Mutation Research, 240 (1990) 267-279 Elsevier
267
MUTGEN 01530
Mutagenicity of isoquinoline alkaloids, especially of the aporphine type Tomio Nozaka 1, Fujio Watanabe l, Shin-ichi Tadaki 1, Masazo Ishino 1, Isao Morimoto 1, Jun-ichi Kunitomo 2, Hisashi Ishii 3 and Shinsaku Natori 4 1 Saitarna Institute of Public Health, 639-1 Kamiokubo, Urawa-shi, Saitarna 338 (Japan), 2 Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien Kyuban-cho, Nishinorniya-shi, Hyogo 663 (Japan), 3 Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba-shi, Chiba 260 (Japan)and 4 Meiji College of Pharmacy, 1-22-1 Yato-cho, Tanashi-shi, Tokyo 188 (Japan) (Received 19 September 1989) (Accepted 9 November 1989)
Keywords: Isoquinoline alkaloids; Aporphine alkaloids; Liriodenine; Mutagenicity; Ames assay; Metabolic activation
Summary The mutagenicity of 44 isoquinoline alkaloids was tested in Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. The alkaloids tested included compounds from the isoquinoline, benzylisoquinoline, bisbenzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphine groups. Among the alkaloids tested, liriodenine was the most potent mutagen for TA100 and roemerine was the most potent for TA98. A clear structure-mutagenicity relationship was observed in a series of aporphine alkaloids (aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-dioxoaporphine), and 10,11-non-substituted aporphines were suggested to exert their mutagenicity through metabolic activation of the 10,11 positions, possibly as the 10,11-epoxides.
In the course of our continuing search for mutagenic principles in crude drugs, we reported the mutagenicity of liriodenine (35), an aporphine alkaloid isolated from sinomeni caulis et rhizoma, which originates from the root, root-stalk and stem of Sinomenium aeutum Rehder et Wilson (Menispermaceae), towards Salmonella typhimurium TA100 and TA98 in the presence of $9 mix (Nozaka et al., 1988). Liriodenine (35) requires microsomal activation for the mutagenicity. The mutagenic potency of liriodenine (35) was comparable to that of the ubiquitous environmental carcinogen, benzo[ a ]pyrene.
Correspondence: Dr. T. Nozaka, Saitama Institute of Public Health, Kamiokubi 639-1, Urawa-shi, Saitama-ken 338 (Japan).
The isoquinoline alkaloids (including aporphine alkaloids) are one of the more numerous groups of natural products and have been isolated from a variety of plants including Papaveraceae, Lauraceae, Magnoliaceae, Menispermaceae and Annonaceae. Some isoquinoline alkaloids such as morphine, papaverine and berberine are of importance because of their medicinal use. However, few studies of the genotoxicity of isoquinoline alkaloids have been reported. Nagao et al. (1977) reported that isoquinoline and 3-methylisoquinoline were n o n - m u t a g e n i c to Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. Suter and Matter-Jaeger (1984) reported the mutagenicity in TA1535 and TA98 of apomorphine, a rearrangement product of morphine, but a clear dose-effect relationship was only found for strain TA1537.
0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 TABLE 1 MUTAGENICITY MIX
OF ISOQUINOLINE
ALKALOIDS
IN SALMONELLA
T E S T E R S T R A I N S i n T H E P R E S E N C E O F $9
M u t a g e n i c i t y was assayed by the p r e i n c u b a t i o n method. C o m p o u n d (No.)
C A S No.
