Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis, in vitro, kinetic and docking studies

Article abstract of DOI:10.1080/14756366.2021.1929949

In the present study, a series of azo derivatives (TR-1 to TR-9) have been synthesised via the diazo-coupling approach between substituted aromatic amines with phenol or naphthol derivatives. The compounds were evaluated for their therapeutic applications against alpha-glucosidase (anti-diabetic) and pathogenic bacterial strains E. coli (gram-negative), S. aureus (gram-positive), S. aureus (gram-positive) drug-resistant strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative) drug-resistant strain and P. vulgaris (gram-negative). The IC₅₀ (μg/mL) of TR-1 was found to be most effective (15.70 ± 1.3 μg/mL) compared to the reference drug acarbose (21.59 ± 1.5 μg/mL), hence, it was further selected for the kinetic studies in order to illustrate the mechanism of inhibition. The enzyme inhibitory kinetics and mode of binding for the most active inhibitor (TR-1) was performed which showed that the compound is a non-competitive inhibitor and effectively inhibits the target enzyme by binding to its binuclear active site reversibly.

Full text of DOI:10.1080/14756366.2021.1929949

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Diaryl azo derivatives as anti-diabetic and
antimicrobial agents: synthesis, in vitro, kinetic
and docking studies
Tehreem Tahir, Mirza Imran Shahzad, Rukhsana Tabassum, Muhammad
Rafiq, Muhammad Ashfaq, Mubashir Hassan, Katarzyna Kotwica-Mojzych &
Mariusz Mojzych
To cite this article: Tehreem Tahir, Mirza Imran Shahzad, Rukhsana Tabassum, Muhammad
Rafiq, Muhammad Ashfaq, Mubashir Hassan, Katarzyna Kotwica-Mojzych & Mariusz Mojzych
(
2021) Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis, inꢀvitro, kinetic
and docking studies , Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1509-1520, DOI:
0.1080/14756366.2021.1929949
1
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
021, VOL. 36, NO. 1, 1509–1520
2
https://doi.org/10.1080/14756366.2021.1929949
RESEARCH PAPER
Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis, in vitro,
kinetic and docking studies
a
a
b
c
b
Tehreem Tahir , Mirza Imran Shahzad , Rukhsana Tabassum , Muhammad Rafiq , Muhammad Ashfaq ,
d
e
f
Mubashir Hassan , Katarzyna Kotwica-Mojzych
and Mariusz Mojzych
a
Institute of Biochemistry, Biotechnology and Bioinformatics, Faculty of
Science, The Islamia University of Bahawalpur, Bahawalpur, Pakistan; Department of Chemistry, Faculty of Science, The Islamia University of
Bahawalpur, Bahawalpur, Pakistan; Department of Physiology and Biochemistry, Faculty of Bio-Sciences, Cholistan University of Veterinary and
b
c
d
Animal Sciences, Bahawalpur, Pakistan; Institute of Molecular Biology & Biotechnology, The University of Lahore (Defense Road Campus),
e
f
Lahore, Pakistan; Department of Histology, Embryology and Cytophysiology, Medical University of Lublin, Lublin, Poland; Department of
Chemistry, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland
ABSTRACT
ARTICLE HISTORY
In the present study, a series of azo derivatives (TR-1 to TR-9) have been synthesised via the diazo-cou-
pling approach between substituted aromatic amines with phenol or naphthol derivatives. The com-
pounds were evaluated for their therapeutic applications against alpha-glucosidase (anti-diabetic) and
pathogenic bacterial strains E. coli (gram-negative), S. aureus (gram-positive), S. aureus (gram-positive)
drug-resistant strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative) drug-resistant strain and
P. vulgaris (gram-negative). The IC50 (mg/mL) of TR-1 was found to be most effective (15.70 ± 1.3 mg/mL)
compared to the reference drug acarbose (21.59 ± 1.5 mg/mL), hence, it was further selected for the kinetic
studies in order to illustrate the mechanism of inhibition. The enzyme inhibitory kinetics and mode of
binding for the most active inhibitor (TR-1) was performed which showed that the compound is a non-
competitive inhibitor and effectively inhibits the target enzyme by binding to its binuclear active
site reversibly.
Received 10 February 2021
Revised 6 May 2021
Accepted 10 May 2021
KEYWORDS
Antibacterial; anti-diabetic;
azo derivatives; molecular
docking; phen-
olic compounds
1
. Introduction
ligands and their metal complexes with Cu(II), Co(II), Ni(II) and
Fe(III) , the synthesised series was therapeutically evaluated for
their anti-cancer activity. It was observed that the metal chelates
of Cu(II) and Fe(III) were proved to be potential anticancer agents
7
Aromatic azo compounds have been extensively studied in recent
years due to their broad-spectrum pharmaceutical applicability
mainly because of their cost-effective, simplistic and reproducible
synthetic procedure. The combination of aromatic substituted
amines with coupling agents could yield a variety of azo com-
pounds having versatile biological properties . The presence of
heterocyclic ring and transition metals in conjugation to azo
pharmacophore enhances the bio-potency of the azo dyes . Ravi
and colleagues studied the anti-mycobacterial and DNA cleaving
efficiency of 2-aminothiazole incorporated azo derivatives , the
most active derivative found to be intervened with the active
amino acids of Mycobacterium tuberculosis. Debnath et al. eval-
uated the antimicrobial efficacy of triorganotin(IV) azo-carboxy-
(
Figure 1).
Diabetes Mellitus (DM) is a well characterised metabolic disease
that results in defects in insulin output i.e. insulin secretion (type-
diabetes) and insulin action (type-2 diabetes) or both. Type-2
1–3
1
diabetes accounts for 70–80% of all diabetic patients worldwide
and it leads to postprandial hyperglycaemia. One of the critical
strategies to prevent postprandial hyperglycaemia is to inhibit the
enzymes which are responsible for the carbohydrate hydrolysis
e.g. a-amylase and a-glucosidase. In this interest, several a-glucosi-
dase inhibitors are being administered in the treatment of type-2
diabetes such as acarbose (valienamine), voglibose (valiolamine)
and miglitol (desoxynojirimycin). However, the prolonged use of
these agents could result in various side effects such as vomiting,
pomposity, diarrhoea and flatulence. Therefore, the discovery and
4
4
5
lates against the bacterium (S. aureus) and the fungus (F.
oxysporum) with significant antimicrobial properties compared to
the standard antibiotics. Similarly, Ispir and colleagues examined
the anti-oxidant and anti-proliferative potential of azo-azomethine development of new and potent a-glucosidase inhibitors have
6
8–10
ligands and their metal complexes with Zn(II), Co(II) and Cu(II) . gained immense attention in pharmaceutical chemistry
. In this
Among the series, the metal complexes bearing azo dyes exhib- regard, a new derivative from a series of sulphonamide containing
ited the best radical scavenging and inhibitory potencies com- diarylpentadienones was found to be a promising inhibitor of
pared to the standards against the studied cell lines. On the a-glucosidase (IC 5.69 ± 0.5 mM) with the competitive mode of
5
0
1
1
similar interest, Matada et al. prepared a series of heterocyclic azo inhibition , some new benzamide derivatives of thiourea (IC50
CONTACT Mariusz Mojzych
mmojzych@yahoo.com
Department of Chemistry, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland; Mirza
Imran Shahzad
mirza.imran@iub.edu.pk Institute of Biochemistry, Biotechnology and Bioinformatics, Faculty of Science, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1
510
T. TAHIR ET AL.
4–7
Figure 1. Some reported bioactive azo derivatives
.
range 20.44 ꢀ 333.41 mM) were evaluated as good candidates for phenolic cinnamic acid derivatives are selective inhibitors of cyclo-
12
22
targeting a-glucosidase enzyme . The literature also suggested oxygenase (COX-1 and COX-2) enzymes , Similarly, phenolic N-
that the derivatives of triazole thiones were found to be effective monosubstituted carbamates have proven to be effective anti-
13
inhibitors of a-glucosidase (IC50 value 36.11 lg/mL) . The recent mycobacterial drug targets against broad-spectrum mycobacterial
research has suggested that polyphenols are excellent anti-dia- strains (Mycobacterium tuberculosis H Ra, H Rv including multi-
3
7
37
betic agents, recently in this interest; the a-glucosidase inhibitory drug and extensively drug-resistant strains, Mycobacterium avium,
potential of flavonoids was compared with acarbose with the Mycobacterium kansasii, Mycobacterium aurum and Mycobacterium
2
3
sequence of inhibitory potential as scutellarein > nepetin > apige- smegmatis) .
