Limonoids from Cipadessa fruticosa and Cedrela fissilis and their Insecticidal Activity

Ana C. Leite, Fabiana C. Bueno, Cintia G. Oliveira, João B. Fernandes*, Paulo C. Vieira, M. Fátima das G. Fernandes da Silva, Odair C. Bueno, Fernando C. Pagnocca, M. José A. Hebling and Maurício Bacci Jr. Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos SP, Brazil Centro de Estudos de Insetos Sociais, Universidade Estadual Paulista, CP 199, 13506-900 Rio Claro SP, Brazil


Introduction
Cipadessa fruticosa Blume (Meliaceae) is widely cultivated in the southwest of China.This plant has been reported to contain ent-clerodanes and labdanes diterpenoids, 1,2 limonoids, sterols, sesquiterpenoids, heneicosene derivatives and one coumarin. 3,4We have recently reported the isolation of six mexicanolide limonoids from this plant. 5edrela fissilis Vell.(Meliaceae) is a valuable tree of timber industry.In Brazil, it can be found from Amazon forest up to north of Espírito Santo State.Previous investigation of fruits and seeds from this species afforded the limonoids fissinolide, mexicanolide and 3β-hydroxyisomexicanolide. 6 In a recent investigation we have isolated several gedunin limonoids from C. fissilis. 7xtracts and fractions from C. fruticosa and C. fissilis have been shown activity on leaf-cutting workers Atta sexdens rubropilosa. 8,9Thus, the aim of this work was the chemical investigation of these Meliaceae species associated with biological assays against Atta sexdens rubropilosa.The study of the fruits of C. fruticosa allowed the isolation of a new mexicanolide limonoid: cipadesin B (1) and the known swietemahonolide (2).The limonoid 3β-acetoxycarapin (3) isolated from fruits of C. fissilis, is new as natural product and its spectral data are being described for the first time.The triterpenes oleanolic acid (4) and oleanonic acid (5) were also isolated from C. fissilis.The evaluation of the activity on leaf-cutting ants of these compounds, along with the following mexicanolide limonoids previously isolated from C. fruticosa: cipadesin A (6), ruageanin A (7), cipadesin (8), khayasin T (9), febrifugin (10) and mexicanolide (11) 5 was performed.

Plant material
The roots of Cedrela fissilis Vell.were collected in São Carlos-SP, Brazil in 01/06/01 and identified by Dr. Maria Inês Salgueiro Lima from the Department of Botany, Universidade Federal de São Carlos, where a voucher specimen (6701) was deposited.The fruits of Cipadessa fruticosa Bl. were collected in Viçosa, Minas Gerais, Brazil, and a voucher specimen (110.664) was deposited in the SPF Herbarium of Instituto de Ciências Biológicas-USP, São Paulo, Brazil.

Bioassay
The A. sexdens rubropilosa workers used in the assays were randomly removed from laboratory nests.They had a body mass of 20-25 mg.Before the assays the nests were supplied daily with leaves of Eucalyptus sp., oat seeds and occasionally with leaves of other plants such as Hibiscus sp., Ligustrum sp. or rose petals.Fifty ants were removed from the nests and put into five Petri dishes (ten ants each) for each treatment.During the assays the ants were maintained on an artificial diet prepared with glucose (50 g L -1 ), bacto-peptone (10 g L -1 ), yeast extract (1.0 g L -1 ) and agar (15 g L -1 ) in distilled water (100 mL). 23The diet (0.4-0.5 g per dish) with the addition of compounds (experiment) or without (control) were offered daily in a small plastic cap.The control was prepared with the diet and the solvent.To ensure that undetectable remaining amounts of the solvent did not affect the ants, a comparison was made with another set of dishes in which water was used instead of solvent.As expected, the same survival rates were obtained with both systems (data not shown).The compounds were poured into the hot diet immediately after it was autoclaved.The final concentration of the compounds added to the diet was 100 μg mL -1 .During the assays the material was maintained in an incubator at a temperature of 25 (±1) o C and relative humidity ranging between 70-80%.The maximum length of observation was 25 days and the number of dead ants was registered daily.
The survival median 50% (S 50 ) was calculated and survival curves were compared by the computer-assisted software Graph-Pad TM using the log-rank test.

