1) The reaction of 3-(tert-butoxycarbonyl)thiazolidine-4(R)-carboxylic acid (I) with tert-butylamine by means of dicyclohexylcarbodiimide (DCC) in THF gives the corresponding protected amide (II), which is deprotected with HCl or methanesulfonic acid to the free amide (III). The condensation of (III) with (2S,3S)-3-(tert-butoxycarbonylamino)-2-hydroxy-4-phenylbutyric acid (IV) by means of 1-hydroxybenzotriazole (HOBT) and DCC in DMF affords the dipeptide (V), which by deprotection with HCl in dioxane and condensation with 2(R)-(tert-butoxycarbonylamino)-3-(methylsulfanyl)propionic acid (VI) by means of HBOT and DCC as before yields the tripeptide (VII). Finally, this compound is deprotected with HCl as before and condensed with 2-(5-isoquinolinyloxy)acetic acid (VIII) by means of HBOT and DCC or HBOT and 5-norbornene-2,3-dicarboxyimide (NDPP) in DMF.
2) The intermediate (V) of the preceding synthesis is submitted to a deprotection - protection sequence [hydrolysis with HCl in dioxane and protection with benzyl succinimidyl carbonate (PhCH2-OSu)] giving the benzyloxycarbonyl protected compound (IX), which is condensed with (VI) by means of DCC and dimethylaminopyridine (DMAP) to yield (X) [the ester isomer of (VII)]. The deprotection of (X) with HCl and condensation with (VIII) by means of HBOT and DCC as before affords (XI) [the benzyloxycarbonyl protected prodrug of KNI-272], which is deprotected with trifluoroacetic acid (TFA)/anisole and HCl affording the ester prodrug (XII). Finally, this compound is converted into KNI-272 by incubation at 37 C in a phosphate buffered saline at pH 7.4.