Syntheses of fused heterocyclic compounds and their inhibitory activities for squalene synthase

Bioorganic & Medicinal Chemistry 10 (2002) 385–400

Syntheses of Fused Heterocyclic Compounds and Their

Inhibitory Activities for Squalene Synthase

Takashi Miki,* Masakuni Kori, Akira Fujishima, Hiroshi Mabuchi, 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 8 June 2001; accepted 12 August 2001

Abstract—A variety of fused heterocyclic compounds (2–11) were synthesized as a modiﬁcation of the lead compound 1a and

evaluated for their inhibition of squalene synthase. 4,1-Benzothiazepine derivative 2, 1,4-benzodiazepine derivative 6, 1,3-benzo-

diazepine derivative 7, 1-benzazepine derivative 9, and 4,1-benzoxazocine derivative 10 potently inhibited squalene synthase activ-

ity, whereas the 4,1-benzoxazepine derivatives 1 was the most potent inhibitor. 4,1-Benzothiazepine S-oxidedreivative 4, 1,4-

benzodiazepine derivative 5, 1,3,4-benzotriazepine derivative 8, and 1,2,3,4-tetrahydroquinoline derivative 11 were found to be

weakly active. Comparison of the X-ray structures of these compounds (1a, 2, 4, 5, 7 and 10) suggests that orientation of the 5- (or

Introduction

other non-steroidal isoprenoids, drugs which inhibit the

step beyond that involving farnesyl pyrophosphate are

desirable as cholesterol biosynthesis inhibitors. Inhibi-

tors of squalene synthase, squalene epoxidase, and oxi-

dosqualene cyclase have therefore been targeted by

many laboratories.²

Adequate control of serum cholesterol level is very

important for prophylaxis and therapy of diseases rela-

ted to atherosclerosis¹such as ischemic cardiopathy and

cerebral infarction, which are the major causes of death

in industrialized countries. Hypercholesterolaemia is

related to the development and progression of coronary

heart disease, and great eﬀorts have been made at dis-

covering agents to lower plasma cholesterol in order to

prevent and treat hypercholesterolemia.²

Squalene synthase [EC2.5.1.21], which catalyzes the

formation of squalene from farnesyl pyrophosphate,

participates in the ﬁrst committed step in sterol synth-

esis. Farnesyl pyrophosphate, the substrate for squalene

synthase, is water-soluble and may be readily metabo-

lized for excretion in urine.⁵It has been reported that

inhibition of squalene synthase plays a role in feedback

regulation of HMG-CoA reductase.⁶We therefore sus-

pected that squalene synthase inhibitors might be safe

and eﬀective in the treatment of hyperlipidemia.

As pharmaceutical preparations for controlling choles-

terol biosynthesis, 3-hydroxy-3-methyl glutaryl-coen-

zymeA (HMG-CoA) rdeuctaseinhibitors such as

pravastatin,^3alovastatin,^3bsimvastatin,^3cﬂuvastatin,^3d

and atorvastatin,^3eareavailablefor clinical use. ⁴HMG-

CoA reductase inhibitors block an early step in the

cholesterol biosynthetic pathway. These agents are

therefore thought to inhibit the biosynthesis of not only

cholesterol but also biologically important isoprenoids

such as dolicol, ubiquinoneand isoprenyl tRNA. Thus,

the occurrence of undesirable side eﬀects arising from

the inhibition of isoprenoid synthesis is feared.

Several classes of squalene synthase inhibitors have

recently been reported,^7,8such as cationic intermediate

analogues (containing ammonium ions or sulfonium

ions), substrateanalogsue (phosphorus-containing

compounds, bisphosphonates, and a-phosphono-

sulfonic acids), quinuclidinederivatives, 2,8-dioxabicy-

clo[3.2.1]octane derivatives^8,9(Squalestatins, Zaragozic

acids, TAN-1607A¹⁰), and others.¹¹

Since farnesyl pyrophosphate is the ﬁnal common pre-

cursor in the biosynthesis pathway of cholesterol and

From random screening for squalene synthase inhibi-

tors, we found that a novel class of inhibitor, benzene

fused lactam derivative, (3,5-trans)-5-phenyl-2-oxo-4,1-

benzoxazepine-3-acetic acid derivative 1a (Fig. 1),

*Corresponding author. Tel.:+81-6-6300-6637; fax:+81-6-6300-6306;

e-mail: Miki_Takashi@takeda.co.jp

PII: S0968-0896(01)00289-9

386

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

exhibited

potent

inhibition

of

rat

enzyme

(NaBH₃CN) to aﬀord thealkylated compounds 14a,b.

After condensation of 14a,b and fumaric acid chloride

monoethyl ester, intramolecular Michael addition of the

obtained amides 15a,b aﬀorded the 4,1-benzoxazepine-

3-acetates 16a,b. In this reaction, thermodynamically

stable3,5- trans-isomers were obtained. Hydrolysis of

the esters 16a,b gavethecarboxylic acids 1a,b.

(IC₅₀=0.072 mM) and HepG2 enzyme (IC₅₀=0.024 mM).

Benzene fused seven-membered heterocycles such as

1,4-benzodiazepines,^12aꢀh1,5-benzothiazepines,¹²ⁱ1,4-

benzothiazepines,^12j1-benzazepines,^12k,land so on,^12mꢀq

are important components of a number of pharmacolo-

gically active compounds. A variety of benzene fused

seven-membered heterocycles have been reported, and

the conformations of these skeletons are likely to be

signiﬁcantly diﬀerent from one another. Since the main

determinant of side-chain orientation is the conforma-

tion of the seven-membered ring, we thought that this

conformation would also determine the selectivity

towards the biological target, and we therefore assumed

that the spatial positions of 5-phenyl, 3-acetic acid and

1-alkyl groups sidechains of 1a were important for

potent inhibitory activity. Thus, as a chemical mod-

iﬁcation of compound 1a, we ﬁrst synthesized het-

erocyclic seven-membered lactam derivatives to ﬁnd

an optimal template for inhibition of squalene syn-

thase.¹³In order to assess the eﬀect of ring size on

the biological activity, six- and eight-membered lac-

tam compounds were also synthesized.

Preparation of 4,1-benzothiazepine-3-acetic acid derivatives

The synthesis of the 4,1-benzothiazepine-3-acetic acid

derivative 2 is outlined in Scheme 2. The dicarboxylic

acid derivative 17 was obtained by treatment of 14a and

thiomalic acid in a mixture of concentrated hydrochlo-

ric acid and acetic acid. Intramolecular cyclization of 17

by reﬂuxing in xylene aﬀorded the 4,1-benzothiazepine-

3-acetic acid derivative 18 as a ca. 1:1 mixtureof cis and

trans isomers.¹⁵Esteriﬁcation of 18 led to the methyl

ester 19, which was epimerized with potassium carbon-

Chemistry

The medium-sized ring systems having acetic acid side

chain prepared in this study are shown in Figure 1. As

seven-membered heterocyclic compounds, the 4,1-ben-

zothiazepine-3-acetic acid derivatives 2, 1,4-benzodiaze-

pine-3-acetic acid derivatives 5 and 6, 1,3-benzodiazepine-

3-acetic acid derivative 7, 1,3,4-benzotriazepine-3-acetic

acid derivative 8 and 1-benzazepine-3-acetic acid deriva-

tive 9 were synthesized. The 4,1-benzoxazocine-3-acetic

acid derivative 10 having an eight-membered ring and

quinoline-3-acetic acid derivative 11 with a six-mem-

bered ring were also prepared (vide infra).

Scheme 1. Reagents and conditions: (a) NaBH₄; (b) isobutylaldehyde

or pivalaldehyde, NaBH₃CN, AcOH; (c) fumaric acid chloridemono-

ethyl ester, NaHCO₃; (d) K₂CO₃, EtOH, (e) NaOH.

Preparation of 4,1-benzoxazepine-3-acetic acid derivatives

The syntheses of the 4,1-benzoxazepine-3-acetic acid

14

derivatives 1a,b areshown in Scheme1.

Reduction of

2-aminobenzophenone 12 with sodium borohydride

(NaBH₄) yielded aminoalcohol 13,¹⁴which were treated

with aldehydes and sodium cyano borohydride

Scheme 2. Reagents and conditions: (a) thiomalic acid, concd HCl–

AcOH; (b) xylene, reﬂux; (c) MeOH, cat H₂SO₄; (d) K₂CO₃, MeOH;

(e) NaOH.

Figure 1. Structures of benzene fused heterocyclic compounds.

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

387

ate in methanol to give the thermodynamically stable

trans-isomer 20. The ester 20 was hydrolyzed to give the

desired 4,1-benzothiazepine-3-acetic acid derivative 2.

Swern’s oxidation of 28, and subsequent reductive ami-

nation of the resulting aldehyde 29 with ethyl glycinate

aﬀorded the aminoacetic acid derivative 30. Acylation

of 30 with triﬂuoroacetic anhydride led to the amide 31.

Theamine 32 was synthesized by reduction of a nitro

group of compound 31. Treatment of 32 with piva-

laldehyde, followed by NaBH₃CN gavethe N-alkylated

compound 33. Deprotection of 33 aﬀorded the diamine 34.

The synthesis of the 1,3-benzodiazepine-3-acetic acid deri-

vative 7 was accomplished via compound 35 by treatment

of 34 with triphosgene and subsequent alkaline hydrolysis.

Oxidation of the methyl ester 20 with 1.0 equivalent of

m-chloroperbenzoic acid (MCPBA), followed by alka-

linehydrolysis of theobtained sulfoxide 21, aﬀorded the

4,1-benzothiazepine-3-acetic acid S-oxide 3. This oxida-

tion proceeded asymmetrically to yield only one isomer,

the stereochemistry of which has not yet been deter-

mined. The S-dioxide 4¹⁶was prepared by oxidation of

thecarboxylic acid derivative 2 with 2.2 equivalents of

MCPBA (Scheme 3).

Preparation of 1,3,4-benzotriazepine-3-acetic acid

derivatives

Preparation of 1,4-benzodiazepine-3-acetic acid derivatives

The preparation of the 1,3,4-benzotriazepine-3-acetic

acid derivative 8 is shown in Scheme 6. Treatment of the

aminobenzophenone derivative 22 and Lawesson’s

reagent gave the thione 36. Reaction of compound 36

with ethyl hydrazineacetate aﬀorded a mixture of two

geometrical isomers 37a,b, which was separated by silica

gel column chromatography to give the less polar iso-

mer 37a and thepolar isomer 37b. Compound 37a was

convertible to the cyclized compound 38 by treatment

with triphosgene. Compound 38 was then hydrolyzed to

give the desired 1,3,4-benzotriazepine-3-acetic acid

derivative 8. On the other hand, treatment of the polar

isomer 37b with triphosgene gave a complex mixture. It

appears that 37a is an (E)-isomer which is advantageous

for cyclization and 37b is a (Z)-isomer which undergoes

only minimal cyclization.

Condensation of b-methyl N-benzyloxycarbonyl-dl-

aspartate¹⁷with the aminobenzophenone derivative 12

by themixed anhydridemethod gavetheamide

23, but

condensation of that dl-aspartic acid derivative with

the N-neopentylated aminobenzophenone 22 did not

proceed. Removal of the benzyloxycarbonyl group by

hydrogenolysis, followed by treatment with acetic acid,

aﬀorded the 1,4-benzodiazepine-3-acetic acid derivative

24. Treatment of compound 24 and isobutyl bromide

with sodium hydride(NaH) gavethe1-isobutyl analo-

gue 25. On theother hand, alkylation of 24 with neo-

pentyl bromide yielded

a trace of an alkylated

compound. Compound 25 was hydrolyzed to give the

desired 1,4-benzodiazepine-3-acetic acid derivative 5.

The2,3,4,5-ttreahydro-1

H-4,1-benzodiazepine-3-acetic

acid derivative 6 was prepared by reduction of the C¼N

doublebond by NaBH ₄(Scheme 4).

Preparation of 1-benzazepine-3-acetic acid derivatives

The synthesis of the 1-benzazepine-3-acetic acid

derivative 9 is outlined in Scheme 7. Condensation

of 2-chlorobenzophenone 39 and diethyl succinate

aﬀorded alkylidenesuccinic acid derivative 40 by Stobbe

condensation.¹⁸Compound 40 was converted succes-

sively to the 4-phenylbutyric acid derivative 41 by dec-

Preparation of 1,3-benzodiazepine-3-acetic acid derivatives

Scheme 5 shows the preparation of the 1,3-benzodia-

zepine-3-acetic acid derivative 7. Treatment of methyl

2-chlorophenylacetate 26 and 4-chloro-1,2-dini-

trobenzene with NaH gave the phenylacetic acid deriva-

tive 27. Reduction of 27 with lithium borotetrahydride

(LiBH₄) led to the 2-phenylethanol derivative 28.

