Synthesis and anti-platelet activity of obovatol derivatives

Article abstract of DOI:10.1007/s12272-011-0708-9

Obovatol derivatives were synthesized and evaluated for anti-platelet activity. Three derivatives (1, 2, 4i) displayed equipotent activity to obovatol in arachidonic acid-induced platelet aggregation. An initial SAR study revealed that the introduction of alkoxy group in B ring could enhance inhibitory activity.

Full text of DOI:10.1007/s12272-011-0708-9

Arch Pharm Res Vol 34, No 7, 1107-1112, 2011
DOI 10.1007/s12272-011-0708-9
Synthesis and Anti-platelet Activity of Obovatol Derivatives
Jae-Hwan Kwak1,*, Seul Lee2,*, Eun-Seok Park1, Jin-Kyung In1, Jiho Song2, Young Jin Kim2, Nam Song Choi3,
Heesoon Lee1, Yeo-Pyo Yun1, Jin Tae Hong1, Young-Shin Kwak4, Kyung Hoon Min2, and Jae-Kyung Jung1
2
1College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju 361-763, Korea, College
3
of Pharmacy, Chung-Ang University, Seoul 156-756, Korea, R&D Center, Huons Co. Ltd., Suwon 440-746, Korea, and
4Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Korea
(Received February 19, 2011/Revised April 25, 2011/Accepted April 25, 2011)
Obovatol derivatives were synthesized and evaluated for anti-platelet activity. Three deriva-
tives (1, 2, 4i) displayed equipotent activity to obovatol in arachidonic acid-induced platelet
aggregation. An initial SAR study revealed that the introduction of alkoxy group in B ring
could enhance inhibitory activity.
Key words: Obovatol, Anti-platelet activity, Derivatives, Small molecules
our interest, obovatol has been reported to show
inhibitory effects on either collagen- or arachidonic
INTRODUCTION
Platelet aggregation is a physiological mechanism acid-induced platelet aggregation (Pyo et al., 2002; Jin
that seals off the damaged blood vessel wall to prevent et al., 2008), as well as other biological effects, such as
blood loss. Various endogenous factors such as colla- antiproliferative activity for vascular smooth muscle
gen, thrombin, ADP, serotonin, vasopressin, and ephi- cells and cancer cells (Kwak et al., 2009; Lee et al.,
neprine induce platelet activation followed by shape 2009; Yu et al., 2009; Lim et al., 2010). Several of the
change and aggregation. In addition, arachidonic acid obovatol derivatives we synthesized were evaluated
is converted to TXA2 and 12-HETE via the COX path- for effect on platelet aggregation activity. Herein, we
way, which plays a major role in aggregation (Cowan, describe anti-platelet activity and structure-activity
1981; Eynard et al., 1986; Pollock, 1986). Under path- relationship of obovatol derivatives.
ological conditions, however, platelets also play a key
role in the pathogenesis of coronary artery disease
and arthrosclerosis. Platelet hyperaggregation has
been observed in pathological conditions such as dia-
betes mellitus, hypercholesterolaemia, and hyperten-
MATERIALS AND METHODS
Saturated obovatol (1)
To a solution of obovatol (20 mg, 0.070 mmol) in
sion, all of which cause significantly coronary artery ethanol was added Pd/C (10 mg). The reaction flask
disease (Willoughby et al., 2002; Michelson, 2010). was purged with hydrogen. The reaction mixture was
Development of platelet activity modulators has been stirred for 1 day at room temperature. The reaction
intensively investigated, as platelet aggregation in- mixture was filtered through celite. The filtrate was
hibition is therapeutically beneficial (Jackson and concentrated in vacuo and purified by flash column
Schoenwaelder, 2003). Among natural resources of chromatography (EtOAc-Hexanes = 1 : 4) to afford
1
saturated compound
NMR (CDCl3, 300 MHz):
6.93 (d, 2H, = 8.4 Hz), 6.56 (s, 1H), 6.28 (s, 1H), 2.57
(t, 2H, = 7.6 Hz), 2.41 (t, 2H, = 7.6 Hz), 1.70 - 1.42
(m, 4H), 0.95 (t, 3H, = 7.3 Hz), 0.88 (t, 3H, = 7.3 Hz).
1
(19 mg, 95%) as an oil. H-
*
These authors contributed equally to this work.
δ
7.14 (d, 2H, J = 8.4 Hz),
Correspondence to: Kyung Hoon Min, College of Pharmacy,
Chung-Ang University, Seoul 156-746, Korea
Tel: 82-2-820-5599, Fax: 82-2-816-7338
E-mail: khmin@cau.ac.kr
Jae-Kyung Jung, College of Pharmacy, Chungbuk National Uni-
versity, Cheongju 361-763, Korea
J
J
J
J
J
Diacetylated obovatol (2)
To a solution of obovatol (30 mg, 0.106 mmol) in
Tel: 82-43-261-2635, Fax: 82-43-268-2732
E-mail: orgjkjung@chungbuk.ac.kr
1107

