Research Article Received: 18 November 2013
Revised: 27 January 2014
Accepted article published: 5 May 2014
Published online in Wiley Online Library: 4 July 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3824
Synthesis and antifungal evaluation of a series of maleimides Xiao-Long Chen,a* Li-Jun Zhang,a Fu-Ge Li,a Yong-Xian Fan,a Wei-Ping Wang,b Bao-Ju Lib and Yin-Chu Shena Abstract: BACKGROUND: Maleimides, both natural and synthesised, have good biological activities. In a continuous effort to discover new maleimides with good antifungal activities, the authors have synthesised a series of 3,4-dichloro-, 3-methyl and non-substituted maleimides based on previous studies. The compounds were biologically evaluated against the fungal pathogen Sclerotinia sclorotiorum. RESULTS: Of the 63 compounds evaluated, 25 compounds had interesting inhibitory potency with EC50 < 10 𝛍g mL−1 . N-(3,5-Dichlorophenyl)-3,4-dichloromaleimide (EC50 = 1.11 𝛍g mL−1 ) and N-octyl-3-methylmaleimide (EC50 = 1.01 𝛍g mL−1 ) were more potent than the commercial fungicide dicloran (EC50 = 1.72 𝛍g mL−1 ). The results showed that compounds exhibiting log P values within the range 2.4–3.0 displayed the best results in terms of fungicidal activity, and this seemed, therefore, to be the optimum range for this physicochemical parameter. CONCLUSION: The present work demonstrates that some maleimides can be used as potential lead compounds for developing novel antifungal agents against S. sclerotiorum. © 2014 Society of Chemical Industry Keywords: maleimides; Sclerotinia sclerotiorum; antifungal agents; fungicide resistance
1
INTRODUCTION
Pest Manag Sci 2015; 71: 433–440
∗
Correspondence to: Xiao-Long Chen, Institute of Fermentation Engineering, College of Biological and Environmental Engineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, China. E-mail: [email protected]
a Institute of Fermentation Engineering, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China b Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
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Sclerotinia sclerotiorum (Lib.) de Bary is a filamentous ascomycete fungus with a wide host range and geographical distribution. It has been one of the most non-specific, omnivorous and successful plant pathogens. Its host range includes approximately 400 primarily dicotyledonous plant species.1,2 Therefore, S. sclerotiorum has been a notorious plant-pathogenic fungus, causing vast economic damage during crop cultivation as well as in harvested products, such as oilseed rape,3 blueberry,4 alfalfa,5 lettuce6,7 and so on, particularly in China and Australia.8 There have been several agrochemicals to control S. sclerotiorum, including carbendazim and the dicarboximide fungicides (procymidone, iprodione and vinclozolin).9 Unfortunately, the extensive use of these fungicides selects for resistant individuals within a population and leads to control failures.10,11 By using mixtures of fungicides with different modes of action, the selection for fungicide resistance could be minimised, so the effectiveness of disease control could be increased.12 However, the resistance problem could not be completely solved. Therefore, it is an urgent challenge to develop novel, highly effective and simple antifungal agents with low toxicities for the disease. In the literature it has been reported that natural products with maleic anhydride, such as tautomycin and tautomycetin, possess good biological activities,13 especially antifungal activities. In addition, natural products with a maleimide core moiety (Fig. 1), such as showdomycin, aqabamycins and arcyruarubins, have been found to have various biological activities.14 – 16 On the other hand, synthesised compounds with maleic anhydride or maleimide
have also been reported to have excellent biological activities.17 For example, N-(4-fluorophenyl)-dichloromaleimide (a commercial fungicide in Japan), N-(4-methylphenyl)-dichloromaleimide and N-cyclohexylmaleimide are well known for effective fungicidal activity against various plant pathogens.18 – 20 Furthermore, the animal toxicity of N-(4-fluorophenyl)-dichloromaleimide is extremely low, with LD50 > 15 000 mg kg−1 orally to mice,21 while it was much higher for procymidone and dicloran (LD50 > 2500 mg kg−1 and >1500 mg kg−1 respectively). Moreover, in recent years, some synthetic maleimides have been confirmed to have potent antifungal activities against human fungal pathogens.22,23 Therefore, compounds with a maleic anhydride or maleimide core could be considered to be attractive prototype compounds for the development of new antifungal agents. However, there have been few studies related to the synthesis of maleimides and the evaluation of their antifungal activities against plant pathogens, except for investigations conducted by the present authors’ research group in recent decades.
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X-L Chen et al. O N CnH2n+1
Cl N CnH2n+1
N CnH2n+1
Cl
O
Figure 1. Antifungal template for maleimide derivatives (the maleimide structure is the main active centre, and R1 , R2 and R3 are different substituents that might influence the bioactivity of the compound).
O
O
O
O
I-1: n = 4
II-1: n = 4
III-1 : n = 4
I-2 : n = 5
II-2: n = 5
III-2 : n = 5
I-3 : n = 6
II-3 : n = 6
III-3 : n = 6
I-4:: n = 8
II-4 : n = 8
III-4 : n = 8
I-5:: n = 12
II-5 : n = 12
III-5 : n = 12
O
O
O Cl
N (CH2)n
N (CH2)n
N (CH2)n Cl
In previous studies,2,13,17,24 some maleic anhydrides and maleimides, including tautomycin and its derivatives, were prepared and screened against S. sclerotiorum and Botrytis cinerea. The results showed that the antifungal activity of compounds with a maleimide moiety was better than that of compounds with maleic anhydride. In the present study, 63 maleimide derivatives (Fig. 2) were synthesised, including N-substituted maleimide derivatives I-1 to I-21, N-substituted 3-methyl-maleimide derivatives II-1 to II-21 and 3,4-dichloro-maleimide derivatives III-1 to III-21. In addition, the structure–activity relationship was also investigated (Scheme 1). This research aims to obtain several maleimide derivatives with higher activity and lower toxicity as novel fungicides against S. sclerotiorum.
2
MATERIALS AND METHODS
2.1 General Dicloran (96%, TC) and procymidone (50%, WP) used as internal reference fungicides were purchased from Sigma-Aldrich (St Louis, MO) and Sumitomo Chemicals (Tokyo, Japan) respectively. Citraconic anhydride and maleic anhydride were purchased from Aladdin (Shanghai, China). 2,3-Dichloromaleic anhydride was synthesised according to the procedure previously described in the literature.25 Alkyamines, phenylalkylamines and substituted anilines were also purchased from Aladdin. All other chemicals used in this work were analytical grade and commercially available on the local market in China (Hangzhou, China). Melting points were taken in open capillary tubes on a WRS-1A electrothermal melting point apparatus (Wuhan, China) and were uncorrected. 1 H NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer (Bruker, Italy) in CDCl3 solutions using tetramethylsilane (TMS) as the internal standard. Electrospray ionisation–mass spectra (ESI-MS) were measured on a mass spectrometer (LCQ/Advantage; Thermo Fisher Scientific, Waltham, MA). IR spectra were recorded on a Nicolet 6700 infrared spectrophotometer (Thermo Fisher Scientific) by the KBr pellet method. Column chromatographic separations were carried out on silica gel (ACME, Mumbai, India) (60–120 mesh).
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2.2 Synthesis of compounds The N-substituted maleimides were synthesised by employing two methods according to an improved procedure based on reported methods2,21,22,24 using maleic anhydrides and amines as the starting materials (Scheme 2). The general procedure for synthesising compounds I-1 to I-5 (Scheme 2, path A) was as follows. In the first step, a three-necked, round-bottomed flask equipped with a magnetic stirrer, a
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O
O
O I-6: n = 1
II-6: n = 1
III-6: n = 1
I-7: n = 2
II-7: n = 2
III-7: n = 2
I-8: n = 3
II-8: n = 3
III-8: n = 3
I-9: R = H
II-9: R = H
III-9: R = H
I-10: R = 4 -CH3
II-10: R = 4 -CH3
III-10: R = 4-CH3
I-11: R = 4-F
II-11: R = 4-F
III-11: R = 4-F
I-12: R = 4 -Cl
II-12: R = 4-Cl
III-12: R = 4-Cl
I-13: R = 2,6-(CH3)2
II-13: R = 2,6-(CH3)2
III-13: R = 2,6-(CH3)2
I-14: R = 2,6-(C2H5)2
II-14: R = 2,6-(C2H5)2
III-14: R = 2,6-(C2H5)2
I-15: R = 2-CH3-3-Cl
II-15: R = 2-CH3-3-Cl
III-15: R = 2-CH3-3-Cl
I-16: R = 2-CH3-5-Cl
II-16: R = 2-CH3-5-Cl
III-16: R = 2-CH3-5-Cl
I-17: R = 2-CH3-3-NO2
II-17: R = 2-CH3-3-NO2 III-17: R = 2-CH3-3-NO2
I-18: R = 3,5-(Cl)2
II-18: R = 3,5-(Cl) 2
III-18: R = 3,5-(Cl)2
I-19: R = 3,4,5-(F)3
II-19: R = 3,4,5-(F)3
III-19: R = 3,4,5-(F)3
I-20
II-20
I-21
II-21
III-20
III-21
Figure 2. Chemical structures of N-substituted maleimides.
dropping funnel and a reflux condenser was charged with maleic anhydride (1.96 g, 0.02 mol) dissolved in 10 mL of toluene. A solution of appropriate alkylamines (1 equiv., 0.02 mol) dissolved in 10 mL of toluene was added dropwise from the dropping funnel to the flask over a period of about 10 min at room temperature with stirring. The reaction mixture was heated to 55–70 ∘ C and maintained at that temperature for 3–4.5 h. For the second step, anhydrous sodium acetate (0.08 g, 0.96 mmol) and triethylamine (1.0 mL, 7.2 mmol) were added sequentially. Then the reaction mixture was heated to 101 ∘ C and was refluxed for an
© 2014 Society of Chemical Industry
Pest Manag Sci 2015; 71: 433–440
Synthesis and antifungal evaluation of maleimides
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O
R1
O
R1 O
+ NH2 R0
N R0
R2
R2
O
O I : R1=H, R2=H II : R1=H, R2=CH3 III : R1=Cl, R2=Cl
Sclerotinia sclerotiorum
63 compounds O Cl
EC50 = 1.01 µg/mL
N C8H17 Cl O Cl
O
EC50 = 1.11 µg/mL
N O
Cl
Scheme 1. Compounds with maleimide with good antifungal activities against S. sclerotiorum.
