Screening of a library of 4-aryl/heteroaryl-4H-fused pyrans for xanthine oxidase inhibition: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1007/s00044-015-1382-0

A series of 4-aryl/heteroaryl-4H-fused pyrans was synthesized via multicomponent reaction in a microwave synthesizer. All the pyrans were evaluated for in vitro xanthine oxidase inhibition. Structure-activity relationship was also established. Among the s

Full text of DOI:10.1007/s00044-015-1382-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1382-0
ORIGINAL RESEARCH
Screening of a library of 4-aryl/heteroaryl-4H-fused pyrans
for xanthine oxidase inhibition: synthesis, biological evaluation
and docking studies
Ramandeep Kaur1 Fatima Naaz1 Sahil Sharma1 Samir Mehndiratta2 Manish Kumar Gupta3
•
•
•
•
•
Preet Mohinder Singh Bedi1 Kunal Nepali1
•
Received: 25 July 2014 / Accepted: 23 May 2015
Ó Springer Science+Business Media New York 2015
Abstract A series of 4-aryl/heteroaryl-4H-fused pyrans
was synthesized via multicomponent reaction in a mi-
crowave synthesizer. All the pyrans were evaluated for
in vitro xanthine oxidase inhibition. Structure–activity re-
lationship was also established. Among the series of 108
compounds, Compound 5n was the most potent displaying
remarkable inhibition against the enzyme with an IC50
value of 0.59 lM. Enzyme kinetic study was carried out
for the compound 5n to determine the type of inhibition.
The study revealed that the compound 5n was a mixed-type
inhibitor. Molecular modelling studies were also performed
to figure out the interactions of both the enantiomers of 5n
with the amino acid residues of the enzyme.
Graphical Abstract
Keywords Pyrans Á Xanthine oxidase Á Inhibition Á
Molecular modelling studies Á Mixed type
& Kunal Nepali
kunal_8855@rediffmail.com
1
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar 143005, Punjab, India
Introduction
2
Present Address: Department of Medicinal Chemistry,
College of Pharmacy, Taipei Medical University, Taipei,
Taiwan
Oxidative hydroxylation of hypoxanthine and xanthine
catalysed by xanthine oxidase to produce uric acid and
reactive oxygen species leads to many diseases such as
gout and at least symptoms of diseases such as oxidative
3
Department of Pharmaceutical Chemistry, ISF College of
Pharmacy, Moga, Punjab, India
123

Med Chem Res
damage to the tissue (Sharma et al., 2014; Stockert et al.,
2002; Borges et al., 2002; Hille, 2006). Therefore, the se-
lective inhibition of XO may result in broad-spectrum
chemotherapeutic for gout, cancer, inflammation and ox-
idative damage (Borges et al., 2002; Hille, 2006; Pacher
et al., 2006). Allopurinol (Hille, 2006; Pacher et al., 2006),
2-alkyl hypoxanthines (Biagi et al., 2001; Robins et al.,
1985), pterin and 6-formylpterin (Oettl and Reibneggar,
1999) represent the class of purine-based xanthine oxidase
inhibitors. All these inhibitors have been successfully uti-
lized 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
patients (Borges et al., 2002; Hille, 2006; Pacher et al.,
2006). Keeping in view these side effects, our research
group has been actively involved in the design of some
non-purine xanthine oxidase inhibitors in the recent past
such as azaflavones (Nepali et al. 2011a, b), n-acetyl
pyrazolines (Nepali et al. 2011a, b), b-acetamido com-
pounds (Dhiman et al., 2012), naphthopyrans (Sharma
et al., 2014) and 4,6-diaryl/heteroarylpyrimidin-2(1H)-ones
(Shukla et al., 2014).
view of some of the potent non-purine xanthine oxidase
inhibitors possessing benzopyran skeleton. The potent in-
hibitory potential of some naphthopyrans was attributed to
the interactions of pyran ring as indicated by molecular
modelling studies (Sharma et al., 2014).
In continuation of our search for non-purine-based xan-
thine oxidase inhibitors (Dhiman et al., 2012; Nepali et al.
2011a, b; Sharma et al., 2014; Singh et al., 2014; Shukla
et al., 2014; Virdi et al., 2014) and motivated by the
promising xanthine oxidase inhibitory potential of naph-
thopyrans, the present study screens a library of fused pyrans
in diverse scaffolds for xanthine oxidase inhibition. A library
of 4-aryl/heteroaryl-4H-fused pyrans was synthesized and
evaluated against the enzyme. The type of inhibition and the
interactions of the most potent inhibitor with the amino acid
residues of the enzyme have also been figured out.
Results and discussion
Synthesis
A library of 4H-pyrans was synthesized as shown in
Scheme 1. The compounds were synthesized by exposing a
mixture of aromatic aldehyde, malononitrile, C–H-acti-
vated acidic compound and catalytic amount of DMAP to
microwave radiation in a microwave synthesizer operating
at 150 °C with the maximum microwave power of 400 W
(Scheme 1). The structures of the synthesized compounds
Polyfunctionalized 4H-pyrans have a unique role in
medicinal chemistry due to their wide range of biological
and pharmacological activities (Elnagdi et al., 1983;
Goldmann and Stoltefus, 1991). These compounds have
been utilized as anticancer agents, anticoagulants, spas-
molytics and antianaphylactics (Andreani and Lapi, 1960;
Bonsignore et al., 1993). 4H-Pyran derivatives containing
heterocyclic rings are extensively used for their pharma-
cological activities (Green et al., 1995; Sanchez et al.,
2012). Fused pyran derivatives also exhibit a wide spec-
trum of pharmacological activities and biological activities,
such as insecticidal (Uher et al., 1994), antiviral and an-
tileishmanial (Perez-Perez et al., 1995; Fan et al., 2010),
anticonvulsant and antimicrobial activities (Aytemir et al.,
2004). Also, many of them are non-peptide human im-
munodeficiency virus (HIV) protease inhibitors (Wang
et al., 1996; Pochet et al., 1996; Mazumder et al., 1996).
Pyrans are also an important structural motif in number of
non-purine xanthine oxidase inhibitors (Nepali et al.
2011a, b; Star and Marby, 1971; Cos et al., 1998). Cou-
marins and flavonoids represent the class of fused pyrans as
non-purine xanthine oxidase inhibitors (Nepali et al.
2011a, b; Cos et al., 1998; Da-Silva et al., 2004; Lin et al.,
2002). Both the classes have been extensively explored for
their xanthine oxidase inhibitory potential and insights
about the structure–activity relationship, and their interac-
tions with the amino acid residues of the enzyme have also
been figured out. Recently working on similar lines, our
research group synthesized and evaluated a series of
naphthopyrans for in vitro xanthine oxidase inhibition in
1
were elucidated by H NMR and 13C NMR. All spectral
data were in accordance with assumed structures.
In vitro xanthine oxidase assay
In vitro screening of the pyrans using bovine milk xanthine
oxidase (grade 1, ammonium sulphate suspension) enzy-
matic assay was performed as described in the literature
(Escribano et al., 1988; Takano et al., 2005). Allopurinol
(Pacher et al., 2006) was employed as reference inhibitor.
The molecules exhibiting % age inhibition of more than
80 % at 50 lM were further tested in triplicate for the
xanthine oxidase inhibitory activity to calculate the IC50
values. Among a series of 108 compounds, 41 compounds
were found to display a % age inhibition of [80 % and
were tested at different concentration against xanthine
oxidase (Table 1; Fig. 1). Compounds 5m and 5n dis-
played significant inhibitory potential with IC50 values, 0.9
and 0.59 lM, respectively (IC50 value of allopuri-
nol = 8.29 lM). Figure 2 shows interesting structure–ac-
tivity relationship for the inhibitory effects against the
enzyme. Careful observation of the IC50 values of the
compounds indicates that nature of Ring A and Ring C
remarkably influences the activity. Few generalizations
123

Med Chem Res
Scheme 1 Synthesis of 4H-
pyrans. *C–H-activated acidic
compound in case of 3 was
prepared from the reaction of
hydrazine hydrate and ethyl
acetoacetate
* C-H activated acidic compound in case of 3 was prepared from the reaction of hydrazine hydrate and ethyl acetoacetate.
about the structure–activity relationship are as follows: (1)
compounds with nitro- and halo-substituted Ring C (1g, 1j,
1s, 2s, 3g, 3j, 3s, 4g, 4j, 4s, 5g, 5j, 5s, 6g, 6j, 6s, 7g, 7j, 7s,
8g, 8j, 8s) exhibited significant inhibition, whereas com-
pounds with methoxy- and hydroxy-substituted phenyl
rings (1e, 1h, 1i, 1k, 1p, 1q, 2e, 2f, 2h, 2i, 2k, 2q, 3c, 3f,
3h, 3i, 3k, 3q, 3r, 4h, 4i, 5h, 5i, 5k, 5p, 5q, 6e, 6h, 6i, 6k,
6q, 7h, 7i, 8h, 8i) did not qualify for the evaluation at
different concentration (i.e. % age inhibition\80). (2) The
influence of placement of substitution was also evident
from Fig. 1 as compounds with para substituted phenyl
rings (halo and nitro groups) were the only one to pass the
initial screening at 50 lM by displaying % age inhibition of
[80 (1g, 1j, 1s, 2s, 3g, 3j, 3s, 4g, 4j, 4s, 5q, 5j, 5s, 6g, 6j,
6s, 7g, 7j, 7s, 8g, 8j, 8s). (3) The compounds with sub-
stituted phenyl rings (substitutions other than nitro and
halo, Ring C) were even less active than compounds
bearing unsubstituted phenyl ring (Ring C) (1a, 3a, 4a, 5a,
6a, 7a, 8a). (4) Replacement of phenyl rings with naphthyl
ring (compare 1a, 3a, 5a with 1l, 3l, 5l) resulted in decline
in the inhibitory potential of the compounds as naphthyl-
substituted (Ring C) inhibitors displayed % age inhibition
of \80 % and did not qualify for evaluation at different
concentrations. (5) Replacement of phenyl rings with het-
eroaryl rings (Ring C) resulted in drastic improvement in
the activity (compare 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a with
1m, 1n, 2n, 3m, 3n, 4n, 5m, 5n, 6n, 7n, 8m, 8n). (6)
Among the heteroaryl-substituted compounds (Ring C),
compounds with thiophenyl Ring C were more active than
the compound with furanyl ring (Ring C) (compare 1n, 3n,
5n, 8n with 1m, 3m, 5m, 8m). This could be attributed to
the higher aromatic character of thiophene ring as com-
pared to Furan ring. Overall the preference order for Ring
C is as follows: thiophene [ furanyl [ phenyl with halo
(preferable chloro at para position) [ phenyl with nitro
(para substituted). (7) Ring A also displayed significant
influence on the inhibitory potential. Coumarin-substituted
(Ring A) compounds were found to be the most active
(compare 5a with 1a, 2a, 3a, 4a, 6a, 7a, 8a). (8) Com-
pounds with bicyclic ring (Ring A) also displayed a sig-
nificant inhibitory potential higher than compounds with
monocyclic rings (compare 5a, 6a, 8a with 1a, 2a, 3a, 4a,
7a). (9) Both 1-naphthyl- and 2-naphthyl-substituted pyr-
ans (Ring A) displayed promising results; however, no
competition was observed between the activity profiles of
two. Overall the preference order for Ring A is as follows:
123

