insect growth inhibitory cardenolide glycosides from anodendron affine

Phyf~~stry, Vol. 32, No. 2, pp. 297-301, 1993 0031-9422,‘93 WM+O.M) Printed in Great Britain. 0 1993 Per~n Press Ltd

INSECT GROWTH INHIBITORY CARDENOLIDE GLYCOSIDES FROM ANODENDRONAFFINE

YOSHIYASU FUKUYAMA, MASAMITS~ OCHI,* HIDEK~ KA~AI* and MIISUAKI KODAMA

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yemashiro-cho, Tokushima 770, Japan; *Faculty of science, Kochi University, Kochi 780, Japan

(Receiued 12 June 1992)

Key Word Index-Am&v&on a&e; Apocynaceae; 12-oxo-aiIinoside E; 4,5-dehydro-12-oxo- affinoside E; 16&hydroxyaffinoside A; afhnoside A, affinoside E, affinoside M; cardenolide glycoside; insect inhibitory activity; Bombyx mori.

A~~~-Bio~say-gods ~actionation of the methanol extract of the stem and bark of Anoahulron a&e kd to the isofation of three new cardenohde glycosides, 4,5-dehydro-12-ox~~noside E, lZoxo-at’hnoside E, and 16& hydroxyaffinoside A along with the previously known cardenolides, affinosides A, E and M, and their structures were elucidated on the basis of spectroscopic data and comparison of spectral data with those of the congeners. Among them, 4,5-dehydro-lZoxo-afIinoside E, affinoside A, and affinoside M exhibit insect growth inhibitory activity against the silkworm, Bombyx mori.

INTRODUCTION

As a part of our search for biolo~~lly active substances [1] in plants growing in Yaeyama Islands which are located further south from the Okinawa Islands in Japan, 30 species collected in Yaeyama islands were primarily screened for insect growth inhibitory effect on the 2nd instar larvae of the silkworm, Eombyx nwri [2,3]. Among them, the methanol extract of the stem and bark of Anodendron a&e D. was found to exhibit potent growth inhibitory activity. Anodendron a&e D. (Apocynaceae), indigenous to the south western part of Japan, elaborates a number of cardenolide [4,5], in particular, cardenolide glycosides with unusual double linkage through acetal and herniketal bonds between the aglycone and a de- oxyhexosulose moiety [6-lo}. It is also of ecological interest that the larvae of Monarch butterflies (Danuus plexippus) and milkweed bugs (Oncopeltus jizsciatus) feed on the leaves of Asclepias host plants containing abundant cardenolides and utilize these chemicals for defense against vertebrate predators [ 11,121. Monitoring growth inhibitory activity of each fraction against Bombyx mori, three new cardenolide glycosides, namely 4,5dehydro- 12-oxo-anti& E (l), 12-oxo-a~o~de E (2) and 16@- hydroxy~noside A (41, were isolated along with the previously known affinosides A (!I), E (3) [A and M (6) [8]. In this paper, we report the isolation and structure of three new cardenolides and their growth inhibitory activ- ities on the 2nd instar larvae of the silkworm.

RESULTS AND DISCUSSION

Bioassay-guided fractionation of the active ethyl acetate-soluble portion by a combination of silica gel and

Sephadex LH-20 column chromatography, and HPLC led to the isolation of three new cardenolide glycosides 1, 2 and 4 with a 3-O-me~yl~,~~xyhexosulopyrano~ moiety which is doubly linked at C-2 and C-3 positions of the cardenolide aglycones through a&al and hemiketal bonds.

Compound 1 had the molecular formula C,,H,,O,, deduced from the quasi-molecular ion peak at m/z 561 [M+I-IJ+ on positive FAB-MS and the presence of 30 carbons observed by 13C NMR spectrum (Table 1). The spectral data for 1 revealed the presence of a butenolide ring [1780, 1740, 1620cm-‘; 6u4.77 (lH, dd, J-18.1, 1.7 Hz), 4.91 (lH, dd, J= 18.1, 1.5 Hz), 5.99 (lH, dd, J = 1.7, 1.5 Hz); &73.7 (d), 119.0(d), 172.5 (s), 174.3 (s)], a carbonyl group (f7OOcm-t; 6,212.5), and hydroxy groups (34OOcrn-‘) which were acetylated by Ac,O/ pyridine to yield a diacetate la. The ‘H NMR spectrum (Table 2) of 1 contained the signals due to two tertiary methyl groups (S, 1.09, 1.37), a methoxy group (6,3.40), and a secondary methyl group [S, 1.22 (d, J=6.3 Hz)] which was shown to be involved in a connectivity of C&-C,. as shown in Fig. 1 by ‘H-‘H and “H-l jC COSYs. Moreover, extensive analyses of 2D ‘H-‘H and ‘H-13C COSYs gave three optional structural fragments as shown by solid lines in Fig. 1. In an HMBC experiment (Fig. l), H-3’ at 6u3.30 showed cross peaks to two oxygen-bearing carbons at 8,90.3 (C-2’) and 96.0 (C-l’) and also an anomeric H-l’ at $4.71 (s) correlated to C-2’ and C-3 b&69.9) giving rise to a 3-0- methyl_4,6_dideoxyhexosulose moiety which is com- monly occurring in cardenolide glycosides [7-j isolated from the title plant. In fact, the 13C NMR data for the deoxy sugar in 1 were in good agreement with those of

297

298

t R=H 2 R=H la R=Ac 2a R=Ac

Table 1. 13C NMR data of cardenoiides 1,2,4 and 5*

c 1 2 4 5

1 42.6 40.5 43.9 43.5 2 68.1 69.2 67.7 67.8 3 69.9 68.8 69.6 69.6 4 120.2 32.4 123.1 122.4 5 144.8 33.3 138.8 138.8 6 31.6 24.7 29.5 29.7 7 28.8 24.5 54.8 54.0 8 39.9 39.2 62.1 62.4 9 53.7 51.0 48.3 48.6 10 41.9 38.2 40.7 40.8 11 73.5 73.0 73.2 73.3 12 212.5 212.5 212.9 212.3 13 62.2 61.9 62.1 63.1 14 85.5 86.9 81.7 81.1 15 33.1 327 43.5 35.9 16 26.9 26.1 73.8 28.3 17 40.6 40.8 49.8. 42.3 18 16.9 16.4 19.4 18.2 19 20.3 20.8 21.1 21.1 20 172.5 172.4 165.5 170.5 21 73.7 73.5 74.9 73.6 22 119.0 119.3 121.2 118.9 23 174.3 174.1 173.9 173.7 1’ 96.0 95.6 95.9 95.9 r 90.3 89.8 PO.2 90.2 3 80.3 80.3 80.3 80.3 4 32.6 33.3 32.6 32.7 5 66.0 66.1 66.1 66.1 6 20.9 21.4 20.9 20.8 OMe 57.4 57.4 57.4 51.4

*In CDC13. All asignments were made by 13C-*H COSY and HMBC.

4 Rr=R3=H,RZ=OCH~,&=OH 4s R,=JH,R~=OCH~,R~=AC,~=OAC 5 RI=R3=R,=H,Rz=OCH~ 6 Rz=R3=%=H,Rt=W3

afEnoside A (?I), isolated as an active substance, and published data for other cardenolides having this type of sugar [7,8]. In addition, the correlations between proton and carbon signals through two and three bonds observed by HMBC (Fig. 1) provided the remaining partial units (C,-C,, C,-C,,, C,,-Ci,) together with the butenolide ring and the five quaternary carbons (C-S, C-10, C-12, C-13 and C-14), resulting in the construction of the cardenolide skeleton. The carbonyl group must be placed at C-12 due to a distinct long range i3C-‘H correlation of a carbon signal at Sc 221.5 to the H-18 signal at 6u 1.09. The relative stereochemistry of the proposed structure for 1 was elucidated on the basis of the J values and NOES as shown in Fig. 2. Thus, the lQ-dioxane ring must be fused tram at C-2 and C-3 due to a large J value (9.7 Hz) between H-2 and H-3, which should take a tram diaxial orientation to each other since there was NOE inter- action present between H-2 and Me-19. It is also evident from a large J value (11.2 Hz) between the H-8 and H-9 that the B and C rings are fused trans. Additionally, H-9 coupled to the H-11 with 11.2 Hz which showed NOE enhancement upon irradiation at Me-18, thereby disclos- ing an a-equatorial orientation of the hydroxyl group attached at C-l 1, The butenolide ring was verified to have the same /I-orientation as the known cardenofides by the observation of NOES for H-21 and H-22 on the butenolide ring upon irradiation of Me-19. The spectral evidence mentioned above, thus determined 1 to be 4,5-dehydro- 12-oxo-athnoside E. The structure of 1 is identical with that reported very recently by Abe et al. [13].

