Discovery And Development Of 2,3-Difluoro-5-(Phthalazinone-4-Methyl)-Benzamides As PARP Inhibitors And Second-Generation FP-2&PfDHFE Dual Inhibitors As Antimalarial Drugs

There are two parts in my dissertation for Ph.D.degreee.Part 1 presents the discovery of 2,3-difluoro-5-(phthalazinone-4-methyl)-benzamides as PARP inhibitors.Breast cancer is a type of cancer with a high incidence of Chinese women.Poly(ADP-ribose)polymerases(PARPs)are ADP-ribosyl transferases that cleave nicotinamide adenine dinucleotide(NAD+)to give nicotinamide and an intermediate oxonium ion,which react with nucleophiles on the target proteins(such as histones,topoisomerases,DNA polymerases,and DNA ligases)or themselves.PARPs are involved in a number of important biological processes,such as the surveillance of genome integrity,progression of the cell cycle,initiation of the DNA damage response,apoptosis,and regulation of transcription.Among the PARP family members,PARP-1 is the most abundant and plays an essential role in the repair of DNA single-strand breaks(SSBs).It accounts for more than 90%of ADP-ribosylation occurring in cells and is evolutionarily conserved in all advanced eukaryotes.PARP-1 is overexpressed in a variety of cancers,and its expression has been associated with overall prognosis in cancers,especially breast and ovarian cancers,so PARP-1 has emerged as an attractive anticancer drug target for effecting DNA damage.Cell lines deficient in breast cancer genes 1/2(BRCA1/2,two known tumor suppressor genes whose mutations are associated with breast,ovary,prostate,and pancreas cancers)were 1000-fold more sensitive to PARP inhibition than wild-type or heterozygous mutant cells.Therefore,the inhibition of PARP-1 can induce highly selective killing of BRCA1/2-deficient tumor cells.In this regard,many small molecules targeting PARP-1 have been developed,either as chemosensitizers in combination with ionizing radiation or DNA-damaging chemotherapeutic agents or as stand-alone(synthetic lethal)therapies to kill cancers defective in DNA repair mechanisms(esp.BRCA 1/2 mutant breast or ovarian tumors).In this study,with the help of molecular docking,40 completely novel 2,3-difluoro-based analogues derived from olaparib(AZD2281)have been synthesized and tested in a PARP-1 enzymatic inhibitory assay.Preliminary SARs were obtained,which showed that the 2,3-difluorophenyl linker in the new analogues inherited the PARP-1 inhibitory activity contributed by the 2-fluorophenyl linker.The molecular docking results also validated that the incorporation of a fluorine atom at the 3-position of the phenyl linker led to formation of two additional hydrogen bonds with Tyr889.Lead optimization led to the identification of A40.which showed high selectivity and high potency against PARP-1 enzyme(IC50=1.3 nM).V-C8 cells(IC50= 0.003 nM),Capan-1 cells(IC50 = 7.1nM)and MDA-MB-436 cells(IC50 ?0.2 nM).In mechanism-based in vitro assays.analogue A40 had a more potent PARP-1-DNA trapping and DSBs-induction activities than AZD2281 in both BRCA1-and BRCA2-deficient cells.Compound A40 also consistently activated the cell cycle checkpoint,induced G2/M arrest in BRCA2-deficient Capan-1 cells,and induced caspase-dependent apoptosis in BRCA1-deficient MDA-MB-436 cells.Compound A40 had potent cell killing capacity in three HR-deficient cells(V-C8,MDA-MB-436 and Capan-1 cells)that was over 50-fold greater than that of AZD2281.Compound A40(50 mg/kg,94.2%)had a more beneficial effect on tumor growth inhibition than AZD2281(100 mg/kg,65.0%)in a BRCAl-mutated xenograft model and significantly inhibited tumor growth(40 mg/kg,48.1%)in a BRCA2-mutated xenograft model,which slightly weaker than the control drug BMN673(0.5 mg/kg,80.0%),but with no negative influence on the body weight of the mice.Compound A40 displayed good transmembrane permeability,plasma stability and plasma protein binding rate.However,it also exhibited faster plasma clearance as well as a shorter terminal elimination half-life,which caused A40 suffered from a poor PK profile with 4.7%oral bioavailability in rats.Quantitative analysis and metabolite identification of A40 by LC-MS/MS gave some clues as to some alternative analogue scaffolds with better PK profiles to investigate in future work.Overall,A40 has the potential to be developed as an antitumor drug,especially against BRCA-deficient tumors,by targeting PARP-1.Part 2 presents the development of second-generation FP-2&PfDHFR dual inhibitors as antimalarial drugsMalaria,a mosquito-borne disease caused by infection with Plasmodium parasites,is a devastating parasitic disease causing widespread mortality and morbidity across many parts of the developing world.In P.falciparum,various proteases catalyze the degradation of human hemoglobin,and the amino acids derived from this process are incorporated into parasite proteins or utilized for energy metabolism.Cysteine protease falcipain-2(FP-2)of P.falciparum is an indispensable protease involved in this metabolic process.FP-2,belonging to the family of cysteine proteases(papain-like enzymes known as clan CA),is expressed during the erythrocytic stage of the life cycle of the parasite.FP-2 inhibitors have been reported to be capable of inactivating the enzyme and could block parasite hemoglobin hydrolysis,halt the development of culture parasites.FP-2 has emerged as a promising target for the development of novel antimalarial drugs.P.falciparum dihydrofolate reductase(PfDHFR)has received considerable attention for the prophylaxes and the treatments of P.falciparum infection.The de novo synthesis of folate is required for DNA synthesis in Plasmodium species.The final step of folate synthesis is the enzymatic reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate,which is catalyzed by the enzyme PfDHFR.Furthermore,PfDHFR is an essential metabolic enzyme that plays a critical role in one-carbon transfer reactions,including the biosynthetic pathways for deoxythymidine monophosphate(dTMP),purines,and several amino acids,which is necessary for the synthesis of DNA.Consequently,a more powerful pesticidal effect could be achieved by inhibiting FP-2 and PfDHFR simultaneously.In this study,based on the structures of uniform fragments of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and PfDHFR,41 completely,two novel series of dual inhibitors through fragments assembly—2,4-diaminopyrimidines and 2,4-diaminoquinazolines analogues derived from B2 have been synthesized and tested in FP-2 and PfDHFR enzymatic inhibitory activities.Lead optimization led to the discovery of C22,which showed high potency against FP-2(IC50= 10.0 ?M),PfDHFR(IC50=84.1 nM),P.falciparum 3D7(IC50= 53.1 nM),P.falciparum Dd2(IC50=1.2 ?M),clinical isolated strains Fab9(IC50=14.2 nM)and GB4(IC50= 23.4 nM).The in vivo inhibition assays against P.berghei in 10 days indicated C22 had a more beneficial effect on the growth inhibition of P.berghei than artemisinin and an identical effect with pyrimethamine.Additionally,C22 moderately inhibited the proliferation of chloroquine-resistant P.falciparum Dd2 strain and the molecular docking results also validated that the 2,4-diaminopyrimidines and 2,4-diaminoquinazolines formed a complicated hydrogen-bond network with Asp54,Ile 14,Cys15 and Ilel64 in the catalytic subpocket of the PfDHFR enzyme.Collectively,these data revealed that C22 could be an excellent lead compound as FP-2 and PfDHFR dual inhibitor for the treatment of malaria.