N-Aryl-N’-ethyleneaminothioureas effectively inhibit acetylcholinesterase 1 from disease-transmitting mosquitoes

Article abstract of DOI:10.1016/j.ejmech.2017.03.050

Vector control of disease-transmitting mosquitoes by insecticides has a central role in reducing the number of parasitic- and viral infection cases. The currently used insecticides are efficient, but safety concerns and the development of insecticide-resistant mosquito strains warrant the search for alternative compound classes for vector control. Here, we have designed and synthesized thiourea-based compounds as non-covalent inhibitors of acetylcholinesterase 1 (AChE1) from the mosquitoes Anopheles gambiae (An. gambiae) and Aedes aegypti (Ae. aegypti), as well as a naturally occurring resistant-conferring mutant. The N-aryl-N’-ethyleneaminothioureas proved to be inhibitors of AChE1; the most efficient one showed submicromolar potency. Importantly, the inhibitors exhibited selectivity over the human AChE (hAChE), which is desirable for new insecticides. The structure-activity relationship (SAR) analysis of the thioureas revealed that small changes in the chemical structure had a large effect on inhibition capacity. The thioureas showed to have different SAR when inhibiting AChE1 and hAChE, respectively, enabling an investigation of structure-selectivity relationships. Furthermore, insecticidal activity was demonstrated using adult and larvae An. gambiae and Ae. aegypti mosquitoes.

Full text of DOI:10.1016/j.ejmech.2017.03.050

Accepted Manuscript
N-aryl-N’-ethyleneaminothioureas effectively inhibit acetylcholinesterase 1 from
disease-transmitting mosquitoes
Sofie Knutsson, Tomas Kindahl, Cecilia Engdahl, Dariush Nikjoo, Nina Forsgren,
Stanley Kitur, Fredrik Ekström, Luna Kamau, Anna Linusson
PII:
S0223-5234(17)30217-9
10.1016/j.ejmech.2017.03.050
EJMECH 9310
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 January 2017
Revised Date: 22 March 2017
Accepted Date: 23 March 2017
Please cite this article as: S. Knutsson, T. Kindahl, C. Engdahl, D. Nikjoo, N. Forsgren, S.
Kitur, F. Ekström, L. Kamau, A. Linusson, N-aryl-N’-ethyleneaminothioureas effectively inhibit
acetylcholinesterase 1 from disease-transmitting mosquitoes, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.03.050.
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ACCEPTED MANUSCRIPT
N-Aryl-N’-ethyleneaminothioureas Effectively Inhibit Acetylcholinesterase 1 from
Disease-Transmitting Mosquitoes
Sofie Knutsson,[a] Tomas Kindahl,[a] Cecilia Engdahl,[a] Dariush Nikjoo,[a] Nina Forsgren,[b] Stanley Kitur,[c] Fredrik
Ekström,[b] Luna Kamau[c] and Anna Linusson[a]*
[a] Dr. S. Knutsson, Dr. T. Kindahl, C. Engdahl, Dr. D. Nikjoo, Prof. A. Linusson
Department of Chemistry
Umeå University
SE-901 82, Umeå (Sweden)
E-mail: anna.linusson@umu.se
[b] Dr. N. Forsgren, Dr. F. Ekström
CBRN Defence and Security
Swedish Defence Research Agency
SE-906 21 Umeå (Sweden)
[c] S. Kitur, Dr. L. Kamau
Centre for Biotechnology Research and Development
Kenya Medical Research Institute
Nairobi (Kenya)
*corresponding author
Abstract: Vector control of disease-transmitting mosquitoes by insecticides has a central role in reducing the
number of parasitic- and viral infection cases. The currently used insecticides are efficient, but safety concerns
and the development of insecticide-resistant mosquito strains warrant the search for alternative compound
classes for vector control. Here, we have designed and synthesized thiourea-based compounds as non-covalent
inhibitors of acetylcholinesterase 1 (AChE1) from the mosquitoes Anopheles gambiae (An. gambiae) and Aedes
aegypti (Ae. aegypti), as well as a naturally occurring resistant-conferring mutant. The N-aryl-N’-
ethyleneaminothioureas proved to be inhibitors of AChE1; the most efficient one showed submicromolar
potency. Importantly, the inhibitors exhibited selectivity over the human AChE (hAChE), which is desirable for
new insecticides. The structure-activity relationship (SAR) analysis of the thioureas revealed that small changes
in the chemical structure had a large effect on inhibition capacity. The thioureas showed to have different SAR
when inhibiting AChE1 and hAChE, respectively, enabling an investigation of structure-selectivity relationships.
Furthermore, insecticidal activity was demonstrated using adult and larvae An. gambiae and Ae. aegypti
mosquitoes.
1. Introduction
Mosquitoes of the Anopheles and Aedes genera act as vectors for parasitic and viral infectious diseases that
affect the health of hundreds of millions of people every year. With 40% of the world’s population at risk,
dengue is now considered one of the fastest spreading vector-borne diseases and poses a serious public health
threat [1-3]. National programs and international initiatives to increase the coverage of mosquito-control
interventions have resulted in a decline in the incidence of the deadliest vector-borne disease, malaria [4]. Still,
in 2016, nearly 1.1 billion people remained at high risk of malaria infection, with young children being among
the most vulnerable [5]. Current mosquito-control interventions are primarily based on insecticide-treated bed
nets (ITN) and indoor residual spraying (IRS). These methods rely on four classes of insecticides, and their
effectiveness is seriously threatened by the development and spread of insecticide-resistant mosquito
populations [5-7]. Accordingly, there is a need for new strategies and chemicals for vector control in order to
maintain or accelerate the current progress of achieving large-scale reduction of vector-borne diseases [8-12].
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The essential enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is pivotal in cholinergic neurotransmission and
a validated insecticidal target [13, 14]. The active site gorge of AChEs is lined with aromatic residues and has
two partly overlapping binding sites, one at the entrance of the gorge, the peripheral anionic site (PAS), and one
at the bottom of the gorge, the catalytic site (CAS) [15]. These two sites in AChE possess similar binding
properties with a preference to bind cationic ligands. The natural substrate of AChE, the neurotransmitter
acetylcholine, is a permanent cation and able to slide from its initial binding at the PAS to the bottom of the
gorge where it is rapidly hydrolyzed by the catalytic triad (Ser203, His447 and Glu334, hAChE numbering is used
throughout the text). Mosquitoes have two genes encoding AChE enzymes: ace-1 and ace-2 [16], where the
enzyme encoded by ace-1 (AChE1) appear to have the central catalytic function [17]. The mosquito AChEs has
been less studied than the vertebrate AChE, although it has been shown to hydrolyze the same substrates with
similar kinetic properties [18-21].
Two of the four chemical classes of insecticides recommended for IRS, namely organophosphates and
carbamates, covalently inhibit the activity of AChE1. These insecticides are efficient at controlling disease-
transmitting mosquitoes, however, their ability to inhibit AChE enzymes from many different species, including
humans, give rise to safety concerns [22, 23]. In addition, some mosquito species have evolved a variant of the
AChE1 enzyme featuring a point mutation in the active site (G122S in human AChE (hAChE), corresponding to
G119S in Torpedo californica numbering) that confers resistance to existing organophosphate- and carbamate-
based insecticides [24]. Consequently, new insecticides targeting AChE need to address both selectivity and
target-site resistance. Encouraging results have been reported in the literature, including a number of covalent
inhibitors of AChE1 displaying either selectivity for the mosquito enzyme over hAChE or retained activity on the
resistant G122S mutant [20, 25-30]. To date, two different design strategies of selective covalent AChE1-
inhibitors have been presented. In the first approach, a cysteine present near the active site gorge of AChE1, but
not present in vertebrate AChE, has been targeted with covalent inhibitors [25, 28, 31, 32]. The second design
strategy targets the Ser203 residue of the catalytic triad with covalent inhibitors, in a similar fashion as the
currently approved and used insecticides. A variety of new potential inhibitors has been used to probe
selectivity and potency profiles [20, 22, 26, 27, 29, 30, 33, 34].
Instead of aiming for new inhibitors that react covalently with AChE, our focus is on non-covalent inhibitors
with the possibility to explore parts of the active site gorge located distant from the catalytic triad. In a recent
differential high-throughput screen (HTS) against the AChE1 enzymes from Anopheles gambiae (An. gambiae;
vector of the malaria parasite; AgAChE1) and Aedes aegypti (Ae. aegypti; vector of the dengue, yellow fever,
chikungunya, and Zika virus infection; AaAChE1), we identified a number of non-covalent hits showing potential
selectivity for the mosquito enzymes over the human enzyme [35]. Herein, one of the compound classes of
AChE1 inhibitors discovered in the screen, viz. N-aryl-N’-ethyleneaminothioureas, has been explored by design,
synthesis, and biochemical evaluation for inhibition of recombinant AgAChE1, AaAChE1, G122S-AgAChE1 and
hAChE. Furthermore, a selection of thioureas was investigated for their inhibitory effect on mosquito extract in
vitro as well as their insecticidal effect on adult and larvae mosquitoes in vivo.
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Figure 1. The chemical structures of the three hits (1-3) that formed the basis for the design of compounds in three sets of molecules (A-
C). X and Y show variation sites of compounds in each of the three sets. The tertiary amine (NR1R2) in the hits was varied cross the three
sets.
