Design, synthesis and biological evaluation of novel indolinedione–coumarin hybrids as xanthine oxidase inhibitors

Article abstract of DOI:10.1007/s00044-020-02589-2

A library of indolinedione–coumarin hybrid molecules was rationally designed and synthesized against hyperuricemia. All of the synthesized hybrid molecules were tested to check their inhibitory activity against xanthine oxidase enzyme by using a spectrophotometric assay. The results revealed that the compound showed IC₅₀ values within the range of 6.5–24.5 μM amongst which compound K-7 was found to be endowed with the most potent IC₅₀ value against xanthine oxidase enzyme. Kinetic studies were also performed to check the mode of inhibition of most potent compound K-7, which revealed its mixed-type inhibition behavior. Structure-activity relationships revealed that electron-donating groups and small alkyl chains between the two active scaffolds might be beneficial in inhibiting xanthine oxidase enzyme. It was also shown that various electrostatic interactions stabilized the compound K-7 within the active site of xanthine oxidase enzyme, which confirmed that it can completely block its catalytic active site. Thus, K-7 is regarded as a potent xanthine oxidase inhibitor and can be served as a promising molecular architectural unit for anti-hyperuricemic drug design. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02589-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02589-2
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of novel
indolinedione–coumarin hybrids as xanthine oxidase inhibitors
Harmandeep Kaur Gulati1 Kavita Bhagat1 Atamjit Singh1 Nitish Kumar1 Arshmeet Kaur1 Akriti Sharma1
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Shilpa Heer1 Harbinder Singh
Jatinder Vir Singh1 Preet Mohinder S. Bedi1
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Received: 21 January 2020 / Accepted: 8 June 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A library of indolinedione–coumarin hybrid molecules was rationally designed and synthesized against hyperuricemia. All
of the synthesized hybrid molecules were tested to check their inhibitory activity against xanthine oxidase enzyme by using a
spectrophotometric assay. The results revealed that the compound showed IC50 values within the range of 6.5–24.5 µM
amongst which compound K-7 was found to be endowed with the most potent IC50 value against xanthine oxidase enzyme.
Kinetic studies were also performed to check the mode of inhibition of most potent compound K-7, which revealed its
mixed-type inhibition behavior. Structure-activity relationships revealed that electron-donating groups and small alkyl
chains between the two active scaffolds might be beneficial in inhibiting xanthine oxidase enzyme. It was also shown that
various electrostatic interactions stabilized the compound K-7 within the active site of xanthine oxidase enzyme, which
confirmed that it can completely block its catalytic active site. Thus, K-7 is regarded as a potent xanthine oxidase inhibitor
and can be served as a promising molecular architectural unit for anti-hyperuricemic drug design.
Graphical Abstract
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Keywords Indolinedione Coumarin Hybrids Xanthine oxidase enzyme Enzyme kinetics Molecular docking studies
Introduction
Xanthine oxidase (XO), a molybdoflavoprotein containing
iron and molybdenum, that promotes the oxidation, espe-
cially of hypoxanthine to xanthine and then to uric acid
(UA) with the release of hydrogen peroxide and superoxide
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02589-2) contains supplementary
anions during the process of purines catabolism in humans.
A well-known relationship exists between XO and hyper-
uricemia, a condition which is characterized by elevated
serum UA level (7 mg/dL) (Wortmann 1998). Impairment
in purine metabolism results in the deposition of sodium
urate crystals in joints, which further causes inflammation
and pain in joints. With this aspect, XO acts as an important
and selective target for sustaining broad-spectrum che-
motherapy in hyperuricemic patients (Ojha et al. 2017;
Kumar et al. 2011).
material, which is available to authorized users.
* Harbinder Singh
singh.harbinder40@gmail.com
* Jatinder Vir Singh
jatindervirsingh1@gmail.com
* Preet Mohinder S. Bedi
bedi_preet@yahoo.com
1
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar, Punjab 143005, India

Medicinal Chemistry Research
In the recent past, numerous successful purine based XO
inhibitors such as allopurinol, pterin and 6-formylpterin
(Oettl and Reibneggar 1999), 2-alkylhypoxanthines (Oettl
and Reibneggar 1999; Biagi et al. 2001; Brien et al. 1985),
and 2-substituted 7Hpyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]-
pyrimidines (Nagamatsu et al. 2000; Nagamatsu et al. 1985;
Nagamatsu et al. 1995; Ali et al. 2010) are used for
the treatment of hyperuricemia. These derivatives were
mainly associated with the number of side effects
(Stevens–Johnson syndrome and drug rash with eosino-
philia and systematic symptoms (Ali et al. 2010; Pacher
et al. 2006; Hille 2006) and also hindered the activities of
both purine and pyrimidine metabolizing enzymes, which
motivate the researchers around the globe to develop non-
purine XO inhibitors (Pacher et al. 2006; Hille 2006; Bor-
ges et al. 2002).
Therefore, researchers paid great attention to the devel-
opment of non-purine XO inhibitors. The compound BOF-
4272, a potent XO inhibitor, its clinical use was limited
because variation occurring in its efficacy which is mainly
due to the difference in hepatic metabolism (Pacher et al.
2006). Febuxostat, a non-purine XO inhibitor that was
approved from European Medicines Evaluation Agency and
USFDA showed efficient efficacy with improved hypour-
icemic effect as compared with Allopurinol (Osada et al.
1993; Komoriya et al. 1993; Becker et al. 2004). Besides
this, it was also reported that Febuxostat showed some side
effects similar to Allopurinol such as headache, diarrhea,
dizziness, abnormalities in liver function, and nausea
(Strilchuk et al. 2019; Love et al. 2010; Becker et al. 2007;
Schumacher et al. 2008; Becker et al. 2007). Furthermore,
pyranostat was found to show higher in vivo efficacy with a
poor pharmacokinetic profile (Ishibuchi et al. 2001;
Sebastian et al. 2016). Whereas, topiroxostat, another non-
purine based XO inhibitor, was also showed significant
activity but with similar side effects as allopurinol (Pascart
and Richette 2018). Based on these particular results, it can
be concluded that researchers that involved in this field
must develop alternate scaffolds that act as potent XO
inhibitors with lesser side effects that can be used for the
treatment of hyperuricemia.
ideal moiety to be included in the novel alternative mole-
cular architecture for the further development of XO
inhibitors.
