Design,Synthesis And Evaluation Of Tumor Targeted Gemcitabine Prodrugs

Gemcitabine is an antitumor drug that has been approved for the treatment of a variety of solid tumors.However,the metabolic stability,drug resistance and poor tumor selectivity of gemcitabine limit its clinical applications.To overcome the issues,prodrug strategy is utilized with 4-N-amino structural modification of gemcitabine based on the study that Cysteine Cathepsin L（CTSL）is highly expressed in a variety of malignant tumor cells and has been validate as anti-tumor drug targets.A series of gemcitabine prodrugs were designed and synthesized by the reaction of carboxyl group from different substituted L-and D-acetylated lysine and the amino group of gemcitabine.Finally,the synthesized prodrugs were selectively cleaved and activated by highly expressed CTSL in tumor cells to release the active gemcitabine.To evaluate the antitumor cell activity and selectivity of gemcitabine prodrugs,tumor cells A549 and Bx PC-3 were selected due to the highly expression of CTSL in these cell lines,while Caco-2 cells showed low expression of the enzymes.Among the prodrugs,compounds 1-3 and 6-8,which were replaced by tert-butylocarbonyl,free amino,and indoce-3-acetyl group,showed significantly enhanced activity against A549 lung cancer cells(IC₅₀=2.23-5.24μM).However,prodrugs 4,5,9 and 10replaced by 1-adamantane carboxyl and octanoyl group showed weak or loss of antitumor activity against A549.In Bx PC-3 pancreatic cancer cells,prodrugs 1-3 and6-7 showed moderate antitumor activity,while compound 8 showed the most significant activity with 1.16 times greater than that of gemcitabine.In contrast,compounds 4-5 and 9-10 generally lost their antitumor activity.For Caco-2 cell line,prodrugs 1 and 6 showed the best inhibitory activity against Caco-2 cells,while the activity of other prodrugs was not obvious,indicating that most of the prodrugs exhibited good antitumor selectivity.In addition,HEK-293T human embryonic kidney cells line was used to test the safety of representative prodrugs 1-3 and 6-8 which showed good antitumor activities.The results showed that the toxicity of representative prodrugs to this normal cell line was generally reduced,and compounds 2、3、7 and 8showed the best safety profiles.According to cell activity data,representative prodrug compounds 1,2,7 and 8were selected to study their chemical stability under physiological conditions,human plasma stability,human liver microsomal stability and metabolic stability in CDA enzymes.The results showed that prodrugs 2 and 7 showed good chemical stability under physiological conditions,and prodrugs 1,2 and 7 showed good plasma stability and liver microsomal stability,and the half-life of prodrug compounds was longer than gemcitabine.In the CDA enzyme stability study,the remaining amount of prodrug incubated in CDA enzyme for 1 h was the same as that of control group without enzyme,both exceeding 95%,while the decomposition amount of gemcitabine only incubated in CDA for 1 h was nearly 23%.Moreover,there was no significant difference in chemical or metabolic stability between the L-type and D-type prodrug compounds.In this study,a series of ten new 4-N-amino modified gemcitabine prodrugs were designed and synthesized and evaluated for their anticancer activity.Among them,prodrugs 2 and 7 showed improved anti-tumor activity,favorable selectivity and safety profiles.In addition,prodrugs 2 and 7 exhibited enhanced chemical stability and metabolic stability as well.Therefore,the designed gemcitabine prodrug in this study could be used as potential selective anticancer agents.