Design and synthesis of 1,4-disubstituted 1,2,3-triazoles: Biological evaluation, in silico molecular docking and ADME screening

Article abstract of DOI:10.1016/j.molstruc.2021.131344

In this study, propargyl compounds were synthesized from 4-hydroxybenzaldehyde and 3?methoxy-4-hydroxybenzaldehyde (2a-2b). As a result of click reactions of synthesized propargyl compounds (2a-b) with organic azides (4a-4e), carbonyl compounds (5a-5 h) h

Full text of DOI:10.1016/j.molstruc.2021.131344

Journal of Molecular Structure 1247 (2022) 131344
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design and synthesis of 1,4-disubstituted 1,2,3-triazoles: Biological
evaluation, in silico molecular docking and ADME screening
a
a
b
a
a,∗
˙
Irfan S¸ ahin , Mustafa Çe s¸ me , Fatma Betül Özgeri s¸ , Özge Güngör , Ferhan Tümer
a
Department of Chemistry, Faculty of Art and Sciences, Kahramanmaras Sutcu Imam University, 46040, Kahramanmaras, Turkey
b
Department of Nutrition and Dietetics, Faculty of Health Sciences, Ataturk University, Erzurum, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, propargyl compounds were synthesized from 4-hydroxybenzaldehyde and 3–methoxy-4-
hydroxybenzaldehyde (2a-2b). As a result of click reactions of synthesized propargyl compounds (2a-b)
with organic azides (4a-4e), carbonyl compounds (5a-5 h) having 1,2,3-triazole skeleton were obtained.
The structures of the synthesized compounds were illuminated by FTIR, 1H/13 C NMR spectroscopies and
elemental analyses. Antioxidant and anti-cancer and α-amylase enzyme inhibition studies of the syn-
thesized compounds were carried out, and the effects of different functional groups in the compounds
on the activity were investigated. When the results of the α-amylase enzyme inhibition studies of the
synthesized compounds were compared with the reference drug acarbose (IC50: 891 μg/mL), it was de-
termined that all compounds (IC50: 165–1471 μg/mL) showed higher activity than acarbose, except for
compounds 5a and 5c. In particular, compound 5 g (IC50: 165 μg/mL) was found to have approximately
Received 5 July 2021
Revised 17 August 2021
Accepted 18 August 2021
Available online 24 August 2021
Keywords:
Triazoles
Anticancer
Antioxidant activity
α-Amylase inhibition
Molecular docking
ADME
5
.5 times higher activity than acarbose. When the DPPH• radical scavenging studies were examined, all
+
compounds showed a higher activity than the standard BHT and β-carotene. According to ABTS• radical
scavenging activity results, all compounds showed more effective activity than Ascorbic acid, and Trolox
used as standard. Compound 5a showed approximately the same scavenging effect with β-carotene and
BHT. Compounds were also screened for anti-cancer activities against the HeLa cell line. According to the
results, 5c (IC50: 50.12 μg/mL) and 5 h (IC50: 57.07 μg/mL) exhibit moderate antitumor activity compared
to cis-platin against the HeLa cell line. The molecules have been studied in detail for their ADME proper-
ties and have not violated any drug-likeness rules. In addition, they exhibited a high oral bioavailability
profile, as their BBB (Blood Brain Barrier) penetration and GI (gastrointestinal) absorption properties were
favorable. Molecular docking results show that all compounds have a high affinity for the active site of
α-amylase.
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
gen, oxygen, and sulfur, but studies of new compounds containing
them are quite large and continue to increase daily. Heterocyclic
compounds are spread over an extensive area in nature and are
essential for life [5]. They play a crucial role in the metabolism of
all living cells. Examples of heterocyclic compounds are purine and
pyrimidine bases of DNA, essential amino acids (proline, histidine,
tryptophan), several vitamins and coenzyme precursors (thiamine,
riboflavin, pyridoxine, folic acid, biotin, vitamin B12 and E fami-
lies), photosynthesizing pigment. In addition, chlorophyll, oxygen-
carrying pigment (hemoglobin), many hormones (kinetin, sero-
tonin, histamine) and most sugars can be given [6–9]. Heterocyclics
with nitrogen atoms in their structure are of great importance in
the pharmaceutical industry and organic chemistry [10,11]. In five-
member ring systems, structures with three nitrogen atoms are
defined as triazoles. Triazole refers to one of a pair of isomeric
Despite remarkable technological advances in diagnosing and
treating diseases in recent years, cancer is one of the chiefly causes
of death worldwide [1,2]. This disease damages different organs
and occurs at various tissue levels. Although outstanding progress
has been made in cancer treatment, ineffective chemotherapy
caused by drug resistance, and many drug’s inabilities to distin-
guish between cancerous cells and normal cells, research studies
of agents with fewer side effects continue intensively [3,4].
Heterocyclic compounds are ringed compounds containing one
or more heteroatoms. The most common heteroatoms are nitro-
∗
Corresponding author at: Department of Chemistry, Faculty of Art and Sciences,
Kahramanmaras Sutcu Imam University, 46040, Kahramanmaras, Turkey.
chemical compounds with the closed formula C H N . These com-
2
3
3
E-mail
addresses:
mustafacesme@msn.com
(M.
Çe s¸ me),
betul.ozgeris@atauni.edu.tr (F.B. Özgeri s¸ ), ftumer@ksu.edu.tr (F. Tümer).
https://doi.org/10.1016/j.molstruc.2021.131344
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
3
–methoxy-4-(prop–2-yn-1-yloxy)benzaldehyde (2b): m.p.:
−
8
3
0–81 °C. Yield: 87%. color: Dark yellow solid. FTIR (υ, cm 1):
249, 2985 (C–H), 2823 (-O-CH ), 2114 (-C≡CH), 1691 (-HC=O),
2
1
589, 1512, 1468, 1426, 1379, 1269, 1221, 1168, 1123, 1008. 1
H
NMR (400 MHz, CDCl ) δ 9.89 (s, 1H), 7.48 (dd, J = 8.2, 1.8 Hz,
3
1
H), 7.45 (d, J = 1.8 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 4.87 (d,
13
J = 2.4 Hz, 2H), 3.96 (s, 3H), 2.58 (t, J = 2.4 Hz, 1H). C NMR
100 MHz, CDCl ) δ 190.84, 152.15, 150.10, 131.00, 126.20, 112.70,
Fig. 1. Structures of 1,2,3-triazole and 1,2,4-triazole.
(
3
109.58, 77.49, 76.64, 56.64, 56.05.
pounds are of two types, 1,2,3-triazoles and 1,2,4-triazoles [12,13].
Fig. 1.)
Molecules with a 1,2,3-Triazole skeleton are highly preferred
1-azido-4-methoxybenzene (4a): Yield: 87%, Colour: Brown
Oily. FTIR (υ, cm 1): 3003 (C–
−
H), 2836, 2105 (-N ), 1609, 1465.
(
3
1-azido-4-isopropylbenzene (4b): Yield: 93%. color: Dark
Brown oily. FTIR (υ, cm 1): 2962 (C–
−
H), 2871, 2096 (-N ), 1605,
in medicinal chemistry. They can be easily bound to biomolecu-
lar targets as they show high stability even in robust oxidative
and reductive environments, increasing their resolution of the ten-
dency to perform hydrogen bonding, dipole-dipole and π stack-
ing interactions. In addition, compounds containing 1,2,3- tria-
zole group are compounds with various biological activities (anti-
cancer, antimicrobial, anti-tubercular, anticonvulsant, antibacterial,
anti-inflammatory, analgesic, antiviral, and anti-HIV agents) that
are extremely important [13].
3
1461.
1-azido-4-methylbenzene (4c): Yield: 90%. color: Dark Brown
oily. FTIR (υ, cm 1): 3029 (C–
−
H), 2864, 2097 (-N ), 1613, 1581,
3
1503, 1452.
