Synthesis, inhibition properties against xanthine oxidase and molecular docking studies of dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate and (N-benzyl-1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives

Article abstract of DOI:10.1016/j.bioorg.2021.104654

This study focused on synthesis various dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate and (N-benzyl-1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives under the conditions of green chemistry without the use of solvent and catalysts. Their inhibition properties were also investigated on xanthine oxidase (XO) activity. All dimethanol and dicarboxylate derivatives exhibited significant inhibition activities with IC₅₀ values ranging from 0.71 to 2.25 μM. Especially, (1-(3-bromobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol (5c) and dimethyl 1-(4-chlorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6 g) compounds were found to be the most promising derivatives on the XO enzyme inhibition with IC₅₀ values 0.71 and 0.73 μM, respectively. Moreover, the double docking procedure was to evaluate compound modes of inhibition and their interactions with the protein (XO) at atomic level. Surprisingly, the docking results showed a good correlation with IC₅₀ [correlation coefficient (R² = 0.7455)]. Also, the docking results exhibited that the 5c, 6f and 6 g have lowest docking scores ?4.790, ?4.755, and ?4.730, respectively. These data were in agreement with the IC₅₀ values. These results give promising beginning stages to assist in the improvement of novel and powerful inhibitor against XO.

Full text of DOI:10.1016/j.bioorg.2021.104654

Bioorganic Chemistry 108 (2021) 104654
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, inhibition properties against xanthine oxidase and molecular
docking studies of dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate
and (N-benzyl-1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives
,
Güler Yagiza, Samir Abbas Ali Nomab, Aliye Altundasa *, Khattab Al-khafajic,
,
,*
Tugba Taskin-Tokc d, Burhan Atesb
a Department of Chemistry, Faculty of Science, Gazi University, 06500 Ankara, Turkey
b Department of Chemistry, Faculty of Science and Arts, Inonu University, 44280 Malatya, Turkey
c Department of Chemistry, Faculty of Arts and Sciences, Gaziantep University, 27310 Gaziantep, Turkey
d Department of Bioinformatics and Computational Biology, Institute of Health Sciences, Gaziantep University, 27310 Gaziantep, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study focused on synthesis various dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate and (N-benzyl-
1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives under the conditions of green chemistry without the use of
solvent and catalysts. Their inhibition properties were also investigated on xanthine oxidase (XO) activity. All
dimethanol and dicarboxylate derivatives exhibited significant inhibition activities with IC50 values ranging from
Triazole derivatives
Green synthesis
Double docking
Xanthine oxidase
Enzyme inhibition
0.71 to 2.25 μM. Especially, (1-(3-bromobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol (5c) and dimethyl 1-(4-
chlorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6 g) compounds were found to be the most promising de-
rivatives on the XO enzyme inhibition with IC50 values 0.71 and 0.73 M, respectively. Moreover, the double
μ
docking procedure was to evaluate compound modes of inhibition and their interactions with the protein (XO) at
atomic level. Surprisingly, the docking results showed a good correlation with IC50 [correlation coefficient (R2
=
0.7455)]. Also, the docking results exhibited that the 5c, 6f and 6 g have lowest docking scores ꢀ 4.790, ꢀ 4.755,
and ꢀ 4.730, respectively. These data were in agreement with the IC50 values. These results give promising
beginning stages to assist in the improvement of novel and powerful inhibitor against XO.
1. Introduction
Uric acid (UA) can pass through the blood to the kidneys and is
excreted from the body by urine. The excess production and accumu-
Triazoles act on bioactive molecules due to their strong dipole mo-
ments, hydrogen bond formation, dipole–dipole, and stacking in-
lation of UA in the body can lead to hyperuricemia, which is the main
role in cases of gout illness [13]. It was reported that high-level of UA
can cause other several diseases such as metabolic syndrome [14],
cardiovascular [15], and diabetes [16]. During purine metabolism, XO
delivers an electron to an oxygen molecule that leads to formation of free
radical (FR) and reactive oxygen species (ROS). The increase in the FR &
ROS levels can cause diverse pathological states inclusive inflammation,
cancer, metabolic disorders, and chronic obstructive pulmonary disease
[17]. XO inhibition could minimize the production of FR & ROS and get
a benefit for treatment from these diseases [18]. Allopurinol is an isomer
structural of hypoxanthine and is used for XO inhibitor in the treatment
of gout since 1966 [19]. However, allopurinol has side effects and can
cause other diseases such as Steven-Johnson syndrome [20], hypersen-
sitivity [21], fatal liver necrosis [22], and renal toxicity [23]. Therefore,
π-π
teractions [1,2]. Organic compounds containing 1,2,3-triazole ring
structure are effected in pharmacophoric units which find enormous
crucial in the drug discovery field owing to their broad fields of phar-
maceutical and therapeutic applications including antibacterial [3],
antifungal [4], anticancer [5], anti-tubercular [6], anti-HIV [7] and
antiviral activity [8]. Triazole rings are also found in some commercially
available drugs consist of cefatrizine (β-lactam antibiotic) [9], tazo-
bactam (β-lactam antibiotic) [10], carboxyamidotriazole (an anti-cancer
agent) [11].
Xanthine oxidase (XO) is a ubiquitous molybdenum-containing
enzyme that existed in the organism which catalyzes the oxidation of
both hypoxanthine and xanthine [12].
* Corresponding authors.
E-mail address: burhan.ates@inonu.edu.tr (B. Ates).
https://doi.org/10.1016/j.bioorg.2021.104654
Received 6 October 2020; Received in revised form 30 December 2020; Accepted 8 January 2021
Available online 12 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
the researchers have investigated to develop novel non-purine-based
compounds that have a powerful effect on XO inhibitors with fewer
side effects [24,25].
