Design, synthesis, herbicidal activity, in vivo enzyme activity evaluation and molecular docking study of acylthiourea derivatives as novel acetohydroxyacid synthase inhibitor

Article abstract of DOI:10.1016/j.molstruc.2021.130627

In order to develop novel herbicides, a series of novel acylthiourea derivatives containing methylsulfone were synthesized and characterized by melting point, elemental analysis, infrared spectroscopy, mass spectrometry and ¹H NMR spectroscopy. Furthermore, the preliminary herbicidal activity of title compounds was determined against Echinochloa crusgal, Polypogon fugax, Digitaria adscendens, Brassica napus, Amaranthus retroflexus and Abutilon theophrasti. The results of the petri dish experiments showed that a small part of title compounds had a good inhibitory effect on Digitaria adscendens and Brassica napus. The results of the greenhouse herbicidal activity experiments indicated that the compounds 4p, 4 w and 4x exhibited more high herbicidal activity against Brassica napus and Digitaria adscendens than the commercial herbicide bensulfuron-methyl. Moreover, except for compound 4i, all test compounds performed well in crop safety evaluation. Next, acetohydroxyacid synthase (AHAS) enzyme activity experiments were carried out, and the results showed that compound 4x had the highest inhibition rate reaching 89.12%. Finally, in order to screen AHAS inhibitor, molecular docking was performed by using AHAS protein and target ligand molecules 4a-4x. The results of molecular docking were similar to bioassay experiments. Compound 4p, 4w and 4x were considered potential AHAS inhibitors.

Full text of DOI:10.1016/j.molstruc.2021.130627

Journal of Molecular Structure 1241 (2021) 130627
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis, herbicidal activity, in vivo enzyme activity
evaluation and molecular docking study of acylthiourea derivatives as
novel acetohydroxyacid synthase inhibitor
Yun-Peng Wua, Yan Wang a,∗, Jia-Hui Lia, Ran-Hong Lia, Jun Wang a, Sui-Xin Lia,
Xin-Yu Gaob, Ling Dongb, An-Qi Lib
a College of Plant Protection, Jilin Agricultural University, Changchun, Jilin, 130000 China
b College of Resources and Environment, Jilin Agricultural University, Changchun, Jilin, 130000 China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop novel herbicides, a series of novel acylthiourea derivatives containing methylsulfone
were synthesized and characterized by melting point, elemental analysis, infrared spectroscopy, mass
spectrometry and 1H NMR spectroscopy. Furthermore, the preliminary herbicidal activity of title com-
pounds was determined against Echinochloa crusgal, Polypogon fugax, Digitaria adscendens, Brassica napus,
Amaranthus retroflexus and Abutilon theophrasti. The results of the petri dish experiments showed that a
small part of title compounds had a good inhibitory effect on Digitaria adscendens and Brassica napus.
The results of the greenhouse herbicidal activity experiments indicated that the compounds 4p, 4 w and
4x exhibited more high herbicidal activity against Brassica napus and Digitaria adscendens than the com-
mercial herbicide bensulfuron-methyl. Moreover, except for compound 4i, all test compounds performed
well in crop safety evaluation. Next, acetohydroxyacid synthase (AHAS) enzyme activity experiments were
carried out, and the results showed that compound 4x had the highest inhibition rate reaching 89.12%.
Finally, in order to screen AHAS inhibitor, molecular docking was performed by using AHAS protein and
target ligand molecules 4a-4x. The results of molecular docking were similar to bioassay experiments.
Compound 4p, 4w and 4x were considered potential AHAS inhibitors.
Received 5 February 2021
Revised 22 April 2021
Accepted 3 May 2021
Available online 9 May 2021
Keywords:
Acetohydroxyacid synthase inhibitor
Acylthiourea derivatives
Synthesis
Herbicidal activity
In vivo enzyme activity
Molecular docking
© 2021 Published by Elsevier B.V.
1. Introduction
and valine, also called BCAAs), and the root cause is AHAS enzyme
did not complete the conversion neither nor 2-ketobutyrate and
Food shortage is one reason why the number of hungry peo-
ple in the world is growing [1]. Therefore employ pesticides to in-
crease grain production is important method to tackle hunger [2].
Pesticides include a wide range of herbicides, insecticides, fungi-
cides, etc. Among them, the herbicide use in the first place ac-
counted for 47.5% of pesticide use [3]. In order to meet the needs
of the agricultural markets, the development of novel herbicides is
very critical.
pyruvate [5]. At present, five AHAS-inhibiting herbicides have been
commercialized (Fig. 1), including sulfonylurea (SU), imidazolinone
(IMI), triazolopyrimidine (TP), pyrimidinylth-thiobenzoates (PTBs)
and sulfonylamino-carbonyl-triazolinones (SCTs) [6]. Due to the
intensive utilization of AHAS inhibitor herbicides, 159 species of
weeds worldwide have generated resistance to these kinds of her-
bicides [7].
The thiourea structure is an important bioactive substructure
in various biomolecules. To the best of our knowledge, thiourea
derivatives are widely used in the agricultural and pharmaceutical
industries with herbicidal [8], insecticidal [9], anti-haemolytic [10],
antithyroid [11], antibacterial [12], antifungal [13] and antioxidant
[14,15]. Also, the thiourea derivatives can be considered as build-
ing blocks in the synthesis of low molecular weight compounds
[16]. The herbicidal properties of sulfonylureas (SUs) were first re-
ported in 1966, and after a decade of exploration, one of the largest
herbicide research programs in the history was began [17]. The
SUs are now the second most familiar kind of herbicides after the
Acetohydroxyacid synthase (AHAS), also known as acetolactate
synthase (ALS), is the target of numerous commercial herbicides
(apply to rice, corn, wheat, and cotton crops). The pesticides as
AHAS inhibitor have three features that low application rates, high
crop selectivity and low toxicity in animals [4]. In plants, AHAS in-
hibitor has an indirect effect on protein synthesis by suppressed
the generation of branched-chain amino acids (isoleucine, leucine
∗
Corresponding author.
E-mail address: wy@jlau.edu.cn (Y. Wang).
https://doi.org/10.1016/j.molstruc.2021.130627
0022-2860/© 2021 Published by Elsevier B.V.

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Fig. 1. Representatives of the five types of AHAS-inhibiting herbicides.
glyphosates, which has been acknowledged as a milestone in her-
bicidal chemistry [18].
silane (TMS) as internal standard. Elemental analysis was per-
formed with an Elemental Vario EL III apparatus (Perkin Elmer,
Waltham, MA, USA). Mass spectra were recorded on an Agilent
1260–6460 high performance liquid chromatography-mass spec-
trometry (Agilent, Santa Clara, California, USA). Enzyme-labeled in-
strument Rayto RT-6100 (Rayto Life and Analytical Sciences Co.,
Ltd., Shenzhen, China). The course of the reactions was monitored
by TLC; analytical TLC was performed on silica gel GF254; column
chromatographic purification was carried out using silica gel.
Highly effective herbicidal activity of SUs is inseparable from
its structure, and urea bridge is one of the active components of
SUs. In this paper, in order to increase the molecular activity, we
modified the urea bridge to a thiourea bridge. Next, we combined
the active structure of the HPPD inhibitor herbicides with the
thiourea bridge, and introduced benzene rings containing differ-
ent substituent groups. The design strategy of compounds shown
in Fig. 2. Finally, the synthesized target compounds (acylthiourea
derivatives) were characterized, and further bioassay and docking
experiments were performed.
2.2. Experimental
2.2.1. General procedure for the synthesis of acylthiourea derivatives
The synthetic route of acylthiourea was in tune with previ-
ous work [19]. The acylthiourea derivatives were synthesized as
shown in Scheme 1. In this paper, the target compounds were
synthesized from 2-chloro-4-methylsulphonyl benzoic acid (CMBA)
or 4-methylsulphonyl benzoic acid (MBA) as starting materials via
three steps. Acylthiourea was prepared by the reaction of 1 (CMBA
or MBA) with SOCl2 to form the intermediate 2-substituted-4-
methylsulfonyl benzoyl chloride (2). Under the condition of PEG-
400 catalysis, benzoyl chloride were treated with an equimolar
quantity of potassium thiocyanate in dichloromethane to afford the
corresponding acylisothiocyanate intermediates (3) which were fil-
tered (retained filtrate). The corresponding aniline was added and
kept on reacting at room temperature. The resulting mixture was
filtered, dried, and recrystallized from DMF/EtOH/H2O. A few of ti-
tle compounds were purified by column chromatography on silica
2. Materials and methods
2.1. General
All reactions were performed with magnetic stirring. Unless
otherwise stated, all reagents were purchased from commercial
suppliers and used without further purification. Melting points
(m.p.) were determined on an X-6 digital display microscope melt-
ing point apparatus (Tech. Instrument Co., Ltd., Beijing, China). In-
frared (IR) spectra were acquired on an FTIR-8400S infrared spec-
trometer (SHIMADZU, Kyoto, Japan) with KBr compression method.
1H NMR spectra were recorded on an Avance-400 spectrome-
ter (Bruker, Bremen, Germany) and an Avance-300 spectrometer
(Bruker, Bremen, Germany) using DMSO-d6 as the solvent, with
chemical shift (δ) values reported in ppm relative to tetramethyl-
2

