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Biotransformation of digitoxigenin by cell suspension cultures of Strophanthus divaricatus
0031-9422/91$3.00+0.00 0 1991Pcrgamon Press plc
Phyrochemisrry,Vol. 30, No. 5, pp. 1503-1506,1991 Printed in Great Britain.
BIOTRANSFORMATION OF DIGITOXIGENIN BY CELL SUSPENSION CULTURES OF STROPHANTHUS DI VARICATUS* KIICHIRO KAWAGUCHI, MASAO HlRoTANIt and TSUTOMU FURUYA~ Medicinal Plant Garden, School of Pharmaceutical Sciences, Kitasato University, Sagamihara, Kanagawa 228, Japan; tSchool of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108, Japan (Receioed
in revisedform
16 October
1990)
Key Word Index-Strophanthus diuaricotus; Apocynaceae; cell cultures; biotransformation; l6ghydroxylation; isomerization of l7/?-butenolide ring; cardenolides; digitoxigenin; 17/?H-gitoxigenin; 3-epi-IIIPH-gitoxigenin.
Abstract-Eight compounds, including a new compound (3-epi-17pH-gitoxigenin) were isolated as biotransformation products of digitoxigenin by cell suspension cultures of Strophanthus diuaricatus. At the same time, three products were identified by TLC and HPLC. 16jGHydroxylation and isomerization of the 17/l-butenolide ring of digitoxigenin molecule were demonstrated using this cell suspension culture.
3-epigitoxigenin (8), 17jH-gitoxigenin (9), 3-epi-17/?Hgitoxigenin (10) and 3-epi-17jYH-periplogenin (12) were isolated as the biotransformation products in the yields shown in Table 1. Because of the small amounts of samples, 3-epidigitoxigenin (3), periplogenin (6) and 3-epiperiplogenin (11)were identified by comparison with authentic samples [l-3, 63 by TLC and HPLC. In the ‘HNMR spectrum of product 9 (C,,H,,O,, HRMS), the methyl proton signal of C-18 was shifted downfield to 6 1.03 (3H, s) and the methine proton signal of H-17 was observed as a doublet (J = 9 Hz) at 63.15 (Table 2). In the 13C NMR spectrum (Table 3), the C-12 signal was shifted upfield to 633.5 (ca 8 ppm) showing that H- I7 has /l-configuration. The H- 16 signal appeared at 64.45 (lH, ddd, J=9, 9, 3 Hz), coupling with the methylene protons of C- 15 [at 6 I .55 (1H, dd, J = 15,3 Hz, H-152) and 62.64 (lH, dd, .I= 15, 9 Hz, H-15/?)]. The values of J, s8. ,6a, J, 5Z,,6a and J,62. 1,= in bufotalin which has a 16/l-acetoxyl group were 9.2 and 9 Hz, respectively [7]. On the other hand, the values of J! 5,=,,68 and J,68, 17a in l6a-hydroxycalactin diacetate which has a 16cx-acetoxyl group were 8.1 and 3.9 Hz, respectively [8]. The values of JL6.. ,,= and .J,68, 171 in 17aH-cardenolides lacking an oxygen function at C-16 were 6 and 9 Hz,
INTRODUCI’IOK In a series of investigations of digitoxigenin (1) biotransformation by plant cells and tissue cultures, IS products, including two new compounds and seven new products, were obtained with cell suspension cultures of Strophanthus gratus Cl], S. amboensis [2] and S. inrermedius [3]. Furthermore three new esters (digitoxigenin stearate, palmitate and myristate) and two new glycosides (3-epidigitoxigenin j?-D-gentiobioside and digitoxigenin fi-D-sophoroside) were isolated as crystals from ginseng hairy root cultures which contained no cardiac glycosides found in the original plant [4]. Strophanthus diuaricatus is indigenous to the southern coastal area of China and the cardiac glycosides from the seeds, leaves, stems and roots were investigated in detail [5]. However, there has been no report on the use of tissue cultures of this plant until now. We report the biotransformation of 1, a precursor of cardiac glycosides, by cell suspension cultures of S. dioaricatus and the structural elucidation of 3-epi17j?H-gitoxigenin (10). RESULTS AND DISCLSSION The calli derived from the seedling of Strophanthus diuaricatus contain no cardenolides; similarly three species of Strophnnthus cultured cells lacked these compounds [l-3]. On the other hand, several cardiac glycosides were detected in the regenerated plants of S. divaricatus obtained from the calli under controlled culture conditions. After incubation of digitoxigenin (1) (725 mg) with the cells (540 g fr. wt) for 18 days, 17j?H-digitoxigenin (2), 3-epi-17/?H-digitoxigenin (4): the main product in this experiment, gitoxigenin (S), l7/?H-periplogenin (7),
Table 1. Biotransformation products of digitoxigenin (1) by cell suspension cultures of Strophanthus diwricatus
*Part 74 in the series ‘Studies on Plant Tissue Cultures’. For Part 73 see Orihara, Y., Miyatake, H. and Furuya, T. (1991) Phytochemistry 30, (in press).
