Design and synthesis of novel imidazo[4,5-b]pyridine based compounds as potent anticancer agents with CDK9 inhibitory activity

Article abstract of DOI:10.1016/j.bioorg.2018.07.006

New imidazo[4,5-b]pyridine derivatives were designed, synthesized and screened for their anticancer activity against breast (MCF-7) and colon (HCT116) cancer cell lines. Nine compounds (I, II, IIIa, IIIb, IV, VI, VIIa, VIII, IX) showed significant activit

Full text of DOI:10.1016/j.bioorg.2018.07.006

Accepted Manuscript
Design and synthesis of novel imidazo[4,5-b]pyridine based compounds as po-
tent anticancer agents with CDK9 inhibitory activity
Nada M. Ghanem, Faten Farouk, Riham F. George, Safinaz E.S. Abbas, Ossama
M. El-Badry
PII:
S0045-2068(18)30337-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.006
YBIOO 2425
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 April 2018
29 June 2018
2 July 2018
Please cite this article as: N.M. Ghanem, F. Farouk, R.F. George, S.E.S. Abbas, O.M. El-Badry, Design and synthesis
of novel imidazo[4,5-b]pyridine based compounds as potent anticancer agents with CDK9 inhibitory activity,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.006
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Design and synthesis of novel imidazo[4,5-b]pyridine based compounds as potent
anticancer agents with CDK9 inhibitory activity
Nada M. Ghanema, Faten Farouka, Riham F. Georgeb, Safinaz E. S. Abbasb, Ossama M. El-
Badrya,c
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt.
bPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
cPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
Abstract
New imidazo[4,5-b]pyridine derivatives were designed, synthesized and screened for their
anticancer activity against breast (MCF-7) and colon (HCT116) cancer cell lines. Nine
compounds (I, II, IIIa, IIIb, IV, VI, VIIa, VIII, IX) showed significant activity against MCF-7,
while six compounds (I, VIIc, VIIe, VIIf, VIII, IX) elicited a remarkable activity against
HCT116. Compounds showing significant anticancer activity revealed remarkable CDK9
inhibitory potential (IC50= 0.63-1.32 μM) relative to sorafenib (IC50= 0.76μM). Moreover, a
molecular docking study was performed to illustrate the binding mode of the most active
compounds in the active site of CDK9 and revealed superior binding affinity relative to the
natural ligand (T3C).
Key words: Imidazo[4,5-b]pyridines, anticancer, CDK9, HCT116, MCF-7.
1

1. Introduction
Cancer is considered as the second leading cause of death after cardiovascular diseases [1].
Targeted chemotherapeutic agents have advantages over the traditional ones due to their
selectivity towards cancer cells and lower side effects [1].
Cyclin Dependent Kinases (CDKs) are a group of serine/threonine kinases. They are
instrumental regulatory enzymes involved in cell cycle progression and cell proliferation. Their
hyperactivity in breast, lung as well as colorectal tumors contributes to cancer cell proliferation,
accordingly their inhibition is a valid targeted anticancer approach [2][3]. Nevertheless, cells
lacking CDK 2, 4 and 6 can proliferate normally [4]. Thus in many cancer cases specifically
targeting those CDKs may not result in optimum activity. This may be due to cell
compensation mechanisms and functional substitution. This drew the attention to targeting
CDKs affecting transcription, namely CDK9/7 [5].
CDK9 regulates the RNA transcription of short-lived anti-apoptotic proteins [6]. Literature
review revealed that the scaffolds showing CDK9 inhibitory action include chromones, thiazole
[7], pyrimidines [5,8,9], macrolides [10], imidazoles, purines, and aminopyrazole [7, 11].
It is worth pointing out that, the imidazoline derivative A is a potent CDK9 inhibitor with
promising anticancer activity against HCT116 colon cancer cell line [12]. Furthermore, the
imidazo[4,5-b]pyridine B was reported to have a potent anti-proliferative activity against MCF-
7 (IC50: 0.58 – 78.66 μM) and HCT116 (IC50: .49 – 87.33μM) cancer cell lines via selective
inhibition of CDK9 [12,13] (Figure 1). Accordingly, the present study deals with the design
and synthesis of new imidazo[4,5-b]pyridines as potential anticancer agents targeting CDK9
enzyme with improved activity.
The synthetic route adopted to prepare the new imidazo[4,5-b]pyridine derivatives was based on
reaction of the key intermediate 4(1H)-imidazo[4,5-b]pyridine-2-yl)aniline I with either
diethylethoxymethylenemalonate to obtain II followed by cyclization to the appropriate
pyrazolidine IIIa,b and IV, pyrimidine Va,b, triazepine VI or via diazotization of the amino
functionality of I followed by coupling with phenol, aniline or active methylene derivatives to
2

afford VIIa-f, VIII and IX, respectively. The biological evaluation of the synthesized
compounds was performed in terms of anti-proliferative activity against MCF-7 and HCT 116
cancer cell lines and CDK9 enzyme inhibitory assay. Furthermore, a docking study of the
synthesized imidazo[4,5-b]pyridine compounds and the native ligand T3C was carried out in the
CDK9 active site in order to explore and compare their binding mode and validate their
mechanism of action.
2. Results and discussion
2.1.Chemistry
The novel imidazo[4,5-b]pyridine derivatives were synthesized according to Schemes 1 and 2. 4-
(1H-imidazo[4,5-b]pyridin-2-yl)aniline I was prepared via condensation of 2,3-diaminopyridine
with 4-aminobenzoic acid (PABA) in the presence of polyphosphoric acid (PPA). IR spectrum of
I showed a forked band at 3344 and 3300 cm-1assigned to NH2 group, a broad band at 3128-3400
cm-1attributed to NH group. 1HNMR spectrum revealed two exchangeable singlet signals at 2.50
and 5.69 ppm attributed to NH and NH2 protons respectively, in addition to doublet, doublet of
doublet and doublet signals at 7.84, 7.90 and 8.20 ppm, respectively corresponding to pyridine
protons. Reaction of I with diethyl ethoxymethylenemalonate in absolute ethanol resulted in the
diethyl methylenemalonate derivative II. IR spectrum showed a strong band at 3402- 3325 cm-1
attributed to NH groups in addition to a band at 1708-1689 cm-1 assigned to C=O of ester.1H
NMR spectrum revealed the presence of the two geometrical isomers appearing as two adjacent
singlet signals assigned to the olefinic proton at 5.92-5.94 ppm equal to 0.5 proton which does
not disappear after D2O, the triplet quartet pattern at 1.22 and 1.28 ppm and 4.11 and 4.22 ppm
corresponding to 2C2H5 protons, two exchangeable singlet signals at 2.51 and 9.05 ppm of 2NH
protons. Furthermore, refluxing the diester II with hydrazine hydrate or phenyl hydrazine in the
presence of Na ethoxide afforded IIIa or IIIb, respectively. Refluxing II with hydrazine hydrate
in glacial acetic acid gave IV. IR spectra of IIIa, IIIb and IV showed a strong absorption band
at 3194-3317 cm-1corresponding to NH groups, a band at 3043-3100 cm-1 attributed to CH
1
aromatic and a band at 1670-1708 cm-1 assigned for C=O groups. H NMR spectra indicated the
disappearance of aliphatic protons of the ethyl groups in addition to the presence of
exchangeable singlet signals at 5.31-9.31 ppm corresponding to NH protons. Compound IV
showed a singlet signal at 2.50 ppm attributed to CH3 protons, whereas compound IIIb revealed
3

