Journal of Chemistry
Volume 2016 (2016), Article ID 8583765, 6 pages
http://dx.doi.org/10.1155/2016/8583765

Research Article

Design Synthesis and Biological Evaluation of Novel N-Nitro Acid Amide Derivatives as Lead Compounds of Herbicide

Xiaojuan Qi,¹ Wenjie Tang,¹ Shan Gao,¹ Min Gao,¹ Changshui Chen,¹ and Qingye Zhang1,2

¹College of Science, Huazhong Agricultural University, Wuhan 430070, China
²Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan 430070, China

Received 4 March 2016; Accepted 7 April 2016

Academic Editor: Teodorico C. Ramalho

Copyright © 2016 Xiaojuan Qi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A series of N-nitro acid amide derivatives compounds were synthesized based on the active site of target acetohydroxyacid synthase (AHAS, EC: 2.2.1.6) enzyme. All the structures of newly prepared compounds were thoroughly characterized by satisfied IR and ¹H NMR spectra. The IC₅₀ values against AHAS enzyme and EC₅₀ values for herbicidal activity against Amaranthus mangostanus L. and Sorghum sudanense of all synthesized target compounds were determined. The compounds II-10, II-21, and II-22 with IC₅₀ values of 7.09 mg/L, 9.07 mg/L, and 9.11 mg/L and the compounds II-8 and II-22 with EC₅₀ values of 9.87 mg/L and 19.88 mg/L against root of Amaranthus mangostanus L. and Sorghum sudanense were illustrated, respectively. Meanwhile, the possible reasons for the lower activity of compounds were analyzed by molecular docking prediction.

1. Introduction

Weed management is a perennial challenge for growers, and continual innovation is essential to maintain the effectiveness of management technologies [1]. With the increasing numbers of herbicide-resistant weeds emergence [2, 3], there is an urgent need of new, more selective, and even more potent herbicides to control the unwanted vegetables.

N-nitro substituted anilines have been demonstrated to display diversely biological activities, including herbicidal properties [4], antifungal effects [5], and plant growth regulating activities [6]. Our laboratory has been engaged over the years to explore N-nitrourea compounds, and some of these compounds have acquired patents [7, 8]. Based on the similarity of structure between sulfonylureas and N-nitrourea compounds, we supposed they have the same targeted enzyme. Our hypothesis has been confirmed combining molecular docking and biological activity assay in our previous study [9]. Urea functionality is an attractive structural unit; it has displayed a broad range of biological activities, and it has been implicated in herbicidal [10], antifungal [11–17], antibacterial [18], plant growth regulator [19], and so on.

To further explore a series of novel structural potential compounds as excellent herbicides, an idea that joined the different trisubstituted N-nitro aniline and benzamides, cinnamic amides together were performed in this study. Twenty-four N-nitro acid amide derivatives compounds were synthesized, and all the target compounds were subject to biological activity tests against AHAS enzyme and herbicidal activity tests against Amaranthus mangostanus L. and Sorghum sudanense. Meanwhile, the possible reasons for the lower activity of target compounds were analyzed by molecular docking prediction.

2. Materials and Methods

2.1. Instrumentation and Chemicals

All chemical reagents were purchased from Shenshi Chemical Instrumentation Network Ltd. (Wuhan, China) at AR grade. ¹H NMR spectra were recorded on an AM-600 MHz spectrometer (Bruker, Bremen, Germany) with tetramethylsilane as interior reference and CDCl₃ as the solvent. Infrared (IR) spectra were acquired on an AVATAR330 infrared spectrometer (Nicolet, Waltham, MA, USA) with KBr compression method. Melting points (m.p.) were determined on an X-4 digital display microscope melting point apparatus (Tech Instrument Co. Ltd., Beijing, China). The progress of the reactions was monitored by thin layer chromatography (TLC) on silica gel plates visualized with UV light.

2.2. General Procedure for Intermediate 1

The AC₂O (30 mmol) was added into a 50 mL round-bottomed flask and the temperature was controlled to 10°C, and HNO₃ (30 mmol) was slowly added over a period of 0.5 h, and then the reaction was stirred for about 0.5 h. Subsequently, the mixture was added into another 100 mL round-bottomed which contained 2,4,6-trisubstituted aniline (20 mmol) and acetic acid (25 mL) solvent over a period of 0.5 h, and the temperature was controlled to 15°C. The reactions were kept and stirred for about 1 h under the controlled temperature whilst monitoring its progress by TLC.

Then the mixtures were poured into ice water (200 mL), and a large amount of orange precipitate appeared. The orange precipitate was filtered under reduced pressure, dissolved into NaHCO₃ (5%, 30 mL) solution, and then HCl (2 mol/L) was added until the PH value of the solution reached 5-6 with a large amount of white precipitate that appeared again. Finally, the crude product was recrystallized from ethanol to get the intermediate 1. The spectra data of intermediate are included in the Supporting Information (see Supplementary Material available online at http://dx.doi.org/10.1155/2016/8583765).

2.3. General Procedure for the Synthesis Target Compounds I1-2 and II1–22

Different substituted carboxylic acid (5 mmol) and SOCl₂ (7.5 mmol) were dissolved into CH₂Cl₂ (10 mL) solvent, two drops of N, N-dimethylformamide (DMF) as catalyst and triethylamine (TEA) (5 mmol) as acid-binding agent were added into the above solution. The reaction was executed for 12 h at room temperature under continuous stirring, which was detected by TLC. Then, the redundant CH₂Cl₂ solvent has been removed by reduced pressure distillation at 40°C. Finally, the crude product of different substituted acryloyl chlorides was obtained.

The intermediate 1 (3.5 mmol) was dissolved into 10 mL ethyl acetate solvent in the 100 mL round-bottomed flask, and the solution was kept into ice water bath. Then the obtained different substituted acryloyl chlorides in the previous step were dropped into the mixture solution over a period of 0.5 h; the reaction was kept and stirred for about 2 h under the controlled temperature whilst monitoring its progress by TLC. At the end of reaction, target compounds were got from silica gel column with eluent which consisted of petroleum ether and ethyl acetate, respectively. The synthetic route for target compounds I1-2 and II1–22 is outlined in Scheme 1. The physicochemical properties of the target compounds were summarized in Table 1, and the spectra data, the ¹H NMR, and IR spectra of target compounds (as shown in Figure S1–Figure S48) are included in the Supporting Information.

Table 1: The structure and physical data summary of all target compounds.

Number
R1,2,3
R′
Yield/%

I-1
2,4,6-tri-Cl
Phenyl
45

I-2
2,4,6-tri-Br
-CH₃
42

Number
R1,2,3
R′′
Yield/%

II-1
2,4,6-tri-F
Phenyl
59

II-2
2,4,6-tri-F
4-OCH₃Ph
63

II-3
2,4,6-tri-F
4-ClPh
51

II-4
2,4,6-tri-F
2,4-di-ClPh
49

II-5
2,4,6-tri-F
3-NO₃Ph
45

II-6
2,4,6-tri-F
3-ClPh
43

II-7
2,4,6-tri-F
2-FPh
53

II-8
2,4,6-tri-F
4-CH₃Ph
49

II-9
2,4,6-tri-F
Thienyl
55

II-10
2,4,6-tri-Cl
Phenyl
57

II-11
2,4,6-tri-Cl
2,4-di-ClPh
62

II-12
2,4,6-tri-Cl
3-NO₃Ph
70

II-13
2,4,6-tri-Cl
3-ClPh
67

II-14
2,4,6-tri-Cl
2-FPh
44

II-15
2,4,6-tri-Cl
4-CH₃Ph
73

II-16
2,4,6-tri-Cl
Thienyl
56

II-17
2,4,6-tri-Br
Phenyl
53

II-18
2,4,6-tri-Br
4-OCH₃Ph
71

II-19
2,4,6-tri-Br
3-NO₂Ph
65

II-20
2,4,6-tri-Br
3-ClPh
41

II-21
2,4,6-tri-Br
2-FPh
57

II-22
2,4,6-tri-Br
Thienyl
50

Scheme 1: The synthetic routes of target compounds.

2.4. Determination of IC₅₀ Values for AHAS Enzyme

The AHAS enzyme assay of the target compounds was performed with the same method as our previously reported work [20]. All the IC₅₀ values of each target compound for AHAS enzyme were calculated as described in the reference.

2.5. Determination of the Herbicidal Activity

With reference to the “Agricultural Industry Standard of the People’s Republic of China Pesticide Indoor Bioassay Test Criteria (herbicides) AGAR Method,” the Amaranthus mangostanus L. and Sorghum sudanense as the target plant, the herbicidal activity of all target compounds were tested.

Each of the target compounds was dissolved in dimethyl sulfoxide (DMSO) solution to the concentration of 10000 mg/L, and then the solution was diluted with distilled water containing 0.1% Tween-80 to the concentration of 10, 50, 100, and 200 mg/L. The concentration of DMSO in all assays was maintained <1.5%. Twenty seeds of each plant including Amaranthus mangostanus L. and Sorghum sudanense were placed into a 9 cm diameter plate containing two pieces of filter paper and 9 mL solution of tested compound (0, 10, 50, 100, and 200 mg/L). The plate was placed in a greenhouse and allowed to germinate for 5 days at a temperature of 25 ± 1°C. The mixture of the same amount of water, DMSO, and Tween-80 was used as the control. Water was a blank control. Each treatment was repeated thrice and the lengths of roots and hypocotyl in each plate were measured and the means were calculated. DPS2000 standard statistical software for data processing system was utilized to perform the regression analysis for the EC₅₀ values of each target compound.

3. Results and Discussion

3.1. Synthesis

The synthetic route for target compounds I1-2 and II1–22 is outlined in Scheme 1. Traditionally, the key trisubstituted phenyl-nitramines intermediate 1 was synthesized using nitric acid in the presence of acetic anhydride with low yield according to the reference method [21, 22]. In this study, the acetyl nitric acid ester was prepared first, and then trisubstituted phenyl-amine was added to obtain the intermediates 1 with high yield.

Then different substituted acryloyl chlorides were prepared with SOCl₂ (7.5 mmol), using DMF as catalyst and TEA as acid-binding agent in the presence of CH₂Cl₂ solvent. The different substituted acryloyl chlorides could not be achieved for their water and air sensitivity. Hence, different trisubstituted phenyl-nitramines intermediates 1 were directly added into the above reaction mixture in the presence of TEA to afford target compounds in 41–73% yields (as shown in Table 1).

3.2. Evaluation IC₅₀ Values of Target Compounds against AHAS Enzyme

To estimate the IC₅₀ values of target compounds against AHAS enzyme, the method of previous reported bioactive assay of AHAS in vitro was utilized by detecting the change in absorbance of acetoin at 525 nm using UV-Vis absorption spectrophotometry. All of the candidate compounds were dissolved in DMSO and its concentration was controlled at 1.5% in final reaction volume. The commercialized herbicide nicosulfuron was taken as a reference. The detailed experimental data were summarized in Table 2.

Table 2: The IC₅₀ values results summary of candidate compounds against AHAS.

Number
IC₅₀ (mg/L)

Nicosulfuron
0.068

I-1
21.06

I-2
29.75

II-1
23.52

II-2
37.6

II-3
28.66

II-4
28.25

II-5
36.2

II-6
10.43

394.41

II-8
24.35

273.38

II-10
7.09

252.81

II-12
14.28

II-13
12.53

II-14
19.93

301.96

509.66

II-17c
14.64

393.12

II-19
17.01

II-20
18.15

II-21
9.07

II-22
9.11

The concentrations of the “” target compounds were 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, and 500 mg/L. The concentrations of the nicosulfuron were 0.01 mg/L, 0.025 mg/L, 0.05 mg/L, 0.075 mg/L, and 0.1 mg/L. The concentrations of the rest of the target compounds were 1 mg/L, 5 mg/L, 10 mg/L, 25 mg/L, and 50 mg/L.

The result showed that the range of IC₅₀ values of all the compounds was 7 mg/L–510 mg/L, and about three-quarters of IC₅₀ values were <40 mg/L. Different trisubstituted phenyl target compounds showed some difference in activity against AHAS enzyme; the activity of 2,4,6-tri-Br and 2,4,6-tri-Cl substituted phenyl were similar and generally better than 2,4,6-tri-F substituted phenyl target compound. The compounds II-10, II-21, and II-22 had the best biological activity in the all candidate compounds with IC₅₀ values of 7.09 mg/L, 9.07 mg/L, and 9.11 mg/L, respectively. Although the inhibition rate of lead compound was lower than the commercial herbicides, we considered it would be able to act as a lead compound for the further study of structural optimization so as to improve the inhibitory activity to AHAS.

3.3. Determination EC₅₀ Values of Target Compounds for Herbicidal Activity

The herbicidal activity of all the candidate compounds against Amaranthus mangostanus L. and Sorghum sudanense has been investigated at the dosages of 0, 10, 50, 100, and 200 mg/L according to the method described in the experimental section. The inhibition rate was increased with the concentration of the compound dose; most of the compounds EC₅₀ did not exceed 100 mg/L. All of the results of bioassay testing were summarized in Table 3. According to the determination EC₅₀ values for herbicidal activity, the target compounds exhibited higher herbicidal activity against both roots and hypocotyls of dicotyledonous plant Amaranthus mangostanus L. than monocotyledonous plant Sorghum sudanense. Most of target compounds showed better inhibition activity against root than that of hypocotyl of the two testing plants.

Table 3: Herbicidal activity summary of target compounds.

Number
Sorghum sudanense
Amaranthus mangostanus L.

Root
EC₅₀ (mg/L)
Hypocotyl
EC₅₀ (mg/L)
Root
EC₅₀ (mg/L)
Hypocotyl
EC₅₀ (mg/L)

I-1
110.25
102.61
48.85
148.20

I-2
36.92
96.34
27.97
99.98

II-1
89.48
70.87
129.06
91.16

II-2
46.76
187.57
18.90
80.82

II-3
62.11
108.38
87.53
129.29

II-4
70.96
113.90
102.56
50.33

II-5
87.88
113.62
25.64
98.45

II-6
63.51
52.78
35.06
81.78

II-7
21.22
152.11
42.37
92.66

II-8
37.12
56.53
9.87
147.68

II-9
85.70
88.28
44.83
108.84

II-10
44.19
106.26
74.19
58.54

II-11
97.25
139.01
35.91
102.58

II-12
58.14
61.84
61.89
108.30

II-13
77.33
80.23
17.46
92.12

II-14
58.56
109.33
53.83
124.35

II-15
84.02
105.05
22.97
126.53

II-16
75.31
61.97
91.90
102.88

II-17
59.59
134.57
64.50
68.14

II-18
80.45
69.28
20.54
253.95

II-19
35.09
104.52
53.44
193.37

II-20
77.62
165.19
20.51
103.68

II-21
82.60
111.93
75.74
156.46

II-22
19.88
111.08
114.64
55.52

The target compounds possessed higher herbicidal activity against root than that of hypocotyl. In particular, compounds II-8 and II-22 exhibited considerable herbicidal activity to root of Amaranthus mangostanus L. and Sorghum sudanense with EC₅₀ values of 9.87 mg/L and 19.89 mg/L, respectively. The activity of 2,4,6-tri-F substituted phenyl target compounds was better than 2,4,6-tri-Br and 2,4,6-tri-Cl substituted phenyl type compounds, such as compounds II-2, II-5, II-6, II-7, II-8, and II-9 with EC₅₀ values of 18.90 mg/L, 25.64 mg/L, 35.06 mg/L, 42.37 mg/L, 9.87 mg/L, and 44.83 mg/L against the root of Amaranthus mangostanus L.

3.4. Analysis for the Lower Activity of the Target Compounds

Most of the synthesized target compounds showed lower activity than the commercial herbicides and the inconsistence between IC₅₀ values and EC₅₀ values in the biological testing. For the discrepancy reason we performed some analysis based on the compound structure and binding conformation in the active site of AHAS target.

First of all, all the target compounds possess N-nitro amides part which is unstable and usually occur rearrangement as reported in [23]. The part of target compound may rearrange during activity testing as shown in Figure 1, and the rearrangement may result in the reduced inhibition activity.

Figure 1: The rearrangement in the N-nitro amides part of the target compound.

The interactions between candidate compounds and AHAS enzyme were predicted by molecular docking analysis. The structures of all compounds were sketched by Sybyl [24]. The Surflex docking module was performed in this study; the docking procedure and parameters were set the same as in our previous reported work [20]. Compared to the binding model of the known commercialized herbicide sulfonylureas and the characteristics of amino acids in the active site of AHAS, it was found that most of synthesized compounds form similar binding conformation in the active site of AHAS, as shown in Figure 2. However, further careful analysis found that the synthesized compounds cannot form fully orthogonal bend at the N-nitro amides group for strong conjugation formed between adjacent the phenyl and carbonyl. Although the N-nitro amides group and the adjacent aromatic ring are located in the entrance of channel leading to the active site, they could not very effectively prevent the substrate entrance. Thus the candidate compounds showed lower inhibition activity compared to sulfonylureas. Next, we would synthesize novel target compounds with canceled strong conjugation formed between adjacent phenyl and carbonyl.

(a)

(b)

Figure 2: The comparison between the docking conformation of the known commercialized herbicide sulfonylureas (a) and the target compound II-10 (b) in the active site of AHAS.

4. Conclusions

Through this study, twenty-four N-nitro acid amide derivatives compounds were synthesized based on the active site of target AHAS enzyme, and all of the target compounds were performed biological testing against AHAS enzyme and herbicidal activity against Amaranthus mangostanus L. and Sorghum sudanense. Compounds II-10, II-21, and II-22 with IC₅₀ values of 7.09 mg/L, 9.07 mg/L, and 9.11 mg/L were confirmed, respectively. Compounds II-8 and II-22 exhibited considerable herbicidal activity to root of Amaranthus mangostanus L. and Sorghum sudanense with EC₅₀ values of 9.87 mg/L and 19.88 mg/L, respectively. Further analysis was carried out based on the structure and interaction conformation in the active site of AHAS predicted by molecular docking to argue possible reason for the lower activity of target compounds. Based on the interaction mechanism analysis, further optimization for the lead compound would be performed to improve inhibition activity in our research group.

Disclosure

Xiaojuan Qi and Wenjie Tang both are co-first authors.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Authors’ Contributions

Xiaojuan Qi and Wenjie Tang contributed equally to this paper.

Acknowledgments

This work was supported by the Natural Science Foundation of Hubei Province (no. 2015CFB442).

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