Design and synthesis of hydroxyethylamines as inhibitors of peptidoglycan biosynthesis

Vse večje število medicinsko pomembnih patogenih bakterij (stafilokokov, streptokokov, enterokokov, Pseudomonas aeruginosa,Mycobacterium tuberculosis), ki so rezistentne na številne protibakterijske učinovine, predstavlja resen problem pri zdravljenju nalezljivih bolezni, zato v zadnjem času potekajo intenzivne raziskave na področju razvoja novih protibakterijskih učinkovin. Eno od pomembnejših in aktualnih tarčnih mest pri iskanju novih protibakterijskih učinkovin je bakterijski peptidoglikan, sestavljen iz mreže polisaharidnih verig, ki so prečno povezane s kratkimi peptidnimi verigami. Kot esencialna komponenta bakterijske celične stene daje celici značilno obliko in je nujen za vzdrževanje notranjega osmotskega pritiska, zato vpletanje v njegovo sintezo ali strukturo vodi v izgubo bakterijske celovitosti in posledično do celične smrti. Pri biosintezi peptidoglikana sodelujejo številni encimi, med katerimi so ključnega pomena od ATP-ja odvisne ligaze (ligaze Mur in ligaza D-alanil-D-alanin), ki so pomembne pri biosintezi UDP-N-acetilmuramil-pentapeptida kot znotrajceličnega prekurzorja peptidoglikana. Ligaze MurC do MurF katalizirajo zaporedno pripenjanje L-alanina, D-glutaminske kisline, L-lizina ali mezo-diaminopimelinske kisline in dipeptida D-alanil-D-alanina, ki ga iz dveh D-alaninov sintetizira ligaza D-alanil-D-alanin (Ddl), na UDP-N-acetilmuraminsko kislino. Iz družine ligaz D-alanil-D-X so poleg Ddl tudi ligaze Van, ki so odgovorne za razvoj rezistence na antibiotik vankomicin pri enterokokih. Ker so se fosfonatni, fosfinatni in sulfonamidni analogi prehodnega stanja že izkazali kot inhibitorji ligaz Mur in ligaze D-alanil-D-alanin, smo v okviru doktorske disertacije načrtovali, sintetizirali in biološko ovrednotili hidroksietilaminske analoge kot nove potencialne inhibitorje encimov MurC do MurF, VanA in Ddl. Pri načrtovanju smo si pomagali s programsko opremo za »de novo« načrtovanje nizkomolekulskih inhibitorjev SPROUT in orodjem za sidranje ligandov eHiTS. Nato smo razvili in optimizirali sintezno pot za pripravo hidroksietilaminskega fragmenta, ki poteka preko oksidacije derivatov alilamina do epoksida in sledečega odpiranja oksiranskega obroča z N-nukleofili ob prisotnosti kalcijevega trifluorometansulfonata kot katalizatorja. Sintetiziranim spojinam smo določili inhibitorno aktivnost na DdlB, MurC, MurD in MurF iz Echerichie coli, MurE iz Staphylococcus aureus in VanA iz Enterococcus faecium. Spojine, ki so poleg hidroksietilaminskega dela vsebovale L-alanin ali D-glutaminsko kislino, se niso izkazale kot inhibitorji encimov MurC oziroma MurD. Hidroksietilaminski derivati piroglutaminske kisline, substituirani s trifluorometilfenilnim substituentom, so zavirali delovanje ligaz Dalanil-D-alanin in VanA v koncetracijah nad 500 M. Najmočnejše inhibitorje pa smo dobili, ko smo na hidroksilno skupino hidroksietilamina uvedli fosfatno skupino. Hidroksietilaminski derivat 1-(4-metoksifenilsulfonamido)-3-morfolinopropan-2-il dihidrogen fosfat se je izkazal kot inhibitor DdlB (IC50 = 110 μM) in VanA (IC50 = 224 μM). Ker je VanA odgovoren za rezistenco na antibiotik vankomicin, je omenjeni hidroksietilaminski inhibitor dobra izhodiščna spojina za razvoj učinkovin, ki bi zavirale pojav rezistence na vankomicin. Omenjena spojina pa je zavirala tudi delovanje ligaze MurE z IC50 vrednostjo v nizkem mikromolarnem območju (IC50 = 6 μM). Z zamenjavo metoksi skupine s trifluorometilno pa smo dobili močan inhibitor MurC in MurF ligaze. Derivati 1-fenilsulfonamido-3-morfolinopropan-2-il dihidrogen fosfata so zato pomembne izhodiščne spojine za razvoj novih multiplih inhibitorjev biosinteze peptidoglikana s potencialnim širokospektralnim protibakterijskim delovanjem. Zaradi zaviranja aktivnosti encima MurE iz Staphylococcus aureus pa bi se lahko uporabljale tudi kot učinkovine pri zdravljenju infekcij z MRSA (na meticilin rezistentno bakterijo S. aureus).The increasing emergence of medically important pathogenic bacteria (Staphylococci, Streptococci, Enterococci, Pseudomonas aeruginosa, Mycobacterium tuberculosis) with high resistance to numerous antibacterial agents constitutes a serious public threat in the treatment of infectious diseases. Therefore, extensive research in the development of novel antibacterial agents has been carried out in the past few years. One of the most important and validated targets for antibacterial drug discovery research is bacterial peptidoglycan, a heteropolymer composed of alternating glycan chains cross-linked by short peptides. As an essential component of bacterial cell wall, peptidoglycan is important for the maintenance of a defined cell shape and withstanding the internal osmotic pressure. Interference with its synthesis or structure leads to loss of cellular integrity and ultimately to bacterial cell death. Biosynthesis of peptidoglycan involves numerous enzymes. Amongst them are ATP-dependant ligases such as Mur ligases and D-alanyl-D-alanine ligase (Ddl), which are essential for the synthesis of UDP-N-acetylmuramyl-pentapetide, a crucial intracellular precursor of peptidoglycan.MurC to MurF ligases catalyze stepwise addition of L-alanine, D-glutamic acid, L-lysine or meso-diaminopimelic acid and dipeptide D-alanyl-D-alanine (prepared by Ddl from two Dalanines) to UDP-N-acetylmuramic acid. In addition to Ddl, the members of D-alanine:D-X ligases superfamily are also the Van enzymes, which are crucial for vancomycin resistance in Enterococci. Phosphinate, phosphonate and sulfonamide transition-state analogs are wellknown inhibitors of Mur ligases and Ddl. Therefore, we decided to design, synthesize and evaluate the hydroxyethylamines as new potential inhibitors of Mur ligases, VanA and Ddl. To design a new class of inhibitors we used »de novo« ligand design software SPROUT and ligand docking tool eHiTS. In the following step we developed and optimized an efficient synthetic approach to hydroxyethylamines via oxidation of the corresponding allylamine derivatives and subsequent opening of epoxide ring with N-nucleophyles in the presence of calcium trifluoromethanesulphonate as a catalyst. The synthesized compounds were evaluated for their inhibitory activities against DdlB, MurC, MurD and MurF from Echerichia coli, MurE from Staphylococcus aureus and VanA from Enterococcus faecium. The compounds composed of hydroxyethylamine moiety and L-alanine or D-glutamic acid did not show any inhibitory activity on MurC or MurD. Hydroxyethylamine derivatives of pyroglutamic acid substituted with trifluoromethylphenyl substituent inhibited the activity of DdlB and VanA in the concentrations above 500 μM. The strongest inhibitors were obtained when a phosphate group was attached to hydroxyl group of hydroxyethylamine moiety. Hydroxyethylamine derivative 1-(4-methoxy-phenylsulfonamido)-3-orpholinopropan-2-yl dihydrogen phosphate was found as an inhibitor of DdlB (IC50 = 110 μM) and VanA (IC50 =224 μM). Since VanA is responsible for vancomycin resistance the aformentioned inhibitor has the potential to be developed into drugs that would reverse bacterial resistance to vancomycin. This compound also showed promising inhibitory activity against MurE with IC50 value in low micromolar range (IC50 = 6 μM). If the metoxy group was replaced by a trifluoromethyl group the inhibitor of MurC and MurF ligases was obtained. The hydroxyethylamine derivatives of 1-morpholino-3-(phenylsulfonamido)propan-2-yl dihydrogen phosphate are therefore an important starting point for development of multiple inhibitors of peptidoglycan biosynthesis with broad-spectrum antibacterial activity. Moreover, due to inhibition of MurE ligase from Staphylococcus aureus this compounds could be developed into antibacterial drugs against MRSA (Methicillin-resistant S. aureus)