Microbial enantioselective ester hydrolysis for the preparation of optically active 4,1-benzoxazepine-3-acetic acid derivatives as squalene synthase inhibitors

Article abstract of DOI:10.1248/cpb.50.59

Microbial enantioselective ester hydrolysis for the preparation of optically active (3R,5S)-(-)-5-phenyl-4,1-benzoxazepine-3-acetic acid derivatives as potent squalene synthase inhibitors was investigated. Pseudomonas diminuta and Pseudomonas taetrolens h

Full text of DOI:10.1248/cpb.50.59

January 2002
Chem. Pharm. Bull. 50(1) 59—65 (2002)
59
Microbial Enantioselective Ester Hydrolysis for the Preparation of
Optically Active 4,1-Benzoxazepine-3-acetic Acid Derivatives
as Squalene Synthase Inhibitors1)
Naoki TARUI,a Kazuo NAKAHAMA,a Yoichi NAGANO,a Motowo IZAWA,a Kiyoharu MATSUMOTO,a
a
Masakuni KORI,a Toshiaki NAGATA,b Takashi MIKI,*,a and Hidefumi YUKIMASA
Takeda Chemical Industries, Ltd., Pharmaceutical Research Divisiona and Pharmaceutical Production Division,b 2–17–85
Juso-Honmachi, Yodogawa-ku, Osaka 532–8686, Japan. Received July 30, 2001; accepted October 1, 2001
Microbial enantioselective ester hydrolysis for the preparation of optically active (3R,5S)-(؊)-5-phenyl-4,1-
benzoxazepine-3-acetic acid derivatives as potent squalene synthase inhibitors was investigated. Pseudomonas
diminuta and Pseudomonas taetrolens hydrolyzed the racemic ethyl ester of the 5-(2-chlorophenyl) analogue to
yield the (؊)-carboxylic acid with excellent enantiomeric excess (Ͼ99% ee). We found that the (؊)-enantiomer
was an active inhibitor. Bulkiness of the ester moiety did not affect the enantioselectivity but did affect reac-
tivity. The racemic ethyl ester of the 5-(2-methoxyphenyl) analogue, 5-(2,3-dimethoxyphenyl) analogue and 5-
(2,4-dimethoxyphenyl) analogue were also hydrolyzed with Pseudomonas taetrolens to afford enantiomerically
pure (؊)-carboxylic acids in large scale. As another route to (3R,5S)-(؊)-7-chloro-5-(2,3-dimethoxyphenyl)-1-
neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid [(؊)-1c], the earlier intermediate (؊)-2-
amino-5-chloro-a-(2,3-dimethoxyphenyl)benzyl alcohol [(؊)-12] was successfully obtained by asymmetric hy-
drolysis of (؎)-5-chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl acetate with Pseudomonas sp. S-13 with
Ͼ99% ee in kilogram scale followed by alkaline treatment. The product (؊)-12 was converted to (؊)-1c without
racemization.
Key words squalene synthase; 4,1-benzoxazepine-3-acetic acid; asymmetric hydrolysis; Pseudomonas taetrolens; Pseudomonas sp.
S-13; hypocholestemic agent
In previous papers,1) we have reported the syntheses of a
practical process for producing the optically active isomer
was required in order to obtain quantities sufficient for ex-
tended biological studies.
Kinetic asymmetric resolution with microorganisms has
received considerable attention because of its high enantiose-
lectivity.2) Since there are currently a great number of differ-
series of 4,1-benzoxazepine-3-acetic acid derivatives, which
led to the identification of potent squalene synthase in-
hibitors, such as (Ϯ)-1a—d (Fig. 1). These compounds were
first prepared as racemates, as shown in Chart 1. Racemic
aminoalcohols (Ϯ)-2 which were obtained by reduction of 2-
aminobenzophenones, were treated with aldehydes and
sodium cyano borohydride (NaBH3CN) to afford the alky-
lated compounds (Ϯ)-3. After condensation of (Ϯ)-3 with
fumaric acid chloride monoethyl ester 4, intramolecular
Michael addition of the resulting amides (Ϯ)-5 afforded ethyl
4,1-benzoxazepine-3-acetates (Ϯ)-6. In this reaction, the
thermodynamically stable 3,5-trans-isomers were obtained,
which were hydrolyzed to give the carboxylic acids (Ϯ)-1. To
clarify which optical isomer is the more active inhibitor, opti-
cal resolution of compound (Ϯ)-1c was carried out via chro-
matographic separation of the diastereomeric L-amino acid
ester amide derivatives. We found the (Ϫ)-(3R,5S)-1c exhib-
ited remarkably potent inhibition of both rat and human
HepG2 enzymes, while the (ϩ)-(3S,5R)-1c exhibited modest
inhibition.1b) However, since this optical resolution method
was not appropriate for large scale synthesis, an efficient and
Reagents: (a) tert-BuCHO, NaBH4 or NaBH3CN, AcOH; (b) NaHCO3, CH2Cl2; (c)
K2CO3, EtOH; (d) K2CO3, MeOH–H2O.
Chart 1
Fig. 1. Structures of 4,1-Benzoxazepine-3-acetic Acid Derivatives
To whom correspondence should be addressed. e-mail: Miki_Takashi@takeda.co.jp
© 2002 Pharmaceutical Society of Japan

60
Vol. 50, No. 1
Table 1. Asymmetric Hydrolysis of (Ϯ)-Ethyl (3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate
[(Ϯ)-6a] with Various Microorganisms
Product
Microorganism
No.
Conversion ratea) (%)
Optical puritya) (% ee)
Pseudomonas diminuta IFO 13182
Pseudomonas taetrolens IFO 12691
Pseudomonas vesicularis IFO 12165
Pseudomonas aeruginosa IFO 3923
Bacillus subtilis IFO 3026
(Ϫ)-1a
(Ϫ)-1a
(Ϫ)-1a
(ϩ)-1a
(ϩ)-1a
27.8
26.7
9.0
3.1
12.9
Ͼ99
Ͼ99
Ͼ99
Ͼ99
Ͼ99
a) Determined by HPLC analysis [column: Ultron ES-OUM (Shinwa Chemical Industries, Ltd.); mobile phase: 20 mM KH2PO4 (pH 3.5)–CH3CN (75 : 25)].
ent readily available microorganisms for this purpose, it
should be possible to find a microorganism suitable for a de-
sired transformation by means of random screening. This
prompted us to examine optical resolution using microorgan-
isms,3) and we describe here the microbial asymmetric hy-
drolysis of racemic 4,1-benzoxazepine-3-acetic acid ethyl es-
ters (Ϯ)-6a—d. We also report the kinetic resolution of an
earlier synthetic intermediate, 2-amino-a-phenylbenzylalco-
hol derivative (Ϯ)-13.
