Mosquito acetylcholinesterase as a target for novel phenyl-substituted carbamates

Article abstract of DOI:10.3390/ijerph16091500

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 stru

Full text of DOI:10.3390/ijerph16091500

International Journal of
Environmental Research
and Public Health
Article
Mosquito Acetylcholinesterase as a Target for Novel
Phenyl-Substituted Carbamates
James M. Mutunga 1,†, Ming Ma 2, Qiao-Hong Chen 2 , Joshua A. Hartsel 2, Dawn M. Wong 2
,
Sha Ding 2, Max Totrov 3, Paul R. Carlier 2 and Jeffrey R. Bloomquist 1,
*
1
Emerging Pathogens Institute, Entomology and Nematology Department, University of Florida,
Gainesville, FL 32610, USA; James.Mutunga@usamru-k.org
2
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA;
gym7981@gmail.com (M.M.); qchen@csufresno.edu (Q.-H.C.); jhartsel@vt.edu (J.A.H.);
dawnwong@vt.edu (D.M.W.); sding@vt.edu (S.D.); pcarlier@vt.edu (P.R.C.)
Molsoft LLC, 11199 Sorrento Valley Road, S209 San Diego, CA 92121, USA; max@molsoft.com
Correspondence: jbquist@epi.ufl.edu; Tel.: +1-352-273-9417
3
*
†
Current Address: Department of Entomology, US Army Medical Research Directorate-Africa, Kenya,
KEMRI CGHR, P.O. Box 54-40100, Kisumu, Kenya.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 February 2019; Accepted: 24 April 2019; Published: 28 April 2019
Abstract: 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–activity
relationships (SAR), but no new significant leads were generated.
Keywords: Anopheles gambiae; anticholinesterase; insecticide; toxicity
1. Introduction
The use of commercial carbamate insecticides dates back to the late 1950s, and numerous efforts
have been made to improve their toxicity to insects. Despite the wide-scale use of carbamates for
crop and urban pest control [
Organization (WHO) for indoor residual spraying of mosquitoes for malaria control [
1
], only propoxur and bendiocarb are authorized by the World Health
]. A major
2
concern for deploying carbamates in or near human dwellings is non-target toxicity, and two primary
factors determine the selectivity of a carbamate: selective detoxification in vertebrate systems and
selective AChE inhibition in insects [3]. Current advances in proteomics and genomics, the availability
of both the Anopheles gambiae and human AChE sequences, and advanced tools of computational
Int. J. Environ. Res. Public Health 2019, 16, 1500; doi:10.3390/ijerph16091500
www.mdpi.com/journal/ijerph

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biology enable fine-scale ligand docking, and virtual ligand library screening aid in the design of new
AChE inhibitors.
This study describes our continuing efforts to improve carbamate insecticides for mosquito
control, building on our previous work with phenyl-substituted analogs [4–7]. Structural modifications
included extending the side-chain branching of alkyl or silyl substituents to explore the effects of
spatial and structural complementarity of compounds to the ammonium group of acetylcholine (ACh),
since Kolbezen et al. [8] suggested that the structural complementarity of the 3-tert-butyl group of
terbam 1a to ACh explained its high potency with bovine AChE. Ring halogenation investigated not
only the size of the halogen substituent, but also the preferred placement on the molecule. Carbamate
side-chains are conventionally thought to be major detoxication sites [9], and halogenation at these
sites typically confers protection against hydroxylation by P450 mono-oxygenases [10]. Inclusion of
thioalkyl substituents sought to explore not only the ACh structural complementarity of these analogs,
but also the possible bioactivation of sulfur to more toxic sulfoxide and sulfone derivatives, in vivo
.
These data help to better understand the interactions of carbamates with insect and human AChE and
inform the future design of a pharmacophore with superior insect toxicity and selectivity.
2. Materials and Methods
2.1. Preparation of Carbamates
Carbamates 1a
, 3a, 4a, and 5a were prepared as previously described by Hartsel et al. [5] and the
synthesis of other experimental compounds is described below.
4-bromo-3-(tert-butyl)phenyl methylcarbamate (1b): 3-t-butylphenol (3 g) was dissolved in 10%
NaOH (10 mL), and iodine (5.6 g, 1.1 equivalent) and sodium iodide (5.6 g, 1.5 equivalent) were added
to afford 5-(t-butyl)-2-iodophenol (4.96 g, 90% yield). Treatment with bromine in CH2Cl2 afforded
4-bromo-5-t-butyl-2-iodophenol; refluxing in N-methylmorpholine for 16 h, followed by column
chromatography afforded 4-bromo-3-t-butylphenol in 50% yield over two steps. This compound (50
mg) was dissolved in dry tetrahydrofuran (THF) (2 mL), and potassium t-butoxide (0.29 mL, 1 M in
THF) was added followed by methylcarbamoyl chloride (2 equiv). After 1 h, aqueous workup and
1
column chromatography afforded 1b as a pale semi-solid (43 mg, 69%). H NMR (400 MHz, CDCl3)
δ
7.53 (d, J = 10.0 Hz, 1H), 7.19 (s, 1H), 6.85 (d, J = 10.0 Hz, 1H), 4.99 (br s, 1H), 2.95 (d, J = 3.0 Hz, 0.15H,
minor carbamate rotamer), 2.88 (d, J = 3.0 Hz, 2.85H, major carbamate rotamer), 1.50 (s, 9H); 13C NMR
(101 MHz, CDCl3) δ 154.9, 150.2, 149.2, 136.2, 121.6, 120.7, 118.4, 36.7, 29.5, and 27.8.
2-bromo-5-(tert-butyl)phenyl methylcarbamate (1d): 3-t-butylphenol (3 g) was dissolved in
dichloromethane (30 mL) and bromine (1.1 mL, 1.1 equivalent) was added dropwise. After stirring for
1 h, aqueous workup and concentration in vacuo afforded 2-bromo-5-t-butylphenol in quantitative
1
yield. This phenol was treated as above for 1b to afford 1d as pale semi-solid (70% yield). H NMR
(400 MHz, CDCl3)
δ 7.44 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.4, 2.0 Hz, 1H),
5.05 (br s, 1H), 3.00 (d, J = 4.8 Hz, 0.15H, minor carbamate rotamer), 2.89 (d, J = 4.8 Hz, 2.85H, major
carbamate rotamer), 1.26 (s, 9H); 13C NMR (101 MHz, CDCl3)
113.0, 34.7, 31.1, and 27.8.
δ 154.3, 152.1, 147.9, 132.4, 124.3, 121.3,
5-(tert-butyl)-2-iodophenyl methylcarbamate (1e): 5-(tert-butyl)-2-iodophenol (described above in
synthesis of 1b) was then dissolved in dry THF (10 mL), treated with NaH (60% dispersion in mineral
oil, 1.6 equivalent), and methylcarbamoyl chloride (2.5 equiv). After 1 h aqueous workup and column
1
chromatography afforded 1e as a pale semi-solid (134 mg 50%). H NMR (400 MHz, CDCl3)
δ 7.68 (d,
J = 8.3 Hz, 1H), 7.17(s, 1H), 6.97 (d, J = 8.3 Hz, 1H), 5.19 (br s, 1H), 3.01 (br s, 0.15H, minor carbamate
rotamer), 2.89 (br s, 2.85H, major carbamate rotamer), 1.28 (s, 9H); 13C NMR (101 MHz, CDCl3)
153.3, 150.8, 138.4, 124.6, 120.6, 86.7, 34.6, 31.1, and 27.8.
δ 154.3,
5-(tert-butyl)-2-hexylphenyl methylcarbamate (1f): The requisite phenol was prepared from
5-(t-butyl)-2-iodophenol (see above) in a four-step sequence (62% overall yield) by (i) acetylation, (ii)
Sonogashira coupling with 1-hexyne, catalytic hydrogenation, and deacetylation. This phenol was
then reacted with NaH and methylcarbamoyl chloride according to the procedure for 1e, to afford

