Design, synthesis and antifungal activity evaluation of coumarin-3-carboxamide derivatives

Article abstract of DOI:10.1016/j.fitote.2018.03.013

A series of coumarin-3-carboxamides/hydrazides have been designed and synthesized, all the target compounds were evaluated in vitro for their antifungal activity against Botrytis cinerea, Alternaria solani, Gibberella zeae, Rhizoctorzia solani, Cucumber anthrax and Alternaria leaf spot, some of the designed compounds 4a-4g exhibited potential activity in the primary assays, this highlighted by the compounds 4a, 4d, 4e and 4f, EC₅₀ values of which against Rhizoctorzia solani were as low as 1.80 μg/mL, 2.50 μg/mL, 2.25 μg/mL and 2.10 μg/mL, respectively, exhibiting more effective control with that of the positive control than Boscalid. Furthermore, compounds 4a and 4e represented equivalent antifungal activity with Boscalid against Botrytis cinerea.

Full text of DOI:10.1016/j.fitote.2018.03.013

Fitoterapiaxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Design, synthesis and antifungal activity evaluation of coumarin-3-
carboxamide derivatives
Xiang Yu, Peng Teng, Ya-Ling Zhang, Zhao-Jun Xu, Ming-Zhi Zhang, Wei-Hua Zhang⁎
Jiangsu Key Laboratory of Pesticide, College of Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Coumarin
Amide
Hydrazide
Synthesis
Antifungal activity
SAR
A series of coumarin-3-carboxamides/hydrazides have been designed and synthesized, all the target compounds
were evaluated in vitro for their antifungal activity against Botrytis cinerea, Alternaria solani, Gibberella zeae,
Rhizoctorzia solani, Cucumber anthrax and Alternaria leaf spot, some of the designed compounds 4a-4g exhibited
potential activity in the primary assays, this highlighted by the compounds 4a, 4d, 4e and 4f, EC50 values of
which against Rhizoctorzia solani were as low as 1.80 μg/mL, 2.50 μg/mL, 2.25 μg/mL and 2.10 μg/mL, re-
spectively, exhibiting more effective control with that of the positive control than Boscalid. Furthermore,
compounds 4a and 4e represented equivalent antifungal activity with Boscalid against Botrytis cinerea.
1. Introduction
commercial amide fungicides have been launched to the market. During
the late 1990s, by the introduction of fluorine atom and active het-
With readily synthetic methods and excellent chemical/physical
characteristics, coumarins have received extensive attention and show
great practical values in many fields, such as medicine discovery, dye
chemistry, materials chemistry and so on [1–3]. Actually, over 1300
kinds of coumarins have been identified, chiefly as secondary meta-
bolites in green plants [4,5], fungi and bacteria [6–9]. The active in-
gredient coumarins also can be found in many traditional Chinese
herbal medicines, such as the former Hu, founder Ma, Cnidium, pepper,
Qin Pekin, etc., and they usually possess a broad scope of pharmaco-
logical and biological activities [10].
As the structural core, coumarin is widely used as a scaffold in
medicinal and agricultural chemicals (Fig. 1). Such as Warfarin and
Acenocoumarol, both are anticoagulant agents that function as vitamin
K antagonists, Coumoxystrobin is a strobilurin fungicide bearing a
coumarin subunit, it displays a broad spectrum of antifungal activity,
and Osthole with a coumarin scaffold has been using as a fungicide for a
long history in China. Inspired by these facts, our group designed and
synthesized several coumarin derivatives with high antifungal activity
[11–13], such as Osthole derivatives, coumarino[8,7-e][1,3]oxazine,
furo[3,2-c]coumarin, and pyrano[3,2-c] chromene-2,5-dione [8,14–16]
(Fig. 1). However, the discovery of coumarin-based fungicides with
high activity still has a long way to go.
erocycle to the target molecules, the research on amide fungicides won
a big step forward. New structures of amide fungicides were reported
with detailed understanding of their mode of action [18]. The newly
developed amide fungicides such as Fluopyram, Bixafen, Sedaxane,
Isopyrazam, Penthiopyrad and Boscalid [19,20] (Fig. 2) with high ac-
tivity and broad antifungal spectrum were successfully used to control a
variety of plant diseases with very limited cross-resistance to most of
the main fungal diseases.
In addition to amide group, molecules with hydrazide scaffolds also
displayed a broad scope of pharmacological and biological activities
[21,22] and have been widely used as frameworks in drug design. For
example, the hydrazide derivative maleic hydrazide [23] has been
found a temporary and inhibiting effect on plant growth, and the in-
dole-coumarin [24] exhibited good antiproliferative profile against
colon HT-2cell line, and the hydrazide derivative tebufenozide [25]
exhibited moderate insecticidal activity as a kind of insect growth
regulators (IGRs) (Fig. 3).
Considering these facts, we envisioned that the combination of the
coumarin framework with carboxamides/hydrazide units may afford
the desired high performance fungicides. Based on our previous work
on structural modification and bioscreening of coumarin based fungi-
cides, which exhibited effective control to certain phytopathogenic
diseases [8,14,16,26], herein we report the design, synthesis and anti-
fungal activities of novel coumarin carboxamides/hydrazides. It was
luckily to discover that a new category of coumarin based fungicides
with excellent performance such as high antifungal activities and low
In the field of fungicide research, the development of carboxylic
acid amide (CAA) fungicides has received a lot of attention. Since the
early report of amide fungicide carbendazim (carboxin) in 1966, amide
fungicides have been used for nearly 50 years [17], and a number of
⁎
Corresponding author.
E-mail address: njzhangwh@126.com (W.-H. Zhang).
https://doi.org/10.1016/j.fitote.2018.03.013
Received 14 February 2018; Received in revised form 14 March 2018; Accepted 31 March 2018
0367-326X/©2018PublishedbyElsevierB.V.
Pleasecitethisarticleas:Yu,X.,Fitoterapia(2018),https://doi.org/10.1016/j.fitote.2018.03.013

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
Fig. 1. Sample structures of coumarin-containing drugs and fungicides.
Fig. 2. Representative structures of carboxylic acid amide fungicides.
Fig. 3. Representative structures of hydrazide.
Fig. 4. Design strategy for target molecules.
drug resistance effect was successfully developed (Fig. 4).
2. Materials and methods
2.1. Chemicals and instruments
All commercially available reagents including substituted salicy-
laldehydes were purchased from Crystal Chemicals and used without
2

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
(a) Meldrum's acid, K2CO3, H2O, rt; (b) T3P, DIEA, DMF, 0 oC; (c) EDCI, DMAP, CH2Cl2, 0 oC; (d) (COCl)2,
DMF, CH2Cl2, rt; (e) DIEA, CH2Cl2, 0 oC.
Scheme 1. Synthetic routes for the target compounds.
