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

Article abstract of DOI:10.1016/j.bmcl.2015.09.019

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.

Full text of DOI:10.1016/j.bmcl.2015.09.019

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Difluoromethyl ketones: Potent inhibitors of wild type and
carbamate-insensitive G119S mutant Anopheles gambiae
acetylcholinesterase
Eugene Camerino a, Dawn M. Wong a, Fan Tong b, Florian Körber a, Aaron D. Gross b, Rafique Islam b,
Elisabet Viayna a, James M. Mutunga b, Jianyong Li c, Maxim M. Totrov d, Jeffrey R. Bloomquist b,
Paul R. Carlier a,
⇑
a Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
b Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA
c Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
d Molsoft LLC, 11199 Sorrento Valley Road, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 a-fluorinated methyl ketones as reversible covalent inhibi-
Received 17 July 2015
Revised 4 September 2015
Accepted 7 September 2015
Available online xxxx
tors 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.
Keywords:
Malaria
Anopheles gambiae
Acetylcholinesterase
Trifluoroketones
Fluorinated ketones
Ó 2015 Elsevier Ltd. All rights reserved.
Malaria is a devastating disease, responsible for an estimated
584,000 deaths world-wide in 2013.1 Efforts to control the vector
Anopheles gambiae have significantly reduced malaria mortality,
through the use of insecticide treated nets (ITN) and indoor
residual spraying (IRS).2 To date these methods rely on only two
biological targets: the voltage-gated sodium ion channel and
acetylcholinesterase (AChE).3 Pyrethroids modulate the sodium
channel and are approved by the World Health Organization
(WHO) for IRS and for use on ITNs.4 Organophosphate and
carbamate AChE inhibitors (AChEI) are approved only for IRS.4
Due to the growing emergence of pyrethroid-resistant strains of
A. gambiae,5 there is increased interest in developing classes of
AChEI that might be safe and effective on ITNs. Our group has
A. gambiae AChE (AgAChE) over human AChE (hAChE).6 We have
also developed five-membered ring heterocycle core carbamates
and carboxamides (e.g., 2 and 3, Fig. 1) that offer good toxicity
against the carbamate-resistant (Akron) strain A. gambiae.7 This
strain of A. gambiae is known to carry a G119S mutant AChE,7a,8
and the smaller core structure of these heterocyclic carbamates
and carboxamides may partly account for their good inhibition of
G119S AgAChE.
To further address the need for new insecticides, we sought to
investigate underexplored AChEI chemotypes 4–8 (Fig. 1). Trifluo-
romethyl ketones (e.g., 4, 5a,b) have been studied as inhibitors of
AChE9 and juvenile hormone esterase.10 Despite the remarkable
(picomolar) potencies that can be achieved,11 with few exceptions
(e.g., 4)10b this class of compounds has received little attention as
insecticides. These highly electrophilic compounds form
tetrahedral intermediates with the catalytic serine of AChE, as
demonstrated by X-ray crystallography.12 Zifrosilone (5b) was
previously reported that c-branched 2-substituted aryl methylcar-
bamates (e.g., 1, Fig. 1) can be highly selective for inhibition of
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.09.019
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Camerino, E.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.019

2
E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 1. Mosquitocidal AChE inhibitors 1–3 and select fluorinated ketones (4–8)
used as AChE or serine/cysteine protease inhibitors.
evaluated as an AChEI for the treatment of memory loss in Alzhei-
mer’s disease,13 suggesting that the CNS penetrance needed for
insecticidal action could be achieved with appropriate structural
modification. Difluoromethyl ketones have received limited atten-
Scheme 1. Synthesis of a-fluorinated ketones. Reagents and conditions: (i) n-BuLi,
THF, À78 °C, 2 h; CH3OC(O)CF3, À78 °C to RT, overnight; (ii) n-BuLi, THF, À78 °C,
2 h; CH3CH2OC(O)CF2H, À78 °C, 5 min; (iii) n-BuLi, THF, À78 °C, 2 h; CH3CH2OC(O)
CFH2, À78 °C, 5 min; (iv) NBS, H2O, 1 h; NaH, DMF, 0 °C, 1 h; R-Br, RT, overnight.
