Design and synthesis of piperazine-1-carbohydrazide inhibitors of human neutrophil elastase and proteinase 3

Človeška nevtrofilna elastaza (HNE) in proteinaza 3 (PR 3) sta serinski proteazi v primarnih granulah nevtrofilcev. S svojo proteolizno aktivnostjo sodelujeta v patogenezi kroničnih vnetnih stanj, uravnavanju imunskega sistema in uničevanju patogenov. Poleg tega imata zaradi neravnovesja med proteazami in antiproteazami pomembno vlogo pri patofiziologiji različnih bolezni, npr. kronični obstruktivni pljučni bolezni, cistični fibrozi in Wegnerjevi granulomatozi. Ravno zaradi njune vpletenosti pri tovrstnih obolenjih predstavljata validirani tarči za razvoj novih biološko aktivnih molekul in je iskanje njunih zaviralcev zelo pomembno. Namen magistrskega dela je bilo načrtovanje in sinteza novih piperazin-1-karbohidrazidnih zaviralcev encimov HNE in PR 3. Na osnovi predhodno sintetiziranih spojin smo v strukturi ohranili N-monosubstituirani piperazinski obroč in amidoksimsko skupino, medtem ko smo namesto naftilne skupine izbrali manjše lipofilne fragmente, s katerimi smo razširili kemijski prostor tovrstnih zaviralcev. V okviru magistrske naloge smo preverili še vpliv kisle karboksilne skupine, ki smo jo vgradili na mesto, kjer je bila v predhodnih spojinah amidoksimska skupina. V prvih dveh sinteznih stopnjah smo na 4-formilbenzonitril in metil 4-formilbenzoat pripeli z Boc zaščiten hidrazin in s sledečim katalitskim hidrogeniranjem dobili izhodni spojini za vse nadaljnje korake sinteze. Pri reakciji s trifosgenom in izbranim terciarnim aminom smo s pripenjanjem dveh piperazinskih derivatov tvorili azafenilalaninsko ogrodje. Temu je sledila odstranitev zaščitne skupine Boc in vezava lipofilnih fragmentov R3. Zadnjo stopnjo sinteze je predstavljala pretvorba ciano skupine v amidoksimsko oziroma hidroliza metilnega estra do karboksilne kisline. Deset novo sintetiziranih končnih spojin smo biokemijsko ovrednotili kot zaviralce proteaz z določanjem encimske kinetike, pri čemer smo uporabili pet serinskih proteaz (HNE, PR 3, Cat G, tripsin in α-kimotropsin). Vse spojine smo uspešno ovrednotili na zaviralno aktivnost HNE, PR 3, tripsina in α-kimotripsina, medtem ko s Cat G nismo ovrednotili dveh spojin. Ena izmed končnih spojin je zavirala nevtrofilne serinske proteaze, medtem ko so bile ostale spojine praktično neaktivne v izbranem testnem sistemu.Human neutrophil elastase (HNE) and proteinase 3 (PR 3) are serine proteases found in the primary granules of neutrophils. Their proteolytic activity contributes to chronic inflammatory conditions, immune regulation and pathogen destruction. In addition, due to the imbalance between proteases and antiproteases, they play important roles in the pathophysiology of various diseases, e.g. chronic obstructive pulmonary disease, cystic fibrosis, and Wegner\u27s granulomatosis. It is their involvement in these diseases that makes them validated targets for the development of biologically active molecules and the search for their inhibitors is an important area. The aim of the MSc thesis was to design and synthesise novel piperazine-1-carbohydrazide inhibitors of HNE and PR 3 enzymes. Based on previously synthesised compounds, we retained the N-monosubstituted piperazine ring and the amidoxime group in the structure, while we replaced the naphthyl group with smaller lipophilic fragments to extend the chemical space of such inhibitors. In addition, the replacement of amidoxime with an acidic carboxylic group was evaluated. In the first two steps of our synthesis, a Boc-protected hydrazine was attached to 4-formylbenzonitrile and methyl 4-formylbenzoate and the subsequent catalytic hydrogenation afforded the starting compounds for all further synthetic steps. In the reaction with triphosgene and a selected tertiary amine, an azaphenylalanine scaffold was formed by the attachment of two piperazine derivatives. This was followed by removal of the Boc protecting group and introduction of lipophilic R3 fragments. The final step of the synthesis was the conversion of the nitrile to an amidoxime group or the hydrolysis of the methyl ester to carboxylic acid. The ten newly synthesised final compounds were biochemically evaluated as protease inhibitors by determining enzyme kinetics using five serine proteases (HNE, PR 3 and Cat G, trypsin and α-chymotrypsin. All compounds were successfully evaluated as HNE, PR 3, trypsin and α-chymotrypsin inhibitors, while two compounds were not evaluated on Cat G. One of the final compounds inhibited neutrophil serine proteases, while the other compounds showed poor ability to inhibit neutrophil serine proteases and were practically inactive in the selected test system