Natural and semi-Synthetic Clerodanes of Croton cajucara and their Cytotoxic Effects against Ehrlich Carcinoma and Human K 562 Leukemia Cells

O diterpeno clerodano trans-desidrocrotonina (1), constituinte majoritário de Croton cajucara, tem sua ocorrência correlacionada com o uso dessa planta na medicina popular. Investigações fitoquímicas levaram ao isolamento dos metabólitos 1, cajucarinolida (6), isocajucarinolida (7), trans-crotonina (2), trans-cajucarina B (3), cis-cajucarina B (4), transcajucarina A (5), N-metiltirosina, ácido vanílico e ácido 4-hidróxi-benzóico. 6 e 7 foram sintetizados em bons rendimentos através da oxidação de 1 com oxigênio-singlete. Todos os clerodanos foram ensaiados frente a células da leucemia humana K562 e do carcinoma de Ehrlich. Os efeitos inibitórios do crescimento celular foram dependentes da dose para os ensaios com o carcinoma de Ehrlich com IC 50 = 166 μM (1), 164 μM (2), 65 μM (6) e 10 μM (7). Além disso, atividade citotóxica moderada foi observada contra as células da leucemia K562 com IC 50 = 38 μM (3), 33 μM (5), 36 μM (6) e 43 μM (7). Os 6 e 7 semi-sintéticos mostraram resultados semelhantes quando comparados com os correspondentes clerodanos naturais.

proved to possess antiinflammatory activity 11 and 2 showed antiinflammatory, antinociceptive and antiulcerogenic effects. 1,2hrough generations, the use of Croton species in the Brazilian Amazon culture is believed to provide health benefits to its users. 1,12,13However, during the 1990s, several cases of toxic hepatitis were reported by public hospitals of Belém city (capital of Pará state) located in the Amazon region.This disease resulted from the abusive use, e.g., extended treatment and high doses of this plant.In spite of the warning about the toxicity effects of its leaves towards hepatitis, many people still use them to reduce weight, catching this disease as a result.In the early years of this new century, a Brazilian TV program called 'Globo Reporter', warned about the toxicity of Croton cajucara, emphasizing that it could cause hepatitis.This folk medicine observation may be correlated to the extended use required for losing weight and slimming, for C. cajucara toxicological effects were not observed by Farias et al. 14 They have found an absence of acute toxicity of a hydroalcoholic extract obtained from its leaves.This observation does not agree with Kubo et al. 15 who had found cytotoxic effects of the methanol extract of the stem bark of C. cajucara in hepatocytes of mice in vitro.Reinforcing the C. cajucara traditional use of the major constituent of its stem bark, 1, was not genotoxic to mice. 5 The folk toxic warning about the use of C. cajucara combined with our early phytopharmacological results prompted us to examine the possible cytotoxicity of 1-7 on the growth of ascitic Ehrlich carcinoma cells and human K562 leukemia cells.We also expanded the stem bark chemical investigations, obtaining two phenolic compounds and an amino acid, which have not been reported yet as constituents of this plant.The semisynthetic 6 and 7 were synthesized by regiospecific oxidation of 1, affording a new tool in the program of chemical transformation of the target molecule 1.

Plant material
The stem bark of Croton cajucara was collected in Jacundá, Pará state (Amazon region-Brazil) and identified by Nelson A. Rosa.A voucher specimen (No. 247) was deposited in the Museu Paraense Emílio Goeldi Herbarium (Belém-Brazil).

General chemical procedures
Melting points were determined with a Kofler (Jasco DIP-370) apparatus and were not corrected.Optical activities were measured in CHCl 3 on a Perkin-Elmer 341 polarimeter.IR spectra (KBr and CHCl 3 ) were taken on a Perkin-Elmer FT-16PC spectrophotometer and UV spectra (MeOH) on a GBC UV/VIS 911A CG instrument.The 13 C( 1 H)/DEPT135 spectra were recorded on Varian-Gemini and Bruker-Advance spectrometers (300 MHz for 1 H and 75 MHz for 13 C).Mass spectra were run on a CG/MS Finnigan-4000 and VG Auto Spec Q instruments at 70 eV.TLC was carried out on 0.25 mm layers of silica gel PF 254 (Merck).Classical column chromatography was performed with silica gel 60 (70-230 mesh).

