An introduction, background and recent advances in the areas of microtubule-binding agents and heat shock protein 90 (HSP90) inhibitors as anticancer agents are briefly reviewed. The work in this dissertation is centered on the synthesis of substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines as potential anticancer agents that act via microtubule inhibition or HSP90 inhibition.<br>Microtubule-binding agents are effective against a broad range of tumors and lymphomas and have been common components of combination cancer-chemotherapy in the clinic. Despite the unparalleled success, drawbacks among microtubule-binding agents such as multi-drug resistance, dose-limiting toxicity, poor pharmacokinetic profile and high cost have supported the sustaining momentum in searching for novel agents of this class.<br>The research on microtubule-binding agents in this dissertation was initiated by an unexpected discovery. The lead compound, a 4-<italic>N</italic>-methyl-4'-methoxyaniline-substituted pyrrolo[2,3-<italic>d</italic>]pyrimidine, was found to inhibit the majority cancer cell lines in the NCI-60 panel at sub-micromolar concentration. The COMPARE analysis based on the activity profile indicated microtubule inhibition as the main mechanism of action of this compound, and was later confirmed through multiple assays. Further, the lead compound displaced 70% of [<super>3</super>H]colchicine from tubulin at a concentration of 5 μM, and was identified as a colchicine-site binder. The compound has also shown unabated or even increased activities against several drug-resistant cancer cell lines, especially the cell lines overexpressing P-glycoprotein or βIII-tubulin. In addition, the compound has favorable physicochemical properties such as high water solubility as its hydrochloride salt.<br>Based on the preliminary data and molecular modeling, a hypothesis on the relationship between binding affinity and the lowest-energy conformation of pyrrolo[2,3-<italic>d</italic>]pyrimidines was proposed. To test the hypothesis and search for compounds with improved potency, 38 pyrrolo[2,3-<italic>d</italic>]pyrimidine analogs in six series were designed and synthesized. The biological evaluations of these compounds are currently in progress at the time this dissertation is submitted.<br>HSP90 is one the molecular chaperones that assist the proper folding of the newly synthesized polypeptides and proteins. The majority of its client proteins are signal transducers with unstable conformations, which play critical roles in growth control, cell survival and development. The expressions of these proteins in normal cells were much less than cancer cell, making HSP90 a viable target for cancer chemotherapy. As of 2012, there are 16 HSP90 inhibitors in clinical trial, among which four are based on the purine-scaffold. All the compounds in clinical trials bind to or overlap with the ATP site on the N-terminal of HSP90.<br>The pyrrolo[2,3-<italic>d</italic>]pyrimidine scaffold is structurally close to purines. In the design of receptor tyrosine kinase (RTK) inhibitors, Gangjee et al. have shown that properly functionalized pyrrolo[2,3-<italic>d</italic>]pyrimidines bind to the ATP site and achieve high degrees of selectivity. This was partly attributed to the incorporation of substitution patterns that are impossible on the purine scaffold. Based on these previous findings and the established SAR of the two purine derivatives in clinical trials (<bold>PU-H71</bold> and <bold>BIIB021</bold>), 18 substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines in three series (in connection with this dissertation) were designed and synthesized. The biological evaluations of these compounds are currently in progress. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Les LIMKs sont deux protéines kinases responsables de la régulation de la dynamique du cytosquelette d’actine et des microtubules. Leur dérégulation peut jouer un rôle prépondérant dans l’apparition de certaines maladies telles que la neurofibromatose de type 1, le cancer ou la sclérose latérale amyotrophique.Au cours de ce projet collaboratif, nous avons cherché à concevoir des inhibiteurs à la fois puissants et sélectifs des LIMKs en nous inspirant du composé le plus performant de l’époque développé par Lexicon Pharmaceuticals.Nous avons, dans un premier temps, travaillé sur la base hétérocyclique en remplaçant la pyrrolo[2,3-d]pyrimidine par différentes pyridopyrimidines et une triazolopyridopyrimidine. Cette dernière modification a ouvert la voie à un sujet de méthodologie autour du réarrangement de Dimroth. Nous avons ensuite modifié le cycle central pipérazine par une 3,6-dihydropyridine ou une pipéridine différemment substituée et avons fait varier la substitution au niveau de l’arylurée. Enfin, nous sommes revenus apporter quelques modifications structurales à la pyrrolo[2,3-d]pyrimidine de départ.Finalement, nous avons créé, à l’aide de méthodologies de synthèse efficaces, un large panel de composés originaux qui ont été testés in vitro et in cellulo sur nos cibles.Au travers de ce projet de chimie médicinale, nous avons pu approfondir les relations structure-activité afin de concevoir des inhibiteurs des LIMKs très prometteurs. / LIMKs are two protein kinases involved in regulating cytoskeleton and microtubule actin dynamics. Their deregulation can play a major role in the onset of several diseases such as neurofibromatosis type 1, cancer or amyotrophic lateral sclerosis.During this collaborative project, we have sought to design both potent and selective LIMKs inhibitors based on one of the most efficient compounds of the day developed by Lexicon Pharmaceuticals.We first worked on the heterocyclic base by replacing the pyrrolo[2,3-d]pyrimidine by different pyridopyrimidines and a triazolopyridopyrimidine. This last modification lead to a directed synthetic methodology study around the Dimroth rearrangement. We then modified the piperazine central ring with a differently substituted 3,6-dihydropyridine or piperidine and varied the substitution at the arylurea. Lastly, we made some further structural changes to the starting pyrrolo[2,3-d]pyrimidine.In conclusion, we have created, using effective synthetic methodology, a wide range of original compounds that have been tested in vitro and in cellulo on our targets.Through this medicinal chemistry project, we now have a better understanding of the structure-activity relationships needed in order to design very promising LIMK inhibitors.

