Synthesis and antiplatelet activity of phenyl quinolones

Article abstract of DOI:10.1016/S0968-0896(98)00141-2

In our search for novel antiplatelet agents, seven positional phenyl quinolone isomers were synthesized. Preliminary screening confirmed their inhibitory effects against arachidonic acid (AA)-induced platelet aggregation. Varying the substitutional position of the phenyl group had a profound effect on the antiplatelet activity of these isomers. 3-Phenyl-4-quinolone showed the greatest potency and was superior to indomethacin, although the two structures are quite different. The mechanism and pharmacological action of 3-phenyl-4-quinolone are currently under investigation. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00141-2

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1657±1662
Synthesis and Antiplatelet Activity of Phenyl Quinolones
Li-Jiau Huang,a Ming-Chieh Hsieh,a Che-Ming Teng,b Kuo-Hsiung Leec
and Sheng-Chu Kuoa,*
aGraduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan
bPharmacological Institute, College of Medicine, National Taiwan University, Taiwan
cNatural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599, USA
Received 6 November 1997; accepted 2 March 1998
AbstractÐIn our search for novel antiplatelet agents, seven positional phenyl quinolone isomers were synthesized.
Preliminary screening con®rmed their inhibitory e€ects against arachidonic acid (AA)-induced platelet aggregation.
Varying the substitutional position of the phenyl group had a profound e€ect on the antiplatelet activity of these iso-
mers. 3-Phenyl-4-quinolone showed the greatest potency and was superior to indomethacin, although the two struc-
tures are quite di€erent. The mechanism and pharmacological action of 3-phenyl-4-quinolone are currently under
investigation. # 1998 Elsevier Science Ltd. All rights reserved.
Chemistry
Introduction
The goal of our research is to develop new antiplatelet
agents. Theoretically, antiplatelet agents can be devel-
oped that target each step in the platelet activation or
inhibition mechanisms. For example, although the anti-
platelet activity of aspirin was discovered while studying
cyclo-oxygenase inhibitors, numerous new antiplatelet
agents were developed based on their inhibitory e€ects
on platelet activation. Nonetheless, the number of anti-
platelet agents ready for clinical trials is still insucient,
and deleterious side e€ects are also associated with most
of the existing agents. Therefore, the search for an ideal
antiplatelet agent is still an important goal for the med-
ical and pharmaceutical industries.
Scheme 1 illustrates the synthetic methods for three tar-
get compounds: 2-phenyl-4-quinolone (3),1 N-methyl-2-
phenyl-4-quinolone (4),1 and 4-phenyl-2-quinolone (6).2
Compounds 3 and 4 were previously reports by us.
Compound 6 was synthesized by the method reported
by B. Staskun in 1961;2 however, we have included
spectral data not given in the previous report.
Although the synthesis of 3-phenyl-4-quinolone (10) is
known in the literature,3,4 prior methods resulted in low
yields. We have developed a new, higher yield synthetic
route as presented in Scheme 2. The starting material,
ethyl phenylacetate (7) was formylated with ethyl for-
mate in the presence of NaH to a€ord intermediate 8,
which was condensed with aniline in EtOH to yield 9. A
thermally-induced cyclization of 9 in polyphosphoric
acid (PPA) resulted in target compound 10.
During routine antiplatelet screening, we recently dis-
covered that 2-phenyl-4-quinolone (3) shows aspirin-like
antiplatelet activity. Since the physiochemical properties
of 3 are quite di€erent from those of aspirin and the
antiplatelet activity of 3 has not been previously repor-
ted, we synthesized a series of positional isomers of 3.
We report herein on the synthesis and antiplatelet
activity of these compounds.
6-, 7-, and 8-Phenyl-4-quinolones (24±26) were prepared
as shown in Scheme 3. A Michael reaction between the
appropriate aminobiphenyl and diethyl ethoxymethyl
malonate (EMME), followed by thermal cyclization in
diphenyl ether gave the corresponding ethyl phenyl-4-
quinolone-3-carboxylates (18±20). These compounds
then underwent basic (NaOH) hydrolysis and thermal
decarboxylation to give the target compounds 24±26.
