Synthesis and structure-activity studies of novel orally active non-terpenoic 2,3-oxidosqualene cyclase inhibitors

3354

J . Med. Chem. 2003, 46, 3354-3370

Syn th esis a n d Str u ctu r e-Activity Stu d ies of Novel Or a lly Active Non -Ter p en oic

2,3-Oxid osqu a len e Cycla se In h ibitor s

Henrietta Dehmlow, J ohannes D. Aebi,* Syne`se J olidon, Yu-Hua J i,^†Elisabeth M. von der Mark,

J acques Himber, and Olivier H. Morand^‡

Pharmaceuticals Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124,

CH-4070 Basel, Switzerland

Received December 17, 2002

New orally active non-terpenoic inhibitors of human 2,3-oxidosqualene cyclase (hOSC) are

reported. The starting point for the optimization process was a set of compounds derived from

a fungicide project, which in addition to showing high affinity for OSC from Candida albicans

showed also high affinity for human OSC. Common structural elements of these inhibitors are

an amine residue and an electrophilic carbonyl C atom embedded in a benzophenone system,

which are at a distance of about 10.7 Å. Considering that the keto moiety is in a potentially

labile position, modifications of the substitution pattern at the benzophenone as well as

annelated heteroaryl systems were explored. Our approach combined testing of the compounds

first for increased binding affinity and for increased stability in vitro. Most promising compounds

were then evaluated for their efficacy in lowering plasma total cholesterol (TC) and plasma

low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic hamsters. In this respect, the

most promising compounds are the benzophenone derivative 1‚fumarate and the benzo[d]-

isothiazol 24‚fumarate, which lowered TC by 40% and 33%, respectively.

In tr od u ction

tomatic individuals with hypercholesterolemia. Accord-

ing to current guidelines, cholesterol lowering treatment

is recommended for patients who had survived a myo-

cardial infarct or patients suffering from angina pectoris

or another atherosclerotic disease, with a target LDL-C

level of 100 mg/dL.

Standard drug therapies include bile acid seques-

trants, fibrates, nicotinic acid, probucol, and statins

(HMG-Co-A reductase inhibitors). Statins, the most

widely used lipid-modifying drugs, reduce plasma LDL-C

by 30-45% at standard dosage and also plasma tri-

glycerides, a synergistic risk factor, but less effectively.

In contrast, fibrates reduce plasma triglycerides more

effectively but not LDL-C. In humans, statins are well

tolerated at standard dosage, but high doses may be

associated with adverse effects such as elevated liver

enzymes and rhabdomyolisis resulting in part from

the reduction of isoprenoid intermediates of choles-

terol synthesis and their metabolites, e.g., coenzyme

Q10⁷.

Causal risk factors that directly promote the develop-

ment of coronary and peripheral atherosclerosis include

elevated plasma low-density lipoprotein cholesterol

(LDL-C), low plasma high-density lipoprotein choles-

terol (HDL-C), hypertension, cigarette smoking, and

diabetes mellitus. Other synergistic risk factors include

elevated concentrations of plasma triglyceride (TG)-rich

lipoproteins, small, dense, low-density lipoprotein par-

ticles, lipoprotein (a) and homocysteine. Predisposing

risk factors such as obesity, physical inactivity, family

history of premature cardiovascular disease (CVD), and

male gender modify the causal or conditional risk factors

and thus affect atherogenesis indirectly. The strong

correlation between coronary heart disease (CHD) and

high LDL-C levels in plasma, and the clinical benefit

associated with the lowering of LDL-C levels are now

well established.^1-3Cholesterol-rich, sometimes un-

stable, atherosclerotic plaques lead to the occlusion of

blood vessels, resulting in ischemia or infarct.

This has stimulated the search for compounds that

inhibit cholesterol biosynthesis downstream of these

important, non-sterol isoprenoid intermediates. 2,3-

Oxidosqualene-lanosterol cyclase (OSC, EC 5.4.99.7),

a microsomal enzyme, represents a unique target for a

cholesterol-lowering drug.^8,9OSC is located downstream

of farnesyl pyrophosphate, beyond the synthesis of

isoprenoids and coenzyme Q. In hamsters, pharmaco-

logically active doses of an OSC inhibitor showed no

adverse effects, in contrast to a statin that reduced food

intake and body weight and increased plasma bilirubin,

liver weight, and liver triglyceride content. In contrast

to the statin, the OSC inhibitor did not lower liver and

heart coenzyme Q10 tissue levels.⁸

Primary prevention studies have shown that lowering

of plasma LDL-C levels reduces the incidence of nonfatal

CHD, while the overall morbidity remains unchanged.^4,5

The lowering of plasma LDL-C levels in patients with

preestablished CHD (secondary prevention) reduces

CHD mortality and morbidity; meta analysis of different

studies shows that this decrease is proportional to the

reduction of LDL-C.⁶

The clinical benefit of cholesterol lowering is greater

for patients with preestablished CHD than for asymp-

* To whom correspondence should be addressed. Phone: ++41 61

688 7738. Fax: ++41 61 688 6459. E-mail: johannes.aebi@roche.com.

^†Present address: Theravance Inc., 901 Gateway Boulevard, South

San Francisco, CA 94065.

In yeast, OSC inhibition or down-regulation stimu-

lated the degradation of the HMG-CoA reductase pro-

^‡Present address: Actelion Pharmaceuticals Ltd., Gewerbestrasse

16, CH-4123 Allschwil, Switzerland.

Published on Web 06/18/2003

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3355

tein.¹⁰OSC inhibition does not trigger the overexpres-

sion of HMGR because of an indirect, negative feedback

regulatory mechanism involving the production of 24-

(S),25-epoxycholesterol.^11,12This negative feedback regu-

latory mechanism is essential to the concept of OSC

inhibition because (i) it potentiates synergistically the

primary inhibitory effect with an indirect down-regula-

tion of HMGR and (ii) it minimizes the massive ac-

cumulation of the precursor monooxidosqualene in the

liver. In addition, 24(S),25-epoxycholesterol is known to

be a potent natural agonist of the nuclear receptor

LXR.¹³Considering that 24(S),25-epoxycholesterol is a

byproduct of inhibition of OSC, it is theoretically pos-

sible that OSC inhibitors could indirectly activate

LXR-dependent pathways such as the ABC trans-

porters.^14-17This has been recently evidenced in cul-

tured macrophages where incubation with OSC inhibi-

tors resulted in upregulation of ABCA1 and ABCG1,

which led to enhanced cholesterol efflux and reduced

foam cell formation.¹⁸

F igu r e 1.

Several mammalian OSCs have been purified, cloned,

and expressed (for a summary, see Corey¹⁹and Abe²⁰),

but so far only one member of the related squalene

cyclase family could be crystallized and structurally

elucidated.^21,22The squalene-hopene cyclase from the

thermophilic bacterium Alicyclobacillus acidocaldarius

is a dimer of 2 × 631 amino acid residues showing a

peculiar monotopic association with the membrane. The

catalytic center is located in a large cavity that is con-

nected through a channel with the membrane interior

from where the cyclase picks the substrate squalene or

oxidosqualene and through which it releases the product

hopene or lanosterol, respectively. The cyclization is

thought to proceed through three discrete steps:¹⁹(i)

enzymatic binding to induce the correct folding of 2,3-

oxidosqualene; (ii) a conformational change, which cor-

rectly positions an electrophilic group for activation of

the epoxide; (iii) cyclization starting the reaction by

protonation of the epoxide to produce protosterol via

cationic intermediates. Further skeletal rearrangements

through 1,2-shifts of hydride and methyl substituents

yield lanosterol.

energy intermediate of oxidosqualene^23,24to optimize

hydrophobic and ionic interactions with the transi-

tion state of the enzyme. The folded, high-energy

intermediate of oxidosqualene cyclization was modeled

as an opened protosterol, using the modeling program

MOLOC.³⁰The structure of human OSC was later

modeled³¹from the X-ray structure of Alicyclobacillus

acidocaldarius squalene-hopene cyclase (SHC).²¹The

active site residues of SHC showed a similarity of 50%

and an identity of 38% with respect to human OSC.^22,32

If residues forming the bottom (residues close to Trp196)

and entrance tunnel are neglected and only the active

site residues are aligned, the score is even higher (65%

sequence similarity and 50% sequence identity).³¹The

X-ray structure of Ro 48-8071 in SHC³³revealed that

the binding of 1 (Ro 48-8071) is largely identical to the

expected binding site for squalene. Owing to the high

similarity of residues in the active site between the SHC

and OSC, observations from SHC crystals were extrapo-

lated to the OSC model. The major part of the inhibitor

is located in the hydrophobic tunnel with a nearly fixed

amine position. In SHC, the positive charge of the amino

group in 1 forms an ion pair with the DXDD motif and

is stabilized by cation-π interactions with tryptophans

and a phenylalanine. The carbonyl group of the ben-

zophenone lacks a hydrogen-bonding partner. The car-

bonyl carbon is probably stabilized by the π system of

Phe605. The bromophenyl group of the inhibitor oc-

cupies part of the entrance to the active site, which is

surrounded by two phenylalanines and stabilized by

edge-to-face interaction. To optimally fulfill the steric

demands of the active site, inhibitors have to be flexible

at the corner position. Ro 48-8071 features a benzophe-

none that is ideally bent. We investigated the influence

of the substitution pattern of the benzophenone on the

activity against human oxidosqualene cyclase (hOSC)

and on the stability of the inhibitors in rat hepatocytes

(Tables 1 and 2). Different substituted amines were

synthesized to see the influence of polarity and pK_aon

the inhibition of hOSC (Table 3). Finally, the benzophe-

none was replaced with new heterocyclic systems to give

potent and stable hOSC inhibitors (Table 4). The SHC

cocrystals of five heterocyclic inhibitors are discussed

The first compounds that represented a starting point

for medicinal chemistry were found in the chemical

collection of a fungicide project.^23,24These compounds

were further optimized to cholesterol-lowering agents

such as Ro 48-8071 1, active on mammalian OSC.⁸A

variety of OSC inhibitors have been published bearing

amines,⁹quinuclidines,²⁵isoquinolines,²⁶and a lipo-

philic residue. The large lipophilic residues that fit into

the cavity of the cyclase offer a wide range of possibili-

ties for designing new OSC inhibitors. The amine that

is protonated at physiological pH mimics the cation

formed in the acid-catalyzed opening of the 2,3-oxirane

ring of monooxidosqualene (MOS). A wide range of pK_a

values are accepted,²⁷and three major classes of OSC

inhibitors with respect to the amines are known: ter-

tiary amines mentioned before, Boehringer Ingelheim’s

oxazole derivatives such as BIBB-515 2 (pK_a) 6.4),²⁸

and AstraZeneca’s pyridine 3a or pyrimidine 3b (pK_a

) 6.1-9.2)^27,29(Figure 1).

In the absence of an X-ray structure of OSC, our

approach for the design of new OSC inhibitors consisted

of superimposing a prototype inhibitor on the high-

3356 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Sch em e 1. Synthesis of Benzophenone OSC Inhibitors^a

Dehmlow et al.

a

Reagents and conditions: (a) AlCl₃, nitrobenzene, 0 °C to room temp; (b) NaSMe, THF, room temp; (c) 62% aqueous HBr, AcOH, 120

°C or BBr₃, CH₂Cl₂, -78 °C to room temp; (d) 1,6-dibromohexane, 10% aqueous NaOH, n-Bu₄NBr, CH₂Cl₂, room temp or 1,6-dibromohexane,

K₂CO₃, acetone, 60 °C; (e) NHMeAllyl, DMA, room temp; (f) for R¹) NHMe, NH₂Me, EtOH/DMA, 120 °C; for R¹) NH₂, (1) PMB-NH₂/

K₂CO₃, toluene, 120 °C, (2) TFA, room temp; for R¹) OMe, (1) NaOMe, MeOH, 70 °C; (g) for R¹) OH, 62% aqueous HBr, AcOH, 90 °C.

in Lenhart et al.³⁴The best compounds were then tested

in hamsters for their cholesterol-lowering effects.

compounds 8a or 8b, respectively. The methyl ether of

the derivative 8b could be cleaved with 62% aqueous

HBr in acetic acid at 90°C to give the compound 8c. The

aminobenzophenone 8d was synthesized from 1 with

p-methoxybenzylamine in the presence of potassium

carbonate in toluene followed by debenzylation in tri-

fluoroacetic acid.

Syn th esis of Am in e An a logu es. For the synthesis

of the cyclopropyl- and cyclopropylmethylamine ana-

logues, two different synthetic routes were chosen

(Scheme 2). The synthesis of 10a and 10b started with

bromide 6b. Reaction of N-cyclopropyl-2,2,2-trifluoro-

acetamide or 3-aminopropanol (to 9a or 9b) followed by

basic deprotection of amide 9a and reductive methyla-

tion with 37% aqueous formalin and 1 N NaH₂PO₃in

dioxane at 65 °C³⁵gave the final compounds 10a and

10b.

Cyclopropylmethyl methylamine derivative 13 was

synthesized from bromide 6b with sodium azide in

DMF, followed by Staudinger reduction and protection

of the primary amine as a trifluoroacetamide to give

intermediate 11. N-methylation by treatment with NaH

and MeI in DMF and deprotection of the tertiary amide

under basic conditions gave derivative 12. Alkylation

of 12 with bromomethylcyclopropane in DMA in the

presence of Hunig’s base yielded the cyclopropylmethyl

methylamine 13.

Syn th esis of P yr id in yl An a logu es. The synthesis

of pyridinylphenyl methanone inhibitor 15 is depicted

in Scheme 3. Starting from the chloropyridinecarboxylic

acid III, the benzophenones 14 were prepared via

treatment of the Weinreb amide with monolithiated 1,4-

Ch em istr y

P r ep a r a tion of Ben zop h en on e Der iva tives. For

the preparation of the aminoalkylbenzophenones, a

synthetic route was sought that would give the possibil-

ity of modifying the substitution pattern at either of the

aromatic rings, the spacer moiety, and/or the amine

residue in a rapid manner. This synthesis is outlined

in Scheme 1 for the aminohexylbenzophenones. Starting

from anisole derivatives Ia -d ,f-h , Friedel-Crafts acy-

lation with substituted benzoyl chlorides IIa -d in the

presence of AlCl₃in nitromethane gave the desired

4-methoxybenzophenones 4a -d and 4f-h . Reaction of

the fluoro-substituted benzophenone 4f with sodium

methanethiolate yielded the methylsulfanyl deriva-

tive 4e. Cleavage of the methyl ethers 4a -g was

achieved by treatment with hydrogen bromide in acetic

acid or boron tribromide in dichloromethane to yield the

4-hydroxybenzophenones 5a -f,i. These 4-hydroxyben-

zophenones were alkylated with an excess of 1,6-di-

bromohexane to give the intermediates 6a -f, which

were transformed to the final aminohexylbenzophenone

inhibitor 1 and 7a -e by amination with N-allylmethyl-

amine.

Further modification of the substitution pattern at the

aromatic moiety was possible by nucleophilic substitu-

tion of the fluoro-substituted benzophenone 1. For the

introduction of amine or oxygene moieties, 1 was treated

with the corresponding amine in DMA at 120 °C or with

sodium methanolate in methanol at 70 °C to give the

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3357

Sch em e 2. Synthesis of Different Aminobenzophenones^a

a

Reagents and conditions: (a) N-cyclopropyl-2,2,2-trifluoroacetamide, K₂CO₃, benzyltriethylammonium bromide, MeCN, 80 °C; (b)

3-aminopropanol, DMA, room temp; (c) (1) 20% aqueous KOH, MeOH/THF, room temp (for 9a ), (2) 37% aqueous formaline, 1 N NaH₂PO₃,

dioxane, 65 °C; (d) (1) NaN₃, DMF, 90 °C, (2) Ph₃P, THF/H₂O, room temp, (3) (CF₃CO)₂O, Et₂O, 0 °C to room temp; (e) (1) NaH, MeI,

DMF, -20 °C to room temp, (2) 20% aqueous KOH, MeOH/THF, room temp; (f) bromomethylcyclopropane, i-Pr₂EtN, DMA, 50 °C.