Scheme 4. Reagents and conditions: (a) b-methyl N-benzy-

loxycarbonyl-dl-aspartate, BuⁱOCOCl, N-methylmorpholine; (b) (1)

H₂, Pd–C; (2) AcOH, DMF; (c) Bu-Br, NaH, DMF; (d) K₂CO₃,

MeOH, H₂O; (e) NaBH₄.

Scheme 3. Reagents and conditions: (a) mCPBA; (b) K₂CO₃, MeOH–

H₂O.

388

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

arboxylation, hydrogenation of double bond and treat-

ment with p-toluenesulfonic acid in ethanol. The a-tet-

ralonederivative 42 was prepared by alkaline hydolysis

of 41 and subsequent intramolecular Friedel–Crafts

acylation. Beckmann rearrangement of the oxime com-

pound derived from 42 aﬀorded the 1-benzazepine

derivative 43.¹⁹After alkylation at the 1-position, the

obtained 1-isobutyl derivative 44 was halogenated to

7-chloro derivative 45 by treatment with N-chloro-

succinimide. Alkylation of 43 with neopentyl bromide

yielded a trace of an alkylated compoumd. Treatment of

compound 45 with lithium diisopropylamide(LDA)

and ethyl iodoacetate aﬀorded the ethyl ester 46.

Hydrolysis of 46 gave the desired 3-acetic acid derivative

9 as a ca. 2:1 mixtureof cis and trans isomers.

Preparation of 4,1-benzoxazocine-3-acetic acid derivatives

The preparation of the 4,1-benzoxazocine-3-acetic acid

derivative 10 is shown in Scheme 8. The 2-phenyletha-

nol derivative 28 was led to the amino analogue 47 by

reduction of a nitro group. Reductive alkylation of

compound 47, followed by treatment of the resulting

compound 48 with fumaryl chloride monoethyl ester

aﬀorded the amide 49. Unlike 4,1-benzoxazepine

derivatives, compound 49 was hardly cyclized to a 4,1-

benzoxazocine derivative by treatment with potassium

carbonate in ethanol. When the amide 49 was treated

with potassium carbonate in the presence of 18-crown-6

Scheme 5. Reagents and conditions: (a) 4-chloro-1,2-dinitrobenzene,

NaH, DMF; (b) LiBH₄; (c) (COCl)₂, DMSO, NEt₃; (d) glycinemethyl

ester hydrochloride, AcONa, NaBH₃CN; (e) (CF₃CO)₂O; (f) H₂, Pd–

C; (g) pivalaldehyde, AcOH, NaBH₃CN; (h) concd HCl; (i) tri-

phosgene, NEt₃; (j) NaOH.

Scheme 7. Reagents and conditions: (a) diethyl succinate, ButOK; (b)

(1) HBr–AcOH; (2) H₂, Pd–C; (3) EtOH, p-TsOH; (c) (1) 1 N NaOH;

(2) SOCl₂then AlCl₃; (d) (1) NH₂OH; (2) PPA, 120 ^ꢁC; (e) Bu-ⁱBr,

NaH; (f) NCS; (g) (1) LDA; (2) ICH₂COOEt; (h) NaOH.

Scheme 6. Reagents and conditions: (a) Lawesson’s reagent; (b) ethyl

hydrazinoacetate hydrochloride, K₂CO₃; (c) triphosgene, NEt₃; (d)

NaOH.

Scheme 8. Reagents and conditions: (a) Raney–Ni, NH₂NH₂; (b)

pivalaldehyde, NaBH₃CN, AcOH; (c) fumalyl chloridemonotehyl

ester, NaHCO₃; (d) K₂CO₃, CH₂Cl₂, 18-Crown-6; (e) concd HCl.

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

389

in dichloromethane, the 4,1-benzoxazocine-3-acetic acid

derivative 50 was obtained in 24% yield.¹⁴In this reac-

tion, only oneisomer could beisolated. Acid hydrolysis

of compound 50 gave the desired acid 10. Alkaline

hydrolysis was unableto yield 10 because of ring-open-

ing reaction. The relative conﬁgrations of 10 and 50

were determined to be 3,6-trans by X-ray diﬀraction

analysis of thecompound 10.

liver and human hepatoma (HepG2) cells. Inhibitory

activity was measured according to the method of

Cohen et al. with a slight modiﬁcation.²¹

Table 1 shows inhibitory activities of the fused hetero-

cyclic compounds (1–11). The 4,1-benzoxazepine deri-

vative 1a and its 1-isobutyl analogue 1b showed the

same level of potency. The 4,1-benzothiazepine deriva-

tive 2 was ca. 2.5-fold less potent for rat enzyme than

that of 1a and had the same potency for human enzyme

as 1a. Oxidation of thesulfur atom of 2 led to the S-

oxide 3, which exhibited ca. 10-fold less potent for both

enzymes than 1a. The S-dioxide 4 was found to exhibit

only a moderate inhibitor of rat enzyme, with an IC₅₀

valueof 3.3 mM. The 1-benzazepine derivative 9 [cis–

trans (2:1) mixture] exhibited 5-fold weaker activity for

both enzymes than 1a. Because the cis isomer appeared

to beinactive, ²²weassumed that thepureform of trans

isomer would have 3-fold more potent activity than the

cis–trans (2:1) mixture. The 1,3-benzodiazepine deriva-

tive 7 exhibited 5-fold weaker activity for rat and

human enzymes than 1a. The 1,4-benzodiazepine deri-

vative 5 and 1,3,4-benzotriazepine derivative 8 had poor

activities for rat enzyme, with IC₅₀values of 3.9 and 8.7

mM, respectively. Reduction of a C¼N doublebond of 5

led to 6, theactivity of which was found to bethesame

Preparation of 1,2,3,4-tetrahydroquinoline-3-acetic acid

derivatives

The synthesis of the 1,2,3,4-tetrahydroquinoline-3-acetic

acid derivative 11 was carried out as follows (Scheme 9).

Heating of the aminobenzophenone derivative 12 and

diethyl malonate with 1,8-diazabicyclo-[5. 4. 0]-7-unde-

cene (DBU) aﬀorded the quinoline derivative 51.²⁰

Compound 51 was led to the isobutyl analogue 52 by

alkylation at the 1-position. Treatment of 51 with neo-

pentyl bromide yielded a trace of a neopentyl analogue.

TheC ¼C doublebond in 52 was reduced selectively

by lithium alminium hydrideto givethe trans ester 53.

The diester 54 was prepared by treatment of 53 with

NaH, followed by reaction with ethyl bromoacetate.

Compound 54 was converted to the 1,2,3,4-tetra-

hydroquinoline-3-acetic acid derivative 11 by decar-

boxylation, esteriﬁcation, puriﬁcation by column

chromatography and then alkaline hydrolysis.

as that of 7 and the4,1-bnezoxazocinedreivative

10.

The1,2,3,4-tetrahydroquinolinederivative 11 was found

to be only weakly active. The above results indicate that

the 4,1-benzoxazepine derivatives exhibited the most

potent inhibition of both rat and human enzymes.

Results and Discussion

The compounds synthesized were evaluated for inhibi-

tion of activity of squalene synthase prepared from rat

It is expected that these fused heterocycles will play an

important roles in determination of relative spatial

positions of the substituents which are predicted to

attach to the enzyme binding site. In order to compare

conformations, theX-ray structurse of somecom-

pounds were overlaid (Figs 2–4). We found that the 6–7

ring systems analyzed here have similar overall con-

formations. Weassumethat thseeconformations are

especially stableand very similar to theactiveconforma-

tion. Actually, these conformations are similar to the con-

formation of 5-naphtyl-4,1-benzoxazepine derivative^11aꢀd

in the crystal of protein–inhibitor complex.^11d

The 4,1-benzoxazepine derivative 1a, 4,1-benzothiaze-

pinedreivative 2, 1,3-benzodiazepine derivative 7 and

4,1-benzoxazocine derivative 10 had potent inhibitory

activities [IC₅₀=0.072–0.42 mM (rat), 0.024–0.13 mM

(human)]. We found that the 1-neopentyl groups and

the 5-phenyl rings in the compounds with a seven-

membered ring (1a, 2 and 7) are superimposed well (Fig.

2). The conformation of the eight-membered ring of 10 is

diﬀerent from that of the seven-membered ring. Inter-

estingly, however, the substituents of 10 superimposed

well on those of 1a, 2 and 7. Although thepositions of

the carboxymethyl part diﬀer in the X-ray structures, it

can be seen that this ﬂexible part occupies a speciﬁc

region when the compounds bind to the enzyme.

The 1,4-benzodiazepine derivative 5 exhibited only

moderate activity [IC₅₀=3.9 mM (rat), 2.3 mM

(human)]. Superimposition of the X-ray structures of 1a

Scheme 9. Reagents and conditions: (a) diethyl malonate, DBU; (b)

BuiBr, NaH, KI; (c) LiAlH₄; (d) BrCH₂COOEt, NaH; (e) (1) KOH,

EtOH–H₂O; (2) MeI; (3) K₂CO₃, MeOH–H₂O.

390

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

and 5 may explain this decrease in activity. As shown in

Figure 3, the orientations of the 5-phenyl rings of these

compounds were diﬀerent, because the bond angle of a

sp³carbon is diﬀerent from that of a sp²carbon. Actu-

ally, reduction of the double bond of 5 resulted in 10-

fold increase in activity (Table 1, compound 6). It is

likely that the orientation of the 5-phenyl ring of the

1,3,4-benzotriazepine derivative 8 [IC₅₀=8.7 mM (rat

enzyme)], which has a sp²carbon at the5-position, is

similar to that of 5.

Proton nuclear magnetic resonance (¹H NMR) spectra

were recorded on a Varian Gemini-200 (200 MHz)

spectrometer (with tetramethylsilane as an internal

standard). Infrared (IR) absorption spectra were recor-

ded on a Jasco IR-810. TLC analyses were carried out

on Merck Kieselgel 60 F₂₅₄plates. Elemental analyses

were carried out by Takeda Analytical Laboratories,

Ltd., and arewithin Æ0.4% of the theoretical values

unless otherwise noted. For column chromatography,

Merck Kieselgel 60 (70–230 mesh ASTM) was used.

Yields were not maximized. The following abbrevia-

tions are used: s=singlet, d=doublet, t=triplet,

q=quartet, m=multiplet, br=broad.

Overlaying of the X-ray structures of the 4,1-benzoxa-

zepine derivative 1a and the4,1-benzothiazepine S-dioxide

derivative 4 revealed that the orientation of the 5-phenyl

ring of 4 was diﬀerent from that of 1a due to steric inter-

action with oxygen atoms (Fig. 4). It is assumed that simi-

lar steric hindrance would result in a change in orientation

of the5-substituent of the S-oxide 3. Wealso assumethat

theconformation of 11 with the six-membered ring

would be quite diﬀerent from that of compounds with a

seven-membered ring since 11 was only weakly active.

5-Chloro-ꢀ-(2-chlorophenyl)-2-(neopentylamino)benzyl-

alcohol (14a). NaBH₃CN (0.69 g, 11.2 mmol) was

added to an ice-cooled solution of 2-amino-5-chloro-a-

(2⁰-chlorophenyl)benzyl alcohol 13 (2.0 g, 7.46 mmol),

pivalaldehyde (0.71 g, 8.21 mmol) and AcOH (0.90 g,

14.9 mmol) in MeOH (20 mL). After being stirred for

1.5 h at room temperature, the reaction was quenched

with 5% KHSO₄. The mixture was extracted with

AcOEt (50 mL, twice). The extract was washed with

saturated NaHCO₃and brine, dried over Na₂SO₄and

then concentrated in vacuo to give 14a (2.4 g, 7.09 mmol,

95%) as colorless prisms. Mp 110–111 ^ꢁC. ¹H NMR

(CDCl₃) d 0.92 (9H, s), 2.83 (2H, s), 6.15 (1H, s), 6.61

(1H, d, J=8.4 Hz), 6.97 (1H, d, J=2.6 Hz), 7.12–7.45

(5H, m). Anal. calcd for C₁₈H₂₁Cl₂NO: C, 63.91; H,

6.29; N, 4.14. Found: C,64.12; H, 6.30; N,4.31.

Conclusion

Various fused heterocyclic compounds were prepared

and their inhibitions of squalene synthase were investi-

gated. The 4,1-benzoxazepine nucleus is optimal template

for inhibitory activity. Theresults of superimposition of

the X-ray structures of some compounds prepared in this

study revealed that the orientations of the 5-phenyl (6-

phenyl) group on the rings strongly aﬀected activity.

5-Chloro-ꢀ-(2-chlorophenyl)-2-(isobutylamino)benzyl-

alcohol (14b). 14b (3.6 g, 11.1 mmol, quant) was

obtained in a similar procedure from 13 (2.9 g,

10.8 mmol) and isobutylaldehyde (0.71 g, 7.90 mmol) as

colorless prisms. Mp 87–88 ^ꢁC. ¹H NMR (CDCl₃) d

0.92 (6H, d, J=6.6 Hz), 1.77–1.97 (1H, m), 2.90 (2H, d,

Experimental

All melting points were determined on a Yanagimoto

micro melting point apparatus and are uncorrected.

