Novel indoline-based acyl-CoA:cholesterol acyltransferase inhibitor with antiperoxidative activity: Improvement of physicochemical properties and biological activities by introduction of carboxylic acid

Article abstract of DOI:10.1021/jm800248r

A series of novel indoline derivatives with an ionizable moiety were synthesized to find a bioavailable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor with antiperoxidative activity. [7-(2,2-Dimethylpropanamido)-4,6- dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate (2, pactimibe sulfate) with low lipophilicity and high water solubility showed good oral absorption and inhibitory activity against foam cell formation in THP-1 cells exposed to acetyl-LDL after differentiation (IC₅₀: 0.3 μM) and an antiperoxidative effect in LDL of hypercholesterolemic rabbits (IC₅₀: 1.0 μM). 2 inhibited macrophage, hepatic, and intestinal ACAT activity (IC₅₀: 1.9, 0.7, and 0.7 μM, respectively). Maximal plasma concentration after oral administration of 2 at 10 mg/kg was 0.9 μg/mL in rats, 3.0 μg/mL in rabbits, and 11.2 μg/mL in dogs. Repeated administration of 2 lowered plasma LDL/VLDL cholesterol in hypercholesterolemic rabbits at 1 mg/kg/day, rats and dogs at 3 mg/kg/day, and in normocholesterolemic hamsters at 3 mg/kg/day. 2 is a promising candidate for antihyperlipidemic and antiatherosclerotic drugs.

Full text of DOI:10.1021/jm800248r

J. Med. Chem. 2008, 51, 4823–4833
4823
Novel Indoline-Based Acyl-CoA:Cholesterol Acyltransferase Inhibitor with Antiperoxidative
Activity: Improvement of Physicochemical Properties and Biological Activities by Introduction
of Carboxylic Acid
Kenji Takahashi,† Masayasu Kasai,† Masaru Ohta,† Yoshimichi Shoji,† Kazuyoshi Kunishiro,† Mamoru Kanda,†
Kazuyoshi Kurahashi,‡ and Hiroaki Shirahase*,†
Research Laboratories, Kyoto Pharmaceutical Industries, Ltd., 38 Nishinokyo Tsukinowa-cho, Nakagyo-ku, Kyoto 604-8444, Japan,
Radioisotope Research Center, Kyoto UniVersity, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
ReceiVed March 7, 2008
A series of novel indoline derivatives with an ionizable moiety were synthesized to find a bioavailable
acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor with antiperoxidative activity. [7-(2,2-Dimethylpro-
panamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate (2, pactimibe sulfate) with low lipophilicity
and high water solubility showed good oral absorption and inhibitory activity against foam cell formation
in THP-1 cells exposed to acetyl-LDL after differentiation (IC50: 0.3 µM) and an antiperoxidative effect in
LDL of hypercholesterolemic rabbits (IC50: 1.0 µM). 2 inhibited macrophage, hepatic, and intestinal ACAT
activity (IC50: 1.9, 0.7, and 0.7 µM, respectively). Maximal plasma concentration after oral administration
of 2 at 10 mg/kg was 0.9 µg/mL in rats, 3.0 µg/mL in rabbits, and 11.2 µg/mL in dogs. Repeated administration
of 2 lowered plasma LDL/VLDL cholesterol in hypercholesterolemic rabbits at 1 mg/kg/day, rats and dogs
at 3 mg/kg/day, and in normocholesterolemic hamsters at 3 mg/kg/day. 2 is a promising candidate for
antihyperlipidemic and antiatherosclerotic drugs.
Introduction
Furthermore, ACAT has two isozymes: ACAT-1 expressed in
macrophages and adrenal glands, and ACAT-2 expressed in the
intestine and liver.14–16 Thus, adrenotoxicity is a critical problem
for systemic bioavailable ACAT inhibitors, which may inhibit
adrenal as well as macrophage ACAT. Bioavailable and weak
ACAT inhibitors such as 2,6-bis(1-methylethyl)phenyl [[2,4,6-
tris(1-methylethyl)phenyl]acetyl]sulfamate (CI-1011, avasimibe)
have been reported to show minimal adrenotoxicity;6,11,17
however, these weak ACAT inhibitors may not effectively
prevent cholesterol deposition in atherosclerotic plaques. There-
fore, we hypothesized that bioavailable moderate ACAT inhibi-
tors with inhibitory effects on LDL oxidation would be safe
and effective antiatherosclerotic drugs, the effects of which may
synergistically suppress accumulation of oxidized LDL in
macrophages.
Acyl-CoA:cholesterol acyltransferase (ACAT)ahas been a
promising target for hyperlipidemia and atherosclerosis because
esterification of cholesterol with a long chain fatty acid is
essential for intestinal absorption of dietary cholesterol, incor-
poration of hepatic cholesterol into very low-density lipoprotein
(VLDL) vascular accumulation of cholesterol.1–3
Classical ACAT inhibitors have been focused on as hypo-
lipidemic drugs, which inhibit intestinal absorption of cholesterol
and the secretion of hepatic cholesterol. A number of highly
potent ACAT inhibitors with high lipophilicity and low oral
bioavailability have been reported.4,5 The target of ACAT
inhibitors then shifted from intestinal/hepatic ACAT to mac-
rophage ACAT: hyperlipidemia to atherosclerosis.6 In athero-
sclerotic plaque, denaturized low-density lipoprotein (LDL),
such as oxidized LDL, is taken up by macrophages and
cholesterol is accumulated via esterification by ACAT. There
have been some attempts to improve bioavailability of ACAT
inhibitors by reduction of lipophilicity;7–11 however, no bio-
available ACAT inhibitors have been successfully developed.
It is very difficult to reduce the lipophilicity of ACAT inhibitors
while maintaining their activities, because lipophilicity is the
preferred property for the inhibition of esterification between
two lipophilic substrate molecules (cholesterol and long chain
fatty acid) by membrane-bound enzymes such as ACAT.12,13
An indoline structure is reportedly a pharmacophore respon-
siblefortheinhibitoryeffectofindapamideonlipidperoxidation.18,19
We synthesized a series of indoline-based ACAT inhibitors and
examined their physicochemical properties and biological activi-
ties.20 Among them, N-(4,6-dimethyl-1-octylindolin-7-yl)-2,2-
dimethylpropanamide hydrochloride (1) potently inhibited he-
patic ACAT activity and lipid peroxidation. Compound 1
lowered serum cholesterol not only in dietary hypercholester-
olemic rats but also in normocholesterolemic hamsters, sug-
gesting that it inhibited hepatic ACAT after oral administra-
tion;20 however, 1 is still highly lipophilic and its bioavailability
may not be sufficiently high to inhibit macrophage ACAT and
LDL peroxidation via systemic circulation. Therefore, to
increase oral bioavailability without reducing ACAT inhibitory
and antiperoxidative activities, we attempted to improve lipo-
philicity and water solubility by introducing an ionizable moiety
(a basic or acidic moiety) to the indoline ring of 1. We found
that [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-
5-yl]acetic acid hemisulfate (2, pactimibe sulfate) with car-
boxymethyl moiety at the 5-position of the indoline ring showed
* To whom correspondence should be addressed. Phone: +81-75-812-
2248. Fax: +81-75-812-0906. E-mail: shirahase@kyoto-pharm.co.jp.
†
Research Laboratories, Kyoto Pharmaceutical Industries, Ltd.
Radioisotope Research Center, Kyoto University.
‡
a Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; VLDL,
very low-density lipoprotein; LDL, low-density lipoprotein; AUC, area under
the plasma concentration-time curve; LCAT, lecithin:cholesterol acyltrans-
ferase; HDL, high-density lipoprotein; WHHL rabbit, Watanabe heritable
hyperlipidemic rabbit; Cmax, maximal concentration; TLC, thin-layer
chromatography; IR, infrared spectra; NMR, nuclear magnetic resonance;
MS, mass spectra; MDA, malondialdehyde; EC, esterified cholesterol; FBS,
fetal bovine serum; PMA, phorbol 12-myristate 13-acetate; KHC rabbit,
Kurosawa and Kusanagi hypercholesterolemic rabbit.
10.1021/jm800248r CCC: $40.75
2008 American Chemical Society
Published on Web 07/12/2008

4824 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Scheme 1. Synthesis of Compounds 2, 4, 6, and 7a
Takahashi et al.
a Reagents, conditions and yields: (i) NaOH, MeOH-H2O, 50 °C, 76-94%; (ii) Br(CH2)9CO2Me, i-Pr2NEt, KI, DMF, 60 °C, 87%; (iii) NaOH, MeOH-
H2O, rt, then acidified with HCl, 83%; (iv) formalin, HCl (gas), conc. HCl, 50 °C; (v) potassium phthalimide, DMF, rt, 96% (2 steps); (vi) (a) hydrazine
monohydrate, MeOH-CHCl3, reflux; (b) Boc2O, CHCl3, rt, 71% (2 steps); (vii) NaCN, 18-crown-6, MeCN, reflux, 95% (2 steps); (viii) NaN3, Me3NH+Cl-,
DMF, 120 °C, 72%; (ix) benzyl chloride, NaH, DMF, rt, 93%; (x) octyl iodide, i-Pr2NEt, DMF, 60 °C, 86-91% (when 13c,h,i); (xi) octyl bromide, K2CO3,
KI, DMF, 40 °C, 76% (when 13j); (xii) NaOH, n-PrOH-H2O, reflux, then acidified with H2SO4, 78%; (xiii) (a) HCl in i-PrOH, HCO2H, rt; (b) HCl in
i-PrOH, CHCl3, 0 °C, 73% (2 steps); (xiv) H2, PdO, AcOH, rt, 72%.
low lipophilicity, high water solubility and good oral absorption,
and exerted excellent pharmacological effects.
NaOH by refluxing, followed by acidification with sulfuric acid
to yield 2 as a hemisulfate salt. A derivative with carboxyl
moiety at 7-position (5) was synthesized from 14 using branched
acyl chloride (Scheme 2).20,21 Scheme 3 describes a method to
prepare 3. Intermediate 26 was synthesized according to the
methods reported previously.20,22 26 was hydrogenated, piv-
aloylated, hydrolyzed, and alkylated by reductive amination to
27, which was treated with HCl to give 3 as a hydrochloride
salt.
Chemistry
Indoline derivatives with carboxylic acid moiety at 1- or
5-position (4 or 2) were synthesized from 8, which was prepared
as described previously (Scheme 1).20 Derivatives with amine
(6) and tetrazole (7) at 5-position were also synthesized from
8. For the synthesis of 4, acetyl moiety of 8 was removed,
alkylated with Br(CH2)9CO2Me, and then the ester was hydro-
lyzed to yield 4 as a hydrochloride salt. For the synthesis of 2,
6, and 7, the intermediate 8 was subjected to a chloromethylation
to obtain 11a without purification by column chromatography
because of its instability. To prepare 6, compound 11a was
treated with potassium phthalimide, and it was converted to
amine protected with Boc group (11c). To prepare 2 and 7,
compound 11a was substituted with cyanide to give 11e as an
intermediate of 2, and 11e was reacted with sodium azide,
followed by benzylation, to give a mixture of regioisomers 11h
and 11i (1:1) as intermediates of 7, which were used for further
reaction without isolating each regioisomer. The intermediates
(11c,e,h,i) were hydrolyzed to 12c,h-j and then were alkylated
with octyl iodide at 1-position to afford 13c,h-j. The Boc
protective group of 13c was removed with acid, followed by
treating with HCl to provide 6 as a dihydrochloride salt. Benzyl
groups of the mixture of 13h,i were removed by hydrogenation
to afford 7. Carbamoyl moiety of 13j was hydrolyzed with
Results and Discussion
Among the indoline-based ACAT inhibitors previously
reported, compound 1 potently inhibited hepatic and intestinal
ACAT as well as lipid peroxidation20 but was expected to
show poor oral absorption because of its high lipophilicity
and low water solubility. Although the inhibitory effect of 1
on macrophage ACAT has not been reported, the effect of
N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropana-
mide hydrochloride (KY-455), which is an analogue with a
shorter alkyl chain at the 1-position, was approximately 3-fold
weaker than that on hepatic ACAT.23 Thus, it is unlikely
that 1 is efficiently absorbed and exerts inhibitory effects on
macrophage ACAT and LDL oxidation. In the present study,
an ionizable moiety, including carboxylic acid, primary
amine, and tetrazole, was introduced at various positions on
the indoline ring of 1 to increase oral absorption without
marked reduction of inhibitory activities against ACAT and

Indoline-Based ACAT Inhibitor with AntiperoxidatiVe ActiVity
Scheme 2. Synthesis of Compound 5a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4825
a Reagents, conditions and yields: (i) NaOH, MeOH-H2O, reflux, 97%; (ii) octyl iodide, NaH, DMF, 50 °C, 93%; (iii) H2, Pd-C, MeOH-THF, 79%; (iv)
ClC(O)C(CH3)2CH2CH2CO2Me,21 Et3N, CH2Cl2, rt. 88%; (v) NaOH, MeOH-H2O, rt, 93%; (vi) HCl in i-PrOH, AcOEt, rt, 68%.
Scheme 3. Synthesis of Compound 3a
a Reagents, conditions and yields: (i) NaNO2, 4 M HCl, 5 °C; (ii) Na, EtOH, rt; (iii) AcONa, H2O, rt, 79%; (iv) conc HCl, EtOH, reflux, 85%; (v) NaOH,
EtOH, reflux, 84%; (vi) (a) 220 °C; (b) HCl in i-PrOH, EtOH, reflux, 83% (2 steps); (vii) (a) NaBH3CN, AcOH, 10-15 °C; (b) Ac2O, CHCl3, rt, 94% (2
steps); (viii) Br2, AcOH, rt, 91%; (ix) fum.HNO3, AcOH-conc H2SO4, rt, 94%; (x) (a) H2, Pd-C, MeOH, rt; (b) pivaloyl chloride, Et3N, CH2Cl2, rt; (c)
NaOH, MeOH-H2O, reflux; (d) octyl aldehyde, NaBH3CN, MeOH, rt, 60% (4 steps); (xi) HCl in i-PrOH, AcOEt, rt, 73%.
LDL oxidation. Six derivatives were synthesized and their
lipophilicity (log D7.0) and water solubility were determined
in comparison with compound 1 (Table 1). The effects of
these derivatives on foam cell formation (esterified cholesterol
accumulation by macrophage ACAT), hepatic ACAT activity,
lipid peroxidation of rat brain homogenate, and oral absorp-
tion (AUC) at 10 mg/kg in rats were examined (Table 2).
The effect on foam cell formation was determined as
inhibitory activity against esterified cholesterol accumulation
in THP-1 cells exposed to acetyl-LDL during differentiation
to macrophages. ACAT inhibitory activities were determined
using liver microsomes isolated from normocholesterolemic
Japanese White rabbits. As shown in Tables 1 and 2, 1 was
highly lipophilic and less water soluble, and its AUC was
very low in rats. All derivatives with an ionizable moiety
showed markedly decreased lipophilicity and increased water
solubility. Among them, 5 with a carboxyl group at the end
of the 7-amide moiety showed markedly increased AUC in

4826 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Table 1. Physicochemical Properties of Indoline Derivatives
Takahashi et al.
a Compounds 1, 3-5: hydrochloride salt; compound 2: hemisulfate salt; compound 6: dihydrochloride salt. b Solubility in water at pH 7.0 (µg/mL).
Table 2. In Vitro Activities and Oral Absorption of Indoline
Derivatives
Table 3. Inhibitory Effects of 2 on Foam Cell Formation and ACAT
Activitya
cmpd
foam cell formationa
liver ACATb
anti-Oxc
AUCd
ACAT IC50 (µM)c
foam cell formationb
1
2
3
4
5
6
7
0.64
1.8
3.6
>10
>10
0.33
>10
0.12
1.2
5.0
5.5
>10
1.1
3.1
1.3
4.2
48.3
6.6
4.2
0.2
1.5
1.7
0.1
5.0
ND
6.6
IC50 (µM)
macrophage
1.9
liver
intestine
adrenal
0.30 ( 0.04
0.69 ( 0.23 0.72 ( 0.24 15.9 ( 5.9
a Mean ( SEM. b THP-1 cells were exposed to acetyl-LDL after
differentiation in the presence of 2. Duplicate assay, n ) 4. c Microsome
fractions of macrophages, liver, intestine, and adrenal gland of hypercho-
lesterolemic rabbits were used as enzymes. Duplicate assay, n ) 4,
macrophage; n ) 1.
>10
1.7
a IC50 (µM) against esterified cholesterol accumulation during differentia-
tion in THP-1 cells, n ) 4. b IC50 (µM) against normocholesterolemic rabbit
liver ACAT activity. Duplicate assay. c IC50 (µM) against rat brain
homogenate peroxidation. Duplicate assay, n ) 2. d Area under the plasma
concentration-time curve (µg·h/mL) for 24 h after oral administration at
10 mg/kg in rats, n ) 3. ND: Not detected.
introduction of ionizable moieties. Thus, instead of the
carboxymethyl moiety, aminomethyl and tetrazol-5-ylmethyl
moieties were introduced to the 5-position (6 and 7).
Lipophilicity of 6 was similar to that of 2, but its water
solubility was lower. 6 showed 6-fold stronger antifoam cell
formation activity and 3-fold weaker antiperoxidative activity
than that of 2 and had hepatic ACAT inhibitory activity
comparable to that of 2; however, it was not detected in
plasma after oral administration: a primary amine may have
been metabolized easily. Compound 7 showed good oral
absorption in spite of higher lipophilicity and lower water
solubility, and had potent antiperoxidative activity, but no
antifoam cell formation or hepatic ACAT inhibitory activity.
Although tetrazole is known as a bioisoster of carboxylic
acid, its bulky ring structure may have disturbed the
interaction between 7 and ACAT protein. Avasimibe, an acyl
sulfamate derivative, was also reported to be a bioavailable
ACAT inhibitor.6,11 In our experimental condition, IC50
values of avasimibe for antifoam cell formation and hepatic
ACAT inhibitory activity were 1.5 and 7.6 µM, respectively.
From these results, 2 (pactimibe sulfate) was chosen for
further evaluation.
comparison with 1, while AUC of 4 with carboxyl moiety at
the end of the 1-alkyl chain was very low and similar to that
of 1. Compound 4 may have been easily glucuronized or
metabolized in the liver. 4 and 5 showed weak inhibitory
activity against foam cell formation and hepatic ACAT. We
have reported that the introduction of a basic moiety such as
imidazole, piperazine, morpholine, and dimethylamine to the
end of the 1-alkyl chain markedly decreased hepatic ACAT
inhibitory activity.20 In addition, introduction of a hydroxyl,
pyridinyl, and piperidinyl moiety to the end of 7-alkylamide
of the 1-hexyl analogue also reduced ACAT inhibitory
activity.20 These results imply that the alkyl chain at 1- or
7-position interacts with the lipophilic pocket of the enzyme,
thus an ionizable or polar moiety at these positions may
disrupt the interaction. Introduction of carboxymethyl to the
5-position (2) and carboxyethyl to the 3-position (3) markedly
increased oral absorption. Inhibitory activity against foam
cell formation of 2 and 3 was about 3-fold and 5-fold weaker
than that of 1, respectively, and hepatic ACAT inhibitory
activity was about 10-fold and 40-fold weaker than that of
1, respectively. The antiperoxidative activity of 2 was about
2-fold stronger, and the activity of 3 was slightly weaker
than that of 1. Compound 3 was weaker than 2 in spite of
higher lipophilicity. Compound 3 is racemic, thus an active
enantiomer may show effects comparable to those of 2. These
results demonstrated that the 5-position was suitable for the
The effects of 2 on foam cell formation when THP-1 cells
were exposed to acetyl-LDL after differentiation to mac-
rophages, and on ACAT of intestine, liver, adrenal gland,
and peritoneal macrophages isolated from hypercholester-
olemic rabbits were examined (Table 3). Compound 2 more
potently inhibited foam cell formation after differentiation
than during differentiation (Tables 2 and 3), suggesting that
it can inhibit foam cell formation in mature macrophages

Indoline-Based ACAT Inhibitor with AntiperoxidatiVe ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4827
Table 4. Inhibitory Effects of 2 on Peroxidation of Genetically
termine the efficacy of 2 in hyperlipidemia/atherosclerosis
and to find new clinical uses. Compound 2 is still a potential
candidate as a novel antihyperlipidemic and antiatheroscle-
rotic drug and is also useful to clarify the pathogenetic
mechanism of hyperlipidemia and atherosclerosis and species
difference of the pathogenesis and role of ACAT.
Hypercholesterolemic Rabbit Serum and LDLa
serum (IC50, µM)b
LDL (IC50, µM)c
2
4.4 ( 0.81
21.6 ( 28.9
0.98 ( 0.10
131.9 ( 106.5
probucol
a Mean ( SEM. b Duplicate assay, n ) 2. c Duplicate assay, n ) 4.
more than in differentiating macrophages. Compound 2 more
potently inhibited hepatic ACAT activity in hypercholester-
olemia than normocholesterolemia, indicating its high efficacy
in hyperlipidemia. It also inhibited macrophage, hepatic, and
intestinal ACAT more potently than adrenal ACAT, sug-
gesting its high adrenal safety. Indeed, repeated administra-
tion of 2 had no adrenotoxicity at 100 mg/kg/day for 4 days
in guinea pigs (unpublished data). ACAT is classified into
subtypes: ACAT-1 in macrophages and adrenal glands, and
ACAT-2 in the intestine and liver. The present data indicated
that 2 is a nonselective ACAT inhibitor and thus is expected
to lower plasma cholesterol levels and inhibit foam cell
formation in atherosclerotic plaques. Lecithin:cholesterol
acyltransferase (LCAT) also mediates esterification of cho-
lesterol and plays an important role in reverse cholesterol
transport. Compound 2 did not affect rat LCAT activity up
to 10 µM (unpublished). Compound 2 markedly inhibited
serum and LDL oxidation (Table 4), suggesting its in vivo
inhibitory effects on foam cell formation by inhibition of
ACAT activity as well as LDL oxidation. The maximal
concentration (Cmax) of a free form of 2 after oral
administration at a dose of 10 mg/kg was 0.92 ( 0.02 µg/
mL (2.2 µM) in rats, 2.99 ( 0.75 µg/mL (7.2 µM) in rabbits,
and 11.18 ( 1.39 µg/mL (26.8 µM) in dogs, which are higher
than IC50 values for antifoam cell formation, hepatic ACAT
inhibitory, and anti-LDL-peroxidative activity. Compound 2
significantly reduced plasma LDL/VLDL cholesterol levels
without significantly influencing high-density lipoprotein
(HDL) cholesterol levels at 1 or 3 mg/kg in rats, rabbits,
and dogs fed a high-cholesterol diet and hamsters fed a
regular diet (Table 5). The potent hypolipidemic effect of 2
in normocholesterolemic hamsters indicated that it was
efficiently absorbed and reduced hepatic secretion of cho-
lesterol by hepatic ACAT inhibition.
Experimental Section
General Procedures. Chemicals were obtained from com-
mercial sources and used without purification. Intermediates 8
and 14 were synthesized according to the method previously
reported.20 Reactions were monitored by thin-layer chromatog-
raphy (TLC) on Merck precoated silica gel 60 F254 (0.25 mm)
plates. Column chromatography was performed on silica gel
(Daiso no. 1001W, Daiso, Osaka, Japan). Melting points were
measured on a melting point apparatus (MP-21, Yamato
Scientific, Tokyo, Japan) and are uncorrected. Infrared spectra
(IR) were obtained with an infrared spectrometer (FT-720,
HORIBA, Kyoto, Japan). Proton nuclear magnetic resonance (1H
NMR) spectra were recorded at 400 MHz on a nuclear magnetic
resonance spectrometer (JNM-AL400, JEOL, Tokyo, Japan)
using tetramethylsilane as an internal standard. Mass spectra
(MS) were obtained on a QTRAP LC/MS/MS system (API2000,
Applied Biosystems, Lincoln Centre Drive Foster, CA).
N-(4,6-Dimethylindolin-7-yl)-2,2-dimethylpropanamide (9). To
a solution of 8 (60.0 g, 208 mmol) in MeOH (600 mL) was added
10.5 M NaOH solution (200 mL, 2.1 mol), followed by stirring at
50 °C for 2 h under nitrogen atmosphere. After evaporation of the
solvent under reduced pressure, the product was extracted with
CHCl3 (3 × 500 mL) and the extracts were combined, washed with
brine, dried over Na2SO4, and evaporated under reduced pressure.
The solid residue was rinsed with i-Pr2O to give 9 (48.4 g, 94%
1
yield). H NMR (CDCl3): δ 1.35 (s, 9H), 2.16 (s, 3H), 2.16 (s,
3H), 2.95 (t, J ) 8.4 Hz, 2H), 3.59 (t, J ) 8.4 Hz, 2H), 6.40 (s,
1H), 7.05 (s, 1H). MS m/z: 247 [M + H]+.
Methyl 10-[7-(2,2-Dimethylpropanamido)-4,6-dimethylindolin-
1-yl]decanoate (10). To a solution of 9 (4.35 g, 17.7 mmol) in DMF
(20 mL) were added Br(CH2)9CO2Me (5.5 mL, 24 mmol), diiso-
propylethylamine (4.6 mL, 26 mmol), and KI (2.93 g, 21.2 mmol),
followed by stirring at 60 °C for 4 h. After 10% citric acid solution
(100 mL) was added, the reaction mixture was extracted with AcOEt
(300 mL), washed with water and brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by
column chromatography, eluting with n-hexane:AcOEt (10:1 to 3:1)
The present study proved that the introduction of car-
boxymethyl moiety to the 5-position of 1 remarkably
improved lipophilicity and water solubility, thus increasing
oral bioavailability without marked reduction of ACAT
inhibitory activity. To our knowledge, 2 is the first ACAT
inhibitor in which carboxylic acid moiety was successfully
introduced to increase oral absorption and antihyperlipidemic
effects. The antiatherosclerotic effect of 2 was not investi-
gated in the present study; however, it has been reported that
2 reduced the atherosclerotic area in hamsters and apolipo-
protein E knockout mice and stabilized plaque in Watanabe
1
to give 10 (6.58 g, 87% yield). H NMR (CDCl3): δ 1.25-1.29
(m, 8H), 1.33 (s, 9H), 1.45-1.65 (m, 6H), 2.06 (s, 3H), 2.29 (s,
3H), 2.29 (t, J ) 7.5 Hz, 2H), 2.82 (t, J ) 8.4 Hz, 2H), 3.13 (t, J
) 7.8 Hz, 2H), 3.41 (t, J ) 8.4 Hz, 2H), 3.66 (s, 3H), 6.40 (s, 1H),
6.73 (s, 1H). MS m/z: 431 [M + H]+.
10-[7-(2,2-Dimethylpropanamido)-4,6-dimethylindolin-1-yl]de-
canoic Acid Hydrochloride (4). To a solution of 10 (6.33 g, 14.7
mmol) in MeOH (50 mL) was added 3.6 M NaOH solution (20
mL, 72 mmol), followed by stirring at room temperature for 2.5 h
under nitrogen atmosphere. After water (50 mL) was added, the
solvent was evaporated under reduced pressure, and the residue
was extracted with AcOEt (2 × 250 mL). The extracts were
combined, and the organic layer was extracted with H2O (3 × 500
mL). The aqueous layers were combined, neutralized with 6 M
HCl solution (13 mL), stirred for 10 min, and extracted with CHCl3
(2 × 500 mL). The extracts were combined, dried over Na2SO4,
and evaporated under reduced pressure to give 4 (5.54 g, 83% yield).
The analytically pure sample was obtained by recrystallization from
EtOH-i-Pr2O (1:2), and then CH2Cl2-i-Pr2O (1:2); mp 179 °C
heritable hyperlipidemic rabbits (WHHL rabbits).24–26
A
clinical study using an intravascular-ultrasonography catheter
failed to show the reduction of plaque volume at a dose of
100 mg in patients with coronary artery disease;27 however,
it remains to be determined whether 2 stabilized the plaque
in this study because the catheter used here cannot analyze
the plaque structure. Furthermore, 2 could be effective in
other patient populations with a relatively high plasma HDL
levels or under therapies that increase acceptor particles for
reverse cholesterol transport such as HDL and apolipoprotein
A-I in the plaque.28 ACAT is involved in chronic renal failure
and Alzheimer disease in addition to hyperlipide-
mia/atherosclerosis.29–31 Further studies are needed to de-
(CH2Cl2-i-Pr2O). IR (ATR): 3254, 1738, 1674 cm-1 1H NMR
.
(CDCl3): δ 1.25-1.33 (m, 10H), 1.42 (s, 9H), 1.56-1.73 (m, 3H),
1.96-2.10 (m, 1H), 2.15 (s, 3H), 2.26 (s, 3H), 2.32 (t, J ) 7.3 Hz,
2H), 3.03-3.27 (m, 4H), 3.62-4.14 (m, 2H), 7.12 (s, 1H), 9.20
(s, 1H), 13.80-14.00 (br, 1H). MS m/z: 417 [M + H]+. Anal.
(C25H40N2O3 ·HCl·0.1H2O) C, H, N.

4828 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Takahashi et al.
Table 5. Cholesterol-Lowering Effects of 2 in Rats, Rabbits, Dogs, and Hamstersa
ratb
rabbitc
dogd
hamstere
LDL/VLDL (mg/dL)
HDL (mg/dL)
control
2
control
2
72.9 ( 8.3
9.7 ( 2.7f
30.4 ( 0.5
29.4 ( 1.1
606.4 ( 4.5
119.5 ( 39.1f
21.4 ( 5.3
15.4 ( 3.1
103.6 ( 22.2
31.6 ( 8.4f
150.4 ( 6.5
141.3 ( 9.3
115.6 ( 4.8
70.3 ( 4.5f
66.3 ( 3.1
58.3 ( 4.4
a Mean ( SEM. b 3 mg/kg/day for 3 days, n ) 5. c 1 mg/kg/day for 7 days, n ) 5. d 3 mg/kg/day for 7 days, n ) 3. e 3 mg/kg/day for 3 weeks, n ) 5.
f p < 0.01, vs control, Student’s t-test. LDL/VLDL: LDL/VLDL cholesterol. HDL: HDL cholesterol.
N-[1-Acethyl-5-(phthalimido-2-ylmethyl)-4,6-dimethylindolin-7-
yl]-2,2-dimethylpropanamide (11b). To a stirred solution of 8 (20.0
g, 69.4 mmol) in concentrated hydrochloric acid (100 mL) was
added 35% formalin (8.2 mL, 104 mmol), and the reaction
mixture was bubbled with HCl gas for 30 min, followed by
stirring at 50 °C for 1.5 h. The reaction mixture was poured
into ice water and extracted with CHCl3 (3 × 1 L). The extracts
were combined, washed with water and brine, dried over Na2SO4,
and evaporated to give crude 11a (21.6 g). To a stirred
suspension of the obtained 11a (8.0 g) in DMF (60 mL) was
added potassium phthalimide (4.82 g, 26.0 mmol), followed by
stirring at room temperature for 16 h under nitrogen atmosphere.
After addition of water, the reaction mixture was extracted with
AcOEt (300 mL) and the extract was washed with brine, dried
over Na2SO4, and evaporated under reduced pressure. The residue
was purified by column chromatography, eluting with CHCl3:
MeOH (20:1) to give 11b (10.7 g, 96% yield). 1H NMR (CDCl3):
δ 1.25 (s, 9H), 2.24 (s, 3H), 2.29 (s, 3H), 2.34 (s, 3H), 2.80-3.23
(m, 2H), 3.95-4.30 (m, 2H), 4.80-5.05 (m, 2H), 7.66-7.78
(m, 4H), 9.14 (s, 1H). MS m/z: 448 [M + H]+.
N-[1-Acetyl-5-(1-benzyltetrazol-5-ylmethyl)-4,6-dimethylindolin-
7-yl]-2,2-dimethylpropanamide (11h) and N-[1-Acetyl-5-(2-ben-
zyltetrazol-5-ylmethyl)-4,6-dimethylindolin-7-yl]-2,2-dimethylpro-
panamide (11i). To a stirred suspension of 11g (8.70 g, 23.5 mmol)
in DMF (55 mL) was added a 60% suspension of NaH in mineral
oil (1.17 g, 29 mmol) portionwise in an ice bath. After stirring at
room temperature for 30 min, benzyl chloride (3.9 mL, 34 mmol)
was added, followed by stirring for 17 h. After addition of water
(200 mL), the reaction mixture was extracted with AcOEt (3 ×
500 mL) and the extracts were combined, washed with water and
brine, dried over Na2SO4, and evaporated under reduced pressure.
The residue was purified by column chromatography, eluting with
1
AcOEt to give a mixture of 11h and 11i (10.1 g, 93% yield). H
NMR (CDCl3): δ 11h: 1.27 (s, 9H), 1.80 (s, 3H), 1.91 (s, 3H),
2.31 (s, 3H), 2.65-3.25 (m, 2H), 3.95-4.20 (m, 4H), 5.38-5.53
(m, 2H), 7.26-7.35 (m, 5H), 9.27 (s, 1H); 11i: 1.27 (s, 9H), 2.20
(s, 3H), 2.25 (s, 3H), 2.29 (s, 3H), 2.87-3.16 (m, 2H), 3.95-4.20
(m, 2H), 4.20-4.30 (m, 2H), 5.65 (s, 2H), 7.25-7.40 (m, 5H),
9.17 (s, 1H). MS m/z: 461 [M + H]+.
N-(5-tert-Butoxycarbonylaminomethyl-4,6-dimethylindolin-7-yl)-
2,2-dimethylpropanamide (12c). To a solution of 11c (3.58 g, 8.57
mmol) in MeOH (60 mL) was added a 2.5 M NaOH solution
(18 mL, 45 mmol), followed by stirring at 50 °C for 22 h under
nitrogen atmosphere. After addition of water (50 mL), the solvent
was evaporated under reduced pressure, and the residue was
extracted with CHCl3 (3 × 200 mL). The extracts were
combined, washed with water and brine, dried over Na2SO4, and
evaporated under reduced pressure to give 12c (2.88 g, 89%
N-(1-Acetyl-5-tert-butoxycarbonylaminomethyl-4,6-dimethylin-
dolin-7-yl)-2,2-dimethylpropanamide (11c). A solution of 11b (5.97
g, 13.3 mmol) and hydrazine monohydrate (1.0 mL, 20.6 mmol)
in MeOH (50 mL) and CHCl3 (25 mL) was refluxed for 1 h. After
the solvent was evaporated under reduced pressure, the residue was
diluted with CHCl3 (200 mL), washed with a saturated NaHCO3
solution and brine, dried over Na2SO4, and evaporated. To a solution
of the obtained residue in CHCl3 (50 mL) was added Boc2O (2.99
g, 13.7 mmol), followed by stirring at room temperature for 1 h.
The reaction mixture was washed with water and brine, dried over
Na2SO4, and evaporated under reduced pressure. The residue was
purified by column chromatography, eluting with CHCl3:MeOH
1
yield). H NMR (CDCl3): δ 1.35 (s, 9H), 1.44 (s, 9H), 2.19 (s,
3H), 2.21 (s, 3H), 3.00 (t, J ) 8.3 Hz, 2H), 3.58 (t, J ) 8.3 Hz,
2H), 4.20-4.40 (m, 4H), 7.05 (s, 1H). MS m/z: 374 [M - H]-.
N-[5-(1-Benzyltetrazol-5-ylmethyl)-4,6-dimethylindolin-7-yl]-2,2-
dimethylpropanamide (12h) and N-[5-(2-Benzyltetrazol-5-ylm-
ethyl)-4,6-dimethylindolin-7-yl]-2,2-dimethylpropanamide (12i). A
mixture of 12h and 12i was prepared from the mixture of 11h and
1
(50:1) to give 11c (4.05 g, 71% yield). H NMR (CDCl3): δ 1.28
(s, 9H), 1.44 (s, 9H), 2.19 (s, 3H), 2.24 (s, 3H), 2.31 (s, 3H),
2.82-3.22 (m, 2H), 3.95-4.48 (m, 5H), 9.14 (s, 1H). MS m/z:
416 [M - H]-.
1
11i using the same procedure as for 12c (88% yield). H NMR
N-(1-Acetyl-5-cyanomethyl-4,6-dimethylindolin-7-yl)-2,2-dimeth-
ylpropanamide (11e). A suspension of crude 11a (8.0 g), NaCN
(3.57 g, 71.8 mmol) and 18-crown-6 (330 mg, 1.25 mmol) in
MeCN (50 mL) was refluxed under nitrogen atmosphere for
3.5 h. After the solvent was evaporated under reduced pressure,
the residue was diluted with CHCl3 (300 mL), washed with water
and brine, dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromatography,
(CDCl3): δ 12h: 1.33 (s, 9H), 1.80 (s, 3H), 1.86 (s, 3H), 2.92-3.00
(m, 2H), 3.58 (t, J ) 8.0 Hz, 2H), 4.06 (s, 2H), 5.36 (s, 2H), 6.92
(s, 1H), 7.29-7.38 (m, 5H); 12i: 1.34 (s, 9H), 2.22 (s, 3H), 2.26
(s, 3H), 3.00 (t, J ) 8.3 Hz, 2H), 3.56 (t, J ) 8.3 Hz, 2H), 4.15 (s,
2H), 5.66 (s, 2H), 7.06 (s, 1H), 7.32-7.38 (m, 5H). MS m/z: 419
[M + H]+.
N-(5-Carbamoylmethyl-4,6-dimethylindolin-7-yl)-2,2-dimethyl-
propanamide (12j). Compound 12j was prepared from 11e using
the same procedure as for 12c (76% yield). 1H NMR (DMSO-d6):
δ 1.22 (s, 9H), 1.96 (s, 3H), 2.08 (s, 3H), 2.82-2.98 (m, 2H),
3.30-3.45 (m, 4H), 4.43 (s, 1H), 6.80 (s, 1H), 7.07 (s, 1H), 8.63
(s, 1H). MS m/z: 304 [M + H]+.
1
eluting with CHCl3 to give 11e (7.70 g, 95% yield). H NMR
(CDCl3): δ 1.28 (s, 9H), 2.21 (s, 3H), 2.27 (s, 3H), 2.32 (s,
3H), 2.79-3.28 (m, 2H), 3.62-3.70 (m, 2H), 3.96-4.27 (m,
2H), 9.20 (s, 1H). MS m/z: 328 [M + H]+.
N-[1-Acetyl-4,6-dimethyl-5-(1H-tetrazol-5-ylmethyl)indolin-7-yl]-
2,2-dimethylpropanamide (11g). To a stirred suspension of 11e
(2.00 g, 6.11 mmol) in DMF (40 mL) were added Me3NH+Cl-
(5.83 g, 61.0 mmol) and NaN3 (3.96 g, 60.9 mmol), followed by
stirring at 120 °C for 3 h under nitrogen atmosphere. After cooling,
1 M hydrochloric acid (100 mL) was added and the reaction mixture
was extracted with AcOEt (2 × 150 mL), the extracts were
combined, washed with water and brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by
column chromatography, eluting with CHCl3:MeOH (100:1 to 10:
N-(5-tert-Butoxycarbonylaminomethyl-4,6-dimethyl-1-octylindo-
lin-7-yl)-2,2-dimethylpropanamide (13c). A solution of 12c (2.20
g, 5.86 mmol), octyl iodide (1.27 mL, 7.03 mmol), and diisopro-
pylethylamine (1.53 mL, 8.78 mmol) in DMF (19 mL) was stirred
at 60 °C for 60 h under nitrogen atmosphere. After addition of
10% citric acid solution (50 mL), the reaction mixture was extracted
with AcOEt (3 × 150 mL) and the extracts were combined, washed
with water and brine, dried over Na2SO4, and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy, eluting with n-hexane:AcOEt (5:1) to give 13c (2.47 g, 86%
1
1) to give 11g (1.70 g, 72% yield). H NMR (DMSO-d6): δ 1.15
1
(s, 9H), 1.99 (s, 3H), 2.19 (s, 3H), 2.28 (s, 3H), 2.80-3.20 (m,
2H), 3.80-4.10 (m, 1H), 4.20-4.45 (m, 2H), 9.22 (s, 1H). MS
m/z: 371 [M + H]+.
yield). H NMR (CDCl3): δ 0.87 (t, J ) 6.8 Hz, 3H), 1.22-1.32
(m, 10H), 1.34 (s, 9H), 1.43 (s, 9H), 1.58 (s, 2H), 2.09 (s, 3H),
2.17 (s, 3H), 2.86-2.92 (m, 2H), 3.13 (t, J ) 7.8 Hz, 2H),

Indoline-Based ACAT Inhibitor with AntiperoxidatiVe ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4829
3.38-3.46 (m, 2H), 4.23-4.29 (m, 2H), 4.35 (s, 1H), 6.77 (s, 1H).
MS m/z: 488 [M + H]+.
2H), 4.08-4.26 (m, 2H), 8.28-8.46 (m, 4H), 9.39 (s, 1H). MS
m/z: 388 [M + H]+. Anal. (C24H41N3O · 2HCl · 0.1H2O) C,
H, N.
N-[5-(1-Benzyltetrazol-5-ylmethyl)-4,6-dimethyl-1-octylindolin-
7-yl]-2,2-dimethylpropanamide (13h) and N-[5-(2-Benzyltetrazol-
5-ylmethyl)-4,6-dimethyl-1-octylindolin-7-yl]-2,2-dimethylpropan-
amide (13i). A mixture of 13h and 13i was prepared from the
mixture of 12h and 12i using the same procedure as for 13c (91%
N-[4,6-Dimethyl-1-octyl-5-(1H-tetrazol-5-ylmethyl)indolin-7-yl]-
2,2-dimethylpropanamide (7). A solution of the mixture of 13h
and 13i (4.03 g, 7.59 mmol) in AcOH (80 mL) was hydrogenated
at 0.4 MPa in the presence of PdO (1.0 g) at room temperature for
4 days. After removal of the catalyst by filtration, water (200 mL)
was added and the mixture was neutralized with K2CO3 and
extracted with AcOEt (2 × 400 mL). The extracts were combined,
washed with water and brine, dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by column
chromatography, eluting with n-hexane:AcOEt (5:1 to 1:1) to give
7 as a foam (2.40 g, 72% yield). The analytically pure sample was
obtained by recrystallization from AcOEt; mp 181 °C (AcOEt). IR
(ATR): 3236, 1641, 1601 cm-1. 1H NMR (CDCl3): δ 0.88 (t, J )
6.8 Hz, 3H), 1.22-1.34 (m, 10H), 1.36 (s, 9H), 1.45-1.56 (m,
2H), 1.97 (s, 3H), 2.02 (s, 3H), 2.80 (t, J ) 8.6 Hz, 2H), 3.14 (t,
J ) 7.7 Hz, 2H), 3.39 (t, J ) 8.6 Hz, 2H), 4.08 (s, 2H), 7.16 (s,
1H). MS m/z: 441 [M + H]+. Anal. (C25H40N6O · 0.1H2O) C,
H, N.
5-Bromo-4,6-dimethyl-7-nitroindoline (15). To a stirred suspen-
sion of 14 (6.63 g, 21.2 mmol) in MeOH (66 mL) was added 5.0
M NaOH solution (21 mL, 0.11 mol), followed by refluxing for 30
min under nitrogen atmosphere. After cooling, water was added
and a precipitate was collected by filtration to give 15 (5.57 g, 97%
yield). 1H NMR (CDCl3): δ 2.30 (s, 3H), 2.66 (s, 3H), 3.11 (t, J )
8.5 Hz, 2H), 3.82 (t, J ) 8.5 Hz, 2H), 6.41 (s, 1H). MS m/z: 268,
270 [M - H]-.
5-Bromo-4,6-dimethyl-7-nitro-1-octylindoline (16). To a solution
of 15 (8.00 g, 29.5 mmol) and octyl iodide (8.1 mL, 45 mmol) in
DMF (80 mL) was added a 60% suspension of NaH in mineral oil
(2.84 g, 71 mmol) at 0 °C, followed by stirring at 50 °C for 14 h
under nitrogen atmosphere. Water was added and the mixture was
extracted with AcOEt (320 mL), washed with water and brine, dried
over Na2SO4, and evaporated under reduced pressure. The residue
was purified by column chromatography, eluting with n-hexane:
AcOEt (20:1) to give 16 (10.5 g, 93% yield). 1H NMR (CDCl3): δ
0.88 (t, J ) 7.1 Hz, 3H), 1.20-1.35 (m, 10H), 1.40-1.50 (m, 2H),
2.25 (s, 3H), 2.29 (s, 3H), 2.90-3.00 (m, 4H), 3.57 (t, J ) 8.6 Hz,
2H). MS m/z: 383, 385 [M + H]+.
7-Amino-4,6-dimethyl-1-octylindoline (17). A solution of 16 (13.3
g, 34.7 mmol) in MeOH and THF (3:1) (130 mL) was hydrogenated
at 0.3 MPa in the presence of 10% Pd-C (1.33 g) at room
temperature for 15 h. After removal of the catalyst by filtration,
the filtrate was evaporated under reduced pressure. The residue was
diluted with AcOEt (500 mL), washed with saturated NaHCO3
solution and brine, dried over Na2SO4, and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy, eluting with n-hexane:AcOEt (8:1 to 1:1) to give 17 (7.52
g, 79% yield). 1H NMR (CDCl3): δ 0.88 (t, J ) 6.8 Hz, 3H),
1.20-1.40 (m, 10H), 1.50-1.60 (m, 2H), 2.12 (s, 3H), 2.13 (s,
3H), 2.88 (t, J ) 8.3 Hz, 2H), 2.95-3.02 (m, 2H), 3.25-3.40 (m,
2H), 3.42 (t, J ) 8.3 Hz, 2H), 6.48 (s, 1H). MS m/z: 275 [M +
H]+.
1
yield). H NMR (CDCl3): δ 13h: 0.85-0.90 (m, 3H), 1.22-1.30
(m, 10H), 1.40-1.55 (m, 2H), 1.74 (s, 3H), 1.76 (s, 3H), 2.74-2.82
(m, 2H), 3.05-3.20 (m, 2H), 3.35-3.43 (m, 2H), 4.02 (s, 2H),
5.39 (s, 2H), 6.75 (s, 1H), 7.27-7.37 (m, 5H); 13i: 0.87 (t, J ) 6.6
Hz, 3H), 1.22-1.30 (m, 10H), 1.33 (s, 9H), 1.40-1.58 (m, 2H),
2.11 (s, 3H), 2.20 (s, 3H), 2.88 (t, J ) 8.6 Hz, 2H), 3.10 (t, J )
7.6 Hz, 2H), 3.39 (t, J ) 8.6 Hz, 2H), 4.12 (s, 2H), 5.66 (s, 2H),
6.78 (s, 1H), 7.30-7.40 (m, 5H). MS m/z: 538 [M + H]+.
N-(5-Carbamoylmethyl-4,6-dimethyl-1-octylindolin-7-yl)-2,2-
dimethylpropanamide (13j). To a suspension of 12j (5.00 g, 16.5
mmol) in DMF (25 mL) were added octyl bromide (6.1 g, 32
mmol), K2CO3 (4.3 g, 31 mmol), and KI (0.52 g, 3.1 mmol),
followed by stirring at 40 °C for 23 h. After addition of water (150
mL), the reaction mixture was extracted with AcOEt (150 and 80
mL), the extracts were combined, washed with water and brine,
dried over Na2SO4, and evaporated under reduced pressure. The
residue was suspended in n-hexane, stirred at 60 °C for 10 min,
and then cooled. After the precipitate was collected by filtration,
the product was dissolved in AcOEt (15 mL) by heating, and to
the solution hot n-hexane (24 mL) was added and allowed to
standing at room temperature for 14 h. The precipitate was collected
1
by filtration to give 13j (5.20 g, 76% yield). H NMR (CDCl3): δ
0.88 (t, J ) 6.9 Hz, 3H), 1.20-1.29 (m, 12H), 1.28 (s, 9H), 2.03
(s, 3H), 2.12 (s, 3H), 2.90 (t, J ) 8.6 Hz, 2H), 3.10-3.25 (m, 2H),
3.43 (t, J ) 8.6 Hz, 2H), 3.53 (s, 2H), 5.29 (s, 1H), 5.54 (s, 1H),
6.86 (s, 1H). MS m/z: 416 [M + H]+.
[7-(2,2-Dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-
yl]acetic Acid Hemisulfate (2, pactimibe sulfate). To a suspension
of 13j (4.50 g, 10.8 mmol) in n-PrOH and H2O (3:1) (25 mL) was
added 2.3 M NaOH solution (9.0 mL, 21 mmol), followed by
refluxing for 23 h under nitrogen atmosphere. After evaporation of
the solution under reduced pressure, the residue was dissolved in
AcOEt (60 mL), washed with 1 M NaOH solution (3 × 60 mL),
and extracted with water (120 mL). The aqueous extract was washed
with AcOEt (30 mL), acidified with 4 M H2SO4 solution (15 mL)
to pH 1-2, stirred for 2 h, and allowed to stand for 1 h at room
temperature. The formed precipitate was collected by filtration to
give 2 (3.90 g, 78% yield). The analytically pure sample was
obtained by recrystallization of 2 (3.7 g) from 50% EtOH (60 mL)
containing H2SO4 (0.8 g) and hot water (130 mL); mp 167-169
°C (EtOH-H2O). IR (ATR): 1736, 1682, 1670, 1498 cm-1. 1H NMR
(DMSO-d6): δ 0.82-0.90 (m, 3H), 1.20-1.35 (m, 19H), 1.49-1.60
(m, 2H), 1.98 (s, 3H), 2.14 (s, 3H), 2.95-3.10 (m, 2H), 3.12-3.20
(m, 2H), 3.53-3.67 (m, 4H), 8.83-8.97 (br, 1H). Anal. (C25H40N2O3 ·
1/2H2SO4 ·0.2H2O) C, H, N.
N-(5-Aminomethyl-4,6-dimethyl-1-octylindolin-7-yl)-2,2-dimeth-
ylpropanamide Dihydrochloride (6). To a solution of 13c (5.47
g, 11.2 mmol) in HCO2H (20 mL) was added 9.25 M HCl in
i-PrOH (3.6 mL, 33 mmol) in an ice bath, followed by stirring
at the same temperature for 1 h. n-Hexane (150 mL) was mixed,
and the supernatant was removed by decantation. After the
manipulation was performed 3 times, the residue was diluted in
CHCl3 (100 mL), washed with water and brine, dried over
Na2SO4, and evaporated under reduced pressure. To a solution
of the residue in CHCl3 (20 mL) was added 9.25 M HCl in
i-PrOH (2.4 mL, 22 mmol) in an ice bath, followed by stirring
at the same temperature for 10 min. After i-Pr2O was added to
the reaction solution, the formed precipitate was collected by
filtration to give 6 (4.0 g, 73% yield). The analytically pure
sample was obtained by recrystallization from EtOH-AcOEt (1:
5); mp 198-200 °C (EtOH-AcOEt). IR (ATR): 3342, 1687
cm-1. 1H NMR (CDCl3): δ 0.87 (t, J ) 6.8 Hz, 3H), 1.18-1.36
(m, 10H), 1.41 (s, 9H), 1.64-1.80 (m, 1H), 1.94-2.08 (m, 1H),
2.26 (s, 3H), 2.39 (s, 3H), 2.98-3.44 (m, 4H), 3.64-4.08 (m,
Methyl 4-(4,6-Dimethyl-1-octylindolin-7-yl)carbamoyl-4-meth-
ylpentanoate (18). To a stirred solution of 17 (2.60 g, 9.47 mmol)
and Et3N (3.2 mL, 23 mmol) in CH2Cl2 (26 mL) was added
ClC(O)C(CH3)2CH2CH2CO2Me21 (2.20 g, 11.4 mmol) at 0 °C,
followed by stirring at room temperature for 9 h. The solution was
washed with 5% citric acid solution, saturated NaHCO3 solution
and brine, dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromatography,
eluting with n-hexane:AcOEt (4:1) to give 18 (3.60 g, 88% yield).
1H NMR (CDCl3): δ 0.88 (t, J ) 6.8 Hz, 3H), 1.20-1.30 (m, 10H),
1.33 (s, 6H), 1.45-1.55 (m, 2H), 1.95-2.05 (m, 2H), 2.07 (s, 3H),
2.11 (s, 3H), 2.40-2.50 (m, 2H), 2.83 (t, J ) 8.5 Hz, 2H),
3.08-3.14 (m, 2H), 3.41 (t, J ) 8.5 Hz, 2H), 3.66 (s, 3H), 6.41 (s,
1H), 6.79 (s, 1H). MS m/z: 431 [M + H]+.

4830 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Takahashi et al.
4-(4,6-Dimethyl-1-octylindolin-7-yl)carbamoyl-4-methylpentano-
ic Acid (19). To a stirred solution of 18 (500 mg, 1.16 mmol) in
MeOH (5 mL) was added 3.6 M NaOH solution (2.3 mL, 8.3 mmol)
at room temperature, followed by stirring at the same temperature
for 6 h. After the solvent was evaporated under reduced pressure,
the reaction mixture was extracted with AcOEt (50 mL), washed
with 5% citric acid solution and brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by
column chromatography, eluting with CHCl3:MeOH (1:1) to give
19 (449 mg, 93% yield). 1H NMR (CDCl3): δ 0.85 (t, J ) 7.1 Hz,
3H), 1.15-1.25 (m, 10H), 1.27 (s, 6H), 1.50-1.60 (m, 2H),
2.00-2.10 (m, 2H), 2.11 (s, 3H), 2.16 (s, 3H), 2.35-2.45 (m, 2H),
2.91 (t, J ) 8.3 Hz, 2H), 3.00-3.10 (m, 2H), 3.42 (t, J ) 8.3 Hz,
2H), 6.71 (s, 1H), 8.62 (s, 1H).
Ethyl 3-(4,6-Dimethylindol-3-yl)propionate (23). 22 (27.3 g, 104
mmol) was melted by heating at 220 °C for 10 min. After cooling,
the residue was dissolved with EtOH (500 mL), and 9.25 M HCl
in i-PrOH (16.9 mL, 156 mmol) was added, followed by refluxing
for 30 min. After cooling, the reaction solution was neutralized
with saturated NaHCO3 solution and brine, evaporated under
reduced pressure, and the residue was extracted with AcOEt (1 L).
The extract was washed with water, dried over Na2SO4, and
evaporated under reduced pressure. The solid residue was stirred
in i-Pr2O (100 mL) for 30 min and collected by filtration to give
1
23 (21.3 g, 83% yield). H NMR (DMSO-d6): δ 1.17 (t, J ) 7.1
Hz, 3H), 2.30 (s, 3H), 2.55 (s, 3H), 2.62 (t, J ) 7.6 Hz, 2H), 3.09
(t, J ) 7.6 Hz, 2H), 4.06 (t, J ) 7.1 Hz, 2H), 6.52 (s, 1H),
6.90-6.93 (m, 2H), 10.57 (s, 1H). MS m/z: 246 [M + H]+.
Ethyl 3-(1-Acetyl-4,6-dimethylindolin-3-yl)propionate (24). To
a suspension of 23 (19.3 g, 78.7 mmol) in AcOH (190 mL) was
added NaBH3CN (11.0 g, 175 mmol) between 10-15 °C, followed
by stirring at the same temperature for 1.5 h. The reaction solution
was neutralized with saturated NaHCO3 solution and extracted with
AcOEt (500 mL). The extract was washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. To a solution of
the residue in CHCl3 (290 mL) was added Ac2O (8.9 mL, 94 mmol)
in an ice bath, followed by stirring for 2 h at room temperature.
The reaction solution was washed with saturated NaHCO3 solution
and brine, dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromatography,
eluting with n-hexane:AcOEt (2:1) to give 24 (21.4 g, 94% yield).
1H NMR (CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H), 1.75-1.85 (m, 1H),
1.95-2.10 (m, 1H), 2.22 (s, 3H), 2.29 (s, 3H), 2.30 (s, 3H),
2.25-2.35 (m, 2H), 3.30-3.40 (m, 1H), 3.77 (dd, J ) 10.2, 2.2
Hz, 1H), 4.04 (t, J ) 10.2 Hz, 1H), 4.11 (q, J ) 7.1 Hz, 2H), 6.68
(s, 1H), 7.90 (s, 1H). MS m/z: 290 [M + H]+.
4-(4,6-Dimethyl-1-octylindolin-7-yl)carbamoyl-4-methylpentano-
ic Acid Hydrochloride (5). To a stirred solution of 19 (3.30 g, 7.92
mmol) in AcOEt (5 mL) was added 9.25 M HCl in i-PrOH (1.0
mL, 9.3 mmol) in an ice bath, followed by stirring at the same
temperature for 30 min. The precipitate was collected by filtration
and washed with AcOEt to give 5 as a crystalline prduct (2.40 g,
68% yield); mp 136-139 °C. IR (ATR): 1726, 1651 cm-1 1H
.
NMR (DMSO-d6): δ 0.84 (t, J ) 7.1 Hz, 3H), 1.15-1.25 (m, 10H),
1.27 (s, 6H), 1.55-1.75 (m, 2H), 1.80-1.90 (m, 2H), 2.07 (s, 3H),
2.21 (s, 3H), 2.15-2.25 (m, 2H), 3.00-3.20 (m, 4H), 3.70-3.85
(m, 2H), 7.08 (s, 1H), 9.30 (s, 1H). MS m/z: 417 [M + H]+. Anal.
(C25H40N2O3 ·HCl·0.4H2O) C, H, N.
1-Ethyl 2-[(3,5-Dimethylphenyl)hydrazono]hexanedioate (20).
To a suspension of 3,5-dimethylaniline (25.0 g, 206 mmol) in 4 M
hydrochloric acid (200 mL) was added an aqueous solution of
NaNO2 (14.2 g, 206 mmol/50 mL) below 5 °C, followed by stirring
at room temperature for 30 min. On the other hand, to a solution
of ethyl cyclopentanone-2-carboxylate (32.2 g, 206 mmol) in EtOH
(150 mL) was added Na (4.74 g, 206 mmol) in an ice bath, followed
by stirring at room temperature for 2 h. To an aqueous solution of
AcONa (25.4 g, 309 mmol/150 mL) was added the two prepared
reaction mixtures simultaneously in an ice bath, followed by stirring
at the same temperature for 3 h. The reaction mixture was
neutralized with 5% NaHCO3 solution, extracted with AcOEt (3
× 150 mL), the organic layers were combined, washed with brine,
dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified by column chromatography, eluting with
Ethyl 3-(1-Acetyl-5-bromo-4,6-dimethylindolin-3-yl)propionate
(25). To a solution of 24 (15.0 g, 51.8 mmol) in AcOH (150 mL)
was added Br2 (3.3 mL, 65 mmol) in an ice bath, followed by
stirring at room temperature for 1 h. Then 10% NaHSO3 solution
was added and the precipitate was collected by filtration. The
obtained solid was dissolved with CHCl3 (500 mL), washed with
water and brine, dried over Na2SO4, and evaporated under reduced
pressure. The solid residue was rinsed with i-Pr2O to give 25 (17.4
g, 91% yield). 1H NMR (CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H),
1.70-1.85 (m, 1H), 1.90-2.05 (m, 1H), 2.22 (s, 3H), 2.30 (t, J )
7.3 Hz, 2H), 2.36 (s, 3H), 2.40 (s, 3H), 3.35-3.45 (m, 1H), 3.79
(dd, J ) 10.2, 1.7 Hz, 1H), 4.04 (t, J ) 10.2 Hz, 1H), 4.12 (q, J
) 7.1 Hz, 2H), 8.01 (s, 1H). MS m/z: 368, 370 [M + H]+.
Ethyl 3-(1-Acetyl-5-bromo-4,6-dimethyl-7-nitroindolin-3-yl)pro-
pionate (26). To a solution of fuming HNO3 (3.2 mL, 71 mmol) in
AcOH (120 mL) and concentrated H2SO4 (60 mL) was added 25
(17.4 g, 47.2 mmol) in an ice bath, followed by stirring at the same
temperature for 6.5 h. Ice water was poured into the reaction
mixture, and the precipitate was collected by filtration. The obtained
solid was dissolved in CHCl3 (500 mL), washed with saturated
NaHCO3 solution and brine, dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by column
chromatography, eluting with CHCl3:MeOH (10:1) to give 26 (18.4
g, 94% yield). 1H NMR (CDCl3): δ 1.27 (t, J ) 7.1 Hz, 3H),
1.66-1.78 (m, 1H), 1.92-2.02 (m, 1H), 2.24 (s, 3H), 2.30-2.40
(m, 2H), 2.45 (s, 3H), 2.48 (s, 3H), 3.25-3.35 (m, 1H), 3.90-4.00
(m, 1H), 4.10-4.20 (m, 3H). MS m/z: 448 [M + H]+.
3-[4,6-Dimethyl-7-(2,2-dimethylpropanamido)-1-octylindolin-3-
yl]propionic Acid (27). A solution of 26 (19.8 g, 47.9 mmol) in
MeOH (400 mL) was hydrogenated at 0.3 MPa in the presence of
10% Pd-C (1.98 g) at room temperature for 14 h. After removal
of the catalyst by filtration, the filtrate was evaporated under reduced
pressure. The residue was dissolved in CHCl3 (300 mL), washed
with saturated NaHCO3 solution, dried over Na2SO4, and evaporated
under reduced pressure. To a solution of the residue and Et3N (7.3
mL, 52 mmol) in CHCl3 (200 mL) was added pivaloyl chloride
(5.8 mL, 48 mmol) in an ice bath, followed by stirring at room
temperature for 30 min. The mixture was washed with 5% citric
acid solution and brine, dried over Na2SO4, and evaporated under
1
n-hexane:AcOEt (10:1) to give 20 (49.7 g, 79% yield). H NMR
(CDCl3): δ 1.30 (t, J ) 7.1 Hz, 3H), 1.85-2.00 (m, 1H), 2.00-2.15
(m, 1H), 2.26 (s, 1H), 2.36 (s, 6H), 2.36-2.55 (m, 2H), 2.60-2.80
(m, 2H), 4.25-4.35 (m, 2H), 7.10 (s, 1H), 7.33 (s, 2H). MS m/z:
305 [M - H]-.
Ethyl 3-(2-Ethoxycarbonylethyl)-4,6-dimethylindole-2-carboxy-
late (21). To a solution of 20 (38.6 g, 126 mmol) in EtOH (386
mL) was added concentrated hydrochloric acid (17.2 mL) at room
temperature, followed by refluxing for 17 h. After cooling, the
reaction solution was neutralized with saturated NaHCO3 solution,
concentrated under reduced pressure, and the reaction mixture was
extracted with CHCl3 (1 L). The extract was washed with brine,
dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified by column chromatography, eluting with
1
n-hexane:AcOEt (10:1 to 5:1) to give 21 (34.1 g, 85% yield). H
NMR (CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H), 1.42 (t, J ) 7.3 Hz,
3H), 2.40 (s, 3H), 2.69 (s, 3H), 2.60-2.65 (m, 2H), 3.50-3.60
(m, 2H), 4.15 (t, J ) 7.1 Hz, 2H), 4.40 (t, J ) 7.3 Hz, 2H), 6.71
(s, 1H), 6.98 (s, 1H), 8.63 (s, 1H). MS m/z: 316 [M - H]-.
3-(2-Carboxyethyl)-4,6-dimethylindole-2-carboxylic Acid (22).
To a solution of 21 (39.5 g, 124 mmol) in EtOH (390 mL) was
added NaOH (24.8 g, 620 mmol) at room temperature, followed
by refluxing for 4 h. After cooling, the precipitate was collected
by filtration and dissolved in H2O (300 mL). The aqueous solution
was acidified with 10% citric acid solution and the precipitate was
1
collected to give 22 (27.3 g, 84% yield). H NMR (DMSO-d6): δ
2.31 (s, 3H), 2.60 (s, 3H), 2.40-2.50 (m, 2H), 3.35-3.45 (m, 2H),
6.60 (s, 1H), 7.00 (s, 1H), 11.28 (s, 1H), 12.30-12.60 (br, 2H).
MS m/z: 260 [M - H]-.

Indoline-Based ACAT Inhibitor with AntiperoxidatiVe ActiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4831
reduced pressure. The residue was purified by column chromatog-
raphy, eluting with n-hexane:AcOEt (1:1) to give a mixture of a
methyl ester derivative and an ethyl ester one (17.5 g). To a solution
of the obtained mixture (5.94 g) in MeOH (50 mL) was added 5.0
M NaOH solution (15.5 mL, 78 mmol), followed by refluxing for
3.5 h. After cooling, water (50 mL) was added to the reaction
solution and the solvent was evaporated under reduced pressure.
The residue was acidified with citric acid until pH 4, extracted with
AcOEt (200 mL), washed with brine, dried over Na2SO4, and
evaporated under reduced pressure. To a solution of the residue
and octyl aldehyde (1.99 g, 15.5 mmol) in MeOH (15 mL) was
added NaBH3CN (1.08 g, 15.5 mmol) at room temperature,
followed by stirring for 1.5 h at the same temperature. After addition
of water (20 mL), the solvent was evaporated under reduced
pressure and the reaction mixture was extracted with AcOEt (200
mL) the extract was washed with water and brine. The residue was
purified by column chromatography, eluting with n-hexane:AcOEt
(2:1) to give 27 as a foam (4.23 g, 60% yield). 1H NMR (CDCl3):
δ 0.87 (t, J ) 6.8 Hz, 3H), 1.20-1.50 (m, 21H), 1.80-1.95 (m,
2H), 2.08 (s, 3H), 2.17 (s, 3H), 2.20-2.35 (m, 2H), 2.85-2.95
(m, 1H), 3.20-3.35 (m, 4H), 6.46 (s, 1H), 7.28 (s, 1H).
incubation period in the presence of vehicle or test compounds was
determined. Inhibition (%) of lipid peroxidation by various
concentrations of test compounds was determined, and IC50 values
were calculated.
Esterified Cholesterol (EC) Accumulation in THP-1 Cell-
Derived Macrophages. Effects of test compounds on EC accumula-
tion in THP-1 cells were determined during differentiation and foam
cell formation. THP-1 cells were grown in RPMI-1640 medium
containing 10% fetal bovine serum (FBS), 100 U/mL penicillin,
and 100 mg/mL streptomycin. To differentiate into macrophages
and to form foam cells, the cells were suspended in RPMI-1640
medium containing 10% FBS, phorbol 12-myristate 13-acetate
(PMA, 200 nM), and acetyl-LDL (400 µg/mL), which was prepared
from the plasma of Kurosawa and Kusanagi hypercholesterolemic
(KHC) rabbits (Japan Laboratory Animals, Tokyo, Japan) and
acetylated with acetic anhydride. Then, they were incubated at 4
× 105 cells/well in a humidified atmosphere of 95% air, 5% CO2
at 37 °C for 3 days in the presence or absence of test compounds.
In separate experiments, the effects of 2 on EC accumulation in
THP-1 cell-derived macrophages after differentiation were deter-
mined. The cells were differentiated into macrophages by culture
in RPMI-1640 medium containing 10% FBS, PMA (200 nM), and
2-mercaptoethanol (50 µM) for 3 days. Then, the cells were washed
with RPMI-1640 medium and cultured in RPMI-1640 medium
containing 2-mercaptoethanol (50 µM), acetyl-LDL (400 µg/mL),
and 2 for 2 days. Cellular cholesterol was extracted by hexane/
isopropanol (3:2) and determined by enzymatic methods. EC content
was calculated by subtracting the amount of free cholesterol from
the total amount of cholesterol. Cellular protein was measured by
Lowry’s method.
Plasma Levels of Compounds after Oral Administration in
Rats, Rabbits, and Dogs. Compounds were suspended in 5% arabic
gum and administered orally at 10 mg/kg to male SD rats (n ) 3,
6-7 weeks old, Japan SLC). SD rats were fasted overnight before
administration. Compound 2 was suspended in 5% arabic gum and
administered orally at 10 mg/kg to male Japanese white rabbits (n
) 4, 2.7-3.0 kg, Japan SLC) and male beagles (n ) 4, 9.0-11.6
kg, HRP Inc., Kalamazoo, MI). Blood samples were drawn using
a heparinized syringe from the jugular vein at 0.5, 1, 3, 5, 8, and
24 h after administration in rats and at 0.25, 0.5, 1, 2, 3, 5, 8, and
12 h in beagles, and from the ear vein at 0.25, 0.5, 1, 2, 3, 5, 8,
and 24 h in rabbits. Blood was centrifuged at 3000 rpm for 10 min
at room temperature. Concentrations of a free form of compounds
in the plasma were determined using HPLC, as described above.
In Vitro ACAT Activity. Male Japanese White rabbits (2.5 kg,
Japan SLC) were anesthetized with sodium pentobarbital (30 mg/
kg, iv) and exsanguinated from the common carotid artery, and
then the liver was isolated. Hyperlipidemic male Japanese White
rabbits (2-3 kg) fed a high cholesterol diet (1% cholesterol CR-3,
Clea Japan, Osaka, Japan) for one month were anesthetized and
exsanguinated from the common carotid artery, and then the
intestinal mucosa, liver, and adrenal were isolated. Rabbits fed a
high cholesterol diet were separately injected with thioglycolate
medium (200 mL/animal, ip), and then 4 days later peritoneal
macrophages were isolated under deep anesthesia. Microsomes were
prepared according to the method of Field and Mathur.32 Briefly,
each sample was homogenized in a buffered sucrose solution (250
mM sucrose, 5 mM K2HPO4/KH2PO4, 1 mM EDTA, and 1 mM
dithioerythritol, pH 7.4) using a Teflon-glass homogenizer. The
homogenate was centrifuged at 12000g for 15 min at 4 °C. The
resulting supernatant was centrifuged at 105000g for 30 min at 4
°C. The microsomal fraction was used as the ACAT preparation.
ACAT activity was determined according to the method described
by Heider et al.33 The microsomes were incubated in 154 mM
phosphate buffer (pH 7.4) containing bovine serum albumin. Test
compounds were applied and preincubated at 37 °C for 5 min, then
30 nmol of [1-14C]oleoyl-CoA (PerkinElmer, Waltham, MA) was
added. The reaction mixture was incubated at 37 °C for 7.5 min
for the intestine, 20 min for the liver, 30 min for macrophages,
and 10 min for the adrenal gland. EC was extracted with
chloroform/methanol (2:1) and separated by thin-layer chromatog-
3-[4,6-Dimethyl-7-(2,2-dimethylpropanamido)-1-octylindolin-3-
yl]propionic Acid Hydrochloride (3). To a solution of 27 (3.77 g,
8.75 mmol) in AcOEt (35 mL) was added 9.25 M HCl in i-PrOH
(1.1 mL, 10 mmol) in an ice bath, followed by stirring at the same
temperature for 30 min. The precipitate was collected by filtration
to give 3 as a crystalline product (2.99 g, 73% yield); mp 178-182
°C. IR (ATR): 1713, 1686 cm-1 1H NMR (DMSO-d6): δ
.
0.80-0.90 (m, 3H), 1.15-1.30 (m, 19H), 1.50-1.75 (m, 3H),
1.95-2.05 (m, 1H), 2.04 (s, 3H), 2.27 (s, 3H), 2.30-2.40 (m, 2H),
3.00-3.30 (m, 2H), 3.40-3.80 (m, 3H), 6.94 (s, 1H), 9.15 (s, 1H).
MS m/z: 431 [M + H]+. Anal. (C26H42N2O3 · HCl · 0.2H2O) C,
H, N.
Partition Coefficient at pH 7.0. Octanol-water partition coef-
ficients at pH 7.0 were determined and log D7.0 values were
calculated as an index of lipophilicity. First, 5 mg of compounds
were weighed and dissolved in 5 mL of octanol and mixed with 5
mL of phosphate buffer (1/15 M K2HPO4/KH2PO4, pH 7.0). The
mixed solution was vigorously shaken at room temperature for 30
min and then centrifuged at 3000 rpm for 10 min at room
temperature. Concentrations in the octanol fraction and buffer
fraction were determined using HPLC. The HPLC equipment
consisted of a pump (PU-980, JASCO, Tokyo, Japan), a UV
detector (Shodex EC-1, SHOWA DENKO, Tokyo, Japan), an
autoinjector (AS-950, JASCO), and a Develosil-ODS-UG-5 column
(5 µm, 4.6 mm × 150 mm, NOMURA Chemical, Seto, Japan).
The octanol-water partition coefficent was calculated as the ratio
of concentration in the octanol fraction to that in the buffer fraction.
The log D7.0 value was determined as the logarithm of a partition
coefficient.
Water Solubility. First, 6 mg of compounds were placed in 3
mL phosphate buffer (1/15 M K2HPO4/KH2PO4, pH 7.0), vigorously
shaken for 30 min at room temperature, and then centrifuged at
3000 rpm for 10 min at room temperature. The supernatant was
filtrated using DISMIC-13HP (0.45 µm, Toyo Roshi Kaisha, Ltd.,
Tokyo, Japan). Concentrations of compounds in the filtrate were
determined using HPLC as described above.
Lipid Peroxidation in Rat Brain Homogenate. The production
of lipid peroxide was determined as malondialdehyde (MDA) in
the rat brain homogenate, as reported previously.18,20 Briefly, the
cerebral cortex isolated from male SD rats (6 weeks old, Japan
SLC, Shizuoka, Japan) was homogenized in ice-cold 50 mM
phosphate-buffered saline (pH 7.4). The homogenate was centri-
fuged at 1300g for 10 min at 4 °C. The supernatant was incubated
at 37 °C for 30 min in the presence of vehicle or test compounds.
The reaction was terminated by addition of 20% trichloroacetic acid,
and the mixture was immediately centrifuged at 2000g for 15 min
at 4 °C. The supernatant was heated at 100 °C for 15 min with
thiobarbituric acid solution at pH 7.0. The optical absorbance of
MDA was measured at 532 nm. The MDA level in medium before
the assay was determined, and the production of MDA during the

4832 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Takahashi et al.
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test compounds were determined. IC50 values were calculated using
data from duplicate assay tubes at the concentrations of test
compounds in each experiment, and the mean value was calculated
for 3-5 experiments.
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plasma was isolated and incubated with CuSO4 (2 mM) at 37
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and the vehicle were orally administered at a dose of 3 mg/kg
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days, during which period 2 suspended in a 5% arabic gum
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collected from the ear artery. Male beagles (8.8-12.0 kg, HRP
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were collected 24 h after the final administration from the
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Supporting Information Available: Elemental analysis data of
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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