1108
J.-H. Kwak et al.
methylene chloride, was added 4-dimethylaminopyri- 3.80 (s, 3H).
dine (3.0 mg, 0.021 mmol), triethylamine (53 mg, 0.53
mmol), acetic anhydride (16 mg, 0.159 mmol). The Methyl 3-(4-ethoxyphenoxy)-4,5-dimethoxyben-
reaction mixture was stirred for 20 min at room tem- zoate (4h)
perature. The reaction mixture was neutralized by Yield 71%; 1H-NMR (CDCl3, 300 MHz):
δ
7.37 (s, 1H),
= 9.0 Hz), 6.85 (d, 2H,
= 7.0 Hz), 3.94 (s, 6H), 3.84 (s,
= 7.0 Hz).
sodium bicarbonate, extracted with EtOAc (3
extract was concentrated in vacuo. Purification by 9.0 Hz), 4.01 (q, 2H,
flash column chromatography (EtOAc-Hexanes = 1 : 3H), 1.41 (t, 3H,
×
). The 7.20 (s, 1H), 6.94 (d, 2H,
J
J =
J
J
1
5) gave acetylated obovatol
(CDCl3, 300 MHz): 7.14 (d, 2H,
2H, = 8.4 Hz), 6.76 (s, 1H), 6.65 (s, 1H), 6.03 - 5.82 zoate (4i)
(m, 2H), 5.06 (d, 4H, J = 15.2 Hz), 3.38 (d, 2H,
= 6.6 Yield 80%; IR (KBr) 1722, 1211, 1095 cm−1; 1H-NMR
Hz), 3.29 (d, 2H, = 6.6 Hz), 2.29 (s, 3H), 2.26 (s, 3H). (CDCl3, 300 MHz): 7.37 (s, 1H), 7.20 (s, 1H), 6.94 (d,
2H, = 9.0 Hz), 6.85 (d, 2H, = 9.0 Hz), 4.01 (q, 2H,
= 7.0 Hz), 3.94 (s, 6H), 3.84 (s, 3H), 1.41 (t, 3H,
(2.00 g, 10.86 mmol) 7.0 Hz).
2
(32 mg, 82%). H-NMR
δ
J
= 8.4 Hz), 6.95 (d, Methyl 3,4-dimethoxy-5-(4-propoxyphenoxy)ben-
J
J
J
δ
J
J
J
J =
Methyl 3-hydroxy-4,5-dimethoxybenzoate (3)
To a solution of methyl gallate
7
in DMF anhydrous was added potassium carbonate
(1.20 g, 8.69 mmol) under nitrogen atmosphere. The
Spectral data of 4b-4e, 4g and 5a-5h were reported
mixture was stirred for 30 min at room temperature. in our previous report (Lee et al., 2007).
To the mixture, was added methyl iodide (3.39 g,
23.89 mmol) drop by drop. The reaction mixture was
stirred for 2 h at room temperature and then extract-
3,4-Dimethox-5-(4-propoxyphenoxy)benzoic
acid (6)
ed with ether. The extract was concentrated in vacuo
.
The compound 1 (0.110 mmol) was dissolved in
The crude product was purified by flash column chro- KOH (aq.) (1 N, 2 mL)/ethanol (2 mL). The reaction
matography (EtOAc-Hexanes = 1 : 3) to afford methyl mixture was refluxed for 2 h. After cooling to room
ether
300 MHz):
(s, 3H), 3.89 (s, 3H), 3.88 (s, 3H).
3
(1.84 g, 80%) as a white solid. 1H-NMR (CDCl3, temperature, the mixture was acidified with 1 N HCl,
δ
7.28 (s, 1H), 7.18 (s, 1H), 6.02 (s, 1H), 3.94 extracted with Ethyl Acetate. The extract was dried
over anhydrous MgSO4, and concentrated in vacuo
.
The resulting acid was used in next reaction without
1
further purification. Yield 99%; H-NMR (Acetone-d6
7.43 (d, 1H, = 1.8 Hz), 7.18 (d, 1H,
copper acetate (0.1 eq) and triethyl amine (5.0 eq) in 1.8 Hz), 6.95 (d, 4H, = 3.6 Hz), 3.84-3.95 (m, 8H),
= 7.2 Hz), 1.02 (t, 3H, = 7.2 Hz).
,
General procedure for the preparation of (4a-4i)
To a mixture of
3
(1.0 eq), arylboronic acid (1.2 eq), 300 MHz):
δ
J
J =
J
methylene chloride was added activated 4 molecular 1.78 (s, 2H,
sieve. The reaction flask was sealed with septa stuck
J
J
by a 18 gauge needle. The reaction mixture was stirred
for 2.5 h at room temperature, and then filtered to
remove molecular sieve and copper salt. The filtrate
General procedure for the preparation of com-
pounds (7a-c)
To a solution of
2 (0.040 mmol) in dry methylene
was concentrated in vacuo and purified by flash chloride (3 mL) was added oxalyl chloride (0.400 mmol)
column chromatography (EtOAc-Hexanes = 1 : 3
: 5) to afford biaryl ether.
→
1
and DMF (catalytic amount). The reaction mixture
was stirred for 1 h at room temperature. After com-
pletion, the mixture was evaporated completely and
Methyl 3-(4-cyanophenoxy)-4,5-dimethoxyben- then dissolved in dry methylene chloride (1 mL). To
zoate (4a)
the solution, alcohol (0.040 mmol) was added and
7.60 (d, 2H, stirred for 30 min at room temperature. The reaction
Yield 60%; 1H-NMR (CDCl3, 300 MHz):
δ
J
= 8.8 Hz), 7.52 (s, 1H), 741 (s, 1H), 6.97 (d, 2H,
J
=
mixture was extracted with Ethyl Acetate, washed
with brine, dried over anhydrous MgSO4, filtered and
concentrated. The residue was purified by flash column
8.8 Hz), 3.96 (s, 3H), 3.90 (s, 3H), 3.83 (s, 3H).
Methyl 3,4-dimethoxy-5-(4-vinylphenoxy)benzo- chromatography to afford ester 7.
ate (4f)
Yield 89%; lH-NMR (CDCl3, 300 MHz):
δ
7.47 (s, 1H), Butyl 3,4-dimethoxy-5-(4-propoxyphenoxy)ben-
= 8.7 Hz), 7.25 (s, 1H), 6.88 (d, 2H, zoate (7a)
8.7 Hz), 6.74-6.64 (m, 2H), 5.68 (d, 1H,
= 17.7 Hz), Yield 90%; 1H-NMR (Acetone-d6, 300 MHz):
5.16 (d, 1H, = 10.8 Hz), 3.92 (s, 3H), 3.84 (s, 3H), 1H, = 1.8 Hz), 7.17 (d, 1H, = 1.8 Hz), 6.95 (s, 4H),
7.40 (d, 2H,
J
J =
J
δ 7.41 (d,
J
J
J