Products
Yield, mg (%)
17gH-Digitoxigenin (2) 3-epi-l7j?H-Digitoxigenin (4) Gitoxigenin (5) 17/?H-Periplogenin (7) 3-Epigitoxigenin (8) 17j?HGitoxigenin (9) 3-epi-17/?H-Gitoxigenin (10) 3-epi-17fiH-Periplogenin (12)
70.5 (9.7) 130.0 (17.9) 8.0 (1.1) 5.0 (0.7) 9.5 (1.3) 22.0 (2.9) 17.0 (2.2) 14.0 (1.9)
1503
K. KAWAGUCHIer al.
1504 Table
2. ‘H NMR spectral
data for biotransformation
products
9, IO and their diacetates
(300 MHG CD,OD)
H
9t
IO
9-diacetate*
IO-&acetate*
3 15a
4.05 br s (7.5) 1.55 dd (15, 3)
3.56 m (24) 1.57 dd (IS, 3)
158 Iti 17 H,-18 H,-19
2.64 4.45 3.15 1.03 0.95 4.88 4.95 6.01
2.66 4.46 3.16 1.03 0.93
5.08 br s (7) 1.60 dd (15, 3) 2.80 dd (15, 9) 5.26 ddd (9, 9. 3) 3.42 d (9) 1.08 s 0.95 s 4.73 dd (18, 1.5) 4.89 dd (18, 2) 5.94 dd (3. 1.5) 2.05 s. 2.05 s
4.72 m (24) 1.61 dd (15, 3) 2.80 dd (15, 9)
2la 21b 22
dd (15, 9) ddd (9, 9, 3) d (9) s s
dd (18, 2) dd (18, 2) dd (3, 2)
dd (I 5, 9) ddd (9. 9, 3) d (9) s s 4.87 dd (18, 2) 4.97 dd (18, 2) 6.02 dd (3, 2)
MeCO, *Measured in chloroform-d. t Measured at 400 MHz. The figures in parentheses are coupling
Table 3. ‘% NMR chemical
C
Multiplicity:
I
I
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23 MeCO, MeCO,
d d
s S
i
d
9 9 s > s 9 9 s s
constants
respectively.
Additionally,
9t
10
31.0 28.8 68.0 34.5 37.7 28.0 21.6’ 42.7 36.9 36.6 22.3’ 33.5 51.0 86.3 42.7 74.6 59.3 19.2 24.6 173.3 76.2 117.6 177.0
36.5 31.6 72.6 37.3 42.9 28.4 21.5’ 43.5 37.7 36. I 22.5’ 33.5 51.0 86.2 42.7 74.6 59.3 19.2 24.1 173.3 76.2 117.7 177.0
the values of J,ti,,7B and lacking an oxygen function at C-16 were 9.5 and 9.5 Hz, respectively [I, 21. As the coupling constants of H-15%, H-15/3, H-16 and H-17
J , 68,, ,@ in 17/U-f-cardenolides
ddd (9, 9. 3) d (9) s s dd (18, 1.5) dd ( IX, 2) dd (3. 1.5) s, 2.06 s
m Hz except for IV,,, values in hr s and tn.
shifts of biotransformation (75 MHz. CD,OD)
*Measured in chloroform-d. t Measured at 100 MHz. ZDetermined by DEPT experiments. ‘,bThe signals with the same superscript spectrum.
5.27 3.43 1.08 0.92 4.74 4.89 5.94 2.04
products
9, 10 and their diacetates
9-diacetate*
IO-&acetate*
30.3’ 25.0 70.2 30.4’ 36.7 26.1 20.7b 41.5 35.6 35.0 20.0h 32.2 49.1 84.6 40.0 75.3 54.2 18.0 23.6 167.5 73.6 117.8 173.4 21.0 21.5 170.6 170.9
34.6 26.6 73.8 32.V 41.2 26.6 20.9” 41.6 36.2 34.7 19.Hb 32.2 49.0 84.6 40.0 75.4 54.1 18.1 23.1 167.4 73.6 117.8 173.4 21.1 21.4 170.7 171.0
may be interchanged
within the corresponding
in compounds 9 and 9-diacetate were very similar to each other, the configuration of H-16 was revealed to he a. From these spectral data, the structure of 9 was determined as 3/I,14,16/?-trihydroxy-5/?,14/l,l7r-card-20(22)-
Biotransformation of digitoxigenin enolide (17/W-gitoxigenin) and 9 is a new biotransformation product of 1, though synthesized chemically using sodium tosylate in DMF by heating [9]. The structure of product 10 (C23H3405, HRMS) was established as 3a,14,16/3-trihydroxy-5/I,14/I,l’la-card20(22)-enolide (3-epi-17bH-gitoxigenin) by comparison with the ‘H NMR spectrum of product 9, especially the proton signal of H-3 at 63.56 (lH, m, IV,,, = 24 Hz, H-38). In the 13C NMR spectrum of 10, the data for C-l to C-10 agreed well with the data of 8 and the data for C-11 to C-23 corresponded to the data of 9, respectively. Furthermore, the ‘H and 13CNMR spectra of lO-diacetate, isolated after acetylation, corresponded to the data of 8and Bdiacetate. This compound has not been reported previously as either a naturally occurring or chemically synthesized substance. The possible biotransformation pathway of 1 by cell suspension cultures of Strophanthus dioaricatus is shown in Scheme 1. It was demonstrated that isomerization of the 17/I-lactone ring (1 to 2,3 to 4.5 to 9,6 to 7,8 to 10 and 11to 12), epimerization of the 3fl-hydroxyl to the 3ahydroxyl(1 to 3,2 to 4,s to 8,6 to 11,7 to 12 and 9 to lo), 5/?