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Cite This: ACS Omega 2019, 4, 9−14
Synthesis and Bacteriostatic Activities of Modified Flutriafol Derivatives Haoquan Guo,† Yuguo Dong,† Xuliang Zhou, Xinyu Lu, Xiaojun Zhu, Han Que, Zhipeng Wu, Kanghua Cheng, and Xiaoli Gu*
ACS Omega 2019.4:9-14. Downloaded from pubs.acs.org by 37.44.253.223 on 01/05/19. For personal use only.
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China ABSTRACT: This study aimed to evaluate the preservative ability of modified flutriafol derivatives against decay fungi. The bacteriostatic effect of flutriafol on Trichoderma viride was not efficient as expected. Flutriafol was modified as a parent substrate to improve its broad spectrum performance. Six triazole compounds were synthesized by Friedel−Crafts reaction, oxygen−sulfur−ylide reaction, and ring-opening addition reaction. The structures of the target products were determined by 1H NMR and MS. Antibacterial and antileakage tests were performed to optimize the most efficient agents among triazole target products.
Table 1. 1H NMR and MS of the Target Compounds
1. INTRODUCTION Common wood preservatives include coal phenolic oil wood preservative, oil-borne wood preservative, and waterborne wood preservative. Although phenolic oil wood preservative has some anticorrosion effect, the treated wood would have “penetration” phenomenon with its highly toxic feature, which poses a threat to the safety of human and animals.1 In spite of significant effects, oil-borne wood preservative is relatively expensive because of the large amount of organic solvent, which would also cause environmental pollution in some extent.2 With the advantage of low cost and high efficiency, waterborne wood preservative plays an important role nowadays such as chromium copper arsenate, alkaline copper quaternary, and so on. However, it is an environmental unfriendly process because the preservative contains heavy metal ions, for example, Cu, Cr, and so forth.3 Therefore, it is necessary to develop new heavy-metal-free waterborne wood preservatives. As a potential waterborne wood preservative without metallic elements, it generally refers to a series of compounds with 1,2,4triazole groups on the main chain, mainly including flutriafol, tebuconazole, propiconazole, furconazole-cis, and so on. Generally, triazole fungicides could be applied as pesticides with high efficiency, low toxicity, and broad spectrum, which are also utilized in wood preservatives recently.4−6 Wood treated with triazole could be burned directly without any toxic gas released that is harmful to the environment.7 The antibacterial action of triazole fungicides is derived from the 1,2,4-triazolyl group.8 Ergosterol plays a key role in maintaining cell membrane stability and ensuring membrane permeability. Ergosterol is produced by three-step reaction of lanosterol under the catalysis of cytochrome P450-14α sterol demethylase (ERG11 or CYP51 or P450-14DM).9 The N atom in the 4 position on the 1,2,4-triazolyl group can interact with the cytochrome P450-14α to restrict the synthesis © 2019 American Chemical Society
