Novel Benzotriazole-ß-lactam Derivatives as Antimalarial Agents: Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Many people around the world suffer from malaria, especially in tropical or subtropical regions. While malaria medications have shown success in treating malaria, there is still a problem with resistance to these drugs. Herein, we designed and synthesized some structurally novel benzotriazole-ß-lactams using 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid as a key intermediate. To synthesize the target molecules, the ketene-imine cycloaddition reaction was employed. First, The reaction of 1H-benzo[d][1,2,3]triazole with 2-bromoacetic acid in aqueous sodium hydroxide yielded 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid. Then, the treatment of 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid with tosyl chloride, triethyl amine, and Schiff base provided new ß-lactams in good to moderate yields.The formation of all cycloadducts was confirmed by elemental analysis, FT-IR, NMR and mass spectral data. Moreover, X-ray crystallography was used to determine the relative stereochemistry of 4a compound. The in vitro antimalarial activity test was conducted for each compound against P. falciparum K1. The IC50 values ranged from 5.56 to 25.65 µM. A cytotoxicity profile of the compounds at 200 µM final concentration revealed suitable selectivity of the compounds for malaria treatment. Furthermore, the docking study was carried out for each compound into the P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site to analyze their possible binding orientation in the active site.

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria.

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7-23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14-16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7-23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal "dual action" for ß-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Antimalarial Activity of Human Group IIA Secreted Phospholipase A2 in Relation to Enzymatic Hydrolysis of Oxidized Lipoproteins.

The level of human group IIA secreted phospholipase A2 (hGIIA sPLA2) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA2s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA2 was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA2s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA2 is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA2s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA2 Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA2, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Design and synthesis of simplified speciophylline analogues and ß-carbolines as active molecules against Plasmodium falciparum.

A structure-activity relationship study of active molecules against chloroquine-resistant Plasmodium falciparum K1 strain is reported. Structurally simplified analogues of antiplasmodial active alkaloids presented similar levels of activity as their corresponding natural products extracted from Guiera senegalensis and Mitragyna inermis with IC50 values on chloroquine-resistant P. falciparum K1 strain of up to 10.6 µM for spirooxindoles and 13.8 µM for ß-carbolines. The identification of such simpler and cheaper structural analogues is crucial to efficiently study these natural products' action mode as well as developing new cures against malaria.

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in the Democratic Republic of the Congo: emergence of highly resistant pfdhfr/pfdhps alleles.

Background: In 2005, the Democratic Republic of the Congo (DRC) switched to artesunate/amodiaquine as the first-line antimalarial in response to increasing sulfadoxine/pyrimethamine resistance and adopted intermittent preventive treatment using sulfadoxine/pyrimethamine in pregnancy. Objectives: To determine the prevalence of molecular markers of sulfadoxine/pyrimethamine resistance in southwestern DRC 10 years after the new policy was instituted. Methods: From March 2014 to December 2015, blood samples were collected from symptomatic patients presenting to outpatient centres in urban and rural areas. A total of 2030 confirmed Plasmodium falciparum isolates were genotyped at codons associated with sulfadoxine/pyrimethamine resistance. Results: The prevalence of pfdhfr-N51I, C59R and S108N and pfdhps-A437G mutations was consistently high; the prevalence of the pfdhps-K540E mutation was low but increased since its first report in 2008 in the same region, reaching 17.6% by 2015. The pfdhps-A581G mutation increased from ∼4.5% in 2014 to ∼14.0% in 2015 at urban sites while in rural areas it remained low (∼4.0%). The mutations pfdhfr-I164L and pfdhps-A613S were detected for the first time in DRC. Also, 11 (0.8%) isolates revealed the presence of the newly described pfdhps-I431V mutation. Combining pfdhfr and pfdhps alleles, quintuple and sextuple mutations were observed, with the emergence of septuple (IRNI/IAGEGA)- and octuple (IRNI/VAGKGS)-mutant genotypes. Conclusions: Intermittent preventive treatment using sulfadoxine/pyrimethamine during pregnancy remains warranted in southwestern DRC. However, the expansion of pfdhps-K540E mutation and emergence of mutants that cause higher levels of sulfadoxine/pyrimethamine resistance is concerning and may present a challenge for future preventive interventions in the country.

