Design And Synthesis Of Biotin Polyethylene Glycol 3,5-bis(4-substituent Benzylidene) Piperidin-4-one Derivatives And Antitumor Applications

Objective:The design and synthesis of high-efficiency,low-toxicity,anti-drug resistant chemotherapy drugs is the focus of people’s attention.In this paper,it is expected that based on the target-prodrug strategy,a class of high-efficiency,low-toxicity and anti-drug resistant antitumor compounds targeting STAT3 will be designed and synthesized.Methods:TwoSTAT3targetingbiotin macrogol-3,5-bis（4-substituentylidene）piperidin-4-one derivatives DAP-F and DAP-NO₂ were used as active parent nuclei,and the targeting group biotin was introduced at the N position of DAP-F and DAP-NO₂ by polyethylene glycol（PEG）linker,respectively,and two anti-cancerprodrugbiotin-peg-3,5-bis（4-substituent benzylidene）piperidin-4-one STAT3 inhibitors were designed and synthesized（3a and 3b）,structural characterization by mass spectrometry and nuclear magnetism,among others;The in vitro antiproliferative activity and in vivo antitumor activity of 3a and 3b were evaluated by MTT method and SW480 xenograft model,respectively.Preliminary studies were conducted on the pharmacokinetics of in vitro stability and in vivo stability of 3a and 3b using HPLC;Western blot was used to analyze the effects of 3a and 3b on intracellular STAT3 expression.Results:Two anticancer prodrugs 3a and 3b,targeting STAT3,were synthesized,and the structure of the compound was characterized and confirmed.The results of HPLC purity analysis showed that the purity of3a was 98.0%and the purity of 3b was 99.2%.The results of MTT experiments showed that the compound DAP-F had a strong inhibitory effect on SW480,T24,MGC-803 and MDA-MB-231 cancer cells,with IC₅₀ values of 6.10±2.53,1.14±0.59,4.63±0.54 and 0.13±0.02μM,respectively.The IC₅₀ values of compound DAP-NO₂ against SW480,MGC-803,MDA-MB-231 and MIA-PACA-2 cancer cells were 6.85±2.82,7.392±0.58,3.22±0.33 and 7.53±0.12μM,respectively.The IC₅₀values of compound 3a against SW480,T24,MGC-803 and MDA-MB-231 cancer cells were 10.15±1.33,16.04±0.86,12.24±1.18and 13.75±1.01μM,respectively.The IC₅₀ values of compound 3b against SW480,MGC-803,MDA-MB-231 and MIA-PACA-2 cancer cells were 11.33±1.21,15.58±1.04,10.24±0.80 and 14.48±2.00μM,respectively.3a and 3b were insensitive to A549/cisplatin-resistant strains,with IC₅₀ of 29.72±0.68,6.50±2.87,respectively.Experiments in nude mouse models of tumor cell xenografting showed that the tumor inhibition rate of 3a against SW480 was 54.5%,the tumor inhibition rate of MDA-MB-231 was 67.5%,the tumor inhibition rate of 3b against SW480 was 46.1%,the tumor inhibition rate of MDA-MB-231 was52.3%,the tumor inhibition rate of DAP-F against MDA-MB-231 was39.2%,and the tumor inhibition rate of DAP-NO₂ against MDA-MB-231was 46.4%.In addition,the 3a and 3b dosing groups had stable weight changes compared to the original drug（DAP-F and DAP-NO₂）and the positive drug Dox.The pathological section results showed that compared with the original drug（DAP-F and DAP-NO₂）and the positive drug Dox,there were no obvious pathological changes in the heart,liver,spleen,kidney and lung in the 3a and 3b administration groups.The above results indicate that the cytotoxicity of prodrugs 3a and 3b in vitro is less than that of the original drug,but the antitumor activity and toxicity in vivo are significantly better than that of the original drugs DAP-F and DAP-NO₂.The preliminary pharmacokinetic results showed that compounds 3a and3b slowly released their original drugs DAP-F and DAP-NO₂ within 12 h after intraperitoneal injection,respectively.Western blot analysis and molecular docking simulation experiments show that DAP-F,DAP-NO₂and compounds 3a and 3b are indeed inhibitors of signal sensors and transcriptional activator 3（STAT3）,and the antitumor effects of compounds 3a and 3b are effective in the release of pathogens.Conclusion:Through the target-prodrug strategy,based on the active parent nucleus of STAT3 inhibitors DAP-F and DAP-NO₂,and the introduction of the targeting group biotin with PEG as the linker,high-efficiency,low-toxicity and anti-drug resistance anti-tumor prodrugs can be designed and synthesized.