Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.


E.
Enzyme activity measurements Protein concentrations were determined using the Thermo Scientific Micro BCA TM Protein Assay Kit (#23235) microplate procedure, and linear working range of 2-40 µg/ml. Wild-type enzymes (rhAChE, G3 homogenate, and rAgAChE-WT) were diluted in ice-cold buffer B and observed in the Ellman assay (described below) to give an approximate reaction rate of 0.040 O.D./min (or ~0.0049 U/ml). Resistant enzymes (Akron homogenate and rAgAChE-G119S) were diluted similarly and observed at a reaction rate of approximately 0.028 O.D./min (or 0.0034 U/ml). All diluted enzymes were kept over ice prior to use. Aliquots of the diluted enzymes (10 µl) thus prepared were separately incubated, in triplicate, in a 96-well microplate with buffer A (170 µl) for 10 min. Thereafter, a freshly prepared solution of ATCh and DTNB (4 and 3 mM respectively, in buffer A, 20 µl), was added and mixed manually to start the enzymatic reaction. Thus, a total volume of 200 µl and optical pathlength of 0.60 cm was achieved in each cell, with the following final concentrations: 0.015% (v/v) Triton X-100, 0.05 mg/ml BSA, 0.4 mM ATCh, 0.3 mM DTNB. Based on our protein concentration calculations, final enzyme concentrations employed in the activity and inhibition assays ranged from 29 to 36 pM (rAgAChE-WT), 0.7 to 1.1 nM (rAgAChE-G119S), and 18 to 23 pM (rhAChE). Enzyme activity was monitored continuously at 405 nm at room temperature (23 ± 1°C) for up to 3.4 min on a DYNEX Triad microplate reader (DYNEX Technologies, Chantilly, VA, USA), analyzed using the Concert TRIAD Series Analysis Software (version 2.1.0.17), corrected for spontaneous substrate hydrolysis. A molar extinction coefficient ε 405 of 13,600 M -1 cm -1 was used for 2mercapto-5-nitrobenzoic acid, the chromophore formed from the reaction of thiocholine and DTNB [3]. One unit (U) of AChE activity is defined as that amount of the enzyme that catalyzes the hydrolysis of 1 µmole of ATCh substrate per minute. For determination of k cat and K m , typically 8 concentrations of ATCh were employed (in duplicate). All V max and K m values were calculated by fitting the velocity (v) and substrate concentration ([S]) data to v = V max [S] / (K m + [S]) using Prism 5.0 (GraphPad Software Inc, La Jolla, CA, USA).

F. Determination of apparent second-order rate constants (k i ) of for enzyme inactivation by carbamate inhibitors.
Inhibition potency of carbamate insecticides was assessed by measuring apparent second-order rate constants k i (mM -1 min -1 ) for inactivation of the enzymes. We adopted a progressive inactivation approach [4,5], in which enzymes were incubated with different concentrations of carbamates for differing times before measuring enzyme residual activity (v/v 0 ). The plate loading protocol described in Section E above was used with slight modification. Instead of adding 170 µL of buffer A, 150 µL of buffer A and 20 µL of an inhibitor solution was added. The inhibitor solutions were prepared in buffer A with a DMSO concentration of 1% (v/v); the final assay concentration of DMSO was thus 0.1% (v/v). After waiting for the desired incubation time t, substrate and indicator were added as described in Section E above, and the enzymatic reaction was followed at 405 nm. As described above, enzymes were incubated with typically five concentrations of inhibitors (and an inhibitor-free control) for up to 6 minutes at approximately 1 min intervals. Each concentration was present in the microplate in duplicate, and each experiment was repeated. Note that for the G119S enzymes (recombinant and Akron homogenate) inhibition was typically very low after 10 min at 10 µM, and so incubations with these enzymes were often extended to 60 min (10 min intervals). Residual activities v/v 0 are the ratio of the rate in the presence of inhibitor to a time-matched inhibitor-free control. This method therefore corrects for slow thermal inactivation of the enzyme. As described in the Results Section of this manuscript, these residual activities were used to calculate pseudo firstorder rate constants k obs (in duplicate) for inactivation at three or more inhibitor concentrations ([I]). Plots of k obs vs [I] were then constructed as shown in Figure 5; in each case the slope of the unconstrained linear fit is the apparent second-order rate constant k i (mM -1 min -1 ) for inactivation. The error in k i is estimated as the standard error in the slope of this plot. Note that inhibitor concentrations [I] were chosen to be low enough to remain in the domain where a plot of k obs vs [I] was linear. For fast-inactivating carbamates, such as terbam on rAgAChE-WT or G3 homogenate, we saw signs of saturation behavior above [I] = 0.2 µM. Saturation behavior is expected for a two-step mechanism of inhibition, such as that depicted in Figure 4 [6]. Resistance ratios and Ag/h selectivity for inhibition (Table 3) were calculated from the measured k i values; the error in these ratios was determined using a standard propagation of error method [7].

