Design,Synthesis And Activity Of Melatonin-based Multi-target Pt(Ⅳ)/Gemcitabine Antitumor Drugs

Melatonin is a hormone that is naturally secreted by the human brain.It is easily metabolized and low toxicity.It can effectively inhibit breast cancer,lung cancer,pancreatic cancer and other cancers.It has a synergistic effect with other chemotherapy drugs.Anticancer mechanisms are perplexing and extensive.Hypoxia-inducible factor-1αand signal transduction and transcription activator-3 are highly expressed at the tumor site to reduce the sensitivity of chemotherapeutic drugs,and MT can increase the chemotherapy drugs by downregulating HIF-1αand Stat3 treatment effect.This study intends to introduce melatonin into the structural design of classic first-line drugs for clinical tumor treatment-cisplatin and gemcitabine,to play a multi-target synergy and enhance efficacy.Objective:Tumor microenvironment regulatory factors HIF-1αand Stat3 are overexpressed in most tumors,and can reduce the sensitivity of chemotherapy drugs through a variety of regulatory mechanisms.In order to overcome the chemotherapy resistance caused by HIF-1αand Stat3,to design and synthesize a new multi-target Pt（IV）/gemcitabine antitumor drug molecule based on melatonin,aiming to provide valuable information for clinical chemotherapy sensitization and promote the development of new anti-tumor drugs with high efficiency and low toxicity.Methods:（I）Design,synthesis and activity study of melatonin-based multi-target Pt（IV）antitumor drugs:Aiming at the shortcomings of clinical treatment of classic Pt（II）drugs with high drug resistance and toxicity,and MT molecules and different carbon chains that could bind HSA were introduced at the two axial positions of Pt（IV）to synthesize a new multifunctional melatonin Pt（IV）prodrug molecule,which can release cisplatin and MT,targeting DNA,HIF-1αand Stat3 respectively,thereby increasing platinum drug sensitivity to achieve multi-target treatment.（1）Design and synthesis of compound:Firstly,compounds 1 and 2 modified by melatonin reacted with oxoplatin to prepare 3 and 4,and introduce isocyanate chains into the other hydroxyl site of Pt（IV）to synthesize the target compound 5?12.The structure was characterized by NMR and HR-MS.The purity and Log P were detected by HPLC and UV spectrum.（2）Study on the antitumor activity:MTT were used to perform cytotoxicity studies.ICP-MS was used to study the accumulation of compounds in cells.Comet experiment was used to study the damage of compounds to DNA.Wound healing experiment was performed to study cell migration inhibition rate.Western blot was used to investigate the anti-cancer molecular mechanism of the compound.The antitumor efficacy was further studied through experiments on tumor-bearing nude mice.（II）Design,synthesis and activity of melatonin-based multi-target gemcitabine anti-tumor drugs:Gemcitabine,a clinical first-line drug for pancreatic cancer and non-small cell lung cancer,is poorly soluble in fat and difficult to pass through the cell membrane by passive diffusion.It needs to rely on a specific nucleic acid transport vector to enter tumor cells,but tumor cells can cause gemcitabine resistance because of the lack of nucleic acid transport vectors.In this study,melatonin with potential anti-tumor effects was linked to gemcitabine to increase the fat-solubility of gemcitabine,increase the tumor’s passive drug uptake,overcome the resistance of gemcitabine caused by the lack of nucleic acid transporter.At the same time,it exerts the regulatory effect of MT on HIF-1αand Stat3,sensitizes chemotherapy and improves the therapeutic effect of gemcitabine.（1）Chemical synthesis:First,di-tert-butyl dicarbonate was used to protect the amino group of gemcitabine.The modified melatonin from succinic anhydride and boc-protected gemcitabine were esterified under the action of EDCI and DMAP.The product after the reaction was deprotected by TFA/DCM to synthesis of the target product MT-GEM,the chemical structure was characterized by NMR and HR-MS.（2）Antitumor activity:The MTT experiment was used to study the IC₅₀ value of MT-GEM,to investigate the inhibitory effect of MT-GEM on the growth and proliferation of various cancer cells and normal cells such as A549,MCF-7,etc.,and compared with gemcitabine and co-administration.Results:（I）Design,synthesis and activity study of melatonin-based multi-target Pt（IV）antitumor drugs.Compounds 5?12 were successfully synthesized,and five intermediates oxoplatin and 1?4 were obtained.Structural characterization and purity analysis（>95%）were performed.MTT test found that the IC₅₀ value of 11 was 87 times that of cisplatin in MCF-7 cells,and it was detected that 11 released cisplatin and 2 in the cell.The high Pt uptake was strongly to induce DNA damage,apoptosis and S-phase cell arrest,inhibiting tumor cell metastasis.Western blot show that the compounds can effectively inhibit the expression of HIF-1αand p-Stat3 protein.The results of in vivo antitumor experiments showed that 11 has almost no observable tissue toxicity.（II）Design,synthesis and activity research of melatonin-based multi-target gemcitabine antitumor drugs.Successfully synthesized a compound GEM-MT and conducted MTT study.In A549,GEM-MT and co-administration group showed better cytotoxicity and higher selection index than gemcitabine.Conclusions:In this study,sixteen compounds were successfully synthesized,including eight melatonin-based Pt（IV）prodrug molecules and one melatonin-based gemcitabine antitumor drug,and seven intermediates.This subject gets rid of the traditional design concept.Combining melatonin secreted by itself and first-line anti-tumor drugs in clinical treatment can not only improve the anti-tumor effect but also be safe for the human body.