Syntheses of (4,1-benzoxazepine-3-ylidene)acetic acid derivatives and their inhibition of squalene synthase

Article abstract of DOI:10.1248/cpb.50.53

The (3,5-trans)-7-chloro-5-(2-chlorophenyl)-2-oxo-1,2,3,5-tetrahydro-4,1- benzoxazepine-3-acetic acid derivatives 1 have been previously identified as potent squalene synthase inhibitors. A series of (4,1-benzoxazepin-3-ylidene) acetic acid derivatives we

Full text of DOI:10.1248/cpb.50.53

January 2002
Chem. Pharm. Bull. 50(1) 53—58 (2002)
53
Syntheses of (4,1-Benzoxazepine-3-ylidene)acetic Acid Derivatives and
Their Inhibition of Squalene Synthase1)
Takashi MIKI,* Masakuni KORI, Ryu-ichi TOZAWA, Masahira NAKAMURA, Yasuo SUGIYAMA, and
Hidefumi YUKIMASA
Takeda Chemical Industries, Ltd., Pharmaceutical Research Division, 2–17–85 Juso-Honmachi, Yodogawa-ku, Osaka
532–8686, Japan. Received July 30, 2001; accepted October 1, 2001
The (3,5-trans)-7-chloro-5-(2-chlorophenyl)-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid deriva-
tives 1 have been previously identified as potent squalene synthase inhibitors. A series of (4,1-benzoxazepin-3-yli-
dene)acetic acid derivatives were synthesized and evaluated for their inhibition of rat and human squalene syn-
thase, and the (E)-isomers were found to exhibit potent inhibitory activity, with the same potency as 4,1-benzox-
azepine-3-acetic acid derivatives. In contrast the (Z)-isomers did not exhibit significant inhibitory activity, and
the active conformation of the 4,1-benzoxazepine-3-acetic acid derivatives was deduced from the folded confor-
mation of the (E)-isomers.
Key words squalene synthase; 4,1-benzoxazepine-3-acetic acid; active conformation; conformationally restricted analogue;
(4,1-benzoxazepin-3-ylidene)acetic acid derivative; hypocholestemic agent
Squalene synthase [EC 2.5.1.21] catalyzes the dimeriza- Chemistry
tion of farnesyl diphosphate to squalene in cholesterol
The epoxy-amide derivative 6a was prepared by acylation
biosynthesis. This enzymatic step occurs after the pathway of the racemic 2-aminobenzylalcohol derivative 5a1b) with
branches to other non-steroidal isoprenoids such as dolicol, the acid chloride prepared from DL-trans-epoxysuccinic acid
ubiquinones and isopentenyl t-RNA. Since squalene synthase monoethyl ester3) and dichloromethylenedimethyliminium
inhibitors do not interfere with the biosynthesis of these iso- chloride.4) Intramolucular cyclization5) of the epoxy-amide
prene derivatives, inhibition of this step would arrest only derivative 6a afforded a ca. 1 : 1 mixture of two products,
cholesterol biosynthesis and might be useful for the treat- which were separated by fractional recrystallization to yield
ment of hyperlipidemia.
the compounds 7a and 7b (Chart 1).6) The structures of 7a
In previous papers,1) we have described chemical modifi- and 7b were determined by X-ray diffraction analysis, and
cation of (3,5-trans)-7-chloro-5-phenyl-2-oxo-1,2,3,5-tetrahy- are shown in Figs. 2 and 3. Both of these compounds are 7-
dro-4,1-benzoxazepine-3-acetic acid derivatives and evalua- membered 4,1-benzoxazepine derivatives and the configura-
tion of their inhibition of squalene synthase. Through the tions of 7a and 7b were determined to be (3RS, 5SR, aSR)
structure–activity relationship study, compounds 1a—c, and (3RS, 5RS, aSR), respectively. The fact that 7a and 7b
which have an alkyl group at the 1-position, were found to be have the same configurations at the 3- and a-positions indi-
potent inhibitors (Fig. 1). In these compounds the conforma- cate intramolecular cyclization of 6a proceeded by attack of
tions of fused ring systems are rigid, but that of the car- the oxygen anion on the epoxy carbon bound to the car-
boxymethyl group at the 3-position, which was essential for bamoyl group from the opposite face of the epoxy oxygen
potent activity, is very flexible. Thus we considered that re- (Fig. 4).
striction of the flexibility of the 3-acetic acid moiety would
yield useful information concerning the active conformation
of these compounds. We report here the preparation of the
(4,1-benzoxazepin-3-ylidene)acetic acid derivatives in which
a double bond was introduced between the 3-position and a-
position (Fig. 1, 2—4) and the evaluation of their inhibition
of squalene synthase.2)
Reagents: (a) DL-trans-Epoxysuccinic acid monoethyl ester, dichloromethylenedi-
methyliminium chloride, NaHCO3, CH2Cl2; (b) 1) K2CO3, EtOH, 2) fractional recrys-
tallization.
Fig. 1. Structures of 4,1-Benzoxazepine Derivatives
Chart 1
To whom correspondence should be addressed. e-mail: Miki_Takashi@takeda.co.jp
© 2002 Pharmaceutical Society of Japan

54
Vol. 50, No. 1
Fig. 2. Stereoscopic Molecular View of Compound 7a
Fig. 3. Stereoscopic Molecular View of Compound 7b
Reagents: (a) MsCl, Et3N, AcOEt; (b) 1) CH3CH2COOCs, DMF, 2) chromatographic
separation; (c) 1N NaOH, EtOH.
