Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives: Antidiabetic, antioxidant, β-glucuronidase inhibiton and their molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2016.01.007

6-Chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives 1-26 were synthesized and characterized by various spectroscopic techniques. All these derivatives were evaluated for their antiglycation, antioxidant and β-glucuronidase potential followed their docki

Full text of DOI:10.1016/j.bioorg.2016.01.007

Accepted Manuscript
Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives: Antidia-
betic, antioxidant, β-glucuronidase inhibiton and their molecular docking stud-
ies
Muhammad Taha, Nor Hadiani Ismail, Syahrul Imran, Hesham Rashwan,
Waqas Jamil, Sajjad Ali, Syed Muhammad Kashif, Fazal Rahim, Uzma Salar,
Khalid Mohammed Khan
PII:
S0045-2068(16)30008-6
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.01.007
YBIOO 1875
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 December 2015
29 January 2016
31 January 2016
Please cite this article as: M. Taha, N.H. Ismail, S. Imran, H. Rashwan, W. Jamil, S. Ali, S.M. Kashif, F. Rahim, U.
Salar, K.M. Khan, Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives: Antidiabetic, antioxidant,
β-glucuronidase inhibiton and their molecular docking studies, Bioorganic Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bioorg.2016.01.007
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Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives: antidiabetic, antioxidant, β-
glucuronidase inhibiton and their molecular docking studies
Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Syahrul Imrana,b, Hesham Rashwanc, Waqas Jamild,
Sajjad Alie, Syed Muhammad Kashifd, Fazal Rahimg, Uzma Salarf, Khalid Mohammed Khanf
aAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
b
c
Puncak Alam, Selangor, Malaysia. Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia. Faculty of Pharmacy,
Universiti Teknologi MARA, Puncak Alam, 42300, Selangor, Malaysia, cMalaysian Institute of Pharmaceuticals and Nutraceuticals
d
e
(IPharm), Ministry of Science. Institute of Advance Research Studies in Chemical Sciences, University of Sindh Jamshoro. Department
f
of Chemistry, Karakoram International University, Gilgit-Baltistan, Gilgit 15100, Pakistan. H. E. J. Research Institute of Chemistry,
g
International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. Department of Chemistry,
Hazara University, Mansehra-21120, Pakistan.
Abstract
6-Chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives 1-26 were synthesized and characterized by
various spectroscopic techniques. All these derivatives were evaluated for their antiglycation,
antioxidant and β –glucuronidase potential followed their docking studies. In antiglycation assay,
compound 2 (IC50 = 240.10 ± 2.50µM) and 4 (IC50 = 240.30 ± 2.90 µM) was found to be most active
compound of this series, while compounds 3 (IC50 = 260.10 ± 2.50 µM), 6 (IC50 = 290.60 ± 3.60 µM),
13 (IC50 = 288.20 ± 3.00 µM) and 26 (IC50 = 292.10 ± 3.20 µM) also showed better activities than the
standard rutin (IC50 = 294.50 ± 1.50 µM). In antioxidant assay, compound 1 (IC50 = 69.45 ± 0.25 µM), 2
(IC50 = 58.10 ± 2.50 µM), 3 (IC50 = 74.25 ± 1.10 µM), and 4 (IC50 = 72.50 ± 3.30 µM) showed good
activities. In β–glucuronidase activity, compounds 3 (IC50 = 29.25 ± 0.50µM), compound 1 (IC50
=
30.10 ± 0.60µM) and compound 4 (IC50 = 46.10 ± 1.10 µM) showed a significant activity as compared
to than standard D-Saccharic acid 1,4-lactonec (IC50 = 48.50 ± 1.25 µM) and their interaction with the
enzyme was confirm by docking studies.
Keywords: 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine, spectroscopy, antiglycation, anti-oxidant, β–
glucuronidase
 Correspondence and reprints
E-mail: taha_hej@yahoo.com and muhamm9000@puncakalam.uitm.edu.my, Tel: 0060193098141
2

1. Introduction
Latest study shows that occurrence of type 2 diabetes which represents 90% of all diabetes cases is
rapidly raising throughout the world. International Diabetes Federation estimates an increase in number
of diabetic patient from 366 million to 552 million by 2030. [1] Type-2 diabetes causes harmful effects
such as production of advanced glycation end products (AGEPs) [2]. Considering this fact, study on new
drugs which could reduce production of advanced glycation end products (AGEs) to treat diabetic
patient is extremely crucial. Currently, number of efficient antiglycation agent is very few and effort of
finding novel inhibitors is still unsuccessful [3]. Plenty of research had been carried out to reverse
glycation process using potential new antiglycation agents [4]. It was found that some inhibitors
function by splitting AGEPs cross-links which provide the opportunity of reversing diabetic
complications [5]. Besides that, AGEPs bind to receptor and induces production of reactive oxygen
species (ROS). It causes pleiotropic transcription factor nuclear factor NF-κB to be activated and causes
various pathological alterations in gene expression [6]. Besides involving in glycation process, free
radicals from hydroxyl, hydrogen peroxide, superoxide anions, peroxyl and lipid peroxides play a
significant role in initiating various diseases like respiratory diseases, cardiovascular diseases, gout,
cancer, aging, and atherosclerosis [7,8]. Observation also suggests that oxidation is involved in
development of diabetic complications. It was found that metabolic abnormalities related to diabetes
results in overproduction of mitochondrial superoxide. The increased amount of superoxide anion
induces tissue damage. Superoxide anions are also involved in activating various pathways which leads
to pathogenesis complications and inactivation of antiatherosclerotic enzymes, prostacyclin synthase and
eNOS [9].
6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine and its analogues consist of a versatile heterocyclic
pharmacophore which is extremely important in organic chemistry due to their diverse biological
activities [10]. Several benzimidazole derivatives had been proven to possess bioactivities such as
antiviral [11], antihypertensive [12], vasodilator [13], anticancer [14] antimicrobial [15,16], antioxidant
and antiglycation [17]. Benzimidazoles are also found to be potent inhibitors for numerous protein such
as urease [18], carbonic anhydrase [19] and tyrosine phosphatase [20].
We have been working on heterocyclic compounds [21-24]. In this communication we are reporting the
multiple activity of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives 1-26.
2. Result and Discussion
3

