Design,Syntheses And Antitumor Activity Of Novel Small Photosensitizers Based On Chlorin Skeleton

Malignancy is a kind of common and frequently occurring disease that seriously endangers human health.Although the existing tumor treatment methods,such as surgical resection,radiotherapy,chemotherapy and immunotherapy,have achieved some success,there are huge trauma,toxic side effects,tumor recurrence,etc.numerous complicated problems,and it is of great necessity to explore new-type tumor treatment.Photodynamic therapy(PDT)is a minimally invasive treatment method based on external light,photosensitizer(PS)and oxygen molecules in tissues,in which photosensitizer is the key to PDT.The second generation of photosensitizers,represented by chlorins,has become one of the R&D hot spots of new photosensitive drugs due to their advantages of large absorption coefficient,high absorption wavelength and better photosensitive antitumor activities.However,despite their excellent tumor inhibition potency,chlorins suffer from some defects such as lower tumor selectivity and potential drug resistance.In recent years,researchers have focused on the introduction or adhesion of some functional groups or chemical molecules with biological characteristics or molecular recognition function in the chlorins,so as to obtain photosensitizers with strong photosensitive activity,high tumor selectivity and low drug-resisitance.Based on the above research background,this study focusd on chlorins to carry out the research of design,syntheses and antitumor activity of novel small photosensitizers,mainly including:(1)Design,syntheses and antitumor activity research of ether and amino acid derivatives of chlorin e₄;(2)Design,syntheses and antitumor activity research of ABCG2-mediated photosensitive molecules;(3)Design,syntheses and antitumor activity research of stimulus-responsive photo-chemotherapy coupling molecules.1.Design,syntheses and antitumor activity research of ether and amino acid derivatives of chlorin e₄Reasonable structure modification and optimization of existing photosensitizers are one of the most vital approaches to obtain more efficient and less toxic photosensitizers.In this part,we chose chlorophyll a stable degradation product chlorin e₄ as the leading compound,and introduced the amino acid residue at position 17³-or 13¹-and/or alkoxy side chain at position 3¹-to obtain a series of ether and amino acid derivatives of chlorin e₄.The in vitro antitumor activity results showed that most of the target compounds exhibited favorable antitumor activity towards B16-F10,A549 and He La cells,among which compound 14b had the inhibition activity of nanomolar level towards the three cell lines.Mainly distributed in mitochondria,lysosomes and endoplasmic reticulum(ER),14b with light radiation could significantly improve the cellular ROS level,cause cell apoptosis and autophagy,and block cell cycle in G1 phase,exhibiting a strong antiproliferation capability in vitro.The PDT antitumor efficacy of 14b was evaluated on C57BL/6 mice bearing B16-F10 tumors using Talaporfin as positive control.The experimental results showed that 14b at a dosage of 2 mg/kg showed 95.1%tumor inhibitory activity,significantly improved objective response rate(60%)and time to progression(11 days)of mice,which was superior to that of Talaporfin at the same dosage.In this part,we found a chlorin-based photosensitive candidate molecule that is safe and effective at cell and mouse levels,and it is worth to carry out in-depth pre-clinical pharmacology,toxicology and pharmacokinetics study on it.In addition,this part also provides new ideas and research basis for the research and development of other types of highly active photosensitive molecules.2.Design,syntheses and antitumor activity research of ABCG2 mediated photosensitive moleculesAs one of the key transmembrane transport proteins in tumor multidrug resistance(MDR),ABCG2 can efflux various types of photosensitizers and thus reduce the efficacy of PDT.Some molecular targeted tyrosine kinase inhibitors(TKIs)have been found to reverse resistance by blocking the efflux of photosensitizers by ABCG2.This part firstly verified that lower concentration(10?M)tyrosine kinase inhibitors Imatinib and Dasatinib incubated with HepG2 cells for a long time(>48 h)could significantly down-regulate ABCG2 expression,stimulate