Synthesis and hypolipidemic and antiplatelet activities of α-asarone isomers in humans (in vitro), mice (in vivo), and rats (in vivo)

Article abstract of DOI:10.1021/jm000905n

A series of α-asarone isomers was synthesized and investigated for their hypolipidemic and antiplatelet activity. Considering the hypolipidemic activity in rats at a dose of 80 mg/kg/day, some isomers were more potent than clofibrate at 150 mg/kg. Compoun

Full text of DOI:10.1021/jm000905n

J . Med. Chem. 2000, 43, 3671-3676
3671
Syn th esis a n d Hyp olip id em ic a n d An tip la telet Activities of r-Asa r on e Isom er s
in Hu m a n s (in Vitr o), Mice (in Vivo), a n d Ra ts (in Vivo)
§
§
§
§
§
J anusz Popławski, Bo z˘ ena Łozowicka, Alina T. Dubis, Barbara Lachowska, Stanisław Witkowski,
#
#
#
‡
‡
Danuta Siluk, J acek Petrusewicz, Roman Kaliszan, J acek Cybulski, Małgorzata Strzałkowska, and
Zdzisław Chilmonczyk*
,
†
Institute of Chemistry, University of Białystok, J . Piłsudskiego 11/ 4, 15-443 Białystok, Poland,
Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gda n´ sk, Gen. J . Hallera 107,
8
0-416 Gda n´ sk, Poland, Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warszawa, Poland, and Drug Institute,
Chełmska 30/ 34, 00-725 Warszawa, Poland
Received May 1, 2000
A series of R-asarone isomers was synthesized and investigated for their hypolipidemic and
antiplatelet activity. Considering the hypolipidemic activity in rats at a dose of 80 mg/kg/day,
some isomers were more potent than clofibrate at 150 mg/kg. Compound 3 was one of the
most active agents elevating the HDL cholesterol level by 56% and lowering the LDL cholesterol
level by 46.8% in rats after 7 days of administration. The activities of the platelet aggregation
test in vitro were significant but lower than those of the reference substances (indomethacine
and acetylsalicylic acid). In the pulmonary thromboembolic in vivo test in mice, two compounds
(
R-asarone (6) and compound 4) produced significant antithrombotic effects at 100 mg/kg,
namely 44% and 52% protection against lung microembolia, respectively. R-Asarone derivatives
form a new group of potential hypolipidemic and/or antithrombotic agents. The compounds 3,
4
, and 6 may serve as lead substances whose structural modifications may result in original
drugs.
In tr od u ction
Since it has been discovered that acetylsalicylic acid
inhibits the activity of thrombocytes, new antiplatelet
agents have been of particular interest. Substances
affecting the platelet function belong to different chemi-
cal groups and exhibit diverse mechanisms of action.
Two main groups can be distinguished among antiplate-
let drugs: agents affecting biotransformation of arachi-
donic acid and drugs with other mechanisms of action.6
Cholesterol is required in the assembly of membranes
and performs other important biological functions.
However, when plasma cholesterol exceeds the level
required for these functions, development of atheroscle-
rosis and related diseases takes place. Hyperlipopro-
teinemia, and especially hypercholesterolemia, are two
risk factors connected with the development of athero-
sclerosis and thrombosis. Particularly, a high LDL
cholesterol level plays an important role in atherogen-
Chemical structures of the agents modifying platelet
functions are comprised of phenol, phenylethanol, imi-
dazole, triazole, thiazole, pyrazolpyridine, indole, car-
bamoilpiperidine, piperazine, and pyrazine. Among
substances of plant origin, the flavonoids that are benzo-
γ-pyrone derivatives are of importance because of their
inhibitory effect on capillary permeability and antiin-
flammatory, spasmolytical, diuretic, and antiaggrega-
1
esis process. The dietary and pharmacological lowering
of elevated plasma LDL cholesterol appears to be one
of the methods to reduce the development of athero-
2
sclerosis.
Thrombocytes and their functions are also under
particular investigation from the viewpoint of the
pathogenesis of atherosclerosis, thrombosis, and acute
coronary syndromes.3 It was demonstrated that in
animals with thrombocytopenia, atherosclerotic lesions
7
tory activity. Natural procyanidines have also been
8
shown to exert antiplatelet activity.
4
R-Asarone (6), occurring in nature in the common
asarabacca root Asarum europaeum L. and calamus
Acorus calamus L. roots, exhibits a wide spectrum of
are smaller or plaques are not observed. It was also
shown that in individuals with atherosclerotic alter-
ations, the platelets exhibit a higher aggregability in
5
9
vivo. Excess lipoprotein concentration enhances platelet
biological activity. It was isolated from the Guatteria
sensitivity to the activating agents. It is well-established
that antiplatelet treatment lowers vascular disease
mortality and the risk of myocardial infarction and
stroke incidence in patients with unstable ischemic
heart disease.
guameri plant growing in Southeast Mexico. The plant
has been applied in traditional Mexican human medi-
cine in the treatment of symptoms of hypercholester-
10
olemia and cholelithiasis.
Gomez et al.11 studied the effect of R-asarone in rats
with hypercholesterolemia. They found that R-asarone
administered at the dose of 4-25 mg/kg/day lowered
triglyceride and cholesterol levels in plasma for 40-60%.
Other investigators reported various other biological
effects of R-asarone: sedating, neuroleptic, spasmolytic,
*
To whom correspondence should be addressed. Tel: +48228515229.