Table 2. Squalene Synthase Inhibitory Activities and Optical Rotations of
(Ϯ)-, (Ϫ)-, and (ϩ)-(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-
oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid 1a
IC50 (mM)a)
Results and Discussion
Microbial Asymmetric Hydrolysis of Racemic Esters
(؎)-6a—d Initially over 400 strains of bacteria were
screened in order to identify those microorganisms that were
capable of catalyzing enantioselective hydrolysis of (Ϯ)-6a
and we selected 5 strains of bacteria. The hydrolysis of
(Ϯ)-6a was carried out by incubation for 4.5 h at 28 °C.
Pseudomonas diminuta IFO 13182, Pseudomonas taetrolens
IFO 12691, and Pseudomonas vesicularis IFO 12165 hy-
drolyzed the substrate stereospecifically to yield (Ϫ)-acid
[(Ϫ)-1a] in excellent enantiomeric excesses (Ͼ99% ee), and
Pseudomonas aeruginosa IFO 3923 and Bacillus subtilis
IFO 3026 hydrolyzed the substrate to yield pure (ϩ)-acid
[(ϩ)-1a] in high enantiomeric excess (Table 1).4) The prod-
ucts (Ϫ)-1a and (ϩ)-1a were isolated, and evaluated for inhi-
bition of squalene synthase prepared from rat liver and
human hepatoma (HepG2) cells according to the method of
Cohen et al. with slight modification.5) Compound (Ϫ)-1a
exhibited more potent inhibition of both rat and human
HepG2 enzymes than racemate (Ϯ)-1a, while (ϩ)-1a exhib-
ited weak inhibition of rat enzyme (Table 2). The above re-
sults indicate that the compound (Ϫ)-1a is an eutomer.
Next, we examined the effect of the ester moiety on enan-
tioselectivity as well as reactivity with Pseudomonas dimin-
uta IFO 13182 and Pseudomonas taetrolens IFO 12691,
which smoothly produced the eutomer (Ϫ)-1a. All reactions
of ethyl, methyl, isopropyl, butyl, phenyl and benzyl esters
[(Ϯ)-6a, (Ϯ)-7—11] with these microorganisms for 16 h at
28 °C yielded (Ϫ)-1a with high optical purities (Ͼ99% ee).
Small esters, ethyl ester (Ϯ)-6a and methyl ester (Ϯ)-7, were
more reactive than other bulky esters [(Ϯ)-8—11] (Table
Compound
[a]D in MeOH
Rat enzyme
HepG2 enzyme
(Ϯ)-1a
(Ϫ)-1a
(ϩ)-1a
0.072b)
0.042
0.024b)
0.016
NTd)
Ϫ252.2° (cϭ0.50)
ϩ244.8° (cϭ0.52)
Ͼ10 (2.8)c)
a) IC50 values were determined by a single experiment run in duplicate. b) Refer-
ences 1a, b. c) % Inhibition at 10Ϫ5 M. d) Not tested.
3).4)
Based on the results obtained above, we applied these mi-
crobial reactions to compounds with other substituents on the
5-phenyl group for large scale synthesis. The ethyl esters
(Ϯ)-6b—d (200—400 g) hydrolysed with Pseudomonas
taetrolens IFO 12691 afforded (Ϫ)-1b—d with excellent
enantioselectivities (Ͼ99% ee) and reasonable yields (23—
37%) (Table 4).4)
The compounds obtained here were evaluated for inhibi-
tion of squalene synthase prepared from HepG2 cells. These
(Ϫ)-isomers were shown to be eutomers, since compounds
(Ϫ)-1b—d were more potent than the corresponding race-
mates (Table 5). Optical rotation of (Ϫ)-1c prepared here
[Ϫ246° (cϭ0.39, MeOH)] was consistent with that of the au-
thentic sample (Ϫ)-(3R,5S)-1c [Ϫ247° (cϭ0.43, MeOH)] re-
ported in a previous paper.1b) Since the absolute configration
of (Ϫ)-1c was determined to be 3R,5S, the stereochemistry
of the other eutomers (Ϫ)-1a, b, d should also be 3R,5S.
Preparation of 2-Amino-a-phenylbenzylalcohol Deriv-
ative (؊)-2c Our next approach to obtain the chiral com-
pound 1 was by optical resolution of an earlier synthetic in-

January 2002
61
Table 3. Asymmetric Hydrolysis of Various Esters with Pseudomonas taetrolens IFO 12691 and Pseudomonas diminuta IFO 13182
Substrate
Pseudomonas taetrolens IFO 12691
Conversion ratea) (%) Optical puritya) (% ee)
Pseudomonas diminuta IFO 13182
Conversion ratea) (%) Optical puritya) (% ee)
R
No.
Methyl
Ethyl
Isopropyl
n-Butyl
Phenyl
Benzyl
(Ϯ)-7
(Ϯ)-6a
(Ϯ)-8
(Ϯ)-9
(Ϯ)-10
(Ϯ)-11
43.6
Ͼ99
48.2
Ͼ99
45.6
10.4
4.1
24.7
0.7
Ͼ99
Ͼ99
Ͼ99
Ͼ99
Ͼ99
45.0
14.6
8.9
32.0
6.5
Ͼ99
Ͼ99
Ͼ99
Ͼ99
Ͼ99
a) Determined by HPLC analysis [column: Ultron ES-OUM (Shinwa Chemical Industries, Ltd.); mobile phase: 20 mM KH2PO4 (pH 3.5)–CH3CN (75 : 25)].
Table 4. Asymmetric Hydrolysis of 4,1-Benzoxazepine Derivatives by Pseudomonas taetrolens IFO 12691
Substrate
No.
Product
Optical puritya) (% ee)
Condition
R
Amount
No.
Conversion ratesa) (%)
Yield
2-OMe
(Ϯ)-6b
(Ϯ)-6c
(Ϯ)-6c
(Ϯ)-6d
200 g
200 g
400 g
220 g
54 h, 28 °C
42 h, 28 °C
48 h, 28 °C
72 h, 28 °C
(Ϫ)-1b
(Ϫ)-1c
(Ϫ)-1c
(Ϫ)-1d
45.0
44.2
44.7
43.3
Ͼ99
Ͼ99
Ͼ99
Ͼ99
44 g (23%)
67 g (35%)
138 g (37%)
76 g (37%)
2,3-diOMe
2,3-diOMe
2,4-diOMe
a) Determined by HPLC analysis [column: Ultron ES-OUM (Shinwa Chemical Industries, Ltd.); mobile phase: 20 mM KH2PO4 (pH 3.5)–CH3CN (75 : 25)].
Table 5. Squalene Synthase Inhibitory Activities and Optical Rotations
of (Ϫ)- and (Ϯ)-(3,5-trans)-7-Chloro-5-phenyl-1-neopentyl-2-oxo-1,2,3,5-
tetrahydro-4,1-benzoxazepine-3-acetic Acid [(Ϫ)- and (Ϯ)-1a—d]
Reagents: (a) PivCl, NaHCO3, DMAP; (b) Ac2O, DMAP, Pyridine. PivϭPivaloyl.
