Synthesis of linezolid metabolites PNU-142300 and PNU-142586 toward the exploration of metabolite-related events

Article abstract of DOI:10.1248/cpb.c16-00831

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.

Full text of DOI:10.1248/cpb.c16-00831

194
Chem. Pharm. Bull. 65, 194–199 (2017)
Vol. 65, No. 2
Regular Article
Synthesis of Linezolid Metabolites PNU-142300 and PNU-142586 toward
the Exploration of Metabolite-Related Events
Kengo Hanaya,* Kazuaki Matsumoto, Yuta Yokoyama, Junko Kizu, Mitsuru Shoji, and
Takeshi Sugai
Faculty of Pharmacy, Keio University; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan.
Received October 25, 2016; accepted November 15, 2016
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 metabo-
lites and lead to improved clinical use of linezolid.
Key words linezolid; metabolite; oxazolidinone; tetrabutylammonium salt; thrombocytopenia
Linezolid (1) is a novel antimicrobial agent that possesses synthetic intermediate, which may encourage the further ex-
an oxazolidinone moiety; it exhibits a broad antibacterial ploration of metabolite-related events and improve the clinical
spectrum against Gram-positive bacteria, including methicil- use of linezolid.
lin-resistant Staphylococcus aureus (MRSA) and vancomycin-
resistant Enterococcus faecium (VRE)1–5) (Chart 1). Linezolid
Results and Discussion
selectively binds to the 50s subunit of bacterial ribosomes to
prevent the formation of the 70s ribosome, resulting in inhibi- noalcohol derivative 4, bearing tert-butyldimethylsilyl (TBS)
tion of bacterial protein synthesis.6)
and benzyl (Bn) groups was designed as a common synthetic
For the convenient synthesis of metabolites 2 and 3, ami-
The major adverse event associated with linezolid treatment intermediate (Chart 2). We planned the selective removal of
is reversible myelosuppression, which may cause anemia and the protecting groups in 4 and subsequent alkylation with bro-
thrombocytopenia. Wu et al.,7) Lin et al.,8) and Matsumoto moacetate derivatives to form 2 and 3, respectively.
et al.9) independently reported that linezolid-associated throm-
bocytopenia 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
Chart 2. The Common Synthetic Intermediate 4 of 2 and 3
in the blood of patients with renal insufficiency. The toxic-
ity of metabolites 2 and 3 has not been fully elucidated; it is
suspected that accumulation of 2 and 3 leads to thrombocyto-
The synthesis of 4 was conducted by a procedure similar to
penia in patients with renal insufficiency. Unfortunately, the the linezolid synthesis developed by Brickner et al.12) Nucleo-
poor availability of 2 and 3 has hindered further investigation philic substitution of difluoride 5 with N-benzylethanolamine
of the clinical significance of the accumulation of these me- and subsequent protection of the alcohol with a TBS group
tabolites. In addition, these metabolites are known to be P450- gave 6 (Chart 3). In the nucleophilic substitution step, high
independent,5,11) and P450 enzyme-mediated direct synthesis temperature was required due to the higher bulkiness and
of 2 and 3 on a preparative scale is likely impossible. Herein, lower reactivity of N-benzylethanolamine compared to mor-
we synthesized the major metabolites 2 and 3 via a common pholine in Brickner’s linezolid synthesis. Reduction of the
nitro group with hydrogen in the presence of a palladium cata-
lyst caused undesired debenzylation. Alternatively, complete
selectivity in the reduction of the nitro group was achieved
by using copper hydride.13) The oxazolidinone ring was con-
structed 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.
For PNU-142300 (2), O-alkylation was attempted after re-
moval of the TBS group in 4. Using sodium hydride (NaH)
as a base and benzyl bromoacetate as the alkylating agent in
Chart 1. Linezolid (1) and Its Major Metabolites, 2 and 3
*To whom correspondence should be addressed. e-mail: hanaya-kn@pha.keio.ac.jp
© 2017 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
195
(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.
Chart 3. Synthesis of Common Synthetic Intermediate 4
(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 5. Synthesis of PNU-142586 (3)
cleavage of the TBS ether and successive lactonization due
to its acidity. Thus, 14 was immediately neutralized with
tetrabutylammonium hydroxide to afford its tetrabutylammo-
nium salt, whose TBS group was removed by treatment with
tetrabutylammonium fluoride (TBAF) to furnish the tetrabu-
tylammonium 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).
t
(a) TBAF, THF, r.t., 99%. (b) BrCH2CO2 Bu, nBu4NHSO4, NaOH, toluene, H2O,
r.t., quant. (c) PPh3, H2O, THF, r.t. (d) AcCl, Et3N, CH2Cl2, 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.
Chart 4. Synthesis of PNU-142300 (2)
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 sup-
press 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 quantita-
tively. Successive Staudinger reaction and acetylation followed
by the stepwise removal of the protecting groups provided
Chart 6. Cyclic Byproduct 15
Finally, the purity of 2 and 3 was evaluated by HPLC
trifluoroacetic acid (TFA) salt of PNU-142300 (2). TFA salt of analysis (Chart 7) and was proved to be well-tolerated for ref-
2 was gradually decomposed in a solution via acid-catalyzed erence standards for HPLC analysis.
cyclization and was neutralized to obtain 2.