2.1. Chemistry
6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine 1-26 were obtained by reacting together with commercially
available 5-chloropyridine-2,3-diamine and different aldehydes in N,N-dimethylformamide. The
products were appeared in significant yields (Scheme-1). In this study, the reaction was carried out by
mixing sodium metabisulfite (Na2S2O5) with mixture of 5-chloropyridine-2,3-diamine (3.12 mmol) and
different substituted aromatic aldehydes (3.16 mmol) in N,N-dimethylformamide. Reaction mixture was
refluxed for 2 h. Upon completion, water (30 mL) was added to the crude reaction mixture to afford
solid mass as precipitates, after filtration the solid 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine
derivatives 1-26 were obtained in high yields. The structures of synthetic were confirmed by
spectroscopic techniques including 1H NMR and EI MS.
Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine 1-26 derivatives Scheme-1.
Table-1: Antiglycation, Antioxidant and β –glucuronidase activities of 6-chloro-2-Aryl-1H-imidazo[4,5-
b]pyridine derivatives 1-26.
Antioxidant
Antiglycation
β- glucuronidase
(µM)
Comp. No.
1
R
(µM)
(µM)
69.45 ± 0.25
58.10 ± 2.50
74.25 ± 1.10
320.20 ± 3.20
240.12 ± 2.40
260.10 ± 2.50
30.10 ± 0.60
49.60 ± 1.20
29.25 ± 0.50
2
3
4

72.50 ± 3.20
240.30 ± 2.90
46.10 ± 1.10
4
N. A.
N.A.
N.A.
N.A.
5
6
98.10 ± 0.30
290.60 ± 3.60
N. A.
N.A
N.A.
7
8
145.50 ± 1.90
310.40 ± 3.80
68.10 ± 1.30
N. A.
N. A.
N. A.
N. A.
N. A.
N. A.
9
10
N. A.
N. A.
N. A.
N. A.
N. A.
N. A.
11
12
120.80±1.60
288.20 ± 3.00
128.60 ± 2.10
13
92.40 ± 1.00
243
N. A.
N. A.
N. A.
N. A.
14
15
5

230
275
N. A.
N. A.
N. A.
N. A.
16
17
265
N. A.
N. A.
18
19
130.30 ± 1.50
298.30 ± 3.20
113.60 ± 2.40
265
230
N. A.
N. A.
N. A.
N. A.
20
21
230
247
N. A.
N. A.
N. A.
N. A.
22
23
247
N. A.
N. A.
24
289
N. A.
N. A.
25
26
146.20 ± 2.20
292.10 ± 3.20
52.60 ± 1.60
Propyl gallte
Rutin
29.20 ± 0.25
-
-
-
-
294.50 ± 1.50
-
D-Saccharic acid 1,4-lactone
48.50 ± 1.25
-
2.2. Antiglycation study
6

All the synthesized compounds 1-26 were evaluated for their antiglycation properties and compared
with the standard Rutin (IC50 = 294.46 ± 1.50 µM) respectively.
In antiglycation assay, all -OH group containing derivative were found to be active and marked as
efficient antiglycating agents. Compound 2 (IC50 = 240.10 ± 2.40µM) and 4 (IC50 = 240.30 ± 2.90 µM)
was found to be most active compound of this series, while compounds 3 (IC50 = 260.10 ± 2.50 µM), 6
(IC50 = 290.60 ± 3.60 µM), 13 (IC50 = 288.20 ± 3.00 µM) and 26 (IC50 = 292.10 ± 3.20 µM) also
showed excellent activity. All of these derivatives were found to be better active than the standard Rutin
(IC50 = 294.50 ± 1.50 µM). Mostly di-OH group containing derivatives found be more active than mono-
OH derivatives but surprisingly 3,4-di -OH compound 1 (IC50 = 320.20 ± 3.20 µM) was found to be less
active than di as well as mono –OH group containing derivatives. It was further observed that all
compounds which showed better activity than the standard bear at least one ortho-OH group, although
compound 26 have –OH group at position 4 (para) was found to be slight better active than the
standard. So the ortho position of –OH group is also matter for better antiglycation activity. So, the
active compounds have competent capability to bind protein or glucose and inhibit the further
progression of glycation. All other derivatives were found to be inactive.
2.3. Antioxidant potential
In anti-oxidant assay, all the hydroxyl derivatives showed good reducing capability. Among hydroxyl
group containing derivatives, compound 1 (IC50 = 69.45 ± 0.25 µM), 2 (IC50 = 58.10 ± 2.50 µM), 3 (IC50
= 74.25 ± 1.10 µM), and 4 (IC50 = 72.50 ± 3.30 µM) showed better activity than other molecules. It was
observed that all of these most active compounds containing more than hydroxyl group, so the activity is
directly related with numbers of hydroxyl group. On comparing these active derivatives, it was found
that compound 2, a 2,4-dihydroxy derivative is most active compound of this series, whereas compound
1, 3 and 4 also exhibited good anti-oxidant properties. A decline in anti-oxidant activity was observed
when one of the hydroxyl was replaced by–H or OCH3 group, as in case of compound numbers 6 (IC50 =
98.10 ± 0.30 µM), 8 (IC50 = 145.50 ± 1.90 µM), 13 (IC50 = 120.80 ± 1.60 µM), 19 (IC50 = 130.30 ± 1.50
µM), and 26 (IC50 = 146.20 ± 2.20 µM). It was also seen that OCH3 group produced more suppressing
effect on activity than -H. All of these compound were found be less active than the standard but slight
modification in structure may result better anti-oxidant compounds. All other derivatives 5, 7, 9, 10, 11,
12, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25 were showed no activities [Table-1].
7

2.4. β – glucuronidase activity
All the synthesized compounds 1-26 were evaluated for β- -glucuronidase and compared with standard
D-Saccharic acid 1,4-lactone (IC50 = 48.50 ± 1.25 µM). In this activity compounds 3 (IC50 = 29.25 ±
0.50µM), compound 1 (IC50 = 30.10 ± 0.60µM) and compound 4 (IC50 = 46.10 ± 1.10 µM) showed
excellent activity. It was observed that all of these most active compounds contain more than one
hydroxyl group, so the activity is directly related with numbers of hydroxyl group. Compound 3, having
2,3-dihydroxy, compound 1, having 3,4-dihydroxy and compound 4, having 2,5-dihydroxy derivatives
are most active compounds of this series. Other compound 2 (IC50 = 49.60 ± 1.20µM) and compound 26
(IC50 = 52.60 ± 1.60 µM) also showed good activity as when compared with standard D-Saccharic acid
1,4-lactonec. In this series compound 8, 13 and 19 showed good to moderate β –glucuronidase activity.
All other derivatives 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24 and 25 were found to be
inactive against β –glucuronidase.
2.5 Docking studies
In this study, molecular docking studies were performed using Human β-D-glucuronidase protein
structure to predict favorable binding mode of imidazo[4,5-b]pyridine derivatives (1-26) [25]. Docking
studies were done to obtain more insight on how imidazo[4,5-b]pyridine derivatives could bind within
the active site of β-D-glucuronidase and to validate the experimental results. In this study, x-ray crystal
structure of human β-glucuronidase enzyme at 2.6 Å resolution (PDB ID: 1BHG) was employed to
further identify binding modes involved in the inhibition activity [25].
Based on previous docking studies on human β-D-glucuronidase, the modeled substrate-bound structure
suggests that the glycoside bond of p-nitrophenyl β-D-glucuronide is oriented towards the catalytic
residues Glu451, Glu540, and Tyr504 (Fig. 1). It has been proposed for human β-D-glucuronidase that
during catalysis, Glu451 acts as the acid/base catalyst while Glu540 serves as the nucleophilic residue
[54], while Tyr504 was found to contribute significantly towards catalysis even though its function is
still unclear. It is also claimed that Asp207-Glu451 pair might form the nucleophile-acid-base catalyst
pair in human β-D-glucuronidase, analogous to the Glu35-Asp52 pair in lysozyme. Docking studies
result displayed in Figure 1 showed that the compounds fit perfectly in the binding site and interact
through various hydrogen bonding interactions with the active site residues that includes Asp207,
His385, Glu451, Tyr504, and Glu540. It was observed that the phenyl-end of all compounds are oriented
8

towards the inner-core of the active binding cavity, while the imidazo[4,5-b]pyridine moiety is sandwich
between polar residues Glu451, Asn484, Asn502, Tyr504, and Tyr508.
Figure 1. Binding of derivatives in active site of β-glucuronidase.
Docking studies for two most active compounds 1 and 3 (Figure 2) revealed that these
compounds are sharing some common interactions with the active site’s residues, which may be the
reason for these compounds to be the most active compounds for this series. Visually inspecting the
docking results obtained for these active compounds suggest that the amine (NH) on imidazo[4,5-
b]pyridine moiety for both compounds 1 and 3 interacts with backbone (OE1) of Glu451 at the distance
of 2.33 and 2.39 Å, respectively. Other common interactions were observed between hydroxyl on
phenyl-end of both compounds with Asp207 and Lys606. For compound 1, hydroxyl substituent at para
position forms a hydrogen bond with backbone (OD2) of Asp207 at a distance of 2.16 Å, while its meta
substituted hydroxyl group forms hydrogen bond with OD1 of Asn604 at a distance of 2.76 Å. As for
compound 3, hydroxyl group at meta position interacts with backbone (OD2) of Asp207 at a distance of
1.86 Å. Hydroxyl group substituted at ortho position for compound 3 forms a hydrogen bond with OH
of Tyr504. Another common interaction that can be observed between these two compounds is the
hydroxyl at meta position of compound 1 and hydrogen bond between oxygen of hydroxyl at ortho
position of compound 3 with the backbone (NZ) of Lys606 at the distance of 2.60 and 2.83 Å. In this
case, the results clear suggest that two hydroxyls on phenyl-end of imidazo[4,5-b]pyridine derivatives
have to be position on adjacent carbon to each other as this will enable them to interact with active
residues like Asp207 and Lys606.
9

Figure-2. Binding position for (a) compounds 1 and (b) compound 3.
Unlike compounds 1 and 3, predicted binding mode for compounds 2 (2,4-dihydroxy) and 4
(2,5-dihydroxy), displayed that these compounds are oriented in such a way that the amine (NH) on
imidazo[4,5-b]pyridine moiety for both compounds 2 and 4 were not able to interact with backbone
(OE1) of Glu451 (Figure-3), as being observed for compounds 1 and 3. Instead, the amine (NH) on
imidazo[4,5-b]pyridine moiety for compounds 2 and 4 were found to interact with backbone (OE1) of
Glu451 at a distance of 2.07 and 2.43 Å, respectively. Despite being able to form hydrogen bonding
with Asp207 and Lys606, compound 2 was unable to form hydrogen bonding with Glu415, which may
be main reason for the low activity observed. As for compound 4, besides not being able to form
hydrogen bonding with catalytic residue Glu451, one of its hydroxyl was positioned in such a way that it
was too far to able form any hydrogen bonding with the surrounding residues and making it less
effective in inhibiting the activity. Observation on docking results for compounds 13 and 19 (Figure-4)
showed that their binding modes are quite similar to compounds 2 and 4. Compounds 13 and 19 interact
with backbone (OE1) of Glu451, as being observed for compounds 13 and 19. Methoxy group on
phenyl-end may be introducing steric effect that causes hydroxyl to form hydrogen bonding with Lys606
within a longer distance (2.52 and 2.67 Å). No hydrogen bonding is formed between these compounds
with catalytic residue Asp207.
10

