Synthesis of acetamide derivatives of 1,2,4-triazole bearing azinane and their binding interactions with bovine serum albumin using spectroscopic techniques

Article abstract of DOI:10.3906/kim-1801-95

A new series of acetamide derivatives containing 1,2,4-triazole and azinane moieties has been synthesized and characterized using¹ H NMR,¹³ C NMR, IR, and EI-MS spectroscopic analysis. The intermediate triazole was synthesized through a sequential synthesis of carboxylate and carbohydrazide. The bovine serum albumin (BSA) binding of the newly synthesized 1,2,4-triazole derivatives was evaluated along with thermodynamics, site-selective binding, and synchronous study. The results obtained by BSA binding as well as thermodynamic studies justify that all the compounds show spontaneous interaction with BSA and could be effectively distributed and eliminated from the body. Therefore, the triazole-based analogs might be a useful strategy for designing new drug systems.

Full text of DOI:10.3906/kim-1801-95

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 1459 – 1478
© TÜBİTAK
http://journals.tubitak.gov.tr/chem/
doi:10.3906/kim-1801-95
Research Article
Synthesis of acetamide derivatives of 1,2,4-triazole bearing azinane and their
binding interactions with bovine serum albumin using spectroscopic techniques
1
1
Javed IQBAL ,, Aziz UR-REHMAN1,∗,, Muhammad Athar ABBASI ,,
1
2
3
Sabahat Zahra SIDDIQUI ,, Hira KHALID , Sabina Jhaumeer LAULLOO ,,
Nausheen JOONDAN ,, Aniisah Banu TAUPASS ,, Shahid RASOOL ,,
3
3
1
4,5
Syed Adnan Ali SHAH
,
1
Department of Chemistry, Government College University, Lahore, Pakistan
2
Department of Chemistry, Forman Christian College University, Lahore, Pakistan
3
Department of Chemistry, University of Mauritius, Reduit, Mauritius
4
Faculty of Pharmacy, MARA University of Technology, Puncak Alam Campus, Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia
5
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), MARA University of Technology,
Puncak Alam Campus, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
Received: 30.01.2018
•
Accepted/Published Online: 12.06.2018
•
Final Version: 06.12.2018
Abstract: A new series of acetamide derivatives containing 1,2,4-triazole and azinane moieties has been synthesized
1
13
and characterized using H NMR,
C NMR, IR, and EI-MS spectroscopic analysis. The intermediate triazole was
synthesized through a sequential synthesis of carboxylate and carbohydrazide. The bovine serum albumin (BSA) binding
of the newly synthesized 1,2,4-triazole derivatives was evaluated along with thermodynamics, site-selective binding, and
synchronous study. The results obtained by BSA binding as well as thermodynamic studies justify that all the compounds
show spontaneous interaction with BSA and could be effectively distributed and eliminated from the body. Therefore,
the triazole-based analogs might be a useful strategy for designing new drug systems.
Key words: Acetamides, 1,2,4-triazole, azinane, sulfonamide, bovine serum albumin binding
1. Introduction
Proteins are the most abundant macromolecules in cells and play a vital role in maintaining normal cell functions,
which are in turn responsible for the origin, evolution, and metabolism of life. Among the different types of
proteins in the body, human serum albumin (HSA) is the principal extracellular protein found in blood plasma,
corresponding to a concentration of 42 mg/mL. It exhibits several crucial physiological and pharmacological
functions such as maintaining osmotic blood pressure, buffering pH, and serving in the transportation and
distribution of a variety of substances such as fatty acids, amino acids, hormones, and pharmaceuticals. Serum
albumin has the ability to bind with most endogenous and exogenous compounds to form stable complexes.
The binding of proteins and drugs greatly influences the absorption, distribution, metabolism, and excretion of
1
typical drugs.
Bovine serum albumin (BSA) has been extensively used as a model protein for binding studies with
biologically active compounds due to its stability and solubility in aqueous media, its low speci?city, and its
1
strong resemblance to HSA in terms of homology sequence (76%) and 3D structures. The main regions of
∗Correspondence: rehman@gcu.edu.pk
1459
This work is licensed under a Creative Commons Attribution 4.0 International License.

IQBAL et al./Turk J Chem
ligand binding sites on BSA are located in subdomains IIA and IIIA, namely site I and site II. It has been
reported that some small molecules including hybrids based on azole heterocyclic systems bind exclusively to
2
specific sites, either to the tryptophan residue (site I) or to the tyrosine residue (site II) on BSA molecules.
Synthetic chemists are in continuous effort to design and synthesize versatile variety of hybrids based on
3−5
azole heterocyclic systems.
Nitrogen-based heterocyclic systems have been reported to have various phar-
6
macological applications. The 1,2,4-triazole core has been found to possess interesting biological activities,
namely antiinflammatory, anticancer, insecticidal, antiviral, antioxidant, analgesic, antimicrobial,
anticonvulsant, and antidepressant activities. Triazole derivatives have also been used as fungicides in agri-
chemicals as well as in pharmaceutical applications for the treatment of topical and systematic fungal infections.
The widespread use of triazole derivatives has generated extensive concerns regarding their distribution, free
concentration, and metabolism within the body, which are affected by the ligand/protein interactions in the
7
8
9
10
11
12
13
14
15
blood stream.
Based on these findings, it was of interest to synthesize a new series of triazole derivatives and evaluate
their interaction with BSA by studying the effect of varying substituents on the binding strength of the
triazole derivatives. The binding mode and binding site of the triazoles to BSA molecules were also studied.
These findings may provide basic data on the binding mechanism of the synthesized triazole derivatives with
blood carrier proteins, which can be helpful for the security of human health when applied in pharmaceutical
formulations.
2. Results and discussion
2.1. Chemistry
N -(substituted)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H -1,2,4-triazol-3-ylthio)acetamides
(8a–8i; Table 1; Scheme) were synthesized in good yield. Reaction of 4-nitrobenzene sulfonyl chloride
with ethyl isonipecotate produced ethyl 1-[(4-nitrophenyl)sulfonyl]piperidin-4-carboxylate (3), which on re-
fluxing with hydrazine monohydrate yielded 1-(4-nitrophenylsulfonyl)piperidin-4-carbohydrazide (4). Com-
pound 4 was first refluxed with phenyl isothiocyanate and then cyclized by KOH into 5-(1-(4-nitrophenylsul-
fonyl)piperidin-4-yl)-4-phenyl-4H -1,2,4-triazole-3-thiol (5). Nine different compounds of N -(substituted)-2-
(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H -1,2,4-triazol-3-ylthio)acetamide (8a–8i) were synthe-
sized by stirring compound 5 with N -aryl/aralkyl-2-bromoacetamides (7a–8i) in DMF at room temperature.
Table 1. Different aryl substitutions for 8a–8i.
Comp. R1 & R2
Comp. R1 & R2
8a
8b
8c
8d
8e
R1 =R2 = H
R1 = 2Et; R2 = H
R1 = 4Et; R2 = H
R1 = 2Et; R2 = 6Me 8i
R1 = 2Me; R2 = 3Me
8f
8g
8h
R1 = 2Me; R2 = 5Me
R1 = 2Me; R2 = 6Me
R1 = 3Me; R2 = 4Me
R1 = 3Me; R2 = 5Me
Compound 8e, a yellowish amorphous solid, was synthesized in good yield with the molecular formula
1
C29 H30 N6 O5 S2 as established by EI-MS through molecular ion peak at m/z 606 and by H NMR by counting
the number of protons. In the IR spectrum the values calculated at 3340 cm−1 , 3075 cm−1 , 1678 cm−1 , 1665
1460