From the last 5 years, we are continuously working on
the similar lines to develop non-purine based XO inhibitors
(Dhiman et al. 2012; Shukla et al. 2014; Sharma et al. 2014;
Virdi et al. 2014; Kaur et al. 2015; Kaur et al. 2015, Kaur
et al. 2017; Singh et al. 2019; Kaur et al. 2019) and now, we
have come up with a new series of coumarin hybrid
molecules clubbed with indolinedione with the help of
triazole moiety as a linker. The rationale for the inclusion of
indolinedione to the designed molecules is very clear. The
two carbonyl groups and the nitrogen atom of the indole
ring could act as hydrogen bond acceptor in the active site
of the XO enzyme, which was confirmed through molecular
modeling studies in the current evaluation. 1,2,3-triazole is
also an active moiety owing to its three H-bond acceptor-
Nitrogen atoms, the inclusion of which could also be ben-
eficial for its better interactions within the XO enzyme.
Results and discussion
Indolinedione–coumarin hybrids were synthesized by fol-
lowing the synthetic Scheme 1. At first, substituted indoli-
nediones were stirred with 1,2-dibromoalkanes (1 eq) at
room temperature using Dimethylformamide (DMF) as a
solvent, and potassium carbonate (1.5 eq) as a base. The
resulting product was then dissolved in DMF and then
sodium azide (1 eq) was added. The reaction mixture was
stirred at room temperature to form 1-(4-azidoalkyl)indo-
line-2,3-diones.
Then, 4-hydroxycoumarin and propargyl bromide (1.2
eq) were stirred in DMF under basic conditions (K2CO3; 1.5
eq) to obtain 4(prop-2-ynyloxy)-2H-chromen-2-one (PHC).
This 4-(prop-2-ynyloxy)-2H-chromen-2-one (PHC) was
further reacted with various 1-(4-azidoalkyl)indoline-2,3-
dione analogues in the presence of pentahydrate CuSO4
(catalytic amount) and sodium ascorbate (as a reducing
agent of CuSO4), in DMF at room temperature to obtain the
desired hybrid compounds. All compounds were monitored
by thin-layer chromatography in the reaction, purified by
Besides these, many researchers have also focussed on
XO inhibitors derived from natural compounds-flavonoids,
hydroxycinnamic acids, tannins, chalcones, saponins, ter-
penoids, stilbenes, phenylethanoid glycosides, since these
can act as lead compounds for the discovery of new syn-
thetics (Malik et al. 2018; Mehmood et al. 2019).
1
column chromatography. H NMR and 13C NMR spectro-
scopic techniques were used to characterize the synthesized
compounds and all the spectral data was found in accor-
dance with assumed structures.
Coumarin is a natural moiety that is known to its various
biological activities, also found to be a potent XO inhibitor.
Numerous reports on the XO inhibitory potential of cou-
marin derivatives are available (Fais et al. 2018; Chen et al.
2014). Figure 1 depicts recently reported coumarin deriva-
tives with XO inhibitory potential. Therefore, it could be an
In vitro xanthine oxidase assay
All the synthesized compounds were evaluated to test their
inhibition against XO enzyme at five different concentra-
tions ranging from 1 to 50 µM. The formation of UA was
determined by using UV spectrophotometer at 292 nm and

Medicinal Chemistry Research
OH
OH
OH
O
O
HO
O
HO
HO
O
O
OH
HO
HO
HO
O
O
OH
1; IC50 = 2.13 µM (XO)
2; IC50 = 7.49 µM (XO)
3; IC50 = 57.4 µM (XO)
Fig. 1 Recently reported potent coumarin derivatives as XO inhibitors
O
Scheme 1 Synthesis of
indolinedione–coumarin
hybrids. Reagents and
R
O
conditions: (a) dibromoalkanes,
K2CO3, DMF, 2 h, stir, rt; (b)
NaN3, DMF, 1 hr, stir, rt; (c)
Propargyl bromide, K2CO3,
DMF, 2 h, stir, rt; (d) Sodium
ascorbate, CuSO4, DMF,
15 min, rt
N
H
Code
n
R
a
K-1
K-2
K-3
K-4
K-5
K-6
K-7
1
1
1
1
1
1
1
H
F
Cl
O
Br
I
R
OH
NO2
OCH3
O
N
n
O
c
O
Br
b
Code
n
R
L-1
L-2
L-3
L-4
L-5
L-6
L-7
2
2
2
2
2
2
2
H
O
F
R
Cl
Br
O
O
I
N
NO2
OCH3
n
O
O
N3
d
Code
n
R
O
M-1
M-2
M-3
M-4
M-5
M-6
M-7
3
3
3
3
3
3
3
H
O
O
O
F
Cl
Br
O
N
N
N
I
n
N
NO2
OCH3
Indolinedione-coumarin hybrids
n = 1, 2, 3, 4
R = H, F, Cl, Br, I, NO2, OCH3
R
percentage inhibition was calculated for each compound.
