Effect of Isoquinoline Alkaloids of Different Structural Types on Antiplatelet Aggregation in Vitro

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

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.

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 aggregation assays and, in our past report, described the target activity of thirty-seven aporphine alkaloids [4], [5]. In our continuing investigation on the activities of different types of isoquinoline alkaloids, we chose and tested forty-one compounds of different structural types, including six protoberberines: berberine chloride (1) [6], berberine iodine (2) [7], jatrorrhizine picrate (3) [8], (±)-tetrahydroberberine (4) [8], (-)-tetrahydropalmatine (5) [9], and (-)-discretamine (6) [10]; three protopines: protopine (7) [11], protopine N-oxide (8) [1], and α-allocryptopine (9) [12]; eight pavines: pavine (10) [13], N-methylpavine (11) [13], L-caryachine (12) [14], dl-caryachine (13) [14], d-O-methylcaryachine HCl (14) [14], (+)-O-methylcaryachine N-oxide (15) [1], crychine picrate (16) [14], and (-)-crychine N-oxide (17) [1]; four spirobenzylisoquinolines: ochotensimine (18) [15], dihydroochotensimine (19) [16], yenhusomidine (20) [15], and yenhusomine (21) [15]; three stilbene alkaloids: O-methylarmepavinemethine MeI (22) [17], O-methylarmepavinemethine (23) [3], and O,O-diethylcoclaurine methine (24) [18]; three bisbenzylisoquinolines: lindoldhamine (25) [19], isotetrandrine (26) [8], and thalicarpine (27) [20]; nine benzylisoquinolines: papaverine (28) [16], papaverine N-oxide · HCl (29) [21], (±)-armepavine (30) [2], D-(-)-armepavine (31) [22], d-(+)-N-norarmepavine (32) [22], L-(-)-N-norarmepavine (33) [17], N,O-dimethyl-N-norarmepavine MeI (34) [22], O,O,N-trimethylcoclaurine · MeI (35) [18], and (+)-laudanosine (36) [23]; three proaporphines: litsericine (37) [24], N-methyllitsericine (38) [24], and N-methyllitsericine · HBr (39) [24], as well as adlumidine (40) [15] and (+)-O-methylflavinandine (41) [25], which were isolated from Formosan plants or were semi-synthetic derivatives.

These alkaloids were studied for their effects on the aggregation of washed rabbit platelets as induced by adenosine 5′-diphosphate (ADP, 20 µM), arachidonic acid (AA, 100 µM), collagen (10 µg/mL), and platelet-activating factor (PAF, 2 ng/mL).

As shown in Table [1], (-)-discretamine (6) showed complete inhibition of AA-induced platelet aggregation, while the other protoberberines 1 - 5 were inactive. The protoberberine 6 possesses a completely different structural skeleton from acetylsalicylic acid (ASA), but it exhibits ASA-like activity to prevent AA-induced platelet aggregation [26]. Thus, the precise mechanism of action still needs to be explored. The phenolic moieties may play an important role in activity, because when the C-3 and C-10 hydroxy groups of 6 were changed to two methoxy groups in 5, the antiplatelet effects were eliminated.

Protopine 7, which contains a methylenedioxy group at C-9 and C-10, completely inhibited collagen- and PAF-induced platelet aggregation, and significantly prevented that by induced AA. Modification of the methylenedioxy functionality to two methoxy groups as in 9 totally abolished the antiplatelet activities. Thus, we suggest that the C-9,10 methylenedioxy group is critical to the action of protopines.

As shown in the assay results for pavine alkaloids 10 - 17, only 13 exhibited significant inhibition of platelet aggregation induced 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.

We also assayed the unusual spirobenzylisoquinolines 18 - 21. Interestingly, compound 18, which possesses an exo-methylene group at C-13, demonstrated excellent activity against platelet aggregation induced by PAF. Converting the methylene group of 18 to the methyl group of 19 eliminated the antiplatelet activity.

