Bioorganic & Medicinal Chemistry Letters 24 (2014) 1812–1814

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Antifungal activity of alkyl gallates against plant pathogenic fungi Shinsaku Ito  , Yasutaka Nakagawa  , Satoru Yazawa  , Yasuyuki Sasaki, Shunsuke Yajima ⇑ Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

a r t i c l e

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Article history: Received 22 October 2013 Revised 28 January 2014 Accepted 7 February 2014 Available online 20 February 2014 Keywords: Drug design Gallic acid Structure–activity relationship study

a b s t r a c t The antifungal activity of alkyl gallates against plant pathogenic fungi was evaluated. All of the fungi tested in this study were susceptible to some alkyl gallates, and the effect of linear alkyl gallates against plant pathogenic fungi was similar to the previously reported effects against Gram-negative and Gram-positive bacteria. We found that branched alkyl gallates showed stronger activity than did linear alkyl gallates with similar log P values. In addition, the antifungal activity of alkyl gallates was correlated with gallate-induced inhibition of the activity of mitochondrial complex II. The antifungal activity of alkyl gallates likely originates, at least in part, from their ability to inhibit the membrane respiratory chain. Ó 2014 Elsevier Ltd. All rights reserved.

Gallic acid and its alkyl esters such as propyl, octyl, and dodecyl gallates are used as food preservatives owing to their antioxidant activity. To date, it has been reported that these chemicals exhibit various biological activities, including cardiovascular protection, tyrosinase inhibition, as well as antibacterial, antifungal, anticancer, anti-inflammatory, and antioxidant activities. With respect to antifungal activity, Kubo et al. reported that octyl gallate was effective against Saccharomyces cerevisiae, Zygosaccharomyces bailii, Candida albicans, and Aspergillus niger; the length of the alkyl group played an important role in determining the antifungal activity.1 In a subsequent study by Kubo et al., nonyl gallate showed the highest activity against S. cerevisiae among linear gallates.2 Leal et al. performed a similar study, which demonstrated that gallates with linear alkyl chains of 7–9 carbons showed the highest antifungal activity against yeasts, hialohyphomycetes, and dermatophytes.3 These studies showed the antifungal activity of linear alkyl gallate against human opportunistic pathogenic fungi likely depend on the hydrophobic alkyl chain. However, structure– activity relationship studies of alkyl gallates against plant pathogenic fungi have not yet been performed. Furthermore, the target site(s) of gallates is unknown. In this study, we synthesized 14 alkyl gallates and investigated the antifungal activity of these gallates against the following four plant pathogenic fungi: Fusarium solani, Colletotrichum acutatum, Colletotrichum dematium, and Alternaria brassicicola, which cause serious damage to economically important crops and fruits worldwide. Here, we investigated structure–activity relationships and the mode of action of gallates against these four fungi. ⇑ Corresponding author. Tel.: +81 3 5477 2768.  

E-mail address: [email protected] (S. Yajima). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bmcl.2014.02.017 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

The chemicals were prepared as described previously.2 Gallic acid and alkanols dissolved in tetrahydrofuran and dichloromethane (CH2Cl2) were stirred at room temperature in the presence of N,N’-dicyclohexylcarbodiimide. The reaction mixture was quenched with distilled water. The aqueous layer was extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The resulting oil was purified by column chromatography. Figure 1 shows the structures and log P values of the alkyl gallates used in this Letter. Log P values were calculated by ChemBioDraw Ultra software. Plant pathogenic fungi were gifts from Professor Natsuaki (Tokyo Univ. of Agric.). First, we estimated the effect of alkyl gallates against four plant pathogenic fungi (Table 1A). Strains were grown on potato dextrose agar (PDA) plates. The antifungal assay was performed as described below. Small agar blocks containing hyphal tips were cut out from the colony margins and placed onto glycerol peptone medium (0.12% KH2PO4, 3% glycerol, 0.2% polypeptone, 0.02% MgSO4, 0.00012% CuSO4, 0.00021% FeCl3, 0.000102% NaMoO4, 0.000021% FeSO4, 0.000006% CoCl2 and 0.000006% CaCl2) supplemented with each gallate dissolved by dimethyl sulfoxide and cultured at 30 °C for one day. Antifungal activities were determined by measuring the minimum inhibitory concentration (MIC). Gallic acid was not effective against these four fungi. Previously, it was reported that gallic acid purified from Terminalia nigrovenulosa bark possessed high antifungal activity against F. solani.4 However, the F. solani strain used in this study was different from that used by Nguyen et al, which may be why gallic acid did not inhibit the growth of F. solani in this study. Some of the linear alkyl gallates tested here (7–12) showed antifungal activity against all fungi strains (Table 1A). Alkyl gallates possessing alkyl chains of 8–12 carbon atoms were particularly strong activity. While the length of the alkyl group was generally associated with antifungal activ-

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Figure 1. Chemical structures and log P values of gallates.

ity, this result was in contrast to previous reports showing that hexyl, heptyl, octyl, and nonyl gallates, but not decyl, undecyl, and dodecyl gallates, effectively inhibited fungal growth.2,3 However, our data were more consistent with the results of

antibacterial activities of alkyl gallates that have been reported previously.5,6 Leal et al. reported that the antifungal activities differed mainly between groups of fungi (yeast, hialohyphomycetes or dermatophytes).3 As we tested the antifungal effects of gallates on the different fungi group (plant pathogenic fungi) in this Letter, our data were consistent with the results of antibacterial activities, rather than those of previously reported antifungal activities. Moreover, some branched alkyl gallates (13–17) showed stronger activity than linear alkyl gallates with a similar log P value (Table 1A), even though it has been previously reported that antifungal activities of gallates were partially dependent on the log P values of each gallate.3 Among the branched alkyl gallates we synthesized in this Letter,7 3,3-dimethylbutyl gallate (14) had the highest antifungal activity. Some gallates (8 and 9 against Colletotrichum acutatum, and 9 and 10 against Colletotrichum dematium) showed the stronger activities than benomyl, which is fungicide practically used all over the world. This result suggests the possibility of gallates as novel antifungal agents. Esters can be easily hydrolyzed and yield alcohols and carboxylic acids. Moreover, primary alkanols from C9 to C12 exhibit antifungal activity.2 To reveal whether the antifungal activity of branched alkyl gallates originated from alkanols, we tested the antifungal activity of 3,3-dimethylbutanol and primary alkanols (Table 1B). All alkanols were completely inactive against these fungi. These results suggest that the mode of action of branched alkyl gallates against plant pathogenic fungi was different from that of alkanols. It has been proposed that the mechanism of antifungal action of alkyl gallates involves their ability to disrupt the native membrane due to their hydrophobicity, as shown for nonionic surfactants;1 however, alkyl gallates possessing alkyl chains of 6–9 carbon atoms were shown to have a further mode of action against Microsporum gypseum.3 Dodecyl gallate has been shown to inhibit oxygen consumption and NADH oxidase in the membrane fraction of Pseudomonas aeruginosa.8 Therefore, to identify a potentially novel mode of antifungal action for alkyl gallates, we prepared mitochondrial membrane fractions and examined their ability to

Table 1 MIC values (lM) of gallic acid derivatives (A) and alkanols (B) against plant pathogenic fungi Fusarium solani

Colletotrichum acutatum

Colletotrichum dematium

Alternaria brassicicolla

A Gallic acid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Benomyl

>50 >50 >50 >50 >50 >50 >50 50 25 6.25 6.25 6.25 12.5 50 12.5 25 50 >50 3.13

>50 >50 >50 >50 >50 >50 >50 12.5 6.25 6.25 6.25 25 50 25 25 50 >50 >50 12.5

>50 >50 >50 >50 >50 >50 >50 50 25 12.5 12.5 6.25 6.25 >50 25 50 50 >50 25

>50 >50 >50 >50 >50 >50 >50 25 25 6.25 6.25 6.25 12.5 >50 50 50 50 >50 6.25

B Heptanol Octanol Nonanol Decanol Undecanol Dodecanol 3,3-Dimethyl-1-butanol

>50 >50 >50 >50 >50 >50 >50

>50 >50 >50 >50 >50 >50 >50

>50 >50 >50 >50 >50 >50 >50

>50 >50 >50 >50 >50 >50 >50

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Figure 2. Inhibition of complex II by alkyl gallates. Alkyl gallates (5–9) were added at a concentration of 100 lM. The data are means ± SD of three samples.

inhibit mitochondrial respiratory chain complexes. The isolation of mitochondrial membrane fraction and the measurement of the activities of mitochondrial respiratory chain complexes were preformed according to a previously described method.9 Briefly, the harvested F. solani cells were homogenized with quartz sand in homogenization buffer (0.8 M sucrose, 20 mM Tris–HCl [pH 7.2], 10 nM EDTA [pH 8.0], and 1 mM phenylmethanesulfonyl fluoride). The homogenate was centrifuged at 1500g for 15 min, and the supernatant was further separated by centrifugation at 10,000g for 60 min. The resulting pellet was resuspended with homogenization buffer and fractionated by sucrose density gradient centrifugation. The interface between the 1.12 and 1.5 M sucrose layers was collected and used as the mitochondrial fraction. In contrast to gallic acid, alkyl gallates that showed antifungal activity inhibited the activity of complex II (succinate dehydrogenase; Fig. 2). In addition, the inhibitory activity of complex II was

dependent on the chain length of linear gallates, which is in agreement with the antifungal activity of gallates. This result suggests the possibility that the antifungal activity of gallates against plant pathogenic fungi is, in part, due to inhibition of complex II activity. In this Letter, we demonstrated that alkyl gallates (6–17) showed antifungal activity against plant pathogenic fungi. In particular, branched alkyl gallates (13–17) inhibited fungal growth more strongly than did linear alkyl gallates with similar log P values. In addition, unhydrolyzed gallates are needed to show the antifungal activities, as neither gallic acid nor any alkanols showed antifungal activities (Table 1) and inhibitory activities of succinate dehydrogenase (data not shown). Moreover, these gallates inhibited the activity of the mitochondrial complex II. Previous studies have suggested the possibility that the major antifungal action of alkyl gallates comes from their ability to act as a nonionic surfactant, although several exceptions have been reported1–3 Indeed, the results in this Letter indicate a new mechanism of action for alkyl gallates through the inhibition of complex II activity in plant pathogenic fungi. References and notes 1. Kubo, I.; Xiao, P.; Fujita, K. Bioorg. Med. Chem. Lett. 2001, 11, 347. 2. Kubo, I.; Xiao, P.; Nihei, K.; Fujita, K.; Yamagiwa, Y.; Kamikawa, T. J. Agric. Food Chem. 2002, 50, 3992. 3. Leal, P. C.; Mascarello, A.; Derita, M.; Zuljan, F.; Nunes, R. J.; Zacchino, S.; Yunes, R. A. Bioorg. Med. Chem. Lett. 2009, 19, 1793. 4. Nguyen, D. M.; Seo, D. J.; Lee, H. B.; Kim, I. S.; Kim, K. Y.; Park, R. D.; Jung, W. J. Microb. Pathog. 2013, 56, 8. 5. Kubo, I.; Xiao, P.; Fujita, K. Bioorg. Med. Chem. Lett. 2002, 12, 113. 6. Kubo, I.; Fujita, K.; Nihei, K. J. Agric. Food Chem. 2002, 50, 6692. 7. 1H NMR spectra were recorded with a JEOL superconducting magnet 400 MHz. High-resolution mass spectra were recorded on an AB SCIEX TripleTOF 5600 System. 1H NMR (CD3OD): 0.97 (9H, s), 1.64 (2H, t J = 7.12), 4.27 (2H, t J = 7.16), 7.01 (2H, s); ESI-HRMS m/z (M H+): calculated for C13H17O5: 253.1081; found: 253.1089. 8. Kubo, I.; Fujita, K.; Nihei, K.; Masuoka, N. Bioorg. Med. Chem. 2003, 11, 573. 9. Takaya, N.; Kuwazaki, S.; Adachi, Y.; Suzuki, S.; Kikuchi, T.; Nakamura, H.; Shiro, Y.; Shoun, H. J. Biochem. 2003, 133, 461.

Antifungal activity of alkyl gallates against plant pathogenic fungi.

The antifungal activity of alkyl gallates against plant pathogenic fungi was evaluated. All of the fungi tested in this study were susceptible to some...
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