Design, synthesis and biological evaluation of organophosphorous-homodimers as dual binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.10.030

The cluster effect is an effective strategy to explore new lead compounds, and has been successfully applied in rational drug design and screening. A series of novel organophosphorous-homodimers were designed and synthesized based on the dual-site structure characteristics of acetylcholinesterase (AChE). The compounds were evaluated in vitro for their inhibitory activity to AChE extracted from Drosophila melanogaster and Musca domestic. Compound 4H showed an excellent inhibitor activity to both Drosophila melanogaster and Musca domestic with the corresponding IC₅₀ values of 23 and 168 nM, respectively. Meanwhile, its activities against Drosophila melanogaster and Musca domestic AChE were more than 10,00,000 and 100,000-fold higher compared with the parent compound (MH), and was up to 245 and 107-fold higher than those of the positive control omethoate. The molecular docking study revealed that 4H possessed an optimal spacer length and can perfectly fit into the central pocket, active gorge, and peripheral site of DmAChE, and consequently exhibited highly improved inhibitor potency to DmAChE. The bioassay tests showed that 4 series compounds showed prominent insecticidal activities against both Lipaphser erysimi and Tetranychus cinnbarinus at a concentration of 200 mg/L. The insecticide activity of compound 4H was particularly significant that can cause 96% mortality to Tetranychus cinnbarinus after 24 h of treatment.

Full text of DOI:10.1016/j.bmc.2012.10.030

Bioorganic & Medicinal Chemistry 21 (2013) 278–282
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of organophosphorous-homodimers
as dual binding site acetylcholinesterase inhibitors
Ruliang Xie a, , Qianfei Zhao a, , Tao Zhang a, Jing Fang b, Xiangdong Mei a, Jun Ning a, , Yun Tang b,
⇑
⇑
a State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
b School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The cluster effect is an effective strategy to explore new lead compounds, and has been successfully
applied in rational drug design and screening. A series of novel organophosphorous-homodimers were
designed and synthesized based on the dual-site structure characteristics of acetylcholinesterase (AChE).
The compounds were evaluated in vitro for their inhibitory activity to AChE extracted from Drosophila
melanogaster and Musca domestic. Compound 4H showed an excellent inhibitor activity to both Drosophila
melanogaster and Musca domestic with the corresponding IC50 values of 23 and 168 nM, respectively.
Meanwhile, its activities against Drosophila melanogaster and Musca domestic AChE were more than
10,00,000 and 100,000-fold higher compared with the parent compound (MH), and was up to 245 and
107-fold higher than those of the positive control omethoate. The molecular docking study revealed that
4H possessed an optimal spacer length and can perfectly fit into the central pocket, active gorge, and
peripheral site of DmAChE, and consequently exhibited highly improved inhibitor potency to DmAChE.
The bioassay tests showed that 4 series compounds showed prominent insecticidal activities against both
Lipaphser erysimi and Tetranychus cinnbarinus at a concentration of 200 mg/L. The insecticide activity of
compound 4H was particularly significant that can cause 96% mortality to Tetranychus cinnbarinus after
24 h of treatment.
Received 12 September 2012
Accepted 16 October 2012
Available online 30 October 2012
Keywords:
Acetylcholinesterase
Dual binding site
Organophosphorous-homodimers
Molecular docking
Insecticidal activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
AChE through nucleophilic attack. The enzymes are phosphory-
lated and deactivated due to the attack of these agents, and conse-
The cluster effect, the multivalent ligands simultaneously bind-
ing to the multiple receptors of a biological system,1–3 has been
shown being an effective strategy for improving drug potency
and selectivity. Particularly, when both the multiple ligands and
the multiple receptors are on the same molecular entities,
respectively, the homo-dimers can exhibit the excellent affinity
binding4–8 as well as the excellent selectivity9–11 to the corre-
sponding monomers. The cluster effect has been successfully ap-
plied in rational drug design, it is therefore reasonable to expect
that the theoretical hypothesis underlying the cluster effect can sim-
ilarly serve as the guidance for the discovery of new agrochemicals.
