Synthesis, antiplatelet and vasorelaxing effects of monooxygenated flavones and flavonoxypropanolamines

Article abstract of DOI:10.1211/0022357011778205

A series of flavones and flavonoxypropanolamines were synthesized and tested in-vitro for their ability to inhibit aggregation of washed rabbit platelets and human platelet-rich plasma (PRP), and for vasoconstriction of rat thoracic aorta. The various substituted positions of the hydroxyl group in flavone ring B and the various oxypropanolamine side chains substituted at position C-2′ of flavone modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline (epinephrine), suggesting that the antiplatelet effect of these compounds is mainly due to an inhibitory effect on thromboxane formation. Compounds 11 and 12 also had potent vasorelaxant effects in rat thoracic aorta. Phenylephrine- and high-K⁺-induced ⁴⁵Ca²⁺ influx in aorta were both inhibited by the selected compound 11. This result indicates that the inhibitory effect of 11 on the contractile response caused by high-K⁺ medium and noradrenaline (norepinephrine) in rat thoracic aorta is mainly due to inhibition of Ca²⁺ influx through both voltage-dependent and receptor-operated Ca²⁺ channels.

Full text of DOI:10.1211/0022357011778205

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 1601–1609
# 2001 The Authors
Received February 21, 2001
Accepted July 7, 2001
ISSN 0022-3573
Synthesis, antiplatelet and vasorelaxing effects
of monooxygenated flavones and
flavonoxypropanolamines
Mei-Ing Chung, Feng-Wen Han, Hsien-Cheng Lin, Shorong-Shii Liou, Pao-
Ling Huang, Horng-Huey Ko, Ya-Ling Chang, Jaw-Jou Kang, Che-Ming
Teng and Chun-Nan Lin
Abstract
A series of flavones and flavonoxypropanolamines were synthesized and tested in-vitro for
their ability to inhibit aggregation of washed rabbit platelets and human platelet-rich plasma
(PRP), and for vasoconstriction of rat thoracic aorta. The various substituted positions of the
hydroxyl group in flavone ring B and the various oxypropanolamine side chains substituted at
position C-2h of flavone modified the antiplatelet effects. All the compounds tested in human
PRP showed significant inhibition of secondary aggregation induced by adrenaline (epi-
nephrine), suggesting that the antiplatelet effect of these compounds is mainly due to an
inhibitory effect on thromboxane formation. Compounds 11 and 12 also had potent
School of Pharmacy, Kaohsiung
Medical University, Kaohsiung,
Taiwan 807, Republic of China
Mei-Ing Chung, Feng-Wen Han,
Horng-Huey Ko, Chun-Nan Lin
School of Technology of Medical
Sciences, Kaohsiung Medical
University, Kaohsiung, Taiwan
807, Republic of China
vasorelaxant effects in rat thoracic aorta. Phenylephrine- and high-K -induced 45Ca2 influx in
+
+
Hsien-Cheng Lin
aorta were both inhibited by the selected compound 11. This result indicates that the inhibitory
Ta-Jen Institute of Technology,
Ping Tung Hsieng, Taiwan 907,
Republic of China
+
effect of 11 on the contractile response caused by high-K medium and noradrenaline
(norepinephrine) in rat thoracic aorta is mainly due to inhibition of Ca2 influx through both
+
voltage-dependent and receptor-operated Ca2 channels.
+
Shorong-Shii Liou, Pao-Ling
Huang
Pharmacological Institute,
College of Medicine, National
Taiwan University, Taipei,
Taiwan 100, Republic of China
Introduction
Ya-Ling Chang, Che-Ming Teng
Flavonoids isolated from natural sources have been shown to be potent inhibitors
of platelet aggregation (Teng et al 1988; Lin et al 1996) and also vasorelaxing agents
(Ko et al 1991). In the course of continued screening work on antithrombotic herbs,
especially those having antiplatelet and vasorelaxing effects, a potent antiplatelet
agent, 2h-hydroxyflavone (1) from Polygonatum alte-lobatum has been isolated
(Huang et al 1997). As part of ongoing work on the discovery of drugs with
biological activity, we synthesized 2h-hydroxyflavone (1) and its analogues and
evaluated their antiplatelet and vasorelaxing actions.
Institute of Toxicology, College
of Medicine, National Taiwan
University, Taipei, Taiwan 100,
Republic of China
Jaw-Jou Kang
Correspondence: M.-I. Chung,
School of Pharmacy, Kaohsiung
Medical University, Kaohsiung,
Taiwan 807, Republic of China.
E-mail: meinch!cc.kmu.edu.tw
Materials and Methods
Funding: This work was
supported by the National
Science Council of the Republic
of China (NSC89-2314-B-037-
024).
Platelet aggregation
Washed rabbit platelets were obtained from ethylene diamine tetraacetate (EDTA)-
anticoagulated platelet-rich plasma (PRP) according to procedures described
1601

1602
Mei-Ing Chung et al
previously (Teng et al 1987). Human PRP was obtained placed in an organ bath containing 5 mL Krebs solution
from the supernatant after centrifugation of a 1:9 maintained at 37mC and bubbled with a 95% O2–5%
mixture of blood and sodium citrate solution (3.8%). CO2 mixture. Two stainless-steel hooks were inserted
Platelet numbers were counted by using a Coulter into the aortic lumen, one was fixed while the other was
Counter (Model ZM) and adjusted to 4.5i108 platelets connected to a transducer. The aorta was equilibrated in
1
−
mL . The platelet pellets were suspended in Tyrode’s the medium for 90 min with three changes of Krebs
solution containing (m): NaCl 136.8, KCl 2.8, solution and maintained under an optimal tension of 1 g
NaHCO3 11.9, MgCl2 2.1, NaH2PO4 0.33, CaCl2 1.0, before specific experimental protocols were initiated.
and glucose 11.2 with 0.35% bovine serum albumin Contractions were recorded isometrically via a force
(BSA). All glassware was siliconized. PRP or the platelet displacement transducer connected to a Gould poly-
suspension was stirred at 1200 rev min 1 for 1 min before graph(Model2400). Thefinalconcentrationofdimethyl
−
addition of the aggregation inducer. Aggregation was sulfoxide (DMSO) was fixed at 0.1%.
measured by a turbidimetric method (O’Brien 1962).
