Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure–activity relationship, in vitro and in vivo evaluation as antitumor agents



Document Type

Journal Article

Publication Date


Publication Title

Bioorganic and Medicinal Chemistry Letters






Colchicine site, Microtubule targeting agents, Nuclear Overhauser Effect spectroscopy, Pyrazolo[4, 3-d]pyrimidine, Structure–activity relationship


The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC50s near ∼1 μM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.

Open Access

Green Accepted