Effect of bioisosteric replacement of thiadiazole with triazole on the inhibitory activity of thiadiazole type InhA inhibitor

Tuberkuloza, ki jo povzroča okužba z bacili Mycobacterium tuberculosis, je najpogostejša nalezljiva kronična bolezen na svetu in po umrljivosti zaostaja le za okužbo s SARS-CoV-2 (COVID-19). Ocenjuje se, da je z bacili tuberkuloze okužena kar četrtina svetovnega prebivalstva, vse večjo skrb pa vzbuja pojav vedno novih odpornih oblik tuberkuloze. Zdravljenje bolezni poteka z različnimi režimi terapije s kombinacijo več zdravilnih učinkovin. Najbolj znan in razširjen antituberkulotik je izoniazid, ki zavira delovanje encima InhA, s čimer zavira sintezo mikobakterijske celične stene. Učinkovina deluje kot predzdravilo, aktivira se s pomočjo mikobakterijskega encima KatG. Večina na izoniazid odpornih oblik sevov ima mutacijo v genu, ki kodira KatG, kar posledično zmanjša aktivacijo in učinkovitost zdravljenja. Prav slednje je usmerilo razvoj učinkovin v iskanje novih direktnih zaviralcev encima. Obetavna skupina novejših direktnih zaviralcev encima InhA so spojine iz serije tiadiazolov. V okviru magistrske naloge smo sintetizirali analoge spojine vodnice I, ki je predstavnik teh tiadiazolnih zaviralcev. Z bioizosterno zamenjavo smo namesto tiadiazola (obroč C) uvedli triazol, pri čemer smo zaradi poenostavitve sinteze odstranili sekundarni amin med obročema B in C. Nato smo pripravili vrsto analogov, pri kateri smo spreminjali obroč D. Sinteza je potekala v dveh stopnjah, pri čemer smo v prvi pripravili različne terminalne alkine. Te smo nato v drugi stopnji s klik reakcijo (z bakrom (I) katalizirano azid-alkin 1,3-dipolarno cikloadicijo) združili s 3-(azidometil)-1-(2,6-difluorobenzil)-1H-pirazolom. Pripravljenim analogom smo z in vitro biološkim testiranjem določili učinkovitost. Rezultati so pokazali, da je delovanje ohranil le neposreden analog spojine vodnice I, ki smo mu nato določili tudi vrednost IC50. V primerjavi s spojino vodnico I je bila ta občutno višja, kar nakazuje na zmanjšano učinkovitost analoga. Sklepamo, da je to posledica odsotnosti vmesnega sekundarnega amina med obročema B in C, ki zagotavlja ključno interakcijo in optimalno umestitev spojine v vezavno mesto encima. Menimo, da bi bilo smotrno pripraviti analog z omenjeno skupino ter nato poskusiti ovrednotiti spremembe obroča D.Tuberculosis, which is caused by infection with Mycobacterium tuberculosis bacteria, is the most frequent chronical infectious illness globally and is right after SARS-CoV-19 (COVID-19) regarding mortality rates. It is estimated that about a quarter of the global population is infected with tuberculosis bacilli, while the rise of new resistant types of tuberculosis is progressively worrisome. Treatment of tuberculosis includes a variety of regimens with a combination of numerous drugs. The most well-known and prevalent antitubercular drug is isoniazid, which inhibits the InhA enzyme, resulting in the suppression of mycobacterial cell growth. The drug works as a prodrug, in order to activate it needs to be conversed by the mycobacterial KatG enzyme. Most of the isoniazid resistant strains of tuberculosis have a mutation in the KatG encoding gene, which results in the decrease of activation and its effectiveness. The latter has directed the development of antitubercular drugs to discovering new direct enzyme inhibitors. A promising group of newer direct InhA inhibitors are compounds from the thiadiazole series. In this master\u27s thesis we synthesized analogs of the lead compound I, which is a member of the thiadiazole series. We introduced a triazole ring instead of thiadiazole (ring C) with a bioisosteric replacement and we eliminated the secondary amine group between rings B in C in order to simplify the synthesis. Afterwards we prepared a series of analogs with different rings D. The synthesis consisted of two stages, in the first one we prepared various terminal alkynes. Then, in the second stage, we combined these with 3-(azidomethyl)-1-(2,6-difluorobenzyl)-1H-pirazole via click reaction (copper (I) catalysed azide-alkyne 1,3- dipolar cycloaddition). The synthesised analogs were then in vitro biologically tested to determine their efficiency. Results indicated, that only the direct analog of lead compound I retained activity, for this reason we then determined its IC50 value. It was considerably higher then the one of lead compound I, which indicates a decreased activity of the analog. We presume that this is the result of the elimination of secondary amine group between rings B and C, that provides the key interaction and optimal position in the binding site of the enzyme. In our opinion it would be sensible to synthesize the direct analog with the mentioned group and only then to try to evaluate changes in ring D