Design, Synthesis, and Biological Evaluation of 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidines as Microtubule Targeting Agents

Abstract

A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and S_NAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC₅₀ values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC₅₀ values of 53-125 nM). Additionally, compounds 4-8, 10 and 12-13 circumvented Pgp and βIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI₅₀ of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.

Keywords: Gewald reaction; colchicine site; microtubule targeting agents; microtubules.

Figure 1

Representative MTAs.

Figure 2

Structures and activities of Lead compounds 1–3.

Figure 3

Systematic incorporation of structural strategies to restrict the rotation around bonds a and b.

Figure 4

(a) Docked pose of compound 4 (cyan) and colchicine (pink) in the CS (PDB: 6BS2, 2.65 Å) [43] in Maestro 2020-2 (docked score of compound 4 was −10.89 kcal/mol.); (b) structures of 4 and colchicine.

Scheme 1

Synthesis of intermediates 20–22 from cyclohexanone.

Scheme 2

Synthesis of intermediate 26.

Scheme 3

Synthesis of target compounds 4–6, 7, 9, 11, 12 and 14.

Scheme 4

Synthesis of target compounds 8, 10 and 13.

Figure 5

Effects of 4 and paclitaxel on the growth of MDA-MB-435 tumors. MDA-MB-435 tumors were implanted into the flanks of nude mice and allowed to grow until they reached a volume of ~200 mm³. The mice were treated i.p. with 4 (75 mg/kg) or paclitaxel (15 mg/kg) three times a week. Tumor volumes and mouse weights were measured 2–3 times a week. Statistical significance was evaluated by two-way ANOVA (time vs. drug) with Dunnett’s post-hoc tests. A statistically significant difference in tumor volume was detected on day 14 between mice treated with 4 (* p = 0.0384) and untreated control tumors.