Mutagenicity Conclusions TA100
Revertants//xg/plate ~ TA98
TAI00
TA98
lsoquinoline-type alkaloids (8-Amino-N- formyl-7-methoxy-6-methyl1 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (1) (N-Formyl-5,7,8-trimethoxy-6-methyl-l,2,3,4t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h y l b e n z o a t e (2) ( N-Formyl-5,7,8-trimethoxy-6-methyl1 , 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (3) (N-Formyl-7-methoxy-6-methyl-5,8-dioxo-l,2,3,4,5,8hexahydro- 1-isoquinolyl)methanol (4)
*
-
nc
nc
*
-
nc
nc
*
-
nc
DC
*
-
nc
nc
61-25-6 524-20-9 85-64-3
+ -
3 nc nc
nc nc nc
481-49-2 548-40-3 518-34-3 518-94-5 57-94-3
-
316-42-7
-
633-65-8 522-97-4 483-14-7
-
Benzylisoquinoline-type alkaloids P a p a v e r i n e h y d r o c h l o r i d e (5) A r m e p a v i n e (6) L a u d a n i n e (7)
Bisbenzylisoquinoline-type alkaloids C e p h a r a n t h i n e (8) O x y a c a n t h i n e (9) T e t r a n d r i n e (10) C y c l e a n i n e (11) d - T u b o c u r a r i n e chloride (12)
i
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
fie
Monoterpeneisoquinoline-type alkaloid E m e t i n e h y d r o c h l o r i d e (13)
Berberine-type alkaloids Berberine h y d r o c h l o r i d e (14) T e t r a h y d r o b e r b e r i n e (15) ( - ) - T e t r a h y d r o p a l m a t i n e (16)
nc
tic
nc
nc
nc
nc
4090-18-0 115-53-7
nc
nc
nc
nc
24148-86-5 1805-85-2
nc
nc
nc
nc
I
I
Morphinane-type alkaloids S i n o a c u t i n e (17)
Sinomenine (18) Hasubanan-type alkaloids A k n a d i n i n e (19) H a s u b a n o n i n e (20)
Benzo[c]phenanthridine-type alkaloids C h e l e r y t h r i n e (21) chloride O x y c h e l e r y t h r i n e (22)
34316-15-9 28342-33-8
nc
NC
+
2
nc
+
4
5
nc
nc
nc 2 29 2O8 2O 97
2 4 103 4 17
Aporphine-type alkaloids D i c e n t r i n e (23) D o m e s t i c i n e (24) Laurifoline (25) O - M e t h y l d o m e s t i c i n e (26) O - N o r n u c i f e r i n e (27) R o e m e r i n e (28) Steporphine (29) U s h i n s u n i n e (30)
517-66-8 476-71-5 7224-61-5 2565-01-7 3153-55-7 548-08-3 24191-98-8 3175-89-1
+ + + + +
nc
269 T A B L E 1 (continued) C o m p o u n d (No.)
CAS No.
Mutagenicity Conclusions
Revertants/~g/plate a
TA100
TA98
TA100
TA98
77784-22-6 55898-15-2 111017-06-2 * 475-75-2 15444-20-9 23740-25-2 70403-81-5 * * 96681-50-4
+ + + + + + + -
+
18
23
-
nc
nc
19 nc 330 107 114 37
2 nc 40 nc 10 nc
nc
nc
35 nc
8
88741-67-7 83287-02-9
+
+ +
nc 5
2 5
+
nc
26
Aporphine-type alkaloids Dehydrocrebanine (31) Dehydronantenine (32) N-Demethyl-N- formyldehydronuciferine (33) 1,2,10-Trimethoxydehydroaporphine (34) Liriodenine (35) Lysicamine (36) 9-Methoxy-l,2-methylenedioxy-7-oxoaporphine (37) 1,2,9-Tfimethoxy-7-oxoaporphine (38) 1,2,10-Trimethoxy-7-oxoaporphine (39) 4,5-Dioxodehydrocrebanine (40) Bianfugecine (41) 6-Hydroxy-5,10-dimethoxy-7Hdibenzo[ de, h ]quinolin-7-one (42) Menisporphine (43) 5,6,10-Trimethoxy-7H-dibenzo[ de, h ]quinolin-7-one (44)
88741-68-8
+ -
+ -
+ -
-
+ -
nc
a All results are single points (each point is the average of 3 plates) from the linear region of the dose-response curve and each value is the m a x i m u m number of revertants//~g/plate that a c o m p o u n d induced. Spontaneous revertants have been subtracted and were 113 (TA100, + $9), 36 (TA98, + $9) per plate (average of values in 10 experiments). *, CAS No. not yet available; - , not mutagenic; + , mutagenic (the samples giving over 250 or 100 revertant colonies per plate with strain TA100 or TA98, respectively, were concluded to be mutagenic); nc, not calculated.
The aim of this work was to elucidate the structure-mutagenicity relationship in isoquinoline alkaloids and the possible modes of action of potently mutagenic aporphines such as liriodenine (35), as well as to obtain basic data on the genotoxicity of isoquinoline alkaloids. The mutagenicity of 44 isoquinoline alkaloids, including isoquinoline, benzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphlne alkaloids, was tested on Salmonella typhimurium TA100 or TA98 in the presence or absence of S9 from rats treated with phenobarbital and fl-naphthoflavone. Materials and methods
d-tubocurarine chloride (12) and emetine hydrochloride (13) were obtained from Tokyo Kasei Kogyo Co., Ltd., Tokyo. Four isoquinoline derivatives (all synthetic) (1-4) were provided by Prof. A. Kubo (Kubo et al., 1986), Meiji College of Pharmacy (Tokyo). Two benzo[c]phenanthridine alkaloids (chelerythrine (21) chloride and oxychelerythrine (22)) were obtained by one of the authors (H.I.) (Ishii et al., 1977). Two benzylisoquinoline (6, 7), 4 bisbenzylisoquinoline (8-11), 2 berberine (15, 16), 1 morphinane (17), 2 hasubanan (19, 20) and 20 aporphine alkaloids (23-32, 34, 36-44) (among these 31 alkaloids, 1,2,9-trimethoxy-7-oxoaporphine (38), 1,2,10-trimethoxy7-oxoaporphine (39), 6-hydroxy-5,10-dimethoxy7H-dibenzo[de,h]quinolin-7-one (42) and 5,6,10-
Chemicals
trimethoxy-7H-dibenzo[ de, h ]quinoline-7-one (44)
Table 1 gives the list of chemicals tested and their Chemical Abstract Services (CAS) registry numbers. The structures of the isoquinoline alkaloids tested are shown in Fig. 1. Papaverine hydrochloride (5), berberine hydrochloride (14),
were synthetic and the others had been isolated from plants) were obtained by one of us (J.K.) (Kunitomo et al., 1970, 1979, 1980, 1981, 1982, 1986). Liriodenine (35), sinomenine (18) and Ndemethyl-N-formyldehydronuciferine (33) were
270 Isoquinoline-type C H 3 ~
0 C H 3 ~
~CH3 C H 3 ~
C H OCH3 3 ~
CH30~N-CHOcH30 ~ N - C H O
~
C H 3 0 ~ , I I N - C H 0 CH30@~f'N-CHO 0 CH20H OCH3 CH20H o~ ~o~o~
~o~
(Z)
(i)
(l)
(i)
Benzylisoquinoline-type CH30~ ~ . ~ CH30~
CH30"~ HCI
N-CH3CH30~ ~ N - C H
HO" ~"
CH30
OCH3 (5_)
(!)
3
I OH (i)
Bisbenzylisoquinoline-type OCH3
~
HO(8)
v
(9)
CH30
CH30~
K
II +N'-c"3
~O~o~~ 0
~o~\~
"'H
~ ~ v
(I__0 )
v
OCH3
(I__i ) Fig.l. Structuresof isoquinolinealkaloidstested.
