Synthesis, characterization, α-glucosidase inhibition and molecular modeling studies of some pyrazoline-1H-1,2,3-triazole hybrids

Article abstract of DOI:10.1016/j.molstruc.2020.128253

A series of molecular hybrids based on pyrazoline and 1,2,3-triazole pharmacophores were designed and synthesized as antidiabetic agents. The structures of all the derivatives were confirmed using ¹H NMR, ¹³C NMR and HRMS. Moreover, the structure of one of the intermediate precursor was confirmed using single crystal X-ray diffraction. The anti-diabetic potential of all the synthesized compounds was explored in terms of α-glucosidase inhibition studies. All the compounds exhibited remarkable inhibition of α-glucosidase. The inhibition of enzyme by compound TPZ2 (IC₅₀ = 41.29 ± 0.123) and TPZ8 (IC₅₀ = 47.94 ± 0.246) was found to be more promising as compared to the reference drug i.e., Acarbose (IC₅₀ = 60.68 ± 0.123). The inhibition of α-glucosidase was further supported by in silico docking studies. In order to explore the most favorable binding interactions the binding pose of lowest energy was then subjected to molecular dynamics studies. Both the ligands have reasonable interactions with the protein active site with average interaction energy of ?270.88 kcal/mol and ?273.90 kcal/mol for TPZ8 and TPZ2, respectively.

Full text of DOI:10.1016/j.molstruc.2020.128253

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Synthesis, characterization, α-glucosidase inhibiton and molecular modeling studies
of some pyrazoline-1H-1,2,3-triazole hybrids
Lokesh Kumar, Kashmiri Lal, Pinki Yadav, Ashwani Kumar, Avijit K. Paul
PII:
S0022-2860(20)30578-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.128253
MOLSTR 128253
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 February 2020
Revised Date: 10 April 2020
Accepted Date: 11 April 2020
Please cite this article as: L. Kumar, K. Lal, P. Yadav, A. Kumar, A.K. Paul, Synthesis, characterization,
α-glucosidase inhibiton and molecular modeling studies of some pyrazoline-1H-1,2,3-triazole hybrids,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128253.
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Credit author statement
Lokesh Kumar: Validation, Formal analysis, Investigation Writing - original draft, Writing
review; Kashmiri Lal: Conceptualization, Methodology, Resources, Data curation,
Supervision; Pinki Yadav: Methodology, Data curation, Visualization; Ashwani Kumar:
Software, Data curation. Avijit K. Paul: Single crystal analysis, Investigation.

Synthesis, Characterization,
α-Glucosidase inhibiton and Molecular
Modeling studies of some pyrazoline-1H-1,2,3-Triazole hybrids
Graphical Abstract

Synthesis, Characterization,
α-Glucosidase inhibition and Molecular
Modeling studies of some pyrazoline-1H-1,2,3-Triazole hybrids
Lokesh Kumar,1 Kashmiri Lal,1,* Pinki Yadav,1 Ashwani Kumar,2 and Avijit K. Paul3
1Department of Chemistry, Guru Jambheshwar University of Science & Technology, Hisar, Haryana 125001, India
2Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology, Hisar, Haryana
125001, India
3Department of Chemistry, National Institute of Technology, Kurukshetra, Haryana 136119, India
*Corresponding author: klal_iitd@yahoo.com (Kashmiri Lal)
ABSTRACT
A series of molecular hybrids based on pyrazoline and 1,2,3-triazole pharmacophore were
designed and synthesized as antidiabetic agents. The structures of all the derivatives were
1
confirmed using H NMR, 13C NMR and HRMS. Moreover, the structure of one of the
intermediate precursor was confirmed using single crystal X-ray diffraction. The anti-diabetic
potential of all the synthesized compounds and their precursors was explored in terms of α-
glucosidase inhibition studies. All the compounds exhibited remarkable inhibition of α-
glucosidase. The inhibition of enzyme by compound TPZ2 (IC50= 41.29±0.123) and TPZ8
(IC50=47.94±0.246) was found to be more promising as compared to the reference drug i.e.,
Acarbose (IC50=60.68±0.123). The inhibition of α-glucosidase was further supported by in silico
docking studies. In order to explore the most favorable binding interactions the binding pose of
lowest energy was then subjected to molecular dynamics studies. Both the ligands have
reasonable interactions with the protein active site with average interaction energy of -270.88
Kcal/mol and -273.90 Kcal/mol for TPZ8 and TPZ2, respectively.
Keywords: 1,2,3-Triazole, Pyrazoline, α-glucosidase, Docking, Molecular dynamics
1
Introduction
Diabetes mellitus is one of the most common and serious diseases caused by high blood glucose
level worldwide [1]. As per W.H.O reports, occurrence of diabetes in adult has increased 4.7% in
1980 to 8.5% in 2014 [2, 3]. Diabetes caused 1.6 million deaths in 2016 worldwide and would be
1

largest cause of death by 2040 [4, 5]. Most of the people with diabetes are diagnosed with type 2
diabetes which is non-insulin dependent. Type 2 diabetes is typically a polygenic disease that
results from a complex interplay between genetic predisposition and environmental factors such
as diet, degree of physical activity, and age.
α-Glucosidase is a key enzyme responsible for digestion of carbohydrates and
biosynthesis of glycoproteins and plays a significant role in the treatment of degenerative
diseases including type 2 diabetes. α-Glucosidase inhibitors can reduce the complications of
diabetes by decreasing the liberation of glucose in the bloodstream via interfering with the
enzymatic action of intestinal α-glucosidase [3]. Currently used drugs are associated with some
disadvantages including flatulence, diarrhea and low efficacy. To combat this problem, there is
an urgent need to develop novel alternative, safe and potent drugs with a broader spectrum of
anti-diabetic activity.
Pyrazolines represents a privileged structural class containing five membered rings with
two nitrogen atoms and a double bond. Pyrazolines are even more special due to their marked
physiological and pharmacological activity. This class is associated with numerous
pharmacological activities such as antidiabetic [6], antimicrobial [7, 8], antimalarial [9],
antitumor [10], antitubercular [11], anticancer [12, 13], Monoamine oxidase inhibitors [12], anti-
inflammatory [14-16], antidepressant [17] and anticonvulsant [18]. Similarly, 1,2,3-triazole also
represents a well-established five membered heterocyclic scaffold which has gained significant
attention in the drug-discovery field since the introduction of the “click” chemistry concept by
Sharpless [19]. Due to active participation in hydrogen bonding and dipole-dipole interactions
1,2,3-triazoles are extremely stable to hydrolysis and oxidative/reductive conditions. Moreover,
contrary to other aza-heterocycles, 1,2,3-triazole are less basic owing to which their protonation
at physiological pH is not feasible. 1,2,3-Triazoles are associated with diverse biological
activities including sensing [20], anticancer [21], antiHIV [22], antimicrobial [23-27], antiviral
[28], antiproliferative [29] and insecticides [30]. Moreover, 1,2,3-triazole namely benzotriazole
is also used as one of the reagent to synthesize peptide and peptide conjugates [31, 32].
Recently, molecular hybridization approach has been explored in medicinal chemistry
[33]. This approach enables the synthesis of molecular hybrids by joining two or more
biologically active compounds. These hybrid molecules are supposed to have high efficacy and
novel mechanism of action in comparison to their parent pharmacophoric units. There are very
2

few reports on hybrid containing 1,2,3-triazole showing α-glucosidase inhibition. For instance,
Mina Saeedia et al. synthesized quinazolinone-1,2,3-triazole hybrids compounds and reported
their alpha-glucosidase inhibitory activity [34]. All the synthesized compounds exhibited good
inhibitory activity against yeast α-glucosidase (IC50 = 181.0-474.5 μM) and were found to be
more potent than standard drug acarbose (IC50 = 750.0 μM). Among them, quinazolinone-1,2,3-
triazoles possessing 4-bromobenzyl moiety connected to 1,2,3-triazole ring demonstrated the
most potent inhibitory activity towards α-glucosidase. Likewise, Avula et al. also synthesized
(R)-4-fluorophenyl-1H-1,2,3-triazole derivatives with remarkable contribution in the overall
activity (Figure 1) [35].
Figure 1: Structures of some hybrids triazoles showing α-glucosidase inhibition
Based on above observations, we planned that molecular hybrids containing 1,2,3-
triazole and a pyrazoline moiety may act as good inhibitors of glucosidase enzyme. Therefore, on
the basis of above cited and our efforts towards the development of novel 1,2,3-triazole based
hybrid molecules [36-42], herein, we have designed and synthesized some 1,2,3-triazole-
pyrazoline hybrids as α-glucosidase inhibitors.
3

