Substitution at the indole 3 position yields highly potent indolecombretastatins against human tumor cells

Abstract

Resistance to combretastatin A-4 is mediated by metabolic modification of the phenolic hydroxyl and ether groups of the 3-hydroxy-4-methoxyphenyl (B ring). Replacement of the B ring of combretastatin A-4 by a N-methyl-5-indolyl reduces tubulin polymerization inhibition (TPI) and cytotoxicity against human cancer cell lines but cyano, methoxycarbonyl, formyl, and hydroxyiminomethyl substitutions at the indole 3-position restores potent TPI and cytotoxicity against sensitive human cancer cell lines. These highly potent substituted derivatives displayed low nanomolar cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies, and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was suggested by molecular modeling studies. Substituted combretastatins displayed higher potencies than the isomeric isocombretastatins and the highest potencies were achieved for the hydroxyiminomethyl (21) and cyano (23) groups, with TPI values in the submicromolar range and cytotoxicities in the nanomolar and subnanomolar range. Dose-response and time-course studies showed that drug concentrations as low as 1 nM (23) or 10 nM (21) led to a complete G/M cell cycle arrest after 15 h treatment followed by a high apoptosis-like cell response after 48-72 h treatment. The P-glycoprotein antagonist verapamil increased 21 and 23 cytotoxicity to IC values of 10 M, and highly potentiated the cytotoxic activity in 100-fold of the CHO derivative (17), in A-549 human non-small cell lung cancer cells. The cyano substituted indolecombretastatin 23 is by itself highly potent against rather resistant HT-29 and A-549 cell lines. A 3,4,5-trimethoxyphenyl ring always afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.

The Masked Polar Group Incorporation (MPGI) Strategy in Drug Design: Effects of Nitrogen Substitutions on Combretastatin and Isocombretastatin Tubulin Inhibitors

“…This ratio is improved to more than 50% (16 out of 31) if the ammonium salts are not considered, as they are all inactive. This high success rate is the result of a combination of favorable scaffolds and substituents, and it is similar to those of previous reports on related series [7,10,13,16,23]. Of the three cell lines, HeLa and MCF-7 showed similar sensitivities, while HT-29 cells were less susceptible.…”

“…Of the three cell lines, HeLa and MCF-7 showed similar sensitivities, while HT-29 cells were less susceptible. We have previously observed the same trend for related compounds acting at the colchicine site of tubulin [10,23]. For this pyridine series, we observed a different potency trend when we compare the 4-amino-3-pyridine and the 3-amino-4-pyridine derivatives.…”

“…However, the 3-hydroxy group of combretastatin A4 that provides an anchor for phosphate prodrug formulation is used by tumor cells for conjugation and development of resistance [9,19]. The replacement of the 3-hydroxy-4-methoxyphenyl ring of combretastatins, isocombretastatins and phenstatins by indoles [11,12,13,14,20,21], naphthalenes [13], and 4-dimethylaminophenyl rings [15,16,22], results in potent tubulin inhibitory and cytotoxic compounds, but require further elaboration in order to achieve improved water solubility [10,23]. An alternative to prodrug formation is the introduction of polar functions, which has been attempted by replacing the phenyl rings of combretastatins, isocombretastatins, and related compounds, with pyridine rings, yielding better results as A than as B rings [15,22,24,25,26].…”

“…With the aim of testing the aforementioned proposal in different scaffold contexts which have been shown to result in different structure–activity relationships [7,10,11,23,27], we have synthesized a new family of combretastatins ( 8 ), phenstatins ( 1 ), isocombretastatins ( 3 ), 1,1-diarylethanes ( 4 ), and diaryloxime derivatives ( 2 ) with a pyridine B–ring ortho substituted with dialkylamino groups and their phenyl counterparts for the sake of comparison. As a further extension of the strategy, ammonium salts of the dialkylamino substituents have been prepared ( 5–7 , 9 ), as they have polar (charged) groups surrounded by a hydrophobic cap.…”

“…We have previously shown that the potency ordering isocombretastatins similar to oximes higher than benzophenones could be explained by the higher conjugation of the ketones with the aromatic rings, which imposes a more planar disposition than those required for high affinity colchicine site binding [10]. For the 3-amino-4-pyridine series, more planar structures are required in order to avoid the bumping of the amino substituents with tubulin out the plane of the aromatic ring, an effect which we have previously shown to impact the potency of 3-substituted indolecombretastatins and dimethylaminophenyl phenstatins [16,23]. This different geometrical disposition of the amino substituents, and the different adaptability of their corresponding pockets in the two series, can also account for the preference of bulkier substituents at position 3, such as the pyrrolidine rather than the smaller dimethylamino.…”