Design, synthesis and biological evaluation of α-aminophosphonate derivatives containing a pyrazole moiety

Article abstract of DOI:10.1248/cpb.c16-00622

Acetylcholinesterase (AChE) is a key enzyme which present in the central nervous system of living organisms. Organophosphorus pesticides (OPs) that serve as insecticides are AChE inhibitors which have been used widely in agriculture. A series of novel OPs containing pyrazole moiety have been designed and synthesized. The biological evaluation indicated compound 4e appeared 81% larvicidal activity against Plutella xylostella at the concentration of 0.1 mg/L and the inhibition of AChE by compound 4e was distinctly enhanced with the increasing doses. Molecular docking of compound 4e into the three dimensional X-ray structure of the Drosophila melanogaster AChE (DmAChE, PDB code: 1QO9) was carried out utilizating the Discovery Studio (DS), the binding model revealed that the title structure was tightly embedded in the binding sites of DmAChE. Therefore, we suggest that compound 4e may serve as a novel AChE inhibitor that can be utilized as a new insecticidal drug.

Full text of DOI:10.1248/cpb.c16-00622

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c16-00622
Advance Publication
October 1, 2016
Chem. Pharm. Bull.
Regular Article
Design, synthesis and biological evaluation of α-aminophosphonate derivatives
containing a pyrazole moiety
Zi-Li Ren a, Jing Zhang b, Hai-dong Li c, Ming-Jie Chu a, Li-Song Zhang a,
Xiao-Kang Yao a, Yong Xia a, Xian-Hai Lv a,*, Hai-Qun Cao a,*
aSchool of Plant Protection, Anhui Agricultural University, Hefei 230036, P.R. China
bSchool of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038, P.R. China
cSchool of Materials, The University of Manchester, Oxford Road, Manchester, M13 9PL,
UK.
*
Authors to whom correspondence should be addressed; E-Mails: lvxianhai@ahau.edu.cn (X.-
H.L.);haiquncao@163.com (H.-Q.C.); Tel./Fax: +86-551-6578-6906 (X.-H.L.).
Ⓒ 2016 The Pharmaceutical Society of Japan

Acetylcholinesterase (AChE) is a key enzyme which present in the central nervous system
of living organisms. Organophosphorus pesticides (OPs) that serve as insecticides are AChE
inhibitors which have been used widely in agriculture. A series of novel OPs containing
pyrazole moiety have been designed and synthesized. The biological evaluation indicated
compound 4e appeared 81% larvicidal activity against P. xylostella at the concentration of
0.1 mg/L and the inhibition of AChE by compound 4e was distinctly enhanced with the
increasing doses. Molecular docking of compound 4e into the three dimensional X-ray
structure of the Drosophila Melanogaster Acetylcholinesterase (DmAChE, PDB code: 1QO9)
was carried out utilizating the Discovery Studio (DS), the binding model revealed that the
title structure was tightly embedded in the binding sites of DmAChE. Therefore, we suggest
that compound 4e may serve as a novel AChE inhibitor that can be utilized as a new
insecticidal drug.
Keywords: α-aminophosphonate; pyrazole; single-crystal; molecule docking
Chemical and Pharmaceutical Bulletin Advance Publication

The synthesis of organophosphorus compound began in early 1930s1. Since then,
organophosphorus insecticides represent one of the major classical insecticides that have
been used over the past several decades around the world. However, organophosphorus
insecticides have been excessively used over the past decades, and insects have became
difficult to control due to their resistance to the traditional organophosphorus insecticides that
cause enormous losses of crop production. The undifferentiated acts of organophosphorus on
acetylcholinesterase (AChE) of organism are threatening to human health2. Thus, the
discovery of new organophosphorus insecticides that have low toxicity and can inhibit the
inhibition of AChE is particularly important.
AChE is a key enzyme present in the central nervous system of living organisms. The
crucial function of AChE is to terminate impulse transmission at cholinergic synapses
through rapid hydrolysis of acetylcholine (ACh), thus to keep the normal transmission of
nerve impulses3-5, while the catalytic mechanism of ACh hydrolysis is a successive two-step
process, namely, acylation and deacylation6. Inhibition of AChE cannot lead to the hydrolysis
of neurotransmitter ACh or accumulation of ACh in the synaptic cleft that results in that
impeded neurotransmission is the primary mechanism of acetylcholinesterase inhibitors7-10
.
Especially, many Organophosphorus pesticides (OPs) that serve as insecticides are
acetylcholinesterase (AChE: E. C. 3.1.1.7) inhibitors that are widely used in agriculture
(Figure 1)11-13. Those organophosphorus compounds prevent ACh hydrolysis, which results
in overstimulation of the cholinergic synapses and leads to neuromuscular paralysis. As a
consequence, this will contribute to the death of the insect14-16. The electrophilic phosphorous
atom of OPs can react with the hydroxyl group of the serine in the AChE through
nucleophilic attack, which leads to the enzymes’ phosphorylation and deactivation, so these
organophosphorus compounds are highly toxic.
Figure 1. The products of organophosphorus pesticides target at AchE
If we introduce some heterocyclic(e.g., Pyridine, Furan, Pyrimidine and Thiazole) into
the organophosphorus scaffold, the resulting compounds may have an interesting structure to
Chemical and Pharmaceutical Bulletin Advance Publication

