Design, synthesis, and safener activity of novel methyl (r)-n-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates

Article abstract of DOI:10.3390/molecules23010155

A series of novel methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates were designed by active substructure combination. The title compounds were synthesized using a one-pot route from L-cysteine methyl ester hydrochloride, acyl chloride, and ketones. All compounds were characterized by IR,¹H NMR,¹³C NMR, and HRMS. The structure of 4q was determined by X-ray crystallography. The biological tests showed that the title compounds protected maize from chlorimuron-ethyl injury to some extent. The ALS activity assay showed that the title compounds increased the ALS activity of maize inhibited by chlorimuron-ethyl. Molecular docking modeling demonstrated that Compound 4e competed against chlorimuron-ethyl to combine with the herbicide target enzyme active site, causing the herbicide to be ineffective.

Full text of DOI:10.3390/molecules23010155

molecules
Article
Design, Synthesis, and Safener Activity of Novel
Methyl (R)-N-Benzoyl/Dichloroacetyl-
Thiazolidine-4-Carboxylates
Li-Xia Zhao, Hao Wu ID , Yue-Li Zou, Qing-Rui Wang, Ying Fu ID , Chun-Yan Li and Fei Ye *
College of Science, Northeast Agricultural University, Harbin 150030, China; zhaolixia@neau.edu.cn (L.-X.Z.);
wuhao2015ji@163.com (H.W.); zouyueli@163.com (Y.-L.Z.); qingruiwang@hotmail.com (Q.-R.W.);
fuying@neau.edu.cn (Y.F.); chunyanli@neau.edu.cn (C.-Y.L.)
* Correspondence: yefei@neau.edu.cn; Tel.: +86-451-5519-1507
Received: 20 December 2017; Accepted: 12 January 2018; Published: 12 January 2018
Abstract: A series of novel methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates were
designed by active substructure combination. The title compounds were synthesized using a one-pot
route from L-cysteine methyl ester hydrochloride, acyl chloride, and ketones. All compounds
were characterized by IR, 1H NMR, 13C NMR, and HRMS. The structure of 4q was determined by
X-ray crystallography. The biological tests showed that the title compounds protected maize from
chlorimuron-ethyl injury to some extent. The ALS activity assay showed that the title compounds
increased the ALS activity of maize inhibited by chlorimuron-ethyl. Molecular docking modeling
demonstrated that Compound 4e competed against chlorimuron-ethyl to combine with the herbicide
target enzyme active site, causing the herbicide to be ineffective.
Keywords: methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates; active substructure
combination; safener activity
1. Introduction
Acetolactate synthase (ALS) is an essential enzyme in the biosynthesis of branched-chain
amino acids. It is the target of ALS-inhibiting herbicides, including imidazolinone, sulfonylurea,
sulfonylamino-carbonyl-triazolinone, triazolopyrimidine, and pyrimidinyl-thiobenzoate herbicides [
ALS-inhibiting herbicides prevent the synthesis of isoleucine, leucine, and valine, resulting in
subsequent weed death [ ]. In addition, these herbicides have played a significant role in controlling
weeds since the early 1980s due to their low toxicity, low cost, high activity, and safety [ ]. As a type
of ALS-inhibiting herbicide, chlorimuron-ethyl has been widely used to control a range of broadleaf
weeds in farmland, especially in soybean fields over the past decade [ ]. However, chlorimuron-ethyl
treatments have been reported to cause phytotoxicity in beans, leading to reduced plant height and
shoot dry matter [ ]. Residues of this herbicide in soil may also inhibit the growth of succeeding
crops, such as maize [ ]. In order to reduce the injury, various methods have been reported, including
restricted use of long residual herbicides, developing new herbicides and so on [ ]. In addition to these
1].
2
3
4,5
6
7
8
methods, herbicide safeners, a class of agrochemicals that can reduce the negative effects of a herbicide
on crops, have been commercialized [9,10].
Over the past decade, novel herbicide safeners have been synthesized using a structure-based
bioisosterism design, which is a useful strategy for structural modification [11]. On the other
hand, active substructure combinations have also proved to be very significant for synthesizing
novel safeners, and these combinations can provide useful information about chemical substituents.
Novel acylsulfamoylbenzamide safeners with excellent bioactivity have been designed using the
previously developed safener cyprosulfamide, as the leading compounds based on similar active
Molecules 2018, 23, 155; doi:10.3390/molecules23010155
www.mdpi.com/journal/molecules

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substructures (Scheme 1) [12]. Two compounds based on acylsulfamoylbenzamide have better
bioactivities than cyprosulfamide and could serve as leading compounds in the design of new safeners.
In addition, isoxadifen-ethyl was designed by combining the active substructures of known active
molecules [13]. Furthermore, molecular docking has been performed to compare the binding affinities
of herbicides and the target compound with the target enzyme (i.e., ALS) [14]. The binding energy
was calculated, which helped researchers predict the mechanism of the herbicide safener [15].
Scheme 1. Design of new molecules as potential safeners.
As a mature safener, R-28725 (2,2-dichloro-1-(2,2-dimethyloxazolidin-3-yl)ethan-1-one) shows
good safener biological activity [9], and thiazolidine may possess similar chemical properties to
R-28725 due to bioisosterism [16]. Recently, researchers reported some thiazolidine compounds with
favorable biological activities in protecting the maize from herbicide phytotoxicity [11]. According to
the facts mentioned above and continuing our previous research on the design of nitrogen-containing
heterocyclic herbicide safeners [17], a series of thiazolidine-4-carboxylates combined with different
groups at the N-3 position were designed and synthesized based on bioisosterism and active
substructure combinations retaining the thiazolidine ring as the parent skeleton structure (Scheme 2).
Scheme 2. Design of the title compounds.
Due to the importance of thiazolidine derivatives, various synthetic routes to these compounds
have been reported in many fields. The most frequently used method is the condensation of

