Antimalarial acridines: Synthesis,in vitro activity against P. falciparum and interaction with hematin

A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure–activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC₅₀ ? 0.07 μM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC₅₀ ? 0.3 μM. These acridine derivatives inhibited the formation of β-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.     

Synthesis and evaluation of the antiplasmodial activity of novel indeno[2,1-c]quinoline derivatives

With the aim to explore the potentiality of new chemical scaffolds for the design of new antimalarials, a set of new indeno[2,1-c]quinolines bearing different basic heads has been synthesized and tested in vitro against chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains of Plasmodium falciparum. Most of the synthesized compounds exhibited a moderate antiplasmodial activity, inhibiting the growth of both CQ-S and CQ-R strains of P. falciparum with IC₅₀ ranging from 0.24 to 6.9 μM and with a very low resistance index. The most potent compounds (1.2–1.3-fold the CQ on the W-2 strain) can be considered as promising ‘lead compounds’ to be further optimized to improve efficacy and selectivity against Plasmodia.     

Novel tacrine–ebselen hybrids with improved cholinesterase inhibitory,hydrogen peroxide and peroxynitrite scavenging activity

A series of tacrine–ebselen hybrids were synthesised and evaluated as possible multifunctional anti-Alzheimer’s disease (AD) agents. Compound 6i, which is tacrine linked with 5,6-dimethoxybenzo[d][1,2]selenazol-3(2H)-one by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) and butylcholinesterase (BuChE) inhibitor, with IC₅₀ values of 2.55 and 2.80 nM, respectively. Furthermore, this compound demonstrated similar hydrogen peroxide and peroxynitrite scavenging activity as ebselen by horseradish peroxidase assay and peroxynitrite scavenging activity assay, indicating that this hybrid is a good multifunctional drug candidate for the treatment of AD.     

Novel oxazine skeletons as potential antiplasmodial active ingredients: Synthesis,in vitro and in vivo biology of some oxazine entities produced via cyclization of novel chalcone intermediates

A novel series of 6-(2-chloroquinolin-3-yl)-4-substituted-phenyl-6H-1,3-oxazin-2-amines were synthesized and evaluated for in vitro antimalarial efficacy against chloroquine sensitive (MRC-02) as well as chloroquine resistant (RKL9) strains of Plasmodium falciparum. The activity tested was at nanomolar concentration. β-Hematin formation inhibition activity (BHIA₅₀) of oxazines were determined and correlated with antimalarial activity. A reasonably good correlation (r?=?0.49 and 0.51, respectively) was observed between antimalarial activity (IC₅₀) and BHIA₅₀. This suggests that antimalarial mode of action of these compounds seems to be similar to that of chloroquine and involves the inhibition of hemozoin formation. Some of the compounds were showing better antimalarial activity than chloroquine against resistant strain of P. falciparum and were also found to be active in the in vivo experiment.     

Synthesis,physicochemical and biological evaluation of tacrine derivative labeled with technetium-99m and gallium-68 as a prospective diagnostic tool for early diagnosis of Alzheimer’s disease

Design, physicochemical and biological studies of novel radioconjugates for the early diagnosis of Alzheimer's disease, based on the newly synthesized tacrine derivatives were performed. Novel tacrine analogues were labeled with technetium-99m and gallium-68. For all obtained radioconjugates ([^99mTc]Tc-Hynic-(tricine)₂NH(CH₂)_ntacrine and [⁶⁸Ga]Ga-DOTA-NH(CH₂)₉tacrine, where n = 2–9 denotes the number of methylene groups CH₂) the studies of physicochemical properties (lipophilicity, stability in the presence of an excess of standard amino acids cysteine or histidine, human serum and in cerebrospinal fluid) were performed. For two selected radioconjugates [^99mTc]Tc-Hynic-(tricine)₂NH(CH₂)₉Tac and [⁶⁸Ga]Ga-DOTA-NH(CH₂)₉tacrine (characterized with the highest lipophilicity values) the biological tests (inhibition of cholinesterases action, molecular docking and biodistribution studies) have been performed. All novel radioconjugates showed high stability in biological solutions used. Both selected radioconjugates proved to be good inhibitors of cholinesterases and be able to cross the blood-brain barrier. Radioconjugates [^99mTc]Tc-Hynic-(tricine)₂NH(CH₂)₉tacrine and [⁶⁸Ga]Ga-DOTA-NH(CH₂)₉tacrine fulfil the conditions for application in nuclear medicine. Radiopharmaceutical [⁶⁸Ga]Ga-DOTA-NH(CH₂)₉tacrine, due to increased accuracy and improved sensitivity in PET imaging, may be better potential diagnostic tool for early diagnosis of Alzheimer’s disease.     

