Microwave-assisted synthesis and antifungal activity of novel fused Osthole derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.08.012

Based on the microwave-assisted synthetic protocol developed in our previous work, we have synthesized a series of novel furo[3,2-c]coumarins as fused Osthole derivatives, via the reaction of 4-hydroxycoumarins and β-ketoesters catalyzed by DMAP. All the target compounds were evaluated in?vitro for their antifungal activity against six phytopathogenic fungi, some compounds exhibited potential activity in the primary assays. Especially compounds 6c, 7b, 8b and 8c (shown in Fig.?1) were the most active ones, EC₅₀values of these four compounds against Colletotrichum capsica, Botrytis cinerea and Rhizoctonia solani were further investigated. 6c was identified as the most promising candidate with the EC₅₀value at 0.110?μM against Botrytis cinerea and 0.040?μM against Colletotrichum capsica, respectively, representing better antifungal activity than that of the commonly used fungicide Azoxystrobin.

Full text of DOI:10.1016/j.ejmech.2016.08.012

Accepted Manuscript
Microwave-assisted synthesis and antifungal activity of novel fused osthole
derivatives
Ming-Zhi Zhang, Rong-Rong Zhang, Jia-Qun Wang, Xiang Yu, Ya-Ling Zhang, Qing-
Qing Wang, Wei-Hua Zhang
PII:
S0223-5234(16)30655-9
10.1016/j.ejmech.2016.08.012
EJMECH 8808
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 June 2016
Revised Date: 29 July 2016
Accepted Date: 8 August 2016
Please cite this article as: 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 derivatives,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.012.
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ACCEPTED MANUSCRIPT
Microwave-assisted synthesis and antifungal activity of novel fused
Osthole derivatives
Ming-Zhi Zhang, Rong-Rong Zhang, Jia-Qun Wang, Xiang Yu, Ya-Ling Zhang,
Qing-Qing Wang, Wei-Hua Zhang*
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing
Agricultural University, Nanjing 210095, P.R. China
A series of novel furo[3,2-c]coumarins as fused Osthole derivatives were designed
and synthesized through an optimized microwave-assisted protocol. Some target
compounds exhibited potential activity in antifungal screening, and 6c was identified
as the most promising candidate with better antifungal activity than Azoxystrobin.

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Microwave-assisted synthesis and antifungal activity of novel fused Osthole
derivatives
Ming-Zhi Zhang, Rong-Rong Zhang, Jia-Qun Wang, Xiang Yu, Ya-Ling Zhang, Qing-Qing Wang,
Wei-Hua Zhang*
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University,
Nanjing 210095, P.R. China
Corresponding Author:
Prof. Wei-Hua Zhang
Nanjing Agricultural University, Nanjing 210095, P.R. China
E-mail: njzhangwh@126.com
Abstract: Based on the microwave-assisted synthetic protocol developed in our previous work, we
have synthesized a series of novel furo[3,2-c]coumarins as fused Osthole derivatives, via the
reaction of 4-hydroxycoumarins and β-ketoesters catalyzed by DMAP. All the target compounds
were evaluated in vitro for their antifungal activity against six phytopathogenic fungi, some
compounds exhibited potential activity in the primary assays. Especially compounds 6c, 7b, 8b and
8c (shown in Figure 1) were the most active ones, EC50 values of these four compounds against
Colletotrichum capsica, Botrytis cinerea and Rhizoctonia solani were further investigated. 6c was
identified as the most promising candidate with the EC50 value at 0.110 µM against Botrytis cinerea
and 0.040 µM against Colletotrichum capsica, respectively, representing better antifungal activity
than that of the commonly used fungicide Azoxystrobin.
Figure 1. Structures of the most active synthetic furo[3,2-c]coumarin derivatives
Key words : Osthole; Microwave-assisted; Furo[3,4-c]coumarin; Synthesis; Antifungal activity.

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1. Introduction
Osthole, a natural occurring coumarin derivative, was reported as the biologically active
ingredient of Cnidium Monnieri, a traditional Chinese herbal medicine. As shown in Figure 2,
Osthole is chemically known as an O-methylated coumarin, which is also found in many plants and
possesses a broad scope of pharmacological and biochemical activities[1-4], including
antiarrhythmia, antidiabetic, anticancer, antiosteoporosis, antiinflammatory, hepatoprotection and
neuroprotection. It has been widely used as a fungicide in China for a long history[5], displaying a
range of antifungal activities against Rhizoctonia solani and other phytopathogenic fungi[6].
Figure 2. Structures of osthole and furocoumarins
Recently, some novel structures and their antifungal activity have been reported[7-9], osthole
and its analogues have attracted great research interest for agricultural chemists, as it could be a
promising lead structure for exploiting new pesticides for the treatment of agricultural diseases[10,
11]. Meanwhile, osthole derivatives (Figure 2), such as furo[3,2-c]coumarin, exhibited interesting
biological acivities because of their capability to form covalent bonds with DNA and other
biological macromolecules[12], such as Wedelolactone, is a antihepatotoxic coumestan from
Eclipta prostrata[13], Neo-tanshinlactone showed significant inhibition against two estrogen
receptor positive human breast cancer cell lines[14, 15], while Angelicin is a furocoumarin
naturally occurring tricyclic aromatic compound, it is reported to exhibit anticancer, antiviral,
antiinflammatory activities[16-18].
In our previous work, we have accomplished various structural modifications to osthole,
including coumarino[8,7-e][1,3]oxazine derivatives[19], and novel fused coumarin analogues
pyrano[3,2-c]chromene-2,5-diones[20]. Bioscreening results showed that some of the synthesized
compounds exhibited effective control to certain phytopathogenic diseases. However, their potency
is too low to be used as agricultural fungicide. In a continuation of our studies aimed at the