Oxidation of 1 using singlet oxygen
Using the regioselective oxidation method previously described for furanic compounds, 17 a solution of 1 (0.3 g) in dichloromethane (30 mL) containing polystyrene-bound rose bengal catalyst (2 mL) was stirred at -78 °C under oxygen atmosphere and irradiated with a 500 W Tungsten incandescent lamp.Aliquots of the reaction mixture were periodically analyzed by TLC until reaction appeared to be completed (6 h).The reaction mixture was allowed to warm to room temperature and filtered through a pad of cotton, and the solvent was evaporated under a vacuum.The synthetic material after evaporation was submitted to chromatography on a silica gel column eluted with a mixture of hexane/EtOAc (1:1) and then MeOH to obtain the semi-synthetic derivatives 6 and 7.The methanolic fraction afforded 0.2 g (65%) of colorless needles, mp 204-205 o C corresponding to 7 isomeric with 6 (20% of colorless needles, mp 202-204 °C, literature 11 202-203 °C).The semi-synthetic derivatives 6 and 7 were purified by similar techniques to those used for the natural cajucarinolides.

Spectroscopic data of the isolated and semi-synthetic compounds
The characterization of the natural 1-7 was previously performed using spectroscopic methods such as IR, UV, MS and 1 H and 13 C NMR. 1,10,18 The semi-synthetic derivatives 6 and 7 were compared with authentic samples and shown to be identical.Aromatic acids (mixture of vanillic acid and 4-hydroxy-benzoic acid) and its derivatives 1 and 2, and also N-methyltyrosine were characterized by spectroscopic methods such as IR, UV, MS and NMR.

Assays
The assays against human K562 leukemia and murine Ehrlich carcinoma were performed in three independent experiments.The cells were grown on RPMI-1640 medium supplemented with 5 % fetal bovine serum, 2 mmol L -1 glutamine, 100 μg mL -1 streptomycin, and 100 U mL -1 penicillin, and were incubated at 37 o C under 5% CO 2 atmosphere.In these experiments, cells were seeded in quadruplicate onto 96-wells plates (2×10 4 cell mL -1 for K562, and 5×10 5 cell mL -1 for Ehrlich) with the tested compounds dissolved in DMSO (final DMSO concentration in culture of 0.2% v/v) at various concentrations.The cultures were incubated for 96 h (for K562) and 48 h (for Ehrlich).Upon incubation, MTT was added and after three hours, formazan was dissolved in 100 mL acidified isopropanol.Tumor cell growths were quantified by the ability of living cells to reduce the yellow dye MTT to a purple formazan product. 19The absorbance was measured at 550 nm using an Elisa microplate reader, and for each tested compound the concentration required to reduce the absorbance by 50% (IC 50 ) in comparison to control cells, was determined.
Two of the aromatic acids vanillic and 4-hydroxybenzoic acid have shown remarkable antioxidant activity in other species. 20,21Based on such results, C. cajucara could be expected to possess antioxidant properties.Reinforcing this suggestion, several kaempferol metabolites have proved to be antioxidant agents [22][23][24] and C. cajucara leaves also contain two of them, e.g.kaempferol 3,4',7-trimethyl ether and 3,7-dimethyl ether. 1 Within the research program for the major biological constituents of 1, we are improving its chemical transformation, which means preparing derivative compounds for screening as pharmacological agents. 1 In the present work, using the method of regioselective oxidation 17 the 7-derivative was synthesized in good yields (67%) by synthetic transformation of the furan moiety of 1, with singlet oxygen generated from molecular oxygen by irradiating a polymer-bound rose bengal catalyst in dichloromethane solution at -78 o C (in the presence of a hindered base, such as diisopropylethylamine).In the absence of a hindered base, the reaction of 1 with singlet oxygen yielded an isomeric mixture of 6 and 7, which was purified by TLC chromatography.The structures of both 6 and 7-derivatives were determined by spectroscopy, including 2D-NMR experiments (COSY 45, HSQC, HMBC) and the observed data corresponded to those of the natural isolated compounds. 10,11

Pharmacological profiling
In order to identify the antiproliferative action of Croton cajucara constituents we studied the effects of natural clerodanes 1, 3, 4, 5, 6 and 7 and also the semisynthetic 6 and 7-derivatives against ascitic Ehrlich carcinoma and human leukemia K562 cells.The experiments were performed using the MTT assay, 19 with quercetin and vincristine as positive controls for Ehrlich and leukemia K562, respectively.
In vitro tests using Ehrlich carcinoma cells, natural 6, 7, 1 and 2, showed a significant cytotoxicity with dose dependent responses over a 48 h culture period.The IC 50 values were 166 μM for 1, 164 μM for 2, 65 μM for 6, and 10 μM for 7, compared to quercetin with an IC 50 = 44 μM (Table 1).The semi-synthetic cajucarinolide derivatives (6 and 7) showed similar antiproliferative activities compared to natural 6 and 7.The remaining natural clerodanes 3, 4 and 5 do not show cytotoxic activity against Ehrlich carcinoma cells.
Natural clerodanes 1-7 and also the semi-synthetic 2, 6 and 7 were also tested for their cytotoxic effects against human K562 leukemia cell line using the Mossman assay 19 and 48 h of cell culture.Among the natural clerodanes assayed, only 3, 4, 6 and 7 (natural and semi-synthetic) showed concentration-dependent growth inhibiting activities on cultured K562 leukemia cells.The IC 50 values were 33 μM (3), 38 μM (5), 36 μM (6) and 43 μM (7).The cajucarinolide-derivatives indicated similar significant antiproliferative activity.Figure 1 shows the comparative cytotoxic effects of the tested clerodanes against both Ehrlich carcinoma and human K562 leukemia cells.The phenolic acids were also assayed against Ehrlich and K562 leukemia cells, but were inactive, under the same experimental conditions.
3 structure has only one α,β-insaturated ketone and its cytotoxicity on K562 cells was similar to that observed for 6, which has this moiety in addition to the conjugated lactone carbonyl in the hydroxybutenolide ring.The similar cytotoxic effects on K562 cells observed for 3, 5, 6 and 7, suggested no significant contribution of hydroxybutenolide ring.Meanwhile, 4, the diastereoisomer of 3, lacked efficacy indicating the importance of the decaline system stereochemistry.Further, 1 and 2 did not present significant cytotoxic effect against K562 cultured cells until 50 μM.This result was in accordance with the lower cytotoxic activity of 1 against promyelocytic HL60 cells with IC 50 = 300 μM, after 24 h, and 180 μM, after 96 h of culture. 26ccording to Wattenberg 25 compounds containing an α,βunsaturated carbonyl moiety have been shown to bind to receptors that induce increased activities of phase II enzymes responsible for metabolizing xenobiotic agents.Thus, the hydroxybutenolide moiety present in 6 and 7, probably led to increase of activities in the Ehrlich carcinoma assay.Taking into account the cytotoxic effect of 7 (10 μM), we suggest that its higher antiproliferative action on growth Ehrlich cells compared to 6 (65 μM) is related to the stereoposition of the hydroxyl group in the hydroxybutenolide moiety (7 with OH at position 16 and its isomeric stereoisomer 6 with OH at position 15).Further, the clerodanes 3, 5 and 6 showed comparable effects against human K562 leukemia cells.

Figure 1 .
Figure 1.Graphic of the IC 50 values of Croton cajucara metabolites against Ehrlich carinoma and human K562 leukemia cells.