14
nin > hispidulin > acarbose . Dihydropyridine derivatives (IC50
Hydroxytriazenes are a class of compounds containing alpha
range 2.21 ± 0.06 ꢀ 9.97 ± 0.08 lM) have been reported as poten- hydroxyl group relative to the diazo group, have versatile pharma-
tial a-glucosidase inhibitors.
cological properties including lipid-lowering, antidiabetic, antioxi-
15
Phenolic compounds are simple and naturally occurring dant, anti-inflammatory, antimicrobial and analgesic agents. In
organic compounds bearing an aromatic ring with one or more view of this, an attempt has been made to study the anti-diabetic,
hydroxyl groups (with or without side chain), received consider- anti-inflammatory and antioxidant effects of sulpha drugs based
2
4
able attention due to their biological functions such as anti-car- hydroxytriazenes . It was suggested that the compounds showed
cinogen, antimicrobial, anti-ageing, anti-diabetic, anti-mutagen significant a-amylase and a-glucosidase inhibition potential with
1
6,17
and immunostimulatory roles
administered phenolic drugs, salbutamol sulphate (SLB), terbuta- inflammatory (89% after 4 h of treatment) and radical scavenging
line sulphate (TBT) and thymol (THY) are highly recognised b
potential (IC50 54.12 mg/mL) highlighted the multifunctional role of
adrenergic receptor agonists, antiseptic and antimicrobial agents hydroxytriazenes. It was also concluded that the polar functional-
. Among the most extensively IC50 values ranging from 122–341 mg/mL. The promising anti-
2
-
1
8
respectively . Drug resistance (DR) has become a worldwide ities like –OH around the heterocyclic rings of triazene compounds
threat challenging a wide range of disciplines, including; infection enhanced the inhibition potential against glucosidase and amyl-
2
4
control, drug design and virulence genes. The antimicrobial drug ase . The azo fused with synthetic phenolic molecules such as
resistance (AMR) develops the manipulative properties of bacteria butylated hydroxyanisole, phlorogucinal, 2,4-di-tert-butylphenol
to counter the standard antibiotics and hence obstructing the and 2,6-di-tert-butylphenol were proved to be remarkable antibac-
2
5
treatment of infection. The classes of antibacterial agents which terial, antioxidant and antitumor agents . Similarly, a series of
offer resistance to certain bacterial infections constitute the ser- new phenol ether derivatives were found to be excellent prote-
2
6
ious therapeutic challenge urging the need for the discovery and asome inhibitors . Some of the reported and commercially avail-
1
9,20
development of novel antibiotics
. In this regard, a new series able phenolic drugs are given in Figure 2.
Keeping in view the synthetic feasibility of azo derivatives and
of azo-phenol derivatives was synthesised and evaluated in vitro
against six clinical bacterial strains including the drug-resistant P. diverse therapeutic properties of phenolic compounds fused with
aeruginosa. It was revealed from the experimental data that some azo moiety, the present study aims at the synthesis, spectroscopic
of the azo-phenol compounds significantly inhibited the growth characterisation, biological evaluation, kinetic and molecular dock-
of drug-resistant bacteria compared to the standard drug tri- ing studies of diaryl azo-phenol derivatives. The structural identifi-
21
methoprim-sulfamethoxazole . The studies also suggested that cation of the synthesised azo-phenol derivatives was done by

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1511
2
1–25
Figure 2. Some of the commercially available and reported phenolic drugs in literature
.
2
7
spectroscopic techniques (IR, NMR and HRMS) and the detailed resorcinol and naphthol derivatives (Scheme 1). Briefly, equimo-
screening was carried out by various in vitro biological assays with lar concentration (10 mmol) of substituted amines (4-nitro aniline,
kinetic and in silico structure-activity relationship studies.
p-toluidine and 3-amino,2-chloro pyridine) were mixed with the
cold aqueous solution of sodium nitrite (10 mmol) in a 100 mL
ꢁ
round bottom flask at 0–5 C. To this reaction mixture, 3 M HCl
2
. Materials and methods
was added drop-wise with continuous stirring for 20 min until the
formation of the diazonium salt at the same temperature. Then
immediately, the cold basic solution of respective phenol deriva-
tives (resorcinol, a-naphthol and b-naphthol) (10 mmol) was slowly
added to the diazonium salt mixture and the stirring was contin-
2.1. Synthesis
2
.1.1. Chemicals and equipment
All solvents and chemicals were of analytical grade and purchased
from Sigma-Aldrich and Merck. Melting points were adjusted using a
calibrated thermometer by digital melting point apparatus (SMA10
of Stuart Scientific Bibby Sterilin Ltd., UK) and stated in degrees centi-
grade ( C). Pre-coated silica TLC plates (60F254 Merck Ltd., Japan) for
determining purification, magnetic stirrer (VELP Scientific England)
for stirring and micropipettes (ZX58143 and ZX57677) for quantita-
tive transfer of liquid were used. The NMR spectra ( H and C) were
recorded in DMSO-d using Varian spectrometer 400MHz. The high-
resolution accurate-mass (HRAM) detection was performed on the
Thermo Scientific Q Exactive apparatus. ELISA reader (BioTek) Epoch
ꢁ
ued for another 3 h at 0–5 C. TLC was employed to monitor the
progress of the reaction by the combination of various solvent
systems. Once the reaction is completed, the resulting crude azo
derivatives were filtered off, washed several times with deionised
water, air-dried and purified by recrystallization.
ꢁ
1
13
2
.1.2.1.
(E)-4-((4-nitrophenyl)diazenyl)benzene-1,3-diol
Orange crystals, yield 83%, melting point: 200–204 C; R 0.78
(TR-1).
6
ꢁ
f:
1
(H
2
O/methanol,7:3); H-NMR (400 MHz, DMSO-d
6
) d: 6.37 (d, 1H,
J ¼ 2.4 Hz), 6.51 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.8 Hz), 7.69 (d, 1H,
1
2
2
by Agilent technologies, USA.
J ¼ 8.8 Hz), 8.05 (d, 2H, J ¼ 6.8 Hz), 8.36 (d, 2H, J 6.8 Hz), 10.87 (s,
1
D
H, OH exchange with D O), 12.12 (s, 1H, OH exchanged with
2
1
3
2
.1.2. General method for the synthesis of azo-compounds
2 6
O); C-NMR (100 MHz, DMSO-d ) d: 103.10 (Ar-C), 110.17 (Ar-C),
The general procedure for the synthesis of asymmetric aromatic 122.48 (Ar-C), 125.04 (Ar-C), 133.35 (Ar-C), 147.12 (C), 154.64 (C),
ꢀ
1
azo compounds followed the traditional approach i.e. the coupling 158.73 (C), 164.93 (C); IR (KBr, cm ): 3441.43 (OH, CH stretch),
of aryl diazonium salts with electron-rich aromatics such as 1627.41 (C¼C), 1514.81 (N¼N), 1336.05 (C–N). HR–MS (ESI, m/z)

1
512
T. TAHIR ET AL.
Scheme 1. Synthetic scheme employed for the synthesis of azo derivatives (TR-1 to TR-9).
Calcd.
For
C H N O
2 9 3 4
[M þ H]
260.06658.
Found 2.1.2.4. (E)-4-(p-tolyldiazenyl)naphthalen-1-ol (TR-4). Red crystals,
1
ꢁ
[M þ H] 260.06653.
yield 60%; melting point: 99–100 C; R : 0.77 (acetone/n-hexane,
f
1
1
:1); H-NMR (400MHz, DMSO-d
6
) d: 2.38 (s, 3H), 7.01 (d, 1H,
J ¼ 9.6 Hz), 7.37 (d, 2H, J ¼ 8.4 Hz), 7.46 (t, 1H, J ¼ 8.0 Hz), 7.82 (d,
2
.1.2.2. (E)-4-((4-nitrophenyl)diazenyl)naphthalen-1-ol (TR-2). Red
ꢁ
28
3H, J ¼ 8.4 Hz), 7.97 (d, 1H, J ¼ 9.2 Hz), 15.60 (s, 1H, OH exchanged
crystals, yield 55%, melting point: 260–265 C ; R : 0.82 (H O/
methanol, 7:3); H-NMR (400 MHz, DMSO-d ) d: 6.72 (d, 1H,
f
2
1
3
1
2
with D O); C-NMR (100 MHz, DMSO-d6) d: 20.89 (–CH), 119.69 (Ar-
6
C), 121.23 (Ar-C), 122.94 (Ar-C), 125.47 (Ar-C), 127.10 (Ar-C), 128.77
J ¼ 9.6 Hz), 7.50 (t, 1H, J ¼ 7.2 Hz), 7.61 (t, 1H, J ¼ 9.6 Hz), 7.72 (d,
(
1
Ar-C), 128.87 (Ar-C), 130.33 (Ar-C), 132.93 (Ar-C), 138.14 (Ar-C),
38.90 (Ar-C), 147.13 (Ar-C), 164.14 (C–OH); IR (KBr, cm ): 3441.80
1
1
H, J ¼ 9.6 Hz), 7.93–7.98 (m, 3H), 8.32 (d, 2H, J ¼ 8.8 Hz), 8.42 (d,
13
ꢀ
1
H, J ¼ 8.0 Hz), 15.68 (s, 1H, OH exchanged with D O); C-NMR
2
(
OH), 1625.00 (C¼C), 1511.02 (N¼N), 1280 (C–N). HRMS (ESI, m/z)
(100 MHz, DMSO-d
6
) d: 117.28 (Ar-C), 122.30 (Ar-C), 125.65 (Ar-C),
Calcd. For C17
14 2
H N
O [Mþ H] 263.11789. Found [Mþ H] 263.11796.