Figure-3. Binding position for (a) compounds 2 and (b) compound 4.
Figure-4.
Binding position for (a) compounds 13 and (b) compound 19.
3. Conclusion
Imidazo[4,5-b]pyridine derivatives 1-26 were synthesized and evaluated for their antiglycation and
antioxidant activities. Compound 2, 3, 4, 6, 13 and 26 showed good antiglycating potential. On the other
hand all OH groups containing derivatives showed moderate antioxidant activity. These compounds can
be further studied for the anti-aging.
4. Experimental section
4.1. Materials and methods
Melting points were measured in open glass capillaries using Stuart Scientific SMP11 Analog melting
point apparatus. NMR spectras of imidazo[4,5-b]pyridine derivatives which had been synthesized were
11

collected using Bruker Ultra Shield FT NMR 500 MHz spectrometer operating at 500 MHz. EI-MS was
used to determine mass and fragmentation patterns were determined by analysis carried out using
Finnigan-MAT-311-A instrument. Thin layer chromatography was used to monitor progress of reactions
was monitored using precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.20 mm) and visualized
using UV lamp at 254 nm (UVGL-58; Upland, USA).
4.2. General Procedure for synthesis of compounds (1-26)
Preparation of 6-chloro-2-phenyl-1H-imidazo[4,5-b]pyridine derivatives 1-26 were synthesized by
mixing 3.12 mmol of 5-chloropyridine-2,3-diamine with 3.16 mmol of substituted aromatic
benzaldehyde. 3 mmol of sodium metabisulphite (Na₂S₂O₅) and 10 ml dimethylformamide (DMF) were
then added to the reaction mixture. The reaction mixture was stirred and refluxed for 6 hours. Upon
completion, 30 ml of water was added to allow precipitation. The precipitate was filtered and subjected
through crystallization in ethyl acetate to afford pure product.
4.2.1 4-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)benzene-1,2-diol (1)
Yield: 79 %, 1H NMR (500 MHz, DMSO-d6): 10.29 (s , 1H), 8.38 (d, J = 2.0 Hz, 1H), 8.14 (d, J = 1.5
Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6):  161.6, 150.2, 147.8, 147.4, 145.8, 135.7, 131.6, 127.5, 124.7, 123.2,116.1, 114.2; Anal.
Calcd for C12H8ClN3O2 C = 55.08, H = 3.08, N = 16.06. Found: C = 55.11, H = 3.06, N = 16.08; EI MS
m/z (% rel. abund.): 263 (M+2, 8), 261 (M+, 25).
4.2.2. 4-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)benzene-1,3-diol (2)
Yield: 76 %, 1H NMR (500 MHz, DMSO-d6): 8.33 (d, J = 2.5 Hz, 1H), 8.09 (d, J = 2.5 Hz, 1H), 7.64
13
(d, J = 2.0 Hz, 1H), 7.54 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H); C NMR (75 MHz,
DMSO-d6):  159.8, 156.7, 151.1, 150.2, 147.8, 135.4, 131.7, 130.2, 127.7, 110.8, 109.1, 105.5; Anal.
Calcd for C12H8ClN3O2 C = 55.08, H = 3.08, N = 16.06. Found: C = 55.07, H = 3.08, N = 16.04; EI MS
m/z (% rel. abund.): 263 (M+2, 14), 261 (M+, 37).
4.2.3. 3-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)benzene-1,2-diol (3)
Yield: 75 %, 1H NMR (500 MHz, DMSO-d6):  9.30 (s, 1H), 8.42 (s, 1H), 8.32 (s, 1H), 7.57 (d, J = 7.0
13
Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 6.86 (t, J = 2.0 Hz, 1H); C NMR (75 MHz, DMSO-d6):  151.1,
150.3, 147.7, 145.7, 143.8, 135.4, 131.7, 127.5, 124.4, 123.1, 119.8, 117.2; Anal. Calcd for
12