(a) Meldrum's acid, K2CO3, H2O, rt.; (b) T3P, DIEA, DMF, 0 °C; (c) EDCI, DMAP, CH2Cl2, 0 °C; (d) (COCl)2, DMF, CH2Cl2, rt.; (e) DIEA, CH2Cl2, 0 °C.
further purification unless otherwise stated. The melting points of the
amide of coumarin derivatives were determined on an X-4 apparatus
(uncorrected), which was purchased from Shanghai Tech. Infrared (IR)
spectra were recorded on a Bruker Tensor 27 spectrometer, and samples
were prepared as KBr plates. 1H NMR and 13C NMR spectra were ob-
tained using a Bruker Avance 400 or 300 MHz spectrometer in CDCl3,
DMSO-d6, Acetone-d6 or TFA-d solution with TMS as an internal stan-
dard. HR-MS (ESI) spectra were carried out with a Thermo Exactive
spectrometer, and X-rays were measured at 296 K on a Bruker SMART
APEX2 CCD area detector diffractometer. Most reaction yields were not
optimized.
stirred for 1 h at room temperature. The reaction mixture was took off
the solvent, and then dissolved in dry DMF (30 mL) 4-chloroaniline
(6 mmol, 1 mL) was added and the mixture was stirred at iced water,
extracted with water for three times until the ether layer was colorless.
And then the organic phase layer was took off the solvent, the resulting
precipitate was filtered off and recrystallized from ethanol to give a
green solid.
2.1.3.1. Coumarin-3-carboxylic acid (2). A white solid; mp:
193.2–193.5 °C; 1H NMR (400 MHz, Acetone‑d6) δ 12.13 (s, 1H), 8.98
(s, 1H), 8.06 (dd, J = 8.1, 1.5 Hz, 1H), 7.89 (ddd, J = 9.0, 7.5, 1.6 Hz,
1H), 7.69–7.40 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 164.45,
157.15, 154.93, 148.85, 134.74, 130.65, 125.27, 118.79, 118.45,
116.59; IR (KBr) ν (cm−1) 3057, 1670, 1206; HR-MS (ESI): m/z calcd
for C10H6O4 ([M + H]+) 191.0344, found 191.0336.
2.1.1. General procedure for the preparation of compounds 3a-3f, 3j-3q
(Scheme 1)
To compound 2 (5.0 mmol, 0.95 g) dissolved in dry DMF (30 mL)
aniline (6.0 mmol, 1 mL) was added and the mixture was stirred at iced
water. Once 2 was completely dissolved, DIEA (15.0 mmol, 1.94 g) and
T3P (propyl phosphoric acid anhydride, 5.5 mmol, 1.75 g) were added
gradually with stirring and cooling for 40 min. After mixed with the
appropriate water, the solution was extracted with ethyl acetate, and
then washed with water for three times, and the product was purified
by column chromatography to give a white solid.
2.1.3.2. 2-Oxo-2H-chromene-3-carboxylic acid phenylamide (3a). A
green solid; mp: 255.4–256.6 °C; 1H NMR (400 MHz, CDCl3) δ 10.87
(s, 1H), 9.05 (s, 1H), 7.74 (dd, J = 19.1, 7.7 Hz, 4H), 7.60–7.34 (m,
4H), 7.19 (t, J = 7.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 161.84,
159.30, 154.49, 148.97, 137.67, 134.38, 129.96, 129.08, 125.50,
124.83, 120.57, 118.72, 118.65, 116.74; IR (KBr) ν (cm−1) 3221,
3030, 1705, 1203; HR-MS (ESI): m/z calcd for C16H11NO3 ([M + H]+
266.0817, found 266.0808.
)
2.1.2. General procedure for the preparation of compounds 4a-4g (Scheme
1)
To compound 2 (5.0 mmol, 0.95 g) dissolved in anhydrous CH2Cl2
(30 mL) (2-chlorophenyl) hydrazine hydrochloride (6.0 mmol, 1.08 g)
was added and the mixture was stirred at iced water. Once 2 was
completely dissolved, DMAP (4-dimethylaminopyridine, 77.2 mmol,
1.53 g) and EDCI (carbodiimide hydrochloride, 6.0 mmol, 1.15 g) were
added gradually with stirring and cooling for 4 h. The reaction mixture
was extracted with saturated brine for three times. After took off the
solvent, the resulting precipitate was purified by column chromato-
graphy to give an orange solid.
2.1.3.3. 2-Oxo-2H-chromene-3-carboxylic acid o-tolylamide (3b). A
green solid; mp: 232.5–233.3 °C; 1H NMR (400 MHz, CDCl3) δ 10.82
(s, 1H), 9.07 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.81–7.69 (m, 2H),
7.51–7.41 (m, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H),
7.13 (t, J = 7.2 Hz, 1H), 2.45 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
162.00, 159.30, 154.48, 148.97, 136.14, 134.34, 130.50, 129.96,
128.59, 126.75, 125.49, 124.97, 121.90, 118.86, 118.75, 116.74,
18.12; IR (KBr) ν (cm−1) 3276, 3051, 2920, 1699, 1201; HR-MS
(ESI): m/z calcd for C17H13NO3 ([M + H]+) 280.0974, found 280.0968.
2.1.3. General synthetic procedure for the preparation of compounds 3g-3i
(Scheme 1)
To a solution of 2 (5.0 mmol, 0.95 g) in anhydrous CH2Cl2 (10 mL)
in a 100 mL two-neck flask, was followed by a few drops of DMF. After
stirring for 10 min, 1 mL of oxalyl chloride in anhydrous CH2Cl2
(10 mL) was added dropwise during 30 min, and the mixture was
2.1.3.4. 2-Oxo-2H-chromene-3-carboxylic acid p-tolylamide (3c). A
green solid; mp: 234.7–235.4 °C; 1H NMR (400 MHz, CDCl3) δ 10.80
(s, 1H), 9.05 (s, 1H), 7.81–7.69 (m, 2H), 7.65 (d, J = 8.4 Hz, 2H),
7.53–7.36 (m, 2H), 7.21 (d, J = 8.3 Hz, 2H), 2.37 (s, 3H);13C NMR
(100 MHz, TFA-d) δ 164.47, 154.16, 152.40, 138.20, 136.07, 131.92,
130.34, 129.61, 126.44, 122.79, 118.39, 116.42, 19.09; IR (KBr) ν
3

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
Fig. 5. X-ray single crystal structures of compounds 3d, 3t and 4f.
Table 1
Table 2
Antifungal activity of target compounds (inhibitory rate, %).
Structures and yields of coumarin-3-carboxamides.
Speciesa
BOT
100
ALT
100
GIB
100
RHI
100
CUC
100
ALS
100
Compd
Rate (μg/mL)
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
76b
51
58
51
51
13
34
12
10
10
59
47
41
59
29
32
11
64
75
64
54
37
47
100d
64
68
92
82
82
57
66
100
15
10
10
10
10
10
10
10
10
10
10
10
10
10
10
15
10
10
10
10
10
10
10
15
37
53
82
75
100
29
30
73
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
12
20
10
10
10
10
10
21
28
10
10
28
28
67
23
22
37
30
20
14
10
17
10
10
10
32
22
39
52
10
10
31
41
34
20
30
20
17
85
88
72
100
100
72
52
70
92
13
10
10
10
10
10
11
10
10
10
10
10
30
10
10
10
10
13
11
10
10
10
10
90
10
74
100
10
74
10
92
10
10c
10
10
10
11
10
10
10
10
10
10
10
22
10
10
10
10
10
13
25
10
10
10
10
10
30
14
30
62
79
51
90
Compound
R1
R2
Yield (%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Bn
H
Et
H
H
H
Ph
48
56
52
o-Methylphenyl
p-Methylphenyl
2,6-Dimethylphenyl
m-Methoxyphenyl
p-Methoxyphenyl
o-Chlorophenyl
m-Chlorophenyl
p-Chlorophenyl
2-Biphenyl
58
56
58
38
35
42
64
68
60
61
63
59
47
71
54
58
75
55
42
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
4a
4b
4c
4d
4e
4f
4g
1-Naphthyl
Bn
4-Methylbenzyl
4-Methoxybenzyl
2,4-Dimethoxybenzyl
4-Fluorobenzyl
Bn
Me
Et
Cyclohexyl
Adamantyl
3s
3t
3u
3v
1-Methyl-1H-pyrazol-3-yl
Table 3
Structures and yields of coumarin-3-carboxhydrazides.