Chart 2
Compounds
IC50 (mM)a) HepG2 enzyme
[a]D in MeOH
termediate, since this is more efficient and economical, and
we decided to examine the preparation of chiral 2-amino-5-
chloro-a-(2,3-dimethoxyphenyl)benzyl alcohol 2c.6) We
chose 5-chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminoben-
zyl acetate (Ϯ)-13 as a substrate for microbial asymmetric
hydrolysis.
Racemic 2-amino-5-chloro-a-(2,3-dimethoxyphenyl)ben-
zyl alcohol (Ϯ)-2c was converted to N-pivaloyl derivative
(Ϯ)-12, which was reacted with acetic anhydride to provide
the benzyl acetate derivative (Ϯ)-13 (Chart 2). Asymmetric
hydrolysis of the compound (Ϯ)-13 was carried out using 3
No.
X
(Ϫ)-1b
(Ϫ)-1c
(Ϫ)-1d
(Ϯ)-1bc)
(Ϯ)-1cc)
(Ϯ)-1dc)
2-OMe
0.018
0.011b)
0.0086
0.023
0.018
0.020
Ϫ246° (cϭ0.42)
Ϫ246° (cϭ0.39)
Ϫ233° (cϭ0.41)
2,3-diOMe
2,4-diOMe
2-OMe
2,3-diOMe
2,4-diOMe
a) IC50 values were determined by a single experiment run in duplicate. b) The
IC50 value of (Ϫ)-(3R,5S)-1c reported in the reference 1b was 0.013 mM. c) Reference
1b.

62
Vol. 50, No. 1
Table 6. Asymmetric Hydrolysis of (Ϯ)-5-Chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl Acetate [(Ϯ)-13] with Microorganisms
Product
Microorganism
Condition
No.
Conversion ratea) (%)
Optical puritya) (% ee)
[a]D In MeOH
Streptomyces sp. 121-39
Pseudomonas sp. S-13
Bacillus subtilis IFO 14117
48 h, 28 °C
24 h, 28 °C
24 h, 28 °C
(Ϫ)-12
(Ϫ)-12
(ϩ)-12
49
35
49
88
Ͼ98
Ͼ99
Ϫ51.7°b)
(cϭ0.64)
ϩ50.1°c)
(cϭ0.46)
a) Determined by HPLC analysis [column: CHIRALCEL OD (Daicel Chemical Industries, Ltd.); mobile phase: hexane–iso-PrOH (9 : 1)]. b) Value of optically pure (Ϫ)-
12 obtained after recrystallization (tree times) from MeOH–H2O (see Experimental section). c) Value of optically pure (ϩ)-12 obtained after recrystallization from MeOH–H2O
(see Experimental section). PivϭPivaloyl.
Table 7. Physicochemical Data of (Ϫ)- and (ϩ)-(3,5-trans)-7-Chloro-5-
phenyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid Deriva-
tives [(Ϫ)- and (ϩ)-1a—d]
Analysis (%)
mp
Compd.
X
Formula
Calcd (Found)
(°C)
C
H
N
(Ϫ)-1a 2-Cl
(ϩ)-1a 2-Cl
242—245 C22H23Cl2NO4 60.56 5.31 3.21
(60.32 5.51 2.82)
241—245 C22H23Cl2NO4 59.94 5.37 3.18
Chart 3. Conversion of (Ϫ)-12 to (Ϫ)-1c
PivϭPivaloyl.
·1/4H2O
176—180 C23H26ClNO5
·3/2H2O
(60.19 5.47 2.97)
60.19 6.37 3.05
(60.05 5.88 3.22)
62.19 6.22 2.97
(62.40 6.11 3.03)
62.40 6.11 3.03
(62.39 6.20 2.81)
strains selected from about. 400 strains of bacteria (Table
6).4) The reaction with Streptomyces sp. 121-39 for 48 h at
28 °C and with Pseudomonas sp. S-13 for 24 h at 28 °C
yielded (Ϫ)-alcohol [(Ϫ)-12] in 88% ee and Ͼ98% ee, re-
spectively. Bacillus subtilis IFO 14117 hydrolyzed the sub-
strate to yield (ϩ)-alcohol [(ϩ)-12] in a high enantiomeric
ratio (Ͼ99% ee).
(Ϫ)-1b 2-OMe
(Ϫ)-1c 2,3-diOMe 230—233 C24H28ClNO6
(Ϫ)-1d 2,4-diOMe 234—235 C24H28ClNO6
Although hydrolysis using Pseudomonas sp. S-13 yielded
(Ϫ)-12 with a high degree of stereoselectivity, this method
was not satisfactory because of a low conversion rate (35%),
probably due to foaming in the culture inhibiting the micro-
bial reaction. In fact, when large scale hydrolysis [15 kg of
(Ϯ)-13] was carried out using Pseudomonas sp. S-13 for
24 h at 28 °C with the addition of an appropriate amount of
antifoaming agent (Actocol®) to the culture, the conversion
rate was improved to 44% (99% ee).
In order to determine the stereochemistry of the product
(Ϫ)-12, (Ϫ)-12 was converted to (Ϫ)-1c (Chart 3). Removal
of the pivaloyl group of (Ϫ)-12 by alkaline hydrolysis af-
forded the aminoalcohol (Ϫ)-2c and conversion of (Ϫ)-2c to
(Ϫ)-1c was carried out according to a method reported previ-
ously (cf. Chart 1).1) Thus compound (Ϫ)-12 was shown to
have the (S)-configration by its conversion into (Ϫ)-1c, the
stereochemistry of which was previously determined to be
(3R,5S).1b) As determined by HPLC analysis, the optical pu-
rity of (Ϫ)-1c was greater than 99% ee and conversion of
(Ϫ)-12 to (Ϫ)-1c was achieved without racemization.
Conclusions
In this study, we have demonstrated the preparation via mi-
crobial hydrolysis of optically active 4,1-benzoxazepine-3-
acetic acid derivatives [(Ϫ)-1a—d] as potent squalene syn-
thase inhibitors. We investigated optical resolution at the
final step as well as at an earlier step in the synthesis route of
(Ϫ)-1a—d, and found that both of the final intermediates
(Ϯ)-6a—d and the early intermediate (Ϯ)-13 were hydro-
lyzed enantioselectively by the treatment with Pseudomo-
nas, Bacillus, and Streptomyces. The microbial hydrolyses
described here were found to be efficient and practical for
large scale preparation of optically active 4,1-benzoxazepine-
3-acetic acid derivatives [(Ϫ)-1a—d], and in particular the
most potent squalene synthase inhibitor (3R,5S)-(Ϫ)-1c.