Chart 2
Fig. 4. Mechanism of Cyclization of 6a
Compound 7a was methanesulfonylated, and the resulting
mesylate 8a was treated with cesium propionate7) to yield a
mixture of two products (Chart 2). Elimination of the mesyl-
oxy group with sodium acetate instead of cesium propionate
proceeded slowly and required excess amount of sodium ac-
etate. Separation of the mixture by column chromatography
gave 9a (more polar isomer) and 9b (less polar isomer) in
1 : 2 ratio. Elemental analysis of these compounds indicated
that they have the same composition and mass spectra of
both compounds showed a molecular ion peak at m/z 433
(Mϩ). Their IR spectra exhibited very similar patterns and in
Fig. 5. Stereoscopic Molecular View of Compound 2a
1
The syntheses of the (4,1-benzoxazepin-3-ylidene)acetic
acid derivatives 3 and 4, which have an n-propyl and isobutyl
group at the 1-position, are outlined in Chart 4. Acylation of
racemic aminobenzylalcohol derivatives 5b1b) and 5c1b) with
the acid chloride obtained from DL-trans-epoxysuccinic acid
monoethyl ester afforded the epoxy-amide 6b, c. Intramolec-
ular cyclization of 6b, c gave a diastereomeric mixture of a-
hydroxyacetic acid derivatives 10a, b which were mesylated
to give 11a and 11b. Subsequent treatment with cesium pro-
pionate, followed by chromatographic separation afforded
12a, b and 13a, b, respectively. The configurations of 12a, b
and 13a, b were determined by comparison of the chemical
shifts of signals due to olefin protons in the 1H-NMR spectra
of 9, 12 and 13. Alkaline hydrolysis of 12a, b and 13a, b
yielded the carboxylic acid derivatives 3a, b and 4a, b, re-
the H-NMR spectra singlet signals due to olefin protons
were observed at d 5.41 and 5.48 ppm of 9a and 9b, respec-
tively. On the basis of these results, 9a and 9b were deter-
mined to be geometrical isomers of (4,1-benzoxazepin-3-yli-
dene)acetic acid derivatives. Compounds 9a and 9b were
then hydrolyzed to the acids 2a and 2b, respectively and con-
figuration of the double bond was established by X-ray dif-
fraction analysis of compound 2a. As shown in Fig. 5, com-
pound 2a (9a) was determined to be the (E)-isomer, and thus
by inference, compound 2b (9b) was the (Z)-isomer.
Compound 7b, which is a diastereoisomer of 7a, could
also be converted to 9a, b by mesylation, and subsequent
treatment of the resulting 8b with cesium propionate. The
ratio of 9a and 9b (1 : 2) prepared from 7b was identical to
that obtained from 7a (Chart 3).8)

January 2002
55
Table 1. Squalene Synthase Inhibitory Activities of (Ϯ)-[7-Chloro-5-(2-
chlorophenyl)-2-oxo-1,5-dihydro-4,1-benzoxazepin-3-ylidene]acetic Acid
Derivatives (2—4) and (Ϯ)-(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-2-oxo-
1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic Acid Derivatives (1)
IC50 (mM)a)
Compd.
Config.
R
Rat enzyme
Human enzyme
Reagents: (a) MsCl, Et3N, AcOEt; (b) 1) CH3CH2COOCs, DMF, 2) chromatographic
separation.
2a
2b
3a
3b
E
Z
E
Z
E
Z
Pri
0.31
Ͼ10 (21.6)b)
0.076
0.17
—
c)
Pri
Prn
Prn
Bui
Bui
Pri
0.098
Chart 3
c)
8.4
0.11
—
4a
0.041
c)
4b
Ͼ10 (14.5)b)
0.71
—
1ad)
1bd)
1cd)
0.54
Prn
Bui
0.14
0.061
—
c)
0.034
a) IC50 values were determined by a single experiment run in duplicate. b) % Inhi-
bition at 10Ϫ5 M. c) Not tested. d) Reference 1b.
of squalene synthase prepared from rat liver and human he-
patoma (HepG2) cells. Inhibitory activities were measured
according to the method of Cohen et al. with slight modifica-
tion.9)
Inhibitory activities of the (4,1-benzoxazepin-3-ylidene)-
acetic acid derivatives 2—4 for squalene synthase are shown
in Table 1. It can be seen that the inhibitory activity of the
(E)-isomers 2a, 3a and 4a are of similar potency as the cor-
responding acetic acid derivatives 1a—c. On the other hand,
the (Z)-isomers 2b, 3b and 4b were found to be only weakly
active against rat enzyme.
In the case of the (E)-isomers, we assume that the double
bonds holds the carboxyl groups in a position favorable for
interaction with the enzyme, and conversely that the spatial
positions of the carboxyl groups of the (Z)-isomers are unfa-
vorable. Since the activities of 1a—c and the (E)-isomers 2a,
3a and 4a are almost identical, the active conformation of the
4,1-benzoxazepine-3-acetic acid derivatives is presumed be
similar to the folded conformation of the (E)-(4,1-benzox-
azepin-3-ylidene)acetic acid derivatives.10) However, since all
compounds described in this paper are racemates, it is not
clear which enantiomer is the active inhibitor, and optical
resolution of these compounds is currently in progress and
will be reported in due course.
Conclusion
The (E)- and (Z)-(4,1-benzoxazepin-3-ylidene)acetic acid
derivatives 2—4 were prepared and evaluated for their inhibi-
tion of squalene synthase. Interestingly, the (E)-isomers were
much more potent than the (Z)-isomers against rat enzyme,
and exhibited equivalent potency to the 4,1-benzoxazepine-3-
acetic acid derivatives. Thus we assume that the active con-
formation of the 4,1-benzoxazepine-3-acetic acid derivatives
is similar to the folded conformation of the (E)-(4,1-benzox-
azepin-3-ylidene)acetic acid derivatives.