Fax: +48228410652. E-mail: chilmon@il.waw.pl.
§
University of Białystok.
Medical University of Gda n´ sk.
Pharmaceutical Research Institute.
#
‡
†
Drug Institute.
1
0.1021/jm000905n CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/09/2000

3
672 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Popławski et al.
Sch em e 1. Synthesis of Asarone Derivatives 1-3 and
Sch em e 3. Synthesis of Compound 5a
a
6
a
Reagents and conditions: (i) n-BuLi, TMEDA; (ii) CH3CH2CHO;
(iii) CuSO4, toluene, 2 h, 110 °C.
chromatography or recrystallization from petroleum
ether separated the two isomers completely.
a
Reagents and conditions: (i) CH3CH2Br, Mg, THF(anhyd), 3
h; (ii) CuSO4(anhyd), toluene, 2 h, 110 °C.
Sch em e 2. Synthesis of Compound 4a
Biologica l Resu lts
Hyp olip id em ic Activity. The hypolipidemic activity
study in rats at 80 mg/kg/day showed that some isomers
of R-asarone were more active than the reference
clofibrate at its therapeutic dose (150 mg/kg/day; the
results were in agreement with the results obtained by
Diaz et al. ). The cholesterol and trigliceride levels
were decreased by 24.1% and LDL cholesterol increased
by 6.7% (Table 1).
v
1
0b
a
Reagents and conditions: (i) H2CdCHCH2Br, K2CO3, acetone,
h, 50 °C; (ii) oil bath, 8 h, 180 °C; (iii) H2O2, KOH, H2O, 24 h;
2
(CH3)2SO4, 10% water solution KOH, 1 h, 80 °C; (iv) T, KOH,
R-Asarone at a dose of 80 mg/kg caused 2.3% increase
of the total cholesterol level. At the same time HDL
cholesterol was increased by 56.7% and LDL cholesterol
was decreased by 43.2%. Compounds 1-4 showed
similar hypolipidemic effects affording 15% reduction
of serum cholesterol. Compound 2 exhibited the maxi-
mum hypotriglycemic activity. The data from the rat
serum lipoprotein fractions study (Table 1) for com-
pounds 1-4 show that HDL cholesterol levels were
elevated and LDL cholesterol levels were reduced. The
compounds increased the HDL cholesterol level signifi-
cantly higher than clofibrate. The isomer 3 exhibited
the best hypolipidemic activity as measured by 56%
elevation and 56% reduction of HDL and LDL choles-
terol levels, respectively.
ethanol(anhyd), 24 h, 70 °C.
antiulcerogenic, and antiatherogenic.12,9b Also antihel-
minthic, teratogenic, and carcinogenic actions were
noted.
It appeared interesting to test a series of R-asarone
isomers with regard to their influence on HDL and LDL
cholesterol levels, as well as their in vitro antiaggrega-
tory and in vivo antithrombotic activity.
1
3
Ch em istr y
The compounds employed in this study were synthe-
sized according to Schemes 1-3. Scheme 1 illustrates
the synthesis of 4-(prop-1-enyl)-1,2,3-trimethoxybenzene
(
5
1), 3-(prop-1-enyl)-1,3,5-trimethoxybenzene (2), and
-(prop-1-enyl)-1,2,3-trimethoxybenzene (3) by using
typical conditions of the Grignard reaction. Aldehydes
a , 2a , or 3a were treated with ethylmagnesium bro-
Effect on P la telet Aggr ega tion . R-Asarone and two
of its derivatives were tested in the study. For the sake
of comparison, selected nonsteroidal antiinflammatory
drugs were also tested. Indomethacine and acetylsali-
cylic acid served as such reference drugs.
1
mide in dry THF to afford appropriate trimethoxy(1-
hydroxypropyl)benzene. Resulting alcohols were then
subjected to dehydration in the next step. The yield of
reaction strongly depends on the temperature and
duration of the elimination process.
The preparation of compound 4 was achieved by
formation of allyl ether from vaniline (4a ), Claisen
rearrangement followed by the Dakin reaction, and
methylation of the phenol formed. Isomerization of the
double bond catalyzed by base (KOH) was the final step
Indomethacine was used at concentrations of 5, 10,
15, 20, and 25 µM. At the concentration of 25 µM it
inhibited ADP-induced aggregation by 55%. The mean
-5
IC50 value for indomethacine was 2.4 × 10 M. Ace-
tylsalicylic acid was used at concentrations of 100, 150,
200, and 250 µM. At the highest studied concentration
it inhibited platelet aggregation by 56% (Table 2). Its
-
4
15
IC50 value was 2.12 × 10 M.
Asarones were tested at five concentrations: 100, 150,
200, 250, and 300 µM. Similar to the indomethacine test,
the aggregation-inducing substance was ADP. R-Asa-
rone at 300 µM concentration caused 47.0 ( 19.0%
inhibition of aggregation. Compounds 2 and 4 added at
analogous concentrations gave 17.5 ( 4.0% and 38.3 (
13.8% inhibition, respectively (Table 2).
(Scheme 2). The best result was achieved using ethanol
as a solvent in the isomerization process.
The isomer 5 was synthesized by lithiation of 1,2,4-
1
4
trimethoxybenzene (5a ) by means of n-buthyllithium
in the presence of N,N,N′,N′-tetramethylethylenedi-
amine (TMEDA), followed by treatment with propional-
dehyde. The resulting alcohol was subjected to dehy-
dration by anhydrous CuSO4 (Scheme 3).
