Antiplatelet activity of synthetic pyrrolo-benzylisoquinolines

Article abstract of DOI:10.1016/S0960-894X(03)00003-9

Pyrrolo-benzylisoquinolines were prepared as target compounds and their antiplatelet aggregation activity, adreno-receptor affinity, and cytotoxicity were screened. Compounds 1d-9d showed specific antiplatelet aggregation activity induced by arachidonic acid and collagen. Among them, 8d and 9d exhibited better activity than the reference drug, aspirin and 9d also showed inhibition of platelet aggregation by all four inducers.

Full text of DOI:10.1016/S0960-894X(03)00003-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 821–823
Antiplatelet Activity of Synthetic Pyrrolo-Benzylisoquinolines
Reen-Yen Kuo,a Chin-Chung Wu,a Fang-Rong Chang,a Jwu-Lai Yeh,b Ing-Jun Chenb
and Yang-Chang Wua,*
aGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
bDepartment of Pharmacology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Received 11 November 2002;revised 20 December 2002;accepted 23 December 2002
Abstract—Pyrrolo-benzylisoquinolines were prepared as target compounds and their antiplatelet aggregation activity, adreno-
receptor affinity, and cytotoxicity were screened. Compounds 1d–9d showed specific antiplatelet aggregation activity induced by
arachidonic acid and collagen. Among them, 8d and 9d exhibited better activity than the reference drug, aspirin and 9d also showed
inhibition of platelet aggregation by all four inducers.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
were treated with thionyl chloride in dry dichloro-
methane to form the active acyl chloride and then com-
Benzylisoquinolines, a class of the most commonly iso-
lated alkaloids in higher plants, have been studied for
their rich structural and pharmacological diversity.1ꢀ5
They also play an important role in the biosynthetic
pathway of morphines and papaverines.6 Benzylisoqui-
noline derivatives have been extensively studied for their
interaction with various biological functions, such as
analgesic,7,8 muscle relaxation,9 anticancer activity,10,11
and cardiovascular activities.12ꢀ15 Among the benzyl-
isoquinoline derivatives, pyrrolo-benzylisoquinolines
were prepared as intermediates in the total synthesis of
aporphines and protoberberines.16,17 However, the bio-
logical activities of these compounds have never been
discussed. In our ongoing research of Annonaceous
alkaloids, pyrrolo-benzylisoquinolines, possessing an
interesting skeleton, were prepared and their biological
activities were studied.
pound
2-(30,40-dimethoxyphenyl)ethylamines
were
added to yield the amide derivatives 1b–9b. The amides
were purified and poured into a mixture of dry aceto-
nitrile and excess phosphoryl chloride. The resulting
mixture was refluxed for 3 h to give the products, 3,4-
dihydrobenzylisoquinolines 1c–9c via Bischler–Napier-
alski reaction. Compounds 1c–9c were not purified
because of their instability. Diluted oxalyl chloride in
CH2Cl2 was added dropwise to the mixture of crude 1c–9c
and CH2Cl2 under N2 at ꢀ20 ꢁC. The deep red final
products, 1d–9d, were purified and identified by spectral
data to confirm the structures.
Pharmacological Evaluation and Discussion
As we mentioned above, benzylisoquinolines are asso-
ciated with various pharmacological functions. Based
on these surveys, we studied the antiplatelet aggregation
activity of compounds 1d–9d. In the platelet aggregation
assays, four inducers were employed, including AA
(arachidonic acid), Col (collagen), PAF (platelet acti-
vating factor), and Thr (thrombin). Table 1 shows the
results of the experiments.
Chemistry
All compounds generally showed inhibitory effects on
platelet aggregation induced by AA and Col in a con-
centration-dependent manner. This specific inhibitory
effect shows similarity to the reference drugs, aspirin. In
comparison of the reference drug with these tested
compounds, 8d and 9d showed better antiaggregatory
Target compounds 1d–9d18 were synthesized as illu-
strated in Scheme 1. Substituted phenylacetic acids 1a–9a
*Corresponding author. Tel.: +886-7-3121101x2197;fax: +886-7-
3114773;e-mail: yachwu@kmu.edu.tw
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00003-9

822
R.-Y. Kuo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 821–823
Table 1. Antiplatelet aggregation activity of compounds 1d–9d19
IC50 (mM)a,b
AA (100mM) COL (10mg/mL) PAF (2ng/mL) Thr (0.1U)
Aspirin 44.6ꢃ9.8
22.4ꢃ4.1
44.9ꢃ10.8
37.5ꢃ5.6
30.0ꢃ1.7
39.8ꢃ6.1
23.3ꢃ3.9
29.8ꢃ0.8
35.8ꢃ12.7
6.1ꢃ1.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1d
2d
3d
4d
5d
6d
7d
8d
9d
78.3ꢃ3.9
53.8ꢃ10.7
52.8ꢃ8.6
>100
84.8ꢃ8.7
>100
>100
45.5ꢃ10.5
72.7ꢃ4.3
65.5ꢃ4.1
21.0ꢃ2.6
15.2ꢃ3.8
>100
>100
>100
33.2ꢃ1.3
9.15ꢃ2.4
30.7ꢃ1.1
aPlatelet were preincubated with DMSO (0.5%, control), aspirin or
tested compounds at 37 ꢁC for 3 min, then four inducers were added.
bThe IC50 values were presented as meansꢃS.E. (n=3).