-hydroxylation (1 to 6,2 to 7,3 to 11 and 4 to 12) and 16/?-hydroxylation (1 to 5, 2 to 9, 3 to 8 and 4 to 10) proceed in these cells. Although a new reaction for the biotransformation of 1 was not found, the formation of 9 and 10 by a combination of known reactions (16jhydroxylation and isomerization of the 1‘I/&lactone ring) was demonstrated for the first time in these cells. On the other hand the original plants of S. diuaricatus contain 12
v A01 0
x
1505
cardenolide glycosides: divaricoside, musaroside, sarmentoloside and so on [S], but I II-, 12/I- and 19hydroxylation and glycosylation of 1 with deoxysugars or glucose could not be demonstrated in the cultured cells. Moreover, there is an interesting relationship between the enzymes for 5fi- and 16jLhydroxylation of 1 in this culture and the digitoxin 12/?-hydroxylase from cell cultures of Digitalis la~ta [lo]. Additionally, &diacetate possessed the highest inotropit activity among the biotransformation products obtained with Struphanthus cells Cl-33 in a preliminary experiment using a guinea-pig left atrial preparation (Furuya, T., Kawaguchi, K. and Hirotani, M., unpublished observations). Similarly, Humber et al. [l l] have reported the synthesis and biological activity of the four diastereomeric 3,16-diacetates related to gitoxigenin (5) and found high inotropic activity of g-diacetate which is 7.5 times more potent than digoxin. EXPERIMENTAL
Mps: uncorr. NMR: 300 and 400 MHz (CDCI, or CD,OD). MS: direct inlet. Culture methods.The seeds of Strophanthus diuaricarus Hook and Am., were collected at the Botanical Gardens, Faculty of Science, University of Tokyo in February 1983. They were aseptically placed on Murashige and Skoog’s (MS) medium containing 3% sucrose and 0.9% agar at 25” in the dark. After 3 weeks, the cotyledons of the seedlings were obtained and callus tissue induced from them was established and maintained on MS
0 0
-
8
OH
5
9
10
“o~,,\~-“o&~o& :o$,\/& OH
OH
II
6
OH
OH 7
12
Scheme 1. Possible pathway for the biotransformation of digitoxigenin (I) by cell suspension cultures of Strophanthus diuaricatus.
K. KAWACXJCHIet al.
1506
agar medium containing I ppm 2,4-dichlorophenoxyacetic acid. 0.1 ppm kinetin and 3% sucrose at 25’ in the dark. The shoots were regenerated from the calli by transferring onto MS agar medium containing 0.5 ppm kinetin and 3% sucrose at 25’ with 3OOC-5000 lux for 16 hr. The regenerated plants were obtamed from the shoots by transferring onto MS basal medium. After that the regenerated plants were cultivated in soil in pots. Detection oJ cardenolides in the regenerated p/anr.s. In the CHCI, and CHCI,-[so-PrOH (312) extracts from the aerial parts of the regenerated plants (340 g fr. wt. 50. 130cm height), 6 Kedde-positive spots (R, 0.37, 0.23, 0.19, 0.1 I, 0.08, 0.04; CHCI,-EtOH. 10: I) were detected on TLC. Isolution and identification oJ hiotransformutio~~ products. Digitoxigenin (I) (725 mg: 30 mg x 23 and 35 mg x 1) suspended with Tween 80(5%) was added to the calli (540 g fr. wt). and after 18 days incubation in a shaker (9Ospm). the CHCI, and the CHCI,- MeOH (2: 1) extracts from the calli and the medium were obtamcd. as previously reported Ll-31. After detection of biotransformation products on TLC with Kedde’s reagent and 10% H,SO,. purilication ofthe products from these extracts was achieved by repeated chromatography on a silica gel column (Wako gel C-200 and 300) using CHCI,-EtOH and CHCI,-MeOH solvent systems and HPLC (Nucleosil 5C18, solvent 60% MeOH in H,O). Products 2.4,s. 7--10 and 12 were isolated in the yields shown in Table I and identified with authentic samples by TLC, HPLC and ‘H and “CNMR spectral data. Products 3.6 and 11 were idenhfied with authentic samples by TLC and HPLC [l-3, 61. 