In conclusion, we have successfully prepared linezolid
PNU-142586 (3) was prepared as illustrated in Chart 5. The metabolites 2 and 3 via a common synthetic intermediate,
Staudinger reaction, acetylation, and debenzylation of 4 were 4. The establishment of a synthetic method for 2 and 3 will
conducted to afford 12. N-Alkylation progressed to afford 13 contribute to further exploration of the pharmacokinetics of
as an inseparable mixture with unreacted amine 12. After re- these metabolites in patients with renal insufficiency, which
moval of the benzyl group on 13, the resulting carboxylic acid may elucidate the roles of linezolid metabolites in thrombo-
14 was easily separated from the contaminant 12. Carboxylic cytopenia.
acid 14 was unstable under storage even at −20°C, owing to

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Chem. Pharm. Bull.
Vol. 65, No. 2 (2017)
70.0 (d, J=2.3Hz), 113.2 (d, J=27.5Hz), 116.4 (d, J=4.6Hz),
121.2 (d, J=2.3Hz), 126.8, 127.6, 128.9, 136.7, 138.3 (d,
J=8.4Hz), 143.9 (d, J=6.9Hz), 150.8 (d, J=246Hz); 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 (4435mg, 15.28mmol) and imidazole
(1249mg, 18.35mmol) in CH2Cl2 (40mL) was added tert-bu-
tyldimethylsilyl chloride (2543mg, 16.87mmol) at 0°C. After
stirring for 40min at room temperature, the reaction mixture
was washed with 0.1M phosphate buffer (pH 7, 40mL), water
(40mL), and brine (40mL). The organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure to
1
afford 6 as a yellow oil (5780mg, 94%). H-NMR (400MHz,
CDCl3/TMS) δ: 0.01 (6H, s), 0.89 (9H, s), 3.49 (2H, dt, J=2.0,
5.5Hz), 3.86 (2H, t, J=5.5Hz), 4.73 (2H, s), 6.79 (1H, dd,
J=8.8, 9.0Hz), 7.21–7.36 (5H, m), 7.85 (1H, dd, J=2.7, 8.8Hz),
7.89 (1H, dd, J=2.7, 14.3Hz). 13C-NMR (100MHz, CDCl3) δ:
−5.5, 18.1, 25.8, 54.7 (d, J=6.8Hz), 56.5 (d, J=3.8Hz), 61.8 (d,
J=2.3Hz), 113.2 (d, J=27.4Hz), 116.1 (d, J=4.5Hz), 121.2 (d,
J=2.3Hz), 126.7, 127.4, 128.8, 137.0, 137.9 (d, J=8.4Hz), 143.9
(d, J=6.9Hz), 150.4 (d, J=245.7Hz). 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)
Conditions: column, SHISEIDO Capcell Pak C18 MG-II (4.6×250mm); mobile
phase, acetonitrile–0.1% TFA in H2O, 10:90; flow rate, 1.0mL/min; column tem-
perature, 40°C; detect, 254nm.
Chart 7. HPLC Analysis Data of (a) 2 (tR 6.23min) and (b) 3 (tR
6.21 min)
Experimental
General Remarks All reagents and solvents purchased
To a solution of NaBH4 (603mg, 1.23mmol) and copper(II)
were of the highest commercial quality and were used with- acetylacetonate (Cu(acac)2) (257mg, 0.981mmol) in EtOH
1
out further purification. H-NMR spectra were measured at (3mL) was added a solution of 6 (643mg, 1.59mmol) in
400MHz on a VARIAN 400-MR spectrometer or at 500MHz THF (3mL). After stirring for 30min at room temperature,
on an Agilent INOVA-500 spectrometer, and 13C-NMR spectra sat. NaHCO3 solution (20mL) was added, and the mixture
were measured at 100MHz on a VARIAN 400-MR spec- was stirred for 20min. The organic materials were extracted
trometer or at 125MHz on an Agilent INOVA-500 spectrom- with AcOEt (20mL ×3). The organic layer was washed with
eter. High resolution mass spectra were recorded using a Jeol brine (20mL), dried over Na2SO4, filtered, and concentrated
JMS-T100LP AccuTOF instrument. IR spectra were recorded under reduced pressure. The crude mixture was purified
using a JASCO FT/IR-4700 spectrometer in attenuated total using the Isolera One system (hexane/AcOEt) to obtain 7 as a
1
reflection (ATR) mode at room temperature. Silica gel column purple oil (563mg, 95%). H-NMR (500MHz, CDCl3/TMS) δ:
chromatography was performed using Silica Gel 60 (spherical −0.03 (6H, s), 0.85 (9H, s), 3.15 (2H, t, J=6.6Hz), 3.64 (2H,
and neutral; 100 to 210µm, 37560–79, Kanto Chemical Co., t, J=6.6Hz), 4.25 (2H, s), 6.29 (1H, ddd, J=0.8, 2.7, 8.8Hz),
Japan) and the Isolera One flash purification system (Biotage, 6.38 (1H, d, J=2.7, 13.4Hz), 6.81 (1H, dd, J=8.8, 8.8Hz),
Sweden).
Synthesis
2-[N-Benzyl-N-(2-fluoro-4-nitrophenyl)amino]ethanol (16)
7.18–7.31 (5H, m). 13C-NMR (100MHz, CDCl3) δ: −5.4, 18.3,
25.9, 54.8 (d, J=2.2Hz), 58.7 (d, J=1.5Hz), 61.5, 103.8 (d,
J=24.4Hz), 110.6 (d, J=2.3Hz), 124.6 (d, J=4.6Hz), 126.8,
To a solution of 3,4-difluoronitrobenzene (5) (2983mg, 128.1, 128.4, 129.9 (d, J=9.9Hz), 139.3, 142.6 (d, J=10.7 Hz),
18.75mmol) in n-butanol (10mL) were added N-benzylethanol- 157.9 (d, J=244.9Hz). IR (neat) cm−1: 3461, 3373, 2927,
amine (2959 µL, 20.74mmol) and N,N-diisopropylethylamine 2855, 1511, 1254, 1092. ESI-MS m/z: 397.2075 (Calcd for
(3613 µL, 20.74mmol). After stirring overnight at reflux tem- C21H31FN2NaOSi: 397.2087).
perature, the reaction mixture was concentrated under reduced
N-Benzyl-4-benzyloxycarbonylamino-N-[2-(tert-butyl-
pressure. The residue was dissolved in AcOEt (60mL), and dimethylsilyloxy)ethyl]-2-fluoroaniline (8)
the solution was washed with water (40mL×2) and brine
To a solution of 7 (501mg, 1.34mmol) and NaHCO3 (226mg,
(40mL). The organic layer was dried over Na2SO4, filtered, 2.69mmol) in THF (3mL) was added a solution of benzyl-
and concentrated under reduced pressure. The resulting chloroformate (248mg, 1.45mmol) in THF (0.5mL). After
residue was purified using the Isolera One system (hexane/ stirring for 2h at room temperature, benzylchloroformate
1
AcOEt) to afford 16 as a yellow oil (4435mg, 81%). H-NMR (50mg, 0.29mmol) was added, and the mixture was stirred
(400MHz, CDCl3/tetramethylsilane (TMS)) δ: 3.66 (2H, dt, for another 30min. Water (20mL) was added to the mixture,
J=1.9, 5.7Hz), 3.89 (1H, dt, J=0.8, 5.7Hz), 4.71 (2H, s), 6.80 and the organic materials were extracted with AcOEt (20mL).
(1H, dd, J=9.0, 9.0Hz), 7.21–7.35 (5H, m), 7.84 (1H, ddd, The organic layer was washed with brine (20mL), dried over
J=0.7, 2.5, 9.0Hz), 7.87 (1H, dd, J=2.5, 14.2Hz). 13C-NMR Na2SO4, filtered, and concentrated under reduced pressure.
(100MHz, CDCl3) δ: 54.2 (d, J=6.9Hz), 56.6 (d, J=3.8Hz), The crude mixture was purified using the Isolera One system

Vol. 65, No. 2 (2017)
Chem. Pharm. Bull.
197
(hexane/AcOEt) to afford 8 as a colorless oil (563mg, quant.). night at 50°C, the mixture was diluted with AcOEt (20mL),
1H-NMR (500MHz, CDCl3/TMS) δ: −0.02 (6H, s), 0.85 (9H, and the solution was washed with water and brine. The
s), 3.28 (2H, t, J=6.4Hz), 3.69 (2H, t, J=6.4Hz), 4.39 (2H, s), organic layer was washed with brine (30mL), dried over
5.18 (2H, s), 6.51 (1H, br), 6.82–6.87 (2H, m), 7.19–7.41 (10H, Na2SO4, filtered, and concentrated under reduced pressure.
m). 13C-NMR (100MHz, CDCl3) δ: −5.5, 18.2, 25.9, 54.2 (d, The crude mixture was purified using the Isolera One system
J=3.1Hz), 57.5 (d, J=2.3Hz), 61.5, 67.0, 108.1, 114.4, 121.5, (hexane/AcOEt) to afford 4 as a yellow oil (340mg, 75% for 2
1
126.9, 128.0, 128.2, 128.3, 128.4, 128.6, 131.8, 134.3, 136.0, steps from 9). H-NMR (500MHz, CDCl3/TMS) δ: 0.00 (6H,
138. 8, 153.4, 155.6 (d, J=244.1Hz). IR (neat) cm−1: 2952, s), 0.86 (9H, s), 3.34 (2H, t, J=6.1Hz), 3.54 (1H, dd, J=4.6,
2927, 2854, 1732, 1702, 1515, 1254, 1209, 1091. ESI-MS m/z: 13.2Hz), 3.66 (1H, dd, J=4.6, 13.2Hz), 3.73 (2H, t, J=6.1Hz),
531.2447 (Calcd for C29H37FN2NaO3Si: 531.2455).
