Synthesis and insecticidal activity of novel carbamate derivatives as potential dual-binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1021/jf1032284

In biological systems, bivalent ligands often possess increased functional affinity for their receptors compared with monovalent ligands. On the basis of the structure of acetylcholinesterase (AChE), a series of novel carbamate heterodimetic derivatives w

Full text of DOI:10.1021/jf1032284

J. Agric. Food Chem. 2010, 58, 12817–12821 12817
DOI:10.1021/jf1032284
Synthesis and Insecticidal Activity of Novel Carbamate
Derivatives as Potential Dual-Binding Site
Acetylcholinesterase Inhibitors
†
H
ONG-J
U
MA
,
†,‡ R
U
-LIANG
X
IE,† QIAN-FEI
,†
Z
HAO,† XIANG-DONG
MEI
,
AND
J
UN
NING
*
†Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China, and ‡College of Plant
Science and Technology of Huazhong Agricultural University, Wuhan 430070, China
In biological systems, bivalent ligands often possess increased functional affinity for their receptors
compared with monovalent ligands. On the basis of the structure of acetylcholinesterase (AChE), a
series of novel carbamate heterodimetic derivatives were designed and synthesized with the aim of
increasing the potency toward AChE inhibition. The AChE inhibitory ability of all the novel
compounds was tested using AChE obtained from the brain of the housefly. The bioassay results
showed that compounds 6j, 6k, 6m, 6n, 6p, and 6q had increased inhibitory activities in comparison
with parent phenyl N-methylcarbamate (MH) at the concentration of 100 mg/L. Among them, the
most potent AChE inhibitor of these compounds was 6q (IC50 = 12 μM), which showed 62-fold
greater AChE inhibitory activity than that of MH and 12-fold greater activity than metolcarb (MT),
which suggested that the 3-nitrophenoxy moiety of compound 6q was able to interact with the
aromatic amino acid residues lining the gorge and the phenyl N-methylcarbamate moiety was able
to interact with the catalytic sites of AChE, simultaneously. The insecticidal activities of compounds
6j, 6k, 6m, 6n, 6p, and 6q were further evaluated. Consistent with the result in vitro bioassay, those
compounds demonstrated better activities against Lipaphis erysimi than parent compound MH at the
concentration of 300 mg/L, and compound 6q showed the best insecticidal activity, causing 98%
mortality after 24 h of treatment.
KEYWORDS: Acetylcholinesterase; carbamate heterodimetic derivatives; inhibitory activity; phenyl
N-methylcarbamate
INTRODUCTION
linesterase (AChE: EC 3.1.1.7), a key enzyme in the nervous
system of insects, by covalently carbamylating the serine residue
within the active site gorge (15). AChE terminates nerve impulses
by catalyzing the hydrolysis of the neurotransmitter acetylcho-
line. The remarkable features of AChE are its deep and narrow
gorge, referred to as the “active site gorge”, and a peripheral site
existing at the gorge mouth. Fourteen aromatic residues lined a
substantial portion of the surface of the gorge (∼40%). Those
residues and their flanking sequences are highly conserved in
AChEs among different species (16), providing multiple hydro-
phobic binding sites (17-19). On the basis of the unique structure
of AChE, many potential dual-site binding inhibitors of AChE
have been synthesized and tested as drugs to treat or alleviate
Alzheimer’s disease (AD) (20-24). For example, Donepezil,
approved in 1996 by the U.S. FDA and marketed as Aricept,
features a unique orientation along the active site gorge, extend-
ing from the anionic subsite at the bottom of the gorge to the
peripheral anionic site at the top of the gorge, via aromatic π-π
stacking interactions with conserved aromatic acid residues (25).
Recently, several AChE-serotonin transporter (SERT) dual
inhibitors were reported; the increased AChE inhibitory potency
was attributed to the simultaneous binding of the units to the
Multivalent ligand-receptor interactions, known as the cluster
effect, are defined as specific simultaneous associations of multi-
ple ligands presented on a molecule that bind to multiple recep-
tors presented on a biological entity (1-3). It is well-known that
multivalent binding interactions could enhance functional
affinity (4-7) and receptor selectivity (8-10) compared to the
monomeric ones, thus providing a broad range of benefits and
unique roles that are not easily achievable with monovalent
interactions.