Molecules 2018, 23, 155
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cysteine methyl ester hydrochloride with ketones in the presence of K2CO3
[
18
,19]. As for
N-benzoylthiazolidines, they are usually prepared by the acylation of the corresponding thiazolidine
compound, which is treated with an acyl chloride in the presence of the base [20]. However, these
reactions, which involve cyclization and acylation, are time-consuming and complicated. According
to our previous research [13], the final products were directly synthesized by a one-pot reaction
(Scheme 3), representing an efficient and time saving route. The aim of this study was to synthesize
new methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates and determine their biological
activities using biological tests. Molecular docking was also performed to research the mechanism of
the safener.
Scheme 3. Route for the synthesis of the title compounds.
2. Results and Discussion
2.1. Chemistry
In this paper, a one-pot synthesis route was designed (Scheme 3) and the effects of solvents
and cyclization temperature were investigated. L-cysteine methyl ester hydrochloride,
cyclized with ketones, , to generate thiazolidines, , with Et3N as the attaching acid agent under
nitrogen atmosphere. The title compounds, , were prepared by direct acylation of the corresponding
2, was
1
3
4
thiazolidine compound with an acyl chloride. Notably, use of toluene as the solvent provided better
yields than CH2Cl2, THF, and CHCl3. In addition, the effect of temperature variation on the cyclization
◦
was determined by increasing the temperature from 25 to 75 C. It was found that the best yields were
obtained when controlling the cyclization temperature at 65 ◦C.
As shown in Table 1, the structure of ketone greatly affected the yield. When cyclohexanone was
used, the formation of spiro compounds made the product more stable than other products. Thus,
the yields of Compounds 4a–f with spiro structures were higher than those of the other compounds,
which were 69–91%. The yields were also considerably affected by the substituent structure on the
benzene ring. For p-substituted phenyl with –NO2, the yields were significantly increased, especially
the yield of Compound 4a, which was increased by approximately 91%. Compounds 4d, 4j, and 4o
with Me-substituted phenyl were obtained in low yields; for example, the yield of Compound 4j was
decreased by 65%. Notably, the yields of Compounds 4r and 4s, with dichloromethyl substituents at
R3, were among the lowest of all compounds, at 48% and 41%, respectively. Finally, side reactions
affected the yield to some extent. These results were likely due to a chemical equilibrium between the
thiazolidine and Schiff base [21], which resulted in the acyl chloride combining with the mercapto
group, resulting in a decreased yield (Scheme 4).
The structures of all compounds, 4a–s
, were confirmed by 1H NMR, 13C NMR, and HRMS. All the
compounds showed similar spectroscopic characteristics because of their structural similarity. In the
IR spectra, two characteristic carbonyl bands at approximately 1630–1740 cm−1 proved the presence

Molecules 2018, 23, 155
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of the amide and ester groups. In the 1H NMR spectra of 4a
–q, the aromatic protons appeared in the
region of 7.00–7.40 ppm, which also confirmed the successful acylation. The measured HRMS data
also confirmed the proposed structures.
Table 1. Yields of title compounds.
Compound
R1
R2
R3
Yield/% Compound
R1
R2
R3
Yield/%
4a
4b
4c
4d
4e
4f
4g
4h
4i
(CH2)5
p-NO2C6H4
2,4-Cl2C6H3
p-ClC6H4
m-CH3C6H4
o-OCH3C6H4
C6H5
91
81
77
69
71
75
67
83
72
65
4k
4l
(CH2)4
CH3 CH3 2,4-Cl2C6H3
CH3 CH3 p-ClC6H4
CH3 CH3 p-NO2C6H4
CH3 CH3 m-CH3C6H4
CH3 CH3 o-OCH3C6H4
CH3 CH3
(CH2)5
o-ClC6H4
70
78
72
87
65
69
71
48
41
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)4
(CH2)4
(CH2)4
(CH2)4
4m
4n
4o
4p
4q
4r
p-ClC6H4
o-ClC6H4
CHCl2
CHCl2
p-NO2C6H4
2,4-Cl2C6H3
m-CH3C6H4
4s
CH3 CH3
4j
Scheme 4. Mechanistic outline for the ring-opening reaction.
2.2. Crystal Structure of Compound 4q
As shown in Figure 1, in the structure of methyl (R)-3-(o-chlorobenzoyl)-2,2-dimethylthiazolidine-4-carboxylate,
the dihedral angle of the phenyl (C1/C2/C3/C4/C5/C6) and thiazolidine (N1/C8/S1/C11/C12)
is 70.25◦. The X-ray structure indicated that Compound 4q contained a chiral carbon, C12, with
R configuration. However, no obvious intermolecular hydrogen bonds and π–π interactions were
observed in the structure of Compound 4q (Figure 2).
Figure 1. Molecular structure for Compound 4q at 30% probability level.

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Figure 2. Packing view of Compound 4q. The unit cell axes are labelled as o, a, b, c.
2.3. Biological Activity Tests
According to our previous research on the biological activity of herbicide safeners [
9
], the
residual concentration of chlorimuron-ethyl in soil was determined as 24 µg/kg. Before the tests, a
preliminary screening was carried out to determine the best concentration of the title compounds, and
Compound 4a was selected for the preliminary screening. The best concentration of Compound 4a
was determined by varying the concentration from 5 to 100 mg/kg. It was found that the best growth
index was obtained when controlling the concentration at 25 mg/kg. Thus, the best concentration was
applied to the biological activity tests.
The protective effects of Compounds 4a
evaluated, as shown in Table 2. Significant recovery of maize growth was observed when the title
compounds were used as safeners and when the chlorimuron-ethyl concentration in soil was 24 g/kg.
–s to maize from injury of chlorimuron-ethyl were
µ
All title compounds showed varying recovery rates for root length, root fresh weight, plant height,
and plant fresh weight, indicating the successful design of the title compounds.
Comparing the protective effects of Compounds 4a
played a significant role in the safener activity of all title compounds. As shown in Table 2, biological
activity tests revealed that Compounds 4a
with ring substituents at R1 and R2 displayed better
safener activity than Compounds 4l
with Me substituents at R1 and R2. Similarly, Compound 4r
–s
showed that substituents R1, R2, and R3
–
k
–
q
with ring substituents at R1 and R2 also showed increasing safener activity compared to Compound 4s
with Me substituents at R1 and R2. These results were likely due to the formation of spiro compounds,
resulting in good safener activities. Furthermore, another crucial result was revealed regarding R3
substitution. For example, when substitution was introduced at different positions on the benzene
ring, the title compounds showed varying recovery rates. Compound 4e with methoxy at o-position
on the benzene ring would show higher recovery rates for maize growth than Compounds 4a
4d, and 4f. Additionally, Compound 4c with a chloro substituent at the p-position on the benzene ring
also showed better safener activity than Compounds 4a 4b 4d, and 4f. Thus, it was found that the
introduction of o-methoxybenzoyl and p-chlorobenzoyl at R3 did have an effect on safener activities. By
comparison, Compounds 4c 4e, and 4g showed better recovery rates for maize growth than the other
, 4b, 4c,
,
,
,
compounds. The protective effects of 4e were superior to the effects of R-28725, which is a commercial
safener and effectively reduced injury from chlorimuron-ethyl herbicides.