Reagents: (a) DL-trans-Epoxysuccinic acid monoethyl ester, dichloromethylenedi-
methyliminium chloride, NaHCO3, CH2Cl2; (b) K2CO3, EtOH; (c) MsCl, Et3N, AcOEt;
(d) 1) CH3CH2COOCs, DMF, 2) chromatographic separation; (e) 1 N NaOH, EtOH.
Chart 4
spectively.
Biological Results and Discussion
The compounds synthesized were evaluated for inhibition

56
Vol. 50, No. 1
Experimental
(2-chlorophenyl)-1-isopropyl-2-oxo-1,5-dihydro-4,1-benzoxazepin-3(2H)-
All melting points were determined on a Yanagimoto micro melting point ylidene]acetate (9b) A mixture of propionic acid (0.23 g, 3.11 mmol) and
apparatus and are uncorrected. Proton nuclear magnetic resonance (1H- Cs2CO3 (0.34 g, 1.04 mmol) in MeOH (10 ml) was stirred for 30 min at room
NMR) spectra were recorded on a Varian GEMINI-200 (200 MHz) spec- temperature, and then concentrated under reduced pressure to give cesium
trometer (with tetramethylsilane as an internal standard). Infrared (IR) ab- propionate as a colorless powder. A mixture of 8a (1.0 g, 1.89 mmol) and ce-
sorption spectra were recorded on a JASCO IR-810. [a]D values were deter- sium propionate in N,N-dimethylformamide (DMF, 10 ml) was stirred for 2 h
mined in the indicated solvents on a JASCO DIP-370 polarimeter. TLC at 80 °C, diluted with water, and extracted with AcOEt. The extract was
analyses were carried out on Merck Kieselgel 60 F254 plates. Elemental
washed with 1 N HCl and saturated NaHCO3, dried over MgSO4, and then
analyses were carried out by Takeda Analytical Laboratories, Ltd., and are concentrated in vacuo. The residue was chromatographed [eluent:
within Ϯ0.4% of the theoretical values. For column chromatography, Merck hexane–AcOEt (10 : 1 then 5 : 1)] to give 9b (0.31 g, 0.714 mmol, 38%) as
Kieselgel 60 (70—230 mesh ASTM) was used. Yields were not maximized. colorless prisms from the first fraction and 9a (0.13 g, 0.299 mmol, 16%) as
The following abbreviations are used: sϭsinglet, dϭdoublet, tϭtriplet, qϭ a colorless prisms from the second fraction.
quartet, mϭmultiplet, brϭbroad.
9a: mp 143—144 °C (Et2O–hexane). IR nmax (KBr) cmϪ1: 1710, 1670
(CϭO), 1650 (CϭC). 1H-NMR (CDCl3) d: 1.23 (3H, t, Jϭ7.2 Hz), 1.34
(3H, d, Jϭ7.0 Hz), 1.66 (3H, d, Jϭ7.0 Hz), 4.05—4.3 (2H, m), 4.9—5.1
Ethyl (2,3-trans)-3-[N-[4-Chloro-2-(2-chloro-a-hydroxybenzyl)phenyl]-
N-isopropyl]carbamoyl]-2,3-epoxybutyrate (6a) A mixture of DL-trans-2,3-
epoxysuccinic acid monoethyl ester3) (3.1 g, 19.3 mmol) and dichlorometh- (1H, m), 5.41 (1H, s), 6.49 (1H, s), 6.53 (1H, s), 7.3—7.6 (5H, m), 7.78 (1H,
ylenedimethyliminium chloride (3.14 g, 19.3 mmol) in CH2Cl2 (90 ml) was d, Jϭ6.8 Hz). Anal. Calcd for C22H21Cl2NO4: C, 60.84; H, 4.87; N, 3.22.
stirred for 1 h with ice-cooling, followed by addition of (Ϯ)-5a (4.0 g, 12.9
mmol) and NaHCO3 (5.42 g, 64.5 mmol). The solution was stirred for 1 h at
Found: C, 60.59; H, 4.84; N, 3.30.
9b: mp 194—195 °C (Et2O–hexane). IR nmax (KBr) cmϪ1: 1700, 1650
0 °C, washed with water, dried over MgSO4, and then concentrated in vacuo. (CϭO), 1630 (CϭC). 1H-NMR (CDCl3) d: 1.27 (3H, t, Jϭ7.0 Hz), 1.30
The residue was chromatographed [eluent: hexane–AcOEt (2 : 1)] to give 6a (3H, d, Jϭ6.8 Hz), 1.61 (3H, d, Jϭ6.8 Hz), 4.05—4.3 (2H, m), 4.7—4.95
(5.6 g, 12.4 mmol, 96%) as a colorless powder. mp 143—146 °C (AcOEt– (1H, m), 5.48 (1H, s), 6.52 (1H, s), 6.57 (1H, d, Jϭ2.2 Hz), 7.3—7.6 (5H,
hexane). IR nmax (KBr) cmϪ1: 3450, 3380 (NH, OH); 1750, 1660 (CϭO). m), 8.16 (1H, d, Jϭ7.4 Hz). Anal. Calcd for C22H21Cl2NO4: C, 60.84; H,
1H-NMR (CDCl3) d: 0.95—1.5 (9H, m), 2.5—3.9 (3H, m), 4.0—4.6 (3H, 4.87; N, 3.22. Found: C, 60.76; H, 4.99; N, 3.32.
m), 6.25—6.6 (1H, m), 6.95—7.8 (7H, m). Anal. Calcd for C22H23Cl2NO5:
8b (1.0 g, 1.89 mmol) was converted to a mixture of 9a and 9b in a similar
C, 58.42; H, 5.13; N, 3.10. Found: C, 58.55; H, 5.31; N, 3.12.
manner as described above. The mixture was chromatographed [eluent:
Ethyl (3RS,5SR,aSR)-7-Chloro-5-(2-chlorophenyl)-1-isopropyl-2-oxo- hexane–AcOEt (10 : 1—5 : 1)] to give 9b (0.33 g, 0.760 mmol, 40%) as col-
1,2,3,5-tetrahydro-4,1-benzoxazepine-3-glycolate (7a) and Ethyl (3RS, orless prisms from the first fraction and 9a (0.17 g, 0.391 mmol, 21%) as a
5RS,aSR)-7-Chloro-5-(2-chlorophenyl)-1-isopropyl-2-oxo-1,2,3,5-tetrahy-
dro-4,1-benzoxazepine-3-glycolate (7b) A mixture of 6a (10.0 g, 22.1
colorless prisms from the second fraction.
