W2 Clone Research Articles

Chemometric modelling of antimalarial activity of aryltriazolylhydroxamates

We have performed quantitative structure–activity relationship (QSAR) and quantitative activity–activity relationship (QAAR) studies for aryltriazolylhydroxamates having antimalarial activity data against both chloroquine-sensitive (D6 clone) and chloroquine-resistant (W2 clone) strains of Plasmodium falciparum to understand the relationships between the biological activity and molecular properties for the design of new compounds. The QSAR studies were performed using 35 compounds among which 26 molecules were taken using k-means clustering technique in the training set for the derivation of the QSAR models and nine molecules were kept as the test-set compounds to evaluate the predictive ability of the derived models. The chemometric tool used for the analysis was the genetic function approximation. The developed models were analysed in terms of their predictive ability, and comparable results were obtained for cross-validated predictive variance (Q 2) and externally predicted variance (R 2 pred) values (0.761 and 0.829, respectively, for the D6 model, 0.708 and 0.748, respectively, for the W2 model and 0.984 and 0.982, respectively for the QAAR model). The QSAR models suggest that the number of methylene groups (between the triazolyl and hydroxamate moieties) and partially negatively charged surface areas of the molecules are important parameters for the antimalarial activity.

Antimalarial Activity ofAspilia pruliseta, a Medicinal Plant from Uganda

Aspilia pruliseta Schweinf. (Asteraceae) is a medicinal plant indigenous to Uganda and the neighboring countries of East Africa. It has been used extensively by the rural population for the treatment of fevers and malaria. During the antimalarial evaluation of this plant, four nontoxic diterpenes were isolated that possessed moderate activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) clones of Plasmodium falciparum, with IC(50) values ranging from 14 to 23 µM. These moderately active compounds included the previously undescribed diterpene, ENT-15 β-senecioyloxy-16,17-epoxy-kauran-18-oic acid that demonstrated an IC(50) value of 23.4 µM against clone D6, but was devoid of activity against clone W2. Four additional diterpenes were obtained from the aerial parts of A. pruliseta, but these known compounds were essentially inactive. The moderate activities of select diterpenes of A. pruliseta could account collectively for the historical and enduring use of this plant in traditional African medicine.

Identification of gut-associated amylase, cellulase and protease-producing bacteria in three species of Indian major carps

Isolation and enumeration of amylase, cellulase and protease-producing autochthonous bacteria in the proximal intestine (PI) and distal intestine (DI) of three species of Indian major carps, catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita), were investigated using the conventional culture-based technique. Population levels of amylolytic strains were the highest in the PI of catla and the lowest in the DI of rohu. The highest viable count of cellulase and protease-producing bacteria was recorded in the DI and PI of mrigal respectively. Among the bacteria isolated, 10 strains (five from PI and five from DI) were selected as potent enzyme producers according to a quantitative enzyme assay. The chosen strains were further identified by 16S rRNA gene sequence analysis. The five strains isolated from catla showed high similarity to Citrobacter sp. clone W2, Enterobacter sp. JA24, Bacillus coagulans strain TR, uncultured bacterial clone Hel3bc04 and Bacillus cereus strain UST2006-BC004. The four strains isolated from mrigal were most closely related to Bacillus sp. KCd2, uncultured bacterial clone Hel3bd09, B. cereus strain BU040901-020 and Citrobacter freundii strain YRL11, while the strain isolated from rohu probably belonged to Bacillus sp. GV.

Constituents of Baccharis dracunculifolia DC (Asteraceae) with in vitro antileishmanial, antiplasmodial and cytotoxic activities

Baccharis dracunculifolia D.C. (Asteraceae) is the most important plant source of the Brazilian green propolis. The aim of this work was to evaluate the antileishmanial and antiplasmodial activities of B. dracunculifolia and its isolated compounds. The leave rinse extract of B. dracunculifolia (BdE) showed in vitro antileishmanial activity against Leishmania donovani, displaying an IC50 value of 45µg/mL, while green propolis hydroalcoholic extract (GPE) showed an IC50 value of 49µg/mL. Among the isolated compounds, ursolic acid and hautriwaic acid lactone, showed the highest antileishmanial activities, displaying IC50 values of 3.7µg/mL and 7.0µg/mL, respectively. The pentacyclic triterpene Uvaol, as well as the flavonoids acacetin and ermanin showed IC50 values of 15.0µg/mL, 18.0µg/mL and 40.0µg/mL, respectively. Regarding the antiplasmodial assay against Plasmodium falciparum, BdE and GPE showed similar IC50 values (about 20µg/mL). Hautriwaic acid lactone displayed moderate antiplasmodial activity, with IC50 values of 0.8µg/mL (D6 clone) and 2.2µg/mL (W2 clone). In order to compare the effect on the parasites with toxicity with mammalian cells, the cytotoxic activity of the samples were evaluated against the Vero cells, showing that all evaluated compounds exhibited no cytotoxicity in the maximum dose tested.

Constituents of Leonotis leonurus flowering tops

Phytochemical investigation of flowering tops of Leonotis leonurus, yielded a new diterpene ester, 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecan-1-yl-palmitate along with five known metabolites. The structures of all compounds were determined by spectroscopic methods including 1D- and 2D NMR spectroscopy. All the isolated compounds were evaluated for antimalarial, cytotoxicity and for antimicrobial activities. Antimalarial activity for luteolin 7- O-β- d-glucopyranoside ( 4) (IC 50 = 2.2 μg/mL for the D6 clone and 1.8 μg/mL for the W2 clone) was observed. Chloroquine and artemisinin were used as positive controls which showed IC 50 of 0.016 and 0.0048 μg/mL for the D6 clone, respectively, and IC 50 of 0.14 and 0.0047 μg/mL for the W2 clone, respectively. None of the compounds were cytotoxic to Vero cells up to a concentration of 4.76 μg/mL.

Synthesis and Antimalarial Activities of Cyclen 4-Aminoquinoline Analogs

In an attempt to augment the efficacy of 7-chloro 4-aminoquinoline analogs and also to overcome resistance to antimalarial agents, we synthesized three cyclen (1,4,7,10-tetraazacyclododecane) analogs of chloroquine [a bisquinoline derivative, 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline HBr, and a 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline-Zn(2+) complex]. The bisquinoline displays the most potent in vitro and in vivo antimalarial activities. It displays 50% inhibitory concentrations (IC(50)s) of 7.5 nM against the D6 (chloroquine-sensitive) clone of Plasmodium falciparum and 19.2 nM against the W2 (chloroquine-resistant) clone, which are comparable to those of artemisinin (10.6 and 5.0 nM, respectively) and lower than those of chloroquine (10.7 and 87.2 nM, respectively), without any evidence of cytotoxicity to mammalian cells, indicating a high selectivity index (>1,333 against D6 clone and >521 against W2 clone). Potent antimalarial activities of the bisquinoline against chloroquine- and mefloquine-resistant strains of P. falciparum were also confirmed by in vitro [(3)H]hypoxanthine incorporation assay. The in vivo antimalarial activity of the bisquinoline, as determined in P. berghei-infected mice, is comparable to that of chloroquine (50% effective dose, <or=1.1 mg/kg when given orally); no apparent toxicity has been observed up to the highest dose tested (3 x 30 mg/kg). The bisquinoline inhibits in vitro hemozoin (beta-hematin) formation with an IC(50) of 1.1 microM, which is about 10-fold more potent than chloroquine (IC(50) 9.5 microM). Overall, this article describes the discovery of a new class of cyclen 4-aminoquinoline analogs as potent antimalarial drugs.

Synthesis, Antimalarial Activity, and Intracellular Targets of MEFAS, a New Hybrid Compound Derived from Mefloquine and Artesunate

A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca(2+) at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H(+) pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria.