1
compounds a4
b4
c4
d4
e4
f4
H NMR and MS
H NMR (CDCl3): δ (ppm) 2.34 (3H, s, −CH3), 4.95−5.58 (2H, dd, −CH2−), 6.85−7.53 (6H, m, Ar-H), 7.84 (1H, s, Tr-H), 7.93 (2H, d, Ar-H), 8.13 (1H, s, Tr-H), MS (m/z): 324 [M + H]+ 1 H NMR (CDCl3): δ (ppm) 4.91−5.60 (2H, dd, −CH2−), 7.21−7.60 (6H, m, Ar-H), 8.03 (1H, s, Tr-H), 8.15 (2H, m, Ar-H), 8.27 (1H, s, Tr-H), MS (m/z): 345 [M + H]+ 1 H NMR (CDCl3): δ (ppm) 4.93−5.51 (2H, dd, −CH2−), 7.11−7.25 (1H, m, Ar-H), 7.27−7.34 (1H, m, Ar-H), 7.57−7.60 (2H, m, Ar-H), 7.63 (1H, m, Ar-H), 7.82 (1H, s, Tr-H), 7.90−7.93 (2H, m, Ar-H), 8.31 (1H, s, Tr-H), MS (m/z): 377 [M + H]+ 1 H NMR (CDCl3): δ (ppm) 2.35 (3H, s, −CH3), 5.01−5.12 (2H, s, −CH2−), 6.85−6.91 (2H, d, Ar-H), 7.24−7.31 (2H, d, Ar-H), 7.41−7.47 (2H, m, Ar-H), 7.69−7.73 (2H, d, Ar-H), 7.96 (1H, s, Tr-H), 8.17 (1H, s, Tr-H), MS (m/z): 359[M + H]+ 1 H NMR (CDCl3): δ (ppm) 4.68 (2H, s, −CH2−), 7.30−7.42 (4H, m, Ar-H), 7.64−7.79 (4H, m, Ar-H), 7.98 (1H, s, Tr-H), 8.25 (1H, s, Tr-H), MS (m/z): 381[M + H]+ 1 H NMR (CDCl3): δ (ppm) 4.67 (2H, s, −CH2−), 7.24−7.30 (2H, m, Ar-H), 7.61−7.65 (2H, m, Ar-H), 7.73−7.69 (3H, m, Ar-H), 7.97 (1H, s, Tr-H), 8.21 (1H, s, Tr-H), MS (m/z): 415 [M + H]+ 1
of ergosterol.10 Once the synthesis of ergosterol is blocked, the stability of bacterial cell membrane is drastically reduced, and the permeability is enhanced. The cell sap overflows and the cell dies, thus achieving the bactericidal effect.11 As an important kind of triazole fungicides, flutriafol has good bacteriostatic efficiency and broad spectrum, but its bacteriostatic ability to the Trichoderma viride is relatively poor. In order Received: September 28, 2018 Accepted: December 17, 2018 Published: January 2, 2019 9
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Table 2. 1H NMR Spectra of the Target Compound
Table 3. Antibacterial Test Data of Target Compoundsa bacterial growth diameter/mm target compounds
wt/%
flutriafol
0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5
a4
b4
c4
d4
e4
f4
1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0
Coriolus versicolor
Gloeophyllum trabeum
18.3 11.7 no growth no growth 56.4 35.3 23.1 22.7 17.3 14.1 11.9 19.4 17.2 no growth no growth 43.5 37.1 26.3 21.9 19.8 15.5 no growth no growth 12.5 no growth no growth no growth
37.1 26.5 20.7 15.2 59.3 47.4 38.7 45.8 33.7 29.8 22.3 30.7 25.5 10.3 no growth 53.7 41.9 36.7 27.1 38.3 21.4 no growth no growth 13.2 no growth no growth no growth
Table 4. Antibacterial Activity of 1.5% Mass Concentration Target Compounds
bacteriostat diameter/mm T. viride
Aspergillus niger
8.2 13.1 15.7 19.3 4.1 6.3 10.7 15.4 17.3 23.3 28.4 16.9 23.7 27.9 30.8
18.3 25.1 30.7 39.5 12.8 16.0 22.5 11.3 13.0 15.7 19.5 13.7 15.1 17.1 29.7 8.4 17.1 19.9 23.2 10.2 18.5 26.5 31.2 11.6 19.3 30.9 36.7
““ means no bacteriostatic effect.
