Effect of isoquinoline alkaloids of different structural types on antiplatelet aggregation in vitro

Article abstract of DOI:10.1055/s-2006-947196

Forty-one isoquinoline alkaloids were tested for antiplatelet aggregation effects. Among them, (-)-discretamine (6), protopine (7), ochotensimine (18), O-methylarmepavinemethine (23), lindoldhamine (25), isotetrandrine (26), thalicarpine (27), papaverine (28), and D-(+)-N-norarmepavine (32) exhibited significant inhibitory activity towards adenosine 5′-diphosphate (ADP)-, arachidonic acid (AA)-, collagen-, and/or platelet-activating factor (PAF)-induced platelet aggregation. The results are discussed on the basis of structure-activity relationships. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2006-947196

Effect of Isoquinoline Alkaloids of
Different Structural Types on
Antiplatelet Aggregation in Vitro
1
2
2
3
Yi-Chen Chia , Fang-Rong Chang , Chin-Chung Wu , Che-Ming Teng ,
1
2
Keh-Shaw Chen , Yang-Chang Wu
Abstract
Forty-one isoquinoline alkaloids were tested for antiplatelet ag-
gregation effects. Among them, (±)-discretamine (6), protopine
(
7), ochotensimine (18), O-methylarmepavinemethine (23), lin-
doldhamine (25), isotetrandrine (26), thalicarpine (27), papaver-
ine (28), and -(+)-N-norarmepavine (32) exhibited significant
D
inhibitory activity towards adenosine 5¢-diphosphate (ADP)-,
arachidonic acid (AA)-, collagen-, and/or platelet-activating fac-
tor (PAF)-induced platelet aggregation. The results are discussed
on the basis of structure-activity relationships.
Supportinginformation available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
Platelets play an important role in the haemostatic process, and
their aggregation can cause arterial thrombosis. Accordingly,
compounds with antiplatelet aggregation activity can be useful
therapeutic agents. Isoquinoline alkaloids were well known to
display numerous biological activities [1], [2], [3]. Therefore, we
evaluated various isoquinoline alkaloids in antiplatelet aggrega-
tion assays and, in our past report, described the target activity of
thirty-seven aporphine alkaloids [4], [5]. In our continuing inves-
tigation on the activities of different types of isoquinoline alka-
loids, we chose and tested forty-one compounds of different
structural types, including six protoberberines: berberine chlori-
de (1) [6], berberine iodine (2) [7], jatrorrhizine picrate (3) [8],
1
238
(
)-tetrahydroberberine (4) [8], (±)-tetrahydropalmatine (5) [9],
and (±)-discretamine (6) [10]; three protopines: protopine (7)
11], protopine N-oxide (8) [1], and a-allocryptopine (9) [12] ;
eight pavines: pavine (10) [13] ,N-methylpavine (11) [13] , -car-
[
L
yachine (12) [14], dl-caryachine (13) [14] , d -O -methylcaryachine
HCl (14) [14], (+)-O-methylcaryachine N-oxide (15) [1], crychine
Affiliation: 1 Department of Food Science & Technology, Tajen Institute of
2
Technology, Ping Tung Hsien, Taiwan ´ Graduate Institute of Natural Pro-
ducts, Kaohsiung Medical University, Kaohsiung, Taiwan ´ 3 Pharmacological
Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
Correspondence: Prof. Dr. Yang-Chang Wu ´ Graduate Institute of Natural Pro-
ducts ´ Kaohsiung Medical University ´ Kaohsiung 807 ´ Taiwan ´ Republic of
China ´ Phone: +886-7-312-1101-2197 ´ Fax: +886-7-311-4773 ´ E-mail: yach-
wu@kmu.edu.tw
Prof. Dr. Keh-Shaw Chen ´ Department of Food Science & Technology ´ Tajen In-
stitute of Technology ´ Ping Tung Hsien 907 ´ Taiwan ´ Phone: +886-8-
762400p359 ´ Fax: +886-8-7621572 ´ E-mail: kschen@mail.tajen.edu.tw
Received: December 13, 2005 ´ Accepted: June 1, 2006
Bibliography: Planta Med 2006; 72: 1238±1241 ꢀ Georg Thieme Verlag KG
Stuttgart ´ New York ´ DOI 10.1055/s-2006-947196 ´ Published online Septem-
ber 18, 2006 ´ ISSN 0032-0943

