Synthesis and bio-evaluation of natural butenolides-acrylate conjugates

Article abstract of DOI:10.3390/molecules24071304

A series of novel 3-aryl-4-hydroxy-2(5H) furanone-acrylate hybrids were designed and synthesized based on the natural butenolides and acrylates scaffolds. The structures of the prepared compounds were characterized by ¹H-NMR, ¹³C-NMR and electrospray ionization mass spectrometry (ESI-MS), and the bioactivity of the target compounds against twelve phytopathogenic fungi was investigated. The preliminary in vitro antifungal activity screening showed that most of the target compounds had moderate inhibition on various pathogenic fungi at the concentration of 100 mg·L^?1, and presented broad-spectrum antifungal activities. Further studies also indicated that compounds 7e and 7k still showed some inhibitory activity against Pestallozzia theae, Sclerotinia sclerotiorum and Gibberella zeae on rape plants at lower concentrations, which could be optimized as a secondary lead for further research.

Full text of DOI:10.3390/molecules24071304

molecules
Article
Synthesis and Bio-Evaluation of Natural
Butenolides-Acrylate Conjugates
Longzhu Bao 1, Shuangshuang Wang 1, Di Song 1, Jingjing Wang 1, Xiufang Cao 1,* and
Shaoyong Ke 2,
*
1
College of Science, Huazhong Agricultural University, Wuhan 430070, China;
baolz@webmail.hzau.edu.cn (L.B.); 1140291490@webmail.hzau.edu.cn (S.W.);
songdi@webmail.hzau.edu.cn (D.S.); 13523041961@163.com (J.W.)
National Biopesticide Engineering Research Center, Hubei Biopesticide Engineering Research Center,
Hubei Academy of Agricultural Science, Wuhan 430064, China
2
*
Correspondence: caoxiufang@mail.hzau.edu.cn (X.C.); shaoyong.ke@nberc.com (S.K.);
Tel.: +86-027-5910-1919 (S.K.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 March 2019; Accepted: 1 April 2019; Published: 3 April 2019
Abstract: A series of novel 3-aryl-4-hydroxy-2(5H) furanone-acrylate hybrids were designed and
synthesized based on the natural butenolides and acrylates scaffolds. The structures of the prepared
compounds were characterized by 1H-NMR, 13C-NMR and electrospray ionization mass spectrometry
(ESI-MS), and the bioactivity of the target compounds against twelve phytopathogenic fungi was
investigated. The preliminary in vitro antifungal activity screening showed that most of the target
−1
compounds had moderate inhibition on various pathogenic fungi at the concentration of 100 mg
·
L
,
and presented broad-spectrum antifungal activities. Further studies also indicated that compounds
7e and 7k still showed some inhibitory activity against Pestallozzia theae, Sclerotinia sclerotiorum and
Gibberella zeae on rape plants at lower concentrations, which could be optimized as a secondary lead
for further research.
Keywords: 3-aryl-4-hydroxy-2(5H)-furanone; butenolides; acrylate; antifungal activity; natural product
1. Introduction
Natural products play an important role in the field of pesticides, and structural modification
and transformation using natural products as a precursor are important ways to create new candidate
pesticides. It has the advantages of a high target, high efficiency and low toxicity [1]. As a typical
example, synthetic strobilurins are now an important class of fungicides for crop protection, they were
derived from structural optimization by taking the sterilizing active natural antibiotic Strobilurin A as
a precursor. During the period of 2011–2017, up to five Strobilurin fungicides have been registered,
and which are Coumoxystrobin, Fluoxastrobin, Mandestrobin, Metominostrobin, Picoxystrobin [2,3].
This class of fungicides has good activity for almost all fungal diseases, such as powdery mildew,
rust, blight, net blotch, downy mildew, rice blast, etc, and can not only effectively prevent and treat
resistance to other fungicides but also has the effect of prolonging the vegetative growth of crops
and increasing yields [
of fungicides.
4]. More and more researchers are involved in the development of this kind
The structure of traditional strobilurin fungicide includes pharmacophore, aromatic bridge
and side chain. Many studies have shown that the modification of the aromatic bridge of this
fungicide does not significantly affect their biological activity, therefore the structural modification
is mainly focused on the side chain. The core structure of this fungicide, the acrylate structure
unit, often remains unchanged [5,6]. 3-Aryl-4-hydroxy-2(5H)-furanone belongs to the class of natural
Molecules 2019, 24, 1304; doi:10.3390/molecules24071304
www.mdpi.com/journal/molecules

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butenolides, and the core structure of these compounds is a five-membered lactone ring unit, and which
is not only a structural fragment contained in many natural products [ ] but also the core structure
of insecticides [ ]. As shown in Figure 1, many natural products such as ascorbic acid (vitamin C) and
pulvinic acid possess the -keto- -butyrolactone motif, and a lot of butenolide compounds containing
this moiety have anti-tumor, anti-inflammatory, antioxidant and other biological activities [10 17].
