Design, synthesis and biochemical evaluation of 3-substituted N-benzylpiperazine-1-carbohydrazide inhibitors of human neutrophil elastase

Človeška nevtrofilna elastaza (HNE) je encim, ki sodi v skupino serinskih proteaz. V organizmu ima pomembno vlogo pri vzdrževanju homeostaze in regulaciji celičnih procesov. Povečana aktivnost HNE lahko povzroči poškodbe tkiva, ki so povezane z infekcijami in vnetji. Iz tega lahko sklepamo, da je HNE udeležena v raznih hujših kroničnih boleznih. Udeležena je tudi v regulaciji celične smrti, tako apoptoze kot nekroptoze. Namen magistrske naloge je bil na podlagi že sintetiziranih spojin sintetizirati novo serijo zaviralcev encima HNE. Pripravili smo derivate spojin, ki temeljijo na 3-substituiranem N-benzilpiperazin-1-karbohidrazidnem ogrodju, pred tem pa smo že pripravili eno končno spojino, ki je bila 4-substituirana. Od že sintetiziranih azafenilalaninskih derivatov smo ohranili amidoksimski fragment na fenilnem obroču, 2-naftoilno skupino na drugem dušiku hidrazinskega dela in ustrezen ciklični derivat (piperidinski oziroma piperazinski derivat). V šeststopenjski sintezni poti smo najprej 3-formilbenzonitril pretvorili v hidrazon, zaščiten z BOC zaščitno skupino. Tega smo katalitsko hidrogenirali in dobili hidrazin. Na prvi dušik spojine smo vezali ustrezni piperazinski oziroma piperidinski derivat. Nato smo odstranili zaščitno skupino BOC z drugega dušika in nanj pripeli naftoilno skupino. V zadnji stopnji pa smo nitrilno skupino pretvorili v amidoksim. Uspešno smo sintetizirali le dve od petih načrtovanih končnih spojin. Za ovrednotenje istovetnosti in čistosti intermediatov in končnih spojin smo uporabili tankoplastno kromatografijo, jedrsko magnetno resonanco, masno spektrometrijo, masno spektrometrijo visoke ločljivosti, infrardečo spektroskopijo in določitev temperature tališča. Vseh pet spojin predzadnje in eno spojino zadnje sintezne stopnje smo biokemijsko vrednotili z meritvijo encimske kinetike. Kot serinske proteaze smo poleg tripsina in α-kimotripsina želeli uporabiti še tri nevtrofilne serinske proteaze (HNE, katepsin G in proteinaza 3), vendar smo izmed teh spojine lahko uspešno biokemijsko vrednotili le na HNE. Najmočnejše zaviranje HNE dosežeta spojini predzadnje stopnje, ki imata na piperazinu vezan fenilni obroč s klorom oziroma fluorom na α-mestu. Tudi ostale spojine so pri nekoliko višjih vrednostih IC50 kazale sposobnost zaviranja. Spojine niso dosegle visoke selektivnosti med serinskimi proteazami.Human neutrophil elastase (HNE) is an enzyme that belongs to the serine protease family. It has an important role in human organism in the preservation of homeostasis and regulation of cell processes. Increased activity of HNE can induce tissue damage that may be linked to infections and inflammation. Therefore, it can be concluded, that HNE is involved in a variety of severe chronic diseases. It is also involved in the regulation of cell death, both apoptosis and necroptosis. The purpose of this master\u27s thesis was to synthesize a new series of HNE inhibitors, based on previously synthesized compounds. We prepared derivatives of compounds that are based on a 3-substituted N-benzylpiperazine-1-carbohydrazide scaffold. A 4-substituted final compound was previously prepared. From already synthesized azaphenylalanine derivatives, we kept the amidoxime group on a phenyl ring, 2-naphtoyl group on the second nitrogen of hydrazine fragment and an adequate cyclic derivate (piperazine or piperidine). In the six-step synthetic pathway, 3-formylbenzonitrile was first converted into hydrazone, protected with a BOC group. Hydrazone was catalytically hydrogenated to hydrazine. The piperazine or piperidine derivative was attached to the first nitrogen. Subsequently, the BOC group was removed from the second nitrogen and the naphthoyl group was introduced. In the last step, the nitrile group was converted to amidoxime. We managed to synthesize only two of five final compounds. The identity and purity of all intermediates and final compounds were evaluated by thin layer chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, high-resolution mass spectrometry, infrared spectroscopy and melting point determination. All five compounds of the fifth and one compound of the sixth step were biochemically evaluated with enzyme kinetics measurements. Besides serine proteases trypsin and α-chymotrypsin, three additional neutrophil serine proteases (HNE, cathepsin G and proteinase 3) were planned to be used, but we were only able to make biochemical evaluations on the HNE enzyme. The strongest inhibition of HNE was shown by two compounds of the fifth step with phenyl ring, with chlorine or fluorine on α-position, bound to piperazine. The ability to inhibit HNE was also shown by other tested compounds at slightly higher IC50 values. High selectivity between serine proteases was not achieved