Med Chem Res
Enzyme kinetic study
Table 1 % age inhibition of the synthesized compounds at 50 lM
Code
% age
inhibition
(50 lM)
Code
% age
inhibition
(50 lM)
Code
% age
inhibition
(50 lM)
Compound 5n was further investigated for the type of in-
hibition, and enzyme kinetics study was carried out. The
Lineweaver–Burk plot (Fig. 3) revealed that the compound
5n was 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 concentration of the
inhibitor. The inhibitor has increased the Km and slope
(Km/Vmax) while decreasing the Vmax. Moreover, Figure 3
shows 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 that defines the degree to which
inhibitor binding affects the affinity of the enzyme for
substrate) is[1. Mixed-type inhibitors are those which are
capable of binding to both the free enzyme and the en-
zyme–substrate complex. However, keeping in view the
pattern of intersecting lines on the graph, it can be assumed
that the inhibitor preferentially binds to the free enzyme
and not the enzyme–substrate complex (Copeland, 2005).
1a
1b
1d
1e
1g
1h
1i
81
62
59
42
88
55
56
84
66
42
84
89
46
59
88
43
56
68
75
35
50
73
59
37
58
23
83
63
87
26
83
45
24
62
27
85
48
3i
38
82
45
54
84
89
44
55
52
88
79
74
83
31
84
10
63
85
90
88
37
66
89
75
89
42
37
85
34
49
93
96
66
65
90
52
15
5w
6a
6b
6d
6e
6g
6h
6i
25
87
43
61
68
82
64
41
86
47
88
63
89
35
80
46
85
32
70
81
89
83
68
70
85
33
87
66
51
83
85
87
86
44
3j
3k
3l
3m
3n
3o
3q
3r
3s
1j
1k
1l
6j
6k
6n
6q
6s
1m
1n
1p
1q
1s
3t
3u
4a
4d
4g
4h
4i
6v
7a
7d
7g
7h
7i
1u
1v
2a
2b
2e
2f
Molecular modelling study
4j
4n
4s
Molecular docking study was performed to get structural
insights into the binding behaviour of the potent compound
5n. A flexible docking study was performed using Gold
Software (GOLD 2012). Compound 5n has a chiral centre;
therefore, both R and S conformations of 5n were docked.
The binding poses with highest fitness score were selected,
and their binding interactions were studied.
7j
4u
4v
5a
5b
5g
5h
5i
7n
7s
2g
2h
2i
7u
7v
8a
8b
8g
8h
8i
2j
The docking study reveals that S-enantiomer of 5n fits
well in the binding site, while R-enantiomer was not able to
get in the cavity (Fig. 4). The binding interactions of S-
enantiomer with highest score were studied. In binding
pose, S-enantiomer of 5n fits well in the binding cavity and
gets stabilized by various molecular interactions. The
chromene ring gets sandwiched in Phe914 and Phe1009
showing ‘‘face-to-face’’ and ‘‘edge-to-face’’ pie-stacking,
respectively. The carbonyl group of chromene ring was
found to involve in hydrogen bonding with Thr1010.
Another hydrogen bonding was observed between Glu802
and oxygen of 5n. The sulphur of thiophene ring was found
to be involved in van der Waals interactions with Ser876.
The above interactions provided an insight behind the in-
hibition of XO by 5n (Fig. 4).
2k
2n
2q
2s
5j
5k
5l
2v
3a
3b
3c
3d
3f
8j
5m
5n
5o
5q
5s
8m
8n
8s
8v
3g
3h
5p
5v
coumarin [ 1-naphthyl = 2-naphthyl [ pyrazole [ cyclo-
hexanedione [ thiobarbituric acid [ barbituric acid [ 5,5-
dimethyl cyclohexanedione. Figure 2 represents the struc-
ture–activity relationship.
Conclusion
Allopurinol, a well-known xanthine oxidase inhibitor, is a
competitive inhibitor and has been employed as standard
for the in vitro and in vivo studies over the years. However,
its use has been associated with some complications. The
Compound 5n, the most active of the series with an IC50
value 0.59 lM, was further investigated for enzyme ki-
netics study and molecular modelling studies.
123

Med Chem Res
C
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A
NH2
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NH2
CN
CN
I
NH2
O
O
O
S
O
I
H3C
HN
I
CN
CN
CN
CN
CN
NH
O
I
HN
HN
a =
I
O
N
I
O
NH2
O
NH2
O
N
H
O
NH2
O
6a
NH2
O
N
H
O
7a
2I
I
8a
IC50= 9.7µM
5a
IC50= 7.43 µM
4a
3a
IC50= 12.4 µM
1a
IC50= 22.2 µM
IC50= 34.3 µM
IC50= 9.5 µM
IC50= 26.4 µM
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Br
Br
Br
Br
Br
Br
CN
NH2
NH2
CN
O
O
O
O
S
O
H3C
HN
CN
CN
CN
HN
CN
CN
HN
g =
O
Br
O
N
H
O
NH2
O
NH2
N
O
NH2
O
N
H
O
NH2
O
NH2
8g
IC50 = 7.57 µM
5g
IC50= 5.5 µM
3g
4g
6g
IC50= 7.5 µM
7g
IC50 = 16.4 µM
1g
IC50= 10.9 µM
IC50= 14.4 µM
IC50= 6.4 µM
_______________________________________________________________________________________________________________________________________________________________
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NO2
NO2
NO2
NO2
NO2
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O N
2
I
NH2
CN
I
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CN
I
I
NH2
O
O
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O
O
S
I
H3C
HN
I
O
CN
NH
CN
CN
I
I
CN
CN
HN
HN
I
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j =
O
NO2 I
I
O
N
H
O
O
NH2
N
2I
I
O
NH2
O
N
H
O
NH2
O
NH2
I
I
I
8j
IC50= 8.39 µM
5j
IC50 = 4.5 µM
3j
4j
7j
6j
1j
IC = 13.4 µM
I
IC50= 17.4 µM
IC50= 8.4 µM
IC50= 19.87 µM
IC50= 8.5 µM
___5_0___________________________________________________________________________________________________________________________________________________________
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NH2
CN
O
O
CN
NH2
O
O
O
H3C
HN
CN
CN
O
O
m =
N
O
NH2
O
NH2
O
5m
O
1m
3m
8m
IC50= 1.97 µM
IC50= 3.58 µM
IC50= 3.2 µM
IC = 0.9 µM
__________________________________________________________________________________5_0____________________________________________________________________________
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NH2
CN
S
S
I
S
S
S
CN
O
S
NH2
I
O
O
O
S
S
O
H3C
HN
CN
I
CN
NH
CN
CN
CN
HN
S
O
CN
I
HN
I
n =
O
NH2
I O
N
H
O
2I
I
N
O
NH2
O
NH2
O
O
NH2
N
H
O
O
NH2
I
I
I
2n
5n
IC50= 0.59 µM
6n
IC50= 1.9 µM
8n
IC50= 1.67 µM
3n
4n
7n
1n
I
IC50= 12.58 µM
IC50= 7.4 µM
IC50= 10.87 µM
IC50= 3.08 µM
IC50= 2.24 µM
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Cl
Cl
Cl
Cl
Cl
Cl
Cl
NH2
CN
CN
NH2
O
O
S
O
O
O
H3C
HN
CN
CN
O
CN
CN
CN
CN
HN
HN
s =
N
O
Cl
O
N
H
O
NH2
O
NH2
O
NH2
O
N
H
O
NH2I
O
NH2
O
NH2
I
I
8s
IC50= 3.01 µM
1s
IC50= 4.32 µM
2s
7s
3s
IC50= 4.01 µM
5s
IC50= 1.3 µM
6s
IC50 = 2.9 µM
4s
IC50= 33.12 µM
IC50= 12.6 µM
IC50= 9.4 µM
I
_______________________________________________________________________________________________________________________________________________________________
I
I
I
I
I
I
I
I
Fig. 1 IC50 values of selected compounds (IC50 value of allopurinol = 8.29 lM)
competitive inhibitors are basically purine-based struc-
tures, and the interactions of purine analogue XO inhibitors
with the activities of purine and pyrimidine metabolism
enzymes such as guanine deaminase, HGPRT (hypoxan-
thine–guanine phosphoribosyltransferase), PNP (purine
nucleoside phosphorylase), OPRT (orotate phosphoribo-
syltransferase) and OMPDC (orotidine-5-monophosphate
decarboxylase) leading to the hypersensitivity (Steven–
Johnson) a syndrome characterized by fever, skin rash,
hepatitis, leukocytosis with eosinophilia and worsening
renal function induced in some of the patients has basically
C
N
A
B
O
NH2
PREFERENCE ORDER
RING A = coumarin > 1-naphthyl = 2-naphthyl > pyrazole > cyclohexanedione > thiobarbituric
acid > barbituric > 5,5-dimethyl cyclohexanedione
RING C= thiophene > furanyl > phenyl with halo (preferable chloro at para position) > phenyl
with nitro (para substituted)
Fig. 2 Structure–activity relationship
123

