Synthesis of sulpha drug based hydroxytriazene derivatives: Anti-diabetic, antioxidant, anti-inflammatory activity and their molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2020.103642

Herein, we report synthesis, characterization, anti-diabetic, anti-inflammatory and anti-oxidant activities of hydroxytriazenes derived from sulpha drugs, namely sulphanilamide, sulphadiazine, sulphapyridine and sulphamethazine. Before biological screening of the compounds, theoretical prediction using PASS was done which indicates probable activities ranging from Pa (probable activity) values 65–98% for anti-inflammatory activity. As per the predication, experimental validation of some of the predicted activities particularly anti-diabetic, anti-inflammatory and anti-oxidant was done. Anti-diabetic activities have been screened using two methods namely α-amylase and α-glucosidase inhibition method and IC₅₀ values were ranging from 66 to 260 and 148 to 401 μg/mL, while for standard drug acarbose the values were 12 μg/mL and 70 μg/mL, respectively. Docking studies have also been done for antidiabetic target pancreatic alpha amylase. The molecular docking studies in α-amylase enzyme reveal that the middle phenyl ring of all the compounds mainly occupies in the small hydrophobic pocket formed by the Ala198, Trp58, Leu162, Leu165 and Ile235 residues and sulphonamide moiety establish H-bond interaction by two water molecules. Further, anti-inflammatory activity has been evaluated using carrageenan induced paw-edema method and results indicate excellent anti-inflammatory activity by hydroxytriazenes (71 to 97%) and standard drug diclofenac 94% after 4 h of treatment. Moreover, antioxidant effect of the compounds was tested using DPPH and ABTS methods. All the compounds displayed good results (24–488 μg/mL) against ABTS radical and many compounds are more active than ascorbic acid (69 μg/mL) while all other compounds showed moderate activity against DPPH radical (292–774 μg/mL) and ascorbic acid (29 μg/mL). Thus, the studies reveal potential of sulfa drug based hydroxytriazenes as candidates for antidiabetic, anti-inflammatory and antioxidant activities which have been experimentally validated.

Full text of DOI:10.1016/j.bioorg.2020.103642

BioorganicChemistry96(2020)103642
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of sulpha drug based hydroxytriazene derivatives: Anti-diabetic,
antioxidant, anti-inflammatory activity and their molecular docking studies
⁎
Poonam Sharmaa, Varsha Daymaa, Aparna Dwivedib, Prabhat K. Baroliyaa, , I.P. Tripathib,
⁎
Murugesan Vanangamudic, R.S. Chauhana, A.K. Goswamia,
a Department of Chemistry, Mohanlal Sukhadia University, Udaipur, Rajasthan, India
b Department of Environmental Sciences, Mahatma Gandhi Chitrakoot Vishwvidhyalay, Chitrakoot, Satna, Madhya Pradesh, India
c Department of Medicinal and Pharmaceutical Chemistry, Sree Vidyanikethan College of Pharmacy, Tirupathi, Andhra Pradesh, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Herein, we report synthesis, characterization, anti-diabetic, anti-inflammatory and anti-oxidant activities of
hydroxytriazenes derived from sulpha drugs, namely sulphanilamide, sulphadiazine, sulphapyridine and sul-
phamethazine. Before biological screening of the compounds, theoretical prediction using PASS was done which
indicates probable activities ranging from Pa (probable activity) values 65–98% for anti-inflammatory activity.
As per the predication, experimental validation of some of the predicted activities particularly anti-diabetic, anti-
inflammatory and anti-oxidant was done. Anti-diabetic activities have been screened using two methods namely
α-amylase and α-glucosidase inhibition method and IC50 values were ranging from 66 to 260 and 148 to 401 µg/
mL, while for standard drug acarbose the values were 12 µg/mL and 70 µg/mL, respectively. Docking studies
have also been done for antidiabetic target pancreatic alpha amylase. The molecular docking studies in α-
amylase enzyme reveal that the middle phenyl ring of all the compounds mainly occupies in the small hydro-
phobic pocket formed by the Ala198, Trp58, Leu162, Leu165 and Ile235 residues and sulphonamide moiety
establish H-bond interaction by two water molecules. Further, anti-inflammatory activity has been evaluated
using carrageenan induced paw-edema method and results indicate excellent anti-inflammatory activity by
hydroxytriazenes (71 to 97%) and standard drug diclofenac 94% after 4 h of treatment. Moreover, antioxidant
effect of the compounds was tested using DPPH and ABTS methods. All the compounds displayed good results
(24–488 µg/mL) against ABTS radical and many compounds are more active than ascorbic acid (69 µg/mL)
while all other compounds showed moderate activity against DPPH radical (292–774 µg/mL) and ascorbic acid
(29 µg/mL). Thus, the studies reveal potential of sulfa drug based hydroxytriazenes as candidates for anti-
diabetic, anti-inflammatory and antioxidant activities which have been experimentally validated.
Hydroxytriazenes
Sulpha drugs
Antioxidant
Anti-diabetic
Anti-inflammatory activity
1. Introduction
concentration of low molecular weight antioxidant and metabolic ab-
normalities [7,8]. This can lead to increased lipid peroxidation, devel-
Nowadays diabetes is considered as most common chronic diseases
and leading cause of death and disability worldwide [1,2]. Diabetes
mellitus is a group of metabolic disorders in which increased oxidative
stress plays an important role in long term damages, dysfunctions and
failure of different organs such as kidneys, eyes nerves, heart and blood
vessels [3–5]. Oxidative stress is mainly due to imbalance between free
radicals generation and scavenging systems. Some experimental studies
have reported that increased production of free radicals and defeat
antioxidant protection of free radicals involved in pathogenesis of
diabetes [6]. Another source of oxidative stress in diabetic patient
might be auto-oxidation of glucose, redox imbalance, decreased
opment of insulin resistance and damages of cellular organelles and
enzymes.
Further, the antioxidant therapy provide a therapeutic strategy to
defend the β-cells against oxidative stress and prevent related diabetic
vascular complication. An antioxidant diminishes free radicals, enhance
scavenging of free radicals and antioxidant defense mechanism.
Although, previous studies demonstrated benefit of antioxidants with
diabetic drugs in prevention of diabetic complications [9]. Antioxidant
may inhibit formation of reactive oxygen species (ROS) or scavenge free
radicals or enhance capabilities of enzymes which prevent oxidative
stress. It is also reported that administration or dietary vitamin E as
⁎ Corresponding authors at: Department of Chemistry, University College of Science, Mohanlal Sukhadia University, Udaipur-313001, India.
E-mail addresses: prabhatkbaroliya@mlsu.ac.in (P.K. Baroliya), akumargoswami56@gmail.com (A.K. Goswami).
https://doi.org/10.1016/j.bioorg.2020.103642
Received 29 January 2019; Received in revised form 21 January 2020; Accepted 29 January 2020
Availableonline31January2020
0045-2068/©2020PublishedbyElsevierInc.

P. Sharma, et al.
BioorganicChemistry96(2020)103642
supplement significantly decreases blood glucose by inhibiting the se-
quence of oxidative stress [10,11].
for α-glucosidase and α-amylase inhibitory activity varies from 148 to
361 and 31–214 μg/mL respectively, which can be concluded as mod-
erate to excellent activity. It is interesting to note that out of this series,
SFN-2 (66 μg/mL), SFN-3 (31 μg/mL) SFN-4 (52 μg/mL) and SFN-7
(72 μg/mL) have shown excellent α-amylase activity as compared to
standard acarbose (69.74 μg/mL) which warrants future investigation
on these compounds. Remaining all series namely SFD, SFP and SMT
have shown moderate to good α-glucosidase (IC50, 175–401 μg/mL)
and α-amylase (IC50, 149–260 μg/mL) inhibition activity. Looking at
the results of all the four series, it can be concluded that these com-
pounds inhibit α-amylase enzyme better than α-glucosidase enzyme.
The investigation of Structure-activity relationship (SAR) was based
on the two ending substituents placed on amino group of 4-sulphona-
midophenyl ring and another one at nitrogen atom of the hydroxy
triazene moiety. The amplification of cyclic and/or long chain bulky
substitutions at both ends as in compounds SFN-2 – SFN-8, SFD-2 – SFD-
7, SFP-1 – SFP-5 and SMT-1 to SMT-6 significantly reduce the α-glu-
cosidase inhibitory activity. The presence of small steric substituent like
methyl at the end of the triazene moiety made the molecules moderate
potent (SFN-1 & SFD-1; IC50 = 148.19 & 175.64 μg/ml, respectively).
Hence, concurrently occurrence of two steric substituents (pyridyl,
pyrimidinyl, alkyl & aralkyl) at both ends is not significant for in-
hibiting the α-glucosidase enzyme. In the SFN series, four compounds
(SFN-2, SFN-3, SFN-4 and SFN-7) possess good α-amylase enzyme in-
hibition activity. Among the four compounds, the increase of non-polar
alkyl carbons from ethyl to isopropyl substituents in compounds SFN-2
Computer aided drug designing (CADD) has attracted attention of
chemists and its application has been gaining importance of late. PASS
is an online freely accessible web resource (http://www.way2drug.
com/passonline), designed for the prediction of the biological activity
spectra of organic compounds based on their structural formulas which
predicts more than 4000 types of biological activities [12]. The pre-
diction is mainly based on an examination of the structure activity re-
lationships with the training set containing information on the structure
and biological activity of more than 300,000 organic compounds. In our
study, the PASS for 26 compounds with hydroxytriazene moiety have
shown Pa values for the validated activities around ≥40%. Further, not
only theoretical prediction has been done but also molecular docking
studies with particular targets for each of the representative series
compound has been done. The docking studies describe the DNA mi-
crogroove binding throwing light on the drug action of these com-
pounds.
