Bivalent carbamates as novel control agents of the malaria mosquito, Anopheles gambiae

Article abstract of DOI:10.2533/chimia.2016.704

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.

Full text of DOI:10.2533/chimia.2016.704

704 CHIMIA 2016, 70, No. 10
doi:10.2533/chimia.2016.704
Malaria Vector control
Chimia 70 (2016) 704–708 © Swiss Chemical Society
Bivalent Carbamates as Novel Control
Agents of the Malaria Mosquito,
Anopheles gambiae
James M. Mutungaae, Qiao-Hong Chenb, Dawn M. Wongb, Polo C-H. Lamd, Jianyong Lic,
Maxim M. Totrovd, Aaron D. Grossa, Paul R. Carlierb, and Jeffrey R. Bloomquista*
Abstract: 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.
Keywords: Anticholinesterase · Catalytic site · Molecular docking · Peripheral site
areas, some reports show that ITNs pro-
vide superior protection against malaria. tethered ligand that binds to the periph-
[5] A combination of both IRS and ITNs has eral site[15–18] or that bears a chemically
been shown to provide enhanced protec- reactive group targeting thiols.[15,19,20]
tion against malaria.[6,7]
Using this approach, selective and cova-
Pyrethroids are the only insecticides lent inhibition of AChE was demonstrated.
currently approved by WHO for deploy- Bivalent organophosphorus[21] and carba-
to the catalytic site atttached to another
1. Introduction
Chemical use of insecticides for mos-
quito control has remained an important
tool and component for the integrated
vector management of malaria and oth-
er vector-borne diseases. The Roll Back
ment with ITNs and continued use has
mate[17,18] inhibitors were shown in some
Malaria Abuja declaration in the year
2000[1] set forth an ambitious plan to re-
duce mortality and morbidity due to ma-
laria in Africa through an array of ways
and means, among them use of insecticide
led to the emergence of resistance.[8] cases to be potent inhibitors of Drosophila
Current reports indicate that insecticide melanogaster and Musca domesticaAChE,
resistance in malaria vectors is increasing and have moderate toxicity to Tetranychus
all over Africa,[9] hence the need for ur- cinnbarinus and Lipaphis erysimi. The
gent measures to mitigate possible control present study was undertaken to explore
treated nets (ITNs) and indoor residual
failure. In semi-field studies, the use of further the potential of bivalent carba-
carbamate-treated nets has shown good mate AChE inhibitors to provide selec-
spraying (IRS). Since then, success in re-
ducing malaria mortality and morbidity
efficacy in controlling mosquitoes, even tive, resistance-breaking insecticides for
due to expansive ITN use has been widely
in pyrethroid-resistant populations.[10–12] malaria vector control.
However, current commercial carbamates
documented.[2–4] Although IRS has been
widely used, especially in malaria endemic
are not effective for control of ace-1 resis-
tantmosquitoes, which express a modified 2. Materials and Methods
acetylcholinesterase possessing a G119S
mutation.[13,14] Conventional carbamates 2.1 Insects
can also have appreciable side effects in
An. gambiae strains G3 (a reference
exposed humans,[11] thereby limiting their susceptible strain) and Akron (insecti-
*Correspondence: Dr. J. R. Bloomquista
E-mail: jbquist@epi.ufl.edu
use. There is a need to develop new insec-
cide-resistant) were obtained from BEI
aDepartment of Entomology and Nematology
Emerging Pathogens Institute
ticides that are less toxic to humans and ef- Resources, through the CDC-MR4. Akron
fective on resistant mosquitoes to be used mosquitoes have both knockdown resis-
as alternatives in mixtures and/or rotations tance (kdr) to pyrethroids (L1014F) and the
University of Florida, Gainesville, FL 32610, USA
bDepartment of Chemistry, Virginia Tech
Blacksburg, VA 24061, USA
with pyrethroids.