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discovery of diversified analogues with improved antifungal activity, we now describe our
research which has focused on the synthesis and antifungal activity of novel furo[3,2-c]coumarins
(Figure 3).
Figure 3. Design strategy for target molecules
2. Materials and methods
2.1 Chemicals and Instruments
All chemicals including 2-Hydroxyacetophenone were purchased from commercial sources
(e.g., Crystal Chemicals) and used without further purification unless otherwise stated. The
microwave treatment was carried out in a microwave oven (WBFY-201, Gongyi Yuhua Instrument
Co., Ltd.) with an emission frequency of 2450 MHz and a maximum output power of 800 W. 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 derivatives were determined on an X-4 apparatus
(uncorrected), which was bought from Shanghai Tech. Infrared (IR) spectra were recorded on a
1
13
Bruker Tensor 27 spectrometer, and samples were prepared as KBr plates. H NMR and C NMR
spectra were obtained with a Bruker Avance 400 MHz spectrometer in CDCl3 solution with TMS as
an internal standard. 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.
2.1.1 General procedure for the preparation of compounds 4a-d (Scheme 1)
4-Hydroxycoumarins 4a-4d were synthesized through the reported synthetic route[20], yields for
4b, 4c, and 4d are 85%, 86%, and 82%, respectively.

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Scheme 1. Synthetic route for 4-Hydroxycoumarins 4a-4d
2.1.2 Microwave-assisted synthetic procedure for the preparation of compounds 5a-8d (Scheme 2)
Based on the method reported in our previous work[20], compounds 5-8 could be prepared in
moderate yields with toluene as the solvent, using 640W microwave irradiation, and DMAP as the
catalyst. The yields vary from 61% to 77% considerably depending upon the irradiative time, which
determined by the TLC monitor. After the reaction was complete, toluene was removed by
rotavapor, and the resulting residue was recrystallized from ethanol and water (10:1 in volume) or
purified by column chromatography. The experimental results were summarized in Table 1 reaction,
reaction yields were not optimized.
Scheme 2. Synthetic routes for the target compounds
Table 1. The structures and yields of target compounds 5a-8d
Product
Reaction Time
10min
R
Yield (%)
5a
5b
5c
H
65
77
67
15min
Methoxy
Allyloxy
10min

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5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
10min
10min
15min
15min
15min
10min
15min
15min
15min
15min
15min
15min
15min
n-Butoxy
H
63
67
73
65
61
69
73
65
62
67
73
65
61
Methoxy
Allyloxy
n-Butoxy
H
Methoxy
Allyloxy
n-Butoxy
H
Methoxy
Allyloxy
n-Butoxy
Yields after recrystallization or isolation by column chromatography.
2.1.3 Compounds data
3-Methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid ethyl ester（5a）：a white solid, mp:
140.2-140.6℃，1H NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 7.9, 1.4 Hz, 1H), 7.69 – 7.58 (m, 1H),
7.56 – 7.37 (m, 2H), 4.50 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.50 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, CDCl3 ) δ 159.01, 158.07, 157.79, 153.50, 141.85, 132.09, 130.31, 124.74, 121.90, 117.40,
112.18, 111.52, 61.45, 14.36, 10.15; IR (KBr) ν (cm-1) 1708, 1631, 1446, 1321, 1139, 1087;
HR-MS (ESI): m/z calcd for C15H12O5([M+H]+) 273.07630, found 273.07575.
7-Methoxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid ethyl ester（5b）：a white
solid, mp: 186.4-187.1℃，1H NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 7.9, 1.4 Hz, 1H), 7.69 – 7.58
13
(m, 1H), 7.56 – 7.37 (m, 2H), 4.50 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.50 (t, J = 7.1 Hz, 3H). C
NMR (101 MHz, CDCl3) δ 163.07, 159.14, 158.71, 158.10, 155.37, 141.03, 130.45, 122.95, 113.03,
109.18, 105.41, 101.37, 61.32, 55.85, 14.37, 10.17; IR (KBr) ν (cm-1) 1739, 1627, 1442, 1323, 1241,
963; HR-MS (ESI): m/z calcd for C16H14O6 ([M+H]+ ) 303.08686, found 303.08631.
7-Allyloxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid ethyl ester（5c）：a white
solid, mp: 155.5-156.2℃, 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.7 Hz, 1H), 7.00 (dd, J = 12.6,
3.8 Hz, 2H), 6.10 (ddd, J = 16.6, 10.4, 5.2 Hz, 1H), 5.45 (dd, J = 39.7, 13.9 Hz, 2H), 4.67 (d, J =
13
5.2 Hz, 2H), 4.49 (q, J = 7.1 Hz, 2H), 2.73 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H). C NMR (101 MHz,
CDCl3) δ 162.01, 159.14, 158.70, 158.11, 155.30, 141.08, 132.02, 130.45, 122.97, 118.66, 113.57,
109.25, 105.55, 102.25, 69.35, 61.31, 14.37, 10.17; IR (KBr) ν (cm-1) 1720, 1607, 1450, 1317, 1244,
1083, 994; HR-MS (ESI): m/z calcd for C18H16O6 ( [M + H]+ ) 329.10252, found 329.10196.