Osthole
Boscalid
a
BOT, Botrytis cinerea; ALT, Alternaria solani; GIB, Gibberella zeae; RHI,
Rhizoctorzia solani; CUC, Cucumber anthrax; ALS, Alternaria leaf spot.
b
All the data was the average value of three replications.
10 indicate the data 10% and below 10% inhibitory.
The bold means data equal to or above 80% inhibitory.
Compound
R4
Yield (%)
c
d
4a
4b
4c
4d
4e
4f
o-Chlorophenyl
m-Chlorophenyl
p-Chlorophenyl
o-Fluorophenyl
m-Fluorophenyl
p-Fluorophenyl
p-Methoxyphenyl
68
54
48
52
43
72
66
(cm−1) 3271, 2918, 1701, 1200; HR-MS (ESI): m/z calcd for C17H13NO3
([M + H]+) 280.0974, found 280.0966.
4g
2.1.3.5. 2-Oxo-2H-chromene-3-carboxylic acid (2,6-dimethyl-phenyl)-
amide (3d). A white solid; mp: 222.6–223.8 °C; 1H NMR (400 MHz,
CDCl3) δ 10.20 (s, 1H), 9.06 (s, 1H), 7.79–7.69 (m, 2H), 7.45 (ddd,
J = 11.7, 8.5, 4.7 Hz, 2H), 7.20–7.12 (m, 3H), 2.32 (s, 6H); 13C NMR
1H), 7.57 (d, J = 8.3 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.43 (s, 1H),
7.33–7.22 (m, 2H), 6.74 (dd, J = 7.9, 1.1 Hz, 1H), 3.78 (s, 3H); 13C
NMR (100 MHz, TFA-d) δ 164.45, 162.29, 161.86, 161.78, 161.49,
161.42, 161.35, 160.98, 158.41, 154.15, 152.59, 136.27, 136.10,
130.33, 130.19, 130.09, 126.42, 116.37, 108.93, 55.27; IR (KBr) ν
(cm−1) 3273, 3046, 2962, 1699, 1234; HR-MS (ESI): m/z calcd for
(100 MHz, DMSO-d6)
δ 160.89, 160.38, 154.42, 147.77, 135.36,
134.86, 134.61, 130.70, 128.27, 127.26, 125.68, 120.44, 118.95,
116.69, 18.71; IR (KBr) ν (cm−1) 3306, 3098, 2930, 1701, 1155; HR-
MS (ESI): m/z calcd for C18H15NO3 ([M + H]+) 294.1130, found
294.1123.
C
17H13NO4 ([M + Na]+) 318.0743, found 318.0733.
2.1.3.6. 2-Oxo-2H-chromene-3-carboxylic acid (3-methoxy-phenyl)-amide
(3e). A yellow solid; mp: 186.6–187.3 °C; 1H NMR (400 MHz, DMSO-
d6) δ 10.66 (s, 1H), 8.93 (s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.85–7.74 (m,
2.1.3.7. 2-Oxo-2H-chromene-3-carboxylic acid (4-methoxy-phenyl)-amide
(3f). A yellow solid; mp: 221.4–221.9 °C; 1H NMR (400 MHz, CDCl3) δ
4

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
Table 4
EC50 determination of compounds 4a-4g.
Pathogen
Compound
Toxic regression
R
EC50 (μg/mL)
95% Confidence interval
Botrytis cinerea
4a
4b
4c
4d
4e
4f
4g
Osthole
Boscalid
4d
4e
4f
Boscalid
4a
4b
4c
4d
4e
4f
Boscalid
4a
4d
4e
Y = 4.7001 + 1.5332x
Y = 3.5532 + 2.0429x
Y = 3.7370 + 1.3387x
Y = 4.0105 + 1.9952x
Y = 4.8562 + 0.6595x
Y = 4.4011 + 1.2183x
Y = 3.3664 + 1.7554x
Y = 3.7194 + 1.1772x
Y = 4.0833 + 1.7989x
Y = 4.6597 + 0.7402x
Y = 4.8753 + 0.4022x
Y = 2.4088 + 3.2917x
Y = 3.5223 + 1.0390x
Y = 4.7709 + 0.8944x
Y = 4.7849 + 0.4068x
Y = 4.8026 + 0.3899x
Y = 4.1872 + 2.0445x
Y = 4.3434 + 1.8676x
Y = 4.7826 + 0.6765x
Y = 4.0772 + 1.9480x
Y = 3.9617 + 1.1657x
Y = 4.3293 + 1.7802x
Y = 4.1668 + 1.3828x
Y = 2.2553 + 2.3373x
Y = −1.0626 + 1.7594x
Y = −4.1368 + 1.4440x
Y = −3.4533 + 1.4168x
Y = 5.5725 + 1.2370x
0.9998
0.9948
0.9945
0.9938
0.9993
0.9961
0.9986
0.9959
0.9814
0.9955
0.9978
1.0000
0.9996
0.9996
0.9997
0.9998
0.9962
0.9923
0.9960
0.9950
0.9908
0.9955
0.9727
0.9909
0.9887
0.9974
0.9949
0.9966
1.5690
5.1074
8.7792
3.1327
1.6523
3.1014
8.5235
12.2400
0.5096
2.8822
2.0424
6.1265
25.4399
1.8034
3.3787
3.2079
2.4977
2.2468
2.0961
2.9767
7.7756
2.3810
3.9468
1.1743
172.9260
3.9606
12.3503
0.3445
1.4508–1.6968
4.0956–6.3691
3.7370–6.9622
2.2587–4.3450
1.4167–1.9271
2.8115–3.4212
7.9700–9.1154
10.3776–14.4366
0.3924–0.6269
2.2341–3.7182
1.7411–2.3959
6.0764–6.1771
25.5210–27.3918
1.6129–2.0164
3.1774–3.5927
3.1945–3.2214
2.07533.0061
1.6879–2.9908
1.5970–2.7513
2.4754–3.5796
5.6352–10.7289
2.1493–2.6376
2.8761–5.4656
1.0858–1.2627
95.0799–314.5081
3.6468–4.3014
10.3579–14.7260
0.3013–0.3939
Alternaria solani
Rhizoctorzia solani
Cucumber anthrax
Alternaria leaf spot
4f
Boscalid
4f
4g
Boscalid
All the data was the average value of three replications.
Fig. 6. Structures of the most active synthetic compounds.
10.75 (s, 1H), 9.03 (s, 1H), 7.79–7.70 (m, 2H), 7.68 (d, J = 9.0 Hz, 2H),
7.45 (dd, J = 18.2, 8.0 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 162.93, 161.75, 160.76, 154.36,
148.04, 135.70, 135.67, 134.56, 130.73, 129.98, 129.89, 125.59,
(cm−1
16H10ClNO3 ([M + H]+) 300.0427, found 300.0417.