an increase in the integration of aromatic protons 6.63-8.11 ppm due to additional phenyl ring,
moreover, the mass spectrum of compound IV displayed a molecular ion peak M+ at of 396 m/z.
Similarly, Pyrimidinetrione Va and thioxopyrimidinedione Vb were prepared via the
condensation of diethylmalonate derivative II with urea or thiourea in the presence of sodium
ethoxide. IR spectra of Va,b showed bands at 3275,3170 cm-1corresponding to NH groups, and
a band at 1650-1678 cm-1 assigned to C=O, while compound Vb showed additional band at 1261
cm-1 due to C=S. 1H NMR spectra of compounds Va,b revealed an exchangeable singlet signal at
10.0 ppm corresponding to additional NH,a singlet signal at 5.42 ppm attributed to olefinic CH
proton in addition to doublet, triplet and doublet signals of pyridine protons at 7.38, 8.16 and
8.26 ppm respectively. On the other hand, reaction of diethylmalonate ester II with
semicarbazide in the presence of sodium ethoxide led to cyclization and formation of triazepine
ring followed by the loss of hydrogen to form a more stable form of triazepinetrione derivative
VI. IR spectrum showed a broad band at 3500- 3336 cm-1assigned to NH groups in addition to a
1
band at 1681cm-1 attributed to 3C=O. H NMR spectra of VI displayed a singlet signal at 10.8
ppm exchanged with D2O corresponding to additional NH proton and a singlet signal at 6.35
ppm attributed to olefinic CH proton (Scheme 1). Proposed mechanisms are illustrated in
figure 2.
4

Scheme 1: Synthesis of 1H-imidazo[4,5-b]pyridine derivatives I-VI
5

Scheme 2: Synthesis of substituted 1H-imidazo[4,5-b]pyridine derivatives VIIa-f, VIII and
IX
Additionally, compounds VIIa-f were prepared by reacting the diazonium salt of I with the
appropriate phenolic compounds namely, phenol, resorcinol, salicylic acid, p- re ol - a t ol
a - a t ol i 1 o i ro i e at - C (Scheme 2). IR spectra lacked the NH2 band
and showed the appearance of a broad band at 3394-3420 cm-1 assigned to OH group.
1
Additionally, compound VIIc showed a band at 1680 cm-1attributed to C=O group. H NMR
spectra displayed the disappearance of the singlet signal of NH2 protons and indicated the
presence of an exchangeable singlet signal at 4.59-7.12 ppm of the OH proton, while compound
VIIc revealed additional exchangeable singlet signal at 16.09 ppm corresponding to COOH
proton. Compound VIId showed a singlet signal at 2.30 ppm attributed to CH3 protons.
Moreover, compounds VIIe, VIIf elicited additional aromatic protons at 6.64-8.49 ppm
corresponding to naphthalene ring. The mass spectrum of VIIf showed a molecular ion peak M+
6

at 365 m/z. Similarly, VIII was obtained via reaction of the diazonium salt of I with aniline at 0-
5ºC.IR spectrum showed the appearance of NH2band at 3317 cm-1in addition to NH band of
1
imidazopyridine ring at 3197 cm-1. H NMR spectra revealed the appearance of a singlet signal
of NH2 protons at 4.05 ppmand a singlet signal at 4.37 ppm assigned to NH protons that were
exchanged with D2O. Finally, reaction of the diazonium salt of I with acetylacetone in ethanol in
the presence of sodium hydroxide afforded compound IX. IR spectrum showed the appearance
of NH band at 3402 cm-1and the C=O band at 1670 cm-1in addition to aliphatic CH band at 2924
1
cm-1. H NMR spectra indicated two singlet signals at 2.46 and 2.48 ppm corresponding to two
CH3protons in addition to two exchangeable signals at 4.46 and 13.70 assigned to 2NH protons.
2.2 Anticancer activity
2.2.1. In- vitro MTT assay
The anticancer activity of the newly synthesized compounds was performed against breast cancer
cell line (MCF-7) and colon cancer cell line (HCT-116) using MTT assay according to the
Mosmann's method [14,15]. Each experiment was performed in triplicate. The assay was
performed in a concentration range of 0.01 .1 1 1 1 a 2 μM. Cisplatin was used
as an in-house internal standard while sorafinib was used as a positive control. There was a
good reproducibility between replicate wells with standard errors below 10 %. The results were
expressed in terms of IC50 [μM] (Table 1).
Concerning MCF-7 breast cancer cell line, imidazopyridin-2-ylaniline derivative I and its
diethylmethylenemalonateester II elicited potent anti-breast cancer activity (IC50 = 0.71 and
.78μM) re e tivel compared to cisplatin (IC50 = 2.84 μM) and sorfinib (IC50=4.16 μM).
Cyclization of II to un/substituted pyrazolidinedione IIIa, IIIb and IV resulted in remarkable
activity with IC50 of 1. 9 .97 a .86 μM, respectively. Pyrimidine derivatives Va, Vb (IC50=
4.80, 3.24 µM) were less active than the pyrazolidinedione (IIIa and IIIb). 2-
Thioxopyrimidinedione Vb was slightly more active than its trione congener Va. In addition,
further ring expansion to triazepine significantly increased the activity resulting in the most
potent compound VI (IC50 = .63 μM).
Coupling of diazonium salt with monohydroxy phenol resulted in compound VIIa with a
promising activity (IC50 =1.23 μM) w ile i ro e ol (re or i ol) VIIb greatly decreased
the activity (IC50 = 8.9 μM). Additionally, the bioisosteric substitution of 4-OH with NH2 VIII
7

