Bioactivity and structure-activity relationship of cinnamic acid esters and their derivatives as potential antifungal agents for plant protection

Article abstract of DOI:10.1371/journal.pone.0176189

A series of cinnamic acid esters and their derivatives were synthesized and evaluated for antifungal activities in vitro against four plant pathogenic fungi by using the mycelium growth rate method. Structure-activity relationship was derived also. Almost all of the compounds showed some inhibition activity on each of the fungi at 0.5 mM. Eight compounds showed the higher average activity with average EC₅₀ values of 17.4-28.6 μg/mL for the fungi than kresoxim-methyl, a commercial fungicide standard, and ten compounds were much more active than commercial fungicide standards carbendazim against P. grisea or kresoxim-methyl against both P. grisea and Valsa Mali. Compounds C1 and C2 showed the higher activity with average EC₅₀ values of 17.4 and 18.5 μg/mL and great potential for development of new plant antifungal agents. The structure-activity relationship analysis showed that both the substitution pattern of the phenyl ring and the alkyl group in the alcohol moiety significantly influences the activity. There exists complexly comprehensive effect between the substituents on the phenyl ring and the alkyl group in the alcohol moiety on the activity. Thus, cinnamic acid esters showed great potential the development of new antifungal agents for plant protection due to high activity, natural compounds or natural compound framework, simple structure, easy preparation, low-cost and environmentally friendly.

Full text of DOI:10.1371/journal.pone.0176189

RESEARCH ARTICLE
Bioactivity and structure-activity relationship
of cinnamic acid esters and their derivatives
as potential antifungal agents for plant
protection
Kun Zhou1☯, Dongdong Chen1☯, Bin Li2, Bingyu Zhang1, Fang Miao2*, Le Zhou1*
1 College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, People’s Republic of
China, 2 College of Life Science, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China
a1111111111
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☯ These authors contributed equally to this work.
* zhoulechem@nwsuaf.edu.cn (LZ); miaofangmf@163.com (FM)
Abstract
A series of cinnamic acid esters and their derivatives were synthesized and evaluated for
antifungal activities in vitro against four plant pathogenic fungi by using the mycelium growth
rate method. Structure−activity relationship was derived also. Almost all of the compounds
showed some inhibition activity on each of the fungi at 0.5 mM. Eight compounds showed
the higher average activity with average EC50 values of 17.4–28.6 μg/mL for the fungi than
kresoxim-methyl, a commercial fungicide standard, and ten compounds were much more
active than commercial fungicide standards carbendazim against P. grisea or kresoxim-
methyl against both P. grisea and Valsa mali. Compounds C1 and C2 showed the higher
activity with average EC50 values of 17.4 and 18.5 μg/mL and great potential for develop-
ment of new plant antifungal agents. The structure−activity relationship analysis showed
that both the substitution pattern of the phenyl ring and the alkyl group in the alcohol moiety
significantly influences the activity. There exists complexly comprehensive effect between
the substituents on the phenyl ring and the alkyl group in the alcohol moiety on the activity.
Thus, cinnamic acid esters showed great potential the development of new antifungal
agents for plant protection due to high activity, natural compounds or natural compound
framework, simple structure, easy preparation, low-cost and environmentally friendly.
OPEN ACCESS
Citation: Zhou K, Chen D, Li B, Zhang B, Miao F,
Zhou L (2017) Bioactivity and structure-activity
relationship of cinnamic acid esters and their
derivatives as potential antifungal agents for plant
protection. PLoS ONE 12(4): e0176189. https://doi.
org/10.1371/journal.pone.0176189
Editor: Beom Seok Kim, Korea University,
REPUBLIC OF KOREA
Received: February 3, 2017
Accepted: April 6, 2017
Published: April 19, 2017
Copyright: © 2017 Zhou et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
Plant mycosis is an important problem of agricultural production worldwide [1] and often
results in severe yield losses and quality decrease of agricultural products. Furthermore, many
of the fungi can harm animal and human health due to their mycotoxins [2]. Therefore, vari-
ous fungicides have been extensively used to control fungal plant diseases in current agricul-
ture. However, the prolonged usage of some antifungal agents can lead to drug resistance,
environmental problems and residue toxicity [3]. Thus, it is necessary to develop environmen-
tally friendly plant fungicides. In the past decades, researchers have already put attention to
Funding: This work was financially supported by
the National Natural Science Foundation of China
(31172365, 31572038), http://www.nsfc.gov.cn/,
Zhou, Geng. This study was also supported by the
Agricultural Science and Technology Innovation
Project of Shaanxi Province (2016NY-171). The
funders had no role in study design, data collection
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
and analysis, decision to publish, or preparation of
the manuscript.
natural product-based or -derived plant protectants due to lower environmental and mamma-
lian toxicity of natural products [4].
Cinnamic acid esters and their derivatives are widely distributed in plants including cereals,
legumes, oilseeds, fruits, vegetables and tea or coffee beverages[5]. Due to the common occur-
rence in plants and the low toxicity for humans, animals and environment [6,7], cinnamic acid
derivatives have attracted much attention of many pharmacologists. In the past decades, cin-
namic acid derivatives including natural and non-natural compounds had been proved to
possess diverse pharmacological actions such as antimicrobial [8–11], anti-Mycobactrium
tuberculosis [12], anticancer [13], anti-inflammatory [14], anti-human immunodeficiency
virus (HIV) [15], antiparasitic [16,17], inducing neural progenitor cell proliferation [18] and
so on. Athough cinnamic acid itself and its derivative cinnaldehyde had been found to have
inhibition activity against some plant pathogenic fungi [19–21], few reports have been found
on systematic investigation on the activity of cinnamic acid esters against plant pathogenic
fungi. The purpose of the present research is to explore the bioactivity of a series of cinnamic
acid esters and their derivatives against phyto-pathogenic fungi and structure-activity relation-
ship (SAR), and meanwhile discover new potent antifungal compounds.
Competing interests: The authors have declared
that no competing interests exist.
This study involves three series of the target compounds: ethyl cinnamates with various
substituents on the phenyl ring (A), cinnamic acid esters with various alkyl groups in the alco-
hol moiety (B) and t-butyl or t-amyl cinnamates with various substituents on the phenyl ring
(C).
Materials and methods
Chemicals
Kresoxim-methyl (>99%) and carbendazim (>99%) were purchased from Aladdin Co. Ltd.
(Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from J&K Chemical Ltd. (Bei-
jing, China). Other reagents and solvents were obtained locally and of analytical grade or puri-
fied according to standard methods before use. The water used was redistilled and ion-free.
Fungi
Plant pathogenic fungi, Fusarium solani, Pyricularia grisea, Valsa mali and Botryosphaeria
dothidea, were provided by the Center of Pesticide Research, Northwest A&F University,
China. The fungi were grown on potato dextrose agar (PDA) plates at 25˚C and maintained at
4˚C with periodic subculturing.
Instruments
Melting points were determined on an XT-4 micro-melting point apparatus. Nuclear magnetic
resonance spectra (NMR) were performed on an Avance III 500 MHz instrument (Bruker,
Karlsruhe, Germany). Chemical shifts (δ values) and coupling constants (J values) are given
in ppm and Hz, respectively. High resolution mass spectra (HR-MS) were carried out with a
microTOF-QⅡ instrument (Bruker, Karlsruhe, Germany).
