Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 3. Discovery of a novel series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas with weak toxicological effects on adrenal glands

Article abstract of DOI:10.1021/jm980399q

A series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas were prepared and evaluated for their ability to inhibit intestinal acyl-CoA:cholesterol O- acyltransferase and to inhibit accumulation of cholesteryl esters in macrophages in vitro. In vivo hypocholesterolemic activity was assessed in cholesterol-fed rats by oral administration as a dietary admixture and/or by gavage in a PEG400 vehicle. Modification of the alkyl substituent on the N'- aryl moiety and on the urea nitrogen significantly influenced macrophage assay in vitro. Toxicological study revealed a distinct relationship between macrophage assay and the toxicity observed in adrenal glands of rabbits treated with representatives of this series of compounds. Investigations utilizing the macrophage assay as an indicator for adrenal toxicity led to the identification of compounds 1g (FR190809) and 1k (FR186485, or FR195249 as its hydrochloride salt) as potent, nonadrenotoxic, orally efficacious ACAT inhibitors irrespective of the administration method.

Full text of DOI:10.1021/jm980399q

4408
J . Med. Chem. 1998, 41, 4408-4420
In h ibitor s of Acyl-CoA:Ch olester ol O-Acyltr a n sfer a se. 3. Discover y of a Novel
Ser ies of N-Alk yl-N-[(flu or op h en oxy)ben zyl]-N′-a r ylu r ea s w ith Wea k
Toxicologica l Effects on Ad r en a l Gla n d s
Akira Tanaka,*,† Takeshi Terasawa,† Hiroyuki Hagihara,‡ Noriko Ishibe,‡ Masae Sawada,‡ Yuri Sakuma,§
Masaharu Hashimoto,| Hisashi Takasugi,† and Hirokazu Tanaka
Medicinal Chemistry Research Laboratories, Medicinal Biology Research Laboratories, Toxicology Research
Laboratories, and New Drug Research Laboratories, Fujisawa Pharmaceutical Company Ltd., 2-1-6 Kashima,
Yodogawa-ku, Osaka 532-8514, J apan, and Exploratory Research Laboratories, Fujisawa Pharmaceutical
Company Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, J apan
Received J uly 7, 1998
A series of N-alkyl-N-[(fluorophenoxy)benzyl]-N′-arylureas were prepared and evaluated for
their ability to inhibit intestinal acyl-CoA:cholesterol O-acyltransferase and to inhibit ac-
cumulation of cholesteryl esters in macrophages in vitro. In vivo hypocholesterolemic activity
was assessed in cholesterol-fed rats by oral administration as a dietary admixture and/or by
gavage in a PEG400 vehicle. Modification of the alkyl substituent on the N′-aryl moiety and
on the urea nitrogen significantly influenced macrophage assay in vitro. Toxicological study
revealed a distinct relationship between macrophage assay and the toxicity observed in adrenal
glands of rabbits treated with representatives of this series of compounds. Investigations
utilizing the macrophage assay as an indicator for adrenal toxicity led to the identification of
compounds 1g (FR190809) and 1k (FR186485, or FR195249 as its hydrochloride salt) as potent,
nonadrenotoxic, orally efficacious ACAT inhibitors irrespective of the administration method.
In tr od u ction
various classes of ACAT inhibitors have consistently
been observed in the adrenal glands of certain species10-17
and are considered to be dose-limiting effects and thus
make the development of ACAT inhibitors more dif-
ficult. With respect to the mechanisms of this toxicity,
although some reports have been published,11b-e,13b,15,16
it still remains unclear whether this adrenotoxicity is
related to ACAT inhibition13b,15 or not.11c,14d-f In our
previously reported series,17 FR182980 showed adrenal
toxicity in rabbits and dogs. In contrast, drug-related
histopathologic alterations to the adrenal glands of
rabbits and/or dogs were not observed with the related
compounds FR179254, FR186054, and FR180734 (Chart
1). The IC50 values of these compounds for the ACAT
enzyme obtained from rabbit intestinal microsomes
were 30, 25, 99, and 14 nM, respectively, and the toxicity
of FR182980 was deemed to be unrelated to ACAT
inhibitory activities (Table 2). However, in the mac-
rophage assay, these compounds showed IC50’s of 48,
80, 350, and 7400 nM (Table 2). Though the precise
reason is unclear at this time, the compound with potent
macrophage ACAT inhibitory activity induced adrenal
toxicity, and the possibility was therefore suggested that
macrophage ACAT inhibition and adrenotoxicity are
related. With regards to reported ACAT inhibitors,
though it has mostly been concluded that ACAT inhibi-
tion and adrenotoxicity are not mechanistically
related,11c,14d-f when examining the published data in
detail, the same tendency as our observations can be
seen; thus, compounds possessing quite potent activity
in the macrophage assay are found to be toxic.14b-d
Therefore, our interests were directed at identifying
ACAT inhibitors with high in vivo potency and no
adrenotoxicity by chemical modification of FR182980
utilizing the macrophage assay as an indicator for
Recent epidemiological studies have shown hyperc-
holesterolemia to be associated with increased risk for
the development of atherosclerosis.1 Since therapeutic
reduction of serum cholesterol levels has been proven
to be an effective treatment of atherosclerotic disease,2
agents to control plasma lipid levels have been sought
as potential therapy.3 Acyl-CoA:cholesterol O-acyl-
transferase (ACAT, EC 2.3.1.26)4 is an intracellular
enzyme responsible for catalyzing the esterification of
free cholesterol with fatty acyl-CoA to produce choles-
teryl esters. This enzyme plays an important role in
the absorption of dietary and biliary cholesterol, the
secretion of hepatic very low-density lipoprotein (VLDL),
and the accumulation of cholesteryl esters in arterial
lesions. Furthermore, ACAT is implicated in the stor-
age of cholesteryl esters to be used as substrate for
steroidogenesis as cytosolic droplets in steroid hormone-
producing tissues, such as the adrenal gland. Inhibition
of ACAT would be expected to reduce the absorption of
cholesterol, lower serum lipid levels,5 and prevent
progression and promote regression of atherosclerotic
lesions.6 Therefore, ACAT inhibitors are very attractive
targets for development of new treatments for hyperc-
holesterolemia and atherosclerosis.7
Despite numerous studies on potent ACAT inhibi-
tors,8 in clinical trials poor efficacy has been found, and
up to the present time none have shown clinical suc-
cess.9 In addition, toxicological effects induced by
* To whom correspondence should be addressed. Phone: 06-390-
5497. Fax: 06-304-5435.
† Medicinal Chemistry Research Laboratories.
‡ Medicinal Biology Research Laboratories.
§ Exploratory Research Laboratories.
| Toxicology Research Laboratories.
New Drug Research Laboratories.
10.1021/jm980399q CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/07/1998

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4409
Ch a r t 1. ACAT Inhibitors
Sch em e 1. Preparation of Target Moleculesa
a
Method A reagents: (1) R3NH2; (2) NaBH4, EtOH. Method B reagents: (1) ketone; (2) NaBH4, EtOH. Method C reagents: phenyl
N-arylcarbamate (6), Et3N. Reagents: (a) ClCO2Ph, PhNMe2, CH2Cl2; (b) ClCO2Ph, ClCH2CH2Cl.
adrenal toxicity. Thus we designed, prepared, and
evaluated the novel series of N-alkyl-N-[(fluorophen-
oxy)benzyl]-N′-arylureas represented by 1 as a related
series to FR182980 (Chart 1). Since replacement of the
N′-aryl moiety reduced the toxicological effect exempli-
fied by the conversion of FR182980 to FR179254, these
modifications might be expected to strongly influence
physicochemical characteristics and biological profile as
well as adrenotoxicity. In this paper, we wish to disclose
the synthesis, structure-activity relationships, and
toxicological evaluation of this novel series of ACAT
inhibitors.
1 was carried out by treatment with phenyl N-arylcar-
bamates 6a ,17ab-i in various solvents (toluene, DMF,
and CH2Cl2) (method C). Carbamates 6a -g were
readily obtained from the corresponding arylamines
5a ,18b-g utilizing N,N-dimethylaniline as a base. How-
ever, in the case of 5h ,i,19 the main product was a urea
compound resulting from self-dimerization under the
reaction conditions. Therefore, carbamates 6h ,i were
prepared as hydrochloride salts under nonbasic condi-
tions. In the free form, these carbamates 6h ,i gradu-
ally convert to the dimerized urea compounds.
The synthetic method for benzaldehydes 2a ,b was
described previously.17a Meta-substituted benzaldehyde
2c was prepared by the condensation of 3-hydroxybenzyl
alcohol (7) with 1-chloro-4-fluorobenzene under the
reported conditions,20 followed by MnO2 oxidation
(Scheme 2). Benzylamine 3 was obtained from reaction
of 4-fluorobenzonitrile (9) with 4-fluorophenol, followed
by LiAlH4 reduction (Scheme 2).
Ch em istr y
The basic synthetic route to the novel trisubstituted
urea compounds 1a -f,j-k ,n -a a prepared in this work
is summarized in Scheme 1 and Table 1. Reductive
amination of benzaldehydes 2a ,b,17ac or benzylamine
3 with various alkylamines or carbonyl compounds
(methods A, B) provided the secondary amines 4a -o
(Table 1). Transformation to the final urea compounds
The synthetic routes to highly substituted aminopy-
ridines 5b-f,h or pyrimidine 5g, the precurser of