2.1. General procedure of benzyl azide derivatives
Benzyl azide derivatives were synthesized in three steps known
procedure in the literature [34]. The reduction of the benzaldehydes
gave benzyl alcohols and they were converted to benzyl bromides with
PBr3 via SN2 reaction condition. In the final step, benzyl azide de-
rivatives were prepared from benzyl bromides by reaction with sodium
azide salts (Fig. 3). A total of ten benzyl azide derivatives were syn-
thesized in this way (yields: 75–99%). Characterization data of com-
pounds were given in Figure S1-S51 in terms of 1H NMR 13C NMR and
HRMS TOF spectrum.
On the other hand, substituted 1,2,3-triazoles have three various
isomers 1,4-, 1,5- and 2,4-disubstituted-1,2,3-triazole depending on the
position of the substituents. Among the isomers of 1,2,3-triazoles espe-
cially 1,4-disubstituted-1,2,3-triazoles are important molecules that
have similar properties such as distance and planarity as amide bonds in
the peptide bond [1]. In the literature, triazole ring and modifications
are included in the enzyme inhibition studies such as potent inhibitors of
urease, carbonic anhydrase, and cholinesterase (acetylcholinesterase
(AChE), butyrylcholinesterase (BuChE)) [26], cyclooxygenase-2 and 15-
lipoxygenase [27].and XO [28,29].
2.2. General procedure of (N-benzyl-1H-1,2,3-triazole-4,5-diyl)
dimethanol derivatives
Some of the important functional groups in biological molecules
include strongly hydrophilic groups such as carboxyl and hydroxyl.
Hydroxyl and carboxyl groups readily form hydrogen bonds and
contribute to making molecules soluble in water. Hydrogen bonds are
important to the function of many molecules, help them to flection
properly, and maintain the suitable shape needed to function correctly
involved in the binding of an enzyme to its substrate. Esters can also
hydrogen bond with water, although not as efficiently as alcohols, and
thus they are less soluble in water [30]. For investigate the inhibition
effect on XO of alcohol and ester functional groups in triazole ring, we
initially synthesized 17 compounds of 1,2,3-triazole derivatives (5a-g
and 6a-k), containing carboxylate and alcohol functional groups. Than,
theirs inhibitory effects on the XO enzyme were screened. Finally, mo-
lecular docking studies were also carried out to determine the binding
mode of the active compounds inside the active site of XO. Molecular
docking is one of the most generally exploited techniques because of its
ability to anticipate the conformation and affinity of ligand binding to
the target site, with a substantial accuracy [31,32]. Docking methods
effectively search high-dimensional spaces for possible interaction and
use a scoring function that properly ranks the candidate [33].
The substituted benzyl azides (1 eq) and but-2-yne-1,4-diol (1 eq)
were heated at 120 0C without solvent (Table 1). The solid products
were crystallized from hexane/dichloromethane.
(1-(4-Methoxybenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol
(5a).
Cream solid, m.p: 154 ◦C, yield: 99%. IR vmax (cmꢀ 1): 3336, 3128, 2832,
1613, 1584, 1513, 1452, 1019. 1H NMR (δ (ppm), d6-DMSO): 7.21 (d, J
= 8.5 Hz, 2H, H2), 6.89 (d, J = 8.7 Hz, 2H, H3), 5.50 (s, 2H, H4), 5.41 (t,
J = 5.3 Hz, 1H, H8), 5.00 (t, J = 5.6 Hz, 1H, H7), 4.55 (d, J = 5.5 Hz, 2H,
H5), 4.48 (d, J = 5.6 Hz, 2H, H6), 3,73 (s, 3H, H1). 13C NMR (δ (ppm), d6-
DMSO): 159.4, 145.5, 134.4, 129.7, 128.3, 114.5, 55.5, 54.7, 51.4, 51.1.
HRMS: m/z calcd for C12H15N3O3: 250.11838; found: 350.11850 [M +
H]+
(1-(2-Hydroxybenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol
(5b).
Brown oil, yield: 81%. IR vmax (cmꢀ 1): 3256, 2866, 1600, 1457, 1125.
1H NMR (δ (ppm), d6-DMSO): 9.74 (s, 1H, H1) 7.11–6.75 (ddd, 4H,
H
2,3,4,5), 5.44 (s, 2H, H6), 5.14 (t, 1H, H9), 5.08 (t, 1H, H10), 4.52 (d, 2H,
H7), 4.10 (d,2H, H8). 13C NMR (δ (ppm), d6-DMSO): 154.1, 146.7,
145.0, 129.6, 129.2, 122.0, 119.2, 115.6, 55.1, 53.0, 49.8. HRMS: m/z
calcd for C11H13N3O3: 236.10444; found: 236.10590 [M + H]+
2. Materials
(1-(3-Bromobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol
(5c).
Cream solid, m.p: 153 ◦C, yield: 88%. IR vmax (cmꢀ 1): 3365, 3178, 2851,
1596, 1569, 1474, 1025. 1H NMR (δ (ppm), d6-DMSO): 7.55–7.44 (m,
2H, H1,2), 7.38–7.25 (m, 2H, H3,4), 5.60 (s, 2H, H5), 5.44 (t, J = 5.4 Hz,
1H, H9), 5.07 (t, J = 5.6 Hz, 1H, H8), 4.59 (d, J = 5.4 Hz, 2H, H6), 4.51
(d, J = 5.4 Hz, 2H, H7). 13C NMR (δ (ppm), d6-DMSO): 145.4, 139.2,
134.8, 131.3, 130.9, 127.3, 122.2, 54.6, 51.2, 50.7. HRMS: m/z calcd for
All the reagents utilized in the synthesis were purchased from com-
mercial suppliers. The solvents of purification were used analytical
grade. XO from bovine milk was obtained from Sigma-Aldrich. (St.
