Cross resistance to brevetoxin-3 by kdr and super-kdr mutations in house flies.

The dinoflagellate Karenia brevis is a causative agent of red tides in the Gulf of Mexico and generates a potent family of structurally related brevetoxins that act via the voltage-sensitive Na+ channel. This project was undertaken to better understand the neurotoxicology and kdr cross-resistance to brevetoxins in house flies by comparing the susceptible aabys strain to ALkdr (kdr) and JPskdr (super-kdr). When injected directly into the hemocoel, larvae exhibited rigid, non-convulsive paralysis consistent with prolongation of sodium channel currents, the known mechanism of action of brevetoxins. In neurophysiological studies, the firing frequency of susceptible larval house fly central nervous system preparations showed a > 200% increase 10 min after treatment with 1 nM brevetoxin-3. This neuroexcitation is consistent with the spastic paralytic response seen after hemocoel injections. Target site mutations in the voltage-sensitive sodium channel of house flies, known to confer knockdown resistance (kdr and super-kdr) against pyrethroids, attenuated the effect of brevetoxin-3 in baseline firing frequency and toxicity assays. The rank order of sensitivity to brevetoxin-3 in both assays was aabys > ALkdr > JPskdr. At the LD50 level, resistance ratios for the knockdown resistance strains were 6.9 for the double mutant (super-kdr) and 2.3 for the single mutant (kdr). The data suggest that knockdown resistance mutations may be one mechanism by which flies survive brevetoxin-3 exposure during red tide events.

Sodium channel-directed alkaloids synergize the mosquitocidal and neurophysiological effects of natural pyrethrins.

We explored the potential of two sodium channel activators, veratrine and aconitine, as both insecticides and synergists of natural pyrethrins (NP) on Aedes aegypti adults and larvae. Aconitine was more toxic than veratrine, with an LD50 of 157 ng/mg compared to 376 ng/mg, on the pyrethroid-susceptible Orlando strain, but only aconitine showed significant resistance in the pyrethroid-resistant Puerto Rico strain (RR = 14.6 in topical application and 8.8 in larval bioassay). When applied in mixtures with piperonyl butoxide (PBO) and NP, large synergism values were obtained on the Orlando strain. Aconitine + PBO mixture synergized NP 21.8-fold via topical adult application and 10.2-fold in larval bioassays, whereas veratrine + PBO synergized NP 5.3-fold via topical application and 30.5-fold in larval bioassays. Less synergism of NP was observed on the resistant Puerto Rico strain, with acontine + PBO synergizing NP only 4.1-fold in topical application (8-fold in larval bioassays) and veratrine + PBO synergizing NP 9.5-fold in topical application (13.3-fold in larval bioassays). When alkaloids were applied directly to the mosquito larval nervous system, veratrine was nearly equipotent on both strains, while aconitine was less active on pyrethroid-resistant nerve preparations (no block at 10 µM compared to block at 1 µM on the susceptible strain). The nerve blocking effect of NP was significantly synergized by both compounds on the pyrethroid-susceptible strain by about 10-fold, however only veratrine synergized NP block on the pyrethroid-resistant strain, also showing 10-fold synergism). These results highlight the potential of site II sodium channel activators as insecticides and their ability to synergize pyrethroids, which may extend the commercial lifetime of these chemistries so essential to public health vector control.

Mode of action and toxicological effects of the sesquiterpenoid, nootkatone, in insects.

Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD50 and LD50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 µM, whereas chloride current was inhibited 4.5-fold less at 100 µM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism.

Development of an a priori computational approach for brain uptake of compounds in an insect model system.

Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure-property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r2 > 0.5, with the descriptors log(P*MW-0.5) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW-0.5) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines.

Enhanced pyrethroid potency in Drosophila melanogaster expressing voltage-gated potassium channel mutants: Insecticidal activity and neuronal action.