(E)-[7-Chloro-5-(2-chlorophenyl)-1-isopropyl-2-oxo-1,5-dihydro-4,1-
mmol), K2CO3 (3.06 g, 2.21 mmol) and EtOH (200 ml) was stirred overnight benzoxazepin-3(2H)-ylidene]acetic Acid (2a) and (Z)-[7-Chloro-5-(2-
at room temperature. The reaction mixture was diluted with water, extracted chlorophenyl)-1-isopropyl-2-oxo-1,5-dihydro-4,1-benzoxazepin-3(2H)-
with AcOEt. The extract was washed with brine, dried over MgSO4, and
ylidene]acetic Acid (2b) One normal NaOH (5 ml) was added to a solu-
then concentrated under reduced pressure. The residue was chroma- tion of 9a (0.16 g, 0.368 mmol) in MeOH (20 ml). The mixture was stirred
tographed [eluent: hexane–AcOEt (2 : 1)] to give a 1 : 1 mixture of 7a and 7b for 1 h at room temperature, diluted with water, acidified, and extracted with
(7.2 g, 15.9 mmol, 72%). The mixture was recrystallized from AcOEt– AcOEt. The extract was washed with brine, dried over MgSO4, and then
hexane to give the (3RS,5SR,aSR)-isomer 7a (1.75 g, 3.87 mmol, 24%) as
concentrated under reduced pressure to give 2a (0.14 g, 0.345 mmol, 94%)
colorless prisms. mp 188—189 °C (AcOEt–hexane). 1H-NMR (CDCl3) d: as colorless plates.
1.29 (3H, t, Jϭ7.1 Hz), 1.30 (3H, d, Jϭ7.0 Hz), 1.56 (3H, d, Jϭ7.0 Hz), 4.07
Compound 2b (0.26 g, 0.640 mmol, 93%) was prepared from 9b (0.3 g,
(1H, d, Jϭ9.0 Hz), 4.19 (1H, d, Jϭ4.8 Hz), 4.26 (2H, dq, Jϭ7.1, 1.8 Hz), 0.691 mmol) in a similar manner as described for the preparation of 2a as a
4.5—4.65 (1H, m), 4.7—4.9 (1H, m), 6.04 (1H, s), 6.54 (1H, d, Jϭ2.6 Hz), colorless plates.
7.2—7.8 (6H, m).
2a: mp 209—214 °C (dec.) (AcOEt–hexane). IR nmax (KBr) cmϪ1: 1720,
Mother liquid was concentrated in vacuo. The residue was recrystallized 1650 (CϭO), 1630 (CϭC). 1H-NMR (CDCl3) d: 1.34 (3H, d, Jϭ7.0 Hz),
from AcOEt–hexane to give the (3RS,5RS,aSR)-isomer 7b (1.2 g, 2.65 1.62 (3H, d, Jϭ7.0 Hz), 4.8—5.0 (1H, m), 5.41 (1H, s), 6.51 (1H, s), 6.54
mmol, 17%) as colorless prisms. mp 141—142 °C (AcOEt–hexane). 1H- (1H, d, Jϭ1.8 Hz), 7.3—7.6 (5H, m), 7.78 (1H, d, Jϭ7.0 Hz). Anal. Calcd
NMR (CDCl3) d: 0.4—0.6 (3H, m), 1.24 (3H, d, Jϭ7.4 Hz), 1.26 (3H, t, Jϭ for C20H17Cl2NO4: C, 59.13; H, 4.22; N, 3.45. Found: C, 58.97; H, 4.24; N,
7.4 Hz), 3.95—4.25 (2H, m), 4.23 (2H, q, Jϭ7.4 Hz), 4.38 (1H, d, Jϭ 3.42.
5.0 Hz), 4.4—4.6 (1H, m), 6.09 (1H, s), 7.1—7.85 (7H, m).
Ethyl (3RS,5SR,aSR)-a-Methanesulfonyloxy-7-chloro-5-(2-
2b: mp 234—239 °C (dec.) (AcOEt–hexane). IR nmax (KBr) cmϪ1: 1670
(CϭO), 1655 (CϭC). 1H-NMR (CDCl3) d: 1.31 (3H, d, Jϭ7.0 Hz), 1.61
chlorophenyl)-1-isopropyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine- (3H, d, Jϭ7.0 Hz), 4.7—4.9 (1H, m), 5.48 (1H, s) , 6.54 (1H, s), 6.57 (1H, d,
3-acetate (8a) and Ethyl (3RS,5RS,aSR)-a-Methanesulfonyloxy-7- Jϭ2.2 Hz), 7.3—7.6 (5H, m), 8.05 (1H, d, Jϭ7.8 Hz). Anal. Calcd for
chloro-5-(2-chlorophenyl)-1-isopropyl-2-oxo-1,2,3,5-tetrahydro-4,1-benz- C20H17Cl2NO4: C, 59.13; H, 4.22; N, 3.45. Found: C, 59.02; H, 4.21; N,
oxazepine-3-acetate (8b) MsCl (0.13 ml, 1.72 mmol) and Et3N (0.24 ml, 3.37.