Scheme 1. Synthesis of N-aryl-N’-ethyleneamino substituted thioureas. MWI: microwave irradiation.
2. Results
2.1. Design and synthesis of thioureas
In a previously reported HTS against AChE1, we identified thiourea-based compounds that displayed potential
selectivity towards inhibition of Ag/AaAChE1 over inhibition of hAChE [35] (Figure 1). The compounds
constituted a group of asymmetric thioureas, all carrying both an aryl substituent as well as one tertiary amine
connected via a carbon chain linker. Three of these hits (1-3) constituted the basis for the design, resulting in
three sets of molecules A-C (Figure 1). Set A consists of twelve molecules, and set B and C consist of five and
four molecules, respectively (Tables 1-3).
Re-synthesis of the HTS hits 1-3 and synthesis of the designed N-aryl-N’-ethyleneamino substituted
thioureas 4-21 were accomplished by one of three methods (A-C, Scheme 1) depending on commercial
availability of the starting materials, ease of purification, and safety. Method A (Scheme 1) proved to be the
most straightforward approach in case of commercially available phenylisothiocyanate, for which heating
together with a primary amine for 10 or 15 minutes yielded the desired thioureas in good yields. The other
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synthetic routes involved in situ formation of the isothiocyanate from the corresponding aniline, achieved by
using thiocarbonyldiimidazole (TCDI, method B) or thiophosgene (method C) as the thiocarbonyl source. In a
one-pot two-step reaction, the substituted aniline was first allowed to react with the thiocarbonyl source, after
which a primary amine was added to give the thiourea. TCDI, a solid, is regarded as a safer and easier to handle
alternative to thiophosgene. However, TCDI also has lower reactivity than thiophosgene and required heating to
promote the reaction (Method B, Scheme 1), leading in some cases to the formation of undesired byproducts.
Therefore, if TCDI gave poor results, thiophosgene was used instead (Method C, Scheme 1). Thiophosgene is a
toxic, volatile liquid, but reacted quickly at room temperature, and in our hands produced less byproducts than
TCDI, which facilitated the isolation and purification of the resulting thioureas. In case a synthesized thiourea
proved difficult to isolate by crystallization or chromatography, salt formation was used as a means of
purification (compounds 19 and 20). The 21 synthesized thioureas were subjected to biochemical evaluations
where the half-maximal inhibitory concentrations (IC50) for AgAChE1, AaAChE1, hAChE and G122S-AgAChE1
were determined.
2.2. Structure-activity relationships of AChE1 inhibition
The potencies of the compounds were assessed by determining their IC50 values using the colorimetric Ellman
assay and four different AChEs (i.e AgAChE1, AaAChE1, hAChE and G122S-AgAChE1). The compounds potencies
varied several orders of magnitude (Tables 1-3), with IC50 values ranging from 90 nM to inactive (IC50>1 mM) for
AgAChE1 and/or AaAChE1. Seven compounds displayed promising AChE1 potencies with IC50 values below 20
μM, five had IC50 values of 20-100 μM, while nine compounds were weak inhibitors (IC50 values > 100 μM) or
inactive. The inhibition profiles based on the IC50 values for AgAChE1 and AaAChE1 showed that the two
mosquito enzymes behaved in a very similar manner when being probed with this class of compounds. The
three HTS hits 1-3 all inhibited AgAChE1 and AaAChE1 in a dose-dependent manner (Figure 2a-c, Tables 1-3). Hit
1, which displayed 90% inhibition of AChE1 in the HTS, proved to be the most potent inhibitor of the three with
IC50 values of 0.93 and 0.63 μM for AgAChE1 and AaAChE1, respectively (Figure 2a). Compounds 2 and 3, which
in the HTS showed 70% and 50% inhibition of AChE1, respectively, were also fairly potent inhibitors with a
factor ten higher IC50 values compared to 1 (Figure 2b-c, Table 2 and 3).
The inhibitory effect of 1 in set A was very sensitive to changes in the 3-chloro-4-methoxy substitution
pattern on the phenyl ring of 1; a large drop in potency was seen when exchanging any of the substituents for
hydrogens, independent of the tertiary amines (Table 1). Also, switching the position of the 3-chloro-4-methoxy
substituents on the phenyl ring to 4-chloro-3-methoxy resulted in a substantial reduction in inhibitory effect
(11). Variations of the tertiary amines of the analogues in set A were more tolerated; replacing the 4-
methylpiperidine moiety for a the more polar morpholine resulted in the most potent inhibitor (8) with IC50
values of 120 nM and 90 nM for AgAChE1 and AaAChE1, respectively, while the dimethyl analogue 12 led to
decreased IC50 values. Interestingly, the SAR of 2 in set B suggests a different AChE1 binding mode of the 4-
sulfamoyl phenyl thioureas compared to the analogues of 1 (Table 2), as compound 16 with the morpholine
moiety shows very low inhibitory effects of AChE1. It also seems, within this limited set of substituents on the
phenyl ring, that inhibitory effect of the 4-sulfamoyl substituent could be due to hydrogen bond donor and
acceptor capabilities, and not electronic effects of the aromatic ring, since both electron donating (methoxy)
and electron withdrawing (trifluoromethyl) substituents resulted in reduced inhibition. The SAR of the ortho
substituted phenyls in the limited set C, suggests that small amines are preferred for potent inhibitors, in
contrast to the analogs in sets A-B (Table 3), as the dimethyl amine moiety was more active than the piperidine-
and morpholine moieties. These results suggests a third binding mode of the thiourea inhibitors.
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Table 1. Chemical structures and IC50 values for compounds in set A.
IC50a (µM)
S.R.b
IC50a (µM)
hAChE/
No
Structure
G122S-
AgAChE1
AaAChE1
hAChE
AgAChE1
AChE1
0.96
0.63
12
24
1
4
13
(0.67-1.4)
(0.27-1.5)
(8.5-17)
(9.6-58)
>200
>200
>200
>200
>200
>200
nad
>200
>200
5
6
7
nad
29
28
120
94
4
(19-45)
(18-44)
(82-160)
(58-150)
140
120
190
22
1
(110-180)
(86-170)
(121-302)
(16-30)
0.12
0.090
14
84
8
9
117
>18
>3
(0.11-0.14)
(0.083-0.098)
(13-15)
(33-212)
11
11
>200
>200
>200
>200
(8.7-13)
(9.2-14)
68
53
10
(41-110)
(45-62)
11
>200
>200
>200
nad
>200
Cl
O
18
16
46
17
S
12
13
14
3
nad
1
(17-20)
(14-19)
(39-53)
(6.0-49)
N
N
N
H
H
>200
>200
>200
>200
>200
96
83
140
(85-110)
(70-99)
(120-170)
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a IC50 values were determined using 2-3 replicates, the 95% confidence interval is given in parentheses. b S.R. = selectivity ratio; were
c
d
computed by taking the compound’s hAChE IC50 value divided by the higher IC50 value for Ag or Aa. inactive up to 1 mM. nd = not
determined. e na = not applicable.
Table 2. Chemical structures and IC50 values for compounds in set B.
IC50a (µM)
S.R.b
IC50a (µM)
hAChE/
No
Structure
G122S-
AgAChE1
AaAChE1
hAChE
AgAChE1
AChE1
7.8
5.5
90
33
2
12
nad
nad
nad
>2.7
(6.9-8.7)
(4.8-6.2)
(72-110)
(17-65)
15
16
17
18
>200
>200
>200
>200
>200
>200
>200
Inactivec
>200
Inactivec
Inactivec
Inactivec
Inactivec
Inactivec
74
56
(66-84)
(43-73)
a IC50 values were determined using 2-3 replicates, the 95% confidence interval is given in parentheses. b S.R. = selectivity ratio; were
c
d
computed by taking the compound’s hAChE IC50 value divided by the higher IC50 value for Ag or Aa. inactive up to 1 mM. nd = not
determined. e na = not applicable.
Table 3. Chemical structures and IC50 values for compounds in set C.
IC50a (µM)
S.R.b
IC50a (µM)
hAChE/
No
Structure
G122S-
AgAChE1
AaAChE1
hAChE
AgAChE1
AChE1
7.1
6.0
120
50
3
17
(6.5-7.6)
(5.6-6.5)
(100-130)
(29-86)
17
14
19
>200
>12
>200
(14-22)
(13-16)
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29
22
32
66
20
21
1
(26-33)
(19-24)
(29-35)
(20-220)
>200
>200
>200
nad
Inactivec
a IC50 values were determined using 2-3 replicates, the 95% confidence interval is given in parentheses. b S.R. = selectivity ratio; were computed by taking the compound’s
hAChE IC50 value divided by the higher IC50 value for Ag or Aa. c inactive up to 1 mM. d nd = not determined. e na = not applicable.
2.3. Structure-selectivity relationship for AChE1 versus hAChE
The biochemical evaluation of the compounds’ potency for AgAChE1, AaAChE1 and hAChE showed potent
AChE1 inhibitors that also displayed selectivity for AChE1 over hAChE (Table 1). For example, the most potent
AChE1 inhibitor (8) had a reduced potency of a factor 100 for hAChE (Figure 2d). Overall, the compounds
showed a much lower inhibitory effect on hAChE compared to the mosquito enzymes. Only two compounds
had IC50 values below 20 μM for hAChE (compared to seven for AChE1) and the most potent hAChE inhibitor (1)
had an IC50 of 12 μM. The selectivity ratios based on the IC50 values (S.R.; hAChE/AChE1) ranged from 1 to over
100 (Tables 1-3).
In set A, the largest SSR effect was seen for the variations in the tertiary amine moiety. Three inhibitors all
having the same 3-chloro-4-methoxy phenyl ring but with different tertiary amines (1, 8 and 12) differed
significantly in their S.R. values; the 4-methylpiperidine moiety (1) resulted in S.R. of 13, while the morpholine
(8) and the dimethylamino (12) moieties gave S.R. values of 117 and 3, respectively. The increased S.R. for the
morpholine analogue 8 compared to 1 was due to the increased potency of 8 for AChE1, which was not seen for
hAChE. The same trend was observed for all morpholine analogues (8, 9 and 10). The introduction of the small
dimethylamine moiety (12) instead of 4-methylpiperidine (1) led to a smaller loss of potency for the human
form compared to the mosquito form (1/4 vs. 1/20), resulting in decreased selectivity. In addition, the
substituents on the phenyl ring were of importance for the selectivity as seen in sets B and C. The 4-sulfamoyl
substituent in 2 showed a higher selectivity toward AChE1 over hAChE (S.R. = 12) than its corresponding
methoxy analogue (6; S.R. = 4). In set C, the ortho-methoxy phenyl thiourea with dimethylamine as tertiary
amine had a S.R. of 17, while the dimethylamine analogues of set A showed low or no selectivity towards AChE1
(12, 13, 14). It was also clear that replacing the dimethylamine moiety for a piperidine lead to loss of selectivity
(cf. 3 vs. 20). These results clearly demonstrate that it is possible to design and synthesize selective non-
covalent AChE1 inhibitors.
2.4. Structure-activity and structure-selectivity relationships of the G122S-AgAChE1 mutant
Our data clearly show that the compounds in sets A-C had a reduced potency of G122S-AgAChE1 compared to
wild type; only five compounds had IC50 values equal or below 50 μM (1, 8, 12, 2 and 3), compared to seven
compounds displaying IC50 values below 20 µM on wild type AgAChE1. The overall G122S-AgAChE1 inhibition
profiles of the compounds were more similar to the effect on hAChE than to Ag/AaAChE1. For example, the
morpholine analogues in set A did not lead to an increase in potency compared to the methylpiperidine
analogues (cf. 1 vs. 8). Nevertheless, three of the compounds (12, 2 and 3) showed moderate selectivity
towards G122S-AgAChE1 compared to hAChE (2-3 times difference). Both 12 and 3 have the smaller
dimethylamine moiety, which seems to result in stronger inhibition effect on G122S-AgAChE1 compared to
hAChE. Also, the 4-sulfamoyl substituent of 2 shows promising selectivity towards G122S-AgAChE1 over hAChE.
Interestingly, one compound (7) displayed stronger potency on the G122S mutant than on either of the wild
type AChE1 tested or the hAChE.
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Figure 2. Dose-response curves showing the relative inhibition of recombinant AgAChE1 (dots), AaAChE1 (squares) and hAChE (triangles)
at different concentrations of compounds 1-3 (a-c) and 8 (d), where the highest compound concentration used was 1 mM. IC50 values for
inhibition of AgAChE1, AaAChE1 and hAChE are presented in Tables 1-3.
2.5. Inhibitory effect of thioureas on mosquito extract
The inhibitory effect of three compounds (8, 3 and 13) with different in vitro IC50 values for recombinant
AaAChE1 was subsequently tested ex vivo on pooled extracts from 30 adult female Ae. aegypti mosquitoes
(Figure 3). Homogenized mosquito extracts from either the head or the rest of the body were used as the
enzyme source. The compounds’ inhibitory effect observed in these mosquito extract experiments
corresponded well with the IC50 values determined on recombinant expressed AaAChE1. The IC50 value of 8,
being the most potent and selective compound in all sets, was determined to 0.19 (0.12-0.29) µM and 0.15
(0.076-0.30) µM for head and body extract respectively (cf. IC50 value of 0.090 (0.083-0.098) µM for
recombinant AaAChE1). Compound 3 displayed IC50 values of 7.1 (4.1-12) µM and 7.6 (5.0-11) µM for head and
body extract respectively, in comparison to 6.0 (5.6-6.5) µM for recombinant AaAChE1. The inactive compound
13 was shown to be inactive up to 1 mM also on mosquito extracts. Our results show no significant difference
between enzyme extracted from the mosquito head or from the body.
The good agreement of the IC50 values for recombinant AChE1 and for mosquito extract shows that the
Ellman assay using recombinant AChE is a valid model for acetylcholine hydrolysis in Ae. aegypti. The results
also suggest that other enzymes, on the timescale of the experiment, do not degrade either acetylthiocholine or
the tested compound.
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Figure 3. Dose-response curves showing the inhibition of esterase activity obtained from homogenized adult mosquitoes of the head
(filled symbols) and body (empty symbols) for compound 8 (squares), compound 3 (circles) and compound 13 (triangles).
2.6. Insecticidal effect of thioureas exposed to mosquitoes
The three compounds assayed in the mosquito extract experiment (8, 3 and 13) were also tested for their
insecticidal effect on living mosquitoes. In vivo experiments were performed on five days old adults and 3rd
instar larvae of An. gambiae and Ae. aegypti. The in vivo potency of the compounds was investigated through
topical application on adults and treated water for compound exposure to larvae. All three thioureas killed adult
An. gambiae, although very high concentrations of the compounds were needed (Tables 4 and 5). The most
efficient compound, 8, showed 70% mortality of An. gambiae at a 1 nmol dose, which can be compared to the
commercial insecticide propoxur with a topical LD50 of 0.015 nmol/mosquito [26]. The thioureas 3 and 13 had a
less potent effect on An. gambiae with approximately 55% mortality at a 10 nmol dose. The potency of these
inhibitors on AgAChE1 differs; compound 3 has an IC50 value of 6 µM, while 13 showed only 30% inhibition at 1
mM. No effect was seen on adult Ae. aegypti for any of the tested compounds at the tested concentrations. A
more prominent effect was seen on Ae. aegypti larvae where exposure of compound 8 at 500 µM gave 97%
mortality. The larvicidal effect was even stronger on An. gambiae larvae, where exposure of compound 8 at 300
µM gave 100% mortality. The other tested compounds did not have any effect on the larvae.
Table 4. Mortality of adult mosquitoes 24 hours after topical applications of thioureasa
Compound
An. gambiae (% mortality)
Ae. aegypti (% mortality)
0.01 nmol
0.2 nmol
1 nmol
10 nmol
0.01 nmol
0.2 nmol
1 nmol
10 nmol
8
3
0
-
22
16
0
70
0
n.d.b
57
-
-
-
5
8
1
0
10
0
n.d.b
10
13
-
32
55
12
a 68 and 40 mosquitoes on average for each treated and control experiment, respectively. The mortality was adjusted based on acetone
b
controls using Abbott’s formula[36]. Compounds were administered in an acetone solution (0.1 µl/mosquito). n.d. = not determined
due to solubility issues.
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Table 5. Mortality of larvae mosquitoes 24 hours after treatments of thioureas
Compound
An. gambiae
(% mortality)a, b
Ae. aegypti
(% mortality) a, b
50
300
µM
500
µM
50
300
µM
500
µM
µM
µM
8
3
1
5
4
100
100
3
8
7
4
64
-
97
5
-
-
13
3
-
1
a75 and 50 larvae for each treated and control experiment, respectively, < 5 % mortality in controls; b Compounds dissolved in de-
chlorinated tap water to the given concentration in 100 mL.
3. Discussion
Analogues were made of the hits 1-3 to give sets A-C, with local variations around the aromatic moieties, and a
selection of amines included in all three sets. It was clear from the SAR analysis that the AChE1 inhibitory effect
of thioureas was dependent on specific combinations of the aromatic moiety and the amine. For instance, the
morpholine was preferred in combination with the 3-chloro-4-methoxy phenyl but not with the ortho-methoxy
phenyl (cf. 8 and 3). The best inhibition effect for the ortho-methoxy phenyl was instead seen in combination
with dimethyl amine (cf. 3 and 12). The lack of common SAR suggests that the three most potent inhibitors, one
from each set of compounds, have three different binding modes in the active site gorge of AChE1. Furthermore,
based on the SAR, the aromatic moiety appears to dictate the binding mode of the molecule, and thus the
positioning of the amines is dependent on that binding mode. Comparisons of the thioureas with other known
AChE inhibitors [35, 37, 38], suggests that the aromatic moiety bind in PAS and the amine moiety is directed
towards CAS. One hypothesis would then be that it is aromatic interactions and hydrogen bonding between the
aromatic moieties of 8, 2 and 3 and residues in PAS that position the inhibitors 8, 2 and 3 differently in the
active site gorge.