3-Epigiroxigenin (8). Compound 8, mp 220. 222- (from EtOH-H,O). C23H340s (required 390.2406, [Ml’ at m/z 390.2390). ‘H NMR (300 MHz. CD,OD): (iO.91 (3H, s. Me-18). 0.93 (3H. s, Me-19). 1.71 (IH, dd, J= 15, 2 HI, H-151), 2.63 (IH. dd, J= 15. 8.5 HI, H-15/1), 3.13 (IH. d, 5=8.5 Hq H-17u), 3.55 (IH. m, W,:,= 24 Hz, H-3/Q 4.65 (IH, ddd, J=8.5, 8.5, 2 Hz, H162),5.09(1H,dd.5=18.5, 1.5Hz,H-21a),5.17(1H.dd,J=18.5, I.5 Hz, H-21b), 5.93 (IH, dd. J= 1.5, 1.5 HI H-22). “CNMR (75 MHz: CD,OD): 6 17.3 (4). 22.2 (t), 22.9 (r), 24.1 (q), 28.5 (I), 31.6 (f), 36.2 (5). 36.5 (I). 37.3 (I). 37.7 (d), 41.2 (1). 43.3 (d), 43.4 (d), 44.1 (t), 51.6 (s), 59.9 (d). 72.6 (d), 73.3 (d), 78.2 (I), 85.9 (s). 120.9 (d). 174.0 (s), 177.6 (s). EIMS m!z (rel. int.): 390 [M] + (I), 372 [M -H,O](22), 354 [M-2xH,O]+ (24). 336 [M-3xH,O]+ (9),246[C,,H2~0]* (21).204[C,,H,,]+ (21),203[C,,H,,](100). 192 (21). 124 (22). 17bH-Giroxlgenin (9). Compound 9, mp 204-210” (from MeOH-H,O), C,,H,,O, (required 390.2406, [Ml+ at m/z 390.2393). ‘H and “C NMR: see Tables 2 and 3. EIMS m/z (ret. int.): 390 [M] ’ (3). 372 [M - H,O] (29). 354 [M -2 x H,O]’ (80),336[M-3xH,OJ(29),228(21),221 (23),204[C,,H,,]+ (21). 203 LC,,H,,]+ (100). 192 (21), 146 (23). 124 (26). 17/?HGifoxigenin (9) diocerafe. After acetylation of 9 (10 mg), 9-diacetate (9 mg) was isolated after purification by HPLC (R, 19.9 min: solvent 70% MeOH in H,O. Row rate: 3 ml min- I). Compound 9-diacetate, mp 224-229‘ (from l
MeOH), C2,H3s0, (required 474.2617, [M]’ at m/z 474.2617). ‘H and 13C NMR: see Tables 2 and 3. EIMS m/z (rel. int.): 474 [Ml’ (I), 414 [M-HOAc]’ (7). 354 [M-ZxHOAc]’ (16). 336 [M-2xHOAc-H,O](5). 204 [C,,H,,]’ (21), 203 [C,,H,,]+ (100). 124 (26). 3epi- 17/fH-Gitoxiyenin (10). Compound IO, amorphous solid, C 23H 340 5 (required 390.2406. [M] + at m/‘z 390.2403). ‘H and ‘3CNMR:sceTables 2and 3. ElMSmlz(rel. int.):390LM]+ (1). 372 [M-H>O]+ (22). 354 [M--2xH,O](64). 336 [M--3 xH,O]+ (32). 204 [C,,H,,]+ (19). 203 [C,,H2,]+ (100). 124 (19). 3-epl-17bH-Giroxiyenin (10) diocerare. After acetylatlon of IO (9 mg), IO-diacetate (7.5 mg) was obtained as an amorphous solid after purification by HPLC (R, 19.4 min: solvent 70% MeOH in H,O, flow rate’ 3 ml mm ‘). Compound IO-diacetate, C2,HjS07 (required 474.2617, LM]’ at m:‘; 474.2615). ‘H and ‘~CNMR:sceTablesZand3.EIMSm:‘;(rel.int.):474LMJ’(l), 414 [M-HOAc]’ (9). 354 [M-ZxHOAc]’ (16). 336 [M-2 x HOAc-HZ<)]’ (7). 204 [C,,H,,]+ (22). 203 [C,,H,,]+ (100). 124 (21). Acknowledgemenrs--We thank Prof. Dr T. Iino (Head) and Mr E. lshii (Botamcal Gardens. Faculty of Science, University of Tokyo) for Srrophonthus divoricatus seeds and plant. We also express our appreciation to the members of the Analytical Centrc of this Umversity for 300 and 400 MHz NMR spectra and mass spectra. This work was supported by a Grant-in-Aid for Scientific Research (Project-2) from the School of Pharmaceutical Sciences, Kitasato Univcrslty.
REFERENCES 1. Furuya, T., Kawaguchi, K. and Hirotani, M. (1988) Phytochemistry 27, 2129. 2. Kawaguchi. K., Hirotani, M. and Furuya, T. (1988) Phytochemistry 27, 3475. 3. Kawaguchi, K., Htrotani, M and Furuya, T. (1989) Phyrochemistry 28, 1093. 4. Kawaguchi, K., Hirotam, M., Yoshikawa. T. and Furuya. T. (1990) Phyrochemisrry 29. 837. 5. Chen, R.-F., Abe, F.. Yamauchi, T. and Taki, M. (1987) Phyrochemistrp
26, 235
I.