Louis, MO, USA). Thin-layer chromatography was applied on silica gel
plates (SiO2, Merck 60 F254) using a UV light for visualization in order
to monitor the reaction completion. For column chromatography Silica
gel (Merck60, particle size 0.040–0.063 mm) was used. 1H and 13C
spectra were recorded on a Bruker spectrometer 300 MHz and 75 MHz
with tetramethylsilane (TMS) as the internal standard, respectively.
Chemical shifts (δ) were expressed in ppm as unit parts per million.
Singlet, doublet, triplet, and multiplet, which are spin split expressions,
are given as s, d, t, m respectively. The other instrument of Fourier-
transform infrared (FTIR) spectra were obtained on a Bruker ATR-FT-IR
spectrometer. Melting points were recorded with STUART (SMP-30)
device. High-Resolution Mass Spectrometry (HRMS) data were recorded
by Agilent Technologies, 6224 TOF LC/MS (see Figs. 1 and 2).
C
11H12N3O2Br: 298.01760; 300.01563; found: 298.07063; 300.01466
[M + H]+
(1-(2-Chlorobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol (5d). Yel-
low solid, m.p: 150 ◦C, yield: 97%. IR vmax (cmꢀ 1): 3436, 3369, 3158,
2948, 1593, 1471, 1003. 1H NMR (δ (ppm), d6-DMSO): 7.50 (dd, J =
7.7, 1.6 Hz, 1H, H1), 7.31 (dtd, J = 16.7, 7.4, 1.7 Hz, 2H, H2-3), 6.88 (dd,
J = 7.4, 1.9 Hz, 1H, H4), 5.69 (s, 2H, H5), 5.40 (t, J = 5.4 Hz, 1H, H9),
5.10 (t, J = 5.6 Hz, 1H, H8), 4.60 (d, J = 5.4 Hz, 2H, H6), 4.56 (d, J = 5.6
Hz, 2H, H7). 13C NMR (δ (ppm), d6-DMSO): 145.4, 135.1, 133.9, 132.4,
130.2, 129.9, 129.7, 127.9, 54.7, 51.4, 49.5. HRMS: m/z calcd for
C11H12N3O2Cl: 254.09731; 255.07042; 256.06446; found: 254.06554;
255.06864; 256.06268 [M + H]+
(1-(4-Hydroxybenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol
(5e).
White solid, m.p: 159 ◦C, yield: 83%. IR vmax (cmꢀ 1): 3468, 3130, 2855,
1613, 1515, 1431, 1021. 1H NMR (δ (ppm), d6-DMSO): 7.52 (dd, J =
7.7, 1.6 Hz, 1H, H1), 7.34 (dtd, J = 16.7, 7.4, 1.7 Hz, 2H, H2,3), 6.90 (dd,
J = 7.4, 1.9 Hz, 1H, H4), 5.69 (s, 2H, H5), 5.40 (t, J = 5.4 Hz, 1H, H9),
5.10 (t, J = 5.6 Hz, 1H, H8), 4.59 (d, J = 5.4 Hz, 2H, H6), 4.55 (d, J = 5.6
Hz, 2H, H7). 13C NMR (δ (ppm), d6-DMSO): 157.6, 145.4, 134.3, 129.7,
126.5, 115.8, 54.7, 51.3, 51.2. HRMS: m/z calcd for C11H13N3O3:
236.10152; found: 236.10007 [M + H]+
NH2
O
S
NH2
O
H
N
O
Cl
Cl
N
N
N
N
N
O
N
OH
O
Cl
Carboxyamidotriazole
Fig. 1. Commercially available drugs of tazobactam and carboxyamidotriazole.
O
(1-(4-Chlorobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol (5f). Pale
yellow solid, m.p: 157 ◦C, yield: 99%. IR vmax (cmꢀ 1): 3371, 3292, 3199,
2867, 1595, 1493, 1475, 1013. 1H NMR (δ (ppm), d6-DMSO): 7.41 (d, J
= 8.5 Hz, 2H, H1), 7.25 (d, J = 8.5 Hz, 2H, H2), 5.57 (s, 2H, H3), 5.39 (t,
Tazobactam
2

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
Fig. 2. Schematic diagram of XO reaction and its inhibition by Allopurinol.
Fig. 3. Synthesis of dicarboxylate and dimethanol derivatives i) NaBH4/MeOH, ii) PBr3/CH2Cl2, iii) NaN3/H2O–(CH3)2CO.
