Microwave-assisted synthesis and antifungal activity of novel coumarin derivatives: Pyrano[3,2-c]chromene-2,5-diones

Article abstract of DOI:10.1016/j.ejmech.2016.03.069

A series of novel fused coumarin analogues pyrano[3,2-c]chromene-2,5-diones have been synthesized through an optimized microwave-assisted protocol. All target compounds were tested and evaluated for their antifungal activity against Botrytis cinerea, Colletotrichum copsica, Alternaria solani, Gibberella zeae and Rhizoctorzia solani. The bioassay results indicated that some of the compounds exhibited potent antifungal activities at concentration less than 50 ppm. For the compounds 5d, 6c and 7b, EC₅₀ values against B. cinerea were as low as 0.141, 0.082 and 0.091 μM, respectively, which represents better antifungal activity than that of the commonly used fungicide Azoxystrobin. Compounds 5d (57%) and 6c (55%) also exhibited more effective control than Azoxystrobin (44%) against Colletotrichum capsica.

Full text of DOI:10.1016/j.ejmech.2016.03.069

Accepted Manuscript
Microwave-Assisted Synthesis and Antifungal Activity of Novel Coumarin Derivatives:
Pyrano[3,2-c]chromene-2,5-diones
Rong-Rong Zhang, Jia Liu, Yu Zhang, Meng-Qing Hou, Ming-Zhi Zhang, Fenger
Zhou, Wei-Hua Zhang
PII:
S0223-5234(16)30251-3
10.1016/j.ejmech.2016.03.069
EJMECH 8494
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2015
Revised Date: 22 March 2016
Accepted Date: 25 March 2016
Please cite this article as: 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, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.069.
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ACCEPTED MANUSCRIPT
Microwave-Assisted Synthesis and Antifungal Activity of Novel
Coumarin Derivatives: Pyrano[3,2-c]chromene-2,5-diones
Rong-Rong Zhang,a Jia Liu,a Yu Zhang,a Meng-Qing Hou,a Ming-Zhi Zhang,a,*
Fenger Zhou,b Wei-Hua Zhang a,*
A series of novel fused coumarin derivatives were designed and synthesized through
an optimized microwave-assisted protocol. Antifungal activity screening against five
phytopathogenic fungi led to the identification of compound 5d as the most promising
candidate for further study.

ACCEPTED MANUSCRIPT
Microwave-Assisted Synthesis and Antifungal Activity of Novel Coumarin
Derivatives: Pyrano[3,2-c]chromene-2,5-diones
Rong-Rong Zhang,a Jia Liu,a Yu Zhang,a Meng-Qing Hou,a Ming-Zhi Zhang,a,* Fenger Zhou,b
Wei-Hua Zhang a,*
a Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing
210095, P.R. China
b Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843, United States
Corresponding Authors:
Dr. Ming-Zhi Zhang, E-mail: mzzhang@njau.edu.cn
Prof. Wei-Hua Zhang, E-mail: njzhangwh@126.com
Nanjing Agricultural University, Nanjing 210095, P.R. China
Abstract:
A series of novel fused coumarin analogues pyrano[3,2-c]chromene-2,5-diones have been
synthesized through an optimized microwave-assisted protocol. All target compounds were tested
and evaluated for their antifungal activity against Botrytis cinerea, Colletotrichum copsica,
Alternaria solani, Gibberella zeae and Rhizoctorzia solani. The bioassay results indicated that
some of the compounds exhibited potent antifungal activities at concentration less than 50 ppm.
For the compounds 5d, 6c and 7b, EC50 values against Botrytis cinerea were as low as 0.141, 0.082
and 0.091 µM, respectively, which represents better antifungal activity than that of the commonly
used fungicide Azoxystrobin. Compounds 5d (57%) and 6c (55%) also exhibited more effective
control than Azoxystrobin (44%) against Colletotrichum capsica.
KEY WORDS: coumarin derivatives; microwave-assisted; synthesis; antifungal activity
1. Introduction
Coumarin, also known as benzopyran-2-one, is a fragrant organic compound which is found
naturally in some plants. These include the Tonka bean, which is known by the French as
coumarou, and is one of the sources from which coumarin was first isolated as a natural product by