Sch em e 3. Synthesis of Pyridinyl Analogues^a

a

Reagents and conditions: (a) (1) SOCl₂, reflux, (2) MeNHOMe‚HCl, Et₃N, CH₂Cl₂, 0 °C to room temp, (3) 1,4-dibromobenzene, n-BuLi,

THF, -78 to 0 °C; (b) 6-(allylmethylamino)hexan-1-ol, KOH, K₂CO₃, dicyclohexano-18-crown-6, toluene, 80 °C.

Sch em e 4. Synthesis of Chromene, Quinazoline, and Indazole OSC Inhibitors^a

a

Reagents and conditions: (a) vinylmagnesium chloride (1.7 M in THF), THF/Et₂O, 0 °C to room temp; (b) o-xylene (Dean-Stark), 170

°C; (c) HCOOH, HCONH₂, 165 °C; (d) MeHNNH₂, K₂CO₃, DMA, 120 °C.

dibromobenzene. Reaction of 14 with allylmethylami-

nohexanol, KOH, K₂CO₃, and dicyclohexano-18-crown-6

in toluene at 80 °C gave the final pyridinyl inhibitor

15.

the envisaged “amine-spacer-heteroaryl” systems. This

approach would allow a rapid modification and optimi-

zation of both the spacer and the amine part of the

products.

Syn th esis of Heter oa r yl System s. For the replace-

ment of the benzophenone moiety by heteroaryl systems,

two alternative approaches were elaborated. The first

approach used the fluoro-substituted benzophenone 1

as an intermediate, from which the new heteroaryl

analogues were derived in one or two steps (Scheme 4).

In the second one, the heteroaryl systems were as-

sembled first and then modified according to the pro-

cedures described for compounds in Scheme 1 to yield

The first approach was used for the preparation of

the heteroaryl compounds 17-19. Hydroxy-substituted

benzophenone 8c (Scheme 4) was treated with vinyl-

magnesium chloride to yield the tertiary alcohol 16.

Cyclization and dehydration was achieved by heating

the compound 16 in a Dean-Stark apparatus in o-

xylene at 120 °C to give the 2H-chromene 17.³⁶Reaction

of the o-aminobenzophenone 8d in formamide and

formic acid at 165 °C gave the quinazoline 18.³⁷Treat-

3358 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Sch em e 5. Synthesis of Isoxazole OSC Inhibitors^a

Dehmlow et al.

a

Reagents and conditions: (a) (1) NH₂OH‚HCl, NaOAc, EtOH, 85 °C, (2) p-TosOH, EtOH, 85 °C; (b) (1) 1,6-dibromohexane, K₂CO₃,

acetone, 60 °C, (2) NHMe-allyl, K₂CO₃, acetone, room temp.

Sch em e 6. Synthesis of Benzo[d]isothiazole, Dioxobenzo[d]isothiazole, and Benzo[b]thiophene OSC Inhibitors^a

a

Reagents and conditions: (a) for R¹) F, (1) KOt-Bu, BnSH, THF, room temp, (2) SO₂Cl₂, CH₂Cl₂, room temp, (3) NH₃, EtOH, room

temp; (b) for R¹) SMe, (1) KOt-Bu, THF, reflux, (2) TFA, toluene, 0 °C; (c) (1) BBr₃, CH₂Cl₂, -78 °C to room temp, (2) 1,6-dibromohexane,

10% aqueous NaOH, n-Bu₄NBr, CH₂Cl₂, room temp; (d) KMnO₄on SiO₂, CH₂Cl₂, room temp; (e) NHMeAllyl, DMA, room temp.

ment of fluoro-substituted benzophenone 1 with meth-

ylhydrazine and potassium carbonate in DMA at 120

°C³⁸gave indazole 19.

Analogously to compound 24, the benzo[d]isothiazole 31

was derived from benzophenone 30.

To investigate the importance of the sulfur in the

heteroaryl system, the corresponding benzofuran 34 was

synthesized (Scheme 7). Alkylation of 3-methoxyphenol

with p-bromophenacylbromide gave compound 32, which

by heating in polyphosphoric acid at 80°C was converted

to benzofuran 33. Following the procedures already

discussed for Scheme 1, compound 33 was transformed

to benzofuran 34. To explore the influence of the

heteroaryl moiety further, dihydro- and isoquinolines

were prepared (Scheme 8). Following the Bischler-

Napieralski cyclization of the benzoylamide 35 gave the

dihydroisoquinoline 36,⁴⁰which was then converted to

the final OSC inhibitor 37. Oxidation of the intermedi-

ate 36 with activated manganese(IV) oxide in the

presence of anhydrous sodium sulfate gave isoquinoline

38, which was transformed into the final inhibitor 39

(Scheme 8).

For the preparation of benzo[d]isoxazole 21, the

second approach was chosen (Scheme 5). Dihydroxy-

benzophenone 5i was treated with hydroxylamine‚

hydrochloride and sodium acetate in ethanol at reflux,

followed by cyclization with p-toluenesulfonic acid to

give benzo[d]isoxazole 20. Transformation using the

standard conditions described in Scheme 1 gave the

final OSC inhibitor 21.

Benzo[d]isothiazole 24 was synthesized from the

fluorobenzophenone 4f by reaction with potassium ben-

zylthiolate followed by S-chlorination and cleavage of

the benzyl group with sulfuryl chloride to the sulfenyl

chloride, which was quenched with ammonia to give

benzo[d]isothiazole 22³⁹(Scheme 6). Methoxy deprotec-

tion and alkylation with an excess of 1,6-dibromohexane

gave the intermediate 23 that was converted to the final

amine 24 by amination with N-allylmethylamine. Fur-

thermore, the intermediate 23 could be oxidized with

potassium permanganate to give the bromohexyloxy

dioxobenzo[d]isothiazole 25, which was further con-

verted to the N-allylmethylamine 26.

Other six-membered electron-poor heteroaryl com-

pounds such as 1H-benzo[d][1,2]oxazines 43 were syn-

thesized also (Scheme 9). The ortho-methylated benzo-

phenone 4h was selectively monobrominated with 1.2

equiv of N-bromosuccinimide in the presence of a cata-

lytic amount of benzoyl peroxide and irradiation (λ

g 390 nM) in tetrachloromethane to give 40. Alkylation

with N-hydroxycarbamide acid tert-butyl ester in the

presence of sodium hydride in DMF gave 41, which was

Ortho thiomethylated derivative 4e was cyclized by

treatment with potassium tert-butylate in THF at reflux

to the benzo[b]thiophene 27 and further transformed

via bromide 28 to the final inhibitor 29 (Scheme 6).

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3359

Sch em e 7. Synthesis of Benzofuran OSC Inhibitors^a

a

Reagents and conditions: (a) polyphosphoric acid, 80 °C; (b) (1) BBr₃(1 M), CH₂Cl₂, 0°C to room temp, (2) 1,6-dibromohexane, K₂CO₃,

acetone, 60 °C, (3) NHMe-allyl, DMA, room temp.

Sch em e 8. Synthesis of Dihydroisoquinoline and Isoquinoline OSC Inhibitors^a

a

Reagents and conditions: (a) POCl₃, MeCN, 80 °C; (b) MnO₂, Na₂SO₄, toluene, 110 °C; (c) (1) 62% aqueous HBr, AcOH, 120 °C, (2)

1,6-dibromohexane, K₂CO₃, acetone, 60 °C, (3) NHMe-allyl, DMA, room temp.

Sch em e 9. Synthesis of 1H-Benzo[d][1,2]oxazine OSC Inhibitors^a

a

Reagents and conditions: (a) 1.2 equiv of NBS, catalytic (PhCOO)₂, hν, λ g 390 nM, CCl₄, room temp; (b) BocNHOH, DMF, 0 °C to

room temp; (c) 62% aqueous HBr, AcOH, 120 °C; (d) (1) 1,6-dibromohexane, K₂CO₃, acetone, 60 °C, (2) NHMe-allyl, K₂CO₃, acetone, room

temp.

deprotected and cyclized with hydrogen bromide to give

1H-benzo[d][1,2]oxazine 42. Standard reaction with 1,6-

dibromohexane and N-allylmethylamine gave 43.

to testing efficacy in reducing cholesterol production

while increasing MOS levels. The measurement of the

substance metabolic stability was achieved by compar-

ing the residual inhibitory activity after a preincubation

of 8 or 24 h with rat hepatocytes with the activity

following co-incubation of only 2 h; hence, reduction of

cholesterol production was used there as a surrogate

marker for the stability of the parent compound and/or

the conversion to active metabolites. From each class

of compounds, the most active and stable inhibitors were

tested in hamsters for 10 days for plasma total choles-

terol (TC) and LDL cholesterol lowering.

Biologica l Resu lts a n d Discu ssion

The strategy to optimize the hOSC inhibitors was as

follows. First, we determined the percent inhibition of

hOSC in the presence of 100 nM concentration of the

compound; compounds exhibiting >50% inhibition at

100 nM were tested for IC₅₀and in a stability assay in

rat hepatocytes. This stability assay was performed to

determine the activity in the cells as well as the

duration of action of the compound and its metabolites.

The stability test was performed as a bioassay to select

compounds retaining sufficient activity over time prior

Compounds inhibiting OSC from Candida albicans

were tested on human OSC. Although there was no

correlation between the IC₅₀for human OSC and that

3360 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Dehmlow et al.

Ta ble 1. Influence of the Modification of the Substituent R¹on

the Inhibitory Activity of the Compounds against hOSC and on

the Stability of the Compounds in Rat Hepatocytes and

Correlation between σ_pand IC₅₀of hOSC

Ta ble 3. Influence of the Modification of the Amino Groups on

the Inhibitory Activity of the Compounds against hOSC and on

the Stability of the Compounds in Rat Hepatocytes

residual inhibitory

activity in

inhib

rat hepatocytes^b

hOSC^a

c

compd

7a ‚HCl

R¹IC₅₀[nM] 2 h [%] 8 h [%] 24 h [%] σ_p

NO₂

1.9

5.4

6.7

95

92

76

87

91

93

75

54

41

78

23

3

0.81

0.26

0.15

0

7b‚fumarate Br

7c‚HCl

7d ‚HBr

F

H

22.5

a

All potency measurements were performed in triplicate. The

b

SEM ranged between 10% and 20%. All stability assays were

performed at 3 µM in duplicate. The SEM did not exceed 4% of

the mean value when the mean value was >50% and did not

exceed 15% when the mean value was <50%. ^cHansch et al.⁴¹

Ta ble 2. Influence of the Modification of the Substitution

Pattern at the Left Aromatic Ring of the Benzophenone on the

Inhibitory Activity of the Compounds against hOSC and on the

Stability of the Compounds in Rat Hepatocytes

a

All potency measurements were performed in triplicate. The

b

SEM ranged between 10% and 20%. All stability assays were

performed at 3 µM in duplicate. The SEM did not exceed 4% of

the mean value when the mean value was >50% and did not

exceed 15% when the mean value was <50%. ^cMeasured at 22

°C in 0.1 M KNO₃containing 43 vol % methanol, following

d

literature procedure.⁵³% inhibition of hOSC at 100 nM.

residual inhibitory

benzophenone system using various substituted benzoyl

chlorides as starting material for the Friedel-Crafts

acylation. With these modifications, it was possible to

polarize the aromatic ring of the inhibitor concomitantly

and thereby to change the properties of the interaction

with the amino acid residues in this region of the

enzyme. The changes observed can now be explained

by the interactions seen in the crystal structure of 1 (Ro

48-8071) in SHC.³³In SHC, the benzophenone extends

into the putative substrate uptake channel and the

bromophenyl is stabilized by two phenylalanines by

edge-to-face interactions. A similar interaction probably

occurs in hOSC, hence the higher degree of inhibition

with compounds bearing electron-deficient aryl groups.

It appeared that electron-withdrawing substituents

such as nitro-7a ‚HCl (IC₅₀) 1.9 nM), bromo-7b ‚

fumarate (IC₅₀) 5.4 nM), and fluoro groups of 7c‚HCl

(IC₅₀) 6.7 nM) in the para position increased potency

compared to the unsubstituted 7d ‚HBr (IC₅₀) 22.5

nM). This could also be seen by comparing the corre-

sponding σ values.⁴¹The modification of the substitution

pattern at the aromatic ring also influenced the stability

of these inhibitors in rat hepatocytes. So the residual

inhibitory activity of the substance after a preincubation

of 24 h with rat hepatocytes increased from 3% for 7d ‚

HBr to 78% for 7b‚fumarate. Subsequently, the most

stable bromo-substituted 7b‚fumarate was taken for

further optimization.

activity in

inhib

rat hepatocytes^b

hOSC^a

compd

R¹

X

IC₅₀[nM] 2 h [%] 8 h [%] 24 h [%]

8a ‚fumarate NHCH₃

8b‚fumarate OCH₃

C

N

4.1

4.6

5.4

6.2

6.3

6.5

8.7

98

89

92

98

95

98

95

98

90

93

97

95

96

90

99

81

78

95

91

80

38

7b‚fumarate

H

7e‚fumarate SCH₃

8c‚fumarate OH

1‚fumarate

F

H

15‚fumarate

a

All potency measurements were performed in triplicate. The

SEM ranged between 10% and 20%. All stability assays were

performed at 3 µM in duplicate. The SEM did not exceed 4% of

the mean value when the mean value was >50% and did not

exceed 15% when the mean value was <50%.

b

for the fungal OSC, several of these compounds had

motifs that were essential to the inhibition of both

enzymes.^23,24These are an amine function and an

electrophilic carbonyl C atom embedded in a benzophe-

none system that are separated by about 10.7 Å. The

best spacers between these two residues were either

rigid benzyl or less rigid trans-butenyl or flexible alkyl

chains. Hypothetically, the amine of the aminohexyl-

oxybenzophenone inhibitors would interact with the

catalytic aspartate of the enzyme in the epoxide-opening

region. The carbonyl of the benzophenone system would

then interact with the region of the OSC stabilizing the

last protosterol cation. Because of chemical feasibility,

we initially focused our synthetic program on alkyl

spacers. In this report, only compounds with a hexyl

spacer are discussed.

In a second step, the influence of the substitution

pattern at the left aromatic ring of the benzophenone

on the inhibitory activity of the compounds was inves-

tigated (Table 2). The electron density in this aryl part

of the inhibitor was modified in order to improve the

interaction with the phenylalanine-rich OSC channel.

Introduction of a fluoro substituent in the ortho position

of 7b to give 1 did not increase the potency or the

First, we investigated the influence of the substitution

pattern at the right aromatic ring of the benzophenone

(see description in Scheme 1) on the potency of the

compounds against hOSC, (Table 1). Carbonyl-activat-

ing or deactivating groups were introduced in the

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3361

Ta ble 4. Influence of the Modification of Heteroaryl Systems

on the Inhibitory Activity of the Compounds against hOSC and

on the Stability of the Compounds in Rat Hepatocytes

the methoxy-8b‚fumarate, and the hydroxy derivatives

8c‚fumarate were potent inhibitors, all as active as the

nonsubstituted 7b‚fumarate. This might be explained

by an improved interaction of the polarized benzophe-

none system with the amino acids of the cavity and

enough space in this region of the enzyme to accom-

modate the sterically demanding residues. The same

was true for the electron-deficient pyridine compound

15‚fumarate (IC₅₀) 8.7 nM). All compounds with

modified ortho substitution did not show a major

improvement in terms of potency but led to, with the

exception of fluoro substitution (1‚fumarate) and meth-

oxy substitution (8b‚fumarate), more stable inhibitors

as determined in the hepatocyte stability assay.

