Evaluation of xanthine oxidase inhibitor properties on isoindoline-1,3-dion derivatives and calculation of interaction mechanism

Article abstract of DOI:10.1016/j.molstruc.2019.127523

The aim of the present study was to synthesis of isoindole-1,3(2H)-dione (Phthalimides) derivatives and to investigation the inhibition of xanthine oxidase (XO). In study, xanthine oxidase inhibitory activities of complexes were observed in the range from

Full text of DOI:10.1016/j.molstruc.2019.127523

Journal Pre-proof
Evaluation of xanthine oxidase inhibitor properties on isoindoline-1,3-dion derivatives
and calculation of interaction mechanism
Özlem Gunduğdu, Samir Abbas Ali Noma, Tugba Taskin-Tok, Burhan Ateş, Nurhan
Kishali
PII:
S0022-2860(19)31632-1
DOI:
https://doi.org/10.1016/j.molstruc.2019.127523
MOLSTR 127523
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 August 2019
Revised Date: 18 November 2019
Accepted Date: 2 December 2019
Please cite this article as: Ö. Gunduğdu, S.A.A. Noma, T. Taskin-Tok, B. Ateş, N. Kishali, Evaluation of
xanthine oxidase inhibitor properties on isoindoline-1,3-dion derivatives and calculation of interaction
mechanism, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2019.127523.
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Evaluation of Xanthine Oxidase Inhibitor Properties on isoindoline-1,3-
dion derivatives and Calculation of Interaction Mechanism
1
2
3
2
Özlem GUNDUĞDU , Samir Abbas Ali NOMA , Tugba TASKIN-TOK , Burhan ATEŞ *,
4
Nurhan KISHALI *
1
Ahi Evran University, Kaman Vocational School, Department of Food Technology, Kırsehir,
TURKEY
2
İnönü University, Faculty of Science, Department of Chemistry, Malatya, TURKEY
3
Gaziantep University, Faculty of Arts and Sciences, Department of Chemistry, 27310 Gaziantep,
TURKEY
4
Ataturk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, TURKEY

Evaluation of Xanthine Oxidase Inhibitor Properties on isoindoline-1,3-
dion derivatives and Calculation of Interaction Mechanism
1
2
3
2
Özlem GUNDOĞDU , Samir Abbas Ali NOMA , Tugba TASKIN-TOK , Burhan ATEŞ *,
4
Nurhan KISHALI *
1
Ahi Evran University, Kaman Vocational School, Department of Food Technology, Kırsehir,
TURKEY
2
İnönü University, Faculty of Science, Department of Chemistry, Malatya, TURKEY
3
Gaziantep University, Faculty of Arts and Sciences, Department of Chemistry, 27310
Gaziantep, TURKEY
4
Ataturk University, Faculty of Science, Department of Chemistry, 25240 Erzurum,
TURKEY
1

Abstract
The aim of the present study was to synthesis of isoindole-1,3(2H)-dione (Phthalimides)
derivatives and to investigation the inhibition of xanthine oxidase (XO). In study, xanthine
oxidase inhibitory activities of complexes were observed in the range from 7.15 to 22.56 ꢀM
for isoindole-1,3-dione (2a-c and 3a-c). N-phenyl isoindole-1,3-dione derivatives (2c, 3c)
showed better activity (almost two times) than the other two derivatives (N-methyl (2a, 3a),
N-ethyl (2b, 3b). It means that phenyl ring (R) remarkably enhances the xanthine oxidase
inhibitory effect of complexes. In the meantime, molecular docking studies of these
compounds against XO were also investigated by providing the inhibitory efficiency and
estimating the interaction mechanisms of isoindol-1,3-dion derivatives with XO.
Keywords: Isoindoline-1,3-dione, Phthalimide, Xanthine Oxidase, Molecular Docking,
Biological Activity
2