(Table 1). The results of the assay revealed that compounds
showed good to moderate inhibition. Two compounds (K-1
& K-7) showed above 80% inhibition against the enzyme at
50 µM concentration, while some displayed below 30%
inhibition even at 50 µM concentration. The concentration
at which 50% of the enzyme was inhibited, was calculated
only for those compounds which inhibited the enzyme
above 50% at 50 µM concentration. Two compounds (K-1
& K-7) were found to be endowed with the most prominent
inhibition with the IC50 values of 8.9 µM and 6.5 µM,
respectively. The IC50 values of all the compounds were
ranging from 6.5 to 24.5 µM. The results revealed that the
structural architecture of the compounds greatly influences
the inhibitory activity. For instance, the compounds bearing
electron-donating groups (-OCH3) on 5th position of

Medicinal Chemistry Research
Table 1 Xanthine oxidase
inhibitory activity of all the
synthesized compounds
O
O
O
O
O
N
N
N
n
N
R
Percent inhibition
IC50
Compound
R
n
1 (µM)
5 (µM)
10 (µM)
25 (µM)
50 (µM)
(µM)
K-1
K-2
K-3
K-4
K-5
K-6
K-7
L-1
L-2
L-3
L-4
L-5
L-6
L-7
M-1
M-2
M-3
M-4
M-5
M-6
M-7
H
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
32
20
24
23
23
21
35
25
12
11
19
21
10
23
9
42
29
30
34
39
30
49
29
19
17
28
30
17
40
15
5
62
39
40
45
48
38
69
41
25
28
34
40
26
49
21
9
79
52
56
60
59
53
85
57
34
39
51
53
32
60
30
18
22
27
28
24
51
85
57
61
65
70
59
92
63
45
49
57
60
44
69
44
30
38
39
41
29
55
8.9 0.12
F
18.4 1.9
Cl
Br
I
15.6 1.43
14.3 1.23
11.4 1.08
NO2
12.5 1.09
OCH3
H
6.5 0.09
15.7 1.45
F
–
Cl
–
Br
24.5 2.87
I
17.8 1.98
NO2
OCH3
H
–
12.4 0.98
–
F
3
–
Cl
4
8
13
20
21
17
37
–
Br
8
14
13
8
–
I
7
–
NO2
OCH3
2
–
19
30
20 1.76
8.16 1.27
Allopurinol
indolinedione moiety enhanced the activity as compared
with the compounds bearing electron-withdrawing groups
(halogen atoms). The chain length between indolinedione
and triazole moieties also affects the activity, as the chain
length increases the inhibitory activity gradually decreases.
Therefore, the activity order for substitution on 5th position
of indolinedione followed as: -OCH3 > H > I > Br > Cl > F ≈
NO2 and for chain length (n): 1 > 2 > 3. From these results,
it has been cleared that relatively compounds with high
molecular weight are less potent than that of small mole-
cules. This can be explained on the basis of the size of the
active pocket of the enzyme XO, which is relatively very
small and those compounds which can well occupy in the
cavity showed the inhibitory activity (Table 1). This was
also confirmed through molecular modeling studies in sec-
tion (Molecular modelling studies).
Fig. 2 Lineweaver–Burk plot of K-7
Enzyme kinetics
Compound K-7 was further investigated for the type of
inhibition by performing enzyme kinetic studies (Fig. 2).
The pattern of the Lineweaver–Burk plot graph shows that

Medicinal Chemistry Research
Fig. 3 3D representation of K-7 (blue) within the active site of XO enzyme (Left-hand side), 2D representation of K-7 within the active site of the
enzyme (right-hand side)
it is a form of mixed inhibition scenario where Km, Vmax
,
cavity and is well stabilized by various electrostatic inter-
actions. Major interactions with XO enzyme include π-
sigma, π-alkyl, carbon-hydrogen bond, π-donor hydrogen
bond, and conventional hydrogen bond interaction. Indoli-
nedione moiety is well positioned in a cavity formed by
hydrophobic residues Ser876, Leu873, Phe1009, Phe914,
Ala1079, and Ala1078. The methoxy group present on the
5th position of indolinedione forms the conventional
hydrogen bond with the amine group of Ala1079. Three
additional hydrogen bonds are formed as expected with the
two carbonyl groups of indolinedione with amino acid
residues Arg880 and Thr1010. On the other end, coumarin
moiety is inserted into the cavity formed by various
hydrophobic amino acids Pro1076, Leu648, Val1011,
Lys771, Leu1014, and Phe1013 and also stabilized by
H-bond interaction with Lys771 through an oxygen atom of
pyran ring (Fig. 3). Therefore, docking studies suggest that
the K-7 completely blocks the binding pocket of the XO
enzyme to exert its inhibitory action which also supports its
potent in vitro activity.
and slope are all affected by the inhibitor. The inhibitor has
increased the Km and slope (Km/Vmax) while decreasing the
Vmax. Moreover, on careful observation, 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 α
(a constant that defines the degree to which inhibitor
binding affects the affinity of the enzyme for substrate) is
>1 (Copeland 2005). This confirms that the inhibitor pre-
ferentially binds to the free enzyme and not the enzyme-
substrate complex. Therefore, the mode of inhibition of K-7
is mixed-type but it seems that it has a strong competitive
component.
Molecular modeling studies
Molecular modeling studies were performed to elucidate
interactions of the most potent compound K-7 within the
active site of XO enzyme. The X-ray crystallographic
structure of XO, complexed with febuxostat (PDB entry:
1N5X; resolution 2.8 Å) was employed. The accuracy of the
docking program was validated by docking co-crystallized
ligand febuxostat in its binding pocket. The program was
able to reproduce best fit confirmation of febuxostat with a
root mean square deviation of 0.523 indicating the relia-
bility of docking protocol. After that, compound K-7 was
docked into febuxostat binding site and its top best pose
with dock score of −32.2563 was selected for discussion
(Fig. 3).
Conclusion
Coumarin and indolinedione are multifunctional products
and in the current study, we have rationally designed and
synthesized various Indolinedione–coumarin molecular
hybrids. Their bioactivities were evaluated in terms of their
XO inhibitory potential. Almost all of the compounds were
found active while two compounds K-1 and K-7 showed
better results in vitro and were potent ones as compared
Overall binding mode of compound K-7 with residues of
the binding site suggests that compound fits well in the

Medicinal Chemistry Research
with standard drug allopurinol. Structure-activity relation-
ship revealed that the compounds with electron-donating
groups (-OCH3) increased the activity and length of two
carbon chain between indolinedione and triazole moieties
are most favorable, one for the inhibitory potential. Kinetic
studies indicated that K-7 showed its mixed-type inhibition
scenario against the XO enzyme. Various binding interac-
tions of K-7 with the active site of the XO enzyme were
also streamlined by using docking studies. Therefore, these
compounds could act as hit lead molecules for the further
development of potent XO inhibitors.
76.50, 79.32, 91.71, 115.39, 116.60, 123.16, 124.28,
132.83, 153.17, 162.02, 164.24.