To evaluate the effect of basicity and polarity of the

amine part on the activity of the inhibitors, different

substituted amines were synthesized (Table 3). Tertiary

amines were much more potent than the secondary

amine 12‚HCl, which has a pK_a> 10. Good activities

were obtained with hydroxypropylmethylamine 10b‚

fumarate (pK_a) 9.05), though not as good as the

N-allylmethylamine 7b‚fumarate (pK_a) 8.18). In ad-

dition, the hydroxyalkyl group of 10b‚fumarate in-

creased the lability of the compounds in rat hepatocytes.

Cyclopropylmethyl methylamine 13‚fumarate (pK_a

)

9.37) with a slightly higher pK_aand cyclopropylmeth-

ylamine 10a ‚HCl (pK_a) 7.48) with a lower pK_awere

not as active as the original compound 7b‚fumarate.

This indicates that for compounds from this benzophe-

none series an optimal pK_avalue would be around 8.2.

This is similar to the series described by Brown et al.,

in which inhibitors with a range of different pK_avalues

were tolerated by the enzyme, but for the optimal

inhibition, only a small range of pK_aseemed accept-

able.²⁷The stability of the two cyclopropyl derivatives

10a ‚HCl and 13‚fumarate was slightly lower compared

to the one observed for 7b‚fumarate.

The wide variety of groups that were tolerated in the

ortho position of the benzophenone led us to explore the

potential of annelated heterocyclic systems as hOSC

inhibitors that are devoid of the potentially metabolic

labile carbonyl moiety (Table 4).^42-44The best compound

of this series was the benzo[d]isothiazole 24‚fumarate,

which had an IC₅₀of 2.9 nM. It was more potent and

also more stable in rat hepatocytes than the correspond-

ing benzophenone derivatives 7b‚fumarate. The benzo-

[b]thiophene 29‚fumarate and the dioxobenzo[d]isothi-

azole 26‚fumarate were slightly less potent but as stable

as the benzo[d]isothiazole 24‚fumarate. A completely

different system, the arylisoquinoline 39‚HCl, was also

a stable inhibitor with an IC₅₀of 3.5 nM. The same was

true for dihydroisoquinoline 37 (IC₅₀) 7.9 nM). The

diaza analogue, namely, the quinazoline 18‚fumarate,

exhibited lower inhibitory activity with an IC₅₀of 12.3

nM. 2H-Chromene 17 with an IC₅₀of 11 nM had an

intermediate activity. The potency of 1H-benzo[d][1,2]-

oxazine 43‚fumarate (IC₅₀) 4.1 nM) was again com-

parable to the one of arylisoquinoline 39‚HCl. The

N-methylated indazole 19‚fumarate (IC₅₀) 20 nM) was

less potent but still better than the corresponding benzo-

[d]isoxazole 21‚fumarate (21% inhibition at 100 nM) and

the benzofuran 34‚fumarate (21% inhibition at 100 nM).

a

All potency measurements were performed in triplicate. The

b

SEM ranged between 10% and 20%. All stability assays were

performed at 3 µM in duplicate. The SEM did not exceed 4% of

the mean value when the mean value was >50% and did not

exceed 15% when the mean value was <50%. ^c% inhibition of

hOSC at 100 nM.

stability. Compound 1 was an interesting synthetic

intermediate for the preparation of derivatives bearing

substituents such as amines, oxygens, and sulfurs. It

was used later for the synthesis of new heteroaromatic

systems. Surprisingly, the corresponding N-methyl-

amine-8a ‚fumarate, the methansulfanyl-7e‚fumarate,

The way of annelation was essential for optimal

inhibition of hOSC. Thus, the compounds with the

3362 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Dehmlow et al.

Ta ble 5. In Vitro Data and Total Plasma Cholesterol (TC) and

LDL Cholesterol (LDL-C) Levels in Fat-Fed Hamsters (n ) 5

per Group) Treated for 10 Days with OSC Inhibitors at 200

µmol/(kg‚day)^a

had a similar efficacy profile in hamsters. The 2-fluoro

analogue 1‚fumarate with a comparable activity and

stability profile reduced TC by 27% and LDL-C by 41%.

The heteroaryl compounds were very potent and also

very stable in vitro. Three of these compounds, the

benzo[d]isothiazole 24‚fumarate, the arylisoquinoline

39‚HCl, and the 1H-benzo[d][1,2]oxazine 43‚fumarate

were tested in hyperlipidemic hamsters. The benzo[d]-

isothiazole 24‚fumarate was quite efficacious in vivo,

showing a lowering of TC of 33% and LDL-C of 36%.

The modest efficacy of arylisoquinoline 39‚HCl (14%

decrease of TC) and 1H-benzo[d][1,2]oxazine 43‚fuma-

rate (3% decrease of TC) could not be explained, but

might be due to a lower intestinal absorption.

residual inhibitory

activity in

rat hepatocytes^c

inhib

hOSC^b

2 h

IC₅₀[nM] [%]

8 h

[%]

24 h

[%]

LDL-C

[%]

TC

[%]

compd

24‚fumarate

1‚fumarate

7b‚fumarate

7c‚HCl

2.9

6.5

5.4

6.7

3.5

22.5

4.1

d

99

98

92

76

97

87

84

d

97

96

93

75

98

54

90

d

98

80

78

23

96

3

-36 ( 14 -33 ( 4

-41 ( 4 -27 ( 6

-27 ( 10 -25 ( 8

-40 ( 4 -24 ( 2

39‚HCl

ND^e

-14 ( 7

-12 ( 5

-3 ( 4

7d ‚HBr

43‚fumarate

simvastatin

94

d

-41 ( 9 -24 ( 4

Con clu sion

a

TC and LDL-C lowering is expressed as a percent ( SEM of

day 0 (before compound administration) and corrected with respect

to the variation of the untreated animals (n ) 10). Simvastatin

was used as a reference lipid-lowering agent at 20 µmol/(kg‚day).⁸

^bAll potency measurements were performed in triplicate. The SEM

ranged between 10% and 20%. ^cAll stability assays were per-

formed at 3 µM in duplicate. The SEM did not exceed 4% of the

mean value when the mean value was >50% and did not exceed

Modification of the substitution pattern of the ben-

zophenone at either of the aromatic rings led to the

identification of the potent and stable hOSC inhibitors

7a ‚HCl and 1‚fumarate. The most potent hOSC inhibi-

tors in these series have tertiary amines, a pK_aof

around 8.2, and small residues at the amine. On the

basis of the X-ray structure of the inhibitors in SHC,

the keto moiety of the benzophenone was not involved

in hydrogen bonding. It is a potentially metabolic labile

position, and in addition, a large variety of substituents

in the ortho position of the benzophenones were toler-

ated for potent hOSC inhibition. These observations led

us to explore annelated heteroaryl systems in more

detail. The most active compounds in vitro were benzo-

[d]isothiazole 24‚fumarate, arylisoquinoline 39‚HCl,

1H-benzo[d][1,2]oxazine 43‚fumarate, and benzo[b]-

thiophene 29‚fumarate. For the benzophenones and the

benzo[d]isothiazole 24‚fumarate, the cholesterol-lower-

ing effect observed in hyperlipidemic hamsters reflects

the in vitro potency against hOSC and the stability

determined in rat hepatocytes. Thus, the nonsubstituted

benzophenone 7d ‚HBr lowered TC by only 12%. Better

efficacy in TC lowering could be achieved with the

appropriate substitution pattern at the benzophenone

system, so 1‚fumarate showed a reduction of TC of

27% and LDL-C of 41%. Further improvement of sta-

bility of the compounds on rat hepatocytes led to the

best heteroaryl derivative, the benzo[d]isothiazole

24‚fumarate, which lowered TC by 33% and LDL-C

by 36%.

d

15% when the mean value was <50%. Not applicable. ^eND: not

determined.

central heteroaryl moiety bearing a phenyl substituent

were more potent than the compounds with a central

phenyl group bearing a heteroaryl substituent. This

is exemplified by the derivatives 24‚fumarate and

31‚fumarate. This can be explained by spatial restric-

tions of the cavity of hOSC and is in contrast to

observations made for HSC, which are discussed in ref

34 In general, the six-membered rings tested were more

potent than the five-membered ones with the exception

of the thio-heteroaryl derivatives. This can be explained

by the similar size of the corresponding thio-heteroaryl

system and the six-membered rings, which is due to the

larger van der Waals radius of sulfur compared to the

radii of oxygen, nitrogen, and carbon. The heteroaryl

compounds with two nitrogens were less potent, which

might be attributed to a less optimal electron distribu-

tion in these systems.

There was a good correlation between IC₅₀for human

and hamster OSC (not shown), allowing the testing of

the efficacy on lipid lowering in vivo. Selected inhibitors

were tested in hyperlipidemic hamsters⁸at a dose of

200 µmol/(kg‚day), corresponding to about 70-90

mg/(kg‚day) (Table 5). The compounds were given as

food admixture for 10 days. Plasma TC and LDL-C

levels were measured and compared to the control

group. The inhibitors were well tolerated; no effect on

food consumption was seen.²⁹In the series of benzophe-

nones, the plasma cholesterol lowering in vivo reflected

the determined in vitro profile. The nonsubstituted

benzophenone 7d ‚HBr, with an IC₅₀of 22.5 nM and low

stability in rat hepatocytes (54% and 3% residual

inhibitory activity after 8 and 24 h), lowered TC by only

12%. Para fluoro substitution on the right aromatic ring

(7c‚HCl) increased the potency in vitro (IC₅₀) 6.7 nM)

and the residual inhibitory activity in rat hepatocytes

after 8 and 24 h (75% and 23%). This resulted in a more

efficacious compound in vivo that decreased TC by 24%

and LDL-C by 40%. The bromo-substituted 7b‚fumarate

that had a similar potency but better stability (93% and

78% residual inhibitory activity after 8 and 24 h) in vitro

Exp er im en ta l Section

Gen er a l Meth od s. Reactions were carried out under an

atmosphere of argon. Unless otherwise noted, NH₄Cl,

NaHCO₃, and Na₂CO₃solutions were saturated aqueous

1

solutions. All compounds were characterized by 250 MHz H

NMR, IR, MS, and microanalyses. Melting points (uncorrected)

were determined using a Bu¨chi 510 apparatus and are

uncorrected. Proton NMR spectra were recorded on a Bruker

AC250 spectrometer, and δ values are given in ppm relative

to tetramethylsilane. In the ¹H NMR spectra, the acidic

protons of the fumarate, HCl, and HBr ammonium salts were

not seen unless listed. IR spectra of KBr pellets were recorded

using a Nicolet 7199 FT-IR spectrometer. ATR (attenuated

total reflection) spectra were recorded with an FT-IR spec-

trometer equipped with an IR microscope and a ZnSe ATR

objective. The spectrometer is a Nicolet Magna 550/860 or

equivalent (resolution of 2 cm^-1, 200 or 500 coadded scans,

MCT detector). Mass spectra were obtained using the pneu-

matically assisted electrospray technique (Perkin-Elmer Sciex,

type API-III). Results of elemental analyses were within 0.4%

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3363

of theoretical values. Unless indicated otherwise, commercially

available chemicals and solvents were used without further

purification. Silica gel 60 (0.04-0.063 mm; Merck) was used

for flash chromatography.

Gen er a l P r oced u r e for th e F or m a tion of F u m a r a tes.

A solution of 1 equiv of the final amine‚hydrobromide in

methylene chloride was extracted with a NaHCO₃solution,

dried (Na₂SO₄), and concentrated. The residue was dissolved

in ethanol with 1.04 equiv of fumaric acid and crystallized or

precipitated with ether to give the amine‚fumarate (1:1).

Following the general procedure for the formation of fuma-

rates, 7b‚HBr (1:1) was converted to 7b‚fumarate (1:1) in 58%

yield as colorless crystals: mp 85-86 °C; IR (ATR microscope)

1714, 1638, 1601, 1581 cm^-1; ¹H NMR (DMSO-d₆) δ 1.25-1.60

(m, 4H), 1.70-1.80 (m, 4H), 2.23 (s, 3H), 2.40-2.55 (m, 2H),

3.09 (d, J ) 6.5, 2H), 4.09 (t, J ) 6.3 Hz, 2H), 5.45-5.62 (m,

2H), 5.82-6.05 (m, 1H), 6.57 (s, 2H), 7.09 and 7.74 (AA′-BB′-

system, J _AB) 8.8 Hz, 4H), 7.62 and 7.77 (AA′-BB′-system, J _AB

) 8.4 Hz, 4H); HRMS calcd for C₂₃H₂₈NO₂Br (M + H⁺, 1Br)

430.1381, found 430.1385.

Analogously to the general procedure A described above, the

following compounds were synthesized. [4-[6-(Allylm eth -

yla m in o)h exyloxy]p h en yl](4-n it r op h en yl)m et h a n on e‚

Hyd r och lor id e (1:1) (7a ‚HCl). 7a ‚HCl was obtained from

4-nitrobenzoyl chloride (IIa ) and anisole (Id ) in 33% yield: mp

79 °C; ¹H NMR (DMSO-d₆) δ 1.28-1.56 (m, 4H), 1.65-1.86

(m, 4H), 2.65 (s, 3H), 2.85-3.15 (m, 2H), 3.58-3.82 (m, 2H),

4.10 (t, J ) 6.4 Hz, 2H), 5.42-5.60 (m, 2H), 5.92-6.15 (m, 1H),

7.11 and 7.77 (AA′BB′, J ) 8.8 Hz, 4H), 7.91 and 7.37 (AA′BB′,

J ) 8.8 Hz, 4H), 10.98 (br s, 1H); MS m/z 396 (M⁺). Anal.

(C₂₃H₂₈N₂O₄‚HCl) H, N, Cl. C: calcd, 63.81; found, 64.22.

[4-[6-(Allylm et h yla m in o)h exyloxy]p h en yl]-(4-flu or o-

p h en yl)m eth a n on e‚HCl (1:1) (7c‚HCl). 7c‚HCl was ob-

tained from (4-fluorophenyl)(4-hydroxyphenyl)methanone

(5c) in 47% yield: mp 84 °C; ¹H NMR (DMSO-d₆) δ 1.26-1.56

(m, 4H), 1.65-1.86 (m, 4H), 2.65 (d, J ) 4.6 Hz, 3H), 2.85-

3.12 (m, 2H), 3.58-3.84 (m, 2H), 4.09 (t, J ) 6.4 Hz, 2H), 5.42-

5.60 (m, 2H), 5.92-6.15 (m, 1H), 7.09 (d, J ) 8.9 Hz, 2H), 7.37

(d, J ) 8.9 Hz, 2H), 7.41 (d, J ) 8.9 Hz, 2H), 7.65-7.85 (m,

4H), 11.10 (br s, 1H); MS m/z 369 (M⁺). Anal. (C₂₃H₂₈NO₂F‚

HCl) H, N, Cl, F. C: calcd, 68.05; found, 67.33.

