Synthesis and evaluation of essential oil-derived β-methoxyacrylate derivatives as high potential fungicides

Article abstract of DOI:10.3390/molecules22050763

Essential oils (EOs) are plant-derived aroma compounds with a wide range of biological activity, but their actions are slow, and they are typically unstable to light or heat, difficult to extract and so on. To find highly potential fungicides derived from natural EOs, a series of essential oil-based β-methoxyacrylate derivatives have been designed and synthesized. The target compounds have been screened for their potential fungicidal activity against eleven species of plant pathogen fungi, including Alternaria alternata, Phomopsis adianticola, Pestalotiopsis theae, Sclerotinia sclerotiorum, etc. Compared with intermediates I, the parent essential oils and azoxystrobin, almost all of essential oil-based β-methoxyacrylate derivatives exhibited significantly better fungicidal activity. Further investigation revealed that some compounds showed remarkable inhibitory activities against Pestalotiopsis theae, Phomopsis adianticola, Sclerotinia sclerotiorum and Magnapothe grisea at different concentrations in contrast to the commercial product azoxystrobin. Compound II-8 exhibited particularly significant fungicidal activity.

Full text of DOI:10.3390/molecules22050763

molecules
Article
Synthesis and Evaluation of Essential Oil-Derived
β-Methoxyacrylate Derivatives as High
Potential Fungicides
Haihuan Su, Wenda Wang, Longzhu Bao, Shuangshuang Wang and Xiufang Cao *
College of Science, Huazhong Agricultural University, Wuhan 430070, China; 13026346321@163.com (H.S.);
hzauwangwenda@163.com (W.W.); joblongzhu@163.com (L.B.); hzaushuang@163.com (S.W.)
* Correspondence: caoxiufang@mail.hzau.edu.cn
Academic Editors: Daniela Barlocco and Fiorella Meneghetti
Received: 31 March 2017; Accepted: 5 May 2017; Published: 8 May 2017
Abstract: Essential oils (EOs) are plant-derived aroma compounds with a wide range of biological
activity, but their actions are slow, and they are typically unstable to light or heat, difficult to extract
and so on. To find highly potential fungicides derived from natural EOs, a series of essential
oil-based β-methoxyacrylate derivatives have been designed and synthesized. The target compounds
have been screened for their potential fungicidal activity against eleven species of plant pathogen
fungi, including Alternaria alternata, Phomopsis adianticola, Pestalotiopsis theae, Sclerotinia sclerotiorum,
etc. Compared with intermediates
I, the parent essential oils and azoxystrobin, almost all of
essential oil-based -methoxyacrylate derivatives exhibited significantly better fungicidal activity.
β
Further investigation revealed that some compounds showed remarkable inhibitory activities against
Pestalotiopsis theae, Phomopsis adianticola, Sclerotinia sclerotiorum and Magnapothe grisea at different
concentrations in contrast to the commercial product azoxystrobin. Compound II-8 exhibited
particularly significant fungicidal activity.
Keywords: essential oils; β-methoxyacrylate; hybrids; synthesis; fungicidal activity
1. Introduction
Essential oils (EOs) obtained by steam distillation are plant-derived aroma compounds, which
are natural, lipophilic substances known for their diverse antibacterial, antifungal, anticancer,
antimutagenic, antidiabetic, antiviral, antiinflammatory and antiprotozoal properties [
numerous studies during last few years have indicated that EOs and their major components exhibited
non-mammalian or less mammalian toxicity according to different toxicity testing methods [ ].
Besides, there are certain advantages associated with the use of EOs, including less genotoxicity,
the ability to act on multiple cellular targets and low cost of production [ ]. EOs possess various
applications, mainly in the health, agriculture, cosmetic and food industries [ ]. Many plant EOs
are composed of a range of bioactive compounds that are associated with antifungal activities [ ].
Muller-Riebau et al. screened nine essential oils against four species of plant pathogenic fungi including
Fusarium moniliforme, Rhizoctonia solani, Sclerotinia sclerotiorum and Phytophthora capsici [ ]. Wilson et al.
1,2], and
1,3,4
1
5
2
6
screened 49 essential oils against the fruit pathogen Botrytis cinerea, and among all the 49 essential
oils tested, some demonstrated the most antifungal activity against Botrytis cinerea, such as red thyme
(Thymus zygis) and clove buds (Eugenia caryophyllata) [
confirmed that many of eleven tested plant essential oils possessed in vitro antifungal activity against
Lecanicillium fungicola var. fungicola and Agaricus bisporus [ ]. In former papers, this antifungal activity
was strongly associated with the presence of monoterpenic phenols, especially thymol and carvacrol,
in the essential oils [ 10]. Paeonol, a major phenolic component of Paeonia suffruticosa and
7]. Moreover, Mostafa and coworkers’ study
8
4,6,8–
Molecules 2017, 22, 763; doi:10.3390/molecules22050763
www.mdpi.com/journal/molecules

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Paeonia lactiflora, also has various biological activities [11]. However, the molecules of EOs are generally
volatile, unstable to light or heat and difficult to extract, having the disadvantages of short-term
fungicidal efficacy and slow action, which restrict their possible practical application.
On the other hand, the
plants, and many natural molecules with this unit have been discovered (such as the strobilurins
A and B seen in Figure 1). These -methoxyacrylate derivatives have been demonstrated to
β-methoxyacrylate unit is an important pharmacophore from natural
β
exhibit good fungicidal activities, and many such compounds have been developed as commercial
fungicides (such as kresoxim-methyl, azoxystrobin, pyraclostrobin, trifloxystrobin, picoxystrobin,
fluoxastrobin, orysastrobin, dimoxystrobin, coumoxystrobin, etc.). Currently, β-methoxyacrylate class
is an important kind of broad-spectrum fungicides, which have a unique fungicidal mechanism,
belonging to mitochondrial respiration inhibitors [12]. Kresoxim-methyl (Figure 1), which has good
fungicidal activity, was the first commercial fungicide of this class [13]. However, following the
commercial introduction of the strobilurin fungicides in 1996 resistant isolates were quickly detected
in several plant pathogens. For example, Erysiphe graminis showed obvious resistance to stobilurins
in Germany in 1998 [14], and, by 1999, resistant wheat isolates were found in other countries,
including France, Belgium, UK and Denmark [15–17]. After this other resistant strains were also
found successively [18 19]. Therefore, pursuing safe and eco-friendly fungicides is always a hot topic,
,
and the research into botanical pesticides is an important aspect to prevent the emergence of resistant
plant pathogenic fungi [12].
Figure 1. The discovery process of β-methoxyacrylates derivatives.
During the course of our research aimed at discovering novel EO derivatives as potential
agrochemicals, several series of EOs-derived chiral esters were previously investigated, which
indicated their obvious insecticidal activities [20,21]. In order to improve the possibility of the
application of EOs, a series of EO-methoxyacrylate derivatives have now been designed and
prepared, and their potential fungicidal activities have been fully investigated. Thus, based on
the aforementioned precedents, in this study natural EOs (marked in pink in Figure 2) were introduced
into the strobilurin derivatives’ common skeleton. We hoped that the combination of these two moieties
could result in functional synergy, and make target molecules have better biological properties, making
up for the shortcomings of essential oils at the same time. Setting the kresoxim-methyl molecule as the
structure model (Figure 2), we selected some phenols from essential oils to splice with the strobilurin
moieties. Thereby, a series of new strobilurins derivatives have been synthesized by etherification
reactions, and all newly synthesized compounds II, intermediates I, and EOs have then been screened
for their potential fungicidal activity against eleven species of plant pathogen fungi.
Figure 2. Design strategy of EOs-based β-methoxyacrylate derivatives.

