Water bridges are essential to neonicotinoids: Insights from synthesis, bioassay and molecular modelling studies

Article abstract of DOI:10.1016/j.cclet.2018.05.013

Water-bridged H-bonds have been observed in many cases of ligand-receptor recognitions. To explore the roles of water bridges in the binding of neonicotinoids with receptors, twenty-four neonicotinoid compounds with nine fragments, including 1H-1,2,3-tria

Full text of DOI:10.1016/j.cclet.2018.05.013

Accepted Manuscript
Title: Water bridges are essential to neonicotinoids: Insights
from synthesis, bioassay and molecular modelling studies
Authors: Chengchun Zhu, Guanglong Li, Keya Xiao, Xusheng
Shao, Jiagao Cheng, Zhong Li
PII:
DOI:
Reference:
S1001-8417(18)30200-6
https://doi.org/10.1016/j.cclet.2018.05.013
CCLET 4545
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
6-4-2018
3-5-2018
4-5-2018
Please cite this article as: Zhu C, Li G, Xiao K, Shao X, Cheng J, Li Z, Water bridges are
essential to neonicotinoids: Insights from synthesis, bioassay and molecular modelling
studies, Chinese Chemical Letters (2010), https://doi.org/10.1016/j.cclet.2018.05.013
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Communication
Water bridges are essential to neonicotinoids: Insights from synthesis, bioassay and
molecular modelling studies
Chengchun Zhua,1, Guanglong Lia,1, Keya Xiaoa, Xusheng Shaoa, Jiagao Chenga,* , Zhong Lia,b
aShanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
bShanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China
 Corresponding author.
E-mail address: jgcheng@ecust.edu.cn (J. Cheng).
1 These authors contributed equally to this article.
Graphical abstract
To identify the detailed roles of water bridges in neonicotinoids recognition, twenty-four neonicotinoids compounds were designed, synthesized, bioassayed and
modelled. Of all nine fragments mimicking water bridges, cyano group was the optimal one. The insecticidal activities indicated that the water bridge might be
stable in the active site and was not suitable to be replaced by other groups, which highlighted the significance of water bridges for neonicotinoids.
ARTICLE INFO
ABSTRACT
Water-bridged H-bonds have been observed in many cases of ligand-receptor recognitions. To
explore the roles of water bridges in the binding of neonicotinoids with receptors, twenty-four
neonicotinoid compounds with nine fragments, including 1H-1,2,3-triazole, CN, COOMe,
CONHNH2, CONHMe, NO2, NH2, NHCOMe and NHCSNH2 were synthesized and evaluated,
of which, compounds with cyano group showed the best activities against Aphis craccivora.
Accordingly, the cyano group is the optimal fragment mimicking the water bridge. Two cyano-
substituted cis-nitromethylene compounds display good insecticidal activities, whereas the
LC50 values are lower than those of their corresponding prototypes. Docking study showed that
the cyano group acts only as H-bond acceptor, while the water bridge can act as both donor and
acceptor. It revealed that the water bridge might be stable in the active site and was not suitable
to be replaced by other groups. The findings illustrated that the water bridge is necessary for
high insecticidal activities of neonicotinoids, which should be also helpful in better
understanding the binding mode of neonicotinoids.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Neonicotinoids
Water bridge
Insecticidal activity
Cyano group
Molecular design
Water molecules in ligand binding pockets can bridge interactions between ligands and proteins through H-bonds, namely water-
bridged H-bonds. Water bridges have been reported in numerous protein-ligand complex structures, and observed playing crucial roles
in many cases of ligand-receptor recognitions [1-3]. If a water bridge is flexible in the binding pocket, it can be mimicked or replaced
by other fragments. In drug discovery and optimization, such a strategy has been fully applied to get novel ligand scaffolds or improve
binding affinities [4-7]. For example, by replacing the role of water bridge, a number of pharmaceutical instances have been
successfully achieved in lead optimization of EGFR [8,9], HIV-1 protease [10], etc.
In the cases of pesticides, water-bridged H-bonds also played key roles in the binding of insect nicotinic acetylcholine receptors
(nAChRs) with neonicotinoids [11-14]. Neonicotinoids were agonists of insect nAChRs [14,15], which occupied the largest
insecticides market share of the world due to their excellent wide-spectrum insecticidal activity and safety to mammals [16-18], and
gained continuous research concern [19-22]. In crystal structures of neonicotinoids with acetylcholine binding proteins (AChBP), good
surrogates for the extracellular domain of nAChRs, a water molecule was captured next to the pyridine/thiazole nitrogen atom of
neonicotinoids in the binding pocket, which bridged crucial H-bonding network with the relevant surrounding residues [23,24]. The
pyridine/thiazole nitrogen atom served as H-bond acceptor in the binding pocket (Fig. 1), whose role could also be replaced by a
fluorine or a carbonyl oxygen of alkyl substituents in neonicotinoid analogues [11]. QSAR study revealed that the insecticidal activities
of neonicotinoids were deeply correlated with the water-bridged H-bonds abilities [12]. When the water-bridged H-bonds were broken
in neonicotinoids N-oxide analogues, the insecticidal activities would be weak [13].

Fig. 1. Design strategy of neonicotinoid derivatives by mimicking water bridge.
Although water-bridged H-bonds are reported in the recognition between neonicotinoids and nAChRs, few studies are focused on
the water bridge itself. The detailed roles of water bridges on neonicotinoids are still open questions. It is still unknown whether the
water bridge could be replaced by other structure fragments in pesticide molecular design. In this work, nine structure fragments that
may mimic the roles of water molecule, including 1H-1,2,3-triazole, CN, COOMe, CONHNH2, CONHMe, NO2, NH2, NHCOMe and
NHCSNH2 were selected to occupy the binding site of water bridge, and then twenty-four neonicotinoid compounds were designed,
synthesized, bioassayed and modeled to capture the detailed roles of water bridge.
The synthesis of target compounds A1-a and A1-b was shown in Scheme S1 in Supporting information. The compound 2 was
obtained in 80% yield from commercially available compound 1 as described in the literature [25]. Intermediate 3 was obtained in 65%
yield through deoxidation of compound 2, then was subjected to a Sonogashira coupling reaction and desilylation to provide compound
4 in 91% yield. Conversion of compound 4 to compounds 6-a and 6-b was accomplished through chlorination and then aminolysis
with relevant key intermediates a and b respectively. Lastly, the triazole substituted compounds A1-a and A1-b were gained by a
modified Click reaction with excellent yields.
The synthesis of target compounds A2-a, A2-b was shown in Scheme 1. Intermediate 8 was obtained in 80% yield from compound
7 through a palladium-catalyzed ortho-halogenation. Based on compound 8, the target compounds were readily obtained through two
steps involving a bromination reaction and a substitution reaction.
Scheme 1. Synthesis route of cyano-substituted compounds A2-a and A2-b.
The synthesis route of target compounds A3-a, A3-b, A4-a, A4-b, A5-a and A5-b was shown in Scheme S2 in Supporting
information, and synthesis of target compounds A6-a, A6-b, A7-a, A7-b, A8-a, A8-b, A8-b, A9-a and A9-b were shown in Scheme
S3 in Supporting information.
The synthesis of target compounds A10~A15 were depicted in Scheme 2. Compounds A10~A13 were gained through the similar
routes with previous compounds A2-a and A2-b. Compounds A14 and A15 were prepared according to the procedures in our previous
studies [26,27]. Compound A14 containing dihydroxy-substituted pyrrole fused ring was generated by treatment A13 with
methylglyoxal. Conjugated diene compound A15 was afforded Stirring the mixture of intermediate A13 and furfural in the presence of
a catalyst amount of hydrogen chloride gas. Compounds P1 [26] and P2 [27] have been reported in our previous studies, which were
also generated in this work for comparison. P3 and P4 were synthesized according to the procedures of P1 [26] and P2 [27],
respectively.