1
. Introduction
Phthalimides are systematically known as isoindole derivatives. Phthalimides are molecules
of interest in organic and medicinal chemistry due to their biological activity. They are
building blocks of some biologically active compounds. Interest in the synthesis of
isoindoline-1,3-dione derivatives is intensely increased due to its recently discovered action
against inflammatory diseases and cancer [1-4].
Researchers have generally investigated the activity of isoindole in two different classes. The
first group: Derivatives via N-atom, The second group: Derivatives via isoindole-aromatic
ring. Activity studies on N-methyl and ethyl isoindole derivatives have been performed and
efficient results have been obtained [5-11]. In this study, we tried to investigate the XO
enzyme inhibition effect by synthesizing an aromatic N-derivative (phenyl) to compare with
aliphatic –N derivatives. In addition, several groups have also studied various activity studies
of many derivatives in the aromatic ring in N-substituted isoindole compounds [12-13]. In this
study, derivatives with high H-binding capacity (acid group) and lower hydrogen bonding
capacity (ester group) with enzyme were synthesized and XO enzyme activity was
investigated. For XO enzyme inhibition, the presence of groups capable of H-bonds (weak
intermolecular bond generally formed between oxygen or nitrogen heteroatoms and protons)
is preferred. As is known, non-covalent interactions between inhibitors and enzymes include
hydrogen bonds, hydrophobic interactions, and ionic bonds. Many of these weak bonds bind
to produce strong and specific binding between enzyme and the inhibitor. Enzymes prefer
hydrogen bonds to make the transition state more stable. Thus, isoindole derivatives (2a-c, 3a-
c) were preferred since they contain both the N atom and the carboxyl group [14].
The biological activities of a great deal of isoindole-1,3-dione derivatives have been studied
wide range of such as anti-inflammatory, anti-convulsant, anticancer activity, and anti-
microbial activity, antioxidant, and analgesic activities. Furthermore, different modifications
of the phthalimide structure have been found to affect biological activity [15-19]. These effect
is seen in drugs such as Lenalidomide (Revlimid ™), Thalidomide and Pomalidomide
(Actimid ™) [20-21].
Also, 2-(3-diethyl aminoalkyl)-isoindoline-1,3-dione derivatives could be described as
selective acetylcholinesterase inhibitors by Malawska et. all. They suggested that these
compounds could be used as a potential drug to treat Alzheimer's disease [22].
3

Scheme 1. Some drugs of the isoindole-1,3-dione derivative.
Recently, we synthesized isoindole-1,3-dione amine derivatives via 1,2-addition reaction of
amines to α, β-unsaturated double bond in cyclohexane system and investigated their
fluorescence properties. [23].
In this paper, we describe the synthesis a series of N-substituted isoindoline-1,3-dione
derivatives and their inhibitory activity on xanthine oxidase. Furthermore, the docking
calculations were exerted to define docking interactions of isoindole-1,3-dione derivatives-
xanthine oxidase (XO) complexes.
2
2
. Results and discussion
.1. Chemistry
The chemical structure of isoindole-1,3-dione have hydrophobic character. This structure
increases their potential to cross different biological membranes in in vivo [24]. Similarly,
enzyme inhibition is indicative of biological activity. For this reason, derivatives of isoindole-
1
,3-dione were synthesized to examine XO inhibition activity. Isoindoline-1,3-dione
derivatives were synthesized by starting from 1,3-dioxo-1,3-dihydro-isobenzofuran-5-
carboxylic acid (1). For this purpose, corresponding amide derivatives were obtained by
reacting 1,3-dioxo-1,3-dihydro-isobenzofuran-5-carboxylic acid (1) with methyl, ethyl,
phenyl amine in the presence of NEt in catalytic amounts (Scheme 1). Thus, 5-hydroxy-N-
3
(
methyl / ethyl / phenyl) isoindoline-1,3-dione derivatives (2a, 2b, 2c) were synthesized and
then converted to the methyl ester derivative methyl 2-(R)-1,3-dioxoisoindoline-5-carboxylate
3a, 3b, 3c)[25].
(
Scheme 2. Isoindoline-1,3-dione derivatives synthesis.
4