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2. Results and Discussion
2.1. Synthesis
In the present study, a series of EOs-based
β-methoxyacrylate derivatives were prepared via
multi-step transformations. The general methods for the preparation of these EO-methoxyacrylate
derivatives II are outlined in Scheme 1.
Scheme 1. The synthetic route of EO-methoxyacrylate derivatives. Reagents and Conditions: (a) MeOH,
H2SO4, reflux; (b) HCO2CH3, NaH, toluene, r.t.; (c) Me2SO4, K2CO3, DMF, r.t.; (d) NBS, AIBN, CCl4,
reflux; (e) MeOH, (30–33%) methylamine water solution, reflux; (f) KMnO4, NaOH, H2O, ice water
◦
bath; (g) MeOH, H2SO4, reflux; (h) CH3ONH2-HCl, EtOH; (i) K2CO3, DMF, 80 C, 8–12 h.
According to references [22–24], three typical β-methoxyacrylates units were selected as
pharmacophores to combine with various natural EOs to investigate the effect of these substitution
patterns on their fungicidal activity. First, the easily available 2-(o-tolyl)acetic acid was selected as
starting substance, which was conveniently transferred to the corresponding intermediates I1 and I2 in
four and five steps, respectively. For the synthesis of intermediate I3, the selected starting compound
was 1-(o-tolyl)ethanone, which was first converted into the 2-oxo-2-(o-tolyl)acetic acid via an classic
oxidation reaction, followed by a sequence including esterification, condensation and bromination
reactions to afford target intermediate I3. Then the intermediates
the presence of a base catalyst to form the target EO-based -methoxyacrylate derivatives II. All these
prepared EO-methoxyacrylate derivatives II were obtained in medium to good yields, summarized
in Table 1, and all compounds II and the intermediates were characterized by NMR spectroscopy,
I were reacted with various EOs in
β
I
MS data, and gave satisfactory chemical analyses. The chemical structures of the target compounds
are also listed in Table 1.
Table 1. The chemical structure of target compounds II-1~II-17.
Substituents
◦
Yield (%) a
Compd. No.
II-1
Appearance
m.p. ( C)
G
X
Y
Yellowish
solid
CH
O
76–77
66–67
-
42.98
Yellowish
solid
II-2
II-3
CH
CH
O
O
78.50
37.72
Yellowish
liquid

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Table 1. Cont.
Substituents
X
◦
Yield (%) a
Compd. No.
Appearance
m.p. ( C)
G
Y
II-4
II-5
CH
O
White solid
112–113
126–127
82.83
Yellowish
solid
CH
O
O
38.40
56.56
II-6
CH
Yellow solid
87–88
Yellowish
solid
II-7
II-8
CH
CH
CH
CH
NH
NH
NH
NH
73–74
76–77
-
37.60
47.28
31.68
90.72
Yellowish
solid
Yellow
liquid
II-9
II-10
White solid
105–106
Yellowish
solid
II-11
II-12
CH
CH
NH
NH
122–123
83–84
37.60
62.00
Yellow solid
Yellowish
solid
II-13
II-14
II-15
N
N
N
O
O
O
80–81
48–49
70.23
74.54
83.12
Yellowish
solid
Yellowish
solid
100–101

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Table 1. Cont.
Substituents
X
◦
Yield (%) a
Compd. No.
Appearance
m.p. ( C)
G
Y
Light pink
solid
II-16
II-17
N
N
O
97–98
81.83
Yellowish
solid
O
103–104
68.02
a
Isolated yield.
2.2. Spectroscopy
The analytical data for all target compounds, including 1H-NMR, 13C-NMR and ESI-MS gave
satisfactory results. The 1H-NMR spectra of the target compounds indicated distinctive signals of aryl
moiety protons, which presented several groups of peaks in the 7.88–6.39 ppm range. The general
1
signals at higher fields in the H-NMR spectra were assigned to the alkyl protons, respectively,
as shown in the representative spectrum shown in Figure 3. The 13C-NMR spectra presented obvious
signals of different alkyl carbons (72.2–16.0), and the other signals at about 198.1–99.7 ppm in the
13C-NMR spectra were assigned to the C=C carbons, aromatic carbons and C=O carbons, as shown
in the representative spectrum of Figure 4. The mass spectra also showed molecular ion peaks at the
appropriate m/z values.
Figure 3. Representative 1H-NMR spectrum of compound II-1.

