Design, synthesis and pharmacology of aortic-selective acyl-CoA: Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.06.024

We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also abbreviated as SOAT) inhibitors, their structure–activity relationships (SARs) and their pharmacokinetic (PK) and pharmacological profiles. The connection of two weak ligands—N-(2,6-diisopropylphenyl)acetamide (50% inhibitory concentration [IC₅₀] = 8.6 μM) and 2-(methylthio)benzo[d]oxazole (IC₅₀ = 31 μM)—via a linker comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC₅₀ = 0.004 μM) toward aortic ACAT. This head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3 mg/kg, 3h significantly decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the plasma total cholesterol level in high fat- and cholesterol-fed F₁B hamsters. Here, we demonstrate the antiatherosclerotic effect of 3h in vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 overexpression.

Full text of DOI:10.1016/j.bmc.2018.06.024

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacology of aortic-selective acyl-CoA:
Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors
Kimiyuki Shibuya⁎, Katsumi Kawamine1, Toru Miura, Chiyoka Ozaki, Toshiyuki Edano2,
Ken Mizuno, Yasunobu Yoshinaka, Yoshihiko Tsunenari
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Co., Ltd., 2-17-43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also
abbreviated as SOAT) inhibitors, their structure–activity relationships (SARs) and their pharmacokinetic (PK)
and pharmacological profiles. The connection of two weak ligands—N-(2,6-diisopropylphenyl)acetamide (50%
inhibitory concentration [IC50] = 8.6 μM) and 2-(methylthio)benzo[d]oxazole (IC50 = 31 μM)—via a linker
comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-
diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC50 = 0.004 μM) toward aortic ACAT. This
head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate
the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3 mg/kg, 3h significantly
decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the
plasma total cholesterol level in high fat- and cholesterol-fed F1B hamsters. Here, we demonstrate the anti-
atherosclerotic effect of 3h in vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol
level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 over-
expression.
Acyl-CoA:cholesterol O-acyltransferase (ACAT/
SOAT)
ACAT-1 (aortic ACAT)
ACAT-2 (intestinal ACAT)
Isoform-selectivity
Double-induced fit
Cholesterol esters (CEs)
Lipid-accumulation areas
Atherosclerosis
1. Introduction
targets for the treatment of hyperlipidemia and atherosclerosis.11,12
However, studies on most of these inhibitors were discontinued because
Acyl-coenzyme A:cholesterol O-acyltransferase (ACAT), also known
as sterol O-acyltransferase (SOAT), is an intracellular enzyme that
catalyzes the acylation of cholesterol with long-chain fatty acids to
afford cholesteryl esters (CEs) and plays several important physiological
roles, including storing CEs as lipid droplets, absorbing dietary
cholesterol and providing the CEs acting as part of the core lipid for
lipoprotein synthesis and assembly in various mammals.1 Two isoforms
of ACAT have been identified in mammals and have been shown to
have different substrate specificities and potential functions. ACAT-1 is
ubiquitously expressed in various human tissues, predominantly in
human liver (Kupffer cells), macrophage, adrenal gland and brain
tissue, whereas ACAT-2 is found in human hepatocytes and intestinal
tissue.2–8 ACAT-1 is closely associated with detrimental effects
on the arterial wall and macrophage-derived foam cell formation in
atherosclerosis. ACAT-2 is responsible for absorption of cholesterol
from the intestine and for lipoprotein production by the liver.9,10
Therefore, several ACAT inhibitors have been developed as post-statin
of their toxicity,13–18 side-effects19 and failure to demonstrate any
beneficial effect on adverse cardiovascular outcomes.20–24 Considering
the unsuccessful clinical trial of non-selective ACAT inhibitors, we fo-
cused on the development of an ACAT-1 inhibitor that can directly act
on the arterial wall without lowering the plasma total cholesterol for
the treatment of atherosclerosis, which is still pending. Recently, sev-
eral potential applications have been demonstrated for the drug-re-
positioning of ACAT-1 inhibition in the treatment of the following
diseases: 1) In multiple neurodegenerative diseases, including Alzhei-
mer’s disease,25–29 blocking ACAT-1 can stimulate autophagy to clear
amyloid β peptides and suppress 24(S)-hydroxycholesterol-induced
neuronal cell death; 2) In non-alcoholic steatohepatitis (NASH),30
ACAT-1 is involved in the progression of steatosis and liver inflamma-
tion via Kupffer cells activated by a high level of CE; 3) In cancer,31–34
the increased CE level is well-known in breast cancer, glioma and ag-
gressive prostate cancer; therefore, ACAT-1 expression could serve as a
potential prognostic marker. Furthermore, ACAT-1 inhibition
⁎
Corresponding author.
E-mail address: k-sibuya@kowa.co.jp (K. Shibuya).
Research Strategy Department, Pharmaceutical Division, Kowa Company, Ltd., 3-4-14, Nihonbashihoncho, Chuoku, Tokyo 103-8433, Japan.
T. Edano: Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women’s Hospital, Harvard Medical School, 3 Blackfan Cir, CLS, 17th Floor, Boston, MA 02115, USA.
1
2
https://doi.org/10.1016/j.bmc.2018.06.024
Received 16 March 2018; Received in revised form 14 June 2018; Accepted 16 June 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Shibuya,K.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.06.024

K. Shibuya et al.
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Figure 2. Molecular design used to discriminate between aortic and intestinal
ACATs based on a combination of two distinctly different ligands connected
with a linker of appropriate length.
Figure 1. Three different types of ACAT inhibitors.
potentiates the antitumor response of CD8+ T cells by modulating
cholesterol metabolism. Consequently, ACAT-1 inhibition has attracted
significant attention. These fascinating trends prompted us to report our
basic concept of molecular design and disclose our proprietary data
related to aortic-selective ACAT inhibitors, which led to the develop-
ment of the ACAT-1 inhibitor, 2-(4-(2-((1H-benzo[d]-imidazol-2-yl)
thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-
yl)acetamide hydrochloride (K-604).35–38 In accordance with the con-
cept of differentiating between the isoforms ACAT-1 (aortic ACAT) and
ACAT-2 (intestinal ACAT),39 we first characterized the reported ACAT
inhibitors in patents and the literature and then extracted the structural
moieties that are essential for ACAT inhibitory action. Focusing on the
chemical structures of the representative code-numbered ACAT in-
hibitors, we identified three different types of ACAT inhibitors: the 4,5-
diphenylimidazole type, such as (S)-1-(5-((4,5-diphenyl-1H-imidazol-2-
yl)sulfinyl)pentyl)-4,5-dimethyl-1H-pyrazole (RP-73163);40 the anilide
type, such as 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide
(CI-976);41 and the hybrid type (a combination of 4,5-diphenylimida-
zole and phenylurea), such as 3-(2,4-difluorophenyl)-1-(5-((4,5-di-
phenyl-1H-imidazol-2-yl)thio)pentyl)-1-heptylurea (Dup-128),42,43 as
shown in Fig. 1.
RP-73163 was reported to have IC50 = 0.245 μM against rabbit
aortic ACAT and IC50 = 0.37 μM against rabbit intestinal ACAT
whereas CI-976 showed IC50 = 0.073 μM against rabbit intestinal
ACAT. Two distinctly different ligands may be accommodated by dif-
ferent binding sites in the two ACAT enzymes. Indeed, Dup-128 was
reported to have IC50 = 1.0 μM against J774 macrophage cells (corre-
sponding to aortic ACAT) and 0.01 μM against rat hepatic microsomes
(corresponding to intestinal ACAT).43 This finding inspired our postu-
lation that a hybrid molecule may potentially differentiate the ACAT
isoforms. Therefore, considering the putative binding sites of the re-
ceptor,44,45 we speculated that connecting two different ligands—A (for
the orthosteric site of the receptor) and B (for the allosteric binding site
of the receptor)46–49—with a linker of appropriate length will generate
a new molecule that enables the discrimination between aortic and
intestinal ACATs. We envisaged that an optimal combination of two
ligands connected with a suitable length linker could bring about an
induced-fit in aortic ACAT and differentiate it from intestinal ACAT. To
study the structure-activity relationships (SARs) between the ligand
structures and their ACAT inhibitory activities, we chose two re-
presentative small ligands: N-(2,6-diisopropylphenyl)acetamide unit A,
which is commonly used for the development of ACAT inhibitors, and
2-(alkylthio)-1,3-benzazole unit B, which can act as an isostere50–52 of
4,5-diphenylimidazole, as shown in Fig. 2. In this paper, we report our
molecular design of an aortic-selective ACAT inhibitor and its SARs and
pharmacology.
2. Chemistry
2.1. Synthesis of compounds 3a-j, 4a-j and 5a-j
To determine the desired atom and optimal bridging distance be-
tween N-(2,6-diisopropylphenyl)acetamide unit A and 2-(alkylthio)-
1,3-benzazole unit B, the basic compounds 3a-j, 4a-j, and 5a-j were
prepared. The synthetic routes used in this work are summarized in
Scheme 1. The acylation of 2,6-diisopropylaniline (1) with bromoalk-
anoic acid provided anilides 2a-j. The transformation to thioether
compounds 3a-j, 4a-j, and 5a-j was achieved by alkylating bromo
compounds 2a-j with three varieties of 2-mercapto-1,3-benzazole
(X = O, NH and S) in the presence of a base and a phase-transfer cat-
alyst. Compound 4b could not be obtained because of the retro-Michael
reaction of 4b, which yielded N-(2,6-diisopropylphenyl)acrylamide.
2.2. Synthesis of compounds 12a-d to 17a-c, 18, 19 and 22a-d
Treatment of aminohydroxybenzoates with thiophosgene or po-
tassium O-ethyl dithiocarbonate yielded 2-mercapto-1,3-benzo[d]ox-
azole[4, or 5, 6, 7]carboxylates 6a-d, and subsequent esterification of
6a-d provided the corresponding methyl ester 7a-d. These compounds
were reacted with 6-bromo-N-(2,6-diisopropylphenyl)hexanamide (2e)
to afford 12a-d, and the methoxycarbonyl group of 12a-d was then
transformed into the carboxyl group of 13a-d, the hydroxymethyl
group of 14a-d and the N,N-dimethylaminomethyl group of 15a-d ac-
cording to the method detailed in Scheme 2. Similarly, the substituted
2-mercaptobenzo[d]oxazoles 8a-d, 9a-c, 10 and 11 were prepared and
converted into 16a-d, 17a-c, 18, 19 and 20a-d, respectively. 2-
Scheme 1. aReagents and conditions: i) Br(CH2)nCOOH (n = 1～9 or 14), 1-(3-
dimethylaminopropy1)-3-ethylcarbodiimide hydrochloride (WSC), hydro-
xylbenzotriazole (HOBt), dimethylformamide (DMF), room temperature (rt); ii)
2-mercapto-1,3-benzazole (X = O, NH or S), K2CO3, 18-crown-6, DMF, 80 °C.
2

K. Shibuya et al.
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Scheme 2. Reagents and conditions: i) KS(C = S)OEt, EtOH, reflux or Et3N, Cl(C = S)Cl, CH2Cl2, rt; ii) p-TsOH, sat.HCl/MeOH, reflux; iii) 2e, K2CO3, 18-crown-6,
DMF, 80 °C; iv) LiOH, t-BuOH/H2O/THF (1:1:2.5), rt; v) Et3N, methyl chloroformate, THF, 0 °C and then NaBH4, H2O, rt; vi) Et3N, MsCl, THF, 0 °C and then Me2NH
(40% aq. solution), THF, reflux. vii) Zn dust, AcOH, rt; viii) 37% HCHO, NaBH3CN, MeCN and then AcOH, rt.
