An extensive research on aldose reductase inhibitory effects of new 4H-1,2,4-triazole derivatives

Article abstract of DOI:10.1016/j.molstruc.2020.129446

Aldose reductase (AR) is a key enzyme, which triggers the excessive accumulation of sorbitol in insulin independent tissues leading to severe diabetes-induced microvascular complications. Substantial evidence has proven that AR inhibition is a well-establ

Full text of DOI:10.1016/j.molstruc.2020.129446

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An extensive research on aldose reductase inhibitory effects of new
4
H-1,2,4-triazole derivatives
a,∗
a
b
a
Belgin Sever , Mehlika Dilek Altıntop , Yeliz Demir , Muhammed Pekdo g˘ an ,
c
c,d
a
Gül s¸ en Akalın Çiftçi , S¸ ükrü Beydemir , Ahmet Özdemir
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470, Eski s¸ ehir, Turkey
Department of Pharmacy Services, Nihat Delibalta Gole Vocational High School, Ardahan University, 75700, Ardahan, Turkey
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470, Eski s¸ ehir, Turkey
The Rectorate of Bilecik S¸ eyh Edebali University, 11230, Bilecik, Turkey
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Aldose reductase (AR) is a key enzyme, which triggers the excessive accumulation of sorbitol in in-
sulin independent tissues leading to severe diabetes-induced microvascular complications. Substantial
evidence has proven that AR inhibition is a well-established strategy to attenuate these complica-
tions. In the current work, new 2-[(4-amino-5-aryl-4H-1,2,4-triazol-3-yl)thio]-N-(thiazol/benzothiazol-2-
yl)acetamides (1–18) were synthesized and evaluated for their inhibitory capacities on AR. 2-[(4-Amino-
5-(4-methylphenyl)-4H-1,2,4-triazol-3-yl)thio]-N-(5-nitrothiazol-2-yl)acetamide (12) and 2-[(4-amino-5-
(3-pyridyl)-4H-1,2,4-triazol-3-yl)thio]-N-(6-nitrobenzothiazol-2-yl)acetamide (17) were identified as the
most effective AR inhibitors in this series with the Ki values of 0.04 ± 0.01 μM and 0.08 ± 0.02 μM,
respectively as compared to quercetin (Ki = 5.66 ± 0.66 μM). These two compounds displayed compet-
itive AR inhibition. MTT assay, a tetrazolium-based cell viability assay, was performed to determine the
cytotoxic effects of compounds 1–18 on L929 mouse fibroblast (healthy) cell line. Compounds 1–18, ex-
cept for compounds 10, 13, 14, 15 and 16, were found nontoxic against healthy cells. Besides, molecular
docking studies were fundamentally in agreement with the biological data with regard to essential π-π
interactions with Trp219, Phe122 and Trp111 residues in the active site of AR. Eventually, in vitro and in
silico assays ascertain that in particular compounds 12 and 17 will attract a great notice as drug-like AR
inhibitors for further investigations related to amelioration of long-term diabetic complications.
Received 8 September 2020
Revised 2 October 2020
Accepted 12 October 2020
Available online xxx
Keywords:
Triazole
Thiazole
Benzothiazole
Aldose reductase
Diabetic complications
Cytotoxicity
Molecular docking
©
2020 Elsevier B.V. All rights reserved.
1
. Introduction
healing [8]. There are several mechanisms implicated in the pro-
gression of these microvascular disorders such as elevated activity
of polyol and hexosamine pathways, excessive advanced glycation
end products (AGEs) and protein kinase C (PKC) isoforms and in-
adequate antioxidant defense [9,10].
Diabetes Mellitus (DM) is a chronic and multi-factorial disease
characterised by impaired glucose homeostasis due to abnormal
insulin action [1–3]. Among two major forms of DM, type 1 or
insulin dependent DM (T1DM) is the result of immune mediated
genetically programmed devastation of the β-cells, whereas type 2
or non-insulin dependent DM (T2DM) stems from the insufficient
insulin secretion and/or the insulin resistance [4,5].
Polyol pathway is normally an alternative route for glucose
metabolism, whereas in hyperglycemia, there is a marked rise in
flux through this pathway detrimentally affecting tissues such as
retina, kidney, peripheral nerves and blood vessels where glu-
cose entry is independent of insulin [11,12]. Aldose reductase (AR)
drives the nicotinamide adenine dinucleotide phosphate (reduced
form) (NADPH)-dependent conversion of glucose to sorbitol as a
first and rate limiting enzyme in the polyol pathway (Fig. 1). Sor-
bitol, in turn is oxidized to fructose by nicotinamide adenine din-
It is well known that patients with DM show a tendency to
develop one or more of the secondary diabetic complications in-
cluding cataract, retinopathy, nephropathy and neuropathy in the
course of their disease [6,7]. These long-term microvascular com-
plications are also associated with the formation of blindness, end
stage renal failure, defective nerve conduction and impaired wound
+
ucleotide (NAD) -dependent sorbitol dehydrogenase (SDH) [13].
Augmented AR levels along with the depletion of NADPH have
been reported to lead to oxidative stress and the accumulation
∗
+
Corresponding author.
of intracellular sorbitol along with the increased free NADH/NAD
E-mail address: belginsever@anadolu.edu.tr (B. Sever).
https://doi.org/10.1016/j.molstruc.2020.129446
0
022-2860/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: B. Sever, M.D. Altıntop, Y. Demir et al., An extensive research on aldose reductase inhibitory effects of new
H-1,2,4-triazole derivatives, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2020.129446
4

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Fig. 1. The polyol pathway.
Fig. 2. Some AR inhibitors.
ratio, which triggers osmotic stress [14–16]. Based upon substantial
experimental data linking the AR inhibition with the alleviation of
secondary diabetic lesions, research efforts have focused on under-
standing the structure and function of AR and developing effective
anti-AR interventions [17,18].
π stacking interactions and hydrogen bonding capability, rigidity,
improved solubility and stability to metabolic degradation and re-
dox conditions. These properties of triazole scaffold also play im-
portant roles in high-affinity binding with the biological targets
[23–33]. On the other hand, compounds carrying a benzothiazole
ring just as in zopolrestat have been enunciated to present higher
selectivity for AR fitting into the hydrophobic pocket. It is also well
ascertained that some thiazole derivatives show very potent AR in-
hibition [34–37].
On the basis of safety and efficacy issues and poor pharmacoki-
netic profiles, the development of many AR inhibitors (Fig. 2) was
terminated. Epalrestat, which was launched into Japanese market
in 1992, is the only currently commercially available AR inhibitor.
Sorbinil has been reported to show in vitro and in vivo AR in-
hibitory activity after comprehensive tests, but it was pulled from
the market owing to emerging hypersensitivity reactions. Besides,
among flavonoids quercetin exhibited dual AR inhibitory and an-
tioxidant effects, whereas its oral bioavailability was found quite
low due to its high hydrophilicity [19–22]. In order to design and
synthesize new potent AR inhibitors, some physicochemical pa-
rameters related to crossing biological membranes easily and se-
lectivity due to the coexistence of AR with aldehyde reductase in
most tissues must be enlightened [19].
Such challenging observations mentioned above have prompted
us to synthesize new 4H-1,2,4-triazole derivatives clubbed with
thiazole/benzothiazole scaffolds (1–18), to investigate their AR in-
hibitory effects, to evaluate their cytotoxicity against L929 mouse
fibroblast (healthy) cells and to in silico explore their possible bind-
ing modes in the active site of AR along with their pharmacoki-
netic profiles.
2. Experimental
2
.1. Chemistry
Triazole is a five-membered heterocyclic pharmacophore sys-
tem bearing three nitrogen and two carbon atoms. 1,2,3-Triazole
and 1,2,4-triazole are two isomeric forms of triazole. Triazoles have
been reported to possess assorted pharmacological activites includ-
ing antidiabetic activity based on its moderate dipole character, π-
All reagents purchased from commercial suppliers were used
without additional purification. The Electrothermal IA9200 digital
melting point apparatus (Staffordshire, UK) was used for the deter-
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mination of the melting points (M.p.) of the compounds. IR spectra
were recorded on an IRPrestige-21 Fourier Transform Infrared spec-
trophotometer (Shimadzu, Tokyo, Japan). 1H NMR and 13C NMR
spectra were recorded on a Bruker 400 MHz spectrometer (Bruker,
Billerica, MA, USA) using DMSO–d6 as a solvent. Chemical shifts
were reported in parts per million (ppm). Tetramethylsilane (TMS)
was used as an internal standard. In the NMR spectra, the split-
ting patterns were designated as follows: s: singlet; d: doublet;
t: triplet; m: multiplet. Coupling constants (J) were reported in
Hertz (Hz). HRMS spectra were recorded on a Shimadzu LCMS-IT-
TOF system (Shimadzu, Kyoto, Japan) using methanol as a solvent.
Thin Layer Chromatography (TLC) was performed on TLC Silica gel
C–O stretching, aromatic C–H in plane bending), 966, 883, 825,
802, 765, 738, 688 (Aromatic C–H out of plane bending and C-S
stretching) (Supplementary Material Fig. S5). 1H NMR (400 MHz,
DMSO–d ): 3.77 (s, 3H), 4.25 (s, 2H), 6.16 (s, 2H), 7.03 (s, 3H), 7.41
6
(s, 2H), 7.71–8.07 (m, 2H), 12.74 (s, 1H) (Supplementary Material
Fig. S6). 13C NMR (100 MHz, DMSO–d ): 34.8 (CH ), 55.2 (CH ),
6
2
3
113.9 (2CH), 119.0 (CH), 121.3 (CH), 121.7 (C), 126.4 (CH), 127.5 (C),
129.0 (CH), 129.1 (CH), 133.1 (C), 147.4 (C), 152.5 (C), 154.0 (C),
158.9 (C), 160.2 (C), 167.9 (C) (Supplementary Material Fig. S7).