Key words: Phenyl quinolone; antiplatelet activity; platelet
aggregation; arachidonic acid; antiin¯ammatory.
*Corresponding author.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00141-2

1658
L.-J. Huang et al./Bioorg. Med. Chem. 6 (1998) 1657±1662
Scheme 1.
Scheme 2.
Scheme 3.

L.-J. Huang et al./Bioorg. Med. Chem. 6 (1998) 1657±1662
1659
Scheme 4.
The synthesis of 3-phenyl-2-quinolone (30) is also
known in the literature,5,6 but again, was achieved only
in low yields. As shown in Scheme 4, we adapted the
method of H. Hubner7 by reacting isatin (27) with phe-
nyl acetic anhydride (28) in nitrobenzene at high tem-
perature to a€ord 3-phenyl-2-quinolone-4-carboxylic
acid (29). Decarboxylation in quinoline in the presence
of active copper then gave target compound 30.
Changing the relative position of the carbonyl group
was also studied. 4-Phenyl-2-quinolone (6) had similar
potency (IC50=12.19 mM) to that of 2-phenyl-4-quino-
lone (3). However, 3-phenyl-2-quinolone (30) was much
less activity (IC50=35.00 mM) than either its 4-phenyl
(6) or 4-quinolone (10) positional isomers.
The preliminary antiplatelet activity±chemical structure
relationships of these seven positional phenyl quinolone
isomers, thus, show a clear e€ect of structure on activ-
ity. The role of physiochemical properties is still under
investigation.
Antiplatelet Activity
Using thrombin (0.1 unit/mL), arachidonic acid (AA)
(100 mM), collagen (10 mg, mL), and platelet activating
factor (PAF) (2 ng/mL) as platelet aggregation inducers,
all synthesized phenyl quinolone analogues (3, 4, 6, 10,
24±26, and 30) were screened for antiplatelet activity in
washed rabbit platelets. Most target compounds demon-
strated antiplatelet activity, especially against AA-
induced platelet aggregation. Table 1 lists these results
as IC50 values.
Table 1. Inhibitory e€ects of phenyl quinolone derivatives on
platelet aggregation induced by arachidonic acid
2-Phenyl-4-quinolone (3) inhibited AA-induced platelet
aggregation in a concentration-dependent manner. Its
IC50 value was 9.63 mM, twice the potency of aspirin
(IC50=20.0 mM). The antiplatelet potency dropped
considerably when the amine (3: NH) was methylated
(4: N-CH3, IC50=40.0 mM).
Compd
IC50 (mM) R2
R3
R6
R7
R8
3
9.63 Phenyl
H
Phenyl
H
H
H
H
H
H
H
10
0.17
34.99
24.90
7.05
H
H
24
H
H
Phenyl
H
25
H
H
H
Phenyl
H
26
4a
6b
H
H
Phenyl
Ð
H
The antiplatelet activity also varied signi®cantly with
the position of the phenyl group. 6-Phenyl-4-quinolone
(26) was slightly more potent (1.4 times) than 3, but
phenyl substitution at position 7 (25) or 8 (24) weakened
potency as seen by higher IC50 values of 24.9 mM
and 34.99 mM, respectively. The antiplatelet activity of
3-phenyl-4-quinolone (10), however, was signi®cantly
enhanced; its IC50 value of 0.17 mM is 57 times that of
3, 118 times that of aspirin, and 1.5 times that of
indomethacin. Thus, 10 has extremely high antiplate-
let potency, higher than that commonly seen in the
literature.