The absorbance of platelet-poor plasma or platelet-free
Tyrode’s solution was taken as 100% aggregation.
The aggregation was measured by means of a Lumi-
45Ca2M influx
2
+
Ca influx was measured in a manner similar to that
described by Meisheri et al (1980). Aortic rings were
placed in test tubes containing Krebs solution with
aggregometer (Chrono-Log Co., USA) connected to
dual-channel recorders.
1 µCi mL 1 of 45Ca in the presence of DMSO (0.1%)
2
−
+
Aortic contraction
or various concentrations of 11 (Table 1) and incubated
+
Wistar rats of either sex, 250–300 g, were killed by a for 20 min. Phenylephrine (3 µ) or K (60 m) was
blow to the head. The thoracic aorta was isolated and then added and incubated for another 15 min. After the
excess fat and connective tissue removed. The aorta was incubation period, the tissues were transferred into test
Table 1 Flavonoxypropanolamines.
Compound
R
Rh
mp(L) °CL Crystallization % yield
Formula
Anal.
solvent

1603
Antiplatelet and vasorelaxant monooxygenated flavones and flavonoxypropanolamines
2
tubes containing 2 mL of ice-cold Ca -free Krebs sol-
+
Melting points (uncorrected) were determined with a
ution with 2 m EGTA for 40 min to remove extra- Yanaco Micro-Melting Point apparatus. IR spectra
45
cellular Ca . The tissues were then removed, lightly were determined with a Hitachi model 260-30 IR spec-
2
+
1
blotted with No. 5 Whatman filter paper, weighed, and trophotometer. H (400 or 200 MHz) and 13C (100 or
dissolved in 37% perchloric acid (0.1 mL) at 75mC. 50 MHz) NMR spectra (δ (ppm), J (Hz)) were deter-
The radioactivity was counted in a liquid scintillation mined with a Varian Unity-400 spectrometer. Mass
counter (Packard Model 2200 CA).
spectra were determined with a Jeol JMS-D-100 mass
spectrometer. Elemental analyses were withinp0.4%
of the theoretical values, unless otherwise noted.
Chromatography was performed using a flash-column
technique on silica gel 60 supplied by E. Merck.
2h-Hydroxyflavone (1), 3h-hydroxyflavone (2), and 4h-
hydroxyflavone (3) were synthesized by genenal syn-
thetic method (Wu et al 1989) and the synthetic products
were identical by spectral methods.
Data analysis
Data are presented as the meansps.e.m. A one-way
analysis of variance was used for multiple comparison.
If there was significant variation between treatment
groups, then the inhibitor-treated groups were com-
pared with the control group by Student’s t-test, and P
values of less than 0.05 were considered to be statistically
significant.
General procedure for synthesis of
flavonoxypropanolamines 4–12
To a solution of 0.1g (2 mmol) sodium hydroxide in
water (1 mL) were added 2-propanol (20 mL) and 2h-
hydroxyflavone(1;0.5 g, 2 mmol). Tothe abovemixture
was then added excess of the appropriate epichloro-
hydrin (2 mL, 20 mmol), and the mixture was heated at
70mC for 2 h with stirring. The hot reaction mixture was
filtered to remove a dimeric byproduct (a glycidyl ether).
The clear filtrate, on cooling, yielded a solid (1h) (Liou et
al 1994), purified by column chromatography (silica gel
and CHCl3).
Chemistry
Monooxygenated flavones (1–3; Figure 1) and a series
of new flavonoxypropanolamines (4–12; Table 1) were
synthesized by general synthetic routes as shown in
Figure 2 for 2h-hydroxyflavone (1). In general, mono-
oxygenated flavones were synthesized from Baker-
Venkataraman rearrangement of 2h-acetylphenyl-
monomethoxygenated benzoate which was prepared
from 2h-hydroxyacetophenone and appropriate mono-
methoxygenated benzoyl chloride, to the diketone com-
pounds. Cyclization of the diketone compounds yielded
monomethoxygenated flavones. After demethylation,
monohydroxyflavones were obtained. 2h-Hydroxy-
flavone was allowed to react with excess epichlorohydrin
to yield the epoxide derivative. Ring opening of epoxide
with the corresponding amines afforded the correspond-
ing 2h-flavonoxypropanolamines (Wu et al 1989).
To the former purified solid (1h), (0.6 g, 2 mmol) in
absolute ethanol (60 mL) the appropriate amine
(40 mmol) was added and the mixture was heated at
50–55mC for 5 h with stirring. The reaction mixture was
clarified by filtration and the filtrate was concentrated
under reduced pressure. The product was filtered,
washed with absolute ethanol, and purified by column
chromatography, yielding the flavonoxypropanola-
mines, 4–12. The yields of 4–12 are reported in Table 1.