1
26.24 (Ar-C), 127.71 (Ar-C), 128.49 (Ar-C), 129.50 (Ar-C), 129.89
(Ar-C), 131.19 (Ar-C), 132.49 (Ar-C), 143.63 (Ar-C), 143.93 (C), 179.28
ꢀ
1
(
1
C
C); IR (KBr, cm ): 3437.49 (OH, CH stretch), 1624.21 (C¼C), 2.1.2.5. (E)-1-(p-tolyldiazenyl)naphthalen-2-ol (TR-5). Orange-red
ꢁ
30
507.92 (N¼N), 1402 (C–N). HR–MS (ESI, m/z) Calcd. For crystals, yield 46%, melting point: 110 C ; R
f
: 0.73 (acetone/n-hex-
) d: 2.38 (s, 3H), 7.01 (d, 1H,
J ¼ 9.6 Hz), 7.37 (d, 2H, J ¼ 8.4 Hz), 7.47 (t, 1H, J ¼ 8.0 Hz), 7.62 (t,
H, J ¼ 8.0 Hz), 7.82 (d, 3H, J ¼ 8.4 Hz), 7.97 (d, 1H, J ¼ 9.2 Hz), 8.60
1
16
H
11 3
N O
3
[M þ H] 294.08732. Found [M þ H] 294.08729.
ane, 1:1); H-NMR (400 MHz, DMSO-d
6
1
2
.1.2.3. (E)-1-((4-nitrophenyl)diazenyl)naphthalen-2-ol (TR-3). Red
1
3
ꢁ
29
(d, 1H, J ¼ 8.4 Hz), 15.60 (s, 1H, OH exchanged with D O); C-NMR
crystals, yield 78%, melting point: 245–252 C ; Rf: 0.86 (H
methanol, 7:3); H-NMR (400 MHz, DMSO-d
2
O/
2
1
(100 MHz, DMSO-d ) d: 20.89 (–CH), 119.69 (Ar-C), 121.23 (Ar-C),
6
) d: 6.72 (d, 1H,
6
1
22.94 (Ar-C), 125.47 (Ar-C), 127.82 (Ar-C), 128.77 (Ar-C), 128.87
J ¼ 9.6 Hz), 7.50 (t, 1H, J ¼ 8.0 Hz), 7.61 (t, 1H, J ¼ 8.0 Hz), 7.72 (d,
(
1
Ar-C), 130.33 (Ar-C), 132.64 (Ar-C), 138.67 (Ar-C), 138.90 (Ar-C),
44.13 (Ar-C), 164.69 (C-OH); IR (KBr, cm ): 3391.78 (OH, CH
1
1
H, J ¼ 8.0 Hz), 7.93–7.98 (m, 3H), 8.32 (d, 2H, J ¼ 6.8 Hz), 8.43 (d,
13
ꢀ
1
H, J ¼ 7.6 Hz) 15.69 (s, 1H, OH exchanged with D
2
O); C-NMR
stretch), 1629.61 (C¼C), 1463.84 (N¼N), 1281 (C–N). HRMS (ESI, m/
z) Calcd. For 263.11789. Found
[M þ H]
M þ H] 263.11780.
(100 MHz, DMSO-d ) d: 117.29 (Ar-CH), 122.30 (Ar-C), 125.66 (Ar-C),
6
17 14 2
C H N O
1
26.24 (Ar-C), 127.72 (Ar-C), 128.49 (Ar-C), 129.50 (Ar-C), 129.90
[
(
(
(
Ar-C), 131.19 (Ar-C), 132.58 (Ar-C), 143.63 (Ar-C), 143.94 (C), 148.01
C), 179.28 (C); IR (KBr, cm ): 3432.58 (OH, CH stretch), 1600.21
ꢀ
1
C¼C), 1507.41 (N¼N), 1207.01 (C–N). HRMS (ESI, m/z) Calcd. for 2.1.2.6. (E)-4-(p-tolyldiazenyl)benzene-1,3-diol (TR-6). Reddish
ꢁ
C
16
H
11 3
N O
3
[M þ H] 294.08732. Found [M þ H] 294.08736.
orange crystals, yield 60%, melting point: 98 C; R
f
: 0.8 (acetone/n-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1513
1
6
hexane, 1:1); H-NMR (400 MHz, DMSO-d ) d: 2.38 (s, 3H), 6.34 to the aboveprepared mixture and incubated again under the
(
1
(
d, 1H, J ¼ 2.4 Hz), 6.49 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.8 Hz), 7.34 (d, same experimental conditions. The reaction was stopped with the
H, J ¼ 8.8 Hz), 7.66 (d, 1H, J ¼ 8.8 Hz), 7.75 (d, 1H, J ¼ 8.0 Hz), 10.50 addition of 0.1 M Na CO and OD
405
was taken by a 96-well ELISA
2
3
s, 1H, OH exchanged with D O), 12.44 (s, 1H, OH exchanged with reader. The experiment was performed in triplicate. Acarbose was
2
1
3
D O); C-NMR (100 MHz, DMSO-d ) d: 20.96 (–CH), 102.97 (Ar-C), used as positive control while DMSO was a negative control. The
2
6
1
08.94 (Ar-C), 121.64 (Ar-C), 129.96 (Ar-C), 130.04 (Ar-C), 132.08 extent of inhibition showed by the tested azo derivatives was cal-
(
Ar-C), 140.24 (Ar-C), 148.63 (Ar-C), 156.07 (C), 162.64 (C); IR (KBr, culated by % inhibition formula and IC50 (mg/mL) was determined
ꢀ
1
cm ): 3476.11 (OH, CH stretch), 1627.19 (C–C¼C), 1458.76 (N¼N). by Graph pad Prism 6.0.
HRMS (ESI, m/z) Calcd. For C13
12
H N
O
2 2
[M þ H] 229.09715. Found
[
M þ H] 229.09710.
2.3. Kinetic analysis of a-glucosidase inhibition
2
.1.2.7. 4-((2-chloropyridin-3-yl)diazenyl)benzene-1,3-diol (TR-7). A series of kinetic assays was performed to determine the inhib-
ꢁ
Orange-brown crystals, yield 98%; melting point: 187–189 C; R
f
ition kinetics of the most active inhibitor from the series of tested
1
32,33
(
6
(
methanol/n-hexane, 7:3); 0.80; H-NMR (400 MHz, DMSO-d
.38 (d, 1H, J ¼ 2.8 Hz), 6.56 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 9.2 Hz), 7.58 concentrations 0.00, 0.062, 0.125, 0.25, 0.5 mM was selected for
dd, 1H, J ¼ 4.8 Hz, J2 ¼ 8.0 Hz), 7.73 (d, 1H, J ¼ 8.8 Hz), 8.25 (d, 1H, kinetic studies. The substrate (pNPG) concentration was optimised
6
) d: compounds by following method
. The potential inhibitor with
J ¼ 8.0 Hz), 8.47 (d, 1H, J ¼ 8.8 Hz), 10.94 (s, 1H, OH exchanged from 5 to 40 mM in all kinetic studies. Pre-incubation and meas-
1
3
with D O), 12.61 (s, 1H, OH exchanged with D O); C-NMR urement time was the same as discussed in a-glucosidase inhib-
2
2
(100 MHz, DMSO-d
6
) d: 103.00 (Ar-C), 110.15 (Ar-C), 124.41 (Ar-C), ition assay. The assay was continuously monitored at 405 nm for
1
1
26.05 (Ar-C), 130.57 (Ar-C), 133.46 (Ar-C), 142.85 (C), 148.07 (C), 5 min at 30 s intervals in the microplate reader after the addition
50.19 (C), 157.46 (C), 164.76 (C); IR (KBr, cm ): 3403.94 (OH, CH of the enzyme. The inhibition type on the enzyme was assayed by
ꢀ
1
stretch), 1635.34 (C–C), 1575.56 (N¼N), 1407 (C–N). HRMS (ESI, m/ Lineweaver–Burk plots of the inverse of velocities (1/V) versus the
ꢀ
1
z) Calcd. For C H ClN O
[M þ H] 250.03778. Found
11
8
3
2
inverse of substrate concentration 1/[S] mM , and the inhibition
constant K was determined by two ways (Dixon plot of 1/V versus
inhibitor concentrations) as well as secondary replot of slope ver-
[M þ H] 250.03790.
i
2
.1.2.8. 4-((2-chloropyridin-3-yl)diazenyl)naphthalen-1-ol (TR-8). sus inhibitor concentrations from Lineweaver–Burk plot.