In all cases the elimination reaction gave a 93:7
mixture of the E and Z isomers of olefins 1-5. Column
Effect on P u lm on ar y Th r om boem bolism in Mice.
At first the reference substances acetylsalicylic acid
(ASA) at a dose of 25 mg/kg and ketorolac at a dose of
100 mg/kg mouse body weight were tested after ip

Synthesis and Activities of R-Asarone Isomers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3673
Ta ble 1. Alteration of Blood Lipoprotein Levels by R-Asarone Isomers
dose
total cholesterol LDL cholesterol HDL cholesterol
atherogenic
compound
normal dieta
cholesterol diet (CD)
clofibrate + CD
R-asarone + CD
(mg/kg)
(mg/dL)
(mg/dL)
(mg/dL)
triglicerides
LDL/HDL
index
66.8 ( 2.00**
110.9 ( 5.70
8.9 ( 1.11**
43.2 ( 2.91
46.1 ( 5.44
24.4 ( 6.70**
30.1 ( 8.69
36.9 ( 6.90
19.8 ( 5.60
35.8 ( 4.22
48.2 ( 4.30
45.2 ( 1.43**
29.0 ( 1.43
76.1 ( 3.90**
125.1 ( 6.80
**
0.20
1.49
2.08
0.54
0.70
0.97
0.44
0.91
1.59
0.52
2.82
2.87
1.45
1.26
1.58
1.06
1.50
2.80
*
*
**
150
80
80
80
80
80
80
85.8 ( 5.50
113.6 ( 9.20
96.6 ( 6.80
98.1 ( 6.42
93.4 ( 11.0
97.9 ( 3.70
115.1 ( 9.30
22.1 ( 2.26
91.6 ( 12.20
**
**
45.5 ( 2.37*
219.3 ( 26.7
124.6 ( 10.5
116.2 ( 5.84
135.7 ( 14.01
140.2 ( 10.31
161.5 ( 18.40*
*
1
2
3
4
5
+ CD
+ CD
+ CD
+ CD
+ CD
42.8 ( 3.20
**
37.9 ( 2.75
45.3 ( 2.43*
*
39.2 ( 2.43**
30.2 ( 2.81
a
Group receiving laboratory chow and the vehicle oil. *p < 0.05 and **p < 0.01 vs cholesterol diet group, n ) 9-10.
Ta ble 2. Percent Inhibition of Human Platelet Aggregation
Induced by ADP after Incubation with Acetylsalicylic Acid,
R-Asarone, and Its Isomersa in Vitro
cholesterol and LDL cholesterol as compared to the
controls. One of the major features of these agents is
that they lower LDL and elevate HDL contents. HDL
cholesterol was significantly increased in the cases of
compounds 1-4 which appear to be more active than
%
inhibition (mean ( SEM)c
concn of
agent (µM)
acetylsalicylic
b
16
acid
compd 2
compd 4
compd 6
4.0 ( 2
clofibrate at its therapeutic dose of 150 mg/kg/day.
1
1
2
2
3
00
50
00
50
00
31.2 ( 5.1
37.1 ( 4.9
48.5 ( 6.7
56.4 ( 5.3
15.9 ( 4.3 13.0 ( 5.4
The HDL fraction leads cholesterol out of the tissue
19.0 ( 6.1 25.4 ( 10.8 14.7 ( 10
31.0 ( 6.4 35.4 ( 10.3 40.3 ( 17
7.5 ( 3.6 38.3 ( 15.2 30.3 ( 17
17.5 ( 4.0 38.3 ( 13.8 47.0 ( 19
17
and protects against atherosclerosis. The LDL fraction
is generally thought to carry cholesterol to the tissues
and is responsible for the atherogenesis process. De-
creasing the ratio of the plasma level of LDL to that of
HDL seems to play an important role in reducing the
risk of atherosclerosis. Compound 3 showed a 5 times
lower value of this ratio than clofibrate (Table 1).
Similarly, compounds 1, 2, and 4 had that value in the
range of 0.7-1, whereas the LDL/HDL ratio for clofi-
brate is 2.08.
a
The percentage inhibition of platelet aggregation ) [(C - A)/
C] × 100%, where C is the percentage aggregation of PRP induced
by 10 µM ADP after incubation with the vehicle (control) and A is
the percentage aggregation of PRP induced by 10 µM ADP after
incubation with the studied compounds. Data from ref 15. c The
data are means from 4-5 experiments.
b
Ta ble 3. Protective Effect of Acetylsalicylic Acid, Ketorolac,
R-Asarone (6), and Its Isomers on Mice after Thrombotic
Challenge Induced by a Mixture of Collagen and Epinephrine
The analysis of the atherogenic index (total choles-
18
terol - LDL/HDL) (Table 1) showed that compound 3
dose
(mg/kg)
no. killed or paralyzed/
no. tested
%
was endowed with potent hypolipidemic activity. Its
derivatives will be the subject of a further study.
Three asarone isomers were investigated (R-asarone
agent
control
protected
4/24
15
sodium acetate
acetylsalicylic acid
control
(
6) and compounds 2 and 4) in the in vitro platelet
25
28/51
27/35
45a
23
aggregation test. The compounds did not exert evident
antiaggregatory activity in vitro against the ADP-
induced platelet aggregation. The observed effects are
much weaker as compared to those of indomethacine.
