Synthesis,Biological Evaluation Of Isatin Derivatives And 1,3-disubstituted Pyrazole Derivatives As Anticancer Drugs

1.Design,synthesis and biological evaluation of novel substituted pyrazole derivatives as AR antagonistsNowadays,prostate cancer is the most common malignancy in men,and it is extremely difficult to cure.Although the situation of PCa in China is not so serious when comparing to developed countries,the prevalence of PCa in China have increased a lot in these years.Androgens are a group of steroid hormones,which is common in males.Testosterone is the most prevalent among different androgens.Testosterone production is regulated through the hypothalamic-pituitary-gonadal axis,most of which(90%)is produced by the Leydig cells,and the other(10%)is produced by the adrenal glands.Androgen receptor(AR)is a nuclear steroid hormone receptor.The AR gene is located on the human X chromosome.AR contains 919 amino acid residues.It has four functionally domain.They are an N-terminal domain(NTD),a DNA-binding domain(DBD),a ligand-binding domain(LBD)and a small hinge region between DBD and LBD.Testosterone is catalyzed by 5a-reductase enzymes in the prostate,thus testosterone is converted to dihydrotestosterone(DHT).DHT plays a key role in AR signalling ways through binding to AR.It should be noted that DHT has a 10-fold higher binding affinity for the AR than testosterone in the prostate.Binding of DHT to the ligand binding domain(LBD)of the AR results in the phosphorylation of AR,then AR translocate to nucleus.The activated AR collects co-activators to androgen response elements(AREs)in the promoter regions of target genes and trigger transcriptional activities of target genes,which are crucial to cell growth and survival.Except for AR genomic signalling pathway,the action of AR can be conducted in the cytoplasm.Activated AR can interact directly with P13K,Src,thus induce the activation of MAPK/ERK(mitogen-activated protein kinase/extracellular signal regulated kinase).Moreover,AR phosphorylation occurs even in the absence of ligand binding.The normal development of the prostate is dependent on androgen and androgen receptor,and it is well known that androgens and AR play a key role in the development and progression of prostate cancer.Huggins and Hodges were the first to realize the androgen dependency of PCa growth and progression in 1941.The androgens and AR have been crucial players for therapeutic treatment of PCa since then.The current treatment strategies for early stages of PCa are radical prostatectomy and radiation therapy.As for advanced metastatic PCa,chemotherapy with docetaxel andr androgen deprivation therapy(ADT)are better choices.Urgically removing the testicles,suppressing the production of testosterone and using AR antagonists are used to treat PCa.AR antagonists,such as biclutamide,enzalutamide,work by blocking AR signalling pathway.Unfortunately,progression of disease is inevitable after a few years of ADT,and patients develop castration-resistant prostate cancer(CRPC).Numerous efforts have been done to study the mechanisms of CRPC.AR over-expression and mutations of AR ligand binding domain will lead to progression to CRPC.AR over-expression could be achieved by enhanced transcription,translation,gene amplification and abnormal splicing.There are second generation AR antagonists that are discovered for treatment of CRPC.Enzalutamide as the most familiar second generation AR antagonist could contribute to its effectiveness in treating CRPC,but drug resistance frequently happened.In a word,AR remains to be the crucial target in the treatment of PCa,and it is important to explore novel AR antagonists.Compound G01 synthesized by our research group didn't show antiproliferation property in LNCaP cells and PC-3 cells.But it could inhibit PSA secretion by 25.40%at 10 ?M.We optimized G01 by changing over 1,5 substituted of pyrozole ring to get Z series,and prolonged the coupling chains between pyrazole and benzene ring to get J series.Modeling conducted by SYBLE-X 2.0 suggested a good docking profile.The J-compounds were prepared by following scheme:we used substituted benzaldehyde as material,and got pyrazole derivatives by aldol condensation