+
HRMS (ESI) (m/z) [M + H] calcd. for C18 H15ClN O S : 447.0459,
6
2 2
found: 447.0460 (Supplementary Material Fig. S8).
2.1.1.3.3. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-3-
6
0 F254 aluminum sheets (Merck, Darmstadt, Germany) to control
yl)thio]-N-(6-bromobenzothiazol-2-yl)acetamide (3). M.p.: 243–
244 °C. Yield: 84%. IR νmax (cm 1): 3379, 3336 (N–H stretching),
−
the purity of the compounds.
3
149, 3115, 3061 (Aromatic C–H stretching), 2999, 2960, 2920,
2
.1.1. General procedure for the synthesis of the compounds
2833 (Aliphatic C–H stretching), 1664 (Amide C=O stretching),
1595, 1556, 1539, 1479, 1462 (N–H bending, C=N and C=C stretch-
ing), 1440, 1388, 1323, 1305, 1251, 1226, 1174, 1111, 1085, 1037
(C–H bending, C–N and C–O stretching, aromatic C–H in plane
bending), 964, 869, 825, 802, 731 (Aromatic C–H out of plane
2.1.1.1. 4-Amino-5-aryl-4H-1,2,4-triazole-3-thiol (A). A mixture of 5-
aryl-1,3,4-oxadiazole-2-thiol (0.01 mol) and hydrazine hydrate
0.01 mol) in absolute ethanol was refluxed for 12 h. Then, the sol-
(
vent was removed under reduced pressure. 100 mL of cold water
was added to residue and later, acidified with concentrated HCl to
pH = 5. Finally, the obtained product was filtered off and crystal-
lized from ethanol [38].
bending and C-S stretching) (Supplementary Material Fig. S9). 1
H
NMR (400 MHz, DMSO–d ): 3.81 (s, 3H), 4.29 (s, 2H), 6.21 (s,
6
2H), 7.06 (d, J = 9.20 Hz, 2H), 7.56 (d, J = 8.80 Hz, 1H), 7.69 (d,
J = 8.80 Hz, 1H), 7.93 (d, J = 9.20 Hz, 2H), 8.23 (s, 1H), 12.72
(s, 1H) (Supplementary Material Fig. S10). 13C NMR (100 MHz,
DMSO–d6): 34.9 (CH2), 55.2 (CH3), 113.9 (2CH), 115.4 (C), 119.1
(CH), 122.1 (CH), 124.2 (C), 129.1 (CH), 129.2 (2CH), 133.7 (C),
147.7 (C), 152.5 (C), 154.0 (C), 159.0 (C), 160.3 (C), 168.0 (C) (Sup-
2.1.1.2. 2-Chloro-N-(aryl)acetamides. Chloroacetyl
chloride
(
(
(
0.03 mol) was added dropwise to a mixture of aromatic amine
0.025 mol) and triethylamine (TEA) (0.025 mol) in toluene
50 mL) at 0–5 °C. The solvent was evaporated under reduced
+
pressure. The residue was washed with water to remove TEA and
crystallized from ethanol. The products were crystallized from
ethanol [39].
plementary Material Fig. S11). HRMS (ESI) (m/z) [M + H] calcd.
for C18 H15BrN6O2S2: 490.9954, found: 490.9967 (Supplementary
Material Fig. S12).
2
.1.1.3.4. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-3-
2
.1.1.3. 2-[(4-Amino-5-aryl-4H-1,2,4-triazol-3-yl)thio]-N-
thiazol/benzothiazol-2-yl)acetamides (1–18). mixture of com-
pound A (0.0015 mol) and appropriate 2–chloro–N-(aryl)acetamide
0.0015 mol) in acetone (25 mL) was stirred at room temperature
yl)thio]-N-(6-methylbenzothiazol-2-yl)acetamide (4). M.p.: 252–
253 °C. Yield: 92%. IR νmax (cm 1): 3342, 3265 (N–H stretching),
3199, 3003 (Aromatic C–H stretching), 2968, 2931, 2837 (Aliphatic
C–H stretching), 1687 (Amide C=O stretching), 1614, 1554, 1481,
1456 (N–H bending, C=N and C=C stretching), 1415, 1390, 1307,
1282, 1259, 1226, 1168, 1099, 1037 (C–H bending, C–N and C–O
stretching, aromatic C–H in plane bending), 997, 968, 893, 813,
744, 713, 680 (Aromatic C–H out of plane bending and C-S
stretching) (Supplementary Material Fig. S13). 1H NMR (400 MHz,
DMSO–d6): 2.34 (s, 3H), 3.81 (s, 3H), 3.90 (s, 2H), 6.45 (s, 2H),
7.01–7.10 (m, 3H), 7.36 (d, J = 7.60 Hz, 1H), 7.46 (s, 1H), 7.99 (d,
J = 8.40 Hz, 2H), 12.65 (s, 1H) (Supplementary Material Fig. S14).
−
(
A
(
for 8 h in the presence of potassium carbonate (0.0015 mol). The
solvent was evaporated under reduced pressure. The residue was
washed with water and crystallized from ethanol [40].
2
.1.1.3.1. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-
3
-yl)thio]-N-(benzothiazol-2-yl)acetamide (1). M.p.: 208–209 °C.
Yield: 80%. IR νmax (cm 1): 3350, 3280 (N–H stretching), 3176,
062 (Aromatic C–H stretching), 2972, 2935, 2839 (Aliphatic C–H
stretching), 1680 (Amide C=O stretching), 1602, 1564, 1556, 1471
N–H bending, C=N and C=C stretching), 1436, 1392, 1336, 1294,
253, 1178, 1097, 1026 (C–H bending, C–N and C–O stretching,
aromatic C–H in plane bending), 970, 893, 873, 833, 800, 752,
29, 694 (Aromatic C–H out of plane bending and C-S stretching)
Supplementary Material Fig. S1). 1H NMR (400 MHz, DMSO–d ):
−
3
13
(
C NMR (100 MHz, DMSO–d6): 20.9 (CH3), 40.6 (CH2), 55.2 (CH3),
1
113.8 (2CH), 118.4 (CH), 119.6 (CH), 120.5 (C), 125.6 (CH), 129.3
(2CH), 129.6 (C), 132.9 (C), 148.1 (C), 153.1 (C), 153.5 (C), 160.1 (C),
166.7 (C), 171.6 (C) (Supplementary Material Fig. S15). HRMS (ESI)
7
+
(
(m/z) [M + H] calcd. for C19 H18 N6O2S2: 427.1005, found: 427.1006
6
3
2
.81 (s, 3H), 4.15 (s, 2H), 6.32 (s, 2H), 7.06 (d, J = 8.40 Hz,
H), 7.18 (t, J = 7.20, 7.60, 14.80 Hz, 1H), 7.34 (t, J = 7.20, 7.60,
(Supplementary Material Fig. S16).
2.1.1.3.5. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-
3-yl)thio]-N-(6-nitrobenzothiazol-2-yl)acetamide (5). M.p.: 212–
213 °C. Yield: 86%. IR νmax (cm 1): 3332, 3211 (N–H stretching),
3091 (Aromatic C–H stretching), 2972, 2927, 2839 (Aliphatic
C–H stretching), 1693 (Amide C=O stretching), 1610, 1575, 1552,
1504, 1460 (N–H bending, NO2, C=N and C=C stretching), 1435,
1323, 1286, 1255, 1226, 1168, 1124, 1045, 1028 (C–H bending,
C–N and C–O stretching, aromatic C–H in plane bending), 975,
881, 825, 800, 750, 732 (Aromatic C–H out of plane bending
and C-S stretching) (Supplementary Material Fig. S17). 1H NMR
(400 MHz, DMSO–d6): 3.81 (s, 3H), 4.11 (s, 2H), 6.31 (s, 2H), 7.06
(d, J = 8.40 Hz, 2H), 7.64 (d, J = 9.20 Hz, 1H), 7.96 (d, J = 8.40 Hz,
2H), 8.14 (d, J = 8.40 Hz, 1H), 8.74 (s, 1H), 12.71 (s, 1H) (Supple-
mentary Material Fig. S18). 13C NMR (100 MHz, DMSO–d6): 36.6
(CH2), 55.2 (CH3), 113.8 (2CH), 117.8 (CH), 118.6 (CH), 119.4 (C),
121.0 (CH), 129.3 (2CH), 132.8 (C), 141.1 (C), 152.9 (C), 153.7 (C),
1
4.80 Hz, 1H), 7.64 (d, J = 7.60 Hz, 1H), 7.83 (d, J = 7.60 Hz, 1H),
−
7
.97 (d, J = 8.80 Hz, 2H), 12.56 (s, 1H) (Supplementary Material
Fig. S2). 13C NMR (100 MHz, DMSO–d ): 37.4 (CH ), 55.3 (CH ),
6
2
3
113.9 (2CH), 119.3 (CH), 119.8 (CH), 121.3 (C), 122.3 (CH), 125.4
(
CH), 129.3 (2CH), 132.0 (C), 149.3 (C), 152.9 (C), 153.9 (C), 160.3
(
C), 162.2 (C), 169.7 (C) (Supplementary Material Fig. S3). HRMS
+
(
ESI) (m/z) [M + H] calcd. for C18 H16 N O S : 413.0849, found:
6
2 2
4
13.0858 (Supplementary Material Fig. S4).