6. Hirotani,
M. and Furuya, T. (1980) Phytochemistry 19, 531. 7. Pettit, G. R., Brown, P., Bruschweiler, F. and Houghton, L. E. (1970) J. Chem. Sot., Chem. Commun. 1566. 8. Jolad, S. D., Bates, R. B., Cole. J. R., Hoffmann. J. J., Siahaan, T. J. and Timmermann, B. N. (1986) Phpchemlsrry 25,258 I, 9. Russel, J. H., Schindler. 0. and Reichstein, T. (1960) Hrlc. Chim. Acta 43, 167.
10. Petersen,
M. and Seitz, H. U. (1988) Biochrm. J. 252, 537. D. C.. Jones, P. S. and Phillipps. G. H. (1985) Steroids 45, 19.
1I. Hum&r.
Phyrochemisrry,Vol. 30, No. 5, pp. 1503-1506,1991 Printed in Great Britain.
BIOTRANSFORMATION OF DIGITOXIGENIN BY CELL SUSPENSION CULTURES OF STROPHANTHUS DI VARICATUS* KIICHIRO KAWAGUCHI, MASAO HlRoTANIt and TSUTOMU FURUYA~ Medicinal Plant Garden, School of Pharmaceutical Sciences, Kitasato University, Sagamihara, Kanagawa 228, Japan; tSchool of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108, Japan (Receioed
in revisedform
16 October
1990)
Key Word Index-Strophanthus diuaricotus; Apocynaceae; cell cultures; biotransformation; l6ghydroxylation; isomerization of l7/?-butenolide ring; cardenolides; digitoxigenin; 17/?H-gitoxigenin; 3-epi-IIIPH-gitoxigenin.
Abstract-Eight compounds, including a new compound (3-epi-17pH-gitoxigenin) were isolated as biotransformation products of digitoxigenin by cell suspension cultures of Strophanthus diuaricatus. At the same time, three products were identified by TLC and HPLC. 16jGHydroxylation and isomerization of the 17/l-butenolide ring of digitoxigenin molecule were demonstrated using this cell suspension culture.
3-epigitoxigenin (8), 17jH-gitoxigenin (9), 3-epi-17/?Hgitoxigenin (10) and 3-epi-17jYH-periplogenin (12) were isolated as the biotransformation products in the yields shown in Table 1. Because of the small amounts of samples, 3-epidigitoxigenin (3), periplogenin (6) and 3-epiperiplogenin (11)were identified by comparison with authentic samples [l-3, 63 by TLC and HPLC. In the ‘HNMR spectrum of product 9 (C,,H,,O,, HRMS), the methyl proton signal of C-18 was shifted downfield to 6 1.03 (3H, s) and the methine proton signal of H-17 was observed as a doublet (J = 9 Hz) at 63.15 (Table 2). In the 13C NMR spectrum (Table 3), the C-12 signal was shifted upfield to 633.5 (ca 8 ppm) showing that H- I7 has /l-configuration. The H- 16 signal appeared at 64.45 (lH, ddd, J=9, 9, 3 Hz), coupling with the methylene protons of C- 15 [at 6 I .55 (1H, dd, J = 15,3 Hz, H-152) and 62.64 (lH, dd, .I= 15, 9 Hz, H-15/?)]. The values of J, s8. ,6a, J, 5Z,,6a and J,62. 1,= in bufotalin which has a 16/l-acetoxyl group were 9.2 and 9 Hz, respectively [7]. On the other hand, the values of J! 5,=,,68 and J,68, 17a in l6a-hydroxycalactin diacetate which has a 16cx-acetoxyl group were 8.1 and 3.9 Hz, respectively [8]. The values of JL6.. ,,= and .J,68, 171 in 17aH-cardenolides lacking an oxygen function at C-16 were 6 and 9 Hz,
INTRODUCI’IOK In a series of investigations of digitoxigenin (1) biotransformation by plant cells and tissue cultures, IS products, including two new compounds and seven new products, were obtained with cell suspension cultures of Strophanthus gratus Cl], S. amboensis [2] and S. inrermedius [3]. Furthermore three new esters (digitoxigenin stearate, palmitate and myristate) and two new glycosides (3-epidigitoxigenin j?-D-gentiobioside and digitoxigenin fi-D-sophoroside) were isolated as crystals from ginseng hairy root cultures which contained no cardiac glycosides found in the original plant [4]. Strophanthus diuaricatus is indigenous to the southern coastal area of China and the cardiac glycosides from the seeds, leaves, stems and roots were investigated in detail [5]. However, there has been no report on the use of tissue cultures of this plant until now. We report the biotransformation of 1, a precursor of cardiac glycosides, by cell suspension cultures of S. dioaricatus and the structural elucidation of 3-epi17j?H-gitoxigenin (10). RESULTS AND DISCLSSION The calli derived from the seedling of Strophanthus diuaricatus contain no cardenolides; similarly three species of Strophnnthus cultured cells lacked these compounds [l-3]. On the other hand, several cardiac glycosides were detected in the regenerated plants of S. divaricatus obtained from the calli under controlled culture conditions. After incubation of digitoxigenin (1) (725 mg) with the cells (540 g fr. wt) for 18 days, 17j?H-digitoxigenin (2), 3-epi-17/?H-digitoxigenin (4): the main product in this experiment, gitoxigenin (S), l7/?H-periplogenin (7),
Table 1. Biotransformation products of digitoxigenin (1) by cell suspension cultures of Strophanthus diwricatus
*Part 74 in the series ‘Studies on Plant Tissue Cultures’. For Part 73 see Orihara, Y., Miyatake, H. and Furuya, T. (1991) Phytochemistry 30, (in press).
Products
Yield, mg (%)
17gH-Digitoxigenin (2) 3-epi-l7j?H-Digitoxigenin (4) Gitoxigenin (5) 17/?H-Periplogenin (7) 3-Epigitoxigenin (8) 17j?HGitoxigenin (9) 3-epi-17/?H-Gitoxigenin (10) 3-epi-17fiH-Periplogenin (12)
70.5 (9.7) 130.0 (17.9) 8.0 (1.1) 5.0 (0.7) 9.5 (1.3) 22.0 (2.9) 17.0 (2.2) 14.0 (1.9)
1503
K. KAWAGUCHIer al.
1504 Table
2. ‘H NMR spectral
data for biotransformation
products
9, IO and their diacetates
(300 MHG CD,OD)
H
9t
IO
9-diacetate*
IO-&acetate*
3 15a
4.05 br s (7.5) 1.55 dd (15, 3)
3.56 m (24) 1.57 dd (IS, 3)
158 Iti 17 H,-18 H,-19
2.64 4.45 3.15 1.03 0.95 4.88 4.95 6.01
2.66 4.46 3.16 1.03 0.93
5.08 br s (7) 1.60 dd (15, 3) 2.80 dd (15, 9) 5.26 ddd (9, 9. 3) 3.42 d (9) 1.08 s 0.95 s 4.73 dd (18, 1.5) 4.89 dd (18, 2) 5.94 dd (3. 1.5) 2.05 s. 2.05 s
4.72 m (24) 1.61 dd (15, 3) 2.80 dd (15, 9)
2la 21b 22
dd (15, 9) ddd (9, 9, 3) d (9) s s
dd (18, 2) dd (18, 2) dd (3, 2)
dd (I 5, 9) ddd (9. 9, 3) d (9) s s 4.87 dd (18, 2) 4.97 dd (18, 2) 6.02 dd (3, 2)
MeCO, *Measured in chloroform-d. t Measured at 400 MHz. The figures in parentheses are coupling
Table 3. ‘% NMR chemical
C
Multiplicity:
I
I
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23 MeCO, MeCO,
d d
s S
i
d
9 9 s > s 9 9 s s
constants
respectively.
Additionally,
9t
10
31.0 28.8 68.0 34.5 37.7 28.0 21.6’ 42.7 36.9 36.6 22.3’ 33.5 51.0 86.3 42.7 74.6 59.3 19.2 24.6 173.3 76.2 117.6 177.0
36.5 31.6 72.6 37.3 42.9 28.4 21.5’ 43.5 37.7 36. I 22.5’ 33.5 51.0 86.2 42.7 74.6 59.3 19.2 24.1 173.3 76.2 117.7 177.0
the values of J,ti,,7B and lacking an oxygen function at C-16 were 9.5 and 9.5 Hz, respectively [I, 21. As the coupling constants of H-15%, H-15/3, H-16 and H-17
J , 68,, ,@ in 17/U-f-cardenolides
ddd (9, 9. 3) d (9) s s dd (18, 1.5) dd ( IX, 2) dd (3. 1.5) s, 2.06 s
m Hz except for IV,,, values in hr s and tn.
shifts of biotransformation (75 MHz. CD,OD)
*Measured in chloroform-d. t Measured at 100 MHz. ZDetermined by DEPT experiments. ‘,bThe signals with the same superscript spectrum.