The three stilbene alkaloids 22 - 24 were effective against platelet aggregation induced by ADP, AA, collagen, and PAF. Among them, the most potent compound 23 completely inhibited platelet aggregation induced by all four activators. By changing the stilbene-type base to the methyl iodide salt (22) or converting the C-5,4′-dimethoxy groups to C-5,4′-diethoxy groups (24), the antiplatelet effects were reduced.

Among the series of bisbenzylisoquinoline alkaloids 25 - 27, compound 25 completely inhibited platelet aggregations induced by AA, collagen and PAF, and significantly inhibited that by ADP. On the other hand, 26 completely inhibited platelet aggregation induced by only AA and collagen, while 27 partially inhibited collagen-, AA- and PAF-induced platelet aggregation.

Furthermore, in the class of the benzylisoquinoline alkaloids 28 - 36, compound 28 showed a wide range of antiplatelet aggregation effects. Another active member of this class is 29, which showed strong inhibition of both ADP- and AA-induced aggregation, and significant inhibition of collagen-induced platelet aggregation. In addition, platelet aggregation induced by collagen was completely inhibited by 32, strongly inhibited by 36, and significantly inhibited by 30. The other members of this class were inactive. Two interesting SAR observations were made. Benzylisoquinoline alkaloid 28, which has four methoxy groups at C-6, 7, 3′, 4′, showed strong inhibition of platelet aggregation; however, modification to the N-methylbenzyltetrahydroisoquinoline 36 reduced the antiplatelet effects. Also saturation of the benzylisoquinoline B ring sharply reduced the antiplatelet activities.

The proaporphines 37 - 39 as well as the two miscellaneous alkaloids 40 and 41 did not exhibit any significant activity.

Among all structural types investigated, some compounds, e. g., 23 and 28, exhibited potent antiplatelet activities against aggregation induced by all four activators, while others, such as 18 and 32, demonstrated highly selective inhibition toward specific targets. The mechanism(s) of antiplatelet actions of those potent 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 of different sub-types of isoquinolines will cause significant changes in anti-platelet aggregation activity.

Materials and Methods

Compounds: Alkaloids 1 - 41 were isolated or semi-synthesized from various Formosan plants in our past investigation. All references were cited. The preparation of compounds 34 and 35 has been detailed in the Supporting Information.

Assay methods for platelet aggregation: The assay protocol is detailed in the Supporting Information.

Data analysis: The experimental results are expressed as means ± S.E. and accompanied by the number of observations. A one-way analysis of variance (ANOVA) was used for multiple comparisons, and if there was significant variation between treatment groups, then the mean values for inhibitors were compared with those for control by Student′s t test, and P values of less than 0.05 were considered to be statistically significant.

Acknowledgements

This investigation was supported by a grant from the National Science Council of the Republic of China awarded to Y.C. Wu.

References

• 1 Wu Y C, Liou Y F, Lu S T, Chen C H, Chang J J, Lee K H. Cytotoxicity of isoquinoline alkaloids and their N-oxides.  Planta Med. 1989;  55 163-5
  Thieme ConnectPubMedSearch in Google Scholar
• 2 Tzeng C C, Wu Y C, Su T L, Watanabe K A, Lu S T, Chou T C. Inhibitory effects of isoquinoline-type alkaloids on leukemic cell growth and macromolecule biosynthesis.  Kaohsiung J Med Sci. 1990;  6 58-65
  PubMedSearch in Google Scholar
• 3 Tsai I L, Liou Y F, Lu S T. Screening of isoquinoline alkaloids and their derivatives for antibacterial and antifungal activities.  Kaohsiung J Med Sci. 1989;  5 132-45
  PubMedSearch in Google Scholar
• 4 Chen K S, Ko F N, Teng C M, Wu Y C. Antiplatelet and vasorelaxing actions of some aporphinoids.  Planta Med. 1996;  62 133-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Chia Y C, Chen K S, Chang Y L, Teng C M, Wu Y C. Antiplatelet actions of aporphinoids from Formosan plants.  Bioorg Med Chem Lett. 1999;  9 3295-300
  CrossrefPubMedSearch in Google Scholar
• 6 Ukita T, Mizuno D, Tamura T. Studies on the antibacterial properties of berberinium chloride.  Jpn J Exp Med. 1949;  20 103-8
  PubMedSearch in Google Scholar
• 7 Awe W U, Gottingen G. Berberine derivatives. VII. Cleavage of 9-benzyl-16,17-dihydrodesoxypalmatine by iodine or mercuric acetate.  Arch Pharm. 1944;  282 97-100
  PubMedSearch in Google Scholar
• 8 Yang T H, Lu S T. Studies on the alkaloids of Berberidaceous plants XXVI. Alkaloids of Berberis mingetsensis Hayata.  Yakugaku Zasshi. 1960;  80 849-51
  PubMedSearch in Google Scholar
• 9 Sun T T, Loh S H, Kyi Z Y. Synthesis of compound related to corydalis B (tetrahydropalmatine). IV. The synthesis of some acyloxy- and alkoxyberbines.  Yao Xue Xue Bao. 1965;  12 314-8
  PubMedSearch in Google Scholar
• 10 Richter W J, Brochmann-Hanssen E. The structure of discretamine, a tetrahydroprotoberberine alkaloid.  Helv Chim Acta. 1975;  58 209-11
  CrossrefPubMedSearch in Google Scholar
• 11 Debska W. Separation and determination of the dissociation constants of chelidonine and protopine by paper chromatography.  Nature. 1958;  182 666-7
  PubMedSearch in Google Scholar
• 12 Lu S T, Wang S J, Su T L. Studies on the alkaloids of Formosan Corydalis species. I. Alkaloids of Corydalis campulicarpa Hayata.  Yakugaku Zasshi. 1971;  91 778-81
  PubMedSearch in Google Scholar
• 13 Lu S T, Lan P K. Studies on the alkaloids of Formosa Lauraceous plants. VIII. Alkaloids of Crypyocarya chinensis Hemsl. (1). Structures of the new alkaloids ”crychine” and ”caryachine”.  Yakugaku Zasshi. 1966;  86 177-84
  PubMedSearch in Google Scholar
• 14 Lu S T. Studies on the alkaloids of Formosan Lauraceous plants IX. Alkaloids of Cryptocarya chinensis Hemsl and Cryptocarya konishii Hayata.  Yakugaku Zasshi. 1966;  86 296-9
  PubMedSearch in Google Scholar
• 15 Lu S T, Su T L, Kametani T, Ihara M. Structural elucidation of two new spirobenzylisoquinoline alkaloids, yenhusomine and yenhusomidine.  Heterocycles. 1975;  3 301-5
  PubMedSearch in Google Scholar
• 16 Lu S T, Wu Y C, Leou S P. The oxidation of isoquinoline alkaloids with m-chloroperbenzoic acid.  J Chin Chem Soc. 1987;  34 33-42
  PubMedSearch in Google Scholar
• 17 Tomita M, Yang T H, Lu S T. Studies on the alkaloids of Formosan Lauraceous plants. I. Alkaloids of Machilus kusanoi Hayata. (1). The isolation of L-(-)-N-norarmepavine.  Yakugaku Zasshi. 1963;  83 15-8
  PubMedSearch in Google Scholar
• 18 Lu S T. Studies on the alkaloids of Formosan Lauraceous plants II. Alkaloids of Machilus kusanoi Hayata. (2). The isolation of dl-coclaurine.  Yakugaku Zasshi. 1963;  83 19-21
  PubMedSearch in Google Scholar
• 19 Lu S T, Chen I S. Structure of a new bisbenzylisoquinoline alkaloids, lindoldhamine.  Heterocycles. 1976;  4 1073-6
  PubMedSearch in Google Scholar
• 20 Tomita M, Furukawa H, Lu S T. The constitution of thalicarpine.  Chem Pharm Bull. 1967;  15 959-63
  PubMedSearch in Google Scholar
• 21 Wu Y C, Liou Y F, Lu S T. Antimicrobial activity of isoquinoline alkaloids and their N-oxide derivatives.  Kaohsiung Med Sci. 1988;  4 336-44
  PubMedSearch in Google Scholar
• 22 Yang T H, Lu S T. Studies on the alkaloids of Magnoliaceous plants XXXV. Alkaloids of Magnolia kachirachirai Dandy. (2). The isolation of D-(+)-N-norarmepavine.  Yakugaku Zasshi. 1963;  83 22-5
  PubMedSearch in Google Scholar
• 23 Lu S T, Tsai I L. Hofmann elimination with diazomethane on quaternary benzyltetrahydroisoquinoline related alkaloids.  Heterocycles. 1988;  27 751-68
  PubMedSearch in Google Scholar
• 24 Lu S T, Su T L, Wang E C. Studies on the alkaloids of Formosan Lauraceous plants XVIII. Alkaloids of Neolitsea buisanensis Yamamato et Kamikoti and Neolitsea aurata (Hay.) Koidz.  J Chin Chem Soc. 1975;  22 349-53
  PubMedSearch in Google Scholar
• 25 Lu S T, Wu Y C, Leou S P. Alkaloids of Formosan Fissistigma and Goniothalamus species.  Phytochemistry. 1985;  24 1829-34
  CrossrefPubMedSearch in Google Scholar
• 26 Vane J R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs.  Nat New Biol. 1971;  231 232-5
  PubMedSearch in Google Scholar