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1
1f as a pale semi-solid (110 mg, 58%). H NMR (400 MHz, CDCl3)
δ
7.23–7.13 (m, 2H), 7.06 (s, 1H)
5.01 (br s, 1H), 2.94 (d, J = 4.8 Hz, 0.15 H, minor carbamate rotamer) 2.88 (d, J = 4.8 Hz, 2.85H, major
carbamate rotamer), 2.50 (t, J = 7.7 Hz, 2H), 1.61–1.52 (m, 2H), 1.36–1.23 (m, 15H), 0.88 (t, J = 6.8 Hz
,
1H); 13C NMR (101 MHz, CDCl3)
29.6, 29.2, 27.7, 22.5, and 14.1.
δ 155.4, 150.0, 148.7, 131.8, 129.4, 122.5, 119.4, 34.4, 31.6, 31.2, 29.9,
4-(allylcarbamoyl)-3-(tert-butyl)phenyl methylcarbamate (1h): 4-bromo-3-t-butylphenol (above
in 1b) was converted to the triiisopropylsilyl derivative (TIPS-Cl, imidazole, DMF), dissolved in
diethyl ether, metalated with t-BuLi and trapped with ethylchloroformate, and then hydrolyzed
(KOH, aq THF; HCl aq). Treatment with TBS-Cl and imidazole in dichloromethane afford
2-(t-butyl)-4-t-butyldimethylsilyloxybenzoic acid in 57% yield over four steps. Treatment with
oxalyl chloride, followed by allyl amine afforded the allyl amide. Deprotection with HF/pyridine,
deprotonation with potassium t-butoxide and acylation with methylcarbamoyl chloride afforded 1h as
1
a pale oil (46% over three steps). H NMR (400 MHz, CDCl3): Note: chiral axis C4-C(O) renders the
four CH2 protons diastereotopic
δ 7.22 (d, J = 9.0 Hz, 1H), 7.15 (s, 1H), 6.88 (d, J = 9.0 Hz, 1H) 5.88 (ddt,
J = 15.0, 10.0, 6.2 Hz, 1H), 5.81 (br s, 1H), 5.25 (dd, J = 15.0, 2.0 Hz, 1H), 5.22 (dd, J = 10.0, 2.0 Hz, 1H),
5.20 (br s, 1H), 4.00 (t, J = 6.2 Hz, 2H), 2.94 (d, J = 4.0 Hz, 0.15H, minor carbamate rotamer), 2.82 (d,
J = 4.0 Hz, 2.85H, major carbamate rotamer), 1.45 (s, 9 H); 13C NMR (101 MHz, CDCl3)
δ
172.3, 155.2,
151.6, 149.7, 133.7, 133.2, 129.4, 120.6, 118.8, 117.2, 42.5, 36.4, 31.4, and 27.8.
3-(tert-butyl)-4-(methylcarbamoyl)phenyl
methylcarbamate
(1i):
2-(t-butyl)-4-t-
butyldimethylsilyloxybenzoic acid (from 1b above) was treated with oxalyl chloride, followed
by methylamine. Deprotection with HF/pyridine, deprotonation with potassium t-butoxide and acylation
with methylcarbamoyl chloride afforded 1i as a pale oil (42% over three steps). 1H NMR (400 MHz,
CDCl3): Note: chiral axis C4-C(O) renders the four CH2 protons diastereotopic
J = 9.0 Hz, 1H), 5.77 (br s, 1H), 5.18 (br s, 1H), 2.93 (d, J = 5.0 Hz, 3H), 2.85 (d, J = 4.4 Hz, 3H), 1.37 (s, 9H);
13C NMR (101 MHz, CDCl3)
173.2, 155.2, 151.5, 149.6, 133.9, 129.3, 120.5, 118.8, 36.3, 31.3, 27.8, and 26.9.
3-(tert-butyl)-4-(isobutylcarbamoyl)phenyl methylcarbamate 1j): 2-(tert-butyl)-4-t-
δ 7.22-7.13 (m, 2H), 6.89 (d,
δ
(
butyldimethylsilyloxybenzoic acid (from 1b above) was treated with oxalyl chloride, followed
by isobutylamine. Deprotection with HF/pyridine, deprotonation with potassium t-butoxide and
1
acylation with methylcarbamoyl chloride afforded 1j as a pale oil (28% over three steps). H NMR
(400 MHz, CDCl3): Note: chiral axis C4-C(O) renders the four CH2 protons diastereotopic
δ 7.22 (d, J =
8.3 Hz, 1H), 7.18 (s, 1H), 6.95 (d, J = 8.3 Hz, 1H), 5.80 (br s, 1H), 5.19 (br s, 1H), 3.23 (dd, J = 6.2, 5.8 Hz,
2H), 2.85 (d, J = 3.8 Hz, 0.15H, minor carbamate rotamer), 2.79 (d, J = 3.8 Hz, 2.85H, major carbamate
rotamer), 1.90 (t sep, J = 6.8, 6.2 Hz, 1H), 1.44 (s, 9H), 0.92 (d, J = 6.8 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ 172.6, 155.2, 151.5, 149.6, 134.1, 129.4, 120.5, 118.8, 47.6, 36.3, 31.3, 28.3, 27.8, and 20.3.
3-(tert-butyl)-4-(diethylcarbamoyl)phenyl
methylcarbamate
(1k):
2-(t-butyl)-4-t-butyldimethylsilyloxybenzoic acid (from 1b above) was treated with oxalyl
chloride, followed by diethylamine. Deprotection with HF/pyridine, deprotonation with potassium
t-butoxide and acylation with methylcarbamoyl chloride afforded 1k as a pale oil (29% over three steps).
1H NMR (400 MHz, CDCl3): Note: chiral axis C4-C(O) renders the four CH2 protons diastereotopic
δ
7.20 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 5.05 (br s, 1H), 3.72 (dq, J = 15.2, 6.2 Hz,
1H), 3.28 (dq, J = 15.6, 6.2 Hz, 1H), 3.20 (dq, J = 15.2, 6.2 Hz, 1H), 3.01 (dq, J = 15.6, 6.2 Hz, 1H), 2.95 (d,
J = 4.0 Hz, 0.15 H, minor carbamate rotamer), 2.78 (d, J = 4.0 Hz, 2.85H, major carbamate rotamer), 1.29
(s, 9H), 1.23 (dd, J = 6.2, 6.2 Hz, 3H), 1.02 (dd, J = 6.2, 6.2 Hz, 3H); 13C NMR (101 MHz, CDCl3)
δ 172.3,
154.6, 150.8, 147.0, 133.5, 127.7, 120.9, 118.1, 44.2, 38.6, 36.1, 32.0, 27.5, 13.8, and 12.7.
3-(tert-butyl)-4-(ethyl(methyl)carbamoyl)phenyl methylcarbamate
(
1l):
2-(t-butyl)-4-t-
butyldimethylsilyloxybenzoic acid (from 1b above) was treated with oxalyl chloride, followed by
ethylmethylamine. Deprotection with HF/pyridine, deprotonation with potassium t-butoxide and
1
acylation with methylcarbamoyl chloride afforded 1l as a pale oil (59% over three steps). H NMR
(400 MHz, CDCl3): Note: chiral axis C4-C(O) renders the four CH2 protons diastereotopic
δ 7.13 (d,
J = 1.8 Hz, 1H), 7.02 (dd, J = 8.2, 1.8 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 5.32 (br s, 1H), 3.69 (dt, J = 15.2,