4410 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Tanaka et al.
Sch em e 2a
a
Reagents: (a) 4-F-C6H4Cl, K2CO3, CuCl, 8-quinolinol (cat.), DMI; (b) MnO2, CHCl3; (c) 4-F-C6H4OH, K2CO3, DMF; (d) LiAlH4, THF.
Ta ble 1. Structures and Synthetic Methods of Secondary
Hydrogenation of 13 provided 17, which showed two
singlet peaks at 7.29 and 7.75 ppm in the 1H NMR
Amines 4
spectrum. The structure of 12 was determined indi-
rectly by desulfurization of ureas 1c,e, which provided
1l,m , respectively (Scheme 6). 1l showed two coupled
doublet signals at 6.65 and 8.24 ppm (J ) 7.9 Hz), and
1m showed a doublet signal derived from ortho coupling
on the pyridine ring at 8.44 ppm (J ) 8.3 Hz). These
data suggested that the methylthio group on the pyri-
dine ring of 12 was at the 4-position. Aminopyrimidine
5g was prepared from 1821 by treatment with NaSMe,
followed by reduction of the nitro group (Scheme 4). 5h
was synthesized from 4H-pyran-4-one 20, which was
converted to 1H-pyridin-4-one 22 in two steps, followed
by nitration, chlorination, substitution with NaSMe, and
hydrogenation (Scheme 5).
Ureas 1g,i bearing sulfone substituents on the pyri-
dine ring were obtained by m-CPBA oxidation of
FR182980 and 1e, respectively (Scheme 6). 1h was
prepared by selective monosubstitution of the methyl-
sulfonyl group at the 4-position of the pyridine ring of
1g as a leaving group, the structure of which was
determined by desulfurization to provide 1a b, which
showed a doublet signal derived from ortho coupling on
1
the pyridine ring at 8.81 ppm (J ) 8.8 Hz) in the H
NMR spectrum. In this case also, the 4-position of the
pyridine ring was more reactive.
Resu lts a n d Discu ssion
carbamates 6b-h which were the reagents for urea
formation, are shown in Schemes 3-5. 5b-f were
synthesized from 1118 as a key intermediate (Scheme
3). Reaction of this intermediate with an excess amount
of NaOMe followed by reduction of the nitro function
provided 5b. Of the two chlorine atoms on the pyridine
ring of 11, the 4-chloro was more reactive; this is the
same as the reactivity of 2,4-dichloropyridine without
any other substituent such as a nitro group. Therefore,
the reaction of 11 with 1 equiv of NaSMe provided
4-(methylthio)pyridine 12 which was converted to 5c by
substitution of the 2-position chlorine atom with NaOMe,
followed by hydrogenation, or to 5d by reduction of the
nitro function. The regiospecific synthesis of aminopy-
ridine 5e, which has the same substituents as 5d but
in the opposite positions, was accomplished as follows.
Highly substituted nitropyridine 14 was prepared by a
similar procedure to the synthesis of 5c and was then
hydrolyzed to 15. Subsequent chlorination with POCl3
and reduction provided 5e. 5f was readily obtained
from 11 by reduction of the nitro function. The reactiv-
ity of the two chlorine atoms of nitropyridine 11 was
confirmed by structure determination of 12 and 13.
The compounds prepared were evaluated for their
ability to inhibit rabbit intestinal ACAT and to inhibit
acetylated LDL-induced accumulation of cholesteryl
esters in mouse peritoneal macrophages in vitro.17 In
vivo hypocholesterolemic activity was assessed in cho-
lesterol-fed rats by oral administration of the test
compounds as a dietary admixture in a cholesterol-
enriched diet.17 Certain of the compounds possessing
potent in vivo effects were also evaluated in a different
administration model, i.e., dosing by gavage in poly-
(ethylene glycol) (PEG400) as a vehicle,17 since it has
been shown that the bioavailability of ACAT inhibitors
can be markedly influenced by dosing mode.10,18,22 The
in vitro activity is expressed as the nanomolar concen-
tration of a compound required to inhibit 50% of the
enzyme activity (IC50), the in vivo cholesterol-lowering
activity is presented in terms of percent reduction at
the dose or ED50, the effective dose to reduce plasma
total cholesterol level by 50%. Furthermore, selected
compounds were examined for adrenal toxicity in rab-
bits (n ) 2) at a single dose of 5 mg/kg iv which is the

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4411
Sch em e 3a
a
Reagents: (a) NaOMe, MeOH; (b) H2, 10% Pd/C; (c) H2, Raney-Ni; (d) NaSMe, MeOH; (e) concd HCl, EtOH; (f) POCl3.
Sch em e 4a
and drug-induced necrosis of the adrenal glands was
assessed at 24 h after dosing and is expressed as
observed (+) or not observed (-), compared to the
controls.
Biological data for urea compounds with various N′-
aryl moieties are shown in Table 2. Although these
modifications did not have a large effect on in vitro
inhibitory activity of rabbit intestinal ACAT, the inhibi-
tory activity of acetylated LDL-induced accumulation
of cholesteryl esters in mouse peritoneal macrophages
was markedly influenced. Conversion of the pyridine
to a pyrimidine did not affect activity in the macrophage
assay (1a ). However, replacement of the bis(meth-
ylthio) group in FR182980 by a bis(methyloxy) group
reduced the inhibitory activity against macrophage
ACAT (1b). On the other hand, conversion of only R2
into a methoxy group (1c), leaving the R1 methylthio
a
Reagents: (a) NaSMe, DMF; (b) H2, Raney-Ni, dioxane,
MeOH.
dose our previously reported FR145237 induced signifi-
cant adrenal toxicity.16 PEG400 was used as a vehicle,
Sch em e 5a
a
Reagents: (a) BnNH2; (b) HCO2H, Pd-black, MeOH; (c) HNO3, H2SO4; (d) POCl3; (e) NaSMe, MeOH; (f) H2, 10% Pd/C, EtOH.