2h-(2,3-Epoxypropoxy)flavone (1h). White power (0.4 g,
1
1
−
1.4 mmol, yield 60%). IR (KBr) 1638, 1258 cm ; H
NMR(CDCl3) δ 2.77 (dd, J l 4.8, 2.4 Hz, 1H, CHH in
the epoxide ring), 2.91 (t, J l 4.8 Hz, 1H, CHH in the
epoxide ring), 3.39 (m, 1H, CH of epoxide ring), 4.13
(dd, J l 11.6, 5.6 Hz, 1H, OCHH), 4.37 (dd, J l 11.6,
3.2 Hz, 1H, OCHH), 7.02 (s, 1H, H-3), 7.07 (m, 1H, H-
3h), 7.14 (dt, J l 7.6, 12 Hz, 1H, H-5h), 7.41 (m, 1H, H-
6), 7.48 (m, 1H, H-4h), 7.53 (m, 1H, H-8), 7.68 (m, 1H,
H-7), 7.84 (dd, J l 8.0, 2.0 Hz, 1H, H-6h), 8.24 (dd, J l
8.0, 2.0 Hz, 1H, H-5);13C NMR (CDCl3) δ 44.7 (CH2 in
the epoxide ring), 49.9 (CH in the epoxide ring), 69.5
(OCH2), 112.7 (C-3), 113.3 (C-3h), 118.0 (C-8), 121.4
Figure 1 Structure of monooxygenated flavones 1–3.

1604
Mei-Ing Chung et al
1
1h
Figure 2 Synthesis of flavonoxypropanolamines 4–12.
4–12
(C-5h), 121.7 (C-1h), 123.9 (C-4a), 124.9 (C-6), 125.7 (-CH2NH-), 65.5 (-CH2CHOH-), 71.8 (-OCH2-), 112.9
(C-5), 129.6 (C-6h), 132.4 (C-4h), 133.6 (C-7), 156.5 (C-3), 114.3 (C-3h), 119.6 (C-8), 122.0 (C-1h), 122.7 (C-
(C-8a), 156.7 (C-2h), 161.2 (C-2), 178.6 (C-4); MS m\z 5h), 124.4 (C-4a), 126.1 (C-6), 126.7 (C-5), 130.7 (C-6h),
+
294 (M , 49). Anal. (C18H14O4 ) C, H.
134.3 (C-4h), 135.8 (C-7), 158.1 (C-8a), 158.3 (C-2h),
163.7 (C-2), 180.9 (CO); MS m\z 340 ((Mj1) , 5).
,
+
2h-[3-(N, N-Dimethylamino)-2-hydroxypropoxy]fla-
1
1
−
vone (4). IR (KBr) 3450, 1658 cm ; H NMR (CD3OD) 2h-[3-(n-Propylamino)-2-hydroxypropoxy]flavone (6).
1
1
−
δ 2.29 (s, 6H, -N(CH3)2), 2.52 (m, 2H, -CH2N(CH3)2), IR (KBr) 3410, 1621 cm ; H NMR (CD3OD) δ 0.94 (t,
4.01 (dd, J l 9.6, 6.0 Hz, 1H, -OCHH-), 4.08 (dd, J l J l 7.6 Hz, 3H, -CH3), 1.57 (m, 2H, -CH2CH3), 2.70
9.6, 4.0 Hz, 1H-OCHH-), 4.14 (m, 1H, -HCOH-), 7.06 (m, 2H, -NHCH2-), 2.88 (dd, J l 12.4, 8.8 Hz, 1H,
(m, 2H, H-3h and -5h), 7.13 (s, 1H, H-3), 7.42 (m, 2H, H- -CHHNH-), 2.97(dd, J l 12.4, 3.8 Hz, 1H, -CHHNH-),
6 and -4h), 7.56 (m, 1H, H-8), 7.71 (m, 1H, H-7), 7.82 4.15 (d, Jl5.2Hz, 2H, -OCH2-), 4.22 (m, 1H, -HCOH-),
(dd, J l 8.0, 1.6 Hz, 1H, H-6h), 8.05 (dd, J l 8.0, 1.6 Hz, 7.17 (m, 2H, H-3h and -5h), 7.16 (s, 1H, H-3), 7.52 (m,
1H, H-5); 13C NMR (CD3OD) δ 46.2 (Mei2), 63.4 2H, H-6 and –4h), 7.68 (m, 1H, H-8), 7.81 (m, 1H, H-7),
(-CH2l), 68.2 (-CHOH-), 72.6 (-OCH2-), 112.8 (C-3), 7.93 (dd, J l 8.0, 2.0 Hz, 1H, H-6h), 8.14 (dd, J l 8.0,
114.1 (C-3h), 119.4 (C-8), 121.5 (C-1h), 122.1 (C-5h), 2.0 Hz, 1H, H-5); 13C NMR (CD3OD) δ 11.7 (-CH3),
124.2 (C-4a), 126.0 (C-6), 126.4 (C-5), 130.2 (C-6h), 22.6 (-CH2CH3), 51.9 (-NH-CH2-), 52.6 (-CHOH-CH2-),
134.1 (C-4h), 135.4 (C-7), 157.8 (C-8a), 158.7 (C-2h), 68.5 (-CHOH-), 72.5 (-OCH2-), 112.8 (C-3), 114.3 (C-
+
163.3 (C-2), 180.8 (CO); MS m\z 340 ((Mj1) , 3).
3h), 119.5 (C-8), 121.9 (C-1h), 122.4 (C-5h), 124.4 (C-4a),
126.1 (C-6), 126.6 (C-5), 130.6 (C-6h), 134.2 (C-4h), 135.7
(C-7), 158.0 (C-8a), 158.6 (C-2h), 163.8 (C-2), 181.0
2h-[3-(Ethylamino)-2-hydroxypropoxy]flavone (5). IR
+
(CO); MS m\z 353 (M , 7).