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Table 2. Protective effects of Compound 4 to maize a,b,c
.
Recovery of Plant
Height (%)
Recovery of Root
Length (%)
Recovery of Plant
Recovery of Root
Compound
Weight (%)
Weight (%)
R-28725
4a
86.22 ± 1.37
42.90 ± 1.22
59.96 ± 1.09
80.33 ± 0.82
67.97 ± 1.02
89.08 ± 0.65
62.05 ± 0.21
85.86 ± 0.97
57.08 ± 0.69
45.64 ± 1.62
57.41 ± 1.26
41.82 ± 0.68
20.60 ± 0.69
48.99 ± 0.57
25.02 ± 0.92
34.12 ± 1.32
76.00 ± 1.16
23.20 ± 0.53
57.74 ± 1.31
33.22 ± 1.65
84.73 ± 0.98
60.52 ± 1.37
45.53 ± 1.27
86.48 ± 0.87
41.97 ± 1.31
91.20 ± 0.89
64.07 ± 0.33
87.53 ± 1.43
46.83 ± 1.07
55.60 ± 0.55
66.61 ± 1.92
46.24 ± 0.23
15.09 ± 0.32
66.04 ± 1.13
29.16 ± 0.91
38.30 ± 0.45
61.09 ± 1.75
14.56 ± 0.32
39.46 ± 1.44
36.30 ± 1.44
87.47 ± 1.12
45.96 ± 1.11
47.16 ± 1.24
94.61 ± 1.43
78.25 ± 0.98
98.92 ± 1.09
73.75 ± 1.39
85.62 ± 1.26
41.26 ± 0.73
53.93 ± 1.29
71.15 ± 1.42
55.83 ± 1.05
15.04 ± 1.22
48.27 ± 1.06
17.01 ± 0.61
39.58 ± 1.02
68.93 ± 1.26
19.04 ± 1.42
31.46 ± 0.88
45.75 ± 1.54
83.61 ± 1.98
68.11 ± 1.82
68.48 ± 0.63
85.79 ± 1.62
41.98 ± 1.05
93.70 ± 1.22
51.56 ± 0.52
83.39 ± 0.92
59.74 ± 0.62
57.56 ± 0.54
67.59 ± 1.65
55.13 ± 0.13
25.27 ± 1.24
47.83 ± 0.36
24.94 ± 0.85
56.35 ± 1.13
55.57 ± 1.44
25.25 ± 0.54
46.34 ± 1.32
37.89 ± 0.74
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
a
Data
are
means
of
three
replicates,
mean
±
standard
deviation;
Treatment with compounds Treatment with Chlorimur on−ethyl
b
c
Recovery Rate (%) =
; Contrast was treated by water.
Contrast Treatment with Chlorimuron−ethyl
2.4. Effect of Safeners on ALS Activity
Chlorimuron-ethyl controls weeds by inhibiting ALS, which is important in the biosynthesis of the
branched-chain amino acid. A direct assay on the ALS activity was carried out to confirm the positive
effect of the title compounds on ALS activity. As shown in Table 3, ALS activity was expressed as the
amount of acetylmethylcarbinol formed per hour per milligram protein. Comparing the ALS activity
values of the control treatment and chlorimuron-ethyl treatment showed that chlorimuron-ethyl
provoked an obvious decrease in the ALS activity; however, a significant increase was observed after
the treatment of R-28725 and Compounds 4a–s. The tested compounds showed a varying degree
of positive effects with ALS activity values between 0.055 and 0.085 nmol h−1 mg−1 protein when
compared with the chlorimuron-ethyl treatment (0.046 nmol h−1 mg−1 protein). In contrast to other
tested compounds, Compounds 4c and 4e almost reversed the inhibition caused by chlorimuron-ethyl,
which showed similar effects as R-28725. Among the series, Compound 4e revealed the best effects
with an ALS activity value of 0.85 nmol h−1 mg−1 protein. These data could indicate that the title
compounds can increase ALS activity of maize inhibited by chlorimuron-ethyl significantly.

