Aryl and Heterocyclic Derivatives of Combretastatin A-4 Endowed with Anticancer Properties

Combretastatins are natural antimitotic agents isolated from the bark of the South African tree Combretum caffrum. Among these compounds, combretastatin A-4 (CA-4) possesses the most potent and interesting antitumor activity. Its mechanism of action is thought to be related to tubulin-binding properties that result in rapid tumour endothelial cell damage, neovascular shutdown and subsequent haemorrhagic necrosis. A large number of combretastatin A-4 analogues has been obtained and studied for cell-growth inhibition activity. These structural derivatives may be classified taking into account A-ring, B-ring or bridge modifications. Following our previous experience in arotinoid field, we envisaged novel bridge modified combretastatins taking into consideration six-membered aromatic rings as bioisosteric conformationally locked double bond analogs. Terphenyl backbone is the simplest scaffold emerged from our project, and despite already known and used in other synthetic and medicinal chemistry fields, nothing was reported in the antineoplastic research. We then adopted a versatile double Suzuki coupling strategy to easily reach a series of terphenyl or diphenyl pyridine combretastatin analogs. Besides six-membered rings, the synthesis of novel isoxazole and isoxazoline derivatives at the bridge position was envisaged to give precious information on conformationally locked combretastatins. Several possible isomers were obtained with different substitutions. The antiproliferative activity of bridge modified combretastatins, tested on HL60 cells showed that terphenyl compounds were scarcely active, having IC50 and AC50 over 10 and 20 μM, but pyridine derivatives demonstrated much more interesting, in special way having the nitrogen placed near to ring A junction. The isoxazole and isoxazoline derivatives had also a very variable behaviour, being the 3,5 disubstituted not much active, but having the 4,5 isoxazolines the most interesting antiproliferative activity in the series of our new analogues. It is worth noting that high potency was found for compounds bearing both cis and trans substitutions at the isoxazoline ring. Both intrinsic (as evidenced through investigation on mitochondrial membrane potential and confirmed in experiments with a caspase-9 inhibitor) and extrinsic (interaction with Fas receptor and reduced activity towards Fas-ligand resistant cell lines) proapoptotic pathways appeared to be influenced, finally resulting in a block into G2 cell-cycle phase. We studied different derivatives bearing the terphenyl structure, varying the substitution pattern of rings substitution. A peculiar behaviour was observed for compounds bearing a 4-carboxy group at ring B. Indeed, when tested in HL60 culture for antiproliferative activity, we observed an unexpected increase of cell population with respect to control. Following further studies, these compounds appeared to support cell viability and decrease apoptotic occurrence even in serum-free media, behaving in similar way as natural 14-hydroxy-retro-retinol (14-HRR). Moreover, these terphenyls were able to protect cultured neuronal cells treated with staurosporin (apoptotic inducer), but no protection was observed towards necrotic stimulations with glutamate. Taking into account that many neurodegenerative pathologies involve the apoptosis of neuronal populations, we think that terphenyl structures could be considered as leads for a novel protective therapeutic strategy.