Synthesis of the 4,5,6,7-tetrahydrobenzothiazole-based heat shock protein 90 C-terminal domain inhibitors

Protein toplotnega šoka 90 (Hsp90) je eden najpomembnejših molekulskih šaperonov, ki pomaga ohranjati proteostazo v fizioloških in stresnih pogojih. Sodeluje pri pravilnem zvijanju in aktivaciji več kot 400 proteinov, ki lahko le v nativni konformaciji opravljajo svoje funkcije. Ker imajo klienti Hsp90 pomembno vlogo v napredovanju kancerogeneze, je omenjeni šaperon postal obetavna tarča v protirakavi terapiji. Prvi zaviralci Hsp90 so preko vezave v ATP-vezavno mesto na N-končni domeni zavrli ATPazno aktivnost in tako zmanjšali nivo od Hsp90 odvisnih onkoproteinov. Pojavljanje številnih neželenih učinkov je omejilo terapevtski potencial N-končnih zaviralcev, kar je vodilo v natančnejše raziskovanje alosteričnega ATP-vezavnega mesta na C-končni domeni (CKD). V sklopu eksperimentalnega dela smo sintetizirali trinajst potencialnih zaviralcev C-končne domene Hsp90 s 4,5,6,7-tetrahidrobenzotiazolnim osnovnim skeletom. Osnovnemu skeletu smo na mesto 6 uvedli 3,4-diklorofenilni obroč in se osredotočili na optimizacijo aminskega dela spojin, vezanega na mesto 2, kamor smo uvedli substituente različnih dolžin. S tem smo želeli ovrednotiti vpliv razdalje med aromatskim obročem in kationskim centrom, opazovali pa smo tudi vpliv njihove velikosti in rigidizacije na zaviralno aktivnost. Sintetizirali smo pare analognih spojin, ki so se razlikovali le v konfiguraciji stereogenega centra na mestu 6, zato smo jih ločili v knjižnici A in B. S primerjavo knjižnic smo želeli odkriti morebiten vpliv konfiguracije na prileganje v vezavno mesto in posledično na delovanje analogov. Naš namen je bil proučiti odnos med strukturo sintetiziranih spojin in njihovim zaviralnim delovanjem na celični liniji raka dojke MCF-7. Na podlagi rezultatov bioloških testiranj smo potrdili pomembnost razdalje med aromatskim obročem in kationskim centrom. Pri enajstih aktivnih spojinah je veriga ogljikovih atomov med amidno in aminsko skupino dolga več kot dva atoma, medtem ko je pri neaktivnih spojinah 7e in 11e dolga le en atom. Spojina z najmočnejšim zaviralnim delovanjem, ki je dosegla najnižjo vrednost IC50 = 2,0 μM, je spojina 7d iz knjižnice A. Z neznatno razliko ji sledi direkten analog 11d iz knjižnice B s spremenjeno konfiguracijo na stereogenem centru. Tudi primerjava drugih parov spojin knjižnice A in B je izkazala primerljivo aktivnost, zato lahko sklepamo, da konfiguracija na mestu 6 osnovnega skeleta ne vpliva bistveno na jakost vezave v vezavnem mestu. Vzrok za to bi lahko bila simetričnost vezavnega mesta Hsp90. Nova spoznanja v magistrski nalogi tako omogočajo boljše razumevanje SAR zaviralcev CKD Hsp90 s tem skeletom.The 90-kDa heat shock protein (Hsp90) is one of the most important molecular chaperones involved in proteostasis regulation under both physiological and stress conditions. It is involved in the folding and activation of more than 400 proteins that must fold into the native conformation to attain their function. Because Hsp90 client proteins play crucial roles in tumor progression, Hsp90 has emerged as a promising target for tumor therapy. First Hsp90 inhibitors induced lower oncoprotein levels by suppressing the ATPase activity of Hsp90 through binding to the ATP pocket in the N-terminal domain. However, several adverse effects limited the therapeutic potential of the N-terminal inhibitors, which led to a detailed exploration of the allosteric ATP-binding site in the C-terminal domain. In this master\u27s thesis, we designed and synthesized thirteen potential 4,5,6,7-tetrahydrobenzothiazole-based Hsp90 C-terminal domain inhibitors. We introduced the 3,4-dichlorophenyl ring to position 6 of the central scaffold, however, our main focus was optimization of the amine-bearing moiety attached to position 2. By varying substituents of different lengths, we aimed to determine the optimal distance between the aromatic ring and the cationic center. We also observed the effects of size and rigidity on antiproliferative activity. The designed pairs of analogous compounds were divided into two libraries with different configurations of the stereocenter on position 6. Comparison of the libraries evaluated the potential impact on fitting to the binding site and consequently on the antiproliferative activity of the analogs. Our aim was to investigate the structure-activity relationships of the synthesized molecules in the breast cancer cell line MCF-7. With the collected results, we were able to confirm the importance of the distance between the aromatic ring and the cationic center. A chain of carbon atoms between an amide and an amine group is more than two atoms long in eleven active compounds, however, it is only one atom long in inactive compounds 7e and 11e. Compound 7d (library A) showed the lowest IC50 value of 2.0 μM and therefore the strongest inhibitory activity, followed by its direct analog 11d (library B) with only slightly weaker activity. Comparison of other pairs of compounds revealed comparable activity between the analogs, therefore, it can be concluded that configuration on position 6 of the main scaffold does not significantly affect the strength of binding to Hsp90. A possible reason for this could be the symmetry of the binding site of Hsp90. Overall, the new findings of this master’s thesis contribute to a better understanding of SAR of the Hsp90 inhibitors with given scaffold