~
v
(12)
-OCH3
271
Monoterpene isoquinoline-type C H 3 0 ~ H~ ~ " 2
5
H'"L. H CH2
OCH3
2HCI (13)
Berberine-type O- .-C,..A
< 0-.-~/~
A
CH30~ ~
o~~,
267
MUTGEN 01530
Mutagenicity of isoquinoline alkaloids, especially of the aporphine type Tomio Nozaka 1, Fujio Watanabe l, Shin-ichi Tadaki 1, Masazo Ishino 1, Isao Morimoto 1, Jun-ichi Kunitomo 2, Hisashi Ishii 3 and Shinsaku Natori 4 1 Saitarna Institute of Public Health, 639-1 Kamiokubo, Urawa-shi, Saitarna 338 (Japan), 2 Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien Kyuban-cho, Nishinorniya-shi, Hyogo 663 (Japan), 3 Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba-shi, Chiba 260 (Japan)and 4 Meiji College of Pharmacy, 1-22-1 Yato-cho, Tanashi-shi, Tokyo 188 (Japan) (Received 19 September 1989) (Accepted 9 November 1989)
Keywords: Isoquinoline alkaloids; Aporphine alkaloids; Liriodenine; Mutagenicity; Ames assay; Metabolic activation
Summary The mutagenicity of 44 isoquinoline alkaloids was tested in Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. The alkaloids tested included compounds from the isoquinoline, benzylisoquinoline, bisbenzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphine groups. Among the alkaloids tested, liriodenine was the most potent mutagen for TA100 and roemerine was the most potent for TA98. A clear structure-mutagenicity relationship was observed in a series of aporphine alkaloids (aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-dioxoaporphine), and 10,11-non-substituted aporphines were suggested to exert their mutagenicity through metabolic activation of the 10,11 positions, possibly as the 10,11-epoxides.
In the course of our continuing search for mutagenic principles in crude drugs, we reported the mutagenicity of liriodenine (35), an aporphine alkaloid isolated from sinomeni caulis et rhizoma, which originates from the root, root-stalk and stem of Sinomenium aeutum Rehder et Wilson (Menispermaceae), towards Salmonella typhimurium TA100 and TA98 in the presence of $9 mix (Nozaka et al., 1988). Liriodenine (35) requires microsomal activation for the mutagenicity. The mutagenic potency of liriodenine (35) was comparable to that of the ubiquitous environmental carcinogen, benzo[ a ]pyrene.
Correspondence: Dr. T. Nozaka, Saitama Institute of Public Health, Kamiokubi 639-1, Urawa-shi, Saitama-ken 338 (Japan).
The isoquinoline alkaloids (including aporphine alkaloids) are one of the more numerous groups of natural products and have been isolated from a variety of plants including Papaveraceae, Lauraceae, Magnoliaceae, Menispermaceae and Annonaceae. Some isoquinoline alkaloids such as morphine, papaverine and berberine are of importance because of their medicinal use. However, few studies of the genotoxicity of isoquinoline alkaloids have been reported. Nagao et al. (1977) reported that isoquinoline and 3-methylisoquinoline were n o n - m u t a g e n i c to Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. Suter and Matter-Jaeger (1984) reported the mutagenicity in TA1535 and TA98 of apomorphine, a rearrangement product of morphine, but a clear dose-effect relationship was only found for strain TA1537.
0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 TABLE 1 MUTAGENICITY MIX
OF ISOQUINOLINE
ALKALOIDS
IN SALMONELLA
T E S T E R S T R A I N S i n T H E P R E S E N C E O F $9
M u t a g e n i c i t y was assayed by the p r e i n c u b a t i o n method. C o m p o u n d (No.)
C A S No.