January 2002
63
Experimental
Sodium chloride (17 kg) was added to the above reaction mixture after
All melting points were determined on a Yanagimoto micro melting point completion of the reaction and the mixture was adjusted to pH 4 with 2 N
apparatus and are uncorrected. Proton nuclear magnetic resonance (1H- HCl and extracted with AcOEt (80 l and then 60 l). The organic layers were
NMR) spectra were recorded on a Varian GEMINI-300 (300 MHz) spec- combined, washed with 0.5% NaHCO3 (15 lϫ2) and 2% NaCl (75 l), and
trometer (with tetramethylsilane as an internal standard). Infrared (IR) ab- concentrated under reduced pressure to give an oil (295 g). This oil was
sorption spectra were recorded on a Shimazu FT-IR-8200PC. Optical rota- washed with hexane (500 ml and then 200 ml) and suspended in 50% MeOH
tions were determined in the indicated solvents on a JASCO DIP-370 po- (30 l). The suspension was adjusted to pH 7 with concentrated HCl and
larimeter. TLC analyses were carried out on Merck Kieselgel 60 F254 plates. stirred at room temperature for 3 h. This mixture was filtered, and the filtrate
Elemental analyses were carried out by Takeda Analytical Laboratories, was applied to a column of ion exchange resin IRA-68 (1 l, acetate-form).
Ltd., and are within Ϯ0.4% of the theoretical values unless otherwise noted.
The column was washed with 6 l of 70% MeOH, and then 1 N NaOH/70%
For column chromatography, Merck Kieselgel 60 (70—230 mesh ASTM) MeOH was passed through the column to recover 7 l of a crude fraction. The
was used. Yields were not maximized. The following abbreviations are used: fraction was adjusted to pH 4 with concentrated HCl (486 ml), diluted with
sϭsinglet, dϭdoublet, tϭtriplet, qϭquartet, mϭmultiplet, brϭbroad.
Microorganisms and Materials Pseudomonas taetrolens IFO 12691,
Pseudomonas diminuta IFO 13182, Pseudomonas aeruginosa IFO 3923,
water (3 l), and cooled at 7 °C for 12 h. The precipitate was collected by fil-
tration to give (Ϫ)-1c (66.7 g, 0.144 mol, 35%) as colorless powder.
(ii) (Ϯ)-6c (400 g, 0.816 mol) was hydrolysed with Pseudomonas
Pseudomonas vesicularis IFO 12165, Bacillus subtilis IFO 3026, and Bacil- taetrolens IFO 12691 in the similar manner as described above. After incu-
lus subtilis IFO 14117 were purchased from the Institute for Fermentation, bation for 48 h at 28 °C, a portion of the reaction mixture was extracted with
Osaka. Pseudomonas sp. S-13 and Streptomyces sp. 121-39 were isolated AcOEt. The extract was subjected to HPLC analysis. The results are shown
from soils and identified in Discovery Research Division. Trypticase Soy in Table 4.
Broth was purchased from Becton Dickinson. Ion exchange resin IRA-68
After completion of reaction, NaCl (34 kg) was added to the reaction mix-
ture (340 l). The mixture was adjusted to pH 4 with 2 N HCl (8 l) and ex-
was supplied by Rohm and Haas Co. Actocol® was supplied by Takeda
Chemical Industries. Ceramic filter was purchased from Toshiba Ceramics. tracted with AcOEt (170 l). The organic layers were combined, washed with
Cation PB-40 was purchased from NOF Corporation.
0.5% NaHCO3 (18 l, twice) and 2% NaCl (100 l), and concentrated under re-
duced pressure. The residue was suspended in water (2 l), washed with
hexane (4 l) and suspended in 50% MeOH (12 l). The suspension was ad-
justed to pH 7.9 with 10 N NaOH (25 ml). After being stirred for 3 h at
Asymmetric Hydrolysis of Ethyl (3,5-trans)-7-Chloro-(2-chlorophenyl)-
1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate [(؎)-
1a] by Various Microorganisms Pseudomonas taetrolens IFO 12691,
Pseudomonas diminuta IFO 13182, Pseudomonas vesicularis IFO 12165, 30 °C, this mixture was filtered. The filtrate (12 l) was adjusted to pH 4.1
and Pseudomonas aeruginosa IFO 3923 were respectively inoculated into with 6 N HCl (61 ml), cooled at 6 °C for 14 h, and then filtered to give (Ϫ)-1c
20 ml of Trypticase Soy Broth in a 200-ml Erlenmeyer flask and grown for
24 h at 28 °C on a rotary shaker. The culture (0.2 ml) was transferred to
(138.1 g, 0.299 mol, 37%) as a colorless powder.
(؊)-(3,5-trans)-7-Chloro-5-(2,4-dimethoxyphenyl)-1-neopentyl-2-oxo-
20 ml of casein medium (pH 7.0) containing 2% glucose, 2.5% casein, 0.1% 1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid [(؊)-1d] (Ϯ)-Ethyl
KH2PO4, 0.1% NaNO3, and 0.05% MgSO4·7H2O in a 200-ml Erlenmeyer
flask. The mixture were incubated for 42 h at 28 °C on a rotary shaker. Solu-
(3,5-trans)-7-chloro-5-(2,4-dimethoxyphenyl)-1,2,3,5-tetrahydro-1-
neopentyl-2-oxo-4,1-benzoxazepine-3-acetate [(Ϯ)-6d, 220 g, 0.449 mol]
tions of ethyl (3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo- was dissolved in DMSO (12 l). This solution was added to the culture (150 l)
1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate [(Ϯ)-6a, 20 mg] in dimethyl of Pseudomonas taetrolens IFO 12691. The incubation was carried out for
sulfoxide (DMSO) (2 ml) were added to the culture (20 ml). After incubation 72 h at 28 °C. A portion of the reaction mixture was extracted with AcOEt.
for 4.5 h at 28 °C with shaking, the reaction mixtures were extracted with The extract was subjected to HPLC analysis. The results are shown in Table
AcOEt. The extracts were analyzed by HPLC to determine hydrolytic con-
version rate, optical purity of the hydrolysate. The results are shown in Table 1.
4.
The above reaction mixture was purified by the procedure similar to the
Bacillus subtilis IFO 3026 was inoculated into 2 ml of medium containing preparation of the compound (Ϫ)-1c [method (i)] to give (Ϫ)-1d (76.1 g,
1% dextrin, 1% glucose, 1% glycerol, 0.5% polypeptone, 0.5% yeast extract,
0.5% meat extract, 0.3% NaCl, and 0.5% precipitated calcium carbonate in a
test tube. At the same time, a solution of (Ϯ)-6a (2 mg) in DMSO (50 ml)
0.165 mol, 37%) as a colorless powder.