Table 1. Inhibitory activities for squalene synthase of the fused heterocyclic compounds

Compounds

A–B–C

R

IC₅₀(mM)^a

Rat enzyme

HepG2 enzyme

1a (3,5-trans)

1b (3,5-trans)

2 (3,5-trans)

3 (3,5-trans)

4 (3,5-trans)

5

6 (3,5-trans)

7

8

CH–O–CH

CH–S–CH

CH₂Bu^t

Buⁱ

0.072

0.061

0.18

0.70

3.3

3.9

0.43

0.42

8.7

0.024

0.034

0.039

0.25

CH₂Bu^t

Buⁱ

CH–SO–CH

CH–SO₂–CH

C¼N–CH

b

—

2.3

0.16

0.13

—

0.12

0.083

—

CH–NH–CH

CH–CH₂–N

C¼N–N

Buⁱ

CH₂Bu^t

Buⁱ

9 (cis–trans=2:1)

10 (3,6-trans)

11 (3,4-trans)

CH–CH₂–CH

CH–CH₂O–CH

CH–CH

0.39

0.13

>10 (10.6)^c

CH₂Bu^t

Buⁱ

^aIC₅₀values were determined by a single experiment run in duplicate.

^bNot tested.

^cValueof % inhibition at 10 mM is given in parentheses.

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

391

J=7.0 Hz), 6.13 (1H, s), 6.58 (1H, d, J=8.6 Hz), 6.91

(1H, d, J=2.6 Hz), 5.99 (1H, s), 7.12–7.47 (5H, m).

Anal. calcd for C₁₇H₁₉Cl₂NO: C, 62.97; H, 5.91; N,

4.32. Found: C, 62.97; H, 6.05; N, 4.44.

3ꢂ1H, d, J=13.4 Hz), 4.10 (2/3ꢂ2H, q, J=7.0 Hz), 4.27

(1/3ꢂ2H, q, J=7.0 Hz), 4.44 (1/3ꢂ1H, d, J=13.4 Hz),

4.57 (2/3ꢂ1H, d, J=13.4 Hz), 6.10 (1/3ꢂ1H, s), 6.22 (2/

3ꢂ1H, d, J=15.0 Hz), 6.30 (2/3ꢂ1H, s), 6.40 (2/3ꢂ1H,

d, J=15.0 Hz), 6.75–7.71 (7H+1/3ꢂ2H, m). Anal.

calcd for C₂₄H₂₇Cl₂NO₄: C, 62.07; H, 5.86; N, 3.02.

Found: C, 62.18; H, 6.12; N,3.14.

Ethyl 3-[N-[4-chloro-2-(2-chloro-ꢀ-hydroxybenzyl)phe-

nyl]-N-neopentylcarbamoyl]acrylate (15a). A solution of

fumaric acid chloride monoethyl ester (1.3 g, 7.96 mmol)

in CH₂Cl₂(10 mL) was added dropwise to a solution of

14a (2.4 g, 7.09 mmol) and NaHCO₃(0.91 g, 10.9 mmol)

in CH₂Cl₂(50 mL). The reaction mixture was stirred for

2 h at room temperature and ﬁltered. The ﬁltrate was

washed with water, dried over Na₂SO₄and then con-

centrated under reduced pressure to give 15a (3.0 g, 6.46

mmol, 91%) as colorless prisms. Mp 153–154 ^ꢁC. IR

n_max(KBr) cm^ꢀ1: 3420 (OH); 1720, 1650, 1625 (C¼O,

Ethyl 3-[N-[4-chloro-2-(2-chloro-ꢀ-hydroxybenzyl)phe-

nyl]-N-isobutylcarbamoyl]acrylate (15b). 15b (4.2 g,

9.33 mmol, 84%) was obtained in a similar procedure

from 14b (3.6 g, 11.1 mmol) as colorless prisms. Mp

136–138 ^ꢁC. IR n_max(KBr) cm^ꢀ1: 3360 (OH); 1725,

1

1655, 1610 (C¼O, C¼C). H NMR (CDCl₃) d 0.75 (1/

3ꢂ3H, d, J=6.6 Hz), 0.89–0.98 (3H+2/3ꢂ3H, m), 1.23

(2/3ꢂ3H, t, J=7.0 Hz), 1.25 (1/3ꢂ3H, t, J=7.0 Hz),

1.7–1.9 (1H, m), 2.65 (1/3ꢂ1H, dd, J=4.8, 13.2 Hz),

3.03 (2/3ꢂ1H, dd, J=4.6, 13.2 Hz), 4.34–4.47 (4H, m),

6.11 (2/3ꢂ1H, d, J=15.0 Hz), 6.12 (1/3ꢂ1H, d,

J=5.0 Hz), 6.27 (2/3ꢂ1H, d, J=5.0 Hz), 6.40 (2/3ꢂ1H,

d, J=15.0 Hz), 6.69 (1/3ꢂ1H, d, J=15.0 Hz), 6.81 (1/

3ꢂ1H, d, J=15.0 Hz), 6.96–7.72 (7H, m). Anal. calcd

for C₂₃H₂₅Cl₂NO₄: C, 61.34; H, 5.59; N, 3.11. Found:

C,61.26; H, 5.71; N,3.06.

1

C¼C). H NMR (CDCl₃) d 0.86 (1/3ꢂ9H, s), 0.93 (2/

3ꢂ9H, s), 1.23 (2/3ꢂ3H, t, J=7.0 Hz), 1.25 (1/3ꢂ3H, t,

J=7.0 Hz), 2.87 (1/3ꢂ1H, d, J=13.4 Hz), 3.14 (2/

Ethyl (3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-neopen-

tyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate

(16a). A mixtureof 15a (3.0 g, 6.46 mmol) and K₂CO₃

(1.1 g, 7.73 mmol) in EtOH (60 mL) was stirred for 24

h. The reaction mixture was diluted with AcOEt,

washed with water, dried over Na₂SO₄and then con-

centrated. The residue was subjected to column chro-

matography [eluent: hexane–AcOEt (3:1, v/v)] and

recrystallized from AcOEt–hexane (1:3, v/v) to give 16a

(2.5 g, 5.438 mmol, 83%) as colorless prisms. Mp 101–

102 ^ꢁC. IR n_max(KBr) cm^ꢀ1: 1740, 1670 (C¼O). ¹H

NMR (CDCl₃) d 0.94 (9H, s), 1.25 (3H, t, J=7.2 Hz),

2.80 (1H, dd, J=16.6, 6.2 Hz), 3.04 (1H, dd, J=16.6,

7.2 Hz), 3.40 (1H, d, J=14.0 Hz), 4.14 (2H, dq, J=7.2,

1.8 Hz), 4.43 (1H, dd, J=7.2, 6.2 Hz), 4.52 (1H, d,

J=14.0 Hz), 6.26 (1H, s), 6.52 (1H, s), 7.37–7.74 (6H,

m). Anal. calcd for C₂₄H₂₇Cl₂NO₄: C, 62.07; H, 5.86; N,

3.02. Found: C,62.36; H, 5.87; N,2.99.

Figure 2. Overlay of the X-ray structures of 1a (red), 2 (blue), 7

(green) and 10 (black).

Ethyl (3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-isobutyl-2-

oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetate (16b).

16b (4.0 g, 8.88 mmol, 95%) was obtained in a similar

procedure from 15b (4.2 g, 9.33 mmol) as a colorless oil.

IR n_max(KBr) cm^ꢀ1: 1740, 1670 (C¼O). ¹H NMR

(CDCl₃) d 0.93 (3H, d, J=6.6 Hz), 1.03 (3H, d,

J=6.6 Hz), 1.25 (3H, t, J=7.2 Hz), 1.90–2.10 (1H, m),

2.80 (1H, dd, J=16.6, 6.2 Hz), 3.06 (1H, dd, J=16.6,

7.2 Hz), 3.44 (1H, dd, J=5.6, 14.0 Hz), 4.14 (2H, q,

J=7.2 Hz), 4.32 (1H, dd, J=14.0, 8.4 Hz), 4.44 (1H, dd,

J=7.2, 6.2 Hz), 6.14 (1H, s), 6.51 (1H, d, J=2.2 Hz),

7.26–7.72 (6H, m).

Figure 3. Overlay of the X-ray structures of 1a (red) and 5 (blue).

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-

oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid

(1a). A mixtureof 16a (2.5 g, 5.38 mmol), K₂CO₃

(0.91 g, 6.56 mmol), MeOH (30 mL) and water (10 mL)

was stirred overnight at room temperature. The reaction

mixture was diluted with water, acidiﬁed, extracted with

Figure 4. Overlay of the X-ray structures of 1a (red) and 4 (purple).

392

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

AcOEt. The extract was washed with water, dried over

Na₂SO₄and then concentrated under reduced pressure

to give 1a (1.8 g, 4.13 mmol, 77%) as colorless prisms.

Mp 247–248 ^ꢁC. IR n_max(KBr) cm^ꢀ1: 3600–2200 (br,

Hz), 7.27-7.92 (6H, m). Anal. calcd for C₂₃H₂₅Cl₂NO₃S:

C, 59.23; H, 5.40; N, 3.00; S, 6.87. Found: C,59.36; H,

5.30; N,2.84; S, 6.86.

1

COOH), 1710, 1685 (C¼O). H NMR (CDCl₃) d 0.94

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-

oxo-1,2,3,5-tetrahydro-4,1-benzothiazepine-3-acetic acid

(2). An aqueous solution of NaOH (1 N, 0.4 mL) was

added to a solution of 20 (0.15 g, 0.322 mmol) in MeOH

(4 mL). After stirring for 3 h at 60 ^ꢁC, thereaction mix-

ture was concentrated. The residue was diluted with

H₂O (20 mL), acidiﬁed, and extracted with CH₂Cl₂

(20 mL, twice). The extracts were washed with saturated

NH₄Cl, dried over Na₂SO₄and then concentrated in

vacuo. The residue was recrystallized from CH₂Cl₂–

petroleum ether (1:2, v/v) to give 2 (0.11 g, 0.243 mmol,

75%) as colorless needles. Mp 269–271 ^ꢁC (CH₂Cl₂–

petroleum ether). IR n_max(KBr) cm^ꢀ1: 3600–2400 (br,

COOH), 1750 (C¼O), 1645 (C¼O). ¹H NMR (CDCl₃) d

0.98 (9H, s), 2.51 (1H, dd, J=3.8, 16.8 Hz), 3.12 (1H,

dd, J=10.2, 16.8 Hz), 3.30 (1H, d, J=13.8 Hz), 3.73

(1H, dd, J=3.8, 10.2 Hz), 4.42 (1H, d, J=13.8 Hz), 6.33

(1H, s), 6.75 (1H, s), 7.33–7.48, 7.86–7.91 (total 6H, m).

Anal. calcd for C₂₂H₂₃Cl₂NO₃S: C, 58.41; H, 5.12; N,

3.10; S, 7.09. Found: C, 58.39; H, 5.19; N, 2.84; S, 6.78.

(9H, s), 2.86 (1H, dd, J=16.8, 5.8 Hz), 3.09 (1H, dd,

J=16.8, 7.4 Hz), 3.41 (1H, d, J=14.0 Hz), 4.39 (1H, dd,

J=7.4, 5.8 Hz), 4.52 (1H, d, J=14.0 Hz), 6.26 (1H, s),

6.54 (1H, d, J=1.4 Hz), 7.36–7.74 (6H, m). Anal. calcd

for C₂₂H₂₃Cl₂NO₄: C, 60.56; H, 5.31; N, 3.21. Found:

C, 60.55; H, 5.47; N,3.11.

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-

1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid (1b).

1b (3.2 g, 7.58 mmol, 85%) was obtained in a similar

procedure from 16b (4.0 g, 8.88 mmol) as colorless

prisms. Mp 220–221 ^ꢁC. IR n_max(KBr) cm^ꢀ1: 3600–

2200 (br, COOH), 1720, 1680 (C¼O). ¹H NMR

(CDCl₃) d 0.93 (3H, d, J=6.6 Hz), 1.03 (3H, d,

J=6.6 Hz), 1.91–2.05 (1H, m), 2.86 (1H, dd, J=16.6,

5.8 Hz), 3.10 (1H, dd, J=16.8, 7.4 Hz), 3.46 (1H, dd,

J=14.0, 5.4 Hz), 4.32 (1H, dd, J=14.0, 8.4 Hz), 4.38

(1H, dd, J=7.4, 5.8 Hz), 6.13 (1H, s), 6.53 (1H, d,

J=2.4 Hz), 7.26–7.74 (6H, m). Anal. calcd for

C₂₁H₂₁Cl₂NO₄: C, 59.73; H, 5.01; N, 3.32. Found:

C,59.98; H, 5.24; N,3.14.