IQBAL et al./Turk J Chem
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Scheme. General scheme for the synthesis of N -(substituted)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-
4H -1,2,4-triazol-3-ylthio)acetamide (8a–8i). Reagents and conditions: (A) 10 % Na2 CO3 (aq), H2 O, 8 h. (B)
N2 H4 •H2 O, EtOH, reflux, 5 h. (C) i. PhNCS, MeOH, reflux, 5 h. ii. 10% KOH, reflux, 6 h. (D) BrCH2 COBr,
15% Na2 CO3 (aq), H2 O, 1 h. (E) DMF, LiH, 2–4 h.
cm−1 , 1596 cm−1 , 1523 cm−1 , 1435 cm−1 , 1076 cm−1 , and 605 cm−1 were assigned to (N-H) stretching,
(Ar C-H) stretching, (C=N) stretching, (C=O) stretching, (Ar C=C) stretching, (N=O) stretching, (S=O)
1
stretching, (C-N) stretching, and (C-S) stretching, respectively. The spectrum of H NMR showed signals at
7.50 (d, J =8.7 Hz, 2H) and 6.64 (d, J =8.6 Hz, 2H) due to aromatic protons of the 4-nitrophenylsulfonyl
group. The signals at 7.56 (d, J =8.8 Hz, 2H), 7.47 (t, J =10.44 Hz, 1H), and 7.19 (d, J =7.62 Hz, 2H)
were due to aromatic protons of the phenyl group attached with a triazole ring. The signals for protons of the
N -substituted aromatic ring of acetamide were obtained at 7.60 (d, J =8.04 Hz, 1H), 7.19 (t, J =10.2 Hz, 1H),
and 6.98 (d, J =7.5 Hz, 1H). An amidic proton showed the signal at 8.56 (s, 1H, N-H) and piperidine showed
signals at 3.71–3.69 (m, 2H), 2.27–2.22 (m, 2H), 2.11 (br.s, 1H), and 1.83–1.81 (m, 4H). The signal at 3.94 (s,
2H) was due to a -CH2 group directly attached with a heteroatom and the signals at 2.29 (s, 3H) and 2.18
13
(s, 3H) showed the presence of two methyl groups. In the C NMR spectrum, peaks of aromatic quaternary
carbons were obtained at δ 167.1158.1, 137.3, 135.7, 132.8, and 132.4 for the whole molecule and two quaternary
carbons of the five-membered triazole ring appeared at 152.6 and 150.5. The carbonyl carbon of the acetamoyl
1461

IQBAL et al./Turk J Chem
group appeared at 168.9. The signals of aromatic carbons of phenyl attached to the sulfonyl group resonated
at 130.8 and 129.8 while the signals of two aromatic rings attached with the 1,2,4-triazole ring appeared at
127.6, 126.9, 125.7, 124.2, 122.8, and 113.9. Peaks of carbon atoms of the piperidine ring appeared at 45.5,
35.6, and 29.2. The carbons of methylene and two methyl groups appeared at 32.1, 20.6, and 13.8, respectively.
On the basis of these accumulative spectral data, the structure of compound 8e was determined as N -(2,3-
dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H -1,2,4-triazol-3-ylthio)acetamide.
2.2. Biological activities
2.2.1. BSA binding study
Triazole and its derivatives have been known for their wide range of biological activities, such as antibacterial,
antispasmodic, antiinflammatory, antiplatelet agglomeration and proliferation inhibition, and induction of
16
differentiation and apoptosis of tumor cells.
In this study, the interaction of selected triazole derivatives
with BSA (BSA) was investigated in order to provide some useful information on the therapeutic effects of
these compounds in pharmacology and pharmacodynamics.
The binding studies of selected triazole derivatives with BSA were evaluated in view of investigating the
effect of various substituents attached to the triazole ring on the interactions with the globular protein. The
binding of the triazole derivatives with BSA was carried out by fluorometric titrations of BSA with varying
concentration of the triazole derivatives (0–5.57 × 10−4 M). The binding abilities and binding site of the
synthesized derivatives were compared to that of fluorescent markers warfarin and ibuprofen. Fluorometric
titrations of BSA with and without compounds 8a–8i or markers were carried out at an emission of 336 nm
(excitation: 295 nm). The emission spectra of BSA in the presence of the different synthesized derivatives
are given in Figure 1. It was observed that BSA showed maximum emission at λem = 336 nm. Addition of
the synthesized compounds quenched the fluorescence intensity of BSA, showing that the 1,2,4-triazole-based
analogs were able to bind to BSA, causing some modification in the microenvironment around the amino acid
residues situated in the subdomain of BSA leading to a hypochromic shift in the fluorescence spectra. The
plots in Figures 2a–2e show the Stern–Volmer plots for the binding of BSA and compounds 8a–8i and the Ksv
values obtained from the slopes are summarized in Table 2.
Table 2. Stern–Volmer quenching constant and quenching rate constant of the different compound-BSA systems.
10)
Entry Compound Ksv (L mol−1
)
Kq (L mol−1s−1) (× 10
1
2
3
4
5
6
8a
8c
8d
8h
902.5
666.2
406.8
713.3
1633
3502
9
6
4
7
16
35
8i
Ibuprofen
Fluorescence quenching can be classified as either a static or dynamic process and is caused by different
mechanisms including excited state reactions, molecular rearrangements, energy transfer, formation of ground
17
state complexes, and collisional quenching. The type of quenching mechanism is usually interpreted by the
1462

IQBAL et al./Turk J Chem
Figure 1. a–e) Fluorescence spectra of BSA in presence of different amounts of selected compounds (8a, 8c, 8d, 8h,
8i) at 295 nm and 298 K (inset graph corresponding to the Stern–Volmer plot).
1463

IQBAL et al./Turk J Chem
Figure 2. a–e) Stern–Volmer plots of the interactions of selected compounds (8a, 8c, 8d, 8h, 8i) with BSA at 298 K.
18
Stern–Volmer equation:
Fo/F = 1 + KSV [Q] = Kqτo[Q] + 1,
(1)
1464

IQBAL et al./Turk J Chem
where Fo and F are the fluorescence intensities of BSA before and after addition of quencher, respectively;
Ksv is the Stern–Volmer quenching constant; [Q] is the concentration of the quencher; Kq is the apparent
bimolecular quenching rate constant; and τo is the average lifetime of the biomolecule without the quencher
19
and its value is 10−8 s.
In all cases, the calculated Kq values were found to be larger than the maximum scattering collision
10
quenching rate constant (2 × 10 M−1 s−1) in dynamic quenching. This indicated that the fluorescence
quenching process of BSA with the triazole compounds, as well as warfarin and ibuprofen, was primarily
20
governed by a static quenching mechanism, rather than a dynamic one.
The binding constant Ka and the number of binding sites n of synthesized compounds can be calculated
by using the following double logarithm equation:
log[(F0 − F)/F] = log Ka + n log[Q].
(2)
Ka and n are obtained from the intercept and slope of a linear plot of log (Fo – F)/F versus log [Q], respectively
(Figure 3), and the values are summarized in Table 3.
Table 3. Binding constant and number of binding site of compounds 8a–8i, warfarin, and ibuprofen with BSA at 298
K.
Entry Compound Ka (L mol−1
)
N
1
2
3
4
5
6
7
8a
8c
8d
8h
859
288.1
1175
1051
1820
1.03
0.89
1.23
0.94
0.96
1.07
1.26
8i
Warfarin
Ibuprofen
641,000
56,870
The different substituents on the triazole ring were found to affect the binding ability of the 1,2,4-triazole-
based derivatives with BSA. The presence of the methyl group at the meta position enhanced the affinity of the
triazole derivative to BSA (entries 4 and 5), giving rise to higher Ka values. This might be due to an increase
in molecular size, providing a larger hydrophobic area that can interact with the hydrophobic surface on the
21
protein molecule. However, when the methyl group was at the para position, a decrease in binding constant
was observed (entry 4).
2.2.2. Thermodynamics of BSA binding
The interactions between the triazole compounds and BSA may involve hydrophobic, electrostatic, or van der
Waals forces as well as hydrogen bonding. In order to elucidate the types of interaction occurring between
triazole compounds and BSA, thermodynamic parameters such as free energy (∆G), enthalpy (∆H), and
entropy (∆S) were determined and these were used to demonstrate the type of binding taking place.
The four binding modes through which small molecules can bind to biological macromolecules are
hydrogen bonds, hydrophobic forces, electrostatic forces, and van der Waals interactions. These interactions can
be described by thermodynamic parameters of enthalpy and entropy energies. If ∆H <0 and ∆S <0, van der
1465