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6.8 Hz, 0.5 H), 3.48 (dt, J = 15.2, 6.8 Hz, 0.5 H), 3.25 (dt, J = 15.2, 6.8 Hz, 0.5 H), 3.05 (s, 1.5 H), 2.98 (dt, J
= 15.2, 6.8 Hz, 0.5 H), 2.97 (d, J = 3.8 Hz, 0.15 H, minor carbamate rotamer), 2.82 (d, J = 3.8 Hz, 2.85
H, major carbamate rotamer), 2.75 (s, 1.5 H), 1.32 (s, 9 H), 1.20 (dd, J = 6.8, 6.8 Hz, 1.5 H), 0.97 (dd,
J = 6.8, 6.8 Hz, 1.5 H); 13C NMR (101 MHz, CDCl3)
δ 176.0, 175.0, 155.3, 151.3, 151.2, 148.1, 148.0, 132.6,
131.9, 128.5, 127.8, 120.9, 119.1, 118.9, 46.0, 41.8, 36.9, 36.4, 31.7, 31.2, 27.7, 13.0, and 11.4 (two equally
populated rotamers (ethymethylamide); 24 of a possible 28 resonances seen).
5-(tert-butyl)-2-(methylthio)phenyl methylcarbamate (1m): 3-t-butylphenol was treated with
chlorosulfonic acid (10 equivalent) in dichloromethane for 4 h, and then poured into ice. Extractive
workup, reduction with stannous chloride in acetic acid (18 h), and column chromatography
afforded 6,6’-disulfanediylbis(3-(t-butyl)phenol). Reduction with sodium borohydride in THF gave
5-(t-butyl)-2-mercaptophenol in 64% yield over three steps. Treatment with methyl iodide (1.1 equiv)
and sodium bicarbonate in DMF, followed by deprotonation with potassium t-butoxide in THF and
acylation with methylcarbamoyl chloride gave 1m as a pale semi-solid in 37% yield over two steps.
1H NMR (400 MHz, CDCl3)
δ 7.23 (dd, J = 6.6, 1.6 Hz, 1H), 7.19 (d, J = 6.6 Hz, 1H), 7.12 (d, J = 1.6 Hz,
1H), 5.04 (br s, 1H), 3.07 (d, J = 4.0 Hz, 0.15 H, minor carbamate rotamer), 2.91 (d, J = 4.0 Hz, 2.85 H,
major carbamate rotamer), 2.42 (s, 3H), 1.30 (s, 9H); 13C NMR (101 MHz, CDCl3)
128.1, 127.3, 123.5, 120.2, 34.6, 31.4, 28.0, 15.7.
δ 154.9, 150.1, 148.3,
5-(tert-butyl)-2-(isobutylthio)phenyl methylcarbamate (1n): 5-(t-butyl)-2-mercaptophenol (from 1m
above) was treated with isobutyl iodide (2.9 equivalents) and sodium bicarbonate in DMF at 55 ◦C for 6
h, followed by deprotonation with potassium t-butoxide in THF and acylation with methylcarbamoyl
1
chloride to gave 1n as a pale semi-solid in 63% yield over two steps. H NMR (400 MHz, CDCl3)
δ
7.27 (d,
J = 6.6 Hz, 1H), 7.18 (d, J = 6.6, 1.6 Hz, 1H), 7.12 (d, J = 1.6 Hz, 1H), 5.04 (br s, 1H), 3.02, (d, J = 3.9 Hz,
0.15H, minor carbamate rotamer), 2.92 (d, J = 3.9 Hz, 2.85H, major carbamate rotamer), 2.73 (d, J = 5.5 Hz,
2H), 1.81 (nonet (9-let), J = 5.5 Hz, 1H), 1.29 (s, 9H), 1.02 (J = 5.3 Hz, 6H); 13C NMR (101 MHz, CDCl3)
δ
155.0, 150.9, 149.5, 130.1, 127.0, 123.4, 120.3, 42.5, 34.7, 31.3, 28.4, 28.0, 22.2.
5-(tert-butyl)-2-(isopropylthio)phenyl methylcarbamate (1o): 5-(t-butyl)-2-mercaptophenol (from
1m above) was treated with isopropyl iodide (3.0 equiv) and sodium bicarbonate in DMF at 55 ◦C for
6 h, followed by deprotonation with potassium t-butoxide in THF and acylation with methylcarbamoyl
1
chloride to give 1o as a pale semi-solid in 60% yield over two steps. H NMR (400 MHz, CDCl3)
δ
7.36 (d, J = 6.6 Hz, 1H), 7.19 (dd, J = 6.6, 1.6 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H), 5.03 (br s, 1H), 3.33 (sept,
J = 5.3 Hz, 1H), 3.02 (d, J = 3.9 Hz, 0.15H, minor carbamate rotamer), 2.92 (d, J = 3.9 Hz, 2.85H, major
carbamate rotamer), 1.30 (s, 9H), 1.27 (d, J = 5.3 Hz, 6H); 13C NMR (101 MHz, CDCl3)
δ 155.1, 152.1,
150.7, 133.2, 125.3, 134.3, 120.4, 37.9, 34.8, 31.3, 28.0, and 23.4.
3-(trifluoromethyl)phenyl methylcarbamate (2a): 3-trifluoromethylphenol (1.09 g) was
deprotonated with potassium t-butoxide in THF and treated with methylcarbamoyl chloride to
1
give 2a as a pale semi-solid (1.16 g, 81% yield). H NMR (400 MHz, CDCl3)
δ
7.47-7.44 (m, 2H), 7.40 (br
s, 1H), 7.33-7.31 (m, 1H), 5.17 (br s, 1H), 2.95 (d, J = 4.0 Hz, 0.15H, minor carbamate rotamer), 2.88 (d,
J = 4.0 Hz, 2.85H, major carbamate rotamer); 13C NMR (101 MHz, CDCl3)
125.3, 123.3 (q, 1JCF = 238 Hz), 122.1, 118.9, and 27.8.
δ 154.8, 151.3, 132.3, 129.9,
4-methyl-3-(trimethylsilyl)phenyl methylcarbamate (3c): 3-bromo-4-methylphenol (0.70 g) was
◦
dissolved in dry THF (5 mL), cooled to -78 C, treated with BuLi (3.4 mL, 2.5 M in hexanes, 2.2
equivalent) and trimethylsilyl chloride (1.2 mL, 1.1 g, 2.6 equivalent). After stirring for 30 min the
reaction was allowed to warm to 25 ◦C and the reaction was quenched with aqueous HCl. Extractive
workup and column chromatograph afforded 4-methyl-3-trimethylsilylphenol as a colorless oil (630 mg,
93%). Applying the procedure above, the phenol (630 mg) was converted to the methyl carbamate,
1
affording 3c as a yellow oil (591 mg, 71% yield). H NMR (400 MHz, CDCl3)
δ 7.12 (s, 1H), 7.11 (d,
J = 9.6 Hz, 1H), 6.98 (d, J = 9.6 Hz, 1H), 4.97 (br s, 1H), 2.93 (d, J = 4.0 Hz, 0.15H, minor carbamate
rotamer), 2.85 (d, J = 4.0 Hz, 2.85H, major carbamate rotamer), 2.40 (s, 3H), 0.30 (s, 9H); 13C NMR
(101 MHz, CDCl3) δ 155.8, 148.7, 140.6, 140.1, 130.8, 127.1, 122.4, 27.9, 22.5, and −0.13.

