Design,Synthesis And Biological Evaluation Of Novel Pyrazolopyrimidines As Potent Modulators For TRPC3/6/7 Channels

Transient receptor potential canonical(TRPC)channels are nonselective cation channels and play a vital role in many tissues and organs,including excitation-contraction coupling in smooth muscles,protein filtration in the kidney,synaptic formation and transmission,myofibroblast transdifferentiation and wound healing,regulation of vascular tone,cell growth and proliferation.TRPC channels are important for a plethora of cellular functions in many physiological systems.The seven mammalian TRPC members can be further divided into four subgroups(TRPC1;TRPC2;TRPC4/5 and TRPC3/6/7)based on their amino acid sequences and functional similarities.The homology of TRPC3/6/7 is as high as 65%-78%.The research on TRP has made great progress since its first member TRPC1 identified in 1995.However,the exact roles played by TRPC channels in kidney and cardiovascular functions and the underlying mechanisms concerning how they contribute to these functions and/or pathogenesis have proven difficult to elucidate due to the lack of specific compounds that modulate these channels.While selectivity remains lacking among TRPC3/6/7 channels,identification of their pharmacophores through systematic structure-activity approaches can be expected to lead to development of subtype-selective TRPC3/6/7 agonists or antagonists,and may perhaps lead to development of novel treatment strategies for kidney and cardiovascular diseases or cancer.In the early stage of study,we focused on the TRPC3/6/7 agonists'research.Using a cell-based high throughput screening approach,a lead compound 4o with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist.Synthetic schemes the lead and its analogs were established and structural-activity relationship studies were carried out using fluorescence membrane potential and intracellular calcium measurements,as well as whole-cell electrophysiological recordings.A series of potent and direct agonists of TRPC3/6/7 channels were identified and among all the 16 newly synthesized compounds,4m-4p act well.Their EC50 on TRPC6 ranged from 1.39±0.01 to 7.79±0.05 ?M,and on TRPC3 ranged from 19±2 to 236±47 nM,while on TRPC7 ranged from 90±12 to 497±91 nM.Their potency order was TRPC3>C7>C6,with 4n being the most potent with an EC50 of<20 nM on TRPC3.Importantly,these compounds exhibited no stimulatory activity on related TRP channels.Functional TRPC6 was found to be widespread overexpressed in many prostate,breast,glioma,liver,renal,oesophagus,stomach and lung tumour entities,in opposition to low or undetectable levels in the corresponding normal tissues.TRPC6 plays a vital role in cancer-related cell signaling pathways,and increased proliferation,induced differentiation or apoptosis.TRPC6 may simultaneously have an important role in most liver and gastric cancers given its up/mis-regulation in tumourigenic tissues.A recent report further supports that Ca2+elevation regulated by TRPC6 was critical for G2/M phase transition in gastric cancer cells and contributed to the development of human gastric cancer.Our advanced SAR exploration effort has led to the identification of a variety of novel pyrazolo[1,5-a]pyrimidine antagonists with excellent selectivity and activities on both TRPC6 and gastric cancer cells,MKN45 and AGS through further chemical modification of agonists 4o.Compound 19c was obtained with excellent TRPC6 inhibitory activity(IC50=2.20±0.18 ?M)and selectivity.It was found that 19c inhibit the growth of gastric cancer cell lines AGS and MKN45.It can also inhibit the tube formation and migration of HUVEC.In vivo toxicity of compound 19c was examined and did not cause toxic reactions towards normal cells.Body weight showed no significant difference between control and 19c groups.Tmax of 19c liposome was 4 h,Cmax was 5.35±2.89 ?g/mL,and the MRT was 3.55±0.38 h,AUC was 28.31 ±14.78 h*?g/mL.The inhibiton on AGS tumor in vivo was 38.5%and 61.5%in two weeks at a dose of 100 mg/kg or 200 mg/kg respectively.The potent and selective compounds,including both the agonists and antagonists,described here should be suitable for the evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases,including glomerulosclerosis and cancer.