Malaria, a disease caused by the parasite Plasmodium, continues to be one of the deadliest diseases worldwide. The WHO reported over 627,000 deaths in 2020, and over 1 billion people are at risk of infection. Even though Artemisinin-based Combination Therapies (ACT) are the current standard of care for malaria, the emergence of drug resistance generates a constant need to develop and synthesize new drugs. Tetrahydro-β-carboline acid (THβC) 1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium-3-carboxylate (MMV008138) has promising antimalarial properties; it was discovered by screening the Malaria Box with the so-called IPP Rescue assay. This assay identified MMV008138 as an inhibitor of the MEP pathway, which produces essential isoprenoid precursors (IPP and DMAPP) in the malaria parasite P. falciparum (EC50 250 ± 70 nM, IPP rescue 100% @ 2.5 μM). Subsequent investigation revealed that (1R,3S)-configuration and 2',4'-dihalogen substitution were critical for the activity of this compound, and that substitution of the non-aromatic ring was not tolerated. To search for new antimalarial structures, our collaborator Dr. Max Totrov constructed a generalized 3D pharmacophore-based on MMV008138 and 92 of its analogs and used it for a virtual ligand screen (VLS) of the 13K compound hit set from which MMV008138 had been selected. This exercise identified TCMDC-140230, a THβC, 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxamide (undefined stereochemistry) reported having nearly the same potency of MMV008138. Synthesis of the stereoisomers of compound TCMDC-140230 was accomplished via Pictet-Spengler reaction of (S)- and (R)-7-methyl tryptophan methyl ester and 3,4-dichlorobenzaldehyde. The individual stereoisomeric esters were converted to the corresponding amides, but none of the stereoisomers of TCMDC-140230 were potent antimalarials (IC50 = 1,300 – 3,700 nM). However, a significant amount of oxidized byproduct 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-9H-pyrido[3,4-b]indole-3-carboxamide (MMV1803522) was observed in the synthesis of (1S,3S)- and (1R,3R)-TCDMC-140230. This achiral β-carboline amide (PRC1584, IC50 = 108 ± 7 nM) proved more potent towards P. falciparum than MMV008138 and its toxicity was not reversed by co-application of IPP. Thus, the antimalarial target of MMV1803522 is distinct from that of MMV008138. Most importantly, MMV1803522 at 40 mg/kg/day (oral) cured P. berghei malaria infection in mice. The lead compound also was found to have a good safety profile. Medicines for Malaria Venture (MMV) has expressed interest in this compound which is now also known as MMV1803522. The results from these biological assays gave the insight to develop new analogs that have better asexual blood stage inhibition potency. Extensive structure-activity relationship studies were conducted by synthesizing analogs of the compound MMV1803522. The studies were mainly focused on analyzing the effect of aliphatic substitutions, how well the potency can be improved with different D-ring substitutions, and amide substitutions. In addition to this structural optimization, several metabolism studies were also conducted on this new lead compound. The potency study results of C1 alkyl-substituted analogs of MMV1803522 showed that aromatic substitutions are required at C1 for maintaining good inhibition potency. The heteroaryl substituents at C1 were found to be slightly less potent than the lead compound MMV1803522. Synthesis of analogs without C8 methyl group as in lead compound showed an EC50 < 100 nM is possible with a C8 hydrogen substitution. Most noteworthy is 3,4,5-trichlotophenyl-bearing compound 3.20a, which had an EC50 of 54 ± 8 nM. This compound is twice as potent as MMV1803522. Equipotent analogs to MMV1803522 were also synthesized with different amide substituents. The metabolism studies showed low solubility for compounds having an EC50 less than or close to 100 nM. Unfortunately, the intrinsic clearance rate of several selected compounds was found to be higher than MMV1803522. These results left us with scope for the development of new analog compounds. The emerging structure-activity relationship within this scaffold and outline of remaining challenges to improve potency sub-100 nM without compromising moderate solubility and good metabolic stability are in progress. / Doctor of Philosophy / Malaria is a global health problem that causes significant sickness and death annually in the developing world. The emergence of resistant parasite strains of malaria massively challenges efforts to eliminate this threat. To control the spread of malaria, there is a continuous need for the development of new antimalarial drugs that ideally offer a single-dose cure and new mechanism of action. One such promising target, called, Methyl Erythrytol Phosphate (MEP) pathway which produces IPP and DMAPP, are important isoprenoid precursors required in living beings. A compound MMV008138 was identified from a collection of compounds that exhibited antimalarial activity, the so-called "Malaria Box", and this compound was further analyzed for several biological assays. Unfortunately, MMV008138 was unsuccessful Since it was found toxic in mice when ingested orally. The efforts to develop structurally similar analogs of MMV008138 resulted in the accidental discovery of a compound that inhibits the parasites' growth much better than the former compound. This compound has a similar molecular structure to MMV008138, and the Medicines for Malaria organization (MMV) has designated it as MMV1803522. The newly obtained compound and its analogs were investigated and found to have promising potency to inhibit the growth of the malarial parasite Plasmodium falciparum. Multiple biological assays were conducted and found that even though MMV1803522 is toxic to malarial parasites, it does not show toxicity to other cells. The studies in mice showed that it was not toxic orally. Also, it was found to be non-toxic towards several mammalian cell lines. The development of structurally similar analogs can help in improving the potency of the compound, make a better orally bioavailable compound, and improve oral efficacy. Analyzing these results will help to determine the mechanism of action of the compound.