Mercaptobenzo[d]oxazol-7-ol of 9d could not be prepared because of
the lack of available starting materials.53 The treatment of 2e with the
commercially available 2-mercaptooxazolo[5,4-b] (or [5,4-c], [4,5-c],
[4,5-b])pyridine 21a-d afforded the corresponding thioethers 22a-d,
respectively.
types of benzoxazole, benzimidazole and benzothiazole derivatives are
presented as scatter plots in Figs. 4, 5 and 6. These figures show that
benzoxazole derivatives 3a-j were much more potent and selective than
benzimidazole derivatives 4a-j. In contrast, the behavior of ben-
zothiazole derivatives 5a-j was similar to that of 3a-j, and distinctive
bimodal peaks (n = 2 and 8) of the aortic ACAT inhibitory activity were
observed. The appearance of bimodal peaks is presumed to stem from
the formation of the dimeric ACAT-1.61 (See Fig. 7).
3. Results and discussion
Interestingly, four independent ligands—N-(2,6-disopropylphenyl)
acetamide (A) (IC50 = 8.6 μM), N-(2,6-disopropylphenyl)hexanamide
The prepared compounds were evaluated for their in vitro ACAT
inhibitory activity according to the modified method of Heider et al.54
using two enzyme sources: rabbit aorta homogenate and rabbit small
intestine microsomes. The J774 macrophage cell culture assay was
performed to assess the inhibition of the target cells by systemic anti-
atherosclerotic agents.43 The ACAT inhibitory activities of 3a-j, 4a-j,
and 5a-j are described and compared with those of the well-known
ACAT inhibitors RP-73163, CI-976, Dup-128 and 2,6-diisopropyl-
phenyl(2-(2,4,6-triisopropylphenyl)acetyl)sulfamate (CI-1011, Avasi-
mibe)55–59 in Table 1.
CI-1011 exhibited remarkably weak potencies toward ACATs in the
conventional assays. Two findings were later reported to amend its
inhibitory activities (IC50) in rat liver microsomes and IC-21 murine
macrophages after the problems of protein binding were carefully ad-
dressed55,59 as shown in Fig. 3. Parke- Davis researchers demonstrated
hypolipidemic and anti-atherosclerotic effects in several animal models
and Pfizer Inc. conducted Phase III clinical trials. We used it as a
comparative compound for in vitro and in vivo test.
(A’) (IC50 = 0.17 μM), 2-(methythio)benzo[d]oxazole (B) (IC50
=
31 μM) and 2-(butylthio)benzo[d]oxazole (B’) (IC50 = 26 μM)—showed
weak or moderate potencies against the aortic ACAT, respectively.
However, compound 3a (n = 1) exhibited remarkably enhanced ac-
tivity (IC50 = 0.035 μM) and 5.7-fold isoform selectivity. Compound 3h
(n = 8) not only had improved activity (IC50 = 0.004 μM) (i.e., by up to
7750-fold) against aorta ACAT but also exhibited the highest potency
(IC50 = 0.007 μM) against J774 cell macrophages while maintaining its
isoform-selectivity. Considering the relationships between the cLog P
and IC50 in the four ligands, two types of ligands notably exhibit dif-
ferent behavior when increasing the same degree of cLog P value; the
anilide type ligand A’ increases in potency by 50-fold whereas the
benzo[d]oxazole type ligand B’ shows nearly no change. Since B’ and
3a possessing a similar value (cLog P = 4.15), ACAT inhibition is not
simply dependent on lipophilicity, reflecting not only different struc-
tural requirement for binding mode but also the existence of two dif-
ferent binding sites. Consequently, we established a basic molecular
design in which the connection of two independently weak ligands with
The relationships between the ACAT inhibitory activity (1/IC50) and
the length of the methylene chain (n = number of CH2 units) for three
3

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Table 1
Effects of benzazole heterocycles (X = O, NH, or S) and methylene chain length (n = number of CH2 units) on ACATs inhibitory activities.
Compound
n (number of
CH2 units)
X (atom)
Aortaa
IC50 (μM)
Intestineb
Intestine/Aorta Ratio
J774 cellsc
cLog Pe
d
IC50 (μM)d
IC50 (μM)d
RP-73163
0.047f
0.19
0.036
31
0.035
0.015
0.036
0.084
0.01
0.005
0.004
0.004
0.006
0.04
0.13f
0.03
0.02
13
2.8f
0.2
0.6
0.4
5.7
1.7
1.8
2.6
6.5
6.0
5.5
5.3
2.3
1.1
2.5
0.58
0.31
0.82
0.59
1.2
0.47
2.8
3.6
4.30
6.09
9.75
9.69
4.15
4.48
4.90
5.11
5.66
6.19
6.72
7.25
7.78
10.42
4.30
4.62
5.05
5.28
5.81
6.34
6.87
7.40
7.93
10.57
4.83
5.15
5.58
5.81
6.34
6.87
7.40
7.93
8.46
11.10
CI-976
Dup-128
CI-1011
3a
3b
3c
3d
3e
3f
3g
1
2
3
4
5
6
7
8
9
14
1
2
3
4
5
6
7
8
9
14
1
2
3
4
5
6
7
O
O
O
O
O
O
O
O
0.2
0.025
0.066
0.22
0.065
0.03
0.022
0.021
0.014
0.044
5.8
1.9
0.31
0.04
0.007
0.1
3h
3i
3j
O
O
0.41
0.18
4a
4b
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
S
S
S
S
S
S
S
S
2.3
NT
g
h
h
h
h
NT
NT
NT
4c
4d
4e
4f
4g
4h
4i
0.3
0.39
0.8
0.43
2.7
1.1
1.5
1.4
1.7
2.2
1.5
0.4
1.5
4.8
3.6
2.1
1.3
4.0
5.1
4.5
2.2
1.1
3.1
5.2
0.026
0.024
0.018
0.014
0.019
0.12
0.031
0.004
0.028
0.062
0.026
0.007
0.007
0.004
0.005
0.088
0.038
0.034
0.03
0.031
0.029
0.044
0.047
0.019
0.1
0.35
0.048
0.03
0.3
0.46
1.7
0.18
0.28
5.6
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.13
57
5.2
0.033
0.028
0.036
0.018
0.011
0.093
0.41
0.059
0.043
0.57
1.8
8
9
14
5j
S
a
IC50 (μM) against rabbit aorta ACAT activity. bIC50 (μM) against rabbit intestinal microsome ACAT activity. cIC50 (μM) against esterified cholesterol accumulation
in J774 cells. dAll samples were assayed in duplicate or triplicate. ecLogP was calculated via ChemDraw/Pro 15.1. fThe discrepancy between the reported value and
our measured value for the ACAT inhibitory activities of RP-73163 was attributed to the microsomal concentration in assay conditions60 (Supporting Information).
g4b could not be synthesized; refer to the discussion in the chemistry section. hNT: not tested.
Figure 3. Structure and biological properties of CI-1011.
a linker of optimal length can yield a highly potent molecule based on
the double-induced fit mechanism.62–64Next, we investigated the elec-
tron-donating or electron-withdrawing inductive effects of the sub-
stituted groups and the incorporated nitrogen atom at each position on
the benzene ring of 1,3-benzoxazole based on template 3e toward the
ligand-binding pocket or cleft of aortic and intestinal ACAT receptors,
respectively. The ACAT inhibitory activities of 12a-d to 22a-d are
summarized in Table 2.
Figure 4. Relationships between the ACAT inhibitory activity (Y-axis: 1/IC50
)
Interestingly, methyl esters 12a and 12d exhibited much higher
values (31- and 10-fold, respectively) of the intestine IC50/aorta IC50
selectivity ratios than the other compounds. However, we detected
carboxylates 13a and 13d during incubation with methyl esters 12a
and 12d in intestinal microsomes over 30 min (data not shown)65 and
determined that these results were false positives. Conclusively, in-
testinal microsomes served as esterase and hydrolyzed methyl esters.
The isoform-selectivity resulted in the ratio value (intestinal IC50 of a
mixture of 13a and 12a/ aortic IC50 of 12a). The substituents at the 4-
position of the benzoxazole, such as the hydroxymethyl group of 14a
and the length of the methylene chain (X-axis: n = number of CH2 units) in
benzoxazole derivatives 3a-j.
(4.0-fold), the N,N-dimethylaminomethyl group66 of 15a (7.6-fold)
provided moderate selectivity compared to the substituents at other
(i.e., 5, 6, and 7-) positions, although the N,N-dimethylamino group-
containing compounds67 20a (4.1-fold), 20b (5.8-fold) and 20c (6.3-
fold) retained some selectivity regardless of the group’s position. It is
unclear why the N,N-dimethylamino substituents in compounds such as
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substituted with a N atom on the aromatic ring of the benzoxazole, the
resulting oxazolo[4,5-b]pyridine derivative 22a (5.6-fold) behaved si-
milarly to the compounds with substituents at the 4-position. However,
these substituents did not improve the activity against J774 cell mac-
rophages. These polar functional groups and substituted nitrogen atom
have the potential to improve drug absorption through the following
effects: 1) introduction of a strong hydrogen bond acceptor, 2) in-
creased polar surface area, 3) decreased lipophilicity (cLogP < 5.6),
and 4) enhanced aqueous solubility. Consequently, the polar-ionic in-
teractions around the binding site not only obstruct the molecular
permeability of the J774 cell membrane but also negatively affect the
adaptability of the ligand-binding pocket or cleft. Based on the SARs
results, we chose compound 3h as the candidate aortic ACAT inhibitor
and evaluated its atherosclerotic efficacy to demonstrate its pharma-
cological behavior compared with the profile of CI-1011. To investigate
the optimal administration conditions in vivo, we conducted a phar-
macokinetic (PK) study in which 3h was orally administered to fasted
and non-fasted dogs.
Figure 5. Relationships between the ACAT inhibitory activity (Y-axis: 1/IC50
and the length of the methylene chain (X-axis: n = number of CH2 units) in
benzimidazole derivatives 4a-j.
)
As anticipated, compound 3h showed extremely low water solubi-
lity (0.03 μg/mL in the first fluid used for the dissolution test [pH 1.2])
due to its high lipophilicity (cLog P = 7.25), which resulted in poor
absorption (Cmax 5.0
displayed a relatively good absorption (Cmax was approximately 100-
fold higher: 582 85 ng/mL) in the non-fasted dogs. We assumed that
6.0 ng/mL) in the fasted dogs. Conversely, 3h
the effect of food intake was a major cause of this phenomenon. Thus,
the secretion of bile juice into the gastrointestinal tract may be ac-
celerated by food intake, which would enhance the solubility and dis-
solution rate and lead to good absorption of 3h under fed condi-
tions.68,69 To select the dosage of the test compounds, we next
measured the drug exposures of 3h and CI-1011 when orally ad-
ministered to non-fasted rats (See Table 3).
As shown in Table 4, the drug concentration of CI-1011 was sig-
nificantly greater (Cmax: 43-fold higher and AUC: 134-fold higher)
than that of 3h (cLog P 7.25) despite its much greater lipophilicity
(cLog P 9.69). In contrast, calculation of the log D values revealed that
CI-1011 (cLog D = 3.94) is considerably more likely to distribute in
water than 3h (cLog D = 7.29). In addition, the sulfamate structure
reportedly contributes to improved oral absorption and bioavail-
ability.70 Considering these compounds’ ACAT inhibitory activities (3h:
IC50 = 7 nM vs CI-1011: IC50 = 590 nM) toward J774 cell macrophages
(i.e., anti-foaming action), we must compensate for the effective capa-
city (F) of ACAT inhibitors using the index expressed in the following
equation: F = AUC/MW/IC50. The F value of 3h was calculated to be
109.7, whereas that of CI-1011 is 162.4. Based on these results, we
determined the dosages of CI-1011 (0.3, 1, 3 and 10 mg/kg) and 3h (1,
Figure 6. Relationships between the ACAT inhibitory activity (Y-axis: 1/IC50
and the length of the methylene chain (X-axis: n = number of CH2 units) in
benzothiazole derivatives 5a-j.
)
20b and 20c contributing higher selectivity. The methyl group at the 6-
position of 18 and trifluoromethyl group at the 5-position of 19 with
higher lipophilicity (cLogP values of 6.16 and 6.66, respectively) en-
hanced the activity against J774 cell macrophages without diminishing
aortic ACAT inhibition and selectivity. When the CeH group was
Figure 7. A highly potent molecule obtained by connecting two weak ligands with a linker of optimal length.
5

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Table 2
Effects of the substituted groups and incorporated nitrogen at each position of the 1,3-benzoxazole moiety on ACAT inhibitory activities.
c
e
Compound
Position
R
Aortaa
IC50 (μM)
Intestineb
IC50 (μM)
Intestine/Aorta Ratio
J774 cell
IC50 (μM)
cLog P
d
d
d
12a
12b
12c
12d
13a
13b
13c
13d
14a
14b
14c
14d
15a
15b
15c
15d
17a
17b
17c
17d
18
4
5
6
7
4
5
6
7
4
5
6
7
4
5
6
7
4
5
6
7
6
5
4
5
6
7
4
5
6
7
COOMe
COOMe
COOMe
COOMe
COOH
COOH
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2OH
CH2NMe2
CH2NMe2
CH2NMe2
CH2NMe2
OH
0.026
0.07
0.81f
0.21f
0.11f
0.33f
NT
4.5
31.2
3.0
3.8
0.9
0.6
0.5
0.1
11
11
4.4
11
5.75
5.75
5.75
5.75
5.18
5.18
5.18
5.18
4.62
4.62
4.62
4.62
5.50
5.50
5.50
5.50
5.71
5.71
5.71
5.71
6.16
6.66
6.23
6.23
6.23
6.23
4.61
4.61
4.61
4.61
0.029
0.032
10.3
g
g
g
NT
NT
3.6
2.1
11
1.3
2.4
1.0
4.0
1.2
2.4
2.0
7.6
0.7
2.8
1.4
1.0
2.8
1.5
5.0
11
0.045
0.064
0.076
0.056
0.042
0.25
0.13
0.096
0.24
0.043
0.053
0.18
0.074
0.18
0.11
0.32
0.17
0.37
0.13
0.24
0.12
0.081
11
1.1
7.0
2.2
1.3
1.8
2.4
5.6
8.8
2.8
2.3
OH
OH
OH
Me
g
g
g
g
NT
NT
NT
NT
0.034
0.028
0.029
0.043
0.015
0.036
0.025
0.045
0.041
0.024
0.11
0.14
0.12
0.25
0.094
0.077
0.14
0.15
0.16
0.036
3.2
5.0
4.1
5.8
6.3
2.1
5.6
3.3
3.9
1.5
0.4
0.1
0.5
0.5
1.1
0.8
1.3
2.8
4.1
3.0
19
CF3
20a
20b
20c
20d
22a
22b
22c
22d
NMe2
NMe2
NMe2
NMe2
N
N
N
N
a
IC50 (μM) against rabbit aorta ACAT activity. bIC50 (μM) against rabbit intestinal microsomes ACAT activity. cIC50 (μM) against esterified cholesterol accu-
mulation in J774 cells. dAll samples were assayed in duplicate or triplicate. cLogP was calculated by ChemDraw/Pro 15.1. Methylester was hydrolyzed during
e
f
bioassay conditions. gNT: not tested.
Table 3
Comparison of the PK propertiesa of 3h under fasted and non-fasted conditions.b
Condition
Tmax (hr)
Cmax (ng/mL)
Area under the curve (AUC) 0–8 h
(ng·hr/mL)
Non-fasted
Fasted
3.0
1.0
2.6
0.0
582
5.0
85
6.0
2958
14
454
22
a
Data are shown as means
the standard deviation (SD). bA 30-mg/kg
dose in 0.5% methyl cellulose (MC) suspension solution was orally adminis-
tered to male dogs (n = 3) under fasted and non-fasted conditions.
Table 4
Comparison of the PK propertiesa between 3h and CI-1011 under non-fasted
conditions.b
Compound
Tmax (hr)
Cmax (ng/mL)
AUC 0–8 h (ng·hr/mL)
3h
CI-1011
3.2
3.3
1.4
0.6
106.7
4580.0
21.7
1520.1
358
48075.8
97.6
4978.7
a
Data are shown as means
SD. bA 30-mg/kg dose (3h and CI-1011) in
Figure 8. Effects of CI-1011 on the plasma TC levels and lipid-accumulation
areas. CI-1011 was administered at doses of 0.3, 1, 3, 10 mg/kg via the diet to
Bio F1B hamster fed a diet that was high in fat and cholesterol. * p < 0.05;
statistical analysis was conducted using Dunnett’s test.
0.5% MC suspension solution was orally administered to male rats (n = 3)
under non-fasted conditions.
3, 10 and 30 mg/kg) for the dietary administration study, which uti-
lized Bio F1B hamsters fed a diet that was high in fat and cholesterol for
10 weeks. Bio F1B hamsters are more responsive to diet-induced
atherosclerosis than mice, and an adequate atherosclerosis model was
used to evaluate the lipid-modulating agents.71 The non-selective ACAT
inhibitor, CI-1011 at doses of 1, 3 and 10 mg/kg remarkably decreased
the lipid-accumulation areas in the aortic arch (33%, 14% and 0% re-
lative to the control, respectively), and this effect was accompanied by a
dramatic reduction in the plasma total cholesterol (TC) level (55%, 25%
and 10% relative to the control, respectively), as shown in Fig. 8. In
contrast, because of its poor PK profile, at three-fold higher doses (i.e.,
1, 3, 10 and 30 mg/kg), our aortic-selective ACAT inhibitor 3h sig-
nificantly decreased the lipid-accumulation areas (74%, 69%, 42% and
7% relative to the control, respectively), as shown in Fig. 9. Notably,
molecule 3h at doses of 1 and 3 mg/kg decreased the lipid-accumula-
tion areas without significantly lowering the plasma TC level (i.e.,
without changing the CE/free cholesterol [FC] ratio which is described
6

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
essentially the difference in these compounds’ mechanism of action in
atherosclerosis.
As shown, only a trace amount of unbound drug can participate in
inhibiting aortic ACAT due to strong plasma binding. For accurate
prediction and discussion, independent measurements of the protein
binding ratio of ACATs microsomes with bovine serum albumin (BSA)
for ACAT assay and hamster serum albumin to monitor the unbound
drug concentration are desirable; however, these measurements are too
troublesome and complicated. Accordingly, the IC50 value (12 μM) of
CI-1011 toward rat liver microsomes can be calculated into the realistic
value (0.5 μM) using the rat fp (3.9%), which is consistent with the
measured value (0.7 μM). Additionally, the IC50 value (4100 nM ac-
cording to our measurement data) toward human monocyte-macro-
phages, can be calculated into an estimated value (13 nM) if using the
human fp (0.319%), which is not substantially different from the
measured value (25 nM) in the absence of albumin. Accordingly, if this
simulation is applied to the aortic IC50 value (4 nM) of 3h using the rat
fp (2.7%) in place of the rabbit or hamster fp, the value can be de-
creased to 0.11 nM. Since the plasma protein binding between inhibitor
3h and the intestinal membrane in the digestive tract is negligible, the
isoform-selectivity (5.3-fold) can also be increased 191-fold. Therefore,
the pharmacological effect based on the aortic-selective ACAT inhibi-
tion by 3h is supported when protein binding is considered.
Figure 9. Effects of 3h on the plasma TC levels and lipid-accumulation areas.
Compound 3h was administered at doses of 1, 3, 10, 30 mg/kg via the diet to
Bio F1B hamsters fed a diet that was high in fat and cholesterol. * p < 0.05;
statistical analysis was conducted using Dunnett’s test.
To investigate the toxicological properties of 3h, we conducted a
safety test in rats and dogs. In male rats over 2 weeks, the no-observed-
adverse-effect level (NOAEL) of 3h was 30 mg/kg. At a dose of 100 mg/
kg, the drug exposure reached saturation. In male dogs at doses of 30
and 100 mg/kg over 2 weeks, the NOAEL could not be determined be-
cause of poor absorption. At a dose of 100 mg/kg, 3h induced the ne-
crosis of adrenal reticular zona cells in all dogs. Although there is a high
probability of toxicity due to ACAT-1 expression in the adrenal gland,
tolerance is also possible, which is relevant to lowering FC level in the
adrenal gland dose-dependently (Supporting Information). Fazio et al.
argued that adrenal toxicity is attributed to FC accumulation in adrenal
cells.23 However, the lipophilic 3h could be accumulated in the adrenal
gland in a similar manner to many ACAT inhibitors that exhibited
adrenal toxicity.13–19
Table 5
Simulation of the unbound fraction in the plasma (fp) ratio of 3h and CI-1011
in human and rat using the ADMET predictor (Northern Science Consulting).
Compound
Human fp (%)a
Rat fp (%)a
CI-1011
3h
0.319
0.626
3.963
2.721
a
The (fp) ratio of CI-1011 and 3h in human and rat are simulated by ADMET
predictor Ver-8.5.
in the Supporting Information). These results indicated that the aortic-
selective ACAT inhibitor 3h did not substantially affect the intestinal
ACAT’s ability to absorb cholesterol or cause the liver to suppress the
secretion of very low-density lipoprotein (VLDL) at lower doses. To
elucidate these interesting results of 3h and CI-1011, we verified the
potency and isoform-selectivity between the rabbit aorta homogenate
and small intestine microsomes, as shown in Table 1. Although CI-1011
exhibited significantly weak potencies toward ACATs in the conven-
tional assays, two findings were later reported to amend its inhibitory
activities in rat liver microsomes and IC-21 murine macrophages after
the problem of protein binding were carefully addressed as shown in
Fig. 3. In response to these reports,55,59 we first simulated the unbound
fraction in the plasma (fp) of 3h and CI-1011 in human and rat using
the ADMET predictor as shown in Table 5.
4. Conclusion
We established our molecular design based on the double-induced
fit mechanism for the feasible development of an ACAT-1-selective in-
hibitor. Based on the SARs study, we selected an aortic-selective ACAT
inhibitor, 3h, that significantly decreased the lipid-accumulation areas
in the aortic arch without lowering the plasma TC level in Bio F1B
hamsters fed a diet that was high in fat and cholesterol. In this study, we
revealed that the plasma protein binding with substrates is crucial to
measure ACAT inhibitory activities in the assay. This information is
useful for interpreting the masked isoform-selectivity of 3h in the direct
inhibition of aortic ACAT. Compound 3h is a potential ACAT-1-selec-
tive inhibitor; however, some problems must be addressed, including its
high lipophilicity and extremely low water solubility, resulted in poor
oral absorption and a poor PK profile. The further development of the
ACAT-1-selective inhibitor, which exhibited much improved physical
and biological properties, will be reported in the next paper.
Hence, 3h can directly suppress atherosclerotic lesion at doses of 1
and 3 mg/kg, whereas CI-1011 affected atherosclerosis via its hypoli-
pidemic action even at a dose of 0.3 mg/kg as shown Fig. 10. This is
5. Experimental section
5.1. Chemistry
5.1.1. General methods
Commercially available reagents and solvents were used without
further purification. Thin-layer chromatography (TLC) analyses were
conducted using silica gel 60 F254 plates (Merck). 1H-nuclear magnetic
Figure 10. Comparison the line graphs of CI-1011 and 3h reflecting the re-
lationship between the plasma TC levels and lipid-accumulation areas. Their
ratios were calculated relative to that of the control.
resonance (NMR) spectra were recorded using
a JEOL JNM-LA
400 MHz and JEOL GSX-270. Tetramethylsilane was used as an internal
7

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
standard for spectra obtained in deuterated dimethyl sulfoxide
(DMSO‑d6) and CDCl3. Chemical shifts (δ) are given in parts per million
(ppm), and coupling constants (J values) are given in hertz (Hz) and
reported to the nearest 0.1 Hz. Infrared (IR) spectra were recorded
using a JASCO ALOR-III. Mass spectra were obtained on a JEOL MS-
BU20 mass spectrometer (MS). Low-resolution mass spectra were re-
corded on a JEOL JMS-D-300 MS. Elemental analyses (C, H, and N)
were performed using Yanaco MT-5. Melting points were determined in
open glass capillaries using a Buchi B-545 melting point apparatus.
5.1.3.3. 4-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
butanamide (3c). Compound 3c was prepared from
1 and 4-
bromobutyric acid in manner similar to that described for
a
compound 3a with a yield of 67% as colorless needles (acetone/
Et2O/n-hexane). Mp 141–142 °C; IR (KBr) cm−1: 3429, 3237, 1648,
1501, 1454; 1H NMR (DMSO‑d6) δ: 1.22 (12H, d, J = 6.8 Hz),
2.23–2.34 (2H, m), 2.61–2.72 (2H, m), 3.19 (2H, sept, J = 6.8 Hz),
3.55 (2H, t, J = 7.3 Hz), 7.21 (2H, m), 7.32 (1H, dd, J = 8.5, 6.8 Hz),
7.39 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.43 (lH, ddd, J = 8.0, 7.3, 1.5 Hz),
7.64–7.73 (2H, m), 8.95 (lH, br s); EIMS m/z 396 (M+); Anal. Calcd for
5.1.2. Materials
C23H28N2O2S: C, 69.66; H, 7.12; N, 7.06. Found: C, 69.86; H, 7.32; N,
2-mercaptooxazolo[5,4-b]pyridine, 2-mercaptooxazolo[5,4-c]pyr-
idine, 2-mercaptooxazolo[4,5-c]pyridine, 2-mercaptooxazolo[4,5-b])
7.06.
pyridine,
N-(2,6-disopropylphenyl)acetamide,
N-(2,6-dis-
5.1.3.4. 5-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
opropylphenyl)hexanamide, 2-(methythio)benzo[d]oxazole and 2-(bu-
tylthio)benzo[d]oxazole were commercially available. The comparative
compounds—RP-73163, CI-976, Dup-128 and CI-1011—were pre-
pared according to known procedures.40–42,55 These purities were de-
termined using high-performance liquid chromatography (HPLC) and
were > 97%.
CI-1011; HPLC purity: 98.97%. Column:Eclipse ZDB-C8; column
size 5 μm, 4.6 mm × 150 mm; mobile phase 10 mM SDS/5mM H3PO4/
CH3CN=25:75; flow rate 1.5 mL/min; column temperature 40 °C; wa-
velength 210 nm; retention time 7.91 min.
pentanamide (3d). Compound 3d was prepared from
1 and 5-
bromopentanoic acid in manner similar to that described for
a
compound 3a with a yield of 70% as colorless needles (acetone/
Et2O/n-hexane). Mp 122–123 °C; IR (KBr) cm−1: 3424, 3352, 1651,
1501, 1454; 1H NMR (DMSO‑d6) δ: 1.12 (12H, d, J = 6.8 Hz),
1.79–2.01 (4H, m), 2.40–2.50 (2H, m), 3.09 (2H, sept, J = 6.8 Hz),
3.41 (2H, t, J = 6.8 Hz), 7.11 (2H, m), 7.22 (1H, dd, J = 8.5, 6.8 Hz),
7.33 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.39 (lH, ddd, J = 8.0, 7.3, 1.5 Hz),
7.59–7.74 (2H, m), 8.76 (lH, br s); EIMS m/z 410 (M+); Anal. Calcd for
C
24H30N2O2S: C, 70.21; H, 7.36; N, 6.82. Found: C, 70.06; H, 7.49; N,
6.90.
5.1.3. Representative synthetic procedures for 3a-3j, 6a-6d, 7a, 8a, 9a,
10a, 11a, 12, 13, 14a, 15a, 17a, 18, 19, 20a, 22a
5.1.3.5. 6-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
5.1.3.1. 2-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)acetamide
(3a). To a stirred solution of 2,6-diisopropylaniline (1) (1.97 g, 10 mmol)
in DMF (30 mL) were added bromoacetic acid (2.08 g, 15 mmol), 1-(3-
dimethylaminopropy1)-3-ethylcarbodiimide hydrochloride (WSC) (2.10 g,
11 mmol) and hydroxylbenzo-triazole (HOBt) (1.49 g, 11 mmol) at 0 °C.
The mixture was stirred at rt for 12 h and then extracted with AcOEt.
The organic layer was washed with water, l-N HCl, saturated NaHCO3
solution and brine; dried over MgSO4; and evaporated under reduced
pressure. The residue was purified by column chromatography and
hexanamide (3e). Compound 3e was prepared from
1 and 6-
bromohexanoic acid in manner similar to that described for
a
compound 3a with a yield of 83% as colorless needles (Et2O/n-
hexane). Mp 116–118 °C; IR (KBr) cm−1: 3231, 2965, 1645, 1454; 1H
NMR (DMSO‑d6) δ: 1.12 (12H, d, J = 6.8 Hz), 1.40–1.95 (6H, m),
2.39–2.49 (2H, m), 3.09 (2H, sept, J = 6.8 Hz), 3.35 (2H, t, J = 6.8 Hz),
7.21 (1H, d, J = 8.3 Hz), 7.22 (1H, d, J = 7.1 Hz), 7.33 (lH, dd, J = 8.3,
7.1 Hz), 7.41 (lH, ddd, J = 8.1, 7.1, 2.4 Hz), 7.44 (lH, ddd, J = 8.1, 7.1,
2.4 Hz), 7.69 (lH, ddd, J = 8.1, 2.4, 0.9 Hz), 7.71 (lH, ddd, J = 7.1, 2.4,
0.9 Hz), 8.70 (lH, br s); EIMS m/z 424 (M+); Anal. Calcd for
eluted with n-hexane/acetone (5:1) to give
a solid, which was
recrystallized from n-hexane/Et2O to afford 2-bromo-N-(2,6-
diisopropylphenyl)acetamide (2a) (1.26 g, 42%) as colorless needle-
like crystals. To a solution of the obtained bromide (149 mg, 0.5 mmol)
and 2-mercaptobenzoxazole (83 mg, 0.55 mmol) in acetone (2 mL)
were added K2CO3 (76 mg, 0.55 mmol) and 18-crown-6 (13 mg,
0.05 mmol). The reaction mixture was stirred at rt for 3 h and then
diluted with saturated NH4Cl solution and extracted with AcOEt. The
organic layer was washed with water, dried over MgSO4 and
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography and eluted with n-hexane/Et2O (3:1) to
give crystals, which were recrystallized from CH2Cl2/Et2O/n-hexane to
afford 3a (156 mg, 85%) as colorless needles. Mp 156–158 °C; IR (KBr)
cm−1: 3272, 2963, 1665, 1135, 742; 1H NMR (CDC13) δ: 1.07 (12H, br
d, J = 6.8 Hz), 3.01 (2H, sept, J = 6.8 Hz), 4.11 (2H, s), 7.14 (2H, d,
J = 7.3 Hz), 7.23–7.34 (3H, m), 7.46–7.59 (2H, m), 8.67 (1H, br s);
electron impact MS (EIMS) m/z: 368 (M+); Anal. Calcd for
C
6.49.
25H32N2O2S: C, 70.72; H, 7.60; N, 6.60. Found: C, 70.42; H, 7.71; N,
5.1.3.6. 7-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
heptanamide (3f). Compound 3f was prepared from
bromoheptanoic acid in manner similar to that described for
1 and 7-
a
compound 3a with a yield of 79% as colorless needles (acetone/
Et2O/n-hexane). Mp 127–128 °C; IR (KBr) cm−1: 3425, 3249, 1648,
1526, 1454; 1H NMR (DMSO‑d6) δ: 1.26 (12H, d, J = 6.8 Hz),
1.56–2.05 (8H, m), 2.46–2.54 (2H, m), 3.23 (2H, sept, J = 6.8 Hz),
3.49 (2H, t, J = 7.3 Hz), 7.20–7.24 (2H, m), 7.34 (1H, dd, J = 8.5,
6.8 Hz), 7.42 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.46 (lH, ddd, J = 8.0, 7.3,
1.5 Hz), 7.66–7.77 (2H, m), 8.82 (lH, br s); EIMS m/z 438 (M+); Anal.
Calcd for C26H34N2O2S: C, 70.62; H, 7.84; N, 6.33. Found: C, 70.70; H,
7.97; N, 6.24.
C
21H24N2O2S: C, 68.45; H, 6.56; N, 7.60; S, 8.70. Found: C, 68.59; H,
6.55; N, 7.60; S, 8.57.
5.1.3.7. 8-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
octanamide (3g). Compound 3 g was prepared from
1 and 8-
5.1.3.2. 3-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
bromooctanoic acid in manner similar to that described for
a
propanamide (3b). Compound 3b was prepared from
1
and 3-
compound 3a with a yield of 75% as colorless needles (acetone/
Et2O/n-hexane). Mp 126–127 °C; IR (KBr) cm−1: 3433, 3263, 1652,
1505, 1454; 1H NMR (DMSO‑d6) δ: 1.27 (12H, d, J = 6.8 Hz),
1.51–2.02 (10H, m), 2.45–2.52 (2H, m), 3.24 (2H, sept, J = 6.9 Hz),
3.50 (2H, t, J = 7.3 Hz), 7.20–7.24 (2H, m), 7.35 (1H, dd, J = 8.5,
6.8 Hz), 7.43 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.46 (lH, ddd, J = 8.0, 7.3,
1.5 Hz), 7.65–7.77 (2H, m), 8.81 (lH, br s); EIMS m/z 452 (M+); Anal.
Calcd for C27H36N2O2S: C, 71.64; H, 8.02; N, 6.19. Found: C, 71.65; H,
8.15; N, 6.35.
bromopropanoic acid in manner similar to that described for
a
compound 3a with a yield of 55% as colorless needles (acetone/Et2O/n-
hexane). Mp 174–176 °C; IR (KBr) cm−1: 3432, 3244, 1652, 1502, 1454;
1H NMR (DMSO‑d6) δ: 1.14 (12H, d, J = 6.8 Hz), 2.97 (2H, t, J = 6.6 Hz),
3.13 (2H, sept, J = 6.8 Hz), 3.64 (2H, t, J = 6.6 Hz), 7.10–7.16 (2H, m),
7.23 (lH, dd, J = 8.5, 6.8 Hz), 7.27–7.37 (2H, m), 7.56–7.64 (2H, m), 8.96
(lH, br s); EIMS m/z 382 (M+); Anal. Calcd for C22H26N2O2S: C, 69.08; H,
6.85; N, 7.32. Found: C, 69.24; H, 6.91; N, 7.29.
8

K. Shibuya et al.
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5.1.3.8. 9-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
nonanamide (3h). To a stirred solution of 9-bromononanol (28.6 g,
128 mmol) in acetone (570 mL) was added dropwise 2-N Jones' reagent
(420 mL) at 0 °C. The reaction mixture was stirred at rt for 10 min and
then extracted with n-hexane (3 × 400 mL). The combined organic
layers were washed with water and extracted with a saturated K2CO3
solution (3 × 400 mL). The combined aqueous layers were made acidic
(pH l) with a 10% H2SO4 solution and extracted with AcOEt. The
organic layer was washed with water and brine and dried over MgSO4.
After filtration, the solvent was evaporated in vacuo to give 9-
bromononanoic acid (21 g, 71%) as a colorless solid. Compound 3h
was prepared from 1 and 9-bromononanoic acid in a manner similar to
that described for compound 3a with a yield of 77% as colorless needles
(acetone/Et2O/n-hexane). Mp 76–77 °C; IR (KBr) cm−1: 3424, 3253,
1650, 1504, 1455; 1H NMR (DMSO‑d6) δ: 1.28 (12H, d, J = 6.8 Hz),
1.46–2.05 (12H, m), 2.44–2.56 (2H, m), 3.25 (2H, sept, J = 6.9 Hz),
3.50 (2H, t, J = 7.3 Hz), 7.20–7.24 (2H, m), 7.37 (1H, dd, J = 8.5,
6.8 Hz), 7.43 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.47 (lH, ddd, J = 8.0, 7.3,
1.5 Hz), 7.66–7.77 (2H, m), 8.82 (lH, br s); 13C NMR (100 MHz, CDCl3)
δ:22.6, 23.6, 24.5(2C), 25.9, 28.4. 28.5, 28.8, 28.9, 29.1, 29.2, 29.3,
31.9, 32.3, 36.9, 109.8, 118.3, 123.4, 123.8, 123.9, 124.3, 128.4,
129.1, 131.1, 141.8, 146.2, 146.9, 151.8, 165.3, 172.4; EIMS m/z 466
(M+); Anal. Calcd for C28H38N2O2S: C, 72.06; H, 8.21; N, 6.00. Found:
C, 72.15; H, 8.39; N, 5.91.
chloride (TsCl) (1.65 g, 6.7 mmol) and N,N-dimethyl-4-aminopyridine
(DMAP) (24 mg 0.2 mmol) at 0 °C. The resulting mixture was stirred at
rt for 12 h. Water was added to the reaction mixture and extracted with
Et2O. The organic layer was washed with 5% KHSO4 solution, brine,
saturated NaHCO3 solution and brine, and dried over MgSO4. After
filtration, the solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography and eluted
with Et2O/n-hexane (1:3) to give 15-(4-toluenesulfonyloxy)-N-(2,6-
diisopropyl-phenyl)pentadecanamide (622 mg, 29%) and 15-chloro-N-
(2,6-diisopropylphenyl)penta-decanamide (177 mg, 10%). To
a
solution of the thus obtained chloride (164 mg, 0.38 mmol) and 2-
mercaptobenzoxazole (55 mg, 0.36 mmol) in DMF (2 mL) were added
K2CO3 (55 mg, 0.4 mmol) and 18-crown-6 (10 mg, 0.04 mmol). The
reaction mixture was stirred at 80 °C for 1 h, and then diluted with
water and extracted with Et2O. The organic layer was washed with
brine, and dried over MgSO4. After filtration, the solvent was
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography, and eluted with acetone/n-hexane (1:15)
to give a solid, which was recrystallized from Et2O/n-hexane to afford
3j (165 mg, 83%) as colorless needles. Mp 64–65 °C; IR (KBr) cm−1
:
3429, 3248, 1649, 1503, 1455; 1H NMR (DMSO‑d6) δ: 1.13 (12H, d,
J = 6.8 Hz), 1.25–1.72 (22H, m), 1.91 (2H, quint, J = 7.3 Hz),
2.29–2.38 (2H, m), 3.10 (2H, sept, J = 6.8 Hz), 3.33 (2H, t,
J = 7.3 Hz), 7.08–7.14 (2H, m), 7.21 (1H, dd, J = 8.5, 6.8 Hz),
7.25–7.35 (2H, m), 7.53–7.63 (2H, m), 8.67 (lH, br s); EIMS m/z 550
(M+); Anal. Calcd for C34H50N2O2S: C, 74.14; H, 9.15; N, 5.09. Found:
C, 74.10; H, 9.25; N, 5.09.
HPLC purity: 99.47%. Column:CAPCELPAK C18 UG120; column
size 3 μm, 4.6 mm × 100 mm; mobile phase H2O/CH3CN=40:60; flow
rate 1.0 mL/min; column temperature 40 °C; wavelength 210 nm; re-
tention time 16.72 min.
5.1.3.11. Methyl 2-((6-((2,6-diisopropylphenyl)amino)-6-oxohexyl)-thio)
5.1.3.9. 10-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
decanamide (3i). Compound 3i was prepared from 10-bromodecanol in
a manner similar to that described for compound 3h with a yield of
58% as colorless needles (acetone/Et2O/n-hexane). Mp 61–63 °C; IR
(KBr) cm−1: 3441, 3241, 1647, 1501, 1455; 1H NMR (DMSO‑d6) δ: 1.28
(12H, d, J = 6.8 Hz), 1.46–2.02 (14H, m), 2.44–2.56 (2H, m), 3.24 (2H,
sept, J = 6.9 Hz), 3.50 (2H, t, J = 7.3 Hz), 7.23–7.27 (2H, m), 7.37 (1H,
dd, J = 8.5, 6.8 Hz), 7.44 (lH, ddd, J = 8.0, 7.3, 1.5 Hz), 7.47 (lH, ddd,
J = 8.0, 7.3, 1.5 Hz), 7.69–7.79 (2H, m), 8.93 (lH, br s); EIMS m/z 480
(M+); Anal. Calcd for C29H40N2O2S: C, 72.46; H, 8.39; N, 5.83. Found:
C, 72.24; H, 8.65; N, 5.82.
benzo[d]oxazole-4-carboxylate
(12a). To
a
solution
of
3-
hydroxyanthranilic acid (1.93 g, 12.6 mmol) in saturated HCl MeOH
(40 mL) was added p-toluenesulfonic acid (p-TsOH) monohydrate
(95 mg, 0.5 mmol). The reaction mixture was stirred at reflux
temperature for 12 h. The solvent was evaporated under reduced
pressure. The residue was dissolved in AcOEt. The organic layer was
washed with saturated KHCO3 solution, water and brine, and dried over
MgSO4. After filtration, the solvent was evaporated under reduced
pressure. The residue was dissolved in Et2O. The insoluble material was
filtered off. The filtrate was concentrated in vacuo. To a solution of
methyl 3-hydroxyanthranilate (1.22 g, 7.37 mmol) in CH2Cl2 (10 mL)
was added Et3N (1.85 g, 18.25 mmol), followed by dropwise addition of
a solution of thiophosgene (923 mg, 8.03 mmol) in CH2Cl2 (2 mL) with
stirring at ambient temperature for 5 min. The reaction mixture was
concentrated in vacuo. The residue was dissolved in AcOEt. The organic
layer was washed with 1-N HCl, water and brine and dried over MgSO4.
After filtration, the solvent was evaporated under reduced pressure to
give a solid, which was crystallized from AcOEt/n-hexane to afford
methyl 2-mercaptobenzo[d]oxazole-4-carboxylate (7a) (1.28 g, 84%).
2-bromo-N-(2,6-diisopropylphenyl)hexanamide (2e) was obtained from
1 using 6-bromohexanoic acid in place of bromoacetic acid in a manner
similar to that described for compound 3a. To a stirred solution of the
thus obtained thiol (1.05 g, 5.0 mmol) and 2e (1.77 g, 5.0 mmol) in
DMF (20 mL) were added K2CO3 (1.04 g, 7.5 mmol) and 18-crown-6
(132 mg, 0.5 mmol). The reaction mixture was stirred at 80 °C for 2 h
and diluted with water. The organic layer was extracted with Et2O,
washed with water and brine, and dried over MgSO4. After filtration,
the filtrate was concentrated in vacuo. The residue was purified by silica
gel column chromatography and eluted with acetone/n-hexane (1:5) to
give a solid, which was recrystallized from acetone/n-hexane to afford
5.1.3.10. 15-(Benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)-
pentadecanamide (3j). 15-Pentadecanolactone (10.0 g, 42 mmol), KOH
(10.0 g, 0.18 mmol) and n-Bu4NBr (1.34 g, 4.2 mmol) were dissolved in
a mixed solvent of tetrahydrofuran (THF) (100 mL) and water (30 mL).
The reaction mixture was heated under reflux for 12 h·THF solvent was
evaporated under reduced pressure. The aqueous layer was made acidic
(pH = l) with 10% H2SO4 solution and then extracted with Et2O. The
organic layer was washed with water and brine and dried over MgSO4.
After filtration, the solvent was evaporated under reduced pressure. The
residue was crystallized from acetone to give 15-hydroxypentadecanoic
acid (10.4 g, 97%) as colorless crystals. To a solution of the thus
obtained carboxylic acid (15.2 g, 20 mmol) and 1 (5.3 g, 30 mmol) in
DMF (40 mL) were added WSC (4.2 g, 22 mmol) and HOBt (3.0 g,
22 mmol) at rt. The resulting mixture was stirred at rt for 12 h. Water
was added to the reaction mixtures, and the formed precipitate was
filtered off and extracted with CHCl3. The organic layer was washed
with 1-N HCl solution and brine and dried over MgSO4. After filtration,
the solvent was evaporated under reduced pressure. The residue was
extracted with Et2O and the insoluble material was filtered off. The
filtrate was concentrated in vacuo. The residue was purified by silica gel
column chromatography and eluted with acetone/n-hexane (1:5) to
give 15-hydroxy-N-(2,6-diisopropylphenyl)pentadecanamide (1.9 g,
23%) as a white solid. To a solution of the thus obtained alcohol
(1.64 g, 3.9 mmol) in pyridine (15 mL) were added p-toluenesulfonyl
12a (1.84 g, 76%) as colorless needles. Mp 131–132 °C; IR (KBr) cm−1
:
3408, 3221, 3172, 2965, 1713, 1641; lH-NMR (DMSO‑d6) δ: 1.13 (12H,
d, J = 6.8 Hz), 1.53–1.97 (6H, m), 2.37–2.46 (2H, m), 3.10 (2H, sept,
J = 6.8 Hz), 3.40 (2H, t, J = 7.2 Hz), 3.91 (3H, s), 7.08 (lH, d,
J = 8.8 Hz), 7.09 (lH, d, J = 6.6 Hz), 7.19 (lH, dd, J = 8.8, 6.6 Hz),
7.34 (lH, t, J = 8.1 Hz), 7.74 (lH, dd, J = 8.1, 1.2 Hz), 7.82 (lH, dd,
9

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
J = 8.1, 1.2 Hz), 8.60 (lH, br s); EIMS m/z: 482 (M+); Anal. Calcd for
C27H34N2O4S: C, 67.19; H, 7.10; N 5.80; S, 6.64. Found: C, 67.29; H,
7.21; N, 5.71; S, 6.62.
EIMS m/z: 482 (M+); Anal. Calcd for C27H34N2O4S: C, 67.19; H, 7.10; N
5.80; S, 6.64. Found: C, 67.42; H, 7.21; N, 5.84; S, 6.49.
5.1.3.15. 2-((6-((2,6-Diisopropylphenyl)amino)-6-oxohexyl)thio)-benzo
5.1.3.12. Methyl 2-((6-((2,6-diisopropylphenyl)amino)-6-oxohexyl)-thio)
benzo[d]oxazole-5-carboxylate (12b). Compound 6b was prepared in
the same manner as in preparation of 6a except for the use of 3-amino-
4-hydroxybenzoic acid instead of 3-hydroxyanthranic acid, with a yield
of 89% as colorless needles.
[d]oxazole-4-carboxylic acid (13a). To a stirred solution of 12a
(965 mg, 2.0 mmol) in THF (10 mL) were added t-BuOH (4 mL) and a
solution of LiOH (336 mg, 8 mmol) in water (4 mL), followed by stirring
at rt for 12 h. The solvent was evaporated under reduced pressure. The
residue was extracted with AcOEt. The organic layer was washed with
0.5-N HCl solution, water and brine, and dried over MgSO4. After
filtration, the solvent was evaporated under reduced pressure to give a
solid, which was recrystallized from acetone/n-hexane to afford 13a
l
Mp 104–106 °C; IR (KBr) cm−1: 3417, 3251, 2960, 1720, 1651; H-
NMR (DMSO‑d6) δ: 1.11 (12H, d, J = 6.8 Hz), 1.47–1.95 (6H, m),
2.34–2.38 (2H, m), 3.07 (2H, sept, J = 6.8 Hz), 3.38 (2H, t, J = 7.1 Hz),
3.88 (3H, s), 7.08 (lH, d, J = 8.5 Hz), 7.09 (lH, d, J = 7.1 Hz), 7.20 (lH,
dd, J = 8.5, 7.1 Hz), 7.67 (lH, d, J = 8.5 Hz), 7.93 (lH, dd, J = 8.5,
2.4 Hz), 8.12 (lH, dd, J = 2.4, 1.2 Hz), 8.68 (lH, br s); EIMS m/z: 482
(M+); Anal. Calcd for C27H34N2O4S: C, 67.19; H, 7.10; N 5.80; S, 6.64.
Found: C, 67.32; H, 7.11; N, 5.81; S, 6.62.
(799 mg, 85%) as colorless crystals. Mp 174–176 °C; IR (KBr) cm−1
:
l
3421, 3244, 2963, 1649, 1493; H-NMR (DMSO‑d6) δ: 1.12 (12H, d,
J = 6.9 Hz), 1.47–1.88 (6H, m), 2.33–2.40 (2H, m), 3.09 (2H, sept,
J = 6.8 Hz), 3.34 (2H, t, J = 7.1 Hz), 7.08 (1H, d, J = 8.5 Hz), 7.09 (lH,
d, J = 6.6 Hz), 7.19 (lH, dd, J = 8.5, 6.6 Hz), 7.34 (1H, t, J = 7.9 Hz),
7.74 (1H, dd, J = 7.9, 0.5 Hz), 7.80 (1H, dd, J = 7.9, 0.5 Hz), 8.76 (lH,
br s); EIMS m/z: 468 (M+); Anal. Calcd for C26H32N2O4S: C, 66.64; H,
6.68; N 5.98; S, 6.84. Found: C, 66.36; H, 6.86; N, 6.03; S, 6.64.
5.1.3.13. Methyl 2-((6-((2,6-diisopropylphenyl)amino)-6-oxohexyl)-thio)
benzo[d]oxazole-6-carboxylate (12c). Compound 6c was prepared in
the same manner as in preparation of 6a except for the use of 4-amino-
3-hydroxybenzoic acid instead of 3-hydroxyanthranic acid, with a yield
of 82% as colorless needles.
5.1.3.16. N-(2,6-Diisopropylphenyl)-6-((4-(hydroxymethyl)benzo[d]-
oxazol-2-yl)thio)hexanamide (14a). To
a stirred solution of 13a
l
Mp 157–158 °C; IR (KBr) cm−1: 3412, 3236, 2959, 1714, 1647; H-
(613 mg, 1.5 mmol) in THF (10 mL) was added Et3N (200 mg,
1.98 mmol), followed by dropwise addition of methyl chloroformate
(195 mg, 1.80 mmol) at 0 °C for 30 min. The appeared Et3N%HCl salt
was filtered off. To the resulting filtrate was dropwisely added a
solution of NaBH4 (227 mg, 6.0 mmol) in water (1.5 mL). The
reaction mixture was stirred at rt for 30 min, diluted with water and
extracted with AcOEt. The organic layer was washed with water and
brine, and dried over MgSO4. After filtration, the solvent was
concentrated in vacuo. The residue was purified by silica gel column
chromatography, and eluted with acetone/n-hexane (2:5 to 3:5) to give
a solid, which was recrystallized from acetone/n-hexane to afford 14a
(468 mg, 69%) as colorless crystals. Mp 93–95 °C; IR (KBr) cm−1: 3358,
3243, 2963, 1646, 1506; lH-NMR (DMSO‑d6) δ: 1.13 (12H, d,
J = 6.8 Hz), 1.50–1.92 (6H, m), 2.33–2.41 (2H, m), 3.09 (2H, sept,
J = 6.8 Hz), 3.35 (2H, t, J = 7.1 Hz), 4.67 (1H, t, J = 5.5 Hz), 4.83 (2H,
d, J = 5.5 Hz), 7.08 (1H, d, J = 8.1 Hz), 7.09 (1H, d, J = 6.6 Hz), 7.19
(lH, dd, J = 8.1, 6.6 Hz), 7.24 (lH, t, J = 7.8 Hz), 7.37 (1H, dd, J = 7.8,
1.4 Hz), 7.40 (1H, dd, J = 7.8, 1.4 Hz), 8.66 (lH, br s); EIMS m/z: 454
(M+); Anal. Calcd for C26H34N2O3S: C, 68.69; H, 7.54; N 6.16; S, 7.05.
Found: C, 68.70; H, 7.57; N, 6.15; S, 7.01.
NMR (DMSO‑d6) δ: 1.12 (12H, d, J = 6.8 Hz), 1.51–1.94 (6H, m),
2.32–2.38 (2H, m), 3.09 (2H, sept, J = 6.8 Hz), 3.39 (2H, t, J = 7.1 Hz),
3.88 (3H, s), 7.08 (lH, d, J = 8.6 Hz), 7.09 (lH, d, J = 6.6 Hz), 7.20 (lH,
dd, J = 8.6, 6.6 Hz), 7.65 (lH, dd, J = 8.3, 0.5 Hz), 7.95 (lH, dd,
J = 8.3, 1.7 Hz), 8.09 (lH, dd, J = 1.7, 0.5 Hz), 8.67 (lH, br s); EIMS m/
z: 483 (M+ +1); Anal. Calcd for C27H34N2O4S: C, 67.19; H, 7.10; N
5.80; S, 6.64. Found: C, 67.35; H, 7.13; N, 5.78; S, 6.47.
5.1.3.14. Methyl 2-((6-((2,6-diisopropylphenyl)amino)-6-oxohexyl)-thio)
benzo[d]oxazole-7-carboxylate (12d). To a solution of 3-aminosalicylic
acid (1.53 g, 10 mmol) in EtOH (70 mL) was added potassium O-ethyl
dithiocarbonate (3.2 g, 20 mmol). The reaction mixture was stirred at
reflux temperature for 2.5 h and allowed to cool to rt. The solvent was
evaporated under reduced pressure. The residue was adjusted to acidity
by adding 1-N HCl solution. The resulting deposited precipitate was
filtered and dried to give 2-mercaptobenzo[d]oxazole-7-carboxylic acid
(6d) (1.3 g, 67%) as a brown powder. To a solution of the thus obtained
carboxylic acid (976 mg, 5.0 mmol) in MeOH (50 mL) was added p-
TsOH (95 mg, 0.5 mmol). The reaction mixture was stirred at reflux
temperature for 72 h. The solvent was evaporated under reduced
pressure. The residue was extracted with AcOEt. The organic layer
was washed with water and brine, and dried over MgSO4. After
filtration, the solvent was evaporated under reduced pressure to give
a solid, which was crystallized from acetone/n-hexane to afford methyl
2-mercaptobenzo[d]oxazole-7-carboxylate (7d) (0.955 g, 91%) as a
brown powder. To a stirred solution of the thus obtained thiol
(837 mg, 4.0 mmol) and 2e (1.42 g, 4.0 mmol) in DMF (20 mL) were
added K2CO3 (608 mg, 4.4 mmol) and 18-crown-6 (106 mg, 0.4 mmol).
The reaction mixture was stirred at 80° C for 2 h and diluted with water
and AcOEt. The organic layer was washed with water and brine, and
dried over MgSO4. After filtration, the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography, and eluted with acetone/n-hexane (1:5) to give a
solid, which was crystallized from acetone/n-hexane to afford 12d
5.1.3.17. N-(2,6-Diisopropylphenyl)-6-((4-((N,N-dimethylamino)-methyl)
benzo[d]oxazol-2-yl)thio)hexanamide (15a). To a stirred solution of 14a
(182 mg, 0.4 mmol) in THF (5 mL) were added Et3N (81 mg, 0.8 mmol)
and DMAP (9.8 mg, 0.08 mmol), followed by dropwise addition of MsCl
(64 mg, 0.56 mmol) at 0 °C. The reaction mixture was stirred for 30 min
and diluted with AcOEt. The organic layer was washed with 0.5-N HCl
solution, water and brine and dried over MgSO4. After filtration, the
solvent was evaporated under reduced pressure. To a solution of the
resulting residue (209 mg) in THF (7 mL) was added aqueous 40% N,N-
dimethylamine solution (180 mg, 1.6 mmol). The reaction mixture was
stirred at reflux temperature for 1 h and diluted with AcOEt. The
organic layer was washed with saturated NaHCO3 solution, water and
brine, and dried over MgSO4. After filtration, the solvent was
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography, and eluted with acetone/n-hexane (3: 5),
followed by eluting with CHCl3: saturated NH3/MeOH (19:1) to give a
solid, which was recrystallized from acetone/n-hexane to afford 15a
(1.93 g, 99%) as colorless needles. Mp 118–119 °C; IR (KBr) cm−1
:
l
3420, 2963, 1719, 1645, 1507; H-NMR (DMSO‑d6) δ: 1.12 (12H, d,
J = 6.9 Hz), 1.56 (2H, quint, J = 7.3 Hz), 1.73 (2H, quint, J = 7.3 Hz),
1.90 (2H, quint, J = 7.3 Hz), 2.37 (2H, t, J = 7.3 Hz), 3.08 (2H, sept,
J = 6.8 Hz), 3.39 (2H, t, J = 7.3 Hz), 3.93 (3H, s), 7.09 (2H, d,
J = 7.6 Hz), 7.19 (lH, t, J = 7.6 Hz), 7.42 (lH, t, J = 7.9 Hz), 7.80
(1H, dd, J = 7.5, 0.9 Hz), 7.81 (1H, dd, J = 7.5, 0.9 Hz), 8.68 (lH, br s);
(137 mg, 71%) as colorless crystals. Mp 113–115 °C; IR (KBr) cm−1
3435, 3237, 2964, 1647, 1506; H-NMR (DMSO‑d6) δ: 1.12 (12H, d,
J = 6.8 Hz), 1.52–1.93 (6H, m), 2.24 (6H, s), 2.32–2.40 (2H, m), 3.09
(2H, sept, J = 6.8 Hz), 3.35 (2H, t, J = 7.2 Hz), 3.77 (2H, s), 7.08 (1H,
:
l
10

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
d, J = 8.3 Hz), 7.09 (1H, d, J = 6.8 Hz), 7.19 (lH, dd, J = 8.3, 6.8 Hz),
7.22 (lH, t, J = 7.6 Hz), 7.28 (1H, dd, J = 7.6, 1.7 Hz), 7.41 (1H, dd,
J = 7.6, 1.7 Hz), 8.65 (lH, br s); EIMS m/z: 481 (M+); Anal. Calcd for
(538 mg, 65%) as a yellowish brown crystal. To a solution of the thus
obtained thiol (453 mg, 2.5 mmol) and 2e (885 mg, 2.5 mmol) in DMF
(8 mL) were added K2CO3 (387 mg, 2.8 mmol) and 18-crown-6 (66 mg,
0.25 mmol). The reaction mixture was stirred at 80° C for 3 h and
diluted with water and AcOEt. The organic layer was washed
sequentially with water and brine, and dried over MgSO4. The
solvent was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography, and eluted with
C
28H39N3O2S: C, 69.82; H, 8.16; N 8.72; S, 6.66. Found: C, 69.76; H,
8.23; N, 8.64; S, 6.72.
5.1.3.18. N-(2,6-Diisopropylphenyl)-6-((4-hydroxybenzo[d]oxazol-2-yl)
thio)hexanamide (17a). To
a stirred solution of 3-benzyloxy-2-
nitrophenol (334 mg, 1.36 mmol) in AcOH (3 mL) was added
portionwisely Zn dust (1.78 g, 27.2 mmol) at rt. The reaction mixture
was stirred at rt for 1 h and diluted with water and AcOEt. The organic
layer was washed with saturated NaHCO3 solution, water and brine,
and dried over anhydrous MgSO4. After filtration, the solvent was
concentrated in vacuo. The residue was purified by silica gel column
chromatography, and eluted with acetone/n-hexane (3:20 to 1:5) to
give an oil, which was solidified from acetone/n-hexane to afford 2-
amino-3-(benzyloxy)phenol (155 mg, 53%) as brown powdery crystals.
To a solution of the aminophenol (135 mg, 0.627 mmol) in EtOH
(10 mL) was added potassium O-ethyl dithiocarbonate (151 mg,
0.941 mmol) at rt. The reaction mixture was stirred at reflux tem-
perature for 24 h and allowed to cool to rt. The solvent was evaporated
under reduced pressure. The residue was adjusted to acidity by adding
with 1-N HCl solution. The resulting precipitate was filtered and dried
to give 4-(benzyloxy)benzo[d]oxazole-2-thiol (9a) (143 mg, 89%). To a
stirred solution in DMF (3 mL) of the obtained thiol (135 mg,
0.525 mmol) and 2e (186 mg, 0.525 mmol) were added K2CO3 (110 mg,
0.788 mmol) and 18-crown-6 (14 mg, 0.053 mmol). The reaction mix-
ture was stirred at 80° C for 90 min and diluted with water and Et2O.
The organic layer was washed with water and brine, and dried over
MgSO4. After filtration, the solvent was evaporated under reduced
pressure. The residue was purified by silica gel column chromato-
graphy, and eluted with acetone/n-hexane (3:20) to give a solid, which
was recrystallized from acetone/n-hexane to afford 6-((4-(benzyloxy)
benzo[d]oxazol-2-yl)thio)-N-(2,6-diisopropylphenyl)hexanamide
(206 mg, 74%) as colorless crystals. The obtained benzyl ether (188 mg,
0.354 mmol) was dissolved in CF3COOH (5 mL) with cooling in an ice
bath, followed by addition of thiophenol (440 mg, 3.54 mmol) with
stirring for 2 min. The reaction mixture was stirred at rt for 12 h.
Furthermore, thiophenol (440 mg, 3.54 mmol) was added to this mix-
ture with stirring for 24 h. The solvent was evaporated under reduced
pressure. The residue was diluted with water and extracted with AcOEt.
The organic layer was washed sequentially with saturated NaHCO3
solution, water and brine, and dried over MgSO4. After filtration, the
solvent was concentrated in vacuo. The residue was purified by silica gel
column chromatography, and eluted with acetone/n-hexane (1:5 to
3:10) to give a solid, which was recrystallized from acetone/n-hexane
to afford 17a (108 mg, 69%) as colorless crystals. Mp 160–161 °C; IR
(KBr) cm−1: 3226, 2963, 1652, 1480, 1036; lH-NMR (DMSO‑d6) δ: 1.12
(12H, d, J = 6.8 Hz), 1.50–1.91 (6H, m), 2.30–2.40 (2H, m), 3.09 (2H,
sept, J = 6.8 Hz), 3.33 (2H, t, J = 7.1 Hz), 6.72 (1H, dd, J = 8.1,
1.2 Hz), 6.97 (lH, dd, J = 8.1, 1.2 Hz), 7.06 (lH, t, J = 8.1 Hz), 7.09
(1H, d, J = 8.5 Hz), 7.09 (1H, d, J = 6.6 Hz), 7.19 (1H, dd, J = 8.5,
6.6 Hz), 8.66 (lH, br s), 9.57 (lH, br s); EIMS m/z: 440 (M+); Anal. Calcd
for C25H32N2O3S: C, 68.15; H, 7.32; N 6.36; S, 7.28. Found: C, 68.05; H,
7.33; N, 6.34; S, 7.18.
acetone/n-hexane (1:5 to 3:10) to give a solid, which was
recrystallized from acetone/ether/n-hexane to afford 18 (908 mg,
82%) as colorless needle-like crystals. Mp 112–114 °C; IR (KBr) cm−1
:
l
3427, 3230, 2964, 1644, 1502; H-NMR (DMSO‑d6) δ: 1.12 (12H, d,
J = 6.8 Hz), 1.46–1.92 (6H, m), 2.36 (2H,m), 2.41 (3H, s), 3.05 (2H,
sept, J = 6.8 Hz), 3.32 (2H, t, J = 7.1 Hz), 7.08 (lH, d, J = 8.8 Hz), 7.09
(lH, d, J = 6.6 Hz), 7.12 (lH, dd, J = 8.1, 2.4 Hz), 7.20 (lH, dd, J = 8.8,
6.6 Hz), 7.36 (lH, dd, J = 2.4, 0.7 Hz), 7.43 (lH, d, J = 8.1 Hz), 8.68
(lH, br s); EIMS m/z (relative intensity): 438 (M+); Anal. Calcd for
C
26H34N2O2S: C, 71.20; H, 7.81; N, 6.39; S, 7.31. Found: C, 71.08; H,
7.99; N, 6.27; S, 7.04.
5.1.3.20. N-(2,6-diisopropylphenyl)-6-((5-(trifluoromethyl)benzo[d]-
oxazol-2-yl)thio)hexanamide (19). To
a
solution of 2-nitro-4-
(trifluoromethyl)phenol (1.25 g, 6.0 mmol) in EtOH (30 mL) was
added portionwisely 10% palladium-carbon (Pd/C) (0.5 g). The
reaction mixture was stirred at rt. for 3 h under a hydrogen gas
atmosphere. After completion of the reaction, the Pd/C catalyst was
filtered
off.
To
the
resulting
solution
of
2-amino-4-
trifluoromehylphenol was added potassium O-ethyl dithiocarbonate
(1.06 g, 6.6 mmol). The reaction mixture was stirred at reflux
temperature for 4 h and allowed to cool to rt. The solvent was
evaporated under reduced pressure. The residue was adjusted to
acidity by adding 1-N HCl solution, and extracted with AcOEt. The
organic layer was washed with brine and dried over MgSO4. The solvent
was evaporated under reduced pressure to give 7-(trifluoromethyl)-
benzo[d]oxazole-2-thiol (0.78 g, 59%).
Compound 19 was prepared from 7-(trifluoromethyl)benzo[d]-ox-
azole-2-thiol (11) in the same manner as in the preparation of 18 with a
yield of 86% as colorless powdery crystals. Mp 98-99° C; IR (KBr) cm−1
:
3232, 2964, 1648 1500; lH-NMR (DMSO‑d6) δ: 1.12 (12H, d,
J = 6.8 Hz), 1.56 (2H, m), 1.72 (2H, m), 1.88 (2H, m), 2.36 (2H, m),
3.08 (2H, sept, J = 6.8 Hz), 3.40 (2H, t, J = 7.0 Hz), 7.10 (2H, d,
J = 7.3 Hz), 7.20 (lH, t, J = 7.3 Hz), 7.63 (lH, d, J = 8.6 Hz), 7.79 (lH,
d, J = 8.6 Hz), 7.92 (lH, s), 8.70 (lH, br s); EIMS m/z (relative in-
tensity): 492 (M+); Anal. Calcd for C26H31F3N2O2S: C, 63.40; H, 6.34;
N, 5.69. Found: C, 63.47; H, 6.62; N, 5.45.
5.1.3.21. N-(2,6-Diisopropylphenyl)-6-((4-(N,N-dimethylamino)benzo-[d]
oxazol-2-yl)thio)hexanamide (20a). To a stirred solution of 2-amino-3-
nitrophenol (1.54 g, 10 mmol) in THF (50 mL) was added portionwise a
60% suspension of NaH in mineral oil (528 mg, 22 mmol) at 0 °C,
followed by heating at 60° C for 5 min. To the resulting mixture was
added portionwisely thiophosgene (1.38 g, 12 mmol) at rt. The reaction
mixture was stirred at 60° C for 5 min and concentrated in vacuo. The
residue was acidified with 1-N HCl solution. The resulting solid was
filtered off and purified by silica gel column chromatography, and
eluted with CHCl3, followed by MeOH/CHCl3 (l:100 to 1:50) to give a
solid, which was crystallized from acetone/n-hexane to afford 4-
nitrobenzo[d]oxazole-2-thiol (8a) (807 mg, 41%) as yellow crystals.
To a stirred solution in DMF (6 mL) of the thus obtained thiol (216 mg,
1.1 mmol) and 2e (390 mg, 1.1 mmol) were added K2CO3 (228 mg,
1.65 mmol) and 18-crown-6 (29 mg, 0.11 mmol). The resulting mixture
was stirred at 80° C for 2 h and diluted with water and AcOEt. The
organic layer was washed with water and brine, and dried over MgSO4.
After filtration, the solvent was concentrated in vacuo. The residue was
purified by silica gel column chromatography, and eluted with acetone/
n-hexane (1:5 to 3:10) to give a solid, which was recrystallized from
5.1.3.19. N-(2,6-diisopropylphenyl)-6-((6-methylbenzo[d]oxazol-2-yl)-
thio)hexanamide (18). To
a solution of 2-amino-6-methylphenol
(636 mg, 4.57 mmol) in EtOH (10 mL) was added potassium O-ethyl-
dithiocarbonate (801 mg, 5 mmol). The reaction mixture was stirred at
reflux temperature for 2 h and allowed to cool to rt. The solvent was
evaporated under reduced pressure. The residue was adjusted to acidity
by adding 1-N HCl solution, and extracted with AcOEt. The organic
layer was washed with brine and dried over MgSO4. The solvent was
evaporated under reduced pressure. The resulting solid was crystallized
from acetone/n-hexane to give 6-methylbenzo[d]oxazole-2-thiol (10)
11

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
acetone/n-hexane to afford N-(2,6-diisopropylphenyl)-6-((4-nitrobenzo
[d]oxazol-2-yl)thio)hexanamide (16a) (304 mg, 59%) as pale yellow
crystals (mp 132–133 °C). To a solution of the thus obtained nitro
compound (386 mg, 0.82 mmol) in AcOH (8 mL) was added Zn dust
(1.07 g, 16.4 mmol) with cooling in an ice bath. The reaction mixture
was stirred at rt for 5 min and diluted with AcOEt. The insoluble
material was filtered through a Celite pad. The filtrate was neutralized
by adding with saturated NaHCO3 solution. The organic layer was
washed with saturated NaHCO3 solution, water and brine, and dried
over MgSO4. After filtration, the solvent was concentrated in vacuo. The
residue was purified by silica gel column chromatography, and eluted
with acetone/CH2Cl2/n-hexane (1:4:4) to give a solid, which was
for 8 weeks and intestinal microsomes from normal NZW rabbits via the
method of Heider et al. (J. Lipid Res., 1983, 24, 1127–1134) with some
modifications. The test compound was dissolved in DMSO (Dojindo
Molecular Technologies) to yield the final desired concentrations
(0.001, 0.01, 0.1, 1 and 10 μmol/L). The compound solution (2 μL) was
added to 88 μL of 0.15-M phosphate buffer (pH 7.4) containing [14C]-
oleoyl-CoA (Amersham International, 40 μM, 60,000 dpm) and bovine
serum albumin (BSA) (Tissue Culture Biologicals, 2.4 mg/mL) complex
and allowed to incubate at 37 °C for 5 min. After the microsomal sus-
pension in phosphate buffer (10 μL) was added, the reaction mixture
was incubated at 37 °C for 5 min (arterial) or 3 min (intestinal). The
reaction was terminated by the addition of 3 mL of CHCl3/MeOH (2:1)
and 0.5 mL of 0.04-N HCl. After shaking, the organic layer was col-
lected, and the solvent was evaporated. The extracted lipids were se-
parated on a silica gel TLC plate (Merck) using a mixed solution of n-
hexane/Et2O/AcOH (75:25:1). The labeled cholesteryl oleate was de-
tected by a BAS2000 (Fujifilm Corporation). The inhibitory activity was
determined as the concentration at which the inhibitors reduced the
formation of cholesteryl oleate by 50% (IC50).
recrystallized
from
acetone/n-hexane
to
afford
N-(2,6-
diisopropylphenyl)-6-(4-aminobenzoxazol-2-ylthio)hexanamide
(231 mg, 64%) as pale yellow crystals (mp 167–168 °C). To a stirred
solution of the thus obtained amino compound (273 mg, 0.621 mmol)
in MeCN (7.5 mL) were added a solution of aqueous 37% HCHO
solution (504 mg, 5.78 mmol) in MeCN (7.5 mL) and a suspension of
NaBH3CN (156 mg, 2.48 mmol) in MeCN (1 mL), followed by adding
with AcOH (48 μL) at rt. The reaction mixture was stirred at rt for
30 min and concentrated in vacuo. The residue was diluted with water
and extracted with AcOEt. The organic layer was washed with water
and brine, and dried over MgSO4. After filtration, the solvent was
concentrated in vacuo. The residue was purified by silica gel column
chromatography, and eluted with acetone/n-hexane (2:5) to give a
solid, which was recrystallized from acetone/n-hexane to afford 20a
6.1.2. Cellular assay
J774A.1 cells (Dainippon Pharmaceutical Company) were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich
Company) containing 10% fetal bovine serum (FBS, Tissue Culture
Biomedical) in a 24-well plate (Nalge Nunc International Corporation).
The cells were incubated with a test compound dissolved in DMSO (5
μL) and 25-hydroxycholesterol (Sigma-Aldrich Company, 10 μg/mL) in
0.5 mL of the medium for 18 h. After removing the medium and
washing twice with phosphate-buffered saline, lipids were extracted
with 1.5 mL of i-PrOH/n-hexane (3:2). The extracts were dried, and the
residues were dissolved in 0.2 mL of i-PrOH containing 10% Triton X-
100 (Sigma-Aldrich Company). The FC and TC contents were measured
by a Cholesterol E-test and a Free Cholesterol E-test (Wako Pure
Chemical Industries), respectively. Proteins were solubilized with
0.25 mL of 2-N NaOH at 37 °C for 30 min and determined via the bi-
cinchoninic acid (BCA) protein assay (Pierce). To calculate the CE
contents, FC was subtracted from TC. The inhibitory activity was de-
termined as the concentration at which the accumulation of CE was
reduced by 50% (IC50).
(177 mg, 61%) as colorless crystals. Mp 129–130 °C; IR (KBr) cm−1
:
3435,3226, 2967, 1645, 1524; lH-NMR (DMSO‑d6) δ: 1.13 (12H, d,
J = 7.1 Hz), 1.51–1.92 (6H, m), 2.32–2.40 (2H, m), 3.09 (2H, sept,
J = 7.1 Hz), 3.16 (6H, s), 3.30 (2H, t, J = 7.1 Hz), 6.49 (1H, dd,
J = 8.8, 0.7 Hz), 6.84 (lH, dd, J = 8.1, 0.7 Hz), 7.07 (lH, d,
J = 8.1 Hz), 7.08 (1H, dd, J = 8.6 Hz), 7.09 (1H, d, J = 6.4 Hz), 7.19
(1H, dd, J = 8.6, 6.4 Hz), 8.63 (lH, br s); EIMS m/z: 467 (M+); Anal.
Calcd for C27H37N3O2S: C, 69.34; H, 7.97; N 8.99; S, 6.86. Found: C,
69.42; H, 8.10; N, 8.85; S, 6.77.
5.1.3.22. N-(2,6-Diisopropylphenyl)-6-(oxazolo[4,5-b]pyridin-2-ylthio)-
hexanamide (22a). To a stirred solution of oxazolo[4,5-b]-pyridine-2-
thiol (21a) (64 mg, 0.42 mmol) and 2e (150 mg, 0.42 mmol) in DMF (3
mL) were added K2CO3 (64 mg, 0.47 mmol) and 18-crown-6 (11 mg,
0.04 mmol). The reaction mixture was stirred at 80° C for 4 h and
diluted with water and AcOEt. The organic layer was washed with
water and dried over MgSO4. After filtration, the solvent was
concentrated in vacuo. The residue was purified by silica gel column
chromatography, and eluted with AcOEt/n-hexane (1:2) to give a solid,
which was recrystallized from AcOEt/n-hexane to afford 22a (49 mg,
27%) as colorless needles. Mp 94–95 °C; IR (KBr) cm−1: 3230, 2965,
All procedures performed on animals in this study were in ac-
cordance with established guidelines and regulations and were re-
viewed and approved by the Committee on the Ethics of Animal
Experiments, Kowa Company Ltd.
6.1.3. Pharmacokinetics
Compound 3h at a dose of 30 mg/kg in 0.5% MC suspension was
orally administered to fasted and non-fasted beagle dogs (n = 3).
Compounds 3h and CI-1011 at a dose of 30 mg/kg in 0.5% MC sus-
pension were orally administered to non-fasted Sprague-Dawley (SD)
rats (male, 8 weeks old, n = 3). Blood samples (heparin plasma) were
collected from the forearm vein 0.5, 1, 2, 3, 4, 6 and 8 h after compound
administration. To the plasma samples (0.2 mL) were added NH4OH
(1.0 mL), the internal standard solution (0.2 mL, 1.0 μg/mL) and a
mixed solvent (6 mL, i-PrOH/n-hexane = 5/95). The resulting mixture
was stirred for 10 min. The supernatant (organic layer) was collected
and evaporated under a nitrogen stream. Then, the residue was dis-
solved in a mobile phase (0.2 mL, 0.1% HCOOH/MeCN = 1/5). The
concentrations of 3h and CI-1011 were measured via liquid chroma-
tography-tandem MS (LC/MS/MS). The MS was equipped with an
electrospray ion source and operated in positive ion mode. The HPLC
l
1646, 1497, 1403; H-NMR (DMSO‑d6) δ: 1.14 (12H, d, J = 6.8 Hz),
1.52–1.68 (2H, m), 1.68–1.82 (2H, m), 1.82–1.97 (2H, m), 2.33–2.45
(2H, m), 3.11 (2H, sept, J = 6.8 Hz), 3.43 (2H, t, J = 7.0 Hz), 7.11 (1H,
d, J = 8.1 Hz), 7.12 (lH, d, J = 6.6 Hz), 7.22 (lH, dd, J = 8.1, 6.6 Hz),
7.31 (1H, dd, J = 8.1, 4.8 Hz), 7.98 (1H, dd, J = 8.1, 1.5 Hz), 8.42 (1H,
d, J = 4.8 Hz), 8.72 (lH, br s); EIMS m/z: 425 (M+); Anal. Calcd for
C
24H31N3O2S: C, 67.73; H, 7.34; N 9.87; S, 7.53. Found: C, 67.68; H,
7.33; N, 9.86; S, 7.57.
The characterization of all compounds except for those in the
Experimental section are described in the Supporting information.
6. Biology
6.1. In vitro ACAT activity
conditions
were
as
follows:
column,
Inertsil-ODS-3
(3.0 mm × 150 mm); mobile phase; flow rate, 1.0 mL/min; and column
6.1.1. Microsomal assay
temperature, 40 °C.
ACAT activities were determined using arterial homogenates from
New Zealand White (NZW) rabbits (Oriental Yeast Co.) fed a diet sup-
plemented with 1% cholesterol (Wako Pure Chemical Industries) diet
6.1.4. In vivo study
Male Bio F1B hamsters (8 weeks old, Charles River Japan, Inc.,
12

K. Shibuya et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Kanagawa, Japan) were used for the animal experiments. The animal
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room was controlled at 23
3 °C with relative humidity of
50 20%. The animals were fed a CE-2 chow diet (CLEA Japan Inc.,
Tokyo, Japan), followed by supplementation with CE-2 containing
0.3% cholesterol and 10% coconut oil for 10 weeks. During fat loading,
3h was added to the chow at 1, 3, 10, or 30 mg/kg/day (n = 7 for each
dose group). The comparative agent, CI-1011, was added to the chow
at 0.3, 1, 3, or 10 mg/kg/day (n = 6 for each dose group). Blood
samples were collected for the determination of the plasma TC levels
using a commercially available kit (Cholesterol E-Test Wako; Wako
Pure Chemical Industries, Osaka, Japan). For atherosclerotic lesion
analysis, the animals were anesthetized via an intraperitoneal injection
of pentobarbital sodium (50 mg/kg) followed by vascular perfusion for
5 min with saline containing 4% paraformaldehyde with a perfusion
pressure of 120 mm H2O. The aorta was separated from the heart at the
upper portion 15 mm from the aortic origin. The isolated thoracic aorta
was cut open, mounted on a rubber plate, fixed with 4% paraf-
ormaldehyde, and stained with oil red O. The lipid accumulation areas
were measured with an image analysis system (SP500F; Olympus,
Tokyo, Japan).
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The manuscript was written through contributions of all authors. All
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authors have reviewed the final version of the manuscript.
Acknowledgments
We are very grateful to Dr. Yasuomi Urano (Doshisha University)
and Dr. Masaya Iwashita for helpful discussions about ACAT inhibitory
assay. We thank Ms. Noriko Inoue for performing the in vitro enzyme
assays.
A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2018.06.024.
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Mercaptobenzo-[d]oxazol-7-ol was prepared in 12% yield by reacting a mixture of 3-
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