CARACTERISATION IN VITRO ET IN VIVO DE NOUVEAUX AGENTS PYRROLO-PYRIMIDINE CIBLANT LES MICROTUBULES POUR LE TRAITEMENT DES CANCERSEn dépit de l’émergence des thérapies ciblées et de l’immunothérapie, la chimiothérapie reste un gold-standard pour le traitement de nombreux cancers. Parmi les agents chimiothérapeutiques conventionnels, les poisons du fuseau interférant avec la dynamique des microtubules (taxanes, vinca-alcaloïdes) sont très largement utilisés. Cependant, leurs nombreux effets indésirables et l’émergence de chimiorésistance limitent leur efficacité et soulignent la nécessité d’identifier de nouveaux inhibiteurs de la tubuline.Notre équipe a réalisé un criblage cellulaire à haut-débit sur plus de 7500 molécules chimiques et identifié une famille de composés pyrrolo-pyrimidine, active sur les formes de cancer pulmonaire résistantes à l’apoptose et aux thérapies ciblées. Notre objectif était de caractériser in vitro et in vivo 15 molécules de cette famille afin d’identifier à terme un potentiel candidat-médicament pour le traitement des cancers résistants.Des essais préliminaires de cytotoxicité et d’apoptose sur différentes lignées cancéreuses pulmonaires ont permis de sélectionner, parmi les 15 composés pyrrolo-pyrimidine, 2 molécules prometteuses : PP-2 et PP-13. PP-2 et PP-13 ont des effets cytotoxiques sur de nombreuses lignées cancéreuses humaines, incluant les lignées résistantes aux thérapies ciblées. En perturbant l’organisation et la dynamique des microtubules, PP-2 et PP-13 induisent le blocage transitoire des cellules en prométaphase puis aboutissent aux phénomènes de catastrophe mitotique, de glissement mitotique ou de division asymétrique. Des études mécanistiques avancées montrent que PP-2 et PP-13 sont des agents poisons du fuseau et entrent en compétition avec la colchicine pour la liaison sur la tubuline. Contrairement aux antimitotiques conventionnels, PP-2 et PP-13 ne sont pas sensibles aux mécanismes de chimiorésistance par surexpression de pompes d’efflux. De plus, à l’IC50, ces 2 composés n’affectent pas le réseau microtubulaire des cellules à l’interphase suggérant un effet toxique (et principalement neurotoxique) moindre. La molécule PP-13 semble être la molécule anticancéreuse la plus prometteuse en raison de son IC50 10 fois inférieure à celle de PP-2 dans les différentes lignées cancéreuses étudiées et d’une affinité pour la tubuline 2 fois plus élevée. In vivo, PP-13 réduit significativement la croissance tumorale et étastatique. L’ensemble de ces résultats suggère que PP-13 pourrait être une alternative intéressante pour le traitement de nombreux cancers y compris des cancers chimiorésistants. / CHARACTERIZATION OF NEW MICROTUBULE-TARGETING AGENTS WITH A PYRROLO-PYRIMIDINE STRUCTURE FOR THE TREATMENT OF CANCERSDespite the emergence of targeted therapies and immunotherapy, chemotherapy remains a gold-standard for the treatment of numerous malignancies. Spindle poisons that interfere with microtubule dynamics (taxanes, vinca alkaloids) are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of resistance highlight the need for identifying alternative agents. Thanks to a high throughput cell-based assay, we screened agents able to restore apoptosis in apoptosis-resistant lung cancer cells. We selected 15 molecules belonging to the pyrrolopyrimidine family and investigated their anti-cancer effects in vitro and in vivo. Our aim was to identify a potential drug-candidate for the treatment of resistant cancers.From cytotoxicity and apoptosis preliminary assays, we selected the 2 most promising molecules (PP-2, PP-13) among the 15 pyrrolopyrimidine compounds. PP-2 and PP-13 exert cytotoxic effects on a large panel of human cancer cell lines, including targeted therapy-resistant cell lines. By interfering with mitotic spindle organization and microtubule dynamics, PP-2 and PP-13 impair the congression of the chromosomes, promote spindle assembly checkpoint-dependent mitotic blockade and finally lead to asymmetric division, mitotic slippage and direct apoptotic death. PP-2 and PP-13 directly target tubulin and compete with colchicine for the binding to tubulin. Unlike conventional antimitotic agents, PP-2 and PP-13 are not sensitive to chemoresistance mechanisms involving the overexpression of efflux pumps. Moreover, at IC50, these two compounds do not affect the microtubule network during interphase suggesting a less toxic (mainly neurotoxic) effect. Among these two compounds, the PP-13 molecule appears to be the most interesting anti-cancer molecule because of its IC50 10-fold lower than PP-2 and its 2-fold higher affinity for tubulin. In vivo, PP-13 significantly reduces tumor and metastasis growth. All these results suggest that PP-13 might be a potential alternative for the treatment of many cancers including chemoresistant cancers.