1.72 mmol) was added to a solution of 7a (0.6 g, 1.33 mmol) in AcOEt
Ethyl (2,3-trans)-3-[N-[4-Chloro-2-(2-chloro-a-hydroxybenzyl)phenyl]-
(30 ml) with ice-cooling. After stirring for 1 h, the reaction mixture was N-propyl]carbamoyl]-2,3-epoxybutyrate (6b) and Ethyl (2,3-trans)-3-[N-
washed with brine, dried over MgSO4, and then concentrated. The residue [4-Chloro-2-(2-chloro-a-hydroxybenzyl)phenyl]-N-isobutyl]carbamoyl]-
was chromatographed [eluent: hexane–CH2Cl2–AcOEt (10 : 5 : 1)] to give 8a
2,3-epoxybutyrate (6c) Compound 6b (5.3 g, 11.7 mmol, 91%) and 6c
(0.63 g, 1.19 mmol, 89%) as colorless prisms. mp 159—160 °C (hexane– (14.0 g, 30.0 mmol, 97%) were prepared from (Ϯ)-5b (4.0 g, 12.9 mmol) and
AcOEt). IR nmax (KBr) cmϪ1: 1745, 1680 (CϭO). 1H-NMR (CDCl3) d: 1.31 (Ϯ)-5c (10.0 g, 30.8 mmol) in a similar manner as described for the prepara-
(3H, t, Jϭ7.1 Hz), 1.31 (3H, d, Jϭ7.0 Hz), 1.56 (3H, d, Jϭ7.0 Hz), 3.17 (3H,
s), 4.18 (1H, d, Jϭ8.0 Hz), 4.32 (2H, q, Jϭ7.1 Hz), 4.7—4.9 (1H, m), 5.37
(1H, d, Jϭ8.0 Hz), 6.04 (1H, s), 6.50 (1H, d, Jϭ2.4 Hz), 7.2—7.6 (6H, m). 0.75—1.05 (3H, m), 1.1—1.8 (5H, m), 2.6—3.9 (4H, m), 4.0—4.5 (3H, m),
tion of 6a.
6b: An oil. IR nmax (neat) cmϪ1: 1745, 1660 (CϭO). 1H-NMR (CDCl3) d:
Anal. Calcd for C23H25Cl2NO7S: C, 52.08; H, 4.75; N, 2.64. Found: C,
52.25; H, 4.80; N, 2.74.
6.1—6.4 (1H, m), 6.9—7.9 (7H, m).
6c: An oil. IR nmax (neat) cmϪ1: 3390 (OH); 1745, 1660 (CϭO). 1H-NMR
Compound 8b (0.82 g, 1.55 mmol, 88%) was prepared from 7b (0.80 g, (CDCl3) d: 0.75—1.05 (6H, m), 1.15—1.4 (3H, m), 1.7—2.0 (1H, m), 2.7—
1.77 mmol) in a similar manner as described for the preparation of 8a as a 3.3 (2H, m), 3.3—3.9 (2H, m), 4.0—4.45 (3H, m), 6.1—6.4 (1H, m), 7.0—
colorless prisms. mp 143—144 °C (AcOEt–hexane). IR nmax (KBr) cmϪ1
:
7.8 (7H, m).
1750, 1670 (CϭO). 1H-NMR (CDCl3) d: 0.5—0.8 (3H, m), 1.26 (3H, d, Jϭ
Ethyl (3,5-trans)-7-Chloro-5-(2-chlorophenyl)-2-oxo-1-propyl-1,2,3,5-
6.8 Hz), 1.29 (3H, t, Jϭ7.0 Hz), 3.16 (3H, s), 4.28 (2H, q, Jϭ7.0 Hz), 4.1— tetrahydro-4,1-benzoxazepine-3-glycolate (10a) and Ethyl (3,5-trans)-7-
4.35 (1H, m), 4.45—4.6 (1H, m), 5.1—5.4 (1H, m), 6.09 (1H, s), 7.1—7.75 Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-1,2,3,5-tetrahydro-4,1-ben-
(7H, m). Anal. Calcd for C23H25Cl2NO7S: C, 52.08; H, 4.75; N, 2.64. Found: zoxazepine-3-glycolate (10b) A mixture of 6c (13.0 g, 27.9 mmol) and
C, 52.38; H, 4.89; N, 2.77.
Ethyl (E)-[7-Chloro-5-(2-chlorophenyl)-1-isopropyl-2-oxo-1,5-dihydro-
K2CO3 (3.85 g, 27.9 mmol) in EtOH (150 ml) was stirred overnight at room
temperature, diluted with water, and extracted with AcOEt. The extract was
4,1-benzoxazepin-3(2H)-ylidene]acetate (9a) and Ethyl (Z)-[7-Chloro-5- washed with brine, dried over MgSO4, and then concentrated. The residue

January 2002
57
was chromatographed [eluent: hexane–AcOEt (2 : 1)] to give 10b (a mixture C20H17Cl2NO4: C, 59.13; H, 4.22; N, 3.45. Found: C, 58.88; H, 4.34; N,
of diastereomers) (7.0 g, 15.0 mmol, 54%) as colorless crystals.
3.27.
3b: Prisms. Yield 0.23 g (0.566 mmol, 82%). mp 208—210 °C (dec.)
10b: IR nmax (KBr) cmϪ1: 3400 (OH); 1740, 1670, 1640 (CϭO). 1H-NMR
(CDCl3) d: 0.7—1.1 (6H, m), 1.2—1.4 (5H, m), 1.7—2.1 (1H, m), 3.35—
(AcOEt–hexane). IR nmax (KBr) cmϪ1: 1690, 1655 (CϭO), 1635 (CϭC).