40.00
12.19
35.00
0.25
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
30c
Ð
Ð
Ð
Ð
Indomethacin
Aspirin
Ð
Ð
Ð
Ð
20.00
Ð
Ð
Ð
Ð
Platelets were incubated with a test sample or 0.5% DMSO at
37 ꢀC for 1 min, then AA (100 mM) was added to trigger aggre-
gation. Aspirin is a positive control. Values are expressed as
mean‹S.D. from four to six separations.
a4: N-methyl-2-phenyl-4-quinolone.
b6: 4-Phenyl-2-quinolone.
c30: 3-Phenyl-2-quinolone.

1660
L.-J. Huang et al./Bioorg. Med. Chem. 6 (1998) 1657±1662
In summary, we have discovered the superb inhibitory
e€ect of 3-phenyl-4-quinolone (10) against AA-induced
platelet aggregation. This compound demonstrated an
inhibitory e€ect superior to that of indomethacin and
aspirin, which are well known for their potent anti-
platelet activity, yet 10 is distinctly di€erent in structure
from these compounds. Therefore, we have selected 10
as a promising, new lead compound for further devel-
opment of antiplatelet agents. The mechanism and
pharmacological action of this compound are currently
under investigation. However, it has been reported that
the antiin¯ammatory action of compound 3 may be due
to inhibition of prostaglandin formation.9
1H NMR (300 MHz) d 6.39 (1H, s, H-3), 7.13 (1H, ddd,
J=1.2, 7.2, 7.5 Hz, H-6), 7.36±7.57 (8H, m, H-5, 7,
8, 20, 30, 40, 50, 60), 11.89 (1H, br, NH); 13C
NMR (75.43 MHz) d 115.99 (C-8), 118.56 (C-4a), 121.40
(C-3), 122.05 (C-6), 126.32 (C-7), 128.85 (C-20, C0-60),
128.88 (C-30, C-50), 128.96 (C-40), 120.75 (C-5), 136.90
(C-10), 139.50 (C-8a), 151.69 (C-4), 161.51 (C-2). Anal.
calcd for C15H11NO2: C, 81.42; H, 5.01; N, 6.33. Found:
C, 81.40; H, 4.98; N, 6.28.
3-Phenyl-4-quinolone (10).3,4 To a mixture of ethyl
phenylacetate (1.64 g, 0.01 mol) and ethyl formate
(1.48 g, 0.02 mol) was added NaH (80% in oil, 0.6 g,
0.02 mol). This mixture was stirred for 1.5 h, poured into
ice water, acidi®ed with 2 N HCl, then extracted with
EtOAc. The EtOAc extract was dried over MgSO4 and
concentrated under reduced pressure to yield 8 as an oil.
Aniline (0.93 g, 0.01mol) and EtOH (100 mL) were added,
and the mixture stirred at room temperature for 10 h, then
concentrated in vacuo to give 9 as an oily product. A
mixture of 9 and PPA (4 g) was stirred at 110 ꢀC for
20 min, cooled to room temperature, and treated with
10% NaOH to a€ord compound 10 as a white powder
(0.96 g, 43% yield) melting point 268±269 ꢀC; MS m/z:
220 [M+], 192 [M+ CO]; UV (MeOH): lmax 261.0 nm
Experimental
General
All melting points are uncorrected. IR spectra were
recorded on Shimadzu IR-440 and Nicolet Impact 400
FT-IR spectrophotometers as KBr pellets. NMR spec-
tra were obtained on Bruker ARX-300 and Varian
VXR-300 FT-NMR spectrometers in DMSO-d6. The
following abbreviations are used: s, singlet; d, doublet, t,
triplet, q, quartet; m, multiplet, and br, broad. MS were
measured with and HP 5995 GC-MS instrument. The
UV spectra were recorded on a Shimadu UV-160A UV-
Vis recording spectrophotometer. Elemental analyses
(C, H, and N) were performed by National Cheng Kung
University and National Chung Hsing University, Tai-
wan and were within 0.4 ppm.
(log e=4.41); IR (KBr): nmax 3218 (NH), 1616 (C ˆ O)
1
cm
.
1H NMR (200 MHz) d 7.22±7.42 (4H, m, H-6,
30, 40, 50), 7.56±7.66 (2H, m, H-7, H-8), 7.71 (2H,
dd, J=1.1, 8.14 Hz, H-20, 60), 8.15 (1H, s, H-2), 8.21
(1H, d, J=8.2 Hz, H-5), 12.10 (1H, br, NH); 13C NMR
(50.33 MHz) d 118.43 (C-8), 119.98 (C-10), 123.52 (C-6),
125.82 (C-5), 126.01 (C-4a), 126.60 (C-40), 128.01 (C-30,
C-50), 128.64 (C-20, C-60), 131.81 (C-7), 136.37 (C-3),
138.37 (C-2), 139.50 (C-8a), 174. 96 (C ˆ O). Anal. calcd
for C15H11NO: C, 81.43; H, 5.01; N, 6.33. Found: C,
81.39, H, 5.03; N, 6.27.
Benzoylacetanilide (5).1 A mixture of aniline (9.3 g,
0.1 mol) and ethyl benzoylacetate (38.44 g, 0.2 mol) was
stirred at 140±150 ꢀC for 1 h, cooled to room tempera-
ture, then treated with 10% NaOH to yield a yellow
precipitate. After ®ltration, the ®ltrate was acidi®ed with
HOAc to a€ord a yellow solid, which was recrystallized
from 50% EtOH to yield 5 as colorless needles (14.3 g,
60% yield). Melting point 109±110 ꢀC; MS m/z: 239
[M+]; UV (MeOH): lmax 244 nm (log e=4.28); IR
Ethyl 8-, 7-, 6-phenyl-4-quinolone-3-carboxylate (18±20).8
Diethyl ethoxymethylene malonate (2.16 g, 0.01 mol)
was heated to 135 ꢀC, then the appropriate amino-
biphenyl (1.69 g, 0.02 mol) was added. This mixture was
stirred for 5 min before adding diphenyl ether (50 mL).
The mixture was then heated to 240 ꢀC, stirred for
5 min, and cooled to room temperature. n-Hexane
(80 mL) was added, and the mixture was stirred and ®l-
tered. The precipitate was washed with CHCl3 to give a
gray solid. The product was chromatographed on a
silica gel column (CHCl3-EtOH, dry method).
(KBr): nmax 3265 (NH), 1695 (C ˆ O), 1693 (C ˆ O)
1
cm
;
1H NMR (90 MHz) d 4.08 (2H, s, CH2), 7.08±
7.61 (8H, m, H-2, 3, 4, 5, 6, 30, 40, 50), 7.98
(2H, d, J=8.4 Hz, H-20, H-60), 9.31 (1H, br, NH). Anal.
calcd for C13H13NO2: C, 75.30; H, 5.48; N, 5.85. Found:
C, 75.26; H, 5.45; N, 5.87.
4-Phenyl-2-quinolone (6).1
A mixture of 5 (4.78 g,
0.02 mol) and PPA (50 g) was stirred at 140±150 ꢀC for
20 min, cooled to room temperature, then treated with
10% NaOH to yield a brown precipitate. Recrystalliza-
tion from dil EtOH gave 6 as colorless needles (3.32 g,
75% yield). Melting point 200±202 ꢀC; MS m/z: 221
Ethyl 8-phenyl-4-quinolone-3-carboxylate (18). From 2-
amino biphenyl, 2.5 g, 85% yield, white powder. Melt-
ing point 238±240 ꢀC; MS m/z: 293 [M+]; IR (KBr):
1
nmax 3167 (NH), 1709 (C ˆ O), 1619 (C ˆ O) cm
.
1H NMR (90 MHz) d 1.25 (3H, t, J=7.2 Hz, CH3), 4.20
(2H, q, J=7.2 Hz, OCH2), 7.2±7.8 (7H, m, Ar-H), 8.21
(1H, dd, J=1.6, 7.8 Hz, H-5), 8.35 (1H, s, H-2), 11.07
[M+], 193 [M+ CO]; UV (MeOH): lmax 224.4 nm (log
1
e=4.37): IR (KBr); nmax 3300 (NH), 1666 (C ˆ O) cm
;

L.-J. Huang et al./Bioorg. Med. Chem. 6 (1998) 1657±1662
1661
(1H, br, NH). Anal. calcd for C18H15NO3: C, 73.71; H,
5.15; N, 4.78. Found: C, 73.60; H, 5.10; N, 4.81.