1-azido-4-chlorobenzene (4d): Yield: 97%. color: Light Brown
oily. FTIR (υ, cm 1): 3472, 3381 (C–
−
H), 2091 (-N ), 1614, 1485.
3
1-azido-4-fluorobenzene (4e): Yield: 83%. color: Dark Brown
oily. FTIR (υ, cm 1): 3229 (C–
−
H), 2986, 2108 (-N ), 1737, 1598,
3
Organic compounds with an aldehyde group in their structure
are found in combined or non –combined forms in many plants
in nature and function significantly as an intermediate. Aldehydes
have effective antimicrobial properties and are typically used for
high-level disinfection in health care environments [14–16]. It is
also used in agriculture for the preparation of herbicides and plant
growth regulators [17,18].
1498.
4-((1-(p-tolyl)−1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde
(5a): m.p.: 128–129 °C. color: Cream solid. Yield: 87%. FTIR (υ,
−
1
cm ): 3163 (C
–
H), 2839–2756 (C–H), 1682 (C=O), 1602–1508
1
(C=C), 1427 (N = N), 1302 (C
–
N), 1242 (C–O), 814 (=C–H).
H
NMR: (400 MHz, CDCl3) δ 9.91 (s, 1H), 8.07 (s, 1H), 7.87 (d,
J = 8.5 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.4 Hz,
13
Oxygen is an essential element for life [19]. When cells use oxy-
gen to produce energy, free radicals form ATP production (adeno-
sine triphosphate) by the mitochondria [20]. Free radical molecules
are unstable and highly reactive molecules containing unpaired
electrons [21]. Free radicals can damage the nucleus, DNA, proteins,
carbohydrates and many biological molecules such as lipids [22].
They also cause cell damage. Free radicals are usually removed or
inactivated by antioxidants in vivo environments [23,24].
2H), 7.16 (d, J = 8.6 Hz, 2H), 5.39 (s, 2H), 2.44 (s, 3H). C NMR:
(100 MHz, CDCl3) δ 190.74, 163.12, 143.91, 139.24, 134.61, 132.04,
130.47, 130.32, 121.24, 120.55, 115.13, 62.18, 21.10. Anal. calcd. for
C17 H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C, 68.13; H, 5.36;
N, 14.09.
4-((1-(4-chlorophenyl)−1H-1,2,3-triazol-4-
yl)methoxy)benzaldehyde (5b): m.p.: 143–144 °C. color: Light
−1
yellow solid. Yield: 94%. FTIR (υ, cm ): 3151, 3081 (C
–
H),
2842–2758 (C
–
H), 1686 (C=O), 1601–1500 (C=C), 1442 (N = N),
In this work, we report the design and synthesis of various
1
1302 (C
–
N), 1243–1228 (C–O), 819 (=C–H). H NMR: (400 MHz,
1
,2,3-triazole compounds (5a-h) with aldehyde groups. Structures
of the compounds were characterized by FTIR, 1H/13C NMR and
elemental analyses. Then antioxidant, anti-cancer and α-amylase
enzyme activity studies were investigated. Finally, computational
parameters like molecular docking study and ADME prediction of
synthesized compounds were performed.
CDCl3) δ 9.91 (s, 1H), 8.23 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.73
(d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.5 Hz,
13
2H), 5.38 (s, 2H). C NMR: (100 MHz, CDCl3) δ 190.73, 163.00,
134.87, 132.06, 130.53, 130.09, 121.86, 115.10, 61.99. Anal. calcd.
for C16 H12ClN3O2: C, 61.25; H, 3.86; N, 13.39. Found: C, 61.25; H,
3
.83; N, 13.01.
-((1-(4-fluorophenyl)−1H-1,2,3-triazol-4-
yl)methoxy)benzaldehyde (5c): m.p.: 134–135 °C. color: Dark
4
2. Materials and methods
yellow solid. Yield: 92%. FTIR (υ, cm 1): 3155–3074 (C–H), 2837–
−
2
.1. Materials and instrumentation
2
755 (C–H), 1686 (C=O), 1601–1575 (C=C), 1421 (N = N), 1302
1
(
C–N), 1255- 1225 (C–O), 814 (=C–H). H NMR: (400 MHz, CDCl )
3
All reagents and solvents were of reagent-grade quality and ob-
δ 9.90 (s, 1H), 8.14 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.78 – 7.71
tained from commercial suppliers (Aldrich or Merck) and used as
received unless noted otherwise. Elemental analyses (C, H, N) were
performed using a Costech ECS 4010 (CHN). Infrared spectra were
recorded on a Perkin-Elmer FT-IR spectrometer (Spectrum 400) kit-
ted with an ATR apparatus. 1H/13C NMR spectra were recorded on
a Bruker 400 MHz instrument, and TMS was used as an internal
standard. The 1H/13C NMR spectra of the compounds were inves-
tigated in CDCl3 solvent. α-Amylase enzyme inhibition activity de-
termined by Hitachi U3900H Spectrophotometer.
(
m, 2H), 7.23 (t, J = 12.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 5.38 (s,
13
2
H). C NMR: (100 MHz, CDCl ) δ 190.73, 163.82, 163.03, 161.33,
3
132.04, 130.50, 122.73, 122.65, 116.97, 116.74, 115.10, 62.04. Anal.
calcd. for C16 H12FN O : C, 64.64; H, 4.07; N, 14.13. Found: C,
3
2
6
2.58; H, 4.52; N, 13.93.
3
–methoxy-4-((1-(4-methoxyphenyl)−1H-1,2,3-triazol-4-
yl)methoxy)benzaldehyde (5d): m.p.: 111–112 °C. color: Light
Brown solid. Yield: 89%. FTIR (υ, cm ): 3080 (C–H), 2947–2832
−1
(
C–H), 1677 (C=O), 1512 (C=C), 1471 (N = N), 1306 (C–N), 1257-
1
1
221 (C–O), 830 (=C–H). H NMR: (400 MHz, CDCl ) δ 9.87 (s,
3
2
.2. Synthesis method of compounds (5a-h)
1H), 8.13 (s, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 7.9 Hz, 1H),
7
.44 (s, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 5.46
13
All compounds were obtained according to our previous work
(s, 2H), 3.94 (s, 3H), 3.87 (s, 3H). C NMR: (100 MHz, CDCl3)
δ 190.88, 160.01, 153.02, 149.97, 130.69, 126.71, 122.31, 114.86,
112.59, 109.31, 62.83, 56.04, 55.63. Anal. calcd. for C18 H17 N3O4: C,
63.71; H, 5.05; N, 12.38. Found: C, 63.42; H, 5.06; N, 11.98.
[
25]. 1H/13C NMR, FT-IR spectra of the compounds are given in the
supplementary. The general synthesis procedure was depicted in
Scheme 1.
2

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Scheme 1. Synthetic route for compounds (5a-h).
1
4
-((1-(4-isopropylphenyl)−1H-1,2,3-triazol-4-yl)methoxy)−3-
1222 (C–O), 804 (=C–H). H NMR: (400 MHz, CDCl ) δ 9.89 (s,
3
methoxybenzaldehyde (5e): m.p.: 95–96 °C. color: Powder solid.
Yield: 86%. FTIR (υ, cm ): 3139, 3102 (C–H), 2960- 2834 (C–H),
1H), 8.13 (s, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.53 (d, J = 8.9 Hz, 2H),
−1
13
7.50 – 7.45 (m, 2H), 7.27 (s, 1H), 5.49 (s, 2H), 3.97 (s, 3H).
C
1
685–1672 (C=O), 1585- 1508 (C=C), 1461 (N = N), 1347 (C–N),
NMR: (100 MHz, CDCl ) δ 190.90, 152.87, 149.94, 144.29, 135.34,
134.87, 130.77, 130.03, 126.72, 121.78, 121.29, 112.49, 109.29, 62.79,
3
1
1
263 (C–O), 833 (=C–H). H NMR: (400 MHz, CDCl ) δ 9.88 (s,
3
1
H), 8.23 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 10.7 Hz,
H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (s, 1H), 5.48 (s, 2H), 3.96 (s,
56.06. Anal. calcd. for C17 H14 ClN O : C, 59.40; H, 4.11; N, 12.22.
3
3
2
3
Found: C, 61.21; H, 4.02; N, 11.37.
