Chloroquine-resistant K1 Research Articles

In vitro antimalarial activity, β-haematin inhibition and structure–activity relationships in a series of quinoline triazoles

A novel series of quinoline triazole amide analogues (38–51) has been synthesized. Analogues 38–44 had a Cl substituent at the 7-position of the quinoline ring, while 45–51 had a CN substituent at this position. Compounds 40, 45 and 49 were found to be the most active in the series against the Plasmodium falciparum chloroquine-sensitive D10 strain, with IC50 values in the range of 349–1247 nM, with 40 and 45, but not 49 also exhibiting similar activity against the chloroquine-resistant K1 strain of parasite. Quinoline triazoles 40 and 44 were the most active β-haematin inhibitors, with 50% inhibitory concentrations of 14.7 and 8.9 μM respectively. In vitro antimalarial activity of the 7-Cl bearing analogues 38–44 exhibited a strong linear dependence of log(1/IC50) on log P. Thus, the more lipophilic, the more active it was found be. The 7-CN series 45–51 showed no such dependence. The resistance index (IC50 K1/IC50 D10) also exhibited a linear dependence on log P, with a substantially steeper slope in the case of the 7-Cl series. The findings demonstrate the feasibility of producing hydrophilic analogues with strong activity and low cross-resistance with chloroquine.

Novel Conjugated Quinoline–Indoles Compromise Plasmodium falciparum Mitochondrial Function and Show Promising Antimalarial Activity

A novel class of antimalarial compounds, based on an indol-3-yl linked to the 2-position of a 4-aminoquinoline moiety, shows promising activity against the malaria parasite, Plasmodium falciparum . Compounds with a quaternary nitrogen on the quinoline show improved activity against the chloroquine-resistant K1 strain. Nonquaternerized 4-aminoquinolines retain significant potency but are relatively less active against the K1 strain. Alkylation of the 4-amino group preferentially improves the activity against the chloroquine-sensitive 3D7 strain. The quinoline-indoles show only weak activity as inhibitors of β-hematin formation, and their activities are only weakly antagonized by a hemoglobinase inhibitor. The compounds appear to dissipate mitochondrial potential as an early event in their antimalarial action and therefore may exert their activity by compromising Plasmodium mitochondrial function. Interestingly, we observed a structural relationship between our compounds and the anticancer and anthelminthic compound, pyrvinium pamoate, which has also been proposed to exert its action via compromising mitochondrial function.

Tetrazole-based deoxyamodiaquines: Synthesis, ADME/PK profiling and pharmacological evaluation as potential antimalarial agents

A series of new deoxyamodiaquine-based compounds was synthesized via the modified TMSN3-Ugi multi-component reaction and evaluated in vitro for antiplasmodial activity. The most potent compounds, 6b, 6c and 6j, showed IC50 values in the range of 6–77nM against chloroquine-resistant K1- and W2-strains of Plasmodium falciparum. In vitro ADME characterization of frontrunner compounds 6b and 6c indicates that these two compounds are rapidly metabolized and have a high clearance rate in human and rat liver microsomes. This result correlated well with an in vivo pharmacokinetics study, which showed low bioavailability of 6c in rats. Tentative metabolite identification was determined by LC–MS and suggested metabolic lability of groups attached to the tertiary nitrogen. Preliminary studies on 6b and 6c suggested strong inhibitory activity against the major CYP450 enzymes. In silico docking studies were used to rationalize strong inhibition of CYP3A4 by 6c. Full characterization and biological evaluation of the metabolites is currently underway in our laboratories.

Synthesis and antiplasmodial and antimycobacterial evaluation of new nitroimidazole and nitroimidazooxazine derivatives.

The synthesis and antiplasmodial and antimycobacterial evaluation of two new series of nitroimidazole and nitroimidazooxazine derivatives is described. The majority of these compounds, especially hybrids 9d, 9f, and 14b, exhibited potent activity against the chloroquine-resistant K1 strain of Plasmodium falciparum. Furthermore, a notable number from the tetrazole series were significantly more active against M. tuberculosis than kanamycin, a standard TB drug.