The thioureas had generally a much lower potency on the human isoform, which opens up for selective
inhibitors towards the mosquito enzyme. Unfortunately, the inhibitors were also less effective on the
resistance-conferring mutant, an important aspect to consider in potentially further chemical optimizations into
insecticides. Still, the SAR and SSR analysis (c.f. 2 and 3) reveal potential to develop divergent SARs for human
AChE and the G122S-AgAChE1 mutant while maintaining efficient inhibition of the wild type, an acknowledged
challenge [29, 30]. We have previously published a potent AChE1 inhibitor from a different compound class
that also efficiently inhibit the resistance-conferring mutant and is selective over the human AChE [35].
The inhibition effects of the thioureas based on IC50 values observed for recombinant AChE1 were confirmed
ex vivo using mosquito extract. Although few compounds were tested, the ex vivo experiments showed that the
thioureas also efficiently inhibited AChE1 from mosquitoes, and thus indicated their potential as insecticides.
Indeed, the most potent thiourea, 8, killed both adults and larvae of the An. gambiae species at high doses.
Similarly, compounds 3 and 13 also had mortal effects on adult An. gambiae mosquitoes, although at even
higher doses. AChE1 enzymes from An. gambiae and Ae. aegypti appear to have very similar functionally and
inhibitory properties in vitro [21, 35]. Nevertheless, in our in vivo studies, we observed differences between the
inhibitors’ effect on the two mosquito species, as none of the thiourea inhibitors tested on Ae. aegypti showed
adult mortality, and only the most potent compound 8 had an insecticidal effect on Ae. aegypti larvae. The
difference in inhibitor mortality of An. gambiae and Ae. aegypti is most likely due to differences in the
pharmacokinetic properties of the tested compounds in the two mosquito species.
The thiourea-based inhibitors are among the first non-covalent selective inhibitors of AChE1 shown to kill
mosquitoes. The discrepancy seen between the in vitro potency and in vivo efficacy may be due to issues
10

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related to penetration through the mosquitoes´ exoskeleton or to metabolism, an issue suggested also for
covalent inhibitors of AChE1 tested on mosquitoes [20, 26, 27, 29] Thus, future development should pay
.
attention not only to in vitro and in vivo potency, but also include pharmacokinetic properties, such as
penetration and metabolism.
4. Conclusions
New tools for vector control of disease-transmitting mosquitoes are needed to successfully combat diseases like
malaria and dengue. The results presented here are encouraging for further development of non-covalent
inhibitors of AChE1 towards the discovery of new insecticides. Highly potent and selective AChE1 inhibitors
were designed that displayed in vivo insecticidal activity in mosquitoes of the An. gambiae and Ae. aegypti
species. The SAR for inhibition of AChE1 was complex and could be explained by multiple binding poses and/or
multiple key interactions formed by the analogues. The complex molecular recognition of AChEs was also
evident by the single point mutation G122S of AgAChE1 that not only led to loss in potency of the thioureas, but
also led to a different SAR. The present work highlight some of the challenges associated with the discovery and
development of insecticide-candidates of AChE1 inhibitors; potency to wild type and mutant enzyme, selectivity
over higher species as well as optimization of the pharmacokinetic properties. In this multi-objective task, we
believe that careful designs of sets of compounds, guided by SARs and SSRs of AChE1 and related targets, have
an important role to play.
5. Experimental section
5.1. General aspects of the synthesis of thioureas
All reactions were carried out under inert atmosphere unless otherwise stated. Dry CH2Cl2 was obtained by
passing through neutral alumina using a solvent drying system, and was freshly collected prior to the reaction,
and MeCN was dried over 4Å molecular sieves for >48 h. All microwave reactions were carried out in a
monomode reactor using Smith process vials sealed with a Teflon septa and an aluminum crimp top. The
temperature was measured with an IR sensor and reaction times refer to the irradiation time at the target
temperature. Reactions were monitored using TLC (silica gel matrix, layer thickness 200 µm, particle size 25 µm)
with UV-detection (254 nm) or developed using KMnO4 solution, or using LC/MS. Flash column chromatography
(eluents given in brackets) was performed on normal phase silica gel (Merck, 60 Å, 40-63 µm). High resolution
mass spectrometry (HRMS) data was obtained on Agilent Technologies 6230 TOF LC/MS in ESI mode. 1H and 13C
NMR spectra were recorded on a Bruker DRX-400 or DRX-600 instrument at 298 K in CDCl3 using residual CHCl3
(δH = 7.26 ppm) or CDCl3 (δC = 77.16 ppm) as an internal standard, (CD3)2SO using residual (CD3)(CD2H)SO (δH =
2.50 ppm) or (CD3)2SO (δC = 39.52 ppm) as an internal standard, or CD3OD using residual CD2HOD (δH = 3.31
ppm) or CD3OD (δC = 49.0 ppm) as an internal standard. 19F NMR spectra were calibrated against an external
standard of CF3COOH in DMSO-d6 (δF = −76.55 ppm).
5.2. Synthesis of building blocks
tert-Butyl (2-(4-methylpiperidin-1-yl)ethyl)carbamate. 2-Bromoethylamine hydrobromide (1.11 g, 5.43 mmol,
1.1 eq.) was added in one portion to di-tert-butyl dicarbonate (1.07 g, 4.88 mmol, 1.0 eq.) in 26 ml of dried
o
CH2Cl2 at 0 C while stirring. Triethylamine (1.02 ml, 7.29 mmol, 1.5 eq.) was added dropwise to the mixture,
and upon complete addition the reaction mixture was stirred at rt for 24 h. The mixture was diluted with CH2Cl2,
washed twice with NH4Cl (aq., sat.), NaHCO3 (aq., sat.) and brine, and the aqueous layers were extracted once
with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced
11

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pressure. The resulting crude product showed remaining di-tert-butyl dicarbonate and was dissolved in CH2Cl2,
washed with water, dried over Na2SO4, filtered and concentrated. The resulting clear oil was dissolved in 10 ml
CH2Cl2 and 4-methylpiperidine (1.12 ml, 9.76 mmol, 2.0 eq.) was added dropwise at rt while stirring. The
reaction mixture was heated at reflux for 20 h. After being allowed to attain rt, the reaction mixture was diluted
with CH2Cl2, washed once with water followed by brine, and the aqueous layers were extracted once with
CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure.
Purification by column chromatography (CH2Cl2:MeOH 90:10) gave the title compound (801 mg, 68% yield over
two steps) as a clear oil, which solidified upon cooling. 1H NMR (400 MHz, CDCl3) δ 5.06 (br s, 1H), 3.22-3.18 (m,
2H), 2.84-2.91 (m, 2H), 2.42-2.39 (m, 2H), 1.93 (dt, J1 = 11.9 Hz, J2 = 1.9 Hz, 2H), 1.60-1.57 (m, 2H), 1.42 (s, 9H),
1.37-1.28 (m, 1H), 1.27-1.15 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 156.1, 79.1, 57.6, 53.9,
37.4, 34.3, 30.9, 28.6, 22.0.
2-(4-Methylpiperidin-1-yl)ethan-1-amine. tert-Butyl (2-(4-methylpiperidin-1-yl)ethyl)carbamate (759 mg, 3.13
mmol) was stirred in 3 ml of HCl (aq., 3 M) at rt for 20 h. The mixture was made basic by addition of NaOH (aq.,
2 M) before being extracted three times with CH2Cl2. The combined organic layers were dried over Na2SO4,
filtered and concentrated under reduced pressure to give the title compound (306 mg, 69% yield) as a light
yellow oil. 1H NMR (400 MHz, CDCl3) δ 2.81-2.79 (m, 2H), 2.74 (t, J = 6.4 Hz, 2H), 2.33 (t, J = 6.4 Hz, 2H), 1.88 (t, J
= 11.5 Hz, 2H), 1.58-1.54 (m, 2H), 1.50 (br s, 2H), 1.34-1.26 (m, 1H), 1.23-1.13 (m, 2H), 0.87 (d, J = 6.4 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 61.8, 54.3, 39.2, 34.5, 30.9, 22.0.
4-Chloro-3-methoxyaniline. Pd(C) (10%, 142 mg, 0.13 mmol, 0.1 eq. Pd) was added to a solution of 1-chloro-2-
methoxy-4-nitrobenzene (250 mg, 1.33 mmol, 1.0 eq.) in 10 ml of EtOAc. The vessel was evacuated, backfilled
with H2, fitted with a H2 balloon, and stirred for 16 h. The suspension was filtered through a pad of Celite and
concentrated. NMR indicated 5-10% of the dehalogenated product; therefore, the crude product mixture was
recrystallized from heptane with a minimal amount of CH2Cl2 to give the title compound as white crystals (111
mg, 0.70 mmol, 53% yield). The 1H spectrum was in accord with literature data [39].
5.3. General procedure for method A for synthesis of thioureas
To the appropriate phenyl isothiocyanate (1 eq) in dry CH2Cl2 (0.1 mmol/ml) the corresponding amine (1-1.2 eq)
was added dropwise at rt while stirring. The reaction mixture was heated at 70 °C for 15 minutes in a sealed vial
purged with N2 using microwave irradiation. After attaining rt, the reaction mixture was diluted with EtOAc and
washed with sat NaHCO3 (aq) followed by brine and the aqueous layers were extracted with EtOAc x3. The
combined organic layers were dried over Na2SO4, filtered and concentrated. The crude product was purified by
recrystallization from heptane:CH2Cl2 (5:1) and the formed crystals were washed with a small amount of
heptane.