Gen er a l P r oced u r e for th e F or m a tion of Hyd r och lo-

r id es. A solution of 1 equiv of the final amine‚hydrobromide

in methylene chloride was extracted with a NaHCO₃solution,

dried (Na₂SO₄), and concentrated The residue was dissolved

in methylene chloride, treated with 1 equiv of a 4.8 M HCl

solution in ether, and crystallized to give the amine‚hydro-

chloride (1:1).

Gen er a l P r oced u r e A. [4-[6-(Allylm eth yla m in o)h ex-

yloxy]p h e n yl](4-b r om op h e n yl)m e t h a n on e ‚F u m a r a t e

(1:1) (7b‚F u m a r a te). Nitrobenzene (225 mL) was cooled in

an ice bath and treated portionwise with aluminum chloride

(72.5 g, 544.5 mmol) and 4-bromobenzoyl chloride (IIb) (109.5

g, 500 mmol) in nitrobenzene (100 mL) at a maximum of 8 °C.

The mixture was stirred for 10 min, whereupon anisole (Id )

(52.0 mL, 476.5 mmol) was added in such a manner that the

temperature did not exceed 6 °C. The solution was left to warm

to room temperature overnight, then poured onto ice-water

and extracted with methylene chloride. The organic phase was

washed with water and 10% NaCl solution, dried (Na₂SO₄),

and concentrated. After crystallization from cyclohexane, (4-

bromophenyl)(4-methoxyphenyl)methanone (4b) (128 g, 88%)

was obtained: ¹H NMR (CDCl₃) δ 3.89 (s, 3H), 6.96 and 7.79

(AA′-BB′-system, J _AB) 8.8 Hz, 4H), 7.62 (br s, 4H).

A solution of 4b (128 g, 440 mmol) in acetic acid (870 mL)

and 62% aqueous HBr solution (500 mL) was refluxed for 5 h,

subsequently evaporated, reevaporated with toluene, taken up

in ethyl acetate, washed with a NaHCO₃solution and 10%

NaCl solution, dried (MgSO₄), and evaporated. After crystal-

lization from toluene, (4-bromophenyl)(4-hydroxyphenyl)-

methanone (5b) (108.7 g, 89%) was obtained: ¹H NMR

(DMSO-d₆) δ 6.90 and 7.66 (AA′-BB′-system, J _AB) 8.7 Hz, 4H),

7.61 and 7.75 (AA′-BB′-system, J _AB) 8.5 Hz, 4H), 10.52 (br s,

1H).

A 10% NaOH solution (80 mL) was added to 1,6-dibromo-

hexane (13.7 mL, 90 mmol), 5b (8.3 g, 30 mmol), and tetra-

n-butylammonium bromide (1.8 g, 6 mmol) in methylene

chloride (80 mL). The mixture was stirred at room temperature

for 36 h, tetra-n-butylammonium bromide (1.8 g, 6 mmol) was

added twice. Then the two phases were poured into water and

extracted with ethyl acetate. The organic phase was washed

with 10% NaCl solution, dried (MgSO₄), filtered, and evapo-

rated. The residue was suspended in n-hexane, filtered,

dissolved in methylene chloride/methanol, and treated with

40 g of Amberlite MB3 for 15 min. After filtration and

evaporation of the solvents, [4-[6-bromohexyloxy]phenyl](4-

bromophenyl)methanone (6b) (9.3 g, 71%) was obtained: ¹H

NMR (CDCl₃) δ 1.46-1.62 (m, 4H), 1.79-1.97 (m, 4H), 3.44

(t, J ) 6.7 Hz, 2H), 4.05 (t, J ) 6.3 Hz, 2H), 6.95 and 7.78

(AA′-BB′-system, J _AB) 8.9 Hz, 4H), 7.62 (br s, 4H).

[4-[6-(Ally lm e t h y la m in o )h e x y lo x y ]p h e n y l]p h e n -

ylm eth a n on e‚Hyd r obr om id e (1:1) (7d ‚HBr ). 7d ‚HBr was

obtained from (4-hydroxyphenyl)phenylmethanone (5d ) in

29% yield: mp 89-91 °C; IR 1645, 1602 cm^-^{1 1}H NMR

;

(DMSO-d₆) δ 1.26-1.56 (m, 4H), 1.65-1.86 (m, 4H), 2.72 (s,

3H), 2.92-3.15 (m, 2H), 3.65-3.90 (m, 2H), 4.09 (t, J ) 6.3

Hz, 2H), 5.48-5.62 (m, 2H), 5.86-6.15 (m, 1H), 7.09 (d, J )

8.9 Hz, 2H), 7.51-7.61 (m, 2H), 7.63-7.82 (m, 4H), 9.85 (br s,

1H); MS m/z 351 (M⁺). Anal.(C₂₃H₂₉NO₂‚HBr) C, H, N, Br.

Gen er a l P r oced u r e B. [4-[6-(Allylm eth yla m in o)h ex-

y lo x y ]-2-m e t h y ls u lfa n y lp h e n y l](4-b r o m o p h e n y l)-

m eth a n on e‚F u m a r a te (1:1) (7e‚F u m a r a te). NaSMe (95%,

1.45 g, 19.6 mmol) was suspended in THF (80 mL) and treated

over a period of 1.5 h with (4-bromophenyl)(2-fluoro-4-meth-

oxyphenyl)methanone (4f) (5.51 g, 17.82 mmol)⁸in THF (100

mL). The solution was stirred at room temperature, again

treated with NaSMe (264 mg, 3.56 mmol), stirred, and treated

after an interval of 10 min with a NH₄Cl solution (50 mL) and

a NaHCO₃solution (100 mL). The aqueous phase was ex-

tracted with methylene chloride, and the organic phases were

washed with a NaHCO₃solution as well as brine and dried

(Na₂SO₄). After evaporation, the crude product was purified

by column chromatography (silica gel, ethyl acetate/n-hexane

1:2). (4-Bromophenyl)(4-methoxy-2-methylsulfanylphenyl)-

methanone (4e) (5.88 g, 89%) was obtained as a yellow oil: ¹H

NMR (CDCl₃) δ 2.44 (s, 3H), 3.89 (s, 3H), 6.64 (dd, 1H), 6.89

(d, 1H), 7.43 (dd, 1H), 7.59 (m, 4H).

Compound 4e was deprotected in a manner similar to 4b

to give 96% (4-bromophenyl)(4-hydroxy-2-methylsulfanyl-

phenyl)methanone (5e) as brown crystals: ¹H NMR (DMSO-

d₆) δ 2.37 (s, 3H), 6.55-6.66 (m, 1H), 6.83 (d, 1H), 7.33 (d, J

) 8.5 Hz, 1H), 7.54 (d, J ) 8.5 Hz, 1H), 7.72 (d, J ) 8.5 Hz,

1H), 10.42 (s, 1H).

5e (450 mg, 1.39 mmol) was taken up in acetone (7 mL)

and treated with K₂CO₃(1.24 g, 8.98 mmol) and 1,6-dibromo-

hexane (0.53 mL, 3.48 mmol). The suspension was heated

under reflux overnight, cooled, filtered, and concentrated. After

removal of the excess 1,6-dibromohexane under reduced pres-

sure, [4-[6-(bromo)hexyloxy]-2-methylsulfanylphenyl](4-bro-

mophenyl)methanone (6e) (710 mg, quantitative) was obtained

as a brown oil: ¹H NMR (CDCl₃) δ 1.42-1.60 (m, 6H), 1.74-

1.96 (m, 2H), 2.43 (s, 3H), 3.43 (t, 2H), 4.04 (t, 3H), 6.62 (dd,

1H), 6.86 (d, 1H), 7.40 (d, 1H), 7.58 (m, 4H).

6b (6.2 g, 14.1 mmol) was taken up in DMA (47 mL) and

stirred at room temperature for 3 days, adding every day

N-allylmethylamine (2.7 mL, 28.2 mmol) and following the

reaction by TLC (Silicagel, CH₂Cl₂/MeOH 98:2, R_f(starting

material) ) 0.73, R_f(product) ) 0.27, UV). The solution was

concentrated, and the residual oil was taken up in water and

lyophilized. Crystallization from methylene chloride/ethyl

acetate gave 7b‚HBr (1:1) (4.3 g, 60%) as colorless crystals:

mp 117-119 °C; IR (KBr) 1640, 1602 cm^-1; ¹H NMR (DMSO-

d₆) δ 1.30-1.55 (m, 4H), 1.60-1.85 (m, 4H), 2.71 (s, 3H), 2.92-

3.15 (m, 2H), 3.62-3.85 (m, 2H), 4.09 (t, J ) 6.3 Hz, 2H), 5.45-

5.62 (m, 2H), 5.82-6.05 (m, 1H), 7.09 and 7.74 (AA′-BB′-

system, J _AB) 8.8 Hz, 4H), 7.62 and 7.77 (AA′-BB′-system, J _AB

) 8.4 Hz, 4H); MS m/z 429 (M⁺, 1Br). Anal. (C₂₃H₂₈NO₂Br‚

HBr) C, H, N, Br.

3364 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Dehmlow et al.

Following the general procedure A described for 6b, 6e and

N-allylmethylamine gave 7e‚HBr, which was converted with

fumaric acid to 7e‚fumarate and isolated after precipitation

from EtOH with Et₂O as a viscous oil in a total yield of 84%:

stirred at room temperature for 45 h and evaporated, and the

residue was converted into the free base with methylene

chloride/NaHCO₃solution. After purification over silica gel

with methylene chloride/methanol (9:1), 8d (13.23 g, 96%) was

obtained.

IR (KBr) 2484, 1703, 1587, 1391, 1276, 845 cm^-^{1 1}H NMR

;

(DMSO-d₆) δ 1.22-1.56 (m, 6H), 1.76 (m, 2H), 2.24 (s, 3H),

2.42 (s, 3H), 3.11 (d, J ) 6.5 Hz, 2H), 4.09 (t, J ) 7 Hz, 2H),

5.19 (d, J ) 9.9 Hz), 5.24 (d, J ) 15.4 Hz), 5.85 (m, 1H), 6.58

(s, 1.6H), 6.81 (dd, J ) 8.6 Hz, 1H), 6.92 (d, 1H), 7.40 (d, J )

8.6 Hz, 1H), 7.55 (d, J ) 8.5 Hz, 2H), 7.74 (d, J ) 8.5 Hz, 1H),

13.0 (s, br, 2H); MS m/ z 476 (M + H⁺, 1Br). Anal. free base

(C₂₄H₃₀NO₂BrS‚0.8C₄H₄O₄) C, H, N.

8d (9.1 g, 20.43 mmol) was dissolved in methylene chloride/

ether and stirred with fumaric acid (2.25 g, 19.38 mmol)

overnight to give after filtration 8d ‚fumarate (7.28 g, 64%):

mp 78 °C (with decomposition); IR (KBr) 1698, 1616, 1585

cm^-1; ¹H NMR (DMSO-d₆) δ 1.25-1.55 (m, 4H), 1.65-1.82 (m,

4H), 2.24 (s, 3H), 2.35-2.50 (m, 2H), 3.12 (d, J ) 6.0 Hz, 2H),

4.05 (t, J ) 6.0 Hz, 2H), 5.14-5.32 (m, 2H), 5.78-5.94 (m, 1H),

6.57 (s, 2H), 6.49-6.65 (m, 2H), 7.40 (d, J ) 8.4 Hz), 7.59 and

7.75 (AA′-BB′-system, J _AB) 8.5, 4H); MS m/z 445 (M + H⁺,

1Br). Anal. (C₂₃H₂₈NO₃Br‚1.1C₄H₄O₄) C, H, N, Br.

[6-[6-(Cyclop r op ylm et h yla m in o)h exyloxy]p h en yl](4-

br om op h en yl)m eth a n on e‚HCl (10a ‚HCl). A suspension of

6b (1 g, 2.27 mmol), N-cyclopropyl-2,2,2-trifluoroacetamide (0.7

g, 4.54 mmol)⁴⁵in acetonitrile (50 mL) was heated at reflux

for 19 h. Additional N-cyclopropyl-2,2,2-trifluoroacetamide

(0.7 g, 4.54 mmol), K₂CO₃(0.63 g, 4.54 mmol), and benzyltri-

ethylammonium bromide (52 mg, 0.23 mmol) were added, and

the mixture was heated for a further 23 h. After cooling to

room temperature, the suspension was filtered. The filtrate

was concentrated, and the residue was dissolved in a mix-

ture of methylene chloride (45 mL) and methanol (5 mL) and

the mixture was stirred with Amberlite MB3 (4.5 g) for 15

min, subsequently filtered and washed with methylene chlo-

ride. The organic phase was washed with water, dried

(Na₂SO₄), concentrated, and purified by chromatography on

silica gel (n-hexane/ethyl acetate 8:2) to afford N-{6-[4-(4-

bromobenzoyl)phenoxy]hexyl}-N-cyclopropyl-2,2,2-trifluoro-

acetamide (9a ) (0.56 g, 48%) as a yellowish solid: MS m/z 512

(M + H⁺, 1Br).

A solution of 9a (0.78 g, 1.52 mmol) in methanol (30 mL)

and THF (10 mL) was treated with 20% aqueous potassium

hydroxide solution (2.13 mL) while cooling with ice. The

mixture was brought to room temperature and stirred for 2

days and concentrated under reduced pressure. The residue

was treated with water/methylene chloride (3×). The organic

extracts were washed with brine, dried (Na₂SO₄), and evapo-

rated to give (4-bromophenyl)-[4-(6-cyclopropylaminohexyloxy)-

phenyl]methanone (0.32 g, 50%): ¹H NMR (CDCl₃) δ 0.29-

0.37 (m, 2H), 0.39-0.50 (m, 2H), 1.33-1.61 (m, 7H), 1.76-

1.91 (m, 2H), 2.08-2.17 (m, 1H), 2.70 (t, J ) 7.2 Hz, 2H), 4.04

(t, J ) 6.5 Hz, 2H), 6.95 (d, J ) 8.8 Hz, 2H), 7.62 (s, br, 4H),

7.78 (d, J ) 8.8 Hz, 2H); MS m/z 415 (M⁺, 1Br).

This product (0.25 g, 0.6 mmol) was taken up in dioxane (5

mL), treated with 1 N NaH₂PO₃(5 mL) solution and a 37%

aqueous solution of formaldehyde (5 mL). The mixture was

heated to 65 °C for 5 h. The mixture was adjusted to pH >11

with 10% aqueous NaOH and extracted with ether. Evapora-

tion and chromatography of the residue on silica gel with ethyl

acetate/n-hexane/triethylamine (40:60:1) gave 10a (0.22 g,

85%), which was converted following the general procedure

for the formation of hydrochlorides into the hydrochloride 10a ‚

HCl: ¹H NMR (DMSO-d₆) δ 0.82 (m, 2H), 1.01, 1.08 (2m, 2H),

1.43 (m, 4H), 1.77 (m, 4H), 2.79 (m, 4H), 3.13 (m, 2H), 4.10 (d,

J ) 6.3 Hz, 2H), 7.09 (d, J ) 8.8 Hz, 2H), 7.62 (d, J ) 8.4 Hz,

2H), 7.74 (d, J ) 8.8 Hz, 2H), 7.77 (d, J ) 8.4 Hz, 2H), 10.22

(br s, HCl); MS m/z 430 (M + H⁺, 1Br). Anal. (C₂₃H₂₈BrNO₂‚

HCl) C, H, N, Br, Cl.