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A. Vogel in 1820[1, 2]. Since coumarin was first synthesized by the English chemist William
Henry Perkin in 1868[3], the application of coumarin derivatives as bioactive molecules against
different kinds of diseases has gained great interest from medicinal chemists. Coumarin derivatives
demonstrate a wide spectrum of biological activities such as anticancer, anticoagulant,
anti-inflammatory, and are usually associated with low toxicity[4-9]. The diversity of bioactivities
among coumarin compounds is so huge that coumarin is described as one of the “privileged
scaffolds”, a term first introduced by Evans to define structures that can bind more than one
receptor[10].
As the structural core, coumarin is used regularly as a scaffold in medicinal and agricultural
chemistry. This is highlighted by Warfarin and Acenocoumarol (shown in Figure 1), which are
anticoagulant agents that function as vitamin K antagonists, and are normally used in the
prevention of thrombosis and thromboembolism[11, 12]. Owing to the vast number of
coumarin-containing molecules in the literature, this paper is primarily focused on antifungal
agents, and serves as a mini-review of currently published coumarin antifungal agents, as limited
reports have been published in this area.
As shown in Figure 1, Osthole is a natural O-methylated coumarin found in many plants and
has a broad scope of antifungal activities against Rhizoctonia solani and other phytopathogenic
fungi[13]. It has also been widely used as a fungicide in China for a long history[14]. The recently
reported compound Cou-NO2 (7-hydroxy-6-nitro-2H-1-benzopyran-2-one), a biosynthetic
coumarin derivative, has been reported for its antifungal activity, and exhibits bioactivity against
Aspergillus mycelial growth and spore germination[15]. Hemiarin (7-methoxycoumarin, or
ayapanin) and Scopoletin are inducible antifungal compounds, and can be considered as defense
tools for plants against pathogenic fungi. Xanthotoxin, a common methoxylated furanocoumarin,
is reported to exhibit activity in darkness against Candida albicans and Cryptococcus laurentii[16].
Mammeisin, a coumarin derivative isolated from Kielmeyera elata, is reported to present a
minimum inhibitory concentration (MIC) value similar to ketoconazole and display a better result
than fluconazole against Candida tropicalis, which is the most common cause of human fungal
infections worldwide[17]. Coumoxystrobin (shown in Figure 1) is a coumarin derivative
containing (E)-methyl 3-methoxy-2-phenylacrylate with a broad spectrum of antifungal

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activity[18]. Excavarin-A, a new γ-lactone coumarin, shows antifungal activity against fifteen
fungal strains that are pathogenic against plants and humans. It is also stronger than a standard
antimicrobial nystatin, against some clinically important pathogens, such as A. fumigatus, C.
tropicalis and M. circinelloides. Additionally, it is more potent than the standard fungicide bavistin
against some plant pathogenic fungi, such as R. solani and F. oxysporum[19].
The presence of the coumarin moiety is known to confer resistance in plant tissues against
microbial attack. Since the antifungal activity of coumarin and its derivatives are mainly
determined by the functional groups and positions of substituents on the benzopyran ring, various
structural modifications to coumarin and their biological activities have been reported. For example,
alkylcoumarins (3-, 4- and 6-alkyl) are reported to display antifungal activities[20, 21], and the
derivatives of 4-methylcoumarin (Compound A in Scheme 1) especially showed good antifungal
activities against different kinds of plant pathogens. 7-hydroxycoumarin (Umbelliferone) and its
derivatives (Compound B in Scheme 1) have also been reported to possess antifungal activities
against spore germination and hyphal growth of many fungi[22]. 4-Phenoxymethylcoumarins and
their in vitro growth-inhibiting activities against various plant pathogenic fungi have been
presented in a series of studies on coumarin based pesticides, and 4-(4-tert-butylphenoxy)methyl
analogues (Compound C in Scheme 1) revealed the highest toxicity towards a majority of the
tested fungi[23].
Only Bharat B. Gupta and coworkers have previously described the synthesis and antifungal
activity
of
some
fused
coumarin
compounds,
such
as
2-ethyl-7,8-dihydro-3-hydroxynaphtho[l,2-c]chromen-6-one (Compound D in Scheme 1), which
possessed effective control with EC50 values ranging from 0.2 to 2.5 µg/mL against five tested
phytopathogenic fungi in culture[24]. Other than this, no further structural modification and
antifungal activity of fused coumarin analogues have been reported so far. The demand to improve
antifungal activity necessity the discovery of novel fused coumarin analogues. Herein we report
our work focused on a series of fused coumarin analogues: pyrano[3,2-c]chromene-2,5-diones.
2. Materials and methods
2.1 Chemicals and Instruments

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All chemicals including 2'-Hydroxyacetophenone (3a) were purchased from commercial
sources (e.g., Adamas, Crystal Chemicals) and used without further purification unless otherwise
stated. The melting points of the ester derivatives of coumarin derivatives were determined on an
1
13
X-4 apparatus (uncorrected), which was purchased from Shanghai Tech. H NMR and C NMR
spectra were obtained using 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. Most reaction yields were not optimized.
General procedure for the preparation of compounds 2a, 3b-d (Scheme 2)
2,4-dihydroxyacetophenone 2a was synthesized through the reported method[25].
To a solution of 50 mL acetone in 250 mL one-neck flask, was added KI (0.1g) and K2CO3
(0.1g), followed by 13 mmol of 2,4-dihydroxyacetophenone 2a. Once 2a was completely dissolved,
13 mmol of methyl iodide (or allyl bromide, 1-chlorobutane) was added to the reaction solution to
afford 3b, 3c, 3d respectively, and the mixture was refluxed with stirring for 3 hours. After the
mixture cooled to room temperature, the precipitate was filtered off and washed with 5 mL of
acetone. The combined acetone solutions were concentrated in vacuum to obtain a residue, and the
resulting residue was purified by column chromatography to give a white solid below 20 .Yields
for 3b, 3c, 3d are 85%, 75%, 82%, respectively.
General procedure for the preparation of compounds 4b-d (Scheme 2)
To a solution of anhydrous toluene (80 mL) in a 250 mL two-neck flask under N2, was added
1.56g of NaH (26 mmol, 60%), followed by 13 mmol of 2,4-dihydroxyacetophenone 3a. After
stirring for 10 mins, 6 mL of diethyl carbonate in anhydrous toluene (20 mL) was added dropwise
during 30 mins, and the mixture was refluxed with stirring for 3 hours. The reaction mixture was
cooled to room temperature, and then poured into water, then extracted with diethyl ether for three
times until the ether layer was colorless. The aqueous layer was acidified with 100 mL of 2 N
hydrochloric acid, and the resulting precipitate was filtered off and recrystallized from ethanol to
give a white solid. Yields for 4b, 4c, and 4d were 85%, 86%, and 82%, respectively.