Many organophosphorus compounds that serve as nerve agents
and insecticides (e.g., parathion and malathion) are acetylcholines-
terase (AChE: E. C. 3.1.1.7) inhibitors. The electrophilic phospho-
rous atom can react with the hydroxyl group of the serine in the
quently, these organophosphorus agents are pretty toxic. The
inhibition of AChE usually leads to an increase in the amount of
neurotransmitter acetylcholine at the central and peripheral sites
of the nervous system, and induces the excessive stimulation to
muscarinic and nicotinic receptors.12,13 Organophosphorus insecti-
cides represent a class of classical agrochemicals that have been
used over the past several decades around the world. They are
one of the major pesticides that target insects. However, organo-
phosphorus insecticides have been excessively used over the past
decade, Consequently, insects have developed strong resistances
to them. Therefore, it is urgent to develop a new action mechanism
by which organophosphorus insecticides can defeat the resistance
that insects have been developed over the time.
The molecular configuration of AChE has three key characteris-
tics. First, it has a deep and narrow gorge, referred to as the ‘active
site gorge’, where fourteen aromatic residues occupied a substan-
tial portion of the surface of the gorge (ꢀ40%). These residues as
well as their flanking sequences are highly conserved in AChEs
among different species.14 Second, at the bottom of the active site
gorge located a catalytic anionic site. Finally, there is a second
binding site that known as peripheral anion site (PAS) in the entry
⇑
Corresponding authors. Tel./fax: +86 10 62899142 (J.N.); tel./fax: +86 21
64251052 (Y.T.).
E-mail addresses: jning@ippcaas.cn (J. Ning), ytang234@ecust.edu.cn (Y. Tang).
These authors equally contributed to this work and should be considered co-first
authors.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.030

R. Xie et al. / Bioorg. Med. Chem. 21 (2013) 278–282
279
of the active site gorge.15 It is located around 18 Å away from the
active site.6 Because AChE has two binding sites (so called the biva-
lent fashion), many compounds with dual binding site have been
selected and tested for AChE as drug candidates to treat
AD.7,8,12,16 They showed the interesting activities toward the corre-
sponding monomers tacrine, huperzine A, huperzine B and nor-
meptazinol, etc., and have been considered as drug candidates
for AD due to their high AChE inhibition activities. The high AChE
inhibition activities of these compounds are very probably due to
their bi-function structure.
In this paper, we report the design and synthesis of a new series
of dual binding site AChEIs 4A–I. They all possessed an important
pharmacophore, that is, the organophosphorous moiety linked by
linear alkanoic acid amides of different lengths, and proved to be
endowed with a strong inhibitory activity due to the increased
capability to interact with the active sites of the target AChE.
One AChEI binding unit binds to the catalytic site via the classical
mode of action,12 while the other unit binds to the active site gorge
or the PAS due to the presence of a spacer of appropriate length
and flexibility. The synthesized AChEIs were tested in vitro against
AChE from the head of Drosophila melanogaster AChE (DmAChE)
and Musca domestic AChE (MdAChE).
of the salts of potassium. The optimal ratio for the intermediate 2
and 3 was 1.05:2, by which the title compound 4 can be conve-
niently produced. The purity of the synthesized compounds was
less than 45%. The structures of the target compounds were con-
firmed by element analysis and 1H NMR.
2.2. AChE inhibitory activity
Newly synthesized compounds were tested in vitro for their
AChE inhibitory activity, in which the AChE extracts from Drosoph-
ila melanogaster and Musca domestic brain were adopted. The re-
sults are shown in Table 1, Those results indicated that all the
compounds except 4A and 4B were the potent inhibitors to both
Drosophila melanogaster and Musca domestic AChE. Compound 4H
exhibited the best inhibitor activity to both Drosophila melanogas-
ter and Musca domestic AChE with the corresponding IC50 value of
23 and 168 nM, respectively, which were 1000,000 and 100,000
times larger than their corresponding IC50 values to the parent
compound (MH) and 245 and 107 time larger than those to the po-
sitive control, respectively. The inhibitory activities of other com-
pounds, however, were significantly lower, which is very
probably due to the difference in the length of the alkyl linker be-
tween these compounds and 4H. Particularly, the inhibitory activ-
ities of compound 4H to both AChEs were 79- and 56-fold higher
than those of the commercial insecticide chlorpyrifos. These obser-