IQBAL et al./Turk J Chem
Figure 3. a–e) Linear double logarithm plot for BSA and 8a, 8c, 8d, 8h, and 8i bindings.
1466

IQBAL et al./Turk J Chem
Waals forces and hydrogen bonds play major roles in the binding reaction. If ∆H >0 and ∆S >0, hydrophobic
22
interactions are dominant. Electrostatic forces are more prominent when ∆H <0 and ∆S >0.
The thermodynamic parameters of selected triazole analogs binding to BSA were studied at three different
temperatures. ∆G, ∆H, and ∆S values were estimated using Eqs. (3) and (4):
∆G = −RT ln K,
(3)
where T is the experimental temperature, Ka is the binding constant at T, and R is the gas constant. The
temperature-dependent thermodynamic parameters, ∆H and ∆S, can be analyzed using the van’t Hoff formula:
ln Ka = −(∆H/RT) + (∆S/R),
(4)
where Ka is the binding constant, R is the gas constant, and T is experimental temperature. To obtain values
of ∆H and ∆S, the graph of ln Ka against 1/T was plotted (Figure 4) and the results are tabulated in Table
4. From the slope and intercept of the linear van’t Hoff plot, values of ∆H and ∆S were obtained and the free
1
energy change, ∆G, was also estimated using the following relationship:
Figure 4. van’t Hoff plots for BSA-(8a, 8c, 8h) systems.
∆G = ∆H − T∆S.
(5)
From Figure 3, it can be observed that there is good linearity of the van’t Hoff plots. The negative ∆H and
negative ∆S indicated that the interactions with 8a–8i are enthalpically driven but not entropically favorable.
Moreover, the negative ∆G values at all temperatures for 8a–8i proved that the interactions between the
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IQBAL et al./Turk J Chem
Table 4. Binding constants and thermodynamic parameters obtained at three temperatures (298, 303, and 308 K).
System Temp. (T) K 1/T × 10−3 K−1 Ka L mol−1 ln Ka ∆G kJmol−1 ∆H kJmol−1 ∆S Jmol−1K−1
4
298
303
308
298
303
308
298
303
308
3.356
3.300
3.247
3.356
3.300
3.247
3.356
3.300
3.247
8.350 × 10
1.114 × 10
3.364 × 10
1.318 × 10
4.080 × 10
1.687 × 10
1.967 × 10
1.135 × 10
1.826 × 10
11.33 –28.07
–420.13
–156.98
–180.83
–1313.45
–448.24
–541.96
4
2
4
3
3
3
3
2
BSA-8a
BSA-8c
BSA-8i
9.32
8.12
9.49
8.31
7.43
7.58
7.03
5.21
–23.48
–20.79
–23.51
–20.93
–19.03
–18.78
–17.71
–13.34
synthesized compounds with BSA were spontaneous. From the thermodynamic parameters, the negative ∆H
and ∆S values indicated that van der Waals forces and hydrogen bonding play major roles in the binding of
compounds 8a–8i to BSA and have a great contribution to the stability of the complex formed. The triazole
derivatives contain electronegative atoms and therefore can form hydrogen bonds with the amino acid residues
in BSA.
2.2.3. Site-selective binding of selected compounds on BSA in the presence of site markers
It is reported that the structure of BSA consists of three homologous domains named I, II, and III and each
domain includes two subdomains called A and B. The major regions of ligand-binding sites on BSA are situated
in hydrophobic cavities known as subdomains IIA (Sudlow’s site 1) and IIIA (Sudlow’s site II).
The binding locations are usually studied with site markers that have specific binding sites and that can
help determine the drug-binding sites on BSA by performing competitive binding experiments. To determine
the binding site of compounds 8a–8i on BSA, warfarin and ibuprofen were selected as site markers for site I
and site II, respectively. A varied amount of compounds 8a–8i was added to a solution of site marker and BSA
and the resulting mixture was excited at 295 nm.
The fluorescence quenching data of the 8a–8i and BSA system in the presence of site markers were
analyzed using Eq. (2) and the results are tabulated in Table 5. The binding constants significantly decreased
in the presence of ibuprofen but to a lesser extent in the presence of warfarin. Therefore, this showed that
ibuprofen competed with the triazole derivatives in the subdomain IIIA of BSA, demonstrating that the binding
of the triazole derivatives with BSA primarily takes place at site II.
2.2.4. Synchronous study of the binding of 1,2,4-triazole analogs to BSA
Synchronous fluorescence spectroscopy was carried out to confirm the binding site of compounds 8a–8i with
BSA by monitoring the change in the microenvironment surrounding Trp and Tyr residues. The shift in the
position of the fluorescence emission maximum corresponded to modification of the polarity around Trp and
Tyr residues. A blue shift of λ
indicated that the amino acid residues were situated in a more hydrophobic
max
environment while a red shift of λ
implied that the amino acid residues were in a polar environment. It is
max
possible to study the change in the conformation of BSA induced by triazole-based synthesized compounds by
measuring the fluorescence intensity of BSA before and after the addition of the compound. From the literature
information, the synchronous fluorescence spectra obtained at ∆λ = 15 nm and ∆λ = 60 nm corresponded to
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IQBAL et al./Turk J Chem
Table 5. Binding constant of selected triazole compounds with BSA in presence of site markers.
System
Site marker
Blank (without marker) 3.70 × 10
Warfarin
Ibuprofen
Blank (without marker) 1.60 × 10
Warfarin
Ibuprofen
Blank (without marker) 9.42 × 10
Warfarin
Ibuprofen
Ka (L mol−1
)
N
3
1.06
0.95
0.92
0.64
1.05
0.94
1.21
0.86
1.11
3
2
3
3
2
3
3
2
BSA-8a
2.70 × 10
5.15 × 10
BSA-8c
BSA-8i
1.24 × 10
9.60 × 10
3.10 × 10
5.49 × 10
23
the Tyr and Trp residues, respectively. The synchronous spectra for BSA-TDs are displayed in Figure 5 for
∆λ = 15 nm and in Figure 6 for ∆λ = 60 nm.
393.1
582.5
350
300
250
200
150
100
50
550
500
450
400
350
300
250
200
150
100
50
-5.0
1.6
270.0
280
290
300
nm
310
320
327.5
270.0
280
290
300
nm
310
320
327.5
BSA+8a
BSA+8c
480.8
450
400
350
300
250
200
150
100
50
-5.0
270.0
280
290
300
nm
310
320
327.5
BSA+8i
Figure 5. Synchronous fluorescence spectra of BSA in the presence of 8a, 8c, and 8i at ∆λ = 15 nm (red shift).
1469

