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An insect antifeedant limonoid from seed of khaya ivorensis
Phytochemistry, Vol. 31, No. 9, pp. 30034004,
Printedin Great
1992
0031~9422,92 $5.00+0.1X3 Q 1992 PergamonPressLtd
Britain.
AN INSECT ANTIFEEDANT LIMONOID FROM SEED OF
KHAYA
IVORENSIS
C. VANUCCI,C. LANGE,G. LHOMMET,*B. DUWNT,~ D. DAVOUST,~B. VAUCHOT,~J. L. CLEMENT~and F. BRUNCK~ Universitk P. et M. Curie, Laboratoire de Chimie Organique Structurale, AssoeiCau CNRS, 4 Place Jussieu, 752252Paris cedex 05, France; tUnivetsite de Rouen, Laboratoire des Composes Az.ot&set Oxygen&s,Service RMN, Associt au CNRS, 76134 Mont-StAignan, France; fCNRS, UPR 27, ~o~unication Chimique, 31 Chemin J. Aiguier, 13402 Marseille eedex 09, France; $CTFT, Dkpartement Forestier du CIRAD, 45 Av. de la BelIe Gabrielle, 94736 Nogent-sur-Mame, France (Received 28 Nouember 1991) Key Word Index-Khayu
iuorensis;Meliaceae; seed; limonoid; antifeedant; inseet.
Abstract-A novel limonoid which is an insect antifeedant has been isolated from a methanolic extract of seeds of Khaya ivorensis. Its structure was determined on the basis of one and two-dimensional NMR.spectroswpy and
chemical evidence.
In our search for new natural insecticides, the observation of an antifeeding activity of seeds of Khuyu ivorensis was
of interest. This led us to identify the principles responsible by separating and testing of the natural substances contained in seed extracts. We report the isolation and structural elucidation of a novel limonoid (la) from the seeds of Khaya ivorensis.
a-
1
O-R
RESULTSAND DIS0.JSSION The IR spectrum of la (CHBr,) showed absorptions in particular at 2940,172O (ester), 1500 and 875 (furan ring) cm-l. Its mass spectrum exhibited a [MH]+ ion at m/z 541 in the positive mode and [M - H] - ion at m/z 539 in the negative mode. They correspond to a M, of 540 with a molecular formula of C,,H,,Os according to the HR mass spectrum (CM]” 540.2716; talc. 540.2723). The EI, PICI and MCI mass spectra provided much structural information. Under EI-MS conditions, the loss of 96 led to m/z 444, which can be attributed to the loss of FuCOH having regard to the IR spectrum. The ion at m/z 416 resulted from the loss of CO from m/z 444. PICIMS (NH:) showed the presence of a Me-CH,-CH,COOsubstituant as indicated by the [MI-i - MeCH,CH2C0J%+ ion at m/z 453. This su~tituant was also observed under NICIMS (NH;) at m/z 451 @i - H - MeCH,CH,CO,m - . The structural anion at m/z 309 corresponds to the cleavage of the moiety including the MeCH,CH,CO, and MeOCO groups, as shown in Scheme 1. The ‘H NMR spectrum indicated the presence of four tertiary methyl groups (60.68, 0.78, 1.08 and l.lS), one methoxy singlet (63.66), three downfield shifted signals attributable to a p-substituted furan ring (67.52; 7.38 and 6.451, two signals characteristic of protons attached to a carbon adjacent to an oxygen atom (65.65, sand 4.95, d, J *Author to whom correspondence should be addresscd.
