Synthesis of 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamide InhA inhibitors

Tuberkuloza je infekcijska bolezen, ki jo povzroča mikobakterija M. tuberculosis in najpo-gosteje prizadene pljuča. Tuberkuloza v svetovnem merilu predstavlja velik zdravstveni problem, saj zaradi nje vsako leto umre 1,3 milijona ljudi. Mikobakterijska celična stena je bogata z lipidi, ključni gradnik pa so mikolne kisline. Encimi, ki sodelujejo pri sintezi mi-kolnih kislin, so ravno zaradi tega glavne tarče učinkovin, ki jih uporabljamo za zdravljenje okužb z M.Tuberculosis. Encim InhA (enoil acilni prenašalni protein) sodeluje pri sintezi mikolnih kislin in je tarča izoniazida, ki je zdravilo prvega izbora. Izoniazid se mora za svoje delovanje aktivirati s peroksidazo KatG. Če pride na genu za KatG do mutacije, postane izoniazid neučinkovit. Zaradi vse pogostejšega pojava sevov, odpornih na izoniazid, razvi-jajo nove direktne zaviralce encima InhA, ki za svoje delovanje ne potrebujejo aktivacije. Eden izmed strukturnih razredov direktnih zaviralcev InhA, ki so jih odkrili z visoko zmo-gljivostnim rešetanjem in so izkazali dober potencial za nadaljnji razvoj, so benzamidni za-viralci encima InhA. V magistrski nalogi smo sintetizirali sedem benzamidnih zaviralcev InhA. V prvem delu magistrske naloge smo sintetizirali 4-((3,5-dimetil-1H-pirazol-1-il)metil)-N-(2-(4-feniltiazol-2-il)ciklopentil)benzamid in 4-((3,5-dimetil-1H-pirazol-1-il)metil)-N-(2-(4-feniltiazol-2-il)cikloheksil)benzamid. Obe molekuli imata kiralni center, zato smo sintezo načrtovali tako, da smo dobili dva diastereomera. Pri biološkem testiranju zaviralne aktivnosti se je izkazalo, da imajo cis in trans derivata spojine s petčlenskim obro-čem in spojina s šestčlenskim obročem zelo podobno zaviralno aktivnost v nizkem mikro-molarnem območju. V drugem delu magistrske naloge smo sintetizirali pet analogov 4-((3,5-dimetil-1H-pirazol-1-il)metil benzamidnih zaviralcev, tako da smo sistematično menja-li substituente, in sicer smo uvedli 2-fenilpiperidin, 2-fenilpirolidin, 2-fenilazetidin, 1-metil-3-fenilpiperazin in 3-fenilmorfolin. Trije analogi so izkazali zaviralno aktivnost v mikromo-larnem območju. Spojini trans 4-((3,5-dimetil-1H-pirazol-1-il)metil)-N-(2-(4-feniltiazol-2-il)ciklopentil)benzamid in (4-((3,5-dimetil-1H-pirazol-1-il)metil)fenil)(2-fenilpiperidin-1-il)metanon pa imata tudi zmerno protibakterijsko aktivnost. Z našim delom smo raziskali odnos med strukturo in delovanjem ter preverili, kako različni substituenti vplivajo na zavi-ranje encima InhA.Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which most commonly affects the lungs. Tuberculosis is a major healthcare burden causing 1.3 million deaths every year. Mycobacterial cell wall is rich in lipids, and the major part of lipid com-ponents are fatty acids called mycolic acids. Enzymes within mycolic acid synthesis path-way are drug targets. One of those enzymes is InhA (enoyl acyl carrier protein), an enzyme which is targeted by isoniazid, a drug of first choice for tuberculosis treatment. Isoniazid is a prodrug and requires activation by KatG peroxidase. Since KatG is essential for activa-tion, mutation on KatG gene causes resistance of M. tuberculosis to isoniazid. Therefore research is conducted on direct InhA inhibitors, which do not require activation. One of direct InhA inhibitor classes discovered through high through-put screening are benzamide inhibitors, which show a good potential for further optimization. In the master thesis our goal we synthesized seven benzamide inhibitors. Firstly, we syn-thesized compounds 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N-((2-(4-phenylthiazol-2-yl)cyclopentyl)benzamide and 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N-((2-(4-phenylthiazol-2-yl)cyclohexyl)benzamide. Since both compounds have a chiral center, we planed the synthesis to prepare both diastereomeres. Both diastereomeres of compound with cyclopentane ring and compound with cyclohexane ring exhibited very similar inhibi-tor potency in micro molar range. In the second part of the master thesis we focused on a synthesis of five different analogues of 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl ben-zamide inhibitors. We used 2-phenilpiperidine, 2-phenilpirolidine, 2-phenilazetidine, 1-methyl-3-phenilpiperazine in 3-phenilmorpholine as substituents. Three of synthesized ana-logues exhibited inhibitor potency in low micro molar range. Compounds trans 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N-((2-(4-phenylthiazol-2-yl)cyclopentyl)benzamide and (4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)(2-phenylpiperidin-1-yl)methanone also showed modest antibacterial activity. In this thesis, we researched structure-activity rela-tionship between different analogues of benzamide InhA inhibitors and their potency