J = 5.4 Hz, 1H, H7), 5.03 (t, J = 5.6 Hz, 1H, H6), 4.54 (d, J = 5.5 Hz, 2H,
H5), 4.49 (d, J = 5.6 Hz, 2H, H4). 13C NMR (δ (ppm), d6-DMSO): 145.4,
135.5, 134.7, 133.0, 130.1, 129.1, 54.6, 51.2, 50.7. HRMS: m/z calcd for
Dimethyl
1-(4-methoxybenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate
(6a). Yellow solid, m.p: 68–70 C, yield: 88%. IR vmax (cmꢀ 1): 3001,
2955, 1721, 1613, 1564, 1513, 1464, 1228. 1H NMR (δ (ppm), CDCl3):
7.23 (d, J = 8.8 Hz, 1H, H2), 6.85 (d, J = 8.8 Hz, 1H, H3), 5.73 (s, 2H,
H4), 3.95 (s, 3H, H1), 3.89 (s, 3H, H5), 3.78 (s, 3H, H6). 13C NMR (75
MHz, CDCl3) δ 160.5, 159.9, 158.9, 140.2, 129.7, 125.9x2, 114.26,
55.29, 53.49, 53.31, 52.67. HRMS: m/z calcd for C14H15N3O5:
306.10847; found: 306.10846 [M + H]+
◦
C
11H12N3O2Cl: 254.06826; 255.07138; 256.06551; found: 254.06744;
255.07056; 256.06468 [M + H]+
(1-(5-Chloro-2-hydroxybenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol
(5 g). Cream solid, m.p: 151 ◦C, yield: 67%. IR vmax (cmꢀ 1): 3175, 2959,
1
1595, 1513, 1494, 1105. H NMR (δ (ppm), d6-DMSO): 10.19 (s, 1H,
H1), 7.17 (dd, J = 8.6, 2.7 Hz, 1H, H4), 6.92 – 6.72 (m, 2H, H2,3), 5.47 (s,
2H, H5), 5.36 (s, 1H, H9), 5.06 (s, 1H, H8), 4.60 (s, 2H, H6), 4.52 (d, 2H,
H7). 13C NMR (δ (ppm), d6-DMSO): 154.1, 145.2, 135.0, 129.1, 128.7,
125.0, 122.8, 117.2, 54.7, 51.2, 46.2. HRMS: m/z calcd for
C11H12N3O3Cl: 270.06374; 271.06678; 272.06074; found: 270.06348;
271.06652; 272.06699 [M + H]+
Dimethyl 1-(4-bromobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6b).
White solid, m.p: 84 ◦C, yield: 92%. IR vmax (cmꢀ 1): 3094, 3001, 2952,
1724, 1587, 1547, 1489, 1213. 1H NMR (δ (ppm), CDCl3): 7.41 (d, J =
8.4 Hz, 1H, H1), 7.12 (d, J = 8.4 Hz, 1H, H2), 5.72 (s, 2H, H3), 3.90 (s,
3H, H4), 3.86 (s, 3H, H5). 13C NMR (δ (ppm), CDCl3): 160.4, 158.7,
140.4, 132.9 132.1, 129.8, 129.4, 123. 0, 53.4, 53.2, 52.8. HRMS: m/z
calcd for C13H12N3O4Br: 354.01055; 356.00884; found: 354.01299;
356.01130 [M + H]+
2.3. General procedure of dimethyl N-benzyl-1H-1,2,3-triazole-4,5-
dicarboxylate derivatives
Dimethyl
1-(2-hydroxybenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate
(6c). Brown liquid, yield: 81%. IR vmax (cmꢀ 1): 3203, 3024, 2950, 1729,
1600, 1458, 1227. 1H NMR (δ (ppm), CDCl3): 7.28–6.83 (m, 4H, H1,4),
5.80 (s, 2H, H5), 3.94 (s, 6H, H6,7). 13C NMR (δ (ppm), CDCl3): 160.4,
159.2, 154.6, 139.8, 139.7, 130.9, 130.7, 121.0, 120.4, 117.1, 53.6,
52.8, 49.7. HRMS: m/z calcd for C13H13N3O5: 292.09551; found:
Benzyl azides (1 eq.) and dimethyl acetylenedicarboxylate (1 eq.)
were heated at 120 0C solvent-free conditions (Table 2). The crude
product was purified by column chromatography on silica gel eluting
with 6:4 hexane/ethyl acetate.
3

G. Yagiz et al.
Table 1
Bioorganic Chemistry 108 (2021) 104654
Table 2
Structure of dimethanol derivatives.
Structure of dicarboxylate derivatives.
Compound Code
Compound structure
Compound Code
6a31
Compound structure
5a
6b32
5b
5c
5d
6c
6d32
6e32
5e30
5f
6f33
5 g
6 g32
6 h
292.09822 [M + H]+
Dimethyl 1-(3-bromobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6d).
Yellow solid, m.p: 69–71 ◦C, yield: 81%. IR vmax (cmꢀ 1): 3029, 2995,
2948, 1721, 1596, 1565, 1469, 1191. 1H NMR (δ (ppm), CDCl3):
7.48–7.41 (m, 2H, H1,2), 7.25–7.18 (m, 2H, H3,4), 5.78 (s, 2H, H5), 3.96
(s, 3H, H6), 3.92 (s, 3H, H7). 13C NMR (δ (ppm), CDCl3): 160.2, 158.5,
140.2, 136.1, 131.8, 131.0, 130.5, 129.5, 126.7, 122.7, 53.4, 53.0, 52.6.
HRMS: m/z calcd for C13H12N3O4Br: 354.01118; 356.00932; found:
354.01397; 356.01212 [M + H]+
6i
6 k
Dimethyl 1-(2-chlorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6e).
White solid, m.p: 89–91 C, yield: 88%. IR vmax (cmꢀ 1): 3067, 3009,
◦
2954, 1727, 1594, 1561, 1468, 1243, 1228. 1H NMR (δ (ppm), CDCl3):
7.43 (dt, J = 7.8, 1.1 Hz, 1H, H1), 7.36–7.17 (m, 2H, H2,3), 6.94–6.85 (m,
1H, H4), 5.94 (s, 2H, H5), 3.99 (s, 3H, H6), 3.90 (s, 3H, H7). 13C NMR (δ
(ppm), CDCl3): 160.3, 158.6, 140.0, 133.0, 131.9, 130.6, 130.0, 129.8,
128.9, 127.4, 53.5, 52.8, 51.4. HRMS: m/z calcd for C13H12N3O4Cl:
310.05848; 311.06182; 312.05578; found: 310.05805; 311.06139;
312.05535 [M + H]+
140.4, 134.9, 132.4x2, 129.5, 129.1, 53.4, 53.2, 52.8. HRMS: m/z calcd
for C13H12N3O4Cl: 310.05941; 311.06265; 312.05759; found:
310.05991; 311.06315; 312.05809 [M + H]+
Dimethyl
1-(5-chloro-2-hydroxybenzyl)-1H-1,2,3-triazole-4,5-dicar-
boxylate (6 h). Cream solid, m.p: 132–134 ◦C, yield: 88%. IR vmax
(cmꢀ 1): 3025, 2956, 1757, 1726, 1604, 1575, 1497, 1489, 1221. 1H
NMR (δ (ppm), CDCl3): 7.28 (d, J = 2.6 Hz, 1H, H3), 7.19 (dd, J = 8.6,
2.6 Hz, 1H, H2), 6.84 (d, J = 8.6 Hz, 1H, H1), 5.76 (s, 2H, H4), 4.00 (s,
3H, H5), 3.96 (s, 3H, H6). 13C NMR (δ (ppm), CDCl3): 160.2, 159.1,
153.3, 140.0, 130.8, 130.4, 129.7, 125.8, 121.9, 118.8, 53.8, 52.9, 49.1.
