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Structure-activity relationships of raphanusanins and their analogues
Phytochemistry, Vol. 32, No. 6, pp. 1371-1373, 1993 Printedin Great Britain.
STRUCTURE-ACTIVITY
MASAKO
SAKODA,
003 1 9422/93 $6.00 + 0.00 Q 1993 PergamonPress Ltd
RELATIONSHIPS OF RAPHANUSANINS THEIR ANALOGUES
KENJI USUI, Kozo
ISHIZUKA, NOBUYUKI
HARADA,*
HIROSHI ONO,*
HISASHI UDA*
AND
and
KOJI
HASEGAWA~S
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan; *Institute for Chemical Reaction Science, Tohoku University, 2-l-l Katahira, Aoba, Sendai 980, Japan; tMizutani Plant Ecochemicals Project, Eniwa RBP Center Building, Megumino-Kita, Eniwa 061-13, Japan
(Receivedin revised form 21 September 1992) Key Word Index-Raphanus sativus; Cruciferae; plant growth inhibitor; 2-pyrrolidinethione; usanin; structure-activity relationship
raphan-
Abstract-The structure-activity relationship of raphanusanins A and B {(3R*,6R*)-and (3R*,6S*)-3-[methoxy(methylthio)methyl]-2_pyrrolidinethione, respectively} and their analogues has been studied for the inhibition of the growth of lettuce hypocotyls and Amaranthus roots. Pyrrolidinethione compounds showed higher activity than pyrrolidinones. The presence of a methoxy(methylthio)methyl group or of a methylthiomethylene moiety at C-3 of pyrrolidinethione was a dominant factor for higher activity. The bis(methylthio)methyl derivative also gave a similar inhibitory activity. It was established that the active sites in the chemical structure of the raphanusanins and their analogues were the thioamide moiety at C-2 of the skeleton and a methoxy(methylthio)methyl, bis(merhylthio)methyl, or methylthiomethylene group in the C-3 position of the side chain.
INTRODUCTION
Four light-induced plant growth inhibitors have been isolated from light-grown Sakurajima radish (Ruphanus sativus L. var. hortensis f. gigantissimus Makino) seedlings, and their structures identified as (3R*,6R*)and (3R*,6S*)-3-[methoxy(methylthio)methyl]-Zpyrrolidinethione (named raphanusanins A and B, respectively) [l-3], 3-(E)-methoxymethylene-2-pyrrolidinethione (raphanusamide) [2, 41, and 3-(Q-methylthiomethylene-2-pyrrolidinethione [S], respectively. Among these growth inhibitors, raphanusanins A and B are the major phototropism-regulating substances in radish seedlings; it was found that they accumulate on the illuminated side of hypocotyls subjected to phototropic stimulation, and caused growth inhibition of the illuminated side through interference with auxin-mediated microtubule orientation [6-121. Furthermore, it was clarified that lateral application of these compounds caused differential growth, which resulted in bending toward the applied side. In this paper, the structure-activity relationship of raphanusanins and their analogues was investigated to clarify the chemical structure required for the inhibitory activity. RESULTS AND DISCUSSION
The structure-activity relationship of the raphanusanins and their analogues shown in Fig. 1 was studied by means of the lettuce hypocotyl growth test and the $Author to whom correspondence should be addressed.
Amaranthus root growth test. There was no significant difference between the two bioassays, except that compounds 6 and 9 showed higher activity in the Amaranthus bioassay than in the lettuce test (Table 1). Pyrrolidinethione derivatives such as raphanusanins had strong inhib-
Table 1. Inhibitory activity of raphanusanins and their analogues on the growth of etiolated lettuce hypocotyls and Amaranthus caudatus roots
I,, (M)* Compounds
Lettuce bioassay
Amaranthus bioassay
1 2 3 4 s 6 7 8 9
5.3 x 4.8 x 4.9 x 3.9 x 5.2 x 4.7 x 1.7 x 1.2 x 1.4 x
2.0 x 2.4 x 5.4 x 3.1 x 3.9 x 1.6 x 1.1 x 1.4 x 6.6 x
10 11 12 13 14
> 1o-2 >10-2 > lo-* 7.0 x 10-a > lo-*
lo-4 1O-4 1o-4 10-b 10-d 10-4 10-J 10-3 10-S
10-b 1O-4 10-d 10-d 10-d lO-4 10-J 10-a 10-s
> 10-Z >10-2 > 10-a 1.2x 10-a 3.4 x 1o-4
*I,, represents the concentration of sample which causes 50% inhibition of the growth of lettuce hypocotyls or Amaranthus roots, respectively.