Past and new challenges for malaria control and elimination: the role of operational research for innovation in designing interventions.

This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response.

Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis.

High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Development of New Antimicrobial Oleanonic Acid Polyamine Conjugates.

A series of oleanolic acid derivatives holding oxo- or 3-N-polyamino-3-deoxy-substituents at C3 as well as carboxamide function at C17 with different long chain polyamines have been synthesized and evaluated for antimicrobial activities. Almost all series presented good to moderate activity against Gram-positive S. aureus, S. faecalis and B. cereus bacteria with minimum inhibitory concentration (MIC) values from 3.125 to 200 µg/mL. Moreover, compounds possess important antimicrobial activities against Gram-negative E. coli, P. aeruginosa, S. enterica, and EA289 bacteria with MICs ranging from 6.25 to 200 µg/mL. The testing of ability to restore antibiotic activity of doxycycline and erythromycin at a 2 µg/mL concentration in a synergistic assay showed that oleanonic acid conjugate with spermine spacered through propargylamide led to a moderate improvement in terms of antimicrobial activities of the different selected combinations against both P. aeruginosa and E. coli. The study of mechanism of action of the lead conjugate 2i presenting a N-methyl norspermidine moiety showed the effect of disruption of the outer bacterial membrane of P. aeruginosa PA01 cells. Computational ADMET profiling renders this compound as a suitable starting point for pharmacokinetic optimization. These results give confidence to the successful outcome of bioconjugation of polyamines and oleanane-type triterpenoids in the development of antimicrobial agents.

Differential association of Plasmodium falciparum Na+/H+ exchanger polymorphism and quinine responses in field- and culture-adapted isolates of Plasmodium falciparum.

Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na(+)/H(+) exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC(50)) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.

Polymorphism of Plasmodium falciparum Na(+)/H(+) exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam.

BACKGROUND: The Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam. METHOD: Ninety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay. RESULTS: Significant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC(50) of 682 nM versus median IC(50) of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC(50) of 704 nM versus median IC(50) of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC(50) showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7. CONCLUSIONS: The haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.

High-performance liquid chromatographic method for the quantification of Mitragyna inermis alkaloids in order to perform pharmacokinetic studies.

In Africa, Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) is commonly used in traditional medicine to treat malaria. Antimalarial activity is mostly due to the hydromethanolic extract of M. inermis leaves and especially to the main alkaloids, uncarine D and isorhynchophilline. In the present study, we describe for the first time an HPLC method for the simultaneous quantification of uncarine D and isorhynchophylline in biological matrices. SPE was used to extract the components and the internal standard naphthalene from human and pig plasma samples. Chromatographic separation was performed on a C-18 reversed column at a flow rate of 1 mL/min, using methanol-phosphate buffer (10:90, pH 7), as a mobile phase. Good linearity was observed over the concentration ranges of 0.0662-3.31 microg/mL for uncarine D and 0.0476-2.38 microg/mL for isorynchophylline. The precision was less than 12% and the accuracy was from 86 to 107% without any discrepancy between the two species. Uncarine D and isorhynchophylline recoveries were over 80%. These results allowed the quantification of both uncarine D and isorhynchophylline in pig plasma after intravenous administration of M. inermis extract.

Synthesis, characterization, and in vitro antimalarial and antitumor activity of new ruthenium(II) complexes of chloroquine.

The new Ru(II) chloroquine complexes [Ru(eta(6)-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(eta(6)-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(eta(6)-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(eta(6)-p-cymene)(eta(6)-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: In 1-4, chloroquine binds to ruthenium in the eta(1)-N mode through the quinoline nitrogen atom, whereas in 5 an unprecedented eta(6) bonding through the carbocyclic ring is observed. 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1, and W2) and CQ-sensitive (FcB1, PFB, F32, and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to 1 (IC50 8 microM); this is significant because this type of tumor does not respond to currently employed chemotherapies.

Diastereoselective Synthesis of Potent Antimalarial Cis-ß-lactam Agents.

Fifteen novel ß-lactams bearing N-ethyl tert-butyl carbamate group 5a-o and fifteen N-(2- aminoethyl) ß-lactams 6a-o were synthesized by [2+2] ketene-imine cycloaddition reaction (Staudinger). The cycloaddition reaction was found to be totally diastereoselective leading exclusively to theformation of cis-ß-lactam derivatives. These newly synthesized ß-lactams were evaluated for their antimalarial activity against p. falciparum K14 resistant strain and showed good to excellent EC50 values. Of the thirty ß-lactams tested, 5 h, 6a and 6c showed IC50 < 20 µM while 5b, 5c, 5e, 5f, 5g, 5i, 5j, 6d, 6g and 6h exhibited IC50 <50 . Compounds 5c, 5h, and 5q-t were examined for their anticancer properties against K562 Leukemia cell line and 5s showed the best activity. Compounds 3a-j, 5a-o, 6a-o, were tested against S. aureus , E. coli, C. albicans and showed no activity below 125 µg/mL.

Diversity and biting patterns of Anopheles species in a malaria endemic area, Umphang Valley, Tak Province, western Thailand.

Malaria is highly endemic in Umphang Valley, a district in the western edge of Tak Province, along the boundary with Kayin State of Myanmar. Although there are high indigenous malaria cases in this area every year, nothing about malaria vectors and their transmission role have been investigated before this study. The objective of this work is to characterize the Anopheles species diversity and trophic behavior of malaria vectors in the transmission area of Umphang Valley. Females of Anopheles mosquitoes were collected every two months during a two-year period. Mosquito collections were using standard collection technique, indoor and outdoor human landing collections and outdoor cattle bait collection. Anopheles mosquitoes were identified using morphological characters and multiplex AS-PCR assay for the identification of sibling species within groups and complexes present. From a total of 16,468 Anopheles females, 2723 specimens (16.54%) were collected from humans and 13,745 specimens (83.46%) were captured from cattle. From human landing collections, 2447 specimens (89.86%) of Anopheles minimus were obtained, followed by 119 Anopheles peditaeniatus (4.37%), 62 Anopheles maculatus (2.28%), 17 Anopheles dirus (0.6%), 15 Anopheles aconitus (0.5%) and 6 Anopheles sawadwongporni (0.2%) respectively. Seven putative malaria vectors, including An. minimus, An. dirus, An. baimaii, An. sawadwongporni, An. maculatus, An. pseudowillmori and An. aconitus were documented from this study and trophic behavior of each respective species were observed. Such information is definitely crucial for defining the vector capacity of each single species and for designing appropriate vector prevention and control strategies against target vector species.

Synthesis of New ß-Lactams Bearing the Biologically Important Morpholine Ring and POM Analyses of Their Antimicrobial and Antimalarial Activities.

Some new ß-lactams bearing biologically important morpholine ring have been synthesized by acylation of amino ß-lactams in the presence of morpholine-4-carbonyl chloride. These novel ß-lactams were prepared under mild reaction conditions without any solvent in short reaction times. Their biological activities have been examined against microbial agents such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and fungi such as Candida albicans (C. albicans) and Candida glabrata (C. glabrata). They have been also tested against Plasmodium falciparum K14 resistant strain and showed moderate to good IC50 values.

Pharmacokinetics of artesunate in the domestic pig.