The inflammation is caused in animals as a protective response to a
cell injury. The clinical signs such as edema, erythema, hyperalgesia,
pain and loss of function at microscopic level are exhibited in this re-
sponse. The non-steroidal anti-inflammatory drug (NSAID) normally
acts at the periphery and not at CNS. These drugs block the synthesis of
ecosanoids at the site of injury which results in blocking the cytokines
and cyclooxygenase pathway (COX) and thereby reducing the produc-
tion of prostaglandins [13] and inhabiting release of histamine [14].
Hydroxytriazenes have occupied an important role in analytical and
medicinal chemistry due to their easy synthesis, good yield and atom
economy. However, biological activities of hydroxytriazenes have
hardly been attempted and only few reports on anti-microbial [15–17],
wound-healing [18], anti-inflammatory [19,20], antidiabetic [21], an-
tioxidant [21,22], anti-dyslipidemic [22] and insecticidal [23,24] ac-
tivities are reported in literature. Thus, in the present study four series
of sulfa drug based hydroxytriazenes have been synthesized, char-
acterized and evaluated for antidiabetic, anti-inflammatory and anti-
oxidant activities.
(IC50
= 66. μg/mL), SFN-3 (IC50 = 31.71 μg/mL), SFN-4
(IC50 = 51.77 μg/mL) enhance the potency of the α-amylase inhibition.
It suggests that the α-amylase enzyme inhibition is related with lipo-
philicity attachment on the nitrogen atom of the hydroxy triazene
moiety. The presence of aromatic/alkyl group in the compounds SFN-5,
SFN-6 and SFN-8 showed moderate α-amylase enzyme inhibitory ac-
tivity except the compound SFN-7 which bearing m-methylphenyl
substituent. The insertion of pyrimidine (in SFD series), pyridine (in
SFP series) and dimethylpyrimidine (in SMT series) at the amino group
of 4-sulphonamidophenyl exhibited moderate biological potency.
Therefore, heteroaromatic incorporation is not crucial for improving
the α-amylase enzyme inhibition activity. The SAR analysis suggests
that, only hydrophobic modification tolerable at hydroxy triazene
portion in all series would be a promising lead for the further devel-
opment of antidiabetic agents.
2. Result and discussion
2.1. Chemistry
Hydroxytriazenes incorporated sulpha drugs namely sulphanila-
mide (SFN-1 to 8), sulphadiazine (SFD-1 to 7), sulphapyridine (SFP-1 to
5) and sulphamethazine (SMT-1 to 6) were synthesized by using the
method reported by Elkins and Hunter [25] and further modified by
Sogani and Bhattacharya [26,27]. This method involves diazocoupling
reaction of alkyl or aryl hydroxylamine with diazonium salt obtained
from sulfa drugs at 0–5 °C in acetate buffer medium of pH 5–6. All the
synthesized compounds were characterized by IR, 1H NMR, 13C NMR
and EI MS techniques (Scheme 1 and Supporting data, S2). The struc-
tures of hydroxytriazenes were confirmed by appearance of a singlet for
N-OH proton around 12 ppm in 1H NMR spectra followed by the pre-
sence of peak around 3400 cm−1for (N-OH) and 1500 cm-1for (N]N),
in the IR spectrum.
It is known that treatments of type 2 diabetes mellitus (T2DM) in-
clude improvement of insulin sensitivity or reducing the rate of car-
bohydrate absorption from the gastrointestinal tract. α-Amylase and α-
glucosidase are the key enzymes taking part in carbohydrate metabo-
lism. Both enzymes are the key players in the postprandial hypergly-
cemia noticed in case of non-insulin dependent diabetes mellitus
(NIDDM; T2DM). So, inhibition of α-amylase and α-glucosidase en-
zymes can act as potential targets that can serve as a powerful strategy
in the treatment of NIDDM [28,29].
2.3. Antiradical activity
Antiradical activity of sulpha drug based hydroxytriazenes by DPPH
and ABTS methods are depicted in Table 1. The results were expressed
by IC50 values and compared with standard reference ascorbic acid
(29.12 μg/mL Y = 0.1371 X + 46.017, R2 = 0.9609 for DPPH method
and 69.133 μg/ml Y = 0.599x + 8.589 R2 = 0.991 for ABTS method).
In case of SFN series, the IC50 values vary from 292 to 523 μg/mL
against DPPH free radical, which can be considered as poor to moderate
antiradical activity. The IC50 values for this series vary from 24 to
359 μg/mL for ABTS free radical, which can be considered as moderate
to excellent activity. The compounds SFN-5,6,7,8 exhibited excellent
antiradical activity with IC50 values 24, 31, 27 and 28 μg/mL respec-
tively, which are better than standard reference ascorbic acid (69, μg/
mL). The SFD, SFP and SMT series showed moderate to poor anti-
2.2. Antidiabetic study
In this investigation all the synthesized compounds were evaluated
for their antidiabetic action via two in-vitro assays namely, α-amylase
and α-glucosidase inhibition method and results were compared with
acarbose standard reference in both α-glucosidase (viz 12.21 μg/mL,
y = 0.226x + 47.24, R2 = 0.939) and α-amylase (69.74 μg/mL,
y = 0.435x + 19.66, R2 = 0.995) methods. The α-glucosidase and α-
amylase activity of four different series of hydroxytriazenes (SFN, SFD,
SFP and SMT series) are present in Table 1, and results are expressed as
IC50 values. As revealed from Table 1, in case of SFN series, IC50 values
2

P. Sharma, et al.
BioorganicChemistry96(2020)103642
Scheme 1. Synthesis of sulpha drug based hydroxytriazene derivatives.
radical activity by DPPH method with IC50 values 302–774 μg/mL. In
the SFD series, compound SFD-5 (42.65 μg/mL), SFD-6 (34.53 μg/mL)
and SFD-7 (78.51 μg/mL) exhibited excellent activity in ABTS method
while all the remaining compounds from this series showed moderate
activity as compare to standard reference ascorbic acid (69 μg/mL). The
compound SMT-4 (33.06 μg/mL) and SMT-5 (33.53 μg/mL) from SMT
series showed excellent activity by ABTS method, which is also better
than standard reference ascorbic acid (69 μg/mL) and remaining
compounds from this series showed moderate activity. Only compound
SMT-4 (IC50 265 μg/mL) showed moderate DPPH radical scavenging
activity among all the reported compounds and standard reference with
IC50 value 29 μg/mL.
Table 1
Antidiabetic and antioxidant activities sulpha drug based hydroxytriazene de-
rivatives.
S.No. Compound
α-Glucosidase α-Amylase
DPPH
ABTS
Inhibition
Inhibition
IC50 (µg/
mL)
Inhibition
IC50 (µg/
mL)
Inhibition
IC50 (µg/
mL)
IC50 (µg/mL)
1.
SFN-1
SFN-2
SFN-3
SFN-4
SFN-5
SFN-6
SFN-7
SFN-8
SFD-1
SFD-2
SFD-3
SFD-4
SFD-5
SFD-6
SFD-7
SFP-1
SFP-2
SFP-3
SFP-4
SFP-5
SMT-1
SMT-2
SMT-3
SMT-4
SMT-5
SMT-6
148.19
190.83
231.5
111.42
66
305
265
2
337
277
3
31.28
51.71
113.28
122
337
359
4
204.71
264.98
361.26
278.82
319.75
175.64
238.26
282.65
321.33
353
348
316
5
523
24.18
31.08
27.52
28.24
488.25
291.76
363.25
402.16
42.65
34.53
78.51
249.07
38.04
36.85
44.95
43.13
277
6
292
The measurement of radical scavenging activity of any antioxidant
is frequently associated with using of DPPH and ABTS methods just
because their reliable, quick and reproducible quality to search the in
vitro general antioxidant activity of compounds [30]. These methods
depend on the reduction of the purple DPPH radical and blue ABTS
radical by accepting electron or radical hydrogen from antioxidant
(A–H) to a stable diamagnetic molecule as described in the following
reactions:
7
71.77
214.39
154.86
149.52
184.67
168.16
213.86
170.78
201.86
125.67
223.19
218.45
260.13
214.53
148.3
298
8
457
9
641
10
11
12
13
14
15
16
17
18
29
20
21
22
23
24
25
26
27
774
365
302
406
325.2
367
205
341.83
370
256.22
366.43
357.97
393.68
328.16
230.47
183.93
231.8
%
%
357.5
355.57
383.8
345.04
331
(DPPH) + A-H → DPPH-H+(A) (Purple to yellow)
%
%
(ABTS) + A-H → ABTS-H+(A) (Blue to colorless)
The intensity of color change from purple to yellow and blue to
colorless at variable concentrations are measured spectro-
photometrically at 517 nm and 734 nm, respectively. The degree of
discoloration shows the scavenging potential of the antioxidant com-
pounds in the term of hydrogen donating ability.