4412 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Tanaka et al.
Sch em e 6a
a
Reagents: (a) m-CPBA, CH2Cl2; (b) NaSMe, MeOH; (c) Raney-Ni, EtOH.
group intact, significantly increased potency (IC50 ) 13
nM). Furthermore, replacement of the methylthio
group of R1 by chlorine reduced the activity (1f), but a
similar modification at R2 retained high activity (1e).
Oxidation of sulfide in 1e to sulfone resulted in reduced
activity (1i). Although 1g, which is a bis(methylsulfo-
nyl) compound derived from FR182980, also showed
relatively weak activity, the monomethylsulfonyl com-
pound 1h had increased activity. Replacement of the
methylthio group at R2 by a simple methyl group still
retained high activity (1j); however, conversion of the
remaining methylthio group in 1j to a methyl group
showed relatively reduced activity (1k ). On the basis
of these results, it was considered that modification of
R1 significantly influenced macrophage ACAT activity,
as compared to R2. As a consequence of these modifica-
tions, we obtained many FR182980 derivatives possess-
ing various macrophage activity as tools to investigate
the relationship between macrophage assay and adreno-
toxicity.
With respect to in vivo hypocholesterolemic activity,
most compounds showed potent activity (ED50 e 0.1 mg/
kg) dosing as a dietary admixture, with the exception
of 1d ,h ,l,m . Furthermore, in a different administration
model, i.e., by gavage in PEG400 as a vehicle, several
compounds exhibited potent cholesterol-lowering effects
(1a ,e,g,k ). The selected five compounds, i.e., 1b and the
four compounds described above, were next evaluated
for adrenal toxicity in rabbits (n ) 2) at a single dose of
5 mg/kg iv as a solution in PEG400. Necrosis of the
zona fasciculata cortical cells in the adrenal cortex was
observed in the animals treated with compounds 1a ,e,
which exhibit potent inhibitory activity of foam cell
formation. However, drug-induced necrosis was not
observed in the adrenal glands taken from rabbits
treated with compounds 1b,g,k , possessing relatively
weak activity in the macrophage assay. Moreover, the
hydrochloride salt of compound 1k was assesed for
adrenotoxicity in dog (n ) 1), which has been reported
to be the most sensitive species to the adrenal effects
of ACAT inhibitors,23 at a single dose of 10 mg/kg po as
a suspension in PEG400. Though FR182980 induced
severe toxicological effects under these conditions,17c
drug-related histopathologic alterations to the adrenal
gland of dog were not observed for the hydrochloride
salt of compound 1k , irrespective of its comparable
plasma drug level (the maximum plasma concentrations
(Cmax) for this compound and FR182980 were 144 and
180 ng/mL, respectively). Among these compounds 1g,k
showed an excellent biological profile comparable to that
of the previously reported FR186054, lacked adrenal
toxicity, and were selected for further development. As
a consequence of these toxicological evaluations, a
distinct relation between macrophage assay and adreno-
toxicity was observed in this series, though the reason
and significance are still unclear at this point. In
addition, it was deemed that the borderline in the
macrophage assay for induction of adrenotoxicity was
about 100 nM in this series. It should be noted that a
correlation between toxicity and ClogP values (Table 2)
or partial charge on the nitrogen atom of the aryl moiety
(data not shown) was not observed.
Effects of the alkyl substituent (R3) on the urea
nitrogen were next examined (Table 3), since modifica-
tion at this position was postulated to markedly influ-
ence the conformation of the molecule and as a result
its biological activity. As described in the modification
of the N′-aryl moiety, these modifications did not have
a large effect on in vitro inhibitory activity of rabbit
intestinal ACAT. Compounds 1n ,q,r showed very po-
tent foam cell formation inhibitory activity (IC50 e 10
nM). In vivo hypocholesterolemic activity of compounds
1n ,o,q-s,v were very potent when dosed as a dietary
admixture; however, 1r ,v only showed moderate effects

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4413

4414 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Ta ble 3. Effect of Varying N-Alkyl Group on Biological Activities
Tanaka et al.
h
a-g,*See corresponding footnotes of Table 2. Different lot of enzyme was used. IC50 is an estimated value based on the relative IC50
i
of FR182980 for this batch of enzyme. For example, 1n showed 0.78 × IC50 of FR182980. Values in parentheses denote percent inhibition
at the concentration indicated. ND denotes not determined.
when dosed by gavage in a PEG400 vehicle. Compound
1t was also weak in vivo in comparison to the unsub-
stituted 1q; this may possibly be due to metabolism or
poor bioavailability. Interestingly, although 1u ,v, a pair
of enantiomers, exhibited about the same in vitro
activity in both assays, the in vivo activity of 1v (R-
isomer) was higher. On the basis of the results dis-
cussed previously, 1u ,v were suggested to be nontoxic;

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4415
Ta ble 4. Effect of Varying Substituent on Terminal Phenyl and Position of Phenoxy Moiety on Biological Activities
h,i
a-g,*See corresponding footnotes of Table 2. See corresponding footnotes of Table 3. ND denotes not determined.
however, further evaluation of toxicity was not per-
formed because of their insufficient in vivo activity. Of
particular note, the introduction of a methyl group onto
the N-benzyl of 1q reduced in vitro activity in the
macrophage assay (1u ,v); however, the activity was
restored by introduction of a dimethyl group (1w ,x).
In our previous report, a bromine atom had been
identified as an alternative substituent for fluorine on
the terminal phenoxy moiety.17a In addition, a benzyl
group as an alkyl substituent on the urea nitrogen has
been shown to give a good biological profile, both in this
series (1q) and our previous series represented by
FR186054.17b,c Therefore, we opted to examine the
combination of bromine atom and a cycloheptyl or
benzyl group (Table 4). These modifications gave
compounds 1y,z possessing excellent biological activity
both in vitro and in vivo. In particular, 1z was still
efficacious when dosed by gavage. However, as pre-
dicted from its potent inhibitory activity of foam cell
formation, this compound induced significant adreno-
toxicity in treated rabbits at a single dose of 5 mg/kg
iv.
For the purpose of investigation of the position of the
4-fluorophenoxy moiety, N-3-(4-fluorophenoxy)benzyl
compound (1a a ) was synthesized and evaluated (Table
4). This compound showed potent hypocholesterolemic
effect; however, its macrophage ACAT inhibitory activ-
ity was extremely potent (IC50 ) 2.9 nM), and it was
rejected for development based on its predicted adreno-
toxicity.
On the basis of the results discussed above, com-
pounds 1g and 1k (or its crystalline hydrochloride salt,
since 1k was an amorphous solid) were identified as
potent, nontoxic, orally efficacious ACAT inhibitors
comparable to our previously reported FR186054,17b,c
independent of the dosing mode, and selected for further
development as a new treatment for hypercholester-
olemia and atherosclerosis.
is then transferred to a membrane pool and induces a
loss of cell viability leading to the cell toxicity.15 Since
adrenal gland is a steroid hormone-producing tissue and
is replete with cholesteryl esters, it is considered that
the effect of ACAT inhibition is direct. Furthermore,
the possibility of the existence of isozymes of ACAT, or
multiple cholesterol esterification enzymes, has been
suggested recently.24-28 It was also reported that
ACAT-deficient (Acact-/-) mice exhibited a marked
reduction in cholesteryl ester levels in the adrenal
glands and peritoneal macrophages. However, the livers
of these mice still contained significant amounts of
cholesteryl esters as well as esterification activity, and
sterol absorption in the intestine was unaffected,26
which was consistent with the results of tissue expres-
sion studies in wild-type mice that the expressions of
mouse ACAT mRNA and protein were at a high level
in steroidogenic tissues, sebaceous glands, peritoneal
macrophages, and atherosclerotic lesions, but not in the
liver or the intestine.27 In this connection, recently an
ACAT homologue, highly expressed in liver and intes-
tine, has been cloned and designated ACAT II or ACAT-
related gene product (ARGP1).28 If adrenotoxicity is
derived from ACAT inhibition, it must be related to
inhibitory activity of adrenal ACAT by these inhibitors.
Unfortunately, we do not yet have data for the adrenal
ACAT assay. However, based on the reports described
above, there is a possibility that ACAT in adrenal glands
and ACAT in peritoneal macrophages are the same
isoform, or derived from the same ACAT gene, and are
different from that of the liver- or intestine-derived
ACAT; it was thus considered that a relationship
between macrophage assay and adrenotoxicity would
indirectly be shown. If the adrenal toxicity observed is
actually mechanism-based, drug-design directed at the
development of inhibitors that cannot reach undesirable
tissues (adrenal gland) or isozyme-specific inhibitors is
a suitable objective for future research. Data from the
adrenal ACAT assay, as well as more detailed molecular
biological aspects of the ACAT enzyme, must be inves-
tigated further.
In this series, a distinct relationship between mac-
rophage assay and adrenotoxicity was observed. Al-
though the precise reason remains unclear at this time,
it has been reported that ACAT inhibition results in the
intracellular buildup of free cholesterol, some of which