2
Results and Discussion
2.1 Chemistry
Synthesis of target hybrid compounds was carried out in various steps as presented in scheme 1
and 2. Firstly, propargylated aldehydes (1a-1b) prepared using reported procedure [43] were
subjected to Claisen-Schmidt reaction under microwave with 4-methoxy and 4-
bromoacetophenone in presence of NaOH to furnish propargylated chalcones (2a-2d) in high
yield. In next step, propargylated chalcones (2a-2d) were refluxed with hydrazine hydrate in
acetic acid to yield pyrazoline containing a terminal alkyne (APZ1-APZ4) in very good yield
(Scheme 1, Table 1). In the final step, pyrazolines with a terminal alkyne were then subjected to
copper (I)-catalyzed azide-alkyne cycloaddition with organic bromides (TPZ1-TPZ16) in the
presence of copper sulphate pentahydrate and sodium ascorbate using DMF: Water (8:2, (v/v)) as
solvent to yield pyrazoline-1,2,3-triazole hybrids in very good yield (Scheme 2, Table 2).
Scheme 1: Synthesis of 1-(3-aryl-5-(2/4-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-
1-ones (APZ1-APZ4).
Table 1: Synthesis of 1-(3-aryl-5-(2/4-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-
ones (APZ1-APZ4).
S.No
Compound
APZ1
Ar1
R1
R2
Yield, %
M. Pt (°C)
110-112
114-117
94-98
1
2
3
4
4-OMeC6H4
4-BrC6H4
4-OMeC6H4
4-BrC6H4
H
70
64
60
62
H
APZ2
H
H
APZ3
103-105
APZ4
4

Scheme 2: Synthesis of 1,2,3-triazole linked pyrazoline based molecular hybrids (TPZ1-TPZ16)
Table 2: Synthesis of 1,2,3-triazole linked pyrazoline based molecular hybrids (TPZ1-TPZ16)
M. Pt (°C)
120-122
70-72
S. No.
1
Compound
TPZ1
Ar1
Ar2
Yield, %
87
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
-C6H5
2
4-MeC6H4
4-BrC6H4
4-NO2C6H4
-C6H5
86
TPZ2
3
84
90-93
TPZ3
4
90
130-132
140-143
119-112
125-127
120-123
65-67
TPZ4
5
82
TPZ5
6
4-MeC6H4
4-BrC6H4
4-NO2C6H4
-C6H5
80
TPZ6
7
78
TPZ7
8
86
TPZ8
9
82
TPZ9
10
11
12
13
14
15
16
4-MeC6H4
4-BrC6H4
4-NO2C6H4
-C6H5
78
70-74
TPZ10
TPZ11
TPZ12
TPZ13
TPZ14
TPZ15
TPZ16
84
128-130
120-122
95-97
86
82
4-MeC6H4
4-BrC6H4
4-NO2C6H4
79
168-170
180-183
130-132
81
85
All the synthesized pyrazolines and triazole derivatives were fully characterized by various
analytical and spectral techniques including FTIR, 1H NMR, 13C NMR, MS techniques.
5

2.1.1 IR Analysis
The IR spectrum of propargylated pyrazoline (APZ1) showed two absorption bands at 1683 cm-1
and 1651 cm-1 due to -C=O stretching of amide and C=N stretching of pyrazoline ring. Two
bands due to alkyne ≡C-H stretching and C≡C stretching were also observed at 3197 cm-1 and
2123 cm-1 which were absent in the IR spectra of all triazole hybrids (TPZ1-TPZ16). The
presence of characteristic bands in the region 3128 cm-1 in the IR spectra of the triazole (TPZ1)
evidenced the formation of triazole ring.
2.1.2 1H NMR analysis
The 1H NMR spectra of the synthesized propargylated pyrazolines (APZ1) exhibited doublet at
δ 4.67 (J = 2.3 Hz) and triplet at δ 2.52 (J = 2.3 Hz), which confirms the formation of terminal
alkyne. An ABX system was found in the structure of pyraozline due to three doublets attributed
for Ha, Hb and Hx. Ha appeared at δ 3.16 (J=17.6 & 4.3 Hz), Hb at δ 3.72 (J=17.6,11.7Hz) and Hx
resonated at δ 5.56 (J =11.6, 4.3 Hz) with characteristic coupling constants. Aromatic protons
1
appeared in the region at δ 6.95-7.71. In the H NMR of triazole (TPZ1) peaks at δ 4.67 and δ
2.52 were disappeared and a sharp peak at δ 7.52 has been appeared due to the proton present in
triazole ring.
2.1.3 13C NMR analysis
13
In the C NMR of pyrazoline (APZ1) due to ABX system C-Ha and C-Hb signal appears at δ
42.4 and C-Hx signal arises at δ 55.4. While carbonyl carbon appears at δ 168.68 confirms the
13
acetylation during the reaction and C=N appears at δ 156.7. In the C NMR of final compound
(TPZ1) a peak at δ 144.0 confirms the formation of triazole ring. Peaks at δ 168.7 and δ 157.6
appeared due to carbonyl group and C=N of pyrazoline ring.
2.1.4 HRMS analysis
The HRMS data of all the synthesized products were in good agreement with their calculated
values
2.2 X-Ray Crystallographic study
The X-ray single crystal data revealed that the compound APZ2 crystallized in monoclinic cell
with
space
group
C2/c
(
6

Table 3). The crystal structure for APZ2 is shown in Figure 2. It is clearly revealed that APZ2 is
a bromo derivative. The resultant crystal structure of APZ2 shows that the pyrazoline substituted
five membered ring is perpendicular to benzene ring which has attached to propargyl units via
oxygen atom. The propargyl unit is parallel or trans to pyrazole ring. Different intermolecular
interactions present in case of APZ2 are shown in Figure 3. The packing diagram for APZ2
along c-axis has shown in Fig S45.
Figure 2: Ortep diagram of the compound APZ2.
Figure 3: Different intermolecular interactions present in case of APZ2.
7