Prof. Dr. Yang-Chang Wu

Graduate Institute of Natural Products

Kaohsiung Medical University

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-312-1101-2197

Fax: +886-7-311-4773

Email: yachwu@kmu.edu.tw

References

• 1 Wu Y C, Liou Y F, Lu S T, Chen C H, Chang J J, Lee K H. Cytotoxicity of isoquinoline alkaloids and their N-oxides.  Planta Med. 1989;  55 163-5
  Thieme ConnectPubMedSearch in Google Scholar
• 2 Tzeng C C, Wu Y C, Su T L, Watanabe K A, Lu S T, Chou T C. Inhibitory effects of isoquinoline-type alkaloids on leukemic cell growth and macromolecule biosynthesis.  Kaohsiung J Med Sci. 1990;  6 58-65
  PubMedSearch in Google Scholar
• 3 Tsai I L, Liou Y F, Lu S T. Screening of isoquinoline alkaloids and their derivatives for antibacterial and antifungal activities.  Kaohsiung J Med Sci. 1989;  5 132-45
  PubMedSearch in Google Scholar
• 4 Chen K S, Ko F N, Teng C M, Wu Y C. Antiplatelet and vasorelaxing actions of some aporphinoids.  Planta Med. 1996;  62 133-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Chia Y C, Chen K S, Chang Y L, Teng C M, Wu Y C. Antiplatelet actions of aporphinoids from Formosan plants.  Bioorg Med Chem Lett. 1999;  9 3295-300
  CrossrefPubMedSearch in Google Scholar
• 6 Ukita T, Mizuno D, Tamura T. Studies on the antibacterial properties of berberinium chloride.  Jpn J Exp Med. 1949;  20 103-8
  PubMedSearch in Google Scholar
• 7 Awe W U, Gottingen G. Berberine derivatives. VII. Cleavage of 9-benzyl-16,17-dihydrodesoxypalmatine by iodine or mercuric acetate.  Arch Pharm. 1944;  282 97-100
  PubMedSearch in Google Scholar
• 8 Yang T H, Lu S T. Studies on the alkaloids of Berberidaceous plants XXVI. Alkaloids of Berberis mingetsensis Hayata.  Yakugaku Zasshi. 1960;  80 849-51
  PubMedSearch in Google Scholar
• 9 Sun T T, Loh S H, Kyi Z Y. Synthesis of compound related to corydalis B (tetrahydropalmatine). IV. The synthesis of some acyloxy- and alkoxyberbines.  Yao Xue Xue Bao. 1965;  12 314-8
  PubMedSearch in Google Scholar
• 10 Richter W J, Brochmann-Hanssen E. The structure of discretamine, a tetrahydroprotoberberine alkaloid.  Helv Chim Acta. 1975;  58 209-11
  CrossrefPubMedSearch in Google Scholar
• 11 Debska W. Separation and determination of the dissociation constants of chelidonine and protopine by paper chromatography.  Nature. 1958;  182 666-7
  PubMedSearch in Google Scholar
• 12 Lu S T, Wang S J, Su T L. Studies on the alkaloids of Formosan Corydalis species. I. Alkaloids of Corydalis campulicarpa Hayata.  Yakugaku Zasshi. 1971;  91 778-81
  PubMedSearch in Google Scholar
• 13 Lu S T, Lan P K. Studies on the alkaloids of Formosa Lauraceous plants. VIII. Alkaloids of Crypyocarya chinensis Hemsl. (1). Structures of the new alkaloids ”crychine” and ”caryachine”.  Yakugaku Zasshi. 1966;  86 177-84