In vitro antileishmanial and antimalarial activities of tetrahydrofuran lignans isolated from Nectandra megapotamica (Lauraceae)

Seven tetrahydrofuran lignans, isolated from Nectandra megapotamica (Lauraceae), were evaluated for their in vitro antileishmanial and antimalarial activities. Among the evaluated compounds, machilin-G (1a) and veraguensin (2a) showed the highest antileishmanial activities, displaying for both compounds an IC(50) value of 18 microg/mL and an IC(90) value of 36 microg/mL, while galgravin (1b), nectandrin-A (1c), nectandrin-B (1d), calopeptin (2b) and ganshisandrine (3) were inactive against Leishmania donovani. In the antimalarial assay against Plasmodium falciparum, it was observed that calopeptin (2b) displayed moderate activity, with IC(50) values of 3800 ng/mL (D6 clone) and 3900 ng/mL (W2 clone), while the lignans 1a-1d, 2a and 3 were inactive. In order to compare the effect on the parasites with toxicity to mammalian cells, the cytotoxic activity of the isolated compounds were evaluated against the Vero cells, showing that all evaluated tetrahydrofuran lignans exhibited no cytotoxicity at the maximum dose tested.

Antileishmanial, Antimalarial and Antimicrobial Activities of the Extract and Isolated Compounds from Austroplenckia populnea (Celastraceae)

Austroplenckia populnea (Celastraceae), known as "marmelinho do campo", is used in Brazilian folk medicine as antimicrobial, anti-inflammatory, and antitumoural agent. The aim of the present work was to evaluate the antimicrobial, antileishmanial and antimalarial activities of the crude hydroalcoholic extract of A. populnea (CHE) and some of its isolated compounds. The phytochemical study of the CHE was carried out affording the isolation of methyl populnoate (1), populnoic acid (2), and stigmast-5-en-3-O-beta-(D-glucopyranoside) (3). This is the first time that the presence of compound 3 in A. populnea is reported. The results showed that the CHE presents antifungal and antibacterial activities, especially against Candida glabrata and Candida albicans, for which the CHE showed IC50 values of 0.7 microg mL(-1) and 5.5 microg mL(-1), respectively, while amphotericin B showed an IC50 value of 0.1 microg mL(-1) against both microorganisms. Compounds 1-3 were inactive against all tested microorganisms. In the antileishmanial activity test against Leishmania donovani, the CHE showed an IC50 value of 52 microg mL(-1), while compounds 2 and 3 displayed an IC50 value of 18 microg mL(-1) In the antimalarial assay against Plasmodium falciparum (D6 and W2 clones), it was observed that all evaluated samples were inactive. In order to compare the effect on the parasites with the toxicity to mammalian cells, the cytotoxicity activity of the isolated compounds was evaluated against Vero cells, showing that all evaluated samples exhibited no cytotoxicity at the maximum dose tested.

New Labdane Diterpenes fromLeonurus cardiaca

Leonurus cardiaca L. has been used in oriental medicine against several types of disorders. The ethanolic extract of leaves of Leonurus cardiaca yielded three new labdane-type diterpenes: 15- O-ethylleopersin C (1), 15- O-methylleopersin C (2), and 15- EPI- O-methylleopersin C (3). Their structures were determined using 1 D and 2 D NMR including 1H-1H COSY, HMQC, HMBC, and ROESY spectroscopic techniques. Compounds (1 - 3) were evaluated for in vitro antiplasmodial activity (D6 and W2 clones) and cytotoxicity (Vero cells).

Synthesis and antimalarial activity of hydroxyethylpiperazine derivatives

The antimalarial activity of hydroxyethylpiperazine derivatives, synthesized from the reaction of (2 S,3 S)Boc-phenylalanine epoxide with benzylpiperazines in good yields (76–96%), has been evaluated in vitro against the Plasmodium falciparum W2 clone (chloroquine resistant). The results show that some compounds have moderate activity against this parasite and none of the active compounds showed cytotoxicity at high concentration (100 μg/ml).

Constituents of Nelumbo nucifera leaves and their antimalarial and antifungal activity

From the leaves of Nelumbo nucifera (an aquatic plant), one new compound, 24( R)-ethylcholest-6-ene-5α-ol-3- O-β- d-glucopyranoside ( 1), along with 11 known metabolites ( 2– 12), were isolated and identified by spectroscopic methods including 1D- and 2D NMR. Antifungal activity for ( R)-roemerine ( 3) (IC 50/MIC = 4.5/10 μg/mL against Candida albicans) and antimalarial activity for ( R)-roemerine ( 3) and N-methylasimilobine ( 5) (IC 50 = 0.2 and 4.8 μg/mL for the D6 clone, respectively, and 0.4 and 4.8 μg/mL for the W2 clone, respectively) was observed. None of the compounds were cytotoxic to Vero cells up to a concentration of 23.8 μg/mL. NMR data for 10-eicosanol ( 7) and 7,11,15-trimethyl-2-hexadecanone ( 10) are presented for the first time. An analysis of the structure–activity relationship shows that the substituents in position C-1 and C-2 of aporphine alkaloids are crucial for the antimalarial activity.

Constituents of Leonotis leonurus Flowering Tops

Leonotis leonurus L., also known as Lion's Tail or Wild Dagga, is a member of the Mint family (Lamiaceae). The plant is reported to have anticonvulsant [1], antinociceptive, anti-inflammatory and hypoglycemic activities [2]. Several labdane diterpenoids were reported in the leaves and aerial parts. However, no work has been reported on the flowers of this plant. A phytochemical study of the flowering tops of L. leonurus yielded a new diterpene ester 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecan-1-palmitate (1) along with providing new source for five known compounds identified as succinic acid (2), uracil (3), luteolin 7-O-glucoside (4), acteoside (5), and geniposidic acid (6). The structures of 1–6 were determined by spectroscopic methods including 1D- and 2D-NMR, IR, UV, MS etc. All six compounds were evaluated for their antimalarial and cytotoxic activities. Antimalarial activity was observed only for compound 4 (IC50 s = 2.2 µg/mL for D6 clone, and 2.6 µg/mL for W2 clone) [3]. Cytotoxicity was not observed for any of these compounds up to a concentration of 4.76 µg/mL. Acknowledgements: This work was supported in part by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No 58-6408-2-0009 and Grant Number P20RR021929 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

Chemical transformation and biological studies of marine sesquiterpene ( S)-(+)-curcuphenol and its analogs

Chemical transformation studies of the marine sesquiterpene phenol ( S)-(+)-curcuphenol ( 1), isolated from the Jamaican sponges Myrmekioderma styx, were accomplished. In order to optimize the activity and better understand the SAR of ( S)-(+)-curcuphenol, nineteen semisynthetic analogs were prepared and evaluated for activity against infectious diseases. A number of analogs showed significant activity against Mtb and Leishmania donovani, while showed good to moderate activities in antibacterial and antifungal assays as well as against Plasmodium falciparium (D6 clone) and (W2 clone). The analogs a, c, h, and r exhibited Mtb activity with MICs of 24.6, 41.2, 6.90, and 50.5 μM, respectively. Analog f showed enhanced activity against L. donovani with an IC 50 of 0.6 μM and IC 90 of 40 μM respectively.

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

ObjectiveThe main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine.MethodsThe study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake.ResultsFrom 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2.ConclusionBased on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.

Antioxidant, Antimalarial and Antimicrobial Activities of Tannin-Rich Fractions, Ellagitannins and Phenolic Acids from Punica granatum L.

The Punica granatum L. (pomegranate) by-product POMx was partitioned between water, EtOAc and n-BuOH, and the EtOAc and n-BuOH extracts were purified by XAD-16 and Sephadex LH-20 column chromatography to afford ellagic acid (1), gallagic acid (2), punicalins (3), and punicalagins (4). Compounds 1 - 4 and the mixture of tannin fractions (XAD-16 eluates) were evaluated for antioxidant, antiplasmodial, and antimicrobial activities in cell-based assays. The mixture of tannins (TPT), XAD-EtOAc, XAD-H2O, XAD-PJ and XAD-BuOH, exhibited IC50 values against reactive oxygen species (ROS) generation at 0.8 - 19 microg/mL. Compounds 1 - 4 showed IC50 values of 1.1, 3.2, 2.3 and 1.4 microM, respectively, against ROS generation and no toxicity up to 31.25 microg/mL against HL-60 cells. Gallagic acid (2) and punicalagins (4) exhibited antiplasmodial activity against Plasmodium falciparum D6 and W2 clones with IC50 values of 10.9, 10.6, 7.5 and 8.8 microM, respectively. Fractions XAD-EtOAc, XAD-BuOH, XAD-H2O and XAD-PJ compounds 1 - 4 revealed antimicrobial activity when assayed against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Cryptococcus neoformans, methicillin-resistant Staphylococcus aureus (MRSA), Aspergillus fumigatus and Mycobacterium intracellulare. Compounds 2 and 4 showed activity against P. aeruginosa, C. neoformans, and MRSA. This is the first report on the antioxidant, antiplasmodial and antimicrobial activities of POMx isolates, including structure-activity relationships (SAR) of the free radical inhibition activity of compounds 1 - 4. Our results suggest a beneficial effect from the daily intake of POMx and pomegranate juice (PJ) as dietary supplements to augment the human immune system's antioxidant, antimalarial and antimicrobial capacities.

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-( tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2 S/2 R)-2-aminosubstitutedamides ( 21– 33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2 S/2 R)-2-aminosubstitutedamides ( 51– 63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs ( 21– 24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21– 24, 26– 32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.

The effects of α1-acid glycoprotein on the reversal of chloroquine resistance inPlasmodium falciparum

An in-vitro model based on the semi-automated microdilution technique has been developed for selecting compounds that might be used clinically for the reversal of chloroquine resistance. This was used initially to test the susceptibility of Plasmodium falciparum clone W2 to chloroquine (CQ). The model was then employed to investigate the effects of each of four resistance-reversing agents (verapamil, desipramine, chlorpheniramine and promethazine, at 1 microM) on this parasite's susceptibility to CQ, with and without alpha(1)-acid glycoprotein (AGP), at a patho-physiological concentration (1.25 g/litre), in the culture medium. In the absence of AGP, each of the resistance-reversing agents reduced the median inhibitory concentrations of CQ by 82%-97%, from a baseline value of about 94 ng/ml. In the presence of AGP, however, most of the resistance-reversing agents had much less effect. There appears to be competitive interaction between CQ, the resistance-reversing agents and AGP. The binding kinetics between CQ, resistance-reversing agents, AGP and other plasma proteins will clearly need to elucidated if clinically effective resistance-reversing agents are to be selected in vitro.

Indoloquinazoline alkaloids from Araliopsis tabouensis

Three new indoloquinazolidine-type alkaloids, 2-methoxy-8-13-dihydro-7H-indolo[pyrido[2,1-b]quinazolin-5-one (1) , 2-methoxy-13-methyl-8-13-dihydro-7H-indolo[2',3',3,4]pyrido[2,1-b]quinazolin-5-one (2) , and 2-methoxy-14-methyl-7,8,13,14-tetrahydroindolo[2',3',4]pyrido[2,l-b]quinazolin-5-one (3) were isolated from Araliopsis tabouensis, together with three known compounds. The structures of the new compounds were determined primarily from 1D- and 2D-NMR spectroscopy. The antimalarial activities of compounds 1-5 were evaluated against Plasmodium falciparum D6 and W2 clones. The IC 50 values in antimalarial bioassay for compounds 2-5 varied from 1.8 to 4.7 μg/ml. Nig. J. Pharm. Res. 3(1) 2004: 42-48

Alkamides from the Leaves of Zanthoxylum syncarpum

Three alkamides (1-3) were isolated from the leaves of Zanthoxylum syncarpum. The structures of the new compounds 1 and 2 were established by spectroscopic data and chemical conversion, and by the X-ray crystallography of 1. Compound 3, the racemic form of the known compound syncarpamide, showed moderate antiplasmodial activity, with IC50 values of 4.2 and 6.1 microM against Plasmodium falciparum D6 clone and W2 clone, respectively.