Scheme 2. Synthesis route of cyan-substituted compounds.
Then insecticidal activities against Aphis craccivora of all compounds were evaluated by leaf-dip method (Supporting information).
Compounds A2-a and A2-b with cyano group showed the best insecticidal activities as compared with the other compounds. The
cyano group has been also successfully used in rational design of inhibitors of scytalone dehydratase [28], EGFR kinase [29] and p38α

MAP kinase [30], through displacing the key water molecule in active sites. Accordingly, the cyano group was considered as the
optimal fragment that could well mimic the role of water bridge and was chosen in further structure derivations.
Six cyano-substituted compounds A10~A15 with different scaffolds were synthesized and bioassayed. Insecticidal activities of
compounds A13~A15 exhibited 100% mortality at 500 mg/L against cowpea aphids. Further bioassay showed that the LC50 values of
two cis-nitromethylene compounds A14 and A15 were 3.71 mg/L and 7.33 mg/L, respectively, which were similar to that of
imidacloprid (3.12 mg/L). Compounds A14 and A15 showed relative low insecticidal activities when compared with their
corresponding chlorine pyridine prototypes P1 (2.04 mg/L) and P2 (1.37 mg/L), respectively. In addition, A14 and A15 displayed
stronger insecticidal activities than the phenyl structures without cyano substitution P3 (6.31 mg/L) and P4 (11.32 mg/L), respectively.
It indicated that the cyano substitution could contribute to the insecticidal activities, while the contribution was not as large as a
pyridine ring.
Physical-chemical properties (logP, logS, pKa and TPSA) of all compounds were calculated (methods were shown in Supporting
information) and then analyzed between the new cyano-substituted compounds versus imidacloprid. As shown in Table 1, the
calculated logP, logS, pKa and TPSA values of new compounds are similar to those of their prototypes without cyano group (P1–P4).
Accordingly, the difference in insecticidal activities could not be resulted only from the pesticide-like properties.
Table 1
Physicochemical property and insecticidal activities of the target compounds and imidacloprid against cowpea aphids (Aphis craccivora).
Compd.
X-R1
NR2
Aphis craccivora
LogP
LogS
pKa
TPSA (A2)
Mortality (%)
(48 h, 500 mg/L)
21
52
13
24
31
35
18
12
37
25
83
33
25
49
13
18
12
13
25
35
17
100
100
100
100
100
100
100
100*
0
LC50
(mg/L )
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
N.t.
6.83
3.71
7.33
2.04
1.37
6.31
11.32
3.12
0.00
A1-a
A2-a
A3-a
A4-a
A5-a
A6-a
A7-a
A8-a
A9-a
A1-b
A2-b
A3-b
A4-b
A5-b
A6-b
A7-b
A8-b
A9-b
A10
A11
A12
A13
A14
A15
P1
P2
P3
C-1H-1,2,3-triazole-4-yl
C-CN
C-COOMe
C-CONHNH2
C-CONHMe
C-NO2
a
a
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
c
d
e
f
2.19
2.00
1.78
0.74
1.40
1.60
1.13
2.04
1.66
2.16
2.22
2.38
1.57
2.13
2.72
2.08
2.29
2.15
2.52
2.07
2.02
1.08
0.99
2.02
0.59
1.72
1.19
2.00
0.57
-3.61
-3.25
-3.28
-3.33
-3.16
-3.80
-2.93
-3.24
-3.84
-3.82
-3.70
-3.82
-3.37
-3.52
-3.73
-3.28
-3.54
-4.25
-3.41
-3.51
-3.25
-3.14
-2.95
-4.02
-1.76
-4.20
-2.09
-4.61
-2.87
7.86
115.02
97.27
99.75
128.57
102.55
119.27
99.47
102.55
143.59
80.96
63.18
65.69
94.51
68.49
80.21
65.41
68.49
109.53
63.18
97.68
91.69
84.88
116.55
99.96
105.65
89.06
92.76
76.17
86.34
16.83
16.62
11.88
14.29
15.32
11.34
14.13
15.75
7.79
14.83
14.64
11.71
14.20
15.28
11.69
14.01
15.73
15.65
14.88
11.02
10.63
13.11
7.09
C-NH2
C-NHCOMe
C-NHCSNH2
C-1H-1,2,3-triazole-4-yl
C-CN
C-COOMe
C-CONHNH2
C-CONHMe
C-NO2
C-NH2
C-NHCOMe
C-NHCSNH2
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
N
N
CH
CH
N
g
h
g
h
g
h
a
11.37
7.68
11.41
6.66
P4
Imidacloprid
CK
13.74
N.t.: Not test.
*Test at 10 mg/L.
Fig. 2. The docking poses of compounds within Ac-AChBP. The binding poses of A2-a (a), A14 (b), and molecular alignment diagram of A2-a and
imidacloprid (c).

Docking studies were performed on compounds A2-a and A14 with Ac-AChBP to illustrate the difference of insecticidal activities
and detailed interactions between compounds and target (method was shown in Supporting information). As displayed in Fig. 2, a N-
H···O H-bond, instead of the water bridged H-bond network, was observed between residue Ile-118 and the cyano groups of A2-a and
A14, respectively. The binding pose of A2-a was very similar to imidacloprid and aligned well with imidacloprid (Fig. 2c). In the
crystal structures of imidacloprid with AChBP, the water molecule could act as both donor and acceptor, and bridged two H-bonds
with Ile-106 and Ile-118, respectively [23,24]. Although the insecticidal activities indicated that the cyano group was the optimal group
mimicking the water bridge among all nine tested fragments, it could act only as H-bond acceptor and formed a single H-bond with
target, which might be one reason why the new cyano-substituted compounds displayed relative low insecticidal activities when
compared with their corresponding leads.
On the other hand, the fragments 1H-1,2,3-triazole, CONHNH2, CONHMe, NHCSNH2 and NHCOMe could act both as H-bond
donor and acceptor, however, they were larger than the cyano in size. The weak activities of such fragment-substituted compounds
indicated that the active water site might be small in volume. In the complex crystal structure of AChBP-imidacloprid, the water bridge
located in a deep inner site of the binding pocket [23,24], implying that the water site was difficult to be occupied by other groups,
even by the cyano group. It illustrated that the water bridge was essential for high insecticidal activities of neonicotinoids.
In summary, to explore the key roles of water bridges in neonicotinoids recognition with receptor, nine fragments including 1H-
1,2,3-triazole, CN, COOMe, CONHNH2, CONHMe, NO2, NH2, NHCOMe and NHCSNH2 were introduced into the molecular design
of neonicotinoids, and then twenty-four neonicotinoid compounds were synthesized, bioassayed and evaluated. The cyano-substituted
compounds showed better insecticidal activities than other fragment-substituted compounds, thus the cyano group was considered as
the optimal fragment mimicking water bridge. Although, the cyano-substituted compounds displayed similar pesticide-like properties
and binding poses with their corresponding prototype structures, they showed relative weak insecticidal activities. It revealed that the
water bridge might be stable in the active site and was difficult to replace by other groups, which illustrated again the significance of
water-bridged H-bonds in the recognition of neonicotinoids with insect nAChRs.
Acknowledgments
We thank for the financial supports from the National Natural Science Foundation of China (Nos. 21572059, 21172070), Innovation
Program of Shanghai Municipal Education Commission (No. 201701070002E00037), and the Fundamental Research Funds for the
Central Universities.
References
[1] J. Yin, J.C. Mobarec, P. Kolb, D.M. Rosenbaum, Nature 519 (2015) 247-250.
[2] M. Nagae, A. Ikeda, M. Hane, et al., J. Biol. Chem. 288 (2013) 33784-33796.