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Microwave-assisted synthetic procedure for the preparation of compounds 5a-8d (Scheme 2)
In our initial study, the reaction of 4-hydroxycoumarin (4a) with ethyl 2-methylacetoacetate in
the presence of toluene was chosen as a model to optimize the reaction conditions, and the
experimental results were summarized in Table 1.
Compounds Data
1
4-Methyl-pyrano[3,2-c]chromene-2,5-dione (5a): a white solid; mp: 257.6-262.5 ; H NMR
13
(400 MHz, CDCl3) δ 8.16 (s, 1H), 7.71 (s, 1H), 7.44 (s, 2H), 6.27 (s, 1H), 2.69 (s, 3H); C NMR
(101 MHz, DMSO) δ 135.09, 125.63, 123.99, 117.14, 114.29, 40.51, 40.40, 40.19, 39.91, 39.78,
39.59, 39.46, 22.31; IR (KBr) ν (cm-1) 1711, 1623, 1446, 1272; HR-MS (ESI): m/z calcd for
C13H9O4 ([M + H]+) 229.05009, found 229.04944.
3-Fluoro-4-methyl-pyrano[3,2-c]chromene-2,5-dione (5b):
a
white solid; mp: 247.3
-247.5 ; 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.69 (s, 1H), 7.41 (s, 2H), 2.64 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ 158.03, 157.69, 152.61, 145.45, 142.95, 134.91, 134.34, 125.30, 123.69,
116.96, 112.73, 103.25, 12.84; IR (KBr) ν (cm-1) 1695, 1616, 1446, 1268; HR-MS (ESI): m/z calcd
for C13H9FO4 ([M + H]+) 247.04066, found 247.03992.
3,4-Dimethyl-pyrano[3,2-c]chromene-2,5-dione (5c): a white solid; mp: 223.5-223.5 ; 1H NMR
13
(400 MHz, CDCl3) δ 8.15 (s, 1H), 7.69 (s, 1H), 7.41 (s, 2H), 2.69 (s, 3H), 2.26 (s,3H); C NMR
(101 MHz, CDCl3) δ 159.52, 159.36, 158.65, 152.73, 149.46, 133.86, 124.90, 123.64, 122.41,
116.67, 113.20, 104.18, 18.18, 13.23; IR (KBr) ν (cm-1) 1727, 1660, 1446, 1191; HR-MS (ESI):
m/z calcd for C14H10O4 ([M + H]+) 243.06573, found 243.06502.
1
3-Ethyl-4-methyl-pyrano[3,2-c]chromene-2,5-dione (5d): a white solid; mp: 143.4-144.3 ; H
NMR (400 MHz, CDCl3) δ 8.14 (d, J = 7.9 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.42 (dd, J = 13.8, 8.0
13
Hz, 2H), 2.73 (dd, J = 15.2, 7.7 Hz, 2H), 2.70 (s, 3H), 1.21 (q, J = 7.7 Hz, 3H); C NMR (101
MHz, CDCl3) δ 159.54, 159.13, 158.71, 152.75, 149.08, 133.86, 128.15, 124.88, 123.67, 116.66 ,
113.24, 104.31, 20.76, 17.67, 12.52; IR (KBr) ν (cm-1) 1715, 1620, 1537, 1449; HR-MS (ESI): m/z
calcd for C15H12O4 ([M + H]+) 257.08139, found 257.08069.
1
4-Trifluoromethyl-pyrano[3,2-c]chromene-2,5-dione (5e): a white solid; mp: 200.5-203.0 ; H
NMR (400 MHz, CDCl3) δ 8.13 (dd, J = 8.0, 1.4 Hz, 1H), 7.80-7.72 (m, 1H), 7.49-7.41 (m, 2H),

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6.88 (d, J = 0.7 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 163.80, 156.30, 154.66, 153.70, 142.35,
141.93, 135.75, 125.38, 124.15, 117.16, 115.97 (q, J = 7.3 Hz, 1C), 112.37, 99.63; IR (KBr) ν
(cm-1) 1724, 1607, 1530, 1458, 1389, 1099, 833; HR-MS (ESI): m/z calcd for C13H5F3O4 ([M +
H]+) 283.02182, found 283.02127.
8-Methoxy-4-methyl-pyrano[3,2-c]chromene-2,5-dione (6a): a white solid; mp: 242.0-242.1 ;
1H NMR (400 MHz,CDCl3)δ 8.02(s, 1H),6.99(s, 1H),6.87(s, 1H),6.17(s, 1H),3.97(s, 3H),2.66(s,
3H); 13C NMR (101 MHz, CDCl3) δ 165.13, 162.71, 158.69, 158.18, 156.45, 155.36, 125.40,
113.70, 112.72, 106.30, 101.51, 100.55, 56.04, 22.67; IR (KBr) ν (cm-1) 1709, 1611, 1541, 1281;
HR-MS (ESI): m/z calcd for C14H10O5 ([M + H]+) 259.06065, found 259.05988.
8-Methoxy-3,4-dimethyl-pyrano[3,2-c]chromene-2,5-dione (6b):
a
white solid; mp:
1
212.3-212.9 ; H NMR(400 MHz,CDCl3) δ 8.01(s, 1H), 6.97 (s, 1H), 6.85 (s, 1H), 3.95 (s, 3H),
2.67 (s, 3H), 2.23 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.54, 159.80, 159.70, 159.01, 154.74,
149.83, 124.85, 120.69, 113.44, 106.33, 101.83, 100.34, 55.98, 18.14, 13.06; IR (KBr) ν (cm-1)
1702, 1609, 1550, 1275; HR-MS (ESI): m/z calcd for C15H12O5 ([M + H]+) 273.07630, found
273.07553.
3-Ethyl-8-methoxy-4-methyl-pyrano[3,2-c]chromene-2,5-dione (6c): a white solid; mp:
143.1-143.3 ; 1H NMR (400 MHz,CDCl3) δ 8.01 (dd, J= 18.7,8.9 Hz,1H), 7.00-6.94 (m,1H), 6.84
(dd, J= 12.5, 2.2 Hz, 1H), 3.95 (s,3H), 2.71 (dd, J= 14.6,7.1 Hz, 2H), 2.68 (s, 3H), 1.18 (t, J= 7.5
13
Hz, 3H); C NMR (101 MHz, CDCl3) δ 164.56, 160.10, 159.36, 159.12, 154.80, 149.47, 126.48,
124.92, 113.40, 106.41, 101.97, 100.35, 55.97, 20.63, 17.64, 12.60; IR (KBr) ν (cm-1) 1715, 1607,
1397, 1275, 1022; HR-MS (ESI): m/z calcd for C16H13O5 ([M + H]+) 287.09195, found 287.09115.
8-Methoxy-4-trifluoromethyl-pyrano[3,2-c]chromene-2,5-dione (6d): a white solid; mp:
1
225.6-225.8 ; H NMR (400 MHz, CDCl3) δ 8.01 (dd, J = 21.3, 8.4 Hz, 1H), 7.09-6.94 (m, 1H),
6.94-6.74 (m, 2H), 3.98 (d, J = 8.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.08, 164.17, 156.66,
156.05, 155.09,143.37, 142.29, 125.49, 115.09, 114.38-113.99 (m, 1C), 105.54, 100.57, 97.02,
56.21, IR (KBr) ν (cm-1) 1765, 1731, 1609, 1376, 1150; HR-MS (ESI): m/z calcd for C14H7F3O5
([M + H]+) 313.03238, found 313.03183.
8-Allyloxy-4-methyl-pyrano[3,2-c]chromene-2,5-dione (7a): a white solid; mp: 178.1-178.3 ;
1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.01 (s, 1H), 6.88 (s, 1H), 6.17 (s, 1H), 6.09 (s, 1H),
13
5.46 (s, 2H), 4.70 (s, 2H), 2.66 (s, 3H); C NMR (101 MHz, CDCl3) δ 164.02, 162.62, 158.63,

