Promising fungicides from allelochemicals: Synthesis of umbelliferone derivatives and their structure–activity relationships

Article abstract of DOI:10.3390/molecules23113002

Umbelliferone was discovered to be an important allelochemical in our previous study, but the contribution of its activity and structure has not yet been revealed. In this study, a series of analogues were synthesized to determine the skeleton of umbelliferone and examine its fungicidal activity. Furthermore, targeted modifications were conducted with three plant parasitic fungi to examine the lead compounds. Among those tested, compounds 2f and 10 were found to show excellent antifungal activity with an inhibitory rate over 80% at 100 ug/mL. The study proves that umbelliferone can be a promising skeleton for fungicides discovery. In addition, the primary structure–activity relationship provides a good guidance for the discovery of novel fungicides based on natural products in the future.

Full text of DOI:10.3390/molecules23113002

molecules
Article
Promising Fungicides from Allelochemicals:
Synthesis of Umbelliferone Derivatives and Their
Structure–Activity Relationships
Le Pan 1,
*
, Dongyu Lei 2, Lu Jin 1,*, Yuan He 1 and Qingqing Yang 1
1
Chemical Engineering College, Xinjiang Agricultural University, Urumqi 830052, China;
hy941813@163.com (Y.H.); yqq1509914@sina.com (Q.Y.)
Department of Physiology, Preclinical School, Xinjiang Medical University, Urumqi 830011, China;
leidongyu0109@163.com
2
*
Correspondence: inmail911@sina.com (L.P.); chemkimlu@xjtu.edu.cn (L.J.);
Tel.: +86-180-992-783-61 (L.P.); +86-189-099-263-89 (L.J.)
Academic Editor: Jean-Marc Sabatier
Received: 20 October 2018; Accepted: 12 November 2018; Published: 16 November 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Umbelliferone was discovered to be an important allelochemical in our previous study,
but the contribution of its activity and structure has not yet been revealed. In this study, a series of
analogues were synthesized to determine the skeleton of umbelliferone and examine its fungicidal
activity. Furthermore, targeted modifications were conducted with three plant parasitic fungi to
examine the lead compounds. Among those tested, compounds 2f and 10 were found to show
excellent antifungal activity with an inhibitory rate over 80% at 100 ug/mL. The study proves
that umbelliferone can be a promising skeleton for fungicides discovery. In addition, the primary
structure–activity relationship provides a good guidance for the discovery of novel fungicides based
on natural products in the future.
Keywords: allelochemical; umbelliferone derivatives; antifungal activity; EC50 values
1. Introduction
Plant pathogenic fungi have jeopardized crop yields and food security globally. Fungicides have
always been an efficient way of minimizing the cost of plant diseases [1]. However, the widespread
use of traditional fungicides has led to increased resistance, mammalian toxicity, and a threat to the
environment. This has stimulated the demand for integrated control measures with more selective,
lower dose agents that have improved resistance [
of novel fungicide development either with direct application or serving as a model for advanced
modification [ ]. However, with the explosive growth in NPs, it has become increasingly more difficult
2–5]. Natural products (NPs) have been a rich source
6
to pick the appropriate product from such a large range of compounds, and the introduction of novel
molecules to the market has recently declined. Therefore, there is an urgent need for cost-effective
tactics, instead of “blind search”, in the development of new fungicides [7].
In nature, allelochemicals play an important role in plant ecological competition by inhibiting
or enhancing the growth of the target organism. Allelochemicals have therefore been proposed as
a promising means in the discovery of growth regulators and agrochemicals [8–10]. Coumarins
are widely distributed in plants, and they have been proven to play a key role in plant
allelopathic effect [11,12]. In our previous study, umbelliferone was identified as an important
allelochemical of Stellera chamaejasme, and its analogs were synthesized to investigate antifungal
activity. The structure–activity relationship (SAR) indicated that the modification of umbelliferone at
the C7 hydroxy not only enhanced fungicidal activity but also decreased phytotoxicity [13]. In light of
Molecules 2018, 23, 3002; doi:10.3390/molecules23113002
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3002
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the above findings, coumarin derivatives were proposed to be an optimal skeleton (Figure 1). In this
study, a series of compounds were designed to confirm the possible application of umbelliferone,
and C7-substituted umbelliferones derivatives (UDs) were designed and synthesized to investigate
their fungicidal activity against three devastating fungi: Alternaria alternata, Botrytis cinerea, and
Fusarium oxysporum.
