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[Image: see text] Virtual ligand screening of a publicly available database of antimalarial hits using a pharmacophore derived from antimalarial MMV008138 identified TCMDC-140230, a tetrahydro-β-carboline amide, as worthy of exploration. All four stereoisomers of this structure were synthesized, but none potently inhibited growth of the malaria parasite Plasmodium falciparum. Interestingly, 7e, a minor byproduct of these syntheses, proved to be potent in vitro against P. falciparum and was orally efficacious (40 mg/kg) in an in vivo mouse model of malaria.

Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, are devastating diseases in the elderly world, which are closely associated with progressive neuronal loss induced by a variety of genetic and/or environmental factors. Unfortunately, currently available treatments for neurodegenerative disorders can only relieve the symptoms but not modify the pathological processes. Over the past decades, our group by collaborating with Profs. Yuan-Ping Pang and Paul R. Carlier has developed three series of homo/hetero dimeric acetylcholinesterase inhibitors derived from tacrine and/or huperzine A. The representative dimers bis(3)-Cognitin (B3C), bis(12)-hupyridone, and tacrine(10)-hupyridone might possess disease-modifying effects through the modulation of N-methyl-d-aspartic acid receptors, the activation of myocyte enhancer factor 2D gene transcription, and the promotion of neurotrophic factor secretion. In this review, we summarize that the representative dimers, such as B3C, provide neuroprotection against a variety of neurotoxins via multiple targets, including the inhibitions of N-methyl-d-aspartic acid receptor with pathological-activated potential, neuronal nitric oxide synthase, and β-amyloid cascades synergistically. More importantly, B3C might offer disease-modifying potentials by activating myocyte enhancer factor 2D transcription, inducing neuritogenesis, and promoting the expressions of neurotrophic factors in vitro and in vivo. Taken together, the novel dimers might offer synergistic disease-modifying effects, proving that dimerization might serve as one of the strategies to develop new generation of therapeutics for neurodegenerative disorders.

Alzheimer's disease (AD) is a neurodegenerative disorder with multiple pathological features. Therefore, a multitarget-directed ligands (MTDLs) strategy has been developed to treat AD. We have previously designed and synthesized dimeric tacrine(10)-hupyridone (A10E), a novel tacrine derivative with acetylcholinesterase (AChE) inhibition and brain-derived neurotrophic factor (BDNF) activation activity, by linking tacrine and a fragment of huperzine A. However, it was largely unknown whether A10E could act on other AD targets and produce cognitive-enhancing ability in AD animal models. In this study, A10E could prevent cognitive impairments in APP/PS1 transgenic mice and β-amyloid (Aβ) oligomers-treated mice, with higher potency than tacrine and huperzine A. Moreover, A10E could effectively inhibit Aβ production and deposition, alleviate neuroinflammation, enhance BDNF expression, and elevate cholinergic neurotransmission in vivo. At nanomolar concentrations, A10E could inhibit Aβ oligomers-induced neurotoxicity via the activation of tyrosine kinase receptor B (TrkB)/Akt pathway in SH-SY5Y cells. Furthermore, Aβ oligomerization and fibrillization could be directly disrupted by A10E. Importantly, A10E at high concentrations did not produce obvious hepatotoxicity. Our results indicated that A10E could produce anti-AD neuroprotective effects via the inhibition of Aβ aggregation, the activation of the BDNF/TrkB pathway, the alleviation of neuroinflammation, and the decrease of AChE activity. As MTDLs could produce additional benefits, such as overcoming the deficits of drug combination and enhancing the compliance of AD patients, our results also suggested that A10E might be developed as a promising MTDL lead for the treatment of AD.

[Image: see text] The tetrahydro-β-carboline scaffold has proven fertile ground for the discovery of antimalarial agents (e.g., MMV008138 (1) and cipargamin (2)). Similarity searching of a publicly disclosed collection of antimalarial hits for molecules resembling 1 drew our attention to N2-acyl tetrahydro-β-carboline GNF-Pf-5009 ((±)-3b). Compound purchase, “analog by catalog”, and independent synthesis of hits indicated the benzofuran-2-yl amide portion was required for in vitro efficacy against P. falciparum. Preparation of pure enantiomers demonstrated the pharmacological superiority of (R)-3b. Synthesis and evaluation of D- and F-ring substitution variants and benzofuran isosteres indicated a clear structure–activity relationship. Ultimately (R)-3b was tested in Plasmodium berghei-infected mice; unfavorable physicochemical properties may be responsible for the lack of oral efficacy.