IQBAL et al./Turk J Chem
637.8
600
457.5
500
400
300
200
400
350
300
250
200
150
100
100
8.2
50
6.8
270.0
280
290
300
nm
310
320
327.5
270.0
280
290
300
nm
310
320
327.5
BSA+8i
BSA+8a
571.8
500
450
400
350
300
250
200
150
100
50
5.4
270.0
280
290
300
nm
310
320
327.5
BSA+8c
Figure 6. Synchronous fluorescence spectra of BSA in the presence of 8a, 8c, and 8i at ∆λ = 60 nm.
The results revealed that the fluorescence intensity of both Trp and Tyr decreased with a notable red
shift at maximum emission ∆λ = 15 nm in the presence of compounds 8a–8i, whereas no significant shift was
observed at ∆λ = 60 nm for 8a–8i. This observation confirmed that the triazole derivatives were bound to the
tyrosine residue of BSA, in a similar manner to that of ibuprofen.
Infrared spectroscopy is considered to be a useful technique to investigate the secondary structure of
proteins. In the FT-IR region, amide I, II, and III vibrations are related to the frequencies of the bands,
especially that of amide I, as it is more sensitive to change of the secondary structure. It was reported that the
amide I peak is situated in the region of 1600–1700 cm−1 (mainly C=O stretching) and the amide II peak at
1
1548 cm−1 (C-N stretch coupled with N-H bending mode). Comparing the FT-IR spectra of BSA with and
without triazole-derived compounds 8a–8i, it was found that the peak of amide I, which corresponds to the
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IQBAL et al./Turk J Chem
(C=O) stretching, shifted from 1651.72 cm−1 to 1652.05 cm−1 , indicating that the secondary structure of BSA
was altered due to the interaction between compounds 8a–8i and BSA. From FT-IR analysis, a change in the
absorbance of BSA in the presence of compound 8a–8i was observed (Figure 7). The spectrum of BSA deforms
as the ratio of peak heights in the absence and in the presence of compounds 8a–8i (diluted and concentrated)
is different. This result indicates that the secondary structure of BSA has been altered due to the interaction
of BSA with compounds 8a–8i.
Figure 7. FT-IR difference spectra of free BSA and BSA + compound.
2.3. Conclusions
Results of the fluorescence studies showed that the triazole derivatives were able to interact with BSA, causing
a static quenching of the fluorescence intensity of BSA in a similar way to that of the well-known drugs warfarin
and ibuprofen. The presence and position of different substituents on the triazole ring was found to affect
the binding affinity of the synthesized compounds for BSA. The thermodynamic parameters indicated that the
binding between the triazole analogs and BSA was spontaneous and involved mostly van der Waals forces and
hydrogen bonding. Synchronous fluorescence data and competitive binding of compounds 8a–8i in the presence
of warfarin and ibuprofen illustrated that compounds 8a–8i were bound to the same binding site as that of
ibuprofen, i.e. on tyrosine residues of BSA. This study showed that the 1,2,4-triazole-based compounds were
able to bind to BSA and therefore can be effectively transported and eliminated in the body, which can be a
useful guideline to open new avenues for the designing of the most suitable triazole derivatives for further drug
design.
3. Experimental
3.1. General
All the chemicals were of analytical grade and were obtained from Sigma Aldrich and Alfa Aesar Ltd. and
were provided by local chemical trading companies in Pakistan. Phosphate buffer (20 mM, pH 7.4), BSA,
1471

IQBAL et al./Turk J Chem
calf-thymus DNA, Tris-buffered saline (0.05 M, pH 7.6), and ethidium bromide were purchased from Sigma
Aldrich (St. Louis, MO, USA). IR spectra were recorded on a Bruker Alpha FT-IR spectrometer. Aluminum
plates coated with silica gel were used to confirm the purity of synthesized compounds with an n-hexane : ethyl
1
13
acetate mobile phase on TLC and visualized with a UV254 lamp. H NMR (600 MHz, CDCl3), C NMR (150
MHz, CDCl3), and EI-MS data were recorded using a Bruker spectrometer and JMS-HX-110 spectrometer,
respectively.
3.2. Synthesis
3.2.1. Synthesis of ethyl 1-(4-nitrophenylsulfonyl)piperidin-4-carboxylate (3)
Ethyl 1-[(4-nitrophenyl)sulfonyl]piperidin-4-carboxylate (3) was synthesized by stirring 4-nitrobenzene sulfonyl
chloride (1; 0.05 mol) with ethyl isonipecotate (2; 0.05 mol) at room temperature for 8 h in distilled water (30
mL). The pH of the reaction was maintained at 9–10 using 10% Na2 CO3 solution. Completion of the reaction
was confirmed by TLC and then chilled distilled water was added to precipitate the product, which was filtered,
washed, and dried.
3.2.2. Synthesis of 1-(4-nitrophenylsulfonyl)piperidine-4-carbohydrazide (4)
Ethyl 1-[(4-chlorophenyl)sulfonyl]piperidin-4-carboxylate (3; 0.043 mol) was refluxed with hydrazine monohy-
drate (0.043 mol) using ethanol as a solvent for 5 h. TLC was used to monitor the reaction. At the completion
of reaction, an excess of chilled distilled water was added with continuous shaking and the reaction mixture
was kept at ice-cold temperature for 5 h. The obtained precipitate was filtered, washed, and dried at room
temperature.
3.2.3. Synthesis of 5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol (5)
Compound 4 (0.030 mol) was refluxed with phenyl isothiocyanate (0.030 mol) in methanol for 5 h. The
uncyclized precipitates obtained were filtered, washed, dried, and refluxed with 10% KOH for 6 h in distilled
water. At the completion of the reaction as monitored by TLC, diluted HCl was added to the mixture drop by
drop with continuous shaking until the pH was 5. After 30 min an off-white solid, compound 5, was obtained,
which was filtered, washed with distilled water, and dried at room temperature.
3.2.4. Synthesis of N-(substituted)-2-bromoacetamides (7a–8i)
Alkylaryl/aryl amines (6a–6i; 0.02 mol) were stirred with 2-bromoacetyl bromide (0.02 mol) in distilled water for
1 h. The pH was adjusted to 9–10 using 15% Na2 CO3 solution. The formed precipitates were filtered, washed
with distilled water, and dried. Those products that were not obtained in precipitate form were extracted using
chloroform.
3.2.5. Synthesis of N-(substituted)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-
1,2,4-triazol-3-ylthio)acetamide (8a–8i)
Compound 5 (0.0044 mol) was stirred with LiH in DMF (15 mL) for 30 min, and then an equimolar amount of
N -(substituted)-2-bromoacetamides (7a–7i) was added. The whole mixture was stirred at room temperature
for 2–4 h. The completion of the reaction was confirmed with TLC. Chilled distilled water was added after the
completion of the reaction to obtain the targeted products, which were filtered, washed, and dried.
1472