Figure 1. Umbelliferone and its potential modification sites.
2. Results and Discussion
The preparation of umbelliferone (1) and 7-(4-bromoalkoxy)-4-methyl benzopyran-2-one (2c–g)
was performed as per our previous methods. The rest of the UDs were synthesized according to
the following method by an alkylation reaction, Friedel-Crafts reaction, and Claisen rearrangement
reaction (Schemes 1–6) [14–16].
a: n=2
Br
Br
b: n=3
c: n=4
d: n=5
n
Br
reflux
reflux
reflux
n
O
O
O
O
2a~d
O
O
F
Br
O
F
K2CO3
O
O
H2SO4
Acetone
HO
OH
2e
HO
O
O
1
Br
Br
Br
O
O
O
2f
Br
reflux
O
O
O
2g
Scheme 1. Synthesis of compounds 1–2g.
Br
Br
Br
R
O
3a,4a
reflux
F
F
Br
Br
R
R
O
3b,4b
3a~d: R=H
4a~d: R=NH2
K2CO3
reflux
reflux
Acetone
R
OH
Br
Br
O
3c,4c
Br
reflux
R
O
3d,4d
Scheme 2. Synthesis of compounds 3a–4d.

Molecules 2018, 23, 3002
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◦
5 and 6. Reagents and conditions: (a) 200 C; (b) 1,4-dibromobutane;
Scheme 3. Synthesis of compounds
KOH; KI and TBAB; reflux in acetonitrile.
Scheme 4. Synthesis of compound
(b) methanol; H2SO4; reflux; (c) compound 2c; AlCl3; 80 C.
7
. Reagents and conditions: (
a) HCl, stirring at room temperature;
◦
Scheme 5. Synthesis of compound
8.
Reagents and conditions: KOH; KI and TBAB; reflux
in acetonitrile.
◦
Scheme 6. Synthesis of compound
(b) ethyl acetoacetate; phosphoric acid; 80 C.
9
and 10. Reagents and conditions: (
a
) para-dioxane; stirred at 0 C;
◦
Fungicidal activity and structure-activity relationship: The fungicidal activity of the compounds
is shown in Table 1. As we had predicted, 4-methylumbelliferone ( ) and its 7-subsituted derivatives
1
exhibited significant inhibitory effects at the concentration of 100 mg/L. In particular, compound 2f
exhibited a significant activity with inhibitory rate up to 90%, and compounds 2a−e and 2g displayed
moderate to strong activity against all tested fungi. The EC50 values of derivatives were evaluated at a
series of lower concentration, which are shown in Table 2. The results indicated that compound 2f had
the lowest EC50 values of 50.5 µmol/L, 30.7 µmol/L, and 58.9 µmol/L against A. alternata, B. cinerea,
and F. oxysporum, respectively.

Molecules 2018, 23, 3002
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Table 1. In vitro fungicidal activities of the prepared compounds at 100 ug/mL (96 h of incubation).
The Inhibition Rate (%; mean ± SD; N = 3)
Compd.