N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3- 4.44 (2H, s), 4.71–4.75 (1H, m), 6.91 (1H, dd, J=9.1, 9.1Hz),
fluorophenyl]-(5R)-hydroxymethyl-2-oxazolidinone (9)
3.76 (1H, dd, J=6.4, 9.1Hz), 3.99 (1H, dd, J=9.1, 9.1Hz),
6.98 (1H, dd, J=2.7, 9.1Hz), 7.20–7.31 (5H, m), 7.39 (1H, dd,
To a solution of 8 (2550mg, 5.196mmol) in THF (10mL) J=2.7, 14.7Hz). 13C-NMR (125MHz, CDCl3) δ: −5.4, 18.2,
was added n-butyllithium in hexane (1.56M, 3.7mL) dropwise 25.9, 47.5, 53.0, 54.4 (d, J=3.9Hz), 57.1, 61.5, 70.6, 107.7 (d,
over 20min at −78°C. After stirring for 1h at −78°C, (R)- J=26.9Hz), 113.8 (d, J=2.8Hz), 120.9 (d, J=4.8Hz), 127.0,
glycidyl butyrate (99% enantiomeric excess (ee), [α]D22 29.7 127.8, 128.3, 131.3 (d, J=10.5Hz), 135.0 (d, J=9.6Hz), 138.7,
(c=1.0, CHCl3)) (792µL, 5.72mmol) was added, and the mix- 153.9, 155.0 (d, J=244.8Hz). IR (neat) cm−1: 2952, 2928, 2855,
ture was allowed to warm to room temperature. After stirring 2104, 1747, 1515, 1217, 1086. ESI-MS m/z: 522.2308 (Calcd for
overnight at room temperature, 0.1M phosphate buffer (pH C25H34FN5NaO3Si: 522.2313).
7) was added to the mixture, and the organic materials were N-[4-[Benzyl(2-hydroxyethyl)amino]-3-fluorophenyl]-(5R)-
extracted with AcOEt (30mL). The organic layer was washed azidemethyl-2-oxazolidinone (18)
with brine (30mL), dried over Na2SO4, filtered, and concen-
To a solution of 4 (302mg, 0.604mmol) in THF (4mL) was
trated under reduced pressure. The crude mixture was purified added 1.0M tetrabutylammonium fluoride in THF (910µL,
using the Isolera One system (hexane/AcOEt) to afford 9 as a 0.910mmol). After stirring for 30min at room temperature,
1
yellow oil (1689mg, 71%). H-NMR (400MHz, CDCl3/TMS) the solution was diluted with AcOEt (40mL), and the solu-
δ: −0.02 (6H, s), 0.85 (9H, s), 3.33 (2H, t, J=6.2Hz), 3.71 (2H, tion was washed with water and brine. The organic layer
t, J=6.2Hz), 3.70–3.73 (1H, m), 3.89 (1H, dd, J=7.0, 8.4Hz), was dried over Na2SO4, filtered, and concentrated under
3.95 (2H, dd, J=8.8, 8.8Hz), 4.43 (2H, s), 4.67–4.73 (1H, m), reduced pressure. The crude mixture was purified using the
6.89 (1H, dd, J=9.0, 9.0Hz), 6.99 (1H, dd, J=2.7, 9.0Hz), Isolera One system (hexane/AcOEt) to afford 18 as a yellow
1
7.19–7.28 (5H, m), 7.38 (1H, dd, J=2.7, 14.8Hz). 13C-NMR oil (231mg, 99%). H-NMR (400MHz, CDCl3/TMS) δ: 3.28
(100MHz, CDCl3) δ: −5.5, 18.2, 25.9, 46.5, 54.3 (d, J=3.8Hz), (2H, t, J=5.3Hz), 3.58 (1H, dd, J=4.3, 13.3Hz), 3.63 (2H, t,
57.2 (d, J=2.2Hz), 61.5, 62.8, 72.8, 107.7 (d, J=26.7Hz), 113.8 J=5.3Hz), 3.70 (1H, dd, J=4.7, 13.3Hz), 3.81 (1H, dd, J=6.3,
(d, J=3.1Hz), 120.9 (d, J=4.6Hz), 126.9, 127.8, 128.3, 131.6 9.0Hz), 4.03 (1H, dd, J=9.0, 9.0Hz), 4.31 (2H, s), 4.74–4.80
(d, J=10.0Hz), 134.8 (d, J=9.1Hz), 138.7, 154.7, 155.1 (d, (1H, m), 6.97 (1H, dd, J=9.0, 9.0Hz), 7.06 (1H, ddd, J=1.0,
J=244.9Hz). IR (neat) cm−1: 3400, 2854, 1736, 1514, 1218, 2.5, 9.0Hz), 7.21–7.31 (5H, m), 7.44 (1H, dd, J=2.5, 13.9Hz).
1087. ESI-MS m/z: 429.2238 (Calcd for C25H35FN2NaO4Si: 13C-NMR (100MHz, CDCl3) δ: 47.3, 53.0, 54.3, 57.8, 59.6,
497.2248).
