RESUMEN
Isocombretastatins are the not isomerizable 1,1-diarylethene isomers of combretastatins. Both families of antimitotics are poorly soluble and new analogs with improved water solubility are needed. The ubiquitous 3,4,5-trimethoxyphenyl ring and most of its replacements contribute to the solubility problem. 39 new compounds belonging to two series of isocombretastatin analogs with 2-chloro-6-methylsulfanyl-4-pyridinyl or 2,6-bis(methylsulfanyl)-4-pyridinyl moieties replacing the 3,4,5-trimethoxyphenyl have been synthesized and their antimitotic activity and aqueous solubility have been studied. We show here that 2-chloro-6-methylsulfanylpyridines are more successful replacements than 2,6-bis(methylsulfanyl)pyridines, giving highly potent tubulin inhibitors and cytotoxic compounds with improved water solubilities. The optimal combination is with indole rings carrying polar substitutions at the three position. The resulting diheteroaryl isocombretastatin analogs showed potent cytotoxic activity against human cancer cell lines caused by tubulin inhibition, as shown by in vitro tubulin polymerization inhibitory assays, cell cycle analysis, and confocal microscopy studies. Cell cycle analysis also showed apoptotic responses following G2/M arrest after treatment. Conformational analysis and docking studies were applied to propose binding modes of the compounds at the colchicine site of tubulin and were in good agreement with the observed SAR. 2-Chloro-6-methylsulfanylpyridines represent a new and successful trimethoxyphenyl ring substitution for the development of improved colchicine site ligands.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Piridinas/química , Estilbenos/farmacología , División Celular/efectos de los fármacos , Colchicina/metabolismo , Fase G2/efectos de los fármacos , Humanos , Solubilidad , Estilbenos/química , Tubulina (Proteína)/metabolismoRESUMEN
Colchicine site antimitotic agents typically suffer from low aqueous solubilities and are formulated as phosphate prodrugs of phenolic groups. These hydroxyl groups are the aim of metabolic transformations leading to resistance. There is an urgent need for more intrinsically soluble analogues lacking these hydroxyl groups. The 3,4,5-trimethoxyphenyl ring of combretastatin A-4 is a liability in terms of solubility but it is considered essential for high cytotoxic and tubulin polymerization inhibitory (TPI) activity. We have synthesized 36 new analogues of combretastatin A-4 replacing the trimethoxyphenyl moiety with more polar pyridine based moieties, measured their aqueous solubility, and studied their anti-proliferative effects against 3 human cancer cell lines. We show here that pyridine rings can be successful replacements for the trimethoxyphenyl ring, resulting in potent and more soluble analogues. The more straightforward replacement, a 2,6-dimethoxypyridine ring led to inactive analogues, but a 2-methoxy-6-methylsulfanylpyridine moiety led to active analogues when combined with different B rings. This replacement led to potent cytotoxic activity against sensitive human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies. Cell cycle analysis also showed apoptotic responses following treatment. Docking studies suggested binding at the colchicine site of tubulin and provided a good agreement with the observed SAR. A 2-methoxy-6-methylsulfanylpyridine moiety is a good trimethoxyphenyl ring replacement for the development of new colchicine site ligands.
Asunto(s)
Antineoplásicos/farmacología , Colchicina/farmacología , Piridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colchicina/síntesis química , Colchicina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ligandos , Estructura Molecular , Polimerizacion/efectos de los fármacos , Piridinas/química , Solubilidad , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
Colchicine site ligands suffer from low aqueous solubility due to the highly hydrophobic nature of the binding site. A new strategy for increasing molecular polarity without exposing polar groups-termed masked polar group incorporation (MPGI)-was devised and applied to nitrogenated combretastatin analogues. Bulky ortho substituents to the pyridine nitrogen hinder it from the hydrophobic pocket while increasing molecular polarity. The resulting analogues show improved aqueous solubilities and highly potent antiproliferative activity against several cancer cell lines of different origin. The more potent compounds showed moderate tubulin polymerization inhibitory activity, arrested the cell cycle of treated cells at the G2/M phase, and subsequently caused apoptotic cell death represented by the cells gathered at the subG0/G1 population after 48 h of treatment. Annexin V/Propidium Iodide (PI) double-positive cells observed after 72 h confirmed the induction of apoptosis. Docking studies suggest binding at the colchicine site of tubulin in a similar way as combretastatin A4, with the polar groups masked by the vicinal substituents. These results validate the proposed strategy for the design of colchicine site ligands and open a new road to increasing the aqueous solubility of ligands binding in apolar environments.
Asunto(s)
Bibencilos/química , Nitrógeno/química , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Colchicina/química , Diseño de Fármacos , Células HT29 , Células HeLa , Humanos , Ligandos , Células MCF-7 , Simulación del Acoplamiento Molecular , Piridinas/química , Solubilidad/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We have synthesized and assayed dimethylaminophenyl, pyrrolidin-1-ylphenyl and carbazole containing phenstatins and isocombretastatins as analogues of the highly potent indoleisocombretastatins with extended or reduced ring sizes. This is an attempt to explore beyond the structural constraints of the X-ray crystal structures the zone of the colchicine site where the tropolone ring of colchicine binds to tubulin (zone 1). The isocombretastatins display up to 30 fold increased water solubility when compared with combretastatin A-4, potent inhibition of tubulin polymerization, and nanomolar cytotoxicities against several human cancer cell lines irrespective of the size of the B ring. On the other hand, substitutions ortho to the nitrogen cause an important reduction in potency. We have also shown that representative compounds inhibit autophagy. These results show that zone 1 can adapt to systems of different size as far as they stay in a common plane, but does not tolerate substituents protruding above or below it. These results can help in the understanding of the binding modes of structures with similar systems and in the design of new colchicine site ligands.