5.4. General procedure for method B for synthesis of thioureas
To thiocarbonyldiimidazole (1 eq.) in dried MeCN (0.1-0.2 M), the appropriate aniline (1 eq.) was added. The
reaction vial was purged with N2, sealed, and stirred at 100 °C for 10 minutes in a microwave reactor. The
appropriate alkylamine was added, and the reaction vessel was heated to 100 °C for 10 minutes. After attaining
rt, the reaction mixture was diluted with EtOAc and washed three times with NaHCO3 (aq., sat.), dried over
Na2SO4, filtered and concentrated. Recrystallization from an appropriate solvent or solvent mixture gave the
desired compound.
5.5. General procedure for method C for synthesis of thioureas
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To thiophosgene (1 eq.) in CHCl3 (0.5-1 M), the appropriate aniline (1 eq.) was added. After adding Et3N (2 eq.),
the appropriate alkylamine (1 eq.) was added. After stirring for 2 h, the reaction mixture was concentrated in
vacuo, dissolved in EtOAc, washed three times with NaHCO3 (aq., sat.), dried over Na2SO4, filtered and
concentrated. Recrystallization from an appropriate solvent or solvent mixture gave the desired compound.
5.6. Synthesis of thioureas 1-21
1-(3-Chloro-4-methoxyphenyl)-3-(2-(4-methylpiperidin-1-yl)ethyl)thiourea (1). Method B. 3-Chloro-4-
methoxyaniline (45 mg, 0.29 mmol, 1.0 eq.) was added in one portion to 1,1'-thiocarbonyldiimidazole (51 mg,
0.29 mmol, 1.0 eq.) in 1 ml of dried MeCN at rt while stirring. The reaction mixture was heated to 100 °C for 10
min using microwave irradiation. 2-(4-Methylpiperidin-1-yl)ethan-1-amine (vide supra, 41 mg, 0.29 mmol, 1.0
eq.) in 0.5 ml of dried MeCN was added dropwise and the reaction mixture was heated at 100 °C for an
additional 10 min using microwave irradiation. The resulting brown solution was cooled to rt upon which a
white precipitation formed. The precipitate was collected by filtration, and washed with MeCN and heptane to
1
give thiourea 1 as off-white crystals (41 mg, 42% yield). H NMR (600 MHz, DMSO-d6) δ 9.56 (br s, 1H), 7.50 (s,
1H), 7.45 (br s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 3.83 (s, 3H), 3.54-3.48 (m, 2H), 2.83-2.77 (m,
2H), 2.44-2.42 (m, 2H), 1.91-1.88 (m, 2H), 1.55 (d, J = 12.1 Hz, 2H) 1.33-1.29 (m, 1H), 1.10-1.04 (m, 2H), 0.87 (d, J
= 6.5 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) δ 180.2, 151.7, 132.3, 125.5, 124.9, 120.5, 112.7, 56.2, 56.1 (br),
53.2, 41.2, 40.0, 33.9, 30.3, 21.8. HRMS calcd for [C16H24ClN3OS+H]+ 342.1402, found 342.1402.
1-(2-(Piperidin-1-yl)ethyl)-3-(4-sulfamoylphenyl)thiourea (2). Modified method A. 1-(2-Aminoethyl)piperidine
(105 mg, 0.49 mmol, 1.0 eq.) was added to 4-isothiocyanatobenzenesulfonamide[40] (0.07 ml, 0.49 mmol, 1.0
eq.) in 3 ml of CH2Cl2 in a thick-walled vial. The vial was sealed and heated to 70 °C for 10 min in a microwave
reactor. The white suspension was concentrated in vacuo, and carefully recrystallized from EtOH/water (6:1.5
ml) to give thiourea 2 as white crystals (104 mg, 0.304 mmol, 62% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.00
(br s, 1H), 7.91 (br s, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.27 (s, 2H), 3.69-3.44 (m, 2H), 2.48-
2.43 (m, 2H), 2.43-2.26 (m, 4H), 1.61-1.45 (m, 4H), 1.45-1.31 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 179.9,
142.7, 138.4, 126.3, 121.2, 56.4, 53.9, 41.2, 25.5, 24.0. HRMS calcd for [C14H22N4O2S2+H]+ 343.1257, found
343.1254.
1-(2-(Dimethylamino)ethyl)-3-(2-methoxyphenyl)thiourea (3). Method C. 2-Methoxyaniline (1.13 ml, 10.0
mmol, 1.0 eq.) was added to thiophosgene (0.77 ml, 10.0 mmol, 1.0 eq.) in 20 ml of CHCl3. The formed thick
suspension was stirred for 5 minutes, after which N,N-dimethylethylenediamine (1.09 ml, 10.0 mmol, 1.0 eq.)
was added over 5 minutes. Et3N (2.8 ml, 20 mmol, 2.0 eq.) was added, and the suspension cleared. The reaction
was stirred at r.t. for 2 h, and was then concentrated in vacuo. The crude reaction mixture was partitioned
between EtOAc and NaHCO3 (aq., sat.), the phases were separated, and the organic phase was washed twice
with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered and concentrated to give a brown oil.
The oil was dissolved in 6 ml of toluene/heptane 1:1, and upon slow cooling and swirling, thiourea 3
precipitated as an off-white powder (1.430 g, 5.64 mmol, 56% yield). 1H NMR (400 MHz, CDCl3, 323 K) δ 7.88 (br
s, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.19-7.13 (m, 1H), 6.95 (br s, 1H), 6.97-6.89 (m, 2H), 3.83 (s, 3H), 3.69-3.60 (m,
2H), 2.46 (t, J = 5.8 Hz, 2H), 2.8 (s, 6H); 13C NMR (100 MHz, CDCl3, 323 K) δ 180.7, 152.1, 126.8, 126.5, 124.2,
120.9, 111.9, 57.5, 55.8, 45.0, 43.1. HRMS calcd for [C12H19N3OS+H]+ 254.1322, found 254.1323.
1-(4-Methoxyphenyl)-3-(2-(4-methylpiperidin-1-yl)ethyl)thiourea (4). Following the general method A and
starting from 4-metoxyphenyl isothiocyanate (48 µl, 0.35 mmol) and 2-(4-methyl-1-piperidinyl)ethanamine (50
mg, 0.35 mmol), thiourea 4 was obtained as an off-white solid (86 mg, 80% yield). 1H NMR (400 MHz, DMSO-d6)
δ 9.47 (br s, 1H), 7.27 (br s, 1H), 7.22 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 3.74 (s, 3H), 3.52-3.45 (m, 2H),
2.77 (d, J = 9.8 Hz, 2H), 2.41 (t, J = 6.2 Hz, 2H), 1.89 (t, J = 11.2 Hz, 2H), 1.54 (d, J = 12.5, 2 H), 1.34-1.23 (m, 1H),
13

ACCEPTED MANUSCRIPT
1.01 (dq, J1 = 3.7 Hz, J2 = 12.5 Hz, 2H), 0.86 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 180.2, 156.7,
131.4, 126.0, 114.1, 55.9, 55.3, 53.1, 41.2, 34.0, 30.3, 21.9. HRMS calcd for [C16H25N3OS+H]+ 308.1791, found
308.1782.
1-(3-Chlorophenyl)-3-(2-(4-methylpiperidin-1-yl)ethyl)thiourea (5). Following the general method A and
starting from 3-chlorophenyl isothiocyanate (46 µl, 0.35 mmol) and 2-(4-methyl-1-piperidinyl)ethanamine (50
mg, 0.35 mmol), thiourea 5 was obtained as an off-white solid (81 mg, 74% yield). 1H NMR (400 MHz, DMSO-d6)
δ 9.82 (br s, 1H), 7.74 (br s, 1H), 7.70 (s, 1H), 7.33-7.32 (m, 2H), 7.15-7.13 (m, 2H), 3.55 (m, 2H), 2.84 (d, J = 9.6
Hz, 2H), 2.46 (t, J = 6.2 Hz, 2H), 1.91 (t, J = 10.9 Hz, 2H), 1.58 (d, J = 11.3, 2 H), 1.37-1.27 (m, 1H), 1.17-1.09 (m,
2H), 0.88 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 179.8, 140.0, 132.8, 130.3, 123.5, 121.8, 120.8,
56.1, 53.3, 41.3, 33.9, 30.3, 22.9. HRMS calcd for [C15H22ClN3S+H]+ 312.1296, found 312.1288.
1-(4-Methoxyphenyl)-3-(2-(piperidin-1-yl)ethyl)thiourea (6). Following the general method A and starting from
4-metoxyphenyl isothiocyanate (69 µl, 0.5 mmol) and 1-(2-aminoethyl)piperidine (71 µl, 0.5 mmol), thiourea 6
was obtained as a light brown solid (103 mg, 70% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.46 (br s, 1H), 7.29 (br
s, 1H), 7.22 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 3.73 (s, 3H), 3.55-3.45 (m, 2H), 2.40 (t, J = 6.2 Hz, 2H),
2.36-2.29 (m, 4H), 1.47-1.42 (m, 4H), 1.38-1.34 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 180.3, 156.6, 131.5,
125.8, 114.1, 56.5, 55.3, 53.8, 41.2, 25.6, 24.0. HRMS calcd for [C15H23N3OS+H]+ 294.1635, found 294.1628.
