Synthesis of stable isotope-labeled chloroquine and amodiaquine and their metabolites.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

Synthesis of novel amodiaquine analogs and evaluation of their in vitro and in vivo antimalarial activities.

BACKGROUND & OBJECTIVES: Due to the rapid increase of drug resistance in Plasmodium parasites, there is a pressing need of developing new antiplasmodial drugs. In this study, new amodiaquine (AQ) analogs were synthesized, followed by an evaluation of their antiplasmodial activity. METHODS: A new series of quinoline derivatives containing N-alkyl (piperazin-1-yl)methyl benzamidine moiety was synthesized by reacting 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl]benzonitrile with appropriate primary amines. The synthesized compounds were investigated for inhibitory activity by inhibition test of heme detoxification (ITHD). Their antiplasmodial activity was then evaluated using the classical 4-day suppressive test (Peter's test) against Plasmodium berghei-infected mice (ANKA strain). RESULTS: The results showed that the percentage of heme detoxification inhibition in the active compounds was 90%. The most promising analogs, N-butyl-4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl]benzamidine (compound 1e), and 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)]-N-(4-methylpentan-2-yl)benzamidine (compound 1f) displayed 97.65 and 99.18% suppressions at the doses of 75 and 50 mg/kg/day, respectively. Further, the mean survival time of the mice treated with these compounds was higher than that of the negative control group. INTERPRETATION & CONCLUSION: The newly synthesized amodiaquine analogs presented sufficient antiplasmodial activity with excellent suppressions and high in vitro heme detoxification inhibition. Higher mean survival time of the mice treated with synthetic compounds further confirmed the in vivo antimalarial activity of these new AQ analogs. Therefore, these compounds have the potential to replace common drugs from 4-aminoquinoline class. However, further investigations such as pharmacokinetic evaluations, cytotoxicity, toxicity, and formulation seem to be necessary.

Novel amodiaquine derivatives potently inhibit Ebola virus infection.

Ebola virus disease is a severe disease caused by highly pathogenic Ebolaviruses. Although it shows a high mortality rate in humans, currently there is no licensed therapeutic. During the recent epidemic in West Africa, it was demonstrated that administration of antimalarial medication containing amodiaquine significantly lowered mortality rate of patients infected with the virus. Here, in order to improve its antiviral activity, a series of amodiaquine derivatives were synthesized and tested for Ebola virus infection. We found that multiple compounds were more potent than amodiaquine. The structure-activity relationship analysis revealed that the two independent parts, which are the alkyl chains extending from the aminomethyl group and a halogen bonded to the quinoline ring, were keys for enhancing antiviral potency without increasing toxicity. When these modifications were combined, the antiviral efficacy could be further improved with the selectivity indexes being over 10-times higher than amodiaquine. Mechanistic evaluation demonstrated that the potent derivatives blocked host cell entry of Ebola virus, like the parental amodiaquine. Taken together, our work identified novel potent amodiaquine derivatives, which will aid in further development of effective antiviral therapeutics.

Design, Synthesis and Evaluation of New Fluoroamodiaquine Analogues.

Malaria is one of the most important tropical diseases; the use of amodiaquine as a current chemotherapy in the treatment of malaria has shown some problems such as hepatotoxicity and agranulocytosis. In this work we present the rational design, synthesis, and biological evaluation (antimalarial activity, cytotoxicity and genotoxicity) of four new fluoroamodiaquine analogues. The results showed significant correlation between MolDock score and IC50 values. The molecules 7b and c were the most active of the planned compounds, with lower IC50 against Plasmodium falciparum W2 strain (0.9 and 0.8 µM, respectively) and an excellent cytotoxicity profile. The present study revealed no mutagenicity or genotoxicity for the analogues. Confirming our docking results, the molecular dynamics showed that compound 7b remains stably bound to the heme group by means of π-stacking interactions between quinoline and the porphyrin ring. Based on these findings, this study may prove to be an efficient approach for the rational design of hemozoin inhibiting compounds to treat malaria.

Benzoheterocyclic amodiaquine analogues with potent antiplasmodial activity: synthesis and pharmacological evaluation.

The synthesis and evaluation of antiplasmodial activity of benzothiazole, benzimidazole, benzoxazole and pyridine analogues of amodiaquine is hereby reported. Benzothiazole and benzoxazole analogues with a protonatable tertiary nitrogen atom possessed excellent activity against the W2 and K1 chloroquine resistant strains of Plasmodium falciparum, with IC(50)s ranging from 7 to 22 nM.

Buchwald reaction as the key step for the synthesis of metabolically more stable analogs of amodiaquine.

Amodiaquine is one of the most active anti-malarial 4-aminoquinoline but its metabolization is believed to generate hepatotoxic derivatives. Previously, we described new analogs of amodiaquine and amopyroquine, in which hydroxyl group was replaced by various amino groups and identified highly potent compounds with lower toxicity. We describe here the synthesis of new analogs that have been modified on their 4'- and 5'-positions in order to reduce their metabolization. A new synthetic strategy was developed using Buchwald coupling reaction as the key step.

Amodiaquine analogues containing NO-donor substructures: synthesis and their preliminary evaluation as potential tools in the treatment of cerebral malaria.

The synthesis and physico-chemical properties of novel compounds obtained by conjugation of amodiaquine with moieties containing either furoxan or nitrooxy NO-donor substructures are described. The synthesised compounds were tested in vitro against both the chloroquine sensitive, D10 and the chloroquine resistant, W-2 strains of Plasmodium falciparum (P. falciparum). Most of the compounds showed an antiplasmodial activity comparable to that of the parent drug. By comparing the activities of simple related structures devoid of the ability to release NO, it appears that the contribution of NO to the antiplasmodial action in vitro is marginal. All the compounds were able to relax rat aorta strips with a NO-dependent mechanism, thus showing their capacity to release NO in the vessels. A preliminary in vivo study using Plasmodium berghei ANKA-infected mice showed a trend for prolonged survival of mice with cerebral malaria treated with compound 40, which is potent and fast amodiaquine-derived NO-donor, when compared with amodiaquine alone or with compound 31, a milder NO-donor. The two compounds showed in vivo antiplasmodial activity similar to that of amodiaquine.

Synthesis, antimalarial activity, and preclinical pharmacology of a novel series of 4'-fluoro and 4'-chloro analogues of amodiaquine. Identification of a suitable "back-up" compound for N-tert-butyl isoquine.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

Replacement of the 4'-hydroxy group of amodiaquine and amopyroquine by aromatic and aliphatic substituents: synthesis and antimalarial activity.

The prophylactic administration of amodiaquine (AQ), a 4-aminoquinoline antimalarial drug, has been associated with side effects such as agranulocytosis and liver damage. The toxicity of this drug is mediated by amodiaquine quinone-imine, an electrophilic metabolite. Replacement of the 4'-hydroxy function of AQ with various alkyl, aryl, or heteroaryl substituents would provide analogues that avoid metabolism to potentially toxic derivatives. Following a multistep procedure, 33 compounds containing hydrophobic groups at the 4'-position were synthesized using Csp(2)-Csp(2) and Csp(2)-Csp(3) Suzuki-Miyaura cross-coupling reactions as the key step. The new derivatives were found to be active against both chloroquine (CQ)-sensitive and CQ-resistant strains of P. falciparum, with IC(50) values in the range of 7-200 nM. Alkyl analogues are more efficient than aryl or heteroaryl derivatives. All compounds were also assessed for their cytotoxicity and ability to inhibit beta-hematin formation in vitro. A detailed investigation of the structure-activity relationships for these new compounds was carried out; the 4'-methyl compound showed interesting in vivo antimalarial activity.

Synthesis and antimalarial activity of new amino analogues of amodiaquine.

Amodiaquine remains one of the most prescribed antimalarial 4-aminoquinoline. To assess the importance of the 4'-hydroxyl group and subsequent hydrogen bond in the antimalarial activity of amodiaquine (AQ), a series of new analogues in which this functionality was replaced by various amino groups was synthesized. The incorporation of a 3'-pyrrolidinamino group instead of the 3'-diethylamino function of AQ allowed the development of a parallel series of amopyroquine derivatives. The compounds were screened against both chloroquine (CQ)-sensitive and -resistant strains of Plasmodium falciparum and their cytotoxicity evaluated upon the MRC5 cell line. Antimalarial activity in a low nanomolar range was recorded showing that the 4'-hydroxy function can be successfully replaced by various amino substituents in terms of activity without any influence of the level of CQ-resistance of the strains. Furthermore the ability of the compounds to inhibit beta-hematin formation was measured in order to discuss the mechanism of action of these new compounds. Compounds 7d and 8d exhibit a high selectivity index and may be considered as promising leads for further development.

Investigation of amodiaquine bulk drug impurities by liquid chromatography/ion trap mass spectrometry.

Three unknown impurities in an amodiaquine bulk drug sample were detected by reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC/UV). A liquid chromatography/tandem mass spectrometry (LC/MS(n)) method is described for the investigation of these impurities. Mass spectral data were acquired on an LCQ ion trap mass analyzer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The fragmentation behavior of amodiaquine and its impurities has been studied. Based on the mass spectral data and the specifics of the synthetic route, the possible structures of these impurities were elucidated as 4-[(5-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol (impurity I), 4-[(7-chloroquinolin-4-yl)-amino]phenol (impurity II) and 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-N(1)-oxy]phenol (impurity III). The structures were confirmed by their independent synthesis and NMR spectral assignment.

Synthesis and antimalarial activity of new analogues of amodiaquine.

In order to determine the real significance of the 4'-phenolic group in the antimalarial activity and/or cytotoxicity of amodiaquine (AQ), analogues for which this functionality was shifted or modified were synthesized. Good in vitro antimalarial activity was obtained for compounds unable to form intramolecular hydrogen bond. Among the compounds synthesized, new amino derivative 5 displayed the greatest selectivity index towards the most CQ-resistant strain tested and was active in mice infected by Plasmodium berghei.

[[1]Benzofuro[3,2-b]pyridin-4-yl-amines - synthesis and investigation of activity against malaria]. / [1]Benzofuro[3,2-b]pyridin-4-yl-amine - Synthese und Prüfung auf Wirksamkeit gegen Malaria.

The ethyl 4-chlorobenzofuro[3,2-b]pyridine-3-carboxylate (2) reacted with the hydrochlorides of the mono- and bis-phenol Mannich bases 3 to yield the amodiaquine and pyronaridine analogues 4. The chloroquine analogue 6 was formed by melting 2 with the novaldiamine base (5) in phenol. The most active compound 4c inhibited the growth of the malaria parasite Plasmodium falciparum with an IC50 of 500 nM.

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.

The effect of fluorine substitution on the metabolism and antimalarial activity of amodiaquine.

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial which causes adverse side effects such as agranulocytosis and liver damage. The observed drug toxicity is believed to be related to the formation of an electrophilic metabolite, amodiaquine quinone imine (AQQI), which can bind to cellular macro-molecules and initiate hypersensitivity reactions. 5'-Fluoroamodiaquine (5'-FAQ, 3), 5',6'-difluoroamodiaquine (5',6'-DIFAQ,4), 2',6'-difluoroamodiaquine (2',6'-DIFAQ,5), 2',5',6'-trifluoroamodiaquine (2',5',6'-TRIFAQ, 6) and 4'-dehydroxy-4'-fluoroamodiaquine (4'-deOH-4'-FAQ,7) have been synthesized to assess the effect of fluorine substitution on the oxidation potential, metabolism, and in vitro antimalarial activity of amodiaquine. The oxidation potentials were measured by cyclic voltammetry, and it was observed that substitution at the 2',6'- and the 4'-positions (2',6'-DIFAQ and 4'-deOH-4'-FAQ) produced analogues with significantly higher oxidation potentials than the parent drug. Fluorine substitution at the 2',6'-positions and the 4'-position also produced analogues that were more resistant to bioactivation. Thus 2',6'-DIFAQ and 4'-deOH-4'-FAQ produced thioether conjugates corresponding to 2.17% (SD: +/- 0.27%) and 0% of the dose compared with 11.87% (SD: +/- 1.31%) of the dose for amodiaquine. In general the fluorinated analogues had similar in vitro antimalarial activity to amodiaquine against the chloroquine resistant K1 strain of Plasmodium falciparum and the chloroquine sensitive T9-96 strain of P. falciparum with the notable exception of 2',5',6'-TRIFAQ (6). The data presented indicate that fluorine substitution at the 2',6'-positions and replacement of the 4'-hydroxyl of amodiaquine with fluorine produces analogues (5 and 7) that maintain antimalarial efficacy in vitro and are more resistant to oxidation and hence less likely to form toxic quinone imine metabolites in vivo.

Synthesis of some novel amodiaquine analogues as potential antimalarial and antifilarial compounds.

Ten amodiaquine analogues, which are hybridized molecules of amodiaquine and diethylcarbamazine, were designed and synthesized. Six analogues, all bearing a basic tertiary amino function at their side chain, were active against Plasmodium berghei in mice and inhibited the mobility of adult worms and microfilariae of Breinlia booliati in vitro. They were inactive against Litomosoides carinii in Mastomys natalensis. The most active antimalarial compound, 7-chloro-4-[alpha-[[N-(4-methyl-1-piperazinyl)carbonyl]amino]-4-hydroxy-m-toluidino]quinoline, had twice the activity of amodiaquine. O-Methylation and N-ethylation generally reduced antimalarial activity. Analogues which lack a basic tertiary amino function at their side chain were also lacking in both antimalarial and antifilarial activities.