7,8
9
β
γ
–
Studies have shown that in the process of bacterial cell wall synthesis, these butenolide derivatives
can inhibit the activity of Mur enzyme to achieve antibacterial action, so it is an antibacterial lead
compound with research and development potential [18,19].
Figure 1. Design strategy of natural butenolides-acrylate conjugates.
In this paper, to develop compounds with better antifungal activity, we combined different
substituted butenolide structural units with the acrylate structural unit to obtain a series of
3-aryl-4-hydroxy-2(5H)-furanone-acrylate hybrids, which was shown in Figure 1. In addition, the target
compounds were tested for their antifungal activity against twelve pathogenic fungi, and the results
showed that the target compounds had moderate antifungal activities.
2. Results and Discussion
2.1. Synthesis of Target Compounds
The synthetic route for target compounds is shown in Scheme 1. Firstly, 2-(O-tolyl)acetic acid was
used as a raw material to obtain key intermediates I by esterification, aldol condensation, methylation
and bromination, that is methyl-2-(2-(bromomethyl)phenyl)-3-methoxyacrylate. Next, the key
intermediate II (6a–6k) was obtained by Dickman condensation reaction using substituted methyl
2-phenylacetate and methyl 2-hydroxyacetate as substances. Finally, intermediate I is reacted with
5
intermediates II (6a–6k) to obtain the target compounds 7a–7k.
2.2. Speculated Synthetic Mechanism of the Key Intermediate II
Based on the above experimental results, the possible mechanism for the formation of the key
intermediates II is presumed as follows (Scheme 2): it mainly involves a transesterification and a
Dieckmann condensation as the two main steps. The transesterification of methyl 2-phenylacetate
5
(with different substituents on the phenyl ring) and methyl 2-hydroxyacetate
derived from methyl glycolate to obtain ester 10, which is then converted to the key intermediates II
by the usual Dieckmann condensation.
8 by the alkoxide 9
8

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Scheme 1. Synthetic route of the target compounds. 7a: R = 2-Me, 7b: R = 2,4-Cl2, 7c: R = 4-F, 7d
:
R = H, 7e: R = 2-Cl, 7f: R = 4-Cl, 7g: R = 2,6-Cl2, 7h: R = 2-MeO, 7i: R = 4-Me, 7j: R = 2-F, 7k: R =
2,4,5-F3. Reagents and conditions: (a) CH3OH, concentrated sulfuric acid, reflux; (b) NaH, HCOOCH3,
toluene; (c) K2CO3, (CH3)2SO4, N,N-dimethylformamide (DMF); (d) CCl4, N-bromosuccinimide (NBS),
azodiisobutyronitrile (AIBN); (e) t-BuOK, THF, reflux; (f) K2CO3, CH3CN, room temperature.
Scheme 2. Speculated synthetic mechanism of the key intermediates II (6a–6k).
2.3. Spectral Analysis of Target Compounds
1
The target compounds were identified by H-NMR, 13C-NMR and ESI-MS, the results of
these spectra indicate that the structure of these compounds is correct (Supplementary Material).
The compound 7g was used as an example for analysis. In the 1H-NMR spectra of this compound,
the chemical shift value of the hydrogen on the methylene group in benzylic position and the hydrogen
on the methylene group on the saturated carbon atom in the five-membered ring is similar, 4.76 and
4.85 respectively. The
δ values of hydrogens on the benzene ring are in region 7.00–7.50 ppm, and the
signal multiplicity, difficult to be assigned, has been indicated as multiplet. In the 13C-NMR spectra
of this compound, the chemical shifts of the carbon of the methoxy groups of the methoxy acrylate
skeleton appear in the high field region, at 62.15 and 51.83 ppm, while the 13C signals of the carbonyl
groups mainly appear in the low field region, at 173.51 and 171.45 ppm. In addition, the mass spectra
also showed molecular ion peaks at the appropriate m/z values, which further confirmed the structure
of target compounds.
2.4. Biological Activity
2.4.1. Primary Fungicidal Activities
The in vitro fungicidal activities of all compounds and some intermediates against 12 different
phytopathogenic fungi at a concentration of 100 mg
bioassays were compared with those of Azoxystrobin, Carbendazim, intermediates I and II.
·
L
−1 are listed in Table 1. The results of preliminary

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Table 1. Preliminary inhibitory effect of target compounds and intermediates on twelve common plant pathogenic fungi (100 mg·L−1).