4416 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Tanaka et al.
filtrate was evaporated. The residue was purified by silica
gel column chromatography (hexane-EtOAc, 4:1 elution) to
give 2c (44.2 g, 90%) as an oil: 1H NMR (CDCl3) δ 6.93-7.17
(4H, m), 7.18-7.32 (1H, m), 7.35-7.64 (3H, m), 9.96 (1H, s);
Con clu sion
In summary, we have prepared a novel series of
N-alkyl-N-[(fluorophenoxy)benzyl]-N′-arylureas as a re-
lated series to FR182980 and evaluated them as ACAT
inhibitors. The SAR study in this series of compounds
revealed the following features: (1) Modification of the
substituent at the 4-position on the N′-pyridine ring
does not influence in vitro inhibitory activity against
intestinal ACAT; however, it did show a large effect on
the macrophage assay. (2) Varing the alkyl substituent
on the urea nitrogen also influences macrophage assay
rather than the intestinal ACAT assay. (3) Changing
the position of the 4-fluorophenoxy moiety to the 3-posi-
tion on the inner phenyl ring retains activity. (4)
Toxicological study revealed a distinct relationship
between macrophage assay and adrenotoxicity in this
series.
Utilizing the macrophage assay as an indicator for
adrenotoxicity, N-cycloheptyl-N-[4-(4-fluorophenoxy)-
benzyl]-N′-[2,4-bis(methylsulfonyl)-6-methylpyridin-3-
yl]urea (1g, FR190809) and N-cycloheptyl-N-[4-(4-
fluorophenoxy)benzyl]-N′-(2,4,6-trimethylpyridin-3-
yl)urea (1k, FR186485, or FR195249 as its hydrochloride
salt) were identified as potent, nonadrenotoxic, orally
efficacious ACAT inhibitors irrespective of the admin-
istration method and were selected for further develop-
ment. The details of pharmacological studies on these
compounds will be the subject of further communica-
tions from these laboratories.
IR (neat) 3074, 2837, 2731, 1701, 1585, 1502, 1481, 1450 cm-1
;
MS m/z 231 (MH+ + MeOH - H2O).
4-(4-F lu or op h en oxy)ben zyla m in e (3). To a solution of
4-fluorobenzonitrile (9) (50.0 g, 413 mmol) and 4-fluorophenol
(50.9 g, 454 mmol) in DMF (400 mL) was added powdered K2-
CO3 (62.8 g, 454 mmol), and the mixture was stirred at 150
°C for 6 h. The reaction mixture was cooled and poured into
ice water (2.5 L). The resulting precipitate was collected by
filtration, washed with water, and dried to give 4-(4-fluorophe-
noxy)benzonitrile (10) (87.6 g, 99%) as a solid: 1H NMR
(DMSO-d6) δ 7.08 (2H, d, J ) 9.0 Hz), 7.13-7.40 (4H, m), 7.84
(2H, d, J ) 9.0 Hz); IR (KBr) 3188, 3076, 2220, 1649, 1608,
1483 cm-1; MS m/z 214 (MH+).
To a suspension of LiAlH4 (5.69 g, 150 mmol) in THF (300
mL) was added dropwise a solution of 10 (21.3 g, 100 mmol)
in THF (200 mL) at 5 °C, and the mixture was stirred at room
temperature for 4 h. To the mixture were added anhydrous
NaF (16.8 g, 400 mmol) and water (5.41 mL) at 5 °C, and the
mixture was stirred at room temperature for 45 min. The
insoluble materials were removed by filtration and washed
with THF. The filtrate was evaporated, and the residue was
purified by silica gel column chromatography (CH2Cl2-MeOH,
10:1 elution) to give 3 (21.4 g, 99%) as a solid: 1H NMR
(DMSO-d6) δ 3.69 (2H, s), 6.86-7.13 (4H, m), 7.13-7.40 (4H,
m); IR (KBr) 3352, 3269, 3043, 1645, 1606, 1495, 1217 cm-1
;
MS m/z 201 (MH+ - NH3).
3-Am in o-2,4-d im eth oxy-6-m eth ylp yr id in e (5b). To a
solution of 2,4-dichloro-6-methyl-3-nitropyridine (11) (30.3 g,
147 mmol) in MeCN (100 mL) was added NaOMe (28% MeOH
solution) (85 mL, 440 mmol) at 5 °C, and the mixture was
stirred at 80 °C for 6 h. The mixture was poured into water
and extracted with EtOAc. The organic layer was washed with
water and brine, dried (MgSO4), and evaporated, and the
residue was purified by silica gel column chromatography
(hexane-EtOAc, 4:1 elution) to give 2,4-dimethoxy-6-methyl-
3-nitropyridine (28.2 g, 97%) as a solid: 1H NMR (DMSO-d6)
δ 2.44 (3H, s), 3.92 (3H, s), 3.94 (3H, s), 6.97 (1H, s); IR (KBr)
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were measured on
a Bu¨chi 535 apparatus in open capillaries and are uncorrected.
IR spectra were recorded on a Horiba Spectradesk FT-210
spectrometer as KBr disks, neat, or films as indicated. NMR
spectra were measured on a Bruker AC200P (1H, 200 MHz)
spectrometer. Chemical shifts are given in parts per million,
and tetramethylsilane was used as the internal standard for
spectra obtained in DMSO-d6 and CDCl3. All J values are
given in hertz. Mass spectra were measured on a Hitachi
model M-1000H mass spectrometer using APCI for ionization.
Elemental analyses were carried out on a Perkin-Elmer 2400
CHN elemental analyzer. Reagents and solvents were used
as obtained from commercial suppliers without further puri-
fication. Column chromatography was performed using silica
gel, and reaction progress was determined by TLC analysis
on silica gel-coated glass plates. Visualization was with UV
light (254 nm) or iodine. The term “evaporated” or “evapora-
tion” refers to removal of solvent on a rotary evaporator at
reduced pressure.
3035, 3005, 2960, 2868, 1601, 1581, 1531, 1460, 1375 cm-1
;
MS m/z 199 (MH+).
To a solution of the above nitropyridine (28.1 g, 142 mmol)
in 1,4-dioxane (200 mL) and MeOH (100 mL) was added 10%
palladium on carbon (14 g), and the mixture was hydrogenated
for 4.5 h under atmospheric pressure. The catalyst was
filtered off, and the filtrate was evaporated. The residue was
purified by silica gel column chromatography (hexane-
EtOAc, 2:1 elution) to give 5b (23.4 g, 98%) as an oil: 1H NMR
(DMSO-d6) δ 2.26 (3H, s), 3.79 (3H, s), 3.82 (3H, s), 3.96 (2H,
br s), 6.52 (1H, s); IR (neat) 3458, 3373, 2945, 2856, 1605, 1446,
1345 cm-1; MS m/z 169 (MH+).
3-Am in o-2-m eth oxy-6-m eth yl-4-(m eth ylth io)p yr id in e
(5c). To a solution of 11 (20.7 g, 100 mmol) in MeOH (250
mL) was added dropwise a solution of NaSMe (7.01 g, 100
mmol) in MeOH (150 mL) at room temperature, and the
mixture was stirred for 5 h. The mixture was evaporated, and
the residue was poured into water and extracted with EtOAc.
The organic layer was washed with water and brine, dried
(MgSO4), and evaporated, and the residue was purified by
silica gel column chromatography (hexane-EtOAc, 3:1 to 1:1
elution) to give 2-chloro-6-methyl-4-(methylthio)-3-nitropyri-
dine (12) (16.6 g, 76%) as a solid: 1H NMR (DMSO-d6) δ 2.53
(3H, s), 2.65 (3H, s), 7.53 (1H, s); IR (KBr) 3091, 2993, 2926,
1574, 1531, 1435, 1346, 1234 cm-1; MS m/z 219, 221 (MH+).
3-(4-F lu or op h en oxy)ben za ld eh yd e (2c). To a solution
of 3-hydroxybenzyl alcohol (7) (51.0 g, 411 mmol) and 1-chloro-
4-fluorobenzene (65.7 mL, 617 mmol) in 1,3-dimethyl-2-imi-
dazolidinone (DMI) (200 mL) were added powdered K2CO3
(34.1 g, 247 mmol), CuCl (814 mg, 8.2 mmol), and 8-quinolinol
(1.19 g, 8.2 mmol). The mixture was stirred at 150 °C for 3
days. The mixture was poured into water and extracted with
EtOAc. The organic layer was washed with water and brine,
dried (MgSO4), and evaporated, and the residue was purified
by silica gel column chromatography (hexane-EtOAc, 3:1 to
2:1 elution) to give 3-(4-fluorophenoxy)benzyl alcohol (8) (49.7
g, 55%) as an oil: 1H NMR (CDCl3) δ 4.66 (2H, s), 6.83-7.15
(7H, m), 7.31 (1H, dd, J ) 8.0, 8.0 Hz); IR (neat) 3352, 3074,
To a solution of 12 (13.3 g, 60.6 mmol) in MeOH (150 mL)
was added NaOMe (28% MeOH solution) (23.4 mL, 121 mmol),
and the mixture was refluxed for 7 h. After cooling, the
resulting solid was collected by filtration and washed with
MeOH-iPE to give 2-methoxy-6-methyl-4-(methylthio)-3-ni-
tropyridine (10.3 g, 79%) as a solid: 1H NMR (DMSO-d6) δ
2.46 (3H, s), 2.57 (3H, s), 3.94 (3H, s), 7.07 (1H, s); IR (KBr)
2875, 1610, 1585, 1502, 1448 cm-1
To a solution of 8 (49.6 g, 227 mmol) in CHCl3 (500 mL)
was added activated MnO2 (98.8 g, 1.14 mol), and the mixture
was refluxed for 3.5 h. The mixture was filtered, and the
.

