The emergence of drug resistance towards existing drugs is a constant challenge in the fight against many diseases including Malaria and gonorrhoeae. To evade resistance, new targets must be engaged, and to do that, new structural classes of anti-infective must be prepared and evaluated. During the course of my PhD journey, I had the opportunity to investigate and optimize the antimalarial candidate (±)-2-3b, and salicylic acid (4-1a) as an anti-gonorrhea treatment. Malaria is a life-threatening mosquito-borne disease. In 2021, there were 247 million cases of malaria and the estimated number of malaria deaths stood at 619,000. Because of the rapid development of resistance to all current antimalarials, discovery of antimalarials with unexploited mechanisms of action is critical to reduce malaria mortality. In the Carlier group, our initial approach focused on discovery of inhibitors of the methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis, since this pathway is essential for Plasmodium falciparum and absent in human. Application of the isopentenyl pyrophosphate (IPP) chemical rescue screen to the compounds of the Malaria Box, a collection of 400 antimalaria candidates with unknown mechanisms of action, identified tetrahydro-β-carboline 2-1 (MMV008138) as an inhibitor of the MEP pathway. Chapter 2 of this work discusses similarity searching of the Novartis portion of the hit set (5K compounds), from the original 20K compound hit set of the Malaria Box, and identifying tetrahydro-β-carboline GNF-Pf-5009, designated as (±)-2-3b. Preparation of pure enantiomers, by resolution, demonstrated the pharmacological superiority of (R)-2-3b over (S)-2-3b, which was found to have good asexual blood stage (ABS) inhibition potency against malarial parasites P. falciparum, and low general cytotoxicity. However, (R)-2-3b was found not orally efficacious in a P. berghei mouse model of malaria. We concluded that the lack of oral efficacy of (R)-2-3b was due to its poor drug-like qualities, in particular its high molecular weight and low solubility. Chapter 3 of this work explores modifications of (R)-2-3b ((R)-3-5Aa) that were expected to improve its properties. We show that the new compounds (R)-3-5Gm and (R)-3-5Gk not only are more potent in vitro than (R)-2-3b ((R)-3-5Aa), but also have molecular weights < 500 g/mol. Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. Due to the increased rates of infection as well as the prevalence of multidrug-resistant N. gonorrhoeae strains worldwide, the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) list N. gonorrhoeae at the highest possible threat level to public health. Dual therapy of azithromycin (AZM) and ceftriaxone has been the standard-of-care for treatment of gonococcal infections. However, due to increasing resistance to azithromycin (>33% in some regions) the CDC removed AZM from the treatment regimen for gonorrhea in 2020. Therefore, ceftriaxone remains the only recommended antibiotic for treatment of gonococcal infections. However, increasing resistance to this treatment option has been reported, consequently there is an urgent need to identify novel therapeutics against N. gonorrhoeae. Drug repurposing is a popular strategy that explores new therapeutic opportunities for approved drugs with available information on their pharmacokinetic data, dosages, and toxicity. Salicylic acid is a highly privileged chemical scaffold. Also, the use of salicylic acid to treat sexually transmitted diseases (including gonorrhea) was reported as early as the 19th century. Recently, Dr. Mohamed N. Seleem reported that salicylic acid (4-1a) exhibited modest activity against N. gonorrhoeae strains including the AZM-resistant strain (CDC-181). Chapter 4 of this work illustrates how the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution. However, we found that substituted naphthyl analogs (4-3b,o,p) have superior activity to salicylic acid itself. In addition, the three analogs showed high selectivity, compared to AZM, against N. gonorrhoeae over the vaginal microbiota. / Doctor of Philosophy / In the fight against malaria and gonorrhea, two different diseases, we face one common challenge, which is the emergence of drug resistance towards existing drugs. Therefore, there is a pressing need for new antimalarial and anti-gonorrhea compounds with novel mechanisms of action. This dissertation encompasses my research on the investigation and optimization of the antimalarial candidate (±)-2-3b and Salicylic acid (4-1a) as anti-gonorrhea treatment. Malaria is a