6-Substituted imidazo[1,2-a]pyridines: Synthesis and biological activity against colon cancer cell lines HT-29 and Caco-2

Article abstract of DOI:10.1016/j.ejmech.2011.07.036

A range of 6-substituted imidazo[1,2-a]pyridines were synthesized using a multicomponent coupling reaction. Most of these compounds were found to exhibit excellent activity against the colon cancer cell lines HT-29 and Caco-2, whilst not showing significa

Full text of DOI:10.1016/j.ejmech.2011.07.036

European Journal of Medicinal Chemistry 46 (2011) 4573e4583
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
6-Substituted imidazo[1,2-a]pyridines: Synthesis and biological activity against
colon cancer cell lines HT-29 and Caco-2
Nurit Dahan-Farkas a, Candice Langley b, Amanda L. Rousseau b, Dharmendra B. Yadav b,
,
c
,
b,
*
Hajierah Davids a
, Charles B. de Koning
**
a Division of Pharmacology, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa
b Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, P.O. Wits 2050, South Africa
c Department of Biochemistry & Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth 6031, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A range of 6-substituted imidazo[1,2-a]pyridines were synthesized using a multicomponent coupling
reaction. Most of these compounds were found to exhibit excellent activity against the colon cancer
cell lines HT-29 and Caco-2, whilst not showing significant toxicity against white blood cells. Our
studies have shown that the proteolytic phase of apoptosis was initiated 2 h after treatment with these
imidazoe[1,2-a]pyridines. The data suggests that the imidazo[1,2-a]pyridine-induced cell death in
HT-29 and Caco-2 cells is mediated via pathway(s) that include the release of cytochrome c from the
mitochondria to the cytosol and the activation of caspase 3 and caspase 8.
Received 23 May 2011
Received in revised form
20 July 2011
Accepted 20 July 2011
Available online 27 July 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
6-substituted imidazo[1,2-a]pyridines
Multicomponent coupling reactions
Colon cancer
HT-29 and Caco-2
Apoptosis
Caspases
1. Introduction
Cellular homeostasis is maintained by a balance between cell
proliferation and cell death, with the latter constituting one of the
Cancer continues to pose significant health problems world-
wide, despite medical advances that have, over the last decade,
increased our understanding of the genetics of cancer and the
application of novel drug therapy. Approximately 782 000 people
are diagnosed with colon cancer annually. In the United States,
colon cancer is the third most common cause of cancer-related
deaths with an estimated annual incidence of 146 970 and
a mortality of 49 920 reported in 2009 [1]. Conventional treatment
of colon cancer is by surgical ablation, with chemotherapy and/or
radiotherapy used as adjunctive treatment, as over 40% of colon
cancer patients develop metastases. These approaches are not
always effective against the highly metastasised disease and hence,
new therapeutic methods are essential for treatment [2].
major targets in medical research. Cell injury can lead to cell death
by necrosis or apoptosis. Apoptosis is characterised by various
morphological changes, including plasma membrane blebbing,
chromatin condensation, nuclear fragmentation and formation of
apoptotic bodies [3]. In addition, biochemical features of apoptosis
such as the exposure of phosphatidylserine (PS) on the outer leaflet
of the plasma membrane [4], changes to the permeability of the
mitochondrial membrane [5], and the release of proteins such as
cytochrome c from the mitochondrial intermembrane space are
noted [6].
There are several factors involved in the apoptotic process,
including the co-ordinated action of a cascade of initiator or
effector caspases (cysteine aspartate-specific proteases) [7]. Cas-
pase activation leads to the various morphological and biochemical
features of apoptosis. Caspase 3 is the key effector caspase
responsible for the cleavage of multiple protein substrates in the
cells, ultimately leading to apoptosis. Opening of the mitochondrial
permeability transition (MPT) pore causes a loss of the inner
mitochondrial membrane potential (Djm), leading to the release of
* Corresponding author. Tel.: þ27 11 7176724; fax: þ27 11 7176749.
** Corresponding author. Division of Pharmacology, Department of Pharmacy and
Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York
Road, Parktown 2193, South Africa. Tel.: þ27 41 504 2019; fax: þ27 41 504 2814.
