Synthesis Of ADC Peptide Linkers And Their Conjugation With Antibody And Toxin

Antibody drug conjugate（ADC）is a combination of monoclonal antibody and small molecule toxin through chemical linker.It has been developed into a new generation of targeted anticancer drugs.As an important component of ADC,the stability and physicochemical properties of the linker play an important role in the effectiveness and toxicity of ADC.Enzymatic lysable peptide linkers have good stability in the blood and can release the toxin specifically in tumor cells by cathepsin B degradation,which are widely used in marketed drugs.In this paper,the synthesis of the tetrapeptide linker（MC-GGFG）and the dipeptide linker（MC-VC-PABOH）and the coupling of the two linkers with antibody and toxin were studied.N-fluorenyl methoxycarbonyl glycine（SM1）reacted with lead tetraacetate to produce acetic acid（N-[（9H-fluorenyl）methoxycarbonyl]glyimyl amino）methyl ester（M1）.As the reaction solvent,the volume of anhydrous tetrahydrofuran was reduced from 27（in the literature）to 15 times without the addition of toluene.In the work-up,the simple washing could get M1 with a purity of 96%and a yield of 90～93%（data from three batches）,avoiding the column chromatography.M2 was obtained by nucleophilic substitution of M1 with benzyl glycolate.The amount of benzyl glycolate was reduced to 8 eq and the reaction solvent was changed from DCM to THF.In the work-up,M2 was precipitated in dichloromethane at low temperature with a purity of more than 97%,avoiding column chromatography.Maleimide caproic acid（SM2）was condensed with N-hydroxysuccinimide（NHS）to obtain N-Succinimidyl 6-maleimidohexanoate（M4）which was separated from n-butanol with a purity of more than 98%without column chromatography.M5 was condensed with L-phenylalanine（L-Phe）to obtain M6 without the addition of water.The charge of L-Phe and base was increased,resulting in a 10%increase in yield.MC-GGFG was obtained by condensation of M8 and M4 in DMF and H₂O without the addition of organic base,which avoided the formation of salt between the product and organic base.Finally,the product was purified by recrystallization of 1,2-dimethoxy-ethane with a purity of more than 98%.With SM1 as the starting material,the route included 6 steps and the total yield was 33.6%.MC-GGFG was coupled with DX8951f to obtain MC-GGFG-Dxd with a yield of 55.4%and a purity of 98.66%.And then,MC-GGFG-Dxd was coupled with trastuzumab to obtain IgG-GGFG-Dxd whose DAR was determined to be 6.2 by UV-Vis and 7.0 by LC-MS.Fmoc-Cit was condensed with p-aminobenzyl alcohol to obtain Fmoc-Cit-PABOH in the presence of EEDQ instead of HATU and the mixed solvent of dichloromethane and methanol instead of DMF was used as the reaction solvent.In the work-up,the product was purified in dichloromethane by stirring with a yield of more than 90%and a purity of more than 93%,avoiding the column chromatography.After removing the Fmoc group,Fmoc-Cit-PABOH was condensed with Fmoc-Val-OSu to obtain Fmoc-Val-Cit-PABOH.dichloromethane and methanol were used as the reaction solvent instead of DMF,and dichloromethane was used to purify by stirring with a yield of more than 92%,avoiding the column chromatography.Fmoc-Val-Cit-PABOH was underwent the de-protection and condensation with MC-OSu to obtain MC-VC-PABOH.The reaction solvent was changed from NMP to DMF.In the work-up,the product was precipitated in methyl tert-butyl ether and purifying in dichloromethane by stirring with a yield of more than 90%and a purity of more than 97%,avoiding the column chromatography.With Fmoc-Cit as starting material,the route included 5 steps and the total yield was 72.7%.MC-VC-PABOH reacted with bis（4-nitrophenyl）carbonate to produce MC-VC-PAB-PNP which was condensed with exatecan to obtain MC-VC-PAB-Exa without the addition of organic base and condensation agent.and the product could be precipitated in tert-Butyl methyl ether with a yield of 47.4%and a purity of 95.34%,avoiding column chromatography.Finally,IgG-VC-Exa was obtained by coupling of MC-VC-PAB-Exa and trastuzumab,and the DAR was determined to be 4.98 by UV-Vis.