vations suggested that both the linker type and linker length can
impose significant influences on compound potency. Given the un-
ique structure of AChE, it is reasonable to expect that the linker
length is probably the most important factor affecting the inhibi-
tory activity, for the compounds can reach both the catalytic and
the peripheral binding sites of AChE only when the linker length
is right. Surprisingly, our results revealed that the linker type also
imposed significant influences on the compound potency. It can be
ascribed to the specific interaction between the linkers and the res-
idues consisting the gorge of the AChE. In addition, it was found
that the activities of compound 4I to DmAChE and MdAChE were
62 and 75 times higher than those of omethoate. Despite the only
structural difference between compound 4H and 4I is that com-
pound 4I has one more methylene group than 4H does, the activity
of compound 4I to AChE was remarkably lower compared to com-
pound 4H. Even though, both 4H and 4I exhibited the ideal inhib-
itory activities with the IC50 values of 23 and 91 nM to DmAChE
and 168 and 238 nM to MdAChE, respectively. In addition, the data
in Table 1 revealed a general tendency. The inhibitory activities of
the synthesized compounds towards both DmAChE and MdAChE
increased as the linker length increased, in spite the increase in
the activity toward MdAChE being more gentle than that toward
DmAChE. This is reasonable, for the DmAChE was obviously more
susceptible to the synthesized compounds than MdAChE did. Nev-
ertheless, other factors such as the temperature, the humidity and
other environmental factors during the evolution may also get in-
volved. The activities of compound 4J were also lower than those of
2. Results and discussion
2.1. Synthesis
The derivatives were prepared following the synthetic routines
outlined in Schemes 1 and 2. As depicted in Scheme 1, the interme-
diates 2A–I were synthesized by using linear chain di-amines of
various lengths and methyl chloroacetate as the starting materials,
all can be purchased from most chemical companies. It was easy to
obtain the 2 series compounds via a continuing violent stir for 6 h
with the temperature below ten centigrade, and it was better to
use cold methanol for filtering the 2 series to reduce the product
loss by dissolving in the methanol. Moreover, the good yield was
achieved by slowly adding the di-amines into the cold methyl chlo-
roformate methanol solution. In order to get the title compounds,
it was crucial to choose the optimal ratio, that is, the 2.4 equiv of
methyl clhlorofonmate to the diamines. In short, the yield up to
90% was achieved by setting the suitable temperature, selecting
the correct chemical addition order, taking the optimal reactant ra-
tio, and reacting over an appropriate time of period. The desired
compounds were got through the reaction between the intermedi-
ate 2 and the 3 in the presence of potassium iodide in methanol for
24–48 h under refluxing. The yield of 20–40% was achieved after
purification by chromatography. The yields were below 20% when
the di-amine spacer length was 2–4 carbon atoms. The low yield is
very probably due to the steric hindrance between the intermedi-
ate 2 and 3. Besides, 5% of the potassium iodide or potassium bro-
mine was necessary for obtaining the title compounds, indicating
that the reaction may be facilitated by the possible catalytic action
O
O
O
n
H2N
NH2
methanol
+
Cl
Cl
n
Cl
n=2-10
O
HN
NH
1
2
n=2-10
Scheme 1. Synthesis of intermediate 2A–I n = 2–10 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I.

280
R. Xie et al. / Bioorg. Med. Chem. 21 (2013) 278–282
O
P
O
O
C2H5O
C2H5O
Cl
SK
Cl
n
+
HN
NH
2
3
n=2-10
O
P
O
P
O
O
C2H5O
C2H5O
OC2H5
methanol
KI
S
S
n
OC2H5
HN
NH
4
n=2-10
Scheme 2. Synthesis of target compounds 4A–I n = 2–10 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I.
both the PAS and the CAS of 1QO9 which was the entry code in pro-
Table 1
In vitro inhibition of AChE activities
tein database of X-ray crystal structure of native DmAChE. Mean-
while, 4H took an extended molecular conformation within the
active-site gorge, as shown in Figure 1. The primary interactions
between 4H and 1QO9 were electrostatic interactions, hydrogen
bonding interaction and hydrophobic interaction. At the CAS, there
was a hydrogen bond between 4H and the substrate involving the
ethoxy oxygen atom of crucial functional phosphate group of 4H as
well as the side chain imidazole ring nitrogen atom of His 480
(O. . .N distance: 3.11 Å). Meanwhile, the nitrogen atom of the low-
er amide group approached to the PAS and formed a hydrogen
bond with hydroxyl oxygen of Tyr71 (N. . .O distance: 3.04 Å).