tion as AChE inhibitors, although
a,a-difluoroalkyl ketone 6 was
shown to be quite potent (Ki = 1.6 nM) at electric eel AChE.9b
More commonly, these difluorinated ketones have been explored
as inhibitors of serine proteases such as chymotrypsin,14
a-lytic
protease,15 human leukocyte elastase,15 and thrombin.16 Perhaps
due to the expectation that reduced electrophilicity would
adversely impact inhibition potency, fluoromethyl ketones have
(to our knowledge) not been reported as AChE inhibitors. Although
7 proved to be a very weak inhibitor of the serine protease chy-
motrypsin,14 dipeptidyl aspartyl fluoromethyl ketones such as 8
can be potent inhibitors of cysteine proteases.17
We thus synthesized a series of tri-, di-, and (mono)fluo-
romethyl ketones bearing substituted benzene and pyrazol-4-yl
substituents (Scheme 1). Trifluoromethyl ketones 5b–i were
prepared by the literature route for 5c: metal–halogen exchange
of the appropriate aryl/heteroaromatic bromide and trapping with
CF3CO2Me (Scheme 1).9d The requisite N-alkyl-4-bromopyrazoles
11d–i were prepared in two steps from pyrazole.18 The
Scheme 2. Possible side reactions in the synthesis of fluorinated ketones 5, 9, 10.
preponderance of
a-branched alkyl groups selected reflects the
observation that these substituents increase AgAChE inhibition
potency of pyrazol-4-yl7a carbamates and 3-oxoisoxazole-2
(3H)carboxamides (e.g., 2, 3).7b Difluoromethyl ketones 9c–i and
fluoromethyl ketones 10c–i were prepared by trapping with
CF2HCO2Me and CFH2CO2Et, respectively. Yields of the
trifluoromethyl ketones 5b–i were moderate, and in part reflects
the high volatilities of these compounds. However, yields of the
difluoro- and fluoromethyl ketones 9c–i and 10c–i were only
poor to fair. We attribute these poor yields to partial collapse of
tetrahedral adducts 13 and 14 to the fluorinated methyl ketones
9 and 10 prior to quench, and reaction with Ar–Li (Scheme 2).
Based on the relative electrophilicity of the fluorinated ketones,
the extent of collapse prior to protic quench should be
14 > 13 > 12, which could account for the trend in chemical yield.
Finally to assess the structure of these compounds in aqueous
solution, 19F NMR spectroscopic studies9c were performed at pH
7.7. Pyrazol-4-yl trifluoromethyl ketone 5g was 67% hydrated,
difluoromethyl ketone 9i was 22% hydrated, and fluoromethyl
ketone 10i was <5% hydrated (24 h, see Supplementary data).19
Enzyme inhibitory activity of the compounds were assessed
using a modified Ellman assay20 in a 96-well microtiter plate
format previously reported.7a Because time-dependent inhibition
of AChE by trifluoromethyl ketones is well-documented,9a,c,e
enzyme velocities (v/v0) were measured as a function of inhibitor
concentrations [I] at incubation times of 10 min and 60 min.
Sigmoidal plots of residual enzyme activity (v/v0) versus [I] were
constructed, from which the IC50 values were obtained. For the
purpose of comparison we also examined the commercial
carbamate propoxur, since it has excellent contact activity
against susceptible (G3) strain A. gambiae, but poor toxicity
against carbamate-resistant (Akron) strain A. gambiae.6b,7a Like all
carbamate insecticides, propoxur carbamoylates the active site
serine of AChE,21 and its time-dependent inhibition of hAChE and
WT AgAChE is evident in Table 1. However this compound was a
very weak inhibitor of G119S AgAChE at 10 or 60 min incubation
times (IC50 > 10,000 nM), consistent with the carbamate resistance
phenotype this mutation confers. The organophosphate inhibitor
dichlorvos was also examined, and the potency and time-
dependence of its inhibition of hAChE and WT AgAChE was
similar to that of propoxur. However unlike propoxur, dichlorvos
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E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Table 2
Inhibition IC50 values for propoxur, dichlorvos and trifluoromethyl ketones 5b–i
against hAChE and AgAChE (WT and G119S)
Inhibition IC50 values for difluoromethyl ketones 9c–i against hAChE and AgAChE (WT
and G119S)
a
Compound Incubation
time (min)
hAChE IC50