elicited significant activity (IC50= 2.11 μM) b t le t a t at e re e b t e 4-OH analogue.
Moreover, Coupling of diazonium salt with phenol bearing an electron withdrawing group
(COOH) VIIc showed moderate activity (IC50 = .6 μM) w ile ele tro o ati g gro (m-
CH3) VIId markedly decreased the activity (IC50 = 27. 4 μM). Co li g wit 1-naphthol gave
VIIe exhibiting moderate activity (IC50= .77 μM) w erea 2-naphthol derivative VII revealed a
sharp
decrease
in
activity (IC50
=
12.7
μM).
Al o
b tit tio of
diethylaminomethylenemalonate moiety of II by hydrazono pentane-2,4-dione produced IX
showing a slight decrease in the anticancer activity (IC50= 2.11 μM) relative to t at expressed by
II ( .78 μM) owever it wa till ore ote t t a i lati (IC50 = 2.84 μM) and sorfinib
(IC50=4.16 μM).
Regarding HCT116 colon cancer cell line, six compounds I, VIIc, VIIe, VIIf, VIII and IX
elicited promising anticancer activity as denoted by their IC50 values (1.69-3. 6 μM) o are
with cisplatin (IC50= 2.82 μM) and sorfinib (IC50=6.12). The most active ones were
hydrazonopentadione IX, diazenylbenzeneamine VIII, diazenyl naphthalen-2-ol VIIe,
diazenylnaphthalen-1-ol VIIf and diazenyl 2-hydroxybenzoic acid VIIc with IC50 values 1.69,
1.83 2.14 2.31 a 2. 2μM re e tivel . Figure 3 illustrates the structural activity
relationship of the designed compounds.
Finally, we can conclude that 9 out of 16 tested compounds exhibited promising anticancer
activity against MCF-7. Five compounds I, II, IIIb, IV, VI elicited superior activity at a
submicromolar level (IC50= 0.63-0.97 µM) where compound VI is the most active one
(IC50=0.63 µM). It’s worth pointing out that each of these compounds was selectivity potent
against one of the two tested cell lines. Nine compounds (I, II, IIIa, IIIb, IV, VI, VIIa, VIII,
IX) showed significant activity against MCF-7, while six compounds (I, VIIc, VIIe, VIIf,
VIII, IX) elicited a remarkable activity against HCT116 (Table 1).
Table 1: In vitro growth inhibitory activity (IC50μM) of o o I-IX against MCF-7 breast
cancer cell line and HCT-116 colon cancer cell line (MTT method):
Antiproliferation (IC50, μM )
Antiproliferation (IC50, μM )
Compound
HCT-116
MCF-7
2.84
0.71
2.82
3.06
Cisplatin
I
8

0.78
1.59
0.97
0.86
4.80
3.24
0.63
1.23
8.90
5.60
27.04
5.77
12.7
2.84
2.11
4.16
4.06
4.65
5.45
4.03
3.94
6.06
5.30
5.45
6.43
2.52
3.77
2.14
2.31
1.83
1.69
6.12
II
IIIa
IIIb
IV
Va
Vb
VI
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIII
IX
Sorafinib
2.2.2. CDK9 Enzyme inhibition assay
Compounds (I, II, IIIa, Vb, VI, VIIa and IX) which elicited superior anticancer activity against
one or both tested cancer cell lines were further screened for their CDK9 enzyme inhibitory
activity in order to validate their proposed mechanism of action. Sorafinib was used as a
reference compound due to its kinase inhibitory potential. Results were expressed in IC50
[μM] (Table 2). The tested compounds were active inhibitors of CDK9 (IC50 = 0.50-2. 6 μM).
The un-substituted aniline I, triazepine VI and hydrazonopentane-2,4-dione IX exhibited
superior CDK9 inhibitory activity with IC50 ranging from 0.50 to 1. 2 μM.
These results strongly agree with the in-vitro anti-proliferative assay where compounds I and VI
showed a superior anticancer activity at a submicromolar level against MCF7 with (IC50=0.71
and 0.63 μM) re e tivel . Al o o o IX was the most active against HCT116 cell line
(IC50 = 1.69 μM).
Table 2. IC50 μM of t e o t a tive o o as CDK9 enzyme inhibitors
Compound
IC50 (μM)
0.95
I
2.06
II
9

1.32
0.50
1.10
1.002
0.76
IIIa
VI
VIIa
IX
Sorafinib
2.3.Docking study
In order to validate the CDK9 inhibitory activity, docking study was performed and results were
compared to those of the native ligand (T3C) and the multi-kinase inhibitor sorafinib as a
reference compound. The data revealed that most of compounds had acceptable binding affinity
as compared to T3C (Table 3).
The energy scores (Kcal/mol) of the native ligand binding affinity with the CDK9 was -21.96.
This binding resulted from interaction between the amino group, phenyl ring, 1,4-diazepine,
carbonitrile group, aminothiazole ring and pyrimidine moieties of the T3C native ligand and the
ATP pocket of the CDK9. The main amino acid residues involved in the interaction were Cys
106, Ile 25, Val 33, Asp 109, Leu 156, Asp 167 and Ala 46.
Our designed compounds showed comparable affinity to CDK9 ranging from -16.84 to -
26.72Kcal/mol. The interacting chemical groups of the proposed compounds and the
corresponding amino acid residues of the CDK9 binding site are described in table 3.
Furthermore, docking study against CDK2/4 and 6 was performed in order to compare the
selectivity of the test compounds against different CDKs. Results revealed that the
compounds showed stronger selectivity towards CDK9 over other CDKs ( Data are
presented in supplementary materials tables 1 and 2).
Table 3. Docking Study of the native ligand, and the designed compounds:
Hydrogen
Bond
Length (Å)
Energy Score
S (Kcal/mol)
Amino Acids
Interaction
Compound ID
Interacting Groups
Cys 106
Ile 25
NH
Phenyl ring
2.05
Val 33
Phenyl ring
1,4-diazepine
Carbonitrile
T3C
Native ligand
Asp 109
Leu 156
Asp 167
Ala 46
-21.96
-16.84
Aminothiazole ring
Pyrimidine ring
Phenyl ring
Asp 109
I
10