(؎)-(3,5-trans)-7-Chloro-5-(2-methoxyphenyl)-1-neopentyl-2-oxo-
1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid [(؊)-1b] (Ϯ)-Ethyl
was added to the medium. The mixture was incubated for 48 h at 28 °C with (3,5-trans)-7-chloro-5-(2-methoxyphenyl)-1,2,3,5-tetrahydro-1-neopentyl-2-
shaking, the products were extracted with AcOEt. The extract was subjected oxo-4,1-benzoxazepine-3-acetate [(Ϯ)-6b, 200 g, 0.435 mol] was dissolved
to HPLC. The results are shown in Table 1.
Asymmetric Hydrolysis of Various Esters with Pseudomonas Pseudomonas taetrolens IFO 12691. The incubation was carried out for 54 h
taetrolens IFO 12691 and Pseudomonas diminuta IFO 13182 The methyl at 28 °C. A portion of the reaction mixture was extracted with AcOEt. The
isopropyl, n-butyl, and benzyl ester (Ϯ)-7—9 and (Ϯ)-11 were prepared by extract was subjected to HPLC analysis. The results are shown in Table 4.
in DMSO (13.5 l). The solution was added to the culture (170 l) of
,
the treatment of (Ϯ)-1a with iodomethane, isopropyl iodide, butyl bromide,
and benzyl bromide. The condensation of (Ϯ)-1a and phenol afforded
phenyl ester (Ϯ)-10.
After completion of the reaction, NaCl (6 kg) was added to the reaction
mixture. The mixture was adjusted to pH 4 with 2 N HCl (4.0 l) and extracted
with AcOEt (60 lϫ2). The organic layers were combined, washed with 2%
The various esters (Ϯ)-6a or (Ϯ)-7—11 (0.5 mg) were dissolved in NaCl (60 l) and 1% NaHCO3 (30 lϫ2), and concentrated under reduced
DMSO (50 ml). The solutions were respectively added to each of the cultures pressure. The residue was extracted with 3% NaHCO3 (20 lϫ4) at 0 °C. The
(0.5 ml) of Pseudomonas taetrolens IFO 12691 and Pseudomonas diminuta aqueous solutions were pooled, adjusted to pH 7 with 63% H2SO4 (1.9 l),
IFO 13182. After incubation for 16 h at 28 °C, each reaction mixture was ex- and extracted with AcOEt (25 lϫ2). The organic layers were combined,
tracted with AcOEt and the extracts were analyzed by HPLC. It was found
washed with 0.1 N H2SO4 (25 lϫ2) and water (25 lϫ2), and concentrated
that all the substrates used were asymmetrically hydrolyzed by each of the under reduced pressure. The residue was subjected to a column chromatog-
cultures to give (Ϫ)-1a. The hydrolytic conversion rates and the optical puri- raphy on silica gel [eluent: CHCl3 then CHCl3–MeOH (20 : 1)] and recrys-
ties of (Ϫ)-1a are shown in Table 3.
tallized from AcOEt to give (Ϫ)-1b (43.8 g, 0.101 mol, 23%) as colorless
Asymmetric Hydrolysis of 4,1-Benzoxazepine Derivatives with crystals.
Pseudomonas taetrolens (IFO 12691). (؊)-(3,5-trans)-7-Chloro-5-(2,3-
dimethoxyphenyl)-1,2,3,5-tetrahydro-1-neopentyl-2-oxo-4,1-benzox-
(؎)-5-Chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl Alco-
hol [(؎)-12] A mixture of (Ϯ)-2c (3 g, 10.2 mmol), NaHCO3 (1.3 g, 15.5
azepine-3-acetic Acid [(؊)-1c] i) Pseudomonas taetrolens IFO 12691 mmol), pivaloyl chloride (1.35 g, 11.2 mmol) and 4-dimethylaminopyridine
was inoculated into 1-l Erlenmeyer flasks containing 200 ml of Trypticase (DMAP) (0.12 g, 0.982 mmol) in AcOEt (30 ml) was stirred for 1 h at room
Soy Broth, and grown for 48 h at 28 °C on a rotary shaker. The culture (1.6 l) temperature. The mixture was diluted with AcOEt (50 ml), washed with
was transferred to a 200-l fermenter containing 160 l of the casein medium water (50 ml), 5% KHSO4 (50 ml), saturated NaHCO3 (50 ml) and brine
described above. The cultivation was carried out for 42 h at 28 °C with aera- (50 ml), dried over Na2SO4, and then concentrated under reduced pressure.
tion and stirring. (Ϯ)-Ethyl (3,5-trans)-7-chloro-5-(2,3-dimethoxyphenyl)- The residue was recrystallized from AcOEt–hexane (1 : 5) to give (Ϯ)-12
1,2,3,5-tetrahydro-1-neopentyl-2-oxo-4,1-benzoxazepine-3-acetate [(Ϯ)-6c, (3.6 g, 9.53 mmol, 93%) as colorless prisms. mp 122—123 °C. IR nmax
200 g, 0.408 mol] was dissolved in DMSO (18 l). This solution was added to
the culture (150 l). The mixture was incubated for 48 h at 28 °C with stirring. 1.12 (9H, s), 3.90 (3H, s), 3.91 (3H, s), 4.3 (1H, d, Jϭ4.4 Hz), 6.5 (1H, dd,
A portion of the reaction mixture was extracted with AcOEt. The extract Jϭ1.8, 7.0 Hz), 6.9—7.03 (3H, m), 7.31 (8H, dd, Jϭ2.4, 8.8 Hz), 8.17 (1H,
(KBr) cmϪ1: 3600—3200 (br, OH, NH), 1665 (CϭO). 1H-NMR (CDCl3) d:
was subjected to HPLC analysis. The results are shown in Table 4.
d, Jϭ8.8 Hz), 9.22 (1H, br s). Anal. Calcd for C17H15Cl2NO3: C, 59.97; H,

64
Vol. 50, No. 1
4.29; N, 3.98. Found: C, 57.79; H, 4.29; N, 4.13.
(؎)-5-Chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl
mixture was filtered through a ceramic filter with a pore diameter of 0.2 mm.
60% EtOH (1800 l) was added and the mixture was concentrated to 160 l.
After addition of Cation PB-40 (6.4 l), the mixture was extracted with
AcOEt (300 l). The extract was washed with 0.1 N H2SO4, 3% Na2CO3, and
water, and then concentrated to 63 l. The residue was stirred for 1 h with ac-
tivated carbon (1.2 kg). After filtration, the filtrate was concentrated. The
residue was diluted with AcOEt (3 l), and then hexane (103 l) was added for
fractional crystallization to give (Ϫ)-12 (5.6 kg, 14.8 mol, 41%). A solution
of KOH (221 g) in MeOH (12.3 l) was added to the mother liquor of frac-
tional crystallization, and the hydrolysis reaction was carried out for 30 min.
This hydrolysate was crystallized from water (24.6 l) to give (ϩ)-12 (6.3 kg,
16.7 mol, 47%).
(R)-5-Chloro-a-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl Alcohol
[(؉)-12] Bacillus subtilis IFO 14117 was grown for 24 h at 28 °C in 40 ml
of a medium containing 2% sucrose, 2.5% corn steep liquor, 0.1% KH2PO4,
0.05% (NH4)2SO4 and 0.5% MgSO4 in 200-ml Erlenmeyer flasks on a rotary
shaker. The culture (3 ml) was transferred to 1-l Erlenmeyer flasks contain-
ing 200 ml of the same medium, and grown for 48 h at 28 °C on a rotary
shaker. The culture (2.1 l) was centrifuged, and the cells were suspended in
0.1 M Tris–HCl buffer (pH 7.5) to give 2.1 l of cell suspension. A solution of
(Ϯ)-13 (4.2 g, 10.0 mmol) in DMF (105 ml) was added to the cell suspen-
sion. After incubation for 24 h at 28 °C with shaking, a portion of this reac-
tion mixture was taken and stirred with AcOEt. The organic layer was ana-
lyzed by HPLC. The hydrolytic conversion rate was found to be 49% and the
optical purity of (ϩ)-12 was not less than 99% ee (Table 6).
Ac-
etate [(؎)-13] A mixture of (Ϯ)-12 (1 g, 2.65 mmol), Ac2O (0.48 g, 3.94
mmol) and DMAP (32 mg, 0.264 mmol) in pyridine (10 ml) was stirred for
1 h at room temperature. The mixture was duluted with AcOEt (100 ml),
washed with 1 N HCl (140 ml), saturated NaHCO3 (100 ml), and brine
(100 ml), dried over Na2SO4, and then concentrated under reduced pressure.
The residue was chromatographed [eluent: hexane–AcOEt (5 : 1)] and crys-
tallized with hexane to give (Ϯ)-13 (0.96 g, 2.29 mmol, 86%) as colorless
prisms. mp 111—168 °C. IR nmax (KBr) cmϪ1: 3364 (NH), 1724, 1685 (Cϭ
1
O). H-NMR (CDCl3) d: 1.38 (9H, s), 2.17 (3H, s), 3.34 (3H, s), 3.83 (3H,
s), 6.97 (1H, dd, Jϭ1.5, 8.4 Hz), 7.11—7.27 (5H, m), 7.8 (1H, d, Jϭ8.7 Hz),
9.0 (1H, br s). Anal. Calcd for C22H26ClNO5: C, 62.93; H, 6.24; N, 3.34.
Found: C, 62.96; H, 6.14; N, 3.24.
Asymmetric Hydrolysis of 5-Chloro-a-(2,3-dimethoxyphenyl)-2-pival-
oylaminobenzyl Acetate with Microorganisms. (؊)-5-Chloro-a-(2,3-
dimethoxyphenyl)-2-pivaloylaminobenzyl Alcohol [(؊)-12] i) Strepto-
myces sp. 121-39 was grown in a 200-ml Erlenmeyer flask containing 40 ml
of a medium consisting of 0.5% glucose, 5% dextrin, 3.5% raw soybean
flour and 0.7% CaCO3 for 48 h at 28 °C on a rotary shaker. The culture (10
ml) was transferred to 1-l Erlenmeyer flasks containing 200-ml of the same
medium. The cultivation was carried out for 48 h at 28 °C on a rotary shaker.
A solution of (Ϯ)-13 (3 g, 7.14 mmol) in N,N-dimethylformamide (DMF)
(150 ml) was added to the culture (3 l) thus obtained. The mixture was incu-
bated for 48 h at 28 °C with the shaking. A portion of the reaction mixture
was extracted with AcOEt. The extract was subjected to HPLC analysis. The
conversion rate was 49% and the optical purity of (Ϫ)-12 was 88% ee (Table
6).
The reaction mixture obtained above was extracted with AcOEt (2.0 l).
The solvent was removed under reduced pressure. The residue was purified
by flash chromatography [eluent: hexane–CH2Cl2–AcOEt (6 : 3 : 1), internal
pressure 0.2 kg/cm2)], and recrystallized from CH2Cl2–hexane (1 : 5). Crude
yield 0.79 g (optical purity 91.8% ee). The crystals were recrystallized from
MeOH–H2O (1 : 1). Yield 0.54 g (optical purity 99.2%). The crystals were
recrystallized twice from MeOH–H2O (3 : 1) to give (Ϫ)-12 (0.124 g, 0.328
mmol, 100% ee) as colorless needles. mp 107—109 °C. [a]D Ϫ51.7° (cϭ
0.64, MeOH). Anal. Calcd for C20H24ClNO4: C, 63.57; H, 6.40; N, 3.71.
Found: C, 63.34; H, 6.20; N, 3.51.
ii) Pseudomonas sp. S-13 was grown in a 2-l Sakaguchi flask containing
500 ml of Trypticase Soy Broth for 24 h at 28 °C on a reciprocal shaker. The
resulting culture was transferred to a 200-l fermenter containing 120 l of a
medium consisting of 2% cottonseed meal, 0.25% K2HPO4, 0.5% NaCl, and
0.25% glucose, and the cultivation was carried out for 48 h at 28 °C with
aeration and stirring. The culture (24 l) was centrifuged to give 3 l of a cell
suspension. A solution of (Ϯ)-13 (30 g, 71.4 mmol) in MeOH (300 ml) was
added to the cell suspension. After incubation for 24 h at 28 °C with stirring,
a portion of the reaction mixture was extracted with AcOEt and the extract
was analyzed by HPLC. The hydrolytic conversion rate was 35% and the op-
tical purity of (Ϫ)-12 was not less than 98% ee (Table 6).
The reaction mixture obtained above was extracted with AcOEt (3 l). The
extract was concentrated under reduced pressure. The residue was purified
by flash chromatography [eluent: hexane–isopropyl ether–AcOEt (16 : 3 : 1,
v/v), internal pressure 0.2 kg/cm2)], and crystallized with n-hexane, and the
crystals were collected by filtration to give (Ϫ)-12 (6.73 g, 17.8 mmol, 25%)
as colorless needles.
The reaction mixture obtained above was extracted with AcOEt (2 l), and
concentrated under reduced pressure. The residue was purified by flash chro-
matography [eluent: hexane–isopropyl ether–AcOEt (7 : 2 : 1, v/v), internal
pressure: 0.2 kg/cm2] to give crude crystals. Yield 1.5 g (optical purity
99.3% ee). The crude crystals were recrystallized from MeOH–H2O (1 : 2)
to give (ϩ)-12 (0.85 g, 2.25 mmol, 100% ee) as colorless needles. mp 105—
106 °C. [a]D ϩ50.1° (cϭ0.46, MeOH). Anal. Calcd for C20H24ClNO4: C,
63.57; H, 6.40; N, 3.71. Found: C, 63.53; H, 6.47; N, 3.64.