BACKGROUND Varroa destructor is among the greatest threats to honey bee health worldwide. Acaricides used to control Varroa are becoming increasingly ineffective due to resistance issues, prompting the need for new compounds that can be used for control purposes. Ideally, such compounds would exhibit high toxicity to Varroa while maintaining relatively low toxicity to bees and beekeepers. We characterized the lethal concentrations (LC50 ) of amitraz, matrine, FlyNap®, the experimental carbamates 2-((2-ethylbutyl)thio)phenyl methylcarbamate (1) and 2-(2-ethylbutoxy)phenyl methylcarbamate (2), and dimethoate (positive control) for Varroa using a glass vial assay. The test compounds also were applied to honey bees using an acute contact toxicity assay to determine the adult bee LD50 for each compound. RESULTS Amitraz was the most toxic compound to Varroa, but carbamate 2 was nearly as active (within 2-fold) and the most selective due to its lower bee toxicity, demonstrating its promise as a Varroa control. While carbamate 1 was less toxic to honey bees than was amitraz, it was also 4.7-fold less toxic to the mites. Both matrine and FlyNap® were relatively ineffective at killing Varroa and were moderately toxic to honey bees. CONCLUSION Additional testing is required to determine if carbamate 2 can be used as an effective Varroa control. As new chemical treatments are identified, it will be necessary to determine how they can be utilized best alongside other control techniques as part of an integrated pest management program.

Nucleophilic ring opening of cyclohexene oxides is known to proceed preferentially through the trans-diaxial pathway (the Fürst-Plattner rule). This preference, however, is not absolute, and can be affected by substituents on the cyclohexene oxide ring, as illustrated by LiAlH4 ring-opening of the cis- and trans-isomers of 4-t-butyl- and 3-methylcyclohexene oxide (cis- and trans-1, cis- and trans-2). We performed B3LYP/6-31+G*(PCM) geometry optimizations to locate the chair-like and twist-boat-like transition structures for the hydride attacks on the pseudoaxial and pseudoequatorial conformers of these epoxides. Our calculations are consistent with the experimental observation of effective Fürst-Plattner control of AlH4--opening of cis-1, trans-1, and cis-2, but low selectivity in ring-opening of trans-2. Our data at B3LYP/6-31+G*(PCM) suggests this reduction in selectivity is due to a diminished pseudoequatorial preference of the 3-methyl group in trans-2 relative to that in cis-2. The two calculated chair-like transition structures for hydride opening of trans-2 differ in activation energy free energy (ΔΔG‡) by only 0.4 kcal mol-1. Thus, these calculations account for the reduced regioselectivity of ring opening seen for trans-2 by AlH4- and other nucleophiles.

The Pictet-Spengler reaction of tryptophan esters and aldehydes has been widely applied in natural product synthesis and medicinal chemistry. To date, the trans- or cis-configuration of 1,3-disubstituted tetrahydro-β-carbolines (THβCs) formed in this reaction has most often been assigned based on the relative 13C chemical shifts of C1 and C3 in the diastereomers. Although the upfield shifts of C1 and C3 in trans-THβCs relative to cis-THβCs has been attributed to steric compression associated with the "γ-gauche" effect, we show that this effect is not borne out experimentally for other carbons that should suffer this same compression. Thus we developed a robust alternative method for stereochemical assignment based on 1H NMR coupling constants (31 examples) and supported by extensive DFT-based conformational analysis and calculation of 1H-1H coupling constants. DFT calculations of 13C NMR chemical shifts also cast doubt upon the role of the "γ-gauche" effect on C1 and C3 chemical shifts in trans-THβCs.

Background Alzheimer’s disease (AD) is a neurodegenerative disorder with multiple pathological features. Therefore, multi-target-directed ligands (MTDLs) strategy has been developed to combat this disease. We have previously designed and synthesized dimeric tacrine (10)-hupyridone (A10E), a novel tacrine derivative with acetylcholinesterase (AChE) inhibition and brain-derived neurotrophic factor (BDNF) activation activity, by linking tacrine and a fragment of huperzine A. However, it was largely unknown whether A10E could act on other AD targets and produce cognition-enhancing ability in AD animal models. Methods Behavioral and biochemical methods were applied to evaluate multi-target cognitive-enhancing effects and mechanisms of A10E in APP/PS1 transgenic mice and β-amyloid (Aβ) oligomers-treated mice. The neuroprotective mechanisms of A10E were explored in SH-SY5Y cells. And the anti-aggregation effects of A10E on Aβ were directly investigated in vitro. Results A10E could prevent cognitive impairments in both APP/PS1 mice and Aβ oligomers-treated mice, with higher potency than tacrine and huperzine A. Moreover, A10E could effectively inhibit Aβ production and deposition, reduce neuroinflammation, enhance brain derived brain-derived neurotrophic factor (BDNF) expression, and elevate cholinergic neurotransmission in vivo. A10E, at nanomolar concentrations, could also inhibit Aβ oligomers-induced neurotoxicity via the activation of the TrkB/Akt pathway. Furthermore, Aβ oligomerization and fibrillization could be directly disrupted by A10E. Conclusion A10E could produce anti-AD neuroprotective effects via multi-target mechanisms, including the inhibition of Aβ aggregation, the activation of the BDNF/TrkB pathway, the reduction of neuroinflammation and the decrease of AChE activity. As MTDLs could produce additional benefits, such as overcoming the deficits of drug combination and enhancing the compliance of AD patients, our results suggested that A10E might be developed as a promising MTDL lead for the treatment of AD.