scheme
1. Fragmentation ions observed under EIMS, PICIMS and NICIMS.
= 10 Hz) and a triplet methyl group (60.95, J = 7 Hz). Some molecular fragments were established using homonuclear bidimensional ‘H NMR techniques (COSY, COSY RCT, long-range COSY). Thus, the observed correlations between the proton signals at 57.52, 7.38, 6.45 and 5.65 determined the position of the furan ring. In the same way, the sequence H-3-H-2-H-30 and H-30’ emerged from the correlations between ‘H signals at 64.95,3.13 and 2.75-2.10, while the correlations between the ‘H s&nals at 60.95, 1.66 and 2.38 (Me-27, CH,-26, CH,-25) showed the presence of a propyl chain. Moreover, the ‘H-“H long-range correlation experiment showed that the proton signals at 60.68 and 0.78 can be attributed to a gem&methyl moiety and that the methyl group at S 1.08 (Me-18) is attached to a carbon adjacent to the proton signal at 6 1.75 (CH-12) (long-range correlation due to a trrms-diaxial geometry) [l]. The 13CNMR
3003
3004
C. VANUCCI et al.
spectrum showed the presence of a ketone (6218.1) and three carboxylic groups (6 174.1, 173.1 and 169.9). The total assignment of the 13C signals as well as of the remaining non attributed ‘H shifts was established by heteronuclear bidimensional techniques such as 13C-lH COSY and ’ 3C-‘H long-range COSY (COLOC), the 13C parameters having been obtained through the observation of the proton (inverse detection). The first technique allowed us to attribute to each proton the carbon it is attached to, whereas the COLOC experiment led to the determination of the molecular skeleton, all the quaternary carbons being unambigously assigned. Thus, the observed correlation between the “C signal at 6174.2 and the ‘H signals at 62.38 and 3.18 established the sequence CO-7-CH,-6-CH-5. In the same way, the existence of correlations between the methyl proton at 61.15 (Me-19) and the “C signals at 652.9,52.1 and 40.8 determined the fragment C-10 = [C-9, C-S, Me-191. The complete and unambiguous ‘H and 13C assignments are reported in Table 1. On the basis of this spectroscopic evidence, compound la was then shown to be the la-butyric derivative of the known limonoid methyl 3P-hydroxy-l-oxomeliac8(14)-enate (lb), also named proceranolide [;?I. It should be noted that this compound was also isolated from seed of Kkaya iuorensix (unpublished observations). In order to confirm the proposed structure, and in particular the relative stereochemistry, the n-butyrate of proceranolide was prepared and shown to be identical to la by IR and NMR spectroscopic comparison. The latter can thus be named methyl 3P-nbutyryloxy-1-oxomeliac-8( 14)-enate c31. Compound la showed a good antifeeding activity against the larvae of the lepidopteran Agrotis segetum. For a concentration of 100 ppm incorporated into the
Table
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1. ‘H and 13CNMR
S,
3.13 4.95 3.18 2.38 2 2.03 1.67 1.05 1.75 3.45 3.70
spectral CDCI,)
data for compound
6,
Position
218.1 s 48.0 d 78.1 d 38.2 s 40.8 d 33.4 t 174.1 s 127.7 s 52.1 d 52.9 s 18.7 t 29.0 t
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
38.1 s 131.7 s 33.3 t
31
6,
5.65 1.08 1.15 7.52 6.45 7.38 2.38 1.66 0.95 0.78 0.68 2.10 2.75 3.66
la (in
& 169.9 80.7 17.7 16.7 120.6 141.7 109.9 142.8 173.1 36.3 18.5 13.8 20.5 23.2 33.3
s d q q s d d d s
t t q q q t
52.1 q
