Discovery of a potent and orally available acyl-CoA: Cholesterol acyltransferase inhibitor as an anti-atherosclerotic agent: (4-Phenylcoumarin) acetanilide derivatives

Article abstract of DOI:10.1248/cpb.59.1268

Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3- yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC₅₀=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os ( p.o.).

Full text of DOI:10.1248/cpb.59.1268

1268
Regular Article
Chem. Pharm. Bull. 59(10) 1268—1273 (2011)
Vol. 59, No. 10
Discovery of a Potent and Orally Available Acyl-CoA: Cholesterol
Acyltransferase Inhibitor as an Anti-atherosclerotic Agent: (4-
Phenylcoumarin)acetanilide Derivatives
Masaki OGINO,* Seiji FUKUI, Yoshihisa NAKADA, Ryosuke TOKUNOH, Shigekazu ITOKAWA,
Yuichi KAKOI, Satoshi NISHIMURA, Tsukasa SANADA, Hiromitsu FUSE, Kazuki KUBO,
Takeo WADA, and Shogo MARUI
Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd.; 2–26–1 Muraokahigashi, Fujisawa, Kanagawa
251–8555, Japan. Received June 22, 2011; accepted July 27, 2011; published online July 29, 2011
Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterifi-
cation. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A se-
ries of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of
the structure–activity relationships of a substituent on this scaffold, with an emphasis on improving the pharma-
cokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-
chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50ꢀ12 nM)
and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic
plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).
Key words acyl-CoA: cholesterol acyltransferase inhibitor; cholesterol; atherosclerosis; (4-phenylcoumarin)acetanilide
Atherosclerosis is a risk factor for stroke and coronary
heart diseases. Current medical management of atherosclero-
sis is based on the control of the risk factors and predisposed
conditions through lipid-lowering therapy involving diet or
drugs.1) However, additional therapies to act directly on
atherosclerotic lesions would provide a powerful treatment
method.2)
Atherosclerotic lesions are typically composed of large
lipid cores with underlying thin fibrous caps, so-called vul-
nerable plaques.3) The progression of atherosclerotic lesions
is associated with the accumulation of esterified cholesterol
in macrophage-derived foam cells.3)
Acyl-CoA: cholesterol acyltransferase (ACAT) is an intra-
cellular enzyme that catalyzes cholesterol esterification and
is highly expressed in macrophage-derived foam cells.4—7)
Therefore, inhibiting ACAT may directly reduce atheroscle- Fig. 1. Structures of ACAT Inhibitors, Avasimibe, Pactimibe and Lead
Compound 1
rotic lesions. Approaches targeting arterial plaques by using
ACAT inhibitors have been investigated for 2 decades.8) Ex-
perimental evidence suggested that ACAT inhibitors reduced
and stabilized atherosclerotic plaques in animals.9—13)
Two ACAT inhibitors, avasimibe and pactimibe, failed to
reduce surrogate markers for coronary artery disease in re-
cent clinical trials (Fig. 1).14—17) Several explanations were
proposed, including insufficient ACAT inhibitory activity
(IC50: avasimibe, 1980 nM; pactimibe, 216 nM).18) Thus, our
initial effort focused on identifying ACAT inhibitors with
Fig. 2. Optimization Plan of the Substituent on the (4-Phenylcoumarin)-
acetanilide Scaffold
both potent activity and a good pharmacokinetic (PK) pro-
file.
A screening of library compounds identified a (4-phenyl-
coumarin)acetanilide derivative 1 as a novel potent ACAT in- atherosclerotic plaques in mice are also explained.
hibitor (IC50ꢀ5 nM) (Fig. 1). However, the PK profile of this
Chemistry The general synthetic approach for the
compound was poor, and the anti-atherosclerotic efficacy was preparation of (4-phenylcoumarin)acetanilide derivatives is
not evaluated. For optimization, we divided (4-phenyl- shown in Chart 1. The starting 2-hydroxybenzophenones 3,
coumarin)acetanilide scaffold (2) into 3 aromatic moieties, which can be easily prepared from the substituted anisoles
an anilide, a coumarin, and a pendant phenyl (Fig. 2). Herein, using known methods,19) were converted into the correspond-
we describe the structure–activity relationship (SAR) study ing coumarinacetates 4 by esterification with ethyl succinyl
of substituents on these moieties with an emphasis on im- chloride and successive cyclization mediated by 1,8-diazabi-
proving the PK profile. Effects of an optimized compound on cyclo[5.4.0]undec-7-ene. After the hydrolysis of the ester
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: Ogino_Masaki@takeda.co.jp

October 2011
1269
3–5
R4
R5
R6
R7
Compds
R1
R2
R3
R4
R5
R6
R7
a
b
c
d
e
–(CH2)3–
–(CH2)4–
Me Me
H
H
H
H
H
H
H
H
H
H
Me
Cl
OCF3
H
H
Cl
Me
H
1
7
8
OMe
OMe
Me
OMe
H
H
H
F
H
H
H
H
H
F
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)4–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
OCF3
H
H
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
F
F
9
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Cl
H
Cl
Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
g
h
i
j
k
l
m
n
o
p
q
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
OMe
Me
Me
Me
Me
Me
Me
Me
Me
F
Me
F
Cl
H
Cl
Cl
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Me
OMe
Me
Me
Me
Me
Me
Me
Me
F
Me
F
Me
Reagents and conditions: (a) EtO2CCH2CH2COCl, Et3N, THF, 0 °C, 1 h; (b) DBU, toluene, reflux, 2.5 h; (c) c HCl, AcOH, reflux, 1 h; (d) (COCl)2, DMF (cat.), THF, rt,
0.5 h; (e) 6, Et3N, THF, rt, 1 h; (f) 6, NaH, THF, rt, overnight.
Chart 1. Synthesis of (4-Phenylcoumarin)acetanilides Derivatives
Table 1. Effects of Substituent on the Anilide Moiety of the (4-Phenyl-
group, resulting acid 5 was converted to the acid chloride and
coupled with aniline 6 to produce (4-phenylcoumarin)acet-
coumarine)acetanilide on ACAT Inhibitory Activitya) and PK Profileb)
anilides 1 and 7—28.
Results and Discussion
The synthesized compounds were examined for ACAT in-
hibitory activity, which was measured using human
macrophage homogenates derived from THP-1 cell lines.
b)
ACATa)
IC50 (nM)
C2 h
First, the SAR of R1, R2, and R3 groups on the anilide moi-
ety was explored (Table 1). The methoxy groups and the para
position were thought to be metabolically vulnerable sites of
the anilide moiety. Removal of one of the methoxy groups
(compound 7) and replacement by an ethyl group (compound
8) led to reduced ACAT inhibitory activity. 2-Trifluoromethyl
analogue 9 showed more potent ACAT inhibitory activity
than compounds 7 and 8. Further introduction of a fluorine
group improved ACAT inhibitory activity (compounds 10,
11). These results suggested that the trifluoromethyl group
acts as not only a bulky substituent but also an electron-with-
drawing substituent to decrease electron density of the
anilide moiety, leading to enhanced ACAT inhibitory activity.
Compound
R1
R2
R3
(mg/ml)
1
7
8
OMe
OMe
Et
CF3
CF3
CF3
CF3
OMe
H
H
H
F
H
H
H
H
H
F
5
100
86
36
6
0.007
ND
ND
0.032
0.082
0.159
3.109
9
10
11
12
H
H
19
96
Cl
a) ACAT inhibitory activity was determined by using homogenized THP-1 cells.
b) Compounds were orally administrated in C57/BL/6 mice (nꢀ3) at a dose of
10 mg/kg (suspension in 0.5% methylcellulose). The values of drug concentration in
plasma at 2h after oral administration (C2 h) were measured. ND: not determined.
On the other hand, 4-chloro analogue 12 showed a weak tration (10 mg/kg) to C57/BL/6 mice (Table 1). Compounds
ACAT inhibitory activity (IC50ꢀ96 nM). This showed that the 9, 10, and 11 showed slightly improved oral availability rela-
introduction of a substituent at the 4-position on the anilide tive to compound 1, potentially due to blockage of the meta-
moiety, except small molecules such as a fluorine, does not bolic site by the trifluoromethyl group and fluorine atom.
result in potent ACAT inhibition.
Surprisingly, compound 12 exhibited significantly high C2 h
We also evaluated oral absorption of these compounds in values. The electron withdrawing property of the chlorine
mice and plasma concentrations 2 h (C2 h) after oral adminis- atom is thought to contribute to the resistive effect on oxida-

1270
Vol. 59, No. 10
Table 2. Effects of Substituent on the Coumarin Moiety of the (4-Phenyl-
coumarine)acetanilide on ACAT Inhibitory Activitya)
Table 3. Effects of Substituent on the Pendant Phenyl Moiety of the (4-
Phenylcoumarine)acetanilide on ACAT Inhibitory Activitya) and PK Profileb)
ACATa)
IC50 (nM)
Compound
R4
R5
ACATa)
IC50 (nM)
Cmax
AUC0—8 h
F
(%)
Compound
R6
R7
(ng/ml) (ng·h/ml)
12
13
14
15
16
17
18
19
20
21
–(CH2)3–
–(CH2)4–
96
ꢁ100
ꢁ100
ꢁ100
ꢁ100
ꢁ100
100
90
44
ꢁ100
20
22
23
24
25
26
27
28
H
Me
Cl
OCF3
H
H
H
H
H
H
Cl
F
44
4
12
ND
503.5
1734.6
80.7
ND
ND
ND
2839.6
8771.1
355.7
ND
ND
2587.7
633.1
ND
15.9
34.5
5.7
ND
ND
7.4
Me
F
Cl
H
Me
Cl
Cl
Cl
Me
F
Cl
Cl
Cl
F
Me
OMe
14
ꢁ100
33
Cl
Me
F
Me
9
6
477.6
118.7
2.2
a) ACAT inhibitory activity was determined by using homogenized THP-1 cells.
b) Compounds were administrated as a cassette dosing to nonfasted mice at 0.1 mg/kg,
i.v. and 1 mg/kg, p.o. ND: not determined.
a) ACAT inhibitory activity was determined by using homogenized THP-1 cells.
tive metabolism on the anilide moiety. After modification of
the anilide moiety, we found that the 2-trifluoro-4-
chloroanilide analogue has an attractive PK profile and mod-
erate ACAT inhibitory activity.
Next, we focused on the modification of R4 and R5 groups
on the coumarin ring based on compound 12 (Table 2) to im-
prove the potency of the ACAT inhibition. In most cases, the
potencies were reduced; however, compound 20 showed im-
proved potency (IC50ꢀ44 nM). These results suggested that a
chloro group and a methyl group are the most effective com-
bination of R4 and R5 groups, respectively, on the coumarin
ring for potent ACAT inhibition.
Introduction of some substituents on the pendant phenyl
moiety was examined for further potency improvement
(Table 3). Most compounds showed more potent inhibitory
activity than compound 20. However, the potency of ACAT
inhibition of compound 25 was weak. Introduction of a hy-
drophobic substituent at the meta-position (R6) led to in-
creased ACAT inhibitory activity (compounds 22—24). This
hydrophobic substituent (R6) might interact with ACAT pro-
teins directly and/or create the proper torsional angle be-
tween the coumarin moiety and the pendant phenyl moiety. Fig. 3. Anti-atherosclerotic Effects of Compound 23 in apoE KO Mice
Six-week-old, male apoE KO mice were fed atherogenic diet for 7 weeks and nomal
On the other hand, only a small group such as a fluorine
chow for one week. Compound 23 was then administered orally once daily for 4 week
group or a methyl group was tolerable for potent ACAT inhi-
at a dose of 0.3, 1, 3 and 10 mg/kg (solution in Gelucire).21) (nꢀ3—10). Regressive ef-
fect on (A) the atherosclerotic lesion and (B) the aortic cholesteryl ester (CE) contents
was determined. Data represent meanꢂS.E. ∗ pꢃ0.025 vs. control by Williams test.
bition as an R7 group (compounds 26—28).
PK profile of compounds 22—24, 27 and 28, which exhib-
ited potent ACAT inhibitory activity, were also examined in
mice (Table 3). Among them, compound 23 showed the best Compound 23 exhibited regressive effects in a dose-depen-
bioavailability. dent manner, particularly on aortic CE contents at a dose of
As a result of the optimization of the substituent on the (4- 0.3 mg/kg.
phenylcoumarin)acetanilide moiety, we chose compound 23,
which exhibited potent ACAT inhibitory activity and an im- Conclusion
proved PK profile (at a dose of 10 mg/kg, per os (p.o.),
maxꢀ6.83 mg/ml, AUCꢀ61.4 mg·h/ml)20) in comparison as a novel potent and oral available ACAT inhibitors. Evalua-
with lead compound 1. tion of the structure–activity relationships of various sub-
We developed (4-phenylcoumarin)acetanilide derivatives
C
Finally, compound 23 was examined anti-atherosclerotic stituents on the (4-phenylcoumarin)acetanilide scaffold with
effects in apolipoprotein E knock out mice (apoE KO an emphasis on improving the PK profile led to the discovery
mice).21,22) After daily oral administration of compound 23 at of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-
doses of 0.3, 1, 3, and 10 mg/kg for 4 weeks, regressive ef- chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]-
fects were estimated by reduction of the lesion area (Fig. 3A) acetamide (23), which exhibits a potent ACAT inhibitory ac-
and the content of aortic cholesteryl ester (CE) (Fig. 3B). tivity and good PK profile in mice. Compound 23 also show-

October 2011
1271
1
Colorless crystals (92%): mp 209—210 °C; H-NMR (CDCl3) d: 3.44 (2H,
s), 6.82 (1H, d, Jꢀ9.2 Hz), 7.21—7.32 (2H, m), 7.48 (1H, d, Jꢀ5.8 Hz),
7.52—7.62 (3H, m); Anal. Calcd for C17H10ClFO4: C, 61.37; H, 3.03.
Found: C, 61.35; H, 2.88.
ed the regressive effects on atherosclerotic plaques in apoE
KO mice at a dose of 0.3 mg/kg p.o.
Experimental
Melting points (mp) were determined on a Yanagimoto micro melting
point apparatus and are uncorrected. Proton nuclear magnetic resonance
(1H-NMR) spectra were recorded on a Varian Gemini 200 (200 MHz) or
(7-Chloro-6-methyl-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5i)
Colorless crystals (64%): mp 222—224 °C; H-NMR (CDCl3) d: 2.28 (3H,
s), 3.41 (2H, s), 6.86 (1H, s), 7.20—7.30 (2H, m), 7.42 (1H, s), 7.50—7.65
1
Varian Mercury 300 (300 MHz) spectrometer. Chemical shifts are reported (3H, m); Anal. Calcd for C18H13ClO4: C, 65.76; H, 3.99. Found: C, 65.72; H,
as d values (ppm) downfield from internal tetramethylsilane of the indicated
organic solution. Peak multiplicities are expressed as follow: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; br s, broad singlet; m,
multiplet. Coupling constants (J values) are given in hertz (Hz). Elemental
analyses were carried out by Takeda Analytical Research Laboratories Ltd.
Reaction progress was determined by thin layer chromatography analysis on
silica gel 60 F254 plate (Merck). Chromatographic purification was carried
out on silica gel columns 60 (0.063—0.200 mm, Merck). Commercial
reagents and solvents were used without additional purification. Abbrevia-
3.90.
(7-Chloro-6-methoxy-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid
(5j) Colorless crystals (61%): mp 254—257 °C; 1H-NMR (CDCl3) d: 3.43
(2H, s), 3.66 (3H, s), 6.47 (1H, s), 7.24—7.33 (2H, m), 7.47 (1H, s), 7.50—
7.61 (3H, m); Anal. Calcd for C18H13ClO5·0.1H2O: C, 62.39; H, 3.84.
Found: C, 62.30; H, 3.91.
[7-Chloro-6-methyl-4-(3-methylphenyl)-2-oxo-2H-chromen-3-yl]acetic
Acid (5k) Colorless crystals (80%): mp 250—253 °C; H-NMR (CDCl3)
d: 2.28 (3H, s), 2.44 (3H, s), 3.41 (2H, s), 6.87 (1H, s), 7.05 (2H, m), 7.40
1
tions are used as follows: CDCl3, deuterated chloroform; DMSO-d6, di- (3H, m); Anal. Calcd for C19H15ClO4: C, 66.58; H, 4.41. Found: C, 66.41; H,
methyl sulfoxide-d6; EtOAc, ethyl acetate; AcOH, acetic acid; DMF, N,N-di- 4.24.
methylformamide; MeOH, methanol; THF, tetrahydrofuran; EtOH, ethanol;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.
2-Hydroxybenzophenones 3a—q were prepared from anisoles and ben-
zoyl chlorides by the Friedel–Crafts acylation according to known meth-
ods.19) These products were subjected to the next recation without further
purification.
2-(2-Oxo-4-phenyl-2,6,7,8-tetrahydrocyclopenta[g]chromen-3-yl)acetic
Acid (5a) To a solution of 3a (1.00 g, 4.20 mmol) in THF (20 ml) was
added dropwise triethylamine (0.98 ml, 7.03 mmol) and ethyl succinyl chlo-
ride (0.55 ml, 3.89 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h and
diluted with water. The reaction mixture was extracted with EtOAc, washed
with brine, dried over MgSO4 and concentrated in vacuo. The residue was
dissolved in toluene (10 ml) and DBU (0.25 ml, 1.66 mmol) was added. The
mixture was refluxed for 2.5 h with a Dean–Stark apparatus to remove water
[7-Chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]acetic
Acid (5l) Colorless crystals (58%): mp 262—264 °C; 1H-NMR (CDCl3) d:
2.30 (1H, s), 3.35 (1H, d, Jꢀ17.0 Hz), 3.46 (1H, d, Jꢀ16.8 Hz), 6.81 (1H, s),
7.14—7.20 (1H, m), 7.25—7.29 (1H, m), 7.43 (1H, s), 7.50—7.55 (1H, m);
Anal. Calcd for C18H12Cl2O4: C, 59.53; H, 3.33. Found: C, 59.42; H, 3.18.
{7-Chloro-6-methyl-2-oxo-4-[3-(trifluoromethoxy)phenyl]-2H-
chromen-3-yl}acetic Acid (5m) Colorless crystals (71%): mp 187—
1
191 °C; H-NMR (CDCl3) d: 2.29 (3H, s), 3.36 (1H, d, Jꢀ17.2 Hz), 3.46
(1H, d, Jꢀ17.2 Hz), 6.81 (1H, s), 7.21 (2H, m), 7.42 (2H, m), 7.64 (1H, t,
Jꢀ7.6 Hz); Anal. Calcd for C19H12ClF3O5: C, 55.29; H, 2.93. Found: C,
55.37; H, 2.89.
[7-Chloro-4-(4-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]acetic
1
Acid (5n) Colorless crystals (87%): mp 238—241 °C; H-NMR (CDCl3)
d: 2.29 (3H, s), 3.40 (2H, s), 6.82 (1H, s), 7.22 (2H, d, Jꢀ8.4 Hz), 7.41 (1H,
azeotropically. After cooling, the reaction mixture was diluted with water, s), 7.56 (2H, d, Jꢀ8.4 Hz); Anal. Calcd for C18H12Cl2O4: C, 59.53; H, 3.33.
extracted with EtOAc, washed with 1 M HCl and brine, dried over MgSO4
and concentrated in vacuo. The residue was dissolved in c HCl aq. (10 ml)
and AcOH (20 ml) and the mixture was refluxed for 1 h. The reaction mix-
ture was evaporated and the residue was extracted with EtOAc, washed with
water and brine, dried (MgSO4), and evaporated. The residue was crystal-
lized from EtOAc to give 5a (0.95 g, 70%) as colorless crystals: mp 216—
218 °C; H-NMR (CDCl3) d: 2.09 (2H, m), 2.81 (2H, d, Jꢀ7.0 Hz), 2.99
(2H, d, Jꢀ7.0 Hz), 3.41 (2H, s), 6.82 (1H, s), 7.20—7.30 (3H, m), 7.50— NMR (CDCl3) d: 2.31 (3H, s), 3.32 (2H, s), 6.82 (1H, s), 7.22 (1H, m), 7.37
7.60 (3H, m); Anal. Calcd for C20H16O4: C, 74.99; H, 5.03. Found: C, 74.75; (3H, m); Anal. Calcd for C18H11Cl2FO4: C, 56.72; H, 2.91. Found: C, 56.76;
Found: C, 59.47; H, 3.07.
[7-Chloro-4-(4-fluorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]acetic
Acid (5o) Colorless crystals (80%): mp 253—257 °C; H-NMR (CDCl3)
d: 2.29 (3H, s), 3.33 (2H, s), 6.83 (1H, s), 7.20—7.40 (5H, m); Anal. Calcd
for C18H12ClFO4: C, 62.35; H, 3.49. Found: C, 62.13; H, 3.29.
1
[7-Chloro-4-(3-chloro-4-fluorophenyl)-6-methyl-2-oxo-2H-chromen-3-
yl]acetic acid (5p) Colorless crystals (88%): mp ꢁ258 °C (decomp.); H-
1
1
H, 5.13.
Compounds 5b—q were prepared in a manner similar to that described
for 5a.
2-(2-Oxo-4-phenyl-6,7,8,9-tetrahydro-2H-benzo[g]chromen-3-yl)acetic
Acid (5b) Colorless crystals (52%): mp 196—197 °C; H-NMR (CDCl3) s), 6.94—7.01 (2H, m), 7.29 (1H, d, Jꢀ7.0 Hz), 7.40 (1H, s); Anal. Calcd
d: 1.64—1.96 (4H, m), 2.50—2.76 (2H, m), 2.80—3.00 (2H, m), 3.40 (2H, for C20H17ClO4: C, 67.32; H, 4.80. Found: C, 67.33; H, 4.83.
H, 2.83.
[7-Chloro4-(3,4-dimethylphenyl)-6-methyl-2-oxo-2H-chromen-3-
yl]acetic Acid (5q) Colorless crystals (86%): mp 225—227 °C; H-NMR
(CDCl3) d: 2.28 (3H, s), 2.34 (3H, s), 2.37 (3H, s), 3.43 (2H, s), 6.91 (1H,
1
1
s), 6.69 (1H, s), 7.09 (1H, s), 7.20—7.40 (2H, m), 7.40—7.70 (3H, m);
Anal. Calcd for C21H18O4: C, 75.43; H, 5.43. Found: C, 75.18; H, 5.43.
(6,7-Dimethyl-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5c) Col-
N-(2,6-Dimethoxyphenyl)-2-(2-oxo-4-phenyl-2,6,7,8-tetrahydrocyclo-
penta[g]chromen-3-yl)acetamide (1) To solution of 5a (150 mg,
0.47 mmol) in THF (10 ml) and DMF (one drop) was added oxalyl chloride
a
1
orless crystals (43%): mp 228—229 °C; H-NMR (CDCl3) d: 2.16 (3H, s),
(60 ml, 0.69 mmol) at room temperature. After stirring for 0.5 h, the mixture
2.34 (3H, s), 3.41 (2H, s), 6.75 (1H, s), 7.18 (1H, s), 7.20—7.36 (2H, m), was concentrated in vacuo. The residue was dissolved with THF (10 ml) and
7.40—7.66 (3H, m); Anal. Calcd for C19H16O4: C, 74.01; H, 5.23. Found: C, a solution of 2,6-dimethoxyaniline (79.0 mg, 0.52 mmol) and triethylamine
73.96; H, 5.39.
(100 ml, 0.72 mmol) in THF (5 ml) was added. After stirring for 1 h at room
temperature, the mixture was diluted with water and extracted with EtOAc.
The organic layer was separated, washed with 1 M HCl aq., sat. NaHCO3 aq.
(6,7-Difluoro-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5d) Col-
orless crystals (64%): mp 164—166 °C; H-NMR (CDCl3) d: 3.43 (2H, s),
4.86 (2H, dd, Jꢀ8.0 Hz, 10.0 Hz), 7.15—7.35 (3H, m), 7.50—7.65 (3H, m); and water successively, dried over MgSO4 and concentrated in vacuo. The
Anal. Calcd for C17H10F2O4·0.5H2O: C, 62.77; H, 3.41. Found: C, 62.71; H, residue was crystallized from EtOAc–THF to give 1 (146 mg, 64%) as color-
3.33.
(6,7-Dichloro-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5e) Col- t, Jꢀ7.0 Hz), 2.99 (2H, t, Jꢀ7.0 Hz), 3.46 (2H, br s) 3.78 (6H, s), 6.54 (2H,
orless crystals (62%): mp 222—223 °C; H-NMR (CDCl3) d: 3.43 (2H, s),
7.10 (1H, s), 7.20—7.36 (2H, m), 7.50—7.64 (4H, m); Anal. Calcd for m), 7.50 (1H, m); Anal. Calcd for C28H25NO5·0.2H2O: C, 73.25; H, 5.58; N,
1
less crystals: mp 213—215 °C; 1H-NMR (CDCl3) d: 2.09 (2H, m), 2.81 (2H,
1
d, Jꢀ8.0 Hz), 6.85 (1H, s), 7.14 (1H, t, Jꢀ8.0 Hz), 7.26 (1H, s), 7.43 (2H,
C17H10Cl2O4·0.3H2O: C, 57.59; H, 3.01. Found: C, 57.44; H, 2.99.
3.05. Found: C, 73.04; H, 5.79; N, 3.14.
(6-Chloro-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5f) Color-
Compounds 7 and 8 were prepared in a manner similar to that described
less crystals (59%): mp 174—177 °C; 1H-NMR (CDCl3) d: 3.44 (2H, s), for 1.
7.01 (1H, d, Jꢀ2.4 Hz), 7.20—7.60 (7H, m); Anal. Calcd for C17H10ClFO4:
N-(2-Methoxyphenyl)-2-(2-oxo-4-phenyl-2,6,7,8-tetrahydrocyclo-
C, 64.88; H, 3.52. Found: C, 65.13; H, 3.54.
penta[g]chromen-3-yl)acetamide (7) Colorless crystals (40%): mp 219—
(6-Chloro-7-methyl-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5g) 222 °C; 1H-NMR (CDCl3) d: 1.22 (3H, t, Jꢀ8.0 Hz), 2.09 (2H, m), 2.66
1
Colorless crystals (50%): mp 216—217 °C; H-NMR (CDCl3) d: 2.45 (3H, (2H, q, Jꢀ8.0 Hz), 2.82 (2H, t, Jꢀ7.0 Hz), 3.00 (2H, t, Jꢀ7.0 Hz), 3.46 (2H,
s), 3.43 (2H, s), 7.00 (1H, s), 7.20—7.30 (3H, m), 7.50—7.62 (3H, m); s), 6.86 (1H, s), 7.1—7.2 (4H, m), 7.26 (1H, s), 7.39 (2H, m), 7.56 (3H, m),
Anal. Calcd for C18H13ClO4: C, 65.76; H, 3.99. Found: C, 65.62; H, 4.12.
8.43 (1H, br s); Anal. Calcd for C25H18Cl2N2O3·0.3H2O: C, 74.64; H, 5.57;
(7-Chloro-6-fluoro-2-oxo-4-phenyl-2H-chromen-3-yl)acetic Acid (5h) N, 3.22. Found: C, 74.55; H, 5.66; N, 3.08.

1272
N-(2-Ethylphenyl)-2-(2-oxo-4-phenyl-2,6,7,8-tetrahydrocyclopenta-
Vol. 59, No. 10
2.59.
[g]chromen-3-yl)acetamide (8) Colorless crystals (46%): mp 234—
2-(6-Chloro-7-methyl-2-oxo-4-phenyl-2H-chromen-3-yl)-N-[4-chloro-
237 °C; 1H-NMR (CDCl3) d: 2.11 (2H, m), 2.83 (2H, t, Jꢀ7.0 Hz), 3.02 2-(trifluoromethyl)phenyl]acetamide (18) Colorless crystals (93%): mp
1
(2H, t, Jꢀ7.0 Hz), 3.54 (2H, s), 6.89 (1H, s), 7.30 (1H, s), 7.35 (2H, m),
7.56 (3H, m), 8.49 (2H, s), 8.74 (1H, br s); Anal. Calcd for
214—216 °C; H-NMR (CDCl3) d: 2.47 (3H, s), 3.47 (2H, s), 7.04 (1H, s),
7.32 (3H, m), 7.55 (5H, m), 8.09 (1H, d, Jꢀ9.0 Hz), 8.25 (1H, br s); Anal.
C28H25NO3·0.3H2O: C, 78.41; H, 6.02; N, 3.27. Found: C, 78.11; H, 6.04; Calcd for C25H16Cl2F3NO3: C, 59.31; H, 3.19; N, 2.77. Found: C, 59.15; H,
N, 3.28.
N-[4-Chloro-2-(trifluoromethyl)phenyl]-2-(2-oxo-4-phenyl-2,6,7,8-
tetrahydrocyclopenta[g]chromen-3-yl)acetamide (12) To a solution of
5a (150 mg, 0.47 mmol) in THF (10 ml) and DMF (one drop) was added ox-
3.21; N, 2.65.
2-(7-Chloro-6-fluoro-2-oxo-4-phenyl-2H-chromen-3-yl)-N-[4-chloro-2-
(trifluoromethyl)phenyl]acetamide (19) Colorless crystals (63%): mp
199—203 °C; H-NMR (CDCl3) d: 3.49 (2H, s), 6.86 (1H, d, Jꢀ9.2 Hz),
1
alyl chloride (60 ml, 0.69 mmol). The mixture was stirred at room tempera- 7.30—7.40 (2H, m), 7.45—7.62 (6H, m), 8.07 (1H, d, Jꢀ8.8 Hz), 8.15 (1H,
ture for 0.5 h. After concentration of the solvent, the residue was dissolved
with THF (10 ml). To the solution was added 4-chloro-2-(trifluoro-
methyl)aniline (6a) (80 mg, 0.41 mmol) and the mixture was stirred at room
br s); Anal. Calcd for C24H13Cl2F4NO3: C, 56.49; H, 2.57; N, 2.74. Found: C,
56.35; H, 2.50; N, 2.60.
2-(7-Chloro-6-methyl-2-oxo-4-phenyl-2H-chromen-3-yl)-N-[4-chloro-
temperature overnight. The mixture was diluted with water and extracted 2-(trifluoromethyl)phenyl]acetamide (20) Colorless crystals (74%): mp
1
with EtOAc. The organic layer was separated, washed with water, dried over
MgSO4 and concentrated in vacuo. The residue was crystallized from
EtOAc–THF to give 12 (125 mg, 55%) as colorless crystals: mp 198—
200 °C; H-NMR (CDCl3) d: 2.00—2.20 (2H, m), 2.82 (2H, d, Jꢀ8.0 Hz),
3.00 (2H, d, Jꢀ8.0 Hz), 3.47 (2H, s), 6.86 (1H, s), 7.20—7.35 (3H, m),
7.40—7.60 (5H, m), 8.08 (1H, d, Jꢀ9.0 Hz), 8.43 (1H, br s); Anal. Calcd for
236—239 °C; H-NMR (CDCl3) d: 2.29 (3H, s), 3.46 (2H, s), 6.90 (1H, s),
7.27—7.60 (8H, m), 8.08 (1H, d, Jꢀ8.4 Hz), 8.25 (1H, br s); Anal. Calcd for
C25H16Cl2F3NO3: C, 59.31; H, 3.19; N, 2.77. Found: C, 59.10; H, 3.01; N,
2.68.
1
2-(7-Chloro-6-methoxy-2-oxo-4-phenyl-2H-chromen-3-yl)-N-[4-
chloro-2-(trifluoromethyl)phenyl]acetamide (21) Colorless crystals
1
C27H19ClF3NO3: C, 65.13; H, 3.85; N, 2.81. Found: C, 64.88; H, 3.98; N: (81%): mp 219—221 °C; H-NMR (CDCl3) d: 3.48 (2H, s), 6.50 (1H, s),
2.73.
7.33—7.40 (2H, m), 7.43—7.52 (1H, m), 7.49 (1H, s), 7.52—7.61 (4H, m),
The compounds 9—11 and 13—29 were prepared in a manner similar to 8.07 (1H, d, Jꢀ8.8 Hz), 8.30 (1H, br s); Anal. Calcd for C25H16Cl2F3NO4: C,
that described for 12.
57.49; H, 3.09; N, 2.68. Found: C, 57.51; H, 3.35; N, 2.67.
2-(2-Oxo-4-phenyl-2,6,7,8-tetrahydrocyclopenta[g]chromen-3-yl)-N-
2-[7-Chloro-6-methyl-4-(3-methylphenyl)-2-oxo-2H-chromen-3-yl]-N-
[2-(trifluoromethyl)phenyl)]acetamide (9) Colorless crystals (79%): mp [4-chloro-2-(trifluoromethyl)phenyl]acetamide (22) Colorless crystals
1
191—193 °C; 1H-NMR (CDCl3) d: 2.02—2.17 (2H, m), 2.82 (2H, t, (85%): mp 235—237 °C; H-NMR (CDCl3) d: 2.30 (3H, s), 2.44 (3H, s),
Jꢀ7.5 Hz), 3.00 (2H, t, Jꢀ7.5 Hz), 3.48 (2H, s), 6.86 (1H, s), 7.16—7.39 3.46 (2H, s), 6.92 (1H, s), 7.12 (2H, m), 7.30—7.50 (4H, m), 7.57 (1H, m),
(4H, m), 7.46—7.62 (5H, m), 8.09 (1H, d, Jꢀ8.4 Hz), 8.35 (1H, br s); Anal.
8.08 (1H, d, Jꢀ8.8 Hz), 8.21 (1H, br s); Anal. Calcd for C26H18Cl2F3NO3: C,
Calcd for C27H20F3NO3·0.1H2O: C, 69.70; H, 4.38; N, 3.01. Found: C, 60.02; H, 3.49; N, 2.69. Found: C, 60.07; H, 3.62; N, 2.67.
69.64; H, 4.36; N, 2.87.
2-[7-Chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-
N-[2-Fluoro-6-(trifluoromethyl)phenyl]-2-(2-oxo-4-phenyl-2,6,7,8-
[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23) Colorless crystals
1
tetrahydrocyclopenta[g]chromen-3-yl)acetamide (10) Colorless crystals (51%): mp 202—204 °C; H-NMR (CDCl3) d: 2.31 (3H, s), 3.39 (1H, d,
(19%): mp 194—196 °C; 1H-NMR (CDCl3) d: 2.00—2.20 (2H, s), 2.83 (2H, Jꢀ14.0 Hz), 3.51 (1H, d, Jꢀ14.4 Hz), 6.86 (1H, s), 7.23—7.30 (2H, m),
t, Jꢀ7.0 Hz), 3.01 (2H, t, Jꢀ7.0 Hz), 3.50 (2H, s), 6.89 (1H, s), 7.25—7.60 7.33—7.37 (1H, m), 7.45 (1H, s), 7.46—7.57 (2H, m), 7.58 (1H, d,
(9H, m), 8.32 (1H, br s); Anal. Calcd for C27H19F4NO3: C, 67.36; H: 3.98; N,
2.91. Found: C, 67.58; H, 3.82; N, 2.59.
Jꢀ2.6 Hz), 8.06 (1H, d, Jꢀ8.8 Hz), 8.21 (1H, br s); Anal. Calcd for
C25H15Cl3F3NO3: C, 55.53; H, 2.80; N, 2.59. Found: C, 55.83; H, 2.97; N,
2.42.
N-[4-Fluoro-2-(trifluoromethyl)phenyl]-2-(2-oxo-4-phenyl-2,6,7,8-
tetrahydrocyclopenta[g]chromen-3-yl)acetamide (11) Colorless crystals
2-{7-Chloro-6-methyl-2-oxo-4-[3-(trifluoromethoxy)phenyl]-2H-
(57%): mp 186—187 °C; 1H-NMR (CDCl3) d: 2.00—2.20 (2H, m), 2.82 chromen-3-yl}-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide
(24)
1
(2H, d, Jꢀ8.0 Hz), 3.00 (2H, d, Jꢀ8.0 Hz), 3.46 (2H, s), 6.86 (1H, s), 7.10— Colorless crystals (40%): mp 171—175 °C; H-NMR (CDCl3) d: 2.31 (3H,
7.40 (5H, m), 7.50—7.60 (3H, m), 8.00 (1H, dd, Jꢀ9.0, 5.0 Hz), 8.36 (1H, s), 3.39 (1H, d, Jꢀ14.4 Hz), 3.52 (1H, d, Jꢀ14.4 Hz), 6.85 (1H, s), 7.20—
br s); Anal. Calcd for C27H19F4NO3: C, 67.36; H, 3.98; N, 2.91. Found: C, 7.50 (5H, m), 7.63 (2H, m), 8.06 (1H, d, Jꢀ9.0 Hz), 8.20 (1H, br s); Anal.
67.35; H, 3.74; N, 2.91.
N-[4-Chloro-2-(trifluoromethyl)phenyl]-2-(2-oxo-4-phenyl-6,7,8,9-
tetrahydro-2H-benzo[g]chromen-3-yl)acetamide (13) Colorless crystals
Calcd for C26H15Cl2F6NO4: C, 52.90; H, 2.56; N, 2.37. Found: C, 52.71; H,
2.55; N, 2.35.
2-[7-Chloro-4-(4-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-
1
(85%): mp 232—234 °C; H-NMR (CDCl3) d: 1.78 (4H, m), 2.65 (2H, m),
[4-chloro-2-(trifluoromethyl)phenyl]acetamide (25) Colorless crystals
1
2.87 (2H, m), 3.46 (2H, s), 6.73 (1H, s), 7.12 (1H, s), 7.32 (2H, m), 7.40- (80%): mp 284—286 °C; H-NMR (CDCl3) d: 2.30 (3H, s), 3.45 (2H, s),
7.60 (5H, m), 8.08 (1H, d, Jꢀ8.8 Hz), 8.42 (1H, br s); Anal. Calcd for
6.87 (1H, s), 7.31 (2H, d, Jꢀ8.4 Hz), 7.45 (1H, s), 7.54 (4H, m), 8.05 (1H, d,
C28H21ClF3NO3: C, 65.69; H, 4.13; N, 2.74. Found: C, 65.67; H, 4.27; N, Jꢀ9.2 Hz), 8.29 (1H, br s); Anal. Calcd for C25H15Cl3F3NO3: C, 55.53; H,
2.68.
N-[4-Chloro-2-(trifluoromethyl)phenyl]-2-(6,7-dimethyl-2-oxo-4-
2.80; N, 2.59. Found: C, 55.64; H, 3.00; N, 2.54.
2-[7-Chloro-4-(4-fluorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-
phenyl-2H-chromen-3-yl)acetamide (14) Colorless crystals (65%): mp [4-chloro-2-(trifluoromethyl)phenyl]acetamide (26) Colorless crystals
1
1
201—203 °C; H-NMR (CDCl3) d: 2.18 (3H, s), 2.35 (3H, s), 3.47 (2H, s), (73%): mp 272—273 °C; H-NMR (CDCl3) d: 2.30 (3H, s), 3.46 (2H, s),
6.80 (1H, s), 7.22 (1H, s), 7.34 (2H, m), 7.56 (5H, m), 8.09 (1H, d, 6.88 (1H, s), 7.20—7.40 (4H, m), 7.45 (1H, s), 7.48 (1H, m), 7.58 (1H, m),
Jꢀ8.0 Hz), 8.34 (1H, br s); Anal. Calcd for C26H19ClF3NO3: C, 64.27; H,
3.94; N, 2.88. Found: C, 64.46; H, 4.03; N, 2.59.
8.05 (1H, d, Jꢀ8.8 Hz), 8.30 (1H, br s); Anal. Calcd for C25H15Cl2F4NO3: C,
57.27; H, 2.88; N, 2.67. Found: C, 57.30; H, 2.97; N, 2.65.
N-[4-Chloro-2-(trifluoromethyl)phenyl]-2-(6,7-difluoro-2-oxo-4-
phenyl-2H-chromen-3-yl)acetamide (15) Colorless crystals (55%): mp
172—174 °C; 1H-NMR (CDCl3) d: 3.48 (2H, s), 6.90 (1H, dd, Jꢀ8.0,
2-[7-Chloro-4-(3-chloro-4-fluorophenyl)-6-methyl-2-oxo-2H-chromen-
3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (27) Colorless
1
crystals (83%): mp 240—241 °C; H-NMR (CDCl3) d: 2.32 (3H, s), 3.38
11.0 Hz), 7.30—7.40 (3H, m), 7.49 (1H, m), 7.57 (4H, m), 8.08 (1H, d, (1H, d, Jꢀ14.2 Hz), 3.51 (1H, d, Jꢀ14.2 Hz), 6.85 (1H, s), 7.29 (2H, m),
Jꢀ9.0 Hz), 8.14 (1H, br s); Anal. Calcd for C24H13ClF5NO3: C, 58.37; H,
2.65; N, 2.84. Found: C, 58.12; H, 2.52; N, 2.68.
N-[4-Chloro-2-(trifluoromethyl)phenyl]-2-(6,7-dichloro-2-oxo-4-
phenyl-2H-chromen-3-yl)acetamide (16) Colorless crystals (61%): mp
203—206 °C; 1H-NMR (CDCl3) d: 3.47 (2H, s), 7.15 (1H, s), 7.34 (2H, m),
7.45 (1H, s), 7.46 (2H, m), 7.59 (1H, m), 8.08 (1H, d, Jꢀ8.8 Hz), 8.25 (1H,
br s); Anal. Calcd for C25H14Cl3F4NO3: C, 53.74; H, 2.53; N, 2.51. Found: C,
53.81; H, 2.73; N, 2.46.
2-[7-Chloro-4-(3,4-dimethylphenyl)-6-methyl-2-oxo-2H-chromen-3-
yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide
(28) Colorless
1
7.57 (6H, m), 8.07 (2H, m); Anal. Calcd for C24H13Cl3F3NO3: C, 54.73; H, crystals (67%): mp 253—254 °C; H-NMR (CDCl3) d: 2.30 (3H, s), 2.34
2.49; N, 2.66. Found: C, 55.10; H, 2.51; N, 2.34.
(3H, s), 2.37 (3H, s), 3.48 (2H, s), 6.96 (1H, s), 7.00—7.10 (2H, m), 7.30
(1H, d, Jꢀ7.0 Hz), 7.43 (1H, s), 7.48 (1H, dd, Jꢀ3.0, 8.0 Hz), 7.57 (1H, d,
Jꢀ3.0 Hz), 8.09 (1H, d, Jꢀ8.0 Hz), 8.23 (1H, br s); Anal. Calcd for
C27H20Cl2F3NO3: C, 60.69; H, 3.77; N, 2.62. Found: C, 60.75; H, 3.89; N,
2-(6-Chloro-2-oxo-4-phenyl-2H-chromen-3-yl)-N-[4-chloro-2-(trifluo-
romethyl)phenyl]acetamide (17) Colorless crystals (46%): mp 178—
180 °C; 1H-NMR (CDCl3) d: 3.49 (2H, s), 7.05 (1H, d, Jꢀ2.6 Hz), 7.34 (3H,
m), 7.57 (6H, m), 8.09 (1H, d, Jꢀ9.2 Hz), 8.20 (1H, br s); Anal. Calcd for 2.53.
C24H14Cl2F3NO3: C, 56.49; H, 3.16; N, 2.74. Found: C, 56.39; H, 2.93; N,
Inhibitory Effects of Compounds on ACAT Activity ACAT activities

October 2011
1273
were determined by incorporation of [3H] oleoyl-CoA into cholesteryl esters
using various enzyme sources. These were prepared as homogenates or mi-
crosome fractions from human THP-1 macrophages or mouse peritoneal
macrophages. Inhibitory effects of compounds on ACAT activity were deter-
mined by measuring the radioactivities of [3H] cholesteryl oleate fraction
with or without compounds and these IC50 values was calculated from
means of ACAT inhibition in duplicate measurements.
Pharmakokinetic Study in Mouse Cassette Dosing Compounds 22—
24, 27 and 28 were administrated as a cassette dosing to nonfasted mice. Af-
teroral and intravenous administration, blood samples were collected. The
blood samples were centrifuged to obtain the plasma fraction. The plasma
samples were deproteinized with acetonitrile. After centrifugation, the ob-
tained supernatant was diluted and centrifuged again. The compound con-
centration in the supernatant was measured by LC/MS/MS.
Pharmakokinetic Study in Mice Compound 23 was administrated to
nonfasted C57BL6 mice (male, 8 weeks old, nꢀ3) orally (10 mg/kg, suspen-
sion in 0.5% methylcellulose). At 15 and 30 min and 1, 2, 4, 8 and 24 h after
oral administration, blood samples were collected from tail vein. The blood
samples were centrifuged to obtain the plasma fraction. The plasma samples
were deproteinized with acetonitrile. After centrifugation, the obtained su-
pernatant was diluted with 0.01 mol/l CH3CO2NH4 and acetonitrile. The
compound concentration in the supernatant was measured by HPLC system
equipped with a UV detector. The HPLC conditions were as follows: col-
umn, L-column ODS (4.6 mmꢄ150 mm); mobile phase, 0.01 mol/l
CH3CO2NH4/acetonitrileꢀ24/76; flow rate, 1 ml/min; column temperature,
40 °C.
Preliminary Test for Oral Absorption Test compounds were adminis-
trated to nonfasted C57BL6 mice (male, 8 weeks old, nꢀ3) orally
(10 mg/kg, suspension in 0.5% methylcellulose). At 2 h after oral adminis-
tration, blood samples were collected from tail vein. The concentration of
the compounds was measured by similar manner as PK study.
Anti-atherosclerotic Effects in apoE KO Mice Six weeks old male
apoE KO mice were fed atherogenic diet containing of 1.25% cholesterol
and 0.5% cholate for 8 weeks and then normal chow for 1 week. Mice were
divided into 5 groups based on plasma total cholesterol level and adminis-
tered compound 23 orally (Gelucire solution) at doses of 0.3, 1, 3 and
10 mg/kg once daily for 4 weeks. Aorta was dissected after 4 weeks adminis-
tration and the atherosclerotic lesion area was estimated as the area of oil red
O staining quantified by image analysis software. Aortic cholesteryl ester
contents were determined by measurements of free cholesterol and total cho-
lesterol contents. Free and total cholesterol concentrations were determined
using commercial kits (Wako Pure Chemical Industries). Cholesteryl ester
content was calculated by subtracting free cholesterol contents from total
cholesterol contents.
2) Braunwald E., N. Engl. J. Med., 337, 657—662 (1997).
3) Libby P., J. Intern. Med., 263, 517—527 (2008).
4) Buhman K. F., Accad M., Farese R. V., Biochim. Biophys. Acta, 1529,
142—154 (2000).
5) Chang T. Y., Chang C. C., Lin S., Yu C., Li B. L., Miyazaki A., Curr.
Opin. Lipidol., 12, 289—296 (2001).
6) Chang T. Y., Li B. L., Chang C. C., Urano Y., Am. J. Physiol. En-
docrinol. Metab., 297, E1—E9 (2009).
7) Oelkers P., Behari A., Cromley D., Billheimer J. T., Sturley S. L., J.
Biol. Chem., 273, 26765—26771 (1998).
8) Miyazaki A., Kanome T., Watanabe T., Curr. Drug Targets Cardio-
vasc. Haematol. Disord., 5, 463—469 (2005).
9) Bocan T. M., Krause B. R., Rosebury W. S., Mueller S. B., Lu X.,
Dagle C., Major T., Lathia C., Lee H., Arterioscler. Thromb. Vasc.
Biol., 20, 70—79 (2000).
10) Delsing D. J., Offerman E. H., van Duyvenvoorde W., van Der Boom
H., de Wit E. C., Gijbels M. J., van Der Laarse A., Jukema J. W.,
Havekes L. M., Princen H. M., Circulation, 103, 1778—1786 (2001).
11) Terasaka N., Miyazaki A., Kasanuki N., Ito K., Ubukata N., Koieyama
T., Kitayama K., Tanimoto T., Maeda N., Inaba T., Atherosclerosis,
190, 239—247 (2007).
12) Yoshinaka Y., Shibata H., Kobayashi H., Kuriyama H., Shibuya K.,
Tanabe S., Watanabe T., Miyazaki A., Atherosclerosis, 213, 85—91
(2010).
13) Ikenoya M., Yoshinaka Y., Kobayashi H., Kawamine K., Shibuya K.,
Sato F., Sawanobori K., Watanabe T., Miyazaki A., Atherosclerosis,
191, 290—297 (2007).
14) Tardif J. C., Gregoire J., L’Allier P. L., Anderson T. J., Bertrand O.,
Reeves F., Title L. M., Alfonso F., Schampaert E., Hassan A., McLain
R., Pressler M. L., Ibrahim R., Lesperance J., Blue J., Heinonen T.,
Rodes-Cabau J., A-PLUS Investigators, Circulation, 110, 3372—3377
(2004).
15)
Nissen S. E., Tuzcu E. M., Brewer H. B., Sipahi I., Nicholls S. J., Ganz
P., Schoenhagen P., Waters D. D., Pepine C. J., Crowe T. D., Davidson
M. H., Deanfield J. E., Wisniewski L. M., Hanyok J. J., Kassalow L.
M., N. Engl. J. Med., 354, 1253—1263 (2006).
16) Meuwese M. C., de Groot E., Duivenvoorden R., Trip M. D., Ose L.,
Maritz F. J., Basart D. C. G., Kastelein J. J. P., Habib R., Davidson M.
H., Zwinderman A. H., Schwocho L. R., Stein E. A., JAMA, 301,
1131—1139 (2009).
17) Hiatt W. R., Klepack E., Nehler M., Regensteiner J. G., Blue J., Imus
J., Criqui M. H., Vasc. Med., 9, 271—277 (2004).
18) In house data.
19) Terashita Z., Nakamura M., Marui S., Ogino M., European Patent
1471064A1 (2004).
20) Compound 23 was dosed orally 10 mg/kg/5 ml, suspension in 0.5%
methylcellulose, in C57/BL/6 mice (nꢀ3).
21) Koga K., Kawashima S., Murakami M., Eur. J. Pharm. Biopharm., 54,
311—318 (2002).
Acknowledgment We would like to thank Dr. M. Shiraishi, Dr. Z.
Terashita for their encouragement and help advice. We would like to thank
Dr. N. Amano, Ms. M. Ono for PK study in mice. We would like to thank
Mr. M. Nakamura for pharmacological directions.
22) The IC50 value of ACAT inhibitory activity in mouse peritoneal
macrophage of compound 23 was determined to be 20 nM in the same
manner as human.
References and Notes
1) Streja L., Packard C. J., Shepherd J., Cobbe S., Ford I., Am. J. Cardiol.,
90, 731—736 (2002).