General procedure for the synthesis of 1-(2-bromoalkyl)
indoline-2,3-diones
Indoline-2,3-dione (1 eq) was dissolved in DMF (in mini-
mum amount), dibromoalkane (1 eq) and K2CO3 (1.5 eq)
were added. The reaction mixture was stirred at room
temperature. After the completion of the reaction as con-
firmed by TLC, the reaction mixture was poured on crushed
ice. The impure product so obtained was filtered, air-dried,
and subjected to column chromatography (hexane/ethyl
acetate as eluent) to gain the desired product. All the other
1-bromoalkylindoline-2,3-diones were synthesized via
the same procedure as mentioned above using various
dibromoalkanes.
Experimental
Material and measurements
The chemical reagents were procured from CDH, Sigma-
Aldrich, and Loba, India. All yields refer to isolated pro-
ducts after purification. Products were characterized by
comparing with authentic samples and by spectroscopic
techniques i.e., 1H and 13C NMR, Elemental Analysis).
Avance III HD 500 MHz Bruker Biospin and JEOL AL
300 MHz machines were used to record the NMR spectra.
The spectra were recorded by dissolving in CDCl3 and
General procedure for the synthesis of 1-(4-azidoalkyl)
indoline-2,3-diones (IBA)
In a minimum amount of DMF, 1-(4-Bromoalkyl)indoline-
2,3-dione (1 eq) were dissolved and sodium azide (1 eq)
was added and the mixture was stirred at room temperature.
After the completion of the reaction, the reaction mixture
was poured on crushed ice and precipitates (1-(4-azi-
doalkyl)indoline-2,3-diones) thus obtained were collected
by simple filtration.
All the other 1-azidoalkylindoline-2,3-dione analogues
were synthesized by following the above-mentioned
procedure.
1
DMSO-d6 relative to TMS (0.00 ppm). In H NMR che-
mical shifts were reported in δ values using an internal
standard (tetramethylsilane) with a number of protons,
multiplicities (s-singlet, d-doublet, t-triplet, q-quartet, m-
multiplet) and coupling constants (J) in Hertz (Hz). Melting
points were determined in open capillaries and were
uncorrected.
General procedure for the synthesis of triazole linked
indoline-2,3-dione-coumarin hybrids
General procedure for the synthesis of 4-(prop-2-ynyloxy)-
2H-chromen-2-one (PGC)
In DMF, 1-azidoalkylindoline-2,3-dione(1 eq) and 4-(prop-
2-ynyloxy)-2H-chromen-2-one (PHC) (1 eq) were dis-
solved. The catalytic amount of pentahydrate copper sul-
phate (CuSO4.5H2O) and its reducing agent, sodium
ascorbate were added in it. Reaction mixture was kept aside
for some time, at room temperature. After the completion of
reaction as confirmed by TLC, the reaction mixture was
filtered directly on crushed ice to remove the excess of
CuSO4 and sodium ascorbate. The solidified final product
thus obtained was filtered and air-dried. The physical data
of all the synthesized bi-functional hybrids is given below:
In 50 mL of DMF, 4-Hydroxy coumarin (20 g) was dis-
solved with the addition of propargyl bromide (1.2 eq) and
K2CO3 (1.5 eq). The reaction mixture was stirred at room
temperature and the reaction was continuously monitored
by TLC. After the completion of the reaction, the reaction
mixture was poured on crushed ice. The solid product of
propargylated coumarin thus obtained was filtered, washed
with cold water, and air-dried. The physical data of pro-
pargylated coumarin is given below:
4-(prop-2-ynyloxy)-2H-chromen-2-one (PGC)
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
Yield 80%; mp 103–107 °C.1H NMR (CDCl3, 500 MHz, δ,
TMS = 0): 3.22–3.24 (m, 1H, -CH-propargylic), 4.92–4.94
(m, 2H, -CH2-), 5.84 (d, 1H, J = 12 Hz, -CH-), 7.25–7.27
(m, 2H, ArH), 7.56 (s, 1H, ArH), 7.77 (d, 1H, J = 8 Hz,
ArH). 13C NMR (CDCl3, 125 MHz, δ, TMS = 0): 57.38,
zol-1-yl)ethyl)indoline-2,3-dione (K-1): Yield 78%, mp
1
104–108 °C. H NMR (d6-DMSO, 300 MHz, δ, TMS = 0):
4.21 (s, 2H, -CH2-), 4.77 (s, 2H, -CH2-), 5.38 (s, 2H, -CH2-),
6.11 (s, 1H, -CH-), 6.91 (d, 1H, J = 7.8 Hz, ArH), 7.08 (t,
1H, ArH, J = 7.5 Hz), 7.41–7.46 (m, 2H, ArH), 7.54–7.56

Medicinal Chemistry Research
(m, 2H, ArH), 7.68–7.71 (m, 2H, ArH), 8.49 (s, 1H, ArH).
13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.64, 62.93,
91.50, 110.52, 115.43, 116.84, 117.64, 123.29, 123.79,
124.64, 125.00, 133.33, 138.59, 150.54, 153.11, 158.55,
162.14, 164.73, 183.32. Anal.Calcd for C22H16N4O5: C,
63.46; H, 3.87; N, 13.46 Found: C, 63.26; H, 3.98; N, 13.15.
7.84–7.87 (m, 2H, ArH), 8.61 (s, 1H, ArH). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 47.59, 63.23, 91.74,
112.86, 115.57, 116.91, 119.59, 123.24, 124.70, 126.53,
127.30, 133.34, 140.19, 149.52, 153.27, 155.90, 161.97,
164.73, 182.53. Anal.Calcd for C22H15IN4O5: C, 48.73; H,
2.79; I, 23.40; N, 10.33 Found: C, 48.93; H, 2.68; I, 23.44;
N, 10.22.
