Acyl-CoA: Cholesterol acyltransferase inhibitory activities of fatty acid amides isolated from Mylabris phalerate Pallas

Article abstract of DOI:10.1016/j.bmcl.2004.05.086

Unsaturated fatty acid amides, 9(Z)-octadecenamide (2) and 9(Z),12(Z)-octadecadienamide (4) as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT) were isolated from the ethyl acetate extracts of the insect, Mylabris phalerate Pallas, and elucidated by their spectroscopic data analysis. Compounds 2 and 4 inhibited rat liver microsomal ACAT, hACAT-1, and hACAT-2 with IC₅₀ values of 170, 85, and 63μM for 2 and of 151, 53, and 45μM for 4, respectively.

Full text of DOI:10.1016/j.bmcl.2004.05.086

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4277–4280
Acyl-CoA: cholesterol acyltransferase inhibitory activities of
fatty acid amides isolated from Mylabris phalerate Pallas
Ming-Zhe Xu,a,c Woo Song Lee,b Mi Jeong Kim,b Doo-Sang Park,a Hana Yu,b
Guan-Rong Tian,c Tae-Sook Jeongb,* and Ho-Yong Parka,*
aInsect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology, 52 Oun, Yusong,
Daejeon 305-333, Republic of Korea
bLipid Metabolism Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, 52 Oun,
Yusong, Daejeon 305-333, Republic of Korea
cDepartment of Chemistry, University of Yanbian, Yanji, China
Received 11 May 2004; revised 28 May 2004; accepted 31 May 2004
Available online 19 June 2004
Abstract—Unsaturated fatty acid amides, 9(Z)-octadecenamide (2) and 9(Z),12(Z)-octadecadienamide (4) as inhibitors of acyl-CoA:
cholesterol acyltransferase (ACAT) were isolated from the ethyl acetate extracts of the insect, Mylabris phalerate Pallas, and elu-
cidated by their spectroscopic data analysis. Compounds 2 and 4 inhibited rat liver microsomal ACAT, hACAT-1, and hACAT-2
with IC50 values of 170, 85, and 63 lM for 2 and of 151, 53, and 45 lM for 4, respectively.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Atherosclerosis is a progressive disease characterized by
the accumulation of lipids and fibrous elements in the
large arteries.1 In early atherogenesis, acyl-coenzyme A:
cholesterol acyltransferase (ACAT, EC.2.3.1.26) accu-
mulated cholesteryl esters with macrophages and
smooth muscle cells to result in foam cell formation.2 In
both experimental and clinical atherosclerosis, the for-
mation of foam cells derived from macrophages and
smooth muscle cells is an important event.3 Since the
propagation of foam cells in the arterial walls is directly
related to the increment of ACAT activity, the inhibitors
of ACAT as new type of medicine are needed to treat or
prevent atherosclerosis and hypercholesterolemia.4
Inhibiting ACAT within the arterial wall may result in
the inhibition of atherosclerotic lesion progression
without lowering total plasma cholesterol levels.5 Many
of these inhibitors showed promise in animal studies
inhibiting intestinal or hepatic ACAT and lowering
plasma cholesterols levels.6 In mammals, ACAT was
found to exist as two isoforms, namely, ACAT-1 is more
involved in macrophage foam cell formation and
ACAT-2 is associated with intestinal cholesterol
absorption.3 ACAT-1 and ACAT-2 respond differently
to ACAT inhibitors of differing structures and classes.7
Insects are the largest and most diverse groups of
organisms. They are evolutionarily distant from verte-
brates, plants, and microbial origin. For these reasons,
the study of insects promises to reveal novel bioactive
compounds. In order to search for human ACAT-1
(hACAT-1) and human ACAT-2 (hACAT-2) inhibitors
from insect origin, especially traditional Asian medicinal
insects, several solvent extracts of insects were screened
and the ethyl acetate extracts of M. phalerate Pallas
showed significant hACAT-1 and hACAT-2 inhibitory
activities with 85% and 89% at 100 lg/mL, respectively.
M. phalerate Pallas (order Coleopteran) is a dried beetle,
which is abundant in China and Eastern India. As an
aphrodisiac, an abortifacient, a vesicant, and a veteri-
nary medicine diuretic,8 M. phalerate Pallas has been
used as traditional medicine in Korea, Japan, and China
for hundreds of years. It has been found that M. phal-
erate Pallas possesses antitumor activity, increases the
number of leucocytes, and has irritant effects on
the urinary organs.9 Activity-guided fractionation of the
ethyl acetate extracts of M. phalerate Pallas led to the
Keywords: ACAT inhibitors; The insect; Mylabris phalerate Pallas.
* Corresponding authors. Tel.: +82-42-860-4650/4558; fax: +82-42-
860-4659/861-2675; e-mail addresses: hypark@kribb.re.kr; tsjeong@
kribb.re.kr
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.086

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M.-Z. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4277–4280
R1
5.24, whereas trans-geometry exhibited a set of two
apparent overlapping doublet split triplets at dH 5.28.11
In the IR spectrum, trans-isomer exhibited an additional
O
strong and characteristic absorption peak at 960 cmꢀ1 12
.
Compound 4, yellow oil, exhibited a molecular formula
of C18H33NO from its positive HREIMS data (m=z
[M]þ, 279.2564, calcd 279.2531 for C18H33NO). The IR
spectrum of 4 exhibited strong amide group at 3387 and
3186 cmꢀ1 and amide carbonyl group at 1642 cmꢀ1. The
1H NMR spectrum of 4 showed characteristic protons
of two olefinic moieties at dH 5.24 (4H, m), methylene
protons between two olefinic bonds at dH 2.68 (2H, t
like, J ¼ 6:5 Hz), a-methylene protons at dH 2.09 (2H, t,
J ¼ 7:5 Hz), and methyl protons at dH 0.81 (3H, t,
J ¼ 6:6 Hz), respectively. The 13C NMR data indicated
amide carbonyl carbon at 179.3 ppm and four olefinic
carbons at 130.9, 130.8, 129.1, and 129.0 ppm, respec-
tively.
1 R1 = OH
2 R1 = NH2
R2
O
3 R2 = OH
4 R2 = NH2
Figure 1. Chemical structures of commercial reagents 1 and 3, and
compounds 2 and 4 isolated from M. phalerate Pallas.
In order to confirm more exact structures and activities
of compounds 2 and 4, commercial available oleic acids
1 and 3 were converted to compounds 2 and 4, respec-
tively. Namely, oxalyl chloride (3.0 equiv) was added
slowly to a solution of 1 in CH2Cl2 at 0 ꢁC and stirred
for 4 h at room temperature. The reaction mixture was
concentrated under reduced pressure, cooled down to
0 ꢁC, treated with saturated aqueous NH4OH for 5 min,
and extracted with EtOAc. The organic layer was dried
over anhydrous MgSO4, filtered, concentrated, and
chromatographed on SiO2 (n-hexane–EtOAc ¼ 2:1) to
produce the fatty acid amide 2 in 97% yield. Linoleic
acid 3 was treated with oxalyl chloride and NH4OH
under similar reaction condition to give compound 4 in
98% yield (Scheme 1). The synthetic compounds 2 and 4
were identical with spectroscopic data of compounds 2
and 4 that were isolated from M. phalerate. Therefore,
the structure of compounds 2 and 4 was elucidated to be
9(Z)-octadecenamide (2) and 9(Z),12(Z)-octadecadien-
amide (4). Also, their structures were determined by
comparing previously reported data.11;13
isolation of bioactive compounds 2 and 4 (Fig. 1) using
silica gel column chromatography, thin layer chroma-
tography, and HPLC.
The powder of dried M. phalerate Pallas (200 g) was
suspended twice with ethyl acetate (2 L) at room tem-
perature. After stirring for 24 h and filtration using
Whatman no. 2 filter papers, the ethyl acetate extracts
was concentrated in vacuo to yield a brown oily material
(24.0 g). This material was chromatographed on a silica
gel column (E. Merck, Kieselgel 60, 230–400 mesh) with
a gradient of chloroform–methanol (100:0–1:1, v/v). The
active fractions were combined and concentrated in
vacuo yielding an oily residue (2.0 g). The residue was
subjected to flash silica gel column chromatography
with a gradient of n-hexane–ethyl acetate (1:1–1:5, v/v).
After combination of active fractions and concentration
in vacuo, the active constituents were finally purified by
preparative HPLC using an ODS column (Hydrosphere
C-18, 250 · 20 mm, YMC, Kyoto, Japan; MeOH/
H2O ¼ 9:1; 4.0 mL/min; k ¼ 220 nm) to obtain com-
pounds 2 (12.0 mg) and 4 (10.0 mg).10
The compounds 1–4 were evaluated for ACAT inhibi-
tory activities. The rate of incorporation of [1-14C]
oleoyl-CoA into cholesteryl ester was determined using
the expressed hACAT-1 and hACAT-2 from Hi5 cells14
Compound 2, white powder, showed a base peak at m=z
281 [M]þ in the EIMS. The molecular formula of com-
pound 2 was determined to be C18H35NO on the basis of
HREIMS (m=z [M]þ, 281.2719, calcd 281.2719 for
C18H35NO) and has mp 72.0–73.0 ꢁC (CHCl3). The IR
(CHCl3) gave two signals at 3393 (symmetric stretching
of N–H) and 3181 (H-bonded symmetric stretching of
HO
i
O
N–H) with a strong signal at 1650 (amide C@O) cmꢀ1
,
1
respectively. The H NMR (CDCl3, 500 MHz) showed
two olefinic methines at dH 5.24 (2H, m, CH@CH), a-
methylene protons of oleamide at dH 2.09 (2H, t,
J ¼ 7:4 Hz, CH2CONH2), and methyl protons at dH 0.80
(3H, t, J ¼ 6:7 Hz), respectively. The 13C NMR data
indicated amide carbonyl carbon at 179.3 ppm and two
olefinic carbons at 130.9 and 130.8 ppm, respectively.
Then, the double bond of oleamide 2 was located on C-9,
which was determined by the fragmentation patterns of
EIMS data. Additionally, cis-geometry of 1 was showed
peaks overlapped with triplet and triplet each other at dH
H2N
1 9 (Z)
3 9(Z), 12 (Z)
O
2 9 (Z)
4 9 (Z), 12 (Z)
Scheme 1. Reagents and conditions: (i) (COCl)2, NH4OH, CH2Cl2, rt.

M.-Z. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4277–4280
Table 1. ACAT inhibitory activities of compounds 1–4
4279
2. Ross, R. Nature 1993, 362, 801.
3. Chang, T. Y.; Chang, C. C. Y.; Lin, S.; Yu, C.; Li, B. L.;
Miyazaki, A. Curr. Opin. Lipidol. 2001, 12, 289.
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Chem. 2002, 39, 121.
5. Chang, T. Y.; Chang, C. C. Y.; Cheng, D. Annu. Rev.
Biochem. 1997, 66, 613.
6. (a) Krause, B. R.; Anderson, M.; Bisgaier, C. L.; Bocan,
T.; Bousley, R.; Dehart, P.; Essenburg, A.; Hamelehle, K.;
Homan, R.; Kieft, K.; Ncnally, W.; Stanfield, R.; Newton,
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L.; Homan, R.; Speyer, C.; Stanfield, R. L.; Krause, B. R.
J. Med. Chem. 1996, 39, 5031.
Compounds
IC50 (lM)a
Rat liver
microsomal
ACAT
hACAT-1
hACAT-2
1
>1000
170
>1000
85
>1000
63
2
3
>1000
150
>1000
53
>1000
45
4
Oleic acid anilideb
Pyripyropene Ab
0.027
0.17
0.14
0.17
0.64
>1000
a In vitro ACAT inhibitory activity was measured using the rat liver
microsomal ACAT, expressed hACAT-1 and -2. Data are shown as
mean values of two independent experiments performed in duplicate.
b Oleic acid anilide and pyripyropene A were used as positive controls.
Data from Ref. 14.
7. Burnett, J. R.; Wilcox, L. J.; Huff, M. W. Clin. Chim. Acta
1999, 286, 231.
8. Wang, C. C.; Wu, C. H.; Hsieh, K. J.; Yen, K. Y.; Yang,
L. L. Toxicology 2000, 147, 77.
and rat liver microsomes.15;16 Oleic acid anilide and
pyripyropene A were used as positive controls.16;17 In
general, polyunsaturated fatty acid analogs have been
known to exhibit anti-inflammatory activity,18 in vitro
or in vivo antioxidant activity,19 and beneficial effects on
risk factors of coronary heart disease (CHD).20 Based
on these results, oleic acid (1) and linoleic acid (3) were
tested tentatively for their ACAT inhibitory activities.
The unsaturated fatty acids 1 and 3 not inhibited hA-
CAT-1 and hACAT-2, and rat liver microsomal ACAT,
whereas the unsaturated fatty acid amides 2 and 4
showed ACAT inhibitory activities. Compounds 2 and 4
inhibited rat liver microsomal ACAT, hACAT-1, and
hACAT-2 with IC50 values of 170, 85, and 63 lM for 2,
of 151, 53, and 45 lM for 4, respectively (Table 1).
Compounds 2 and 4 inhibited both isoforms with simi-
lar degree of inhibitory activities; whereas rat liver
microsomal ACAT fraction was inhibited with two- to
threefold lower activities compared to each human
ACAT isoform. On the other hand, oleic acid anilide
and pyripyropene A,16;17 known ACAT inhibitors,
showed more potent inhibitory activities against rat liver
microsomal ACAT than both hACAT-1 and hACAT-2
as previously known.14 These results suggest that the
specificity of inhibitor was highly different, depending
on enzyme sources.
9. Wang, G. S. J. Ethnopharmacol. 1989, 26, 147.
10. Physical and spectroscopic data: 9(Z)-octadecenamide:
white powder; it has mp 72–73 ꢁC (CHCl3); IR (CHCl3)
mmax 3393, 3181, 2914, 2840, 1650, 1462, 1396, 1146, 810,
664 cmꢀ1 1H NMR (CDCl3, 500 MHz) d 5.24 (2H, m),
;
2.09 (2H, t, J ¼ 7:4 Hz), 1.93 (4H, m), 1.50 (2H, m), 1.23–
1.19 (20H, m), 0.81 (3H, t, J ¼ 6:6 Hz); 13C NMR (CDCl3,
125 MHz) d 179.3, 130.9, 130.8, 36.5, 33.1, 30.84, 30.81,
30.62, 30.64, 30.61, 30.45, 30.35, 30.24, 28.1, 28.1, 26.9,
14.5; EIMS m=z 281 [M]þ, 264 (25), 246 (12), 126 (30), 98
(25), 83 (20), 72 (90), 59 (100); HREIMS m=z calcd for
C18H35NO 281.2719, found 281.2719. 9(Z), 12(Z)-Octa-
decadienamide: a yellow oil; IR (CHCl3) mmax 3387, 3186,
2927, 2583, 1642, 1468, 1410, 1289, 1124, 829, 664 cmꢀ1
;
1H NMR (CDCl3, 500 MHz) d 5.24 (4H, m), 2.67 (2H, t,
J ¼ 6:5 Hz), 2.09 (2H, t, J ¼ 7:5 Hz), 1.96 (4H, m), 1.50
(2H, m), 1.28–1.19 (14H, m), 0.81 (3H, t, J ¼ 6:5 Hz); 13C
NMR (CDCl3, 125 MHz) d 179.3, 130.9, 130.8, 129.1,
129.0, 36.5, 32.7, 30.7, 30.5, 30.4, 30.3, 30.2, 28.1, 28.1,
26.9, 26.5, 23.5, 14.4; EIMS m=z 279 [M]þ 262 (11), 244
(10), 220 (20), 150 (25), 109 (35), 95 (60), 81 (80), 67 (90),
59 (100); HREIMS m=z calcd for C18H33NO 279.2562,
found 279.2531.
11. Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem.
Soc. 1996, 118, 580.
12. Griffiths, L.; Horton, R. Magn. Reson. Chem. 1998, 36,
104.
13. Jain, M. K.; Ghomashchi, F.; Yu, B. Z.; Bayburt, T.;
Murphy, D.; Houck, D.; Brownell, J.; Reid, J. C.;
Solowiej, J. E.; Wong, S. M.; Mocek, U.; Jarrell, R.;
Sasser, M.; Gelb, M. H. J. Med. Chem. 1992, 35,
3584.
14. Cho, K. H.; An, S. J.; Lee, W. S.; Paik, Y. K.; Jeong, T. S.
Biochem. Biophys. Res. Commun. 2003, 309, 864.
15. (a) Jeong, T. S.; Kim, S. U.; Son, K. H.; Kwon, B. M.;
Kim, Y. K.; Choi, M. U.; Bok, S. H. J. Antibiot. 1995, 48,
751; (b) Lee, C. H.; Jeong, T. S.; Choi, Y. K.; Hyun, B.
W.; Oh, G. T.; Kim, E. H.; Kim, J. R.; Han, J. I.; Bok, S.
H. Biochem. Biophys. Res. Commun. 2001, 284, 681.
16. ACAT activity assay: microsomal fractions of Hi5 cells
containing baculovirally expressed hACAT-1 or -2 and rat
liver microsomes were used as sources of the enzyme. The
activity of the hACAT-1 and hACAT-2 was measured
according to the method of Brecher and Chan21 with slight
modification.15 The reaction mixture, containing 4 lL of
microsomes (8 mg/mL protein), 20 lL of 0.5 M potassium–
phosphate buffer (pH 7.4) with 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),
The fatty acid amides 2 and 4 were isolated firstly from
the EtOAc extracts of M. phalerate Pallas and moder-
ately inhibited rat liver microsomal ACAT, hACAT-1,
and hACAT-2. Further studies on confirmation of the
inhibitory activity of ACAT using cells stably expressing
hACAT-1 or -2 and the in vivo efficacy test of choles-
terol-lowering and anti-atherogenic activities of com-
pounds 2 and 4 are underway.
Acknowledgements
This work is supported by grants from KRIBB Research
Initiative Program (KGS 3220311) and the Ministry of
Science and Technology (M1-0302-00-0033), Korea.
References and notes
1. Lusis, A. J. Nature 2000, 407, 233.

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M.-Z. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4277–4280
41 lL of water, and 10 lL of test sample in a total volume
Finland). Background values were obtained by preparing
heat inactivated microsomes or normal insect cell lysate
microsomes, usually background value was 200–250 cpm,
while 8000 cpm of ACAT reaction. The ACAT activity
was expressed as a defined unit, cholesteryl oleate pmol/
min/mg protein.
of 92 lL, was preincubated for 20 min at 37 ꢁC with brief
vortexing and sonication. 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 0.4 mL of 0.1 M potassium–
phosphate buffer (pH 7.4) with 2 mM dithiothreitol 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 0.9ꢁ1.0 mL). The
radioactivity in 100 lL of the upper phase was measured in
a 3 mL liquid scintillation viral with 3 mL of scintillation
cocktail (Lipoluma, Lumac Co.) using a liquid scintilla-
tion counter (1450 Microbeta Trilux Wallac Oy, Turku,
17. (a) Roth, B. D.; Blankley, J.; Hoefle, M. L. J. Med. Chem.
1992, 35, 1609; (b) Kim, Y. K.; Tomoda, H.; Nishida, H.
J. Antibiot. 1994, 47, 154.
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