Inhibitors of acyl-CoA:cholesterol O-acyltransferase. Synthesis and pharmacological activity of (±)-2-dodecyl-α-phenyl-N-(2,4,6- trimethoxyphenyl)-2H-tetrazole-5-acetamide and structurally related tetrazole amide derivatives

Article abstract of DOI:10.1021/jm960170f

A series of tetrazole amide derivatives of (±)-2-dodecyl-α-phenyl-N- (2,4,6-trimethoxyphenyl)-2H-tetrazole-5-acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O- acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo. For this series of compounds, our objective was to systematically replace substituents appended to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess the effect that these changes have on biological activity. The ensuing structure-activity relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30 mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia. Replacement of the α-phenyl moiety with electron-withdrawing substituents (13e-h), however, significantly reduced liver microsomal ACAT inhibitory activity (IC₅₀ > 1 μM). This is in contrast to electron-donating substituents (13ij,m-q), which produce IC₅₀ values ranging from 5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n,o), reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring (36a- d), in general, improved macrophage ACAT inhibitory activity and provided excellent cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted methylene derivatives (13n and 36a) caused adrenocortical cell degeneration in guinea pigs treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with the corresponding α-phenyl-substituted analogs (7b and 36c) were essentially unchanged compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay showed that both compounds and/or their metabolites were present in plasma after oral dosing. Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d) showed poor oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically available (7b, 36c) and poorly absorbed inhibitors (1, 36d) were more effective in lowering plasma total cholesterol than the fatty acid amide CI-976.

Full text of DOI:10.1021/jm960170f

2354
J . Med. Chem. 1996, 39, 2354-2366
In h ibitor s of Acyl-CoA:Ch olester ol O-Acyltr a n sfer a se. Syn th esis a n d
P h a r m a cologica l Activity of
(()-2-Dod ecyl-r-p h en yl-N-(2,4,6-tr im eth oxyp h en yl)-2H-tetr a zole-5-a ceta m id e a n d
Str u ctu r a lly Rela ted Tetr a zole Am id e Der iva tives
Patrick M. O’Brien,* Drago R. Sliskovic, J oseph A. Picard, Helen T. Lee, Claude F. Purchase, II, Bruce D. Roth,
Andrew D. White, Maureen Anderson,† Sandra Bak Mueller,† Thomas Bocan,† Richard Bousley,†
Katherine L. Hamelehle,† Reynold Homan,† Peter Lee,† Brian R. Krause,† J . F. Reindel,‡
Richard L. Stanfield,† and Daniel Turluck§
Departments of Chemistry, Atherosclerosis Therapeutics, Pathology and Experimental Toxicology, and Pharmacokinetics/ Drug
Metabolism, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road,
Ann Arbor, Michigan 48105
Received March 4, 1996X
A series of tetrazole amide derivatives of (()-2-dodecyl-R-phenyl-N-(2,4,6-trimethoxyphenyl)-
2H-tetrazole-5-acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA:
cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo.
For this series of compounds, our objective was to systematically replace substituents appended
to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess
the effect that these changes have on biological activity. The ensuing structure-activity
relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-
trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and
exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30
mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia.
Replacement of the R-phenyl moiety with electron-withdrawing substituents (13e-h ), however,
significantly reduced liver microsomal ACAT inhibitory activity (IC50 > 1 µΜ). This is in
contrast to electron-donating substituents (13i,j,m -q), which produce IC50 values ranging from
5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n ,o),
reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring
(36a -d ), in general, improved macrophage ACAT inhibitory activity and provided excellent
cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol
at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted
methylene derivatives (13n and 36a ) caused adrenocortical cell degeneration in guinea pigs
treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with
the corresponding R-phenyl-substituted analogs (7b and 36c) were essentially unchanged
compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay
showed that both compounds and/or their metabolites were present in plasma after oral dosing.
Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d ) showed poor
oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically
available (7b, 36c) and poorly absorbed inhibitors (1, 36d ) were more effective in lowering
plasma total cholesterol than the fatty acid amide CI-976.
In tr od u ction
particular, inhibition of acyl-CoA:cholesterol O-acyl-
transferase (ACAT). ACAT catalyzes the intracellular
esterification of cholesterol in most mammalian tissues
by using CoA-activated fatty acids to produce cholesteryl
ester.3 Experimental evidence suggests that the ACAT-
mediated esterification of cholesterol plays a key role
in intestinal cholesterol absorption, hepatic production
of very low density lipoproteins (VLDL), and the un-
regulated accumulation of cholesteryl esters within the
arterial wall.4,5 In animal models, the fatty acid amide
ACAT inhibitor CI-976 has been shown to inhibit
intestinal cholesterol absorption, reduce the hepatic
cholesteryl ester content necessary for VLDL synthesis
and secretion, and prevent the accumulation of choles-
teryl ester-rich foam cells within the arterial wall, with
or without a reduction in plasma total cholesterol.6,7
Despite impressive lipid-lowering activity in animal
models, the hypocholesterolemic effects of ACAT inhibi-
tors in human trials have been disappointing.8 This
Epidemiological studies have shown that elevated
plasma total and low-density lipoprotein cholesterol
(LDL-C) levels are associated with increased risk for
premature coronary heart disease (CHD).1 Currently,
the most effective approach for regulating plasma
cholesterol concentrations in hypercholesterolemic pa-
tients is by inhibiting cholesterol biosynthesis with
HMG-CoA reductase (HMGR) inhibitors. Recent clini-
cal studies have demonstrated that reducing LDL-C
with HMGR inhibitors alone, or in combination with bile
acid sequestrants, translate into a decreased incidence
of cardiovascular events.2 However, the clinical success
of HMGR inhibitors has overshadowed the considerable
research into other potential lipid-regulating targets, in
† Department of Atherosclerosis Therapeutics.
‡ Department of Pathology and Experimental Toxicology.
§ Department of Pharmacokinetics/Drug Metabolism.
X Abstract published in Advance ACS Abstracts, May 15, 1996.
S0022-2623(96)00170-7 CCC: $12.00 © 1996 American Chemical Society

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2355
synthesized using reaction conditions previously de-
scribed (method A, Scheme 1). The penultimate car-
boxylic acid derivative 6 was synthesized in three steps
utilizing commercially available ethyl phenylcyano-
acetate 2. Amides 7a -j were then prepared by coupling
appropriately substituted amines with 6 using dicyclo-
hexylcarbodiimide (DCC) or carbonyldiimidazole (CDI)
as the coupling agent.10
Several methods were used to vary substituents
positioned R with respect to the amide moiety (method
B, Scheme 2, and method C, Scheme 3). The methylene
analogs, 13n and 13o (Scheme 2), were synthesized
from ethyl cyanoacetate 8n (R1 ) R2 ) H) using the
reaction conditions previously described for amides 7a -
j. Alternatively, treatment of 8n with NaH and a
stoichiometric amount of an appropriate electrophile
gave substituted cyanoacetate derivatives 8i,j,p,q, which,
like 8n , were converted to the corresponding amides
13i,j,p ,q utilizing reaction conditions previously de-
scribed in Scheme 1. Similarly, deprotonation of an R
hydrogen atom in 10n with lithium hexamethyldisila-
zane at -20 °C, followed by quenching the resulting
anion with N-fluoro-O-benzenedisulfonimide,11 gave
10e, an intermediate used in the synthesis of 13e. The
cyclohexyl derivative, 13m , was prepared by catalytic
hydrogenation of 1 over 10% Rh/C.
F igu r e 1. Structures of some known amide ACAT inhibitors.
result, coupled with the recent success of HMGR inhibi-
tors, contributed to the evolution of ACAT inhibitors
from nonabsorbed hypocholesterolemic agents to sys-
temically available agents capable of inhibiting the
enzyme in the liver and artery wall and thus directly
affecting the atherosclerotic process.9
The first bioavailable ACAT inhibitor reported to
show direct antiatherosclerotic effects was CI-976 (Fig-
ure 1).7 This result prompted extensive SAR studies
based on CI-976, from which structurally novel di- and
trisubstituted ureas, amino acid anilides, malondi-
amides, malonester amides, and â-keto amides emerged
as potent ACAT inhibitors.9 Further modification of the
fatty acid side chain led to the discovery of (()-2-dodecyl-
R-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5-
acetamide (1), a potent inhibitor of rabbit intestinal and
rat liver ACAT in vitro (IC50 ) 8 and 23 nM, respec-
tively), that is significantly more efficacious in lowering
non HDL-C in a cholesterol-fed (ED50 ) 6.4 vs 19 mg/
kg) rat model in vivo, compared to CI-976. This
improvement in efficacy may in part be attributed to
the ability of 1 to inhibit cholesterol absorption, since
in the lymph fistula model, 1 reduced cholesterol
absorption, i.e. the appearance of lymph cholesteryl
esters, by 79% at 10 mg/kg, whereas with CI-976 the
lowest effective dose is 30 mg/kg.6 Subsequent evalu-
ation of the individual stereoisomers showed that the
majority of biological activity observed for 1 resides in
the (+)-enantiomeric form. In this study, the 2-regio-
isomer (1) was shown to be 3 times more potent in vitro
than the corresponding 1-regioisomer.10
As an extension of these studies, we investigated the
SAR around the amide moiety of 1 (Figure 1), utilizing
(()-2-phenyl-(2-dodecyl-2H-tetrazol-5-yl)acetic acid and
a variety of aryl- and heteroaryl-substituted amines. A
second approach modified the electronic and steric
environment about the tetrazole moiety, varying sub-
stituents in either the 2- or 5-positions on the hetero-
cyclic ring. In this paper, we assess the effects these
structural changes have on inhibiting hepatic and
macrophage ACAT in vitro, hypocholesterolemic activity
in vivo, and, for selected compounds, oral bioactivity ex
vivo.
Commercially available acetonitrile derivatives 14a -
d ,f-h ,k ,l were also instrumental in functionalizing the
R position (Scheme 3). Refluxing the starting nitriles
with tri-n-butyltin azide, followed by alkylation of the
resulting tetrazole with 1-bromododecane, gave a mix-
ture of 1- and 2-regioisomers, from which the 2-regio-
isomers, 15a -d ,f-h ,k ,l, were isolated pure by silica gel
chromatography. Compounds 15a -d ,f-h ,k ,l were
deprotonated in tetrahydrofuran using n-butyllithium,
with subsequent quenching of the anion with 2,4,6-
trimethoxyphenyl or 2,6-diisopropylphenyl isocyanates
to give amides 13a -d ,f,k and 13g,l, respectively. For
13g, treatment with tri-n-butyltin azide in refluxing
p-dioxane yielded the corresponding tetrazole derivative
13h .
As shown in Scheme 4 (method D), the tetrazoles
derived from ethyl cyanoformate 16a and the methyl
ester of 3-cyanopropanoic acid 16b provided variation
in the linkage between the amide and tetrazole moieties.
Compound 16a was converted to the carbethoxy-
substituted tetrazole 17a , when treated with sodium
azide in a mixture of pyridine/trifluoroacetic acid at 60
°C. Alkylation of 17a gave a 1.3:1 mixture of regio-
isomers 18a and 19a , both of which were separated by
silica gel chromatography. The 2-regioisomer, 18a , was
hydrolyzed with KOH in ethanol and then coupled in
refluxing tetrahydrofuran with 2,4,6-trimethoxyaniline
and 2,6-diisopropylaniline, using CDI as the coupling
agent, to give compounds 20 and 21, respectively.
Reaction conditions employed for amides 22 and 23 were
similar to those described for 20 and 21.
The C12 functionality in 1 was replaced with a variety
of lipophilic side chains. Side chains incorporating
unsaturation were prepared as shown in Scheme 5. The
mesylate of 11-dodecen-1-ol (28) was synthesized in five
steps via homologation of commercially available 10-
undecen-1-ol (24). Alcohol 24 was activated with meth-
anesulfonyl chloride to give mesylate 25 which under-
went nucleophilic displacement to nitrile 26 upon
Ch em istr y
The synthetic strategies used to prepare the com-
pounds employed in this study are illustrated in Schemes
1-7. Our first synthetic targets, amides 7a -j, were