Synthesis
Compounds A1−A7, A16, A19, A23−A28, C1−C4, C11 and C12 were prepared by our previ-
ously reported methods (method A in Fig 1) [16]. The general procedure is as follows. In brief,
the solution of triphenylphosphanylidene acetate (Ph3P = CHCO2R, R = ethyl, t-butyl or t-amyl)
(12 mmol) and aromatic aldehyde (10 mmol) in 50 mL toluene was refluxed for 1–4 h. After
removal of the solvent, the resulting residue was subjected to a silica gel column chromatography
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
Fig 1. Synthetic routes of compounds A, B and C. Reagents and conditions. (a) Ph3P = CHCO2R3, EtOH or toluene, reflux, 1−4 h; (b) acetic
anhydride, Et3N, r.t., 1 h; (c) SOCl2, reflux, 2 h; (d) R0OH, DCM, 0˚C, 1 h.
https://doi.org/10.1371/journal.pone.0176189.g001
(ϕ 40 mm×L 40 cm) using petroleum ether–ethyl acetate as eluent to yield the desired
compounds.
Compounds A1−A7, A16, A19, A25−A28. The NMR data of the compounds were con-
sistent with those previously reported by us [16].
Compounds A23, C1 and C3. Ethyl 2,4-dihydroxycinnamate (A23) [22], t-butyl 2-hydro-
xycinnamate (C1) [23] and t-butyl 4-hydroxycinnamate (C3) [24] were confirmed by compar-
ison of NMR data with those in literature.
Ethyl 2,5-dihydroxycinnamate (A24). Yield: 54% yield; a pale brown crystal; mp: 118–
120˚C; 1H NMR (500 MHz, CD3OD): δ 7.91 (1H, d, J = 16.0 Hz), 6.90 (1H, s), 6.69 (2H, s),
6.46 (1H, d, J = 16.0 Hz), 4.20 (2H, q, J = 7.2 Hz), 1.30 (3H, t, J = 7.2 Hz); 13C NMR (125 MHz,
CD3OD): δ 169.5, 151.5, 151.3, 141.9, 122.9, 120.2, 118.1, 117.9, 114.6, 61.5, 14.6; Negative
ESI-MS m/z: 206.9 [M-H]-.
t-Amyl 2-hydroxycinnamate (C2). Yield: 60%; a yellow oil; 1H NMR (500 MHz, CDCl3): δ
7.99 (1H, d, J = 16.1 Hz), 7.45 (1H, dd, J = 7.6, 1.2 Hz), 7.30 (1H, s, OH), 7.22 (1H, 2×t, J = 7.9,
1.4 Hz), 6.90 (2H, q, J = 7.6 Hz), 6.59 (1H, d, J = 16.1 Hz), 1.90 (2H, q, J = 7.4 Hz), 1.52 (6H, s),
0.95 (3H, t, J = 7.4 Hz); 13C NMR (125 MHz, CDCl3): δ 167.9, 155.6, 139.7, 131.1, 129.0, 121.9,
120.4, 118.3, 116.3, 83.2, 33.6, 25.7, 8.2; HR-ESI-MS [M+Na]+ calcd for C14H18NaO3 , 257.1148,
+
found 257.1157.
t-Amyl 4-hydroxycinnamate (C4). Yield: 64%; a yellow oil; 1H NMR (500 MHz, CDCl3):
δ 7.54 (1H, d, J = 15.9 Hz), 7.39 (2H, d, J = 8.6 Hz), 6.85 (2H, d, J = 8.5 Hz), 6.60 (1H, s, OH),
6.24 (1H, d, J = 15.9 Hz), 1.88 (2H, q, J = 7.4 Hz), 1.50 (6H, s), 0.94 (3H, t, J = 7.4 Hz); 13
C
NMR (125 MHz, CDCl3): δ 167.2, 158.0, 143.6, 129.8, 127.0, 115.8, 114.9, 83.1, 33.5, 25.7, 8.2;
HR-ESI-MS [M+Na]+ calcd for C14H18NaO3 , 257.1148, found 257.1159.
+
t-Butyl 2-hydroxy-3-methoxycinnamate (C11). Yield: 79%; a yellow powder; mp: 76–
77˚C; 1H NMR (500 MHz, CDCl3): δ 7.87 (1H, d, J = 16.1 Hz), 7.07 (1H, dd, J = 6.9, 2.3 Hz),
6.82–6.83 (2H, m), 6.53 (1H, d, J = 16.1 Hz), 6.18 (1H, s, OH), 3.89 (3H, s), 1.53 (9H, s); 13
C
NMR (125 MHz, CDCl3): δ 166.8, 146.8, 145.1, 138.4, 121.1, 121.0, 120.8, 119.5, 111.4, 80.2,
56.1, 28.2; HR-ESI-MS [M+Na]+ calcd for C14H18NaO4 , 273.1097, found 273.1132.
+
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
t-Amyl 2-hydroxy-3-methoxycinnamate (C12). Yield: 63%; a white powder; mp: 76.2–
77˚C; 1H NMR (500 MHz, CDCl3): δ 7.87 (1H, d, J = 16.1 Hz), 7.08 (1H, dd, J = 7.1, 2.1 Hz),
6.82–6.85 (2H, m), 6.53 (1H, d, J = 16.1 Hz), 6.15 (1H, s, OH), 3.90 (3H, s), 1.88 (2H, q, J = 7.4
Hz), 1.50 (6H, s), 0.94 (3H, t, J = 7.4 Hz); 13C NMR (125 MHz, CDCl3): δ 166.7, 146.8, 145.1,
138.3, 121.16, 121.11, 120.8, 119.5, 111.4, 82.6, 56.1, 33.5, 25.7, 8.2; HR-ESI-MS [M+Na]+ calcd
+
for C15H20NaO4 , 287.1254, found 287.1254.
Compounds A11−A15, A17, A18, A20−A22, B1−B12, C7−C10 and C13−C20 were pre-
pared according to method B in Fig 1 by reaction of the corresponding acyl chloride and the
corresponding alcohol. The general procedure was as follows. The mixture of cinnamic acid or
cinnamic acids with substituents on the phenyl ring (0.10 mol) and 30 mL thionyl chloride
was refluxed at 75˚C for 2 h. The excess thionyl chloride was removed under reduced pressure.
After the residue was dissolved in 10 mL DCM, the corresponding alcohol (10 mmol) was
added at 0˚C. The solution was stirred at 0˚C for 1 h, and then washed with water (3 × 30 mL)
followed by 5% Na2CO3 aqueous solution, and dried over anhydrous sodium sulfate. After fil-
tration, the solvent was removed under reduced pressure. The residue was purified by silica gel
column chromatography (ϕ 40 mm × L 40 cm) to afford the desired product.
Compounds A11-A15, A17, A18, A20-A22, B1-B5, B7-B12, C7, C9, C13, C15, C17 and
C19. Compounds ethyl ethyl 2-fluorocinnamate (A11) [25], ethyl 3-fluorocinnamate (A12)
[26], ethyl 4-fluorocinnamate (A13) [27], ethyl 2-chlorocinnamate (A14) [28], ethyl 3-chloro-
cinnamate (A15) [29], 2-bromocinnamate (A17) [25], ethyl 3-Bromocinnamate (A18) [30],
ethyl 2-trifluoromethylcinnamate (A20) [25], ethyl 3-trifluoromethylcinnamate (A21) [26],
ethyl 4-trifluoromethylcinnamate (A22) [27], methyl cinnamate (B1) [25], n-propyl cinnamate
(B2) [31], n-butyl cinnamate (B3) [31], t-butyl cinnamate (B4) [32], n-amyl cinnamate (B5)
[33], n-hexyl cinnamate (B7) [34], n-heptyl cinnamate (B8) [35], n-octyl cinnamate (B9) [36],
cyclohexyl cinnamate (B10) [32], benzyl cinnamate (B11) [37], phenyl cinnamate (B12) [37],
t-butyl 2-methoxycinnamate (C7) [38], t-butyl 4-methoxycinnamate (C9) [38], t-butyl 4-fluor-
ocinnamate (C13) [39], t-butyl 4-bromocinnamate (C15) [39], t-butyl 4-trifluoromethylcinna-
mate (C17) [39] and t-butyl 3,4-methylenedioxy cinnamate (C19) [40] were confirmed by
comparison of NMR data with those in literature.