Methyl (3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-neopen-

tyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzothiazepine-3-ace-

tate S-oxide (21). MCPBA (0.37 g, 2.14 mmol) was

added to an ice-cooled solution of 20 (1 g, 2.14 mmol) in

CH₂Cl₂(10 mL). Themixturewas stirred for 10 min at

room temperature. The reaction mixture was diluted

with CH₂Cl₂(50 mL), washed with saturated NaHSO₃,

saturated NaHCO₃and brine, dried over Na₂SO₄and

then concentrated under reduced pressure. The residue

was recrystallized from CH₂Cl₂–hexane (1:3, v/v) to give

21 (0.59 g, 1.22 mmol, 57%) as a colorless powder. Mp

166–169 ^ꢁC (CH₂Cl₂–hexane). IR n_max(KBr) cm^ꢀ1: 1730,

Methyl (3,5-trans)-7-chloro-5-(2-chlorophenyl)-1-neopen-

tyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzothiazepine-3-ace-

tate (20).

A

mixtureof

14a (6.5 g, 19.2 mmol),

thiomalic acid (2.85 g, 19.0 mmol), concentrated HCl

(10 mL) and AcOH (10 mL) was stirred for 30 min at

100 ^ꢁC. The reaction mixture was cooled, and a 10%

NaOH (200 mL) was added. The mixture (pH=3) was

extracted with CH₂Cl₂–THF (9:1, v/v) (100 mL, twice).

The extracts were washed with saturated NH₄Cl

(150 mL), dried over Na₂SO₄, and then concentrated

under reduced pressure. The residue was dissolved in

xylene (200 mL) and the solution was reﬂuxed over-

night. The reaction mixture was concentrated in vacuo.

The residue was dissolved in MeOH (100 mL), and

concentrated H₂SO₄(0.5 mL) was added. This mixture

was reﬂuxed for 3 h and concentrated in vacuo. The

residue was dissolved in CH₂Cl₂(100 mL), washed with

saturated NaHCO₃(100 mL) and brine, dried over

Na₂SO₄, and then concentrated under reduced pressure.

The residue was subjected to column chromatography

[eluent: hexane–AcOEt (1:1, v/v)] to give 19 as 3,5-cis

and -trans mixtures. K₂CO₃(1.4 g, 10.1 mmol) was

added to a solution of 19 in MeOH (50 mL). After stir-

ring overnight at room temperature, the reaction mix-

turewas dilutde with AcOEt (150 mL). Thesolution

was washed with brine, dried over Na₂SO₄, and then

concentrated under reduced pressure. The residue was

chromatographed [eluent: hexane–AcOEt (3:1, v/v)] and

recrystallized from CH₂Cl₂–petroleum ether (1:10, v/v)

to give 20 (4.4 g, 9.43 mmol, 49%) as colorless prisms.

1

1670 (C¼O), 1055 (S⁺ꢀO^ꢀ). H NMR (CDCl₃) d 1.01

(9H, s), 2.83 (1H, dd, J=5.2, 17.6 Hz), 3.38 (1H, dd,

J=9.6, 17.6 Hz), 3.44 (1H, d, J=14.2 Hz), 3.68 (3H, s),

3.81 (1H, dd, J=5.2, 9.6 Hz), 4.50 (1H, d, J=14.2 Hz),

5.91 (1H, s), 6.93–6.95, 7.26–7.55, 7.85–7.89 (7H, m).

.

Anal. calcd for C₂₃H₂₅Cl₂NO₄S 1.7H₂O: C, 53.85; H,

5.58; N, 2.73. Found: C, 53.70; H, 5.27; N, 2.36.

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-

oxo-1,2,3,5-tetrahydro-4,1-benzothiazepine-3-acetic acid

S-oxide (3). An aqueous solution (5 mL) of K₂CO₃

(0.17 g, 1.23 mmol) was added to a solution of 21 (0.5 g,

1.04 mmol) in MeOH (10 mL). The mixture was stirred

for 2 h at 60 ^ꢁC. The reaction mixture was diluted with

water (50 mL), acidiﬁed, and then extracted with

CH₂Cl₂(50 mL, twice). The extracts were washed with

brine, dried over Na₂SO₄, and then concentrated under

reduced pressure. The residue was recrystallized from

CH₂Cl₂–hexane (1:1, v/v) to give 3 (0.38 g, 0.811 mmol,

78%) as a colerless powder. Mp 230–235 ^ꢁC (dec)

(CH₂Cl₂–hexane). IR n_max(KBr) cm^ꢀ1: 3600–2400 (br,

Mp 133–136 ^ꢁC (CH₂Cl₂-petroleum ether). IR

1

n

_max(KBr) cm^ꢀ1: 1730 (C¼O), 1680 (C¼O). H NMR

COOH), 1740, 1660 (C¼O), 1050 (S⁺–O^ꢀ). H NMR

(CDCl₃) d 0.98 (9H, s), 2.42 (1H, dd, J=3.8, 17.0 Hz),

3.13 (1H, dd, J=10.4, 17.0 Hz), 3.30 (1H, d, J=14.0

Hz), 3.66 (3H, s), 3.78 (1H, dd, J=3.8, 10.4 Hz), 4.42

(1H, d, J=14.0 Hz), 6.34 (1H, s), 6.75 (1H, d, J=1.6

(CDCl₃) d 1.00 (9H, s), 2.86 (1H, dd, J=4.8, 17.2 Hz),

3.41 (1H, dd, J=9.6, 17.2 Hz), 3.45 (1H, d, J=13.6

Hz), 3.78 (1H, dd, J=4.8, 9.6 Hz), 4.51 (1H, d, J=13.6

Hz), 5.93 (1H, s), 6.96 (1H, brs), 7.27–7.56, 7.87–7.91

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

393

(6H, m). Anal. calcd for C₂₂H₂₃Cl₂NO₄S: C, 56.41; H,

4.95; N, 2.99. Found: C, 56.36; H, 5.04; N, 3.04.

and brine, dried over Na₂SO₄, and then concentrated

under reduced pressure. The residue was crystallized

with Et₂O to give 24 (2.7 g, 7.16 mmol, quant) as a col-

orless amorphous powder. IR n_max(KBr) cm^ꢀ1: 3280

(NH), 1720, 1690 (C¼O), 1610 (C¼N). ¹H NMR

(CDCl₃) d 3.22 (1H, dd, J=7.0, 16.8 Hz), 3.44 (1H dd,

J=7.4, 16.8 Hz), 3.74 (3H, s), 4.23 (1H, t, J=7.1 Hz),

7.08 (1H, d, J=2.2 Hz), 7.38–7.48 (6H, m), 8.72 (1H, br).

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-

oxo-1,2,3,5-tetrahydro-4,1-benzothiazepine-3-acetic acid

S-dioxide (4). MCPBA (0.25 g, 1.46 mmol) was added

to an ice-cooled solution of 2 (0.3 g, 0.663 mmol) in

CH₂Cl₂(10 mL). Themixturewas stirred for 2h at room

temperature. The reaction mixture was diluted with

CH₂Cl₂(50 mL), washed with saturated NaHCO₃

(50 mL) and brine, dried over Na₂SO₄, and then con-

centrated under reduced pressure. The residue was

recrystallized from CH₂Cl₂–hexane (1:2, v/v) to give 4

(0.14 g, 0.289 mmol, 44%) as a colerless powder. Mp

245–249 ^ꢁC (dec) (CH₂Cl₂–hexane). IR n_max(KBr)

cm^ꢀ1: 3600–2400 (br, COOH), 1710, 1680 (C¼O), 1315,

.

Anal. calcd for C₁₈H₁₄Cl₂N₂O₃0.75H₂O: C, 55.33; H,

4.00; N, 7,17. N, 7.17. Found: C, 54.92; H, 3.60; N, 7.21.

Methyl 7-chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-2,3-

dihydro-1H-1,4-benzodiazepine-3-acetate (25). NaH

(36 mg, 1.50 mmol) was added to a solution of 24

(0.52 g, 1.38 mmol) in DMF (5 mL) at 0 ^ꢁC. Themixture

was stirred for 5 min at 0 ^ꢁC, followed by addition of

isobutyl bromide(0.23 g, 1.7 mmol). Thereaction mix-

ture was stirred for 1 h at room temperature, and dilu-

ted with AcOEt (50 mL). The solution was washed with

5% KHSO₄, saturated NaHCO₃and brine, dried over

Na₂SO₄, and then concentrated under reduced pressure.

The residue was subjected to column chromatography

[eluent: hexane–AcOEt (4:1, v/v)] to give 25 (0.3 g,

0.692 mmol, 50%) as a paleyellow oil. IR n_max(neat)

cm^ꢀ1: 1740, 1680 (C¼O), 1600 (C¼N). ¹H NMR

(CDCl₃) d 0.80 (3H, d, J=6.4 Hz), 0.88 (3H, d, J=6.6

Hz), 1.76 (1H, m), 3.22 (1H, dd, J=7.0, 16.8 Hz), 3.44

(1H, dd, J=7.4, 16.8 Hz), 3.53 (1H, qd, J=4.8, 14.2

Hz), 3.72 (3H, s), 4.17 (1H, t, J=7.1 Hz), 4.33 (1H, dd,

J=10.0, 14.2 Hz), 7.08 (1H, d, J=2.4 Hz), 7.37–7.53

(6H, m).

1

1135 (SO₂). H NMR (CDCl₃+1drop of DMSO-d₆) d

0.93 (9H, s), 2.88 (1H, dd, J=3.0, 17.2 Hz), 3.44 (1H, d,

J=14.0 Hz), 3.46 (1H, dd, J=10.6, 17.2 Hz), 4.40 (1H,

dd, J=3.0, 10.6 Hz), 4.51 (1H, d, J=14.0 Hz), 6.37 (1H,

s), 7.40–7.59, 8.29–8.34 (7H, m). Anal. calcd for

.

C₂₂H₂₃NO₅SCl₂0.2H₂O: C, 54.15; H, 4.83; N, 2.87.

Found: C, 54.08; H, 4.83; N, 2.65.

Methyl 3-benzyloxycarbonylamino-3-[N-[4-chloro-2-(2-

chlorobenzoyl)phenyl]carbamoyl]propionate (23). N-

Methylmorpholine (1.6 g, 16 mmol) and isobutyl-

chloroformate (2.2 g, 16 mmol) was added to a stirred

solution of b-methyl N-benzyloxycarbonyl-dl-aspartate

(4.3 g, 15.3 mmol) in CH₂Cl₂(50 mL) at 0 ^ꢁC. Themix-

ture was stirred for 10 min at room temperature and

then warmed to reﬂux. A solution of 12 (4.1 g,

15.4 mmol) in CH₂Cl₂(20 mL) was added to the reﬂux-

ing reaction mixture. Heating was continued for 20 min.

The mixture was then cooled to room temperature,

stirred for 2 days, and diluted with CH₂Cl₂(100 mL).

The mixture was washed with 10% citric acid, saturated

NaHCO₃and brine, dried over Na₂SO₄, and then con-

centrated under reduced pressure. The residue was

chromatographed [eluent: hexane–AcOEt (3:1)] to give

23 (3.7 g, 6.99 mmol, 45%) as a colorless amorphous

powder. IR n_max(KBr) cm^ꢀ1: 3400 (NH), 1735, 1710,

7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-2,3-dihy-

dro-1H-1,4-benzodiazepine-3-acetic acid (5). An aqu-

eous solution (2 mL) of K₂CO₃(0.11 g, 0.82 mmol) was

added to a solution of 25 (0.23 g, 0.531 mmol) in MeOH

(4 mL). Themixturewas stirred for 1 h at 60 ^ꢁC, diluted

with water (50 mL), acidiﬁed with 1 N HCl, and extrac-

ted with CH₂Cl₂(50 mL, twice). The extracts were

washed with brine, dried over Na₂SO₄, and then con-

centrated under reduced pressure. The residue was

recrystallized from CH₂Cl₂–petroleum ether (1:3, v/v) to

give 5 (0.11 g, 0.262 mmol, 49%) as a colorless powder.

Mp 175–178 ^ꢁC (CH₂Cl₂–petroleum ether). IR n_max

(KBr) cm^ꢀ1: 3600–2400 (br, COOH), 1710 (C¼O), 1670

1

1690, 1650 (C¼O). H NMR (CDCl₃) d 2.82 (1H, dd,

J=4.8, 17.6 Hz), 3.31 (1H, dd, J=4.4, 17.6 Hz), 3.69

(3H, s), 4.84 (1H, m), 5.15 (1H, d, J=12.2 Hz), 5.27

(1H, d, J=12.2 Hz), 7.24–7.56 (11H, m), 8.76 (1H, d,

J=8.8 Hz). Anal. calcd for C₂₆H₂₂Cl₂N₂O₆: C, 58.99;

H, 4.19; N, 5.29. Found: C, 58.81; H, 4.19; N, 5.16.

