Design, Synthesis And Bioactivity Study Of Novel [1,2,4]Triazolo[4,3-A]Pyrazines As PARP-1 Inhibitors

Poly (ADP-ribose) polymerases (PARPs) were the crucial enzymes in the DNA repair pathway. It could block the damaged DNA repair and finally lead the cell death when inhibiting the activities of enzymes. And its inhibitors were widely used as plant growth regulators, fungicides, pesticide, anticancer, stress response diseases and so on. Although PARP inhibitors extensive applications, we concerned was the application in fungicides and insecticides. The reported PARP inhibitors were designed to mimic the nicotinamide of NAD+to compete with NAD+to bind to the donor site of the PARP catalytic domain, like olaparib or niraparib. Our initial work was focused on the design of aminoglycosides analogues by mimicking nicotinamide mononucleotide (NMN) of the NAD+that could improve the potency of PARP inhibition and obtain good PK profiles.We initially attempted to use 3-aminobenzamide and glycoside 3-8a to synthesize the key intermediate 3-7. To our surprise, intermediate 3-7 was not observed, but cyclization products 3-9a and 3-9a' were obtained. The cyclization products 3-9a and 3-9a'had the pivotal scaffold of the antitumor natural product Marmycin A. This unique scaffold could obtain from glycal and aniline which had been reported previously. The high reactivity of the glycal substrate is also the major reason for several side reactions. 2,3-Unsaturated glycosides 3-8 which retained sufficient reactivity and possessed better stability to construct both C-and N-glycoside linkages simultaneously without side reactions caused by the high reactivity and moisture/alcohol-sensitive glycals.At the same time, we used a cyclopropane 3-13a to react with 4-chloroaniline and hypothesized that the ring-opening of cyclopropane will lead to a 7-member ring aminoglycoside. After carefully examined the'H NMR, we found what we got actually the pyrrole derivative 3-14a. After condition optimizion, we synthesized a series derivative of products 3-14. The cytotoxicity of representative compounds 3-14b,3-14n, 3-14o and 3-14t, as well as clinical anticancer drug etoposide (as positive control), was evaluated against several cancer cell lines, including human lung cancer A549 cells, squamous carcinoma KB cells and vincristine-resistant KB/VCR cells. Compared to the potency of etoposide, our new synthetic compounds 3-14 showed moderate activity in all the three cancer cells, but slightly higher sensitivity toward vincristine-resistant KB/VCR cell was observed for 3-14, opposite from the selectivity profile of etoposide. In a word, we found a new method for synthesis of 3-14, which show moderate cytotoxicity against several cancer cell lines.In the meanwhile, in order to find new PARP-1 inhibitors we screened our inner compounds library and find a 2-arylaminobenzothiazole 4-1 with the excellent PARP-1 inhibition activity (ICso= 200 nM). However, we found that it does not have the cell activity (CC50= 10>?M, BRCA2m, V-C8 cells). At the same time, we also found that the compound 4-1 had a certain inhibitory activity on PARP-14. PARP14, another member of PARPs super family, might be another potential target for prevention of tumor migration and invasion according to recent intensive studies. Accordingly, we envisioned that novel dual-target inhibitors against PARP14 could not only inhibit cancer cells proliferation, but also prevent their migration and invasion, which would be much beneficial for cancer treatment. After condition optimizion, we developed a palladium-catalyzed N-arylation of the biologically interesting, but synthetically rather challenging 2-arylaminobenzothiazoles bearing multiple functionalities. This protocol was successfully used to readily synthesize our initial PARP14 inhibitor followed by a limited structural optimization. More potent PARP14 inhibitor with an IC50 value of 1.69 ?M, respectively was identified, and the interaction was ascertained by the X-ray co-crystal structure of the catalytic domain of PARP14 in complex with compound 4-8. The binding mode of PARP14 with 4-8 was analyzed through co-crystallization of the complexes?We chose the reported PARP-1 inhibitor 1-2 as the lead compound for further structural optimization for improving the efficience. First, we designed and syntheised a series of tetrahydroisoquinoline PARP-1 inhibitors 5-10. The tetrahydroisoquinoline PARP-1 inhibitors 5-10 only showed moderate PARP-1 inhibition, at least 12-fold less potentcy than compound 1-2. Then, we designed and synthesized several series of heterpyrazine PARP-1 inihibitors, i.e., [1,2,3]triazolo[1,5-a]pyrazine 5-23, [1,2,4]triazolo[4,3-a]pyrazine, thieno[3,2-c]pyridine and imidazo[4,5-c]pyridine derivatives 5-25. All the series of heterpyrazine PARP-1 inhibitors (except [1,2,3]triazolo[1,5-a]pyrazine) were identified with high PARP-1 inhibitory potency with low nanomolar range of enzyme activities Further cellular activities evaluation tests exhibited that only compound 5-25c displayed cytotoxicity against BRCA deficient cells (compounds 5-25 failed to show any cytotoxicity against BRCA1 deficient cells inspite of their high level enzyme activities). Unfortunately, compound 5-25c showed significant toxicity during our preclinical investigation.Compound 5-25c was selected as the hit compound for further structural optimization to increase the cell permeability. When we introduced substituents on the triazole ring afforded a compound 5-34n which much improved cellular activity against BRCA deficient cells. It is worth noting that compound 5-34n also exhibited high cellular potency against BRCA1 deficient cells which resistant to current PARP-1 inhibitors 1-2 and 1-6. However, the compound 5-34n suffered from moderate hERG inhibitory activity (ICso= 17.4 ?M). To address this issue, we developed a compound 5-41c with retained high cellular activity against both BRCA deficient cells and resistant BRCA2 deficient cells. More importantly, this compound did not show any inhibition of hERG at the concentration of 40 ?M. At the oral dose of 20 mg/kg once daily for 21 days, compound 5-41c showed partial inhibit against the growth (T/C= 57%) of Capan-1 tumor in mice xenograft models bearing the BRCA-2 mutant and without body weight loss. In summary, we developed a class of [1,2,4]triazolo[4,3-a]pyrazine PARP-1 inhibitors, and validated by in vitro and in vivo experiments. Next, we will investigate the bactericidal and insecticidal activities.