2356 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Sch em e 1. Method Aa
O’Brien et al.
a
(a) (n-Bu)3SnN3, p-dioxane, reflux; (b) 1-bromododecane, NEt3, CH3CN, reflux; (c) NaOH, EtOH; (d) RNH2, CDI/THF or DCC/CH2Cl2.
Sch em e 2. Method Ba
a
(a) (n-Bu)3SnN3, p-dioxane, reflux; (b) 1-bromododecane, NEt3, CH3CN, reflux; (c) NaOH, EtOH; (d) RNH2, CDI/THF or DCC/CH2Cl2.
Sch em e 3. Method Ca
described, the esters 33a ,c were hydrolyzed to the
corresponding acids 35a ,c and coupled with 2,4,6-
trimethoxy- or 2,6-diisopropylaniline to give amides
36a -d .
Resu lts a n d Discu ssion
a
(a) (n-Bu)3SnN3, p-dioxane, reflux; (b) 1-bromododecane, NEt3,
CH3CN, reflux; (c) RNCO, n-BuLi, THF.
Compounds prepared were initially tested for ACAT
inhibition in hepatic microsomes isolated from choles-
terol-fed rats and in a cell culture assay (see the
Experimental Section) using murine IC-21 macrophages
(Tables 1-4).6 The primary in vivo assay was per-
formed in rats given a single dose of test compound by
gavage suspended in carboxymethylcellulose (CMC) and
Tween-20 in water, followed by a single high-fat, high-
cholesterol meal. This model measures the ability of
the test compound to inhibit the overnight rise in
plasma total cholesterol (Tables 1-4).12 In the chronic
cholesterol-fed rat model (Table 5), rats were fed the
same diet as above but for 14 days. During the second
week, the test compound was administered daily by
gavage using the CMC/Tween vehicle. Efficacy in this
model is defined as the ability to reduce plasma total
cholesterol (TC), lower the amount of cholesterol in
apoB-containing lipoproteins (non HDL-C), and to el-
evate the diet-induced low levels of HDL cholesterol
(HDL-C).12
treatment with potassium cyanide in dimethyl sulfoxide.
Hydrolysis of 26 using aqueous NaOH in refluxing
methanol afforded the carboxylic acid intermediate 27,
which reduced to the corresponding alcohol upon treat-
ment with lithium aluminum hydride (LAH) in tetrahy-
drofuran. The alcohol was then activated with meth-
anesulfonyl chloride in dichloromethane to give mesylate
28 (Scheme 5). Compounds 25 and 28, and the com-
mercially available saturated alkyl halides were then
utilized (R3) in the synthesis of amides 30a -d as shown
in Scheme 6 (method E). These were coupled to tetra-
zole 3 using the standard conditions previously de-
scribed. The 2-isomers, 29a -d , were hydrolyzed to the
carboxylic acids, activated with DCC, and coupled with
2,4,6-trimethoxyaniline to give amides 30a -d .
In order to further explore SAR around the 2- and
5-positions of the tetrazole ring, a series of reverse
tetrazole amides (i.e. the C12 in the 5-position and the
acetamide coupled to the 2-position) were prepared
utilizing the 2,6-diisopropyl- and the 2,4,6-trimethoxy-
anilide substitution patterns shown in Scheme 7 (method
F). Lauryl cyanide 31 was converted to 5-dodecyl-
tetrazole 32 by cycloaddition with tri-n-butyltin azide
in refluxing dioxane. The resulting tetrazole was alky-
lated with the appropriately substituted R-bromoacetate
to give a mixture of regioisomers 33a ,c and 34a ,c, from
which 33a ,c were isolated pure using silica gel chro-
matography. Employing reaction conditions previously
The most potent and efficacious tetrazole amides were
evaluated for adrenal and liver toxicity in chow-fed
guinea pigs. The test compounds were dosed (100 mg/
kg) orally by gavage (oleic acid vehicle) over a 2-week
period, followed by the microscopic examination of
hepatocytes and adrenal cortical cells for drug-related
pathologic alterations, compared to the untreated con-
trols.13 Compounds classed as nontoxic in this model
were further evaluated for oral bioactivity in chow-fed

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2357
Sch em e 4. Method Da
a
(a) NaN3, TFA, pyridine, 60 °C, or NaN3, NH4Cl, DMF, 100 °C; (b) 1-bromododecane, NEt3, CH3CN, reflux; (c) KOH, EtOH; (d) RNH2,
CDI, THF, 60 °C.
Sch em e 5a
In order to further define the structural features
necessary for potent inhibition of ACAT in vitro and
efficacy in vivo, we systematically replaced the R-phenyl
moiety of 1 with substituents capable of affecting the
electronic and steric environment about the methylene
bridge connecting the amide carbonyl and the tetrazole
ring (Table 2). For the 4-substituted phenyl analogs
(13a -d ), only the 4-fluoro derivative (13a ) was as
effective as 1 in the biological screens. Electron-
donating groups, 13b-d , were at least 10-fold less
potent in inhibiting hepatic ACAT in vitro, but 13b was
more potent than 1 in the cellular assay. Replacement
of the R-phenyl moiety with smaller electron-withdraw-
ing substituents (i.e. F, CN, tetrazolyl), reduced potency
in both microsomal (IC50 > 1 µM) and cellular assays
for both the 2,4,6-trimethoxy- and 2,6-diisopropylanilide
derivatives (13e-h ), whereas the 2-pyridyl analogs,
13k ,l, maintained potent inhibitory activity. In con-
trast, electron-donating substituents (13i,j,m -q) vary-
ing in size and lipophilicity were generally well tolerated
in the R-position. Among these, the benzyl derivative,
13j, potently inhibited macrophage ACAT in vitro and
maintained excellent hypocholesterolemic activity in
vivo. Substituting hydrogen for phenyl yielded the most
a
(a) MsCl, NEt3, CH2Cl2, 0 °C; (b) KCN, DMSO, 80 °C; (c)
NaOH, H2O/CH3OH, reflux; (d) LAH, THF; (e) MsCl, NEt3,
CH2Cl2, 0 °C.
Sch em e 6. Method Ea
a
(a) R3OMs or R3Br, NEt3, CH3CN, reflux; (b) NaOH, EtOH;
(c) RNH2, DCC, CH2Cl2, 0 °C.
rabbits (see the Experimental Section for the experi-
mental protocol). In this bioassay, systemic availability
was determined ex vivo utilizing the hepatic microsomal
assay previously described.6
potent inhibitor of hepatic ACAT activity, 13n (IC50
)
5 nM), which unlike 1 does not possess an asymmetric
center. The in vitro activity for other achiral analogs,
however, decreased as the bulk of the substituents
increased (compare 13o-q).
The biological activity for amides derived from (()-
2-phenyl-(2-dodecyl-2H-tetrazol-5-yl)acetic acid is shown
in Table 1. Consistent with previous fatty acid amide
SAR,14 optimal in vitro and in vivo activity was observed
for compounds bearing phenyl (1, 7b) or heterocyclic
rings (7f,g) disubstituted with alkyl or alkoxy substit-
uents flanking the amide moiety. For this series of
compounds, the steric bulk is required for good microso-
mal activity, since the unsubstituted anilide 7a and the
heterocyclic derivatives having a nitrogen atom occupy-
ing one of the ortho positions in the heteroaryl ring
(7d ,e,h ), in general, are less active at inhibiting hepatic
ACAT activity. However, weak inhibitors in the micro-
somal assay (7a ,h ) demonstrated potency comparable
to 1 in the cellular assay. The most potent ACAT
inhibitor in the cellular assay, 7g, provided the best
overall activity in our preliminary in vitro and in vivo
biological screens. Other modifications such as replac-
ing the 2,4,6-trimethoxyphenyl moiety of 1 with tert-
butyl (7j) or cyclopropyl (7i) functionalities failed to
provide increases in potency or efficacy.
Additional SAR studies focused on substituent effects
in both the 2- and 5-positions of the tetrazole ring. From
Table 3, it can be seen that coupling the amide carbonyl
directly to the 5-position of the heterocylic ring (20, 21)
significantly reduced in vitro and in vivo activity
compared to the corresponding acetamides, 13n ,o. Ho-
mologation of the methylene linkage connecting the
amide and tetrazole moieties (22 and 23) also reduced
potency and efficacy in the respective biological screens,
albeit to a lesser extent than that observed for carbox-
amides 20 and 21. In order to evaluate the role of the
dodecyl side chain appended to the 2-position in the
tetrazole ring, we replaced the C12 functionality in 1
with a variety of saturated (C8, C16) and unsaturated
(C11, C12) aliphatic chains and assessed the effects these
changes have on biological activity. As shown in Table
3, inhibiton of hepatic ACAT activity decreased on the
order of C12 > C16 > C8 for the saturated side chains