artificial feeding medium during five days, the percentage of the difference of growth for each tested sampling compared with the control was 56% (0.99 signification level for the t-test of Student and Fisher, n = 30). No mortality was observed. The percentage of the difference of food consumed by each larva compared with the control was 36% (0.99 signification level for the t-test). In the same test, azadirachtin, one of the most efficient antifeedants of plant origin [4], exhibited a growth inhibition of 93% and a mortality of 60%. EXPERIMENTAL General. Mp: uncorr. EI and CIMS were obtained with a quadrupole mass spectrometer. NMR: CDCI, was used as the solvent and TMS as the int. reference. Plant material. Seeds of Khaya ivorensis were collected in the Ivory Coast and authenticated by the CTFT of Yapo Sud. They were stored in closed containers at 4”. Extraction and isolation. Seeds (150 g) of Khaya iuorensu were finely ground and extracted with MeOH (5 1)at room temp. for 4 days. The coned extracts were washed with n-hexane and evapd to give a brown semi-solid residue (15 g). CC on silica gel using nhexane-EtOAc (3:2) as the eluting solvent led to the isolation of compound la (ca 8% in weight related to the crude extract) as a solid. Compound la. Mp 65-68” [a]~” - 135” (CHCI,; c 1.025). EIMS m/z (rel. int.): 43 (100); 56 (40); 71 (25); 117 (12); 149 (6); 209 (5); 299 (3); 328 (3); 357 (3), 416 (7); 444 (3); 540 (1). PICIMS m/z (rel. int.): 558 ([M+NH,]+, 25); 541 ([MH]+, 100); 453 (25). NICIMS m/z (rel. int.): 539 ([M-q-, 100), 451 (90), 450 (50), 309 (75). IR v,,,*, cm- I: 2940,1720,1455,1480,1295,1245,1165,1090, 1025,990, 875, 755. 1H and 13C NMR: see Table 1. Ester@cation ofcompound lb. Proceranolide (lb) (0.25 g) was esterikd with n-butyric anhydnde in the presence of pyridine. Purification by TLC on silica gel (EtOAc-hexane, 1: 1) of the crude product yielded the n-butyrate (0.09 g, 31%). Antgeedant bioassays. The compound was dissolved in CH,CI, and incorporated into the artificial feeding medium of larvae of the lepidopteran Agrotis segetum at a concn of 100 ppm. Thirty 6-day-old larvae were individually reared and weighed at the beginning of the test and after a 5-day diet. The nutritious medium was equally weighed. The antifceding activity was determined by calculating the ratio of the difference of growth of the tested larvae compared with the control. The average quantity of food consumed by each larva was equally evaluated and the ratio of the difference observed between the tested and control sampling compared.
REFERENCES 1. Platzer, N., Goasdoue, N. and Davoust, D. (1987) Magn. Reson. C/tern. 25, 3 11, 2. Sodengam, B. L., Kanga, C. S. and Connoly, J. D. (1980) Phytochemistry 19,2488. 3. Adegosan, A. K. and Taylor, D. A. H. (1974) J. Chem. Sot. C. 1968. 4.
Zanno, P. R., Miura, I. and Nakanishi, K. (1975) J. Am. Chem. sot. 97, 1975.
Printedin Great
1992
0031~9422,92 $5.00+0.1X3 Q 1992 PergamonPressLtd
Britain.
AN INSECT ANTIFEEDANT LIMONOID FROM SEED OF
KHAYA
IVORENSIS
C. VANUCCI,C. LANGE,G. LHOMMET,*B. DUWNT,~ D. DAVOUST,~B. VAUCHOT,~J. L. CLEMENT~and F. BRUNCK~ Universitk P. et M. Curie, Laboratoire de Chimie Organique Structurale, AssoeiCau CNRS, 4 Place Jussieu, 752252Paris cedex 05, France; tUnivetsite de Rouen, Laboratoire des Composes Az.ot&set Oxygen&s,Service RMN, Associt au CNRS, 76134 Mont-StAignan, France; fCNRS, UPR 27, ~o~unication Chimique, 31 Chemin J. Aiguier, 13402 Marseille eedex 09, France; $CTFT, Dkpartement Forestier du CIRAD, 45 Av. de la BelIe Gabrielle, 94736 Nogent-sur-Mame, France (Received 28 Nouember 1991) Key Word Index-Khayu
iuorensis;Meliaceae; seed; limonoid; antifeedant; inseet.
Abstract-A novel limonoid which is an insect antifeedant has been isolated from a methanolic extract of seeds of Khaya ivorensis. Its structure was determined on the basis of one and two-dimensional NMR.spectroswpy and
chemical evidence.
