Acanthoic Acid and other Constituents from the Stem of Annona amazonica (Annonaceae)

O presente trabalho descreve o isolamento, a partir do caule de Annona amazonica, do ácido acantóico, um diterpeno do tipo pimaradieno que possui várias e importantes atividades biológicas descritas na literatura. Neste estudo foi verificado que este composto apresenta significante atividade tripanocida contra as formas epimastigotas de Trypanosoma cruzi. Também foi constatado que este diterpeno é o constituinte majoritário da planta, encontrado em cerca de 65% do extrato hexânico, demonstrando que A. amazonica é uma nova fonte natural renovável desta substância. Além do ácido acantóico, a investigação química resultou no isolamento dos alcalóides liriodenina e cassiticina, entre outros compostos, tais como terpenos, esteróides e ácidos graxos. Adicionalmente, é descrita a completa e inequívoca atribuição dos deslocamentos químicos de RMN de H e C da cassiticina.


Introduction
The family Annonaceae, comprised of tropical and subtropical species with about 135 genera and more than 2500 species and widely distributed in South and Central America, Africa, Asia, and Australia, 1 is known for its edible fruits and the medicinal properties of several species. 2 In Brazil, there are 26 genera with about 260 species, including the genus Annona, which contains approximately 120 species. 3 Chemical investigations of species of Annonaceae have revealed their high chemical diversity in terms of secondary metabolites, such as alkaloids, acetogenins, terpenoids and lactones. These compounds have shown important biological activities, including antiparasitic, in particular against Leishmania sp., Plasmodium falciparum, and Trypanosoma cruzi. [4][5][6][7][8][9][10][11][12] Annona amazonica R.E. Fries is a tropical tree that grows up to 20-25 m tall and 35-60 cm in diameter, and is found from Panama to South America. In Brazil this species commonly occurs in the Amazon, mainly in the states of Amazonas and Pará. 13 To the best of our knowledge, only one previous phytochemical study has described the isolation and identification of cyanogenic constituents from this species. 14 Here we describe the isolation and structural identification of the chemical constituents from the stems of A. amazonica, including acanthoic acid (1), a promising compound with several important biological activities described in the literature. This compound was found to be the principal component of A. amazonica, and showed trypanocidal activity against epimastigote forms of Trypanosoma cruzi, the causative agent of Chagas' disease. In addition, the full NMR analyses of the alkaloid cassythicine (3) are included.

General procedures
1D and 2D NMR experiments were recorded on a Bruker AVANCE 400 spectrometer operating at 9.4 T, equipped with a 5 mm multinuclear direct detection probe with z-gradient, observing 1 H and 13 C at 400. 13 and 100.61 MHz, respectively. 1 H-13 C correlation (HSQC and HMBC) experiments were performed with the average coupling constant 1 J(C,H) and LR J(C,H) optimized for 140 and 8 Hz, respectively. The 1D nOe experiments were obtained by selective excitation of each 1 H NMR frequency and gradient selection using the double-pulsed field gradient spin-echo (DPFGSE)-nOe experiment with constant mixing time of 500 ms. IR spectra were recorded in KBr pellets on a Perkin-Elmer Spectrum 2000 spectrometer. Low resolution ESI-MS and ESI-MS/MS data were taken in positive ion mode, on a Micromass Quatro LC mass spectrometer, equipped with an ESI/APCI "Z-spray" ion source. GC-MS analyses were performed on an Agilent Technologies 6896 N instrument equipped with a HP-5MS (29.6 m × 0.25 mm; 0.25 µm film thickness) fusedsilica capillary-column. The following conditions were used: temperature program: 70 o C for 5 min; 5 o C min -1 until 300 o C; 300 o C for 20 min; injector temperature: 250 o C; carrier gas: helium, adjusted to a linear rate of 32 cm s -1 (measured at 100 o C); injection type: split flow, adjusted to give a 1:20 ratio; septum sweep constant at 1.0 mL min -1 ; EI-MS: electron energy at 70 eV. Melting points were obtained on a Quimis Q-340S23 micromelting apparatus. Optical rotations were measured on a Rudolph Research Autopol III polarimeter. Gravity-column chromatography (CC) was carried out on silica gel 60 (70-230 mesh, Merck), while analytical and preparative thin-layer chromatography (TLC) was run on 0.25 mm thick aluminum-backed silica-gel 60 plates type F/UV 254 and 366 (Merck) and 2 mm thick glass-backed silica gel 60 plates type P/UV 254 (Macherey-Nagel), respectively. The spots were detected by exposure to UV light at 254 or 366 nm, as well as by spraying with Dragendorff's reagent or 5% H 2 SO 4 in EtOH and then heating on a hot plate.