[4-[6-(Allylm e t h yla m in o)h e xyloxy]-2-m e t h yla m in o-

p h en yl](4-br om op h en yl)m eth a n on e‚F u m a r a te (1:1) (8a ‚

F u m a r a te). A solution of 1 (2.6 g, 5.8 mmol)⁸in DMA (115

mL) was boiled at 120 °C for 3 h with methylamine (7.2 mL,

58 mmol, 8.03 M in ethanol). The solution was concentrated

and the residue was chromatographed over silica gel with

methylene chloride/methanol (2.5-10%) to give 8a (2.21 g,

83%). The resulting oil (0.5 g, 1.09 mmol) was dissolved in

methylene chloride and stirred with fumaric acid (0.12 g, 1.03

mmol) overnight to give 8a ‚fumarate (0.33 g, 53%): mp 72 °C,

1

dec; IR (KBr) 1680, 1617, 1580, 1566 cm^-1; H NMR (DMSO-

d₆) δ 1.25-1.55 (m, 6H), 1.65-1.85 (m, 2H), 2.23 (s, 3H), 2.36-

2.60 (m, 2H), 2.90 (d, J ) 6 Hz, 3H), 2.90 (d, J ) 6 Hz, 3H),

4.06 (t, J ) 6.6, 2H), 5.15-5.32 (m, 2H), 5.74-5.94 (m, 1H),

6.10-6.25 (m, 2H), 6.57 (s, 2H), 7.25 (d, J ) 8.4 Hz), 7.42 and

7.69 (AA′-BB′-system, J _AB) 8.4, 4H); MS m/z 429 (M⁺, 1Br).

Anal. (C₂₄H₃₁N₂O₂Br‚C₄H₄O₄) C, H, N, Br.

[4-[6-(Allylm eth yla m in o)h exyloxy]-2-m eth oxyp h en yl]-

(4-br om op h en yl)m eth a n on e‚F u m a r a te (8b‚F u m a r a te).

Analogously to 8a , 1 (8.97 g, 20 mmol) in THF (450 mL) was

stirred with 5.4 M sodium methanolate (37 mL, 200 mmol) in

methanol at room temperature for 14 h and under reflux for

1 h. The solution was evaporated, and the residue was taken

up in methylene chloride/10% NaCl solution. The organic

phase was dried (Na₂SO₄), evaporated, dissolved in ether, and

stirred overnight with fumaric acid (2.08 g, 18 mmol). 8b‚

fumarate (8.17 g, 71%) was obtained: mp 108-113 °C; IR

1

(KBr) 1649, 1602 cm^-1; H NMR (DMSO-d₆) δ 1.28-1.55 (m,

4H), 1.70-1.85 (m, 4H), 2.21 (s, 3H), 2.35-2.50 (m, 2H), 3.08

(d, J ) 6.0 Hz, 2H), 3.64 (s, 3H), 4.06 (t, J ) 6.0 Hz, 2H), 5.14-

5.28 (m, 2H), 5.75-5.94 (m, 1H), 6.57 (s, 2H), 6.61-6.71 (m,

2H), 7.32 (d, J ) 8.4 Hz), 7.57 and 7.70 (AA′-BB′-system, J _AB

) 8.6, 4H); MS m/z 460 (M + H⁺, 1Br). Anal. (C₂₄H₃₀NO₃Br‚

0.95C₄H₄O₄) H, N, Cl, Br.

[4-[6-(Allylm eth yla m in o)h exyloxy]-2-h yd r oxyp h en yl]-

(4-b r om op h en yl)m et h a n on e‚F u m a r a t e (8c‚F u m a r a t e).

8b‚fumarate was taken up in methylene chloride/NaHCO₃

solution, and the organic phase was dried and concentrated.

The thus-obtained 8b (3.09 g, 6.72 mmol) was boiled in acetic

acid (13 mL)/62% HBr solution (7.7 mL) at 90 °C for 2 h. The

reaction mixture was concentrated, and the residue was

converted into the free base with methylene chloride/NaHCO₃

solution. The residue (3.14 g, quantitative) was dissolved in

ethanol with fumaric acid (0.74 g, 6.4 mmol) and precipitated

with ether. 8c‚fumarate (2.05 g, 49%) was obtained as a

yellowish oil: IR (KBr) 1705, 1624, 1586 cm^-^{1 1}H NMR

;

(DMSO-d₆) δ 1.25-1.55 (m, 4H), 1.65-1.82 (m, 4H), 2.24 (s,

3H), 2.35-2.50 (m, 2H), 3.12 (d, J ) 6.0 Hz, 2H), 4.05 (t, J )

6.0 Hz, 2H), 5.14-5.32 (m, 2H), 5.78-5.94 (m, 1H), 6.57 (s,

2H), 6.49-6.65 (m, 2H), 7.40 (d, J ) 8.4 Hz), 7.59 and 7.75

(AA′-BB′-system, J _AB) 8.5, 4H); MS m/z 446 (M + H⁺, 1Br).

Anal. (C₂₃H₂₈NO₃Br‚1.1C₄H₄O₄) C, H, N, Br.

[2-Am in o-4-[6-(Allylm eth yla m in o)h exyloxy]p h en yl](4-

br om op h en yl)m eth a n on e‚F u m a r a te (1:1) (8d‚F u m a r a te).

Analogous to 8a , 1 (17.55 g, 39.03 mmol) was boiled under

reflux for 23 h with 4-methoxybenzylamine (50.7 mL, 307.55

mmol) and K₂CO₃(6.5 g, 46.83 mmol) in toluene (600 mL).

After filtration, evaporation, and purification over silica gel

with methylene chloride/methanol (2.5-10%), [4-[6-(allyl-

methylamino)hexyloxy]-2-(4-methoxybenzylamino)phenyl]-

(4-bromophenyl)methanone (17.43 g, 79%) was obtained. A

solution of this material in trifluoroacetic acid (200 mL) was

(4-Br om oph en yl)[4-[6-[(3-h ydr oxypr opyl)m eth ylam in o]-

h exyloxy]p h en yl]m eth a n on e‚F u m a r a te (10b‚F u m a r a te).

10b‚fumarate was synthesized from (4-bromophenyl)-[4-[6-[(3-

hydroxypropyl)amino]hexyloxy]phenyl]methanone 9b (which

was obtained by treatment of 6b with 3-aminopropanol in

analogy to 7b (general procedure A) in 99% yield) followed by

N-methylation (analogous to 10a ) in a yield of 68%. Following

the general procedure for the formation of fumarates, 10b‚

fumarate was obtained: ¹H NMR (CDCl₃) δ 1.36-1.56 (m, 4H),

1.67-1.98 (2m, 6H), 2.65 (s, 3H), 2.90 (t, J ) 6 Hz, 2H), 3.07

(t, J ) 4.8 Hz, 2H), 3.75 (d, J ) 5 Hz, 2H), 4.24 (d, J ) 6.3 Hz,

2H), 5.3 (s, br, 3H), 6.77 (s, 2H), 6.94 (d, J ) 8.8 Hz, 2H), 7.61

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3365

(s, 4H), 7.77 (d, J ) 8.8 Hz, 2H); MS m/z 448 (M + H⁺, 1Br).

Anal. (C₂₃H₃₀BrNO₃‚C₄H₄O₄) C, H, N.

°C for 24 h and concentrated, and the residue was treated with

a NaHCO₃solution and extracted with methylene chloride.

The organic phases were washed with brine, dried, and

evaporated. The residue was purified over silica gel with

methylene chloride/MeOH/NH₄OH (94:5.4:0.6 to 85:12.5:2.5)

to obtain 13 (0.77 g, 76%), which was converted into 13‚

fumarate, following the general procedure for the formation

of fumarates: ¹H NMR of the free amine (CDCl₃) δ 0.12, 0.53

(2m, 4H), 0.88 (m, 1H), 1.40, 1.52, 1.82 (3m, 8H), 2.25 (d, J )

6.5 Hz, 2H), 2.32 (s, 3H), 2.42 (d, J ) 7.1 Hz, 2H), 4.04 (t, J )

6.4 Hz, 2H), 6.95 (d, J ) 8.8 Hz, 2H), 7.62 (s, 4H), 7.78 (d, J

) 8.8 Hz, 2H); MS m/z 443 (M⁺, 1Br). Anal. (C₂₄H₃₀BrFNO₂‚

C₄H₄O₄) C, H, N.

6-[6-(Ally lm e t h y la m in o )h e x y lo x y ]p y r id in -3-y l](4-

br om op h en yl)m eth a n on e‚F u m a r a te (1:1) (15‚F u m a r a te).

1-Bromo-6-hexanol (1 mL, 7.6 mmol) was taken up in DMA

(26 mL), treated with N-allylmethylamine (1.47 mL, 15.3

mmol) and stirred at room temperature for 16 h. The reaction

mixture was concentrated, and the residue was lyophilized

overnight. 6-(Allylmethylamino)hexan-1-ol‚hydrobromide (1.9

g, 100%) was obtained as the crude product: ¹H NMR of the

HBr salt (DMSO-d₆) δ 1.23-1.50 (m, 4H), 1.58-1.75 (m, 2H),

2.70 (s, 3H), 2.95-3.10 (m, 2H), 3.30-3.46 (m, 2H), 3.70-3.82

(m, 2H), 4.32-4.45 (m, 1H), 5.46-5.62 (m, 2H), 5.88-6.08 (m,

1H), 9.70 (m, 1H), MS m/z 171 (M⁺).

6-Chloronicotinic acid (III) (10 g, 63.4 mmol) was dissolved

in thionyl chloride (50 mL), and the mixture was refluxed for

3.5 h. The solution was cooled, and the excess thionyl chloride

was removed under reduced pressure. The crude product was

taken up in methylene chloride (60 mL) and treated with N,O-

dimethylhydroxylamine‚hydrochloride (7.22 g, 73.96 mmol).

Triethylamine (25 mL) in methylene chloride (30 mL) was

added while cooling with ice, and the solution was stirred at

room temperature for 1.5 h. The suspension was filtered, and

the filtrate was washed with a diluted NaOH solution and

brine and dried (Na₂SO₄). After concentration of the solution,

crude 6-chloro-N-methoxy-N-methylnicotinamide (13.12 g,

quantitative) was obtained: ¹H NMR (CDCl₃) δ 3.39 (s, 3H),

3.56 (s, 3H), 7.39 (d, 1H), 8.02 (dd, 1H), 8.78 (d, 1 H).

(4-Br om op h en yl)[4-(6-m eth yla m in oh exyloxy)p h en yl]-

m eth a n on e‚HCl (1:1) (12‚HCl). A solution of 6b (10 g, 22.6

mmol) in DMF (200 mL) was treated with sodium azide (14.7

g, 22.6 mmol) and stirred at 90 °C for 24 h. After filtration

and concentration, the residue was taken up in ethyl acetate

(200 mL) and washed with a 10% aqueous NaHCO₃solution.

The organic phase was dried (Na₂SO₄) and concentrated. [4-(6-

Azidohexyloxy)phenyl](4-bromophenyl)methanone (9.12 g,

99%) was obtained: ¹H NMR (CDCl₃) δ 1.51 (m, 4H), 1.66 (m,

2H), 1.85 (m, 2H), 3.30 (t, J ) 6.8 Hz, 2H), 4.05 (t, J ) 6.4 Hz,

2H), 6.90 (d, J ) 8.9 Hz, 2H), 7.62 (s, 4H), 7.80 (d, J ) 8.9 Hz,

2H).

Triphenylphosphine (7.1 g, 27 mmol) was added to a solution

of the derived azide (8.7 g, 21.6 mmol) in THF/water (4:1) (100

mL). The mixture was stirred at room temperature for 5 h and

subsequently evaporated. The residue was taken up in meth-

ylene chloride and treated with hydrochloric acid in ether. The

hydrochloride was filtered off and washed with ether. [4-(6-

Aminohexyloxy)phenyl](4-bromophenyl)methanone‚hydro-

chloride (8.0 g, 90%) was obtained: ¹H NMR (DMSO-d₆) δ 1.44

(m, 4H), 1.60 (m, 2H), 1.77 (m, 2H), 2.78 (m, 2H), 4.09 (t, J )

6.4 Hz, 2H), 7.09 (d, J ) 8.8 Hz, 2H), 7.63 (d, J ) 8.5 Hz, 2H),

7.74 (d, J ) 8.8 Hz, 2H), 7.77(d, J ) 8.5 Hz, 2H), 8.03 (s, br,

3H).

The free amine (1.1 g, 3 mmol) (obtained from the hydro-

chloride described above by extraction with a NaHCO₃solu-

tion/methylene chloride) was suspended in ether (30 mL), and

trifluoroacetic anhydride (1.25 mL) was slowly added dropwise

at 4 °C. The ice bath was removed, and the reaction mixture

was stirred at room temperature for 24 h. The solution was

poured into water (100 mL) and neutralized with a NaHCO₃

solution. The aqueous phase was separated and extracted with

ethyl acetate. The organic phases were washed with brine,

dried, and evaporated. The residue was taken up in methylene

chloride and filtered off. The filtrate was concentrated, dis-

solved in methylene chloride, and treated with n-hexane. After

filtration of the precipitated crystals, N-[6-[4-(4-bromobenzoyl)-

phenoxy)]hexyl]-2,2,2-trifluoroacetamide (11) (0.8 g, 56%) was

obtained: ¹H NMR (CDCl₃) δ 1.38-1.60 (m, 4H), 1.64 (m, 2H),

1.83 (m, 2H), 3.39 (q, J ) 6.8 Hz, 2H), 4.04 (t, J ) 6.4 Hz,

2H), 6.42 (s, br, 1H), 6.94 (d, J ) 8.9 Hz, 2H), 7.62 (s, 4H),

7.79 (d, J ) 8.9 Hz, 2H).

At -20 °C, 11 (0.65 g, 1.4 mmol) was added to a suspension

of sodium hydride (55% in oil, 0.08 g, 1.7 mmol) in DMF (20

mL). The mixture was stirred for 30 min at that temperature.

Subsequently, methyl iodide (0.24 g, 1.7 mmol) was added at

room temperature and the mixture was stirred for 1 h. The

reaction mixture was treated with an NH₄Cl solution, adjusted

to pH ∼4 with 1 M hydrochloric acid solution, and extracted

with methylene chloride. The organic extracts were dried and

concentrated. After chromatography of the residue on silica

gel with ethyl acetate/n-hexane (2:8), N-[6-[4-(4-bromobenzoyl)-

phenoxy)]hexyl]-N-methyl-2,2,2-trifluoroacetamide (0.45 g,

65%) was obtained: ¹H NMR (CDCl₃) δ 1.40, 1.54, 1.64,

1.83 (4m, 8H), 3.02, 3.12 (2s, 3H), 3.45 (m, 2H), 4.04 (t, J )

6.4 Hz, 2H), 6.94 (d, J ) 8.9 Hz, 2H), 7.62 (s, 4H), 7.78 (d, J

) 8.9 Hz, 2H).

This amide (6.7 g, 33.3 mmol) in THF (40 mL) was added

dropwise at -78 °C to a solution of n-BuLi (1.6M in n-hexane,

40 mL, 64 mmol) and 1,4-dibromobenzene (15 g, 63.58 mmol)

in THF (140 mL), which was previously stirred at -78 °C (20

min). The solution was stirred at -78 °C for 2 h and at 0 °C

for 1 h and then treated with 2 M HCl (60 mL). The phases

were separated, the aqueous phase was extracted with ether

(50 mL), and the organic phases were washed with a NaHCO₃

solution and brine and dried. The crude product was purified

over silica gel with ethyl acetate/n-hexane (6:1) and recrystal-

lized from ethyl acetate/n-hexane. (4-Bromophenyl)(6-chloro-

pyridin-3-yl)methanone (14) (6.75 g, 68%) was obtained: mp

127.4 °C; IR (KBr) 1652, 1582, 1458, 851 cm^-^{1 1}H NMR

;

(DMSO) δ 7.68-7.88 (m, 5H), 8.17 (dd, 1H), 8.72 (d, 1 H); MS

m/z 295 (M⁺, 1Br).