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Table 3. Effect of Compounds 4a–s and chlorimuron-ethyl on the acetolactate synthase (ALS) activity
of maize.
ALS Activity (nmol
h
ALS Activity (nmol
h
ALS Activity (nmol
Treatment
Treatment
Treatment
−1
−1
−1
−1
−1
−1
mg Protein) a
mg Protein) a
h
mg Protein) a
0.059 ± 0.003
0.064 ± 0.001
0.071 ± 0.001
0.055 ± 0.002
0.064 ± 0.002
0.062 ± 0.001
chlorimuron-ethyl
+ 4f
chlorimuron-ethyl
chlorimuron-ethyl
+ 4n
chlorimuron-ethyl
+ 4o
chlorimuron-ethyl
+ 4p
chlorimuron-ethyl
+ 4q
chlorimuron-ethyl
+ 4r
chlorimuron-ethyl
+ 4s
Control b
0.091 ± 0.002
0.046 ± 0.003
0.084 ± 0.002
0.064 ± 0.001
0.062 ± 0.004
0.081 ± 0.002
0.065 ± 0.002
0.085 ± 0.002
0.075 ± 0.003
0.079 ± 0.003
0.071 ± 0.002
0.068 ± 0.002
0.077 ± 0.002
0.068 ± 0.001
0.058 ± 0.001
0.068 ± 0.003
chlorimuron-ethyl
+ 4g
chlorimuron-ethyl
+ R-28725
chlorimuron-ethyl
+ 4a
chlorimuron-ethyl
+ 4b
chlorimuron-ethyl
+ 4c
chlorimuron-ethyl
+ 4d
chlorimuron-ethyl
+ 4e
chlorimuron-ethyl
+ 4h
chlorimuron-ethyl
+ 4i
chlorimuron-ethyl
+ 4j
chlorimuron-ethyl
+ 4k
chlorimuron-ethyl
+ 4l
chlorimuron-ethyl
+ 4m
a
Data are means of three replicates, mean ± standard deviation; b Control was treated by water.
2.5. Molecular Docking Studies
ALS is a key enzyme in the biosynthesis of branched-chain amino acids, which is the target
of chlorimuron-ethyl. The crystal structure of ALS was provided by the Protein Data Bank (PDB
ID 1N0H). In molecular docking studies, the binding modes of chlorimuron-ethyl to ALS were
clarified (Figure 3A). Compound 4e was selected to dock with ALS due to its superior safener activity
(Figure 3B). Obviously, both chlorimuron-ethyl and Compound 4e bound to the target active site
of ALS. Molecular docking revealed the mechanism of the herbicidal activity: chlorimuron-ethyl
prevented the substrate from binding with the active pocket by clogging the entrance to the channel.
In comparison, Compound 4e sufficiently bound to the target active site and prevented the combination
of chlorimuron-ethyl with the target active site. Additionally, Compound 4e had a smaller structure
than chlorimuron-ethyl, hardly blocking the entrance to the channel. Therefore, there was an increased
opportunity for the small substrate to enter into the channel and catalyze the active site.
Figure 3. Docking modeling of chlorimuron-ethyl (A) and 4e (B) with ALS.
The docking modeling is revealed in greater detail in Figure 4. Chlorimuron-ethyl docked to ALS
between the two active site residues (Arg 380 and Trp 586) and binding interactions included two
hydrogen bonds between the oxygen atom and Arg 380. Compound 4e also bound to the target site by
hydrogen bonds. Obviously, three hydrogen bonds were formed between Compound 4e and Arg 380,
resulting in a stable combination. Upon application, 4e possibly competed with chlorimuron-ethyl
at the target active site by stopping the herbicide from acting on the ALS active pocket, which

Molecules 2018, 23, 155
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caused chlorimuron-ethyl to lose its effect. This function may be the detoxification mechanism of the
title compound.
Figure 4. Zoomed-in view of the docking modeling of chlorimuron-ethyl (
atoms are shown in yellow ( ) and green ( ); the sulfur atoms are shown in brown (
); the nitrogen atoms are shown in blue (A,B
A
) and 4e
) and yellow (
), and the chlorine
(
B
). The carbon
A
B
A
B
);
the oxygen atoms are shown in red (
A,
B
atoms are shown in green (A).
3. Materials and Methods
3.1. Reagents and Analysis
All the reagents were of analytical grade and used without further purification. Melting points
were determined on a Beijing Taike melting point apparatus (X-4) (Taike, Beijing, China) and were
uncorrected. IR spectra were obtained on a Bruker ALPHA-T spectrometer (BRUKER Inc., Beijing,
1
China). H NMR and 13C NMR spectra were recorded on a Bruker AV400 spectrometer (BRUKER
Inc., Beijing, China) with CDCl3 (Energy Chemical., Shanghai, China) as the solvent and TMS (Energy
Chemical., Shanghai, China) as the internal standard. HRMS spectra were recorded on an FTICR-MS
spectrometer (BRUKER Inc.). X-ray diffraction data were recorded on a BRUKER D8 VENTURE
X-diffractometer (BRUKER Inc.) with Mo Kα radiation (λ = 0.71073 Å) at 273(2) K.
3.2. General Procedure for the Preparation of 4
The synthetic route of the title compound 4, is shown in Scheme 3. L-cysteine methyl ester
hydrochloride (4.29 g, 25 mmol), an appropriate ketone (25 mmol), and Et3N (5.05 g, 50 mmol)
◦
were stirred for 2 h in toluene (20 mL) at 65 C under nitrogen atmosphere. Benzoyl chloride or
dichloroacetyl chloride (25 mmol) was added dropwise to the reaction mixture at 0 ◦C and reacted
for 1 h. The mixture was washed with saturated NaCl solution (3
anhydrous sodium sulfate. The solvent was removed under reduced pressure to yield crude methyl
(R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylate ( ). The title compounds were purified by
are presented in Supplementary Materials
×
20 mL) and dried using
4
column chromatography. The spectra data of compound
(Figures S2–S77).
4
Methyl (R)-4-(p-nitrobenzoyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4a). Yellow solid, m.p. 148 ◦C.
Yield 91%. IR (KBr):
(cm−1) 2914 (C-H), 1727, 1642 (C=O), 1515 (C=C); 1H NMR (400 MHz, CDCl3):
(ppm) 8.26 (d, J = 8.8 Hz, 2H, Ar-H), 7.52 (d, J = 8.8 Hz, 2H, Ar-H), 4.66 (br, 1H, N-CH), 3.72 (s, 3H,
O-CH3), 3.19–3.20 (m, 2H, S-CH2), 1.34–3.18 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
(ppm)
ν
δ
δ
170.38, 167.47, 147.98, 144.59, 126.85, 124.08, 81.14, 67.34, 53.06, 35.77, 35.18, 30.75, 25.60, 25.36, 24.49;
HRMS calcd. for [M + Na+] C17H20N2O5S: 387.0985, found 387.0990.
Methyl (R)-4-(2,4-dichlorobenzoyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4b). White solid, m.p.
◦
151–152 C. Yield 81%. IR (KBr):
ν
(cm−1) 2843–2931 (C-H), 1734, 1633 (C=O), 1437–1575 (C=C);
1H NMR (400 MHz, CDCl3):
δ
(ppm) 7.26–7.40 (m, 3H, Ar-H), 4.50 (dd, J = 4.9, 1.7 Hz, 1H, N-CH), 3.71