Scheme 1. Synthesis of pyrrolo-benzylisoquinoline derivatives 1d–9d.
pyrrolobenzylisoquinolines possessed lower antiplatelet
activity than 30 or 40-substituted ones. The presence of
the methoxy group on the benzyl group exhibited better
activity than halogenated ones. Compound 9d, the
derivative that fits in with the two conditions showed
the best activity among these compounds.
Besides the antiaggregation activity, compounds 2d, 3d, 6d,
7d, and 9d were submitted to screen the b1 and b2 adreno-
receptor binding affinities. However, all of them demon-
strate mild affinity to both b1 and b2 receptors (Fig. 1).
Cytotoxic activity was also evaluated for compounds
1d–9d.21 Preliminary results of cytotoxicity toward
HONE-1 (human nasopharyngeal carcinoma) and
NUGC (human gastric cancer) cell lines revealed that
all compounds showed no activity against two cell lines
in a concentration of 10 mM-with survival percentage
86–105% and 93–104%, respectively, in comparison
with the DMSO vehicle control.
In conclusion, we synthesized nine pyrrolo-benzyliso-
quinolines that displayed a specific activity toward pla-
telet aggregation and have provided a new active
skeleton in the development of antiplatelet aggregation
drugs. Compound 9d, the most potent alkaloid, could
be further investigated as the lead compound and the
relationship between 3-dimensional structure and activ-
ity of these derivatives should be the focus in a
continuing study.
Figure 1. Inhibition of [3H]CGP-12177 specific binding to guinea pig
b1 (ventricular membrane) and b2 (lung membrane) adrenoreceptor by
2d, 3d, 6d, 7d, and 9d.20
activity with IC50 15–21 mM and 6ꢂ9 mM induced by
AA and Col, respectively, than aspirin and 5d demon-
strated almost equal potency as aspirin. Compound 9d,
the most potent derivative, showed additional inhibition
on platelet aggregation induced by the other two indu-
cers, PAF and Thr. This result revealed that 9d may act
by a different mechanism from other derivatives.
Acknowledgements
This investigation was supported by a grant from the
National Science Council of the Republic of China. We
also thank the Division of Biotechnology and Pharma-
ceutical Research, National Health Research Institute,
Republic of China for the cytotoxicity studies.
In the structure–activity relationship of these synthe-
sized compounds, the substitution variation at the ben-
zyl group slightly changed the activity: 20-substituted

R.-Y. Kuo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 821–823
823
References and Notes
cmꢀ1. 4d: 1H NMR: d 7.68 (1H, d, J=7.8 Hz, Ar-H), 7.26
(3H, m, Ar–H), 6.76 and 6.60 (each 1H, s, Ar–H), 3.90 and
3.25 (each 3H, s, 2ꢄOMe), 3.83 (2H, t, J=6.2 Hz, N-CH2–),
3.06 (2H, t, J=6.2 Hz, Ar-CH2–); 13C NMR: d 182.3, 158.5,
157.7, 153.9, 148.0, 134.5, 133.4, 132.3, 130.1, 130.0, 128.4,
127.9, 116.2, 111.9, 111.3, 106.6, 56.3, 55.4, 36.3, 28.6;EI–MS
m/z: 415 [M]+, 413 [Mꢀ2]+;UV: 260, 288(sh), 317, 379 nm;
IR (KBr): 1743, 1701 cmꢀ1. 5d: 1H NMR: d 7.52 (1H, d,
J=1.2 Hz, Ar–H), 7.46 (1H, m, Ar–H), 7.30 (2H, m, Ar–H),
6.94 and 6.77 (each 1H, s, Ar–H), 3.93 and 3.39 (each 3H, s,
2ꢄOMe), 3.80 (2H, t, J=6.2 Hz, N-CH2–), 3.06 (2H, t, J=6.2
Hz, Ar-CH2–); 13C NMR: d 182.2, 157.9, 157.6, 153.8, 147.8,
133.6, 132.6, 130.7, 130.2, 129.1, 122.4, 116.1, 111.7, 111.2,
108.0, 106.6, 56.2, 55.4, 36.2, 27.1;EI–MS: m/z: 415 [M]+, 413
[Mꢀ2]+;UV: 234 (sh), 262, 284 (sh), 319, 381 nm;IR (KBr):
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1
1742, 1697 cmꢀ1. 6d: H NMR: d 7.53 (2H, d, J=8.4 Hz, Ar–
H), 7.24 (2H, d, J=8.4 Hz, Ar–H), 6.92 and 6.77 (each 1H, s,
Ar–H), 3.95 and 3.40 (each 3H, s, 2xOMe), 3.83 (2H, t, J=5.8
Hz, N-CH2–) and 3.07 (2H, t, J=5.8 Hz, Ar-CH2–); 13C
NMR: d 182.6, 158.2, 157.6, 153.9, 148.0, 133.4, 132.0 (2 sig-
nals), 131.9 (2 signals), 129.5, 122.0, 116.4, 111.9, 111.4, 107.1,
56.4, 55.5, 36.4, 28.7;EI–MS m/z: 415 [M]+, 413 [Mꢀ2]+;
UV: 240, 284 (sh), 322 nm;IR (KBr): 1739, 1697 cm ꢀ1. 7d: 1H
NMR: d 7.33 (1H, td, J=7.8, 1.8 Hz, Ar-H), 7.22 (1H, dd,
J=7.8, 1.8 Hz, Ar–H), 7.02 (1H, brt, J=7.6 Hz, Ar–H), 6.99
(1H, brd, J=7.6 Hz, Ar–H), 6.86 and 6.73 (each 1H, s, Ar–H),
3.93, 3.68, and 3.30 (each 3H, s, 3ꢄOMe), 3.84 (2H, t, J=6.2
Hz, N-CH2–), 3.07 (2H, t, J=6.2 Hz, Ar-CH2–); 13C NMR: d
182.9, 158.7, 157.7 (2 signals), 153.1, 147.8, 132.3, 132.1, 129.8,
121.1, 119.5, 117.4, 111.5, 111.3, 110.9, 105.0, 56.1, 55.6, 55.2,
36.3, 29.4;EI–MS m/z: 365 [M]+;UV: 256, 284 (sh), 324 (sh),
336 nm;IR (KBr): 1742, 1697 cm ꢀ1. 8d: 1H NMR: d 7.27 (1H,
m, Ar–H), 6.96 and 6.74 (each 1H, s, Ar–H), 6.84 (3H, m, Ar–
H), 3.91, 3.73 and 3.21 (each 3H, s, 3ꢄOMe), 3.77 (2H, t,
J=5.8 Hz, N-CH2–), 3.04 (2H, t, J=5.8 Hz, Ar-CH2–); 13C
NMR: d 182.6, 159.8, 158.1, 157.3, 153.4, 147.6, 133.0, 131.6,
129.7, 122.3, 116.4, 115.4, 113.5, 112.0, 111.1, 108.1, 56.1, 55.2
(2 signals), 36.1, 28.3;EI–MS m/z: 365 [M]+;UV: 256, 330
(sh), 336 nm;IR (KBr): 1739, 1695 cm ꢀ1. 9d: 1H NMR: d 7.26
and 6.93 (each 2H, d, J=8.4 Hz, Ar–H), 7.03 and 6.75 (each
1H, s, Ar–H), 3.95, 3.80 and 3.38 (each 3H, s, 3ꢄOMe), 3.80
(2H, t, J=5.8 Hz, N-CH2–), 3.07 (2H, t, J=5.8 Hz, Ar-CH2–);
13C NMR: d 183.1, 159.0, 158.0, 156.7, 153.1, 147.4, 132.8,
131.1 (2 signals), 122.1, 116.4, 114.0 (2 signals), 111.6, 111.0,
107.7, 56.0, 55.1 (2 signals), 36.0, 28.2;EI–MS m/z: 365[M]+;
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18. General Experimental Procedure for the synthesis of com-
pounds 1d–9d: A mixture of 1b–9b (each 1 mmol) and POCl3
(1.2 mL) in dry MeCN (6mL) was heated at reflux for 4 h.