70.8, 107.5 (d, J=26.7Hz), 113.7 (d, J=3.1Hz), 122.8 (d,
N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3- J=4.6Hz), 127.2, 128.0, 128.3, 128.4, 133.1 (d, J=9.9Hz),
fluorophenyl]-(5R)-methanesulfonyloxymethyl-2-oxazolidinone 134.3 (d, J=9.9Hz), 138.1, 154.0, 156.4 (d, J=245.6Hz). IR
(17)
To a solution of 9 (413mg, 0.904mmol), Et3N (151µL, m/z: 408.1457 (Calcd for C19H20FN5NaO3: 408.1448).
1.09mmol), and N,N-dimethyl-4-aminopyridine (DMAP) tert-Butyl [2-[[4-[(5R)-Azidemethyl-2-oxazolidinon-3-yl]-2-
(10mg, 0.082mmol) in CH2Cl2 (3mL), was added methane- fluorophenyl]benzylamino]ethoxy]acetate (10)
(neat) cm−1: 3449, 2930, 2893, 2103, 1737, 1514, 1212. ESI-MS
sulfonyl chloride (78µL, 1.0mmol). After stirring overnight
To a solution of 18 (357mg, 0.926mmol) in toluene (5.5mL)
at room temperature, the reaction mixture was concentrated were added tert-butyl bromoacetate (997µL, 7.41mmol), tet-
under reduced pressure. The residue was dissolved in AcOEt rabutylammonium hydrogen sulfate (251mg, 0.741mmol), and
(30mL), and the solution was washed with water and brine. 5M NaOH in water (18mL). After stirring vigorously for 2h
The organic layer was dried over Na2SO4, filtered, and con- at room temperature, the organic layer was separated. The or-
centrated under reduced pressure to afford 17 as a yellow oil ganic materials were further extracted with AcOEt (30mL×2).
(526mg, quant.). The crude 17 was used in the next step with- The combined organic layer was washed with water and
1
out further purification. H-NMR (500MHz, CDCl3/TMS) δ: brine, dried over Na2SO4, filtered, and concentrated under
−0.01 (6H, s), 0.85 (9H, s), 3.08 (3H, s), 3.35 (2H, t, J=6.1Hz), reduced pressure. The crude mixture was purified using the
3.73 (2H, t, J=6.1Hz), 3.86 (1H, dd, J=6.1, 9.1Hz), 4.06 (1H, Isolera One system (hexane/AcOEt) to afford 10 as a yellow
1
dd, J=9.1, 9.1Hz), 4.39 (1H, dd, J=4.4, 11.7Hz), 4.45 (2H, s), oil (469mg, quant.). H-NMR (400MHz, CDCl3/TMS) δ: 1.46
4.46 (1H, dd, J=3.6, 11.7Hz), 4.85–4.89 (1H, m), 6.90 (1H, dd, (9H, s), 3.43 (2H, t, J=6.0Hz), 3.58 (1H, dd, J=4.5, 13.3Hz),
J=9.0, 9.0Hz), 6.97 (1H, dd, J=2.7, 9.0Hz), 7.19–7.29 (5H, m), 3.64 (2H, t, J=6.0Hz), 3.68 (1H, dd, J=4.7, 13.3Hz), 3.79 (1H,
7.37 (1H, dd, J=2.7, 14.5Hz). dd, J=6.2, 9.0Hz), 3.89 (2H, s), 4.02 (1H, dd, J=9.0, 9.0Hz),
N-[4-[Benzyl[2-(tert-butyldimethylsilyloxy)ethyl]amino]-3- 4.44 (2H, s), 4.72–4.78 (1H, m), 6.93 (1H, dd, J=9.0, 9.0Hz),
fluorophenyl]-(5R)-azidemethyl-2-oxazolidinone (4)
6.99 (1H, dd, J=2.5, 9.0Hz), 7.19–7.30 (5H, m), 7.31 (1H, dd,
To a solution of crude 17 (526mg) in DMF (3mL) was J=2.5, 14.5Hz). 13C-NMR (100MHz, CDCl3) δ: 28.1, 47.4,
added sodium azide (88mg, 1.4mmol). After stirring over- 51.7 (d, J=3.8Hz), 53.0, 57.1, 69.0, 69.8, 70.6, 81.5, 107.6 (d,

198
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Vol. 65, No. 2 (2017)
J=26.7Hz), 113.8 (d, J=3.8Hz), 121.4 (d, J=4.6Hz), 127.0, (500MHz, CD3OD) δ: 1.97 (3H, s), 3.34 (2H, br), 3.54 (2H,
128.0, 128.3, 131.8 (d, J=9.9Hz), 134.7 (d, J=9.1Hz), 138.4, d, J=5.1Hz), 3.72–3.73 (2H, m), 3.74 (1H, dd, J=6.3, 9.1Hz),
153.9, 155.3 (d, J=244.7Hz), 169.5. IR (neat) cm−1: 2977, 2931, 3.99 (2H, s), 4.07 (1H, dd, J=8.7, 9.1Hz), 4.75 (1H, ddt, J=5.1,
2897, 2103, 1740, 1514, 1223, 1129. ESI-MS m/z: 522.2121 6.3, 8.7Hz), 6.78 (1H, dd, J=8.5, 9.3Hz), 7.02 (1H, dd, J=2.2,
(Calcd for C25H30FN5NaO5: 522.2129).