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Fig. 2. Design approach for novel target molecules.
Scheme 1. The synthetic route of title compounds.
3

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
gel using dichloromethane/hexane (2: 1) as eluent. All the struc-
tures of newly synthesized compounds were elucidated on the ba-
sis of their elemental analysis, mass spectrometry and spectro-
scopic data.
(20 mL), 4e was isolated as white solid, yield 82%, m.p: 229–
231°C. FT-IR (KBr) cm−1: 3211, 3161 (N-H), 3029 (=C-HAr), 1683
(C=O), 1525 (C=CAr), 1247 (C=S), 1155 (C-N). 1H NMR (400 MHz,
DMSO-d6): δ, ppm 12.46 (s, 1H), 12.06 (s, 1H), 8.14 (d, J = 1.5 Hz,
1H), 8.04–7.91 (m, 4H), 7.73 (d, J = 8.6 Hz, 1H), 3.36 (s, 3H). Anal.
Calcd. for C16 H11 BrClF3N2O3S2 (MW 515.74): C, 37.26; H, 2.15; N,
5.43. Found: C, 37.24; H, 2.41; N, 5.31. LC-MS (ESI) m/z: 514.90
[M+H]+.
2.2.2. Characterization of acylthiourea derivatives(4a-4x)
2.2.2.1. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-fluoro-5-
trifluoromethylphenyl) thiourea (4a). Starting with 2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-fluoro-5-
trifluoromethylaniline (1.79 g, 10 mmol) and dichloromethane
(20 mL), 4a was isolated as white solid, yield 78%, m.p: 216–
218°C. FT-IR (KBr) cm−1: 3367, 3222 (N-H), 3012 (=C-HAr), 1699
(C=O), 1521 (C=CAr), 1253 (C=S), 1166 (C-N). 1H NMR (400 MHz,
DMSO-d6): δ, ppm 12.45 (s, 1H), 12.17 (s, 1H), 8.43 (d, J = 6.6 Hz,
1H), 8.14 (s, 1H), 8.05–7.92 (m, 2H), 7.83–7.73 (m, 1H), 7.61 (t,
J = 9.3 Hz, 1H), 3.36 (s, 3H). Anal. Calcd. for C16 H11 ClF4N2O3S2
(MW 454.84): C, 42.25; H, 2.44; N, 6.16. Found: C, 42.06; H, 2.32;
N, 6.38. LC-MS (ESI) m/z: 454.98 [M+H]+.
2.2.2.6. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-bromo-5-
fluorophenyl)
thiourea
(4f). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-bromo-5-
fluoroaniline (1.90 g, 10 mmol) and dichloromethane (20 mL),
4f was isolated as yellow solid, yield 83%, m.p: 236–237°C. FT-IR
(KBr) cm−1: 3174, 3068 (N-H), 3020 (=C-HAr), 1693 (C=O), 1531
(C=CAr), 1240 (C=S), 1155 (C-N). 1H NMR (400 MHz, DMSO-d6): δ,
ppm 12.42 (s, 1H), 12.23 (s, 1H), 8.14 (s, 1H), 8.04–7.92 (m, 3H),
7.81 (dd, J = 9.1, 5.7 Hz, 1H), 7.20 (td, J = 8.4, 2.9 Hz, 1H), 3.36 (s,
3H). Anal. Calcd. for C15H11 BrClFN2O3S2 (MW 465.74): C, 38.68; H,
2.38; N, 6.01. Found: C, 38.30 H, 2.49; N, 5.87. LC-MS (ESI) m/z:
464.91 [M+H]+.
2.2.2.2. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(3-trifluoromethyl-
4-fluorophenyl)
thiourea
(4b). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 4-fluoro-3-
trifluoromethylaniline (1.79 g, 10 mmol) and dichloromethane
(20 mL), 4b was isolated as white solid, yield 76%, m.p: 219–
221°C. FT-IR (KBr) cm−1: 3218, 3178 (N-H), 3033 (=C-HAr), 1701
(C=O), 1533 (C=CAr), 1251 (C=S), 1151 (C-N). 1H NMR (300 MHz,
DMSO-d6): δ, ppm 12.28 (s, 1H), 12.12 (s, 1H), 8.15–8.12 (m, 2H),
8.04–7.91 (m, 3H), 7.62–7.56 (m, 1H), 3.36 (s, 3H). Anal. Calcd. for
C16 H11 ClF4N2O3S2 (MW 454.84): C, 42.25; H, 2.44; N, 6.16. Found:
C, 42.01; H, 2.51; N, 6.00. LC-MS (ESI) m/z: 454.98 [M+H]+.
2.2.2.7. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-bromo-4,6-
difluorophenyl)
thiourea
(4g). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-bromo-4,6-
difluoroaniline (2.08 g, 10 mmol) and dichloromethane (20 mL),
4g was isolated as white solid, yield 79%, m.p: 220–222°C. FT-IR
(KBr) cm−1: 3207, 3188 (N-H), 3089 (=C-HAr), 1697 (C=O), 1521
(C=CAr), 1244 (C=S), 1155 (C-N). 1H NMR (300 MHz, DMSO-d6): δ,
ppm 12.42 (s, 1H), 11.50 (s, 1H), 8.15 (d, J = 1.5 Hz, 1H), 8.05–7.94
(m, 2H), 7.65 (ddd, J = 8.3, 2.8, 1.7 Hz, 1H), 7.52 (td, J = 9.3,
2.8 Hz, 1H), 3.37 (s, 3H). Anal. Calcd. for C15H10BrClF2N2O3S2 (MW
483.73): C, 37.25; H, 2.08; N, 5.79. Found: C, 37.07; H, 2.41; N,
5.45. LC-MS (ESI) m/z: 482.90 [M+H]+.
2.2.2.3. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-chloro-5-
trifluoromethylphenyl) thiourea (4c). Starting with 2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-Amino-5-
chlorobenzotrifluoride (1.96 g, 10 mmol) and dichloromethane
(20 mL), 4c was isolated as yellowish solid, yield 78%, m.p: 215–
217°C. FT-IR (KBr) cm−1: 3245, 3174 (N-H), 3076 (=C-HAr), 1691
(C=O), 1533 (C=CAr), 1247 (C=S), 1159 (C-N). 1H NMR (300 MHz,
DMSO-d6): δ, ppm 12.47 (s, 1H), 12.31 (s, 1H), 8.44 (d, J = 2.2 Hz,
1H), 8.13 (d, J = 1.6 Hz, 1H), 8.05–7.92 (m, 2H), 7.86 (d, J = 8.5 Hz,
1H), 7.72 (dd, J = 8.5, 2.2 Hz, 1H), 3.35 (s, 3H). Anal. Calcd. for
C16 H11 Cl2F3N2O3S2 (MW 471.29): C, 40.78; H, 2.35; N, 5.94. Found:
C, 40.51; H, 2.22; N, 5.76. LC-MS (ESI) m/z: 470.95 [M+H]+.
2.2.2.8. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(4-bromo-2,6-
difluorophenyl)
thiourea
(4h). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 4-bromo-2,6-
difluoroaniline (2.08 g, 10 mmol) and dichloromethane (20 mL),
4h was isolated as white solid, yield 72%, m.p: 241–243°C. FT-IR
(KBr) cm−1: 3213, 3174 (N-H), 3076 (=C-HAr), 1703 (C=O), 1529
(C=CAr), 1247 (C=S), 1155 (C-N). 1H NMR (300 MHz, DMSO-d6):
δ, ppm 12.45 (s, 1H), 11.37 (s, 1H), 8.12 (d, J = 1.1 Hz, 1H), 7.99
(d, J = 1.1 Hz, 2H), 7.64–7.58 (m, 2H), 3.35 (s, 3H). Anal. Calcd.
for C15H10BrClF2N2O3S2 (MW 483.73): C, 37.25; H, 2.08; N, 5.79.
Found: C, 37.34; H, 2.21; N, 5.40. LC-MS (ESI) m/z: 482.90 [M+H]+.
2.2.2.4. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(3-trifluoromethyl-
4-chlorophenyl)
thiourea
(4d). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 5-amino-2-
chlorobenzotrifluoride (1.96 g, 10 mmol) and dichloromethane
(20 mL), 4d was isolated as white solid, yield 74%, m.p: 227–
229°C. FT-IR (KBr) cm−1: 3269, 3217 (N-H), 3066 (=C-HAr), 1697
(C=O), 1527 (C=CAr), 1274 (C=S), 1157 (C-N). 1H NMR (400 MHz,
DMSO-d6): δ, ppm 12.30 (s, 1H), 12.20 (s, 1H), 8.27 (d, J = 2.4 Hz,
1H), 8.13 (d, J = 1.9 Hz, 1H), 8.05–7.89 (m, 3H), 7.78 (d, J = 8.7 Hz,
1H), 3.35 (s, 3H). Anal. Calcd. for C16 H11 Cl2F3N2O3S2 (MW 471.29):
C, 40.78; H, 2.35; N, 5.94. Found: C, 40.49; H, 2.50; N, 6.21. LC-MS
(ESI) m/z: 470.95 [M+H]+.
2.2.2.9. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-iodophenyl)
thiourea (4i). Starting with 2-chloro-4-methylsulphonyl ben-
zoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL),
KSCN (0.97 g, 10 mmol), 2-iodoaniline (2.19 g, 10 mmol) and
dichloromethane (20 mL), 4i was isolated as yellowish solid, yield
85%, m.p: 205–207°C. FT-IR (KBr) cm−1: 3195, 3170 (N-H), 3058
(=C-HAr), 1689 (C=O), 1529 (C=CAr), 1242 (C=S), 1153 (C-N). 1H
NMR (300 MHz, DMSO-d6): δ, ppm 12.30 (s, 1H), 11.97 (s, 1H),
8.14 (d, J = 1.6 Hz, 1H), 8.04–7.91 (m, 3H), 7.67 (dd, J = 8.0, 1.6 Hz,
1H), 7.47 (td, J = 7.6, 1.4 Hz, 1H), 7.11 (td, J = 7.6, 1.6 Hz, 1H), 3.36
(s, 3H). Anal. Calcd. for C15H12ClIN2O3S2 (MW 494.75): C, 36.42;
H, 2.44; N, 5.66. Found: C, 36.42; H, 2.38; N, 5.67. LC-MS (ESI)
m/z: 494.90 [M+H]+.
2.2.2.5. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-trifluoromethyl-
4-bromophenyl)
thiourea
(4e). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-Amino-5-
bromobenzotrifluoride (2.40 g, 10 mmol) and dichloromethane
4