(؊)-2-Amino-5-chloro-a-(2,3-dimethoxyphenyl)benzyl Alcohol [(؊)-
2c]
A solution of (Ϫ)-12 (0.15 g, 0.397 mmol) and NaOH (0.16 g,
3.97 mmol) in EtOH (2 ml) was refluxed for 3 h. the reaction mixture was di-
luted with water, extracted with AcOEt (50 ml). The extract was washed
with water (50 ml), dried over MgSO4, and then concentrated under reduced
pressure. The residue was chromatographed [eluent: hexane–AcOEt (3 : 1)]
and recrystallized from hexane–AcOEt (1 : 1) to give (Ϫ)-2c (0.1 g,
0.340 mmol, 86%) as colorless prisms. mp 128—129 °C. [a]D Ϫ75.9°
(cϭ0.47, MeOH). IR nmax (KBr) cmϪ1: 3600—3200 (br, NH2, OH). 1H-
NMR (CDCl3) d: 3.1—3.2 (1H, br), 3.84 (3H, s), 4.1—4.3 (2H, br), 6.03
(1H, s), 6.58 (1H, d, Jϭ8.4 Hz), 6.86—6.92 (2H, m), 7.01—7.10 (3H, m).
Anal. Calcd for C15H16ClNO3: C, 61.33; H, 5.49; N, 4.77. Found: C, 61.34;
H, 5.45; N, 4.67.
Synthesis of (؊)-(3R,5S)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-
oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid [(؊)-1c] from
(؊)-2c NaBH4 (50 mg, 1.32 mmol) was added to an ice-cooled solution of
(Ϫ)-2c (0.1 g, 0.340 mmol) and pivalaldehyde (40 mg, 0.464 mmol) in
AcOH (3 ml). After being stirred for 20 min at room temperature, the mix-
ture was diluted with water (50 ml). The solution was extracted with AcOEt
(50 ml, twice). The extracts were washed with saturated NaHCO3 (50 ml)
and brine (50 ml), dried over Na2SO4, and then concentrated under reduced
pressure. The residue was chromatographed [eluent: hexane–AcOEt (8 : 1)]
to give an oil (90 mg, 0.247 mmol, 73%). The oil was dissolved in AcOEt
(5 ml). NaHCO3 (0.1 g, 1.19 mmol) was added to the solution, followed by
addition of monoethyl fumaryl chloride (50 mg, 0.308 mmol). After being
stirred for 40 min at room temperature, the mixture was diluted with AcOEt
(50 ml). The solution was washed with water (50 ml), dried over Na2SO4,
and then concentrated under reduced pressure. The residue was chro-
matographed [eluent: hexane–AcOEt (3 : 1)] to give an oil (0.12 g, 0.245
mmol, 99%). The oil was dissolved in EtOH (5 ml). K2CO3 (50 mg,
0.362 mmol) was added to the solution, and the mixture was stirred for 3 h at
room temperature. The mixture was diluted with water (50 ml), extracted
with AcOEt (50 ml). The extract was washed with water (50 ml), dried over
Na2SO4, and then concentrated under reduced pressure to give a colorless
powder (90 mg, 0.184 mmol, 75%). The powder was dissolved in EtOH (1
ml), followed by addition of 1 N NaOH (0.2 ml). After being stirred for
30 min at 60 °C, the mixture was diluted with water (50 ml). The solution
was acidified with 1 N HCl (0.2 ml), extracted with AcOEt (50 ml). The ex-
tract was washed with brine (50 ml), dried over Na2SO4, and then concen-
trated under reduced pressure. The residue was recrystallized from
AcOEt–hexane (1 : 1) to give (Ϫ)-1c (80 mg, 0.173 mmol, 94%) as colorless
(iii) Pseudomonas sp. S-13 was grown in a 500-ml Erlenmeyer flask con-
taining 60 ml of Trypticase Soy Broth for 24 h at 28 °C on a rotary shaker.
The culture was transferred to 120 l of a medium containing 2% corn steep
liquor, 0.25% K2HPO4, 0.5% NaCl, 0.25% sucrose, and Actocol® in a 200-l
fermenter, and grown for 24 h at 28 °C with aeration and stirring. The cul-
ture (15 l) was transferred to 1500 l of a medium containing 2% corn steep
liquor, 0.25% K2HPO4, 0.5% NaCl, 2% sucrose, 3% (NH4)2SO4, and Acto-
col® [0.005%—0.01% (v/v) of the culture medium] in a 2000-l fermenter.
The cultivation was carried out for 45 h at 28 °C with aeration and stirring.
A solution of (Ϯ)-13 (15 kg, 35.7 mol) in MeOH (150 l) was mixed with
above culture. After incubation for 14 h at 28 °C, a portion of the reaction
mixture was extracted with AcOEt. The extract was analyzed by HPLC. The
hydrolytic conversion rate was found to be 44% and the optical purities of
(Ϫ)-12 and unreacted acetate were found to be 99% ee and 96% ee, respec-
tively.
The reaction mixture was washed with water, acidified (pH 5.0) with
H2SO4, and concentrated to 360 l by means of a ceramic filter with a pore di-
ameter of 0.2 mm (Toshiba Ceramics, Japan). After addition of EtOH (540 l),
the mixture was stirred for 1 h at 50 °C in order to dissolve (Ϫ)-12. This

January 2002
65
Acknowledgments The authors wish to thank Dr. Kanji Meguro for his
encouragement and helpful discussion throughout this study. We also thank
Dr. Ryu-ichi Tozawa and Dr. Masahira Nakamura for assay of squalene syn-
thase inhibitory activities.
prisms. [a]D Ϫ250.3° (cϭ0.41, MeOH).
Animals and Materials Animals were supplied by Clea, Japan, Inc.
RS-[2-14C]Mevalonolactone and [1-3H]farnesyl pyrophosphate were pur-
chased from New England Nuclear. [2-14C]Mevalonic acid was synthesized
from [2-14C]Mevalonolactone by saponification with potassium hydroxide.
[2-14C]Sodium acetate was purchased from Amersham. Farnesyl pyrophos-
phate was synthesized by the method described by V. J. Davisson and
coworkers7) (Nemoto & Co.). HepG2 cells were supplied by The American
Type Culture Collection (ATCC). Fetal bovine serum (FBS) and Dulbecco’s
modified Eagle’s medium (DMEM) were purchased from GIBCO. Human
lipoprotein deficient serum (human LPDS) was purchased from Sigma. All
other reagents were supplied by Wako Pure Chemical Industries.
References and Notes
1) Part 4 in the series of studies on 4,1-benzoxazepine derivatives as
squalene synthase inhibitors; a) Part 1: Miki T., Kori M., Fujishima
A., Mabuchi H., Tozawa R., Nakamura M., Sugiyama Y., Yukimasa
H., Bioorg. Med. Chem., 10, 385—400 (2002); b) Part 2: Miki T., Kori
M., Mabuchi H., Banno H., Tozawa R., Nakamura M., Itokawa S.,
Sugiyama Y., Yukimasa H., ibid., 10, 401—414 (2002); c) Part 3: Miki
T., Kori M., Tozawa R., Nakamura M., Sugiyama Y., Yukimasa H.,
Chem. Pharm. Bull., 50, 53—58 (2002).
2) a) For review see: Santaniello E., Ferraboschi P., Grisenti P., Manzoc-
chi A., Chem. Rev., 92, 1071—1140 (1992); Mori K., Synlett, 1995,
1097—1109; Robert S. M., J. Chem. Soc., Perkin Trans. 1, 1998,
157—169 and 1999, 1—12; Whitesides G. M., Wong C.-H., Angew.
Chem., Int. Ed. Engl., 24, 617—718 (1985); b) Sakamoto K., Tsuzaki
K., Shima M., JP 09308497 (1997) [Chem. Abstr., 128, 74400 (1997)];
c) Ozaki E., Uragaki T., JP 09059217 (1997) [Chem. Abstr., 126,
277200 (1997)]; d) Idem, JP 09059211 (1997) [Chem. Abstr., 126,
277199 (1997)]; e) Miyadera A., Imura A., JP 08070887 (1996)
[Chem. Abstr., 125, 56411 (1996)]; f) Ozaki E., Uragaki T., Sakashita
K., Ikemoto T., Kobayashi Y., Sakimae A., WO 9534525 (1995)
[Chem. Abstr., 124, 260386 (1995)]; g) Isshiki K., Nakajima T., Tera-
sawa T., Watanabe Y., Tanaka H., Yoshioka T., JP 06319593 (1994)
[Chem. Abstr., 122, 131151 (1994)]; h) Isshiki K., Nakashima T.,
Yoshioka T., Tsunekawa H., Adachi T., Ota T., WO 9405637 (1994)
[Chem. Abstr., 121, 9161 (1994)]; i) Yanase H., Arishima T., Kita K.,
Nagai J., Mizuno S., Takuma Y., Endo K., Kato N., Biosci. Biotech.
Biochem., 57, 1334—1337 (1993).
3) Nakahama K., Izawa M., Nagano Y., Tarui N., Matsumoto K., Kori
M., Kanamaru T., Nagata T., JP 08205889 (1996) [Chem. Abstr., 125,
32076 (1996)].
4) The optical purities of the unreacted esters obtained simultaneously
were not determined.
5) Cohen L. H., Griffioen A. M., Wanders R. J. A., Van Roermund C. W.
T., Huysmans C. M. G., Princen H. M. G., Biochem. Biophys. Res.
Commun., 138, 335—341 (1986).
6) Although optically active 2-amino-5-chloro-a-(2,3-dimethoxyphenyl)-
benzyl alcohol derivatives are valuable intermediates for the produc-
tion of drugs and other chemicals, few reports are available on their
synthesis. a) Oishi T., Akita H., Kato M., JP 03-22992 (1991) [Chem.
Abstr., 115, 157162 (1991)]; b) Kato M., Sasahara K., Ochi K., Akita
H., Oishi T., Chem. Pharm. Bull., 39, 2498—2501 (1991); c) Shieh
W.-C., Cantrell W. R., Carlson J. A., Tetrahedron Lett., 36, 3797—
3800 (1995); d) Nozaki K., Kobori K., Uemura T., Tsutsumi T.,
Takaya H., Hiyama T., Bull. Chem. Soc. Jpn., 72, 1109—1113 (1999);
e) Sakaguchi T., Imai T., Miura T., Yamazaki T., Okada T., JP 09-
235255 (1997) [Chem. Abstr., 127, 262513 (1997)].
Preparation of Rat Squalene Synthase An Sprague-Dawley (SD)
male rat (6-week old) was killed by bleeding, and its liver was excised.
About 10 g of the liver was washed with a saline solution cooled with ice,
which was then homogenized in 15 ml of an ice-cooled buffer [100 mM
potassium phosphate (pH 7.4), 15 mM nicotinamide, 2 mM MgCl2], followed
by centrifugation for 20 min at 10000ϫg (4 °C). The supernatant layer was
obtained and subjected to further centrifugation for 90 min at 105000ϫg
(4 °C). The sediment was then suspended in an ice-cooled 100 mM potassium
phosphate buffer solution (pH 7.4), which was again subjected to centrifuga-
tion for 90 min at 105000ϫg (4 °C). The sediment thus obtained (micro-
some fraction) was suspended in an ice-cooled 100 mM potassium phosphate
buffer (pH 7.4) (about 40 mg/ml protein concentration, determined using
Bicinchoninic acid (BCA) protein assay kit of Pierce Co., Ltd.). This sus-
pension was used as the enzyme solution.
Preparation of Human Squalene Synthase HepG2 cells (about 1ϫ109
cells) obtained by incubation (37 °C in the presence of 5% CO2) in a DMEM
contains 10% FBS, penicillin G (100 units/ml) and streptomycin (10 mg/ml)
were suspended in 10 ml of ice-cooled buffer solution [100 mM potassium
phosphate buffer (pH 7.4), 30 mM nicotinamide and 2.5 mM MgCl2]. The
cells were crashed by means of ultrasonication (for 30 s, twice). From the
sonicate thus obtained, the microsome fraction was obtained by the same
procedure as in preparation of rat-derived enzyme, which was suspended in
an ice-cooled 100 mM potassium phosphate buffer (pH 7.4) (about 4 mg/ml
protein concentration). This suspension was used as the enzyme solution.
Assay of Squalene Synthase Inhibitory Activity Squalene synthase
activity was monitored by the formation of [3H]squalene from [1-3H]farne-
syl pyrophosphate. Fifty microliters of assay mixture included 5mM [1-
3H]farnesyl pyrophosphate (25 mCi/mol), 1 mM NADPH, 5 mM MgCl2, 6 mM
glutathione, 100 mM buffer solution of potassium phosphate (pH 7.4), the
test compound dissolved in DMSO (a final concentration of DMSO was 2%)
and enzyme solution prepared from rat or HepG2 cells (protein content
0.8 mg). The assay ran 45 min at 37 °C and stopped by adding 150 ml of
CHCl3–MeOH (1 : 2) containing 0.2% cold squalene as carrier. Aqueous so-
lution of 3 N NaOH (50 mM) and CHCl3 (50 mM) were added to the mixture.
The chloroform layer containing the reaction mixture having squalene as the
principal component and 3 ml of toluene-based liquid scintillator were
mixed, and its radioactivity was determined by means of a liquid scintillation
counter. The squalene synthase inhibitory activity was expressed in terms of
the concentration of the test compound inhibiting by 50% the radioactivity
taken into the chloroform layer (IC50, molar concentration (M)).
7) Davisson V. J., Woodside A. B., Poulter C. D., Methods Enzymol., 110,
130—144 (1985).