Structure based design, synthesis and evaluation of novel indoleamine 2,3-dioxygenase inhibitors and toll-like receptors 7 and 8 modulators

Pri raziskovalnem delu v okviru doktorske disertacije smo se ukvarjali s tremi tarčami imunskega sistema - indolamin 2,3-dioksigenazo 1 (IDO1) in Toll-u podobnima receptorjema tipa 7 in 8 (TLR7 in TLR8). Medtem ko IDO1 sodeluje v imunomodulaciji preko metabolizma triptofana, sta TLR7 in TLR8 senzorja nukleinskih kislin patogenih organizmov. Vse tri tarče so udeležene v patologiji številnih bolezenskih stanj, med drugim tudi rakavih obolenj. Na tarči IDO1 smo se na podlagi spojine zadetka lotili sinteze analogov, za kar smo razvili večstopenjski sintezni postopek, s pomočjo katerega smo osnovni izoksazolo[5,4-d]pirimidin-4(5H)-onski skelet modificirali na dveh različnih mestih. Vsem pripravljenim spojinam smo ovrednotili zaviralno aktivnost s fluorescenčnim testom na izoliranem IDO1 ter nadalje preverili še selektivnost napram ostalima izoformama - indolamin 2,3-dioksigenazi 2 in triptofan 2,3-dioksigenazi. Spojine so izkazovale selektivno zaviranje IDO1 v nizkem mikromolarnem območju, najmočnejše med njimi so v svoji strukturi posedovale anilinski fragment, na para mestu substituiran s trifluorometilno, cikloheksilno ali metoksikarbonilno skupino. Aktivnim spojinam smo določili tudi citotoksični profil, spojini 23 kot najmočnejšemu zaviralcu IDO1 z IC50 vrednostjo 23 [mikro]M pa smo aktivnost ovrednotili še v celičnem zaviralnem testu. V sklopu raziskovalnega dela na TLR7 smo na podlagi treh spojin zadetkov pripravili dve seriji nizko molekularnih selektivnih agonistov TLR7 s kromeno[3,4-d]imidazol-4(1H)-onskim in 2-(trifluorometil)kinolin/ kinazolin-4-aminskim skeletom. Z uvedbo različnih substitucij na osnovni kromenoimidazolonski skelet smo dosegli premik iz antagonističnega v agonistično delovanje, spojina 26 pa predstavlja najmočnejšega agonista (EC50 = 1,8 [mikro]M) prve serije. Druga serija agonistov je na mestu 4 osnovnega skeleta posedovala sekundarni ali terciarni amin z vezano krajšo alkilno verigo ali večjim in rigidnejšim alifatskim obročem. Najboljši predstavnik te serije, spojina 46, je nadalje signifikantno inducirala sproščanje provnetnega citokina IL8 iz enojedrnih celic periferne krvi. Lotili smo se tudi sistematične študije značilnosti med strukturo in delovanjem negativnih modulatorjev TLR8. Za pripravo večje knjižnice analogov spojine zadetka s pirimidinskim skeletom, ki smo jo strukturno modificirali na treh različnih mestih, smo razvili dve sintezni poti. Potrdili smo, da so trifluorometilna skupina na mestu 6 in ostala dva substituenta na mestih 2 in 4 osnovnega 4-(furan-2-il)-6-(trifluorometil)pirimidinskega skeleta pomembni strukturni elementi pripravljene serije modulatorjev TLR8. Na mestu 2 je bila za modulacijo TLR8 ključna prisotnost benzilaminskega substituenta s para hidroksi ali hidroksimetilno skupino. Večje število sintetiziranih spojin je v nizkem mikromolarnem območju izkazovalo selektivno in koncentracijsko odvisno zaviranje TLR8-signalne poti na celični liniji HEK293. Najbolj perspektivni spojini 4-(furan-2-il)-2-(metilsulfonil)-6-(trifluorometil)pirimidin (15a) in (4-(((4-(furan-2-il)-6-(trifluorometil)pirimidin-2-il)amino)metil)fenil)metanol (53) predstavljata pomembni kemijski probi za nadaljnjo optimizacijo do spojine vodnice z močnim imunomodulatornim delovanjem. V okviru raziskovalnega dela na TLR8 smo se lotili tudi računalniško-podprtega načrtovanja novih modulatorjev. Več kupljenim zadetkom iz virtualnega rešetanja smo ovrednotili jakost delovanja na TLR8 in preverili selektivnost napram TLR7. Pri tem smo prišli do nove spojine zadetka 3-(1-hidroksiheksan-2-il)-5-metilkinazolin-4(3H)-ona (T91), dualnega agonista TLR7 in TLR8, ki smo mu na enojedrnih celicah periferne krvi preverili sposobnost indukcije ekspresije provnetnih citokinov. Spojina T91 je signifikantno inducirala ekspresijo IL8, s čemer smo potrdili njeno imunomodulatorno delovanje. Raziskovalno delo smo zaključili z optimizacijo pogojev Suzuki reakcije, ene najpogosteje uporabljenih reakcij za tvorbo C-C vezi, ki smo jo sami uporabili za pripravo nekaterih negativnih modulatorjev TLR8. Osredotočili smo se na reakcijo med 2,4-dikloropirimidini in različnimi aril- ali heteroaril boronskimi kislinami. Med poglobljenim študijem reakcijskih pogojev smo preverili vpliv topila, katalizatorja, temperature in časa na potek reakcije, z uporabo mikrovalovnega sevanja pa smo razvili hiter in visoko učinkovit sintezni postopek za pripravo C4-substituiranih pirimidinov. Zaradi kratkega reakcijskega časa (15 min) in predvsem nizke porabe (0,5 mol %) katalizatorja tetrakis(trifenilfosfino)paladija (0) je postopek primeren za hitro in poceni regioselektivno pripravo substituiranih pirimidinov. V okviru doktorske disertacije smo uspešno načrtovali in sintetizirali nizkomolekularne zaviralce IDO1 ter modulatorje TLR7 in TLR8 z jakostjo delovanja v nizkem mikromolarnem območju. Za pripravo substituiranih pirimidinov smo razvili metodo Suzuki reakcije v mikrovalovnem reaktorju. Več modulatorjev TLR7 in TLR8 je induciralo sproščanje provnetnih citokinov. S pripravljenimi serijami spojin smo pomembno prispevali k razvoju spojin vodnic z imunomodulatornim delovanjem.The doctoral thesis research was focused on three targets of the immune system - indoleamine 2,3-dioxygenase 1 (IDO1) and Toll-like receptors 7 and 8 (TLR7 and TLR8). While IDO1 is responsible for immunomodulation through the metabolism of tryptophan, TLR7 and TLR8 act as sensors for nucleic acids of different pathogens. All three targets are involved in the pathology of several diseases, among them also in different cancers. Our research work on IDO1 started with the synthesis of a focused library of analogs based on the previously discovered hit compound. Therefore, a multistep synthetic procedure for the preparation of isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold was developed, enabling substitutions on the main scaffold in two different positions. Inhibitory IDO1 activity of all synthesized compounds was evaluated in a fluorescence-based assay, with selectivity over two isoforms, i.e., indoleamine 2,3-dioxygenase 2 and tryptophan 2,3-dioxygenase, being further assessed. Prepared compounds selectively inhibited IDO1 in low micromolar range, with the most potent among them possessing aniline fragment, which was substituted with the trifluoromethyl, cyclohexyl or methoxycarbonyl group on the para position. The cytotoxicity profile of inhibitors was further determined and for the most potent compound 23 (IC50 of 23 [micro]M) we also evaluated its cellular activity. Two series of low micromolar and selective TLR7 agonists were prepared based on three hit compounds with chromeno[3,4-d]imidazol-4(1H)-one and 2-(trifluoromethyl)quinoline/ quinazoline-4-amine scaffolds. Substitutions on the chromenoimidazolone scaffold led to the shift from antagonist to agonist activity, with compound 26 representing the best-in-class agonist with EC50 value of 1.8 [micro]M. The agonists from the second series possessed secondary or tertiary amines with smaller alkyl substituents or bulkier rigid aliphatic ring on the position 4 of the main scaffold. Furthermore, 46 as the most potent compound from this series significantly induced the expression of proinflammatory cytokine IL8 from peripheral blood mononuclear cells. A systematic structure -activity relationship study of TLR8 negative modulators was further performed. Two synthetic pathways were developed for the preparation of a focused library of analogs based on the hit compound with pyrimidine scaffold, which were modified in three different positions. We confirmed the imperative presence of 6-trifluoromethyl group and other two substituents on positions 2 and 4 of the main 4-(furan-2-yl)-6-(trifluoromethyl)pyrimidine scaffold of our TLR8 modulators. Furthermore, para hydroxy or hydroxymethyl groups on benzylamine substituent at position 2 were found to be essential for potent modulation of TLR8. Several of the prepared compounds exhibited concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells in low micromolar range. The most perspective compounds 4-(furan-2-yl)-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine (15a) and (4%(((4%(furan-2-yl)-6-(trifluoromethyl)pyrimidin-2-yl)amino)methyl)phenyl)methanol (53) represent promising chemical probes for further optimization to a lead compound with potent immunomodulatory properties. During our research work on TLR8 we also performed computer-aided design of novel modulators. The potency of purchased hit compounds was evaluated on TLR8, with selectivity over TLR7 also being determined. A dual TLR7 and TLR8 agonist 3%(1-hydroxyhexan-2-yl)-5-methylquinazolin-4(3H)-one (T91) was discovered and its ability to induce the expression of proinflammatory cytokines on peripheral blood mononuclear cells was also assessed. The compound T91 significantly induced the expression of IL8, thus confirming its immunomodulatory activity. Research work was concluded with the optimization of conditions for Suzuki coupling, which is one of the most common reactions for the formation of C-C bonds and was used for the synthesis of some TLR8 negative modulators. We focused on the coupling between different 2,4-dichloropyrimidines and aryl- or heteroaryl boronic acids. A thorough screening of reaction conditions (e.g., solvent, catalyst, temperature, and time) and microwave irradiation led to a very efficient and straightforward synthetic procedure for the preparation of C4-substituted pyrimidines. Due to short reaction time (15 min) and extremely low catalyst loading (0,5 mol %) of vii tetrakis(triphenylphosphine)palladium (0), the established procedure could be used for quick and low-cost regioselective preparation of substituted pyrimidine rings. During our research work we successfully designed and synthesized low-molecular IDO1 inhibitors, as well as TLR7 and TLR8 modulators with low micromolar potency. For the preparation of substituted pyrimidines we developed a method for Suzuki coupling using a microwave reactor. Several TLR7 and TLR8 modulators induced the expression of proinflammatory cytokines. Prepared series of compounds crucially contributed to the development of lead compounds with immunomodulatory properties