Design, synthesis and evaluation of 2,4-diarylpyrano[3,2-c]chromen-5(4H)-one as a new class of non-purine xanthine oxidase inhibitors

Article abstract of DOI:10.3109/14756366.2014.961446

Keeping in view the recent success of molecular hybridization technique in drug design, 2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones as conjugates of coumarins and chalcones have been designed and synthesized in the present study. The catalytic efficiency of

Full text of DOI:10.3109/14756366.2014.961446

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J Enzyme Inhib Med Chem, Early Online: 1–7
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.961446
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ORIGINAL ARTICLE
Design, synthesis and evaluation of 2,4-diarylpyrano[3,2-c]chromen-
5(4H)-one as a new class of non-purine xanthine oxidase inhibitors
Harpreet S. Virdi1, Sahil Sharma1, Samir Mehndiratta2, P. M. S. Bedi1, and Kunal Nepali1
1Department of Pharmaceutical sciences, Guru Nanak Dev University, Amritsar, Punjab, India and 2Department of Medicinal Chemistry, College of
Pharmacy, Taipei Medical University, Taipei, Taiwan
Abstract
Keywords
Keeping in view the recent success of molecular hybridization technique in drug design,
2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones as conjugates of coumarins and chalcones have
been designed and synthesized in the present study. The catalytic efficiency of various
Lewis acids for the synthesis of designed conjugates under neat conditions was investigated,
and SiO2 (200–400 mesh)-ZnCl2 was optimized as the best catalyst among the tested ones. The
conjugates were evaluated for in-vitro xanthine oxidase activity. The results of the in-vitro assay
were quite promising as some conjugates were endowed with remarkable inhibitory potential
against the enzyme. HV-8, 11 and 12 were found to be high-potent inhibitors with HV-11 (the
most potent inhibitor) possessing an IC50 value of 2.21 mM. The most active conjugate HV-11
was evaluated for the type of inhibition and was found to be a mixed type inhibitor. The
compliance of some selected conjugates to the Lipinski rule was also calculated.
Conjugates, inhibition, lipinski rule, mixed,
silica, xanthine oxidase
History
Received 6 May 2014
Revised 2 August 2014
Accepted 1 September 2014
Published online 30 September 2014
Introduction
and xanthine oxidase inhibitory activities24–26 makes it a
privileged structure. Among the diverse array of biological
activities possessed by coumarins, we are particularly interested
towards their xanthine oxidase inhibitory potential which has
also been extensively explored24–26. Esculetin represents the most
potent xanthine oxidase inhibitor of this class24.
Chalcones (1,3-diaryl-2-propen-1-ones) with an enone system
between two aromatic rings constitute an important class of
natural products which serve as precursors for the preparation of
various flavonoids and exhibit interesting pharmacological
activities27,28. Chalcones are also known as open chain flavonoid
derivatives which are extensively reported as xanthine oxidase
inhibitors29–31. Recently 3,5,20,40-tetrahydroxychalcone was found
to be potent competitive inhibitor of the enzyme as indicated
by the kinetic study. In vivo, intragastric administration of the
chalcone was able to significantly reduce serum uric acid levels
and inhibited hepatic xanthine oxidase activities of hyperuricemic
mice in a dose-dependent manner32.
Oxidative hydroxylation of hypoxanthine and xanthine catalyzed
by xanthine oxidase to produce uric acid and reactive oxygen
species leads to many diseases like gout and at least symptoms
of diseases like oxidative damage to the tissue1–3. Therefore,
the selective inhibition of XO may result in broad spectrum
chemotherapeutic for gout, cancer, inflammation and oxidative
damage2–4. Allopurinol3,4, 2-alkyl hypoxanthines5,6, pterin and
6-formylpterin7 represents the class of purine-based xanthine
oxidase inhibitors. All these inhibitors have been successfully
utilized and have proved their inhibitory potential towards the
enzyme. However, these purine-based inhibitors have been
reported to be associated with Steven Johnson syndrome and
worsening of renal function induced in some of the patients2–4
.
Keeping in view these side effects, we have recently designed
some non-purine xanthine oxidase inhibitors such as azaflavones8,
n-acetyl pyrazolines9, b-acetamido compounds10, naphthopyr-
ans11 and 4,6-diaryl/heteroarylpyrimidin-2(1H)-ones12.
Keeping in view the promising xanthine oxidase inhibitory
potential of these non-purine chemical architectures, the present
study explores xanthine oxidase inhibitory evaluation of the
designed 2,4-diarylpyrano[3,2-c]chromen-5(4H)-one as couma-
rin–chalcone conjugates (Figure 1).
Coumarins form an important class of compounds, which
occupy a special role in nature13,14. The presence of coumarin
architecture in naturally occurring phytoconstituents with anti-
cancer15,16, anti-HIV17, antituberculosis18, anti-influenza19, anti-
alzheimer20,21, anti-inflammatory22, antiviral23, antimicrobial13
Thus, the proved potential of coumarin and chalcones as
xanthine oxidase inhibitors and recent success of molecular
hybridization technique in drug design33 tempted us to design
such conjugates. Pyran was selected as a linker for tethering
coumarin with chalcones. The presence of pyran as an structural
motif in various non-purine xanthine oxidase inhibitors supports
the suitability of its selection as a linker10,28–31
.
Results and discussion
Chemistry
The usual strategy for the synthesis of 2,4-diaryl pyrano[3,2-
c]coumarins is to react 4-hydroxy coumarin with a,b-unsaturated
compounds. There are some methodologies34–36 utilizing various
Addres for correspondence: Kunal Nepali, Guru Nanak Dev University,
Amritsar, India. Email: kunal_8855@rediffmail.com

2
H. S. Virdi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
catalysts reported for the synthesis of 2,4-diaryl pyrano[3,2-
In an attempt to investigate the catalytic efficiency of various
c]coumarins, but each one suffers from some drawbacks such as Lewis acids (Table 1), a model reaction was performed with
environmentally hazardous (POCl3), expensive catalysts (AuCl3/ different catalyst for the synthesis of target compound
3AgOTf) and some unavailable bronsted acids. Recently, Bagdi (Scheme 1). All reactions were carried out using 1 mmol of
et al. reported an efficient regioselective methodology for the hydroxy coumarin and chalcone.
synthesis of 2,4-diaryl pyrano[3,2-c]coumarins via copper (II)
Table 1 presents the yield of the model reaction at 100 ꢀC and a
triflate37. However, in view of limited efficient and environmental reaction time of 4 h with various Lewis acids. Further increase in
friendly synthetic schemes and in continuation of our efforts to temperature and time resulted in slight decrease in the yields with
develop cost effective, highly yielding and environmental friendly all the catalyst. Among the various Lewis acids tested, ZnCl2 was
methodologies for non-purine xanthine oxidase inhibitors, we found to be the best catalyzing model reaction in 50% yield. Thus
investigated the effects of some inexpensive and easily available catalytic efficiency of ZnCl2 was further investigated by adsorb-
Lewis acids under neat conditions for the synthesis of desired ing it over different grades of silica. The yields of the model
compounds.
reaction were higher with ZnCl2 adsorbed on SiO2 (200–400
mesh) than the other grades of silica. The results clearly
demonstrates the effect of increased effective surface area on
the catalytic efficiency of silica-supported zinc chloride as the
yield of the model reaction with SiO2 (200–400 mesh)-ZnCl2 was
around 1.5-folds higher than silica (60–120 mesh). Thus the
optimum reaction conditions involve the use of SiO2 (200–400
mesh)-ZnCl2 at 100 ꢀC for 4 h. In order to examine the substrate
scope of this reaction, variety of chalcone derivatives was used.
Table 2 indicates that all the reactions with electronically and
sterically diverse chalcone derivatives proceeded smoothly to
afford the target compounds in moderate to good yields. It was
observed that the reaction of 4-hydroxy coumarin with chalcones
possessing nitro and halo-substituted phenyl rings resulted in
higher yields as compared to chalcones (methyl and methoxy
substitutions). This could be attributed to the stronger ꢁI effect of
the halogens and nitro group as comparison to methoxy and
methyl groups with stronger +R effect. The reaction of hydroxy
coumarin and chalcone (synthesized from heteroaryl aldehyde)
resulted in a clean reaction and afforded to yield the pyrano[3,2-
c]coumarins in moderate yield; however, the reaction with
chalcones synthesized from heteroaryl ketones did not produce
the desired compound, rather an unidentified product was
obtained. The characterization of the unidentified product is
being dealt separately in our laboratory. The yields of the target
compound obtained by our investigations were quite similar to
one of the earlier reported methodology by Bagdi et al.37 Thus,
this protocol presents an alternative methodology employing the
use of silica supported Lewis acid with an increased surface area
under solvent-free condition as the advantages over the other
existing methodologies.
O
A
B
O
O
Chalcone
Framework
Coumarin
Skeleton
MOLECULAR
HYBRIDIZATION
Chalcone Frame work
A
O
O
B
O
Coumarin
Skeleton
2,4-diarylpyrano[3,2-c]chromen-5(4H)-one
as pyran tethered coumarin-chalcone conjugates
Figure 1. Design strategy.
Table 1. Percentage yield of the model reaction with various catalysts.
Entry
Catalyst (10 mol%)
AlCl3
FeCl3
I2
BF3
ZnCl2
SnCl2
H3BO3
Silica (200–400 #)
SiO2-ZnCl2 (60–120 #)
SiO2-ZnCl2 (100–200 #)
SiO2-ZnCl2 (200–400 #)
Yield (%)
In-vitro xanthine oxidase assay
1
2
3
4
5
6
7
8
9
38
35
32
25
50
41
13
18
55
62
82
In vitro screening of the conjugates using bovine milk xanthine
oxidase (grade 1, ammonium sulfate suspension) enzymatic assay
was performed as described in the literature38. Allopurinol was
employed as reference inhibitor39. The results of the in-vitro assay
(Table 2) indicated that HV-8, HV-11 and HV-12 were endowed
with significant inhibitory potential against the enzyme. HV-11
with an unsubstituted phenyl ring (Ring A) and thiophenyl ring
(Ring B) was the most potent among the series with an IC50 value
of 2.21 mM followed by HV-12 (unsubstituted phenyl ring – Ring
A and 1-naphthyl – Ring B, IC50 value 4.31 mM) and HV-8
10
11
OH
O
O
catalyst
O
O
neat , 1000 C, 4h
Scheme 1. Synthesis of 2,4–diphenyl pyrano[3,2-c]coumarins.
O
O

DOI: 10.3109/14756366.2014.961446
New class of non-purine xanthine oxidase inhibitors
3
Table 2. Isolated yields, IC50 values and type of inhibition.
Table 2. Continued
Yield
(%)
IC50
(mM)
Yield
(%)
IC50
(mM)
Code
Structure
Code
Structure
82
25.42
HV-7
72
75
84
21.31
HV-1
O
O
O
O
O
O
OCH3
HV-2
84 78.31HV-3
HV-8
8.21
O
O
O
O
O
O
Cl
O
O
83
85
83
68
80.21
HV-9
75.21
O
O
O
O
O
Br
Cl
OCH3
HV-4
HV-5
HV-6
65.32
HV-10
69
56.21
O
O
O
O
S
F
O
72.1
HV-11
75
2.21
O
O
O
O
O
NO2
S
O
16.71
O
HV-12
81
4.31
O
O
O
OCH3
OCH3
O
O
(continued )
(continued )

4
H. S. Virdi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 2. Continued
Table 2. Continued
Yield
(%)
IC50
(mM)
Yield
(%)
IC50
(mM)
Code
Structure
Code
Structure
NO2
Cl
HV-13
85
85.32
HV-18
80
33.21
O
O
O
O
O
OCH3
O
HV-14
71
26.43
HV-19
78
43.21
36.81
8.79
O
O
OCH3
O
O
O
OCH3
Cl
O
HV-15
60
59.21
H3CO
HV-20
53
O
O
O
O
O
OH
OCH3
O
HV-16
81
86.32
Allopurinol
O
O
(unsubstituted phenyl – Ring A and 2-naphthyl – Ring B, IC50
value 8.21 mM). Conjugates HV-11 and HV-12 were found to be
more active than allopurinol (IC50 ¼ 8.79 mM), whereas HV-8
displayed a similar inhibitory profile to allopurinol. Enzyme
kinetics study was performed for HV-11 (most active inhibitor).
The Lineweaver–Burk plot (Figure 2) revealed that compound
HV-11 was a mixed-type XO inhibitor. The pattern of graph
shows that it is a form of mixed inhibition scenario. The Km, Vmax
and slope are all affected by the inhibitor. The inhibitor has
NO2
O
HV-17
70
27.51
increased the Km and slope (Km/Vmax) while decreasing the Vmax
.
O
Moreover, carefully observing Figure 2, it was found that
intersecting lines on the graph converge to the left of the y-axis
and above the x-axis which indicates that the value of a (a
constant which defines the degree to which inhibitor binding
affects the affinity of the enzyme for substrate) is greater than 1.
This confirms that the inhibitor preferentially binds to the free
enzyme and not the enzyme substrate complex. Table 2 also
revealed some interesting generalizations about the structure–
activity relationship (SAR). SAR study revealed (i) placement of
any substituent on Ring A irrespective of its electronic effects
resulted in decreased activity profile (compare HV-1 with HV-13,
O
O
(continued )

DOI: 10.3109/14756366.2014.961446
New class of non-purine xanthine oxidase inhibitors
5
20 and HV-10 with HV-11. (ii) Conjugates with phenyl rings molecular properties. Among the six conjugates, only HV-6 and
possessing electron donating substituents (Ring B) were more -11 are completely consistent with Lipinski’s rule.
active than the conjugates with unsubstituted phenyl rings and
phenyl rings possessing electron withdrawing substituents (Ring
B) (compare HV-6, HV-7, HV-14 and HV-17 with HV-2, 3, 4, 5,
Conclusion
The present study reports the xanthine oxidase inhibitory potential
of 2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones designed and
synthesised as coumarin–chalcone conjugates for the first time.
Some of the conjugates were endowed with potent inhibitory
potential. The most potent inhibitor was found to be a mixed type
inhibitor. Overall HV-11 and -12 seems to be good hits among the
series. Further detailed investigation on HV-11 and -12 is under
progress.
9, 16, 19. Over all the preference order of substituents for Ring B
is as follows: OCH34CH34H4halo and nitro. (iii) Placement of
a heteroaryl ring as Ring B significantly enhanced the inhibitory
profile (compare HV-1 with HV-11). (iv) Placement of a bicyclic
ring as Ring B also led to enhancement of activity (HV-1 with
HV-8 and HV-12). (v) 1-Naphthyl was favored over 2-naphthyl as
Ring B (Compare HV-8 with HV-12).
We also calculated the compliance of compounds to the
Lipinski’s rule of five40. Briefly, this simple rule is based upon the
observation that most biological active drugs have a molecular
weight (MW) of 500 or less, a log P not higher than 5, five or
fewer hydrogen bond donor sites and ten or fewer hydrogen bond
acceptor sites (N or O atoms). Conjugates HV-6 (moderately
active) HV-8, 11, 12 (potent), HV-13, 16 (least active) were
selected for the calculation of molecular properties on the basis of
the degree of variation of the activity profile. The three most
potent conjugates, i.e. HV-8, 11, 12, possessed an exactly same
total polar surface area, number of rotatable bonds, and number of
OH and NH bonds. Among HV-8, 11 and 12, the most potent
inhibitor, i.e. HV-11 possessed a lower ClogP as well as molecular
volume. Thus the increase in ClogP value and molecular volume
could be responsible for the variation in XO inhibitory activity
among the three most potent inhibitors. The increase in TPSA,
nON, nrotb, volume and the ClogP by introducing the nitro group
either on Ring A and B (HV-13, -16) in comparison to the value
displayed by HV-11 (the most potent inhibitor) might be
responsible for their diminished activity profile. HV-6, a conju-
gate with moderate inhibitory profile possessed TPSA value in
between HV-11 (the most potent) and HV-16 (the least potent),
significantly increased molecular volume, nON as well as nrotb as
compared to HV-11. Thus, Table 3 indicates a clear correlation
between the inhibitory potential of the conjugates with the
Experimental
The reagents were purchased from Sigma Aldrich, Merck, CDH,
Loba chem., Spectro chem., India, and used without further
purification. All yields refer to isolated products after purifica-
tion. Biotage Microwave Synthesizer (Model: Initiator) operating
at 150 ꢀC with the microwave power maximum level of 400 W
was used for the fries rearrangement. Products were characterized
by spectral data. 1H NMR and 13C NMR spectra were recorded on
Bruker Advance II 400 NMR Spectrometer and JEOL AL 300
NMR Spectrometer. The spectra were measured in CDCl3 relative
to TMS (0.00 ppm). Melting points were determined in open
capillaries and were uncorrected.
Procedure for the synthesis of 2,4-diaryl
pyrano[3,2-c]coumarins
The mixture of 4-hydroxy coumarin (1 mmol), differently
substituted chalcones (1 mmol) and SiO2 (200–400 mesh)-
ZnCl2 (10 mol%), was heated on an oil bath for 4 h. The
reaction mixture was extracted with water and ethyl acetate. The
ethyl acetate fraction was concentrated and subjected to column
chromatography. The product was eluted with increasing % age
of ethyl acetate in hexane. The remaining reactions were carried
out following these general procedures. In each occasion, the
spectral data (1H NMR, 13C NMR and MASS) of known
compounds are 2,4-diphenylpyrano[3,2-c]chromen-5(4H)-one37,
4-(3-nitrophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one37,
2-phenyl-4-p-tolylpyrano[3,2-c]chromen-5(4H)-one37, 2-(4-chlor-
ocyclohexa-2,4-dienyl)-4-(4-methoxyphenyl)pyrano[3,2-c]chro-
men-5(4H)-one37, 4-(4-chlorophenyl)-2-(4-methylcyclohexa-2,4-
dienyl)pyrano[3,2-c]chromen-5(4H)-one37,
2-(4-methylcyclo-
hexa-2,4-dienyl)-4-phenylpyrano[3,2-c]chromen-5(4H)-one37.
The physical data of the 14 new compounds are provided
below.
Characterization data
The characterization data for the 14 newly synthesized com-
pounds are given below.
4-(4-Chlorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one
(HV-2): m.p.: 87–88 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ,
Figure 2. Lineweaver–Burk plot of HV-11.
Table 3. Molecular properties of some selected hybrids.
Entry
Compound
TPSA
Mol. wt.
ClogP
nOHNH
nON
nrotb
Volume
No. of violations
1
2
3
4
5
6
HV-11
HV-12
HV-8
HV-16
HV-13
HV-6
35.539
35.539
35.539
81.363
81.363
81.363
360
404
404
399
399
414
4.602
5.863
6.36
5.111
5.135
4.823
0
0
0
0
0
0
3
3
3
6
6
5
2
2
2
3
3
4
310.742
364.022
364.022
343.364
343.364
371.121
0
1
1
1
1
0

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H. S. Virdi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
TMS ¼ 0): 8.01 (1H, d, J ¼ 7.8 Hz), 7.73 (2H, d, J ¼ 7.8 Hz), 7.60 7.59 (1H, m), 7.19–7.31 (5H, m), 7.31–7.46 (4H, m), 5.80 (1H, d,
(1H, m), 7.25–7.48 (9H, m), 5.79 (1H, d, J ¼ 4.8 Hz), 4.68 (1H, d, J ¼ 3.0 Hz), 4.69 (1H, d, J ¼ 3.00 Hz): 13C NMR (CDCl3,
J ¼ 4.8 Hz). 13C NMR (CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 31.172, 75 MHz, ꢀ, TMS ¼ 0): 36.422, 102.858, 102.995, 114.317,
102.923, 103.480, 114.560, 116.862, 122.761, 124.237, 124.905, 116.800, 122.745, 124.321, 124.676, 126.716, 127.397, 128.451,
125.532, 127.028, 128.736, 129.465, 132.148, 147.646, 161.482. 128.705, 129.434, 129.841, 132.272, 134.431, 145.574, 147.152,
Anal. Calc. for C24H15ClO3: C, 74.52; H, 3.91; Found: C, 74.68; 152.726, 156.025, 161.308. Anal. Calc. for C24H15ClO3: C, 74.52;
H, 4.07.
4 -(4-Bromophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one
H, 3.91; Found: C, 74.68; H, 3.82.
2-(4-Methoxyphenyl)-4-(thiophen-2-yl)pyrano[3,2-c]chromen-
(HV-3): m.p.: 168–169 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ, 5(4H)-one (HV-10): m.p.: 134–135 ꢀC; 1H NMR (CDCl3,
TMS ¼ 0): 8.02 (1H, d, J ¼ 6.6 Hz), 7.73 (2H, d, J ¼ 6.3 Hz), 300 MHz, ꢀ, TMS ¼ 0): 8.04 (1H, d, J ¼ 7.8 Hz), 7.92 (2H, d,
7.57 (1H, m), 7.39–7.45 (5H, m), 7.26–7.36 (4H, m), 5.80 (1H, d, J ¼ 8.7 Hz), 7.67 (1H, d, J ¼ 9.00 Hz), 7.54 (1H, m), 7.32–7.38
J ¼ 4.8 Hz), 4.68 (1H, d, J ¼ 4.8 Hz). 13C NMR (CDCl3, 75 MHz, (3H, m), 7.11–7.18 (2H, m), 6.91–6.99 (2H, m), 6.93 (2H, d,
ꢀ, TMS ¼ 0): 36.207, 103.114, 103.220, 114.428, 116.905, J ¼ 9.00 Hz), 5.81 (1H, d, J ¼ 5.1 Hz), 5.02 (1H, d, J ¼ 5.1 Hz).
121.207, 122.718, 124.274, 124.723, 128.744, 129.443, 130.242, 13C NMR (CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 31.118, 55.435,
131.720, 132.204, 132.449, 142.564, 147.255, 152.798. Anal. 101.146, 102.864, 103.523, 113.695, 114.109, 114.607, 116.724,
Calc. for C24H15BrO3: C, 66.84; H, 3.51; Found: C, 67.02; 116.853, 122.751, 124.031, 124.215, 124.839, 125.081, 125.442,
H, 3.76.
126.360, 127.026, 130.329, 130.622, 132.107, 132.606, 147.445,
4-(4-Fluorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one 147.981, 152.326, 152.685, 155.551, 160.573, 161.595, 164.396.
(HV-4): m.p.: 142–143 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ, Anal. Calc. for C23H16SO4: C, 71.12; H, 4.15; S, 8.25; Found: C,
TMS ¼ 0): 8.03 (1H, d, J ¼ 7.5 Hz), 7.75 (2H, d, J ¼ 7.8 Hz), 70.92; H, 3.96; S, 7.99.
7.59 (1H, m), 7.34–7.49 (7H, m), 7.00 (2H, m), 5.83 (1H, d,
2-Phenyl-4-(thiophen-2-yl)pyrano[3,2-c]chromen-5(4H)-one
J ¼ 4.8 Hz), 4.72 (1H, d, J ¼ 4.8 Hz). Anal. Calc. for C24H15FO3: (HV-11): m.p.: 168–169 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ,
C, 77.83; H, 4.08; Found: C, 78.11; H, 3.95.
TMS ¼ 0): 8.00 (1H, d, J ¼ 7.5 Hz), 7.76 (2H, d, J ¼ 6.3 Hz), 7.58
4-(4-Nitrophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (1H, m), 7.34–7.48 (5H, m), 7.19 (1H, d, J ¼4.5 Hz), 7.12 (1H, d,
(HV-5): m.p.:190–191 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ, J ¼ 3.6 Hz), 6.95 (1H, dd, J ¼ 3.6 Hz and 5.1 Hz), 5.95 (1H, d,
TMS ¼ 0): 8.18 (2H, d, J ¼ 9.00 Hz), 8.04 (1H, d, J ¼7.5 Hz), J ¼ 5.1 Hz), 5.06 (1H, d, J ¼ 5.1 Hz). 13C NMR (CDCl3, 75 MHz,
7.73–7.81 (2H, m), 7.58–7.61 (3H, m), 7.35–7.48 (5H, m), 5.78 ꢀ, TMS ¼ 0): 31.161, 102.907, 103.452, 114.539, 116.846,
(1H, d, J ¼ 4.8 Hz), 4.85 (1H, d, J ¼ 4.8 Hz). 13C NMR (CDCl3, 122.762, 124.254, 124.894, 125.522, 127.028, 128.736, 129.468,
75 MHz, ꢀ, TMS ¼ 0): 36.721, 102.141, 102.425, 114.200, 132.162, 132.498, 147.575, 147.643, 152.700, 155.539, 161.491.
116.986, 117.586, 122.807, 123.013, 123.251, 123.489, 123.929, Anal. Calc. for C22H14SO3: C, 73.72; H, 3.94; S, 8.95; Found: C,
124.070, 124.220, 124.466, 124.795, 125.041, 128.026, 128.308, 73.93; H, 4.12; S, 9.11.
128.556, 128.826, 128.939, 129.427, 129.740, 130.283, 130.536,
131.566, 132.119, 132.560, 133.077, 147.101, 147.834, 150.613, one (HV-12): m.p.: 192–193 ꢀC; H NMR (CDCl3, 300 MHz, ꢀ,
152.867, 156.315, 161.289. Anal. Calc. for C24H15NO5: C, 72.54; TMS ¼ 0): 8.39 (1H, d, J ¼ 8.7 Hz), 8.08 (1H, d, J ¼7.8 Hz), 7.89
4-(Naphthalen-1-yl)-2-phenylpyrano[3,2-c]chromen-5(4H)-
1
H, 3.80; N, 3.52; Found: C, 72.48; H, 4.02; N, 3.76.
4-(3,4-Dimethoxyphenyl)-2-phenylpyrano[3,2-c]chromen- 7.32–7.45 (7H, m), 5.96 (1H, d, J ¼ 4.5 Hz), 5.57 (1H, d,
(1H, d, J ¼ 7.8 Hz), 7.74 (1H, d, J ¼ 7.2 Hz), 7.50–7.68 (5H, m),
1
5(4H)-one (HV-6): m.p.: 85–86 ꢀC; H NMR (CDCl3, 300 MHz, J ¼ 4.5 Hz). 13C NMR (CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 32.100,
ꢀ, TMS ¼ 0): 8.02 (1H, d, J ¼ 7.8 Hz), 7.74 (2H, d, J ¼ 6.3 Hz), 103.029, 103.775, 114.538, 116.964, 122.702, 122.939, 124.270,
7.57 (1H, m), 7.36–7.46 (5H, m), 6.99 (1H, s), 6.93 (1H, d, 124.640, 125.799, 125.931, 126.584, 127.736, 128.613, 129.022,
J ¼ 8.4 Hz), 6.80 (1H, d, J ¼ 8.1 Hz), 5.85 (1H, d, J ¼ 5.1 Hz), 129.181, 130.839, 132.153, 132.665, 134.084, 140.024, 146.361,
4.66 (1H, d, J ¼ 4.8 Hz), 3.86 (6H, s), 5.85 (1H, d, J ¼ 5.1 Hz), 152.892, 156.972, 161.420. Anal. Calc. for C28H18O3: C, 83.57;
4.66 (1H, d, J ¼ 4.8 Hz). Anal. Calc. for C26H21O5: C, 75.72; H, H, 4.51; Found: C, 83.49; H, 4.66.
4.89; Found: C, 75.84; H, 5.05.
2-(4-Nitrophenyl)-4-phenylpyrano[3,2-c]chromen-5(4H)-one
4-(4-Methoxyphenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)- (HV-13): m.p.: 120–124 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ,
1
one (HV-7): m.p.: 132–133 ꢀC; H NMR (CDCl3, 300 MHz, ꢀ, TMS ¼ 0): 8.31 (1H, d, J ¼ 9.00 Hz), 7.92 (1H, d, J ¼ 8.7 Hz),
TMS ¼ 0): 8.00 (1H, d, J ¼ 7.5 Hz), 7.72 (2H, d, J ¼ 7.8 Hz), 7.42 7.25–7.61 (11H, m), 6.06 (1H, d, J ¼ 5.4 Hz), 4.76 (1H, d,
(1H, m), 7.41 (2H, d, J ¼ 7.5 Hz), 7.15–7.35 (5H, m), 6.84 (2H, d, J ¼ 4.5 Hz). Anal. Calc. for C24H15NO5: C, 72.54; H, 3.80; N,
J ¼ 8.4 Hz), 5.81 (1H, d, J ¼ 5.1 Hz), 4.66 (1H, d, J ¼ 5.1 Hz), 3.52; Found: C, 72.44; H, 4.04; N, 3.78.
3.84 (3H, s). 13C NMR (CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 35.745,
4-(2-Methoxyphenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-
1
55.299, 103.873, 114.005, 114.613, 116.816, 122.685, 124.182, one (HV-14): m.p.: 168–169 ꢀC; H NMR (CDCl3, 300 MHz, ꢀ,
124.650, 126.130, 128.475, 128.697, 129.230, 129.587, 131.964, TMS ¼ 0): 8.03 (1H, d, J ¼ 7.5 Hz), 7.70 (2H, d, J ¼ 7.5 Hz),
132.685, 135.832, 146.770, 152.715, 155.510, 158.754, 161.568. 7.36–7.49 (5H, m), 7.17–7.25 (3H, m), 6.87–6.98 (2H, m), 5.86
Anal. Calc. for C25H18O4: C, 78.52; H, 4.74; Found: C, 78.69; H, (1H, d, J ¼ 5.4 Hz), 5.12 (1H, d, J ¼ 4.5 Hz), 3.89 (3H, s) 13C
4.99.
NMR (CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 27.225, 30.624, 32.969,
4-(Naphthalen-2-yl)-2-phenylpyrano[3,2-c]chromen-5(4H)- 55.785, 102.480, 103.468, 111.091, 114.646, 116.371, 116.717,
1
one (HV-8): m.p.: 178–179 ꢀC; H NMR (CDCl3, 300 MHz, ꢀ, 116.860, 121.004, 122.134, 122.611, 122.793, 124.118, 124.545,
TMS ¼ 0): 7.74–8.14 (4H, m), 7.43–7.58 (5H, m), 7.28–7.36 (7H, 125.225, 125.686, 128.177, 128.543, 128.638, 128.929, 129.430,
m), 5.90 (1H, d, J ¼ 3.9 Hz), 4.89 (1H, d, J ¼ 3.9 Hz). 13C NMR 131.658, 131.909, 132.012, 132.968, 146.401, 152.872, 156.802,
(CDCl3, 75 MHz, ꢀ, TMS ¼ 0): 18.405, 36.823, 58.455, 103.706, 157.128, 161.414, 192.114. Anal. Calc. for C25H18O4: C, 78.52;
116.882, 122.743, 124.220, 124.735, 125.810, 126.103, 126.511, H, 4.74; Found: C, 78.69; H, 4.56.
127.199, 127.616, 127.940, 128.428, 128.706, 129.304, 132.068,
132.710. Anal. Calc. for C28H18O3: C, 83.57; H, 4.51; Found: C, ano[3,2-c]chromen-5(4H)-one (HV-15): m.p.: 122–123 ꢀC; 1H
83.33; H, 4.77.
4-(4-Hydroxy-3-methoxyphenyl)-2-(3,4-dimethoxyphenyl)pyr-
NMR (CDCl3, 300 MHz, ꢀ, TMS ¼ 0): 7.98 (1H, bs), 7.36–7.56
4-(3-Chlorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (6H, m), 6.75–7.00 (4H, m), 6.06 (1H, d, J ¼ 4.5 Hz), 4.76 (1H, d,
(HV-9): m.p.: 132–133 ꢀC; 1H NMR (CDCl3, 300 MHz, ꢀ, J ¼ 4.5 Hz), 3.95 (3H, s), 3.93 (3H, s), 3.87 (3H, s). Anal. Calc. for
TMS ¼ 0): 8.02 (1H, d, J ¼ 8.1 Hz), 7.72 (2H, d, J ¼ 7.8 Hz), C27H22O7: C, 70.73; H, 4.84; Found: C, 71.01; H, 5.06.

DOI: 10.3109/14756366.2014.961446
New class of non-purine xanthine oxidase inhibitors
7
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Xanthine oxidase assay
Bovine milk xanthine oxidase (grade 1, ammonium sulfate
suspension, Sigma–Aldrich) activity was assayed spectrophoto-
metrically by measuring the uric acid formation at 293 nm using a
Hitachi U-3010 UV–visible spectrophotometer at 25 ꢀC. The
reaction mixture contained 50 mM potassium phosphate buffer
(pH 7.6), 75 mM xanthine and 0.08 units of xanthine oxidase.
Inhibition of xanthine oxidase a ctivity by various inhibitors was
measured by following the decrease in the uric acid formation at
293 nm at 25 ꢀC. The enzyme was pre-incubated for 5 min, with
test compound, dissolved in DMSO (1% v/v), and the reaction was
started by the addition of xanthine. Final concentration of DMSO
(1% v/v) did not interfere with the enzyme activity. All the
experiments were performed in triplicate, and values were
expressed as means of three experiments38.
Declaration of interest
23. Curini M, Epifano F, Maltese F, et al. Synthesis of collinin, an
antiviral coumarin. Aust J Chem 2003;56:59–60.
The authors declare no conflict of interest.
24. Chang WS, Chiang HC. Structure-activity relationship of coumarins
in xanthine oxidase inhibition. Anticancer Res 1995;15:1969–73.
25. Lin HC, Tsai SH, Chen CS, et al. Structure-activity relationship of
coumarin derivatives on xanthine oxidase-inhibiting and free radical-
scavenging activities. Biochem Pharmacol 2008;75:1416–25.
26. Umamaheswari M, Madeswaran A, Asokkumar K, et al. Discovery
of potential xanthine oxidase inhibitors using in silico docking
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