Ile 25
Val 33
Glu 107
Val 33
Gly 112
Asp 109
Leu 156
Ile 25
Phenyl ring
Pyridine ring
NH imidazole
Phenyl ring
Pyridine ring
Pyridine ring
C=O
1.75
-23.81
II
CH3
Ala 153
Thr 29
Phenyl ring
Phenyl ring
Phenyl ring
His 108
Lys 48
Leu 156
Phe 105
C=O
C=CH
C=O
2.55
-21.03
IIIa
Phenyl ring,
NH imidazole
Pyridine ring
Imidazole ring
C=O
Ile 25
Glu 107
Asp 167
Lys 48
2.78
1.88
Asp 109
Leu 156
Ile 25
Gly 112
Asp 167
Glu 107
Asp 109
Ile 25
Imidazopyridine ring
C=CH
Phenyl ring
Imidazopyridine ring
C=O
-26.72
-24.73
VI
NH Imidazole
Pyridine ring
Phenyl ring
Phenyl ring
VIIa
Leu 156
2.85
1.75
Lys 48
Ile 25
N pyridine
NH amino
Asp 109
Diester group
-21.44
IX
Thr 29
Phenyl ring
Phenyl ring
Asp 167
Val 33
Ile 25
Asp 109
Glu 107
His 108
Leu 156
Phenyl ring
Amide group
Pyridine ring
Phenyl ring
Methyl group
Methyl group
Trifluoromethyl
group
Sorafinib
-20.9152
Asp 167
11

2.4. Assessment of physicochemical properties and drug likeness
The Swiss-Adme tool [16] was used to determine physicochemical properties of the novel
compounds. The molecular properties of the synthesized compounds, the theoretical lipophilicity
and water solubility of the compounds are shown in Tables 3- 6 of supplementary materials.
Theoretical assessment of pharmacokinetics and cytochrome inhibition potential of the
synthesized molecules is displayed in table 7 of the supplementary materials. The data show
that the molecular attributes of the designed molecules have high potential to be absorbable from
GIT except for compounds IIIa, Vb and VIIc. Apart from compound I, all the molecules cannot
cross blood brain barrier. The designed compounds are not susceptible to Pgp efflux except I,
IIIa and IV. Compounds IV, VI and VIIc are not expected to inhibit any cytochrome enzyme.
Collectively compound VI is predicted to have best pharmacokinetic attributes.
Compound I show desirable drug-likeness attributes according to Lipinski, Ghose, Veber, Egan
and Muegge rules. From medicinal chemistry perspective, the compound can be easily
synthesized.
Similarly compound II had desirable drug-likeness however the compound molecular weight is
more than 350 with more than 7 rotatable bonds and elevated lipophilicity. These characteristics
slightly weaken its lead likeness.
Compound IIIa is compatible with Lipinski, Ghose, Veber and Muegge filters. It is considered
as a lead like compound.
Compound IIIb has desirable drug likeness as per all medicinal chemistry filters. The lead
likeness is compromised because its molecular weight (MW) is more than 350. Similarly was
compound IV.
Compound Va attributes make it a drug-like compound. Additionally, the compound may act as
a perfect lead like compound.
Compound Vb attributes make it a good drug like compound except for its elevated topological
polar surface area which makes it incompatible with Veber and Egan rule. Its lead likeness may
be compromised due to its larger molecular weight (>350).
Drug likeness of compound VI is similar to those of compound Vb. The large molecular weight
(>350) is a manageable concern against its lead-likeness.
12

CompoundsVIIa-e are perfect drug like compounds however their lead-likeness is compromised,
while compound VIIf has an elevated LogP which hinders its compatibility with Muegge, Egan
and Ghose as well as its drug-likeness.
Compounds VIII and IX have good drug-likeness where IX shows an additional good lead
likeness.
Collectively the good predicted pharmacokinetic profile and drug-likeness attributes along with
the improved docking profile of the proposed compounds strongly support their biological
evaluation.
13

3. Conclusion
In this study, novel imidazo[4,5-b]pyridine derivatives were designed and synthesized. The
synthesized derivatives had promising anticancer activity against either breast or colon cancer
cell lines. Three compounds elicited remarkable activity against both cell lines I, VIII, IX. It is
worth pointing out that, compound VI was the most potent one exhibiting a superior activity at a
submicromolar level against breast cancer cell line (IC50= .63 μM) a CDK9 e z e a a
(IC50= 0.50 μM). Notabl t e ole lar attrib te of o o VI predict optimum
pharmacokinetic profile and acceptable drug likeness. Finally, the promising anticancer activity
of diazenyl benzamine VIII against MCF-7 and HCT116 cell lines (IC50= 2.84 a 1.83 μM) a
hydrazono pentane-2,4-dione IX (IC50 = 2.11 a 1.69 μM) along with its desirable drug-
likeness attributes directed our attention to perform a future study by coupling diazonium salt of
I with appropriate primary amines and methylene derivatives.
14

4. Experimental
4.1.Chemistry
2,3-Diaminopyridine and diethyl ethoxymethylenemalonate were purchased from Acros
Organics. All reactions were monitored by TLC (aluminum sheet) using CHCl3/CH3OH (9:1)
and all spots were visualized at 254, 366 nm by UV Vilber Lourmat 77202 (Vilber, Marne La
Vallee, France). All melting points were determined by open capillary tube method using Gallen
Kamp melting point apparatus MFB-595-010M (Gallen Kamp, London, England) and were
uncorrected. Elemental microanalysis was carried out at the Regional Center for Mycology and
Biotechnology, Al-Azhar University. Infrared spectra (IR) were recorded as potassium bromide
discs on Schimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan) and expressed in
1
13
wave ber (υmax) cm-1. HNMR and CNMR spectra were recorded on Varian Mercury VX-
300 NMR spectrometer at 300 MHz and Bruker NMR spectrometer at 400 MHz. Chemical
ift are q ote i δ a art er illio ( ) ow fiel fro tetra et l ila e (TMS) a
internal standard. Mass spectra were recorded using Schimadzu Gas Chromatograph Mass
spectrometer- QP 100 Ex (Schimadzu).
4.1.1. 4-(1H-imidazo[4,5-b]pyridin-2-yl)aniline(I)
2,3-Diaminopyridine (5.45 g, 0.05 mole), 4-aminobenzoic acid (6.85 g, 0.05 mole) in
polyphosphoric acid (PPA) (85 g) were heated in an oil bath at 220 ºC for 3 h. The reaction
mixture was cooled, and then poured onto ice-cooled 10% sodium carbonate solution (1 Liter).
The formed solid product was filtered, washed with water. The crude product was crystallized
from aqueous methanol. Yield (86%), m.p 328-33 ºC. IR υmax, cm-1: 3344, 3221 (NH2), 3128
1
(NH), 3067 (CH Ar), 1630 (NH), 1604 (C=N), 1543, 1473 (C=C). H NMR (400 MHz, DMSO-
d6) δ 3.50 (s, 1H, NH exchanged with D2O), 5.69 (s, 2H, NH2, exchanged with D2O), 6.67 (d, J =
8.64 Hz, 2H, H Ar), 7.12 (dd, J = 4.84, 7.84 Hz, 1H, H imidazopyridine), 7.84 (dd, J = 1.36, 7.88
Hz, 1H, H imidazopyridine), 7.90 (d, J = 7.52 Hz, 2H, H Ar), 8.20 (dd, J = 1.40, 4.80 Hz, 1H, H
imidazopyridine). 13CNMR (101 MHz, DMSO-d6): δ 154.85, 152.02, 151.63, 142.80, 128.62,
122.75, 117.58, 117.20, 113.95, 111.96.MS m/z %:210 (M+). Anal. calcd for C12H10N4; C, 68.56;
H, 4.79; N, 26.65; Found: C, 68.79; H, 4.88; N, 26.94.
15

2-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}malonate (II).
Equimolar amounts of compound I (4.2 g, 0.02 mole) and diethyl ethoxymethylenemalonate
(4.32 g, 4.04 ml, 0.02 mole) in absolute ethanol (60 ml) were refluxed for 6 h. The solvent was
evaporated under reduced pressure. The crude product was crystallized from ethanol. Yield
(73%), m.p 151-1 2 ºC. IR υmax, cm-1: 3402, 3325 (2NH), 3063 (CH Ar), 2931 (CH Aliph), 1708
1
(2C=O), 1654 (NH), 1600 (C=N), 1593, 1481 (C=C). H NMR (400 MHz, DMSO-d6): δ 1.22 (t,
J = 7.08 Hz, 3H, CH3), 1.28 (t, J = 7.24 Hz, 3H, CH3), 4.11 (q, J = 7.04 Hz, 2H, CH2), 4.22 (q,
J = 7.08 Hz, 2H, CH2), 5.92 (s, 1H, H olefinic, =CH), 6.66-6.69 (dd, J = 4.92, 7.68 Hz, 1H, H
imidazopyridine), 7.48 (dd, J = 1.28, 7.68 Hz, 1H, H imidazopyridine), 7.86 (dd, J = 1.48, 4.88
Hz, 1H, H imidazopyridine), 8.169 (d, J = 13.36 Hz, 2H, H Ar), 9.04 (s, 1H, NH exchanged with
D2O), 9.07 (s, 1H, NH exchanged with D2O), 10.18 (d, J = 13.2, 2H, H Ar). 13CNMR (101 MHz,
DMSO-d6): δ 167.63, 165.33, 164.01, 152.77, 150.56, 144.09, 141.56, 128.62, 126.05, 123.84,
118.43, 118.14, 94.96, 60.29, 60.10, 14.70. MS m/z: 380 (M+). Anal. calcd for C20H20N4O4; C,
63.15; H, 5.30; N, 14.73; Found: C, 63.39; H, 5.37; N, 14.59.
4.1.2. General procedure for preparation of IIIa,b
Compound II (3.80 g, 0.01mole) was added to sodium ethoxide solution prepared by dissolving
Na metal (0.23g, 0.01 mole) in absolute ethanol (10 ml), followed by drop-wise addition of
hydrazine hydrate or phenyl hydrazine (0.01 mole) while stirring. The mixture was refluxed for 6
h. The solvent was removed under reduced pressure. The crude product was crystallized from
ethanol.
4.1.2.1.4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}pyrazolidine-3,5-dione
(IIIa)
Yield (78%), m.p. 168-169 ºC. IR υmax, cm-1: 3317, 3267, 3213, 3194 (4NH), 3070 (CH Ar),
1
1670 (2C=O), 1631 (NH), 1573 (C=N), 1523, 1473 (C=C). H NMR (400 MHz, DMSO-d6): δ
4.50 (s, 1H, NH exchanged with D2O), 5.31 (s, 2H, 2NH exchanged with D2O), 6.34-6.37 (m,
3H, 2H imidazopyridine, 1H, =CH), 6.67 (d, J= 8.84 Hz, 2H, ArH), 7.26 (dd, J = 1.40, 4.88, 1H,
16

H imidazopyridine), 7.39 (s, 2H, Ar H), 8.40 (s, 1H, NH exchanged with D2O). Anal. calcd for
C16H12N6O2; C, 60.00; H, 3.78; N, 26.24; Found: C, 59.78; H, 3.89; N, 26.17.
4.1.2.2.4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}-1-phenylpyrazolidine-
3,5-dione (IIIb).
Yield (88%), m.p. 89-91 ºC. IR υmax, cm-1: 3255 (2NH), 3186 (NH), 3024 (CH Ar), 1685
(2C=O), 1597 (NH), 1543 (C=N), 1520, 1496 (C=C). 1H NMR (400 MHz, DMSO-d6): δ 5.71 (s,
1H, NH exchanged with D2O), 6.61 (s, 1H, H olefinic, =CH), 6.68-6.71 (m, 2H, Ar H), 6.85 (d,
J = 7.84, 2H, Ar H), 6.99 (d, J = 7.84, 2H, Ar H), 7.17-7.72 (m, 2H, H imidazopyridine ), 7.28
(d, J = 7.28, 2H, Ar H), 7.38 (t, 1H, Ar H), 8.11-8.16 (m, 1H, H imidazopyridine), 9.03 (s, 1H,
NH exchanged with D2O), 13.21 (s, 1H, NH exchanged with D2O). MS m/z: 396 (M+). Anal.
calcd for C22H16N6O2; C, 66.66; H, 4.07; N, 21.20; Found: C, 66.38; H, 3.97; N, 21.43.
4.1.3. 4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}-1-acetylpyrazolidine-3,5-
dione (IV).
Compound II (3.80g, 0.01 mole) was dissolved in glacial acetic acid (10 ml) and then hydrazine
hydrate (0.32 g, 0.316 ml, 0.01 mole) was added while stirring. The reaction mixture was heated
under reflux for 6 h, then cooled and the formed precipitate was filtered, washed with ethanol (5
ml) and dried. The crude product was crystallized from ethanol. Yield (67%), m.p. 289-290 ºC.
IR υmax, cm-1: 3332 (NH), 3224 (2NH), 3043 (CH Ar), 2893 (CH Aliph), 1697 (2C=O), 1660
1
(C=O), 1651 (NH), 1608 (C=N), 1558, 1504 (C=C). H NMR (400 MHz, DMSO-d6): δ 2.25 (s,
3H, CH3), 5.73 (s, 1H, NH, exchanged with D2O), 5.90 (s, 1H, H olefinic , =CH), 6.67 (d, J =
8.24 Hz, 2H, H Ar), 7.20-7.24 (m, 1H, H imidazopyridine), 7.76 (d, J = 8.28 Hz, 2H, H Ar), 7.89
(d, J = 8.28 Hz, 1H, H imidazopyridine), 8.15 (d, J = 8.52 Hz, 1H, H imidazopyridine), 8.87 (s,
1H, NH exchanged with D2O), 9.25 (s, 1H, NH exchanged with D2O). 13CNMR (101 MHz,
DMSO- 6) δ 174.63 169.63 168. 9 1 1.67 1 .63 141.82 128.63 127.92 124.64 124. 7
119.41, 113.98, 59.34, 24.97. Anal. calcd for C18H14N6O3; C, 59.67; H, 3.89; N, 23.19; Found:
C, 59.29; H, 3.95; N, 23.57.
17