Conventional carbamate insecticides, such as metolcarb, meth-
omyl, thiodicarb, carbofuran, carbosulfan, and aldicarb have
been used to control insect pests throughout the world during the
past several decades. However, the intensive use of carbamate
insecticides has resulted in the development of resistance to these
insecticides (11-14). As the discovery of new modes of action
insecticides has become ever-increasingly challenged, develop-
ment of new insecticides with known modes of action remains an
important strategy. The carbamate insecticides inhibit acetylcho-
*Corresponding author (phone þ86-10-62899142; e-mail jning502@
yahoo.com.cn).
© 2010 American Chemical Society
Published on Web 11/29/2010
pubs.acs.org/JAFC

12818 J. Agric. Food Chem., Vol. 58, No. 24, 2010
Ma et al.
active and the hydrophobic siteofthe AChE (19). It was proposed
that the phenoxy moiety interacted efficiently with the conserved
aromatic amino acid residues lining the gorge and resulted in
the increasing AChE inhibitory ability.
Continuing with our research on dual-site inhibitors of AChE
in the pesticide field (26, 27) and hypothesizing that the phenoxy
moiety would be able to interact with aromatic amino acid
residues through π-π interactions, we have designed and synthe-
sized a series of novel carbamate derivatives incorporated with a
phenoxy moiety as potential AChE inhibitors to enhance the
insecticidal activity and to overcome the resistance via additional
interaction in the gorge of the AChE.
comparison with phenyl N-methylcarbamate (MH) and metolcarb
(MT). AChE was prepared from heads of adult houseflies (female/ male =
1:1). Inhibitory activities of these compounds were measured against
AChE according to a previously reported method (26, 27).
In Vivo Insecticidal Activities Assay. To evaluate the insecticidal
activity of synthesized compounds 6j, 6k, 6m, 6n, 6p, and 6q, insecti-
cidal activities were carried out with Lipaphis erysimi. These insects
were reared in a room maintained at 25 ((1) °C, 60 ((5)% relative
humidity, and 14 h light photoperiod. Stock solutions of test com-
pound were prepared in acetone at a concentration of 10 g/L and then
diluted to the required test concentrations with water (containing 0.1%
Triton X-100).
Disks of Chinese cabbage leaves with 35 apterous adult L. erysimi were
prepared. Leaf disks with insects were separately dipped in a test solution
for 10 s. After drying, the leaves were placed in a plastic dish (6 cm
diameter). The dishes were placed into an incubator at 25 °C, and
mortality was determined after 24 h by the number in the treated dishes
relative to that in the untreated controls. The untreated control experi-
ments were carried out under the same conditions without test com-
pounds. This insecticidal activity of MT was also evaluated against
L. erysimi and utilized as positive control. All experiments and the
respective controls were carried out in three replicates, and the corrected
mortality was calculated (Table 2).
MATERIALS AND METHODS
Synthesis. 1H NMR spectra were recorded in CDCl3 with a Bruker
DPX300 spectrometer, using tetramethylsilane as internal standard
(performed at China Agricultural University). Elemental analyses (C, H, N)
were performed at the Institute of Chemistry, Chinese Academy of Sciences.
Melting points were measured on a WRS-2A melting point apparatus and are
uncorrected. The solvents and reagents were used as received or were dried
prior to use as needed.
1-(3-Bromopropoxy)-4-(trifluoromethyl)benzene (2b). To a solu-
tion of 1,3-dibromopropane (46.5 g, 0.23 mol) in acetone (200 mL) were
added 4-trifluoromethylphenol (15 g, 0.092 mol) and potassium carbonate
(19 g, 0.138 mol). After 16 h of refluxing, the reaction mixture was cooled
to room temperature and filtered. The filtrate was concentrated, and the
resulting mixture was purified by column chromatography on silica gel
(ethyl acetate/hexane = 1:70) to give 1-(3-bromopropoxy)-4-(trifluoro-
methyl)benzene (22.88 g, 88% yield) as a yellow liquid. Intermediates
2a-2k were synthesized via the same procedures accordingly.
4-(3-(4-(Trifluoromethyl)phenoxy)propoxy)phenol (3b). A mix-
ture of p-hydroquinone (4.8 g, 0.044 mol), compound 2b (5 g, 0.018 mol),
and potassium carbonate (7.3 g, 0.053 mol) in acetone (100 mL) was
refluxed for 24 h. The reaction mixture was cooled to room temperature
and filtered. The filtrate was concentrated, and the resulting mixture was
purified by column chromatography on silica gel (ethyl acetate/hexane=
1:10) to give 4-(3-(4-(trifluoromethyl)phenoxy)propoxy)phenol (3.9 g,
71% yield) as a yellow liquid. Intermediates 3a-3d, 4a-4d, and 5a-5k
were synthesized via the same procedures accordingly.