Table 3: Crystal data and structure refinement parameters for APZ2.
Compound
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
APZ2
C20H17BrN2O2
397.27
Monoclinic
C2/c
16.267(5)
b (Å)
13.859(5)
c (Å)
16.767(6)
α (°)
90.00
β (°)
103.698(8)
γ (°)
90.00
V (Å3)
3672(2)
Z
8
D (calc/gcm-3)
μ (mm-1)
1.437
2.254
0.71073
λ (Mo Kα/Å)
θ range (°)
1.95-25.00
Total data collected
Unique data
Rint
14730
3242
0.0991
R1 = 0.0605; wR2 = 0.1588
R1 = 0.1146; wR2 = 0.1882
R indexes [ I > 2σ (I)]
R indexes (all data)
2.3 α-Glucosidase inhibitory activity
All the synthesized pyrazoline alkynes (APZ1-APZ4), and 1H-1,2,3-triazole hybrid derivatives
(TPZ1-TPZ16) were evaluated for their α-glucosidase inhibitory activity and the results are
presented in Table 4. It has been revealed that most of the compounds exhibited very good α-
glucosidase inhibition potential with IC50 values ranging from 41.281 to 106.71 μM. Moreover,
some of the compounds displayed even better inhibitory profile than acarbose. Some of the
8

compounds i.e., TPZ2 (IC50 = 41.29 μM), TPZ4 (IC50 = 60.03 μM), TPZ4 (IC50= 50.73 μM),
and TPZ8 (IC50= 47.94 μM) displayed better inhibitory potencies against α-glucosidase than
acarbose (IC50 = 60.68μM). The activity of triazole hybrids (TPZ1-TPZ16) was found to be
more promising when compared with their precursors (pyrazoline-alkynes; APZ1-APZ4)
thereby it can be concluded that the attachment of triazole ring to pyrazoline has led to increased
activity. From structure activity relationship studies, it was observed that all the pyrazoline-
triazoles hybrids derived from para propargylated chalcones exhibited better activity those
derived from ortho isomers. Apparently, the activity of the potent compounds is likely due to the
presence of central pyrazoline ring which interacts with the active site of the enzyme. The Ar1
substitutions in the pyrazoline influence the activities too. Compound containing methoxy group
at pyrazoline are more active than bromo, methyl and nitro.
Table 4: α-Glucosidase inhibition (IC50) activity of synthesized pyrazoline-alkynes (3a-3d) and triazole
hybrids (4a-4p)
Entry
1
Compound
APZ1
APZ2
APZ3
APZ1
TPZ1
TPZ2
TPZ3
TPZ4
TPZ5
TPZ6
TPZ7
TPZ8
TPZ9
TPZ10
TPZ11
TPZ12
IC50 value*
84.90±0.060
94.00±0.061
101.67±0.123
106.71±0.246
64.19±0.185
41.29±0.123
63.84±0.370
60.03±0.061
50.73±0.246
65.65±0.123
63.89±0.863
47.94±0.246
74.66±0.185
87.87±0.061
79.13±0.185
76.22±0.246
Docking score
-8.4
2
-7.6
3
-7.4
4
-7.0
5
-9.7
6
-10.7
-10
7
8
-9.8
9
-10.4
-9.8
10
11
12
13
14
15
16
-9.6
-10.3
-8.8
-8.3
-8.8
-9.4
9

17
18
19
20
21
78.22±0.061
80.51±0.678
87.7± 0.123
74.03± 0.493
60.68± 0.123
-9.8
-9.9
-8.4
-8.4
-7.4
TPZ13
TPZ14
TPZ15
TPZ16
Acarbose
*
Values are means ± SD (n: 3); p < 0.05
2.4 Molecular modeling studies
2.4.1 Docking simulations
The 3D structure of Saccharomyces cerevisiae α-glucosidase was predicted by homology
modeling technique as till now no crystal structure for this enzyme has been reported. The
primary structure was assessed in FASTA form from Uniprot database (accession ID: P53341)
and SWISS-MODEL Homology Modelling web server was used for homology modeling [44].
The services of BLAST and HHBlits were utilized for template search which resulted in Oligo-
1,6-glucosidase (3axh.1.A) structure as best template for protein alignment [45-48]. The latter
function was performed with ProMod3 while quality of structure was judged using QMEAN
scoring function (QMEAN value = 0.04) [49-51]. More than 96 % of the residues were in
favored region and 100 % residues were in permitted area in the Mol Probity Ramachandran plot
[52]. Most preferred 3D conformations of compounds TPZ2 and TPZ8 in the binding site of the
enzyme are shown in Figure 4.
10

(a)
(b)
Figure 4: Binding interactions of compounds (a) TPZ2; (b) TPZ8 (green: hydrogen bond; purple: pi
sigma; yellow: electrostatic; blue: halogen bond; dark pink: pi-pi interaction; light pink: hydrophobic)
Methoxy oxygen of the compound TPZ2 created a hydrogen bond with Arg439 while phenoxy
oxygen atom made a hydrogen bond with His279. Two carbon hydrogen bonds were observed
with Asp68 and Phe310. Phenyl group attached with triazole ring displayed one Pi-Donor
hydrogen bond with Arg312. Pyrazoline ring showed sigma-pi interactions with Phe157 and
Phe300. In molecule TPZ8, oxygen atom of nitro group and nitrogen atom of triazole displayed
hydrogen bond interactions with Arg312 and Asn421 respectively. Bromine atom made halogen
bond with Leu437. Pi orbitals of bromophenyl ring exhibited pi-anion electrostatic interactions
with Asp349 while pi electrons of triazole were involved in T-shaped pi-pi interactions with pi
orbitals of His245. Pyrazoline ring, again, put on view sigma-pi interactions with Phe157 and
Phe300.Surface diagram of protein along with docked molecules is shown in Figure 5. It can be
seen that the molecules went deep in the binding pocket.
Figure 5: Surface diagram of alpha glucosidase containing docked molecules TPZ2 (cyan) and TPZ8
(magenta)
2.4.2 Molecular dynamics studies
Further, to ascertain the stability of the docked complexes of the molecules with protein in the
physiological conditions, molecular dynamics studies were carried out (Fig 6 and Fig 7). Three
systems were investigated by using Newtonian mechanics-based molecular dynamic simulations.
The first system contains protein while the other two consists of one of each ligand along with
the protein. For each system Cartesian coordinates were placed in the center of the cubic box, the
remaining volume of the box was filled by water molecules. Solvation is followed by the
11

neutralization where a respective number of ions were added to each system to mimic
physiological conditions. Both solvation and neutralization steps were processed by using
GROMACS 5.4 [53]. To remove unusual contacts between atoms energy minimization was
performed using the steepest descendant method followed by 10 ns equilibration. The potential
energy of the system is calculated by using OPLS force fields integrated into GROMACS 5.4
package [54]. Whereas, Extended Simple Point Charge (SPCe) potential is used to describe the
molecular mechanics of the water molecules [55].
Classical simulations consider each atom as a hard-sphere with a user-defined charge. Whereas,
the motion of each atom is governed by the newton`s equation of motion through estimation of
the force on each atom from potential energy function. Before making any conclusion out of the
simulated system especially protein one needs to assure that the system must be equilibrated.
Therefore, we check the protein backbone RMSD for all the three systems which shows a stable
behavior after an initial surge. Both the ligands have reasonable interaction with the protein
active site with average interaction energy of -270.88 Kcal/mol and -273.90 Kcal/mol for TPZ8
and TPZ2, respectively. We also calculated the diffusion coefficient for both the ligands to
compare their displacement from the initial docked positions. The calculated diffusion
coefficient shows that throughout the MD run both the ligands are tightly restrained within the
active site with a diffusion coefficient of 0.14e-5 cm2/sec and 0.15e-5 cm2/sec for TPZ8 and
TPZ2, respectively.
12

(a)
(b)
Figure 6: Qualitative analysis of the Molecular dynamics trajectory. (a) Radius of gyration; RMSD
plot of protein and ligand.
13

