Leaf-Cutting Ants Toxicity of Limonexic Acid and Degraded Limonoids from Raulinoa echinata . X-Ray Structure of Epoxy-fraxinellone

A investigação fitoquímica das raízes de Raulinoa echinata, planta endêmica do Vale do Itajaí/ SC, levou ao isolamento e identificação dos limonóides degradados: fraxinelona, fraxinelonona e epoxi-fraxinelona. O último foi anteriormente isolado dos caules, porém sua configuração relativa não foi resolvida por experimentos de RMN. Este trabalho apresenta a análise de difração de raios-X da epoxi-fraxinelona, descreve o isolamento da fraxinelonona e fraxinelona pela primeira vez em Raulinoa, e a atividade tóxica da primeira sobre formigas cortadeiras (Atta sexdens rubropilosa). Pouca atividade foi observada para o limonóide degradado testado; entretanto, o limonóide ácido limonéxico isolado dos caules, apresentou alta toxicidade contra as formigas cortadeiras, reduzindo consideravelmente a longevidade das mesmas, em comparação com o controle.


Introduction
Raulinoa is a monospecific genus and the species Raulinoa echinata Cowan (Rutaceae) is endemic in the Itajaí Valley, SC, Brazil. 1 This endangered perennial woody shrub is characterized by the presence of spines (popularly know as Cutia-de-espinho) and has only been found in a short interval (1000 m) on the frequently inundated banks of Itajaí river in an approximate altitude of 100 m, showing a high degree of adaptation to the environment.
We have previously reported the isolation and biological activities of some compounds of R. echinata. 2 continuation of our search for compounds and natural products to be used in the control of leaf-cutting ants, we have investigated the hexane extracts of roots of R. echinata leading to the isolation of typical rutaceous degraded limonoids: fraxinellone (1), fraxinellonone (2) and new epoxy-fraxinellone (3).The extracts of the stems furnished the known limonin precursor, limonexic acid (4).
Leaf-cutting ants are one of the most serious agricultural pests in South American countries and the biological control of these insects has been the aim of many studies.Their control can be achieved by means of intoxicating the leaf-cutting ants 3 or inhibiting the growth of their symbiotic fungus.
In this paper we describe the isolation, structural determination and evaluation of the activity of degraded limonoids and limonoids as toxicants to the leaf-cutting ants Atta sexdens rubropilosa.

General experimental procedures
NMR spectra: a Bruker DRX-400 spectrometer, operating at 400.13 MHz for 1 H and 100.62 MHz for 13 C was used.2All spectra were run in CDCl 3 using TMS as internal standard.

Single crystal X-ray analysis
Low temperature X-ray diffraction data collection was performed at 120(2) K, on an Enraf-Nonius Kappa-CCD diffractometer equipped with an Oxford Cryosystem liquid N 2 device, using graphite-monochromated MoKα radiation (0.71073 Å).Data were collected up to 52.7° in 2θ, with a redundancy of 4. The final unit cell parameters were based on all reflections.Data collections were made using the COLLECT program; 4 integration and scaling of the reflections were performed with the HKL Denzo-Scalepack system of programs. 5No absorption corrections were applied.
The structure was solved by direct methods with SHELXS-97. 6The model was refined by full-matrix least squares on F 2 with SHELXL-97. 7All the hydrogen atoms were set isotropic and freely refined.Data collections and experimental details for the complexes are summarized in Table 1.The programs SHELXL-97, 7 and ORTEP-3 8 were used within WinGX 9 to prepare materials for publication.Data collections and experimental details for compound 3 are summarized in Table 1.

Atta sexdens bioassay
The toxicity of the compounds to Atta sexdens rubropilosa was determined by ingestion bioassay, according to established protocol described in Bueno et  al. (1997). 14ach treatment consisted of 50 ants distributed in 5 Petri dishes.The leaf-cutting ants were isolated from their nest and fed with an artificial diet.The ants from the treatment received a diet added with epoxy-fraxinellone or limonexic acid, at a concentration of 200 μg.The ants from the control treatment were fed with a diet without this compound.
During 25 days, the number of dead ants for each Petri dish was counted and a new portion of artificial diet given.It was estimated the survival curve of leaf-cutting ants of each treatment and calculated the median longevity.The test used for the statistic analyze of data was the Log rank-test (α=0.05).

Results and Discussion
][12][13] Epoxy-fraxinellone has a quaternary epoxy group attached to C8-C14, instead of the double bond as in fraxinellone.The spectroscopic data published 13 could not furnish clear evidence of the spatial configuration of this moiety.In an attempt to confirm the epoxy group position, nOe difference experiments were performed, but the defining signals were superimposed (in d-chloroform and benzene-d 6 ), frustrating clear conclusions.In order to solve these indistinguishable findings, an X-ray analysis (Figure 1) was performed to confirm the epoxy group position.Single crystal of epoxy-fraxinellone was produced by recrystallization followed by a crystal-growing process.The recrystallization involved dissolution of the material in proper amount of ethyl acetate-acetone followed by drops of water.Suitable crystals were selected and analysed by single crystal X-ray diffraction which established undoubtedly the structure of the compound 3. Figure 1 shows an Ortep 8 type view of 3 showing the thermal ellipsoids and atoms labels.This study shows that the molecular conformation of 3 is fixed by the presence of three main intramolecular interactions (Table 2).The analyses of the crystal packing showed the presence of others three intermolecular interactions in 3 (Table 3).These interactions give rise to the formation of infinite chains along the c axis (Figure 2).All geometrical details of the intermolecular contacts were interpreted as intermolecular interactions on geometrical grounds, 15 and the Table 2 show their selected geometrical parameters.1][12] The EI-mass spectra of both compounds shown to be formed mainly from the molecular ion by the consecutive exclusion of furan-3aldehyde, CH3, HCHO and CO fragments.
As previously reported, crude extracts of leaves and stems as well as fractions of R. echinata showed good inhibitory activity on L. gongylophorus growth, the fungus cultured by the leaf-cutting ant Atta sexdens rubropilosa. 16poxy-fraxinellone showed not be toxic to the leafcutting ants, although limonexic acid displayed a significant reduction on lifetime (11 days) of leaf cutting ants, when compared with the control group (22 days) (Figure 3).
Limonexic acid was also isolated from Dictamnus angustifolius 17 a traditional plant used in China and from Tetradium trichotomum 18 root barks, and together with isolimonexic acid are the main constituents of the fruit of Citrus nippokoreana. 19Despite being known since 1948, 20 no significant reference of its bioactivity was found.
Just a dozen degraded limonoids have been published, mainly from Rutaceae and Meliaceae plants.As the parent limonoids, some of them have been tested for biological activities including antifeeding and growth regulating activities against insects, and some relationship between the structures and biological activity has been studied.

Figure 1 .
Figure 1.ORTEP-3 view of the asymmetric unit of 3, showing the atoms labelling and the 50% probability ellipsoids.

Figure 3 .
Figure 3. Survival curve of leaf-cutting ants for treatment and control, calculated the median longevity.Statistic analyze of data: log rank-test (a=0.05).

Figure 2 .
Figure 2. Intermolecular interactions in 3 that form an infinite chain along the c axis.

Table 1 .
Crystal data and structure refinement of compound 3 Data collection, data processing, structure solution and structure refinement respectively. 11