13
H), 3.05 – 2.95 (m, 1H), 1.30 (d, J = 6.9 Hz, 6H). C NMR:
4-((1-(4-fluorophenyl)−1H-1,2,3-triazol-4-yl)methoxy)−3-
(
100 MHz, CDCl ) δ 190.90, 153.00, 150.13, 149.99, 135.16, 130.72,
methoxybenzaldehyde (5 h): m.p.: 113–114 °C. color: Cream
3
solid. Yield: 67%. FTIR (υ, cm 1): 3155, 3099 (C–H), 3007–2959
−
127.80, 126.76, 120.79, 112.62, 109.29, 62.79, 56.06, 33.85, 23.86.
Anal. calcd. for C20H21N O : C, 68.36; H, 6.02; N, 11.96. Found: C,
(C–H), 1685 (C=O), 1584 (C=C), 1424 (N = N), 1262- 1236 (C–O),
3
3
1
6
9.24; H, 5.99; N, 11.35.
–methoxy-4-((1-(p-tolyl)−1H-1,2,3-triazol-4-
yl)methoxy)benzaldehyde (5f): m.p.: 118–119 °C. color: Dark
831(=C–H). H NMR: (400 MHz, CDCl ) δ 9.87 (s, 1H), 8.15 (s,
3
3
1H), 7.77 – 7.68 (m, 2H), 7.49 – 7.43 (m, 2H), 7.27 – 7.19 (m, 3H),
5.47 (s, 2H), 3.95 (s, 3H). 13C NMR: (100 MHz, CDCl ) δ 190.88,
3
−1
red solid. Yield: 83%. FTIR (υ, cm ): 3151, 3077 (C–H), 2913–2853
163.80, 161.32, 152.91, 149.95, 133.30, 130.76, 126.68, 122.72,
122.63, 116.95, 116.71, 112.54, 109.34, 62.75, 56.04. Anal. calcd. for
C17 H14 FN O : C, 62.38; H, 4.31; N, 12.84. Found: C, 63.37; H, 4.40;
(
C–H), 1682 (C=O), 1598–1586 (C=C), 1470 (N = N), 1344 (C–N),
1
1
280- 1221 (C–O), 801 (=C–H). H NMR: (400 MHz, CDCl ) δ 9.88
3
3
3
(
s, 1H), 8.11 (s, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.48 (s, 1H), 7.46 (d,
N, 12.34.
J = 3.7 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 6.5 Hz, 1H),
5
.48 (s, 2H), 3.96 (s, 3H), 2.44 (s, 3H). 13C NMR: (100 MHz, CDCl )
3
2
2
.3. Biological assays
δ 190.96, 152.99, 149.93, 139.19, 134.67, 134.64, 130.66, 130.32,
26.80, 120.55, 112.52, 109.22, 62.88, 56.05, 21.12. Anal. calcd. for
C18 H17 N O : C, 66.86; H, 5.30; N, 13.00. Found: C, 66.38; H, 5.32;
1
.3.1. Evaluation of antioxidant activity
3
3
The total radical scavenging capacity of the tested compounds
was determined by the DPPH• scavenging method as per the re-
ported procedure compared to that of BHT, β-Carotene, Trolox
and ascorbic acid [26]. The solution of DPPH• was daily prepared,
stored in a flask coated with aluminum foil and kept in the dark
N, 12.06.
-((1-(4-chlorophenyl)−1H-1,2,3-triazol-4-yl)methoxy)−3-
methoxybenzaldehyde (5 g): m.p.: 120–121 °C. color: Yellow
4
−1
solid. Yield: 93%. FTIR (υ, cm ): 3137, 3076 (C–H), 3004- 2942
C–H), 1690 (C=O), 1588 (C=C), 1464 (N = N), 1346 (C–N), 1262-
(
at 4 °C. In brief, a fresh solution of DPPH• (0.1 mM) was prepared
3

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
in ethanol. Then, 1.5 mL of each compound in ethanol was added
an aliquot (0.5 mL) of this solution (6.25 – 200 μg/mL). These mix-
tures were mixed firmly and incubated in the dark for 30 min. Fi-
nally, the absorbance value was logged at 517 nm in a spectropho-
tometer [27].
used as the positive control, while cells not treated with 5a-h were
used as the negative control [31].
2
.3.4. α-Amylase enzyme inhibition activity
The stock solution of α-amylase enzyme purchased from Sigma
•
+
Aldrich was prepared in DMSO solvent with a concentration of
0 mg/10 mL. Reagent solution was used as control, and en-
ABTS
scavenging method is based on the ability of the
1
method described by Re et al. [26]. The ABTS solution (2 mM) in
zyme activity was checked at different concentrations (50, 100,
200 μg/mL) without test sample. Starch solution (0.5%) was pre-
pared by stirring and boiling starch (0.5 g) in deionized water
water with the oxidizing agent of potassium persulfate (2.3 mM)
•
+
yielded the ABTS cation radical (ABTS ), which was soluble in
both aqueous and organic solvents. It was diluted with phos-
phate buffer (0.1 mM, pH 7.4) to adjust inquired absorbance
(
100 mL) for 15 min. To prepare the enzyme solution, 100 mg
of the enzyme solution (1 unit/mL) was taken and mixed with
00 mL of 20 mM sodium phosphate buffer (pH 6.9). The color
(
0.700 ± 0.025) at 734 nm. Finally, tested samples solution (3 mL)
1
at various concentrations (6.25–200 μg/mL) has interacted with
•
+
reagent was prepared by mixing 2 M NaOH (8 mL), deionized wa-
ter (12 mL) and 96 mM 3,5-dinitrosalicylic acid (DNSA). Acarbose
(1 mg/mL) was used as reference. Test solution (25 μL) and en-
zyme solution (50 μL) were mixed in vials and incubated for half
an hour at 25 °C. Starch solution (50 μL) was added after incuba-
tion. It was then incubated for another 15 min. Color reagent (100
μL) was added to the mixture, and the mixture was heated in a
water bath at 85 °C for 15 min. The reaction mixture was then
cooled by removing it from the water bath, and absorbance values
at 540 nm were recorded. Measurements were made triplicate for
each sample. The control experiment was performed by replacing
the drug sample (25 μL) with DMSO [32]. Inhibition values were
calculated using the following equation: (Eq. 2).
ABTS
(1 mL) and the remaining absorbance was spectrophoto-
metrically recorded at 734 nm.
•
+
The capability to scavenge the DPPH• and ABTS
radical was
(1)
calculated using the following equation (Eq. 1).
ꢀ
ꢁ
Asample
Acontrol
RSE (%) = 1 −
× 100
where RSE is radical scavenging effects, AC is the absorbance value
of the control and AS is the absorbance value of the sample [28].
The half-maximal scavenging concentration of the sample (IC50
)
was determined from the graph plotted inhibition percentage
against all compounds concentrations (μg/mL) [29].
Cupric ions (Cu2 ) reducing power was used as the reduc-
+
ꢂ
ꢃ
ing ability method for compounds 5a-h. Cu2 reducing capa-
bility was performed according to the reported method [30].
For this purpose, aliquots of CuCl2 solution (0.25 mL, 0.01 M),
+
Aexample
%
Inhibition = Acontrol − Acontrol
× 100
(2)
ethanolic neocuproine solution (0.25 mL, 7.5 × 10− M), and NH Ac
3
4
2.3.5. Statistical analysis
buffer solution (0.25 mL, 1 M) were transferred to a test tube,
Statistical analysis was performed using SPSS Statistics for
Windows, statistical program SPSS software (version 20.0, SPSS,
Chicago, IL, USA. Duncan’s test was used to determine whether any
treatment significantly differed from controls or each other. Statis-
tical decisions were made with a significance level of 0.05.
which contains compounds 5a-h at different concentrations (6.25–
2
2
4
00 μg/mL). Total volume was completed with distilled H O to
2
mL and shaken. The absorbance of samples was recorded at
50 nm after 30 min.