The present study focused on the toxicity of the aphid anti-feedant flonicamid and its main metabolite, 4-trifluoromethylnicotinamide (TFNA-AM) to Aedes aegypti and Anopheles gambiae mosquitoes. The compounds were toxic to both species via topical application, resulting in un-coordinated locomotion and leg splaying, with a favorable An. gambiae LD50 value of 35 ng/mg for TFNA-AM, but no significant lethality to Ae. aegypti at 10 μg/female. There was mild cross resistance in the Akron-kdr (Akdr) strain of An. gambiae. Both compounds were non-toxic to intact larvae (LC50 > 300 ppm); however, headless Ae. aegypti larvae displayed spastic paralysis, with PC50 values of 2–4 ppm, indicating that the cuticle is a significant barrier to penetration. TFNA-AM showed low mammalian toxicity, with an LD50 of >2000 mg/kg in mice. Electrophysiological experiments showed larval Aedes muscle depolarization and Kv2 channel blocking activity that required near mM concentrations, suggesting that this potassium channel is not the main target for flonicamid nor its metabolite. However, TFNA-AM was a potent blocker of evoked body wall sensory discharge in dipteran larvae, suggesting that some component of the chordotonal organ system may be involved in its toxicity. Finally, flonicamid and TFNA-AM showed about 2-fold synergism of permethrin toxicity against An. gambiae adult females whose mechanism should become more clear once the mode of action of these compounds is better defined.

The widespread emergence of pyrethroid-resistant Anopheles gambiae has intensified the need to find new contact mosquitocides for indoor residual spraying and insecticide treated nets. With the goal of developing new species-selective and resistance-breaking acetylcholinesterase (AChE)-inhibiting mosquitocides, in this report we revisit the effects of carbamate substitution on aryl carbamates, and variation of the 1-alkyl group on pyrazol-4-yl methylcarbamates. Compared to aryl methylcarbamates, aryl dimethylcarbamates were found to have lower selectivity for An. gambiae AChE (AgAChE) over human AChE (hAChE), but improved tarsal contact toxicity to G3 strain An. gambiae. Molecular modeling studies suggest the lower species-selectivity of the aryl dimethylcarbamates can be attributed to a less flexible acyl pocket in AgAChE relative to hAChE. The improved tarsal contact toxicity of the aryl dimethylcarbamates relative to the corresponding methylcarbamates is attributed to a range of complementary phenomena. With respect to the pyrazol-4-yl methylcarbamates, the previously observed low An. gambiae-selectivity of compounds bearing α-branched 1-alkyl groups was improved by employing β- and γ-branched 1-alkyl groups. Compounds 22a (cyclopentylmethyl), 21a (cyclobutylmethyl), and 26a (3-methylbutyl) offer 250-fold, 120-fold, and 96-fold selectivity, respectively, for inhibition of AgAChE vs. hAChE. Molecular modeling studies suggests the high species-selectivity of these compounds can be attributed to the greater mobility of the W84 sidechain in the choline-binding site of AgAChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC50 = 269 μg/mL) and low cross-resistance to Akron strain (LC50 = 948 μg/mL), which bears the G119S resistance mutation.

New public health insecticides are urgently required to prevent the spread of vector-borne disease. With the goal of identifying new K+-channel-directed mosquitocides, analogs of the RH-5849 family of diacyl t-butylhydrazines were synthesized and tested for topical toxicity against adult Anopheles gambiae, the African vector of malaria. In total, 80N'-monoacyl and N, N'-diacyl derivatives of benzyl- and arylhydrazines were prepared. Three compounds (2bo, 2kb, 3ab) were identified that were more toxic than RH-5849 and RH-1266. The potencies of these compounds to block K+ currents in An. gambiae and human Kv2.1 channels were assessed to address their possible mechanism of mosquitocidal action. Selectivity for inhibition of An. gambiae Kv2.1 vs human Kv2.1 did not exceed 3-fold. Furthermore, no correlation was seen between the potency of insecticidal action and K+ channel blocking potency. These observations, combined with the minimal knockdown seen with 2bo near its LD50 value, suggests a mode of action outside of the nervous system.