Anti-platelet Activity of New Obovatol Derivatives
1109
4.25 (t, 2H,
(s, 3H), 1.66-1.79 (m, 4H), 1.42 (s, 2H,
(t, 3H, = 7.2 Hz), 0.93 (t, 3H, = 7.5 Hz).
J
= 6.6 Hz), 3.94 (t, 6H,
J
J
= 6.6 Hz), 3.83 the ear artery of conscious rabbits and collected direct-
= 7.8 Hz), 1.02 ly in the vacutainer tubes containing anticoagulant
J
J
solution (comprised of 0.8% citric acid, 2.2% trisodium
citrate, and 2% dextrose (w/v)). PRP was prepared by
Allyl 3,4-dimethoxy-5-(4-propoxyphenoxy)benzo- centrifugation at 230 g for 10 min at room tempera-
ate (7b)
ture. Platelets were sedimented by centrifugation of
7.39 (d, PRP at 2100 g for 10 min and then washed twice with
J = 1.8 Hz), 6.92 (d, 2H, HEPES buffer (137 mM NaCl, 2.7 mM KCl, 1 mM
Yield 55%; 1H-NMR (Acetone-d6, 300 MHz):
δ
1H,
= 6.3 Hz), 6.87 (d, 2H,
(d, 1H, = 17.1 Hz), 5.25 (d, 1H,
2H, = 5.7 Hz), 3.88-3.94 (m, 8H), 1.80 (s, 2H,
13.8 Hz), 1.04 (t, 3H, = 7.2 Hz).
J
= 1.8 Hz), 7.25 (d, 1H,
= 6.3 Hz), 5.97 (m, 1H), 5.34 MgCl2, 5.6 mM glucose, 0.35% bovine serum albumin,
= 10.5 Hz), 4.76 (d, and 3.8 mM HEPES, pH 6.5) containing 0.4 mM
J
J
J
J
J
J
=
EGTA. After centrifugation, the pellets were re-sus-
pended in HEPES buffer (pH 7.4). The platelet con-
centration was counted using a Coulter Counter
J
Benzyl 3,4-dimethoxy-5-(4-propoxyphenoxy)ben- (Coulter Electronics) and adjusted to 4 × 108 platelets
zoate (7c)
per milliliter. Platelet aggregation was mea sured as
7.31- previously described (Jin et al., 2008). Briefly, washed
= 6.6 platelet suspensions were incubated at 37oC in the
1
Yield 50%; H-NMR (Acetone-d6, 300 MHz):
7.40 (m, 5H), 7.26-7.27 (m, 2H), 6.92 (d, 2H,
δ
J
Hz), 6.85 (d, 2H,
(m, 8H), 1.80 (s, 2H,
Hz).
J
= 6.6 Hz), 5.30 (s, 2H), 3.84-3.94 aggregometer with stirring at 1000 rpm. After incuba-
= 14.4 Hz), 1.04 (t, 3H, = 7.5 tion with various concentration of obovatol (50
and 10 M), DMSO as a control, for 5 min, platelet
aggregation was induced by the addition of collagen
(10 g/mL), arachidonic acid (100 M), respectively.
The resulting aggregation measured as the change in
J
J
µM
µ
µ
µ
Washed rabbit platelet preparation and aggre-
gation assay
Rabbits were fixed in a cage. Blood was drawn from light transmission was recorded for 10 min.
Scheme 1. Synthesis of acetylated and saturated obovatol derivatives
Scheme 2. Synthesis of obovatol derivatives