IQBAL et al./Turk J Chem
3.3. Spectral characterization of synthesized compounds
3.3.1. 2-(5-(1-(4-Nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-pheny-
lacetamide (8a)
Yellowish amorphous solid; yield: 79%; mp: 140–141 °C; molecular formula C27 H26 N6 O5 S2 ; molecular mass:
578.66 g mol−1 ; IR (KBr, υmax , cm−1): 3343 (N-H), 3109 (Ar C-H), 1654 (C=N), 1639 (C=O), 1584 (Ar
1
C=C), 1538 (N=O), 1445 (S=O), 1150, 1146 (C-N), 619 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 10.27
(s, 1H, N-H), 7.57 (d, J = 7.8 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.54 (d, J =8.8 Hz, 2H, H-2′′′ , H-6′′′ ), 7.49 (d, J =8.9
Hz, 2H, H-3′′ , H-5′′ ), 7.45 (d, J =7.7 Hz, 2H, H-3′′′ , H-5′′′ ), 7.31 (t, J = 8.2 Hz, 1H, H-4′′′′ ), 7.21 (d, J =
7.7 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.10 (t, J = 7.4 Hz, 1H, H-4′′′ ), 6.66 (d, J = 8.6 Hz, 2H, H-2′′ , H-6′′ ), 4.11 (s,
2H, H-1′′′′′ ), 3.67–3.65 (m, 2H, Heq -2′ , Heq -6′ ), 2.50–2.46 (m, 1H, H-4′ ), 2.27–2.23 (m, 2H, Hax -2′ , Hax -6′ ),
13
2.04–2.00 (m, 2H, Heq -3′ , Heq -5′ ), 1.97–1.83 (m, 2H, Hax -3′ , Hax -5′ );
C NMR (150 MHz, CDCl3): δ
168.2 (C-2′′′′′ ), 166.6 (C-4′′ ), 158.0 (C-1′′ ), 150.5 (C-1′′′′′′ ), 138.2 (C-2′′′′′′ ), 132.4 (C-1′′′′ ), 130.7 (C-1′′′ ),
130.2 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 128.8 (C-2′′′′ , C-6′′′′ ), 126.6 (C-2′′′ , C-6′′′ ), 124.1 (C-3′′′ , C-5′′′ ),
122.9 (C-4′′′ ), 119.7 (C-4′′′′ ), 113.9 (C-3′′′′ , C-5′′′′ ), 45.5 (C-2′ , C-6′ ), 36.1 (C-4′ ), 32.1 (C-1′′′′′ ), 29.4 (C-3′ ,
+
+
+
+
C-5′ ); EI-MS (m/z): 578 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] , 186
+
+
+
+
[C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 78 [C6 H6 ] , 51 [C4 H4 ] ; Anal. Calcd. for C27 H26 N6 O5 S2 : C 56.04,
H 4.53, N 14.52, O 13.82, S 11.08; found C 56.01, H 4.31, N 14.29, O 13.56, S 11.02.
3.3.2. N-(2-Ethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-triazol-
3-ylthio)acetamide (8b)
Light yellowish amorphous solid; yield: 85%; mp: 137–138 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular
mass: 606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3341 (N-H), 3134 (Ar C-H), 1652 (C=N), 1638 (C=O), 1596
1
(Ar C=C), 1546 (N=O), 1441 (S=O), 1125 (C-N), 633 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 9.66
(s, 1H, N-H), 7.66 (s, 1H, H-6′′′ ), 7.54 (d, J = 9.1 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.49 (d, J =8.5 Hz, 2H, H-3′′ ,
H-5′′ ), 7.39–7.38 (m, 1H, H-4′′′′ ), 7.20 (d, J = 7.7 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.17 (d, J = 10.9 Hz, 1H, H-3′′′ ),
7.10 (t, J = 8.2 Hz, 1H, H-5′′′ ), 7.02 (t, J = 7.9 Hz, 1H, H-4′′′ ), 6.66 (d, J = 8.6 Hz, 2H, H-2′′ , H-6′′ ), 4.11
(s, 2H, H-1′′′′′ ), 3.69–3.67 (m, 2H, Heq -2′ , Heq -6′ ), 2.63 (q, J = 8 Hz, 2H, H-2′′′′′ ), 2.50–2.48 (m, 1H, H-4′ ),
2.33–2.29 (m, 2H, Hax -2′ , Hax -6′ ), 1.98 (dq, J = 3.6, 13.8 Hz, 2H, Heq -3′ , Heq -5′ ), 1.84–1.82 (m, 2H, H-3′ ,
13
H-5′ ), 1.14 (t, J = 7.5 Hz, 3H, H-3′′′′′ ); C NMR (150 MHz, CDCl3): δ 168.8 (C-4′′′′′ ), 167.1 (C-4′′ ), 158.2
(C-1′′ ), 152.7 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 135.5 (C-1′′′′ ), 132.3 (C-1′′′ ), 130.7 (C-3′′′ ), 130.4 (C-3′′ , C-5′′ ), 129.8
(C-2′′ , C-6′′ ), 128.7 (C-6′′′ ), 127.5 (C-2′′′ ), 126.9 (C-2′′′′ , C-6′′′′ ), 126.4 (C-5′′′ ), 125.3 (C-4′′′ ), 123.3 (C-4′′′′ ),
113.9 (C-3′′′′ , C-5′′′′ ), 45.4 (C-2′ , C-6′ ), 35.5 (C-4′ ), 31.9 (C-1′′′′′ ), 29.4 (C-3′ , C-5′ ), 24.4 (C-2′′′′′ ), 14.2
+
+
+
+
(C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] ,
+
+
+
+
186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 106 [C8 H10 ] , 51 [C4 H4 ] ; Anal. Calcd. for C29 H30 N6 O5 S2 : C
57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.41, H 4.66, N 13.67, O 13.06, S 10.39.
3.3.3. N-(4-Ethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-triazol-
3-ylthio)acetamide (8c)
Yellowish amorphous solid; yield: 78%; mp: 111-112 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular mass:
606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3349 (N-H), 3138 (Ar C-H), 1657 (C=N), 1630 (C=O), 1598 (Ar
1473

