Design, Synthesis, and Safener Activity of Novel Methyl (R)-N-Benzoyl/Dichloroacetyl-Thiazolidine-4-Carboxylates

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.

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 [1]. ALS-inhibiting herbicides prevent the synthesis of isoleucine, leucine, and valine, resulting in subsequent weed death [2]. 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 [3]. 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 [4,5]. However, chlorimuron-ethyl treatments have been reported to cause phytotoxicity in beans, leading to reduced plant height and shoot dry matter [6]. Residues of this herbicide in soil may also inhibit the growth of succeeding crops, such as maize [7]. In order to reduce the injury, various methods have been reported, including restricted use of long residual herbicides, developing new herbicides and so on [8]. In addition to these 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 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].

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).

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 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.

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, 2, was cyclized with ketones, 1, to generate thiazolidines, 3, with Et₃N as the attaching acid agent under nitrogen atmosphere. The title compounds, 4, were prepared by direct acylation of the corresponding 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. Yields of title compounds.

Compound	R1	R2	R3	Yield/%	Compound	R1	R2	R3	Yield/%
4a	(CH2)5		p-NO2C6H4	91	4k	(CH2)4		o-ClC6H4	70
4b	(CH2)5		2,4-Cl2C6H3	81	4l	CH3	CH3	2,4-Cl2C6H3	78
4c	(CH2)5		p-ClC6H4	77	4m	CH3	CH3	p-ClC6H4	72
4d	(CH2)5		m-CH3C6H4	69	4n	CH3	CH3	p-NO2C6H4	87
4e	(CH2)5		o-OCH3C6H4	71	4o	CH3	CH3	m-CH3C6H4	65
4f	(CH2)5		C6H5	75	4p	CH3	CH3	o-OCH3C6H4	69
4g	(CH2)4		p-ClC6H4	67	4q	CH3	CH3	o-ClC6H4	71
4h	(CH2)4		p-NO2C6H4	83	4r	(CH2)5		CHCl2	48
4i	(CH2)4		2,4-Cl2C6H3	72	4s	CH3	CH3	CHCl2	41
4j	(CH2)4		m-CH3C6H4	65

, 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 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.

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).

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–s to maize from injury of chlorimuron-ethyl were evaluated, as shown in

Table 2. Protective effects of Compound 4 to maize ^a,b,c.

Compound	Recovery of Plant Height (%)	Recovery of Root Length (%)	Recovery of Plant Weight (%)	Recovery of Root Weight (%)
R-28725	86.22 ± 1.37	84.73 ± 0.98	87.47 ± 1.12	83.61 ± 1.98
4a	42.90 ± 1.22	60.52 ± 1.37	45.96 ± 1.11	68.11 ± 1.82
4b	59.96 ± 1.09	45.53 ± 1.27	47.16 ± 1.24	68.48 ± 0.63
4c	80.33 ± 0.82	86.48 ± 0.87	94.61 ± 1.43	85.79 ± 1.62
4d	67.97 ± 1.02	41.97 ± 1.31	78.25 ± 0.98	41.98 ± 1.05
4e	89.08 ± 0.65	91.20 ± 0.89	98.92 ± 1.09	93.70 ± 1.22
4f	62.05 ± 0.21	64.07 ± 0.33	73.75 ± 1.39	51.56 ± 0.52
4g	85.86 ± 0.97	87.53 ± 1.43	85.62 ± 1.26	83.39 ± 0.92
4h	57.08 ± 0.69	46.83 ± 1.07	41.26 ± 0.73	59.74 ± 0.62
4i	45.64 ± 1.62	55.60 ± 0.55	53.93 ± 1.29	57.56 ± 0.54
4j	57.41 ± 1.26	66.61 ± 1.92	71.15 ± 1.42	67.59 ± 1.65
4k	41.82 ± 0.68	46.24 ± 0.23	55.83 ± 1.05	55.13 ± 0.13
4l	20.60 ± 0.69	15.09 ± 0.32	15.04 ± 1.22	25.27 ± 1.24
4m	48.99 ± 0.57	66.04 ± 1.13	48.27 ± 1.06	47.83 ± 0.36
4n	25.02 ± 0.92	29.16 ± 0.91	17.01 ± 0.61	24.94 ± 0.85
4o	34.12 ± 1.32	38.30 ± 0.45	39.58 ± 1.02	56.35 ± 1.13
4p	76.00 ± 1.16	61.09 ± 1.75	68.93 ± 1.26	55.57 ± 1.44
4q	23.20 ± 0.53	14.56 ± 0.32	19.04 ± 1.42	25.25 ± 0.54
4r	57.74 ± 1.31	39.46 ± 1.44	31.46 ± 0.88	46.34 ± 1.32
4s	33.22 ± 1.65	36.30 ± 1.44	45.75 ± 1.54	37.89 ± 0.74

^a Data are means of three replicates, mean ± standard deviation; ^b $\mathrm{Recovery}\mathrm{Rate}(\%)=\frac{\mathrm{Treatment}\mathrm{with}\mathrm{compounds}—\mathrm{Treatment}\mathrm{with}\mathrm{Chlorimur}\mathrm{on}-\mathrm{ethyl}}{\mathrm{Contrast}—\mathrm{Treatment}\mathrm{with}\mathrm{Chlorimuron}-\mathrm{ethyl}}$; ^c Contrast was treated by water.

. 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 μg/kg. 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–s showed that substituents R¹, R², and R³ played a significant role in the safener activity of all title compounds. As shown in Table 2, biological activity tests revealed that Compounds 4a–k with ring substituents at R¹ and R² displayed better safener activity than Compounds 4l–q with Me substituents at R¹ and R². Similarly, Compound 4r 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, 4b, 4c, 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 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.

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. Effect of Compounds 4a–s and chlorimuron-ethyl on the acetolactate synthase (ALS) activity of maize.

Treatment	ALS Activity (nmol h−1 mg−1 Protein) a	Treatment	ALS Activity (nmol h−1 mg−1 Protein) a	Treatment	ALS Activity (nmol h−1 mg−1 Protein) a
Control b	0.091 ± 0.002	chlorimuron-ethyl + 4f	0.075 ± 0.003	chlorimuron-ethyl + 4n	0.059 ± 0.003
chlorimuron-ethyl	0.046 ± 0.003	chlorimuron-ethyl + 4g	0.079 ± 0.003	chlorimuron-ethyl + 4o	0.064 ± 0.001
chlorimuron-ethyl + R-28725	0.084 ± 0.002	chlorimuron-ethyl + 4h	0.071 ± 0.002	chlorimuron-ethyl + 4p	0.071 ± 0.001
chlorimuron-ethyl + 4a	0.064 ± 0.001	chlorimuron-ethyl + 4i	0.068 ± 0.002	chlorimuron-ethyl + 4q	0.055 ± 0.002
chlorimuron-ethyl + 4b	0.062 ± 0.004	chlorimuron-ethyl + 4j	0.077 ± 0.002	chlorimuron-ethyl + 4r	0.064 ± 0.002
chlorimuron-ethyl + 4c	0.081 ± 0.002	chlorimuron-ethyl + 4k	0.068 ± 0.001	chlorimuron-ethyl + 4s	0.062 ± 0.001
chlorimuron-ethyl + 4d	0.065 ± 0.002	chlorimuron-ethyl + 4l	0.058 ± 0.001
chlorimuron-ethyl + 4e	0.085 ± 0.002	chlorimuron-ethyl + 4m	0.068 ± 0.003

^a Data are means of three replicates, mean ± standard deviation; ^b Control was treated by water.

, 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.

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.

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 caused chlorimuron-ethyl to lose its effect. This function may be the detoxification mechanism of the title compound.

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, China). ¹H 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 × 20 mL) and dried using anhydrous sodium sulfate. The solvent was removed under reduced pressure to yield crude methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylate (4). The title compounds were purified by column chromatography. The spectra data of compound 4 are presented in Supplementary Materials (Figures S2–S77).

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): ν (cm⁻¹) 2914 (C-H), 1727, 1642 (C=O), 1515 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 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 (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): ν (cm⁻¹) 2937 (C-H), 1747, 1639 (C=O), 1442–1550 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 170.78, 168.66, 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): ν (cm⁻¹) 2834–2951 (C-H), 1737, 1637 (C=O), 1436 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): 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): ν (cm⁻¹) 2844–2931 (C-H), 1728,1637 (C=O), 1372 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 170.68, 168.12, 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): ν (cm⁻¹) 2842–3079 (C-H), 1723, 1639 (C=O), 1521 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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, 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): ν (cm⁻¹) 2842–2989 (C-H), 1733, 1632 (C=O), 1437–1574 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 170.27, 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.

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): ν (cm⁻¹) 2846–2984 (C-H), 1727, 1626 (C=O), 1407 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (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.

Methyl (R)-4-(o-chlorobenzoyl)-1-thia-4-azaspiro[4.4]nonane-3-carboxylate (4k). White solid, m.p. 111–112 °C. Yield 70%. IR (KBr): ν (cm⁻¹) 2844–2936 (C-H), 1727, 1628 (C=O), 1423 (C=C); ¹H 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): ν (cm⁻¹) 2906–3066 (C-H), 1723, 1643 (C=O), 1573 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 170.23, 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): ν (cm⁻¹) 2906–3087 (C-H), 1728, 1630 (C=O), 1428–1589 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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, 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): ν (cm⁻¹) 2914–3034. (C-H), 1713, 1628 (C=O), 1421.85 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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, 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): ν (cm⁻¹) 2843–2979 (C-H), 1740, 1644 (C=O), 1437–1599 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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 (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): ν (cm⁻¹) 2927–3004 (C-H), 1743, 1645 (C=O), 1393–1437 (C=C); ¹H NMR (400 MHz, CDCl₃): δ (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): δ (ppm) 170.45, 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): ν (cm⁻¹) 2937 (C-H), 1734, 1682 (C=O); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 5.92 (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, 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): ν (cm⁻¹) 2927–3039 (C-H), 1736, 1685 (C=O); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 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 (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: [email protected] 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 Kα) = 0.399 mm⁻¹, D_c = 1.386 g/cm³, 19190 reflections measured (6.18° ≤ 2θ ≤ 56.578°), 3722 unique (R_int = 0.0228, R_σ = 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 4 are presented in Supplementary Materials (Figure S78 and Tables S79–S100).

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 added to the extraction medium (3.0 mL) and (NH₄)₂SO₄ (1.5 g). The mixture was centrifuged at 25,000× g for 20 min after staying at 4 °C for 2 h. The enzyme solution was obtained by dissolving the 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.