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4417
3024, 2997, 2951, 2924, 2856, 1587, 1541, 1495, 1452 cm-1
;
(1H, s); IR (KBr) 3417, 3300, 3207, 2922, 1618, 1558, 1443,
1367 cm-1; MS m/z 189, 191 (MH+).
MS m/z 215 (MH+).
3-Am in o-2,4-d ich lor o-6-m eth ylp yr id in e (5f). To a solu-
tion of 11 (4.14 g, 20 mmol) in 1,4-dioxane (50 mL) and MeOH
(50 mL) was added Raney nickel (2 g), and the mixture was
hydrogenated for 4 h under atmospheric pressure. Raney
nickel was filtered off, and the filtrate was evaporated. The
residue was purified by silica gel column chromatography
(hexane-EtOAc, 4:1 elution) to give 5f (3.53 g, 100%) as an
oil: 1H NMR (DMSO-d6) δ 2.28 (3H, s), 5.52 (2H, br s), 7.23
(1H, s); IR (neat) 3479, 3385, 3188, 2924, 1616, 1576, 1543,
1471, 1367, 1309 cm-1; MS m/z 177, 179, 181 (MH+).
To a suspension of the above nitropyridine (12.1 g, 56.5
mmol) in 1,4-dioxane (100 mL) and MeOH (100 mL) was added
10% palladium on carbon (9 g), and the mixture was hydro-
genated for 3 h under atmospheric pressure. The catalyst was
filtered off, and the filtrate was evaporated. The residue was
purified by silica gel column chromatography (hexane-EtOAc,
4:1 elution) to give 5c (9.07 g, 87%) as an oil: 1H NMR (DMSO-
d6) δ 2.26 (3H, s), 2.43 (3H, s), 3.84 (3H, s), 4.39 (2H, br s),
6.64 (1H, s); IR (neat) 3444, 3350, 2983, 2947, 2922, 1585,
1558, 1462, 1387 cm-1; MS m/z 185 (MH+).
5-Am in o-4,6-bis(m eth ylth io)-2-m eth ylp yr im id in e (5g).
To a solution of 4,6-dichloro-2-methyl-5-nitropyrimidine (18)
(7.95 g, 38.2 mmol) in DMF (150 mL) was added NaSMe (5.89
g, 84.0 mmol), and the mixture was stirred at room temper-
ature for 6 h and then stirred at 150 °C for 10 h. After cooling,
the mixture was poured into ice water and extracted with
EtOAc. The organic layer was washed with water and brine,
dried (MgSO4), and evaporated, and the residue was purified
by silica gel column chromatography (hexane-EtOAc, 4:1
elution) to give 4,6-bis(methylthio)-2-methyl-5-nitropyrimidine
(19) (5.19 g, 59%) as a solid: 1H NMR (DMSO-d6) δ 2.54 (6H,
s), 2.66 (3H, s); IR (KBr) 3007, 2926, 1518, 1425, 1329, 1201
cm-1; MS m/z 232 (MH+).
To a suspension of 19 (5.17 g, 22.4 mmol) in 1,4-dioxane
(120 mL) and MeOH (50 mL) was added Raney nickel (10 g),
and the mixture was hydrogenated for 2.5 h under atmospheric
pressure. Raney nickel was filtered off, and the filtrate was
evaporated. The residue was purified by silica gel column
chromatography (hexane-EtOAc, 4:1 elution) to give 5g (4.11
g, 91%) as a solid: 1H NMR (DMSO-d6) δ 2.45 (3H, s), 2.52
(6H, s), 4.57 (2H, br s); IR (KBr) 3363, 3284, 3197, 2922, 1625,
1531, 1417, 1363 cm-1; MS m/z 202 (MH+).
3-Am in o-2,6-d im eth yl-4-(m eth ylth io)p yr id in e (5h ). A
mixture of 2,6-dimethyl-4H-pyran-4-one (20) (100 g, 806 mmol)
and benzylamine (88 mL, 806 mmol) was heated at 180 °C for
10 h. After cooling, the reaction mixture was purified by silica
gel column chromatography (EtOAc-MeOH, 10:1 to 5:1 elu-
tion) to give 1-benzyl-2,6-dimethyl-1H-pyridin-4-one (21) (135.2
g, 79%) as a solid: 1H NMR (DMSO-d6) δ 2.19 (6H, s), 5.22
(2H, s), 6.04 (2H, s), 6.92-7.02 (2H, m), 7.23-7.45 (3H, m);
IR (KBr) 3377, 3167, 1641, 1550, 1477, 1450, 1371, 1346, 1207
cm-1; MS m/z 214 (MH+).
To a suspension of 21 (135 g, 633 mmol) in MeOH (800 mL)
were added palladium-black (40 g) and HCO2H (72.2 mL), and
the mixture was refluxed for 10 h. The catalyst was removed
by filtration, and the filtrate was evaporated. The residue was
purified by silica gel column chromatography (CH2Cl2-MeOH,
10:1 to 5:1 elution) to give 2,6-dimethyl-1H-pyridin-4-one (22)
(68.0 g, 87%) as a solid: 1H NMR (DMSO-d6) δ 2.15 (6H, s),
5.84 (2H, s); IR (KBr) 3365, 2902, 2777, 1651, 1628, 1504, 1437,
1394, 1367, 1200 cm-1; MS m/z 124 (MH+).
To ice-cooled fuming H2SO4 (30% SO3) (98 mL) was added
fuming HNO3 (d ) 1.52) (90 mL), and the mixture stirred at
5 °C for 40 min. To the mixture was added 22 (66.0 g, 536
mmol) carefully at 5 °C, followed by stirring at room temper-
ature for 3 days. The mixture was poured into ice water (600
mL) and adjusted to pH 3 with 5 N NaOH solution. The
resulting precipitate was collected by filtration, washed with
ice water, and dried to give 2,6-dimethyl-3-nitro-1H-pyridin-
4-one (23) (67.2 g, 75%) as a solid: 1H NMR (DMSO-d6) δ 2.22
(3H, s), 2.28 (3H, s), 6.18 (1H, s), 11.83 (1H, br); IR (KBr) 3271,
3068, 2949, 1647, 1620, 1572, 1508, 1383, 1346 cm-1; MS m/z
169 (MH+).
To POCl3 (260 mL, 2.8 mol) was added 23 (67.2 g, 400
mmol), and the mixture stirred at 100 °C for 10 h. The
mixture was poured into water (1.2 L), adjusted to pH 8 with
5 N NaOH solution, and extracted with EtOAc. The organic
layer was washed with water and brine, dried (MgSO4), and
evaporated, and the residue was purified by silica gel column
chromatography (hexane-EtOAc, 1:1 elution) to give 4-chloro-
2,6-dimethyl-3-nitropyridine (24) (48.6 g, 65%) as a solid: 1H
NMR (DMSO-d6) δ 2.49 (3H, s), 2.52 (3H, s), 7.67 (1H, s); MS
m/z 187, 189 (MH+).
3-Am in o-2-ch lor o-6-m eth yl-4-(m eth ylth io)pyr idin e (5d).
To a suspension of 12 (16.0 g, 73.2 mmol) in 1,4-dioxane (200
mL) and MeOH (50 mL) was added Raney nickel (30 g), and
the mixture was hydrogenated for 3 h under atmospheric
pressure. Raney nickel was filtered off, and the filtrate was
evaporated. The residue was purified by silica gel column
chromatography (hexane-EtOAc, 6:1 to 4:1 elution) to give
5d (12.9 g, 93%) as an oil: 1H NMR (DMSO-d6) δ 2.29 (3H, s),
2.51 (3H, s), 4.93 (2H, br s), 6.98 (1H, s); IR (neat) 3429, 3350,
2922, 1606, 1570, 1529, 1468, 1444 cm-1; MS m/z 189, 191
(MH+).
3-Am in o-4-ch lor o-6-m eth yl-2-(m eth ylth io)pyr idin e (5e).
To a solution of 11 (41.4 g, 200 mmol) in MeOH (400 mL) was
added NaOMe (28% MeOH solution) (38.6 mL, 200 mmol), and
the mixture was stirred at 60 °C for 1 h. The mixture was
evaporated, and the residue was poured into water and
extracted with EtOAc. The organic layer was washed with
water and brine, dried (MgSO4), and evaporated, and the
residue was purified by silica gel column chromatography
(hexane-EtOAc, 4:1 elution) to give 2-chloro-4-methoxy-6-
methyl-3-nitropyridine (13) (30.4 g, 75%) as a solid: 1H NMR
(DMSO-d6) δ 2.51 (3H, s), 4.01 (3H, s), 7.42 (1H, s); IR (KBr)
3088, 2987, 2953, 2883, 1601, 1552, 1524, 1471 cm-1; MS m/z
203, 205 (MH+).
To a solution of 13 (30.4 g, 150 mmol) in MeOH (300 mL)
was added dropwise a solution of NaSMe (12.6 g, 180 mmol)
in MeOH (200 mL) at room temperature, and the mixture was
stirred at 50 °C for 4 h. The mixture was evaporated, and
the residue was poured into water and extracted with EtOAc.
The organic layer was washed with water and brine, dried
(MgSO4), and evaporated, and the residue was purified by
silica gel column chromatography (hexane-EtOAc, 4:1 elution)
to give 4-methoxy-6-methyl-2-(methylthio)-3-nitropyridine (14)
(30.2 g, 94%) as a solid: 1H NMR (DMSO-d6) δ 2.51 (3H, s),
2.53 (3H, s), 3.95 (3H, s), 7.11 (1H, s); IR (KBr) 3066, 2997,
2956, 2933, 2858, 1585, 1549, 1514, 1466, 1352 cm-1; MS m/z
215 (MH+).
To a suspension of 14 (30.2 g, 141 mmol) in EtOH (300 mL)
was added concentrated HCl (58.6 mL), and the mixture was
refluxed for 10 h. After cooling to 5 °C, the resulting
precipitate was collected, washed with EtOH and iPE, and
dried to give 6-methyl-2-(methylthio)-3-nitro-1H-pyridin-4-one
(15) (19.8 g 70%) as a solid: 1H NMR (DMSO-d6) δ 2.39 (3H,
s), 2.50 (3H, s), 6.62 (1H, s); IR (KBr) 2989, 2920, 1551, 1518,
1309, 1246, 1225 cm-1
.
A suspension of 15 (30.7 g, 153 mmol) in POCl3 (85.6 mL,
918 mmol) was stirred at 100 °C for 10 h. The mixture was
poured into water, neutralized with 5 N NaOH solution, and
extracted with EtOAc. The organic layer was washed with
water and brine, dried (MgSO4), and evaporated, and the
residue was purified by silica gel column chromatography
(hexane-EtOAc, 5:1 elution) to give 4-chloro-6-methyl-2-
(methylthio)-3-nitropyridine (16) (11.9 g, 36%) as a solid: 1H
NMR (DMSO-d6) δ 2.55 (3H, s), 2.59 (3H, s), 7.55 (1H, s); IR
(KBr) 3103, 3053, 2933, 1560, 1518, 1402, 1344 cm-1; MS m/z
219, 221 (MH+).
To a suspension of 16 (11.9 g, 54.3 mmol) in EtOH (200 mL)
was added Raney nickel (20 g), and the mixture was hydro-
genated for 4 h under atmospheric pressure. Raney nickel was
filtered off, and the filtrate was evaporated. The residue was
purified by silica gel column chromatography (hexane-
EtOAc, 6:1 elution) to give 5e (9.83 g, 96%) as a solid: 1H NMR
(DMSO-d6) δ 2.31 (3H, s), 2.51 (3H, s), 4.96 (2H, br s), 6.96

