furnished 10 quantitatively. Successive Staudinger reaction and acetylation followed by the stepwise removal of the protecting groups provided trifluoroacetic acid (TFA) salt of PNU-142300 (2). TFA salt of 2 was gradually decomposed in a solution via acid-catalyzed cyclization and was neutralized to obtain 2.

Chart 4. Synthesis of PNU-142300 (2)

(a) TBAF, THF, r.t., 99%. (b) BrCH₂CO₂^tBu, ⁿBu₄NHSO₄, NaOH, toluene, H₂O, r.t., quant. (c) PPh₃, H₂O, THF, r.t. (d) AcCl, Et₃N, CH₂Cl_2, r.t. (e) H₂, Pd(OH)₂/C, THF, r.t., 83% for 3 steps. (f) TFA, CH₂Cl₂, r.t. (g) NaOH, H₂O, r.t., 73% for 2 steps.

PNU-142586 (3) was prepared as illustrated in Chart 5. The Staudinger reaction, acetylation, and debenzylation of 4 were conducted to afford 12. N-Alkylation progressed to afford 13 as an inseparable mixture with unreacted amine 12. After removal of the benzyl group on 13, the resulting carboxylic acid 14 was easily separated from the contaminant 12. Carboxylic acid 14 was unstable under storage even at −20°C, owing to cleavage of the TBS ether and successive lactonization due to its acidity. Thus, 14 was immediately neutralized with tetrabutylammonium hydroxide to afford its tetrabutylammonium salt, whose TBS group was removed by treatment with tetrabutylammonium fluoride (TBAF) to furnish the tetrabutylammonium salt of PNU-142586 (3). In contrast, when the TBS group on 13 was removed first with TBAF, the generated alkoxide attacked the adjacent ester moiety to form lactone 15 as a major product (Chart 6).

Chart 5. Synthesis of PNU-142586 (3)

(a) PPh₃, H₂O, THF, r.t. (b) AcCl, Et₃N, CH₂Cl₂, r.t. (c) H₂, Pd(OH)₂/C, THF, r.t., 55% for 3 steps. (d) BrCH₂CO₂Bn, K₂CO₃, NaI, acetonitrile, reflux, 88%. (e) H₂, Pd(OH)₂/C, THF, r.t., 55%. (f) ⁿBu₄NOH, H₂O, r.t. (g) TBAF, THF, r.t., 66% for 2 steps.

Chart 6. Cyclic Byproduct 15

Finally, the purity of 2 and 3 was evaluated by HPLC analysis (Chart 7) and was proved to be well-tolerated for reference standards for HPLC analysis.

Chart 7. HPLC Analysis Data of (a) 2 (t_R 6.23 min) and (b) 3 (t_R 6.21 min)

Conditions: column, SHISEIDO Capcell Pak C18 MG-II (4.6×250 mm); mobile phase, acetonitrile–0.1% TFA in H₂O, 10 : 90; flow rate, 1.0 mL/min; column temperature, 40°C; detect, 254 nm.

In conclusion, we have successfully prepared linezolid metabolites 2 and 3 via a common synthetic intermediate, 4. The establishment of a synthetic method for 2 and 3 will contribute to further exploration of the pharmacokinetics of these metabolites in patients with renal insufficiency, which may elucidate the roles of linezolid metabolites in thrombocytopenia.

Experimental

General Remarks

All reagents and solvents purchased were of the highest commercial quality and were used without further purification. ¹H-NMR spectra were measured at 400 MHz on a VARIAN 400-MR spectrometer or at 500 MHz on an Agilent INOVA-500 spectrometer, and ¹³C-NMR spectra were measured at 100 MHz on a VARIAN 400-MR spectrometer or at 125 MHz on an Agilent INOVA-500 spectrometer. High resolution mass spectra were recorded using a Jeol JMS-T100LP AccuTOF instrument. IR spectra were recorded using a JASCO FT/IR-4700 spectrometer in attenuated total reflection (ATR) mode at room temperature. Silica gel column chromatography was performed using Silica Gel 60 (spherical and neutral; 100 to 210 µm, 37560–79, Kanto Chemical Co., Japan) and the Isolera One flash purification system (Biotage, Sweden).