reduce the toxicity to human health while maintaining its insecticidal activity, and the
introduction of further structurally diversity to OPs is also conducive to delay the onset of
resistance. The constitution of pyrazole derivatives is very important in pesticide field due to
their broad range of biological activities, high efficiency and low toxicity17-21, we thus choose
pyrazole ring introduced into the α-aminophosphonate skeleton to synthesize a series of title
compounds (Figure 2), and some single-crystal structures were obtained. All compounds
have been evaluated for their insecticidal activities against P. xylostella and were performed
molecular docking, and the binding model revealed that the title structure was tightly
embeded in the binding sites of DmAChE (PDB code: 1QO9). The structure–activity
relationship for the inhibitory effects of the title structure on AChE was discussed.
Figure 2. The structure of the target compound.
Results and Discussion
Chemistry All the title compounds 4a-4t were synthesized by using the modified
Thirumurugan’s method27 and the general synthetic route was depicted in Chart 1.
The requisite intermediates 3-(4-substituented phenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde 3a-3e were prepared by the reaction of the Vilsmeier–Haack reagent
(DMF/POCl3), respectively. The available methods for the synthesis of the target compounds
are based on the synthesis of imines followed by a Lewis acid-catalyzed nucleophilic addition
of phosphates to imines, it was obtained from substituted amines, pyrazole aldehyde, and
diethyl phosphite using BF3·Et2O as catalyst under acetonitrile conditions. Then the title
compounds were obtained by subsequent purification with recrystallization in ethanol. All the
synthetic compounds presented the satisfactory analytical and spectroscopic data, which were
full accordance with their depicted structures.
Chemical and Pharmaceutical Bulletin Advance Publication

O
R1
a
b
NHNH2
R1
N
2
N
H
1
R2
R1
NH
O
NH2
O
c
N
N
P
P
H
N
O
O
O
O
R2
N
OHC
3
4
R1
R1=H; CH3; Cl; OCH3; F
R2=CH3; OCH3; F; Cl
Chart 1. General synthesis of compounds 4a-4t. Reagents and conditions: (a)sodium acetate,
ethanol, rt; (b) DMF, POCl3, 80–85 °C, 5h; (c)BF3·Et2O, acetonitrile, 3h, reflux.
X-ray crystallography The crystal data and refinement parameter for part of the title
compounds are listed in Table 1, the crystal structure is shown in Figure 3. The structure was
solved by direct methods and refined on F2 by full-matrix least-squares methods using
SHELXTL25. The unit cell dimensions and intensity data were recorded at 293 K. The
program SAINT/XPREP was used for data reduction and APEX2/SAINT for cell
refinement28. All non-hydrogen atoms were refined with anisotropic thermal parameters. All
hydrogen atoms with the exception of those on nitrogen atoms were geometrically fixed and
refined using a riding model. Molecular graphics employed were MERCURY and
PLATON29. Crystallographic data (excluding structure factors) for the structure were
deposited with the Cambridge Crystallographic Data Center as supplementary publication No.
CCDC-1414578 (4e)，CCDC-1414579 (4b)， CCDC-1414580 (4i)，CCDC-1414581 (4j)，
CCDC-1414582 (4k)，CCDC-1414583 (4n)，CCDC-1414584 (4r).
Chemical and Pharmaceutical Bulletin Advance Publication