ATP enzyme activity,and increase the amount of intracellular chlorin e₆,thereby enhancing the efficacy of PDT.Subsequently,we chose chlorin e₆ as the structural framework,and synthesized 18 ABCG2-mediated low-resistance photosensitive molecules by introducing Imatinib or Dasatinib or its active pharmacophore.Besides,we also synthesized a polymer micelle conjugating with Dasatinib and loading chlorin e₆.In vitro antitumor activity results showed that the photodynamic antitumor activity of most of the target compounds and macromolecule micelle was improved compared with that of clinical-approved drug Talaporfin,among which compound 29b exhibited a strong inhibitory effect towards B16-F10 and HepG2cells.Subsequent mechanism studies showed that 29b not only had a certain down-regulation effect on ABCG2,but also significantly reduced the amount of ABCG2excreted by it.On the other hand,29b could widely distribute in mitochondria,lysosomes,endoplasmic reticulum and Golgi apparatus,effectively induce a large amount of ROS production,significantly induce cell apoptosis and autophagy,and block cell cycle in S phase.In BALB/c nude mice bearing HepG2 xenograft model,29b could significantly inhibit tumor growth,delay tumor progression,and obviously prolong overall survival(53days),median survival(43 days),and progression-free survival(12 days)at a dose of 2mg/kg,without causing significant adverse drug reactions.Based on in vitro and in vivo antitumor activity results,we believe that compound 29b can be used as a candidate molecule for the development of low-resistance anti-tumor photosensitive drugs,which is worthy of further study on the mechanism of its activity.3.Design,syntheses and antitumor activity research of stimulation-responsive photo-chemotherapeutic dual-mode antitumor moleculesThe combination of photodynamic therapy and chemotherapy is one of the most common methods for cancer therapy at present.However,this"cocktail"therapy has some defects,such as uncontrollable pharmacokinetic properties of photosensitizers and chemotherapy drugs.In contrast,coupling photosensitizers with chemotherapeutic drugs using Linker,which breaks down at tumor tissue specifically,enables the simultaneous release of photosensitizers and chemotherapeutic drugs at the tumor site to play a synergistic role.Relevant studies have shown that both 5-Fluorouracil and Histone deacetylase(HDAC)inhibitors exhibited synergistic effects when combined with photodynamic therapy.To enhance the tumor selectivity of photosensitizers and reduce the uncontrollable pharmacokinetic properties caused by simple drug combination,based on pharmacophore fusion and prodrug design strategy of traditional medicinal chemistry,in this part we chose chlorin e₆ as the structural framework,and synthesized 11photochemotherapy molecules coupling 5-Fluorouracil,SAHA or Chidamide,which contains tumor microenvironment stimulation response Linker hydrazone bond or disulfide bond molecules including compound 32,36a and 36b.In vitro drug release experiments showed that both Linkers were prone to fracture under in vitro conditions simulating the tumor microenvironment in vivo,in which 5-Fluorouracil and SAHA were more easily released than Chidamide.The results of in vitro antitumor activity indicated that compound32,36a,43a and 45a exhibited powerful cytotoxicities against B16-F10 and HepG2 cells,which were superior to the positive drugs SAHA and Talaporfin.Subsequent experiments on the cytotoxic mechanism showed that compounds 32,36a and 43a could significantly increase the level of ROS in B16-F10 cells,induce cell apoptosis of tumor cells,and block cell cycle in S phase.In addition,compound 36a could up-regulate Acetly-H3 and Acetly-H4 in B16-F10 cells.The PDT antitumor efficacy of 36a was evaluated on C57BL/6 mice bearing B16-F10 tumors.Results showed that compound 36a at a low dose(2 mg/kg)under light condition could not only significantly inhibit the proliferation of melanoma,prolong the survival time of mice,but also effectively inhibit the lung metastasis of melanoma.Experimental results of antitumor activity mechanism in vitro and in vivo indicated that compound 36a can be used as a photo-chemotherapeutic dual-mode antitumor molecule that could effectively inhibit the proliferation and metastasis of melanoma,and it is worthy of further study on its mechanism.