G119S mutation (ace-1R), which confers
cDepartment of Biochemistry, Virginia Tech
Blacksburg, VA 24061, USA
One proposed strategy to achieve this resistance to carbamates, in addition to el-
goal is to design bivalent inhibitors of evated levels of carboxylesterase and cyto-
cholinesterases that are able to bind si- chromeP450monooxygenaseactivities.[22]
multaneously and selectively to the pe- Mosquito colonies were maintained at the
ripheral and catalytic sites of AChE. FralinLifeScienceCenterinsectary(Virginia
dMolsoft LLC
11199 Sorrento Valley Road
San Diego, CA 92121, USA
eCurrent address: Human Health Division
International Centre of Insect Physiology and Ecology
(ICIPE)
This approach has been attempted in Tech) or reared from eggs at the Emerging
insects through compounds that bind Pathogens Institute, University of Florida),
P. O. Box 30772-00100, Nairobi, Kenya.

Malaria Vector control
CHIMIA 2016, 70, No. 10 705
under 75% relative humidity and 28 oC, with
at which 50% of treated mosquitoes were
dead.
2.3 Mosquito Toxicity Assays
a 12:12 dark:light cycle. Treated mosquitoes
were held in environmental chambers
maintained under similar conditions.
All bioassays were run on 2–5 day
old An. gambiae, non-blood fed females.
A WHO-type protocol, slightly modi-
fied,[23,24] was used to assess paper contact
toxicity. Solutions of each compound (up
to 1 mg/mL) were applied to a 12 cm ×
15 cm paper, with 95% ethanol as sol-
vent, which was allowed to evaporate.
Mosquitoes were anesthetized on ice, and
2.4 AChE Inhibition Assays
Enzyme was prepared from ho-
mogenates of frozen Anopheles gam-
biae (AgAChE) as described in previous
studies.[15,24] Recombinant human AChE
(hAChE) was purchased from Sigma-
Aldrich and recombinant constructs of the
2.2 Chemicals and Solvents
Propoxur (technical grade), ethanol,
piperonyl butoxide (PBO), and Ellman
reagents were purchased from Sigma-
Aldrich Chemical Co. (St. Louis, MO,
USA). Terbam was synthesized according
catalytic subunit of wild type (LiN) and
acclimatized for 1 h in a WHO cylindri-
cal holding chamber. Mosquitoes were
then moved to the treatment chamber lined
with treated paper and left for 1 h, after
which they were placed in paper cups and
provided with 10% sugar solution ad libi-
to published procedures.[23] Phthalimide-
tethered bivalent carbamates were syn-
thesized as shown in Scheme 1. In brief,
pyrazole (1) was converted to 4-iodo-
pyrazole (2), and phthalimide was re-
acted with α,ω-dibromoalkanes to afford
phthalimide-tethered 1° alkyl bromides
4a–d. N-alkylation of 2 with 4a–d afford-
ed 5a–d. Palladium-catalyzed borylation
afforded 6a–d, and oxidative deborylation
afforded pyrazol-4-yl-ols 7a–d. Reaction
with methylcarbamoyl chloride afforded
a mixture of the anticipated methylcarba-
mates 8a–d, and the rarely seen allophan-
ates 9a–d. Careful chromatographic sepa-
its cognate resistant mutant (G119S) were
prepared as described in a prior publica-
tion.[24] AChE activity was measured using
the Ellman method adapted for a 96-well
microplate assay on a 96-well plate reader
(Dynex, Chantilly, VA, USA), with inhibi-
tum for 24 h. Topical treatments utilized
tion rate constants for each inhibitor and
compound determined as described previ-
ously.[24]
200 nL of chemical solution (dissolved in
95% ethanol) applied to the insect prono-
tum using a handheld Hamilton® microap-
plicator. Synergism studies with PBO were
performed as a topical 4 h pretreatment
(100ng)beforetopicalapplicationofcarba-
mate. For injection, 100 nL was introduced
into the thorax in insect saline containing
2.5 Molecular Modeling
Inhibitor molecule was docked into a
homology model of AgAChE, constructed
as reported by Carlier et al.[15] All in silico
modeling steps were performed in Molsoft
insecticide dissolved in ethanol (no more
[25]
ration afforded pure samples of each. To
ICM software (Molsoft, San Diego, CA
than 5% ethanol final concentration).