Antifungal Activities In Vitro (100 µg·mL−1, Inhibitory Rate %)
Rhizoctonia
solani
Phomopsis
adianticola
Rap Sclerotinia
stem rot
Pestallozzia
theae
Sclerotinia
Altermaria
alternata
Pestalotiopsis
theae
Monilinia
fructicola
Phytophthora
capsici
Colletotrichum
gloeosporioides
Magnapothe
grisea
Compd. No.
Gibberella zeae
sclerotiorum
a
6c
6i
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
-
7.9
12.24
41.49
72.84
74.18
73.28
73.28
72.99
35.07
20.45
13.43
31.94
27.31
73.58
60.60
93.43
99.40
12.27
34.33
33.17
48.92
37.81
45.94
49.25
33.00
22.06
37.31
36.65
39.64
45.11
63.18
100.00
100.00
/
17.65
/
4.12
51.03
14.71
43.82
20.29
12.94
24.26
13.53
19.12
8.53
10.88
6.32
17.94
97.65
100.00
100.00
7.08
21.77
9.56
37.79
47.16
0.33
33.61
19.73
30.77
23.41
/
/
38.13
8.36
34.28
32.27
60.03
100.00
100.00
16.70
64.09
35.91
37.88
36.09
41.47
52.42
24.06
9.16
30.52
36.80
33.57
33.93
77.74
93.00
100.00
/
10.25
/
/
2.65
24.91
13.60
17.31
/
-
-
/
5.61
-
2.46
7.72
3.86
/
7.87
28.32
25.52
38.46
29.37
29.72
27.62
29.55
38.81
28.67
26.05
24.48
38.11
29.02
100.00
100.00
-
13.75
64.43
58.25
43.64
43.64
66.15
38.14
86.94
29.55
36.08
55.50
51.03
73.88
95.19
100.00
b
/
32.31
-
1.15
46.73
23.85
/
/
-
/
1.92
33.08
88.65
100.00
16.34
29.20
23.20
45.10
29.90
28.76
14.87
32.68
5.07
44.28
66.99
68.63
100.00
20.18
20.00
20.00
21.59
19.82
17.52
21.42
27.08
17.88
23.19
39.82
59.65
17.70
/
/
4.21
-
19.08
10.60
0.00
24.03
6.36
5.96
10.18
21.93
58.95
100.00
7k
Intermediate I
Azoxystrobin
Carbendazim
a
b
denotes that the compound was not tested. denotes that the compound has no inhibitory activity.

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As indicated in Table 1, under dosage condition of 100 mg
·
L
−1, most of the compounds showed
potent fungicidal activities against 12 different phytopathogenic fungi, especially for the Phomopsis
adianticola and Rap Sclerotinia stem rot. Compound 7e has the most obvious inhibitory effect on all
fungi, and the inhibition effect on Rhizoctonia solani, Phomopsis adianticola, Rap Sclerotinia stem rot,
Pestallozzia theae and Monilinia fructicola is better than on those of other fungi, the inhibition rate against
Rap Sclerotinia stem rot is up to 72.99% that better than the intermediate I. Compound 7g has a good
selectivity for the inhibition of Phomopsis adianticola. Both compounds 7a and 7k have more prominent
inhibition on the Phomopsis adianticola and Rap Sclerotinia stem rot. Compounds 7b
on the inhibition against Rap Sclerotinia stem rot.
, 7c, 7d perform well
From the data in Table 1, we can also see that compared with the intermediate II (6c), the antifungal
activity for most of the fungi except for Pestalotiopsis theae of the target compound 7c is significantly
improved after hybridization. Compared with the intermediate I, the antifungal activity of the
compounds after hybridization has not been improved, which also indicates that the antifungal activity
of these compounds may depend mainly on the methoxy acrylate skeleton structure.
2.4.2. Further Fungicidal Activities
Based on the above antifungal activity, we launched a series of screening test work in a lower
concentration range.
The inhibitory effect of compounds 7k and 7e on four common plant pathogenic fungi can be
seen in Table 2. From the table, we can see that as the concentration gradient decreases, the inhibition
rate of compounds 7k and 7e against most fungi is generally reduced, which is consistent with our
expected results. What surprised us was that under the same concentration conditions, the inhibitory
effect of them on Gibberella zeae and Sclerotinia sclerotiorum fungi strains are significantly better than the
control of intermediate I. For other strains, these two compounds did not show good inhibitory activity,
indicating that the compounds have good selectivity for Gibberella zeae and Sclerotinia sclerotiorum
fungi strains.
Table 2. In vitro antifungal activities of compounds 7k and 7e on four common plant pathogenic fungi.
Compd. No.
Concentration
Phomopsis adianticola
Pestallozzia theae
Gibberella zeae Sclerotinia sclerotiorum
100
50
25
12.5
6.25
36.91
36.76
29.71
30.15
4.12
39.14
38.97
37.93
31.03
20.00
46.62
10.44
12.66
7.47
33.14
27.44
23.32
12.07
8.90
7k
3.91
a
100
50
25
12.5
6.25
35.44
35.00
28.38
13.53
10.59
42.59
40.17
22.59
24.31
14.66
/
43.28
44.07
50.10
11.28
19.04
22.60
5.99
/
7e
1.54
100
50
25
12.5
6.25
100.00
50.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
87.69
89.77
89.92
82.21
100.00
100.00
100.00
100.00
100.00
Carbendazim
Intermediate I
100
50
25
12.5
6.25
69.26
41.76
30.29
12.94
21.18
32.76
23.62
17.07
/
1.84
/
/
/
/
26.96
14.29
/
/
/
6.38
a
denotes that the compound has no inhibitory activity.
2.5. Analysis of Structure-Activity Relationships
From the perspective of compound structure, this paper mainly discusses the effect of different
substituents on the benzene ring on the antifungal activity of the target compounds. As can
be seen from Table 1, when the substituent on the benzene ring is an electron-donating group,
such as the compound 7i, the antifungal activity of the compound is adversely affected, and the

Molecules 2019, 24, 1304
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electron-donating group weakens the activity of the compound. Conversely, when a substituent
is an electron-withdrawing group, such as compound 7c, the activity of the compound is much
higher than that of the intermediates. Therefore, the antifungal action of the novel synthesized
3-aryl-4-hydroxy-2(5H) furanone-acrylate hybrid strobilurin derivatives is mainly caused by the
methoxy acrylate structural unit, and the introduction of this main structural unit can enhance the
antifungal action of the intermediate II.
3. Conclusions
A series of novel 3-aryl-4-hydroxy-2(5H) furanone-acrylate hybrids containing a five-membered
lactone structure were designed and synthesized based on the structural characteristics of
methoxyacrylate backbone. The test for fungicidal activity indicated that these compounds have
a moderate inhibitory effect on most plant pathogenic fungi, and showing good selectivity for Rap
Sclerotinia stem rot fungi strain, which could be further optimized as a secondary lead.
4. Experimental Section
4.1. Instruments and Reagents
All chemicals or reagents used for syntheses were commercially available, were of AR grade,
and were used as received without further purification except as indicated. Anhydrous CH2Cl2 and
CH3CN were dried according to standard methods. All other solvents and reagents were analytical
reagent and used directly without purification. All melting points (m. p) were obtained with a
digital model X-4 apparatus (Shanghai Instrument Physical Optics Instrument Co., LTD, Shanghai,
China) and are uncorrected. 1H-NMR spectra were recorded on a Brucker spectrometer (Bruker,
Bremen, Germany) at 600 MHz with CDCl3 as the solvent and TMS as the internal standard; 13C-NMR
spectra were recorded on a Brucker spectrometer at 150 MHz with CDCl3 as the solvent. Chemical
shifts are reported in δ
(parts per million) values. Coupling constants nJ are reported in Hz. Liquid
chromatography–mass spectrometry analysis was performed on a Waters ACQUITY UPLC® H-CLASS
PDA (Waters®, Milford, MA, USA) instrument. Analytical thin-layer chromatography (TLC) was
carried out on precoated plates, and spots were visualized with ultraviolet light.
4.2. Experimental Method
4.2.1. Synthesis of Methoxyacrylate Intermediates
Methyl-2-(2-(bromomethyl)phenyl)-3-methoxyacrylate was prepared according to known
methods as shown in Scheme 1 [20–22].
4.2.2. General Procedure for Synthesis of the Key Lactone Intermediate II (6a–6k)
General synthetic procedure for the key lactone intermediates II (6a–6k), for example 6a.
2-(O-tolyl) acetic acid (0.15 g, 1 mmol) was dissolved in 15 mL of methanol, slowly add 2–3 drops of
concentrated sulfuric acid, then refluxing for 8 h and monitored by TLC, after the reaction is completed,
the solvent methanol was removed by rotary evaporation, 30 mL of water was added to the residue
and stirred, extracted three times with ethyl acetate, washed with water, dried and concentrated to give
compound 5a. The obtained compound 5a was placed in a round bottom flask, and 1.2 eq of methyl
glycolate, 20 mL of tetrahydrofuran, and 2.2 eq of potassium t-butoxide were added, refluxing and
stirring the reaction and monitored by TLC, after the reaction is completed, 50 mL of water was added
to the residue and stirred, adjust the pH of the solution to 5–6, then extracted three times with ethyl
acetate, washed with water, dried and concentrated to obtain key lactone intermediates II (6a) [10–14].

Molecules 2019, 24, 1304
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4.2.3. General Procedure for Synthesis of Target Compounds (7a–k)
General synthetic procedure for the target compounds, take 7a for example.
4-Hydroxy-3-(O-tolyl)furan-2(5H)-one (0.19 g, 1 mmol) was dissolved in 10 mL of acetonitrile, and
anhydrous potassium carbonate (0.21 g, 1.5 mmol) was added to the solution in a round bottom flask.
The solution was stirred for 0.5 h, and methyl-2-(2-(bromomethyl)phenyl)-3-methoxyacrylate (0.29 g,
1 mmol) was then added. The reaction is carried out at room temperature and monitored by TLC.
After 10 h, the mixture was diluted with 50 mL water and extracted with ethyl acetate (3
×
60 mL). The
combined extracts were washed with brine, dried (anhydrous sodium sulfate) and filtered. The filtrate
was evaporated, and the crude product was purified via silica gel column chromatography using a
1:5 (v/v) mixture of ethyl acetate and petroleum ether (boiling point range 60–90 ◦C) as the eluting
solution to obtain the pure target compound 7a. The specific experimental result data is as follows:
Data for Methyl 3-methoxy-2-(2-(((5-oxo-4-(O-tolyl)-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)acrylate (7a).
Yield: 37.1%; light yellow oil; 1H-NMR (600 MHz, chloroform-d)
δ 7.41 (s, 1H, CH3OCH), 7.34–7.30
(m, 3H, ArH), 7.27 (d, J = 7.2 Hz, 1H, ArH), 7.26–7.18 (m, 3H, ArH), 7.15–7.11 (m, 1H, ArH), 4.80 (s,
2H, ArCH2), 4.70 (s, 2H, CH2), 3.67 (s, 3H, CH3OCH), 3.64 (s, 3H, CH3OCO), 2.26 (s, 3H, ArCH3).
13C-NMR (150 MHz, chloroform-d)
δ 173.32, 172.01, 167.38, 160.29, 138.12, 133.89, 131.64, 131.33, 131.11,
130.24, 129.32, 128.79, 128.64, 128.29, 127.69, 125.74, 109.46, 104.09, 72.00, 66.83, 62.12, 51.82, 20.26. MS
(ESI) m/z 417.38 [M + Na]+, calcd. For C23H22O6 m/z = 394.14.
Data for Methyl 2-(2-(((4-(2,4-dichlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
(
7b). Yield: 31.2%; yellow oil; 1H-NMR (600 MHz, chloroform-d)
δ 7.49 (d, J = 1.9 Hz, 1H, CH3OCH),
7.47 (s, 1H, ArH), 7.35 (s, 3H, ArH), 7.30 (d, J = 1.9 Hz, 1H, ArH), 7.30 (s, 1H, ArH), 7.15 (d, J = 2.7 Hz,
1H, ArH), 4.89 (s, 2H, ArCH2), 4.71 (s, 2H, CH2), 3.74 (s, 3H, CH3OCH), 3.66–3.66 (s, 3H, CH3OCO).
13C-NMR (150 MHz, chloroform-d)
δ 173.47, 172.39, 167.35, 160.33, 135.86, 135.39, 133.48, 133.30, 131.62,
131.44, 129.55, 128.82, 128.40, 127.89, 127.73, 127.26, 109.48, 101.17, 72.00, 66.91, 62.20, 51.87. MS (ESI)
m/z 471.31 [M + Na]+, calcd. For C22H18Cl2O6 m/z = 448.05.
Data for Methyl 2-(2-(((4-(4-fluorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
(
7c). Yield: 35.4%; yellow oil; 1H-NMR (600 MHz, chloroform-d)
δ 7.95 (dd, J = 8.9, 5.5 Hz, 2H, ArH),
7.57 (s, 1H, CH3OCH), 7.45–7.35 (m, 3H, ArH), 7.23–7.17 (m, 1H, ArH), 7.08 (t, J = 8.8 Hz, 2H, ArH),
5.12 (s, 2H, ArCH2), 4.70 (s, 2H, CH2), 3.76 (s, 3H, CH3OCH), 3.67 (s, 3H, CH3OCO). 13C-NMR (150
MHz, chloroform-d) δ
172.54, 172.26, 167.44, 162.01 (d, 1JC-F = 246 Hz), 160.62, 133.80, 131.86, 131.69,
129.47 (d, 3JC-F = 7.5 Hz), 129.00, 128.68, 127.49, 125.58 (d, 4JC-F = 3 Hz), 115.32 (d, 2JC-F = 18 Hz), 109.66,
102.26, 71.36, 64.89, 62.31, 51.96. MS (ESI) m/z 421.35 [M + Na]+, calcd. For C22H19FO6 m/z = 398.12.
Data for Methyl 3-methoxy-2-(2-(((5-oxo-4-phenyl-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)acrylate (7d).
Yield: 42.1%; dark yellow solid; m.p.103–106 ◦C. 1H-NMR (600 MHz, chloroform-d)
δ 7.92 (d, J = 7.2
Hz, 2H, ArH), 7.55 (s, 1H, CH3OCH), 7.46–7.41 (m, 1H, ArH), 7.43–7.34 (m, 4H, ArH), 7.29 (t, J = 7.4 Hz,
1H, ArH), 7.19 (d, J = 5.3 Hz, 1H, ArH), 5.11 (s, 2H, ArCH2), 4.70 (s, 2H, CH2), 3.74 (s, 3H, CH3OCH),
3.67 (s, 3H, CH3OCO). 13C-NMR (150 MHz, chloroform-d)
δ 172.67, 172.54, 167.46, 160.63, 133.92,
131.78, 131.66, 129.52, 128.89, 128.62, 128.37, 127.83, 127.66, 127.51, 109.61, 103.15, 71.36, 64.98, 62.28,
51.94. MS (ESI) m/z 403.34 [M + Na]+, calcd. For C22H20O6 m/z = 380.13. ESI-HRMS: calcd. for
C22H20O6 ([M + Na]+), 403.3804; found, 403.4203.
Data for Methyl 2-(2-(((4-(2-chlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
1
(
7e). Yield: 23.7%; light yellow oil; H-NMR (600 MHz, chloroform-d)
δ
7.47 (d, J = 7.3 Hz, 1H,
CH3OCH), 7.44 (s, 1H, ArH), 7.40–7.27 (m, 6H, ArH), 7.17–7.10 (m, 1H, ArH), 4.88 (s, 2H, ArCH2),
4.71 (s, 2H, CH2), 3.71 (s, 3H, CH3OCH), 3.64 (s, 3H, CH3OCO). 13C-NMR (150 MHz, chloroform-d)
δ
173.00, 172.78, 167.41, 160.31, 135.16, 133.68, 132.66, 131.77, 131.36, 130.08, 129.68, 129.41, 128.77,

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128.36, 127.93, 126.85, 109.48, 102.02, 72.03, 66.97, 62.16, 51.84. MS (ESI) m/z 437.28 [M + Na]+, calcd.
For C22H19ClO6 m/z = 414.09.
Data for Methyl 2-(2-(((4-(4-chlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
(
7f). Yield: 30.2%; light yellow solid; m.p.149–152 ◦C. 1H-NMR (600 MHz, chloroform-d)
δ
7.92 (dd,
2H, ArH), 7.57 (s, 1H, CH3OCH), 7.45–7.32 (m, 5H, ArH), 7.24–7.16 (m, 1H, ArH), 5.13 (s, 2H, ArCH2),
4.70 (s, 2H, CH2), 3.76 (s, 3H, CH3OCH), 3.67 (s, 3H, CH3OCO). 13C-NMR (150 MHz, chloroform-d)
δ
172.86, 172.30, 167.42, 160.65, 133.70, 133.30, 131.89, 131.70, 131.06, 129.04, 128.87, 128.69, 128.56, 127.49,
109.63, 102.05, 71.48, 64.88, 62.32, 54.43, 51.97. MS (ESI) m/z 437.19 [M + Na]+, calcd. For C22H19ClO6
m/z = 414.09. ESI-HRMS: calcd. for C22H19ClO6 ([M + Na]+), 437.0768; found, 437.4438.
Data for Methyl 2-(2-(((4-(2,6-dichlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
(
7g). Yield: 53.2%; light yellow solid; m.p.124–127 ◦C. 1H-NMR (600 MHz, chloroform-d)
δ 7.44 (s,
1H, CH3OCH), 7.42–7.32 (m, 5H, ArH), 7.30 (t, J = 8.1 Hz, 1H, ArH), 7.15–7.12 (m, 1H, ArH), 4.85 (s,
2H, ArCH2), 4.76 (s, 2H, CH2), 3.73 (s, 3H, CH3OCH), 3.64 (s, 3H, CH3OCO). 13C-NMR (150 MHz,
chloroform-d)
δ 173.51, 171.45, 167.36, 160.33, 137.01, 133.31, 131.92, 131.33, 130.70, 129.14, 128.88,
128.37, 128.10, 128.02, 109.40, 99.98, 71.77, 67.29, 62.15, 51.83. MS (ESI) m/z 471.13 [M + Na]+, calcd. For
C22H18Cl2O6 m/z = 448.05. ESI-HRMS: calcd. for C22H18Cl2O6 ([M + Na]+), 471.0378; found, 471.3771.
Data for Methyl 3-methoxy-2-(2-(((4-(2-methoxyphenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)acrylate
(
7h). Yield: 34.3%; yellow oil; 1H-NMR (600 MHz, chloroform-d)
δ 7.40 (s, 1H, CH3OCH), 7.39–7.32 (m,
4H, ArH), 7.28 (dd, J = 7.5, 1.7 Hz, 1H, ArH), 7.14–7.11 (m, 1H, ArH), 7.00 (td, J = 7.5, 1.1 Hz, 1H, ArH),
6.94 (d, J = 8.3 Hz, 1H, ArH), 4.89 (s, 2H, ArCH2), 4.68 (s, 2H, CH2), 3.81 (s, 3H, ArOCH3), 3.64 (s, 3H,
CH3OCH), 3.62 (s, 3H, CH3OCO). 13C-NMR (150 MHz, chloroform-d)
δ 173.77, 172.63, 167.45, 160.25,
157.70, 134.27, 132.12, 131.47, 131.21, 130.03, 128.43, 128.24, 127.65, 120.63, 119.07, 111.08, 109.45, 100.39,
71.21, 66.96, 62.08, 55.61, 51.79. MS (ESI) m/z 433.32 [M + Na]+, calcd. For C23H22O7 m/z = 410.14.
Data for Methyl 3-methoxy-2-(2-(((5-oxo-◦4-(p-tolyl)-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)acrylate (7i).
Yield: 38.6%; yellow solid; m.p.104–107 C. 1H-NMR (600 MHz, chloroform-d)
δ 7.81 (d, J = 8.2 Hz, 2H,
ArH), 7.55 (s, 1H, CH3OCH), 7.43 (t, J = 4.7, 4.1 Hz, 1H, ArH), 7.42–7.34 (m, 2H, ArH), 7.19 (t, J = 8.9 Hz,
3H, ArH), 5.09 (s, 2H, ArCH2), 4.67 (s, 2H, CH2), 3.75 (s, 3H, CH3OCH), 3.67 (s, 3H, CH3OCO), 2.36 (s,
3H, ArCH3). 13C-NMR (150 MHz, chloroform-d)
δ 172.81, 171.93, 167.47, 160.61, 137.49, 134.04, 131.74,
131.59, 129.08, 128.84, 128.62, 127.74, 127.45, 126.60, 109.64, 103.23, 71.22, 64.99, 62.28, 51.94, 21.46.
MS (ESI) m/z 417.29 [M + Na]+, calcd. For C23H22O6 m/z = 394.14. ESI-HRMS: calcd. for C23H22O6
([M + Na]+), 417.4069; found, 417.5518.
Data for Methyl 2-(2-(((4-(2-fluorophenyl)-5-oxo-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)-3-methoxyacrylate
(
7j). Yield:41.3%; light yellow solid; m.p.102–105 ◦C. 1H-NMR (600 MHz, chloroform-d)
δ 7.49 (s, 1H,
CH3OCH), 7.47–7.37 (m, 2H, ArH), 7.40–7.31 (m, 3H, ArH), 7.24–7.17 (m, 1H, ArH), 7.19–7.09 (m, 2H,
ArH), 4.99 (s, 2H, ArCH2), 4.69 (s, 2H, CH2), 3.73 (s, 3H, CH3OCH), 3.65 (s, 3H, CH3OCO). 13C-NMR
(150 MHz, chloroform-d)
δ
173.66, 172.70, 167.43, 160.43 (d, 1JC-F = 247.5 Hz), 160.35, 133.81, 131.98 (d,
4JC-F = 3 Hz), 131.63, 131.47, 130.32 (d, 3JC-F = 7.5 Hz), 128.77, 128.47, 127.75, 124.22 (d, 4JC-F = 3 Hz),
117.55 (d, 2JC-F = 15 Hz), 115.80 (d, 2JC-F = 21 Hz), 109.54, 98.64, 71.62, 66.56, 62.18, 51.86. MS (ESI) m/z
421.26 [M + Na]+, calcd. For C22H19FO6 m/z = 398.12. ESI-HRMS: calcd. for C22H19FO6 [M + Na]+,
421.3708; found, 421.5121.
Data for Methyl 3-methoxy-2-(2-(((5-oxo-4-(2,4,5-trifluorophenyl)-2,5-dihydrofuran-3-yl)oxy)methyl)phenyl)acrylate
◦
1
(
7k). Yield: 33.2%; yellow solid; m.p. 92–95 C. H-NMR (600 MHz, chloroform-d) δ 7.55 (s, 1H,
CH3OCH), 7.41–7.35 (m, 3H, ArH), 7.32–7.27 (m, 1H, ArH), 7.19–7.16 (m, 1H, ArH), 7.03–6.97 (m, 1H,
ArH), 5.04 (s, 2H, ArCH2), 4.69 (s, 2H, CH2), 3.80 (s, 3H, CH3OCH), 3.68 (s, 3H, CH3OCO). 13C-NMR
(150 MHz, chloroform-d) δ 174.38, 171.92, 167.36, 160.45, 133.53, 131.66, 131.56, 131.45, 128.92, 128.62,

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127.44, 119.31 (d, JC-F = 6 Hz), 119.18 (d, JC-F = 4.5 Hz), 109.59, 105.96 (d, JC-F = 27 Hz), 105.82 (d,
C-F = 27 Hz), 97.45, 71.61, 66.25, 62.29, 51.91. MS (ESI) m/z 457.17 [M + Na]+, calcd. For C22H17F3O6
J
m/z = 434.10.
4.3. Biological Activity Assay
All fungal phytopathogenic strains were obtained from the Hubei Biopesticide Engineering
Research Center at the Hubei Academy of Agricultural Sciences and College of Plant Science
and Technology of Huazhong Agricultural University. Commercial fungicides Azoxystrobin and
Carbendazim as a positive fungicide control for bioassay were bought from Aladdin Reagent Database
Co. (Shanghai, China).
All compounds were tested for common phytopathogenic fungi from six major crops, mainly
including tea, rape, peach, wheat, rice and pepper, to control the Phomopsis adianticola, Rap Sclerotinia
stem rot, Pestallozzia theae, Gibberella zeae, Sclerotinia sclerotiorum, Altermaria alternate, Pestalotiopsis theae,
Monilinia fructicola, Phytophthora capsica, Magnapothe grisea.
The growth inhibition rates were calculated by means of the following equation:
I = [(D0 − Dt)/D0] × 100%, where I is the growth inhibition rate (%), D0 is the blank control colony
growth diameter (mm) and Dt is the pharmacy treatment colony growth diameter (mm) [21–23].
4.3.1. Primary Screening Test for Inhibiting Mycelial Growth of Pathogenic Fungi
Each of the test compounds (25 mg) was first dissolved in 2.5 mL acetone to generate a 104 mg·L−1
stock solution, then the stock solution was added to 250 mL of Potato Dextrose Agar (Medium) that is
PDA to prepare a drug-loaded PDA medium plate having a concentration of 100 mg
2.5 mL acetone was added to the PDA as a blank control, Azoxystrobin and Carbendazim as positive
·
L
−1, and only
fungicides control. The above 12 pathogens were activated (prepared by a punch with Φ = 0.50 cm)
and they were cut from the edge of the colony, inoculated into the above PDA plate, and each strain
and blank control were repeated twice, sealing with a sealing film to prevent contamination of other
,
bacteria, the poisonous PDA culture dishes were placed in a constant temperature incubator at the
◦
temperature condition of 30
±
1 C. After the blank control group culture was grown to about
two-thirds of the culture dish, the colony diameter was measured by the cross method in millimeters
(mm) with a cross and a vertical method, and the average value was taken, and the inhibition rate of
each compound against the fungus was calculated.
4.3.2. Further Screening Test for Inhibiting Mycelial Growth of Pathogenic Fungi
According to the results of the primary screening test, compounds 7e and 7k with good inhibition
rates and 4 strains were selected for a further screening test for inhibiting mycelial growth of pathogenic
fungi in the lower concentration range sequentially. The method used is still the same as the primary
screening. The difference is that the selected compounds have five different concentration gradients
for the growth inhibition of each strain, that is, 100, 50, 25, 12.5, and 6.25 mg·L−1
.
1
Supplementary Materials: The following are available online. H-NMR, 13C-NMR, MS of the target compounds.
Author Contributions: L.B. performed the experiments and writing; S.W. and L.B. performed the bioassay and
analyses; D.S. and J.W. carried out data collection and interpretation; X.C. conceived and designed the experiments,
analyses for NMR spectrum, and review the manuscript; S.K.: conceived and designed this project, and revised
the manuscript.
Funding: We gratefully acknowledge the support of this work by the Fundamental Research Funds for the Central
Universities (2662016PY112), and the authors also gratefully acknowledge the support of this work by the Program
for Leading Talents of Hubei Academy of Agricultural Sciences and Hubei Agricultural Science Innovation Centre
(2016-620-000-001-039).
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the target compounds 7a–7k are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).