Resolution of cis-2-fluorocyclopropanecarboxylic acid by a microbial enantioselective hydrolysis

Article abstract of DOI:10.1016/S0957-4166(98)00321-8

The important key intermediate of quinolone analogue synthesis, (1S,2S)- 2-fluorocyclopropanecarboxylic acid, was prepared enantioselectively by a microbial resolution. One of the strains with the highest enzymatic specificity was selected from soil and when lyophilized cells were treated with corresponding ester, the remaining (1S,2S)-ester was obtained with high enantiomeric purity (98% e.e.).

Full text of DOI:10.1016/S0957-4166(98)00321-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 3047–3052
Resolution of cis-2-fluorocyclopropanecarboxylic acid by a
microbial enantioselective hydrolysis
Akihiro Imura,∗ Motohiro Itoh and Akihiko Miyadera
Chemical Technology Research Laboratories, Daiichi Pharmaceutical Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku,
Tokyo 134-8630, Japan
Received 29 June 1998; accepted 3 August 1998
Abstract
The important key intermediate of quinolone analogue synthesis, (1S,2S)-2-fluorocyclopropanecarboxylic acid,
was prepared enantioselectively by a microbial resolution. One of the strains with the highest enzymatic specificity
was selected from soil and when lyophilized cells were treated with corresponding ester, the remaining (1S,2S)-
ester was obtained with high enantiomeric purity (98% e.e.). © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
Utilization of optically active fluoroorganic compounds in biological and medicinal chemistry1 con-
tinues to expand and requires the development of a method of synthesis with high stereoselectivity,
because of the unique physical and biological features resulting from fluorine. Hayakawa et al. dis-
covered 7-[7(S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-quinolinecarboxylic acid 1,2
a new-generation antibacterial quinolone carboxylic acid with broad-spectrum antibacterial activity.
A number of syntheses of the important key intermediate, cis-2-fluorocyclopropylamine 2 and cis-2-
fluorocyclopropanecarboxylic acid 3, have been reported (Scheme 1).3 Although a few methods of
resolution of 3 have also been reported, an expensive chiral reagent is used4 and chromatography is
required for the separation of diastereomers.5 Therefore, practical access to (1S,2S)-3 is limited in the
above two syntheses.
On the other hand, an enzymatic approach to the synthesis of monofluorinated compounds has been
reported.6 Furthermore, it is particularly interesting to compare fluorine with other halogens or alkyl
groups, to confirm the mimic effect of the fluorine atom.7
In this paper, we describe, as shown in Scheme 2, the first synthesis of the key chiral compound 7
using a microbial resolution.
∗
Corresponding author. E-mail: imuraycm@daiichipharm.co.jp
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00321-8
tetasy 2470 Article

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A. Imura et al. / Tetrahedron: Asymmetry 9 (1998) 3047–3052
Scheme 1.
2. Results and discussion
The synthesis of cis-ethyl-2-fluorocyclopropanecarboxylate 4a was achieved by Kusuyama’s route,8
derived from the cyclopropanation of butadiene with bromofluorocarbene as the intermediate. We
selected this cis-ethyl ester 4a as the substrate for attempted resolution by a microbial enantioselective
hydrolysis.
Scheme 2.
In our first trial of an enantioselective hydrolysis reaction, about 100 commercially available enzymes
such as esterases, lipases and proteases were screened for their ability to hydrolyze the ester 4a. Although
many enzymes especially esterases and lipases hydrolyzed 4a, they had poor specificity, and the acid
5 was formed as a racemate. We therefore attempted to hydrolyze 4a by use of microorganisms. A
few strains from cultures were found to afford good enantiomeric excess of acetate 6a. However, the
configuration of acetate was R, and the e.e. value of the (S)-acid, which is the desirable configuration,
was only 27–68% (runs 1–4, Table 1). We directed our efforts to obtain the highest e.e. of the (S)-
acetate. One of the strains, which was selected from soil, exhibited better enantiomeric discrimination,
and hydrolyzed the (R)-enantiomer. We found the DSC 4011 strain yielded good to excellent e.e. of the
(S)-acetate in a reasonable time (run 5).
The microbial reaction was applied to other aliphatic esters 4b–4f to study the effect of chain length
on reaction rate and enantioselectivity. The reaction was monitored by GC analysis and stopped when ca.
55% conversion was achieved. As shown in Table 2, 4b was the most reactive substrate for the DSC 4011
among the six substrates examined, and longer reaction times were required for substrates with longer
chains. The reaction time required to achieve 54% conversion was 144 h for 4e, and 10 times longer
than that for 4b. For 4f, the conversion ratio was only 24% at 144 h and never reached 50%, even after
a prolonged reaction time. For 4a–4e, excellent e.e. values of the (S)-ester were obtained. Therefore the
chain length of the alkyl group was found to have little effect on enantioselectivity.