Chemical and Bioactivity Evaluation of the Bark of Neonauclea purpurea

Bioassay-guided fractionation of the MeOH extract from the stem bark of Neonauclea purpurea used in traditional medicine, resulted in the isolation of 2 indole alkaloids, cadambine (1) and alpha-dihydrocadambine (2), as well as a quinolic compound, 2,6-dimethoxy-1,4-benzoquinone (3). Antimalarial activity evaluation showed that compounds 2 and 3 exhibited mild in vitro antimalarial activity against Plasmodium falciparum, the chloroquine-resistant strain K1 with IC50 values of 6.6 and 11.3 microM, respectively. Compounds 1 and 2 showed no cytotoxicity to monkey (Vero) cells, but compound 3 showed weak cytotoxicity with an IC50 value of 1.19 microM.

Synthesis of hybrid 4-anilinoquinoline triazines as potent antimalarial agents, their in silico modeling and bioevaluation as Plasmodium falciparumtransketolase and β-hematin inhibitors

Analogues of a novel class of hybrid 4-anilinoquinoline triazines have been synthesized with the aim of identifying the compounds with improved antimalarial activity preserving the potency of parent drug chloroquine (CQ). All the synthesized molecules were evaluated in vitro for their antimalarial activity against chloroquine-sensitive 3D7 and chloroquine-resistant K1 strains of P. falciparum. Molecules were also screened for their cytotoxicity towards VERO cell line. Sixteen compounds (17, 19, 26, 27, 29, 31, 32, 33, 35, 36, 37, 39, 40, 49, 50, and 52) exhibited excellent antimalarial activity with IC50 values ranging from 1.36–4.63 ng ml−1 and were also found to be nontoxic with good selectivity index. In silico activity prediction as well as enzyme inhibitory activity against P. falciparumtransketolase reveals that the molecules are also good inhibitors of the enzymeP. falciparumtransketolase. The compound 52 showed good in vivo activity by oral route and resulted in survival of 3 out of 5 mice till day 28.

Synthesis and Evaluation of 7-Substituted 4-Aminoquinoline Analogues for Antimalarial Activity

We previously reported that substituted 4-aminoquinolines with a phenyl ether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine's alkyl substituents exhibited potent antimalarial activity against the multidrug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogues in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain as well as for cytotoxicity against mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1 strain and good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.

Synthesis and evaluation of phenylequine for antimalarial activity in vitro and in vivo

Synthesis of the potent antiplasmodial 4-aminoquinoline, phenylequine (PQ), is reported for the first time. PQ and the two analogues show increased efficacy in moving from the chloroquine sensitive D10 to the chloroquine resistant K1 strain in vitro. The in vivo efficacy of PQ, and salts thereof, have been determined in Plasmodium berghei ANKA and Plasmodium yoelii. Phenylequine hydrochloride has shown an ED 50 of 0.81 in P. yoelii (cf chloroquine ED 50 = 1.31).

A new series of amodiaquine analogues modified in the basic side chain with in vitro antileishmanial and antiplasmodial activity

The synthesis and the study of new amodiaquine derivatives bearing modified lateral basic chains as new agents with both antimalarial and antileishmanial activities are reported. The compounds were tested in vitro against Leishmania donovani MHOM/ET/67/HU3 and 2 strains of Plasmodium falciparum, 3D7 and K1. All the compounds show complex ionisation profiles. At physiological pH the ionised form(s) are in equilibrium with the uncharged form, while at acid pH all the products exist largely as protonated forms. The antiprotozoal profile indicates that all derivatives are endowed with both antimalarial and antileishmanial activity. Interestingly amodiaquine, together with some synthesised derivatives ( 11, 12, 15, 27, 34), displayed antileishmanial activity in the low micromolar range, although these compounds were also cytotoxic and have a narrow therapeutic window, most of the synthesised compounds proved to be potent antimalarials, a few of them showing a good activity against the chloroquine resistant K1 strain.

Growth-inhibitory effect of a fucoidan from brown seaweed Undaria pinnatifida on Plasmodium parasites.

The present study was undertaken to investigate the inhibitory effects of fucoidan, a sulfated polysaccharide isolated from the edible brown seaweed Undaria pinnatifida, on the growth of Plasmodium parasites. In order to assess the anti-malarial activity of fucoidan, growth inhibition activities were evaluated using cultured Plasmodium falciparum parasites in vitro and on Plasmodium berghei-infected mice in vivo. Fucoidan significantly inhibited the invasion of erythrocytes by P. falciparum merozoites, and its 50% inhibition concentration was similar to those for the chloroquine-sensitive P. falciparum 3D7 strain and the chloroquine-resistant K1 strain. Four-day suppressive testing in P. berghei-infected mice with fucoidan resulted in a 37% suppressive effect versus the control group and a delay in death associated with anemia (P < 0.05). In addition, fucoidans had no toxic effect on RAW 264.7 cells. These findings indicate that fucoidans from the Korean brown algae U. pinnatifida inhibits the invasion of Plasmodium merozoites into erythrocytes in vitro and in vivo.

Plasmodium vivax trophozoites insensitive to chloroquine

BackgroundPlasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax.MethodsThe in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment.ResultsPlasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM.ConclusionData from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

Isoquine and related amodiaquine analogues: a new generation of improved 4-aminoquinoline antimalarials.

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects including agranulocytosis and liver damage. The observed drug toxicity is believed to involve the formation of an electrophilic metabolite, amodiaquine quinoneimine (AQQI), which can bind to cellular macromolecules and initiate hypersensitivity reactions. We proposed that interchange of the 3' hydroxyl and the 4' Mannich side-chain function of amodiaquine would provide a new series of analogues that cannot form toxic quinoneimine metabolites via cytochrome P450-mediated metabolism. By a simple two-step procedure, 10 isomeric amodiaquine analogues were prepared and subsequently examined against the chloroquine resistant K1 and sensitive HB3 strains of Plasmodium falciparum in vitro. Several analogues displayed potent antimalarial activity against both strains. On the basis of the results of in vitro testing, isoquine (ISQ1 (3a)) (IC(50) = 6.01 nM +/- 8.0 versus K1 strain), the direct isomer of amodiaquine, was selected for in vivo antimalarial assessment. The potent in vitro antimalarial activity of isoquine was translated into excellent oral in vivo ED(50) activity of 1.6 and 3.7 mg/kg against the P. yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine. Subsequent metabolism studies in the rat model demonstrated that isoquine does not undergo in vivo bioactivation, as evidenced by the complete lack of glutathione metabolites in bile. In sharp contrast to amodiaquine, isoquine (and Phase I metabolites) undergoes clearance by Phase II glucuronidation. On the basis of these promising initial studies, isoquine (ISQ1 (3a)) represents a new second generation lead worthy of further investigation as a cost-effective and potentially safer alternative to amodiaquine.

Antiplasmodial activity of a series of 1,3,5-triazine-substituted polyamines.

Polyamine biosynthesis and function has been shown to be a good drug target in some parasitic protozoa and it is proposed that the pathway might also represent a target in the malaria parasite Plasmodium falciparum. A series of 1,3,5-triazine-substituted polyamine analogues were tested for activity against Plasmodium falciparum in vitro. The series showed activity against the parasites and were generally more active against the chloroquine-resistant line K1 than the chloroquine-susceptible line NF54. Simple unbranched analogues had better activity than analogues carrying branched or cyclic central chains. Addition of multiple triazine units in general led to increased activity of the compounds.

Novel short chain chloroquine analogues retain activity against chloroquine resistant K1 Plasmodium falciparum.

A series of short chain chloroquine (CQ) derivatives have been synthesized in one step from readily available starting materials. The diethylamine function of CQ is replaced by shorter alkylamine groups (4-9) containing secondary or tertiary terminal nitrogens. Some of these derivatives are significantly more potent than CQ against a CQ resistant strain of Plasmodium falciparum in vitro. We conclude that the ability to accumulate at higher concentrations within the food vacuole of the parasite is an important parameter that dictates their potency against CQ sensitive and the chloroquine resistant K1 P. falciparum.

Synthesis, Stability, and Antimalarial Activity of New Hydrolytically Stable and Water-Soluble (+)-Deoxoartelinic Acid

(+)-Deoxoartelinic acid (13), a new hydrolytically stable, water-soluble, and potent non-acetal-type antimalarial drug candidate, was successfully prepared from artemisinic acid by using sulfur ylide and photooxygenative cyclization in seven steps. This compound showed superior in vitro antimalarial activity against the chloroquine-resistant K1 strain of Plasmodium falciparum and higher suppression (98.7%) than arteether in vivo against Plasmodium chabaudi infected mice. (+)-Deoxoartelinic acid also showed remarkable stability with a half-life of 258.66 h, 23 times more stable than clinically useful arteether in simulated stomach acid, and improved solubility, 4 times more soluble than artemisinin in water.

Optimisation of the allylsilane approach to C-10 deoxo carba analogues of dihydroartemisinin: synthesis and in vitro antimalarial activity of new, metabolically stable C-10 analogues

An optimised protocol has been developed for the coupling of dihydroartemisinin benzoate with a range of aromatic allylsilanes to provide a number of new C-10 deoxo derivatives (11a–11g) in yields ranging from 70 to 94%. These compounds were up to ten times more potent than artemisinin in in vitro tests against the chloroquine resistant K1 strain of Plasmodium falciparum. Ferrous mediated degradation of these analogues produces as the main product, a dicarbonyl formate 12, which is not seen when the same reaction is carried out with artemisinin or artemether. This finding may indicate that analogues in this class have a subtly different “antimalarial” mechanism of action.

Regioselective Mukaiyama hydroperoxysilylation of 2-alkyl- or 2-aryl-prop-2-en-1-ols: application to a new synthesis of 1,2,4-trioxanes

Co(II)-mediated peroxysilylation of allylic alcohols 6 or 9 regioselectively provides peroxysilyl alcohols 7 and 10 in good yield. Reaction of these peroxysilyl alcohols with aldehydes or ketones provides target 1,2,4-trioxanes in good to excellent yields. The sequence of Markovnikov hydroperoxysilylation and subsequent reaction with a carbonyl compound can also be readily achieved in a one-pot procedure. Significantly, easily prepared trioxanes 11a and 11b have potent in vitro antimalarial activity versus chloroquine resistant K1 Plasmodium falciparum.

Deletion of the parasite-specific insertions and mutation of the catalytic triad in glutathione reductase from chloroquine-sensitive Plasmodium falciparum 3D7

The flavoenzyme glutathione reductase (GR; NADPH+glutathione disulphide+H +→NADP ++2 glutathione-SH) of Plasmodium falciparum is a promising drug target against tropical malaria. As P. falciparum genes are assumed to be highly polymorphic we have cloned and expressed the GR cDNA of the chloroquine-sensitive strain 3D7. In comparison to the known GR of the chloroquine-resistant K1 strain there are three base exchanges all of them leading to amino acid substitutions (residues 281, 285 and 335). The catalytic efficiency k cat/ K m of the 3D7 enzyme is 5-fold lower than for the K1 enzyme. In contrast, vis-à-vis the drugs carmustine, methylene blue and fluorophenyliso-alloxazine the two enzyme species exhibited identical inhibition kinetics. Two structural motifs which are specific for P. falciparum GR were studied by mutational deletion analysis of 3D7 GR. Loop 126–138 appears to be important for folding and stability of the enzyme, whereas the subdomain 318–350 was found to be involved in FAD-binding. The subdomain has no major influence on the known functions of the catalytic triad Cys-40, Cys-45 and His-485′. Flavin absorption spectroscopy of inactive point mutants showed that Cys-45 forms a thiolate charge transfer complex and Cys-40 is the interchange thiol, which reduces glutathione disulphide. The mutant His-485→Gln had a normal K m for glutathione disulphide reduction but only 0.8% residual catalytic activity when compared with wild-type GR, which confirms its function as an acid/base catalyst. The parasite-specific domains in combination with the reactive catalytic residues appear to be a suitable target matrix for inhibiting GR in vivo.

Synergy between two calcium channel blockers, verapamil and fantofarone (SR33557), in reversing chloroquine resistance in Plasmodium falciparum

This study describes the synergistic interaction of two calcium channel blockers, verapamil (VR) and SR33557 or fantofarone (SR), in reversing chloroquine resistance in Plasmodium falciparum, the causative agent of human malaria. The two calcium channel blockers exhibited an intrinsic antimalarial activity at 10 and 1 μM for verapamil and fantofarone, respectively. Isobolograms revealed that chloroquine and verapamil, and chloroquine and fantofarone, acted synergistically against chloroquine-resistant strains of P. falciparum. When used at subinhibitory concentrations, verapamil appeared 2 to 3 times more potent than fantofarone in reversing chloroquine resistance. Indeed, verapamil completely reversed the chloroquine resistance in P. falciparum, while fantofarone did so only partially. In the highly chloroquine-resistant strain FcB1, VR and SR acted synergistically to reverse CQ resistance, and the concentrations of VR used in these combinations could be reduced 10- or 100-fold (e.g. 100 nM and 10 nM) those required when this drug was used alone. In the moderately chloroquine-resistant strain K1, a combination of VR and SR for CQ resistance reversal allowed us to reduce the concentration of these chemosensitizers 1000- and 100-fold, respectively. The maximum tolerable plasma level beyond which side-effects occurred when using verapamil is 2.5 μM. Thus, the approach described, which allowed us to lower the doses of chemosensitizers, could well prevent toxic effects in humans and enlighten the advantages of polychemotherapy.

Synthesis, antimalarial activity, and molecular modeling of tebuquine analogues.

Tebuquine (5) is a 4-aminoquinoline that is significantly more active than amodiaquine (2) and chloroquine (1) both in vitro and in vivo. We have developed a novel more efficient synthetic route to tebuquine analogues which involves the use of a palladium-catalyzed Suzuki reaction to introduce the 4-chlorophenyl moiety into the 4-hydroxyaniline side chain. Using similar methodology, novel synthetic routes to fluorinated (7a, b) and a dehydroxylated (7c) analogue of tebuquine have also been developed. The novel analogues were subjected to testing against the chloroquine sensitive HB3 strain and the chloroquine resistant K1 strain of Plasmodium falciparum. Tebuquine was the most active compound tested against both strains of Plasmodia. Replacement of the 4-hydroxy function with either fluorine or hydrogen led to a decrease in antimalarial activity. Molecular modeling of the tebuquine analogues alongside amodiaquine and chloroquine reveals that the inter-nitrogen separation in this class of drugs ranges between 9.36 and 9.86 A in their isolated diprotonated form and between 7.52 and 10.21 A in the heme-drug complex. Further modeling studies on the interaction of 4-aminoquinolines with the proposed cellular receptor heme revealed favorable interaction energies for chloroquine, amodiaquine, and tebuquine analogues. Tebuquine, the most potent antimalarial in the series, had the most favorable interaction energy calculated in both the in vacuo and solvent-based simulation studies. Although fluorotebuquine (7a) had a similar interaction energy to tebuquine, this compound had significantly reduced potency when compared with (5). This disparity is possibly the result of the reduced cellular accumulation (CAR) of fluorotebuquine when compared with tebuquine within the parasite. Measurement of the cellular accumulation of the tebuquine analogues and seven related 4-aminoquinolines shows a significant relationship (r = 0.98) between the CAR of 4-aminoquinoline drugs and the reciprocal of drugs IC50.