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158.12, 156.41, 155.21, 131.70, 125.36, 118.89, 114.14, 112.72, 106.34, 101.51, 101.35, 69.53,
22.65; IR (KBr) ν (cm-1) 1711, 1609, 1544, 1489, 1279, 1089; HR-MS (ESI): m/z calcd for
C16H11O5 ([M + H]+) 285.07630, found 285.07554.
8-Allyloxy-3,4-dimethyl-pyrano[3,2-c]chromene-2,5-dione (7b):
a
white solid; mp:
192.7-193.3 ; 1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.15 (s, 1H), 6.32 (s, 1H), 6.10 (s, 1H),
5.39 (d, J = 40.7 Hz, 2H), 4.78 (s, 2H), 3.33 (s, 3H), 2.21 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
163.44, 159.77, 159.02, 154.63, 149.81, 131.80, 124.86, 120.72, 118.80, 113.92, 106.41, 101.86,
101.17, 69.45, 18.15, 13.07. IR (KBr) ν (cm-1) 1718, 1605, 1541, 1489, 1291, 1105; HR-MS (ESI):
m/z calcd for C17H14O5 ([M + H]+) 299.09195, found 299.09109.
8-Allyloxy-3-ethyl-4-methyl-pyrano[3,2-c]chromene-2,5-dione (7c):
a
white solid; mp:
143.1-143.3 ; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.9 Hz, 1H), 7.00-6.88 (m, 1H), 6.82 (d,
J = 2.3 Hz, 1H), 6.05 (ddd, J = 22.5, 10.6, 5.3 Hz, 1H), 5.41 (ddd, J = 13.9, 11.6, 1.1 Hz, 2H), 4.63
(d, J = 5.3 Hz, 2H), 2.67 (dd, J = 14.9, 7.4 Hz, 2H), 2.63 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 163.46, 160.04, 159.31, 159.09, 154.67, 149.43, 131.81, 126.52, 124.90,
118.79, 113.92, 106.47, 101.98, 101.17, 69.46, 20.63, 17.63, 12.59; IR (KBr) ν (cm-1) 1704, 1616,
1544, 1503, 1277, 1205; HR-MS (ESI): m/z calcd for C18H16O5 ([M + H]+) 313.10760, found
313.10669.
8-Butoxy-4-methyl-pyrano[3,2-c]chromene-2,5-dione (8a): a white solid; mp: 183.0-183.9 ，
1HNMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.9 Hz, 1H), 6.99 (dd, J = 8.9, 2.2 Hz, 1H), 6.85 (d, J =
2.2 Hz, 1H), 6.17 (d, J = 1.0 Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 2.66 (d, J = 0.9 Hz, 3H), 1.91 – 1.79
13
(m, 2H), 1.55 (dt, J = 14.7, 7.4 Hz, 2H), 1.04 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, CDCl3) δ
164.74, 162.73, 158.73, 158.21, 156.48, 155.35, 125.30, 114.07, 112.59, 106.02, 101.36, 100.93,
68.73, 30.90, 22.67, 19.14, 13.77; IR (KBr) ν (cm-1) HR-MS (ESI): m/z calcd for C17H16O5 ([M +
H]+) 301.10760, found 301.10675.
8-Butoxy-3,4-dimethyl-pyrano[3,2-c]chromene-2,5-dione (8b): a white solid; mp: 172.5-172.7℃;
1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.9 Hz, 1H), 6.93 (dd, J = 8.9, 2.3 Hz, 1H), 6.79 (d, J =
2.2 Hz, 1H), 4.05 (t, J = 6.5 Hz, 2H), 2.63 (s, 3H), 2.19 (s, 3H), 1.88 – 1.73 (m, 2H), 1.52 (dq, J =
14.7, 7.4 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 164.17, 159.98, 159.77,
159.13, 154.78, 149.89, 124.80, 120.56, 113.84, 106.11, 101.73, 100.76, 68.62, 30.92, 19.15, 18.15,
13.77, 13.05; HR-MS (ESI): m/z calcd for C18H18O5 ([M + H]+) 315.12325, found 315.12239.

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8-Butoxy-3-ethyl-4-methyl-pyrano[3,2-c]chromene-2,5-dione (8c): a white solid; mp: 144,3
1
-144.7 ; H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 8.9, 1.3 Hz, 1H), 6.92 (dd, J = 8.9, 2.1 Hz,
1H), 6.79 (s, 1H), 4.05 (t, J = 6.4 Hz, 2H), 2.67 (dd, J = 13.2, 5.7 Hz, 2H), 2.63 (s, 3H), 1.88 – 1.76
(m, 2H), 1.51 (dt, J = 14.9, 7.4 Hz, 2H), 1.26 (s, 1H), 1.14 (t, J = 7.5 Hz, 3H), 1.00 (t, J = 7.4 Hz,
3H); 13C NMR (101 MHz, CDCl3) δ 164.17, 160.15, 159.38, 159.18, 154.80, 149.51, 126.35,
124.83, 113.83, 106.15, 101.83, 100.74, 68.61, 30.93, 20.61, 19.15, 17.64, 13.77, 12.60; HR-MS
(ESI): m/z calcd for C19H20O5 ([M + H]+)329.13890, found 329.13809.
1
7,8,9,10-Tetrahydro-5,12-dioxa-chrysene-6,11-dione (9a): a white solid; mp: 211.2-212.3 ; H
NMR (400MHz,CDCl3) δ 8.09 (s,1H), 7.65 (s,1H), 7.37 (s,2H), 3.14 (s,2H), 2.59 (s,2H), 1.80
13
(s,4H); C NMR (101 MHz, CDCl3) δ 159.29, 159.17, 158.40, 152.73, 150.76, 133.77, 124.87,
123.79, 123.56, 116.67, 113.27, 103.83, 28.55, 24.50, 21.53, 20.84; IR(KBr) ν (cm-1) 1720, 1620,
1605, 1541, 1189; HR-MS (ESI): m/z calcd for C16H12O4 ([M + H]+) 269.08139, found 269.08072.
2-Methoxy-7,8,9,10-tetrahydro-5,12-dioxa-chrysene-6,11-dione (9b): a white solid, mp:
238.2-240.8 ; 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 6.95 (s, 1H), 6.82 (s, 1H), 3.91 (s, 3H),
13
3.12 (s, 2H), 2.56 (s, 2H), 1.78 (s, 4H); C NMR (101 MHz, CDCl3) δ 164.48, 159.88, 159.42,
158.81, 154.77, 151.13, 124.81, 122.11, 113.41, 106.44, 101.49, 100.39, 55.96, 28.55, 24.37, 21.57,
20.93; IR (KBr) ν (cm-1) 1702, 1605, 1528, 1275; HR-MS (ESI): m/z calcd for C17H14O5 ([M + H]+)
299.09195, found 299.09109.
2-Allyloxy-7,8,9,10-tetrahydro-5,12-dioxa-chrysene-6,11-dione (9c): a white solid, mp: 206.9
1
-207.3 ; H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.9 Hz, 1H), 7.07-6.93 (m, 1H), 6.86 (d, J =
2.2 Hz, 1H), 6.09 (ddd, J = 22.4, 10.6, 5.3 Hz, 1H), 5.45 (dd, J = 36.7, 13.9 Hz, 2H), 4.68 (d, J =
5.3 Hz, 2H), 3.16 (s, 2H), 2.60 (s, 2H), 1.87 – 1.71 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 163.40,
159.85, 159.40, 158.81, 154.67, 151.11, 131.83, 124.82, 122.15, 118.78, 113.89, 106.53, 101.53,
101.23, 69.45, 28.54, 24.37, 21.57, 20.92; IR(KBr)ν (cm-1) 1702, 1611, 1550, 1277, 1110; HR-MS
(ESI): m/z calcd for C19H16O5 ([M + H]+) 325.10760, found 325.10675.
2-Butoxy-7,8,9,10-tetrahydro-5,12-dioxa-chrysene-6,11-dione (9d):
a
white solid; mp:
1
146.7-147.1℃; H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.9 Hz, 1H), 6.92 (dd, J = 8.9, 2.3 Hz,
1H), 6.79 (d, J = 2.2 Hz, 1H), 4.05 (t, J = 6.5 Hz, 2H), 3.11 (s, 2H), 2.56 (s, 2H), 1.89-1.69 (m, 6H),
1.60-1.42 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 162.71, 159.17, 158.84,
158.19, 155.43, 141.00, 130.49, 122.90, 113.44, 109.11, 105.24, 101.85, 68.45, 61.27, 30.98 ,

