Ceramides and Cytotoxic Constituents from Ficus glumosa Del. (Moraceae)

A investigação química da casca do caule de Ficus glumosa (Moraceae) deu origem a duas novas ceramidas (2R,7E)-2-hidróxi-N-[(2S,3S,4R)-1,3,4-trihidróxihexadecano-2-il]hexacos-7enamida e (2R)-N-{(2S,3S,4R,9Z)-1-O-[(β-D-glucopiranosil]-3,4-dihidróxiheptadec-9-eno-2-il}2-hidróxipentacosanamida, em conjunto com vinte e um compostos conhecidos. As estruturas foram estabelecidas usando-se dados de RMN, espectrometria de massas, transformação química e por comparação com dados relatados. Vinte e um compostos foram posteriormente testados contra células de câncer de próstata PC-3 e seis deles revelaram efeito citotóxico. Dongnósido E foi o composto mais ativo, com IC50 de 0,75 μmol L -1 contra células de câncer PC-3, enquanto o medicamento de referência doxorrubicina, apresentou IC50 de 0.91 μmol L . Este composto também demonstrou inibir o crescimento de células do câncer de fibrossarcoma HT1080 (IC50 0,7 μmol L).


Introduction
Ficus glumosa is a small to medium-sized tree with 5 to 10 m tall; it may become a large tree reaching 20 m with a trunk of about 2 m of girth.Ficus species also produce white latex like commonly see in the Moraceae. 1In Ivory Coast, the aqueous decoction of the leaves of F. glumosa is prescribed in traditional pharmacopeia to relieve chest pain and to treat lung diseases such as bronchitis, pneumonia and cough. 2 The aqueous decoction of its stem bark is rather used in Central African Republic as oral solution to cure gingivitis, caries, and tooth aches. 3Additionally, the methanolic extract of the stem bark of F. glumosa has demonstrated in vivo antidiabetic and in vitro antioxidant activities. 4The positive reactions of the diluted crude extract to FeCl 3 , Liebermann Burchard, and Molish reagent associated to the traditional uses prompted us to look for the secondary metabolites of this plant and to evaluate their cytotoxicity.
We herein report the structure elucidation of two new ceramides and the cytotoxicity of some compounds isolated from F. glumosa.

Plant material
The stem bark of F. glumosa was collected in March 2009 in Makenene, central region of Cameroon and identified by the national herbarium where a specimen was deposited under the registration 28151/SRF/Cam (Makenene 1972).

Acetylation and oxidative cleavage of 1
An amount of 3 mg of 1 was dissolved in 3 mL of pyridine and treated with 3 mL acetic anhydride under magnetic stirring for 6 h at room temperature.The acetylated compound was obtained by concentration of medium under vacuum.Comparative TLC revealed the formation of a non polar compound which was further dissolved in 4 mL of THF-H 2 O (9:1) and treated with 5 equiv. of KMnO 4 and NaIO 4 each.The medium was stirred at room temperature overnight and poured onto water.The organic layer obtained by extraction with n-butanol was subjected to de-acetylation using MeONa in MeOH overnight at room temperature.The medium was concentrated under vacuum, treated with aqueous solution of 1 mol L -1 HCl and extracted with EA yielding compound 1a which was characterized by ESI-MS which gave the pseudo-molecular ion at m/z 448.

Methanolysis of 1 and 2
An amount of 1.5 mg of 1, and 2 mg of 2 were separately refluxed in 2 mL of MeOH and 2 mL of aqueous solution of 1 mol L -1 HCl for 18 h under magnetic stirring at 70 °C.An aqueous solution of NaHCO 3 was used to neutralize the medium which was further extracted with DCM.The organic fractions were purified by CC on silica gel eluted with Hex-EA (4:1).The residual oil of both compounds were identified by EI-MS from peaks at m/z 424 [C 27 H 52 O 3 ] +. and 426.3 [C 27 H 54 O 3 ] +. , corresponding to the fatty acid methyl esters (2R)-2-hydroxyhexacos-7-enoic acid methyl ester 1b and (2R)-2-hydroxypentacos-7-enoic acid methyl ester 2a.