Molecules 2017, 22, 763
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Figure 4. Representative 13C-NMR spectrum of compound II-1.
2.3. The In Vitro Antifungal Activity
The bioactivities of the newly prepared essential oil-based
β-methoxyacrylate derivatives
II-1~II-17, intermediates I and the corresponding essential oil molecules were all screened for their
potential fungicidal activity against Alternaria alternata, Mucor, Phomopsis adianticola, Phoma adianticola,
Pestalotiopsis theae and Colletotrichum fructicola sinensis Miyake isolated from Camelia sinensis; Sclerotinia
sclerotiorum isolated from rape; Magnapothe grisea isolated from Oryza sativa; Monilinia fructicola
isolated from peach; Botrytis cinerea isolated from strawberry; Gibberella zeae isolated from wheat
at the concentration of 100 mg/L, and the bioassay primary screening results are listed in the Table 2.
Generally, as shown in Table 2, the preliminary assay illustrated that most of the synthesized
EO-based
fungal strains, and we also can find that most of the target compounds have better inhibitory activities
than that of the corresponding intermediates and the typical EO molecules (Entries 18–23) at the
concentration of 100 mg/L. Notably, the inhibition rates of compounds II-2 II-8, and II-13 on
Pestalotiopsis theae were 92.39%, 86.45% and 93.43%, respectively, where II-2 and II-13 had better
inhibitory activity than azoxystrobin (86.73%). Compounds II-1 II-3, and II-13 presented 82.54%,
β-methoxyacrylate derivatives II displayed good inhibitory activities against all tested
I
,
,
80.40%, and 83.77% inhibition rates against Sclerotinia sclerotiorum, respectively, and all exhibited
obviously better inhibitory activities than the positive control azoxystrobin (31.8%). Compound II-13
exhibited 99.72% inhibition rate against Monilinia fructicola, while the inhibitory activity of azoxystrobin
was 96.84%. Compounds II-2, II-3, II-7, II-8, II-11 and II-13 displayed 86.55%, 82.83%, 86.12%, 82.83%,
84.19%, 95.71% fungicidal activity, respectively, against Magnapothe grisea and all had better inhibitory
activity than azoxystrobin (83.83%), except compounds II-3 and II-8. Compounds II-13 displayed an
83.88% inhibition rate against Gibberella zeae, better than that of azoxystrobin (47.69%).

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Table 2. The fungicidal activity of EOs-oriented β-methoxyacrylates derivatives II-1~II-17 at a concentration of 100 mg/L.
Inhibition Activity (%)
Colletotrichum
fructicola
sinensis Miyake
Entry
Compd. No.
Pestalotiopsis
theae
Botrytis
cinerea
Sclerotinia
sclerotiorum
Phoma
adianticola
Monilinia
fructicola
Alternaria
alternata
Phomopsis
adianticola
Magnapothe
grisea
Gibberella
zeae
Mucor
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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
I1
75.50 ± 4.45
92.39 ± 1.30
71.39 ± 2.03
51.71 ± 5.18
60.16 ± 3.61
45.42 ± 2.48
76.93 ± 6.70
86.45 ± 1.69
73.43 ± 0.17
45.10 ± 5.40
55.26 ± 4.90
49.44 ± 0.06
93.43 ± 1.41
78.61 ± 7.71
33.35 ± 1.75
39.16 ± 0.96
40.00 ± 4.73
25.34 ± 0.00
23.98 ± 1.35
16.10 ± 5.18
28.92 ± 1.69
31.71 ± 2.70
9.80 ± 3.38
86.73 ± 0.17
57.50 ± 4.82
66.73 ± 13.56
45.59 ± 2.76
42.77 ± 3.15
76.14 ± 2.12
17.73 ± 0.00
48.27 ± 2.31
54.91 ± 3.86
41.18 ± 10.03
48.91 ± 0.00
64.41 ± 0.19
24.27 ± 0.00
82.00 ± 5.40
67.68 ± 1.35
−11.45 ± 0.26
40.23 ± 7.52
64.36 ± 0.00
4.09 ± 0.00
54.23 ± 3.52
58.94 ± 2.24
53.92 ± 0.67
35.66 ± 2.24
38.62 ± 7.48
33.49 ± 0.52
41.27 ± 7.33
50.58 ± 1.95
53.92 ± 0.67
31.85 ± 3.44
41.85 ± 5.91
36.19 ± 4.49
55.77 ± 8.83
52.96 ± 6.81
16.72 ± 6.58
18.73 ± 1.35
22.06 ± 0.00
25.56 ± 0.52
16.30 ± 3.14
9.37 ± 0.00
47.78 ± 2.32
8.57 ± 0.00
−2.70 ± 0.37
58.89 ± 3.07
82.54 ± 4.66
70.43 ± 10.60
80.40 ± 1.40
65.82 ± 19.22
72.98 ± 2.10
54.37 ± 9.79
79.57 ± 3.73
75.04 ± 4.31
76.85 ± 1.05
63.34 ± 14.56
72.16 ± 4.66
67.13 ± 0.12
83.77 ± 0.35
66.72 ± 5.36
−76.28 ± 0.00
50.58 ± 6.99
65.65 ± 2.45
−162.93 ± 3.26
−155.11 ± 24.35
11.53 ± 1.40
−167.46 ± 0.82
−168.53 ± 4.66
−172.65 ± 0.00
31.80 ± 7.22
44.02 ± 1.98
48.99 ± 3.35
46.86 ± 1.37
31.99 ± 0.11
51.24 ± 6.15
28.53 ± 1.81
41.03 ± 2.25
47.71 ± 1.70
57.96 ± 7.52
44.29 ± 4.67
48.06 ± 0.33
32.88 ± 2.91
67.24 ± 8.45
56.06 ± 6.15
53.34 ± 4.83
26.94 ± 3.51
22.86 ± 4.78
6.75 ± 0.00
60.23 ± 1.38
70.99 ± 0.23
68.14 ± 0.81
19.65 ± 3.92
31.96 ± 5.88
28.94 ± 1.84
51.11 ± 0.23
65.94 ± 1.84
69.77 ± 1.27
16.88 ± 2.07
38.80 ± 0.81
31.06 ± 3.23
58.77 ± 0.46
73.27 ± 1.61
27.80 ± 2.54
34.32 ± 4.38
15.17 ± 0.35
19.57 ± 4.26
17.78 ± 3.11
18.27 ± 1.73
18.02 ± 1.38
55.83 ± 1.15
8.57 ± 0.00
71.32 ± 1.38
70.35 ± 8.02
92.29 ± 7.23
90.89 ± 1.05
64.41 ± 2.23
85.51 ± 3.55
80.39 ± 4.34
78.16 ± 9.59
81.97 ± 19.45
91.08 ± 1.05
87.55 ± 1.84
87.73 ± 3.15
95.54 ± 3.42
99.72 ± 0.39
85.87 ± 4.47
93.77 ± 1.45
64.22 ± 3.29
−15.52 ± 0.00
17.47 ± 2.37
2.60 ± 0.00
38.98 ± 1.03
47.33 ± 1.83
38.98 ± 0.57
17.10 ± 3.09
44.98 ± 4.01
38.49 ± 3.09
33.95 ± 0.11
49.11 ± 0.23
52.19 ± 0.00
39.06 ± 2.52
50.24 ± 1.38
41.25 ± 1.03
62.56 ± 1.38
25.61 ± 4.35
41.82 ± 1.60
38.09 ± 0.23
32.58 ± 2.98
31.28 ± 0.69
32.98 ± 1.03
7.78 ± 2.29
9.32 ± 2.64
14.26 ± 0.69
7.21 ± 0.34
51.46 ± 0.11
64.22 ± 1.23
64.14 ± 7.15
52.37 ± 2.57
55.61 ± 3.13
53.08 ± 0.22
41.07 ± 10.50
42.26 ± 5.77
68.72 ± 2.23
52.76 ± 2.01
45.34 ± 14.30
49.37 ± 2.79
43.05 ± 5.25
42.02 ± 6.70
27.49 ± 0.00
26.07 ± 5.14
31.44 ± 0.00
32.23 ± 2.52
15.64 ± 5.92
13.74 ± 0.00
3.24 ± 0.00
74.39 ± 4.86
86.55 ± 0.61
82.83 ± 0.00
46.92 ± 2.43
47.28 ± 6.78
65.45 ± 2.33
86.12 ± 0.81
82.83 ± 1.01
77.90 ± 4.96
60.80 ± 15.58
84.19 ± 0.10
70.17 ± 0.91
95.71 ± 1.01
19.24 ± 3.34
50.79 ± 11.73
77.25 ± 3.64
45.35 ± 2.43
36.48 ± 0.61
34.19 ± 1.21
1.65 ± 0.00
15.48 ± 0.00
44.13 ± 0.34
34.72 ± 3.50
14.13 ± 5.87
22.35 ± 3.50
35.67 ± 8.92
36.27 ± 1.19
38.51 ± 3.56
31.36 ± 3.84
6.86 ± 8.24
19.63 ± 7.22
48.40 ± 0.06
83.88 ± 8.80
36.15 ± 3.39
28.53 ± 5.47
26.26 ± 3.39
19.03 ± 9.06
37.07 ± 2.71
35.47 ± 1.86
24.06 ± 6.04
13.93 ± 0.00
28.81 ± 0.00
−6.98 ± 0.00
47.69 ± 14.45
I2
I3
−6.09 ± 0.00
9.14 ± 4.18
11.14 ± 2.58
9.86 ± 0.00
66.64 ± 0.39
40.99 ± 1.45
65.15 ± 12.49
6.69 ± 1.05
Eugenol
Paoneol
Syringol
Azoxystrobin
37.27 ± 8.61
82.14 ± 0.96
−7.95 ± 0.32
54.32 ± 0.45
42.82 ± 5.11
16.69 ± 7.80
−15.68 ± 1.76
63.51 ± 2.09
43.44 ± 0.00
9.56 ± 0.00
22.82 ± 3.54
8.30 ± 1.62
9.32 ± 4.32
0.00 ± 1.62
96.84 ± 0.00
60.19 ± 17.20
83.83 ± 1.42

Molecules 2017, 22, 763
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According to the primary screening results, compounds II-2
Pestalotiopsis theae; compounds II-2 II-5
compounds II-1 II-3 II-7 II-8 II-9 II-13 had good activity against Sclerotinia sclerotiorum; compounds
II-2 II-3 II-7 II-8 II-9 II-11 II-13 and compounds II-2 II-3 II-5 II-9 II-10 II-11 II-12 II-13
II-14 II-15, had good bioactivity against Magnapothe grisea and Monilinia fructicola respectively; and
,
II-8
,
II-13 had good activity against
,
, II-14 had good fungicidal activity against Botrytis cinerea;
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
Phomopsis adianticola and Gibberella zeae, while finally compound II-8 and compound II-13 had better
bioactivity, respectively.
Furthermore, the preliminary assay indicated many of the target compounds exhibited good
fungicidal activities compared to the commercial fungicide azoxystrobin (Table 2), so in order to
further investigate the potential fungicidal activities, the efficient concentration values (EC50) for
typical compounds were used to further evaluate the fungicidal activity at different concentrations,
including 6.25, 12.5, 25, 50 and 100 mg/L. The fungicidal activities expressed as EC50 values for
highly potential compounds are listed in Table 3, and further prove that some of these EO-based
β-methoxyacrylate derivatives II exhibited higher fungicidal activity than the commercial compound
azoxystrobin under the same conditions.
Table 3. The EC50 values of the selective compounds against several strains of fungi.
EC50 (mg/L)
Compd. No.
Pestalotiopsis
theae
Phomopsis
adianticola
Sclerotinia
sclerotiorum
Magnapothe
grisea
Gibberella zeae
II-1
II-2
II-3
-
-
-
-
-
-
-
-
-
6.75 ± 0.12
0.64 ± 0.06
0.1 ± 0.01
-
-
0.25 ± 0.02
0.96 ± 0.08
-
-
2.05 ± 0.13
II-5
-
II-7
II-8
II-9
II-13
-
-
-
-
-
0.25 ± 0.05
0.003 ± 0.00
0.79 ± 0.06
0.1 ± 0.02
18.75 ± 1.10
-
0.04 ± 0.01
3.06 ± 0.29
0.95 ± 0.08
-
-
-
-
-
-
15.48 ± 1.76
Azoxystrobin
2.54 ± 0.38
35.3 ± 3.65
36.69 ± 3.69
2.41 ± 0.30
As shown in Table 3, the EC50 values indicated that many target compounds had better fungicidal
activity compared with the positive control azoxystrobin. For Pestalotiopsis theae, Phomopsis adianticola,
Sclerotinia sclerotiorum and Magnapothe grisea the EC50 values of compound II-8, which were 0.04, 3.06,
0.003 nd 0.95 mg/L, respectively, were the smallest; for Gibberella zeae, compound II-13 with an EC50
value of 15.48 mg/L had better fungicidal activity than azoxystrobin. On the whole, the compound
II-8 exhibited obviously better activity against Pestalotiopsis theae, Phomopsis adianticola, Sclerotinia
sclerotiorum and Magnapothe grisea than azoxystrobin.
In addition, the effects of the compound II-8, azoxystrobin and blank control on mycelial growth
of Pestalotiopsis theae were investigated. As shown in Figure 5, under the desired concentration
of 6.25 mg/L, 12.5 mg/L, 25 mg/L and 50 mg/L, the diameter
18.50 ± 0.71, 12.23 0.99, 10.50 0.71 and 9.75 2.12 mm; 25.68
10.15 1.56 mm; 61.90 0.14, 60.55 0.78, 57.00 2.47 and 61.98
Compound II-8 presented significant inhibition compared to the positive control azoxystrobin and
blank control.
±
SD of inhibition zone reached
0.60, 17.08 5.06, 11.25 1.06 and
0.11 mm, respectively.
±
±
±
±
±
±
±
±
±
±
±
According to the above results, considering the EO portion, when natural essential oil molecules
such as thymol and carvacrol were introduced, many compounds indicated wonderful activity, which
maybe because thymol and carvacrol themselves display fungicidal activity. In addition, considering
the strobilurin derivatives’ common skeleton, compounds indicated better fungicidal activity when an
(E)-carboxamide methoxypropenoate acrylate unit was introduced into the molecules instead of an
(E)-methoxyacrylate, so maybe the presence of N atoms allows the N-H bond to form hydrogen bonds
with other electron-rich atoms or groups (such as O, F, N, or benzene rings), which could improve
the bioactivity.

Molecules 2017, 22, 763
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Figure 5. Mycelial growth of Pestalotiopsis theae using compound II-8 and azoxystrobin at the different
concentration of 6.25, 12.5, 25 and 50 mg/L.
3. Experimental
3.1. General Information
Melting points (m.p.) were determined on an X-4 digital display microscopic melting point
1
apparatus (Shanghai Instrument Physical Optics Instrument Co., Ltd., Shanghai, China). H-NMR
spectra were recorded on a Bruker spectrometer at 600 or 400 MHz (Bruker, Bremen, Germany)
with CDCl3 as the solvent and TMS as the internal standard; 13C-NMR spectra were recorded on a
Bruker spectrometer at 150 MHz with CDCl3 as the solvent. Chemical shifts are reported in
δ
(parts
n
per million) values. Coupling constants J are reported in Hz. Mass spectra were performed on
a Waters ACQUITY UPLC® H-CLASS PDA (Waters®, Milford, MA, USA) instrument. Analytical
thin-layer chromatography (TLC) was carried out on pre-coated plates, and spots were visualized
with ultraviolet light. Column chromatography was carried out on silica gel (200–300 mesh, Qingdao
Haiyang Chemical, Qingdao, China). Agar (Shanghai Regal Chemical, Shanghai, China), D-(+)-glucose
(Shanghai Hushi Chemical, Shanghai, China). All other solvents and reagents were analytical reagent
and used directly without purification.
3.2. General Synthetic Procedure for Intermediates I
2-(o-Tolyl)acetic acid or 1-(o-tolyl)ethanone as starting materials were prepared according to
the patent literature [22–24]. Intermediates I were synthesized via esterification, ester condensation,
methylation, and bromination reactions. All intermediates
analytical data.
I (Scheme 1) have satisfactory chemical
3.3. General Synthetic Procedure for Target Compounds II
A mixture of essential oil phenol (2.11 mmol) and anhydrous potassium carbonate (2.64 mmol) in
DMF (8 mL) was stirred at room temperature for 0.5 h◦. Then intermediates
I (1.76 mmol) were added
and the reaction mixtures were stirred for 8–12 h at 80 C. The resulting mixtures were cooled to room
temperature and filtered, and then the reaction mixtures were extracted with ethyl acetate (3
The combined organic layers were washed with brine (3 20 mL), dried over anhydrous sodium sulfate
and concentrated with a rotary evaporator. The crude products were purified by chromatography
×
10 mL).
×

Molecules 2017, 22, 763
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on silica using a mixture of petroleum ether and ethyl acetate as an eluent to give the target essential
oils-oriented -methoxyacrylate derivatives II. All the characterization data are as follows and the
β
corresponding spectra of the target molecules are presented in the Supplementary Materials.
(E)-2-(2-((2-Isopropyl-5-methyl)phenoxymethyl)phenyl)-3-methoxyacrylate (II-1). This compound was
◦
1
obtained as a yellowish solid following the abovementioned method, m.p. 76–77 C. H-NMR
(400 MHz, CDCl3): = 7.61 (s, 1H), 7.59 (d, J = 4 Hz, 1H), 7.37–7.29 (m, 2H), 7.16 (dd, 1H), 7.10 (d,
J = 4 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.60 (s, 1H), 4.94 (s, 2H), 3.82 (s, 3H), 3.70 (s, 3H), 3.41–3.34 (m, 1H),
2.28 (s, 3H), 1.23 (s, 3H), 1.21 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 168.0, 160.2, 155.8, 136.7, 136.2,
δ
δ
134.3, 130.8, 130.6, 128.1, 127.2, 127.0, 125.8, 121.2, 112.6, 110.0, 67.7, 62.0, 51.7, 26.5, 22.8, 21.4; MS (ESI)
m/z 377.4 (M + Na)+, calcd. for C22H26O4 m/z = 354.2.
(E)-2-(2-((5-Isopropyl-2-methyl)phenoxymethyl)phenyl)-3-methoxyacrylate (II-2). This compound was
obtained as a yellowish solid following the abovementioned method, m.p. 66–67 ◦C. 1H-NMR (400
MHz, CDCl3):
δ = 7.60 (s, 1H), 7.58 (d, J = 8 Hz, 1H), 7.36–7.29 (m, 2H), 7.17 (dd, 1H), 7.06 (d, J = 8 Hz,
1H), 6.75–6.71 (m, 1H), 6.62 (s, 1H), 4.96 (s, 2H), 3.82 (s, 3H), 3.70 (s, 3H), 2.96–2.77 (m, 1H), 2.23 (s, 3H),
1.20 (s, 3H), 1.18 (s, 3H); 13C-NMR (150 MHz, CDCl3):
δ = 168.0, 160.1, 156.8, 147.8, 136.6, 130.8, 130.7,
130.4, 128.0, 127.2, 124.3, 117.9, 110.0, 67.7, 61.9, 51.7, 34.1, 24.1, 16.0; MS (ESI) m/z 377.3 (M + Na)+,
calcd. for C22H26O4 m/z = 354.2.
(E)-2-(2-((4-Allyl-2-methoxy)phenoxymethyl)phenyl)-3-methoxyacrylate (II-3). This compound was
obtained as a yellowish liquid following the abovementioned method, 1H-NMR (400 MHz, CDCl3):
δ
= 7.59 (s, 1H), 7.56 (d, J = 8 Hz, 1H), 7.32–7.26 (m, 2H), 7.14 (dd, 1H), 6.71 (s, 1H), 6.66 (d, J = 8 Hz,
1H), 6.58 (dd, 1H), 5.98–5.88 (m, 1H), 5.09–5.04 (m, 4H), 3.88 (s, 3H), 3.82 (s, 3H), 3.70 (s, 3H), 3.30
(d, J = 8 Hz, 2H); 13C-NMR (150 MHz, CDCl3):
= 168.0, 160.1, 149.4, 146.5, 137.7, 136.4, 132.9, 130.8,
δ
130.5, 128.1, 127.1, 126.8, 120.4, 115.6, 113.9, 112.3, 109.9, 68.8, 62.0, 55.9, 51.7, 39.8; MS (ESI) m/z 391.4
(M + Na)+, calcd. for C22H24O5 m/z = 368.2.
(E)-2-((2-Carboxylate-phenoxymethyl)phenyl)-3-methoxyacrylate (II-4). This compound was obtained as
a white solid following the abovementioned method, m.p. 112–113 ◦C. 1H-NMR (400 MHz, CDCl3):
δ
= 7.79 (dd, 1H), 7.70 (d, J = 8 Hz, 1H), 7.61 (s, 1H), 7.36–7.27 (m, 3H), 7.15 (dd, 1H), 6.96–6.93 (m, 1H),
6.86 (d, J = 8 Hz, 1H), 5.09 (s, 2H), 3.92 (s, 3H), 3.82 (s, 3H), 3.70 (s, 3H); 13C-NMR (150 MHz, CDCl3):
δ = 167.8, 166.9, 160.2, 158.0, 135.9, 133.3, 131.6, 130.8, 130.2, 128.2, 127.2, 126.6, 120.5, 120.2, 113.7, 109.8,
68.3, 62.0, 52.0, 51.7; MS (ESI) m/z 379.3 (M + Na)+, calcd. for C20H20O6 m/z = 356.1.
(E)-2-(2-((2-Acetyl-4-methoxy)phenoxymethyl)phenyl)-3-methoxyacrylate (II-5). This compound was
◦
1
obtained as a yellowish solid following the abovementioned method, m.p. 126–127 C. H-NMR
(600 MHz, CDCl3): = 7.82 (d, J = 6 Hz, 1H), 7.61 (s, 1H), 7.52 (t, J1 = 3.6 Hz, J2 = 6 Hz, 1H), 7.36–7.32
(m, 2H), 7.20–7.18 (m, 1H), 6.50 (dd, 1H), 6.40 (d, J = 2.4 Hz, 1H), 5.06 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H),
3.70 (s, 3H), 2.57 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 198.1, 167.7, 164.4, 160.4, 160.2, 135.2, 132.6,
δ
δ
131.2, 128.2, 127.8, 127.6, 121.4, 109.8, 105.1, 99.8, 68.6, 62.0, 55.5, 51.7, 32.1; MS (ESI) m/z 393.4 (M +
Na)+, calcd. for C21H22O6 m/z = 370.1.
(E)-2-(2-(2,6-Dimethoxyphenoxymethyl)phenyl)-3-methoxyacrylate (II-6). This compound was obtained as
a yellow solid following the abovementioned method, m.p. 87–88 ◦C. 1H-NMR (600 MHz, CDCl3):
δ
= 7.88 (d, J = 12 Hz, 1H), 7.56 (s, 1H), 7.36 (t, J1 = 6 Hz, J2 = 12 Hz, 1H), 7. 26 (t, J1 = J2 = 6 Hz, 1H),
7.12 (d, J = 12 Hz, 1H), 6.96 (t, J1 = 6 Hz, J2 = 12 Hz, 1H), 6.56 (d, J = 6 Hz, 2H), 4.88 (s, 2H), 3.79 (s, 6H),
3.74 (s, 3H), 3.64 (s, 3H); 13C-NMR (150 MHz, CDCl3):
δ = 168.1, 160.1, 153.9, 137.5, 137.3, 131.0, 130.4,
128.4, 127.8, 127.1, 123.6, 110.1, 105.5, 72.2, 61.8, 56.1, 51.5; MS (ESI) m/z 381.3 (M + Na)+, calcd. for
C20H22O6 m/z = 358.1.
(E)-2-(2-((2-Isopropyl-5-methyl)phenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide
compound was obtained as yellowish solid following the abovementioned method, m.p. 73–74 ◦C.
1H-NMR (400 MHz, CDCl3):
= 7.62 (s, 1H), 7.60 (s, 1H), 7.39–7.30 (m, 2H), 7.18 (dd, 1H), 7.12 (d,
(
II-7).
This
δ

Molecules 2017, 22, 763
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J = 8 Hz, 1H), 6.75 (d, J = 8 Hz, 1H), 6.62 (s, 1H), 4.96 (s, 2H), 3.84 (s, 3H), 3.72 (s, 3H), 3.43–3.36 (m,
1H), 2.29 (s, 3H), 1.25 (s, 3H), 1.23 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 168.0, 160.2, 155.8, 136.7,
δ
136.2, 134.3, 130.8, 130.6, 128.1, 127.2, 127.0, 125.8, 121.2, 112.6, 110.0, 67.7, 62.0, 51.7, 26.5, 22.9, 21.4; MS
(ESI) m/z 377.4 (M + Na + H)+, calcd. for C22H27 NO3 m/z = 353.2.
(E)-2-(2-((5-Isopropyl-2-methyl)phenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide
compound was obtained as a yellowish solid following the abovementioned method, m.p. 76–77 ◦C.
1H-NMR (400 MHz, CDCl3):
= 7.60 (s, 1H), 7.58 (d, J = 4 Hz, 1H), 7.37-7.29 (m, 2H), 7.18 (dd, 1H),
7.06 (d, J = 8 Hz, 1H), 6.72 (dd, 1H), 6.62 (s, 1H), 4.97 (s, 2H), 3.82 (s, 3H), 3.70 (s, 3H), 2.85–2.78 (m, 1H),
2.24 (s, 3H), 1.21 (s, 3H), 1.19 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 168.0, 160.1, 156.8, 147.8, 136.6,
(
II-8).
This
δ
δ
130.9, 130.7, 130.4, 128.0, 127.2, 124.3, 117.9, 110.0, 67.7, 62.0, 51.7, 34.1, 24.1, 16.0; MS (ESI) m/z 377.4
(M + Na + H)+, calcd. for C22H27NO3 m/z = 353.2.
(E)-2-(2-((4-Allyl-2-methoxy)phenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide (II-9). This compound
was obtained as a yellow liquid following the abovementioned method, 1H- NMR (400 MHz, CDCl3):
δ
= 7.59 (s, 1H), 7.55 (d, J = 4 Hz, 1H), 7.32–7.26 (m, 2H), 7.14 (dd, 1H), 6.71 (s, 1H), 6.66 (d, J = 4 Hz,
1H), 6.58 (dd, 1H), 5.98–5.88 (m, 1H), 5.09–5.04 (m, 4H), 3.88 (s, 3H), 3.82 (s, 3H), 3.70 (s, 3H), 3.30 (d,
J = 8 Hz, 2H); 13C-NMR (150 MHz, CDCl3):
= 167.9, 160.1, 149.4, 146.5, 137.7, 136.4, 132.9, 130.8, 130.5,
δ
128.1, 127.1, 126.8, 120.4, 115.6, 113.9, 112.3, 109.9, 68.8, 62.0, 55.9, 51.7, 39.8; MS (ESI) m/z 391.4 (M +
Na + H)+, calcd. for C22H25 NO4 m/z = 367.2.
(E)-2-((2-Carboxylatephenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide (II-10). This compound was
◦
obtained as a white solid following the abovementioned method, m.p. 105–106 C. 1H-NMR (400 MHz,
CDCl3):
δ = 7.79 (dd, 1H), 7.70 (d, J = 8 Hz, 1H), 7.61 (s, 1H), 7.37–7.27 (m, 3H), 7.15 (d, J = 8 Hz, 1H),
6.94 (t, J1 = J2 = 8 Hz, 1H), 6.86 (d, J = 8 Hz, 1H), 5.09 (s, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 3.70 (s, 3H);
13C-NMR (150 MHz, CDCl3):
δ = 167.8, 166.9, 160.2, 158.1, 135.9, 133.3, 131.7, 130.8, 130.2, 128.2, 127.2,
126.6, 120.6, 120.3, 113.7, 109.8, 68.3, 62.0, 52.0, 51.7; MS (ESI) m/z 379.4 (M + Na + H)+, calcd. for
C20H21NO5 m/z = 355.1.
(E)-2-(2-((2-Acetyl-4-methoxy)phenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide (II-11). This compound
was obtained as a yellowish solid following the abovementioned method, m.p. 122–123 ◦C. 1H-NMR
(600 MHz, CDCl3):
(m, 2H), 7.20–7.18 (m, 1H), 6.49 (d, J = 6 Hz, 1H), 6.39 (d, J = 6 Hz, 1H), 5.06 (s, 2H), 3.81 (s, 3H), 3.77 (s,
3H), 3.69 (s, 3H), 2.57 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 198.0, 167.7, 164.4, 160.2, 135.2, 132.6,
δ = 7.82 ( d, J = 6 Hz, 1H), 7.60 (s, 1H), 7.51 (t, J1 = 3 Hz, J2 = 6 Hz, 1H), 7.35–7.32
δ
131.2, 128.2, 127.8, 127.5, 121.4, 109.8, 105.1, 99.8, 68.6, 62.0, 55.4, 51.7, 32.1; MS (ESI) m/z 393.4 (M + Na
+ H)+, calcd. for C21H23NO5 m/z = 369.2.
(E)-2-(2-(2,6-Dimethoxyphenoxymethyl)phenyl)-3-methoxy-N-methylacrylamide (II-12). This compound was
◦
obtained as a yellow solid following the abovementioned method, m.p. 83–84 C. 1H-NMR (600 MHz,
CDCl3):
δ = 7.88 (d, J = 6 Hz, 1H), 7.55 (s, 1H), 7.37 (t, J1 = J2 = 6 Hz, 1H), 7. 28 (t, J1 = 6 Hz, J2 = 12 Hz,
1H), 7.12 (d, J = 6 Hz, 1H), 6.98 (t, J1 = 12 Hz, J2 = 6 Hz, 1H), 6.56 (d, J = 6 Hz, 2H), 4.88 (s, 2H), 3.80 (s,
6H), 3.77 (s, 3H), 3.65 (s, 3H). 13C-NMR (150 MHz, CDCl3):
δ = 168.2, 160.1, 153.9, 137.5, 137.3, 131.0,
130.4, 128.5, 127.8, 127.1, 123.6, 110.1, 105.5, 72.2, 61.8, 56.1, 51.5; MS (ESI) m/z 381.4 (M + Na + H)+,
calcd. for C20H23NO5 m/z = 357.2.
(E)-2-(2-((2-Isopropyl-5-methyl)phenoxymethyl)phenyl)-2-(methoxyimino)acetate (II-13). This compound
was obtained as a yellowish solid following the above mentioned method, m.p. 80–81 ◦C. 1H-NMR
(600 MHz, CDCl3):
δ = 7.62 (d, J = 6 Hz, 1H), 7.46 (t, J1 = 12 Hz, J2 = 6 Hz, 1H), 7.38 (t, J1 = J2 = 6 Hz,
1H), 7.20 (d, J = 12 Hz, 1H), 7.10 (d, J = 6 Hz, 1H), 6.76 (d, J = 6 Hz, 1H), 6.62 (s, 1H), 4.92 (s, 2H), 4.05 (s,
3H), 3.84 (s, 3H), 3.36–3.31 (m, 1H), 2.29 (s, 3H), 1.20 (d, J = 6 Hz, 6H); 13C-NMR (150 MHz, CDCl3):
δ = 163.3, 155.5, 149.3, 136.3, 135.9, 134.3, 129.7, 128.7, 128.3, 127.4, 125.9, 121.6, 112.7, 68.0, 63.9, 53.0,
26.4, 22.9, 21.3; MS (ESI) m/z 378.4 (M + Na)+, calcd. for C21H25NO4 m/z = 355.2.

Molecules 2017, 22, 763
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(E)-2-(2-((4-Allyl-2-methoxy)phenoxymethyl)phenyl)-2-(methoxyimino)acetate (II-14). This compound was
◦
1
obtained as a yellowish solid following the abovementioned method, m.p. 48–49 C. H-NMR
(600 MHz, CDCl3): = 7.58 (d, J = 6 Hz, 1H), 7.40 (t, J1 = J2 = 6 Hz, 1H), 7.34 (t, J1 = 12 Hz, J2 = 6 Hz,
δ
1H), 7.16 (d, J = 6 Hz, 1H), 6.71 (d, J = 12 Hz, 2H), 6.62 (d, J = 12 Hz, 1H), 5.97-5.90 (m, 1H), 5.06 (t,
J1 = 18 Hz, J2 = 6 Hz, 2H), 4.98 (s, 2H), 4.02 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.30 (d, J = 6 Hz, 2H);
13C-NMR (150 MHz, CDCl3):
δ = 163.3, 149.6, 149.4, 146.3, 137.6, 135.7, 133.5, 129.6, 129.0, 128.2, 127.5,
120.4, 115.6, 114.3, 112.5, 69.2, 63.8, 55.9, 52.9, 39.8; MS (ESI) m/z 392.4 (M + Na)+, calcd. for C21H23NO5
m/z = 369.2.
(E)-2-((2-Carboxylate phenoxymethyl)phenyl)-2-(methoxyimino)acetate (II-15). This compound was
◦
1
obtained as yellowish solid following the abovementioned method, m.p. 100–101 C. H-NMR
(600 MHz, CDCl3): = 7.81 (dd, 1H), 7.74 (d, J = 6 Hz, 1H), 7.46 (t, J1 = 6 Hz, J2 = 12 Hz, 1H), 7.40–7.36
(m, 2H), 7.18 (d, J = 6 Hz, 1H), 6.98 (t, J1 = 6 Hz, J2 = 12 Hz, 1H), 6.90 (d, J = 6 Hz, 1H), 5.05 (s, 2H), 4.03
δ
(s, 3H), 3.90 (s, 3H), 3.85 (s, 3H); 13C-NMR (150 MHz, CDCl3):
δ = 166.7, 163.2, 157.8, 149.2, 135.1, 133.4,
131.8, 129.7, 128.4, 128.2, 127.5, 127.1, 120.6, 113.7, 68.5, 63.8, 53.0, 52.0; MS (ESI) m/z 380.4 (M + Na)+,
calcd. for C19H19NO6 m/z = 357.1.
(E)-2-(2-((2-Acetyl-4-methoxy)phenoxymethyl)phenyl)-2-(methoxyimino)acetate (II-16). This compound
was obtained as a light pink solid following the abovementioned method, m.p. 97–98 ◦C. 1H-NMR
(600 MHz, CDCl3):
7.42 (t, J1 = J2 = 6 Hz, 1H), 7.22 (d, J = 6 Hz, 1H), 6.52 (dd, 1H), 6.40 (d, J = 1.8 Hz, 1H), 5.02 (s, 2H), 4.04
(s, 3H), 3.83 (s, 3H), 3.79 (s, 3H), 2.54 (s, 3H); 13C-NMR (150 MHz, CDCl3):
= 197.8, 164.3, 163.2, 159.8,
δ = 7.83 (d, J = 12 Hz, 1H), 7.56 (d, J = 6 Hz, 1H), 7.46 (t, J1 = 12 Hz, J2 = 6 Hz, 1H),
δ
149.0, 134.4, 132.7, 129.8, 129.2, 128.6, 128.1, 127.8, 121.4, 105.3, 99.7, 68.6, 63.9, 55.5, 53.1, 32.0; MS (ESI)
m/z 394.4 (M + Na)+, calcd. for C20H21NO6 m/z = 371.1.
(E)-2-(2-(2,6-Dimethoxyphenoxymethyl)phenyl)-2-(methoxyimino)acetate (II-17). This compound was
◦
1
obtained as a yellowish solid following the abovementioned method, m.p. 103–104 C. H-NMR
(600 MHz, CDCl3): = 7.88 (d, J = 12 Hz, 1H), 7.46 (t, J1 = J2 = 6 Hz, 1H), 7.36 (t, J1 = J2 = 6 Hz, 1H), 7.14
(d, J = 6 Hz, 1H), 6.98 (t, J1 = 6 Hz, J2 = 12 Hz, 1H), 6.56 (d, J = 6 Hz, 2H), 4.83 (s, 2H), 4.00 (s, 3H), 3.82
δ
(s, 3H), 3.80 (s, 6H); 13C-NMR (150 MHz, CDCl3):
δ = 163.3, 153.8, 149.3, 137.0, 136.4, 129.4, 129.2, 129.0,
127.8, 127.4, 123.8, 105.3, 72.1, 63.7, 56.0, 52.8; MS (ESI) m/z 382.5 (M + Na)+, calcd. for C19H21NO6
m/z = 359.1.
3.4. Biological Assays
The fungi used in this study include Alternaria alternata, Mucor, Phomopsis adianticola, Phoma
adianticola, Pestalotiopsis theae and Colletotrichum fructicola sinensis Miyake isolated from Camellia sinensis;
Sclerotinia sclerotiorum isolated from rape; Magnapothe grisea isolated from Oryza sativa; Monilinia
fructicola isolated from peach; Botrytis cinerea isolated from strawberry; Gibberella zeae isolated from
wheat as well as. Target compounds II, intermediates
in acetone and added to Potato Dextrose Agar (PDA) culture medium according to desired final
concentrations [ ], pouring into Petri plates (9-mm diameter). Each sample was screened at a
concentration of 100 mg/L, after that further evaluation was performed based on the a preliminary
results, involving 6.25, 12.5, 25, 50 and 100 mg/L, acetone solutions (less than 1% v/v [ ]). Then,
I and essential oil molecules were diluted
2,8,9
9
each dish was inoculated in the center with a 5 mm diameter stipe, containing mycelia of the fungus
culture. Positive control sets were run simultaneously, using the medium containing azoxystrobin,
and which is a representative strobilurin fungicide [12
PDA medium supplemented with the corresponding concentration of acetone. Dishes were sealed and
incubated at a temperature of 27
1 ◦C for four to six days in a culture chamber, and then diameter
,17], and blank control consisted of unamended
±
mycelium was measured. The percentage of relative inhibition was computed after comparison with
the blank control using the following formula [25]:
I = [(C − T)/C] × 100
(1)

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where I is growth inhibition (%), C and T represent mycelial growth diameter in control and target
compounds II, intermediates I and essential oil molecules in the treated Petri plates, respectively.
EC50 values (mg/L concentrations inhibiting radial mycelial growth by 50%) for the studied
compounds were determined by interpolation from computer-generated log-probit plots of compounds
concentrations and relative inhibition (Probit SPSS version 22).
4. Conclusions
In summary, seventeen new essential oil-based β-methoxyacrylate derivatives have been designed,
synthesized and evaluated as potential fungicides. The structures of all obtained molecules were
1
characterized by H-NMR, 13C-NMR and ESI-MS spectra analyses. The preliminary bioassays
indicated that almost of target compounds exhibited significant fungicidal activity compared with
the commercial compound azoxystrobin, and some compounds presented good fungicidal activity
at lower concentrations. These results also demonstrated that the combination of β-methoxyacrylate
units with essential oils could lead to new active molecules, which may have potential application
for plant fungi treatment. Further structural optimization and specific structure-activity relationship
studies are still under investigation in our laboratory.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: We gratefully acknowledge the support of this work by the Fundamental Research Funds for
the Central Universities (2662015PY034, 2662016PY112).
Author Contributions: Xiufang Cao initiated the idea and supervised the whole experiment and provided
technical guidance, and Haihuan Su conducted the experiment and wrote the paper; Wenda Wang and Longzhu
Bao assisted in the completion of synthesis of the title compounds; Shuangshuang Wang took part in the
experiments on activity evaluation.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds II-1–II-17 are available from the authors.
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