4.1.4. General procedure for preparation of compoundsVa,b and VI
Compound II (3.80g, 0.01 mole), urea/ thiourea or thiosemicarbazide (0.01 mole) were added to
sodium ethoxide solution prepared by dissolving Na metal (0.23g, 0.01 mole) in absolute ethanol
(10 ml) while stirring and the mixture was refluxed for 6 h. The solvent was removed under
reduced pressure. The crude product was crystallized from ethanol.
4.1.4.1.5-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}pyrimidine
2,4,6(1H,3H,5H)-trione (Va).
Yield (58%), m.p. 213-214 ºC. IR υmax, cm-1: 3444 (2NH), 3348, 3209 (2NH), 3093 (CH Ar),
1
1678 (2C=O), 1620 (NH), 1608 (C=N), 1546, 1492 (C=C). H NMR (400 MHz, DMSO-d6): δ
5.42 (s, 2H, 2NH exchanged with D2O), 5.70 (s, 1H, NH exchanged with D2O ), 7.17-7.18 (m,
1H, H imidazopyridine), 7.22 (d, J = 8.72 Hz, 2H, H Ar), 7.89-7.93 (m, 2H, 1H imidazopyridine,
1H, olefinic, =CH ), 8.15 (d, J = 8.72 Hz, 2H, H Ar), 8.26 (dd, J = 1.28,4.88 Hz , 1H, H
imidazopyridine), 10.0 (s, 1H, NH exchanged with D2O). Anal. calcd for C17H12N6O3; C, 58.62;
H, 3.47; N, 24.13; Found: C, 58.94; H, 3.61; N, 24.38.
4.1.4.2.5-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}-2-thioxodihydro-
pyrimidine-4,6(1H,5H)-dione (Vb).
Yield (74%), m.p. 350-3 2 ºC. IR υmax, cm-1: 3383 (2NH), 3275, 3170 (2NH), 3097 (CH Ar),
1
1650 (2C=O), 1610 (NH), 1604 (C=N), 1490, 1465 (C=C) 1261 (C=S). H NMR (400 MHz,
DMSO-d6): δ 5.17 (s, 1H, H olefinic, =CH), 7.56(s, 1H, NH exchanged with D2O), 8.07 (d, J =
7.36 Hz, 1H, H imidazopyridine), 8.17 (d, J = 8.48 Hz, 2H, H Ar), 8.32-8.34 (m, 2H , H Ar).
8.39-8.43 (m, 2H, H imidazopyridine), 8.92 (s, 2H, NH exchanged with D2O), 13.59 (s, 1H, NH
exchanged with D2O). Anal. calcd for C17H12N6O2S; C, 56.04; H, 3.32; N, 23.06; Found: C,
56.53; H, 3.62; N, 23.49.
18

4.1.4.3.(Z)-6-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenylamino]methylene}-4H-1,2,4-triazepine-
3,5,7(6H)-trione (VI).
Yield (58%), m.p. 223-224 ºC. IR υmax, cm-1: 3500, 3363, 3336 (NH), 3062 (CH Ar), 1681
1
(3C=O), 1640 (NH), 1604 (C=N), 1580, 1496 (C=C). H NMR (400 MHz, DMSO-d6) δ: 5.72
(s,1H, 1NH exchanged with D2O), 6.35 (s, 1H, =CH), 6.67 (d, J = 8.64 Hz, 2H, H Ar), 7.11-
7.22 (m, 1H, H imidazopyridine), 7.54 (d, J = 8.76, 2H, H Ar), 7.77 (d, J = 8.68 Hz, 1H, H
imidazopyridine), 8.19-8.29 (m, 1H, H imidazopyridine), 10.20 (s, 1H, NH exchanged with
D2O), 10.80 (s, 1H, NH exchanged with D2O). Anal. calcd for C17H11N7O3; C, 56.51; H, 3.07; N,
27.14; Found: C, 56.81; H, 3.17; N, 27.45.
4.1.5. General procedure for preparation of Substituted 1-{4-(1H-imidazo[4,5-b]pyridin-2-
yl)phenyl}diazene (Azocoupling products) VIIa-f.
A solution of sodium nitrite (0.89 g, 0.013 mole) in distilled water (5 ml) was added portion wise
to a stirred ice-cooled solution of compound I (2.10g, 0.01 mole) in HCl (2.5 ml) and distilled
water (5 ml). The cold diazonium solution was added portion wise to a solution of the
appropriate phenolic compound (0.01 mole) in an aqueous solution of 10% sodium hydroxide (4
ml) while stirring and cooling. The reaction mixture was kept in ice for 2 h, filtered, dried. The
crude product was crystallized from ethanol.
4.1.5.1.4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}phenol (VIIa).
Yield (65%), m.p. 350-3 1 ºC. IR υmax, cm-1: 3400 (OH), 3363 (NH), 3062 (CH Ar), 1660 (NH),
1
1589 (C=N), 1480, 1454 (C=C). H NMR (400 MHz, DMSO-d6): δ 6.77-6.80 (m, 3H, 2 Ar H,
1H, NH exchanged with D2O), 7.12 (s, 1H , OH exchanged with D2O), 7.18 (dd, J = 4.72, 7.88
Hz,1H, H imidazopyridine), 7.75 (d, J = 8.92 Hz, 2H, Ar H), 7.88 (d, J = 8.56 Hz, 2H, H Ar),
7.96 (dd, J = 7.92,1.44 Hz, 1H, H imidazopyridine), 8.30 (dd, J = 1.40,4.72 Hz, 1H, H
imidazopyridine), 8.41 (d, J = 8.56 Hz, 2H, Ar H). MS m/z: 315 (M+). Anal. calcd for
C18H13N5O; Calcd: C, 68.56; H, 4.16; N, 22.21; Found: C, 68.90; H, 4.27; N, 22.51.
19