2
.2. Biological activity
Xanthine oxidase is known to play an important role in the synthesis of uric acid. XO is
widely distributed throughout various organs including the liver, gut, lung, kidney, heart,
brain and plasma. High levels of xanthine oxidase in some tissues have been associated with
tissue damage and serious illnesses due to the relationship with production cytotoxic oxygen
-
-
metabolites (H O , O and OH ) cause oxidative damage on the cells [26]. Allopurinol, a
2
2
2
purine derivative with the aim of inhibiting XO, has also been used in therapy for many years.
However, it is not desirable that allopurinol may be converted to 4,6-dihydroxy pyrazole (3,4-
δ) pyrimidine, which is high in toxicity level and is not as effective as itself in vivo.
Moreover, allopurinol has to compete with xanthine in order to be able to inhibit the enzyme
xanthine oxidase, depending on its chemical structure, and thus the yield decreases [27]. In
addition, several studies have shown that hypersensitivity reactions of allopurinol and
inducing side effects of Stevens-Johnson syndrome are present [28]. Therefore, there is a need
for the synthesis of new non-purine compounds which are more efficient and have low side
effects which will inhibit XO. For this reason, it has been decided to the investigated of XO
activity of isoindole-1,3-dione derivatives.
2
.2.1. Xanthine Oxidase Inhibition Properties of Complexes
The IC values for isoindoline-1,3-dione derivatives ( 2a, 2b, 2c, 3a, 3b, 3c) and allopurinol
5
0
as a positive control were given in Table 1. The isoindoline-1,3-dione derivatives ( 2a, 2b, 2c,
a, 3b, 3c derivatives showed a good inhibition activity toward against XO, the lowest IC50
3
ratio was for 1,3-dioxo-2-phenylisoindoline-5-carboxylic acid (2c) compound as 7.15 ꢀM and
the highest one was 22.56 ꢀM for 2-methyl-1,3-dioxoisoindoline-5-carboxylic acid (2a), and
for allopurinol positive control was 6.34 ꢀM. All the isoindoline-1,3-dione derivatives ( 2a,
2
b, 2c, 3a, 3b, 3c) derivatives showed inhibition of XO enzyme by decreasing the uric acid
formation. Zafar H., et al prepared isonicotinohydrazides compounds and investigated their
inhibitory effect on XO enzyme [29]. They found the range of IC value from 0.9 ± 0.01 ꢀM
5
0
to 330.4 ± 2.1 ꢀM. The inhibition activities of benzimidazole derivatives against XO enzyme
were determined in the range from 4.3 ꢀM to 9.7 ꢀM [30].
Ting-jian Z. et al. synthesized N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide
derivatives as novel xanthine oxidase inhibitors, and the IC50 value range was from 0.3 ±
0
.03 ꢀM to 19.2 ± 1.2 ꢀM [31]. Aktas et al. synthesized 2-hydroxyethyl substituted N-
heterocyclic carbine to investigate the enzymes inhibition effect and the IC value range for
50
5

XO was found in between 1.253 ± 0.084 ꢀM and 4.992 ± 0.113 ꢀM [32]. In other study by
Burmaoglu et al. [33], they tested novel bis-chalcone derivatives to determine their biological
activity toward enzyme and cancer cells. They found IC for XO inhibition in the range from
5
0
0
.728 ± 0.009 to 6.058 ± 0.095 ꢀM. Kıbrız et al. [34] investigated the effect of pyrrole
carboxamide rings on XO activity and it was reported that the IC values were of 4.608 -
5
0
7
.084 ꢀM.
Table 1. IC50 values of complexes on XO activities.
2
Compounds
IC µM
r
5
0
2
a
a
22.56
21.19
20.788
15.794
7.15
0.9902
0.9447
0.9912
0.976
0.982
0.9755
0.986
3
2
b
b
3
2
c
c
3
10.94
6.34
Allopurinol
2
.2.2. Structure-activity relationship
In order to determine the relationship between the structures of the synthesized compounds
and their XO enzyme inhibition, it would be rational to classify these compounds into two
main groups: i) The substituted groups bonded to the nitrogen atom of the imide. There are
three different groups bonded to the nitrogen atom. These are methyl, ethyl and phenyl
groups. ii) The substituted groups bonded to aromatic ring. For this purpose, two derivatives
were prepared for each group bonded to the nitrogen atom. These are 5-carboxylic acid (2a-c)
and 5- methyl-carboxylate (3a, 3b, 3c) derivatives. In literature [35] observation revealed that
substitution of phenyl ring R remarkably enhances the xanthine oxidase inhibitory effect,
whereas the placement of phenyl ring with aliphatic groups diminishes this effect. Similar
results were observed in this study; It was found that N-phenyl isoindole-1,3-dione derivatives
(2c, 3c) showed better activity than the other two derivatives (N-methyl (2a, 3a), N-ethyl (2b,
3
b). In addition, it was observed that compounds (2a, 2b, 2c) with acid groups in the aromatic
ring exhibited better activity than esters derivatives (3a, 3b, 3c). This circumstance indicates
that the interaction between the enzyme and the isoindole derivative is more hydrophobic. As
6

the hydrophobic interactions in the ester derivatives are reduced according to the acid, it can
be thought that there is a decrease in activity.
2
.3. In silico details
In this study molecular docking applications were performed with DS 2018 SP1 to predict the
interaction mechanisms of isoindol-1,3-dion derivatives with XO. Additionally, allopurinol
compound for XO was used positive control in this research. Not only the docking results
were evaluated based on docking score and binding energy values, but also compared with the
selected positive controls.
2
.3.1. Docking results between isoindol-1,3-dion derivatives and xantine oxidase (XO)
In this part, control compound, allopurinol was docked with XO. The results demonstrate that
interaction mechanism and which type of non-bonding interactions involve in the binding site
of the target protein, XO. In docking studies, the defined compounds, isoindol-1,3-dion
derivatives were applied same process based on the positive control. Their binding affinity
were likewise calculated and compared with allopurinol as XO inhibitor.
The positive control, allopurinol forms three hydrogen bonds with Ser876 (2.949 Å), Glu879
(2.224 Å) and Arg880 (2.706 Å) residues of the enzyme and five hydrophobic interactions
with Phe914, Phe1009, Ala1078 and Ala1079 residues. The three dimensional (3D)
orientation of the allopurinol and detail interaction types were given in Figure S7 and Table
S7 of the supporting information part.
As known that selected compounds, isoindol-1,3-dion derivatives involve six structures were
docked with XO. 1,3-dioxo-2-phenylisoindoline-5-carboxylic acid (2c) which is one of the
isoindol-1,3-dion derivatives, has six hydrogen bonds with Asn768, Ser876, Arg880, Glu802
and Pro1076 residues of the XO, (Figure 1). Beside hydrogen bonds, there are two
hydrophobic interactions with Phe914 and Val1011. Furthermore, the residue compounds
(five structures) which display different non-bonding interactions with residues in the active
site of the same enzyme. The docking calculations and views showed that a compounds form
was more affinity than others that were given in Figure S8 and Table S8 of the supporting
information part.
Especially, binding energy values of isoindol-1,3-dion derivatives are also reinforced the
compound 1,3-dioxo-2-phenylisoindoline-5-carboxylic acid (2c) (-16.1342 kcal/mol) is better
interaction and affinity than others. Even this binding energy value of the 1,3-dioxo-2-
phenylisoindoline-5-carboxylic acid (2c) is exhibited better tendency than the positive control,
allopurinol (-14.0382 kcal/mol) in Table 2. Moreover, orientations of each structure are very
7

important to do good interaction and show the biological functionality. Figure 2 shows the
orientation of the ligands in XO as compared with the positive control. The results displayed
that 1,3-dioxo-2-phenylisoindoline-5-carboxylic acid (2c) has almost more similar orientation
than other ones with positive control, allopurinol. Thus, the data obtained in the molecular
docking calculations help to reveal the results of the XO inhibition analysis.
Figure 1. The best docking orientations of 1,3-dioxo-2-phenylisoindoline-5-carboxylic acid
(2c) (yellow and stick) and allopurinol (green and stick) in XO.
Table 2. The binding energy analysis of ligands and allopurinol* as positive control with that
of XO.
Ligand
Binding
Energy
Ligand
Energy
Protein
Energy
Complex
Energy
Entropic
Energy
CDOCKER
intr
Name-
CDOCKER
CDOCKER
(kcal/mol)
-5.1495
(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)
2a
3a
2b
3b
-9.99882
-5.84289
-9.05045
-6.82023
-10.8283
-3.98577
-17.3939
-28.7439
-27.9546
-27.0582
-25.0198
-30.1796
-27.0448
-19.1738
-10.8867
6.9048
-32,554.60 -32,570.70
-32,554.60 -32,553.10
-32,554.60 -32,563.50
-32,554.60 -32,574.30
-32,554.60 -32,588.30
-32,554.60 -32,567.30
-57,006.30 -56,978.60
18.6678
19.0259
18.4715
18.2815
18.4968
19.2069
17.0079
-5.3659
-6.5306
-2.3801
-8.8453
-17.5508
-1.5854
41.7355
-10.8327
-16.1342
-11.0454
-14.0382
2
c
3
c
Allopurinol*
8