Table 1 Effects of test compounds on the platelet aggregation induced by ADP, AA, collagen and PAF in washed rabbit platelets a
Compoundb
Aggregation (%)
Collagen (10 mg/mL)
ADP (20 mM)
AA (100 mM)
PAF (2 ng/mL)
1
2
3
4
5
6
7
69.2  3.0*
90.2  4.5
84.7  2.5
85.6  0.7
73.2  4.3*
91.2  0.5
80.7  2.9**
86.6  0.8
83.3  3.6
89.6  1.1
92.4  1.2
87.5  2.4
85.6  1.2*
91.7  0.8
88.8  1.3
82.0  0.7**
78.0  6.9
91.4  1.4
89.0  0.9
90.0  1.9
65.0  4.3**
83.6  0.5***
81.8  1.1
0.0  0.0***
48.5  0.7*
88.0  0.7
76.4  8.4
84.3  2.8
0.0  0.0***
89.5  2.4
0.0  0.0***
77.6  10.7
77.6  0.4***
80.6  4.6
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
(25 mM)
86.3  0.5*
90.2  1.6
87.6  1.5
80.5  1.7**
74.9  5.8
0
80.2  1.8**
87.6  1.7
1
86.2  2.2
78.3  3.5*
53.8  5.3***
31.7  16.9***
87.7  1.1
73.5  3.3***
85.3  1.8**
84.8  2.3
2
88.5  1.0
70.8  4.9**
67.6  2.3**
87.6 1.9
3
88.4  2.4
4
78.2  2.0
87.5  1.4
5
65.7  4.1**
76.5  1.1
85.8  1.1
89.7  0.9
90.8  0.9
6
93.2  0.5**
86.0  0.8
87.2  2.0
83.7  4.7
7
73.3  1.7
89.3  2.4
87.9  0.6
8
85.4  2.9
81.9  1.9
71.8  2.8***
87.9  1.3
0.0  0.0***
87.9  1.1
9
79.3  3.4
89.3  1.8
0
91.6  1.9
84.4  3.2
61.8  7.0*
85.1  4.2
87.0  0.8
1 (50 mM)
90.0  0.5
63.2  14.8
59.9  6.7*
0.0  0.0***
26.2  3.3***
3.2  1.7***
0.0  0.0***
35.2  4.7***
0.0  0.0***
25.3  12.4***
62.4  8.3**
67.3  8.1**
50.8  15.7*
84.4  0.8
80.3  3.6*
13.8  6.2***
0.0  0.0***
22.6  6.1***
0.0  0.0***
79.8  2.0***
34.9  3.0***
0.0  0.0***
75.5  13.8
82.2  2.9
2
51.0  7.2***
0.0  0.0***
67.2  6.5***
30.2  10.9***
90.2  1.3
9.5  8.2***
0.0  0.0***
37.0  7.9***
0.0  0.0***
0.0  0.0***
2.8  2.3***
7.6  4.6***
42.0  8.6***
44.4  17.9*
53.2  16.2*
0.0  0.0***
78.9  3.6**
79.8  6.6
3
4 (50 mM)
5
6
7
78.5  2.2***
10.1  3.0***
18.7  7.1***
90.2  1.5
8
9 (25 mM)
1239
0
1
2
3
4
5
6
7
8
9
0
1
91.2  1.9
82.1  2.6
91.6  1.4
71.1  4.4**
92.9  1.0
91.6  0.7
89.2  0.6
85.9  0.8
81.8  2.7*
74.6  2.9**
70.8  8.0
93.5  0.7
86.8  3.0
86.5  1.4
90.3  2.1
70.2  4.1***
81.5  3.3
27.6  10.8***
86.3  0.4
92.4  3.0
88.5  1.8
90.0  1.7
67.1  7.8*
88.1  1.1
53.0  14.6*
89.5  3.8
85.9  2.6
93.4  1.7
91.0  1.3
88.1  0.7**
53.5  11.0*
77.9  1.9
56.0  8.7***
79.8  5.5
84.4  1.5*
77.3  9.8
84.9  0.7***
85.5  4.3
Aspirin
Control
0.0  0.0***
88.4 1.1
87.8  1.5
90.4  1.1
92.9 0.3
88.5  0.4
90.5  1.1
a
Platelets were preincubated with DMSO (0.5%, control), aspirin or test compounds at 37 8C for 3 min, then ADP (20 mM), AA (100 mM), collagen (10 mg/mL) or PAF (3.6 nM) was added.
Percentage of aggregation are presented as means  S.E.(n = 3~5). * P < 0.05, ** P < 0.01,*** P < 0.001 as compared with the respective control.
The concentration of each test compound was 100 mM, aspirin was 25 mM.
b
picrate (16) [14], and (±)-crychine N-oxide (17) [1]; four spiro- (27) [20]; nine benzylisoquinolines: papaverine (28) [16], papa-
benzylisoquinolines: ochotensimine (18) [15], dihydroochotensi- verine N-oxide HCl (29) [21], ()-armepavine (30) [2],
mine (19) [16], yenhusomidine (20) [15], and yenhusomine (21) mepavine (31) [22], d-(+)-N-norarmepavine (32) [22],
D
-(±)-ar-
-(±)-N-
L
[
(
15]; three stilbene alkaloids: O-methylarmepavinemethine MeI norarmepavine (33) [17] ,N,O-dimethyl-N-norarmepavine MeI
22) [17] ,O-methylarmepavinemethine (23) [3], and O,O-di- (34) [22], O,O,N-trimethylcoclaurine MeI (35) [18], and (+)-lau-
ethylcoclaurine methine (24) [18]; three bisbenzylisoquinolines: danosine (36) [23]; three proaporphines: litsericine (37) [24], N-
lindoldhamine (25) [19], isotetrandrine (26) [8], and thalicarpine methyllitsericine (38) [24], and N-methyllitsericine HBr (39)
Letter¼ Planta Med 2006; 72: 1238±1241