2
.4. Cheminformatics studies
2
.3.2. Preparation of Hela cells culture
The human cervix Hela cell line was purchased from ATCC
The compound’s physicochemical and pharmacological prop-
(
American Type Culture Collection, Manassas, VA, USA). The cells
erties were calculated using the Molinspiration cheminformatics
online software [33] (Molinspiration Cheminformatics, SK 90,026
Slovensky Grob, SR) and swissADME [34] web tool to monitor
the compounds against the Lipinski RO5. Chemical structures and
smiles notations of the synthetic compounds were obtained by
BIOVIA Draw 2019 software. The smiles notations of compounds
were then fed in Molinspiration and swissADME to calculate many
molecular and ADME properties. Absorption (% ABS) of compounds
from the intestine was assessed by the following formula:
were grown and maintained in Dulbecco’s Modified Eagle Medium/
Nutrient Mixture F-12 (DMEM-F12) medium containing 10% fe-
tal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Life
Technologies GmbH, Germany). Cells were cultured in a humidified
atmosphere at 37 °C in 5% CO . The medium was refreshed every
2
two days.
2
.3.3. Measurement of cell growth by MTT assay
Anti-proliferative effects of 5a-h on Hela were determined by
MTT cell proliferation assay. MTT (3-[4,5-dimethylthiazol-2-yl]−2,5-
diphenyltetrazolium bromide) is a yellow auxiliary agent that re-
duced formazan crystals in living cells. MTT was purchased from
% ABS = 109 × (0.345 × TPSA)
2.5. Molecular docking studies
4
Sigma Chemical Company (St. Louis, Missouri, USA). 2 × 10 cells
were inoculated in each well of 96- well plates. The cells were in-
2.5.1. Ligand preparation for docking
cubated in the absence or presence of 5a-h (6.25,12.5, 25, 50, 100,
In this study, 3D structures for synthesized compounds were
sketched in drawing BIOVIA Draw 2019 software. All the com-
pound’s 3D structures were energy minimized using the MM2
minimization algorithm by Chem3D 20. All the minimized struc-
tures were converted into PDB or SDF file format before carrying
out molecular docking analysis. Molecular electrostatic potential
map calculations of compounds have been performed by utilizing
Schrödinger Maestro Release 2021–1 [35].
2
00, 400,800 μg/ml) for 48 h. After incubation periods, 20 μl of
MTT solution (MTT stock solution: 5 mg/ml in phosphate-buffered
saline) was added to each well, and the cells were incubated at
37 °C, 5% CO2 containing incubator for 3 h. Then, plates were cen-
trifuged at 1800 rpm for 10 min. The supernatant was removed,
and 150 μl DMSO (Sigma-Aldrich®, USA) was added to each well to
dissolve the formazan crystals. Finally, the absorbance values were
measured at 570 nm by a spectrophotometer [31]. The viability
was determined as the percentage of absorbance of 5a-h treated
cultures compared with untreated control cultures. In the exper-
iment, cells treated with 1% Triton X-100 (Sigma-Aldrich®) were
2.5.2. Receptor preparation for docking
The X-ray crystal structure protein, the Human pancreatic
alpha-amylase complexed with nitrite and acarbose (PDB code:
4

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
˚
2
QV4, 1.97 A), was retrieved from the RCSB protein data bank PDB’
(
http://www.rcsb.org/pdb). Water molecules, ions, heteroatoms,
and other ligands existing in the protein target were removed us-
ing Discovery Studio 2020 software. The downloaded structure of
the protein target was converted into PDB format for molecular
docking analysis.
2
.5.3. Molecular docking process
In silico docking studies, compounds were docked against the
QV4, and the docking scoring function was conducted using PyRx
2
docking (academic licensed version 0.9.8) software. In this analy-
sis, flexible-ligand:rigid-receptor docking was performed, and accu-
rate docking conditions were selected [36]. The interactions were
analyzed for the docking of ligands into active protein sites and
estimating the binding affinities of docked compounds. For the
generation of the grid, studies were executed using the PyRx
Autodock Vina wizard. The grid on the ligand-binding site of
the protein 2QV4 was centered at the binding site of X:12.385,
Y:48.136, Z:26.209, and the grid dimensions size were 25×25×25
Fig. 2. DPPH free radical scavenging activity of different concentrations (6.25-
200 μg/mL) of compound 5a-h and reference antioxidants; Trolox, BHT, β-Carotene
and ascorbic acid.
1H/13C NMR spectra of compounds were investigated, and the
resulting spectral data were given in the experimental section and
spectra were provided in supplementary documents (Fig. S15-S32).
The 1H/13C NMR signals of the triazole and phenyl rings are sim-
ilar in all compounds and the spectra of compound 5e were dis-
cussed as representative. In the 1H NMR spectrum of 5e (Fig. S25),
the methyl protons in the isopropyl group resonated as a doublet
at δ 1.30 ppm. Again, the -CH- proton in the isopropyl group res-
onated as heptet at δ 3.00 ppm. The methoxy group in the ben-
zaldehyde ring is singlet at δ 3.96 ppm, and the signal of etheric
˚
A
3
˚
dimensions with 0.375 A. The exhaustiveness value of the pro-
tein has been set at 8. Nine poses were estimated for each lig-
and/compound with the protein. The binding energies of nine
docked conformations of each ligand against the protein have been
recorded for further assessment and visualization. The results of
the best docking poses of the interactions were visualized and an-
alyzed using Discovery Studio 2020. The obtained scoring func-
tion (binding energies (kcal/mol)) gives a score based on the best-
docked ligand complex is picked out. The more negative the bind-
ing energy results from the interaction, the more stable the small
molecule and the complex formed by the protein [36].
methylene protons (–OCH -) is singlet at δ 5.48 ppm. Singlet signal
2
at δ 7.31 ppm and doublet at δ 7.47 (J = 10.7 Hz) ppm belong to
the 3 protons in the aldehyde substituted benzene ring. The dou-
blets observed at δ 7.39 (J = 8.2 Hz) and δ 7.66 (J = 8.4 Hz) ppm
are signals of protons in the isopropyl substituted benzene ring.
The -CH- proton in the triazole ring resonated as a singlet at δ
3
. Results and discussion
3
.1. Chemistry
8
.23 ppm. The singlet observed at δ 9.88 ppm belongs to the alde-
This study aimed to change the substituents in two different
hyde proton. In the 13C NMR spectrum of 5e (Fig. S25), the signals
regions of 1,2,3-triazole molecules containing aldehyde groups. The
H and -OMe groups have been changed in the aryl ring at position
, where it is in the aldehyde group relative to the triazole ring. On
of methyl carbon (2 x -CH ) atoms belong to the isopropyl group
-
3
are seen at δ 23.86 ppm. The signal observed at δ 33.85 ppm is the
resonance signal of the -CH- carbon atom in the isopropyl group.
The carbon atom signal of the methoxy group in the benzaldehyde
ring was observed at δ 56.06 ppm, and the signal belonging to the
4
i
the ring at position 1 -OMe, - Pr, Me, -Cl and -F groups have been
changed and the effect of the groups on the molecule has been
investigated.
carbon atom (O–CH ) at the place where the triazole ring was at-
This study aims to determine the effects of 1,2,3 triazole com-
pounds on antioxidant, anti-cancer, ADME, molecular docking, ac-
cording to functional groups. Azides (4a-e), propargyl compounds
2
tached to oxygen was observed at δ 62.79 ppm. At δ 120.79 ppm,
the signal for the -C-H carbon in the triazole ring was observed
[
39,40]. The signals of the aromatic carbon atoms and the carbon
(
2a-b) and 1,2,3-triazole compounds (5a-h) were synthesized in
atom in the triazole ring are observed in the range of δ 109.29–
high yields concerning our previous study [25]. All compounds ex-
cept compound 5e and 5 h were synthesized previously by Rao
et al. [37] and Bistrovic et al. [38] (Table S2). The melting points,
1H/13C NMR spectra of the compounds agree with the literature
value. The synthesized compounds structures were described by
elemental analysis, FT-IR, 1H/13C NMR (Scheme 1).
1
53.0 ppm. The carbonyl carbon of the aldehyde (-C=O) group was
observed at δ 190.90 ppm.
3.3. Antioxidant activity
Antioxidant properties, especially radical scavenging activities,
are significant due to removing the harmful effects of free radi-
3
.2. Structural characterization analysis of the compounds
•
•+
cals in biological systems. DPPH and ABTS
have been widely
Spectroscopic data of all synthesized triazole compounds are
used to analyze the free radical scavenging efficacy of various an-
tioxidant substances [41]. In this study, the antioxidant activity of
the newly synthesized compounds 5a-h and standard antioxidants
such as Trolox, BHT, β-Carotene and Ascorbic acid were deter-
given in the experimental section. FTIR spectra of all compounds
are given in Fig. S1-S14. The characteristic vibrational bands of the
N3 group in azide compounds and the alkyne group in propargyl
compounds were observed around ν 2100 cm 1. These bands van-
ish as a result of the “click reaction” of azide and propargyl com-
pounds. The vibrational band of the characteristic aldehyde group
−
mined using DPPH and ABTS
•
•+
methods. The samples were ex-
amined for their radical scavenging capabilities ranging between
6.25-200 μg/mL concentrations. Fig. 2 defines a significant decrease
•
(
HC=O) in propargyl and triazole compounds are detected as a
(P<0.05) in the concentration of DPPH radical due to the scaveng-
−
1
sharp peak around ν 1685 cm . The presence of characteristic
bands in the FT-IR spectra of the compounds confirms their struc-
ture.
ing ability of compounds and standards.
The scavenging effect of compounds 5a-h and standards on the
•
DPPH radical decreased in the order of Ascorbic acid > Trolox >
5

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Table 1
Table 2
Determination of half-maximal concentrations (IC50, μg/mL) of
Determination of reducing power of same
concentration (200 μg/mL) of compounds
compounds 5a-h and standards for DPPH• and ABTS•+ scaveng-
ing.
and standards by cupric ions (Cu2 ) re-
+
ducing capacity by Cuprac method.
•
•+
scavenging
DPPH scavenging
ABTS
Cu2 - Cu reducing
+
+
Compounds
IC50 (μg/mL)
IC50 (μg/mL)
Antioxidants
∗
λ
450
Ascorbic acid
Trolox
3.05± 0.001
6.76± 0.002
10.73± 0.001
9.74± 0.003
8.91± 0.004
8.92± 0.002
9.11± 0.001
9.31± 0.002
9.15± 0.005
9.48± 0.001
8.94± 0.003
9.59± 0.002
35.15± 0.007
19.20± 0.004
4.82± 0.001
4.78± 0.002
4.99± 0.005
5.98± 0.001
5.93± 0.003
5.96± 0.004
5.99± 0.002
6.01± 0.001
5.81± 0.002
5.91± 0.003
Trolox
0.1735 ± 0.002
0.1512 ± 0.001
0.0875 ± 0.001
0.9605 ± 0.002
0.0738 ± 0.001
0.0667 ± 0.002
0.0626 ± 0.001
0.0823 ± 0.004
0.0925 ± 0.002
0.0634 ± 0.004
0.0632 ± 0.003
0.0687 ± 0.005
BHT
BHT
β-Carotene
β-Carotene
Ascorbic acid
5
5
5
5
5
5
5
5
a
b
c
5a
5b
5c
5d
5e
5f
d
e
f
g
h
5 g
5
h
Fig. 4. Cytotoxic effects of 5a-h on Hela cells (p < 0.05) (Error bars represents stan-
dard deviations (SD), p < 0.05 was considered as significant).
Table 3
Fig. 3. ABTS radical scavenging activity of different concentrations (6.25–
00 μg/mL) of compound 5a-h and reference antioxidants; Trolox, BHT, β-Carotene
and ascorbic acid.
Anti-cancer activity of the com-
pounds against the HeLa cell line.
2
Comp.
MTT IC50 (μg/mL)
5
5
a
77.73± 0.003
75.02± 0.005
50.12± 0.002
129.12± 0.003
112.71± 0.002
1394.12± 0.001
181.87± 0.004
57.07± 0.005
16.30± 0.003
5
a > 5b > 5d > 5 g > 5c > 5e > 5f > 5 h > BHT > β-Carotene
b
which were 94.95%, 94.41%, 70.63%, 70.12%, 70.06%, 69.87%, 68.55%,
5c
6
2
8.55%, 65.92%, 65.17%, 64.96%, 63.23% at the concentration of
00 μg/mL, respectively. Free radical scavenging activity of these
5d
5
5
5
e
f
samples also increased with an increasing concentration.
g
•
IC50 values of DPPH radical scavenging are as shown as
5h
•
Table 1. A lower IC50 value indicates a higher DPPH free radical
Cis-platin
•
scavenging effect (Table 1). The DPPH activity of 5a-h are higher
than BHT and β-Carotene standards.
All tested compounds exhibited effective radical scavenging ac-
tivity against ABTS•+ radicals (p>0.001). As seen in Fig. 3, all com-
compound 5e showed higher activity than β-Carotene. At the same
concentration (200 μg/mL), reducing capacities decreased in the or-
der of Ascorbic acid > Trolox > BHT > 5e > β-Carotene > 5d >
5a > 5 h > 5b > 5 g > 5f > 5c.
pounds had effective ABTS+ radical scavenging activity in a con-
centration dependent manner (6.25 - 200 μg/mL). The scavenging
effect of compounds and standards on the ABTS•+ radicals de-
creased in the order of BHT > β-Carotene > 5a > 5 g > 5 h >
3.4. Anti-cancer activity
5
9
6
c > 5d > 5b > 5e > 5f > Ascorbic acid > Trolox, which were
9.31%, 99.17%, 86.62%, 68.31%, 67.97%, 66.75%, 64.93%, 64.91%,
3.98%, 63.98%, 35.47%, 31.99% at the concentration of 200 μg/mL,
The synthesized compounds 5a-h were screened for cytotoxic
activity against the Hela cell line. Hela cells were treated with in-
creasing concentrations of 5a-h for 48 h, and MTT cell proliferation
assays were carried out to determine the anti-proliferative effects
of the agent on these cells [42]. The cells were exposed to com-
pounds 5a-h from 6.25 to 800 μg/ml. Cis-platin, one of the most
effective anti-cancer agents, was used as the positive control. The
results showed that there were dose-dependent decreases in cell
proliferation compared to untreated controls (p < 0.05) (Fig. 4.)
IC50 values for compounds 5a-h were given in Table 3.
respectively. Free radical scavenging activity of these samples also
increased with an increasing concentration.
IC50 values for compounds 5a-h and standards are shown
Table 1. As with the DPPH• method, a lower IC50 value indicates
a higher ABTS•+ radical scavenging ability. All compounds showed
+
better ABTS• radical scavenging ability than Ascorbic acid and
Trolox standards. Especially compound 5a has almost the same ac-
tivity as BHT and β-Carotene.
The values of the CUPRAC method, in which the chromogenic
neocuproine was used as the oxidizing agent, are shown in Table 2.
The highest CUPRAC value was observed in the ascorbic acid. The
All compounds showed anti-cancer activity. The IC50 values
of compounds 5a-h are as follows, 77.73, 75.02, 50.12, 129.12,
112.71, 1394.12, 181.87, 57.07, respectively. Especially when the IC50
6

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Table 4
more specific expression, a complex balance of different molecu-
lar properties and structural features that decide whether a spe-
cific molecule is comparable to known drugs can be described as
drug similarity. These properties primarily influence any living or-
ganism’s molecular behaviors, including hydrophobicity, electronic
dispersion, hydrogen bonding properties, molecular size, flexibility,
and the existence of different pharmacophoric properties, bioavail-
ability, protein affinity, reactivity, toxicity, metabolic stability, and
transport properties.
α-amylase inhibition IC50 values of
synthesized compounds.
Comp.
IC50 (μg/mL)
5
5
5
5
5
5
5
5
a
b
c
d
e
f
1471± 0.003
840± 0.008
1391± 0.006
315± 0.005
239± 0.001
248± 0.003
165± 0.005
248± 0.001
891
g
Molecular properties such as partition coefficient (Log Po/w),
topological polar surface area (TPSA), hydrogen bond donors and
acceptors, rotatable bonds, number of atoms, molecular weight,
and violations of Lipinski’s rule of five were calculated to assess
the drug-likeness of the compounds and represented in Table 5.
The Lipinski rules recommend no more than one violation of an
orally bioactive drug or drug candidate. ADME data showed that all
values calculated for synthetic compounds fell within anticipated
ranges as described in Lipinski’s rule of five. All synthesized com-
pounds fully complied with the Lipinski rules and did not exhibit
any violations of these rules.
h
Acarbose
values
aromatic ring, were examined, it was seen that they had moder-
ate activity against Hela cell line compared to cis-platinum (IC50
6.30 μg/mL).
of 5c and 5 h, which have a fluorine group in 1-position
:
1
3
.5. α-Amylase enzyme activity
α-Amylase enzyme inhibition is of great importance in the
Bioavailability radar chart screened for compounds for prompt
assessment of drug similarity. This radar chart illustrates six dif-
ferent physicochemical properties: lipophilicity, size, polarity, sol-
ubility, flexibility, and saturation. On each axis, a physicochemical
range is depicted as a pink area, defined by default with identi-
fiers adapted from SwissADME, which must fall on the radar plot
of the molecule to be considered fully drug-like. Consistent with
this model, all compounds exhibited a high oral bioavailability pro-
file, as seen in the bioavailability radar charts (Fig. 5). On the other
treatment of diabetes, as it delays glucose absorption. One way to
reduce high sugar in the blood (hyperglycemia) is to delay the di-
gestion of carbohydrates in the intestine through digestive enzyme
inhibitors; it is envisaged as a new solution for the treatment of
diabetes. Acarbose is used as an α-amylase inhibitor in the liter-
ature [43,44]. The α-amylase activity of the compounds synthe-
sized in this study was determined using the 3,5-dinitrosalicylic
acid (DNSA) method. Results were compared with acarbose used
as standard (Table 4). The α-amylase inhibition IC50 values of the
compounds are as follows, 1471, 840, 1391, 315, 239, 248, 165,
hand, if the topological polar surface area (TPSA) values are greater
than 130 A˚ 2, the compound’s oral bioavailability is considered low.
The TPSA value is between 57.01 and 75.47 for all compounds.
The compounds BOILED-Egg diagram is shown in Fig. 6. The
BOILED-Egg is the main feature of this diagram, which is an in-
tuitive way for predicting two major ADME characteristics at the
same time: passive gastrointestinal absorption (HIA) and brain ac-
cess (BBB: Blood-Brain Barrier). Although this classification model
is conceptually basic because it only uses two physicochemical de-
scriptors (WLOGP and TPSA, for lipophilicity and apparent polarity,
respectively), it was designed with great care in terms of statis-
tical significance and robustness. BOILED-Egg diagram consists of
gray and yellow area. Both compartments are not one another ex-
clusive. The outside gray area stands for molecules with properties
implying predicted low absorption and limited brain penetration.
In practice, the BOILED-Egg diagram has proven straightforward in-
terpretation and efficient translation to molecular design in vari-
ous drug discovery studies [34]. We can conclude from the red dot
position for our compounds that BBB penetration and GI absorp-
tion property are positive, and the PGP effect on the molecule is
negative. All molecules are positioned within the range within the
specified limits.
2
48 from 5a to 5 h, respectively. In general, compounds with a
methoxy (–OCH ) group on the aromatic ring at the 4-position
3
(
5d-h), showed higher inhibitory activity compared to the com-
pounds without the methoxy group (5a-c). In particular, compound
g, which has a methoxy (–OCH ) group in the 4-position aro-
5
3
matic ring and a chlorine group in the 1-position aromatic ring,
exhibited the highest activity. Also, compound 5 g has about 5.5
times higher activity than the standard acarbose (IC50: 891) [45].
3
.6. In-silico adme results
ADME data improves the selection and identification at the
therapeutic dose of molecules with an optimal safety profile, to-
gether with the drug discovery process, instead of the final phase.
This assessment prevents waste of time and valuable resources on
medication molecules that are disposable over time.
Also known as Pfizer’s rule of five or merely the rule of five
(
RO5), Lipinski’s five rules are a practical rule for assessing drug
similarity or figuring out whether a chemical compound with
a particular pharmacological or biological activity has favorable
chemical properties and physical properties which would make it
an orally active drug in humans. Christopher A. Lipinski formulated
the rule [46] based on the observation that most orally adminis-
tered drugs are relatively small to moderately lipophilic molecules.
The rule describes the molecular properties essential to the hu-
man body’s pharmacokinetics, including their absorption, distribu-
tion, metabolism, and excretion (ADME). The rule states that if a
ligand molecule violates Lipinski’s rule 5, it has more than 5 hy-
drogen bond donors, the molecular weight is above 500, the log P
is above 5, and N and O atoms are more than 10. As a result, in-
adequate absorption or permeability is more likely for compounds
and drug candidates. Drug likeness is a qualitative term about how
a chemical compound or drug candidate molecule is “drug-like” re-
garding factors such as bioavailability used in drug design. With a
In order to elucidate the qualitative structure-activity relation-
ships of synthetic compounds (5a-h), physicochemical properties
and calculations were carried out using molinspiration chemin-
formatics software. A molecule’s lipophilic character be subject
to two critical factors, i.e., hydrophobicity and polarity, which
assist the molecule cross or irreversibly detriment the cellular
membrane. The molecular lipophilicity potential (MLP) map and
polar surface areas (PSAs) of synthetic compounds are given in
Fig. S33.
In the present study, compounds 5a, 5b and 5c (PSA:57.01) were
the most active, followed by compounds 5e, 5f, 5 g, 5 h and 5d
with PSAs of 66.24, and 75.47, respectively. On the other hand,
compounds 5a, 5b, and 5c showed similar PSAs, while compound
5e showed a greater lipophilic area than compounds 5b and 5 g.
Similarly, although the PSA of compound 5d was higher than that
7

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Table 5
Physicochemical and drug-likeliness properties of synthetic compounds (5a-h) according to the rule of five.
MW
%Abs
TPSA( A˚ ²)
RB
HBA
HBD
Rules
≤ 5
Log Po/w
LV
BS
SA
WS
Comp.
