Design,Synthesis,and Biological Evaluation Of Akt Inhibitors And ?,?-unsaturated Ketone Derivatives In Mantle Cell Lymphoma

Mantle cell lymphoma(MCL)is a subtype of B cell Non-Hodgkin's Lymphoma(NHL)with poor prognosis,with a median age of 60 years old.Although MCL typically responds to conventional chemotherapy,most patients eventually experience disease progression,and survival outcomes following relapse are the poorest of all NHL subtypes.Recent research has uncovered new knowledge about signaling networks which drive MCL pathogenesis,such as B-cell receptor(BCR),PI3K/Akt,NF-?B and apoptotic pathways.Discovery of the importance of these various signaling axes is prompting development of molecular therapies targeting these oncogenic signaling pathways.Ibrutinib is the first oral covalent BTK inhibitor to obtain FDA approval in 2013 for treatment of MCL.Its breakthrough designation was spurred by clinical evaluation of Ibrutinib for treatment of replased/refractory MCL,demonstrating remarkable responses,with a 68%ORR.Despite the success of ibrutinib,cases of resistance typically emerge following prolonged treatment.Survival outcomes are especially poor for patients who relapse from ibrutinib therapy within 12 months.Therefore.exploration of novel target therapy is urgently needed.Investigation of mechanisms for IBN resistance suggests that constitutive Akt and ERK,but not BTK activation,correlates with cellular responses of primary MCL tumour cells to ibrutinib.On basis of these findings,Akt may be a potential therapeutic target in MCL.Based on the crystal structure of Akt and the binding modes of Akt and ATP-competitive inhibitors,we performed structure optimization of a previously confirmed Akt inhibitor 1,designed and synthesized benzyl substituted pyrrolopyrimidines and substituted aminopyrimidines,and evaluated their activity in mantle cell lymphoma.To synthesize benzyl substituted pyrrolopyrimidines,4-chloro-pyrrolopyrimidine was used as a starting material.Electrophilic substitution,nucleophilic substitution,and Boc-deprotection gave the intermediate amino.Benzyl substituted acetic acid was synthesized through methylester formation,free radical reaction,nucleophilic substitution,and esterolysis.These steps were followed by acid amine condensation and Boc-deprotection to afford the target compounds.Substituted aminopyrimidines were obtained using bis(2-chloroethyl)amine as a material,undergoing amino protection,substitution cyclization,and cyan hydrolysis to obtain an intermediate,and followed by acid amine condensation and Boc-deprotection if necessary to afford the target compounds.In vitro enzyme assays of the benzyl substituted pyrrolopyrimidines showed that many compounds exhibited potent Akt inhibitory effects.Notably,activities of compounds 8 and 14g were comparable to Akt inhibitor GSK690693.Compounds 8,14a,14b,14c,14g demonstrated potent antiproliferative effects in MCL cell lines and primary patient cells.Further experiments found that compounds 8 and 14g dose-dependently induced cell apoptosis and cell cycle arrest,and decreased the viability of MCL primary patient cells.Western blotting of Jeko-1 and primary cells treated with these compounds displayed potent downregulation of the Akt downstream effectors GSK3? and S6.Although substituted aminopyrimidines lost Akt inhibitory acitivity,they maintained the antiproliferative effects in MCL cells.Of note,the antiproliferative activity of compound 25h was similar to that of ibrutinib.25h similarly induced cell apoptosis and cell cycle arrest,and downregulated Akt and downstream S6.Interestingly,25h also suppressed phosphorylation of MEK1/2?ERK1/2,suggesting that 25h may be a dual Akt/ERK signaling inhibitor.These results warrant further evaluation and could identify this compound as a promising candidate for the treatment of MCL.In another approach,in vitro screening identified three ?,?-unsaturated ketones with promising antiproliferative effects in MCL.Further evaluation of these compounds in a MCL Jeko-1 xenograft mouse model showed that all the three compounds inhibited tumor growth in mice and prolonged survival comparable to ibrutinib.Using the three compounds as lead compounds,maintaining the structural nucleus and ?,?-unasaturated ketone moiety,series ? was designed and synthesizedTarget compounds using 2,3-dichlorophenol as a starting material were synthesized through alkylation,Friedel-Crafts acylation,and aldol condensation.In vitro MCL cell viability assay showed that 29o was the most potent compound in this series.Series ? was designed based on analysis of structure-activity relationship for series ?I.Similarly,target compounds were synthesized through alkylation,Friedel-Crafts acylation,electrophilic substitution,nucleophilic substitution,acid amine condensation and aldol condensation.From Series ?,compounds 29o,32a,32b,32c and 40 demonstrated potent antiproliferative effects in MCL cell lines and decreased the viability of MCL primary cells.Further experiments revealed that representatives 32c and 40 induced cell apoptosis and cell cycle arrest.Notably,these compounds were able to inhibit viability of BTK-KO Jeko cells,demonstrating antitumor activity irrespective of BTK expression.In vivo treatment in a Mino xenograft mouse model showed that 29o and 40 inhibited tumor growth in mice and provided significant survival benefit compared to control mice.This thesis describes the design and synthesis of two series of Akt inhibitors and two series of ?,?-unsaturated ketones.62 compounds in total were confirmed by 1H NMR and 13C NMR,which showed that all the target compounds have not been previously reported.All the designed compounds were evaluated in mantle cell lymphoma.We found that a promising number of compounds displayed potent activity in MCL,which was either comparable to or better than that of ibrutinib.This study may serve as a basis for further design of target therapy,study of antitumor mechanisms,and antitumor drug screening.