1-(3-Chlorophenyl)-3-(2-(piperidin-1-yl)ethyl)thiourea (7). Following the general method A and starting from 3-
chlorophenyl isothiocyanate (77 µl, 0.5 mmol) and 1-(2-aminoethyl)piperidine (80 µl, 0.5 mmol), thiourea 7 was
1
obtained as an off-white solid (191 mg, 96% yield). H NMR (400 MHz, DMSO-d6) δ 9.82 (br s, 1H) , 7.75 (br s,
1H), 7.71 (s, 1H), 7.33-7.30 (m, 2H), 7.15-7.12 (m, 1H), 3.59-3.51 (m, 2H), 2.45 (t, J = 6.2 Hz, 2H), 2.40-2.33 (m,
4H), 1.53-1.48 (m, 4H), 1.41-1.36 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 179.8, 141.0, 132.7, 130.2, 123.4,
121.7, 120.7, 56.4, 53.8, 41.1, 25.5, 24.0. HRMS calcd for [C14H20ClN3S+H]+ 298.1139, found 298.1136.
1-(3-Chloro-4-methoxyphenyl)-3-(2-morpholinoethyl)thiourea (8). Method C. 3-Chloro-4-methoxyaniline (514
mg, 3.26 mmol, 1.0 eq.) was added to a solution of thiophosgene (0.25 ml, 3.3 mmol, 1.0 eq.) in 3 ml of CHCl3 at
0 °C. Et3N (0.91 ml, 6.5 mmol, 2.0 eq.) was added, and the reaction was stirred for 5 min. 2-(4-
Morpholino)ethylamine (0.43 ml, 3.3 mmol, 1.0 eq.) was added, and the reaction was stirred at 0 °C for 2 h. The
reaction mixture was diluted with EtOAc and extracted four times with HCl (aq., 1 M). The combined aqueous
phases were made basic by the addition of NaOH (aq., 2 M), extracted three times with CH2Cl2, dried over
Na2SO4, filtered, and concentrated to give a brown oil. The oil was dissolved in 12 ml of hot toluene/heptane 1:1
to give thiourea 8 as off-white microcrystals after cooling (672 mg, 2.04 mmol, 63% yield). 1H NMR (600 MHz,
DMSO-d6) δ 9.57 (br s, 1H), 7.54 (br s, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.23 (dd, J = 8.8, 2.4 Hz, 1H), 7.11 (d, J = 8.8
Hz, 1H), 3.84 (s, 3H), 3.59-3.53 (m, 6H), 2.47 (t, J = 6.2 Hz, 2H), 2.43-2.34 (m, 4H); 13C NMR (150 MHz, DMSO-d6)
δ 180.3, 151.6, 132.4, 125.4, 123.7, 120.5, 112.7, 66.2, 56.4 (br), 56.2, 53.1, 40.7. HRMS calcd for
[C14H20ClN3O2S+H]+ 330.1038, found 330.1033.
1-(4-Methoxyphenyl)-3-(2-morpholinoethyl)thiourea (9). Modified method A. To 4-methoxyphenyl
isothiocyanate (70 µl, 0.51 mmol, 1.0 eq.) in 5 ml of dried CH2Cl2, 1-(2-aminoethyl)morpholine (67 µl, 0.51 mmol,
1.0 eq.) was added dropwise at rt while stirring. The reaction vial was purged with N2, sealed, and stirred at
70 °C for 15 minutes in a microwave reactor. After attaining rt, the reaction mixture was washed with sat
NaHCO3 (aq.) followed by brine, and the aqueous layers were extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4, filtered and concentrated. The crude product was dissolved in CH2Cl2 and
precipitated by the addition of heptane, and the formed crystals were washed with a small amount of heptane
to give thiourea 9 (131 mg, 88% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.47 (br s, 1H), 7.33 (br s, 1H), 7.23 (d, J =
8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.74 (s, 3H), 3.58-3.50 (m, 6H), 2.45 (t, J = 6.3 Hz, 2H), 2.42-2.33 (m, 4H); 13
C
14

ACCEPTED MANUSCRIPT
NMR (150 MHz, DMSO-d6) δ 180.3, 156.6, 131.5, 125.7, 114.1, 66.2, 56.4, 55.3, 53.0, 40.7. HRMS calcd for
[C14H21N3O2S+H]+ 296.1427, found 296.1419.
1-(3-Chlorophenyl)-3-(2-morpholinoethyl)thiourea (10). Following the general method A and starting from 3-
chlorophenyl isothiocyanate (77 µl, 0.6 mmol) and 1-(2-aminoethyl)morpholine (77 µl, 0.6 mmol), thiourea 10
was obtained as an off-white solid (173 mg, 96% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.82 (br s, 1H), 7.81 (br s,
1H), 7.71 (s, 1H), 7.36-7.13 (m, 2H), 7.15-7.12 (m, 1H), 3.61-3.58 (m, 6H), 2.50 (m, 2H), 2.45-2.38 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) δ 178.9, 140.9, 132.8, 130.2, 123.5, 121.7, 120.7, 66.2, 56.3, 53.1, 40.7. HRMS calcd
for [C13H18ClN3OS+H]+ 300.0932, found 300.0926.
1-(4-Chloro-3-methoxyphenyl)-3-(2-morpholinoethyl)thiourea (11). Method B. Thiocarbonyldiimidazole (81 mg,
0.46 mmol, 1.0 eq.) and 4-chloro-3-methoxyaniline (72 mg, 0.46 mmol, 1.0 eq.) were stirred in 2 ml of dried
MeCN in a thick-walled vial. The vial was sealed and heated to 100 °C for 10 min in a microwave reactor. 2-(4-
Morpholino)ethylamine (0.06 ml, 0.5 mmol, 1 eq.) was added, and the vessel was heated to 100 °C for 10 min.
The solution was concentrated in vacuo, and the resulting solid was taken up in EtOAc and washed three times
with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude product
was recrystallized from toluene, but a remaining contaminant required further purification by dissolution in
CH2Cl2 (3 ml) and precipitation of the product by addition of heptane (10 ml). After washing with heptane,
thiourea 11 was obtained as white microcrystals (74 mg, 0.22 mmol, 49% yield). 1H NMR (600 MHz, DMSO-d6) δ
9.77 (br s, 1H), 7.70 (br s, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 6.99 (dd, J = 8.5, 2.0 Hz, 1H), 3.82
(s, 3H), 3.62-3.54 (m, 6H), 2.49 (t, J = 6.3 Hz, 2H), 2.44-2.35 (m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 179.9,
154.3, 139.3, 129.5, 115.8, 115.3, 107.5, 66.2, 56.4, 56.0, 53.2, 40.7. HRMS calcd for [C14H20ClN3O2S+H]+
330.1038, found 330.1033.
1-(3-Chloro-4-methoxyphenyl)-3-(2-(dimethylamino)ethyl)thiourea (12). Method B. Thiocarbonyldiimidazole
(113 mg, 0.63 mmol, 1.0 eq.) and 3-chloro-4-methoxyaniline (100 mg, 0.63 mmol, 1.0 eq.) were stirred in 2 ml
of dried MeCN in a thick-walled vial. The vial was sealed and heated to 100 °C for 10 min in a microwave reactor.
N,N-Dimethylethylenediamine (0.07 ml, 0.6 mmol, 1 eq.) was added, and the vessel was heated to 100 °C for 10
min. The solution was concentrated in vacuo, and the resulting solid was taken up in EtOAc and washed three
times with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude
product was recrystallized from a mixture of 1.5 ml of CH2Cl2 and 12 ml of heptane to give thiourea 12 as off-
white flakes (134 mg, 0.47 mmol, 73% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.57 (br s, 1H), 7.59 (d, J = 2.6 Hz,
1H), 7.57 (br s, 1H), 7.23 (dd, J = 8.8, 2.6 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 3.83 (s, 3H), 3.59-3.45 (m, 2H), 2.41 (t, J
= 6.2 Hz, 2H), 2.18 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 180.5, 151.4, 132.8, 125.2, 123.4, 120.3, 112.6, 57.3,
56.2, 45.0, 41.7. HRMS calcd for [C12H18ClN3OS+H]+ 288.0932, found 288.0929.
1-(2-(Dimethylamino)ethyl)-3-(4-methoxyphenyl)thiourea (13). Following the general method A and starting
from 4-metoxyphenyl isothiocyanate (69 µl, 0.5 mmol) and N,N-dimethylethylenediamine (55 µl, 0.5 mmol),
thiourea 13 was obtained as an off-white solid (110 mg, 87% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.46 (br s,
1H), 7.37 (br s, 1H), 7.25 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.73 (s, 3H), 3.55-3.48 (m, 2H), 2.39 (t, J = 6.2
Hz, 2H), 2.16, (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 180.6, 156.4, 131.9, 125.5, 113.9, 57.5, 55.3, 45.1, 41.8.
HRMS calcd for [C12H19N3OS+H]+ 254.1322, found 254.1315.