<
500
>80
<130
≤ 9
≤ 10
≤ 5
≤ 1
–
–
–
5
5
5
5
5
5
5
5
a
b
c
293.32
313.74
297.28
339.35
351.40
323.35
343.76
327.31
89.33
89.33
89.33
82.96
86.15
86.15
86.15
86.15
57.01
57.01
57.01
75.47
66.24
66.24
66.24
66.24
5
5
5
7
7
6
6
6
4
4
5
6
5
5
5
6
0
0
0
0
0
0
0
0
2.67
2.90
2.65
2.30
3.27
2.66
2.83
2.63
0
0
0
0
0
0
0
0
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
2.56
2.49
2.42
2.82
2.96
2.78
2.70
2.64
−3.53
−3.80
−3.25
−3.40
−4.48
−3.69
−3.96
−3.50
d
e
f
g
h
Key: MW: Molecular weight (gr/mol),%Abs: Percentage of absorption, TPSA: Topological polar surface area, RB:
number of rotatable bonds, HBA: number of hydrogen bond acceptors, HBD: number of hydrogen bond donors,
LV: number of Lipinski rule of 5 violations, Log Po/w (iLOGP): Lipophilicity, BS: Bioavailability score, SA: Synthetic
accessibility [From 1 (very easy) to 10 (very difficult)], WS: Water solubility, Log S (Insoluable < −10 < Poorly <
−6 < Moderately < −4 < Soluable < −2 Very < 0 < Highly).
Fig. 5. Bioavailability radar related to physicochemical properties of molecules. The pink area symbolizes the ideal range for each properties (lipophilicity: (Criteria’s:
Lipophilicity: - 0.7 < XLOGP3 < ++5.0, Size: 150 MW 500 g/mol, Polarity: 20 < TPSA < 130 A˚ 2, Insolubility: 0 < log S < 6, Insaturation, Flexibility: 0.25 < rotatable
bonds < 9). POLAR (polarity), LIPO (lipophilicity), INSOLU (solubility), FLEX (flexibility), and INSATU (saturation).
of other compounds, their MLP maps were almost the same apart
from compound 5e. In the light of the qualitative data obtained
from PSA and MLP maps, it is possible to say that there is a com-
mon correlation between the structure-activity relationship, con-
sidering the biological activity tests performed. It is seen that these
results also overlap with the data obtained from molecular docking
studies.
ters can be an essential tool to predict and interpret the molec-
ular structure of the investigated drugs and their physicochemi-
cally structure-activity and property relationships. Moreover, the
MEP is a helpful tool for predicting drug reactivity to electrophilic
and nucleophilic attacks [48-49]. The MEP of the total 8 com-
pounds within the study’s scope was calculated using the Adaptive
Poisson-Boltzmann Solver (APBS) method and is given in Fig. 7.
The maximum negative region in the MEP is the favored site for
the electrophilic attack, as indicated by the red color. As a re-
sult, an attacking electrophile will be drawn to the negatively
charged sites, while the blue regions will be saturated in the re-
verse direction [50]. Electrostatic potential increases are identi-
fied in the colors in red < orange < yellow < green < blue. Be-
cause the receptor and the corresponding ligands recognize each
other at the molecular surfaces, the electrostatic potential value is
mainly responsible for binding a substrate to the receptor bind-
ing sites. As a result, small red, yellow-orange, and blue patches
on the larger green surface of compounds balance the hydrophilic
and hydrophobic parts required for good quality protein-enzyme
binding and interaction. The electrostatic potential scales of
3
.7. Molecular electrostatic potential (MEP)
In such studies, it is essential to see the electrophilic and
nucleophilic regions of the studied compounds before starting
the molecular docking process to make a preliminary predic-
tion and interpretation of which possible atoms or functional
groups may interact with the relevant receptors. Calculation of
the molecular electrostatic potential (MEP) plays an important
role in drug research and development studies to verify the ev-
idence for the reactivity of molecules as inhibitors [46,47]. Al-
though MEP indicates the molecular size and shape of the pos-
itive, negative, and neutral electrostatic potential, these parame-
8

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Fig. 8. Validation of molecular docking protocol.
5
a-h compounds were calculated in the range between −4.951 and
−
4.951.
3
.8. Molecular docking results
Fig. 6. The BOILED-Egg ADME diagram of the compounds (5a-h) WLOGP vs. TPSA.
(Points located in the BOILED-Egg’s yolk (yellow) signify the molecules predicted to
passively permeate through the blood-brain barrier (BBB), whereas the ones in the
egg white are relative to the molecules predicted to be passively absorbed by the
gastrointestinal tract. The red ones point out to the molecules predicted not to be
effluated from the CNS (Central Nervous System- by the P-glycoprotein). Yolk and
white regions are not reciprocally exclusive.
3.8.1. Validation of docking protocol
The molecular docking procedure was validated by assessing
the binding poses of ligands in the protein-ligand complex crystal
structure of the 2QV4 protein, which we used in the study. In the
structure of Human pancreatic alpha-amylase (PDB ID: 2QV4), co-
ligand acarbose (native ligand) is covalently linked to the receptor
residues HIS-101, VAL-107, ALA-106, ASN-105, GLY-164, THR-163,
HIS-299, TYR-62, ASP-300, ARG-195, GLU-233, GLN-63 and TRP-59.
Therefore, in docking procedures, the amylase’s receptor grid was
selected by limiting it to the residues occupied by the acarbose
ligand and interacting with the ligand area. For this process, the
ligand (acarbose) in the X-ray crystal structure of the amylase en-
zyme was removed from the structure and re-docked to the pro-
tein’s binding site. In the next step, the ligand’s pose in the en-
zyme’s crystal structure was compared with the pose obtained by
redocking, and the mean square deviation (RMSD) value was cal-
culated. According to the results, the co-crystallized ligand showed
similar orientations and conformational poses as in the crystal
structure when re-docked at the active binding sites of the tar-
get enzyme. The ligand in the crystal structure of amylase and the
re-docked ligand were almost superimposed. We found that the
RMSD value between crystal structure pose and re-docked acar-
˚
bose pose in active α-amylase residues was 0.7980 A (Fig. 8). Ac-
cordingly, since the calculated RMSD value was significantly lower
˚
than the maximum allowable value of 2.0 A, it showed that the
docking procedure performed in this study was valid.
3
.8.2. Docking analysis results
The investigation of the binding interaction mechanisms of
biomacromolecules with small molecules such as drugs or drug
candidates has been caused a significant rise in molecular dock-
ing studies. In addition to being a powerful tool in rational drug
design, molecular docking may predict the most stable structure
and mode of interaction of receptor-ligand complex formation and
allows for proper identification and investigation of interaction in-
formation during the production of new drugs [36].
This study investigated the affinity of 8 compounds we syn-
thesized against human pancreatic α-amylase enzyme (HPA). The
results of molecular docking on α-amylase are given in Table 6.
The binding affinities obtained from the molecular docking re-
sults show that all compounds have
the active site of α-amylase. These values ranged from −8.9 to
8.2 kcal/mol for all compounds, with the highest binding affin-
ity being −8.9 kcal/mol for compound 5a. In general, GLU233 and
a high affinity towards
Fig. 7. The molecular electrostatic potential (MEP) surface of compounds.
−
9

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Table 6
Interaction and amino acid residues involved in the inhibition of a-amylase enzyme summary.
Comp.
Binding affinity (kcal/mol)
Type of interaction bond
Involved receptor residues
5
5
5
5
5
5
5
5
a
b
c
−8.9
−8.7
−8.7
−8.4
−8.3
−8.4
−8.2
−8.6
HB, EC, HP
HB,EC,HP
HB, HP
ALA198, GLN63, GLU233, TYR62, LYS200, ILE235, HIS201, TYR151, LEU162
ALA198, HIS201, GLU233
GLN63, LEU165, ILE235, HIS201, LEU162, ALA198,
d
e
f
HB, EC, HP
HB, EC, HP
HB, EC, HP
HB, HP
GLN63, LYS200, GLU233, ASP197, ILE235, TYR62, HIS201, LEU165, TRP59, LEU162, ALA198
HIS305, ASP197, GLU233, ASP300, ILE235, TRP59, LEU165, LEU162
GLU233, ASP197, ASP300, ASP197, ILE235, HIS201, LYS200, TYR151, ALA198, LEU162
GLN63, THR163, LEU162, ILE235, HIS201, LYS200, ALA198, LEU165
TRP59, ASP197, HIS299, ASP300, TYR62, LEU165
g
h
HB, HL, HP
Key: HB:hydrogen bond; C–HB: Carbon-Hydrogen Bond, EC:electrostatic interaction, HP:hydrophobic interaction, HL:halogen bond.
