Discovery Of Thieno[2,3-d]pyrimidine Based Dual Target Drugs Against Carcinoma In Vitro And In Vivo

As the bioelectronic isoforms of purines,Thieno[2,3-d]pyrimidines have been widely used in the molecular composition of anticancer drugs in recent years due to their diverse pharmacological effects and biological activities.Among various protein kinase inhibitors,thieno[2,3-d]pyrimidine-based small molecule drugs are not uncommon.Due to its excellent biological activity and great anti-tumor potential,I completed the research of dual-target antitumor drugs based on thieno[2,3-d]pyrimidine during my doctoral study.Part I was about the design,synthesis and anti-bladder cancer activity evaluation of HSP90/m TOR dual target inhibitors.Part II was about the design,synthesis and anti-ovarian cancer activity evaluation of BRD4inhibitor equipped with NO donor.Part I:Discovery of Thieno[2,3-d]pyrimidine-Based HSP90/m TOR dual Inhibitors Against Bladder cancer in vitro and in vivo.Bladder cancer,as the most common tumor in the urinary system,seriously endangers people’s health and survival with increasing morbidity and mortality year by year.The over-expression and activation of HSP90 and m TOR in bladder cancer are closely related to cancer proliferation,metastasis,invasion and angiogenesis,indicating a poor prognosis.In this study,a series of thieno[2,3-d]pyrimidine-based potential dual-target Hsp90/m TOR inhibitors were designed after analyzing and simulating the interaction pattern between inhibitors and target proteins.The HSP90and m TOR inhibitor fragments were synthesized respectively,and the total synthesis was completed by 13-15 steps of chemical reaction.In the structure-activity relationship study,we introduced different cyclic aliphatic amine substituents at pyrimidine 2 and 4 sites,and demonstrated the importance of pyrimidine 4-position morpholine substitution for m TOR inhibitor activity.Under the premise that the introduction of aniline at pyrimidine can significantly enhance the activity,the amino group of aniline was transformed into different substituted ureas,and the activity of the compounds were further enhanced.Of all the compounds synthesized,24q showed the strongest inhibitory activity against target proteins,could inhibit the activities of HSP90(IC₅₀=69±8 n M)and m TOR(IC₅₀=29±4 n M)effectively.the IC₅₀values of 24q against J82,T24 and SW780 were 360±30 n M,410±60 n M and 160±3 0 n M,respectively,better than positive controls.We docked 24q into the crystal structures of Hsp90 and m TOR respectively to explore the interaction mode between 24q and target protein.The resorcinol fragment of 24q occupies the ATP binding pocket of the HSP90 N domain.The hydroxyl in the hydrophilic region of the pocket formed a hydrogen bond with the ASN51 of HSP90,while the isopropyl in the hydrophobic region of the pocket extended into the hydrophobic cavity composed of MET98、LEU107、PHE138 and other residues,MET98 also formed S-Pi interaction with phenyl.Moreover,the van der Waals force and the hydrogen bond between the 7-carbonyl and the THR184 also enhanced the binding of 24q to the HSP90.Similarly,the hydroxy,carbonyl and fluorine can form hydrogen bonds with Gln2161,Asp2357 and Gln2194 of m TOR,respectively.Moreover,the tetrahydropyridinothiophene skeleton and chlorine atoms can form hydrophobic interactions with protein hydrophobic residues of m TOR.In addition,fluorine atoms can form halogen bonds with Asp2191 of m TOR.Annexin V-FITC/PI double staining was used to test the ability of 24q to induce apoptosis of SW780 cells,8.1%and 14.2%of the cancer cells underwent apoptosis when the 24q concentration was 0.2 and 0.5μM,respectively.The autophagosomes were observed by transmission electron microscope.Western Blot analysis showed that the expression levels of HSP90 client proteins（CDK4,c-Raf and CDC2）were significantly decreased in bladder cells treated with 24q,p-m TOR（S2448）and p-Akt（S473）,the downstream substrates of m TOR,was significantly down-regulated,indicating that 24q could effectively inhibit HSP90 and m TOR.Moreover,the increase of LC-II/I,cleaved caspase-3,cytochrome C and the decrease of SQSTM-1 indicated that 24q could induce apoptosis and autophagy.In vivo,24q could significantly inhibit tumor growth without affecting body weight,and the therapeutic effect was better than AUY922 and AZD8055.Finally,the immunohistochemical analysis of Ki67,HSP70,p-m TOR and LC3-II and the immunofluorescence analysis of TUNEL in tumor tissue showed that 24q could inhibit the proliferation of bladder cancer cells,induce apoptosis and autophagy through inhibiting HSP90 and m TOR.In conclusion,24q was an effective dual inhibitor of HSP90/m TOR,which showed promising anti-tumor effect on bladder cancer in vitro and in vivo and deserved further study.Part II:Design,synthesis and anti-ovarian cancer activity evaluation of BRD4inhibitor equipped with NO donor.The development of ovarian cancer has been a serious threat to women’s health and life.BRD4 which was amplified and up-regulated in ovarian cancer regulated the proliferation and development of cancer cells,While different concentrations of NO could promote or inhibit the development of ovarian cancer.In this part,we designed,synthesized a series of heterozygotes of BRD4 inhibitor and NO donor,and evaluated their efficacy against ovarian cancer.Firstly,4l which was developed by us previously,was selected as the BRD4 inhibitor,hybridized with four different types of NO donors,11 compounds included.Phenylfuroxans owned the highest inhibitory activity against BRD4,while phenylsulfonylfuroxans released the highest amount of NO in vitro.With the strongest inhibitory activity of BRD4(IC₅₀=0.82±0.13),n5 could release NO effectively.The docking results showed that n5 could stably bind to the target protein through hydrogen bonding and hydrophobic interaction by mimicking Kac.Annexin V-FITC/PI double staining confirmed n5 could induce apoptosis of ovarian cancer cells in a dose-dependent manner.The formation of autophagosomes was observed in GFP-LC3 transfected cells treated with n5.Western Blot analysis showed BRD4 and its downstream protein c-Myc were down-regulated.The decrease of BCL-2,increase of cytochrome C and cleaved caspase-3,confirmed the apoptosis-inducing ability of n5.The increase of autophagy-related proteins LC3-II and Beclin1,accompanied by the increase of p62,suggested that n5 could induce autophagy and inhibit autophagy flux.Combined with the autophagy inhibitor 3-MA,the cytotoxicity of n5 was reversed.The co-localization analysis of LC3 and LAMP1showed that n5 could inhibit the fusion of autophagosome and lysosome.In conclusion,n5,through inhibiting BRD4 and releasing NO,could induce apoptosis of cancer cells via mitochondrial pathway,activating autophagy of cancer cells and blocking autophagy flux.As a heterozygote of BRD4 inhibitor and NO donor,n5 showed excellent anti-ovarian cancer activity in vitro,deserving further study.This project provided new ideas and directions for the hybridization of targeted inhibitors and NO donors.