In Vivo and Mechanistic Studies on Antitumor Lead 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one and Its Modification as a Novel Class of Tubulin-Binding Tumor-Vascular Disrupting Agents

Abstract

7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2), a promising anticancer lead previously identified by us, inhibited tumor growth by 62% in mice at 1.0 mg/kg without obvious signs of toxicity. Moreover, compound 2 exhibited extremely high antiproliferative activity in the NIH-NCI 60 human tumor cell line panel, with low to sub-nanomolar GI₅₀ values (10^-10 M level). It also showed a suitable balance between aqueous solubility and lipophilicity, as well as moderate metabolic stability in vivo. Mechanistic studies using Mayer's hematoxylin and eosin and immunohistochemistry protocols on xenograft tumor tissues showed that 2 inhibited tumor cell proliferation, induced apoptosis, and disrupted tumor vasculature. Moreover, evaluation of new synthetic analogues (6a-6t) of 2 indicated that appropriate 2-substitution on the quinazoline ring could enhance antitumor activity and improve druglike properties. Compound 2 and its analogues with a 4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one scaffold thus represent a novel class of tubulin-binding tumor-vascular disrupting agents (tumor-VDAs) that target established blood vessels in tumors.

Figure 1.

Colchicine and some clinical trial VDA candidates.

Figure 2.

Leads and new derivatives.

Figure 3.

Dose-dependent anticancer effects of compound 2 on NCI-H460 lung cancer xenografts in nu/nu mice. Treatments started when tumors reached a mean volume of around 100 mm³. Mice were treated with 2 at an iv dose of 0.25, 0.5, or 1 mg/kg or paclitaxel at 15 mg/kg as reference or the vehicle as control (n = 8), every 5 days for 3 weeks. (A) Growth difference of tumor volumes at different time points. (B) Images of resected NCI-H460 xenograft tumors. (C) Tumors were resected and weighed at the end of experiment, (●) indicates the weight value of each tumor; (—) indicates average value of tumor weights. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle controls (one-way analysis of variance with Tukey’s post hoc method).

Figure 4.

Predicted binding mode of 2 (brown stick) and 6e (green stick) with tubulin (PDB code 5LYJ) and overlapping with CA-4 (pink, the bound of ligand of 5LYJ). Surrounding amino acid side chains are shown in gray stick format and are labeled. Hydrogen bonds are shown by green dashed lines, and the distance between ligands and protein is less than 3 Å.

Figure 5.

Effects of compounds on cell cycle. A549 cells were treated for 24 h with 6d or 6e at 15 nM. CA-4 (20 nM) or DMSO was used as a tubulin polymerization inhibitor or control, respectively. Fixed and propidium iodide (PI)-stained cells were analyzed by flow cytometry.

Figure 6.

Compound 2 treatment resulted in apoptosis and vascular disruption of NCI-H460 xenograft tumors. Representative images of HE staining and IHC staining of CD31 (endothelial marker), cleaved caspase-3 (apoptosis marker), and Ki67 (proliferation marker) in different treatment groups. Sections were counterstained with hematoxylin. Red arrows in CD31 staining images indicate micro-vessels. Scale bar, 100 μm.

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