HRMS: m/z calcd for C13H12N3O5Cl: 326.05564; 327.05797; 328.06043;
found: 326.05746; 327.05979; 328.06226 [M + H]+
Dimethyl
1-(4-hydroxybenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate
(6f). White solid, m.p: 108–110 ◦C, yield: 88%. IR vmax (cmꢀ 1): 3174,
2947, 1726, 1615, 1597, 1574, 1466, 1224. 1H NMR (δ (ppm), CDCl3):
7.17 (d, J = 8.5 Hz, 2H, H2), 6.77 (d, J = 8.5 Hz, 2H, H3), 5.72 (s, 2H,
H4), 3.96 (s, 3H, H5), 3.8990 (s, 3H, H6). 1.55 (s, 1H, H1). 13C NMR (δ
(ppm), DMSO): 160.5, 159.0, 158.1, 139.3, 130.7, 130.1, 125.2, 115.9,
54.1, 53.4, 52.0. HRMS: m/z calcd for C13H13N3O5: 292.09747; found:
292.09214 [M + H]+
Dimethyl
1-(2,4-difluorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate
(6i). Orange solid, m.p: 67 ◦C, yield: 88%. IR vmax (cmꢀ 1): 3044, 2954,
1741, 1720, 1618, 1604, 1461, 1106. 1H NMR (δ (ppm), CDCl3): 7.19
(td, J = 8.6, 8.2, 6.2 Hz, 1H, H1), 6.86 (ddt, J = 10.8, 8.6, 2.7 Hz, 2H,
Dimethyl 1-(4-chlorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6 g).
Yellow solid, m.p: 52–55 ◦C, yield: 88%. IR vmax (cmꢀ 1): 3097, 2952,
1725, 1595, 1547, 1434, 1214. 1H NMR (δ (ppm), CDCl3): 7.30 (d, J =
8.6 Hz, 2H, H1), 7.25 (d, J = 8.6 Hz, 2H, H2), 5.78 (s, 2H, H3), 3.96 (s,
3H, H4), 3.91 (s, 3H, H5). 13C NMR (δ (ppm), CDCl3): 160.4, 158.7,
H
2,3), 5.82 (s, 2H, H4), 3.97 (s, 3H, H5), 3.95 (s, 3H, H6). 13C NMR (δ
(ppm), CDCl3): 165.1, 164.9, 160.6 (d, J = 263.7 Hz), 160.4 (d, J =
261.4 Hz), 140.5, 131.1 (dd, J = 9.9, 4.6 Hz), 130.1, 117.4 (dd, J = 14.6,
4

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
4.0 Hz), 112.1 (dd, J = 21.6, 3.8 Hz), 104.4 (d, J = 50.8 Hz), 53.5, 52.8,
47.0. HRMS: m/z calcd for C13H11N3O4F: 312.07958; found: 312.08012
[M + H]+
default values [37]. The AutoGrid 4.2 and AutoDock 4.2 programs
employed to produce grid maps and to obtain results from generated
DLG files. The lowest binding energy conformers along with the lowest
RMSD were captured of 50 diversified conformers from gross docking
GA runs. To visualize the double docking results and interpret the pro-
tein–ligand interactions, we utilized the Discovery Studio 3.5 program
[36,38,39].
Dimethyl 1-(3,4-difluorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6
k). Yellow liquid, yield: 88%. IR vmax (cmꢀ 1): 3002, 2956, 1727, 1611,
1553, 1519, 1463, 1180. 1H NMR (δ (ppm), CDCl3): 7.10 (m, 3H, H1,2,3),
5.74 (s, 2H, H4), 3.94 (s, 3H, H5), 3.91 (s, 3H, H6). 13C NMR (δ (ppm),
CDCl3): 160.3, 158.7, 150.5 (dd, J = 249.8, 18.6 Hz), 150.4 (dd, J =
249.8, 18.7 Hz), 140.6, 130.80 (dd, J = 5.7, 4.0 Hz), 129.2, 124.6 (dd, J
= 6.6, 3.8 Hz), 117.6 (d, J = 24.2 Hz), 117.8 (d, J = 23.6 Hz), 53.4, 52.8,
52.7. HRMS: m/z calcd for C13H11N3O4F: 312.07992; found: 312.08080
[M + H]+
3. Results and discussion
3.1. Chemistry
The 1,2,3-triazole ring system has become remarkable units due to its
interaction with various biological systems. In recent years, many syn-
thetic methods have been developed for the synthesis of this ring system.
The first method developed for the synthesis of the 1,2,3-triazole ring is
the 1,3-dipolar cycloaddition is also known as Huisgen. In our study, the
synthesis of ester-containing derivatives was performed by heating in a
solvent-free metalless environment within 1–2 min, while dimethanol
derivatives were synthesized in 9 h taked a longer time under the same
conditions.