L’objectif de ces travaux est de découvrir et de valoriser des petites molécules organiques inhibant l’influx de l’iode dans les cellules thyroïdiennes. Ces composés présentent en effet un double intérêt : à court terme, ils peuvent être dérivés en biosondes afin de mieux caractériser les protéines impliquées dans les mécanismes de transport d’iode par génétique chimique directe. A plus long terme, ces inhibiteurs représentent des candidats-médicaments potentiels pour le traitement de pathologies thyroïdiennes et/ou pour la protection de populations exposées aux radioisotopes de l’iode. Pour chacune des deux familles d’inhibiteurs considérées, nous avons donc tout d’abord synthétisé une chimiothèque d’une centaine d’analogues ; puis ces derniers ont été évalués biologiquement afin de fournir un ensemble de relations structure-activité. Par ailleurs, la configuration absolue des centres stéréogènes nécessaire à l’activité biologique a été déterminée : dans chacun des cas, une stéréochimie particulière est responsable du pouvoir inhibiteur des composés. A partir de ces informations, une dizaine d’analogues « de seconde génération » a été synthétisée dans chaque famille, en combinant plusieurs modifications structurales contribuant à l’activité biologique. Après évaluation biologique, neuf d’entre eux possèdent des IC50 < 6 nM et des propriétés physico-chimiques satisfaisantes pour des candidats-médicaments. Enfin, dans chaque famille, une biosonde photoactivable biotinylée a été synthétisée et utilisée en photomarquage d’affinité. Plusieurs protéines marquées spécifiquement ont été repérées, qui correspondraient à des protéines-cibles de chacun des inhibiteurs et dont l’identification reste à achever. / This work was intended to discover small organic molecules acting as iodide uptake inhibitors in thyroid cells. These compounds can indeed be derivatized into biochemical probes for further characterization of proteins involved in iodide transport mechanisms. On the long term, these inhibitors also appear as attractive drug candidates for treatment of thyroid pathologies or radioprotection against iodine isotopes. A similar strategy was adopted for both of the two inhibitor families. First, we synthesized a chemical library of around 100 analogues; we measured their IC50 against iodide uptake in FRTL-5 cells to get structure-activity relationships. Absolute configuration of stereogenic centers was also investigated, and a preferential stereochemistry was found to be responsible for activity. From this basis, around twenty « second-generation » analogues were synthesized by combining fragments contributing to biological activity. Biological evaluation indicated that nine were very potent inhibitors, with IC50 < 6 nM and satisfying physicochemical properties required for drug candidates. Finally, one photoactivatable biotinylated probe was developed in each family and used for photoaffinity labeling. Several specifically labeled proteins are still under identification and constitute new potential therapeutic targets.

L'objectif de ces travaux est de découvrir et de valoriser des petites molécules organiques inhibant l'influx de l'iode dans les cellules thyroïdiennes. Ces composés présentent en effet un double intérêt : à court terme, ils peuvent être dérivés en biosondes afin de mieux caractériser les protéines impliquées dans les mécanismes de transport d'iode par génétique chimique directe. A plus long terme, ces inhibiteurs représentent des candidats-médicaments potentiels pour le traitement de pathologies thyroïdiennes et/ou pour la protection de populations exposées aux radioisotopes de l'iode. Pour chacune des deux familles d'inhibiteurs considérées, nous avons donc tout d'abord synthétisé une chimiothèque d'une centaine d'analogues ; puis ces derniers ont été évalués biologiquement afin de fournir un ensemble de relations structure-activité. Par ailleurs, la configuration absolue des centres stéréogènes nécessaire à l'activité biologique a été déterminée : dans chacun des cas, une stéréochimie particulière est responsable du pouvoir inhibiteur des composés. A partir de ces informations, une dizaine d'analogues " de seconde génération " a été synthétisée dans chaque famille, en combinant plusieurs modifications structurales contribuant à l'activité biologique. Après évaluation biologique, neuf d'entre eux possèdent des IC50 < 6 nM et des propriétés physico-chimiques satisfaisantes pour des candidats-médicaments. Enfin, dans chaque famille, une biosonde photoactivable biotinylée a été synthétisée et utilisée en photomarquage d'affinité. Plusieurs protéines marquées spécifiquement ont été repérées, qui correspondraient à des protéines-cibles de chacun des inhibiteurs et dont l'identification reste à achever.

Malaria is a serious mosquito-borne disease affecting the majority of Earth's southern hemisphere. While consistent efforts to curb malaria spread throughout 20th and early 21st century were largely successful, the recent rise in resistance to antimalarial treatments resulted in an increasing incidence rate and stalling mortality rate. This trend clearly signifies the need for the development of novel antimalarial agents able to circumvent current drug-resistance mechanisms. In 2014, in collaboration with Prof. Maria Belen Cassera from the University of Georgia, our group found that compound 1a (1R,3S-MMV008138), discovered from the publicly available Malaria Box, targets an essential biosynthetic pathway (MEP pathway) of malaria-causing parasite Plasmodium falciparum. Analogs of 1a synthesized in our laboratory were found effective against multi-resistant Dd2 strain of P. falciparum which, together with an absence of MEP pathway in humans, suggests that potent analogs of 1a may be safe and efficient antimalarial drug candidates. The initial bioassay studies determined that only one of four possible MMV008138 stereoisomers satisfactorily inhibits the target PfIspD enzyme. Thus a secure determination of stereochemistry in 1a analogs was of utmost importance to the structure-activity relationship studies performed in our group. The second chapter of this work discusses the validation of the previously known empirical stereoassignment method based on analysis of relative shift of 13C NMR resonances between cis and trans diastereomers and compares it to a new method based on 3JHH coupling constants developed in our laboratory. We demonstrate that the new method relying on the analysis of 1H-1H coupling is reliable over large samples of experimental data and suitable even when only a single diastereomer is produced in the synthetic process. Importantly, the origin of 3JHH coupling constants is well understood, unlike the source of relative differences in 13C NMR shifts observed in the older method. The empirical observations for both stereoassignment methods are supported by extensive density-functional theory calculations, which validate the new 1H-1H coupling-based assignment but do not provide a conclusive explanation for the origin of the 13C NMR-based method. In the third chapter, we discuss the replacement of the carboxylic acid moiety in 1a by alternative functional groups promising improved toxicity and bioavailability profile. The total synthesis of tetrazole (trans-23a) and phosphonic acid ((±)-62a) derivatives of 1a is discussed in detail. The tetrazole analog 23a was previously synthesized in the Carlier group as a diastereomeric mixture of cis and trans isomers (dr = 3:7), and it was tested for growth inhibition of multi-resistant P. falciparum with promising results. Later, the synthesis was revisited to obtain a stereochemically pure sample of trans-23a, which was expected to show improved potency compared to the original sample. Furthermore, the synthesis of pure trans-23a confirmed the accuracy of the previous assignment of cis and trans diastereomers in the mixture. Unfortunately, neither analog showed an improvement in potency relative to 1a. / Doctor of Philosophy / The most severe form of malaria disease is caused by the parasite, Plasmodium falciparum, which gives rise to over 200 million infections and more than 400 thousand deaths every year, the majority of which affect young children. In recent years, the effectiveness of clinically used antimalarial medicines decreased due to an increase in drug-resistant strains of P. falciparum. Therefore, there is an urgent need for new antimalarial agents that could bypass the emerging resistance. A promising candidate for a new antimalarial drug is a molecule named MMV008138. This molecule exists in four distinct forms called stereoisomers. Stereoisomers are molecules with the same chemical formula, but the atoms in each molecule are positioned differently. Only one of MMV008138's four stereoisomers (1a) was effective in killing the P. falciparum. The second chapter of this work discusses a new method for identifying stereoisomers in molecules like MMV008138. We demonstrate that the new method is both reliable and simpler than the previously used procedures. The third chapter of this dissertation discusses the preparation of two new compounds based on the structure of 1a that contain modifications promising improved biological activity. Unfortunately, neither of these two molecules was able to kill the P. falciparum efficiently.

<p>The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. </p><p>In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis.</p><p>The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[<i>a</i>]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. </p><p>These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.</p>

The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis. The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.