1

(C¼O), 1600 (C¼N). H NMR (CDCl₃) d 0.79 (3H, d,

J=6.6 Hz), 0.89 (3H, d, J=6.8 Hz), 1.75 (1H, m), 3.23

(1H, dd, J=6.4, 16.8 Hz), 3.36 (1H, dd, J=6.4, 16.8

Hz), 3.55 (1H, qd, J=4.6, 14.0 Hz), 4.11 (1H, t,

J=6.4 Hz), 4.34 (1H, dd, J=9.8, 14.0 Hz), 7.10 (1H, d,

J=2.4 Hz), 7.37–7.55 (6H, m). Anal. calcd for

Methyl 7-chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-

1H-1,4-benzodiazepine-3-acetate (24). A 10% Pd–C cat-

alyst (0.5 g) and concentrated HCl (0.59 mL) was added

to a stirred solution of 23 (3.7 g, 6.99 mmol) in MeOH

(60 mL). The apparatus was ﬁlled with hydrogen and

the mixture was stirred for 30 min at room temperature.

Thecatalyst was removed by ﬁltration and theﬁltrate

was concentrated in vacuo. The residue was dissolved in

CH₂Cl₂–THF (9:1, v/v) (100 mL) and washed with

saturated NaHCO₃and brine, dried over Na₂SO₄, and

then concentrated in vacuo. The residue was dissolved

in DMF (20 mL). After addition of AcOH (1 mL), the

mixturewas stirred for 2 h at 60 ^ꢁC, diluted with AcOEt

(50 mL), washed with 5% KHSO₄, saturated NaHCO₃

.

C₂₁H₂₀Cl₂N₂O₃0.2H₂O: C, 59.65; H, 4.86; N, 6.62.

Found: C, 59.65; H, 4.96; N, 6.62.

(3,5-trans)-7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-

2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-3-acetic acid

(6). NaBH₄(10 mg) was added to a solution of 5

(30 mg, 0.0715 mmol) in MeOH–H₂O (6:1, v/v)

(0.7 mL). The mixture was stirred for 2 h at room tem-

perature, diluted with water, neutralized with 1 N HCl,

and extracted with CH₂Cl₂(50 mL, twice). The extracts

were washed with brine, dried over Na₂SO₄and then

concentrated under reduced pressure. The residue was

394

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

recrystallized from CH₂Cl₂–petroleum ether (1:10, v/v)

to give 6 (17 mg, 0.0403 mmol, 56%) as a colorless

powder. Mp 184–188 ^ꢁC (dec) (CH₂Cl₂–petroleum

ether). IR n_max(KBr) cm^ꢀ1: 3600–2400 (br, COOH),

added to a solution of 29 (1.7 g, 5.48 mmol) in MeOH

(15 mL). After stirring for 30 min at room temperature,

NaBH₃CN (0.35 g, 5.5 mmol) was added to the mixture.

After stirring for 3 h at 50 ^ꢁC, 1 N NaOH (50 mL) was

added and the solution was extracted with CH₂Cl₂

(50 mL, twice). The extracts were washed with brine,

dried over Na₂SO₄and then concentrated under

reduced pressure. The residue was subjected to column

chromatography [eluent: hexane–AcOEt (3:1, v/v)] to

give 30 (1.1 g, 2.87 mmol, 52%) as a paleyellow oil. IR

n_max(neat) cm^ꢀ1: 3600–3200 (br, NH), 1740 (C¼O),

1520 (NO₂), 1345 (NO₂). ¹H NMR (CDCl₃) d 3.20 (1H,

dd, J=6.8, 12.2 Hz), 3.37 (1H, dd, J=7.6, 12.2 Hz), 3.48

(2H, s), 3.73 (3H, s), 5.26 (1H, t, J=7.2 Hz), 7.22–7.44

(6H, m), 7.85 (1H, d, J=8.6 Hz).

1

1705, 1665 (C¼O). H NMR (CDCl₃) d 0.918 (3H, d,

J=6.6 Hz), 1.035 (3H, d, J=6.6 Hz), 1.940 (1H, m),

2.668 (1H, dd, J=5.4, 16.4 Hz), 2.877 (1H, dd, J=7.8,

16.4 Hz), 3.419 (1H, dd, J=5.2, 13.6 Hz), 3.685 (1H, dd,

J=5.4, 7.8 Hz), 4.245 (1H, dd, J=8.8, 13.6 Hz), 5.634

(1H, s), 5.56–5.92 (1H, br), 6.479 (1H, d, J=2.2 Hz),

7.22–7.44, 7.93–7.96 (6H, m). Anal. calcd for

.

C₂₁H₂₂Cl₂N₂O₃H₂O: C, 57.41; H, 5.50; N, 6.38. Found:

C, 57.56; H, 5.16; N, 6.40.

Methyl 5-chloro-ꢀ-(2-chlorophenyl)-2-nitrophenylacetate

(27). A mixture of NaH (oil-free, 3.4 g, 0.141 mol), 26

(28 g, 0.152 mol) and 4-chloro-1,2-dinitrobenzene (27 g,

0.133 mol) in DMF (100 mL) was stirred for 1 h at 0 ^ꢁC.

The reaction mixture was poured into 1 N HCl

(300 mL) and the resulting aqueous solution was

extracted with AcOEt (200 mL, twice). The extracts

were washed with brine, dried over Na₂SO₄and evapo-

rated to give an oil, which was chromatographed [elu-

ent: hexane–AcOEt (10:1, v/v)] to give 27 (26.3 g,

77.3 mmol, 58%) as a yellow powder. Mp 96–97 ^ꢁC

(hexane). IR n_max(KBr) cm^ꢀ1: 1740 (C¼O), 1515, 1340

Methyl N-[2-(2-chlorophenyl)-2-(5-chloro-2-nitrophenyl)-

ethyl]-N-triﬂuoroacetylglycinate (31). Triﬂuoroacetic

anhydride(3.0 g, 14.2 mmol) was added to a solution of

30 (4.9 g, 12.8 mmol) and pyridine(3.0 g, 38.4 mmol) in

CH₂Cl₂(50 mL). This mixturewas stirred for 10 min at

room temperature, duluted with CH₂Cl₂(50 mL). The

solution was washed with 1 N HCl, saturated NaHCO₃

and brine, dried over Na₂SO₄and then concentrated

under reduced pressure. The residue was subjected to

column chromatography [eluent: hexane–AcOEt (5:1,

v/v)] to give 31 (5.9 g, 12.3 mmol, 96%) as a yellow oil.

IR n_max(neat) cm^ꢀ1: 1750, 1700 (C¼O), 1525, 1350

1

(NO₂). H NMR (CDCl₃) d 3.80 (3H, s), 6.07 (1H, s),

6.90 (1H, d, J=2.2 Hz), 7.22–7.49 (5H, m), 8.06 (1H, d,

J=8.4 Hz). Anal. calcd for C₁₅H₁₁Cl₂NO₄: C, 52.96; H,

3.26; N, 4.12. Found: C, 53.04; H, 3.34; N, 4.06.

1

(NO₂). H NMR (CDCl₃) d: 3.79 (3H, s), 4.14 (1H, dd,

J=7.0, 14.0 Hz), 4.22 (2H, s), 4.43 (1H, dd, J=8.8,

14.0 Hz), 5.47 (1H, dd, J=7.0, 8.8 Hz), 7.27–7.52 (6H,

m), 7.87 (1H, d, J=8.4 Hz).

2-(2-Chlorophenyl)-2-(5-chloro-2-nitrophenyl)ethanol (28).

A mixtureof 27 (26 g, 76.4 mmol) and LiBH₄(2 g) in

THF (200 mL) was stirred for 4 h at room temperature.

Themixturewas poured into 20% AcOH (50 mL) and

the resulting aqueous solution was extracted with

AcOEt (200 mL, twice). The extracts were washed with

brine, dried over Na₂SO₄and evaporated to give an oil,

which was chromatographed [eluent: hexane–AcOEt

(3:1, v/v)] to give 28 (11 g, 35.2 mmol, 46%) as a brown

Methyl N-[2-(2-amino-5-chlorophenyl)-2-(2-chlorophenyl)-

ethyl]-N-triﬂuoroacetylglycinate (32). A 10% Pd–C cat-

alyst (100 mg) was added to a solution of 31 (1 g, 2.09

mmol) in AcOEt (20 mL). Theapparatus was ﬁlled with

hydrogen and the mixture was stirred at room tem-

perature for 8 h. The catalyst was removed by ﬁltration

and the ﬁltrate was concentrated in vacuo. The residue

was subjected to column chromatography [eluent: hex-

ane–AcOEt (4:1, v/v)] to give 32 (0.39 g, 0.868 mmol,

42%) as a yellow oil. IR n_max(neat) cm^ꢀ1: 3460 (NH₂),

1

oil. H NMR (CDCl₃) d 1.90 (1H, br), 4.15–4.33 (2H,

m), 5.27 (1H, t, J=6.2 Hz), 7.20–7.40 (6H, m), 7.87 (1H,

d, J=8.4 Hz).

1

3380 (NH₂), 1750, 1695 (C¼O). H NMR (CDCl₃) d

5-Chloro-ꢀ-(2-chlorophenyl)-2-nitrophenylacetaldehyde

(29). A solution of DMSO (6.7 mL, 94.2 mmol) in

CH₂Cl₂(30 mL) was added to a solution of oxalyl chloride

(6.2 mL, 70.8 mmol) in CH₂Cl₂(300 mL) at ꢀ78 ^ꢁC, and

the resulting mixture was stirred for 10min at ꢀ78^ꢁC.

After addition of a solution of 28 (11 g, 35.2 mmol) in

CH₂Cl₂(100 mL), thewholemixturewas stirred for 15 min

at ꢀ78 ^ꢁC. After addition of NEt₃(37 mL, 0.26 mol), the

mixturewas allowde to warm to 0 ^ꢁC, washed with

saturated NH₄Cl (124 mL) and brine, and dried over

Na₂SO₄, and then concentrated. The residue was chro-

matographed [eluent: hexane–AcOEt (3:1, v/v)] to give

29 (9.1 g, 29.3 mmol, 83%) as a brown oil. ¹H NMR

(CDCl₃) d 6.30 (1H, s), 6.84 (1H, d, J=2.2 Hz), 7.14–

7.65 (5H, m), 8.10 (1H, d, J=8.8 Hz), 9.89 (1H, s).

3.71 (1/4ꢂ3H, s), 3.74 (3/4ꢂ3H, s), 3.47–4.25 (4H, m),

4.72–4.80 (1/4ꢂ1H, m), 4.87 (3/4ꢂ1H, dd, J=5.8,

9.4 Hz), 6.57–6.63 (1H, m), 7.05–7.44 (6H, m).

Methyl N-[2-(5-chloro-2-neopentylaminophenyl)-2-(2-chlo-

rophenyl)ethyl]-N-triﬂuoroacetylglycinate (33). A solu-

tion of 32 (0.39 g, 0.868 mmol), AcOH (0.05 mL), and

pivalaldehyde (78 mg, 0.90 mmol) in MeOH (5 mL) was

stirred for 30 min at room temperature, followed by

addition of NaBH₃CN (57 mg, 0.90 mmol) at 0 ^ꢁC. The

mixture was stirred for 1 h at room temperature, diluted

with CH₂Cl₂(50 mL), washed with 1 N NaOH and

brine, dried over Na₂SO₄and then concentrated under

reduced pressure. The residue was subjected to column

chromatography [eluent: hexane–AcOEt (5:1, v/v)] to

give 33 (0.27 g, 0.520 mmol, 60%) as a paleyellow oil. IR

1

Methyl N-[2-(2-chlorophenyl)-2-(5-chloro-2-nitrophenyl)-

ethyl]glycinate (30). Methyl glycinate hydrochloride

(0.69 g, 5.5 mmol) and AcONa (0.45 g, 5.5 mmol) was

n_max(neat) cm^ꢀ1: 3420 (br, NH), 1755, 1700 (C¼O). H

NMR (CDCl₃) d 0.85 (9H, s), 2.65–2.83 (2H, m), 3.32 (1/

4ꢂ1H, d, J=17.4 Hz), 3.51–3.60 (1H, m), 3.70 (1/4ꢂ3H,

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

395

s), 3.75 (3/4ꢂ3H, s), 3.87 (1H, dd, J=9.8, 13.4 Hz), 4.06

(3/4ꢂ1H, d, J=17.8 Hz), 4.27 (1H, dd, J=5.8, 13.4 Hz),

4.72–4.86 (1H, m), 6.53–6.60 (1H, m), 7.11–7.40 (6H, m).

tion was washed with water and brine, dried over

Na₂SO₄, and then concentrated under reduced pressure

to give 36 (0.54 g, 1.53 mmol, quant) as a red oil. IR

n_max(neat) cm^ꢀ1: 1235 (C¼S).

Methyl N-[2-(5-chloro-2-neopentylaminophenyl)-2-(2-chl-

orophenyl)ethyl]glycinate (34). Concentrated HCl

(0.6 mL) was added to a solution of 33 (0.2 g,

0.385 mmol) in MeOH (3 mL). The mixture was reﬂuxed

overnight, followed by addition of 1 N NaOH (8 mL).