E-mail addresses: Candice.Langley@gmail.com (C. Langley), Amanda.Rousseau@
wits.ac.za (A.L. Rousseau), Dharmendra.Yadav@wits.ac.za (D.B. Yadav), Hajierah.
Davids@nmmu.ac.za (H. Davids), Charles.deKoning@wits.ac.za (C.B. de Koning).
cytochrome
c from the mitochondrial intermembrane space.
Cytochrome c then binds to apoptotic protease activating factor 1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.036

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N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
(Apaf-1) and activated caspase 3, which results in mitochondrial-
R2
dependent apoptosis [8,9].
N
In cancerous cells, there are very low levels of apoptosis and
DNA repair seldom occurs. Several chemotherapeutic agents have
been shown to be effective inducers of apoptosis through under-
lying cellular mechanisms. The identification of inducers of
apoptosis presents a strong basis for the development of potential
anticancer agents and through the induction of apoptosis, novel
compounds may reduce the resistance of colon cancer cells to
current therapeutic regimes [10].
OHC
R1NC, catalyst
NH2
N
N
R
R
NH
R2
R1
Scheme 1. Three component coupling reaction for the synthesis of imidazo[1,2-a]
pyridines.
OMe
HO
HO
N
N
N
OMe
OH
OH
N
N
N
O2N
Br
NH
NH
NH
3 (42%)
2 (74%)
1 (11%)
MeO
OMe
OBn
OBn
N
N
N
OMe
N
N
N
Br
Br
Br
OMe
NH
NH
NH
6 (44%)
4 (67%)
5 (39%)
BnO
HO
OBn
OBn
N
N
N
OBn
OH
H2N
N
N
N
O2N
NH
NH
O
NH
9 (19%)
8 (43%)
7 (85%)
MeO
HO
MeO
N
N
N
OMe
OH
N
OMe
N
O2N
N
Br
NH
NH
NH
11 (68%)
12 (84%)
10 (15%)
MeO
N
OMe
N
NH
13 (84%)
Fig. 1. Imidazo[1,2-a]pyridines 1e13 and percentage yields.

N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
Table 2
4575
OMe
OMe
OMe
IC50 values for the imidazopyridines and camptothecin on the Caco-2 and HT-29 cell
lines.
N
N
OMe
(i)
N
N
Compound
IC50
(
m
M)
Ph
Br
NH
NH
HT-29
Caco-2
6
3
4
6
7
12
13
14
12.89 ꢁ 2.41
6.57 ꢁ 1.91
9.14 ꢁ 1.02
9.20 ꢁ 0.83
21.98 ꢁ 1.17
10.03 ꢁ 2.69
8.56 ꢁ 1.22
10.00 ꢁ 1.41
11.91 ꢁ 1.10
6.43 ꢁ 1.01
9.03 ꢁ 0.99
17.38 ꢁ 1.13
20.28 ꢁ 3.45
15.02 ꢁ 1.98
8.73 ꢁ 1.28
9.55 ꢁ 2.21
14
Scheme 2. Reagents and conditions (i) 10% Pd(PPh3)4, PhB(OH)2, CsF, DME, Discovery
microwave, 150 ꢀC and 150 W, 20 min, 95%.
Camptothecin
In recent times, the use of multicomponent coupling reactions
[11] in the synthesis of compounds displaying interesting phar-
macological properties has been an area of prolific research. One of
the classes of compounds that are accessible by multicomponent
coupling reactions are the imidazo[1,2-a]pyridines [12e20].
In the present study, we synthesized a number of novel imidazo
[1,2-a]pyridines and then assessed the anticancer properties of
these imidazopyridines against two colon cancer cell lines, aiming
to investigate any cytotoxic effects on the cells, the mode of cell
death, and to explore the pathways by which cell death was
induced.
catalytic Pd(PPh3)4 in a microwave afforded 14 in a yield of 95%
(Scheme 2).
2.2. Pharmacology
A number of cell viability and flow cytometry assays were used
to determine whether imidazo[1,2-a]pyridines 1e14 induced cell
death in colon cancer cell lines.