1
(KBr) 3410, 1629 cm ; H NMR (CD3OD) δ 1.26 (t,
1
−
J l 7.6 Hz, 3H, -CH2CH3), 2.99 (m, 2H, -CH2CH3), 3.06
(dd, J l 12.8, 9.6 Hz, 1H, -CHHNHCH2-), 3.16 (dd, 2h-[3-(Cyclopropylamino)-2-hydroxypropoxy]flavone
1
1
−
J l 12.8, 3.4 Hz, 1H- CHHNHCH2-), 4.19 (m, 2H, (7). IR (KBr) 3280, 1620 cm ; H NMR (CD3OD) δ
-OCH2-), 4.30 (m, 1H, -HCOH-), 7.18 (s, 1H, H-3), 7.20 0.33 (m, 2H, -CHH- and -CHH in the cyclopropyl ring),
(m, 2H, H-3h and -5h), 7.53 (m, 2H, H-6 and -4h), 7.70 0.44 (m, 2H, -CHH- and -CHH- in the cyclopropyl
(m, 1H, H-8), 7.83 (m, 1H, H-7), 7.96 (dd, J l 8.0, ring), 2.18 (m, 1H, -CH- in the cyclopropyl ring), 2.81
2.0 Hz, 1H, H-6h), 8.15 (dd, J l 8.0, 2.0 Hz, 1H, H-5); (dd, J l 12.6, 8.2 Hz, 1H, -CHHNH-), 2.92 (dd, J l
13C NMR (CD3OD) δ 11.5 (CH3), 43.5 (-CH2CH3), 55.6 12.6, 3.8 Hz, 1H, -CHHNH-), 4.13 (m, 2H, -OCH2-),

1605
Antiplatelet and vasorelaxant monooxygenated flavones and flavonoxypropanolamines
4.18 (m, 1H, -CHOH-), 7.17 (s, 1H, H-3), 7.18 (m, 2H, H-8), 7.84 (m, 1H, H-7), 7.95 (dd, J l 8.0, 1.6 Hz, 1H,
H-3h and -5h), 7.50 (m, 2H, H-6 and -4h), 7.69 (m, 1H, H-6h), 8.17 (dd, J l 8.0, 1.6 Hz, 1H, H-5); 13C NMR
H-8), 7.82 (m, 1H, H-7), 7.92 (dd, J l 8.0, 1.6 Hz, 1H, (CD3OD) δ 20.7 (-CH3), 20.8 (-CH3), 28.5 (-CH(CH3)2),
H-6h), 8.15 (dd, J l 8.0, 1.6 Hz, 1H, H-5); 13C NMR 52.9 (-NH-CH2), 58.0 (-CHOHCH2NH-), 68.6
(CD3OD) δ 6.08 (-CH3), 6.34 (-CH2-), 31.2 (lCHNH-), (-CHOH-), 72.5 (-OCH2-), 112.8 (C-3), 114.3 (C-3h),
53.1 (-CH2NH-), 69.3 (-CHOH-), 72.8 (-OCH2-), 112.8 119.5 (C-8), 121.9 (C-1h), 122.4 (C-5h), 124.4 (C-4a),
(C-3), 114.3 (C-3h), 119.5 (C-8), 121.9 (C-1h), 122.2 (C- 126.1 (C-6), 126.6 (C-5), 130.6 (C-6h), 134.2 (C-4h), 135.7
5h), 124.4 (C-4a), 126.1 (C-6), 126.6 (C-5), 130.6 (C-6h), (C-7), 158.1 (C-8a), 158.7 (C-2h), 163.9 (C-2), 181.0
+
134.2 (C-4h), 135.6 (C-7), 158.1 (C-8a), 158.8 (C-2h), (CO); MS m\z 367 (M , 2).
164.0 (C-2), 181.0 (CO); MS m\z 351 (M , 4).
+
2h-[3-(sec-Butylamino)-2-hydroxypropoxy]flavone
2h-[3-(Isopropylamino)-2-hydroxypropoxy]flavone (8).
(11). Compound 11 was prepared from (p) sec-butyl-
1
1
1
1
−
−
IR (KBr) 3275, 1620 cm ; H NMR (CD3OD) δ 1.06 (d,
J l 6.4 Hz, 6H, -CH(CH3)2), 2.83 (dd, J l 12.6, 6.0 Hz,
1H, -CHHNH-), 2.88 (dd, J l 12.6, 3.6 Hz, 1H,
-CHHNH-), 4.15 (m, 3H, -OCH2CHOH-), 7.16 (s, 1H,
H-3), 7.18 (m, 2H, H-3h and -5h), 7.50 (m, 2H, H-6 and
-4h), 7.69 (m, 1H, H-8), 7.82 (m, 1H, H-7), 7.93 (dd,
J l 8.0, 1.6 Hz, 1H, H-6h), 8.16 (dd, J l 8.0, 1.6 Hz,
1H, H-5); 13C NMR (CD3OD) δ 22.4 (2 i-CH3),
49.9 (-CHl), 50.8 (-CH2NH-), 69.5 (-CHOH-), 72.8
(-OCH2-), 112.8 (C-3), 114.3 (C-3h), 119.5 (C-8), 122.0
(C-1h), 122.3 (C-5h), 124.4 (C-4a), 126.1 (C-6), 126.6 (C-
5), 134.2 (C-4h), 135.6 (C-7), 158.1 (C-8a), 158.8 (C-2h),
amine. IR (KBr) 3300, 1630 cm ; H NMR (CD3OD) δ
0.88 (t, J l 7.2 Hz, 3H, -CH2 CH3), 1.05 (d, J l 6.4Hz,
3H, -CHCH3), 1.31 (m, 1H, -CHHCH3), 1.55 (m, 1H,
-CHHCH3), 2.64 (m, 1H, lCHHCH3), 2.78 (m, 1H,
-CHHNH-), 2.90 (m, 1H, -CHHNH-), 4.12 (m, 3H,
-OCH2-and-CHOH-), 7.04(m, 2H, H-3hand-5h), 7.09(s,
1H, H-3), 7.40 (m, 2H, H-6 and -4h), 7.50 (d, J l 8.0 Hz,
1H, H-8), 7.69 (m, 1H, H-7), 7.78 (dd, J l 7.6, 1.6 Hz,
1H, H-6h), 8.01 (dd, J l 7.6, 1.6 Hz, 1H, H-5); 13C
NMR (CD3OD) δ 10.2 (-CH3), 18.2 (lCHCH3), 55.4
(-NHCHCH3-), 49.8 (-CH2NH-), 67.9 (-CHOH-), 72.2
(-OCH2-), 113.0 (C-3), 113.6 (C-3h), 118.7 (C-8), 121.2
(C-5h), 121.5 (C-1h), 124.4 (C-4a), 125.3 (C-6), 125.6
(C-5), 132.9 (C-4h), 134.0 (C-7), 156.8 (C-8a), 163.8
+
164.0 (C-2), 181.0 (CO); MS m\z 353 (M , 1).