C12H8ClN3O2 C = 55.08, H = 3.08, N = 16.06. Found: C = 55.10, H = 3.07, N = 16.04; EI MS m/z (%
rel. abund.): 263 (M+2, 7), 261 (M+, 20).
4.2.4. 2-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)benzene-1,4-diol (4)
Yield: 78 %,1H NMR (500 MHz, DMSO-d6):  9.18 (s, 1H), 8.40 (d, J = 2.5 Hz, 1H), 8.20 (s, 1H), 7.53
13
(s, 1H), 6.91 (d, J = 7.0 Hz, 2H); C NMR (75 MHz, DMSO-d6):  151.1, 150.5, 150.0, 147.8, 146.5,
135.4, 131.7, 127.5, 119.6, 117.9, 117.2, 114.1; Anal. Calcd for C12H8ClN3O2 C = 55.08, H = 3.08, N =
16.06. Found: C = 55.05, H = 3.07, N = 16.09; EI MS m/z (% rel. abund.): 263 (M+2, 17), 261 (M+, 56).
4.2.5. 6-chloro-2-(3-methoxyphenyl)-1H-imidazo[4,5-b]pyridine (5)
Yield: 75 %,1H NMR (500 MHz, DMSO-d6):  8.37 (d, J = 2.0 Hz, 1H), 8.18 (s, 1H), 7.83-7.80 (m,
2H), 7.50 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 3.87 (s, 3H); 13C NMR (75 MHz, DMSO-d6): 
161.6, 161.0, 150.3, 147.7, 135.4, 131.9, 131.5, 130.1, 127.5, 119.7, 114.2, 111.2, 55.7; Anal. Calcd for
C13H10ClN3O C = 60.12, H = 3.88, N = 16.18. Found: C = 60.10, H = 3.86, N = 16.20; EI MS m/z (%
rel. abund.): 261 (M+2, 23), 259 (M+, 63).
4.2.6. 2-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)phenol (6)
Yield: 80 %,1H NMR (500 MHz, DMSO-d6):  8.42 (d, J = 2.5 Hz, 1H), 8.32 (s, 1H), 8.18 (d, J = 8.0,
1H), 7.45 (t, J = 8.5 Hz, 1H), 7.09-7.04 (m, 2H); 13C NMR (75 MHz, DMSO-d6):  154.2, 150.3, 151.1,
147.8, 135.4, 131.7, 131.6, 130.0, 127.7, 121.7, 118.2, 117.9; Anal. Calcd for C12H8ClN3O C = 58.67, H
= 3.28, N = 17.10. Found: C = 58.69, H = 3.30, N = 17.08; EI MS m/z (% rel. abund.): 247 (M+2, 18),
245 (M+, 59).
4.2.7. 6-chloro-2-(2,6-dimethoxyphenyl)-1H-imidazo[4,5-b]pyridine (7)
Yield: 75 %,1H NMR (500 MHz, DMSO-d6):  8.38 (d, J = 2.0 Hz, 1H), 8.19 (s, 1H), 7.36 (t, J = 7.5
Hz, 1H), 7.22-7.21 (m, 2H), 2.12 (s, 6H); 13C NMR (75 MHz, DMSO-d6):  158.2, 158.2, 150.3, 147.8,
147.2, 135.4, 131.7, 130.8, 127.7, 108.7, 108.7, 107.6, 56.0, 56.0; Anal. Calcd for C14H12ClN3O2 C =
58.04, H = 4.17, N = 14.50. Found: C = 58.06, H = 4.19, N = 14.53; EI MS m/z (% rel. abund.): 291
(M+2, 8), 289 (M+, 26).
4.2.8. 3-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)phenol (8)
Yield: 79 %,1H NMR (500 MHz, DMSO-d6):  9.82 (s, 1H), 8.35 (d, J = 2.0 Hz, 1H), 8.14 (s, 1H), 7.66-
7.65 (m, 2H), 7.37 (t, J = 8.5 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6):  161.8,
157.4, 150.3, 147.7, 135.4, 132.1, 131.6, 130.5, 127.4, 120.0, 115.7, 112.8; Anal. Calcd for C12H8ClN3O
13

C = 58.67, H = 3.28, N = 17.10. Found: C = 58.65, H = 3.31, N = 17.12; EI MS m/z (% rel. abund.): 247
(M+2, 14), 245 (M+, 43).
4.2.9. 6-chloro-2-(4-nitrophenyl)-1H-imidazo[4,5-b]pyridine (9)
Yield: 87 %,1H NMR (500 MHz, DMSO-d6):  8.37 (d, J = 2.5 Hz, 1H), 8.27-8.25 (m, 2H), 8.10 (d, J =
13
2.5 Hz, 1H), 7.62-7.60 (m, 2H); C NMR (75 MHz, DMSO-d6):  161.8, 150.3, 147.8, 147.8, 140.7,
135.4, 131.7, 127.5, 127.1, 127.1, 124.2, 124.2; Anal. Calcd for C12H7ClN4O2 C = 52.47, H = 2.57, N =
20.40. Found: C = 52.49, H = 2.55, N = 20.37; EI MS m/z (% rel. abund.): 276 (M+2, 14), 274 (M+, 50).
4.2.10. 6-chloro-2-(4-fluorophenyl)-1H-imidazo[4,5-b]pyridine (10)
Yield: 85 %,1H NMR (500 MHz, DMSO-d6):  8.35 (d, J = 1.5 Hz, 1H), 8.29-8.27 (m, 2H), 8.15 (d, J =
1.5, 1H), 7.46-7.43 (m, 2H); 13C NMR (75 MHz, DMSO-d6):  162.8, 161.6, 150.3, 147.8, 135.4, 131.7,
130.2, 129.0, 129.0,127.7, 116.1, 116.1; Anal. Calcd for C12H7ClFN3 C = 58.20, H = 2.85, N = 16.97.
Found: C = 58.21, H = 2.83, N = 16.99; EI MS m/z (% rel. abund.): 249 (M+2, 24), 248 (M+1, 11), 247
(M+, 76).
4.2.11. 6-chloro-2-(3-nitrophenyl)-1H-imidazo[4,5-b]pyridine (11)
Yield: 84 %,1H NMR (500 MHz, DMSO-d6):  9.05 (d, J = 1.0 Hz, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.41
(d, J = 1.5, 1H), 8.39 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.24 (s, 1H), 7.88 (t, J = 8.0 Hz, 1H); 13C NMR (75
MHz, DMSO-d6):  161.6, 150.3, 148.3, 147.8, 135.4, 133.7, 131.6, 131.5, 130.0, 127.5, 123.8, 122.7;
Anal. Calcd for C12H7ClN4O2 C = 52.47, H = 2.57, N = 20.40. Found: C = 52.45, H = 2.55, N = 20.42.;
EI MS m/z (% rel. abund.): 276 (M+2, 13), 274 (M+, 42).
4.2.12. 6-chloro-2-(p-tolyl)-1H-imidazo[4,5-b]pyridine (12)
Yield: 83 %,1H NMR (500 MHz, DMSO-d6):  8.33 (s, 1H), 8.17 (s, 1H), 8.13-8.11 (m, 2H), 7.40-7.39
(m, 2H), 2.40 (s, 3H); 13C NMR (75 MHz, DMSO-d6):  161.6, 150.3, 147.8, 135.4, 131.7, 131.6, 131.5,
129.4, 129.4, 128.8, 128.8, 127.5, 21.2; Anal. Calcd for C13H10ClN3 C = 64.07, H = 4.14, N = 17.24.
Found: C = 64.09, H = 4.12, N = 17.25; EI MS m/z (% rel. abund.): 245 (M+2, 15), 243 (M+, 52).
4.2.13. 2-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)-4-methoxyphenol (13)
Yield: 86 %,1H NMR (500 MHz, DMSO-d6):  8.42 (d, J = 2.5 Hz, 1H), 8.27 (s, 1H), 7.72 (d, J = 3.0
Hz, 1H), 7.07 (dd, J = 2.5 Hz, 9.0 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 3.81 (s, 3H); 13C NMR (75 MHz,
DMSO-d6):  153.5, 151.1, 150.3, 147.7, 146.3, 135.4, 131.7, 127.5, 119.2, 117.3, 115.8, 112.8, 55.7;
Anal. Calcd for C13H10ClN3O2 C = 56.64, H = 3.66, N = 15.24. Found: C = 56.66, H = 3.67, N = 15.22;
EI MS m/z (% rel. abund.): 277 (M+2, 18), 275 (M+, 58).
4.2.14. 6-chloro-2-(o-tolyl)-1H-imidazo[4,5-b]pyridine (14)
14

Yield: 90 %,1H NMR (500 MHz, DMSO-d6):  8.25 (s, 1H), 8.17 (s, 1H), 7.62-7.60 (m, 3H), 7.40 (d, J
= 1.5 Hz, 1H), 2.41 (s, 3H); 13C NMR (75 MHz, DMSO-d6):  150.3, 147.7, 147.0, 138.4, 136.7, 135.4,
131.7, 130.0, 129.6, 129.4, 128.5, 127.5, 18.8; Anal. Calcd for C13H10ClN3 C = 64.07, H = 4.14, N =
17.24. Found: C = 64.09, H = 4.12, N = 17.21; EI MS m/z (% rel. abund.): 245 (M+2, 17), 243 (M+, 48).
4.2.15. 6-chloro-2-(m-tolyl)-1H-imidazo[4,5-b]pyridine (15)
Yield: 82 %, 1H NMR (500 MHz, DMSO-d6):  8.33 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 8.02 (d, J = 8.0
Hz, 1H), 7.46 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 2.43 (s, 3H); 13C NMR (75 MHz, DMSO-d6):
 161.6, 150.3, 147.8, 137.4, 137.2, 135.4, 131.7, 131.1, 129.2, 129.0, 128.8, 127.5, 21.5; Anal. Calcd
for C13H10ClN3 C = 64.07, H = 4.14, N = 17.24. Found: C = 64.05, H = 4.13, N = 17.27; EI MS m/z (%
rel. abund.): 245 (M+2, 23), 243 (M+, 68).
4.2.16. 6-chloro-2-(pyridin-4-yl)-1H-imidazo[4,5-b]pyridine (16)
Yield: 88 %,1H NMR (500 MHz, DMSO-d6):  8.93 (d, J = 5.0 Hz, 2H), 8.49 (d, J = 2.0 Hz, 1H), 8.36
13
(d, J = 6.0 Hz, 2H), 8.33 (d, J = 2.5 Hz, 1H); C NMR (75 MHz, DMSO-d6):  161.6, 150.3, 149.7,
149.7, 147.8, 144.5, 135.4, 131.7, 127.5, 121.2, 121.2; Anal. Calcd for C11H7ClN4 C = 57.28, H = 3.06,
N = 24.29. Found: C = 57.29, H = 3.05, N = 24.26; EI MS m/z (% rel. abund.): 232 (M+2, 7), 230 (M+,
24).
4.2.17. 6-chloro-2-(3-chlorophenyl)-1H-imidazo[4,5-b]pyridine (17)
Yield: 87 %,1H NMR (500 MHz, DMSO-d6):  8.43 (d, J = 2.5 Hz, 1H), 8.39 (s, 1H), 8.28 (s, 1H), 8.26
13
(s, 1H), 8.21 (d, J = 5.0 Hz, 1H), 7.62 (s, 2H); C NMR (75 MHz, DMSO-d6):  161.6, 150.3, 147.8,
139.5, 135.4, 134.7, 131.7, 130.2, 130.0, 129.5, 128.7, 127.7; Anal. Calcd for C12H7Cl2N3 C = 54.57, H
= 2.67, N = 15.91. Found: C = 54.56, H = 2.69, N = 15.93; EI MS m/z (% rel. abund.): 265 (M+2, 21),
263 (M+, 72).
4.2.18. 6-chloro-2-(2-chlorophenyl)-1H-imidazo[4,5-b]pyridine (18)
Yield: 89 %,1H NMR (500 MHz, DMSO-d6):  8.39 (s, 1H), 8.28 (s, 1H), 8.26 (s, 1H), 8.21 (d, J = 5.5
Hz, 1H), 7.64 (s, 2H); 13C NMR (75 MHz, DMSO-d6):  150.3, 147.7, 147.0, 138.4, 135.3, 132.1, 131.7,
130.0, 129.2, 128.8, 127.5, 127.2; Anal. Calcd for C12H7Cl2N3 C = 54.57, H = 2.67, N = 15.91. Found: C
= 54.55, H = 2.68, N = 15.89; EI MS m/z (% rel. abund.): 265 (M+2, 15), 263 (M+, 46).
4.2.19. 2-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)-5-methoxyphenol (19)
Yield: 87 %,1H NMR (500 MHz, DMSO-d6):  8.37 (d, J = 1.5 Hz, 1H), 8.14 (d, J = 1.5 Hz, 1H), 7.73
13
(d, J = 2.0 Hz, 1H), 7.64 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 6.82 (d, J = 7.5 Hz, 1H); C NMR (75 MHz,
DMSO-d6):  162.1, 156.3, 151.1, 150.5, 147.8, 135.4, 131.7, 129.7, 127.5, 110.4, 107.3, 104.3, 55.6;
15