Synthesis

2-[N-Benzyl-N-(2-fluoro-4-nitrophenyl)amino]ethanol (16)

To a solution of 3,4-difluoronitrobenzene (5) (2983 mg, 18.75 mmol) in n-butanol (10 mL) were added N-benzylethanolamine (2959 µL, 20.74 mmol) and N,N-diisopropylethylamine (3613 µL, 20.74 mmol). After stirring overnight at reflux temperature, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in AcOEt (60 mL), and the solution was washed with water (40 mL×2) and brine (40 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was purified using the Isolera One system (hexane/AcOEt) to afford 16 as a yellow oil (4435 mg, 81%). ¹H-NMR (400 MHz, CDCl₃/tetramethylsilane (TMS)) δ: 3.66 (2H, dt, J=1.9, 5.7 Hz), 3.89 (1H, dt, J=0.8, 5.7 Hz), 4.71 (2H, s), 6.80 (1H, dd, J=9.0, 9.0 Hz), 7.21–7.35 (5H, m), 7.84 (1H, ddd, J=0.7, 2.5, 9.0 Hz), 7.87 (1H, dd, J=2.5, 14.2 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: 54.2 (d, J=6.9 Hz), 56.6 (d, J=3.8 Hz), 70.0 (d, J=2.3 Hz), 113.2 (d, J=27.5 Hz), 116.4 (d, J=4.6 Hz), 121.2 (d, J=2.3 Hz), 126.8, 127.6, 128.9, 136.7, 138.3 (d, J=8.4 Hz), 143.9 (d, J=6.9 Hz), 150.8 (d, J=246 Hz); IR (neat) cm⁻¹: 3399, 2925, 2880, 1599, 1519, 1494, 1317, 1278, 1230, 1071; Electrospray ionization (ESI)-MS m/z: 603.2026 (Calcd for C₃₀H₃₀F₂N₄NaO₆ (2M+Na): 603.2031).

N-Benzyl-N-[2-(tert-butyldimethylsilyloxy)ethyl]-2-fluoro-4-nitroaniline (6)

To a solution of 5 (4435 mg, 15.28 mmol) and imidazole (1249 mg, 18.35 mmol) in CH₂Cl₂ (40 mL) was added tert-butyldimethylsilyl chloride (2543 mg, 16.87 mmol) at 0°C. After stirring for 40 min at room temperature, the reaction mixture was washed with 0.1 M phosphate buffer (pH 7, 40 mL), water (40 mL), and brine (40 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford 6 as a yellow oil (5780 mg, 94%). ¹H-NMR (400 MHz, CDCl₃/TMS) δ: 0.01 (6H, s), 0.89 (9H, s), 3.49 (2H, dt, J=2.0, 5.5 Hz), 3.86 (2H, t, J=5.5 Hz), 4.73 (2H, s), 6.79 (1H, dd, J=8.8, 9.0 Hz), 7.21–7.36 (5H, m), 7.85 (1H, dd, J=2.7, 8.8 Hz), 7.89 (1H, dd, J=2.7, 14.3 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.5, 18.1, 25.8, 54.7 (d, J=6.8 Hz), 56.5 (d, J=3.8 Hz), 61.8 (d, J=2.3 Hz), 113.2 (d, J=27.4 Hz), 116.1 (d, J=4.5 Hz), 121.2 (d, J=2.3 Hz), 126.7, 127.4, 128.8, 137.0, 137.9 (d, J=8.4 Hz), 143.9 (d, J=6.9 Hz), 150.4 (d, J=245.7 Hz). IR (neat) cm⁻¹: 2952, 2927, 2855, 1601, 1519, 1501, 1281, 1237, 1093, 1072. ESI-MS m/z: 427.1852 (Calcd for C₂₁H₂₉FN₂NaO₃Si: 427.1829).

4-Amino-N-benzyl-N-[2-(tert-butyldimethylsilyloxy)ethyl]-2-fluoroaniline (7)