2358 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Sch em e 7. Method Fa
O’Brien et al.
a
(a) (n-Bu)3SnN3, p-dioxane, reflux; (b) HCl, Et2O; (c) ethyl 2-bromophenylacetate or ethyl bromoacetate, NEt3, CH3CN, reflux; (d)
NaOH, EtOH/H2O; (e) RNH2, DCC, CH2Cl2, 0 °C.
(compounds 1, 30b,a ), which correlates well with the
decrease in hypocholesterolemic activity seen in the
acute cholesterol-fed rat model. Of the compounds
prepared, only the unsaturated C12 derivative, 30d ,
exhibited comparable in vitro and in vivo activity,
relative to 1, in our preliminary pharmacological screens.
However, 30b and 30c are more potent at inhibiting
macrophage ACAT than 1 and 30d .
modify the R-position (13a ,j) failed to attain the efficacy
levels established by 1, 30c, and 36a in this biological
screen.
Having identified structural features necessary for
potent inhibition of ACAT in vitro and excellent lipid-
regulating activity in vivo, we further assessed the most
potent and efficacious compounds for adrenal toxicity,
since this toxicity has been reported for urea and
imidazole-containing ACAT inhibitors.13,15 In this study,
the 2,6-diisopropylanilides substituted with phenyl (7b,
36c) or unsubstituted (13n , 36a ) in the R-position were
selected for further evaluation in the in vivo guinea pig
tox assay. Similarly, the corresponding 2,4,6-trimethoxy-
anilides (1, 36d , 13o, 36b) were also tested in this
screen. After 2 weeks of oral dosing (100 mg/kg) of
compound 13n using an oleic acid vehicle to facilitate
absorption, increased coarse vacoulation of the zona
fasciculata cortical cells with adrenocortical cell degen-
eration was observed in the drug-treated animals.
Similar drug-related pathologic alterations to the ad-
renal gland were observed for all R-methylene unsub-
stituted anilides (36a , 13o, 36b), the toxicity being less
severe for the 2,4,6-trimethoxyphenyl derivatives. In-
terestingly, the adrenal glands taken from guinea pigs
treated with tetrazole amides bearing an R-phenyl
moiety (1, 7b, 36c, 36d ) were essentially unchanged
compared to those of controls. Neither the methylene
nor the R-phenyl-substituted anilides caused drug-
related alterations in the livers of the treated guinea
pigs.
Having identified methylene as the optimal linkage
connecting the amide carbonyl and the tetrazole ring,
we further investigated substituent effects influencing
the R-position by appending the acetamide, substituted
with R-phenyl or unsubstituted, to the nitrogen in the
2-position of the heterocyclic ring (Table 4). Despite
these changes, the biological activity for these com-
pounds (36a -d ) parallels that previously observed for
the parent tetrazole amide derivatives (1, 7b, 13n ,o).
Once again, the 2,6-disopropylanilides (36a and 36c)
potently inhibit ACAT in vitro, compound 36a being
equipotent to 13n in the microsomal assay with an IC50
of 6 nM. Interestingly, compounds 36b-d are more
potent than the parent tetrazole amides (13o, 7b, 1) in
inhibiting macrophage ACAT activity. When dosed (30
mg/kg) to cholesterol-fed rats, both 2,4,6-trimethoxy-
and 2,6-diisopropylanilides significantly reduce the
overnight rise in plasma total cholesterol (-65% to
-77%).
Compounds demonstrating potency comparable to 1
for inhibiting hepatic microsomal and macrophage
ACAT in vitro and cholesterol-lowering activity in vivo
were selected for further evaluation in the chronic
cholesterol-fed rat model (Table 5). This model more
directly mimics the situation found in the clinic, i.e. pre-
established hypercholesterolemia, than the acute screen
previously described. The R-phenyl-2,6-diispropyl-
anilide derivatives (7b, 36c) previously shown to be as
efficacious as the methylene analogs (13n , 36a ) in the
acute rat screen were considerably less effective at
lowering non HDL-C in the chronic screen (-40% and
-27% for 7b an 36c, respectively, vs -63% and -81%
for 13n and 36a , respectively). The most efficacious of
these, 36a , also elevated HDL-C by 165%, an increase
nearly 2 times that produced by compound 1 (+83%).
The opposite effect was observed for the 2,4,6-tri-
methoxyanilide pair, 1 and 13o, the R-phenyl derivative
providing superior in vivo activity (-68% vs -30%).
Replacing the dodecyl side chain of 1 with the unsatu-
rated C11 hydrocarbon chain (30c) further enhanced
hypocholesterolemic activity (-68% vs -77%). Other
analogs designed to replace the anilide moiety (7g) or
To ensure that the nontoxic compounds 1, 7b, and
36c,d (or bioequivalents thereof) were systemically
available to peripheral tissues, we assessed the bio-
activity upon oral dosing to New Zealand white rabbits
utilizing the bioassay (Table 6). Employing CI-976 as
the reference standard, compounds were dosed to rab-
bits (25 mg/kg) using an oil vehicle. Plasma samples
were taken at time zero (before drug), 1, 2, and 4 h
postdrug meal. The plasma was extracted and evalu-
ated for ACAT inhibitory activity in the microsomal
assay. For CI-976, optimal inhibition was observed at
the 4-h timepoint (45%). Suprisingly, compound 1 was
inactive, even at a dose as high as 50 mg/kg. The
corresponding 2,6-diisopropylanilide (7b), however, was
very effective in this biological screen (96% at 25 mg/
kg). This was consistent with the results obtained for
the reverse tetrazoles 36c and 36d (91% vs 0%). To
assess whether other efficacious trimethoxyanilides
were inactive, compounds 13j and 30c,d were screened.
Similar to 1, the higher homolog, 13j, was also shown

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2359
Ta ble 1. Physical and Biological Properties of Analogs of 1 Bearing a Variety of Amide Substituents
a
b
Analytical results are within 0.4% of the theoretical values unless otherwise noted. Melting points are uncorrected. c ACAT inhibition
in vitro, liver microsomes isolated from cholesterol-fed rats. Each determination performed in triplicate. See ref 6 for the complete
d
protocol. Cell culture assay using murine IC-21 macrophages. See the Experimental Section for the complete protocol. e Denotes percent
change in total cholesterol in cholic acid (0.5%)-cholesterol (1.5%)-peanut oil (5.5%)-fed rats. See ref 12 for the complete protocol. The
standard dose was 30 mg/kg of inhibitor. f Anal. C: calcd, 67.61; found, 67.17. An asterisk (*) denotes significantly different from control,
p < 0.05 using analysis of variance followed by Fisher’s multiple range test. ND denotes not determined.
to be inactive in this assay. For the unsaturated side
chains, 30c and 30d , ACAT was inhibited by 26% and
37%, respectively, when dosed at 50 mg/kg, considerably
more effective than 1, but significantly less active
compared to the 2,6-diisopropylanilide derivatives.
In preparation for evaluation in a rabbit model of
atherosclerosis, an additional efficacy study was carried
out in cholesterol-fed rabbits for selected nonadrenotoxic
tetrazole amide derivatives. In this model, we compared
the hypocholesterolemic activity for 1 and 36d , com-
pounds previously shown to be inactive in the bioassay,
to that of their bioactive 2,6-diisopropylphenyl-substi-
tuted counterparts, 7b and 36c. As shown in Table 7,
all of the inhibitors significantly lowered rabbit plasma
total cholesterol (-55% to -78%) after 2 weeks of oral
dosing at 5 and 25 mg/kg compared to the untreated
progression controls. However, the tetrazole amides 1
and 7b were the most efficacious in lowering preestab-
lished hypercholesterolemia (-38% and -41% at 25 mg/
kg, respectively) 24-h postdrug meal (T0). When com-
pared to CI-976 (Figure 2) in
a dose-response
experiment, 1 was the most potent compound by far,
having a calculated ED50 (dose required to lower plasma
total cholesterol 50%) value of 0.1 mg/kg, 218 times
more potent than CI-976. The 2,6-diisopropylanilide 7b
was 18 times more potent than CI-976 (ED50 1.2 vs 21.8
mg/kg) in this pharmacological screen.

2360 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
O’Brien et al.
Ta ble 2. Physical and Biological Properties of Tetrazole Amides Bearing R-Substituents Other Than Phenyl
ACAT inhibition
liver
macrophage
no.
Rf
R1
R2
method
formulaa
mp (°C)b
(IC50, µM)c
(% inhibn, 0.1 µM)d
% change in TCe
1
TMP
TMP
TMP
TMP
TMP
TMP
TMP
DIP
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
A
C
C
C
C
B
C
C
C
B
B
C
C
A
B
B
B
B
C30H43N5O4
C30H42FN5O4
C31H45N5O5
C32H48N6O4
119-120
124-125
131-133
106-107
135-137
oil
0.023
0.036
0.200
0.659
0.221
1.76
13.6
>5.0
>5.0
0.031
0.049
0.059
0.026
0.028
0.005
0.024
0.033
0.063
40
46
61
0
10
ND
7
-63*
-68*
-57*
-62*
-43*
-44*
-27
-75*
-37*
-55*
-73*
-60*
-64*
-61*
-64*
-60*
-69*
-56*
13a
13bg
13c
13d
13e
13f
13g
13h
13i
4-FPh
4-CH3OPh
4-(NMe)2Ph
p-biphenyl
F
CN
CN
5-tetrazolyl
CH3
CH2Ph
2-pyridyl
2-pyridyl
cyclohexyl
H
C
C
C
C
C
36H47N5O4
24H38FN5O4
25H38N6O4
28H44N6O
28H45N9O
112-113
72-75
7
8
DIP
147-148
113-114
86-87
TMP
TMP
TMP
DIP
TMP
DIP
TMP
TMP
TMP
C25H41N5O4
C31H45N5O4
C29H42N6O4
49
77
23
24
8
91
66
34
16
13j
13kh
13l
70-72
70-71
C
C
C
C
32H48N6O
30H49N5O4
27H45N5O
24H39N5O4
13m
13n
13o
13p
13q
140-141
75-79
H
CH3
108-109
C26H43N5O4
C28H45N5O4
oil
oil
-(CH2)4-
a-e,* Refer to the footnotes in Table 1. f TMP ) 2,4,6-trimethoxyphenyl; DIP) 2,6-diisopropylphenyl. ND denotes not determined. g Anal.
h
C: calcd, 65.58; found, 66.14. Anal. C: calcd, 64.66; found, 64.20.
Ta ble 3. Modification of Substituents Appended to the 2- and 5-Positions in the Tetrazole Ring
ACAT inhibition
liver
macrophage
no.
Rf
R3
m
n
method
formulaa
mp (°C)b
(IC50, µM)c (% inhibn, 0.1 µM)d
% change in TCe
1
20
TMP
TMP
DIP
TMP
DIP
TMP
TMP
TMP
TMP
(CH2)11CH3
(CH2)11CH3
(CH2)11CH3
(CH2)11CH3
(CH2)11CH3
(CH2)7CH3
(CH2)15CH3
(CH2)9CHdCH2
(CH2)10CHdCH2
1
0
0
0
0
1
1
1
1
0
0
0
2
2
0
0
0
0
A
D
D
D
D
E
E
E
E
C30H43N5O4
C23H37N5O4
C26H43N5O
C25H41N5O4
C28H47N5O
C26H35N5O4
C34H50N5O4
C29H39N5O4
C30H41N5O4
119-120
83-85
0.023
35.58
1.057
0.480
0.030
0.270
0.053
0.054
0.025
40
ND
33
9
17
13
55
54
37
-63*
-21
21
39-43
NC
22g
23
86-88
41-43
113-116
134-135
115-116
114-115
-21
-47*
-39*
-47*
-56*
-63*
30a
30b
30c
30d
g
a,b,c,d,e,* Refer to footnotes in Table 1. f Refer to footnote in Table 2. ND denotes not determined. NC denotes no change. Anal. H:
calcd, 8.69; found, 9.12.
Con clu sion
kg. For the corresponding R-phenyl derivatives (1, 7b,
36c,d ), histopathologic alterations to the adrenal or liver
of guinea pigs were not observed. Further evaluation
of these compounds showed that only the 2,6-diisopro-
pylanilides 7b and 36c (or bioactive metabolites) were
present in the plasma of rabbits dosed orally with these
inhibitors. Compared to CI-976, both 2,4,6-trimethoxy-
phenyl- and 2,6-diisopropylphenyl-substituted tetrazole
amides (1, 7b) displayed superior efficacy in cholesterol-
fed rabbits (ED50 ) 21.8, 0.1, 1.2 mg/kg, respectively).
In conclusion, the cumulative data from this series
of compounds reveals two drug profiles, one represented
by 7b and 36c which would be expected to have direct
antiatherosclerotic activity by virtue of their ability to
reach the arterial wall to inhibit macrophage ACAT
while being nontoxic to steroidogenic tissues and one
represented by 1 which preferentially targets intestinal
ACAT, thus reducing plasma cholesterol, but yet is
intrinsically nontoxic due to the lack of systemic expo-
In summary, we have examined the structure-
activity relationship for a series of tetrazole amides by
systematically replacing substituents appended to the
amide and tetrazole moieties of 1. For this series of
compounds, the highest potency for both microsomal
and cellular ACAT inhibition is obtained with 2,6-
diisopropyl substitution in the anilide ring. We have
shown that the methylene bridge connecting the amide
carbonyl and tetrazole ring is required for good activity,
since the higher and lower homologs are considerably
less active in our preliminary biological screens. The
best profile of in vivo activity was found with 36a , a
reverse tetrazole amide which lowered non HDL-C 81%
and elevated HDL-C 165% in rats with pre-established
hypercholesterolemia. Compound 36a and other meth-
ylene-substituted tetrazole amides (13n ,o, 36b), how-
ever, exhibited drug-related adrenal toxicity in guinea
pigs following oral administration at a dose of 100 mg/

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2361
Ta ble 4. Physical and Biological Properties of Reverse Tetrazole Amides
ACAT inhibition
liver
macrophage
no.
Rf
R1
R2
method
formulaa
mp (°C)b
(IC50, µM)c
(% inhibn, 0.1 µM)d
% change inTCe
36a
36b
36c
36d
DIP
TMP
DIP
H
H
Ph
Ph
H
H
H
H
F
F
F
F
C27H45N5O
91-93
144-146
93-95
0.006
0.056
0.015
0.021
95
61
67
52
-68*
-69*
-77*
-65*
C
C
C
24H39N5O4
33H49N5O
30H43N5O4
TMP
141-145
a-f,* Refer to the footnotes in Tables 1 and 2.
Ta ble 5. Efficacy Data for Selected Tetrazole Amides in the
Ta ble 7. Efficacy Data for Selected Tetrazole Amides in the
Chronic Cholesterol-Fed Rat Modela
Chronic Cholesterol-Fed Rabbit Modela
% change
in TC
% change
in HDL
% change
in non-HDL
dose
(mg/kg)
% change in
TC vs T0
% change in
TC vs PC
no.
no.
1
7b
7g
-60*
-38*
-38
+83*
+27
-68*
-40*
-41
CI-976
25
5
25
5
25
5
25
5
+19**
+96**
-38**
-14
-43**
-14**
-20**
+18
-20
-11**
-54*
-28*
-78*
-71*
-73*
-61*
-68*
-55*
-68*
-63*
+28
1
13a
13j
13n
13o
30b
30c
30d
36a
36b
36c
36d
-47*
-48*
-42*
-29
+12
+27
-53*
-50*
-63*
-30
7b
+82*
-9
36c
36d
-8
+16
-9
-72*
-52*
-72*
-44*
-25
+102*
+98*
+165*
+3
-77*
-58*
-81*
-46*
-27
25
5
a
Progression control (PC) are the animals maintained on the
cholesterol diet (see Experimental Section) without drug interven-
tion. *Significantly different from untreated hypercholesterolemic
control at p < 0.05. Time zero (T0) is the time at which drug
intervention begins (plasma cholesterol determined 24 h postdrug
meal.). **Significantly different from initiation of drug adminis-
tration at p < 0.05.
+50*
-3
-42
-44
a
Compounds were dosed at 30 mg/kg. For experimental details,
see ref 12. An asterisk (*) denotes significantly different from
control, p < 0.05 using analysis of variance followed by Fisher’s
multiple range test.
Ta ble 6. Oral Bioactivity for Selected Tetrazole Amides in the
Rabbit Bioassaya
dose
(mg/kg)
% inhibition 4 h
postdose (mean)
no.
CI-976
1
1
25
25
50
25
25
50
50
25
25
45
0
4
96
0
26
37
91
0
7b
13j
30c
30d
36c
36d
a
See the Experimental Section for the complete protocol.
sure. The latter may have indirect antiatherosclerotic
activity due to reduction of plasma cholesterol, but as
with other poorly absorbed ACAT inhibitors, may not
be efficacious in humans.8 Studies in atherosclerotic
lesion models to differentiate these structurally-related
but pharmacokinetically distinct potent inhibitors of
ACAT will be the subject of future reports from these
laboratories.
F igu r e 2. Dose responses for CI-976, 1, and 7b in cholesterol-
fed rabbits. Each point (dose) represents the mean reduction
of plasma cholesterol (n ) 8 animals/dose). The ED50 values
were calculated using these mean responses at each dose and
the median-effect equation (BIOSOFT Software, Cambridge,
U.K.).
Exp er im en ta l Section
on either a Varian EM-390 or a Varian XL-200 spectrometer.
Chemical shifts (δ) are expressed in ppm, relative to internal
tetramethylsilane. Mass spectra were obtained on a VG
Masslab Trio-2A mass spectrometer. Elemental analyses were
determined on a Perkin-Elmer 240C elemental analyzer and
were within (0.4% unless otherwise noted.
Unless otherwise noted, the starting materials were ob-
tained from commercial suppliers and used without further
purification. All organic extracts were dried over MgSO4,
except when otherwise noted. Melting points were determined
on a Thomas-Hoover melting point apparatus and are uncor-
rected. Nuclear magnetic resonance spectra were determined