2
.1.1.3.2. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-
3
-yl)thio]-N-(6-chlorobenzothiazol-2-yl)acetamide (2). M.p.: 248–
49 °C. Yield: 79%. IR νmax (cm 1): 3381, 3338 (N–H stretching),
−
2
3
118, 3062, 3005 (Aromatic C–H stretching), 2964, 2920, 2835
Aliphatic C–H stretching), 1664 (Amide C=O stretching), 1600,
558, 1479, 1462 (N–H bending, C=N and C=C stretching), 1442,
388, 1327, 1305, 1251, 1174, 1099, 1037 (C–H bending, C–N and
(
1
1
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(Supplementary Material Fig. S33). 1H NMR (400 MHz, DMSO–d ):
1
55.7 (C), 160.2 (C), 170.4 (C), 172.6 (C) (Supplementary Material
6
+
Fig. S19). HRMS (ESI) (m/z) [M + H] calcd. for C18 H15N O S :
2.36 (s, 3H), 3.98 (s, 2H), 6.38 (s, 2H), 7.31 (d, J = 8.40 Hz, 1H),
7.35 (d, J = 8.80 Hz, 2H), 7.43 (d, J = 8.40 Hz, 1H), 7.92 (d,
J = 8.00 Hz, 3H), 12.79 (s, 1H) (Supplementary Material Fig. S34).
7
4 2
4
58.0700, found: 458.0677 (Supplementary Material Fig. S20).
2
.1.1.3.6. 2-[(4-Amino-5-(4-methoxyphenyl)−4H-1,2,4-triazol-3-
13
yl)thio]-N-(5-nitrothiazol-2-yl)acetamide (6). M.p.: 184–185 °C.
C NMR (100 MHz, DMSO–d ): 20.9 (CH ), 40.7 (CH ), 112.5 (C),
6
3
2
Yield: 55%. IR νmax (cm 1): 3352, 3307 (N–H stretching), 3149,
093, 3008 (Aromatic C–H stretching), 2929, 2835 (Aliphatic C–H
stretching), 1672 (Amide C=O stretching), 1610, 1575, 1504, 1481,
−
120.4 (CH), 123.0 (CH), 124.3 (CH), 124.4 (C), 127.7 (2CH), 128.9
(2CH), 134.9 (C), 139.1 (C), 149.3 (C), 153.3 (C), 153.7 (C), 167.3
(C), 171.9 (C) (Supplementary Material Fig. S35). HRMS (ESI) (m/z)
3
+
1
460 (N–H bending, NO , C=N and C=C stretching), 1417, 1381,
[M + H] calcd. for C18 H15BrN OS : 475.0005, found: 475.0006
2
6
2
1
286, 1253, 1172, 1122, 1029 (C–H bending, C–N and C–O stretch-
ing, aromatic C–H in plane bending), 962, 935, 904, 827, 732,
(Supplementary Material Fig. S36).
2.1.1.3.10. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-
6
78, 646 (Aromatic C–H out of plane bending and C-S stretching)
Supplementary Material Fig. S21). 1H NMR (400 MHz, DMSO–d ):
3-yl)thio]-N-(6-methylbenzothiazol-2-yl)acetamide (10). M.p.:
−1
(
206–207 °C. Yield: 90%. IR νmax (cm ): 3340, 3269 (N–H stretch-
6
3
2
.81 (s, 3H), 4.06 (s, 2H), 5.77 and 6.28 (2s, 2H), 7.05–7.09 (m,
H), 7.95–8.00 (m, 2H), 8.48 (s, 1H), 12.75 (s, 1H) (Supplementary
ing), 3188, 3030 (Aromatic C–H stretching), 2918 (Aliphatic C–H
stretching), 1685 (Amide C=O stretching), 1598, 1558, 1481, 1456
(N–H bending, C=N and C=C stretching), 1419, 1390, 1323, 1282,
1226, 1180, 1120, 1105, 1043 (C–H bending, C–N and C–O stretch-
ing, aromatic C–H in plane bending), 999, 974, 923, 893, 815,
746, 721 (Aromatic C–H out of plane bending and C-S stretching)
(Supplementary Material Fig. S37). 1H NMR (400 MHz, DMSO–
d6): 2.34 (s, 3H), 2.37 (s, 3H), 3.86 (s, 2H), 6.45 (s, 2H), 7.00 (d,
J = 7.60 Hz, 1H), 7.32 (t, J = 8.00, 16.00 Hz, 3H), 7.43 (s, 1H),
7.94 (d, J = 7.60 Hz, 2H), 12.81 (s, 1H) (Supplementary Material
Material Fig. S22). 13C NMR (100 MHz, DMSO–d ): 39.4 (CH ),
6
2
5
5.3 (CH ), 113.9 (CH), 113.9 (CH), 119.4 (C), 129.3 (CH), 129.6
3
(
(
[
(
CH), 137.0 (C), 145.6 (CH), 152.8 (C), 153.7 (C), 160.2 (C), 166.5
C), 172.0 (C) (Supplementary Material Fig. S23). HRMS (ESI) (m/z)
+
M + H] calcd. for C14 H13N O S : 408.0543, found: 408.0538
7
4 2
Supplementary Material Fig. S24).
2
.1.1.3.7. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-3-
yl)thio]-N-(benzothiazol-2-yl)acetamide (7). M.p.: 204–205 °C.
Yield: 75%. IR νmax (cm 1): 3338, 3275 (N–H stretching), 3176,
−
13
Fig. S38). C NMR (100 MHz, DMSO–d ): 20.8 (2CH ), 40.0 (CH ),
6
3
2
3
059 (Aromatic C–H stretching), 2918, 2841 (Aliphatic C–H stretch-
121.1 (CH), 124.3 (CH), 127.4 (CH), 128.3 (C), 129.6 (2CH), 130.7
(2CH), 132.1 (C), 135.9 (C), 140.9 (C), 150.2 (C), 154.3 (C), 154.5 (C),
168.2 (C), 171.8 (C) (Supplementary Material Fig. S39). HRMS (ESI)
ing), 1668 (Amide C=O stretching), 1614, 1556, 1485, 1460 (N–H
bending, C=N and C=C stretching), 1438, 1381, 1321, 1282, 1265,
+
1
224, 1172, 1126, 1028 (C–H bending, C–N and C–O stretching,
(m/z) [M + H] calcd. for C19 H18 N OS : 411.1056, found: 411.1068
6
2
aromatic C–H in plane bending), 975, 943, 879, 821, 750, 721,
80 (Aromatic C–H out of plane bending and C-S stretching)
Supplementary Material Fig. S25). 1H NMR (400 MHz, DMSO–d ):
(Supplementary Material Fig. S40).
6
2.1.1.3.11. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-3-
(
yl)thio]-N-(6-nitrobenzothiazol-2-yl)acetamide (11). M.p.: 217–
6
218 °C. Yield: 87%. IR νmax (cm 1): 3336, 3236 (N–H stretching),
3176, 3095 (Aromatic C–H stretching), 2922, 2837 (Aliphatic C–H
stretching), 1683 (Amide C=O stretching), 1653, 1616, 1575, 1558,
−
2
.36 (s, 3H), 4.23 (s, 2H), 6.28 (s, 2H), 7.20–7.47 (m, 4H), 7.70 (d,
J = 8.40 Hz, 1H), 7.89 (d, J = 7.60 Hz, 3H), 12.78 (s, 1H) (Supple-
13
mentary Material Fig. S26). C NMR (100 MHz, DMSO–d ): 20.9
6
(
CH ), 36.1 (CH ), 120.1 (CH), 121.4 (CH), 122.9 (CH), 124.0 (CH),
1541, 1506, 1456 (N–H bending, NO , C=N and C=C stretching),
3
2
2
127.4 (C), 127.6 (2CH), 129.0 (2CH), 131.7 (C), 139.3 (C), 148.9 (C),
1435, 1321, 1280, 1224, 1166, 1124, 1045 (C–H bending, C–N
and C–O stretching, aromatic C–H in plane bending), 974, 885,
819, 752, 719, 680, 651 (Aromatic C–H out of plane bending
and C-S stretching) (Supplementary Material Fig. S41). 1H NMR
153.0 (C), 154.1 (C), 160.1 (C), 168.6 (C) (Supplementary Material
+
Fig. S27). HRMS (ESI) (m/z) [M + H] calcd. for C18 H16 N OS :
6
2
3
97.0900, found: 397.0904 (Supplementary Material Fig. S28).