t-Amyl cinnamate (B6). Yield: 72%; a colorless oil; 1H NMR (500 MHz, CDCl3): δ 7.59
(1H, d, J = 16.0 Hz), 7.50–7.52 (2H, m), 7.36–7.37 (3H, m), 6.38 (1H, d, J = 16.0 Hz), 1.86 (2H,
q, J = 7.5 Hz), 1.50 (6H, s), 0.93 (3H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3): δ 166.3,
143.5, 134.7, 130.0, 128.8, 128.0, 120.2, 83.0, 33.6, 25.7, 8.3.
t-Amyl 2-methoxycinnamate (C8). Yield: 76%; a yellow oil; 1H NMR (500 MHz, DMSO-
d6): δ 7.82 (1H, d, J = 16.1 Hz), 7.68 (1H, dd, J = 7.7, 1.3 Hz), 7.38–7.42 (1H, m), 7.09 (1H, d,
J = 8.3 Hz), 6.99 (1H, t, J = 7.4 Hz), 6.51 (1H, d, J = 16.1 Hz), 3.86 (3H, s), 1.82 (2H, q, J = 7.5
Hz), 1.44 (6H, s), 0.89 (3H, t, J = 7.4 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.2, 158.2,
138.7, 132.2, 128.9, 122.8, 121.1, 120.3, 112.1, 82.5, 56.1, 33.2, 25.8, 8.5; HR-ESI-MS [M+Na]+
+
calcd for C15H20NaO3 , 271.1305; found 271.1312.
t-Amyl 4-methoxycinnamate (C10). Yield: 87%; a yellow oil; 1H NMR (500 MHz,
CDCl3): δ 7.54 (1H, d, J = 16.0 Hz), 7.45 (2H, dt, J = 8.8, 1.9 Hz), 6.88 (2H, d, J = 8.8 Hz), 6.25
(1H, d, J = 16.0 Hz), 3.82 (3H, s), 1.86 (2H, q, J = 7.5 Hz), 1.49 (6H, s), 0.93 (3H, t, J = 7.5 Hz);
13C NMR (125 MHz, CDCl3): δ 166.6, 161.4, 143.2, 129.6, 127.4, 117.7, 114.3, 82.7, 55.3, 33.6,
25.7, 8.3; HR-ESI-MS [M+Na]+ calcd for C15H20NaO3 , 271.1305, found 271.1310. [M+K]+
+
+
calcd for C15H20KO3 , 287.1044; found 287.1048.
t-Amyl 4-fluorocinnamate (C14). Yield: 93%; yellow oil; 1H NMR (500 MHz, DMSO-
d6): δ 7.77–7.78 (2H, m), 7.58 (1H, d, J = 16.0 Hz), 7.22–7.25 (2H, m), 6.51 (1H, d, J = 16.0 Hz),
1.83 (2H, q, J = 7.4 Hz), 1.45 (6H, s), 0.90 (3H, t, J = 7.4); 13C NMR (125 MHz, CDCl3): δ 166.1,
163.7 (d, J = 250.6 Hz), 142.2, 130.9 (d, J = 3.5 Hz), 129.8 (d, J = 8.3), 120.0 (d, J = 2.2. Hz),
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
116.0 (d, J = 21.9 Hz), 83.1, 33.5, 25.7, 8.3; HR-ESI-MS [M+Na]+ calcd for C14H17FNaO2 ,
+
259.1105; found 259.1113.
t-Amyl 4-bromocinnamate (C16). Yield: 95%; a yellow oil; 1H NMR (500 MHz, DMSO-
d6): δ 7.59–7.66 (4H, m), 7.55 (1H, d, J = 16.0 Hz), 6.58 (1H, d, J = 16.0 Hz), 1.83 (2H, q, J = 7.5
Hz), 1.45 (6H, s), 0.90 (3H, t, J = 7.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 165.7, 142.6, 133.9,
132.2, 130.5, 124.0, 121.1, 82.8, 33.2, 25.8, 8.5; HR-ESI-MS [M+Na]+ calcd for C14H1779BrNaO2 ,
+
+
319.0304, found 319.0302, calcd for C14H1781BrNaO2 , 321.0284; found 321.0280.
t-Amyl 4-trifluoromethylcinnamate (C18). Yield: 92%; a yellow oil; 1H NMR (500 MHz,
DMSO-d6): δ 7.93 (2H, d, J = 8.2 Hz), 7.76 (2H, d, J = 8.3 Hz), 7.65 (1H, d, J = 16.0 Hz), 6.70
(1H, d, J = 16.0 Hz), 1.86 (2H, q, J = 7.5 Hz), 1.46 (6H, s), 0.91 (3H, t, J = 7.5 Hz); 13C NMR (125
MHz, DMSO-d6): δ 165.5, 142.1, 138.6, 130.3 (q, J = 31.9 Hz), 129.3, 126.1 (q, J = 3.6 Hz), 124.5
(q, J = 271.8 Hz), 123.2, 83.1, 33.2, 25.8, 8.5; HR-ESI-MS [M+Na]+ calcd for C15H17F3NaO2 ,
+
309.1073; found 309.1068.
t-Amyl 3,4-methylenedioxy cinnamate (C20). Yield: 85%; a white powder; mp: 47–48˚C;
1H NMR (500 MHz, CDCl3): δ 7.49 (1H, d, J = 15.9 Hz), 7.01 (1H, d, J = 1.6 Hz), 6.97 (1H, dd,
J = 8.0, 1.6 Hz), 6.79 (1H, d, J = 8.0 Hz), 6.20 (1H, d, J = 15.9 Hz), 5.99 (2H, s), 1.85 (2H, q,
J = 7.5 Hz), 1.49 (6H, s), 0.92 (3H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3): δ 166.5, 149.3,
148.3, 143.2, 129.1, 124.1, 118.2, 108.5, 106.5, 101.5, 82.8, 33.5, 25.7, 8.3; HR-ESI-MS [M+Na]+
calcd for C15H18NaO4 , 285.1097, found 285.1099; [M+K]+ calcd for C15H18KO4 , 301.0837;
+
+
found 301.0841.
Synthesis of compounds A8-A10, C5 and C6. According to our previously reported
method [16], compounds A8−A10, C5 and C6 were prepared by acetylation reaction of the
corresponding phenols. Briefly, the solution of compounds A2, A3, A4, C1 or C2 (2.1 mmol)
and acetic anhydride (0.6 g, 6.2 mmol) in 20 mL triethylamine was stirred for 1 h at room tem-
perature to provide the desired compounds.
Compounds A8-A10 and C5. The NMR and MS data of compounds A8-A10 [26] and
C5 [41] were consistent with those in the literature.
t-Amyl 2-acetoxyl cinnamate (C6). Yield: 98%; a yellow oil; 1H NMR (500 MHz, CDCl3):
δ 7.67 (2H, m), 7.39 (1H, 2×t, J = 8.0, 1.6 Hz), 7.24 (1H, d, J = 7.7 Hz), 7.11 (1H, dd, J = 8.1, 0.9
Hz), 6.40 (1H, d, J = 16.0 Hz), 2.36 (3H, s), 1.86 (2H, q, J = 7.5 Hz), 1.49 (6H, s), 0.94 (3H, t,
J = 7.4 Hz); 13C NMR (125 MHz, CDCl3): δ 169.1, 165.9, 149.2, 136.6, 130.8, 127.4, 127.3,
126.3, 123.0, 122.1, 83.1, 33.6, 25.6, 20.9, 8.2; HR-ESI-MS [M+Na]+ calcd for C16H20NaO4 ,
+
299.1254; found 299.1260.
