Design, synthesis and antifungal activity of coumarin ring-opening derivatives

Article abstract of DOI:10.3390/molecules21101387

Based on our initial design, we synthesized two series of coumarin ring-opening derivatives by the reactions of hydrolysis and methylation. Results of antifungal screening in vitro showed that the target compounds exhibited potent activity against the six

Full text of DOI:10.3390/molecules21101387

molecules
Article
Design, Synthesis and Antifungal Activity of
Coumarin Ring-Opening Derivatives
Ming-Zhi Zhang, Yu Zhang, Jia-Qun Wang and Wei-Hua Zhang *
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University,
Nanjing 210095, China; mzzhang@njau.edu.cn (M.-Z.Z.); 2014811023@njau.edu.cn (Y.Z.);
2014111011@njau.edu.cn (J.-Q.W.)
* Correspondence: njzhangwh@126.com; Tel.: +86-025-8439-5255
Academic Editor: Kamal Kumar
Received: 3 September 2016; Accepted: 13 October 2016; Published: 17 October 2016
Abstract: Based on our initial design, we synthesized two series of coumarin ring-opening derivatives
by the reactions of hydrolysis and methylation. Results of antifungal screening in vitro showed
that the target compounds exhibited potent activity against the six common pathogenic fungi.
Compounds 6b
values of these active compounds were further tested. Compared to the commonly used fungicide
Azoxystrobin (0.0884 M), compounds 6b (0.0544 M) and 6e (0.0823 M) displayed improved
activity against Botrytis cinerea.
, 6e, 6g, 6i, 7b and 7c were identified as the most active ones, and the EC50
µ
µ
µ
Keywords: coumarin; strobilurin; synthesis; ring-opening reaction; antifungal activity
1. Introduction
Coumarin derivatives are widely distributed throughout nature as secondary metabolites from
plants, and the structural modification of coumarin derivatives is a hotspot in the field of natural
product chemistry [
agrochemicals (Figure 1), these important applications have generated considerable interest in this
ring system and various fused coumarin derivatives have been reported [ ]: Osthole, a natural
1,2]. As the structural core, coumarin occurs widely in natural products, drugs and
3–6
O-methylated coumarin found in Cnidium Monnieri, a traditional Chinese herbal medicine that has been
used as as a fungicidein China for a long history, and shows antifungal activity against Rhizoctonia solani
and a broad spectrum of other phytopathogenic fungi [
anticoagulants normally used in the prevention of thrombosis and thromboembolism, function as the
vitamin K antagonists [10 12]; and Coumoxystrobin (SYP-3375) is a coumarin-containing strobilurin
7–9]; Warfarin and Acenocoumarol are
–
fungicide that contains an (E)-methyl 3-methoxy-2-phenylacrylate substructure and displays a broad
spectrum of antifungal activity [13–15].
O
O
O
OH
O
OH
O
O
O
O
O
O
n-Bu
NO2
O
O
O
O
O
Coumoxystrobin
Osthole
Warfarin
Acenocoumarol
Figure 1. Structures of coumarin-containing drugs and agrochemicals.
In our previous work, Osthole was used as the lead structure to carry out structural optimization,
and some synthesized furan[3,2-c]coumarin derivatives showed potent antifungal activity [16 18].
–
Molecules 2016, 21, 1387; doi:10.3390/molecules21101387
www.mdpi.com/journal/molecules

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In this study, the chemical structure of coumarin can be treated as O-hydroxyphenylacrylate lactone,
its ring-opening product contains a substructural unit of strobilurin fungicide, the pharmacophore
of which is (E)-methyl 3-methoxy-2-phenylacrylate (Figure 2). According to our initial design,
we synthesized two series of coumarin ring-opening derivatives differentially substituted on the
benzene ring, starting from furan[3,2-c]coumarin reported as an active lead structure in our previous
research [17].
Figure 2. Design strategies of target molecules.
2. Results and Discussion
2.1. Synthetic Chemistry
Based on the reported synthetic route (Scheme 1) [19],three kinds of substituted phenols
1 and
Meldrum’s acid were employed as the starting materials to generate the intermediate maleate 3,
2
the resulting acetone was removed by rotavapor, then the Eaton’s reagent (phosphoric anhydride +
methylsulfonic acid) was added to the residue of the reaction mixture that was continuously stirred
◦
at 70 C for 3 h. Then, the parent structure 4-hydroxycoumarins
4 were prepared in a two-step
process, which involved initial transesterification and following oxidative cyclization. Afterward,
the reaction of 4-hydroxycoumarin and -haloketone generated furo[3,2-c]coumarin derivatives via an
efficient tandem O-alkylation/cyclisation protocol [17 20]. Then, furan[3,2-c]coumarin was used as
an intermediate to generate a compound that contains the substructure of the strobilurin fungicide
by hydrolysis and methylation [21].Two series of coumarin ring-opening derivatives and were
synthesized efficiently in moderate to good yields (from 48% to 75%). The reaction yields were not
optimized. The structures of all target compounds and have been confirmed by NMR, IR and
α
,
5
6
7
6
7
HRMS. The yields and structures are also summarized in Chemicals and Methods section.
Scheme 1. Synthetic routes for target compounds.

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2.2. Antifungal Activity and the Structure-Activity Relationships (SAR)
In general, data presented in Table 1 indicate that the coumarin ring-opening derivatives exhibit
certain activities against Botrytis cinerea, Alternaria solani and Rhizoctorzia solani at 50 M, especially
µ
effective to Botrytis cinerea, almost half of the synthesized compounds displayed better activity than
the positive control Azoxystrobin. Although compound 7c demonstrated similar activity against
Cucumber anthrax to Azoxystrobin, most of the target compounds showed rather poor activity against
Gibberella zeae, Cucumber anthrax and Alternari amali.