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7-Butoxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid ethyl ester（5d）：a white
solid, mp :189.3-192.3℃, 1H NMR (400 MHz, CDCl3) δ 8.07 (dd, J = 7.9, 1.4 Hz, 1H), 7.69 – 7.58
13
(m, 1H), 7.56 – 7.37 (m, 2H), 4.50 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.50 (t, J = 7.1 Hz, 3H). C
NMR (101 MHz, CDCl3) δ 173.09, 165.25, 162.73, 155.77, 155.45, 126.39, 122.94, 113.73, 113.48,
108.54, 101.87, 100.77, 68.54, 61.30, 30.95, 19.16, 14.38, 13.78, 10.20; IR (KBr) ν (cm-1) 1732,
1696, 1623, 1442, 1309, 1135, 962; HR-MS (ESI): m/z calcd for C19H20O6 ([M + H]+) 345.13382,
found 345.13331.
3-Methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid methyl este（r 6a）：a white solid, mp:
1
152.2-152.4℃, H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 26.3 Hz, 1H),
7.43 (dt, J = 34.3, 17.6 Hz, 2H), 4.04 (t, J = 16.4 Hz, 3H), 2.76 (t, J = 16.6 Hz, 3H). 13C NMR (101
MHz, CDCl3 ) δ 159.34, 158.12, 157.72, 153.50, 141.59, 132.16, 130.64, 124.78, 121.85, 117.41,
112.11, 111.49, 52.21, 10.12; IR (KBr) ν (cm-1) 1700, 1623, 1446,1325, 1232, 1131, 1083; HR-MS
(ESI): m/z calcd for C14H10O5 ([M+H]+) 259.06065, found 259.06010.
7-Methoxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid methyl ester（6b）：a
white solid, mp: 216.0℃, 1H NMR (500 MHz, CDCl3) δ 7.91 (d, J = 8.6 Hz, 1H), 7.31 (t, J = 14.7
Hz, 1H), 6.96 (d, J = 11.6 Hz, 1H), 4.01 (dd, J = 17.9, 11.4 Hz, 3H), 3.95 – 3.88 (m, 3H), 2.72 (dd,
J = 17.9, 11.4 Hz, 3H). 13C NMR (101 MHz, CDCl3 ) δ 163.15, 159.51, 158.83, 158.09, 155.45,
140.83, 130.83, 122.95, 113.10, 109.21, 105.41, 101.43, 55.87, 52.12, 10.16; IR (KBr) ν (cm-1)
1736, 1699, 1617, 1445, 1325, 1235, 1082, 960; HR-MS (ESI): m/z calcd for C15H10O5 ([M + H]+)
289.07122, found 289.07066.
7-Allyloxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid methyl ester（6c）: a
white solid, mp: 151.0-154.9℃, 1H NMR (500 MHz, CDCl3) δ 7.98 – 7.85 (m, 1H), 7.04 – 6.89 (m,
2H), 6.12 – 5.99 (m, 1H), 5.43 (dd, J = 49.7, 13.8 Hz, 2H), 4.65 (s, 2H), 4.07 – 3.94 (m, 3H), 2.77 –
2.65 (m, 3H). 13C NMR (101 MHz, CDCl3 ) δ 162.05, 159.46, 158.73, 158.02, 155.31, 140.82,
132.00, 130.79, 122.91, 118.74, 113.55, 109.22, 105.47, 102.25, 69.46, 52.09, 10.13; IR (KBr) ν
(cm-1) 1732, 1623, 1446, 1333, 1260, 1091, 994; HR-MS (ESI): m/z calcd for C17H14O6 ([M + H]+)
315.08686, found 315.08631.
7-Butoxy-3-methyl-4-oxo-4H-furo[3,2-c]chromene-2-carboxylic acid methyl ester（6d）：a white
solid, mp: 145.9-146.8℃, 1H NMR (500 MHz, CDCl3) δ 7.90 (dd, J = 8.6, 3.5 Hz, 1H), 6.94 (dd, J
= 11.6, 5.9 Hz, 2H), 4.10 – 4.03 (m, 2H), 4.00 (d, J = 3.5 Hz, 3H), 2.70 (t, J = 9.4 Hz, 3H), 1.84 (s,
13
2H), 1.56 (dd, J = 16.8, 5.8 Hz, 3H), 1.10 – 0.96 (m, 3H). C NMR (101 MHz, CDCl3) δ 159.34,