Table 1. Crystal data of partly of the title compounds
compound
Empirical formula C27H30N3O4P
4b
4e
4i
4j
4k
4n
4r
C28H32N3O3P
C27H29ClN3O3 C27H29ClN3O4 C26H26ClFN3
C28H32N3O5P
C27H29FN3O4P
P
P
O3P
Molecular weight
Temperature(K)
Radiation
491.51g/mol
273(2)
489.54g/mol
273(2)
509.95g/mol
273(2)
525.95g/mol
273(2)
513.92g/mol
273(2)
Mo-
521.54g/mol
273(2)
509.50g/mol
273(2)
Mo-
Mo-
Mo-
Mo-
Mo-
Mo-
Kα(0.71073Å) Kα(0.71073Å)
Kα(0.71073Å) Kα(0.71073Å) Kα(0.71073Å) Kα(0.71073Å) Kα(0.71073Å)
Crystal system
Space group
a(Å)
orthorhombic
Pbca
monoclinic
P2(1)/n
monoclinic
P2(1)/c
monoclinic
P2(1)/c
monoclinic
P2(1)/c
monoclinic
P2(1)/c
8.247(5)
13.643(5)
24.492(5)
90.000(5)
93.122(5)
90.000(5)
2752(2)
4
triclinic
P-1
19.970(5)
11.280(5)
23.426(5)
90.000(5)
90.000(5)
90.000(5)
5276.9(3)
8
14.570(5)
11.953(5)
17.058(5)
90.000(5)
112.905(5)
90.000(5)
2736.3 (8)
4
14.889(5)
12.064(5)
17.420(5)
90.000(5)
109.169(5)
90.000(5)
2955.4(6)
4
14.729(5)
12.243(5)
17.468(5)
90.000(5)
112.688(5)
90.000(5)
2906.1(5)
4
14.977(5)
11.766(5)
17.486(5)
90.000(5)
108.706(5)
90.000(5)
2918.5(2)
4
9.234(5)
11.744(5)
13.031(5)
81.400(5)
72.404(5)
79.395(5)
1317.3(10)
2
b(Å)
c(Å)
α (°)
β(°)
γ(°)
V(Å3)
Z
Dc (g cm–3)
μ (mm–1)
absort.coeff
F(000)
1.237
1.188
1.146
1.202
1.170
1.259
1.284
0.141
0.133
0.213
0.221
0.221
0.141
0.149
2080
1040
1072
1104
1072
1104
536
θ rang (deg)
Reflections
collected
Indep. reflns
Refns obs.
[I > 2σ(I)]
Data/restr./paras
2.25-25.20
49287
2.35-25.20
13899
2.30-25.20
15930
2.27-25.20
28098
2.25-25.20
28353
2.24-25.20
26506
2.30-30.75
13623
(Rint=0.0302)
4721
(Rint=0.0564)
4737
Rint=0.0453)
5122
(Rint=0.0325)
5219
(Rint=0.0266)
5243
(Rint=0.0248)
4929
(Rint=0.0154)
4671
3863
3131
3210
3677
4006
3908
4050
4721/0/316
4737/0/317
1.031
5122/0/316
1.014
5219/0/334
10.018
5243/0/316
1.023
4929/0/334
1.008
4671/0/343
1.054
Goodness-of-fit on 10.23
F2
R1, wR2
(all data)
0.0714/0.1819 0.1590/0.3186
0.0590/0.1686 0.1092/0.2820
0.1323/0.2422 0.0851/0.1854 0.0652/0.1588 0.0730/0.1928 0.0666/0.1929
0.0889/0.2242 0.0569/0.1584 0.0496/0.1476 0.0585/0.1777 0.0600/0.1847
R1, wR2
[I > 2σ(I)]
Larg.peak/hole
(e. Å)
0.558/-0.403
0.580/-0.356
0.302/-0.264
0.334/-0.258
0.288/-0.282
1.259/-0.245
0.671/-0.463
Chemical and Pharmaceutical Bulletin Advance Publication

Figure 3. The molecular structure of title compounds showing 30% probability displacement ellipsoids.
Chemical and Pharmaceutical Bulletin Advance Publication

Insecticidal activity All the compounds 4a-4t were evaluated for their insecticidal
activities against P. xylostella using procedures reported previously30. Standard insecticidal
agent chlorpyrifos were also screened under identical conditions for comparison. The results
revealed that some of the synthetic compounds exhibited significant insecticidal activities in
Table 2.
Table 2. Insecticidal activity of synthesised compounds against P. xylostella.
Larvicidal activity (%) at a concentration of (mg/L)
compound
R1
R2
10
5
1
0.5
64
0
0.1
21
0
H
CH3
OCH3
F
100
84
100
54
100
12
4a
H
4b
H
100
100
100
100
100
100
63
100
100
100
100
100
100
40
92
37
67
100
54
90
85
0
36
15
81
16
44
34
0
4c
H
Cl
84
4d
CH3
CH3
CH3
CH3
Cl
CH3
OCH3
F
100
85
4e
4f
100
100
11
4g
Cl
4h
CH3
OCH3
F
4i
Cl
74
56
28
0
0
4j
Cl
100
100
100
100
100
79
100
86
100
57
87
34
46
27
84
0
57
0
4k
Cl
Cl
4l
OCH3
OCH3
OCH3
OCH3
F
CH3
OCH3
F
95
72
21
15
55
0
4m
94
67
4n
100
51
100
18
4o
Cl
4p
CH3
OCH3
F
100
100
100
89
100
88
84
56
34
79
0
25
14
44
0
4q
F
75
4r
F
100
46
100
13
4s
4t
F
Cl
100
100
100
100
89
chlorpyrifo
As Table 2 showed, some of the synthetic compounds exhibited the nearly equal
inhibitory effects to chlorpyrifos at 10 or 5 mg/L, such as compounds 4g and 4e. Especially,
compound 4e showed the most potent insecticidal activity against P. xylostella, which could
be comparable with chlorpyrifos. In addition, introducing different substituent groups on
benzene ring could lead to a remarkable change in bioactivity. For instance, when compound
introduced electron-donating substituent groups on benzene ring, such compound insecticidal
activity significantly increased, and when introduced electron-withdrawing substituent
groups, compound insecticidal activity significantly decreases. In electron-withdrawing
substituent groups, F showed the most potent insecticidal activity such as compounds 4c, 4g,
4k and 4o. However, not all compounds which containing methyl are beneficial such as 4i
and 4m. Similarly, not all compounds which containing methyl are harmful such as 4g and
Chemical and Pharmaceutical Bulletin Advance Publication