Results and Discussion
Compound 1, isolated from fruits of C. fruticosa, had a molecular formula C 32 H 42 O 11 determined from the pseudo-molecular ion peak at m/z 601 (M -H) -in the negative ESI mass spectrum and elemental analysis.The 1 H and 13 C NMR spectral data (Table 1) of 1 are similar to that cipadesin A, a mexicanolide-type limonoid previously isolated from this plant. 5They differed to each other only with respect to the γ-hydroxybutenolide moiety at C-17 of 1.The 1 H NMR spectral data (Table 1) of limonoid 1 indicated the presence of four tertiary methyl groups (δ H 0.79, 0.80, 1.05 and 1.06), one methoxyl group (δ H 3.72), two signals characteristic of carbinolic hydrogen (δ H 5.09, brs, H-17 and δ H 5.10, d, J 8.9 Hz, H-3) and of the proton on the epoxide ring (δ H 3.30, d, J 1.9 Hz, H-30).
In the HMBC correlation map, the signal at δ C 214.3 (C-1) showed correlations with the signals at δ H 1.06 (s, H-19, 3H), 3.30 (d, J 1.9 Hz, H-30) and 3.56 (m, H-2).It was observed long-range correlations of δ The 2-methylbutyryloxyl ester moiety at C-3 was characterized by the signals at δ H 2.58 (m, H-2'); 1.55 and 1.76 (m, H-3'); 0.97 (t, J 7.5 Hz, H-4') and 1.25 (d, J 7.0 Hz, H-5') and which showed correlations in the HSQC correlation map with δ C 41.5 (C-2'), 26.7 (C-3'), 17.3 (C-4') and 11.9 (C-5'), respectively.The presence of this group in 1 was confirmed by comparison of its spectral data with those published for swietenin E, isolated from Swietenia mahagoni. 10Its β-orientation was defined by the large coupling constant of H-3 (J 8.9 Hz). 11he characteristic signals for a furan ring at C-17, typical for limonoids of Meliaceae, were not observed in the 1 H and 13 C NMR spectral data of 1.However, it showed two broad one-proton singlets at δ H 6.19/6.25 (H-23) and 7.34/7.37(H-22), which showed cross peaks in the 1  Compound 3 was isolated from roots of C. fissilis and its complete spectral data are being described for the first time.3][14] It showed the pseudo molecular ion peak  15 The position of the trisubstituted olefin at ∆ The mexicanolide limonoid swietemahonolide (2), 16 from fruits of C. fruticosa and the triterpenes oleanolic acid (4) 17 and oleanonic acid (5), 18 from roots of C. fissilis were characterized by comparison of their NMR spectral data with those previously reported in the literature.
Recently, we reported the biological activity of extracts and fractions of C. fissilis and C. fruticosa against the leaf-cutting ants Atta sexdens rupropilosa. 8,9These studies showed that the fruits of C. fruticosa and roots of C. fissilis were active.In the continuation of these studies, the compounds 1-5 and six mexicanolide limonoids previously isolated from fruits of C. fruticosa (6-11) 5 were evaluated on the ants.
The results (Table 2) showed that all compounds assayed presented significant difference as compared to the control, according to the log-rank test (p < 0.05), which consider all tested period.In a recent work, we verified that several gedunin limonoids isolated from roots and leaves of C. fissilis were not active on A. sexdens rubropilosa workers. 7Thus, the insecticidal activity of C. fissilis seems to be related to the presence of the oleanane triterpenoids.The activity previously verified for fruits of C. fruticosa 9 is probably due to the presence of mexicanolide-type limonoids, since these compounds showed significant activity on ants.
Limonoids have attracted considerable interest because of their biological properties.Indeed, C-seco limonoids, such as azadirachtin, are the most active insecticidal compound, possessing both antifeedant and growthregulating activities. 19,20The mexicanolide limonoids have been showed high activity against insects of Lepidoptera order, 15,21 but this is the first report of their activity on ants.The activity of oleanane triterpenes on leaf-cutting ants was already reported, showing their repellent action on Atta cephalotes workers in a feeding preferences assay. 22he results presented here suggest that the limonoids and triterpenes isolated from C. fissilis and C. fruticosa are promising in controlling leaf-cutting ants.In continuation of this research, these active compounds should be further evaluated in the field assays against leacutting ants.

Table 1 .
13 and13C NMR spectral data for compounds 1 and 3 in CDCl 3 .Resonances for 1 and 3 were confirmed by 1 H-1 H COSY, HSQC and HMBC spectra. Cog constants (J in Hz) in parentheses Na] + , in the positive ESI mass spectrum, according to the molecular formula C 29 H 36 O 8 , which was confirmed by elemental analysis.Their 1 H and13C NMR spectral data (Table1) indicated that it was also a mexicanolide-type limonoid.The signals at δ H 7.49, 6.43 and 7.42 in the 1 H NMR spectra indicated the presence of a β-substituted furan-ring.The signal at δ H 5.78 (d, J 2.2