Nevertheless, the absence or a low activity in vitro does
not disqualify the substances tested as potential anti-
platelet agents. Ticlopidine, one of the most frequently
used antiplatelet drugs, also does not show any in vitro
DMSO buffer
ketorolac
control
100
16/24
45/55
33
18
Miglyol-812
R-asarone (6)
1
1
2
3
4
50
10/18
100
100
100
13/13
0
a
00
44
14/14
30/40
17/21
12/25
0
25
19
19
activity.
b
100
52
On the basis of the pharmacological tests carried out,
we conclude that R-asarone (6) and isomer 4 exert
antithrombotic action in vivo, although the activity of
the agents is significantly lower than that of acetylsali-
cylic acid. Compounds 1-3 did not show any antithrom-
botic activity in our in vivo studies.
R-Asarone derivatives appear to be a new promising
original group of potential hypolipidemic and anti-
thrombotic drugs and deserve further investigation. The
compounds 3, 4, and 6 may serve as lead substances
whose structural modifications may result in potent
original antithrombotic drugs.
a
Significantly different from controls, p < 0.05; data from ref
1
5. b Significantly different from controls, p < 0.01.
administration. ASA prevented pulmonary microembo-
lism in mice at 45%, while ketorolac gave 33% protection
Table 3).
(
R-Asarone administered intraperitoneally at a dose
of 100 mg/kg gave 44% protection (p < 0.05), while a
dose of 50 mg/kg did not give any statistically significant
protection (Table 3). The strongest antithrombotic activ-
ity among the asarone isomers was found for compound
4
(
(100 mg/kg) which showed 52% (p < 0.01) protection
Table 3).
Exp er im en ta l Section
Discu ssion
Ch em ica ls a n d Rea gen ts. A series of newly synthesized
R-asarone isomers (1-5) was subjected to pharmacological
studies. Other substances used in the study were purchased
from the following sources: R-asarone from Aldrich-Chemie
(Steiheim, Germany), acetylsalicylic acid (ASA) and ketorolac
from Polpharma S.A. (Starogard Gda n´ ski, Poland), indometha-
Our data demonstrated that a series of R-asarone
isomers are potent hypolipidemic agents in rats (Table
1
). The present study shows that some R-asarone
isomers decrease and some increase the level of total

3
674 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Popławski et al.
cine from Polfa Krak o´ w (Krak o´ w, Poland), clofibrate from Polfa
Grodzisk (Grodzisk Mazowiecki, Poland), miglyol 812 from
Caelo (Hilden, Germany), and ADP, collagen and epinephrine
from Sigma (St. Louis, MO). Sodium chloride and sodium
acetate were obtained from Polish Chemical Reagents (Gliwice,
Poland), dimethyl sulfoxide from Ubichem Ltd. (Hampshire,
U.K.), sodium cholate from Aldrich (Pozna n´ , Poland) and
murigram from Lomianki (Poland). All used kits were pur-
chased from BioMerieux (Poland).
The resulting mixture was refluxed for 2 h. After cooling the
mixture was diluted by addition of water and the inorganic
material was filtered off. The filtrate was extracted with ether.
The organic extract was washed with 10% solution of NaOH
4
and with water and next dried over MgSO . After the evapora-
tion of the solvent, crude vanillin allyl ether was subjected to
Claisen rearrangement. The ether was heated for 8 h at 185
°C in an oil bath. After cooling, the product was distilled under
a reduced pressure to give 3 g of 3-methoxy-4-prop-2-enylben-
zaldehyde (bp 100-107 °C, 5-7 mmHg). The resulting com-
pound was oxidized with 50 mL of 3% solution (0.021 mol) of
hydrogen peroxide. After 24 h, the mixture was neutralized
Ch em ica l Meth od s. Melting points were determined on a
1
K o¨ ffler apparatus of the B o¨ etius type and are uncorrected. H
1
3
and C NMR spectra were recorded on a Bruker AC 200F
spectrometer using CDCl solution with TMS as internal
2 4
with a 10% H SO solution. Yellow precipitates were collected
by filtration and recrystallized from hexane to give 2.9 g of
3
standard (chemical shifts in δ, ppm). IR spectra were recorded
on a Nicolet Magna 550 FTIR spectrometer in chloroform
solutions. The UV spectra were collected on a Hewlett-Packard
UV-vis diode array spectrophotometer 8452A. Mass spectra
were obtained at 70 eV with an AMD-604 spectrometer. The
reaction products were isolated by column chromatography
performed on silica gel 70-230 mesh ASTM (Merck). Thin-
layer chromatograms were developed on aluminum TLC sheets
precoated with silica gel F254. The spots were visualized with
2
-methoxy-6-prop-2-enylbenzene-1,4-diol.
A solution of 2.9 g (0.016 mol) of this product and 2.3 g (0.04
mol) of KOH in 40 mL of water was cooled to 10 °C and 1.5
mL (0.016 mol) of dimethyl sulfate was added dropwise. The
mixture was warmed for 1 h at 80 °C. After cooling the organic
layer was separated. The aqueous layer was extracted with
ether. Organic extracts were combined and washed with 10%
solution of NaOH and with water. After drying, the solvent
was removed and the residue (2 g) was subjected to the next
step.
5
0% sulfuric acid after heating. All the solvents were dried
and freshly distilled prior to use.
Syn th esis of 4-((E)P r op -1-en yl)-1,2,3-tr im eth oxyben -
zen e (1), 2-((E)P r op -1-en yl)-1,3,5-tr im eth oxyben zen e (2),
a n d 5-((E)P r op -1-en yl)-1,2,3-tr im eth oxyben zen e (3). 9.1
mL (0.1 mol) of 1-bromoethane in 20 mL of THF was added
dropwise to a mixture of 2.4 g (0.1 mol) of magnesium turnings
in 15 mL of dry THF. The solution of 0.02 mol of trimethoxy-
benzaldehyde (1a , 2a or 3a ) in 20 mL of THF was added
dropwise after the formation of Grignard reagent. After 3 h of
stirring, 50 mL of methanol was added and the resulting
solution was diluted with water and extracted with ether. The
A solution of 2 g (0.01 mol) of 1,3,5-trimethoxy-3-prop-2-
enylbenzene in 25 mL of anhydrous ethanol and 32 g (0.6 mol)
of KOH was heated 24 h under reflux. The mixture was diluted
with water and extracted with ether. The ethereal solution
was dried over MgSO , filtered and evaporated. The residual
4
solid was treated with hexane, separated by filtration and
purified by recrystallization (petroleum ether bp 40-60 °C)
to give 4 (1.4 g, 70%): mp 44-45 °C; IR (CHCl
3
) 3015, 1605,
-
1
1
1090 cm ; H NMR (200 MHz, CDCl
1.68 Hz, J
3
) δ 6.71 (dq, 1H, J )
1
2
) 15.9 Hz); 6.55 (d, 1H, J ) 2.83 Hz); 6.53 (d, 1H,
organic extract was washed with water and dried over MgSO
4
.
ArH); 6.38 (d, 1H, J ) 2.84 Hz); 6.25 (dq, 1H, J ) 6.6 Hz, J )
The mixture was filtered and concentrated under a reduced
pressure. The crude product was used without any purification
in the next step.
15.9 Hz); 3.84 (s, 3H); 3.79 (s, 3H); 3.75 (s, 3H); 1.92 (dd, 3H,
J
1
) 1.67 Hz, J
140.3 (C); 131.7 (C); 127 (C); 100.2 (CH); 125 (CH); 98.9 (CH);
0.9 (CH ); 55.7 (CH ); 55.4 (CH ); 18.7 (CH ); UV λmax (nm)
2
) 6.59 Hz); 13C NMR 156.0 (C); 153.5 (C);
6
3
3
3
3
A solution of crude alcohol (0.02 mol) in 50 mL of dry toluene
2
0
220 (ꢀ ) 40262), 254 ((ꢀ ) 16073), 304 (ꢀ ) 4145); MS m/z )
08 (M , 100), 193 (91%), 178 (10%), 165 (40%), 150 (21%),
05 (18%).
was treated with 4.8 g (0.03 mol) of CuSO
4
.
The reaction
+
2
1
mixture was refluxed 2 h. An inorganic material was filtered
off and washed with toluene. After the evaporation of the
solvent, the residue was purified by silica gel column chroma-
tography (elution with hexane/ether, 3:7, v/v) giving 1, 2 or 3.
Compound 2 was recrystallized from petroleum ether (bp 60-
Syn th esis of 2-((E)P r op -1-en yl)-1,3,4-tr im eth oxyben -
zen e (5). To a solution of 1,2,4-trimethoxybenzene (3.024 g,
18 mmol) was added n-butyllithium (21.0 mL of 15% hexane
solution) in 100 mL of dry hexane under neutral gas via
syringe. After 1 h TMEDA (2.45 mL) and propanal (1.94 mL,
27 mmol) were added dropwise. The mixture was stirred for
8
0 °C).
3
Compound 1: mp 25-26 °C; IR ν(CHCl ) 3020, 1600, 1100
-
1 1
cm ; H NMR (CDCl , 200 MHz) δ 7.10 (d, 1H, J ) 8.70 Hz);
3
1
0 min and 2 mL of water was carefully added. The resulting
6
1
3
6
.63 (d, 1H, J ) 8.70 Hz); 6.58 (dq, 1H, J
5.9 Hz); 6.13 (1H, dq, J ) 6.57 Hz, J ) 15.9 Hz); 3.88 (s,
H); 3.86 (s, 3H); 3.85 (s, 3H); 1.89 (3H,dd, J ) 1.67, J
.57); C NMR 152.4 (C); 150.8 (C); 142.2 (C); 125.0 (C); 124.9
); 60.6
); UV λmax (nm) 264 (ꢀ ) 15300);
1
) 1.66 Hz; J )
2
mixture was evaporated to dryness and the residue was
1
2
extracted with methylene chloride (3 × 50 mL). The combined
1
2
)
1
3
2 4
extracts were washed with 15% solution of H SO and dried
4
over MgSO . After evaporation to dryness the crude product
(
CH); 124.8 (CH); 120.3 (CH); 107.6 (CH); 60.8 (CH
CH ); 55.8 (CH ); 18.6 (CH
3
was purified by column chromatography using carbon tetra-
chloride-ethyl acetate mixture (30:1, v/v) as an eluent. 1.8 g
of pure 1-(2,3,6-trimethoxyphenyl)-1-propanol was obtained.
(
3
3
3
+
MS m/z ) 208 (M , 100%), 193 (18%), 179 (24%), 133 (31%).