4-(3-(4-(Trifluoromethyl)phenoxy)propoxy)phenyl Methylcar-
bamate (6b). Compound 3b (1.05 g, 3.36 mmol) and methylcarbamic
chloride (0.63 g, 6.72 mmol, 90%) were dissolved in dichloromethane
(50 mL) in an ice bath, and then 20% sodium hydroxide aqueous solution
(2 mL) was added to the solution for 5 min at 0-5 °C. After the mixture
had been stirred for 20 min in an ice bath, 5% brine (50 mL) was added,
and the mixture was stirred for another 20 min at room temperature. The
organic layer was isolated and washed with water twice and dried over
anhydrous sodium sulfate. The solvent was removed in vacuo, and the
residue was purified by column chromatography on silica gel (ethyl
acetate/hexane=1:3) to give 4-(3-(4-(trifluoromethyl)phenoxy)propoxy)-
phenyl methylcarbamate (0.8 g, 65% yield) as a white solid. Target
compounds 6a-6s were synthesized via the same procedures accordingly.
4-(3-(4-(Trifluoromethyl)phenoxy)propoxy)phenyl Dimethylcar-
bamate (7b). Compound 3b (1.25 g, 4 mmol) and N,N-dimethylcarbamic
chloride (0.64 g, 6 mmol) were dissolved in N,N-dimethylformamide
(10 mL), and then potassium carbonate (0.83 g, 6 mmol) was added.
After the mixture had been stirred at room temperature for 8 h, the
reaction mixture was poured into water (10 mL) and then extracted with
ethyl acetate (2 ꢀ 20 mL). The combined organic extracts were washed
with brine and dried over anhydrous sodium sulfate. The solvent was
removed in vacuo, and the residue was purified by column chromatogra-
phy on silica gel (ethyl acetate/hexane=1:3) to give 4-(3-(4-(trifluoro-
methyl)phenoxy)propoxy)phenyl dimethylcarbamate (7b) (1.13 g, 73.8%
yield). Target compounds 7a-7l were synthesized via the same procedures
accordingly.
RESULTS AND DISCUSSION
On the basis of the dual-site binding strategy in rational design
of AChE inhibitors, phenyl carbamate was selected as a parent
compound to bind to the active site gorge, and the substituted
phenoxy moiety was chosen as a pharmacophoric element corre-
sponding to the hydrophobic sites. Therefore, we designed a series
of compounds containing phenyl carbamate and substituted
phenoxy linked by different carbon numbers of alkylene to
simultaneously bind to the catalytic site and the hydrophobic
site as potential dual-site binding inhibitors of AChE. The
synthesis, AChE inhibitory assays, and insecticidal activities
against L. erysimi are reported here.
Synthesis. During the preparation of the intermediate (3-
bromoalkoxy)benzene (Scheme 1), both bromine atoms of the
R,ω-dibromoalkanes could possibly be replaced by the substituted
phenoxy to generate the bis-substituted byproduct. However, the
reactions provided the monosubstituted products (2) as the major
products in excellent isolated yields by using an excess of R,ω-
dibromoalkanes. The reaction of intermediates 2 with different
dihydroxybenzenes (Scheme 2) also gave the desired monosubstituted
products by using an excess of dihydroxybenzene. Target compounds
6 and 7 were synthesized by reacting N-methylcarbamoyl chloride
and N,N-dimethylcarbamoyl chloride with intermediates 3, 4, and 5,
respectively (Scheme 3).
In Vitro Inhibition of AChE Activities. The AChE inhibitory
ability of all the new compounds was tested using AChE obtained
from the brain of the housefly. As shown in Table 1, compounds
6j, 6k, 6m, 6n, 6p, and 6q exhibited increased inhibitory activities
in comparison with parent phenyl N-methylcarbamate (MH) at
the concentration of 100 mg/L. These six compounds had three
common structural features. First, these compounds possessed
the structure of N-methylcarbamate; second, the N-methylcarba-
mate group was in the meta-position of the aromatic ring; third,
the linker between phenyl carbamate and the substituted phenoxy
moiety was a chain ofthree or four carbon atom alkoxy units. The
IC50 values of compounds 6j, 6k, 6m, 6n, 6p, and 6q (Table 2)
demonstrated higher inhibitory activity for AChE than MH. The
most potent AChE inhibitor of these compounds was 6q (IC50=
12 μM), which showed 62-fold greater AChE inhibitory activity
than MH and 12-fold greater activity than MT.
The structures, appearance, melting points, and yields of target
compounds 6a-6s and 7a-7l are listed in Table 1, and their 1H NMR
and elemental analysis are given in the Supporting Information.
In Vitro AChE Inhibitory Bioassay. To evaluate the biological
profiles of these novel compounds, AChE inhibiton was assayed in
The data in Table 1 show that all N,N-dimethylcarbamate
heterodimetic derivatives exhibited the same low inhibition of

Article
J. Agric. Food Chem., Vol. 58, No. 24, 2010 12819
Table 1. Melting Point, Yield, and in Vitro Inhibition of AChE for Target Compounds at 100 mg/L
compd
R
Y
-OOCNR1CH3
R1
appearance
mp (°C)
yield (%)
inhibition rate of AChE (%)
MH
6a
6b
6c
6d
6e
6f
46
2
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-NO2
4-NO2
4-NO2
2-NO2
3-NO2
2-OCH3
4-Cl
CH2CH2
CH2CH2CH2
p-
H
white solid
white solid
163-164
152-154
99-101
151-152
142-143
91-93
94-95
103-106
86-88
94-96
92-95
105-107
103-105
123-126
153-154
66-67
66-67
70-74
78-81
109-112
119-121
113-114
99-101
64-67
_
55.5
40.6
52.5
48.4
49.3
48.7
46.6
48.6
52.5
52.0
45.5
46.5
50.2
52.5
47.8
49.2
49.2
45.2
49.5
52.4
46.1
47.5
45.8
50.4
46.5
50.8
54.1
47.4
48.8
48.5
49.8
p-
H
2
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2
p-
H
white solid
white solid
4
p-
H
3
o-
H
white solid
white solid
13
25
19
17
20
61
62
12
69
80
45
80
92
46
30
6
CH2CH2CH2
o-
H
6g
6h
6i
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2
o-
H
white solid
white solid
o-
H
m-
m-
m-
m-
m-
m-
m-
m-
m-
m-
m-
p-
H
white solid
white solid
6j
CH2CH2CH2
H
6k
6l
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2CH2
H
white solid
white solid
H
6m
6n
6o
6p
6q
6r
H
white solid
white solid
white solid
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2CH2CH2
CH2CH2CH2CH2
CH2CH2CH2CH2
CH2CH2CH2CH2
CH2CH2
H
H
H
pale yellow solid
pale yellow solid
pale yellow solid
pale yellow solid
white solid
H
H
6s
7a
7b
7c
7d
7e
7f
H
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH2CH2
p-
pale yellow solid
white solid
white solid
1
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2
p-
6
p-
0
o-
pale yellow solid
pale yellow viscous oil
yellow viscous oil
white solid
15
25
7
CH2CH2CH2
o-
7g
7h
7i
CH2CH2CH2CH2
CH2CHCHCH2
CH2CH2
o-
_
o-
_
7
m-
m-
m-
m-
white solid
_
8
7j
CH2CH2CH2
CH2CH2CH2CH2
CH2CHCHCH2
pale yellow viscous oil
pale yellow viscous oil
white solid
_
27
20
13
7k
7l
_
69-72
Table 2. In Vitro Inhibition of AChE and in Vivo Insecticidal Activity of Some Compounds
in vitro inhibition of AChE activities
in vivo insecticidal activity (mortality, %)
Lipaphis erysimi (300 mg/L)
compd
IC50 (95% confidence intervals) (μM)
slope ((SE)
ratio to MH
1
ratio to MT
MH
MT
6j
766 (711-826)
150 (68-330)
105 (73-149)
107 (99-115)
70 (40-118)
46 (25-81)
1.3 ((0.2)
0.8 ((0.3)
1.1 ((0.2)
1.2 ((0.2)
0.9 ((0.2)
0.6 ((0.2)
1.5 ((0.3)
1.3 ((0.2)
76 ea
97 ab
85 d
7
7
1
1
6k
92 c
6m
6n
6p
6q
11
17
13
62
2
89 cd
92 c
3
61 (50-72)
12 (11-14)
2
93 bc
98 a
12
a Treatments were grouped into statistically distinct classes by applying analysis of variance (ANOVA) at P = 0.05 based on Fisher’s LSD.