Previous studies have shown that blockers of voltage-gated potassium (Kv) channels, such as 4-aminopyridine (4-AP) and 2-methoxy-N-((1-phenylcyclopentyl)methyl)benzamide (2-MPB) synergized pyrethroid toxicity as well, or better than, piperonyl butoxide. The present study assessed the involvement of different Kv channels as possible pyrethroid synergist targets in Drosophila melanogaster. Three Kv1 mutants (Sh5, Sh133, and ShM) and one Kv2 mutant (Shab3) were tested. All Kv1 mutant flies showed increased sensitivity to permethrin in topical and glass contact toxicity assays, of 2- to 11-fold. Central nervous system (CNS) recordings of larval D. melanogaster showed a similar pattern of increased sensitivity. Potentiated effects were also observed with deltamethrin on the mutants Sh5 (30- to 35-fold) and Sh133 (33- to 47-fold), but the mutant ShM showed little change in sensitivity. In contrast, the Shab3 strain showed toxicity and physiological effects of both pyrethroids that were similar to the susceptible OR strain. Thus, some K+ channel mutations mimicked the synergistic effect of channel blockers. Additional studies showed that Shab3 had the highest sensitivity to 4-AP in topical assays, and the Shaker-null mutants, ShM and Sh133 showed greater sensitivity to 2-MPB in CNS recordings of larval D. melanogaster. These results suggest that Kv1 channels are a useful synergist target for pyrethroids, as assessed both in whole insects and at the level of the nervous system. Thus, Kv1-targeting compounds can potentially serve as insect control tools to reduce pyrethroid use via synergistic action.

Mosquitocidal activity of p,p'-difluoro-diphenyl-trichloroethane (DFDT).

New insecticides are urgently needed for the control of arthropod vectors of public health diseases. As resistance to many insecticides used for the control of public health pests is ubiquitous, all available chemistries should be evaluated for their potential to effectively control both insecticide-susceptible and insecticide-resistant strains of mosquitoes. This study aimed to evaluate p-p'-difluoro-diphenyl-trichloroethane (DFDT) as a mosquito control technology and relate its activity to that of DDT. We found that topical DFDT was significantly less toxic than DDT to both pyrethroid-susceptible and pyrethroid-resistant strains of Anopheles gambiae and Aedes aegypti. Direct nervous system recording from Drosophila melanogaster CNS demonstrated that DFDT is approximately 10-times less potent than DDT at blocking nerve firing, which may explain its relatively lower toxicity. DFDT was shown to be at least 4500 times more vapor-active than DDT, with an LC50 in a vapor toxicity screening assay of 2.2 µg/cm2. Resistance to DFDT was assessed in two mosquito strains that possess target-site mutations in the voltage-gated sodium channel and upregulated metabolic activity. Resistance ratios for Akdr (An. gambiae) and Puerto Rico (Ae. aegypti) strains were 9.2 and 12.2, respectively. Overall, this study demonstrates that DFDT is unlikely to be a viable public health vector control insecticide.

Voltage-gated chloride channel blocker DIDS as an acaricide for Varroa mites.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

Insecticidal and repellent properties of novel trifluoromethylphenyl amides III.

The adulticidal, larvicidal, and repellent activity of 18 trifluoromethylphenyl amides (TFMPAs) was determined against Aedes aegypti mosquitoes. The compounds studied are the third generation designed from active structures of the previous two generations. N-(3,5-Bis(trifluoromethyl)phenyl)-2-chloroacetamide (8f) and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (8h) were most active against 1st stage Ae. aegypti larvae with LC50 values of 125 and 2.53 µM; for comparative purposes, the published LC50 for fipronil is 0.014 µM. Compound 8h was the most toxic against adult female Ae. aegypti with an LD50 = 2.12 nmol/mg, followed by 8f, and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (8g) with LD50 values of 4.27 and 4.73 nmol/mg, respectively, although these compounds were significantly less toxic than fipronil against adult female Ae. aegypti. Compounds N-(2-(trifluoromethyl)phenyl)butyramide (9c), N-(2-(trifluoromethyl)phenyl)pentanamide (9d) and N-(2-(trifluoromethyl)phenyl)hex-5-enamide (9e) were the best repellents for female Ae. aegypti, with minimum effective dosages (MEDs) of 0.026, 0.052, and 0.091 µmol/cm2, respectively, compared to DEET at 0.052 µmol/cm2. Out of 52 TFMPAs (total number of compounds from three generations of this research) compound 9c was the most active repellent along with two synthesized in our previous studies, 2-chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) and 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c).

The impacts of chlorothalonil and diflubenzuron on Apis mellifera L. larvae reared in vitro.