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
2.2.2.10. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(4-iodophenyl)
thiourea (4j). Starting with 2-chloro-4-methylsulphonyl ben-
zoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL),
KSCN (0.97 g, 10 mmol), 4-iodoaniline (2.19 g, 10 mmol) and
dichloromethane (20 mL), 4j was isolated as white solid, yield
83%, m.p: 227–229°C. FT-IR (KBr) cm−1: 3228, 3180 (N-H), 3060
(=C-HAr), 1691 (C=O), 1535 (C=CAr), 1249 (C=S), 1157 (C-N). 1H
NMR (300 MHz, DMSO-d6): δ, ppm 12.18 (s, 1H), 12.14 (s, 1H),
8.12 (d, J = 1.6 Hz, 1H), 8.00 (dd, J = 8.0, 1.7 Hz, 1H), 7.92 (d,
J = 8.0 Hz, 1H), 7.81–7.75 (m, 2H), 7.56–7.50 (m, 2H), 3.35 (s, 3H).
Anal. Calcd. for C15H12ClIN2O3S2 (MW 494.75): C, 36.42; H, 2.44;
N, 5.66. Found: C, 36.25; H, 2.36; N, 5.45. LC-MS (ESI) m/z: 494.90
[M+H]+.
2.2.2.15. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-methyl-
4-fluorophenyl)
thiourea
(4o). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 4-fluoro-2-
methylaniline (1.25 g, 10 mmol) and dichloromethane (20 mL),
4o was isolated as white solid, yield 86%, m.p: 237–239°C. FT-IR
(KBr) cm−1: 3251, 3172 (N-H), 3029 (=C-HAr), 1685 (C=O), 1539
(C=CAr), 1216 (C=S), 1151 (C-N). 1H NMR (400 MHz, DMSO-d6): δ,
ppm 12.18 (s, 1H), 11.75 (s, 1H), 8.13 (s, 1H), 8.04–7.92 (m, 2H),
7.50 (dd, J = 8.8, 5.5 Hz, 1H), 7.19 (dd, J = 9.5, 2.9 Hz, 1H), 7.09
(td, J = 8.5, 2.8 Hz, 1H), 3.36 (s, 3H), 2.27 (s, 3H). Anal. Calcd. for
C16 H14 ClFN2O3S2 (MW 400.87): C, 47.94; H, 3.52; N, 6.99. Found:
C, 47.95; H, 3.59; N, 7.08. LC-MS (ESI) m/z: 401.01 [M+H]+.
2.2.2.11. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2,4,5-
2.2.2.16. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-nitro-4-
trifluorophenyl)
thiourea
(4k). Starting
with
2-chloro-4-
fluorophenyl)
thiourea
(4p). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2,4,5-trifluoroaniline
(1.47 g, 10 mmol) and dichloromethane (20 mL), 4k was isolated
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 4-fluoro-2-nitroaniline
(1.56 g, 10 mmol) and dichloromethane (20 mL), 4p was isolated
as yellow solid, yield 72%, m.p: 211–212°C. FT-IR (KBr) cm−1: 3234,
3184 (N-H), 3028 (=C-HAr), 1681 (C=O), 1537 (C=CAr), 1201 (C=S),
1153 (C-N). 1H NMR (400 MHz, DMSO-d6): δ, ppm 12.40 (s, 1H),
12.26 (s, 1H), 8.14 (s, 1H), 8.08 (dd, J = 8.5, 2.9 Hz, 1H), 8.01 (d,
J = 8.1 Hz, 1H), 7.98–7.87 (m, 2H), 7.72 (td, J = 8.3, 2.9 Hz, 1H),
3.36 (s, 3H). Anal. Calcd. for C15H11 ClFN3O5S2 (MW 431.84): C,
41.72; H, 2.57; N, 9.73. Found: C, 41.91; H, 2.67; N, 9.57. LC-MS
(ESI) m/z: 431.98 [M+H]+.
as white solid, yield 77%, m.p: 210–212°C. FT-IR (KBr) cm−1
:
3244, 3180 (N-H), 3033 (=C-HAr), 1687 (C=O), 1535 (C=CAr),
1218 (C=S), 1153 (C-N). 1H NMR (400 MHz, DMSO-d6): δ, ppm
12.41 (s, 1H), 11.98 (s, 1H), 8.15–8.04 (m, 2H), 8.04–7.92 (m,
2H), 7.75 (td, J = 10.5, 7.4 Hz, 1H), 3.35 (s, 3H). Anal. Calcd. for
C15H10ClF3N2O3S2 (MW 422.82): C, 42.61; H, 2.38; N, 6.63. Found:
C, 42.77; H, 2.23; N, 6.60. LC-MS (ESI) m/z: 422.98 [M+H]+.
2.2.2.12. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2,3,4-
trifluorophenyl)
thiourea
(4l). Starting
with
2-chloro-4-
2.2.2.17. N-(4-methylsulfonylbenzoyl)-N’-(2-chloro-5-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2,3,4-trifluoroaniline
(1.47 g, 10 mmol) and dichloromethane (20 mL), 4l was isolated
as white solid, yield 79%, m.p: 237–239°C. FT-IR (KBr) cm−1: 3271,
3199 (N-H), 3074 (=C-HAr), 1695 (C=O), 1529 (C=CAr), 1238 (C=S),
1153 (C-N). 1H NMR (400 MHz, DMSO-d6): δ, ppm 12.40 (s, 1H),
11.85 (s, 1H), 8.13 (d, J = 1.7 Hz, 1H), 8.04–7.93 (m, 2H), 7.62–7.54
(m, 1H), 7.39 (q, J = 10.1, 9.1 Hz, 1H), 3.35 (s, 3H). Anal. Calcd. for
C15H10ClF3N2O3S2 (MW 422.82): C, 42.61; H, 2.38; N, 6.63. Found:
C, 42.69; H, 2.40; N, 6.39. LC-MS (ESI) m/z: 422.98 [M+H]+.
trifluoromethylphenyl)
thiourea
(4q). Starting
with
4-
methylsulphonyl benzoic acid (2.00 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-Amino-5-
chlorobenzotrifluoride (1.96 g, 10 mmol) and dichloromethane
(20 mL), 4q was isolated as white solid, yield 82%, m.p: 195–
197°C. FT-IR (KBr) cm−1: 3242, 3174 (N-H), 3004 (=C-HAr), 1693
(C=O), 1539 (C=CAr), 1245 (C=S), 1161 (C-N). 1H NMR (400 MHz,
DMSO-d6): δ, ppm 12.66 (s, 1H), 12.26 (s, 1H), 8.48 (d, J = 2.0 Hz,
1H), 8.20 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.86 (d,
J = 8.4 Hz, 1H), 7.72 (dd, J = 8.4, 2.1 Hz, 1H), 3.32 (s, 3H). Anal.
Calcd. for C16 H12ClF3N2O3S2 (MW 436.85): C, 43.99; H, 2.77; N,
6.41. Found: C, 43.90; H, 2.76; N, 6.51. LC-MS (ESI) m/z: 436.99
[M+H]+.
2.2.2.13. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(3,4-difluorophenyl)
thiourea (4m). Starting with 2-chloro-4-methylsulphonyl benzoic
acid (2.35 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 3,4-difluoroaniline (1.29 g, 10 mmol) and
dichloromethane (20 mL), 4m was isolated as yellowish solid,
yield 78%, m.p: 224–226°C. FT-IR (KBr) cm−1: 3228, 3109 (N-H),
3055 (=C-HAr), 1691 (C=O), 1521 (C=CAr), 1240 (C=S), 1152 (C-N).
1H NMR (400 MHz, DMSO-d6): δ, ppm 12.23 (s, 1H), 12.11 (s,
1H), 8.12 (d, J = 2.1 Hz), 8.04–7.97 (m, 1H), 7.92 (dq, J = 10.0,
6.6, 4.9 Hz, 2H), 7.57–7.44 (m, 2H), 3.35 (s, 3H). Anal. Calcd. for
C15H11 ClF2N2O3S2 (MW 404.83): C, 44.50; H, 2.74; N, 6.92. Found:
C, 44.19; H, 2.86; N, 6.81. LC-MS (ESI) m/z: 404.99 [M+H]+.
2.2.2.18. N-(4-methylsulfonylbenzoyl)-N’-(2-iodophenyl) thiourea (4r).
Starting with 4-methylsulphonyl benzoic acid (2.00 g, 10 mmol),
SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 2-
iodoaniline (2.19 g, 10 mmol) and dichloromethane (20 mL), 4r
was isolated as yellowish solid, yield 91%, m.p: 218–219°C. FT-IR
(KBr) cm−1: 3195, 3159 (N-H), 3018 (=C-HAr), 1679 (C=O), 1527
(C=CAr), 1234 (C=S), 1151 (C-N). 1H NMR (400 MHz, DMSO-d6): δ,
ppm 12.28 (s, 1H), 12.10 (s, 1H), 8.21 (d, J = 8.1 Hz, 2H), 8.09 (d,
J = 8.3 Hz, 2H), 7.95 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H),
7.47 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 3.33 (s, 3H). Anal.
Calcd. for C15H13IN2O3S2 (MW 460.30): C, 39.14; H, 2.85; N, 6.09.
Found: C, 39.20; H, 2.79; N, 6.18. LC-MS (ESI) m/z: 460.94 [M+H]+.
2.2.2.14. N-(2-chloro-4-methylsulfonylbenzoyl)-N’-(2-fluoro-
4-chlorophenyl)
thiourea
(4n). Starting
with
2-chloro-4-
methylsulphonyl benzoic acid (2.35 g, 10 mmol), SOCl2 (1.79 g,
15 mmol, 1.09 mL), KSCN (0.97 g, 10 mmol), 4-chloro-2-
fluoroaniline (1.46 g, 10 mmol) and dichloromethane (20 mL),
4n was isolated as white solid, yield 76%, m.p: 228–229°C. FT-IR
(KBr) cm−1: 3203, 3168 (N-H), 3072 (=C-HAr), 1695 (C=O), 1504
(C=CAr), 1242 (C=S), 1155 (C-N). 1H NMR (400 MHz, DMSO-d6): δ,
ppm 12.36 (s, 1H), 12.01 (s, 1H), 8.13 (d, J = 1.6 Hz, 1H), 8.04–7.91
(m, 3H), 7.60 (dd, J = 9.9, 2.3 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H),
3.35 (s, 3H). Anal. Calcd. for C15H11 Cl2FN2O3S2 (MW 421.28): C,
42.77; H, 2.63; N, 6.65. Found: C, 42.63; H, 2.55; N, 6.45. LC-MS
(ESI) m/z: 420.96 [M+H]+.
2.2.2.19. N-(4-methylsulfonylbenzoyl)-N’-(2-bromo-5-fluorophenyl)
thiourea (4s). Starting with 4-methylsulphonyl benzoic acid
(2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 2-bromo-5-fluoroaniline (1.90 g, 10 mmol) and
dichloromethane (20 mL), 4s was isolated as yellow solid, yield
91%, m.p: 199–200°C. FT-IR (KBr) cm−1: 3296, 3153 (N-H), 3024
(=C-HAr), 1674 (C=O), 1531 (C=CAr), 1234 (C=S), 1157 (C-N). 1H
NMR (400 MHz, DMSO-d6): δ, ppm 12.56 (s, 1H), 12.21 (s, 1H),
8.20 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.3 Hz, 2H), 8.01–7.93 (m, 1H),
7.80 (dd, J = 9.0, 5.8 Hz, 1H), 7.19 (td, J = 8.4, 2.9 Hz, 1H), 3.33
5