Botanical material
The stems of A. amazonica weres collected in May 2003 in the Adolpho Ducke Forest Reserve (coordinates: 02º54'26'' to 03º00'22"S; 59º52'40" to 59º58'40"W), situated 26 km northeast of the city of Manaus in the state of Amazonas, and identified by a specialist in Annonaceae, Prof. Dr. A.C. Webber. A voucher specimen (# 1796) was deposited in the Herbarium of the Instituto Nacional de Pesquisas da Amazônia (INPA).

Hexane extract: isolation of 1 and GC-MS analysis
The hexane extract yielded large crystals in very high quantity. These were manually collected, washed in hexane, and submitted directly to NMR and other spectroscopic investigations. They were identified as a pimaradiene-type diterpene, (-)-ent-pimara-9(11),15-dien-19-oic acid or acanthoic acid (1, 6.9 g). After the acanthoic acid crystals were removed, 10 mg of the remaining hexane extract was methylated with diazomethane and submitted to GC-MS analyses according to the conditions described in the general procedures, above. The chemical constituents were identified on the basis of their GC retention indices with reference to a homologous series of C 8 -C 26 n-alkanes, as well as by matching their mass spectra with those from the NIST 98 MS and Wiley 7n MS libraries, and by comparing the fragmentation patterns of the mass spectra with those reported in the literature. 15 Dichloromethane extract: isolation of 1-10 TLC investigations indicated the presence of alkaloids in the CH 2 Cl 2 extract by exposure to Dragendorff's reagent. Therefore, this extract was redissolved in CH 2 Cl 2 (100 mL) and subjected to an acid-base extraction with 3% aqueous HCl (6 × 200 mL), resulting in organic CH 2 Cl 2 and aqueous fractions. The organic CH 2 Cl 2 fraction was submitted to solvent removal under reduced pressure to yield a neutral fraction (4.9 g). The aqueous fraction was adjusted to pH 12 with NH 4 OH and submitted to a new extraction with CH 2 Cl 2 (7 × 200 mL). Following this, the CH 2 Cl 2 parts were combined and the solvent evaporated under reduced pressure to yield the alkaloid fraction (0.8 g). 9 Vol. 20, No. 6, 2009 The alkaloid fraction (0.7 g) was initially subjected to silica-gel column chromatography (CC), previously treated with a 10% NaHCO 3 solution 9 and eluted with increasing concentrations of CH 2 Cl 2 in hexane, followed by EtOAc in CH 2 Cl 2 , and MeOH in EtOAc. The eluted fractions (30 mL) were evaluated and pooled by TLC analysis, affording 10 subfractions. Subfraction 5 (200 mg) was further purified by silica gel CC using the same methodology as above, and subsequently on preparative TLC eluted with CH 2 Cl 2 -MeOH (95:05, v/v), resulting in the oxoaporphine alkaloid liriodenine (2, 4 mg), and in the aporphine alkaloid cassythicine (3, 6 mg).
The neutral fraction (4.5 g) was initially subjected to silica-gel CC, and eluted with increasing concentrations of CH 2 Cl 2 in hexane, followed by EtOAc in CH 2 Cl 2 and MeOH in EtOAc. The eluted fractions (10 mL) were evaluated and pooled by TLC analysis, giving 18 groups (A-M). Group F (700 mg) was purified by silica-gel CC eluted with increasing concentrations of CH 2 Cl 2 in hexane, to give five subfractions (If-Vf). Subfraction IIf yielded a mixture of three methyl esters of the fatty acids, oleic, linoleic, and linolenic (4, 5 and 6, 44 mg). Subfractions IIIf-Vf afforded acanthoic acid (1, 385 mg), and the sesquiterpene, caryophyllene oxide (7, 11 mg) after being purified by preparative TLC eluted with hexane-EtOAc (90:10, v/v). Group K (300 mg) was further purified by silica-gel CC eluted with the same gradient system described previously, to give seven subfractions (Ik-VIIk). Subfraction IIIk was purified by preparative TLC eluted with hexane-EtOAc (80:20, v/v) to give a mixture of two steroids, β-sitosterol and stigmasterol (8 and 9, 66 mg). Subfraction IVk yielded a mixture containing acanthoic acid (1, 22 mg). Group N (558 mg) was further purified by using silica-gel CC eluted with the same gradient system described previously, to give eight subfractions. Subfractions Vn and VIIIn yielded one monounsaturated fatty acid, oleic acid (10, 36 mg), and in a mixture of three methyl esters of the fatty acids, oleic, linoleic, and linolenic (4, 5, and 6, 36 mg), respectively. Additionally, all fractions that showed the presence of high amounts of acanthoic acid as evidenced by 1 H NMR analysis were pooled and purified according to the conditions described above, to give 1.8 g of this compound.

In vitro trypanocidal activity assay Parasite
The epimastigote form of Trypanosoma cruzi strain Y was grown in liver infusion tryptose (LIT) supplemented with 10% fetal-calf serum (FCS, Gibco) at 28 ºC for 96 h.

Antiproliferative activity on epimastigote form
The epimastigote form of T. cruzi in the logarithmic phase was used for this assay. Acanthoic acid was redissolved in DMSO and LIT medium to obtain concentrations of 3, 16, 33, 165, and 331 µmol L -1 at the well. The final concentration of DMSO did not exceed 1%. For each experiment, there was a growth control with and without DMSO.
Cells at a density of 1×10 6 epimastigotes mL -1 were cultured in a 24-well microplate to obtain a final volume of 1 mL. The cells were incubated at 28 ºC and their growth was determined by counting the parasites with a hemocytometer chamber after 96 h. The IC 50 value (50% inhibition concentration) was determined using linear regression analysis from the inhibition percentage. These tests were performed in triplicate on separate occasions. 16

Cytotoxicity assay
The cellular toxicity was evaluated against the LLCMK 2 cell line in 96-well plates. The cells were seeded onto microplates at a concentration of 2.5 cells × 10 5 cells mL -1 , and incubated in DMEM supplemented with 10% FCS. The monolayer obtained was treated with different concentrations of acanthoic acid (20; 41; 82; 165; 331; 1655; 3311 µmol L -1 ). DMSO was used as the negative control. After incubation at 37 °C with 5% CO 2 for 96 h, cell viability was evaluated by the sulforhodamine B technique. 17 The absorbance was read at 530 nm in a microplate spectrophotometer (Biotek-Power wave XS). The CC 50 of the substance (the concentration that lysed 50% of the cells) was calculated. These tests were performed in duplicate on separate occasions.