Additionally, due to its long alky spacer (having nine methylene
groups), part of 4H structure extended to the outside of the active
site gorge and formed two hydrogen bonds with 1QO9 in the
peripheral region of the active site. The first was formed between
Entry
Chain length n
DmAChE
IC50 (nM)
MdAChE
IC50 (nM)
MH
>400,00,000
68,791
4575
1063
427
1138
109
1862
23
>250,00,000
6083
10,70,000
3681
572
722
564
7252
168
238
4A
2
4B
3
4C
4
4D
5
4E
6
4F
7
4G
8
4H
9
4I
10
91
5633
1825
Omethoate
Chlorpyrifos
18,028
9514
Treatments were grouped into statistically distinct classes by applying analysis of
variance (ANOVA) at P = 0.05 based on Fisher’ s LSD.
4H. Ikuya et al. reported that the optimal configuration of alkyl
spacers, although relatively nonspecific, was determined by the re-
gional binding domain involving various stabilization interactions.
Amongst all the compounds studied here, compound 4B–C exhib-
ited the lowest potency, implying that either their linkers may be
too short to reach both the catalytic and peripheral anion sites or
the fitted residues simultaneously occupy the gorge of the AChE.
In contrast, compounds 8D–J demonstrated moderate potencies,
that is, their activities to DmAChE and MdAChE were 5 and 240
times larger than those of omethoate to the same AChEs, indicating
that their conformation may be more better fit to the structure of
DmAChE.
2.3. Molecular docking studies
Molecular docking study has been conducted and the results
were reported elsewhere.5 The purpose was to ascertain at the
molecular level that compounds 4H can bind to both the catalytic
and peripheral sites of DmAChE, and meanwhile to provide a
molecular explanation for the surprisingly good activity of com-
pound 4H.
The AChE active sites including the central catalytic pocket, the
binding gorge and peripheral pocket were taken as the target in the
docking simulations. The docking results showed that compound
4H demonstrated the highest inhibitory activity (IC50: 23 nM)
among all the 4-series compounds. It can simultaneously bind at
Figure 1. Two representations of 4H (C atoms colored magentas and green
respectively, shown as stick models) docked into the binding sites of DmAChE
1QO9. Interaction details between 4H and 1QO9, the hot pink dash lines represent
H-bonds formed between the two linking atoms. The side chains of active-site
residues involved in interactions are all displayed (C atoms colored gray), of which
the critical residues shown in stick and others shown in line.

R. Xie et al. / Bioorg. Med. Chem. 21 (2013) 278–282
281
Table 2
In vivo insecticide activities
Entry
Chain length n
Lipaphis erysimi
Tetranychus cinnbarinus
(Mortality %, 200 mg/L)
(Mortality %, 200 mg/L)
MH
12 i
10 g
4A
4B
4C
4D
4E
4F
4G
4H
2
3
4
5
6
7
8
9
44 h
83 cd
85 c
80 de
92 b
84 cd
90 b
77 ef
60 g
75 f
80 e
95 ab
85 d
99 a
69 f
92 bc
93 b
96 ab
83 de
94 ab
89 c
4I
10
Omethoate
Chlorpyrifos
99 a
Treatments were grouped into statistically distinct classes by applying analysis of variance.
the ethoxy oxygen of 4H phosphate group and the side chain ami-
no nitrogen atom of Asn322 (O. . .N distance: 2.87 Å), while the sec-
ond involved the amide nitrogen at the upper part of 4H and the
side chain carbonyl oxygen of Asn322 (N. . .O distance: 3.01 Å). Be-
sides, the binding free energy of 4H to 1QO9 was found to be
ꢁ57.04 kcal/mol.
excellent insecticidal activities against L. erysimi and tetranychus
cinnbarinus. The results present in this work demonstrate the
application potential of the homo-dimer strategy in the field of
agrochemical.
It has been expected that the homo-dimers that linked by dis-
tinct types of spacers of different lengths may show potential
inhibitory activity to AChE by acting on its CAS and PAS.4,7 Our
docking study have revealed that, compound 4H, which consists
of monomer 3 and the linker ononanediamine, possessed the opti-
mal spacer length that allows it strongly interacting with 1QO9. As
a result, compound 4H is expected to be able to better bind to the
central pocket, the active gorge, and peripheral site of DmAChE,
and therefore show the improved inhibitory potency to AChE.
The homo-dimer 4H could take an optimal conformation to best
fit into the binding sites of AChE.
4. Experimental section
4.1. Chemicals and characterizations
All chemicals were purchased from commercial suppliers. 1H
NMR spectra were recorded on a Bruker DPX300 spectrometer in
CDCl3 with TMS as an internal standard. Elemental analysis (C, H,
N, S, P) was conducted at the Institute of Chemistry, Chinese Acad-
emy of Sciences. Analytical thin-layer chromatography (TLC) was
carried out on precoated plates (silica gel 60 F 254), and spots were
visualized under UV light.
2.4. Insecticidal activities
4.2. General synthetic procedure for 2A–I
The insecticide activities of 4 series compounds against L. ery-
simi and Tetranychus cinnbarinus were investigated. The results
are shown in Table 2. All the homo-dimers, except 4B and 4I,
exhibited the excellent insecticide activity against L. erysimi at a
concentration of 200 mg/L. The compounds 4C–H showed a higher
insecticide activity than the positive control omethoate, while 4E–
G were at least as active as the commercial insecticide chlorpyrifos.
Meanwhile, all the compounds also showed a high insecticidal
activity against Tetranychus cinnbarinus, and the activities of these
compounds were at least as active as or even more active than that
of chlorpyrifos at the concentration of 200 mg/L. The in vivo results
of insecticide activity of these compounds, as well as their further
structure optimization and the detailed structure–activity relation-
ships will be reported in the future.
To a solution of methyl 2-chloroacetate (13.0 g, 120.0 mmol) in
methanol (20.0 ml), ethane-1, 2-diamine (1A) (3.0 g, 50.0 mmol) of
methanol was added dropwise at room temperature, and stirred
for 10 h. The white solid was filtered, washed, dried and collected.
The solution was condensed and cooled to crystallize the residual
Table 3
1H NMR data of organophosphorus compounds 4A–I
Compounds 1H NMR (CDCl3/TMS)(d), J (Hz)
4A
1.35–1.39 (m, 12H), 3.40 (d, 4H, J = 3.3 Hz), 3.48 (d, 4H,
J = 18.9 Hz),
4.17–4.23 (m, 8H), 7.57 (s, 2H)
4B
4C
4D
4E
4F
4G
4H
4I
1.35–1.40 (m, 12H), 1.70 (t, 2H, J = 6.3 Hz), 3.30–3.36 (m, 4),
3.48(d, 4H, J = 19.2 Hz) 4.13–4.24 (m, 8H), 7.50 (s, 2H)
1.26–1.40 (m, 12H), 1.57–1.60 (m, 4H), 3.28 (t, 4H, J = 11.1 Hz),
3.43 (d, 4H, J = 21.0 Hz) 4.12–4.24 (m, 8H), 7.29 (s, 2H)
1.35–1.40 (m, 14H), 1.50–1.60 (m, 4H), 3.22–3.28 (m, 4H),
3.44 (d, 4H, J = 19.5 Hz) 4.11–4.24 (m, 8H), 7.20 (s, 2H)
1.36–1.40 (m, 16H), 1.53 (t, 4H, J = 13.2 Hz), 3.20–3.27 (m, 4H),
3.39 (d, 4H, J = 1.5 Hz), 4.11–4.24 (m, 8H), 7.17(s, 2H)
1.33–1.40 (m, 18H), 1.52 (t, 4H, J = 13.5 Hz), 3.20–3.27 (m, 4H),
3.36 (d, 4H, J = 16.8 Hz), 4.1–4.24 (m, 8H), 7.12 (s, 2H)
1.35–1.40(m, 20H), 1.52 (d, 4H, J = 6.3 Hz), 3.23–3.31 (m, 4H),
3.43 (d, 4H, J = 19.5 Hz), 4.15–4.21 (m, 8H), 7.15 (s, 2H)
1.25–1.40 (m, 22H), 1.49–1.53 (m, 4H), 3.20–3.27 (m, 4H),
3.43 (d, 4H, J = 21.0 Hz), 4.11–4.25 (m, 8H), 7.19 (s, 2H)
1.24–1.37 (m, 24H), 1.39 (t, 4H, J = 7.2 Hz), 3.22–3.24 (m, 4H),
3.42 (d, 4H, J = 19.5 Hz), 4.13–4.22 (m, 8H), 7.19 (s, 2H)
3. Conclusions
A series of homo-organophosphorous compounds with various
spacer lengths were designed on the basis of homo-dimer strategy
that has been successfully applied in rational drug design. The
compounds were prepared by using a group of inactive organo-
phosphorous compounds as the monomers. The prepared com-
pounds were evaluated for their inhibitory activity against
DmAChE and MdAChE, and most exhibited a pretty good activity.