Mutagenicity Conclusions TA100
Revertants//xg/plate ~ TA98
TAI00
TA98
lsoquinoline-type alkaloids (8-Amino-N- formyl-7-methoxy-6-methyl1 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (1) (N-Formyl-5,7,8-trimethoxy-6-methyl-l,2,3,4t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h y l b e n z o a t e (2) ( N-Formyl-5,7,8-trimethoxy-6-methyl1 , 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (3) (N-Formyl-7-methoxy-6-methyl-5,8-dioxo-l,2,3,4,5,8hexahydro- 1-isoquinolyl)methanol (4)
*
-
nc
nc
*
-
nc
nc
*
-
nc
DC
*
-
nc
nc
61-25-6 524-20-9 85-64-3
+ -
3 nc nc
nc nc nc
481-49-2 548-40-3 518-34-3 518-94-5 57-94-3
-
316-42-7
-
633-65-8 522-97-4 483-14-7
-
Benzylisoquinoline-type alkaloids P a p a v e r i n e h y d r o c h l o r i d e (5) A r m e p a v i n e (6) L a u d a n i n e (7)
Bisbenzylisoquinoline-type alkaloids C e p h a r a n t h i n e (8) O x y a c a n t h i n e (9) T e t r a n d r i n e (10) C y c l e a n i n e (11) d - T u b o c u r a r i n e chloride (12)
i
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
fie
Monoterpeneisoquinoline-type alkaloid E m e t i n e h y d r o c h l o r i d e (13)
Berberine-type alkaloids Berberine h y d r o c h l o r i d e (14) T e t r a h y d r o b e r b e r i n e (15) ( - ) - T e t r a h y d r o p a l m a t i n e (16)
nc
tic
nc
nc
nc
nc
4090-18-0 115-53-7
nc
nc
nc
nc
24148-86-5 1805-85-2
nc
nc
nc
nc
I
I
Morphinane-type alkaloids S i n o a c u t i n e (17)
Sinomenine (18) Hasubanan-type alkaloids A k n a d i n i n e (19) H a s u b a n o n i n e (20)
Benzo[c]phenanthridine-type alkaloids C h e l e r y t h r i n e (21) chloride O x y c h e l e r y t h r i n e (22)
34316-15-9 28342-33-8
nc
NC
+
2
nc
+
4
5
nc
nc
nc 2 29 2O8 2O 97
2 4 103 4 17
Aporphine-type alkaloids D i c e n t r i n e (23) D o m e s t i c i n e (24) Laurifoline (25) O - M e t h y l d o m e s t i c i n e (26) O - N o r n u c i f e r i n e (27) R o e m e r i n e (28) Steporphine (29) U s h i n s u n i n e (30)
517-66-8 476-71-5 7224-61-5 2565-01-7 3153-55-7 548-08-3 24191-98-8 3175-89-1
+ + + + +
nc
269 T A B L E 1 (continued) C o m p o u n d (No.)
CAS No.
Mutagenicity Conclusions
Revertants/~g/plate a
TA100
TA98
TA100
TA98
77784-22-6 55898-15-2 111017-06-2 * 475-75-2 15444-20-9 23740-25-2 70403-81-5 * * 96681-50-4
+ + + + + + + -
+
18
23
-
nc
nc
19 nc 330 107 114 37
2 nc 40 nc 10 nc
nc
nc
35 nc
8
88741-67-7 83287-02-9
+
+ +
nc 5
2 5
+
nc
26
Aporphine-type alkaloids Dehydrocrebanine (31) Dehydronantenine (32) N-Demethyl-N- formyldehydronuciferine (33) 1,2,10-Trimethoxydehydroaporphine (34) Liriodenine (35) Lysicamine (36) 9-Methoxy-l,2-methylenedioxy-7-oxoaporphine (37) 1,2,9-Tfimethoxy-7-oxoaporphine (38) 1,2,10-Trimethoxy-7-oxoaporphine (39) 4,5-Dioxodehydrocrebanine (40) Bianfugecine (41) 6-Hydroxy-5,10-dimethoxy-7Hdibenzo[ de, h ]quinolin-7-one (42) Menisporphine (43) 5,6,10-Trimethoxy-7H-dibenzo[ de, h ]quinolin-7-one (44)
88741-68-8
+ -
+ -
+ -
-
+ -
nc
a All results are single points (each point is the average of 3 plates) from the linear region of the dose-response curve and each value is the m a x i m u m number of revertants//~g/plate that a c o m p o u n d induced. Spontaneous revertants have been subtracted and were 113 (TA100, + $9), 36 (TA98, + $9) per plate (average of values in 10 experiments). *, CAS No. not yet available; - , not mutagenic; + , mutagenic (the samples giving over 250 or 100 revertant colonies per plate with strain TA100 or TA98, respectively, were concluded to be mutagenic); nc, not calculated.