Chemical studies
Members of the genus Annona have a good ability to produce alkaloids, a class of compounds typically found in species of the family Annonaceae. However, A. amazonica produces as its major secondary metabolite, the pimaradiene-type diterpene acanthoic acid (1), as evidenced by the surprising and extraordinary appearance of large glassy prism crystals in very high quantities, practically 65% of the hexane extract, which allowed their direct collection and investigation by spectrometric methods, mainly by NMR analysis, without the need of any additional purification procedure. 8 Hz) that is characteristic for the ring C position ∆ 9(11) of pimaradienetype diterpenes. 18 These findings, confirmed by 13 C NMR data as well as by one-bond and long-range heteronuclear 1 H-13 C correlations from HSQC and HMBC experiments, along with the MS fragmentation pattern, mainly by the ion at m/z 234, attributed to an elimination of 2-methylbutadiene by a retro-Diels-Alder process, allowed us to establish the structure of 1 as acanthoic acid. 18  These results are in full accordance with those previously described for acanthoic acid. 18 Moreover, almost all of the investigated fractions of the CH 2 Cl 2 extract contained high amounts of 1, as evidenced by 1 H NMR analysis. Acanthoic acid (1) was first isolated from Acanthopanax koreanum (Araliaceae) 18 and has also been reported from Mikania sp. (Asteraceae), 19 Rollinia pittieri and R. exsucca (Annonaceae). 20 Therefore, this is the fifth description of 1 in the literature, the first in the genus Annona, and the third in the family Annonaceae, which is important from a chemotaxonomic point of view.
Several important in vitro and in vivo biological activities against a wide range of diseases and medical conditions, mainly antitumor and anti-inflammatory, have been described in the literature for 1. [21][22][23][24][25][26][27][28][29][30][31][32][33] These findings clearly show the medicinal potential of 1. However, almost all of the biological activities described in the literature for 1 have been shown by the compound extracted from Acanthopanax koreanum, which resulted in the protection of the process for its extraction from this plant by an international patent, 21 and has attracted interest in its synthetic preparation and modification. 26,29,34,35 Therefore, according to the results of the present study, a new renewable natural source for Vol. 20, No. 6, 2009 acanthoic acid (1) from Annona amazonica has been demonstrated, which can advance research for new drugs based on this compound.
Because cassythicine (3) was isolated a long time ago, only partial 1 H NMR data are described in the literature. 46 Therefore, this study reports the complete and unequivocal 1 H and 13 C NMR chemical shift assignments for cassythicine 3, as well as their heteronuclear 1 H-13 C correlations, nOe observations, and accurate 1 H-1 H scalar coupling constants ( Table 1). The NMR data for 3 were initially collected in CDCl 3 as the solvent. However, in this solvent it was too difficult to observe both 1 H and 13 C signals of the aliphatic system, probably due to a dynamic process, mainly for the aliphatic moiety, and consequently it was too difficult to identify all the signals, measure the 1 H-1 H coupling constants, and recognize the heteronuclear 1 H-13 C correlations in the HSQC and HMBC experiments. For this reason, cassythicine (3) was redissolved in methanol-d 4 and resubmitted to NMR analysis. In contrast to CDCl 3 , the signals were much more evident in both 1 H and 13 C{ 1 H} NMR spectra obtained in methanol-d 4 and it was possible to recognize all signals in the spectra as well as to perform all NMR chemical shift assignments and accurately measure the 1 H-1 H coupling constants. Moreover, by taking the NMR measurements of 3 in methanol-d 4 it was possible to observe the nuclear Overhauser effect, which was essential for the relative 1 (Table 1). In contrast, in the 1 H NMR spectrum obtained in methanol-d 4 the HO-9 signal was not evident as in CDCl 3 . Here, we report all NMR data obtained in both CDCl 3 and methanol-d 4 for cassythicine (3), which can aid in further elucidation of the chemical structure (Table 1).

Biological studies
Acanthoic acid (1) showed anti-proliferative activity against epimastigote forms of Trypanosoma cruzi. After 96 h this compound considerably reduced the number of parasites, by causing progressive injury compared with untreated cells. A dose-dependent effect was observed, and at this time the IC 50 value was 59 µmol L -1 (Figure 1), whereas the standard drug benznidazole showed an IC 50 of 7 µmol L -1 . 47 Other terpene derivatives showed some trypanocidal activity, such as dehydrozaluzanin C isolated from Munnozia maronii   (Asteraceae), that showed IC 90 between 10-205 µmol L -1 against different strains of T. cruzi. 48 The potential toxic effect of 1 on the LLCMK 2 cell line was also evaluated. After 96 h of treatment, the 50% cytotoxic concentration was 347 µmol L -1 ( Figure 2). Therefore, by the selectivity index (SI) ratio (CC 50 for LLCMK 2 /IC 50 for parasite), acanthoic acid (1) was more selective against the parasite than against mammal cells, with a SI of 5.9.
Because of the present inefficient chemotherapy available for Chagas' disease, new active compounds need to be found, and natural products may play an important role. Some previous studies have demonstrated the therapeutic potential of compounds derived from natural sources. 16,[48][49] Several synthetic compounds have also shown trypanocidal activity. 50,51 Acanthoic acid (1) at 3 µmol L -1 (the lowest concentration tested) displayed a growth inhibition of parasites of 13.6%, while at the highest concentration (331 µmol L -1 ), over 90% of the cells were affected. Therefore, the 50% inhibition concentration was determined at 59 µmol L -1 . In contrast, the toxic effect on mammalian cells occurs only in high concentrations, with a CC 50 of 347 µmol L -1 , showing that 1 is 5.9 times more toxic to the epimastigote forms of T. cruzi than the LLCMK 2 cell line.

Conclusions
Our results demonstrate that Annona amazonica, in addition to containing alkaloids, a class of compounds typically found in species of the family Annonaceae, produces as its major secondary metabolite, the pimaradienetype diterpene, acanthoic acid (1). Therefore, this plant is a new renewable natural source of this promising compound for drug development. Knowledge of the medicinal potential of 1 was augmented by demonstrating its trypanocidal activity, together with its low toxicity. In addition, the complete and unequivocal 1 H and 13 C NMR chemical shift assignments for cassythicine are now available.