life-threatening mosquito-borne disease. It is transmitted through the bites of infected female Anopheles mosquitoes. In 2021, there were 247 million cases of malaria and the estimated number of malaria deaths stood at 619,000. Children under 5 accounted for about 80% of all malaria deaths. In the Carlier group, compound 2-1 (MMV008138) was identified and thoroughly studied as antimalaria candidate. It was found to be effective in killing the malaria parasite, P. falciparum, in red blood cells, in vitro. However, when tested on P. berghei mouse model of malaria, it was found not effective. Chapter 2 of this work discusses the discovery of (±)-2-3b after searching for a structurally similar analog of 2-1. The compound (±)-2-3b can exist in two distinct spatial arrangements (enantiomers) R and S. After preparation of pure enantiomers, we confirmed the pharmacological superiority of (R)-2-3b over (S)-2-3b. The compound (R)-2-3b showed good activity in vitro against malarial parasites P. falciparum, and low general cytotoxicity. However when tested orally on P. berghei mouse model of malaria, it was found not effective. We concluded that the lack of oral efficacy of (R)-2-3b was due to its poor drug-like qualities, such as high molecular weight and low solubility. Chapter 3 of this work explore modifications of (R)-2-3b ((R)-3-5Aa) that will improve its properties. We show that the new compounds (R)-3-5Gm and (R)-3-5Gk not only are more active in vitro than (R)-2-3b ((R)-3-5Aa), but also have lower molecular weights (< 500 g/mol). Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. The World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) list N. gonorrhoeae at the highest possible threat level to public health because of the increased rates of infection and the appearance of multidrug-resistant N. gonorrhoeae strains worldwide. Dual therapy of azithromycin (AZM) and ceftriaxone has been the standard-of-care for treatment of gonococcal infections. However, due to increasing resistance to azithromycin the CDC removed AZM from the treatment regimen. Therefore, ceftriaxone remains the only recommended antibiotic for treatment of gonococcal infections. However, increasing resistance to this treatment option has been reported. Drug repurposing is a popular strategy that explores new therapeutic opportunities for approved drugs with available information on their pharmacokinetic data, dosages, and toxicity. Salicylic acid is a highly privileged chemical scaffold that has been used to treat sexually transmitted diseases (including gonorrhea) since the 19th century. Chapter 4 of this work illustrates our efforts to enhance the potency of salicylic acid (4- 1a). I performed chemical modifications on (4-1a) and concluded that anti-gonococcal activity is easily lost by inopportune substitution. However, we found that substituted naphthyl analogs (4-3b,o,p) have superior activity to salicylic acid itself. In addition, the three analogs showed high selectivity, compared to AZM, against N. gonorrhoeae over the vaginal microbiota.

The war between human and malaria has never stopped, and the development and application of antimalarial drugs has not eradicated this terrible disease. To fight drug-resistant malaria, many leads have been studied over the years. (1S,3R)-MMV008138 and MMV1803522 are two compounds that have been studied in the Carlier Group. My research focused on the structural variation of each of these compounds, in the hope that greater potency could be realized. Chapter 2 describes my work on (1S,3R)-MMV008138, which inhibits the enzyme PfIspD in the methylerythritol phosphate (MEP) pathway. This compound shows good in vitro potency against the drug resistant Dd2 strain of Plasmodium falciparum. However, this lead showed no activity in mouse models. This lack of activity may be due to poor metabolic stability of the compound. However, a significant increase in in vitro potency could also improve in vivo activity. Towards that end, I focused on further variation of the D-ring and A-rings. With the regard to the D-ring, we made five analogs of MMV008138 that replaced the 2,4-dichlorophenyl ring with dihalogenated thiophen-3-yl and thiophen-2-yl rings. We also explored the effect of installing a cyano group on the A-ring of MMV008138. Unfortunately, none of these new compounds were potent growth inhibitors of Dd2 strain P. falciparum. We conclude that the lead goes into a well-defined pocket within the PfIspD enzyme that only accommodates 2,4-dihalogenated phenyl D-rings. This pocket also cannot accept any substitution larger than F on the A-ring. Interestingly, the crystal structure of 5-cyano-substituted MMV008138 was obtained ((±)-2-50c). This is the first compound out of more than 100 analogs of MMV008138 family to be amenable to crystallization. The solid state conformation of the (±)-2-50c revealed that the C3-carboxyl group was in a pseudoequatorial orientation, and the C1-aryl group was thus in a pseudoaxial orientation. 1H NMR spectroscopic studies in CD3OD-D2O were carried out to determine the solution conformation. As expected from previous studies of ester derivatives of MMV008138, these studies indicated that in solution, 2-5 would adopt both the C3-carboxyl pseudoequatorial and pseudoaxial conformations. In Chapter 3, I describe the synthesis of analogs of the antimalarial drug candidate MMV1803522. This β-carboline-3-carboxamide shows good in vitro growth inhibition potency of Dd2 strain P. falciparum, operating by a still unknown mechanism. To investigate the pharmacophore of this lead, I first sought to determine whether the pyridine N (i.e. N2) of the β-carboline was important for in vitro potency. I prepared series of carbazole analogs of MMV1803522, which replace N2 with a CH. These compounds potently inhibited the growth of Dd2 strain P. falciparum. These results suggest that N2 of MMV1803522 is not involved in any energetically significant interactions with its target protein. To further identify the pharmacophore, we prepared truncated analogs lacking the A- and B- rings (biphenyl analogs), and tricyclic analogs that feature a reversed indole moiety. Unfortunately, the biphenyl analogs and reversed indole analogs show no growth inhibition at 10,000 nM the highest concentration tested. Lastly, I describe analogs of MMV1803522 in which the 3,4-dichlorophenyl ring of MMV1803522 was replaced with halogenated thiophene. This substitution was tolerated, but compounds were roughly half as potent as MMV1803522. / Doctor of Philosophy / Malaria, mainly caused by the infection of P. falciparum, is a serious worldwide disease. In 2020, there were 241 million cases of malaria infections and over 600,000 deaths from malaria. Combinations of commercially available antimalarial compounds, such as chloroquine, mefloquine and artemisinin, are commonly used as combination therapies to treat malaria. Since different antimalarial compounds have different mechanisms of action, this combination strategy can greatly slow down the spread of drug-resistant parasites. However, multiple drug-resistant strains of P. falciparum have been reported. Therefore, there is an urgent need for new antimalarial compounds with novel mechanisms of action. This dissertation involves my research on the investigation and optimization of two novel antimalarial compounds, MMV008138 and MMV1803522. MMV008138 is an inhibitor of the MEP pathway, which is an essential metabolic pathway and attractive target for antimalarial therapies, in malaria parasites. The parasites cannot survive, with the MEP pathway inhibited. Since the MEP pathway is not present in human, the MMV008138 molecule is unlikely to have toxicity to human. The MMV008138 molecule has been demonstrated to have great in vitro performance of inhibiting the MEP pathway in several studies, however, the in vivo performance in mouse models is yet to improve. This may be due to the poor metabolic stability of this compound. The compound decomposes in the mouse body before it takes effect. To enhance the metabolic stability and potency, I performed chemical modifications on the A- and D-rings of the MMV008138 compound. An X-ray crystal structure was obtained to help elucidate the conformer distribution of MMV008138. This crystal structure can be used to guide our understanding of the docking of this compound to the target enzyme in the future. MMV1803522 is another compound that shows great potency in vitro and in vivo. This compound is fully oxidized and contains four aromatic rings. However, the target enzyme and the mechanism of action of MMV1803522 is yet to be discovered, and the structure-activity relationship between the chemical structure and the biological activity of this molecule is still unknown. Therefore, I have developed synthetic methods to synthesize a series of compounds that are structurally similar to the MMV1803522 and found that potency of this molecule is not due to the nitrogen on the C-ring. Also, the number and size of the ring structures in the MMV1803522 may be crucial for this molecule to exhibit great potency in vitro and in vivo.

Malaria continues to threaten human beings, causing a staggering number of more than 400,000 deaths each year. Although effective treatment and prevention methods are available, rapidly emerging resistance towards existing drugs is of great concern, and the need for novel antimalarial compounds are still urgent. The Malaria Box lead molecules MMV008138 and MMV665831 are promising in this regard, due to their apparently novel antimalarial mechanisms of action. The target of MMV008138 is the PfIspD enzyme in the MEP pathway, which is absent in humans. This difference makes the PfIspD a great target. However, while MMV008138 shows potency against Plasmodium falciparum-infected human erythrocytes in vitro, no efficacy was observed in a humanized mouse model or a P. berghei infected mouse in vivo. In order to block potential metabolic spots and to probe for steric demand, a series of analogous featuring C1-deuteration, methyl substitution on B- and C-ring, and an ethylene bridge were prepared. The effect of various substitution on the tetrahydro-β-carboline conformation and D-ring orientation was studied. In the course of preparing the C1-Me analog of MMV008138 featuring 2',4'- dichloro substitution, unexpected ring-expanded azepane products were isolated. Later it was found that the desired product could be isolated when the imine formed was treated with acid at lower temperature. Other intermediates possessing a 2ʹ- substituent were also isolated under the low temperature acid treatment protocol, which upon heating in acid gave the ring-expanded azepane we initially isolated. A mechanism was proposed to account for the formation of the azepane as well as other intermediates. The driving force of the expansion reaction was explored, and the hypothesis that the steric interaction between the 2ʹ-substituent and the C1-Me was supported via DFT calculation and conformational analysis. MMV665831 is another potent hit from the Malaria Box, and it appears to inhibit the hemoglobin endocytosis process of P. falciparum. The structure–activity relationship of MMV665831 was studied with analogues featuring modifications on C2-benzamide, C3-ester, C-7 phenol, as well as the phenolic Mannich base moiety. Modifications at phenolic Mannich base moiety leads to the discovery of an analogue that is twice as potent toward cultured P. falciparum compared to MMV665831. We were worried the phenolic Mannich base moiety might act as the precursor of toxic quinone methide intermediates, and designed two analogs to block this potential toxicophore. Although the modification resulted in reduced potency, this result proved that the potency of MMV665831 does not stem from the formation of quinone methides. Unfortunately, MMV665831 did not reduce parasitemia in P. berghei- infected mice. Fast hepatocyte metabolism was observed for MMV665831, and the loss of in vivo efficacy was discussed in comparison with other phenolic Mannich bases with similar hepatocyte stability. / Doctor of Philosophy / In the fight against malaria, one concerning issue is the rapidly emerging resistance towards existing drugs. The continuous development of antimalarials with novel mechanism of action is greatly needed. To accelerate the development of novel antimalarials, an open access ensemble of 400 compounds that are toxic to the malaria parasite known as the Malaria Box, has been made available. My work involves the optimization of two compounds from this ensemble, MMV008138 and MMV665831. MMV008138 kills the malaria parasite by inhibiting an enzyme named PfIspD, which is absent in human. In the parasite an enzyme called PfIspD is responsible for the biosynthesis of IPP and DMAPP, two chemical building blocks that are essential for all cells. It is unlikely that MMV008138 will interrupt with the biosynthesis of IPP and DMAPP in human, since we use another enzyme to synthesize them. Although MMV008138 shows great in vitro potency, but did not protect a live mouse from malaria infection. The lack of in vivo efficacy could stem from the rapid metabolism of MMV008138, and analogs aimed to prevent metabolism were designed and prepared. While preparing analogs featuring 2ʹ-substitution, the desired product was not found, but other unexpected by-products were isolated. The conditions that leads to both the desired products and the by-products were found, and the mechanistics detail of this unexpected reaction were studied. During the blood-stage, which causes malaria symptoms in human, the Plasmodium falciparum parasite invades and feeds on human red blood cells (erythrocytes). The parasite destroys human hemoglobin through a multistep process that begins by transporting the hemoglobin from the red blood cell into itself, a process called endocytosis. MMV665831 appears to interfere with this endocytosis process of P. falciparum, thus starving the parasite of its food. Analogs of MMV665831 were prepared to probe for the effect on potency, and one compound that is twice as potent in cultured parasites was found. The structure of MMV665831 contains a potentially unstable moiety, which might lead to toxicity in humans. Two analogs with the problematic moiety removed were designed and prepared, and one still shows antimalarial activity, showing that the reactivity of the potentially unstable moiety is not the reason for the antimalarial activity of MMV665831. However, MMV665831did not protect P. berghei-infected mice (murine malaria) in vivo, and the reason for the loss of efficacy was discussed.