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29.70, 19.17, 14.24, 13.78, 10.17; HR-MS (ESI): m/z calcd for C20H20O5 ([M + H]+) 341.13890,
found 341.13835.
2.2. X-ray Diffraction Analysis
White crystals of compound 5b (0.130 × 0.130 × 0.050 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 mono-chromated MoKα radiation (λ=
0.71073 Å); θmax = 25.010; 1578 independent reflections (Rint = 0.0334). 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) + (0.0659P)2 + 0.0500P] gave final values of R = 0.0334, ωR = 0.1054, in
the title compound, the moiety formula is C13H7FO4.
White crystals of compound 6b (0.520 × 0.480 × 0.210 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 Mo Kα radiation (λ=
0.71073 Å); θmax = 27.559;1838 independent reflections (Rint = 0.0487). Data were corrected for
Lorentz and polarization effects and for absorption (Tmin = 0.942; Tmax = 0.975). 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 = 0.0868, ωR =
0.1424, 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 C15H12O5.
The crystallographic data have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication number: CCDC 1047356 (5b), CCDC 1419461 (6b).
Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB12 1EZ, UK (Fax +44 1223 336033 or E-mail: deposit@ccdc.cam.ac.uk).
2.3. Biological Assays

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The antifungal activities of all the synthesized compounds were carried out at a concentration
of 50 ppm using a mycelia growth inhibitory rate method, including Azoxystrobin used as a
positive control. The tested fungi were provided by the Laboratory of Plant Disease Control,
Nanjing Agricultural University, the fungal strains and experimental methods used in the biological
assays are the same as those reported in the literature[26]. After retrieval from the storage tube, the
strains were incubated in PDA at 25 for a week to get new mycelia for the antifungal assay. All
the tested compounds were first dissolved in 5 mL N,N-dimethylformamide to generate a 100 ppm
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, and Rhizoctorzia solani are listed in Table 3.
3. Results and discussion
3.1 Synthetic Chemistry
Most synthetic procedures are costly, time consuming and not environmentally friendly,
usually giving relatively poor yields. The application of microwave irradiation in synthetic
chemistry has become increasingly popular in modern combinatorial and medicinal chemistry.
Compared with traditional heating for organic reactions, the microwave irradiation used in this
paper is an very efficient and green synthetic approach which dramatically reduces reaction time
(reduced from days and hours to minutes and seconds) and improves the reaction yields[27]. As
shown in Table 1, compound 5c could be prepared in a moderate yield with toluene as the best
solvent (Entry 3, 5, 6), using 640W microwave irradiation (Entry 6, 7, 8), and DMAP as the
catalyst (Entry 4, 6). The yields increased consistently with time, from 34% (10 min) to a
maximum of 69% (15min) under these conditions, however, when extending the reaction time
longer than 20 min, the yield did not increase significantly (shown in Entry 5, 6 and 9). Therefore,
we can conclude that the optimal conditions include using toluene as the solvent in the presence of
DMAP under microwave irradiation (640 W) for 15 mins. This optimized method was used to
synthesize the designed coumarin derivatives. Furthermore, compared with conventional heating
(Entry 13-14), the reaction times for the target compounds can be reduced from about 6 hours to 15
mins at minimum (Entry 6), and the isolated yields can be improved from 30% to 76% at
maximum (Compound 7a).

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With this efficient method, a focused library of 19 compounds including 3-(trifluoro)methyl
(5a-5e), 8-alkoxy (6a-6d, 8a-8c), 8-allyloxy (7a-7c) pyrano[3,2-c]chromene-2,5-diones and
3,4-cyclohexane pyrano[3,2-c]chromene-2,5-diones (9a-9d) have been designed and synthesized.
All of the target molecules listed in Table 2 were characterized on the basis of physical and spectral
data. In addition, compounds 5b and 6b were further confirmed by X-ray diffraction
crystallography (Figure 2).
3.2 Antifungal Activity and the Structure-Activity Relationships (SAR)
The results of the biological testing against seven phytopathogenic fungi are given in Table 3.
For the purposes of analysis of structure-activity relationships, the antifungal activities of all target
compounds were compared to the positive control Azoxystrobin, a marketed broad spectrum
fungicide developed by Syngenta.
Data presented in Table 3 reveal that the synthesized coumarin derivatives exhibit differential
activities against five plant pathogenic fungi at 50 ppm. All of the compounds, excluding 6a and 8a,
showed better antifungal activity against Botrytis cinerea than Azoxystrobin (34%). Compounds 5d
(64%), 5e (60%), 6b (60%), 7b (64%) and 9b (63%) demonstrate a two-fold increase in potency
against Botrytis cinerea compared to Azoxystrobin. Compounds 5d (57%) and 6c (55%) exhibited
better activity than Azoxystrobin (44%) against Colletotrichum capsica. However, these coumarin
derivatives showed weak antifungal activity against Alternaria solani, though 5d (22%) and 9a
(19%) showed improved antifungal activity compared to Azoxystrobin (16%). The bioscreening
data also showed that all of these target compounds did not show effective control against
Gibberella zeae and Rhizoctorzia solani, though compound 6c (64%) showed activity equivalent to
the positive control Azoxystrobin (69%) against Rhizoctorzia solani.
As 5d, 6c and 7b showed effective control against Botrytis cinerea and Colletotrichum
capsica, we further tested the EC50 values of these compounds together with the positive control.
As shown in Table 4, we noticed that the EC50 values of compounds 6c and 7b were as low as
0.082 and 0.091 µM, respectively, which proves they are much more effective than Azoxystrobin
(0.158 µM). Compound 5d also exhibited much better activity than the control against
Collecterichum capsica. The EC50 value was almost half that of Azoxystrobin, which was as low as

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0.115 µM.
Although the antifungal activity of most of the fused coumarin derivatives has been proven to
be very poor, making it difficult to extract a clear structure-activity relationship analysis, some
broad conclusions can still be drawn. These compounds were noticeably more active against
Botrytis cinerea and Colletotrichum capsici, but lacked potency against Alternaria solani,
Alternaria mali and Rhizoctonia solani, as illustrated by the absence of activity against these three
kinds of fungi. Compounds 5d, 5e and 9a showed equivalent activity with the positive control
Azoxystrobin against Alternaria mali. Secondly, the spectrum of antifungal activity is generally
improved if R2 and R3 are Me, Et or CF3, highlighted by 5d and 6c, both of which displayed a
broad spectrum of activity and are particularly effective against Botrytis cinerea.
4. Conclusions
In summary, we have efficiently designed and synthesized a novel series of fused coumarin
analogues through an optimized microwave-assisted protocol. The reaction times for the target
compounds can be reduced from about 6 hours to 15 mins at minimum, and the isolated yields can
be improved from 30% to 76% at maximum. Biological testing data showed that some of the fused
coumarin analogues exhibited good antifungal activity against Botrytis cinerea and Collecterichum
capsica. Compounds 5d, 6c and 7b were identified as the most active ones, and EC50 values shows
against Botrytis cinerea were as low as 0.141, 0.082 and 0.091 µM, respectively, which improved
antifungal activity compared to Azoxystrobin. Compound 5d (EC50 = 0.115 µM) also exhibited
much better activity than the control (EC50 = 0.222 µM) against Collecterichum capsica.
Compound 5d was identified as the most active and therefore the most promising candidate for
further study. Further structural optimization of fused coumarin analogues is well underway, with
the aim of preparing analogues with improved 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 (KYZ201426). We wish
especially to mention our gratitude to Dr. Andrew Jeffries from Chemistry Department at Texas
A&M University for his great contribution on the manuscript revision.

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Figure 1. Structures of coumarin-containing drugs and antifungal agents
Figure 2. X-ray structures of compounds 5b and 6b
Scheme 1. The design of coumarin-containing antifungal compounds
Scheme 2. Synthetic route for target compounds 5a-9d
Table 1. Reaction Condition Optimization for compound 5c
Table 2. The structures and yields of compounds 5a-9d
Table 3. Antifungal activity of coumarin analogues (inhibitory rate, %)
Table 4. EC50 determination of compounds 5d, 6c and 7b

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O
O
OH
OH
O2N
HO
O
O
O
O
O
O
O
NO2
O
O
Cou-NO2
Osthole
Warfarin
Acenocoumarol
O
OH Ph
O
O
HO
O
O
O
O
O
O
HO
O
O
O
O
Herniarin
Scopoletin
Xanthotoxin
Mammeisin
O
O
O
O
O
O
O
O
O
O
O
Excavarin-A
Coumoxystrobin
Figure 1. Structures of coumarin-containing drugs and antifungal agents
5b
6b
Figure 2. X-ray structures of compounds 5b and 6b