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3-fluoro-5-(trimethylsilyl)phenyl methylcarbamate (3g): 3-bromo-5-fluorophenol (0.19 g) was
◦
dissolved in dry THF (9 mL), cooled to
−
78 C, treated with BuLi (1.4 mL, 2.5 M in hexanes, 3.3
equivalent) and trimethylsilyl chloride (0.45 mL, 0.39 g, 3.6 equivalent). After stirring for 30 min the
reaction was allowed to warm to 25 ◦C and the reaction was quenched with aqueous HCl. Extractive
workup and column chromatograph afforded 5-fluoro-3-trimethylsilylphenol as a colorless oil (66 mg,
66%). Applying the procedure above, the phenol (66 mg) was converted to the methyl carbamate,
1
affording 3g as a yellow oil (78 mg, 92% yield). H NMR (400 MHz, CDCl3)
δ
7.08-6.99 (m, 2H), 6.87
(ddd, J = 9.6, 2.3, 2.1 Hz, 1H), 5.12 (br s, 1H), 2.92 (d, J = 4.7 Hz, 0.15H, minor carbamate rotamer), 2.87
(d, J = 4.7 Hz, 2.85H, major carbamate rotamer), 0.25 (s, 9H); 13C NMR (101 MHz, CDCl3)
δ 162.5 (d,
1JCF = 248.4 Hz) 154.9, 151.5, 144.2 (d, 3JCF = 4.7 Hz), 121.6 (d, 3JCF = 2.9 Hz), 116.5 (d, 2JCF = 18.2 Hz),
110.0 (d, 2JCF = 24.1 Hz), 27.7, and −1.4.
3-(ethyldimethylsilyl)-4-methylphenyl methylcarbamate (4c): 3-bromo-4-methylphenol (0.70 g)
was dissolved in dry THF (5 mL), cooled to
78 ◦C, treated with BuLi (3.4 mL, 2.5 M in hexanes, 2.2
−
equivalent) and ethyldimethylsilyl chloride (1.36 mL, 1.19 g, 2.6 equivalent). After stirring for 30
min, the reaction was allowed to warm to 25 ◦C and the reaction was quenched with aqueous HCl.
Extractive workup and column chromatograph afforded 4-methyl-3-trimethylsilylphenol as a colorless
oil (665 mg, 91%). Applying the procedure above, the phenol (419 mg) was converted to the methyl
1
carbamate, affording 4c as a yellow oil (419 mg, 77% yield). H NMR (400 MHz, CDCl3)
δ 7.13 (s, 1H),
7.11 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 5.05 (br s), 2.90 (d, J = 4.9 Hz, 0.15H, minor carbamate
rotamer), 2.83 (d, J = 4.9 Hz, 2.85H, major carbamate rotamer), 2.40 (s, 3H), 0.93 (t, J = 8.0 Hz, 3H), 0.79
(q, J = 8.0 Hz, 2H), 0.28 (s, 6H); 13C NMR (101 MHz, CDCl3)
122.3, 27.9, 22.5, 7.75, 7.74, and −2.35.
δ 155.9, 148.7, 140.7, 139.2, 130.8, 127.5,
3-(ethyldimethylsilyl)-5-fluorophenyl methylcarbamate (4g): 3-bromo-5-fluorophenol (0.27 g)
was dissolved in dry THF (9 mL), cooled to 78 Ç, treated with BuLi (2.1 mL, 2.5 M in hexanes, 3.5
−
equivalent) and ethyldimethylsilyl chloride (0.80 mL, 0.70 g, 4.0 equivalent). After stirring for 30
min the reaction was allowed to warm to 25 ◦C and the reaction was quenched with aqueous HCl.
Extractive workup and column chromatograph afforded 3-ethyldimethylsilyl-5-fluorophenol as a
colorless oil (178 mg, 62%). Applying the procedure above, the phenol (58 mg) was converted to
1
the methylcarbamate, affording 4g as a yellow oil (55 mg, 74% yield). H NMR (400 MHz, CDCl3)
δ
7.08–6.98 (m, 2H), 6.87 (ddd, J = 9.6, 2.2, 2.2 Hz, 1H), 5.09 (br s, 1H), 2.93 (d, J = 4.0 Hz, 0.15H, minor
carbamate rotamer), 2.87 (d, J = 4.0 Hz, 2.85Hz, major carbamate rotamer), 0.94 (t, J = 7.8 Hz, 3H), 0.70
(q, J = 7.8 Hz, 2H), 0.23 (s, 9H); 13C NMR (101 MHz, CDCl3)
δ
162.5 (d, 1JCF = 248.4 Hz), 154.8, 151.5 (d,
2JCF = 9.7 Hz), 143.2 (d, 3JCF = 4.6Hz), 121.8 (d, 3JCF = 2.9 Hz), 116.6, 109.9 (d, 2JCF = 24.2), 27.7, 7.23,
7.13, and −3.73.
2-(propylthio)phenyl methylcarbamate (6a) 2-mercaptophenol (300 mg, 2.5 mmol) was dissolved
in dry DMF (5 mL) and NaHCO3 (630 mg, 3 equivalent) and propyl bromide (0.45 g, 5.0 equivalent)
◦
were added. After heating to 55 C for 16 h, aqueous workup, and column chromatography,
2-propylthiophenol was isolated as a pale oil (391 mg, 98%). 2-propylthiophenol (354 mg, 2.11 mmol)
was dissolved in dry THF (20 mL), treated with NaH (60% in mineral oil, 110 mg, 2.75 equivalent),
and methylcarbamoyl chloride (394 mg, 4.2 equivalent) was added. Aqueous workup and column
1
chromatography afforded 6a as a pale oil (360 mg, 76%). H NMR (400 MHz, CDCl3)
δ
7.33 (dd, J = 5.6,
1.8 Hz, 1H), 7.21–7.15 (m, 2H), 7.11 (dd, J = 5.3, 1.9 Hz, 1H), 5.08 (br s, 1H), 3.02 (d, J = 4.0 Hz, 0.15H,
minor carbamate rotamer), 2.91 (d, J = 4.0 Hz, 2.85Hz, major carbamate rotamer), 2.85 (t, J = 5.8 Hz,
2H), 1.66 (hextet, J = 5.9 Hz, 2H), 1.02 (t, J = 5.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
δ 154.8, 149.3,
130.5, 129.5, 126.7, 126.2, 123.1, 34.9, 28.0, 22.5, and 13.6.
3-(tert-butyl)phenyl methyl(phenylthio)carbamate (7a): Compound 1a (150 mg) was treated with
triethylamine (4 equiv) and benzenesulfenyl chloride (1.5 equvalent) in carbon tetrachloride (4 mL) at
45 ◦C for 18 h. Following aqueous workup the residue was chromatographed in 30:1 hexanes:ethyl
1
acetate to afford 7a as a yellow oil (186 mg, 82% yield). H NMR (400 MHz, CDCl3)
δ
7.40–7.36 (m, 2H),
7.34 (d, J = 9.5 Hz, 2H), 7.30–7.22 (m, 3H), 7.08 (s, 1H), 6.92 (d, J = 8.2 Hz, 1H), 3.43 (s, 3H), 1.30 (s, 9H);

