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  • Pyrazolopyrimidines are bioisostere of purine exhibit promis

    2020-11-23

    Pyrazolopyrimidines are bioisostere of purine exhibit promising antitumor activity by competitively binding to the ATP active site of different kinase gpr109a inhibitor [16,17]. Several compounds of this family were found to induce apoptosis and/or reduce cell proliferation in various solid tumour and leukaemia cell lines [[18], [19], [20], [21]]. Their anticancer properties being accredited to the inhibition of various key enzymes such as mammalian target of rapamycin (mTOR) [22], glycogen synthase kinases 3 (GSK3) [23], Src/Abl tyrosine kinases [24,25], cyclin-dependent kinases (CDK) [26], xanthine oxidase [27], and other kinases [28]. Fig. 2 depicts several reported derivatives of pyrazolo [3,4-d]pyrimidine displaying potential anticancer activities by inhibiting different protein kinases [25,[29], [30], [31], [32], [33], [34]]. Based on the above-mentioned facts and in continuation of our research work on anticancer drug discovery [[35], [36], [37], [38], [39]], we envisaged to further exploit the pyrazolo [3,4-d]pyrimidine scaffold to synthesize novel CDK2 inhibitors. In our present study, a novel series of pyrazolo [3,4-d]pyrimidine derivatives (Scheme 1, Scheme 2) were synthesized through a molecular hybridization approach by substituting various chemical entities at C-4 and C-6 positions and evaluated against a couple of kinases (CDK2 and Abl kinase) as well as cancer cell lines (K-562 and MCF-7). Further, in silico molecular docking studies were performed to analyze the binding energies and orientations of these compounds with respect to the active site of the CDK2 protein.
    Results and discussion
    Conclusion In summary, we have successfully synthesized and characterized different analogues of mono and disubstituted pyrazolo [3,4-d]pyrimidine with good yields. The key intermediates 6-mercapto-1H-pyrazolo [3,4-d]pyrimidin-4-ol (4), 6-(pentylthio)-1H-pyrazolo [3,4-d]pyrimidin-4-ol (10), 6-(phenethylthio)-1H-pyrazolo [3,4-d]pyrimidin-4-ol (11) and 6-(hexylthio)-1H-pyrazolo [3,4-d]pyrimidin-4-ol (12) allowed us to generate a library of 34 fused pyrimidine derivatives (9a-9s, 13a-13h, 14a-14d and 15a-15c). All synthesized compounds were evaluated for in vitro enzymatic inhibitory activity against CDK2/cyclin E, Abl kinases as well as anti-proliferative activity against K-562 and MCF-7 cancer cell lines. Interestingly, it was observed that compounds 14c, 13a, 15a, 13c and 15b displayed best CDK2/cyclin E activity with IC50 values ranging from 6.8 to 21.2 μM. Further, compounds 14b (IC50 = 20.4, 25 μM), 14c (IC50 = 19.8, 18.9 μM) and 14d (IC50 = 23.2, 18.9 μM) displayed appreciable anti-proliferative activity at specific IC50 values. From the SAR study, it was clear that the presence of the benzofuran at C-4 of the scaffold led to prominent activity. In addition, the binding interaction and energies (in silico) of the best active compound (14c) were in agreement with the experimental data and supported the SAR studies. Moreover, the cytotoxicity profile of the most active compounds demonstrated that the compounds are safe to the normal cells. Thus, these research findings could further guide the researchers in developing novel pyrazolo [3,4-d]pyrimidinebased CDK2 inhibitors as potential anticancer agents.
    Experimental section
    Biological activity protocol
    Molecular docking simulation Molecular docking experiments were performed using Glide software package [46] implemented in Schrodinger Suite (2017–2) (Schrödinger, Inc., USA) [47] running on Intel CORE i7 based hpZ230 workstation with the Microsoft Windows 10 OS. In this protocol, the protein was kept rigid, while the ligands were allowed to be flexible throughout the docking simulation.
    Conflicts of interest
    Acknowledgments Authors are thankful to the Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal (UKZN), South Africa, for their constant support, encouragement and financial assistance. The corresponding author RK is also thankful to National Research Foundation- South Africa (NRF-SA) for funding this project (Grant No. 103728; 112079). Authors wish to thank Dr Vladimir Krystof (Palacký University Olomouc, Czech Republic) for performing biochemical and cellular assays () and Director, Indian Institute of Chemical Technology (IICT-Hyderabad) for cytotoxicity studies. One of the authors (CB) gratefully acknowledges the National Research Foundation (DST-NRF), South Africa for research funding in the form of an Innovation Post-Doctoral Research Fellowship (UID: 99546). Authors also sincerely thank the Centre for High Performance Computing (CHPC), Cape Town, South Africa for computational resources. Authors express heartfelt thanks to Mr Dilip Jagjivan and Dr Caryl Janse Van Rensburg (UKZN, South Africa) for their assistance in the NMR and HRMS experiments.