ꢁ
Orange crystals, yield 71%, melting point: 170–175 C; R
methanol/n-hexane, 7:3); H-NMR (400 MHz, DMSO-d
6
f
¼ 0.81
) d: 6.78 (d,
H, J ¼ 9.6 Hz), 7.47 (t, 1H, J ¼ 9.6 Hz), 7.56–7.60 (m, 2H), 7.71 (d,
H, J ¼ 9.6 Hz), 7.95 (d, 1H, J ¼ 9.6 Hz), 8.30 (d, 1H, J ¼ 8.0 Hz), 8.47
1
(
1
1
(
1
(
1
(
(
[
2.4. Inhibition mechanism of potential inhibitor
The inhibitory mechanism against a-glucosidase by most active
inhibitor from the tested series of azo derivatives was investi-
gated. The plot of the remaining enzyme activity versus the con-
centrations of the enzyme at different inhibitor concentrations
1
3
t, 2H, J ¼ 8.0 Hz); C-NMR (100 MHz, DMSO-d
24.84 (Ar-C), 125.14 (Ar-C), 125.24 (Ar-C), 127.29 (Ar-C), 128.29
Ar-C), 129.34 (Ar-C), 129.73 (Ar-C), 131.58 (Ar-C), 132.31 (Ar-C),
37.16 (Ar-C), 139.87 (Ar-C), 142.81 (C), 146.46 (C), 175.47 (C); IR
6
) d: 122.11 (Ar-C),
(0.00, 0.062, 0.125, 0.25 and 0.5 mM) was calculated.
ꢀ
1
KBr, cm ): 3301.93 (OH, CH stretch), 1631.42 (C¼C), 1513.94
N¼N), 1332 (C–N). HRMS (ESI, m/z) Calcd. For C H ClN O
1
5
10
3
M þ H] 284.05852. Found [M þ H] 284.05845.
2.5. In silico molecular docking
.5.1. Retrieval of a-glucosidase from protein data bank (PDB)
The crystal structure of a-glucosidase having PDBID: 4J5T was
accessed from the Protein Data Bank (PDB) (http://www.rcsb.org/
2
2
.1.2.9. 4-((2-chloropyridin-3-yl)diazenyl)naphthalen-2-ol (TR-9).
ꢁ
Reddish orange crystals, yield 70%; melting point: 180–182 C; R
.75 (methanol/n-hexane, 7:3); H-NMR (400 MHz, DMSO-d ) d:
f
:
1
0
6
7
6
4
j5t). The retrieved protein structure was minimised by using the
.79 (d, 1H, J ¼ 9.6 Hz), 7.47 (t, 1H, J ¼ 8.0 Hz), 7.57–7.60 (m, 2H),
conjugate gradient algorithm and amber force field in UCSF
.72 (d, 1H, J ¼ 7.2 Hz), 7.95 (d, 1H, J ¼ 9.6 Hz), 8.30 (d, 1H,
34
Chimaera 1.10.1 . The stereo-chemical properties, Ramachandran
13
J ¼ 8.4 Hz), 8.48 (t, 2H, J ¼ 8.0 Hz); C-NMR (100 MHz, DMSO-d
6
) d:
35
graph and values of a-glucosidase were assessed by Molprobity
1
(
1
1
1
2
22.14 (Ar-C), 124.87 (Ar-C), 125.17 (Ar-C), 125.27 (Ar-C), 127.33
36
server , while the Ramachandran graph was generated by
Ar-C), 128.31 (Ar-C), 129.38 (Ar-C), 129.78 (Ar-C), 131.61 (Ar-C),
32.33 (Ar-C), 137.19 (Ar-C), 139.89 (Ar-C), 142.85 (C), 146.50 (C),
Discovery Studio 2.1 Client. The protein architecture and statistical
percentage values of helices, beta-sheets, coils and turns were
ꢀ
1
75.51 (C); IR (KBr, cm ): 3475 (OH), 1626 (C¼C), 1456 (N¼N),
37
accessed by VADAR 1.8 .
208 (C–N). HRMS (ESI, m/z) Calcd. for C H ClN O [M þ H]
15
10
3
84.05852. Found [M þ H] 284.05862.
2.5.2. In silico drugs designing
The designed ligands (TR1-9) were sketched in drawing ACD/
ChemSketch tool and retrieved in PDB format. Furthermore, the
2.2. In vitro a-glucosidase inhibition assay
The a-glucosidase inhibition assay was performed as described UCSF Chimaera 1.10.1 tool was employed to energy minimisation
3
1
previously . In summary, the stock solution of the enzyme from of each drug separately having default parameters such as steep-
Saccharomyces Cerevisiae (0.2 U/mL) was prepared in 0.05 M est descent steps 100 with step size 0.02 (Å), conjugate gradient
sodium phosphate buffer pH 6.8. In microtitre plate, 70 mL of steps 100 with step size 0.02 (Å) and update interval was fixed at
phosphate buffer, 10 mL of enzyme and 10 mL of varying concen- 10. Finally, Gasteiger charges were added using Dock Prep in
trations of azo derivatives (25 mg, 12.5 mg, 6.25 mg, 3.12 mg and drugs to obtain a good structural conformation for docking ana-
ꢁ
1
.56 mg/mL) were mixed and incubated at 37 C for 15 min. After lysis. A molecular docking experiment was employed on synthes-
incubation, 10 mL of the substrate, p-nitrophenyl a-D-glucopyrano- ised ligands against a-glucosidase by using PyRx virtual screening
3
8
side (pNPG, 5 mM solution in 0.05 M phosphate buffer) was added tool with AutoDock VINA Wizard approach . The grid box centre

1
514
T. TAHIR ET AL.
values were adjusted as for X¼ ꢀ19.2552, Y¼ ꢀ21.0275 and achieved through re-crystallization and TLC was employed to con-
Z ¼ 4.2779, respectively. We have adjusted sufficient grid box size firm the purification status. The structures of the purified diaryl
1
13
big enough on biding pocket residues to allow the ligand to azo-phenol derivatives were elucidated by FTIR, H-NMR, C-NMR
move freely in the search space. The default exhaustiveness value and mass spectrometry (HRMS). The FTIR spectra were recorded in
ꢀ
1
¼
8 was adjusted in both dockings to maximise the binding con- the range of 4000–600 cm , the characteristic prominent peaks
formational analysis. In all docked complexes, the drug’s conform- of synthesised azo-phenol derivatives were observed. The dis-
ꢀ
1
ational poses were keenly observed to obtain the best docking appearance of NH
2
stretch at ꢂ3500 cm , a broad OH stretch
results. The docked complexes were evaluated on the lowest bind- overlapped with the aromatic C–H stretching vibrations around
ꢀ
1
ing energy (Kcal/mol) values and structure-activity relationship 3300–3000 cm and a characteristic N¼N peak at ꢂ1500 have
analyses. The graphical depictions of all the docking complexes been observed in all FTIR spectra indicating the presence of the
1
were carried out using Discovery Studio (2.1.0).
said functional groups. In the H-NMR spectra, recorded in deuter-
ated DMSO, the disappearance of NH signal in the range of
2
5
.0ꢀ4.0 ppm characteristic for the used amines was observed,
2
.6. Antibacterial activity
while the amount and type of signals in the range of 6.0ꢀ9.0 ppm
correspond exactly to the number of aromatic protons. Similarly,
The prepared azo derivatives were screened for their antibacterial
activity against six bacterial strains obtained from hospital isolates
i.e. gram positive (S. aureus, S. aureus (DR)) and gram-negative (E.
coli, P. aeruginosa, P. aeruginosa (DR), P. vulgaris). All the strains
were confirmed by biochemical identification and drug suscepti-
bility methods. The antibacterial evaluation was done by agar disc
1
the numbers of signals in the C-NMR spectra for the obtained
derivatives were consistent with their structure. High-resolution
mass spectrometry (HRMS) was also used to identify and con-
firmed the structure of azo compounds. The obtained spectro-
scopic data was found consistent with the reported literature in
this regard
are given in Supplementary material.