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
(s, 3H). Anal. Calcd. for C15H12BrFN2O3S2 (MW 431.29): C, 41.77;
H, 2.80; N, 6.50. Found: C, 41.67; H, 2.74; N, 6.39. LC-MS (ESI) m/z:
430.95 [M+H]+.
NMR (400 MHz, DMSO-d6): δ, ppm 12.54 (s, 1H), 12.20 (s, 1H),
8.20 (d, J = 8.2 Hz, 2H), 8.13–8.02 (m, 3H), 7.94 (dd, J = 9.1, 5.3 Hz,
1H), 7.73 (td, J = 8.3, 2.9 Hz, 1H), 3.34 (s, 3H). Anal. Calcd. for
C15H12FN3O5S2 (MW 397.40): C, 45.34; H, 3.04; N, 10.57. Found: C,
45.60; H, 3.29; N, 10.46. LC-MS (ESI) m/z: 367.05 [M+H]+.
2.2.2.20. N-(4-methylsulfonylbenzoyl)-N’-(4-bromo-2,6-
difluorophenyl) thiourea (4t). Starting with 4-methylsulphonyl
benzoic acid (2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL),
KSCN (0.97 g, 10 mmol), 4-bromo-2,6-difluoroaniline (2.08 g,
10 mmol) and dichloromethane (20 mL), 4t was isolated as white
solid, yield 88%, m.p: 218–220°C. FT-IR (KBr) cm−1: 3176, 3072
(N-H), 3010 (=C-HAr), 1687 (C=O), 1521 (C=CAr), 1242 (C=S), 1157
(C-N). 1H NMR (400 MHz, DMSO-d6): δ, ppm 12.25 (s, 1H), 11.61
(s, 1H), 8.20 (d, J = 8.2 Hz, 2H), 8.08 (d, J = 8.0 Hz, 2H), 7.62 (d,
J = 8.2 Hz, 2H), 3.32 (s, 3H). Anal. Calcd. for C15H11 BrF2N2O3S2
(MW 449.28): C, 40.10; H, 2.47; N, 6.24. Found: C, 40.22; H, 2.41;
N, 6.44. LC-MS (ESI) m/z: 448.94 [M+H]+.
2.2.3. Herbicidal activity assay
2.2.3.1. Petri dish tests. The preliminary herbicidal activity determi-
nation of 24 compounds against six kinds of plant seeds in petri
dishes. The seeds of monocotyledon such as Echinochloa crusgali
(EC), Polypogon fugax (PF) and Digitaria adscendens (DA), and di-
cotyledon such as Brassica napus (BN), Amaranthus retroflexus (AR)
and Abutilon theophrasti (AT) were collected from experimental plot
of Jilin Agricultural University in 2020. The title compound (10 mg)
was dissolved in DMF (0.25 mL) with the addition of Tween 80
(0.05 mg) and diluted with water to give the tested solutions of
various concentrations (100 and 10 mg/L). The same concentra-
tion of bensulfuron-methyl solution and nicosulfuron solution were
prepared by the same method and dosage for positive control. The
seeds were germinated at 29 1 °C, with 24 h dark cultivation and
relative humidity (80%), and every twenty germinated plant seeds
were placed in a petri dish covered with two pieces of filter paper
and specific tested solution (5 mL). The seeds in the petri dishes
were cultured under the same circumstances for 48 h. Three repe-
titions were completed in each concentration. The results were ex-
pressed as averages of three independent experiments. The stan-
dard deviation ( SD) was calculated based on three repetitions.
The percent rate of weed inhibition was calculated using the for-
mula:
2.2.2.21. N-(4-methylsulfonylbenzoyl)-N’-(2,3,4-trifluorophenyl)
thiourea (4u). Starting with 4-methylsulphonyl benzoic acid
(2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 2,3,4-trifluoroaniline (1.47 g, 10 mmol) and
dichloromethane (20 mL), 4u was isolated as white solid, yield
87%, m.p: 210–212°C. FT-IR (KBr) cm−1: 3168, 3089 (N-H), 3033
(=C-HAr), 1703 (C=O), 1519 (C=CAr), 1234 (C=S), 1147 (C-N).
1H NMR (400 MHz, DMSO-d6): δ, ppm 12.18 (s, 1H), 12.12 (s,
1H), 8.19 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.58 (q,
J = 7.7 Hz, 1H), 7.40 (q, J = 9.2 Hz, 1H), 3.33 (s, 3H). Anal.
Calcd. for C15H11 F3N2O3S2 (MW 388.38): C, 46.39; H, 2.85; N,
7.21. Found: C, 46.38; H, 2.81; N, 7.11. LC-MS (ESI) m/z: 389.02
[M+H]+.
Inhibition rate % = [the blank control cm − the treatment group(cm)]/
( )
(
)
2.2.2.22. N-(4-methylsulfonylbenzoyl)-N’-(3,4-difluorophenyl)
thiourea (4v). Starting with 4-methylsulphonyl benzoic acid
(2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 3,4-difluoroaniline (1.29 g, 10 mmol) and
dichloromethane (20 mL), 4v was isolated as yellowish solid,
yield 91%, m.p: 215–216°C. FT-IR (KBr) cm−1: 3236, 3184 (N-H),
3047 (=C-HAr), 1668 (C=O), 1527 (C=CAr), 1215 (C=S), 1145 (C-
N). 1H NMR (400 MHz, DMSO-d6): δ, ppm 12.37 (s, 1H), 11.98
(s, 1H), 8.17 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.92
(dd, J = 11.7, 8.0 Hz, 1H), 7.55–7.43 (m, 2H), 3.31 (s, 3H). Anal.
Calcd. for C15H12F2N2O3S2 (MW 370.39): C, 48.64; H, 3.27; N,
7.56. Found: C, 48.55; H, 3.23; N, 7.61. LC-MS (ESI) m/z: 371.03
[M+H]+.
the blank control cm .
( )
2.2.3.2. Greenhouse herbicidal activity. Pre-emergence and post-
emergence herbicidal activity of the tested compounds against
Brassica napus and Digitaria adscendens were evaluated in green-
house. The greenhouse herbicidal activity tested was performed in
a pot experiment of sandy loam soil (100 g, 60% sand and 40% clay)
with a diameter of 8 cm. Grow 20 sprouted seeds of each plant to
a depth of 0.6 cm and placed in a greenhouse. All test compounds
dissolved in DMF was added to a 10 ml volumetric flask. Next,
Tween-80 was added as an emulsifying agent and added water to
volume to 10 ml. Then dilute the prepared 100 mg/L mother liquor
with water to the required concentration. For pre-emergence treat-
ments, the prepared solution were sprayed on the surface of soil
after the seeds were cultivated for 24 h. For another treatments,
the seeds of Digitaria adscendens were treated at the one-leaf stage
and the seeds of Brassica napus were treated at the two-leaf stage
used a small hand-held spray bottle with 5 ml drug reagent at a
rate of 1.5 a.i. kg/ha with three replicates. The same concentration
of DMF and Tween 80 solution was used as control. After 20 days,
the control effect was calculated by confirmed the number of live
weeds in the pot. Furtherly, calculate the standard deviation ( SD)
of three replicates. The percent rate of weed inhibition was calcu-
lated using the formula:
2.2.2.23. N-(4-methylsulfonylbenzoyl)-N’-(2-methyl-4-fluorophenyl)
thiourea (4w). Starting with 4-methylsulphonyl benzoic acid
(2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 4-fluoro-2-methylaniline (1.25 g, 10 mmol) and
dichloromethane (20 mL), 4w was isolated as yellowish solid, yield
88%, m.p: 214–215°C. FT-IR (KBr) cm−1: 3249, 3170 (N-H), 3028
(=C-HAr), 1685 (C=O), 1537 (C=CAr), 1215 (C=S), 1153 (C-N). 1H
NMR (400 MHz, DMSO-d6): δ, ppm 12.01 (s, 1H), 11.94 (s, 1H),
8.20 (d, J = 8.2 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.51 (dd, J = 8.8,
5.7 Hz, 1H), 7.19 (dd, J = 9.6, 2.9 Hz, 1H), 7.09 (td, J = 8.6, 2.9 Hz,
1H), 3.32 (s, 3H), 2.27 (s, 3H). Anal. Calcd. for C16 H15FN2O3S2 (MW
366.43): C, 52.45; H, 4.13; N, 7.65. Found: C, 52.26; H, 4.01; N,
7.57. LC-MS (ESI) m/z: 367.05 [M+H]+.
Inhibitionrate % = [the blank control quantity − the treatment group
( )
(
)
(quantity)]/(the blank control quantity .
(
)
2.2.2.24. N-(4-methylsulfonylbenzoyl)-N’-(2-nitro-4-fluorophenyl)
thiourea (4x). Starting with 4-methylsulphonyl benzoic acid
(2.00 g, 10 mmol), SOCl2 (1.79 g, 15 mmol, 1.09 mL), KSCN
(0.97 g, 10 mmol), 4-fluoro-2-nitroaniline (1.56 g, 10 mmol) and
dichloromethane (20 mL), 4x was isolated as yellow solid, yield
89%, m.p: 214–216°C. FT-IR (KBr) cm−1: 3269, 3168 (N-H), 3016
(=C-HAr), 1674 (C=O), 1521 (C=CAr), 1205 (C=S), 1151 (C-N). 1H
2.2.3.3. AHAS activity determination in vivo. The germinated Bras-
sica napus seeds were cultivated first, and then continue to cul-
tured in the dark by the method of above-mentioned petri dish
tests for 48 h. Later, all the tested seeds were fully grinded with
PBS solution, centrifuged at 4000 r/min for 15 min, obtained the
supernatant. Finally, the samples were processed through the Elisa
6

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
3.2. FT-IR
The FT-IR spectra of the respective acylthiourea derivative con-
firmed the synthesis by the appearance of absorption bands for N-
H (secondary amine) in the region of 3367–3068 cm−1, the lower
wave numbers of N-H absorption were attributable to intramolec-
ular hydrogen bonding interactions between the hydrogen atom
of the N-H group and the oxygen atom of the carbonyl group
(Fig. 4), as reported in the literature [21]. The C=O strong absorp-
tion band appeared at a range of 1745–1668 cm−1 in all com-
pounds. The presence of a strong band ranging from 3089 to 3004
cm−1 confirmed the presence of =C-HAr bond. The bands of 1554–
1496 cm−1 were associated with C=CAr of phenyl. Furthermore, the
other significant absorption bands for C=S and C-N appeared rang-
ing from 1274 to 1205 cm−1 and 1166 to 1145 cm−1 respectively.
All of feature absorptions were similar to previously reported [22].
Fig. 3. The reaction mechanism of solid-liquid phase transfer catalysis.
3.3. 1H NMR
kit, and the AHAS enzyme activity was determined by enzyme la-
beled instrument at 450 nm.
The structure of the particular acylthioureas (4a-4x) were fur-
ther confirmed by 1H NMR spectroscopy analyses by the appear-
ance of characteristic signals an extremely down field one proton
singlet in the range of δ = 12.01–12.66 ppm for CONH and an-
other characteristic down field singlet in the range of δ = 11.37–
12.31 ppm for CSNH. The CSNH signal was displayed higher chem-
ical shift because of the downfield effect caused by possible hy-
drogen bonds [23]. And in the δ = 7.09–8.48 ppm range of signal
peaks corresponding to the aromatic protons were observed. The
δ = 3.31–3.37 ppm was recognized as CH of methylsulfone. The
appearance of signal at δ = 2.27 ppm belongs to the methyl pro-
tons of aromatic ring.
2.2.3.4. Crop safety. Conventional dicotyledonous crop soybean
(Glycine max) and gramineous crop wheat (Triticum aestivum)
were planted by the method as above (pre-emergence and post-
emergence herbicidal activity tests) for 20 days. The tested com-
pounds were 4g, 4h, 4i, 4o, 4p, 4r, 4s, 4t, 4v, 4w and 4x, and
the final evaluation for crop safety of 11 title compounds was per-
formed.
2.2.4. Molecular docking preparation
Twenty four ligand molecules were optimized to the mini-
mum energy by MM2 method in the Chem3D 18.0, and con-
verted into mol2 format. Then Gasteiger charges were added to
each atom, torsion angles were determined, and saved PDBQT for-
mat for all ligand molecules. The protein structure file was ob-
tained from RSCB Protein Data Bank (acetylhydroxylate synthase,
PDB ID: 1YHZ), furthermore, removed metal molecules, solvent
and co-crystal ligand, and added polar hydrogen atoms. Both lig-
and molecules and receptor were organized with AutoDock 4.2.6
software, and docking study was carried out using a Lamarckian
genetic algorithm method, here, a total of 50 independent dock-
ing computations were performed. The docking results of ligand
molecules 4a-4x with AHAS protein were analyzed via PyMOL
software.
3.4. Herbicidal activity assay
3.4.1. Petri dish tests
Through the preliminary herbicidal activity test of compound
4a-4x, the compounds with higher activity and the corresponding
species of tested plants were screened out. It was obvious from
the Table 1 that the title compounds had significant inhibitory ef-
fects on Digitaria adscendens and Brassica napus. As an initial eval-
uation, the compounds generally had higher inhibitory activity at
a dosage of 100 mg/L on most tested plant species. For example,
at the concentration of 100 mg/L, compounds 4b, 4d, 4e, 4g, 4l,
4m, 4o, 4p, 4q, 4w and 4x inhibited growth of Digitaria adscendens
by 62–87%, which is higher than bensulfuron-methyl and nicosul-
furon. And compounds 4a, 4b, 4d, 4e, 4f, 4g, 4i, 4o, 4p, 4q, 4s, 4w
and 4x exhibited higher herbicidal activity (71–99%) against Bras-
sica napus. Unfortunately, all of the compounds showed extremely
poor inhibition of Echinochloa crusgalli, Polypogon fugax, Amaran-
thus retroflexus and Abutilon theophrasti, among which compound
4w had a perfect effect on Abutilon theophrasti.
3. Results and discussion
3.1. Chemistry
In present study, we had conducted our reaction using
polyethylene glycol-400 (PEG-400) as solid-liquid phase transfer
catalyst (Fig. 3). The phase transfer catalyst PEG-400 was indis-
pensable for these solid-liquid phase reactions; it can easily react
with KSCN to form a [PEG-400-K+] SCN− complex, which made it
possible for SCN− to readily reacted with acid chloride and led to
the formation of intermediates 3 [20].
3.4.2. Greenhouse herbicidal activity and structure-activity
relationship analyses (SAR)
On the basis of the preliminary petri dish tests, 13 compounds
with significant inhibitory activity against weeds were selected and
Fig. 4. Intramolecular hydrogen bonds of acylthioureas.
7

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Table 1
The preliminary herbicidal activity results of the title compounds.
Inhibition rate (%)h
ECa
DAb
PFc
BNd
ARe
ATf
No.
R1 R2
100 mg/L 10 mg/L 100 mg/L 10 mg/L 100 mg/L 10 mg/L 100 mg/L 10 mg/L
100 mg/L 10 mg/L 100 mg/L 10 mg/L
4a
Cl 2-F,5-CF3
Cl 3- CF3,4-F
27
13
1
1
1
0
1
1
1
11
14
16
22
5
1
2
1
1
55
65
53
79
62
57
77
53
53
50
41
66
63
59
71
87
80
57
55
57
49
53
76
82
61
56
1
1
0
1
1
1
1
2
1
1
0
1
1
0
1
1
1
1
1
1
0
1
2
1
1
1
13
44
18
50
20
16
31
−17
7
1
1
1
1
1
1
2
6
0
0
0
76 0.5
80 1.5
67 1.0
64 1.9
56 1.9
18 1.0
44 1.7
0
6
7
0.4
1.1
0.2
11 1.7
25 1.6
35 0.8
4b
19
23
52
−18
22
0
0
12
1
6
4
1.7
1.1
4c
Cl 2-Cl,5- CF3 23
Cl 3- CF3,4-Cl 52
Cl 2- CF3,4-Br 27
1
2
18
2
9
1.2
−6
1.5 16 2.0
4d
8
1
74 1.0
75 1.2
70 1.0
80 1.3
42 1.6
79 0.9
41 1.3
71 0.7
60 1.6
73 0.8
65 1.3
47 2.1
63 1.6
41 2.2
22 1.6
44 2.0
15 1.1
17 1.2
35 1.5
13 0.1
63 1.1
18 1.8
3 1.6
39 1.9
17 0.8
39 0.2
17 1.7
15 1.8
6
4
1.7
1.4
4e
1
0
15
2
2
1
4f
Cl 2-Br,5-F
34
26
10
3
1
1
1
−10
17
2
0
15 1.5
18 1.2
26 0.9
23 2.5
40 1.6
34 2.1
20 0.2
34 1.2
4g
Cl 2-Br,4-F,6-F 22
1
4h
Cl 2-F,4-Br,6-F
Cl 2-I
3
1
0
1
1
−4
11
3
0
−17
19
−3
1.7
4i
16
27
9
2
1
8
1
0
0
4
2.0
12 1.1
−15 1.0 −22 2.3
0.7
2.1
1.4 33 1.1
18 2.2
4j
Cl 4-I
16
0
1
0
1
0
−33
−28
−18
15
1
1
1
5
1.4
4k
Cl 2-F,4-F,5-F
Cl 2-F,3-F,4-F
Cl 3-F,4-F
0
2
−14
35
27
5
−30
−27
15
3
1
1
−22 2.2 −25 1.5 56 1.1
−56 1.2 −61 0.5 32 0.9
4l
21
37
−2
21
43
1
1
15
15
0
1
1
−11 1.6 −10 1.0 −1
4m
1
0
1
1
29 2.6
23 1.1
47 1.1
27 1.5
20 1.2
32 1.7
1
4n
Cl 2-F,4-Cl
Cl 2-CH3,4-F
Cl 2-NO2,4-F
0
0
1
1
−18
11
1
2
−17 1.5 −11 1.2 42 2.0
−6
0
4o
1
1
1
1
1
2
1
2
1
0
1
1
−5
29
6
1
36
39
37
30
−1
14
−5
35
45
45
55
49
1
0
1
0
16
−15
2
1
0
2
76
80
9
0
1
67
1
1
56
18
16
22
44
15
17
35
13
63
56
32
1
1
2
1
2
1
1
1
0
1
1
0
44
6
1
0
1
0
11
25
35
6
1
1
0
4p
1
0
2
14
64
1
6
4q
H
H
H
H
H
H
H
H
–
2-Cl,5- CF3 25
1
0
−15
7
1
−6
71
1
4
1
7
4r
2-I
24
24
8
2
0
−13
31
−5
−20
17
−38
23
21
26
1
74
75
70
80
42
79
41
−22
−56
1
1
1
1
1
0
1
0
1
0
1
2
1
2
18
3
1
39
17
39
17
15
4
1
1
4s
2-Br,5-F
5
1
2
1
39
0
60
1
0
0
1
1
4
1
4t
2-F,4-Br,6-F 15
12
5
1
1
0
−4
−2
−7
−39
4
0
73
15
18
26
23
40
34
−11
1
20
34
−3
18
5
0
4u
2-F,3-F,4-F
3-F,4-F
2-CH3,4-F
2-NO2,4-F
–
22
29
32
41
33
86
1
1
1
1
2
0
65
1
0
2
1
2
1
4v
−16
9
2
1
0
2
0
0
47
1
4w
1
1
63
2
2
4x
22
29
69
1
2
0
0
0
41
12
−15
−10
1
1
Bensulfuron-Mg
0
2
6
2
2
1
−25
−61
1
1
1
−22
−1
2
Nicosulfuron
–
–
11
1
0
1
0
a
Abbreviations of Echinochloa crusgalli.
Abbreviations of Digitaria adscendens.
Abbreviations of Polypogon fugax.
Abbreviations of Brassica napus.
b
c
d
e
f
Abbreviations of Amaranthus retroflexus.
Abbreviations of Abutilon theophrasti.
Abbreviations of bensulfuron-methyl.
g
h
“
” mean Standard Deviation.
further bioassay were carried out. The results of bioassay showed
that a few compounds had powerful herbicidal activity, which was
higher than the bensulfuron-methyl. As shown in the Fig. 5, the
compounds 4p, 4w and 4x had well-herbicidal activity against di-
cotyledonous species Brassica napus, with >81% inhibitory rate at
dosages of 1.5 a.i. kg/ha, but other compounds showed only low
herbicidal activity. Surprisingly, compounds 4l and 4q exhibited
good plant growth-regulating activity, which was promoted the
growth of weeds. The inhibitory effect of the most of compounds
on the Digitaria adscendens was not as expected. Fortunately, com-
pound 4x still displayed excellent herbicidal activity against di-
cotyledon weed Digitaria adscendens by pre-emergence treatment,
with 93% inhibitory rate. At the same time, the compounds 4a and
4o showed different degree of promoting effect on Digitaria adscen-
dens. The SAR revealed that the species of substituent groups on
the benzene ring near the thiourea bridge was crucially important
to inhibitory effect against weeds. The herbicidal activity of acylth-
iourea compounds containing nitro group were significantly higher
than the other title compounds.
Expectedly, the highest activity value was compound 4x (89.12%),
and compounds 4p (86.95%) and 4w (87.70%) had the same activity
as the positive control. The results of inhibition of enzyme activity
tests were similar with the tests of herbicidal activity.
3.4.4. Crop safety evaluation
The crop safety evaluation results were showed in Fig. 7 and
Fig. 8. Most of the tested compounds were safety to soybean and
wheat by spraying treatment at 1.5 a.i. kg/ha, and the inhibition
rates of 11 compounds were less than 20%. Interestingly, the pro-
motive effect of compound 4i on soybean was as high as 13–14%,
and the other compounds did not show significant promotional ef-
fect on crop growth. In the tests of crop fresh weight, compound
4i also showed better growth-enhancing effect than other com-
pounds, and the other 10 compounds had no significant effect on
crop fresh weight.
3.5. Molecular docking study
Molecular docking had gradually developed into an effective
tool to explore the mechanisms of the enzyme-herbicide interac-
tions, and this method was widely applied to screening of novel
compounds study [24, 25]. Jingqian Xie et al. had provided a log-
ical structural basis for the enantioselectivity of AHAS activity of
the IMIs through molecular docking [26]. Yanzhen He et al. had
verified the binding sites of pyrimidinyl thiobenzoates was simi-
lar to sulfonylureas [27]. Thus, we had carried out molecular dock-
ing studies. The docking results were listed in Table 2, includ-
ing docking score and amino acid residues that have H-bond in-
teractions with the ligands. As can be seen from the results, the
It was possible that strong electron-withdrawing group (nitro)
enhance the bioactivity of the compounds 4p and 4x. The com-
pound 4w also showed good herbicidal activity, but the reasons
for this need to be further explored.
3.4.3. AHAS activity determination in vivo
Due to the title compounds displayed an excellent herbicidal
activity against Brassica napus, AHAS enzyme inhibitory activity of
acylthioureas was tested. The inhibited activity of AHAS in vivo at
100 mg/L was showed in Fig. 6. The commercial AHAS inhibitor
bensulfuron-methyl inhibited the in vivo AHAS activity by 87.4%.
8