Anal. Calcd for C13H10ClN3O2 C = 56.64, H = 3.66, N = 15.24. Found: C = 56.66, H = 3.64, N = 15.26;
EI MS m/z (% rel. abund.): 277 (M+2, 22), 275 (M+, 62).
4.2.20. 6-chloro-2-(4-chlorophenyl)-1H-imidazo[4,5-b]pyridine (20)
Yield: 82 %,1H NMR (500 MHz, DMSO-d6):  8.37 (d, J = 2.0 Hz, 1H), 8.25-8.22 (m, 2H), 8.18 (s,
1H), 7.68-7.65 (m, 2H); 13C NMR (75 MHz, DMSO-d6):  161.5, 150.5, 147.8, 135.4, 132.7, 131.7,
130.3, 129.2, 129.2, 128.8, 128.8, 127.4; Anal. Calcd for C12H7Cl2N3 C = 54.57, H = 2.67, N = 15.91.
Found: C = 54.54, H = 2.65, N = 15.93; EI MS m/z (% rel. abund.): 265 (M+2, 11), 263 (M+, 35).
4.2.21. 6-chloro-2-(pyridin-2-yl)-1H-imidazo[4,5-b]pyridine (21)
Yield: 79 %,1H NMR (500 MHz, DMSO-d6):  8.78 (d, J = 4.0 Hz, 1H), 8.45-8.28 (m, 2H), 8.06-7.97
(m, 2H), 7.61 (s, 1H); 13C NMR (75 MHz, DMSO-d6):  166.3, 155.1, 150.3, 149.1, 147.8, 137.1, 135.4,
131.7, 127.5, 124.1, 123.5; Anal. Calcd for C11H7ClN4 C = 57.28, H = 3.06, N = 24.29. Found: C =
57.26, H = 3.04, N = 24.31; EI MS m/z (% rel. abund.): 232 (M+2, 14), 230 (M+, 38).
4.2.22. 6-chloro-2-(pyridin-3-yl)-1H-imidazo[4,5-b]pyridine (22)
Yield: 85 %,1H NMR (500 MHz, DMSO-d6):  9.38 (d, J = 2.0 Hz, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.55
(d, J = 8.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.23 (s, 1H), 7.63 (dd, J = 4.5 Hz, 8.0 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6):  150.3, 147.8, 147.7, 147.0, 135.4, 135.3,132.8, 131.7, 127.5, 124.1, 115.2;
Anal. Calcd for C11H7ClN4 C = 57.28, H = 3.06, N = 24.29. Found: C = 57.30, H = 3.04, N = 24.27; EI
MS m/z (% rel. abund.): 232 (M+2, 14), 230 (M+, 46).
4.2.23. 6-chloro-2-(3-fluorophenyl)-1H-imidazo[4,5-b]pyridine (23)
Yield: 86 %,1H NMR (500 MHz, DMSO-d6):  8.39 (s, 1H), 8.25 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 8.02
13
(d, J = 10.0 Hz, 1H), 7.65 (d, J = 6.0 Hz, 1H), 7.43 (td, J = 2.0 Hz, 8.5 Hz, 1H); C NMR (75 MHz,
DMSO-d6):  162.1, 161.7, 150.3, 147.7, 135.4, 132.1, 131.7, 127.7, 127.4, 123.0, 115.8, 115.4; Anal.
Calcd for C12H7ClFN3 C = 58.20, H = 2.85, N = 16.97. Found: C = 58.18, H = 2.87, N = 16.94; EI MS
m/z (% rel. abund.): 249 (M+2, 13), 248 (M+1, 6), 247 (M+, 46).
16

4.2.24. 4,6-dichloro-2-(2-fluorophenyl)-1H-benzo[d]imidazole (24)
Yield: 89 %,1H NMR (500 MHz, DMSO-d6):  8.42 (d, J = 2.0 Hz, 1H), 8.20 (s, 1H), 7.67-7.62 (m,
1H), 7.50-7.42 (m, 3H); 13C NMR (75 MHz, DMSO-d6):  158.4, 150.3, 147.7, 147.0, 135.4, 131.6,
130.2, 129.0,127.5, 124.7, 123.4, 114.6; Anal. Calcd for C12H7ClFN3 C = 58.20, H = 2.85, N = 16.97.
Found: C = 58.23, H = 2.83, N = 16.95; EI MS m/z (% rel. abund.): 249 (M+2, 27), 248 (M+1, 13), 247
(M+, 83).
4.2.25. 6-chloro-2-(3,4-dimethoxyphenyl)-1H-imidazo[4,5-b]pyridine (25)
Yield: 78 %,1H NMR (500 MHz, DMSO-d6):  8.33 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.84-
7.82 (m, 2H), 7.18-7.16 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H); 13C NMR (75 MHz, DMSO-d6):
 161.5, 150.3, 150.1, 149.7, 147.8, 135.6, 131.7, 127.5, 123.8, 123.7, 122.4, 111.1, 56.2, 56.0; Anal.
Calcd for C14H12ClN3O2 C = 58.04, H = 4.17, N = 14.50. Found: C = 58.02, H = 4.20, N = 14.48; EI MS
m/z (% rel. abund.): 291 (M+2, 15), 289 (M+, 48).
4.2.26. 4-(6-chloro-1H-imidazo[4,5-b]pyridin-2-yl)phenol (26)
Yield: 86 %,1H NMR (500 MHz, DMSO-d6):  8.34 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.09-
13
8.07 (m, 2H), 6.97-6.96 (m, 2H); C NMR (75 MHz, DMSO-d6):  161.6, 158.4, 150.3, 147.8, 135.4,
131.7, 130.6, 130.6, 127.5, 127.4, 116.3, 116.3; Anal. Calcd for C12H8ClN3O C = 58.67, H = 3.28, N =
17.10. Found: C = 58.66, H = 3.26, N = 17.13; EI MS m/z (% rel. abund.): 247 (M+2, 11), 245 (M+, 39).
3.5. Antiglycation Assay
For the eval;uation of antiglycation activity of compounds Bovine Serum Albumin (BSA), and Glucose
system was employed with certain modifications from earlier reported [26-30] The total volume of
reaction mixture was 60 L, containing 20 L of 10 mg/mL BSA 20 L of 3 mM test compound and 20
L of 50 mg/mL anhydrous glucose. Reagents were prepared in 67 mM phosphate buffer (pH 7.40) and
inhibitor was dissolved in DMSO. An appropriate amount of sodium azide was added to avoid any
microbial growth. A positive control, containing standard inhibitor (Rutin) and a negative control,
containing only BSA, were also set for comparison with the test compounds. High throughput
experiment was carried out in 96-well microtiter plate in triplicate for each sample. Then the reaction
mixture was incubated for one week at 37o C. After seven days, incubation mixture was removed from
the incubator and allowed to cool at room temperature. In order to initiate precipitation, 6 µL of 100%
trichloro acetic acid (TCA) was added to each sample well. Centrifugation at 14,000 rpm for 4 minutes
was carried out to remove the unreacted glucose in the supernatants and to obtain a pellet of AGEs at the
17

bottom. After removal of the supernatants, the pellets were dissolved by adding 60 L of basic buffer
(PBS, pH: 10). Spectrofluorimetry was performed to record the intensity of fluorescence at excitation
370 nm and emission at 440 nm. For the determination of IC50 values the compound was tested at
specified concentration levels against glycation (1-500µM). The obtained values were used for
determination of IC50 by using kinetic program, EZ-Fit. % inhibition was calculated by.
% Inhibition=(1-AS/AC)*100
Where, AS is the absorbance of sample and AC is absorbance of control
3.6. Antioxidant Assay
For the evaluation of antioxidamt activity of compounds DPPH radical scavenging menthod was
employed with slighty modification in method used by Donata and his co-workers in 2002 [31-33]. The
method was performed by taking 95 µL of DPPH solution and 5 µL of test compound in a micro titer
plate. DPPH solution was prepared in ethanol and sample was dissolved in DMSO. The final well
concentration for DPPH was maintained at 300 µM and for sample at 500 µM. Reaction mixture was
incubated at 37o C for half an hour and the final absorbance was recorded on microplate reader (Spectra
Max-384) at 515 nm. Radical quenching efficacy of compounds was determined by comparison with
DMSO treated blank (control). Following formula was used for calculation of radical scavenging
activity.
%RSA=(1-AS/ AC)*100
3.7 β – Glucuronidase Inhibition Assay
The protocol; was followed in this study was earlier reported [34,35]. β-Glucuronidase activity was
evaluated by measuring the absorbance at 405 nm of p-nitrophenol formed from the substrate by the
spectrophotometeric method. The total reaction volume was set at 250 µL. The reaction mixture
comprises of 185 µL of 0.1 M acetate buffer, 5 µL of test compound solution, 10 µL of enzyme solution
and incubated at 37 oC for 30 min. The readings were recorded on a multiplate reader (SpectraMax plus
384) at 405 nm after the addition of 50 µL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were
carried out in triplicate. Beside this, to prevent precipitation, the concentration of the compounds was
decreased and the reaction volume was high (200 µL) so the probabitlity of precipitation was attenuated
hence the addition of detergents was not required.
% Inhibition=(1-AS/AC)*100
18

3.8. Docking studies
The structures of all compounds were prepared using Chem3D by CambridgeSoft. The human β-D-
glucuronidase crystal structure was first retrieved from the protein data bank (PDB code: 1BHG) [25].
Docking had been carried out using AutoDock 4.2 [36]. In Genetic Algorithm (GA) search parameter,
number of runs was set at 150, while the other settings were left as default. Docking parameter was also
left with its default settings. The 3D docking results was visualized using Discovery Studio visualizer
4.1.
3.9. Statistical Analysis
The experimental data of biological activity was expressed as ± standard error of mean (±SEM).
Statistical
analysis
was
performed
by
using
one-way
analysis
of
variance
(ANOVA).Acknowledgement:
Authors would like to acknowledge The Ministry of Agriculture (MOA) Malaysia and Universiti
Teknologi MARA for the financial support under MOA grant file No. 100-RMI/ MOA 16/6/2 (1/2013)
and Atta-ur-Rahman Institute for Natural product Discovery (RiND) to provide excellent designed lab
and facility for the research and all technical and non-technical staff for a lot of support for this work.
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Graphical Abstract
Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine
derivatives: antidiabetic, antioxidant, β-glucuronidase
Inhibition and their molecular docking studies
Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Syahrul Imrana,b, Hesham Rashwanc, Waqas Jamild, Sajjad
Alie, Syed Muhammad Kashifd, Fazal Rahimg, Uzma Salarf, Khalid Mohammed Khanf
aAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam,
c
Selangor, Malaysia. bFaculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia. Faculty of Pharmacy, Universiti Teknologi MARA,
c
d
Puncak Alam, 42300, Selangor, Malaysia, Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm), Ministry of Science. Institute of
e
Advance Research Studies in Chemical Sciences, University of Sindh Jamshoro. Department of Chemistry, Karakoram International University,
f
Gilgit-Baltistan, Gilgit 15100, Pakistan. H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan. gDepartment of Chemistry, Hazara University, Mansehra-21120, Pakistan.
Synthesis and Evaluation of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine (1-26) for Antidiabetic, Antioxidant and β-
glucuronidase inhibitory activity.

Research Highlights
 Synthesis of 1-26 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine
 In-vitro antioxidant, antiglycation, and β-glucuronidase inhibitory activity.
 Docking studies were carried out to confirm binding of active compounds with enzyme
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