The solution was extracted with CH₂Cl₂(50 mL, twice).

The extracts were washed with brine, dried over Na₂SO₄

and then concentrated in vacuo to give 34 (72 mg,

Ethyl [2-[5-chloro-2-(neopentylamino)phenyl](2-chloro-

phenyl)methylene]hydrazino]acetate

(37a,b).

Ethyl

hydrazinoacetate hydrochloride (0.23 g, 1.49 mmol) and

K₂CO₃(0.11 g, 0.765 mmol) was added to a solution of

36 (0.54 g, 1.53 mmol) in EtOH (7 mL). Themixturewas

stirred overnight at 70 ^ꢁC. Theraection mixturewas

diluted with CH₂Cl₂(50 mL), washed with water and

brine, dried over Na₂SO₄, and then concentrated in

vacuo. The residue was chromatographed [eluent: hex-

ane–AcOEt (15:1, v/v)] to give 37a (less polar isomer)

(0.16 g, 0.367 mmol, 24%) as a colorless oil from the

ﬁrst fraction and 37b (polar isomer) (0.26 g, 0.596 mmol,

39%) as colorless prisms from the second fraction.

1

0.170 mmol, 44%) as a brown oil. IR n_max(neat) cmꢀ :

3420 (NH), 1740 (C¼O). ¹H NMR (CDCl₃) d 0.82 (9H,

s), 2.66 (1H, d, J=11.2 Hz), 2.77 (1H, d, J=11.2 Hz),

3.03 (1H, dd, J=5.2, 11.8 Hz), 3.28 (1H, dd, J=8.4,

11.8 Hz), 3.43 (1H, d, J=17.6 Hz), 3.53 (1H, d,

J=17.6 Hz), 3.74 (3H, s), 4.56 (1H, dd, J=5.2, 8.4 Hz),

6.54 (1H, d, J=8.8 Hz), 6.99–7.42 (6H, m).

Methyl 7-chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo-

1,2,4,5-tetrahydro-3H-1,3-benzodiazepine-3-acetate (35).

Triphosgene (0.14 g, 0.472 mmol) was added to a solu-

tion of 34 (0.47 g, 1.11 mmol) and NEt₃(0.21 g,

2.08 mmol) in toluene (5 mL). The mixture was stirred

for 5 h at 70 ^ꢁC, diluted with CH₂Cl₂(50 mL), washed

with 1 N HCl and brine, dried over Na₂SO₄, and then

concentrated under reduced pressure. The residue was

crystallized with hexane to give 35 (0.30 g, 0.668 mmol,

60%) as a colorless powder. Mp 142–148 ^ꢁC (hexane).

IR n_max(KBr) cm^ꢀ1: 1750 (C¼O), 1640 (C¼O). ¹H

NMR (CDCl₃) d 0.94 (9H, s), 3.49 (1H, d, J=14.4 Hz),

3.64 (1H, d, J=17.2 Hz), 3.72 (3H, s), 3.88 (2H, d,

J=8.6 Hz), 4.04 (1H, d, J=17.2 Hz), 4.31 (1H, d,

J=14.4 Hz), 5.32 (1H, t, J=8.6 Hz), 6.65 (1H, d,

J=1.8 Hz), 7.14–7.50 (6H, m).

37a. IR n_max(neat) cm^ꢀ1: 3400 (NH), 1740 (C¼O). ¹H

NMR (CDCl₃) d: 0.95 (9H, s), 1.27 (3H, t, J=7.2 Hz),

2.96 (2H, d, J=5.8 Hz), 4.04–4.27 (4H, m), 5.97 (1H, t,

J=5.8 Hz), 6.68 (1H, d, J=9.0 Hz), 6.88 (1H, d,

J=2.6 Hz), 7.15–7.50 (5H, m).

37b. Mp 116–118 ^ꢁC (CH₂Cl₂–petroleum ether). IR

1

n_max(KBr) cm^ꢀ1: 3280 (NH), 1735 (C¼O). H NMR

(CDCl₃) d 1.07 (9H, s), 1.25 (3H, t, J=6.8 Hz), 3.01

(2H, d, J=4.8 Hz), 3.89 (1H, dd, J=3.8, 17.6 Hz), 4.09

(1H, dd, J=7.8, 17.6 Hz), 4.16 (2H, q, J=6.8 Hz), 5.25

(1H. dd, J=4.4, 7.4 Hz), 6.50 (1H, d, J=2.6 Hz), 6.62

(1H, d, J=8.8 Hz), 7.06 (1H, dd, J=2.6, 8.8 Hz), 7.30–

7.62 (4H, m), 8.38 (1H, br).

Ethyl 7-chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo-1,2-

dihydro-3H-1,3,4-benzotriazepine-3-acetate (38). Tri-

phosgene (54 mg, 0.184 mmol) was added to a solution

of 37a (0.16 g, 0.367 mmol) and NEt₃(90 mg,

0.891 mmol) in toluene (2 mL). After being stirred for

1 h at 70 ^ꢁC, the reaction mixture was diluted with

AcOEt (50 mL), washed with water and brine, dried

over Na₂SO₄and then concentrated under reduced

pressure. The residue was subjected to column chroma-

tography [eluent: hexane–AcOEt (3:1, v/v)] to give 38

(0.16 g, 0.346 mmol, 94%) as a paleyellow oil. IR n_max

7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo-1,2,4,5-

tetrahydro-3H-1,3-benzodiazepine-3-acetic acid (7). A

mixtureof 35 (0.3 g, 0.668 mmol) and 1 N NaOH

(0.7 mL) in MeOH (3 mL) was stirred for 30 min at

70 ^ꢁC. The reaction mixture was diluted with water

(50 mL), acidiﬁed with 1 N HCl, extracted with CH₂Cl₂

(50 mL, twice). The extracts were washed with brine,

dried over Na₂SO₄, and then concentrated under

reduced pressure. The residue was recrystallized from

CH₂Cl₂–hexane (1:10, v/v) to give 7 (0.21 g, 0.482 mmol,

72%) as colorless prisms. Mp 228–231 ^ꢁC (CH₂Cl₂–

hexane). IR n_max(KBr) cm^ꢀ1: 3400–2400 (br, COOH),

1

(neat) cm^ꢀ1: 1750, 1680 (C¼O). H NMR (CDCl₃) d

0.88 (9H, s), 1.24 (3H, t, J=7.0 Hz), 3.38 (1H, d,

J=14.0 Hz), 4.18 (2H, q, J=7.0 Hz), 4.32 (1H, d,

J=16.8 Hz), 4.36 (1H, d, J=14.0 Hz), 4.48 (1H, d,

J=16.8 Hz), 6.86 (1H, d, J=2.6 Hz), 7.20–7.50 (6H, m).

1

1755 (C¼O), 1600 (C¼O). H NMR (CDCl₃) d 0.94

(9H, s), 3.41 (1H, d, J=14.2 Hz), 3.44 (1H, d,

J=16.0 Hz), 3.84 (2H, d, J=8.8 Hz), 4.24 (1H, d,

J=16.0 Hz), 4.53 (1H, d, J=14.2 Hz), 5.28 (1H, t,

J=8.8 Hz), 6.62 (1H, s), 7.20–7.53 (6H, m). Anal. calcd

for C₂₂H₂₄Cl₂N₂O₃: C, 60.70; H, 5.56. N, 6.43. Found:

C, 60.37; H, 5.49; N, 6.15.

7-Chloro-5-(2-chlorophenyl)-1-neopentyl-2-oxo-1,2-dihy-

dro-3H-1,3,4-benzotriazepine-3-acetic acid (8). An aqu-

eous solution of NaOH (1 N, 0.3 mL) was added to a

solution of 38 (0.16 g, 0.346 mmol) in EtOH (3 mL).

After being stirred at room temperature for 4 h, the

reaction mixture was diluted with water (50 mL), acidi-

ﬁed with 1 N HCl, and concentrated. The residue was

extracted with CH₂Cl₂(50 mL, twice). The extracts were

washed with brine, dried over Na₂SO₄and concentrated

under reduced pressure. The residue was recrystallized

[5-Chloro-2-(neopentylamino)phenyl](2-chlorophenyl)me-

thanethione (36). Lawesson’s reagent [2,4-bis(4-methoxy-

phenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulﬁde] (0.3

g, 0.743 mmol) was added to a solution of 22 (0.5 g, 1.49

mmol) in toluene (5 mL). After being reﬂuxed for 2 h,

themixturewas diluted with AcOEt (50 mL). Thesolu-

396

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

from CH₂Cl₂-petroleum ether (1:10, v/v) to give 8

(91 mg, 0.21 mmol, 61%) as a colorless powder. Mp

181–183 ^ꢁC (CH₂Cl₂–petroleum ether). IR n_max(KBr)

then concentrated. The residue was crystallized with

hexane–Et₂O to givetheoxime(3.9 g, 82%) as colorless

needles (mp 114–115 ^ꢁC). A mixtureof theoxime(3.9 g,

27.7 mmol) and polyphosphoric acid (30 g) was heated

for 20 min at 120 ^ꢁC. Water was added to the mixture

and the deposited powder was collected, chromato-

graphed [eluent: hexnae–CH₂Cl₂–AcOEt (1:1:1, v/v)] to

give 43 (3.0 g, 11.0 mmol, 63%) as colorless prisms. Mp

226–227 ^ꢁC (hexane–AcOEt). IR n_max(KBr) cm^ꢀ1: 1670

1

cm^ꢀ1: 3600–2400 (br, COOH), 1720, 1680 (C¼O). H

NMR (CDCl₃) d 0.86 (9H, s), 3.39 (1H, d, J=14.6 Hz),

4.43–4.41 (3H, m), 6.88 (1H, d, J=2.6 Hz), 7.22–7.47

(6H, m). Anal. calcd for C₂₁H₂₁Cl₂N₃O₃: C, 58.07; H,

4.87. N, 9.67. Found: C, 57.90; H, 5.13; N, 9.46.

1

Ethyl 4-(2-chlorophenyl)-4-phenylbutyrate (41). A mix-

tureof 40¹⁹(27 g, 78.3 mmol) and 47% HBr (80 mL) in

AcOH (100 mL) was reﬂuxed overnight. The resulting

mixturewas dilutde with AcOEt. Thesolution was

washed with brine, dried over Na₂SO₄and then con-

centrated to give ethyl 4-(2-chlorophenyl)-4-phenylbut-

3-enoate (21 g). A solution of this compound in AcOEt

(150 mL) was hydrogenated over 10% Pd–C (50% wet,

2 g) under atomospheric pressure until the absorption of

hydrogen stopped. The catalyst was removed by ﬁltra-

tion and the ﬁltrate was concentrated in vacuo. A mix-

(C¼O). H NMR (CDCl₃) d 2.4–2.7 (4H, m), 4.7–4.9

(1H, m), 6.55–6.7 (1H, m), 6.95–7.6 (8H, m). Anal.

calcd for C₁₆H₁₄ClNO: C, 70.72; H, 5.19; N, 5.15.

Found: C, 70.94; H, 5.20; N, 5.20.

5-(2-Chlorophenyl)-1-isobutyl-1,3,4,5-tetrahydro-2H-1-

benzazepin-2-one (44). NaH (0.82 g, 60% in oil,

20.6 mmol) was added to an ice-cooled solution of 43

(2.8 g, 10.3 mmol) and isobutylbromide(2.2 mL,

20.6 mmol) in DMF (20 mL). After being stirred for 4 h

at room temperature, the mixture was diluted with

AcOEt. Thesolution was washed with 1 N HCl, satu-

rated NaHCO₃and brine, dried over Na₂SO₄, and then

concentrated. The residue was chromatographed [elu-

ent: hexane–AcOEt (5:1, v/v)] to give 44 (3.0 g,

9.15 mmol, 89%) as cololess prisms. Mp 139–140 ^ꢁC

.

ture of the residue, p-TsOH H₂O (0.5 g) and EtOH

(150 mL) was reﬂuxed overnight. After removal of the

sovent under reduced pressure, the mixture was dis-

solved in AcOEt. The solution was washed with satu-

rated NaHCO₃and brine, dried over Na₂SO₄, and then

concentrated. The residue was chromatographed [elu-

ent: hexane–AcOEt (10:1, v/v)] to give 41 (8.0 g,

1

(hexane–AcOEt). IR n_max(KBr) cm^ꢀ1: 1660 (C¼O). H

NMR (CDCl₃) d 0.92 (3H, d, J=6.7 Hz), 1.08 (3H, d,

J=6.5 Hz), 1.8–2.1 (1H, m), 2.3–2.6 (4H, m), 3.44 (1H,

dd, J=13.7, 4.9 Hz), 4.23 (1H, dd, J=13.7, 9.0 Hz),

4.65–4.8 (1H, m), 6.54 (1H, d, J=7.3 Hz), 6.95–7.10

(1H, m), 7.2–7.6 (6H, m). Anal. calcd for C₂₀H₂₂ClNO:

C, 73.27; H, 6.76; N, 4.27. Found: C, 73.08; H, 6.69; N,

4.36.