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-fluoroindoline-2,3-dione
(K-2): Yield
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-nitroindoline-2,3-dione (K-6): Yield 83%,
mp 104–108 °C. 1H NMR (d6-DMSO, 500 MHz, δ, TMS =
0): 4.14 (s, 2H, -CH2-), 4.65 (s, 2H, -CH2-), 5.37 (s, 2H,
-CH2-), 6.11 (s, 1H, -CH-), 6.83 (s, 1H, ArH), 7.41–7.44
(m, 2H, ArH), 7.61–7.64 (m, 2H, ArH), 7.96–7.99 (m, 2H,
ArH), 8.76 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz,
δ, TMS = 0): 47.71, 63.32, 91.65, 112.82, 115.55, 116.96,
119.62, 123.43, 124.76, 126.52, 127.13, 135.33, 146.56,
152.43, 153.86, 155.95, 162.45, 164.78, 182.72. Anal.
Calcd for C22H15N5O7: C, 57.27; H, 3.28; N, 15.18Found:
C, 57.42; H, 3.13; N, 15.45.
79%, mp 74–78 °C. 1H NMR (d6-DMSO, 300 MHz, δ,
TMS = 0): 4.22 (s, 2H, -CH2-), 4.76 (s, 2H, -CH2-), 5.39 (s,
2H, -CH2-), 6.14 (s, 1H, -CH-), 6.94 (s, 1H, ArH),
7.40–7.47 (m, 4H, ArH), 7.69–7.71 (m, 2H, ArH), 8.51 (s,
1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
47.61, 63.06, 79.06, 79.32, 79.58, 91.60, 111.80, 112.00,
112.07, 115.45, 116.87, 118.58, 118.64, 123.22, 124.60,
133.28, 146.86, 153.16, 158.59, 162.03, 164.72, 182.73.
Anal.Calcd for C22H15FN4O5: C, 60.83; H, 3.48; F, 4.37; N,
12.90 Found: C, 60.52; H, 3.50; F, 4.21; N, 12.96.
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-chloroindoline-2,3-dione
(K-3): Yield
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-methoxyindoline-2,3-dione (K-7): Yield
75%, mp 70–74 °C. 1H NMR (d6-DMSO, 300 MHz, δ,
TMS = 0): 3.68 (s, 3H, -OCH3), 4.12 (s, 2H, -CH2-), 4.69
(s, 2H, -CH2-), 5.32 (s, 2H, -CH2-), 6.06 (s, 1H, -CH-), 6.80
(d, 1H, J = 8.0 Hz, ArH), 7.07–7.09 (m, 2H, ArH),
7.33–7.39 (m, 2H, ArH), 7.63–7.66 (m, 2H, ArH), 8.42
(s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS =
0): 47.61, 56.24, 63.06, 91.57, 109.50, 111.73, 112.08,
115.45, 116.87, 118.19, 123.26, 124.44, 124.64, 126.59,
133.30, 144.45, 153.67, 156.15, 158.61, 162.08, 164.77,
183.56. Anal.Calcd for C23H18N4O6: C, 61.88; H, 4.06; N,
12.55 Found: C, 60.99; H, 4.23; N, 12.32.
1
79%, mp 160–164 °C. H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 4.16 (s, 2H, -CH2-), 4.70 (s, 2H, -CH2-), 5.34 (s,
2H, -CH2-), 6.09 (s, 1H, -CH-), 6.88 (d, 1H, J = 8.5 Hz,
ArH), 7.35–7.40 (m, 2H, ArH), 7.56–7.57 (m, 2H, ArH),
7.64–7.67 (m, 2H, ArH), 8.45 (s, 1H, ArH). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 47.59, 63.15, 91.68,
112.35, 115.49, 116.90, 119.13, 123.23, 124.48, 124.66,
126.67, 128.02, 133.26, 137.35, 149.19, 153.20, 158.34,
161.99, 164.74, 182.25. Anal.Calcd for C22H15ClN4O5: C,
58.61; H, 3.35; Cl, 7.86; N, 12.43 Found: C, 58.33; H, 3.54;
Cl, 7.77; N, 12.55.
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-bromoindoline-2,3-dione
(K-4): Yield
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
83%, mp 74–78 °C. 1H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 4.15 (s, 2H, -CH2-), 4.70 (s, 2H, -CH2-), 5.34 (s,
2H, -CH2-), 6.10 (s, 1H, -CH-), 6.84 (d, 1H, J = 8 Hz,
ArH), 7.37–7.41 (m, 2H, ArH), 7.65–7.71 (m, 4H, ArH),
8.44 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ,
TMS = 0): 47.55, 63.15, 91.69, 112.80, 115.52, 116.92,
119.52, 123.26, 124.71, 126.56, 127.24, 133.29, 140.17,
149.57, 153.21, 155.86, 162.02, 164.77, 182.12. Anal.
Calcd for C22H15BrN4O5: C, 53.35; H, 3.05; Br, 16.13; N,
11.31 Found: C, 53.44; H, 3.01; Br, 16.33; N, 11.12.
zol-1-yl)propyl)indoline-2,3-dione (L-1): Yield 76%, mp
122–125 °C. H NMR (d6-DMSO, 300 MHz, δ, TMS = 0):
1
2.23 (s, 2H, -CH2-), 3.75 (s, 2H, -CH2-), 4.52 (s, 2H, -CH2-
), 5.41 (s, 2H, -CH2-), 6.15 (s, 1H, -CH-), 7.11–7.18
(m, 2H, ArH), 7.32–7.41 (m, 2H, ArH), 7.54–7.75 (m, 4H,
ArH), 8.37 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz,
δ, TMS = 0): 28.00, 37.36, 47.71, 63.29, 91.78, 110.97,
115.54, 116.94, 118.19, 123.25, 123.61, 124.70, 124.89,
125.81, 133.31, 138.45, 150.84, 153.23, 158.82, 162.03,
164.82, 183.74. Anal.Calcd for C23H18N4O5: C, 64.18; H,
4.22; N, 13.02 Found: C, 64.33; H, 4.11; N, 13.41.