After treatments, mosquitoes were held
for 24 h as described above. A solvent-
only treatment was included in each ex-
periment as a negative control, and 24 h
mortality data were analyzed by log-probit
using Poloplus® (LeOra software Co. CA,
USA) to generate the 24 h LC50 or LD50 of
each chemical, the concentration or dose
assess the effect of N,N-disubstitution on
the reactivity and toxicity of the bivalent
carbamates, 7a was treated with dimethyl-
carbamoyl chloride to afford 10a. All com-
USA), as described previously.[26,27]
3. Results and Discussion
pounds were characterized by 1H and 13
C
NMR, and HRMS and were purified to >
95% before enzyme and mosquito toxicity
assays.
Chemical synthetic efforts (Scheme 1)
were directed towards the assembly of
structurally novel bivalent inhibitors that
might occupy both the peripheral and cata-
lytic sites on the mosquito enzyme. These
compounds utilized a pyrazole as the
central moiety, because its smaller size
relative to the phenyl ring was shown previ-
ously to alleviate the high cross-resistance
values found among typical carbamate in-
secticides.[24] The pyrazoles were tethered
to a phthalimido group via alkyl tethers of
varying length (8a–d), along with matched
tether length allophanate analogs (9a–d)
and a single N,N-dimethyl carbamate
(10a). Initial screening for contact insecti-
cidal activity was by WHO paper assay to
adult female mosquitoes. All the bivalents
showed low and variable contact toxicity
to mosquitoes in this assay, regardless of
changes in tether length, or derivatization
to allophanate and N,N-dimethyl analogs
(Table 1). This activity stands in contrast
to the performance of propoxur and terbam
in this same paper assay, where both com-
pounds showed LC50 values of 39 and 37
µg/mL, respectively.[24]
Hypothesizing that the variable toxic-
ity observed for these compounds might be
due to poor transfer from filter paper, topi-
cal application studies were undertaken of
compounds 8a–d (Table 2). While com-
pounds 8b–d were inactive, 8a had signifi-
cant toxicity by this route of exposure, but
was 14-fold and 20-fold less active than the
Scheme 1. Synthesis of pyrazol-4-yl bivalent carbamates.

706 CHIMIA 2016, 70, No. 10
Malaria Vector control
Table 1. Toxicity of pyrazol-4-yl/phthalimide
bivalent compounds (n = the number of
methylene groups in the linker) observed at
24 h when susceptible G3 mosquitoes were
exposed to 1 mg/mL in the WHO paper assay.
Table 2. Topical and injection toxicity studies of bivalent N-methylcarbamates, compared to
established carbamate insecticides, propoxur and terbam.
Compound
n
G3 Topical G3 Injection
bRR
Akron Topical
aLD50 aLD50,
aLD50
8a
3
63 (55-72)
386 (340-430) 0.24 (0.16-0.30)
6
Compound
n
3
3
3
5
5
6
6
7
7
Toxicity
10a
3
5
6
168 (118-205)
0% @ 200 ng
0% @ 200 ng
–
–
–
–
–
–
–
–
–
8a
9a
10a
8b
9b
8c
ca. 16%
ca. 12%
ca. 28%
ca. 20%
0%
8b
8c
8d
7
0% @ 200 ng
c3.2 (2.4-4.2)
c4.5 (3.6-5.4)
–
–
–
propoxur
terbam
d201 (138-295)
d223 (202-245)
63
50
0.24 (0.17-0.34)
0.30 (0.17-0.50)
0%
9c
0%
aLD50 in ng/female or % mortality at the indicated concentration; 95% confidence limits in
parentheses; bRR = Resistance Ratio: Akron LD50 / G3 LD50: Values taken from cHartsel et al.[23]
and dMutunga et al.[22]
8d
9d
10%
10%
established carbamates terbam and pro- genase-inhibiting synergist[28] was tested greater activity than 8a. In terms of hAChE
poxur, respectively. Similarly, 10a showed with 8a and 10a (Fig. 1) to ascertain the selectivity ratios, 8a > 8b = 8d > 9d (Table
topical lethality to An. gambiae females, impact of oxidative metabolism on toxicity. 3). Compared to 8a, terbam and propoxur
albeit 2.7-fold less than 8a. Topical assays
These studies demonstrated a significant had 2.6-fold and 3.6-fold, respectively
with 8a on resistantAkron mosquitoes also increase in toxicity for 8a following 4 h greater selectivity for mosquito enzyme
gave encouraging results. Only mild resis- pretreatment with PBO (P = 0.015), with compared to human. There were variable
tance was observed in Akron when treated a slight decrease (P = 0.17) in the toxicity levels of resistance observed to bivalent
with 8a, in contrast to much greater levels of 10a (Fig. 1). The findings suggest that
of resistance observed for propoxur and metabolism is playing a limited role in the and engineered G119S enzymes, but in all
terbam (Table 2). Interestingly, 8a, ter- toxicity of these compounds. cases was much lower than that expressed
bam, and propoxur are roughly equitoxic Enzyme inhibition studies were per- against propoxur and terbam.
to G3 An. gambiae by injection (Table 2). formed on selected compounds to assess The intrinsic potency of the bivalent
Toxicity is increased 13-fold and 15-fold the intrinsic potency of the bivalent carba- carbamates at the target site, as assessed
for propoxur and terbam, respectively, fol- mates at the target site (Table 3) and com- using mosquito homogenates as enzyme
carbamates by the Akron (homogenate)
lowing injection, while for 8a, the effect pare their inhibitory activities to those of sources, was also compared with results
is more pronounced (263-fold), suggesting propoxur and terbam. Comparing respons- for recombinant enzymes (Table 3). We
that cuticular penetration is an important es on G3 homogenate, it was observed that reported previously on both recombinant
factor impacting its insecticidal activity. 8a is a more potent inhibitor than 8b and Anopheles gambiae AChE and its G119S
The extent to which penetration could be
8d (8- to 9-fold ratio of ki values). The anti- mutant, and verified that the recombinant
enhanced by different adjuvant formula- cholinesterase action of 9b was much less, wild type and resistant enzymes behaved
tions of 8a remains to be investigated. while propoxur had about one third the ac- identically in their sensitivity to conven-
Finally, piperonyl butoxide, a monooxy- tivity of 8a and terbam approached a 2-fold tional aryl-substituted carbamates as the
native enzymes from G3 and Akron ho-
mogenates, respectively.[24] The present
data indicate that inhibitory constants of
bivalent carbamates were also essentially
Fig. 1. Synergism
experiments with
8a and 10a with
PBO, which caused
no lethality in the
mosquitoes at the
dosed used (100 ng).
Asterisk indicates a
significant increase
in mortality for 8a by
PBO (paired T-test,
P = 0.015).
the same for both susceptible and resistant
mosquito homogenates and recombinant
enzymes, with the same rank order of po-
tencies, resistance ratios, and selectivity
ratios (Table 3). These results suggest that
the catalytic mechanism and gorge geom-
etry do not differ significantly between ho-
mogenates and recombinant enzymes.
Molecular modeling studies suggest
that compound 8a probably interacts
with both the catalytic and peripheral site
on AgAChE, as intended (Fig. 2). In the
catalytic site, carbamate interacts with the
oxyanion hole, forming hydrogen bonds
to backbone N–H groups of residues G118
and A200. These interactions position car-
bamate for the nucleophilic attack on S199
hydroxyl, and the pyrazole nitrogen forms
a hydrogen bond to Y121 sidechain. The
phthalimido moiety is mostly occupying

Malaria Vector control
CHIMIA 2016, 70, No. 10 707
Table 3. Enzyme inhibition constants of pyrazol-4-yl/phthalimide bivalents on mosquito strain
homogenates (hmg), recombinant mosquito AChE constructs (bold), and human AChE.
The phthalimide moiety has been used
previously to affect binding of inhibitors to
the peripheral site of AChE. For example,
several phthalimide heterodimers were
prepared and based upon their enzyme
inhibition potencies, molecular modeling
Cmpd
aSusceptible ki:
G3 hmg
aResistant ki:
Akron hmg
G119S
bRR
ahAChE
cSR
LiN
studies suggested that they interacted with
8a
951 ± 39
831 ± 24
16.8 ± 0.7
6.5 ± 0.5
57
128
174 ± 7
91.5 ± 4.2
0.16 ± 0.04
87.5 ± 3.7
17.0 ± 0.4
126 ± 3
5.5
4.8
both the catalytic and peripheral sites of
Torpedo californica AChE.[30] In addition,
8b
109 ± 2
105 ± 1
7.0 ± 0.5
5.3 ± 0.1
16
20
1.2
1.1
a series of N-methyl[16] N,N-dimethyl[17]
carbamate phenyl-phthalimide heterodi-
9b
0.15 ± 0.02
0.16 ± 0.02
0.05 ± 0.02
0.08 ± 0.02
3
2
0.9
1.0
mers (tethers of C3–C ) were synthesized
and assayed for inhibi1t0ion potency against
M. domesticaAChE. The most potent com-
pound in each series was connected to the
phthalimido group via a three-carbon teth-
er, with IC50 values in the low micromolar
range. Little insect toxicity data were re-
ported for these compounds, and no shorter
tethers were synthesized. Of the bivalent
8d
101 ± 3
108 ± 13
4.7 ± 0.4
4.9 ± 0.2
21
22
1.2
1.2
dpropoxur
dterbam
323 ± 8
266 ± 9
0.040 ± 0.005
0.037 ± 0.007
8075
7189
19
16
1710 ± 20
1510 ± 100
0.65 ± 0.06
0.4 ± 0.03
2630
3775
14
12
carbamates reported here, a three-carbon
tether was optimal for both anticholines-
terase activity and lethality, but analogs
having two and four carbons should be
aki values (mM–1min–1) ± SEM determined according to the method of Hartsel et al.[23]; bRR =
Resistance Ratio: G3 hmg ki / Akron hmg ki or LiN ki / G119S ki; cSR = Selectivity Ratio: G3 AChE
ki / hAChE ki or LiN ki / hAChE ki; dData for propoxur and terbam taken from Wong et al.[24]
evaluated.
4. Conclusions
Compound 8a is the first bivalent anti-
cholinesterase that can potently inhibit An.
gambiae AChE and cause demonstrated
lethality to both susceptible and carba-
mate- and pyrethroid-resistant mosquitoes.
The smaller core structure of pyrazol-4-yl
relative to a phenyl ring is in large part
responsible for the ability to link a large
ligand without abrogating AChE inhibito-
ry potency, and the lack of basic nitrogen
groups allows for permeation of the blood-
brain barrier, as well as facilitating contact
insecticidal activity. Based upon these ob-
servations, it should be possible to design
additional bivalent carbamate inhibitors
with improved contact toxicity, low cross
Fig. 2. Molecular homology model of 8a docked into the catalytic site and gorge of susceptible
AChE. See text for explanation.
resistance, and target site selectivity.
Acknowledgments
whole mosquitoes. In particular, the in vi-
tro high activity of 8a is consistent with its
potent injected toxicity, although its topical
activity is significantly less than propoxur
and terbam. Given the lower, but signifi-
cant anticholinesterase activity of 8b and
8d (about one third that of propoxur), topi-
cal doses above 200 ng would be expected
the ‘gorge’ that connects peripheral and
catalytic sites, making a hydrogen bond
to Y328 and contacts with Y121 and Y332
side-chains as well as V71 and D72 back-
bone. It does not reach as far as the key
W280 peripheral site side-chain, which
was observed to interact extensively with
the corresponding moiety in the X-ray
structure of the galanthamine-phthalimide
bivalent inhibitor complex (PDB ID
We thank Paul Howell of the MR4 as part
of the BEI Resources Repository, NIAID,
NIH, for providing eggs of the G3 (MRA-112)
and Akron (MRA-913) strains of Anopheles
gambiae. We also thank the FNIH, Grand
Challenges in Global Health (#1497, to J.R.B.),
the Innovative Vector Control Consortium
(to P.R.C), and USDA Hatch Project (FLA-
to produce mortality. There is somewhat
ENY-005237, to J.R.B.) for financial support.
of a disconnect, however, for comparisons
involving the G119S enzyme and in vivo
toxicity. We observed a 6-fold difference in
toxicity, but a 55- to 128-fold difference in
1W4L).[29] This novel interaction mode is
Received: July 20, 2016
in agreement with the much shorter linker
in our inhibitor, -(CH2)3- as compared to
-(CH2)8- in the previously reported bivalent
compound.[29]
A partial correspondence was observed
between in vitro AChE inhibition and in
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