reaction,cyclization reaction.Other material substituted aniline reacted with chloroacetyl chloride to get amide.The amides reacted with pyrazole derivatives to obtain target compounds.The pyrazole derivatives reacted with acids to obtain Z-compounds.The stuctures of these compounds were identified by 1H-nuclear magnetic resonance(1H-NMR)and 13C-nuclear magnetic resonance(13C-NMR).Biological evaluations included transcriptional activation assay and cells growth inhibition assays.Antiproliferation properties were tested by MTT assay to inhibit AR positive LNCap cell line and AR negative PC-3 cell line.Luciferase reporter gene assay was conducted in MDA-Kb2 cell line to evaluate AR antagonistic activity.The results showed that most of these compounds have good antiproliferation properties in LNCap cell line,but PC-3 cell growth was not inhibited.The results conformed to the original intension of our design.Compounds J13 and J14 showed better antiproliferation activities than G01 and biclutamide since they induced antiproliferation of LNCap cell with IC50 values of 13.86 ?M and 21.09 ?M,respectively,and showed poor antiproliferation of PC-3 cell as IC50 values were all greater than 80 p.M.All compounds showed AR antagonitic activities in luciferase reporter gene assay.AR antagonitic activities of compounds J5,J6,J8,J9,J10 were better than bialutamide.The antagonism rates of compounds J8 and J10 in 1 ?M were 49.77%,59.74%,respectively.In conclusion,results showed that these novel pyrazole derivatives as androgen receptor antagonists could provide potential prospect in treatment of PCa,and we want to change different coupling chains to optimize the compounds.2.Synthesis and biological evaluation of isatin derivatives as anticancer drugsCancer is a global health problem.According to the data from Ministry of Health,cancer is the leading cause of death in China.Carcinogenesis involves complex biological processes,and is considered as a kind of genetic disease.The activation of oncogene,deactivation of anti-oncogene and mutations of other related genes might lead to abnormal cell proliferation.Surgery,radiotherapy and chemotherapy are three conventional cancer therapy strategies.Strategies for early cancer are surgery and chemotherapy,and the advanced cancer may require a variety of methods including different methods.Drugs targeting to signaling pathway of cancer cell proliferation and survival are common in the treatment of cancer,such as VEGFR inhibitors,PI3K inhibitors,AR antagonists and so on.Since BCL2 inhibitors ABT-199 was approved by FDA,Targeting apoptosis will become more important for cancer drug discovery.A series of isatin derivatives synthesized by our research group could inhibit proliferation of many cancer cells,similar structures were unclosed in reference could induce cancer cell apoptosis and could inhibit proliferation of cancer cells.Thus we decided to optimize the structure of these compounds,benzyl in 1-substituted of isatin was replaced with 3,4-dichlorobenzyl and substituted 1,2,4-oxadiazole.Compounds were obtained from isatin.Compounds of series Ty were prepared by nitratlon reaction,protection for 3-carbonyl group,substitution reaction with 3,4-dichlorobenzyl,nitroreduction,amide condensation and deprotection of carbonyl.Compounds of series Yd were prepared by nitratlon reaction,protection for 3-carbonyl group,nitroreduction,amide condensation,substitution reaction with substituted 1,2,4-oxadiazole,deprotection of carbonyl.The stuctures of these compounds were identified by 1HNMR and 13CNMR.These compounds could strongly inhibit antiproliferation of PC-3 cell.Except for two compounds,series Ty induced antiproliferation of PC-3 cell with IC50 values range from 0.2-1.5 ?M which were all lower than lead compound Tii-6p(1.64 ?M).Specifically,Ty5?Ty9 and Tyl3 inhibited PC-3 cell proliferation with IC50 values of 240 nM?210 nM and 290 nM,respectively.Series Yd had slightly lower antiproliferation properties of PC-3 cell with IC50 values range from 2-10 ?M.In conclusion,we had synthesized novel isatin derivatives as anticancer drugs.Further biological evaluations should focus on proapoptosis effect(for example,activation of caspase-3)of synthesized compounds.