Pyrazolo[4,3-d]pyrimidine Bioisostere of Roscovitine: Evaluation of a Novel Selective Inhibitor of Cyclin-Dependent Kinases with Antiproliferative Activity

Abstract

Inhibition of cyclin-dependent kinases (CDKs) with small molecules has been suggested as a strategy for treatment of cancer, based on deregulation of CDKs commonly found in many types of human tumors. Here, a new potent CDK2 inhibitor with pyrazolo[4,3-d]pyrimidine scaffold has been synthesized, characterized, and evaluated in cellular and biochemical assays. 7-Benzylamino-5(R)-[2-(hydroxymethyl)propyl]amino-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine, compound 7, was prepared as a bioisostere of the well-known CDK inhibitor roscovitine. An X-ray crystal structure of compound 7 bound to CDK2 has been determined, revealing a binding mode similar to that of roscovitine. Protein kinase selectivity profile of compound 7 and its biological effects (cell cycle arrest, dephosphorylation of the retinoblastoma protein, accumulation of the tumor suppressor protein p53, induction of apoptosis, inhibition of homologous recombination) are consistent with CDK inhibition as a primary mode of action. Importantly, as the anticancer activities of the pyrazolo[4,3-d]pyrimidine 7 exceed those of its bioisostere roscovitine, compound 7 reported here may be preferable for cancer therapy.

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“…The cells were cultivated according to the instructions provided by suppliers. Cell viability assay was performed using calcein AM (Invitrogen, Carlsbad, CA, USA), the fluorescence of viable cells was measured at 485/538 nm (ex/em) using a Fluoroskan Ascent Plate Reader (Thermo Labsystems, Waltham, MA, USA) according to Jorda et al [19]. …”

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“…Lysates were then incubated for 3 h with 100 µM Ac-DEVD-AMC as a substrate of caspases 3 and 7 in the assay buffer (25 mM PIPES, 2 mM EGTA, 2 mM MgCl 2 , 5 mM DTT, pH 7.3). The fluorescence of the product was measured using a Fluoroskan Ascent microplate reader (Labsystems) at 355/460 nm (excitation/emission) as described previously [39,40]. …”

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“…The fluorescence of the product was measured using a Fluoroskan Ascent microplate reader (Labsystems) operated at 355/460 nm (excitation/emission) as described previously. 31 …”