4h. Therefore, the activity of compounds is determined by R1 and R2. Further to say, as to the
R1, R2 place, the contribution of electron-donating substituents is greater than electron-
withdrawing substituents’, Wherein CH3 has the greatest contribution. The results provided
valuable information for the design of insecticidal agents.
Inhibition assay against AchE In order to determine the relationship between
compounds and insecticidal effect, AChE inhibitory activity assays were performed. The
results were given in Table 3. Chlorpyrifos showed the best inhibitory ability against AChE
enzymes with IC50 (inhibitory concentration, 50%) values of 2.83 mg/L, and compound 4e
showed slightly weak activity with IC50 values of 3.16 mg/L. Furthermore, these compounds
show different inhibitory abilities with IC50 values from 3 to 10 mg/L exhibited the same
tendency as the data acquired by using the insecticidal activity assay in vitro. AChE
inhibitory activity assays fully proved that the target compounds displayed as good inhibitory
effects as other successfully developed α-aminophosphonate insecticides. Therefore, we
assumed that compound 4e could affect the function of AChE then lead to insect death.
Table 3. IC50 Values of AChE Inhibition Activity (in Vitro)
Compounds
IC50 (mg/L)a
9.04 ± 0.2
4a
4e
4g
4h
4o
4s
3.16 ± 0.1
4.56 ± 0.3
6.47 ± 0.2
5.89 ± 0.4
8.94 ± 0.3
2.83 ± 0.2
chlorpyrifos
aValues are the mean ± standard deviation (SD) of three replicates.
Molecular Docking The purpose was to ascertain at the molecular level that title compounds
can provide a molecular explanation on the surprisingly good activity. Molecular docking of
compound 4e into the three dimensional X-ray structures of the DmAChE (PDB code: 1QO9)
was carried out using the Discovery Studio (DS) (version 3.5, NeoTrident Corporation,
Beijing, China) software as implemented through the graphical user interface DS-CDOCKER
protocol (Figure 4). The three-dimensional structures of the aforementioned compounds were
constructed using Chem. 3D ultra 12.0 software (Chemical Structure Drawing Standard,
Chemical and Pharmaceutical Bulletin Advance Publication

CambridgeSoft Corporation, Cambridge, MA, USA), then they were energetically minimized
by using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of protein complex were retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do) and prepared by Discovery Studio 3.5 with all
bound waters and ligands eliminated from the protein and the polar hydrogen added to the
protein. The molecular docking procedure was performed by using CDOCKER protocol for
receptor-ligand interactions section of DS 3.5.
Figure 4. Docking model of compound 4e with DmAChE 1QO9. It is nicely bound to the
DmAChE via one H-bonds (ARG 17-O: 2.32Å, 103.31o) and a Pi-cation interaction (4.5 Å).
Experimental
General All of the synthesized compounds were chemically characterized by thin layer
chromatography (TLC), proton nuclear magnetic resonance (1H NMR) and elemental
microanalyses (CHN). 1H NMR and 13C NMR spectra were measured on a Bruker AV-300 or
a Agilent DD2 600Hz spectrometer AV-600 spectrometer at 25 ºC and referenced to Me4Si.
Chemical shifts were reported in ppm (δ) using the residual solvent line as internal standard.
Splitting patterns were designated as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS
spectra were recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument and were within ±0.4% of the theoretical
values. Melting points were measured on a XT4 MP apparatus (Taike Corp, Beijing, China).
Chemical and Pharmaceutical Bulletin Advance Publication

Analytic thin-layer chromatography was performed on the glass backed silica gel sheets
(silica gel 60A GF254). All compounds were detected using UV light (254 nm or 365 nm).
Preparation of the Intermediates 3-(4- substituented phenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde derivatives (3a-3e)
The intermediates 3-(4-substituented phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(3a-3e) were synthesized as following: para-substituted acetophenone (1a-1e) (20 mmol)
interacted with phenylhydrazine hydrochloride (20 mmol) couple with sodium acetate (40
mmol) in anhydrous ethanol to form 1-phenyl-2-(1-phenylethylidene) hydrazine (2a-2e),
which was then dissolved in a cold mixed solution of DMF (20 mL) and POCl3 (16 mL),
stirred at 80-85 ºC for 5 h. The resulting mixture was poured into ice-cold water, a saturated
solution of sodium hydroxide was added to neutralize the mixture, and the solid precipitate
was filtered, washed with water, dried and recrystallized from ethanol to give the compounds
3a-3e22, 23
.
Preparation of diethyl ((1,3-diphenyl-1H-pyrazol-4-yl)(p-tolylamino)methyl)
phosphonate derivatives (4a–4t)
The intermediates 3-(4-substituented phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3a-
3e (1mmol), para-substituted aniline (1mmol) and diethyl phosphite (1mmol) were added to
acetonitrile (8 mL). Catalytic amount of BF3·Et2O solution in was also added. The mixture
was stirred for 3h at reflux temperature then cooled to ambient temperature. The solvent was
concentrated under reduced pressure, and the solid was obtained, then the crude product was
purified preparative TLC with a mixture of petroleum ether and ethyl acetate (v/v = 1:1) as
the developing solvent to yield the title compounds 4a-4t.
diethyl ((1,3-diphenyl-1H-pyrazol-4-yl)(p-tolylamino)methyl)phosphonate (4a)
1
White crystal, yield 88%; mp 184-185°C; H NMR (300 MHz, CDCl3, δ ppm): 1.08 (t,
J=7.2Hz, 3H), 1.25 (t, J=7.2Hz, 3H), 2.23 (s, 3H), 3.80-4.20 (m, 4H), 4.86 (d, J=21Hz, 1H),
6.35 (d, J=8.4Hz, 2H), 6.80 (d, J=8.4Hz, 2H), 7.18-7.68 (m, 11H), 8.23 (d, J=2.04Hz, 1H).
MS (ESI): 476.5 (C27H30N3O3P, [M+H]+). Anal. Calcd for C27H30N3O3P: C, 68.15; H, 6.46;
N, 8.92; Found: C, 68.20; H, 6.36; N, 8.84%.
diethyl ((1,3-diphenyl-1H-pyrazol-4-yl)((4-methoxyphenyl)amino)methyl)phosphonate (4b)
1
White crystal, yield 90%; mp 190-192°C; H NMR(300 MHz, CDCl3, δ ppm): 1.09 (t,
J=7.2Hz, 3H), 1.26 (t, J=7.2Hz, 3H), 3.68 (s, 3H), 3.80-4.18 (m, 4H), 4.80 (d, J=21Hz, 1H),
Chemical and Pharmaceutical Bulletin Advance Publication