1371
1312
M. SAKOVA
1
11
et al.
10
12
14
No biological activity
a
0+
it
H
N ’ d0
l3 0 7
1
’
J
In*’
Iso (M) Effect Frg. 1. Structure-activity
on lettuce
relationship of raphanusanms and their analogues. Compound 1, raphanusanin (3R6R);2,raphanusanin B (3R,6S); 3, raphanusamlde.
itory activity in both bioassays, whereas pyrrolidinone compounds showed no inhibitory activity (Table 1 and Fig. 1). The most striking examples are seen in the comparisons of the 2-pyrrolidinethione derivatives 3 and 7 with the 2-pyrrolidinones 10 and 12, respectively; the former showed strong or fair inhibitory activity in both bioassays, but the latter were inactive. Therefore, the skeleton of 2-pyrrolidinethione is necessary for inhibitory activity. Another active site was shown to be the side chain at the C-3 position. The activity of pyrrolidinethiones having a methoxy(methylthio)methyl, a bis(methylthio)methyl, methylthiomethylene, or a methoxymethylene side chain at C-3 (l-6) was much higher than that of the 2pyrrolidinethione 9. However, the activity of 3(dimethoxy)methyl-2-pyrrolidinethione (7) was lower than that of the other 2-pyrrolidinethione compounds. Therefore, the presence of a dimethoxy group at C-6 led
1o-2
hypocotyls A
to a reduction in activity. The biological activity of 2pyrrolidinethiones bearing a bis(methylthio) group or a methoxy(methylthio) group at C-6 was higher than those of other pyrrolidinethiones. The presence of thioacetal or dithioacetal group at C-6 is thus important and leads to an increase in activity. On the other hand, acetal 7 showed weaker activity than the thioacetals and dithioacetal. The activity of methylthiomethylene and methoxymethylene compounds (S5) was almost comparable to those of thioacetal and dithioacetal compounds. Furthermore, there was not much difference in the inhibitory activity between the geometrical isomers trans-4 and cis5. It was thus clarified that the active sites in the chemical structure of the raphanusanins and their analogues are the thioamide moiety at the C-2 position and the methoxy(methylthio)methyl, bis(methylthio)methyl, or methylthiomethylene groups at the C-3 position.
Structure-activity
relationship of raphanusanins
EXPERIMENTAL Materials. Raphanusamide
[3,3-(Q-methoxymethylene-2-pyrrolidinethione] was prepared according to the procedure reported in the literature [4, 13). Treatment of 3 with MeSNa in aq. MeOH at room temp. for 15 min gave a mixt. of raphanusanins A (l), B (2), 3, 4,6 and 7, which were sepd by a combination of HPLC on silica gel (hexane-EtOAc 2: 1) and HPLC on silica gelODS (MeOH-H,O 3 : 2) to afford 1 (21%), 2 (29%), 3 (3%), 4 (1 l%), 6 (5%) and 7 (trace). Compounds 1 and 2 are racemates, respectively. The c&isomer 5 was synthesized by photo-isomerization of the trans-isomer 4; a MeOH soln was illuminated by a tungsten-lamp for 6 hr. The ratio of E and Z isomers of the products obtained was 1: 3; the isomers were sepd by HPLC on silica gel (hexane-EtOAc 3 : 1). Compound 7 was obtained by treatment of raphanusamide (3) with 5% KOH-MeOH (room temp., 4 hr). 2-Pyrrolidinethione (9) was easily prepared from 2-pyrrolidinone [P,S,, in tetrahydrofuran (THF)]. Compounds 12-14 are synthetic intermediates of raphanusamide (3). Hydrogenation of 12 with Pd-C in MeOH gave 10 and 11; the major product 11 was then converted to 8 by treatment with P,S,, in THF. The details of the synthesis and the spectroscopic data of these analogues will be reported elsewhere. Bioassays. A soln of the test compound in Me&O was absorbed on to a filter paper (Toyo filter No. 1) and dried at room temp., and 0.4 ml H,O added. Twelve lettuce seeds (Lactuca sadiua L. cv. Mikado Great) were spread on the filter paper in a Petri dish (27 mm), and were cultured for 4 days in the dark at 25”. The length of the hypocotyls was measured. Fifteen seeds of Amaranthus caudatus L. were spread in a Petri dish (33 mm) containing a sheet of filter paper and 0.6 ml test soln, and were cultured for 6 days in the dark at 25”. The length of the roots was measured. The experiments were each repeated twice. The results were reproducible. Acknowledgements-This
work was supported in part by grants from the Ministry of Education, Science and
1373
Culture, Japan [(General (B) No. 01470026 to N.H., Priority Areas Nos. 02250103 and 03236101 to N.H. and K.H., and International Joint Research No. 02044014 to N.H.] and Suntory Institute for Bioorganic Research (SUNBOR grants to N.H.).
REFERENCES Hasegawa, K., Shiihara, S., Iwagawa, T. and Hase, T. (1982) Plant Physiol. 70, 626.
Hasegawa, K., Noguchi, H., Iwagawa, T. and Hase, T. (1986) Plant Physiol. 81, 976. Harada, N., Hagiwara, H., Ono, H., Uda, H., Ohba, S., Kubota, M., Nishiyama, S., Yamamura, S., Hasegawa, K. and Sakoda, M. (1991) Tetrahedron Letters 32, 6757.
4. Harada, N., Ono, H., Hagiwara, H., Uda, H., Hasegawa, K. and Sakoda, M. (1991) Tetrahedron Letters 32, 6761.
Sakoda, M., Hase, T. and Hasegawa, K. (1990) Phytochemistry 29, 1031.
Noguchi, H., Nishitani, K., Bruinsma, J. and Hasegawa, K. (1986) Plant Physiol. 81, 980. Noguchi, H. and Hasegawa, K. (1987) Plant Physiol. 83, 672.
Hasegawa, K., Noguchi, H., Tanoue, C., Sando, S., Takada, M., Sakoda, M. and Hashimoto, T. (1987) Plant Physiol. 85, 379.
9. Sakoda, M., Matsuoka, T., Sando, S. and Hasegawa, K. (1988) J. Plant Physiol. 133, 110. 10. Bruinsma, J. and Hasegawa, K. (1990) Physiol. Plant. 79,700.
11. Sakoda, M., Hasegawa, K. and Ishizuka, K. (1991) Phytochemistry 30, 57.
12. Sakoda, M., Hasegawa, K. and Ishizuka, K. (1992) Physiol. Plant. 84, 509.
13. Shibayama, F., Yamada, S., Kikuchi, H. and Yamada, Y. (1981) Jpn. Kokai Tokyo Koho JP 81, 104, 864; (1982) Chem. Abstr. 96, 35076~.
STRUCTURE-ACTIVITY
MASAKO
SAKODA,
003 1 9422/93 $6.00 + 0.00 Q 1993 PergamonPress Ltd
RELATIONSHIPS OF RAPHANUSANINS THEIR ANALOGUES
KENJI USUI, Kozo
ISHIZUKA, NOBUYUKI
HARADA,*
HIROSHI ONO,*
HISASHI UDA*
AND
and
KOJI
HASEGAWA~S
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan; *Institute for Chemical Reaction Science, Tohoku University, 2-l-l Katahira, Aoba, Sendai 980, Japan; tMizutani Plant Ecochemicals Project, Eniwa RBP Center Building, Megumino-Kita, Eniwa 061-13, Japan
(Receivedin revised form 21 September 1992) Key Word Index-Raphanus sativus; Cruciferae; plant growth inhibitor; 2-pyrrolidinethione; usanin; structure-activity relationship
raphan-
Abstract-The structure-activity relationship of raphanusanins A and B {(3R*,6R*)-and (3R*,6S*)-3-[methoxy(methylthio)methyl]-2_pyrrolidinethione, respectively} and their analogues has been studied for the inhibition of the growth of lettuce hypocotyls and Amaranthus roots. Pyrrolidinethione compounds showed higher activity than pyrrolidinones. The presence of a methoxy(methylthio)methyl group or of a methylthiomethylene moiety at C-3 of pyrrolidinethione was a dominant factor for higher activity. The bis(methylthio)methyl derivative also gave a similar inhibitory activity. It was established that the active sites in the chemical structure of the raphanusanins and their analogues were the thioamide moiety at C-2 of the skeleton and a methoxy(methylthio)methyl, bis(merhylthio)methyl, or methylthiomethylene group in the C-3 position of the side chain.
INTRODUCTION
Four light-induced plant growth inhibitors have been isolated from light-grown Sakurajima radish (Ruphanus sativus L. var. hortensis f. gigantissimus Makino) seedlings, and their structures identified as (3R*,6R*)and (3R*,6S*)-3-[methoxy(methylthio)methyl]-Zpyrrolidinethione (named raphanusanins A and B, respectively) [l-3], 3-(E)-methoxymethylene-2-pyrrolidinethione (raphanusamide) [2, 41, and 3-(Q-methylthiomethylene-2-pyrrolidinethione [S], respectively. Among these growth inhibitors, raphanusanins A and B are the major phototropism-regulating substances in radish seedlings; it was found that they accumulate on the illuminated side of hypocotyls subjected to phototropic stimulation, and caused growth inhibition of the illuminated side through interference with auxin-mediated microtubule orientation [6-121. Furthermore, it was clarified that lateral application of these compounds caused differential growth, which resulted in bending toward the applied side. In this paper, the structure-activity relationship of raphanusanins and their analogues was investigated to clarify the chemical structure required for the inhibitory activity. RESULTS AND DISCUSSION
The structure-activity relationship of the raphanusanins and their analogues shown in Fig. 1 was studied by means of the lettuce hypocotyl growth test and the $Author to whom correspondence should be addressed.
Amaranthus root growth test. There was no significant difference between the two bioassays, except that compounds 6 and 9 showed higher activity in the Amaranthus bioassay than in the lettuce test (Table 1). Pyrrolidinethione derivatives such as raphanusanins had strong inhib-
Table 1. Inhibitory activity of raphanusanins and their analogues on the growth of etiolated lettuce hypocotyls and Amaranthus caudatus roots
I,, (M)* Compounds
Lettuce bioassay
Amaranthus bioassay
1 2 3 4 s 6 7 8 9
5.3 x 4.8 x 4.9 x 3.9 x 5.2 x 4.7 x 1.7 x 1.2 x 1.4 x
2.0 x 2.4 x 5.4 x 3.1 x 3.9 x 1.6 x 1.1 x 1.4 x 6.6 x
10 11 12 13 14
> 1o-2 >10-2 > lo-* 7.0 x 10-a > lo-*
lo-4 1O-4 1o-4 10-b 10-d 10-4 10-J 10-3 10-S
10-b 1O-4 10-d 10-d 10-d lO-4 10-J 10-a 10-s
> 10-Z >10-2 > 10-a 1.2x 10-a 3.4 x 1o-4
*I,, represents the concentration of sample which causes 50% inhibition of the growth of lettuce hypocotyls or Amaranthus roots, respectively.
1371
1312
M. SAKOVA
1
11
et al.
10
12
14
No biological activity
a
0+
it
H
N ’ d0
l3 0 7
1
’
J
In*’
Iso (M) Effect Frg. 1. Structure-activity
on lettuce
relationship of raphanusanms and their analogues. Compound 1, raphanusanin (3R6R);2,raphanusanin B (3R,6S); 3, raphanusamlde.
itory activity in both bioassays, whereas pyrrolidinone compounds showed no inhibitory activity (Table 1 and Fig. 1). The most striking examples are seen in the comparisons of the 2-pyrrolidinethione derivatives 3 and 7 with the 2-pyrrolidinones 10 and 12, respectively; the former showed strong or fair inhibitory activity in both bioassays, but the latter were inactive. Therefore, the skeleton of 2-pyrrolidinethione is necessary for inhibitory activity. Another active site was shown to be the side chain at the C-3 position. The activity of pyrrolidinethiones having a methoxy(methylthio)methyl, a bis(methylthio)methyl, methylthiomethylene, or a methoxymethylene side chain at C-3 (l-6) was much higher than that of the 2pyrrolidinethione 9. However, the activity of 3(dimethoxy)methyl-2-pyrrolidinethione (7) was lower than that of the other 2-pyrrolidinethione compounds. Therefore, the presence of a dimethoxy group at C-6 led
1o-2
hypocotyls A
to a reduction in activity. The biological activity of 2pyrrolidinethiones bearing a bis(methylthio) group or a methoxy(methylthio) group at C-6 was higher than those of other pyrrolidinethiones. The presence of thioacetal or dithioacetal group at C-6 is thus important and leads to an increase in activity. On the other hand, acetal 7 showed weaker activity than the thioacetals and dithioacetal. The activity of methylthiomethylene and methoxymethylene compounds (S5) was almost comparable to those of thioacetal and dithioacetal compounds. Furthermore, there was not much difference in the inhibitory activity between the geometrical isomers trans-4 and cis5. It was thus clarified that the active sites in the chemical structure of the raphanusanins and their analogues are the thioamide moiety at the C-2 position and the methoxy(methylthio)methyl, bis(methylthio)methyl, or methylthiomethylene groups at the C-3 position.
