Cytotoxic Sesquiterpene Lactones and other Constituents of Centaurea omphalotricha

A investigação fitoquímica das partes aéreas de Centaurea omphalotricha levou ao isolamento de três lactonas sesquiterpênicas novas, 4’-acetilcinaropicrina, 4’-acetilcebelina F e 15-acetil desidromelitensina, juntamente com doze compostos conhecidos, sendo sete lactonas sesquiterpênicas, dois isoprenóides e três flavonóides. As estruturas dos novos compostos foram elucidadas por meio de RMN 1D e 2D, e espectrometria de massas, e por comparação com os dados descritos na literatura. O efeito de lactonas sesquiterpênicas sobre a viabilidade das células tumorais humanas, linhagens HL-60 e U937, também foi investigado e 3-acetilcinaropicrina e 4’-acetilcinaropicrina foram os compostos mais citotóxicos contra células de leucemia humana com valores de IC50 de 2,0 ± 0,9 e 5,1 ± 0,4 μmol L , respectivamente.


General
Optical rotations were measured using a Perkin-Elmer model 343 polarimeter using CHCl 3 as solvent.UV spectra were recorded using a SHIMADZU model UV-1700 spectrophotometer.IR spectra were recorded as CHCl 3 films on NaCl plates, using a Bruker model IFS-55 and Perkin-Elmer model FTIR-8400S spectrophotometer. 1 H and 13 C NMR spectra were obtained on a Bruker model Avance 400 and AMX-500 spectrometer with standard pulse sequences, operating at 400 and 500 MHz for 1 H and 100 and 125 MHz for 13

Cell culture and cytotoxicity assays
The human leukemia HL-60 and U937 cells (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 containing 2 mmol L -1 L-glutamine supplemented with 10% (v/v) heat-inactivated fetal bovine serum as previously described. 19tock solutions of 25 mmol L -1 sesquiterpene lactones were made in dimethyl sulfoxide (DMSO) and further dilutions were made in culture media just before use.In all experiments, the final concentration of DMSO did not exceed 0.4% (v/v), a concentration which is non toxic to the cells.The cytotoxicity of sesquiterpene lactones on human tumor cells was analyzed by colorimetric 3-[4,5-dimethylthiazol-2-yl-]2,5-diphenyl tetrazolium bromide (MTT) assay as previously described. 20Concentrations inducing a 50% inhibition of cell growth (IC 50 ) were determined graphically using the curve fitting algorithm of the computer software Prism 4.0 (GraphPad).Values are means ± SE from at least three independent experiments, each performed in triplicate.The antitumor agent etoposide was used as a positive control in both HL-60 (IC 50 = 0.4 ± 0.1 mmol L -1 ) and U937 cells (IC 50 = 1.4 ± 0.3 mmol L -1 ).
Sesquiterpene lactones have attracted much attention during the last three decades, because they display a wide range of biological activities, including antitumor and antiinflammatory properties. 8,23The structural requirement for the biological activities of these compounds is associated with α-methylene-γ-butyrolactone moiety, which acts as alkylating agent in a Michael-type reaction with nucleophiles. 24Thus, sesquiterpene lactones are believed to exert their numerous biological activities through inhibition of enzymes and other functional proteins by forming covalent bonds with free cysteine residues in these macromolecules or by conjugation with glutathione. 25,26revious studies have shown that sesquiterpene lactones display cytotoxic properties in tumor cells 8 and that the sesquiterpene lactone of the guaianolide type cynaropicrin induces cytotoxicity in U937 and Jurkat T cell lines. 27A quantitative structure-activity relationships (QSAR) study including four different skeletons of sesquiterpene lactones revealed the most active among the guaianolides and pseudoguainolides, and steric properties and electronic features as the most important descriptors. 23owever, antiproliferative studies of the naturally occurring sesquiterpene lactones described in this paper in human leukemia cells have not yet been assessed.
Sesquiterpene lactones 1-6 were found to inhibit the growth and cell viability of HL-60 and U937 cells in culture as determined by the 3-[4,5-dimethylthiazol-2-yl]2,5- diphenyl tetrazolium bromide (MTT) dye-reduction assay (Table 3).In contrast, the sesquiterpene lactone 7 is not an effective antiproliferative agent showing an IC 50 value higher than 100 mmol L -1 in leukemia cells, in accordance with the absence of the alkylating group, the exocyclic conjugated double bond.
Among the different sesquiterpene lactones, the presence of the exocyclic, conjugated double bond is essential for the cytotoxic activity against HL-60 and U937 cells.Compounds 1, 2, 4, 5 and 6 displayed similar potency in both cell lines.The potency of these sesquiterpene lactones might be explained by their lipophilicity.However, other factors, such as molecular geometry and the chemical environment of the target sulfhydryl may also influence the activity of sesquiterpene lactones.All these guaianolides contain an ester functional group at C-8.The sesquiterpene lactone 8-(4-hydroxymethacrylate)-dehydromelitensin 6 also contains an ester near from the exocyclic methylene bond.The presence of this group appears to be important, since compound 3 (15-acetyl dehydromelintensin) -which does not contain this functional group -was less cytotoxic than 6.
In conclusion, the Algerian plant Centaurea omphalotricha has been chemically studied for the first time, and three new sesquiterpene lactones have been identified along with twelve known compounds.The naturally occurring sesquiterpene lactones evaluated in the present study were strongly cytotoxic against human leukemia cell lines and the results of the present study may lead to the discovery of new and highly specific antitumor agents against leukemia cells.

Table 1 .
13 NMR data of compounds 1-3 in CDCl 3 (d in ppm and J in Hz) suggesting that both compounds are closely related in structure.The major variation is that compound 2 has a different acyl group.A detailed comparison of the 1 H (Table1) and13C NMR (Table2) signals of 2 and 1 revealed an extra carbon signal in13C NMR, besides the presence of a methyl group at d H 1.98 (d, 3H, J 1.2 Hz, H 3 -5') and a vinylic proton signal at d H 6.85 (tq, 1H, J 6.1, 1.2 Hz, H-3') in1H NMR suggesting that the substituent group in 2 is the 4-acetoxy-2-methyl butenoyl.The relative stereochemistry of the double bond in the acyl group was determined as E based on the observed correlation between the signals at d H 4.83 and 1.98 in the ROESY spectrum.The combination of all the above data and the HRESIMS experiment led us to assign the structure of 2 as 4'-acetyl cebellin F.

Table 3 .
Effects of compounds 1-7 on the growth of the human leukemia cell lines Cells were cultured for 72 h and the IC 50 values were calculated as described in the Experimental section.The data shown represent the means ± SEM of 3-5 independent experiments with three determinations in each.