(nM)
WT AgAChE
IC50 (nM)
G119S AgAChE
IC50 (nM)
a
a
9c
9d
9e
9f
10
60
10
60
10
60
10
60
10
6.05 0.11
8.69 0.18
802 30
9.12 0.31
9.79 0.32
1650 100
996 39
8290 400
8380 420
452 12
Compound Incubation
time (min)
hAChE
IC50 (nM)
WT AgAChE
IC50 (nM)
G119S AgAChE
IC50 (nM)
354
430
8
7
a
a
a
869 20
110
2
26.3 0.4
25.2 0.6
23.4 0.4
29.1 0.4
1.01 0.02
1.22 0.03
ND
Propoxur
10
60
2300 50
590 19
2310 60
627 12
182
43.6 1.0
324 7
3
>10,000
>10,000
1650 44
85.9 1.6
185
797 20
297
680 43
125
7
149
158
2
3
Dichlorvos 10
60
6
60.1 0.9
848 44
257 15
53.4 0.8
18.1 0.4
300
7
9g
28.8 0.7
35.2 0.7
ND
5b
5c
5d
5e
5f
10
60
10
60
10
60
10
60
10
60
10
60
10
60
10
60
285 15
>10,000
10,100 500
>10,000
20,800 1900
>10,000
>10,000
8180 450
2340 90
19,900 2400
2000 140
>10,000
1730 140
>10,000
3520 140
>10,000
60
6
92.9 5.8
14.6 0.2
5.00 0.16
77.5 2.2
330
1380
10
60
10
36.7 1.7
25.1 1.2
3220 150
3390 110
>10,000
ND
ND
9h
9i
208
244
5
5
106
134
2
2
142
285
5
7
121
3
9780 470
11,000 400 3950 80
3000 80
6.47 0.15 2.47 0.04
8.84 0.28 2.29 0.17
7.15 0.11 3.74 0.08
8.37 0.16 2.06 0.06
3.77 0.06 2.75 0.04
2.27 0.04 0.68 0.01
5.20 0.15 2.87 0.07
6.11 0.18 1.54 0.08
60
>10,000
a
Measured at 23 1 °C, pH 7.7, 0.1% (v/v) DMSO;. ND signifies not determined.
Average Hill slopes at WT AgAChE, hAChE, and G119S AgAChE (9c–9g only for this
enzyme) are 0.9 0.1.
5g
5h
5i
Difluoromethyl ketones 9c–i were then examined for their
enzyme inhibition properties (Table 2). As expected, IC50 values
for the difluoromethyl ketones at hAChE and WT AgAChE were gen-
erally higher than the values for the corresponding trifluoromethyl
ketones (Table 1), with a few noteworthy exceptions. Difluo-
romethyl ketone 9c was more potent than trifluoromethyl analog
5c at both hAChE and WT AgAChE, and difluoromethyl ketone 9g
was similar in potency to trifluoromethyl analog 5g at WT AgAChE.
Both compounds have rather large alkyl substituents (t-Bu and
3-pentyl, respectively), which suggests that in some cases the smal-
ler size of the –CF2H group compared to –CF3 can compensate for its
lower electron-withdrawing ability. This effect also appears to be
operative in the inhibition of G119S AgAChE, which has a more
crowded oxyanion hole than WT AgAChE. As can be seen in Table 2,
G119S AgAChE IC50 values of difluoromethyl ketones 9c–h are uni-
formly lower than those of the corresponding trifluoromethyl
ketones (Table 1). In addition, time-dependent inhibition is seen
for the G119S enzyme: after a 60 min incubation the G119S AgAChE
IC50 value of 9g is 125 nM, 13-fold lower than that of trifluo-
romethyl ketone 5g, and 2-fold lower than that of dichlorvos.
Steady-state inhibition of G119S AgAChE by 9g is nearly attained
after 330 min incubation, but after 1380 min (23 h) an IC50 value
of 25.1 1.2 nM was measured (Table 2). Thus in the case of 9g,
the smaller size of the –CF2H group effectively compensates for
its lower electron-withdrawing power, creating a slow, tight-
binding inhibitor of G119S AgAChE.
591 11
678 15
950 19
1040 20
>10,000
a
Measured at 23 1 °C, pH 7.7, 0.1% (v/v) DMSO; all enzymes are recombinant.
Average Hill slopes at WT AgAChE and hAChE are 1.0 0.2.
exhibited significant inhibition of G119S AgAChE, likely due to the
smaller structure of its enol leaving group. The 5-fold ratio of
G119S and WT IC50 values is very similar to that measured in a
previous study.22
Trifluoromethyl ketones 5b–i showed varying degrees of time-
dependence to their inhibition of hAChE and WT AgAChE; those
bearing a phenyl group (5b,c) showed time-dependent inhibition
of both hAChE and AgAChE. However, those trifluoromethyl
ketones bearing a pyrazol-4-yl substituent approach steady-state
inhibition within 10 min, and 5e–h gave single-digit nanomolar
IC50 values. In contrast, compound 5i, which bears a b-branched
isobutyl group, was considerably less potent at hAChE and WT
AgAChE than any of the pyrazol-4-yl compounds bearing
a
-branched substituents (5d–h). Although potent inhibition of
WT AgAChE can be achieved with a pyrazol-4-yl trifluoromethyl
ketone, this structure confers no inhibition selectivity against
hAChE (Table 1).