To a solution of NaBH₄ (603 mg, 1.23 mmol) and copper(II) acetylacetonate (Cu(acac)₂) (257 mg, 0.981 mmol) in EtOH (3 mL) was added a solution of 6 (643 mg, 1.59 mmol) in THF (3 mL). After stirring for 30 min at room temperature, sat. NaHCO₃ solution (20 mL) was added, and the mixture was stirred for 20 min. The organic materials were extracted with AcOEt (20 mL ×3). The organic layer was washed with brine (20 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to obtain 7 as a purple oil (563 mg, 95%). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: −0.03 (6H, s), 0.85 (9H, s), 3.15 (2H, t, J=6.6 Hz), 3.64 (2H, t, J=6.6 Hz), 4.25 (2H, s), 6.29 (1H, ddd, J=0.8, 2.7, 8.8 Hz), 6.38 (1H, d, J=2.7, 13.4 Hz), 6.81 (1H, dd, J=8.8, 8.8 Hz), 7.18–7.31 (5H, m). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.4, 18.3, 25.9, 54.8 (d, J=2.2 Hz), 58.7 (d, J=1.5 Hz), 61.5, 103.8 (d, J=24.4 Hz), 110.6 (d, J=2.3 Hz), 124.6 (d, J=4.6 Hz), 126.8, 128.1, 128.4, 129.9 (d, J=9.9 Hz), 139.3, 142.6 (d, J=10.7 Hz), 157.9 (d, J=244.9 Hz). IR (neat) cm⁻¹: 3461, 3373, 2927, 2855, 1511, 1254, 1092. ESI-MS m/z: 397.2075 (Calcd for C₂₁H₃₁FN₂NaOSi: 397.2087).

N-Benzyl-4-benzyloxycarbonylamino-N-[2-(tert-butyldimethylsilyloxy)ethyl]-2-fluoroaniline (8)

To a solution of 7 (501 mg, 1.34 mmol) and NaHCO₃ (226 mg, 2.69 mmol) in THF (3 mL) was added a solution of benzylchloroformate (248 mg, 1.45 mmol) in THF (0.5 mL). After stirring for 2 h at room temperature, benzylchloroformate (50 mg, 0.29 mmol) was added, and the mixture was stirred for another 30 min. Water (20 mL) was added to the mixture, and the organic materials were extracted with AcOEt (20 mL). The organic layer was washed with brine (20 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to afford 8 as a colorless oil (563 mg, quant.). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: −0.02 (6H, s), 0.85 (9H, s), 3.28 (2H, t, J=6.4 Hz), 3.69 (2H, t, J=6.4 Hz), 4.39 (2H, s), 5.18 (2H, s), 6.51 (1H, br), 6.82–6.87 (2H, m), 7.19–7.41 (10H, m). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.5, 18.2, 25.9, 54.2 (d, J=3.1 Hz), 57.5 (d, J=2.3 Hz), 61.5, 67.0, 108.1, 114.4, 121.5, 126.9, 128.0, 128.2, 128.3, 128.4, 128.6, 131.8, 134.3, 136.0, 138. 8, 153.4, 155.6 (d, J=244.1 Hz). IR (neat) cm⁻¹: 2952, 2927, 2854, 1732, 1702, 1515, 1254, 1209, 1091. ESI-MS m/z: 531.2447 (Calcd for C₂₉H₃₇FN₂NaO₃Si: 531.2455).

N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-(5R)-hydroxymethyl-2-oxazolidinone (9)

To a solution of 8 (2550 mg, 5.196 mmol) in THF (10 mL) was added n-butyllithium in hexane (1.56 M, 3.7 mL) dropwise over 20 min at −78°C. After stirring for 1 h at −78°C, (R)-glycidyl butyrate (99% enantiomeric excess (ee), [α]^D₂₂ 29.7 (c=1.0, CHCl₃)) (792 µL, 5.72 mmol) was added, and the mixture was allowed to warm to room temperature. After stirring overnight at room temperature, 0.1 M phosphate buffer (pH 7) was added to the mixture, and the organic materials were extracted with AcOEt (30 mL). The organic layer was washed with brine (30 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to afford 9 as a yellow oil (1689 mg, 71%). ¹H-NMR (400 MHz, CDCl₃/TMS) δ: −0.02 (6H, s), 0.85 (9H, s), 3.33 (2H, t, J=6.2 Hz), 3.71 (2H, t, J=6.2 Hz), 3.70–3.73 (1H, m), 3.89 (1H, dd, J=7.0, 8.4 Hz), 3.95 (2H, dd, J=8.8, 8.8 Hz), 4.43 (2H, s), 4.67–4.73 (1H, m), 6.89 (1H, dd, J=9.0, 9.0 Hz), 6.99 (1H, dd, J=2.7, 9.0 Hz), 7.19–7.28 (5H, m), 7.38 (1H, dd, J=2.7, 14.8 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.5, 18.2, 25.9, 46.5, 54.3 (d, J=3.8 Hz), 57.2 (d, J=2.2 Hz), 61.5, 62.8, 72.8, 107.7 (d, J=26.7 Hz), 113.8 (d, J=3.1 Hz), 120.9 (d, J=4.6 Hz), 126.9, 127.8, 128.3, 131.6 (d, J=10.0 Hz), 134.8 (d, J=9.1 Hz), 138.7, 154.7, 155.1 (d, J=244.9 Hz). IR (neat) cm⁻¹: 3400, 2854, 1736, 1514, 1218, 1087. ESI-MS m/z: 429.2238 (Calcd for C₂₅H₃₅FN₂NaO₄Si: 497.2248).

N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-(5R)-methanesulfonyloxymethyl-2-oxazolidinone (17)

To a solution of 9 (413 mg, 0.904 mmol), Et₃N (151 µL, 1.09 mmol), and N,N-dimethyl-4-aminopyridine (DMAP) (10 mg, 0.082 mmol) in CH₂Cl₂ (3 mL), was added methanesulfonyl chloride (78 µL, 1.0 mmol). After stirring overnight at room temperature, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in AcOEt (30 mL), and the solution was washed with water and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford 17 as a yellow oil (526 mg, quant.). The crude 17 was used in the next step without further purification. ¹H-NMR (500 MHz, CDCl₃/TMS) δ: −0.01 (6H, s), 0.85 (9H, s), 3.08 (3H, s), 3.35 (2H, t, J=6.1 Hz), 3.73 (2H, t, J=6.1 Hz), 3.86 (1H, dd, J=6.1, 9.1 Hz), 4.06 (1H, dd, J=9.1, 9.1 Hz), 4.39 (1H, dd, J=4.4, 11.7 Hz), 4.45 (2H, s), 4.46 (1H, dd, J=3.6, 11.7 Hz), 4.85–4.89 (1H, m), 6.90 (1H, dd, J=9.0, 9.0 Hz), 6.97 (1H, dd, J=2.7, 9.0 Hz), 7.19–7.29 (5H, m), 7.37 (1H, dd, J=2.7, 14.5 Hz).

N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-(5R)-azidemethyl-2-oxazolidinone (4)

To a solution of crude 17 (526 mg) in DMF (3 mL) was added sodium azide (88 mg, 1.4 mmol). After stirring overnight at 50°C, the mixture was diluted with AcOEt (20 mL), and the solution was washed with water and brine. The organic layer was washed with brine (30 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to afford 4 as a yellow oil (340 mg, 75% for 2 steps from 9). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 0.00 (6H, s), 0.86 (9H, s), 3.34 (2H, t, J=6.1 Hz), 3.54 (1H, dd, J=4.6, 13.2 Hz), 3.66 (1H, dd, J=4.6, 13.2 Hz), 3.73 (2H, t, J=6.1 Hz), 3.76 (1H, dd, J=6.4, 9.1 Hz), 3.99 (1H, dd, J=9.1, 9.1 Hz), 4.44 (2H, s), 4.71–4.75 (1H, m), 6.91 (1H, dd, J=9.1, 9.1 Hz), 6.98 (1H, dd, J=2.7, 9.1 Hz), 7.20–7.31 (5H, m), 7.39 (1H, dd, J=2.7, 14.7 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ: −5.4, 18.2, 25.9, 47.5, 53.0, 54.4 (d, J=3.9 Hz), 57.1, 61.5, 70.6, 107.7 (d, J=26.9 Hz), 113.8 (d, J=2.8 Hz), 120.9 (d, J=4.8 Hz), 127.0, 127.8, 128.3, 131.3 (d, J=10.5 Hz), 135.0 (d, J=9.6 Hz), 138.7, 153.9, 155.0 (d, J=244.8 Hz). IR (neat) cm⁻¹: 2952, 2928, 2855, 2104, 1747, 1515, 1217, 1086. ESI-MS m/z: 522.2308 (Calcd for C₂₅H₃₄FN₅NaO₃Si: 522.2313).

N-[4-[Benzyl(2-hydroxyethyl)amino]-3-fluorophenyl]-(5R)-azidemethyl-2-oxazolidinone (18)