2
.1.1.3.8. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-3-
(400 MHz, DMSO–d ): 2.35 (s, 3H), 4.13 (s, 2H), 6.25 (s, 2H), 7.28
6
yl)thio]-N-(6-chlorobenzothiazol-2-yl)acetamide (8). M.p.: 258–
59 °C. Yield: 83%. IR νmax (cm 1): 3329, 3250 (N–H stretching),
(d, J = 6.80 Hz, 2H), 7.65 (d, J = 8.00 Hz, 1H), 7.88 (d, J = 6.40 Hz,
2H), 8.13 (d, J = 7.60 Hz, 1H), 8.73 (s, 1H), 12.80 (s, 1H) (Supple-
−
2
mentary Material Fig. S42). 13C NMR (100 MHz, DMSO–d ): 20.8
3
182 (Aromatic C–H stretching), 2981, 2922, 2856 (Aliphatic C–H
stretching), 1681 (Amide C=O stretching), 1622, 1591, 1550, 1483
N–H bending, C=N and C=C stretching), 1442, 1423, 1404, 1323,
280, 1238, 1178, 1130, 1103, 1043 (C–H bending, C–N and C–O
stretching, aromatic C–H in plane bending), 975, 941, 894, 858, 827,
06, 744 (Aromatic C–H out of plane bending and C-S stretching)
Supplementary Material Fig. S29). 1H NMR (400 MHz, DMSO–d ):
6
(CH ), 38.3 (CH ), 117.8 (CH), 118.8 (CH), 121.0 (CH), 124.1 (C),
3
2
(
127.6 (2CH), 128.9 (2CH), 132.7 (C), 139.2 (C), 141.3 (C), 153.0 (C),
1
153.9 (C), 155.3 (C), 169.4 (C), 172.0 (C) (Supplementary Material
+
Fig. S43). HRMS (ESI) (m/z) [M + H] calcd. for C18 H15N O S :
7
3 2
8
442.0751, found: 442.0772 (Supplementary Material Fig. S44).
(
2.1.1.3.12. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-3-
6
2
1
.33 (s, 3H), 3.88 (s, 2H), 6.38 (s, 2H), 7.16–7.38 (m, 4H), 7.67 (s,
H), 7.89 (s, 2H), 12.83 (s, 1H) (Supplementary Material Fig. S30).
C NMR (100 MHz, DMSO–d ): 20.92 (CH ), 40.77 (CH ), 119.51
yl)thio]-N-(5-nitrothiazol-2-yl)acetamide (12). M.p.: 182–183 °C.
Yield: 65%. IR νmax (cm 1): 3361, 3282 (N–H stretching), 3095,
3043 (Aromatic C–H stretching), 2924, 2837 (Aliphatic C–H
stretching), 1672 (Amide C=O stretching), 1612, 1575, 1537, 1456
−
13
6
3
2
(
(
CH), 120.05 (CH), 124.20 (CH), 124.37 (2C), 127.72 (2CH), 128.89
2CH), 134.60 (C), 139.05 (C), 149.31 (C), 153.45 (C), 153.65 (C),
(N–H bending, NO , C=N and C=C stretching), 1413, 1381, 1321,
2
1
68.75 (C), 172.70 (C) (Supplementary Material Fig. S31). HRMS
1288, 1244, 1182, 1145, 1120, 1101, 1045 (C–H bending, C–N and
C–O stretching, aromatic C–H in plane bending), 962, 906, 819,
742, 719 (Aromatic C–H out of plane bending and C-S stretching)
(Supplementary Material Fig. S45). 1H NMR (400 MHz, DMSO–
+
(
ESI) (m/z) [M + H] calcd. for C18 H15ClN OS : 431.0510, found:
6
2
4
31.0519 (Supplementary Material Fig. S32).
2
.1.1.3.9. 2-[(4-Amino-5-(4-methylphenyl)−4H-1,2,4-triazol-3-
yl)thio]-N-(6-bromobenzothiazol-2-yl)acetamide (9). M.p.: 247–
48 °C. Yield: 87%. IR νmax (cm 1): 3329 (N–H stretching), 3188,
d ): 2.37 (s, 3H), 4.03 (s, 2H), 6.27 (s, 2H), 7.31 (d, J = 8.40 Hz,
6
−
2
2H), 7.91 (d, J = 8.00 Hz, 2H), 8.44 (s, J = 7.60 Hz, 1H), 12.76
(s, 1H) (Supplementary Material Fig. S46). 13C NMR (100 MHz,
DMSO–d ): 20.8 (CH ), 38.3 (CH ), 124.2 (C), 127.6 (2CH), 128.8
3
113, 3043 (Aromatic C–H stretching), 2922, 2837 (Aliphatic C–H
stretching), 1681 (Amide C=O stretching), 1616, 1585, 1548, 1481
N–H bending, C=N and C=C stretching), 1440, 1423, 1404, 1323,
276, 1236, 1172, 1130, 1101, 1035 (C–H bending, C–N and C–O
stretching, aromatic C–H in plane bending), 977, 941, 894, 881, 825,
04, 738 (Aromatic C–H out of plane bending and C-S stretching)
6
3
2
(
(2CH), 136.3 (C), 139.0 (C), 145.7 (CH), 153.0 (C), 153.7 (C), 173.1
1
(C), 175.0 (C) (Supplementary Material Fig. S47). HRMS (ESI) (m/z)
+
[M + H] calcd. for C14 H13N O S : 392.0594, found: 392.0592
7
3 2
8
(Supplementary Material Fig. S48).
4

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Journal of Molecular Structure xxx (xxxx) xxx
2
.1.1.3.13. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
out of plane bending and C-S stretching) (Supplementary Material
N-(benzothiazol-2-yl)acetamide (13) (CAS registry number 901,151–
Fig. S61). 1H NMR (400 MHz, DMSO–d ): 2.40 (s, 3H), 4.33 (s, 2H),
6
9–3). M.p.: 221–222 °C. Yield: 62%. IR νmax (cm 1): 3317 (N–H
−
9
6.29 (s, 2H), 7.25 (d, J = 8.00 Hz, 1H), 7.54–7.57 (m, 1H), 7.65 (d,
J = 8.80 Hz, 1H), 7.75 (s, 1H), 8.35 (d, J = 8.40 Hz, 1H), 8.69 (s,
1H), 9.15 (s, 1H), 12.57 (s, 1H) (Supplementary Material Fig. S62).
stretching), 3172, 3062 (Aromatic C–H stretching), 2924, 2848
Aliphatic C–H stretching), 1681 (Amide C=O stretching), 1633,
(
13
1
598, 1568, 1548, 1504, 1483 (N–H bending, C=N and C=C stretch-
C NMR (100 MHz, DMSO–d ): 20.8 (CH ), 34.7 (CH ), 120.1 (CH),
6 3 2
ing), 1440, 1392, 1323, 1267, 1226, 1174, 1124, 1026 (C–H bending,
C–N and C–O stretching, aromatic C–H in plane bending), 975,
121.1 (CH), 122.9 (CH), 123.4 (CH), 127.4 (C), 131.5 (C), 133.0 (C),
134.9 (CH), 146.4 (C), 148.1 (CH), 150.3 (CH), 152.2 (C), 153.5 (C),
156.8 (C), 167.1 (C) (Supplementary Material Fig. S63). HRMS (ESI)
9
37, 879, 812, 752, 698, 680 (Aromatic C–H out of plane bending
and C-S stretching) (Supplementary Material Fig. S49). 1H NMR
400 MHz, DMSO–d ): 4.32 (s, 2H), 6.32 (s, 2H), 7.29 (t, J = 8.00,
+
(m/z) [M + H] calcd. for C17 H15N OS : 398.0852, found: 398.0860
7
2
(
(Supplementary Material Fig. S64).
6
7
.60, 15.60 Hz, 1H), 7.42 (t, J = 7.60, 15.20 Hz, 1H), 7.54–7.57 (m,
H), 7.75 (d, J = 8.40 Hz, 1H), 7.94 (d, J = 8.00 Hz, 1H), 8.36 (d,
J = 8.40 Hz, 1H), 8.69 (s, 1H), 9.16 (s, 1H), 12.60 (s, 1H) (Supple-
2.1.1.3.17. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
N-(6-nitrobenzothiazol-2-yl)acetamide (17). M.p.: 232–233 °C.
Yield: 82%. IR νmax (cm 1): 3338, 3280 (N–H stretching), 3165,
3105 (Aromatic C–H stretching), 2968, 2924, 2900 (Aliphatic C–H
stretching), 1668 (Amide C=O stretching), 1612, 1575, 1556, 1506
1
−
13
mentary Material Fig. S50). C NMR (100 MHz, DMSO–d ): 35.3
6
(
CH ), 120.4 (CH), 121.5 (CH), 123.0 (CH), 123.3 (CH), 123.5 (CH),
2
1
25.9 (C), 131.5 (C), 135.0 (CH), 148.1 (C), 148.6 (CH), 150.3 (CH),
(N–H bending, NO , C=N and C=C stretching), 1435, 1392, 1323,
2
1
52.2 (C), 153.6 (C), 158.5 (C), 167.7 (C) (Supplementary Material
1278, 1261, 1228, 1168, 1122, 1043 (C–H bending, C–N and C–O
stretching, aromatic C–H in plane bending), 972, 891, 846, 827, 812,
752, 702 (Aromatic C–H out of plane bending and C-S stretching)
+
Fig. S51). HRMS (ESI) (m/z) [M + H] calcd. for C16 H13N OS :
7
2
3
84.0696, found: 384.0696 (Supplementary Material Fig. S52).
2
.1.1.3.14. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
(Supplementary Material Fig. S65). 1H NMR (400 MHz, DMSO–d ):
6
N-(6-chlorobenzothiazol-2-yl)acetamide (14). M.p.: 258–259 °C.
Yield: 82%. IR νmax (cm 1): 3315 (N–H stretching), 3140, 3061
Aromatic C–H stretching), 2958, 2922, 2837 (Aliphatic C–H
4.29 (s, 2H), 6.33 (s, 2H), 7.54–7.57 (m, 1H), 7.80 (d, J = 9.20 Hz,
1H), 8.22 (d, J = 8.40 Hz, 1H), 8.36 (d, J = 8.00 Hz, 1H), 8.68
(s, 1H), 8.91 (s, 1H), 9.15 (s, 1H), 12.57 (s, 1H) (Supplementary
Material Fig. S66). 13C NMR (100 MHz, DMSO–d ): 36.3 (CH ),
−
(
stretching), 1660 (Amide C=O stretching), 1597, 1564 (N–H bend-
ing, C=N and C=C stretching), 1446, 1421, 1379, 1330, 1255, 1228,
6
2
120.1 (CH), 119.8 (CH), 121.3 (CH), 123.0 (CH), 123.4 (C), 132.4 (C),
134.9 (CH), 142.2 (C), 148.1 (C), 150.3 (CH), 152.1 (CH), 153.6 (C),
154.2 (C), 166.0 (C), 169.8 (C) (Supplementary Material Fig. S67).
1
178, 1128, 1095, 1026 (C–H bending, C–N and C–O stretching,
aromatic C–H in plane bending), 972, 921, 866, 800, 761, 746, 704
Aromatic C–H out of plane bending and C-S stretching) (Supple-
mentary Material Fig. S53). 1H NMR (400 MHz, DMSO–d ): 4.33 (s,
+
(
HRMS (ESI) (m/z) [M + H] calcd. for C16 H12N O S : 429.0547,
8
3 2
found: 429.0549 (Supplementary Material Fig. S68).
6
2
H), 6.29 (s, 2H), 7.45 (d, J = 6.80 Hz, 1H), 7.54–7.57 (m, 1H), 7.75
2.1.1.3.18. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
(
d, J = 8.40 Hz, 1H), 8.10 (s, 1H), 8.35 (d, J = 7.60 Hz, 1H), 8.52
N-(5-nitrothiazol-2-yl)acetamide (18). M.p.: 181–182 °C. Yield:
52%. IR νmax (cm 1): 3325, 3224 (N–H stretching), 3151, 3078
(Aromatic C–H stretching), 2926, 2833 (Aliphatic C–H stretching),
1687 (Amide C=O stretching), 1633, 1593, 1573, 1492 (N–H bend-
−
(
s, 1H), 9.14 (s, 1H), 12.62 (s, 1H) (Supplementary Material Fig.
S54). 13C NMR (100 MHz, DMSO–d ): 34.8 (CH ), 121.2 (CH), 121.6
6
2
(
(
(
[
(
CH), 122.9 (CH), 123.4 (CH), 126.3 (C), 127.4 (C), 133.1 (C), 134.9
CH), 147.3 (C), 148.0 (CH), 150.3 (CH), 152.2 (C), 153.4 (C), 158.8
C), 167.7 (C) (Supplementary Material Fig. S55). HRMS (ESI) (m/z)
ing, NO , C=N and C=C stretching), 1429, 1384, 1355, 1294, 1251,
2
1186, 1138, 1118, 1026 (C–H bending, C–N and C–O stretching,
aromatic C–H in plane bending), 968, 894, 821, 738, 700, 659,
628 (Aromatic C–H out of plane bending and C-S stretching)
+
M + H] calcd. for C16 H12ClN OS : 418.0306, found: 418.0312
7
2
Supplementary Material Fig. S56).
2
.1.1.3.15. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
(Supplementary Material Fig. S69). 1H NMR (400 MHz, DMSO–d ):
6
N-(6-bromobenzothiazol-2-yl)acetamide (15) (CAS registry num-
ber 901,615–40–5). M.p.: 261–262 °C. Yield: 83%. IR νmax (cm 1):
315 (N–H stretching), 3143, 3057 (Aromatic C–H stretching), 2960,
4.09 (s, 2H), 6.39 (s, 2H), 7.54–7.58 (m, 1H), 8.38 (d, J = 7.60 Hz,
1H), 8.47 (s, 1H), 8.68 (d, J = 6.40 Hz, 1H), 9.15 (s, 1H), 12.63
(s, 1H) (Supplementary Material Fig. S70). 13C NMR (100 MHz,
DMSO–d ): 38.5 (CH ), 123.2 (CH), 123.5 (C), 135.1 (CH), 136.8
−
3
2
927, 2831 (Aliphatic C–H stretching), 1660 (Amide C=O stretch-
6
2
ing), 1597, 1562, 1487 (N–H bending, C=N and C=C stretching),
(C), 145.7 (CH), 148.2 (CH), 150.3 (CH), 151.9 (C), 154.1 (C), 172.5
1
444, 1419, 1377, 1330, 1257, 1226, 1176, 1128, 1083, 1026 (C–H
bending, C–N and C–O stretching, aromatic C–H in plane bending),
74, 923, 840, 800, 742, 704, 680 (Aromatic C–H out of plane
bending and C-S stretching) (Supplementary Material Fig. S57). 1
NMR (400 MHz, DMSO–d ): 4.34 (s, 2H), 6.29 (s, 2H), 7.54–7.59
(C), 174.5 (C) (Supplementary Material Fig. S71). HRMS (ESI) (m/z)
+
[M + H] calcd. for C12H10N O S : 379.0390, found: 379.0390
8
3 2
9
(Supplementary Material Fig. S72).
H
6
2.2. Biochemistry
(
m, 2H), 7.70 (d, J = 8.80 Hz, 1H), 8.24 (s, 1H), 8.35 (d, J = 8.00 Hz,
H), 8.69 (d, J = 6.40 Hz, 1H), 9.14 (s, 1H), 12.76 (s, 1H) (Supple-
1
2.2.1. AR activity assay
13
mentary Material Fig. S58). C NMR (100 MHz, DMSO–d ): 34.7
AR activity was assessed by means of the absorbance decrease
of NADPH at 340 nm spectrophotometrically. Enzymatic reaction
mixture (1 mL total volume) contains 0.1 M sodium phosphate
buffer (pH = 5.5), 4.7 mM DL-glyceraldehyde, 0.11 mM NADPH and
enzyme solution [41].
6
(
CH ), 115.4 (CH), 122.1 (C), 122.9 (CH), 123.4 (CH), 124.1 (CH),
2
1
29.0 (C), 133.6 (C), 134.9 (CH), 147.6 (C), 148.0 (CH), 150.3 (CH),
1
52.2 (C), 153.5 (C), 158.5 (C), 167.6 (C) (Supplementary Material
+
Fig. S59). HRMS (ESI) (m/z) [M + H] calcd. for C16 H12BrN OS :
7
2
4
61.9801, found: 461.9810 (Supplementary Material Fig. S60).
2
.1.1.3.16. 2-[(4-Amino-5-(3-pyridyl)−4H-1,2,4-triazol-3-yl)thio]-
2.2.2. Homogenate preparation and purification of AR from bovine
liver
N-(6-methylbenzothiazol-2-yl)acetamide (16). M.p.: 252–253 °C.
Yield: 76%. IR νmax (cm 1): 3311 (N–H stretching), 3151, 3053 (Aro-
matic C–H stretching), 2956, 2922, 2843 (Aliphatic C–H stretching),
−
The homogenate was centrifuged at 13.500 x g for 60 min af-
ter approximately 15 g of bovine liver was homogenized in 45 mL
of 10 mM sodium phosphate buffer (pH = 7.4). The supernatant
was used for the next experiments. The supernatant suspension
was precipitated with ammonium sulfate. The precipitation inter-
vals for AR were detected as 0%−70%. The precipitate was gath-
1
656 (Amide C=O stretching), 1610, 1573, 1456 (N–H bending, C=N
and C=C stretching), 1421, 1377, 1336, 1253, 1226, 1178, 1128, 1028
C–H bending, C–N and C–O stretching, aromatic C–H in plane
bending), 972, 898, 866, 800, 748, 704, 682, 661 (Aromatic C–H
(
5

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Journal of Molecular Structure xxx (xxxx) xxx
ered by centrifugation at 13.500 × g for 30 min and redissolved in
a 10 mM sodium phosphate buffer (pH = 7.4). The solution was
dialyzed against 10 mM sodium phosphate buffer (pH = 7.4) con-
taining 5 mM 2-mercaptoethanol.
and 100 IU/mL penicillin-streptomycin (Gibco, Paisley, Scotland)
and 1% glutamin. Then, cells were incubated at 37 °C in a humidi-
fied atmosphere of 95% air and 5% CO . Exponentially growing cells
2
4
were plated at 2 × 10 cells/mL into 96-well microtiter tissue cul-
The dialyzed enzyme solution was loaded onto the DE-52 cel-
lulose anion exchange column formerly equilibrated with 10 mM
sodium phosphate buffer (pH= 7.4). AR was not allowed to at-
tach to the column at pH = 7.4 and left the column with the
same buffer. The purpose herein was to separate AR from other
enzymes attached to the column. The fractions were collected, and
the enzyme activity was controlled at 340 nm. After the fractions
displaying the enzyme activity were pooled and mixed with glyc-
erol (10%), this enzyme solution was loaded onto the Sephadex
G-100 column equilibrated with 10 mM sodium phosphate buffer
ture plates (Nunc, Roskilde, Denmark). The optimum cell number
for cytotoxicity assays was determined in preliminary experiments.
The stock solutions of the compounds were prepared in DMSO and
further dilutions were made with a fresh culture medium (the con-
centration of DMSO in the final culture medium was 0.1% which
had no effect on the cell viability) [37].
2
.3.2. MTT assay
The level
of
cellular
3-(4,5-dimethylthiazol-2-yl)−2,5-
diphenyltetrazolium bromide (MTT) reduction was quantified as
(
(
pH = 7.4). The fractions were analyzed for both protein amount
previously described in the literature [55] with small modifications
at 280 nm) and enzyme activity (at 340 nm). Then, the fractions
4
[
37]. L929 cells (2 × 10 cells/mL) were seeded on 96-well plates
ꢀ
ꢀ
from the Sephadex G-100 column were loaded onto the 2 ,5 -ADP
Sepharose 4B affinity column equilibrated with 10 mM sodium
phosphate buffer (pH = 7.4). The column was washed with 10 mM
sodium phosphate buffer (pH = 7.4) and then elution was per-
formed with linear gradient of 50–250 mM NaCl. The enzyme ac-
tivity was controlled at 340 nm in collected fractions, and the
tubes showing enzyme activity were combined. All purification
steps were performed at 4 °C [42].
and incubated for 24 h at 37 °C. Then, the tested compounds were
added to give final concentration in the range of 0.195–25 μM
and the cells were incubated for 24 h. At the end of this period,
MTT was added to final concentration of 0.5 μg/mL and the cells
were incubated for 2 h at 37 °C. After removal of medium, the
formazan crystals formed by MTT metabolism were solubilized by
the addition of 200 μL DMSO to each well and absorbance was
read at 540 nm with a microplate spectrophotometer (Bio-Tek,
Winooski, VT, USA). Every concentration was repeated in three
wells and the IC50 values (μM) for cell viability were defined as
the concentrations of compounds that reduced absorbance to 50%
of control (untreated cells) values and represented as mean ± SD.
2
.2.3. Protein determination
During the purification steps, Bradford method was used to de-
termine the quantitative protein by measuring the absorbance at
5
95 nm using bovine serum albumin as a standard [43].
2
.2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
2
2
.4. In silico studies
(SDS-PAGE)
The enzyme purity was checked according to Laemmli’s proce-
.4.1. Molecular docking studies
dure [44]. This method consists of 3% and 8% acrylamide concen-
trations for running and stacking gel, respectively according to pre-
vious studies [45–47]. SDS-PAGE gel was stained with silver and
the electrophoretic pattern was photographed.
The X-ray crystallographic structure of AR was obtained from
the PDB server (PDB ID code: 1T41) [37,56] and optimized for
docking studies in protein preparation module of Schrödinger soft-
ware (Schrödinger Release 2016–2: Schrödinger, LLC, New York, NY,
USA). Ligands were prepared with energy minimization using Opti-
mized Potential Liquid Simulations (OPLS_2005) force field at phys-
iological pH in ligand preparation module of Schrödinger software.
In molecular docking simulations: Grid Generation and Glide/XP
docking protocols were applied for the prediction of topologies of
compounds 1–18, quercetin and sorbinil in the active site of AR
2
.2.5. In vitro inhibition studies
The AR activity was measured in the presence of different
concentrations of compounds 1–18. Half maximal inhibitory con-
centration (IC50) was defined as the concentration of compound
causing 50% inhibition and it was calculated from Activity%-
[Compounds] graphs for each compound [48–50]. In order to de-
[
37].
termine the Vmax and Km values for the enzyme, activity mea-
surements were performed at five different concentrations of DL-
glyceraldehyde (0.070, 0.141, 0.230, 0.320 and 0.420 mM) and
Lineweaver–Burk graphs were plotted [51] according to the pre-
vious studies [52–54]. The inhibition types and inhibition constant
2.4.2. ADME prediction
Some physicochemical properties and the bioavailability
of compounds 1–18 were predicted by QikProp module of
Schrödinger software (Schrödinger Release 2016–2: QikProp,
Schrödinger, LLC, New York, NY, 2016). This module computes
physically significant descriptors and pharmaceutically relevant
properties compared to the determined range or recommended
values.
(
Ki) values were found from graphs with three different concentra-
tions of tested compounds. All measurements were repeated three
times.
2
.2.6. Statistical analyses
Analysis of the data, calculation of IC50 and K values, and draw-
i
ing of graphs were carried out using GraphPad Prism 7 software.
Differences between data sets were examined using the Kruskal-
Wallis test and considered statistically significant when the p-value
was less than 0.05. For significant p-values, post hoc Dunn’s multi-
3
. Results and discussion
3
.1. Chemistry
ple comparisons tests were performed. The K constants were pre-
i
In this work, the synthesis of the desired new triazoles
sented as mean ± standard deviation (95% confidence intervals).
(
1–18) was accomplished as described in Scheme 1. Initially,
2
.3. Cytotoxicity studies
4-amino-5-aryl-4H-1,2,4-triazole-3-thiol (A) was synthesized via
the reaction of 5-aryl-1,3,4-oxadiazole-2-thiol with hydrazine
2
.3.1. Cell culture and drug treatment
hydrate
2-yl)acetamide derivatives were obtained via the reaction of
2-aminothiazoles/benzothiazoles with chloroacetyl chloride
[38].
Meanwhile,
2–chloro–N-(thiazol/benzothiazol-
L929 mouse fibroblast cells were incubated in 90% DMEM, sup-
plemented with 10% fetal bovine serum (Gibco, Paisley, Scotland)
6

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Journal of Molecular Structure xxx (xxxx) xxx
Scheme 1. The synthetic route for the preparation of compounds 1–18. Reagents and conditions: (i) NH2NH2.H2O, ethanol, reflux, 12 h; (ii) ClCOCH2Cl, TEA, toluene, 0–5 °C;
iii) K2CO3, acetone, rt, 8 h.
(
in the presence of TEA [39]. Then, the subsequent nucle-
zothiazole scaffold present in compounds 4, 10 and 16 emanated
at 2.34–2.40 ppm. All other aromatic and heteroaromatic protons
were consistent with the proposed structures of the compounds.
In the 13C NMR spectra of compounds 1–18, the important S-
CH2 and C=O carbons at 34.7–40.7 ppm and 167.1–175.0 ppm, re-
spectively also substantiated the formation of the thioacetamide
moieties of final compounds. The methoxy carbons of compounds
1–6 and the methyl carbons at the 4th position of the phenyl
ring of compounds 7–12 came in sight at 55.2–55.3 ppm and
20.8–20.9 ppm, respectively. Besides, the methyl carbons of the
6-methylbenzothiazoles of compounds 4, 10 and 16 appeared at
20.8–20.9 ppm. The C3 and C5 carbons of triazoles gave rise to
peaks at 153.0–155.7 ppm and 151.9–154.3 ppm, respectively. All
other carbon peaks belonging to benzothiazole, thiazole, phenyl
and pyridine scaffolds also approved the proposed structures of the
final compounds. Once and for all, the HRMS data of compounds
1–18 matched with their molecular formulas.
ophilic substitution reaction of compound A with 2–chloro–N-
(
thiazol/benzothiazol-2-yl)acetamides in the presence of potassium
carbonate led to the formation of the target compounds (1–18).
The structures of title compounds (1–18) were verified by dif-
ferent spectroscopic techniques, such as Infrared (IR), 1H Nu-
clear Magnetic Resonance (NMR), 13C NMR and High Resolution
Mass Spectrometry (HRMS). In the IR spectra of compounds 1–
1
8, the N–H stretching bands belonging to the amino group at
the 4th position of 1,2,4-triazole scaffold and the acetamide group
−
1
were observed at 3211–3381 cm as expected [39,40]. The amide
C=O stretching bands appearing at 1656–1693 cm− confirmed
the formation of the final compounds. Moreover, the aromatic
and aliphatic C–H stretching vibrations gave rise to the bands
1
−
1
−1
at 3003–3188 cm
C=C stretching and N–H bending bands were detected at 1456–
and 2831–2999 cm , respectively. The C=N,
−1
1
653 cm . Finally, the C–H bending, C–N and C–O stretching, aro-
matic C–H in plane and out of plane bending and C-S stretching
bands occurred at prospective regions.
3.2. Biochemistry
In the 1H NMR spectra of compounds 1–18, the observation
of the characteristic singlet peaks at 3.86–4.34 ppm and 12.56–
In this study, diverse chromatographic methods, namely DE-52
cellulose anion exchange chromatography, Sephadex G-100 gel fil-
12.83 ppm of S-CH2 and NH protons, respectively is a clear evi-
ꢀ
ꢀ
dence for the formation of the thioacetamides of final compounds.
Along with this data, the singlet NH2 protons belonging to 1,2,4-
triazoles appeared at 5.77–6.45 ppm. In the 1H NMR spectra of
compounds 1–18, the singlet peak belonging to the methoxy pro-
tons attached to the phenyl moiety of compounds 1–6 was de-
tected at 3.77–3.81 ppm, whereas the singlet peak belonging to
the methyl protons attached to the phenyl moiety of compounds
tration chromatography and 2 ,5 -ADP-Sepharose 4B affinity col-
umn chromatography were applied to purify AR from bovine liver.
It was purified 338.98-fold with a yield of 1.12% and a specific ac-
tivity of 1.70 EU/mg (Table 1). SDS-PAGE was performed to check
the purity of AR and a single band at around 39 kDa was observed
(Fig. 3). Both the IC50 and the K values of compounds 1–18 were
i
obtained from the Activity%-[Inhibitor] and Lineweaver-Burk (1/V-
1/[S]) graphs, respectively. The inhibition types of compounds 1–18
as competitive or non-competitive inhibition were also determined
7
–12 became apparent at 2.33–2.37 ppm. Moreover, the singlet
peak belonging to the methyl protons at the 6th position of ben-
Table 1
Summary of the AR purification procedure.