4.0 (2H, m), 4.0—4.8 (5H, m), 6.15, 6.19 (1H, each s, ca. 1 : 1), 6.55, 6.96 1H-NMR (CDCl3) d: 0.98 (3H, t, Jϭ7.4 Hz), 1.6—1.9 (2H, m), 3.4—3.7
(1H, each d, Jϭ2.4 Hz), 7.15—7.8 (6H, m). Anal. Calcd for C23H25Cl2NO5: (1H, m), 4.4—4.6 (1H, m), 5.51 (1H, s), 6.56 (1H, s), 6.58 (1H, d,
C, 59.24; H, 5.40; N, 3.00. Found:C, 59.14; H, 5.37; N, 2.98.
Jϭ2.4 Hz), 7.2—7.6 (5H, m), 8.07 (1H, d, Jϭ7.0 Hz). Anal. Calcd for
Compound 10a (2.3 g, 5.08 mmol, 43%) was prepared from 6b (5.3 g, C20H17Cl2NO4: C, 59.13; H, 4.22; N, 3.45. Found: C, 59.20; H, 4.39; N,
11.7 mmol) in a similar manner as described for the preparation of 10b.
10a: An oil. IR nmax (KBr) cmϪ1: 3380 (OH); 1745, 1650 (CϭO). 1H-
3.32.
4a: Prisms. Yield 0.25 g (0.595 mmol, 95%). mp 207—209 °C (dec.)
NMR (CDCl3) d: 0.7—1.05 (3H, m), 1.15—1.4 (3H, m), 1.45—1.8 (2H, m), (AcOEt–hexane). IR nmax (KBr) cmϪ1: 1720 (CϭO), 1645 (CϭC). 1H-NMR
3.4—3.9 (2H, m), 4.0—4.7 (4H, m), 6.08, 6.10 (1H, each s), 6.5—7.8 (7H, (CDCl3) d: 0.97 (3H, d, Jϭ6.7 Hz), 1.08 (3H, d, Jϭ6.6 Hz), 1.95—2.2 (1H,
m). Anal. Calcd for C22H23Cl2NO5: C, 58.42; H, 5.12; N, 3.10. Found : C, m), 3.63 (1H, dd, Jϭ13.9, 5.6 Hz), 4.23 (1H, dd, Jϭ13.9, 8.8 Hz), 5.46 (1H,
58.40; H, 5.23; N, 3.01.
s), 6.55 (1H, d, Jϭ2.2 Hz), 6.58 (1H, s), 7.3—7.9 (6H, m). Anal. Calcd for
Ethyl a-Methanesulfonyloxy-(3,5-trans)-7-chloro-5-(2-chlorophenyl)- C21H19Cl2NO4: C, 60.01; H, 4.56; N, 3.33. Found: C, 59.95; H, 4.64; N,
2-oxo-1-propyl-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate (11a) and 3.45.
Ethyl a-Methanesulfonyloxy-(3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-
isobutyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetatel (11b)
Compounds 11a (1.9 g, 3.58 mmol, 81%) and 11b (2.0 g, 3.67 mmol, 95%)
4b: Prisms. Yield 0.25 g (0.595 mmol, 89%). mp 236—238 °C (dec.)
(AcOEt–hexane). IR nmax (KBr) cmϪ1: 1690, 1655 (CϭO), 1630 (CϭC).
1H-NMR (CDCl3) d: 0.95 (3H, d, Jϭ6.6 Hz), 1.02 (3H, d, Jϭ6.6 Hz), 1.85—
were prepared from 10a (2.0 g, 4.42 mmol) and 10b (1.8 g, 3.86 mmol) in a 2.1 (1H, m), 3.45 (1H, dd, Jϭ13.8, 5.8 Hz), 4.46 (1H, dd, Jϭ13.8, 8.6 Hz),
similar manner as described for the preparation of 8a.
5.52 (1H, s), 6.57 (1H, d, Jϭ2.2 Hz), 6.64 (1H, s), 7.2—7.6 (5H, m). Anal.
1
11a: An oil. IR nmax (neat) cmϪ1: 1750, 1670 (CϭO). H-NMR (CDCl3) Calcd for C21H19Cl2NO4: C, 60.01; H, 4.56; N, 3.33. Found: C, 60.16; H,
d: 0.7—1.1 (3H, m), 1.2—1.4 (3H, m), 1.5—1.85 (2H, m), 3.07, 3.16 (3H, 4.56; N, 3.50.
each s), 3.4—3.8 (2H, m), 4.2—5.45 (4H, m), 6.07, 6.10 (1H, each s), 6.5—
7.7 (7H, m).
Animals and Materials Animals were supplied by Clea, Japan, Inc. un-
less otherwise mentioned. RS-[2-14C]Mevalonolactone and [1-3H]farnesyl
11b: A mixture of crystals and an oil. IR nmax (Nujol) cmϪ1: 1765, 1670, pyrophosphate were purchased from New England Nuclear. [2-14C]meval-
1
1630 (CϭO). H-NMR (CDCl3) d: 0.75—1.1 (6H, m), 1.25—1.4 (3H, m), onic acid was synthesized from [2-14C]mevalonolactone by saponification
1.7—2.1 (1H, m), 2.98, 3.16 (3H, each s), 3.35—3.6 (1H, m), 3.9—5.5 (5H,
m), 6.18 (1H, s), 6.5—7.85 (7H, m). Anal. Calcd for C24H27Cl2NO7S: C,
52.95; H, 5.00; N, 2.57. Found: C, 52.92; H, 5.07; N, 2.72.