14 (1.11 g, 3.74 mmol), 6-(allylmethylamino)hexan-1-ol (696

mg, 3.96 mmol) (the hydrobromide described above was

converted into the free base with methylene chloride/NaHCO₃

solution), KOH (880 mg, 15.6 mmol), K₂CO₃(552 mg, 3.96

mmol), and dicyclohexano-18-crown-6 (200 mg, 0.51 mmol)

were dissolved in toluene (50 mL), and the mixture was heated

to 80 °C overnight. The suspension was treated with additional

6-(allylmethylamino)hexan-1-ol (323 mg, 1.87 mmol) and

heated for a further 5 h. The reaction mixture was treated

with water and extracted with methylene chloride. The organic

phases were washed with a NaHCO₃solution and brine and

dried. The crude product obtained was chromatographed in

methylene chloride/MeOH (95:5). 15 (1.02 g) was obtained as

a yellow-brown oil, which was dissolved in ethanol (15 mL)

and treated with fumaric acid (261 mg, 2.25 mmol) in ethanol

(5 mL). The solution was stirred at room temperature for 1 h

and concentrated, and the oil was evaporated several times

and lyophilized. 15‚fumarate (1.2 g, 59%) was obtained as a

yellow oil: IR (KBr) 2509, 1707, 1652, 1593, 1394, 1284, 982,

Analogous to the hydrolysis of 9a , (4-bromophenyl)-[4-(6-

methylaminohexyloxy)phenyl]methanone (12) was obtained

from N-[6-[4-(4-bromobenzoyl)phenoxy)]hexyl]-N-methyl-2,2,2-

trifluoroacetamide in a yield of 89%. For the in vitro test, it

was converted to the 12‚HCl following the general procedure

for the formation of hydrochlorides: ¹H NMR of free amine

(CDCl₃) δ 1.50 (m, 6H), 1.83 (m, 2H), 2.04 (s, br, 1H), 2.46 (s,

3H), 2.62 (d, J ) 7.1 Hz, 2H), 4.04 (t, J ) 6.4 Hz, 2H), 6.95 (d,

J ) 8.8 Hz, 2H), 7.62 (s, 4H), 7.78 (d, J ) 8.8 Hz, 2H), MS m/z

389 (M + H⁺). Anal. (C₂₀H₂₄BrNO₃) C, H, N, Br.

(4-B r o m o p h e n y l)[4-[6-(c y c lo p r o p y lm e t h y lm e t h -

yla m in o)h exyloxy]p h en yl]m eth a n on ‚F u m a r a te (1:1) (13‚

F u m a r a te). A mixture of 12 (0.1 g, 0.26 mmol), diisopropyl-

ethylamine (0.13 mL, 0.78 mmol), and bromomethylcyclopro-

pane (0.06 mL, 0.63 mmol) in DMA (20 mL) was stirred at 50

1

845 cm^-1; H NMR (DMSO-d₆) δ 1.25-1.55 (m, 6H), 1.74 (m,

3366 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Dehmlow et al.

2H), 2.26 (s, 3H), 2.44-2.54 (m, 2H), 3.14 (d, J ) 5 Hz, 2H),

4.36 (t, J ) 5 Hz, 2H), 5.21 (d, J ) 9 Hz), 5.27 (d, J ) 17.5

Hz), 5.83 (m, 1H), 6.58 (s, 2H), 6.96 (d, 1H), 7.63-7.85 (m,

4H), 8.07 (dd, 1H), 8.53 (d, 1H); MS m/z 430 (M⁺, 1Br). Anal.

(C₂₂H₂₇N₂O₂Br‚C₄H₄O₄) C, H, N, Br.

Allyl[6-[3-(4-br om op h en yl)ben zo[d ]isoxa zol-6-yloxy]-

h exyl]m eth yla m in e (21). A mixture of (4-bromophenyl)(2,4-

dihydroxyphenyl)methanone (5i) (4.25 g, 14.5 mmol) (synthe-

sized from 1,3-dimethoxybenzene and 4-bromobenzoyl chloride

followed by deprotection with HBr in AcOH; see general

procedure A, 5b), hydroxylamine‚hydrochloride (3.22 g, 46.4

mmol), and sodium acetate (2.85 g, 34.8 mmol) in ethanol (100

mL) was refluxed. The reaction mixture was concentrated,

treated with a NaHCO₃solution (100 mL), and extracted with

ethyl acetate. The organic extracts were washed with brine,

dried (Na₂SO₄), and evaporated. The residue was dissolved

in ethanol (200 mL), treated with p-toluenesulfonic acid

(1.1 g, 5.8 mmol), and heated to 85 °C for 24 h. The reac-

tion mixture was concentrated, and the residue was redis-

solved in ethyl acetate (250 mL) and washed with a NaHCO₃

solution and with brine. The organic phase was dried

(Na₂SO₄) and evaporated. 3-(4-Bromophenyl)benzo[d]isoxazol-

6-ol (20) (3.7 g, 98%) was obtained: ¹H NMR (DMSO-d₆) δ

6.87 (dd, J ₁) 8.6 Hz, J ₂) 2.3 Hz, 1H), 7.10 (d, J ) 2.3 Hz,

1H), 7.60 (d, J ) 8.6 Hz, 1H), 7.79 (d, J ) 8.58 Hz, 2H), 8.05

(d, J ) 8.58 Hz, 2H), 9.95 (s, br, ArOH, 1H); MS m/z 289 (M

+ H⁺, 1Br).

A mixture of 20 (1.82 g, 6.27 mmol), 1,6-dibromohexane

(2.96 mL, 18.82 mmol), and K₂CO₃(2.6 g, 18.82 mmol) in

acetone (80 mL) was stirred at 65 °C for 18 h. The reaction

mixture was filtered, concentrated, and purified by chroma-

tography (flash column on silica gel, toluene). 6-(6-Bromo-

hexyloxy)-3-(4-bromophenyl)benzo[d]isoxazole (3.27 g, quan-

titative) was obtained: ¹H NMR (CDCl₃) δ 1.47-1.62 (m, 4H),

1.79-1.98 (m, 4H), 3.44 (t, J ) 6.4 Hz, 2H), 4.03 (t, J ) 6 Hz,

2H), 6.96 (1H, dd, J ₁) 8.8 Hz, J ₂) 2.3 Hz), 7.10 (1H, d, J )

2.3 Hz), 7.61 (1H, d, J ) 8.8 Hz), 7.65 (d, J ) 8.7 Hz, 2H),

8.08 (d, J ) 8.7 Hz, 2H); MS m/z 451 (M⁺, 2Br).

This compound (2.0 g, 4.41 mmol) was dissolved in acetone

(40 mL) and treated at room temperature with N-allylmeth-

ylamine (1.27 mL, 13.24 mmol) and K₂CO₃(1.83 g, 13.24

mmol). After 6 h, additional N-allylmethylamine (4.22 mL,

44.13 mmol) was added. The mixture was stirred at room

temperature for 29 h, filtered, concentrated, and chromato-

graphed over silica gel with toluene/acetone (8:2) and 1% of

triethylamine as eluent. 21 (0.99 g, 51%) was obtained, which

was converted to the fumarate 21‚fumarate, following the

general procedure for the formation of fumarates: ¹H NMR

(DMSO-d₆) δ 1.30-1.52 (6H, m), 1.78 (2H, m), 2.20 (3H, s),

2.43 (2H, m), 3.08 (2H, m), 4.08 (2H, m), 5.15-5.26 (2H, m),

5.82 (1H, m), 6.57 (2H, s), 7.02 (1H, dd, J ₁) 8.8 Hz, J ₂) 2.3

Hz), 7.42 (1H, d, J ) 2.3 Hz), 7.71 (1H, d, J ) 8.8 Hz), 7.83

(2H, d, J ) 8.6 Hz), 8.08 (2H, d, J ) 8.6 Hz); MS m/z 443 (M

+ H⁺, 1Br). Anal. (C₂₃H₂₇N₂O₂Br‚0.9C₄H₄O₄) C, H, N.

Gen er a l P r oced u r e C. Allyl[6-[3-(4-br om op h en yl)ben -

zo[d ]isot h ia zol-6-yloxy]h exyl]m et h yla m in e‚F u m a r a t e

(1:1) (24‚F u m a r a te). Potassium tert-butylate (4.3 g, 38.3

mmol) was dissolved in THF (160 mL) and treated slowly with

benzyl mercaptan (4.5 mL, 38.3 mmol).³⁹The suspension was

stirred at room temperature for 30 min and then treated with

4f (10 g, 32.35 mmol) in THF (200 mL). The solution was

stirred at room temperature (1.5 h) and treated with NH₄Cl

solution (100 mL) as well as a NaHCO₃solution (200 mL). The

phases were separated, the aqueous phase was extracted with

ethyl acetate, and the organic phases were washed with a

NaHCO₃solution and brine and dried (Na₂SO₄). (2-Benzyl-

sulfanyl-4-methoxyphenyl)(4-bromophenyl)methanone (15.8 g,

quantitative) was obtained, which was taken up in methylene

chloride (160 mL), treated with sulfuryl chloride (3.4 mL, 41.9

mmol), and stirred at room temperature (1 h). After distillation

of the sulfuryl chloride, the residue was taken up in THF (160

mL), treated with a saturated ammonia solution in ethanol

(120 mL), and stirred at room temperature overnight. A 10%

NaHCO₃(100 mL) solution was added. The solvent was

removed, and the residue was again taken up in NaHCO₃solu-

tion and ethyl acetate. The phases were separated, and the

aqueous phase was extracted with ethyl acetate and ether. The

organic phases were washed with brine and dried (Na₂SO₄).

Recrystallization from ethyl acetate/ethanol yielded 3-(4-bro-

Allyl[6-[4-(4-b r om op h en yl)-2H -ch r om en -7-yloxy]h ex-

yl]m eth yla m in e (17). 8c (1.0 g, 2.34 mmol) in THF/ether (1:

1) (9 mL) was added dropwise over a period of 45 min to

vinylmagnesium chloride solution (1.7 M in THF, 4.8 mL,

8.19 mmol) at 0 °C. The solution was left to warm to room

temperature overnight, treated with acetic acid/water (1:1) (3

mL), and worked up with NaHCO₃solution/methylene chlo-

ride. After the mixture was dried (Na₂SO₄), the organic phase

was concentrated and the residue was purified over silica gel

with methylene chloride/methanol (95:5). (RS)-5-[6-(Allyl-

methylamino)hexyloxy]-2-[1-(4-bromophenyl)-1-hydroxyallyl]-

phenol (16) (0.82 g, 73%) was isolated: mp 78 °C (with

decomposition); IR (KBr) 1621, 1584, 1508 cm^-^{1 1}H NMR

;

(DMSO-d₆) δ 1.21-1.51 (m, 6H), 1.60-1.76 (m, 2H), 2.14 (s,

3H), 2.32 (t, J ) 6.6 Hz, 2H), 2.98 (d, J ) 6.0 Hz, 2H), 3.86 (t,

J ) 6.0 Hz, 2H), 5.09-5.25 (m, 4H), 5.71-5.91 (m, 1H), 6.29

(d, J ) 3 Hz, 1H), 6.35 (dd, J ) 8.4 Hz, J ) 3 Hz, 1H), 6.55

(dd, J ) 16.8 Hz, J ) 11.4 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 1H),

7.18 and 7.48 (AA′-BB′-system, J _AB) 8.5 Hz, 4H); MS m/z 474

(M + H⁺, 1Br). Anal. (C₂₅H₃₂NO₃Br) C, H, N.

A solution of 16 (0.26 g, 0.5 mmol) in o-xylene (50 mL) was

boiled at 170 °C in a Dean-Stark apparatus for 2 h and

evaporated, and the residue was purified over silica gel with

methylene chloride/methanol 95:5 to give 17 (0.18 g, 72%) as

a yellow oil: IR 1612, 1566 cm^-1; ¹H NMR (DMSO-d₆) δ 1.23-

1.50 (m, 6H), 1.60-1.75 (m, 2H), 2.12 (s, 3H), 2.31 (t, J ) 10.8

Hz, 2H), 2.95 (d, J ) 6 Hz, 2H), 3.92 (t, J ) 6 Hz, 2H), 4.75 (d,

J ) 3.6 Hz, 2H), 5.08-5.25 (m, 2H), 5.70-5.81 (m, 2H), 6.44-

6.52 (m, 2H), 6.81 (d, J ) 9 Hz, 1H), 7.28 and 7.65 (AA′BB′, J

) 9 Hz, 4H); MS m/z 456 (M + H⁺, 1Br). Anal. (C₂₄H₃₀N₃OBr)

C, H, N.

Allyl[6-[4-(4-br om op h en yl)qu in a zolin -7-yloxy]h exyl]-

m eth yla m in e‚F u m a r a te (1:1) (18‚F u m a r a te). In analogy

to Uff et al.,³⁷a solution of 8d (222 mg, 0.5 mmol) in formic

acid (0.5 mL) and formamide (2 mL) was boiled at 165 °C for

25 min and then concentrated and converted into the free

amine (title compound) with methylene chloride/NaHCO₃

solution. After purification (silica gel, methylene chloride/

methanol 2.5-10%), 18 (103 mg, 0.23 mmol) was dissolved in

methylene chloride/ether and treated with fumaric acid (23.7

mg, 0.20 mmol). After the mixture was stirred overnight and

filtered, 18‚fumarate (30 mg, 11%) was obtained: mp 90-95

°C; IR 1706, 1614, 1590, 1569 cm^-^{1 1}H NMR (DMSO-d₆) δ

;

1.25-1.62 (m, 6H), 1.72-1.92 (m, 2H), 2.31 (s, 3H), 2.50-2.55

(m, 2H), 3.21 (d, J ) 6 Hz, 2H), 4.24 (t, J ) 6 Hz, 2H), 5.20-

5.34 (m, 2H), 5.74-5.94 (m, 1H), 6.60 (s, 2H), 7.32 (dd, J ) 9

Hz, J ) 2.4 Hz, 1H), 7.45 (d, J ) 2.4 Hz, 1H), 7.72 and 7.85

(AA′BB′, J ) 8.4 Hz, 4H), 7.94 (d, J ) 9 Hz, 1H), 9.22 (s, 1H);

MS m/z 454 (M + H⁺, 1Br). Anal. (C₂₄H₂₈N₃OBr‚C₄H₄O₄) C,

H, N.

Allyl[6-[3-(4-br om oph en yl)-1-m eth yl-1H-in dazol-6-yloxy]-

h exyl]m et h yla m in e‚F u m a r a t e (1:1.6) (19‚F u m a r a t e). A

mixture of methylhydrazine (4.61 mL, 87.5 mmol) and K₂CO₃

(1.45 g, 10.5 mmol) was stirred in DMA (40 mL) at room

temperature. 1 (3.92 g, 8.75 mmol) in DMA (30 mL) was added,

and the mixture was heated for 1.5 h at 120 °C. After filtration

and concentration at 80 °C/0.3 Torr, the residue was taken

up in methylene chloride, filtered over Na₂SO₄, and concen-

trated. The residue was dissolved in ethanol, treated with

fumaric acid (1.02 g, 8.75 mmol), and stirred. After the mixture

was filtered and dried (Na₂SO₄), 19‚fumarate (1:1.6) (3.1 g,

78%) was obtained: mp 135-137 °C, dec; IR 1705, 1623 cm^-1

;

¹H NMR (DMSO-d₆) δ 1.28-1.60 (m, 6H), 1.65-1.80 (m, 2H),

2.25 (s, 3H), 2.35-2.60 (m, 2H), 3.13 (d, J ) 5.5 Hz, 2H), 4.04

(s, 3H), 4.08 (d, J ) 5.5 Hz, 1H), 5.20-5.30 (m, 2H), 5.76-

5.95 (m, 1H), 6.58 (s, 2H), 6.87 (dd, J ) 9 Hz, J ) 1 Hz, 1H),

7.20 (d, J ) 1, 1H), 7.67 and 7.90 (AA′BB′, J ) 8 Hz, 4H), 7.91

(d, J ) 9 Hz, 1H); MS m/z 456 (M + H⁺, 1Br). Anal. (C₂₄H₃₀N₃-

OBr‚1.6C₄H₄O₄) C, H, N, Br.

