Design And Synthesis Of Bleomycin(BLM) Disaccharide-Camptothecin Conjugates And Their Antitumor Activity

Bleomycin(BLM)disaccharide is an indispensable part for recognizing tumor when bleomycin was clinically used as a first-line antitumor drug.It is composed of 3-O-carbamoyl-D-mannose and L-gulose,which is linked with α-1,2-glucosidic bond.Camptothecin is a cytotoxic anticancer drug isolated from Camptotheca acuminata Decne,and it exerts antitumor effect by inhibiting topoisomerase Ⅰ.Due to its rigid planar structure and six-ring lactone,its poor water-solubility,poor stability and non-specificity leading to toxic-side effects in vivo were observed,limiting its clinical application.Although many domestic and foreign teams had modified it to obtain first-line drugs such as Irinotecan,Topotecan,to cure intestinal cancer,head and neck cancer etc.,lack of specificity to tumor is still needed to be solved.Herein,in combination with the structure-activity relationship of camptothecin,we used the bleomycin disaccharide or some monosaccharides with the property to distinguish the tumor cells from normal cells,to modify 20-hydroxyl or 10-hydroxyl of camptothecin via a suitable linker,aiming to improve the water solubility,stability and targeting of camptothecin,and reduce the toxicity towards normal tissues.This will open a new approach to developing old drug in new use.Applying a privileged fragment from one clinical anticancer drug to modify another anticancer drug will increase the probability of druggabity.Generally speaking,natural bleomycin disaccharide and its derivatives were first multi-gramm synthesized,and a series of bleomycin disaccharide-camptothecin conjugates were designed and synthesized,and ultimately antitumor activities,solubility and stability of all the compounds obtained were assessed,in hope to find a class of targeted camptothecin derivatives with good water solubility,high-stability,and low toxicity.(1)Multi-gram synthesis of natural BLM disaccharide and its derivativesAs for multi-gram scale synthesis of bleomycin disaccharide synthesis,donor 3-O-carbamoyl-D-mannose and receptor L-gulose were first synthesized separately,and then the condensation reaction proceeded between the donor and receptor to obtain BLM disaccharide and its derivatives.The donor 3-O-carbamoyl-D-mannose diphenyl phosphate was synthesized starting from D-mannose in several steps with the total yield of 48%.The highlight of the method is that starting material is cheap and easy to obtain,the synthetic route is short,the yield is high,and the repeatability is good.Meantime,D-galactose was used as a starting material to prepare receptor L-gulose with 2-hydroxyl group in a total 51%yield.The synthesis of bleomycin disaccharide was carried out in a 3-step reaction in total 38.5%yield after the glycosidation coupling reaction of donor 3-O-carbamoyl-D-mannose diphenyl phosphate and receptor 2-OH-L-gulose linked withα-1,2-glucosidic bond.In the synthesis of the bleomycin disaccharide derivative,donor 3-O-carbamoyl-D-mannose diphenyl phosphate donor and receptor L-gulose with free hydroxyl grounp were first coupled in the presence of trimethylsilyl trifluoromethanesulfonate,and then the bleomycin disaccharide derivative was obtained via a series of reactions including acylation,debenzylation,activation,and etherification.(2)Design and synthesis of BLM disaccharide-camptothecin conjugatesIn order to explore the effect BLM disaccharide on cytotoxic drugs,we designed and synthesized BLM disaccharide-camptothecin conjugates,and D-glucose,D-galactose,Dmannose,3-O-cambamyl-D-mannose were selected as reference compounds,and glycol with different sizes was used as a linkage because of good biological compatibility and strong water solubility.Three-step synthesis of BLM disaccharide-camptothecin conjugates was adopted,firstly,20-OH of camptothecin or 10-OH of 10-HCPT was activated with 4-nitrophenyl carbonochloridate(PNPCOCl),and ethylene glycols with different chain lengthes were introduced to the reduced end of BLM disaccharide derivatives,which was further reacted with activated camptothecin to produce a series of BLM disaccharidecamptothecin conjugates,the globe route included a seven-step synthesis with a total yield of 6.0%to 8.2%.(3)Evaluation of antitumor activity,stability and water solubility of conjugates preparedHuman pancreatic cancer cell(SW1990),human liver cancer cell(HEPG2),and human colon cancer cell(HCT116)were selected as tumor evalution models,and human embryonic kidney cell(HEK293)was used as normal cells,Using MTT assay antitumor activity of 36 conjugates we prepared were evaluated against the three cancer cells and one normarl.The results showed that the conjugates with BLM disaccharide,mannose and galactose,carbamyl mannose showed bettter cytotoxicity in the presence of the same linker,IC50 was at 0.2-5.0μM against cancer cells,but more than 100μM towards normal cells.It was noted that with the same sugar ligand,the conjugates with a two-carbon linker had more cytotoxic effect on normal cell than on tumor one,while the compounds with a four-carbon linker showed the stronger killing ability to tumor cells than normal one,the conjugates with a six-carbon linkage was similar to that lead compound CPT,strongly suggesting that the antitumor activity of simple sugar-CPT conjugates was highly related to the type of simple sugar and length of a linker.Stability tests showed that in pH=7.4 PBS buffer(simulated human blood),the stability of sugar-20-camptothecin conjugates improved significantly in comparison with lead compound CPT.With the incubated time was 50 mins,CPT was converted to the form of salt.However,sugar-20-camptothecin conjugates were fairly stable,no trace of degradation product was observed when the incubated time was extended to 72 hours.Noticeablely,sugar-10-camptothecin was unstable.Solubility tests showed that under phosphate buffers that was pH=7.4,water solubility of the sugar-camptothecin conjugates reached a soluble range.Among them,conjugate 31,48 and 60 were respectively 21.2 mg/mL,17.1mg/mL,and 17.4 mg/mL in PBS solution.In conclusion,this paper developed a multi-gram scale synthesis of naturally occurring bleomycin disaccharide and its derivatives,which provided a solid foundation for exploring the antitumor structure-activity relationship of bleomycin disaccharide and the discovery of its target.Also 36 oligosaccharide-camptothecin conjugates have been synthesized,and the results from in vitro cell-based assay displayed that BLM disaccharide,mannose,carbamyl mannose is advantageous to the antitumor activity of camptothecin,which was closely related with the length of the linker.This will provide an experimental support for finding a class of camptothecin derivatives with low toxicity,high efficiency,good selectivity,and open a new avenue to new uses of old drugs.