2. Results and discussion
2.2.1. Effect of imidazo[1,2-a]pyridines on HT-29 and Caco-2 cell
proliferation
2.1. Chemistry
The effect of imidazo[1,2-a]pyridines 1e14 on HT-29 and Caco-2
cell proliferation was accomplished by exposing a cell population to
compounds 1e14 and then monitoring the mitochondrial enzy-
matic reduction of MTT to formazan. The colon cancer cells were
Using methodology previously described [14], a series of novel
6-substituted imidazo[1,2-a]pyridines were prepared from
a variety of commercially available 5-substituted 2-aminopyridines
with either benzyl isocyanide, cyclohexyl isocyanide or 2,6-
dimethylphenyl isocyanide, and a variety of substituted aromatic
aldehydes in the presence of montmorillonite clay K10 (Scheme 1).
In addition, two imidazo[1,2-a]pyridines lacking a substituent in
the 6-position were also prepared by this method (R ¼ H, Scheme
1). This facile, one-pot reaction afforded imidazo[1,2-a]pyridines
1e13 in the unoptimized yields shown in Fig. 1. All products were
characterised by 1H, 13C NMR spectroscopy as well as by HRMS.
A number of the imidazo[1,2-a]pyridines prepared contain an
aromatic bromine substituent, and we envisaged using this as
a handle for carbonecarbon coupling reactions such as the Suzu-
kieMiyaura reaction. Using this methodology, further variety could
be introduced onto the imidazo[1,2-a]pyridine nucleus. For
example, exposure of 6 to phenylboronic acid in the presence of
initially exposed to all imidazo[1,2-a]pyridines at 100
mM (final
concentration in the well) at 37 ꢀC for 24 h. The MTT assay was used
to quantify cell viability after exposure (Table 1). A compound was
considered active if it reduced the growth of the cell lines to 50% or
less. Interestingly, structureeactivity relationships became
apparent from our small set of imidazo[1,2-a]pyridines. All of the
compounds bearing a nitro substituent in the 6-position of the
imidazopyridine nucleus and all those bearing
a
2,5-
dihydroxyphenyl substituent attached to the imidazopyridine
showed little reduction of cell growth of both colon cancer cell
lines. Conversely, imidazopyridines containing protected hydroxyl
substituents on the benzene ring generally displayed good activi-
ties. Therefore, only imidazo[1,2-a]pyridines 3, 4, 6, 7 and 12e14
showing >50% reduction in cell viability were tested further.
The IC50 values (micromolar concentration that inhibited growth
by 50%) obtained from the active imidazo[1,2-a]pyridines 3, 4, 6, 7
and 12e14 are reported in Table 2. IC50 values showed no significant
Table 1
difference (p ¼ 0.665) between compounds 6 (9.14 ꢁ 1.02
(9.20 ꢁ 0.83 M), 13 (10.03 ꢁ 2.69 M), 14 (8.56 ꢁ 1.22
camptothecin (9.99 ꢁ1.41 M) on the HT-29 cells, while compounds
mM), 7
Percentage cell viability of HT-29 and Caco-2 treated with 100
pyridines and camptothecin, for 24 h.
m
M of the imidazo-
m
m
mM) and
m
Compound
Cell Viability % (100
HT-29
mM)
12 and 3 showed significantly (p ¼ 0.005) higher IC50 values of
Caco-2
21.98 ꢁ 1.17
mM and 12.89 ꢁ 2.41
mM respectively.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
118.79 ꢁ 1.13
108.30 ꢁ 0.69
17.08 ꢁ 1.03
8.64 ꢁ 0.84
113.43 ꢁ 1.01
109.77 ꢁ 1.42
15.68 ꢁ 2.27
9.47 ꢁ 1.41
Table 3
Percentage cell viability of the white blood cells treated with 100
dazopyridines or camptothecin, for 24 h.
