Sinteza derivatov zaviralca encima InhA pirolnega tipa ter kiralna ločba izbranih zaviralcev InhA in imunoproteasoma

Sinjur, Anja (author), Pajk, Stane (mentor)

Tuberkuloza je stara, nalezljiva in lahko tudi smrtna bolezen. Povzroča jo okužba z bakterijo Mycobacterium tuberculosis. Zanjo je značilna nenavadna struktura celične ovojnice, ki je zgrajena iz mikolne kisline. Vse večji problem pri zdravljenju predstavlja odpornost M. tuberculosis proti antituberkulotikom, ki se uporabljajo v standardni terapiji. Izoniazid, zdravilna učinkovina prvega izbora, je predzdravilo. Za njegovo delovanje je potrebna in vivo aktivacija z encimom KatG. Nastali adukt je močan zaviralec encima InhA, ki je udeležen pri biosintezi mikolnih kislin. Ker prihaja pogosto do mutacij na encimu KatG, so zaviralci, katerih tarča je neposredno encim InhA, obetavni kandidati za razvoj novih zdravilnih učinkovin proti vse večji grožnji sevov M. tuberculosis, odpornih na zdravila. V okviru magistrske naloge smo sintetizirali štiri potencialne direktne zaviralce encima InhA (7, 8, 15 in 16). Izhajali smo iz spojine ?, kjer smo namesto tetrahidropiranskega dela uvedli 1,2,3,4-tetrahidropirolo[1,2-a]pirazin z različnimi substituenti na mestu 1 in 6 in tako raziskovali kemijski prostor v aktivnem mestu encima InhA, kjer se tvorijo lipofilne interakcije med učinkovino in vezavnim mestom. Sintetiziranim spojinam so na Fakulteti za farmacijo na Univerzi v Ljubljani določili IC50. Spojina 15 je izkazala največjo aktivnost, IC50 znaša 830 nM. Ker gre za molekulo s kiralnim centrom, bi bila v prihodnje smiselna kiralna ločba, saj predvidevamo, da je za aktivnost odgovoren le en enantiomer. V nadaljevanju smo ugotovili, da je potrebno na mestu 6 v 1,2,3,4-tetrahidropirolo[1,2a]pirazinu uvesti substituent zaradi drugače nestabilnega pirola. V drugem delu magistrske naloge smo izvedli kiralno ločbo racemne zmesi tiadiazolnega tipa zaviralca InhA M1155 in racemne zmesi zaviralca imunoproteasoma LR114P. Izvajali smo direktno kiralno ločevanje spojin s HPLC. Pri prvi spojini enantiomerov nismo uspeli ločiti na bazni liniji, vendar smo dobili zadovoljive rezultate za nadaljnje potrebe. Z optimizacijo pogojev ločbe druge spojine smo prišli do ločbe enantiomerov na bazni liniji. Zbrane posamezne frakcije enantiomerov bodo uporabili na Fakulteti za farmacijo za nadaljnja testiranja.

• Language: Slovenian
• Keywords: tuberkuloza, direktni zaviralci InhA, kiralna kromatografija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107725
• Publication date in RUL: 17.05.2019

Secondary language

• Language: English
• Title: Synthesis of pyrrole type derivatives as InhA inhibitors and chiral separation of selected InhA and immunoproteasome inhibitors

Abstract

Tuberculosis is an ancient, contagious and possibly deadly disease caused by the infection with bacterium Mycobacterium tuberculosis. The bacterial cell wall has an unusual structure and is composed of mycolic acids. The immunity of M. tuberculosis to antituberculosis drugs used in standard therapy poses a serious problem. Isoniazid, first-line agents, is a prodrug and needs to be activated in vivo by KatG enzyme. The formed adduct is a strong inhibitor of the enzyme InhA, present in biosynthesis of mycolic acids. Due to common mutations on KatG enzyme, the inhibitors directly targeting the InhA enzyme become auspicious candidates for the development of active substances against the growing threat of M. tuberculosis strains that are immune to medications. In the first part of our research we synthesised four potential direct inhibitors of enzyme InhA (7, 8, 15 and 16). We used the compound ? in which we replaced the tetrahydropyran part with 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine with different substituents in positions 1 and 6. This way we could explore chemical space in the active site of InhA enzyme where lipophilic interactions between the substance and the binding site take place. At the Faculty of Pharmacy, they defined the synthesised compounds as IC50. The compound 15 exhibited the highest activity with IC50 totalling 830 nM. According to our predictions that only one enantiomer is responsible for the activity, we concluded that a chiral separation would be more reasonable for the molecule with a chiral centre. Furthermore, we found out that in 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine the replacement of the substituent in position 6 is essential due to the instability of pyrrole. In the second part of our research we performed chiral separation of the racemic mixture of thiadiazole inhibitor InhA M1155 and of immunoproteasome inhibitor LR114P. We carried out the direct chiral separation by HPLC. We did not manage to separate the enantiomers of the first compound on baseline. Nevertheless, we obtained adequate results for future research. The optimisation of the conditions for the separation of the second compound enabled the baseline separation of enantiomers. Gathered individual fractions of enantiomers will be used at the Faculty of Pharmacy for further testing.

• Keywords: tuberculosis, direct inhibitors InhA, chiral chromatograph