-
1 1
Compound 2: mp
NMR (200 MHz, CDCl
H, J
s, 3H); 1.91 (d, 3H, J ) 4,9 Hz); C NMR 159.3 (C); 158.7
C); 127.8 (CH); 120.9 (CH); 108.8 (C); 90.6 (CH); 55.6(CH );
); UV λmax (nm) 268 (ꢀ ) 28953), 222 (ꢀ )
.
72-73 °C; IR (CHCl
) δ 6.59 (d, 1H, J ) 16.1 Hz); 6.49 (dq,
) 16.1 Hz); 6.15 (s, 2H); 3.83 (s, 6H) 3.82
3
) 3022, 1605 cm ; H
3
4
The alcohol and anhydrous CuSO (1.50 g, 9.3 mmol) in 50
1
1
) 5.45 Hz, J
2
mL of dry toluene were refluxed for 2 h. The inorganic salt
was filtered off and the filtrate was evaporated to dryness.
The residue was purified by column chromatography (carbon
tetrachloride-ethyl acetate mixture, 20:1, v/v) to give com-
1
3
(
(
3
5
3
5.2 (CH
8248); MS m/z ) 208 (M , 100%), 193 (13%), 179 (61%), 151
17-18 °C; IR (CHCl ) 3022, 1610
cm ; H NMR (200 MHz, CDCl ) δ 6.56 (s, 2H); 6.34 (dq, 1H,
) 15.7 Hz); 6.17 (dq, 1H, J ) 6.2 Hz, J
5.7 Hz); 3.87 (s, 6H); 3.84 (s, 3H); 1.89 (dd, 3H, J ) 1.23 Hz,
) 6.22 Hz); C NMR 153.2 (C); 133.7 (C); 130.8 (CH); 125.2
); 55.9 (CH ); 18.3 (CH );
3 3
); 20.0 (CH
+
-1
-1
3
pound 5 (1.4 g): IR (CHCl , cm ) 3020, 1610, 1050 cm ; UV
1
(
25%). Compound 3: mp
.
3
λ
max (nm) 264 (ꢀ ) 15300); H NMR (200 MHz, CDCl
3
) δ 6.72
-
1 1
3
(d,1H, J ) 9.0 Hz); 6.67 (dq, 1H, J ) 5.57 Hz, J ) 16.0 Hz);
1
2
J
1
J
1
) 1.28 Hz, J
2
1
2
)
6.58 (d, 1H, J ) 9.0 Hz); 6.57 (d, 1H, J ) 9.0 Hz); 3.83 (s, 3H);
13
1
3.80 (s, 3H); 3.78 (s, 3H); 1.94 (d, 3H, J ) 5.1 Hz); C NMR
1
3
2
152.0 (C); 147.6 (C); 147.2 (C); 131.3 (CH); 121.3 (CH); 121.1
(
CH); 129.8 (C); 102.7 (CH); 60.8 (CH
3
3
+
3
3 3 3
(C); 110.2 (CH), 105.8 (CH), 60.1 (CH ); 56.2 (CH ); 55.8 (CH );
+
UV λmax (nm) 266 (25932); MS m/z ) 208 (M , 100%), 193
15%), 179 (57%), 151 (20%), 121 (16%).
3
20.0 (CH ); MS m/z ) 208 (M , 100), 193 (32%), 179 (6%), 165
(
(13%), 150 (14%).
Syn th esis of 3-((E)P r op -1-en yl)-1,2,5-tr im eth oxyben -
Hyp olip id em ic Activity. Wistar male rats weighing 200-
250 g were purchased from the Pharmaceutical Research
Institute, Warsaw, Poland. The animals were equally divided
into groups of about 10 animals each and maintained at a
zen e (4). To a stirred solution of 5 g (0.033 mol) of vanillin
4a ) and 5.5 g (0.04 mol) of anhydrous K CO in 50 mL of dry
acetone was added allyl bromide (3.4 mL, 0.04 mol) dropwise.
(
2
3

Synthesis and Activities of R-Asarone Isomers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3675
temperature of 24 ( 2 °C, 45% relative humidity, and 12 h
periods changing of light and darkness. All rats were fed a
high-cholesterol diet (Murigram enriched with cholesterol 1%,
sodium cholate 0.2%, and olive oil 5%) for 7 days. Simulta-
neously, compounds diluted in oil were administered through
a gastric intubation at 80 mg/kg once a day for the duration
of the experiment. The group receiving clofibrate (150 mg/kg)
served as a positive control. Compounds, diluted in oil, were
adjusted so that the rats were administered a volume of 5 mL/
kg of body weight. Rats fed with laboratory chow for the same
duration as above were used as the non-cholesterol control
group. The control group received a similar volume of vehicle
oil. At the end of a 7-day period, each animal was fasted for
Refer en ces
(
1) (a) Russel, R. The pathogenesis of atherosclerosis - an update.
N. Engl. J . Med. 1986, 314, 488-500. (b) Gammill, R. B.; Day,
Ch. E.; Schurr, P. E.; Khellin analogues. 1. General topological
requirements for lipid-altering activity in furochromones. J . Med.
Chem. 1983, 26, 1672-1674.
(2) Marx, J . L. The HDL: The good cholesterol carriers? Science
1
979, 205, 677-679.
(
3) (a) Mann, K. G.; Tracy, P. B.; Krishnaswamy, S.; J enny, R. J .;
Odegaard, B. H.; Nesheim, M. E. Platelets and coagulations.