Med Chem Res
Fig. 3 Lineweaver–Burk plot
encouraged us to focus on XO inhibitors with structurally
diverse and novel non-purine isosteres. Moreover, the
success of febuxostat has further motivated us to focus on
non-purine isosters. Febuxostat is a non-purine selective
inhibitor of xanthine oxidase. It works by non-com-
petitively blocking the molybdenum pterin centre which is
the active site on xanthine oxidase. Many long- and short-
term clinical trials have proved the efficacy of febuxostat in
the treatment of gout and lowering uric acid levels. In these
studies, febuxostat was found to be superior to allopurinol
in reducing the serum uric acid levels. Thus, all these
reasons have collectively led us to investigate non-com-
petitive chemical architectures for xanthine oxidase inhi-
bition. Keeping in view the success of some non-purine
xanthine oxidase inhibitors, a library of 4H-pyrans was
designed in the present study and the compounds were
evaluated for inhibitory effects against the enzyme xan-
thine oxidase. All the compounds were first screened at
50 lM, and the compounds displaying a % age inhibition of
[80 were further evaluated at different concentrations.
Structure–activity relationship revealed that Ring A as well
as Ring C remarkably influences the inhibitory potential.
The most potent compound 5n was investigated to explore
the type of inhibition it was exerting, and thus, enzyme
kinetics study was carried out on 5n. The Lineweaver–
Burk plot revealed that compound 5n was a mixed-type
inhibitor. The compound was studied for its interactions
with the amino acid residues. The 3D structural coordinates
of XO were obtained from protein databank (PDB ID:
1VDV) for the docking study. The docking study reveals
that S-enantiomer of 5n fits well in the binding site, while
R-enantiomer was not able to get in the cavity (Fig. 4). S-
enantiomer of 5n fits well in the binding cavity and gets
stabilized by various molecular interactions, i.e. ‘‘face-to-
face’’ and ‘‘edge-to-face’’ pie-stacking and hydrogen
bonding.
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 purification. Biotage Microwave Synthesizer (Model:
Initiator) operating at 150 °C with the microwave power
maximum level of 400 W. Products were characterized by
spectral data. 1H NMR and 13C NMR spectra were
recorded on Bruker Avance II 500 NMR Spectrometer and
123

Med Chem Res
flask was exposed to microwave radiation for 20 min in a
microwave reactor operating at 150 °C with the maximum
microwave power of 400 W. Cold methanol was added to
the reaction mixture, and the solid precipitates were filtered
off to obtain the desired product.
The structures of the synthesized compounds were elu-
1
cidated by H NMR and 13C NMR. All spectral data were
in accordance with assumed structures. In each occasion,
the spectral data (1H and 13C NMR) of known compounds
were compared with that reported in the literature.
2-Amino-5,6,7,8-tetrahydro-5-oxo-4-phenyl-4H-chro-
mene-3-carbonitrile (1a) (Yu and Da-Ming, 2012; Xu
et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(4-fluor-
ophenyl)-5-oxo-4H-chromene-3-carbonitrile (1b) (Yu
and Da-Ming, 2012), 2-amino-5,6,7,8-tetrahydro-4-(4-
bromophenyl)-5-oxo-4H-chromene-3-carbonitrile (1c)
(Yu and Da-Ming, 2012), 2-amino-5,6,7,8-tetrahydro-4-
(2-methoxyphenyl)-5-oxo-4H-chromene-3-carbonitrile
(1h) (Xu et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(4-
methoxyphenyl)-5-oxo-4H-chromene-3-carbonitrile (1i)
(Xu et al., 2011; Rostamnia and Morsali, 2014),
2-amino-5,6,7,8-tetrahydro-4-(4-nitrophenyl)-5-oxo-4H-
chromene-3-carbonitrile (1j) (Yu and Da-Ming, 2012;
Xu et al., 2011; Rostamnia and Morsali, 2014; Hosseini-
Monfared et al., 2013), 2-amino-5,6,7,8-tetrahydro-4-(4-
hydroxyphenyl)-5-oxo-4H-chromene-3-carbonitrile (1k)
(Xu et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(furan-2-
yl)-5-oxo-4H-chromene-3-carbonitrile (1m) (Xu et al.,
2011), 2-amino-5,6,7,8-tetrahydro-4-(3,4-dimethoxyphenyl)-
5-oxo-4H-chromene-3-carbonitrile (1q) (Yu and Da-Ming,
2012; Xu et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(4-
chlorophenyl)-5-oxo-4H-chromene-3-carbonitrile (1s) (Yu
and Da-Ming, 2012; Rostamnia and Morsali, 2014; Hosseini-
Monfared et al., 2013), 2-amino-5,6,7,8-tetrahydro-4-(3-ni-
trophenyl)-5-oxo-4H-chromene-3-carbonitrile (1v) (Xu et al.,
2011), 2-amino-5,6,7,8-tetrahydro-7,7-dimethyl-5-oxo-4-phe-
nyl-4H-chromene-3-carbonitrile (2a) (Kumar et al., 2009;
Sadegh and Ali, 2014; Yu and Da-Ming, 2012; Gao et al.,
2008; Hasaninejad et al., 2013; Jiang-Cheng et al., 2011;
Bihani et al., 2013; Khaksar et al., 2012; Banerjee et
al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(4-fluorophenyl)-7,7-
dimethyl-5-oxo-4H-chromene-3-carbonitrile (2b) (Sadegh
and Ali, 2014; Gao et al., 2008; Khaksar et al., 2012),
2-amino-5,6,7,8-tetrahydro-4-(4-bromophenyl)-7,7-dimethyl-
5-oxo-4H-chromene-3-carbonitrile (2g) (Sadegh and Ali,
2014; Khaksar et al., 2012), 2-amino-5,6,7,8-tetrahydro-4-(2-
methoxyphenyl)-7,7-dimethyl-5-oxo-4H-chromene-3-carboni-
trile (2h) (Yu and Da-Ming, 2012; Jiang-Cheng et al., 2011),
2-amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7,7-dime-
thyl-5-oxo-4H-chromene-3-carbonitrile (2i) (Kumar et al.,
2009; Jiang-Cheng et al., 2011; Bihani et al., 2013; Khaksar
et al., 2012; Banerjee et al., 2011), 2-amino-5,6,7,8-te-
trahydro-4-(4-nitrophenyl)-7,7-dimethyl-5-oxo-4H-chromene-
Fig. 4 a Binding poses of R-enantiomer (brown) and S-enantiomer
(green) in binding cavity of XO. b Binding interactions of S-
enantiomer of 5n (green) (Color figure online)
JEOL AL 300 NMR Spectrometer. The spectra were
measured in DMSO-d6 relative to TMS (0.00 ppm).
Melting points were determined in open capillaries and
were uncorrected.
Experimental procedure for the synthesis of 4H-
pyrans (1, 2, 3, 4, 5, 6, 7, 8)
A mixture of aromatic aldehyde (1 mmol), malononitrile
(1 mmol), C–H-activated acidic compound (1 mmol) and
catalytic amounts of DMAP (5 mol%) in a 50-ml conical
123