1-(2-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)ethyl)-5-iodoindoline-2,3-dione (K-5): Yield 81%,
mp 88–91 °C. H NMR (d6-DMSO, 500 MHz, δ, TMS =
0): 4.17 (s, 2H, -CH2-), 4.68 (s, 2H, -CH2-), 5.37 (s, 2H,
-CH2-), 6.08 (s, 1H, -CH-), 6.82 (d, 1H, J = 8 Hz, ArH),
7.35–7.39 (m, 2H, ArH), 7.63–7.65 (m, 2H, ArH),
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
1
zol-1-yl)propyl)-5-fluoroindoline-2,3-dione
(L-2): Yield
79%, mp 94–97 °C. 1H NMR (d6-DMSO, 300 MHz, δ,
TMS = 0): 2.18 (s, 2H, -CH2-), 4.19 (s, 2H, -CH2-), 4.77 (s,
2H, -CH2-), 5.37 (s, 2H, -CH2-), 6.15 (s, 1H, -CH-), 6.93

Medicinal Chemistry Research
(s, 1H, ArH), 7.43–7.48 (m, 4H, ArH), 7.70–7.74 (m, 2H,
ArH), 8.50 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz,
δ, TMS = 0): 28.87, 47.56, 63.21, 79.32, 79.31, 79.54,
91.66, 111.83, 112.12, 112.21, 115.43, 116.82, 118.52,
118.59, 123.32, 124.64, 133.19, 146.82, 153.14, 158.73,
162.23, 164.69, 183.74. Anal.Calcd for C23H17FN4O5: C,
61.61; H, 3.82; F, 4.24; N, 12.49 Found: C, 61.45; H, 3.95;
F, 4.06; N, 12.53.
TMS = 0): 2.14 (s, 2H, -CH2-), 4.15 (s, 2H, -CH2-), 4.65 (s,
2H, -CH2-), 5.34 (s, 2H, -CH2-), 6.10 (s, 1H, -CH-), 6.83 (s,
1H, ArH), 7.42–7.45 (m, 2H, ArH), 7.61–7.67 (m, 3H,
ArH), 7.97–7.99 (m, 1H, ArH), 8.76 (s, 1H, ArH). 13C
NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.56, 47.73,
63.31, 91.64, 112.83, 115.56, 116.92, 119.65, 123.43,
124.77, 126.52, 127.16, 135.34, 146.53, 152.46, 153.82,
155.94, 162.55, 164.74, 182.72. Anal.Calcd for
C23H17N5O7: C, 58.11; H, 3.60; N, 14.73Found:C, 58.19;
H, 3.51; N, 14.99.
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)propyl)-5-chloroindoline-2,3-dione
(L-3): Yield
1
71%, mp 166–169 °C. H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 2.20 (s, 2H, -CH2-), 3.74 (s, 2H, -CH2-), 4.51 (s,
2H, -CH2-), 5.41 (s, 2H, -CH2-), 6.15 (s, 1H, -CH-), 7.03–7.04
(m, 1H, ArH), 7.34–7.41 (m, 2H), 7.66–7.96 (m, 4H, ArH),
8.35 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS =
0): 27.87, 37.40, 47.65, 63.30, 86.47, 91.78, 113.48, 115.54,
116.95, 123.35, 124.70, 125.76, 132.53, 133.32, 145.86,
150.17, 153.23, 158.17, 162.03, 164.83, 182.41. Anal.Calcd
for C23H17ClN4O5: C, 59.43; H, 3.69; Cl, 7.63; N, 12.05
Found: C, 59.60; H, 3.55; Cl, 7.75; N, 12.01.
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)propyl)-5-methoxyindoline-2,3-dione (L-7): Yield
76%, mp 87–90 °C. 1H NMR (d6-DMSO, 300 MHz, δ, TMS
= 0): 2.19–2.24 (m, 2H, -CH2-), 3.71–3.76 (m, 3H, -OCH3),
4.50–4.53 (m, 2H, -CH2-), 5.41 (s, 2H, -CH2-), 6.15 (s, 1H,
-CH-), 7.10–7.14 (m, 2H, ArH), 7.21–7.23 (m, 1H, ArH),
7.32–7.41 (m, 2H, ArH), 7.64–07.75 (m, 2H, ArH), 8.37 (s,
1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
27.99, 36.26, 37.34, 47.71, 56.36, 63.30, 91.77, 109.76,
112.04, 115.54, 116.93, 118.68, 123.35, 124.09, 124.69,
133.31, 144.59, 153.22, 156.16, 158.82, 162.03, 164.83,
183.98. Anal.Calcd for C24H20N4O6: C, 62.60; H, 4.38; N,
12.17 Found: C, 62.66; H, 4.13; N, 12.34.
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)propyl)-5-bromoindoline-2,3-dione
(L-4): Yield
80%, mp 74–78 °C. 1H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 2.16 (s, 2H, -CH2-), 4.17 (s, 2H, -CH2-), 4.72 (s,
2H, -CH2-), 5.35 (s, 2H, -CH2-), 6.08 (s, 1H, -CH-), 6.86
(d, 1H, J = 8 Hz, ArH), 7.38–7.42 (m, 2H, ArH), 7.64–7.70
(m, 4H, ArH), 8.46 (s, 1H, ArH). 13C NMR (d6-DMSO,
125 MHz, δ, TMS = 0): 28.66, 47.59, 63.12, 91.71,
112.89, 115.56, 116.97, 119.57, 123.29, 124.76, 126.58,
127.28, 133.32, 140.19, 149.59, 153.28, 155.89, 162.06,
164.79, 182.56. Anal.Calcd for C23H17BrN4O5: C, 54.24;
H, 3.36; Br, 15.69; N, 11.00 Found: C, 54.28; H, 3.15; Br,
15.78; N, 11.03.