Thrombosis and Haemostasis; Leuven University Press: Brus-
sels, 1987; pp 505-523. (b) Arniral, J ., Farud, J . Thromboembolic
diseases: biochemical mechanisms and new possibilities of
biological diagnosis. Semin. Thromb. Hemostas. 1996, 22 (Suppl.
1
), 41-48. (c) Fuster, V.; Badimon, L.; Cohen, M.; Ambrose, J .
1
6 h and anesthetized with ether. Blood samples were collected
A.; Badimon, J . J .; Chesebro, J . H. Insights into the pathogenesis
through ocular puncture and centrifuged at 3000 rpm. Total
cholesterol, HDL cholesterol, LDL cholesterol and triglicerides
were determined using BioMerieux kits on a Shimadzu UV
spectrophotometer.
Acu te P u lm on a r y Th r om boem bolism Test in Mice.
Male Swiss mice were obtained from the Pharmaceutical
Institute, Warsaw, Poland. Mice were fed with conventional
food and water ad libitum. The experiments were performed
as described by DiMinno and Silver.21 A pulmonary throm-
boembolic death or severe paralysis was induced in mice
weighing 29.4 ( 2.6 g by a rapid administration into the tail
vein of a mixture of collagen (25 µg/mouse) and epinephrine
of acute ischemic syndromes. Circulation 1988, 77, 1213-
1
220.
(
(
(
4) Raines, E.; Ross, R. Smooth muscle cells and the pathogenesis
of the lesions of atherosclerosis. Br. Heart J . 1993, 69 (Suppl.),
30-37.
5) Elwood, P.; Renaud, S.; Sharp, D.; Besewiick, A.; O’Brien, J .;
Yarnel, W. Ischaemic heart disease and platelet aggregation.
Circulation 1991, 83, 38-44.
6) (a) Fitzgerald, G. A.; Meagher, E. A. Antiplatelet drugs. Eur. J .
Clin. Invest. 1994, 24 (Suppl. I), 46-49. (b) Fareed, J . Current
trends in antithrombotic drug and device development. Semin.
Thromb. Hemost. 1996, 22 (Suppl. I), 3-8.
(7) Swies, J .; Robak, J .; Dıbrowski, L.; Dunies, Z.; Michalski, Z.;
Gryglewski, R. J . Antiaggregatory effects of flavonoids in vivo
and their influence on lipooxygenase and cyclooxygenase in vitro.
Pol. J . Pharm. 1984, 36, 455-461.
(
1
27 µg/mouse). Mortality or paralysis was registered within
0 min after injection. Tested substances were given intra-
peritoneally as single doses 1 h before the thrombotic challenge
in 0.1 mL of solvent/10 g of animal mass. Asarones were
dissolved in miglyol 812, ketorolac in the mixture of DMSO (1
mL) and S o¨ erensen phosphate buffer, pH ) 7.0 (1.5 mL),
acetylsalicylic acid in 0.3 M sodium acetate, collagen and
epinephrine in 0.154 M sodium chloride. The vehicles were
administered to animals in the same way as the substances
studied, and their protection activities were treated as control
values. The magnitude of the control data is similar to that
(
8) Chang, W.-Ch.; Hsu, F.-L. Inhibition of platelet aggregation and
arachidonate metabolism in platelets by procyanidins. Prostag-
land. Leuk. Essent. Fatty Acids 1989, 38, 181-188.
(9) (a) Gracza, L.; Spaich, W. Analytische und biopharmazeutische
Untersuchung transisoasaronhaltiger Preparate. Planta Med.
1
978, 33, 160-169. (b) Belova, L. F.; Alibekov, S. D.; Baginskaya,
A. I.; Sokolov, S. Ya.; Pokrovskaya, G. V. Asarone and its
biological properties. Farmakol.-Toksikol. 1985, 48, 17-20. (c)
Shirokova, E. A.; Segal, G. M.; Torgov, I. V. Synthesis (E) and
(
Z)-asarones and its analogues. Bioorg. Chim. 1985, 2, 270-
1
5
reported previously.
272. (d) Hernandez, A.; Lopez, M. L.; Chamorro, G.; Mendoza-
Figueroa, T. Inhibition of lipid synthesis and secretion in long-
term culture of adult rat hepatocytes by R-asarone. Planta Med.
The results of the test were expressed as mortality (paraly-
sis) rate, i.e., the total number of animals used and the
percentage of those surviving.
1
993, 59, 121-124.
(
10) (a) Enqiquez, R. G.; Ch a´ vez, M. A.; J a´ uregui, F. Propenylben-
zenes from Guatteria guameri. Phytochemistry 1980, 19, 2024-
2025. (b) Diaz, F.; Mu o` oz, H.; Labarrios, F.; Chamorro, G.;
Salazar, M.; Morelos, M. E.; Tamariz, J . Synthesis and hypo-
lipidemic activity of some R-asarone analogues. Med. Chem. Res.
In Vitr o P la telet Aggr ega tion . Platelet aggregation was
2
2
measured by the Born method. Human blood was collected
by venipuncture from healthy male volunteers into 3.8%
sodium citrate solution (volume ratio 9:1). The blood was
centrifuged at 150g for 20 min to obtain platelet-rich plasma
1
993, 3, 101-109.
(
11) Gomez, C.; Chamorro, G.; Ch a´ vez, M. A.; Martinez, G.; Salazar,
M.; Pages, N. Effet de l′ R-asarone sur l′hypercholesterolemie
et la cholelithiasis experimentales. Plant. Med. Phytother. 1987,
21, 279-284.