Table 1. Antifungal activity of coumarin ring-opening derivatives (inhibitory rate, %).
Species a
BOT
ALS
GIB
RHI
RHI
ALM
Compound
50 Rate (µM)
50 Rate (µM)
50 Rate (µM)
50 Rate (µM)
50 Rate (µM)
50 Rate (µM)
c
60.3 b
81.0
41.3
49.2
74.1
58.6
77.8
33.3
77.6
51.7
70.7
67.2
25.9
32.7
65.5
−
6a
38.0
24.0
12.0
30.0
7.3
16.7
28.3
0
31.2
42.2
6.2
7.8
−
6b
0
9.8
6c
0
−
6d
5.0
23.4
25.9
12.1
34.4
20.3
15.5
37.9
51.7
51.7
34.5
50.8
27.6
39.6
13.8
8.6
12.1
18.2
0
19.6
14.8
−
6e
7.1
6f
5.5
0
6g
40.0
24.0
30.9
20.0
23.6
14.5
14.5
0
13.3
8.3
−
17.6
−
6h
0
6i
26.8
28.6
44.6
30.4
19.6
29.5
28.6
32.1
17.8
14.3
58.2
14.5
21.8
14.5
84.2
14.5
5.8
38.2
14.5
0
38.9
22.2
33.3
25.9
7.4
7a
7b
7c
7d
7e
11.1
24.1
16.7
0
16.7
44.5
7f
20.0
9.1
7g
7h
10.3
0
−
7i
0
0
Azoxystrobin
50.4
31.3
60.7
89.9
a
BOT, Botrytis cinerea; ALS, Alternariasolani; GIB, Gibberellazeae; RHI, Rhizoctorziasolani; CUC, Cucumber anthrax;
ALM, Alternariamali. All data are the average value of three replications. Test failure or not test.
b
c
As eight compounds (6a, 6b, 6e, 6g, 6i, 7b, 7c and 7f) showed relatively effective control against
Botrytis cinerea and/or Rhizoctorzia solani, we further tested the EC50 values of these compounds
together with Azoxystrobin. As shown in Table 2, we noticed that the EC50 values of compounds 6b
and 6e were as low as 0.0544 and 0.0823
more effective than Azoxystrobin (0.0884
µ
M against Botrytis cinerea, respectively, which proves it is
M). Compound 7b (0.137 M) and 7c (0.182 M) exhibited
M) against Rhizoctorzia solani. Compounds
7b and 7c were identified as the most active ones among the synthesized coumarin
ring-opening derivatives, as shown in Figure 3.
µ
µ
µ
equivalent antifungal activity with Azoxystrobin (0.122
6b 6e 6g 6i
µ
,
,
,
,
Table 2. EC50 determination of some active compounds.
Pathogen
Compound
Toxic Regression
R
EC50 (µM)
95% Confidence Interval
BOT
BOT
BOT
BOT
BOT
BOT
BOT
BOT
BOT
RHI
RHI
RHI
6a
Y = 2.4538 + 1.7909x
Y = 3.3419 + 1.4690x
Y = 2.6307 + 1.7429x
Y = 2.1175 + 2.1486x
Y = 1.9225 + 2.1902x
Y = 2.0301 + 2.0457x
Y = 1.7715 + 2.0569x
Y = 0.9264 + 2.6215x
Y = 3.5635 + 0.9256x
Y = 3.3881 + 1.0377x
Y = 2.0840 + 1.6967x
Y = 3.4242 + 0.9321x
0.9905
0.9994
0.9932
0.9768
0.9779
0.9942
0.9980
0.9954
0.9998
0.9994
0.9950
0.9981
0.1130
0.0544
0.0823
0.0889
0.0901
0.1090
0.1290
0.1120
0.0884
0.1370
0.1820
0.1220
0.0966–0.1330
0.0523–0.0566
0.0724–0.0942
0.0696–0.1130
0.0699–0.1160
0.0964–0.1230
0.1200–0.1380
0.1010–0.1250
0.0858–0.0910
0.1320–0.1430
0.1610–0.2050
0.1050–0.1410
6b
6e
6g
6i
7b
7c
7f
Azoxystrobin
7b
7c
Azoxystrobin
The EC50 value was the average value of three replications.

Molecules 2016, 21, 1387
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Figure 3. Structures of the most active coumarin ring-opening derivatives.
Although the antifungal activity of most of the coumarin ring-opening derivatives has been
proven to be rather poor, making it difficult to extract a clear SAR analysis, some broad conclusions
still can be drawn from the presented data in Table 1. Firstly, these coumarin ring-opening compounds
were noticeably more active against Botrytis cinerea than against the five other phytopathogenic
fungi. Half of the target compounds displayed better or equivalent activity to the positive control
Azoxystrobin. This is highlighted by compounds 6b, 6e, 6g and 6i. Secondly, the antifungal activity of
target compounds varied with the alternation of substituting groups at the benzene ring, as a tentative
conclusion, it has a beneficial effect when the R1 was H rather than the other substituents, illustrated by
the fact that compounds 6a–6c, and 7a–7c generally showed better control against most of the tested
fungi than the other compounds.
