Synthesis and In vitro Efficacy of Tetracyclic Benzothiazepines Against Blood-Stage Plasmodium falciparum and Liver-Stage P. berghei.

OBJECTIVE: A series of novel, substituted tetracyclic benzothiazepines were designed and prepared in an effort to optimize the potency of this chemical class against drug-resistant strains of the malaria parasite. METHODS: Tetracyclic benzothiazepines bearing structural modification at seven distinct positions within the structure were synthesized in Knoevenagel condensation reactions followed by sequential intermolecular thio-Michael and then intramolecular imine formation reactions. Following purification and chemical characterization, the novel compounds were tested for in vitro efficacy against blood-stage P. falciparum and liver-stage P. berghei and also for in vivo efficacy against P. berghei. RESULTS: Benzothiazepines bearing structural modification at the sulfur atom and at the three carbocycles within the molecule were successfully synthesized. The majority of analogs inhibited bloodstage P. falciparum with submicromolar IC50 values. The potency of an 8-methoxy-substituted analog 12 exceeded that of chloroquine in all three P. falciparum strains tested. The parent benzothiazepine 1 possessed liver-stage activity, inhibiting P. berghei sporozoites infecting HepG2 cells with an IC50 of 106.4 nM and an IC90 of 408.9 nM, but failed to enhance the longevity of P. berghei infected mice compared to the controls. Compounds displayed modest toxicity toward HepG2 cells and were tolerated by mice at the highest dose tested, 640 mg/kg/dose once daily for three days. CONCLUSION: The tetracyclic benzothiazepine described, which inhibits P. berghei infected hepatic cells with an IC50 of 106.4 nM, would appear to warrant further investigation. Optimization of ADME properties may be required since the most active analogs are probably excessively lipophilic.

Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model.

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model.

In vitro efficacy of 2,N-bisarylated 2-ethoxyacetamides against Plasmodium falciparum.

Investigation of a series of 2,N-bisarylated 2-ethoxyacetamides resulted in the identification of four inhibitors 5, 20, 24, 29 with single-digit micromolar in vitro efficacy against two drug-resistant Plasmodium falciparum strains. These compounds are analogs of structurally-related 1,3-bisaryl-2-propen-1-ones (chalcones), the latter showing efficacy in vitro but not in a malaria-infected mouse. The 2,N-bisarylated 2-ethoxyacetamides (e.g., 2, 5, 20) were shown to possess significantly greater stability in the presence of metabolizing enzymes than the corresponding 1,3-bisaryl-2-propen-1-ones (e.g., 1, 3, 18).

Antimalarial activity of 4-amidinoquinoline and 10-amidinobenzonaphthyridine derivatives.

Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug due to its being long-acting and having a high potency against blood stage P. falciparum (Pf). However, serious CNS toxicity of MQ has compromised its clinical value as a prophylaxis drug. Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS toxicity are urgently needed to combat this deadly human disease. In this study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were designed, prepared, and assessed to search for new therapeutic agents to replace CQ and MQ. The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ, and none were toxic in mice up to 160 mpk × 3. The best compound shows IC50 < 1 ng/mL against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K(+) channel blockage testing, negativity in the Ames test, and 5/5 cure @ <15 mpk × 3 in mice infected with Plasmodium berghei. In addition to these desirable pharmacological profiles, compound 13b, one of the most active compounds, is metabolically stable in both human and mouse liver microsomal preparations and has a plasma t(1/2) of 50 h in mice, which made it a good MQ replacement candidate.

Structure-activity relationships of 4-position diamine quinoline methanols as intermittent preventative treatment (IPT) against Plasmodium falciparum.

A library of diamine quinoline methanols were designed based on the mefloquine scaffold. The systematic variation of the 4-position amino alcohol side chain led to analogues that maintained potency while reducing accumulation in the central nervous system (CNS). Although the mechanism of action remains elusive, these data indicate that the 4-position side chain is critical for activity and that potency (as measured by IC(90)) does not correlate with accumulation in the CNS. A new lead compound, (S)-1-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-(2-(cyclopropylamino)ethylamino)ethanol (WR621308), was identified with single dose efficacy and substantially lower permeability across MDCK cell monolayers than mefloquine. This compound could be appropriate for intermittent preventative treatment (IPTx) indications or other malaria treatments currently approved for mefloquine.