5.27 3.43 1.08 0.92 4.74 4.89 5.94 2.04
products
9, 10 and their diacetates
9-diacetate*
IO-&acetate*
30.3’ 25.0 70.2 30.4’ 36.7 26.1 20.7b 41.5 35.6 35.0 20.0h 32.2 49.1 84.6 40.0 75.3 54.2 18.0 23.6 167.5 73.6 117.8 173.4 21.0 21.5 170.6 170.9
34.6 26.6 73.8 32.V 41.2 26.6 20.9” 41.6 36.2 34.7 19.Hb 32.2 49.0 84.6 40.0 75.4 54.1 18.1 23.1 167.4 73.6 117.8 173.4 21.1 21.4 170.7 171.0
may be interchanged
within the corresponding
in compounds 9 and 9-diacetate were very similar to each other, the configuration of H-16 was revealed to he a. From these spectral data, the structure of 9 was determined as 3/I,14,16/?-trihydroxy-5/?,14/l,l7r-card-20(22)-
Biotransformation of digitoxigenin enolide (17/W-gitoxigenin) and 9 is a new biotransformation product of 1, though synthesized chemically using sodium tosylate in DMF by heating [9]. The structure of product 10 (C23H3405, HRMS) was established as 3a,14,16/3-trihydroxy-5/I,14/I,l’la-card20(22)-enolide (3-epi-17bH-gitoxigenin) by comparison with the ‘H NMR spectrum of product 9, especially the proton signal of H-3 at 63.56 (lH, m, IV,,, = 24 Hz, H-38). In the 13C NMR spectrum of 10, the data for C-l to C-10 agreed well with the data of 8 and the data for C-11 to C-23 corresponded to the data of 9, respectively. Furthermore, the ‘H and 13CNMR spectra of lO-diacetate, isolated after acetylation, corresponded to the data of 8and Bdiacetate. This compound has not been reported previously as either a naturally occurring or chemically synthesized substance. The possible biotransformation pathway of 1 by cell suspension cultures of Strophanthus dioaricatus is shown in Scheme 1. It was demonstrated that isomerization of the 17/I-lactone ring (1 to 2,3 to 4.5 to 9,6 to 7,8 to 10 and 11to 12), epimerization of the 3fl-hydroxyl to the 3ahydroxyl(1 to 3,2 to 4,s to 8,6 to 11,7 to 12 and 9 to lo), 5/?-hydroxylation (1 to 6,2 to 7,3 to 11 and 4 to 12) and 16/?-hydroxylation (1 to 5, 2 to 9, 3 to 8 and 4 to 10) proceed in these cells. Although a new reaction for the biotransformation of 1 was not found, the formation of 9 and 10 by a combination of known reactions (16jhydroxylation and isomerization of the 1‘I/&lactone ring) was demonstrated for the first time in these cells. On the other hand the original plants of S. diuaricatus contain 12
v A01 0
x
1505
cardenolide glycosides: divaricoside, musaroside, sarmentoloside and so on [S], but I II-, 12/I- and 19hydroxylation and glycosylation of 1 with deoxysugars or glucose could not be demonstrated in the cultured cells. Moreover, there is an interesting relationship between the enzymes for 5fi- and 16jLhydroxylation of 1 in this culture and the digitoxin 12/?-hydroxylase from cell cultures of Digitalis la~ta [lo]. Additionally, &diacetate possessed the highest inotropit activity among the biotransformation products obtained with Struphanthus cells Cl-33 in a preliminary experiment using a guinea-pig left atrial preparation (Furuya, T., Kawaguchi, K. and Hirotani, M., unpublished observations). Similarly, Humber et al. [l l] have reported the synthesis and biological activity of the four diastereomeric 3,16-diacetates related to gitoxigenin (5) and found high inotropic activity of g-diacetate which is 7.5 times more potent than digoxin. EXPERIMENTAL
Mps: uncorr. NMR: 300 and 400 MHz (CDCI, or CD,OD). MS: direct inlet. Culture methods.The seeds of Strophanthus diuaricarus Hook and Am., were collected at the Botanical Gardens, Faculty of Science, University of Tokyo in February 1983. They were aseptically placed on Murashige and Skoog’s (MS) medium containing 3% sucrose and 0.9% agar at 25” in the dark. After 3 weeks, the cotyledons of the seedlings were obtained and callus tissue induced from them was established and maintained on MS
0 0
-
8
OH
5
9
10
“o~,,\~-“o&~o& :o$,\/& OH
OH
II
6
OH
OH 7
12
Scheme 1. Possible pathway for the biotransformation of digitoxigenin (I) by cell suspension cultures of Strophanthus diuaricatus.
K. KAWACXJCHIet al.
1506