157.54
171.93
161.4
425
218.71
279.02
33.06
33.53
131.08
69.13
(Ascorbic
acid)
344
344.22
401.28
320
265.75
440
253
222.5
562
Standard drug 12.21
69.74
29.12
(Ascorbic
acid)
Structure-activity relationship: In ABTS radical scavenging activity,
most of the compounds exhibited potent activities with IC50 range from
24 to 45 μg/mL (Examples: compound SFN-5 to 8, SFD-5, 6, SFP-2 to 5
and SMT-4,5) in comparison with reference compound, ascorbic acid
(Acarbose)
(Acarbose)
3

P. Sharma, et al.
BioorganicChemistry96(2020)103642
(IC50 = 69.13 μg/mL). The ABTS radical scavenging properties depend
on the incorporation of phenyl and o/m-alkyl substituted phenyl groups
at the hydroxytriazene moiety. Inclusion of only 2–3 alkyl groups sig-
nificantly reduces the ABTS radical scavenging activity as in com-
pounds SFN-1, SFD-2, SFP-1, SMT-1 etc (IC50 = > 218 μg/mL). The
addition of pyridine & pyrimidine at -SO2-NH- portion did not influence
the scavenging activity in all series of most active compounds. Hence
the heteroaromatic substituents are not vital for the ABTS radical
scavenging activity.
2.4.1. Correlation of antidiabetic, antioxidant and anti-inflammatory
activities in type 2 diabetes mellitus
Current studies have demonstrated that oxidative stress plays an
important role in the pathogenesis of type 2 diabetes [31,32] and may
diminish the antioxidative defense system of the body. This can lead to
damage of cellular organelles and enzymes, increased lipid peroxida-
tion and development of insulin resistance and after damage has oc-
curred, preventing further cell degeneration [33]. Type 2 diabetes is
also associated with endothelial dysfunction, which may develop into
macro and microvascular diseases such as retinopathy, nephropathy,
lower extremity amputations, coronary artery and cardiovascular dis-
eases [34,35].
The compound SMT-4 revealed moderate DPPH radical scavenging
activity (IC50 = 265 μg/mL) due the presence of presence of un-
substituted phenyl at the basic skeleton of sulfonamide linked triazene.
The drastic reduction of DPPH radical scavenging activity appears in
remaining compounds by means of existence of the -SO2-NH-substituted
heteroaromatic rings like pyridine & pyrimidine along with triazene N-
alkyl or alkyl substituted phenyl ring at either side of the basic skeleton
of the compounds, respectively.
Many studies have demonstrated that inhibition of intracellular free
radicals generation would offer an alternate way to prevent oxidative
stress and related diabetic complications. Source of oxidative stress in
diabetic patient is related to leaking of ROS from mitochondria, auto-
xidation of glucose, redox imbalance etc. Bajaj et al. [36] reviewed the
effect of antioxidant therapy in management of diabetes. Supple-
mentation with antioxidants may potentially improve endothelial dys-
function in T2DM by recoupling of RNS, mitochondrial function as well
as macro/microvascular complications. The non-enzymatic antioxidant
such as biogenic and synthetic substance and natural cellular defense
mechanism may work by neutralizing free radicals by producing glu-
tathione, ubichinol and uric acid. The enzymatic antioxidants particu-
larly dismutase, glutathione peroxidase, glutathione reductase and
catalase may remove ROS directly or sequentially preventing their ex-
cessive accumulation and consequent adverse effect. Rajendiran et al.
[11] studied that vitamin C and E, alpha-lipoic acid, selenium play
important role in controlling in hyperglycemia. These antioxidants may
work by significantly increasing SOD and GSH-Px activity and in-
hibiting sequence of oxidative stress. The mechanism of inhibition of
oxidative stress is not very clear but the plasma glucose level decreased
by increasing glucose metabolism in peripheral tissue. Aforementioned
findings suggest that intake of antioxidant with hypoglycemic drugs
may have cumulative/synergistic antioxidant effects alleviating the
progression of conditions caused by oxidative stress. More studies are
warranted to better understand the biological mechanisms of simulta-
neous intake of hypoglycaemic drug with antioxidants.
2.4. Anti-inflammatory study
Biological activity spectra of all the synthesized compounds were
calculated using PASS web tool. PASS tool will interpret the biological
active spectra using 2D structure of molecules. Interpretation of results
of PASS as described in supporting information reveled that all the
hydroxytriazenes displayed good anti-inflammatory probable activity
upto 96%. The probable anti-inflammatory activity of the compounds
has been experimentally validated by carrageenan induced hind paw
edema method.
Results of anti-inflammatory activity are presented as % inhibition of
paw-edema after 2 and 4 h. As revealed from Table 2, all the compound
showed good to excellent anti-inflammatory activity in terms of percent
reduction of paw edema volume which were 32–62% after 2 h and
71–97% after 4 h. This is explicitly clear from the value that all the
compounds screened for anti-inflammatory activity have shown ≈97%
inhibition of paw-edema after 4 h which are comparable to or better than
the standard drug (diclofenac). It has been observed that presence of alkyl
group attached to triazene moiety may enhance the anti-inflammatory
activity. If we compare results of anti-inflammatory activity of all the
three series reported, sulphamethazine containing hydroxytriazenes (SMT
series) has exhibited best results since it has two additional methyl group
attached to the molecule in addition to the other functional group. The
tentative mechanism of action exhibited by hydroxytriazenes is proposed
to be NSAID like as described under introduction.
The association between hyperglycemia, inflammation and vascular
complications in diabetes is now well established [37]. In diabetes,
hyperglycemia and elevated free fatty acids may promote inflammation
by stimulating glucose utilization. Glucotoxicity and lipotoxicity might
also exert oxidative and endoplasmic reticulum stress, which in turn
elicits an inflammatory response. Metabolic stress (hyperglycemia) and
elevated free fatty acid levels, may initiate insulin dysfunction [38].
Multiple mechanisms are thought to contribute to β-cell dysfunction,
insulin resistance and vascular complications of diabetes. Thus, the
regulation of inflammation play a major role in the management of
diabetic mellitus complications. Furthermore, under the condition of
oxidative stress by generation of ROS and RNS species also play crucial
role in activation of signaling pathway which affect intra and extra
cellular pathway. At the site of inflammation accumulation of ROS
occurs because mast cells and leukocytes are recruited, which leads to a
‘respiratory burst’ due to an increased uptake of oxygen. This sustained
inflammatory/oxidative environment leads to a vicious circle, which
can damage healthy neighboring epithelial and stromal cells and over a
long period of time may lead to carcinogenesis [39,40].
Table 2
Anti-inflammatory activity of sulpha drug based hydroxytriazene derivatives.
S. No.
Code
% Inhibition of paw edema
% Inhibition of paw edema
after 2 h
after 4 h
1
SFN-1
SFN-2
SFN-3
SFN-4
SFN-5
SFN-6
SFN-7
SFP-1
32.50
45.00
47.51
57.53
55.00
62.52
50.00
55.00
52.53
55.00
55.00
47.51
55.00
55.00
50.00
62.52
55.00
50.00
57.50
71.42
85.71
91.42
88.57
88.71
85.71
88.57
80.0
2
3
4
5
6
7
8
9
SFP-2
94.28
77.14
91.42
71.42
88.57
82.85
94.28
94.28
94.28
97.14
94.28
10
11
12
13
14
15
16
17
18
19
SFP-3
Xie et al. described the multifunctional role of hydroxytyrosol-fe-
nofibrate with antidiabetic, antihyperlipidemic, antioxidant and anti-
inflammatory activities towards the defense of pancreatic β cells from
oxidative stress damage, preserve liver function and death induced by
sustained oxidative stress in type 2 diabetes mellitus disorder (T2DM)
complications [41]. Similarly, our synthesized compounds also possess
the triple functional biological activities (antidiabetic, antioxidant and
anti-inflammatory) as shown in Tables 1 and 2. Such a combination of
activities present in the sulpha drug based hydroxytriazene derivatives
SFP-4
SFP-5
SMT-1
SMT-2
SMT-3
SMT-4
SMT-5
SMT-6
Diclofenac
4

P. Sharma, et al.
BioorganicChemistry96(2020)103642
could suppress the progress of T2DM by inhibiting the damage of
pancreatic β cells and liver tissues through their antioxidant and anti-
inflammatory biological properties.
than standard ascorbic acid. All the compounds were also exhibited
71–97% reduction in inflammation while standard drug diclophenac
reduce by 94% after 4 h of treatment. However, the SAR as well as
prediction studies indicated that, particularly anti-inflammatory ac-
tivity can be explored further with more such compounds. Docking
results indicated that the best binding was with middle phenyl ring of
all compounds with small hydrophobic pocket formed by the Ala198,
Trp58, Leu162, Leu165 and Ile235 residues and hydrogen bonding with
hydroxyl group responsible for inhibition of α-amylase enzyme. Thus,
this study explores, theoretical prediction, antidiabetic, antioxidant,
anti-inflammatory activities and docking studies of hydroxytriazenes
for development of potential multifunctional medicinal candidates.