tert-Butyl [2-[[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3- CD3OD) δ: 21.0, 41.8, 43.0, 48.3, 48.4, 69.2, 72.0, 107.0 (d,
yl]-2-fluorophenyl]benzylamino]ethoxy]acetate (11) J=23.0Hz), 112.1 (d, J=4.7Hz), 115.5 (d, J=2.9Hz), 127.7 (d,
8.5Hz), 7.34 (1H, dd, J=2.2, 13.6Hz). 13C-NMR (125MHz,
To a solution of 10 (358mg, 0.717mmol) in THF/H2O (10:1, J=9.6Hz), 134.1 (d, J=12.5Hz), 151.2 (d, J=239.0Hz), 155.7,
3.3mL) was added triphenylphosphine (210mg, 0.800mmol). 172.6, 175.3. IR (neat) cm−1: 3314, 2931, 2877, 1735, 1654,
After stirring for 1h at room temperature, H2O (0.3mL) was 1637, 1524, 1420, 1226, 1117. ESI-MS m/z: 392.1221 (Calcd for
added to the solution, and the solution was stirred overnight C16H20FN3NaO6: 392.1234).
at room temperature. The reaction mixture was concentrated
(5S)-Acetylaminomethyl-N-[4-[benzyl[2-(tert-butyl-
under reduced pressure. The residue was dissolved in CH2Cl2 dimethylsilyloxy)ethyl]amino]-3-fluorophenyl]-2-oxazolidinone
(4mL), and Et3N (150µL, 1.08mmol) and acetylchloride (20)
(67 µL, 0.93mmol) were added to the solution. After stirring
To a solution of 4 (351mg, 0.702mmol) in THF/H2O (10:1,
for 25min at room temperature, the solution was concentrated 3.3mL) was added triphenylphosphine (203mg, 0.773mmol).
under reduced pressure. The crude mixture was purified by After stirring overnight at room temperature, the reaction
silica gel column chromatography (CHCl3/MeOH, 100:0 to mixture was concentrated under reduced pressure to afford a
100:1) to afford an inseparable mixture of 11 and triphe- pale yellow oil. The residue was dissolved in CH2Cl2 (4mL),
nylphosphine oxide (100:1) as a colorless amorphous solid and Et3N (147µL, 1.05mmol) and acetylchloride (99µL,
1
(339mg). H-NMR (500MHz, CDCl3/TMS) δ: 1.46 (9H, s), 0.913mmol) were added to the solution. After stirring for
2.02 (3H, s), 3.42 (2H, t, J=6.1Hz), 3.56 (1H, ddd, J=6.1, 6.1, 10min at room temperature, the solution was concentrated
14.7Hz), 3.64 (2H, t, J=6.1Hz), 3.69 (1H, dd, J=6.6, 9.0Hz), under reduced pressure. The crude mixture was purified by
3.69–3.74 (1H, m), 3.89 (2H, s), 3.99 (1H, dd, J=9.0, 9.0Hz), silica gel column chromatography (CHCl3/MeOH, 100:0 to
4.44 (2H, s), 4.72–4.77 (1H, m), 5.97 (1H, dd, J=6.1, 6.1Hz), 100:1) to obtain an inseparable mixture of 20 and triphen-
6.92 (1H, dd, J=8.8, 8.8Hz), 6.95 (1H, dd, J=2.5, 8.8Hz), ylphosphine oxide (6.25:1) as a colorless amorphous solid
7.19–7.29 (5H, m), 7.40 (1H, dd, J=2.5, 14.4Hz).
tert-Butyl [2-[(4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3- further purification. H-NMR of 20 (500MHz, CDCl3/TMS)
yl]-2-fluorophenyl)amino]ethoxy]acetate (19)
δ: −0.02 (6H, s), 0.85 (9H, s), 2.02 (3H, s), 3.33 (2H, t,
(300mg). This mixture was used in the next step without
1
To a solution of a 100:1 mixture of 11 and triphe- J=6.1Hz), 3.56 (1H, ddd, J=6.1, 6.1, 15.3Hz), 3.68–3.71 (2H,
nylphosphine oxide (339mg) in THF (3mL) was added 20% m), 3.71 (2H, t, J=6.1Hz), 3.98 (1H, dd, J=8.8, 9.0Hz), 4.44
Pd(OH)2/C (92mg). After stirring for 3.5h under H2 atmo- (2H, s), 4.74 (1H, m), 5.98 (1H, dd, J=6.1, 6.1Hz), 6.89 (1H,
sphere at room temperature, the mixture was filtered through dd, J=9.2, 9.2Hz), 6.95 (1H, dd, J=2.7, 9.2Hz), 7.37 (1H, dd,
a Celite pad. The filtrate was concentrated under reduced J=2.7, 14.7 Hz).
pressure, and the crude mixture was purified by silica gel
column chromatography (CHCl3/MeOH, 200:1 to 100:1) to silyloxy)ethyl]amino]-3-fluorophenyl]-2-oxazolidinone (12)
afford 19 as a colorless amorphous solid (253mg, 83% based To a solution of a mixture of 20 and triphenylphosphine
(5S)-Acetylaminomethyl-N-[4-[[2-(tert-butyldimethyl-
1
on 10). H-NMR (500MHz, CDCl3/TMS) δ: 1.49 (9H, s), 2.05 oxide (300mg) in THF (3mL) was added 20% Pd(OH)2/C
(3H, s), 3.35 (2H, dd, J=4.6, 9.1Hz), 3.58 (1H, ddd, J=6.1, 6.1, (43mg). After stirring overnight under H2 atmosphere at room
12.2Hz), 3.70 (1H, dd, J=5.8, 9.1Hz), 3.69–3.74 (1H, m), 3.78 temperature, the mixture was filtered through a Celite pad.
(2H, t, J=9.1Hz), 4.00 (1H, dd, J=9.1, 9.1Hz), 4.01 (2H, s), The filtrate was concentrated under reduced pressure, and the
4.46 (1H, br), 4.72–4.77 (1H, m), 6.08 (1H, dd, J=6.1, 6.1Hz), crude mixture was purified by silica gel column chromatogra-
6.67 (1H, dd, J=8.8, 8.8Hz), 6.98 (1H, dd, J=2.4, 8.8Hz), 7.35 phy (CHCl3/MeOH, 100:0 to 100:1) to afford 12 as a colorless
1
(1H, dd, J=2.4, 13.2Hz). 13C-NMR (100MHz, CDCl3) δ: 22.9, solid (165mg, 55% from 4). H-NMR (500MHz, CDCl3/TMS)
28.1, 41.9, 43.5, 48.0, 68.7, 69.8, 71.9, 81.8, 107.1 (d, J=23.7Hz), δ: 0.07 (6H, s), 0.90 (9H, s), 2.02 (3H, s), 3.24 (2H, dt, J=5.6,
111.9 (d, J=4.5Hz), 115.0 (d, J=3.0Hz), 127.7 (d, J=9.1Hz), 5.6Hz), 3.57 (1H, ddd, J=6.3, 6.3, 14.6Hz), 3.69–3.76 (2H,
133.8 (d, J=11.6Hz), 151.1 (d, J=239.6Hz), 154.8, 169.6, 171.4. m), 3.82 (2H, t, J=5.3Hz), 4.00 (1H, dd, J=9.0, 9.0Hz), 4.25
IR (neat) cm−1: 3292, 2978, 2931, 1733, 1654, 1526, 1255, 1129. (1H, br), 4.72–4.76 (1H, m), 5.94 (1H, br), 6.68 (1H, dd, J=9.2,
ESI-MS m/z: 448.1871 (Calcd for C20H28FN3NaO6: 448.1860).
[2-[[4-[(5S)-Acetylaminomethyl-2-oxazolidinone-3-yl]-2- 13.2Hz). 13C-NMR (100MHz, CDCl3) δ: −5.4, 18.2, 23.0,
fluorophenyl]amino]ethoxy]acetic Acid (2) 25.8, 41.9, 45.7, 48.1, 61.4, 71.9, 107.1 (d, J=23.6Hz), 112.3
9.2Hz), 6.98 (1H, dd, J=2.4, 9.2Hz), 7.35 (1H, dd, J=2.4,
To a solution of 19 (50mg, 0.12mmol) in CH2Cl2 (1.8mL) (d, J=4.6Hz), 115.0 (d, J=3.0Hz), 127.6 (d, J=9.1Hz), 134.1
was added TFA (200µL, 2.61mmol). After stirring for 4h (d, J=12.4Hz), 151.3 (d, J=239.6Hz), 154.8, 171.3. IR (neat)
at room temperature, the solution was concentrated under cm−1: 3404, 3355, 2952, 2929, 2858, 1739, 1669, 1523, 1216,
reduced pressure. The crude mixture was dissolved in H2O 1075. mp: 120.6−121.1°C. ESI-MS m/z: 448.2038 (Calcd for
(1.8mL). The solution was neutralized with aqueous NaOH C20H32FN3NaO4Si: 448.2044).
solution and was washed with CHCl3. The aqueous layer was
Benzyl [[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2-
concentrated under reduced pressure. The crude mixture was fluorophenyl][2-(tert-butyldimethylsilyloxy)ethyl]amino]acetate
purified by silica gel column chromatography (CHCl3/MeOH, (13)
5:1 to 1:1) and using the Isolera One system (MeOH/H2O)
to afford 2 as a gray amorphous solid (32mg, 73%). H-NMR were added benzyl bromoacetate (39µL, 0.25mmol), K2CO3
To a solution of 12 (87mg, 0.20mmol) in acetonitrile (2mL)
1

Vol. 65, No. 2 (2017)
Chem. Pharm. Bull.