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3367

mophenyl)-6-methoxy[d]benzisothiazole (22) (7.52 g, 73%): IR

(KBr) 2840, 1600, 1499, 1236, 1124, 1017, 829 cm^-1; ¹H NMR

(DMSO-d₆) δ 3.94 (s, 3H), 7.07 (dd, J ) 8.8 Hz, 1H), 7.36 (d,

1H), 7.62-7.78 (m, 4H), 7.97 (d, J ) 9 Hz, 1H); MS m/z 318

(M⁺, 1Br).

Allyl[6-[3-(4-br om op h en yl)ben zo[b]th iop h en -6-yloxy]-

h exyl]m eth yla m in e‚F u m a r a te (1:1) (29‚F u m a r a te). Po-

tassium tert-butylate (2.55 g, 22.7 mmol) was placed in THF

(15 mL), and the solution was treated with 4e (2.50 g, 7.4

mmol) in THF (12 mL). The mixture was heated under reflux

and then treated with water and NH₄Cl solution. The water

phases were extracted with ethyl acetate. The organic phases

were washed with a NaHCO₃solution and brine and dried

(Na₂SO₄). The crude product obtained after concentration (2.08

g) was dissolved in toluene (14 mL) and treated with trifluo-

roacetic acid (6 mL) at 0 °C (2 h). The mixture was neutralized

with a NaHCO₃solution. The aqueous phase was extracted

with ethyl acetate. The organic phases were washed with a

NaHCO₃solution and brine and dried (Na₂SO₄). The crude

product obtained was purified by column chromatography

(silica gel, ethyl acetate/n-hexane 1:6) and crystallized from

ethyl acetate/n-hexane. 3-(4-Bromophenyl)-6-methoxybenzo-

[b]thiophene (27) (476 mg, 20%) was obtained as white

crystals: mp 102.5-104.5 °C; IR (KBr) 2830, 1601, 1460, 1267,

1049, 825 cm^-1; ¹H NMR (CDCl₃) δ 3.89 (s, 3H), 7.02 (dd, J )

8.9, 2.4 Hz, 1H), 7.21 (s, 1H), 7.37 (d, J ) 2.4 Hz, 1H), 7.43

(m, 2H), 7.60 (m, 2H), 7.72 (d, J ) 8.9 Hz, 1H); MS m/z 318

(M⁺, Br).

22 (6.32 g, 19.7 mmol) was dissolved in methylene chloride

(395 mL), and the mixture was cooled to -78 °C and treated

with a boron tribromide solution (1 M in methylene chloride,

49.3 mL, 49.3 mmol). The solution was thawed overnight and

then stirred at room temperature. The solution was added to

a NaHCO₃solution, the phases were separated, and the

aqueous phase was extracted with methylene chloride (150

mL). The organic phases were washed with brine, dried

(Na₂SO₄), and evaporated. (4-Bromophenyl)benzo[d]isothiazol-

6-ol (1.5 g, 25%) was crystallized from the crude product using

methylene chloride as solvent. The mother liquor was concen-

trated, dissolved in methylene chloride (320 mL), and treated

at -78 °C with boron tribromide solution (1 M in methylene

chloride, 40 mL, 40 mmol). After workup and crystallization,

further 3-(4-bromophenyl)benzo[d]isothiazol-6-ol (2.24 g, 37%)

was obtained: ¹H NMR (DMSO-d₆) δ 7.04 (dd, J ) 11 Hz, 1H),

7.51 (d, 1H), 7.79 (m, 4H), 8.0 (d, J ) 8.9 Hz, 1H), 10.38 (s,

1H).

This compound (3.0 g, 9.8 mmol) was treated as discribed

for 6e in general procedure B to give 6-(6-bromohexyloxy)-3-

(4-bromophenyl)benzo[d]isothiazole (23) (4.18 g, 91%) as a

pale-brown substance: ¹H NMR (CDCl₃) δ 1.56 (m, 4H), 2.9

(m, 4H), 3.44 (t, 3H), 4.08 (t, 3H), 7.07 (dd, 1H), 7.47 (d, 1H),

7.69 (m, 4H), 7.96 (d, J ) 9 Hz, 1H), 10.38 (s, 1H).

23 (4.15 g, 8.84 mmol) was treated as described for 7b in

general procedure A to give colorless crystals of 24‚fumarate

(3.43 g, 67%): mp 122-123.5 °C; IR (KBr) 2591, 2504, 1671,

1589, 1391, 1237, 960 cm^-1; ¹H NMR (DMSO-d₆) δ 1.31-1.58

(m, 4H), 1.66 (m, 2H), 1.81 (m, 2H), 2.69 (s, 3H), 3.05 (t, 2H),

3.70 (d, 2H), 4.12 (t, 2H), 5.50 (d, 1H), 5.54 (d, 1H), 5.90 (m,

1H), 6.62 (s, 2H), 7.15 (dd, J ) 8.9 Hz, 1H), 7.80 (m, 5H), 8.06

(d, J ) 9 Hz, 1H); MS m/z 459 (M + H⁺, 1Br). Anal. (C₂₃H₂₇N₂-

OBr‚C₄H₄O₄) C, H, N, Br, S.

Analogous to general procedure C, 29‚fumarate was ob-

tained from 27 in 35% yield: IR (KBr) 2496, 1704, 1270, 1233,

1068, 984, 824 cm^-1; ¹H NMR (DMSO-d₆) δ 1.20-1.58 (m, 6H),

1.76 (m, 2 H), 2.25 (s, 3H), 2.38-2.56 (m, 2H), 3.12 (d, J ) 6.4

Hz, 2H), 4.05 (t, J ) 5 Hz, 2 H), 5.19 (d, J ) 10 Hz, 1H), 5.25

(d, J ) 17.5 Hz, 1H), 5.82 (m, 1H), 6.58 (s, 2H), 7.05 (dd, J )

8.9 Hz, 1H), 7.5-7.77 (m, 2H), 13.0 (s, br, 2H); MS m/z 457

(M + H⁺, Br). Anal. (C₂₄H₂₈NOBrS‚C₄H₄O₄) C, H, N, S.

Allyl[6-[3-(4-br om op h en yl)ben zofu r a n -6-yloxy]h exyl]-

m eth yla m in e‚F u m a r a te (34‚F u m a r a te). 4-Bromophenacyl

bromide (11.5 g, 41.5 mmol) was dissolved in acetone (250 mL),

and after the addition of K₂CO₃(5.75 g, 41.5 mmol) and

3-methoxyphenol (4.5 mL, 41.5 mmol), the reaction mixture

was stirred at room temperature for 18 h and the mixture was

subsequently filtered. The filtrate was concentrated and

chromatographed over silica gel with ethyl acetate/n-hexane

(5:95 to 10:90). 1-(4-Bromophenyl)-2-(3-methoxyphenoxy)etha-

none (32) (7.3 g, 55%) was isolated as an off-white solid: ¹H

NMR (CDCl₃) δ 3.78 (s, 3H), 5.19 (s, 2H), 6.50-6.57 (m, 3H),

7.19 (t, J ) 8.8 Hz, 1H), 7.65 (d, J ) 8.6 Hz, 2H), 7.90 (d, J )

8.6 Hz, 2H); MS m/z 320 (M⁺, 1Br).

Ally l[6-[4-(6-b r o m o b e n zo [d ]is o t h ia zo l-3-y l)p h e n -

oxy]h exyl]m eth yla m in e‚F u m a r a te (1:1) (31‚F u m a r a te).

31‚fumarate was synthesized from 4-bromo-2-fluorobenzoyl

chloride and anisole (Id ) via (4-bromo-2-fluorophenyl)(4-meth-

oxyphenyl)methanone (30) following general procedure A (63%

yield) and general procedure C (38% yield): mp 134-135 °C;

IR (KBr) 2643, 1696, 1608, 1581, 1516, 1462, 1252, 840 cm^-1

;

32 (5.0 g, 15.6 mmol) was dissolved in polyphosphoric acid

(53 g), and the mixture was heated for 1.5 h at 80 °C. The

mixture was then adjusted to pH ∼8 with a Na₂CO₃solution,

cooled, and subsequently extracted with ethyl acetate. The

organic extracts were dried (Na₂SO₄) and concentrated. 3-(4-

Bromophenyl)-6-methoxybenzofuran (33) (4.6 g, 98%) was

obtained: ¹H NMR (CDCl₃) δ 3.88 (s, 3H), 6.95 (dd, J ₁) 8.6

Hz, J ₂) 2.1 Hz, 1H), 7.07 (d, J ) 2.1 Hz, 1H), 7.49 (d, J ) 8.4

Hz, 2H), 7.58 (d, J ) 8.4 Hz, 2H), 7.63 (d, J ) 8.6 Hz, 1H),

7.70 (s, 1H); MS m/z 302 (M⁺, 1Br).

¹H NMR (DMSO-d₆) δ 1.28-1.58 (m, 6H), 1.76 (m, 2H), 2.24

(s, 3H), 2.38-2.58 (m, 2H), 3.10 (d, J ) 5 Hz, 2H), 4.06 (t, J )

8 Hz, 2H), 5.18 (d, J ) 9 Hz, 1H), 5.25 (d, J ) 15.4 Hz, 1H),

5.82 (m, 1H), 6.57 (s, 2H), 7.13 (d, J ) 8.7 Hz, 2H), 7.70 (dd,

J ) 8.7 Hz, 1H), 7.81 (d, J ) 8.7 Hz, 2H), 8.12 (d, J ) 8.7 Hz,

1H), 8.61 (dd, 1H), 13.0 (s, br, 2H); MS m/z 458 (M⁺, 1Br).

Anal. (C₂₃H₂₇N₂OBrS‚2C₄H₄O₄) C, H, N, Br, S.

Allyl[6-[4-(6-br om o-1,1-d ioxoben zo[d ]isoth ia zol-3-yl)-

p h en oxy]h exyl]m eth yla m in e‚F u m a r a te (1:1) (26‚F u m a -

r a te). 23 (2.34 g, 4.99 mmol) was taken up in methylene

chloride (40 mL) and treated with potassium permanganate

(8.5 g, 5.38 mmol) adsorbed on silica gel.⁴⁶The suspension was

stirred at room temperature and then treated with Na₂SO₄

and filtered through silica gel. After concentration, the crude

product was crystallized from ethyl acetate and n-hexane to

give 6-(6-bromohexyloxy)-3-(4-bromophenyl)benzo[d]isothiaz-

ole 1,1-dioxide (25) (650 mg, 28%) and starting material 23

(1.23 g, 52%). 25: ¹H NMR (CDCl₃) δ 1.54 (m, 4H), 1.9 (m,

4H), 3.44 (t, 2H), 4.13 (t, 2H), 7.14 (dd, 1H), 7.47 (d, 1H), 7.67-

7.77 (m, 3H), 7.84 (d, 2H); MS m/z 499 (M⁺, 1Br).

Analogous to general procedure A for compound 7b, 26‚

fumarate was isolated as an oil in a yield of 55%: IR (KBr)

2640, 1714, 1604, 1169, 1011, 963, 844 cm^-1; ¹H NMR (DMSO-

d₆) δ 1.33-1.59 (m, 4H), 1.68 (m, 2H), 1.85 (m, 2H), 2.50 (s,

3H), 2.74 (m, 2H), 3.40 (d, J ) 8 Hz, 2H), 4.10 (t, J ) 6 Hz,

2H), 5.32 (d, J ) 6 Hz, 1H), 5.36 (d, J ) 10 Hz, 1H), 5.93 (m,

1H), 6.79 (s, 2H), 7.16 (dd, 1H), 7.46 (d, 1H), 7.69-7.77 (m,

3H), 7.79-7.86 (m, 2H)); MS m/z 491 (M + H⁺, 1Br). Anal.

(C₂₃H₂₇N₂O₃BrS‚C₄H₄O₄) C, H, N, Br, S.

33 (3.0 g, 9.9 mmol) in methylene chloride (100 mL) was

treated at 0 °C with a boron tribromide solution (1 M in

methylene chloride, 100 mL, 100 mmol). The ice bath was

removed, and the reaction mixture was stirred for 4 h,

subsequently poured into a Na₂CO₃solution, and extracted

with methylene chloride. The organic phases were washed with

brine, dried (Na₂SO₄), and evaporated to give 3-(4-bromophen-

yl)benzofuran-6-ol (2.83 g, 99%): ¹H NMR (CDCl₃) δ 6.86 (dd,

J ₁) 8.6 Hz, J ₂) 2.1 Hz, 1H), 7.04 (d, J ) 2.1 Hz, 1H), 7.48

(d, J ) 8.5 Hz, 2H), 7.58 (d, J ) 8.5 Hz, 2H), 7.60 (d, J ) 8.6

Hz, 1H), 7.69 (s, 1H); MS m/z 288 (M⁺, 1Br).

Analogous to general procedure B, 34 was obtained in a total

yield of 37% and the compound was converted into 34‚

fumarate, following the general procedure for the formation

of fumarates: ¹H NMR (DMSO-d₆) δ 1.37 (m, 2H), 1.50 (m,

4H), 1.75 (m, 2H), 2.28 (s, 3H), 2.50 (m, 2H), 3.17 (d, J ) 6.6

Hz, 2H), 4.03 (t, J ) 6.5 Hz, 2H), 5.20-5.30 (m, 2H), 5.77-

5.92 (m, 1H), 6.57 (s, 2H), 6.97 (dd, J ₁) 8.6 Hz, J ₂) 2.1 Hz,

1H), 7.27 (d, J ) 2.1 Hz, 1H), 7.68 (s, 4H), 7.77 (d, J ) 8.6 Hz,

3368 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15

Dehmlow et al.

1H), 8.31 (s, 1H); MS m/z 442 (M + H⁺, 1Br). Anal. (C₂₄H₂₈

BrNO₂‚C₄H₄O₄) C, H, N, Br.

-

6.4 Hz, 1H), 10.89 (s, br, 2H); MS m/z 453 (M + H⁺, 1Br). For

39: Anal. (C₂₅H₂₉BrN₂O) C, H, N, Br.

Allyl[6-[1-(4-b r om op h en yl)-3,4-d ih yd r oisoq u in olin -6-

yloxy]h exyl]m eth yla m in e‚F u m a r a te (37‚F u m a r a te). To

a solution of 4-bromobenzoyl chloride (43.9 g, 200 mmol) in

methylene chloride (500 mL) was added dropwise 2-(3-meth-

oxyphenyl)ethylamine (29.2 mL, 200 mmol) at 0 °C followed

by the addition of diisopropylethylamine (34.2 mL, 200 mmol).

