Research Article | Open Access
Design Synthesis and Biological Evaluation of Novel N-Nitro Acid Amide Derivatives as Lead Compounds of Herbicide
A series of N-nitro acid amide derivatives compounds were synthesized based on the active site of target acetohydroxyacid synthase (AHAS, EC: 184.108.40.206) 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.
Weed management is a perennial challenge for growers, and continual innovation is essential to maintain the effectiveness of management technologies . 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 , antifungal effects , and plant growth regulating activities . 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 . Urea functionality is an attractive structural unit; it has displayed a broad range of biological activities, and it has been implicated in herbicidal , antifungal [11–17], antibacterial , plant growth regulator , 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.
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 . 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
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.
|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.
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 . 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.
The interactions between candidate compounds and AHAS enzyme were predicted by molecular docking analysis. The structures of all compounds were sketched by Sybyl . The Surflex docking module was performed in this study; the docking procedure and parameters were set the same as in our previous reported work . 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.
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.
Xiaojuan Qi and Wenjie Tang both are co-first authors.
The authors declare that there are no competing interests regarding the publication of this paper.
Xiaojuan Qi and Wenjie Tang contributed equally to this paper.
This work was supported by the Natural Science Foundation of Hubei Province (no. 2015CFB442).
The spectra data, the 1H NMR, and IR spectra of target compounds are shown as below. 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.
- J. M. Green, “Review of glyphosate and ALS-inhibiting herbicide crop resistance and resistant weed management,” Weed Technology, vol. 21, no. 2, pp. 547–558, 2007.
- J. Hattori, R. Robert, D. Brown, G. Sunohara, and B. Miki, “Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance,” Molecular Genetics and Genomics, vol. 232, pp. 167–173, 1991.
- G. W. Haughn and C. R. A. Somerville, “A mutation causing imidazolinone resistance maps to the Csr1 locus of Arabidopsis thaliana,” Plant Physiology, vol. 92, no. 4, pp. 1081–1085, 1990.
- B. Cross, R. Hill, and W. H. Gastrock, “Phenylnitramine herbicides,” U.S. Patent 3844762, 1974.
- B. Cross, R. Hill, and H. D. Dawe, “Fungicidal phenylnitramines and new phenylnitramines,” U.S. Patent 4130645, 1978.
- T. D. O'Neal, P. R. Bhalla, and B. Cross, “Method for the control of stem growth and stem stiffness of graminaceous crops,” U.S. Patent 4643755, 1987.
- C. S. Chen, S. Z. Xu, X. G. Li, M. H. Cao, and J. P. Xiong, “Antimicrobial activity of N-nitro-N-phenyl-N′-pyridyl urea derivative and synthesize,” C.N. Patent 101584338, 2009.
- C. S. Chen, S. Z. Xu, X. L. Yue, H. J. Ma, J. H. Li, and Z. J. Wei, “N-nitro-2,4,6-Trichloroaniline of synthesis and application,” C.N. Patent 102311361, 2012.
- J. N. Jin, H. J. Ma, X. F. Cao et al., “The discovery of the novel lead compound of N-nitroureas target on acetohydroxyacid synthase,” Pesticide Biochemistry and Physiology, vol. 104, no. 3, pp. 218–223, 2012.
- P. A. Yonova and G. M. Stoilkova, “Synthesis and biological activity of urea and thiourea derivatives from 2-aminoheterocyclic compounds,” Journal of Plant Growth Regulation, vol. 23, no. 4, pp. 280–291, 2004.
- R. H. Tale, A. H. Rodge, G. D. Hatnapure, and A. P. Keche, “The novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of N-aryl urea: synthesis, anti-inflammatory, antibacterial and antifungal activity evaluation,” Bioorganic and Medicinal Chemistry Letters, vol. 21, no. 15, pp. 4648–4651, 2011.