199
(44mg, 0.32mmol), and NaI (5mg, 0.03mmol). After stir- t, J=8.5Hz), 3.57–3.69 (6H, m), 3.72 (1H, dd, J=6.6, 9.1Hz),
ring overnight at reflux temperature, an additional amount 3.86 (2H, d, J=1.5Hz), 3.96 (1H, dd, J=9.0, 9.1Hz), 4.73–4.78
of benzyl bromoacetate (32µL, 0.20mmol), K2CO3 (44mg, (1H, m), 6.85 (1H, dd, J=9.1, 9.5Hz), 6.90 (1H, dd, J=2.5,
0.32mmol), and NaI (12mg, 0.080mmol) were added to the 9.1Hz), 7.02 (1H, dd, J=5.6, 5.6Hz), 7.26 (1H, dd, J=2.5,
reaction mixture. After stirring for 3d at reflux temperature, 15.7Hz). 13C-NMR (125MHz, CDCl3) δ: 13.6, 19.7, 23.0, 23.9,
the mixture was filtered through a Celite pad. The filtrate was 41.9, 48.1, 56.5 (d, J=4.8Hz), 58.7, 58.8, 59.9, 71.6, 108.2 (d,
concentrated under reduced pressure, and the crude mixture J=26.8Hz), 114.7 (d, J=2.9Hz), 118.5 (d, J=5.8Hz), 129.1 (d,
was purified by silica gel column chromatography (CHCl3/ J=10.6Hz), 134.5 (d, J=8.7Hz), 152.8 (d, J=241.8Hz), 154.7,
MeOH, 100:0 to 100:1) to afford an inseparable mixture of 171.4, 177.0. IR (neat) cm−1: 3412, 3266, 2963, 2935, 2876,
1
13 and 12 (10:1) as a pale yellow oil (111mg, 88%). H-NMR 1744, 1660, 1595, 1521, 1225. ESI-MS m/z: 392.1227 (Calcd for
of 13 (500MHz, CDCl3/TMS) δ: 0.01 (6H, s), 0.86 (9H, s), C16H20FN3NaO6: 392.1234).
2.02 (3H, s), 3.44 (2H, t, J=5.8Hz), 3.58 (1H, ddd, J=6.1, 6.1,
14.6Hz), 3.69–3.76 (2H, m), 3.78 (2H, t, J=5.8Hz), 3.99 (1H,
Acknowledgments This study is supported by the Plat-
dd, J=9.0, 9.1Hz), 4.16 (2H, s), 4.73–4.77 (1H, m), 5.13 (2H, s), form Project for Supporting in Drug Discovery and Life
5.97 (1H, dd, J=6.1, 6.1Hz), 6.91 (1H, dd, J=9.1, 9.5Hz), 6.98 Science Research (Platform for Drug Discovery, Informatics,
(1H, dd, J=2.1, 9.1Hz), 7.30–7.38 (6H, m).
and Structural Life Science) from the Ministry of Education,
[[4-[(5S)-Acetylaminomethyl-2-oxazolidinon-3-yl]-2- Culture, Sports, Science and Technology (MEXT) of Japan
fluorophenyl][2-(tert-butyldimethylsilyloxy)ethyl]amino]acetic and Japan Agency for Medical Research and Development
Acid (14)
(AMED), the Sasakawa Scientific Research Grant from The
To a solution of a 10:1 mixture of 13 and 12 (86mg) in Japan Science Society, and Grants-in-Aid for Scientific Re-
THF (2mL) was added 20% Pd(OH)2/C (21mg). After stirring search.
for 2h under H2 atmosphere at room temperature, the mixture
was filtered through a Celite pad. The filtrate was concentrat-
Conflict of Interest The authors declare no conflict of
ed under reduced pressure, and the crude mixture was purified interest.
by silica gel column chromatography (CHCl3/MeOH, 50:1
to 30:1) to afford 14 as a colorless amorphous solid (42mg,
55%). Compound 14 was unstable and was immediately used
in the next reaction. H-NMR (500MHz, CDCl3/TMS) δ: 0.04
(6H, s), 0.87 (9H, s), 2.02 (3H, s), 3.40 (2H, t, J=4.9Hz), 3.61
(1H, ddd, J=6.1, 6.3, 14.9Hz), 3.68–3.76 (4H, m), 3.89 (2H,
d, J=1.9Hz), 4.02 (1H, dd, J=8.8, 9.1Hz), 4.75–4.80 (1H, m),
6.10 (1H, dd, J=6.1, 6.3Hz), 7.01 (1H, dd, J=8.8, 9.1Hz), 7.11
(1H, dd, J=2.6, 8.8Hz), 7.45 (1H, dd, J=2.6, 13.9Hz).
References
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To a solution of 14 (42mg, 0.087mmol) in H2O (1mL) was
added 40% tetrabutylammonium hydroxide in water (56µL,
0.087mmol). After stirring at room temperature, the reac-
tion mixture was diluted with water (5mL), and the organic
materials were extracted with CHCl3 (10 mL×5). The or-
ganic layer was dried over Na2SO4, filtered, and concentrated
under reduced pressure to obtain the tetrabutylammonium
salt of 14 (59mg). To a solution of the tetrabutylammonium
salt of 14 in THF (1.5mL) was added 1.0M tetrabutylam-
monium fluoride in THF (130µL, 0.130mmol). After stirring
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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 (35mg, 66%).
1H-NMR (500MHz, CDCl3/TMS) δ: 0.98 (12H, t, J=7.4Hz),
1.36–1.43 (8H, m), 1.59–1.66 (8H, m), 2.01 (3H, s), 3.25 (8H,