A. Alternata
B. Cinerea
F. Oxysporum
1
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
4a
4b
4c
4d
38.5 ± 4.38
38.1 ± 5.9
42.9 ± 6.7
70.6 ± 5.5
40.3 ± 3.7
63.7 ± 3.9
81.5 ± 3.7
48.4 ± 4.0
49.6 ± 3.6
3.8 ± 2.8
58.2 ± 3.6
29.6 ± 5.3
30.5 ± 5.2
35.3 ± 5.6
12.6 ± 2.6
10.2 ± 3.5
22.4 ± 4.7
27.6 ± 8.3
20.8 ± 7.7
26.6 ± 3.9
58.5 ± 3.4
77.3 ± 5.5
0
25.8 ± 3.8
39.6 ± 6.2
36.3 ± 5.8
68.3 ± 4.6
46.2 ± 5.8
72.9 ± 3.5
90.99 ± 2.3
70.6 ± 5.2
41.2 ± 5.4
10.2 ± 2.6
63.0 ± 7.0
32.5 ± 6.2
32.1 ± 4.5
46.2 ± 3.7
15..0 ± 6.4
12.6 ± 4.5
26.4 ± 2.1
35.93 ± 1.73
21.9 ± 3.0
18.9 ± 8.5
70.9 ± 1.7
84.4 ± 3.5
0
41.2 ± 2.5
33.2 ± 4.4
28.6 ± 3.4
60.4 ± 5.3
35.5 ± 7.2
62.8 ± 5.6
84.8 ± 3.2
53.5 ± 4.2
50.2 ± 6.3
8.4 ± 4.1
61.3 ± 3.8
26.2 ± 4.9
28.4 ± 5.8
33.2 ± 3.1
13.7 ± 4.2
9.8 ± 2.6
32.6 ± 5.6
29.8 ± 3.8
18.7 ± 4.1
25.3 ± 5.2
77.5 ± 3.4
80.5 ± 5.7
0
5
6
7
8
9
10
DMSO (1%)
carbendazim
90.7 ± 8.18
94.3 ± 6.4
91.4 ± 5.4
Table 2. EC50 values and log P of effective compounds against three phytopathogenic fungi.
EC50 (95% CI µmol/L)
Log P
Compound
A. Alternata
B. Cinerea
F. Oxysporum
1
2c
2e
2f
2g
9
10
1.61
3.26
2.62
3.22
3.11
2.87
3.23
1.29
922.4 (827.6–1098.9) 1037.1 (955.3–1201.1)
1165.9 (1076.2–1268.1)
245.2 (180.9–298.2)
405.9 (311.3–505.9)
58.9 (30.4–89.3)
207.1 (149.8–279.2)
182.9 (145.9–248.2)
60.8 (34.6–120.6)
33.5 (14.6–49.7)
193.5 (145.6–258.7)
381.6 (282.1–466.7)
50.5 (28.5–82.2)
221.1 (137.2–270.9)
360.4 (274.1–462.7)
30.7 (15.5–47.5)
232.1 (158.8–336.9)
185.8 (117.6–254.0)
105.1 (69.5–176.2)
18.8 (6.8–30.0)
280.8(206.7–350.4)
130.7 (76.0–195.7)
66.6 (50.7–115.1)
20.6 (7.8–32.4)
Carbendazim
It is worth noting that the side chain was important to the activity exhibition. As the length of the
side chain increased from two to five carbon atoms, the activity varied with nonlinear changes. It was
clearly seen that four carbon atoms was optimum on activity exhibition, which was in accordance with
our previous study. In order to further confirm the contribution of the coumarin skeleton, its core
structure of benzopyran-2-one was substituted by benzene (3a–d) and aniline (4a–d). The activity
was found to be significantly decreased compared with the related umbelliferone derivatives, except
(4-bromobutoxy) benzene (3a). Therefore, the results proved that taking umbelliferone as the main
skeleton, together with our proposed modification strategy, was evidently effective.
Based on the above findings, a series of coumarin derivatives were designed and screened for their
antifungal activity. The results indicated that the functional groups played an important role in activity
exhibition. Halides, especially fluoro compounds, are widely applied in lead compound modification
and have been proven to show good biocompatibility and activity [17]. In this study, it was found that
the inhibition rate increased significantly with compound 2e (about 65%) compared to compound
1
(about 30%) when the terminal bromine was substituted with fluorine. We also deemed it meaningful to

Molecules 2018, 23, 3002
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introduce different fluoro moieties in our studies. Isopentenyl has been reported to play an important
role in membrane binding and activity expression of biomolecules [18 19]. Our results showed
similar results, with activity being enhanced when isopentenyl was introduced as a terminal moiety.
Methoxyacrylate, originated from strobilurin A, is a key skeleton in commercial fungicides [20 21].