AChE as parent phenyl N,N-dimethylcarbamate (IC50 = 2961 μg
mL-1) (26), whereas N-methylcarbamate heterodimeric mole-
cules showed higher inhibition against AChE than N,N-dimethyl-
carbamate heterodimetic molecules. The AChE inhibition
potency of the carbamate heterodimetic derivatives varied evi-
dently with the length of the tether chain. Heterodimetic deriva-
tives with a two-carbon-atom chain showed the lowest inhibition
of AChE, which may result from too short of a tether length to
bind well the dual site of the anzyme. The introduction of
butylene with a less flexible linker led to reduced activity,
indicating that a flexible chain was favorable for AChE inhibitory
activities. Consequently, the variation of the tether chain among
the heterodimetic derivatives affected the interaction on binding
the dual site in AChE. The optimal tether length among our
heterodimetic derivatives was a chain of four-carbon-atom
alkoxy moiety. We reasoned that a four-carbon-atom linking
chain bearing a phenyl ring might favorably interact with the
aromatic residues lining the wall of the AChE gorge. As shown in
Table 1, the effect of electron-donating (;OCH3) and electron-
withdrawing (;CF3, ;NO2, ;Cl) substituents R at the phenyl
ether did not show good regularity to the inhibition of AChE, and
nitro-substituted derivatives demonstrated high potency against
AChE, which is consistent with the previous study (19). This
might be due to hydrogen-bond formation between the NO2

12820 J. Agric. Food Chem., Vol. 58, No. 24, 2010
Ma et al.
group and hydrogen-bond donor from residues lining the gorge
of the AChE.
Compounds 6k, 6n, 6p, and 6q with four-carbon-atom chains
demonstrated increased inhibitory activities, with >90% mor-
tality in comparison with compounds 6j and 6m containing three-
carbon-atom chains. This suggested the variation of the length
of the tether chain had the same effect on insecticidal activities
as shown in the in vitro inhibition. Consistent with the in vitro
bioassay, compound 6q showed the best insecticidal activity
against L. erysimi, exhibiting an activity similar to that of the
commercial insecticide MT. This result indicated that those
carbamate heterodimetic derivatives not only showed excellent
activities in vitro AChE inhibition but also had whole insect
activity for L. erysimi. Further structural optimization and detailed
structure-insecticidal activity relationships will be reported in
the future.
In summary, we describe the design, synthesis, and structure-
activity relationships (SAR) of a series of carbamate heterodi-
metic derivatives prepared with the aim of increasing the potency
toward AChE inhibition. Heterodimetic derivative 6q showed
62-fold higher inhibitory activity than that of parental compound
and 12-fold higher activity than MT. In addition, compound 6q
exhibited the best insecticidal activity against L. erysimi. The
results obtained from these studies were further confirmed on the
dual-site binding design strategy in the pesticide field.
The mode of action of conventional phenyl carbamate insecti-
cides is that the carbamate part of the molecule is attached to the
esteratic site and the substituted aromatic part to the anionic sites
of the AChE during the inhibitory process. As the distance
between the esteratic and anionic sites was 0.5 nm in the AChE
molecule, carbamate insecticides would be the most efficient if the
distance between the two groups to be bound to the two sites of
the anzyme was also 0.5 nm (28-30). Compound 6q showed
higher activity than MT, in which the substituent on the phenyl
carbamate was longer than 0.5 nm to extend halfway into the
gorge. It suggested that the 3-nitrobenzene group of this com-
pound interacted well with the hydrophobic site lining the gorge
to promote the affinity of the molecules.
Insecticidal Activities. The insecticidal activities of compounds
6j, 6k, 6m, 6n, 6p, and 6q against L. erysimi were investigated,
and the results are shown in Table 2. As shown in Table 2,
all compounds showed better activities against L. erysimi
than parent compound MH at the concentration of 300 mg/L.
Scheme 1. Synthesis of Intermediates 2
ABBREVIATIONS USED
AChE, acetylcholinesterase; AD, Alzheimer’s disease; SERT,
serotonin transporter; MH, N-methylcarbamate; MT, metolcarb.
Supporting Information Available: Experiment data are
provided forcompounds 6a-s and 7a-l. Thismaterial is available
free of charge via the Internet at http://pubs.acs.org.
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Received for review August 22, 2010. Revised manuscript received
November 2, 2010. Accepted November 8, 2010. This work was
financially supported by the National Basic Research Program of
China (No. 2010CB126106 and No. 2006CB101907).
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