ACCEPTED MANUSCRIPT
Scheme 1. The design of coumarin-containing antifungal compounds
Scheme 2. Synthetic route for target compounds 5a-9d

ACCEPTED MANUSCRIPT
Table 1. Reaction Condition Optimization for compound 5c
Entry
1
Solvent
DMF
Catalyst
NH4OAc
NH4OAc
DMAP
MW
Temperature
Time
15 min
20 min
15 min
15 min
10 min
15 min
15 min
15 min
20 min
20 min
20 min
6h
Yield a
45%
40%
35%
45%
34%
69%
55%
20%
65%
48%
54%
30%
40%
45%
640 W
640 W
640 W
640 W
640 W
640 W
560 W
720 W
640 W
640 W
640 W
r.t.
r.t
2
DMF
3
DMF
r.t.
4
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Ethanol
Ethanol
DMF
NH4OAc
DMAP
r.t
5
r.t.
6
DMAP
DMAP
r.t.
7
r.t.
8
DMAP
r.t.
9
DMAP
r.t.
10
11
12
13
14
NH4OAc
DMAP
r.t.
r.t.
b
NH4OAc
DMAP
/
Reflux
Reflux
Reflux
b
DMF
/
6h
b
Toluene
DMAP
/
6h
a Yields after recrystallization from ethanol. b Conventional heating, no microwave irradiation.

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Table 2. The structures and yields of compounds 5a-9d
Compd.
R1
R2
R3
Yield (%) a Compd.
R1
R2
R3
Yield (%) a
5a
5b
5c
5d
5e
6a
6b
6c
6d
7a
H
H
F
CH3
CH3
72
13
68
64
20
75
71
69
31
76
7b
7c
8a
8b
8c
9a
9b
9c
9d
OCH2CH=CH2 CH3 CH3
66
69
70
62
64
74
65
67
75
H
OCH2CH=CH2
O(CH2)3CH3
O(CH2)3CH3
O(CH2)3CH3
H
Et
H
CH3
CH3
H
H
CH3 CH3
Et
H
H
CH3
CF3
CH3
CH3 CH3
Et CH3
(CH2)4
H
OCH3
OCH3
OCH3
OCH3
OCH2CH=CH2
CH3 CH3
OCH3
(CH2)4
(CH2)4
(CH2)4
Et
H
H
CH3
CF3
CH3
OCH2CH=CH2
O(CH2)3CH3
a Yields after recrystallization or column chromatography

ACCEPTED MANUSCRIPT
Table 3. Antifungal activity of coumarin analogues (inhibitory rate, %)
Species a
BOT
50
COL
50
ALT
50
5
GIB
50
15
4
RHI
50
Compound Rate(ppm)
5a
47b
47
47
64
60
31
60
67
50
41
64
54
23
41
36
35
63
54
40
34
21
12
15
57
10
15
15
55
22
15
40
13
36
27
34
14
15
15
21
44
21
14
16
30
14
19
53
64
44
26
28
29
26
31
21
41
21
21
21
69
5b
4
5c
5
3
5d
22
16
4
30
6
5e
6a
6
6b
13
13
5
16
44
38
8
6c
6d
7a
4
7b
4
57
4
7c
13
7
8a
23
15
18
13
6
8b
5
8c
10
19
11
11
5
9a
9b
9c
6
9d
15
68
Azoxystrobin
16
a BOT, Botrytis cinerea; COL, Collecterichum capsica; ALT, Alternaria solani; GIB, Gibberella zeae; RHI, Rhizoctorzia solani.
b All the data was the average of three replications.

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Table 4. EC50 determination of compounds 5d, 6c and 7b
95% confidence
interval
Compound
Pathogen a
Toxic regression
R
EC50 ( µM) b
5d
BOT
BOT
BOT
BOT
COL
COL
COL
COL
Y=4.2485+0.4824x
Y=3.8914+0.8086x
Y=3.1704+1.2718x
Y=3.0886+1.0598x
Y=4.3614+0.4345x
Y=3.4096+1.0175x
Y=0.9851+2.2526x
Y=3.9631+0.5312x
0.9988
0.9959
0.9986
0.9997
0.9960
0.9995
0.9988
0.9985
0.141
0.082
0.091
0.158
0.115
0.128
0.203
0.222
0.131-0.151
0.074-0.091
0.085-0.097
0.145-0.171
0.103-0.128
0.122-0.136
0.184-0.225
0.194-0.254
6c
7b
Azoxystrobin
5d
6c
7b
Azoxystrobin
a BOT, Botrytis cinerea; COL, Collecterichum capsica. b The EC50 value was the average of three replications.

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Highlights
The microwave-assisted synthesis of novel fused coumarin derivatives was described.
Antifungal activity screening of the designed compounds against five fungi was
screened.
Some compounds exhibited potent antifungal activity with 5d as the most promising
candidate for further study.
The structure-activity relationships of the fused coumarin derivatives were
summarized.