Figure 2. (a) The relative orientation of ligands, 1,3-dioxo-2-phenylisoindoline-5-carboxylic
acid (2c) (yellow, stick) and allopurinol (green, stick) in XO. (b) A different view,
superimposed form of the all ligands (2a, orange; 3a, light blue; 2b, light pink; 3b,
purple; 3c, dark straw color, stick) and allopurinol (green, stick) in XO.
3
3
. Experimental
.1. General Procedure for 2
Onto the solutions of starting compound (1 equiv.) in toluene Et N (3 equiv.) was added at rt.
3
Then R-NH (1.2 equiv.) was added and refluxed for 24 hours. After that the reaction was
2
cooled to room temperature. EtOAc (50mL) was added to the mixture and it was extracted
with saturated NH Cl solution (3 x 50 mL). The organic layer was dried over Na SO and the
4
2
4
solvent was removed in evaporator. The crude product was filtered from a small column with
EtOAc / petroleum ether (60:40). The product was crystallized from EtOAc / petroleum ether
(Yield: 90-95%) [25].
o
o
2
a; M.p: 232-233 C (lit: 240 C) [36], IR (KBr): 3775, 3444, 3185, 1773, 1717, 1695, 1607,
1
1
570, 1478, 1449, 1378, 1253, 1214, 1164. H-NMR (400 MHz, CDCl ) δ: 8.57 (s, 1H); 8.48
3
1
3
(
dd, 1 H, J=7.7, 1.5 Hz); 7.97 (d, 1 H, J=7.7 Hz); 3.23 (s, 3 H). C-NMR (100 MHz, CDC )
l3
δ: 169.4 (2C); 167.5; 136.5; 136.2; 134.7; 132.8; 125.1; 123.6; 24.5. Anal. calc. for C H NO
4
1
0
7
(205.17): C, 58.54; H, 3.44; N, 6.83; found: C 58.20; H 3.66; N 6.41.
9

