Discovery and preclinical evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-4-amines as single agents with microtubule targeting effects along with triple-acting angiokinase inhibition as antitumor agents

Abstract

The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR), along with microtubule targeting in single molecules are described. These compounds also inhibited blood vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In addition, some of the analogs circumvent the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to microtubule targeting agents (MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three digit nanomolar GI50 values across the entire NCI 60 tumor cell panel and one of these, compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.

Sterically induced conformational restriction: Discovery and preclinical evaluation of novel pyrrolo[3,2-d]pyrimidines as microtubule targeting agents

“…The orientation of the phenyl ring relative to the pyrimidine fused heterocycle shown in Fig. 2B and 3B and in previously reports 11, 15 are distinct from the modeling reported by Banerjee et al . 14 that show a 180-degree rotation of the C4-N bond .…”

“…The additional 2’-OCH 3 moiety incorporated in 7 to mimic the C-ring of colchicine did not improve activity compared to 6 , thus suggesting that N5-methylation alone is not sufficient to significantly improve the microtubule depolymerizing activities of 6 and 7 in comparison with 2 and 3 , reiterating the importance of the N4 methyl group for inhibition of tubulin polymerization. 8, 10, 11 Compounds 9–13 were about 20- to 60- fold more potent than 1 for microtubule depolymerizing activity, but less potent than 5 in this assay. The N5-alkyl groups can have two effects: allowing additional hydrophobic interactions with tubulin and/or restricting the rotation around the N-phenyl bond due to steric bulk, thus providing conformational rigidity.…”

“…The importance and designation of the orientation for the phenyl ring has been previously described in detail. 11 Poses similar to 2A/3A and 2B/3B were revealed from the molecular modeling of 8 . In keeping with the recently reported crystal structure 14 and the molecular modeling of 1 and 5 , the pose with the phenyl ring oriented on top of the 5-N of the pyrrole ring was also obtained for 8 .…”

“…In previous studies, we discovered that the methyl group on the N4 is essential for potent activity as a MTA. 8, 10, 11 Compounds 6 and 7 with a N5-CH 3 moiety were designed to restrict conformation about bonds a and b in the absence of the N4-CH 3 , in an attempt to determine if the N5-CH 3 could sterically mimic the functions of the N4-CH 3 and compensate for its absence and its conformational influence on bonds a and b. The number of low energy conformations of 6 and 7 within 1 kcal/mol were found to be 87 and 65, respectively, suggesting a restricted conformation, compared to 1 (88 low energy conformations), at least for 7 .…”

Recent Progress on Tubulin Inhibitors with Dual Targeting Capabilities for Cancer Therapy

“…Hence, targeting multiple main angiokinases (such as EGFR, VEGFR, and PDGFR) may be a more effective approach. Accordingly, Gangjee et al 58 further investigated the effects of conformational changes on compound 3 to develop new molecules that can simultaneously target multiple angiokinases and microtubule (Figure 3A). Compound 4 was 44-fold more potent for inhibition of tubulin polymerization (IC 50 = 0.48 μM) than compound 3 and significantly inhibited VEGFR, EGFR, and PDGFR-β with IC 50 values of 33, 2.3, and 10.3 nM, respectively.…”