Antifungal assay
The mycelium linear growth rate method was used to screen the antifungal activities in vitro
of compounds against four strains of plant pathogenic fungi [42]. Commercial fungicide stan-
dards kresoxim-methyl or carbendazim were used as positive controls.
Briefly, the solution of the test compound (50 μmol) in 0.5 mL of DMSO was added to 9.5
mL of sterile water. The resulting solution was added to 90 mL of melted PDA agar at 50˚C
and quickly and completely mixed to provide the mixture containing 0.5 mM of the com-
pounds and 0.5% (v/v) DMSO. The mixture was poured into a sterilized Petri dish (ca. 15 mL
each plate) in a laminar flow chamber. The culture medium containing only 0.5% DMSO was
used as a blank without observable effect on the growth of the fungi. When the medium in the
plate was partially solidified, a fungus disk (d = 5 mm) cut beforehand from subcultured Petri
dishes was placed at the center of the medium. The dishes were kept in an incubator at 25˚C
for 72 h. Each experiment was carried out in triplicate. The diameters (in mm) of a fungal
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
colony were measured in three different directions, and the growth inhibition rates were calcu-
lated according to the method reported previously [42].
The compounds with higher initial activities were further assayed for median effective con-
centrations (EC50) according to the method described above. A set of stock solutions of test
compounds in 5% DMSO aqueous solution was prepared by double-fold dilution method. Each
stock solution (10 mL) was mixed with autoclaved liquid PDA medium (190 mL) to provide a
series of mediums with various concentrations of the test compound. The culture medium con-
taining only 0.5% DMSO was used as a blank control. Each test was performed in triplicate. The
probit value of the average inhibition rate for each test concentration and the corresponding lg
[concentration (μg/mL)] were used to establish antifungal toxicity regression equation by the
linear least-square fitting method. The EC50 value of each compound and its confidence inter-
vals at 95% probability were calculated from the corresponding toxicity regression equation by
using PRISM software ver. 5.0 (GraphPad Software Inc., San Diego, CA,USA) [42].
Duncan multiple comparison test was used to evaluate significant difference between the aver-
age inhibition rates or EC50 values of various compounds by using PRISM software ver. 5.0 [42].
Results
Design of compounds
In order to know the effect of substitution patterns of the phenyl ring and the type of alkyl
groups in the alcohol moiety on the activity, we firstly designed both A and B series of target
compounds. A series consisting of 28 compounds is a class of ethyl cinnamate derivatives con-
taining various substituents on the phenyl ring. B series including 12 compounds is a class of
cinnamic acid esters formed by cinnamic acid and various alcohols or phenol. Next, under the
guidance of SAR of compounds A and B, we further designed C series including 20 com-
pounds by combination of preponderant active groups in order to further explore the compre-
hensive effect of the substituents on the phenyl ring and the alkyl groups in the alcohol moiety
on the activity and find more potential compounds. Compounds C are a class of t-butyl or t-
amyl cinnamates with various substituents on the phenyl ring (Fig 1).
Synthesis of compounds
Compounds A1–A7, A16, A19, A23–A28, C1-C4, C11 and C12 were prepared by Wittig
reaction of triphenylphosphanylidene acetate [Ph3P = CHCO2R0] and aromatic aldehyde in
ethanol or toluene. Compounds A2–A4, C1 and C2 were acetylated with acetic anhydride to
afford compounds A8–A10, C5 and C6, respectively. Substituted cinnamoyl chlorides reacted
with the corresponding alcohols to obtain compounds A11–A15, A17, A18, A20–22, B1–12,
C7–C10 and C13–C20. The synthetic route of compounds A, B and C is outlined in Fig 1. The
substitution patterns of the compounds are listed in Tables 1, 2 and 3.
The synthesized compounds include 11 new compounds and 49 known compounds. The
known compounds were confirmed by comparison of NMR data and those reported in litera-
ture. The new compounds (C2, C4, C6, C8, C10–C12, C14, C16, C18, C20) were identified by
1H NMR, 13C NMR and HRMS analysis. In positive or negative ESI-MS spectra, each of the
compounds showed its corresponding pseudo-molecular ion peak [M+Na]+. In 1H and 13
C
NMR spectra, all the compounds revealed two doublet signals in the ranges of 7.5 to 8.1 and
δH 6.2 to 6.7 ppm due to the protons of the ethylene moiety and one signal in the range of δC
165 to 167 ppm due to C (= O). The coupling constant of ca. 16 Hz between the protons of the
ethylene moiety showed E configuration of the compounds. The NMR data of the known com-
pounds were agreement with the corresponding literature data.
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
Table 1. Substitution patterns of compounds A1-A28 and initial antifungal activity at 0.5 mM (72 h).
Compound
R1, R2
H
Average inhibition rate (%) (around 100 μg/mL, 72 h)a
No.
A1
F. solani
P. grisea
V. mali
B. dothidea
40.4 ± 1.2lm
91.4 ± 1.3b
42.8 ± 0.9l
65.8 ± 2.2g
77.0 ± 3.5e
46.8 ± 1.2k
73.6 ± 1.2f
88.5 ± 1.2c
48.3 ± 1.0k
70.6 ± 1.1f
28.0 ± 1.5o
56.9 ± 1.6i
65.0 ± 1.9gh
62.5 ± 0.3h
38.1 ± 2.0mn
58.3 ± 2.5i
73.2 ± 1.0f
36.3 ± 2.6n
53.3 ± 4.0j
52.3 ± 0.9j
46.5 ± 1.9k
56.3 ± 0.8i
40.9 ± 1.4lm
21.1 ± 1.0p
83.3 ± 2.1d
49.1 ± 2.3k
49.1 ± 1.2k
62.4 ± 1.2h
88.2 ± 1.5c
94.4 ± 0.1a
Mean
20.1
84.6
43.1
68.9
80.7
43.7
60.2
76.4
43.3
49.8
39.3
54.8
63.9
45.2
30.8
59.5
57.1
32.3
69.6
48.0
57.0
74.3
42.9
24.8
83.1
42.6
49.1
62.6
70.2
72.4
8.9 ± 2.6t
14.4 ± 0.7n
80.1 ± 0.9a
47.9 ± 4.4j
67.2 ± 1.1cde
53.9 ± 1.3h
49.1 ± 0.9ij
53.6 ± 0.5h
72.6 ± 0.0b
70.5 ± 1.2bc
55.1 ± 1.8h
38.0 ± 1.3k
51.7 ± 1.7hi
33.3 ± 1.2l
34.4 ± 3.0l
17.4 ± 0.7n
48.8 ± 4.1ij
62.3 ± 1.0fg
29.4 ± 0.5m
68.9 ± 1.1cd
32.2 ± 1.1lm
64.4 ± 1.1ef
79.7 ± 0.6a
54.2 ± 0.8h
35.3 ± 1.0kl
68.5 ± 2.9cd
78.2 ± 1.6a
60.1 ± 6.3g
73.2 ± 0.6b
66.8 ± 1.1de
5.3 ± 0.8o
16.8 ± 1.8q
82.1 ± 0.1e
48.8 ± 0.0jk
54.4 ± 0.7i
97.6 ± 1.2a
40.6 ± 3.5lm
53.3 ± 3.1i
66.0 ± 2.2g
25.9 ± 4.0o
38.1 ± 1.3m
25.0 ± 1.3o
42.2 ± 2.6l
74.2 ± 2.0f
23.3 ± 1.7op
50.0 ± 1.3jk
85.0 ± 1.3d
60.7 ± 1.4h
50.6 ± 0.6j
84.8 ± 1.3de
30.2 ± 1.3n
47.4 ± 1.3k
85.7 ± 0.7d
23.8 ± 0.3op
21.7 ± 0.4p
88.5 ± 0.6c
24.6 ± 0.4o
42.6 ± 2.1l
74.8 ± 1.0f
54.3 ± 0.7i
93.9 ± 0.7b
A2
2-OH
84.7 ± 0.1d
32.9 ± 0.5no
88.2 ± 1.5c
94.4 ± 2.5ab
38.1 ± 1.0m
60.4 ± 0.8j
78.3 ± 2.0e
28.5 ± 2.7p
35.3 ± 2.5n
66.1 ± 3.0i
68.2 ± 1.4hi
83.1 ± 1.5d
60.4 ± 0.8j
18.0 ± 0.8r
45.8 ± 2.2l
32.0 ± 0.8o
12.8 ± 3.5s
71.2 ± 1.6g
77.1 ± 1.2ef
69.7 ± 2.3gh
75.3 ± 2.4f
52.5 ± 0.6k
21.2 ± 0.8q
91.9 ± 0.5b
18.5 ± 0.8qr
44.7 ± 1.1l
40.1 ± 0.8m
71.3 ± 0.1g
96.1 ± 0.1a
A3
3-OH
A4
4-OH
A5
2-OMe
3-OMe
4-OMe
2-OAc
3-OAc
4-OAc
2-F
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
2,4-(OH)2
2,5-(OH)2
2-OH-3-OMe
4-OH-3-OMe
3,4-(OMe)2
3,4-OCH2O
Kresoxim-methyl
Carbendazim
a The difference between the data with the different lowercase letters within a column is significant (P < 0.05).