2.5. Docking study
A molecular operating environment (MOE 2015.1001) software
package [42,43] was used for docking study. This was attempted to
bring better understanding of mechanism of action of sulpha drug based
hydroxytriazene compounds (SFN-2, SFD-1, SFP-2 & SMT-2) toward the
human pancreatic alpha-amylase enzyme. Analysis was done for in-
duced-fit docking in to the active site of co-crystal structure of PDB:
2QV4. The results showed dissimilarity in binding sites of hydro-
xytriazenes and reference acarbose due to the different geometries with
short length and/or greater hindrance of the compounds. The binding
energies have been predicted within the range of 6.97–7.29 Kcal/mol (S
score) compared to standard drug of acarbose (−9.95 Kcal/mol). The
middle phenyl ring of all compounds occupies in the small hydrophobic
pocket formed by the Ala198, Trp58, Leu162, Leu165 and Ile235 re-
sidues. The sulphonamide moiety of SFN-2 compound forms three hy-
drogen bond interactions: with the NHe of His201 residue, Ile235 and
Lys200 residues through two water molecules. The negative charges of
hydroxytriaz-1-enyl of all compounds interact electrostatically with the
acidic residues of Asp197, Glu233 and Asp300. The ethyl end of SFN-2
makes hydrophobic contact with the Trp58 and Trp59 residues. The
hydroxytriazene moiety of SFD-1 and SFP-2 compounds forms H-
bonding interactions with the His201 residue. In compound SFD-1,
eNH and eS]O of sulphonamide groups make strong double H-
bonding interactions with the Asp300, His299 & Arg195 residues. The
position of the pyridine and pyrimidine (SFD-1, SFP-2 & SMT-2) moi-
eties is stabilized by π-stacking interactions with aromatic side chains of
Trp58, Trp59 and Tyr62. The alkyl fragments of isopropyl and propyl in
SFP-2 and SMT-2 make a small hydrophobic contact with Tyr151 and
Ile235 (Figs. 1 & 2). Molecular docking results indicated that double
hydrogen bonding contacts of sulphonamide moiety and the middle
phenyl hydrophobic interactions play roles in the binding of these
compounds within the binding site of alpha-amylase enzyme. It gives
clue to optimize the derivatives with strong potency in further lead
development.
4. Experimental section
4.1. Material and methods
Commercially available Sulpha drugs of Sigma-Aldrich have been
used as it is, and other chemicals and solvents of analytical reagent
grade have been used. Melting point was measured in open glass ca-
pillaries using SONAR apparatus. NMR spectra of sulpha drug based
hydroxytriazene derivatives which had been synthesized were collected
using Bruker Avance II 400 NMR spectrometer operating at 400 MHz in
deuterated DMSO-D6 solvent at SAIF Chandigarh. XEVO-G2SQTOF-EI-
MS was used to determine mass at MNIT, Jaipur. IR spectra were col-
lecting using Bruker Optik GmbH Alpha Sample Compartment RT-
DLaTGS ZnSe HR 0.8 IR instrument. Thin layer chromatography was
used to confirm single spot of pure compound using precoated silica gel
plate and visualized using UV lamp at 254 nm.
4.2. General procedure for synthesis of compounds
Sulpha drug based hydroxytriazene derivatives were obtained in
three steps. First one is reduction, in which 0.02 mol of nitro com-
pounds (aromatic as well as aliphatic) reduced with 0.02 mol NH4Cl
and 0.04 gm of zinc dust at 50–60 °C to get hydroxylamine. Then
second one is diazotization of various sulpha drugs taken as an amine
group through diazotization procedure with HNO2/HCl at 0–5 °C. Third
one is coupling, in which products of above two steps were coupled at
0–5 °C and pH 5–6. Upon completion of coupling, product was obtained
as precipitation which was filtered and crystallized using in appropriate
solvent to afford pure product.
3. Conclusion
It is apparent from the present studies that hydroxytriazenes based
on sulpha drugs have shown excellent anti-diabetic, anti-oxidants as
well as anti-inflammatory activity. Compounds SFN-2, SFN-3, SFN-4
and SFN-7 were found to be most potent α-amylase inhibitors among all
the tested compounds with IC50 value of 66 μg/mL, 31.28 μg/mL
51.71 μg/mL and 71.77 μg/mL respectively, comparable to standard
acarbose (69.74 μg/mL). Many compounds from all series were dis-
played excellent antioxidant activities by ABTS method, even better
4.2.1. 4-(3-hydroxy-3-methyltriaz-1-en-1-yl)benzenesulfonamide (SFN-1)
4-(3-hydroxy-3-methyltriaz-1-en-1-yl)benzenesulfonamide (SFN-1)
m.p. 198–200 °C; Crystallization: methanol; IR (KBr): ѴOe
H
(3559 cm−1), ѴNH2 (3322, 3185 cm−1), ѴN] (1588 cm−1), ѴCe
N
N
(1222 cm−1), ѴS] (1145 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
3.96 (3H, S, H1), 11.59 (1H, S, H2), 7.17–7.38 (2H, d, AreH4,5
,
J = 6.92 Hz), 7.70–7.73 (2H, d, AreH6–7, J = 6.92 Hz), 7.174 (2H, S,
Fig. 1. Predicted binding modes of SFN-2 and SFD-1with the human pancreatic alpha-amylase active site.
5

P. Sharma, et al.
BioorganicChemistry96(2020)103642
Fig. 2. Predicted binding modes of SFP-2 & SMT-2with the human pancreatic α-amylase active site.
H9); 13C NMR (400 MHz, DMSO‑d6): δ 50.68 (1C, C1), 143.62 (1C, C3),
113.49 (2C, C4,5), 127.22 (2C, C6–7), 136.43 (1C, C8); ESI-HRMS for
C7H10N4O3S, calcd. 230.244, found 231.2469.
(400 MHz, DMSO‑d6): δ142.93 (1C, C1), 119.92 (2C, AreC2,6), 130.20
(2C, AreC3,5), 129.27 (1C, AreC4), 142.20 (1C AreC8), 114.49 (2C,
AreC9,13), 127.24 (2C, AreC10,12), 137.47 (1C, AreC11); ESI-HRMS for
C12H12N4O3S, calcd. 292.3137, found 292.3223.
4.2.2. 4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)benzenesulfonamide (SFN-2)
4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)benzenesulfonamide
(SFN-2)
4.2.6. 4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]
benzenesulfonamide (SFN-6)
m.p. 194–196 °C; Crystallization: methanol; IR (KBr): ѴOe
H
N
(3410 cm−1), ѴNH2 (3301, 3226 cm−1), ѴN] (1595 cm−1), ѴCe
N
(1217 cm−1), ѴS] (1149 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]benzenesulfona-
mide (SFN-6) m.p.189–191 °C; Crystallization: ethylalcohol; IR (KBr):
ѴOe (3391 cm−1), ѴNH2 (3313, 3263 cm−1), ѴN] (1583 cm−1),
O
1.44 (3H, t, H1, J = 7.28 Hz), 4.18 (2H, q, H2, J = 7.28 Hz), 11.53 (1H,
H
N
N
S, H3), 7.39–7.73 (2H, d, AreH5,8), 7.71–7.73 (2H, d, AreH6,7
,
ѴCe (1301 cm−1), ѴS] (1147 cm−1); 1H NMR (400 MHz,
J = 8.8 Hz), 7.174 (2H, S, H10); 13C NMR (400 MHz, DMSO‑d6): δ 12.37
(1C, C1), 58.31 (1C, C2), 143.73 (1C, C3), 113.51 (2C, C4,8), 127.21 (2C,
C5,7), 136.39 (1C, C6); ESI-HRMS for C8H12N4O3S, calcd. 244.2709,
found 245.2770.
O
DMSO‑d6): δ 2.42 (3H, S, H1), 7.39–7.50 (4H, m, AreH3–6), 12.18 (1H,
S, H8), 7.65–7.67 (2H, d, AreH10,14, J = 7.84 Hz), 7.75–7.78 (2H, d,
AreH11,13, J = 7.84 Hz), 7.22 (2H, S, H15); 13C NMR (400 MHz,
DMSO‑d6):δ 17.99 (1C, C1), 131.40 (1C, AreC2), 131.68 (1C, AreC3),
126.83 (1C, AreC4), 129.94 (1C, AreC5), 123.87 (1C, AreC6), 143. 63
(1C, AreC7), 143.68 (1C, AreC9), 114.22 (2C, AreC10,14), 127.28 (2C,
4.2.3. 4-(3-hydroxy-3-propyltriaz-1-en-1-yl)benzenesulfonamide (SFN-3)
4-(3-hydroxy-3-propyltriaz-1-en-1-yl)benzenesulfonamide (SFN-3)
AreC11,13), 137.21 (1C, AreC12), ESI-HRMS for C13
H N4O3S, calcd.
14
m.p. 169–171 °C; Crystallization: methanol; IR (KBr): ѴOe
H
306.3403, found 307.3417.
(3410 cm−1), ѴNH2 (3324, 3214 cm−1), ѴN] (1592 cm−1), ѴCe
N
N
(1215 cm−1), ѴS] (1149 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
0.916 (3H, t, H1, J = 7.2 Hz), 1.9202 (2H, m, H2, J = 7.2 Hz, 7.5 Hz),
4.12 (2H, t, H3, J = 7.5 Hz), 11.55 (1H, S, H4), 7.36–7.39 (2H, d,
AreH6,10, J = 8.84 Hz), 7.71–7.74 (2H, dd, AreH7,9, J = 8.84 Hz),
7.178 (2H, S, H11); 13C NMR (400 MHz, DMSO‑d6): δ 10.64 (1C, C1),
20.21 (1C, C2), 64.59 (1C, C3), 143.71 (1C, AreC5), 113.53 (2C,
AreC6,10), 127.22 (2C, AreC7,9), 136.41 (1C, AreC8); ESI-HRMS for
C9H14N4O3S, calcd. 258.2975, found 259.2971.
