“Design, Synthesis, and Biological Evaluation of Pyrazolopyrimidine Derivatives"

Abstract

By adopting fragmonic and structural hybrid approache, a model of structural hybrid between pyrazolopyrimidine heterocylic cores and p-substituted diaryl shiffs base fragment was designed. The design of small molecule was based on biologically multitarget cytotoxic lead structures like roscovitine, resveratrol to decrease drug resistance of cancer cells. The model reprsented by synthetic molecules VIIa-t and IXa-i were prepared by direct arylation of 4-chloropyrimidine derivatives Va-f with p-substituted phenolic aldehyde or m-substituted phenolic aldehyde either by direct nucleophilic substitution or under metal catalyzed assisted synthesis then condensation of the aldehyde products VIa-f and VIIIa-f with different aromatic amines, another model Xa-t was designed through alkylation of NH group of pyrazolopyrimidinone IVa-f. All synthetic steps were performed under open vessel microwave assisted synthesis conditions. Five compounds (VIIf, VIIm, VIIo, Xj and Xr) were selected by National Cancer Institute “NCI” (www.dtp.nci.nih.gov); under the Developmental Therapeutic Program (DTP) USA for single dose screening program at 10 uM in the full 60 cell panel. One compound (VIIo) was further screened at five different concentrations. All the target synthesized molecules (VIIa-t and IXa-i) were evaluated in vitro for their anti-proliferative activity against colon cancer cell line HCT-116 while synthetic molecules Xa-t screened against breast cancer MCF-7 at National cancer institute Egypt. Most of the synthesized compounds VIIa-t and IXa-i showed excellent antiproliferative activity ranging from 7.97 μM to 74.43 μM against HCT-116 cell line, while N-alkylated pyrazlopyrimidinone (Xa-t) derivatives showed a range from 8.22 and 34.78µM against MCF-7. In order to explore the mechanism of cytotoxicity, DNA-flow cytometry analysis for the effect of the molecules (VIIo, IXi) on HCT-116 cancer cell, which showed that these molecules arrested the cancer cells in G0/G1 phase, in addition, measurment of apoptotic regulator PUMA, BAX, caspase-3 level in colon cancer cell line after treatment with the molecule (VIIo, IXi) showed that they have a pro-apoptoitic activity compared with control. Also two compounds (Xj, Xr) from the second series were analyzed by cell cycle flow analysis where they showed cell cycle arrest of breast cancer MCF-7 mainly in Go/G1 phase. Furthermore, CDK2 kinases enzyme inhibition % assay was done for molecules (VIIe, VIIo, VIIt, IXd, IXe, IXh, IXi) and IC50 for the molecules (IXd, IXe, IXh, IXi) showed selective inhibition CDK2. Finally,to investigate cell cycle arrest of HCT-116 due to effect of synthetic molecule, western blot analysis for expression of CDK-2, cyclin A, P53, P21 and P27 was performed in dose dependant manner that suggested this molecule (VIIo) may be CDK pathway inhibitor one of its targets. As conclusion, in this investigation, a different series of pyrazolopyrimidine derivatives were discovered as potential cytotoxic agents against HCT-16 and MCF-7 cancer cells.