Synthesis and isolation of diastereomeric forms of InhA inhibitors

Tuberkuloza je stara nalezljiva bolezen, ki jo povzročajo bacili bakterije Mycobacterium tuberculosis in najpogosteje prizadene pljuča. Po podatkih Svetovne zdravstvene organizacije se letno s tuberkulozo okuži približno 10 milijonov ljudi, za posledicami bolezni pa letno umre okoli 1,5 milijonov obolelih. Posebnost mikobakterij je njihova z lipidi bogata celična stena, poglavitni del katere sestavljajo mikolne kisline. Za sintezo teh je odgovornih več encimov, med drugim tudi InhA, ki je esencialen in prisoten le pri bakterijah, kar zagotavlja veliko selektivno toksičnost. Izoniazid je predzdravilo, ki ga encim KatG pretvori do produktov, ki so močni zaviralci encima InhA. Mutacije gena katG so v glavnem odgovorne za odpornost na izoniazid. Seve, ki so hkrati odporni na zdravila prvega izbora izoniazid in rifampin, imenujemo na zdravila odporna tuberkuloza. Zdravljenje te oblike bolezni je zahtevno in dolgotrajno, uporabljena zdravila drugega izbora pa izkazujejo več toksičnih učinkov, zato je smiselno iskanje direktnih zaviralcev InhA. V magistrski nalogi smo resintetizirali že znane zaviralce InhA ciklopentanskega in cikloheksanskega tipa, ki pa so bili optično čisti. Za optimizacijo sinteznih postopkov smo izhajali iz večje količine racemnega terc-butil (2-karbamoilciklopentil)karbamata. Na eni izmed vmesnih stopenj nam je uspelo s kolonsko kromatografijo ločiti syn in anti racemata. Iz obeh racematov nam je uspelo pripraviti končna anti (4A) in syn (4B) racemata. Po podobnem postopku smo nato iz optično čistih snovi poskusili sintetizitati posamezne optično čiste končne produkte, kar nam je uspelo s spojino 14, pri spojinah 7 in 18 pa zaradi slabih izkoristkov s sintezo nismo nadaljevali do konca. Izoliranim končnim spojinam so na Katedri za farmacevtsko kemijo Fakultete za farmacijo Univerze v Ljubljani testirali sposobnost zaviranja encima InhA. Proti pričakovanju so za spojino 4A določili IC50 3,7 μM in za 4B 2,9 μM, kar pomeni, da razlik med spojinama praktično ni. Sposobnost zaviranja encima InhA so testirali tudi za spojino 14. Slednja ima IC50 2 μM, kar je v istem velikostnem razredu kot 4A in 4B. Na podlagi teh rezultatov lahko zaključimo, da nadaljnja resolucija syn in anti racematov oz. sinteza čistih enantiomerov ni smiselna.Tuberculosis is an old infectious disease caused by Mycobacterium tuberculosis that most often affects the lungs. According to data by the World Health Organization, every year 10 million people fall ill with tuberculosis and 1,5 million die each year. A distinguishing characteristic of mycobacteria is their lipid rich cell wall, comprised predominantly of mycolic acids. The synthesis of the latter involves several enzymes, among which InhA is essential and limited to mycobacteria, which provides high selective toxicity. Isoniazid is a prodrug, which after activation by the enzyme KatG forms several products that are strong inhibitors of InhA. Resistance to isoniazid is most often caused by mutations of the katG gene. Multi-Drug Resistant Tuberculosis (MDR-TB) refers to strains which exhibit resistance to both isoniazid and rifampin simultaneously. Treatment of MDR-TB is demanding and extensive, since second line drugs used in the treatment exhibit several toxic side effects. Therefore, it is worthwhile to search for new direct InhA inhibitors. In our work we resynthesized known InhA inhibitors of the cyclopentane and cyclohexane type that were optically pure. In order to optimize the synthetic procedures we used a larger amount of racemic tert-butyl (2-carbamoylcyclopentyl)carbamate as our starting compound. During one of the intermediate stages of the synthesis we managed to separate the syn and anti racemates via column chromatography. We used both of those racemates to synthesize the final compounds of anti (4A) and syn (4B) racemates. We then tried using similar procedures to synthesize optically pure final compounds, which we managed to do with compound 14, whereas we had to discontinue the synthesis of compounds 7 and 18 due to low yields. The inhibiting potential of the enzyme InhA of our final compounds was tested at the Faculty of pharmacy, University of Ljubljana. Against our expectations, the results determined the IC50 of 3,7 μM and 2,9 μM for compounds 4A and 4B, respectively, indicating little difference between the two compounds. Similarly, InhA inhibiting potential was also tested for compound 14. The latter had an IC50 of 2 μM, putting it in the same class of inhibition potency as compounds 4A and 4B. Based on these results we can conclude that further resolution of syn and anti racemates or synthesis of pure enantiomers is not worthwhile