mM imi-
86.46 ꢁ 1.09
11.44 ꢁ 0. 20
6.81 ꢁ 0.36
90.32 ꢁ 0.74
10.77 ꢁ 0.17
6.45 ꢁ 1.76
Compound
White Blood Cell Viability %
93.71 ꢁ 1.12
79.48 ꢁ 0.93
86.92 ꢁ 1.33
82.46 ꢁ 0.74
17.19 ꢁ 0.95
11.37 ꢁ 1.20
10.67 ꢁ 0.80
12.79 ꢁ 1.56
89.27 ꢁ 0.45
82.53 ꢁ 2.16
82.95 ꢁ 0.84
88.62 ꢁ 1.03
19.78 ꢁ 1.32
13.58 ꢁ 0.56
12.94 ꢁ 0.2
3
4
6
7
12
13
14
76.176 ꢁ 0.567%
88.943 ꢁ 1.996%
83.762 ꢁ 1.389%
93.834 ꢁ 0.271%
96.311 ꢁ 5.023%
97.479 ꢁ 1.178%
77.345 ꢁ 1.005%
33.782 ꢁ 2.031%
Camptothecin
13.94 ꢁ 1.54
Camptothecin

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N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
Fig. 2. Analysis of apoptosis induced by compound 7 in the HT-29 colon cancer cell line.
IC50 values determined for imidazopyridines 3, 7, 12 and 13 on
the Caco-2 cell line were significantly higher (p ¼ 0.001) than for
camptothecin (9.55 2.21 M), while those obtained for
compounds and 14 were comparable with camptothecin.
percentage of each subpopulation of cells in both cell lines exposed
to the imidazo[1,2-a]pyridines is summarized in Table 4.
ꢁ
m
6
2.2.4. Enzyme activity assays
Compound 4 was more effective than camptothecin in inhibiting
both Caco-2 and HT-29 growth.
During the process of apoptosis, the initiation of all intracel-
lular events is linked to the activation of caspases. All of the
caspases promote apoptosis through the proteolytic degradation
of cellular components. Caspase 8, a member of the caspase
family, is crucial for cell death induction. Impaired expression or
function of caspase 8 can promote tumour formation, progression
and treatment resistance in several types of cancers [22]. Caspase
8 is an initiator caspase which cleaves and activates the effector
caspases (e.g. caspase 3). Once activated, caspase 3, the key
executioner of apoptosis, is responsible for the cleavage and
breakdown of several cellular components related to DNA repair
and regulation, and cleaves polypeptides that ultimately undergo
proteolysis in apoptotic cells. To determine the involvement of
caspase 3 and caspase 8 in the imidazo[1,2-a]pyridine-induced
apoptosis, caspase-specific colorimetric assays were used to
measure caspase activity. The level of caspase activity in the cell
lysate is directly proportional to the absorbance of p-nitroaniline
(pNA) for caspase 3 and caspase 8 at 405 nm.
2.2.2. Effects on cell viability of white blood cells
Freshly isolated white blood cells were exposed to a range of
active imidazopyridines at a final well concentration of 100 mM for
24 h. Cell viability was determined by the MTT assay. The results
obtained are summarized in Table 3. The prepared imidazopyr-
idines showed significantly lower cytotoxicity towards the white
blood cells than 100
that the imidazo[1,2-a]pyridines considered in this study may be
m
M camptothecin (p ¼ 0.0001). This suggests
more selective than camptothecin.
2.2.3. Effects on cell death of HT-29 and Caco-2 cells
Vermes et al. [21] reported that translocation of phosphati-
dylserine (PS) from the inner side of the plasma membrane to the
outer layer occurs in the early stage of apoptosis. The apoptosis
assay monitors cell membrane translocation events and the
accessibility of nuclear material in response to extracellular
disruptions, with the various stages of apoptosis being quantified
by flow cytometry.
In this study, it was noted that treatment with the selected
imidazo[1,2-a]pyridines induced caspase 8 and caspase 3 activity in
both colon cancer cell lines at a concentration of 100
mM. A time-
Our results show that apoptosis was induced after addition of
the imidazopyridines in both colon cancer cell lines, as shown in
the representative example of compound 7 compared to campto-
thecin in the HT-29 colon cancer cell line, shown in Fig. 2.
Apoptosis differs from necrosis in that during the initial stages
of apoptosis, the cell membrane remains intact, whilst the cell
membrane loses its integrity during necrosis. The apoptotic nature
of the imidazo[1,2-a]pyridine-induced cell death was confirmed by
annexin V-FITC labelling of PS exposed on the plasma membrane.