After the reaction was finished, the resulting mixture was
diluted with H2O (40 mL), made basic, and then extracted
with CH2Cl2 (3ꢄ50 mL). The CH2Cl2 was evaporated in
vacuo to give viscous yellowish oil, the crude 1c–9c. The oil
was dissolved in a mixture of dry CH2Cl2 (3 mL) and pyridine
(0.2 mL) cooled at ꢀ20 ꢁC. Excess oxalyl chloride (0.5 mL) in
dry CH2Cl2 (3 mL) was added dropwise to the mixture. After
5 min, the temperature was raised to 40 ꢁC, and the mixture
was stirred for 1 h. The deep red precipitate was collected by
filtration and purified by column chromatography. The 1H
and 13C NMR spectra were recorded at 200 and 50 MHz,
respectively, using CDCl3 as solvent. 1d: 1H NMR: d 7.42 (1H,
m, Ar-H), 7.26 (3H, m, Ar-H), 6.76 and 6.57 (each 1H, s, Ar-
H), 3.85 and 3.20 (each 3H, s, 2ꢄOMe), 3.77 (2H, t, J=5.8 Hz,
N-CH2ꢀ) and 3.02 (2H, t, J=5.8 Hz, Ar-CH2ꢀ); 13C NMR:
d 181.3, 158.3, 158.2, 153.5, 147.7, 134.8, 133.0, 132.4, 129.9,
129.5, 129.4, 127.0, 116.2, 111.0, 110.8, 105.4, 55.9, 54.8, 36.0,
27.9;EI–MS m/z: 371 [M]+, 369 [Mꢀ2]+;UV: 233, 280(sh),
318 nm;IR (KBr): 1744, 1700 cm ꢀ1. 2d: 1H NMR: d 7.31 (4H,
m, Ar-H), 6.96 and 6.77 (each 1H, s, Ar-H), 3.95 and 3.40
(each 3H, s, 2ꢄOMe), 3.83 (2H, t, J=5.8 Hz, N-CH2–), 3.08
(2H, t, J=5.8 Hz, Ar-CH2–); 13C NMR: d 181.4, 158.3, 158.2,
153.6, 148.0, 133.0, 132.9, 132.7, 132.3, 129.8, 127.9, 125.7,
116.5, 111.1 (2 signals), 107.7, 56.1, 55.0, 36.2, 28.2;EI–MS m/
z: 371[M]+, 369 [Mꢀ2]+;UV: 260, 288 (sh), 317, 379 nm;IR
ꢀ1
UV: 229, 281, 325 (sh) nm;IR (KBr): 1744, 1696 cm
.
19. Antiplatelet aggregation assays: see: Chen, K. S.;Ko,
F. N.;Teng, C. M.;Wu, Y. C. Planta Med. 1996, 62, 133.
20. (a) Adrenoreceptor (b1 and b2) binding affinity assays:
see: Huang, Y. C.;Yeh, J. L.;Wu, B. N.;Lo, Y. C.;Liang,
J. C.;Lin, Y. T.;Sheu, S. H.;Chen, I. J. Drug Develop. Res.
1999, 47, 77. (b) Lin, Y. T.;Wu, B. N.;Horng, C. H.;Huang,
Y. C.;Horng, S. J.;Lo, Y. C.;Cheng, C. J.;Chen, I. J. Jpn. J.
Pharmacol. 1999, 80, 127.
21. (a) Cytotoxicity assay: Cancer cells were seeded in 96-well
microtiter plates at a density of 6000/well in 100 mL culture
medium. After an overnight adaptation period, 10 mM/mL
(final concentration) of test compounds in serium-free medium
were added to individual wells. Cells were treated with test
compounds for three days. Cell viability was determined by
the 5-(3-carbonylmethoxyphenyl)-2-(4,5-dimethylthazolyl)-3-
(4-silfophenyl)tetrazolium salt (MTS) reduction. Actinomycin
D 5mM (final concentration) and DMSO 0.1% (final concen-
tration) were used as positive and vehicle controls. Results
were expressed as percent of DMSO control. For details of the
assay protocols, see: Gieni, R. S.;Li, Y.;HayGlass, K. T. J.
Immunol. Meth. 1995, 187, 85. (b) Malich, G.;Markovic, B.;
Winder, C. Toxicology 1997, 124, 179.
1
(KBr): 1740, 1695 cmꢀ1. 3d: H NMR: d 7.39 (2H, d, J=8.4
Hz, Ar–H), 7.30 (2H, d, J=8.4 Hz, Ar–H), 6.94 and 6.77
(each 1H, s, Ar–H), 3.95 and 3.40 (each 3H, s, 2ꢄOMe), 3.83
(2H, t, J=5.8 Hz, N-CH2–) and 3.07 (2H, t, J=5.8 Hz, Ar-
CH2–); 13C NMR: d 182.6, 158.1, 157.5, 153.8, 147.9, 133.7,
133.3, 131.4 (2 signals), 129.0 (2 signals), 128.9, 116.3, 111.8,
111.2, 107.0, 56.2, 55.4, 36.2, 28.5;EI–MS m/z: 371[M]+, 369
[Mꢀ2]+;UV: 232, 267 (sh), 318 nm;IR (KBr): 1741, 1696