The Pictet-Spengler reaction of tryptophan esters and aldehydes has been widely applied in natural product synthesis and medicinal chemistry. To date, the trans- or cis-configuration of 1,3-disubstituted tetrahydro-β-carbolines (THβCs) formed in this reaction has most often been assigned based on the relative (13)C chemical shifts of C1 and C3 in the diastereomers. Although the upfield shifts of C1 and C3 in trans-THβCs relative to cis-THβCs has been attributed to steric compression associated with the “γ-gauche” effect, we show that this effect is not borne out experimentally for other carbons that should suffer this same compression. Thus we developed a robust alternative method for stereochemical assignment based on (1)H NMR coupling constants (31 examples) and supported by extensive DFT-based conformational analysis and calculation of (1)H-(1)H coupling constants. DFT calculations of (13)C NMR chemical shifts also cast doubt upon the role of the “γ-gauche” effect on C1 and C3 chemical shifts in trans-THβCs.

New insecticides are needed for control of disease-vectoring mosquitoes and this research evaluates the activity of new carbamate acetylcholinesterase (AChE) inhibitors. Biochemical and toxicological characterization of carbamates based on the parent structure of terbam, 3-tert-butylphenyl methylcarbamate, was performed. In vitro enzyme inhibition selectivity (Anopheles gambiae versus human) was assessed by the Ellman assay, as well as the lethality to whole insects by the World Health Organization (WHO) paper contact assay. Bromination at the phenyl C6 position increased inhibitory potency to both AChEs, whereas a 6-iodo substituent led to loss of potency, and both halogenations caused a significant reduction of mosquitocidal activity. Similarly, installation of a hexyl substituent at C6 drastically reduced inhibition of AgAChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against AgAChE and generally showed more activity against hAChE than AgAChE. Replacement of the 3-t-buyl group with CF3 resulted in poor anticholinesterase activity, but this compound did have measurable mosquitocidal activity. A series of methyl- and fluoro- analogs of 3-trialkylsilyl compounds were also synthesized, but unfortunately resulted in disappointing activity. Finally, a series of sulfenylated proinsecticides showed poor paper contact toxicity, but one of them had topical activity against adult female Anopheles gambiae. Overall, the analogs prepared here contributed to a better understanding of carbamate structure&ndash, activity relationships (SAR), but no new significant leads were generated.

The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed.

Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure–activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC50 ≤ 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials.

Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria.

Malaria is a devastating disease in sub-Saharan Africa, and current vector control measures are threatened by emerging resistance mechanisms. With the goal of developing new, selective, resistance-breaking insecticides we explored α-fluorinated methyl ketones as reversible covalent inhibitors of Anopheles gambiae acetylcholinesterase (AgAChE). Trifluoromethyl ketones 5 demonstrated remarkable volatility in microtiter plate assays, but 5c,e-h exhibited potent (1–100 nM) inhibition of wild type (WT) AgAChE and weak inhibition of resistant mutant G119S mutant AgAChE. Fluoromethyl ketones 10c-i exhibited submicromolar to micromolar inhibition of WT AgAChE, but again only weakly inhibited G119S AgAChE. Interestingly, difluoromethyl ketone inhibitors 9c and 9g had single digit nanomolar inhibition of WT AgAChE, and 9g had excellent potency against G119S AgAChE. Approach to steady-state inhibition was quite slow, but after 23 h incubation an IC50 value of 25.1 ± 1.2 nM was measured. We attribute the slow, tight-binding G119S AgAChE inhibition of 9g to a balance of steric size and electrophilicity. However, toxicities of 5g, 9g, and 10g to adult An. gambiae in tarsal contact, fumigation, and injection assays were lower than expected based on WT AgAChE inhibition potency and volatility. Potential toxicity-limiting factors are discussed. 2015 Elsevier Ltd. All rights reserved.

Insecticide resistance in the malaria vector, Anopheles gambiae, is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a "pseudo-pyrethroid." There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety.

Compounds that target isoprenoid biosynthesis in Plasmodium falciparum could be a welcome addition to malaria chemotherapy, since the methylerythritol phosphate (MEP) pathway used by the parasite is not present in humans. We previously reported that MMV008138 targets the apicoplast of P. falciparum and that its target in the MEP pathway differs from that of Fosmidomycin. In this Letter, we determine that the active stereoisomer of MMV008138 is 4a , which is (1 R ,3 S )-configured. 2′,4′-Disubstitution of the D ring was also found to be crucial for inhibition of the parasite growth. Limited variation of the C3-carboxylic acid substituent was carried out, and methylamide derivative 8a was found to be more potent than 4a ; other amides, acylhydrazines, and esters were less potent. Finally, lead compounds 4a , 4e , 4f , 4h , 8a , and 8e did not inhibit growth of Escherichia coli , suggesting that protozoan-selective inhibition of the MEP pathway of P. falciparum can be achieved.