26.4 mmol, 34%) as a colorless oil. IR n_max(neat) cm^ꢀ1

:

1730 (C¼O). ¹H NMR (CDCl₃) d: 1.23 (3H, t,

J=7.1 Hz), 2.2–2.5 (4H, m), 4.10 (2H, q, J=Hz), 4.45–

4.6 (1H, m), 7.0–7.4 (9H, m).

4-(2-Chlorophenyl)-1-tetralone (42). An aqueous solu-

tion of NaOH (1 N, 40 mL) was added to a solution of

41 (8.0 g, 26.4 mmol) in EtOH (80 mL). After being

stirred for 1 h, the mixture was concentrated in vacuo.

The residue was dissolved in water (100 mL). The solu-

tion was washed with Et₂O (50 mL). The aqueous layer

was acidiﬁed with concentrated HCl and extracted with

AcOEt (150 mL). The extract was washed with water,

dried over MgSO₄, and concentrated. A mixture of the

residue, SOCl₂(4.0 mL) and DMF (0.05 mL) in toluene

(50 mL) was heated for 1 h at 80 ^ꢁC, and then con-

centrated in vacuo. AlCl₃(2.9 g, 21.8 mmol) was added

to an ice-cooled solution of the residue in 1,2-dichloro-

ethane (50 mL) in portions. After being stirred for 1 h at

room temperature, the reaction was quenched with 1 N

HCl. The solution was washed with 1 N NaOH, dried

over MgSO₄, and concentrated in vacuo. The residue

was chromatographed [eluent: hexane–AcOEt (10:1, v/

v)] to give 42 (4.5 g, 17.5 mmol, 66%) as a colorless oil.

7-Chloro-5-(2-chlorophenyl)-1-isobutyl-1,3,4,5-tetrahy-

dro-2H-1-benzazepin-2-one (45). A mixtureof 44 (2.7 g,

8.24 mmol) and N-chlorosuccinimide(1.7 g, 12.4 mmol)

in DMF (10 mL) was stirred for 7 h at 70 ^ꢁC. Themix-

ture was diluted with AcOEt. The solution was washed

with 1 N HCl, saturated NaHCO₃and brine, dried over

Na₂SO₄, and then concentrated. The residual solid was

recrystallzed from hexane–AcOEt to give 45 (2.4 g,

6.62 mmol, 80%) as colorless prisms. Mp 152–154 ^ꢁC

1

(hexane–AcOEt). IR n_max(KBr) cm^ꢀ1: 1655 (C¼O). H

NMR (CDCl₃) d 0.92 (3H, d, J=6.8 Hz), 1.07 (3H, d,

J=6.6 Hz), 1.8–2.1 (1H, m), 2.3–2.6 (4H, m), 3.38 (1H,

dd, J=13.7, 4.8 Hz), 4.71 (1H, dd, J=13.7, 9.1 Hz), 4.6–

4.8 (1H, m), 6.51 (1H, d, J=2.0 Hz), 7.2–7.55 (6H, m).

Anal. calcd for C₂₀H₂₁Cl₂NO: C, 66.30; H, 5.84; N,

3.87. Found: C, 66.59; H, 5.88; N, 4.12.

1

IR n_max(neat) cm^ꢀ1: 1685 (C¼O). H NMR (CDCl₃) d

Ethyl 7-chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-2,3,4,5-

(46).

2.2–2.5 (2H, m), 2.6–2.8 (2H, m), 4.85 (1H, t,

J=5.9 Hz), 6.7–7.5 (7H, m), 8.05–8.20 (1H, m).

tetrahydro-1H-1-benzazepine-3-acetate

n-BuLi

(1.14 mL, 1.58 M in hexane, 1.79 mmol) was added to a

solution of Prⁱ₂NH (0.25 mL, 1.79 mmol) in dry THF

(5 mL) at ꢀ15 ^ꢁC. Themixturewas stirred for 45 min at

ꢀ15 ^ꢁC. After addition of 45 (0.5 g, 1.38 mmol) in THF

(5 mL), themixturewas stirred for 15 min at 0 ^ꢁC. The

5-(2-Chlorophenyl)-1,3,4,5-tetrahydro-2H-1-benzazepin-

2-one (43).

.

solution of NH₂OH HCl (1.3 g,

A

19.2 mmol) and AcONa (2.2 g, 26.3 mmol) in water

(30 mL) was added to a solution of 42 (4.5 g, 17.5 mmol)

in EtOH (100 mL). After being reﬂuxed for 2 h, the

mixture was concentrated in vacuo. The residue was

dissolved in AcOEt. The solution was washed with

saturated NaHCO₃and brine, dried over Na₂SO₄, and

mixturewas coodle to

ꢀ78 ^ꢁC and ICH₂COOEt

(0.25 mL, 2.07 mmol) was added. After being stirred for

15 min at ꢀ78 ^ꢁC and for 1 h at 0 ^ꢁC, themixturewas

quenched by 1 N HCl (50 mL). The mixture was extrac-

ted with AcOEt (50 mL). The extract was washed with

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

397

saturated NaHCO₃, dried over MgSO₄, and con-

centrated in vacuo. The residue was puriﬁed by column

chromatography [eluent: hexane–AcOEt (2:1, v/v)] to

give 46 (0.10 g, 0.223 mmol, 16%, cis:trans=ca. 2:1) as a

colorless oil. IR n_max(neat) cm^ꢀ1: 1730, 1660 (C¼O). ¹H

NMR (CDCl₃) d 0.5–1.15 (6H, m), 1.15–1.4 (3H, m),

1.7–3.1 (5H, m), 3.1–3.9 (2H, m), 4.0–4.2 (2H, m), 4.4–

4.8 (1H, m), 6.4–7.6 (7H, m). SIMS (m/z): 449 (MH⁺).

In this reaction, the starting material (0.38 g, 76%) was

recovered.

being stirred for 1 h, the reaction mixture was diluted with

CH₂Cl₂(100 mL). The solution was washed with water

and brine, dried over Na₂SO₄, and then concentrated

under reduced pressure. The recidue was chromato-

graphed [eluent: hexane–AcOEt (5:1, v/v)] to give 49 (6.8 g,

14.2 mmol, 91%) as a brown oil. IR n_max(neat) cm^ꢀ1

:

1

3600–3200 (br, OH), 1720, 1660, (C¼O), 1625 (C¼C). H

NMR (CDCl₃) d 0.68 (1/2ꢂ9H, s), 0.94 (1/2ꢂ9H, s),

1.21 (1/2ꢂ3H, t, J=7.2Hz), 1.25 (1/2ꢂ3H, t, J=7.2 Hz),

2.30 (1/2ꢂ1H, d, J=13.8Hz), 2.88 (1/2ꢂ1H, d,

J=13.4Hz), 3.96–4.29 (4H+1/2ꢂ1H, m), 4.60–4.67 (1H,

m), 4.82 (1/2ꢂ1H, t, J=7.0 Hz), 5.86 (1/2ꢂ1H, d,

J=15.2 Hz), 6.17 (1/2 1H, d, J=15.2 Hz), 6.69–7.81

(8H, m).

7-Chloro-5-(2-chlorophenyl)-1-isobutyl-2-oxo-2,3,4,5-tet-

rahydo-1H-1-benzazepine-3-acetic acid (9). An aqueous

solution of NaOH (1 N, 1 mL) was added to a solution

of 46 (0.09 g, 0.201 mmol) in EtOH (3 mL). After stir-

ring for 20 min, the mixture was diluted with water

(30 mL), washed with Et₂O (20 mL), acidiﬁed with 1 N

HCl (30 mL), and extracted with AcOEt (30 mL). The

extract was washed with water, dried over MgSO₄, and

concentrated in vacuo to give 9 (50 mg, 0.119 mmol,

59%) as colorless crystals. Mp 165–171 ^ꢁC (hexane–

AcOEt). IR n_max(KBr) cm^ꢀ1: 1730, 1710, 1650, 1625

Ethyl (3,6-trans)-8-chloro-6-(2-chlorophenyl)-1-neopentyl-

2-oxo-2,3,5,6-tetrahydro-1H-4,1-benzoxazocine-3-acetate

(50). A mixtureof 49 (6.8 g, 14.2 mmol), 18-crown-6

(3.8 g, 14.3 mmol) and K₂CO₃(2.0 g, 14.3 mmol) in

CH₂Cl₂(70 mL) was stirred for 3 days at room tem-

perature. After removal of the solvent, the residue was

chromatographed [eluent: hexane–AcOEt (5:1, v/v)] to

give 50 (1.6 g, 3.34 mmol, 24%) as a colorless amor-

phous powder. IR n_max(KBr) cm^ꢀ1: 1725, 1670 (C¼O).

¹H NMR (CDCl₃) d 1.03 (9H, s), 1.23 (3H, t,

J=7.2 Hz), 2.74 (1H, dd, J=6.6, 17.2 Hz), 2.97 (1H, dd,

J=7.8, 17.2 Hz), 3.72 (1H, d, J=13.4 Hz), 3.94–4.14

(5H, m), 4.42 (1H, dd, J=1.4, 11.4 Hz), 4.67 (1H, dd,

J=1.4, 8.8 Hz), 7.01 (1H, d, J=2.2 Hz), 7.23–7.43 (6H,

m).

1

(C¼O). H NMR (CDCl₃) d 0.5–1.15 (6H, m), 1.6–2.0

(1H, m), 2.1–3.1 (5H, m), 3.1–4.3 (2H, m), 4.4–4.8 (1H,

m), 6.5–7.65 (7H, m). Anal. calcd for C₂₂H₂₃Cl₂NO₃: C,

62.86; H, 5.51; N, 3.33. Found: C, 62.77; H, 5.61; N,

3.29.

2-(2-Amino-5-chlorophenyl)-2-(2-chlorophenyl)ethanol (47).

Hydrazinehydrate(3.4 g, 67.2 mmol) and Ranye Ni

(0.1 g) was added to a solution of 28 (7.0 g, 22.4 mmol)

in EtOH (70 mL). After being stirred for 30 min at

room temperature, the catalyst was removed by ﬁl-

tration and the solvent was removed in vacuo. The

residue was chromatographed [eluent: hexane–AcOEt

(2:1, v/v)] to give 47 (4.4 g, 15.6 mmol, 70%) as a brown

oil. IR n_max(neat) cm^ꢀ1: 3450 (NH₂), 3370 (NH₂), 3600-

3200 (br, OH). ¹H NMR (CDCl₃) d 4.11 (2H, d,

J=6.6 Hz), 4.59 (1H, t, J=6.6 Hz), 6.60 (1H, d,

J=8.4 Hz), 7.01–7.43 (6H, m).

(3,6-trans)-8-Chloro-6-(2-chlorophenyl)-1-neopentyl-2-

oxo-2,3,5,6-tetrahydro-1H-4,1-benzoxazocine-3-acetic

Acid (10). Concentrated HCl (10 mL) was added to a

solution of 50 (1.2 g, 2.51 mmol) in dioxane(20 mL).

After being reﬂuxed for 3 h, the mixture was diluted

with CH₂Cl₂(100 mL), washed with water and brine,

dried over Na₂SO₄and then concentrated in vacuo. The

residue was recrystallized from CH₂Cl₂–petroleum ether

(1:10, v/v) to give 10 (0.23 g, 0.511 mmol, 20%) as col-

orless needles. Mp 127–133 ^ꢁC (CH₂Cl₂–petroleum

ether). IR n_max(KBr) cm^ꢀ1: 3600–2400 (br, COOH),

2-(5-Chloro-2-neopentylaminophenyl)-2-(2-chlorophenyl)-

ethanol (48). AcOH (1.4 mL), and pivalaldehyde (2.0 g,

23.6 mmol) was added to a solution of 47 (4.4 g,

15.6 mmol) in MeOH (50 mL). After being stirred for 30

min at room temperature, NaBH₃CN (1.5 g, 23.6 mmol)

was added. The reaction mixture was stirred for 1 h at

room temperature, diluted with CH₂Cl₂(100 mL). The

solution was washed with 1 N NaOH and brine, dried

over Na₂SO₄, and then concentrated under reduced

pressure. The residue was chromatographed [eluent:

hexane–AcOEt (4:1, v/v)] to give 48 (5.5 g, 15.6 mmol,

quant) as a brown oil. IR n_max(neat) cm^ꢀ1: 3600–3100

1

1710, 1660 (C¼O). H NMR (CDCl₃) d 1.03 (9H, s),

2.81 (1H, dd, J=6.6, 17.4 Hz), 2.97 (1H, dd, J=7.0,

17.4 Hz), 3.73 (1H, d, J=13.8 Hz), 3.93–4.09 (3H, m),

4.43 (1H, d, J=11.0 Hz), 4.66 (1H, d, J=8.2 Hz), 7.00

(1H, d, J=2.6 Hz), 7.23–7.44 (6H, m). Anal. calcd for

.