Chlorothalonil is a broad-spectrum fungicide and diflubenzuron is an insect growth regulator used to control many insect larvae feeding on agricultural, forest and ornamental plants. Honey bee larvae may be exposed to both via contaminated pollen, in the form of beebread, added to their diet by their adult nurse sisters. In this study, we determined how single (acute: 72 h mortality) and repeated (chronic: mortality until emergence as adults) exposure to chlorothalonil and diflubenzuron in their diet affected honey bee larvae reared in vitro. The tested doses of chlorothalonil (20, 100, or 200 mg/L) did not impact 72 h larval mortality acutely relative to that of the solvent control. The 72 h mortality of larvae exposed to 1.6 mg/L and higher doses of diflubenzuron acutely in their diet (47.2-63.9% mortality) was significantly higher than that of larvae fed the solvent control, with no predictable dose dependent pattern observed. In the chronic toxicity tests, consuming an artificial diet with 30 or 100 mg/L chlorothalonil and 0.8, 1.3 or 2 mg/L diflubenzuron significantly lowered the survival of honey bee larvae over that of larvae feeding on the solvent control diet. We calculated risk quotients (RQs) for both compounds using the data we generated in our experiments. Collectively, the RQs suggest that neither compound is likely to affect larval mortality directly at field relevant doses given that pollen composes only a fraction of the total larval diet. Nevertheless, our data do not preclude any sublethal effects that chronic exposure to either compound may cause.

Pharmacology of central octopaminergic and muscarinic pathways in Drosophila melanogaster larvae: Assessing the target potential of GPCRs.

G-Protein-Coupled Receptors (GPCRs) are an underdeveloped target in the search for agrochemicals with octopamine receptors, a GPCR, being the target of a single insecticide/acaricide class (formamidines). The evolution of insecticide resistance has resulted in the need to identify new or underutilized targets for the development of agrochemicals, with the goal of controlling arthropod pests that affect agriculture or human and animal health. The insect cholinergic system has been a fruitful target for the development of insecticides/acaricides viz. acetylcholinesterase inhibitors and agonists/modulators of the nicotinic acetylcholine receptor. However, the muscarinic acetylcholine receptors (mAChRs), which are GPCRs, have not been successfully developed as a target for agrochemicals. Others have recently identified three subtypes of insect mAChRs in Drosophila melanogaster, and extracellular recordings from transected D. melanogaster larval central nervous system (CNS) were performed to investigate the electrogenesis of the octopaminergic and muscarinic systems. Octopamine (10 µM) resulted in a sustained neuroexcitation during a 30 min exposure, and neuroexcitation after 21 min was blocked by octopamine receptor antagonist, phentolamine (100 µM). Exposure of this preparation to the non-selective mAChR agonist, pilocarpine (10 µM), resulted in a biphasic response, characterized by neuroexcitation followed by a decrease in the CNS firing rate below initial control levels. This biphasic effect was antagonized by the classical mAChR antagonist atropine (10 µM). It was also found that atropine (10 µM) blocked octopamine's sustained neuroexcitation, indicating the possibility of cross-talk between these two GPCR pathways.

Toxicity, mode of action, and synergist potential of flonicamid against mosquitoes.

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.

Select ß- and γ-branched 1-alkylpyrazol-4-yl methylcarbamates exhibit high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

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 ß- 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 side chain 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.

Insecticidal and repellent properties of novel trifluoromethylphenyl amides II.

This project focused on the design, synthesis, and testing of trifluoromethylphenyl amides (TFMPAs) as potential mosquitocides and repellents. Fourteen compounds were evaluated for toxicity against larvae and adults of Aedes aegypti. Several compounds were toxic against Aedes aegypti (larval, adult and feeding bioassays) and Drosophila melanogaster (glass-surface contact assay), but were much less toxic than fipronil, with toxicity ratios ranging from 100-fold in the larval assay to 100,000-fold for topical application to adult insects. In repellency bioassays to determine minimum effective dosage (MED), compound N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (7b) repelled Ae. aegypti females at lower concentration, 0.017 (±0.006) µmol/cm2, than N, N-diethyl-meta-toluamide (DEET) 0.026 (±0.005) µmol/cm2. 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) performed better than DEET against two species of mosquitoes: it repelled Ae. aegypti females at 0.013 (±0.006) µmol/cm2 and Anopheles gambiae females (in a warm body repellent assay), at a standard exposure of 2 nmol/cm2. These studies revealed novel active structures that could further lead to compounds with better repellent activity.

Amitraz and its metabolite modulate honey bee cardiac function and tolerance to viral infection.

