Sinteza derivatov 1,2,3,4-tetrahidropirolo[1,2-a]pirazina kot zaviralcev mikobakterijskega encima InhA

Grabnar, Andreja (author), Pajk, Stane (mentor)

Vse pogostejše pojavljanje sevov bakterij odpornih na protimikrobne učinkovine postaja resen javnozdravstveni problem na svetovnem nivoju. Eden od povzročiteljev okužb, ki vztrajno razvija odpornost, je tudi bakterija Mycobacterium tuberculosis. Povzroča infekcijsko bolezen tuberkulozo, ki je kljub vsemu vloženemu trudu še vedno med 10 vodilnimi vzroki smrti v manj razvitem delu sveta. Po ocenah WHO se letno okuži 10 milijonov ljudi, zaradi tuberkuloze in njenih posledic pa jih 1,5 milijonov letno umre. Zdravljenje tuberkuloze je dolgotrajno in zahteva kombinacijo več zdravilnih učinkovin. Ena od učinkovin je izoniazid, ki preko inhibicije InhA (trans-enoil-(acilni prenašalni protein) reduktaza) zavira sintezo gradnikov bakterijske celične stene - mikolnih kislin. Za njegovo delovanje mora in vivo poteči aktivacija, ki jo katalizira encim KatG. Pojav mutacij na tem encimu je vodilni vzrok za razvoj odpornosti proti izoniazidu. Posledično se preko spojin vodnic, pridobljenih z metodami rešetanja visoke zmogljivosti, išče nove direktne zaviralce InhA. V sklopu eksperimentalnega dela magistrske naloge smo sintetizirali derivate zaviralca InhA, ki ima v svoji strukturi 1,2,3,4-tetrahidropirolo[1,2-a]pirazin. Pirol je pri tej spojini podvržen oksidaciji, zato smo ga zamenjali z drugimi aromatskimi obroči (benzen in tiofen) oziroma zmanjšali občutljivost na oksidacijo z uvedbo elektron-privlačnih skupin (karboksamid, trifluoroacetamid). Načrtovane sinteze smo uspešno izvedli. V prvih stopnjah posameznih sintez smo uporabili ariletilamin, ki smo ga pripravili sami ali pa je bil komercialno dostopen, nato smo amin pretvorili v benzilamid. Sledila je ciklizacija s POCl3 in redukcija cikličnega imida z NaBH4. V zadnji stopnji smo med pripravljenimi amini in kislino 24 tvorili amid. Izolirali smo 5 končnih spojin, ki smo jim poleg stopnje čistote, temperature tališča, molske mase in NMR spektrov, določili vrednost IC50 na encimu InhA. Vseh 5 izoliranih spojin je imelo primerljive vrednosti IC50. Najnižjo vrednost IC50 je imela spojina 22 z uvedeno trifluoroacetilno skupino na pirolni obroč z vrednostjo 12 µM. Ugotovili smo, da z zamenjavo pirola ali uvedbo bioizosterne zamenjave (tiofen, benzen) ne vplivamo na afiniteto vezave na encim InhA.

• Language: Slovenian
• Keywords: tuberkuloza, mikolne kisline, direktni zaviralci InhA
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-148331
• Publication date in RUL: 16.08.2023

Secondary language

• Language: English
• Title: Synthesis of 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine type derivatives as mycobacterial InhA enzyme inhibitors

Abstract

The increasing bacterial resistance to chemoterapeutics is becoming a serious public health problem at the global level. One of the causative agents of infections that steadily develops resistance is the bacterium Mycobacterium tuberculosis. It causes the infectious disease tuberculosis, which is, despite all efforts, still among the 10 leading causes of death in the less developed part of the world. According to WHO approximately 10 million people are infected annually, and 1.5 million die annually from tuberculosis and its consequences. The duration of treatment is long and it requires a combination of several drugs. One of the first line drugs is isoniazid, which through the inhibition of InhA (trans-enoyl-(acyl carrier protein) reductase) inhibits the synthesis of the component of the bacterial cell wall - mycolic acids. For its action in vivo, activation catalyzed by the enzyme KatG must take place. The occurrence of mutations in this enzyme is the leading cause of the development of resistance to isoniazid. Consequently, new direct inhibitors of InhA are being sought through lead compounds obtained by high-throughput screening method. In the experimental part of the master's thesis, we synthesized derivatives of the InhA inhibitor, which has 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine in its structure. The pyrrole is subject to oxidation in this compound, so we replaced it with other aromatic rings (benzene and thiophene) or reduced the sensitivity to oxidation by introducing electron-withdrawing groups (carboxamide, trifluoroacetamide). We successfully performed the planned syntheses. In the first stages of individual syntheses, we used arylethylamine, which we prepared ourselves or which was commercially available, then the amine was converted into benzylamide. This was followed by cyclization with POCl3 and reduction of the cyclic imide with NaBH4. In the last step, an amide was formed between the prepared amines and the acid 24. We isolated 5 final compounds and in addition to their degree of purity, melting temperature, molar mass and NMR spectra, we determined their IC50 value against InhA enzyme. All 5 isolated compounds have comparable IC50 values. Compound 22 with an introduced trifluoroacetyl group on the pyrrole ring had the lowest IC50 value at 12 µM. We found that the substitution of pyrrole or the introduction of a bioisosteric substitution (thiophene, benzene) does not affect the binding affinity to the InhA enzyme.

• Keywords: tuberculosis, mycolic acids, direct InhA inhibitors