a
required to modify according to the action mechanism of triazole bactericides. To our knowledge, this is the most detailed report of modified flutriafol derivatives to date.12,13
to improve the bacteriostatic performance and broad spectrum of flutriafol and its derivatives further, the original flutriafol is 10
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Table 5. Losses and Loss Rates of Different Agents under 200−500 g/m3 Drug Loading flu
c4
e4
f4
drug loading (g/m3)
loss (mg)
loss rate (%)
loss (mg)
loss rate (%)
loss (mg)
loss rate (%)
loss (mg)
loss rate (%)
200 300 400 500
0.902 1.049 1.294 1.594
14.08 10.59 10.11 9.96
0.869 0.937 1.208 1.489
13.58 9.76 9.44 9.30
0.941 1.245 1.602 2.103
14.70 12.97 12.52 13.15
0.920 1.320 1.808 2.271
14.37 13.75 14.13 14.19
2. RESULTS AND DISCUSSION 2.1. Structure Characterization of Target Compounds. Analysis of 1H NMR and MS, the structures of target products were consistent with the design structures (see Tables 1 and 2). 2.2. Antibacterial Performance of Target Compounds. The target compounds were immersed into methanol solution with mass concentration ranging from 0.5 to 2%, and the bacteriostatic test was carried out by the filter paper diffusion method (see Table 3). In order to visually investigate the antibacterial effect of the experimental synthetic products, the bacteriostatic test was photographed, and Table 4 indicated the bacteriostatic effect when the concentration of the drug was 1.5%. Compared with flutriafol, c4, e4, and f4 have better inhibitory effects on T. viride, and f4 is particularly effective against T. viride. In addition, c4, e4, and f4 also have good inhibitory effects on Coriolus versicolor and Gloeophyllum trabeum. It could be found that the antibacterial activities of c4, e4, and f4 are better than that of flutriafol. The results indicated that there were electronegative elements on the benzene ring, which was more conducive to improving the antibacterial ability of the products, which might be caused by the substituent group inducing effect on the benzene ring. The electron-withdrawing action on the benzene ring changed the electron cloud density of the triazolyl group, and the atom at the 4 position can be fully exposed to the complex with iron, thereby blocking the synthesis of ergosterol until the bacterial strain inactivated and bactericidal effect was achieved. In addition, it can be found that the stronger the electronwithdrawing effect on the benzene ring of the drug, the better the bacteriostatic effect of the drug. A4, b4, and c4 all contain a carboxyl group, which is a weak electron-withdrawing group. The bactericidal performance of products (d4, e4, and f4) was improved significantly because the weakly electron-withdrawing group was replaced. It also evidenced that the strong electronwithdrawing on the benzene ring enhanced the antibacterial ability of the compounds. 2.3. Antileakage Test of Target Compounds. Antileakage performance is also an important indicator of wood preservatives besides anti-mold and anti-corrosion properties. The effective agents c4, e4, and f4 were screened for the antileakage test.14 The anti-leakage properties of wood preservatives determine the effective life span of the treated wood in wet and rainy environments. According to the AWPA standard E11-06 (2011), the anti-leakage ability of target products was determined (see Table 5, Figures 1, and 2). It could be found that the anti-leaking performance of c4 was slightly better than other agents. The presence of carboxyl groups in c4 had a certain improvement on the anti-leaking ability of triazole fungicides to some extent. In detail, the carboxyl group could form a hydrogen bond with the water molecule, with the benzene ring structure. The hydrophilicity of the carboxyl group was not as strong as the hydrophobicity of the benzene ring, thus causing it to be insoluble in water. Otherwise,
Figure 1. Losses of different drug loadings.
Figure 2. Loss rates of different drug loadings.
the carboxyl group might form chemical bonds with the abundant hydroxyl groups in the wood which results in low loss. The sulfonic acid group in e4 and f4 belonged to a hydrophilic group, and its hydrophilic ability was stronger than that of the benzene ring, thereby causing a large loss of the agent.