+
(C-2h), 178.1 (CO); MS m\z 367 (M , 2).
2h-[3-(n-Butylamino)-2-hydroxypropoxy]flavone (9).
1
1
−
IR (KBr) 3390, 1630 cm ; H NMR (CDCl3) δ 0.87
(t, J l 7.2 Hz, 3H, -CH2CH3), 1.31 (m, 2H, -CH2CH3),
1.47 (m, 2H, -CH2CH2CH2), 2.64 (m, 2H, -NHCH2),
2.81 (dd, J l 12.0, 8.0 Hz, 1H, -CHOHCHHNH-), 2.90
(dd, J l 12.0, 3.8 Hz, 1H, -CHOHCHHNH), 3.40 (bs,
1H, NH), 4.12 (m, 2H, -OCH2-), 4.13 (m, 1H, -CHOH-),
7.06 (m, 2H, H-3h and -5h), 7.10 (s, 1H, H-3), 7.74 (m,
2H, H-6 and -4h), 7.50 (m, 1H, H-8), 7.66 (m, 1H, H-7),
7.82 (dd, J l 8.0, 1.6 Hz, 1H, H-6h), 8.18 (dd, J l 8.0,
1.6 Hz, 1H, H-5); 13C NMR (CDCl3) δ 13.8 (-CH3), 20.3
(-CH2CH3), 31.5 (-CH2CH2CH3), 49.4 (-NHCH2), 51.9
(-CHOHCH2NH-), 67.6 (-CHOH-), 71.3 (-OCH2-),
112.4 (C-3), 112.8 (C-3h), 118.0 (C-8), 121.0 (C-1h and
-5h), 123.6 (C-4a), 125.0 (C-6), 125.6 (C-5), 129.3 (C-
6h), 132.5 (C-4h), 133.7 (C-7), 156.4 (C-8a), 157.1 (C-2h),
2h-[3-(tert-Butylamino)-2-hydroxypropoxy]flavone
1
1
−
(12). IR (KBr) 3390, 1636 cm ; H NMR (CD3OD) δ
1.27 (s, 9H, 3iMe), 2.97 (dd, J l 12.0, 9.2 Hz, 1H,
-CHHNH-), 3.10 (dd, J l 12.0, 3.2Hz, 1H, -CHHNH-),
4.16 (m, 2H, -OCH2-), 4.06, (m, 1H, -CHOH-), 7.09 (m,
2H, H-3h and -5h), 7.09 (s, 1H, H-3), 7.42 (m, 2H, H-6
and -4h), 7.55 (dd, J l 8.4, 0.4 Hz, 1H, H-8), 7.72 (m,
1H, H-7), 7.80 (dd, J l 8.0, 1.6 Hz, 1H, H-6h), 8.02 (dd,
J l 8.0, 1.6 Hz, 1H, H-5); 13C NMR (CD3OD) δ 26.8
(-C(CH3)3), 45.9 (-CH2NH-), 55.8 [-C(CH3)3], 68.1
(-CHOH-), 72.1 (-OCH2-), 112.6 (C-3), 114.3 (C-3h),
119.4 (C-8), 121.4 (C-1h), 122.3 (C-5h), 124.1(C-4a),
125.9 (C-6), 126.5 (C-5), 130.3 (C-6h), 134.2 (C-4h), 135.6
(C-7), 157.7 (C-8a), 158.4 (C-2h), 163.3 (C-2), 180.7
+
+
161.4 (C-2), 178.9 (CO); MS m\z 367 (M , 5).
(CO); MS m\z 367 (M , 2).
2h-[3-(Isobutylamino)-2-hydroxypropoxy]flavone (10).
1
IR (KBr) 3410, 1620 cm ; H NMR (CD3OD) δ 0.95 (d,
1
−
Results and Discussion
J l 6.7 Hz, 6H, -CH(CH3)2), 1.86 (m, 1H, -CH(CH3)2),
2.60 (m, 2H, -NHCH2CH-), 2.97 (m, 2H, The antiplatelet effects of 1–12 were studied in the
-CHOHCH2NH-), 4.17 (m, 2H, -OCH2-), 4.23 (m, 1H, aggregation of washed rabbit platelets induced by
1
−
-CHOH-), 7.17 (s, 1H, H-3), 7.19 (m, 2H, H-3h and -5h), thrombin (0.1 IU mL ), arachidonic acid (100 µ),
1
−
7.55 (m, 2H, H-6 and -4h), 7.71 (dd, J l 8.0, 1.6 Hz, 1H, collagen (10 µg mL ), and platelet-activating factor

1606
Mei-Ing Chung et al
Table 2 Effect of flavones and flavonoxypropanolamines on the aggregation induced by thrombin, arachidonic acid, collagen and platelet-
activating factor (PAF) in washed rabbit platelets.