To a solution of 4 (302 mg, 0.604 mmol) in THF (4 mL) was added 1.0 M tetrabutylammonium fluoride in THF (910 µL, 0.910 mmol). After stirring for 30 min at room temperature, the solution was diluted with AcOEt (40 mL), and the solution was washed with water and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to afford 18 as a yellow oil (231 mg, 99%). ¹H-NMR (400 MHz, CDCl₃/TMS) δ: 3.28 (2H, t, J=5.3 Hz), 3.58 (1H, dd, J=4.3, 13.3 Hz), 3.63 (2H, t, J=5.3 Hz), 3.70 (1H, dd, J=4.7, 13.3 Hz), 3.81 (1H, dd, J=6.3, 9.0 Hz), 4.03 (1H, dd, J=9.0, 9.0 Hz), 4.31 (2H, s), 4.74–4.80 (1H, m), 6.97 (1H, dd, J=9.0, 9.0 Hz), 7.06 (1H, ddd, J=1.0, 2.5, 9.0 Hz), 7.21–7.31 (5H, m), 7.44 (1H, dd, J=2.5, 13.9 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: 47.3, 53.0, 54.3, 57.8, 59.6, 70.8, 107.5 (d, J=26.7 Hz), 113.7 (d, J=3.1 Hz), 122.8 (d, J=4.6 Hz), 127.2, 128.0, 128.3, 128.4, 133.1 (d, J=9.9 Hz), 134.3 (d, J=9.9 Hz), 138.1, 154.0, 156.4 (d, J=245.6 Hz). IR (neat) cm⁻¹: 3449, 2930, 2893, 2103, 1737, 1514, 1212. ESI-MS m/z: 408.1457 (Calcd for C₁₉H₂₀FN₅NaO₃: 408.1448).

tert-Butyl [2-[[4-[(5R)-Azidemethyl-2-oxazolidinon-3-yl]-2-fluorophenyl]benzylamino]ethoxy]acetate (10)

To a solution of 18 (357 mg, 0.926 mmol) in toluene (5.5 mL) were added tert-butyl bromoacetate (997 µL, 7.41 mmol), tetrabutylammonium hydrogen sulfate (251 mg, 0.741 mmol), and 5 M NaOH in water (18 mL). After stirring vigorously for 2 h at room temperature, the organic layer was separated. The organic materials were further extracted with AcOEt (30 mL×2). The combined organic layer was washed with water and brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude mixture was purified using the Isolera One system (hexane/AcOEt) to afford 10 as a yellow oil (469 mg, quant.). ¹H-NMR (400 MHz, CDCl₃/TMS) δ: 1.46 (9H, s), 3.43 (2H, t, J=6.0 Hz), 3.58 (1H, dd, J=4.5, 13.3 Hz), 3.64 (2H, t, J=6.0 Hz), 3.68 (1H, dd, J=4.7, 13.3 Hz), 3.79 (1H, dd, J=6.2, 9.0 Hz), 3.89 (2H, s), 4.02 (1H, dd, J=9.0, 9.0 Hz), 4.44 (2H, s), 4.72–4.78 (1H, m), 6.93 (1H, dd, J=9.0, 9.0 Hz), 6.99 (1H, dd, J=2.5, 9.0 Hz), 7.19–7.30 (5H, m), 7.31 (1H, dd, J=2.5, 14.5 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: 28.1, 47.4, 51.7 (d, J=3.8 Hz), 53.0, 57.1, 69.0, 69.8, 70.6, 81.5, 107.6 (d, J=26.7 Hz), 113.8 (d, J=3.8 Hz), 121.4 (d, J=4.6 Hz), 127.0, 128.0, 128.3, 131.8 (d, J=9.9 Hz), 134.7 (d, J=9.1 Hz), 138.4, 153.9, 155.3 (d, J=244.7 Hz), 169.5. IR (neat) cm⁻¹: 2977, 2931, 2897, 2103, 1740, 1514, 1223, 1129. ESI-MS m/z: 522.2121 (Calcd for C₂₅H₃₀FN₅NaO₅: 522.2129).

tert-Butyl [2-[[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-fluorophenyl]benzylamino]ethoxy]acetate (11)

To a solution of 10 (358 mg, 0.717 mmol) in THF/H₂O (10 : 1, 3.3 mL) was added triphenylphosphine (210 mg, 0.800 mmol). After stirring for 1 h at room temperature, H₂O (0.3 mL) was added to the solution, and the solution was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH₂Cl₂ (4 mL), and Et₃N (150 µL, 1.08 mmol) and acetylchloride (67 µL, 0.93 mmol) were added to the solution. After stirring for 25 min at room temperature, the solution was concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 100 : 0 to 100 : 1) to afford an inseparable mixture of 11 and triphenylphosphine oxide (100 : 1) as a colorless amorphous solid (339 mg). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 1.46 (9H, s), 2.02 (3H, s), 3.42 (2H, t, J=6.1 Hz), 3.56 (1H, ddd, J=6.1, 6.1, 14.7 Hz), 3.64 (2H, t, J=6.1 Hz), 3.69 (1H, dd, J=6.6, 9.0 Hz), 3.69–3.74 (1H, m), 3.89 (2H, s), 3.99 (1H, dd, J=9.0, 9.0 Hz), 4.44 (2H, s), 4.72–4.77 (1H, m), 5.97 (1H, dd, J=6.1, 6.1 Hz), 6.92 (1H, dd, J=8.8, 8.8 Hz), 6.95 (1H, dd, J=2.5, 8.8 Hz), 7.19–7.29 (5H, m), 7.40 (1H, dd, J=2.5, 14.4 Hz).