4.2.7. 4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]
benzenesulfonamide (SFN-7)
4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]benzenesulfona-
mide (SFN-7) m.p.178–180 °C; Crystallization: ethylalcohol; IR (KBr):
ѴOe (3418 cm−1), ѴNH2 (3335, 3275 cm−1), ѴN] (1598 cm−1),
H
N
N
ѴCe (1296 cm−1), ѴS] (1148 cm−1); 1H NMR (400 MHz,
O
DMSO‑d6): 2.44 (3H, S, H1), 7.61–7.64 (2H, d, AreH10,14, J = 8.84 Hz),
7.77–7.80 (2H, d, AreH11,13, J = 8.84 Hz), 7.94 (1H, d, AreH5,
J = 8.12 Hz), 7.90 (1H, m, AreH4), 7.48 (1H, d, AreH3, J = 7.8 Hz),
7.36 (1H, d, AreH7, J = 7.6 Hz); 13C NMR (400 MHz, DMSO‑d6):δ
20.88 (1C, C1), 129.01 (1C, C3), 139.08 (1C, C2), 130.82 (1C, C4),
117.13 (1C, C5), 120.18 (1C, C7) 142.95 (1C, C6), 114.57 (2C, C10,14),
127.23 (2C, C11,13), 143.22 (1C, C9), 137.42 (1C, C12); ESI-HRMS for
C13H14N4O3S, calcd. 306.3403, found 307.3470.
4.2.4. 4-[3-hydroxy-3-(propan-2-yl)triaz-1-en-1-yl] benzenesulfonamide
(SFN-4)
4-[3-hydroxy-3-(propan-2-yl)triaz-1-en-1-yl] benzenesulfonamide
(SFN-4) m.p. 190 °C; Crystallization: methanol; IR (KBr): ѴOe
H
(3344 cm−1), ѴNH2 (3289, 3209 cm−1), ѴN] (1590 cm−1), ѴCe
N
N
(1208 cm−1), ѴS] (1150 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
1.146 (6H, d, H1,2, J = 6.8 Hz), 4.53 (1H, m, H3, J = 6.8 Hz), 11.29
(1H, S, H4), 7.18–7.358 (2H, m, AreH6,10), 7.70–7.731 (2H, m,
AreH7,9), 7.185 (2H, S, H11); 13C NMR (400 MHz, DMSO‑d6): δ 20.12
(2C, C1,2), 65.20 (1C, C3), 143.90 (1C AreC5), 113.50 (2C, AreC6,10),
127.19 (2C, AreC7,9), 136.31 (1C, AreC8); ESI-HRMS for C9H14N4O3S,
calcd. 258.2975, found 259.2971.
4.2.8. 4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]
benzenesulfonamide (SFN-8)
4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]benzenesulfona-
mide (SFN-8) m.p. 112 °C; Crystallization: ethylalcohol; IR (KBr) ѴOe
H
(3444 cm−1), ѴNH2 (3361, 3257 cm−1), ѴN] (1595 cm−1), ѴCe
N
N
4.2.5. 4-(3-hydroxy-3-phenyltriaz-1-en-1-yl) benzene sulfonamide (SFN-
(1237 cm−1), ѴS] (1156 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
5)
2.32 (3H, S, H1), 7.30–7.32 (2H, d, AreH3,7, J = 8.24 Hz), 7.54–7.52
(2H, dd, AreH4,6, J = 8.24 Hz), 12.12 (1H, S, H8), 7.69–7.71 (2H, d,
AreH10,14, J = 8.84 Hz), 7.91–7.93 (2H, d, AreH11,13, J = 8.84 Hz),
7.16 (2H, S, H15); 13C NMR (400 MHz, DMSO‑d6):δ 20.67 (1C, C1),
129.64 (2C, C3,7), 119.76 (2C, C4,6), 140.110 (1C, C2), 140.78 (1C, C5),
114.48 (2C, C10,14), 127.23 (2C, C11,13), 143.26 (1C, C9), 137.31 (1C,
4-(3-hydroxy-3-phenyltriaz-1-en-1-yl) benzene sulfonamide (SFN-5)
m.p.170 °C; Crystallization: ethylalcohol; IR (KBr): ѴOe (3356 cm−1),
H
ѴNH2 (3260, 3199 cm−1), ѴN] (1597 cm−1), ѴCe (1252 cm−1),
N
N
ѴS] (1150 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ 7.58–7.65 (5H,
O
m, H2–6), 12.29 (1H, S, H7), 7.79–7.82 (2H, d, AreH9,13, J = 8.84 Hz),
8.12–8.15 (2H, d, AreH10,12, J = 8.84 Hz), 7.26 (2H, S, H14); 13C NMR
C12); ESI-HRMS for C13H14N4O3S, calcd. 306.3403, found 307. 3474.
6

P. Sharma, et al.
BioorganicChemistry96(2020)103642
4.2.9. 2-{[4-(3-hydroxy-3-methyltriaz-1-en-1-yl)phenyl]sulfonyl}
pyrimidine (SFD-1)
(1578 cm−1), ѴCe (1223 cm−1), ѴS] (1157 cm−1); 1H NMR
N
O
(400 MHz, DMSO‑d6): δ.2.47 (3H, S, H1), 7.26–7.41 (4H, m, AreH3–6),
2-{[4-(3-hydroxy-3-methyltriaz-1-en-1-yl)phenyl]sulfonyl}pyr-
imidine (SFD-1) m.p. 238–240 °C; Crystallization: methanol; IR (KBr):
ѴOe (3421 cm−1), ѴNe (3252 cm−1), ѴN] (1579 cm−1), ѴCe
7.58–7.62 (2H, d, AreH10,14, J = 8.1 Hz), 8.09–8.11 (2H, d, AreH11,13,
J = 8.1 Hz), 11.54 (1H, S, H8), 8.63–8.65 (2H, d, AreH16,18), 7.26 (1H,
m, AreH17); 13C NMR (400 MHz, DMSO‑d6): 18.80 (1C, C1), 130.89
(1C, C2), 131.94 (1C, C3), 126.73 (1C, C4), 130.26 (1C, C5), 123.94 (1C,
C6), 142.3 (1C, C7), 113.99 (2C, C10,14), 127.40 (2C, C10,13), 132.89
(1C, C12), 143.53 (1C, C9), 158.60 (1C, C15), 156.78 (2C, C16,18), 112.40
(1C, C17); ESI-HRMS for C17H16N6O3S calcd. 384.4123, found 385.4145
H
H
N
N
(1259 cm−1), ѴS] (1152 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
4.14 (3H, S, H1),11.56 (1H, S, H2), 7.20–7.41 (2H, d, AreH4,8
,
J = 8.24 Hz), 7.71–7.92 (2H, d, AreH5,7, J = 8.24 Hz), 10.03 (1H, S,
H
10), 8.65 (2H, d, AreH12,14), 6.99 (1H, d, AreH13); 13C NMR
(400 MHz, DMSO‑d6): δ 50.68 (1C, C1), 143.62 (1C, C3), 113.49 (2C,
C
4,8), 127.47 (2C5,7), 137.43 (1C, C6), 157.62 (1C, C10), 149.2 (2C,
4.2.14. 2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyrimidine (SFD-6)
C11,13), 112.80 (1C, C12); ESI-HRMS for C11H12N6O3S, calcd. 308.3164,
found 309.3172.
2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyrimidine (SFD-6) m.p. 165–167 °C; Crystallization: ethy-
lalcohol; IR (KBr): ѴOe (3392 cm−1), ѴNe (3201 cm−1), ѴN]
4.2.10. 2-{[4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)phenyl]sulfonyl}
pyrimidine (SFD-2)
H
H
N
(1579 cm−1), ѴCe (1224 cm−1), ѴS] (1157 cm−1); 1H NMR
N
O
2-{[4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)phenyl]sulfonyl}pyr-
imidine (SFD-2) m.p. 160–162 °C; Crystallization: methanol; IR (KBr):
ѴOe (3464 cm−1), ѴNe (3227 cm−1), ѴN] (1583 cm−1), ѴCe
(400 MHz, DMSO‑d6): δ 2.46 (3H, S, H1), 7.26–7.39 (3H, m, AreH4,6),
7.21 (1H, d, AreH3, J = 3.2 Hz), 11.31 (1H, S, H8), 7.86–7.88 (2H, d,
AreH10,14, J = 7.99 Hz), 8.12–8.14 (2H, d, AreH11,13, J = 7.99 Hz),
10.25 (1H, S, H19), 8.61–8.63 (2H, d, AreH16,18), 6.97–6.99 (1H, d,
AreH17); 13C NMR (400 MHz, DMSO‑d6): δ 20.66 (1C, C1), 120.54 (1C,
C3), 139.66 (1C, C2), 129.28 (1C, C4), 130.22 (1C, C5), 117.13 (1C, C7),
113.99 (2C, C10,14), 127.20 (2C, C11,13), 149.34 (1C, C9), 132.81 (1C,
H
H
N
N
(1224 cm−1), ѴS] (1153 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
1.58 (3H, t, H1, J = 6.66 Hz), 4.19 (2H, q, H2, J = 6.66 Hz),11.06 (1H,
s, H3), 7.15–7.18 (2H, d, AreH5,9, J = 8.1 Hz), 7.88–7.90 (2H, d, AreH
6,8, J = 8.1 Hz), 10.26 (1H, S, H10), 8.60–8.62 (2H, d, AreH12,14
,
J = 5.5 Hz), 8.05–8.08 (1H, d, AreH13, J = 5.5 Hz); 13C NMR
(400 MHz, DMSO‑d6): δ 12.92 (1C, C1), 58.96 (1C, C2), 145.14 (1C, C4),
113.59 (2C, C5,9), 130.67 (2C, C6,8), 136.76 (1C, C7), 158.63 (1C, C11),
142.13 (2C, C12,14), 117.91 (1C, C13); ESI-HRMS for C12H14N6O3S,
calcd. 322.3430, found 323.3471.
