Synthesis, antimitotic and antivascular activity of 1-(3',4',5'-trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles

Abstract

A new class of compounds that incorporated the structural motif of the 1-(3',4',5'-trimethoxtbenzoyl)-3-arylamino-5-amino-1,2,4-triazole molecular skeleton was synthesized and evaluated for their antiproliferative activity in vitro, interactions with tubulin, and cell cycle effects. The most active agent, 3c, was evaluated for antitumor activity in vivo. Structure-activity relationships were elucidated with various substituents on the phenyl ring of the anilino moiety at the C-3 position of the 1,2,4-triazole ring. The best results for inhibition of cancer cell growth were obtained with the p-Me, m,p-diMe, and p-Et phenyl derivatives 3c, 3e, and 3f, respectively, and overall, these compounds were more or less as active as CA-4. Their vascular disrupting activity was evaluated in HUVEC cells, with compound 3c showing activity comparable with that of CA-4. Compound 3c almost eliminated the growth of syngeneic hepatocellular carcinoma in Balb/c mice, suggesting that 3c could be a new antimitotic agent with clinical potential.

Figure 1

ORTEP view of compound 3c displaying the thermal ellipsoids at 30% probability.

Figure 2

(A) Proposed binding for 3c (in gray) in the colchicine site. Co-crystallized DAMA–colchicine is shown in green. (B) Representation of the binding mode of 3c in the colchicine site, with a summary of the SARs observed for the reported series of compounds. (C) Superposition of the conformation obtained from the docking simulations (in gray) and the crystal structure (in yellow) of 3c.

Figure 3

Percentage of cells in each phase of the cell cycle in HeLa (A) and Jurkat cells (B) treated with 3c at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the Experimental Section. Data are represented as mean ± SEM of three independent experiments.

Figure 4

Effects of 3c on G2/M regulatory proteins (A) and on p53, p21, and γH2AX expression (B). HeLa cells were treated for 24 or 48 h with the indicated concentration of 3c. The cells were harvested and lysed for the detection of cyclin B1, p-cdc2^Tyr15, and cdc25C (A) or p53, p21, and γH2AX expression (B) by Western blot analysis. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 5

Flow cytometric analysis of apoptotic cells after treatment of HeLa cells (A) or Jurkat cells (B) with 3c at the indicated concentrations after incubation for 24 or 48 h. The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are represented as mean ± SEM of three independent experiments.

Figure 6

Western blot analysis of caspase-3, cleaved caspase-9, PARP Bcl-2, and Bax after treatment of HeLa cells with 3c at the indicated concentrations and for the indicated times. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Figure 7

Compound 3c has antivascular activity in vitro. (A) Confluent HUVECs in a monolayer were wounded, and cells treated with different concentrations of 3c and at different times were photographed, 7× magnification; bar = 100 µm. The dotted lines define the areas lacking cells. (B) The graph shows the quantitative effect of 3c. Migration was quantified by measuring the gap closure at the indicated times as shown in A. Data are represented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 vs control. (C) Inhibition of endothelial cell capillary-like tubule formation by 3c. Tubule formation on Matrigel was carried out as described in the Experimental Section. Representative pictures (10× magnification; bar = 100 µm) of preformed capillary-like tubules treated with increasing concentrations of 3c for 1 or 3 h. (D) Quantitative analysis of the effects of 3c on the dimensional and topological parameters of the preformed capillary-like tubule networks after a 3 h treatment. Data were represented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 vs. control.

Figure 8

Inhibition of mouse allograft tumor growth in vivo by compound 3c. (A) Male mice were injected subcutaneously in their dorsal region with 10⁷ BNL 1MEA.7R.1 cells, a syngeneic hepatocellular carcinoma cell line. Tumor-bearing mice were administered the vehicle, as control, or the indicated doses of 3c or CA-4P as reference compound at the concentration of 5 mg/kg. Daily injections were given intraperitoneally starting on day 1. The figure shows the average measured tumor volumes (A) and body weights of the mice (B) recorded at the beginning and at the end of the treatments. Data are presented as mean ± SEM of tumor volume and body weight at each time point for five animals per group. *p < 0.05, **p < 0.01 vs. control.

Scheme 1a

^a Reagents: (a) ArNH₂, i-PrOH, reflux; (b) NH₂NH₂·H₂O, THF, reflux; (c) 3’,4’,5’-(OMe)₃C₆H₂COCl, pyridine, 0 °C.

Chart 1

Inhibitors of Tubulin Polymerization