Design And Synthesis Of Procarbazine-based Nobel Anti-tumor Drugs And Mechanism Studies

Objectives:Procarbazine is mainly used for the treatment of malignant lymphoma,lung cancer,melanoma and so on,but it also has side effects such as gastrointestinal disorders,myelosuppression,and reproductive system damages and so on,These shortcomings limit its clinical application.In order to reduce the toxic side effects and enhance Pharmacodynamic effect of procarbazine,we want to design and synthesize the anti-tumor prodrug which consists of procarbazine targeting fibroblast activation protein-??FAP??according to the design theory of enzyme-activated anti-tumor prodrugs,we also study its chemical properties,anti-tumor efficacy and toxicity.At the same time,based on the fact that phenolic compounds accelerate the dehydrogenation of terpenoids under the action of tyrosinase,These results further explore the anti-tumor synergistic effect and preliminary mechanism of combining procarbazine with N-acetyl-L-tyrosine,and lay a scientific foundation for the development of new anti-tumor drugs based on procarbazine.Methods:1.Synthesis of anti-tumor prodrugs:Using the p-tolualdehyde as a starting material,the parent drug procarbazine was obtained by amidation,bromination oxidation and reduction amination in three steps.Starting from glycine,the FAP?specific target Z-GP-OH was obtained by benzyloxycarbonyl protection,amino acid condensation and ester hydrolysis in three steps.Finally,the parent drug and the specific target are linked by a amide bond to obtain the final product Z-GP-Pcb.2.Using HPLC technology as an analytical tool,the standard curve of Z-GP-Pcb and Pcb was established,and the drug-forming properties of prodrugs were studied,such as enzymatic hydrolysis rate,solubility,stability and oil-water distribution coefficient.3.Establish a cell MTT assay to study the cytotoxicity of Pcb and Z-GP-Pcb on human lung cancer cell line NCI-H460,human liver cell line HL-7702 and human embryonic kidney cell HEK293T,and the cytotoxicity of Pcb and combination group on murine melanoma cell B16/F10.4.Mouse tumor model was established by using mouse hepatoma cell line H22and murine melanoma cell line B16/F10,.the anti-tumor efficacy,toxicity and targeted distribution of the synthesized compound and the anti-tumor efficacy,toxicity and synergistic mechanism of the group combining Pcb with N-acetyl-L-tyrosine were explored in vivo.5.Flow cytometry was used to study the effects of Pcb and Pcb/NAT combination on apoptosis,cell cycle and mitochondrial membrane potential of murine melanoma B16/F10,and the expression level of proteins related to apoptosis-related and proteins related to mechanisms were detected by Western blot experiment.Results:1.A high-efficiency and simple seven-step synthetic route was successfully developed for the synthesis of Z-GP-Pcb which is the anti-tumor prodrug of procarbazine targeting FAP?enzyme,A total of 6 intermediate compounds and 1target compound were obtained,the total yield was 20.8%.All compounds were identified by ESI-HRMS,¹H NMR,¹³C NMR,and so on.2.Z-GP-Pcb can release Pcb and exert anti-tumor activity under the action of rh FAP?enzyme,and it can also maintains good stability under various environments,and its oil-water distribution coefficient is 1.3402.3.The IC₅₀ of Pcb for human lung cancer cell line NCI-H460 is 27.2±3.5?M.The prodrug Z-GP-Pcb doesn't have cytotoxic in the absence of FAP?enzyme.While in the presence of the FAP?enzyme,the prodrug recovered cytotoxicity and IC₅₀ was comparable to Pcb equally.The IC₅₀ of Pcb for murine melanoma cell line B16/F10 is 31.88±1.12?M.When Pcb combines with N-acetyl-L-tyrosine,the tumor cytotoxicity of Pcb is greatly increased,The IC₅₀ of Pcb is 14.21±1.08?M.Furthermore,Z-GP-Pcb is less toxic against human embryonic hepatocyte HL-7702and human embryonic kidney cell HEK293T than Pcb.4.Compared to Pcb,Z-GP-Pcb showed comparable antitumor activity in the mouse tumor-bearing model,but it had less myelosuppressive activity and reproductive toxicity,and it also had a higher accumulation in tumor tissue.5.Compared to Pcb,the combination group had a better inhibition of the proliferation activity of murine melanoma cells B16/F10,and both of them showed a good synergistic effect.In addition,the combination group has a more significant effect on the apoptosis,cycle and mitochondrial membrane potential of murine melanoma cells B16/F10.Its mechanism of action is to enhance the release of methyl or aryl radicals by Tyrosinase and Glyceraldehydes-3-phosphate dehydrogenase,and promote the apoptosis of tumor cells through proapoptotic proteins such as p53 and Bcl-2/Bax.Conclusion:Compared to the parent drug Pcb,the prodrug Z-GP-Pcb showed comparable antitumor activity in the presence of the FAP?enzyme at the cellular level,and in H22 and B16 tumor-bearing mice,but its myelosuppressive activity and reproductive toxicity are greatly reduced.After combining Pcb with N-acetyl-L-tyrosine,the inhibitory activity of B16/F10 cell proliferation of Pcb was enhanced,and the combination group has a more pronounced effect on apoptosis,cell cycle and mitochondrial membrane potential.Its mechanism of action is to enhance the release of methyl or aryl radicals by Tyrosinase and Glyceraldehydes-3-phosphate dehydrogenase,and promote the apoptosis of tumor cells through proapoptotic proteins such as p53 and Bcl-2/Bax.