Particularly, the activities of homo-dimer 4H were 100,000-fold
and 245-fold higher than those of monomer and ometheoate,
respectively. In addition, the homo-dimer 4H exhibited the

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R. Xie et al. / Bioorg. Med. Chem. 21 (2013) 278–282
Table 4
Physical properties, yields and elemental analyses of compounds 4A–I
Compound
Physical properties
Yield (%)
Elemental analyses (found)
C
H
N
P
S
4A
4B
4C
4D
4E
4F
4G
4H
4I
Colorless oil
Colorless oil
Colorless oil
Colorless oil
Colorless oil
Colorless oil
Colorless oil
Colorless oil
Colorless oil
15.3
16.5
12.7
24.3
27.0
35.8
42.6
40.4
38.8
35.00 (34.87)
36.43 (36.32)
37.79 (37.66)
39.07 (39.14)
40.29 (40.42)
41.45 (41.59)
42.54 (42.46)
43.59 (43.51)
44.58(44.49)
6.29 (6.20)
6.52 (6.62)
6.74 (6.65)
6.94 (7.04)
7.14 (7.08)
7.32 (7.26)
7.50 (7.41)
7.66 (7.57)
7.82 (7.71)
5.83 (5.94)
5.66 (5.54)
5.51 (5.64)
5.36 (5.28)
5.22 (5.31)
5.09 (5.14)
4.96 (5.04)
4.84 (4.94)
4.73 (4.82)
12.8 9(12.81)
12.53 (12.60)
12.18 (12.30)
11.85 (1196)
11.54 (11.66)
11.25 (11.32)
10.9 7(11.04)
10.71 (10.80)
10.45 (10.56)
13.3 5(13.44)
12.97 (13.06)
12.61 (12.72)
12.27 (12.36)
11.95 (12.04)
11.65 (11.73)
11.3 6(11.54)
11.08 (11.20)
10.82 (10.91)
product. The leading white solid was washed, dried and collected.
Two products were combined to get N0,N0-(ethane-1, 2-diyl) bis(2-
chloroacetamide) (2A). The same procedure was employed in the
preparation of the products 2B–I by using the corresponding al-
kane diamine.
4.4.2. Insecticidal test for Tetranychus cinnbarinus
The insecticide activities of the title compounds against Tetr-
anychus cinnbarinus were evaluated using the method similar to
that against Lipaphis erysimi as described. Cotton leaves with
approximately 80 Tetranychus cinnbarinus adults were selected,
and dipped into the solutions containing the prepared compounds
for 5 s. The omethoate and chlprpyrifos were taken as the positive
controls. The aqueous solution of Triton X-100 (0.1 mg/L) was used
as the control. Percentage mortalities were evaluated 24 h after
treatment, and three replicates were carried out.
To
a solution of O,O,-diethylthiophosphate, potassium (3)
(2.18 g, 10.5 mmol) in methanol (20.0 ml), N0,N0-(ethane-1,2-diyl)
bis(2-chloroacetamide) (2A) (1.06 g, 5.0, mmol) was added, and
the mixture was refluxed for 16 h. The reaction mixture was fil-
tered and the filtrate was concentrated under vacuum. The viscous
residue was dissolved with 20 ml ethyl acetate, then washed with
water and the ethyl acetate layer was dried over anhydrous so-
dium sulfate. The dried solution was concentrated under reduced
pressure, the crude compound was chromatographed on silica gel
to give S0,S0-2,2-(ethane-1,2-diylbis(azanediyl)) bis(2-oxoethane-
2,1-diyl) O,O,O0,O0-tetraethyl diphosphorodithioate (4A). The same
procedure was employed in the preparation of the products 4A–I
Acknowledgments
This research was supported by the 973 high-tech Key Project of
China (2010CB126106) and National Basic Research Program of
China (2011BAE06B03, 2012BC1140X).
by using the corresponding N0,N0-(alkane-
a,x-diyl) bis(2-
chloroacetamide).
References and notes
The structures, appearance, and yields of target compounds 4A–
I are listed in Table 4, and their 1H NMR and elemental analysis
data are given in the Table 3 and Table 4.
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erysimi were tested by using leaf-dip method reported previ-
ously.19–21 Chinese cabbage leaves with 45 apterous adults were
dipped in the diluted Triton X-100 (0.2 g/L) solution for 10 s, the
leaves were then placed in a plastic dish until they were dry. The
dried leaves were transported to a conditioned room (25 1 °C,
50% RH). The aqueous solution of Triton X-100 (0.1 mg/L) was used
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against L. erysimi and utilized as the positive controls. Each test
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