The reaction mixture was left to warm to room temperature

and stirred for 26 h, subsequently diluted with methylene

chloride and treated with a NaHCO₃solution. The organic

phase was washed with a NaHCO₃solution and brine, dried

(Na₂SO₄), and concentrated. 4-Bromo-N-[2-(3-methoxyphenyl)-

ethyl]benzamide (35) (66.1 g, 99%) was obtained: ¹H NMR

(CDCl₃) δ 2.91 (t, J ) 6.8 Hz, 2H), 3.71 (q, J ) 6.8 Hz, 2H),

3.79 (s, 3H), 6.11 (s, br, 1H), 6.78-6.84 (m, 3H), 7.25 (t, J )

7.8 Hz, 1H), 7.55 (s, 4H); MS m/z 333 (M⁺, 1Br).

35 (50 g, 150 mmol) was dissolved in acetonitrile (750 mL)

and treated under argon with phosphorus oxychloride (55 mL,

598 mmol). After the mixture was heated at 80 °C for 4 h, the

solvent was removed, adjusted to pH >12 with concentrated

ammonia solution and extracted with ethyl acetate. The

organic extracts were washed with brine, dried, and concen-

trated. 1-(4-Bromophenyl)-6-methoxy-3,4-dihydroisoquinoline

(36) (46.4 g, 98%) was obtained: ¹H NMR (CDCl₃) δ 2.77 (m,

2H), 3.81 (m, 2H), 3.86 (s, 3H), 6.75 (dd, J ₁) 8.5 Hz, J ₂) 2.4

Hz, 1H), 6.79 (d, J ) 2.4 Hz, 1H), 7.16 (d, J ) 8.5 Hz, 1H),

7.47 (d, J ) 8.7 Hz, 2H), 7.56 (d, J ) 8.7 Hz, 2H); MS m/z 314

(M⁺, 1Br).

Treatment of 36 (15 g, 47.4 mmol) in acetic acid (95 mL)

with 62% aqueous HBr solution (65 mL) at 100 °C (46 h)

(analogous to general procedure A, compound 5b) gave 1-(4-

bromophenyl)-3,4-dihydroisoquinolin-6-ol (13.7 g, 96%): ¹H

NMR (CDCl₃) δ 2.72 (m, 2H), 3.75 (m, 2H), 4.83 (s, br, 1H),

6.56 (m, 2H), 7.03 (d, J ) 8.5 Hz, 1H), 7.41 (d, J ) 8.5 Hz,

2H), 7.54 (d, J ) 8.5 Hz, 2H); MS m/z 301 (M⁺, 1Br).

Analogous to general procedure B for compound 7e, 37 was

obtained in a yield of 50% and converted into the 37‚fumarate,

following the general procedure for the formation of fuma-

rates: ¹H NMR of free base (CDCl₃) δ 1.35-1.59 (m, 6H), 1.82

(m, 2H), 2.21 (s, 3H), 2.34 (m, 2H), 2.76 (m, 2H), 2.99 (d, J )

6.5 Hz, 2H), 3.80 (m, 2H), 4.00 (t, J ) 6.5 Hz, 2H), 5.11-5.20

(m, 2H), 5.87 (m, 1H), 6.73 (dd, J ₁) 8.5 Hz, J ₂) 2.1 Hz, 1H),

6.78 (d, J ) 2.1 Hz, 1H), 7.14 (d, J ) 8.5 Hz, 1H), 7.46 (d, J )

8.5 Hz, 2H), 7.55 (d, J ) 8.5 Hz, 2H); MS m/z 455 (M + H⁺,

1Br). Anal. (C₂₅H₃₁BrN₂O) C, H, N, Br.

Allyl[6-[1-(4-br om op h en yl)isoqu in olin -6-yloxy]h exyl]-

m eth yla m in e‚2HCl (39‚2HCl). 36 (10 g, 31.6 mmol) was

dissolved in toluene (500 mL), treated with anhydrous

Na₂SO₄(60 g) and γ-manganese dioxide (15 g, 172.5 mmol),^47,48

and boiled under reflux for 24 h. After cooling, the reaction

mixture was filtered and concentrated to give 1-(4-bromo-

phenyl)-6-methoxyisoquinoline (38) (9.2 g, 92%) as an off-

white solid: ¹H NMR (CDCl₃) δ 3.97 (s, 3H), 7.13 (d, J ) 2.1

Hz, 1H), 7.17 (dd, J ₁) 9 Hz, J ₂) 2.2 Hz, 1H), 7.56 (m, 3H),

7.66 (d, J ) 8.5 Hz, 2H), 7.94 (d, J ) 9 Hz, 1H), 8.52 (d, J )

5.7 Hz, 1H); MS m/z 313 (M⁺, 1Br).

Allyl-6-[4-(4-br om op h en yl)-1H-ben zo[d ][1,2]oxa zin -7-

yloxy]h exylm eth yla m in e‚F u m a r a te (43‚F u m a r a te). A so-

lution of (4-bromophenyl)(2-methyl-4-methoxyphenyl)metha-

none (4h ) (3 g, 9.8 mmol) (prepared from 3-methylanisole (Ih )

and 4-bromobenzoyl chloride (IIb) by Friedel-Crafts reaction,

analogous to general procedure A, compound 4b) in tetrachlo-

romethane (100 mL) was treated with N-bromosuccinimide

(2.1 g, 11.8 mmol) and a spatula tip of dibenzoyl peroxide. The

reaction mixture was stirred at room temperature for 6 h

under irradiation with a Hg lamp TQ 150 (150 W, Heraeus,

Hanau, FRG) with light λ g 390 nM, subsequently diluted with

methylene chloride (100 mL), and washed in succession with

a NaHCO₃solution and brine. The organic phase was dried

(Na₂SO₄), filtered, and immediately reacted. The solution of

40 was added dropwise to a mixture of tert-butyl N-hydroxy-

carbamate (2.13 g, 16 mmol) and sodium hydride (55% in oil,

0.38 g, 8.7 mmol) in DMF (30 mL), previously stirred at 0 °C

(20 min). The reaction mixture was warmed to room temper-

ature and stirred overnight. The mixture was treated with an

NH₄Cl solution (60 mL) and extracted with methylene chloride.

The organic phase was washed with brine, dried (Na₂SO₄), and

concentrated. After chromatography of the residue (silica gel,

ethyl acetate/n-hexane, 10:90 to 25:75), tert-butyl [2-(4-bro-

mobenzoyl)-5-methoxybenzyloxy]carbamate (41) (2.65 g, 61%)

was obtained: ¹H NMR (CDCl₃) δ 1.43 (9H, s), 3.89 (3H, s),

5.08 (2H, s), 6.85 (1H, dd, J ₁) 8.5 Hz, J ₂) 2.5 Hz), 7.13 (1H,

s, br), 7.21 (1H, d, J ) 2.5 Hz), 7.36 (1H, d, J ) 8.5 Hz), 7.61

(4H, m). MS m/z 436 (M + H⁺, 1Br).

A solution of 41 (1.9 g, 4.36 mmol) was treated with 62%

aqueous HBr solution and acetic acid as described in procedure

A (compound 5b) for 3.5 days to give 4-(4-bromophenyl)-1H-

benzo[d][1,2]oxazin-7-ol (42) (1.2 g, 88%): ¹H NMR (CDCl₃) δ

4.97 (s, 2H), 5.52 (s, br, 1H), 6.72 (d, J ) 2.5 Hz, 1H), 6.80

(dd, J ₁) 8.4 Hz, J ₂) 2.5 Hz, 1H), 7.09 (d, J ) 8.4 Hz, 1H),

7.50 (d, J ) 8.5 Hz, 2H), 7.61 (d, J ) 8.5 Hz, 2H); MS m/z 302

[(M - H)^-, 1Br].

Analogously to compound 21, 43 was obtained from 42 in

56% yield. For the in vitro test, it was converted to the 43‚

fumarate, following the general procedure for the formation

of fumarates: ¹H NMR of the free amine (CDCl₃) δ 1.35-1.56

(m, 6H), 1.82 (m, 2H), 2.21 (s, 3H), 2.35 (m, 2H), 3.00 (m, 2H),

4.01 (t, J ) 6.2 Hz, 2H), 4.98 (s, 2H), 5.11-5.21 (m, 2H), 5.86

(m, 1H), 6.75 (d, J ) 2.5 Hz, 1H), 6.84 (dd, J ₁) 8.4 Hz, J ₂

)

2.5 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 1H), 7.50 (d, J ) 8.5 Hz, 2H),

7.60 (d, J ) 8.5 Hz, 2H); MS m/z 457 (M + H⁺, 1Br). Anal. for

43 (C₂₄H₂₉BrN₂O₂) C, H, N, Br.

Biologica l Assa ys. In h ibition of Hu m a n Liver Mi-

cr osom a l 2,3-Oxid osqu a len e-La n oster ol Cycla se (OSC).

A section of liver from an 18-year-old healthy donor was

obtained at the hepatic transplantation unit of Hoˆpital Biceˆtre,

Paris, and frozen at -80 °C.⁸Liver microsomes were prepared

in sodium phosphate buffer (pH 7.4). The OSC activity was

measured in the same buffer, which also contained 1 mM

EDTA and 1 mM dithiothreitol. The microsomes were diluted

to 0.8 mg/mL protein in cold phosphate buffer. [¹⁴C]R,S-

monooxidosqualene (MOS, 12.8 mCi/mmol) was diluted to 20

nCi/µL with ethanol and mixed into phosphate buffer/1% BSA

(bovine serum albumin). A stock solution of 1 mM test

substance in DMSO was diluted to the desired concentration

with phosphate buffer/1% BSA. An amount of 40 µL of

microsomes was mixed with 20 µL of a solution of the test

substance, and the reaction was subsequently started with 20

µL of the [¹⁴C]R,S-MOS solution. The final conditions were

the following: 0.4 mg/mL of microsomal proteins and 30 µL

of [¹⁴C]R,S-MOS in phosphate buffer, pH 7.4, containing 0.5%

albumin, <0.1% DMSO, and <2% ethanol for a total volume

of 80 µL.

38 (8.1 g, 25.8 mmol) was deprotected to give 1-(4-bro-

mophenyl)isoquinolin-6-ol (6.7 g, 86%) following procedure A

(compound 5b): ¹H NMR (DMSO-d₆) δ 7.48 (dd, J ₁) 9.2 Hz,

J ₂) 2 Hz, 1H), 7.52 (d, J ) 2.0 Hz, 1H), 7.77 (d, J ) 8.5 Hz,

2H), 7.95 (d, J ) 8.5 Hz, 2H), 8.15 (d, J ) 9.2 Hz, 1H), 8.23 (d,

J ) 6.6 Hz, 1H), 8.48 (d, J ) 6.6 Hz, 1H), 11.75 (s, br, 1H);

MS m/z 298 [(M - H)^-, 1Br].

Analogous to general procedure B for compound 7e, 1-(4-

bromophenyl)isoquinolin-6-ol gave 39 in 72% yield as a yellow

oil, which was converted into the 39‚2HCl, following the

general procedure for the formation of hydrochlorides: ¹H

NMR as dihydrochloride (DMSO-d₆) δ 1.47 (m, 4H), 1.74 (m,

2H), 1.85 (m, 2H), 2.66 (d, J ) 4.9 Hz, 3H), 3.02 (m, 2H), 3.71

(m, 2H), 4.28 (t, J ) 6.5 Hz, 2H), 5.47-5.56 (m, 2H), 6.02 (m,

1H), 7.51 (dd, J ₁) 9.3 Hz, J ₂) 2.1 Hz, 1H), 7.75 (d, J ) 8.5

Hz, 2H), 7.78 (d, J ) 2.1 Hz, 1H), 7.92 (d, J ) 8.5 Hz, 2H),

8.01 (d, J ) 9.3 Hz, 1H), 8.22 (d, J ) 6.4 Hz, 1H), 8.55 (d, J )

After 1 h at 37 °C, the reaction was stopped by the addition

of 0.6 mL of 10% KOH/methanol, 0.7 mL of water, and 0.1

mL of n-hexane/ether (1:1), which contained 25 µg of nonra-

dioactive MOS and 25 µg of lanosterol as carriers. After

shaking, 1 mL of n-hexane/ether (1:1) was added to each test

2,3-Oxidosqualene Cyclase Inhibitors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3369

tube, and these were again shaken and then centrifuged. The

upper phase was transferred into a glass test tube, and the

lower phase was again extracted with n-hexane/ether and

combined with the first extract. The entire extract was

evaporated to dryness with nitrogen, the residue was sus-

pended in 50 µL of n-hexane/ether (1:1) and applied to a silica

gel plate for chromatographic separation using n-hexane/ether

(1:1) as the eluent. The R_fvalues for the MOS substrate and

the lanosterol product were 0.91 and 0.54, respectively. After

the sample was dried, radioactive MOS and lanosterol were

detected on the silica gel plate using a phosphor imager

(Molecular Dynamics). The ratio of MOS to lanosterol was

determined from the radioactive bands in order to determine

the yield of the reaction and OSC inhibition.

exclusion Superose-6 gel chromatography⁵¹and total choles-

terol was quantified in each 50 µL elution fraction using a

fluorometric enzymatic assay.⁵²The effect of each compound

on plasma total cholesterol and LDL cholesterol is expressed

as a percent of day 0 (prior to compound administration) and

corrected with respect to the variation of the untreated animals

(Table 5).

Ack n ow led gm en t. We sincerely thank Daniel Eg-

ger, Gisela Flach, Corinne Handschin, Marie-Paule

Imhoff, Isabelle Kaufmann, Hans-J akob Krebs, Anke

Kurt, Bettina Roser, and Heidi Stoll for their excellent

and dedicated assistance, and Wolf Arnold, Andre´

Bubendorf, Walter Meister, Roger Rueher, and Bjo¨rn

Wagner for spectroscopic determination and analysis.

In h ibition of Ch olester ol Syn th esis in P r im a r y Ra t

Hep a tocytes. This assay was performed to determine the

cellular activity and the metabolic stability of the compounds

prior to testing in hyperlipidemic hamsters. The stability test

was performed as a bioassay and to assess the efficacy and

potency of each test substance in reducing cholesterol produc-

tion while increasing monooxidosqualene (MOS) levels. Also,

it allowed the measurement of substance stability by compar-

ing the residual inhibitory activity after preincubation for 8

or 24 h or after co-incubation for 2 h. Hepatocytes were isolated

from collagenase perfused rat liver and seeded in collagen-

coated cell-culture plates in medium containing 10% FCS. At

4 h after seeding, the medium was replaced with medium

containing 5% lipoprotein deficient serum (LPDS), and the

cells incubated overnight. In one set of wells, hepatocytes were

incubated for 2 h with 3 µM test substance in the presence

of ¹⁴C-acetate to trace cholesterol biosynthesis. In another

set of wells, test compound was applied for 8 and 24 h and

tracing of cholesterol biosynthesis was performed by adding

¹⁴C-acetate to these wells for the last 2 h. At the end of the

2-h labeling period, cellular lipids were extracted twice from

each well using 2-propanol followed by n-hexane/2-propanol

(3:2). After evaporation of solvent under nitrogen, lipids were

saponified in KOH/methanol/water, and nonsaponifiable lipids

were extracted with n-hexane/ether (1:1). After drying under

nitrogen, nonsaponifiable lipids were separated by thin-layer

chromatography (TLC) using n-hexane/ether/acetic acid

(60:40:1). Radioactive lipids were identified using radioactive

standards running in separate lanes, and radioactivity was

quantified using a phosphor imager (Molecular Dynamics). The

results are given as the percentage of inhibition of cholesterol

synthesis after 2, 8, or 24 h of incubation in the presence of

test compound.

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J M021120F

Article Doi

Synthesis and structure-activity studies of novel orally active non-terpenoic 2,3-oxidosqualene cyclase inhibitors

DOI: 10.1021/jm021120f

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