The health and survival of managed honey bee (Apis mellifera) colonies are affected by multiple factors, one of the most important being the interaction between viral pathogens and infestations of the ectoparasitic mite Varroa destructor. Currently, the only effective strategy available for mitigating the impact of viral infections is the chemical control of mite populations. Unfortunately, the use of in-hive acaricides comes at a price, as they can produce sublethal effects that are difficult to quantify, but may ultimately be as damaging as the mites they are used to treat. The goal of this study was to investigate the physiological and immunological effects of the formamidine acaricide amitraz and its primary metabolite in honey bees. Using flock house virus as a model for viral infection, this study found that exposure to a formamidine acaricide may have a negative impact on the ability of honey bees to tolerate viral infection. Furthermore, this work has demonstrated that amitraz and its metabolite significantly alter honey bee cardiac function, most likely through interaction with octopamine receptors. The results suggest a potential drawback to the in-hive use of amitraz and raise intriguing questions about the relationship between insect cardiac function and disease tolerance.

N'-mono- and N, N'-diacyl derivatives of benzyl and arylhydrazines as contact insecticides against adult Anopheles gambiae.

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.

Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line.

The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1µM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50=116nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening.

Impact of Topical Application Site On the Efficacy of Permethrin and Malathion To Culex quinquefasciatus.

Although insecticide spray droplets will potentially impinge on many exoskeletal body regions, traditional mosquito topical bioassays focus insecticide application to the mesothoracic pleural or dorsal area. Concentrations of permethrin and malathion found in droplets from ultra-low volume and low-volume sprays were evaluated for efficacy against adult Culex quinquefasciatus using a topical application bioassay. Results document nonuniform insecticide sensitivity across body regions, which has not been previously assessed in mosquitoes. Insecticide contact with appendages, such as the leg and the wing, returned much lower mortality from both insecticides than exposure to the primary body (i.e., head, thorax, and abdomen). No difference was observed in percent mortality 24 h after exposure to different insecticides to the same body region. Sublethal behaviors were also observed and discussed for both insecticides. Our findings provide valuable information for those performing topical bioassays, and may help explain insecticide effectiveness wherever droplets impinge upon the mosquito body during laboratory or field applications.

Alkaloids with Activity against the Zika Virus Vector Aedes aegypti (L.)-Crinsarnine and Sarniensinol, Two New Crinine and Mesembrine Type Alkaloids Isolated from the South African Plant Nerine sarniensis.

Two new Amaryllidaceae alkaloids, belonging to the mesembrine- and crinine-types, named crinsarnine (1) and sarniensinol (2), were isolated from the dried bulbs of Nerine sarniensis together with bowdensine (3), sarniensine (4), hippadine (5) and 1-O-acetyl-lycorine (6). Crinsarnine (1) and sarniensinol (2) were characterized using spectroscopic and chiroptical methods as (1S,2S,4aR,10bS)-2,7-dimethoxy-1,2,3,4,4a,6-hexahydro-5,11b-ethano[1,3]dioxolo-[4,5-j]phenanthridin-1-yl acetate and (6-(3aR,4Z,6S,7aS)-6-methoxy-1-methyl-2,3,3a,6,7,7a-hexa-hydro-1H-indol-3a-yl)benzo[d][1,3]dioxol-5-yl)methanol, respectively. Furthermore, the complete spectroscopic characterization of bowdensine (3) is reported for the first time. Compounds 1-6 were evaluated against the Orlando reference strain of Aedes aegypti. None of compounds showed mortality against 1st instar Ae. aegypti larvae at the concentrations tested. In adult topical bioassays, only 1 displayed adulticidal activity with an LD50 = 2.29 ± 0.049 µg/mosquito. As regards the structure-activity relationship, the pretazettine and crinine scaffold in 2 and 4 and in 1 and 3 respectively, proved to be important for their activity, while the pyrrole[de]phenanthridine scaffold present in 5 and 6 was important for their reactivity. Among the pretazettine group compounds, opening of the B ring or the presence of a B ring lactone as well as the trans-stereochemistry of the A/B ring junction, appears to be important for activity, while in crinine-type alkaloids, the substituent at C-2 seems to play a role in their activity.

Bivalent Carbamates as Novel Control Agents of the Malaria Mosquito, Anopheles gambiae.

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.

Difluoromethyl ketones: Potent inhibitors of wild type and carbamate-insensitive G119S mutant Anopheles gambiae acetylcholinesterase.

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 A. 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.