6.38 (d, J=8.4Hz, 2H), 6.57 (d, J=8.4Hz, 2H), 7.18-7.68 (m, 11H), 8.23 (d, J=2.04Hz,1H).
MS (ESI): 492.7 (C27H30N3O4P, [M+H]+). Anal. Calcd for C27H30N3O4P: C, 66.14; H, 6.27;
N, 8.46; Found: C, 65.98; H, 6.15; N, 8.55%.
diethyl ((1,3-diphenyl-1H-pyrazol-4-yl)((4-fluorophenyl)amino)methyl)phosphonate (4c)
1
White crystal, yield 75%; mp 166-167°C; H NMR(300 MHz, CDCl3, δ ppm): 1.08 (t,
J=7.2Hz, 3H), 1.19 (t, J=7.2Hz, 3H), 3.82-4.17 (m, 4H), 4.80 (d, J=21Hz, 1H), 6.38 (q, 2H),
6.69 (q, 2H), 7.19-7.68 (m, 11H), 8.24 (d, J=2.04Hz,1H). MS (ESI): 480.8 (C26H27FN3O3P,
[M+H]+). Anal. Calcd for C26H27FN3O3P: C, 65.25; H, 5.72; N, 8.68; Found: C, 65.13; H,
5.68; N, 8.76%.
diethyl (((4-chlorophenyl)amino)(1,3-diphenyl-1H-pyrazol-4-yl)methyl)phosphonate (4d)
White crystal, yield 83%; mp 188-190°C; 1H NMR(300 MHz, CDCl3, δ ppm): 1.08 (t,
J=7.2Hz, 3H), 1.19 (t, J=7.2Hz, 3H), 3.80-4.17 (m, 4H), 4.81 (d, J=21Hz, 1H), 6.35 (d,
J=8.79Hz, 2H), 6.94 (d, J=6.78Hz, 2H), 7.19-7.67 (m, 11H), 8.24 (d, J=2.04Hz,1H). MS
(ESI): 497.3 (C26H27ClN3O3P, [M+H]+). Anal. Calcd for C26H27ClN3O3: C, 63.12; H, 5.47; N,
8.58; Found: C, 62.97; H, 5.49; N, 8.47%.
diethyl ((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)(p-tolylamino)methyl)phosphonate (4e)
1
White crystal, yield 89%; mp 172-174°C; H NMR (300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz, 3H), 1.25 (t, J=7.2Hz, 3H), 2.19 (s, 3H), 2.43 (s, 3H), 3.70-4.28 (m, 4H), 4.91 (d,
J=21Hz, 1H), 6.42 (d, J=8.4Hz, 2H), 6.88(d, J=8.4Hz, 2H), 7.24-7.75 (m, 10H), 8.28 (d,
J=2.04Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 152.66, 152.60, 139.80, 138.09, 129.81,
129.61, 129.38, 129.30, 128.47, 127.58, 127.56, 126.40, 118.89, 116.64, 114.41, 63.49 63.34,
47.32 (d, J=159.8 Hz, C-P), 21.34, 20.37, 16.46, 16.26. MS (ESI): 490.5 (C28H32N3O3P,
[M+H]+). Anal. Calcd for C28H32N3O3P: C, 68.78; H, 6.67; N, 8.49; Found: C, 68.70; H,
6.59; N, 8.58%.
diethyl(((4-methoxyphenyl)amino)(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)phosphonate
(4f)
1
White crystal, yield 69%; mp 156-158°C; H NMR(300 MHz, CDCl3, δ ppm): 1.08 (t,
J=7.2Hz, 3H), 1.23 (t, J=7.2Hz，3H), 2.35 (s, 3H), 3.62 (s, 3H), 3.80-4.18 (m, 4H), 4.79 (d,
J=21Hz, 1H), 6.39 (d, J=8.4Hz, 2H), 6.65(d, J=8.4Hz, 2H), 7.19-7.67 (m, 10H), 8.21 (d,
J=2.04Hz, 1H). MS (ESI): 506.6 (C28H32N3O4P, [M+H]+). Anal. Calcd for C28H32N3O4P: C,
66.54; H, 6.27; N, 8.21; Found: C, 66.52; H, 6.38; N, 8.31%.
Chemical and Pharmaceutical Bulletin Advance Publication