4
8–50
1
13
3
9,40
41–43
. All detailed spectra (FTIR, H and C-NMR, HRMS)
diffusion method
and MIC was determined
. In summary, a
4
8-h old inoculum having ꢂ0.48 OD600, of the above-mentioned
bacterial strains was spread on the solidified trypticase soy agar
media and Whatman no. 1 filter paper discs (5 mm in diameter)
soaked with the title compounds (10 mL/disc of 10 mg/mL) were
3
.2. Inhibitory potential against a-glucosidase
placed on the plates. Ampicillin and moxifloxacin were used as All the title compounds (TR-1 to TR-9) were evaluated for their
positive controls and DMSO as negative control respectively. The inhibitory potential against a-glucosidase enzyme. At a concentra-
ꢁ
plates were incubated at 37 C for 48 h, post-inoculation and tion of 250 mg/mL, almost all the tested azo-phenol derivatives
diameters for the zone of inhibition were calculated in millimetres exhibited 60–98% anti-diabetic activity, except TR-4 (43%) and
(
mm) and MIC (mg/mL) was determined for the most TR-5 (56%). It was noticed that the diaryl azo derivatives with two
hydroxyl groups at ortho and meta-positions (TR-1, TR-6, TR-7)
have enhanced a-glucosidase inhibition potential with IC50 values
active compounds.
1
5.70 ± 1.3, 53.7 ± 3.2 and 29.7 ± 2.8 respectively, compared to the
2
.7. Antibiofilm assay
other active derivatives (TR-2, TR-3, TR-4 and TR-5) with one
hydroxyl group at any position on the phenyl ring. The present
results were found to be in good agreement with the previous
studies establishing the fact that the number and position of
hydroxyl groups on the phenyl ring could increase or decrease
the inhibitory activity of the compounds against a-glucosidase by
effecting the magnitude of hydrogen bond interactions with the
protein . On the other hand, in the case of diaryl azo derivatives
TR-7, TR-8 and TR-9, the notable anti-diabetic potential could
possibly be due to the introduction of the nitrogen-containing
The antibiofilm activity of the synthesised azo derivatives was
assessed by microtitre plate-based crystal violet method against
respective bacterial strains i.e. gram-positive (S. aureus, S. aureus
4
4
(DR)) and gram-negative (P. aeruginosa, P. aeruginosa (DR)). In
brief, 10 mL bacterial suspension, 100 mL trypticase soy broth and
1
0 mL (10 mg/mL) of test compounds were added in the wells in
5
1
ꢁ
duplicates and incubated at 37 C overnight. Ampicillin and moxi-
floxacin were used as positive controls. After the aspiration of
planktonic cells, the biofilm was fixed with 99% methanol. The
wells were then washed with 0.1 M sterile PBS (pH ¼ 6.9) thrice
and air-dried. Then, 200 mL of crystal violet solution (2%) was
added to all wells. After 15 min, the excess of the crystal violet
dye was removed and the wells were washed again with PBS.
Then, the cell-bound crystal violet was dissolved in 33% acetic
acid. The biofilm growth was quantified in terms of OD575 nm
using an ELISA plate reader and biofilm hydrolysis was calculated
by % inhibition formula.
1
3
heterocyclic pharmacophore with 4–Cl group. The promising
anti-diabetic potential exerted by the derivatives TR-7, TR-8 and
TR-9 is also justified by the minimum binding energy values
ꢀ
7.50, ꢀ8.20 and ꢀ8.30 Kcal/mol respectively. The following trend
in the decreasing order of IC50 (mg/mL) was observed: TR-5>TR-
>TR-2>TR-9>TR-6>TR-8>TR-7>TR-1. The IC50 of TR-1 was
3
found to be effective (15.70 ± 1.3 mg/mL) compared to the refer-
ence drug acarbose (21.59 ± 1.5 mg/mL) (Table 1), hence, it was fur-
ther selected for the kinetic and docking studies in order to
illustrate the mechanism of inhibition.
3
. Results and discussion
3.1. Chemistry
3.3. Kinetic analysis
The targeted diaryl aromatic azo compounds were synthesised by To understand the inhibitory mechanism of the most potent com-
the facile diazo-coupling approach (Scheme 1) described previ- pound against a-glucosidase, kinetic studies were conducted.
4
5–48
ously in literature
. The combination of the substituted amines Kinetic studies showed a concentration-dependent inhibition of
(4-nitro aniline, p-toluidine and 3-amino,2-chloro pyridine) with a-glucosidase by the inhibitors. Continuous monitoring of the
phenol derivatives (resorcinol, a-naphthol and b-naphthol) pro- reaction showed a marked decrease in reaction rate in the pres-
duced azo-phenol compounds in moderate to good yields ence of the inhibitors, which is ultimate, indicated the decrease in
(40–80%). The purification of the synthesised compounds was the final absorbance when compared with controls containing no

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1515
Table 1. In vitro a-glucosidase inhibitory potential of diaryl azo-phenol derivatives
Inhibition
%
No.
Sample code
250 (mg/mL)
125 (mg/mL)
62.5 (mg/mL)
31.2 (mg/mL)
15.62 (mg/mL)
IC50 (mg/mL)
Binding energy (Kcal/mol)
1
2
3
4
5
6
7
8
9
TR-1
TR-2
TR-3
TR-4
TR-5
TR-6
TR-7
TR-8
TR-9
95
60
62
43
56
87
87
98
78
92
88
55
54
35
49
63
77
75
63
85
78
42
38
27
35
55
68
63
58
73
68
30
25
13
22
38
52
45
39
62
49
16
12
7
14
22
34
29
22
40
15.70 ± 1.3
110.2 ± 7.8
116.9 ± 8.6
nt
153.4 ± 11.5
53.7 ± 3.2
29.7 ± 2.8
36.7 ± 3.1
54.4 ± 3.3
21.59 ± 1.5
–7.80
–8.20
–8.00
nt
–8.40
–7.60
–7.50
–8.20
–8.30
nt
1
0
Acarbose
Nt: mean not determined. IC50: 50% inhibitory concentration (means ± SEM of triplicate experimental values).
Figure 3. TR-1 a) Lineweaver-Burk plots for the inhibition of a-Glucosidase by various concentrations 0.00, 0.062, 0.125, 0.25 and 0.5mM of compound TR-1 in the
presence of different concentrations 5, 10, 20 and 40 mM of substrate pNPG (p-nitrophenyl-a-D-glucopyranoside). b) Secondary replot of the Lineweaver–Burk plot
between the slopes of each line on Lineweaver–Burk plot vs. different concentrations of inhibitor. c) Dixon plot of reciprocal of rate of reaction (velocities) versus dif-
ferent concentrations of compounds TR-9 in the presence of various concentrations (5, 10, 20 and 40 mM) of pNPG.
inhibitor. The potency of inhibition exhibited by the compounds
varied depending on the presence and position of different substi-
tutions and on the class of compounds Inhibition kinetics was
Table 2. Kinetic parameters of a-glucosidase inhibition activity for the com-
pound TR-1.
Dose
V
max
K
m
K
i
analysed by Lineweaver–Burk plot and Dixon plots to determine Compounds (mM) (DA/S) (mM)
Inhibition type
Inhibition (mM)
the type of inhibition and inhibition constant (K
i
).
TR-1
0.00
0.062
0.25
20
20
20
20
20
Non-competitive reversible
0.22
From the kinetic analyses, the Lineweaver–Burk plot of 1/V ver-
sus 1/[S] in the presence of different concentrations of TR-1
showed that all the straight line intersects on the X-axis in the
second quadrant, the analysis showed that Vmax decreased with
0.166
0.125
0.090
0.071
0
.125
0
0
.25
.50
V
max is the reaction velocity.
constant K
m
in the presence of increasing concentrations of com-
K
K
m
is the Michaelis–Menten constant.
is the EI dissociation constant.
pounds TR-1. This behaviour of the compound indicated that the
compound TR-1 is a non-competitive inhibitor against a-glucosi-
i
3
2
dase (Figure 3(a)) . The inhibition constant K
i
for compounds TR-
1
was calculated by two methods, secondary replot of slope from
Lineweaver-Burk plot versus inhibitor concentrations (Figure 3(b))
and by Dixon plot as shown in (Figure 3(c)) respectively. All the
kinetics parameters are written in Table 2. The inhibitory mechan-
ism of a-glucosidase by varying concentrations of the most potent
compound TR-1 by the plots of the remaining enzyme activity
versus the enzyme concentration was also studied. The results
5
2
suggested that the compound behaves reversibly (Figure 4) .