Total
Total
Total
Specific
activity
(EU/mg)
Activity
(EU/mL)
volume
(mL)
Protein
protein
(mg)
activity
(EU)
Yield
(%)
Purification
fold
Purification Steps
(mg/mL)
Homogenate
0.265
0.285
0.180
0.085
0.017
40
30
24
10
7
32.44
21.13
7.22
1297.60
633.90
173.28
1.02
10.60
8.55
0.0081
0.0135
0.0249
0.8333
1.70
100
1
Ammonium sulfate precipitation and dialysis
DE-52 Cellulose anion exchange chromatography
Sephadex G-100 gel filtration chromatography
80.66
40.75
8.02
1.12
1.67
3.07
102.88
209.88
4.32
0.102
0.010
0.85
ꢀ
ꢀ
2
,5 -ADP-Sepharose 4B Affinity chromatography
0.070
0.119
7

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Journal of Molecular Structure xxx (xxxx) xxx
Fig. 3. The SDS-PAGE analysis of purified AR.
[
57,58]. Competitive inhibition is proportional to the amount of in-
hibitor bound in the active site and is therefore proportional to
inhibitor concentration. Because the inhibitor binds reversibly, the
substrate can compete with it at high substrate concentrations.
Thus a competitive inhibitor does not change the Vmax of an en-
zyme. On the other hand, competitive inhibitors raise the Km of
an enzyme since higher concentrations of substrate would be re-
quired to achieve half-maximal activity. Non-competitive inhibition
is a type of enzyme inhibition where the inhibitor reduces the ac-
tivity of the enzyme and binds equally well to the enzyme whether
or not it has already bound the substrate. When a non-competitive
inhibitor is added the Vmax is changed, while the Km remains un-
changed. According to the Lineweaver-Burk plot the Vmax is re-
duced during the addition of a non-competitive inhibitor, which
is shown in the plot by a change in both the slope and y-intercept
when a non-competitive inhibitor is added. Km and Vmax values
were determined for competitive and non-competitive inhibition.
Km values were found as 10.04 and 17.20 mM for non-competitive
and competitive inhibition respectively. Vmax values were found
as 0.056 and 0.035 mM/min for non-competitive and competitive
Fig. 4. The Lineweaver-Burk plots of compounds 12, 17 and quercetin (All measure-
ments were repeated three times).
inhibition, respectively. Quercetin was used as a standard AR in-
hibitor depending on relevant studies [59–61].
Ki denotes the equilibrium constant of the dissociation of the
inhibitor-bound enzyme complex, whereas IC50 quantifies the con-
centration of inhibitor necessary to halve the reaction rate of an
enzyme-catalyzed reaction observed under specified assay condi-
tions. Since Ki is an important constant associated with enzyme
kinetics, the AR inhibitory effects of compounds 1–18 were com-
pared based on their K values [62]. The results indicated that the
i
compounds displayed excellent AR inhibitory activity according to
the IC50 and the Ki values in the range of 0.205 μM-0.346 μM
and 0.04 ± 0.01 μM–0.81 ± 0.11 μM, respectively when com-
8

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Table 3
The IC50 values of compounds 1–18 on L929 cell line for 24 h.
Compound
IC50 (μM)
Compound
IC50 (μM)
1
2
3
4
5
6
7
8
9
10.67 ± 1.53
>25
10
11
12
13
14
15
16
17
18
0.58 ± 0.03
11.5 ± 1.73
1.07 ± 1.12
<0.195
>25
>25
>25
<0.195
7.5 ± 1.32
10.67 ± 1.53
>25
<0.195
<0.195
20.5 ± 0.71
6.55 ± 1.34
>25
Table 4
Docking score (kcal/mol), glide gscore (kcal/mol) and glide
emodel (kcal/mol) results of compounds 1–18 for AR (PDB ID
code: 1T41).
Fig. 5. Docking poses of compounds 1–18 along with quercetin and sorbinil in the
active site of AR (Blue and green dashes: π-π interactions, yellow dashes: hydrogen
bonding).
1
T41
Compound
Docking score
Glide score
Glide emodel
1
2
3
4
5
6
7
8
9
−4.99
−4.76
−6.41
−4.34
−4.88
−4.20
−4.88
−2.91
−3.22
−1.81
−4.64
−6.74
−2.68
−5.65
−5.61
−5.81
−6.61
−6.13
−6.34
−5.94
−6.74
−6.41
−6.48
−4.38
−6.18
−5.85
−6.18
−2.94
−3.33
−3.56
−5.95
−6.78
−2.71
−5.72
−6.66
−7.50
−6.64
−6.20
−8.68
−6.59
−47.82
−44.43
−35.55
−47.14
−39.24
−45.86
−39.24
−32.09
−47.14
−42.04
−37.80
−39.59
−36.48
−31.79
−46.84
−50.37
−33.57
−35.55
−43.64
−46.80
termined for their AR inhibitory activities pointing out their safety
as AR inhibitors.
3.3. Molecular docking studies
In an attempt to understand the mechanistic details of biolog-
ical activity, molecular docking studies were employed for com-
pounds 1–18 along with quercetin and sorbinil in the active site
of AR (PDB ID code: 1T41) [37,56]. Highly hydrophobic active site
pocket of AR consists of residues such as Trp20, Trp79, Trp111,
Trp219, Phe122 and Leu300 [63,64]. In general, compounds 1–
18 displayed high affinity forming proper interactions and only
compound 10 formed no observable intermolecular interactions
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
(
Fig. 5). Compounds 1–18, excluding compound 10, presented π-π
Quercetin
Sorbinil
stacking interactions with Trp219 and Phe122 through their tria-
zole moieties. The substitutions at 5th position of the triazole ring
also played a crucial role in the formation of π-π stacking inter-
actions with Trp111. N-(substituted)acetamide moieties were en-
gaged with water molecules via hydrogen bonds. Quercetin and
sorbinil exhibited π-π stacking interactions with Trp111 but they
missed key interactions with Trp219 and Phe122. The docked pose
and interactions of compounds 12 and 17 were depicted in de-
tail in Fig. 6. In order to compare the different conformations of
the same ligand, the emodel score is generally preferred, while
the different ligands are compared using the docking score [65].
All docking scores of the compounds were determined to range
from −2.68 to −6.74 kcal/mol. In general, the docking scores of
compounds were consistent with the biological data. The lowest
docking score, belonging to compound 10 explained its low in-
hibitory potency and binding capacity to the active site of AR. The
docking scores of compounds 12 and 17 were calculated as −6.74
and −6.61 kcal/mol as compared to quercetin (−6.34 kcal/mol) and
sorbinil (−5.94 kcal/mol) (Table 4).
pared to quercetin (IC50 = 3.86 μM and Ki = 5.66 ± 0.66 μM)
Table 2). The inhibitory potencies varied with the modifications
(
at the 5th position of the triazole and N-(substituted)acetamide
moieties. 4-Methylphenyl and 5-nitrothiazole carrying compound
(
(
12) and 3-pyridyl and 6-nitrobenzothiazole substituted compound
17) were found as the most potential AR inhibitors in this series
with the Ki values of 0.04± 0.01 μM and 0.08± 0.02 μM, respec-
tively. These compounds showed competitive inhibition behaviours
against AR (Fig. 4). Aside from compounds 12 and 17, compounds
3
, 6, 14 and 15 exhibited competitive inhibition in this series. 4-
Methylphenyl and 6-methylbenzothiazole bearing compound (10)
was determined to exhibit the lowest AR inhibitory activity with
the K value of 0.81± 0.11 μM indicating that two methyl moieties
i
substituted at the exact opposite sides together diminished the AR
inhibitory potency. Compounds 12 and 17 were also identified as
the most significant AR inhibitors among compounds 7–12 and
compounds 13–18, respectively. In the same manner, among com-
pounds 1–6, compound 3 showed the most obvious enhancement
3.4. In silico ADME studies
in AR inhibition with the K value of 0.19 ± 0.01 μM.
The Absorption, Distribution, Metabolism and Excretion (ADME)
profiles of compounds 1–18 were predicted by means of QikProp,
a predictive ADME module within the Maestro suite produced
by Schrödinger (Table 5). The conformation-independent pre-
dicted aqueous solubility (CIQPlogS), brain/blood partition coeffi-
cient (QPlogBB), human serum albumin binding (QPlogKhsa), pre-
dicted octanol/water partition coefficient (QPlogPo/w) of all com-
pounds were found within the acceptable range. The compliance
of compounds 1–18 to Lipinski’s rule of five and Jorgensen’s rule of
three was also investigated. Lipinski’s rule of five states that drug
i
One of the most significant drawback to available AR inhibitors
is toxicity and untoward side effects. Therefore, the cytotoxic ef-
fects of compounds 1–18 on L929 mouse fibroblast (healthy) cell
line were evaluated using MTT assay. Compounds 10, 13, 14, 15
and 16 were determined as toxic at their effective concentrations.