By coupling methoxyacrylate with coumarin with four carbon chain ( ), we managed to achieve
,
,
7
good activity. The type of linkage also played an important role in antifungi activity. The results
indicated that the inhibitory effect of compound 2f was much better than compound 2c due to the
slight structural difference between the two, with compound 2f having an olefinic linkage instead of
the alkoxy side chain of compound 2c. We propose that the increase in activity may have been induced
by the conjugation of double bond, O atom, and aromatic ring in compound 2f.
3. Experimental Section
All starting chemicals were purchased from commercial vendors, were of AR grade, and were
used without purification. Reactions were monitored by thin-layer chromatography (Silica Gel 60 F254).
Target compounds were purified with column chromatography using silica gel (200–300 mesh).
1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Bruker AM-400BB
spectrometer (Bruker, Karlsruhe, Germany) in CDCl3/DMSO-d6 solution with TMS as the internal
standard. The chemical shift values (δ) below are listed in ppm and the coupling constant values (J)
are in Hertz. The melting points were determined with a X-4 melting point apparatus. The purity of
all test compounds was above 95%. Compounds 2a and 2b were prepared as per our previous study
and used directly [13].
3.1. General Procedure for the Preparation of Compounds 2c–g
1,4-Dibromobutane, 1,5-dibromopentane, 1-bromo-4-fluorobutane, (2E)-1,4-dibromo-2-butene
(3.0 mmol), or 1-bromo-3-methyl-2-butene (5.0 mmol) was added to a solution of 4-methylcoumarin
(3.5 mmol), KOH (1 mmol), and K2CO3 (4 mmol) in acetone (20 mL), respectively. The reaction
was heated at reflux detected with TLC for 5 h. Then, the crude products were prepared by
filtering the mixture and evaporating the solvent and were further purified by silica chromatography
(CHCl3/MeOH 50/1) to give compounds 2c–g (Scheme 1).
7-(4-Bromobutoxy)-4-methylbenzopyran-2-one (2c): White solid; yield: 65%; m.p., 42–44 ◦C; ESI-MS m/z:
1
312.2 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ
:1.98–2.02 (m, 2H), 2.06–2.12 (m, 2H), 2.40 (s, 3H),
3.50 (t, 2H, J = 8.0 Hz), 4.06 (t, 2H, J = 8.0 Hz), 6.14 (s, 1H), 6.80 (d, 1H, J = 4.0 Hz), 6.82 (dd, 1H,
J = 8.0 Hz), 7.49 (d, 1H, J = 8.0 Hz); 13C-NMR (CHCl3-d, 100 MHz) δ: 27.69, 29.34, 32.16, 67.56, 101.41,
112.61, 112.87, 113.18, 128.79, 143.36, 155.93, 161.15, 162.09.
7-(4-Bromopentyloxy)-4-methylbenzopyran-2-one (2d): White solid; yield: 60%; m.p., 60–62 ◦C; ESI-MS
1
m/z: 326.2 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ:1.30–1.34 (m, 2H), 1.92–1.98 (m, 2H), 2.02–2.06
(m, 2H), 2.42 (s, 3H), 3.51 (t, 2H, J = 8.0 Hz), 4.08 (t, 2H, J = 8.0 Hz), 6.15 (s, 1H), 6.82 (d, 1H, J = 2.4 Hz),
6.88 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 7.50 (d, 1H, J = 8.4Hz).
7-(4-Fluorobutoxy)-4-methylbenzopyran-2-one (2e): White solid; yield: 63%; m.p., 116–118 ◦C; ESI-MS
m/z: 251.3 [M + H]+. 1H-NMR (CHCl3-d, 400 MHz)
δ: 1.86–1.98 (m, 4H), 2.40 (s, 3H), 4.07 (t, 2H,
J = 8.0 Hz), 4.79 (t, 2H, J = 8.0 Hz), 6.13 (s, 1H), 6.80 (d, 1H, J = 2.4 Hz), 6.85 (dd, 1H, J = 8.8 Hz, 2.4 Hz),
7.49 (d, 1H, J = 8.8 Hz).
7-(4-Bromobutenyloxy)-4-methylbenzopyran-2-one (2f): White solid; yield: 65%; m.p., 116–118 ◦C ESI-MS
1
m/z: 310.2 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ
: 2.40 (s, 3H), 4.00 (d, 2H, J = 7.2 Hz), 4.62 (d, 2H,
J = 4.8 Hz), 6.01–6.15 (m, 2H), 6.15 (s, 1H), 6.80 (d, 1H, J = 2.0 Hz), 6.87 (dd, 1H, J = 8.8 Hz, 2.4 Hz),
7.55 (d, 1H, J = 8.8 Hz).

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7-(4-Isopentenyloxy)-4-methylbenzopyran-2-one (2g): White solid; yield: 75%; ESI-MS m/z: 247.3 [M + H]+
1H-NMR (CHCl3-d, 400 MHz)
:1.77 (s, 3H), 1.81 (s, 3H), 2.49 (s, 3H), 4.58 (d, 2H, J = 8.0 Hz), 5.47–5.50
.
δ
(m, 1H), 6.13 (s, 1H), 6.82 (d, 1H, J = 2.0 Hz), 6.87 (dd, 1H, J = 8.8 Hz, J = 2.4Hz), 7.49 (d, 1H, J = 8.8 Hz).
3.2. General Procedure for the Preparation of Compounds 3a–d and 4a–d
1,4-Dibromobutane, 1-bromo-4-fluorobutane, (2E)-1,4-dibromo-2-butene or 1-bromo-3-methyl-
2-butene(3.0 mmol) was added to a solution of benzene (3a–d) or aniline (4a–d) (3.5 mmol), KOH
(1 mmol), and K2CO3 (4 mmol) in acetone (20 mL), respectively. The reaction was heated at
reflux detected with TLC. Then, the crude products were further purified by silica chromatography
(CHCl3/MeOH 50/1) to give compounds 3a–d and 4a–d (Scheme 2).
◦
1
(4-Bromobutoxy) benzene (3a): White oil; yield: 75%; m.p., 40–41 C; H-NMR (CHCl3-d, 400 MHz)
δ:1.74–1.78 (m, 2H), 1.80–1.86 (m, 2H), 3.51 (t, 2H, J = 8.0 Hz), 4.08 (t, 2H, J = 8.0 Hz), 6.99 (dd, 2H,
mboxemphJ = 8.0 Hz, 2.4 Hz), 7.03 (dd, 1H, J = 8.0 Hz, 2.4 Hz), 7.37–7.40 (m, 2H).
(4-Fluorobutoxy) benzene (3b): White oil; yield: 65%; m.p., 55–57 ◦C; 1H-NMR (CHCl3-d, 400 MHz)
δ:
1.70–1.86 (m, 4H), 4.07 (t, 2H, J = 8.0 Hz), 4.50 (t, 2H, J = 8.0 Hz), 6.96 (dd, 2H, J = 8.0 Hz, 2.4 Hz),
7.02(dd, 1H, J = 8.0 Hz, 2.4 Hz), 7.33–7.36 (m, 2H).
◦
1
7-(4-Bromobutenyloxy) benzene (3c): White solid; yield: 72%; m.p., 46–48 C; H-NMR (CHCl3-d,
400 MHz) : 4.02 (d, 2H, J = 7.2 Hz), 4.65 (d, 2H, J = 4.8 Hz), 6.00–6.12(m, 2H), 7.03 (dd, 1H, J = 8.8 Hz,
δ
J = 2.0 Hz), 7.05 (dd, 2H, J = 8.8 Hz, J = 2.0 Hz), 7.34–7.38 (m, 1H).
(4-Isopentenyloxy) benzene (3d): Yellow oil; yield: 76%; 1H-NMR (CHCl3-d, 400 MHz)
δ: 1.75 (s, 3H),
1.80 (s, 3H), 4.59 (d, 1H, J = 8.0 Hz), 5.46 (m, 1H), 7.02 (dd, 1H, J = 8.8 Hz, J = 2.4Hz), 7.04 (dd, 2H,
J = 8.8 Hz, J = 2.4Hz), 7.34–7.37 (m, 2H).
(4-Bromobutoxy) aniline (4a): White solid; yield: 63%; m.p., 116–118 ◦C; 1H-NMR (CHCl3-d, 400 MHz)
δ
:1.74–1.82 (m, 4H), 3.52 (t, 2H, J = 8.0 Hz), 3.68 (s, 2H), 4.09 (t, 2H, J = 8.0 Hz), 6.16–6.19 (m, 2H),
6.35 (dd, 1H, J = 8.0 Hz, 2.4 Hz), 7.09 (dd, 1H, J = 8.4 Hz, 8.2 Hz).
(4-Fluorobutoxy) aniline (4b): Yellow solid; yield: 60%; m.p., 76–78 ◦C; 1H-NMR (CHCl3-d, 400 MHz)
δ:
1.62–1.78 (m, 4H), 3.66 (s, 2H), 4.06 (t, 2H, J = 8.0 Hz), 4.52 (t, 2H, J = 8.0 Hz), 6.21 (dd, 1H, J = 8.0 Hz,
2.4 Hz), 6.25 (dd, 1H, J = 2.4 Hz, 2.0 Hz), 6.28–6.31 (m, 1H), 7.02 (dd, 1H, J = 8.4 Hz, 8.0 Hz).
◦
(4-Bromobutenyloxy) aniline (4c): Yellow solid; m.p., 121–123 C; 1H-NMR (CHCl3-d, 400 MHz)
δ: 3.62 (s,
2H), 4.02 (d, 2H, J = 7.2 Hz), 4.68 (d, 2H, J = 4.8 Hz), 6.00–6.12 (m, 2H), 6.16–6.20 (m, 1H), 6.22 (dd, 1H,
J = 2.4 Hz, 2.0 Hz), 6.38 (m, 1H), 7.05 (dd, 1H, J = 8.4 Hz, 8.0 Hz).
◦
(4-Isopentenyloxy) aniline (4d): Yellow solid; yield: 68%; m.p.,68–70 C; 1H-NMR (CHCl3-d, 400 MHz)
δ:
1.72 (s, 3H), 1.84 (s, 3H), 3.70 (s, 2H), 4.56 (d, 1H, J = 8.0 Hz), 5.44 (m, 1H), 6.14–6.21 (m, 1H), 6.23 (dd,
1H, J = 8.4 Hz, 2.0 Hz), 6.35–6.41 (m, 1H), 7.07 (dd, 1H, J = 8.4 Hz, 8.0 Hz).
3.3. General Procedure for the Preparation of Compounds 5 and 6
Compound 2g was added into single-necked flask and heated to 200 ◦C. Then, the mixture was
dissolved into methanol and purified by silica chromatography to get compound 5. Then, compound 5
(1.5 mmol) was added to a solution of 1, 4-dibromobutane (2 mmol), KOH (1 mmol), and K2CO3
(2 mmol) in acetone (10 mL). The reaction was heated at reflux for 3 h. The crude products were
prepared by filtering the mixture and evaporating the solvent and were further purified by silica
chromatography to give compound 6.

Molecules 2018, 23, 3002
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7-Hydroxy-6-isopentenyl-4-methylbenzopyran-2-one (
5
): White solid; yield: 30%; m.p., 83–84 ◦C; ESI-MS
1
m/z: 245.3 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ: 1.70 (s, 3H), 1.83 (s, 3H), 2.41 (s, 3H), 3.30 (d, 2H,
J = 6.0 Hz), 4.08 (t, 1H, J = 6.0 Hz), 6.09 (s, 1H), 6.69 (d, 1H, J = 2.0 Hz), 6.79–6.82 (dd, 1H, J = 8.8 Hz,
J = 2.4Hz), 7.58 (d, 1H, J = 8.8 Hz); 13C-NMR (CHCl3-d, 100 MHz)
: 17.24, 19.34, 22.4, 24.62, 102.06,
109.82, 112.44, 112.94, 115.1, 125.20, 126.03 154.56, 155.15, 161.63, 162.46.
δ
◦
1,4-Di(6-isopentenyl-4-methylbenzopyran-2-one-7-oxy)butane (
6
): White solid; yield: 20%; m.p.,162–164 C;
:1.28 (s, 6H), 1.35 (s, 6H), 2.03–2.07 (m,
1
ESI-MS m/z: 529.6 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ
4H), 2.44 (s, 6H, J = 6.0 Hz), 3.26 (d, 4H, J = 6.0 Hz), 4.05 (d, 2H, J = 8.0 Hz), 4.60 (d, 2H, J = 8.0 Hz),
6.06 (s, 2H), 6.16 (s, 2H), 6.90 (d, 2H, J = 8.8 Hz), 6.98 (dd, 2H, J = 8.8 Hz, J = 2.0 Hz), 7.68 (d, 2H,
J = 9.6 Hz); 13C-NMR (CHCl3-d, 100 MHz)
δ: 17.25, 19.11, 19.33, 24.92, 30.68, 63.69, 67.78, 101.25, 112.69,
125.95, 127.62, 128.17, 128.98, 129.84, 154.29, 155.00, 162.11
3.4. General Procedure for the Preparation of Compound 7
Benzoylcyanide (3 mmol) was dissolved in hydrochloric acid (10 mL) with stirring at room
temperature for 2h. Then, the products were filtered out and added into methanol. Concentrated
H2SO4 (1 mL) was added to the mixture and refluxed. After that, the mixture was extracted
with ethyl acetate and evaporated to yield the crude products, which reacted with compound 2c
without purification catalyzed by AlCl3. The crude products were further purified by means of silica
chromatography to provide compound 7 (Scheme 2).
1-(4-Methylbenzopyran-2-one-7-oxy)-4-(3-methylphenylglyoxylate)butane (
7
): White solid; yield: 30%;
1
m.p.,117–119 ◦C; ESI-MS m/z: 395.4 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ: 1.22–1.27 (m, 2H),
1.71–1.79 (m, 2H), 2.40 (s, 3H), 2.86–2.91 (m, 2H), 3.68 (s, 3H), 4.14–4.18 (m, 2H), 6.16 (s, 1H), 6.85 (dd,
1H, J = 8.4 Hz, 2.4 Hz), 7.48 (t, 1H, J = 8.4 Hz), 7.57(dd, 1H, J = 8.0 Hz, 2.4 Hz), 7.62 (t, 1H, J = 8.4 Hz),
7.70–7.73 (m, 2H), 8.11(d, 1H, J = 8.0 Hz); 13C-NMR (CHCl3-d, 100 MHz)
δ: 18.73, 22.73, 29.71, 31.68,
52.34, 68.70, 103.39, 111.63, 112.50, 113.07, 113.57, 125.41, 125.96, 128.51, 130.20, 133.72, 145.2, 152.7,
153.15, 155.12, 159.71, 161.89, 170.43.
3.5. General Procedure for the Preparation of Compound 8
Compound 2c (2.0 mmol) was added to a solution of 3-aminophenol (2.5 mmol), KOH (1 mmol),
and K2CO3 (3 mmol) in acetone (10 mL). The mixture was heated at reflux for 3 h and the reaction
was detected with TLC. The mixture was extracted with ethyl acetate and evaporated to get crude
product. The crude products were prepared by filtering the mixture and evaporating the solvent and
were further purified by silica chromatography to give compound 8 (Scheme 3).
1-(4-Met◦hylbenzopyran-2-one-7-oxy)-4-(3-aminophenol) butane (
8
): White solid; yield: 54%; m.p.,
1
123–125 C; ESI-MS m/z: 340.4 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ
: 1.23–1.28 (m, 2H), 2.02–2.10
(m, 2H), 2.40 (s, 3H), 4.11 (s, 2H), 4.51–4.57 (m, 2H), 4.62–4.66 (m, 2H), 6.02–6.09 (m, 1H), 6.14 (s, 1H),
6.19–6.34 (m, 2H), 6.85–6.89 (m, 1H), 7.05 (t, 1H, J = 8.4 Hz), 7.48 (t, 2H, J = 8.8 Hz); 13C-NMR (CHCl3-d,
100 MHz) δ: 18.69, 27.8(2), 67.32(2), 101.74, 104.69, 108.29, 112.08, 113.73, 126.88, 128.43, 129.56, 130.16,
147.81, 152.59, 155.12, 159.71, 161.34, 161.50.
