Chemoenzymatic synthesis of all four cytoxazone stereoisomers

Article abstract of DOI:10.1055/s-2001-17711

Racemic cytoxazone (±-5) was synthesized starting from easily available glycidic ester (±)-1 by nucleophilic epoxide ring opening, followed by 2-oxazolidinone ring construction and calcium chloride/sodium borohydride reduction of the intermediary ester (±

Full text of DOI:10.1055/s-2001-17711

PAPER
1989
Chemoenzymatic Synthesis of All Four Cytoxazone Stereoisomers
C
hemoenzymat
d
ic Synthesis
e
of
A
ll Four
n
Cytoxazone
k
S
tereoisome
o
rs Hamer ak,a Edina Ljubovi ,a Mladen Mer ep,b Milan Mesi ,b Vitomir unji *a
a
Ru er Bo kovi Institute, P.O.B. 180, HR-10002, Zagreb, Croatia
b
PLIVA d.d., Research Institute, Prilaz baruna Filipovi a 25, HR-10000, Zagreb, Croatia
Fax +385(1)4680195; E-mail: sunjic@rudjer.irb.hr
Received 6 April 2001; revised 12 July 2001
Abstract: Racemic cytoxazone ( -5) was synthesized starting from
easily available glycidic ester ( )-1 by nucleophilic epoxide ring
opening, followed by 2-oxazolidinone ring construction and calci-
um chloride/sodium borohydride reduction of the intermediary ester
( )-4. Kinetic resolution of ( )-5 performed by acetylation with vi-
nyl acetate catalyzed by Penicillium camemberti lipase (PcamL) af-
forded, on hydrolysis of acetate (–)-6, cytoxazone (–)-5 in 33%
overall yield and 88.2% enantiomeric excess (ee), and its enanti-
omer (+)-5 (38% yield, 89.3% ee). Base-catalyzed epimerization of
intermediary ( )-4 to ( )-7, and reduction and kinetic resolution
with Candida antarctica lipase (CAL) led to epi-cytoxazones (–)-8
and (+)-8.
Key words: oxazolidinones, epoxide ring opening, lipases, kinetic
resolution
(–)-Cytoxazone (–)-5, (4R,5R)-5-(hydroxymethyl)-4-(4-
methoxyphenyl)-1,3-oxazolidin-2-one), a novel cytokine
modulator produced by Streptomyces sp. was isolated
from the fermentation broth in a low yield,1 and its abso-
lute configuration has been determined by X-ray crystal-
lographic analysis and CD-spectroscopy.2 Cytoxazone
and its analogues have become a new subject of synthetic
studies because of their immunostimulating activity.1 As
yet, three syntheses of cytoxazone stereoisomers have
been published. Two of them, reported by Nakata’s
group3 and by Mori’s group4, employed Sharpless asym-
metric dihydroxylation of p-methoxy cinnamic deriva-
tive, while the third route involved imino-1,2-Wittig
rearrangement of hydroximate and conventional optical
resolution of racemates in the final step.5 4-epi-Cytox-
azone has been prepared by asymmetric dihydroxylation
of p-methoxy cinnamic ester followed by cyclic iminocar-
bonate rearrangement.6
Scheme 1 Synthesis of (–)- and (+)-cytoxazone; Reagents and con-
ditions: a) aq NaN3, dioxane, 50 °C, 3 h, 56%; b) ClCO2Ph, CH2Cl2,
–5 °C, 1 h, 100%; c) Ph3P, aq THF, 50 °C, 90 min, 88%; d) NaBH4,
CaCl2, absolute EtOH, r.t., 20 min, 79%; e) PcamL, vinyl acetate,
30 °C; f) KOH, MeOH, r.t., 1 h
accomplished with calcium chloride/sodium borohydride
in absolute ethanol; in the absence of calcium chloride,
slow reaction and poor selectivity was observed. Correla-
Here, we describe the chemoenzymatic synthesis of cy-
toxazone and its stereoisomers starting from readily avail-
able glycidic ester ( )-1,7 Scheme 1.