The extent to which annexin V-FITC binds to the cell surface was
measured in conjunction with a propidium iodide (PI) exclusion
test to investigate the integrity of the cell membrane. The untreated
cells (control) were annexin V-FITC and PI negative, indicating that
they were viable and not undergoing apoptosis.
After a 24 h exposure to the imidazo[1,2-a]pyridines or camp-
tothecin, there were primarily two populations of cells: viable, non-
apoptosing cells (annexin V-FITC and PI negative) and cells
undergoing apoptosis (annexin V-FITC positive and PI negative). A
small population of cells was observed to be annexin V-FITC and PI
positive, indicating that they were in end-stage apoptosis. The
dependent activation of caspase 3 was found through 24 h of
exposure to the imidazo[1,2-a]pyridines after which the activity
diminished. The reduction of the caspase 3 activity at 24 h is
possibly due to the cytotoxic effects of the imidazopyridines
resulting in the death of cells.
Table 4
Analysis of apoptosis induced by the imidazo[1,2-a]pyridines in the HT-29 and Caco-
2 cell lines.
Compound
Viable cells (%)
Early
Late
apoptosis (%)
apoptosis (%)
HT-29
Caco-2
HT-29
Caco-2
HT-29
Caco-2
3
4
6
7
12
13
14
12.53
16.53
12.11
14.48
38.57
18.51
50.18
47.95
13.21
23.88
17.47
20.27
44.40
13.97
15.55
50.18
18.11
28.18
18.29
22.96
42.71
43.28
27.06
27.27
21.85
25.87
15.97
18.67
25.02
19.93
18.99
27.06
60.98
47.22
66.54
59.05
15.75
30.8
56.31
44.77
57.23
53.8
18.67
59.81
60.05
17.78
17.78
19.64
Camptothecin

N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
4577
For example, the exposure of the HT-29 cell line to imidazo-
pyridine 7 resulted in maximal caspase 8 activity within 2 h, as
shown in Fig. 3. After 2 h, the activity of caspase 8 declined whereas
that of caspase 3 increased, indicating that the proteolytic phase of
apoptosis was initiated. By comparison, maximal caspase 8 activity
was observed in the HT-29 cells within 2 h of exposure to
compounds 4 (Fig. 3), 6 and 14, and within 4 h of exposure to
compounds 3, 12 and 13.
Similar trends were observed with the executioner caspase,
caspase 3, with maximum levels in HT-29 cells observed after 2 h of
exposure to compounds 4 (Fig. 3), 6 and 14. Caspase 3 activity in
HT-29 cells treated with compound 3 reached a maximum after 4 h
of exposure, while maximum levels were observed between 2 and
4 h after exposure to compound 13, and between 2 h and 8 h after
exposure to compound 12 (figures not shown). These high levels of
caspase 3 activity indicate that apoptosis was initiated fairly early
after exposure to these compounds. Camptothecin was also tested
in the time-dependent induction of caspase 3 and 8 activity (Fig. 3)
and these results showed that compounds 14, 4 and 6 initiated
apoptosis at an earlier stage than camptothecin.
Maximal caspase 8 activity was observed in the Caco-2 cells
within 4 h of exposure to compounds 3, 7, 12 and 13 (Fig. 3); and
within 2 h of exposure to compounds 4 (Fig. 3), 6 and 14. Maximal
caspase 3 activity was noted in the Caco-2 cells after 2 h of exposure
to compounds 4 (Fig. 3), 6 and 14 and after 4 h of exposure to
compound 3. Caspase 3 activity in Caco-2 cells increased between 2
and 4 h after exposure to compounds 7, 13 (Fig. 3) and between 2
and 8 h after exposure to compound 12. The effects of these imi-
dazopyridines on caspase 8 and caspase 3 activities in Caco-2 cells
produced similar effects to that observed in the HT-29 cells. As
before, the high levels of caspase 3 activity shows that apoptosis
was initiated shortly after exposure to the imidazo[1,2-a]pyridines.