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Pre-emergence treatment of Brassica napus
Post-emergence treatment of Brassica napus
Pre-emergence treatment of Digitaria adscendens
Post-emergence treatment of Digitaria adscendens
120
100
80
60
40
20
0
-20
-40
4i
4l
4e
M
4a
4g
4o
4x
4b
4d
4p
4q
4w
4m
ron-
u
f
Bensul
Fig. 5. Herbicidal activities of 13 compounds under pre-emergence (post-emergence) conditions at 1.5 a.i. kg/ha. Vertical bars represented mean
Standard Deviation (SD).
The Negative values on the Y axis in the figure indicated that the compound had a promoting effect on the growth of the tested plants.
100
80
60
40
20
0
v
4i
4l
4f
4j
4t
4s
4c
4e
4r
4a 4b
4d
4g
4k
4o 4p 4q
4
4x
4h
4n
4u
4w
4m
n-M
o
r
u
f
l
u
s
n
Be
Fig. 6. The inhibited activity of AHAS in vivo in Brassica napus with 100 mg/L compounds treatments.
9

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Pre-emergence treatment of Glycine max
Post-emergence treatment of Glycine max
Pre-emergence treatment of Triticum aestivum
Post-emergence treatment of Triticum aestivum
30
20
10
0
-10
4i
4t
4s
4r
M
4g
4o
4v
4x
4h
4p
4w
ron-
u
f
ul
Bens
Fig. 7. The inhibition rates of 11 compounds under pre-emergence (post-emergence) conditions at 1.5 a.i. kg/ha. Vertical bars represented mean
Standard Deviation (SD).
The Negative values on the Y axis in the figure indicated that the compound had a promoting effect on the growth of the tested crops.
Pre-emergence treatment of Glycine max
Post-emergence treatment of Glycine max
Pre-emergence treatment of Triticum aestivum
Post-emergence treatment of Triticum aestivum
20
15
10
5
0
-5
-10
-15
-20
x
4i
4t
4s
4r
M
4g
4o
4v
4
4h
4p
4w
ron-
ulfu
ns
Be
Fig. 8. The fresh weight inhibition rates of 11 compounds on 2 crops at 1.5 a.i. kg/ha.
hydrogen bonds interactions were formed among all ligand
molecules and amino acid residues. All the compounds were
docked into the binding pockets of a developed homology model
of AHAS enzyme [28] (Fig. 9A).
NH groups linked to acyl group formed hydrogen bond with ASN-
312. In addition, oxygen atoms played a significant part in molec-
ular affixation of compound 4p (the second most potent deriva-
tive). By acting as an acceptor, one of oxygen atom of methyl sul-
fone formed two hydrogen bonds with SER-186 and ARG-246, re-
spectively. Additionally, the acyl of compound 4p also formed a
hydrogen bond with GLY-309. Furthermore, two oxygen atoms of
nitro group located at position 2 of the phenyl ring interacted
with four amino acid residues, namely, GLY-309, ASP-414, ASN-
312 and SER-313 which resulted five hydrogen bonds with AHAS
acceptor.
The binding mode of compounds 4p and 4x at active site of
AHAS enzyme were described in Fig. 9B and Fig. 9C respectively.
Among all the acylthiourea molecules, compound 4x was the most
active compound. Methyl sulfone of the aromatic ring in com-
pound 4x showed two hydrogen bonds formation with ARG-502.
Moreover, the oxygen atom located at acyl group rendered hy-
drogen bonds with GLY-308 and GLY-309. The hydrogen atom of
10

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
Fig. 9. A) The binding pocket containing most of ligands. B) Binding mode of ligand 4x at active site of AHAS enzyme. C) Binding mode of ligand 4p at active site of AHAS
enzyme. Yellow dash lines indicate hydrogen bonds.
Table 2
drogen bonds and introducing nitro substituents are terrific ideas
The list of intermolecular interactions between the ligand molecules docked with
in the design and optimization of novel compounds.
1YHZ using AutoDock 4.2.6 software.
Ligand
4. Conclusion
moleculeDocking score
(No.)
(kcal/mol) Interacting groups
In this paper, a series of unreported acylthiourea derivatives
were synthesized and characterized. The biological activity of
acylthioureas were evaluated including petri dish experiments,
greenhouse herbicidal activity experiments, AHAS activity determi-
nation in vivo, crop safety tests and molecular docking simulations.
A few compounds had a good inhibitory effect against Digitaria
adscendens and Brassica napus in the petri dish test. Similarly, in
the pot experiments, the ability of title compounds to block the
growth of weeds was still formidable, and the inhibition effect of
acylthiourea on Brassica napus was better. The results of enzyme
activity determination indicated that 4p, 4w and 4x were potential
AHAS-inhibiting herbicide, and through the crop safety assessment
to determined crops were safety by acylthiourea treatments. Com-
pounds 4p and 4x performed better than others in molecular dock-
ing study, the possible reason was the larger number of hydrogen
bonds. The docking results were roughly the same as the bioassay
results. In addition, the minimum energy conformations of 4p and
4x looked save more space than other compounds. So, we assumed
that 4p and 4x were easier to enter the binding pocket due to their
smaller size. In our future research, we will explore whether the
hypothesis is true.
4a
4b
4c
4d
4e
4f
−6.82
−5.87
−5.58
−6.81
−5.45
−7.1
SER-186, THR-331, ARG-373
SER-186, ARG-246, GLY-309, ASN-312
SER-186, ARG-246, ASN-312
THR-331, LEU-332, MET-333
GLY-245, ASP-414
SER-186, ARG-246, GLY-309, ASP-395
SER-186, GLY-245, ASP-395
4g
4h
4i
−6.27
−6.2
SER-186, GLY-309, ASP-395, ASP-414, VAL-415
GLN-465, GLU-645, HIS-646, THR-662, GLY-664
HIS-352, ARG-377
−7.34
−6.49
−6.98
−6.41
−6.12
−7.21
−7.42
−7.58
−6.99
−6.83
−6.42
−6.33
−6.73
−6.73
−7.5
4j
4k
4l
LYS-456, GLU-645, GLU-663
SER-186, GLY-245, ARG-246, GLY-309
SER-186, GLY-245, ARG-246, GLY-309
SER-186, ARG-246, THR-331, ARG-373
SER-454, LYS-456, GLU-645, GLU-663
SER-186, ARG-246, GLY-309, ASN-312, SER-313, ASP-414
ARG-373, ASP-395, ILE-396
4m
4n
4o
4p
4q
4r
GLY-245
4s
4t
SER-186, ARG-246, GLY-309, ASP-395
GLN-110, GLU-236
4u
4v
4w
4x
LYS-456, GLN-465,GLU-645, HIS-646, GLU-663
SER-186, THR-331, ARG-373
LYS-456, GLN-465, GLU-645, HIS-646, THR-662, GLU-663
GLY-308, GLY-309, ASN-312, ARG-502
−8.01
In summary, part of the acylthiourea compounds synthesized
in this article had a certain inhibitory activity on weeds. Through
enzyme activity experiments and molecular docking experiments,
we confirmed that the herbicidal activity mechanism of this class
of compounds was related to the inhibition of AHAS.
According to the molecular docking results, compound 4x
showed the highest activities, and it may be considered as good in-
hibitor of AHAS enzyme. Combined with the relationship between
the docking score and the number of hydrogen bonds, arrive at a
conclusion that increasing the number of hydrogen bonds can im-
prove biological activities. And the compounds contain nitro group
performed better in the docking results. Therefore, increasing hy-
Declaration of Competing Interest
All authors declare that no conflict of interest exists.
11

Y.-P. Wu, Y. Wang, J.-H. Li et al.
Journal of Molecular Structure 1241 (2021) 130627
CRediT authorship contribution statement
[13] K. Ghazal, S. Shoaib, M. Khan, S. Khan, N.Khan M.K.Rauf, A. Badshah,
M.N. Tahir, I. Ali, A.-.U. Rehman, Synthesis, characterization, X-ray diffraction
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Yun-Peng Wu: Conceptualization, Methodology, Software, For-
mal analysis, Investigation, Data curtion, Writing – original draft.
Yan Wang: Conceptualization, Methodology, Validation, Formal
analysis, Resources, Writing
– review & editing, Supervision,
Project administration. Jia-Hui Li: Conceptualization, Methodology,
Software, Data curtion, Writing – review & editing. Ran-Hong Li:
Methodology, Validation, Resources, Writing – original draft, Su-
pervision. Jun Wang: Validation, Formal analysis, Visualization.
Sui-Xin Li: Methodology, Software, Investigation. Xin-Yu Gao: In-
vestigation, Formal analysis, Visualization. Ling Dong: Investiga-
tion, Software, Visualization. An-Qi Li: Investigation, Formal anal-
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[17] J.V. Hay, Chemistry of sulfonylurea herbicides, Pest Manag. Sci. 29 (3) (1990)
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