tert-Butyl [2-[(4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-fluorophenyl)amino]ethoxy]acetate (19)

To a solution of a 100 : 1 mixture of 11 and triphenylphosphine oxide (339 mg) in THF (3 mL) was added 20% Pd(OH)₂/C (92 mg). After stirring for 3.5 h under H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 200 : 1 to 100 : 1) to afford 19 as a colorless amorphous solid (253 mg, 83% based on 10). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 1.49 (9H, s), 2.05 (3H, s), 3.35 (2H, dd, J=4.6, 9.1 Hz), 3.58 (1H, ddd, J=6.1, 6.1, 12.2 Hz), 3.70 (1H, dd, J=5.8, 9.1 Hz), 3.69–3.74 (1H, m), 3.78 (2H, t, J=9.1 Hz), 4.00 (1H, dd, J=9.1, 9.1 Hz), 4.01 (2H, s), 4.46 (1H, br), 4.72–4.77 (1H, m), 6.08 (1H, dd, J=6.1, 6.1 Hz), 6.67 (1H, dd, J=8.8, 8.8 Hz), 6.98 (1H, dd, J=2.4, 8.8 Hz), 7.35 (1H, dd, J=2.4, 13.2 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: 22.9, 28.1, 41.9, 43.5, 48.0, 68.7, 69.8, 71.9, 81.8, 107.1 (d, J=23.7 Hz), 111.9 (d, J=4.5 Hz), 115.0 (d, J=3.0 Hz), 127.7 (d, J=9.1 Hz), 133.8 (d, J=11.6 Hz), 151.1 (d, J=239.6 Hz), 154.8, 169.6, 171.4. IR (neat) cm⁻¹: 3292, 2978, 2931, 1733, 1654, 1526, 1255, 1129. ESI-MS m/z: 448.1871 (Calcd for C₂₀H₂₈FN₃NaO₆: 448.1860).

[2-[[4-[(5S)-Acetylaminomethyl-2-oxazolidinone-3-yl]-2-fluorophenyl]amino]ethoxy]acetic Acid (2)

To a solution of 19 (50 mg, 0.12 mmol) in CH₂Cl₂ (1.8 mL) was added TFA (200 µL, 2.61 mmol). After stirring for 4 h at room temperature, the solution was concentrated under reduced pressure. The crude mixture was dissolved in H₂O (1.8 mL). The solution was neutralized with aqueous NaOH solution and was washed with CHCl₃. The aqueous layer was concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 5 : 1 to 1 : 1) and using the Isolera One system (MeOH/H₂O) to afford 2 as a gray amorphous solid (32 mg, 73%). ¹H-NMR (500 MHz, CD₃OD) δ: 1.97 (3H, s), 3.34 (2H, br), 3.54 (2H, d, J=5.1 Hz), 3.72–3.73 (2H, m), 3.74 (1H, dd, J=6.3, 9.1 Hz), 3.99 (2H, s), 4.07 (1H, dd, J=8.7, 9.1 Hz), 4.75 (1H, ddt, J=5.1, 6.3, 8.7 Hz), 6.78 (1H, dd, J=8.5, 9.3 Hz), 7.02 (1H, dd, J=2.2, 8.5 Hz), 7.34 (1H, dd, J=2.2, 13.6 Hz). ¹³C-NMR (125 MHz, CD₃OD) δ: 21.0, 41.8, 43.0, 48.3, 48.4, 69.2, 72.0, 107.0 (d, J=23.0 Hz), 112.1 (d, J=4.7 Hz), 115.5 (d, J=2.9 Hz), 127.7 (d, J=9.6 Hz), 134.1 (d, J=12.5 Hz), 151.2 (d, J=239.0 Hz), 155.7, 172.6, 175.3. IR (neat) cm⁻¹: 3314, 2931, 2877, 1735, 1654, 1637, 1524, 1420, 1226, 1117. ESI-MS m/z: 392.1221 (Calcd for C₁₆H₂₀FN₃NaO₆: 392.1234).

(5S)-Acetylaminomethyl-N-[4-[benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-2-oxazolidinone (20)

To a solution of 4 (351 mg, 0.702 mmol) in THF/H₂O (10 : 1, 3.3 mL) was added triphenylphosphine (203 mg, 0.773 mmol). After stirring overnight at room temperature, the reaction mixture was concentrated under reduced pressure to afford a pale yellow oil. The residue was dissolved in CH₂Cl₂ (4 mL), and Et₃N (147 µL, 1.05 mmol) and acetylchloride (99 µL, 0.913 mmol) were added to the solution. After stirring for 10 min at room temperature, the solution was concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 100 : 0 to 100 : 1) to obtain an inseparable mixture of 20 and triphenylphosphine oxide (6.25 : 1) as a colorless amorphous solid (300 mg). This mixture was used in the next step without further purification. ¹H-NMR of 20 (500 MHz, CDCl₃/TMS) δ: −0.02 (6H, s), 0.85 (9H, s), 2.02 (3H, s), 3.33 (2H, t, J=6.1 Hz), 3.56 (1H, ddd, J=6.1, 6.1, 15.3 Hz), 3.68–3.71 (2H, m), 3.71 (2H, t, J=6.1 Hz), 3.98 (1H, dd, J=8.8, 9.0 Hz), 4.44 (2H, s), 4.74 (1H, m), 5.98 (1H, dd, J=6.1, 6.1 Hz), 6.89 (1H, dd, J=9.2, 9.2 Hz), 6.95 (1H, dd, J=2.7, 9.2 Hz), 7.37 (1H, dd, J=2.7, 14.7 Hz).

(5S)-Acetylaminomethyl-N-[4-[[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-2-oxazolidinone (12)

To a solution of a mixture of 20 and triphenylphosphine oxide (300 mg) in THF (3 mL) was added 20% Pd(OH)₂/C (43 mg). After stirring overnight under H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 100 : 0 to 100 : 1) to afford 12 as a colorless solid (165 mg, 55% from 4). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 0.07 (6H, s), 0.90 (9H, s), 2.02 (3H, s), 3.24 (2H, dt, J=5.6, 5.6 Hz), 3.57 (1H, ddd, J=6.3, 6.3, 14.6 Hz), 3.69–3.76 (2H, m), 3.82 (2H, t, J=5.3 Hz), 4.00 (1H, dd, J=9.0, 9.0 Hz), 4.25 (1H, br), 4.72–4.76 (1H, m), 5.94 (1H, br), 6.68 (1H, dd, J=9.2, 9.2 Hz), 6.98 (1H, dd, J=2.4, 9.2 Hz), 7.35 (1H, dd, J=2.4, 13.2 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.4, 18.2, 23.0, 25.8, 41.9, 45.7, 48.1, 61.4, 71.9, 107.1 (d, J=23.6 Hz), 112.3 (d, J=4.6 Hz), 115.0 (d, J=3.0 Hz), 127.6 (d, J=9.1 Hz), 134.1 (d, J=12.4 Hz), 151.3 (d, J=239.6 Hz), 154.8, 171.3. IR (neat) cm⁻¹: 3404, 3355, 2952, 2929, 2858, 1739, 1669, 1523, 1216, 1075. mp: 120.6−121.1°C. ESI-MS m/z: 448.2038 (Calcd for C₂₀H₃₂FN₃NaO₄Si: 448.2044).

Benzyl [[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-fluorophenyl][2-(tert-butyldimethylsilyloxy)ethyl]amino]acetate (13)

To a solution of 12 (87 mg, 0.20 mmol) in acetonitrile (2 mL) were added benzyl bromoacetate (39 µL, 0.25 mmol), K₂CO₃ (44 mg, 0.32 mmol), and NaI (5 mg, 0.03 mmol). After stirring overnight at reflux temperature, an additional amount of benzyl bromoacetate (32 µL, 0.20 mmol), K₂CO₃ (44 mg, 0.32 mmol), and NaI (12 mg, 0.080 mmol) were added to the reaction mixture. After stirring for 3 d at reflux temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 100 : 0 to 100 : 1) to afford an inseparable mixture of 13 and 12 (10 : 1) as a pale yellow oil (111 mg, 88%). ¹H-NMR of 13 (500 MHz, CDCl₃/TMS) δ: 0.01 (6H, s), 0.86 (9H, s), 2.02 (3H, s), 3.44 (2H, t, J=5.8 Hz), 3.58 (1H, ddd, J=6.1, 6.1, 14.6 Hz), 3.69–3.76 (2H, m), 3.78 (2H, t, J=5.8 Hz), 3.99 (1H, dd, J=9.0, 9.1 Hz), 4.16 (2H, s), 4.73–4.77 (1H, m), 5.13 (2H, s), 5.97 (1H, dd, J=6.1, 6.1 Hz), 6.91 (1H, dd, J=9.1, 9.5 Hz), 6.98 (1H, dd, J=2.1, 9.1 Hz), 7.30–7.38 (6H, m).