8-, 7-, and 6-Phenyl-4-quinolone (24±26). Compound 21,
22, or 23 (2.5 g, 0.01 mol) was suspended in diphenyl
ether (50 mL) and heated at 240 ꢀC for 24 h to a€ord a
black liquid. After cooling, n-hexane (80 mL) was added
and the liquid mixture stirred, then ®ltered. The pre-
cipitate was recrystallized from MeOH to give com-
pound 24 (1.9 g, 86% yielded), 25 (2.0 g, 90% yield),
or 26 (1.9 g, 86% yield), respectively, as pale-yellow
needles.
Ethyl 7-phenyl-4-quinolone-3-carboxylate (19). From 3-
amino biphenyl, 1.8 g, 61% yield, white powder. Melt-
ing point 254±256 ꢀC; MS m/z: 293 [M+]; IR (KBr):
1
nmax 3137 (NH), 1694 (C ˆ O), 1616 (C ˆ O) cm 1. H
NMR (90 MHz) d 1.58 (3H, t, J=7 Hz, CH3), 4.72 (2H,
q, J=7 Hz, OCH2), 7.43±8.0 (5H, m, Ar-H), 8.17±8.43
(2H, m, H-6, H-8), 8.85 (1H, d, J=9 Hz, H-5), 9.20 (1H,
s, H-2). Anal. calcd for C18H15NO3: C, 73.71; H, 5.15;
N, 4.78. Found: C, 73.78; H, 5.21; N, 4.83.
8-Phenyl-4-quinolone (24). Melting point 186±188 ꢀC;
MS m/z: 221 [M+]; UV (MeOH): lmax 227.6 nm (log
1
e=4.54); IR (KBr): nmax 3191 (NH), 1620 (C ˆ O) cm
.
Ethyl 6-phenyl-4-quinolone-3-carboxylate (20). From 4-
amino biphenyl, 2.5 g, 85% yield, white powder. Melt-
ing point 294±296 ꢀC; MS m/z: 293 [M+]; IR (KBr):
1H NMR (200 MHz) d 6.07 (1H, d, J=7.46 Hz, H-3),
7.34±7.56 (7H, m, Ar-H), 7.72 (1H, d, J=7.42 Hz, H-2),
8.15 (1H, dd, J=1.6, 7.94 Hz, H-5), 10.59 (1H, br, NH);
13C NMR (50.33 MHz) d 108.82 (C-3), 123.20 (C-6),
124.86 (C-8), 126.43 (C-4a), 128.45 (C-5), 129.38 (C-20,
C-60), 129.66 (C-30, C-50), 131.67 (C-40), 132.96 (C-7),
136.87 (C-10), 137.30 (C-8a), 140.34 (C-2), 177.28
(C ˆ O). Anal. calcd for C15H11NO: C, 81.43; H, 5.01;
N, 6.33. Found: C, 81.40, H, 5.09; N, 6.38.
1
nmax 3164 (NH), 1701 (C ˆ O), 1620 (C ˆ O) cm 1. H
NMR (90 MHz) d 1.56 (3H, t, J=7 Hz, CH3), 4.71 (2H,
q, J=7 Hz, OCH2), 7.2±8.0 (5H, m, Ar-H), 8.16 (1H, d,
J=9 Hz, H-8), 8.81 (1H, d, J=2 Hz, H-5), 9.26 (1H, s,
H-2). Anal. calcd for C18H15NO3: C, 73.71; H, 5.15; N,
4.78. Found: C, 73.61; H, 5.18; N, 4.75.
6-, 7-, and 8-Phenyl-4-quinolone-3-carboxylic acid (21±23).