Design, synthesis and biological evaluation of new allosteric Hsp90 inhibitors with anticancer activity

Hsp90 je obetavna tarča za odkrivanje novih učinkovin za zdravljenje raka, saj so njegovi proteini substrati vpleteni v številne mehanizme nastanka in razvoja te bolezni. Večina do sedaj odkritih zaviralcev Hsp90 se veže v ATP-vezavno mesto na N-končni domeni. ATP-kompetitivni zaviralci Hsp90 v kliničnih študijah niso bili uspešni zaradi številnih neželenih učinkov, njihova največja pomanjkljivost pa je indukcija odziva toplotnega šoka, ki ima za posledico preživetvene in antiapoptotične učinke. Zaradi teh pomanjkljivosti raziskujemo nove strategije zaviranja Hsp90, kot je vezava zaviralcev v C-končno domeno (CKD), ki ne sproža odziva toplotnega šoka. V odsotnosti ko-kristalne strukture CKD in nekovalentnega zaviralca so možnosti strukturno podprtega načrtovanja omejene, kar se kaže v majhni strukturni raznolikosti do sedaj znanih zaviralcev CKD Hsp90. V okviru doktorske disertacije smo se posvetili odkrivanju in optimizaciji alosteričnih zaviralcev Hsp90 z novimi strukturnimi skeleti in razvili tri strukturno različne serije novih zaviralcev CKD Hsp90. Pri načrtovanju in odkrivanju novih spojin smo si pomagali z in silico metodami, in sicer smo razvili nov pristop za odkrivanje alosteričnih zaviralcev Hsp90, ki vključuje sidranje v predpostavljeno vezavno mesto na CKD, simulacije molekulske dinamike (MD), iskanje najbolj reprezentativnega farmakofornega modela v trajektoriji MD in virtualno rešetanje. Z našim pristopom smo odkrili tri strukturno različne spojine zadetke in jih optimizirali do analogov s protitumornim delovanjem v nizkem mikromolarnem območju. Sintetizirane spojine so izkazale antiproliferativno aktivnost na celičnih linijah različnih tipov raka dojke in celični liniji Ewingovega sarkoma. Prav tako so sprožale razgradnjo onkogenih proteinov substratov, vpletenih v različne mehanizme razvoja in napredovanja raka in niso inducirale odziva toplotnega šoka. Spojinam iz serije piperidinskih zaviralcev smo dokazali citotoksično in citostatično delovanje na celični liniji trojno negativnega raka dojke. Učinkovitost najobetavnejše spojine TMT11 smo ovrednotili in vivo v BALB/c mišjem modelu in ugotovili, da zavira rast tumorja trojno negativnega raka dojke primerljivo z N-končnim zaviralcem AUY922, ki je bil predhodno vrednoten v fazi II kliničnih študij. Prav tako je spojina TMT11 izkazala ustrezno varnost v uporabljenem mišjem modelu. Novi strukturni razredi alosteričnih zaviralcev Hsp90, razviti v okviru te doktorske disertacije, predstavljajo pomemben doprinos k strukturni raznolikosti zaviralcev CKD Hsp90, prav tako pa so dobro izhodišče za nadaljnji razvoj novih učinkovin za zdravljenje raka.Hsp90 client proteins play critical roles in all aspects of cancer development, highlighting Hsp90 as a potential target for novel anticancer drugs. However, current inhibitors primarily target the N-terminal domain (NTD) of Hsp90 and are associated with significant therapeutic limitations, particularly the induction of the heat shock response. Therefore, alternative strategies for inhibiting Hsp90 are needed, such as targeting the C-terminal domain (CTD). Unfortunately, due to the absence of crystal structure of Hsp90 CTD in complex with inhibitor, developing structure-based design approaches for Hsp90 CTD inhibitors remains challenging, leading to limited structural diversity in this class. In this dissertation, we focused on the discovery and optimization of new allosteric Hsp90 inhibitors with unique scaffolds. We successfully developed three distinct structural classes of Hsp90 CTD inhibitors. To identify potential allosteric Hsp90 CTD inhibitors, we employed in silico methods, combining docking into the proposed binding site, molecular dynamics simulations, pharmacophore modeling and virtual screening. Through this approach, we obtained three virtual screening hits that were further optimized into analogs exhibiting anticancer activity in the low micromolar range. The synthesized analogues demonstrated significant anticancer effects in multiple breast cancer cell lines and Ewing sarcoma cell line. Importantly, these novel allosteric Hsp90 inhibitors induced degradation of oncogenic Hsp90 client proteins associated with various cancer pathways without inducing a heat shock response. Notably, compounds belonging to the piperidine inhibitor series exhibited both cytotoxic and cytostatic effects in triple-negative breast cancer cell line. Among the tested compounds, the most promising candidate TMT11 was evaluated for its in vivo efficacy using the BALB/c nude mouse model. This compound displayed similar antitumor activity to Hsp90 NTD inhibitor, which has undergone clinical trials, and was well tolerated. The discovery of new structural classes of Hsp90 CTD inhibitors in this doctoral thesis expands the diversity of available options and represents a promising starting point for the development of anticancer agents with new mode of action in the future

