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Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

[Image: see text] Synthetic endoperoxide antimalarials, such as 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes, are promising successors for current front-line antimalarials, semisynthetic artemisinin derivatives. However, limited solubility of second-generation analogues in biological-relevant media represents a barrier in clinical development. We present methodology for the synthesis of nonlinear analogues of second-generation tetraoxane antimalarials E209 and N205 to investigate reduced molecular symmetry on in vitro antimalarial activity and physicochemical properties. While maintaining good antimalarial activity and metabolic stability, head-to-head comparison of linear and nonlinear counterparts showed up to 10-fold improvement in FaSSIF solubility for three of the four analogues studied. Pharmacokinetic studies in rats comparing a selected nonlinear analogue 14a and its parent N205 showed improvement on oral absorption and exposure in vivo with more than double the AUC and a significant increase in oral bioavailability (76% versus 41%). These findings provide support for further in vivo efficacy studies in preclinical animal species.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

The rapidly growing COVID-19 pandemic is the most serious global health crisis since the "Spanish flu" of 1918. There is currently no proven effective drug treatment or prophylaxis for this coronavirus infection. While developing safe and effective vaccines is one of the key focuses, a number of existing antiviral drugs are being evaluated for their potency and efficiency against SARS-CoV-2 in vitro and in the clinic. Here, we review the significant potential of nitazoxanide (NTZ) as an antiviral agent that can be repurposed as a treatment for COVID-19. Originally, NTZ was developed as an antiparasitic agent especially against Cryptosporidium spp.; it was later shown to possess potent activity against a broad range of both RNA and DNA viruses, including influenza A, hepatitis B and C, and coronaviruses. Recent in vitro assessment of NTZ has confirmed its promising activity against SARS-CoV-2 with an EC50 of 2.12 μM. Here we examine its drug properties, antiviral activity against different viruses, clinical trials outcomes, and mechanisms of antiviral action from the literature in order to highlight the therapeutic potential for the treatment of COVID-19. Furthermore, in preliminary PK/PD analyses using clinical data reported in the literature, comparison of simulated TIZ (active metabolite of NTZ) exposures at two doses with the in vitro potency of NTZ against SARS-CoV-2 gives further support for drug repurposing with potential in combination chemotherapy approaches. The review concludes with details of second generation thiazolides under development that could lead to improved antiviral therapies for future indications.

[Image: see text] A series of 2-pyrazolyl quinolones has been designed and synthesized in 5–7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC(50) (concentration of drug required to achieve half maximal growth suppression) values in the range of 15–33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Q(i) site of the parasite bc(1) complex, which is supported by crystallographic studies of bovine cytochrome bc(1) complex.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Significance Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are neglected tropical diseases that cause severe disability and affect more than 157 million people globally. Current control efforts are hindered by the lack of a safe macrofilaricidal drug that can eliminate the parasitic adult nematodes safely. A clinically validated approach for delivering macrofilaricidal activity is to target the Wolbachia bacterial endosymbiont of the causative nematodes. This first-in-class and highly potent and specific anti-Wolbachia preclinical candidate molecule, AWZ1066S, has the potential to significantly impact current global onchocerciasis and lymphatic filariasis elimination programs and reduce elimination time frames from decades to years., Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

A series of 2-pyridylquinolones has been prepared in 5–7 steps and through lead optimisation, antimalarial activity as low as 12 nM against Plasmodium falciparum (Pf) has been achieved. Compared with previous analogues in this series, selected molecules have improved solubility, a reduced potential for off-target toxicity and improved metabolic stability profiles. Docking studies performed with a homology model of the Pfbc1 complex target demonstrate a key role for the Tyr16 residues in the recognition of highly active quinolone based inhibitors.

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified similar to 100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further similar to 90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC50 = 525 nM, Mtb Wayne IC50 = 76 nM, and MDR Mtb patient isolates IC50 = 140 nM) and favorable pharmacokinetic and toxicological profiles. National Institute of Health Research (NIHR, BRC Liverpool); Medical Research Council [MRC DPFS-G1002586]; Medical Research Council (MRC CiC) info:eu-repo/semantics/publishedVersion

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase ( Plasmodium falciparum NDH2) and cytochrome bc 1 . Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites ( Plasmodium cynomolgi ) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 μM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria.

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies.

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress.

Using the Gould-Jacobs methodology a small array of 6- and 7-substituted quinolones have been prepared. Analogues in the 7-series express activity as low as 0.46 nM versus Plasmodium falciparum malaria parasites and docking studies performed in silico at the yeast Qo site demonstrate a key role for residues His182 and Glu 272 in the recognition of high potency inhibitors.

In two steps from dihydroartemisinin, a small array of 16 semisynthetic C-10 pyrrole Mannich artemisinin derivatives (7a−p) have been prepared in moderate to excellent yield. In vitro analysis against both chloroquine sensitive and resistant strains has demonstrated that these analogues have nanomolar antimalarial activity, with several compounds being more than 3 times more potent than the natural product artemisinin. In addition to a potent antimalarial profile, these molecules also have very high in vitro therapeutic indices. Analysis of the optimal Mannich side chain substitution for in vitro and in vivo activity reveals that the morpholine and N-methylpiperazine Mannich side chains provide analogues with the best activity profiles, both in vitro and in vivo in the Peter’s 4 day test.