Sinteza in biokemijsko vrednotenje 2-klorobenzotiazolnih zaviralcev encima MurA

Klobučar, Ema (author), Hrast, Martina (mentor), Knez, Damijan (comentor)

Odpornost bakterij proti protibakterijskim učinkovinam je globalni problem, pri čemer patogeni mikroorganizmi razvijejo odpornost na te učinkovine predvsem zaradi nepravilne in prekomerne uporabe le-teh, zaradi česar je potreba po novih protibakterijskih zdravilnih učinkovinah vse večja. Dober potencial za razvoj novih protibakterijskih učinkovin predstavljajo encimi, ki sodelujejo pri sintezi peptidoglikana, ključnega gradnika bakterijske celične stene. Encim MurA (UDP-N-acetilglukozamin enolpiruvil transferaza) je znotrajcelični bakterijski encim, ki ima ključno vlogo v prvi stopnji sinteze peptidoglikana in predstavlja dobro potencialno tarčo, saj je njegova aktivnost nujno potrebna za preživetje tako gramnegativnih kot tudi grampozitivnih bakterij, hkrati pa ni prisoten pri človeku. V sklopu magistrske naloge smo izhajali iz spojine 2-klorobenzo-1,3-tiazola (spojina 1b), ki je v predhodnih raziskavah pokazala dobro zaviralno delovanje na MurA encimu iz bakterije Escherichia coli (IC50 = 120 ± 12 µM) in Staphylococcus aureus (IC50 = 123 ± 12 µM). Sintetizirali smo različno substituirane derivate spojine 1b ter z biokemijskim testiranjem ugotavljali, kako s substitucijo na aromatskem obroču vplivamo na zaviralno delovanje spojin. Pri dvostopenjski sintezi končnih spojin smo pri prvi stopnji reakcije dobili intermediate (analoge benzo-1,3-tiazol-2-amina), katerim smo prav tako določili zaviralno aktivnost na MurA. Zaviralno delovanje spojin lahko bistveno izboljšamo z meta (glede na dušik) substitucijo na aromatskem obroču. Izmed vseh sintetiziranih spojin sta najmočnejše zaviralno delovanje pokazali spojina 6 (2-amino analog) in spojina 24 (2-kloro analog) s substitucijo na meta in para mestu (IC50: 6 = 1,2 μM; 24 = 3,62 µM). Sledijo spojine z meta substitucijo – 2-kloro produkti (IC50: 25 = 12,7 µM; 27 = 50 µM; 29 = 7,6 µM) ter 2-amino analoga (IC50: 7 = 3,1 µM; 9 = 23 µM). Tudi para substituirani 2-amino analogi so v primerjavi s spojino 1b pokazali izboljšano zaviralno delovanje (IC50: 2 = 82 μM; 5 in 8 = 43 µM). Spojine 19, 20, 21, 22, 26, 35, 36 (IC50: 92–615 μM) s substitucijo na mestu para in disubstituirana spojina 30 (IC50 = 116 µM) pa v primerjavi s spojino 1b nimajo bistveno izboljšanega zaviralnega delovanja. Zanimiva je bila ugotovitev, da so tudi 2-amino derivati pokazali dobro zaviralno delovanje na MurA encim, iz česar lahko sklepamo, da klor ni ključen za zaviralno delovanje benzotiazolov.

• Language: Slovenian
• Keywords: encim MurA, benzotiazoli, protibakterijske učinkovine
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-116831
• Publication date in RUL: 12.06.2020

Secondary language

• Language: English
• Title: Synthesis and biochemical evaluation of 2-chlorobenzothiazole MurA inhibitors

Abstract

Antibacterial resistance is a global problem, where pathogenic microorganisms develop resistance to many active substances, mainly due to improper use and overuse of antibacterial agents, increasing the need for new antibacterials. Enzymes involved in the synthesis of peptidoglycan, a key element of the bacterial cell wall, represent promising targets for development of new antibacterial agents. MurA enzyme (UDP-N-acetylglucosamine enolpyruvyl transferase) is an intracellular bacterial enzyme that plays an essential role in the first stage of peptidoglycan synthesis and is a good target, as its activity is essential for the survival of both Gram-negative and Gram-positive bacteria, and it is not found in humans. We started with 2-chlorobenzo-1,3-thiazole (compound 1b), which in previous studies showed a good inhibition of MurA enzymes from Escherichia coli (IC50 = 120 ± 12 µM) and Staphylococcus aureus (IC50 = 123 ± 12 µM). Differently substituted derivatives of compound 1b were synthesized and biochemical testing was performed to determine how substitution on the aromatic ring effects the inhibitory potency of the compounds. Two-step synthesis of the final compounds yielded intermediates (benzo-1,3-thiazol-2-amines), for which inhibitory potencies were also determined. The inhibitory activity of the compounds was significantly improved by introduction of substituents on the meta position (relative to nitrogen) on aromatic ring. Of all the synthesized compounds, compounds 6 (2-amino analogue) and 24 (2-chloro analogue) were the most potent inhibitors with meta and para substituents (IC50: 6 = 1,2 μM; 24 = 3,62 µM), followed by meta substituted compounds – 2-chloro products (IC50: 25 = 12,7 µM; 27 = 50 µM; 29 = 7,6 µM) and 2-amino analogues (IC50: 7 = 3,1 µM; 9 = 23 µM). Additionally, para substituted 2-amino analogues showed improved inhibition of MurA (IC50: 2 = 82 μM; 5 and 8 = 43 µM) in comparison to compound 1b. Derivatives 19, 20, 21, 22, 26, 35, 36 (IC50: 92–615 μM) with substituents at para position and the disubstituted compound 30 (IC50 = 116 µM), did not improve the inhibitory activity significantly in comparison to compound 1b. Interestingly, 2-amino derivatives also showed promising inhibition of MurA enzymes, which suggests that chlorine in benzothiazoles is not essential for the inhibition.

• Keywords: MurA enzyme, benzothiazoles, antibacterial agents