Structure-activity
relationship of raphanusanins
EXPERIMENTAL Materials. Raphanusamide
[3,3-(Q-methoxymethylene-2-pyrrolidinethione] was prepared according to the procedure reported in the literature [4, 13). Treatment of 3 with MeSNa in aq. MeOH at room temp. for 15 min gave a mixt. of raphanusanins A (l), B (2), 3, 4,6 and 7, which were sepd by a combination of HPLC on silica gel (hexane-EtOAc 2: 1) and HPLC on silica gelODS (MeOH-H,O 3 : 2) to afford 1 (21%), 2 (29%), 3 (3%), 4 (1 l%), 6 (5%) and 7 (trace). Compounds 1 and 2 are racemates, respectively. The c&isomer 5 was synthesized by photo-isomerization of the trans-isomer 4; a MeOH soln was illuminated by a tungsten-lamp for 6 hr. The ratio of E and Z isomers of the products obtained was 1: 3; the isomers were sepd by HPLC on silica gel (hexane-EtOAc 3 : 1). Compound 7 was obtained by treatment of raphanusamide (3) with 5% KOH-MeOH (room temp., 4 hr). 2-Pyrrolidinethione (9) was easily prepared from 2-pyrrolidinone [P,S,, in tetrahydrofuran (THF)]. Compounds 12-14 are synthetic intermediates of raphanusamide (3). Hydrogenation of 12 with Pd-C in MeOH gave 10 and 11; the major product 11 was then converted to 8 by treatment with P,S,, in THF. The details of the synthesis and the spectroscopic data of these analogues will be reported elsewhere. Bioassays. A soln of the test compound in Me&O was absorbed on to a filter paper (Toyo filter No. 1) and dried at room temp., and 0.4 ml H,O added. Twelve lettuce seeds (Lactuca sadiua L. cv. Mikado Great) were spread on the filter paper in a Petri dish (27 mm), and were cultured for 4 days in the dark at 25”. The length of the hypocotyls was measured. Fifteen seeds of Amaranthus caudatus L. were spread in a Petri dish (33 mm) containing a sheet of filter paper and 0.6 ml test soln, and were cultured for 6 days in the dark at 25”. The length of the roots was measured. The experiments were each repeated twice. The results were reproducible. Acknowledgements-This
work was supported in part by grants from the Ministry of Education, Science and
1373
Culture, Japan [(General (B) No. 01470026 to N.H., Priority Areas Nos. 02250103 and 03236101 to N.H. and K.H., and International Joint Research No. 02044014 to N.H.] and Suntory Institute for Bioorganic Research (SUNBOR grants to N.H.).
REFERENCES Hasegawa, K., Shiihara, S., Iwagawa, T. and Hase, T. (1982) Plant Physiol. 70, 626.
Hasegawa, K., Noguchi, H., Iwagawa, T. and Hase, T. (1986) Plant Physiol. 81, 976. Harada, N., Hagiwara, H., Ono, H., Uda, H., Ohba, S., Kubota, M., Nishiyama, S., Yamamura, S., Hasegawa, K. and Sakoda, M. (1991) Tetrahedron Letters 32, 6757.
4. Harada, N., Ono, H., Hagiwara, H., Uda, H., Hasegawa, K. and Sakoda, M. (1991) Tetrahedron Letters 32, 6761.
Sakoda, M., Hase, T. and Hasegawa, K. (1990) Phytochemistry 29, 1031.
Noguchi, H., Nishitani, K., Bruinsma, J. and Hasegawa, K. (1986) Plant Physiol. 81, 980. Noguchi, H. and Hasegawa, K. (1987) Plant Physiol. 83, 672.
Hasegawa, K., Noguchi, H., Tanoue, C., Sando, S., Takada, M., Sakoda, M. and Hashimoto, T. (1987) Plant Physiol. 85, 379.
9. Sakoda, M., Matsuoka, T., Sando, S. and Hasegawa, K. (1988) J. Plant Physiol. 133, 110. 10. Bruinsma, J. and Hasegawa, K. (1990) Physiol. Plant. 79,700.
11. Sakoda, M., Hasegawa, K. and Ishizuka, K. (1991) Phytochemistry 30, 57.
12. Sakoda, M., Hasegawa, K. and Ishizuka, K. (1992) Physiol. Plant. 84, 509.
13. Shibayama, F., Yamada, S., Kikuchi, H. and Yamada, Y. (1981) Jpn. Kokai Tokyo Koho JP 81, 104, 864; (1982) Chem. Abstr. 96, 35076~.