Figure 7: (a) Energy plot of ligand TPZ8; (b) Energy plot of ligand TPZ2; (c) The plot of hydrogen
bonds formed between protein and ligand TPZ8; (d) The plot of hydrogen bonds formed between
protein and ligand TPZ2.
2.5 Conclusion
In summary, we have successfully designed, synthesized and evaluated the α-glucosidase
inhibition potential of some pyrazoline-1,2,3-triazole hybrids. All the synthesized compounds
were fully characterized with the help of various spectral techniques. The α-glucosidase
inhibition studies showed that most of the compounds exhibited better or comparable activity to
that of standard drug Acarbose. It was also observed that the attachment of triazole ring to
pyrazoline has increased the activity as suggested by molecular hybridization approach. Further,
the docking results also showed that the two most potent compounds bind more strongly than the
Acarbose with α-glucosidase enzyme. Moreover, the molecular dynamics studies also supported
14

the wet experiment results. Therefore, it can be said that these kind of pyrazoline-triazole hybrids
can be useful for the development of lead molecules for diabetes.
3
Experimental
3.1 Chemistry
All chemicals and solvents used in the present work were purchased from Sigma Aldrich, Alfa-
Aesar and Hi-media and used without further purification. Melting point of the synthesized
1
13
compounds was recorded in open capillaries and is uncorrected. The H and C were recorded
on Bruker Avance II 400 MHz at 400 MHz and 100 MHz respectively, using deuterated
chloroform as solvent (CDCl3) and tetramethylsilane (TMS) as an internal standard (chemical
shift in del, ppm). The IR spectra were recorded on Shimazdu IR Affinity-I FT-IR
spectrophotometer using potassium bromide (KBr) and the values are given in cm-1. High
resolution mass spectra (HRMS) were recorded on SCIEX-QTOF spectrometer
spectrophotometer. The completion of the reaction and the purity of the compounds were
monitored by thin layer chromatography (TLC) using silica gel plates (SIL G/UV254,
ALUGRAM) and visualized under ultraviolet lamp.
3.1.1 Synthesis of pyrazolines from chalcones with terminal alkyne. (3a-3d)
The synthesis of pyrazolines from chalcones with terminal alkyne were achieved via sequential
synthesis. For this, firstly 2-(prop-2-yn-1-yloxy) benzaldehyde (1a)/ 4-(prop-2-yn-1-yloxy)
benzaldehyde (1b) were synthesized using reported procedure. After synthesis of propargylated
aldehyde they were subjected to Claisen Schmidt reaction with differently substituted
acetophenones (2) under microwave. The chalcones (2a-2d) (1.0 mml), thus formed were reacted
with hydrazine hydrate (3.0 mml) in acetic acid (20 mL) and the mixture was refluxed under
stirring for 6 h. The progress of the reaction was monitored using TLC. After completion of the
reaction as indicated by TLC the reaction was quenched using ice cold water and precipitate thus
formed was filtered under suction. The solid so obtained was recrystallized using ethanol.
4.1.1. 1-(3-(4-methoxyphenyl)-5-(4-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1
yl)ethanone (APZ1): Appearance: solid; Brown solid; FT-IR (KBr, υmax cm-1): 3197 (
str.), 3020 (C-H str., aromatic ring), 2935, 2123 (C C str) 1683 (C=O str, Amide), 1608 (C=N
H-pyrazol-1-
≡
C-H
≡
str); 1H NMR (400 MHz, CDCl3):
δ
7.71 (d,
J
= 8.8 Hz, 2H), 7.20 (d,
15
J = 8.7 Hz, 2H), 6.95 (t, J

= 9.2 Hz, 4H), 5.56 (dd,
J
= 11.6, 4.3 Hz, 1H), 5.56 (dd,
J
= 11.6, 4.3 Hz, 1H), 4.67 (d,
= 17.6, 11.7 Hz, 1H), 3.23-3.08 (m,
168.6 (s), 161.3 (s),
J = 2.3
Hz, 2H), 4.67 (d,
1H), 2.52 (t,
J
= 2.3 Hz, 2H), 3.87 (s, 3H), 3.72 (dd, J
13
J
= 2.4 Hz, 1H), 2.43 (s, 3H); C NMR (100 MHz, CDCl3):
δ
156.9 (s), 153.7 (s), 135.1 (s), 128.1 (s), 126.9 (s), 124.1 (s), 115.1 (s), 114.1 (s), 78.5 (s), 75.5
(s), 59.2 (s), 55.8 (s), 55.4 (s), 42.3 (s), 21.9 (s); HRMS (m/z) [M+H]+calculated for C21H20N2O3
349.1552 found: 349.1582
3.2.3
yl)ethanone (APZ2): Appearance: solid; Brown solid; FT-IR (KBr υmax, cm-1): 3219 (
str.), 3049 (=C-H str., aromatic ring), 2951, 2113 (C
NMR (400 MHz, CDCl3): 7.62 (d, = 8.6 Hz, 3H), 7.55 (d,
Hz, 1H), 7.07 (d, = 9.1 Hz, 1H), 7.02 (d,
11.9, 4.8 Hz, 1H), 4.75 (dd,
17.7, 4.8 Hz, 1H), 2.49 (t,
1-(3-(4-bromophenyl)-5-(4-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1H-pyrazol-1-
≡
C-H
1
≡
C str), 1658 (C=O str), 1608(C=N str); H
δ
J
J
= 8.6 Hz, 2H), 7.25 (d,
= 7.9 Hz, 1H), 6.97 (t, = 7.5 Hz, 1H), 5.86 (dd,
= 17.7, 11.9 Hz, 1H), 3.08 (dd,
J = 8.3
J
J
J
J
J
=
=
J
= 4.3, 2.4 Hz, 2H), 3.74 (dd, J
J
= 2.4 Hz, 1H), 2.48 (s, 3H);13C NMR (100 MHz, CDCl3):
δ
168.8
(s), 154.1 (s), 153.6 (s), 131.8 (s), 130.6 (s), 129.6 (s), 128.6 (s), 128.0 (s), 126.2 (s), 124.4 (s),
121.7 (s), 112.5 (s), 78.5 (s), 75.6 (s), 56.1 (s), 41.3 (s), 21.9 (s); HRMS (m/z) [M+H]+ calculated
for C20H17BrN2O2 : 397.0552 found: 397.0587
3.2.2 1-(3-(4-methoxyphenyl)-5-(2-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1
yl)ethanone (APZ3)
H-pyrazol-1-
Appearance: Brown solid; FT-IR (KBr, υmax, cm-1): 3213 (
str., aromatic ring), 2941, 2115 (-C
C str), 1651 (C=O str), 1593 (C=N str); 1H NMR (400 MHz,
CDCl3): 7.69 (d, = 8.9 Hz, 1H), 7.25 (t, = 7.8 Hz, 1H), 7.07 (d, = 9.1 Hz, 1H), 7.03 (d,
7.7 Hz, 1H), 6.97 (d, = 7.5 Hz, 1H), 6.93 (d, = 8.9 Hz, 1H), 5.86 (dd, = 11.7, 4.6 Hz, 1H),
4.76 (dd, = 4.7, 2.4 Hz, 1H), 3.86 (s, 2H), 3.74 (dd, = 17.6, 11.7 Hz, 1H), 3.08 (dd, = 17.6,
4.6 Hz, 1H), 2.51 (t, 168.7 (s),
= 2.3 Hz, 1H), 2.49 (s, 1H); 13C NMR (100 MHz, CDCl3):
≡C-H str., triazole ring), 3041 (C-H
≡
δ
J
J
J
J =
J
J
J
J
J
J
J
δ
161.2 (s), 154.6 (s), 154.2 (s), 130.0 (s), 128.4 (s), 128.2 (s), 126.0 (s), 124.3 (s), 121.7 (s), 114.0
(s), 112.5 (s), 78.6 (s), 75.5 (s), 56.2 (s), 55.6(s), 55.4(s), 41.6(s), 21.9 (s); HRMS (m/z)
[M+H]+:calculated for C21H20N2O3 349.1552 found: 349.1582
3.2.4
1-(3-(4-bromophenyl)-5-(2-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (APZ4)
16

Appearance: Brown solid ; FT-IR (KBr, υ max, cm-1): 3250 (
ring), 2943, 2113 (C
7.60 (dd, = 20.7, 8.6 Hz, 1H), 7.18 (d,
11.8, 4.5 Hz, 1H), 4.67 (d, = 2.3 Hz, 1H), 3.72 (dd,
4.6 Hz, 1H), 2.52 (t,
157.0 (s), 152.7 (s), 134.8 (s), 131.9 (s), 130.1 (d,
≡
C-H str.), 3211 (C-H str., aromatic
1
≡
C str), 1681 (C=O str), 1599 (C=N str); H NMR (400 MHz, CDCl3):
δ
J
J
= 8.7 Hz, 1H), 6.94 (d,
J
= 8.7 Hz, 1H), 5.58 (dd,
J
=
J
J
= 17.7, 11.8 Hz, 1H), 3.14 (dd,
J
= 17.7,
168.8 (s),
= 54.7 Hz), 128.0 (s), 126.9 (s), 124.6 (s),
J
= 2.4 Hz, 1H), 2.43 (s, 1H); 13C NMR (100 MHz, CDCl3):
δ
J
115.2 (s), 78.5 (s), 75.6 (s), 59.5 (s), 55.8 (s), 42.1 (s), 21.9 (s); HRMS (m/z) [M+H]+calculated
for C20H17BrN2O2: 397.0552 found: 397.0587
3.3 General procedure for the synthesis pyrazoline-1,2,3-triazol hybrids (TPZ1-TPZ16):
To a stirred solution of benzyl bromide (1.0 mmol) in dimethylformamide, aqueous solution of
sodium azide (3.0 mmol) was added. Then, after 1-2 h, pyrazoline alkyne (APZ1-APZ4), (1.0
mmol), copper sulphate pentahydrate (0.1mmol) and sodium ascorbate (0.4 mmol) were added
successively [10]. Stirring of the reaction content was continued at 40-45°C for 3-5 h (Scheme
2). The progress of reaction was monitored by thin layer chromatography. On completion of the
reaction, aqueous cold ammonia solution was added to the reaction mixture and filtered the
precipitated solid. The solid product was washed with water. The crude products obtained above
were purified by washing with ethyl acetate and dried under vacuum to get the target compounds
(TPZ1-TPZ16)
3.3.1
dihydro-1
cm-1): 3126 (C-H str., triazole ring), 3078 (C-H str., aromatic ring), 2941, 1664 (C=O str), 1606
(C=N str pyrazoline); 1H NMR (400 MHz, CDCl3):
7.70 (d, = 8.4 Hz, 2H), 7.52 (s, 1H), 7.38
(s, 2H), 7.29 (d, = 8.1 Hz, 2H), 7.16 (d, = 8.4 Hz, 2H), 6.93 (dd, = 18.6, 8.5 Hz, 4H), 5.54
(s, 2H), 5.16 (s, 2H), 3.87 (s, 3H), 3.71 (dd, = 17.4, 11.9 Hz, 1H), 3.13 (d, = 22.0 Hz, 1H);
13C NMR (100 MHz, CDCl3):
168.7 (s), 161.3 (s), 157.6 (s), 153.7 (s), 144.6 (s), 134.8 (s),
1-(5-(4-((1-benzyl-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-methoxyphenyl)-4,5-
H
-pyrazol-1-yl)ethanone (TPZ1): Appearance: Light yellow solid; FT-IR (KBr, υmax
,
δ
J
J
J
J
J
J
δ
134.4 (s), 129.1 (s), 128.8 (s), 128.2 (s), 128.1 (s), 126.9 (s), 124.1 (s), 122.6 (s), 115.1 (s), 114.2
(s), 62.1 (s), 59.2 (s), 55.4 (s), 54.3 (s), 42.4 (s), 22.0 (s). HRMS (m/z) [M+H]+calculated for
C28H27N5O3: 482.2194 found: 482.1875
3.3.2 1-(3-(4-methoxyphenyl)-5-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-4,5-dihydro-1
H
-pyrazol-1-yl)ethanone (TPZ2)
17

Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3134 (C-H str., triazole ring), 3007 (C-
1
H str., aromatic ring), 2939, 1664 (C=O str), 1606 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
= 6.9 Hz, 2H), 6.91 (d,
2H), 3.87 (s, 3H), 3.70 (dd,
2.37 (s, 3H); 13C NMR (100 MHz, CDCl3):
δ
7.70 (d,
J
= 8.5 Hz, 2H), 7.50 (s, 1H), 7.20 (s, 4H), 7.16 (d,
= 8.4 Hz, 2H), 5.54 (dd, = 11.6, 4.0 Hz, 1H), 5.49 (s, 2H), 5.15 (s,
= 17.4, 11.8 Hz, 1H), 3.12 (dd, = 17.5, 4.1 Hz, 1H), 2.41 (s, 3H),
168.7 (s), 161.4 (s), 157.5 (s), 153.6 (s), 144.6 (s),
J = 8.4 Hz, 2H), 6.95 (t, J
J
J
J
J
δ
134.7 (s), 134.4 (s), 129.1 (s), 128.7 (s), 128.2 (s), 128.2 (s), 126.8 (s), 124.2 (s), 122.5 (s), 115.1
(s), 114.1 (s), 62.1 (s), 59.3 (s), 55.4 (s), 54.3 (s), 42.4 (s), 22.0 (s) 21.3 (s). HRMS (m/z)
[M+H]+calculated for C29H29N5O3: 496.2349 found: 496.1265
3.3.3 1-(5-(4-((1-(4-bromobenzyl)-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-methoxy
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ3)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3122 (C-H str., triazole ring), 3080 (C-
1
H str, aromatic ring), 2935, 1666 (C=O str), 1606 (C=N str Pyrazoline); H NMR (400 MHz,
CDCl3):
= 7.7 Hz, 3H), 6.96 (d, 2H), 6.91 (d, 2H), 5.53 (dd,
2H), 3.87 (s, 3H), 3.71 (dd, 1H), 3.13 (dd,
MHz, CDCl3):
δ 7.77–7.65 (d, 2H), 7.55-7.52 (m, 2H), 7.52 (s, 1H), 7.21 (d, J = 7.3 Hz, 1H), 7.17 (d, J
J
= 10.1, 5.0 Hz, 1H), 5.49 (s, 2H), 5.17 (s,
13
J
= 17.5, 5.6 Hz, 1H), 2.42 (s, 3H); C NMR (100
δ
168.6 (s), 161.3 (s), 157.5 (s), 153.7 (s), 144.8 (s), 134.9 (s), 133.4 (s), 132.3 (s),
131.9 (s), 129.8 (s), 129.7 (s), 128.1 (s), 126.9 (s), 124.0 (s), 122.9 (s), 122.5 (s), 115.1 (s), 114.1
(s), 62.1 (s), 59.2 (s), 55.4 (s), 53.5 (s), 42.4 (s), 21.9 (s); HRMS (m/z) [M+H]+ calculated for
C28H26 BrN5O3: 560.1297 found: 560.1477
3.3.4 1-(3-(4-methoxyphenyl)-5-(4-((1-(4-nitrobenzyl)-1
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ4)
H-1,2,3-triazol-4-yl)methoxy)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3124 (C-H str., triazole ring), 3086 (C-
H str, aromatic ring), 2939, 1635 (C=O str), 1606 (C=N str pyrazoline),1517 (N=O str), 1342
1
(N=O str); H NMR (400 MHz, CDCl3):
δ
8.25 (d, 2H), 7.70 (d,
= 8.6 Hz, 2H), 6.96 (d, = 8.8 Hz, 2H), 6.91 (d,
= 11.6, 4.3 Hz, 1H), 5.20 (s, 2H), 3.87 (s, 3H), 3.72 (dd,
= 17.5, 4.4 Hz, 1H), 2.41 (s, 3H); 13C NMR (100 MHz, CDCl3):
J
= 8.8 Hz, 2H), 7.60 (s, 1H),
= 8.7 Hz,
= 17.5,
7.43 (d,
2H), 5.65 (s, 1H), 5.54 (dd,
11.7 Hz, 1H), 3.12 (dd,
J
= 8.6 Hz, 2H), 7.17 (d,
J
J
J
J
J
J
δ
168.8 (s), 161.3 (s), 157.6 (s), 153.7 (s), 144.7 (s), 134.9 (s), 133.5 (s), 132.4 (s), 132.0 (s), 129.7
18

(s), 129.7 (s), 128.2 (s), 126.8 (s), 124.2 (s), 122.0 (s), 122.4 (s), 115.1 (s), 114.1 (s), 62.2 (s),
59.3 (s), 55.4 (s), 53.4 (s), 42.4 (s), 21.9 (s). HRMS (m/z) [M+H]+ calculated for C28H26N6O5:
527.2043 found: 527.2082
3.3.5 1-(5-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-bromophenyl)-4,5-
dihydro-1 -pyrazol-1-yl)ethanone (TPZ5)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3122 (C-H str., triazole ring), 3066 (C-
H str, aromatic ring), 2992, 1670 (C=O str), 1585 (C=N str, pyrazoline).1H NMR (400 MHz,
CDCl3):
δ
7.60 (q,
= 8.7 Hz, 2H), 6.93 (d,
= 17.7, 11.8 Hz, 1H), 3.12 (dd,
NMR (100 MHz, CDCl3): 168.8 (s), 157.7 (s), 152.7 (s), 144.5 (s), 134.5 (s), 134.4 (s), 131.9
J
= 8.7 Hz, 4H), 7.52 (s, 1H), 7.39 (d,
= 8.7 Hz, 2H), 5.61-5.55 (dd, 1H), 5.55 (s,
= 17.7, 4.6 Hz, 1H), 2.41 (s, 3H).13C
J
= 7.2 Hz, 2H), 7.32-7.28 (m, 2H),
7.16 (d,
J
J
J
= 5.1 Hz, 2H),
5.17 (s, 2H), 3.72 (dd,
J
J
δ
(s), 130.4 (s), 129.1 (s), 128.8 (s), 128.1 (s), 128.0 (s), 126.9 (s), 124.6 (s), 122.5 (s), 115.1 (s),
62.1 (s), 59.5 (s), 54.2 (s), 42.1 (s), 30.3(s), 21.9 (s); HRMS (m/z) calculated for C27H24BrN5O2
[M+H]+: 530.1192 found: 530.1352.
3.3.6
1-(3-(4-bromophenyl)-5-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-4,5-dihydro-1
H
-pyrazol-1-yl)ethanone(TPZ6)
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3120 (C-H str., triazole ring), 3072
(C-H str, aromatic ring), 2995, 1672 (C=O str), 1595 (C=N str., pyrazoline).1H NMR (400 MHz,
CDCl3):
1H), 6.93 (t,
3.13 (dd,
= 17.7, 5.7 Hz, 1H), 2.98 (s, 3H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3):
δ
8.03 (s, 1H), 7.64-7.59 (d, 2H), 7.56 (d, 2H), 7.50 (s, 1H), 7.20 (d, 3H), 7.18-7.13 (m,
= 8.9 Hz, 1H), 5.62-5.53 (dd, 1H), 5.49 (s, 2H), 5.15 (s, 2H), 3.76-3.65 (dd, 1H),
168.9
J
J
δ
(s), 162.5 (s), 158.1 (s), 152.8 (s), 144.4 (s), 138.8 (s), 134.4 (s), 131.9 (s), 129.8 (s), 128.2 (s),
128.0 (s), 126.9 (s), 124.6 (s), 122.4 (s), 115.2 (s), 115.1 (s), 62.1 (s), 59.5 (s), 54.6 (s), 42.1 (s),
36.5 (s), 31.4 (s), 25.3 (s), 21.9 (s), 21.1 (s); HRMS: (m/z) [M+H]+ calculated for
C28H26BrN5O2: 544.1348 found: 544.1515
3.3.7 1-(5-(4-((1-(4-bromobenzyl)-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-
bromophenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ7)
H
Appearance: Light yellow solid; FT-IR (KBr, υ max, cm-1):3122 (C-H str., triazole ring), 3078 (C-
1
H str, aromatic ring), 2943, 1664 (C=O str), 1591 (C=N str, pyrazoline); H NMR (400 MHz,
19

CDCl3):
4.8 Hz, 4H), 6.92 (d,
3.72 (dd, = 17.7, 11.8 Hz, 1H), 3.12 (dd,
MHz, CDCl3): 168.9 (s), 157.6 (s), 152.8 (s), 144.7 (s), 134.5 (s), 133.4 (s), 132.3 (s), 131.9 (s),
δ
7.62 (d,
J
= 8.7 Hz, 2H), 7.57 (d,
= 8.7 Hz, 1H), 5.56 (dd,
= 17.6, 4.6 Hz, 1H), 2.42 (s, 3H); C NMR (100
J
= 8.7 Hz, 2H), 7.55-7.51 (m, 2H), 7.16 (m,
J = 8.5,
J
J
= 11.8, 4.5 Hz, 1H), 5.49 (s, 2H), 5.17 (s, 2H),
13
J
J
δ
130.3 (s), 129.7 (s), 128.0 (s), 126.9 (s), 124.6 (s), 123.0 (s), 122.5 (s), 115.1 (s), 62.1 (s), 59.7
(s), 53.6 (s), 42.5 (s), 22.0 (s). HRMS (m/z) [M+H]+ calculated for C27H23Br2N5O2: 608.0297
found: 608.0483
3.3.8 1-(3-(4-bromophenyl)-5-(4-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ8)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1):3118 (C-H str., triazole ring), 3080 (C-
H str., aromatic ring), 2943, 1651 (C=O str), 1597 (C=N str, pyrazoline), 1523 (N=O str), 1350
(N=O str); 1H NMR (400 MHz, CDCl3):
Hz, 2H), 7.17 (d, = 8.6 Hz, 2H), 6.96 (d,
5.54 (dd, = 11.6, 4.3 Hz, 1H), 5.20 (s, 2H), 3.87 (s, 3H), 3.72 (dd,
(dd,
= 17.5, 4.4 Hz, 1H), 2.41 (s, 3H); 13C NMR (100 MHz, CDCl3):
δ
7.70 (d,
J
= 8.8 Hz, 2H), 7.60 (s, 1H), 7.43 (d,
= 8.7 Hz, 1H), 5.65 (s, 2H),
= 17.5, 11.7 Hz, 1H), 3.12
168.9 (s), 158.5 (s), 152.9
J = 8.6
J
J
= 8.8 Hz, 2H), 6.91 (d,
J
J
J
J
δ
(s), 144.9 (s), 135.5 (s), 134.4 (s), 133.3 (s), 132.8 (s), 131.3 (s), 129.7 (s), 129.0 (s), 127.8 (s),
125.6 (s), 124.0 (s), 123.5 (s), 115.1 (s), 62.1 (s), 60.3 (s), 53.5 (s), 43.2 (s), 22.4 (s); HRMS
(m/z) [M+H]+ calculated for C27H23BrN6O4:575.1042 found: 575.1219
3.3.9 1-(5-(2-((1-benzyl-1
dihydro-1 -pyrazol-1-yl)ethanone (TPZ9)
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3134 (C-H str., triazole ring), 3068
(C-H str., aromatic ring), 2941, 1666 (C=O str), 1604 (C=N str); 1H NMR (400 MHz, CDCl3):
7.68 (d, = 8.6 Hz, 2H), ), 7.65 (s, 1H), 7.22 (m, = 7.7 Hz, 5H), 7.08 (t, = 7.4 Hz, 1H), 7.00
(d, = 8.1 Hz, 1H), 6.97-6.90 (m, 2H), 5.79 (dd, = 11.8, 4.8 Hz, 1H), 5.49 (s, 2H), 5.26 (s,
5.6 Hz, 2H), 3.86 (s,3H), 3.61 (dd, = 17.7, 11.8 Hz, 1H), 3.05 (dd, = 28.4, 15.7 Hz, 1H), 2.43
(s, 3H);13C NMR (100 MHz, CDCl3):
168.6 (s), 161.2 (s), 154.6 (s), 154.5 (s), 144.4 (s), 134.6
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-methoxyphenyl)-4,5-
H
δ
J
J
J
J
J
J =
J
J
δ
(s), 129.8 (s), 129.1 (s), 128.2 (s), 128.1 (s), 128.0 (s), 126.2 (s), 124.1 (s), 122.9 (s), 121.4 (s),
114.1 (s), 112.4 (s), 62.4 (s), 55.7 (s), 55.4 (s), 54.1 (s), 41.6 (s), 21.9 (s); HRMS (m/z)calculated
for C28H27N5O3 [M+H]+: 482.2114 found: 482.1876
20

3.3.10 1-(3-(4-methoxyphenyl)-5-(2-((1-(4-methylbenzyl)-1
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ10)
H-1,2,3-triazol-4-yl)methoxy)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3136 (C-H str., triazole ring), 3068 (C-
1
H str, aromatic ring), 2941, 1662 (C=O str), 1589 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
δ
7.64 (d,
J
= 8.7 Hz, 2H), 7.58 (s, 1H), 7.18 (m, 5H), 7.07 (d,
= 7.4 Hz, 3H), 5.78 (dd, = 11.7, 4.7 Hz, 1H), 5.45 (s, 2H), 5.25 (d,
= 17.6, 11.8 Hz, 1H), 3.03 (dd, = 17.6, 4.8 Hz, 1H),
168.6 (s), 161.2 (s), 154.6 (s), 154.4
J = 7.0 Hz, 1H), 7.00 (d,
J
= 8.2 Hz, 1H), 6.93 (t,
J
J
J
= 5.8 Hz, 2H), 3.86 (s, 3H), 3.61 (dd,
J
J
13
2.43 (s, 3H), 2.35 (s, 3H); C NMR (100 MHz, CDCl3):
δ
(s), 144.3 (s), 138.6 (s), 131.5 (s), 129.7 (s), 129.7 (s), 128.6 (s), 128.1 (s), 126.2 (s), 124.2 (s),
122.7 (s), 121.4 (s), 114.0 (s), 112.5 (s), 62.4 (s), 62.4 (s), 55.7 (s), 55.4 (s), 54.0 (s), 54.0 (s),
41.5 (s), 21.9 (s), 21.1 (s); HRMS m/z calculated for C29H29N5O3 [M+H]+: 496.2270 found:
496.1567
3.3.11 1-(5-(2-((1-(4-bromobenzyl)-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-
methoxyphenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone TPZ11
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1):3136 (C-H str., triazole ring), 3009 (C-
1
H str, aromatic ring), 2939, 1662 (C=O str), 1591 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
8.4 Hz, 2H), 7.08 (d,
= 11.7, 4.8 Hz, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 3.86 (s, 3H), 3.63 (dd,
3.04 (dd,
= 17.6, 4.9 Hz, 1H), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3):
δ
7.64 (d,
J
= 9.1 Hz, 3H), 7.50 (d,
J
= 8.4 Hz, 2H), 7.22 (t,
J
= 7.0 Hz, 1H), 7.14 (d,
= 7.9 Hz, 3H), 5.80 (dd,
= 17.6, 11.8 Hz, 1H),
168.6 (s), 161.2 (s),
J =
J
= 7.6 Hz, 1H), 6.99 (d,
J
= 8.1 Hz, 1H), 6.94 (t,
J
J
J
J
δ
154.4 (s), 154.4 (s), 144.6 (s), 133.6 (s), 132.2 (s), 130.1 (s), 129.6 (s), 128.6 (s), 128.1 (s), 126.2
(s), 124.1 (s), 122.9 (s), 122.8 (s), 121.5 (s), 114.0 (s), 112.6 (s), 62.5 (s), 60.4 (s), 55.5 (s), 55.4
(s), 53.4 (s), 41.5 (s), 21.9 (s); HRMS (m/z) [M+H]+calculated for C28H26BrN5O3: 560.1297
found: 560.1477
3.3.12 1-(3-(4-methoxyphenyl)-5-(2-((1-(4-nitrobenzyl)-1
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ12)
H-1,2,3-triazol-4-yl)methoxy)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1):3134 (C-H str., triazole ring), 3076 (C-
H str., aromatic ring), 2941, 1641 (C=O str), 1606 (C=N str., pyrazoline), 1517 (N=O str), 1346
21

(N=O str); 1H NMR (400 MHz, CDCl3):
Hz, 2H), 7.37 (d, = 8.2 Hz, 2H), 7.28 (s, 1H), 7.21 (t,
6.98 (d, = 8.3 Hz, 1H), 6.92 (d, = 8.6 Hz, 2H), 5.83 (dd,
5.36 (s, 2H), 3.86 (s, 3H), 3.65 (dd, = 17.5, 11.9 Hz, 1H), 3.05 (dd,
(s, 3H); 13C NMR (100 MHz, CDCl3):
168.6 (s), 161.3 (s), 154.4 (s), 154.2 (s), 145.0 (s), 141.7
δ
8.20 (d,
J
= 8.2 Hz, 2H), 7.81 (s, 1H), 7.65 (d,
= 7.5 Hz, 1H), 7.07 (d, = 7.4 Hz, 1H),
= 11.4, 4.2 Hz, 1H), 5.58 (s, 2H),
= 17.6, 4.5 Hz, 1H), 2.42
J = 8.5
J
J
J
J
J
J
J
J
δ
(s), 130.3 (s), 128.6 (s), 128.6 (s), 128.1 (s), 126.2 (s), 124.2 (s), 124.1 (s), 123.4 (s), 121.7 (s),
114.1 (s), 112.8 (s), 62.5 (s), 55.4 (s), 55.2 (s), 53.0 (s), 41.6 (s), 31.5 (s), 22.6 (s), 21.9 (s);
HRMS (m/z) [M+H]+ calculated for C28H26N6O5: 527.2043 found: 527.2082
3.3.13 1-(5-(2-((1-benzyl-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-bromophenyl)-4,5-
dihydro-1 -pyrazol-1-yl)ethanone (TPZ13)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3136 (C-H str., triazole ring), 3064 (C-
1
H str, aromatic ring), 2941, 1666 (C=O str), 1593 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
1H), 7.07 (d,
11.9, 5.0 Hz, 1H), 5.48 (s, 2H), 5.25 (d,
(dd,
= 17.7, 5.0 Hz, 1H), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3):
δ
7.56 (s, 1H), 7.54 (s, 4H), 7.38 (d,
= 7.5 Hz, 1H), 7.01 (d, = 8.1 Hz, 1H), 6.94 (t,
= 3.7 Hz, 2H), 3.61 (dd,
J
= 6.9 Hz, 3H), 7.28 (s,2H), 7.22 (d,
= 7.5 Hz, 1H), 5.79 (dd,
= 17.7, 11.9 Hz, 1H), 3.04
168.8 (s), 154.7 (s),
J = 8.4 Hz,
J
J
J
J =
J
J
J
δ
153.5 (s), 144.2 (s), 134.5 (s), 131.8 (s), 130.5 (s), 129.5 (s), 129.1 (s), 128.8 (s), 128.7 (s), 128.0
(s), 127.9 (s), 126.4 (s), 124.4 (s), 122.8 (s), 121.4 (s), 112.5 (s), 62.3 (s), 56.2 (s), 54.2 (s), 41.2
(s), 31.58 (s), 22.65 (s), 21.94 (s); HRMS (m/z) [M+H]+ calculated for C27H24BrN5O2: 530.1192
found: 530.1352
3.3.14 1-(3-(4-bromophenyl)-5-(2-((1-(4-methylbenzyl)-1
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ14)
H-1,2,3-triazol-4-yl)methoxy)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3130 (C-H str., triazole ring), 3013 (C-
1
H str, aromatic ring), 2931, 1660 (C=O str), 1595 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
δ
7.54 (s, 5H), 7.23 (t,
J
= 7.3 Hz, 1H), 7.18 (s, 4H), 7.06 (d,
J
= 7.4 Hz, 1H), 7.01 (d,
J
= 8.2 Hz, 1H), 6.94 (t,
4.5 Hz, 2H), 3.60 (dd,
J
J
= 7.5 Hz, 1H), 5.79 (dd, = 11.9, 5.0 Hz, 1H), 5.44 (s, 2H), 5.23 (d,
= 17.7, 11.9 Hz, 1H), 3.05 (s, 3H), 2.42 (s, 3H), 2.36 (s, 1H); 13C NMR
J
J
=
(100 MHz, CDCl3):δ 168.8 (s), 154.7 (s), 153.6 (s), 144.1 (s), 138.7 (s), 131.8 (s), 131.5 (s),
130.5 (s), 129.7 (s), 129.4 (s), 128.8 (s), 128.1 (s), 127.9 (s), 126.3 (s), 124.4 (s), 122.7 (s), 121.4
22