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure–activity relationships by probing the ability of MMV008138 analogs to inhibit Pf IspD recombinant enzyme. Here, we report Pf IspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and Pf IspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

BACKGROUND Insecticide resistance in the malaria mosquito Anopheles gambiae is well documented, and widespread agricultural use of pyrethroids may exacerbate development of resistance when pyrethroids are used in vector control. We have developed carbamate anticholinesterases that possess a high degree of An. gambiae:human selectivity for enzyme inhibition. The purpose of this study was to assess the spectrum of activity of these carbamates against other mosquitoes and agricultural pests. RESULTS Experimental carbamates were potent inhibitors of mosquito acetylcholinesterases, with IC50 values in the nanomolar range. Similar potencies were observed for Musca domestica and Drosophila melanogaster enzymes. Although meta-substituted carbamates were potent inhibitors, two ortho-substituted carbamates displayed poor enzyme inhibition (IC50 ≥ 10−6 M) in honey bee (Apis mellifera), Asian citrus psyllid (Diaphorina citri) and lepidopteran agricultural pests (Plutella xylostella and Ostrinia nubilalis). Enzyme inhibition results were confirmed by toxicity studies in caterpillars, where the new carbamates were 2–3-fold less toxic than propoxur and up to tenfold less active than bendiocarb, indicating little utility of these compounds for crop protection. CONCLUSION The experimental carbamates were broadly active against mosquito species but not agricultural pests, which should mitigate selection for mosquito insecticide resistance by reducing agricultural uses of these compounds. © 2014 Society of Chemical Industry

Malaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparum and low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of

Malaria is an urgent world health concern and vector control is one important option for reducing disease prevalence. Increased reports of pyrethroid-resistant mosquito strains have amplified the need for new vector-control chemicals. We compared three commercially available carbamate insecticides (carbofuran, bendiocarb, and propoxur) to eight experimental compounds 1–8 for activity against Anopheles gambiae acetylcholinesterase, as well as enzymes from mammalian, avian, and aquatic species. The experimental compounds (except 7) were less potent than the commercial inhibitors against the mosquito enzyme, but had higher selectivity values (up to near 600-fold, IC50 of non-target species/IC50 An. gambiae) because of their low potency for acetylcholinesterases from nontarget species. Neurotoxic esterase assay showed that none of the experimental carbamates (1 mM) displayed NTE inhibition, while bendiocarb did (24% inhibition at 1 mM), although the effect was much less than that of mipafox. In vivo bioassays using Daphnia magna showed that all novel carbamates were of similar killing potency as bendiocarb (24 h LC50 = 611 nM), with the exception of experimental compound 1 (LC50 = 172 nM). Overall, the results suggested that the novel carbamate insecticides 4–8 presented in this study were safer to mammals than the commercial compounds and were promising insecticides for malaria vector control usage on bednets or indoor residual sprays.

The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.

Conventional insecticides targeting acetylcholinesterase (AChE) typically show high mammalian toxicities and because there is resistance to these compounds in many insect species, alternatives to established AChE inhibitors used for pest control are needed. Here we used a fluorescence method to monitor interactions between various AChE inhibitors and the AChE peripheral anionic site, which is a novel target for new insecticides acting on this enzyme. The assay uses thioflavin-T as a probe, which binds to the peripheral anionic site of AChE and yields an increase in fluorescent signal. Three types of AChE inhibitors were studied: catalytic site inhibitors (carbamate insecticides, edrophonium, and benzylpiperidine), peripheral site inhibitors (tubocurarine, ethidium bromide, and propidium iodide), and bivalent inhibitors (donepezil, BW284C51, and a series of bis(n)-tacrines). All were screened on murine AChE to compare and contrast changes of peripheral site conformation in the TFT assay with catalytic inhibition. All the inhibitors reduced thioflavin-T fluorescence in a concentration-dependent manner with potencies (IC50) ranging from 8 nM for bis(6)-tacrine to 159 μM for benzylpiperidine. Potencies in the fluorescence assay were correlated well with their potencies for enzyme inhibition (R2 = 0.884). Efficacies for reducing thioflavin-T fluorescence ranged from 23–36% for catalytic site inhibitors and tubocurarine to near 100% for ethidium bromide and propidium iodide. Maximal efficacies could be reconciled with known mechanisms of interaction of the inhibitors with AChE. When extended to pest species, we anticipate these findings will assist in the discovery and development of novel, selective bivalent insecticides acting on AChE.