[24], as well as adlumidine (40) [15]and (+)- O-methylflavinan- was completely inhibited by 32, strongly inhibited by 36, and
dine (41) [25], which were isolated from Formosan plants or significantly inhibited by 30. The other members of this class
were semi-synthetic derivatives.
were inactive. Two interesting SAR observations were made.
Benzylisoquinoline alkaloid 28, which has four methoxy groups
These alkaloids were studied for their effects on the aggregation at C-6, 7, 3¢, 4¢, showed strong inhibition of platelet aggregation;
of washed rabbit platelets as induced by adenosine 5¢-diphos- however, modification to the N-methylbenzyltetrahydroisoqui-
phate (ADP, 20 ꢁM), arachidonic acid (AA, 100 ꢁM), collagen noline 36 reduced the antiplatelet effects. Also saturation of the
(
10 ꢁg/mL), and platelet-activating factor (PAF, 2 ng/mL).
benzylisoquinoline B ring sharply reduced the antiplatelet activ-
ities.
As shown in Table 1, (±)-discretamine (6) showed complete inhi-
bition of AA-induced platelet aggregation, while the other proto- The proaporphines 37± 39 as well as the two miscellaneous alka-
berberines 1±5 were inactive. The protoberberine 6 possesses a loids 40 and 41 did not exhibit any significant activity.
completely different structural skeleton from acetylsalicylic acid
(
ASA), but it exhibits ASA-like activity to prevent AA-induced Among all structural types investigated, some compounds, e.g.,
platelet aggregation [26]. Thus, the precise mechanism of action 23 and 28, exhibited potent antiplatelet activities against aggre-
still needs to be explored. The phenolic moieties may play an im- gation induced by all four activators, while others, such as 18 and
portant role in activity, because when the C-3 and C-10 hydroxy 32, demonstrated highly selective inhibition toward specific tar-
groups of 6 were changed to two methoxy groups in 5, the anti- gets. The mechanism(s) of antiplatelet actions of those potent
platelet effects were eliminated.
compounds is(are) not clear at this time. As also observed in the
previous results [4], [5], in general, a tiny change in the structure
Protopine 7, which contains a methylenedioxy group at C-9 and of different sub-types of isoquinolines will cause significant
C-10, completely inhibited collagen- and PAF-induced platelet changes in anti-platelet aggregation activity.
aggregation, and significantly prevented that by induced AA.
Modification of the methylenedioxy functionality to two meth-
oxy groups as in 9 totally abolished the antiplatelet activities. Materials and Methods
Thus, we suggest that the C-9,10 methylenedioxy group is critical
to the action of protopines.
Compounds: Alkaloids 1±41 were isolated or semi-synthesized
from various Formosan plants in our past investigation. All refer-
As shown in the assay results for pavine alkaloids 10±17, only 13 ences were cited. The preparation of compounds 34 and 35 has
exhibited significant inhibition of platelet aggregation induced been detailed in the Supporting Information.
by collagen. If the hydroxy group at C-8 of 13 was converted to a
methoxy group as in 14, no antiplatelet effects were seen.
Assay methods for platelet aggregation: The assay protocol is de-
tailed in the Supporting Information.
1
240
We also assayed the unusual spirobenzylisoquinolines 18 ± 21.
Interestingly, compound 18, which possesses an exo-methylene Data analysis: The experimental results are expressed as means 
group at C-13, demonstrated excellent activity against platelet S.E. and accompanied by the number of observations. A one-way
aggregation induced by PAF. Converting the methylene group of analysis of variance (ANOVA) was used for multiple compari-
18 to the methyl group of 19 eliminated the antiplatelet activity. sons, and if there was significant variation between treatment
groups, then the mean values for inhibitors were compared
The three stilbene alkaloids 22±24 were effective against plate- with those for control by Student¢s t test, and P values of less
let aggregation induced by ADP, AA, collagen, and PAF. Among than 0.05 were considered to be statistically significant.
them, the most potent compound 23 completely inhibited plate-
let aggregation induced by all four activators. By changing the
stilbene-type base to the methyl iodide salt (22) or converting Acknowledgements
the C-5,4¢-dimethoxy groups to C-5,4¢-diethoxy groups (24), the
antiplatelet effects were reduced.
This investigation was supported by a grant from the National
Science Council of the Republic of China awarded to Y.C. Wu.
Among the series of bisbenzylisoquinoline alkaloids 25±27,
compound 25 completely inhibited platelet aggregations in-
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