https://doi.org/10.1371/journal.pone.0176189.t001
Antifungal activity
According to the mycelium linear growth rate method [39], the synthesized compounds were
screened for antifungal activity in vitro at 0.5 mM (ca.100 μg/mL) against four plant patho-
genic fungi. Kresoxim-methyl and carbendazim, two commercial fungicide standards, were
used as positive controls.
The results listed in Tables 1, 2 and 3 showed that all the compounds displayed some inhibi-
tion activity against each of the fungi at 0.5 mM (around 100 μg/mL). Among A and B series,
compounds A2, A5, A8, A22, A25, B4 and B6 showed the higher activity with average inhibi-
tion rates of 73–84% for four tested fungi, superior to kresoxim-methyl and carbendazim
(70.2%, 72.4%). For A series, A2 showed the highest average activity (84.6%), followed by A25
and A5 (83.1%, 80.7%). Among B series, B6 gave the highest average activity (81.1%) followed
by B4 (73.0%). For C series, all the compounds showed the average inhibition rates of >50%
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
Table 2. Substitution patterns of compounds B1−B12 and initial antifungal activity at 0.5 mM.
Compound
R3
Average inhibition rate (%) (around 100 μg/mL, 72 h)a
No.
B1
F. solani
P. grisea
V. mali
B. dothidea
3.6 ± 0.8l
Mean
20.4
20.1
39.4
46.8
73.0
35.4
81.1
35.8
29.8
22.2
51.8
44.7
23.0
70.2
72.4
Methyl
Ethyl
32.6 ± 0.7g
8.9 ± 2.6k
20.6 ± 2.4i
14.4 ± 0.7j
46.0 ± 1.4f
55.1 ± 1.4d
79.3 ± 0.7a
54.0 ± 0.4d
80.5 ± 2.2a
50.8 ± 0.8e
37.9 ± 0.9g
33.3 ± 1.1h
60.3 ± 1.2c
50.8 ± 2.5e
35.7 ± 2.7gh
66.8 ± 1.1b
5.3 ± 0.8k
24.6 ± 2.5h
16.8 ± 1.8j
20.9 ± 0.9i
60.0 ± 0.5c
30.2 ± 1.3f
47.7 ± 1.4e
78.6 ± 1.4b
46.0 ± 1.3e
30.5 ± 2.3f
27.0 ± 0.7g
56.4 ± 1.1d
55.3 ± 1.3d
15.5 ± 0.8j
54.3 ± 0.7d
93.9 ± 0.7d
A1
40.4 ± 1.2f
29.3 ± 0.5h
41.5 ± 1.3f
100.0 ± 0.0a
11.3 ± 0.8k
91.9 ± 0.1c
10.5 ± 1.6k
26.6 ± 0.9i
9.0 ± 0.8k
B2
n-Propyl
n-Butyl
t-Butyl
61.2 ± 1.1d
30.4 ± 0.9gh
82.6 ± 1.3b
28.6 ± 1.7h
73.2 ± 1.2c
35.8 ± 1.7f
24.1 ± 1.5i
19.5 ± 0.9j
39.7 ± 1.0e
38.9 ± 1.0e
18.4 ± 2.1j
71.3 ± 0.1c
96.1 ± 0.1a
B3
B4
B5
n-Amyl
t-Amyl
B6
B7
n-hexyl
n-Heptyl
n-octyl
Cyclohexyl
benzyl
B8
B9
B10
B11
B12
50.9 ± 1.0e
33.8± 2.2g
22.5 ± 3.4j
88.2 ± 1.5 d
94.4 ± 0.1b
Phenyl
Kresoxim-methyl
Carbendazim
a The difference between the data with the different lowercase letters within a column is significant (P < 0.05).
https://doi.org/10.1371/journal.pone.0176189.t002
Table 3. Substitution patterns of compounds C1−C20 and initial antifungal activity at 0.5 mM.
Compound
Substituent
R3
Average inhibition rate (%) (around 100 μg/mL, 72 h)a
R1, R2
F. solani
P. grisea
V. mali
B. dothidea
96.5 ± 0.1a
95.3 ± 0.1ab
93.0 ± 0.1c
93.4 ± 0.6c
93.0 ± 0.1c
94.1 ± 0.1bc
77.5 ± 0.8f
63.8 ± 1.1k
75.2 ± 0.2g
78.1 ± 1.8f
85.0 ± 2.0e
71.3 ± 0.4h
57.5 ± 0.1l
73.6 ± 0.5g
66.4 ± 0.5j
64.7 ± 0.8m
43.6 ± 2.1n
43.0 ± 1.5n
66.5 ± 1.0j
69.0 ± 1.1i
88.2 ± 1.5d
94.4±0.1bc
Mean
88.3
C1
C2
C3
C4
C5
C6
C7
C8
C9
2-OH
t-Butyl
84.7 ± 0.1d
91.9 ± 0.7b
80.9 ± 1.2fg
82.1 ± 0.1ef
83.4 ± 1.2de
89.4 ± 0.7c
71.3 ± 3.7h
55.3 ± 1.1l
58.9 ± 0.4 k
64.6 ± 1.1i
66.5 ± 1.1i
79.7 ± 1.9g
61.4 ± 1.0j
57.5 ± 0.4k
44.2 ± 0.6n
39.4 ± 1.3o
20.2 ± 0.8q
31.9 ± 0.9p
43.4 ± 1.2n
51.3 ± 1.1lm
71.3 ± 0.1h
96.1±0.1a
82.3 ± 1.6b
87.1 ± 0.0ab
91.4 ± 1.9a
82.3 ± 1.6b
82.3 ± 1.6b
89.2 ± 0.9a
60.4 ± 0.7fg
59.1 ± 1.2g
66.0 ± 1.4ef
69.7 ± 1.7de
67.0 ± 0.6e
65.8 ± 1.0ef
50.5 ± 0.9h
57.2 ± 0.7g
57.7 ± 0.9g
57.2 ± 0.7h
59.9 ± 0.2g
46.1 ± 1.0h
74.1 ± 0.2cd
76.2 ± 0.7c
66.8 ± 1.1e
5.3±0.8i
89.8 ± 0.1e
91.1 ± 1.2d
92.3 ± 0.1c
82.1 ± 0.1g
86.0 ± 1.2f
88.9 ± 0.7e
64.1 ± 1.5k
58.6 ± 0.5l
54.9 ± 1.0o
61.3 ± 0.3l
52.0 ± 0.7p
65.1 ± 0.6k
48.8 ± 0.0q
66.5 ± 0.4j
50.0 ± 0.0q
54.4 ± 0.8l
96.4 ± 1.2a
40.3 ± 0.7r
73.6 ± 1.0j
75.8 ± 0.8h
54.3 ± 0.7o
93.9±0.7b
2-OH
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
t-Butyl
t-Amyl
91.4
89.4
85.0
86.2
90.4
68.3
59.2
63.8
68.4
67.6
70.5
54.6
63.7
54.6
53.9
55.0
40.3
64.4
68.1
70.2
72.4
4-OH
4-OH
2-OAc
2-OAc
2-OMe
2-OMe
4-OMe
4-OMe
2-OH-3-OMe
2-OH-3-OMe
4-F
C10
C11
C12
C13
C14
4-F
C15
4-Br
C16
4-Br
C17
4-CF3
C18
4-CF3
C19
3,4-OCH2O
3,4-OCH2O
C20
Kresoxim-methyl
Carbendazim
a The difference between the data with the different lowercase letters within a column is significant (P < 0.05).
https://doi.org/10.1371/journal.pone.0176189.t003
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
Table 4. EC50 values of the compounds with higher initial activity against four strains of fungi.
Compd.
EC50 (confidence intervals at 95% probability) (μg/mL, 72 h)a
F. solani
P. grisea
18.3±0.5b (13.5–24.0)
48.9±2.5f (36.3–63.1)
46.0±1.6e (38.9–55.0)
44.5±1.2e (36.3–55.0)
15.4±0.1a (12.6–18.6)
17.0±0.1ab (12.3–22.9)
23.4±0.6cd (20.0–26.9)
23.0±0.7c (21.4–24.5)
25.7±0.4d (19.1–33.9)
24.8±0.3cd (20.0–30.9)
58.7±2.6g (36.3–128.8)
>100
V. mali
B. dothidea
17.5±0.8e (13.2–22.9)
43.2±1.1i (34.7–52.5)
21.0±1.7f (15.1–28.2)
23.2±1.2g (20.4–26.9)
11.4±0.0c (9.1–13.8)
9.9±0.1b (5.9–14.5)
23.2±0.7g (19.5–26.9)
29.2±1.0h (25.7–33.1)
13.7±0.1d (9.5–18.2)
20.4±0.7f (17.4–24.0)
4.3±0.1a (3.0–5.8)
Mean
26.1
A2
26.1±0.5d (21.4–31.6)
19.0±1.0c (13.8–25.7)
32.8±2.0f (28.8–37.2)
17.8±0.6c (14.8–21.4)
24.1±0.8d (20.9–28.2)
19.8±0.9c (17.0–22.9)
40.6±1.0g (34.7–47.9)
29.3±1.1e (24.5–35.5)
42.2±1.4g (33.9–51.3)
34.1±0.5f (25.1–45.7)
14.2±4.4b (9.8–20.4)
3.3±0.1a (2.5–4.4)
42.6±0.8e (31.6–57.5)
60.8±4.3f (42.7–97.7)
59.1±2.8f (41.7–87.1)
18.4±0.6b (13.5–24.5)
18.6±0.8b (14.8–23.4)
27.4±1.4c (22.9–33.1)
21.1±0.2b (15.8–27.5)
31.0±0.5c (22.9–41.7)
29.2±0.4c (22.4–37.2)
34.9±0.5d (25.1–49.0)
98.5±4.5g (77.6–134.9)
2.8±0.1a (1.8–4.1)
A4
43.0
39.7
26.0
17.4
18.5
27.1
28.1
27.7
28.6
43.9
A8
A25
C1
C2
C3
C4
C5
C6
KMb
CBDc
3.6±0.1a (3.3–4.0)
aThe difference between the data with the different lowercase letters within a column is significant (P < 0.05)
b KM: Kresoxim-methyl
c CBD: carbendazim.
https://doi.org/10.1371/journal.pone.0176189.t004
except for 18. Especially, C1-C6 exhibited the very high average inhibition rates of 85–91% for
all the fungi, obviously superior to all compounds A, B, kresoxim-methyl or carbendazim.
In order to explore the antifungal potential in more detail and SAR, some of the compounds
(A2, A4, A8, A25, C1−C6) with the higher initial activity were further determined for median
effective concentrations (EC50) against each of the fungi. The results are listed in Table 4. Sev-
enty percent of all the test items (28/40) revealed good to excellent activity with EC50 values of
9.9−30 μg/mL. Compounds A4, A25 and C2 showed the highest activity against F. solani with
EC50 values of 17.8−19.8 μg/mL (P<0.05). For P grisea, C1 and C2 showed the highest activity
(EC50 = 15.4, 17.0 μg/mL) (P<0.05), followed by A2 (EC50 = 18.3 μg/mL). Compounds A25, C1
and C3 displayed the strongest activity against V mali (EC50 = 18.4, 18.6, 21.1 μg/mL) (P<0.05).
For B. dothidea, C1, C1 and C5 showed the highest activity with EC50 values of 11.4, 9.9 and
13.7 μg/mL (P<0.05), followed by A2 with EC50 values of 17.5 μg/mL. As far as the average
EC50 value of each compound for all the fungi was concerned, C1 had the highest activity (aver-
age EC50 = 17.4 μg/mL), followed by C2 (average EC50 = 18.5 μg/mL). It was worth noting that
all the test compounds were much more active on P. grisea and V. mali than kresoxim-methyl
(EC50 = 58.7, 98.5 μg/mL) or on P. grisea than carbendazim (EC50 > 100 μg/mL).
Discussion—Structure-activity relationship
Comparison of the activities and structures of the compounds in Tables 1 and 2 showed that
both substitution patterns of the benzene ring and the type of alkyl groups in the alcohol moi-
ety significantly influence the activity of cinnamic esters. For ethyl cinnamate derivatives (A
series), in most cases, the introduction of substituents to the benzene ring leads to increase of
the activity. For electron-donating groups like OH, OMe, and OAc, the order of the activity of
various position isomers is o-substituted isomer > p-substituted isomer > m-substituted iso-
mer, whereas for electron-withdrawing groups like halogen atoms and trifluoromethyl, p-
substituted isomers generally have the highest activity. 2,4-Dihydroxy or 2,5-dihydroxy com-
pounds (A23, A24) are less active than the corresponding single hydroxyl compounds (A23 vs
A2 or A4; A24 vs A2 or A3). For 2-OH-substituted compound (A2), the presence of additional
3-OMe has little effect on the activity (A2 vs A25), whereas for 4-OH- or 4-OMe-substituted
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Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
compounds, introduction of 3-OMe leads to significant decrease of the activity (A4 vs A26;
A7 vs A27). Additionally, compared with 3,4-dimethoxy, 3,4-methylenedioxy can improve the
activity (A27 vs A28).
For B series, the activity of the linear alkyl esters (A1, B1−B3, B5, B7−B9) showed an obvi-
ous trend of increase at first and then decrease with elongation of the linear alkyl chain in the
alcohol moiety (Table 2). The n-butyl ester (B3) showed the highest activity with an average
inhibition rates of 46.8% for all the fungi, followed by the n-propyl ester (B2, 39.4%), n-amyl
ester (B5, 35.4%) and n-hexyl ester (B7, 35.8%). Compared with linear alkyl groups, the corre-
sponding tertiary alkyl groups can significantly increase the activity (B3 vs B4; B5 vs B6),
showing that the steric hindrance near the ester group is helpful to improvement of the activ-
ity. The same trend was also observed by comparison of the activity of B7 and B10. Replace-
ment of the cyclohexyl with phenyl leads to decrease of the activity (B12 vs B10), whereas
replacement of the linear alkyl with benzyl (B11) causes slight increase of the activity.
By comparison of the activity of compounds C in Table 3 and that of the corresponding com-
pounds A in Table 1 or B in Table 2, it was found that there exists a complex comprehensive
effect between the substituent on the phenyl ring and the alkyl group in the alcohol moiety on
the activity. The combinations of “2-OH, 4-OH or 2-OAc + t-butyl or t-amyl” (C1-C6) are able
to create significantly synergistic enhancement effect on the activity. On the contrary, the combi-
nations of “2-OMe, 4-OMe, 2-OH-3-OMe, 4-F, 4-Br, 4-CF3 or 3,4-OCH2O + t-butyl or t-amyl”
(C7-C20) generally decrease the activity, compared with t-butyl or t-amyl cinnamates (B4, B6),
or the corresponding substituted cinnamic acid ethyl esters (A5, A7, A13, A19, A22, A25, A28).
From the EC50 values in Table 4, it was further found that the comprehensive effect between
the substituent on the phenyl ring and the alkyl group in the alcohol moiety on the activity var-
ies with the species of the test fungi. The combinations of both “2-OH + t-butyl” (C1) and
“2-OH + t-amyl” (C2) can significantly improve the activity against each of the fungi, relative
to “2-OH + ethyl” (A2). By contrast, the combinations of “4-OH or 2-OAc + t-butyl or t-amyl”
(C1−C6) can increase the activity on P grisea, V mali and B. dothidea, but decrease the activity
on F. solani (C3, C4 vs A4; C5, C6 vs A8).
Although the activity of the compounds against some of the fungi is inferior to carbenda-
zim, this class of compounds showed some attractive advantages, such as natural compounds
or natural compound framework, simple structure, easy synthesis, low-cost as well as environ-
mentally friendly [6,7]. Therefore, cinnamic acid esters possess great potential as new antifun-
gal agents or promising lead compounds for development of new environmentally friendly
plant fungicides. The results of this study can provide direction for the further optimal design
of the structure. Furthermore, it is necessary to determine the in vivo activity and action mech-
anism of these compounds.
Supporting information
S1 File. 1H NMR, 13C NMR, LRMS and HRMS data and spectra of the compounds and
their toxicity regression equations against four fungi.
(PDF)
Acknowledgments
We sincerely thank Hongli Zhang (Northwest A&F University) for the NMR determination of
the compounds and Professor Zhenghui guan (Northwest University, China) the HRMS deter-
mination of NMR.
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10 / 13

Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
Author Contributions
Conceptualization: LZ FM.
Data curation: KZ DDC.
Formal analysis: BL.
Funding acquisition: LZ.
Investigation: KZ DDC BL BYZ FM.
Methodology: LZ FM.
Project administration: LZ FM.
Resources: LZ.
Supervision: LZ FM.
Validation: LZ KZ.
Visualization: LZ KZ.
Writing – original draft: LZ KZ FM.
Writing – review & editing: LZ KZ.
References
1. Savary S, Teng PS. Willocquet L, Nutter FW Jr. Quantification and modeling of crop losses: a review of
purposes. Annu Rev Phytopathol. 2006; 44: 89–112. https://doi.org/10.1146/annurev.phyto.44.
070505.143342 PMID: 16480337
¨
2. Brase S, Encinas A, Keck J, Nising CF. Chemistry and biology of mycotoxins and related fungal metab-
olites. Chem Rev. 2009; 109: 3903–3990. https://doi.org/10.1021/cr050001f PMID: 19534495
3. Delp CJ. Coping with resistance to plant disease. Plant Dis. 1980; 64: 652–657.
4. Wedge DE, Camper ND. Biologically active natural products in agrochemicals and pharmaceuticals.
Cutler HG, Cutler SJ, editors. CRC Press: Boca Raton, FL, USA; 2000. Pp. 1−15.
5. Clifford NM. Chlorogenic acids and other cinnamates—nature, occurrence, dietary burden, absorption
and metabolism. J Sci Food Agric. 2000; 80: 1033–1043.
6. Lafay S, Gil-Izquierdo A. Bioavailability of phenolic acids. Phytochem Rev. 2008; 7: 301–311.
7. Hoskins JA. The occurrence, metabolism and toxicity of cinnamic acid and related compounds. J Appl
Toxicol. 1984; 4: 283–292. PMID: 6394637
8. De Vita D, Friggeri L, D’Auria F D, Pandolfi F, Piccoli F, Panella S, et al. Activity of caffeic acid deriva-
tives against Candida albicans biofilm. Bioorg Med Chem Lett. 2014; 24: 1502–1505. https://doi.org/10.
1016/j.bmcl.2014.02.005 PMID: 24582984
9. Fu J, Cheng K, Zhang Z, Fang R, Zhu H. Synthesis, structure and structure-activity relationship analysis
of caffeic acid amides as potential antimicrobials. Eur J Med Chem. 2010; 45: 2638–2643. https://doi.
org/10.1016/j.ejmech.2010.01.066 PMID: 20181415
10. Sova M. Antioxidant and antimicrobial activities of cinnamic acid derivatives. Mini Rev Med Chem.
2012; 12: 749–767. PMID: 22512578
11. Fabricio B, Laura M, Cecilia S, Francisco G, Fernando G, Marcela K-S, et al. Structure-antifungal activ-
ity relationship of cinnamic acid derivatives. J Agric Food Chem. 2007; 55: 10635–10640. https://doi.
org/10.1021/jf0729098 PMID: 18038998
12. Guzman JD, Mortazavi PN, Munshi T, Evangelopoulos D, McHugh TD, Gibbons S, et al. 2-Hydroxy-
substituted cinnamic acids and acetanilides are selective growth inhibitors of Mycobacterium tuberculo-
sis. MedChemComm. 2014; 5: 47–50.
13. De P, Baltas M, Bedos-Belval F. Cinnamic acid derivatives as anticancer agents-a review. Curr Med
Chem. 2011; 18: 1672–1703. PMID: 21434850
PLOS ONE | https://doi.org/10.1371/journal.pone.0176189 April 19, 2017
11 / 13

Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
14. Debnath B, Samanta S, Roy K, Jha T. QSAR study on some p-arylthio cinnamides as antagonists of
biochemical ICAM-1/LFA-1 interaction and ICAM-1/JY-8 cell adhesion in relation to anti-inflammatory
activity. Bioorg Med Chem. 2003; 11: 1615–1619. PMID: 12659746
15. Lee SU, Shin CG, Lee CK, Lee YS. Caffeoylglycolic and caffeoylamino acid derivatives, halfmers of L-
chicoric acid, as new HIV-1 integrase inhibitors. Eur J Med Chem. 2007; 42: 1309–1315. https://doi.
org/10.1016/j.ejmech.2007.02.016 PMID: 17434650
16. Zhang B-Y, Lv C, Li WB, Cui Z-M, Chen D-D, Cao F-J, et al. Ethyl cinnamate derivatives as promising
high-efficient acaricides against Psoroptes cuniculi: synthesis, bioactivity and structure-activity relation-
ship, Chem Pharm Bull. 2015; 63: 255–262. https://doi.org/10.1248/cpb.c14-00765 PMID: 25739666
´
17. Otero E, Robledo SM, Dıaz S, Carda M, Muñoz D, Paños J, et al. Synthesis and leishmanicidal activity
of cinnamic acid esters: structure-activity relationship. Med Chem Res. 2014; 23: 1378–1386.
18. Yabe T, Hirahara H, Harada N, Ito N, Nagai T, Sanagi T, et al. Ferulic acid induces neural progenitor
cell proliferation in vitro and in vivo. Neuroscience. 2010; 165: 515–524. https://doi.org/10.1016/j.
neuroscience.2009.10.023 PMID: 19837139
19. Hazir S, Shapiro-Ilan D I, Bock C H, Hazir C, Leite L G, Hotchkiss M W. Relative potency of culture
supernatants of Xenorhabdus and Photorhabdus spp. on growth of some fungal phytopathogens. Eur J
Plant Pathol. 2016; 146: 369–381.
20. Lu M, Han Z, Yao L. In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds
against Phytophthora parasitica var. nicotianae. J. Applied Microbiol. 2013; 115: 187–198.
21. Bock H C, Shapiro-Ilan D I, Wedge D E, Cantrell C L. Identification of the antifungal compound, trans-
cinnamic acid, produced by Photorhabdus luminescens, a potential biopesticide against pecan scab. J.
Pest Sci. 2014; 87: 155–162;
22. Gagey N, Neveu P, Benbrahim C, Goetz B, Aujard I, Baudin J-B, et al. Two-photon uncaging with fluo-
rescence reporting: evaluation of the o-hydroxycinnamic platform. J Am Chem Soc. 2007; 129: 9986–
9998. https://doi.org/10.1021/ja0722022 PMID: 17658803
23. Frimer AA, Aljadeff G, Gilinsky-Sharon P. Reaction of coumarins with superoxide anion radical (O2-.):
facile entry to o-coumarinic acid systems. Israel J Chem. 1986; 27: 39–44.
24. Ouimet MA, Stebbins ND, Uhrich KE. Biodegradable coumaric acid-based poly(anhydride-ester) syn-
thesis and subsequent controlled release. Macromol Rapid Commun. 2013; 34: 1231–1236. https://
doi.org/10.1002/marc.201300323 PMID: 23836606
25. Chatterjee AK, Choi T-L., Sanders DP, Grubbs RH. A general model for selectivity in olefin cross
metathesis. J Am Chem Soc. 2003; 125: 11360–11370. https://doi.org/10.1021/ja0214882 PMID:
16220959
26. Fan X, Zhang H-S, Chen L, Long Y-Q. Efficient synthesis and identification of novel propane-1,3-dia-
mino bridged CCR5 antagonists with variation on the basic center carrier. Eur J Med Chem. 2010; 45:
2827–2840. https://doi.org/10.1016/j.ejmech.2010.03.003 PMID: 20347189
27. Cao P, Li C-Y, Kang Y-B, Xie Z, Sun X-L, Tang Y. Ph3As-catalyzed Wittig-type olefination of aldehydes
with diazoacetate in the presence of Na2S2O4. J Org Chem. 2007; 72: 6628–6630. https://doi.org/10.
1021/jo0709899 PMID: 17655362
28. Huang Z-Z, Tang Y. Unexpected catalyst for Wittig-type and dehalogenation reactions. J Org Chem.
2002; 67: 5320–5326. PMID: 12126422
29. Penafiel I, Pastor IM, Yus M. Heck-matsuda reactions catalyzed by a hydroxyalkyl-functionalized NHC
and palladium acetate. Eur J Org Chem. 2012; 43: 3151–3156.
30. Fontan N, Garcia-Dominguez P, Alvarez R, de Lera AR. Novel symmetrical ureas as modulators of pro-
tein arginine methyl transferases. Bioorg Med Chem. 2013; 21: 2056–2067. https://doi.org/10.1016/j.
bmc.2013.01.017 PMID: 23395110
31. Yang W, Chen H, Li J, Li C, Wu W, Jiang H. Palladium-catalyzed aerobic oxidative double allylic C-H
oxygenation of alkenes: a novel and straightforward route to α,β-unsaturated esters. Chem Comm.
(Cambridge, United Kingdom) 2015; 51: 9575–9578.
32. Tyagi V, Fasan R. Myoglobin-catalyzed olefination of aldehydes. Angew Chem Int Ed. 2016; 55: 2512–
2516.
33. Liu J, Shao C, Zhang Y, Shi G, Pan S. Copper-catalyzed highly efficient ester formation from carboxylic
acids/esters and formates. Org Biomol Chem. 2014; 12: 2637–2640. https://doi.org/10.1039/
c4ob00193a PMID: 24668215
34. Zhan S, Tao X, Cai L, Liu X, Liu T. The carbon material functionalized with NH2+ and SO3H groups cat-
alyzed esterification with high activity and selectivity. Green Chem. 2014; 16: 4649–4653.
35. Xin Y-C, Shi S-H, Xie D-D, Hui X-P, Xu P-F. N-heterocyclic carbene-catalyzed oxidative esterification
reaction of aldehydes with alkyl halides under aerobic conditions. Eur J Org Chem. 2011; 2011: 6527–
6531.
PLOS ONE | https://doi.org/10.1371/journal.pone.0176189 April 19, 2017
12 / 13

Inhibition activity of cinnamic acid esters against four plant pathogenic fungi
36. Sinha AK, Sharma A, Swaroop A, Kumar V. Single step green process for the preparation of substituted
cinnamic esters with trans-selectivity. U.S. Pat. Appl. Publ. US 20080045742 A1 20080221, 2008.
37. Sova M, Perdih A, Kotnik M, Kristan K, Rizner T L, Solmajer T, et al. Flavonoids and cinnamic acid
esters as inhibitors of fungal 17β-hydroxysteroid dehydrogenase: A synthesis, QSAR and modelling
study. 2006; 14: 7404–7418. https://doi.org/10.1016/j.bmc.2006.07.027 PMID: 16891119
38. Davies SG, Mulvaney AW, Russell AJ, Smith AD. Synthesis of homochiral β-amino acids. Tetrahedron:
Asymmetry. 2007; 18: 1554–1566.
39. Zhu M-K, Zhao J-F, Loh T-P. Palladium-catalyzed C-C bond formation of arylhydrazines with olefins via
carbon-nitrogen bond cleavage. Org. Lett. 2011; 13: 6308–6311. https://doi.org/10.1021/ol202862t
PMID: 22054076
40. El-Batta A, Jiang C, Zhao W, Anness R, Cooksy AL, Bergdahl M. Wittig reactions in water media
employing stabilized ylides with aldehydes. Synthesis of α,β-unsaturated esters from mixing aldehydes,
α-bromoesters, and Ph3P in aqueous NaHCO3. J Org Chem. 2007; 72: 5244–5259. https://doi.org/10.
1021/jo070665k PMID: 17559278
41. Wong CTT, Lam HY, Song T, Chen G, Li X. Synthesis of constrained head-to-tail cyclic tetrapeptides
by an imine-induced ring-closing/contraction strategy. Angew Chem Int Ed. 2013; 52: 10212–10215.
42. Hou Z, Zhu L-F, Yu X-C, Sun M-Q, Miao F, Zhou L. Design, synthesis, and structure-activity relationship
of new 2-aryl-3,4-dihydro-β-carbolin-2-ium salts as antifungal agents, J Agric Food Chem. 2016; 64:
2847–2854. https://doi.org/10.1021/acs.jafc.6b00505 PMID: 27004437
PLOS ONE | https://doi.org/10.1371/journal.pone.0176189 April 19, 2017
13 / 13