(PRP). A portion of PRP was further centrifuged at 2000g for
1
0 min to obtain platelet-poor plasma (PPP).
Aggregation was induced by ADP. The percentage aggrega-
(
12) Menon, M. K.; Dandiya, P. C. The mechanism of the tranquil-
izing action of asarone from Acorus Calamus Linn. J . Pharm.
Pharmacol. 1967, 9 (3), 170-175.
tion was determined 6 min after the addition of the aggregat-
ing agent and was standardized by assuming that PPP
represented 100% light transmission and PRP represented 0%
light transmission. The agents studied were dissolved in 95%
ethanol. The ethanol was reported not to influence aggregation
(
13) (a) Sagimoto, N.; Goto, Y.; Akao, N.; Kiucki, F.; Kondo, K.
Mobility inhibition and nematocidal activity of asarone and
related phenylpropanoids on second-stage larvae of Toxocara
canis. Biol. Pharm. Bull. 1995, 18, 605-609. (b) Salazar, M.;
Salaz, S.; Ulloa, V.; Mendoza, T.; Pages, N.; Chamorro, G.
Teratogenic action of alpha-asarone in the mouse. J . Toxicol.
Clin. Exp. 1992, 12, 149-154. (c) Chamorro, G.; Salazar, M.;
Salazar, S.; Mendoza, T. Pharmacology and toxicology of Gatteria
Guameri and alpha-asarone. Rev. Invest. Clin. 1993, 45, 592-
1
5
in used volumes. 1.0 µL of the solution of an agent of fixed
concentration was added to 0.45 mL of PRP and placed in an
aggregometer at 37 °C. Stirring rate was 1000 rpm. After
2
-min preincubation ADP was added at a concentration
inducing maximum aggregation and the changes in light
transmission were recorded for 8 min. A maximum aggrega-
tory effect was induced by adding 50 µL of 10 µM ADP in
Tyrode solution to 0.45 mL of PRP.
6
04.
(
14) (a) Gilman, H.; Thirtle, J . Dibenzofuran. XXI. Benzene and
Biphenyl Intermediates for 1,9-Derivatives. J . Am. Chem. Soc.
1
994, 66, 858-859. (b) Shulgin, A. T. The six trimethoxyphen-
ylisopropylamines (Trimethoxyamphetamines). J . Med. Chem.
966, 9, 445-449.
Sta tistics. Student’s t-test was used to determine the
statistical significance of hypolipidemic activity of tested
1
2
compounds. The ø test was used for determining the signifi-
(
15) Petrusewicz, J .; Turowski, M.; Foks, H.; Pilarski, B.; Kaliszan,
R. Comparative studies of antiplatelet activity of nonsteroidal
antiinflammatory drugs and new pyrazine CH- and NH-acids.
Life Sci. 1995, 56, 667-677.
cance of difference in the pharmacological response between
the mice treated with the antithrombotic agents and the
controls. In the platelet aggregation test in vitro arithmetical
means and standard error of the mean (SEM) were deter-
mined.
(16) Griffin, T. S.; Dock, E. L.; Brotherton, R. J .; Hall, I. H. The
hypolipidemic activity of alkylamines and their boranes deriva-
tives: structure-activity relationship in rodents. Eur. J . Med.
Chem. 1991, 26, 517-527.
Su p p or tin g In for m a tion Ava ila ble: Purity data for
R-asarone isomers. This material is available free of charge
via the Internet at http://pubs.acs.org.
(
17) Bisgaier, C.; Pape, M. High-density lipoprotein: are elevated
levels desirable and achievable? Curr. Pharm. Des. 1998, 4, 53-
70.

3
676 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Popławski et al.
(
18) (a) Hughes, T. A.; Elan, M. B.; Applegate, W. B.; Bond, M. G.;
Hughes, S. M.; Wang, X.; Tolley, E. A; Bittle, J . C.; Stentz, F.
B.; Kang, E. S. Postprandial lipoprotein responses in hypertrig-
lyceridemic subjects with and without cardiovascular disease.
Metabolism 1995, 44, 1082-1098. (b) Garduno, L.; Salazar, M.;
Salazar, S.; Morelos, M. E.; Labarrios, F.; Tamariz, J .; Chamorro,
G. A. Hypolipidemic activity of R-asarone in mice. J . Ethno-
pharm. 1997, 55, 161-163.
19) Teitelbaum, P. Pharmacodynamics and Pharmacokinetics of
Ticlopidine. In Ticlopidine, Platelets and Vascular Disease; Hass,
W. K., Easton, J . D., Eds.; Springer-Verlag: New York, 1993;
pp 27-40.
(20) Harushige, F.; Masatore, Y. Novel synthesis of 2,3,4-trimethoxy-
1-propenylbenzene. Yuki Gosei Kagaku Kyokai Shi 1974, 32,
6
47-648.
(
21) DiMinno, G.; Silver, M. J . Mouse antithrombotic assay: a simple
method for the evaluation of antithrombotic agents in vivo.
Potentiation of antithrombotic activity by alcohol. J . Pharmacol.
Exp. Ther. 1983, 225, 57-60.
(
(22) Born, G. V. R. Aggregation of blood platelets by adenosine
diphosphate and its reversal. Nature 1962, 194, 927-929.
J M000905N