4418 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Tanaka et al.
To a solution of 24 (48.3 g, 259 mmol) in MeOH (400 mL)
was added dropwise a solution of NaSMe (21.8 g, 311 mmol)
in MeOH (200 mL) at room temperature, and the mixture was
refluxed for 5 h. The mixture was evaporated, and the residue
was poured into water and extracted with EtOAc. The organic
layer was washed with water and brine, dried (MgSO4), and
evaporated, and the residue was purified by silica gel column
chromatography (hexane-EtOAc, 1:1 elution) to give 2,6-
dimethyl-4-(methylthio)-3-nitropyridine (25) (36.0 g, 70%) as
a solid: 1H NMR (DMSO-d6) δ 2.48 (3H, s), 2.50 (3H, s), 2.57
(3H, s), 7.32 (1H, s); MS m/z 199 (MH+).
ethane (120 mL) at 100 °C, and the mixture was refluxed for
1 h. After cooling to room temperature, to the reaction mixture
was added dropwise a mixture of EtOAc (2 L) and THF (1 L).
The resulting precipitate was collected, washed with EtOAc
and iPE, and dried to give crude 6i (48.8 g, 70%) as a highly
hygroscopic solid: 1H NMR (DMSO-d6) δ 2.49 (3H, s), 2.69 (6H,
s), 7.15-7.75 (6H, m), 9.63 and 10.20 (total 1H, each br s); IR
(KBr) 3413, 1741, 1645, 1541, 1483, 1234, 1201 cm-1; MS m/z
257 (MH+ - HCl).
Compound 6h was also prepared as described for 6i from
5h .
To a suspension of 25 (35.8 g, 181 mmol) in EtOH (400 mL)
was added 10% palladium on carbon (35 g), and the mixture
was hydrogenated for 3 h under atmospheric pressure. The
catalyst was filtered off, and the filtrate was evaporated. The
residue was purified by silica gel column chromatography
(CH2Cl2-MeOH, 20:1 elution) to give 5h (26.2 g, 86%) as a
solid: 1H NMR (DMSO-d6) δ 2.26 (3H, s), 2.27 (3H, s), 2.43
(3H, s), 4.51 (2H, br s), 6.81 (1H, s); IR (KBr) 3429, 3307, 3194,
2927, 1633, 1568, 1552, 1468, 1431, 1389, 1292 cm-1; MS m/z
169 (MH+).
P h en yl N-[2,6-d im et h yl-4-(m et h ylt h io)p yr id in -3-yl]-
ca r ba m a te h yd r och lor id e (6h ): yield 9.76 g (38%); 1H NMR
(DMSO-d6) δ 2.61 (3H, s), 2.66 (3H, s), 2.69 (3H, s), 6.68-7.55
(5H, m), 7.74 (1H, s), 9.65 and 10.14 (total 1H, each br s); IR
(KBr) 3130, 2752, 1743, 1630, 1608, 1481, 1225, 1198 cm-1
;
MS m/z 289 (MH+ - HCl).
Meth od A. Reductive amination of benzaldehyde deriva-
tives 2a -c with various amine compounds was performed as
previously described.17
Meth od B. N-[4-(4-F lu or op h en oxy)ben zyl]tetr a h yd r o-
p yr a n -4-yla m in e (4c). A mixture of 3 (4.35 g, 20 mmol) and
tetrahydropyran-4-one (2.22 mL, 24 mmol) was heated at 120
°C for 4 h. The mixture was then cooled to room temperature
and dissolved in EtOH (80 mL). To the solution was added
carefully NaBH4 (757 mg, 20 mmol), and the mixture was
stirred at ambient temperature for 2 h. The mixture was
poured into water and extracted with CH2Cl2. The organic
layer was washed with water and brine, dried (MgSO4), and
evaporated, and the residue was purified by silica gel column
chromatography (CH2Cl2-MeOH, 20:1 elution) to give 4c (5.15
g, 86%) as an oil: 1H NMR (CDCl3) δ 1.34-1.60 (2H, m), 1.77-
1.95 (2H, m), 2.65-2.85 (1H, m), 3.30-3.50 (2H, m), 3.79 (2H,
s), 3.88-4.08 (2H, m), 6.83-7.13 (6H, m), 7.28 (2H, d, J ) 8.6
Hz); IR (neat) 2930, 2845, 1498, 1464, 1250, 1211 cm-1; MS
m/z 302 (MH+).
3-Am in o-4-m eth oxy-6-m eth ylp yr id in e Hyd r och lor id e
(17). To a solution of 13 (608 mg, 3.0 mmol) in EtOH (40 mL)
was added 10% palladium on carbon (1.0 g), and the mixture
was hydrogenated for 2 h under atmospheric pressure. The
catalyst was filtered off, and the filtrate was evaporated. The
residual solid was collected by filtration and washed with iPE
to give 17 (419 mg, 80%) as a solid: 1H NMR (DMSO-d6) δ
2.51 (3H, s), 4.03 (3H, s), 5.78 (2H, br s), 7.29 (1H, s), 7.75
(1H, s), 14.70 (1H, br); IR (KBr) 3390, 3304, 2661, 1624, 1603,
1500, 1452, 1348, 1319 cm-1; MS m/z 139 (MH+ - HCl).
Syn th esis of P h en yl N-Ar ylca r ba m a tes 6b-i, Rea gen ts
for Ur ea F or m a tion . (1) P h en yl N-[2-Meth oxy-6-m eth yl-
4-(m eth ylth io)p yr id in -3-yl]ca r ba m a te (6c). To a solution
of 5c (9.06 g, 49.2 mmol) and N,N-dimethylaniline (7.48 mL,
59.0 mmol) in CH2Cl2 (120 mL) was added phenyl chlorofor-
mate (6.79 mL, 54.1 mmol) at 5 °C, and the mixture was
stirred at room temperature for 3 h. The reaction mixture was
washed with 1 N HCl, water, saturated NaHCO3 solution,
water, and brine, dried (MgSO4), and evaporated. The result-