Synthesis of chiral chloroquine and its analogues as antimalarial agents

In this investigation, we describe a new approach to chiral synthesis of chloroquine and its analogues. All tested compounds displayed potent activity against chloroquine sensitive as well as chloroquine resistant strains of Plasmodium falciparum in vitro and Plasmodium yoelii in vivo. Compounds S-13b, S-13c, S-13d and S-13i displayed excellent in vitro antimalarial activity with an IC₅₀ value of 56.82, 60.41, 21.82 and 7.94 nM, respectively, in the case of resistant strain. Furthermore, compounds S-13a, S-13c and S-13d showed in vivo suppression of 100% parasitaemia on day 4 in the mouse model against Plasmodium yoelii when administered orally. These results underscore the application of synthetic methodology and need for further lead optimization.     

Dimeric bisindole alkaloids from the stem bark of Strychnos nux-vomica L.

Strychnos nux-vomica L. (Loganiaceae) is famous for its monomeric alkaloid content, such as strychnine, a convulsant poison. The stem bark of the tree is traditionally used to treat intermittent fever in South East Asia. In various studies, it appeared that dimeric indolo-monoterpenic alkaloids possess a promising activity on Plasmodium falciparum. Three bisindolomonoterpenic alkaloids together with strychnochrysine, previously identified in the root bark of S. nux-vomica, were isolated from the stem bark. The structures of these compounds were established using NMR spectroscopy and mass spectrometry.Stereochemistry of the compounds was confirmed by molecular modelling. This then allowed the structural determination of strychnoflavine, a coloured bisindole alkaloid previously isolated from the root bark of the tree. Moreover, the conformational inversion in alkaloids possessing an ether bond in the strychnane moiety could be easily predicted by specific δ ¹³C NMR values.These longicaudatine-type alkaloids were found to display in vitro antiplasmodial activity against a chloroquine resistant strain and a chloroquine sensitive strain. The most interesting was strychnochrysine showing an IC₅₀ value at around 10 μM.     

Evaluation of substituted phenalenone analogues as antiplasmodial agents

A set of derivatives encompassing structural modifications on the privileged phenalenone scaffold were assessed for their antiplasmodial activities against a strain of chloroquine sensitive Plasmodium falciparum F32. Two compounds exhibited considerable effects against the malaria parasite (IC₅₀ ? 1 μg/mL), one of which maintained the same level of activity in a chloroquine-resistant strain. This is the first record of antiplasmodial activity on this type of scaffold, providing a new structural motif as a new lead for antimalarial activity.     

A Process Similar to Autophagy Is Associated with Cytocidal Chloroquine Resistance in Plasmodium falciparum

Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR^CS) as quantified by CQ IC₅₀ shift, account for only 10–20% of cytocidal CQR (CQR^CC) as quantified by CQ LD₅₀ shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR^CS vs CQR^CC phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD₅₀. Candidate genes in these loci are consistent with a role for autophagy in CQR^CC, leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR^CC. Taken together, the data show that an unusual autophagy – like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD₅₀ doses (but not IC₅₀ doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR^CC phenotype.     

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 ED₅₀ of 0.81 in P. yoelii (cf chloroquine ED₅₀ = 1.31).     

Synthesis, characterization and antimalarial activity of new chromium arene-quinoline half sandwich complexes

Organometallic analogs of chloroquine (CQ) are of interest as drug candidates that may be able to overcome the widespread chloroquine resistance developed by malaria parasites. Two new chromium arene CQ-analogs: [η⁶-N-(7-chloroquinolin-4-yl)-N′-(2-dimethylamino-methylbenzyl)-ethane-1,2-diamine]tricarbonylchromium 4 and [η⁶-N-(7-chloroquinolin-4-yl)-N′-(2-dimethylaminobenzyl)-ethane-1,2-diamine]tricarbonylchromium 9 have been synthesized and characterized. In addition, X-ray crystal structures of the intermediates (η⁶-benzyldimethylamine)tricarbonylchromium 2, [η⁶-2-((dimethylamino)methyl) benzaldehyde]tricarbonylchromium 3 and p-(η⁶-dimethylaminobenzaldehyde)tricarbonyl chromium 8 are reported. Compound 4 was more active than chloroquine against both CQ-sensitive and CQ-resistant strains of Plasmodium falciparum when antimalarial activity was tested in vitro. The activity of 4 against the CQ-resistant parasite strain was twice as high as for the organic ligand alone (IC₅₀ values of 33.9 nM versus 63.1 nM).     

Design,synthesis and biological evaluation of novel N-phosphorylated and O-phosphorylated tacrine derivatives as potential drugs against Alzheimer’s disease

In this work, we designed, synthesised and biologically investigated a novel series of 14 N- and O-phosphorylated tacrine derivatives as potential anti-Alzheimer’s disease agents. In the reaction of 9-chlorotacrine and corresponding diamines/aminoalkylalcohol we obtained diamino and aminoalkylhydroxy tacrine derivatives. Next, the compounds were acid to give final products 6–13 and 16–21 that were characterised by ¹H, ¹³C, ³¹P NMR and MS. The results of the docking studies revealed that the designed phosphorus hybrids, in theory can bind to AChE and BChE. All compounds exhibited significantly lower AutoDock Vina scores compared to tacrine. The inhibitory potency evaluation was performed using the Ellman’s method. The most inhibitory activity against AChE exhibited compound 8 with an IC₅₀ value of 6.11 nM and against BChE 13 with an IC₅₀ value of 1.97 nM and they were 6- and 12-fold potent than tacrine. Compound 19 showed the lack of hepatocytotoxicity in MTT assay.     

Syntheses of coumarin–tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase,Aβ aggregation,and β-secretase

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer’s disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine–coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (K_i = 16.7 nM) against human AChE and about 2-fold lower potency (K_i = 16.1 nM) against BChE than tacrine (K_i = 35.7 nM for AChE, K_i = 8.7 nM for BChE), respectively. In addition, some of the tacrine–coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine–coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer’s disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC₅₀ = 33.0 µM; 2, IC₅₀ = 8.5 µM) compared to tacrine (CYP1A2 IC₅₀ = 1.5 µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species.     

Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain

We report the synthesis and in vitro antimalarial activity of several new 4-amino- and 4-alkoxy-7-chloroquinolines carrying a linear dibasic side chain. Many of these chloroquine analogues have submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strain of Plasmodium falciparum) and low resistance indices were obtained in most cases. Importantly, compounds 11–15 and 24 proved to be more potent against Dd2 than chloroquine. Branching of the side chain structure proved detrimental to the activity against the CQR strain.     

Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate,LynxTag-CQGREEN,as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQ_GREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQ_GREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQ_GREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC₅₀s were determined. These data were compared with LynxTag-CQ_GREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC₅₀ of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.     

Search for new pharmacophores for antimalarial activity. Part I: Synthesis and antimalarial activity of new 2-methyl-6-ureido-4-quinolinamides

A total of 80 new 2-methyl-6-ureido-4-quinolinamides were synthesized and evaluated for their antimalarial activity. Several analogs elicited the antimalarial effect at MIC of 0.25 mg/mL against the chlooquine-sensitive P. falciparum strain. The IC₅₀ values of the active compounds were observed to be in ng/mL range and two of the analogs have better IC₅₀ value than the standard chloroquine. In the in vivo assay against mdr CQ resistant P. yoelii N67/P. yoelii nigeriensis, however, none of the compound showed complete suppression of parasitemia on day 7. One of the compounds displayed significant antibacterial effect against several strains of bacteria and was many-fold better than the standard drug gentamicin.     

Chlorinated tacrine analogs: Design,synthesis and biological evaluation of their anti-cholinesterase activity as potential treatment for Alzheimer’s disease

In search of potent acetyl cholinesterase inhibitors with low hepatotoxicity for the treatment of Alzheimer’s disease, introduction of a chloro substitution to tacrine and some of its analogs has proven to be beneficial in maintaining or potentiating the cholinesterase inhibitory activity. Furthermore, it was found to be able to reduce the hepatotoxicity of the synthesized compounds, which is the main target of the study. Accordingly, a series of new 4-(chlorophenyl)tetrahydroquinoline derivatives, was synthesized and characterized. The synthesized compounds were evaluated for their in vitro and in vivo anti-cholinesterase activity using tacrine as a reference standard. Furthermore, they were investigated for their hepatotoxicity compared to tacrine. The obtained biological results revealed that all synthesized compounds displayed equivalent or significantly higher anti-cholinesterase activity and lower hepatotoxicity in comparison to tacrine. In addition, in silico drug-likeness of the synthesized compounds were predicted and their practical logP were assessed indicating that all synthesized compounds can be considered as promising hits/leads. Furthermore, docking study of the compound showing the highest in vitro anticholinesterase activity was performed and its binding mode was compared to that of tacrine.     

Antimalarial compounds from Schefflera umbellifera

The organic extract of the leaves of Schefflera umbellifera exhibited good antimalarial activity when tested against the chloroquine-susceptible strain (D10). Bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity with an IC₅₀ value of 3.2 µg/ml. The reference compound, chloroquine gave an IC₅₀ value of 27.2 ng/ml. Two other compounds were also isolated from the dichloromethane extract namely, 7-hydroxy-6-methoxycoumarin and ent-kaur-16-en-19-oic acid. These two compounds did not exhibit any significant antiplasmodial activity.