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

Bis(12)-hupyridone (B12H), derived from the Chinese medicinal component huperzine A, was originally designed as a novel acetylcholinesterase (AChE) inhibitor. In this paper, we report that B12H (24-h pretreatment) effectively blocked glutamate-induced neuronal excitotoxicity in cerebellar granule neurons (CGNs). However, the huge discrepancy between the EC50 value and IC50 value of B12H, to protect against neuronal toxicity (0.09 μM) and to block the NMDA receptor (21.8 μM) respectively, suggests that the neuroprotection of B12H might be not primarily due to the blockade of the NMDA receptor. Pretreatment by specific antagonists of alpha7-nicotinic acetylcholine receptor (α7nAChR), but not muscarinic acetylcholine receptor (mAChR) or α4β2nAChR, decreased the neuroprotection of B12H. The neuroprotection of B12H could also be abolished by the pretreatment of specific PI3-K inhibitors. Furthermore, B12H restored the suppressed activation of the Akt pathway caused by glutamate as evidenced by the decreased expressions of pSer473-Akt and pSer9-GSK3β. All these results suggest that B12H substantially protected CGNs against glutamate-induced neuronal excitotoxicity via activating α7nAChR/PI3-K/Akt cascade.

Widespread pyrethroid resistance has caused an urgent need to develop new insecticides for control of the malaria mosquito, Anopheles gambiae. Insecticide discovery efforts were directed towards the construction of bivalent inhibitors that occupy both the peripheral and catalytic sites of the mosquito acetylcholinesterase (AChE). It was hypothesized that this approach would yield a selective, high potency inhibitor that would also circumvent known catalytic site mutations (e.g. G119S) causing target site resistance. Accordingly, a series of bivalent phthalimide-pyrazole carbamates were prepared having an alkyl chain linker of varying length, along with other modifications. The most active compound was (1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate, 8a), which has a chain length of three carbons, good mosquito anticholinesterase activity, and ca. 5-fold selectivity compared to human AChE. Moreover, this compound was toxic to mosquitoes by topical application (LD50 = 63 ng/female) with only 6-fold cross resistance in the Akron strain of Anopheles gambiae that showed 50- to 60-fold resistance to conventional carbamate insecticides. However, contact lethality in the WHO paper assay was disappointing. The implications of these results for design of new mosquitocides are discussed.

Great reductions in malaria mortality have been accomplished in the last 15 years, in part due to the widespread roll-out of insecticide-treated bednets across sub-Saharan Africa. To date, these nets only employ pyrethroids, insecticides that target the voltage-gated sodium ion channel of the malaria vector, Anopheles gambiae. Due to the growing emergence of An. gambiae strains that are resistant to pyrethroids, there is an urgent need to develop new public health insecticides that engage a different target and possess low mammalian toxicity. In this review, we will describe efforts to develop highly species-specific and resistance-breaking inhibitors of An. gambiae acetylcholinesterase (AgAChE). These efforts have been greatly aided by advances in knowledge of the structure of the enzyme, and two major inhibitor design strategies have been explored. Since AgAChE possesses an unpaired Cys residue not present in mammalian AChE, a logical strategy to achieve selective inhibition involves design of compounds that could ligate that Cys. A second strategy involves the design of new molecules to target the catalytic serine of the enzyme. Here the challenge is not only to achieve high inhibition selectivity vs human AChE, but also to demonstrate toxicity to An. gambiae that carry the G119S resistance mutation of AgAChE. The advances made and challenges remaining will be presented. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

Potential targets for new vector control insecticides are nerve and muscle potassium channels. In this study, the activities of known potassium channel blockers (4-aminopyridine, quinidine, and tetraethylammonium) and the insecticide propoxur were compared to three experimental catechols and several other compounds against Anopheles gambiae and Aedes aegypti mosquitoes. Experimental catechol 1 was the most toxic experimental compound in all of the mortality assays conducted, but was at least 100-fold and 39-fold less toxic than propoxur against Ae. aegypti and An. gambiae, respectively. Injection treatment and synergist (piperonyl butoxide) bioassays found that catechol toxicity was not unduly impacted by cuticular transport or oxidative metabolism. Electrophysiological studies showed a decrease in amplitude of evoked muscle contractions, along with an increase in twitch duration at concentrations that increased basal muscle tension (mM). High concentration effects on basal muscle tension were matched by complete depolarization of the muscle membrane potential. Effects on muscle physiology and blockage of Kv2.1 potassium channels in patch clamp experiments were generally consistent with in vivo toxicity, except for 4-aminopyridine, which suggest the involvement of other potassium channel subtypes. Extensive melanization of Anopheles larvae, but not Aedes larvae, occurred from exposure to catechol compounds. Interaction with the phenol oxidase system within insects may be the cause of this melanization, but any contribution to toxicity requires further investigation.

To identify potential human-safe insecticides against the malaria mosquito we undertook an investigation of the structure–activity relationship of aryl methylcarbamates inhibitors of acetylcholinesterase (AChE). Compounds bearing a β-branched 2-alkoxy or 2-thioalkyl group were found to possess good selectivity for inhibition of Anopheles gambiae AChE over human AChE; up to 530-fold selectivity was achieved with carbamate 11d . A 3D QSAR model is presented that is reasonably consistent with log inhibition selectivity of 34 carbamates. Toxicity of these compounds to live Anopheles gambiae was demonstrated using both tarsal contact (filter paper) and topical application protocols.

In the course of a β-site APP-cleaving enzyme 1 (BACE1) inhibitor discovery project an in situ synthesis/screening protocol was employed to prepare 120 triazole-linked reduced amide isostere inhibitors. Among these compounds, four showed modest (single digit micromolar) BACE1 inhibition. Our ligand design was based on a potent reduced amide isostere 1, wherein the P2 amide moiety was replaced with an anti-1,2,3-triazole unit. Unfortunately, this replacement resulted in a 1000-fold decrease in potency. Docking studies of triazole-linked reduced amide isostere A3Z10 and potent oxadiazole-linked tertiary carbinamine 2a with BACE1 suggests that the docking poses of A3Z10 and 2a in the active sites are quite similar, with one exception. In the docked structures the placement of the protonated amine that engages D228 differs considerably between 2a and A3Z10. This difference could account for the lower BACE1 inhibition potency of A3Z10 and related compounds relative to 2a.

The cause of many neurodegenerative disorders can be ascribed to the loss of functional neurons, and thus agents capable of promoting neuronal differentiation may have therapeutic benefits to patients of these disorders. In this study, the effects and underlying mechanisms of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase inhibitor modified from huperzine A (HA), on neuronal differentiation were investigated using both the rat PC12 pheochromocytoma cell line and adult rat hippocampus neural stem cells. B12H (3-30 μM), characterized by morphological changes and expression of GAP-43, induced neurite outgrowth in a concentration- and time-dependent manner, with almost 3-fold higher efficacy than that of HA in PC12 cells. Furthermore, B12H (2.5-10 μM), but not HA, promoted neuronal differentiation as shown by the percentage increase of βIII-tubulin positive neurons in neural stem cells. The activities of extracellular signal-regulated kinase (ERK), as well as its downstream transcription factors Elk-1 and cAMP response element-binding protein (CREB) were elevated in the B12H-treated PC12 cells. Mitogen-activated protein kinase kinase inhibitors and alpha7-nicotinic acetylcholine receptor (α7nAChR) antagonist blocked the neurite outgrowth and the activation of ERK induced by B12H. All these findings suggest that B12H potently induces pro-neuronal cells into differentiated neurons by activating the ERK pathway possibly via regulating α7nAChR. These findings support the recent proposition that α7nAChR is required for the neuronal dendritic arborization and differentiation in the adult mice hippocampus, and provide insights into the possible therapeutic potential of B12H in treating neurodegenerative disorders.

Oxidative stress-induced apoptosis plays a critical role in the pathogenesis of various neurodegenerative disorders. In this study, the neuroprotective properties of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase (AChE) inhibitor modified from a naturally occurring monomeric analogue, huperzine A, on H 2 O 2 -induced neurotoxicity were investigated in cerebellar granule neurons (CGNs). Exposure of CGNs to H 2 O 2 resulted in apoptosis which could be attenuated by the pre-treatment of B12H (0.3–5 nM) in a concentration-dependent manner. Moreover, tacrine and neostigmine failed to prevent neurotoxicity, indicating that the neuroprotection of B12H might not be due to its inhibitory property of AChE enzymatic activity. Increased activation of extracellular signal-regulated kinase (ERK) and decreased activation of glycogen synthase kinase (GSK) 3β were observed after H 2 O 2 exposure, and B12H reversed the altered activation of GSK3β, but not that of ERK. Furthermore, using vascular endothelial growth factor (VEGF), phospho-VEGF receptor-2 (VEGFR-2) antibody, a specific VEGFR-2 inhibitor (PTK787/ZK222584) and specific phosphoinositide 3-kinase inhibitors (LY294002 and wortmannin), it was found that VEGF prevented H 2 O 2 -induced neuronal loss from activating the VEGF/VEGFR-2 system and that the observed B12H neuroprotective effects might share the same signaling pathway. These findings strongly suggest that B12H prevents H 2 O 2 -induced neuronal apoptosis independent of inhibiting AChE, but through regulating VEGFR-2/Akt/GSK3β signaling pathway.

Glycine-derived 1H-benzo[e][1,4]diazepin-2(3H)-ones (BZDs) 5d-g featuring C9- and N1- substitution exhibit enantiomerization barriers too high to be measured by (1)H NMR coalescence experiments. To address this problem, we found that room-temperature H/D exchange of these compounds is remarkably selective, affording only the axial-d(1) isotopomers. (1)H NMR spectroscopy was then employed to measure the rate of conformational inversion of these d(1)-compounds at elevated temperatures. These studies reveal the highest enantiomerization barriers (up to 28 kcal/mol) ever determined for a BZD. Density functional theory calculations match the experimental enantiomerization barriers within 1.2 kcal/mol.

Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species-selectivity in the rational design of improved insecticides for malaria vector control.

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C 18 5 μm column (length, 250 mm; inner diameter, 4.6 mm) using a mixture of water–acetonitrile–trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1 mL/min, with detection at 229 nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1–7.5 μg/mL with r 2 = 0.9991. The limit of detection and quantification of this method were 0.05 μg/mL and 0.1 μg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5 mg/kg and intraperitoneal administration of 10 and 20 mg/kg in rats.

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

Due to the putative involvement of dopaminergic circuits in depression, triple reuptake inhibitors are being developed as a new class of antidepressant, which is hypothesized to produce a more rapid onset and better efficacy than current antidepressants selective for serotonin or norepinephrine neurotransmission. ( 1S,2S )-3-(Methylamino)-2-(naphthalen-2-yl)-1-phenylpropan-1-ol (PRC200-SS), a new triple reuptake inhibitor, potently bound to the human serotonin, norepinephrine, and dopamine transporters with K d values of 2.3, 0.63, and 18 nM, respectively. Inhibition of serotonin, norepinephrine, and dopamine uptake by PRC200-SS was also shown in cells expressing the corresponding transporter ( K i values of 2.1, 1.5, and 61 nM, respectively). In vivo, PRC200-SS dose-dependently decreased immobility in the forced-swim test in rats and in the tail-suspension test in mice, models predictive of antidepressant activity, with effects comparable with imipramine. These results in the behavioral models did not seem to result from the stimulation of locomotor activity. Consistent with the in vitro data and behavioral effects, peripheral administration of PRC200-SS (5 and 10 mg/kg i.p.) significantly increased extracellular levels of serotonin and norepinephrine in the medial prefrontal cortex, and of serotonin and dopamine in the core of nucleus accumbens, with reduction of levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid compared with levels for saline control. Furthermore, PRC200-SS self-administration, which was used as a marker of abuse liability, was not observed with rats. Therefore, it seems that PRC200-SS may represent a novel triple reuptake inhibitor and possess antidepressant activity.

The lipophilicity and solubility profiles of bis(12)-hupyridone (B12H) and bis(7)-tacrine (B7T), two novel acetylcholinesterase inhibitors dimerized from huperzine A fragments and tacrine, respectively, were investigated over a broad pH range. Lipophilicity was assessed by both shake flask method with 1-octanol-water system and a reverse-phase HPLC system with methanol-water as mobile phase. The former method was used for determining the lipophilicities of the ionized forms (log D) of the dimers while the latter method was used for that of the neutral forms (log P). The log P values for B12H and B7T were found to be 5.4 and 8.2, respectively, indicating that the two dimers are highly lipophilic. The solubilities of both dimers were found to be affected by pH. The solubility of B12H was1.41 mg/ml when the pH was7, but0.06 mg/ml when the pH was8. The solubility of B7T was0.26 mg/ml when the pH was9, but0.005 mg/ml when the pH was12. The ionic strength of a solution could affect the solubilities considerably (11.16 mg/ml for B12H and 12.71 mg/ml for B7T in water; 2.07 mg/ml for B12H and 0.36 mg/ml for B7T in saline). The ionization constants (pK(a)) of the two dimers were determined by UV spectrophotometry. Both dimers were found to have two pK(a) values: 7.5+/-0.1 (pK(a1)) and 10.0+/-0.2 (pK(a2)) for B12H; and 8.7+/-0.1 (pK(a1)) and 10.7+/-0.4 (pK(a2)) for B7T. Furthermore, an in vivo pharmacological assay conducted in mice showed that a maximum AChE inhibition occurred 15 min after the single-dose and intraperitoneal administration of either dimer. This indicates that the two dimers may easily cross the blood-brain barrier. In summary, these physiochemical characteristics suggest that the two dimers may be promising candidates for the development of better drugs for Alzheimer's disease.

[structure: see text]. Magnesium-bromine exchange on enantiopure cyclopropyl bromonitrile 5 at -100 degrees C for 1 min followed by a D2O quench gives the deuterionitrile in 81% ee (retention); additional trapping experiments establish t(1/2)(rac) = 11.4 h at -100 degrees C. These experiments provide the first glimpse into the stereochemical aspects of Mg-Br exchange. The intermediate formed is the first metalated nitrile demonstrated to possess macroscopic configurational stability.