Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels

Abstract

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated non-selective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural–activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/ 7 channels were identified, and among them, 4m–4p have a potency order of 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. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Pyrazolo[1,5-a]pyrimidine TRPC6 antagonists for the treatment of gastric cancer

“…Pyrazolopyrimidines 4a-4h , 4o, 5a-5c , 9a , 14a-14h , 16 , 17a , 17b , 19a-19j were prepared via similar synthetic routes as reported previously [33] and/or described in the Supporting Information. Human gastric cancer cell lines (AGS and MNK-45) and normal human kidney cells (HK-2) were obtained from Prof. Yizheng Wang and Prof. Xiaoguang Chen cultured in RPMI-1640 media supplemented with 10 % fetal bovine serum.…”

“…HEK293 cells growth and stable cell lines that express human TRPC3, mouse TRPC6 plus M5 muscarinic receptor, mouse TRPC4 plus μ opioid receptor, mouse TRPV3, and mouse TRPM8 were established as described previously [33,34]. For those that co-expressed M5 muscarinic or μ opioid receptor, 100 μg/mL hygromycin B (Calbiochem) was also included in the culture medium.…”

“…For those that co-expressed M5 muscarinic or μ opioid receptor, 100 μg/mL hygromycin B (Calbiochem) was also included in the culture medium. Stable cell lines that inducibly express human TRPC7, TRPA1, and TRPV1 were established as described previously [33,35] and maintained in the medium supplemented with 100 μg/mL hygromycin B and 5 μg/mL blasticidin [26]. The expression of TRP channels was induced by the addition of 0.1 μM doxycycline to the culture medium 24 h prior to the assay.…”

“…The detailed protocols for the Flexstation FMP and Ca 2+ assays have been described previously [33,35]. Briefly, the extracellular solution for all FlexStation assays contained (in mM): 140 NaCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH.…”

“…We have previously reported the identification of a novel pyrazolopyrimidine-based TRPC6 lead agonist ( 4n ) with an EC 50 of ~1 μM [33]. Here, we report further modifications of 4n to provide additional analogues and among them, we identified a potent TRPC6 antagonist, compound 14a , which not only effectively inhibited gastric cancer cell growth in culture, but also suppressed xenograft tumor formation in nude mice with excellent bioavailability, demonstrating the therapeutic potential of TRPC6 antagonists in treating gastric cancer.…”