Central nervous system exposure of next generation quinoline methanols is reduced relative to mefloquine after intravenous dosing in mice.

BACKGROUND: The clinical use of mefloquine (MQ) has declined due to dose-related neurological events. Next generation quinoline methanols (NGQMs) that do not accumulate in the central nervous system (CNS) to the same extent may have utility. In this study, CNS levels of NGQMs relative to MQ were measured and an early lead chemotype was identified for further optimization. EXPERIMENTAL DESIGN: The plasma and brain levels of MQ and twenty five, 4-position modified NGQMs were determined using LCMS/MS at 5 min, 1, 6 and 24 h after IV administration (5 mg/kg) to male FVB mice. Fraction unbound in brain tissue homogenate was assessed in vitro using equilibrium dialysis and this was then used to calculate brain-unbound concentration from the measured brain total concentration. A five-fold reduction CNS levels relative to mefloquine was considered acceptable. Additional pharmacological properties such as permeability and potency were determined. RESULTS: The maximum brain (whole/free) concentrations of MQ were 1807/4.9 ng/g. Maximum whole brain concentrations of NGQMs were 23 - 21546 ng/g. Maximum free brain concentrations were 0.5 to 267 ng/g. Seven (28%) and two (8%) compounds exhibited acceptable whole and free brain concentrations, respectively. Optimization of maximum free brain levels, IC90s (as a measure or potency) and residual plasma concentrations at 24 h (as a surrogate for half-life) in the same molecule may be feasible since they were not correlated. Diamine quinoline methanols were the most promising lead compounds. CONCLUSION: Reduction of CNS levels of NGQMs relative to mefloquine may be feasible. Optimization of this property together with potency and long half-life may be feasible amongst diamine quinoline methanols.

Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation.

BACKGROUND: Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of Plasmodium falciparum. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a head-to-head comparison of the in vitro invasive capabilities of parasites isolated by either of the two methods. METHODS: The P. falciparum laboratory strain isolate 7G8 was grown in vitro using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes. RESULTS: In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation (95% CI, p = 0.00067) A paired t test with two tails was used for these comparisons. CONCLUSIONS: In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation.

In vitro biotransformation, in vivo efficacy and pharmacokinetics of antimalarial chalcones.

4'-n-Butoxy-2,4-dimethoxy-chalcone (MBC) has been described as protecting mice from an otherwise lethal infection with Plasmodium yoelii when dosed orally at 50 mg/kg/dose, daily for 5 days. In contrast, we found that oral dosing of MBC at 640 mg/kg/dose, daily for 5 days, failed to extend the survivability of P. berghei-infected mice. The timing of compound administration and metabolic activation likely contribute to the outcome of efficacy testing in vivo. Microsomal digest of MBC yielded 4'-n-butoxy-4-hydroxy-2-methoxy-chalcone and 4'-(1-hydroxy-n-butoxy)-2,4-dimethoxy-chalcone. We propose that the latter will hydrolyze in vivo to 4'-hydroxy-2,4-dimethoxy-chalcone, which has greater efficacy than MBC in our P. berghei-infected mouse model and was detected in plasma following oral dosing of mice with MBC. Pharmacokinetic parameters suggest that poor absorption, distribution, metabolism and excretion properties contribute to the limited in vivo efficacy observed for MBC and its analogs.

In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones.

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.

Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum.

Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.

Anti-malarial activity of a non-piperidine library of next-generation quinoline methanols.

BACKGROUND: The clinical utility for mefloquine has been eroded due to its association with adverse neurological effects. Better-tolerated alternatives are required. The objective of the present study was the identification of lead compounds that are as effective as mefloquine, but exhibit physiochemical properties likely to render them less susceptible to passage across the blood-brain barrier. METHODS: A library of drug-like non-piperidine analogs of mefloquine was synthesized. These compounds are diverse in structure and physiochemical properties. They were screened in appropriate in vitro assays and evaluated in terms of their potential as lead compounds. The correlation of specific structural attributes and physiochemical properties with activity was assessed. RESULTS: The most potent analogs were low molecular weight unconjugated secondary amines with no heteroatoms in their side-chains. However, these compounds were more metabolically labile and permeable than mefloquine. In terms of physiochemical properties, lower polar surface area, lower molecular weight, more freely rotatable bonds and fewer H-bond acceptors were associated with greater potency. There was no such relationship between activity and LogP, LogD or the number of hydrogen bond donors (HBDs). The addition of an H-bond donor to the side-chain yielded a series of active diamines, which were as metabolically stable as mefloquine but showed reduced permeability. CONCLUSIONS: A drug-like library of non-piperidine analogs of mefloquine was synthesized. From amongst this library an active lead series of less permeable, but metabolically stable, diamines was identified.

Structure-activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum.

Utilizing mefloquine as a scaffold, a next generation quinoline methanol (NGQM) library was constructed to identify early lead compounds that possess biological properties consistent with the target product profile for malaria chemoprophylaxis while reducing permeability across the blood-brain barrier. The library of 200 analogs resulted in compounds that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Herein we report selected chemotypes and the emerging structure-activity relationship for this library of quinoline methanols.

Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquine.

Two novel SF5 analogs of the antimalarial agent mefloquine were synthesized in 5 steps and 10-23% overall yields and found to have improved activity and selectivity against malaria parasites. This work also represents the first report of SF5-substituted quinolines.

Effects of point mutations in Plasmodium falciparum dihydrofolate reductase and dihydropterate synthase genes on clinical outcomes and in vitro susceptibility to sulfadoxine and pyrimethamine.

BACKGROUND: Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G]) and septuplet (BR/51I/108N/164L and 437G/540E/581G) with geometric means of 76 nM (35-166 nM), 582 nM (49-6890- nM) and 4909 (3575-6741 nM) nM for sulfadoxine and 33 nM (22-51 nM), 81 nM (19-345 nM), and 215 nM (176-262 nM) for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L) or dihydropteroate synthase (540E) predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L) vs 23.7% (I164); relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E) vs 37.5% (K540); relative risk = 2.58; 95% CI: 1.88 - 3.73). Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to patients having both wild forms (I164 and K540). CONCLUSIONS: In this part of the Amazon basin, it may be possible to predict treatment failure with sulfadoxine-pyrimethamine equally well by determination of either of the single mutations dihydrofolate reductase 164L or dihydropteroate synthase 540E. TRIAL REGISTRATION: ClinicalTrials.gov NCT00951106.

Selective inhibition of Pfmrk, a Plasmodium falciparum CDK, by antimalarial 1,3-diaryl-2-propenones.

The cyclin dependent protein kinases, Pfmrk and PfPK5, most likely play an essential role in cell cycle control and differentiation in Plasmodium falciparum and are thus an attractive target for antimalarial drug development. Various 1,3-diaryl-2-propenones (chalcone derivatives) which selectivity inhibit Pfmrk in the low micromolar range (over PfPK5) are identified. Molecular modeling shows a pair of amino acid residues within the Pfmrk active site which appear to confer this selectivity. Predicted interactions between the chalcones and Pfmrk correlate well with observed potency. Pfmrk inhibition and activity against the parasite in vitro correlate weakly. Several mechanisms of action have been suggested for chalcone derivatives and our study suggests that kinase inhibition may be an additional mechanism of antimalarial activity for this class of compounds.

Targeting the fatty acid biosynthesis enzyme, beta-ketoacyl-acyl carrier protein synthase III (PfKASIII), in the identification of novel antimalarial agents.

The importance of fatty acids to the human malaria parasite, Plasmodium falciparum, and differences due to a type I fatty acid synthesis (FAS) pathway in the parasite, make it an attractive drug target. In the present study, we developed and a utilized a pharmacophore to select compounds for testing against PfKASIII, the initiating enzyme of FAS. This effort identified several PfKASIII inhibitors that grouped into various chemical classes of sulfides, sulfonamides, and sulfonyls. Approximately 60% of the submicromolar inhibitors of PfKASIII inhibited in vitro growth of the malaria parasite. These compounds inhibited both drug sensitive and resistant parasites and testing against a mammalian cell line revealed an encouraging in vitro therapeutic index for the most active compounds. Docking studies into the active site of PfKASIII suggest a potential binding mode that exploits amino acid residues at the mouth of the substrate tunnel.

Antimalarial activity of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors.

The antimalarial activity and pharmacology of a series of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors (HDACIs) was evaluated. In in vitro growth inhibition assays approximately 50 analogs were evaluated against four drug resistant strains of Plasmodium falciparum. The range of 50% inhibitory concentrations (IC(50)s) was 0.0005 to >1 microM. Five analogs exhibited IC(50)s of <3 nM, and three of these exhibited selectivity indices of >600. The most potent compound, WR301801 (YC-2-88) was shown to cause hyperacetylation of P. falciparum histones, which is a marker for HDAC inhibition in eukaryotic cells. The compound also inhibited malarial and mammalian HDAC activity in functional assays at low nanomolar concentrations. WR301801 did not exhibit cures in P. berghei-infected mice at oral doses as high as 640 mg/kg/day for 3 days or in P. falciparum-infected Aotus lemurinus lemurinus monkeys at oral doses of 32 mg/kg/day for 3 days, despite high relative bioavailability. The failure of monotherapy in mice may be due to a short half-life, since the compound was rapidly hydrolyzed to an inactive acid metabolite by loss of its hydroxamate group in vitro (half-life of 11 min in mouse microsomes) and in vivo (half-life in mice of 3.5 h after a single oral dose of 50 mg/kg). However, WR301801 exhibited cures in P. berghei-infected mice when combined at doses of 52 mg/kg/day orally with subcurative doses of chloroquine. Next-generation HDACIs with greater metabolic stability than WR301801 may be useful as antimalarials if combined appropriately with conventional antimalarial drugs.

Evaluation of broad spectrum protein kinase inhibitors to probe the architecture of the malarial cyclin dependent protein kinase Pfmrk.

We tested Pfmrk against several naphthalene and isoquinoline sulfonamides previously reported as protein kinase A (PKA) inhibitors. Pfmrk is a Cyclin Dependent protein Kinase (CDK) from Plasmodium falciparum, the causative parasite of the most lethal form of malaria. We find that the isoquinoline sulfonamides are potent inhibitors of Pfmrk and that substitution on the 5 position of the isoquinoline ring greatly influences the degree of potency. Molecular modeling studies suggest that the nitrogen atom in the isoquinoline ring plays a key role in ligand-receptor interactions. Structural analysis suggests that even subtle differences in amino acid composition within the active sites are responsible for conferring specificity of these inhibitors for Pfmrk over PKA.

Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening.

Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and approximately 0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z' range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [(3)H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r(2) >/= 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1.

BACKGROUND: The need for improved malaria diagnostics has long been recognized. METHODS: Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of (3)H-hypoxanthine-labeled parasites. RESULTS: The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r(2) = 0.923-0.968) with the parasitemia measured by (3)H-hypoxanthine incorporation assay when parasites variants incubated with various antimalarial drugs. In addition, the low levels of parasitemia (7.9%-21.3%) detected by microscopy than by FCM may be related to a number of tiny schizonts externally attached to the erythrocyte membranes but were not definitely inside the erythrocyte that normally would never be included in microscopy counting. CONCLUSION: On the basis of data reported herein, a rapid, high sensitivity, lower sampling error and reliable identification of human parasitized erythrocytes by the principles of FCM have been established. Published 2007 Wiley-Liss, Inc.