o
2
1
1
b; M.p: 158-159 C, IR (KBr): 3772, 3443, 2982, 2947, 1772, 1702, 1441, 1400, 1385,
1
308, 1257, 1199, 1077. H-NMR (400 MHz, CDCl ) δ: 8.57 (s, 1H); 8.48 (dd, 1 H, J=7.7,
3
13
.1 Hz), 7.97 (d, 1 H, J=7.7 Hz); 3.79 (q, 2 H, J=7.3 Hz); 1.30 (t, 3H, J= 7.3 Hz). C-NMR
(
100 MHz, CDC ) δ: 169.8 (2C); 167.3; 136.6; 136.2; 134.7; 132.8; 125.1; 123.6; 33.6; 14.1.
l3
Anal. calc. for C H NO (219.20): C, 60.28; H, 4.14; N, 6.39; found: C 60.62; H 4.70, N
1
1
9
4
6
.48.
o
o
2
1
7
c; M.p: 259-260 C (lit: 257-259 C [37]), IR (KBr): 3445, 3053, 1787, 1720, 1504, 1392,
1
296, 1221, 1124, 1104, 1072. H-NMR (400 MHz, CD OD) δ: 8.48 (m, 2H); 8.44 (m, 1 H);
3
.64-7.33 (m, 5H).
1
3
C-NMR (100 MHz, CD OD) δ 166.7; 166.6; 166.3; 135.5; 135.0; 132.1; 131.9; 128.6;
3
1
28.4; 127.9; 126.68; 123.90; 123.24. Anal. calc. for C H NO (267.24): C, 67.42; H, 3.39;
1
5
9
4
N, 5.24; found: C 67.62; H 3.21; N 5.70.
3
.2. General Procedure for 3
The starting compound (1 eq.) was dissolved in methanol and cooled to 0 °C. Then SOCl (1.2
2
eq.) was added and refluxed for 18 hours. After that, the reaction mixture was cooled to room
temperature and the solvent removed in evaporator. The solid was extracted by addition of
EtOAc (2 x 20 mL) and saturated NH Cl solution (20 mL). The organic layer was dried over
4
Na SO and the solvent was removed in an evaporator. The crude product was purification
2
4
with column chromatography with EtOAc / petroleum ether (30:70). The product was
crystallized from EtOAc / petroleum ether (Yield: 90-95%) [25].
o
3
1
1
1
a; M.p: 122-123 C, IR (KBr): 3455, 3078, 2956, 1773, 1738, 1717, 1480, 1438, 1383, 1288,
1
261, 1188, 1161. H-NMR (400 MHz, CDCl )δ: 8.47, (dd, 1H, J = 1.5, 0.7 Hz); 8.39 (dd,
3
1
3
H, J = 7.7, 1.5 Hz); 3.98 (s, 3H); 3.21 (s, 3H). C-NMR (100 MHz, CDC ) δ: 167.7; 167.7;
l3
65.5; 135.7; 135.6; 132.7; 129.1; 124.5; 123.5; 53,1; 24.5. Anal. calc. For C H NO
4
1
1
9
(219,20): C, 60.28; H, 4.14; N, 6.39; found: C 60.01; H 4.15; N 5.99.
o
3
1
b; M.p: 96-97 C, IR (KBr) δ: 3457, 3115, 2980, 2960, 1773, 1725, 1459, 1431, 1399, 1380,
1
330, 1295, 1256, 1197, 1183. H-NMR (400 MHz, CDCl ) δ: 8.47 (s, 1H); 8.39 (d, 1 H, J
3
=7.8 Hz); 7.90 (d, 1H, J = 7.7 Hz); 3.98 (s, 3H); 3.76 (q, 1 H, J =7.2 Hz); 1.28 (t, 3H, J = 7.2
1
3
Hz). C-NMR (100 MHz, CDC ) δ: 167.3; 167.2; 165.3; 135.6; 135.5; 135.3; 132.5; 124.2;
l3
1
23.2; 52.8; 33,2; 13.9 Anal. calc. For C H NO (233,22): C, 61.80; H, 4.75; N, 6.01; found:
12 11 4
C 62.22; H 4.21; N 6.31.
1
0

o
3
1
0
3
1
6
c; M.p: 210-211 C, IR (KBr): 3444, 2964, 1776, 1730, 1622, 1597, 1505, 1493, 1458, 1424,
1
386, 1353, 1293, 1278, 1217, 1112, 1097. H-NMR (400 MHz, CDCl ) δ: 8.60 (d, 1H, J =
3
.7 Hz); 8.50 (dd, 1H, J =7.7, 1.5 Hz); 8.04 (d, 1H, J = 7.7 Hz); 7.55-7.43 (m, 5 H); 4.01 (s,
1
3
H); 1.55 (s, 3H). C-NMR (100 MHz, CDC ) δ:166.7; 166.6; 166.2; 136.2; 136.0; 135.3;
l3
32.5; 129.5; 128.6; 126.7; 125.1; 124.1; 123.2; 53.2. Anal. calc. For C H NO (281,27); C,
1
6
11
4
8.33; H, 3.94; N, 4.98; found: C 68.62, H 3.81, N 4.75.
3
.3. Xantine Oxidase (XO) Inhibition Assay
Bovine milk XO activity was determined spectrophotometrically by measuring uric acid
formation at 295 nm at 37 °C, xanthine was used as substrate. For enzyme assay protocol 50
mM (pH=7.5) phosphate buffer, 1mM xanthine, and 0.2 U of XO enzyme were used. The
inhibition of XO by isoindol-1,3-dion compounds derivatives were measured by the reduction
of the uric acid concentration. Different concentrations of isoindol-1,3-dion derivatives were
added for XO inhibition activity. The enzyme was pre-incubated for 10 min, with tested
compounds, then the reaction was started by addition xanthine to reaction mixture. XO
activity was determined by measuring the changes in absorbance of uric acid formation at 295
nm at 37 °C. The tested compounds were dissolved in DMSO, then diluted with phosphate
buffer 50 mM (pH=7.5), the final concentration of DMSO in the reaction mixture was less
than (0.01% v/v) and this ration does not interface with enzyme assay. A blank sample was
measured without tested compounds. All the experiments were performed in triplicates, and
values were expressed as means of three experiments. Allopurinol was used as a positive
control. The IC values were determined for all based compounds were measured as percent
5
0
inhibition of XO was studied in terms of decrease in uric acid formation as compared to the
product formation in absence of inhibitor. The percent inhibition of XO activity was
calculated as the following:
%
Inhibition = (A-B)/A×100
where A = the absorbance at 295 nm without the test compound,
B = the absorbance at 295 nm with the test compound.
3
.4. In silico details
Molecular docking calculations
Discovery Studio (DS) 2018 SP1 [38] were used to arrange and exert the docking calculations
to define docking interactions of isoindol-1,3-dion derivatives-xanthine oxidase (XO)
1
1