3.4. Molecular docking analyses
3
.4.1 a-Glucosidase structural assessment
The a-glucosidase is a class of hydrolase enzyme and is actively
involved in the digestion of food carbohydrates. The a-glucosidase
comprises 811 amino acids with the residual architecture of 35%
helices, 25% b sheets and 38% coils. The X-ray diffraction studies
Figure 4. The catalytic activity of a-glucosidase as a function of enzyme concen-
of a-glucosidase confirmed its resolutions 2.04 Å. The tration at different concentrations of compound TR-1.

1
516
T. TAHIR ET AL.
Figure 5. 3D structure of a-glucosidase enzyme.
Ramachandran plots of a-glucosidase indicated that 97.6% of resi-
dues were present in favored regions. The Ramachandran graphs
values showed the good accuracy of phi (u) and psi (w) angles
among the coordinates of target protein (Figure 5)
3
.4.2. Binding pocket and deltamethrin and permethrin binding
conformations
A molecular docking experiment is the best approach to study
5
3–55
the binding conformation of ligands against target protein
.
Figure 6. Binding pocket prediction and interactions of all ligands (TR1-9)
The binding pocket analysis showed that deltamethrin and per- against a-glucosidase.
methrin were confined in the active region of a-glucosidase.
Results showed that the drugs bound in the active site having dif- compared to all other derivatives. The docking energy values of
ferent conformational poses. The docked complexes of the drug the synthesised ligands (TR1-9) against a-glucosidase are pre-
against tyrosinase were analysed on the basis of the lowest bind- sented in Table 1.
ing energy values (Kcal/mol) and hydrogen/hydrophobic inter-
action pattern. Results showed that all the drugs exhibited good
docking energy values and showed their interaction within the
active region of the target protein (Figure 5). The docking energy 3.4.2. Binding pocket and hydrogen binding analysis
values of all the docking complexes were calculated by using Figure 6 showed the binding pocket prediction and interactions
Equation (1).
DGbinding ¼ DGgauss þ DGrepulsion þ DGhbond
þ DGhydrophobic þ DGtors
Here, DGgauss: attractive term for dispersion of two gaussian
functions, DGrepulsion: square of the distance if closer than a
threshold value, DGhbond: ramp function – also used for interac-
tions with metal ions, DGhydrophobic: ramp function, DGtors: pro-
portional to the number of rotatable bonds. In docking, energy
results compound selected as best having more than 2.5 Kcal/mol
energy value different compared to other compounds. The stand-
of all ligands at conformational positions. All the ligands (TR1-9)
bind within the active region of a glucosidase which ensures the
stability and reliability of docking results. In most of the ligands,
oxygen atoms were confined at the outer region of the binding
pocket whereas, the benzene ring part showed inside the bind-
ing pocket.
Based on in vitro results, TR-1 has selected for detail binding
interactions. The docking results showed that TR-1 formed two at
different residual positions hydrogen bond. The hydroxyl group of
TR-1 formed a hydrogen bond with Gly546 and Trp690 having
bond distances of 1.87 and 1.92 Å, respectively. In docking results,
(1)
ard error for Autodock is reported as 2.5 Kcal/mol (http://auto- TR-1 binds within the active site and the bond lengths were com-
5
6
dock.scripps.edu/) . The present docking results justified that the parable with the standard value (<3 Å) (Figure 7). Our docking
energy value difference among all docking complexes was com- results were well correlated with a previously published article,
5
7
parable with the standard error value. Therefore, based on the confirming the accuracy of our docking results .
basis of both in vitro and in silico docking energy results, delta-
Supplementary data (S1): The a-glucosidase Ramachandran
methrin and permethrin were ranked as the best drugs that graph is presented the residual position in the favour region (pink
showed good inhibitory potential against targeted enzyme as boundary) by evaluating the Psi (u) and Phi (w) angles. Most of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1517
the residues (green circle) lie in favour region while only ten poor (mg/200 mL). The antimicrobial results of synthesised azo deriva-
rotamers are present in the outer region.
tives exhibited good to moderate broad-spectrum activity against
screened pathogens (S. aureus, P. aeruginosa, E. coli and P. vulga-
ris). Among the studied title compounds, TR-1, TR-4, TR-6 dis-
played promising antibacterial potential with an average MIC
value of 125 mg/200 mL and was found to be dose-dependent,
compared to the standard drugs i.e. ampicillin and moxifloxacin
3.5. Antimicrobial potential
The antibacterial results of the prepared azo-phenol derivatives
are displayed in (Table 3) and are expressed quantitatively in MIC
(MIC 62.5 mg/200 mL). It was also observed that the detrimental
effect of derivatives on bacterial strains was due to the electron-
4
withdrawing groups on the phenyl rings .
3.6. Biofilm inhibition potency
The ability to produce biofilm increases the resistance of microbial
cells against harmful environments. Bacterial adhesion to the sur-
face, as well as cell-cell interaction via biological membrane,
occurs due to electrostatic interactions. In order to inhibit biofilm
formation, one strategy is to break the adhesive forces between
bacterial cells and the surface which can be achieved with the use
5
8
of various antimicrobial agents . In this aspect, the synthesised
azo-phenol derivatives were tested for their hydrolysis potential
against biofilm formation. Therefore, in the case of P. aeruginosa
(
DR), P. aeruginosa (DS); it was found that at the concentration of
.5 mg/mL, all the derivatives except TR-4 and TR-7 (Figure 8(a))
were highly active against P. aeruginosa (DS) in biofilm inhibition
>70%) compared to the standard drug penicillin, which showed
0% inhibition at 0.5 mg/mL, however, against P. aeruginosa (DR)
0
(
6
most of the azo derivatives were active (ꢂ70%) in degrading bio-
Figure 7. Molecular docking interaction of TR-1 against target protein.
logical membrane but TR-5 and TR-8 efficacy was found to be
Table 3. Minimum inhibitory concentration (mg/200 mL) of azo-phenol derivatives against below mentioned bacterial strains
S. aureus
DS)
S. aureus
(DR)
P. aeruginosa
P. aeruginosa
(
(DS)
(DR)
E. coli
P. vulgaris
Compound code
250
125
62.5
250
125
62.5
250
125
62.5
250
125
62.5
250
125
62.5
250
125
62.5
TR-1
TR-2
TR-3
TR-4
TR-5
TR-6
TR-7
TR-8
S
R
R
S
R
S
R
S
R
S
–
S
R
R
S
R
S
R
S
R
S
–
R
R
R
S
R
S
R
R
R
S
–
R
S
R
S
S
S
R
R
S
–
S
R
S
R
S
S
S
R
R
S
–
S
R
R
R
R
S
S
R
R
S
–
R
R
S
S
S
S
S
R
S
S
S
–
R
R
R
S
R
S
R
R
R
S
–
R
R
R
R
R
R
R
R
R
S
–
S
R
R
S
S
R
S
R
R
–
S
S
R
R
S
S
R
S
R
R
–
S
S
R
R
R
R
R
S
R
R
–
R
S
S
R
R
S
S
R
R
R
S
–
S
R
R
R
S
R
R
R
R
S
–
S
R
R
R
S
R
R
R
R
S
–
S
R
R
S
R
S
R
R
R
S
–
S
R
R
S
R
S
R
R
R
S
–
S
R
R
S
R
S
R
R
R
S
–
TR-9
Ampicillin
Moxifloxacin
R: resistant; S: sensitive; –: not tested.
Figure 8. Biofilm hydrolysis potential of azo derivatives against P. aeruginosa.

1
518
T. TAHIR ET AL.
Figure 9. Biofilm hydrolysis potential of azo-phenol compounds against S. aureus.
highest (80%) compared to the standard i.e. moxifloxacin (8b). Author contributions
Interestingly, the compound TR-7 showed 55% anti-biofilm
Conceptualisation, M.A. and M.I.S.; Investigation & Methodology,
potency against the drug-resistant strain of P. aeruginosa which
was not found active against P. aeruginosa (DS). However, the rest
of the tested derivatives were found less efficient in this regard.
Similarly, between S. aureus (DS) and S. aureus (DR) biofilm
hydrolysis efficiencies, TR-1, TR-3, TR-8 showed potential greater
T.T., M.R., R.T. and M.M.; Supervision, M.I.S. and M.A.; Writing- ori-
ginal draft, T.T., R.T. and M.R.; Docking studies, M.H.; Writing-
review and editing, M.M., K.K.M., M.R., and M.I.S.
than 70% compared to a penicillin (65%) and all the derivatives Disclosure statement
except TR-5, TR-7 and TR-8 showed efficacy equal to moxifloxacin
No potential conflict of interest was reported by the author(s).