3. CONCLUSIONS In summary, six modified flutriafol derivatives were successfully synthesized, and their structures were determined by 1H NMR and MS. The compounds can be stably stored at room temperature. Antibacterial and anti-leakage experiments were carried out on the target products. The results of antibacterial experiments showed that substituent groups on the benzene ring could affect the antibacterial properties. The presence of electron-donating 11
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and purchased from commercial sources. Monomethyl phthalate monochloride (self-made). Test species: white rot fungi: C. versicolor; brown rot fungus: G. trabeum; mold: A. niger, T. viride, provided by the Institute of Wood Protection of Nanjing Forestry University. Instruments: NMR spectra were obtained in deuterochloroform (CDCl3) and tetramethylsilane as internal standards on Bruker M-500 spectrometer operating at 400 MHz for 1H NMR. Mass spectra were determined on the 1100 ESI/MS instrument (ion source temperature 350 °C). The antibacterial tests were carried out in HHS-250B temperature humidity chambers (Nanjing Hengyu instrument equipment manufacturing Co., Ltd.). 4.2. Synthetic Procedure. The synthesis procedure of the target compound in this experiment was followed by the Friedel−Craft reaction, the oxygen−sulfide−ylide reaction, and the ring-opening addition reaction.
groups on the benzene ring will decrease the antibacterial performance of the products, while the presence of electronwithdrawing groups on the benzene ring will improve the antibacterial performance of the products. The stronger the electron-withdrawing group on the benzene ring, the better is its germicidal performance. The result of the anti-leakage experiment showed that the presence of sulfonic group has slightly decreased the anti-leakage ability of triazole fungicides because of its hydrophilicity.
4. MATERIAL AND METHODS 4.1. Material. Experimental reagents: AlCl3 (anhydrous), toluene, dimethyl sulfoxide, chlorobenzene, 1,3-dichlorobenzene, benzoyl chloride, H2SO4 (98%), dichloromethane, NaSO4 (anhydrous), (CH3)3SOI, NaH (60% paraffin oil dispersion), petroleum ether, ethyl acetate, NaCl, 1,2,4-triazole sodium salt, K2CO3, DMF, methanol, all of which were of analytical grade
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4.3.4. Synthesis of 4-(1-Hydroxy-1-p-tolyl-2-(1,2,4triazole)ethyl)benzoic Acid (a4). Added 13 g of methyl 4-(1hydroxy-1-p-tolyl-2-(1,2,4-triazole)ethyl)benzoate (a3), 300 mL of deionized water and 5 g of sodium hydroxide to 500 mL flask and stirred at about 80 °C for 12 h under reflux. After the reaction completed, the mixture was cooled, filtered, and dilute hydrochloric acid was added dropwise to the filtrate until the solution was weakly acidic, and the residue was filtered and retained. After column chromatography (petroleum ether/ethyl acetate = 1:3), 8 g of white solid was collected. The synthesis method of 4-(1-hydroxy-1-p-chlorophenyl-2(1,2,4-triazole)ethyl)benzoic acid (b4), 4-(1-hydroxy-1-(2,4dichlorophenyl)-2-(1,2,4-triazole)ethyl)benzoic acid (c4) is the same as a4. 4.3.5. Synthesis of 4-(1-Hydroxy-1-p-tolyl-2-(1,2,4triazole)ethyl)benzoic Acid (d4). 1-Phenyl-1-(p-tolyl)-2(1,2,4-triazolyl)ethanol (d3, 10 g), 7 g of concentrated sulfuric acid was added to a 100 mL flask and connected to a water separator. Stirred at 100 °C for 8 h. After the reaction was completed, the mixture was poured into hot water to be cooled, and crystals were precipitated. The filter residue was retained by filtration, and the residue was dissolved in water, and recrystallized by adding HCl to obtain 11 g of white solid. The synthesis method of 4-(1-hydroxy-1-p-chlorophenyl-2(1,2,4-triazole)ethyl)benzoic acid (e4), 4-(1-hydroxy-1-(2,4dichlorophenyl)-2-(1,2,4-triazole)ethyl)benzoic acid (f4) is the same as d4. 4.4. Test of Antibacterial Properties. The target compounds a4, b4, c4, d4, e4, and f4 were subjected to bacteriostatic test, and the experiment was carried out by using the filter paper spreading method, and the comparative test was set. The antibacterial standards are as follows: inhibition zone diameter > 7 mm or hyphal growth diameter < 25 mm, the target compound has an antibacterial effect; inhibition zone diameter ≤ 7 mm or hyphal growth diameter ≥ 25 mm, the target compound has no antibacterial effect.
4.3. Synthesis Method. 4.3.1. Synthesis of Methyl 4-(4Methylbenzophenone)benzoate (a1). Anhydrous aluminum trichloride (8 g) and 27 g of dried toluene were successively added to a 250 mL three-necked flask in turn and stirred. Monomethyl phthalate monochloride (13 g) was slowly added dropwise, and the mixture was refluxed, heated to 70 °C for 3 h, and then heated to 105 °C for 6 h. After the mixture was cooled, it was poured into 100 mL of glacial hydrochloric acid and stirred well, and extracted three times with dichloromethane. The mixture was mixed and washed with saturated brine for 3 times. The phases were dried overnight by adding an appropriate amount of anhydrous sodium sulfate. Filtration, rotary evaporation, and desolvation to give pale yellow thick liquid 15 g. The synthesis method of methyl 4-(p-chlorobenzophenone)benzoate (b1), methyl 4-(2,4-dichlorobenzophenone)benzoate (c1), 1-p-tolyl-1-phenyl methanone (d1), 1-p-chlorophenyl-1phenyl methanone (e1), 1-(2,4-dichlorophenyl)-1-phenyl methanone (f1) is the same as a1. 4.3.2. Synthesis of Methyl 4-(4-p-Tolyl-2-oxiranyl)benzoate (a2).15 Under ice water bath protection conditions, 22 g of trimethylsulfoxonium sulfoxide and 4 g of NaH were sequentially added to a 250 mL three-necked flask through nitrogen gas protection, stirring was started, and an appropriate amount of dimethyl sulfoxide was slowly added dropwise until no bubbles were produced. Methyl 4-(4-methylbenzophenone)benzoate (a1, 15 g) was dissolved in tetrahydrofuran, added dropwise to a three-necked flask, and the nitrogen protection was removed and shaken for 3 min in the ultrasonic. After the shaking was completed, it was placed in 55 °C water bath and stirred for 5 h. After end of the reaction, the mixture was poured into an appropriate amount of distilled water, and the mixture was extracted three times with ethyl acetate. The organic phase was combined, washed three times with saturated brine and filtered, and then added an appropriate amount of anhydrous sodium sulfate. After desolvation, 14 g of dark brown liquid was collected. The synthesis method of methyl 4-(4-p-chlorophenyl-2oxiranyl)benzoate (b2), methyl 4-(2,4-dichlorophenyl-2oxiranyl)benzoate (c2), 2-phenyl-2-p-tolyl oxirane (d2), 2phenyl-2-p-chlorophenyl oxirane (e2), 2-phenyl-2-(2, 4dichlorophenyl)oxirane (f2) is the same as a2. 4.3.3. Synthesis of Methyl 4-(1-Hydroxy-1-p-tolyl-2-(1,2,4triazole)ethyl)benzoate (a3).16 Sodium 1,2,4-triazole (9 g), 13 g of potassium carbonate, and 60 mL of N-N-dimethylformamide (DMF) were added to a 250 mL three-necked flask and stirred well. Methyl 4-(4-p-tolyl-2-oxiranyl)benzoate (a2, 14.32 g) was dissolved in 40 mL of DMF and added dropwise to the mixture. After the reaction ended, the reaction mixture was poured into ice water, hydrochloric acid was added dropwise and stirred until no bubbles were produced. After extracting three times with ethyl acetate, the organic phase was combined, and the fraction washed with saturated brine, and then an appropriate amount of anhydrous sodium sulfate was added. The mixture was allowed to stand overnight, filtered, and evaporated to dissolve. Dark brown thick liquid (13 g) was collected. The synthesis method of methyl 4-(1-hydroxy-1-p-chlorophenyl-2-(1,2,4-triazole)ethyl)benzoate (b3), 4-(1-hydroxy-1(2,4)-dichlorophenyl)-2-((1,2,4-triazole)ethyl)benzoic acid methyl ester (c3), 1-phenyl-1-(p-tolyl)-2-(1,2,4-triazolyl)-ethanol (d3), 1-phenyl-1-(p-chlorophenyl)-2-(1,2,4-triazolyl)ethanol (e3), 1-phenyl-1- The (2,4-dichlorophenyl)-2-(1,2,4triazolyl)ethanol (f3) is the same as a3.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (X.G.). ORCID
Xiaoli Gu: 0000-0001-8588-0358 Author Contributions †
H.G. and Y.D. contributed equally. All authors discussed the results and contributed to the final manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial supports from the National Natural Science Foundation of China (no. 21774059), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the opening funding of Jiangsu Key Lab of Biomassbased Green Fuels and Chemicals, and College Students’ Practice and Innovation Training Project (201810298058Z).
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REFERENCES
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(3) Thompson, R. The Chemistry of Wood Preservation; The Royal Society of Chemistry Press: Cambridge, 1991; pp 193−223. (4) American Wood Protection Association P14-08, Standard for tebuconazole (TEB). (5) American Wood Protection Association P42-08, Standard for propiconazole (PPZ). (6) American Wood Protection Association P46-08, Standard for cyproconazloe (CPZ). (7) Fenner, K.; Canonica, S.; Wackett, L. P.; Elsner, M. Evaluating pesticide degradation in the environment: blind spots and emerging opportunities. Science 2013, 341, 752−758. (8) Lv, X.; Pan, L.; Wang, J.; Lu, W.; Yan, W.; Zhu, Y.; Xu, Y.; Guo, M.; Zhuang, S. Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity. Environ. Pollut. 2017, 222, 504−512. (9) Duff, S. M. G.; Davila-Aponte, J.; Thompson, R. The development of a medium throughput assay for lanosterol synthase from Leptosphaeria nodorum: Comparison of the enzyme from L. nodorum, Saccharomyces cerevisiae, and two species of Fusarium. Pestic. Biochem. Physiol. 2005, 83, 97−106. (10) Pernak, J.; Markiewicz, B.; Łęgosz, B.; Walkiewicz, F.; Gwiazdowski, R.; Praczyk, T. Known triazole fungicidesa new trick. RSC Adv. 2015, 5, 9695−9702. (11) Ren, J.; Jin, X.; Zhang, Q.; Zheng, Y.; Lin, D.; Yu, Y. Fungicides induced triazole-resistance in Aspergillus fumigatus associated with mutations of TR46/Y121F/T289A and its appearance in agricultural fields. J. Hazard. Mater. 2017, 326, 54−60. (12) Chen, L.; Wu, Q.; Fan, Z.; et al. Design, Synthesis and Biological Evaluation of Isothiazole Based 1,2,4-Trizaole Derivatives. Chin. J. Chem. 2018, 36, 731−736. (13) Worthington, P. A. Synthesis of 1,2,4-triazole compounds related to the fungicides flutriafol and hexaconazole. Pestic. Sci. 1991, 31, 457− 498. (14) Chopra, D.; Mohan, T. P.; Rao, K. S. 2-(2, 4-dichlorophenyl)-1(1H-1, 2, 4-triazol -1-yl)-hexan-2-ol (hexaconazole). Acta Crystallogr., Sect. E: Struct. Rep. Online 2004, 60, o2410−o2412. (15) Hatch, M. J. Synthesis of oxiranes from aqueous solutions of simple alkyl, allyl, and benzylsulfonium salts. J. Org. Chem. 1969, 34, 2133−2137. (16) Wang, C.; Wu, Q.; Wu, C. Application of dispersionsolidification liquid-liquid microextraction for the determination of triazole fungicides in environmental water samples by high-performance liquid chromatography. J. Hazard. Mater. 2011, 185, 71−76.
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