Compounds 18, 19, or 20 (2.93 g, 0.01 mol) was treated
with 10% NaOH (100 mL), then heated until completely
dissolved. The reaction mixture was then cooled and
acidi®ed with 10% HCl. The precipitate was ®ltered and
recrystallized from MeOH to yield compound 21 (2.4 g,
90% yield), 22 (2.5 g, 94% yield), or 23 (2.4 g, 90%
yield), respectively, as colorless needles.
7-Phenyl-4-quinolone (25). Melting point 264±266 ꢀC;
MS m/z: 221 [M+]; IR (KBr): nmax 3240 (NH), 1624
1
(C ˆ O) cm
.
1H NMR (90 MHz) d 6.02 (1H, d,
J=8 Hz, H-3), 7.20±8.0 (8H, m, Ar-H, H-2) 8.06 (1H,
dd, J=8 Hz, H-5). Anal. calcd for C15H11NO: C, 81.43;
H, 5.01; N, 6.33. Found: C, 81.38; H, 5.05; N, 6.29.
6-Phenyl-4-quinolone (26). Melting point 264±266 ꢀC;
MS m/z: 221 [M+]; UV (MeOH): lmax: 256.6 nm (log
1
8-Phenyl-4-quinolone-3-carboxylic acid (21). Melting
point 234±236 ꢀC; MS m/z: 265 [M+]; IR (KBr): nmax
3300 (NH), 3300±2300 (OH), 1700 (C ˆ O) 1615 (C ˆ O)
cm 1. 1H NMR (90 MHz) d 7.4±7.9 (7H, m, Ar-H), 8.35
(1H, dd, J=8 Hz, H-5), 8.57 (1H, s, H-2). Anal. calcd
for C16H11NO3: C, 72.45; H, 4.18; N, 5.28. Found: C,
72.55; H, 4.16; N, 5.17.
e=4.48); IR (KBr): nmax 3248 (NH), 1651 (C ˆ O) cm
.
1H NMR (90 MHz) d 6.06 (1H, d, J=7.33 Hz, H-3),
7.42±7.95 (8H, m, Ar-H, H-2) 8.32 (1H, dd, J=2.45 Hz,
H-5). Anal. calcd for C15H11NO: C, 81.43; H, 5.01; N,
6.33. Found: C, 81.35; H, 4.98; N, 6.29.
3-Phenyl-2-quinolone (30).5,6 A mixture of phenyl acetic
acid (7.73 g, 0.05 mol) and acetic anhydride (10.60 g,
0.03 mol) was re¯uxed for 2 h, cooled to room tempera-
ture, and concentrated in vacuo to remove excess acetic
anhydride. Addition of water gave a yellow precipitate,
which was washed with petroleum ether to yield 28
(7.6 g) as a yellow solid. Nitrobenzene (50 mL) and isa-
tin (27) (4.41 g, 0.03 mol) were added, and the mixture
was allowed to react at 185 ꢀC for 5 h. After cooling to
room temperature, a yellow compound precipitated and
was washed successively with water and EtOH to give
29 (2.5 g) as a yellow±brown solid. Quinoline (12 mL)
and activated copper (0.2 g), freshly prepared by react-
ing zinc powder with CuSO4 solution, were added. The
mixture as re¯uxed for 2 h, cooled to room temperature,
poured into cold HCl solution, and then extracted with
CHCl3. The CHCl3 layer was dried over MgSO4 and
concentrated in vacuo to yield a yellow±brown solid,
7-Phenyl-4-quinolone-3-carboxylic acid (22). Melting
point 278±280 ꢀC; MS m/z: 265 [M+]; IR (KBr): nmax
3160 (NH), 3300±2500 (OH), 1680 (C ˆ O) 1630 (C ˆ O)
