3,4-Diarylisoxazoles-Analogues of Combretastatin A-4: Design, Synthesis, and Biological Evaluation In Vitro and In Vivo

Abstract

Focusing on the molecular docking results, a series of 3,4-diarylisoxazoles, analogues of Combretastatin A4, bearing various substituents at the fifth position of the isoxazole ring and pharmacophore groups bioisosteric to methoxy substituent at ring B, were synthesized in good yields and high regioselectivity. Depending on the substituent at C5, three approaches were chosen for the construction of isoxazole ring, including nitrosation of gem-dihalocyclopropanes, nitrile oxide synthesis, and difluoromethoxylation of isoxazolone to afford 5-haloisoxazoles, 5-unsubstituted isoxazoles, and 5-difluoromethoxyisoxazoles, respectively. Isoxazoles 43 and 45 showed selective cytotoxicity and antitubulin inhibition properties in vitro, with pharmacodynamic profiles closely related to that of CA-4. Both of them slow down tumor growth (66-74%) in mouse xenografts and slightly exceed in effectiveness Combretastatin A4-phosphate itself.

Figure 1

Combretastatin A-4 (I), its prodrugs (II,III), and analogues synthesized in our previous work (IV) and in this work (V).

Figure 2

S1–S4 structures have the highest affinity for the colchicine binding site of tubulin.

Scheme 1. Retrosynthetic Scheme for the Synthesis of 5-Chloro-3,4-diarylisoxazoles

Scheme 2. Synthesis of 5-Chloroisoxazoles 1–7 and 5-Methylisoxazole 9

Scheme 3. Synthesis of 5-Unsubstituted Isoxazoles 10–12 and 5-Difluoromethoxyisoxazoles 13–14

Figure 3

Geometry of 3,4-diarylisoxazoles S1–S4 binding with the colchicine binding site of tubulin. Colored marks on the tubulin surface: red is oxygen, blue is nitrogen.

Scheme 4. Panel of Synthesized CA-4 Analogues of the 3,4-Diarylisoxazole Series

Unsubstituted at the fifth position, 3,4-diarylisoxazoles 39 and 40 were obtained by dehydrochlorination of the corresponding 5-chloroisoxazoles with tetrabutylammonium borohydride. Isoxazole 41 was obtained via diazotization of isoxazole 45. Isoxazoles 42–49 with amino group at the ring B were obtained by reduction of the corresponding nitro derivatives (Na₂S₂O₄ in EtOH-H₂O).

Figure 4

Molecular structures of isoxazoles 29 (CCDC deposition number 2286042) and 36 (CCDC deposition number 2286040). Nonhydrogen atoms are shown as 50% thermal ellipsoids.

Figure 5

Concentration–viability dependence obtained for isoxazoles 43, 44, 45, and 48 on A549, VA13, MCF7′, and HEK293T cells in MTT screening assays in vitro.

Figure 6

In vitro tubulin polymerization in the presence of 43, 44, 45, or 48, taken in 10 μM concentration, the same for CA-4 and Paclitaxel. Cntrl is tubulin polymerization in the absence of compounds.

Figure 7

Immunofluorescent microscopy images of the tubulin staining in A549 cells after 24 h of treatment with 43 in comparison with untreated cells and treatment with CA-4 and Paclitaxel; scale bar is 10 μM.

Figure 8

Tumor growth of SW620 human colon cancer xenografts in BALB/c nude mice; a statistical value of p < 0.05 was considered significant (*p < 0.05,** p < 0.001).