(s), 112.5 (s), 62.3 (s), 56.2 (s), 54.0 (s), 41.2 (s), 21.9 (s), 21.1 (s); HRMS (m/z) [M+H]+
calculated for C27H26BrN5O2: 544.1348 found: 544.1515
3.3.15 1-(5-(2-((1-(4-bromobenzyl)-1
H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-(4-
bromophenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ15)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1):3136 (C-H str., triazole ring), 3064 (C-
1
H str, aromatic ring), 2943, 1664 (C=O str), 1593 (C=N str, pyrazoline); H NMR (400 MHz,
CDCl3):
= 8.5 Hz, 2H), 7.14 (d,
4.2 Hz, 1H), 5.18 (s, 2H), 3.70 (dd,
(s, 3H); 13C NMR (100 MHz, CDCl3):
δ
8.24 (t,
J
= 8.7 Hz, 2H), 7.59 (t,
= 8.4 Hz, 2H), 6.90 (d,
= 24.5, 12.3 Hz, 1H), 3.10 (dd,
168.8 (s), 157.5 (s), 152.7 (s), 148.1 (s), 145.1 (s), 141.5
J
= 6.7 Hz, 3H), 7.56 (s, 1H), 7.49 (s, 1H), 7.41 (d,
= 8.4 Hz, 2H), 5.64 (s, 2H), 5.54 (dd, = 11.7,
= 17.7, 4.3 Hz, 1H), 2.39
J
J
J
J
J
J
δ
(s), 134.6 (s), 131.9 (s), 130.3 (s), 128.6 (s), 128.1 (s), 126.9 (s), 124.6 (s), 124.3 (s), 124.0 (s),
122.9 (s), 115.1 (s), 62.0 (s), 59.5 (s), 53.1 (s), 42.1 (s), 21.9 (s); HRMS (m/z) [M+H]+ calculated
for C27H23Br2N5O2: 608.0292 found: 608.0483
3.3.16 1-(3-(4-bromophenyl)-5-(2-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
phenyl)-4,5-dihydro-1 -pyrazol-1-yl)ethanone (TPZ16)
H
Appearance: Light yellow solid; FT-IR (KBr, υmax, cm-1): 3180 (C-H str., triazole ring), 3076 (C-
H str, aromatic ring), 2945, 1662 (C=O str), 1593 (C=N str, pyrazoline), 1521 (N=O str), 1346
1
(N=O str); H NMR (400 MHz, CDCl3):
δ
7.59 (d,
= 8.3 Hz, 3H), 7.16-7.10 (m, 2H), 6.89 (d,
Hz, 1H), 5.47 (s, 2H), 5.14 (s, 2H), 3.70 (dd, = 17.7, 11.8 Hz, 1H), 3.09 (dd,
1H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3):
168.90 (s), 154.21 (d, = 12.0 Hz), 153.62 (d,
= 11.7 Hz), 148.02 (s), 144.8 (s), 141.6 (s), 131.9 (s), 130.6 (s), 129.9 (s), 128.8 (s), 128.6 (s),
J
= 8.6 Hz, 2H), 7.55 (s, 1H), 7.54-7.46 (m,
= 8.6 Hz, 1H), 5.53 (dd, = 11.8, 4.4
= 17.7, 4.5 Hz,
2H), 7.19 (d,
J
J
J
J
J
δ
J
J
128.0 (s), 127.9 (s), 126.2 (s), 124.2 (s), 123.3 (s), 121.7 (s), 78.5 (s), 75.6 (s), 62.4 (s), 56.1 (s),
56.8 (s), 53.0 (s), 41.3 (s), 31.5 (s), 22.6 (s), 21.9 (s); HRMS (m/z) [M+H]+calculated for
C27H23BrN6O4: 575.1042 found: 575.1219.
2.2
α
-Glucosidase inhibitory activity
α-Glucosidase enzyme ((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate
(pnitrophenylglucopyranoside) were purchased from Sigma-Aldrich. All solutions were prepared
in potassium phosphate buffer (
p
H 6.9, 50 mM), and all samples (APZ1-APZ4), (TPZ1-TPZ16)
23

were dissolved in DMSO. The various concentrations of compounds (APZ1-APZ4) (10 µL),
enzyme solution (10 µL) and potassium phosphate buffer (120 µL), were added in the 96-well
plate and incubated at 37 °C for 15 min. Then, the substrate (20 µL) was added to the mentioned
mixture and allowed to incubate at 37 °C for 20 min. Finally, the change in absorbance was
measured at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America).
DMSO and Acarbose were used respectively as control and standard drug. The percentage of
enzyme inhibition was calculated and IC50 values were obtained from non-linear regression.
Statistical analysis was performed using MS Excel. The data were analyzed by one way analysis
of variance (ANOVA). All the results were expressed as mean ± SD for triplicate determinations.
3.2 Computational details
Compounds TPZ2 and TPZ8 were placed in this binding pocket by using AutoDockVina
program. The center of the search box were x = -20.0948507758, y = -14.1248385256, z = -
24.3380527589 and size of the box was x = 34.8708465392, y = 36.1942925778, z =
31.0074280237. The binding interactions and conformations were visualized with discovery
studio visualizer and PyMOL.
3.3 Single crystal X-ray crystallography
Yellow colour single crystals were carefully picked under a polarizing microscope and
pasted to very fine glass fibers with the help of cyanoacrylate (superglue) adhesive. The single-
crystal X-ray diffraction data were collected with Bruker D8 Quest PHOTON II diffractometer
with monochromatic Mo Kα radiation (λ = 0.71073 Å) at 296 (2) K by using ω and ϕ scan. The
X-ray generator was operated at 50 KV and 20 mA. The data were reduced by using APEX3 and
SAINTPLUS program was used for diffraction profiles integration and absorption correction
(multi-scan) has been done by SADABS program [56]. The structure was solved and refined by
full matrix least square method on F
2 using SHELXL1997 program [57] present in the WinGx
package of programs (version 1.63.04a) [58]. All the hydrogen positions were initially located in
the difference Fourier maps and for the final refinement, the hydrogen atoms were placed in
geometrically ideal positions and refined in the riding mode. Final refinement comprised the
atomic positions of all the atoms, isotropic thermal parameters for all the hydrogen atoms and
anisotropic thermal parameters for all the non-hydrogen atoms. The crystallographic data for the
24

compound APZ2 can be found in CCDC No: 1953241 by free of charge from The Cambridge
Crystallographic Data Centre (CCDC) via www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgement
This work was carried out under the financial support from Guru Jambheshwar
University of Science & Technology, Hisar and PURSE program No. SR/PURSE 2/40(G) from
DST, New Delhi. The authors are also thankful to A P J Abdul Kalam Central instrumentation
laboratory, Guru Jambheshwar University of Science & Technology, Hisar, India for NMR and
MS spectra of the compounds.
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