Novel 3,5-diaryl pyrazolines as human acyl-CoA:cholesterol acyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.03.079

A series of pyrazoline derivatives were prepared for evaluating their acyl-CoA:cholesterol acyltransferase activities. 3-(3,5-Di-tert-butyl-4- hydroxyphenyl)-5-(multi-substituted 4-hydroxyphenyl)-2-pyrazolines 4a-i were shown in vitro inhibitory activity on hACAT-1 and -2.

Full text of DOI:10.1016/j.bmcl.2004.03.079

Bioorganic & Medicinal Chemistry Letters 14(2004) 2715–2717
Novel 3,5-diaryl pyrazolines as human
acyl-CoA:cholesterol acyltransferase inhibitors
Tae-Sook Jeong, Kyung Soon Kim, So-Jin An, Kyung-Hyun Cho,
Sangku Lee and Woo Song Lee*
National Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology,
Daejeon 305-333, South Korea
Received 2 March 2004; revised 25 March 2004; accepted 26 March 2004
Abstract—A series of pyrazoline derivatives were prepared for evaluating their acyl-CoA:cholesterol acyltransferase activities. 3-
(3,5-Di-tert-butyl-4-hydroxyphenyl)-5-(multi-substituted 4-hydroxyphenyl)-2-pyrazolines 4a–i were shown in vitro inhibitory
activity on hACAT-1 and -2.
Ó 2004Elsevier Ltd. All rights reserved.
As the understanding of cellular cholesterol metabolism
has advanced, acyl-CoA:cholesterol acyltransferase
(ACAT) has been known to play a crucial role in the
development of atherosclerosis. It catalyzes the forma-
tion of cholesterol esters from cholesterol and long-
chain fatty acyl-coenzyme A, and also modulates
cholesterol absorption from the intestine, secretion of
VLDL from the liver, and steroidogenesis.1 Recently, it
was found to be present as two isoforms in mammals,2
ACAT-1 and ACAT-2, with different tissue distribution
and membrane topology.3 In mammals, such as mouse
and monkey, ACAT-1 is located in various tissues as a
ubiquitous manner, whereas ACAT-2 is found only in
liver and intestine. However, in the adult human,
ACAT-1 is the major ACAT isoform in the liver,
whereas ACAT-2 is the major ACAT isoform in the
intestine.4 ACAT-2 appears to be responsible for chol-
esterol ester formation in intestine and liver, and
ACAT-1 also in peripheral tissue including human liver
has a very important role to synthesize cholesterol ester
for storage in the cell.
ACAT. So, we reported recently mass-production of
hACAT-1 and hACAT-2 individually from Hi5 cells to
screen isoform-specific inhibitors.6 Also, analogues of
pyrazolines have potential antioxidant activities against
low-density lipoprotein (LDL).7 Therefore, pyrazolines
were tentatively applied to ACAT enzyme to search a
specific inhibitor against hACAT-1 or -2. In mammals,
selective inhibition of ACAT-2 would be beneficial for
treating hypercholesterolemia and cholesterol gall-
stones, whereas inhibition of ACAT-1 and -2 might be
an useful strategy for atherosclerosis.8 In this study, we
describe the synthesis and in vitro ACAT inhibitory
activities of a novel series of pyrazoline derivatives 4a–i.
Pyrazolines 4a–i were prepared according to the meth-
ods shown in Scheme 1. Treatment of 1-(3,5-di-tert-
butyl-4-hydroxyphenyl)ethanone (1) with 3,5-di- or
2,3,5-tri-substituted 4-hydroxybenzaldehydes 2a–i gave
a,b-unsaturated ketones 3a–i by a typical acid-catalyzed
aldol condensation in 28–86% yields, as shown in
Scheme 1. Reaction of 3a–i and hydrazine monohydrate
afforded pyrazolines 4a–i via a one-pot addition–cyclo-
condensation process in 50–96% yields (Table 1). The
structures of 4a–i were determined by their spectro-
scopic analysis.9
Although many compounds have been reported as po-
tent ACAT inhibitors,5 thus far, however, ACAT
inhibitors with significant preference for one isoform
over the other have not been described for the human
The potential of compounds 4a–i was evaluated as an
inhibitor of hACAT-1 or -2 that was expressed and
characterized from Hi5 cells by recombinant baculovi-
ruses.6 Then, the rate of incorporation of [1-14C]oleoyl-
CoA into cholesteryl ester was determined using the
expressed hACAT-1 or -2.10 The ACAT inhibitory
Keywords: ACAT inhibitor.
* Corresponding author. Tel.: +82-42-860-4278; fax: +82-42-861-2675;
e-mail: wslee@kribb.re.kr
Both authors contributed equally to the work.
0960-894X/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.079

2716
T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2715–2717
R1
O
R1
O
O
R2
t-Bu
HO
R2
t-Bu
HO
CH3
H
i
OH
OH
R3
t-Bu
R3
t-Bu
3a-i
1
2a-i
3a R1 = H, R2 = R3 = t-Bu, 86% 3b R1 = H, R2 = R3 = i-Pr, 54%
3c R1 = H, R2 = R3 = Me, 66% 3d R1= R2 = R3 = Me, 52%
R1
R2
NH
N
3e R1 = H, R2 = R3 = F, 58%
3f R1 = H, R2 = R3 = Ph, 28%
3g R1 = R3 = H, R2 = OMe, 71% 3h R1 = R2 = H, R3 = NO2, 48%
3i R1 = R2 = R3 = H, 67%
t-Bu
OH
ii
R3
HO
t-Bu
4a-i
Scheme 1. Reagents and conditions: (i) H2SO4, MeOH, reflux; (ii) N2H4, EtOH, rt––reflux.
activities of the compounds 4a–i were confirmed by the
positive control with oleic acid anilide, which inhibited
hACAT-1 and hACAT-2 with IC50 values of 0.14and
0.17 lM, respectively.6 Compounds 4a–d containing
alkyl moieties (t-Bu, i-Pr, and Me) at the 2,3,5-position
of phenol ring showed similar degree of inhibitory
activities in both hACAT-1 and hACAT-2 in vitro
assays, whereas compounds 4e–i having weak inhibitory
activities relative to 4a–d showed selectivity of hACAT-
1 over hACAT-2. According Rudel et al.’s results,11
topology orientation of African green monkey ACAT-1
and ACAT-2 in the membrane of the endoplasmic
reticulum (ER) shows that serine residue is located on
opposite sides of the membrane for either enzyme. This
result means that functional differences between the
enzymes may occur, even though the role of this serine
in enzyme function is not yet known. Also, it may be not
only influenced at substrate binding site but also at
lipophilicity of inhibitors, which are through ER mem-
brane. Therefore, these results may be rationalized that
more specificity of compounds 4e–i against hACAT-1
may be due to hydrogen bonding effect between
hydrophilic groups (fluoro, methoxy, phenyl, and nitro)
and enzyme. In the case of 4i, it also may conclude that
4i includes less hindered phenol to lead to hydrogen
bonding with enzyme, when compared to hindered alkyl
groups that were substituted at 2,3,5-position of phenol
ring of 4a–d. From the results of inhibitory activities of
4a–i, it appeared that the activity was insensitive to the
size of alkyl groups at the 2,3,5-position on 5-phenol
ring, whereas it was influenced by electron density of
substituents on the 5-phenol ring to show specificity
against hACAT-1 and -2.
In conclusion, we have discovered a novel class of
hACAT-1 and hACAT-2 enzyme inhibitors, 3-(3,5-
di-tert-butyl-4-hydroxyphenyl)-5-(di- or tri-substituted
4-hydroxyphenyl)-2-pyrazolines 4a–i. Furthermore, the
structure–activity relationship (SAR), stereospecific
inhibitory activity, and pharmacological studies of this
novel series of pyrazolines will be the subject of future
publications.
Table 1. ACAT inhibitory activities of pyrazoline compounds 4a–i
R1
R2
NH
N
t-Bu
OH
R3
HO
t-Bu
Compound
R1
R2
R3
Yield (%)a
IC50 (lM)b
hACAT2
hACAT1
4a
4b
4c
4d
4e
4f
H
H
H
Me
H
H
H
H
H
t-Bu
i-Pr
Me
Me
F
t-Bu
i-Pr
Me
Me
F
50
50
12.8
14.7
12.416.4
19.9
27.0
8412.8
79
95
31.5
164.3
127.4
64.5
35.6
23.3
Ph
OMe
H
Ph
40
96
4g
4h
4i
OMe
NO2
H
32.1
65.4125.8
32.468.5
96
96
H
a Isolated yield from 3a–i.
b In vitro ACAT inhibitory activity was measured using the expressed hACAT-1 or hACAT-2. Data are shown as mean values of two independent
experiments performed in duplicate.

T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2715–2717
2717
1.46 (18H, s), 3.00 (1H, dd, J ¼ 10:5, 16.2 Hz), 3.43 (1H,
dd, J ¼ 10:2, 15.9 Hz), 4.83 (1H, t like, J ¼ 10:5 Hz), 5.20
(1H, s, –OH), 5.37 (1H, s, –OH), 5.84(1H, br, –NH), 7.23
(2H, s), 7.53 (2H, s); 13C NMR (75 MHz, CDCl3) d 30.2,
30.3, 34.37, 34.4, 42.2, 64.9, 123.1, 123.3, 124.4, 133.1,
136.0, 136.1, 152.6, 153.3, 154.7.
Acknowledgements
This work is supported by a grant from Korea Health 21
R and D project, Ministry of Health and Welfare,
Republic of Korea (No 02-PJ1-PG10-20999-0001).
10. ACAT activity assay: Microsomal fractions of Hi5 cells
containing baculovirally expressed ACAT-1 or -2 were
used as the sources of enzymes.6 The activity of the
hACAT-1 and hACAT-2 was measured according to the
method of Brecher and Chan12 with slight modification.13
The reaction mixture, containing 4 lL of microsomes
(8 mg/mL protein), 20 lL of 0.5 M potassium–phosphate
buffer (pH 7.4, 10 mM dithiothreitol), 15 lL of bovine
serum albumin (fatty acid free, 40 mg/mL), 2 lL of
cholesterol in acetone (20 lg/mL, added last), 41 lL of
water, and 10 lL of test sample in a total volume of 92 lL,
was preincubated for 20 min at 37 °C. The reaction was
initiated by the addition of 8 lL of [1-14C]oleoyl-CoA
solution (0.05 lCi, final concn 10 lM). After 25 min of
incubation at 37 °C, the reaction was stopped by the
addition of 1.0 mL of isopropanol–heptane (4:1; v/v)
solution. A mixture of 0.6 mL of heptane and 0.4mL of
0.1 M potassium–phosphate buffer (pH 7.4, 2 mM dithio-
threitol) was then added to the terminated reaction
mixture. The above solution was mixed and allowed to
phase separation under gravity for 2 min. Cholesterol
oleate was recovered in the upper heptane phase (total
volume 0.9–1.0 mL). The radioactivity in 100 lL of the
upper phase was measured in scintillation vial with 3 mL
of scintillation cocktail (Lipoluma, Lumac Co.) using a
liquid scintillation counter (1450 Microbeta Trilux Wallac
Oy, Turku, Finland). Background values were obtained by
preparing heat inactivated microsomes. The ACAT activ-
ity was expressed as a defined unit, cholesteryl oleate
pmol/min/mg protein.
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9. Physical and spectroscopic data: 4a: colorless prisms, mp
1
243–244 °C, H NMR (300 MHz, CDCl3) d 1.45 (18H, s),