Synthesis and evaluation of 2-chloro-1,3-benzothiazole covalent fragments

Tarčni kovalentni zaviralci so spojine, ki s tvorbo kovalentne vezi z nukleofilnimi aminokislinskimi ostanki selektivno zavirajo aktivnost tarčnega proteina. Njihova uporaba pri načrtovanju novih učinkovin je omejena predvsem zaradi potencialnih neželenih stranskih učinkov. Encimi, ki imajo pomembno vlogo v začetnih stopnjah sinteze peptidoglikana, izkazujejo dober potencial za razvoj novih protibakterijskih učinkovin, saj so nujno potrebni za bakterije, niso pa prisotni v človeškem organizmu. Eden izmed teh znotrajceličnih encimov je MurA (UDP-N-acetilglukozamin enolpiruvil transferaza), ki sodeluje pri prvi stopnji sinteze peptidoglikana. Monoamin oksidaza (MAO) je encim, vezan na zunanjo mitohondrijsko membrano, in ima visoko stopnjo izražanja v nevronih ter prebavnih tkivih. Pri ljudeh obstajata dve izoobliki MAO, ki sta si podobni po tridimenzionalni strukturi, razlikujeta pa se po selektivnosti zaviralcev, substratni specifičnosti in porazdelitvi v tkivih. Magistrsko nalogo smo osnovali na spojini zadetku – 2-kloro-1,3-benzotiazolu, ki zavira encim MurA z IC50 = 91 µM. Pri sintezi analogov smo izhajali iz komercialno dostopnih 1,3-benzotiazol-2-aminov oziroma substituiranih anilinov, ki smo jih v reakciji z amonijevim tiocianatom in bromom pretvorili do derivatov 1,3-benzotiazol-2-amina. 2-Amino derivate smo nato v Sandmeyerjevi reakciji preko tvorbe diazonijeve soli pretvorili v 2-kloro-1,3-benzotiazole. Pripravljene derivate smo nato biokemijsko ovrednotili na izoliranih encimih MurA, MAO-A, MAO-B ter β5i podenoti imunoproteasoma. Določili smo tudi zaviralno aktivnost intermediatov – 1,3-benzotiazol-2-aminov, saj nas je zanimala razlika v aktivnosti 2-amino in 2-kloro analogov. Ugotovili smo, da vezava klora na drugo mesto 1,3-benzotiazola ne poveča zaviralne aktivnosti na encimu MurA, ampak jo v primerjavi z 1,3-benzotiazol-2-amini celo zmanjša. Za 2-kloro derivat 25 smo pokazali, da je zaviranje MurA časovno odvisno, kar nakazuje na kovalentno zaviranje encima. 2-Kloro-1,3-benzotiazoli s substitucijo na mestu 5 zavirajo hMAO-B močneje kot spojine s substitucijo na pozicijah 4, 6 ter 7, zaviranje encima pa je časovno neodvisno. Z nadaljnjimi biokemijskimi študijami bo treba najprej dodatno raziskati mehanizem zaviranja encimov, na podlagi le-tega pa bomo lahko načrtovali nove derivate, ki kovalentno zavirajo encim MurA.Targeted covalent inhibitors are compounds that selectively inhibit the activity of target proteins by forming a covalent bond with nucleophilic amino acid residues. Their use in the development of novel drugs is limited mainly because of possible undesirable side effects. Enzymes that play an important role in the initial stages of peptidoglycan synthesis show promise for the development of novel antibacterials because they are essential for bacteria but are not present in the human body. One of these intracellular enzymes is MurA (UDP-N-acetylglucosamine enolpyruvil transferase), which is involved in the first step of peptidoglycan biosynthesis. Monoamine oxidase (MAO) is an enzyme bound to the outer mitochondrial membrane and is highly expressed in neurons and gastrointestinal tissues. In humans, two isoforms of MAO are present that are similar in their three-dimensional structure but differ in their selectivity for inhibitors, substrate specificity, and tissue distribution. The work was based on the hit compound – 2-chloro-1,3-benzothiazole, which inhibits the MurA enzyme (IC50 = 91 µM). The synthesis of analogs was based on commercial 1,3-benzothiazol-2-amines or substituted anilines, which were converted to 1,3-benzothiazol-2-amine derivatives in a reaction with ammonium thiocyanate and bromine. The obtained 2-amino derivatives were then converted to the desired 2-chloro-1,3-benzothiazoles via a diazonium salt intermediate in the Sandmeyer reaction. The synthesized derivatives were then biochemically assayed on the isolated enzymes MurA, MAO-A, MAO-B and β5i subunit of the immunoproteasome. We also determined the inhibitory activity of intermediates (i.e., 1,3-benzothiazol-2-amines), to investigate the difference in inhibitory potency of 2-amino and 2-chloro derivatives. We found that the binding of chlorine to the second position of 1,3-benzothiazole does not increase the inhibition of the MurA enzyme, rather, the inhibition is decreased compared to 1,3-benzothiazole-2-amines. For 2-chloro derivative 25, MurA inhibition is time-dependent, indicating covalent inhibition of the enzyme. 5-Substituted 2-chloro-1,3-benzothiazoles inhibit hMAO-B with higher potency compared to 4-, 6-, and 7- substituted counterparts, and the enzyme inhibition is not time-dependent. Further biochemical studies will investigate the mechanism of enzyme inhibition in depth, and based on the results, we will develop novel derivatives that inhibit MurA enzyme in a covalent manner