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My graduate work focused on the syntheses and pharmacology of species-selective aryl methylcarbamate acetylcholinesterase inhibitors to combat the malaria-transmitting mosquito, Anopheles gambiae. We identified six novel carbamates that demonstrated levels of target selectivity exceeding our project milestone of 100-fold. Among the C2-substituted phenylcarbamates examined (class II), 2'-(2- ethylbutoxy)phenyl N-methylcarbamate (9bd*) was extraordinarily selective (570-fold ± 72). The high level of selectivity observed for many of the class II carbamates was attributed to a helical displacement within the active site of An. gambiae acetylcholinesterase, able to accommodate carbamates with larger C2-substituted secondary β-branching side chains. Conversely, this type of side chain forms unfavorable interactions within the active site of human acetylcholinesterase. The C3-substituted carbamates (class I), such as terbam (9c), were less selective than many of the class II carbamates; however, class I carbamates related to terbam (9c) were highly toxic to An. gambiae. In particular, the contact toxicity measured for 9c (LC₅₀ = 0.037 mg/mL) was equal to the commonly used agricultural insecticide, propoxur (9a, LC₅₀ = 0.037 mg/mL). In total, seventy aryl carbamates were screened for their inhibition potency and contact toxicity towards An. gambiae. The common final step in all of these syntheses was the carbamoylation of a phenol, which normally proceeded in a 70 to 90% yield. Thirty seven novel carbamates are reported out of the seventy two prepared. Although sixteen of the phenols were commercially available, the others were prepared with known and adapted synthetic methodologies. The emerging structure-activity relationships led us to focus on the synthesis of 3-tert-alkylphenols (Class I) and 2-alkoxy or 2-alkylthio-substituted phenols (Class II). Three methods particularly stand out: First, we applied the methods of Tanaka to prepare 3-tert-alkylphenols wherein a methyl group was replaced by a trifluoromethyl group. Second, we adapted the methods of Tanaka to prepare 3-tert-alkylphenols that lack fluorine substitution. This method is competitive with the little known method of Reetz to convert aryl ketones to the corresponding 1,1-dimethylalkyl group and allows one to access electron rich tert-alkyl-substituted aromatics that are not accessible by the Friedel-Crafts alkylation (Friedel-Crafts restricted). Third, we found a convenient and high-yielding method for selective S-alkylation of 2-mercaptophenol. In addition to the synthesis of carbamates, the preparation of one hundred three intermediates, phenols, and electron rich tert-alkyl arenes are reported. / Ph. D.

Estuaries, an important facet of coastal regions, are highly productive natural systems, frequently acting as drainage basins for various pollutants such as agricultural runoff. The estuarine grass shrimp, Palaemonetes pugio, has been shown to be a sensitive indicator of pesticide exposure. In this study, emphasis was placed on the quantification of the lethal and sublethal effects of aldicarb on three different life stages of P. pugio. Acute 96-h toxicity tests were conducted with newly hatched larvae, 22-d old larvae and adult grass shrimp to determine lethal toxicant ranges. LC₅₀ values were 85.0 ug/L for newly hatched larvae, 70.7 ug/L for 22-d old larvae and 125.4 ug/L for adults. The impact of aldicarb on specific neurological functions in the grass shrimp was examined using an acetylcholinesterase assay. Acetylcholinesterase (AChE) activity was reduced in the larvae after exposure to acute concentrations of aldicarb. Mean whole-body AChE activity for the newly hatched and 22-d larvae was significantly lower from the controls (P=0.009). Conversely, mean whole body AChE activity in the adult shrimp was not significantly different from the controls at any concentration (P=0.401), although there was a trend towards reduced activity at 50 and 100 ug/L exposures. A behavioral study was conducted to examine the ability of adult grass shrimp to detect and avoid aldicarb-treated seawater. Behavioral responses were measured in a modified steep gradient chamber. A partition divided the chamber, creating three distinct areas: 1) seawater 2) aldicarb-treated seawater and 3) mixing. There were significant differences between the control and exposed adult shrimp in the amount of time spent in the mixing area (P<0.05). Aldicarb exposed shrimp spent, on the average, 20% more time in the mixing area than the controls. Additionally, exposed shrimp spent significantly more time facing downstream, away from the toxicant source (P<0.05). Exposed shrimp displayed increasing hyperactivity and attempted to jump out of the chamber. / Master of Science

Pyrethroids are the only class of insecticides approved by the World Health Organization (WHO) for use in insecticide treated nets (ITNs), the first line of malaria vector control. Widespread resistance development to pyrethroids undermines current control efforts, and hence an urgent need for alternative chemistries. I report the evaluation of pharmacological differences between insect and vertebrate acetylcholinesterase (AChE) as well as selectivity and toxicity testing of new carbamate insecticides on Anopheles gambiae, the African malaria mosquito. AChE gorge pharmacology data revealed differences between insect and vertebrate AChE that can be exploited in the design of a bivalent insecticide. Toxicokinetic analysis showed that metabolic detoxication and cuticular penetration affect toxicity of carbamates in a manner dependent on the chemical structure. Structure activity relationships of side-chain branched N-methylcarbamates emphasized the importance of structural complementarity of ligands to the AChE catalytic active site and the substrate, acetylcholine. Monovalent pyrazoles and acetophenone oxime carbamates were toxic to both susceptible and carbamate-resistant mosquitoes carrying a G119S mutation within the catalytic site. A bivalent phthalimide-pyrazole carbamate and sulfenylated phenyl N-methyl carbamates were highly toxic when topically applied onto insect but less toxic by treated filter paper assays. In vitro evaluation of a molecular mosquito-selectivity model using AChE peripheral site ligands confirmed that selectivity of PRC 472 was due to presence of I70 in mosquito, which is Y70 in human AChE. The findings presented here are important steps in the on-going search of a mosquito-selective and resistance mitigating carbamate insecticide for control of malaria mosquitoes. / Ph. D.

Organophosphates are insecticides which result in acute adverse signs when exposed at toxic doses by animals and lead to death if left untreated. The current treatment for organophosphate toxicity includes atropine and the federally approved oxime 2-PAM. However, 2-PAM is not very effective at crossing the blood brain barrier which results in prolonged inactivation of acetylcholinesterase, which is the primary target of organophosphates, in the brain even after administration. The novel oximes, Oxime 15 and Oxime 20, are able to cross the blood brain barrier and reactivate the inhibited acetylcholinesterase. In this experiment with six animal species frequently used in toxicity studies, they were proven to be just as effective and sometimes better than 2-PAM at reactivating acetylcholinesterase or butyrylcholinesterase inhibited by paraoxon, chlorpyrifos-oxon, phorate-oxon, or dicrotophos. The detoxication enzymes butyrylcholinesterase, carboxylesterase, and paraoxonase were also studied as potential influences of the toxicity of the organophosphates in these different species.