IQBAL et al./Turk J Chem
1
C=C), 1544 (N=O), 1449 (S=O), 1124 (C-N), 637 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 10.12 (s,
1H, N-H), 7.53 (d, J = 9.3 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.52 (d, J =9.0 Hz, 2H, H-3′′ , H-5′′ ), 7.50–7.48 (m,
1H, H-4′′′′ ), 7.20 (d, J = 7.8 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.19 (d, J = 8.3 Hz, 2H, H-2′′′ , H-6′′′ ), 7.14–7.13 (m,
2H, H-3′′′ , H-5′′′ ), 6.66 (d, J = 8.6 Hz, 2H, H-2′′ , H-6′′ ), 4.12 (s, 2H, H-1′′′′′ ), 3.72–3.70 (m, 2H, Heq -2′ ,
Heq -6′ ), 2.60 (q, J = 4.3 Hz, 2H, H-2′′′′′ ), 2.49–2.47 (m, 1H, H-4′ ), 2.26–2.22 (m, 2H, Hax -2′ , Hax -6′ ), 1.99
13
(dq, J = 3.8, 9.0 Hz, 2H, Heq -3′ , Heq -5′ ), 1.84–1.82 (m, 2H, H-3′ , H-5′ ), 1.20 (t, J = 4.5 Hz, H-3′′′′′ );
C
NMR (150 MHz, CDCl3): δ 168.9 (C-4′′′′′ ), 167.4 (C-4′′ ), 158.0 (C-1′′ ), 152.8 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 140.3
(C-1′′′′ ), 135.8 (C-1′′′ ), 132.1 (C-4′′′ ), 130.7 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 128.2 (C-2′′′′ , C-6′′′′ ), 126.9
(C-2′′′ , C-6′′′ ), 124.2 (C-4′′′′ ), 119.8 (C-3′′′′ , C-5′′′′ ), 113.9 (C-3′′′ , C-5′′′ ), 45.5 (C-2′ , C-6′ ), 36.0 (C-4′ ), 32.1
+
+
(C-1′′′′′ ), 29.4 (C-3′ , C-5′ ), 28.3 (C-2′′′′′ ), 15.7 (C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] ,
+
+
+
+
+
295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] , 186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 106 [C8 H10 ] , 51
[C4 H4 ] ; Anal. Calcd. for C29 H30 N6 O5 S2 : C 57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.40, H
+
4.66, N 13.67, O 13.07, S 10.39.
3.3.4. N-(2-Ethyl-6-methylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-
1,2,4-triazol-3-ylthio)acetamide (8d)
Light green amorphous solid; yield: 81%; mp: 129–130 °C; molecular formula: C30 H32 N6 O5 S2 ; molecular
mass: 620.74 g mol−1 ; IR (KBr, υmax , cm−1): 3332 (N-H), 3128 (Ar C-H), 1646 (C=N), 1644 (C=O), 1582
1
(Ar C=C), 1534 (N=O), 1443 (S=O), 1133 (C-N), 632 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 8.56
(s, 1H, N-H), 7.56 (d, J = 9.0 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.50 (d, J =7.2 Hz, 2H, H-3′′ , H-5′′ ), 7.21 (t, J =
8.0 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.38–7.35 (m, 1H, H-4′′′′ ), 7.16–7.13 (m, 2H, H-3′′′ , H-5′′′ ), 7.05 (t, J = 6.8
Hz, 1H, H-4′′′ ), 6.65 (d, J = 11.1 Hz, 2H, H-2′′ , H-6′′ ), 4.09 (s, 2H, H-1′′′′′ ), 3.71–3.66 (m, 2H, Heq -2′ ,
Heq -6′ ), 2.40 (s, 3H, H-2′′′′′ ), 2.33–2.29 (m, 2H, Hax -2′ , Hax -6′ ), 2.25–2.24 (m, 1H, H-4′ ), 1.98–1.96 (m,
13
4H, H-3′ , H-5′ ), 1.26 (q, J = 4.9 Hz, 2H, H-3′′′′′ ), 1.08 (t, J = 7.5 Hz, 3H, H-4′′′′′ ); C NMR (150 MHz,
CDCl3): δ 169.2 (C-5′′′′′ ), 167.8 (C-4′′ ), 158.2 (C-1′′ ), 155.8 (C-1′′′′′′ ), 152.1 (C-2′′′′′′ ), 135.3 (C-1′′′′ ), 133.9
(C-1′′′ ), 131.8 (C-2′′′ , C-6′′′ ), 130.8 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 128.6 (C-2′′′′ , C-6′′′′ ), 127.1 (C-4′′′′ ),
126.4 (C-3′′′ , C-5′′′ ), 125.0 (C-4′′′ ), 116.4 (C-3′′′′ , C-5′′′′ ), 45.3 (C-2′ , C-6′ ), 36.1 (C-4′ ), 32.3 (C-1′′′′′ ), 30.2
+
+
(C-3′ , C-5′ ), 21.7 (C-2′′′′′ ), 19.1 (C-3′′′′′ ), 15.8 (C-4′′′′′ ); EI-MS (m/z): 620 [M] , 386 [C18 H18 N4 O4 S] ,
+
+
+
+
+
295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] , 186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 120 [C9 H12 ] , 66
[C5 H6 ] ; Anal. Calcd. for C30 H32 N6 O5 S2 : C 58.05, H 5.20, N 13.54, O 12.89, S 10.33; found C 58.01, H
+
5.09, N 13.32, O 12.78, S 10.24.
3.3.5. N-(2,3-Dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-
triazol-3-ylthio)acetamide (8e)
Yellowish amorphous solid; yield: 78%; mp: 123–124 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular mass:
606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3340 (N-H), 3075 (Ar C-H), 1678 (C=N), 1665 (C=O), 1596 (Ar
1
C=C), 1523 (N=O), 1435 (S=O), 1076 (C-N), 605 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 8.56 (s,
1H, N-H), 7.60 (d, J =8.04 Hz, 1H, H-6′′′ ), 7.56 (d, J =8.8 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.50 (d, J =8.7 Hz, 2H,
H-3′′ , H-5′′ ), 7.47 (t, J =10.44 Hz, 1H, H-4′′′′ ), 7.19 (d, J =10.2 Hz, 1H, H-5′′′ ), 7.07 (t, J =7.62 Hz, 2H,
H-3′′′′ , H-5′′′′ ), 6.98 (d, J =7.5 Hz, 1H, H-4′′′ ), 6.64 (d, J =8.6 Hz, 2H, H-2′′ , H-6′′ ), 3.94 (s, 2H, H-1′′′′′ ),
3.71–3.69 (m, 2H, Heq -2′ , Heq -6′ ), 2.29 (s, 3H, H-2′′′′′ ), 2.27–2.22 (m, 2H, Hax -2′ ,Hax -6′ ), 2.18 (s, 3H, H-
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IQBAL et al./Turk J Chem
13
3′′′′′ ), 2.11 (br.s, 1H, H-4′ ), 1.83–1.81 (m, 4H, H-3′ , H-5′ ); C NMR (150 MHz, CDCl3): δ 168.9 (C-4′′′′′ ),
167.1 (C-4′′ ), 158.1 (C-1′′ ), 152.6 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 137.3 (C-1′′′′ ), 135.7 (C-1′′′ ), 132.8 (C-2′′′ ), 132.4
(C-3′′′ ), 130.8 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 127.6 (C-2′′′′ , C-6′′′′ ), 126.9 (C-6′′′ ), 125.7 (C-5′′′ ), 124.2
(C-4′′′ ), 122.8 (C-4′′′′ ), 113.9 (C-3′′′′ , C-5′′′′ ), 45.5 (C-2′ , C-6′ ), 35.6 (C-4′ ), 32.1 (C-1′′′′′ ), 29.2 (C-3′ , C-5′ ),
+
+
+
20.6 (C-2′′′′′ ), 13.8 (C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270
+
+
+
+
+
[C11 H14 N2 O4 S] , 186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 106 [C8 H10 ] , 51 [C4 H4 ] ; Anal. Calcd. for
C29 H30 N6 O5 S2 : C 57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.23, H 4.78, N 13.66, O 13.11, S 10.36.
3.3.6. N-(2,5-Dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-
triazol-3-ylthio)acetamide (8f)
Light brown amorphous solid; yield: 87%; mp: 122–123 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular
mass: 606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3335 (N-H), 3077 (Ar C-H), 1679 (C=N), 1635 (C=O), 1598
1
(Ar C=C), 1525 (N=O), 1437 (S=O), 1089 (C-N), 609 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 9.62 (s,
1H, N-H), 7.75 (s, 1H, H-6′′′ ), 7.58 (d, J =6.8 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.49 (d, J =8.6 Hz, 2H, H-3′′ , H-5′′ ),
7.20 (d, J =7.8 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.19 (d, J =7.6 Hz, 1H, H-3′′′ ), 7.06–7.04 (m, 1H, H-4′′′′ ), 6.86 (d,
J =7.5, 10.2 Hz, 1H, H-4′′′ ), 6.65 (d, J =8.6 Hz, 2H, H-2′′ , H-6′′ ), 4.11 (s, 2H, H-1′′′′′ ), 3.75–3.70 (m, 2H,
Heq -2′ , Heq -6′ ), 2.90–2.80 (m, 2H, Hax -2′ ,Hax -6′ ), 2.47–2.43 (m, 1H, H-4′ ), 2.30 (s, 3H, H-2′′′′′ ), 2.23 (s,
13
3H, H-3′′′′′ ), 1.99–1.98 (m, 2H, Heq -3′ , Heq -5′ ), 1.83–1.81 (m, 2H, Hax -3′ , Hax -5′ ); C NMR (150 MHz,
CDCl3): δ 166.9 (C-4′′′′′ ), 166.9 (C-4′′ ), 158.1 (C-1′′ ), 152.6 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 136.2 (C-1′′′′ ), 135.9
(C-1′′′ ), 132.3 (C-2′′′ ), 130.7 (C-6′′′ ), 130.4 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 126.9 (C-2′′′′ , C-6′′′′ ), 126.1
(C-3′′′ ), 125.6 (C-4′′′ ), 124.2 (C-5′′′ ), 122.5 (C-4′′′′ ), 113.0 (C-3′′′′ , C-5′′′′ ), 45.4 (C-2′ , C-6′ ), 36.7 (C-4′ ), 31.9
+
+
(C-1′′′′′ ), 29.3 (C-3′ , C-5′ ), 21.1 (C-2′′′′′ ), 17.7 (C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] ,
+
+
+
+
+
295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] , 186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 106 [C8 H10 ] , 51
[C4 H4 ] ; Anal. Calcd. for C29 H30 N6 O5 S2 : C 57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.21, H
+
4.78, N 13.63, O 13.11, S 10.36.
3.3.7. N-(2,6-Dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-
triazol-3-ylthio)acetamide (8g)
Light brown amorphous solid; yield: 77%; mp: 102–103 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular
mass: 606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3421 (N-H), 3074 (Ar C-H), 1672 (C=N), 1637 (C=O), 1592
1
(Ar C=C), 1528 (N=O), 1431 (S=O), 1072 (C-N), 603 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 9.32
(s, 1H, N-H), 7.56 (d, J =7.6 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.49 (d, J = 8.5 Hz, 2H, H-3′′ , H-5′′ ), 7.20–7.19 (m,
3H, H-3′′′′ , H-4′′′′ , H-5′′′′ ), 7.09 (d, J =7.0 Hz, 2H, H-3′′′ , H-5′′′ ), 7.06 (t, J =7.3 Hz, 1H, H-4′′′ ), 6.64 (d,
J =8.5 Hz, 2H, H-2′′ , H-6′′ ), 4.09 (s, 2H, H-1′′′′′ ), 3.68–3.66 (m, 2H, Heq -2′ , Heq -6′ ), 2.32–2.25 (m, 1H, H-4′ ),
2.32–2.25 (m, 2H, Hax -2′ ,Hax -6′ ), 2.21 (s, 3H, H-2′′′′′ ), 2.20 (s, 3H, H-3′′′′′ ), 1.96 (dq, J = 3.7, 11.0 Hz, 2H,
13
Heq -3′ , Heq -5′ ), 1.84–1.82 (m, 2H, Hax -3′ , Hax -5′ ); C NMR (150 MHz, CDCl3): δ 168.7 (C-4′′′′′ ), 167.2
(C-4′′ ), 158.1 (C-1′′ ), 152.8 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 135.3 (C-1′′′′ ), 133.9 (C-1′′′ ), 132.4 (C-2′′′ , C-6′′′ ),
130.5 (C-3′′ , C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 128.1 (C-2′′′′ , C-6′′′′ ), 127.2 (C-4′′′′ ), 126.9 (C-3′′′ , C-5′′′ ), 124.2
(C-4′′′ ), 113.9 (C-3′′′′ , C-5′′′′ ), 45.4 (C-2′ , C-6′ ), 34.9 (C-4′ ), 31.9 (C-1′′′′′ ), 29.3 (C-3′ , C-5′ ), 18.2 (C-2′′′′′
,
+
+
+
+
C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] ,
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IQBAL et al./Turk J Chem
+
+
+
+
186 [C6 H4 NO4 S] , 123 [C6 H5 NO2 ] , 120 [C9 H12 ] , 66 [C5 H6 ] ; Anal. Calcd. for C29 H30 N6 O5 S2 : C
57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.21, H 4.78, N 13.66, O 13.10, S 10.36.
3.3.8. N-(3,4-Dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-
triazol-3-ylthio)acetamide (8h)
Light green amorphous solid; yield: 82%; mp: 94–95 °C; molecular formula: C29 H30 N6 O5 S2 ; molecular mass:
606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3422 (N-H), 3071 (Ar C-H), 1670 (C=N), 1634 (C=O), 1593 (Ar
1
C=C), 1520 (N=O), 1439 (S=O), 1228, 1077 (C-N), 602 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 8.02
(s, 1H, N-H), 7.56 (d, J =7.2 Hz, 1H, H-6′′′ ), 7.55 (d, J =8 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.54 (d, J =8 Hz, 2H,
H-3′′ , H-5′′ ), 7.35 (s, 1H, H-2′′′ ), 7.34–7.32 (m, 1H, H-4′′′′ ), 7.20–7.19 (m, 2H, H-3′′′′ , H-5′′′′ ), 7.06 (d, J =8
Hz, 1H, H-5′′′ ), 6.66 (d, J = 6.8 Hz, 2H, H-2′′ , H-6′′ ), 4.11 (s, 2H, H-1′′′′′ ), 3.73–3.71 (m, 2H, Heq -2′ , Heq -6′ ),
2.47 (dt, J = 2.7, 11.8 Hz, 2H, Hax -2′ , Hax -6′ ), 2.20 (s, 3H, H-2′′′′′ ), 2.13 (s, 3H, H-3′′′′′ ), 2.10 (m, 1H, H-4′ ),
13
1.84–1.82 (m, 4H, H-3′ , H-5′ ); C NMR (150 MHz, CDCl3): δ 167.9 (C-4′′′′′ ), 166.4 (C-4′′ ), 158.0 (C-1′′ ),
152.8 (C-1′′′′′′ ), 150.5 (C-2′′′′′′ ), 137.1 (C-1′′′′ ), 135.9 (C-1′′′ ), 132.5 (C-3′′′ ), 132.4 (C-4′′′ ), 130.7 (C-3′′ , C-
5′′ ), 129.8 (C-2′′ , C-6′′ ), 126.9 (C-2′′′′ , C-6′′′′ ), 121.1 (C-6′′′ ), 120.2 (C-2′′′ ), 119.5 (C-5′′′ ), 117.3 (C-4′′′′ ),
114.0 (C-3′′′′ , C-5′′′′ ), 45.5 (C-2′ , C-6′ ), 36.1 (C-4′ ), 32.2 (C-1′′′′′ ), 29.4 (C-3′ , C-5′ ), 19.8 (C-2′′′′′ ), 19.1
+
+
+
+
(C-3′′′′′ ); EI-MS (m/z): 606 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] ,
+
+
+
+
+
+
186 [C6 H4 NO4 S] , 177 [C11 H15 NO] , 123 [C6 H5 NO2 ] , 106 [C8 H10 ] , 66 [C5 H6 ] , 51 [C4 H4 ] ; Anal.
Calcd. for C29 H30 N6 O5 S2 : C 57.41, H 4.98, N 13.85, O 13.19, S 10.57; found C 57.22, H 4.78, N 13.64, O
13.11, S 10.36.
3.3.9. N-(3,5-Dimethylphenyl)-2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)-4-phenyl-4H-1,2,4-
triazol-3-ylthio)acetamide (8i)
Off-white amorphous solid; yield: 85%; mp: 127–128 °C; molecular formula C29 H30 N6 O5 S2 ; molecular mass:
606.72 g mol−1 ; IR (KBr, υmax , cm−1): 3323 (N-H), 3105 (Ar C-H), 1652 (C=N), 1638 (C=O), 1586 (Ar
1
C=C), 1532 (N=O), 1442 (S=O), 1152, 1143 (C-N), 611 (C-S); H NMR (CDCl3 , 600 MHz, δ (ppm)): 9.62
(s, 1H, N-H), 7.76 (s, 2H, H-2′′′ , H-6′′′ ), 7.56 (d, J =7.4 Hz, 2H, H-2′′′′ , H-6′′′′ ), 7.49 (d, J =8.6 Hz, 2H,
H-3′′ , H-5′′ ), 7.26 (d, J = 8.2 Hz, 2H, H-3′′′′ , H-5′′′′ ), 7.05–7.04 (m, 1H, H-4′′′′ ), 6.87 (s, 1H, H-4′′′ ), 6.66
(d, J = 8.6 Hz, 2H, H-2′′ , H-6′′ ), 4.11 (s, 2H, H-1′′′′′ ), 3.68–3.66 (m, 2H, Heq -2′ , Heq -6′ ), 2.95–2.86 (m, 2H,
Hax -2′ , Hax -6′ ), 2.50–2.47 (m, 1H, H-4′ ), 2.30 (s, 3H, 2′′′′′ ), 2.24 (s, 3H, 3′′′′′ ), 2.01–2.00 (m, 2H, Heq -3′ ,
13
Heq -5′ ), 1.96–1.81 (m, 2H, Hax -3′ , Hax -5′ ); C NMR (150 MHz, CDCl3): δ 168.5 (C-4′′′′′ ), 166.9 (C-4′′ ),
158.1 (C-1′′ ), 152.6 (C-1′′′′′′ ) , 150.5 (C-2′′′′′′ ), 136.2 (C-1′′′′ ), 135.9 (C-1′′′ ), 132.4 (C-3′′′ , C-5′′′ ), 130.2 (C-3′′ ,
C-5′′ ), 129.8 (C-2′′ , C-6′′ ), 126.9 (C-2′′′′ , C-6′′′′ ), 125.6 (C-2′′′ , C-6′′′ ), 122.9 (C-4′′′′ ), 113.9 (C-3′′′′ , C-5′′′′ ),
45.4 (C-2′ , C-6′ ), 35.6 (C-4′ ), 31.9 (C-1′′′′′ ), 29.3 (C-3′ , C-5′ ), 21.1 (C-2′′′′′ ), 17.7 (C-3′′′′′ ); EI-MS (m/z):
+
+
+
+
+
606 [M] , 386 [C18 H18 N4 O4 S] , 295 [C12 H13 N3 O4 S] , 270 [C11 H14 N2 O4 S] , 186 [C6 H4 NO4 S] , 123
+
+
+
+
[C6 H5 NO2 ] , 106 [C8 H10 ] , 66 [C5 H6 ] , 51 [C4 H4 ] ; Anal. Calcd. for C29 H30 N6 O5 S2 : C 57.41, H
4.98, N 13.85, O 13.19, S 10.57; found C 57.21, H 4.78, N 13.62, O 13.10, S 10.36.
3.4. Assays
3.4.1. BSA interactions using fluorescence measurements
For the determination of the BSA quenching constant, fluorometric titration of a solution of BSA (3 mL, 3 µM)
in phosphate buffer (20 mM, pH 7.4) with compounds 8a–8i (1 mg/mL in DMSO solvent) was carried out. The
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solutions were excited at 295 nm and the intensities of the BSA solutions with and without compounds 8a–8i
were recorded at 336 nm at 298 K. For thermodynamic studies of BSA and synthesized derivatives, the above
experiment was repeated at other two temperatures, 303 K and 308 K, respectively, for each of the synthesized
compounds 8a–8i. Thus, three sets of fluorescence spectra were obtained at three temperatures: 298, 303, and
308 K.
For site-selective binding studies, fluorometric titration of BSA solution (3 mL, 3 µM) with and without
site markers (ibuprofen or warfarin) was carried out with the solutions of synthesized compounds (1 mg/mL in
DMSO solvent). The solutions were scanned using an excitation wavelength of 295 nm at 298 K.
For the synchronous binding studies (at ∆λ 15 nm or 60 nm), BSA solution (3 mL, 3 µM) was titrated with
each compound of the synthesized series of triazole derivatives or the site markers at an excitation wavelength
of 295 nm.
3.4.2. FT-IR spectroscopy
The FT-IR analysis was carried out on a Bruker Alpha FT–IR spectrometer. The spectrum of phosphate buffer
in D2 O was collected and subtracted from the spectrum of BSA to obtain the FT-IR spectrum of pure BSA.
The change in the spectra of BSA before and after addition of compounds 8a–8i was analyzed.
Acknowledgment
The authors acknowledge the Higher Education Commission of Pakistan for financial support.
References
1. Rehman, A. U.; Iqbal, J.; Abbasi, M. A,; Siddiqui, S. Z.; Khalid, H.; Laulloo, S. J.; Virk, N. A.; Rasool, S.; Shah,
S. A. A. Cogent Chem. 2018, 4, 1-16.
2. Jiaxin, F. U.; Kaiwei, W.; Yushu, G. E.; Fenglei, J.; Xiaohong, S.; Yang, L.; Yi, L. Sci. China Chem. 2011, 54,
788-796.
3. Qingpeng, W.; Jingqing, Z.; Guri, D.; Kun, W.; Huizhen, Z.; Chenghe, Z. Sci. China Chem. 2012, 55, 2134-2153.
4. Mitsumori, T.; Bendikov, M.; Sedó, J.; Wudl, F. Chem. Mater. 2003, 15, 3759-3768.
5. Zbancioc, G. N.; Mangalagiu, I. I. Tetrahedron 2010, 66, 278-282.
6. Tozkoparan, B.; Kupeli, E.; Yesilada, E.; Ertan, M. Bioorg. Med. Chem. 2007, 15, 1808-1814.
7. Rabea, S. M.; El-Koussi, N. A.; Hassan, H. Y.; Aboul-Fadl, V. Arch. Pharm. 2006, 339, 32-40.
8. Abdel-Aal, M. T.; El-Sayed, W. A.; El-Kosy, S. M.; El-Ashry, S. H. Arch. Pharm. 2008, 341, 307-313.
9. Dumitrascu, F.; Caproiu, M. T.; Georgescu, F.; Draghici, B.; Popa, M. M.; Georgescu, E. Synlett 2010, 2407-2410.
10. Jones, D. H.; Slack, R.; Squires, S.; Wooldridge, K. R. H. J. Med. Chem. 1965, 8, 676-680.
11. Ilango, K.; Valentina, P. Pharm. Chem. 2010, 2, 16-22.
12. Sughen, J. K.; Yoloye, T. Pharm. Acta Helv. 1978, 58, 64-68.
13. Shams, E. S. A.; Hazzaa, A. A. B. Pharmazie 1974, 29, 761-763.
14. Oruc, E. E.; Rollas, S.; Kandemirli, F.; Shvets, N.; Dimoglo, A. S. Bioorg. Med. Chem. Lett. 2004, 12, 5651-5659.
15. Wu, G.; Ouyang, L.; Liu, J.; Zeng, S.; Huang, W.; Han, B.; Wu, F.; He, G.; Xiang, M. Mol. Diversity 2013, 17,
271-283.
16. Duan, Y. Q.; Lei, H. G.; Min, S. G.; Duan, Z. Q. Spectrosc. Spect. Anal. 2009, 29, 2998-3002.
17. Jiang, H.; Chen, R.; Pu, H. J. Lumin. 2012, 132, 592-599.
1477

IQBAL et al./Turk J Chem
18. Ghosh, S.; Dey, J. J. Colloid Interface Sci. 2015, 458, 284-292.
19. Zhang, H. M.; Fei, Z. H.; Tang, B. P.; Chen, J.; Tao, W. H.; Wang, Y. Q. Mol. Biol. Rep. 2012, 39, 4937-4947.
20. Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16, 922-927.
21. Prashant, M. K.; Madaiah, M.; Revanasiddappa, H. D. Organic Chemistry 2013, 2013, 1-12.
22. Teng, Y.; Liu, R.; Yan, S.; Pan, X.; Zhang, P.; Wang, M. J. Fluoresc. 2010, 20, 381-387.
23. Ghosh, S.; Jana, S.; Guchhait, N. J. Phys. Chem. 2012, 116, 1155-1163.
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