1-(3-Chlorophenyl)-3-(2-(dimethylamino)ethyl)thiourea (14). Following the general method A and starting
from 3-chlorophenyl isothiocyanate (77 µl, 0.6 mmol) and N,N-dimethylethylenediamine (64 µl, 0.6 mmol),
1
thiourea 14 was obtained as an off-white solid (130 mg, 86% yield). H NMR (400 MHz, DMSO-d6) δ 9.84 (br s,
1H), 7.81 (s, 1H), 7.80 (br s, 1H), 7.33-7.31 (m, 2H), 7.12-7.10 (m, 1H), 3.59-3.51 (m, 2H), 2.44 (t, J = 6.1 Hz, 2H),
15

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2.19 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 180.0, 141.2, 132.6, 130.1, 123.2, 121.5, 120.5, 57.0, 44.9, 41.6.
HRMS calcd for [C11H16ClN3S+H]+ 258.0826, found 258.0822.
1-(2-(Piperidin-1-yl)ethyl)-3-(4-(trifluoromethyl)phenyl)thiourea (15). Method B. Thiocarbonyldiimidazole (100
mg, 0.56 mmol, 1.0 eq.) and 4-(trifluoromethyl)aniline (0.07 ml, 0.6 mmol, 1 eq.) were stirred in 2 ml of dried
MeCN in a thick-walled vial. The vial was sealed and heated to 100 °C for 10 min in a microwave reactor. 1-(2-
Aminoethyl)piperidine (0.08 ml, 0.6 mmol, 1 eq.) was added, and the vessel was heated to 100 °C for 10 min.
The solution was concentrated in vacuo, and the resulting solid was taken up in EtOAc and washed three times
with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered, and concentrated to give a dark solid.
Careful recrystallization from heptane gave thiourea 15 as a pale yellow powder (61 mg, 0.18 mmol, 33% yield).
1H NMR (400 MHz, DMSO-d6) δ 10.02 (br s, 1H), 7.88 (br s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.5, 2H),
3.64-3.50 (m, 2H), 2.46 (t, J = 6.2 Hz, 2H), 2.43-2.28 (m, 4H), 1.57-1.45 (m, 4H), 1.44-1.35 (m, 2H); 13C NMR (150
MHz, DMSO-d6) δ 179.9, 143.4, 125.7, 124.4 (q, J = 271 Hz), 123.2 (q, J = 32 Hz), 121.6, 56.4, 53.9, 41.2, 25.5,
24.0. 19F NMR (376 MHz, DMSO-d6) δ -60.81. HRMS calcd for [C15H20F3N3S+H]+ 332.1403, found 332.1400.
1-(2-(Morpholin-4-yl)ethyl)-3-(4-sulfamoylphenyl)thiourea
(16).
Modified
method
A.
4-(2-
Aminoethyl)morpholine (0.05 ml, 0.4 mmol, 1.0 eq.) was added to 4-isothiocyanatobenzenesulfonamide[40] (86
mg, 0.40 mmol, 1.0 eq.) in 3 ml of CH2Cl2 in a thick-walled vial. The vial was sealed and heated to 70 °C for 10
min in a microwave reactor. The white suspension was concentrated and recrystallized from 7 ml of EtOH to
1
give thiourea 16 as white crystals (112 mg, 0.33 mmol, 81% yield). H NMR (400 MHz, DMSO-d6, 333 K) δ 9.86
(br s, 1H), 7.82 (br s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.14 (br s, 2H), 3.66-3.56 (m, 6H), 2.53
(t, J = 6.3 Hz, 2H), 2.46-2.40 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 333 K) δ 180.1, 142.5, 138.4, 126.0, 121.3,
66.0, 56.1, 52.9, 40.6. HRMS calcd for [C13H20N4O3S2+H]+ 345.1050, found 345.1046.
1-(4-Chlorophenyl)-3-(2-morpholinoethyl)thiourea (17). Method B. Thiocarbonyldiimidazole (100 mg, 0.56
mmol, 1.0 eq.) and 4-chloroaniline (72 mg, 0.56 mmol, 1.0 eq.) were stirred in 2 ml of dried MeCN in a thick-
walled vial. The vial was sealed and heated to 100 °C for 10 min in a microwave reactor. 2-(4-
Morpholino)ethylamine (0.07 ml, 0.6 mmol, 1 eq.) was added, and the vessel was heated to 100 °C for 10 min.
The solution was concentrated in vacuo, and the resulting solid was taken up in EtOAc and washed three times
with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered, and concentrated. The obtained
crude product was recrystallized from toluene to give thiourea 17 as off-white small flakes (130 mg, 0.43 mmol,
77% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.75 (br s, 1H), 7.69 (br s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.7
Hz, 2H), 3.64-3.51 (m, 6H), 2.49-2.44 (m, 2H), 2.44-2.35 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 180.1, 138.3,
128.5, 127.8, 124.3, 66.2, 56.4, 53.1, 40.7. HRMS calcd for [C13H18ClN3OS+H]+ 300.0932, found 300.0924.
1-(2-(Dimethylamino)ethyl)-3-(4-sulfamoylphenyl)thiourea
(18).
Modified
method
A.
N,N-
dimethylethylenediamine (0.06 ml, 0.55 mmol, 1.1 eq.) was added to 4-isothiocyanatobenzenesulfonamide[40]
(106 mg, 0.49 mmol, 1.0 eq.) in 3 ml of CH2Cl2 in a thick-walled vial. The vial was sealed and heated to 70 °C for
10 min in a microwave reactor. The mixture was allowed to attain rt and concentrated. The resulting crude
mixture was dissolved in dioxane (1 ml) and the product was precipitated by addition of toluene (15ml). The
solvent was decanted and the resulting solid was twice treated with toluene and sonicated to give thiourea 18
1
as white solid (148 mg, 0.49 mmol, 99% yield). H NMR (400 MHz, DMSO-d6) δ 10.05 (br s, 1H), 7.92 (br s, 1H),
7.76-7.67 (m, 4H), 7.25 (br s, 2H), 3.65-3.47 (m, 2H), 2.47 (t, J = 5.9 Hz, 2H), 2.22 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) δ 180.1, 142.8, 138.3, 126.2, 121.2, 57.0, 44.9, 41.5. HRMS calcd for [C11H18N4O2S2+H]+ 303.0944,
found 303.0942.
1-(2-Chlorophenyl)-3-(2-(dimethylamino)ethyl)thiourea sulfate (19). Method C. 2-Chloroaniline (0.14 ml, 1.3
mmol, 1.0 eq.) was added dropwise to a solution of thiophosgene (0.10 ml, 1.3 mmol, 1.0 eq.) in 1.5 ml of CHCl3
16

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at −10 °C. To the formed yellow-white precipitate was added Et3N (0.36 ml, 2.6 mmol, 2.0 eq.), and the vessel
contents cleared. N,N-Dimethylethylenediamine (0.14 ml, 1.3 mmol, 1.0 eq.) was added dropwise, and the
reaction was allowed to reach r.t. over 2 h. The dark red reaction mixture was diluted with EtOAc and extracted
three times with HCl (aq., 1 M). The obtained aqueous phase was made basic by the addition of NaOH (aq., 2 M)
and extracted three times with CH2Cl2. The organic phase was dried over Na2SO4, filtered and concentrated to
give a brown oil. The oil was dissolved in 10 ml of EtOH, and treated with 48 mg of H2SO4 (ca. 0.5 eq.) dissolved
in 1.2 ml of EtOH. The product precipitated slowly as transparent needles, which were triturated with cold EtOH
to give thiourea 19 after removing residual solvent in vacuo (209 mg, 0.68 mmol, 52% yield). 1H NMR (600 MHz,
DMSO-d6, 323 K) δ 9.40 (br s, 1H), 7.97-7.90 (m, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.49 (dd, J = 8.0, 1.3 Hz, 1H), 7.32
(dt, J = 1.3, 7.6 Hz, 1H), 7.24 (dt, J = 1.5, 7.7 Hz, 1H), 3.76-3.67 (m, 2H), 3.26 (v br s, 1H), 2.92-2.86 (m, 2H), 2.54
(s, 6H); 13C NMR (150 MHz, DMSO-d6, 323 K) δ 181.7, 135.9, 129.3, 129.2, 127.1, 127.0, 56.3, 43.7, 40.4. We
were unable to resolve all 6 aromatic carbon peaks in either DMSO-d6 (sulfate) or CDCl3 (freebase). HRMS calcd
for [C11H16ClN3S+H]+ 258.0826, found 258.0825.
1-(2-Methoxyphenyl)-3-(2-(piperidin-1-yl)ethyl)thiourea
hydrochloride
(20).
Method
B.
Thiocarbonyldiimidazole (103 mg, 0.58 mmol, 1.0 eq.) and 2-methoxyaniline (0.07 ml, 0.6 mmol, 1 eq.) were
stirred in 2 ml of dried MeCN in a thick-walled vial. The vial was sealed and heated to 100 °C for 10 min in a
microwave reactor. 1-(2-Aminoethyl)piperidine (0.08 ml, 0.6 mmol, 1 eq.) was added, and the vessel was
heated to 100 °C for 10 min. The solution was concentrated in vacuo, and the resulting solid was taken up in
EtOAc and washed three times with NaHCO3 (aq., sat.). The organic phase was dried over Na2SO4, filtered, and
concentrated to give the crude product as an oil. The oil was dissolved in EtOAc, and the corresponding
hydrochloride salt was precipitated by the addition of HCl in Et2O (1.0 M, 1.5 ml). Concentration in vacuo and
1
recrystallization from 10 ml of EtOH gave thiourea 20 as red crystals (111 mg, 0.34 mmol, 58% yield). H NMR
(600 MHz, DMSO-d6) δ 10.35 (br s, 1H), 9.22 (s, 1H), 8.17 (br s, 1H), 7.63 (br d, J = 7.0 Hz, 1H), 7.18 (t, J = 7.6 Hz,
1H), 7.05 (d, J = 8.2 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 3.92-3.85 (m, 2H), 3.79 (s, 3H), 3.46-3.39 (m, 2H), 3.23-3.16
(m, 2H), 2.99-2.88 (m, 2H), 1.83-1.73 (m, 4H), 1.72-1.65 (m, 1H), 1.44-1.33 (m, 1H); 13C NMR (150 MHz, DMSO-
d6) δ 181.0, 152.5, 126.9, 126.4, 126.3, 120.0, 111.8, 55.6, 54.7, 52.3, 38.4, 22.4, 21.2. HRMS calcd for
[C15H23N3OS+H]+ 294.1635, found 294.1636.
1-(2-Methoxyphenyl)-3-(2-morpholinoethyl)thiourea (21). Method B. Thiocarbonyldiimidazole (100 mg, 0.56
mmol, 1.0 eq.) and 2-methoxyaniline (0.06 ml, 0.6 mmol, 1 eq.) were stirred in 2 ml of dried MeCN in a thick-
walled vial. The vial was sealed and heated to 100 °C for 10 min in a microwave reactor. 2-(4-
Morpholino)ethylamine (0.07 ml, 0.6 mmol, 1 eq.) was added, and the vessel was heated to 100 °C for 10 min.
The vessel contents were taken up in EtOAc and washed three times with NaHCO3 (aq., sat.). The organic phase
was extracted three times with HCl (aq., 1 M); the obtained aqueous phase were made basic by the addition of
NaOH (aq., 2 M) and extracted three times with EtOAc. The organic phase was dried over Na2SO4, filtered and
concentrated to give a clear oil, which was flash chromatographed in heptane/EtOAc 2:8 to 1:9 to give thiourea
1
21 as a transparent, thick oil (123 mg, 0.42 mmol, 74% yield). H NMR (400 MHz, CDCl3) δ 7.79 (br s, 1H), 7.34-
7.21 (m, 1H), 7.15 (dt, J = 7.8, 1.4 Hz, 1H), 7.12-7.05 (m, 1H), 6.94-6.88 (m, 2H), 3.77 (s, 3H), 3.67-3.56 (m, 2H),
3.52-3.41 (m, 4H), 2.47 (t, J = 5.9 Hz, 2H), 2.40-2.27 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 179.8, 152.2, 127.2,
125.6, 124.6, 120.7, 111.8, 66.7, 55.8, 55.6, 52.8, 41.2. HRMS calcd for [C14H21N3O2S+H]+ 296.1427, found
296.1441.
5.7. Protein expression
The expression of recombinant AChE1 enzymes from the mosquitoes An. gambiae [21], the G122S mutant of
AgAChE1 [21], and Ae. aegypti [21], as well as recombinant hAChE [41] was performed as described previously.
17

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5.8. IC50 determinations
In order to study SAR and SSR on thiourea scaffold-based compounds, dose-response experiments were
performed with a small library of analogues of this compound class on the four target proteins (Ag/AaAChE1,
hAChE and G122S-AgAChE1). The recombinant enzymes used in this study have previously been probed with
commercial reference compounds, both covalent and non-covalent inhibitors [21]. Stock solutions of the
synthesized compounds were prepared in DMSO at 100 mM, and working dilutions were prepared in either
sodium phosphate buffer (0.1 M, pH 7.4) or MilliQ water, depending on solubility preferences. Compound
solutions of eight different concentrations up to a maximum of 1 mM were used. The activity measurements
were performed using secreted non-purified proteins in growth medium, and enzymatic activity was measured
using the Ellman assay [42] adapted to a 96-well format. Automatic handling of liquids for assay plate setup was
performed using a QIAgility robotic benchtop instrument (Qiagen). Assays were performed at 30 °C in a final
volume of 200 µL of 0.1 M phosphate buffer (pH 7.4) containing 0.2 mM 5,5′-dithiobis(2-nitrobenzoic acid) and
1 mM acetylthiocholine iodide. The enzymatic reaction was measured by monitoring changes in the absorbance
of individual wells at 412 nm over 60 s in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices).
The average slope determined for eight positive (uninhibited) controls on each plate was taken to represent
100% activity, and the activity observed in the sample wells were quantified in relation to this value. IC50 values
were calculated using non-linear regression (curve fitting) in GraphPad Prism [43], and the log [inhibitor] vs.
response variable slope equation was fitted using three parameters. IC50 values were determined using 2-3
independent replicates. The lower catalytic activity in the dose-response experiments of the G122S mutant of
the AgAChE1 enzyme led to a reduced signal to noise ratio compared to wild type AgAChE1 (cf. CI in Tables 1-3).
The intrinsic reactivities between 5,5’-dithiobis(2-nitrobenzoic acid) and compounds 3 and 13 were tested in
phosphate buffer (pH 7.4), and was found to be negligible on the timescale of the assay.
5.9. Inhibitory effect of thioureas on mosquito extract
The inhibitory effect of thiourea-based compounds on mosquito extract was investigated using a method
modified from [44]. 30 adult Ae. aegypti female mosquitoes stored at -20°C were sorted two per tube, the
heads separate from the bodies. The samples were homogenized by manual grinding in 100 µL phosphate
buffer pH 7.4 containing 1% Triton X-100. The grinder was rinsed with 300 µL phosphate buffer and the samples
were subsequently vortexed and centrifuged at 10 000 rpm for 3 min at 4°C. The supernatant from all samples
were pooled together, still heads separate from bodies. 100 µL of pooled sample were used per measurement
and the Ellman assay described before was used for IC50 determinations of head- and body-samples.
5.10.
In vivo experiments with An. gambiae and Ae. aegypti
An. gambiae s.s. Kisumu strain and Ae. aegypti Mombasa strain, both from Kenya, were used to test the
insecticial activities of the compounds. Mosquito rearing was carried out in an insectary maintained at 27-28 °C
at ca. 80% humidity, on a 12/12 h light/darkness cycle, and maintained at optimal larval concentrations to avoid
possible effects of competition. Mosquito larvae were reared in de-chlorinated tap water, and were fed on
finely ground Sera Vipan staple dietTM (Sera, Germany), while adults were offered a fresh 10% (w/v) glucose
solution meal daily, and were fed on hamster (Mesocricetus auratus) as a source of blood meals when egg
production was desired. Insecticidal activity tests of the compounds were carried out following World Health
Organization guidelines for testing adulticides and larvicides [45, 46].
For the adult mosquito tests, non-blood fed, five day old female mosquitoes were used, and testing was
performed in batches of five mosquitoes each. Each batch of mosquitoes was placed in a 500 ml paper cup and
anesthetized by placing the cup in a -20 °C freezer for 3 minutes. Thereafter, the mosquitoes were gently
18

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poured onto a plate refrigerated at -20 °C overlaid with a paper towel, and the compound solution (acetone, 0.1
µl) was deposited on the upper part of the pronotum using a micropipetter. As a negative control, 0.1 µl of
acetone was used, while for the positive control, 6.25 ng of propoxur (2-isopropoxyphenyl-N-methylcarbamate)
in 0.1 µl of acetone was used [47]. After the topical application, the mosquitoes were returned to the insectary,
where they were supplied with a glucose meal and maintained under standard conditions. The mortality rates
were recorded after 24 h. The mortality rates were adjusted using Abbott’s formula[36] in cases where
mortality in the control was over 5%. No experiment had a >20% mortality in controls.
Larval tests were conducted in the following manner. For each tested compound and concentration, 25 larvae
at third instar were introduced into a cup containing compound dissolved in 100 ml of de-chlorinated water,
and maintained in the insectary under standard conditions. Negative control experiments comprised of larvae
maintained in plain de-chlorinated water while the positive controls were conducted with propoxur at 25 µM.
Mortality rates were recorded after 24 h.
Acknowledgements
This work was funded by grants from the Swedish Research Council (Dnrs 2014-4218 and 2014-2636) and
Centrum för Miljövetenskaplig Forskning (CMF). We thank Stina Bäckman, Swedish Defence Research Agency,
for kindly donating female Ae. aegypti mosquitoes.
Keywords: acetylcholinesterase 1 • vector control • Anopheles gambiae • Aedes aegypti • insecticides •
thiourea
Additional information
1
Supporting information includes H and 13C NMR spectra of synthesized compounds, dose-response curves for
IC50 determination of recombinant AgAChE1, AaAChE1, hAChE and G122S-AgAChE1 and details about the
mosquito experiments.
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Highlights
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21 aryl-ethyleneamino substituted thioureas were synthesized and evaluated
Compounds inhibit mosquito AChE1 with IC50 values from 0.1 µM
Several compounds show selectivity for mosquito AChE1 over human AChE
Insecticidal activity was demonstrated on Aedes aegypti and Anopheles gambiae