Int. J. Environ. Res. Public Health 2019, 16, 1500
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13C NMR (101 MHz, CDCl3)
42.0, 34.8, and 32.2.
δ 156.8, 153.0, 151.3, 137.5, 129.3, 128.8, 127.3, 126.3, 125.3, 122.8, 118.5,
3-(tert-butyl)phenyl methyl(p-tolylthio)carbamate (7b): Following the procedure for 7a
,
1a, and
p-toluylsulfenyl chloride were reacted to afford 7b as a yellow oil (213 mg, 89%). H NMR (400 MHz,
CDCl3)
7.36–7.22 (m, 6H), 7.11 (s, 1H), 6.96 (br s, 1H), 3.42 (s, 3H), 2.38 (s, 3H), 1.32 (s, 9H); 13C NMR
(101 MHz, CDCl3) 156.8, 154.9, 151.4, 138.2, 134.1, 130.0, 128.8, 127.2, 122.7, 118.6, 41.9, 34.8, 31.3, and 21.2.
3-(tert-butyl)phenyl ((4-(tert-butyl)phenyl)thio)(methyl)carbamate (7c): following the procedure
for 7a 1a, and 4-t-butylphenylsulfenyl chloride were reacted to afford 7c as a yellow oil (152 mg (73%).
1H NMR (400 MHz, CDCl3)
7.43 (d, J = 9.7 Hz, 2H), 7.37 (d, J = 9.7 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H),
7.25 (d, 8.0 Hz, 1H), 7.10 (s, 1H), 6.95 (d, J = 8.0 Hz, 1H), 3.42 (s, 3H), 1.34 (s, 9H), 1.31 (s, 9H); 13C NMR
(101 MHz, CDCl3) 156.8, 152.9, 151.4, 151.2, 134.3, 128.8, 126.9, 126.3, 122.7, 118.6, 41.9, 34.8, 34.7, 31.6,
31.3, 31.2, 22.7, and 14.1.
1
δ
δ
,
δ
δ
3-(trimethylsilyl)phenyl methyl(phenylthio)carbamate (7d): Following the procedure for 7a
benzenesulfenyl chloride were reacted to afford 7d as a yellow oil (68 mg, 58%). H NMR (400 MHz,
CDCl3) δ
7.44–7.30 (m, 6H), 7.28–7.23 (m, 1H), 7.19 (s, 1H), 7.09 (s, 1H), 3.47 (s, 3H), 0.27 (s, 9H); 13C NMR
,
3a and
1
(101 MHz, CDCl3) δ 156.9, 151.2, 142.7, 133.7, 130.7, 129.4, 129.0, 127.5, 125.9, 122.0, 42.1, and −1.11.
4-methyl-3-(trimethylsilyl)phenyl methyl(phenylthio)carbamate (7e): Following the procedure for
1
7a
,
4c and benzenesulfenyl chloride were reacted to afford 7e as a yellow oil (166 mg, 58%). H NMR
(400 MHz, CDCl3)
δ
7.40 (d, J = 8.7 Hz, 2H), 7.39–7.35 (m, 2H), 7.29 (t, J = 8.7, Hz, 1H), 7.19–7.10 (m,
2H), 7.00 (d, J = 8.0 Hz, 1H), 3.44 (s, 3H), 2.44 (s, 3H), 0.27 (s, 9H); 13C NMR (101 MHz, CDCl3)
δ 157.0,
149.0, 140.9, 140.1, 130.7, 129.3, 129.0, 127.3, 126.7, 125.6, 122.0, 42.0, 22.3, and −0.4.
2.2. Insects and Reagents
Anopheles gambiae (insecticide-susceptible G3 strain), were taken from colonies cultured in the
Department of Entomology at Virginia Polytechnic Institute and State University, or the University
of Florida, Emerging Pathogens Institute. Acetylthiocholine (ATChI), recombinant human enzyme
(hAChE), 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), and all buffer components were purchased from
Sigma–Aldrich (MO, USA). Whole mosquito homogenate was prepared essentially as described by
Anderson et al. [11], and was used as the source of AgAChE.
2.3. AChE Inhibition Assays
AChE enzyme inhibition assays were performed using the Ellman [12] method adapted for a
96-well microplate assay [11], with a few modifications. Briefly, inhibitor stocks of 0.01 M were freshly
prepared in dimethylsulfoxide (DMSO) followed by serial dilutions in DMSO. A 100-fold dilution
into sodium phosphate buffer (pH 7.8) was made for each of the DMSO dilutions. The final DMSO
concentration was maintained at 0.1% v/v and concentrations of the drug typically ranged from 1 nM
to 0.1 mM in 10-fold steps. After a 10 min pre-incubation of the enzyme and inhibitor, hydrolysis of
the Ellman’s reagents (ATChI and DTNB) was monitored for 10 mins at 405 nm, in a Dynex 96-well
plate reader (Dynex, Chantilly, VA, USA). Percent residual AChE activity values (relative to the control)
were plotted in Prism® (GraphPad, USA) and analyzed by non-linear regression (curve-fit) to generate
IC50 values. Half maximal inhibitory concentration (IC50) is defined as the inhibitor concentration
that blocks 50% of the enzyme activity and is used to define the potency of the inhibitor. Mosquito
selectivity (S) was determined by the IC50 ratio of hAChE/AgAChE for each compound. Statistical
significance of both potency and toxicity of the compounds was assessed based on non-overlapping of
95% confidence intervals (95% CI).
2.4. Insect Bioassays
A standard WHO-treated filter paper assay [13] was used to assess contact toxicity of the structural
analogs, dissolved in ethanol and without any added silicon or other oil, which reduced activity (data
not shown). An initial range finding assay was performed with concentrations of 0.1, 0.5, and 1 mg/mL