C12), 158.05 (1C, C15), 155.15 (2C, C16,18), 115.63 (1C, C17); ESI-HRMS
for C17H16N6O3S calcd. 384.4123, found 385.4125.
4.2.15. 2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyrimidine (SFD-7)
2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyrimidine (SFD-7) m.p. 102–104 °C; Crystallization: acetone; IR
(KBr): ѴOe (3390 cm−1), ѴNe (3218 cm−1), ѴN] (1578 cm−1),
4.2.11. 2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}
pyrimidine (SFD-3)
H
H
N
2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}pyr-
imidine (SFD-3) m.p. 175–177 °C; Crystallization: methanol; IR (KBr):
ѴOe (3465 cm−1), ѴNe (3258 cm−1), ѴN] (1585 cm−1), ѴCe
ѴCe (1215 cm−1), ѴS] (1154 cm−1); 1H NMR (400 MHz,
N
O
DMSO‑d6): δ 2.433 (3H, S, H1), 7.26–7.29 (2H, d, AreH2,5), 7.31–7.33
(2H, d, AreH3,6), 11.46 (1H, S, H8), 7.94–7.96 (2H, d, AreH10,14
,
H
H
N
N
(1223 cm−1), ѴS] (1150 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
J = 8.1 Hz), 8.11–8.14 (2H, d, AreH11,13, J = 8.1 Hz), 10.82 (1H, S,
O
0.9566 (3H, t, H1, J = 7.42 Hz), 2.01 (2H, m, H2, J = 7.42 Hz,
H19), 8.61–8.635 (2H, d, AreH16,18, J = 6.01 Hz), 6.96–6.99 (1H, d,
J = 7.5 Hz), 4.12 (2H, t, H3, J = 7.5 Hz), 7.24–7.54 (2H, d, AreH6,10
,
AreH17, J = 6.01 Hz); 13C NMR (400 MHz, DMSO‑d6): 21.22 (1C, C1),
138.90 (1C, C4), 130.49 (2C, C2,5), 118.81 (2C, C3,6), 143. 75 (1C, C7),
113.64 (2C, C10,14), 129.51 (2C, C11,13), 146.70 (1C, C9), 135.29 (1C,
J = 8.0 Hz), 7.90–8.061 (2H, d, AreH7,9, J = 8.0 Hz), 10.25 (1H, S,
H11), 8.59–8.61 (2H, d, AreH13,15, J = 5.5 Hz), 7.643 (1H, d, AreH14
,
J = 5.5 Hz); 13C NMR (400 MHz, DMSO‑d6): δ10.64 (1C, C1), 20.21
(1C, C2), 64.60 (1C, C3), 143.71 (1C, C5), 113.53 (2C, C6,10), 128.22
(2C, C7,9), 136.41 (1C, C8), 157.68 (1C, C12), 152.77 (2C, C13,15),
115.10 (1C, C14); ESI-HRMS for C13H16N6O3S, calcd. 336.3695, found
337.3670.
C12), 158.31 (1C, C15), 154.13 (2C, C16,18), 112.78 (1C, C17); ESI-HRMS
for C17H16N6O3S calcd. 384.4123, found 385.4178.
4.2.16. 2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}pyridine
2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}pyridine (SFP-1)
(SFP-1)
4.2.12. 2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}
pyrimidine (SFD-4)
2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}pyridine
2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}pyridine
(SFP-1)(SFP-1) m.p. 138–140 °C; Crystallization: acetone; IR (KBr):
ѴOe (3334 cm−1), ѴNe (3244 cm−1), ѴN] (1595 cm−1), ѴCe
2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}pyr-
imidine (SFD-4) m.p. 170–172 °C; Crystallization: methanol; IR (KBr):
ѴOe (3394 cm−1), ѴNe (3212 cm−1), ѴN] (1579 cm−1), ѴCe
H
H
N
N
(1222 cm−1), ѴS] (1136 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
H
H
N
N
O
(1226 cm−1), ѴS] (1163 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
0.898 (3H, t, H1, J = 7.16 Hz), 1.88 (2H m, H2), 4.08 (2H, t, H3,
O
11.036 (1H, S, H7),6.96–6.987 (1H, m, AreH4), 7.33–7.35 (2H, d,
AreH3,5, J = 7.99 Hz) 7.491–7.51 (2H, d, AreH9,13, J = 8.1 Hz),
8.07–8.15 (2H, d, AreH10,12, J = 8.1 Hz), 10.39 (1H, S, H18), 8.59–8.61
(2H, d, AreH15,17, J = 6.1 Hz), 7.00–7.26 (1H, d, AreH16, J = 6.1 Hz);
13C NMR (400 MHz, DMSO‑d6): δ 120.16 (2C, C2,6), 130.54 (2C, C3,5),
124.72 (1C, C4), 139.92 (1C, C1), 114.00 (2C, C9,13), 128.25 (2C,
J = 7.57 Hz), 11.36 (1H, S, H4), 7.32–7.57 (2H, d, AreH6,10,
J = 7.99 Hz), 7.84–7.88 (2H, d, AreH7,9, J = 7.99 Hz), 10.30 (1H, S,
H
11), 8.35–8.38 (1H, m, AreH16), 8.02–8.04 (1H, m, AreH15),
7.62–7.66 (1H, m, AreH14), 8.12–8.16 (1H, m, AreH13); 13C NMR
(400 MHz, DMSO‑d6): δ 11.25 (1C, C1), 20.39 (1C, C2), 67.59 (1C, C3),
113.39 (2H, C6,10), 127.29 (2C, C7,9), 143.21 (1C, C5), 136.12 (1C, C8),
156.41 (1C, C12), 146.31 (1C, C16), 116.89 (1C, C15), 135.20 (1C, C14),
114.50 (1C, C13); ESI-HRMS for C14H17N5O3S, calcd. 335.3815, found
336.3871.
C
10,12), 144.11 (1C, C8), 136.42 (1C, C11), 150.76 (1C, C14), 146.11 (2C,
C15,17), 115.98 (1C, C16); ESI-HRMS for C16H14N6O3S calcd. 370.3858,
found 371.4857.
4.2.13. 2-({4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyrimidine (SFD-5)
4.2.17. 2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}
pyridine (SFP-2)
2-({4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyrimidine (SFD-5) m.p. 148–150 °C; Crystallization: ethy-
lalcohol; IR (KBr): ѴOe (3392 cm−1), ѴNe (3216 cm−1), ѴN]
2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}pyridine
(SFP-2) m.p. 118–120 °C; Crystallization: acetone; IR (KBr): ѴOe
H
(3444 cm−1), ѴNe
(3311 cm−1), ѴN]
(1595 cm−1), ѴCe
H
H
N
H
N
N
7

P. Sharma, et al.
BioorganicChemistry96(2020)103642
4.2.21. 2-{[4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-
(1256 cm−1), ѴS] (1134 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ
O
7.26–7.32 (1H, m, AreH4), 7.39–7.49 (1H, m, AreH5,3), 7.52–7.57 (1H,
dimethylpyrimidine (SMT-1)
m, AreH2,6), 11.32 (1H, S, H7), 7.66–7.70 (2H, d, AreH9,13
,
2-{[4-(3-ethyl-3-hydroxytriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-di-
methylpyrimidine (SMT-1) m.p. 102–104 °C; Crystallization: acetone;
IR (KBr): ѴOe (3458 cm−1), ѴNe (3228 cm−1), ѴN] (1597 cm−1),
J = 8.01 Hz), 7.90–7.92 (2H, d, AreH10,12, J = 8.01 Hz), 8.31–8.33
(1H, d, AreH19, J = 5.53 Hz), 10.86 (1H, S, H15), 7.99–8.01 (1H, m,
AreH18), 8.02–8.06 (1H, m, AreH17), 8.16–8.18 (1H, m, AreH16); 13C
NMR (400 MHz, DMSO‑d6): δ 118.18 (2C, C2,6), 130.81 (2C, C3,5),
129.19 (1C, C4), 114.27 (2C, C9,13), 127.24 (2C, C10,12), 143.46 (1C,
C8), 136.34 (1C, C11), 155.43 (1C, C15), 147.95 (1C, C19), 120.06 (1C,
H
H
N
ѴCe (1218 cm−1), ѴS] (1152 cm−1); 1H NMR (400 MHz,
N
O
DMSO‑d6): δ 1.41 (3H, t, H1, J = 7.28 Hz), 4.17 (2H, q, H2,
J = 7.28 Hz), 11.60 (1H, S, H3), 7.34–7.41 (2H, m, AreH5,9), 7.87–7.90
(2H, m, AreH6,8), 10.31 (1H, S, H10); 2.51 (6H, s, H11,15), 6.74 (1H, s,
AreH13); 13C NMR (400 MHz, DMSO‑d6): 11.73 (1C, C1), 58.38 (1C,
C2), 144.42 (1C, C4), 113.60 (2C, C5,9), 127.86 (2C, C6,8), 132.30 (1C,
C7), 167.29 (1C, C16), 156.29 (2C, 12,14), 112.97 (1C, C13), 22.91 (2C,
C
18), 139.56 (1C, C17), 112.28 (1C, C16); ESI-HRMS for C17H15N5O3S,
calcd. 369.3977, found 370.3974.
C
11,15); ESI-HRMS for C14H18N6O3S, calcd. 350.3961, found 351.3971.