Compound
Aggregation (%)
1
1
1
−
−
−
Thrombin (0.1 IU mL
)
Arachidonic acid (100 µ)
Collagen (10 µg mL
)
PAF (2 ng mL
)
Control
91.5p0.4
85.8p0.6
88.6p0.7
91.0p0.4
1
86.2p2.1 (100)
88.0p2.3
0.0p0.0*** (100)
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.08***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
8.3p4.48*** (100)
14.0p2.5***
10.1p7.5***
0.0p0.0*** (100)
0.0p0.0***(100)
0.0p0.0*** (50)
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0*** (100)
0.0p0.0***
0.0p0.0***
82.4p3.5* (100)
11.4p4.7***
61.4p2.2***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
90.5p1.2
2
3
85.9p2.4*
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
0.0p0.0***
1.2p1.1***
0.0p0.0***
5.1p4.2***
0.0p0.0***
91.9p2.5
4
5
6
7
8
9
10
11
12
Aspirin
85.4p3.9
Platelets were pre-incubated with DMSO (0.5%, control), aspirin (50 µ) or compounds 1–12 (300 µ) at 37mC for 3 min, and the inducer was
then added. Percentages of aggregation are presented as meansps.e.m., n l 3–5. When the concentration of compounds was different from
300 µ, the corrected dose is given in parentheses. *P ! 0.05, ***P ! 0.001 as compared with the respective control values.
Table 3 IC50 values (µ) of flavones and flavonoxypropanolamines on the aggregation induced by
thrombin, arachidonic acid, collagen and platelet-activating factor (PAF) in washed rabbit platelets.
1
1
−
−
Compound
Thrombin (0.1 IU mL
)
Arachidonic acid
Collagen (10 µg mL
)
PAF
(100 µ)
1
2
" 100 (10.1)
" 300 (3.0)
" 300 (5.3)
" 100 (4.6)
" 100 (4.3)
" 100 (3.2)
" 100 (2.3)
183
18.9
170
47.2
" 100 (8.4)
220
! 300 (84.5)
! 300 (88.8)
43.9
3
37.5
85.6
49.0
93.5
18.0
56.9
61.4
48.9
35.1
48.5
! 300 (31.8)
153
4
5
46.0
139
6
20 (5.4)
38.0
221
7
154
8
58.8
71.8
9
164
38.7
51.1
10
11
12
" 100 (7.5)
! 300 (94.4)
! 300 (100)
41.6
90.7
! 300 (100)
! 300 (100)
54.2
62.7
When 50% inhibition could not be reached at the highest concentration, the % of inhibition is given in
parentheses. IC50, 50% inhibition concentration.
1
(PAF, 2 ng mL ). As shown in Table 2, mono- group of 1–3 did not alter the antiplatelet effect at high
−
hydroxyflavones 1–3, at a concentration of 100 or concentration on the aggregation induced by thrombin.
Compound 2 (300 µ) strongly inhibited the platelet
300 µ, showed a potent antiplatelet effect on the ag-
gregation induced by arachidonic acid and collagen aggregation induced by PAF while 1 (100 µ) and 3
while having no significant antiplatelet effect on the (300 µ) did not. This indicated that the flavone with a
aggregation induced by thrombin. It clearly indicated hydroxyl group substituted at C-3h enhanced the anti-
that the substituted position of the B-ring hydroxyl platelet effect at high concentration on the aggregation

1607
Antiplatelet and vasorelaxant monooxygenated flavones and flavonoxypropanolamines
Table 4 Effect of selected flavones and flavonoxypropanolamines
on aggregation of human platelet-rich plasma (PRP) induced by
ADP, collagen or adrenaline.
enhanced the antiplatelet effects on platelet aggregation
induced by thrombin, collagen and PAF (Table 2).
Aspirin was used in this study as a positive control. It
was found that aspirin (50 µ) completely inhibited the
platelet aggregation induced by arachidonic acid but
not by thrombin, collagen or PAF. More experiments
were performed to study the potency of 1–12 on throm-
bin-, arachidonic-acid-, collagen- and PAF-induced
washed rabbit platelet aggregation. As shown in Table
3, in thrombin-induced platelet aggregation, none of the
compounds at low concentration showed any anti-
platelet effect. In arachidonic-acid-induced platelet ag-
gregation, compounds 1 and 7 were the most potent.
This indicated that a 3-cyclopropylamino-2-hydroxy-
propoxy side chain substituted at C-2h of 1 significantly
enhanced the antiplatelet effect. In collagen-induced
platelet aggregation, 1–3, 6–8, 11 and 12 showed a less
potent antiplatelet effect than in arachidonic-acid-
induced platelet aggregation while 4, 5, 9 and 10 showed
a more potent antiplatelet effect than in arachidonic-
acid-induced platelet aggregation. Compounds 4, 5, 7–9
and 10, with an oxypropanolamine side chain substi-
tuted at C-2h of 1, at low concentration significantly
enhanced the antiplatelet effect on collagen-induced
Compound
Aggregation (%)
ADP
Adrenaline
Collagen
Control
91.8p2.6
–
93.0p0.4
–
91.2p2.8
94.7p1.6
97.6p1.2
–
1
(150 µ)
(300 µ)
(450 µ)
(300 µ)
(450 µ)
(300 µ)
(450 µ)
(150 µ)
(300 µ)
(50 µ)