ing solid was triturated with iPE to give 6c (7.83 g, 52%) as a
solid: 1H NMR (DMSO-d6) δ 2.39 (3H, s), 2.45 (3H, s), 3.86
(3H, s), 6.81 (1H, s), 6.85-7.50 (5H, m), 8.76 and 9.17 (total
Typ ica l P r oced u r e for Meth od C. N-Cycloh ep tyl-N-
[4-(4-flu or op h en oxy)ben zyl]-N′-(2,4,6-tr im eth ylp yr id in -
3-yl)u r ea (1k , F R186485). To a solution of N-cycloheptyl-4-
(4-fluorophenoxy)benzylamine (13.9 g, 44.3 mmol) in DMF (150
mL) was added crude carbamate 6i (13.0 g, 44.3 mmol)
followed by Et3N (18.5 mL, 133 mmol), and the mixture was
stirred at 100 °C for 3 h. After cooling to room temperature,
the reaction mixture was poured into water and extracted with
EtOAc. The organic layer was washed with water and brine,
dried (MgSO4), and evaporated, and the residue was purified
by silica gel column chromatography (EtOAc elution) to give
1k (15.4 g, 73%) as a solid: 1H NMR (CDCl3) δ 1.38-2.10 (12H,
m), 2.03 (3H, s), 2.19 (3H, s), 2.42 (3H, s), 4.30-4.50 (1H, m),
4.48 (2H, s), 5.51 (1H, br s), 6.81 (1H, s), 6.87-7.13 (6H, m),
7.36 (2H, d, J ) 8.6 Hz); IR (KBr) 3300, 2926, 2856, 1632,
1H, each br s); IR (KBr) 3217, 1740, 1649, 1541, 1518 cm-1
;
MS m/z 305 (MH+).
The following compounds 6b,d -g were also prepared as
described for 6c from the appropriate starting material.
P h en yl N-(2,4-d im et h oxy-6-m et h ylp yr id in -3-yl)ca r -
1
ba m a te (6b): yield 22.0 g (55%); H NMR (DMSO-d6) δ 2.38
(3H, s), 3.85 (6H, s), 6.72 (1H, s), 7.05-7.30 (3H, m), 7.35-
7.47 (2H, m), 8.83 (1H, br); IR (KBr) 3251, 3147, 2983, 2947,
1713, 1593, 1497, 1454 cm-1; MS m/z 289 (MH+).
1498, 1250, 1213 cm-1; MS m/z 476 (MH+). Anal. (C29H34
-
P h en yl N-[2-ch lor o-6-m eth yl-4-(m eth ylth io)p yr id in -3-
FN3O2) C, H, N.
1
yl]ca r ba m a te (6d ): yield 47.2 g (68%); H NMR (DMSO-d6)
N-Cycloh eptyl-N-[4-(4-flu or oph en oxy)ben zyl]-N′-(2,4,6-
tr im eth ylp yr id in -3-yl)u r ea Hyd r och lor id e (F R195249,
h yd r och lor id e sa lt of F R186485). To a solution of 1k (23.9
g, 50.2 mmol) in EtOAc (100 mL) was added dropwise a
solution of 4 N HCl in EtOAc (37.6 mL) at 5 °C, and the
mixture stirred for 20 min. To the mixture was then added
iPE (100 mL), followed by warming to room temperature, and
then the mixture stirred for 3 h. The resulting precipitate was
collected, washed with iPE, and recrystallized from EtOH-
hexane to give FR195249 (16.7 g, 65%) as a solid: mp 174-
δ 2.45 (3H, s), 2.51 (3H, s), 6.98-7.50 (6H, m), 9.36 and 9.76
(total 1H, each br s); IR (KBr) 3145, 2926, 1751, 1579, 1516,
1489, 1342, 1201 cm-1; MS m/z 309, 311 (MH+).
P h en yl N-[4-ch lor o-6-m eth yl-2-(m eth ylth io)p yr id in -3-
1
yl]ca r ba m a te (6e): yield 11.9 g (62%); H NMR (DMSO-d6)
δ 2.48 (3H, s), 2.51 (3H, s), 6.98-7.53 (6H, m), 9.37 and 9.77
(total 1H, each br s); IR (KBr) 3207, 3026, 2926, 1724, 1597,
1554, 1524, 1489 cm-1; MS m/z 309, 311 (MH+).
P h en yl N-(2,4-d ich lor o-6-m eth ylp yr id in -3-yl)ca r ba m -
1
1
a te (6f): yield 1.96 g (33%); H NMR (DMSO-d6) δ 2.27 (3H,
176 °C; H NMR (DMSO-d6) δ 1.30-1.88 (12H, m), 2.32 (3H,
s), 7.10-7.55 (5H, m), 7.65 (1H, s), 10.10 (1H, br s); IR (KBr)
3282, 3184, 3012, 1718, 1637, 1608, 1524, 1491 cm-1; MS m/z
297, 299, 301 (MH+).
s), 2.52 (3H, s), 2.65 (3H, s), 4.03-4.23 (1H, m), 4.53 (2H, s),
6.90-7.12 (4H, m), 7.14-7.38 (4H, m), 7.61 (1H, s), 8.30 (1H,
br s); IR (KBr) 3369, 2925, 2858, 2619, 1645, 1497, 1248, 1211
cm-1; MS m/z 476 (MH+ - HCl). Anal. Calcd for C29H34
-
P h en yl N-[4,6-b is(m et h ylt h io)-2-m et h ylp yr im id in -5-
1
FN3O2‚HCl: C, 68.02; H, 6.89; N, 8.21. Found: C, 68.15; H,
yl]ca r ba m a te (6g): yield 5.74 g (88%); H NMR (DMSO-d6)
7.03; N, 8.23.
δ 2.49 (6H, s), 2.59 (3H, s), 6.98-7.52 (5H, m), 9.27 and 9.68
(total 1H, each br s); IR (KBr) 3217, 3005, 2924, 1711, 1595,
1485, 1408, 1360, 1300 cm-1; MS m/z 322 (MH+).
N-Cycloh ep tyl-N-[4-(4-flu or op h en oxy)ben zyl]-N′-[2,4-
b is(m et h ylsu lfon yl)-6-m et h ylp yr id in -3-yl]u r ea
(1g,
(2) P h en yl N-(2,4,6-Tr im eth ylp yr id in -3-yl)ca r ba m a te
Hyd r och lor id e (6i). To a solution of phenyl chloroformate
(25 mL, 199 mmol) in 1,2-dichloroethane (250 mL) was added
dropwise a solution of 5i (22.6 g, 166 mmol) in 1,2-dichloro-
F R190809). To a solution of N-cycloheptyl-N-[4-(4-fluorophe-
noxy)benzyl]-N′-[2,4-bis(methylthio)-6-methylpyridin-3-yl]-
urea (FR182980) (26.1 g, 48.4 mmol) in CH2Cl2 (400 mL) was
added dropwise a solution of 80% m-CPBA (41.7 g, 193 mmol)

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4419
in CH2Cl2 (900 mL) at room temperature, and the mixture was
stirred for 3 days. Insoluble materials were removed by
filtration, and the filtrate was washed with saturated NaHCO3
solution, water, and brine, dried (MgSO4), and evaporated. The
residue was purified by silica gel column chromatography
(hexane-EtOAc, 1:1 elution) to give 1g (20.8 g, 71%) as a
solid: mp 129-130 °C; 1H NMR (CDCl3) δ 1.40-2.13 (12H,
m), 2.66 (3H, s), 3.19 (3H, s), 3.30 (3H, s), 4.05-4.30 (1H, m),
4.55 (2H, s), 6.90-7.10 (6H, m), 7.34 (2H, d, J ) 8.6 Hz), 7.85
(1H, s); IR (KBr) 3361, 2927, 2860, 1664, 1500, 1325, 1248,
Ack n ow led gm en t. We especially wish to thank Dr.
David Barrett, Medicinal Chemistry Research Labora-
tories, for his help in preparing the manuscript.
Refer en ces
(1) (a) Kannel, W. B.; Castelli, W. P.; Gordon, T.; McNamara, P. M.
Serum Cholesterol, Lipoproteins, and the Risk of Coronary Heart
Disease. The Framingham Study. Ann. Intern. Med. 1971, 74,
1-12. (b) Martin, M. J .; Hulley, S. B.; Browner, W. S.; Kuller,
L. H.; Wentworth, D. Serum Cholesterol, Blood Pressure, and
Mortality: Implications from a Cohort of 361 662 Men. Lancet
1986, 2, 933-936. (c) Badimon, J . J .; Fuster, V.; Chesebro, J .
H.; Badimon, L. Coronary Atherosclerosis: A Multifactorial
Disease. Circulation 1993, 87 (Suppl. II), 3-16.
1211, 1159, 1128 cm-1; MS m/z 604 (MH+). Anal. (C29H34
-
FN3O6S2) C, H, N.
N-Cycloh ep t yl-N-[4-(4-flu or op h en oxy)b en zyl]-N′-[6-
m et h yl-2-(m et h ylsu lfon yl)-4-(m et h ylt h io)p yr id in -3-yl]-
u r ea (1h ). To a solution of 1g (3.04 g, 5.0 mmol) in MeOH
(100 mL) was added NaSMe (315 mg, 4.5 mmol) at room
temperature, and the mixture was stirred at 50 °C for 1 h.
After cooling, the resulting precipitate was collected, washed
with MeOH and iPE, and dried to give 1h (1.35 g 47%) as a
solid: mp 184-185 °C; 1H NMR (CDCl3) δ 1.38-2.08 (12H,
m), 2.44 (3H, s), 2.54 (3H, s), 3.23 (3H, s), 4.10-4.32 (1H, m),
4.55 (2H, s), 6.74-7.10 (7H, m), 7.36 (2H, d, J ) 8.6 Hz); IR
(KBr) 3377, 3072, 2926, 2858, 1657, 1572, 1498, 1309, 1248,
1209, 1142 cm-1; MS m/z 572 (MH+). Anal. (C29H34FN3O4S2)
C, H, N.
N-Cycloh ep t yl-N-[4-(4-flu or op h en oxy)b en zyl]-N′-[6-
m eth yl-2-(m eth ylsu lfon yl)p yr id in -3-yl]u r ea (1a b). To a
suspension of 1h (172 mg, 0.3 mmol) in EtOH (20 mL) was
added Raney nickel (1.0 g), and the mixture was refluxed for
15 min. Raney nickel was filtered off, and the filtrate was
evaporated. The residue was purified by silica gel column
chromatography (hexane-EtOAc, 4:1 elution) to give 1a b
(127 mg, 81%) as an oil: 1H NMR (CDCl3) δ 1.38-2.00 (12H,
m), 2.50 (3H, s), 3.21 (3H, s), 4.05-4.25 (1H, m), 4.52 (2H, s),
6.87-7.32 (9H, m), 8.81 (1H, d, J ) 8.8 Hz), 9.12 (1H, br s);
MS m/z 526 (MH+).
N-Cycloh ep t yl-N-[4-(4-flu or op h en oxy)b en zyl]-N′-[2-
ch lor o-6-m eth yl-4-(m eth ylsu lfon yl)p yr id in -3-yl]u r ea (1i).
To a solution of N-cycloheptyl-N-[4-(4-fluorophenoxy)benzyl]-
N′-[2-chloro-6-methyl-4-(methylthio)pyridin-3-yl]urea (1e) (1.0
g, 1.90 mmol) in CH2Cl2 (40 mL) was added 80% m-CPBA (1.23
g, 5.69 mmol) at room temperature. After stirring for 2 h, the
reaction mixture was washed with saturated NaHCO3 solution,
water, and brine, dried (MgSO4), and evaporated. The residue
was purified by silica gel column chromatography (hexane-
EtOAc, 3:2 elution) to give 1i (976 mg, 92%) as a solid: 1H
NMR (CDCl3) δ 1.40-2.10 (12H, m), 2.59 (3H, s), 3.09 (3H, s),
4.10-4.35 (1H, m), 4.54 (2H, s), 6.65 (1H, br), 6.90-7.10 (6H,
m), 7.31 (2H, d, J ) 8.6 Hz), 7.58 (1H, s); IR (KBr) 3367, 2927,
2858, 1664, 1498, 1317, 1213, 1149 cm-1; MS m/z 560, 562
(MH+). Anal. (C28H31ClFN3O4S) C, H, N.
N-Cycloh ep t yl-N-[4-(4-flu or op h en oxy)b en zyl]-N′-(2-
ch lor o-6-m eth ylp yr id in -3-yl)u r ea (1m ). To a suspension
of 1e (730 mg, 1.38 mmol) in EtOH (30 mL) was added Raney
nickel (5.0 g), and the mixture was refluxed for 3 h. Raney
nickel was filtered off, and the filtrate was evaporated. The
residue was purified by silica gel column chromatography
(hexane-EtOAc, 4:1 to 2:1 elution) to give 1m (230 mg, 35%)
as a solid: mp 128-129 °C; 1H NMR (CDCl3) δ 1.38-2.05 (12H,
m), 2.43 (3H, s), 4.25-4.45 (1H, m), 4.50 (2H, s), 6.76 (1H, br
s), 6.88-7.12 (7H, m), 7.30 (2H, d, J ) 8.6 Hz), 8.44 (1H, d, J
) 8.3 Hz); IR (KBr) 3386, 2927, 2858, 1676, 1512, 1497, 1460,
1296 cm-1; MS m/z 482, 484 (MH+). Anal. (C27H29ClFN3O2)
C, H, N.
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Compound 1l was also prepared as described for 1m from
1c.
N-Cycloh ep t yl-N-[4-(4-flu or op h en oxy)b en zyl]-N′-(2-
m eth oxy-6-m eth ylp yr id in -3-yl)u r ea (1l): yield 215 mg
1
(64%); mp 132-133 °C; H NMR (CDCl3) δ 1.40-2.05 (12H,
m), 2.34 (3H, s), 3.74 (3H, s), 4.23-4.43 (1H, m), 4.45 (2H, s),
6.65 (1H, d, J ) 7.9 Hz), 6.77 (1H, br s), 6.90-7.10 (6H, m),
7.32 (2H, d, J ) 8.6 Hz), 8.24 (1H, d, J ) 7.9 Hz); IR (KBr)
3394, 2927, 2858, 1666, 1591, 1520, 1497, 1454, 1379 cm-1
;
MS m/z 478 (MH+). Anal. (C28H32FN3O3) C, H, N.

4420 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
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