In silico discovery and optimisation of a novel structural class of Hsp90 C-terminal domain inhibitors

Hsp90 is a promising target for the development of novel agents for cancer treatment. The N-terminal Hsp90 inhibitors have several therapeutic limitations, the most important of which is the induction of heat shock response, which can be circumvented by targeting the allosteric binding site on the C-terminal domain (CTD) of Hsp90. In the absence of an Hsp90—CTD inhibitor co-crystal structure, the use of structure-based design approaches for the Hsp90 CTD is difficult and the structural diversity of Hsp90 CTD inhibitors is limited. In this study, we describe the discovery of a novel structural class of Hsp90 CTD inhibitors. A structure-based virtual screening was performed by docking a library of diverse compounds to the Hsp90β CTD binding site. Three selected virtual hits were tested in the MCF-7 breast cancer cell line, with compound TVS-23 showing antiproliferative activity with an IC50 value of 26.4 ± 1.1 µM. We report here the optimisation, synthesis and biological evaluation of TVS-23 analogues. Several analogues showed significantly enhanced antiproliferative activities in MCF-7 breast cancer and SK-N-MC Ewing sarcoma cell lines, with 7l being the most potent (IC50 = 1.4 ± 0.4 µM MCF-7IC50 = 2.8 ± 0.4 µM SK-N-MC). The results of this study highlight the use of virtual screening to expand the structural diversity of Hsp90 CTD inhibitors and provide new starting points for further development

Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed antiproliferative activity in the SK-N-MC Ewing sarcoma cell line with an IC50 value of 15.9±0.7 µM. The optimised compounds showed enhanced anticancer activity in the SK-N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma

Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations

Structure-activity relationships of benzothiazole-based Hsp90 C-terminal-domain inhibitors

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC$_{50}$, with the most potent being compounds 5g and 9i (IC$_{50}$, 2.8 ± 0.1, 3.9 ± 0.1 µM, respectively). Based on these results, a ligand-based structure–activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response

Homochiral β-CF3, -SCF3 and -OCF3 secondary alcohols: catalytic stereoconvergent synthesis, bioactivity and flexible crystals

An optimized catalytic protocol for enantio- and diastereoselective reduction of racemic α-CF3, α-SCF3 and α-OCF3 aryl ketones is described. The reaction involves a dynamic kinetic resolution (DKR) based on ruthenium catalyzed Noyori–Ikariya asymmetric transfer hydrogenation for simultaneous construction of two contiguous stereogenic centers. A range of previously inaccessible fluorinated secondary alcohols was prepared in excellent stereomeric purity (up to above 99.9% ee, up to above 99.9:0.1 dr) and in high isolated yield (up to 99%). The origin of DKR (exceptional stereoselectivity and racemization mechanism) is rationalized by density functional theory calculations. Pharmaceutically relevant further transformations of the products are demonstrated including incorporation into heat shock protein 90 inhibitor with in vitro anti-cancer activity. Moreover, needle-shaped crystals of representative stereopure products are mechanically responsive: either elastically or plastically flexible, opening the door to novel class of functional materials based on chiral molecular crystals

Discovery of novel Hsp90 C-terminal inhibitors using 3D-pharmacophores derived from molecular dynamics simulations

Hsp90 C-terminal domain (CTD) inhibitors are promising novel agents for cancer treatment, as they do not induce the heat shock response associated with Hsp90 N-terminal inhibitors. One challenge associated with CTD inhibitors is the lack of a co-crystallized complex, requiring the use of predicted allosteric apo pocket, limiting structure-based (SB) design approaches. To address this, a unique approach that enables the derivation and analysis of interactions between ligands and proteins from molecular dynamics (MD) trajectories was used to derive pharmacophore models for virtual screening (VS) and identify suitable binding sites for SB design. Furthermore, ligand-based (LB) pharmacophores were developed using a set of CTD inhibitors to compare VS performance with the MD derived models. Virtual hits identified by VS with both SB and LB models were tested for antiproliferative activity. Compounds 9 and 11 displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound 11 inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 11 offers a unique scaffold that is promising for the further synthetic optimization and development of molecules needed for the evaluation of the Hsp90 CTD as a target for the development of anticancer drugs

Catalytic Stereoconvergent Synthesis of Homochiral β-CF 3 , β-SCF 3 , and β-OCF 3 Benzylic Alcohols

International audienceWe describe an efficient catalytic strategy for enantio- and diastereoselective synthesis of homochiral β-CF3, β-SCF3, and β-OCF3 benzylic alcohols. The approach is based on dynamic kinetic resolution (DKR) with Noyori–Ikariya asymmetric transfer hydrogenation leading to simultaneous construction of two contiguous stereogenic centers with up to 99.9% ee, up to 99.9:0.1 dr, and up to 99% isolated yield. The origin of the stereoselectivity and racemization mechanism of DKR is rationalized by density functional theory calculations. Applicability of the previously inaccessible chiral fluorinated alcohols obtained by this method in two directions is further demonstrated: As building blocks for pharmaceuticals, illustrated by the synthesis of heat shock protein 90 inhibitor with in vitro anticancer activity, and in particular, needle-shaped crystals of representative stereopure products that exhibit either elastic or plastic flexibility, which opens the door to functional materials based on mechanically responsive chiral molecular crystals