Compound 2 showed the quasi-molecular ion peak m/z 563 [M + H] + indicating an increase of two mass units in comparison with 1. The IR, iH NMR and i3CNMR spectra (Tables 1 and 2) for 2 were very similar not only to

Cardenolide glycosides from Anodendron afine 299

Table 2. ‘H NMR spectral data for cardenolides I,2 and 4 (400 MHz in CJX&, Sppm from TMS)

H 1 2 4

la

18 2 3 4a 48 5 6a 68 la 78 8 9 11 15a 15s 16U 168 17 18 19 2la 218 22 1’ 3 4’a

4’8 5 6 OMe OH-11 OH-14 OH-2

1.69 dd (13.4,12.0)* 240 dd (13.4,4.3) 4.15 ddd (12.0,9.7,3.4) 4.55 dd (9.7,1.0) 5.31 d (1.0) - -

2.18 m 218 m 1.15 m 2.20 m 2.04 ddd (12.4,11.2,3.4) 1.28 dd (11.2,11.2) 4.47 dd (11.2,3.7) 1.3Om 1.62 m 2.01 m 1.85 m 4.14 dd (9.0,3.2) 1.09s 1.37 s 4.77 dd (18.1,1.7) 4.91 dd (18.1,1.5) 5.99dd (1.7,1.5) 4.11 s 3.30 dd (2.7,2.7) 1.69 m 1.82 ddd (11.0,2.7,1.5) 3.92 dqd (11.0,6.3,1.5) 1.22 d (6.3) 3.40 s 3.65 d (3.7) 3.48 s 3.71 s

1.44dd (11.7,11.7) 234 dd (11.7,2.7) 4.06ddd (11.7,11.7,2.7) 3.97 ddd (11.7,9.7,2.7) 1.68 m 1.79 m 1.80 m 1.56 m 1.92 m 1.6Om 1.46m 1.53 m 1.22 dd (10.7,10.7) 4.48 d (10.7) 1.62 m 1.81 m 2.01 m 1.78 m 4.23 dd (9.5,4.6) 1.08 s 1.23 s 4.76 dd (18.0,1.7) 4.94 dd (18.0,1.7) 5.97 t (1.7) 4.65 s 3.26 dd (2.4.2.4) 1.62 m 1.80 m 3.91 dqd (11.0,5.6,1.2) 1.22 d (5.6) 3.38 s

2.24 8

3.39 s

1.48 dd (13.2,13.2) 2.41 dd (13.2,3.6) 4.10ddd (13.2,8.9,3.6) 4.51 ddd (8.9,2.0,2.0) 5.38 dd (2.2,20) - -

2.56 dd (17.3,5.6) 2.84 ddd (17.3,2.0,2.0) -

3.51 d (5.6) -

1.78 d (12.9) 4.79 dd (12.9,3.6) 1.94 d (3.4) 1.94 d (3.4) 4.33 dt (5.1,3.4) -

3.79 d (5.1) 1.15 s 1.46s 4.95 dd (18.3,1.7) 5.05 dd (18.3,1.7) 6.37 t (1.7) 4.51 s 3.29 dd (2.4.24) 1.65 ddd (13.2,13.2,24) 1.85 ddd (13.2,2.4,2.4) 3.92 dqd (13.2,6.3,24) 1.23 d (6.3) 3.41 s 3.60 d (3.6)

3.20 s

*Coupling constants (.I in Hz) are given in parentheses.

Fig. 2. Relative stereockmistry for 1 on the basis of NOB indicated by arrows.