- A. P. Keche, G. D. Hatnapure, R. H. Tale, A. H. Rodge, and V. M. Kamble, “Synthesis, anti-inflammatory and antimicrobial evaluation of novel 1-acetyl-3,5-diaryl-4,5-dihydro (1H) pyrazole derivatives bearing urea, thiourea and sulfonamide moieties,” Bioorganic and Medicinal Chemistry Letters, vol. 22, no. 21, pp. 6611–6615, 2012.
- G. D. Hatnapure, A. P. Keche, A. H. Rodge, S. S. Birajdar, R. H. Tale, and V. M. Kamble, “Synthesis and biological evaluation of novel piperazine derivatives of flavone as potent anti-inflammatory and antimicrobial agent,” Bioorganic and Medicinal Chemistry Letters, vol. 22, no. 20, pp. 6385–6390, 2012.
- A. P. Keche, G. D. Hatnapure, R. H. Tale, A. H. Rodge, S. S. Birajdar, and V. M. Kamble, “Synthesis, anti-inflammatory and antimicrobial evaluation of novel N1-(quinolin-4yl)ethane-1,2-diamine phenyl urea derivatives,” Medicinal Chemistry Research, vol. 22, no. 3, pp. 1480–1487, 2013.
- A. P. Keche, G. D. Hatnapure, R. H. Tale, A. H. Rodge, S. S. Birajdar, and V. M. Kamble, “A novel pyrimidine derivatives with aryl urea, thiourea and sulfonamide moieties: synthesis, anti-inflammatory and antimicrobial evaluation,” Bioorganic and Medicinal Chemistry Letters, vol. 22, no. 10, pp. 3445–3448, 2012.
- R. H. Tale, A. H. Rodge, G. D. Hatnapure, A. P. Keche, K. M. Patil, and R. P. Pawar, “The synthesis, anti-inflammatory, and anti-microbial activity evaluation of new series of 4-(3-arylureido)phenyl-1,4-dihydropyridine urea derivatives,” Medicinal Chemistry Research, vol. 22, no. 3, pp. 1450–1455, 2013.
- R. H. Tale, A. H. Rodge, G. D. Hatnapure, A. P. Keche, K. M. Patil, and R. P. Pawar, “The synthesis, anti-inflammatory and antimicrobial activity evaluation of novel thioanalogs of 3,4-dihydrothiopyrimidin-2(1H)-one derivatives of N-aryl urea,” Medicinal Chemistry Research, vol. 21, no. 12, pp. 4252–4260, 2012.
- S. A. Khan, N. Singh, and K. Saleem, “Synthesis, characterization and in vitro antibacterial activity of thiourea and urea derivatives of steroids,” European Journal of Medicinal Chemistry, vol. 43, no. 10, pp. 2272–2277, 2008.
- J. C. Emerson and L. H. Steimel, “Corrosion inhibitor composition enhanced by carbamide addition for scale removal on galvanized metal surfaces,” US 20070164258, 2007.
- J. N. Jin, X. J. Qi, D. D. Yao et al., “Rational design and screening study of novel lead compound based on acetohydroxyacid synthase structure,” Chemical Biology and Drug Design, vol. 84, no. 3, pp. 316–324, 2014.
- X. G. Li and C. S. Chen, “Synthesis of N-nitro urea compounds by non-phosgene method,” Journal of Huazhong Agricultural University, vol. 22, pp. 298–300, 2003.
- Z. J. Wei, C. S. Chen, L. B. Shi et al., “Novel N-nitroacetamide derivatives derived from 2,4-D: design, synthesis, bioevaluation, and prediction of mechanism of action,” Pesticide Biochemistry and Physiology, vol. 106, no. 1-2, pp. 68–74, 2013.
- E. H. White and R. J. Baumgarten, “N-nitroamides and N-nitrocarbamates. II. Amino acid derivatives,” The Journal of Organic Chemistry, vol. 29, no. 12, pp. 3636–3640, 1964.
- J. Ruppert, W. Welch, and A. N. Jain, “Automatic identification and representation of protein binding sites for molecular docking,” Protein Science, vol. 6, no. 3, pp. 524–533, 1997.
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.