diethyl(((4-fluorophenyl)amino)(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)phosphonate
(4g)
1
White crystal, yield 87%; mp 176-178°C; H NMR(300 MHz, DMSO-d6, δ ppm): 1.18 (t,
J=7.2Hz, 3H), 1.28 (t, J=7.2Hz, 3H), 2.43 (s, 3H), 3.80-4.18 (m, 4H), 4.85 (d, J=21Hz, 1H),
6.45 (d, J=8.4Hz, 2H), 6.78(d, J=8.4Hz, 2H), 7.00-7.75 (m, 10H), 8.28 (d, J=2.04Hz, 1H).
MS (ESI): 494.3 (C27H29FN3O3P, [M+H]+). Anal. Calcd for C27H29FN3O3P: C, 65.75; H,
6.02; N, 8.45; Found: C, 65.71; H, 5.92; N, 8.51%.
diethyl(((4-chlorophenyl)amino)(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)phosphonate
(4h)
1
White crystal, yield 85%; mp 194-196°C; H NMR(300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz, 3H), 1.25 (t, J=7.2Hz, 3H), 2.44 (s, 3H), 3.70-4.20 (m, 4H), 4.89 (d, J=21Hz, 1H),
6.45 (d, J=8.4Hz, 2H), 7.02(d, J=8.4Hz, 2H), 7.30-7.75 (m, 10H), 8.31 (d, J=2.04Hz, 1H).
MS (ESI): 510.4 (C27H29ClN3O3P, [M+H]+). Anal. Calcd for C27H29ClN3O3P: C, 63.57; H,
5.82; N, 8.15; Found: C, 63.59; H, 5.73; N, 8.24%.
diethyl((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)(p-tolylamino)methyl)phosphonate(4i)
1
White crystal, yield 83%; mp 182-184°C; H NMR (300 MHz, CDCl3, δ ppm): 1.08 (t,
J=7.2Hz, 3H), 1.21 (t, J=7.2Hz, 3H), 2.11 (s, 3H), 3.84-4.15 (m, 4H), 4.79 (d, J=21Hz, 1H),
6.33 (d, J=8.4Hz, 2H), 6.81 (d, J=8.4Hz, 2H), 7.19-7.66 (m, 10H), 8.21 (d, J=2.04Hz,1H).
MS (ESI): 510.9 (C27H29ClN3O3P, [M+H]+). Anal. Calcd for C27H29ClN3O3P: C, 63.51; H,
5.66; N, 8.29; Found: C, 63.59; H, 5.73; N, 8.24%.
diethyl((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
methoxyphenyl)amino)methyl)phosphonate (4j)
1
White crystal, yield 87%; mp 146-148°C; H NMR(300 MHz, CDCl3, δ ppm): 1.15 (t,
J=7.2Hz，3H), 1.28 (t, J=7.2Hz，3H), 3.78 (s，3H), 3.86-4.25 (m, 4H), 4.80 (d, J=21Hz,
1H), 6.46 (d, J=8.4Hz, 2H), 6.68 (d, J=8.4Hz, 2H), 7.16-7.63 (m, 10H), 8.30 (d,
J=2.04Hz,1H). MS (ESI): 527.2 (C27H29ClN3O4P, [M+H]+). Anal. Calcd for C27H29ClN3O4P:
C, 61.72; H, 5.67; N, 8.05; Found: C, 61.66; H, 5.56; N, 7.99%.
diethyl((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
fluorophenyl)amino)methyl)phosphonate (4k)
1
White crystal, yield 78%; mp 164-166°C; H NMR(300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz, 3H), 1.37 (t, J=7.2Hz, 3H), 3.90-4.25 (m, 4H), 4.80 (d, J=21Hz, 1H), 6.43 (d,
J=8.4Hz, 2H), 6.78 (d, J=8.4Hz, 2H), 7.29-7.73 (m, 10H), 8.30 (d, J=2.04Hz,1H). MS (ESI):
Chemical and Pharmaceutical Bulletin Advance Publication