1
cm 1. H NMR (90 MHz) d 7.51±7.96 (6H, m, Ar-H),
8.19 (1H, s, H-8), 8.7 (1H, J=8.8 Hz, H-5) 8.83 (1H, s,
H-2). Anal. calcd for C16H11NO3: C, 72.45; H, 4.18; N,
5.28. Found: C, 72.51; H, 4.14; N, 5.21.
6-Phenyl-4-quinolone-3-carboxylic acid (23). Melting
point 286±288 ꢀC; MS m/z: 265 [M+]; IR (KBr): nmax
3020 (NH), 3000±2500 (OH), 1670 (C ˆ O) 1630 (C ˆ O)
1
cm 1. H NMR (90 MHz) d 7.41±7.95 (6H, m, Ar-H),
8.22 (1H, q, J=2.44, 8.9 Hz, H-7), 8.48 (1H, d,
J=2.5 Hz, H-5), 8.89 (1H, s, H-2). Anal. calcd for
C16H11NO3: C, 72.45; H, 4.18; N, 5.28. Found: C,
72.39; H, 4.24; N, 5.36.

1662
L.-J. Huang et al./Bioorg. Med. Chem. 6 (1998) 1657±1662
which was chromatographed on a silica gel column
(CHCl3±EtOH) to give 30 (0.2 g, 24% yield) as a dark-
red powder.
without EDTA. After centrifugation at the same condi-
tions, the platelet pellets were ®nally suspended in Tyr-
ode's solution of the following composition (mM): NaCl
(136.8), KCl (2.8), NaHCO3 (11.9), MgCl2 (1.1),
NaH2PO4 (0.33), CaCl2 (1.0), and glucose (11.2). Plate-
let numbers were counted by Coulter Counter (Model
ZM) and adjusted to 4.5Â108 platelets/mM.
8-Phenyl-4-quinolone (24). Melting point 200±202 ꢀC;
MS m/z: 221 [M+]; UV (MeOH): lmax: 226 nm (log
1
e=4.53); IR (KBr): nmax 1657 (C ˆ O) cm 1. H NMR
(300 MHz) d 7.18 (1H, ddd, J=1.2, 7.7 Hz, H-6), 7.32±
7.45 (4H, m, H-8, 30, 40, 50) 7.71 (1H, dd, J=1.2,
7.77 Hz, H-5), 7.73±7.83 (2H, m, H-20, H-60), 8.07 (1H,
s, H-4), 11.86 (1H, s, NH): 13C NMR (74.53 MHz) d
114.62 (C-8), 119.51 (C-4a), 121.80 (C-6), 127.65 (C-5),
127.71 (C-20, C-60), 127.84 (C-7), 128.59 (C-30, C-50),
130.08 (C-40), 131.58 (C-3), 136.31 (C-10), 137.51 (C-4),
138.31 (C-8a), 160.99 (C ˆ O). Anal. calcd for
C15H11NO: C, 81.43; H, 5.01; N, 6.33. Found: C, 81.31;
H, 4.92; N, 6.28.
Platelet aggregation. Aggregation was measured by the
turbidmetric method with a dual-channel Lumiag-
gregometer (Model 1020, Payton, Canada). All glass-
ware was siliconized. One minute before the addition of
the aggregation inducer, the platelet suspension was
stirred at 900 rpm. The percentage of aggregation was
calculated as described previously.
Acknowledgements
This work was supported by a research grant from the
National Council of the Republic of China (NSC81-
0412-B-039-01).
Materials
Evaluation of antiplatelet aggregation activity. Collagen
(type 1, bovine Achilles tendon), obtained from Sigma
Chemical Co., was homogenized in 25 mL HOAc and
stored at 70 ꢀC. Arachidonic acid, bovine serum albu-
min (BSA), EDTA (disodium salt), sodium citrate,
dimethyl sulfoxide (DMSO), and platelet activating
factor (PAF) were purchased from Sigma Chemical Co.
Thrombin (bovine) was obtained from Parke±Davis Co.
and dissolved in 50% (v/v) glycerol to give a stock
solution of 100 NIH units/mL.
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