Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis.

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 ∼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 ∼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.

Artemisinin-polypyrrole conjugates: synthesis, DNA binding studies and preliminary antiproliferative evaluation.

Greater than the sum of its parts: Artemisinins are currently in phase I-II clinical trials against breast, colorectal and non-small-cell lung cancers. In an attempt to offer increased specificity, a series of hybrid artemisinin-polypyrrole minor groove binder conjugates are described. DNA binding/modelling studies and preliminary biological evaluation give insights into their mechanism of action and the potential of this strategy.

Glutathione transport: a new role for PfCRT in chloroquine resistance.

AIMS: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. RESULTS: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. INNOVATION: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. CONCLUSION: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

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.

HDQ, a potent inhibitor of Plasmodium falciparum proliferation, binds to the quinone reduction site of the cytochrome bc1 complex.

The mitochondrial bc(1) complex is a multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c coupled to the vectorial translocation of protons across the inner mitochondrial membrane. The complex contains two distinct quinone-binding sites, the quinol oxidation site of the bc(1) complex (Q(o)) and the quinone reduction site (Q(i)), located on opposite sides of the membrane within cytochrome b. Inhibitors of the Q(o) site such as atovaquone, active against the bc(1) complex of Plasmodium falciparum, have been developed and formulated as antimalarial drugs. Unfortunately, single point mutations in the Q(o) site can rapidly render atovaquone ineffective. The development of drugs that could circumvent cross-resistance with atovaquone is needed. Here, we report on the mode of action of a potent inhibitor of P. falciparum proliferation, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ). We show that the parasite bc(1) complex--from both control and atovaquone-resistant strains--is inhibited by submicromolar concentrations of HDQ, indicating that the two drugs have different targets within the complex. The binding site of HDQ was then determined by using a yeast model. Introduction of point mutations into the Q(i) site, namely, G33A, H204Y, M221Q, and K228M, markedly decreased HDQ inhibition. In contrast, known inhibitor resistance mutations at the Q(o) site did not cause HDQ resistance. This study, using HDQ as a proof-of-principle inhibitor, indicates that the Q(i) site of the bc(1) complex is a viable target for antimalarial drug development.

Identification of novel antimalarial chemotypes via chemoinformatic compound selection methods for a high-throughput screening program against the novel malarial target, PfNDH2: increasing hit rate via virtual screening methods.

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.

Identification, design and biological evaluation of bisaryl quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

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.

Identification, design and biological evaluation of heterocyclic quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

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.

The molecular basis of folate salvage in Plasmodium falciparum: characterization of two folate transporters.

Tetrahydrofolates are essential cofactors for DNA synthesis and methionine metabolism. Malaria parasites are capable both of synthesizing tetrahydrofolates and precursors de novo and of salvaging them from the environment. The biosynthetic route has been studied in some detail over decades, whereas the molecular mechanisms that underpin the salvage pathway lag behind. Here we identify two functional folate transporters (named PfFT1 and PfFT2) and delineate unexpected substrate preferences of the folate salvage pathway in Plasmodium falciparum. Both proteins are localized in the plasma membrane and internal membranes of the parasite intra-erythrocytic stages. Transport substrates include folic acid, folinic acid, the folate precursor p-amino benzoic acid (pABA), and the human folate catabolite pABAG(n). Intriguingly, the major circulating plasma folate, 5-methyltetrahydrofolate, was a poor substrate for transport via PfFT2 and was not transported by PfFT1. Transport of all folates studied was inhibited by probenecid and methotrexate. Growth rescue in Escherichia coli and antifolate antagonism experiments in P. falciparum indicate that functional salvage of 5-methyltetrahydrofolate is detectable but trivial. In fact pABA was the only effective salvage substrate at normal physiological levels. Because pABA is neither synthesized nor required by the human host, pABA metabolism may offer opportunities for chemotherapeutic intervention.

Endoperoxide carbonyl falcipain 2/3 inhibitor hybrids: toward combination chemotherapy of malaria through a single chemical entity.

We extend our approach of combination chemotherapy through a single prodrug entity (O'Neill et al. Angew. Chem., Int. Ed. 2004, 43, 4193) by using a 1,2,4-trioxolane as a protease inhibitor carbonyl-masking group. These molecules are designed to target the malaria parasite through two independent mechanisms of action: iron(II) decomposition releases the carbonyl protease inhibitor and potentially cytotoxic C-radical species in tandem. Using a proposed target "heme", we also demonstrate heme alkylation/carbonyl inhibitor release and quantitatively measure endoperoxide turnover in parasitized red blood cells.

Synthesis, in vitro and in vivo antimalarial assessment of sulfide, sulfone and vinyl amide-substituted 1,2,4-trioxanes prepared via thiol-olefin co-oxygenation (TOCO) of allylic alcohols.

Thiol-Olefin Co-Oxygenation (TOCO) methodology has been applied to the synthesis of a small library of weak base and polar 1,2,4-trioxanes. The 1,2,4-trioxane units synthesised exhibit remarkable stability as they survive base catalysed hydrolysis and mixed anhydride/amine coupling reactions. This unique stability feature has enabled a range of novel substitution patterns to be incorporated within the spiro 1,2,4-trioxane unit. Selected analogues express potent in vitro nM antimalarial activity, low cytotoxicity and oral activity in the Plasmodium berghei mouse model of malaria.

Design, synthesis and antimalarial/anticancer evaluation of spermidine linked artemisinin conjugates designed to exploit polyamine transporters in Plasmodium falciparum and HL-60 cancer cell lines.

A series of artemisinin-spermidine conjugates designed to utilise the upregulated polyamine transporter found in cancer cells have been prepared. These conjugates were evaluated against human promyelocytic leukaemia HL-60 cells and chloroquine-sensitive 3D7 Plasmodium falciparum and several show promising anticancer and antimalarial activity. Although some limitations in this vector-based approach are apparent, a number of high potency Boc-protected analogues were identified with activity against malaria parasites as low as 0.21nM.

Synthesis, antimalarial activity, and preclinical pharmacology of a novel series of 4'-fluoro and 4'-chloro analogues of amodiaquine. Identification of a suitable "back-up" compound for N-tert-butyl isoquine.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

Semi-synthetic and synthetic 1,2,4-trioxaquines and 1,2,4-trioxolaquines: synthesis, preliminary SAR and comparison with acridine endoperoxide conjugates.

A novel series of semi-synthetic trioxaquines and synthetic trioxolaquines were prepared, in moderate to good yields. Antimalarial activity was evaluated against both the chloroquine-sensitive 3D7 and resistant K1 strain of Plasmodium falciparum and both series of compounds were shown to be active in the low nanomolar range. For comparison the corresponding 9-amino acridine analogues were also prepared and shown to have low nanomolar activity like their quinoline counterparts.

Antitumour and antimalarial activity of artemisinin-acridine hybrids.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

Candidate selection and preclinical evaluation of N-tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Piperidine dispiro-1,2,4-trioxane analogues.

Dispiro N-Boc-protected 1,2,4-trioxane 2 was synthesised via Mo(acac)(2) catalysed perhydrolysis of N-Boc spirooxirane followed by condensation of the resulting beta-hydroperoxy alcohol 10 with 2-adamantanone. N-Boc 1,2,4-trioxane 2 was converted to the amine 1,2,4-trioxane hydrochloride salt 3 which was subsequently used to prepare derivatives (4-7). Several of these novel 1,2,4-trioxanes had nanomolar antimalarial activity versus the 3D7 strain of Plasmodium falciparum. Amine intermediate 3 represents a versatile derivative for the preparation of achiral arrays of trioxane analogues with antimalarial activity.

Two-step synthesis of achiral dispiro-1,2,4,5-tetraoxanes with outstanding antimalarial activity, low toxicity, and high-stability profiles.

A rapid, two-step synthesis of a range of dispiro-1,2,4,5-tetraoxanes with potent antimalarial activity both in vitro and in vivo has been achieved. These 1,2,4,5-tetraoxanes have been proven to be superior to 1,2,4-trioxolanes in terms of stability and to be superior to trioxane analogues in terms of both stability and activity. Selected analogues have in vitro nanomolar antimalarial activity and good oral activity and are nontoxic in screens for both cytotoxicity and genotoxicity. The synthesis of a fluorescent 7-nitrobenza-2-oxa-1,3-diazole (NBD) tagged tetraoxane probe and use of laser scanning confocal microscopy techniques have shown that tagged molecules accumulate selectively only in parasite infected erythrocytes and that intraparasitic formation of adducts could be inhibited by co-incubation with the iron chelator desferrioxamine (DFO).