OBJECTIVES: The aim was to study the pharmacokinetic profile of artesunate and its metabolite dihydroartemisinin (DHA) in a pig model. METHODS: Thirteen pigs received either intravenous (iv) or intramuscular (im) artesunate (60 mg), with the alternative preparation given 24 h later in an open crossover design. Five of them also received an additional intra-arterial (ia) artesunate dose (60 mg). The plasma concentrations of artesunate and DHA were determined by high-performance liquid chromatography with electrochemical detection. Population modelling was performed with NONMEM, using a two-compartment model. RESULTS: Plasma concentration-time profiles were comparable to those observed in humans, with a rapid and biphasic decline for both artesunate and DHA. Following an iv bolus, artesunate had a median maximum plasma concentration (C(max)) of 13.8 microM [interquartile range (IQR), 10.4-22.1 microM], elimination half-life (t(1/2)) = 18 min (IQR, 16-22 min), total plasma clearance (CL) = 5.58 L/h/kg (IQR, 3.31-5.91 L/h/kg) and volume of distribution (V(d)) = 1.85 L/kg (IQR, 1.27-3.20 L/kg). The median C(max) value for DHA was 3.30 microM (IQR, 2.08-5.95 microM), t(1/2) = 26 min (IQR, 23-31 min), CL/Fm = 4.37 L/h/kg (IQR, 3.29-6.87 L/h/kg) and V(d)/Fm = 2.56 L/kg (IQR, 1.93-4.49 L/kg). Artesunate and DHA pharmacokinetic parameters were similar after ia administration. Following im dosing, median artesunate C(max) was 4.81 microM (IQR, 3.74-5.40 microM), t(1/2) = 18 min (IQR, 16-28 min), CL = 4.37 L/h/kg (IQR, 4.13-4.68 L/h/kg) and V(d) = 2.07 L/kg (IQR, 1.83-2.79 L/kg); the bioavailability was 100%. For DHA, median C(max) was 1.43 microM (IQR, 1.00-1.92 microM), t(1/2) = 27 min (IQR, 25-37 min), CL/Fm = 4.68 L/h/kg (IQR, 3.35-6.73 L/h/kg) and V(d)/Fm = 3.31 L/kg (IQR, 2.89-4.27 L/kg). CONCLUSIONS: The pharmacokinetic properties of artesunate and DHA in pigs were similar to those reported in humans, suggesting that the swine model is suitable for determining the preclinical pharmacokinetics of artemisinin derivatives.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: synthesis, and in vitro antimalarial activity.

Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1-2 were synthesized from various substituted nitroanilines and tested for in vitro activity upon the erythrocytic development of Plasmodiumfalciparum strains with different chloroquine-resistance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6-8 steps through a regioselective palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(aminopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quinoxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)(3). The best results were observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected compounds 1b, 1f-h, 1l and 2a were tested for their ability to inhibit beta-haematin formation, the synthetic equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested compounds, only 2a showed a beta-haematin formation inhibition, but no inhibition of haem polymerization was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b, 1f-h and 1l, as the IC(50) values were superior to 10 equivalents.

Synthesis and biological evaluation of some novel diastereoselective benzothiazole ß-lactam conjugates.

Highly diastereoselective synthesis of some novel benzothiazole-substituted ß-lactam hybrids was achieved starting from (benzo[d]thiazol-2-yl)phenol as an available precursor. This is the first time (benzo[d]thiazol-2-yl)phenoxyacetic acid has been used as ketene source in synthesizing monocyclic 2-azetidinones. These compounds were evaluated for their antimicrobial activities against a large panel of Gram-positive and Gram-negative bacterial strains and moderate activities were encountered. Antimalarial data revealed that adding methoxyphenyl or ethoxyphenyl group on the ß-lactam ring makes compounds that are more potent. Moreover, hemolytic activity and mammalian cell toxicity survey of the compounds showed their potential as a medicine.

Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travellers.

Real-time PCR diagnosis of malaria has advantages over traditional microscopic methods, especially when parasitaemia is low and when dealing with mixed infections. We have developed a new real-time PCR with specific genes in each Plasmodium species present only in one copy to identify the four pathogenic Plasmodium spp. for humans. The sensitivity was less than 25 parasites/microl. No cross-hybridisation was observed with human DNA or among the four Plasmodium spp. Using LightCycler PCR and conventional microscopy, we compared the diagnosis of malaria in patients from Vietnam and in returned European travellers with suspicion of malaria. In patients from Vietnam with suspicion of malaria, one mixed infection was observed by PCR only; the remaining data (54 of 55 patients) correlated with microscopy. In 79 patients without symptoms, low parasitaemia was detected in 7 samples by microscopy and in 16 samples by PCR. In returned travellers, PCR results were correlated with microscopy for all four species in 48 of 56 samples. The eight discrepant results were resolved in favour of real-time PCR diagnosis. This new real-time PCR is a rapid, accurate and efficient method for malaria diagnosis in returned travellers as well as for epidemiological studies or antimalarial efficiency trials in the field.