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)indoline-2,3-dione(M-1): Yield 74%, mp
1
146–150 °C. H NMR (d6-DMSO, 500 MHz, δ, TMS = 0):
1.58–1.61 (m, 2H, -CH2-), 1.92–1.95 (m, 2H, -CH2-),
3.69–3.72 (m, 2H, -CH2-), 4.44–4.47 (m, 2H, -CH2-), 5.40
(s, 2H, -CH2-), 6.14 (s, 1H, -CH-), 7.11–7.18 (m, 2H, ArH),
7.34–7.41 (m, 2H, ArH), 7.53 (d, 1H, J = 7 Hz, ArH),
7.63–7.66 (m, 2H, ArH), 7.73 (d, 1H, J = 7.5 Hz, ArH),
8.38 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ,
TMS = 0): 24.25, 27.42, 49.53, 63.33, 91.77, 111.06,
115.54, 116.91, 118.01, 123.33, 123.58, 124.66, 124.90,
133.26, 138.51, 151.03, 153.22, 158.67, 161.98, 164.81,
183.86. Anal.Calcd for C24H20N4O5: C, 64.86; H, 4.54; N,
12.61 Found: C, 64.97; H, 4.32; N, 12.85.
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)propyl)-5-iodoindoline-2,3-dione
(L-5): Yield
1
83%, mp 98–102 °C. H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 2.13 (s, 2H, -CH2-), 4.14 (s, 2H, -CH2-), 4.70 (s,
2H, -CH2-), 5.35 (s, 2H, -CH2-), 6.11 (s, 1H, -CH-), 6.81 (s,
1H, ArH), 7.36–7.39 (m, 2H, ArH), 7.65–7.68 (m, 2H,
ArH), 7.80–7.84 (m, 2H, ArH), 8.59 (s, 1H, ArH). 13C
NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.69, 47.65,
63.28, 91.72, 112.81, 115.54, 116.98, 119.63, 123.23,
124.72, 126.57, 127.33, 133.37, 140.14, 149.56, 153.24,
155.94, 161.94, 164.76, 182.63. Anal.Calcd for
C23H17IN4O5: C, 49.66; H, 3.08; I, 22.81; N, 10.07 Found:
C, 49.55; H, 2.88; I, 22.84; N, 10.01.
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)-5-fluoroindoline-2,3-dione
(M-2): Yield
1
77%, mp 110–114 °C. H NMR (d6-DMSO, 300 MHz, δ,
TMS = 0): 1.59 (s, 2H, -CH2-), 1.87 (s, 2H, -CH2-), 3.70 (s,
2H, -CH2-), 4.44 (s, 2H, -CH2-), 5.34 (s, 2H, -CH2-), 6.13
(s, 1H, -CH2-), 6.91 (s, 1H, -CH-), 7.44–7.49 (m, 4H, ArH),
7.72–7.74 (m, 2H, ArH), 8.52 (s, 1H, ArH). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 24.44, 27.34, 47.52, 63.25,
79.32, 79.38, 79.53, 91.65, 111.83, 112.11, 112.29, 115.42,
116.81, 118.55, 118.68, 123.32, 124.61, 133.23, 146.81,
153.15, 158.77, 162.19, 164.71, 183.72. Anal.Calcd for
C24H19FN4O5: C, 62.34; H, 4.14; F, 4.11; N, 12.12 Found:
C, 62.62; H, 4.25; F, 3.99; N, 12.19.
1-(3-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)propyl)-5-nitroindoline-2,3-dione
79%, mp 106–109 °C. H NMR (d6-DMSO, 500 MHz, δ,
(L-6): Yield
1

Medicinal Chemistry Research
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
Calcd for C24H19N5O7: C, 58.90; H, 3.91; N, 14.31 Found:
C, 58.99; H, 3.76; N, 14.42.
zol-1-yl)butyl)-5-chloroindoline-2,3-dione
(M-3): Yield
1
70%, mp 170–172 °C. H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 1.56 (s, 2H, -CH2-), 1.92 (s, 2H, -CH2-), 3.68 (s,
2H, -CH2-), 4.44 (s, 2H, -CH2-), 5.39 (s, 2H, -CH2-), 6.13
(s, 1H, -CH-), 7.20–7.40 (m, 3H, ArH), 7.56–7.73 (m, 4H,
ArH), 8.39 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz,
δ, TMS = 0): 24.14, 27.30, 49.56, 63.37, 91.75, 112.75,
115.52, 116.89, 119.43, 123.33, 124.37, 124.67, 127.79,
133.27, 137.29, 149.53, 153.20, 158.46, 162.01, 164.82,
182.75. Anal.Calcd for C24H19ClN4O5: C, 60.19; H, 4.00;
Cl, 7.40; N, 11.70 Found: C, 60.30; H, 3.88; Cl, 7.47;
N, 11.64.
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)-5-methoxyindoline-2,3-dione (M-7): Yield
1
73%, mp 97–100 °C. H NMR (d6-DMSO, 300 MHz, δ,
TMS = 0): 1.57 (s, 2H, -CH2-), 1.92 (s, 2H, -CH2-), 3.67 (s,
2H, -CH2-), 3.75 (s, 3H, -OCH3), 4.44 (s, 2H, -CH2-), 5.39
(s, 2H, -CH2-), 6.13 (s, 1H, -CH-), 7.09–7.11 (m, 2H, ArH),
7.20–7.22 (m, 1H, ArH), 7.33–7.35 (m, 1H, ArH),
7.39–7.40 (m, 1H, ArH), 7.65–7.67 (m, 1H, ArH),
7.72–7.73 (m, 1H, ArH), 8.37 (s, 1H, ArH). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 24.23, 27.39, 49.53, 56.35,
63.32, 91.74, 109.78, 112.13, 115.52, 116.90, 118.18,
123.33, 124.23, 124.68, 125.58, 133.28, 144.78, 153.20,
156.14, 158.67, 162.02, 164.83, 184.12. Anal.Calcd for
C25H22N4O6: C, 63.29; H, 4.67; N, 11.81 Found: C, 63.45;
H, 4.47; N, 11.92.
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)-5-bromoindoline-2,3-dione
(M-4): Yield
76%, mp 75–80 °C. 1H NMR (d6-DMSO, 500 MHz, δ,
TMS = 0): 1.57 (s, 2H, -CH2-), 1.92 (s, 2H, -CH2-), 3.69 (s,
2H, -CH2-), 4.44 (s, 2H, -CH2-), 5.40 (s, 2H, -CH2-), 6.14
(s, 1H, -CH-), 7.16 (s, 1H, ArH), 7.34–7.41 (m, 2H, ArH),
7.66–7.80 (m, 4H, ArH), 8.38 (s, 1H, ArH). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 24.13, 27.31, 49.55, 63.36,
88.90, 91.77, 113.20, 115.27, 115.53, 116.91, 119.83,
123.34, 124.46, 127.10, 133.27, 140.09, 147.92, 153.21,
158.30, 161.99, 164.81, 182.61. Anal.Calcd for
C24H19BrN4O5: C, 55.08; H, 3.66; Br, 15.27; N, 10.71
Found: C, 55.25; H, 3.44; Br, 15.45; N, 10.55.