[3] M. Adler, D.D. Davey, G.B. Phillips, et al., Biochemistry 39 (2000) 12534-12542.
[4] S.B.A. Beer, N.P.E. Vermeulen, C. Oostenbrink, Curr. Top. Med. Chem. 10 (2010) 55-56.
[5] J. Seo, J. Igarashi, H.Y. Li, et al., J. Med. Chem. 50 (2007) 2089-2099.
[6] G. Rastelli, Z.Q. Tian, Z. Wang, D. Myles, Y. Liu, Bioorg. Med. Chem. Lett. 15 (2005) 5016-5021.
[7] S. Ness, R. Martin, A.M. Kindler, et al., Biochemistry 39 (2000) 5312-5321.
[8] A. Wissner, M.B. Floyd, S.K. Rabindran, et al., Bioorg. Med. Chem. Lett. 12 (2002) 2893-2897.
[9] C.J. Torrance, P.E. Jackson, E. Montgomery, et al., Nat. Med. 6 (2000) 1024-1028.
[10] P.Y.S. Lam, P.K. Jadhav, C.J. Eyermann, et al., Science 263 (1994) 380-384.
[11] I. Ohno, M. Tomizawa, K.A. Durkin, J.E. Casida, S. Kagabu, J. Agric. Food Chem. 57 (2009) 2436-2440.
[12] S. Xia, J.G. Cheng, Y. Feng, et al., Chin. J. Chem. 32 (2014) 324-334.
[13] W.W Zhang, Y.F Fan, Y. Tao, et al., Chin. J. Chem. 30 (2012) 357-361.
[14] M. Tomizawa, J. Casida, Acc. Chem. Res. 42 (2009) 260-269.
[15] K. Matsuda, S.D. Buckingham, D. Kleier, et al., Trends Pharmacol. Sci. 22 (2001) 573-580.
[16] P. Jeschke, R. Nauen, M.E. Beck, Angew. Chem. Int. Edit. 52 (2013) 9464-9485.
[17] X.S. Shao, P.W. Lee, Z.W. Liu, et al., J. Agric. Food Chem. 59 (2011) 2943–2949.
[18] M. Tomizawa, J.E. Casida, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 247-268.
[19] C.W. Sun, T. Fang, J. Wang, Z.B. Hao, S.B. Nan, J. Agric. Food Chem. 60 (2012) 9553−9561.
[20] W.C. Su, Y.H. Zhou, Y.Q. Ma, et al., J. Agric. Food Chem. 60 (2012) 5028−5034.
[21] Q.Q. Hou, Y.F. Jing, X.S. Shao, Chin. Chem. Lett. 28 (2017) 1723-1726.
[22] D.D. Zhang, S.X. Cui, Z.P. Xu, D.M. Li, Z.Z. Tian, Chin. Chem. Lett. 28 (2017) 1743-1745.
[23] M. Ihara, T. Okajima, A. Yamashita, et al., Invertebr. Neurosci. 8 (2008) 71-81.
[24] T.T. Talley, M. Harel, R.E. Hibbs, et al., Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 7606-7611.
[25] L. Kraszkiewicz, M. Sosnowski, L. Skulski, Synthesis 7 (2006) 1195-1199.
[26] Z.J. Ye, L.N. Shi, X.S. Shao, et al., J. Agric. Food. Chem. 61 (2013) 312-319.
[27] X.S. Shao, Z. Li, X.H. Qian, X.Y. Xu, J. Agric. Food Chem. 57 (2009) 951-957.
[28] J.M. Chen, S.L. Xu, Z. Wawrzak, G.S. Basarab, D.B. Jordan, Biochemistry 37 (1998) 17735-17744.
[29] A. Wissner, D.M. Berger, D.H. Boschelli, et al., J. Med. Chem. 43 (2000) 3244-3256.
[30] C.J. Liu, S.T. Wrobleski, J. Lin, et al., J. Med. Chem. 48 (2005) 6261-6270.