Synthesis and antimalarial activity in vitro and in vivo of a new ferrocene-chloroquine analogue

The antimalarial activities of ferrocenic compounds mimicking chloroquine and active upon chloroquine-resistant strains of Plasmodium falciparum were evaluated. Four 7-chloro-4-[[[2-[(N,N-substituted amino)methyl]ferrocenyl]methyl]amino]quinoline derivatives have been synthesized; one of them, 1a, showed high potent antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and was 22 times more potent against schizontocides than chloroquine in vitro against a drug-resistant strain of P. falciparum.     

Antimalarial activity of novel arylene bis(methylketone) compounds

Because of the spread of drug-resistant Plasmodium species, there is an urgent need for novel effective antimalarial agents. A series of arylene bis(methylketone) compounds were screened in vitro against a number of Plasmodium falciparum clones and in vivo against Plasmodium berghei. 2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (Cytokine Network Inc. [CNI]-H0294) was the most effective of the compounds in vitro, with an IC50 of 1.5-4.0 microM against parasite clones with a wide range of sensitivities to chloroquine and pyrimethamine. Other compounds in the series had in vitro IC50 values of 20-25 microM. In a 4-day test for suppression of P. berghei parasitemia in vivo, 50 mg/kg/day CNI-H0294 significantly decreased parasitemia by >90%. The compound was found to have low toxicity in mice, with an LD50 of 590 +/- 66 mg/kg intraperitoneally, and rapid plasma kinetics. These results show that CNI-H0294 has considerable antimalarial activity and merits further study.     

The novel oxygenated chalcone, 2,4-dimethoxy-4'-butoxychalcone, exhibits potent activity against human malaria parasite Plasmodium falciparum in vitro and rodent parasites Plasmodium berghei and Plasmodium yoelii in vivo

Previous studies have shown that licochalcone A, an oxygenated chalcone, exhibits antileishmanial and antimalarial activities. The present study was designed to examine the antimalarial activity of an analog of licochalcone A, 2,4-dimethoxy-4'-butoxychalcone (2,4mbc). 2,4mbc inhibited the in vitro growth of both a chloroquine-susceptible (3D7) and a chloroquine-resistant (Dd2) strain of Plasmodium falciparum in a [3H]hypoxanthine uptake assay. The in vivo activity of 2,4mbc was tested in mice infected with Plasmodium berghei or Plasmodium yoelii and in rats infected with P. berghei. 2,4mbc administered either orally, intraperitoneally, or subcutaneously for 5 days protected the mice from otherwise lethal infections of these parasites. 2,4mbc administered orally for 5 days reduced parasitemia in the rats infected with P. berghei. These results demonstrate that 2,4mbc exhibits potent antimalarial activity and might be developed into a new antimalarial drug.     

Synergism of cimetidine with anti-malarial agents

The histamine type-2 receptor antagonist and cytochrome P450 inhibitor cimetidine was examined for its antimalarial properties in the presence or absence of chloroquine or pyrimethamine. When used alone, cimetidine displayed little activity against a number of Plasmodium falciparum clones in vitro, with an IC50 of 300-700 microM. The compound was found to be highly synergistic in combination with chloroquine and also displayed a degree of synergism when used in combination with pyrimethamine. These synergistic effects were independent of the chloroquine- or pyrimethamine-resistance status of the clones. The cytochrome P450 inhibitor proadifen displayed weak synergism or antagonism with chloroquine, depending on the clone tested, and clear antagonism with pyrimethamine. The results with proadifen indicate that cimetidine was exerting its synergistic activity via a mechanism distinct from inhibition of cytochrome P450. Additional experiments have demonstrated that cimetidine interferes with neither plasmodial sterol metabolism nor heme polymerization.     

Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum

A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions. 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices. A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods. Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position. In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM). Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme. The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM. It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above. In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM. Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay. The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P. falciparum in vitro (IC50 20 nM) and P. falciparum-derived topoisomerase II. However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.     

Novel bisquinoline antimalarials. Synthesis, antimalarial activity, and inhibition of haem polymerisation

We report the synthesis of two series of novel bisquinoline compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. To study the molecular basis of the action of these novel antimalarial drugs, we examined their ability to inhibit haem polymerisation in the presence and absence of parasite extracts. The level of antimalarial potency was correlated with the level of inhibition of haem polymerisation, suggesting that these bisquinolines exert their antimalarial activity by antagonising the sequestration of toxic haem moieties.     

Antiplasmodial triterpene from Vernonia brasiliana

The hexane extract from leaves of Vernonia brasiliana (L.) Druce (Compositae) was active in vitro against Plasmodium falciparum and in vivo in mice infected with Plasmodium berghei. This extract was subjected to a bioassay-guided fractionation protocol based on the in vitro model. Lupeol was identified as a compound responsible for the activity, inhibiting the P.falciparum growth by 45% when tested at 25 micrograms/ml. However, this triterpene was inactive in vivo when 15 mg/kg were administered per os during four consecutive days to mice infected with P.berghei. beta-Amyrin and germanicol, isolated from the same fraction that yielded lupeol, were inactive in the in vitro assay.     

Citalopram enhances the activity of chloroquine in resistant plasmodium in vitro and in vivo

Citalopram, is an extremely potent inhibitor of neuronal serotonin reuptake. It is structurally unrelated to other antidepressants, but it contains the chemical features associated with reversal of drug resistance and exhibits minimal cardiotoxic side effects and fewer of the anticholinergic and adrenolytic side effects associated with other psychotropic agents. Sensitivity tests to citalopram alone and in combination with chloroquine were performed against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum and Plasmodium chabaudi. Citalopram alone showed intrinsic activity against the chloroquine-resistant strains of P. falciparum (IC50 = 1.51 +/- .6 microM) but only limited activity against the chloroquine-sensitive strain (IC50 = 33.27 +/- 5.87 microM) and no activity in vivo. The interaction of chloroquine and citalopram in vitro resulted in a synergistic response in the chloroquine-resistant strain but there was no interaction between the drugs in the chloroquine-sensitive strain--a pattern found with other reversal agents. Citalopram enhanced chloroquine susceptibility in both strains of P. chabaudi, however, the potentiating effect was seen at lower doses in the chloroquine-resistant strain. The results of this study suggest that citalopram may have potential as a chemosensitizer in Plasmodium infections on the basis of the low toxicity of citalopram at concentrations potentiating chloroquine activity both in vitro and in vivo.     

Variations in serum ferritin, serum iron, total iron binding capacity and saturation index in elderly hospitalized patients with iron deficiency anaemia after blood transfusion

Five xanthones from the bark of Garcinia cowa, namely 7-O-methylgarcinone E (1), cowanin (2), cowanol (3), cowaxanthone (4), and beta-mangostin (5), were found to possess in vitro antimalarial activity against Plasmodium falciparum with IC50 values ranging from 1.50 to 3.00 micrograms/ml. Complete 1H- and 13C-NMR assignments of these compounds are also reported.     

Orally active, hydrolytically stable, semisynthetic, antimalarial trioxanes in the artemisinin family

In only three chemical operations, natural trioxane lactone artemisinin (1) was converted into a series of C-10 carbon-substituted 10-deoxoartemisinin compounds 4-9. The three steps involved lactone reduction, replacement of the anomeric lactol OH by F using diethylaminosulfur trifluoride, and finally boron trifluoride-promoted substitution of F by aryl, heteroaryl, and acetylide nucleophiles. All of these C-10 nonacetal, chemically robust, enantiomerically pure compounds 4-9 have high antimalarial potencies in vitro against Plasmodium falciparum malaria parasites, and furans 5a and 5b and pyrrole 7a are antimalarially potent also in vivo even when administered to rodents orally.     

Prognostic value of quantitatively measured AgNORs in ductal mammary carcinoma

Antimalarial activities of tetracycline (TC) and erythromycin (EM), alone or in combination with artemisinin (Qinghaosu, QHS), were studied using chloroquine (CQ)-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum in vitro. The antimalarial potency of TC (IC50 = 9862 nM for the CQ-sensitive parasite, 32414 nM for the CQ-resistant one) or EM (IC50 = 45787 nM for the CQ-sensitive parasite, 33397 nM for the CQ-resistant one) was much less than that of QHS (IC50 ranging from 25 to 40 nM). The CQ-resistant falciparum parasite displayed a cross-resistance to TC, while both the drug-sensitive and -resistant parasites exhibited similar responses to EM. However, antimalarial potency of EM appeared to be less than that of TC against the drug-sensitive parasite. When TC was combined with QHS, an additive interaction was observed against the CQ-sensitive falciparum parasite, while synergism was found with the CQ-resistant parasite. When EM was tested in combination with QHS, a potentiating interaction occurred with both the CQ-sensitive and resistant falciparum parasite. The above results indicated that the QHS combination with either TC or EM may be a promising antimalarial preparation with low recrudescence compared to artemisinin used alone in clinical practice.     

Design, synthesis, derivatization, and structure-activity relationships of simplified, tricyclic, 1,2,4-trioxane alcohol analogues of the antimalarial artemisinin

Novel C4-(hydroxyalkyl)trioxanes 5d and 5e were designed and synthesized based on an understanding of the molecular mechanism of action of similar 1,2,4-trioxanes structurally related to the antimalarial natural product artemisinin (1). In vitro efficacies of these two new pairs of C4-diastereomers against chloroquine-sensitive Plasmodium falciparum support conclusions about the importance to antimalarial activity of formation of a C4 radical by a 1,5-hydrogen atom abstraction. Derivatives 6, 7, and 21 of C4 beta-substituted trioxane alcohols 4a, 5d, and 5e were prepared, each in a single-step, high-yielding transformation. Four of these new analogues, 6a-c and 7, are potent in vitro antimalarials, having 140 to 50% of the efficacy of the natural trioxane artemisinin (1).     

New quinoline di-Mannich base compounds with greater antimalarial activity than chloroquine, amodiaquine, or pyronaridine

We have compared the ex vivo antimalarial activity of 12 new quinoline di-Mannich base compounds containing the 7-dichloroquinoline or 7-trifluoromethylquinoline nucleus with amodiaquine, chloroquine, and pyronaridine using the Saimiri-bioassay model. Each compound was administered orally (30 mg/kg of body weight) to three or more noninfected Saimiri sciureus monkeys, and serum samples were collected at various times after drug administration and serially diluted with drug-free (control) serum. In vitro activity against the multidrug-resistant K1 isolate of Plasmodium falciparum was determined in serum samples by measuring the maximum inhibitory dilution at which the treated monkey serum inhibited schizont maturation in vitro. Of the 12 Mannich bases tested, 8 were associated with levels of ex vivo antimalarial activity in serum greater than those of amodiaquine, chloroquine, or pyronaridine 1 to 7 days after drug administration. Further studies were carried out with four of these compounds, and the results showed that the areas under the serum drug concentration-time curves for the four compounds were between 7- and 26-fold greater than that obtained for pyronaridine. Activity against four multidrug-resistant strains of P. falciparum was also much greater in serum samples collected from monkeys after administration of these four compounds than in serum samples collected after administration of pyronaridine or chloroquine. These findings suggest that these four quinoline Mannich base compounds possess a very marked and prolonged antimalarial activity and that further studies should be performed to determine their value as antimalarial drugs.     

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.     

Adenosine analogues as antimetabolites against Plasmodium falciparum malaria

Analogues of purine nucleosides and deoxynucleosides were tested for toxicity against the intraerythrocytic parasite Plasmodium falciparum in vitro culture. Sangivamycin (7-deaza-7-amido-adenosine) (IC37 of 0.3 microM), tubercidin (7-deaza-adenosine) (IC37 of 0.7 microM), 6-methylamino-deoxyadenosine (IC37 of 10 microM), 8-aza-2-amino-deoxy-adenosine (IC37 of 11 microM) and 2-chloro-adenosine (IC37 of 11 microM) were found to be the most toxic towards the parasite. Structure-activity analysis suggested that alteration of the purine ring at the 7 or 8 position significantly increased the toxicity of the compound against P. falciparum. Analysis by HPLC of parasite lysates which had been subjected to the cytotoxic compounds confirmed that alterations in the flux of the purine salvage pathways of the parasite had occurred. Comparison of the toxicity of these compounds against P. falciparum with the toxicity against a similar intraerythrocytic parasite, Babesia bovis, or human melanoma cell lines indicated a differential toxicity, in that many of the compounds toxic towards P. falciparum were relatively non-toxic towards human melanoma cell lines or B. bovis and vice versa. The mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids appears to vary significantly between P. falciparum, B. bovis and mammalian cells.     

In vitro culture and drug sensitivity assay of Plasmodium falciparum with nonserum substitute and acute-phase sera

The short-term in vitro growth of Plasmodium falciparum parasites in the asexual erythrocytic stage and the in vitro activities of eight standard antimalarial drugs were assessed and compared by using RPMI 1640 medium supplemented with 10% nonimmune human serum, 10% autologous or homologous acute-phase serum, or 0.5% Albumax I (lipid-enriched bovine serum albumin). In general, parasite growth was maximal with autologous (or homologous) serum, followed by Albumax I and nonimmune serum. The 50% inhibitory concentrations (IC50s) varied widely, depending on the serum or serum substitute. The comparison of IC50s between assays with autologous and nonimmune sera showed that monodesethylamodiaquine, halofantrine, pyrimethamine, and cycloguanil had similar IC50s. Although the IC50s of chloroquine, monodesethylamodiaquine, and dihydroartemisinin were similar with Albumax I and autologous sera, the IC50s of all test compounds obtained with Albumax I differed considerably from the corresponding values obtained with nonimmune serum. Our results suggest that Albumax I and autologous and homologous sera from symptomatic, malaria-infected patients may be useful alternative sources of serum for in vitro culture of P. falciparum isolates in the field. However, autologous sera and Albumax I do not seem to be suitable for the standardization of isotopic in vitro assays for all antimalarial drugs.     

Korundamine A, a novel HIV-inhibitory and antimalarial "hybrid" naphthylisoquinoline alkaloid heterodimer from Ancistrocladus korupensis

A unique heterodimeric naphthylisoquinoline alkaloid, korundamine A (2), comprised of two different monomeric biaryl halves, has been isolated from the Cameroonian tropical liana Ancistrocladus korupensis. Korundamine A is the first "hybrid" dimer found in the Ancistrocladaceae; in vitro, it demonstrated anticytopathic activity against HIV-1 and antimalarial activity against Plasmodium falciparum.     

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

Relation between the space flight-induced frequency of dominant lethal mutations and the level of allozyme heterozygosity in Drosophila melanogaster populations

Currently, the treatment of falciparum malaria is seriously compromised by spreading drug resistance. We studied the effects of camptothecin, a potent and specific topoisomerase I inhibitor, on erythrocytic malaria parasites in vitro. In Plasmodium falciparum, camptothecin trapped protein-DNA complexes, inhibited nucleic acid biosynthesis, and was cytotoxic. These results provide proof for the concept that topoisomerase I is a vulnerable target for new antimalarial drug development.     

Integrity and common sense in medicine

Hannoa chlorantha and Hannoa klaineana (Simaroubaceae) are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and four quassinoids from H. chlorantha were examined in vitro against Plasmodium falciparum NF 54. The extracts displayed good activities, while the quassinoids were highly active, with IC50 values well below 1 microgram ml-1, those of chaparrinone and 15-desacetylundulatone being much lower than 0.1 microgram ml-1 (0.037 and 0.047 microgram ml-1, respectively). Chaparrinone is five times more active than 14-hydroxychaparrinone against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity. As 14-hydroxychaparrinone has a seven-times higher cytotoxic activity against P-388 cells than chaparrinone, the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were evaluated in vivo in a standard 4-day test as well. 15-Desacetylundulatone was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinone and 14-hydroxychaparrinone were active for at least 4 days. Quassinoids have ED50 values much lower than 50 mg kg-1 body weight day-1 and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone is apparently of crucial importance for its high activity. 6-alpha-Tigloyloxyglaucarubol was not active at all. Chaparrinone is considered the most interesting of the investigated quassinoids and its in-vivo antimalarial potential will be examined further.