complexes. The crystal structures of target, XO (pdb: 3NVY) was downloaded from the
protein data bank (http://www.rcsb.org) was used for docking processes. The ligands,
isoindol-1,3-dion derivatives were drawn and optimized at DFT/B3LYP/6-31G (d) basis set
using Gaussian 09 (G09) [39] software. The optimized structures and data were also shown at
Figure S1-S6 and Table S1-S6 in supporting information part of this study. Their
conformational analyses were also investigated using DS 2018 SP1. On the other hand, the
enzyme was prepared using DS tools and minimized until the root mean square deviation
2
(
RMSD) reaches the lower value of 0.05 kcal/mol Å . Define and edit binding site tool was
used to detect binding site of the XO against isoindol-1,3-dion derivatives structures. Dock
Ligands (CDOCKER) was performed to predict the conformations and binding interactions
for the ligands. The best pose for each complex was chosen according to the docking score
and energy.
4
. Conclusion
We report an efficient synthesis of 5-carboxylic acid and 5-carboxymethyl ester derivatives of
isoindole-1,3-dion. This method has potential to be widely used to synthesis 5- carboxylic
acid and 5-carboxymethyl ester substituted isoindole-1,3-dione. Inhibitor properties of these
derivatives of isoindole-1,3-dion were then evaluated against XO activity. The isoindoline-
1
,3-dione derivatives ( 2a, 2b, 2c, 3a, 3b, 3c) were showed a good inhibition activity toward
against XO. All the isoindoline-1,3-dione derivatives ( 2a, 2b, 2c, 3a, 3b, 3c) showed
inhibition of XO enzyme by decreasing the uric acid formation. Then, isoindole-1,3-dione
derivatives were applied docking studies. In docking study, their binding affinity were
calculated and compared with allopurinol as XO inhibitor. The results revealed that 1,3-
dioxo-2-phenyl isoindoline-5-carboxylic acid (2c) had more similar orientation with
allopurinol than other synthesized isoindole-1,3-dione derivatives (2a, 2b, 3a, 3b, 3c). These
results in molecular docking are compatible the results of XO inhibition assay. This
interaction studies with biological activity and molecular docking offer the opportunity to
know about the effects of the isoindoline-1,3-dione derivatives in XO target and mechanism
of interaction and it may be useful to design new compounds.
5
. Acknowledgments
We would like to thank Ataturk University (Project number: 2014/178), (Project number:
FBG-2018-6476) for its financial support of this work. The numerical calculations reported in
1
2

this study were performed at TUBITAK ULAKBIM, High Performance and Grid Computing
Center (TRUBA resources).
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1
7

HIGHLIGHTS:
1
2
3
. Isoindoline-1,3-dione derivatives ( 2a, 2b, 2c, 3a, 3b, 3c) were synthesized by starting from
1
,3-dioxo-1,3-dihydro-isobenzofuran-5-carboxylic acid.
. All of the synthesized isoindoline-1,3-dione derivatives showed a good inhibition of XO by
reducing uric acid formation.
. The highest IC ratio was observed in 2-methyl-1,3-dioxoisoindoline-5-carboxylic acid
5
0
(
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50
4
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acid (2c) has almost more similar orientation than other ones with positive control,
allopurinol.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: