Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site

Abstract

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC₅₀ 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC₅₀ 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the βIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

Keywords: Colchicine site agents; Cytotoxins; Microtubule targeting agents; Pyrimidines.

Figure 1.

Tubulin inhibitor 1

Figure 2.

Series I (compounds 2-3)

Figure 3.

Series II

Figure 4.

Series III

Figure 5.

(a) Superimposition of the docked poses of 1 (green) and 12 (magenta) in the colchicine site of tubulin (PDB ID: 4O2B). (b) Superimposition of the docked pose of 12 (magenta) and crystallized ligand colchicine (peach). (c) Structures and docked scores of 1, 12, and colchicine.

Figure 6.

Series IV (compounds 18-19)

Figure 7.

¹³C NMR of compound 3

Figure 8.

Antitumor efficacy of 12 and paclitaxel in an MDA-MB-435 xenograft model. MDA-MB-435 tumors were implanted s.c. into the flanks of nude mice and allowed to grow until they reached an average volume of 200 mm³. Compound 12 (50 mg/kg) and paclitaxel (PTX; 15 mg/kg) were administered i.p. three times a week for 2 weeks (Days 1, 3, 5, 8, 10 and 12). Tumor volumes and mouse weights were assessed 2–3 times a week. Differences in final tumor volume on day 14 were evaluated by one-way ANOVA with Dunnett’s post-hoc tests; control (CTRL).

Scheme 1.

Synthesis of lead compounds 2 and 3 (Series I)

Scheme 2.

Synthesis of N-methylated anilines 24, 26 and 30

Scheme 3.

Synthesis of target compounds 4, 5, 7, 8 and 19

Scheme 4.

Synthesis of target compounds 6 and 9

Scheme 5.

Synthesis of target compounds 10-18 (Series III)

Scheme 6.

Synthesis of compound 18 (Series IV)