This outcome indicated that the existence of 3-pyridyl moiety led
to an increase in cytotoxicity to healthy cells apart from com-
pounds 17 and 18 (Table 3). On the other hand, other compounds
exerted no cytotoxicity towards L929 cells at their IC50 values de-
9

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Fig. 6. Docking poses of compounds 12 and 17, quercetin and sorbinil (A) (coloured in purple, yellow green, red and pink, respectively) and docking interactions of com-
pounds 12 and 17 (B) in the active site of AR (Blue and green dashes: π-π interactions, yellow dashes: hydrogen bonding).
10

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Table 2
The IC50, Ki values and inhibition types for compounds 1–18.
Compound
R
R’
Benzothiazol-2-yl
IC50 (μM)
R2
Ki (μM)
Type of Inhibition
1
4-Methoxyphenyl
0.213
0.205
0.226
0.232
0.273
0.291
0.256
0.330
0.307
0.346
0.243
0.294
0.260
0.302
0.308
0.257
0.311
0.232
3.860
0.9857
0.9812
0.9820
0.9814
0.9903
0.9781
0.9733
0.9747
0.9888
0.9836
0.9833
0.9874
0.9768
0.9760
0.9836
0.9863
0.9737
0.9825
0.9837
0.33 ± 0.08
0.41 ± 0.02
0.19 ± 0.01
0.47 ± 0.06
0.40 ± 0.03
0.50 ± 0.14
0.45 ± 0.02
0.58 ± 0.06
0.54 ± 0.06
0.81 ± 0.11
0.42 ± 0.05
0.04 ± 0.01
0.61 ± 0.02
0.28 ± 0.09
0.32 ± 0.08
0.25 ± 0.07
0.08 ± 0.02
0.23 ± 0.03
5.66 ± 0.66
Non-Competitive
Non-Competitive
Competitive
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
Pyridin-3-yl
6-Chlorobenzothiazol-2-yl
6-Bromobenzothiazol-2-yl
6-Methylbenzothiazol-2-yl
6-Nitrobenzothiazol-2-yl
5-Nitrothiazole-2-yl
Non-Competitive
Non-Competitive
Competitive
Benzothiazol-2-yl
Non-Competitive
Non-Competitive
Non-Competitive
Non-Competitive
Non-Competitive
Competitive
6-Chlorobenzothiazol-2-yl
6-Bromobenzothiazol-2-yl
6-Methylbenzothiazol-2-yl
6-Nitrobenzothiazol-2-yl
5-Nitrothiazole-2-yl
0
1
2
3
4
5
6
7
8
Benzothiazol-2-yl
Non-Competitive
Competitive
Pyridin-3-yl
6-Chlorobenzothiazol-2-yl
6-Bromobenzothiazol-2-yl
6-Methylbenzothiazol-2-yl
6-Nitrobenzothiazol-2-yl
5-Nitrothiazole-2-yl
Pyridin-3-yl
Competitive
Pyridin-3-yl
Non-Competitive
Competitive
Pyridin-3-yl
Pyridin-3-yl
Non-Competitive
Non-Competitive
Quarcetin
–
–
Table 5
Predicted ADME properties of compounds 1–18.
CIQPlogSa
QPlogBBa
QPlogKhsaa
QPlogPo/wa
(–2.0 to 6.5)
Rule of
Five
Rule of
Compound
(–6.5 to 0.5)
(−3 to 1.2)
(–1.5 to 1.5)
Three
1
2
3
4
5
6
7
8
9
−4.208
−4.870
−5.739
−4.476
−4.704
−3.513
−4.172
−4.835
−5.705
−4.441
−4.673
−3.481
−3.395
−4.040
−4.891
−3.656
−3.876
−2.711
−1.088
−0.949
−0.943
−1.136
−2.304
−2.251
−1.014
−0.874
−0.868
−1.060
−2.236
−2.205
−1.283
−1.150
−1.146
−1.314
−2.459
−2.403
−0.063
0.034
2.006
2.480
2.553
2.290
1.312
0.339
2.216
2.693
2.766
2.503
1.527
0.524
1.042
1.509
1.581
1.336
0.362
−0.606
0
0
0
0
1
1
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
0
0
0
1
1
0
0
0
0
1
1
0.054
0.070
−0.107
−0.415
0.063
0.169
0.183
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
0.200
0.023
−0.290
−0.330
−0.239
−0.221
−0.207
−0.380
−0.663
a
CIQPlogS: Conformation-independent predicted aqueous solubility, log S. S in mol dm–3 is the con-
centration of the solute in a saturated solution that is in equilibrium with the crystalline solid, QPlogBB:
brain/blood partition coefficient, QPlogKhsa: Prediction of binding to human serum albumin, QPlogPo/w:
Predicted octanol/water partition coefficient.
like molecules have octanol/water partition coefficient (QPlogPo/w)
5, molecular weight < 500, number of hydrogen bond accep-
ophilic substitution reaction of 2–chloro–N-(thiazol/benzothiazol-
2-yl)acetamides with 4-amino-5-aryl-4H-1,2,4-triazole-3-thiol (A),
which was obtained via the reaction of 5-aryl-1,3,4-oxadiazole-2-
thiol with hydrazine hydrate. The structures of all compounds were
confirmed by IR, 1H NMR, 13C NMR and HRMS data. Compounds 1–
18 were investigated for their inhibitory effects on AR, which was
known as a substantial target in the treatment of some diabetes-
linked abnormalities such as cataract, retinopathy, nephropathy
and neuropathy. According to in vitro studies, compounds 1–18 in-
hibited AR more notably than quercetin. Among them, compounds
12 and 17 were the most pronounced competitive AR inhibitors
with the Ki values of 0.04± 0.01 μM and 0.08± 0.02 μM, respec-
tively. Cytotoxicity studies place a great emphasis on the design
and development of new AR inhibitors. The results of MTT as-
say indicated that apart from compounds 10, 13, 14, 15 and 16,
compounds 1–18 were found nontoxic against L929 mouse fibrob-
<
tors ≤ 10, and number of hydrogen bond donors ≤ 5. Based on
Lipinski’s rule, these compounds can be considered as drug-like
molecules. On the other hand, the three rules of Jorgensen’s rule
are aqueous solubility (QPlogS) > −5.7, apparent Caco-2 cell per-
meability (QPPCaco in nm/s) > 22 nm/s, # primary metabolites <
7. Compounds with fewer (and preferably no) violations of these
rules are more likely to be orally bioavailable agents. On the basis
of these findings, all compounds are expected to have good oral
bioavailability.
4. Conclusion
In this study, new 4H-1,2,4-triazole derivatives carrying thia-
zole/benzothiazole scaffolds (1–18) were synthesized via the nucle-
11

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last (healthy) cells. Molecular docking studies confirmed that com-
pounds 1–18, apart from compound 10, forged fundamental in-
teractions with Trp219, Phe122 and Trp111 in the active site of
AR. According to in silico ADME studies, compounds 1–18 were
expected to possess conceivable pharmacokinetic profiles. Overall,
compounds 1–18, excluding compounds 10, 13, 14, 15 and 16, draw
attention as outstanding AR inhibitors for further investigations
about diabetic complications.
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11] C. Caglayan, Y. Demir, S. Kucukler, P. Taslimi, F.M. Kandemir, I˙ . Gulçin, The ef-
fects of hesperidin on sodium arsenite-induced different organ toxicity in rats
on metabolic enzymes as antidiabetic and anticholinergics potentials: a bio-
chemical approach, J. Food Biochem. 43 (2019) e12720.
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12] C. Ça g˘ layan, P. Taslimi, Y. Demir, S. Küçükler, F.M. Kandemir, I˙ . Gulçin, The ef-
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Declaration of Competing Interest
icol. 33 (2019) e22381.
[
13] Y. Demir, H.E. Duran, L. Durmaz, P. Taslimi, S¸ . Beydemir, I˙ . Gulçin, The influ-
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
ence of some nonsteroidal anti-inflammatory drugs on metabolic enzymes of
aldose reductase, sorbitol dehydrogenase, and α-glycosidase: a perspective for
metabolic disorders, Appl. Biochem. Biotechnol. 190 (2020) 437–447.
[
[
[
14] Y. Demir, M.S. Özaslan, H.E. Duran, Ö. I˙ . Küfrevio g˘ lu, S¸ . Beydemir, Inhibition ef-
fects of quinones on aldose reductase: antidiabetic properties, Environ. Toxicol.
Pharmacol. 70 (2019) 103195.
15] Y. Demir, Z. Köksal, Some sulfonamides as aldose reductase inhibitors: ther-
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CRediT authorship contribution statement
Belgin Sever: Conceptualization, Data curation, Formal anal-
ysis, Funding acquisition, Investigation, Methodology, Project ad-
ministration, Resources, Software, Supervision, Validation, Visu-
alization, Writing - original draft, Writing - review & editing.
Mehlika Dilek Altıntop: Conceptualization, Funding acquisition,
Methodology, Project administration, Resources, Software, Supervi-
sion, Writing - review & editing. Yeliz Demir: Data curation, For-
mal analysis, Methodology, Resources, Software, Validation, Writ-
ing - review & editing. Muhammed Pekdo g˘ an: Methodology, Soft-
ware, Writing - review & editing. Gül s¸ en Akalın Çiftçi: Data cura-
tion, Formal analysis, Methodology, Resources, Software, Validation,
Writing - review & editing. S¸ ükrü Beydemir: Conceptualization,
Methodology, Resources, Supervision, Writing - review & editing.
Ahmet Özdemir: Conceptualization, Resources, Supervision, Writ-
ing - review & editing.
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Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129446.
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