In addition, none of these inhibitors offered potent inhibition of
G119S AgAChE, most likely due to crowding in the oxyanion hole
caused by the glycine to serine mutation. Compound 5g proved
most potent at this enzyme, but its 1730 nM IC50 value after
60 minutes incubation is roughly 2500-fold greater than the
0.7 nM value observed for WT AgAChE. Finally 5d and 5i curiously
exhibit higher hAChE and WT AgAChE IC50 values at 60 min than at
10 min. We attribute this phenomenon to rapid attainment of
steady state, and evaporation of 5d and 5i out of the well plate dur-
ing the longer incubation. As we will illustrate below, fluorinated
compounds can be remarkably volatile, and 5d has the lowest
molecular weight of all the trifluoromethyl ketones tested.
To rationalize the drastically different potencies of trifluo-
romethyl ketone 5g at WT AgAChE and G119S AgAChE (IC50 values
0.68 and 1730 nM, respectively, at 60 min), and the high potency of
difluoromethyl ketone 9g for G119S AgAChE, we examined the
computed structures of these compounds bound to the enzymes
(Fig. 2). As a starting point for docking studies we used previously
developed homology models of WT and G119S AgAChE7a based on
the published X-ray structure of mouse AChE complexed to 5a
(PDB ID 2H9Y).12c Flexible ligand docking of the tetrahedral
intermediates derived from 5g and WT AgAChE, and of 9g and
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E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
with the G119 to S119 mutation, the covalent adduct of 5g with
WT AgAChE was superimposed with the G119S AgAChE apo struc-
ture (Fig. 2B). As can be seen in Figure 2A, trifluoromethyl ketone
5g can easily bind to the catalytic serine (S199) of WT AgAChE,
as expected from the X-ray structures of 5a bound to TcAChE and
mAChE (PDB ID 1AMN12a and 2H9Y,12c respectively). However, as
shown in Figure 2B, replacement of G119 with S119 causes steric
repulsion between the S119 hydroxy group and one of the
fluorine atoms of the CF3 group of 5g: the indicated O–F distance
of 2.31 Å is significantly shorter than the sum of the respective
van der Waals radii (2.99 Å). However in the complex of G119S
AgAChE with the analogous difluoromethyl ketone 9g (Fig. 2C),
this unfavorable interaction is replaced with
a potential
hydrogen bond from the hydrogen of the HCF2 group to the S119
oxygen. The CF2H group is a known H-bond donor;24 in this way
the excellent inhibitory potency of 9g for G119S AgAChE can be
rationalized.
Turning to the fluoromethyl ketones 10c–i, much weaker inhi-
bition of hAChE and WT AgAChE is seen compared to that of diflu-
oromethyl ketones 9c–i and trifluoromethyl ketones 5c–i (Table 3).
This outcome is understandable in view of the low electrophilicity
of the fluoromethyl ketones. Two results stand out, however.
Firstly, fluoromethyl ketone 10g is a ꢀ350 nM inhibitor of WT
AgAChE. At a 10 min incubation time, its IC50 value is roughly
2-fold higher than that of propoxur (Table 1). Secondly, compound
10g also displayed micromolar inhibition of G119S AgAChE. Thus
given the appropriate pyrazol-4-yl substituent, fluoromethyl
ketones evidence inhibition of both WT and G119S AgAChE.
During our microtiter plate inhibition assay development we
were surprised to find that trifluoroketones appeared to migrate
from high [I] wells to the adjacent inhibitor-free control wells.25
We further observed that the spatial extent of this migration was
significantly enhanced when the microtiter plate was covered with
its accompanying ‘non-sealing’ polystyrene lid. We believe the
loose cover provided by the lid serves to direct evaporation to
the neighboring wells rather than to allow it to escape to the
atmosphere. Progressive ‘distillation’ of 5g in such a loosely
covered microtiter plate format is demonstrated in Figure 3:
although the inhibitor was placed only in wells D6–D7, after
Table 3
Inhibition IC50 values for fluoromethyl ketones 10c–i against hAChE and AgAChE (WT
and G119S)
a
Compound Incubation
time (min)
hAChE IC50
(nM)
WT AgAChE
IC50 (nM)
G119S AgAChE
IC50 (nM)
a
a
10c
10d
10e
10f
10g
10h
10i
10
60
10
60
10
60
10
60
10
60
10
60
10
60
578 13
715 15
>10,000
>10,000
16,900 1200 2750 60
18,000 1900 2860 60
24,200 2700 3520 70
17,600 1800 3290 80
5290 250
5190 270
2500 80
1310 30
>10,000
726 12
953 18
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2990 160
3000 160
>10,000
>10,000
>10,000
>10,000
355 11
337 11
5860 160
1540 40
>10,000
Figure 2. (A) Trifluoromethyl ketone 5g bound to S199 of WT AgAChE. (B) 5g bound
to S199 of G119S AgAChE; repulsive non-bonded contact of the S119 hydroxy group
with the CF3 group is evident. (C) Difluoromethyl ketone 9g bound to S199 of G119S
AgAChE; a potential hydrogen bond from the CF2-H of 9g to the oxygen of S119 is
indicated.
>10,000
>10,000
G119S AgAChE were performed in ICM using default settings for
‘covalent’ docking mode (ICM-Docking module, Molsoft) to
generate Figure 2A and C.23 To identify steric conflicts associated
a
Measured at 23 1 °C, pH 7.7, 0.1% (v/v) DMSO; Average Hill slopes at WT
AgAChE and hAChE are 0.9 0.1 and 0.8 0.1, respectively (compounds 10d, 10i
excluded).
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E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Figure 3. Microtiter plate heat maps of WT AgAChE residual activity in which only wells D6–D7 of the microtiter plates were charged with 10,000 nM of inhibitor 5g for the
indicated incubation time (10–60 min, 23 1 °C) before the addition of substrate. Data for Row H (enzyme-free background wells) are not shown. Color coding: red, 610%
residual activity; yellow, 675% residual activity; green P93% residual activity. Progressive vapor phase diffusion of 5g over 60 min is evident. See Supplementary data for
residual activity values.
10 min significant enzyme inhibition is visible up to 3 wells away.
Further spreading is evident at 20 and 40 min, and at 60 min nearly
every well evidences contamination by 5g. In a 10 min incubation
study of a homologous series of compounds, volatility was seen to
decrease from 5g (CF3) to 9g (CF2H) to 10g (CFH2) (Fig. 4). Whereas
trifluoromethyl ketone 5g diffuses over 3 wells from D6–D7 in
10 min, difluoromethyl ketone 9g diffuses only 1 well. In contrast,
no diffusion of fluoromethyl ketone 10g seen over 10 min. Thus the
degree of fluorination plays a dominant role in the volatility of this
series of analogs. Interestingly, no diffusion of dichlorvos was seen
over 10 min, suggesting it is less volatile than 5g and 9g (see
Fig. S1, Supplementary data). Finally, as expected, no diffusion of
the propoxur control is seen.
As noted above, several of the fluorinated methyl ketone inhibi-
tors (5c–h, 9e–h) demonstrated potent inhibition of WT AgAChE.
We thus examined the tarsal contact toxicity of select compounds
to adult susceptible (G3) strain A. gambiae, using the recommended
WHO treated paper protocol26 (Table 4). As can be seen none of the
compounds tested approach the contact toxicity of propoxur,
despite the fact that many (5c, 5e–5h, 9c, 9e–9g) are much more
potent inhibitors of WT AgAChE than propoxur after a 60 min incu-
bation. The most toxic compounds tested were fluoromethyl
dates that treated papers be dried overnight to remove the solvent
vehicle prior to mosquito exposure. Could extensive evaporation of
tri- and difluoroketones (all liquids) from the treated papers prior
to mosquito exposure account for the low and variable toxicities
seen in Table 4? To assess this possibility, we measured the evap-
orative weight loss of 5g at room temperature, and compared it to
propoxur (mp 91 °C) and dichlorvos (liquid at room temperature).
As can be seen in Table 5, water evaporated completely within
1 day, and propoxur showed no weight loss over 28 days. The
liquid organophosphate inhibitor dichlorvos lost 32 1% of its
mass over 12 days, and 51 1% over 28 days. Trifluoromethyl
ketone 5g proved even more volatile than dichlorvos, losing
68 1% of its mass over 12 days, and 99 1% over 28 days. How-
ever over 1 day, 5g lost only 9 1% of its mass. Thus the ꢀ25-fold
lower tarsal contact toxicity of 5g (40% mortality at 1000
l
g/mL)
relative to propoxur (LC50 = 39
to compound evaporation.
lg/mL) cannot be attributed solely
Since poor penetration of the exoskeleton following tarsal con-
tact could impede delivery of these compounds to the mosquito
CNS, we explored the toxicity of the most potent trifluoromethyl,
difluoromethyl and fluoromethyl ketone inhibitors of AgAChE-WT
(5g, 9g, and 10g, respectively) in two other assays. First we exam-
ined the toxicity of these compounds in a fumigation assay,27
whereby A. gambiae were placed in a sealed 1 L vessel containing
a known mass of an AChE inhibitor, but prevented from direct
physical contact with the compound (Table 6). As expected, the
nonvolatile compound propoxur was completely non-toxic at a
high nominal concentration of 1000 ng/mL.
ketones 10d and 10g (85% mortality at 1000 lg/mL). Yet these
compounds differ dramatically in their inhibition of WT AgAChE,
giving IC50 values of >10,000 nM and 337 11 nM, respectively
(60 min incubation). Therefore there is no apparent correlation
between tarsal contact % mortality and AChE inhibition potency
for these compounds. The procedure for this toxicity assay26 man-
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6
E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 4. Microtiter plate heat maps of WT AgAChE residual activity (10 min incubation) in which only wells D6–D7 of the microtiter plates were charged with 10,000 nM of
inhibitor (5g, 9g, 10g, propoxur: 23 1 °C). Data for Row H (enzyme-free background wells) are not shown. Color coding follows that of Figure 3. Vapor phase diffusion of 5g
and 9g is evident. See Supplementary data for residual activity values.
Table 4
Table 5
Tarsal contacta toxicity of select fluorinated methyl ketones to susceptible (G3) strain
Evaporative weight loss of AChE inhibitors (and water control) at room temperaturea
adult A. gambiae
Compound
% mass lost by evaporation
6 days 12 days
Compound
LC50
mortality at
1000 g/mL
(
l
g/mL) or %
Compound
LC50
% mortality at
1000 lg/mL
(lg/mL) or
1 day
28 days
l
Propoxur
Dichlorvos
5g
0
2
1
0
16
33
99
1
1
1
1
1
32
68
1
1
1
1
0
51
99
1
1
1
1
Propoxur
5b
5c
39 (32–45)b
10
8
9f
9g
9h
0
0
0
1
1
1
9
Water
99
100
100
5d
5e
5f
5g
5h
9c
9d
9e
20
30
0
40
40
20
50
50
10c
10d
10e
10f
10g
10h
10i
50
85
65
20
85
0
a
Compounds (starting masses 8–15 mg) were placed in open 1 dram vials in a
fume hood at room temperature.
20
Table 6
a
Papers were treated with ethanolic solutions of fluorinated methyl ketones and
Fumigation and injection toxicity of select AChE inhibitors
allowed to dry overnight. Mosquitoes were exposed (1 h) and mortality was
recorded after 24 h.
Compound Fumigation mortality at
indicated nominal
Injection LD50 (ng/insect) or
LC50 values derive from the concentrations of inhibitor used to treat the paper;
mortality at 50 ng/insectb
b
95% confidence limits are given in parenthesis. Data for propoxur were reported
concentrationa
previously.7a
Propoxur
Dichlorvos 100%@10 ng/mL
0%@1000 ng/mL
0.24 (0.20–0.34)
ND
5g
9g
86 7%@1000 ng/mL
57 25%@1000 ng/mL
83 6%
65 4%
In contrast dichlorvos, which is known for its vapor phase
insecticidal action,27 displayed 100% mortality at a 100-fold lower
nominal concentration (10 ng/mL). However 5g, which is 100-fold
more potent than dichlorvos at WT AgAChE (Table 1), and more
volatile (Table 5), proved to be >100-fold less toxic than dichlorvos
(86 7% mortality at 1000 ng/mL). Thus the low fumigation
10g
8
6%@1000 ng/mL
21 8%
a
Measured % mortality after 24 h in a 1 L sealed vessel, see Supplementary data
for experimental details. Nominal concentration is calculated from the mass of
AChE inhibitor delivered and the volume of the vessel.
See Supplementary data for protocol; the 95% confidence interval for the pro-
poxur LD50 value is given in parenthesis. ND signifies ‘not determined’.
b
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E. Camerino et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
7
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Supplementary data
Supplementary data (experimental protocols, additional figures,
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019.
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Please cite this article in press as: Camerino, E.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.019