Med Chem Res
3-carbonitrile (2j) (Kumar et al., 2009; Sadegh and Ali, 2014;
Yu and Da-Ming, 2012; Hasaninejad et al., 2013; Jiang-
Cheng et al., 2011; Bihani et al., 2013; Khaksar et al., 2012;
Banerjee et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(4-hy-
droxyphenyl)-7,7-dimethyl-5-oxo-4H-chromene-3-carbonitrile
(2k) (Gao et al., 2008; Jiang-Cheng et al., 2011; Banerjee
et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(thiophen-2-yl)-
7,7-dimethyl-5-oxo-4H-chromene-3-carbonitrile (2n) (Hasa-
ninejad et al., 2013), 2-amino-5,6,7,8-tetrahydro-4-(3,4-meth-
oxyphenyl)-7,7-dimethyl-5-oxo-4H-chromene-3-carbonitrile
(2q) (Sadegh and Ali, 2014; Jiang-Cheng et al., 2011),
2-amino-5,6,7,8-tetrahydro-4-(4-chlorophenyl)-7,7-dimethyl-
5-oxo-4H-chromene-3-carbonitrile (2s) (Sadegh and Ali,
2014; Yu and Da-Ming, 2012; Gao et al., 2008; Jiang-Cheng
et al., 2011; Bihani et al., 2013; Khaksar et al., 2012; Ban-
erjee et al., 2011), 2-amino-5,6,7,8-tetrahydro-4-(3-nitro-
phenyl)-7,7-dimethyl-5-oxo-4H-chromene-3-carbonitrile (2v)
(Kumar et al., 2009; Yu and Da-Ming, 2012; Jiang-Cheng
et al., 2011; Bihani et al., 2013; Khaksar et al., 2012; Ban-
erjee et al., 2011), 6-amino-2,4-dihydro-3-methyl-4-
phenylpyrano[2,3-c]pyrazole-5-carbonitrile (3a) (Ali and El-
Remaily, 2013; Paul et al., 2013; Bora et al., 2013; Bolligarla
and Das, 2011; Bihani et al., 2013), 6-amino-2,4-dihydro-4-
(4-fluorophenyl)-3-methylpyrano[2,3-c]pyrazole-5-carbonitrile
(3b) (Ali and El-Remaily, 2013; Bora et al., 2013), 6-amino-
2,4-dihydro-4-(3-hydroxyphenyl)-3-methylpyrano[2,3-c]pyra-
zole-5-carbonitrile (3c) (Bora et al., 2013), 6-amino-2,4-di-
hydro-4-(3-chlorophenyl)-3-methylpyrano[2,3-c]pyrazole-5-
carbonitrile (3d) (Ali and El-Remaily, 2013), 6-amino-2,4-
dihydro-4-(2-hydroxyphenyl)-3-methylpyrano[2,3-c]pyrazole-
5-carbonitrile (3f) (Ali and El-Remaily, 2013), 6-amino-2,4-
dihydro-4-(4-bromophenyl)-3-methylpyrano[2,3-c]pyrazole-5-
carbonitrile (3g) (Ali and El-Remaily, 2013; Paul et al., 2013;
Bora et al., 2013), 6-amino-2,4-dihydro-4-(4-methoxyphenyl)-
3-methylpyrano[2,3-c]pyrazole-5-carbonitrile (3i) (Ali and El-
Remaily, 2013; Paul et al., 2013; Bora et al., 2013; Bolligarla
and Das, 2011; Bihani et al., 2013), 6-amino-2,4-dihydro-4-(4-
nitrophenyl)-3-methylpyrano[2,3-c]pyrazole-5-carbonitrile (3j)
(Ali and El-Remaily, 2013; Bolligarla and Das, 2011; Bihani
et al., 2013), 6-amino-2,4-dihydro-4-(4-hydroxyphenyl)-3-
methylpyrano [2,3-c]pyrazole-5-carbonitrile (3k) (Ali and El-
Remaily, 2013; Bolligarla and Das, 2011; Bihani et al., 2013),
6-amino-2,4-dihydro-4-(naphthalen-2-yl)-3-methylpyrano[2,3-
c]pyrazole-5-carbonitrile (3l) (Bolligarla and Das, 2011; Bihani
et al., 2013), 6-amino-2,4-dihydro-4-(thiophen-2-yl)-3-
methylpyrano[2,3-c]pyrazole-5-carbonitrile (3n) (Paul et al.,
2013), 6-amino-2,4-dihydro-4-(3,4-dimethoxyphenyl)-3-
methylpyrano[2,3-c]pyrazole-5-carbonitrile (3q) (Bolligarla
and Das, 2011; Bihani et al., 2013), 6-amino-2,4-dihydro-4-
(4-chlorophenyl)-3-methylpyrano[2,3-c]pyrazole-5-carbonitrile
(3s) (Ali and El-Remaily, 2013; Bolligarla and Das, 2011;
Bihani et al., 2013), 6-amino-2,4-dihydro-4-(pyridin-4-yl)-3-
methylpyrano[2,3-c]pyrazole-5-carbonitrile (3t) (Colombo
et al., 2003), 2-amino-4,5-dihydro-5-oxo-4-phenylpyrano[3,2-
c]chromene-3-carbonitrile (5a) (Safaei et al., 2012; Jain et al.,
2013; Kidwai and Sexena, 2006), 2-amino-4,5-dihydro-4-(4-
bromophenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5g)
(Safaei et al., 2012; Jain et al., 2013), 2-amino-4,5-dihydro-4-
(2-methoxyphenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile
(5h) (Wang et al., 2010), 2-amino-4,5-dihydro-4-(4-methox-
yphenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5i) (Jain
et al., 2013), 2-amino-4,5-dihydro-4-(4-nitrophenyl)-5-oxopy-
rano[3,2-c]chromene-3-carbonitrile (5j) (Jain et al., 2013),
2-amino-4,5-dihydro-4-(4-hydroxyphenyl)-5-oxopyrano[3,2-c]-
chromene-3-carbonitrile (5k) (Xiang-Shan et al., 2005; Kidwai
and Sexena, 2006; Gong et al., 2009), 2-amino-4,5-dihydro-4-
(furan-2-yl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5m)
(Safaei et al., 2012; Jain et al., 2013), 2-amino-4,5-dihydro-4-
(thiophen-2-yl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile
(5n) (Jain et al., 2013), 2-amino-4,5-dihydro-4-(1H-indol-2-
yl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5o) (Abd-
El-Aziz et al., 2004), 2-amino-4,5-dihydro-4-(4-hydroxy-3-
methoxyphenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile
(5p) (Bihani et al., 2013), 2-amino-4,5-dihydro-4-(4-chlor-
ophenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5s) (Jain
et al., 2013; Kidwai and Sexena, 2006), 2-amino-4,5-dihydro-
4-(3-nitrophenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile
(5v) (Jain et al., 2013), 2-amino-4,5-dihydro-4-(2-bro-
mophenyl)-5-oxopyrano[3,2-c]chromene-3-carbonitrile (5w)
(Wang et al., 2010), 2-amino-4-phenyl-4H-benzo[h] chromene-
3-carbonitrile (6a) (Bihani et al., 2013; Khurana et al., 2010),
2-amino-4-(4-fluorophenyl)-4H-benzo[h]chromene-3-car-
bonitrile (6b) (Khurana et al., 2010), 2-amino-4-(4-bro-
mophenyl)-4H-benzo[h]chromene-3-carbonitrile (6g) (Khurana
et al., 2010), 2-amino-4-(2-methoxyphenyl)-4H-benzo[h]-
chromene-3-carbonitrile (6h) (Maalej et al., 2012), 2-amino-
4-(4-methoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (6i)
(Bihani et al., 2013), 2-amino-4-(4-nitrophenyl)-4H-ben-
zo[h]chromene-3-carbonitrile (6j) (Bihani et al., 2013;
Khurana et al., 2010), 2-amino-4-(4-chlorophenyl)-4H-
benzo[h] chromene-3-carbonitrile (6s) (Bihani et al.,
2013; Khurana et al., 2010), 2-amino-4-(3-nitrophenyl)-
4H-benzo[h]chromene-3-carbonitrile (6v) (Bihani et al.,
2013; Khurana et al., 2010), 7-amino-2,3,4,5-tetrahydro-
2,4-dioxo-5-phenyl-1H-pyrano[2,3-d]pyrimidine-6-car-
bonitrile (7a) (Devi et al., 2003), 7-amino-5-(3-chlor-
ophenyl)-2,3,4,5-tetrahydro-2,4-dioxo-1H-pyrano[2,3-d]
pyrimidine-6-carbonitrile (7d) (Xiang-Shan et al., 2005),
7-amino-5-(2-methoxyphenyl)-2,3,4,5-tetrahydro-2,4-dioxo-
1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (7h) (Safaei
et al., 2012), 7-amino-5-(4-nitrophenyl)-2,3,4,5-tetrahy-
dro-2,4-dioxo-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (7j)
(Safaei et al., 2012), 7-amino-5-(4-chlorophenyl)-2,3,4,5-te-
trahydro-2,4-dioxo-1H-pyrano[2,3-d]pyrimidine-6-car-
bonitrile (7s) (Safaei et al., 2012), 7-amino-5-(3-nitrophenyl)-
2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-
123

Med Chem Res
(CH), 58.88 (C), 113.69 (C), 117.33(CN), 124.52 (Ar–C),
124.96 (Ar–C), 125.43 (Ar–C), 125.67 (Ar–C), 126.73
(Ar–C), 126.86 (Ar–C), 128.94 (Ar–C), 132.66 (Ar–C),
133.57 (Ar–C), 134.01 (Ar–C), 158.67 (C), 165.98 (C–
NH2), 196.77 (C=O). Anal. Calcd. for C20H16N2O2: C,
75.93; H, 5.10; N, 8.86; Found: C, 75.20; H, 5.30; N, 9.10.
carbonitrile (7v) (Safaei et al., 2012), 3-amino-1-phenyl-1H-
benzo[f] chromene-2-carbonitrile (8a) (Bihani et al., 2013;
Wang et al., 2008), 3-amino-1-(4-fluorophenyl)-1H-ben-
zo[f]chromene-2-carbonitrile (8b) (Wang et al., 2008),
3-amino-1-(4-bromophenyl)-1H-benzo[f]chromene-2-carboni-
trile (8g) (Wang et al., 2008), 3-amino-1-(4-methoxyphenyl)-
1H-benzo[f]chromene-2-carbonitrile (8i) (Wang et al., 2008),
3-amino-1-(4-nitrophenyl)-1H-benzo[f]chromene-2-carboni-
trile (8j) (Wang et al., 2008; Bihani et al., 2013), 3-amino-1-
(furan-2-yl)-1H-benzo[f]chromene-2-carbonitrile (8m) (Wang
et al., 2008), 3-amino-1-(4-chlorophenyl)-1H-benzo[f]-
chromene-2-carbonitrile (8s) (Wang et al., 2008; Bihani
et al., 2013), 3-amino-1-(4-chlorophenyl)-1H-benzo[f]chromene-
2-carbonitrile (8v) (Wang et al., 2008; Bihani et al., 2013).
The characterization data for the synthesized new
compounds are given below:
2-Amino-4-(thiophen-2-yl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (1n) Yield 85 %; mp: 158–
159 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS = 0): 7.38
(1H, d, J = 4.8 Hz), 7.119 (2H, s, D2O exchangeable pro-
tons), 6.85–6.92 (2H, m), 4.53 (1H, s), 2.56 (2H, bs), 2.31
(2H, bs), 1.90 (2H, m). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 20.33 (CH2), 25.64 (CH), 26.96 (CH2), 36.87
(CH2), 58.44 (C), 113.74 (C), 120.22 (CN), 123.67 (Ar–C),
126.61 (Ar–C), 126.98 (Ar–C), 139.77 (Ar–C), 158.71 (C),
165.83 (C–NH2), 196.27 (C=O). Anal. Calcd. for C14H12-
N2O2S: C, 61.75; H, 4.44; N, 10.29; S, 11.77; Found: C,
61.90; H, 4.35; N, 10.45; S, 11.81.
2-Amino-4-(3-chlorophenyl)-5,6,7,8-tetrahydro-5-oxo-4H-
chromene-3-carbonitrile (1d) Yield 80 %; mp: 210–
211 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS = 0):
7.27–7.35 (2H, m), 7.12–7.18 (2H, m), 7.08 (2H, bs, D2O
exchangeable protons), 4.22 (1H, s), 2.63 (2H, m), 2.30
(2H, t, J = 6 Hz), 1.93 (2H, m). 13C NMR (DMSO-d6,
500 MHz, d, TMS = 0): 20.22 (CH2), 26.94 (CH2), 35.74
(CH2), 36.73 (CH), 57.97 (C), 113.57 (C), 120.01 (CN),
126.45 (Ar–C), 127.07 (Ar–C), 127.47 (Ar–C), 130.75
(Ar–C), 133.36 (C–Cl), 147.74 (Ar–C), 158.97 (C–C),
165.33 (C–NH2), 196.37 (C=O). Anal. Calcd. for C16H13
ClN2O2: C, 72.18; H, 3.94; Cl, 10.65; N, 8.42; Found: C,
72.33; H, 3.58; Cl, 10.95; N, 8.56.
2-Amino-4-(4-hydroxy-3-methoxyphenyl)-5-oxo-5,6,7,8-te-
trahydro-4H-chromene-3-carbonitrile (1p) Yield 76 %;
mp: 200–201 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS =
0): 8.87 (1H, s, D2O exchangeable proton), 6.91 (2H, bs, D2O
exchangeable proton), 6.67 (2H, m), 6.51 (1H, d, J = 8.1 Hz),
4.09 (1H, s), 3.72 (3H, s), 2.59 (2H, bs), 2.27 (2H, bs), 1.91
(2H, m). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
20.42 (CH2), 26.80(CH2), 36.24(CH2), 37.37 (CH), 56.22
(OCH3), 58.78 (C), 113.11 (C), 114.22 (Ar–C), 116.17
(Ar–C), 120.99 (CN), 122.88 (Ar–C), 135.42 (Ar–C), 142.23
(C–OH), 151.39 (C–OCH3), 158.91 (C), 166.01 (C–NH2),
196.72 (C=O). Anal. Calcd. for C17H16N2O4: C, 65.38; H,
5.16; N, 8.97; Found: C, 64.80; H, 5.02; N, 8.93.
2-Amino-4-(3,4-dihydroxyphenyl)-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (1e) Yield 60 %; mp:
1
200–201 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
2-Amino-4-(3-methylthiophen-2-yl)-5-oxo-5,6,7,8-tetrahy-
0): 6.86 (2H, s, D2O exchangeable protons), 6.61 (1H, d,
J = 8.1 Hz,), 6.54 (1H, s), 6.41 (1H, d, J = 8.1 Hz), 4.01
(1H, s), 2.58 (2H, bs), 2.26 (2H, bs), 1.92 (2H, m). 13C
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 19.39 (CH2),
26.17 (CH2), 36.85 (CH2), 38.63 (CH), 58.19 (C), 113.96
(C), 120.27 (CN), 118.18 (Ar–C), 116.2 (Ar–C), 123.14
(Ar–C), 136.22 (Ar–C), 144.21 (C–OH), 147.27 (C–OH),
158.12 (C), 165.33 (C–NH2), 196.97 (C=O). Anal. Calcd.
for C16H14N2O4: C, 64.42; H, 4.73; N, 9.39; Found: C,
64.24; H, 5.11; N, 9.55.
dro-4H-chromene-3-carbonitrile (1u) Yield 78 %; mp:
1
150–151 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
0): 7.19 (1H, d, J = 4.8 Hz), 7.01 (2H, s, D2O exchange-
able protons), 6.74 (1H, d, J = 5.1 Hz), 4.57 (1H, s), 2.60
(2H, bs), 2.28 (2H, bs), 2.23 (3H, s), 1.96 (2H, bs). 13C
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 12.33 (CH3),
19.10 (CH2), 20.30 (CH), 26.44 (CH2), 36.22 (CH2), 58.65
(C), 113.99 (C), 120.27 (CN), 124.10 (Ar–C), 124.70 (Ar–
C), 135.11 (Ar–C), 135.99 (Ar–C), 158.31 (C), 166.21 (C–
NH2), 196.11 (C=O). Anal. Calcd. for C15H14N2O2S: C,
62.92; H, 4.93; N, 9.78; S, 11.20; Found: C, 63.21; H, 4.58;
N, 9.94; S, 11.32.
2-Amino-4-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (1l) Yield 83 %; mp: 210–
211 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS = 0):
8.39 (1H, d, J = 8.1 Hz), 7.90 (1H, d, J = 1.5 Hz), 7.77
(1H, d, J = 8.1 Hz), 7.57 (1H, d, J = 5.1 Hz), 7.41–7.54
(3H, m), 7.25 (1H, d, J = 6.9 Hz), 6.95 (2H, s, D2O ex-
changeable protons), 5.15 (1H, s), 2.71 (2H, m), 2.30 (2H,
m), 1.98 (2H, m). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 20.31 (CH2), 26.74 (CH2), 35.69 (CH2), 36.78
2-Amino-4-(3,4-dihydroxyphenyl)-7,7-dimethyl-5-oxo-5,6,
7,8-tetrahydro-4H-chromene-3-carbonitrile (2e) Yield
57 %; mp: 185–186 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 8.78 (2H, bs, D2O exchangeable protons), 6.89
(2H, s, D2O exchangeable protons), 6.61 (1H, d, J =
8.1 Hz), 6.52 (1H, d, J = 1.8 Hz), 6.38 (1H, dd, J = 1.8
and 8.1 Hz), 3.97 (1H, s), 2.55 (2H, bs), 2.22 (1H, d,
123

Med Chem Res
J = 16.2 Hz), 2.08 (1H, d, J = 16.2 Hz), 1.06 (3H, s),
0.92 (3H, s). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 27.23 (CH3), 28.95 (CH3), 32.25 (C), 35.27
(CH2), 50.52 (CH2), 59.30 (C), 113.79 (C), 115.12 (Ar–C),
115.74 (Ar–C), 118.36 (CN), 123.1 (Ar–C), 136.27 (Ar–C),
144.40 (C–OH), 145.39 (C–OH), 158.87 (C), 162.31 (C–
NH2), 196.11 (C=O). Anal. Calcd. for C18H18N2O4: C,
66.25; H, 5.56; N, 8.58; Found: C, 65.98; H, 5.75; N, 8.32.
7.48 (1H, d, J = 7.5 Hz), 7.21–7.29 (2H, m), 4.84 (1H, s),
1.76 (3H, s). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 10.88 (CH3), 30.02 (CH), 54.44 (C), 96.03 (C),
105.55 (Ar–C), 110.66 (Ar–C), 119.94 (Ar–C), 120.15
(CN), 120.88 (Ar–C), 122.22 (Ar–C), 128.04 (Ar–C),
135.20 (Ar–C), 136.55 (Ar–C), 142.05 (C), 162.27 (C–
NH2). Anal. Calcd. for C16H13N5O: C, 65.97; H, 4.50; N,
24.04; Found: C, 65.66; H, 4.33; N, 24.13.
2-Amino-4-(2-hydroxyphenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
6-Amino-3-methyl-4-(2,3,4-trimethoxyphenyl)-2,4-dihy-
tetrahydro-4H-chromene-3-carbonitrile (2f) Yield 68 %;
1
dropyrano[2,3-c]pyrazole-5-carbonitrile
(3r) Yield
mp: 80–81 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
79 %; mp: 196–197 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 12.10 (1H, s, D2O exchangeable protons), 6.87
(2H, D2O exchangeable protons), 6.43 (2H, bs), 4.58 (1H,
s), 3.85 (6H, s), 3.84 (3H, s), 1.87 (3H, s). 13C NMR
(DMSO-d6, 500 MHz, d, TMS = 0): 10.53 (CH3), 12.01
(CH), 54.28 (OCH3), 55.63 (C), 56.27 (OCH3), 56.36
(OCH3), 96.75 (C), 105.30 (Ar–C), 110.96 (Ar–C), 120.69
(CN), 123.36 (Ar–C), 136.42 (C), 139.91 (C–OCH3),
142.94 (C–OCH3), 147.71 (C–OCH3), 151.72 (C), 162.76
(C–NH2). Anal. Calcd. for C17H18N4O4: C, 59.64; H, 5.30;
N, 16.37; Found: C, 59.78; H, 5.25; N, 16.55.
0): 8.72 (1H, s, D2O exchangeable proton), 6.32–6.89 (7H,
m), 4.84 (1H, s), 2.55 (2H, bs), 2.22 (1H, d, J = 16.2 Hz),
2.08 (1H, d, J = 16.2 Hz), 1.06 (3H, s), 0.92 (3H, s). 13C
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 27.23 (CH3),
29.05 (CH3), 32.46 (C), 35.78 (C), 50.15 (CH2), 59.36 (C),
113.23 (C), 115.25 (Ar–C), 118.63 (CN), 121.22 (Ar–C),
122.36 (Ar–C), 127.78 (Ar–C), 130.20 (Ar–C), 147.41 (C),
158.68 (C–OH), 162.26 (C–NH2), 196.01 (C=O). Anal.
Calcd. for C18H18N2O3: C, 72.95; H, 6.80; N, 9.45; Found:
C, 72.17; H, 7.10; N, 9.64.
6-Amino-4-(2-methoxyphenyl)-3-methyl-2,4-dihydropyra-
6-Amino-3-methyl-4-(3-methylthiophen-2-yl)-2,4-dihydropy-
no[2,3-c]pyrazole-5-carbonitrile (3h) Yield 84 %; mp:
1
170–171 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
rano[2,3-c]pyrazole-5-carbonitrile (3u) Yield 85 %; mp:
1
179–180 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
0): 12.01 (1H, s, D2O exchangeable proton), 7.19 (1H, m),
6.96–7.01 (2H, m), 6.90 (1H, m), 6.79 (2H, s, D2O ex-
changeable protons), 4.97 (1H, s), 3.78 (3H, s), 1.79 (3H,
s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0): 10.55
(CH3), 11.10 (CH), 54.45 (OCH3), 56.11 (C), 112.75 (Ar–
C), 110.96 (Ar–C), 114.90 (C), 120.99 (Ar–C), 121.00
(CN), 126.80 (Ar–C), 130.01 (Ar–C), 136.82 (C), 142.27
(C–OCH3), 156.09 (C), 162.00 (C–NH2). Anal. Calcd. for
C15H14N4O2: C, 63.82; H, 5.00; N, 19.85; Found: C, 64.09;
H, 4.75; N, 19.76.
0): 12.15 (1H, s, D2O exchangeable proton), 7.02(1H, d,
J = 5.1 Hz), 6.86 (2H, bs, D2O exchangeable protons),
6.78 (1H, d, J = 5.1 Hz), 5.01 (1H, s), 2.16 (3H, s), 1.83
(3H, s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
10.35 (CH3), 11.55 (CH3), 12.70 (CH), 54.90 (C), 110.76
(C), 120.07 (CN), 124.11 (Ar–C), 124.71 (Ar–C), 133.31
(Ar–C), 135.56 (Ar–C), 136.89 (C), 142.24 (C), 162.08 (C–
NH2). Anal. Calcd. for C13H12N4OS: C, 57.34; H, 4.44; N,
20.57; S, 11.77; Found: C, 56.99; H, 4.71; N, 20.34; S,
11.98.
6-Amino-4-(furan-2-yl)-3-methyl-2,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (3m) Yield 67 %; mp: 185–
186 (DEC) °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS =
0): 12.16 (1H, s, D2O exchangeable proton), 7.53 (1H, bs),
6.95 (2H, s, D2O exchangeable proton), 6.37 (1H, d,
J = 1.8 Hz), 6.17 (1H, d, J = 2.7 Hz), 4.77 (1H, s), 1.97
(3H, s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
10.03 (CH3), 30.28 (CH), 54.45 (C), 95.57 (C), 106.09
(Ar–C), 110.69 (Ar–C), 121.04 (CN), 136.28 (C), 142.72
(Ar–C), 155.27 (Ar–C), 156.18 (C), 161.94 (C–NH2). Anal.
Calcd. for C12H10N4O2: C, 59.50; H, 4.16; N, 23.13;
Found: C, 59.81; H, 3.97; N, 23.29.
7-Amino-2-oxo-5-phenyl-4-thioxo-2,3,4,5-tetrahydro-1H-
pyrano[2,3-d]pyrimidine-6-carbonitrile (4a) Yield 82 %;
mp: 180–181 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 7.98 (2H, d, J = 7.5 Hz), 7.19 (2H, d, J =
7.5 Hz), 4.56 (1H, s), 3.89 (3H, s). 13C NMR (DMSO-d6,
500 MHz, v TMS = 0): 36.53 (CH), 59.63 (C), 94.62 (C),
115.17 (CN), 125.88 (Ar–C), 126.71 (Ar–C), 126.77
(Ar–C), 142.25 (Ar–C), 151.99 (C=O), 158.36 (C), 160.36
(C–NH2), 174.62 (C=S). Anal. Calcd. for C14H10N4O2S: C,
56.37; H, 3.38; N, 18.78; S, 10.75; Found: C, 56.60; H,
3.15; N, 18.92; S, 10.82.
7-Amino-5-(3-chlorophenyl)-2-oxo-4-thioxo-2,3,4,5-tetrahy-
6-Amino-4-(1H-indol-2-yl)-3-methyl-2,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (3o) Yield 66 %; mp: 190–191 °C,
1H NMR (DMSO-d6, 300 MHz, d, TMS = 0): 11.69 (1H,
s, D2O exchangeable proton), 8.91 (1H, s, D2O ex-
changeable proton), 8.34 (1H, d, J = 7.2 Hz), 7.92 (1H, s),
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4d) Yield
1
87 %; mp: 190–191 °C, H NMR (DMSO-d6, 300 MHz):
d, TMS = 0): 9.11 (1H, s), 10.91 (1H, s), 7.41 (1H, s),
7.24–7.33 (2H, m), 7.13 (1H, d, J = 8.3 Hz), 7.06 (1H, s),
4.59 (1H, s). 13C NMR (DMSO-d6, 500 MHz, d,
123

Med Chem Res
TMS = 0): 36.86 (CH), 59.97 (C), 94.75 (C), 115.67 (CN),
125.61 (Ar–C), 127.63 (Ar–C), 128.94 (Ar–C), 130.26
(Ar–C), 134.43 (C–Cl), 143.34 (Ar–C), 151.69 (C=O),
158.24 (C), 160.56 (C–NH2), 174.67 (C=S). Anal. Calcd.
for C14H9ClN4O2S: C, 50.53; H, 2.73; Cl, 10.65; N, 16.84;
S, 9.64; Found: C, 50.80; H, 2.51; Cl, 10.76; N, 16.96; S,
9.38.
(CH), 59.23 (C), 94.28 (C), 115.58 (CN), 121.57 (Ar–C),
121.89 (Ar–C), 130.28 (Ar–C), 130.85 (Ar–C), 145.14
(Ar–C), 148.95 (C=O), 151.39 (Ar–C), 158.27 (C), 160.33
(C–NH2), 174.68 (C=S). Anal. Calcd. for C14H9N5O4S: C,
48.98; H, 2.64; N, 20.40; S, 9.34; Found: C, 49.12; 2.36; N,
20.54; S, 9.60.
7-Amino-2-oxo-5-(thiophen-2-yl)-4-thioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4n) Yield
81 %; mp: 212–213 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 12.09 (1H, bs), 11.42 (1H, bs), 7.38 (2H, bs),
7.02 (1H, d), 6.57 (1H, m), 6.49 (1H, d), 4.21 (1H, s). 13C
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 34.33 (CH),
59.65 (C), 94.56 (C), 115.69 (CN), 123.65 (Ar–C), 126.71
(Ar–C), 126.99 (Ar–C), 139.77 (Ar–C), 151.91 (C=O),
158.79 (C), 160.45 (C–NH2), 174.85 (C=S). Anal. Calcd.
for C12H8N4O2S2: C, 47.36; H, 2.65; N, 18.41; S, 21.07;
Found: C, 47.56; H, 2.45; N, 18.19; S, 21.29.
7-Amino-5-(4-bromophenyl)-2-oxo-4-thioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4g) Yield
88 %; mp: 185–186 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 11.17 (1H, s, D2O exchangeable proton), 7.47
(2H, d, J = 8.1 Hz), 7.18 (2H, d, J = 8.4 Hz), 7.15 (2H,
bs, D2O exchangeable proton), 4.23 (1H, s). 13C NMR
(DMSO-d6, 500 MHz, d, TMS = 0): 35.86 (CH), 59.97
(C), 94.75 (C), 115.67 (CN), 120.55 (C–Br), 131.25 (Ar–
C), 131.32 (Ar–C), 131.51 (Ar–C), 131.82 (Ar–C), 141.24
(Ar–C), 151.37 (C=O), 158.59 (C), 160.86 (C–NH2),
174.78 (C=S). Anal. Calcd. for C14H9BrN4O2S: C, 44.58;
H, 2.40; Br, 21.18; N, 14.85; S, 8.50; Found: C, 44.81; H,
2.12; Br, 21.36; N, 14.52; S, 8.39.
7-Amino-5-(4-chlorophenyl)-2-oxo-4-thioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4s) Yield
85 %; mp: 194–195 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 11.1 (1H, s), 9.2 (1H, s), 7.73 (2H, d,
J = 8.0 Hz), 7.17 (2H, d, J = 8.0 Hz), 6.97 (2H, s), 4.37
(1H, s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
35.97 (CH), 59.60 (C), 94.50 (C), 115.11 (CN), 128.48
(Ar–C), 128.84 (Ar–C), 130.01 (Ar–C), 130.10 (Ar–C),
131.53 (C–Cl), 140.30 (Ar–C), 151.97 (C=O), 158.15 (C),
160.17 (C–NH2), 174.19 (C=S). Anal. Calcd. for C14H9-
ClN4O2S: C, 50.53; H, 2.73; Cl, 10.65; N, 16.84; S, 9.64;
Found: C, 50.81; H, 2.39; Cl, 10.54; N, 16.78; S, 9.78.
7-Amino-5-(2-methoxyphenyl)-2-oxo-4-thioxo-2,3,4,5-te-
trahydro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4h)
Yield 80 %; mp: 180–181 °C, 1H NMR (DMSO-d6,
300 MHz, d, TMS = 0): 10.88 (1H, s), 9.01 (1H, s), 7.13
(1H, d, J = 8.0 Hz), 6.87–6.89 (3H, m), 6.84–6.88 (2H, m),
4.29 (1H, s), 3.64 (3H, s). 13C NMR (DMSO-d6, 500 MHz,
d, TMS = 0): 35.42 (CH), 56.31 (OCH3), 59.78 (C), 94.79
(C), 114.41 (Ar–C), 115.99 (CN), 121.58 (Ar–C), 121.89
(Ar–C), 126.88 (Ar–C), 130.12 (Ar–C), 151.98 (C=O),
157.66 (C), 158.37 (C–OCH3), 160.00 (C–NH2), 174.28
(C=S). Anal. Calcd. for C15H12N4O3S: C, 54.87; H, 3.68;
N, 17.06; S, 9.77; Found: C, 54.44; H, 3.92; N, 17.32; S,
9.49.
7-Amino-5-(3-methylthiophen-2-yl)-2-oxo-4-thioxo-2,3,4,
5-tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile
(4u) Yield 80 %; mp: 228–229 °C, 1H NMR (DMSO-d6,
300, d, TMS = 0): 12.04 (1H, bs), 11.02 (1H, bs), 7.26
(2H, bs), 6.48 (1H, d), 6.62 (1H, m), 3.99 (1H, s), 2.20 (3H,
s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0): 13.13
(CH3), 33.03 (CH), 59.32 (C), 94.43 (C), 115.54 (CN),
124.11 (Ar–C), 124.17 (Ar–C), 133.95 (Ar–C), 135.09
(Ar–C), 151.15 (C=O), 158.59 (C), 160.19 (C–NH2),
174.91 (C=S). Anal. Calcd. for C13H10N4O2S2: C, 51.65;
H, 3.33; N, 18.53; S, 10.61; Found: C, 51.39; H, 3.61; N,
18.72; S, 10.31.
7-Amino-5-(4-methoxyphenyl)-2-oxo-4-thioxo-2,3,4,5-te-
trahydro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4i)
Yield 76 %; mp: 200–201 °C, 1H NMR (DMSO-d6,
300 MHz, d, TMS = 0): 7.98 (2H, d, J = 7.5 Hz), 7.19
(2H, d, J = 7.5 Hz), 4.56 (1H, s), 3.89 (3H, s). 13C NMR
(DMSO-d6, 500 MHz, d, TMS = 0): 35.35 (CH), 56.42
(OCH3), 59.36 (C), 94.26 (C), 113.96 (Ar–C), 114.13 (Ar–
C), 115.71 (CN), 129.00 (Ar–C), 129.54 (Ar–C), 136.08
(Ar–C), 151.90 (C=O), 157.76 (C), 158.63 (C–OCH3),
160.63 (C–NH2), 174.26 (C=S). Anal. Calcd. for C15H12-
N4O3S: C, 54.87; H, 3.68; N, 17.06; S, 9.77; Found: C,
54.51; H, 3.88; N, 17.34; S, 9.82.
7-Amino-5-(3-nitrophenyl)-2-oxo-4-thioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4v) Yield
80 %; mp: 200–201 °C, 1H NMR (DMSO-d6, 300, d,
TMS = 0): 11.13 (1H, bs), 9.15 (1H, s), 8.16 (1H, s), 8.01
(1H, d, J = 8.0 Hz), 7.56–7.63 (2H, m), 4.33 (1H, s). 13C
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 35.99 (CH),
59.95 (C), 94.55 (C), 115.16 (CN), 118.28 (Ar–C), 124.34
(Ar–C), 129.29 (Ar–C), 135.45 (Ar–C), 143.53 (Ar–C),
150.60 (C–NO2), 151.06 (C=O), 158.84 (C), 160.48 (C–
NH2), 174.88 (C=S). Calculated Anal. Calcd. for
7-Amino-5-(4-nitrophenyl)-2-oxo-4-thioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4j) Yield
84 %; mp: 268–269 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 10.93 (1H, s), 9.41 (1H, s), 8.13 (2H, d,
J = 8.5 Hz), 7.49 (2H, d, J = 8.5 Hz), 7.08 (2H, s), 3.29
(1H, s). 13C NMR (DMSO, 500 MHz, d, TMS = 0): 36.59
123

Med Chem Res
C14H9N5O4S: C, 48.98; H, 2.64; N, 20.40; S, 9.34; Found:
C, 49.10; H, 2.34; N, 20.68; S, 9.02.
(CN), 121.20 (Ar–C), 124.56 (Ar–C), 126.49 (Ar–C),
126.97 (Ar–C), 127.23 (Ar–C), 127.47 (Ar–C), 127.86
(Ar–C), 128.18 (Ar–C), 131.20 (Ar–C), 133.25 (Ar–C),
133.73 (C–Cl), 143.25 (Ar–C), 148.61 (Ar–C), 160.74 (C–
NH2). Anal. Calcd. for C20H13ClN2O: C, 72.18; H, 3.94;
Cl, 10.65; N, 8.42; Found: C, 71.94; H, 3.63; Cl, 10.86;
8.22.
2-Amino-4,5 dihydro-4-(4-fluorophenyl)-5-oxopyrano[3,2-
c]chromene-3-carbonitrile (5b) Yield 84 %; mp: 190–
191 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS = 0):
7.83 (1H, d, J = 8.1 Hz), 7.65 (1H, dd, J = 8.1 and
7.1 Hz), 7.44 (1H, m), 7.32 (1H, d, J = 8.4 Hz), 7.21 (2H,
d, J = 7.9 Hz), 7.12 (2H, d, J = 8.0 Hz), 4.31 (1H, s). 13
C
2-Amino-4-(3,4-dihydroxyphenyl)-4H-benzo[h]chromene-
3-carbonitrile (6e) Yield 71 %; mp: 150–151 °C, 1H
NMR (DMSO-d6, 300 MHz, d, TMS = 0): 8.30–8.39 (2H,
m), 7.83–7.97 (2H, m), 7.18–7.73 (4H, m), 6.92 (1H, d,
J = 8.4 Hz), 7.18 (2H, s, D2O exchangeable protons), 5.80
(1H, s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
30.10 (CH), 59.22 (C), 115.36 (Ar–C), 117.76 (CN),
117.96 (Ar–C), 120.23 (Ar–C), 120.93 (Ar–C), 121.10
(Ar–C), 122.35 (Ar–C), 125.71 (Ar–C), 125.74 (Ar–C),
127.48 (Ar–C), 132.77 (Ar–C), 134.44 (Ar–C), 143.05
(Ar–C), 144.20 (C–OH), 147.55 (C–OH), 160.31 (C–NH2).
Anal. Calcd. for C20H14N2O3: C, 72.72; H, 4.27; N, 8.48;
Found: C, 72.99; H, 3.98; N, 8.72.
NMR (DMSO-d6, 500 MHz, d, TMS = 0): 37.81 (CH),
58.14 (C), 105.24 (C), 115.21 (Ar–C), 115.41 (Ar–C),
115.45 (Ar–C), 116.40 (Ar–C), 119.14 (CN), 123.32 (Ar–
C), 125.40 (Ar–C), 128.34 (Ar–C), 130.58 (Ar–C), 130.60
(Ar–C), 139.12 (Ar–C), 152.50 (Ar–C), 159.10 (C–NH2),
159.90 (C–F), 160.12 (C), 161.90 (C=O). Anal. Calcd. for
C19H11FN2O3: C, 68.26; H, 3.32; F, 5.68; N, 8.38; Found:
C, 68.32; H, 3.28; F, 5.70; N, 8.44.
2-Amino-4-(naphthalene-2-yl)-5-oxo-4,5-dihydropyrano[3,2-
c]chromene-3-carbonitrile (5l) Yield 88 %; mp: 210–
211 °C, 1H NMR (DMSO-d6, 300 MHz, d, TMS = 0):
8.43 (1H, d, J = 7.8 Hz), 7.94–7.99 (2H, m), 7.82 (1H, d,
J = 8.1 Hz), 7.72 (1H, m), 7.44–7.62 (5H, m),7.32–7.41
(3H, m), 5.47 (1H, s). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 36.87 (CH), 58.16 (C), 104.03 (C), 117.30 (Ar–
C), 119.66 (CN), 121.55 (Ar–C), 125.66 (Ar–C), 125.79
(Ar–C), 126.15 (Ar–C), 126.71 (Ar–C), 127.32 (Ar–C),
127.43 (Ar–C), 127.76 (Ar–C), 127.89 (Ar–C), 131.65
(Ar–C), 133.94 (Ar–C), 135.35 (Ar–C), 152.56 (Ar–C),
153.49 (C–NH2), 158.98 (Ar–C), 160.10 (C=O). Anal.
Calcd. for C23H14N2O3: C, 75.40; H, 3.85; N, 7.65; Found:
C, 75.26; H, 4.00; N, 7.35.
2-Amino-4-(4-hydroxyphenyl)-4H-benzo[h]chromene-3-car-
bonitrile (6k) Yield 85 %; mp: 228–229 °C, 1H NMR
(DMSO-d6, 300 MHz, d, TMS = 0): 9.35 (1H, s, D2O
exchangeable protons), 8.23 (1H, d, J = 8.1 Hz), 7.87 (1H,
d, J = 8.1 Hz), 7.57–7.63 (3H, m), 7.03–7.11 (5H, m),
6.69(2H, d, J = 8.1 Hz), 4.77 (1H, s). 13C NMR (DMSO-
d6, 500 MHz, d, TMS = 0): 29.88 (CH), 57.24 (C), 115.83
(Ar–C), 118.92 (CN), 121.14 (Ar–C), 123.23 (Ar–C),
124.84 (Ar–C), 126.80 (Ar–C), 127.07 (Ar–C), 129.14
(Ar–C), 133.06 (Ar–C), 136.64 (Ar–C), 143.02 (Ar–C),
151.74 (C–OH), 160.43 (C–NH2). Anal. Calcd. for
C20H14N2O2: C, 76.42; H, 4.49; N, 8.91; Found: C, 76.22;
H, 4.64; N, 9.02.
2-Amino-4-(3,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano
[3,2-c]chromene-3-carbonitrile (5q) Yield 76 %; mp:
1
170–171 °C, H NMR (DMSO-d6, 300 MHz, d, TMS =
0): 7.92 (1H, d, J = 8.1 Hz), 7.66 (1H, dd, J = 8.1 Hz and
7.2 Hz), 7.44 (1H, m), 7.37 (1H, d, J = 9 Hz), 6.83–6.86
(2H, m), 6.76 (1H, d, J = 8.7 Hz), 4.43 (1H, s), 3.73 (3H,
s), 3.76 (3H, s). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 36.98 (CH), 55.97 (OCH3), 56.02 (OCH3),
58.61 (C), 104.59 (C), 112.11 (Ar–C), 112.36 (Ar–C),
113.51 (Ar–C), 117.03 (Ar–C), 119.76 (CN), 120.14 (Ar–
C), 122.93 (Ar–C), 125.10 (Ar–C), 133.31 (Ar–C), 136.31
(Ar–C), 148.42 (Ar–C), 148.99 (Ar–C), 152.58 (C), 153.64
(C–NH2), 158.39 (C), 160.00 (C=O). Anal. Calcd. for
C21H16N2O5: C, 67.02; H, 4.28; N, 7.44; Found: C, 67.26;
H, 3.98; N, 7.64.
2-Amino-4-(thiophen-2-yl)-4H-benzo[h]chromene-3-car-
1
bonitrile (6n) Yield 83 %; mp: 231–232 °C, H NMR
(DMSO-d6, 300 MHz, d, TMS = 0): 8.22 (1H, d,
J = 8.1 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.55–7.65 (3H, m),
7.36 (1H, d, J = 4.5 Hz), 7.24–7.28 (3H, m), 7.08 (1H, bs),
6.93 (1H, bs), 5.26 (1H, s). 13C NMR (DMSO-d6,
500 MHz, d, TMS = 0): 30.53 (CH), 59.02 (C), 119.84
(CN), 120.44 (Ar–C), 120.97 (Ar–C), 121.12 (Ar–C),
123.67 (Ar–C), 125.84 (Ar–C), 125.98 (Ar–C), 126.37
(Ar–C), 126.63 (Ar–C), 126.74 (Ar–C), 132.77 (Ar–C),
139.44 (Ar–C), 143.50 (Ar–C), 160.77 (C–NH2). Anal.
Calcd. for C18H12N2OS: C, 71.03; H, 3.97; N, 9.20; S,
10.54; Found: C, 71.38; H, 3.58; N, 8.90; S, 10.82.
2-Amino-4-(3-chlorophenyl)-4H-benzo[h]chromene-3-car-
1
bonitrile (6d) Yield 82 %; mp: 176–177 °C, H NMR
(DMSO-d6, 300 MHz, d, TMS = 0): 8.25 (1H, d,
J = 8.1 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.56–7.67 (3H, m),
7.12 (1H, d, J = 8.4 Hz), 4.97 (1H, s). 13C NMR (DMSO-
d6, 500 MHz, d, TMS = 0): 29.33 (CH), 56.10 (C), 117.67
2-Amino-4-(3,4-dimethoxyphenyl)-4H-benzo[h]chromene-
3-carbonitrile (6q) Yield 73 %; mp: 140–141 °C, 1H
NMR (DMSO-d6, 300 MHz, d, TMS = 0): 8.24 (1H, d,
J = 7.8 Hz), 7.88(1H, d, J = 7.54 Hz), 7.54–7.65 (3H, m),
7.11–7.16 (3H, m), 6.72–6.91 (3H, m), 4.84 (1H, s), 3.82
123

Med Chem Res
(3H, s), 3.80 (3H, s). 13C NMR (DMSO-d6, 500 MHz, d,
TMS = 0): 31.11 (CH), 55.67 (OCH3), 56.66 (OCH3),
59.68 (C), 113.32 (Ar–C), 115.86 (Ar–C), 118.59 (Ar–C),
120.24 (Ar–C), 120.95 (Ar–C), 121.17 (Ar–C), 125.71
(Ar–C), 125.77 (Ar–C), 127.42 (Ar–C), 132.76 (Ar–C),
133.75 (Ar–C), 143.54 (Ar–C), 147.84 (C–OCH3), 150.31
(C–OCH3), 160.34 (C–NH2). Anal. Calcd. for C22H18
N2O3: C, 73.73; H, 5.06; N, 7.82; Found: C, 73.44; H, 5.26;
N, 8.10.
(CH3), 22.30 (CH), 58.10 (C), 80.12 (C), 119.10 (CN),
121.10 (Ar–C), 124.30 (Ar–C), 133.60 (Ar–C), 135.40
(Ar–C), 150.6 (C=O), 159.3 (C–NH2), 160.4 (C), 163.8
(C=O). Anal. Calcd. for C13H10N4O3S: C, 51.65; H, 3.33;
N, 18.53; S, 10.61; Found: C, 50.21; H, 3.20; N, 20.10; S,
12.31.
3-Amino-1-(2-methoxyphenyl)-1H-benzo[f]chromene-2-car-
bonitrile (8h) Yield 70 %; mp: 200–201 °C, 1H NMR
(DMSO-d6, 300 MHz, d, TMS = 0): 7.89–7.92 (2H, m),
7.74 (1H, d, J = 7.8 Hz), 7.42 (2H, m), 7.32 (1H, dd,
J = 1.2 and 6.9 Hz), 7.13 (1H, m), 7.03 (1H, d,
J = 8.4 Hz), 6.77–6.86 (4H, m), 5.60 (1H, s). 13C NMR
(DMSO-d6, 500 MHz, d, TMS = 0): 17.31 (CH), 56.44
(OCH3), 57.41 (C), 112.18 (Ar–C), 117.16 (CN), 121.52
(Ar–C), 123.39 (Ar–C), 125.30 (Ar–C), 127.61 (Ar–C),
128.41 (Ar–C), 128.95 (Ar–C), 129.05 (Ar–C), 129.63
(Ar–C), 131.13 (Ar–C), 134.07 (Ar–C), 151.77 (Ar–C),
156.11 (C–OCH3), 160.69 (C–NH2). Anal. Calcd. for
C21H16N2O2: C, 76.81; H, 4.91; N, 8.53; Found: C, 76.52;
H, 5.12; N, 8.88.
7-Amino-5-(4-bromophenyl)-2,4-dioxo-2,3,4,5-tetrahydro-
1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (7g) Yield
79 %; mp: 170–171 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 11.17 (1H, s, D2O exchangeable proton), 7.47
(2H, d, J = 8.1 Hz), 7.18 (2H, d, J = 8.4 Hz), 7.15 (2H,
bs, D2O exchangeable proton), 4.23 (1H, s). 13C NMR
(DMSO-d6, 500 MHz, d, TMS = 0): 35.72 (CH), 58.72
(C), 88.40 (C), 119.51 (CN), 120.22 (C–Br), 130.15 (Ar–
C), 131.58 (Ar–C), 144.06 (Ar–C), 149.95 (C=O), 152.81
(C–NH2), 158.00 (C), 162.96 (C=O). Anal. Calcd. for
C14H9BrN4O3: C, 46.56; H, 2.51; Br, 22.12; N, 15.51;
Found: C, 46.26; H, 2.86; Br, 22.31; N, 15.23.
3-Amino-1-(thiophen-2-yl)-1H-benzo[f]chromene-2-carboni-
trile (8n) Yield 77 %; mp: 228–229 °C, 1H NMR
(DMSO-d6, 300 MHz, d, TMS = 0): 8.05 (1H, d,
J = 8.1 Hz), 7.93 (2H, d, J = 8.7 Hz), 7.43–7.54 (2H, m),
7.26–7.32 (2H, m), 7.09 (2H, m), 6.86–7.02 (2H, m), 5.71
(1H, s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0):
17.41 (CH), 57.44 (C), 117.3 (CN), 118.81 (Ar–C), 121.25
(Ar–C), 122.15 (Ar–C), 122.51 (Ar–C), 123.22 (Ar–C),
126.38 (Ar–C), 126.61 (Ar–C), 126.95 (Ar–C), 128.57
(Ar–C), 128.79 (Ar–C), 138.81 (Ar–C), 139.18 (Ar–C),
136.81 (Ar–C), 151.75 (Ar–C), 161.09 (C–NH2). Anal.
Calcd. for C18H12N2OS: C, 71.03; H, 3.97; N, 9.20; S,
10.54; Found: C, 71.32; H, 3.66; N, 8.96; S, 10.76.
7-Amino-5-(4-methoxyphenyl)-2,4-dioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (7i) Yield
70 %; mp: 237–238 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 7.98 (2H, d, J = 7.5 Hz), 7.19 (2H, d,
J = 7.5 Hz), 4.56 (1H, s), 3.89 (3H, s). 13C NMR (DMSO-
d6, 500 MHz, d, TMS = 0): 35.27 (CH), 55.55 (OCH3),
58.22 (C), 88.44 (C), 114.22 (Ar–C), 120.15 (CN), 130.11
(Ar–C), 134.50 (Ar–C), 149.59 (C=O), 152.18 (C–OCH3),
156.00 (C–NH2), 158.55 (C), 162.69 (C=O). Anal. Calcd.
for C15H12N4O4: C, 57.69; H, 3.87; N, 17.94; Found: C,
57.99; H, 3.96; N, 17.52.
7-Amino-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-2,4-dioxo-1H-
pyrano[2,3-d]pyrimidine-6-carbonitrile (7n) Yield 74 %;
mp: 172–173 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 11.14 (1H, s, D2O exchangeable proton), 7.40
(1H, d, J = 7.8 Hz), 6.83–6.86 (2H, bs), 6.8 (1H, s, D2O
exchangeable proton), 4.52 (1H, s). 13C NMR (DMSO-d6,
500 MHz, d, TMS = 0): 25.80 (CH), 58.10 (C), 80.10 (C),
119.10 (CN), 123.50 (Ar–C), 125.40 (Ar–C), 127.00 (Ar–
C), 139.80 (Ar–C), 150.40 (C=O), 158.90 (C–NH2), 160.84
(C), 163.68 (C=O). Anal. Calcd. for C12H8N4O3S: C,
50.00; H, 2.80; N, 19.43; S, 11.12; Found: C, 48.10; H,
3.21; N, 20.21; S, 11.21.
Xanthine oxidase assay
Bovine milk xanthine oxidase (grade 1, ammonium sul-
phate suspension, Sigma-Aldrich) activity was assayed
spectrophotometrically by measuring the uric acid forma-
tion at 293 nm using a Hitachi U-3010 UV–visible spec-
trophotometer at 25 °C (Escribano et al., 1988; Takano
et al., 2005). The reaction mixture contained 50 mM
potassium phosphate buffer (pH 7.6), 75 lM xanthine and
0.08 U of xanthine oxidase. Inhibition of xanthine oxidase
activity by various inhibitors was measured by following
the decrease in the uric acid formation at 293 nm at 25 °C.
The enzyme was preincubated for 5 min, with test com-
pound, 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 ac-
tivity. All the experiments were performed in triplicate, and
values were expressed as means of three experiments.
7-Amino-5-(3-methylthiophen-2-yl)-2,4-dioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (7u) Yield
78 %; mp: 171–173 °C, 1H NMR (DMSO-d6, 300 MHz, d,
TMS = 0): 11.21 (1H, s, D2O exchangeable proton), 7.80
(2H, bs, D2O exchangeable proton), 7.28 (1H, d,
J = 7.6 Hz), 6.26 (1H, d, 7.6 Hz), 4.62 (1H, s), 2.30 (3H,
s). 13C NMR (DMSO-d6, 500 MHz, d, TMS = 0): 13.40
123

Med Chem Res
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