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158.12, 157.72, 153.50, 141.59, 132.16, 130.64, 124.78, 121.85, 117.41, 112.11, 111.49, 68.46,
52.21, 30.98, 19.18, 13.80, 10.12; IR (KBr) ν (cm-1) 1758, 1711, 1614, 1453, 1329, 1132, 999;
HR-MS (ESI): m/z calcd for C18H18O6 ([M + H]+) 331.11816, found 331.11761.
2-Acetyl-furo[3,2-c]chromen-4-one（7a）: a white solid, mp: 178.7-179.5℃, 1H NMR (400 MHz,
CDCl3) δ 7.98 (dd, J = 7.8, 1.2 Hz, 1H), 7.66 – 7.56 (m, 1H), 7.52 – 7.35 (m, 2H), 2.75 (s, 3H), 2.65
13
(s, 3H). C NMR (101 MHz, CDCl3 ) δ 188.73, 157.79, 157.33, 153.66, 149.14, 132.28, 129.54,
124.84, 121.57, 117.54, 112.12, 112.04, 27.66, 10.24; IR (KBr) ν (cm-1) 1739, 1683, 1623, 1548,
1442, 1360, 1229, 1185, 994; HR-MS (ESI): m/z calcd for C14H10O4 ([M+H]+) 243.06573, found
243.06519.
1
2-Acetyl-7-methoxy-furo[3,2-c]chromen-4-one(7b): a white solid, mp: 200.9-203.2℃, H NMR
(400 MHz, CDCl3) δ 7.87 (d, J = 8.6 Hz, 1H), 7.03 – 6.90 (m, 2H), 3.96 – 3.87 (m, 3H), 2.74 (s,
3H), 2.62 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 188.56, 163.23, 158.06, 158.03, 155.61, 148.61,
129.77, 122.62, 113.22, 109.69, 105.35, 101.46, 55.95, 27.60, 10.28; IR (KBr) ν (cm-1) 1744, 1627,
1546, 1438, 1276, 1087; HR-MS (ESI): m/z calcd for C15H12O5 ([M+H]+) 273.07630, found
273.007575.
1
2-Acetyl-7-allyloxy-furo[3,2-c]chromen-4-one(7c): a white solid, mp: 146.2-146.3℃, H NMR
(500 MHz, CDCl3) δ 7.83 (d, J = 8.7 Hz, 1H), 6.99 – 6.87 (m, 2H), 4.05 (t, J = 6.5 Hz, 2H), 2.77 –
2.66 (m, 3H), 2.58 (d, J = 17.9 Hz, 3H), 1.87 – 1.76 (m, 2H), 1.52 (dt, J = 14.8, 7.5 Hz, 2H), 1.00 (t,
13
J = 7.4 Hz, 3H). C NMR (101 MHz, CDCl3 ) δ 188.58, 162.14, 158.10, 158.04, 155.50, 148.61,
131.95, 129.76, 122.63, 118.76, 113.73, 109.72, 105.44, 102.30, 69.39, 27.62, 10.29; IR (KBr) ν
(cm-1) 1736, 1607, 1438, 1333, 1252, 1099, 998; HR-MS (ESI): m/z calcd for C17H14O5 ([M+H]+)
289.09195, found 289.09140.
2-Acetyl-7-butoxy-furo[3,2-c]chromen-4-one(7d): a white solid, mp: 149.7℃, 1H NMR (500 MHz,
CDCl3) δ 7.83 (d, J = 8.7 Hz, 1H), 6.99 – 6.87 (m, 2H), 4.05 (t, J = 6.5 Hz, 2H), 2.77 – 2.66 (m,
3H), 2.58 (d, J = 17.9 Hz, 3H), 1.87 – 1.76 (m, 2H), 1.52 (dt, J = 14.8, 7.5 Hz, 2H), 1.00 (t, J = 7.4
13
Hz, 3H). C NMR (101 MHz, CDCl3) δ 188.52, 162.84, 158.12, 158.09, 155.62, 148.56, 129.78,
122.54, 113.59, 109.58, 105.11, 101.89, 68.50, 30.98, 27.58, 19.18, 13.80, 10.28; IR (KBr) ν (cm-1)
1744, 1667, 1627, 1595, 1458, 1256, 1011; HR-MS (ESI): m/z calcd for C18H18O5 ([M+H]+)
315.12325, found 315.12270.
1
3-Phenyl-furo[3,2-c]chromen-4-one(8a): a white solid, mp: 209.1-210.2℃, H NMR (500 MHz,
CDCl3) δ 7.95 (dd, J = 7.8, 1.2 Hz, 1H), 7.80 (dd, J = 6.3, 2.0 Hz, 2H), 7.57 (ddd, J = 8.8, 7.3, 1.5