Molecules 2018, 23, 155
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(s, 3H, O-CH3), 3.23–3.24 (m, 2H, S-CH2), 1.31–3.23 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
δ
(ppm) 170.42, 164.97, 136.42, 135.47, 129.91, 129.29, 129.20, 127.68, 81.09, 67.18, 52.91, 35.99, 34.27, 31.03,
25.51, 25.37, 24.54; HRMS calcd. for [M + Na+] C17H19NO3SCl2: 410.0355, found 410.0359.
Methyl (R)-4-(p-chlorobenzoyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4c). White solid, m.p. 96–97 ◦C.
Yield 77%. IR (KBr):
CDCl3): (ppm) 7.26–7.37 (m, 4H, Ar-H), 4.76 (d, J = 2.1 Hz, 1H, N-CH), 3.70 (s, 3H, O-CH3), 3.13–3.17
(m, 2H, S-CH2), 1.28–3.12 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
(ppm) 170.78, 168.66,
ν
(cm−1) 2937 (C-H), 1747, 1639 (C=O), 1442–1550 (C=C); 1H NMR (400 MHz,
δ
δ
137.45, 135.25, 128.90, 127.18, 80.86, 67.59, 52.87, 35.84, 35.21, 30.69, 25.67, 25.38, 24.53; HRMS calcd. for
[M + H+] C17H20NO3SCl: 354.0925, found 354.0929.
◦
Methyl (R)-4-(m-methylbenzoyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4d). White solid, m.p. 74–75 C.
Yield 69%. IR (KBr):
CDCl3): (ppm) 7.09–7.28 (m, 4H, Ar-H), 4.81 (d, J = 3.9 Hz, 1H, N-CH), 3.70 (s, 3H, O-CH3), 3.15–3.20
(m, 2H, S-CH2), 2.36 (s, 3H, CH3), 3.12–3.13, 1.32–1.86 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
ν
(cm−1) 2834–2951 (C-H), 1737, 1637 (C=O), 1436 (C=C); 1H NMR (400 MHz,
δ
δ
(ppm) 171.08, 169.85, 139.15, 138.47, 129.92, 128.51, 126.10, 122.48, 80.71, 67.70, 52.70, 35.91, 35.19, 30.74,
25.71, 25.44, 24.56, 21.38; HRMS calcd. for [M + Na+] C18H23NO3S: 356.1291, found 356.1289.
Methyl (R)-4-(o-methoxybenzoyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4e). White solid, m.p.
95–96 ◦C. Yield 71%. IR (KBr):
ν
(cm−1) 2851–2935 (C-H), 1744, 1642 (C=O), 1489 (C=C); 1H NMR
(400 MHz, CDCl3):
δ 7.24–7.33, 6.87–6.96 (m, 4H, Ar-H), 4.63 (d, J = 4.6 Hz, N-CH), 3.84 (s, 3H,
O-CH3), 3.65 (s, 3H, O-CH3), 3.17–3.17 (m, 2H, S-CH2), 3.14–3.16, 1.31–1.85 (m, 10H, C6H10); 13C NMR
(100 MHz, CDCl3):
δ (ppm) 171.13, 167.07, 153.93, 130.35, 128.58, 128.16, 121.03, 110.94, 80.78, 67.17,
55.82, 52.56, 36.08, 34.50, 30.98, 25.59, 25.52, 24.63; HRMS calcd. for [M + H+] C18H23NO4S: 350.1421,
found 350.1417.
Methyl (R)-4-benzoyl-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4f). White solid, m.p. 98–99 ◦C. Yield
75%. IR (KBr): ν (cm−1) 2857–2941 (C-H), 1746, 1638 (C=O), 1442 (C=C); 1H NMR (400 MHz, CDCl3):
δ
(ppm) 7.31–7.40 (m, 5H, Ar-H), 4.81 (d, J = 3.4 Hz, 1H, N-CH), 3.69 (s, 3H, O-CH3), 3.15–3.17 (m, 2H,
S-CH2), 3.13–3.14, 1.32–1.86 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
δ
(ppm) 170.00, 169.72,
139.13, 129.24, 128.62, 125.58, 80.75, 67.67, 52.74, 35.92, 35.23, 30.72, 25.71, 25.42, 24.57; HRMS calcd. for
[M + Na+] C17H21NO3S: 342.1134, found 342.1140.
Methyl (R)-4-(p-chlorobenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4g). White solid, m.p. 101 ◦C.
Yield 67%. IR (KBr):
CDCl3): (ppm) 7.28–7.38 (m, 4H, Ar-H), 4.73 (dd, J = 5.7, 3.2 Hz, 1H, N-CH), 3.67 (s, 3H, O-CH3),
3.21–3.25 (m, 2H, S-CH2), 1.68–3.06 (m, 8H, C5H8); 13C NMR (100 MHz, CDCl3):
(ppm) 170.68, 168.12,
ν
(cm−1) 2844–2931 (C-H), 1728,1637 (C=O), 1372 (C=C); 1H NMR (400 MHz,
δ
δ
136.88, 135.52, 128.86, 127.49, 82.21, 67.05, 52.85, 39.29, 38.11, 32.29, 25.16, 25.11; HRMS calcd. for
[M + Na+] C16H18ClNO3S: 362.0588, found 362.0592.
Methyl (R)-4-(p-nitrobenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4h). Yellow solid, m.p. 141 ◦C.
Yield 83%. IR (KBr):
CDCl3): (ppm) 8.26 (d, J = 8.7 Hz, 2H, Ar-H), 7.54 (d, J = 8.7 Hz, 2H, Ar-H), 4.63 (dd, J = 4.7, 3.0 Hz, 1H,
N-CH), 3.69 (s, 3H, O-CH3), 3.25–3.28 (m, 2H, S-CH2), 1.61–3.08 (m, 8H, C5H8); 13C NMR (100 MHz,
ν
(cm−1) 2842–3079 (C-H), 1723, 1639 (C=O), 1521 (C=C); 1H NMR (400 MHz,
δ
CDCl3):
δ (ppm) 170.28, 166.90, 148.09, 144.15, 127.12, 124.02, 82.31, 66.80, 53.09, 39.56, 37.88, 32.33,
25.19, 25.04; HRMS calcd. for [M + Na+] C16H18N2O5S:373.0829, found 373.0832.
Methyl (R)-4-(2,4-dichlorobenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4i). White solid, m.p. 95 ◦C.
Yield 72%. IR (KBr):
CDCl3): (ppm) 7.26–7.40 (m, 3H, Ar-H), 4.47 (t, J = 3.5 Hz, 1H, N-CH), 3.70 (s, 3H, O-CH3), 3.27–3.28
(m, 2H, S-CH2), 2.93–3.13, 1.66–1.96 (m, 8H, C5H8); 13C NMR (100 MHz, CDCl3):
(ppm) 170.27,
ν
(cm−1) 2842–2989 (C-H), 1733, 1632 (C=O), 1437–1574 (C=C); 1H NMR (400 MHz,
δ
δ
164.51, 136.01, 135.63, 130.06, 129.40, 129.29, 127.66, 82.02, 66.52, 52.92, 39.49, 37.42, 32.51, 25.31, 24.94;
HRMS calcd. for [M + Na+] C16H17Cl2NO3S: 396.0198, found 396.0201.

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Methyl (R)-4-(m-methylbenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4j). White solid, m.p. 79 ◦C.
Yield 65%. IR (KBr):
(cm−1) 2846–2984 (C-H), 1727, 1626 (C=O), 1407 (C=C); 1H NMR (400 MHz,
CDCl3): (ppm) 7.11–7.27 (m, 4H, Ar-H), 4.78 (dd, J = 5.7, 2.9 Hz, 1H, N-CH), 3.66 (s, 3H, O-CH3),
3.17–3.24 (m, 2H, S-CH2), 2.36 (s, 3H, CH3), 2.95–3.08, 1.69–1.93 (m, 8H, C5H8); 13C NMR (100 MHz,
CDCl3): (ppm) 170.98, 169.33, 138.52, 138.42, 130.17, 128.46, 126.42, 122.80, 82.09, 67.16, 52.69, 39.34,
38.11, 32.30, 25.16, 25.08, 21.36. HRMS calcd. for [M + Na+] C17H21NO3S: 342.1134, found 342.1140.
ν
δ
δ
Methyl (◦R)-4-(o-chlorobenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4k).
White solid, m.p.
1
111–112 C. Yield 70%. IR (KBr):
ν
(cm−1) 2844–2936 (C-H), 1727, 1628 (C=O), 1423 (C=C); H
NMR (400 MHz, CDCl3):
δ (ppm) 7.28–7.38 (m, 4H, Ar-H), 4.50 (dd, J = 5.0, 1.8 Hz, 1H, N-CH), 3.69
(s, 3H, O-CH3), 3.20–3.32 (m, 2H, S-CH2), 3.19–3.20, 1.69–2.11 (m, 8H, C5H8); 13C NMR (100 MHz,
CDCl3):
δ (ppm) 170.50, 165.39, 137.56, 130.33, 129.39, 129.12, 128.34, 127.19, 82.01, 66.59, 52.78, 39.45,
37.47, 32.59, 25.32, 24.95; HRMS calcd. for [M + Na+] C16H18ClNO3S: 362.0588, found 362.0593.
Methyl (R)-3-(2,4-dichlorobenzoyl)-2,2-dimethylthiazolidine-4-carboxylate (4l). White solid, m.p. 67–68 ◦C.
Yield 78%. IR (KBr):
CDCl3): (ppm) 7.26–7.40 (m, 3H, Ar-H), 4.47 (d, J = 5.1 Hz, 1H, N-CH), 3.71 (s, 3H, O-CH3), 3.25–3.38
(m, 2H, S-CH2), 2.03 (d, J = 14.7 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz, CDCl3):
(ppm) 170.23,
ν
(cm−1) 2906–3066 (C-H), 1723, 1643 (C=O), 1573 (C=C); 1H NMR (400 MHz,
δ
δ
164.86, 135.92, 135.61, 130.02, 129.36, 129.27, 127.67, 73.54, 67.16, 52.94, 31.61, 29.00, 27.77; HRMS calcd.
for [M + Na+] C14H15Cl2NO3S: 370.0042, found 370.0046.
Methyl (R)-3-(p-chlorobenzoyl)-2,2-dimethylthiazolidine-4-carboxylate (4m). White solid, m.p. 53 ◦C. Yield
72%. IR (KBr): ν (cm−1) 2930–3010 (C-H), 1373 (C=C), 1733, 1644 (C=O); 1H NMR (400 MHz, CDCl3):
δ
(ppm) 7.29–7.38 (m, 4H, Ar-H), 4.75 (br, 1H, N-CH), 3.70 (s, 3H, O-CH3), 3.20–3.30 (m, 2H, S-CH2),
2.02 (s, 3H, CH3), 1.97 (d, J = 18.5 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz, CDCl3):
δ (ppm) 170.63,
168.50, 136.79, 135.44, 128.85, 127.38, 73.31, 67.62, 52.90, 31.29, 29.55, 28.04; HRMS calcd. for [M + Na+]
C14H16ClNO3S: 336.0432, found 336.0435.
Methyl (R)-2,2-dimethyl-3-(p-nitrobenzoyl)thiazolidine-4-carboxylate (4n). Yellow solid, m.p. 131 ◦C. Yield
87%. IR (KBr):
CDCl3): (ppm) 8.25 (d, J = 8.7 Hz, 2H, Ar-H), 7.54 (d, J = 8.6 Hz, 2H, Ar-H), 4.62 (br, 1H, N-CH), 3.70
(s, 3H, O-CH3), 3.23–3.32 (m, 2H, S-CH2), 2.00 (d, J = 11.0 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz,
ν
(cm−1) 2906–3087 (C-H), 1728, 1630 (C=O), 1428–1589 (C=C); 1H NMR (400 MHz,
δ
CDCl3):
δ (ppm) 170.22, 167.26, 148.07, 144.08, 127.03, 123.99, 73.65, 67.37, 53.08, 31.38, 29.56, 27.77;
HRMS calcd. for [M + Na+] C14H16N2O5S: 347.0672, found 347.0676.
Methyl (R)-2,2-dimethyl-3-(m-methylbenzoyl)thiazolidine-4-carboxylate (4o). White solid, m.p. 62–63 ◦C.
Yield 65%. IR (KBr):
CDCl3): (ppm) 7.11–7.26 (m, 4H, Ar-H), 4.80 (br, 1H, N-CH), 3.70 (s, 3H, O-CH3), 3.17–3.30 (m, 2H,
ν
(cm−1) 2914–3034. (C-H), 1713, 1628 (C=O), 1421.85 (C=C); 1H NMR (400 MHz,
δ
S-CH2), 1.99 (d, J = 18.5 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz, CDCl3):
δ (ppm) 170.93, 169.73, 138.43,
130.11, 128.46, 126.31, 122.69, 73.21, 67.74, 52.73, 31.32, 29.64, 28.02, 21.37; HRMS calcd. for [M+Na+]
C15H19NO3S: 316.0978, found 316.0976.
◦
Methyl (R)-3-(o-methoxybenzoyl)-2,2-dimethylthiazolidine-4-carboxylate (4p). White solid, m.p. 93 C. Yield
69%. IR (KBr):
CDCl3): (ppm) 7.25–7.34, 6.87–6.97 (m, 4H, Ar-H), 4.61 (d, J = 4.7 Hz, 1H, N-CH), 3.86 (s, 3H,
O-CH3), 3.64 (s, 3H, COOCH3), 3.20–3.32 (m, 2H, S-CH2), 2.03 (d, J = 18.3 Hz, 6H, C-(CH3)2); 13C NMR
ν
(cm−1) 2843–2979 (C-H), 1740, 1644 (C=O), 1437–1599 (C=C); 1H NMR (400 MHz,
δ
(100 MHz, CDCl3):
δ (ppm) 170.96, 166.97, 154.13, 130.48, 128.29, 127.97, 121.01, 110.95, 73.24, 67.11,
55.83, 52.56, 31.57, 29.23, 27.96; HRMS calcd. for [M + H+] C15H19NO4S: 310.1108, found 310.1110.
Methyl (R)-3-(o-chlorobenzoyl)-2,2-dimethylthiazolidine-4-carboxylate (4q). White solid, m.p. 135 ◦C. Yield
71%. IR (KBr):
CDCl3): (ppm) 7.27–7.38 (m, 4H, Ar-H), 4.51 (d, J = 4.9 Hz, 1H, N-CH), 3.68 (s, 3H, O-CH3), 3.22–3.36
(m, 2H, S-CH2), 2.03 (d, J = 14.4 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz, CDCl3):
(ppm) 170.45,
ν
(cm−1) 2927–3004 (C-H), 1743, 1645 (C=O), 1393–1437 (C=C); 1H NMR (400 MHz,
δ
δ