3. Materials and Methods
3.1. Chemicals and Methods
All chemicals including substituted phenols, Meldrum’s acid, Eaton’s reagent and
4-Hydroxycoumarin 4a were purchased from commercial sources (e.g., Crystal Chemicals, Nanjing,
China, and Alfa Aesar, Beijing, China) and used without further purification unless otherwise
stated. The course of reactions and the purity of products were monitored by TLC using silica gel
GF/UV 254. The melting points of these coumarin ring-opening derivatives were determined on an X-4
apparatus (uncorrected), which was bought from Shanghai Tech (Shanghai, China). Nuclear magnetic
resonance (1H- and 13C-NMR) spectra were obtained using a Bruker Avance 400 MHz spectrometer
(Bruker Biospin Co., Stuttgart, Germany) in CDCl3 solution with TMS as an internal standard. Infrared
(IR) spectra were recorded on a Bruker Tensor 27 spectrometer (Bruker Biospin Co.), and samples were
prepared as KBr plates. High Resolution Mass Spectrometer (HRMS) spectra were carried out with a
ThermoExactive spectrometer (ThermoFisher Scientific Inc., Waltham, MA, USA).
3.1.1. General Procedure for the Synthesis of Compound 4 (Scheme 1)
A mixture of substituted phenol (0.094 g, 1.0 mmol) and Meldrum’s acid (0.144 g, 1.0 mmol) was
◦
stirred at 100 C for 3 h (monitored by TLC), then the small remaining amount of acetone was removed
◦
by vacuum. Eaton’s reagent (3 mL) was added to the mixture at 70 C for 4 h. Then, water was added
to this mixture while stirring vigorously. The precipitate was filtered by suction, washed with water,
and dried to give a crude product. It was recrystallized from ethanol to afford compounds 4b
(4-Hydroxycoumarin 4a was obtained from a commercial source).
–4d.
4-Hydroxy-7-methoxy-2H-chromen-2-one (4b): white solid, yield 74%, m.p. 264.0–264.5 ◦C. 1H-NMR
(300 MHz, DMSO-d6)
δ 12.36 (s, 1H), 7.70 (dd, J = 8.4, 0.7 Hz, 1H), 6.96–6.86 (m, 2H), 5.43 (s, 1H), 3.83
(s, 3H).

Molecules 2016, 21, 1387
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◦
4-Hydroxy-6-methyl-2H-chromen-2-one (4c): light yellow solid, yield 54%, m.p. 260.1–261.3 C; 1H-NMR
(400 MHz, DMSO-d6) 12.47 (s, 1H), 7.62 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 8.4, 2.1 Hz, 1H), 7.27 (d,
J = 8.5 Hz, 1H), 5.57 (s, 1H), 2.38 (s, 3H).
δ
◦
1
4-Hydroxy-2H-benzo[g]chromen-2-one (4d): white solid, yield 51%, m.p. 188.5–186.0 C; H-NMR
(400 MHz, Acetone-d6)
δ 8.51–8.43 (m, 1H), 8.08–8.01 (m, 1H), 7.94–7.81 (m, 2H), 7.78–7.69 (m, 2H), 5.79
(s, 1H).
3.1.2. General Procedure for the Synthesis of Compound 5 (Scheme 1)
To a stirring solution of substituted 4-hydroxycoumarin (10 mmol) and ammonium acetate
4
(7.7 g, 100 mmol) in toluene (50 mL) and acetic acid (5 mL) was added cholroacetone (5 mL, 62 mmol)
or 3-chlorobutan-2-one, and 3-chloropentane-2,4-dione (62 mmol) in a single portion via syringe.
The mixture was stirred under reflux for about 10 huntil full conversion of the substrates was
indicated by TLC analysis, and then cooled to room temperature and concentrated at reduced pressure.
Then 50 mL saturated brine solution was added to the mixture and extracted with EtOAc three
times (3 × 50 mL), the extract was washed with 10% NaHCO3 solution, organic phase was dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by
silica gel column chromatography using petroleum ether/acetone (20:1 to 5:1) as eluent to give the
compound 5 (Table 3).
Table 3. The yields and structures of compound 5.
Compound
R1
R2
Yield
Compound
R1
R2
Yield
5a
5b
5c
5d
5e
5f
H
H
H
7-OMe
7-OMe
7-OMe
H
Me
Ac
H
Me
Ac
79%
38%
33%
79%
22%
36%
5g
5h
5i
5j
5k
8-Me
8-Me
7,8-(CH)4
7,8-(CH)4
7,8-(CH)4
H
Ac
H
Me
Ac
67%
34%
55%
37%
45%
◦
1
3-Methyl-4H-furo[3,2-c]chromen-4-one (5a): white solid, m.p. 155.7–156.3 C; H-NMR (400 MHz,
Chloroform-d)
(td, J = 7.5, 1.2 Hz, 1H), 2.39 (d, J = 1.4 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6)
143.13, 131.35, 125.23, 121.11, 119.81, 117.36, 112.83, 110.42, 8.66. IR (KBr)
(cm−1) 3046, 2927, 1742,
δ
7.87 (dd, J = 7.8, 1.6 Hz, 1H), 7.52 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.47–7.40 (m, 2H), 7.35
δ
158.07, 157.30, 152.43,
ν
1632, 1581, 1548, 1502, 1445; HR-MS (ESI): m/z calcd for C12H8O3 ([M + H]+) 201.0552, found 201.0546.