A. Imura et al. / Tetrahedron: Asymmetry 9 (1998) 3047–3052
3049
Table 1
Screening of enzymes to catalyze hydrolysis of racemate 4a
Table 2
Effect of the chain length of alkyl group 4a–4f
3. Conclusion
We have demonstrated a highly enantioselective preparation of the optically active 2-
fluorocyclopropanecarboxylic acid 7 using a microbial hydrolysis with the corresponding esters.
We are interested in the chiral discrimination of the fluorine atom, and the substrate specificity of other
cyclopropyl compounds by the purified enzyme from the DSC 4011. These studies are in progress.

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4. Experimental section
4.1. General procedures
Melting points were determined on a Yanagimoto apparatus and are uncorrected. Infrared spectra
1
were recorded on an FT-720 spectrometer (Horiba). H-NMR spectra were recorded on a JEOL JNM-
EX270 (270 MHz) instrument. Coupling constants are reported in hertz (Hz) and chemical shifts in
ppm downfield from internal TMS. 19F-NMR spectra were recorded on a JEOL JNM-GSX500 (500
MHz) instrument and the values are reported on the δ scale relative to hexafluorobenzene as an internal
standard. Mass spectra were recorded on a JEOL JMS-HX110 or JMS-AX505W mass spectrometer.
Optical rotations were measured with a SEPA-300 polarimeter (Horiba). All chemicals were obtained
from commercial sources and were used without further purification.
4.2. Microorganisms, media and culture conditions
Bacteria were obtained from culture collections, except for the Micrococcus sp. (DSC 4011). This
was selected from soil, cultivating with cis-ethyl-2-fluorocyclopropanecarboxylate 4a as the sole carbon
source at 30°C for 7 days. The identification of bacteria (DSC 4011) was based on Bergey’s Manual of
Determinative Bacteriology. Cultures were performed with a reciprocating shaker (150 rpm/min) on the
following medium; pepton (10 g/L), meat extract (3 g/L), NaCl (5 g/L) at pH 7.0 after sterilization
(120°C, 15 min). At the late exponential growth phase, the cells were harvested by centrifugation
(3000×g), resuspended in phosphate buffer (50 mM, pH 7.0), centrifuged again and lyophilized. The
cells were stored for several months at 5°C without significant loss of activity.
4.3. Measurement of enantiomeric purity
Enantiomeric excesses were analyzed by GLC (Shimadzu GC-14A) on a CP cyclodextrin-β-236M
capillary column (0.25 mm×25 m, Chrompack Co., Ltd.) with flame ionization detection, after derivati-
zation of the carboxylic acid 5 to the methyl ester by diazomethane, at an oven temperature of 70°C. A
typical separation is illustrated in Fig. 1.
4.3.1. cis-Ethyl-2-fluorocyclopropanecarboxylate 4a
cis-2-Fluorocyclopropanecarboxylic acid (12.0 g, 115 mmol) was dissolved in ethanol (150 mL), and
sulfuric acid (0.5 g) was added to the solution. The mixture was refluxed for 8 h, and evaporated in
vacuo. The residue was extracted with dichloromethane (150 mL), and washed with water (3×100 mL).
The organic layer was dried and distilled in vacuo to obtain 4a as a colorless liquid (13.7 g, 90.3%). Bp
1
49–51°C (24 mmHg); H-NMR (CDCl3) δ: 1.04–1.18 (m, 1H), 1.29 (t, J=7.3 Hz, 3H), 1.74–1.89 (m,
2H), 4.18–4.26 (q, J=7.3 Hz, 2H), 4.73 (dm, J=70 Hz); 19F-NMR (CDCl3) δ: −59.4 (m, CHF); m/z 133
(M++1).
4.3.2. cis-Propyl-2-fluorocyclopropanecarboxylate 4b
Yield 92.9%; bp 78–80°C (24 mmHg); 1H-NMR (CDCl3) δ: 0.96 (t, J=7.6 Hz, 3H) 1.01–1.20 (m, 1H),
1.29 (q, J=7.6 Hz, 3H), 1.74–1.89 (m, 2H), 4.18–4.26 (q, J=5.9 Hz, 2H), 4.73 (dm, J=70 Hz); 19F-NMR
(CDCl3) δ: −59.3 (m, CHF); m/z 147 (M++1)