[[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-fluorophenyl][2-(tert-butyldimethylsilyloxy)ethyl]amino]acetic Acid (14)

To a solution of a 10 : 1 mixture of 13 and 12 (86 mg) in THF (2 mL) was added 20% Pd(OH)₂/C (21 mg). After stirring for 2 h under H₂ atmosphere at room temperature, the mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 50 : 1 to 30 : 1) to afford 14 as a colorless amorphous solid (42 mg, 55%). Compound 14 was unstable and was immediately used in the next reaction. ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 0.04 (6H, s), 0.87 (9H, s), 2.02 (3H, s), 3.40 (2H, t, J=4.9 Hz), 3.61 (1H, ddd, J=6.1, 6.3, 14.9 Hz), 3.68–3.76 (4H, m), 3.89 (2H, d, J=1.9 Hz), 4.02 (1H, dd, J=8.8, 9.1 Hz), 4.75–4.80 (1H, m), 6.10 (1H, dd, J=6.1, 6.3 Hz), 7.01 (1H, dd, J=8.8, 9.1 Hz), 7.11 (1H, dd, J=2.6, 8.8 Hz), 7.45 (1H, dd, J=2.6, 13.9 Hz).

[[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-fluorophenyl](2-hydroxyethyl)amino]acetic Acid Tetrabutylammonium Salt (3)

To a solution of 14 (42 mg, 0.087 mmol) in H₂O (1 mL) was added 40% tetrabutylammonium hydroxide in water (56 µL, 0.087 mmol). After stirring at room temperature, the reaction mixture was diluted with water (5 mL), and the organic materials were extracted with CHCl₃ (10 mL×5). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the tetrabutylammonium salt of 14 (59 mg). To a solution of the tetrabutylammonium salt of 14 in THF (1.5 mL) was added 1.0 M tetrabutylammonium fluoride in THF (130 µL, 0.130 mmol). After stirring for 3 h at room temperature, the solution was concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH, 40 : 1 to 3 : 1) to afford 3 as a colorless, viscous oil (35 mg, 66%). ¹H-NMR (500 MHz, CDCl₃/TMS) δ: 0.98 (12H, t, J=7.4 Hz), 1.36–1.43 (8H, m), 1.59–1.66 (8H, m), 2.01 (3H, s), 3.25 (8H, t, J=8.5 Hz), 3.57–3.69 (6H, m), 3.72 (1H, dd, J=6.6, 9.1 Hz), 3.86 (2H, d, J=1.5 Hz), 3.96 (1H, dd, J=9.0, 9.1 Hz), 4.73–4.78 (1H, m), 6.85 (1H, dd, J=9.1, 9.5 Hz), 6.90 (1H, dd, J=2.5, 9.1 Hz), 7.02 (1H, dd, J=5.6, 5.6 Hz), 7.26 (1H, dd, J=2.5, 15.7 Hz). ¹³C-NMR (125 MHz, CDCl₃) δ: 13.6, 19.7, 23.0, 23.9, 41.9, 48.1, 56.5 (d, J=4.8 Hz), 58.7, 58.8, 59.9, 71.6, 108.2 (d, J=26.8 Hz), 114.7 (d, J=2.9 Hz), 118.5 (d, J=5.8 Hz), 129.1 (d, J=10.6 Hz), 134.5 (d, J=8.7 Hz), 152.8 (d, J=241.8 Hz), 154.7, 171.4, 177.0. IR (neat) cm⁻¹: 3412, 3266, 2963, 2935, 2876, 1744, 1660, 1595, 1521, 1225. ESI-MS m/z: 392.1227 (Calcd for C₁₆H₂₀FN₃NaO₆: 392.1234).

Acknowledgments

This study is supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and Japan Agency for Medical Research and Development (AMED), the Sasakawa Scientific Research Grant from The Japan Science Society, and Grants-in-Aid for Scientific Research.

Conflict of Interest

The authors declare no conflict of interest.

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