The aim of this work was to elucidate the structure-mutagenicity relationship in isoquinoline alkaloids and the possible modes of action of potently mutagenic aporphines such as liriodenine (35), as well as to obtain basic data on the genotoxicity of isoquinoline alkaloids. The mutagenicity of 44 isoquinoline alkaloids, including isoquinoline, benzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphlne alkaloids, was tested on Salmonella typhimurium TA100 or TA98 in the presence or absence of S9 from rats treated with phenobarbital and fl-naphthoflavone. Materials and methods
d-tubocurarine chloride (12) and emetine hydrochloride (13) were obtained from Tokyo Kasei Kogyo Co., Ltd., Tokyo. Four isoquinoline derivatives (all synthetic) (1-4) were provided by Prof. A. Kubo (Kubo et al., 1986), Meiji College of Pharmacy (Tokyo). Two benzo[c]phenanthridine alkaloids (chelerythrine (21) chloride and oxychelerythrine (22)) were obtained by one of the authors (H.I.) (Ishii et al., 1977). Two benzylisoquinoline (6, 7), 4 bisbenzylisoquinoline (8-11), 2 berberine (15, 16), 1 morphinane (17), 2 hasubanan (19, 20) and 20 aporphine alkaloids (23-32, 34, 36-44) (among these 31 alkaloids, 1,2,9-trimethoxy-7-oxoaporphine (38), 1,2,10-trimethoxy7-oxoaporphine (39), 6-hydroxy-5,10-dimethoxy7H-dibenzo[de,h]quinolin-7-one (42) and 5,6,10-
Chemicals
trimethoxy-7H-dibenzo[ de, h ]quinoline-7-one (44)
Table 1 gives the list of chemicals tested and their Chemical Abstract Services (CAS) registry numbers. The structures of the isoquinoline alkaloids tested are shown in Fig. 1. Papaverine hydrochloride (5), berberine hydrochloride (14),
were synthetic and the others had been isolated from plants) were obtained by one of us (J.K.) (Kunitomo et al., 1970, 1979, 1980, 1981, 1982, 1986). Liriodenine (35), sinomenine (18) and Ndemethyl-N-formyldehydronuciferine (33) were
270 Isoquinoline-type C H 3 ~
0 C H 3 ~
~CH3 C H 3 ~
C H OCH3 3 ~
CH30~N-CHOcH30 ~ N - C H O
~
C H 3 0 ~ , I I N - C H 0 CH30@~f'N-CHO 0 CH20H OCH3 CH20H o~ ~o~o~
~o~
(Z)
(i)
(l)
(i)
Benzylisoquinoline-type CH30~ ~ . ~ CH30~
CH30"~ HCI
N-CH3CH30~ ~ N - C H
HO" ~"
CH30
OCH3 (5_)
(!)
3
I OH (i)
Bisbenzylisoquinoline-type OCH3
~
HO(8)
v
(9)
CH30
CH30~
K
II +N'-c"3
~O~o~~ 0
~o~\~
"'H
~ ~ v
(I__0 )
v
OCH3
(I__i ) Fig.l. Structuresof isoquinolinealkaloidstested.
~
v
(12)
-OCH3
271
Monoterpene isoquinoline-type C H 3 0 ~ H~ ~ " 2
5
H'"L. H CH2
OCH3
2HCI (13)
Berberine-type O- .-C,..A
< 0-.-~/~
A
CH30~ ~
o~~,