C₂₃H₂₅Cl₂NO₄H₂O: C, 58.98; H, 5.81; N, 2.99. Found:

C, 58.82; H, 5.43; N, 2.97.

Ethyl 6-chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihyroquino-

12 (5 g,

line-3-carboxylate (51).

A

mixtureof

18.8 mmol), diethyl malonate (4.5 mL, 26.3 mmol) and

DBU (0.28 mL, 2.6 mmol) was heated for 8 h at 190–

200 ^ꢁC. Theresulting mixturewas diluted with AcOEt.

The solution was washed with 1 N HCl, saturated

NaHCO₃and brine, dried over Na₂SO₄, and then con-

centrated. The residue was chromatographed [eluent:

hexane–AcOEt (3:2, v/v)] to give 51 (4.3 g, 11.9 mmol,

63%) as colorless crystals. IR n_max(KBr) cm^ꢀ1: 1730,

1710, 1650(C¼O). ¹H NMR (CDCl₃) d: 0.97 (3H, t,

J=7.2 Hz), 4.0–4.2 (2H, m), 7.02 (1H, d, J=2.1 Hz),

7.2–7.6 (6H, m), 12.6 (1H, br).

1

(br, NH, OH). H NMR (CDCl₃) d 0.81 (9H, s), 2.65

(1H, d, J=11.4 Hz), 2.77 (1H, d, J=11.4 Hz), 4.11–4.15

(2H, m), 4.59 (1H, t, J=6.3 Hz), 6.55 (1H, d,

J=8.6 Hz), 7.01–7.44 (6H, m).

Ethyl

3-[N-[4-Chloro-2-[1-(2-chlorophenyl)-2-hydroxye-

thyl]phenyl]-N-neopentylcarbamoyl]acrylate (49). Fumaryl

chloride monoethyl ester (2.6g, 16.0 mmol) was added

dropwiseto a suspnesion of 48 (5.5 g, 15.6 mmol) and

NaHCO₃(1.7 g, 20.2 mmol) in CH₂Cl₂(30 mL). After

398

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

Ethyl 6-chloro-4-(2-chlorophenyl)-1-isobutyl-2-oxo-1,2-

dihydroquinoline-3-carboxylate (52). A mixtureof 51

(2.5 g, 6.90 mmol), isobutylbromide(1.5 mL, 13.8 mmol),

KI (2.2 g, 10.4 mmol), K₂CO₃(1.9 g, 13.8 mmol) and

DMF (20 mL) was stirred for 3 days at room teperature.

The resulting mixture was diluted with AcOEt. The

solution was washed with 1 N HCl, saturated NaHCO₃

and brine, dried over Na₂SO₄, and then concentrated.

The residue was puriﬁed by column chromatography

[eluent: hexane–AcOEt (3:2, v/v)] to give 52 (1.4 g,

3.35 mmol, 49%) as colorless prisms. Mp 117–119 ^ꢁC

(hexane–AcOEt). IR n_max(KBr) cm^ꢀ1: 1730, 1645

DMF (10 mL) was stirred for 1 h. The mixture was

diluted with AcOEt. The mixture was washed with 1 N

HCl, saturated NaHCO₃and brine, dried over Na₂SO₄,

and then concentrated. The residue was chromato-

graphed [eluent: hexane–AcOEt (5:1, v/v)] to give the

methyl ester of 11 (0.31 g, 0.738 mmol). A mixtureof the

methyl ester, K₂CO₃(0.2 g, 1.48 mmol), MeOH (10 mL)

and water (10 mL) was reﬂuxed for 1 h. The resulting

mixture was acidiﬁed with 1 N HCl and extracted with

AcOEt. The extract was washed with brine, dried over

Na₂SO₄, and then concentrated to give 11 (0.20 g,

0.492 mmol, 38%) as colorless crystals. Mp 131–133 ^ꢁC

(hexane–AcOEt). IR n_max(KBr) cm^ꢀ1: 1715, 1665

1

(C¼O). H NMR (CDCl₃) d: 0.9–1.2 (9H, m), 2.1–2.4

1

(1H, m), 3.9–4.35 (4H, m), 7.03 (1H, d, J=2.4 Hz), 7.25–

7.60 (6H, m). Anal. calcd for C₂₂H₂₁Cl₂NO₃: C, 63.17;

H, 5.06; N, 3.35. Found : C, 63.03; H, 5.34; N, 3.17.

(C¼O). H NMR (CDCl₃) d 0.93 (3H, d, J=6.6 Hz),

0.96 (3H, d, J=7.0 Hz), 1.9–2.2 (1H, m), 2.34 (1H, dd,

J=16.4, 4.0 Hz), 2.62 (1H, dd, J=14.4, 8.8 Hz), 3.35–

3.55 (1H, m), 4.08 (1H, dd, J=14.4, 8.8 Hz), 4.75 (1H,

d, J=13.4 Hz), 6.5–6.6 (1H, m), 6.9–7.6 (6H, m). Anal.

calcd for C₂₁H₂₁Cl₂NO₃: C, 62.08; H, 5.21; N, 3.45.

Found : C, 62.18; H, 5.42; N, 3.34.

Ethyl (3,4-trans)-6-chloro-4-(2-chlorophenyl)-1-isobutyl-

2-oxo-1,2,3,4-tetrahydroquinoline-3-carboxylate

(53).

LiAlH₄(0.18 g, 4.84 mmol) was added to an ice-cooled

solution of 52 (1.4 g, 3.35 mmol) in THF (20 mL). The

mixture was stirred for 20 min at ice-bath temperature.

Water (2 mL) was added to the mixture. The resulting

mixturewas dilutde with AcOEt. Themixturewas

Single-crystal X-ray analysis of 1a, 2, 4, 5, 7and 10

Crystals of 1a, 2, 4, 5, 7 and 10 were grown from

methanol. Data were collected on a diﬀractometer,

Rigaku AFC5R, and corrected for Lorentz and polar-

ization factors. Absorption correction was not applied.

The structures were determined by direct methods with

washed with 1 N HCl, saturated NaHCO₃and brine,

dried over Na₂SO₄, and then concentrated. The residue

was puriﬁed by column chromatography [eluent: hex-

ane–AcOEt (10:1, v/v)] to give 53 (0.65 g, 1.55 mmol,

46%) as a colorless oil. IR n_max(neat) cm^ꢀ1: 1740, 1680

23

theaid of TEXSAN and reﬁned by CRYLSQ²⁴in the

1

(C¼O). H NMR (CDCl₃) d 0.95 (3H, d, J=6.8 Hz), 1.00

XTAL package. The parameters reﬁned include the

coordinates and anisotropic thermal parameters for all

non-hydrogen atoms. Hydrogen atoms were included

using a riding model in which the distances from the

bonded carbon atoms were ﬁxed at 1.09 A. Thermal

parameters of hydrogen atoms were taken from their

bonded atoms as U_isoand ﬁxed through the next several

cycles of reﬁnement. The ﬁnal R factors were 0.063,

0.082, 0.048, 0.053, 0.073 and 0.085, respectively. In the

caseof compound 10, the chloro atom existed on both

sides of the 6-phenyl ring in the ratio of 4:1. It was sug-

gested that two comformers resulted from turnover of

the 6-phenyl ring existed in a single crystal, and the ratio

of these comformers was 4:1. Figure 2 shows only the

chloro atom of major comformer. Crystal data, condi-

tions of data collection, atomic coordinates, thermal

parameters, bond distances, bond angles, and torsion

angles are available as supporting information.

(3H, d, J=6.8Hz), 1.11 (3H, t, J=7.1Hz), 2.0–2.25 (1H,

m), 3.76 (1H, dd, J=14.2, 7.2 Hz), 3.94 (1H, dd, J=14.2,

7.4 Hz), 4.02 (1H, d, J=7.4 Hz), 4.0–4.2 (2H, m), 5.09 (1H,

d, J=7.4Hz), 6.9–7.5 (7H, m).

Ethyl 6-chloro-4-(2-chlorophenyl)-3-ethoxycarbony-1-iso-

butyl-2-oxo-1,2,3,4-tetrahydroquinoline-3-acetate (54).

NaH (68 mg, 1.70 mmol) was added to an ice-cooled

solution of 53 (0.65 g, 1.55 mmol) and BrCH₂COOEt

(0.22 mL, 2.17 mmol) in DMF (10 mL). After being

stirred for 8 h at room temperature, the mixture was

diluted with AcOEt. The mixture was washed with 1 N

HCl, saturated NaHCO₃and brine, dried over Na₂SO₄,

and then concentrated. The residue was puriﬁed by col-

umn chromatography [eluent: hexane–AcOEt (5:1, v/v)]

to give 54 (0.65 g, 1.28 mmol, 83%) as a colorless oil. IR

n

_max(neat) cm^ꢀ1: 1730, 1665 (C¼O). ¹H NMR (CDCl₃)

d 0.9–1.1 (9H, m), 1.23 (3H, t, J¼7.1 Hz), 2.0–2.3 (1H,

m), 2.66 (1H, d, J=17.7 Hz), 3.22 (1H, d, J=17.7 Hz),

3.78 (1H, dd, J=14.2, 6.2 Hz), 3.9–4.3 (5H, m), 5.73 (1H,

s), 6.63 (1H, s), 6.95–7.6 (6H, m). MS (m/e) 505 (M+).

Crystallographic data (excluding structure factors) for

the structures in this paper have been deposited with the

Cambridge Crystallographic Data Centre as supple-

mentary publication nos. CCDC 164786 (1a),

CCDC164787 (2), CCDC164788 (4), CCDC164789 (5),

CCDC164790 (7) and CCDC164791 (10). Copies of the

data can be obtained, free of charge, on application to

CCDC, 12 Union Road, CambridgeCB2 1EZ, UK

(fax: +44-1223-336033; e-mail: deposit@ccdc.cam.-

ac.uk).

(3,4-trans)-6-Chloro-4-(2-chlorophenyl)-3-ethoxycar-

bony-1-isobutyl-2-oxo-1,2,3,4-tetrahydroquinoline-3-ace-

tic acid (11). A mixtureof 54 (0.65 g, 1.28 mmol), 85%

KOH (0.42 g, 6.42 mmol), EtOH (10 mL) and water

(10 mL) was reﬂuxed overnight. The mixture was acidi-

ﬁed with 1 N HCl and extracted with AcOEt. The

extract was washed with brine, dried over Na₂SO₄, and

then concentrated. The residue was subjected to column

chromatography [eluent: hexane–CH₂Cl₂MeOH (5:5:1,

v/v)] to givecrude 11. A mixtureof thecrude 11, Me I

(0.08 mL, 1.28 mmol), K₂CO₃(0.18 g, 7.62 mmol) and

Animals and materials

Animals were supplied by Clea, Japan, Inc. RS-[2-¹⁴C]

mevalonolactone and [1-³H]-farnesyl pyrophosphate

T. Miki et al. / Bioorg. Med. Chem. 10 (2002) 385–400

399

were purchased from New England Nuclear. [2-¹⁴C]

mevalonic acid was synthesized from [2-¹⁴C] mevalono-

lactoneby saponiﬁcation with potassium hydroxid.e

[2-¹⁴C] Sodium acetate was purchased from Amersham.

Farnesyl pyrophosphate was synthesized by the method

described by V. J. Davisson and coworkers²⁵(Nemoto

& Co.). HepG2 cells were supplied by 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.

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 radioactivuty was determined by means

of a liquid scintillation counter. The squalene synthase

inhibitory activity was expressed in terms of the con-

centration of the test compound inhibiting by 50% the

radioactivity taken into the chloroform layer [IC₅₀,

molar concentration (M)].

Acknowledgements

Preparation of rat squalene synthase

Theauthors wish to thank Dr. Kanji Mgeuro for his

encouragement and helpful discussion throughout this

study. Wealso thank Ms. Keiko Higashikawa for X-ray

crystallographic analysis.

An SD male rat (6 weeks 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

solution [100 mM potassium phosphate(pH 7.4),

15 mM nicotinamide, 2 mM MgCl₂], followed by cen-

trifugation for 20 minutes at 10,000g (4 ^ꢁC). Thesuper-

natant layer was separated and subjected to further

centrifugation for 90 min at 105,000g (4 ^ꢁC). Thesedi-

ment was then suspended in an ice-cooled 100 mM

potassium phosphatebuﬀre solution (pH 7.4), which

was again subjected to centrifugation for 90 min at

105,000g (4 ^ꢁC). The sediment thus obtained (micro-

some fraction) was suspended in an ice-cooled 100 mM

potassium phosphatebuﬀer (pH 7.4) (about 40 mg/mL

protein concentration, determined using BCA protein

assay kit of Pierce Co., Ltd.). This suspension was used

as theenzymesolution.

References and Notes

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Article Doi

Syntheses of fused heterocyclic compounds and their inhibitory activities for squalene synthase

DOI: 10.1016/S0968-0896(01)00289-9

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