87.3p2.3*
–
53.7p1.9***
48.9p3.6***
91.1p0.7*
57.6p3.7***
90.8p1.5*
46.9p5.5***
91.3p3.4
2
83.5p1.5
–
80.0p7.1
–
3
78.0p3.4*
–
85.6p1.8
–
7
85.0p0.3
88.6p3.5
84.4p1.2
94.0p2.7
88.4p1.6
74.0p3.2
54.3p2.2***
39.6p15.4*
Aspirin
Platelets were pre-incubated with 1–3, 7, aspirin or DMSO (0.5%,
control) at 37mC for 3 min, then ADP (20 µ), adrenaline (5 µ) or
1
−
collagen (10 µg mL ) was added. Percentages of aggregation are
presented as meansps.e.m., n l 3–5. – , not determined. *P ! 0.05,
***P ! 0.001 as compared with the respective control values.
induced by PAF. All the 2h-oxypropanolamines (4–12) platelet aggregation. In PAF-induced platelet aggre-
strongly inhibited the platelet aggregation at high con- gation, none of the compounds at low concentration
centration induced by various inducers as shown in had a potent antiplatelet effect.
Table 2. This showed that the amino side chains of 4–12
The antiplatelet effects of selected compounds 1, 2, 3
did not alter the antiplatelet effects at high concentration and 7 were also tested on the aggregation of human PRP
on the platelet aggregation induced by various inducers. induced by ADP (20 µ), adrenaline (5 µ) and collagen
1
It also clearly indicated that an oxypropanolamine side (10 µg mL ). As shown in Table 4, all selected com-
−
chain substituted at C-2h of the flavone significantly pounds showed significant antiplatelet effects on
A
B
C
Figure 3 Effect of 2h-hydroxyflavone (1) on aggregation of human platelet-rich plasma (PRP) induced by adrenaline. PRP was incubated with
dimethylsulfoxide (0.5%) (A), or 2h-hydroxyflavone, 300 µ (B), 450 µ (C) for 3 min; the inducer adrenaline (5 µ) was then added to trigger
the aggregation.

1608
Mei-Ing Chung et al
2
+
+
Table 5 Effect of selected flavonoxypropanolamines on high-K - and Ca -induced and noradrenaline-
induced contraction of rat thoracic aorta.
2
+
+
Compound
K (80 m)jCa (1.9 m)
Noradrenaline
(3 µ) Phasic
(3 µ) Tonic
Control
100p5.8
–
100p3.9
96.5p2.5
0.0p0.0***
–
100p3.8
80.8p0.7*
25.0p1.3**
–
11
(15 µ)
(45 µ)
(150 µ)
(15 µ)
(45 µ)
(150 µ)
68.4p2.1*
24.9p0.5**
–
12
82.1p6.2
0.0p0.0***
–
81.9p1.0*
10.9p2.7**
–
76.4p6.2
24.6p8.6*
+
Rat aorta was pre-incubated with 11 and 12 or DMSO (0.1%, control) at 37mC for 15 min, then high K
2
+
(80 µ) and Ca (1.9 m) or noradrenaline (3 µ) was added. Percentages of contraction were calculated
and presented as meansps.e.m., n l 3. –, not determined. *P ! 0.05, **P ! 0.01, ***P ! 0.001 as
compared with the respective control values.
adrenaline-induced platelet aggregation. Aspirin was verapamil, a relatively selective inhibitor of voltage-
2
also used in this study as a positive control. It was found dependent Ca channels (Karaki et al 1984). The results
+
that aspirin (50 µ) inhibited the platelet aggregation from this study indicate that 11 is a relatively selective
2
induced by adrenaline but not by ADP and collagen. In inhibitor of receptor-operated Ca channels and its
+
human PRP, these compounds prevented secondary inhibitory effect on the contractile response caused by
+
aggregation induced by adrenaline (e.g. 1; Figure 3). We high K and noradrenaline in rat thoracic aorta is mainly
2
conclude that their mechanism of action is chiefly due to dueto the inhibitionof Ca influxthrough both voltage-
+
2
the inhibition of thromboxane formation (Weiss 1983b). dependent and receptor-operated Ca channels. Thus,
+
In rat thoracic aorta, 11 and 12 (Table 5) markedly 11 and 12 possess antiplatelet and vasorelaxing actions
2
depressed the contractions induced by Ca (1.9 m) in similar to those of norathyriol and apigenin. The activity
+
+
high-K (80 m) medium and the noradrenaline (3 µ)- in PRP represents the functional antagonist property in
induced phasic and tonic contractions, but the tonic a physiologically relevant medium and the activity in
contractions were more resistant to 11 and 12. the rat thoracic aorta represents antagonism on a non-
Compound 11 inhibited, in a concentration-depen- platelet side of the vascular system. These dual activities
+
dent manner, phenylephrine- and high-K -induced show that these compounds may be developed as anti-
45
2
Ca influx of rat aorta (Figure 4).
+
thrombotic agents.
The tonic contraction in response to noradrenaline
2
results primarily from Ca entry through receptor-
2
activated Ca channels with little requirement for Ca
+
Conclusion
2
+
+
2
+
through voltage-dependent Ca channels (Bohr 1963; These results indicate that most of the synthesized
Hudgins & Weiss 1968; Weiss 1983a). Noradrenaline- compounds possessed potent antiplatelet effect on
induced tonic contraction and phenylephrine-induced arachidonic-acid- and collagen-induced platelet aggre-
45
2
+
Ca influx were both inhibited by 11 (Table 5, Figure gation. Their antiplatelet effect is mainly due to an
4), indicating that 11 is a blocker of receptor-operated inhibitory effect on thromboxane formation. The selec-
ted compound 11 is a relatively selective inhibitor of
2
Ca channels.