1
tion of H and 13C NMR spectra of ( )-5 with literature
data for (–)-cytoxazone3 confirmed its structure and cis-
Epoxide ring opening in ( )-1 with sodium azide proceed-
ed, according to a well-known mechanism,8 with com-
plete regio and stereoselectivity affording azido alcohol
( )-2. Construction of the 2-oxazolidinone ring was com-
pleted on conversion of ( )-2 to phenyl carbonate ( )-3
followed by simultaneous reduction and cyclization. Re-
duction of the ester group in cis-oxazolidinone ( )-4 was
configuration.
Kinetic resolution of ( )-5 was attempted by a number of
commercial lipases: Penicillium camambertii (PcamL),
Candida antarctica (CAL) immobilized in Sol-Gel-AK,
Candida antarctica A, Penicillium roqueforti, Humicola
lanuginosa, Pseudomonas fluorescens, Pseudomonas ce-
pacia, Porcine pancreas, Candida cylindracea, Aspergi-
lus niger, Mucor miehei, Mucor iavanicus, Candida
lipolitica, Rhizopus niveus and Rhisopus delemar. Most of
them exhibited low activity and very low enantioselectiv-
Synthesis 2001, No. 13, 08 10 2001. Article Identifier:
1437-210X,E;2001,0,13,1989,1992,ftx,en;Z06201SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

1990
Z. Hamer ak et al.
PAPER
ity (E values < 5); PcamL was selected for the preparative valuable alternative to the earlier reported synthetic ap-
enzymatic resolution (E value = 39).9 At 50.7% conver- proaches to these chiral compounds.
sion, the ee of alcohol (+)-5 was 89.3%, and the ee of ac-
etate (–)-6 was 88.2%. Upon their separation by silica
gel column chromatography and subsequent hydrolysis of
(–)-6, both alcohols were crystallized to obtain (–)-cytox-
azone (–)-5 with 99.0% ee, and (+)-cytoxazone, (+)-5
with 95.2% ee.
1H and 13C NMR spectra were recorded on a Varian Gemini 300 FT
spectrometer; chemical shifts (ppm) are reported downfield from
TMS. IR spectra were recorded on Perkin–Elmer 297 spectrometer.
High resolution mass spectrometry was performed on an Extrel-
FTMS 2001 DD Instrument. Optical rotations were determined on
an AA-10 polarimeter. HPLC analyses were performed on a
Hewlett Packard instrument Series 1050 with UV detector ( = 235
nm) and Hewlett Packard integrator 3396A. Conversions were
( )-epi-Cytoxazone ( )-8 was obtained from common in-
termediate, oxazolidinone ( )-4, which was epimerized at
C(5) using potassium hydroxide in ethanol followed by monitored by HPLC chromatography using Nucleosil C-18 7
m
column (250 4.6 mm); flow rate 0.8 mL/min; using gradient pro-
gram from 10% to 100% MeOH over 20 min. Enzymatic kinetic
resolutions were monitored using Nucleosil C-18 7 m column
(250 4.6 mm) under the following conditions: MeOH–H2O (1:1)
as eluent, flow rate 0.8 mL/min. Determination of the enantiomeric
purity was performed using Chiralcel AS column (250 4.6 mm)
with Chiralcel AS precolumn (50 4.6 mm) in n-hexane–i-PrOH
(1:1) for ( )-5, and (7:4.5) for ( )-8, flow rate 1.0 mL/min, method
accuracy 0.2%. Enzymatic acylations were performed in a thermo-
stated shaker (Tehtnica Co.) at 220 rpm. Lipases from the following
microorganisms (purchased from Amano Co.) were tested: Penicil-
lium camambertii, Candida antarctica immobilized in Sol-Gel-AK,
Candida antarctica A, Penicillium roqueforti, Humicola lanugi-
nosa, Pseudomonas fluorescens, Pseudomonas cepacia,
Pseudomonas cepacia immobilized on ceramics, Porcine pancreas,
Candida cylindracea, Aspergilus niger, Mucor miehei, Mucor ia-
vanicus, Candida lipolitica, Rhizopus niveus and Rhisopus delemar.
esterification with methyl iodide (Scheme 2). Reduction
of ( )-7 with calcium chloride/sodium borohydride af-
forded ( )-8, whose structure was confirmed by correla-
tion of 1H and 13C NMR data with those reported for (–)-
epi-cytoxazone.3
Methyl 3-azido-2-hydroxy-3-(4-methoxyphenyl)propanoate
[( )-2]
To a solution of NaN3 (2.90 g, 52 mmol) in H2O (30 mL), a solution
of epoxide ( )-1 (6.60 g, 31 mmol) in dioxane (80 mL) was added.
The reaction mixture was stirred at 50 °C for 3 hours. EtOAc (80
mL) was added, the solution was washed with H2O (3 50 mL), and
dried. Upon evaporation of the solvent, crude product was purified
by column chromatography on silica gel (EtOAc–hexane, 2:8)
yielding 4.29 g (56%) of oily azide ( )-2, which was 98% pure by
HPLC.
IR (neat): 3500, 2960, 2100, 1745, 1615, 1515, 1250 cm–1.
1H NMR (CDCl3): = 3.03 (d, 1 H, J = 6.5 Hz), 3.72 (s, 3 H), 3.80
(s, 3 H), 4.50 (dd, 1 H, J = 6.5, 4.0 Hz), 4.83 (d, 1 H, J = 4.0 Hz),
6.90 (d, 2 H, J = 8.5 Hz), 7.27 (d, 2 H, J = 8.5 Hz).
13C NMR (CDCl3): = 52.6, 55.1, 66.4, 73.5, 113.9, 126.0, 129.0,
159.8, 171.7.
Scheme 2 Synthesis of (–)- and (+)-epi-cytoxazone; a) KOH,
EtOH, reflux, 1 h; b) Mel, K2CO3, DMF, r.t., 16 h, 46%; c) NaBH4,
CaCl2, absolute EtOH, r.t., 20 min, 82%; d) CAL in SOL-Gel-AK,
vinyl acetate, 30 °C; e) KOH, MeOH, r.t., 1 h
HRMS: m/z calcd. for C11H13N3O4 [M+]: 252.0978. Found:
Interestingly, epi-racemate ( )-8 proved a better substrate
for microbial lipases than ( )-5. Penicillium camambertii,
Candida antarctica immobilized in Sol-Gel-AK, Candida
antarctica A, Penicillium roqueforti, Humicola lanugi-
nosa, Pseudomonas fluorescens and Pseudomonas cepa-
cia immobilized on ceramics exhibited high activity, but
most of them showed relatively low enantioselectivity (E
252.0891.
Methyl-3-azido-3-(4-methoxyphenyl)-2-[(phenoxycarbonyl)-
oxy]propanoate [( )-3]
To a solution of azide ( )-2 (3.80 g, 16 mmol) and pyridine (1.4 mL,
17 mmol) in anhyd CH2Cl2 (60 mL), a solution of phenylchlorofor-
mate (2.2 mL, 2.75 g, 17 mmol) in CH2Cl2 (5.0 mL) was added at -
5 °C over 10 min. After stirring at –5 °C for 1 h, the reaction mix-
values 2–5). With CAL in Sol-Gel-AK, the best selectivi- ture was poured into H2O. The organic layer was washed with 1%
H3PO4, then with 3% NaHCO3, and dried. Upon evaporation of the
solvent, 6.10 g (quantitative) of oily ( )-3 was obtained; HPLC
analysis revealed 97% purity.
IR (neat): 2955, 2100, 1760, 1610, 1510, 1245 cm–1.
1H NMR (CDCl3): = 3.75 (s, 3 H), 3.83 (s, 3 H), 5.05 (d, 1 H,
J = 5.0 Hz), 5.35 (d, 1 H, J = 5.0 Hz), 6.82–7.46 (m, 9 H).
13C NMR (CDCl3): = 52.4, 54.7, 63.7, 76.9, 113.7, 113.9, 120.4,
124.9, 125.8, 128.6, 129.0, 150.4, 152.2, 159.7, 166.6, 166.8.
ty was obtained (E value = 37), so this lipase was selected
for the preparative resolution; at 49.1% conversion, ee of
alcohol (–)-8 was 84.2%, and ee of acetate (+)-9 was
87.3%, which was hydrolyzed to (+)-8 with 79.1% ee.
In conclusion, all four cytoxazone stereoisomers have
been prepared by the chemoenzymatic route starting from
easily available glycidic ester ( )-1. This method is a
Synthesis 2001, No. 13, 1989–1992 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Chemoenzymatic Synthesis of All Four Cytoxazone Stereoisomers
1991
Methyl 4-(4-Methoxyphenyl)-2-oxo-1,3-oxazolidine-5-carb-
oxylate [( )-4]
HRMS: m/z calcd. for C12H13NO5 [M+]: 252.0866. Found:
252.0792.
Azido-ester ( )-3 (6.0 g, 16 mmol) and Ph3P (12.6 g, 48 mmol) were
dissolved in THF (160 mL) and H2O (3.3 mL). The reaction mixture
was heated at 50 °C for 1.5 h. Evolution of N2 was observed during
the first 30 min of the reaction. The solvent was evaporated, the sol-
id residue was dissolved in EtOAc (150 mL), washed with 5% NaCl
(3 50 mL), and dried. Crude product was crystallized from i-Pr2O
to obtain 3.55 g (88%) of oxazolidinone ( )-4, which was 98% pure
by HPLC.
5-epi-5-(Hydroxymethyl)-4-(4-methoxyphenyl)-1,3-oxazolidin-
2-one [( )-8]
5-epi-Cytoxazone [( )-8] was obtained in 82% yield, as described
for ( )-5.
Mp 158–159 °C.
IR (KBr): 3290, 2920, 1720, 1510, 1240, 1025, 830 cm–1.
1H NMR (CD3OD): = 3.39 (br s, 1 H), 3.77 (dd, 1 H, J = 12.0, 3.0
Hz), 3.88 (s, 3 H), 3.90 (dd, 1 H, J = 12.0, 3.0 Hz), 4.35–4.45 (m, 1
H), 4.83 (d, 1 H, J = 6.5 Hz), 7.05 (d, 2 H, J = 8.5 Hz), 7.39 (d, 2 H,
J = 8.5 Hz).
13C NMR (CD3OD): = 55.9, 58.7, 62.6, 87.0, 115.5, 128.7, 133.7,
161.0, 161.5.
Mp 160–161 °C.
IR (KBr): 3240, 2945, 1745, 1720, 1510, 1460, 1215, 1090 cm–1.
1H NMR (CDCl3): = 3.39 (s, 3 H), 3.80 (s, 3 H), 5.19 (d, 1 H,
J = 9.0 Hz), 5.26 (d, 1 H, J = 9.0 Hz), 6.21 (br s, 1 H), 6.87 (d, 2 H,
J = 8.5 Hz), 7.21 (d, 2 H, J = 8.5 Hz).
13C NMR (CDCl3): = 51.9, 55.2, 57.8, 78.0, 113.9, 128.0, 128.5,
158.1, 160.1, 166.7.
Preparative Kinetic Resolution of [( )-5] Catalyzed by Lipase
from Penicillium camembertii
HRMS: m/z calcd. for C12H13NO5 [M+]: 252.0866. Found:
To the solution of ( )-5 (200 mg) in vinyl acetate (140 mL), lipase
from PcamL (4.0 g) was added. The reaction was performed in a
thermostated shaker at 30 °C and 220 rpm. After 9 days, at 50.7%
conversion, the reaction mixture was filtered. The filtrate was evap-
orated and products were separated on a silica gel column (EtOAc–
CH2Cl2, 3:1). Combined fractions yielded pure (+)-5 (81 mg, 89.3%
ee) and (–)-6 (92 mg, 88.2% ee). Acetate (–)-6 was hydrolysed with
KOH/MeOH (1.1 mol equiv of KOH) at r.t. Upon crystallization
from MTBE, (–)-5 was obtained in 99.0% ee, and (+)-5 in 95.2% ee.
252.0811.
5-(Hydroxymethyl)-4-(4-methoxyphenyl)-1,3-oxazolidin-2-one
[( )-5]
Oxazolidinone ( )-4 (50 mg, 0.22 mmol) was dissolved in absolute
EtOH (4 mL), CaCl2 (50 mg, 0.45 mmol) and NaBH4 (40 mg, 1.0
mmol) was added, and reaction mixture was stirred for 20 min at r.t.
The excess of reducing agent was destroyed by the addition of sat.
NH4Cl (0.5 mL), EtOH was evaporated; the residue was dissolved
in EtOAc (30 mL), washed with 5% NaCl (2 20 mL), and dried.
Crude product was crystallized from t-butyl methyl ether (MTBE)–
MeOH (95:5) to give 35 mg (79%) of ( )-5, which was >99% pure
by HPLC.
(–)-5
Mp 122–123 °C (Lit.3 mp 122–123 °C).