In our search for new natural insecticides, the observation of an antifeeding activity of seeds of Khuyu ivorensis was
of interest. This led us to identify the principles responsible by separating and testing of the natural substances contained in seed extracts. We report the isolation and structural elucidation of a novel limonoid (la) from the seeds of Khaya ivorensis.
a-
1
O-R
RESULTSAND DIS0.JSSION The IR spectrum of la (CHBr,) showed absorptions in particular at 2940,172O (ester), 1500 and 875 (furan ring) cm-l. Its mass spectrum exhibited a [MH]+ ion at m/z 541 in the positive mode and [M - H] - ion at m/z 539 in the negative mode. They correspond to a M, of 540 with a molecular formula of C,,H,,Os according to the HR mass spectrum (CM]” 540.2716; talc. 540.2723). The EI, PICI and MCI mass spectra provided much structural information. Under EI-MS conditions, the loss of 96 led to m/z 444, which can be attributed to the loss of FuCOH having regard to the IR spectrum. The ion at m/z 416 resulted from the loss of CO from m/z 444. PICIMS (NH:) showed the presence of a Me-CH,-CH,COOsubstituant as indicated by the [MI-i - MeCH,CH2C0J%+ ion at m/z 453. This su~tituant was also observed under NICIMS (NH;) at m/z 451 @i - H - MeCH,CH,CO,m - . The structural anion at m/z 309 corresponds to the cleavage of the moiety including the MeCH,CH,CO, and MeOCO groups, as shown in Scheme 1. The ‘H NMR spectrum indicated the presence of four tertiary methyl groups (60.68, 0.78, 1.08 and l.lS), one methoxy singlet (63.66), three downfield shifted signals attributable to a p-substituted furan ring (67.52; 7.38 and 6.451, two signals characteristic of protons attached to a carbon adjacent to an oxygen atom (65.65, sand 4.95, d, J *Author to whom correspondence should be addresscd.
scheme
1. Fragmentation ions observed under EIMS, PICIMS and NICIMS.
= 10 Hz) and a triplet methyl group (60.95, J = 7 Hz). Some molecular fragments were established using homonuclear bidimensional ‘H NMR techniques (COSY, COSY RCT, long-range COSY). Thus, the observed correlations between the proton signals at 57.52, 7.38, 6.45 and 5.65 determined the position of the furan ring. In the same way, the sequence H-3-H-2-H-30 and H-30’ emerged from the correlations between ‘H signals at 64.95,3.13 and 2.75-2.10, while the correlations between the ‘H s&nals at 60.95, 1.66 and 2.38 (Me-27, CH,-26, CH,-25) showed the presence of a propyl chain. Moreover, the ‘H-“H long-range correlation experiment showed that the proton signals at 60.68 and 0.78 can be attributed to a gem&methyl moiety and that the methyl group at S 1.08 (Me-18) is attached to a carbon adjacent to the proton signal at 6 1.75 (CH-12) (long-range correlation due to a trrms-diaxial geometry) [l]. The 13CNMR
3003
3004
C. VANUCCI et al.
spectrum showed the presence of a ketone (6218.1) and three carboxylic groups (6 174.1, 173.1 and 169.9). The total assignment of the 13C signals as well as of the remaining non attributed ‘H shifts was established by heteronuclear bidimensional techniques such as 13C-lH COSY and ’ 3C-‘H long-range COSY (COLOC), the 13C parameters having been obtained through the observation of the proton (inverse detection). The first technique allowed us to attribute to each proton the carbon it is attached to, whereas the COLOC experiment led to the determination of the molecular skeleton, all the quaternary carbons being unambigously assigned. Thus, the observed correlation between the “C signal at 6174.2 and the ‘H signals at 62.38 and 3.18 established the sequence CO-7-CH,-6-CH-5. In the same way, the existence of correlations between the methyl proton at 61.15 (Me-19) and the “C signals at 652.9,52.1 and 40.8 determined the fragment C-10 = [C-9, C-S, Me-191. The complete and unambiguous ‘H and 13C assignments are reported in Table 1. On the basis of this spectroscopic evidence, compound la was then shown to be the la-butyric derivative of the known limonoid methyl 3P-hydroxy-l-oxomeliac8(14)-enate (lb), also named proceranolide [;?I. It should be noted that this compound was also isolated from seed of Kkaya iuorensix (unpublished observations). In order to confirm the proposed structure, and in particular the relative stereochemistry, the n-butyrate of proceranolide was prepared and shown to be identical to la by IR and NMR spectroscopic comparison. The latter can thus be named methyl 3P-nbutyryloxy-1-oxomeliac-8( 14)-enate c31. Compound la showed a good antifeeding activity against the larvae of the lepidopteran Agrotis segetum. For a concentration of 100 ppm incorporated into the