Molecules 2018, 23, 155
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165.74, 137.47, 130.31, 129.39, 129.07, 128.30, 127.21, 73.49, 67.24, 52.79, 31.69, 29.02, 27.82. HRMS calcd.
for [M + H+] C14H16ClNO3S: 314.0612, found 314.0616.
Methyl (R)-4-(2,2-dichloroacetyl)-1-thia-4-azaspiro[4.5]decane-3-carboxylate (4r). White solid, m.p. 127 ◦C.
Yield 48%. IR (KBr):
(s, 1H, CHCl2), 5.09 (d, J = 5.3 Hz, 1H, N-CH), 3.78 (s, 3H, O-CH3), 3.13–3.35 (m, 2H, S-CH2), 2.76–3.11,
ν δ (ppm) 5.92
(cm−1) 2937 (C-H), 1734, 1682 (C=O); 1H NMR (400 MHz, CDCl3):
1.16–1.84 (m, 10H, C6H10); 13C NMR (100 MHz, CDCl3):
δ (ppm) 170.00, 161.25, 82.57, 67.33, 64.95,
53.44, 35.38, 33.54, 30.88, 25.53, 25.25, 24.44; HRMS calcd. for [M + H+] C12H17NO3SCl2: 326.0379,
found 326.0376.
◦
Methyl (R)-3-(2,2-dichloroacetyl)-2,2-dimethylthiazolidine-4-carboxylate (4s). White solid, m.p. 105 C.
Yield 41%. IR (KBr):
5.99 (s, 1H, CHCl2), 5.10 (d, J = 5.1 Hz, 1H, N-CH), 3.85 (s, 3H, O-CH3), 3.30–3.42 (m, 2H, S-CH2), 1.89
ν δ (ppm)
(cm−1) 2927–3039 (C-H), 1736, 1685 (C=O); 1H NMR (400 MHz, CDCl3):
(d, J = 17.7 Hz, 6H, C-(CH3)2); 13C NMR (100 MHz, CDCl3):
δ (ppm) 169.84, 161.10, 74.95, 66.88, 65.07,
53.46, 31.40, 28.49, 27.25; HRMS calcd. for [M + H+] C9H13NO3SCl2: 286.0066, found 286.0061.
3.3. X-ray Diffraction
Single crystals were obtained by dissolving Compound 4q in ethyl acetate and n-hexane and by
allowing the solvent to slowly evaporate at 25 ◦C. X-ray data for 4q were collected on a BRUKER D8
VENTURE X-diffractometer equipped with graphite monochromatic Mo Kα radiation (λ = 0.71073 Å)
at 273(2) K. The structure was solved by direct methods and refined by full-matrix least-squares on
F2 using the SHELXL-2014/7 program (2014/7, BRUKER Inc.) [22]. Crystallographic data for the
structural analysis have been deposited with the Cambridge Crystallographic Data Centre (CCDC
No. 1551890). Copies of this information can be obtained free of charge from the Director, CCDC,
12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-336033; E-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk). Selected bond lengths and angles for compound 4q are presented in
Supplementary Materials (Table S1).
Crystal data for Compound 4q: C14H16ClNO3S (M = 313.79 g/mol): orthorhombic, space group
P212121 (no. 19), a = 6.1350(2) Å, b = 13.1870(3) Å, c = 18.5868(5) Å, V = 1503.71(7) Å 3, Z = 4, T = 273(2) K,
µ
(Mo K
α
) = 0.399 mm−1, Dc = 1.386 g/cm3, 19190 reflections measured (6.18◦
≤
2
θ ≤ 56.578◦),
3722 unique (Rint = 0.0228, Rσ = 0.0174), which were used in all calculations. The final R1 was 0.0299
(I > 2σ (I)) and ωR2 was 0.0822 (all data).
3.4. Biological Activity Texts
Maize seeds (Dongnong 253) were soaked in a solution of methyl (R)-N-benzoyl/dichloroacetyl-
thiazolidine-4-carboxylate,
4 (25 mg/kg) overnight, and the untreated seeds were soaked in
water. Afterwards, the seeds were germinated in a growing chamber (26.5 ◦C, 12 h of light,
relative humidity 75%) overnight. Then, the seeds were sown in paper cups in which soil
was mixed with a chlorimuron-ethyl solution (24
µg/kg) or water. Finally, the paper cup was
incubated in a growing chamber (26.5 ◦C, 12 h of light, relative humidity 75%) for 7 days.
The growth level of maize was determined to investigate the biological activity of methyl
(R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates. Each treatment was replicated three times
in a completely randomized design. The physical data of compound
Materials (Figure S78 and Tables S79–S100).
4 are presented in Supplementary
3.5. Determination of ALS Activity
The treatment of maize seeds and soil was the same as that in the biological activity tests.
At 6 days after treatment, leaves of maize were collected to determine the ALS activity. ALS activity
determination was carried out following the procedure of Kobayashi and Sugiyama (1991) with a few
modifications [23]. One gram of maize leaves was grated in liquid nitrogen and extraction medium
(3.0 mL), and the mixture was centrifuged at 25,000
×
g for 20 min. The supernatant (2.0 mL) was