4.1.5.2.4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}benzene-1,3-diol (VIIb).
Yield (76%), m.p. 353-3 4 ºC. IR υmax, cm-1: 3394 (2OH), 3255 (NH), 3097 (CH Ar) 1680
1
(NH), 1610 (C=N), 1604, 1489 (C=C). H NMR (400 MHz, DMSO-d6): δ 6.18-6.23 (m, 2H, Ar
H), 6.47 (s, 1H, Ar H), 6.57 (d, J = 8.84 Hz, 2H, Ar H), 6.89 (t, J = 7.96 Hz , 1H, H
imidazopyridine), 8.09 (d, J =8.24 Hz, 2H, Ar H), 8.40 (d, J =7.76 Hz, 1H, imiazopyridine H),
8.54 (d, J =8.24 Hz, 1H, imidazopyridine H), 9.23 (s, 1H, NH exchanged with D2O), 11.00 (s,
1H, OH, exchanged with D2O), 12.31 (s, 1H, NH exchanged with D2O). MS m/z: 331 (M+).
Anal. calcd for C18H13N5O2; C, 65.25; H, 3.95; N, 21.14; Found: C, 65.43; H, 4.06; N, 21.31.
4.1.5.3.5-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}-2-hydroxybenzoic acid (VIIc).
Yield (67%), m.p. 351-3 2 ºC. IR υmax, cm-1: Broad band 3417- 2500 OH, NH, CH Ar), 1680
1
(C=O), 1650 (NH), 1600 (C=N), 1581, 1485 (C=C). H NMR (400 MHz, DMSO-d6): δ 6.58 (s,
1H, NH exchanged with D2O), 6.60-6.64 (m, 4H, 3 Ar H + 1H imidazopyridine), 7.12 -7.17 (m,
3H, 2 Ar H + 1H imidazopyridine), 7.67-7.69 (m, 3H, 2 Ar H, 1H imidazopyridine), 16.09 (s,
2H, OH exchanged with D2O). MS m/z: 359 (M+). Anal. calcd for C19H13N5O3; C, 63.51; H,
3.65; N, 19.49; Found: C, 63.31; H, 3.78; N, 19.68.
4.1.5.4.2-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}-4-methylphenol (VIId).
Yield (52%), m.p. 258-2 9 ºC. IR υmax, cm-1: 3400 (OH), 3383 (NH), 3097 (CH Ar), 2920 (CH
1
Aliph), 1615 (NH), 1604 (C=N), 1530, 1496 (C=C). H NMR (400 MHz, DMSO-d6): δ 2.30 (s,
3H, CH3), 4.59 (s, 1H, OH exchanged with D2O), 6.64 (d, J = 7.36 Hz, 1H, Ar H), 6.92-7.01 (m,
2H,1 Ar H, 1H imidazopyridine), 7.27 (d, J = 7.20 Hz , 1H, Ar H), 7.56 (s, 1H, Ar H), 8.20 (d,
J = 7.52 Hz, 1H, H imidazopyridine), 8.22-8.24 (m , 2H, 1 Ar H, 1H imidazopyridine), 8.54 (d,
J = 7.76 Hz, 2H, Ar H), 10.85 (s, 1H, NH exchanged with D2O). Anal. calcd for C19H15N5O; C,
69.29; H, 4.59; N, 21.26; Found: C, 69.29; H, 4.72; N, 21.18.
20

4.1.5.5.1-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}naphthalen-2-ol (VIIe)
Yield (91%), m.p. 308-3 9 ºC. IR υmax, cm-1: 3420 (OH), 3390 (NH), 3028 (CH Ar), 1627 (NH),
1
1593 (C=N), 1535, 1450 (C=C). HNMR (400 MHz, DMSO-d6): δ 5.19 (s, 1H, OH exchanged
with D2O), 6.64 (d, J = 10.44 Hz, 1H, Ar H), 7.03 (d, J = 10.08 Hz, 1H, Ar H), 7.11 (d, J = 7.00
Hz, 1H, Ar H), 7.48-7.53 (m, 3H, Ar H), 7.62-7.64 (m, 1H, H imidazopyridine), 7.90 (d, J =
8.84 Hz, 1H, H imidazopyridine), 7.95-8.02 (m, 2H, Ar H), 8.46 (d, J = 8.92 Hz, 2H, Ar H),
8.57 (d, J = 5.32 Hz, 1H, H imidazopyridine), 13.4 (s, 1H, NH exchanged with D2O). Anal.
calcd for C22H15N5O; C, 72.32; H, 4.14; N, 19.17; Found: C, 72.04; H, 4.19; N, 19.03.
4.1.5.6.4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}naphthalen-1-ol (VIIf).
Yield (89%), m.p. 339-34 ºC. IR υmax, cm-1: 3410 (OH), 3390 (NH), 3024 (CH Ar), 1680 (NH),
1
1620 (C=N), 1600, 1496 (C=C). H NMR (400 MHz, DMSO-d6): δ 4.10 (s, 1H, OH, exchanged
with D2O), 6.78 (d, J = 9.52 Hz, 1H , H Ar), 7.09-7.12 (m, 2H, 1H Ar, 1H imidazopyridine),
7.37 (t, J = 7.36 Hz, 1H, H Ar), 7.47 (t, J = 7.36 Hz 1H, H Ar), 7.61-7.67 (m, 3H, 2H Ar, 1H
imidazopyridine), 7.71-7.76 (m, 2H, H Ar), 7.93 (d, J = 9.6 Hz, 1H, H Ar), 8.01 (d, J = 8.68 Hz,
1H, H Ar), 8.42 (d, J = 7.84 Hz, 1H, H Ar), 8.49 (dd, J = 4.08,8.60 Hz, 1H, H imidazopyridine),
15.87 (s, 1H, NH exchanged with D2O). MS m/z: 365 (M+). Anal. calcd for C22H15N5O; C,
65.25; H, 3.95; N, 21.14; Found: C, 65.43; H, 4.06; N, 21.31.
4.1.6. 4-{[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]diazenyl}benzenamine (VIII).
A solution of sodium nitrite (0.98g, 0.013 mole) in water (5ml) was added portion wise to a
stirred ice-cooled solution of compound I (2.10 g, 0.01 mole) in HCl (2.5 ml) and cold distilled
water (5 ml). Aniline (0.01 mole) was added dropwise to the cold diazonium solution while
stirring and cooling. The reaction mixture was kept in ice for 2 h, filtered, dried and crystallized
from ethanol. Yield (92%), m.p332-333 ºC. IR υmax, cm-1: 3317 (NH2), 3197 (NH), 3097 (CH
Ar), 1604 (NH), 1550 (C=N), 1520, 1496 (C=C). 1H NMR (400 MHz, DMSO-d6): δ 4.05(s,2H,
NH2 exchanged with D2O), 4.37 (s, 1H, NH exchanged with D2O), 6.78 (d, J = 8.68 Hz, 2H, H
Ar), 7.37 (d, J = 7.32 Hz, 2H, H Ar), 7.48-7.51 (m, 4H , H Ar), 7.68 (d, J = 8.72 Hz, 1H, H
21

