Optimization of B-Ring-Functionalized Antimalarial Tambjamines and Prodiginines.

Malaria has been a deadly enemy of mankind throughout history, affecting over 200 million people annually, along with approximately half a million deaths. Resistance to current therapies is of great concern, and there is a dire need for novel and well-tolerated antimalarials that operate by clinically unexploited mechanisms. We have previously reported that both tambjamines and prodiginines are highly potent novel antiplasmodial agents, but they required rigor optimizations to enhance the oral efficacy, safety, and physicochemical properties. Here, we launched a comprehensive structure-activity relationship study for B-ring-functionalized tambjamines and prodiginines with 54 novel analogues systematically designed and synthesized. A number of compounds exhibited remarkable antiplasmodial activities against asexual erythrocytic Plasmodium parasites, with improved safety and metabolic profiles. Notably, several prodiginines cured erythrocytic Plasmodium yoelii infections after oral 25 mg/kg × 4 days in a murine model and provided partial protection against liver stage Plasmodium berghei sporozoite-induced infection in mice.

3-Position Biaryl Endochin-like Quinolones with Enhanced Antimalarial Performance.

ELQ-300 is a potent antimalarial drug with activity against blood, liver, and vector stages of the disease. A prodrug, ELQ-331, exhibits reduced crystallinity and improved in vivo efficacy in preclinical testing, and currently, it is in the developmental pipeline for once-a-week dosing for oral prophylaxis against malaria. Because of the high cost of developing a new drug for human use and the high risk of drug failure, it is prudent to have a back-up plan in place. Here we describe ELQ-596, a member of a new subseries of 3-biaryl-ELQs, with enhanced potency in vitro against multidrug-resistant Plasmodium falciparum parasites. ELQ-598, a prodrug of ELQ-596 with diminished crystallinity, is more effective vs murine malaria than its progenitor ELQ-331 by 4- to 10-fold, suggesting that correspondingly lower doses could be used to protect and cure humans of malaria. With a longer bloodstream half-life in mice compared to its progenitor, ELQ-596 highlights a novel series of next-generation ELQs with the potential for once-monthly dosing for protection against malaria infection. Advances in the preparation of 3-biaryl-ELQs are presented along with preliminary results from experiments to explore key structure-activity relationships for drug potency, selectivity, pharmacokinetics, and safety.

Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Long-acting intramuscular injections of ELQ-331, an antimalarial agent.

The overarching premise of this investigation is that injectable, long-acting antimalarial medication would encourage adherence to a dosage regimen for populations at risk of contracting the disease. To advance support for this goal, we have developed oil-based formulations of ELQ-331 (a prodrug of ELQ-300) that perform as long-acting, injectable chemoprophylactics with drug loading as high as 160 mg/ml of ELQ-331. In a pharmacokinetic study performed with rats, a single intramuscular injection of 12.14 mg/kg maintained higher plasma levels than the previously established minimum fully protective plasma concentration (33.25 ng/ml) of ELQ-300 for more than 4 weeks. The formulations were well tolerated by the rats and the tested dose produced no adverse reactions. We believe that by extending the length of time between subsequent injections, these injectable oil-based solutions of ELQ-331 can offer a more accessible, low-cost option for long-acting disease prevention and reduced transmission in malaria-endemic regions and may also be of use to travelers.

Total Synthesis and Antimalarial Activity of 2-(p-Hydroxybenzyl)-prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine MYP1 Isolated from Marine Bacteria.

Highly efficient and straightforward synthetic routes toward the first total synthesis of 2-(p-hydroxybenzyl)-prodigiosins (2-5), isoheptylprodigiosin (6), and geometric isomers of tambjamine MYP1 ((E/Z)-7) have been developed. The crucial steps involved in these synthetic routes are the construction of methoxy-bipyrrole-carboxaldehydes (MBCs) and a 20-membered macrocyclic core and a regioselective demethylation of MBC analogues. These new synthetic routes enabled us to generate several natural prodiginines 24-27 in larger quantity. All of the synthesized natural products exhibited potent asexual blood-stage antiplasmodial activity at low nanomolar concentrations against a panel of Plasmodium falciparum parasites, with a great therapeutic index. Notably, prodiginines 6 and 24-27 provided curative in vivo efficacy against erythrocytic Plasmodium yoelii at 25 mg/kg × 4 days via oral route in a murine model. No overt clinical toxicity or behavioral change was observed in any mice treated with prodiginines and tambjamines.

Acridones Are Highly Potent Inhibitors of Toxoplasma gondii Tachyzoites.

Acridone derivatives, which have been shown to have in vitro and in vivo activity against Plasmodium spp, inhibit Toxoplasma gondii proliferation at picomolar concentrations. Using enzymatic assays, we show that acridones inhibit both T. gondii cytochrome bc1 and dihydroorotate dehydrogenase and identify acridones that bind preferentially to the Qi site of cytochrome bc1. We identify acridones that have efficacy in a murine model of systemic toxoplasmosis. Acridones have potent activity against T. gondii and represent a promising new class of preclinical compounds.

A New Scalable Synthesis of ELQ-300, ELQ-316, and other Antiparasitic Quinolones.

The Endochin-Like Quinolone (ELQ) compound class may yield effective, safe treatments for a range of important human and animal afflictions. However, to access the public health potential of this compound series, a synthetic route needed to be devised that lowers costs and is amenable to large scale production. In the new synthetic route described here, a substituted ß-keto ester, formed by an Ullmann reaction and subsequent acylation, is reacted with an aniline via a Conrad-Limpach reaction to produce 3-substituted 4(1H)-quinolones such as ELQ-300 and ELQ-316. This synthetic route, the first described to be truly amenable to industrial scale production, is relatively short (5 reaction steps), does not require palladium, chromatographic separation or protecting group chemistry, and may be performed without high vacuum distillation.

Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi.

BACKGROUND: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. METHODS: The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. RESULTS: We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. CONCLUSIONS: The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

ELQ-331 as a prototype for extremely durable chemoprotection against malaria.

BACKGROUND: The potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice. METHODS: Four trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P. yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 h to 5½ months after injection. RESULTS: A single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 h) and sustained (4-5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration versus time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60-80 nM). CONCLUSIONS: Extrapolation of these results to humans predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for anti-malarial LAI-C. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection and enable protection for far longer than 3 months. These findings suggest that ELQ-331 warrants consideration as a leading prototype for LAI-C.

Discovery and Structural Optimization of Acridones as Broad-Spectrum Antimalarials.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Boron Trifluoride Etherate Promoted Microwave-Assisted Synthesis of Antimalarial Acridones.

A microwave-assisted, rapid and efficient method using boron trifluoride etherate (BF3.Et2O) for the synthesis of acridones, via an intramolecular acylation of N-phenylanthranilic acid derivatives, has been developed. The reaction proceeds under solvent-free conditions, tolerates a wide range of functional groups, and provides rapid access to a range of acridones in good to excellent yields. Several of the synthesized acridones exhibited potent antimalarial activities against CQ sensitive and multi-drug resistant (MDR) parasites.

Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc1 and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis.

Cytochrome bc1 inhibitors have been broadly studied as human and veterinary medicines and agricultural fungicides. For the most part, cytochrome bc1 inhibitors compete with ubiquinol at the ubiquinol oxidation (Qo) site or with ubiquinone at the quinone reduction (Qi) site. 4(1 H)-Quinolones with 3-position substituents may inhibit either site based on quinolone ring substituents. 4(1 H)-Quinolones that inhibit the Qi site are highly effective against toxoplasmosis, malaria, and babesiosis and do not inhibit human cytochrome bc1. We tested a series of 4(1 H)-Quinolones against wild-type and drug resistant strains of Toxoplasma gondii and Plasmodium falciparum. These experiments identified very potent compounds that inhibit T. gondii proliferation at picomolar concentrations. The most potent compounds target the Qo site, and for these compounds, an alkyl side chain confers potency against T. gondii greater than that of bulkier side chains. Our experiments also show that substituents on the quinolone ring influenced selectivity between T. gondii and P. falciparum and between Qo and Qi site-mediated activity. Comparison of the parasite cytochrome b sequences identified amino acids that are associated with drug resistance in P. falciparum that exist naturally in wild-type T. gondii. These underlying differences may influence drug susceptibility. Finally, a Qo site active 4(1 H)-quinolone-3-diarylether tested in a murine model of toxoplasmosis was superior to atovaquone, resulting in survival from Type I strain T. gondii infection. These experiments identify highly effective compounds for toxoplasmosis and provide valuable insight into the structure-activity relationship of cytochrome bc1 inhibitors.

Diphenylether-Modified 1,2-Diamines with Improved Drug Properties for Development against Mycobacterium tuberculosis.