Molecules 2018, 23, 155
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added to the extraction medium (3.0◦mL) and (NH4)2SO4 (1.5 g). The mixture was centrifuged at
g for 20 min after staying at 4 C for 2 h. The enzyme solution was obtained by dissolving the
25,000
×
precipitation in dissolution medium (3.0 mL). The ALS activity was assayed in mixed solution, which
was composed of 0.1 mL of 0.05 mol/L phosphate buffer (pH 7.0), 0.5 mL of the reaction medium
and 0.4 mL of the enzyme solution. After incubation at 35 ◦C for 1 h, the reaction was terminated
by the addition of 0.1 mL of 3 mol/L H2SO4. Afterwards, the decarboxylation was carried out at
60 ◦C for 15 min. ALS activity was expressed as the amount of acetylmethylcarbinol per hour per
milligram protein and the content of acetylmethylcarbinol was measured at 530 nm. Protein content
was measured by the methods described by Bradford (1976) [24].
3.6. Molecular Docking Studies
The 3D molecular structures of chlorimuron-ethyl and Compound 4e were built by the
sketch module of the SYBYL-X 2.0 program package (Tripos, St. Louis, MO, USA). In addition,
Gasteiger–Huckel charges were calculated after optimizing the molecules. The crystal structure of
ALS was provided by the Protein Data Bank (PDB ID 1N0H). Accelrys Discovery Studio 2.5 provided
the CDOCKER method for the docking modeling. Some co-crystallized small molecules and water
were removed from the protein structure and the CHARMM force field was used to minimize the
protein before docking. After the protein preparation, the active site for the docking studies was
defined with a range of 13.0 Å from the center of the known ligand. The obtained receptor was used
as the “Input Receptor.” Chlorimuron-ethyl and Compound 4e were chosen as the “Input Ligand.”
During the docking process, the top 10 conformations were saved for each ligand based on negative
CDOCKER_ENERGT value after energy minimization. Finally, the default values were used in DS 2.5
if not mentioned otherwise.
4. Conclusions
In conclusion, complete details of the synthesis of methyl (R)-N-benzoyl/dichloroacetyl-
thiazolidine-4-carboxylates from simple materials are presented in this paper. The bioactivities of
these compounds as herbicide safeners were examined in maize. The ALS activity assay confirmed the
positive effect of the title compounds on ALS activity. The bioactivity results showed that 4e exhibited
a significant protective effect to maize from injury of chlorimuron-ethyl, which was also confirmed by
molecular docking modeling.
Supplementary Materials: Supplementary Materials are available online. Table S1; Figures S2–S78 and
Tables S79–S100.
Acknowledgments: This work was supported by the National Nature Science Foundation of China (31401787,
31572042), the China Postdoctoral Science Foundation (2015M571384), the Natural Science Foundation of
Heilongjiang Province of China (C2015014, ZD2017002), and the “Academic Backbone” Project of Northeast
Agricultural University (16XG24). The authors are grateful to Jia-Zhong Li (Lanzhou University) for assistance
with molecular docking analyses.
Author Contributions: For L.-X.Z. and F.Y. developed the concept of the work. H.W. and Y.-L.Z. carried out
the synthetic work. Y.F. developed the molecular docking and comparison. Q.-R.W. and C.-Y.L. conducted the
bioactivity assay. H.W. and L.-X.Z. contributed to the discussion and analysis of the results. F.Y. wrote the paper.
Conflicts of Interest: The authors have no conflicts of interest to declare.
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Sample Availability: Samples of the compounds are available from the authors’ lab.
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