1110
J.-H. Kwak et al.
Synthesis of ester derivatives was started with hydro-
RESULTS AND DISCUSSION
Synthesis of obovatol derivatives
lysis of
4. The resulting carboxylic acid
6 was trans-
formed to other esters
7
via acid chloride intermediate
The obovatol derivatives were prepared as shown in (Scheme 2).
Scheme 1. Most compounds were obtained according
to the known procedure we previously reported (Lee et
al., 2007; Kwak et al., 2009). Copper catalyzed etheri-
Anti-platelet activities
All obovatol derivatives prepared in this study were
fication was used as the key step to prepare diaryl evaluated for their anti-platelet aggregation activity.
ethers. Selective dimethylation of methyl gallate and Collagen and arachidonic acid were used as platelet
subsequent etherification catalyzed by Cu(OAc)2 pro- aggregation inducers. Anti-platelet activity of the
vided the diversed diaryl ethers. Demethylation of obovatol derivatives at 50 µM and 10 µM is summar-
aryl methyl ethers by BBr3 gave catechol derivatives. ized in Table I. Obovatol inhibited collagen- and
Fig. 1. Structures of synthesized compounds

Anti-platelet Activity of New Obovatol Derivatives
1111
Table I. Anti-platelet aggregation activity of obovatol
longer alkoxy group in the B ring resulted in better
activity. The longest propyl derivatives showed the
best inhibitory activities in this study. Therefore,
further structural modifications might lead to the
discovery of more potent anti-platelet aggregation
agents.
derivatives
Aggregation rate (% of control)
obovatol
derivatives
Arachidonic acid
(100 M)
Collagen (10
µg/mL)
µ
at 50 at 10
µ
M
µM
at 50
µM
at 10 µM
obovatol
1
005.8
001.2
009.2
091.2
001.1
002.3
057.7
000.0
086.6
000.0
000.0
000.0
047.3
100.0
100.0
080.8
091.2
071.6
100.0
2.3
009.9
038.8
024.4
ND*
083.1
097.6
ND
079.5
ND
100.0
098.5
022.0
ND
ND
ND
ND
ND
ND
ND
084.9
088.9
095.7
090.4
002.5
002.5
002.5
089.3
000.0
049.1
069.2
000.0
079.2
000.0
001.3
000.0
000.0
083.0
074.2
001.3
002.5
002.5
081.8
6.3
001.2
001.0
002.4
ND
081.9
ND
ND
94.0
ND
091.6
100.0
001.0
088.2
ND
ACKNOWLEDGEMENTS
2
This research was supported by the Medical Research
Center program (2010-0029480), the Regional Core
Research Program (Chungbuk BIT Research-Oriented
University Consortium) through the National Re-
search Foundation of Korea, and by the Ministry of
Land, Transport and Maritime Affairs (Korea Ocean
Research and Development Institute), Republic of
Korea.
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
7c
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