Effect of a new β-sitosterol analogue on plasma lipid concentrations in rats

Article abstract of DOI:10.1248/cpb.52.597

N-Substituted succinamic acid β-sitosteryl ester derivatives were prepared and evaluated. Compounds 1 and 2 were prepared in 76-92% yields by the reaction of β-sitosterol and succinic anhydride, followed by the activation of the resulting acid compound 1 by thionyl chloride or methyl chloroformate, and finally by amination with appropriate amines. Compound 2a (DANA87) was also easily obtained in one step by the direct addition of β-sitosterol to dicyclohexylcarbodiimide (DCC) in 80% yield. Administration of the dietary compound DANA87 resulted in significant decreases in total plasma cholesterol (TC) and low-density lipoprotein (LDL) cholesterol concentrations compared with controls in a rat model. High-density lipoprotein cholesterol and plasma triglyceride levels were not affected. These findings indicate that DANA87 functions as TC and LDL cholesterol-reducing agent.

Full text of DOI:10.1248/cpb.52.597

May 2004
Notes
Chem. Pharm. Bull. 52(5) 597—601 (2004)
597
Effect of a New b-Sitosterol Analogue on Plasma Lipid Concentrations in
1)
Rats
,
a
a
a
a
a
a
Yang-Heon SONG,* Soonil HONG, Hongsan LIM, Jinmoo SEO, Sungjoo CHUNG, Insook NO,
a
b
Kyunghee LEE, and Michung YOON
a
b
Department of Chemistry, Mokwon University; and Department of Biology, Mokwon University; Daejeon 302–729,
Korea. Received August 14, 2003; accepted February 2, 2004
N-Substituted succinamic acid b-sitosteryl ester derivatives were prepared and evaluated. Compounds 1 and
were prepared in 76—92% yields by the reaction of b-sitosterol and succinic anhydride, followed by the activa-
2
tion of the resulting acid compound 1 by thionyl chloride or methyl chloroformate, and finally by amination with
appropriate amines. Compound 2a (DANA87) was also easily obtained in one step by the direct addition of b-
sitosterol to dicyclohexylcarbodiimide (DCC) in 80% yield. Administration of the dietary compound DANA87
resulted in significant decreases in total plasma cholesterol (TC) and low-density lipoprotein (LDL) cholesterol
concentrations compared with controls in a rat model. High-density lipoprotein cholesterol and plasma triglyc-
eride levels were not affected. These findings indicate that DANA87 functions as TC and LDL cholesterol-reduc-
ing agent.
Key words cholesterol-reducing agent; b-sitosteryl ester derivative; plasma lipid concentration
A major risk factor for the development of coronary heart Chemistry
disease (CHD) or atherosclerosis is elevated levels of serum
The synthesis of b-sitosterol analogues 1 and 2 are sum-
cholesterol. Recent clinical trials and studies have confirmed marized Chart 1. The b-sitosterol-3-hemisuccinate ester 1
that lowering of low-density lipoprotein (LDL) cholesterol is was prepared by the reaction of b-sitosterol and succinic an-
2,3)
15)
related to a reduction in CHD risk. The reduction of serum hydride in toluene with a catalytic amount of DMAP. The
cholesterol levels has mainly been accomplished by inhibit- chlorination of the resulting acid compound 1 with SOCl2
ing cholesterol biosynthesis or by blocking the absorption of and then direct amination with appropriate amines afforded
dietary cholesterol. Statins, such as pravastatin, that effec- N-substituted succinamic acid b-sitosteryl ester derivatives 2.
tively inhibit cholesterol biosynthesis are HMG-CoA reduc- Compounds 2 were also synthesized in 76—92% yields by
tase (3-hydroxy-3-methylglutaryl-coenzyme A reductase) in- the reaction of 1 with methyl chloroformate, followed by am-
hibitors and are widely prescribed in the treatment of hyper- ination of the activated intermediate. Interestingly, compound
4
,5)
cholesterolemia.
Several squalene synthesis inhibitors 2a (DANA87) was also easily obtained in one step by the di-
6
,7)
8)
have been also reported. SCH-48461 (a trans-azetidinone)
rect addition of b-sitosterol to dicyclohexylcarbodiimide
and 4ꢀ,6ꢀ-bis((2-fluorophenyl)carbamoyl) b-O-cellobioside (a (DCC) in 80% yield. It was considered that compound
steriodal glycoside) have recently been reported to be cho- DANA87 was prepared by slow thermal degradation of the
lesterol absorption inhibitors.
9
)
Phytosterols, or plant sterols, are an interesting class of
compound due to their application and biological activities.
The phytosterols found most frequently in nature are b-sitos-
terol, campesterol, and stigmasterol. It has been known that
they inhibit the absorption of dietary and endogenously pro-
duced cholesterol from the small intestine, reducing blood
cholesterol concentrations both in animal models and in hu-
10—13)
mans.
b-Sitosterol and b-sitostanol fatty acid esters
were recently commercialized as food additives to reduce
serum cholesterol levels. A novel hydrophilic phytostanol
analogue, FM-VP4, was also developed to reduce serum cho-
14)
lesterol levels.
However, even with the current therapeutic agents, it is not
easy to achieve proper plasma cholesterol levels without side
effect. Therefore the discovery of more effective, well-toler-
ated agents that reduce high cholesterol levels is needed. We
here report the synthesis and interesting pharmacologic prop-
erties of novel succinamic acid ester derivatives that are b-
sitosterol analogues, compounds 1 and 2, that have the ability
to lower plasma cholesterol levels and that can be more effec-
tive cholesterol-lowering alternatives to b-sitosterol and b-
sitostanol fatty acid esters.
Reagents and conditions: (a) succinic anhydride, DMAP, toluene, reflux; (b) SOCl2,
rt and RNH , TEA, THF, reflux; (c) methyl chloroformate, TEA, Et O, rt and RNH ,
TEA, THF, reflux; (d) DCC, toluene, reflux, 2 h; (e) toluene, heat, 24 h
2
2
2
Chart 1
∗
To whom correspondence should be addressed. e-mail: yhsong@mokwon.ac.kr
© 2004 Pharmaceutical Society of Japan

5
98
Vol. 52, No. 5
b-sitosterol-DCC adduct 1a that was initially formed, fol-
lowed by the spontaneous formation of the corresponding
amide compound. For a large-scale reaction, the procedure
using methyl chloroformate gives increased yields compared
with the route with SOCl , because an undesirable side reac-
2
tion like breakage of the ester bond in DANA87 could occur
in the reaction using SOCl2.
Results and Discussion
In a preliminary biological experiment on compounds 2,
2
a (DANA87) was much more effective than b-sitosterol it-
self and other compounds 2 and found to lower the increased
levels of plasma cholesterol potently in a rat model. Rats
Fig. 2. Effect of DANA87 on the Total Plasma Cholesterol (TC) Level in
were fed a hyperlipidemic diet (1% cholesterol and 0.5% the Blood of Rats
cholic acid) containing 1% of each compounds 2 or b-sitos-
terol for 1 week. The percentage by which the total plasma
cholesterol (TC) level was lowered compared with the con-
trol group is shown in Fig. 1. Therefore, to observe the long-
term effect of the most potent compound DANA87 on lower-
ing of the TC level, rats were divided into 4 groups and fed
the hyperlipidemic diet (1% cholesterol and 0.5% cholic
acid) containing various concentrations (control group 0%;
experimental groups 0.1%, 0.5%, 1%) of DANA87 for 1—4
weeks. The TC levels and the percentage by which choles-
terol was lowered in the bloods of rats are shown in Figs. 2
and 3, respectively.
Rats were fed hyperlipidemic powder diets containing 0 (control), 0.1, 0.5, or 1% of
DANA87 for 1 to 4 weeks (nꢂ10). ∗∗ pꢃ 0.01.
TC levels were reduced in a dose-dependent manner with
DANA87 administration. The TC levels in the 0.1% and
Fig. 3. Lowering (%) of TC by DANA87 in the Blood of Rats
0.5% DANA87 groups were lowered by about 19% and 1 %
at 1 week and by 30% and 35% at 2 weeks, respectively. At 3
and 4 weeks when the TC level was also reduced in the con-
trol group fed only the hyperlipidemic diet, it was lowered by
17% and ꢁ3%, respectively, in the 0.1% DANA87 group,
and also by 26% and 24% in the 0.5% DANA87 group, re-
spectively.
The slight decrease in the TCs in the low-dose DANA87
groups at 3—4 weeks compared with 1—2 weeks might have
been due to a physiologic feedback regulation against hyper-
cholesterolemia induced by the hyperlipidemic diet, as
shown in control group.
The TC level in the 1% DANA87 group was markedly and
continuously lowered by 42% at 1 week and by 64% at 4
weeks. This suggests strongly that a high blood concentration
of DANA87 is needed to show an efficient pharmacologic ef-
fect. The level in the 1% DANA87 group significantly and
Fig. 4. Effect of DANA87 on the Ratio of HDL to LDL in the Blood of
Rats Fed a Hyperlipidemic Diet Containing Varying Concentrations of
DANA87 for 1 to 4 Weeks
Rats were fed hyperlipidemic powder diets containing 0 (control), 0.1, 0.5, or 1% of
DANA87 for 1 to 4 weeks (nꢂ10). ∗∗ pꢃ0.0001.
gradually increased from 1 week to 4 weeks. It was also
found that the high-density lipoprotein (HDL) cholesterol
level changed little, but the low-density lipoprotein (LDL)
cholesterol level was markedly decreased in a dose-depen-
dent manner with DANA87 administration. The ratio of
HDL/LDL cholesterol was also investigated, as shown in Fig.
4
.
To examine the effect of DANA87 in normal rats, another
group of rats was fed a normal powder diet containing only
% DANA87 for 1 week. The TC, HDL and triglyceride
TG) levels after DANA87 administration changed little
compared with control group, as shown in Fig. 5, but the
LDL cholesterol level was decreased, which increased the
1
(
Fig. 1. Lowering (%) of Total Plasma Cholesterol (TC) Levels by Com-
pounds 2 or b-Sitosterol (ST) Compared with Controls
Rats were fed hyperlipidemic powder diets containing 0 (control) or 1% of each of
compounds 2 or b-sitosterol (ST), respectively, for 1 week (nꢂ5).

May 2004
599
ester b-sitosterol analogue DANA87 functions as a total
plasma cholesterol- and LDL cholesterol-reducing agent.
Further pharmacologic evaluation and the structure–activity
relationship study of DANA87 and related compounds are in
progress.
Experimental
1
13
Chemistry H-NMR (300 MHz) and C-NMR (125 MHz) spectra were
obtained with a Varian Unity NMR spectrophotometer. Chemical shifts (d)
are reported in ppm relative to tetramethylsilane (TMS) as internal standard.
IR spectra were recorded on a JASCO FT/IR 300E infrared spectrophotome-
ter. Elemental analyses (C, H, N) and MS analyses were performed at the
Korea Research Institute of Chemical Technology. Melting points were ob-
tained on a MEL-TEMPII (Laboratory Devices, U.S.A.) apparatus and are
uncorrected.
Fig. 5. Effect of DANA87 on the Levels of TC, HDL, LDL, and TG in
Rats Fed a Normal Diet Containing 1% DANA87 for 1 Week
Rats were fed normal powder diets containing 0 (control) or 1% DANA87 for 1 week
nꢂ10). ∗∗ pꢃ0.01.
b-Sitosterol-3-hemisuccinate Ester (1) (Succinic Acid Mono-[17-(4-
ethyl-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,
(
16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl] Ester) To a
stirred solution of b-sitosterol (1.5 g, 3.6 mmol) dissolved in distilled
toluene (20 ml), succinic anhydride (0.36 g, 3.6 mmol) and DMAP (10 mg,
ratio of HDL/LDL cholesterol (from 1.08 to 1.42). Western
blotting and RT-PCR analyses showed that the amount of 0.073 mmol) were added and the solution was heated at reflux for 24 h. The
LDL receptor in the liver was also markedly increased (data mixture was left at room temperature for 4 h, then the precipitated solid was
filtered off, and the filtrate was washed with water, hydrochloric acid solu-
tion (0.1 M), and again with water. The organic layer was dried over MgSO4,
not shown).
Administration of compound DANA87 did not show any
changes in the levels of functional biochemical indicators
evaporated, and the residue was recrystallized from hexane–ethyl acetate.
ꢁ1
1
Yield 88%, mp 150—151 °C. IR (KBr) cm : 2945, 1735, 1715. H-NMR
such as alanine transaminase (ALT), albumin, creatine, and (CDCl , 300 MHz) d: 0.68—2.05 (m, 45H), 2.33 (d, 2H), 2.44 (t, 2H), 2.63
3
(t, 2H), 4.60 (m, 1H), 5.36 (d, 1H), 11.05 (s, 1H). Anal. Calcd for C H O :
33 54 4
ꢄ
uric acid in plasma. There was no statistically significant ef-
fect on body weight gain.
It has been suggested that phytosterols lower circulating
plasma cholesterol concentrations by direct competitive
C, 76.99; H, 10.57%. Found: C, 76.21; H, 11.08%. MS (FAB) m/z 514 (M ).
General Procedure for the Preparation of N-Substituted Succinamic
Acid b-Sitosteryl Ester Derivatives (2): Method A (Using Thionyl Chlo-
ride) To a stirred solution of b-sitosterol-3-hemisuccinate ester 4 (1.0 g,
blocking of intestinal cholesterol absorption in animals and 1.94 mmol) and DMF (2 drops) dissolved in THF (20 ml), thionyl chloride
10—13)
(0.17 ml, 2.33 mmol) was added dropwise under cooling with an ice bath.
humans.
The potent ability of DANA87 to reduce
The reaction mixture was stirred at room temperature for 24 h. The solvent
and excess thionyl chloride were removed at reduced pressure, and the crude
chlorinated b-sitosterol-3-hemisuccinate ester (3-chlorocarbonyl-propionic
plasma cholesterol levels could also be explained by inhibi-
tion or displacement of cholesterol by the b-sitosterol part of
DANA87 from cholesterol-containing micelles, formed with acid 17-(4-ethyl-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,
bile acids in the small intestine, which are required for cho- 14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl) ester) was
16)
used for further reaction without purification. The residue was redissolved in
THF (15 ml) and cooled in an ice bath. An amine (2.05 mmol) and TEA
lesterol absorption. It has also been reported recently that
an increase in a sitostanol analogue dose decreased the rate
of cholesterol absorption and the overall cholesterol exposure
(0.7 ml, 5.02 mmol) were added dropwise, and the mixture was heated
slowly. After heating at reflux for 24 h, the reaction mixture was poured into
17)
to the body. The decrease in absorption rate may be due to ice-water. The solid obtained was filtered, washed with water, and dried, and
a decrease in facilitated cholesterol uptake by enterocyte the residue was purified by recrystallization or column chromatography on
18—20)
silica gel.
cholesterol transporters.
testinal mechanism has been widely cited as responsible for
the cholesterol lowering of b-sitosterol, studies in rats have
Although such an external in-
Method B (Using Methyl Chloroformate) To a stirred solution of b-
sitosterol-3-hemisuccinate ester 1 (1.0 g, 1.94 mmol) dissolved in dry ether
20 ml), methyl chloroformate (0.25 ml, 2.0 mmol) and TEA (0.7 ml,
(
suggested that intraperitoneal and subcutaneous injection of 5.02 mmol) were added dropwise under cooling with an ice bath. The reac-
21)
tion mixture was stirred at room temperature for 24 h. The solution was fil-
b-sitosterol lowers circulating cholesterol concentrations.
tered, washed with water, dried over MgSO , and evaporated, and the residue
4
It was speculated that b-sitosterol intrinsically affects the cir-
culating cholesterol concentration, possibly by altering en-
zymes involved in cholesterol metabolism. Decreases in total
was recrystallized from hexane–ethyl acetate to give 4-methoxycarbonyloxy-
4-oxo-butyric acid 17-(4-ethyl-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,
9
,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-
ꢁ1
blood cholesterol levels were due only to decreases in LDL yl ester. Yield 85%, mp 157—158 °C. IR (KBr) cm : 2934, 1735, 1705.
1
H-NMR (CDCl , 300 MHz) d: 0.68—2.05 (m, 45H), 2.33 (d, 2H), 2.61 (t,
cholesterol concentrations, as HDL cholesterol levels did not
change with the DANA87 dose. This suggests that DANA87
may further decrease total blood cholesterol by inhibiting the
3
2
H), 2.79 (t, 2H), 3.73 (s, 3H), 4.62 (m, 1H), 5.38 (d, 1H). Anal. Calcd for
C H O : C, 73.38; H, 9.85%. Found: C, 72.76; H, 10.15%. To a stirred so-
3
5
56
6
lution of 4-methoxycarbonyloxy-4-oxo-butyric acid 17-(4-ethyl-1,5-di-
formation of LDL cholesterol. The decrease in LDL choles- methyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradec-
terol is consistent with previously reported results that apoE- ahydro-1H-cyclopenta[a]phenanthren-3-yl ester (1.14 g, 2.0 mmol) in dry
ether (15 ml) an amine (2.05 mmol) and TEA (0.3 ml, 2.15 mmol) were
added, and the mixture was heated slowly. After heating at reflux for 24 h,
the reaction mixture was poured into ice-water. The solid obtained was fil-
tered, washed with water, and dried, and the residue was purified by recrys-
deficient mice develop severe hypercholesterolemia and ath-
erosclerotic lesions similar to those observed in humans, and
that phytosterol therapy is associated with a significant de-
crease in hepatic and lipoprotein lipase activities in apoE-de- tallization or column chromatography on silica gel.
2
2—24)
ficient mice.
However, further investigations are re-
N-Cyclohexyl-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cy-
clopenta[a]phenanthren-3-yl Ester (2a) (DANA 87): Method B Yield
quired to elucidate the mechanism involved and to explain
why DANA87 has more potent cholesterol-lowering ability
than b-sitosterol and b-sitostanol fatty acid esters.
ꢁ1
1
88%, mp 162—163 °C, IR (KBr) cm : 3305, 2930, 1730, 1645. H-NMR
(
CDCl , 300 MHz) d: 0.65—2.05 (m, 55H), 2.33 (d, 2H, Jꢂ7.5 Hz), 2.44 (t,
3
In conclusion, we have shown that the succinamic acid 2H, Jꢂ7.0 Hz), 2.63 (t, 2H, Jꢂ6.9 Hz), 3.70 (m, 1H), 4.60 (m, 1H), 5.36 (d,

6
00
Vol. 52, No. 5
1
3
1
1
(
H, Jꢂ5.7 Hz), 5.64 (d, 1H, Jꢂ7.5 Hz), C-NMR (CDCl , 150 MHz) d:
N-(2,6-Diethyl-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-
hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahy-
3
22.63, 139.56 (double bond of b-sitosterol), 170.43 (–CONH–), 172.45
–COO–). Anal. Calcd for C H NO : C, 78.60; H, 10.99; N, 2.35%. Found: dro-1H-cyclopenta[a]phenanthren-3-yl Ester (2h): Method A Yield
39 65 3
ꢄ
ꢁ1 1
C, 78.02; H, 11.15; N, 2.59%. MS (FAB) m/z 596 (M ꢄ1).
Method C (Using DCC) To a stirred solution of b-sitosterol-3- (CDCl
78%, mp 132—133 °C, IR (KBr) cm : 3445, 2975, 1715, 1703. H-NMR
, 300 MHz) d: 0.68—2.10 (m, 51H), 2.30 (d, 2H), 2.56 (d, 4H),
2.60—2.72 (m, 4H), 4.63 (m, 1H), 5.35 (d, 1H), 7.01 (s, 1H), 7.05 (d, 1H)
7.20 (d, 1H), 7.25 (dd, 1H). Anal. Calcd for C43 : C, 79.94; H, 10.45;
3
hemisuccinate ester 1 (3.0 g, 5.82 mmol) dissolved in dry ether (40 ml),
methyl chloroformate (0.75 ml, 6.0 mmol) and TEA (0.9 ml, 7.8 mmol) were
added dropwise under cooling with an ice bath. The reaction mixture was
stirred at room temperature for 24 h. The solution was filtered, washed with
H67NO
3
ꢄ
N, 2.16%. Found: C, 80.41; H, 10.66; N, 2.35%. MS (FAB) m/z 645 (M ).
N-(3,4-Dimethyl-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-
hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahy-
water, dried over MgSO , and evaporated, and the residue was recrystallized
from hexane–ethyl acetate to give 4-(N,Nꢅ-dicyclohexyl-carbamimidoyloxy)- dro-1H-cyclopenta[a]phenanthren-3-yl Ester (2i): Method B Yield
-oxo-butyric acid 17-(4-ethyl-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,
,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- (CDCl
yl ester (1a). Yield 92%, mp 50—51 °C. IR (KBr) cm : 3325, 2934, 1735, (t, 2H), 2.72 (t, 2H), 4.63 (m, 1H), 5.36 (m, 1H), 7.09—7.25 (m, 3H). Anal.
707, 1660. H-NMR (CDCl , 300 MHz) 0.64—2.04 (m, 65H), 2.34 (d, 2H, Calcd for C41
Jꢂ7.3 Hz), 2.45 (t, 2H, Jꢂ6.9 Hz), 2.65 (t, 2H, Jꢂ7.0 Hz), 3.69 (m, 1H),
4
ꢁ1
1
4
9
88%, mp 129—130 °C, IR (KBr) cm : 3450, 2975, 1710, 1685. H-NMR
, 300 MHz) d: 0.68—2.05 (m, 45H), 2.30 (d, 2H), 2.56 (d, 6H), 2.64
3
ꢁ1
1
1
H
63NO
3
: C, 79.69; H, 10.27; N, 2.26%. Found: C, 80.02; H,
3
ꢄ
11.01; N, 2.43%. MS (FAB) m/z 617 (M ).
1
3
4
.05 (m, 1H), 4.60 (m, 1H), 5.36 (d, 1H, Jꢂ5.8 Hz), d: 7.15 (br, 1H). C-
N-o-Tolyl-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-10,13-di-
NMR (CDCl , 125 MHz) d: 122.67, 139.57 (double bond of b-sitosterol), methyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
3
1
53.99 (–CꢂN–), 170.94 (–COOCꢂN–), 172.49 (–COO–). Anal. Calcd for penta[a]phenanthren-3-yl Ester (2j): Method A Yield 77%, mp 142—
ꢁ
1
1
C H N O : C, 76.56; H, 10.54; N, 3.88%. Found: C, 76.81; H, 10.75; N, 143 °C, IR (KBr) cm : 3445, 2978, 1715, 1687. H-NMR (CDCl ,
46
76
2
4
3
ꢄ
4
.03%. MS (FAB) m/z 719 (M ꢁ1). A solution of 1a (2.0 g, 2.77 mmol) in
toluene (25 ml) was heated with stirring at 80 °C in a water bath for 24 h.
The solution was evaporated and the residue was purified by column chro-
300 MHz) d: 0.67—2.10 (m, 45H), 2.26 (s, 3H), 2.34 (d, 2H), 2.66 (t, 2H),
2.74 (t, 2H), 4.63 (m, 1H), 5.36 (d, 1H), 7.05 (m, 1H), 7.20 (m, 2H), 7.47 (s,
1H), 7.81 (d, 1H). Anal. Calcd for C40H61NO : C, 79.55; H, 10.18; N, 2.31%.
3
ꢄ
matography on silica gel to afford DANA87. Yield 90%. DANA87 was also Found: C, 79.92; H, 10.33; N, 2.62%. MS (FAB) m/z 603 (M ).
easily obtained in 80% yield without isolation of 1a by the direct addition of
b-sitosterol to DCC under the same conditions.
Pharmacology Four-week-old male Sprague–Dawley rats (160) weigh-
ing 100—120 g were acclimatized for 4 d on a pellet diet in an animal cham-
N-Phenyl-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-10,13-di- ber and subsequently fed a hyperlipidemic diet (Purina Rodent Chow Spe-
methyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo- cial Mix 5001-S) supplemented with 0.5% cholic acid and 1% cholesterol
penta[a]phenanthren-3-yl Ester (2b): Method A Yield 76%, mp 182—
for 3 d, and then the hyperlipidemic diet containing either no DANA87 (con-
trol) or DANA87 at 0.10, 0.50, and 1% w/w for 4 continuous weeks. Blood
00 MHz) d: 0.68—1.95 (m, 45H), 2.48 (d, 2H), 2.66 (t, 2H), 2.73 (t, 2H), was collected from the heart under the ether anesthesia after 1 to 4 weeks,
.61 (m, 1H), 5.37 (d, 1H,) 7.09 (t, 1H), 7.30 (m, 2H), 7.50 (m, 2H), 7.72 (s, and the serum was separated by centrifugation. The lipoproteins (TC, HDL,
H) Anal. Calcd for C H NO : C, 79.40; H, 10.08; N, 2.37%. Found: C, LDL, TG) and ALT, albumin, creatine, and uric acid in plasma were ana-
ꢁ1
1
1
3
4
1
7
83 °C, IR (KBr) cm : 3440, 2965, 1715, 1680. H-NMR (CDCl3,
39
59
3
ꢄ
9.75; H, 10.45; N, 2.72%. MS (FAB) m/z 589 (M ).
lyzed using the Sigma Diagnostics enzymatic kit and the automatic analyzer
Hitachi model 747.
N-(4-Fluoro-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-
0,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
1
cyclopenta[a]phenanthren-3-yl Ester (2c): Method A Yield 79%, mp
References and Notes
ꢁ1
1
1
3
3
58—161 °C, IR (KBr) cm : 3430, 2975, 1715, 1705. H-NMR (CDCl ,
00 MHz) d: 0.68—1.80 (m, 45H), 1.88 (d, 2H), 2.00 (t, 2H), 2.28 (t, 2H),
.60 (m, 1H), 5.42 (d, 1H,) 6.63 (m, 2H), 6.86 (m, 2H), 7.35 (s, 1H). Anal.
1) In memory of the late Professor LeRoy H. Klemm.
2) Jaeger B. R., Seidel D., Herz, 26, 531—544 (2001).
3) Assmann G., Cullen P., Schulte H., Atherosclerosis, 130 (Suppl. 1),
S22 (1997).
3
Calcd for C H FNO : C, 77.05; H, 9.61; N, 2.30%. Found: C, 76.73; H,
3
9
58
3
ꢄ
9
.83; N, 2.66%. MS (FAB) m/z 607 (M ).
4) Shepherd J., Lancet, 359, 2271—2273 (2002).
N-(4-Chloro-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-
0,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
5) Jones P., Kafonek S., Laurora I., Hunniganhake D., Am. J. Cardiol., 81,
582—587 (1998).
1
cyclopenta[a]phenanthren-3-yl Ester (2d): Method B Yield 92%, mp
6) Miki T., Kori M., Mabuchi H., Tozawa R., Nishimoto T., Sugiyama Y.,
Teshima K., Yukimasa H., J. Med. Chem., 45, 4571—4580 (2002).
7) Chan C., Andreotti D., Cox B., Kirk B. E., Lester M. G., McCarthy A.
D., Procopiou P. A., Ross B. C., J. Med. Chem., 39, 207—216 (1996).
8) Burnett D. A., Caplen M. A., Davis H. R., Jr., Burrier R. E., Clader J.
W., J. Med. Chem., 37, 1733—1736 (1994).
ꢁ1
1
1
3
4
99—200 °C, IR (KBr) cm : 3425, 2985, 1725, 1690. H-NMR (CDCl ,
00 MHz) d: 0.68—1.90 (m, 45H), 2.31 (d, 2H), 2.65 (t, 2H), 2.72 (t, 2H),
.63 (m, 1H), 5.35 (d, 1H), 7.25 (d, 2H,) 7.46 (d, 2H), 7.98 (s, 1H). Anal.
3
Calcd for C H ClNO : C, 75.02; H, 9.36; N, 2.24%. Found: C, 75.62; H,
3
9
58
3
ꢄ
1
0.05; N, 2.47%. MS (FAB) m/z 624 (M ꢄ1).
N-(3-Bromo-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)-
0,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
9) McCarthy P. A., DeNinno M. P., Morehouse L. A., Chandler C. E.,
Bangerter F. W., Wilson T. C., Urban F. J., Walinsky S. W., Cosgrove P.
G., Duplantier K., Etienne J. B., Fowler M. A., Lambert J. F., O’Don-
nell J. P., Pezzullo S. L., Watson H. A., Wilkins R. W., Zaccaro L. M.,
Zawistoski M. P., J. Med. Chem., 39, 1935—1937 (1996).
1
cyclopenta[a]phenanthren-3-yl Ester (2e): Method A Yield 77%, mp
ꢁ1
1
1
3
4
7
37—138 °C, IR (KBr) cm : 3430, 2981, 1713, 1670. H-NMR (CDCl ,
3
00 MHz) d: 0.68—2.05 (m, 45H), 2.32 (d, 2H), 2.65 (t, 2H), 2.72 (t, 2H),
.63 (m, 1H), 5.35 (d, 1H), 7.12—7.26 (m, 2H,) 7.39 (d, 1H), 7.77 (s, 1H), 10) Ling W. H., Jones P. J. H., Atherosclerosis, 118, 319—331 (1995).
.91 (s, 1H). Anal. Calcd for C H BrNO : C, 70.04; H, 8.74; N, 2.09%. 11) Becker M., Staab D., von Bergmann K., J. Pediatr., 122, 292—296
3
9
58
3
ꢄ
Found: C, 70.82; H, 9.02; N, 1.83%. MS (FAB) m/z 668 (M ).
N-(3-Iodo-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-hexyl)- 12) Heinemann T., Axtmann G., von Bergmann K., Eur. J. Clin. Invest.,
(1993).
1
0,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
23, 827—831 (1993).
cyclopenta[a]phenanthren-3-yl Ester (2f): Method B Yield 90%, mp
13) Laraki L., Pelletier X., Debry G., Ann. Nutr. Metab., 35, 221—225
ꢁ1
1
1
3
4
7
41—142 °C, IR (KBr) cm : 3440, 2985, 1714, 1705. H-NMR (CDCl ,
(1991).
3
00 MHz) d: 0.67—2.10 (m, 45H), 2.32 (d, 2H), 2.64 (t, 2H), 2.70 (t, 2H), 14) Wasan K. M., Najafi S., Peteherych K. D., Pritchard P. H., J. Pharm.
.61 (m, 1H), 5.36 (d, 1H), 7.01 (s, 1H), 7.40—7.48 (m, 2H) 7.82 (s, 1H),
Sci., 90, 1795—1799 (2001).
.92 (s, 1H). Anal. Calcd. for C H INO : C, 65.44; H, 8.16; N, 1.95%. 15) Habib N. S., Khalil M. A., Arch. Pharm., 323, 401—404 (1990).
3
9
58
3
ꢄ
Found: C, 65.62; H, 8.22; N, 2.32%. MS (FAB) m/z 715 (M ).
16) Heinemann T., Leiss O., von Bergmann K., Atherosclerosis, 61, 219—
N-(4-Methoxy-phenyl)-succinamic Acid 17-(4-Ethyl-1,5-dimethyl-
223 (1986).
hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahy- 17) Wasan K. M., Peteherych K. D., Najafi S., Zamfier C., Pritchard P. H.,
dro-1H-cyclopenta[a]phenanthren-3-yl Ester (2g): Method A Yield
J. Pharm. Pharmaceut. Sci., 4, 207—216 (2001).
9%, mp 164—165 °C, IR (KBr) cm : 3430, 2975, 1715, 1672. H-NMR 18) Berge K. E., Tian H., Graf G. A., Yu L., Grishin N. V., Schultz J.,
ꢁ1
1
7
(
CDCl , 300 MHz) d: 0.67—2.05 (m, 45H), 2.33 (d, 2H), 2.64 (t, 2H), 2.72
Kwitwrrovich P., Shan B., Barnes R., Hobbs H. H., Science, 290,
1771—1775 (2000).
3
(t, 2H), 3.78 (s, 3H), 4.63 (m, 1H), 5.37 (d, 1H), 6.85 (d, 2H), 7.40 (d, 2H,)
7
.57 (s, 1H). Anal. Calcd for C H NO : C, 77.49; H, 9.91; N, 2.25%. 19) Simons K., Ikonen E., Science, 290, 1721—1725 (2000).
4
0
61
4
ꢄ
Found: C, 77.88; H, 10.21; N, 2.44%. MS (FAB) m/z 619 (M ).
20) Repa J. J., Turley S. D., Lobaccaro J.-M. A., Medina J., Li L., Lustig

May 2004
K., Shan B., Heyman R. A., Dietschy J. M., Mangelsdorf D. J.,
601
Frohlich J. J., Circulation, 99, 1733—1739 (1999).
Science, 289, 1524—1529 (2000).
1) Malini T., Vanithakumari G., J. Ethnopharm., 36, 51—55 (1992).
2) Moghadasian M. H., MaManus B. M., Godin D. V., Rodrigues B.,
23) Moghadasian M. H., Frohlich J. J., Am. J. Med., 107, 588—594 (1999).
24) Moghadasian M. H., MaManus B. M., Pritchard P. H., Frohlich J. J.,
Arterioscler. Thromb. Vasc. Biol., 17, 119—126 (1997).
2
2