Synthesis and Cytotoxic Activity of a New Group of Heterocyclic Analogues of the Combretastatins

A series of new analogs of combretastatin A-4 (CA-4, 1) with the A or B-ring replaced by a 3-oxo-2,3-dihydrofurocoumarin or a furocoumarin residue have been designed and synthesized by employing a cross-coupling approach. All the compounds were evaluated for their cytotoxic activity with respect to model cancer cell lines (CEM-13, MT-4, U-937) using conventional MTT assays. Structure-activity relationship analysis reveals that compounds 2, 3, 6–8 in which the (Z)-styryl substituent was connected to the 2-position of the 3-oxo-2,3-dihydrofurocoumarin core, demonstrated increased potency compared to 3-(Z)-styrylfurocoumarins 4, 5, 9–11. The methoxy-, hydroxyl- and formyl- substitution on the aromatic ring of the (Z)-styryl moiety seems to play an important role in this class of compounds. Compounds 2 and 3 showed the best potency against the CEM-13 cell lines, with CTD50 values ranging from 4.9 to 5.1 μM. In comparison with CA-4, all synthesized compounds presented moderate cytotoxic activity to the T-cellular human leucosis cells MT-4 and lymphoblastoid leukemia cells CEM-13, but most of them were active in the human monocyte cell lines U-937.

Herein we describe the first synthesis of combretastatin A-4 analogues 2,3 and 4,5 by replacement of the A or B aromatic ring with linear dihydrofurocoumarins or furocoumarins (psoralens) (Figure 1). The (Z)-styryl moiety was attached to 2-or 3-position of the heterocyclic molecule. We also include other examples of (Z)-arylvinylfurocoumarins 6-11 for obtaining structure-cytotoxicity relationships.
Linear furocoumarins are employed in Psoralen + UVA (PUVA) therapy for the treatment of autoimmune or hyper-proliferative skin diseases, including psoriasis and vitiligo. Activated by UV-A light furocoumarins induce many biological effects, such as photocycloadditions to DNA, immune system modulation, reactions with proteins, RNA and lipids [13][14][15]. In last decades many new potential therapeutic applications for furocoumarins are found. For instance, some psoralen derivatives were found to induce erythroid differentiation in different cellular models [16,17]. Moreover it was shown that annelation of a cyclopentane, cyclohexane, benzene, or pyridazine ring to the furan ring in the psoralen skeleton changed the phototoxicity and, in some cases led to a marked increase in the photo-antiproliferative activity [18]. There was therefore value in a targeted preparation and investigation of novel furocoumarins, containing a (Z)-styryl substituent in the furan ring of the molecules. Compounds 12 and 13, containing an additional N-methylpiperazinyl substituent in the furocoumarin scaffold were also prepared and investigated, since modification in the 9-position has a great influence on the properties of psoralenes as enzymatic inhibition and human keratinocyte proliferation [13].

