Development of new chiral 2-hydroxyiminoacetamide reactivators of phosphylated cholinesterases

Korištenjem računalnih metoda molekulskog modeliranja proučavane su konformacijske promjene u aktivnom mjestu acetilkolinesteraze (AChE; EC 3.1.1.7) nakon vezanja raznih liganada te su definirane strukturne karakteristike učinkovitih oksimskih reaktivatora AChE inhibirane živčanim bojnim otrovima kao i smjernice za njihovu sintezu. Pripravljena su četiri nova kiralna oksima iz skupine N-supstituiranih 2-hidroksiiminoacetamida polazeći od racemičnog 1-fenilprop-2-en-1-amina pripravljenog iz 3-fenilprop-2-en-1-ola. Enantiomeri oksima odijeljeni su tekućinskom kromatografijom visoke djelotvornosti na polisaharidnim kiralnim nepokretnim fazama. Novi oksimi ispitani su kao inhibitori AChE i butirilkolinesteraze (BChE; EC 3.1.1.8) i kao reaktivatori kolinesteraza inhibiranih tabunom, ciklosarinom, sarinom i VX-om. Svi oksimi reverzibilno inihibiraju oba enzima s konstantom inhibicije (KI) u mikromolarnom području. Oba enzima pokazuju najveći afinitet prema N-(3- (4-((2-metilimidazol-1-il)metil)-1,2,3-triazol-1-il)-1-fenilpropil)-2-hidroksiiminoacetamidu prema kojem BChE pokazuje značajnu selektivnost i stereoselektivnost. Isti oksim jedini se pokazao učinkovitijim od vodećih oksima opisanih u literaturi i to u slučaju BChE inhibirane ciklosarinom. Molekulskim pristajanjem utvrđeno je da na razlike u smještavanju novih oksima u AChE i BChE najviše utječu razlike u aminokiselinama na pozicijama Tyr72, Tyr124, Phe297 i Tyr337 u aktivnom mjestu AChE.Using computational methods of molecular modelling conformational changes in the active site of acetylcholinesterase (AChE; EC 3.1.1.7) upon binding of various ligands were investigated and structural characteristics of efficient oxime reactivators of AChE inhibited with warfare nerve agents and gudelines for their synthesis were defined. Four new chiral oxime reactivators from N-substituted 2-hydroxiiminoacetamide group were prepared starting from racemic 1-phenyl-allylamine prepared from cinnamyl alcohol. Enantiomers of oximes were separated using high performance liquid chromatography on polysaharidic chiral stationary phases. New oximes were tested for inhibition of AChE and butyrylcholinesterase (BChE; EC 3.1.1.8) and reactivation of cholinesterases inhibited with tabun, cyclosarin, sarin and VX. New oximes reversibly inhibit both enzymes with inhibition constant (KI) in the micromolar range. Both enzymes showe greatest affinity toward 2-hydroxyimino-N-(3-(4-((2- methylimidazol-1-yl)methyl)-1,2,3-triazol-1-yl)-1-phenylpropyl)acetamide towards which BChE displays significant selectivity and stereoselectivity. Only this oxime showed greater reactivation efficiency than the reference oximes described in literature in the case of BChE inhibited with cyclosarin. Molecular docking studies concluded that differences in binding of new oximes in AChE and BChE largely result from differences in the amino acids at the position of Tyr72, Tyr124, Phe297 and Tyr337 in the AChE active site