2,3-Dimethyl-4H-furo[3,2-c]chromen-4-one (5b): white solid, m.p. 183.8–184.1 ◦C; 1H-NMR (400 MHz,
Chloroform-d)
δ
7.83 (dd, J = 7.8, 1.5 Hz, 1H), 7.5–7.38 (m, 2H), 7.33 (ddd, J = 8.2, 7.2, 1.4 Hz, 1H),
158.84, 155.57,
2.42 (d, J = 1.0 Hz, 3H), 2.30 (d, J = 1.1 Hz, 3H); 13C-NMR (101 MHz, Chloroform-d)
δ
152.22, 150.82, 129.79, 124.24, 120.38, 117.08, 114.06, 113.08, 111.31, 11.51, 8.52; IR (KBr) ν (cm−1) 3062,
2956, 1756, 1634, 1619, 1592, 1568, 1439; HR-MS (ESI): m/z calcd for C13H10O3([M + H]+) 215.0708,
found 215.0702.
2-Acetyl-3-methyl-4H-furo[3,2-c]chromen-4-one (5c): light yellow solid, m.p. 178.6–178.9 ◦C; 1H-NMR
(400 MHz, Chloroform-d)
J = 8.4 Hz, 1H), 7.45–7.38 (m, 1H), 2.76 (d, J = 1.0 Hz, 3H), 2.65 (d, J = 0.9 Hz, 3H); 13C-NMR (101 MHz,
Chloroform-d) 188.67, 157.76, 157.31, 153.67, 149.14, 132.25, 129.53, 124.80, 121.55, 117.54, 112.13,
δ 7.99 (dd, J = 7.8, 1.5 Hz, 1H), 7.63 (ddt, J = 8.5, 7.1, 1.2 Hz, 1H), 7.48 (d,
δ
111.98, 27.63, 10.23; IR (KBr)
ν
(cm−1) 3028, 2922, 1736, 1674, 1625, 1596, 1545, 1427; HR-MS (ESI):
m/z calcd for C14H10O4 ([M + H]+) 243.0657, found 243.0651.
7-Methoxy-3-methyl-4H-furo[3,2-c]chromen-4-one (5d): light yellow solid, m.p.150.7–151.0 ◦C; 1H-NMR
(400 MHz, DMSO-d6) δ 7.84 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.6, 2.2 Hz, 1H),

Molecules 2016, 21, 1387
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5.91 (s, 1H), 4.02 (s, 3H), 2.32 (s, 3H); IR (KBr)
ν
(cm−1) 3076, 2920, 1743, 1625, 1600, 1580, 1553, 1446;
HR-MS (ESI): m/z calcd for C13H10O4([M + H]+) 231.0657, found 231.0652.
◦
1
7-Methoxy-2,3-dimethyl-4H-furo[3,2-c]chromen-4-one (5e): white solid, m.p.143.5–144.0 C; H-NMR
(400 MHz, Chloroform-d) 7.72 (d, J = 8.5 Hz, 1H), 6.96–6.88 (m, 2H), 3.89 (s, 3H), 2.39 (d, J = 1.1 Hz,
3H), 2.28 (d, J = 1.1 Hz, 3H); 13C-NMR (101 MHz, Chloroform-d)
161.34, 159.15, 156.31, 153.94, 149.73,
121.33, 113.65, 112.42, 108.96, 106.60, 101.26, 55.68, 11.42, 8.53; IR (KBr)
(cm−1) 3030, 2950, 1748, 1631,
δ
δ
ν
1602, 1511, 1439; HR-MS (ESI): m/z calcd for C14H12O4 ([M + H]+) 245.0814, found 245.0806.
2-Acetyl-7-methoxy-3-methyl-4H-furo[3,2-c]chromen-4-one (5f): orange solid, m.p. 203.0–204.6 ◦C;
1H-NMR (400 MHz, Chloroform-d)
(s, 3H), 2.62 (s, 3H); 13C-NMR (101 MHz, Chloroform-d)
129.76, 122.61, 113.21, 109.69, 105.35, 101.45, 55.88, 27.60, 10.28; IR (KBr)
1628, 1606, 1551, 1423; HR-MS (ESI): m/z calcd for C15H12O5([M + H]+) 273.0763, found 273.0756.
δ
7.87 (d, J = 8.6 Hz, 1H), 7.01–6.92 (m, 2H), 3.93 (s, 3H), 2.73
188.53, 163.21, 158.07, 158.01, 155.61, 148.60,
(cm−1) 3088, 2942, 1746, 1672,
δ
ν
◦
1
3,8-Dimethyl-4H-furo[3,2-c]chromen-4-one (5g): light yellow solid, m.p. 141.7–143.7 C; H-NMR
(400 MHz, Chloroform-d) 7.67–7.63 (m, 1H), 7.42 (t, J = 1.3 Hz, 1H), 7.34–7.31 (m, 2H), 2.47 (s, 3H),
2.38 (d, J = 1.3 Hz, 3H); IR (KBr)
(cm−1) 3064, 2921, 1735, 1632, 1583, 1558, 1501, 1428; HR-MS (ESI):
m/z calcd for C13H10O3 ([M + H]+) 215.0708, found 215.0702.
δ
ν
◦
1
2-Acetyl-3,8-dimethyl-4H-furo[3,2-c]chromen-4-one (5h): orange solid, m.p. 225.6–226.0 C; H-NMR
(400 MHz, Chloroform-d) 7.78–7.75 (m, 1H), 7.42 (dd, J = 8.5, 2.0 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 2.76
(s, 3H), 2.66 (s, 3H), 2.50 (s, 3H); 13C-NMR (101 MHz, Chloroform-d)
188.66, 157.98, 157.41, 151.91,
δ
δ
149.06, 134.77, 133.32, 129.63, 121.18, 117.27, 111.90, 111.80, 27.64, 20.91, 10.26; IR (KBr) ν (cm−1) 3026,
2925, 1733, 1668, 1630, 1593, 1574, 1548, 1506, 1453; HR-MS (ESI): m/z calcd for C15H12O4 ([M + H]+)
257.0814, found 257.0805.