R1
R1
O
P +
NH2 R
a
O
A ath
3
R
2
O OH N R3 H O
Path
O
O N R3
R2
O
c
2
R
b
R1
B
Scheme 2. Synthesis of N-substituted maleimides. Path A: a – toluene, 25–65 ∘ C, 2–8 h; b – CH3 COONa, Et3 N, 101 ∘ C, 10–24 h. Path B: c – CH3 COOH.
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The compounds I-1 to I-12, I-21, II-6 to II-9 and III-1 to III-12 have been previously reported.22,26 – 31 The structures of all the compounds were confirmed by IR, ESI-MS and 1 H NMR. The physical and spectroscopic data of the new compounds are shown below. N-(2,6-Dimethylphenyl)-maleimide I-13. Yield 71%; light-yellow crystals; mp 188.8–190.4 ∘ C. IR (KBr) cm−1 3465, 3088, 2932, 1714, 1576, 1453, 1386, 1090, 852, 720, 703, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.25 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 6.90 (s, 2H), 2.13 (s, 6H). ESI-MS m/z (%) 201.1 (100) [M]+ , 183.1 (98), 144.1 (40), 118.1 (15), 91.1 (23), 76.1 (18), 54 (15), 39.1 (8). N-(2,6-Diethylphenyl)-maleimide I-14. Yield 81%; brown powder crystals; mp 192.8–194.1 ∘ C. IR (KBr) cm−1 3469, 3091, 2934, 1719, 1570, 1453, 1386, 1090, 853, 722, 700, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (t, J = 7.7 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 6.90 (s, 2H), 2.42 (q, J = 7.6 Hz, 4H), 1.15 (t, J = 9.5, 5.7 Hz, 6H). ESI-MS m/z (%) 229.1 (100) [M]+ , 196.1 (48), 158.1 (30), 132.1 (42), 115.1 (19), 77.1 (20), 54.1 (18), 37.0 (7).
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additional 10–12 h. N-Alkyl maleimides were separated by silica column chromatography to afford products I-1 to I-5 in good yields. The general procedure for synthesising compounds I-6 to I-21, II-1 to II-21 and III-1 to III-21 (Scheme 2, path B) was as follows. A stirred solution of anhydride (maleic anhydride or citraconic anhydride or 2,3-dichloromaleic anhydride) in acetic acid was added with appropriate amines (alkyamines, phenylalkylamines or substituted anilines, 0.01 mol) in 20 mL of acetic acid. The reaction mixture was heated to 115–130 ∘ C, and the mixture was refluxed for 3 h. The reaction mixture was then cooled to 15–25 ∘ C. If products were solids, crystals were collected by filtration and rinsed with water or acetic acid. Then the products were recrystallised from acetone solution. If the products were in oil, the solvent was distilled under reduced pressure. The mixture was separated by silica column chromatography to afford products in good yields. For the sake of clarity, the synthesised compounds were grouped according to their structural features, as shown in Fig. 2.
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N-(2-Methyl-3-chloro-phenyl)-maleimide I-15. Yield 70%; light-yellow powder; crystals; mp 160.5–162.0 ∘ C. IR (KBr) cm−1 3468, 3096, 1702, 1533, 1383, 1363, 1094, 730, 715, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.48 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.91 (s, 2H), 2.19 (s, 3H). ESI-MS m/z (%) 223.0 (29), 221.0 (88) [M]+ , 205.0 (33), 203.0 (100), 186.0 (10), 164.0 (30), 130.1 (23), 102.1 (12), 77.1 (22), 54.0 (28), 37.0(3). N-(2-Methyl-5-chloro-phenyl)-maleimide I-16. Yield 72%; light-yellow powder crystals; mp 164.1–165.4 ∘ C. IR (KBr) cm−1 3468, 3091, 1705, 1530, 1384, 1362, 1092, 731, 714, 516. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.34 (dd, J = 8.3, 2.2 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1 H), 7.15 (d, J = 2.1 Hz, 1H) 6.89 (s, 2H), 2.14 (s, 3H). ESI-MS m/z (%) 223.0 (33), 221.0 (100) [M]+ , 205.0 (33), 203.0 (99), 186.0 (6), 164.0 (28), 130.1 (26), 102.0 (11), 77.1 (17), 54.0 (23), 37.1 (5). N-(2-Methyl-3-nitro-phenyl)-maleimide I-17. Yield 73%; yellow powder crystals; mp 155.8–157.0 ∘ C. IR (KBr) cm−1 3463, 3099, 1709, 1534, 1387, 1363, 1094, 730, 715, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.99 (dd, J = 8.2, 1.1 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.40 (dd, J = 7.9, 1.0 Hz, 1H), 6.95 (s, 2H), 2.32 (s, 3H). ESI-MS m/z (%) 232.1 (3) [M]+ , 215.1 (100), 187.0 (48), 160.0 (32), 131.1 (17), 104.1 (20), 77.1 (42), 54.1 (34), 37.0 (6). N-(3,5-Dichlorophenyl)-maleimide I-18. Yield 75%; yellow powder crystals; mp 182.5–183.0 ∘ C. IR (KBr) cm−1 3463, 3094, 2926, 1716, 1577, 1454, 1387, 1092, 854, 724, 702, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (s, 3H), 6.90 (s, 2H). ESI-MS m/z (%) 245.0 (11), 243.0 (67), 241.0 (100) [M]+ , 197.0 (15), 171.0 (13), 150.0 (7), 124.0 (15), 82.0 (7), 54.0 (26), 32.1 (4). N-(3,4,5-Trifluorophenyl)-maleimide I-19. Yield 72%; white powder crystals; mp 153.4–165.6 ∘ C. IR (KBr) cm−1 3478, 3081, 1718, 1627, 1521, 1458, 1323, 1231, 1097, 1040, 852, 723, 690, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.20–7.13 (m, 2H) 6.89 (s, 2H). ESI-MS m/z (%) 227.0 (100) [M]+ , 183.0 (15), 157.0 (20), 118.0 (7), 95.0 (4), 75.0 (4), 54.0 (18). N-iso-Butyl-maleimide I-20. Yield 70%; light-yellow plate crystals; mp 52.8–53.9 ∘ C. IR (KBr) cm−1 3457, 2963, 2929, 2874, 1711, 1439, 1408, 1375, 1059, 734, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.71 (s, 2H), 3.34 (d, J = 7.4 Hz, 2H), 2.02 (dp, J = 14.0, 6.9 Hz, 1H), 0.90 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 153.1 (49) [M]+ , 110.0 (100), 82.0 (38), 54.1 (21), 41.1 (11). N-Butyl-3-methylmaleimide II-1. Yield 73%; light-yellow oil. IR (KBr) cm−1 3844, 2962, 2936, 2870, 1705, 1441, 1404, 1379, 1055, 730, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.7 Hz, 1H), 3.48 (t, J = 7.3 Hz, 2H), 2.07 (d, J = 1.8 Hz, 3H), 1.58–1.51 (m, 2H), 1.34–1.26 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). ESI-MS m/z (%) 167.1 (35) [M]+ , 138.1 (8), 124.1 (100), 94.0 (12), 69.1 (17), 56.1 (17), 39.1 (18). N-Amyl-3-methylmaleimide II-2. Yield 79%; light-yellow oil. IR (KBr) cm−1 3452, 2952, 2930, 2862, 1701, 1441, 1403, 1373, 1061, 734, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.31 (q, J = 1.7 Hz, 1H), 3.50–3.47 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.61–1.54 (m, 2H), 1.37–1.29 (m, 2H), 1.29–1.22 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). ESI-MS m/z (%) 181.1 (38) [M]+ , 138.1 (18),124.1 (100), 96.1 (15), 70.1 (12), 56.1 (15), 39.1(15). N-Hexyl-3-methylmaleimide II-3. Yield 80%; light-yellow oil. IR (KBr) cm−1 3459, 2952, 2931, 2859, 1702, 1442, 1407, 1377, 1064, 734, 520. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.8 Hz, 1H), 3.50–3.46 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.56 (p, J = 7.3 Hz, 2H), 1.31–1.25 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H). ESI-MS m/z (%) 193.1 (38) [M]+ , 150.1 (50), 112.1 (100), 96.1 (35), 81.1 (38), 67.1 (42), 53.1 (22), 39.1 (47). N-Octyl-3-methylmaleimide II-4. Yield 78%; light-yellow oil. IR (KBr) cm−1 3346, 2929, 2851, 1704, 1440, 1406, 1372, 1072, 733, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.8 Hz, 1H), 3.49–3.46