(85%) at 0.5 mg/mL concentration respectively. It was also
observed that the efficiency of the compound TR-8 was drastically
decreased when it was challenged with the drug-resistant strain
of S. aureus (Figure 9).
ORCID
Katarzyna Kotwica-Mojzych
655-7324
http://orcid.org/0000-0002-
3
Mariusz Mojzych
http://orcid.org/0000-0002-9884-6068
4
. Conclusions
In summary, a series of azo derivatives were synthesised via the
diazo-coupling approach between substituted aromatic amines
with phenol derivatives. All the synthesised compounds were
obtained in good to moderate yields and analytically determined
by spectroscopic methods (IR, NMR and HRMS). To the best of our
knowledge, the anti-diabetic and antimicrobial activities of the
reported derivatives were not evaluated earlier. Hence, the title
compounds were biologically screened in vitro against a-glucosi-
dase and pathogenic bacterial strains i.e. E. coli (gram-negative), S.
aureus (gram-positive), S. aureus (gram-positive) drug-resistant
strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative)
drug-resistant strain and P. vulgaris (gram-negative). The experi-
mental data showed that the compounds under consideration
have promising pharmacological properties to be used as poten-
tial drug leads for the treatment against diabetes mellitus (DM).
The title compounds can also serve as the functional template in
the antimicrobial resistance (AMR) issue as they have been
screened against broad-spectrum bacteria including drug-resistant
strains and exhibited good to moderate antimicrobial potential as
almost all the compounds exhibited dose-dependent broad-spec-
trum activity against screened pathogens (MIC 125 mg/200 mL) and
biofilm hydrolysis potential (>60 ꢀ 80%). The presence of an aro-
matic hydroxyl group and a heterocyclic pharmacophore fused
with the azo moiety confirmed their high inhibitory potency
against the a-glucosidase enzyme. The enzyme inhibitory kinetics,
mode of binding and docking studies of the most potent inhibi-
tors could help out to design good inhibitors against a-glucosi-
dase to overcome the worldwide health problem of diabetes.
References
1
.
Chhetri A, Chettri S, Rai P, et al. Synthesis, characterization
and computational study on potential inhibitory action of
novel azo imidazole derivatives against COVID-19 main pro-
tease (Mpro: 6LU7). J Mol Struct 2021;1225:129230.
2
.
Niluvanji Matada M, Jathi K, Rangappa MM, et al. A new sul-
phur containing heterocycles having azo linkage: synthesis,
structural characterization and biological evaluation. J King
Saud Uni – Science 2020; 32:3313–20.
3. Maliyappa M, Keshavayya J, Mallikarjuna N, Pushpavathi I.
Novel substituted aniline based heterocyclic dispersed azo
dyes coupling with 5-methyl-2-(6-methyl-1, 3-benzothiazol-
2-yl)-2, 4-dihydro-3H-pyrazol-3-one: synthesis, structural,
computational and biological studies. J Mol Struct 2020;
1205:127576.
4. Ravi BN, J K, M MN, et al. Synthesis, characterization and
pharmacological evaluation of 2-aminothiazole incorporated
azo dyes. J Mol Struct 2020;1204:127493.
5. Debnath P, Das A, Singh KS, et al. Synthesis, structural char-
acterization and antimicrobial activities of triorganotin (IV)
azo-carboxylates derived from ortho/para-amino benzoic
acids and b-naphthol. Inorganica Chimica Acta 2019;498:
119172.
6. Ispir E, Ikiz M, Inan A, et al. Synthesis, structural characteriza-
tion, electrochemical, photoluminescence, antiproliferative
and antioxidant properties of Co (II), Cu (II) and Zn (II)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1519
complexes bearing the azo-azomethine ligands. J Mol Struct
019;1182:63–71.
isolates. Comparative Immunol Microbiol Infect Disease
2021;75:101613.
2
7.
8.
9.
Matada MN, Jathi K. A novel azo metal complexes of 5, 5, 7- 21. Ben Mohamed-Smati S, Faraj FL, Becheker I, et al. Synthesis,
trimethyl-4, 5, 6, 7-tetrahydro-1, 3-benzothiazol as potential
pharmacological agents: Synthesis and spectroscopic charac-
terization. J Mol Struct 2019;1180:196–208.
characterization and antimicrobial activity of some new azo
dyes derived from 4-hydroxy-6-methyl-2H-pyran-2-one and
its dihydro derivative. Dye Pigment 2021;188:109073.
Tavaf Z, Dangolani SK, Yousefi R, et al. Synthesis of new cur- 22. Ribeiro D, Poen c¸ a C, Varela C, et al. New phenolic cinnamic
cumin derivatives as influential antidiabetic a-glucosidase
and a-amylase inhibitors with anti-oxidant activity.
Carbohydrate Res 2020;494:108069.
Peytam F, Adib M, Shourgeshty R, et al. Synthesis and bio-
logical evaluation of new dihydroindolizino[8,7-b]indole
derivatives as novel a-glucosidase inhibitors. J Mol Struct
acid derivatives as selective COX-2 inhibitors. Design, syn-
thesis, biological activity and structure-activity relationships.
Bioorg Chem 2019; 91:103179.
2
3. Kr ꢀa tk ꢀy M, Jand'ourek O, Baranyai Z, et al. Phenolic N-mono-
substituted carbamates: antitubercular and toxicity evalu-
ation of multi-targeting compounds. Europ J Med Chem
2
019; 181:111578.
2021;1224:129290.
2
2
4. Dayma V, Chopra J, Sharma P, et al. Synthesis, antidiabetic,
antioxidant and anti-inflammatory activities of novel hydrox-
ytriazenes based on sulpha drugs. Heliyon 2020;6:e04787.
5. Prakash S, Somiya G, Elavarasan N, et al. Synthesis and char-
acterization of novel bioactive azo compounds fused with
benzothiazole and their versatile biological applications. J
Mol Struct 2021;1224:129016.
1
0. Azimi F, Ghasemi JB, Azizian H, et al. Design and synthesis
of novel pyrazole-phenyl semicarbazone derivatives as
potential a-glucosidase inhibitor: kinetics and molecular
dynamics simulation study. Int J Biol Macromolecule 2021;
166:1082–95.
1
1. Abdullah MA, Lee Y-R, Mastuki SN, et al. Development of
diarylpentadienone analogues as alpha-glucosidase inhibitor:
synthesis, in vitro biological and in vivo toxicity evaluations,
and molecular docking analysis. Bioorg Chem 2020;104:
2
6. Yu J, Xu L, Hong D, et al. Design, synthesis, and biological
evaluation of novel phenol ether derivatives as non-covalent
proteasome inhibitors. Europ J Med Chem 2019;161:543–58.
7. Athira L, Balachandran S, Devi RS. Synthesis, crystal struc-
ture, solvatochromic properties and DNA cleaving activity of
azo derivative of naphthalen-2-ol. J Mol Struct 2019;1180:
104277.
2
1
2. Akhter S, Ullah S, Yousuf S, et al. Synthesis, crystal structure
and Hirshfeld Surface analysis of benzamide derivatives of
thiourea as potent inhibitors of a-glucosidase in-vitro. Bioorg
Chem 2021;107:104531.
1
00–9.
2
2
8. Bl u€ mel S, Stolz A. Cloning and characterization of the gene
coding for the aerobic azoreductase from Pigmentiphaga
kullae K24. Applied Microbiol Biotechnol 2003;62:186–90.
9. Valizadeh H, Amiri M, Shomali A, Hosseinzadeh F. Ionic
liquid 1-(3-Trimethoxysilylpropyl)-3-methylimidazolium nitrite
as a new reagent for the efficient diazotization of aniline
derivatives and in situ synthesis of azo dyes. J Iran Chem
Soc 2011;8:495–501.
1
3. Aziz H, Saeed A, Rehman AU, et al. Synthesis, characteriza-
tion, in vitro biological and computational evaluation of 5-
benzyl-4-(benzylideneamino)-2H-1, 2, 4-triazole-3 (4H)-thio-
nes. J Iranian Chem Soc 2021;1–13. DOI:10.1007/s13738-021-
02156-5
1
4. Yang J, Wang X, Zhang C, et al. Comparative study of inhib-
ition mechanisms of structurally different flavonoid com-
pounds on a-glucosidase and synergistic effect with
acarbose. Food Chem 2021;347:129056.
3
3
0. Zellner
H.
1-(2-Nitro-4-methylphenylazo)-2-naphthol.
Fresenius’ Zeitschrift F u€ r Analytische Chemie 1953;140:317.
1. Arshad T, Khan KM, Rasool N, et al. Syntheses, in vitro evalu-
ation and molecular docking studies of 5-bromo-2-aryl ben-
zimidazoles as a-glucosidase inhibitors. Med Chem Res 2016;
1
1
1
5. Yousuf H, Shamim S, Khan KM, et al. Dihydropyridines as
potential a-amylase and a-glucosidase inhibitors: synthesis,
in vitro and in silico studies. Bioorg Chem 2020;96:103581.
6. Ali G, Cuny GD. An efficient synthesis of an 8-phenoxy apor-
phine derivative utilizing mono-ligated palladium ortho-phe-
nol arylation. Tetrahedron 2019;75:4318–24.
7. Rai S, Kureel AK, Dutta PK, Mehrotra GK. Phenolic com-
pounds based conjugates from dextran aldehyde and BSA:
preparation, characterization and evaluation of their anti-
cancer efficacy for therapeutic applications. Int J Biol
Macromolecule 2018;110:425–36.
2
5:2058–69.
3
2. Ur Rehman N, Halim SA, Al-Azri M, et al. Triterpenic acids as
non-competitive a-glucosidase inhibitors from Boswellia
elongata with structure-activity relationship: in vitro and in
silico studies. Biomolecule 2020;10:751.
3
3. Motoshima K, Noguchi-Yachide T, Sugita K, et al. Separation
of a-glucosidase-inhibitory and liver X receptor-antagonistic
activities of phenethylphenyl phthalimide analogs and gen-
eration of LXRa-selective antagonists. Bioorg Med Chem
1
1
2
8. Al Majidi MIH, El-Shaheny R, El-Shabrawy Y, El-Maghrabey M.
Screening and greenness profiling of oxidative-coupling and
electrophilic aromatic substitution reactions for determin-
ation of three phenolic drugs. Microchem J 2019;149:
2
009;17:5001–14.
3
4. Pettersen EF, Goddard TD, Huang CC, Couch GS, et al. UCSF
Chimera—a visualization system for exploratory research
and analysis. J Computation Chem 2004;25:1605–12.
5. Lovell SC, Davis IW, Arendall IIW, et al. Structure validation
by Ca geometry: /, w and Cb deviation. Proteins: Struct
Function Bioinfo 2003;50:437–50.
104051.
9. Okasha RM, Alsehli M, Ihmaid S, et al. First example of Azo-
Sulfa conjugated chromene moieties: synthesis, characteriza-
tion, antimicrobial assessment, docking simulation as potent
3
class I histone deacetylase inhibitors and antitumor agents. 36. Chen VB, Arendall WB, Headd JJ, et al. MolProbity: all-atom
Bioorg Chem 2019;92:103262.
structure validation for macromolecular crystallography. Acta
Crystallographica Section D: Biol Crystallograph 2010;66:
12–21.
0. Obodoechi LO, Carvalho I, Chenouf NS, et al. Antimicrobial
resistance in Escherichia coli isolates from frugivorous
(Eidolon helvum) and insectivorous (Nycteris hispida) bats in 37. Monzavi H, Willard L, Zhang H, et al. VADAR: a web server
Southeast Nigeria, with detection of CTX-M-15 producing
for quantitative evaluation of protein structure quality. 2003.

1
520
T. TAHIR ET AL.
3
8. Dallakyan S, Olson AJ, Small-molecule library screening by
docking with PyRx. Chem Biol. Springer; 2015: 243–50. DOI:
0.1007/978-1-4939-2269-7_19
values, antioxidant, antibacterial and anticancer activity. J
Organometal Chem 2015;783:28–39.
50. Keshavayya J. Synthesis, structural investigations and in vitro
biological evaluation of N, N-dimethyl aniline derivatives
based azo dyes as potential pharmacological agents. J
MolStruct 2019;1186:404–12.
1
3
9. Famuyide IM, Aro AO, Fasina FO, et al. Antibacterial and
antibiofilm activity of acetone leaf extracts of nine under-
investigated South African Eugenia and Syzygium
(Myrtaceae) species and their selectivity indices. BMC 51. Delogu GL, Era B, Floris S, et al. A new biological prospect-
Complement Alternative Med 2019;19:141.
0. Brahma U, Kothari R, Sharma P, Bhandari V. Antimicrobial
and anti-biofilm activity of hexadentated macrocyclic com-
ive for the 2-phenylbenzofurans as inhibitors of a-glucosi-
dase and of the islet amyloid polypeptide formation. Int J
Biol Macromol 2021;169:428–35.
4
4
4
plex of copper (II) derived from thiosemicarbazide against 52. Jenis J, Baiseitova A, Yoon SH, et al. Competitive a-glucosi-
Staphylococcus aureus. Sci Rep 2018;8:1–8.
1. Kaur H, Lim SM, Ramasamy K, et al. Diazenyl schiff bases:
synthesis, spectral analysis, antimicrobial studies and cyto-
dase inhibitors, dihydrobenzoxanthones, from the barks of
Artocarpus elasticus. J Enzyme Inhibition Med Chem 2019;34:
1623–32.
toxic activity on human colorectal carcinoma cell line (HCT- 53. Hassan M, Abbas Q, Ashraf Z, et al. Pharmacoinformatics
1
16). Arab J Chem 2020;13:377–92.
exploration of polyphenol oxidases leading to novel inhibi-
tors by virtual screening and molecular dynamic simulation
study. Computation Biol Chem 2017;68:131–42.
2. Aziz H, Saeed A, Khan MA, et al. Synthesis, characterization,
antimicrobial, antioxidant and computational evaluation of
N-acyl-morpholine-4-carbothioamides. Mol Diversity 2020;25: 54. Hassan M, Ashraf Z, Abbas Q, et al. Exploration of novel
1
–14.
3. Aziz H, Saeed A, Khan MA, et al. Novel N-Acyl-1H-imidazole-
-carbothioamides: design, synthesis, biological and compu-
human tyrosinase inhibitors by molecular modeling, docking
and simulation studies. Interdisciplinary Sci: Computation
Life Sci 2018;10:68–80.
4
1
tational studies. Chem Biodiversity 2020;17:e1900509.
4. Shukla SK, Rao TS. An improved crystal violet assay for bio-
film quantification in 96-well microtitre plate. BioRxiv 2017;
55. Hassan M, Shahzadi S, Seo SY, et al. Molecular docking and
dynamic simulation of AZD3293 and solanezumab effects
against BACE1 to treat Alzheimer’s disease. Frontier
Computation Neurosci 2018;12:34.
4
100214. DOI:10.1101/100214
4
4
5. Xu H, Zeng X. Synthesis of diaryl-azo derivatives as potential 56. Channar PA, Saeed A, Albericio F, et al. Sulfonamide-linked
antifungal agents. Bioorg Med Chem Lett 2010;20:4193–5.
6. Qiu J, Xiao J, Tang B, et al. Facile synthesis of novel disperse
azo dyes with aromatic hydroxyl group. Dye Pigment 2019;
ciprofloxacin, sulfadiazine and amantadine derivatives as a
novel class of inhibitors of jack bean urease; synthesis, kin-
etic mechanism and molecular docking. Molecule 2017;22:
1352.
160:524–9.
4
7. Maliyappa M, Keshavayya J, Mallikarjuna N, et al. Synthesis, 57. Abbas Q, Hassan M, Raza H, et al. In vitro, in vivo and in sil-
characterization, pharmacological and computational studies
of 4, 5, 6, 7-tetrahydro-1, 3-benzothiazole incorporated azo
dyes. J Mol Struc 2019;1179:630–41.
ico anti-hyperglycemic inhibition by sinigrin. Asian Pac J
Trop Med 2017;10:372–9.
58. Sai Saraswathi V, Kamarudheen N, Bhaskara Rao KV,
Santhakumar K. Biofilm inhibition formation of clinical
strains of Pseudomonas aeruginosa mutans, photocatalytic
activity of azo dye and GC–MS analysis of leaves of
Lagerstroemia speciosa. J Photochem Photobiol B: Biol 2017;
169:148–60.
4
4
8. Ghanavatkar CW, Mishra VR, Mali SN, et al. Synthesis, bioac-
tivities, DFT and in-silico appraisal of azo clubbed benzothia-
zole derivatives. J Mol Struct 2019;1192:162–71.
9. Kantar C, Akal H, Kaya B, et al. Novel phthalocyanines con-
taining resorcinol azo dyes; synthesis, determination of pKa