514.7 (C26H26ClFN3O3P, [M+H]+). Anal. Calcd for C26H26ClFN3O3P: C, 60.87; H, 5.17; N,
8.25; Found: C, 60.76; H, 5.10; N, 8.18%.
diethyl((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
chlorophenyl)amino)methyl)phosphonate (4l)
1
White crystal, yield 80%; mp 163-165℃; H NMR(300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz, 3H), 1.37 (t, J=7.2Hz, 3H), 3.94-4.18 (m, 4H), 4.82 (d, J=21Hz, 1H), 6.44 (d,
J=8.4Hz, 2H), 7.03 (d, J=8.4Hz, 2H), 7.29-7.73 (m, 10H), 8.30 (d, J=2.04Hz, 1H). MS (ESI):
531.4 (C26H26Cl2N3O3P, [M+H]+). Anal. Calcd for C26H26Cl2N3O3P: C, 58.97; H, 5.01; N,
7.85; Found: C, 58.88; H, 4.94; N, 7.92%.
diethyl((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)(p-tolylamino)methyl)phosphonate
(4m)
1
White crystal, yield 85%; mp 181-183℃; H NMR (300 MHz, CDCl3, δ ppm): 1.15 (t,
J=7.2Hz, 3H), 1.28(t, J=7.2Hz, 3H), 2.31 (s, 3H), 3.88 (s, 3H), 4.02-4.21 (m, 4H), 4.89 (d,
J=21Hz, 1H), 6.41 (d, J=8.4Hz, 2H), 6.87 (d, J=8.4Hz, 2H), 7.28-7.73 (m, 10H), 8.26 (d,
J=2.04Hz, 1H). MS (ESI): 506.9 (C28H32N3O4P, [M+H]+). Anal. Calcd for C28H32N3O4P: C,
66.43; H, 6.45; N, 8.29; Found: C, 66.52; H, 6.38; N, 8.31%.
diethyl((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
methoxyphenyl)amino)methyl)phosphonate (4n)
1
White crystal, yield 86%; mp 148-150℃; H NMR(300 MHz, CDCl3, δ ppm): 1.15 (t,
J=7.2Hz, 3H), 1.26(t, J=7.2Hz, 3H), 3.68 (s, 3H), 3.86 (s, 3H), 4.02-4.21 (m, 4H), 4.83 (d,
J=21Hz, 1H), 6.45 (d, J=8.4Hz, 2H), 6.64(d, J=8.4Hz, 2H), 6.97-7.73 (m, 10H), 8.26 (d,
J=2.04Hz, 1H). MS (ESI): 522.6 (C28H32N3O5P, [M+H]+). Anal. Calcd for C28H32N3O5P: C,
64.54; H, 6.21; N, 8.12; Found: C, 64.48; H, 6.18; N, 8.06%.
diethyl(((4-fluorophenyl)amino)(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)phosphonate (4o)
1
White crystal, yield 78%; mp 180-181℃; H NMR(300 MHz, CDCl3, δ ppm): 1.15 (t,
J=7.2Hz, 3H), 1.30(t, J=7.2Hz, 3H), 3.87(s, 3H), 3.91-4.11 (m, 4H), 4.83 (d, J=21Hz, 1H),
6.46 (d, J=8.4Hz, 2H), 6.76(d, J=8.4Hz, 2H), 6.97-7.74 (m, 10H), 8.29 (d, J=2.04Hz, 1H).
MS (ESI): 510.6 (C27H29FN3O4P, [M+H]+). Anal. Calcd for C27H29FN3O4P: C, 63.74; H,
5.82; N, 8.21; Found: C, 63.65; H, 5.74; N, 8.25%.
diethyl(((4-chlorophenyl)amino)(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)phosphonate (4p)
Chemical and Pharmaceutical Bulletin Advance Publication