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(m, 2H), 2.07 (d, J = 1.8 Hz, 3H), 1.56 (p, J = 7.3 Hz, 2H), 1.30–1.25 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H). ESI-MS m/z (%) 223.2 (47) [M]+ , 180.1 (16), 152.1 (11), 124.1 (100), 96.1 (18), 69.1 (16), 41.1 (18). N-Dodecyl-3-methylmaleimide II-5. Yield 81%; brown plate crystals; mp 70.8–71.4 ∘ C. IR (KBr) cm−1 3459, 2924, 2858, 1712, 1440, 1403, 1375, 1058, 733, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.7 Hz, 1H), 3.50–3.47 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.59–1.55 (m, 2H), 1.30–1.25 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H). ESI-MS m/z (%) 279.1 (100) [M]+ , 236.2 (14), 152.1 (14), 124.0 (93), 96.1 (14), 69.1 (14), 41.1 (15). N-(4-Tolyl)-3-methylmaleimide II-10. Yield 82%; light-yellow powder crystals; mp 114.1–115.9 ∘ C. IR (KBr) cm−1 3448, 3042, 2921, 1709, 1518, 1397, 1091, 821, 732, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.30 (d, J = 8.2 Hz, 2H), 7.24–7.20 (m, 2H), 6.51 (q, J = 1.7 Hz, 1H), 2.41 (s, 3H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 201.1 (100) [M]+ , 172.1 (8), 157.1 (10), 117.1 (12), 104.0 (8), 91.1 (8) , 68.1 (12), 39.1 (12). N-(4-Fluorophenyl)-3-methylmaleimide II-11. Yield 79%; white powder crystals; mp 155.8–156.4 ∘ C. IR (KBr) cm−1 3468, 1709, 1492, 1397, 1089, 833, 735. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.36–7.32 (m, 2H), 7.18–7.14 (m, 2H), 6.50 (q, J = 1.7 Hz, 1H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 205.1 (100) [M]+ , 161.1 (15), 148.1 (12), 135.1 (13), 121.1 (12), 109.1 (17), 82.1 (9), 68.1 (14), 40.1 (12). N-(4-Chlorophenyl)-3-methylmaleimide II-12. Yield 83%; white powder crystals; mp 142.3–143.9 ∘ C. IR (KBr) cm−1 3464, 1709, 1492, 1395, 1089, 831, 731. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.46–7.42 (m, 2H), 7.35–7.31 (m, 2H), 6.51 (q, J = 1.7 Hz, 1H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 223.0 (33), 221.0 (100) [M]+ , 207.1 (9), 177.1 (16), 153.0 (22), 137.0 (22), 125.1 (22), 90.1 (24), 68.1 (42), 40.1 (51). N-(2,6-Dimethylphenyl)-3-methylmaleimide II-13. Yield 76%; brown powder crystals; mp 190.8–192.0 ∘ C. IR (KBr) cm−1 3460, 3099, 2922, 1711, 1570, 1459, 1380, 1097, 858, 728, 701, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.27–7.22 (m, 1H), 7.15 (d, J = 7.6 Hz, 2H), 6.52 (q, J = 1.8 Hz, 1H), 2.21 (d, J = 1.8 Hz, 3H), 2.13 (s, 6H). ESI-MS m/z (%) 215.1 (100) [M]+ , 197.1 (61), 168.1 (22), 144.1 (31), 131.1 (17), 91. 1 (20), 77.1 (14), 39.1 (18). N-(2,6-Diethylphenyl)-3-methylmaleimide II-14. Yield 80%; brown granular crystals; mp 195.8–197.3 ∘ C. IR (KBr) cm−1 3458, 3081, 2925, 1714, 1575, 1452, 1384, 1090, 857, 720, 704, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.36 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 6.53 (q, J = 1.7 Hz, 1H), 2.42 (q, J = 7.6 Hz, 4H), 2.21 (d, J = 1.8 Hz, 3H), 1.15 (t, J = 7.6 Hz, 6H). ESI-MS m/z (%) 243.1 (100) [M]+ , 214.1 (34), 198.1 (31), 186.1 (14), 172.1 (13), 158.1 (14) , 132.1 (28), 117.1 (27), 91.1 (14), 77.1 (14), 39.1 (15). N-(2-Methyl-3-chloro-phenyl)-3-methylmaleimide II-15. Yield 80%; light-yellow crystals; mp 149.8–151.4 ∘ C. IR (KBr) cm−1 3459, 3111, 1719, 1532, 1384, 1361, 1092, 733, 711, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 6.52 (q, J = 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H), 2.14 (s, 3H). ESI-MS m/z (%): 237.1 (33), 235.1 (100) [M]+ , 219.1 (19), 217.1 (57), 190.0 (17), 164.0 (24), 138.0 (14), 77.0 (22), 39.1 (27). N-(2-Methyl-5-chloro-phenyl)-3-methylmaleimide II-16. Yield 77%; dark-yellow crystals; mp 150.3–152.0 ∘ C. IR (KBr) cm−1 3469, 3090, 1702, 1532, 1384, 1360, 1098, 730, 717, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 6.52 (q, J = 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H), 2.14 (s, 3H). ESI-MS m/z (%) 237.1 (33), 235.1 (100) [M]+ , 219.1 (17), 217.1 (52), 192.0 (14), 154.1 (27), 172.1 (14), 154.1 (26), 138.0 (13), 102.1 (10), 89.1 (13), 77.1 (17), 51.1 (9), 39.1 (25). N-(2-Methyl-3-nitro-phenyl)-3-methylmaleimide II-17. Yield 79%; light-yellow powder crystals; mp 153.1–154.9 ∘ C. IR (KBr)
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Pest Manag Sci 2015; 71: 433–440
J = 8.3 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 2.17 (s, 3H). ESI-MS m/z (%) 295.0 (4), 293.0 (33), 291.0 (99), 289.0 (100) [M]+ , 271.0 (50), 254.0 (15), 226.0 (49), 210.0 (38), 166.0 (34), 132.1 (25), 87.0 (57), 63.0 (10), 51.1 (15). N-(2-Methyl-3-nitro-phenyl)-3,4-dichloromaleimide III-17. Yield 83%; light-yellow powder crystals; mp 168.8–169.9 ∘ C. IR (KBr) cm−1 3468, 3090, 1701, 1538, 1384, 1360, 1099, 730, 715, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 8.03 (d, J = 8.2 Hz, 1H), 7.53–7.48 (m, 1H), 7.43 (dd, J = 7.9, 1.1 Hz, 1H), 2.35 (s, 3H). ESI-MS m/z (%) 300.0 (6) [M]+ , 287.0 (11), 285.0 (67), 283.0 (100), 255.0 (46), 191.0 (60), 161.0 (23), 148.0 (20), 106.0 (21), 87 (86), 77.1 (69), 63.0 (18), 51.1 (34), 39.1 (9). N-(3,5-Dichlorophenyl)-3,4-dichloromaleimide III-18. Yield 90%; yellow crystals; mp 190.2–191.4 ∘ C. IR (KBr) cm−1 3463, 3094, 2926, 1716, 1577, 1454, 1387, 1092, 854, 724, 702, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.43 (t, J = 1.8 Hz, 1H), 7.36 (d, J = 1.8 Hz, 2H). ESI-MS m/z (%) 316.9 (1), 314.9 (11), 312.9 (50), 310.9 (100), 308.9 (75) [M]+ , 229.9 (27), 186.9 (14), 124.0 (28), 87.0 (34). N-(3,4,5-Trifluorophenyl)-3,4-dichloromaleimide III-19. Yield 75%; white plate crystals; mp 158.9–160.4 ∘ C. IR (KBr) cm−1 3478, 3089, 1710, 1624, 1537, 1458, 1329, 1230, 1095, 1040, 856, 722, 694, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.20–7.13 (m, 2H). ESI-MS m/z (%) 299.0 (33), 297.0 (67), 295.0 (100) [M]+ , 216.0 (42), 173.0 (25), 145.0 (22), 121.9 (38), 87.0 (70), 75.0 (9). N-iso-Butyl-3,4-dichloromaleimide III-20. Yield 71%; light-yellow crystals; mp 63.8–64.5 ∘ C. IR (KBr) cm−1 3462, 2957, 2925, 2874, 1711, 1439, 1408, 1375, 1059, 734, 525. 1 H NMR (500 MHz, CDCl3 ) 𝛿 3.43 (d, J = 7.4 Hz, 2H), 2.04 (dp, J = 13.9, 6.9 Hz, 1H), 0.92 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 225.0 (4), 223.0 (24), 221.0 (38) [M]+ , 182.0 (33), 180.0 (67), 178.0 (100), 151.0 (8), 116.0 (28), 87.0 (58), 56.1 (72), 39.1 (38). N-Cyclohexyl-3,4-dichloromaleimide III-21. Yield 71%; brown powder crystals; mp 67.2–68.5 ∘ C. IR (KBr) cm−1 3439, 2925, 2862, 1720, 1398, 1380, 1089, 733, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 4.00 (tt, J = 12.4, 3.9 Hz, 1H), 2.04 (qd, J = 12.5, 3.4 Hz, 2H), 1.86 (dd, J = 16.2, 2.6 Hz, 2H), 1.71–1.65 (m, 2H), 1.38–1.17 (m, 4H). ESI-MS m/z (%) 251.0 (6), 249.0 (36), 247.0 (53) [M]+ , 204.0 (82), 171.0 (33), 169.0 (67), 167.0 (100), 128.0 (15), 81.1 (98), 41.1 (18).
2.3 Bioassays S. sclerotiorum was isolated from sclerotia collected from a diseased plant of oilseed rape in Zhejiang, China, as described before.2 The culture medium was potato dextrose agar (PDA). The in vitro antifungal activities of 63 compounds against S. sclerotiorum were assayed on solid medium PDA using the mycelium growth rate method.2 These maleimides, dicloran and procymidone were dissolved in 0.1% (m/v) Tween-80 while mixing with PDA to generate a series of concentrations in the final test solution of 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25, 50, 100 and 200 μg mL−1 by the twofold broth dilution method. The compound-free agar with 0.1% (m/v) Tween-80 solution was employed as the blank control. The PDA medium was poured into 9 cm diameter petri dishes (10 mL plate−1 ), which were then inoculated with 6 mm diameter mycelial plugs from the edge of 36-hour-old S. sclerotiorum. Plates in three replicates were used for each test concentration. The dishes were incubated at 23 ∘ C for 36 h, the time by which the growth of the control would have reached the edge of the plate. Then the diameter of each colony was measured by making two measurements at right angles. Tests were repeated twice.24 The inhibition of growth against S. sclerotiorum was calculated using
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cm−1 3468, 308, 1704, 1530, 1382, 1361, 1090, 730, 715, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.97 (dd, J = 8.2, 1.1 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 7.8, 1.0 Hz, 1H), 6.57 (q, J = 1.7 Hz, 1H), 2.32 (s, 3H), 2.23 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 246.1(5) [M]+ , 229.1 (100), 201.1 (7), 174.1 (53), 145.1 (19), 133 (9), 117.1 (18), 96.0 (19), 77.1 (40), 55.1 (19), 39.1 (38). N-(3,5-Dichlorophenyl)-3-methylmaleimide II-18. Yield 81%; brown crystals; mp 170.8–173.2 ∘ C. IR (KBr) cm−1 3469, 3091, 2924, 1712, 1573, 1450, 1387, 1092, 854, 724, 701, 525. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.43 (t, J = 1.8 Hz, 1H), 7.36 (d, J = 1.8 Hz, 2H), 6.53 (q, J = 1.7 Hz, 1H), 2.21 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 259.0 (11), 257.0 (67), 255.0 (100) [M]+ , 211.0 (14), 187.1 (15), 124.0 (16), 68.1 (27), 40.1 (20). N-(3,4,5-Trifluorophenyl)-3-methylmaleimide II-19. Yield 72%; white powder crystals; mp 158.8–160.6 ∘ C. IR (KBr) cm−1 3482, 3080, 1710, 1628, 1535, 1458, 1321, 1238, 1090, 1042, 850, 728, 693, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.22–7.15 (m, 2H), 6.52 (dd, J = 3.6, 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 241.1 (100) [M]+ , 197.0 (15), 173.0 (18), 157.0 (7), 145.0 (18), 131.0 (4), 118.0 (6), 95.0 (8), 68.0 (24), 40.1 (23). N-iso-Butyl-3-methylmaleimide II-20. Yield 71%; light-yellow oil. IR (KBr) cm−1 3449, 2959, 2929, 2872, 1721, 1439, 1408, 1375, 1059, 734, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.31 (q, J = 1.8 Hz, 1H), 3.30 (d, J = 7.4 Hz, 2H), 2.08 (d, J = 1.9 Hz, 3H), 2.00 (td, J = 13.9, 6.9 Hz, 1H), 0.87 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 167.1 (35) [M]+ , 134.1 (100), 96.1 (12), 56.1 (15), 39.1 (15). N-Cyclohexyl-3-methylmaleimide II-21. Yield 70%; light-yellow powder crystals; mp 65.6–66.5 ∘ C. IR (KBr) cm−1 3449, 2932, 2862, 1703, 1400, 1381, 1089, 733, 526. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.26 (q, J = 1.8 Hz, 1H), 3.88 (tt, J = 12.3, 3.9 Hz, 1H), 2.05 (d, J = 1.8 Hz, 3H), 2.04 (qd, J = 12.5, 3.4 Hz, 2H), 1.86 (dd, J = 16.2, 2.6 Hz, 2H), 1.71–1.65 (m, 2H), 1.38–1.17 (m, 4H). ESI-MS m/z (%) 193.1 (43) [M]+ , 150.1 (53), 112.0 (100), 96.1 (38), 81.1 (41), 67.1 (45), 53.1 (22), 39.1 (45). N-(2,6-Dimethylphenyl)-3,4-dichloromaleimide III-13. Yield 86%; light-yellow powder crystals; mp 200.8–201.6 ∘ C. IR (KBr) cm−1 3469, 3084, 2923, 1706, 1574, 1464, 1389, 1082, 859, 726, 704, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.30 (d, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 2.15 (s, 6H). ESI-MS m/z (%) 273.0 (11), 271.0 (67), 269.0 (100) [M]+ , 251.0 (42), 226.0 (13), 206.1 (16), 190.0 (63), 146.1 (16), 132.1 (9), 118.1 (16), 105.1 (19), 87.0 (25), 77.1 (18), 65.1 (11), 51.1 (10), 39.1 (10). N-(2,6-Diethylphenyl)-3,4-dichloromaleimide III-14. Yield 87%; brown powder crystals; mp 204.1–205.8 ∘ C. IR (KBr) cm−1 3472, 3098, 2928, 1713, 1579, 1458, 1389, 1098, 859, 726, 706, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.41 (t, J = 7.7 Hz, 1H), 7.23 (d, J = 7.7 Hz, 2H), 2.43 (q, J = 7.6 Hz, 4H), 1.17 (t, J = 7.6 Hz, 6H). EI-MS m/z (%) 181 (32). ESI-MS m/z (%) 301.1 (11), 299.1 (67), 297.1 (100) [M]+ , 282.0 (29), 268.0 (32), 250.0 (8), 232.0 (17), 132.1 (75), 117.1 (50), 105.1 (8), 87.0 (17), 65.1 (4), 39.1 (3). N-(2-Methyl-3-chloro-phenyl)-3,4-dichloromaleimide III-15. Yield 78%; light-yellow powder crystals; mp 162.3–164.1 ∘ C. IR (KBr) cm−1 3473, 3090, 1706, 1539, 1382, 1361, 1090, 730, 715, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.52 (dd, J = 8.1, 0.7 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.9, 0.6 Hz, 1H), 2.22 (s, 3H). ESI-MS m/z (%) 295.0 (4), 293.0 (34), 291.0 (99), 289.0 (100) [M]+ , 271.0 (60), 254.0 (27), 226.0 (33), 210.0 (50), 166.0 (20), 132.0 (17), 87.0 (50), 77.1 (23), 63.0 (10), 51.1 (14), 39.1 (8). N-(2-Methyl-5-chloro-phenyl)-3,4-dichloromaleimide III-16. Yield 77%; yellow powder crystals; mp 160.1–161.4 ∘ C. IR (KBr) cm−1 3460, 3084, 1712, 1537, 1380, 1368, 1090, 734, 715, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (dd, J = 8.3, 2.1 Hz, 1H), 7.30 (d,
www.soci.org the following formula:
However, path B was a facile method by a one-step reaction to prepare 3-methylmaleimides and 3,4-dichloromaleimides with a shorter reaction time, especially for the synthesis of 3,4-dichloromaleimides. Furthermore, path B had higher yields and an easier isolation method when compared with path A.
) ( ) ( Inhibition of growth (%) = Dck − D ∕ Dck − 6 × 100 where Dck represents the average colony diameter of the blank control, D represents the average colony diameter of test compounds and 6 represents the diameter of the inoculum plug (in mm). With a logit-log transformation, the concentration–response curves were linearised by regression and were then used to calculate the effective concentration (EC) values for different control levels. The antifungal activities were assessed with EC50 values.32,33 The log P values were calculated by ChemDraw 8.0.
3.2 Antifungal activities of compounds against S. sclerotiorum In vitro antifungal activities of 63 N-substituted maleimides and dicloran and procymidone were evaluated against S. sclerotiorum, and the EC50 values were calculated. The results are shown in Table 1. In the study, compounds with EC50 > 500 μg mL−1 were considered to be inactive, compounds with EC50 = 100–250 μg mL−1 were considered to be moderately active and compounds with EC50 ≤ 50 μg mL−1 were considered to be highly active. In particular, compounds displaying EC50 ≤ μg mL−1 were considered to be of great interest for further development. Results indicated that most of the 63 compounds showed good inhibition to the mycelial growth of S. sclerotiorum, except for III-5 which possessed marginal or null activity. The starting materials (maleic anhydride, citraconic anhydride and 2,3-dichloromaleic anhydride) were almost inactive against S. sclerotiorum. Of the 63 compounds, 25 compounds had interesting inhibitory potency with EC50 < 10 μg mL−1 . In particular, compounds II-18 and III-4, the EC50 values of which were 1.11 and 1.01 μg mL−1 respectively, were more effective than the commercial fungicide dicloran (EC50 = 1.72 μg mL−1 ) but less active than procymidone WP (EC50 = 0.44 μg mL−1 ). Moreover, only seven compounds (II-11, II-15, III-5, III-7, III-8, III-17, III-19) had EC50 values above 100 μg mL−1 . In summary, the other 56 synthetic compounds displayed moderate to excellent antifungal activities against S. sclerotiorum.
2.4 Statistical analysis Analysis of variance (ANOVA) (SAS Institute, Cary, NC) was applied to determine the statistical significance of differences among treatments in each bioassay. The data on growth inhibition of S. sclerotiorum by the synthesised compounds in each replicate were arcsine transformed to angular data prior to ANOVA. Means for different treatments in each bioassay or trial were separated using the least significant difference test at the P = 0.05 level.
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RESULTS AND DISCUSSION
3.1 Synthesis of compounds Sixty-three compounds were synthesised by path A (I-1 to I-5) and path B shown in Scheme 2 with good yields (above 70%). Among them, there were five novel compounds (II-16, II-17, II-19, III-17 and III-19). As for the two synthetic methods, path A was used to prepare N-alkylmaleimides (I-1 to I-5) by two-step reactions, dehydration and a ring-closing reaction, as reported in the literature.24
Table 1. In vitro antifungal activities of maleimides against S. sclerotiorum Compounds
EC50
log P
I-1 I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9 I-10 I-11 I-12 I-13 I-14 I-15 I-16 I-17 I-18 I-19 I-20 I-21 Maleicanhydride Dicloran
5.55 8.46 9.96 8.42 16.02 6.88 9.19 15.08 15.27 23.41 17.76 7.80 6.03 2.45 11.17 10.46 10.47 6.61 6.01 6.82 13.09 >105 1.72
0.72 1.14 1.56 2.39 4.06 1.21 1.49 1.91 1.14 1.63 1.30 1.70 2.12 2.95 2.19 2.19 1.31 2.26 1.62 0.70 1.03 −0.08 2.25
Compounds II-1 II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-9 II-10 II-11 II-12 II-13 II-14 II-15 II-16 II-17 II-18 II-19 II-20 II-21 Citraconic anhydride Procymidone
EC50 27.44 26.64 11.62 20.06 165.65 27.84 46.28 53.59 27.80 55.20 108.39 73.38 4.27 8.85 18.87 28.50 18.87 1.11 10.83 30.48 65.78 >105 0.44
log P 1.07 1.49 1.91 2.74 4.41 1.56 1.84 2.26 1.49 1.98 1.65 2.05 2.47 3.30 2.54 2.54 1.30 2.61 1.97 1.05 1.38 0.27 3.61
Compounds III-1 III-2 III-3 III-4 III-5 III-6 III-7 III-8 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 III-17 III-18 III-19 III-20 III-21 Dichloromaleic anhydride
EC50
log P
7.38 5.88 8.69 1.01 732.3 29.57 136.8 10.64 45.74 14.06 189.8 48.32 5.77 7.21 41.15 67.72 305.7 7.47 263.0 4.51 4.44 >105
0.80 1.22 1.63 2.47 4.14 1.29 1.57 1.99 1.22 1.71 1.38 1.78 2.19 3.03 2.27 2.27 1.70 2.34 1.69 0.78 1.10 0
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3.3 Structure–activity relationships 3.3.1 Influence of variation in the substituents at the 3and 4-positions of the maleimide ring From the data in Table 1 it is apparent that the introduction of substituents at the 3- and 4-positions of the maleimide ring had different influences on the antifungal activities against S. sclerotiorum, depending on the type of substituents introduced. On the whole, 3,4-non-substituted maleimides (I-1 to I-21) displayed strong antifungal activities, with EC50 values ranging from 2.45 to 23.41 μg mL−1 . In particular, I-1, I-5 to I-7, I-9, I-11, I-12, I-14 to I-17 and I-19 showed more interesting antifungal activities than the corresponding 3-methylmaleimides and 3,4-dichloromaleimides. However, III-4 displayed the strongest antifungal activity (EC50 = 1.01 μg mL−1 ) and II-18 showed the second strongest antifungal activity (EC50 = 1.11 μg mL−1 ). Therefore, there was no obvious regularity for the influences of variation in the substituents at the 3- and 4-positions of the maleimide ring.
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CONCLUSION
The results of the biological assays indicate that most maleimides showed good antifungal activity against S. sclerotiorum. Twenty-five of the 63 compounds had interesting inhibitory potency, with EC50 values of
Revised: 27 January 2014
Accepted article published: 5 May 2014
Published online in Wiley Online Library: 4 July 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3824
Synthesis and antifungal evaluation of a series of maleimides Xiao-Long Chen,a* Li-Jun Zhang,a Fu-Ge Li,a Yong-Xian Fan,a Wei-Ping Wang,b Bao-Ju Lib and Yin-Chu Shena Abstract: BACKGROUND: Maleimides, both natural and synthesised, have good biological activities. In a continuous effort to discover new maleimides with good antifungal activities, the authors have synthesised a series of 3,4-dichloro-, 3-methyl and non-substituted maleimides based on previous studies. The compounds were biologically evaluated against the fungal pathogen Sclerotinia sclorotiorum. RESULTS: Of the 63 compounds evaluated, 25 compounds had interesting inhibitory potency with EC50 < 10 𝛍g mL−1 . N-(3,5-Dichlorophenyl)-3,4-dichloromaleimide (EC50 = 1.11 𝛍g mL−1 ) and N-octyl-3-methylmaleimide (EC50 = 1.01 𝛍g mL−1 ) were more potent than the commercial fungicide dicloran (EC50 = 1.72 𝛍g mL−1 ). The results showed that compounds exhibiting log P values within the range 2.4–3.0 displayed the best results in terms of fungicidal activity, and this seemed, therefore, to be the optimum range for this physicochemical parameter. CONCLUSION: The present work demonstrates that some maleimides can be used as potential lead compounds for developing novel antifungal agents against S. sclerotiorum. © 2014 Society of Chemical Industry Keywords: maleimides; Sclerotinia sclerotiorum; antifungal agents; fungicide resistance
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INTRODUCTION
Pest Manag Sci 2015; 71: 433–440
∗
Correspondence to: Xiao-Long Chen, Institute of Fermentation Engineering, College of Biological and Environmental Engineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, China. E-mail: [email protected]
a Institute of Fermentation Engineering, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China b Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
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Sclerotinia sclerotiorum (Lib.) de Bary is a filamentous ascomycete fungus with a wide host range and geographical distribution. It has been one of the most non-specific, omnivorous and successful plant pathogens. Its host range includes approximately 400 primarily dicotyledonous plant species.1,2 Therefore, S. sclerotiorum has been a notorious plant-pathogenic fungus, causing vast economic damage during crop cultivation as well as in harvested products, such as oilseed rape,3 blueberry,4 alfalfa,5 lettuce6,7 and so on, particularly in China and Australia.8 There have been several agrochemicals to control S. sclerotiorum, including carbendazim and the dicarboximide fungicides (procymidone, iprodione and vinclozolin).9 Unfortunately, the extensive use of these fungicides selects for resistant individuals within a population and leads to control failures.10,11 By using mixtures of fungicides with different modes of action, the selection for fungicide resistance could be minimised, so the effectiveness of disease control could be increased.12 However, the resistance problem could not be completely solved. Therefore, it is an urgent challenge to develop novel, highly effective and simple antifungal agents with low toxicities for the disease. In the literature it has been reported that natural products with maleic anhydride, such as tautomycin and tautomycetin, possess good biological activities,13 especially antifungal activities. In addition, natural products with a maleimide core moiety (Fig. 1), such as showdomycin, aqabamycins and arcyruarubins, have been found to have various biological activities.14 – 16 On the other hand, synthesised compounds with maleic anhydride or maleimide
have also been reported to have excellent biological activities.17 For example, N-(4-fluorophenyl)-dichloromaleimide (a commercial fungicide in Japan), N-(4-methylphenyl)-dichloromaleimide and N-cyclohexylmaleimide are well known for effective fungicidal activity against various plant pathogens.18 – 20 Furthermore, the animal toxicity of N-(4-fluorophenyl)-dichloromaleimide is extremely low, with LD50 > 15 000 mg kg−1 orally to mice,21 while it was much higher for procymidone and dicloran (LD50 > 2500 mg kg−1 and >1500 mg kg−1 respectively). Moreover, in recent years, some synthetic maleimides have been confirmed to have potent antifungal activities against human fungal pathogens.22,23 Therefore, compounds with a maleic anhydride or maleimide core could be considered to be attractive prototype compounds for the development of new antifungal agents. However, there have been few studies related to the synthesis of maleimides and the evaluation of their antifungal activities against plant pathogens, except for investigations conducted by the present authors’ research group in recent decades.
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X-L Chen et al. O N CnH2n+1
Cl N CnH2n+1
N CnH2n+1
Cl
O
Figure 1. Antifungal template for maleimide derivatives (the maleimide structure is the main active centre, and R1 , R2 and R3 are different substituents that might influence the bioactivity of the compound).
O
O
O
O
I-1: n = 4
II-1: n = 4
III-1 : n = 4
I-2 : n = 5
II-2: n = 5
III-2 : n = 5
I-3 : n = 6
II-3 : n = 6
III-3 : n = 6
I-4:: n = 8
II-4 : n = 8
III-4 : n = 8
I-5:: n = 12
II-5 : n = 12
III-5 : n = 12
O
O
O Cl
N (CH2)n
N (CH2)n
N (CH2)n Cl
In previous studies,2,13,17,24 some maleic anhydrides and maleimides, including tautomycin and its derivatives, were prepared and screened against S. sclerotiorum and Botrytis cinerea. The results showed that the antifungal activity of compounds with a maleimide moiety was better than that of compounds with maleic anhydride. In the present study, 63 maleimide derivatives (Fig. 2) were synthesised, including N-substituted maleimide derivatives I-1 to I-21, N-substituted 3-methyl-maleimide derivatives II-1 to II-21 and 3,4-dichloro-maleimide derivatives III-1 to III-21. In addition, the structure–activity relationship was also investigated (Scheme 1). This research aims to obtain several maleimide derivatives with higher activity and lower toxicity as novel fungicides against S. sclerotiorum.
2
MATERIALS AND METHODS
2.1 General Dicloran (96%, TC) and procymidone (50%, WP) used as internal reference fungicides were purchased from Sigma-Aldrich (St Louis, MO) and Sumitomo Chemicals (Tokyo, Japan) respectively. Citraconic anhydride and maleic anhydride were purchased from Aladdin (Shanghai, China). 2,3-Dichloromaleic anhydride was synthesised according to the procedure previously described in the literature.25 Alkyamines, phenylalkylamines and substituted anilines were also purchased from Aladdin. All other chemicals used in this work were analytical grade and commercially available on the local market in China (Hangzhou, China). Melting points were taken in open capillary tubes on a WRS-1A electrothermal melting point apparatus (Wuhan, China) and were uncorrected. 1 H NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer (Bruker, Italy) in CDCl3 solutions using tetramethylsilane (TMS) as the internal standard. Electrospray ionisation–mass spectra (ESI-MS) were measured on a mass spectrometer (LCQ/Advantage; Thermo Fisher Scientific, Waltham, MA). IR spectra were recorded on a Nicolet 6700 infrared spectrophotometer (Thermo Fisher Scientific) by the KBr pellet method. Column chromatographic separations were carried out on silica gel (ACME, Mumbai, India) (60–120 mesh).
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2.2 Synthesis of compounds The N-substituted maleimides were synthesised by employing two methods according to an improved procedure based on reported methods2,21,22,24 using maleic anhydrides and amines as the starting materials (Scheme 2). The general procedure for synthesising compounds I-1 to I-5 (Scheme 2, path A) was as follows. In the first step, a three-necked, round-bottomed flask equipped with a magnetic stirrer, a
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O
O
O I-6: n = 1
II-6: n = 1
III-6: n = 1
I-7: n = 2
II-7: n = 2
III-7: n = 2
I-8: n = 3
II-8: n = 3
III-8: n = 3
I-9: R = H
II-9: R = H
III-9: R = H
I-10: R = 4 -CH3
II-10: R = 4 -CH3
III-10: R = 4-CH3
I-11: R = 4-F
II-11: R = 4-F
III-11: R = 4-F
I-12: R = 4 -Cl
II-12: R = 4-Cl
III-12: R = 4-Cl
I-13: R = 2,6-(CH3)2
II-13: R = 2,6-(CH3)2
III-13: R = 2,6-(CH3)2
I-14: R = 2,6-(C2H5)2
II-14: R = 2,6-(C2H5)2
III-14: R = 2,6-(C2H5)2
I-15: R = 2-CH3-3-Cl
II-15: R = 2-CH3-3-Cl
III-15: R = 2-CH3-3-Cl
I-16: R = 2-CH3-5-Cl
II-16: R = 2-CH3-5-Cl
III-16: R = 2-CH3-5-Cl
I-17: R = 2-CH3-3-NO2
II-17: R = 2-CH3-3-NO2 III-17: R = 2-CH3-3-NO2
I-18: R = 3,5-(Cl)2
II-18: R = 3,5-(Cl) 2
III-18: R = 3,5-(Cl)2
I-19: R = 3,4,5-(F)3
II-19: R = 3,4,5-(F)3
III-19: R = 3,4,5-(F)3
I-20
II-20
I-21
II-21
III-20
III-21
Figure 2. Chemical structures of N-substituted maleimides.
dropping funnel and a reflux condenser was charged with maleic anhydride (1.96 g, 0.02 mol) dissolved in 10 mL of toluene. A solution of appropriate alkylamines (1 equiv., 0.02 mol) dissolved in 10 mL of toluene was added dropwise from the dropping funnel to the flask over a period of about 10 min at room temperature with stirring. The reaction mixture was heated to 55–70 ∘ C and maintained at that temperature for 3–4.5 h. For the second step, anhydrous sodium acetate (0.08 g, 0.96 mmol) and triethylamine (1.0 mL, 7.2 mmol) were added sequentially. Then the reaction mixture was heated to 101 ∘ C and was refluxed for an
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Pest Manag Sci 2015; 71: 433–440
Synthesis and antifungal evaluation of maleimides
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O
R1
O
R1 O
+ NH2 R0
N R0
R2
R2
O
O I : R1=H, R2=H II : R1=H, R2=CH3 III : R1=Cl, R2=Cl
Sclerotinia sclerotiorum
63 compounds O Cl
EC50 = 1.01 µg/mL
N C8H17 Cl O Cl
O
EC50 = 1.11 µg/mL
N O
Cl
Scheme 1. Compounds with maleimide with good antifungal activities against S. sclerotiorum.
R1
R1
O
P +
NH2 R
a
O
A ath
3
R
2
O OH N R3 H O
Path
O
O N R3
R2
O
c
2
R
b
R1
B
Scheme 2. Synthesis of N-substituted maleimides. Path A: a – toluene, 25–65 ∘ C, 2–8 h; b – CH3 COONa, Et3 N, 101 ∘ C, 10–24 h. Path B: c – CH3 COOH.
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The compounds I-1 to I-12, I-21, II-6 to II-9 and III-1 to III-12 have been previously reported.22,26 – 31 The structures of all the compounds were confirmed by IR, ESI-MS and 1 H NMR. The physical and spectroscopic data of the new compounds are shown below. N-(2,6-Dimethylphenyl)-maleimide I-13. Yield 71%; light-yellow crystals; mp 188.8–190.4 ∘ C. IR (KBr) cm−1 3465, 3088, 2932, 1714, 1576, 1453, 1386, 1090, 852, 720, 703, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.25 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 6.90 (s, 2H), 2.13 (s, 6H). ESI-MS m/z (%) 201.1 (100) [M]+ , 183.1 (98), 144.1 (40), 118.1 (15), 91.1 (23), 76.1 (18), 54 (15), 39.1 (8). N-(2,6-Diethylphenyl)-maleimide I-14. Yield 81%; brown powder crystals; mp 192.8–194.1 ∘ C. IR (KBr) cm−1 3469, 3091, 2934, 1719, 1570, 1453, 1386, 1090, 853, 722, 700, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (t, J = 7.7 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 6.90 (s, 2H), 2.42 (q, J = 7.6 Hz, 4H), 1.15 (t, J = 9.5, 5.7 Hz, 6H). ESI-MS m/z (%) 229.1 (100) [M]+ , 196.1 (48), 158.1 (30), 132.1 (42), 115.1 (19), 77.1 (20), 54.1 (18), 37.0 (7).
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additional 10–12 h. N-Alkyl maleimides were separated by silica column chromatography to afford products I-1 to I-5 in good yields. The general procedure for synthesising compounds I-6 to I-21, II-1 to II-21 and III-1 to III-21 (Scheme 2, path B) was as follows. A stirred solution of anhydride (maleic anhydride or citraconic anhydride or 2,3-dichloromaleic anhydride) in acetic acid was added with appropriate amines (alkyamines, phenylalkylamines or substituted anilines, 0.01 mol) in 20 mL of acetic acid. The reaction mixture was heated to 115–130 ∘ C, and the mixture was refluxed for 3 h. The reaction mixture was then cooled to 15–25 ∘ C. If products were solids, crystals were collected by filtration and rinsed with water or acetic acid. Then the products were recrystallised from acetone solution. If the products were in oil, the solvent was distilled under reduced pressure. The mixture was separated by silica column chromatography to afford products in good yields. For the sake of clarity, the synthesised compounds were grouped according to their structural features, as shown in Fig. 2.
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N-(2-Methyl-3-chloro-phenyl)-maleimide I-15. Yield 70%; light-yellow powder; crystals; mp 160.5–162.0 ∘ C. IR (KBr) cm−1 3468, 3096, 1702, 1533, 1383, 1363, 1094, 730, 715, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.48 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.91 (s, 2H), 2.19 (s, 3H). ESI-MS m/z (%) 223.0 (29), 221.0 (88) [M]+ , 205.0 (33), 203.0 (100), 186.0 (10), 164.0 (30), 130.1 (23), 102.1 (12), 77.1 (22), 54.0 (28), 37.0(3). N-(2-Methyl-5-chloro-phenyl)-maleimide I-16. Yield 72%; light-yellow powder crystals; mp 164.1–165.4 ∘ C. IR (KBr) cm−1 3468, 3091, 1705, 1530, 1384, 1362, 1092, 731, 714, 516. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.34 (dd, J = 8.3, 2.2 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1 H), 7.15 (d, J = 2.1 Hz, 1H) 6.89 (s, 2H), 2.14 (s, 3H). ESI-MS m/z (%) 223.0 (33), 221.0 (100) [M]+ , 205.0 (33), 203.0 (99), 186.0 (6), 164.0 (28), 130.1 (26), 102.0 (11), 77.1 (17), 54.0 (23), 37.1 (5). N-(2-Methyl-3-nitro-phenyl)-maleimide I-17. Yield 73%; yellow powder crystals; mp 155.8–157.0 ∘ C. IR (KBr) cm−1 3463, 3099, 1709, 1534, 1387, 1363, 1094, 730, 715, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.99 (dd, J = 8.2, 1.1 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.40 (dd, J = 7.9, 1.0 Hz, 1H), 6.95 (s, 2H), 2.32 (s, 3H). ESI-MS m/z (%) 232.1 (3) [M]+ , 215.1 (100), 187.0 (48), 160.0 (32), 131.1 (17), 104.1 (20), 77.1 (42), 54.1 (34), 37.0 (6). N-(3,5-Dichlorophenyl)-maleimide I-18. Yield 75%; yellow powder crystals; mp 182.5–183.0 ∘ C. IR (KBr) cm−1 3463, 3094, 2926, 1716, 1577, 1454, 1387, 1092, 854, 724, 702, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (s, 3H), 6.90 (s, 2H). ESI-MS m/z (%) 245.0 (11), 243.0 (67), 241.0 (100) [M]+ , 197.0 (15), 171.0 (13), 150.0 (7), 124.0 (15), 82.0 (7), 54.0 (26), 32.1 (4). N-(3,4,5-Trifluorophenyl)-maleimide I-19. Yield 72%; white powder crystals; mp 153.4–165.6 ∘ C. IR (KBr) cm−1 3478, 3081, 1718, 1627, 1521, 1458, 1323, 1231, 1097, 1040, 852, 723, 690, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.20–7.13 (m, 2H) 6.89 (s, 2H). ESI-MS m/z (%) 227.0 (100) [M]+ , 183.0 (15), 157.0 (20), 118.0 (7), 95.0 (4), 75.0 (4), 54.0 (18). N-iso-Butyl-maleimide I-20. Yield 70%; light-yellow plate crystals; mp 52.8–53.9 ∘ C. IR (KBr) cm−1 3457, 2963, 2929, 2874, 1711, 1439, 1408, 1375, 1059, 734, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.71 (s, 2H), 3.34 (d, J = 7.4 Hz, 2H), 2.02 (dp, J = 14.0, 6.9 Hz, 1H), 0.90 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 153.1 (49) [M]+ , 110.0 (100), 82.0 (38), 54.1 (21), 41.1 (11). N-Butyl-3-methylmaleimide II-1. Yield 73%; light-yellow oil. IR (KBr) cm−1 3844, 2962, 2936, 2870, 1705, 1441, 1404, 1379, 1055, 730, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.7 Hz, 1H), 3.48 (t, J = 7.3 Hz, 2H), 2.07 (d, J = 1.8 Hz, 3H), 1.58–1.51 (m, 2H), 1.34–1.26 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). ESI-MS m/z (%) 167.1 (35) [M]+ , 138.1 (8), 124.1 (100), 94.0 (12), 69.1 (17), 56.1 (17), 39.1 (18). N-Amyl-3-methylmaleimide II-2. Yield 79%; light-yellow oil. IR (KBr) cm−1 3452, 2952, 2930, 2862, 1701, 1441, 1403, 1373, 1061, 734, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.31 (q, J = 1.7 Hz, 1H), 3.50–3.47 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.61–1.54 (m, 2H), 1.37–1.29 (m, 2H), 1.29–1.22 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). ESI-MS m/z (%) 181.1 (38) [M]+ , 138.1 (18),124.1 (100), 96.1 (15), 70.1 (12), 56.1 (15), 39.1(15). N-Hexyl-3-methylmaleimide II-3. Yield 80%; light-yellow oil. IR (KBr) cm−1 3459, 2952, 2931, 2859, 1702, 1442, 1407, 1377, 1064, 734, 520. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.8 Hz, 1H), 3.50–3.46 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.56 (p, J = 7.3 Hz, 2H), 1.31–1.25 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H). ESI-MS m/z (%) 193.1 (38) [M]+ , 150.1 (50), 112.1 (100), 96.1 (35), 81.1 (38), 67.1 (42), 53.1 (22), 39.1 (47). N-Octyl-3-methylmaleimide II-4. Yield 78%; light-yellow oil. IR (KBr) cm−1 3346, 2929, 2851, 1704, 1440, 1406, 1372, 1072, 733, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.8 Hz, 1H), 3.49–3.46
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(m, 2H), 2.07 (d, J = 1.8 Hz, 3H), 1.56 (p, J = 7.3 Hz, 2H), 1.30–1.25 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H). ESI-MS m/z (%) 223.2 (47) [M]+ , 180.1 (16), 152.1 (11), 124.1 (100), 96.1 (18), 69.1 (16), 41.1 (18). N-Dodecyl-3-methylmaleimide II-5. Yield 81%; brown plate crystals; mp 70.8–71.4 ∘ C. IR (KBr) cm−1 3459, 2924, 2858, 1712, 1440, 1403, 1375, 1058, 733, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.30 (q, J = 1.7 Hz, 1H), 3.50–3.47 (m, 2H), 2.08 (d, J = 1.8 Hz, 3H), 1.59–1.55 (m, 2H), 1.30–1.25 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H). ESI-MS m/z (%) 279.1 (100) [M]+ , 236.2 (14), 152.1 (14), 124.0 (93), 96.1 (14), 69.1 (14), 41.1 (15). N-(4-Tolyl)-3-methylmaleimide II-10. Yield 82%; light-yellow powder crystals; mp 114.1–115.9 ∘ C. IR (KBr) cm−1 3448, 3042, 2921, 1709, 1518, 1397, 1091, 821, 732, 519. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.30 (d, J = 8.2 Hz, 2H), 7.24–7.20 (m, 2H), 6.51 (q, J = 1.7 Hz, 1H), 2.41 (s, 3H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 201.1 (100) [M]+ , 172.1 (8), 157.1 (10), 117.1 (12), 104.0 (8), 91.1 (8) , 68.1 (12), 39.1 (12). N-(4-Fluorophenyl)-3-methylmaleimide II-11. Yield 79%; white powder crystals; mp 155.8–156.4 ∘ C. IR (KBr) cm−1 3468, 1709, 1492, 1397, 1089, 833, 735. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.36–7.32 (m, 2H), 7.18–7.14 (m, 2H), 6.50 (q, J = 1.7 Hz, 1H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 205.1 (100) [M]+ , 161.1 (15), 148.1 (12), 135.1 (13), 121.1 (12), 109.1 (17), 82.1 (9), 68.1 (14), 40.1 (12). N-(4-Chlorophenyl)-3-methylmaleimide II-12. Yield 83%; white powder crystals; mp 142.3–143.9 ∘ C. IR (KBr) cm−1 3464, 1709, 1492, 1395, 1089, 831, 731. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.46–7.42 (m, 2H), 7.35–7.31 (m, 2H), 6.51 (q, J = 1.7 Hz, 1H), 2.19 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 223.0 (33), 221.0 (100) [M]+ , 207.1 (9), 177.1 (16), 153.0 (22), 137.0 (22), 125.1 (22), 90.1 (24), 68.1 (42), 40.1 (51). N-(2,6-Dimethylphenyl)-3-methylmaleimide II-13. Yield 76%; brown powder crystals; mp 190.8–192.0 ∘ C. IR (KBr) cm−1 3460, 3099, 2922, 1711, 1570, 1459, 1380, 1097, 858, 728, 701, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.27–7.22 (m, 1H), 7.15 (d, J = 7.6 Hz, 2H), 6.52 (q, J = 1.8 Hz, 1H), 2.21 (d, J = 1.8 Hz, 3H), 2.13 (s, 6H). ESI-MS m/z (%) 215.1 (100) [M]+ , 197.1 (61), 168.1 (22), 144.1 (31), 131.1 (17), 91. 1 (20), 77.1 (14), 39.1 (18). N-(2,6-Diethylphenyl)-3-methylmaleimide II-14. Yield 80%; brown granular crystals; mp 195.8–197.3 ∘ C. IR (KBr) cm−1 3458, 3081, 2925, 1714, 1575, 1452, 1384, 1090, 857, 720, 704, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.36 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 6.53 (q, J = 1.7 Hz, 1H), 2.42 (q, J = 7.6 Hz, 4H), 2.21 (d, J = 1.8 Hz, 3H), 1.15 (t, J = 7.6 Hz, 6H). ESI-MS m/z (%) 243.1 (100) [M]+ , 214.1 (34), 198.1 (31), 186.1 (14), 172.1 (13), 158.1 (14) , 132.1 (28), 117.1 (27), 91.1 (14), 77.1 (14), 39.1 (15). N-(2-Methyl-3-chloro-phenyl)-3-methylmaleimide II-15. Yield 80%; light-yellow crystals; mp 149.8–151.4 ∘ C. IR (KBr) cm−1 3459, 3111, 1719, 1532, 1384, 1361, 1092, 733, 711, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 6.52 (q, J = 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H), 2.14 (s, 3H). ESI-MS m/z (%): 237.1 (33), 235.1 (100) [M]+ , 219.1 (19), 217.1 (57), 190.0 (17), 164.0 (24), 138.0 (14), 77.0 (22), 39.1 (27). N-(2-Methyl-5-chloro-phenyl)-3-methylmaleimide II-16. Yield 77%; dark-yellow crystals; mp 150.3–152.0 ∘ C. IR (KBr) cm−1 3469, 3090, 1702, 1532, 1384, 1360, 1098, 730, 717, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 6.52 (q, J = 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H), 2.14 (s, 3H). ESI-MS m/z (%) 237.1 (33), 235.1 (100) [M]+ , 219.1 (17), 217.1 (52), 192.0 (14), 154.1 (27), 172.1 (14), 154.1 (26), 138.0 (13), 102.1 (10), 89.1 (13), 77.1 (17), 51.1 (9), 39.1 (25). N-(2-Methyl-3-nitro-phenyl)-3-methylmaleimide II-17. Yield 79%; light-yellow powder crystals; mp 153.1–154.9 ∘ C. IR (KBr)
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Pest Manag Sci 2015; 71: 433–440
Synthesis and antifungal evaluation of maleimides
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Pest Manag Sci 2015; 71: 433–440
J = 8.3 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 2.17 (s, 3H). ESI-MS m/z (%) 295.0 (4), 293.0 (33), 291.0 (99), 289.0 (100) [M]+ , 271.0 (50), 254.0 (15), 226.0 (49), 210.0 (38), 166.0 (34), 132.1 (25), 87.0 (57), 63.0 (10), 51.1 (15). N-(2-Methyl-3-nitro-phenyl)-3,4-dichloromaleimide III-17. Yield 83%; light-yellow powder crystals; mp 168.8–169.9 ∘ C. IR (KBr) cm−1 3468, 3090, 1701, 1538, 1384, 1360, 1099, 730, 715, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 8.03 (d, J = 8.2 Hz, 1H), 7.53–7.48 (m, 1H), 7.43 (dd, J = 7.9, 1.1 Hz, 1H), 2.35 (s, 3H). ESI-MS m/z (%) 300.0 (6) [M]+ , 287.0 (11), 285.0 (67), 283.0 (100), 255.0 (46), 191.0 (60), 161.0 (23), 148.0 (20), 106.0 (21), 87 (86), 77.1 (69), 63.0 (18), 51.1 (34), 39.1 (9). N-(3,5-Dichlorophenyl)-3,4-dichloromaleimide III-18. Yield 90%; yellow crystals; mp 190.2–191.4 ∘ C. IR (KBr) cm−1 3463, 3094, 2926, 1716, 1577, 1454, 1387, 1092, 854, 724, 702, 521. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.43 (t, J = 1.8 Hz, 1H), 7.36 (d, J = 1.8 Hz, 2H). ESI-MS m/z (%) 316.9 (1), 314.9 (11), 312.9 (50), 310.9 (100), 308.9 (75) [M]+ , 229.9 (27), 186.9 (14), 124.0 (28), 87.0 (34). N-(3,4,5-Trifluorophenyl)-3,4-dichloromaleimide III-19. Yield 75%; white plate crystals; mp 158.9–160.4 ∘ C. IR (KBr) cm−1 3478, 3089, 1710, 1624, 1537, 1458, 1329, 1230, 1095, 1040, 856, 722, 694, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.20–7.13 (m, 2H). ESI-MS m/z (%) 299.0 (33), 297.0 (67), 295.0 (100) [M]+ , 216.0 (42), 173.0 (25), 145.0 (22), 121.9 (38), 87.0 (70), 75.0 (9). N-iso-Butyl-3,4-dichloromaleimide III-20. Yield 71%; light-yellow crystals; mp 63.8–64.5 ∘ C. IR (KBr) cm−1 3462, 2957, 2925, 2874, 1711, 1439, 1408, 1375, 1059, 734, 525. 1 H NMR (500 MHz, CDCl3 ) 𝛿 3.43 (d, J = 7.4 Hz, 2H), 2.04 (dp, J = 13.9, 6.9 Hz, 1H), 0.92 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 225.0 (4), 223.0 (24), 221.0 (38) [M]+ , 182.0 (33), 180.0 (67), 178.0 (100), 151.0 (8), 116.0 (28), 87.0 (58), 56.1 (72), 39.1 (38). N-Cyclohexyl-3,4-dichloromaleimide III-21. Yield 71%; brown powder crystals; mp 67.2–68.5 ∘ C. IR (KBr) cm−1 3439, 2925, 2862, 1720, 1398, 1380, 1089, 733, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 4.00 (tt, J = 12.4, 3.9 Hz, 1H), 2.04 (qd, J = 12.5, 3.4 Hz, 2H), 1.86 (dd, J = 16.2, 2.6 Hz, 2H), 1.71–1.65 (m, 2H), 1.38–1.17 (m, 4H). ESI-MS m/z (%) 251.0 (6), 249.0 (36), 247.0 (53) [M]+ , 204.0 (82), 171.0 (33), 169.0 (67), 167.0 (100), 128.0 (15), 81.1 (98), 41.1 (18).
2.3 Bioassays S. sclerotiorum was isolated from sclerotia collected from a diseased plant of oilseed rape in Zhejiang, China, as described before.2 The culture medium was potato dextrose agar (PDA). The in vitro antifungal activities of 63 compounds against S. sclerotiorum were assayed on solid medium PDA using the mycelium growth rate method.2 These maleimides, dicloran and procymidone were dissolved in 0.1% (m/v) Tween-80 while mixing with PDA to generate a series of concentrations in the final test solution of 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25, 50, 100 and 200 μg mL−1 by the twofold broth dilution method. The compound-free agar with 0.1% (m/v) Tween-80 solution was employed as the blank control. The PDA medium was poured into 9 cm diameter petri dishes (10 mL plate−1 ), which were then inoculated with 6 mm diameter mycelial plugs from the edge of 36-hour-old S. sclerotiorum. Plates in three replicates were used for each test concentration. The dishes were incubated at 23 ∘ C for 36 h, the time by which the growth of the control would have reached the edge of the plate. Then the diameter of each colony was measured by making two measurements at right angles. Tests were repeated twice.24 The inhibition of growth against S. sclerotiorum was calculated using
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cm−1 3468, 308, 1704, 1530, 1382, 1361, 1090, 730, 715, 524. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.97 (dd, J = 8.2, 1.1 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 7.8, 1.0 Hz, 1H), 6.57 (q, J = 1.7 Hz, 1H), 2.32 (s, 3H), 2.23 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 246.1(5) [M]+ , 229.1 (100), 201.1 (7), 174.1 (53), 145.1 (19), 133 (9), 117.1 (18), 96.0 (19), 77.1 (40), 55.1 (19), 39.1 (38). N-(3,5-Dichlorophenyl)-3-methylmaleimide II-18. Yield 81%; brown crystals; mp 170.8–173.2 ∘ C. IR (KBr) cm−1 3469, 3091, 2924, 1712, 1573, 1450, 1387, 1092, 854, 724, 701, 525. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.43 (t, J = 1.8 Hz, 1H), 7.36 (d, J = 1.8 Hz, 2H), 6.53 (q, J = 1.7 Hz, 1H), 2.21 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 259.0 (11), 257.0 (67), 255.0 (100) [M]+ , 211.0 (14), 187.1 (15), 124.0 (16), 68.1 (27), 40.1 (20). N-(3,4,5-Trifluorophenyl)-3-methylmaleimide II-19. Yield 72%; white powder crystals; mp 158.8–160.6 ∘ C. IR (KBr) cm−1 3482, 3080, 1710, 1628, 1535, 1458, 1321, 1238, 1090, 1042, 850, 728, 693, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.22–7.15 (m, 2H), 6.52 (dd, J = 3.6, 1.8 Hz, 1H), 2.20 (d, J = 1.8 Hz, 3H). ESI-MS m/z (%) 241.1 (100) [M]+ , 197.0 (15), 173.0 (18), 157.0 (7), 145.0 (18), 131.0 (4), 118.0 (6), 95.0 (8), 68.0 (24), 40.1 (23). N-iso-Butyl-3-methylmaleimide II-20. Yield 71%; light-yellow oil. IR (KBr) cm−1 3449, 2959, 2929, 2872, 1721, 1439, 1408, 1375, 1059, 734, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.31 (q, J = 1.8 Hz, 1H), 3.30 (d, J = 7.4 Hz, 2H), 2.08 (d, J = 1.9 Hz, 3H), 2.00 (td, J = 13.9, 6.9 Hz, 1H), 0.87 (d, J = 6.7 Hz, 6H). ESI-MS m/z (%) 167.1 (35) [M]+ , 134.1 (100), 96.1 (12), 56.1 (15), 39.1 (15). N-Cyclohexyl-3-methylmaleimide II-21. Yield 70%; light-yellow powder crystals; mp 65.6–66.5 ∘ C. IR (KBr) cm−1 3449, 2932, 2862, 1703, 1400, 1381, 1089, 733, 526. 1 H NMR (500 MHz, CDCl3 ) 𝛿 6.26 (q, J = 1.8 Hz, 1H), 3.88 (tt, J = 12.3, 3.9 Hz, 1H), 2.05 (d, J = 1.8 Hz, 3H), 2.04 (qd, J = 12.5, 3.4 Hz, 2H), 1.86 (dd, J = 16.2, 2.6 Hz, 2H), 1.71–1.65 (m, 2H), 1.38–1.17 (m, 4H). ESI-MS m/z (%) 193.1 (43) [M]+ , 150.1 (53), 112.0 (100), 96.1 (38), 81.1 (41), 67.1 (45), 53.1 (22), 39.1 (45). N-(2,6-Dimethylphenyl)-3,4-dichloromaleimide III-13. Yield 86%; light-yellow powder crystals; mp 200.8–201.6 ∘ C. IR (KBr) cm−1 3469, 3084, 2923, 1706, 1574, 1464, 1389, 1082, 859, 726, 704, 527. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.30 (d, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 2.15 (s, 6H). ESI-MS m/z (%) 273.0 (11), 271.0 (67), 269.0 (100) [M]+ , 251.0 (42), 226.0 (13), 206.1 (16), 190.0 (63), 146.1 (16), 132.1 (9), 118.1 (16), 105.1 (19), 87.0 (25), 77.1 (18), 65.1 (11), 51.1 (10), 39.1 (10). N-(2,6-Diethylphenyl)-3,4-dichloromaleimide III-14. Yield 87%; brown powder crystals; mp 204.1–205.8 ∘ C. IR (KBr) cm−1 3472, 3098, 2928, 1713, 1579, 1458, 1389, 1098, 859, 726, 706, 529. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.41 (t, J = 7.7 Hz, 1H), 7.23 (d, J = 7.7 Hz, 2H), 2.43 (q, J = 7.6 Hz, 4H), 1.17 (t, J = 7.6 Hz, 6H). EI-MS m/z (%) 181 (32). ESI-MS m/z (%) 301.1 (11), 299.1 (67), 297.1 (100) [M]+ , 282.0 (29), 268.0 (32), 250.0 (8), 232.0 (17), 132.1 (75), 117.1 (50), 105.1 (8), 87.0 (17), 65.1 (4), 39.1 (3). N-(2-Methyl-3-chloro-phenyl)-3,4-dichloromaleimide III-15. Yield 78%; light-yellow powder crystals; mp 162.3–164.1 ∘ C. IR (KBr) cm−1 3473, 3090, 1706, 1539, 1382, 1361, 1090, 730, 715, 522. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.52 (dd, J = 8.1, 0.7 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.9, 0.6 Hz, 1H), 2.22 (s, 3H). ESI-MS m/z (%) 295.0 (4), 293.0 (34), 291.0 (99), 289.0 (100) [M]+ , 271.0 (60), 254.0 (27), 226.0 (33), 210.0 (50), 166.0 (20), 132.0 (17), 87.0 (50), 77.1 (23), 63.0 (10), 51.1 (14), 39.1 (8). N-(2-Methyl-5-chloro-phenyl)-3,4-dichloromaleimide III-16. Yield 77%; yellow powder crystals; mp 160.1–161.4 ∘ C. IR (KBr) cm−1 3460, 3084, 1712, 1537, 1380, 1368, 1090, 734, 715, 528. 1 H NMR (500 MHz, CDCl3 ) 𝛿 7.38 (dd, J = 8.3, 2.1 Hz, 1H), 7.30 (d,
www.soci.org the following formula:
However, path B was a facile method by a one-step reaction to prepare 3-methylmaleimides and 3,4-dichloromaleimides with a shorter reaction time, especially for the synthesis of 3,4-dichloromaleimides. Furthermore, path B had higher yields and an easier isolation method when compared with path A.
) ( ) ( Inhibition of growth (%) = Dck − D ∕ Dck − 6 × 100 where Dck represents the average colony diameter of the blank control, D represents the average colony diameter of test compounds and 6 represents the diameter of the inoculum plug (in mm). With a logit-log transformation, the concentration–response curves were linearised by regression and were then used to calculate the effective concentration (EC) values for different control levels. The antifungal activities were assessed with EC50 values.32,33 The log P values were calculated by ChemDraw 8.0.
3.2 Antifungal activities of compounds against S. sclerotiorum In vitro antifungal activities of 63 N-substituted maleimides and dicloran and procymidone were evaluated against S. sclerotiorum, and the EC50 values were calculated. The results are shown in Table 1. In the study, compounds with EC50 > 500 μg mL−1 were considered to be inactive, compounds with EC50 = 100–250 μg mL−1 were considered to be moderately active and compounds with EC50 ≤ 50 μg mL−1 were considered to be highly active. In particular, compounds displaying EC50 ≤ μg mL−1 were considered to be of great interest for further development. Results indicated that most of the 63 compounds showed good inhibition to the mycelial growth of S. sclerotiorum, except for III-5 which possessed marginal or null activity. The starting materials (maleic anhydride, citraconic anhydride and 2,3-dichloromaleic anhydride) were almost inactive against S. sclerotiorum. Of the 63 compounds, 25 compounds had interesting inhibitory potency with EC50 < 10 μg mL−1 . In particular, compounds II-18 and III-4, the EC50 values of which were 1.11 and 1.01 μg mL−1 respectively, were more effective than the commercial fungicide dicloran (EC50 = 1.72 μg mL−1 ) but less active than procymidone WP (EC50 = 0.44 μg mL−1 ). Moreover, only seven compounds (II-11, II-15, III-5, III-7, III-8, III-17, III-19) had EC50 values above 100 μg mL−1 . In summary, the other 56 synthetic compounds displayed moderate to excellent antifungal activities against S. sclerotiorum.
2.4 Statistical analysis Analysis of variance (ANOVA) (SAS Institute, Cary, NC) was applied to determine the statistical significance of differences among treatments in each bioassay. The data on growth inhibition of S. sclerotiorum by the synthesised compounds in each replicate were arcsine transformed to angular data prior to ANOVA. Means for different treatments in each bioassay or trial were separated using the least significant difference test at the P = 0.05 level.
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RESULTS AND DISCUSSION
3.1 Synthesis of compounds Sixty-three compounds were synthesised by path A (I-1 to I-5) and path B shown in Scheme 2 with good yields (above 70%). Among them, there were five novel compounds (II-16, II-17, II-19, III-17 and III-19). As for the two synthetic methods, path A was used to prepare N-alkylmaleimides (I-1 to I-5) by two-step reactions, dehydration and a ring-closing reaction, as reported in the literature.24
Table 1. In vitro antifungal activities of maleimides against S. sclerotiorum Compounds
EC50
log P
I-1 I-2 I-3 I-4 I-5 I-6 I-7 I-8 I-9 I-10 I-11 I-12 I-13 I-14 I-15 I-16 I-17 I-18 I-19 I-20 I-21 Maleicanhydride Dicloran
5.55 8.46 9.96 8.42 16.02 6.88 9.19 15.08 15.27 23.41 17.76 7.80 6.03 2.45 11.17 10.46 10.47 6.61 6.01 6.82 13.09 >105 1.72
0.72 1.14 1.56 2.39 4.06 1.21 1.49 1.91 1.14 1.63 1.30 1.70 2.12 2.95 2.19 2.19 1.31 2.26 1.62 0.70 1.03 −0.08 2.25
Compounds II-1 II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-9 II-10 II-11 II-12 II-13 II-14 II-15 II-16 II-17 II-18 II-19 II-20 II-21 Citraconic anhydride Procymidone
EC50 27.44 26.64 11.62 20.06 165.65 27.84 46.28 53.59 27.80 55.20 108.39 73.38 4.27 8.85 18.87 28.50 18.87 1.11 10.83 30.48 65.78 >105 0.44
log P 1.07 1.49 1.91 2.74 4.41 1.56 1.84 2.26 1.49 1.98 1.65 2.05 2.47 3.30 2.54 2.54 1.30 2.61 1.97 1.05 1.38 0.27 3.61
Compounds III-1 III-2 III-3 III-4 III-5 III-6 III-7 III-8 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 III-17 III-18 III-19 III-20 III-21 Dichloromaleic anhydride
EC50
log P
7.38 5.88 8.69 1.01 732.3 29.57 136.8 10.64 45.74 14.06 189.8 48.32 5.77 7.21 41.15 67.72 305.7 7.47 263.0 4.51 4.44 >105
0.80 1.22 1.63 2.47 4.14 1.29 1.57 1.99 1.22 1.71 1.38 1.78 2.19 3.03 2.27 2.27 1.70 2.34 1.69 0.78 1.10 0
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Synthesis and antifungal evaluation of maleimides
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3.3 Structure–activity relationships 3.3.1 Influence of variation in the substituents at the 3and 4-positions of the maleimide ring From the data in Table 1 it is apparent that the introduction of substituents at the 3- and 4-positions of the maleimide ring had different influences on the antifungal activities against S. sclerotiorum, depending on the type of substituents introduced. On the whole, 3,4-non-substituted maleimides (I-1 to I-21) displayed strong antifungal activities, with EC50 values ranging from 2.45 to 23.41 μg mL−1 . In particular, I-1, I-5 to I-7, I-9, I-11, I-12, I-14 to I-17 and I-19 showed more interesting antifungal activities than the corresponding 3-methylmaleimides and 3,4-dichloromaleimides. However, III-4 displayed the strongest antifungal activity (EC50 = 1.01 μg mL−1 ) and II-18 showed the second strongest antifungal activity (EC50 = 1.11 μg mL−1 ). Therefore, there was no obvious regularity for the influences of variation in the substituents at the 3- and 4-positions of the maleimide ring.
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CONCLUSION
The results of the biological assays indicate that most maleimides showed good antifungal activity against S. sclerotiorum. Twenty-five of the 63 compounds had interesting inhibitory potency, with EC50 values of