with potassium hydroxide. [2-14C]Sodium acetate was purchased from
Amersham. Farnesyl pyrophosphate was synthesized by the method de-
scribed by V. J. Davisson and coworkers11) (Nemoto & Co.). HepG2 cells
Ethyl (E)- and (Z)-[7-Chloro-5-(2-chlorophenyl)-2-oxo-1-propyl-1,5- were supplied by The American Type Culture Collection (ATCC). Fetal
dihydro-4,1-benzoxazepin-3(2H)-ylidene]acetate (12a, b) and Ethyl (E)- bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM)
and (Z)-[7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-1,5-dihydro-4,1- were purchased from GIBCO. Human lipoprotein deficient serum (human
benzoxazepine-3(2H)-ylidene]acetate (13a, b) Compounds 12a, b and LPDS) was purchased from Sigma. All other reagents were supplied by
13a, b were prepared from 11a (1.9 g, 3.58 mmol) and 11b (2.0 g, 3.67
mmol) in a similar manner as described for the preparation of 9a, b.
Wako Pure Chemical Industries.
Preparation of Rat Squalene Synthase An Sprague-Dawley (SD)
12a (Polar): Prisms. Yield 0.20 g (0.461 mmol, 13%). mp 114—115 °C male rat (6-week old) was killed by bleeding, and its liver was excised.
1
(Et2O–hexane). IR nmax (KBr) cmϪ1: 1715, 1670 (CϭO), 1640 (CϭC). H- About 10 g of the liver was washed with a saline solution cooled with ice,
NMR (CDCl3) d: 1.00 (3H, t, Jϭ7.4 Hz), 1.27 (3H, t, Jϭ7.1 Hz), 1.5—1.9 which was then homogenized in 15 ml of an ice-cooled buffer solution
(2H, m), 3.5—3.75 (1H, m), 4.13 (2H, q, Jϭ7.1 Hz), 4.3—4.55 (1H, m),
[100 mM potassium phosphate (pH 7.4), 15 mM nicotinamide, 2 mM MgCl2],
5.45 (1H, s), 6.49 (1H, s), 6.53 (1H, d, Jϭ1.8 Hz), 7.3—7.6 (5H, m), 7.78 followed by centrifugation for 20 min at 10000ϫg (4 °C). The supernatant
(1H, d, Jϭ6.6 Hz). Anal. Calcd for C22H21Cl2NO4: C, 60.84; H, 4.87; N, layer was separated and subjected to further centrifugation for 90 min at
3.22. Found: C, 60.85; H, 5.17; N, 3.13.
105000ϫg (4 °C). The sediment was then suspended in an ice-cooled
12b (Less Polar): Prisms. Yield 0.61 g (1.40 mmol, 39%). mp 171—
100 mM potassium phosphate buffer solution (pH 7.4), which was again sub-
172 °C (Et2O–hexane). IR nmax (KBr) cmϪ1: 1710, 1660 (CϭO), 1630 jected to centrifugation for 90 min at 105000ϫg (4 °C). The sediment thus
(CϭC). 1H-NMR (CDCl3) d: 0.98 (3H, t, Jϭ7.4 Hz), 1.28 (3H, t, Jϭ7.1 Hz),
obtained (microsome fraction) was suspended in an ice-cooled 100 mM
1.6—1.85 (2H, m), 3.45—3.7 (1H, m), 4.1—4.3 (2H, m), 4.4—4.6 (1H, m), potassium phosphate buffer (pH 7.4) (about 40 mg/ml protein concentration,
5.50 (1H, s), 6.53 (1H, s), 6.57 (1H, d, Jϭ2.2 Hz), 7.2—7.6 (5H, m), 8.17 determined using Bicinchoninic acid (BCA) protein assay kit of Pierce Co.,
(1H, d, Jϭ7.2 Hz). Anal. Calcd for C22H21Cl2NO4: C, 60.84; H, 4.87; N, Ltd.). This suspension was used as the enzyme solution.
3.22. Found: C, 60.65; H, 4.73; N, 3.15.
Preparation of Human Squalene Synthase HepG2 cells (about 1ϫ109
13a (Polar): An oil. Yield 0.29 g (0.647 mmol, 18%). IR nmax (Neat) cells) obtained by incubation (37 °C in the presence of 5% CO2) in a DMEM
1
cmϪ1: 1710, 1670 (CϭO), 1645 (CϭC). H-NMR (CDCl3) d: 0.95 (3H, d, contains 10% FBS, penicillin G (100 units/ml) and streptomycin (10 mg/ml)
Jϭ6.6 Hz), 1.07 (3H, d, Jϭ6.6 Hz), 1.9—2.2 (1H, m), 3.57 (1H, dd, Jϭ14.0,
5.8 Hz), 4.0—4.25 (2H, m), 4.37 (1H, dd, Jϭ14.0, 8.6 Hz), 5.44 (1H, s),
6.54 (1H, d, Jϭ1.8 Hz), 6.57 (1H, s), 7.3—7.9 (6H, m).
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
13b (Less Polar): Prisms. Yield 0.89 g (1.99 mmol, 54%). mp 181— sonicate thus obtained, the microsome fraction was obtained by the same
182 °C (Et2O–hexane). IR nmax (KBr) cmϪ1: 1715 (CϭO), 1650 (CϭC). 1H- procedure as in preparation of rat-derived enzyme, which was suspended in
NMR (CDCl3) d: 0.94 (3H, d, Jϭ6.6 Hz), 1.01 (3H, d, Jϭ6.6 Hz), 1.85—2.1
(1H, m), 3.43 (1H, dd, Jϭ13.8, 5.6 Hz), 4.05—4.25 (2H, m), 4.45 (1H, dd,
Jϭ13.8, 8.6 Hz), 5.51 (1H, s), 6.57 (1H, d, Jϭ2.4 Hz), 6.61 (1H, s), 7.2—7.6
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
(5H, m), 8.19 (1H, d, Jϭ8.0 Hz). Anal. Calcd for C23H23Cl2NO4: C, 61.62; activity was monitored by the formation of [3H]squalene from [1-3H]farne-
H, 5.17; N, 3.12. Found: C, 61.54; H, 5.30; N, 3.09.
(E)- and (Z)-[7-Chloro-5-(2-chlorophenyl)-2-oxo-1-propyl-1,5-dihy-
dro-4,1-benzoxazepin-3-ylidene]acetic Acid (3a, b; Table 1) and Ethyl
syl pyrophosphate. Fifty microliters of assay mixture included 5 mM [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
(E)- and (Z)-[7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-1,5-dihydro- test compound dissolved in dimethyl sulfoxide (DMSO) (a final concentra-
4,1-benzoxazepin-3-ylidene]acetic Acid (4a, b; Table 1) Compounds tion of DMSO was 2%) and enzyme solution prepared from rat or HepG2
3a, b and 4a, b were prepared from 12a (0.17 g, 0.391 mmol), 12b (0.30 g, cells (protein content 0.8 mg). The assay ran 45 min at 37 °C and stopped by
0.691 mmol), 13a (0.28 g, 0.625 mmol) and 13b (0.30 g, 0.669 mmol) in a adding 150 ml of CHCl3–MeOH (1 : 2) containing 0.2% cold squalene as car-
similar manner as described for the preparation of 2a, b.
rier. Aqueous solution of 3 N NaOH (50 mM) and CHCl3 (50 mM) were added
3a: Prisms. Yield 0.15 g (0.369 mmol, 94%). mp 192—194 °C (dec.) to the mixture. The chloroform layer containing the reaction mixture having
(AcOEt–hexane). IR nmax (KBr) cmϪ1: 1715, 1645 (CϭO), 1635 (CϭC). squalene as the principal component and 3 ml of toluene-based liquid scintil-
1H-NMR (CDCl3) d: 1.01 (3H, t, Jϭ7.4 Hz), 1.6—1.9 (2H, m), 3.6—3.8 lator were mixed, and its radioactivity was determined by means of a liquid
(1H, m), 4.25—4.5 (1H, m), 5.47 (1H, s), 6.51 (1H, s), 6.54 (1H, d, scintillation counter. The squalene synthase inhibitory activity was ex-
Jϭ2.2 Hz), 7.3—7.6 (5H, m), 7.80 (1H, d, Jϭ6.6 Hz). Anal. Calcd for pressed in terms of the concentration of the test compound inhibiting by

58
Vol. 50, No. 1
50% the radioactivity taken into the chloroform layer (IC50, molar concen-
tration (M)).
3) Tamai M., Yokoo C., Murata M., Oguma K., Sota K., Sato E.,
Kanaoka Y., Chem. Pharm. Bull., 35, 1098—1104 (1987).
4) Reaction of 5a with 4-nitrophenyl ester3) of DL-trans-epoxysuccinic
acid monoethyl ester did not proceed.
5) Yamamoto M., Yoshitake M., Kishikawa K., Kohmoto S., Yuki Gosei
Kagaku Kyokai Shi., 50, 638—651 (1992).
6) Only one spot was detected by TLC analysis of the mixture of 7a and
7b. Separation of the mixture by column chromatography was un-
sucessful, even when this mixture was converted to acetate or ben-
zoate.
7) We initially tried to synthesize the epimer [(3RS,5SR,aRS)-derivative]
of 7a using cesium propionate, which is a reagent for inversion of the
configuration of secondary alcohol. Kruizinga W. H., Strijtveen B.,
Kellogg R. M., J. Org. Chem., 46, 4321—4323 (1981).
Single-Crystal X-Ray Analysis. 7a: Unit cell parameters [aϭ11.245
(1) Å, bϭ11.746 (1) Å, cϭ9.067 (1) Å, aϭ107.26 (1)°, bϭ98.64 (1)°, gϭ
74.94 (1)°], Zϭ2, space group P1, R-factorϭ0.080.
7b: Unit cell parameters [aϭ10.854 (1) Å, bϭ11.083 (1) Å, cϭ9.807 (1)
Å, aϭ108.20 (1)°, bϭ96.53 (1)°, gϭ94.13 (1)°], Zϭ2, space group P1, R-
factorϭ0.083.
2a: Unit cell parameters [aϭ10.018 (1) Å, bϭ11.304 (1) Å, cϭ8.508 (1)
Å, aϭ93.87 (1)°, bϭ98.23 (1)°, gϭ91.83 (1)°], Zϭ2, space group P1, R-
factorϭ0.063.
Acknowledgments The authors wish to thank Dr. Kanji Meguro for his
encouragement and helpful discussion throughout this study. We also thank
Mr. Akira Fujishima and Ms. Keiko Higashikawa for X-ray crystallographic
analysis.
8) The mechanism of this elimination is considered to be E1cB.
9) 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).
References and Notes
1) Part 3 in the series of studies on 4,1-benzoxazepine derivatives as 10) The active conformation of 5-naphtyl-4,1-benzoxazepine derivative in
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).
the crystal of protein–inhibitor complex supports our results. Pandit J.,
Danley D. E., Schulte G. K., Mazzalupo S., Pauly T. A., Hayward C.
M., Hamanaka E. S., Thompson J. F., Harwood H. J., Jr., J. Biol.
Chem., 275, 30610—30617 (2000).
11) Davisson V. J., Woodside A. B., Poulter C. D., Methods Enzymol., 110,
130—144 (1985).
2) Yukimasa H., Kori M., Tozawa R., Sigiyama Y. (Takeda), Eur. Patent,
645377 (1995) [Chem. Abstr., 123, 256777w (1995)].