4.2.18. 2-({4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyridine (SFP-3)
4.2.22. 2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-
dimethylpyrimidine (SMT-2)
2-({4-[3-hydroxy-3-(2-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyridine (SFP-3) m.p. 109–111 °C; Crystallization: acetone; IR
(KBr): ѴOe (3432 cm−1), ѴNe (3229 cm−1), ѴN] (1594 cm−1),
2-{[4-(3-hydroxy-3-propyltriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-di-
methylpyrimidine (SMT-2) m.p. 102–104 °C; Crystallization: acetone;
IR (KBr): ѴOe (3432 cm−1), ѴNe (3225 cm−1), ѴN] (1598 cm−1),
H
H
N
ѴCe (1232 cm−1), ѴS] (1137 cm−1) 1H NMR (400 MHz, DMSO‑d6):
N
O
H
H
N
δ 2.47 (3H, S, H1), 6.79–7.38 (4H, m, AreH3–6), 11.30 (1H, S, H8),
ѴCe (1218 cm−1), ѴS] (1153 cm−1); 1H NMR (400 MHz,
N
O
7.45–7.51 (2H, d, AreH10,14, J
=
8.24 Hz), 7.51–7.60 (2H, d,
DMSO‑d6): δ 0.89 (3H, t, H1, J = 7.42 Hz), 1.85–1.90 (2H, m, H2,
J = 7.42 Hz, J = 7.57 Hz), 4.10 (2H, t, H3, J = 7.57 Hz), 11.58 (1H, S,
H4), 7.36–7.34 (2H, d, AreH6,10, J = 8.84 Hz), 7.90–7.88 (2H, d,
AreH7,9, J = 8.84 Hz), 10.20 (2H, S, H11), 2.26 (6H, S, H14,17), 6.75
(1H, s, AreH15); 13C NMR (400 MHz, DMSO‑d6): δ 10.61 (1C, C1),
22.92 (1C, C2), 64.65 (1C, C3), 144.40 (1C, C5), 113.84 (2C, C6,10),
129.83 (2C, C7,9), 136.40 (1C, C8), 168.29 (1C, C12), 156.29 (2C,
AreH11,13, J = 8.24 Hz), 10.80 (1H, S, H15), 8.31–8.33 (1H, m,
AreH20), 7.91–7.93 (1H, m, AreH19), 7.67–7.69 (1H, m, AreH18),
7.88–7.90 (1H, m, AreH17); 13C NMR (400 MHz, DMSO‑d6): δ 18.83
(1C, C1), 138.37 (1C, C2), 130.18 (1C, C3), 126.33 (1C, C4), 123.94 (1C,
C5), 131.92 (1C, C6), 142.14 (1C, C7), 114.43 (2C, C10,14), 128.81 (2C,
C
C
11,13), 146.31 (1C, C9), 137.58 (1C, C12), 154.75 (1C, C16), 146.31 (1C,
20), 115.18 (1C, C19), 138.87 (1C, C18), 112.03 (1C, C17); ESI-HRMS
C
13,16), 112.98 (1C, C15), 20.20 (2C, C14,17); ESI-HRMS for
15H20N6O3S, calcd. 364.4227, found 365.4325.
for C18H17N5O3S, calcd. 383.4243, found 384.4282.
C
4.2.23. 2-({4-[3-hydroxy-3-(propan-2-yl)triaz-1-en-1-yl]phenyl}
sulfonyl)-4,6-dimethylpyrimidine (SMT-3)
4.2.19. 2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyridine (SFP-4)
2-({4-[3-hydroxy-3-(propan-2-yl)triaz-1-en-1-yl]phenyl}sulfonyl)-
4,6-dimethylpyrimidine (SMT-3) m.p. 164–166 °C; Crystallization:
acetone; IR (KBr): ѴOe (3432 cm−1), ѴNe (3223 cm−1), ѴN]
2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyridine (SFP-4) m.p. 108–110 °C; Crystallization: acetone; IR
(KBr): ѴOe (3423 cm−1), ѴNe (3192 cm−1), ѴN] (1594 cm−1),
H
H
N
(1598 cm−1), ѴCe (1217 cm−1), ѴS] (1154 cm−1); 1H NMR
H
H
N
N
O
ѴCe (1275 cm−1), ѴS] (1137 cm−1); 1H NMR (400 MHz,
(400 MHz, DMSO‑d6): δ 1.05 (6H, d, H1,2, J = 6.43 Hz), 2.97 (1H, m,
H3, J = 6.43 Hz), 7.36–7.34 (2H, m, ArH6,10), 7.83–7.94 (2H, m,
AreH7,9), 11.58 (1H, S, H4), 10.30 (1H, S, H11), 2.18 (6H, S, H14,17),
6.73 (1H, s, AreH15); 13C NMR (400 MHz, DMSO‑d6): δ 20.09 (2C,
C1,22), 65.30 (1C, C3), 145.20 (1C, C5), 112.97 (2C, C6,10), 127.87 (2C,
N
O
DMSO‑d6): δ 2.45 (3H, S, H1), 7.60–7.68 (1H, m, AreH3), 7.79–6.82
(2H, m, AreH7), 7.30–7.34 (1H, m, AreH6), 7.38–7.44 (1H, m, AreH5),
11.45 (1H, S, H8), 7.50–7.54 (2H, d, AreH10,14, J = 8.01 Hz),
7.80–7.90 (2H, d, AreH11,13, J = 8.01 Hz), 10.87 (1H, S, H15),
8.02–8.06 (3H, m, AreH17), 8.31–8.33 (1H, m, AreH20), 13C NMR
(400 MHz, DMSO‑d6): δ 21.45 (1C, C1), 138.94 (1C, C2), 129.49 (1C,
C7), 131.07 (1C, C6), 120.46 (1C, C5), 118.95 (1C, C3), 142.81 (1C, C4),
114.67 (2C, C10,14), 128.75 (2C, C11,13), 144.0 (1C, C9), 135.30 (1C,
C
7,9), 136.21 (1C, C8), 165.13 (1C, C12), 151.50 (2C, C13,16), 110.9 (1C,
15), 22.93 (2C, C14,17); ESI-HRMS for C15H20N6O3S, calcd. 364.4227,
C
found 365.4375.
C
12), 153.82 (1C, C16), 146.98 (1C, C20), 118.95 (1C, C19), 139.46 (1C,
4.2.24. 2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-
dimethylpyrimidine (SMT-4)
C18), 111.92 (1C, C17); ESI-HRMS for C18H17N5O3S, calcd. 383.4243,
found 384.4262.
2-{[4-(3-hydroxy-3-phenyltriaz-1-en-1-yl)phenyl]sulfonyl}-4,6-di-
methylpyrimidine (SMT-4) m.p. 168–170 °C; Crystallization: acetone;
IR (KBr): ѴOe (3446 cm−1), ѴNe (3214 cm−1), ѴN] (1595 cm−1),
H
H
N
4.2.20. 2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)pyridine (SFP-5)
ѴCe (1224 cm−1), ѴS] (1148 cm−1); 1H NMR (400 MHz,
N
O
DMSO‑d6): δ 7.36–7.47 (5H, m, H2e ), 12.26 (1H, S, H7), 7.59–7.61
6
2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)pyridine (SFP-5) m.p. 111–113 °C; Crystallization: acetone; IR
(KBr): ѴOe (3392 cm−1), ѴNe (3192 cm−1), ѴN] (1594 cm−1),
(2H, m, AreH9,13), 7.89–7.96 (2H, m, AreH10,12), 11.45 (1H, S, H14),
2.24 (6H, S, H17,20), 6.71 (1H, dd, AreH18); 13C NMR (400 MHz,
DMSO‑d6): δ 119.23 (2C, C2,6), 124.93 (2C, C3,5), 123.2 (1C, C4),
113.96 (2C, C9,13), 128.93 (2C, C10,12), 142.92 (1C, C8), 132.20 (1C,
H
H
N
ѴCe (1274 cm−1), ѴS] (1134 cm−1); 1H NMR (400 MHz,
N
O
DMSO‑d6): δ2.42 (3H, S, H1), 7.42–7.48 (2H, m, AreH4,6), 7.26–7.30
C
11), 166.87 (1C, C15), 165.01 (2C, C16,19), 110.2 (1C, C18), 22.94 (2C,
17,20); ESI-HRMS for C18H18N6O3S, calcd. 398.4389, found 399.4326.
(2H, m, AreH3,7), 11.45 (1H, S, H8), 7.66–7.70 (2H, d, AreH10,14
,
C
J = 7.89 Hz), 7.870–7.90 (2H, d, AreH11,13, J = 7.89 Hz), 10.82 (1H,
S, H15), 8.32–8.34 (1H, m, AreH20), 7.26 (1H, dd, AreH19), 7.92–7.95
(2H, m, AreH17); 13C NMR (400 MHz, DMSO‑d6): δ 46.09 (1C, C1),
138.91 (1C, C2), 129.61 (2C, C3,7), 119.88 (2C, C4,6), 140.81 (1C, C5),
114.58 (2C, C10,14), 127.18 (2C, C11,13), 146.32 (1C, C9), 137.97 (1C,
4.2.25. 2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)-4,6-dimethylpyrimidine (SMT-5)
2-({4-[3-hydroxy-3-(3-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
fonyl)-4,6-dimethylpyrimidine
(SMT-5)
m.p.
181–183
°C;
C
C
12), 155.46 (1C, C16), 143.11 (1C, C20), 117.27 (1C, C19), 134.32 (1C,
18), 112.32 (1C, C17); ESI-HRMS for C18H17N5O3S, calcd. 383.4243,
Crystallization: acetone; IR (KBr): ѴOe
(3486 cm−1), ѴNe
H
N
H
O
(3187 cm−1), ѴN] (1593 cm−1), ѴCe (1222 cm−1), ѴS]
N
found 384.4274.
(1143 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ 2.43 (3H, S, H1),
8

P. Sharma, et al.
BioorganicChemistry96(2020)103642
7.36–7.47 (3H, m, AreH3–5), 7.59 (1H, m, AreH7), 7.59–7.61 (2H, m,
AreH10,14), 7.89–7.96 (2H, m, AreH11,13), 12.26 (1H, S, H8), 11.59
(1H, S, H15), 6.76 (1H, S, AreH19) 2.26 (); 13C NMR (400 MHz,
DMSO‑d6): δ 22.93 (1C, C1), 139.08 (1C, C2), 143. 87 (1C, C6), 120.22
(1C, C5), 130.89 (1C, C4), 129.05 (1C, C3), 114.09 (2C, C10,14), 127.05
(2C, C11,13), 136.8 (1C, C12), 156.28 (1C, C16), 146.22 (2C, C17,20),
20.87 (2C, C18,21), 110.23 (1C, C19); ESI-HRMS for C19H20N6O3S, calcd.
412.4655, found 413.4689.
absorbance at 540 nm in multimode reader. To afford control absor-
bance 350 µL reaction mixture was prepared with same procedure
without containing test sample. Following formula was used for cal-
culation of enzyme inhibition activity. Experiments were done in tri-
plicates.
(Absorbance control Absorbance sample)
% Inhibition =
× 100
Absorbance control
4.2.26. 2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}
sulfonyl)-4,6-dimethylpyrimidine (SMT-6)
4.5. DPPH inhibition assay
2-({4-[3-hydroxy-3-(4-methylphenyl)triaz-1-en-1-yl]phenyl}sul-
The DPPH radical scavenging activity was performed using reported
method [45]. The method was performed by taking 125 µL of 0.004%
DPPH solution which was prepared in methanol 30 min before use, and
125 µL of various dilution (viz. 20, 60, 120, 180, 240 and 300 µL/mL,
prepared in DMSO) and 50 µL of tris-HCl buffer in 96-well micro plate.
Total reaction mixture was set at 300 µL and was incubated at 37 °C for
half an hour. Final absorbance was recorded on BioTek Synergy H4
hybrid multimode micro plate reader (BioTek instruments, Inc Wi-
nooski, VT, USA) at 517 nm. Radical quenching efficiency of com-
pounds was determined by comparison with 125 µL DMSO, 50 µL of
tris–HCl buffer and 125 µL of DPPH solution treated control absor-
bance. Ascorbic acid was taken as standard in this assay. Following
formula was used for calculation of radical scavenging activity. Ex-
periments were done in triplicates.
fonyl)-4,6-dimethylpyrimidine
(SMT-6)
m.p.
186–188
°C;
Crystallization: acetone; IR (KBr): ѴOe
(3400 cm−1), ѴNe
H
N
H
O
(3208 cm−1), ѴN] (1597 cm−1), ѴCe (1230 cm−1), ѴS]
N
(1152 cm−1); 1H NMR (400 MHz, DMSO‑d6): δ 7.40–7.58 (2H, m,
AreH4,6), 7.34–7.38 (2H, dd, AreH3,7), 2.43 (3H, S, H1), 7.92–7.99
(2H, m, AreH10,14), 78.08–8.14 (2H, m, AreH11,13), 12.30 (1H, S, H4),
11.20 (1H, S, H15), 2.24 (6H, S, H18,21), 6.72 (1H, S, AreH19); 13C NMR
(400 MHz, DMSO‑d6): δ20.77 (1C, C1), 138.48 (1C, C2), 119.77 (2C,
C
4,6), 129.37 (2C, C3,7), 141.25 (1C, C5), 142.22 (1C, C9), 113.86 (2C,
C10,14), 127.23 (2C, C11,13), 139.89 (1C, C12), 165.50 (1C, C16), 152 22
(2C, C17,20), 109.01 (1C, C19), 21.04 (2C, C18,21); ESI-HRMS for
C
19H20N6O3S, calcd. 412.4655, found 413.4687.
4.3. α-Glucosidase inhibition assay
DPPHscavengingeffect(%)
(Absorbancecontrol Absorbancesample)
In this method inhibition of α-glucosidase enzyme was determined
spectrophotmetrically in a 96-well microtiterplate which is based on p-
nitrophenyl-α-D-glucopyranoside (PNPG) as a substrate.
=
× 100
Absorbancecontrol
The concentration of sample required to scavenge 50% of DPPH•
(IC50) were determined. Decreasing of the DPPH• solution absorbance
indicates an increase of the DPPH radical scavenging activity.
Tripathi and his co-worker’s protocol was followed in this study
[44]. In that, rat intestinal powder was dissolved in 100 mL of saline
water and properly sonicated at 4 °C, then the suspension was cen-
trifuged (3000 rpm, at 4 °C for 30 min) and the resulting supernatant
was used as an enzyme solution. Total reaction mixture was set at
250 µL, containing 50 µL of phosphate buffer (50 mM, pH 6.8), 75 µL of
α-glucosidase solution and 50 µL of test samples of different con-
centrations (viz. 20, 60, 120, 180, 240 and 300 µL/mL) which have
been prepared in DMSO. Reaction mixture was pre incubated for 5 min
at 37 °C, and then added 75 µL of 3 mM PNPG as substrate, then again
reaction mixture was incubated for 30 min at 37 °C. On completion of
30 min, enzymatic activity was quantified by measuring the absorbance
at 405 nm. Inhibition efficiency of compounds was determined by
comparison with solution containing 50 µL of phosphate buffer, 75 µL
of α-glucosidase solution, 50 µL DMSO and 75 µL of PNPG treated
control absorbance. Acarbose was taken as standard in this assay. Fol-
lowing formula was used for calculation of enzyme inhibition activity.
Experiments were done in triplicates.
4.6. ABTS inhibition assay
%+
The ability of the synthesized compound to scavenge ABTS
ra-
dical was reported in the literature [46]. Activated ABTS (7 mM con-
centration) solution through ammoniumpersulphate was used and fur-
ther diluted to get required absorbance at 734 nm. Reaction volume
was set on 300 µL, for that took 100 µL of test samples of various
concentration (viz. 20, 60, 120, 180, 240 and 300 µL/mL, prepared in
DMSO) and 200 µL of ABTS%+ solution. For control absorbance, solvent
was used in place of test sample. Absorbance was recorded after 30 min
incubation in dark at 734 nm on multimode micro plate reader. As-
corbic acid was used as positive antioxidant and percentage scavenging
was calculated from the formula-
ABTSscavengingeffect(%)
(Absorbance control Absorbance sample)
(Absorbance control Absorbance sample)
=
× 100
% Inhibition =
× 100
Absorbance control
Absorbance control
4.4. α-Amylase inhibition assay
4.7. Carrageenan induced paw edema assay for Anti-inflammatory activity
The protocol followed in this study was previously reported [44].
Total reaction volume was set at 350 µL. 50 µL of various concentration
(20, 60, 120, 180, 240 and 300 µL/mL in DMSO) of test sample and
50 µL of α-amylase solution (prepared by dissolving 0.005gm α-amylase
in 5 mL sodium phosphate buffer of 0.02 M concentration, pH 6.8) were
taken and incubate for 10 min at 25 °C. After incubation for 10 min,
added 50 µL of 1% starch (1 gm of starch dissolved in 100 mL of so-
dium-phosphate buffer) and reaction mixture was again incubated for
10 min at 25 °C. Then added 100 µL of DNS solution (1 gm of DNS
dissolved in 50 mL of water) and again incubated in boiling water for
5 min. Upon completion of incubation time, added 100 µL of distilled
water and enzymatic activity was quantified by measuring the
In the present study carrageenan induced acute inflammation model
has been used and the results have been compared with standard drug
diclofenac sodium.
Six rats of either sex in each group were taken and starved overnight
with free access to water. The group I which served as control was given
water (1 mL/kg) orally, while group II which served as carrageenan was
given only carrageen. Group III were given diclofenac sodium
(12.5 mg/kg) orally as a standard drug. The remaining groups were
treated with test samples (100 mg/kg) orally. Just above the tibio-torsal
joint of the hind paw was marked to ensure uniform dipping every time.
Diclofenac sodium and test samples were administered 1 h prior to
carrageenan injection. Edema was produced according to the method
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P. Sharma, et al.
BioorganicChemistry96(2020)103642
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tion, paw volume was measured plethysmographically (by mercury
displacement) to note initial volume. Similar procedure was repeated at
intervals of 1 h, 2 h, 3 h and 4 h. The difference between the initial and
subsequent reading at a given time was the actual edema volume. Thus
edema volume in control (Vc) and in-group treated with drugs (Vt)
were calculated. Mean increase in paw volume was measured. % in-
hibition was calculated by this formula
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
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The financial support from UGC INDIA (Poonam Sharma UGC SRF,
23/06/2013(i)EU-V), (Varsha Dayma, RGNF, 2015-17-SC-RAJ-9509),
UGC INDIA (A.K. Goswami, 18-1/2011(BSR), BSR faculty fellowship) is
gratefully acknowledged. Authors are also thankful to SAIF, Chandigarh
and MNIT Jaipur for providing analytical facility.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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