Disulfenylation of 2
To a solution of 2 (1 mg) dissolved in toluene (2 mL) were added 5 mg of FeCl 3 and 2 mL of dimethyl disulfide; the reaction mixture was stirred at room temperature and monitored by TLC.After 24 h, the medium was concentrated under reduced pressure and the residue was treated with a saturated aqueous solution of NaHCO 3 and extracted with EA.The colorless amorphous solid 2b obtained was analyzed by EI-MS.

Cytotoxicity assays
The cytotoxicity of the compounds was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 5 assay for the prostate cancer PC-3 cell line and flow cytometry for the fibrosarcoma cancer HT1080 cell line. 6The compounds were dissolved with DMSO and incubated with the cells for 48 h.The drug references used were doxorubicin and etoposide for PC-3 and HT1080, respectively.Furthermore, the IC 50 were calculated to evaluate the cytotoxicity of each natural product.The concentration of sample required to inhibit 50% of the cell proliferation (IC 50 ) was calculated from a calibration curve by a linear regression 7 using Microsoft Excel.Three independent experiments are carried out for each sample.
The resonances of two overlapped olefin protons in trans-geometry 21 were further observed at d H /d C 5.34 (br s)/129.7 and 5.34 (br s)/130.8 and located in the fatty acid chain, as deduced from the mass spectrum of the methanolysis product of 1, which displayed a molecular ion at m/z 424 [C 27 H 52 O 3 ] +. .The position D 7' of this double bond was assigned from its oxidative cleavage (1a) which formed a fragment with a pseudo-molecular ion at m/z 448 [M+H] + (Scheme 1).NOESY correlations and comparison of 1 H and 13 C NMR data with those of the literature allowed to deduce the relative and absolute configuration at C-2, C-3, C-4, and C-2' to be (S), (S), (R), and (R) respectively. 24he foregoing data led to identify 1 as (2R,7E)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyhexadecan-2-yl]hexacos-7enamide, trivially named glumoamide.
Compound 2 was isolated as a white solid.Its molecular formula C 48 H 93 NO 10 was determined on the basis of HR-ESI-MS which displayed a pseudo-molecular ion peak at m/z 844.6897 [M+H] + , and the NMR data (Table 1) accounting for three double bond equivalents.The presence of OH and NH functions with absorption bands on the IR spectrum at 3402, 1620 and 1544 cm -1 was characteristic of cerebrosides. 25his compound gave a positive test to Molish reagent indicative of glycosides.The NMR spectra of C-3, C-4, and C-2' was determined as being (S), (S), (R), and (R) according to the 1 H and 13 C NMR data compared with those of the literature. 24,25 he above mentioned data led to characterize 2 as (2R)-N-{(2S,3S,4R,9Z)-1-O-[(β-D-glucopyranosyl]-3,4-dihydroxyheptadec-9-en-2-yl}-2-hydroxypentacosanamide, trivially named glumoside.The structures of known compounds were identified by using their NMR data and by comparison of those reported in the literature.Some of compounds were tested against the prostate cancer PC-3 cells (Table 2).Dongnoside E (23) showed a significant antiproliferative activity with an IC 50 of 0.75 µmol L -1 , while the value obtained with the reference drug doxorubicin was 0.91 µmol L -1 .In contrast, lanosta-7,24-dien-3-one (3), β-amyrine (5), lupeol (6), 6-prenylapigenin (12) and luteolin (17) showed moderated activities, whereas other compounds were not active.Compound 23 was further tested against fibrosarcoma cancer HT1080 cells lines and presented an interesting antiproliferative cells growth with 0.7 µmol L -1 as IC 50 after 48 h.This natural product was more active than the reference drug etoposide which showed an IC 50 1 µmol L -1 .

Conclusions
The higher concentration of flavonoids, benzoic acid derivatives, saponine, steroids and triterpenes highlights the use of this plant in traditional remedy against lung diseases, since several compounds belonging to the abovementioned classes are present in plants having antitussive and expectorant activities. 26Besides, some polyphenols are active against viral respiratory infections, bacteria and fungi. 27The foregoing results clearly indicated that Ficus glumosa is a promising source of drug development, and 23 can be a potential antitumour lead.

Table 2 .
Cytotoxicity of isolated compounds and doxorubicin against prostate cancer PC-3 cell line