2.4. Xanthine oxidase (XO) inhibition assay of triazole compounds
The in vitro XO inhibitory properties of triazole complexes were
assayed according to method was described by Sweeney A. P. et al [35].
Enzyme activity was measured by following the formation of uric acid at
294 nm at 37 ◦C. To determine the IC50 values, different concentrations
of triazole complexes were added to the reaction mixture. Briefly, the
enzyme assay medium includes phosphate buffer (50 mM, pH = 7.4), XO
(0.2 U), and xanthine (1 mM) used as a substrate. First, enzyme and
triazole complexes were pre-incubated for 10 min, then the reaction was
started by adding xanthine to the mixture. The triazole complexes were
dissolved in DMSO as a stock and diluted with phosphate buffer. The last
concentration of DMSO in the reaction mixture was less than (0.001%)
and this ratio didn’t effect on the enzyme activity. Whole the experi-
ments were performed in triplicates. Allopurinol was used as a positive
control. The IC50 values of triazole complexes were measured as percent
inhibition of XO in terms of decrease in uric acid formation as compared
to the product formation in the absence of inhibitor. The percent inhi-
bition IC50 of XO activity was calculated as the following:
Compounds 6a-k and 5a-g were characterized using ATR-FTIR, 1H
NMR, 13C NMR, and HRMS spectroscopies. For the synthesis of 1,2,3-tri-
azole derivatives, benzyl azides (4a-k) were prepared from benzalde-
hydes in three steps in the literature. In the IR spectra, there was ѵ
(-N N = N) stretching at 2090–2100 cmꢀ 1 supported the formation of
–
–
azides. Dicarboxylate derivatives (6a-k) were synthesized by heating
the dimethyl acetylenedicarboxylate with benzyl azides (4a-k) without
–
solvent. The –N (-N N = N-) stretching band of azides was disappeared
–
3
at 2090–2100 cmꢀ 1 and the band of the carbonyl group (-C O) bond
–
–
stretching of the 6a-k derivatives was observed at about 1720–1740
cmꢀ 1. These stretching bands were proof of dicarboxylate derivatives
presented. In the 1H NMR spectra of the ester groups in the 4- and 5-po-
sition of the compounds dicarboxylate derivatives (6a-k) were asym-
metric so methyl protons of ester functionality resonate at around 3.80
and 4.00 ppm, respectively. The signals of the olefinic proton of the
aromatic ring moiety were in the range of 7.50–6.50 ppm. In the pres-
ence of groups that deactivate the ring (for example compounds of 6b,
6d, 6e), protons resonated in the lower region in comparison to in the
presence of activating groups (for example derivatives of 6a, 6b, 6 g).
Dimethanol derivatives (5a-g) were prepared by using but-2-yne-
1,4-diol and benzyl azide under heating for 9 h. Observation of the
characteristic –OH stretching vibration band at approximately 3200
cmꢀ 1 showed the formation of alcohol functional groups. In the IR
spectra, at 1100–1000 cmꢀ 1 bands the presence of single bonds of
– –
(A ꢀ B)
Inhibition(%) =
× 100
A
where A is the absorbance at 294 nm without the test compound and B is
the absorbance at 294 nm with the test compound.
2.5. Ligand-protein docking
Therefore, we implemented molecular docking for evaluated the
synthesized compounds combined with wet lab techniques contributes
towards the development of new drug molecules [31]. To enhance the
performance of molecular docking we employed the double docking
concept through dualling two programs [32]. Firstly, we downloaded
the crystal structure of XO from protein data bank (PDB: 3NVY). We
utilized the docking software Autodock Flexible Residue program
(ADFR) [36] in conjunction with the AutoGrid Flexible Residue program
(AGFR) for generating the configuration file of flexible residues of the
active site. The grid maps were set based on the position of reference
ligand (3,5,7,3′,4′-pentahydroxyflavone) inside the binding site of XO.
AGFR calculations were employed to determine flexible side-chains of
XO’s binding site by putting the corresponding residues in the config-
uration file [31]. Furthermore, the best conformation of ligands was
obtained by utilizing the ADFR. In the following section, all obtained
conformations were doubly docked again by employing the Autodock
4.2 for more-precise estimation of ligand pose within the binding site of
XO. Not only, Autodock 4.2 has scoring function that uses the AMBER
force field to estimate the binding energy of ligand-receptor but also
AutoDock 4.2 is an automated and durable docking algorithm based on
the Lamarckian Genetic Algorithm (LGA) [33]. The grid box of XO active
was of grid points on the x, y, and z axes to 40 × 40 × 40 with 0.375 Å
grid spacing. Docking parameters: the number of energy evaluations
were set to 250,000, and the number of generations was 50. Another key
thing to remember, other docking parameters were set to the software’s
–C
O
C group and at around 2090–2100 cmꢀ 1 disappearance of that
of the –N3 stretching prove to formation of alcohol derivatives. The 1H
NMR spectra of dimethanols (5a-g) were consistent with the proposed
structures. In general, it was clear that the methylene protons (–CH2-)
were shifted to the low domain by the inductive effect of the triazole ring
1
and –OH groups. In the H NMR spectrum, it was observed that reso-
nance signals of –CH2-OH protons bound to the 4- and 5-position of the
triazole ring of compound have triplet and doublet splitting. While the
methylene (–CH2-) protons in 5a-g were resonated between 4.0 and 5.0
–
ppm (J = 5.5 Hz), O H protons are resonated as triplet at ranging from
5.00 to 6.00 ppm with the coupling constant of J = 5.5 Hz.
3.2. Xanthine oxidase inhibition properties of compounds
XO plays an essential role in the synthesis of uric acid. XO is exces-
sively distributed throughout various organs such as liver, intestine,
lung, kidney, heart, brain, and plasma. XO levels rise in tissues cause
tissue damage and several illnesses due to the relationship with pro-
̵
-
duction of reactive oxygen metabolites such as (H2O2, O2 , and OH )
[40]. Allopurinol, is one of purine derivatives and widely used to XO
inhibition, has also as a therapeutic agent for many years (Fig. 4).
5

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
Table 4
The IC50 values of xanthine oxidase inhibition of dicarboxylate derivatives 6a-k.
Compound
IC50
(μM)
R2
Docking Score
6a
1.71 ± 0.078
1.40 ± 0.068
1.61 ± 0.093
1.57 ± 0.061
0.80 ± 0.025
0.75 ± 0.047
0.73 ± 0.030
0.87 ± 0.071
1.65 ± 0.091
1.23 ± 0.082
2.56 ± 0.031
0.999
0.994
0.995
0.964
0.998
0.989
0.965
0.982
0.978
0.982
0.989
ꢀ 3.934
ꢀ 4.136
ꢀ 4.154
ꢀ 4.166
ꢀ 4.613
ꢀ 4.755
ꢀ 4.730
ꢀ 4.496
ꢀ 4.574
ꢀ 4.386
6b
6c
6d
6e
6f
6 g
6 h
Fig. 4. Structure of allopurinol, dimethanol (5a-g), and dicarboxylate de-
rivatives (6a-k).
6i
6 k
Allopurinol
However, the disadvantage of allopurinol can be possibly converted to
4,6-dihydroxy pyrazole (3,4-d) pyrimidine, which is very high in
toxicity chemical. Furthermore, Fais et al. demonstrated the side effects
of allopurinol such as hypersensitivity reactions and inducing Stevens-
Johnson syndrome [41]. Therefore, it is urgent to synthesis of new
non-purine compounds which has more efficient and low side effects
[42,43]. For this reason, triazole derivatives containing hydroxyl and
ester functional groups and different substituents in the N-phenyl ring
were evaluated their inhibition properties against xanthine oxidase
(Fig. 4). The IC50 values of compounds were demonstrated in Tables 3
and 4. The results showed that dicarboxylate derivatives have better
effect for inhibiting XO than dimethanol derivatives. The range for IC50
alkoxy-3-cyanophenyl) isonicotinamide/nicotinamide derivatives was
synthesized and determined as 0.3–35.0 μM for IC50 in terms of inhibi-
tory potential against XO [46]. Burmaoglu et al. synthesized a series of
B-ring fluoro substituted bis-chalcone derivatives, the XO inhibition
activities IC50 range were determined as 0.728–6.058 μM [24]. Kıbrız
et al. synthesized a new pyrrole carboxamide derivatives and reported
that the IC50 value range was 4.608–7.084 μM [25]. In a recent study
present by Aktas et al., new 2-hydroxyethyl substituted N-heterocyclic
carbene precursors and reported that the IC50 value range was
1.253–5.342 μM [47]. In a new study reported by Gundugdu et al.,
isoindole-1,3(2H)-dione (phthalimides) derivatives exhibited the inhi-
bition of xanthine oxidase (XO), the IC50 value range was from 7.15 to
values for dimethanol derivatives was founded (0.71–2.25
other hand, the IC50 range for dicarboxylate derivatives founded
(0.73–1.71 M). All dicarboxylate derivatives showed higher inhibition
μM). On the
22.56 μM [48].
μ
activity toward XO, even more than the positive control allopurinol. This
may be based on free carboxylic group on the compound that can easily
compete with xanthine to inhibit XO. However, dimethanol derivatives
relatively showed weaker inhibition against XO, that because hydroxyl
group is weaker than carboxylic group in terms of interaction with
enzyme active center (Table 3 and 4).
3.3. Docking analysis of dicarboxylate derivatives and dimethanol
derivatives
A total of 17 compounds of both dimethanol and dicarboxylate series
were docked into the narrow channel of xanthine oxidase. The docking
results showed a good correlation with the experimental data. The
experimentally top three active compounds were also ranked as top
three based on docking score. pIC50 values were calculated as the
negative log of the IC50 values. The results produce a good correlation
coefficient (R2 = 0.7455) between the docking scores and pIC50 values of
the ligands (Fig. 5). Based on IC50 values and docking scores, the top 3
among the 17 docked compounds were discussed in this study as the
most active (potent).
The 5a-g and 6a-k compounds which were substituted of different
functional groups (F, Cl, Br, OH, and OCH3) on N-benzyl ring exhibited
range from 0.71 to 2.25 μM inhibition value for XO enzymes. Especially,
amidst dimethanol and dicarboxylate compounds of 5c, 5 g, 6e, 6f, 6 g
and 6 h proved to be show the most potent enzyme inhibitory activity.
When the structures of the compounds (5 g, 6e, 6f, 6 g and 6 h) with the
best inhibitory activity were examined, it was seen that they contained
chloride atom and hydroxyl group in the N-benzyl ring except 5c
substituted 3-bromide. The dicarboxylate derivatives (6f and 6 g)
bearing a 4-chloro, and 4-hydroxyl groups on N-benzyl ring were
exhibited more activity when compared with the dimethanol derivatives
(5e and 5f) bearing same groups. According to IC50 values, it was seen
that OH group attached to the N-benzyl ring in ester compounds con-
tributes positively to the inhibition of XO, while the OH group in the N-
benzyl ring in dimethanol derivatives has a decreasing effect on the
inhibition of XO.
