Chromatography-free hydrochloride synthesis of Difelikefalin:
Firstly, the piperidine amino acid 21.1 was coupled with the fully protected d-lysine analogue 21.2 under conventional EDCI/HOBt peptide coupling conditions to produce the fully protected dipeptide 21.3 in 97% yield. Conventional hydrogenation reduction was used to remove the N-terminal benzyloxycarbonyl protection to afford the amine 21.4 in 91% yield. Subsequent treatment of dipeptide 21.4 with protected d-leucine (21.5) and the same peptide coupling method followed by deprotection of the benzyl protecting group afforded tripeptide 21.6 in yields of 99% and 48%, respectively. Notably, the lower yield of tripeptide 21.6 may be due to crystallisation implemented during purification using cyclohexane and ethyl acetate rather than failure of the reaction to complete. Pure tripeptide 21.6 was then treated with protected d-phenylalanine and subjected to the same peptide coupling method followed by deprotection of the benzyl protecting group to give tetrapeptide 21.8 in 99% yield at each step. Treatment of tetrapeptide 21.8 with d-BocPhe under conventional peptide coupling conditions yielded a fully protected pentapeptide in 99% yield. The pentapeptide methyl ester 21.10 was precipitated in 99% yield by deprotection of the global Boc protecting group using 6N hydrochloric acid in isopropanol and addition of toluene to the reaction mixture. Next, the methyl ester 21.10 was treated under standard saponification conditions and the reaction was burst with 1N hydrochloric acid to obtain the pentapeptide 21.11 as a hydrochloride salt. although not disclosed, it is conceivable that the saponification reaction of 21.10 was burst with acetic acid to obtain its difelikefalin as a commercially available acetate salt (Compound No. 21).