Fig. 1. Dotted lines indicate the connectivites of the partial stkctures infermd from ‘H-‘H and lH-13C COSYs. krows denote the correlation between protons (trail) and carbons (bead)

derivative of 1. In fact, extensive analyses of various

in the HMBC (Jc_” = 8 Hz). 2D NMR experiments (DQFCOSY and HMBC) supported this structure. The stereochemistry for the ring junction between A and B rings was assigned as cis on the

those of 1 except for disappearance of the A 4*s double basis of chemical shift value (20.7 ppm) for Me-19. This

bond present in 1 but also to those of alkoside E (3) was consistent with the foilowing diagnostic “C NMR except for the C ring. Acetylation of 2 afforded a mono- shift values for Me-19 in cardenolides fused cis between A acetate 2a. These data suggest that 2 is the 4,5-dihydro- and B rings such as afGnosides C and E, Me-19 carbon

300 Y. FUKUYAMA et al.

signal appeared at lower field than 20 ppm [7, lo], where- as A/% trans cardenolides such as gomphoside [14] and desgluwsyrioside [lS] showed the carbon signal due to Me-19 at around 13 ppm. In addition, the observation of NOES for the H-5 (S, 1.80) and H-4/? (6ul.79) upon irradiation of Me-19 supported the cis A/% fused ring. Accordingly, 2 can be represented by 12-oxo-affinoside E.

Compound 4 showed the quasi molecular ion peaks at m/z613[M+Na]+and591[M+H]+onpositiveFAB- MS giving the molecular formula C,,H,,O,,. Its IR spectrum displayed the absorptions attributable to a butenolide moiety (1780, 1740 and 1620 cm-‘) and hydroxyl groups (3450 cm-‘) two of which could be acetylated (Ac,O/pyridine) to yield a diacetate 4a. The 13C NMR data (Table 1) of 4 were almost identical with those of affinoside A (5) [7] from the same plant, except for the carbon signals on the D ring, in particular, the presence of an oxygenated methine carbon resonance at Sc 73.8. This suggested that C-15 or C-16 is hydroxylated. In fact, the extra carbinyl methine signal (6,4.33) was shifted down field to 6,5.04 on acetylation. Moreover, ‘H-‘H COSY experiment provided a connectivity of C,,(H,)-C,,(H)(OH)-C,,(H) which was further verified to be involved in the D ring by the correlation of the Me- 18 and H-17 signals with the C-17 signal and C-22 signal on the butenolide ring, respectively, in HMBC. The hydroxyl group attached at C-16 should be taking a fi- configuration due to the observation of NOE between H- 17 and H-16, and no NOE detection for H-16 upon irradiation of the Me-19 signal. The remaining 3-0- methyl-4,6-dideoxyhexosulose moiety and the stereochemistry of the functional groups on A, B and C rings were substantiated to be identical with those of affinoside A (5) by spectral comparison of both compounds in addition to the data obtained from HMBC and difference NOE experiments. Hence 4 is 16fl-hydroxyaffinoside A.

The EDSo values for 50% growth inhibition of the cardenolide glycosides 16, isolated from Anodendron afine, against the 2nd larvae of Bombyx mori, are sum- marized in Table 3. Affinoside A (S), 4,5-dehydro-12-oxo- afhnoside E (l), and afflnoside M (6) exhibited potent growth inhibitory activity, but compounds 2-4 showed no inhibitory activity even at a concentration as high as 10 ppm though they have structural units similar to those of the active cardenolides. The larvae of Monarch butterflies are known to utilize some cardenolides as defence substances against vertebrate predators [ 11,121 by feed- ing on the leaves containing abundant cardenolides. By

Table 3. Growth inhibitory activity of the cardenolides l-6 against the 2nd larvae of Bo&yx mori*

1 2 3 4 5 6

J=,,t @pm) 3 > 10 z-10 > 10 1 7.5

*Bioassay was carried out according to Asano’s method [Z]. tThe effective dose for 50% growth inhibition in the 2nd

larvae of the silkworm.

contrast, the present study suggests that the presence of cardenolides in plants can be harmful to certain insects.

EXPERIMENTAL

‘H (4OOMHz) and 13C (1OOMHz) NMR: CDCl, unless otherwise noted, TMS as int. standard. CC: silica gel (Wakogel C-300). TLC: precoated silica gel 60 F,,, and RP-8 FZs4 plates. Spots were visualized by UV (254 nm) and 2% CeSO, in H,SO,. after heating. HPLC: Waters 6000 A for pump and TSK-GEL LS-410 KG (4 10 x 300 mm) for column.

Plant material. The stem and bark of Anode&on a@ne D. was collected in Ishigaki island, Japan and identified by Dr Hiroyuki Murata (Juni-cho, Ibusuki, Kagoshima, Japan). A voucher specimen is deposited at the Herbar- ium of Institute of Pharmacognosy, Tokushima Bunri University.

Extraction and isolation. Dried and powdered stem and bark (3.0 Kg) of A. afine were immersed in MeOH (24 1) at room temp. for 3 weeks. The MeOH extract was evapd in vacua to give a crude extract, which was partitioned between EtOAc and H,O. An EtOAc-soluble portion (21.5 g) was divided by CC on Sephadex LH-20 using MeOH into 4 fractions: Fr. 2 (15 g) was chromatographed on TOYOPEARL HW40F (MeOH) to give 8 fractions: fr. 1 (1.04 gX fr. 2 (1.41 g), fr. 3 (3.99 g), fr. 4 (1.59 g), fr. 5 (840 mg), fr. 6 (1.53 g), fr. 7 (1.48 g) and fr. 8 (842 mg). Fr. 5 (840 mg) was further chromatographed on silica gel using a n-hexane-EtOAc gradient, dividing to 7 fractions. The fourth fraction (49mg) was purified by HPLC (MeOH-H,O, 9: 11) to give athnoside A (5) (14.2 mg). The fifth fraction (157 mg) was purified by CC on silica gel (CHCl,-MeOH, 9: i), followed by HPLC (MeOH-H,O, 1: 1) to give 4,5dehydro- lZoxo-affinoside E (1) (15 mg). Fr. 6 (1.53 g) was again divided by CC on silica gel using a CHCl,-EtOAc-MeOH gradient into 15 fractions. Fr. 13 (331 mg) was purified by CC on silica gel (n- hexane-Me&O, 1: l), followed by HPLC (MeOH-HzO, 1: 1) to afford 12-oxo-attinoside E (2) (33 mg). Fr. 14 (191 mg) was purified by CC on silica gel (CHCl,-MeOH, 49:1), followed by HPLC (MeOH-H,0,9: 11) tdyieldafFmosideE(3)(17.3 mg). Fr. 7 (1.48 g) was again purified by silica gel (CHClz-n- hexane-MeOH gradient) to divide into 12 fractions. Fr. 6 (358 mg) was purified by HPLC on Lobar Si 60 (CHCl,-MeOH, 49: 1) and then ODS (TSK410 GK) to give a@inoside M (6) (25 mg). Fr. 9 (117 mg) was purified by CC on silica gel (n-hexane-Me&O-MeOH, 1: 1 :O.l), followed by HPLC (MeOH-HzO, 1:4) to afford 16/3- hydroxyaffioside A (4) (8.2 mg).

4,5-Dehydro-12-oxo-ajhoside E (1). Amorphous. [ali 12.2” (CHCl,; c 0.27). Positive FAB-MS m/z: 561 [M +H]+. IR v~:“cm- ‘: 3400 (OH), 2850, 1780, 1740 (butenolide), 1700 (C=O), 1620 (C=C). ‘H and “C NMR: see Tables 1 and 2. Acetate la, oil. Positive FAB-MS m/z: 667 [M+Na]+, 645 [M+H]+. ‘H NMR: 6 1.17 (3H, s, H-18), 1.24 (3H, s, H-19), 1.25 (3H, d, J=6.4Hz, H-6’), 2.12 (3H, s, AC), 2.22 (3H, s, AC), 3.30 (3H, s, OMe), 3.85 (1H,ddd,5=11.2,9.0,3.0Hz,H-2),4.04(1H,dqd,5=11.5,

Cardenollde giycosides from Anodendron affiine 301

6.4,2.0Hz,H-5’),4.11(1H,dd,J=7.7,?.7Hz,H-17),4.34 (lH,dd,J=2.1,2.1Hz,H-3’),4.61(1H,dd,~=9.0,1.71&, H-3), 4.74 (lH, dd, J= 17.1, 1.7 Hz, H-21), 4.81 (lH, s, H- l’), 4.85 (lH, dd, 5=17.1, 1.7Hz, H-21), 5.34 (lH, d, J =1.7Hz,H-4),5.47(1H,d,J=l1.7Hz,H-11),5.98(1H,t, J = 1.7 Hz, H-22).

12-Uxo-@noside E (2). Amorphous. [a]1p 36.2” (CHCl,, c 0.11). Positive FAD-MS m/z: 563 [M+H]+, 367 (base peak). IR ~~%m-~: 3450 (OH), 2900,1780, 1740 (bu~olideA 1700 (c--O), 1620 (c--C). ‘H and 13C NMR: see Tables 1 and 2. Acetate 2a, oit. Positive FAB-MS m/z: 605 [M+HJ+, 587 [M+H-H&l+. ‘H NMR: 6 1.08 (3H, s, H-19), l.l6(3H, s, H-18), 1.25 (3H, d,J=6.4 Hz, H-6’), 2.20(3H, s, AC), 2.28 (lH, t,J=2.0 Hz, H-3’), 3.42 (3H, s, OMe), 3.91 (lH, dqd, 5=10.3, 6.4, 1.7 Hz, H-S), 3.97 (lH, ddd, 5=10.5, 8.3, 2.6Hz, H-2), 4.15 (lH, ddd, J=8.3,8.3,4.4 Hz, H-3), 4.64 (lH, s, H-l’), 4.73 (lH, dd, J- 18.0,1.4 Hz, H-21),4.88(1H,dd,J= 18.0, 1.4H~H-21),5.50(1H,d,~~12~0H~H-11~,5.97(1H,~,~ = 1.4 Hz,, H-22).

16~-~yd~o~y~~~si~ A (4). Amorphous. [ali 71.2” (MeGI-& c 0.08). Positive FAB-MS m/z: 613 [M +Na]+, 591 [M+H]+, 573 [M+H-18]+. IR

“,X cHc13 m -

‘: 3450 (OH), 3000, 1780, 1740 (butenolide), 1700 (C=O), 1620 (C=C). ‘H and “C NMR: see Tables 1 and 2. Acetate 3a, oil. Positive FAB-MS m/z: 675 [M fH]+,657[M+H-H1O] +. ‘H NMR: 6 1.21(3H, s, H- 19), 1.27 (3H, s, H-18), 1.26 (3H, d, 5=6.4 Hz, H-6’), 1.70 (lH, dd, J= 14.0, 13.0 Hz, H-lu), 1.70 (lH, m, H-4’), 1.87 (lH, m, H-4’), 1.94 (lH, dd, J=16.6,8.5 Hz, H-15@, 1.99 (3H, s, Ac), 2.17(lH, dd, f= 13.0,3.4 Hz, H-lfl), 2.18 (lH, d,J=l3.7 Hz,H-9),2.24(3H,s,Ac),2.46(1H,dd,5=16.6, 9.3 Hz, H-lSa), 2.56 (lH, dd, J= 17.0, 5.6 Hz., H&), 2.73 (2H, s, 2 x OH), 2.83 (lH, ddd, J = 17.0,2.0,1.2 Hz, H-6@), 3.32(1H, b, J=2.4Hz, H-3’), 3.43 (3H, s, OMe), 3.54(1H, d, J=5.6Hz, H-7), 3.94(1H,dqd, J=lO.O, 6.1, 1.7H2, H- 5’),4.05(lH,ddd, 5=14.0,8.8,3.4 Hz, H-2),4.33 (lH, d, J =7.8 Hz, H-17), 4.57 (lH, ddd, 5=8.8, 2.0, 2.0H2, H-3), 4.71 (lH,s,H-1’),4.81 (lH,dd,J=18.2,2.0Hz,H-21), 5.03

(lH,dd, 5=18.0,2.0Hz, H-21), 5.04(1H,ddd,J=9.3,8.5, 7.8 Hz, H-16), 5.41 (lH, dd, J=2.0, 1.2 Hz, H-4), 5.70 (lH, d, J= 13.7 Hz, H-11), 6.11 (lH, t, J-2.0 Hz, H-22).

Acknowledgement-We thank Dr Hiroyuki Murata for identification of plant material.

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insect growth inhibitory cardenolide glycosides from anodendron affine

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