3.4. Binding interactions between top-three ligands and XO
Most active ligands according to IC50 values, 5c (IC50 0.70 μM) was
the most active in its series, besides it has the lowest docking score from
17-screened compounds as given in Table 3 and 4. Compound 5c
established five different contacts with the active site (position of native
ligand: 3,5,7,3′,4′-pentahydroxyflavone) residues of XO (PDB: 3NVY)
(Fig. 6). Thr1010 formed conventional carbon hydrogen bond with ox-
ygen of methanol group. Ser876 interacted with hydrogen of carbon
linker (benzene ring-triazole ring). Further, linked to the Phe914,
Leu873, Phe1009, Lys771, Val1011, Pro1076 Leu1014 and Leu648 via
different types of interactions such as van der Waals, pi-sigma, pi-pi
stacked, pi-pi T-shaped, alkyl and pi-alkyl. ring through the pi-pi stacked
bond. While 6 g bound with Leu648, Phe649, Leu873, Val1011,
Phe1009, Phe914, Leu1014, Leu1014, Glu802, Pro1076 and Lys771
residues of XO through van der Waals, pi-sigma, pi-pi stacked, pi-pi T-
shaped, alkyl and pi-alkyl interactions (Fig. 7). And 6f established many
different types of interactions include: hydrogen bond with Thr1010,
while other hydrophobic interactions (van der Waals, pi-pi stacked, pi-pi
T-shaped, alkyl and pi-alkyl) were with Leu648, Phe649 Phe914,
Ala1079, Phe1009, Leu873, Leu1014, Val1011 and Phe1013 (Fig. 8).
These interactions were responsible for high docking scores and also
account for low value of IC50 of 5c, 6f and 6 g. Thus, we have drawn
In a recent study, XO inhibition activities (IC50) were demonstrated
as 0.263–20.45 μM for hesperidin derivatives [44]. In another study, a
research group synthesized coumarin derivatives and reported that the
IC50 value range was 14.79–97.65 μM [45]. Besides, a series of N-(4-
Table 3
The IC50 values of xanthine oxidase inhibition of dimethanol derivatives 5a-g.
Compound
IC50
(μ
M)
R2
Docking Score
5a
2.11 ± 0.050
2.25 ± 0.056
0.71 ± 0.059
1.22 ± 0.038
1.60 ± 0.076
1.43 ± 0.097
0.93 ± 0.067
2.56 ± 0.031
0.970
0.976
0.988
0.985
0.993
0.976
0.997
0.989
ꢀ 3.468
ꢀ 3.364
ꢀ 4.790
ꢀ 4.168
ꢀ 4.148
ꢀ 4.288
ꢀ 4.399
5b
5c
5d
5e
5f
5 g
Allopurinol
6

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
Fig. 5. A plot of the experimental pIC50 values versus the docking score values (kcal/mol) of the 17-studied compound.
Fig. 6. A) The binding orientation of 5c (blue-ball and stick) inside the binding site of XO, B) The 2D ligand–protein interaction of the docked 5c and residues of XO.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
several conclusions from the kinetics and molecular docking studies. For
instance, the activity of compounds 5c, 6f and 6 g indicated the
importance of an electron-withdrawing group para and/or meta posi-
tions of benzene ring, thereby making the benzene ring more flexible to
rotate around triazole ring axis, and this enhance the ability of benzene
ring to reach buried positions inside the binding pocket and fit well
inside the pocket this can explain why 5c, 6f and 6 g (Fig. 9) have lowest
docking scores and IC50 values. Also, the presence of bromine atom at
meta position enhances the electron density of the phenyl ring through
its ability to interact with the aromatic electrons. The real secret behind
the activity of 5c compounds return back to its geometry where its less
steric hindered at C4 and C5 of triazole ring if it compared with 6 g and
6f which contain bulky groups at the same positions. To assess the fitness
of studies ligands we investigate the binding mode within the pocket of
XO as shown in Figure S52, it can be seen that 5c, 5 g, 6f, 6 g and 6 h
were well fitted inside the pocket of XO while the rest have no good
binding mode.
4. Conclusion
Evaluation of inhibition effects of the triazole compounds on the XO
enzyme indicated that all derivatives were more promising in compared
with allopurinol, positive standard. Particularly, 5c, 6f and 6 g, com-
pounds exhibited the most effective inhibitory activities on the XO with
IC50 values 0.71 ± 0.059, 0.75 ± 0.047 and 0.73 ± 0.03 μM, respec-
tively, comparable to that of allopurinol as the reference molecule (IC50
= 2.56 ± 0.031 M). While XO inhibition activities of synthesized de-
rivatives have activity in the range of 0.73 to 1.71 M in dicarboxylate
derivatives, it was that in derivatives of dimethanol 0.71 to 2.25 M.
μ
μ
μ
Therefeore, it was also found that the inhibition effect of the dicarbox-
ylate derivatives were higher than those of dimethanol. Docking studies
were also in agreement with the experimental results. According to
docking results, most of the compounds have low docking score as a
result of the multiple substitutions at benzene ring or bulk groups which
makes the entering of these compounds inside the narrow pocket of XO.
7

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
Fig. 7. A) The binding orientation of 6 g (yellow-ball and stick) inside the binding site of XO, B) The 2D ligand–protein interaction of the docked 6 g and residues of
XO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. A) The binding orientation of 6f (cyan-ball and stick) inside the binding site of XO, B) The 2D ligand–protein interaction of the docked 6f and residues of XO.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
This also reduced the docking score as a result of substitution in the
ortho position of benzene, preventing the benzene ring from rotating
around the axis of the triazole ring. It is interesting to mention that, the
most active compounds structurally have a single substituent group
attached to the benzene ring and small groups to the triazole ring. In
general, based on the docking results and in vitro data, it consultable that
the presence of one substituent of -Br, -Cl, or –OH at the para position or
meta of benzene ring facilitates an orientation which enables complex
8

G. Yagiz et al.
Bioorganic Chemistry 108 (2021) 104654
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