Identification of a promising hit from a new series of pyrazolo[1,5-a]pyrimidine based compounds as a potential anticancer agent with potent CDK1 inhibitory and pro-apoptotic properties through a multistep in vitro assessment.

A new series of sixteen new 2-arylamino-5,7-disubstituted-N-aryl-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivatives was designed and synthesized. The antitumor activities of the new compounds were initially screened through the developmental therapeutics program at NCI-USA 60 cell line panel. 2-((2,4-dimethoxyphenyl)amino)-5,7-diphenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7a) was identified as a potential hit with a mean percentage of growth inhibition of 48.5% over the 60-NCI cancer cell lines whereas the other fifteen compounds ranged from 0.5 to 10.72%. In MTT assay, compound 7a exhibited IC50 of 6.28 ± 0.26 µM and 17.7 ± 0.92 µM against HCT-116 colorectal cancer and WI-38 human lung fibroblast normal cell lines, respectively. In cell cycle analysis, compound 7a arrested cell cycle at G2/M phase. It was able to inhibit CDK1 (Cyclin-Dependent Kinase 1)/Cyc B (Cyclin B) complex at IC50 161.2 ± 2.7 nM. The apoptosis-inducing ability of compound 7a was assessed through apoptosis detection flow-cytometry and gene expression analysis of apoptosis markers and caspase cascade which revealed that compound 7a exerts pro-apoptotic effect and increased expression of p53, Bax, cytochrome c, caspases (-3,-8, and-9), and decreased expression of Bcl-2. This suggests that the pro-apoptotic effect is exerted through the intrinsic pathway. The molecular docking study revealed a unique binding mode at the ATP binding pocket of CDK1/Cyc B/Cks2 through its 2,4-dimethoxyphenyl-amino. These results suggest that compound 7a could be a promising hit as a targeted protein kinase inhibitor which exerts its antitumor effect through CDK1 inhibition and pro-apoptotic action.

Synthesis, in vitro biological investigation, and molecular dynamics simulations of thiazolopyrimidine based compounds as corticotrophin releasing factor receptor-1 antagonists.

Corticotrophin releasing factor receptor-1 (CRFR1) is a potential target for treatment of depression and anxiety through modifying stress response. A series of new thiazolo[4,5-d]pyrimidine derivatives were designed, prepared and biologically evaluated as potential CRFR1 antagonists. Four compounds produced more than fifty percent inhibition in the [125I]-Tyr0-sauvagine specific binding assay. Assessment of binding affinities revealed that compound (3-(2,4-dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-one) 8c was the best candidate with highest binding affinity (Ki = 32.1 nM). Further evaluation showed the ability of compound 8c to inhibit CRF induced cAMP accumulation in a dose response manner. Docking and molecular dynamics simulations were used to investigate potential binding modes of synthesized compounds as well as the stability of 8c-CRFR1 complex. These studies suggest similar allosteric binding of 8c compared to that of the co-crystalized ligand CP-376395 in 4K5Y pdb file.

An investigative study of antitumor properties of a novel thiazolo[4,5-d]pyrimidine small molecule revealing superior antitumor activity with CDK1 selectivity and potent pro-apoptotic properties.

New thiazolo[4,5-d]pyrimidine analogues were synthesized and biologically assessed in-vitro for their antineoplastic activity. The growth inhibitory effects of these compounds were assessed through the National Cancer Institute-United States of America (NCI-USA) anticancer screening program. Compound5(7-Chloro-3-(2,4-dimethoxyphenyl)-5-methylthiazolo[4,5-d]pyrimidine-2(3H)-thione) was found to have a potent and broad-spectrum cytotoxic action against NCI panel with GI50 (50% growth inhibition concentration) mean graph midpoint (MG-MID) = 2.88 µM. MTT assay was used to determine IC50 values of the most potent agent against HCT-116 colorectal carcinoma and WI-38 human lung fibroblast cell lines; 5.33 µM ± 0.69 and 21.69 µM ± 1.04, respectively. Flow cytometric analysis revealed that compound5triggered apoptosis and G2/M cell cycle arrest. The ability of compound5to inhibit CDK1 (Cyclin-Dependent Kinase 1)/Cyclin B complex was evaluated, and its IC50 value was 97 nM ± 2.33. Moreover, according to the gene expression analysis, compound5up-regulated p53, BAX, cytochrome c, caspases-3,-8 and-9 besides down-regulated Bcl-2. In conclusion, compound5exerted a potent pro-apoptotic activity through the activation of the intrinsic apoptotic pathway and arrested the cell cycle at the G2/M phase.