Fig. 9. 3D depiction of 5a compound which has maximum docking score with α-amylase.
ASP 300 residues play a role in the main interactions and bonds
in all α-amylase-compounds (5a-h) complexes. These two residues
are known to act as catalytic residues in the hydrolytic reactions
of α-amylase [32]. The data showing the bonds, bond types, and
bond lengths formed between the α-amylase and compounds are
presented in detail, Supporting Table 1. For all molecules, pre-
dominantly hydrophobic interactions were responsible for anchor-
ing at the active site of α-amylase in the formation of stable
enzyme-compound complexes. This phenomenon can be explained
by the predominance of hydrophobic bonding types in the com-
plexation interaction with the active site of the α-amylase, as it
will be associated with the low and moderate lipophilicity of the
compounds, as can be seen from the lipophilicity potential of the
molecules.
atom in the aldehyde group attached to the benzene ring in com-
pound 5a, C–H bond was formed over the second nitrogen atom in
the triazole ring with the ALA 198 residue.
We noted that the interaction between the TRP 59 residue of
the active site of 5b and α-amylase in the 5b-enzyme complex is
hydrophobic interactions in the Pi-Pi Stacked (triazole moiety) and
Pi-Alkyl (-Cl atom) type. In this strong interaction, we can see that
ALA198 and HIS201 residues form a stable complex with the H-
donor and H-acceptor over the oxygen atom in the aldehyde group
attached to the benzene ring. Similarly, an electrostatic Pi-Anion
interaction has occurred between the benzene ring to which the
aldehyde group is attached and the GLU 233 residue. For the α-
amylase-compound complexes, all the docked model illustrations
are given in detail in Fig. 10.
The 3D representation of the complex formed between 5a and
α-amylase is given in Fig. 9, indicating the chemical bonding mode
and bond types. This complex structure (5a- α-amylase) is formed
between twelve different amino acid residues of α-amylase and
When examining the results of molecular docking for com-
pound 5c, it is seen that the resulting complex is primarily due
to hydrophobic interactions. Four different Pi-Alkyl bonds were
formed from the hydrophobic interactions from 5c to the residues
LEU162 and ALA198 via the triazole and the benzene ring attached
to this group. In addition, Pi-sigma interactions were established
with ILE235 and LEU165 residues over the same benzene ring and
the benzene ring attached to the aldehyde group, respectively. The
occurrence of Pi-sigma interactions is generally thought to be due
to hyperconjugation or bending (tilting) of the molecular structure.
In compound 5d, the catalytic residue GLU233 formed a C–H
bond interaction with the etheric methylene group attached to
the triazole ring. In the triazole moiety of the compound, ASP197
formed an electrostatic interaction with the Pi-anion bond, and
TYR62 formed a Pi-Pi stacked interaction. In the ring to which
the aldehyde group is attached, the residues of LEU162, ALA198,
5
a atoms. It can be spotted from Fig. 9 that predominantly hy-
drophobic interactions are responsible for anchoring of compound
a. Nine different hydrophobic interactions occurred through Alkyl,
5
Pi-Alkyl, Pi-Pi Stacked and Pi-Pi T-shaped bonding types. In the Pi-
Alkyl binding modes, the direction of the interaction was formed
from the compound 5a to the ALA198, LEU162 and ILE235 residues
via the Pi-Orbitals, while in TYR151 and HIS201 it was formed
from the residues to 5a. Similarly, 5a formed Pi-interactions (Pi-
Pi stacked, Pi-Pi T-shaped Pi-anion, and alkyl) with amino acid
residues GLU233, TYR62, HIS201, ILE235, and LYS200. In addition,
between the H bonds formed in the structure; While the conven-
tional H bond was formed between GLN63 residue with the oxygen
10

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
Fig. 10. 2D representations of the best pose interactions between the α-amylase and compounds.
11

I˙ . S¸ ahin, M. Çe s¸ me, F.B. Özgeri s¸ et al.
Journal of Molecular Structure 1247 (2022) 131344
HIS201, and ILE205 have interacted over the bonds Pi-Sigma, Pi-Pi
Stacked, Pi-Pi T-shaped, Alkyl, Pi-Alkyl.
BBB (Blood Brain Barrier) penetration and GI (gastrointestinal) ab-
sorption properties were favorable. Furthermore, molecular dock-
ing study was performed to predict possible interaction modes and
binding energies of the compounds for the active site of α-amylase.
Thus, suggesting that compounds from the present series 5a-h
can be further optimized and developed as lead molecules.
In addition to hydrophobic interactions in compound 5e, elec-
trostatic interactions came to the fore a little more. As in com-
pound 5d, the catalytic residue GLU233 exhibited an electrostatic
interaction with the ring to which the methyl groups were at-
tached via the Pi-Anion bond. The triazole ring is also electro-
statically linked with residues ASP197 and ASP300 via the Pi-
Anion bond. While HIS 305 interacted by forming an H bond
over the aldehyde group oxygen, other moiety interactions of the
molecule occurred through hydrophobic bonds (Pi-Sigma, Pi-Pi
Stacked, Alkyl, and Pi-Alkyl) with residues ILE235, TRP59, LEU165,
and LEU162.
Declaration of Competing Interest
The authors declare no conflicts of interest.
CRediT authorship contribution statement
Compound 5f interacted with the catalytic residue GLU233 both
by H bond and electrostatically forming a salt bridge. The salt
bridge formed is due to the 1,3-dipolar structure of the triazole
ring in which the nitrogen atom (middle one) has higher elec-
tron density resulting in dipole-dipole interactions with ALA198,
GLU233, ASP197 and ASP300 residues. The other catalytic residue,
ASP300, on the other hand, formed an electrostatically attractive
charge interaction over the benzene ring in the aldehyde group
and the positively charged nitrogen atom in the triazole moiety.
While the residue ASP197 interacted with the etheric methylene
group attached to the triazole ring via the C–H bond, it also inter-
acted electrostatically with the nitrogen atom in the triazole ring.
The methyl group has attached TYR151, TYR152, LEU162, ALA198,
HIS201, and ILE235 in the ring to which the methyl group has
formed hydrophobic interactions through the bonds Pi-Sigma, Pi-
Pi Stacked, Pi-Pi T-shaped, Alkyl, and Pi-Alkyl.
˙
Irfan S¸ ahin: Visualization, Investigation, Writing – review &
editing. Mustafa Çe s¸ me: Visualization, Investigation, Writing – re-
view & editing. Fatma Betül Özgeri s¸ : Visualization, Investigation,
Writing – original draft. Özge Güngör: Visualization, Investigation.
Ferhan Tümer: Supervision, Project administration, Methodology,
Writing – review & editing.
Acknowledgments
˙
The authors thanks Kahramanmara s¸ Sütçü Imam University
Scientific Research Projects Unit (Project code: BAP-2019/3–20 M)
(
Turkey) for funding. Furthermore, we would like to thank the
Higher Education Council Research Board (YÖK 100/2000 PhD.
Scholarship Priority Areas Program “Molecular Pharmacology and
˙
Drug Research”) for granting the Scholarship (to Irfan S¸ ahin).
When the complex structure formed for the 5 g compound is
examined; The residues in the active site of the enzyme showed
a dominant binding tendency through the triazole moiety of the
molecule, the benzene ring attached to this group, and the Cl
atom in the ring. The resulting stable structure provided the Pi-
Alkyl interaction with the residues ALA198, LEU165, LEU162 and
ALA198 through the triazole region and the benzene ring attached
to this group. The Cl atom is attached to the residues by hydropho-
bic Alkyl interaction with LYS200 and HIS201. While the GLN63
residue interacted by forming an H bond over the aldehyde group
oxygen, the THR163 residue formed an H bond with the oxygen in
the methoxy group in the same ring.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131344.
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