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Hz, 1H), 7.51 – 7.47 (m, 3H), 7.44 – 7.38 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 158.80, 157.86,
152.61, 141.28, 130.97, 129.09, 128.68, 128.59, 128.39, 126.74, 124.51, 120.99, 117.13, 112.77,
108.48; IR (KBr) ν (cm-1) 1732, 1627, 1490, 1438, 1317, 1079, 962; HR-MS (ESI): m/z calcd for
C17H10O3 ([M+H]+) 263.07082, found 263.07027.
1
7-Methoxy-3-phenyl-furo[3,2-c]chromen-4-one(8b): a white solid, mp: 162.1-162.4 , H NMR
(500 MHz, CDCl3) δ 7.95 (dd, J = 7.8, 1.2 Hz, 1H), 7.80 (dd, J = 6.3, 2.0 Hz, 2H), 7.57 (ddd, J =
8.8, 7.3, 1.5 Hz, 1H), 7.51 – 7.47 (m, 3H), 7.44 – 7.38 (m, 3H)，13C NMR (101 MHz, CDCl3) δ
162.18, 159.46, 158.17, 154.39, 140.38, 129.29, 128.62, 128.54, 128.27, 126.47, 121.95, 112.88,
106.14, 106.05, 101.04, 55.78; IR (KBr) ν (cm-1) 1732, 1627, 1454, 1337, 1236, 1131; HR-MS
(ESI): m/z calcd for C18H12O4 ([M + H]+) 293.08139, found 293.08084.
1
7-Allyloxy-3-phenyl-furo[3,2-c]chromen-4-one(8c): a white solid, mp: 167.4-168.5℃, H NMR
(500 MHz, CDCl3) δ 7.80 (ddd, J = 14.9, 11.1, 6.3 Hz, 3H), 7.72 (d, J = 1.7 Hz, 1H), 7.53 – 7.38 (m,
3H), 6.99 (td, J = 4.6, 2.3 Hz, 2H), 6.09 (ddt, J = 17.3, 10.6, 5.3 Hz, 1H), 5.55 – 5.34 (m, 2H), 4.69
– 4.62 (m, 2H), 13C NMR (101 MHz, CDCl3) δ 161.11, 159.44, 158.14, 154.31, 140.40, 132.22,
129.29, 128.62, 128.54, 128.27, 126.49, 121.95, 118.55, 113.41, 106.29, 106.13, 101.96, 69.29; IR
(KBr) ν (cm-1) 1739, 1611, 1451, 1232, 1138, 1068, 941; HR-MS (ESI): m/z calcd for C20H14O4
([M+H]+) 319.09704, found 319.09649.
1
7-Butoxy-3-phenyl-furo[3,2-c]chromen-4-one(8d): a white solid, mp: 182.0-182.1℃, H NMR
(500 MHz, CDCl3) δ 7.84 – 7.77 (m, 3H), 7.71 (d, J = 1.6 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.44 – 7.38
(m, 1H), 6.98 – 6.94 (m, 2H), 4.06 (t, J = 6.5 Hz, 2H), 1.91 – 1.79 (m, 2H), 1.55 (td, J = 14.8, 7.3
Hz, 3H), 1.06 – 0.97 (m, 3H), 13C NMR (101 MHz, CDCl3) δ 161.80, 159.56, 158.20, 154.44,
140.30, 132.17, 129.35, 128.62, 128.52, 128.24, 126.49, 121.87, 113.29, 105.97, 101.54, 68.36,
31.04, 19.21, 13.83; IR (KBr) ν (cm-1) 1728, 1623, 1510, 1446, 1272, 1244; HR-MS (ESI): m/z
calcd for C21H18O4 ([M+H]+) 335.12834, found 335.12779.
2.2. X-ray diffraction analysis
White crystals of compound 5b (0.280 × 0.260 × 0.220 mm3) were mounted on a quartz fiber
with protection oil. Cell dimensions and intensities were measured at 296 K on a Bruker SMART
APEX2 CCD area detector diffractometer with graphite monochromated MoKα radiation (λ=
0.71073 Å); θmax = 25.010; 2016 independent reflections (Rint = 0.0408). The structure was solved
by direct methods using SHELXS-97; all other calculations were performed with Bruker SAINT
System and Bruker SMART programs. Full-matrix least-squares refinement based on F2 using the

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weight of ω= 1/[σ2(Fo2) + (0.0659P)2 + 0.0500P] gave final values of R = 0.0408, ωR = 0.1331, in
the title compound, the moiety formula is C16H14O6.
White crystals of compound 7a (0.500 × 0.480 × 0.360 mm3) were mounted on a quartz fiber
with protection oil. Cell dimensions and intensities were measured at 296 K on a Bruker SMART
APEX2 CCD area detector diffractometer with graphite monochromated MoKα radiation (λ=
0.71073 Å); θmax = 27.517;1993 independent reflections (Rint = 0.0453).Data were corrected for
Lorentz and polarization effects and for absorption (Tmin = 0.942; Tmax = 0.961). The structure was
solved by direct methods using SHELXS-97; all other calculations were performed with Bruker
SAINT System and Bruker SMART programs. Full-matrix least-squares refinement based on F2
using the weight of ω= 1/[σ2(Fo2) + (0.0633P)2 + 0.4557P] gave final values of R = 0.0453, ωR =
0.1377, max/min residual electron density = 0.205 and -0.171 e.Å-3. Hydrogen atoms were observed
and refined with a fixed value of their isotropic displacement parameter. In the title compound, the
moiety formula is C14H10O4.
The crystallographic data have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication number: CCDC 1055787 (5b), CCDC 1417754 (7a),
Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB12 1EZ, UK (Fax + 441223336033 or E-mail: deposit@ccdc.cam.ac.uk). The
structures are shown in Figure 4.
Compound 5b
Compound 7a
Figure 4. X-ray structures of compounds 5b and 7a
2.3. Biological assays

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The antifungal activities of all the synthesized target compounds were carried out at the
concentration of 50 ꢀg/mL using mycelia growth inhibitory rate methods, with Azoxystrobin used
as the positive control. The tested 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 US, the detailed procedure of experimental methods for the antifungal activity refer to
the paper from Department of Plant Pathology, Nanjing Agriculture University[21]. After retrieval
from the storage tube, the strains were then incubated in PDA at 25
for a week to get new
mycelia for the antifungal assay. All the test compounds were first dissolved in 5 mL DMF to
generate a 100 ꢀg/mL stock solution. The needed test solutions were prepared by diluting the above
solution. The antifungal results of all the compounds against Botrytis cinerea, Collecterichu
mcapsica, Alternaria solani, Gibberella zeae, Cucumber anthrax and Rhizoctorzia solani are listed
in Table 2.
Table 2. Antifungal activity of coumarin analogues (inhibitory rate, %)
Species a
BOT
50
COL
50
ALT
50
GIB
50
CUC
50
RHI
50
Compd Rate (ꢀg/mL)
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
10.7 b
11.8
10.1
12.8
20.7
22.6
54.2
14.1
25.7
40.8
22.1
20.7
16.1
27.1
15.7
20.7
31.7
25.7
31.4
20.1
32.9
24.3
65.7
21.4
32.9
35.7
24.5
21.7
30.1
34.7
35.7
20.1
10.0
10.0
10.0
10.0
10.0
10.0
14.2
10.0
10.1
10.1
10.0
10.0
10.0
10.0
10.0
10.0
13.2
11.8
14.7
13.2
16.7
11.8
41.2
11.8
16.2
23.5
11.8
11.8
11.8
11.8
14.7
27.5
20.1
11.4
15.7
18.6
18.6
15.7
27.1
15.7
15.7
21.4
25.7
15.7
22.9
20.1
22.9
20.1
33.4
23.8
30.2
22.8
34.7
37.1
54.7
24.7
32.8
64.3
33.2
27.4
33.8
47.8
42.9
33.8

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Azoxystrobin
33.8
43.6
15.7
67.6
82.9
62.9
a BOT, Botrytis cinerea; COL, Collecterichum capsica; ALT, Alternaria solani; GIB, Gibberella zeae; CUC,
Cucumber anthrax; RHI, Rhizoctorzia solani.
b All the data was the average value of three replications, 10.0 indicate the data below 10% inhibitory.
Table 3. EC50 determination of compounds 6c, 7c, 8b and 8c
95% confidence
Pathogen
Compound
Toxic regression
R
EC50 (µM)
interval
6c
7b
8b
8c
Y=4.0226+0.6349x
Y=3.0842+0.9859x
Y=3.3405+0.7564x
Y=3.2146+0.6974x
0.9996
0.9999
0.9995
0.9994
0.9997
0.9998
0.9998
0.9999
1.0000
0.9985
0.9996
0.9995
0.9999
0.9998
0.9995
0.110
0.322
0.533
1.140
0.158
0.040
0.362
0.584
0.518
0.222
0.110
0.051
0.203
0.316
0.053
0.1069-0.1136
0.3139-0.3312
0.482-0.590
Botrytis cinerea
0.978-1.325
Azoxystrobin Y=3.0886+1.0598x
0.1453-0.1708
0.0385-0.0414
0.3492-0.3947
0.5697-0.5981
0.5112-0.5253
0.1936-0.2543
0.1069-0.1136
0.0485-0.0537
0.1999-0.2065
0.3049-0.3263
0.0506-0.0548
6c
7b
8b
8c
Y=4.2672+0.6668x
Y=3.7545+0.6248x
Y=3.3566+0.7364x
Y=3.4286+0.7088x
Collecterichum
capsica
Azoxystrobin Y=3.9631+0.5312x
6c
7b
8b
8c
Y=3.6804+0.8438x
Y=4.2512+0.6556x
Y=3.5907+0.7947x
Y=3.8061+0.5964x
Rhizoctorzia
solani
Azoxystrobin Y=3.8654+0.8546x
The EC50 value was the average value of three replications.
3. Results and discussion
3.1 Synthetic chemistry
In our previous study, the molecule annulation at the 3, 4 position of coumarin structure
resulting in Pyrano[3,2-c]coumarin derivatives was reported, the application of microwave
irradiation has been employed as an efficient and green synthetic approach which dramatically
reduces reaction time. Based on our aim to discover novel Osthole analogues with improved
antifungal activity, our previous research was focused on a series of fused coumarin analogues:

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pyrano[3,2-c]chromene-2,5-diones. In this study, we design and synthesize furo[3,2-c]coumarin as
the scaffold for the further structural modification. With the optimized microwave-assisted synthetic
method, some β-ketoesters including 3-chloropentane-2,4-dione, ethyl 2-chloroacetoacetate, methyl
2-chloro-3-oxobutanoate and 2-bromoacetophenone were employed to react with
4-Hydroxycoumarins to extend the variety of furocoumarin derivatives, then a focused library of 16
compounds diversified on furan ring and 7-position of coumarin was set up for the antifungal
screening.
All of the target molecules listed in Table 1 were characterized by IR, 1H NMR, 13C NMR and
HR-MS. In addition, the structures of two typical compounds 5b and 7a were further confirmed by
X-ray crystallographic diffraction analysis (Figure 4).
3.2 Antifungal activity and the structure-activity relationships (SAR)
The results of the antifungal activity screening against six phytopathogenic fungi are shown in
Table 2. For the purposes of analysis of structure-activity relationships, the antifungal activity of all
target compounds were compared with the positive control Azoxystrobin, a broad spectrum
fungicide used for protecting plants and food crops from fungal diseases.
In general, data presented in Table 2 indicate that the synthesized furocoumarin derivatives
exhibit certain activities against Botrytis cinerea, Collecterichumcapsica and Rhizoctorzia solani at
50 ꢀg/mL, while, most of the target compounds showed rather poor activity against Alternaria
solani, Gibberella zeae and Cucumber anthrax.
As four compounds (6c, 7b, 8b and 8c) showed relatively effective control against Botrytis
cinerea, Colletotrichum capsica and Rhizoctorzia solani, therefore, we further tested the EC50
values of these compounds together with Azoxystrobin. As shown in Table 3, it was noticed that the
EC50 values of compounds 6c was as low as 0.110 and 0.040 µM against Botrytis cinerea and
Colletotrichum capsica, respectively, which proves it’s more effective than Azoxystrobin (0.158
µM). Compound 7b (0.051 µM) exhibited slightly better activity than the control Azoxystrobin
(0.053 µM) against Rhizoctorzia solani.
Although the antifungal activity for most synthesized derivatives has been proven to be
slightly poor, making it hard to extract a distinct structure-activity relationship analysis, we still can
draw some broad conclusions from the presented data. Firstly, these fused coumarin compounds

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were noticeably more active against Botrytis cinerea, Colletotrichum capsici and Rhizoctorzia
solani, but they usually lacked potency against Alternaria solani, Gibberella zeae and Cucumber
anthrax, as illustrated by the absence of activity against these three kinds of fungi. Secondly, the
alkoxy group on the 7-position of coumarin is essential for the antifungal activity, as compounds
(5a, 6a, 7a and 8a) showed very weak activity or even totally inactive against the tested
phytopathogenic fungi, however, their equivalents with methoxy, allyloxy or n-butoxy on 7-position
of the coumarin ring showed greatly improved antifungal activity, highlighted by compounds 6c, 7b,
8b and 8c. Thirdly, though the influence of substituents on the furan ring is not clear due to the
limited diversity, we still noticed that the antifungal activity of ethyl ester (5a-5d) is usually weaker
than their equivalents with methyl ester (6a-6d) or acetyl group (7a-7d).
4. Conclusions
In summary, aiming to discover novel Osthole analogues with improved antifungal activity, we
have efficiently synthesized a series of novel furo[3,2-c]coumarins through the microwave-assisted
protocol developed in our previous research. Biological testing data showed that the fused Osthole
analogues displayed an altered pattern of biological activity, compounds 6c, 7b, 8b and 8c were
identified as the most active ones, the EC50 values of these compounds together with Azoxystrobin
were further tested. Compared to Azoxystrobin, compound 6c displayed improved activity against
Botrytis cinerea, Collecterichum capsica, and equivalent activity against Rhizoctorzia solani,
compound 7b also showed slightly better activity against Rhizoctorzia solani. Compound 6c was
identified as the most promising candidate for further study. Further structural optimization of fused
Osthole analogues is well underway, alongside more detailed biological testing of the most active
compounds, with the aim to improve their levels of antifungal activity.
Acknowledgement
The project was supported by the National Natural Science Foundation of China (21272116) and
the Fundamental Research Funds for the Central Universities (KYTZ201604). We thank Dr. Fenger
Zhou from Chemistry Department at Texas A&M University (US) for his kind help on the
manuscript revision.