imidazopyridine), 8.14 (d, J = 8.40 Hz, 1H, H imidazopyridine), 8.47 (d, J = 5.40 Hz, 1H, H
imidazopyridine). Anal. calcd for C18H14N6; C, 68.78; H, 4.49; N, 26.74; Found: C, 69.06; H,
4.58; N, 26.89.
4.1.7. 3-{2-[4-(1H-imidazo[4,5-b]pyridin-2-yl)phenyl]hydrazono}pentane-2,4-dione (IX).
The diazonium salt produced from compound I (4.20 g, 0.02 mole) was dissolved in absolute
ethanol (15 ml) in presence of sodium hydroxide (2.40 g, 0.06 mole) and acetylacetone (3.00 g,
3.06 ml, 0.03mole) was added on cold while stirring. The reaction mixture was stirredat room
temperature for 2 h. The obtained solid was filtered, dried and crystallized from ethanol. Yield
(73%), m.p 295-296 ºC. IR υmax, cm-1: 3402 (2NH), 3089 (CH Ar), 2924 (CH Aliph), 1670
(2C=O), 1640 (NH), 1608 (C=N), 1504, 1465 (C=C). 1H NMR (400 MHz, DMSO-d6): δ 2.46 (s,
3H, CH3), 2.48 (s, 3H, CH3), 4.46 (s, 1H, NH exchanged with D2O), 7.54 (dd, J = 7.36, 5.6 Hz,
1H, H imidazopyridine), 7.78 (d, J = 8.64 Hz, 2H, ArH), 8.35 (d, J = 7.52 Hz, 1H, H
imidazopyridine), 8.47 (d, J = 8.60 Hz, 2H, Ar H), 8.52 (d, J = 5 Hz, 1H, H imidazopyridine),
13.70 (s, 1H, NH exchanged with D2O). Anal. calcd for C17H15N5O2; C, 63.54; H, 4.71; N,
21.79; Found: C, 63.19; H, 4.78; N, 22.05.
4.2. Anticancer activity
4.2.1. In-vitro MTT assay
Cell lines were provided by American Type Culture Collection (ATCC) and propagated in
RPMI-1640. Cells were cultured using DMEM (Invitrogen/Life Technologies) supplemented
with 10% FBS (Hyclone,), 10 µg/ml of insulin (Sigma), and 1% penicillin-streptomycin. All
chemicals and reagents were from Sigma, or Invitrogen.
Cells suspended in medium (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of 100µl
complete growth medium were seeded into a 96-well plate and incubated at 37°C in a CO2
i bator over ig t. After 24 t e te t a le (1 μl of the tested compound per well) was
added to the cells in 96- well plates and cultured in fresh new medium at 37 ºC for 48 h. Media
containing test compounds were discarded and the plate was washed with PBS. The cultured
ell were i e wit 2 μl of MTT dye solution and incubated for 4 h at 37 ºC. The supernatant
wa aref ll re ove fro ea well a 1 μl of DMSO were a e to ea well to i olve
22

the formazan crystals which were formed by the cellular reduction of MTT. After mixing with a
mechanical plate mixer, the absorbance of each well was measured by microplate reader using a
test wavelength of 570 nm [12] 14]. The results were expressed in IC50, which induces a 50 %
inhibition of cell growth of treated cells when compared to the growth of control cells. Each
experiment was performed at least 3 times in a concentration of 0.01, 0.1, 1, 5, 10, 100 and 200
μM. T ere wa a goo re ro ibilit betwee re li ate well wit ta ar error below 1 .
4.2.2. CDK9 enzyme inhibition assay
The micro ELISA plate provided has been pre-coated with an antibody specific to CDK-9.
Standards or samples are added to the appropriate micro ELISA plate wells and combined with
the specific antibody. Then the reactants become antibody-antigen-antibody-enzyme complex,
after washing completely, 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution was added
and TMB substrate becomes blue color under HRP enzyme-catalyzed, reaction is terminated by
the addition of a sulphuric acid solution and the color change is measured spectrophotometrically
at a wavelength of 450 nm. The concentration of CDK9 in the samples is then determined by
comparing the optical density (O.D) of the samples to the standard curve.
23

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Graphical abstract
26

Highlights
 New imidazo[4,5-b]pyridine derivatives were designed, synthesized and screened for
their anticancer activity
 The new imidazo[4,5-b]pyridine derivatives had remarkable anti-proliferative activity
against breast and colon cancer cell lines
 Synthesized compounds showed superior binding affinity relative to the natural ligand
(T3C)
 Synthesized compounds had excellent physicochemical properties and drug-likeness
attributes
 Synthesized compounds had excellent pharmacokinetic profile
27