New treatments for tuberculosis infection are critical to combat the emergence of multidrug- and extensively drug-resistant Mycobacterium tuberculosis (Mtb). We report the characterization of a diphenylether-modified adamantyl 1,2-diamine that we refer to as TBL-140, which has a minimal inhibitory concentration (MIC99) of 1.2 µg/mL. TBL-140 is effective against drug-resistant Mtb and nonreplicating bacteria. In addition, TBL-140 eliminates expansion of Mtb in cell culture infection assays at its MIC. To define the mechanism of action of this compound, we performed a spontaneous mutant screen and biochemical assays. We determined that TBL-140 treatment affects the proton motive force (PMF) by perturbing the transmembrane potential (ΔΨ), consistent with a target in the electron transport chain (ETC). As a result, treated bacteria have reduced intracellular ATP levels. We show that TBL-140 exhibits greater metabolic stability than SQ109, a structurally similar compound in clinical trials for treatment of MDR-TB infections. Combined, these results suggest that TBL-140 should be investigated further to assess its potential as an improved therapeutic lead against Mtb.

Discovery of dual function acridones as a new antimalarial chemotype.

Preventing and delaying the emergence of drug resistance is an essential goal of antimalarial drug development. Monotherapy and highly mutable drug targets have each facilitated resistance, and both are undesirable in effective long-term strategies against multi-drug-resistant malaria. Haem remains an immutable and vulnerable target, because it is not parasite-encoded and its detoxification during haemoglobin degradation, critical to parasite survival, can be subverted by drug-haem interaction as in the case of quinolines and many other drugs. Here we describe a new antimalarial chemotype that combines the haem-targeting character of acridones, together with a chemosensitizing component that counteracts resistance to quinoline antimalarial drugs. Beyond the essential intrinsic characteristics common to deserving candidate antimalarials (high potency in vitro against pan-sensitive and multi-drug-resistant Plasmodium falciparum, efficacy and safety in vivo after oral administration, inexpensive synthesis and favourable physicochemical properties), our initial lead, T3.5 (3-chloro-6-(2-diethylamino-ethoxy)-10-(2-diethylamino-ethyl)-acridone), demonstrates unique synergistic properties. In addition to 'verapamil-like' chemosensitization to chloroquine and amodiaquine against quinoline-resistant parasites, T3.5 also results in an apparently mechanistically distinct synergism with quinine and with piperaquine. This synergy, evident in both quinoline-sensitive and quinoline-resistant parasites, has been demonstrated both in vitro and in vivo. In summary, this innovative acridone design merges intrinsic potency and resistance-counteracting functions in one molecule, and represents a new strategy to expand, enhance and sustain effective antimalarial drug combinations.

Synthesis and heme-binding correlation with antimalarial activity of 3,6-bis-(omega-N,N-diethylaminoamyloxy)-4,5-difluoroxanthone.

In this study, the effect of fluorine upon the heme-binding ability of the xanthone nucleus was investigated for 3,6-bis-(omega-N,N-diethylaminoamyloxy)-4,5-difluoroxanthone (F2C5). 2-Fluoro-1,3-dimethoxybenzene was prepared by a new, improved method and used to build up the xanthone nucleus. The interaction of F2C5 with heme was investigated by UV-vis, (1)H NMR, and (19)F NMR spectroscopy. For the first time, NMR studies for the heme-drug interactions are carried out at pH 5.0, physiological for the acidic food vacuole of the malaria parasite.

Design, synthesis, and evaluation of 10-N-substituted acridones as novel chemosensitizers in Plasmodium falciparum.

A series of novel 10-N-substituted acridones, bearing alkyl side chains with tertiary amine groups at the terminal position, were designed, synthesized, and evaluated for the ability to enhance the potency of quinoline drugs against multidrug-resistant (MDR) Plasmodium falciparum malaria parasites. A number of acridone derivatives, with side chains bridged three or more carbon atoms apart between the ring nitrogen and terminal nitrogen, demonstrated chloroquine (CQ)-chemosensitizing activity against the MDR strain of P. falciparum (Dd2). Isobologram analysis revealed that selected candidates demonstrated significant synergy with CQ in the CQ-resistant (CQR) parasite Dd2 but only additive (or indifferent) interaction in the CQ-sensitive (CQS) D6. These acridone derivatives also enhanced the sensitivity of other quinoline antimalarials, such as desethylchloroquine (DCQ) and quinine (QN), in Dd2. The patterns of chemosensitizing effects of selected acridones on CQ and QN were similar to those of verapamil against various parasite lines with mutations encoding amino acid 76 of the P. falciparum CQ resistance transporter (PfCRT). Unlike other known chemosensitizers with recognized psychotropic effects (e.g., desipramine, imipramine, and chlorpheniramine), these novel acridone derivatives exhibited no demonstrable effect on the uptake or binding of important biogenic amine neurotransmitters. The combined results indicate that 10-N-substituted acridones present novel pharmacophores for the development of chemosensitizers against P. falciparum.