Synthesis of piperazine-1-carbohydrazide inhibitors of serine proteases

Serinske proteaze in njihovi endogeni zaviralci imajo pomembno vlogo v življenju vseh organizmov. Njihovo pravilno delovanje je ključnega pomena za kakovostno in pravilno delovanje celic. Majhna napaka lahko pusti trajne posledice na celici in organizmu. Zaviralci serinskih proteaz so nova skupina zdravilnih učinkovin v procesu zdravljenja rakavih bolezni. Spremenijo biokemične in morfološke značilnosti celic, kar lahko vodi v apoptozo. V sklopu predhodnih raziskav na naši fakulteti smo odkrili skupino molekul, ki so vplivale na proces apoptoze rakavih celic. Namen magistrske naloge je bil sintetizirati nove piperazin-1-karbohidrazidne zaviralce serinskih proteaz in s tem optimizirati lastnosti naših predhodnih spojin. Želeli smo izboljšati predvsem vodotopnost spojin, kar posledično lahko vpliva na njihovo biološko uporabnost, olajša pa tudi biokemijsko vrednotenje spojin in vitro. Končne spojine vsebujejo velik hidrofobni fragment (naftalenski obroč), benzamidoksimsko skupino in ustrezen piperazinski derivat, vezane na hidrazidno ogrodje. V šeststopenjski sintezni poti smo izhodno spojino najprej zaščitili z BOC-skupino ter jo s katalitskim hidrogeniranjem pretvorili do intermediata, na katerega smo pripeli ustrezen piperazinski derivat. Kasneje smo BOC-zaščito odstranili ter uvedli naftoilno skupino. V zadnji stopnji smo ciano skupino pretvorili v amidoksim, ki je znana oblika predzdravila za amidine. Zadnja stopnja nam je povzročala težave, saj reakcija po osnovnem predpisu ni potekla, tako da smo bili primorani iskati nove, alternativne poti. Prav tako smo spojine v šesti in po potrebi v peti sintezni stopnji očistili z gravitacijsko kolonsko kromatografijo. Uspešno smo sintetizirali štiri končne spojine. Istovetnost in čistost vseh intermediatov in končnih spojin smo ovrednotili in potrdili s tankoplastno kromatografijo, jedrsko magnetno resonanco, masno spektrometrijo, masno spektrometrijo visoke ločljivosti, z infrardečo spektroskopijo ter z določitvijo talilnega intervala. Vmesne in končne spojine smo poslali na nadaljnje biokemijsko vrednotenje na Katedro za klinično biokemijo. Spojine so pokazale obetavno zaviranje serinskih proteaz človeške nevtrofilne elastaze (HNE) in katepsina G (CatG) tako v manjši kot tudi v večji testirani koncentraciji. Serinsko proteazo proteinaza 3 (PR3) pa je zavirala le spojina 27.Serine proteases and their endogenous inhibitors are very important for the life of all living organisms. Their proper functioning is crucial for the quality and proper functioning of the cells. A minor malfunction may have permanent, even fatal, effects on cells and the organism. Serine protease inhibitors are a new pharmacological group of active substances in the process of cancer treatment. They alter biochemical and morphological characteristics of the cell itself, which may lead to cell apoptosis. As part of previous research at our faculty, we discovered a group of molecules, which modulated the apoptosis of cancer cells. The purpose of my master\u27s thesis was to synthesize new piperazine-1-carbohydrazide derivatives as serine protease inhibitors, thereby optimizing the properties of previously reported compounds. In particular, we tried to improve the water solubility of compounds, which in turn may affect their bioavailability as well as facilitate the biochemical evaluation of the compounds in vitro. Final compounds contain a large hydrophobic fragment (naphthalene ring), benzamidoxime group and suitable piperazine derivative, attached to the hydrazide scaffold. In the six-step synthetic pathway, the starting compound was first protected with a BOC group and converted with catalytic hydrogenation to the intermediate, to which the piperazine derivative was attached. Subsequently, the BOC protection was removed and the naphthoyl group was introduced. In the last step, the cyano group was converted to amidoxime, which is a known prodrug form of amidines. The last synthetic step caused us some problems, because the reaction did not proceed as planned. We were forced to look for a new, alternative approach. Also, compounds in the sixth and, if necessary, in the fifth synthetic step were purified by gravity column chromatography. We successfully synthesized four final compounds. The identity and purity of all intermediates and final compounds were evaluated and confirmed by thin layer chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, high resolution mass spectrometry, infrared spectroscopy and melting range determination. Intermediates and final compounds were sent for further biological evaluation to the Chair of Clinical Biochemistry. The tested compounds showed promising inhibition of serine proteases human neutrophil elastase (HNE) and cathepsin G (CatG) in both, lower and higher, tested concentration. However, serine protease proteinase 3 (PR3) was only inhibited by compound 27