3-Methyl-4H-benzo[g]furo[3,2-c]chromen-4-one (5i): light yellow solid, m.p. 205.0–205.3 ◦C; 1H-NMR
(400 MHz, Chloroform-d)
(m, 2H), 7.46 (q, J = 1.4 Hz, 1H), 2.43 (d, J = 1.3 Hz, 3H); IR (KBr)
1489; HR-MS (ESI): m/z calcd for C16H10O3([M + H]+) 251.0708, found 251.0702.
δ
8.67–8.59 (m, 1H), 7.94–7.84 (m, 2H), 7.77 (d, J = 8.6 Hz, 1H), 7.70–7.61
ν
(cm−1) 3028, 2920, 1730, 1617, 1578,
◦
1
2,3-Dimethyl-4H-benzo[g]furo[3,2-c]chromen-4-one (5j): yellow solid, m.p. 238.5–239.1 C; H-NMR
(400 MHz, Chloroform-d) 8.62 (dd, J = 8.0, 1.5 Hz, 1H), 7.97–7.81 (m, 2H), 7.75 (d, J = 8.6 Hz, 1H),
7.64 (dqd, J = 8.4, 6.9, 1.5 Hz, 2H), 2.45 (d, J = 1.0 Hz, 3H), 2.35 (d, J = 1.0 Hz, 3H); 13C-NMR (101 MHz,
Chloroform-d) 158.85, 156.63, 150.72, 148.77, 133.86, 127.98, 127.77, 127.19, 124.53, 123.46, 122.48,
116.99, 114.04, 111.08, 108.35, 11.59, 8.62; IR (KBr)
(cm−1) 2925, 1724, 1632, 1621, 1601, 1593, 1458;
HR-MS (ESI): m/z calcd for C17H12O3 ([M + H]+) 265.0865, found 265.0859.
δ
δ
ν
◦
2-Acetyl-3-methyl-4H-benzo[g]furo[3,2-c]chromen-4-one (5k): yellow solid, m.p. 248.9–249.4 C; 1H-NMR
(400 MHz, Chloroform-d) 8.66–8.55 (m, 1H), 8.00–7.88 (m, 2H), 7.81 (d, J = 8.6 Hz, 1H), 7.73–7.66
(m, 2H), 2.78 (s, 3H), 2.67 (s, 3H); 13C-NMR (101 MHz, Chloroform-d)
188.65, 158.20, 157.68, 151.01,
149.04, 134.95, 129.63, 128.96, 128.12, 127.66, 125.09, 123.22, 122.70, 116.98, 111.56, 107.31, 27.64, 10.33;
δ
δ
IR (KBr)
ν
(cm−1) 2916, 1737,1673, 1614, 1597, 1556, 1495; HR-MS (ESI): m/z calcd for C18H12O4
([M + H]+) 293.0814 found 293.0807.
3.1.3. General Procedure for the Synthesis of Target Compounds 6 and 7 (Scheme 1)
A mixture of substituted furocoumarin 5 (10 mmol) and 5% KOH solution (45 mL) was stirred
until completely dissolved, then dimethylsulfate (1.0 mL, 10 mmol) was added in a single portion via
syringe. The mixture was refluxed for about 10 h until full conversion (monitored by TLC), then cooled
to room temperature and acidified to pH = 4–5 with 5% HCl solution, then extracted with EtOAc three
times (3
×
50 mL), the extract was dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude product was purified by recrystallized from acetone to give the target compound
6.

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Compound
6
(5 mmol) was dissolved in 20 mL CH3OH along with SOCl2 (30 mmol), the mixture
was stirred at room temperature for about 8 h. After completion of the reaction, the mixture was
concentrated at reduced pressure, and then 50 mL of water was added, and extracted with EtOAc
(
3 × 30 mL) and washed with saturated NaHCO3 solution (30 mL), washed with water (30 mL),
the organic phase was dried over anhydrous Na2SO4, and concentrated under reduced pressure to
afford the target compound 7.
The reaction yields were not optimized, and the structures of target compounds
summarized in Table 4.
6 and 7 are
Table 4. The yields and structures of target compounds 6 and 7.
Compound
R1
R2
Yield
Compound
R1
R2
Yield
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
H
Me
Ac
H
Me
Ac
H
65%
55%
55%
60%
56%
51%
71%
50%
48%
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
H
Me
Ac
H
Me
Ac
H
75%
65%
68%
74%
71%
73%
69%
62%
58%
4-OMe
4-OMe
4-OMe
5-Me
5-Me
4,5-(CH)4
4-OMe
4-OMe
4-OMe
5-Me
5-Me
4,5-(CH)4
Me
H
Me
H
2-(2-Methoxyphenyl)-4-methylfuran-3-carboxylic acid (6a): yellow solid, m.p. 134.0–134.2 ◦C; 1H-NMR
(400 MHz, Chloroform-d3) 7.43 (ddd, J = 15.5, 7.6, 1.7 Hz, 2H), 7.30 (d, J = 1.3 Hz, 1H), 7.04 (td, J = 7.5,
1.0 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.82 (s, 3H), 2.24 (s, 3H); IR (KBr)
(cm−1) 3072, 2965, 1680, 1616,
δ
ν
1580, 1557, 1464; HR-MS (ESI): m/z calcd for C13H12O4 ([M + H]+) 233.0814, found 233.0807.
2-(2-Methoxyphenyl)-4,5-dimethylfuran-3-carboxylic acid (6b): light yellow solid, m.p. 197.0–197.8 ◦C;
1H-NMR (400 MHz, Acetone-d6)
1H), 3.79 (s, 3H), 2.26 (s, 3H), 2.12 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
130.84, 130.70, 120.43, 120.37, 117.49, 115.47, 111.89, 55.72, 11.50, 9.81; IR (KBr)
1683, 1607, 1580, 1565, 1484; HR-MS (ESI): m/z calcd for C14H14O4([M + H]+) 247.0970, found 247.0962.
δ
7.40 (td, J = 7.4, 1.2 Hz, 2H), 7.07 (d, J = 8.5 Hz, 1H), 7.01 (t, J = 7.5 Hz
165.70, 157.25, 151.44, 147.51,
(cm−1) 3050, 2983,
,
δ
ν
◦
5-Acetyl-2-(2-methoxyphenyl)-4-methylfuran-3-carboxylic acid (6c): white solid, m.p. 212.6–213.6 C;
1H-NMR (400 MHz, Acetone-d6)
δ
7.61 (dd, J = 7.5, 1.7 Hz, 1H), 7.55–7.47 (m, 1H), 7.19–7.06 (m, 2H),
188.77, 164.83, 157.53, 154.38,
147.53, 132.18, 130.43, 130.13, 120.75, 120.15, 118.96, 112.25, 55.83, 27.76, 10.84; IR (KBr) ν (cm−1) 3052,
2937, 1703, 1667, 1606, 1581, 1536, 1494; HR-MS (ESI): m/z calcd for C15H14O5 ([M + H]+) 275.0919,
found 275.0912.
3.84 (s, 3H), 2.55 (s, 3H), 2.48 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ
2-(2,4-Dimethoxyphenyl)-4-methylfuran-3-carboxylic acid (6d): light yellow solid, m.p. 140.9–141.2 ◦C;
1H-NMR (400 MHz, Chloroform-d3)
δ
7.38 (d, J = 8.4 Hz, 1H), 7.28–7.25 (m, 1H), 6.57 (dd, J = 8.5, 2.3 Hz,
1H), 6.53 (d, J = 2.3 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 3H), 2.23 (s, 3H); IR (KBr)
ν
(cm−1) 3110, 2962, 1678,
1619, 1597, 1578, 1549, 1481; HR-MS (ESI): m/z calcd for C14H14O5 ([M + H]+) 263.0919, found 263.0911.
2-(2,4-Dimethoxyphenyl)-4,5-dimethylfuran-3-carboxylic acid (6e): light yellow solid, m.p. 156.9–158.3 ◦C;
1H-NMR (400 MHz, Acetone-d6)
2.24 (s, 3H), 2.11 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
116.75, 115.37, 113.18, 105.16, 98.89, 55.80, 55.77, 11.48, 9.89; IR (KBr)
1579, 1445; HR-MS (ESI): m/z calcd for C15H16O5([M + H]+) 277.1076, found 277.1068.
δ
7.31 (d, J = 8.4 Hz, 1H), 6.64–6.55 (m, 2H), 3.86 (s, 3H), 3.78 (s, 3H),
165.79, 161.72, 158.57, 151.95, 147.00, 131.63,
(cm−1) 3076, 2961, 1690, 1615,
δ
ν
5-Acetyl-2-(2,4-dimethoxyphenyl)-4-methylfuran-3-carboxylic acid (6f): orange solid, m.p. 170.0–171.3 ◦C;
1H-NMR (400 MHz, DMSO-d6)
δ
7.46 (d, J = 8.4 Hz, 1H), 6.71–6.63 (m, 2H), 3.84 (s, 3H), 3.76 (s, 3H),
2.45 (s, 3H), 2.43 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ
188.62, 165.01, 162.78, 158.92, 154.72, 147.15,

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131.37, 130.30, 119.37, 111.65, 105.89, 99.07, 55.95, 55.89, 27.71, 10.88; IR (KBr)
ν
(cm−1) 3071, 2930, 1662,
1614, 1580, 1540, 1450; HR-MS (ESI): m/z calcd for C16H16O6 ([M + H]+) 305.1025, found 305.1018.
2-(2-Methoxy-5-methylphenyl)-4-methylfuran-3-carboxylic acid (6g): yellow solid, m.p. 143.7–144.2 ◦C;
1H-NMR (400 MHz, Chloroform-d)
δ
7.29 (d, J = 1.2 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.24–7.19 (m, 1H),
6.87 (d, J = 8.4 Hz, 1H), 3.80 (s, 3H), 2.34 (s, 3H), 2.23 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ
165.52,
155.27, 154.14, 139.98, 131.38, 131.03, 129.11, 121.41, 120.01, 117.01, 111.90, 55.77, 20.36, 9.93; IR (KBr)
ν
(cm−1) 3078, 2998, 1673, 1552, 1502, 1451; HR-MS (ESI): m/z calcd for C14H14O4 ([M + H]+) 247.0970,
found 247.0963.
2-(2-Methoxy-5-methylphenyl)-4,5-dimethylfuran-3-carboxylic acid (6h): yellow solid, m.p. 213.8–214.5 ◦C;
1H-NMR (400 MHz, Acetone-d6)
δ
7.44–7.39 (m, 1H), 7.34–7.28 (m, 1H), 7.04 (d, J = 8.5 Hz, 1H), 3.80
(s, 3H), 2.54 (s, 3H), 2.48 (s, 3H), 2.34 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ
188.72, 164.85, 155.52,
154.42, 147.47, 132.47, 130.50, 130.14, 129.56, 120.14, 118.69, 112.20, 55.84, 27.78, 20.37, 10.84; IR (KBr)
ν
(cm−1) 3062, 2997, 1664, 1619, 1577, 1536, 1501, 1456; HR-MS (ESI): m/z calcd for C16H16O5 ([M + H]+)
289.1076, found 289.1069.
◦
2-(3-Methoxynaphthalen-2-yl)-4-methylfuran-3-carboxylic acid (6i): orange solid, m.p. 137.9–138.6 C;
1H-NMR (300 MHz, DMSO-d6)
δ
8.16–8.08 (m, 1H), 8.00–7.93 (m, 1H), 7.73–7.66 (m, 2H), 7.64–7.55
(m, 2H), 7.46 (d, J = 8.5 Hz, 1H), 3.63 (s, 3H), 2.17 (s, 3H); IR (KBr)
ν
(cm−1) 3059, 2987, 1678, 1608, 1594,
1556, 1501, 1471; HR-MS (ESI): m/z calcd for C17H14O4 ([M + H]+) 283.0970, found 283.0963.
Methyl 2-(2-methoxyphenyl)-4-methylfuran-3-carboxylate (7a): yellow oil, 1H-NMR (400 MHz, DMSO-d6)
δ
7.61 (d, J = 1.4 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.03 (td, J = 7.5, 1.0 Hz, 2H), 3.73 (s, 3H), 3.61 (s, 3H),
2.11 (s, 3H); IR (KBr)
C14H14O4 ([M + H]+) 247.0970, found 247.0963.
ν
(cm−1) 2950, 1770, 1713, 1598, 1582, 1552, 1493; HR-MS (ESI): m/z calcd for
Methyl 2-(2-methoxyphenyl)-4,5-dimethylfuran-3-carboxylate (7b): yellow solid, m.p. 55.5–56.2 ◦C;
1H-NMR (400 MHz, DMSO-d6)
δ
7.52–7.33 (m, 2H), 7.09 (dd, J = 8.4, 1.0 Hz, 1H), 7.01 (td, J = 7.5, 1.0 Hz,
1H), 3.72 (s, 3H), 3.59 (s, 3H), 2.25 (s, 3H), 2.04 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
164.78, 156.76,
151.02, 147.86, 130.97, 130.11, 120.54, 119.88, 116.86, 115.34, 111.82, 55.82, 51.48, 11.49, 9.53; IR (KBr)
ν
(cm−1) 3047, 2924, 1710, 1633, 1602, 1581, 1560, 1495; HR-MS (ESI): m/z calcd for C15H16O4 ([M + H]+)
261.1127, found 261.1121.
◦
Methyl 5-acetyl-2-(2-methoxyphenyl)-4-methylfuran-3-carboxylate (7c): white solid, m.p. 61.4–62.2 C;
1H-NMR (300 MHz, DMSO-d6)
δ
7.58 (dd, J = 7.6, 1.7 Hz, 1H), 7.50 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H),
7.19–7.05 (m, 2H), 3.74 (s, 3H), 3.64 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
188.78, 163.89, 157.13, 153.99, 147.56, 132.44, 130.13, 129.82, 120.94, 119.25, 118.40, 112.14, 56.00, 52.05,
δ
27.77, 10.62; IR (KBr)
C16H16O5 ([M + H]+) 289.1076, found 289.1068.
ν
(cm−1) 2948, 1725, 1665, 1610, 1584, 1534, 1492; HR-MS (ESI): m/z calcd for
Methyl 2-(2,4-dimethoxyphenyl)-4-methylfuran-3-carboxylate (7d): yellow oil; 1H-NMR (400 MHz,
DMSO-d6)
(s, 3H), 3.61 (s, 3H), 2.10 (s, 3H); IR (KBr)
(ESI): m/z calcd for C15H16O5 ([M + H]+) 277.1076, found 277.1069.
δ
7.55 (d, J = 1.5 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.77–6.54 (m, 2H), 3.82 (s, 3H), 3.72
ν
(cm−1) 3052, 2948, 1711, 1618, 1578, 1503, 1455; HR-MS
◦
Methyl 2-(2,4-dimethoxyphenyl)-4,5-dimethylfuran-3-carboxylate (7e): yellow solid, m.p. 71.6–73.2 C;
1H-NMR (300 MHz, DMSO-d6)
7.53 (d, J = 8.6 Hz, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.56 (dd, J = 8.6,
(cm−1) 3068, 2922,
δ
2.4 Hz, 1H), 3.86 (s, 3H), 3.77 (s, 3H), 3.68 (s, 3H) 2.21 (s, 3H), 1.91 (s, 3H). IR (KBr)
ν
1714, 1612, 1582, 1498; HR-MS (ESI): m/z calcd for C16H18O5 ([M + H]+) 291.1232, found 291.1226.
Methyl 5-acetyl-2-(2,4-dimethoxyphenyl)-4-methylfuran-3-carboxylate (7f): yellow solid, m.p. 120.2–12.4 ◦C
;
1H-NMR (400 MHz, DMSO-d6)
δ
7.53 (d, J = 9.1 Hz, 1H), 6.69 (dd, J = 5.2, 2.3 Hz, 2H), 3.85 (s, 3H),
3.76 (s, 3H), 3.67 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
188.61, 164.08,
162.95, 158.56, 154.37, 147.16, 131.13, 130.01, 118.35, 111.17, 106.06, 99.00, 56.09, 56.00, 51.97, 27.73,
δ

Molecules 2016, 21, 1387
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10.66; IR (KBr)
ν
(cm−1) 3020, 2918, 1708, 1662, 1615, 1582, 1439; HR-MS (ESI): m/z calcd for C17H18O6
([M + H]+) 319.1182, found 319.1175.
Methyl 2-(2-methoxy-5-methylphenyl)-4-methylfuran-3-carboxylate (7g): yellow oil; 1H-NMR (300 MHz,
DMSO-d6)
δ 7.57 (q, J = 1.2 Hz, 1H), 7.24–7.16 (m, 2H), 6.97 (d, J = 8.3 Hz, 1H), 3.67 (s, 3H), 3.59 (s, 3H),
2.26 (s, 3H), 2.08 (s, 3H); IR (KBr)
for C15H16O4 ([M + H]+) 261.1127, found 261.1122.
ν
(cm−1) 2950, 1707, 1617, 1553, 1498, 1438; HR-MS (ESI): m/z calcd
Methyl 2-(2-methoxy-5-methylphenyl)-4,5-dimethylfuran-3-carboxylate (7h): yellow solid, m.p. 117.8–118.1 ◦C;
1H-NMR (400 MHz, DMSO-d6)
δ
7.40 (d, J = 2.2 Hz, 1H), 7.32 (dd, J = 8.6, 2.3 Hz, 1H), 7.05 (d, J = 8.5 Hz
1H), 3.72 (s, 3H), 3.65 (s, 3H), 2.47 (s, 3H), 2.44 (s, 3H), 2.32 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
188.73, 163.92, 155.12, 154.04, 147.51, 132.76, 130.20, 129.84, 129.79, 119.24, 118.10, 112.05, 56.00, 52.01,
,
δ
27.80, 20.37, 10.62; IR (KBr)
ν
(cm−1) 3049, 2917, 1708, 1665, 1584, 1505, 1440; HR-MS (ESI): m/z calcd
for C17H18O5 ([M + H]+) 303.1232, found 303.1226.
Methyl 2-(3-methoxynaphthalen-2-yl)-4-methylfuran-3-carboxylate (7i): yellow oil; 1H-NMR (300 MHz,
DMSO-d6)
δ 8.13 (ddd, J = 6.2, 2.3, 1.4 Hz, 1H), 7.97 (dt, J = 5.2, 3.1 Hz, 1H), 7.77–7.70 (m, 2H), 7.65–7.57
(m, 2H), 7.50 (d, J = 8.5 Hz, 1H), 3.60 (s, 3H), 3.59 (s, 3H), 2.16 (s, 3H). IR (KBr)
ν
(cm−1) 2922, 1727,
1618, 1579, 1493; HR-MS (ESI): m/z calcd for C18H16O4 ([M + H]+) 297.1127, found 297.1120.
3.2. Biological Assays
Many experimental protocols for antifungal tests are reported in the literature [22
the antifungal activities of all the synthesized target compounds were carried out at the concentration
of 50 M using mycelia growth inhibitory rate methods, with Azoxystrobin used as the positive control.
,23].In this study,
µ
For the detailed procedure of experimental methods for the antifungal activity, refer to the paper
from Department of Plant Pathology, Nanjing Agriculture University [24]. The assay of antifungal
activity toward Botrytis cinerea, Alternariasolani, Gibberellazeae, Rhizoctorziasolani, Cucumber anthrax and
Alternariamali was carried out on 100 mm
×
15 mm Petri plates each contained 10 mL potato dextrose
agar, under sterile conditions, on a clean bench in a sterile room. Sterile blank paper disks (0.65 cm in
diameter) were placed at a distance 2.5 cm away from the rim of the mycelial colony. The plates were
sealed with parafilm, and incubated at 25 ◦C until mycelial growth had enveloped disks containing
the control and had formed crescents of inhibition around disks containing samples with antifungal
activity. When the mycelia colony of the control had grown to almost fill the plate, the area of the
mycelia colony was measured, and the inhibition of fungal growth in the other plates was determined
by calculating the percent reduction in the area of the mycelia colony. The resulting data were collated
for each compound, and averages across replicates were used to make a judgment of the overall
activity level of the compound.
The antifungal data listed in Table 1 are the screening results of all the compounds against
Botrytis cinerea, Alternaria solani, Gibberella zeae, Rhizoctorzia solani, Cucumber anthrax and Alternaria mali,
which are the most common phytopathogenic fungi in China.
4. Conclusions
In summary, aiming to discover novel Osthole analogs with improved antifungal activity, we have
designed and synthesized two series of coumarin ring-opening derivatives through hydrolysis and
methylation. Biological testing data showed that some target compounds displayed an altered pattern
of biological activity, and compounds 6b
The EC50 values of these compounds together with Azoxystrobin were further tested. Compared to
Azoxystrobin (0.0884 M), compound 6b (0.0544 M) and 6e (0.0823 M) displayed improved activity
, 6e, 6g, 6i, 7b and 7c were identified as the most active ones.
µ
µ
µ
against Botrytis cinerea. Further structural optimization of coumarin ring-opening derivatives is well
underway, with the aim to improve their levels of antifungal activity.

Molecules 2016, 21, 1387
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Acknowledgments: The project was supported by the National Natural Science Foundation of China (21272116)
and the Fundamental Research Funds for the Central Universities (KYTZ201604).
Author Contributions: Ming-Zhi Zhang and Wei-Hua Zhang conceived and designed the experiments; Yu Zhang
and Jia-Qun Wang performed the experiments; Ming-Zhi Zhang analyzed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the all target compounds are available from the authors.
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