Design, Synthesis And Biological Evaluation Of PARP-1 Inhibitors

Cancer is a major public health issue greatly threatening human health, and has aroused wide attention around the world. As a major developing country with aging populations, we are facing with more severe situation that both incidence and mortality of cancer continued to show an upward tendency. Breast cancer and ovarian cancer are common gynecological malignancies. Breast cancer is the most common cancer among women and triple-negative breast cancer with high invasive and poor prognosis lacks effective therapy in clinic. Ovarian cancer has caused more deaths than any other cancers of the female reproductive system, and the symptoms are insidious in onset and difficult in early diagnosis. Relapse and resistance are always the problems in clinic. Epidemiological investigation identified that 10~40% breast cancers and 8~18% ovarian cancers were detected with BRCA1/2 gene mutations. These genetic changes raise the risk of breast and ovarian cancer on the one hand, but provide an opportunity for the prevention and treatment at the same time. Recently, PARP-1 inhibitors used for the treatment of BRCA-1/2 deficient breast and ovarian cancers as monotherapy or in combination with radiotherapy and chemotherapy have become a focus and bring hopes to triple-negative breast cancers and recurrent advanced ovarian cancers.Poly(ADP-ribose)polymerases(PARPs) are nucleus enzymes exist in eukaryotic cells, which play an important role in the process of DNA repair. PARP family composed by 18 members and PARP-1 is the most abundant and best characterized one. Using NAD+ as substrate, PARP-1 can transfer ADP-ribose to nucleus enzymes to facilitate DNA repair, and this is a key process during base excision repair(BER) process. PARP-1 is one of the antitumor targets through clinical validation, but restricted by the understanding of mechanism, the development history of PARP-1 inhibitors moves in zigzags. The initial focus was combination of PARP-1 inhibitors with radio- or chemo-therapy, but this strategy has been challenged by the enhanced toxicity of normal tissues and 30 years of research has not succeed yet. The destiny of PARP-1 inhibitors was changed until the proposal and application of synthetic lethality. PARP-1 inhibitors can induce cell death through blocking the BER pathway of homologous repair(HR) deficient tumors, such as BRCA1/2-deficient breast and ovarian cancers, and are emerging as promising agents as monotherapy. In December 2014, Olaparib was approved by FDA to treat BRCAm advanced ovarian cancer, clinical trials for other indications such as breast cancer, pancreatic cancer and prostatic cancer are in process.As a major breakthrough in cancer treatment, synthetic lethality facilitates the approval of Olaparib and sparked the application potential of PARP-1 inhibitors indeed, and also demonstrated broad prospects in other HR-deficient tumors. Beyond synthetic lethality, PARP-1 inhibitors also face many opportunities and challenges, such as combination with C-Met inhibitors, AKT inhibitors and PD-1 inhibitors. These researches broaden the potential application fields greatly and have great significance.Present PARP-1 inhibitors were designed based on the nicotinamide moiety of NAD+ and demonstrated diverse structures. We analyzed the X-ray structures of PARP with substrates and found that the common structural feathers of PARP-1 inhibitors were the key amide group and rigid plane moiety. Furthermore, we find that a large hydrophobic pocket exists in the active site. This feature allows us to introduce various substituents to enhance potency and improve solubility or other physicochemical properties. Based on the above SAR study, we take Olaparib and Veliparib as templates to design four series of compounds as novel PARP-1 inhibitors, and the four scaffolds are triazolopyrimidine, thienoimidazole, dihydroquinazolinone and triazinone, respectively. Considering the large hydrophobic pocket, we further introduce various side chains to the scaffolds. Molecular dockings were adopted to make sure the rationality of design.We have accomplished the synthesis of triazolopyrimidine, thienoimidazole and dihydroquinazolinone compounds through the rational design of synthetic routes and continuous exploration of reaction condition. According to different side chains, the synthesis of target compounds involved 7 routes, and reaction steps including condensation, cyclization, reduction, ammonolysis, etc. The synthesis of triazinones compounds met with some difficulties and further explorations are in process. 56 compounds were synthesized and confirmed by MS and 1H-NMR.Compounds were evaluated in vitro on PARP-1 enzyme, Hela cells and BRCA1/2 cells. In the PARP-1 enzymatic evaluation, triazolopyrimidine and dihydroquinazolinone compounds were determined the inhibitory rate at 10 μM preliminarily. The triazolopyrimidine compounds showed low inhibitory rate below 10%, but it might due to the poor solubility. We plan to achieve hydrochlorides of these compounds and re-determine them further. The thienoimidazole compounds with inhibitory rates higher than 50% were determined IC50 values. It was disappointed that the compounds with large piperazine side chains showed low micromolar potencies far below the control compounds, but we found that potencies were improved with the reduce of side chains bulk. It was seemed that the large side chains cannot be tolerated on the thienoimidazole scaffold. Then compounds with shorter side chains were synthesized and results showed that potencies were improved overall. Compound 27 l proved to be the most potent compound with IC50 value of 43 nM. The most potent four compounds(27g, 27 i, 27 j, 27l) were further evaluated cell- ular potencies. PARylation assay results showed that the four compounds possessed lower inhibitory activities than control compounds. However, on BRCA1/2 deficient cell lines HCC1937, four compounds demonstrated anti-proliferative activities than Olaparib and Veliparib. We speculated that membrane permeability might be one of the possible reasons, and further evaluation is in process.Through design, synthesis and biological evaluation, we found four compounds(27g, 27 i, 27 j, 27l) with relatively good enzymatic potencies, and they showed better anti-proliferative activities than Veliparib on BRCA1/2 deficient HCC1937 and CAP AN-1 cell lines, which worth further investigation. The structure and activity relationships provide clues for further design and optimization of PARP-1 inhibitors. Our study also pointed out that though present drug design methods provided diverse and rational options, the structure and activity relationships cannot be predicted accurately. The challenges of drug design also exist, but the uncertainty also brings us hopes and motivations.