  PubMedSearch in Google Scholar
• 14 Lu S T. Studies on the alkaloids of Formosan Lauraceous plants IX. Alkaloids of Cryptocarya chinensis Hemsl and Cryptocarya konishii Hayata.  Yakugaku Zasshi. 1966;  86 296-9
  PubMedSearch in Google Scholar
• 15 Lu S T, Su T L, Kametani T, Ihara M. Structural elucidation of two new spirobenzylisoquinoline alkaloids, yenhusomine and yenhusomidine.  Heterocycles. 1975;  3 301-5
  PubMedSearch in Google Scholar
• 16 Lu S T, Wu Y C, Leou S P. The oxidation of isoquinoline alkaloids with m-chloroperbenzoic acid.  J Chin Chem Soc. 1987;  34 33-42
  PubMedSearch in Google Scholar
• 17 Tomita M, Yang T H, Lu S T. Studies on the alkaloids of Formosan Lauraceous plants. I. Alkaloids of Machilus kusanoi Hayata. (1). The isolation of L-(-)-N-norarmepavine.  Yakugaku Zasshi. 1963;  83 15-8
  PubMedSearch in Google Scholar
• 18 Lu S T. Studies on the alkaloids of Formosan Lauraceous plants II. Alkaloids of Machilus kusanoi Hayata. (2). The isolation of dl-coclaurine.  Yakugaku Zasshi. 1963;  83 19-21
  PubMedSearch in Google Scholar
• 19 Lu S T, Chen I S. Structure of a new bisbenzylisoquinoline alkaloids, lindoldhamine.  Heterocycles. 1976;  4 1073-6
  PubMedSearch in Google Scholar
• 20 Tomita M, Furukawa H, Lu S T. The constitution of thalicarpine.  Chem Pharm Bull. 1967;  15 959-63
  PubMedSearch in Google Scholar
• 21 Wu Y C, Liou Y F, Lu S T. Antimicrobial activity of isoquinoline alkaloids and their N-oxide derivatives.  Kaohsiung Med Sci. 1988;  4 336-44
  PubMedSearch in Google Scholar
• 22 Yang T H, Lu S T. Studies on the alkaloids of Magnoliaceous plants XXXV. Alkaloids of Magnolia kachirachirai Dandy. (2). The isolation of D-(+)-N-norarmepavine.  Yakugaku Zasshi. 1963;  83 22-5
  PubMedSearch in Google Scholar
• 23 Lu S T, Tsai I L. Hofmann elimination with diazomethane on quaternary benzyltetrahydroisoquinoline related alkaloids.  Heterocycles. 1988;  27 751-68
  PubMedSearch in Google Scholar
• 24 Lu S T, Su T L, Wang E C. Studies on the alkaloids of Formosan Lauraceous plants XVIII. Alkaloids of Neolitsea buisanensis Yamamato et Kamikoti and Neolitsea aurata (Hay.) Koidz.  J Chin Chem Soc. 1975;  22 349-53
  PubMedSearch in Google Scholar
• 25 Lu S T, Wu Y C, Leou S P. Alkaloids of Formosan Fissistigma and Goniothalamus species.  Phytochemistry. 1985;  24 1829-34
  CrossrefPubMedSearch in Google Scholar
• 26 Vane J R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs.  Nat New Biol. 1971;  231 232-5
  PubMedSearch in Google Scholar

Prof. Dr. Yang-Chang Wu

Graduate Institute of Natural Products

Kaohsiung Medical University

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-312-1101-2197

Fax: +886-7-311-4773

Email: yachwu@kmu.edu.tw

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