A. Imura et al. / Tetrahedron: Asymmetry 9 (1998) 3047–3052
3051
Fig. 1. GC of methyl ester of 7, (ꢀ)-form (left) or (+)-form from microbial resolution (right)
4.3.3. cis-Butyl-2-fluorocyclopropanecarboxylate 4c
1
Yield 90.8%; bp 68–70°C (15 mmHg); H-NMR (CDCl3) δ: 0.94 (t, J=7.6 Hz, 3H), 1.04–1.23 (m,
1H), 1.30–1.69 (m, 4H), 1.73–1.92 (m, 2H), 4.15 (t, J=6.6 Hz, 2H), 4.74 (dm, J=70 Hz); 19F-NMR
(CDCl3) δ: −59.2 (m, CHF).
4.3.4. cis-Pentyl-2-fluorocyclopropanecarboxylate 4d
1
Yield 89.8% (purified by silica); H-NMR (CDCl3) δ: 0.91 (t, J=7.6 Hz, 3H), 1.02–1.19 (m, 1H),
1.29–1.72 (m, 6H), 1.74–1.93 (m, 2H), 4.13 (t, J=6.9 Hz, 2H), 4.74 (dm, J=69 Hz).
4.3.5. cis-Hexyl-2-fluorocyclopropanecarboxylate 4e
1
Yield 87.6% (purified by silica); H-NMR (CDCl3) δ: 0.90 (t, J=7.4 Hz, 3H), 1.01–1.18 (m, 1H),
1.30–1.89 (m, 10H), 4.14 (t, J=6.8 Hz, 2H), 4.74 (dm, J=70 Hz)
4.3.6. cis-Octyl-2-fluorocyclopropanecarboxylate 4f
1
Yield 83.6% (purified by silica); H-NMR (CDCl3) δ: 0.88 (t, J=7.4 Hz, 3H), 1.03–1.17 (m, 1H),
1.31–1.94 (m, 14H), 4.13 (t, J=6.8 Hz, 2H), 4.75 (dm, J=69 Hz).
4.4. Microbial hydrolysis of 4b
Lyophilized microbial cells (DSC 4011, 2.0 g) were suspended in phosphate buffer (500 mL, 0.1 M, pH
7.0) and stirred for 15 min at 40°C. cis-Propyl-2-fluorocyclopropanecarboxylate 4b (20.0 g, 136 mmol)
was then added and the whole mixture was stirred at 40°C, while the reaction was monitored by GC. As
the pH value of the mixture was decreased by formed acid, it was maintained at pH 7.0 by the addition
of 10% aqueous sodium hydroxide with an autotitrator. After an appropriate degree of conversion (0.55)
was reached (14 h), ethyl acetate was added (400 mL) and the cells were filtered through Celite. The
aqueous layer of the filtrate was extracted with ethyl acetate. The combined organic layers were washed
with water, dried and evaporated in vacuo. The residue was dissolved in 50% methanol (100 mL) and
sodium hydroxide (3.2 g, 80 mmol) was added at 0°C. The mixture was left at room temperature (2

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A. Imura et al. / Tetrahedron: Asymmetry 9 (1998) 3047–3052
h) then evaporated to half volume, and adjusted to pH 2 with 10% hydrochloric acid, and extracted
with ethyl acetate. The organic layer was evaporated in vacuo to obtain a colorless solid of (1S,2S)-2-
fluorocyclopropanecarboxylic acid 7 (5.95 g, 38.0%, 98% e.e.). [α]D25 +23.0 (c=0.50, CHCl3). The solid
was crystallized from toluene to obtain colorless crystals (>99% e.e.). Mp 62–63°C; 1H-NMR (CDCl3)
δ: 1.15–1.32 (m, 3H), 1.56–1.98 (m, 2H), 4.76 (dm, J=69 Hz, 1H), 8.76 (brs, 1H); 19F-NMR (CDCl3) δ:
−58.6 (m, CHF); m/z 104 (M+); IR (KBr) 3210, 1727, 1439 and 1190 cm−1; anal. calcd for C4H5FO2:
C, 46.16; H, 4.84; F, 18.26. Found: C, 46.17; H, 4.80; F, 18.23. The aqueous layer of the filtrate was
adjusted to pH 2 and extracted with ethyl acetate. The organic layer was evaporated in vacuo to obtain
25
a colorless solid of (1R,2R)-2-fluorocyclopropanecarboxylic acid 5 (8.45 g, 54.1%, 78% e.e.). [α]D
−18.9 (c=0.50, CHCl3). The solid was crystallized from toluene to obtain colorless crystals (84% e.e.).
1
Mp 59–61°C; H-NMR (CDCl3) δ: 1.15–1.32 (m, 3H), 1.56–1.98 (m, 2H), 4.76 (dm, J=69 Hz, 1H),
8.76 (brs, 1H); 19F-NMR (CDCl3) δ: −58.6 (m, CHF); m/z 104 (M+); IR (KBr) 3210, 1724, 1440 and
1192 cm−1; anal. calcd for C4H5FO2: C4H5FO2: C, 46.16; H, 4.84; F, 18.26. Found: C, 46.27; H, 4.82;
F, 18.20.
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