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of each compound, to identify additional test concentrations needed to obtain an LC50 value. From this
initial assay, only compounds showing greater than 50% mortality at 0.5 mg/mL were tested further,
with up to five concentrations per compound and 25 mosquitoes (2–5-day old sugar-fed females)
per treatment. Assays were repeated at least twice using different batches of mosquitoes to account
for inter-batch variability. Treated mosquitoes and untreated controls were maintained at 75% RH
and 25.6 ◦C. Mortality was assessed after 24 h and corrected for control mortality by the method of
Abbott [14]. Data was plotted in Poloplus® and a probit analysis performed to generate LC50 values;
the carbamate concentration that killed 50% of the exposed mosquitoes. Statistical significance of
different IC50 values was judged by non-overlap of the 95% confidence intervals.
3. Results
3.1. AChE Potency and Mosquito Toxicity of Terbam (1a) Analogs
We have previously disclosed results with terbam (3-t-butylphenyl methylcarbamate) 1a. It displayed
excellent potency for inhibiting AgAChE and excellent contact toxicity to An. gambiae (LC50 = 37
µg/mL),
but enzymatic inhibition selectivity (S) of only 12-fold [ ]. Except for 2a, all modifications using 1a as a
5
template involved holding the 3-t-butyl group constant. Bromination at the 4-position (1b) led to a 16-fold
loss in AgAChE inhibitory potency, compared to a 4-fold loss in hAChE inhibitory potency, relative to 1a
(Table 1). Bromination at the 6-position (1d) increased inhibitory potency to both AChEs (about 2-fold for
AgAChE and 7-fold for hAChE), but reduced toxicity to mosquitoes 7-fold (Table 1). A 6-iodo substituent
(1e) led to 58-fold and 2-fold loss in potency to AgAChE and hAChE, respectively, and a significant loss
of mosquitocidal activity. Finally, addition of a 6-hexyl group (1f) drastically reduced AgAChE inhibitory
potency while only reducing hAChE inhibitory potency 20-fold (Table 1).
Installation of a 4-carboxamido group abrogated AgAChE inhibition and mosquito toxicity (1h
–1l).
For hAChE, however, we observed micromolar IC50 values with the allyl (1h), N-di-ethyl (1k), and
N-ethyl (1l) carbamoyl analogs, but no activity was observed with the methyl (1i) and iso-butyl (1j
)
analogs (Table 1). Addition of a thioether functionality at C6, as exemplified by compounds 1m–1o
,
caused significant loss of AgAChE inhibition potency; 27-fold with 1m and much more with 1n and
1o, and they had little insecticidal activity. Finally, when the tert-butyl group of 1a was replaced
with the smaller and highly electron-withdrawing trifluoromethyl moiety (2a), inhibitory potency to
both mosquito and human AChEs was lost, but surprisingly the compound was toxic to mosquitoes,
although it was less toxic than 1a by about 10-fold (Table 1).

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Table 1. AChE inhibition and mosquito toxicity to adult female An. gambiae by 3-tert-butylphenyl
carbamate 1a and the substituted analogs shown in Figure 1.
S b
Compound
AgAChE, IC50, a nM
hAChE aIC50, nM
LC50 or % Mortality c
36 (34–38) a
580 (260–892) b
14 (12–17) c
2100 (1195–2309) d
>105
* 320 (293-349) a
* 1200 (913–1533) b
* 48 (40–56) c
9
2
3.4
37 (14–60) a
1a d
1b
1d
1e
1f
1h
1i
1j
1k
1l
1m
1n
1o
2a
600 (570–627) b
260 (239–276) c
* 640 (538–752) d
6300 (5126–7460) e
2800 (1719–4677) f,g
>105
0.3
20%
4%
0%
4%
0%
0%
0%
0%
4%
<0.06
<0.03
-
>105
>105
>105
>105
-
>105
2300 (1189–4448) b,f.g
5500 (2830–8128) e,f,g
* 640 (572–719) d
* 4700 (3777–5784) e,f
2200 (1653–2941) g
>105
<0.023
<0.06
0.66
0.15
0.03
-
>105
970 (830–1172) b
32,000 (23,060–63,300) e
75,000 (N/A)
>105
16%
390 (333–471) d
a
IC50 with (95% CI); b S = selectivity ratio (IC50 hAChE/AgAChE), and and asterisk indicates an IC50 value for
hAChE that is significantly different from AgAChE, as judged by non-overlap of the 95% CI; c LC50 in
µ
g/ml or %
mortality at 1 mg/mL; IC50s or LC50s within a column not labeled by the same lower case letter are significantly
different (p > 0.05), as judged by non-overlap of the 95% CI.
Figure 1. Terbam (1a) and structural analogs described in this study.
3.2. SAR of 3-Trialkylsilyl- and 2-Thioalkyl-Substituted Methylcarbamates
Close structural analogs of 1a featured replacement of the t-butyl group with trialkylsilyl groups,
and these analogs had similar inhibition potency and selectivity, but reduced contact toxicity as
exemplified by 3a and 4a, which were reported in a previous study [5]. Thus, analogs of 3a and 4a,
variants featuring 4-methyl (3c 4c) and 5-fluoro (3g 4g) were prepared (Figure 2) and assayed (Table 2).
,
,

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Both substitutions reduced AgAChE inhibition potency, and 5-fluorination had a significantly more
deleterious effect for the 3-SiMe3 derivative than for the 3-SiEtMe2 derivative (3g and 4g). None of
these variants had improved enzymatic selectivity, and reductions in AgAChE inhibition potency
correlated with reduced mosquitocidal action (Table 2).
Figure 2. Analogs of 3-trialkylsilyl- and 2-thioalkyl-substituted methylcarbamates.
One additional 2-thioalkylphenyl methylcarbamate (6a) (Figure 2) was also synthesized based
upon previous studies that identified compound 5a [5], as having good enzyme selectivity (135-fold),
but poor contact toxicity on paper (Table 2). As can be seen, 2-thiopropylphenyl methylcarbamate 6a
had a 3-fold reduction of inhibitory potency to AgAChE, and 7-fold reduced inhibition selectivity, but
had improved paper toxicity to adult An. gambiae (Table 2).
Table 2. Enzyme inhibition potency and toxicity to adult female An. gambiae of chemical structures
shown in Figure 2.
a
c
S b
Compound
AgAChE IC50 nM
hAChE IC50 a, nM
LC50 or % Mortality c
3a
3c
3g
4a
4c
4g
5a
6a
50 (40–64) a
380 (318–450) b
2600 (1260–5453) c
72 (67–78) d
* 490 (452–526) a
* 2400 (2032–2814) b
4200 (3714–4616) c
* 630 (515–761) a,d
* 1400 (1205–1559) e
* 1900 (1649–2245) f
* 5000 (4514–5618) g
* 2300 (2019–2596) f
9.8
6.3
1.6
8.8
2.2
10
170 (162–176) a
240 (154–330) ac
4%
190 (154–229) a
16%
630 (556–714) e
190 (158–225) f
37 (32–43) a
440 (405–474) b
27%
135
21
110 (135–152) g
340 (321–361) c
a
IC50 with (95% CI); b S = selectivity ratio (IC50 hAChE / AgAChE), and and asterisk indicates an IC50 value for
hAChE that is significantly different from AgAChE, as judged by non-overlap of the 95% CI; c LC50 in
µ
g/mL or %
mortality at 1 mg/mL; IC50s or LC50s within a column not labeled by the same lower case letter are significantly
different (p > 0.05), as judged by non-overlap of the 95% CI.
3.3. Toxicity of N-Sulfenylated Methylcarbamates 7a–e
N-Sulfenylated methylcarbamates (Figure 3) were also tested because these pro-insecticides have
been shown to have lower mammalian toxicity; hydrolysis of the labile sulfur-nitrogen bond in vivo
yields the active insecticide [15]. N-Sulfenylated analogs of 1a
, 3a, and 3c were synthesized using
thiophenyl or para-substituted thiophenyl groups (7a 7e). Among the N-sulfenylated compounds,
–
only 7a and 7b showed contact toxicity to mosquitoes in the WHO paper assay. It was observed that
7a killed 70% of G3 mosquitoes in 24 h at 0.5 mg/mL but was not toxic at 0.1 mg/mL. Likewise, 7b
had 100% G3 mortality in 24 h at 1 mg/mL but was not toxic at 0.5 mg/mL. Despite showing modest

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paper contact toxicity, 7a was highly toxic to G3 mosquitoes when topically applied, with an LD50
2.0 ng/female and 95% CI of 1.8–2.3.
=
Figure 3. N-sulfenylated pro-insecticidal analogs of 3-t-butyl-phenyl- and 3-trimethylsilyl-phenyl
methylcarbamates.
4. Discussion
This study initiated a synthesis and testing program with the goal of expanding the existing SAR
as it pertains to carbamate inhibition of AgAChE and hAChE, as well as toxicity. Due to the overall
limited number of analogs made for each chemical series, this work constitutes a search for new leads
as opposed to a thorough exploration of SAR of these two enzymes. It is noteworthy that much of the
classical literature on carbamates focused on activity against housefly AChE and topical toxicity to
this species, as well as larval mosquito bioassays, which makes comparisons to the data of the present
study somewhat difficult.
4.1. Effects of Structural Modification of 1a: Implications for the AgAChE Active Site
The AChE–ligand interactions and molecular docking experiments with structurally diverse
carbamates have been extensively explored in previous studies [
it is clear that AgAChE can accommodate only relatively small substituents at the 6-position of terbam
1a). A 6-bromo-substituent (1d) is favorable, but the slightly larger 6-iodo (1e) and 6-thiomethyl (1m
substituents adversely impact AgAChE inhibitory potency. The larger 6-hexyl (1f), 6-isobutylthio
1n), and 6-isopropylthio (1o) groups all have significantly reduced potency for inhibition of AgAChE.
4,5,11]. Examining the data in Table 1,
(
)
(
Interestingly, hAChE inhibitory potency is less sensitive to steric bulk at the 6-position. In previous
studies, SAR of 3-substituted phenyl carbamates suggested that AgAChE had a slightly larger ligand
binding pocket than hAChE in order to accommodate a 3-t-butyl group [
that in the vicinity of the 6-position, hAChE is more accommodating to steric bulk than AgAChE.
The design of the 4-carboxamido analogs 1h was inspired by preliminary molecular modeling (data
5]; the present study suggests
–
l
not shown) that suggested a hydrogen-bond acceptor at the C4 position of 1a might increase inhibition
potency. Unfortunately, these compounds were even less potent inhibitors of AgAChE than the 4-bromo
analog 1b. The last analog of 1a explored was 2a, which replaced the 3-t-Bu with 3-CF3. We propose
that the drastic decrease in inhibition potency can be attributed to electronic effects. Sterically, the
CF3 group was approximately intermediate in size compared to the isopropyl and t-butyl groups [16],
so there should be plenty of space for it in the AChE active site. Thus, the poor inhibition potency of 2a
may be due to repulsive interaction of the electron-rich CF3 group with one or more of the aromatic
amino acid sidechains that are present in the active site of AgAChE [4]. Reviewing the hAChE potency
of these inhibitors in Table 1, none of these modifications improved enzymatic selectivity, and in
many cases reversed the selectivity to be more potent against hAChE, which was especially true of the
carbamoylated phenyl ring analogs, 1h–l. Lastly, in general, the decreases seen in AgAChE inhibition
potency were reflected by decreased An. gambiae contact toxicity. Interestingly, however, compound
2a exhibited only a 10-fold decrease in contact toxicity relative to 1a, despite its drastically reduced
(>300-fold) AgAChE inhibition potency. A convincing explanation for this discrepancy remains elusive
at present.

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The AChE–ligand interactions and molecular docking experiments with structurally diverse
carbamates have been extensively explored in previous studies [ 11]. Examining the data in Table 1,
it is clear that AgAChE can accommodate only relatively small substituents at the 6-position of terbam
1a). A 6-bromo-substituent (1d) is favorable, but the slightly larger 6-iodo (1e) and 6-thiomethyl (1m
substituents adversely impact AgAChE inhibitory potency. The larger 6-hexyl (1f), 6-isobutylthio
1n), and 6-isopropylthio (1o) groups all have significantly reduced potency for inhibition of AgAChE.
4,5,
(
)
(
Interestingly, hAChE inhibitory potency is less sensitive to steric bulk at the 6-position. In previous
studies, SAR of 3-substituted phenyl carbamates suggested that AgAChE had a slightly larger ligand
binding pocket than hAChE in order to accommodate a 3-t-butyl group [5]; the present study suggests
that in the vicinity of the 6-position, hAChE is more accommodating to steric bulk than AgAChE.
The design of the 4-carboxamido analogs 1h–l was inspired by preliminary molecular modeling (data
not shown) that suggested a hydrogen-bond acceptor at the C4 position of 1a might increase inhibition
potency. Unfortunately, these compounds were even less potent inhibitors of AgAChE than the 4-bromo
analog 1b. The last analog of 1a explored was 2a, which replaced the 3-t-Bu with 3-CF3. We propose
that the drastic decrease in inhibition potency can be attributed to electronic effects. Sterically, the
CF3 group was approximately intermediate in size compared to the isopropyl and t-butyl groups [16],
so there should be plenty of space for it in the AChE active site. Thus, the poor inhibition potency of 2a
may be due to repulsive interaction of the electron-rich CF3 group with one or more of the aromatic
amino acid sidechains that are present in the active site of AgAChE [4]. Reviewing the hAChE potency
of these inhibitors in Table 1, none of these modifications improved enzymatic selectivity, and in
many cases reversed the selectivity to be more potent against hAChE, which was especially true of the
carbamoylated phenyl ring analogs, 1h–l. Lastly, in general, the decreases seen in AgAChE inhibition
potency were reflected by decreased An. gambiae contact toxicity. Interestingly, however, compound
2a exhibited only a 10-fold decrease in contact toxicity relative to 1a, despite its drastically reduced
(>300-fold) AgAChE inhibition potency. A convincing explanation for this discrepancy remains elusive
at present.
4.2. Structural Modification of Trialkylsilylphenyl Methylcarbamate and 2-Thioalkyl Methylcarbamates
Turning to analogs of the 3a and 4a, variants featuring 4-methyl (3c, 4c) and 5-fluoro (3g, 4g) were
prepared and assayed. Both substitutions reduced AgAChE inhibition potency, notably, 5-fluorination
had a significantly more deleterious effect for the 3-SiMe3 derivative than for the 3-SiEtMe2 derivative
(
3g and 4g). None of these variants had improved enzymatic selectivity, and reductions in AgAChE
inhibition potency correlated with reduced mosquitocidal action.
A single variant of 2-thioalkyl-substituted 5a featured a smaller alkyl group (propyl, 6a). This
compound proved less potent and selective for inhibition of AgAChE, relative to 5a. Interestingly, the
insecticidal potency of 6a improved relative to 5a, which may reflect reduced oxidative metabolism in
the mosquito or better transfer off of paper. In a previous study of ortho thioalkyl phenylcarbamates,
compound 6a had essentially optimal activity in this series for both housefly AChE inhibition and Culex
pipiens larval toxicity [3]. Moreover, the addition of piperonyl butoxide to housefly toxicity bioassays
increased toxicity, indicating that sulfur oxidation was not bioactivating as it is for aldicarb [
contrary, these results suggest oxidative detoxication.
3]; on the
4.3. Paper versus Topical Toxicity of N-Sulfenylated N-Methylcarbamates 7a–e
N-arylsulfenyl and N-alkylsulfenyl derivatives of methylcarbamate insecticides have been shown
to possess lower mammalian toxicity and be more effective mosquito larvicides than the parent
methylcarbamates [15]. This same study also reported that the selective toxicity did not relate directly
to anticholinesterase activity. Thus, selectivity of these compounds was likely due to differential
metabolism. Toxicity of arylsulfenylated compounds to mice was lowered up to 50-fold in these
compounds, but was 5- to 17-fold lower for alkylsulfenylated analogs [15]. With such confirmed
improvement of mammalian safety, as well as increased toxicity to insects, a new generation of

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sulfenylated compounds was synthesized for testing. However, sulfenylated analogs of 1a
,
3a, and 3c
did not show good paper contact toxicity to mosquitoes, but 7a was twice as toxic to G3 mosquitoes
topically, compared to 1a, its non-sulfenylated parent. The topical toxicity of the other compounds has
not been determined, and the lack of paper contact toxicity of 7a, even though it is highly toxic when
topically applied, cannot be fully addressed at this point
5. Conclusions
Structural modifications of promising anticholinesterase mosquitocides 1a, 3a, 4a, and 5a were
examined in this paper. A series of 6-substituted analogs of 1a analogs indicated AgAChE cannot
accommodate a substituent larger than bromine in this position. A group of 4-substituted analogs of
1a, 3a, and 4 indicates that AgAChE cannot accommodate any of the attempted substitutions in this
position. Replacement of the t-butyl group of 1a with CF3 resulted in poor anticholinesterase activity,
but surprisingly this compound (2a) did have measurable mosquitocidal activity. The 2-thiopropyl
analog of 5a (6a) possessed lower AgAChE inhibition potency but improved toxicity to adult mosquitoes.
Finally, N-sulfenylated proinsecticidal derivatives of 1a and 3a that were synthesized had poor contact
activity on paper, but one of them (7a) had good topical activity.
Author Contributions: Conceptualization, S.D., M.T., P.R.C., and J.R.B.; methodology, J.M.M.; software, M.T.;
validation, J.M.M.; formal analysis, J.M.M.; investigation, J.M.M.; resources, M.M., Q.-H.C., J.A.H., and D.M.W.;
data curation, J.M.M.; writing—original draft preparation, J.M.M.; writing—review and editing, P.R.C. and J.R.B.;
visualization, J.M.M., M.T., P.R.C., and J.R.B.; supervision, M.T., P.R.C., and J.R.B.; project administration, M.T.,
P.R.C., and J.R.B.; funding acquisition, M.T., P.R.C., and J.R.B.
Funding: This research was funded by the FNIH, grant number 1496, under the Grand Challenges in Global
Health program.
Acknowledgments: The authors are grateful to the MR4 as part of the BEI Resources Repository, NIAID, NIH,
for providing eggs for the Anopheles gambiae G3 (MRA-112) strain.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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