Table
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1. ‘H and 13CNMR
S,
3.13 4.95 3.18 2.38 2 2.03 1.67 1.05 1.75 3.45 3.70
spectral CDCI,)
data for compound
6,
Position
218.1 s 48.0 d 78.1 d 38.2 s 40.8 d 33.4 t 174.1 s 127.7 s 52.1 d 52.9 s 18.7 t 29.0 t
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
38.1 s 131.7 s 33.3 t
31
6,
5.65 1.08 1.15 7.52 6.45 7.38 2.38 1.66 0.95 0.78 0.68 2.10 2.75 3.66
la (in
& 169.9 80.7 17.7 16.7 120.6 141.7 109.9 142.8 173.1 36.3 18.5 13.8 20.5 23.2 33.3
s d q q s d d d s
t t q q q t
52.1 q
artificial feeding medium during five days, the percentage of the difference of growth for each tested sampling compared with the control was 56% (0.99 signification level for the t-test of Student and Fisher, n = 30). No mortality was observed. The percentage of the difference of food consumed by each larva compared with the control was 36% (0.99 signification level for the t-test). In the same test, azadirachtin, one of the most efficient antifeedants of plant origin [4], exhibited a growth inhibition of 93% and a mortality of 60%. EXPERIMENTAL General. Mp: uncorr. EI and CIMS were obtained with a quadrupole mass spectrometer. NMR: CDCI, was used as the solvent and TMS as the int. reference. Plant material. Seeds of Khaya ivorensis were collected in the Ivory Coast and authenticated by the CTFT of Yapo Sud. They were stored in closed containers at 4”. Extraction and isolation. Seeds (150 g) of Khaya iuorensu were finely ground and extracted with MeOH (5 1)at room temp. for 4 days. The coned extracts were washed with n-hexane and evapd to give a brown semi-solid residue (15 g). CC on silica gel using nhexane-EtOAc (3:2) as the eluting solvent led to the isolation of compound la (ca 8% in weight related to the crude extract) as a solid. Compound la. Mp 65-68” [a]~” - 135” (CHCI,; c 1.025). EIMS m/z (rel. int.): 43 (100); 56 (40); 71 (25); 117 (12); 149 (6); 209 (5); 299 (3); 328 (3); 357 (3), 416 (7); 444 (3); 540 (1). PICIMS m/z (rel. int.): 558 ([M+NH,]+, 25); 541 ([MH]+, 100); 453 (25). NICIMS m/z (rel. int.): 539 ([M-q-, 100), 451 (90), 450 (50), 309 (75). IR v,,,*, cm- I: 2940,1720,1455,1480,1295,1245,1165,1090, 1025,990, 875, 755. 1H and 13C NMR: see Table 1. Ester@cation ofcompound lb. Proceranolide (lb) (0.25 g) was esterikd with n-butyric anhydnde in the presence of pyridine. Purification by TLC on silica gel (EtOAc-hexane, 1: 1) of the crude product yielded the n-butyrate (0.09 g, 31%). Antgeedant bioassays. The compound was dissolved in CH,CI, and incorporated into the artificial feeding medium of larvae of the lepidopteran Agrotis segetum at a concn of 100 ppm. Thirty 6-day-old larvae were individually reared and weighed at the beginning of the test and after a 5-day diet. The nutritious medium was equally weighed. The antifceding activity was determined by calculating the ratio of the difference of growth of the tested larvae compared with the control. The average quantity of food consumed by each larva was equally evaluated and the ratio of the difference observed between the tested and control sampling compared.
REFERENCES 1. Platzer, N., Goasdoue, N. and Davoust, D. (1987) Magn. Reson. C/tern. 25, 3 11, 2. Sodengam, B. L., Kanga, C. S. and Connoly, J. D. (1980) Phytochemistry 19,2488. 3. Adegosan, A. K. and Taylor, D. A. H. (1974) J. Chem. Sot. C. 1968. 4.
Zanno, P. R., Miura, I. and Nakanishi, K. (1975) J. Am. Chem. sot. 97, 1975.