) 3190, 3052, 1706, 643; HR-MS (ESI): m/z calcd for
C
2.1.3.9. 2-Oxo-2H-chromene-3-carboxylic acid (3-chloro-phenyl)-amide
(3h). A green solid; mp: 237.0–238.1 °C; 1H NMR (400 MHz, CDCl3)
δ 10.92 (s, 1H), 9.04 (s, 1H), 7.93 (s, 1H), 7.76 (dd, J = 17.0, 7.7 Hz,
2H), 7.58 (d, J = 8.2 Hz, 1H), 7.51–7.42 (m, 2H), 7.31 (dd, J = 15.8,
7.7 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, TFA-d) δ
164.60, 154.18, 152.74, 136.17, 135.97, 135.06, 130.33, 129.93,
127.00, 126.47, 122.53, 120.41, 116.4; IR (KBr) ν (cm−1) 3182,
3056, 1693, 1201, 645; HR-MS (ESI): m/z calcd for C16H10ClNO3
([M + H]+) 300.0427, found 300.0416.
119.51, 118.93, 116.60, 115.65, 115.44, 42.51; IR (KBr) ν (cm−1
)
3277, 3054, 2840, 1698, 1237; HR-MS (ESI): m/z calcd for C17H13NO4
([M + H]+) 296.0923, found 296.0915.
2.1.3.8. 2-Oxo-2H-chromene-3-carboxylic acid (2-chloro-phenyl)-amide
(3g). A white solid; mp: 234.9–235.2 °C; 1H NMR (400 MHz, CDCl3)
δ 11.36 (s, 1H), 9.05 (s, 1H), 8.65–8.53 (m, 1H), 7.75 (dd, J = 17.4,
7.7 Hz, 2H), 7.51–7.41 (m, 3H), 7.35 (t, J = 7.8 Hz, 1H), 7.16–7.09 (m,
1H); 13C NMR (100 MHz, CDCl3) δ 161.55, 159.63, 154.61, 149.21,
149.17, 135.03, 134.53, 134.50, 130.00, 129.37, 127.52, 125.48,
125.46, 125.22, 124.22, 122.32, 118.64, 118.59, 116.82; IR (KBr) ν
2.1.3.10. 2-Oxo-2H-chromene-3-carboxylic acid (4-chloro-phenyl)-amide
(3i). A green solid; mp: 261.6–262.4 °C; 1H NMR (400 MHz, CDCl3) δ
5

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
10.90 (s, 1H), 9.04 (s, 1H), 7.75 (dd, J = 17.8, 8.2 Hz, 4H), 7.46 (dd,
J = 16.1, 8.2 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz,
TFA-d) δ 164.60, 154.17, 152.66, 136.16, 133.38, 132.94, 130.33,
129.12, 126.48, 123.62, 118.35, 116.40; IR (KBr) ν (cm−1) 3191, 3052,
3054, 2834, 1703, 1160; HR-MS (ESI): m/z calcd for C19H17NO5
([M + Na]+) 362.1005, found 362.0993.
2.1.3.17. 2-Oxo-2H-chromene-3-carboxylic acid 4-fluoro-benzylamide
(3p). A white solid; mp: 175.6–176.4 °C; 1H NMR (400 MHz, DMSO-
d6) δ 9.15 (s, 1H), 8.88 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.76 (t,
J = 7.8 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.48–7.36 (m, 3H), 7.17 (t,
J = 8.6 Hz, 2H), 4.53 (d, J = 5.9 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ
161.51 (d, J = 13.1 Hz), 154.45, 148.69, 134.20, 133.76 (d,
J = 3.2 Hz), 129.85, 129.44 (d, J = 8.1 Hz), 125.36, 118.60, 118.30,
116.66, 115.65, 115.44, 43.14; IR(KBr) ν (cm−1) 3328, 3051, 2944,
1694, 639; HR-MS (ESI): m/z calcd for C16H10ClNO3 ([M + H]+
)
300.0427, found 300.0416.
2.1.3.11. 2-Oxo-2H-chromene-3-carboxylic
acid
biphenyl-2-ylamide
(3j). A yellow solid; mp: 200.9–201.6 °C; 1H NMR (300 MHz, DMSO-
d6) δ 10.62 (s, 1H), 8.99 (s, 1H), 8.39 (d, J = 7.5 Hz, 1H), 8.04–7.97 (m,
1H), 7.78–7.70 (m, 1H), 7.52–7.37 (m, 8H), 7.33–7.20 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) δ 160.93, 160.32, 160.12, 154.36, 147.97,
139.54, 134.78, 130.78, 130.31, 125.76, 120.30, 118.96, 116.72,
112.62, 110.27, 106.10, 55.57; IR (KBr) ν (cm−1) 3225, 3052, 1702,
1203; HR-MS (ESI): m/z calcd for C22H15NO3 ([M + H]+) 342.1130,
found 342.1122.
1700, 1157; HR-MS (ESI): m/z calcd for C17H12FNO3 ([M + H]+
)
298.0879, found 298.0871.
2.1.3.18. 2-Oxo-2H-chromene-3-carboxylic acid dibenzylamide (3q). A
white solid; mp: 143.6–144.2 °C; 1H NMR (300 MHz, DMSO-d6) δ
8.29 (s, 1H), 7.73 (dd, J = 7.8, 1.4 Hz, 1H), 7.65 (ddd, J = 8.8, 7.4,
1.6 Hz, 1H), 7.34 (dddd, J = 11.7, 11.0, 7.7, 5.6 Hz, 10H), 7.22–7.16
(m, 2H), 4.51 (d, J = 20.0 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ
142.24, 136.90, 136.89, 133.12, 129.42, 129.07, 129.01, 128.02,
127.89, 127.05, 125.47, 125.29, 118.84, 116.79, 51.60, 47.10; IR
(KBr) ν (cm−1) 3031, 2923, 1709, 1226; HR-MS (ESI): m/z calcd for
2.1.3.12. 2-Oxo-2H-chromene-3-carboxylic acid naphthalen-1-ylamide
(3k). A yellow solid; mp: 228.6–229.0 °C; 1H NMR (400 MHz, CDCl3)
δ 11.52 (s, 1H), 9.13 (s, 1H), 8.45 (d, J = 7.0 Hz, 1H), 8.21 (d,
J = 8.5 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.82–7.72 (m, 3H), 7.64
(ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.60–7.44 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) δ 148.66, 130.96, 129.17, 127.14, 126.76, 126.32, 126.23,
125.86, 119.13; IR (KBr) ν (cm−1) 3248, 3055, 1703, 1207; HR-MS
(ESI): m/z calcd for C20H13NO3 ([M + H]+) 316.0974, found 316.0966.
C
24H19NO3 ([M + H]+) 370.1443, found 370.1436.
2.1.3.19. 2-Oxo-2H-chromene-3-carboxylic acid methylamide (3r). A
white solid; mp: 168.7–169.8 °C; 1H NMR (300 MHz, DMSO-d6) δ
8.85 (s, 1H), 8.62 (d, J = 4.3 Hz, 1H), 7.98 (dd, J = 7.7, 1.6 Hz, 1H),
7.78–7.67 (m, 1H), 7.44 (ddd, J = 11.2, 8.4, 4.7 Hz, 2H), 2.84 (d,
J = 4.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 162.04, 160.72,
154.31, 147.71, 134.45, 130.70, 125.56, 119.45, 118.93, 116.57,
26.79; IR (KBr) ν (cm−1) 3352, 3057, 2943, 1692, 1242; HR-MS
(ESI): m/z calcd for C11H9NO3 ([M + H]+) 204.0661, found 204.0654.
2.1.3.13. 2-Oxo-2H-chromene-3-carboxylic acid benzylamide (3l). A
yellow solid; mp: 139.4–140.2 °C; 1H NMR (400 MHz, CDCl3) δ 9.21
(s, 1H), 8.98 (s, 1H), 7.71 (dd, J = 16.5, 7.7 Hz, 2H), 7.47–7.34 (m,
6H), 4.66 (dd, J = 24.9, 6.1 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ
161.55, 161.43, 154.45, 148.61, 137.89, 134.12, 129.84, 128.73,
127.73, 127.48, 125.33, 118.64, 118.41, 116.66, 43.88; IR (KBr) ν
(cm−1) 3329, 3055, 1700, 1244; HR-MS (ESI): m/z calcd for C17H13NO3
([M + H]+) 280.0974, found 280.0965.
2.1.3.20. 2-Oxo-2H-chromene-3-carboxylic acid diethylamide (3s). A
white solid; mp: 69.6–71.1 °C; 1H NMR (400 MHz, CDCl3) δ 7.80 (d,
J = 5.1 Hz, 1H), 7.60–7.55 (m, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.37–7.27
(m, 2H), 3.55 (q, J = 7.1 Hz, 2H), 3.31 (q, J = 7.1 Hz, 2H), 1.26 (t,
J = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 164.15, 158.28, 153.80, 141.28, 132.96, 129.37, 125.96, 125.32,
118.84, 116.76, 42.99, 39.08, 14.48, 13.17; IR (KBr) ν (cm−1) 3056,
2.1.3.14. 2-Oxo-2H-chromene-3-carboxylic acid 4-methyl-benzylamide
(3m). A white solid; mp: 136.8–137.9 °C; 1H NMR (300 MHz, DMSO-
d6) δ 9.05 (t, J = 5.8 Hz, 1H), 8.87 (s, 1H), 8.03–7.92 (m, 1H),
7.80–7.66 (m, 1H), 7.55–7.36 (m, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.13
(d, J = 7.9 Hz, 2H), 4.48 (d, J = 5.9 Hz, 2H), 2.26 (s, 3H); 13C NMR
(100 MHz, DMSO-d6)
δ
161.62, 154.36, 148.00, 136.56, 136.30,
2943, 1711, 1210; HR-MS (ESI): m/z calcd for C14H15NO3 ([M + H]+
)
246.1130, found 246.1121.
134.57, 130.74, 129.43, 127.91, 125.61, 119.56, 118.97, 116.62,
42.98, 21.16; IR (KBr) ν (cm−1) 3318, 3053, 2920, 1704, 1244; HR-
MS (ESI): m/z calcd for C18H15NO3 ([M + Na]+) 316.0950, found
316.0941.
2.1.3.21. 2-Oxo-2H-chromene-3-carboxylic acid cyclohexylamide (3t). A
white solid; mp: 176.2–177.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.87
(s, 1H), 8.62 (d, J = 7.7 Hz, 1H), 8.00 (dd, J = 7.8, 1.2 Hz, 1H),
7.84–7.66 (m, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H),
3.82 (d, J = 4.3 Hz, 1H), 1.86 (d, J = 9.5 Hz, 2H), 1.69 (dd, J = 9.2,
4.0 Hz, 2H), 1.57 (d, J = 11.9 Hz, 1H), 1.50–0.84 (m, 6H). 13C NMR
2.1.3.15. 2-Oxo-2H-chromene-3-carboxylic acid 4-methoxy-benzylamide
(3n). A white solid; mp: 145.1–145.7 °C; 1H NMR (400 MHz, DMSO-
d6) δ 9.04 (t, J = 5.4 Hz, 1H), 8.88 (s, 1H), 7.99 (d, J = 7.7 Hz, 1H),
7.76 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.5 Hz,
1H), 7.29 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 4.47 (d,
(100 MHz, DMSO-d6)
δ 161.14, 160.52, 154.29, 147.81, 134.50,
130.71, 125.61, 119.51, 118.97, 116.60, 48.22, 32.54, 25.56, 24.67;
IR (KBr) ν (cm−1) 3320, 3051, 2851, 1703, 1248; HR-MS (ESI): m/z
calcd for C16H17NO3 ([M + H]+) 272.1287, found 272.1279.
J = 5.8 Hz, 2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
161.53, 160.84, 158.83, 154.34, 147.99, 134.55, 131.25, 130.72,
129.36, 125.60, 119.52, 118.95, 116.60, 114.27, 55.53, 42.71; IR
(KBr) ν (cm−1) 3311, 3060, 2838, 1721, 1248; HR-MS (ESI): m/z calcd
for C18H15NO4 ([M + Na]+) 332.0899, found 332.0893.
2.1.3.22. 2-Oxo-2H-chromene-3-carboxylic acid adamantan-1-ylamide
(3u). A white solid; mp: 214.5–215.0 °C; 1H NMR (400 MHz, CDCl3)
δ 8.88 (s, 1H), 8.69 (s, 1H), 7.67 (dd, J = 14.7, 7.8 Hz, 2H), 7.40 (dd,
J = 17.0, 8.2 Hz, 2H), 2.16 (s, 9H), 1.80–1.69 (m, 6H); 13C NMR
(100 MHz, CDCl3) δ 161.65, 159.92, 154.37, 147.68, 133.73, 129.69,
125.17, 119.63, 118.79, 116.54, 52.32, 41.41, 36.41, 29.45; IR (KBr) ν
(cm−1) 3353, 3055, 2900, 1694, 1242; HR-MS (ESI): m/z calcd for
2.1.3.16. 2-Oxo-2H-chromene-3-carboxylic
acid
2,4-dimethoxy-
benzylamide (3o). A white solid; mp: 142.9–143.7 °C; 1H NMR
(400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.89 (s, 1H), 7.99 (d, J = 7.7 Hz,
1H), 7.76 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.45 (t,
J = 7.5 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 6.60 (s, 1H), 6.49 (d,
J = 8.2 Hz, 1H), 4.43 (d, J = 5.6 Hz, 2H), 3.84 (s, 3H), 3.75 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 161.27, 161.05, 160.56, 158.72, 154.39,
148.14, 133.85, 130.28, 129.74, 125.18, 118.81, 118.68, 118.56,
116.57, 103.86, 98.61, 55.39, 55.27, 39.38; IR (KBr) ν (cm−1) 3333,
C
20H21NO3 ([M + Na]+) 346.1420, found 346.1409.
2.1.3.23. 2-Oxo-2H-chromene-3-carboxylic acid (1-methyl-1H-pyrazol-3-
yl)-amide (3v). A green solid; mp: 248.0–249.1 °C; 1H NMR (300 MHz,
DMSO-d6) δ 10.89 (s, 1H), 8.96 (s, 1H), 8.02 (dd, J = 7.8, 1.5 Hz, 1H),
6

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
7.78 (ddd, J = 8.8, 7.4, 1.6 Hz, 1H), 7.64 (d, J = 2.2 Hz, 1H), 7.57–7.42
(m, 2H), 6.60 (d, J = 2.2 Hz, 1H), 3.77 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 161.44, 158.84, 154.38, 148.35, 146.16, 134.89, 132.02,
hydrazide (4f). An orange solid; mp: 196.1–196.5 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.15 (d, J = 2.6 Hz, 1H), 8.79 (s, 1H), 8.38
(d, J = 2.4 Hz, 1H), 7.99 (dd, J = 7.8, 1.4 Hz, 1H), 7.82–7.71 (m, 1H),
7.54 (d, J = 8.4 Hz, 1H), 7.49–7.43 (m, 1H), 7.19 (dd, J = 14.9, 8.1 Hz,
1H), 6.66 (dd, J = 8.1, 1.5 Hz, 1H), 6.60 (dt, J = 11.7, 2.2 Hz, 1H),
6.52 (td, J = 8.3, 2.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 162.52,
159.97, 157.68, 155.35, 154.33, 147.36, 145.75, 134.55, 130.59,
125.63, 120.32, 118.78, 116.69, 115.80, 115.58, 114.11 (d,
J = 7.6 Hz); IR (KBr) ν (cm−1) 3353, 3320, 3052, 1704, 1203; HR-
MS (ESI): m/z calcd for C16H11ClN2O3 ([M + H]+) 299.0832, found
299.0820.
130.88, 125.79, 119.18, 119.00, 116.72, 97.19; IR (KBr) ν (cm−1
)
3262, 3132, 1693, 1693, 1123; HR-MS (ESI): m/z calcd for C14H11O3N3
([M + Na]+) 292.0699, found 292.0688.
2.1.3.24. 2-Oxo-2H-chromene-3-carboxylic acid N′-(2-chloro-phenyl)-
hydrazide (4a). A yellow solid; mp: 198.2–199.0 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.24 (s, 1H), 8.81 (s, 1H), 8.01 (dd, J = 7.7,
1.2 Hz, 1H), 7.79 (dd, J = 10.6, 3.4 Hz, 2H), 7.55 (d, J = 8.3 Hz, 1H),
7.47 (t, J = 7.6 Hz, 1H), 7.33 (dd, J = 7.9, 1.1 Hz, 1H), 7.18 (t,
J = 7.4 Hz, 1H), 6.98–6.91 (m, 1H), 6.84–6.76 (m, 1H); 13C NMR
2.1.3.30. 2-Oxo-2H-chromene-3-carboxylic acid N′-(4-methoxy-phenyl)-
hydrazide (4g). An orange solid; mp: 173.2–174.5 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.07 (d, J = 3.6 Hz, 1H), 8.78 (s, 1H), 7.99
(dd, J = 7.8, 1.2 Hz, 1H), 7.80–7.74 (m, 2H), 7.53 (d, J = 8.3 Hz, 1H),
7.46 (t, J = 7.5 Hz, 1H), 6.80 (s, 4H), 3.68 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 162.43, 159.97, 154.38, 147.74, 144.65, 134.69, 130.69,
129.69, 128.25, 125.66, 120.37, 120.02, 118.75, 117.90, 116.71,
113.77, 31.17; IR (KBr) ν (cm−1) 3331, 3285, 3057, 2839, 1709,
1030; HR-MS (ESI): m/z calcd for C17H14N2O4 ([M + Na]+) 333.0852,
found 333.0840.
(100 MHz, DMSO-d6)
δ 162.31, 160.09, 154.30, 153.40, 147.35,
142.97, 134.51, 130.58, 125.60, 120.23, 118.79, 116.66, 114.73,
114.41, 55.73, 31.15; IR (KBr) ν (cm−1) 3348, 3312, 3045, 1718,
1177; HR-MS (ESI): m/z calcd for
C )
16H11ClN2O3 ([M + Na]+
337.0356, found 337.0345.
2.1.3.25. 2-Oxo-2H-chromene-3-carboxylic acid N′-(3-chloro-phenyl)-
hydrazide (4b). An orange solid; mp: 230.2–231.7 °C; 1H NMR
(400 MHz, CDCl3) δ 10.37 (s, 1H), 8.95 (s, 1H), 7.75 (ddd, J = 7.4,
4.2, 1.5 Hz, 2H), 7.53–7.40 (m, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.93 (dd,
J = 7.0, 5.2 Hz, 2H), 6.86–6.80 (m, 1H), 6.44 (s, 1H); 13C NMR
2.2. X-ray diffraction analysis
(100 MHz, DMSO-d6)
δ 162.59, 159.93, 154.36, 150.88, 147.47,
134.59, 134.03, 130.89, 130.62, 125.62, 120.27, 118.78, 118.75,
116.69, 112.11, 111.52; IR (KBr) ν (cm−1) 3398, 3230, 3067, 1726,
1160; HR-MS (ESI): m/z calcd for C16H11ClN2O3 ([M + H]+) 315.0536,
found 315.0529.
Among the synthesized compounds, the crystal structures of com-
pounds 3d, 3t and 4f were determined by X-ray diffraction analysis.
The crystallographic data have been deposited with the Cambridge
Crystallographic Data Centre (CCDC) as supplementary publication
number: CCDC 1511231 (compound 3d), CCDC 1511235 (compound
3t), CCDC 1511236 (compound 4f), copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road, Cambridge
CB12 1EZ, UK (Fax +441,223,336,033 or E-mail: deposit@ccdc.cam.
ac.uk). The structures are shown in Fig. 5.
2.1.3.26. 2-Oxo-2H-chromene-3-carboxylic acid N′-(4-chloro-phenyl)-
hydrazide (4c). A yellow solid; mp: 207.4–208.3 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.14 (d, J = 1.7 Hz, 1H), 8.79 (s, 1H), 8.26
(d, J = 1.9 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.76 (q, J = 7.7 Hz, 1H),
7.54 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.21 (d, J = 8.3 Hz,
2H), 6.84 (d, J = 8.3 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 162.53,
159.93, 154.35, 148.23, 147.46, 134.59, 130.61, 129.01, 125.63,
122.65, 120.24, 118.76, 116.70, 114.36; IR (KBr) ν (cm−1) 3306,
3226, 3041, 1724, 1169, 642; HR-MS (ESI): m/z calcd for
2.3. Biological assays
The antifungal activities of the synthesized compounds were tested
in vitro against six phytopathogenic fungi (Botrytis cinerea, Alternaria
solani, Gibberella zeae, Rhizoctorzia solani, Cucumber anthrax, Alternaria
leaf spot) at the concentration of 100 μg/mL using mycelia growth in-
hibitory rate methods on PDA, with Osthole and Boscalid used as the
positive control. The six phytopathogenic fungi were provided by the
Laboratory of Plant Disease Control, Nanjing Agricultural University,
and they are representative fungal strains in the outbreaks region in
China and USA, the detailed procedure of experimental methods for the
antifungal activity refers to the paper from Department of Plant
Pathology, Nanjing Agriculture University [27]. The compounds were
dissolved in DMF to generate a stock solution. The compounds pos-
sessing good activity (inhibitory rate﹥70% at 100 μg/mL) were further
evaluated using different concentration by diluting the above solution.
DMF served as the negative control. The antifungal results of all the
compounds against Botrytis cinerea, Alternaria solani, Gibberella zeae,
Rhizoctorzia solani, Cucumber anthrax and Alternaria leaf spot are listed
in Table 1.
C
16H11ClN2O3 ([M + Na]+) 337.0356, found 337.0346.
2.1.3.27. 2-Oxo-2H-chromene-3-carboxylic acid N′-(2-fluoro-phenyl)-
hydrazide (4d). An orange solid; mp: 208.8–209.5 °C; 1H NMR
(400 MHz, CDCl3) δ 10.37 (s, 1H), 8.95 (s, 1H), 7.78–7.71 (m, 2H),
7.45 (ddd, J = 10.3, 8.3, 4.9 Hz, 2H), 7.04 (qdd, J = 15.4, 7.9, 1.3 Hz,
3H), 6.89 (dddd, J = 9.5, 7.3, 4.9, 2.0 Hz, 1H), 6.58 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 162.54, 160.00, 154.37, 147.62, 136.79 (d,
J = 10.6 Hz), 134.64, 130.66, 125.65, 125.02 (d, J = 3.1 Hz), 120.13,
119.62 (d, J = 6.8 Hz), 118.76, 116.71, 115.40 (d, J = 17.7 Hz),
114.42; IR (KBr) ν (cm−1) 3333, 3043, 1710, 1191; HR-MS (ESI): m/
z calcd for C16H11FN2O3 ([M + H]+) 299.0832, found 299.0825.
2.1.3.28. 2-Oxo-2H-chromene-3-carboxylic acid N′-(3-fluoro-phenyl)-
hydrazide (4e). An orange solid; mp: 218.1–219.2 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.15 (d, J = 2.6 Hz, 1H), 8.79 (s, 1H), 8.38
(d, J = 2.4 Hz, 1H), 7.99 (dd, J = 7.8, 1.4 Hz, 1H), 7.82–7.71 (m, 1H),
7.54 (d, J = 8.4 Hz, 1H), 7.49–7.43 (m, 1H), 7.19 (dd, J = 14.9, 8.1 Hz,
1H), 6.66 (dd, J = 8.1, 1.5 Hz, 1H), 6.60 (dt, J = 11.7, 2.2 Hz, 1H),
6.52 (td, J = 8.3, 2.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.90,
162.51, 159.92, 154.34, 151.49 (d, J = 10.4 Hz), 147.35, 134.55,
130.84 (d, J = 9.9 Hz), 130.59, 125.62, 120.38, 118.77, 116.70,
108.89, 105.35 (d, J = 21.3 Hz), 99.38 (d, J = 25.8 Hz); IR (KBr) ν
3. Results and discussion
3.1. Synthetic chemistry
Due to the problems of time consuming, not environment friendly,
and complex post-processing, most conventional carboxylic acid and
amine acylation reactions are not satisfactory. In recent years, however,
there are plenty of condensation reagents becoming more and more
popular in modern combinatorial and medicinal chemistry, such as T3P
and EDCI. T3P has the good selectivity, mild conditions, simple post-
(cm−1
16H11FN2O3 ([M + H]+) 299.0832, found 299.0822.
) 3288, 3051, 1703, 1176; HR-MS (ESI): m/z calcd for
C
2.1.3.29. 2-Oxo-2H-chromene-3-carboxylic acid N′-(3-fluoro-phenyl)-
7

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
processing and pollution-free which is our first choice to generate
compounds 3a-3f, 3j-3v. Nonetheless, to increase yield, EDCI is used to
generate compounds 4a-4g. Furthermore, compared with the modern
condensation reagents, conventional acylation reaction reactivity is
higher. It is beneficial for synthesis of carboxylic acid and aniline
bearing electron-withdrawing groups (compounds 3g-3i).
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgement
With the efficient method, we synthesized all target compounds
listed in Tables 2–3, and gave satisfactory analytical and spectroscopic
data (1H NMR, 13C NMR, HRMS, IR), which were full accordance with
their depicted structures, and the structures of three typical compounds
3d, 3t and 4f were further confirmed by X-ray crystallographic dif-
fraction analysis (Fig. 5).
We wish to thank the Program of National Key R&D Program of
China (2017YFD0200500) and the Fundamental Research Funds for the
Central Universities (KYTZ201604) for partially funding this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
3.2. Antifungal activity and the structure-activity relationships (SAR)
doi.org/10.1016/j.fitote.2018.03.013.
The results of the biological testing against six phytopathogenic
fungi are given in Table 1. For the purposes of the structure-activity
relationship analysis, the antifungal activity of all target compounds
were compared with the positive controls, including Osthole, and
Boscalid, a broad spectrum fungicide used for protecting plants and
food crops from fungal diseases.
References
[1] W.C. Yang, S. Yang, L. Wang, Visible-light initiated oxidative cyclization of phenyl
propiolates with sulfinic acids to coumarin derivatives under metal-free conditions,
Chem. Commun. 51 (2015) 7520–7523.
[2] D.D. Zha, H.J. Li, S.Q. Li, L. Wang, Rhodium(III)-catalyzed regioselective dec-
arboxylative cyclization for the synthesis of 4H-Furo[3,2-c]chromen-4-one deriva-
tives, Adv. Synth. Catal. 359 (2017) 467–475.
In general, data presented in Table 1 indicated that the synthesized
compounds exhibit certain activities at 100 μg/mL, while most of the
target compounds showed rather poor activity against all fungi.
As seven compounds 4a-4 g showed relatively effective control
against Botrytis cinerea, Colletotrichum capsica, Rhizoctorzia solani,
Cucumber anthrax and Alternaria leaf spot, therefore, we further tested
the EC50 values of these compounds together with Boscalid. As shown
in Table 4, it was noticed that the EC50 values of compounds 4a and 4e
were as low as 1.57 μg/mL and 1.65 μg/mL, respectively against Botrytis
cinerea, which proves they are equivalent to Boscalid (0.51 μg/mL).
Meanwhile, compound 4a (1.80 μg/mL) exhibited much more effective
activity than the control Boscalid (2.98 μg/mL) against Rhizoctorzia
solani.
Although it is difficult to extract clear structure-activity relation-
ships from the presented biological data, the conclusion still can be
drawn is that the spectrum of antifungal activity is improved by re-
placing the amide bond by the hydrazide bond, or even displayed a
broader spectrum of activity. Firstly, these coumarin amide compounds
were noticeably more active against Botrytis cinerea and Rhizoctorzia
solani, but they usually lacked potency against Alternaria solani,
Gibberella zeae, Cucumber anthrax and Alternaria leaf spot. Secondly, it is
noticeable that almost all the electron-withdrawing groups substituted
on the phenylhydrazine of compounds 4a-4f are inactive, while elec-
tron-donating group substituted compound 4g is active against
Alternaria leaf spot. Furthermore, the introduction of meta-F/Cl sub-
stituted phenylhydrazine to the coumarin core afforded active drugs
such as compounds 4e and 4e against Cucumber anthrax.
[3] S. Yang, H. Tan, W.Q. Ji, X.B. Zhang, P.H. Li, L. Wang, Visible light-induced dec-
arboxylative acylarylation of phenyl propiolates with α-oxocarboxylic acids to
coumarins catalyzed by hypervalent iodine reagents under transition metal-free
conditions, Adv. Synth. Catal. 359 (2017) 443–453.
[4] K.C. Fylaktakidou, D.J. Hadjipavlou-Litina, K.E. Litinas, D.N. Nicolaides, Natural
and synthetic coumarin derivatives with anti-inflammatory/antioxidant activities,
Curr. Pharm. Design. 10 (2004) 3813–3833.
[5] J.R.S. Hoult, M. Payá, Pharmacological and biochemical actions of simple cou-
marins: natural products with therapeutic potential, Gen. Pharmacol. 27 (1996)
713–722.