+
2
Ithasbeenreportedthathigh-K -inducedcontraction receptor-operated Ca channels and its vasorelaxing
+
+
+
in vascular smooth muscle is mediated by an increase in effect on the contraction caused by high K and
2
Ca influx through voltage-dependent Ca channels noradrenaline in rat thoracic aorta is mainly due to
2
+
+
2
(Karaki & Weiss 1979). Since 11 inhibited the Ca - the inhibition of Ca influx through both voltage-
2
+
+
45
2
dependent contraction and Ca influx in high-K
2
+
+
+
dependent and receptor-operated Ca channels. Thus
this series of compounds with novel antiplatelet and
channels (Table 5, Figure 4). As shown in Figure 4, high vasorelaxing effects might be developed as antithrom-
2
+
medium, it may be a blocker of voltage-dependent Ca
45
2
+
+
K -induced Ca influx was more easily inhibited by botic agents.

1609
Antiplatelet and vasorelaxant monooxygenated flavones and flavonoxypropanolamines
Hudgins, P. M., Weiss, G. B. (1968) Differential effects of calcium
removal upon vascular smooth muscle contraction induced by
norepinephrine, histamine and potassium. J. Pharmacol. Exp. Ther.
159: 91–99
Control
Verapamil
11 15 lM
11 45 lM
11 100 lM
11 300 lM
Karaki, H., Weiss, G. B. (1979) Alterations in high and low affinity
binding of 45Ca in rabbit aortic smooth muscle by norepinephrine
and potassium after exposure to lanthanum and low temperature.
J. Pharmacol. Exp. Ther. 211: 86–92
Karaki, H., Nakagawa, H., Urakawa, N. (1984) Comparative effects
of verapamil and sodium nitroprusside on contraction and 45Ca
uptake in the smooth muscle of rabbit aorta, rat aorta, and guinea-
pig taenia coli. Br. J. Pharmacol. 81: 393–400
Ko F. N., Huang T. F., Teng C. M. (1991) Vasodilatory action
mechanisms of apigenin isolated from Apium graveolens in rat
thoracic aorta. Biochim. Biophys. Acta. 1115: 69–74
Control
Lin, C. N., Lu, C. M., Lin, H. C., Fang, S. C., Shieh, B. J., Hsu, M.
F., Wang, J. P., Ko, F. N., Teng, C. M. (1996) Novel antiplatelet
constituents from formosan moraceous plants. J. Nat. Prod. 59:
834–838
Verapamil
11 15 lM
11 45 lM
11 100 lM
11 300 lM
Liou, S. S., Teng, C. M., Ko, F. N., Lin, C. N. (1994) Gamma-Pyrone
compounds. 5. Synthesis and antiplatelet effects of xanthonoxy-
propanolamines and related compounds. J. Pharm. Sci. 83: 391–395
Meisheri, K. D., Palmer, R. F., Van Breemen, C. (1980) The effects of
2
+
amrinone on contractility, Ca uptake and cAMP in smooth
muscle. Eur. J. Pharmacol. 61: 159–165
O’Brien, J. R. (1962) Platelet aggregation. II. Some results from a new
method of study. J. Clin. Pathol. 15: 452–455
Figure 4 Effect of 2h-[3-(sec-Butylamino)-2-hydroxypropoxy]fla-
2
vone (11) on the 45Ca influx induced by phenylephrine and KCl in
+
Teng, C. M., Chen, W. Y., Ko, W. C., Ouyang, C. (1987) Antiplatelet
effect of butylidenephthalide. Biochim. Biophys. Acta 46: 375–382
Teng, C. M., Lee, L. G., Ko, F. N., Huang, T. H. (1988) Inhibition of
platelet aggregation by apigenin from Apium graveolens. Asia
Pacific J. Pharmacol. 3: 85–89
rat aortic rings. Aortic rings were placed in test-tubes containing
45
2
1
+
−
Krebs solution with Ca (1 µCi mL ) and incubated for 20 min
with DMSO (0.1%) or verapamil (2 µ) or various concentrations of
11, then phenylephrine (3 µ, A), KCl (60 m, B) or saline (resting)
45
2
+
was added and incubated for another 15 min. Ca influx into the
Weiss, G. B. (1983a) Delineation of actions of calcium antagonists in
2
vascular smooth muscle. In: Merrill, G. F., Weiss, H. R. (eds) Ca
muscle was measured. Data are expressed as percent increase of
45
+
2
+
Ca uptake over the resting value. Values are expressed as the
entry blockers, adenosine and neurohumors. Urban and Schwarzen-
berg, Baltimore, p. 63
meansps.e.m., n l 3–4, *P ! 0.05, **P ! 0.01, ***P ! 0.001 com-
+
pared with the respective control (phenylephrine or K alone).
Weiss, H. J. (ed.) (1983b) Antiplatelet drugs: pharmacological aspects.
In: Platelets: pathophysiology and antiplatelet drug therapy. Alan R
Liss Inc., New York, p. 46
Wu, E. S. C., Cole, T. E., Davidson, T. A., Dailey, M. A., Doring, K.
G., Fedorchuk, M., Loch, J. T., Thomas, T. L., Blosser, J. C.,
Borrelli, A. R., Kinsolving, C. R., Parker, R. B., Strand, J. C.,
Watkins, B. E. (1989) Flavones. 2. Synthesis and structure-activity
relationship of flavodiol and its analogues, a novel class of anti-
hypertensive agents with catecholamine depleting properties.
J. Med. Chem. 32: 183–192
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