Cytotoxicity Studies
The cytotoxic activity of the synthesized series of 2-(Z)-styryldihydrofurocoumarins 2,3,6-8, 3-(Z)-styrylfurocoumarins 4,5,9-13, the parent oreoselone (14) and combretastatin A-4 (1) was determined by measuring the concentration inhibiting human tumor cell viability by 50% (CTD 50 ). The CTD 50 was determined using the conventional MTT assay, which allows to estimate the number of survived cells spectrophotometrically [27]. The results are presented in Table 1. At first glance, it is evident that there is a difference in activity between the parent compound 14 and (Z)-styryl modified furocoumarins 2-13. All synthesized compounds 2-13 exhibited cytotoxic activity in respect to model cancer cell lines and were more potent than furocoumarin 14. Furocoumarins 4, 5, 9, 10 and 11, in which the (Z)-styryl substituent was located in the 3-position, demonstrated decrease of potency compared to 2-(Z)-styryl-3-oxodihydrofurocoumarins 2, 3, 6, 7 and 8. Compounds 2, 3 possessed the best activity on the lymphoblastoid leukemia cells CEM-13. The phenolic substituent in (Z)-styryl moiety seems to have an important role in this class of compounds; indeed 3,4-dihydroxyphenyl substituent in compounds 7, 10, 12 demonstrated increase of potency against MT-4 cell lines. Compounds having а 2,3,4trimethoxyphenyl moiety (compounds 6, 9) show week cytotoxic activity, while several derivatives containing a 3,4,5-trimethoxyphenyl residue (compounds 2, 4) shown improved activity. The latter effect is unsurprising, since it is well established that the replacement of A-ring in combretastatins [28,29] and phenstatin [30] is highly detrimental for the activity of the compounds. In the 3,9-disubstituted furocoumarins 12,13 cytotoxicity does not seem to greatly influence by the substituent in the aromatic ring, however, the (4-methylpiperazin-1-ylmethyl) substitution on the 9th-position in 3-styrylfurocoumarin core gave increase in cytotoxic activity of compounds (compare 10 and 12 or 11 and 13), especially, on the human monocyte-lines U-937. The activity of all synthesized compounds on the lymphoblastoid cell lines was lower than the activity of СA-4 1, however, compounds 2,7,12 and 13 shown potency in respect to human monocyte-lines U-937 in comparison with СA-4.
The IR spectra were recorded by means of the KBr pellet technique on a Bruker Vector-22 spectrometer. The UV spectra were obtained on an HP 8453 UV-Vis spectrometer (Hewlett-Packard, Waldbronn, Germany). The melting points were determined on a Stuart SMF-38 melting point apparatus (Bibby Scientific, Staffordshire, UK) and are uncorrected. Elemental analysis was carried out on a Carlo-Erba 1106 Elemental analysis instrument (Carlo-Erba, Milan, Italy). Spectral and analytical investigations were carried out at Collective Chemical Service center of Siberian Branch of the Russian Academy of Sciences.
Reaction products were isolated by column chromatography on silica gel 60 (0.063-0.200 mm, Merck KGaA, Darmstadt, Germany) and eluted with chloroform and chloroform-ethanol (100:1; to 25:l). The reaction progress and the purity of the obtained compounds were monitored by TLC on Silufol UV-254 plates (Kavalier, Czech Republic, CHCl 3 -EtOH, 25:1; detection under UV light or by treatment with iodine vapor).

Cell Culture and Cytotoxicity Assay
The human cancer cells of the MT-4, CEM-13 (the cells of T-cellular human leucosis), and U-937 (human monocytes) were used in this study. The cells were cultured in the RPMI-1640 medium that contained 10% embryonic calf serum, L-glutamine (2 mmol/L), gentamicin (80 lg/mL), and lincomycin (30 mg/mL) in a CO 2 incubator at 37 °C. The tested compounds were dissolved in DMSO and added to the cellular culture at the required concentrations. Three wells were used for each concentration. The cells which were incubated without the compounds were used as a control. Cells were placed on 96-well microliter plates and cultivated at 37 °С C in 5% CO 2 /95% air for 72 h. The cell viability was assessed through an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bromide] conversion assay. 1% MTT was added to each well. Four hours later DMSO was added and mixed for 15 min. Optical density (D) of the samples was measured on a BioRad 680 spectrophotometer Microplate Reader (BioRad, Hercules, CA, USA) at the wavelength of 570 nm. The 50% cytotoxic dose (CTD50) of each compound (i.e., the compound concentration that causes the death of 50% of cells in a culture, or decreases the optical density twice as compared to the control wells) was calculated from the data obtained. Statistical processing of the results was performed using the Microsoft Excel-2007, STATISTICA 6.0, and GraphPad Prism 5.0 programs. The results are given as an average value ± a deviation from the average. Reliability of differences (p) was estimated using the Student t test. The differences with p < 0.05 were considered as reliable. The experimental results are given as the data average values obtained from three independently conducted experiments.

Conclusions
A series of original furocoumarin derivatives having 2-(Z)-or 3-(Z)-styryl substitution in their structures have been synthesized. The cytotoxic activity of the resulting compounds against several cancer lines have been determined in the conventional MTT assay. The cytotoxicity data of compounds 2-13 demonstrate that they exhibit anticancer activity in micromolar range. Structure-activity comparison provides evidence that а 2-(Z)-styryl substitution in the furocoumarin scaffold is preferred for cytotoxicity over the subsequent 3-(Z)-styryl substitution; the (4-methylpiperazin-1-ylmethyl) substitution in the 9-position of 3-styrylfurocoumarins increases the cytotoxic activity in MT-4 and U-937 cell lines. The biological results for the furocoumarin analogs of CA-4 1, reported herein, shown that the structural modification of furocoumarins with the introduction of (Z)-styryl moiety may prove of great importance to obtain cytotoxic anti-cancer agents.