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Figure 1. Structures of the most active synthetic furo[3,2-c]coumarin derivatives
Figure 2. Structures of osthole and furocoumarins
Figure 3. Design strategy for target molecules
Figure 4. X-ray structures of compounds 5b and 7a
Scheme 1. Synthetic route for 4-Hydroxycoumarins 4a-4d
Scheme 2. Synthetic routes for the target compounds
Table 1. The structures and yields of target compounds 5a-8d
Table 2. Antifungal activity of coumarin analogues (inhibitory rate, %)
Table 3. EC50 determination of compounds 6c, 7c, 8b and 8c

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Figure 1. Structures of the most active synthetic furo[3,2-c]coumarin derivatives
Figure 2. Structures of osthole and furocoumarins
Figure 3. Design strategy for target molecules
Compound 5b
Compound 7a
Figure 4. X-ray structures of compounds 5b and 7a

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Scheme 1. Synthetic route for 4-Hydroxycoumarins 4a-4d
Scheme 2. Synthetic routes for the target compounds

ACCEPTED MANUSCRIPT
Table 1. The structures and yields of target compounds 5a-8d
Product
5a
Reaction Time
10min
15min
10min
10min
10min
15min
15min
15min
10min
15min
15min
15min
15min
15min
15min
15min
R
Yield (%)
65
H
5b
Methoxy
Allyloxy
n-Butoxy
H
77
5c
67
5d
63
6a
67
6b
Methoxy
Allyloxy
n-Butoxy
H
73
6c
65
6d
61
7a
69
7b
Methoxy
Allyloxy
n-Butoxy
H
73
7c
65
7d
62
8a
67
8b
Methoxy
Allyloxy
n-Butoxy
73
8c
65
8d
61
Yields after recrystallization or isolation by column chromatography.
Table 2. Antifungal activity of coumarin analogues (inhibitory rate, %)
Species a
BOT
50
COL
50
ALT
50
GIB
50
CUC
50
RHI
50
Compd Rate (ꢀg/mL)
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
10.7 b
11.8
10.1
12.8
20.7
22.6
54.2
14.1
25.7
40.8
22.1
20.7
31.7
25.7
31.4
20.1
32.9
24.3
65.7
21.4
32.9
35.7
24.5
21.7
10.0
10.0
10.0
10.0
10.0
10.0
14.2
10.0
10.1
10.1
10.0
10.0
13.2
11.8
14.7
13.2
16.7
11.8
41.2
11.8
16.2
23.5
11.8
11.8
20.1
11.4
15.7
18.6
18.6
15.7
27.1
15.7
15.7
21.4
25.7
15.7
33.4
23.8
30.2
22.8
34.7
37.1
54.7
24.7
32.8
64.3
33.2
27.4

ACCEPTED MANUSCRIPT
8a
16.1
27.1
15.7
20.7
33.8
30.1
34.7
35.7
20.1
43.6
10.0
10.0
10.0
10.0
15.7
11.8
11.8
14.7
27.5
67.6
22.9
20.1
22.9
20.1
82.9
33.8
47.8
42.9
33.8
62.9
8b
8c
8d
Azoxystrobin
a BOT, Botrytis cinerea; COL, Collecterichum capsica; ALT, Alternaria solani; GIB, Gibberella zeae; CUC,
Cucumber anthrax; RHI, Rhizoctorzia solani.
b All the data was the average value of three replications, 10.0 indicate the data below 10% inhibitory.
Table 3. EC50 determination of compounds 6c, 7c, 8b and 8c
95% confidence
Pathogen
Compound
Toxic regression
R
EC50 (µM)
interval
6c
7b
8b
8c
Y=4.0226+0.6349x
Y=3.0842+0.9859x
Y=3.3405+0.7564x
Y=3.2146+0.6974x
0.9996
0.9999
0.9995
0.9994
0.9997
0.9998
0.9998
0.9999
1.0000
0.9985
0.9996
0.9995
0.9999
0.9998
0.9995
0.110
0.322
0.533
1.140
0.158
0.040
0.362
0.584
0.518
0.222
0.110
0.051
0.203
0.316
0.053
0.1069-0.1136
0.3139-0.3312
0.482-0.590
Botrytis cinerea
0.978-1.325
Azoxystrobin Y=3.0886+1.0598x
0.1453-0.1708
0.0385-0.0414
0.3492-0.3947
0.5697-0.5981
0.5112-0.5253
0.1936-0.2543
0.1069-0.1136
0.0485-0.0537
0.1999-0.2065
0.3049-0.3263
0.0506-0.0548
6c
7b
8b
8c
Y=4.2672+0.6668x
Y=3.7545+0.6248x
Y=3.3566+0.7364x
Y=3.4286+0.7088x
Collecterichum
capsica
Azoxystrobin Y=3.9631+0.5312x
6c
7b
8b
8c
Y=3.6804+0.8438x
Y=4.2512+0.6556x
Y=3.5907+0.7947x
Y=3.8061+0.5964x
Rhizoctorzia
solani
Azoxystrobin Y=3.8654+0.8546x
The EC50 value was the average value of three replications.

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Highlights
The microwave-assisted synthesis of novel furo[3,2-c]coumarins was described.
Antifungal activity screening of the designed compounds against six fungi was
screened.
Some compounds exhibited potent antifungal activity with 6c as the most promising
candidate for further study.
The SAR of synthesized furo[3,2-c]coumarins were summarized.