In vitro xanthine oxidase assay
All the synthesized compounds were evaluated against XO
enzyme. Bovine milk XO (grade 1, ammonium sulfate
suspension, Sigma-Aldrich, India) activity was assayed
spectrophotometrically by measuring the UA formation at
293 nm using a Hitachi U-3010 UV-visible spectro-
photometer at 25 °C. The reaction mixture contains 50 mM
potassium phosphate buffer (pH 7.6), 75 µM xanthine, and
0.08 units of XO. Inhibition of XO activity of synthetics at
different concentrations (1, 5, 10, 25, 50 µM) was measured
by following the decrease in the UA formation at 293 nm at
25 °C. The enzyme was pre-incubated for 5 min with a test
compound, dissolved in DMSO (1% v/v), and the reaction
was started by the addition of xanthine. The final con-
centration of DMSO (1% v/v) did not interfere with the
enzyme activity. All the experiments were performed in
triplicate and values were expressed as the mean of three
experiments (Escribano et al. 1988; Takano et al. 2005).
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)-5-iodoindoline-2,3-dione (M-5): Yield 83%,
1
mp 97–102 °C. H NMR (d6-DMSO, 500 MHz, δ, TMS =
0): 1.58 (s, 2H, -CH2-), 1.91 (s, 2H, -CH2-), 3.71 (s, 2H,
-CH2-), 4.44 (s, 2H, -CH2-), 5.38 (s, 2H, -CH2-), 6.08 (s,
1H, -CH-), 6.84 (s, 1H, ArH), 7.34–7.37 (m, 2H, ArH),
7.67–7.69 (m, 2H, ArH), 7.76–7.79 (m, 2H, ArH), 8.61 (s,
1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
24.54, 27.43, 48.99, 63.56, 91.76, 112.82, 115.58, 117.12,
119.67, 123.25, 124.77, 126.53, 127.33, 133.32, 140.11,
149.45, 153.00, 155.95, 161.94, 164.71, 182.73. Anal.
Calcd for C24H19IN4O5: C, 50.54; H, 3.36; I, 22.25; N, 9.82
Found: C, 50.51; H, 3.45; I, 22.12; N, 10.00.
Enzyme kinetics study
Potent XO enzyme inhibitors were further investigated for
the type of inhibition and enzyme kinetics study was carried
out. The Lineweaver–Burk plot was established from which
we could calculate the Km, Vmax of the slope of inhibitor and
the value of α (a constant that defines the degree to which
inhibitor binding affects the affinity of the enzyme for
substrate (Copeland 2005).
1-(4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-tria-
zol-1-yl)butyl)-5-nitroindoline-2,3-dione (M-6): Yield 80%,
mp 110–112 °C. 1H NMR (d6-DMSO, 500 MHz, δ, TMS =
0): 1.57 (s, 2H, -CH2-), 1.92 (s, 2H, -CH2-), 3.66 (s, 2H,
-CH2-), 4.43 (s, 2H, -CH2-), 5.37 (s, 2H, -CH2-), 6.10 (s,
1H, -CH-), 6.84 (s, 1H, ArH), 7.44–7.47 (m, 2H, ArH),
7.62–7.67 (m, 2H, ArH), 7.94–7.96 (m, 2H, ArH), 8.72 (s,
1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
24.34, 27.65, 48.56, 63.65, 91.34, 112.87, 115.52, 116.97,
119.63, 123.46, 124.73, 126.55, 127.12, 135.35, 146.56,
152.43, 153.84, 155.96, 162.53, 164.77, 182.69. Anal.
Molecular modeling studies
Crystal structure of XO (PDB entry: 1N5X; resolution
2.8 Å) was downloaded from Protein Data Bank.

Medicinal Chemistry Research
Preparation of structure was done by using drug design
platform LeadIT. Co-crystalized ligand Febuxostat was
used for defining binding site with the radius of 6.50 Å.
Structure of compound was drawn on ChemDraw Ultra
(2013) and its energy was minimized by employing MM2
force field in Chem 3D Ultra software. Prepared compound
was used as protonated in aqueous solution and docked into
prepared binding site using FlexX docking module in
LeadIT. All FlexX solutions obtained were scored using a
consensus scoring function (CScore) and ranked accord-
ingly. Top best pose with the highest score was selected for
investigation of interactions. 3D enzyme-hybrid interactions
were visualized using Discovery Studio Visualizer: Biovia
2016.
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Acknowledgements Authors are grateful to the University Grants
Commission for providing funds under Rajiv Gandhi National Fel-
lowship (RGNF) and National Fellowships for Other Backword
Classes (NFOBC), Department of Science & Technology (DST-
PURSE), Council of Scientific and Industrial Research (CSIR): Project
no. 02(0319)17/EMR-II, Women Scientists Scheme-A (WOS-A: DST)
and DST-FIST. The authors are also thankful to Guru Nanak Dev
University, Amritsar for providing various basic facilities to carry out
the research work.
Ishibuchi S, Morimoto H, Oe T, Ikebe T, Inoue H, Fukunari A,
Kamezawa M, Yamada I, Naka
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Bedi PMS (2015) Synthesis, screening and docking studies of
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
Kaur M, Kaur A, Mankotia S, Singh H, Singh A, Singh JV, Gupta
MK, Sharma S, Nepali K, Bedi PMS (2017) Synthesis, screening
and docking of fused pyrano[3,2-d]pyrimidine derivatives as
xanthine oxidase inhibitor. Eur J Med Chem 131:14–28
Kaur R, Naaz F, Sharma S, Mehndiratta S, Gupta MK, Bedi PMS,
Nepali K (2015) Screening of a library of 4-aryl/heteroaryl-4H-
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oxidative stress. Curr Top Med Chem 18:2154–2164
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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