2362 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
O’Brien et al.
Gen er a l P r oced u r e for P r ep a r in g r-P h en yl-Su bsti-
tu ted Tetr a zole Aceta m id es. Meth od A: Syn th esis of
(()-2-Dod ecyl-r-p h en yl-N-(2,4,6-tr im eth oxyp h en yl)-2H-
tetr a zole-5-a ceta m id e (1). (a ) (()- P h en yltetr a zole-5-
a cetic a cid (3). To a solution of ethyl phenylcyanoacetate 2
(100 g, 0.52 mol) in p-dioxane (1.8 L) was added tri-n-butyltin
azide (191 g, 0.57 mol) in one portion. The solution was
refluxed for 16 h, cooled to room temperature, and concen-
trated in vacuo. The resulting liquid was diluted with ethyl
ether (2 L), followed by the addition of gaseous HCl over 45
min. The solvent was evaporated and the residue triturated
with hexane to give 3 (84 g, 69%) as a white solid: mp 104-
20H), 1.1 (m, 3H), 0.9 (m, 3H) ppm; CI-MS m/ e 544 (M + 1)+.
Anal. (C30H49N5O4) C, H, N.
Gen er a l P r oced u r e for Va r yin g Su bstitu en ts P osi-
tion ed r w ith Resp ect to th e Am id e Moiety. Meth od B:
Syn t h esis of 2-Dod ecyl-r,r-d im et h yl-N-(2,4,6-t r im et h -
oxyp h en yl)-2H-tetr a zole-5-a ceta m id e (13p ). (a ) Eth yl
2,2-Dim eth ylcya n oa ceta te (8p ). A solution of ethyl cyano-
acetate 8n (20 g, 0.17 mol) in tetrahydrofuran (350 mL) was
cooled to -10 °C followed by the addition of NaH (7.2 g, 0.17
mol) in several portions. The suspension was stirred at -10
°C for 10 min before iodomethane (23.3 g, 0.17 mol) was added.
The ice bath was removed and the solution gradually warmed
to 20 °C for over 45 min. The solution was then recooled to
-10 °C, and a second equivalent of NaH (7.2 g, 0.17 mol) was
added. After 10 min of stirring, iodomethane (23.3 g, 0.17 mol)
was added, and the solution gradually warmed to room
temperature over a 2-h period. The solution was diluted with
water (350 mL), followed by the addition of diethyl ether (500
mL). The layers were separated, and the organic portion was
washed with brine, dried, and concentrated in vacuo. Distil-
lation of the crude product gave 8p as a colorless liquid (16.9
1
105 °C; H NMR (DMSO-d6) δ 7.3 (s, 5H), 5.7 (s, 1H), 4.2 (q,
2H), 1.1 (t, 3H) ppm; CI-MS m/ e 233 (M + 1)+. Anal.
(C11H12N4O2) C, H, N.
(b) (()-2-Dod ecyl-r-p h en yl-2H-tetr a zole-5-a cetic a cid ,
Eth yl Ester (4). The tetrazole 3 (55 g, 0.23 mol) was dissolved
in a solution of triethylamine (23.8 g, 0.23 mol) and acetonitrile
(700 mL). The solution was heated to reflux, followed by the
dropwise addition of 1-bromododecane (58.8 g, 0.23 mol). Upon
completion, the stirred solution was refluxed for 20 h, then
cooled, and concentrated in vacuo. The residue was triturated
with ethyl acetate (400 mL) and filtered, and the filtrate was
evaporated to dryness. The crude liquid containing a mixture
of the 2- and 1-regioisomers (3:1) was purified using silica gel
chromatography (elution with 75% hexane/25% ethyl acetate)
to give 4 (55.4 g, 60%) as a colorless liquid: 1H NMR (CDCl3)
δ 7.5 (d, 2H), 7.3 (m, 3H), 5.3 (s, 1H), 4.5 (t, 2H), 4.2 (m, 2H),
2.0 (m, 2H), 1.2 (s, 18H), 0.8 (t, 3H) ppm; CI-MS m/ e 401 (M
+ 1)+. Anal. (C23H36N4O2) C, H, N.
1
g, 68%): bp 82-85 °C, 15 mm Hg; H NMR (CDCl3) δ 4.3 (q,
2H), 1.6 (s, 6H), 1.3 (t, 3H) ppm; CI-MS m/ e 142 (M + 1)+.
(b ) r,r-Dim et h ylt et r a zole-5-a cet ic Acid , E t h yl E st er
(9p ). A solution of tri-n-butyltin azide (27.3 g, 0.082 mol) in
p-dioxane (240 mL) was added to ethyl 2,2-dimethylcyano-
acetate 8p (11.6 g, 0.082 mol) in one portion. The solution
was refluxed overnight, cooled to room temperature, and
concentrated in vacuo. The resulting liquid was diluted with
ethyl ether (500 mL) and treated with gaseous HCl continu-
ously for 15 min. Evaporation of the solvent yielded a viscous
liquid which solidified when triturated with warm hexane. The
solid was filtered and recrystallized from hexane/ethyl acetate
to give 9p (8.4 g, 55%) as a cream-colored solid: 1H NMR
(CDCl3) δ 12.2 (bs, 1H), 4.2 (q, 2H), 1.8 (s, 6H), 1.3 (t, 3H) ppm;
CI-MS m/ e 185 (M + 1)+.
The 1-regioisomer 5 (17 g, 18%) was isolated pure as a
colorless liquid: 1H NMR (CDCl3) δ 7.4-7.2 (m, 5H), 5.3 (s,
1H), 4.2 (q, 2H), 4.0 (t, 2H), 1.5 (m, 2H), 1.2 (s, 18H), 0.8 (t,
3H) ppm.
(c) (()-2-Dod ecyl-r-p h en yl-2H-tetr a zole-5-a cetic Acid
(6). To a solution of 4 (45.3 g, 0.11 mol) in absolute ethanol
(400 mL) was added sodium hydroxide (8.8 g, 0.22 mol) in one
portion. The solution was stirred at room temperature for 16
h and then concentrated in vacuo. The resulting residue was
dissolved in water (600 mL) and acidified with concentrated
HCl (pH ) 1.0), and the product was partitioned between ethyl
acetate (400 mL) and aqueous HCl. The organic layer was
separated, washed with brine, dried, and concentrated to
dryness to give a colorless liquid. Trituration of the liquid with
hexane provided 6 (36.6 g, 89%) as a white crystalline solid:
mp 55-57 °C; 1H NMR (DMSO-d6) δ 7.4 (d, 2H), 7.3 (m, 3H),
5.4 (s, 1H), 4.6 (t, 2H), 1.8 (m, 2H), 1.2 (m, 18H), 0.8 (t, 3H)
ppm; CI-MS m/ e 373 (M + 1)+. Anal. (C21H32N4O2) C, H, N.
(d) (()-2-Dodecyl-r-ph en yl-N-(2,4,6-tr im eth oxyph en yl)-
2H-tetr a zole-5-a ceta m id e (1). Compound 6 (20 g, 0.053 mol)
was dissolved in tetrahydrofuran (300 mL) and treated with
CDI (8.69 g, 0.053 mol) in one portion. The solution was
stirred for 1 h at room temperature, followed by the dropwise
addition of 2,4,6-trimethoxyaniline (9.8 g, 0.053 mol). After
16 h of stirring, ethyl acetate (1 L) was added, and the solution
was washed with aqueous HCl (1 M), aqueous NaOH (1 M),
and brine and dried. The solution was concentrated in vacuo
leaving a lavendar solid. The crude product was purified using
silica gel chromatography (elution with 97% chloroform/3%
methanol) to give 1 (19.3 g, 67%) as a white solid: mp 119-
(c) 2-Dod ecyl-r,r-d im eth yl-2H-tetr a zole-5-a cetic Acid ,
Eth yl Ester (10p ). The tetrazole (4.0 g, 0.021 mol) 9p was
dissolved in acetonitrile (50 mL) containing 1 equiv of triethyl-
amine (2.3 g, 0.021 mol). The solution was heated to reflux,
followed by the dropwise addition of 1-bromododecane (5.6 g,
0.022 mol). The solution was refluxed for 16 h, cooled to room
temperature, and then concentrated in vacuo. The residue was
triturated with ethyl acetate (250 mL) and filtered, and the
filtrate was washed with aqueous HCl (1 M) and brine, dried,
and filtered. Concentration of the filtrate in vacuo left a
viscous liquid containing both the 1- and 2-regioisomers. Only
the latter isomer 10p (4.5 g, 59%) was obtained, as a colorless
liquid, utilizing silica gel chromatography (elution with 90%
hexane/10% ethyl acetate): 1H NMR (CDCl3) δ 4.5 (t, 2H), 4.1
(q, 2H), 1.9 (m, 2H), 1.7 (s, 6H), 1.2 (s, 18H), 0.9 (t, 3H) ppm;
CI-MS m/ e 353 (M + 1)+.
(d ) 2-Dod ecyl-r,r-d im eth yl-2H-tetr a zole-5-a cetic Acid
(12p ). Hydrolysis of 10p was achieved by stirring the ester
(3.2 g, 9.0 mmol) in a solution of sodium hydroxide (0.38 g,
9.5 mmol) and ethanol (40 mL) at room temperature for 16 h.
The solution was concentrated in vacuo, and the product was
partitioned between ethyl acetate (50 mL) and aqueous HCl
(50 mL, pH ) 1.0). The layers were separated, and the organic
portion was washed with brine, dried, and filtered. The filtrate
was concentrated in vacuo to give 12p (2.0 g, 69%) as a
colorless liquid, which gradually solidified to a wax on stand-
ing: 1H NMR (CDCl3) δ 4.5 (t, 2H), 2.0 (m, 2H), 1.7 (s, 6H),
1.2 (s, 18H), 0.9 (t, 3H) ppm; CI-MS m/ e 325 (M + 1)+. Anal.
(C17H32N4O2) C, H, N.
1
120 °C; H NMR (CDCl3) δ 7.7 (bs, 1H), 7.6 (d, 2H), 7.3 (m,
3H), 6.1 (s, 2H), 5.4 (s, 1H), 4.6 (t, 2H), 3.7 (d, 9H), 2.0 (m,
2H), 1.3 (s, 18H), 0.9 (t, 3H) ppm; CI-MS m/ e 538 (M + 1)+.
Anal. (C30H43N5O4) C, H, N.
Compounds 7a -j were synthesized utilizing similar reaction
conditions.
(e) 2-Dodecyl-r,r-dim eth yl-N-(2,4,6-tr im eth oxyph en yl)-
2H-tetr a zole-5-a ceta m id e (13p ). To a stirred solution of
12p (2.0 g, 6.1 mmol) in tetrahydrofuran (50 mL) was added
CDI (1.0 g, 6.1 mmol) in one portion. The solution was stirred
for 1 h at room temperature, followed by the dropwise addition
of 2,4,6-trimethoxyaniline (1.1 g, 6.1 mmol). After stirring at
room temperature for 5 days, the solution was diluted with
ethyl acetate (100 mL) and washed with aqueous HCl (1 M),
aqueous NaOH (1 M), and brine. The solution was dried and
filtered, and the filtrate was concentrated in vacuo leaving a
maroon-colored liquid. The crude product was purified using
(()-2-Dodecyl-r-cycloh exyl-N-(2,4,6-tr im eth oxyph en yl)-
2H-tetr a zole-5-a ceta m id e (13m ). Compound 1 (0.86 g, 1.5
mmol) was hydrogenated in a Parr shaker containing 10%
Rh/C suspended in acetic acid (5 mL) and THF (100 mL). The
reaction vessel was shaken for 48 h under 50 psi, followed by
filtration of the catalyst. The filtrate was concentrated in
vacuo, and the residue was recrystallized from ethyl acetate
to give 13m (0.38 g, 48%) as a white solid: mp 140-141 °C;
1H NMR (CDCl3) δ 7.7 (s, 1H), 6.1 (s, 2H), 4.6 (t, 2H), 3.7 (d,
9H), 3.8 (d, 1H), 2.2 (m, 1H), 2.0 (m, 3H), 1.6 (m, 6H), 1.2 (s,