Compound 7
a
b
Compound 4
3
2
1
0
Caspas
Caspas
e 3
e 8
2.5
2.0
1.5
1.0
0.5
0.0
Caspa
Caspa
se 3
se 8
0
10
20
30
0
10
20
30
Induction Time (Hours)
Induction Time (Hours)
Compound 4
Compound 13
c
d
Casp
Casp
ase 3
ase 8
4
3
2
1
0
Caspas
e 3
3
2
1
0
Casp
ase 8
0
10
20
30
0
10
20
30
Induction Time (Hours)
Induction Time (Hours)
e
Camptothecin
HT-29 and Caspase 3
HT-29 and Caspase 8
Caco-2 and Caspase 3
Caco-2 and Caspase 8
5
4
3
2
1
0
0
10
20
30
Induction Time (Hours)
Fig. 3. Time-dependent induction of caspase 3 and caspase 8 activity by (a) compound 7 in HT-29 cells, (b) compound 4 in HT-29 cells, (c) compound 13 in Caco-2 cells,
(d) compound 4 in Caco-2 cells and (e) camptothecin in HT-29 and Caco-2 cells. Each point represents the mean and standard deviation of the triplicate values of caspase 3 and
caspase 8 activity after cell exposure to the selected compounds over 2 h, 4 h, 8 h, 12 h and 24 h.

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N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
Fig. 4. JC-1 staining in control and apoptotic cells induced by camptothecin, compounds 4, and 14.
Camptothecin was also tested in these caspase induction studies
and the results are shown in Fig. 3.
Membrane Potential Detection kit (BD Biosciences) was used to
measure mitochondrial membrane depolarisation in viable cells.
There was a significant increase in the number of cells with
decreased fluorescence, indicative of a change in the Djm, in the
populations induced to undergo apoptosis. Compound 4 was
shown to cause the greatest change in Djm in the HT-29 cells
(Fig. 4), with 89.60% of the mitochondria undergoing a change in
the Djm in comparison to 10.11% of viable cells remaining after
treatment. Compound 14 was observed to cause the greatest loss in
the Djm in the Caco-2 cells with an average of 91.85% of cells
2.2.5. Effects on the mitochondrial membrane potential (Djm
)
There is increasing evidence that altered mitochondrial function
is linked to apoptosis. In particular, a decreasing mitochondrial
transmembrane potential is associated with mitochondrial
dysfunction [5] and has been shown to be a vital factor in
controlling the induction of apoptosis. Loss of mitochondrial
membrane potential, Djm, occurs early in the apoptotic process.
The Djm results from the asymmetrical distribution of protons on
both sides of the inner mitochondrial membrane, giving rise to
a chemical (pH) and electrical gradient, which is essential for
mitochondrial function. The collapse of the Djm during apoptosis
was noticed in several studies and it has thus been suggested that
the depolarisation of the mitochondria is one of the first events
which occurs in apoptosis and could be a prerequisite for the
release of cytochrome c. Since the collapse of the Djm does not
always occur in apoptosis, the depolarisation of the Djm may only
be a cause of, or associated with apoptosis in some systems. To
assess whether disruption of the Djm was involved in the apoptotic
action of the imidazo[1,2-a]pyridines in this study, the cationic
fluorescent dye 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimida-
zolcarbocyanine iodide (JC-1)-based flow cytometry Mitochondrial
Table 5
Analysis of mitochondrial membrane depolarisation induced by the imidazo[1,2-a]
pyridines in the HT-29 and Caco-2 colon cancer cell lines.
Compound
% cells with depolarized Djm
HT-29
Caco-2
3
4
6
7
12
13
14
85.92
89.60
63.09
83.33
65.30
79.62
84.29
44.00
85.12
83.33
76.10
81.91
78.00
79.17
91.85
40.88
Camptothecin

N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
4579
Cytosolic fraction
activation and subsequently induces apoptosis. To determine
whether the observed reduction of Djm by the imidazo[1,2-a]
pyridines could lead to cytochrome c release from the mitochondria
into the cytosol, cell lysates were subfractionated and cytosolic and
mitochondrial cytochrome c levels were determined by a human
cytochrome c ELISA assay (Assay Designs).
Mitochondrial fraction
100
75
50
25
0
Consistent with the loss of Djm, cytochrome c release into the
cytosol was clearly identified after exposure to the novel
compounds for 24 h, with cytochrome c levels significantly higher
(p ¼ 0.00001) in the cytosolic fraction than the mitochondrial
fractions of the treated cells, whilst cytochrome c remained
concentrated in the mitochondria in the untreated cells (Fig. 5).
This demonstrated that the compounds induced the loss of Djm
accompanied with increased levels of cytochrome c in the cytosol of
both the HT-29 (Fig. 5) and Caco-2 cells (figure not shown).
The untreated cells had higher cytochrome c levels in the mito-
chondrial fraction of both cell lines than the cytosolic fraction
(p ¼ 0.00001). These results indicate the translocation of cyto-
chrome c from the mitochondria to the cytoplasm after treatment
with the selected compounds. This confirms the results obtained
with the JC-1 dye (Table 5) which suggests that the mitochondria are
implicated in the imidazo[1,2-a]pyridine-induced apoptosis.
As a result of these experimental observations, we have
demonstrated that the observed imidazo[1,2-a]pyridine-induced
caspase 8 activation was the upstream event of the mitochondrial
cytochrome c release, which in turn, led to the activation of caspase
3. It is also suggested that caspase 3 could be activated independent
of the mitochondrial cytochrome c release, as a downstream event
of caspase 8 activation (Fig. 6).
3
4
6
7
12
13
14
Camptothecin Control
Fig. 5. Cytochrome c percentage in the cytosolic and mitochondrial fractions of HT-29
cells after treatment with imidazopyridines 3, 4, 6, 7, 12, 13, 14 and camptothecin for
24 h. The bars represent the mean ꢁ SEM (n ¼ 3).
undergoing a change in the Djm. These findings suggest that
apoptosis induced by the imidazo[1,2-a]pyridine derivatives are
associated with a depolarisation of the mitochondrial membrane.
The percentages of Djm in the imidazo[1,2-a]pyridine-treated
HT-29 and Caco-2 cells compared with the control cells are
summarized in Table 5.
2.2.6. Effects on the release of cytochrome c
Cytochrome c is an electron transport protein which is normally
located between the inner and outer mitochondrial membranes.
Disruption of the Djm results in the opening of the mitochondrial
membrane pores, causing the release of soluble intermembrane
proteins such as cytochrome c, which contributes to caspase 3
3. Conclusions
The biological properties of the imidazo[1,2-a]pyridine class of
compounds have received considerable attention in recent years,
however their effect on colon cancer cell lines has not yet been
established. In the present study, we demonstrated a time- and
dose-dependent induction of apoptosis with a series of novel,
structurally related 6-substituted imidazo[1,2-a]pyridines in HT-29
and Caco-2 colon cancer cells.
Among the selected compounds that were tested against the
HT-29 and Caco-2 cell lines, compounds 4 and 7 are significantly
cytotoxic (p < 0.05) and elicit apoptosis in the cells at very low
micromolar concentrations. Importantly, the compounds showed
a degree of selective cytotoxicity against the cancer cell lines, with
minimal cytotoxicity against white blood cells. The proteolytic
phase of apoptosis was initiated 2 h after treatment with these
compounds. The imidazo[1,2-a]pyridines are also associated with
a marked reduction in the Djm, as well as causing an increase in
cytochrome c levels within the cytosolic fraction of the cells. The
induction of apoptosis (Fig. 6) is considered to be the main mech-
anism underlying the therapeutic efficacy of anticancer drugs,
hence the present results suggest that the imidazo[1,2-a]pyridines
described here may successfully be developed into novel chemo-
therapeutic drugs for the treatment of colon cancer.
4. Experimental procedures
4.1. Chemistry
4.1.1. General experimental procedures
1H NMR and 13C NMR spectra were recorded on a Bruker
AVANCE 300 spectrometer. All spectra were recorded in CDCl3,
MeOD or d6DMSO. All chemical shift values are reported in parts
per million referenced against TMS which is given an assignment of
zero parts per million. Coupling constants (J-values) are given in
Fig. 6. Proposed mechanism of the imidazo[1,2-a]pyridine-induced apoptosis in the
HT-29 and Caco-2 cell lines.

4580
N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
Hertz (Hz). Infrared spectra were recorded on a Bruker Tensor 27
standard system spectrometer. MachereyeNagel Kieselgel 60
(particle size 0.063e0.200 mm) was used for conventional silica gel
column chromatography with various EtOAc and hexane mixtures
as the mobile phase. TLC was performed on aluminium-backed
MachereyeNagel Alugram Sil G/UV254 plates pre-coated with
0.25 mm silica gel 60. HPLC purity of compounds synthesized was
determined using a phenomenex Luna 5u C18 column using an
acetonitrile/water mixture. All compounds were >95% pure by this
HPLC method. Microwave reactions were carried out on
a commercial CEM Discover microwave oven.
Mp ¼ 185e188 ꢀC; IR ¼ nmax (cmꢂ1) ¼ 3370, 3345, 2959, 1587, 1509,
1465, 1409, 1372, 1333, 1300; 1H NMR (300 MHz, CDCl3)
d 7.64 (dd,
J ¼ 8.4, 1.9, 1H), 7.60 (s, 1H), 7.50 (d, J ¼ 9.2, 1H), 7.47 (d, J ¼ 1.9, 1H),
7.01 (dd, J ¼ 9.2, 1.5, 1H), 6.96 (d, J ¼ 7.5, 2H), 6.82 (d, J ¼ 8.5, 1H),
6.77 (t, J ¼ 7.5, 1H), 5.56 (br s, 1H), 3.87 (s, 3H), 3.73 (s, 3H), 2.26 (s,
3H), 2.00 (s, 6H); 13C NMR (75 MHz, CDCl3)
d 149.0, 148.7, 141.0,
140.2, 137.7, 130.2, 128.0, 126.3, 124.7, 122.4, 120.7, 120.2, 119.8,
119.5, 116.4, 111.1, 110.0, 55.9, 55.8, 18.7, 18.6; HRMS (m/z) calculated
for C24H26N3O2, (M þ H), 388.2025, found 388.2023.
4.1.1.4. Benzyl-[6-bromo-2-(2,5-dimethoxy-phenyl)-imidazo[1,2-a]pyr-
idin-3-yl]-amine 4. 2-Amino-5-bromopyridine (0.3026 g, 1.75 mmol)
was dissolved in 1,4-dioxane (5 mL) in a small round-bottomed flask.
2,5-dimethoxybenzaldehyde (0.2975 g, 1.79 mmol), Benzyl iso-
cyanide (0.1500 g, 1.28 mmol), K10 (0.3194 g) were added to the flask
with further 1,4-dioxane (5 mL). The reaction was refluxed for 72 h at
110 ꢀC and after work-up as previously described yielded a brown oil.
The oil was purified by silica gel column chromatography (50% EtOAc/
hexane) to give the product 4 as a yellow oil (0.3760 g, 67%). IR ¼ nmax
(cmꢂ1) ¼ 3339, 2933, 2832, 1586, 1559, 1496, 1453, 1419, 1387, 1322;
4.1.1.1. 4-[3-(2,6-Dimethyl-phenylamino)-6-nitroimidazo[1,2-a]pyr-
idin-2-yl]-benzene-1,3-diol 1. 2-Amino-5-nitropyridine (0.4601 g,
3.31 mmol) was dissolved in 1,4-dioxane (5 mL) 2,4-
Dihydroxybenzaldehyde
(0.4426
g,
3.20
mmol),
2,6-
dimethylphenyl isocyanide (0.4157 g, 3.17 mmol), K10 (0.4175 g)
and a stirrer bar were added to the flask with further 1,4-dioxane
(5 mL). The reaction was refluxed for 72 h at 110 ꢀC while moni-
toring the reaction progress with TLC. The K10 was filtered-off
using boiling hot EtOAc. The filtrate was collected and the solvent
removed in vacuo before further drying on the high-vacuum line to
yield a red-brown solid. The solid was dissolved in dichloro-
methane and on cooling a red powder settled out and was collected
and was then recrystallised with EtOAc to reveal a red powder 1
(0.1344 g,11%). Mp ¼ 288e293 ꢀC (EtOAc); IR ¼ nmax (cmꢂ1) ¼ 3427,
3365, 3111, 2917, 1697, 1593, 1568, 1540, 1472, 1443, 1373, 1352; 1H
1H NMR (300 MHz, CDCl3)
d
8.19 (d, J ¼ 1.1, 1H), 7.53 (d, J ