Synthesis and hypolipidaemic evaluation of a series of α-asarone analogues related to clofibrate in mice

Article abstract of DOI:10.1211/0022357991772015

A series of α-asarone analogues related to clofibrate, containing an acetic acid group at C-2 of the aromatic ring, has been prepared as the acids or as the ethyl and methyl esters. The corresponding alcohols were also synthesized by reduction of the ethy

Full text of DOI:10.1211/0022357991772015

J. Pharm. Pharmacol. 1999, 51: 1±7
Received April 28, 1998
Accepted September 7, 1998
# 1999 J. Pharm. Pharmacol.
Synthesis and Hypolipidaemic Evaluation of a Series of a-
Asarone Analogues Related to Clo®brate in Mice
Ä
FERNANDO LABARRIOS, LETICIA GARDUNO*, MARIA DEL ROSARIO VIDAL, RAUL GARCIA,
MARIA SALAZAR*, ELIZDATH MARTINEZ*, FRANCISCO DIAZ, GERMAN CHAMORRO AND
AND JOAQUIN TAMARIZ,
Department of Organic Chemistry and *Department of Toxicology, (CQB), National School of Biological
 Â
Sciences, National Polytechnic Institute, PO Box 105-314, 11591 Mexico, D. F., Mexico
Abstract
A series of a-asarone analogues related to clo®brate, containing an acetic acid group at C-2
of the aromatic ring, has been prepared as the acids or as the ethyl and methyl esters. The
corresponding alcohols were also synthesized by reduction of the ethyl esters. The com-
pounds were examined in hyperlipidaemic male mice to evaluate their ability to modify
serum lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein
cholesterol and triglycerides after oral administration of 40 and 80 mg kg71 for 6 days.
Except for methyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate at either dose, these
clo®brate-related phenoxyacetic acid derivatives were found to have signi®cant hypocho-
lesterolaemic activity. Levels of low-density lipoprotein cholesterol and triglycerides were
signi®cantly reduced and those of high-density lipoprotein cholesterol were elevated. 2-
Methoxy-5-nitro-4-(2-propenyl)phenoxyacetic acid was active at both doses in all the tests.
Clo®brate (150 mg kg71) was more potent at reducing low-density lipoprotein cholesterol.
No activity was detected for the alcohol derivatives.
These preliminary results suggest that this class of compound might have more promise
as potential hypolipidaemic agents than other a-asarone derivatives. Further investigation
and characterization should be performed to determine the mode of action of these agents
on lipid metabolism.
a-Asarone (Figure 1), the active principle of the
native Mexican plant Guatteria gaumeri (annona-
ceae), has previously been shown to be a potent
hypolipidaemic agent in rodents after oral admin-
teratogenic (Salazar et al 1992) effects have been
also demonstrated in mice. These results prompted
us to undertake the synthesis and evaluation of
several non-conjugated dimethoxypropenyl deri-
vatives of a-asarone (MunÄoz et al 1993; Chamorro
et al 1998). Results of experiments with mice
revealed hypolipidaemic reduction of lipid levels in
the serum, suggesting that the lack of conjugation
of the propenyl chain with the aromatic ring does
not signi®cantly affect the pharmacological pro®le
of these a-asarone analogues.
With the aim of establishing a structure±activity
relationship for this class of promising hypolipi-
daemic agent, in this paper we address the unex-
plored role played by the C-2 methoxy group in the
activity of these compounds. We now present the
synthesis of clo®brate (1)-related phenoxyacetic
derivatives 2a and 2b, and their corresponding
methyl and ethyl esters 2c±2f. The biological
evaluation of these compounds and the alcohol
derivatives 3a±3c is also reported.
Â
istration (Gomez et al 1987; GardunÄo et al 1997).
Moreover, exposure of 3T3-hepatocyte cultures to
micromolar concentrations of a-asarone sig-
ni®cantly inhibited lipid secretion and probably
lipid synthesis, suggesting that at least part of the
hypolipidaemic effect could be a result of reduction
in the secretion of lipids by the hepatocytes (Her-
Â
nandez et al 1993). A toxicity study on long-term
cultures of adult rat hepatocytes revealed morpho-
logical and structural alterations, triacylglycerol
accumulation and inhibition of protein synthesis
Â
and secretion (Lopez et al 1993). The secretion of
proteins was the most sensitive indication of toxi-
Â
city. Mutagenic (Morales-Ramõrez et al 1992) and
Correspondence: J. Tamariz or G. Chamorro, National
School of Biological Sciences, PO Box 105-314, 11591
Â
Mexico, D. F., Mexico.

2
FERNANDO LABARRIOS ET AL
Figure 2. Synthesis of compounds 2a±2f and 3a±3c. (a)
aqueous NaOH, 5, 50ꢀC, 7 h; (b) HNO3, acetic acid, 5ꢀC, 1 h;
(c) methanol, HCl, re¯ux, 4 h; (d) ethanol, HCl, re¯ux, 4 h; (e)
LiAlH4, THF, re¯ux, 4 h; (f) Na2S2O4, methanol.
Figure 1. Chemical structures of a-asarone, clo®brate (1),
clo®brate-related phenoxyacetic derivatives (2a and 2b), the
corresponding methyl and ethyl esters (2c±2f), and the alcohol
derivatives (3a±3c).
to nitration under similar conditions, the nitro
compounds 2e (53%) and 2f (40%) were obtained
in moderate yields.
To improve the preparation of compounds 2e and
2f, an alternative approach was followed. By
esteri®cation of nitro compound 2b, under acidic
conditions (gaseous HCl, under re¯ux) the corre-
sponding alcohol derivatives, 2e and 2f were iso-
lated as pale yellow crystals in better yields (83 and
68%, respectively).
The reduction of esters 2d and 2f by treatment
with lithium aluminium hydride furnished alcohols
3a and 3b, respectively, in good yields (Figure 2).
The amino compound 3c was obtained by reduction
of the nitro alcohol 3b with sodium hydrosulphite
Materials and Methods
Synthesis
Starting materials and reagents were purchased
from Aldrich and were used without further pur-
i®cation. Thin-layer chromatography (TLC) was
performed on precoated silica gel 60 F254 plates
(Merck); short- and long-wavelength ultraviolet
light were used to visualize the spots.
Â
(Dõaz et al 1991, 1993).
Melting points were determined with a Thomas-
Hoover melting-point apparatus and are uncor-
rected. Infrared (IR) spectra were obtained with a
Perkin-Elmer 599B spectrophotometer. 1H and 13
C
Synthesis of 2-methoxy-4-(2-propenyl)phenoxyace-
tic acid, 2a
Nuclear magnetic resonance (NMR) spectra were
recorded with a Varian Gemini (300 MHz) spec-
trometer; TMS was used as internal standard. Mass
spectra were acquired with a Hewlett-Packard
5971A spectrometer. Elemental analyses were
performed by M-H-W Laboratories (Phoenix, AZ).
The preparation of compounds 2 was performed
as depicted in Figure 2. Basic treatment of 4 in the
presence of chloroacetic acid (5) furnished 2-
methoxy-4-(2-propenyl)phenoxyacetic acid (2a) in
75% yield. Nitration of 2a under mild conditions
gave a satisfactory yield of 2b. The methyl and
ethyl esters 2c and 2d were prepared in high yields
by acidic treatment of 2a with methanol and etha-
nol, respectively. When these esters were subjected
An aqueous solution of NaOH (4Á49 g, 0Á112 mol)
and chloroacetic acid (5) (7Á01 g, 0Á074 mol) in
water were successively added dropwise to com-
pound 4 (10Á65 g, 0Á065 mol) at room temperature,
keeping the temperature of the reaction mixture
below 60ꢀC. The mixture was maintained without
stirring for 1 h at room temperature and then stirred
at 50ꢀC for 7 h. A concentrated aqueous solution of
HCl (5 mL) was added and the mixture was ®ltered.
Hexane was then slowly added to this solution until
crystallization. The crystals were removed by ®l-
tration and recrystallized from hexane, giving
10Á81 g (75%) 2a as colourless needles: RF 0Á84
(CHCl3-C2H5OH, 1 : 1); mp 98±99ꢀC (Abraham et

SYNTHESIS AND EVALUATION OF a-ASARONE ANALOGS
3
al 1984, 95±97ꢀC); IR (KBr) 3520, 3400, 3200±
2400, 1750, 1520, 1255, 1230, 1160, 1030, 910,
44ꢀC; IR (CH2Cl2) 1760, 1637, 1593, 1512, 1425,
1281, 1263, 1246, 1207, 1146, 1134, 918, 784,
680 cm71; 1H NMR (CDCl3) d 6Á76 (d, J ˆ 8Á1 Hz,
1H, H-6), 6Á73 (d, J ˆ 1Á9 Hz, 1H, H-3), 6Á68 (dd,
J ˆ 8Á1, 1Á9 Hz, 1H, H-5), 5Á94 (ddt, J ˆ 16Á8,
10Á2, 6Á7 Hz, 1H, ArCH2CH ˆ ), 5Á09 (dm,
J ˆ 16Á8 Hz, 1H, Z-CH2 ˆ ), 5Á07 (dm, J ˆ 10Á2 Hz,
1H, E-CH2 ˆ ), 4Á67 (s, 2H, OCH2), 3Á86 (s, 3H,
OCH3), 3Á78 (s, 3H, CO2CH3), 3Á33 (da, J ˆ 6Á7 Hz,
2H, ArCH2); 13C NMR (75 MHz, CDCl3) d 40Á0
(C-11), 52Á2 (C-10), 55Á9 (C-7), 67Á1 (C-8), 113Á0
(C-3), 115Á0 (C-6), 116Á0 (C-13), 121Á8 (C-5), 134Á9
(C-4), 137Á5 (C-12), 145Á8 (C-1), 149Á8 (C-2), 170Á0
810 cm71
;
1H NMR (CDCl3) d 6Á83 (dm,
J ˆ 8Á0 Hz, 1H, H-6), 6Á75 (d, J ˆ 1Á9 Hz, 1H, H-
3), 6Á72 (dd, J ˆ 8Á0, 1Á9 Hz, 1H, H-5), 6Á40 (br,
1H, CO2H), 5Á94 (m, 1H, ArCH2CH ˆ ), 5Á08 (dm,
J ˆ 16Á0 Hz, 1H, CH2 ˆ ), 5Á07 (dm, J ˆ 11Á0 Hz,
1H, CH2 ˆ ), 4Á68 (s, 2H, CH2O), 3Á87 (s, 3H,
CH3O), 3Á33 (dm, J ˆ 6Á7 Hz, 2H, ArCH2CH ˆ );
13C NMR (75 MHz, CDCl3) d 40Á0 (C-10), 56Á0 (C-
7), 67Á6 (C-8), 112Á8 (C-3), 116Á1 (C-12), 116Á3 (C-
6), 121Á0 (C-5), 135Á8 (C-4), 137Á2 (C-11), 145Á6
(C-1), 149Á6 (C-2), 172Á6 (C-9); MS (70 eV) m/z
‡
‡
222 (M , 100), 207 (1), 163 (40), 147 (7), 131 (11),
(C-9); MS (70 eV) m/z 236 (M , 100), 221 (1), 209
115 (13), 107 (17), 103 (28), 91 (22), 77 (12).
Analysis: calc. for C12H14O4: C, 64Á86; H, 6Á30;
found: C, 64Á67; H, 6Á21.
(2), 177 (12), 163 (56), 131 (22), 115 (26), 103
(42), 91 (37), 77 (20). Analysis: calc. for C13H16O4:
C, 66Á09; H, 6Á82; found: C, 66Á16; H, 6Á71.
Synthesis of 2-methoxy-5-nitro-4-(2-propenyl)phe-
noxyacetic acid, 2b
Synthesis of ethyl 2-methoxy-4-(2-propenyl)phe-
noxyacetate, 2d
Conc. HNO3 (3Á5 mL, 0Á082 mol) was added
dropwise to a solution of 2a (6Á98 g, 0Á0314 mol) in
glacial acetic acid (25 mL, 0Á437 mol) at 5ꢀC,
keeping the temperature constant. The mixture was
stirred for 1 h and poured on to ice (100 g). The
precipitate was removed by ®ltration, washed with
water (3 6 20 mL), and recrystallized from hex-
ane-ethyl acetate, giving 6Á88 g (82%) 2b as a pale
brown powder: RF 0Á79 (hexane-ethyl acetate,
7 : 3); mp 105±106ꢀC; IR (KBr) 3500±2400, 1740,
1590, 1520, 1335, 1280, 1205, 1086, 1050, 930,
860, 795 cm71; 1H NMR (CDCl3) d 7Á62 (s, 1H, H-
6), 6Á95 (br, 1H, CO2H), 6Á80 (s, 1H, H-3), 5Á98
(ddt, J ˆ 17Á0, 10Á2, 6Á4 Hz, 1H, ArCH2CH ˆ ),
5Á13 (ddt, J ˆ 10Á2, 1Á5, 1Á4 Hz, 1H, CH2 ˆ ), 5Á10
(ddt, J ˆ 17Á0, 1Á6, 1Á5 Hz, 1H, CH2 ˆ ), 4Á76 (s,
2H, OCH2), 3Á98 (s, 3H, OCH3), 3Á74 (dm,
J ˆ 6Á4 Hz, 2H, ArCH2); 13C NMR (75 MHz,
CDCl3) d 37Á5 (C-10), 56Á4 (C-7), 66Á1 (C-8), 111Á5
(C-6), 113Á9 (C-3), 117Á1 (C-12), 132Á2 (C-4), 135Á1
(C-11), 141Á0 (C-5), 145Á0 (C-1), 153Á5 (C-2), 172Á2
(C-9). Analysis: calc. for C12H13NO6: C, 53Á93; H,
4Á90; N, 5Á24; found: C, 53Á73; H, 5Á03; N, 5Á29.
The same procedure as for 2c was used, but with a
saturated ethanolic solution of gaseous HCl, to give
2Á74 g (93%) 2d as colourless needles: RF 0Á5
(hexane-ethyl acetate, 7 : 3); mp 34±35ꢀC; IR
(CH2Cl2) 1759, 1639, 1592, 1512, 1464, 1434,
1295, 1279, 1246, 1200, 1146, 1073, 1034, 918,
784 cm71; 1H NMR (CDCl3) d 6Á77 (d, J ˆ 8Á2 Hz,
1H, H-6), 6Á74 (d, J ˆ 1Á8 Hz, 1H, H-3), 6Á69 (d,
J ˆ 8Á2, 1Á8 Hz, 1H, H-5), 5Á95 (ddt, J ˆ 16Á7,
10Á2, 6Á6 Hz, 1H, ArCH2CH ˆ ), 5Á09 (ddt,
J ˆ 16Á7, 3Á4, 1Á4 Hz, 1H, Z-CH2 ˆ ), 5Á07 (ddt,
J ˆ 10Á2, 3Á3, 1Á4 Hz, 1H, E-CH2 ˆ ), 4Á67 (s, 2H,
OCH2), 4Á26 (q, J ˆ 7Á1 Hz, 2H, CO2CH2), 3Á86 (s,
3H, OCH3), 3Á34 (br d, J ˆ 6Á6 Hz, 2H, ArCH2),
1Á28 (t, J ˆ 7Á1 Hz, 3H, COCH2CH3); 13C NMR
(75 MHz, CDCl3) d 14Á2 (C-11), 39Á8 (C-12), 55Á9
(C-7), 61Á2 (C-10), 67Á0 (C-8), 112Á8 (C-3), 114Á9
(C-6), 115Á8 (C-14), 120Á5 (C-5), 134Á6 (C-4), 137Á5
(C-13), 145Á8 (C-1), 149Á7 (C-2), 169Á2 (C-9); MS
‡
(70 eV) m/z 250 (M , 100), 235 (1), 223 (2), 178
(19), 163 (85), 147 (8), 131 (30), 115 (29), 103
(46), 91 (36). Analysis: calc. for C14H18O4: C,
67Á18; H, 7Á25; found: C, 67Á30; H, 7Á27.
Synthesis of methyl 2-methoxy-4-(2-propenyl)phe-
noxyacetate, 2c
Compound 2a (2Á60 g, 0Á0117 mol) was mixed with
a saturated methanolic solution of gaseous HCl
(5 mL) at room temperature. The mixture was
heated to re¯ux for 4 h. The solvent was removed
in-vacuo and the residue was crystallized in hot
hexane. After recrystallization (hexane), 2Á46 g
(89%) of 2c were obtained as colourless crystals:
RF 0Á41 (hexane-ethyl acetate, 7 : 3); mp 42Á5±
Synthesis of methyl 2-methoxy-5-nitro-4-(2-prope-
nyl)phenoxyacetate, 2e
Method A. Compound 2b (4 g, 0Á015 mol) was
mixed with a saturated methanolic solution of
gaseous HCl (20 mL) at room temperature and the
mixture was heated to re¯ux for 4 h. The solvent
was removed in-vacuo and the residue was crys-
tallized by dissolving it in the minimum amount of

4
FERNANDO LABARRIOS ET AL
ethyl acetate, cooling to 0ꢀC and adding hexane
dropwise. Recrystallization was performed simi-
larly, giving 3Á5 g (83%) 2e as pale yellow crystals.
Method B. Conc. HNO3 (2Á64 g, 0Á042 mol) was
added dropwise to a solution of 2c (5Á0 g,
0Á0212 mol) in glacial acetic acid (25 mL,
0Á437 mol) at 5ꢀC (prepared by adding acetic acid
dropwise to 2c at 5ꢀC and stirring the mixture for
15 min at the same temperature), keeping the
temperature constant. The mixture was stirred for
1 h and poured on to ice (100 g). The precipitate
was ®ltered, washed with water (3 6 20 mL) and
recrystallized from ethyl acetate-hexane, giving
3Á15 g (53%) 2e as pale yellow crystals: RF 0Á32
(hexane-ethyl acetate, 7 : 3); mp 107±109ꢀC; IR
(CH2Cl2) 3060, 1761, 1637, 1583, 1519, 1441,
1334, 1261, 1211, 1180, 1081, 1051, 998, 923, 868,
J ˆ 6Á5 Hz, 2H, ArCH2), 1Á31 (t, J ˆ 7Á1 Hz, 3H,
CO2CH2CH3); 13C NMR (75 MHz, CDCl3) d 14Á2
(C-11), 37Á6 (C-12), 56Á4 (C-7), 61Á6 (C-10), 66Á3
(C-8), 110Á8 (C-6), 113Á8 (C-3), 117Á0 (C-14), 131Á6
(C-4), 135Á2 (C-13), 140Á9 (C-5), 145Á4 (C-1), 153Á6
‡
(C-2), 168Á0 (C-9); MS (70 eV) m/z 295 (M , 20),
278 (100), 265 (22), 250 (21), 222 (40), 208 (24),
204 (22), 190 (23), 174 (23), 162 (36), 115 (32), 91
(25), 77 (27), 55 (23). Analysis: calc. for
C14H17NO6 : C, 56Á95; H, 5Á80; found: C, 57Á12; H,
5Á61.
Synthesis of 1-(2-hydroxyethyl)-2-methoxy-4-(2-
propenyl)benzene, 3a
2d (1Á0 g, 4Á0 mmol) was dissolved in dry THF
(50 mL) in a two-necked round-bottom ¯ask. The
solution was stirred and heated to re¯ux and
LiAlH4 (0Á76 g, 20 mmol) was slowly added
(2 h). The mixture was heated to re¯ux for a
further 2 h, then ®ltered, and the residue was
washed with aqueous NaOH (10%, 10 mL) and
the aqueous ®ltrate extracted with CH2Cl2
(3 6 10 mL). The original ®ltrate was con-
centrated in-vacuo, and aqueous NaOH (10%,
10 mL) was added and the mixture extracted with
CH2Cl2 (3 6 10 mL). The organic phases were
mixed, dried (Na2SO4) and evaporated in-vacuo,
giving a white solid residue which was recrys-
tallized (hexane) to give 0Á76 g (92%) 3a as
colourless crystals. RF 0Á57 (hexane-acetone,
6 : 4); mp 38±40ꢀC; IR (KBr) 3390, 1508, 1259,
1
793 cm71; H NMR (CDCl3) d 7Á57 (s, 1H, H-6),
6Á78 (s, 1H, H-3), 5Á98 (ddt, J ˆ 16Á9, 10Á2, 6Á5 Hz,
1H, ArCH2CH ˆ ), 5Á12 (ddt, J ˆ 10Á2, 1Á5, 1Á4 Hz,
1H, E-CH2 ˆ ), 5Á09 (ddt, J ˆ 16Á9, 1Á6, 1Á5 Hz, 1H,
Z-CH2 ˆ ), 4Á74 (s, 2H, OCH2), 3Á96 (s, 3H, OCH3),
3Á82 (s, 3H, CO2CH3), 3Á73 (dm, J ˆ 6Á5 Hz, 2H,
ArCH2); 13C NMR (75 MHz, CDCl3) d 37Á8 (C-
11), 52Á6 (C-10), 56Á4 (C-7), 66Á1 (C-8), 111Á0 (C-
6), 114Á0 (C-3), 117Á1 (C-13), 130Á9 (C-4), 135Á2
(C-12), 141Á0 (C-5), 145Á4 (C-1), 153Á6 (C-2), 168Á4
‡
(C-9); MS (70 eV) m/z 281 (M , 17), 264 (100),
251 (22), 222 (15), 204 (20), 190 (18), 174 (25),
162 (34), 146 (15), 133 (21), 115 (29), 91 (21), 77
(21), 55 (25). Analysis: calc. for C13H15NO6: C,
55Á51; H, 5Á37; found: C, 55Á58; H, 5Á20.
1
NMR and IR spectroscopy and melting-point
determination showed that the products obtained by
methods A and B were identical.
1131, 1075, 1027 cm71; H NMR (DMSO-d6) d
6Á86 (d, J ˆ 8Á2 Hz, 1H, H-6), 6Á78 (d,
J ˆ 1Á7 Hz, 1H, H-3), 6Á67 (dd, J ˆ 8Á2, 1Á7 Hz,
1H, H-5), 5Á94 (ddt, J ˆ 16Á8, 10Á0, 6Á8 Hz, 1H,
ArCH2CH ˆ ), 5Á14±5Á00 (m, 2H, CH2 ˆ ), 4Á59
(br s, 1H, OH), 3Á92 (t, J ˆ 4Á9 Hz, 2H,
CH2OAr), 3Á74 (s, 3H, CH3O), 3Á74±3Á69 (m,
2H, CHH 2OH), 3Á29 (br d, J ˆ 6Á6 Hz, 2H,
CH2Ar); 13C NMR (75 MHz, DMSO-d6) d 39Á1
(C-10), 55Á4 (C-7), 59Á6 (C-9), 70Á3 (C-8), 112Á5
(C-3), 113Á4 (C-6), 115Á5 (C-12), 120Á2 (C-5),
132Á4 (C-4), 138Á0 (C-11), 146Á5 (C-1), 148Á9 (C-
Synthesis of ethyl 2-methoxy-5-nitro-4-(2-prope-
nyl)phenoxyacetate, 2f
Method A. The procedure followed was the same as
for 2e (method A) but 2b was used. A saturated
ethanolic solution of gaseous HCl gave 3Á0 g (68%)
2f as pale yellow crystals.
Method B. The same procedure as for 2e (method
B) was used, but with 2d (5Á0 g, 0Á02 mol) and
2Á52 g (0Á04 mol) conc. HNO3, yielding 2Á36 g
(40%) 2f as pale yellow crystals: RF 0Á40 (hexane-
ethyl acetate, 7 : 3); mp 54±55ꢀC; IR (CH2Cl2)
3060, 1752, 1637, 1612, 1583, 1524, 1463, 1437,
‡
2). MS (70 eV) m/z 208 (M , 42), 164 (68), 149
(65), 131 (57), 115 (30), 103 (71), 78 (87), 77
(100), 65 (52), 55 (77). Analysis: calc. for
C12H16O3: C, 69Á21; H, 7Á74; found: C, 69Á38; H,
7Á51.
1
1332, 1294, 1203, 1050, 1021, 996, 878 cm71; H
NMR (CDCl3) d 7Á57 (s, 1H, H-6), 6Á78 (s, 1H, H-
3), 5Á98 (ddt, J ˆ 16Á9, 10Á2, 6Á5 Hz, 1H, ArCH2-
CH ˆ ), 5Á13 (ddt, J ˆ 10Á2, 1Á6, 1Á4 Hz, 1H, E-
CH2 ˆ ), 5Á09 (ddt, J ˆ 16Á9, 1Á65, 1Á60 Hz, 1H, Z-
CH2 ˆ ), 4Á72 (s, 2H, OCH2), 4Á28 (q, J ˆ 7Á1 Hz,
2H, CO2CH2CH3), 3Á96 (s, 3H, OCH3), 3Á72 (dm,
Synthesis of 1-(2-hydroxyethyl)-2-methoxy-5-nitro-
4-(2-propenyl)benzene, 3b
The same procedure as for 3a was used, but with 2f
(1Á0 g, 3Á4 mmol) and 0Á64 g (17 mmol) LiAlH4 to
give 0Á77 g (90%) 3b as pale yellow crystals: RF

SYNTHESIS AND EVALUATION OF a-ASARONE ANALOGS
5
0Á52 (hexane-ethyl acetate, 8 : 2); mp 90±91ꢀC; IR
darkness. A high-cholesterol diet (cholesterol 1Á0%,
sodium cholate 0Á5%, butter 5%, sucrose 30Á0%,
casein 10%, laboratory chow 53Á5%, prepared from
a powdered basal diet; Lab Rodent Diet 5001) was
made freely available for 6 days (Matsuda et al
1986). Mice fed with laboratory chow for the same
duration as above were used as non-cholesterol
control groups. Compounds, dissolved in 1 : 9
Tween 80-water, were administered orally by an
intubation needle at doses of 40 and 80 mg kg71
once a day for the duration of the experiment.
Concentrations were adjusted so that a 30-g mouse
would receive 0Á15 mL. Mice in the control group
received a similar volume of the vehicle. At the end
of 6 days, each animal was fasted for 12±15 h.
Blood samples were collected by means of an
orbital bleeding technique and centrifuged at 3000 g
for 10 min. After blood collection the animals were
killed and inspected for gross abnormalities. Total
cholesterol, low-density lipoprotein (LDL) choles-
terol, high-density lipoprotein (HDL) cholesterol
and triglycerides were determined by means of an
Abbott VP Bichromatic Analyzer.
(KBr) 3350, 1512, 1328, 1270, 1230, 1073 cm71
;
1H NMR (CDCl3) d 7Á64 (s, 1H, H-6), 6Á75 (s, 1H,
H-3), 5Á98 (ddt, J ˆ 16Á9, 10Á1, 6Á7 Hz, 1H,
ArCHCH ˆ ), 5Á15±5Á00 (m, 2H, CH2 ˆ ), 4Á17 (t,
J ˆ 4Á4 Hz, 2H, CH2OAr), 4Á00 (t, J ˆ 4Á4 Hz, 2H,
CH2OH), 3Á94 (s, 3H, CH3O), 3Á72 (br d,
J ˆ 6Á4 Hz, 2H, ArCH2), 2Á90-2Á65 (br, 1H, OH);
13C NMR (75 MHz, CDCl3) d 37Á5 (C-10), 56Á2 (C-
7), 60Á9 (C-9), 71Á0 (C-8), 110Á3 (C-6), 113Á3 (C-3),
116Á8 (C-12), 130Á7 (C-4), 135Á3 (C-11), 141Á0 (C-
5), 146Á3 (C-1), 153Á4 (C-2); Analysis: calc. for
C12H15NO5: C, 56Á91; H, 5Á97; N, 5Á53; found: C,
56Á77; H, 5Á76; N, 5Á45.
Synthesis of 5-amino-1-(2-hydroxyethyl)-2-meth-
oxy-4-(2-propenyl)benzene, 3c
3b (2Á0 g, 7Á9 mmol) was dissolved in methanol
(50 mL) by heating to re¯ux in a two-necked
round-bottom ¯ask. A solution of Na2S2O4 (5Á22 g,
30 mmol) in H2O (30 mL) was added dropwise, and
the mixture was heated for 2 h. The solution was
®ltered and the residue was washed with CH2Cl2
(2 6 10 mL). The ®ltrate was separated into two
phases and the aqueous phase was extracted with
CH2Cl2 (2 6 10 mL). The organic phase was dried
(Na2SO4) and evaporated in-vacuo, giving dark
brown crystals, which were recrystallized (hexane)
to give 0Á67 g (38%) 3c, as pale brown crystals. RF
0Á54 (hexane-acetone, 1 : 1); mp 84±85ꢀC; IR
(KBr) 3351, 1508, 1259, 1203, 1123, 1083,
1000 cm71; 1H NMR (CDCl3) d 6Á62 (s, 1H, H-3),
6Á12 (s, 1H, H-6), 5Á92 (ddt, J ˆ 16Á6, 10Á2, 6Á1 Hz,
1H, ArCHCH ˆ ), 5Á11 (ddt, J ˆ 10Á2, 1Á7, 1Á5 Hz,
1H, E-CH2 ˆ ), 5Á07 (ddt, J ˆ 17Á0, 1Á7, 1Á6 Hz, 1H,
Z-CH2 ˆ ), 4Á06±4Á03 (m, 2H, CH2O), 3Á89±3Á86
(m, 2H, CH2COOH), 3Á79 (s, 3H, OCH3), 3Á25
(ddd, J ˆ 6Á1, 1Á6, 1Á5 Hz, 2H, ArCH2), 3Á20±3Á10
(br, 3H, NH2, OH); 13C NMR (75 MHz, CDCl3) d
36Á0 (C-10), 56Á9 (C-7), 61Á3 (C-9), 71Á9 (C-8),
105Á1 (C-6), 115Á4 (C-3), 115Á9 (C-12), 117Á4 (C-4),
135Á9 (C-11), 138Á7 (C-5), 143Á1 (C-2), 147Á5 (C-
All data are expressed as meansÆ s.d. (standard
deviation)andwereanalysedstatisticallybyStudent's
t-test (Tallarida & Murray 1987). P values less than
0Á05 were considered as indicative of signi®cance.
Results and Discussion
Owing to interest in the synthesis of new hypoli-
pidaemic agents and as a continuation of our
Â
research (Dõaz et al 1993; MunÄoz et al 1993) we
designed and synthesized a new series of a-asarone
analogues and evaluated their pharmacological
activity. Table 1 list details of data on the lipid-
reducing activity of these compounds.
All animals appeared healthy throughout the
dosing period, maintaining normal food intake. No
gross abnormalities were observed in any treated
mice. Treatment of mice with the hyperlipidaemic
diet for 6 days resulted in a signi®cant elevation in
total serum cholesterol (from 3Á4Æ 0Á2 to
8Á6Æ 0Á4 mM) and LDL cholesterol (from 0Á4Æ 0Á1
to 7Á5Æ 0Á3 mM), reduction of HDL cholesterol
(from 2Á8Æ 0Á3 to 1Á4Æ 0Á1 mM) and total trigly-
cerides (from 1Á4Æ 0Á8 to 1Á4Æ 0Á4 mM). Clo®brate
(1), the reference antihyperlipidaemic drug, at a
dose of 150 mg kg71 day71, reduced cholesterol
by 39Á5%, LDL cholesterol by 49Á9%, triglycerides
by 20Á1%, and HDL by 14Á6%.
‡
1); MS (70 eV) m/z 223 (M , 88), 208 (14), 164
(100), 150 (15), 136 (61), 118 (18), 91 (13), 77 (8).
Analysis: calc. for C12H17NO3: C, 64Á55; H, 7Á67;
N, 6Á27; found: C, 64,71; H, 7Á62; N, 6Á33.
Biological screening
Experiments were performed on CF1 male mice,
Â
25±28 g, from the Instituto Nacional de Virologõa,
Secretarõa de Salud, Mexico City. The animals
Â
The reduced cholesterol levels were accompanied
by a concomitant reduction in LDL cholesterol and
triglyceride levels. Triglyceride levels were also
reduced by both doses of the alcohol derivative 3b.
were housed in hanging metal cages (two in each)
and maintained at a temperature of 24Æ 2ꢀC, 45%
relative humidity and 12-h periods of light and

6
FERNANDO LABARRIOS ET AL
Table 1. Hypolipidaemic effect of derivatives of a-asarone
Compound
Dose
(mg mg 7 1)
Total
cholesterol
Low-density High-density
lipoprotein cholesterol lipoprotein cholesterol
Triglycerides
Normal diet
Cholesterol diet
2a ‡ cholesterol diet
±
±
60Á5Æ 0Á2{
100Æ 0Á4{
24Á4Æ 0Á3{
30Á2Æ 0Á2{
17Á4Æ 0Á2{
41Á9Æ 0Á3{
23Á2Æ 0Á5*
53Á5Æ 0Á2{
41Á9Æ 0Á3{
26Á7Æ 0Á2{
12Á8Æ 0Á4
7Á6Æ 0Á08
100Æ 0Á04§
18Á7Æ 0Á06{
37Á5Æ 0Á06{
25Á0Æ 0Á04{
13Á9Æ 0Á03{
17Á4Æ 0Á08*
25Á7Æ 0Á02{
21Á5Æ 0Á02{
15Á3Æ 0Á04{
19Á4Æ 0Á03{
26Á4Æ 0Á00{
11Á1Æ 0Á04*
28Á5Æ 0Á02{
23Á1Æ 0Á04{
30Á5Æ 0Á02{
6Á2Æ 0Á03
‡ 103Á6Æ 0Á28{
100Æ 0Á11}
94Á9Æ 0Á11{
100Æ 0Á30**
48Á1Æ 0Á25{
54Á9Æ 0Á48{
43Á3Æ 0Á39{
65Á9Æ 0Á46{
52Á7Æ 0Á82{
51Á6Æ 0Á61{
11.1Æ 1Á10
40
80
40
80
40
80
40
80
40
80
40
80
40
80
40
80
40
80
150
‡ 41Á6Æ 0Á06{
‡ 33Á6Æ 0Á23
‡ 46Á7Æ 0Á11{
‡ 91Á2Æ 0Á27{
‡ 45Á2Æ 0Á06{
‡ 12Á4Æ 0Á25
48Á0Æ 0Á13{
25.5Æ 0Á09*
‡ 89Á0Æ 0Á38{
‡ 98Á5Æ 0Á71*
‡ 28Á5Æ 0Á09*
18Á9Æ 0Á16
2b ‡ cholesterol diet
2c ‡ cholesterol diet
2d ‡ cholesterol diet
2e ‡ cholesterol diet
2f ‡ cholesterol diet
3a ‡ cholesterol diet
3b ‡ cholesterol diet
3c ‡ cholesterol diet
Clo®brate (1)
45Á7Æ 0Á39*
33Á1Æ 0Á44{
35Á2Æ 1Á31*
53Á9Æ 0Á56{
45Á4Æ 0Á91{
25Á1Æ 0Á84
17Á4Æ 0Á6
41Á9Æ 0Á3{
48Á8Æ 0Á4{
15Á1Æ 0Á81
‡ 15Á1Æ 0Á60
4Á6Æ 0Á6
15Á3Æ 0Á09
45Á9Æ 0Á08{
0Á00Æ 0Á06
‡ 2Á27Æ 0Á59
28Á7Æ 0Á68*
42Á5Æ 0Á87*
12Á2Æ 1Á32
‡ 3Á9Æ 0Á6
4Á6Æ 0Á9
9Á7Æ 0Á08
‡ 30Á6Æ 0Á27
‡ 27Á0Æ 0Á23
‡ 8Á32Æ 0Á12
14Á6Æ 0Á90{
‡ 4Á2Æ 0Á05
‡ 6Á25Æ 0Á06
49Á9Æ 0Á53{
‡ 40Á7Æ 0Á4{
39Á5Æ 0Á06{
‡ 14Á30Æ 0Á80
20Á1Æ 0Á06{
Data (meanÆ standard error; n ˆ 6) are expressed as percentages of the result for the cholesterol diet group. * P < 0Á05,
{ P < 0Á01 signi®cantly different from result for cholesterol diet group. { 8Á6 mM, § 7Á5 mM; } 1Á4 mM; ** 1Á4 mM.
A statistically signi®cant increase in HDL choles-
terol was discerned after dosing with compounds
2a±2c, 2e and 2f. Analogue 2b was active at 40
and 80 mg kg71 day71 in all tests.
the carboxylic group in compounds 2 is involved in
their activity. In addition, and in agreement with
previous results (Chamorro et al 1998), the hypo-
lipidaemic activity of compounds 2a±2f, which
have the unconjugated propenyl group, also sug-
gests that conjugation of the double bond of the
side-chain with the aromatic ring is not an impor-
tant factor in determining the high hypolipidaemic
activity shown by a-asarone and some of its 4-
Clo®brate-related phenoxyacetic derivatives and
their corresponding methyl and ethyl esters, 2a±2f,
administered orally at doses of 40 and
80 mg kg71 day71, proved to be effective hypo-
lipidaemic agents in mice. In hyperlipidaemic
induced mice the drugs reduced the elevated levels
of serum cholesterol, LDL cholesterol and trigly-
cerides and increased the HDL cholesterol. At
80 mg kg71 day71 the methyl ester 2c was more
active than ethyl ester 2f or the acid 2b. The clo-
®brate-related phenoxyacetic derivative 2a and its
ethyl ester 2d also had signi®cant hypocholester-
olaemic activity. Exceptions were compound 2e,
which reduced total cholesterol, but not sig-
ni®cantly, and compound 2d, which also reduced
HDL cholesterol. However, these compounds were
active in the other tests.
The hypocholesterolaemic effect of compounds
2b, 2c and 2f was comparable with or greater than
that of clo®brate (1) at its therapeutic effective dose
of 150 mg kg71 day71. This difference was more
important for triglycerides, which all compounds
except 2d reduced by a larger amount after a
40 mg kg71 day71 dose. Compound 1 was more
effective at reducing LDL cholesterol than our
synthesized compounds.
Â
substituted derivatives (Dõaz et al 1993).
Various genetic and environmental factors con-
tribute to the development and progression of
atherosclerotic disease (Dart et al 1997). Elevated
blood cholesterol has been conclusively established
in epidemiological studies as a major independent
risk factor for coronary heart disease and other
vascular diseases (Byington et al 1995); in hyper-
lipidaemic patients who suffer from myocardial
infarction or atherosclerosis, or both, LDL choles-
terol is high and HDL cholesterol low (Hall et al
1987; Chapman et al 1989). This relationship
favours the movement of cholesterol to peripheral
tissue, including atherogenic plaques, where cho-
lesterol ester accumulates (Hall et al 1987).
The current results also showed that repeated
administration of these a-asarone analogues, con-
taining an acetic acid group on C-2 of the aromatic
ring, to hyperlipidaemic mice reduced triglyceride
levels. Although the impact of triglyceridaemia on
cardiovascular disease is controversial, recent stu-
dies have suggested that it might contribute to
cardiovascular disease by increasing the risk in
The alcohol derivatives 3a±3c did not have
consistent hypolipidaemic activity, suggesting that

SYNTHESIS AND EVALUATION OF a-ASARONE ANALOGS
7
Chapman, J. M., Sowell, J. W., Abdalla, G., Hall, J. H., Wong,
O. T. (1989) Hypolipidemic activity of cyclic imido alkyl
ethers, thioethers, sulfoxides, and sulfones. J. Pharm. Sci. 78:
903±909
Dart, A., Jerums, G., Nicholson, G., d'Emden, M., Hamilton-
Craig, J. H., Tallis, G., Best, J., West, M., Sullivan, D., Bracs,
P., Black, D. (1997) A multicenter, double-blind, one-year
study comparing safety and ef®cacy of atorvastatin versus
simbastatin in patients with hypercholesterolemia. Am. J.
Cardiol. 80: 39±44
patients who already have an adverse LDL/HDL
ratio (Hughes et al 1995).
The compounds synthesized are members of a
unique structural series which seem to have hypoli-
pidaemic activity. Theroleofdifferent substituents in
eliciting the observed activity might not be rationa-
lized until the mechanism of action is known, a
determination which was beyond the scope of this
investigation. However, on the basis of a previous
study of a-asarone which suggested that at least part
of its hypolipidaemic effect could be because of a
reduction in the secretion of lipids by hepatocytes
Â
Dõaz, F., Contreras, L., Flores, R., Tamariz, J., Labarrios, F.,
Chamorro, G., MunÄoz, H. (1991) An ef®cient synthesis of a-
asarone. Org. Prep. Proced. Int. 23: 133±138
Â
Dõaz, F., MunÄoz, H., Labarrios, F., Chamorro, G., Salazar, M.,
Morelos, M. E., Tamariz, J. (1993) Synthesis and hypolipi-
demic activity of some a-asarone analogues. Med. Chem.
Res. 3: 101±109
GardunÄo, L., Salazar, M., Salazar, S., Morelos, M. E., Labar-
rios, F., Tamariz, J., Chamorro, G. (1997) Hypolipidaemic
activity of a-asarone in mice. J. Ethnopharmacol. 55: 161±
163
Â
(Hernandez et al 1993), a similar mechanism of
action might be expected of these derivatives.
Thus, this class of potential hypolipidaemic
agents warrants further investigation and char-
acterization with regard to the mode of action of the
compounds on lipid metabolism, because the
results of this preliminary study imply that they
might have more promise as a chemically useful
agent than other a-asarone derivatives
Â
Â
Â
Gomez, C., Chamorro, G., Chavez, M. A., Martõnez, G.,
Salazar, M., Pages, N. (1987) Effet de l'a-asarone sur
Â
l'hypercholesterolemie et la cholelithiase experimentales.
Plantes Med. Phytother. 21: 279±284
Â
Â
Â
Â
Hall, J. H., Spielvogel, B. F., Sood, A., Amhed, F., Jafri, S.
(1987) Hypolipidaemic activity of trimethylamine-carbo-
methoxyborane and related derivatives in rodents. J. Pharm.
Sci. 76: 359±364
Acknowledgements
We are grateful to Q. F. I. Marõa E. Morelos
Â
for expert technical assistance and to Dr Hugo
Â
Â
Hernandez, A., Lopez, M. L., Chamorro, G., Mendoza-Fig-
ueroa, T. (1993) Alpha-asarone inhibits lipids synthesis and
secretion by long-term cultures of adult rat hepatocytes.
Planta Med. 59: 121±124
Â
A. Jimenez-Vazquez for helpful comments. J.
Tamariz acknowledges the Direccion de Estudios
Â
Â
Hughes, T. A., Elan, M. B., Applegate, W. B., Bond, M. G.,
Hughes, S. M., Wang, X., Tolley, E. A., Bittle, J. C., Stenz,
F. B., Kang, E. S. (1995) Post-prandial lipoprotein responses
in hypertriglyceridemic subjects with and without cardiovas-
cular disease. Metabolism 44: 1082±1098
Â
Â
de Posgrado e Investigacion/Instituto Politecnico
Nacional, and G. C. Consejo Nacional de Ciencia
Â
y Tecnologõa for ®nancial support. M. R. Vidal
thanks the Ludwig K. Hellweg Foundation
for a scholarship and R. Garcia thanks the
Â
Consejo Nacional de Ciencia y Tecnologõa for a
scholarship.
Â
Â
Lopez, M. L., Hernandez, A., Chamorro, G., Mendoza-Fig-
ueroa, T. (1993) a-asarone toxicity in long-term cultures of
adult rat hepatocytes. Planta Med. 59: 115±120
Matsuda, H., Chisaka, T., Kubomura, Y., Yamahara, J.,
Sawada, J. T., Fujimura, H., Kimkura, H. (1986) Effects of
crude drugs on experimental hypercholesterolemia. I. Tea
and its active principles. J. Ethnopharmacol. 2: 213±224
Â
Morales-Ramõrez, P., Madrigal-Bujaidar, E., Mercader-Martõ-
nez, J., Cassani, M., Gonzalez, G., Chamorro-Cevallos, G.,
Â
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