3.6. General Procedure for the Preparation of Compounds 9 and 10
The amine (5.0 mmol) was dissolved in water, and NaHCO3 (10.0 mmol) was added with stirring.
After being cooled to 5 ◦C, Cbz-Cl (7.5 mmol) was added slowly in para-dioxane. Then, the mixture
◦
was stirred at 0 C for 1 h and allowed to warm to room temperature for 12 h. After the reaction,
water was added and extracted with ethyl acetate. The aqueous layers were acidified to pH of 1 and
extracted three times with ethyl acetate. The organic layers were dried with sodium sulfate, and the
solvent was evaporated to get compound
9
. Ethyl acetoacetate (2 mmol) was added to a mixt◦ure of
compound
9
(1 mmol) and phosphoric acid (10 mL). The mixture was heated with stirring at 80 C for

Molecules 2018, 23, 3002
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5 h. After cooling down, the mixture was quenched with ice-water. The crude product was filtered out,
dried, and purified by recrystallization from ethanol to yield compound 10 (Scheme 4).
N-Carbobenzoxy-3-hydroxyaniline (
9
): White solid; yield: 68%; ESI-MS m/z: 244.3 [M + H]+. m.p.,
204–206 C; 1H-NMR (CHCl3-d, 400 MHz)
: 4.62 (s, 2H), 5.35 (s, 1H), 6.65 (dd, 1H, J = 8.0, Hz, 2.0 Hz),
◦
δ
7.12 (dd, 1H, J = 8.0 Hz, 2.0 Hz), 7.25–7.38 (m, 5H), 7.47 (dd, 2H, J = 8.0 Hz, 2.0 Hz), 9.10 (s, 1H);
13C-NMR (CHCl3-d, 100 MHz)
136.15, 137.30, 153.38, 158.78.
δ: 68.76, 105.10, 113.42, 114.18, 127.26(2), 127.60, 128.92(2), 130.20,
4-Methyl-7-(N-carbobenzoxy)-aminocoumarin (10): White solid; yield: 28%; m.p., 228–230 ◦C; ESI-MS
1
m/z: 310.4 [M + H]+. H-NMR (CHCl3-d, 400 MHz)
δ
: 2.40 (s, 3H), 5.23 (s, 2H), 6.19 (s, 1H), 6.97 (s,
1H), 7.37–7.42 (m, 5H), 7.44 (d, 1H, J = 1.6 Hz), 7.52 (d, 2H, J = 8.4 Hz); 13C-NMR (CHCl3-d, 100 MHz)
δ
: 18.69, 67.62, 106.96, 113.24, 114.36, 115.60, 125.39, 128.45(2), 128.59, 128.72 (2), 135.57, 141.32, 152.19,
152.77, 154.58, 161.06.
3.7. Antifungal Activity
The fungicidal activities were investigated with three plant parasitic fungi (Alternaria alternata,
Botrytis cinerea, and Fusarium oxysporum), and mycelial inhibition of radial growth on PDA media was
employed [22,23].
4. Conclusions
In summary, the umbelliferone skeleton was revealed to play a key role in the antifungal
activity. The structure–activity relationship study showed that coumarin coupled with haloalkane,
methoxyacrylate, and isopentenyl moieties could significantly enhance activity. The research provides
a good guidance for further study of the molecular design and discovery of allelochemical-based
agents for phytopathogenic fungi control.
Author Contributions: Conceptualization, L.P.; Investigation, D.L., Q.Y. and Y.H.; Writing-Original Draft
Preparation, L.P.; Writing-Review & Editing, L.P.; Supervision, L.P. and L.J.
Funding: This work was supported by the Tianchi Talent on Doctor scheme introduced in the Xinjiang Uygur
Autonomous Region of China, Research Foundation for Advanced Talents in Xinjiang Uygur Autonomous Region
of China, and the Prophase-Sustentation Foundation of Xinjiang Agricultural University (No. XJAU201606).
Acknowledgments: The authors wish to thank Xiuzhuang Li and Hui Jin (Lanzhou Institute of Chemical Physics)
for help in bioassay and for fruitful discussions.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–10 are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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