agar medium containing I ppm 2,4-dichlorophenoxyacetic acid. 0.1 ppm kinetin and 3% sucrose at 25’ in the dark. The shoots were regenerated from the calli by transferring onto MS agar medium containing 0.5 ppm kinetin and 3% sucrose at 25’ with 3OOC-5000 lux for 16 hr. The regenerated plants were obtamed from the shoots by transferring onto MS basal medium. After that the regenerated plants were cultivated in soil in pots. Detection oJ cardenolides in the regenerated p/anr.s. In the CHCI, and CHCI,-[so-PrOH (312) extracts from the aerial parts of the regenerated plants (340 g fr. wt. 50. 130cm height), 6 Kedde-positive spots (R, 0.37, 0.23, 0.19, 0.1 I, 0.08, 0.04; CHCI,-EtOH. 10: I) were detected on TLC. Isolution and identification oJ hiotransformutio~~ products. Digitoxigenin (I) (725 mg: 30 mg x 23 and 35 mg x 1) suspended with Tween 80(5%) was added to the calli (540 g fr. wt). and after 18 days incubation in a shaker (9Ospm). the CHCI, and the CHCI,- MeOH (2: 1) extracts from the calli and the medium were obtamcd. as previously reported Ll-31. After detection of biotransformation products on TLC with Kedde’s reagent and 10% H,SO,. purilication ofthe products from these extracts was achieved by repeated chromatography on a silica gel column (Wako gel C-200 and 300) using CHCI,-EtOH and CHCI,-MeOH solvent systems and HPLC (Nucleosil 5C18, solvent 60% MeOH in H,O). Products 2.4,s. 7--10 and 12 were isolated in the yields shown in Table I and identified with authentic samples by TLC, HPLC and ‘H and “CNMR spectral data. Products 3.6 and 11 were idenhfied with authentic samples by TLC and HPLC [l-3, 61. 3-Epigiroxigenin (8). Compound 8, mp 220. 222- (from EtOH-H,O). C23H340s (required 390.2406, [Ml’ at m/z 390.2390). ‘H NMR (300 MHz. CD,OD): (iO.91 (3H, s. Me-18). 0.93 (3H. s, Me-19). 1.71 (IH, dd, J= 15, 2 HI, H-151), 2.63 (IH. dd, J= 15. 8.5 HI, H-15/1), 3.13 (IH. d, 5=8.5 Hq H-17u), 3.55 (IH. m, W,:,= 24 Hz, H-3/Q 4.65 (IH, ddd, J=8.5, 8.5, 2 Hz, H162),5.09(1H,dd.5=18.5, 1.5Hz,H-21a),5.17(1H.dd,J=18.5, I.5 Hz, H-21b), 5.93 (IH, dd. J= 1.5, 1.5 HI H-22). “CNMR (75 MHz: CD,OD): 6 17.3 (4). 22.2 (t), 22.9 (r), 24.1 (q), 28.5 (I), 31.6 (f), 36.2 (5). 36.5 (I). 37.3 (I). 37.7 (d), 41.2 (1). 43.3 (d), 43.4 (d), 44.1 (t), 51.6 (s), 59.9 (d). 72.6 (d), 73.3 (d), 78.2 (I), 85.9 (s). 120.9 (d). 174.0 (s), 177.6 (s). EIMS m!z (rel. int.): 390 [M] + (I), 372 [M -H,O](22), 354 [M-2xH,O]+ (24). 336 [M-3xH,O]+ (9),246[C,,H2~0]* (21).204[C,,H,,]+ (21),203[C,,H,,](100). 192 (21). 124 (22). 17bH-Giroxlgenin (9). Compound 9, mp 204-210” (from MeOH-H,O), C,,H,,O, (required 390.2406, [Ml+ at m/z 390.2393). ‘H and “C NMR: see Tables 2 and 3. EIMS m/z (ret. int.): 390 [M] ’ (3). 372 [M - H,O] (29). 354 [M -2 x H,O]’ (80),336[M-3xH,OJ(29),228(21),221 (23),204[C,,H,,]+ (21). 203 LC,,H,,]+ (100). 192 (21), 146 (23). 124 (26). 17/?HGifoxigenin (9) diocerafe. After acetylation of 9 (10 mg), 9-diacetate (9 mg) was isolated after purification by HPLC (R, 19.9 min: solvent 70% MeOH in H,O. Row rate: 3 ml min- I). Compound 9-diacetate, mp 224-229‘ (from l
MeOH), C2,H3s0, (required 474.2617, [M]’ at m/z 474.2617). ‘H and 13C NMR: see Tables 2 and 3. EIMS m/z (rel. int.): 474 [Ml’ (I), 414 [M-HOAc]’ (7). 354 [M-ZxHOAc]’ (16). 336 [M-2xHOAc-H,O](5). 204 [C,,H,,]’ (21), 203 [C,,H,,]+ (100). 124 (26). 3epi- 17/fH-Gitoxiyenin (10). Compound IO, amorphous solid, C 23H 340 5 (required 390.2406. [M] + at m/‘z 390.2403). ‘H and ‘3CNMR:sceTables 2and 3. ElMSmlz(rel. int.):390LM]+ (1). 372 [M-H>O]+ (22). 354 [M--2xH,O](64). 336 [M--3 xH,O]+ (32). 204 [C,,H,,]+ (19). 203 [C,,H2,]+ (100). 124 (19). 3-epl-17bH-Giroxiyenin (10) diocerare. After acetylatlon of IO (9 mg), IO-diacetate (7.5 mg) was obtained as an amorphous solid after purification by HPLC (R, 19.4 min: solvent 70% MeOH in H,O, flow rate’ 3 ml mm ‘). Compound IO-diacetate, C2,HjS07 (required 474.2617, LM]’ at m:‘; 474.2615). ‘H and ‘~CNMR:sceTablesZand3.EIMSm:‘;(rel.int.):474LMJ’(l), 414 [M-HOAc]’ (9). 354 [M-ZxHOAc]’ (16). 336 [M-2 x HOAc-HZ<)]’ (7). 204 [C,,H,,]+ (22). 203 [C,,H,,]+ (100). 124 (21). Acknowledgemenrs--We thank Prof. Dr T. Iino (Head) and Mr E. lshii (Botamcal Gardens. Faculty of Science, University of Tokyo) for Srrophonthus divoricatus seeds and plant. We also express our appreciation to the members of the Analytical Centrc of this Umversity for 300 and 400 MHz NMR spectra and mass spectra. This work was supported by a Grant-in-Aid for Scientific Research (Project-2) from the School of Pharmaceutical Sciences, Kitasato Univcrslty.
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