[ ]D25 –74.0 (c 0.5, MeOH) [Lit.3 [ ]D –75.7 (c 1.00, MeOH)].
(+)-5
Mp 143–144 °C.
[ ]D25 +70.1 (c 0.5, MeOH).
IR (KBr): 3350, 3240, 2940, 1720, 1215, 1025, 965 cm–1.
1H NMR (acetone-d6): = 3.15–3.26 (m, 3 H), 3.80 (s, 3 H), 4.82
(ddd, 1 H), 5.03 (d, 1 H, J = 8.0 Hz), 6.94 (d, 2 H, J = 8.5 Hz), 6.96
(br s, 1 H), 7.24 (d, 2 H, J = 8.5 Hz).
(–)-6
[ ]D25 –45.2 (c 1.01, MeOH).
13C NMR (acetone-d6): = 54.9, 57.2, 61.9, 80.8, 113.9, 128.3,
129.6, 158.8, 159.9.
Preparative Kinetic Resolution of [( )-8] Catalyzed by Lipase
from Candida antarctica Immobilised in Sol-Gel-AK
To a solution of ( )-8 (157 mg) in vinyl acetate (220 mL), lipase
from CAL in SOL-Gel-AK (455 mg) was added. Using the same
conditions and isolation procedure as described for ( )-5, pure alco-
hol (–)-8 (68 mg, 84.2% ee), and acetyl derivative (+)-9 (90 mg,
87.3% ee) were obtained. Acetyl derivative (+)-9 was hydrolyzed
with KOH–MeOH (1.1 equiv) at r.t. affording 58 mg of (+)-8
(79.1% ee). Upon crystallization from MTBE, both enantiomers
were obtained with >96% ee.
Methyl 4-(4-Methoxyphenyl)-2-oxo-1,3-oxazolidine-5-carb-
oxylate [( )-7]
To a solution of ( )-4 (1.30 g, 5.1 mmol) in EtOH (6 mL), KOH
(320 mg, 5.7 mmol) was added. The resulting mixture was kept at
reflux for 1 h, then cooled to r.t., acidified with 10% HCl, and ex-
tracted with EtOAc (3 30 mL). Combined extracts were washed
with H2O (30 mL), dried (Na2SO4), and evaporated in vacuo. The
residue was dissolved in DMF (15 mL), K2CO3 (700 mg, 5.1 mmol)
and MeI (980 mg, 7.0 mmol) were added. The reaction mixture was
stirred overnight at r.t., then H2O (30 mL) and EtOAc (60 mL) were
added. The organic layer was successively washed with H2O
(5 30 mL), dried, and evaporated. Crude product was purified by
silica gel chromatography (MTBE–hexane, 4:1). Evaporation of the
fractions with ( )-7 afforded 530 mg (46%) of pure product (>99%
by HPLC).
(–)-8
Mp 160–161 °C (Lit.3 mp 161.5–162.5 °C).
[ ]D25 –29.7 (c 1.32, MeOH) [Lit.3 [ ]D –30.4 (c 1.01, MeOH)].
(+)-8
Mp 159–160 °C.
[ ]D25 +28.8 (c 0.59, MeOH).
(+)-9
Mp 123–124 °C.
[ ]D25 +42.7 (c 1.68, MeOH).
IR (KBr): 3270, 2960, 1760, 1710, 1515, 1255, 1205, 1090, 920,
835 cm–1.
1H NMR (CDCl3): = 3.81 (s, 3 H), 3.86 (s, 3 H), 4.72 (d, 1 H,
J = 5.2 Hz), 4.93 (d, 1 H, J = 5.2 Hz), 6.60 (br s, 1 H), 6.92 (d, 2 H,
J = 8.1 Hz), 7.28 (d, 2 H, J = 8.1 Hz).
13C NMR (CDCl3): = 52.9, 55.2, 58.6, 80.3, 114.4, 127.1, 130.6,
157.8, 159.9, 168.7.
Acknowledgement
This work was completed within a collaboration project with PLI-
VA Co., Zagreb.
Synthesis 2001, No. 13, 1989–1992 ISSN 0039-7881 © Thieme Stuttgart · New York

1992
Z. Hamer ak et al.
PAPER
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Synthesis 2001, No. 13, 1989–1992 ISSN 0039-7881 © Thieme Stuttgart · New York