1
White crystal, yield 81%; mp 186-188℃; H NMR(300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz, 3H), 1.28(t, J=7.2Hz, 3H), 3.87(s, 3H), 3.91-4.21 (m, 4H), 4.85 (d, J=21Hz, 1H),
6.43 (d, J=8.4Hz, 2H), 6.99(d, J=8.4Hz, 2H), 7.27-7.74 (m, 10H), 8.28 (d, J=2.04Hz, 1H).
MS (ESI): 527.1(C27H29ClN3O4P, [M+H]+). Anal. Calcd for C27H29ClN3O4P: C, 61.57; H,
5.62; N, 8.02; Found: C, 61.66; H, 5.56; N, 7.99%.
diethyl((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)(p-tolylamino)methyl)phosphonate
(4q)
1
White crystal, yield 79%; mp172-174°C; H NMR (300 MHz, CDCl3, δ ppm): 1.17 (t,
J=7.2Hz, 3H), 1.30 (t, J=7.2Hz, 3H), 2.20 (s, 3H), 3.88-4.28 (m, 4H), 4.85 (d, J=21Hz, 1H),
6.43 (d, J=8.4Hz, 2H), 6.89 (d, J=8.4Hz, 2H), 7.14-7.78(m, 10H), 8.31 (d, J=2.04Hz,1H).
MS (ESI): 510.9 (C27H29FN3O3P, [M+H]+). Anal. Calcd for C27H29FN3O3P: C, 65.71; H,
5.92; N, 8.51; Found: C, 65.84; H, 5.74; N, 8.65%.
diethyl((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
methoxyphenyl)amino)methyl)phosphonate (4r)
White crystal, yield 84%; mp 155-157°C; 1H NMR(300 MHz, CDCl3, δ ppm): 1.16 (t,
J=7.2Hz，3H), 1.30 (t, J=7.2Hz，3H), 3.68 (s，3H), 3.84-4.23 (m, 4H), 4.84 (d, J=21Hz,
1H), 6.46 (d, J=8.4Hz, 2H), 6.78 (d, J=8.4Hz, 2H), 7.17-7.74 (m, 10H), 8.31 (d,
J=2.04Hz,1H). MS (ESI): 527.2 (C27H29FN3O4P, [M+H]+). Anal. Calcd for C27H29FN3O4P: C,
63.65; H, 5.74; N, 8.25; Found: C, 63.74; H, 5.68; N, 8.14%.
diethyl((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)((4-
fluorophenyl)amino)methyl)phosphonate(4s)
1
White crystal, yield 74%; mp 143-145°C; H NMR(300 MHz, CDCl3, δ ppm): 1.14(t,
J=7.2Hz, 3H), 1.36 (t, J=7.2Hz, 3H), 3.94-4.23 (m, 4H), 4.84(d, J=21Hz, 1H), 6.45 (d,
J=8.4Hz, 2H), 6.73 (d, J=8.4Hz, 2H), 7.24-7.71(m, 10H), 8.31 (d, J=2.04Hz,1H). MS (ESI):
514.7 (C26H26F2N3O3P, [M+H]+). Anal. Calcd for C26H26F2N3O3P: C, 62.77; H, 5.27; N, 8.45;
Found: C, 62.85; H, 5.19; N, 8.37%.
diethyl(((4-chlorophenyl)amino)(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)phosphonate(4t)
1
White crystal, yield 82%; mp 168-169℃; H NMR(300 MHz, CDCl3, δ ppm): 1.18 (t,
J=7.2Hz, 3H), 1.27 (t, J=7.2Hz, 3H), 3.90-4.25 (m, 4H), 4.83 (d, J=21Hz, 1H), 6.44 (d,
J=8.4Hz, 2H), 7.03 (d, J=8.4Hz, 2H), 7.17-7.71 (m, 10H), 8.31 (d, J=2.04Hz, 1H). MS (ESI):
531.4 (C26H26ClFN3O3P, [M+H]+). Anal. Calcd for C26H26ClFN3O3P: C, 60.76; H, 5.10; N,
8.18; Found: C, 50.81; H, 4.95; N, 7.99%.
Chemical and Pharmaceutical Bulletin Advance Publication

Insecticidal activity The larvicidal activity of the title compounds and contrast compound
fipronil and chlorantraniliprole against P. xylostella was tested by the leaf-dip method using
the reported procedure. The bioassay was replicated at 25 ± 1℃ according to statistical
requirements. Assessments were made on a dead/alive basis, and mortality rates were
corrected applying Abbott’s formula.
Fresh cabbage discs were dipped into the test solutions containing title compounds and
the control reagent of for 10s, after air-drying, and the treated leaf disks were placed
individually in a petri dish lined with filter paper. Thirty larvae of second-instar P. xylostella
were carefully transferred to the dried treated leaf disk. Percentage mortalities were evaluated
3 days after treatment. Each treatment was performed three times. The insecticidal activity is
summarized in Table 2.
AchE Inhibitory activity The activities of AChE was measured according to the
instructions of the acetyl cholinesterase assay kits (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China)24. The optical density (OD) of AChE was monitored at 412 nm
using a Thermo Fisher Multiskan GO (Thermo Scientific Inc. USA). Three replicates were
maintained for each treatment and per replicate was performed thrice.
Crystal Structure Determination Crystal structure determination of title compounds was
carried out on a Bruker D8 VENTURE PHOTON equipped with graphitemonochromated
MoKa (l ¼ 0.71073 Å) radiation. The structure was solved by direct methods and refined on
F2 by full-matrix leastsquares methods using SHELXTL25. All non-hydrogen atoms were
refined with anisotropic thermal parameters. All hydrogen atoms with the exception of those
on nitrogen atoms were geometrically fixed and refined using a riding model.
Molecular Docking The crystal structures of DmAChE (PDB code: 1QO9) were retrieved
from the Protein Data Bank. The molecular docking procedure was performed by using
CDOCKER protocol for receptor-ligand interactions section of DS 3.1 (Discovery Studio 3.5,
Accelrys, Inc., San Diego, CA)26.
Conclusions
To sum up, a series of α-aminophosphonate derivatives containing a pyrazole moiety
has been designed and the target compounds 4a-4t have been evaluated for their insecticidal
Chemical and Pharmaceutical Bulletin Advance Publication

activity and AChE inhibitory activity. The biological evaluation showed that some of
compounds exhibit excellent larvicidal activities against P. xylostella. Specifically,
compound 4e shows the significant inhibitory effect for AChE. For the binding model of 4e
an H-bond (2.32 Å and 103.31°) is established between its oxygen atomic and ARG17, and
it also forms a Pi-cationinteraction(4.5Å). In short, the results obtained from this study
suggest that compound 4e may serve as a novel AChE inhibitor used as a new insecticidal
drug.
Acknowledgements This work was supported by National Natural Science Foundation of
China (No.21302002) and Anhui Natural Science Foundation(1408085QB33). This work has
been checked by Dr. Deepak Kumar.
Conflict of Interest The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

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