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2363
silica gel chromatography (elution with 75% hexane/25% ethyl
acetate) to give 13p as a pale yellow liquid: 1H NMR (CDCl3)
δ 7.2 (bs, 1H), 6.1 (s, 2H), 4.6 (t, 2H), 3.7 (d, 9H), 2.1 (m, 2H),
1.7 (s, 6H), 1.3 (s, 18H), 0.9 (t, 3H) ppm; CI-MS m/ e 490 (M +
1)+. Anal. (C26H43N5O4) C, H, N.
4.6 (t, 2H), 2.8 (m, 2H), 2.0 (m, 2H), 1.3 (s, 20H), 1.1 (dd, 12H),
0.8 (t, 3H) ppm; CI-MS m/ e 524 (M + 1)+. Anal. (C28H45N8O)
C, H, N.
Gen er a l P r oced u r e for P r ep a r in g Tetr a zole Ca r box-
a m id es. Meth od D: Syn th esis of 2-Dod ecyl-N-(2,4,6-
tr im eth oxyp h en yl)-2H-tetr a zole-5-ca r boxa m id e (20). (a )
1H-Tetr a zole-5-ca r boxylic Acid , Eth yl Ester (17a ). To a
stirred solution of ethyl cyanoformate 16a (19.6 g, 0.19 mol)
in pyridine (78 mL) was added a cold solution (+5 °C) of
trifluoroacetic acid (33 mL, 0.43 mol) in pyridine (128 mL).
The mixture was heated at 60 °C for 48 h, cooled to room
temperature, and then poured over ice (400 g) containing
concentrated HCl (156 mL). The aqueous acid was washed
with several portions of ethyl acetate. The extracts were
combined, dried, and concentrated to an oil. The liquid was
filtered through silica gel (elution with ethyl acetate/methanol
(15:1)), and fractions containing the product were combined
and concentrated in vacuo. The oil obtained was diluted with
dichloromethane and extracted with aqueous NaOH (2M). The
basic solution was washed with dichloromethane, then acidi-
fied to pH ) 1 with concentrated HCl. Ethyl acetate was
added, the layers were separated, the organic portion was dried
and filtered, and the filtrate was concentrated in vacuo to give
17a (9.3 g, 33%) as a white solid: mp 87.5-91.5 °C; 1H NMR
(DMSO-d6) δ 4.4 (q, 2H), 1.3 (t, 3H) ppm; EI-MS m/ e 143 (M+).
Anal. (C4H6N4O2) C, H, N.
(b) 2-Dod ecyl-2H-tetr a zole-5-ca r boxylic Acid , Eth yl
Ester (18a ). The tetrazole 17a (8.9 g, 0.063 mol) was added
to a solution of triethylamine (9.6 mL, 0.069 mol) in acetonitrile
(175 mL). The suspension was heated to 72 °C and treated
with 1-bromododecane (16.5 mL, 0.068 mol) in one portion.
The mixture was refluxed for 24 h, cooled, and filtered.
Concentration of the filtrate in vacuo gave a mixture regio-
isomers (1:1), from which the 2-isomer was separated by silica
gel chromatography (elution with 90% hexane/10% ethyl
acetate) to give 18a (9.2 g, 46%) as a white solid: mp 41-45
°C; 1H NMR (CDCl3) δ 4.7 (t, 2H), 4.5 (q, 2H), 2.0 (m, 2H), 1.5
(t, 2H), 1.33-1.25 (m, 19H), 0.9 (t, 3H) ppm; EI-MS m/ e 311
(M+). Anal. (C16H30N4O2) C, H, N.
Compounds 13e,i,j were synthesized in this manner. Uti-
lizing steps b-e, anilides 13n ,o were prepared.
Alter n a tive P r oced u r e for Va r yin g Su bstitu en ts P o-
sition ed r w ith Resp ect to th e Am id e Moiety. Meth od
C: P r ep a r a tion of (()-N-[2,6-Bis(1-m eth yleth yl)p h en yl]-
2-d od ecyl-r-2-p yr id in yl-2H-tetr a zole-5-a ceta m id e (13l).
(a ) 5-(2-P yr id ylm eth yl)-1H-tetr a zole. To a solution of
2-pyridylacetonitrile 14l (24.8 g, 0.21 mol) in p-dioxane (500
mL) was added tri-n-butyltin azide (100 g, 0.30 mol) in one
portion. The solution was refluxed for 20 h, cooled to room
temperature, and then concentrated in vacuo. Dilution of the
resulting liquid with ethyl ether (500 mL), followed by the
addition of gaseous HCl over 15 min, provided a maroon-
colored precipitate. The solid was collected by filtration and
recrystallized from ethanol to give the tetrazole hydrochloride
(22.4 g, 54%) as amber-colored needles: 1H NMR (DMSO-d6)
δ 10.4 (bs, 1Η), 8.9 (d, 1H), 8.4 (t, 1H), 7.9 (t, 2H), 4.8 (s, 2H)
ppm; CI-MS m/ e 162 (M + 1)+.
(b) 4-(2-P yr id ylm eth yl)-2-d od ecyl-2H-tetr a zole (15l).
The salt (22.4 g, 0.11 mol) obtained in step a was diluted with
a solution of acetonitrile (300 mL) containing 2 equiv of tri-
ethylamine (22.9 g, 0.22 mol). The solution was heated to
reflux, followed by the dropwise addition of 1-bromododecane
(28.2 g, 0.11 mol). After 16 h of reflux, the solution was cooled
to room temperature and concentrated in vacuo. Ethyl acetate
was added (300 mL), the insoluble salts were filtered, and the
filtrate was concentrated in vacuo. The resulting brown liquid
was purified by silica gel chromatography (elution with hex-
ane/ethyl acetate (1:1)), isolating only the faster eluting
2-isomer 15l (15.9 g, 44%) as an orange-colored liquid: 1H
NMR (CDCl3) δ 8.5 (d, 1H), 7.7 (t, 1H), 7.3 (d, 1H), 7.2 (m,
1H), 4.5 (t, 2H), 4.4 (s, 2H), 1.9 (m, 2H), 1.3 (s, 18H), 0.9 (t,
3H) ppm; CI-MS m/ e 330 (M + 1)+. Anal. (C19H31N5) C, H,
N.
(c) 2-Dod ecyl-2H-tetr a zole-5-ca r boxylic Acid . To a
stirred solution of potassium hydroxide (1.9 g, 0.035 mol) in
absolute ethanol (280 mL) was added 18a (8.7 g, 0.028 mol)
in one portion. The suspension was stirred at room temper-
ature for 24 h, at which time the precipitate was collected by
filtration and washed with ethanol. The filter cake was
partitioned between ethyl acetate (160 mL) and 1 M HCl (66
mL). The organic phase was washed with brine, dried, and
filtered, and the filtrate was concentrated in vacuo to give
2-dodecyl-2H-tetrazole-5-carboxylic acid (7.7 g, 98%) as a white
(c) (()-N-[2,6-Bis(1-m eth yleth yl)p h en yl]-2-d od ecyl-r-
2-p yr id in yl-2H-tetr a zole-5-a ceta m id e (13l). A solution of
15l (10.7 g, 0.032 mol) in dry tetrahydrofuran (200 mL) was
cooled to -20 °C and treated dropwise with n-butyllithium
(24.4 mL, 0.039 mol). The solution was stirred for 30 min at
-20 °C and was delivered by cannula to a solution of
2,6-diisopropylphenyl isocyanate (7.9 g, 0.039 mol) in tetrahy-
drofuran (50 mL), also cooled to -20 °C. After warming to
room temperature, the yellow solution was diluted with ethyl
acetate (250 mL) and water (100 mL). The organic phase was
separated, dried, and filtered, and the filtrate was concentrated
in vacuo leaving a yellow liquid. The crude product was
purified by silica gel chromatography (elution with hexane/
ethyl acetate (1:1)) to give a pale yellow liquid. The liquid
obtained crystallized from pentane to give the title compound
1
solid: mp 122-124 °C; H NMR (DMSO-d6) δ 14.2 (bs, 1H),
4.7 (t, 2H), 1.9 (m, 2H), 1.2 (m, 18H), 0.9 (t, 3H) ppm; EI-MS
m/ e 283 (M+). Anal. (C14H26N4O2) C, H, N.
(d) 2-Dodecyl-N-(2,4,6-tr im eth oxyph en yl)-2H-tetr azole-
5-ca r boxa m id e (20). The acid (1.68 g, 5.95 mmol) prepared
in step c was dissolved in tetrahydrofuran (35 mL) and treated
with CDI (1.0 g, 6.17 mmol) in one portion. The solution was
stirred at room temperature for 1 h, followed by the dropwise
addition of a solution of 2,4,6-trimethoxyaniline (1.15 g, 6.28
mmol) in tetrahydrofuran (30 mL). The mixture was stirred
at room temperature for 72 h and then refluxed for an
additional 168 h. The suspension was cooled, filtered, and
concentrated to an oil. Ethyl acetate (40 mL) was added, and
the solution was washed with aqueous NaOH (1 M), aqueous
HCl (1 M), water, and brine. The organic phase was dried
and filtered, and the filtrate was concentrated in vacuo to an
oil. The crude product was purified using silica gel chroma-
tography (elution with petroleum ether/ethyl acetate (4:1)) to
give 20 as a white solid: mp 83-85 °C; 1H NMR (DMSO-d6) δ
9.6 (s, 1H), 6.3 (s, 2H), 4.7 (t, 2H), 3.8 (s, 3H), 3.7 (s, 6H), 1.9
(m, 2H), 1.2 (m, 18H), 0.9 (t, 3H) ppm; EI-MS m/ e 447 (M+).
Anal. (C23H37N5O4) C, H, N.
1
13l (11.9 g, 69%) as a yellow solid: mp 70-71 °C; H NMR
(CDCl3) δ 9.2 (s, 1H), 8.6 (d, 1H), 7.8 (t, 1H), 7.6 (d, 1H), 7.3
(m, 2H), 7.1 (d, 2H), 5.6 (s, 1H), 4.6 (t, 2H), 2.9 (bs, 2H), 2.0
(m, 2H), 1.3 (s, 20H), 1.1 (d, 12H), 0.8 (t, 3H) ppm; CI-MS m/ e
533 (M + 1)+. Anal. (C32H48N6O) C, H, N.
Compounds 13a -d ,f,g,k were also synthesized from the
appropriately substituted acetonitrile utilizing similar reaction
conditions.
N-(2,6-d iisop r op ylp h en yl)-2-(2-d od ecyl-2H-tetr a zol-5-
yl)-2-(1H-tetr a zol-5-yl)a ceta m id e (13h ). To a solution of
13g (1.4 g, 2.8 mmol) in p-dioxane (20 mL) was added, in one
portion, tri-n-butyltin azide (1.1 g, 3.3 mmol). The solution
was refluxed overnight, cooled, and concentrated in vacuo. The
resulting viscous liquid was diluted with diethyl ether, followed
by the addition of gaseous HCl for 15 min. The ethereal
solution was concentrated in vacuo leaving a viscous liquid,
which solidified upon treatment with hexane/ethyl acetate (4:
1). The crude product was recrystallized from hexane/ethyl
acetate to give 13h (0.9 g, 62%) as a white solid: mp 147-148
Compound 21 in Table 3 was synthesized in a similar
manner.
Gen er a l P r oced u r e for P r ep a r in g Tetr a zole P r op a n -
a m id e Der iva tives. Meth od E: Syn th esis of 2-Dod ecyl-
N-[2,6-bis(1-m eth yleth yl)p h en yl]-2H-tetr a zole-5-p r op a n -
1
°C; H NMR (DMSO-d6) δ 7.3 (t, 1Η), 7.1 (d, 2Η), 6.2 (s, 1H),

2364 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
O’Brien et al.
a m id e (23). (a ) 1H-Tetr a zole-5-p r op a n oic Acid , Meth yl
Ester (17b). To a stirred solution of 3-cyanopropanoic acid,
methyl ester (16b) (109 g, 0.96 mol) in N,N-dimethylformamide
(1 L) was added ammonium chloride (51.5 g, 0.96 mol) and
sodium azide (62.7 g, 0.96 mol), respectively. The suspension
was stirred for 6 h at 100 °C, then cooled, filtered, and
concentrated in vacuo, leaving a yellow liquid. Water (500 mL)
was added, and the solution was cooled to 5 °C and acidified
with concentrated HCl (170 mL). The product was extracted
with several portions of ethyl acetate (400 mL), then combined
and washed with brine, dried, and concentrated to give 17b
(102 g, 68%) as a viscous oil: 1H NMR (DMSO-d6) δ 3.6 (s,
3H), 3.5 (bs, 1H), 3.1 (t, 2H), 2.8 (t, 2H) ppm; CI-MS m/ e 157
(M + 1)+. Anal. (C5H8N4O2) C, H, N.
temperature, water (500 mL) was added and the product was
extracted with several portions of ethyl ether (2 × 150 mL).
The extracts were combined, washed with brine, dried, and
filtered. Evaporation of the filtrate gave 26 (14.5 g, 98%) as
a viscous orange liquid: 1H NMR (CDCl3) δ 5.8 (m, 1H), 4.9
(m, 2H), 2.3 (t, 2H), 2.0 (m, 2H), 1.6 (m, 2H), 1.3 (bs, 12H)
ppm.
(c) 11-Dod ecen oic Acid (27). A solution of 26 (14.4 g,
0.080 mol) in methanol (150 mL) was treated with 25%
aqueous NaOH (40 g) in one portion. The solution was
refluxed for 24 h and cooled to room temperature, and the
methanol was concentrated in vacuo. The aqueous concentrate
was diluted with water (150 mL), washed with ethyl ether (1
× 100 mL), and neutralized (pH ) 6.0) with concentrated HCl.
The product was exracted with ethyl acetate (150 mL), washed
with brine, dried, and filtered. Evaporation of the filtrate gave
27 (10.6 g, 67%), isolated in the form of a viscous liquid which
gradually solidified to a wax on standing: 1H NMR (CDCl3) δ
5.8 (m, 1H), 4.9 (m, 2H), 2.3 (m, 2H), 2.0 (m, 2H), 1.6 (m, 2H),
1.3 (s, 12H) ppm.
(d ) 11-Dod ecen -1-ol. Lithium aluminum hydride (2.2 g,
0.058 mol) was suspended in dry tetrahydrofuran (50 mL) and
cooled to 0 °C. The suspension was treated dropwise with a
solution of 27 (10.6 g, 0.053 mol) in dry tetrahydrofuran (160
mL). The reaction mixture gradually warmed to room tem-
perature and was stirred for 16 h. Excess hydride was
cautiously quenched with aqueous HCl (0.1 M), followed by
the addition of ethyl acetate (200 mL). The suspension was
filtered through Celite, and the filtrate was dried and concen-
trated to dryness, isolating the title compound as a yellow
liquid (5.9 g, 60%): 1H NMR (CDCl3) δ 5.8 (m, 1H), 4.9 (m,
2H), 3.6 (t, 2H), 2.0 (m, 2H), 1.9 (bs, 1H), 1.6 (m, 2H), 1.3 (s,
12H) ppm.
(e) 11-Dod ecen yl Meth a n esu lfon a te (28). Following the
procedure previously described for 25, the alcohol (5.9 g, 0.032
mol) prepared from step d yielded mesylate 28 (8.27 g, 99%)
as an orange liquid: 1H NMR (CDCl3) δ 5.8 (m, 1H), 4.9 (m,
2H), 4.2 (t, 2H), 3.0 (s, 3H), 2.0 (m, 2H), 1.7 (m, 2H), 1.3 (bs,
12H) ppm. The product was used in the coupling with 3
without further purification.
Gen er a l P r oced u r e for Va r yin g t h e Tet r a zole Sid e
Ch a in . Met h od F : (()-2-(11-Dod ecen yl)-r-p h en yl-N-
(2,4,6-tr im eth oxyph en yl)-2H-tetr azole-5-acetam ide (30d).
(a ) (()-2-(11-Dod ecen yl)-r-p h en yl-2H-tetr a zole-5-a cetic
Acid , Eth yl Ester (29). The mesylate of 11-dodecen-1-ol 24
(8.2 g, 0.032 mol) was added dropwise to a stirred solution
containing 3 (7.4 g, 0.032 mol), triethylamine (3.3 g, 0.032 mol),
and acetonitrile (100 mL). The solution was refluxed for 16
h, cooled, and concentrated in vacuo. The residue was
triturated with ethyl acetate and filtered, and the filtrate was
washed with aqueous HCl (1 M) and brine, dried, and filtered.
Evaporation of the filtrate gave a viscous liquid containing a
2.5:1 mixture of the 2- and 1-regioisomers, respectively. Only
the faster eluting 2-isomer 29 was isolated from the mixture
as a pale yellow liquid (4.6 g, 36%) using silica gel chroma-
tography (elution with 75% hexane/25% ethyl acetate): 1H
NMR (CDCl3) δ 7.5 (m, 2H), 7.3 (m, 3H), 5.8 (m, 1H), 5.3 (s,
1H), 4.9 (m, 2H), 4.6 (t, 2H), 4.2 (q, 2H), 2.0 (m, 4H), 1.4-1.3
(m, 14H), 1.2 (t, 3H) ppm.
(b) (()-2-(11-Dod ecen yl)-r-p h en yl-2H-tetr a zole-5-a ce-
tic Acid . Using the same procedure as that for compound 4,
hydrolysis of 29 (4.5 g, 0.011 mol) yielded the title compound
(4.1 g, 98%) as a yellow liquid: 1H NMR (CDCl3) δ 7.5 (m,
2H), 7.3 (m, 3H), 5.8 (m, 1H), 5.4 (s, 1H), 4.9 (m, 2H), 4.6 (t,
2H), 2.0 (m, 4H), 1.3 (m, 14H) ppm.
(c) (()-2-(11-Dod ecen yl)-r-p h en yl-N-(2,4,6-t r im et h -
oxyp h en yl)-2H-tetr a zole-5-a ceta m id e (30d ). The acid (4.1
g, 0.011 mol) prepared in step b was dissolved in a solution
containing 2,4,6-trimethoxyaniline (2.0 g, 0.011 mol) and
dichloromethane (75 mL), cooled to 0 °C, and treated with DCC
(2.3 g, 0.011 mol) in one portion. The suspension was stirred
at room temperature for 16 h, then diluted with ethyl acetate,
and filtered. The filtrate was washed with aqueous HCl (1
M), aqueous NaOH (1 M), and brine and dried. The solution
was concentrated in vacuo, and the residue was purified using
silica gel chromatography (elution with 90% hexane/10% ethyl
(b) 2-Dod ecyl-2H-tetr a zole-5-p r op a n oic Acid , Eth yl
Ester (18b). The tetrazole 17b (29.2 g, 0.18 mol) was diluted
with acetonitrile (500 mL) and treated with 1 equiv of
triethylamine (18.1 g, 0.018 mol). The solution was heated to
reflux, followed by the addition of 1-bromododecane (49.5 mL,
0.20 mol). After 24 h of reflux, the solution was cooled and
filtered. Evaporation of the filtrate gave a viscous liquid, from
which the 2-isomer 18b (12.0 g, 20%) was isolated as a white
solid using silica gel chromatography (elution with petroleum
1
ether/ethyl acetate (15:1)): mp 39-42 °C; H NMR (DMSO-
d6) δ 4.7 (t, 2H), 3.6 (s, 3H), 3.1 (t, 2H), 2.8 (t, 2H), 1.9 (m,
2H), 1.2 (m, 18H), 0.9 (t, 3H) ppm; EI-MS m/ e 325 (M+). Anal.
(C17H32N4O2) C, H, N.
(c) 2-Dod ecyl-2H-tetr a zole-5-p r op a n oic Acid . The ester
18b (11.5 g, 0.035 mol) was dissolved in a solution containing
potassium hydroxide (2.5 g, 0.045 mol) and absolute ethanol
(210 mL). The solution was stirred for 72 h at room temper-
ature and then concentrated in vacuo. The residue was
dissolved in water (300 mL), cooled to 5 °C, and acidified with
concentrated HCl (10 mL). Dichloromethane was added, the
layers were separated, and the organic phase was dried,
filtered, and concentrated to give the title compound as a white
1
solid (10.6 g, 96%): mp 63-65 °C; H NMR (DMSO-d6) δ 4.6
(t, 2H), 3.0 (t, 2H), 2.7 (t, 2H), 1.8 (m, 2H), 1.2 (m, 18H), 0.9
(t, 3H) ppm.
(d ) 2-Dod ecyl-N-[2,6-bis(1-m eth yleth yl)p h en yl]-2H-tet-
r a zole-5-p r op a n a m id e (23). A portion of the acid (1.2 g, 3.87
mmol) prepared in step c was dissolved in tetrahydrofuran (35
mL), followed by the addition of CDI (0.65 g, 3.98 mmol). The
mixture was stirred at room temperature for 1 h, before 2,6-
diisopropylaniline (0.70 g, 3.98 mmol) was added in one
portion. After 168 h of reflux, the suspension was cooled and
filtered, and the filtrate was concentrated to dryness. The
resulting oil was diluted with ethyl acetate (40 mL) and
washed with aqueous NaOH (1 M), aqueous HCl (1 M), water,
and brine, respectively. The organic phase was dried and
concentrated to an oil. The crude product was purified using
silica gel chromatography (elution with 75% petroleum ether/
25% ethyl acetate) to give 23 as a tan solid: mp 41-43 °C; 1H
NMR (DMSO-d6) δ 9.3 (s, 1H), 7.2-7.1 (m, 3H), 4.6 (t, 2H),
3.1 (t, 2H), 2.9 (m, 2H), 2.8 (t, 2H), 1.9 (m, 2H), 1.2 (s, 18H),
1.0 (m, 12H), 0.9 (t, 3H) ppm; EI-MS m/ e 469 (M+). Anal.
(C28H47N5O) C, H, N.
Similar conditions were used for preparing 22 in Table 3.
11-Dod ecen yl m eth a n esu lfon a te (28) was utilized in the
synthesis of 30d . (a ) 10-Un d ecen yl Met h a n esu lfon a t e
(25). Alcohol 24 (30.4 g, 0.18 mol) was diluted with dichloro-
methane (360 mL), cooled to 0 °C, and treated with triethyl-
amine (18.1 g, 0.18 mol) in one portion. Methanesulfonyl
chloride (22.4 g, 0.19 mol) was then added at such a rate so as
to maintain a solution temperature of 0 °C. After stirring at
0 °C for 3 h, the product was partitioned between dichloro-
methane and brine. The layers were separated, and the
organic phase was dried and filtered, and the filtrate was
concentrated in vacuo, leaving 25 as a viscous yellow liquid
(35.9 g, 81%): 1H NMR (CDCl3) δ 5.8 (m, 1H), 4.9 (m, 2H), 4.2
(t, 2H), 3.0 (s, 3H), 2.1 (m, 2H), 1.8 (m, 2H), 1.3 (bs, 12H) ppm.
(b) 11-Dod ecen en itr ile (26). To a solution of mesylate
25 (20.5 g, 0.082 mol) in dimethyl sulfoxide (250 mL) was
added potassium cyanide (16.0 g, 0.24 mol). The solution was
stirred at 80 °C for 10 min, resulting in the formation of a
gelatinous precipitate. After the mixture was cooled to room

Inhibitors of ACAT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2365
acetate). The solid obtained was recrystallized from hexane/
ing albumin (20 µM) and phosphatidylserine/cholesterol vesicles.
The latter were prepared by combining phosphatidylserine
(27.4 µmol) and cholesterol (26 µmol) in chloroform and then
evaporating the chloroform in order to disperse the dried lipids
into 5 mL of 150 mM NaCl, 5 mM 3-(N-morpholino)propane-
sulfonic acid (MOPS), and 1 mM EDTA (pH 7.4). This lipid
suspension was sonicated to form vesicles, which were then
sterilized by filtration (0.45 µM filter). Following the pre-
incubation, the media with vesicles was replaced with serum-
free DMEM containing the test compound in DMSO and
ethyl acetate to give 30d (3.2 g, 53%) as a white solid: mp
1
114-115 °C; H NMR (CDCl3) δ 7.6 (m, 3H), 7.3 (m, 3H), 6.1
(s, 2H), 5.8 (m, 1H), 5.5 (s, 1H), 4.9 (m, 2H), 4.6 (t, 2H), 3.8 (d,
9H), 2.0 (m, 4H), 1.3 (bs, 14H) ppm; CI-MS m/e 536 (M + H)+.
Anal. (C30H41N5O4) C, H, N.
The tetrazole side chains for compounds 30a -c were
synthesized from 3 using similar reaction conditions.
Gen er a l P r oced u r e for P r ep a r in g Rever se Tetr a zole
Am id es. Meth od G: Syn th esis of N-[2,6-bis(1-m eth yl-
eth yl)p h en yl]-5-d od ecyl-2H-tetr a zole-2-a ceta m id e (36a ).
Syn t h esis of 5-Dod ecyl-1H-t et r a zole (32). A mixture of
lauryl cyanide (31, 147.1 g, 0.753 mol) and tri-n-butyltin azide
(375.0 g, 1.13 mol) in 900 mL of dioxane was heated to reflux
overnight. The reaction mixture was concentrated in vacuo
and redissolved in 1 L of diethyl ether. HCl gas was passed
through the ether solution for 1 h. The resulting precipitate
was collected by filtration to give 115.5 g of product as a white
solid. Additional ether was added to the filtrate, and HCl gas
was again passed through this solution to give a second crop
of product which was washed with pentane to give an ad-
ditional 30.3 g of product: total yield 145.8 g (81%); mp 68-
incubated for 1 h, after which [14
C]sodium oleate was added
for an additional 4 h. The media was removed, and cells were
washed and extracted with hexane/2-propanol (3:2, v/v) con-
taining internal standard ([14C]cholesterol). Neutral lipids
were separated by TLC followed by isotopic scanning (Phos-
phorImager, Molecular Dynamics, Sunnyvale, CA). Cell pro-
tein concentration was determined by the method of Lowry et
al.16
ACAT Bioa ssa y. Male, New Zealand white rabbits (1.2-
1.5 kg) were conditioned to meal-feeding for 1 week and then
given a meal containing the drug in an oil vehicle (3% peanut
oil/3% coconut oil) at the appropriate test dose level. Blood
samples were obtained at time zero (before drug) and 1-, 2-,
and 4-h postdrug meal. The plasma samples (0.2 mL) were
extacted into hexane, dried, and then taken up into chloroform/
methanol (2:1, v/v) for transfer to tubes used for the microso-
mal liver ACAT assay. Standards were prepared for each test
1
70 °C; H NMR (CDCl3) δ 14.57 (bs, 1H), 3.14 (t, 2H), 1.86-
1.94 (m, 2H), 1.18-1.28 (m, 18H), 0.86 (t, 3H) ppm. Anal.
(C13H26N4) C, H, N.
Syn th esis of Eth yl 5-Dod ecyl-2H-tetr a zole-2-a ceta te
(33a ) a n d Eth yl 5-Dod ecyl-1H-tetr a zole-1-a ceta te (34a ).
A solution of 5-dodecyl-1H-tetrazole (32, 40.4 g, 0.168 mol),
ethyl bromoacetate (30.8 g, 0.185 mol), and triethylamine (25.8
mL, 0.185 mol) in 400 mL of acetonitrile was heated to reflux
for 4 h. This was then cooled to room temperature and stirred
overnight. The solution was concentrated in vacuo and the
residue partitioned between water and dichloromethane. The
organic layer was dried, filtered, and concentrated to leave a
yellow oil. Chromatography (10% ethyl acetate/hexanes) on
silica gel gave 20.16 g (37%) of ethyl 5-dodecyl-2H-tetrazole-
2-acetate 33a as a clear oil which solidified upon standing:
compound by direct addition to rabbit plasma in DMSO.
A
reference control was also included in each assay (i.e. the
ACAT inhibitor, CI-976 at 2.5 µM). These “spiked” plasmas
were extracted together with the test samples.
In Vivo Ch olester ol-F ed Ra bbit Assa y. Male, New
Zealand rabbits (1-1.5 kg) were adapted to a meal-feeding
regimen (50 g/day). The diet consisted of normal rabbit chow
supplemented with cholesterol (0.5%), coconut oil (3%), and
peanut oil (3%). After 1 week the compounds were added to
the diet for a period of 2 weeks at the indicated doses. Blood
was obtained from the heart of fasted animals after nitrogen
asphyxiation for determining plasma total cholesterol concen-
trations. Efficacy is expressed as a percentage change from
the cholesterol-fed controls.
1
mp 38-40 °C; H NMR (CDCl3) δ 5.36 (s, 2H), 4.26 (q, 2H),
2.91 (t, 2H), 1.76-1.83 (m, 2H), 1.25-1.37 (m, 21H), 0.88 (t,
3H) ppm. Anal. (C17H32N4O2) C, H, N.
Also isolated 21.38 g (39%) of ethyl 5-dodecyl-1H-tetrazole-
1-acetate (34a ) as a white solid: mp 48-50 °C; 1H NMR
(CDCl3) δ 5.08 (s, 2H), 4.27 (q, 2H), 2.78 (t, 2H), 1.78-1.88
(m, 2H), 1.25-1.37 (m, 21H), 0.87 (t, 3H) ppm. Anal.
(C17H32N4O2) C, H, N.
Ack n ow led gm en t. We thank Dr. G. A. McClusky
and staff for spectral and analytical determinations.
Syn th esis of 5-Dodecyl-2H-tetr azole-2-acetic Acid (35a).
Ethyl 5-dodecyl-2H-tetrazole-2-acetate (33a , 1.96 g, 0.006 mol)
was dissolved in 50 mL of ethanol, and 9 mL of 1 M NaOH
was added. After 2 h, the reaction was concentrated in vacuo,
and the residue was partitioned between water and diethyl
ether. The aqueous layer was acidified with concentrated HCl
and extracted with diethyl ether. The ether extracts were
dried over magnesium sulfate, filtered, and concentrated to
give 1.02 g (57%) of 5-dodecyl-2H-tetrazole-2-acetic acid (35a )
as a white solid: mp 89-91 °C; 1H NMR (CDCl3) δ 10.18 (bs,
1H), 5.43 (s, 2H), 2.94 (t, 2H), 1.73-1.83 (m, 2H), 1.20-1.33
(m, 18H), 0.87 (t, 3H) ppm. Anal. (C15H28N4O2) C, H, N.
Syn t h esis of N-[2,6-Bis(1-m et h ylet h yl)p h en yl]-5-d o-
d ecyl-2H-tetr a zole-2-a ceta m id e (36a ). N,N-Dicyclohexyl-
carbodiimide (9.14 g, 0.044 mol) was added in one portion to
a solution of 5-dodecyl-2H-tetrazole-2-acetic acid (35a , 12.5 g,
0.042 mol) and 2,6-diisopropylaniline (7.48 g, 0.042 mol) in 300
mL of dichloromethane at -15 °C. This was allowed to warm
to room temperature and stirred overnight. The mixture was
then concentrated in vacuo and partitioned between water and
ethyl acetate. The organic layer was dried, filtered, and
concentrated to leave an orange oil. Trituration with hexanes
gave 15.16 g (79%) of N-[2,6-bis(1-methylethyl)phenyl]-5-
dodecyl-2H-tetrazole-2-acetamide (36a ) as an off-white solid:
Refer en ces
(1) Kannel, W. B.; Castelli, W. P.; Gordon, T.; McNamara, P. M.;
Serum Cholesterol, Lipoproteins, and the Risk of Coronary Heart
Disease. Ann. Intern. Med. 1971, 74, 1-12.
(2) Levine, G. N.; Keaney, J . F., J r.; Vita, J . A. Cholesterol Reduction
in Cardiovascular Disease. Clinical Benefits and Possible Mech-
anisms. N. Eng. J . Med. 1995, 332, 512-521.
(3) Suckling, K. E.; Stange, E. F. Role of Acyl CoA: Cholesterol
Acyltransferase in Cellular Cholesterol Metabolism. J . Lipid Res.
1985, 26, 647-671.
(4) Khan, B.; Wilcox, H. G.; Heimberg, M. Cholesterol is Required
for Secretion of Very-Low-Density Lipoprotein by Rat Liver.
Biochem. J . 1989, 259, 807-816.
(5) Brown, M. S.; Goldstein, J . L. Lipoprotein Metabolism in the
Macrophage: Implication for Cholesterol Depositions in Ath-
erosclerosis. Annu. Rev. Biochem. 1983, 52, 223-261.
(6) Krause, B. R.; Anderson, M.; Bisgaier, C. L.; Bocan, T.; Bousley,
R.; DeHart, P.; Essenburg, A.; Hamelehle, K.; Homan, R.; Kieft,
K.; McNally, W.; Stanfield, R.; Newton, R. S. In Vivo Evidence
that the Lipid-Regulating Activity of the ACAT Inhibitor CI-
976 in Rats is Due to Inhibition of Both Intestinal and Liver
ACAT. J . Lipid Res. 1993, 4, 279-294.
(7) Bocan, T. M. A.; Bak Mueller, S.; Uhlendorf, P. D.; Newton, R.
S.; Krause, B. R. Comparison of CI-976, an ACAT Inhibitor, and
Selected Lipid-Lowering Agents for Antiatherosclerotic Activity
in Iliac-Femoral and Thoracic Aortic Lesions. A Biochemical,
Morphological, and Morphometric Evaluation. Arteriosclerosis
Thrombosis 1991, 11, 1830-1843.
(8) Roark, W. H.; Roth, B. D. ACAT Inhibitors: Preclinical Profiles
of Clinical Candidates. Exp. Opin. Invest. Drugs 1994, 3, 1143-
1152.
1
mp 91-93 °C; H NMR (CDCl3) δ 7.14-7.37 (m, 4H), 5.46 (s,
2H), 2.81-2.96 (m, 4H), 1.74-1.86 (m, 2H), 1.13-1.38 (m,
30H), 0.88 (t, 3H) ppm. Anal. (C27H45N5O1) C, H, N.
Biologica l Meth od s. In Vitr o Ma cr op h a ge Assa y. IC-
21 mouse macrophages (American Type Culture Collection,
Rockville, MD) were preincubated for 16 h in serum-free
medium (Dulbecco’s modified Eagle medium, DMEM) contain-
(9) Sliskovic, D. R.; Trivedi, B. K. ACAT Inhibitors: Potential Anti-
atherosclerotic Agents. Curr. Med. Chem. 1994, 1, 204-225.

2366 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
O’Brien et al.
(10) O’Brien, P. M.; Sliskovic, D. R.; Bernabei, A.; Hurley, T.;
Anderson, M. K.; Bousley, R. F.; Krause, B. R.; Stanfield, R. L.
Inhibitors of Acyl-CoA: Cholesterol O-Acyltransferase (ACAT)
as Hypocholesterolemic Agents. 13. Design, Synthesis and
Biological Evaluation of Tetrazole Anilides as Potent Inhibitors
of ACAT In Vitro and Hypocholesterolemic Agents In Vivo.
Biorg. Med. Chem. Lett. 1995, 5, 295-300.
Cytotoxicity in Guinea Pigs Administered PD 132301-2, an
Inhibitor of Acyl-CoA: Cholesterol Acyltransferase. Toxicol.
Pathol. 1993, 21, 54-62.
(14) Roth, B. D.; Blankley, C. J .; Hoefle, M. L.; Holmes, A.; Roark,
W. H.; Trivedi, B. K.; Essenburg, A. D.; Kieft, K. A.; Krause, B.
R.; Stanfield, R. L. Inhibitors of Acyl-CoA: Cholesterol Acyl-
transferase. 1. Identification and Structure-Activity Relation-
ships of a Novel Series of Fatty Acid Anilide Hypocholesterolemic
Agents. J . Med. Chem. 1992, 35, 1609-1617.
(15) Smith, C.; Ashton, M. J .; Bush, R. C.; Facchini, V.; Harris, N.
V.; Hart, T. W.; J ordan, R.; MacKenzie, R.; Riddell, D. RP
73163: A Bioavailable Alkylsulphinyl-Diphenylimidazole ACAT
Inhibitor. Biorg. Med. Chem. Lett. 1996, 6, 47-50.
(11) Davis, F. A.; Han, W. Diastereoselective Fluorination of Chiral
Imide Enolates Using N-Fluoro-O-Benzenedisulfonimide
(NFOBS). Tetrahedron Lett. 1992, 33, 1153-1156.
(12) Krause, B. R.; Black, A.; Bousley, R. F.; Essenburg, A. D.;
Cornicelli, J . A.; Holmes, A.; Homan, R.; Kieft, K. A.; Sekerke,
C.; Shaw, M. K.; Stanfield, R. L.; Trivedi, B. K.; Woolf, T.
Divergent Pharmacological Activities of PD 132001-2 and CL
277082 Urea Inhibitors of Acyl-CoA: Cholesterol Acyltransferase
(ACAT). J . Pharmacol. Exp. Ther. 1993, 267, 734-743.
(16) Lowry, O. H.; Rosebrough, N. J .; Farr, A. L.; Randell, R. J .
Protein Measurement with the Folin Phenol Reagent. J . Biol.
Chem. 1951, 193, 265-275.
(13) Dominick, M. A.; Bobrowski, W. A.; MacDonald, J . R.; Gough,
A. W. Morphogenesis of
a
Zone-Specific Adrenocortical
J M960170F