[6] K.V. Sairam, B.M. Gurupadayya, V.B. Iyer, C. RS, V.B. Iyer, Cytotoxicity studies of
coumarin analogs: design, synthesis and biological activity, RSC Adv. 6 (2016)
98816–98828.
[7] F.G. Medina, J.G. Marrero, M. Macías-Alonso, M.C. Gonzalez, I. Córdovaguerrero,
G.T. García Ariana, S. Osegueda-Robles, Coumarin heterocyclic derivatives: che-
mical synthesis and biological activity, Nat. Prod. Rep. 32 (2015) 1472–1507.
[8] M.Z. Zhang, R.R. Zhang, J.Q. Wang, X. Yu, Y.L. Zhang, Q.Q. Wang, W.H. Zhang,
Microwave-assisted synthesis and antifungal activity of novel fused Osthole deri-
vatives, Eur. J. Med. Chem. 97 (2016) 275–279.
[9] B.M. Chougala, S. Samundeeswari, M. Holiyachi, L.A. Shastri, S. Dodamani,
S. Jalalpure, S.R. Dixit, S.D. Joshi, V.A. Sunagar, Synthesis, characterization and
molecular docking studies of substituted 4-coumarinylpyrano[2,3- c]pyrazole de-
rivatives as potent antibacterial and anti-inflammatory agents, Eur. J. Med. Chem.
125 (2016) 101–116.
[10] S. Emami, S. Dadashpour, Current developments of coumarin-based anti-cancer
agents in medicinal chemistry, Eur. J. Med. Chem. 102 (2015) 611–630.
[11] A.K. Bhattacharya, H.R. Chand, J. John, M.V. Deshpande, Clerodane type diterpene
as a novel antifungal agent from Polyalthia longifolia var. pendula, Eur. J. Med.
Chem. 94 (2015) 1–7.
[12] X.F. Cao, Y.Y. Xu, Y.B. Cao, R.L. Wang, R. Zhou, W.J. Chu, Y.S. Yang, Design,
synthesis, and structure-activity relationship studies of novel thienopyrrolidone
derivatives with strong antifungal activity against Aspergillus fumigates, Eur. J.
Med. Chem. 102 (2015) (417-416).
[13] A. Ramírez-Villalva, D. Gonzalez-Calderon, C. Gonzalez-Romero, M. Morales-
Rodríguez, B. Jauregui-Rodríguez, E. Cuevas-Yanez, A. Fuentes-Benítes, A facile
synthesis of novel miconazole analogues and the evaluation of their antifungal
activity, Eur. J. Med. Chem. 97 (2015) 275–279.
[14] M.Z. Zhang, R.R. Zhang, W.Z. Yin, X. Yu, Y.L. Zhang, P. Liu, Y.C. Gu, W.H. Zhang,
Microwave-assisted Synthesis and antifungal activity of coumarin[8,7-e] [1,3]ox-
azine derivatives, Mol. Divers. 20 (2016) 611–618.
Totally, compounds 4a and 4e showed effective control to Botrytis
cinerea, and compounds 4a-4g displayed a broader spectrum of activity
in vitro (Fig. 6).
4. Conclusions
[15] R.R. Zhang, J. Liu, Y. Zhang, M.Q. Hou, M.Z. Zhang, F. Zhou, W.H. Zhang,
Microwave-assisted synthesis and antifungal activity of novel coumarin derivatives:
pyrano[3,2-c]chromene-2,5-diones, Eur. J. Med. Chem. 116 (2016) 76–83.
[16] R.R. Zhang, Z.C. Xu, W.Z. Yin, P. Liu, W.H. Zhang, Microwave-assisted synthesis
and antifungal activities of polysubstituted Furo[3,2-c]chromen-4-ones and
7,8,9,10-Tetrahydro-6H-benzofuro[3,2- c]chromen-6-ones, Synth. Commun. 46
(2015) 3257–3263.
[17] N.S. Reddy, M.R. Mallireddigari, S. Cosenza, K. Gumireddy, S.C. Bell, E. Premkumar
Reddy, M.V. Ramana Reddy, Synthesis of new coumarin 3-(N-aryl) sulfonamides
and their anticancer activity, Bioorg. Med. Chem. Lett. 14 (2004) 4093–4097.
[18] W. Peng, Y. Liu, W. Li, Y. Qian, Y.X. Nie, D. Kim, M.C. Wang, Metabolic and dy-
namic profiling for risk assessment of fluopyram, a typical phenylamide fungicide
widely applied in vegetable ecosystem, Sci. Rep.-UK 6 (2016) 33898.
[19] F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Simple coumarins and
analogues in medicinal chemistry: occurrence, synthesis and biological activity,
Curr. Med. Chem. 12 (2005) 887–916.
In conclusion, a series of coumarin-3-carboxamides/hydrazides
were designed, synthesized and evaluated for their antifungal activity
against six important phytopathogens. The in vitro assessment of target
compounds revealed that most of compounds showed certain biological
activity, compounds 4a-4g were identified as the most active ones
among the target compounds (Fig. 4), the EC50 values of these active
compounds together with Boscalid were further tested. Seven most
active compounds 4a-4g were identified as the most promising candi-
dates for further study. Further structural optimization of coumarin-
based hydrazides is well under way, alongside more detailed biological
testing of the most active compounds, aiming to improve their levels of
antifungal activity.
[20] K.D. Wehrstedt, P.A. Wandrey, D. Heitkamp, Explosive properties of 1-
8

X. Yu et al.
Fitoterapiaxxx(xxxx)xxx–xxx
Obaid, Synthesis and anticancer potential of certain novel 2-oxo-N'-(2-oxoindolin-3-
ylidene)-2H-chromene-3 -carbohydrazides, Eur. J. Med. Chem. 70 (2013) 358–363.
[25] G. Smagghe, D. Degheele, Action of a novel nonsteroidal ecdysteroid mimic, te-
bufenozide (RH-5992), on insects of different orders, Pest Manag. Sci. 42 (1994)
85–92.
[26] M.Z. Zhang, Y. Zhang, J.Q. Wang, W.H. Zhang, Design, synthesis and antifungal
activity of coumarin ring-opening derivatives, Molecules 21 (2016) 1387.
[27] Y.F. Liu, Z.Y. Chen, T.B. Ng, J. Zhang, M.G. Zhou, F.P. Song, F. Lu, Y.Z. Liu,
Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities
from Bacillus subtilis strain B-916, Peptides 28 (2007) 553–559.
hydroxybenzotriazoles, J. Hazard. Mater. 126 (2005) 1–7.
[21] F. Grande, F. Aiello, O.D. Grazia, A. Brizzi, A. Garofaloa, N. Neamati, Synthesis and
antitumor activities of a series of novel quinoxalinhydrazides, Bioorg. Med. Chem.
15 (2007) 288–294.
[22] M. Zou, J.R. Lu, H.P. Chen, Synthesis, characterization and antibacterial activities
of N-[1-(substituted phenyl) ethyl]-2-hydroxybenzohydrazide, Chem. J. Chin. Univ.
33 (2012) 2235–2238.
[23] O.L. Hoffmann, A.E. Smith, A new group of plant growth regulators, Science 109
(1949) 588.
[24] H.A. Abdel-Aziz, T. Elsaman, A. Al-Dhfyan, M.I. Attia, K.A. Al-Rashood, A.R.M. Al-
9