G.
Determination of carbamate toxicity to live An. gambiae Adult female non-blood fed An. gambiae (both G3 and Akron strains) 3-5 days old, were used for filter paper assays of toxicity, which were performed in exposure tubes according to the 2006 World Health Organization recommendations, with slight modification [8]. In brief, filter papers (15 x 12 cm) were treated with 2.0 mL of various concentrations of the carbamate in ethanol, and allowed to dry overnight. For the G3 strain, batches of 20-25 mosquitoes (in triplicate) were transferred to a holding tube and allowed to adapt for one hour. Due to lower colony numbers, toxicity measurements with Akron strain used batches of 10-15 mosquitoes in duplicate. Mosquitoes were then transferred to the exposure tube (held horizontally) that contained a treated filter paper. Knockdown was noted after 1 h, and all mosquitoes were transferred back to the holding tube (held upright), and given free access to 10% (w/v) sugar water. Mortality was recorded at 24 h. Both during exposure and the 23 h period following, mosquito tubes were kept in an environmental chamber at 24 ± 1 °C and 75% RH. To determine LC 50 values, typically 5-8 concentrations were examined, and mortality data were used for probit analysis using PoloPlus [9].

H. Synthetic procedures and analytical characterization for pyrazol-4-yl methylcarbamates
Analytical and purification protocols. NMR spectra were obtained at 500 ( 1 H) and 126 ( 13 C) MHz . The chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. High-resolution ESI Mass spectra were obtained on an Agilent 6220 Accurate Mass TOF LC/MS. THF was distilled from sodiumbenzophenone and dichloromethane was distilled from calcium hydride. Other reagents and solvents were purchased from commercial sources and were used without further purification. Silica gel column chromatography was performed using flash silica gel (32-63 µ). Preparative thin-layer chromatography (PTLC) separations were carried out on 1000 µ Uniplate thin layer chromatography plates.

General procedure for the synthesis of 1-alkyl-4-iodopyrazoles (2a-e):
To a mixture of sodium hydride (60% dispersion in mineral oil, 520 mg, 13 mmol) in DMF (4 mL) at 0 °C was added dropwise a solution of pyrazole (680 mg, 10 mmol) in DMF (1 mL), and the subsequent mixture was kept stirring at room temperature for 1 h. To this mixture was added alkyl bromide (15 mmol), and the reaction was allowed to proceed at room temperature (25 °C) for 16 h. The reaction mixture was diluted with diethyl ether, which was rinsed with brine. After removing the diethyl ether, the crude product was directly used for the next step reaction without further purification. To a mixture of 1-alkylpyrazole (nominally 10 mmol) in water (7 mL) was sequentially added iodine (7 mmol) and 35% hydrogen peroxide (8.4 mmol), and the mixture was kept stirring for 24 h at room temperature. The reaction was quenched by the addition of a cold solution of saturated sodium thiosulfate (60 mL), and the subsequent mixture was extracted with ethyl acetate (3 x 50 mL). The combined extracts were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue obtained was chromatographed over silica gel eluting with 5% ethyl acetate in hexanes to furnish the respective 1-alkyl-4-iodopyrazole (2a-e).

General procedure for the synthesis of 1-alkyl-4-hydroxypyrazoles (3a-e):
A thick-walled sealable reaction tube was charged with CuI (9.5 mg, 0.05 mmol), 3,4,7,8-tetramethyl-1,10phenantholine (24 mg, 0.10 mmol), Cs 2 CO 3 (490 mg, 1.5 mmol), N-alkyl-4-iodopyrazole (1.0 mmol), benzyl alcohol (0.16 mL, 1.5 mmol), toluene (0.50 mL), and a magnetic stir bar. The was purged with dry nitrogen and then quickly sealed with a teflon screwcap. The vessel was immersed in a 80 °C oil bath and stirred vigorously for 18 h. The reaction mixture was allowed to cool to room temperature, diluted with ethyl acetate (15 mL), filtered through a plug of silica gel, and further eluted with additional ethyl acetate (30 mL). The filtrate was concentrated and the resulting residue was purified by column chromatography over silica gel (10% ethyl acetate in hexanes) to provide the desired 1-alkyl-4-benzyloxypyrazole. This material (nominally 1 mmol) was then dissolved in methanol (7 mL), to which was sequentially added 10% Pd-C (50% wet, 230 mg) and ammonium formate (315 mg, 5 mmol), and the reaction was allowed to proceed under reflux for 16 h. After cooling to room temperature the reaction was filtered through a silica gel pad, which was then rinsed with 100 mL of ethyl acetate. The filtrate was evaporated under reduced pressure to afford the desired products 3a-e. The physical and spectroscopic data of the 1-alkyl-4-hydroxypyrazoles are listed below:

General procedure for the synthesis of 1-alkylpyrazol-4-yl methylcarbamates (4a-e):
This procedure is loosely based on the 7-hydroxycoumarin alanine methyl ester carbamate protocol of Seto [11]. To a solution of triphosgene (99 mg, 0.33 mmol, 0.33 equiv) in dichloromethane (10 mL) was slowly added a solution of 1-alkyl-4-hydroxypyrazole (1 mmol, 1 equiv) and diisopropylethylamine (1 mmol, 1 equiv) in THF (10 mL), and the mixture was stirred at room temperature for 30 min. To the reaction mixture, a solution of methylamine (2M in THF, 1.5 mmol, 1.5 equiv) and diisopropylethylamine (2.25 mmol, 2.25 equiv) in dichloromethane (2 mL) was added, and the reaction was allowed to proceed for an additional 30 min. The subsequent reaction mixture was partitioned between dichloromethane and water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue obtained was purified by column chromatography eluting with 20% ethyl acetate in hexanes to give the respective carbamate. The physical and spectroscopic data of the carbamates are listed below: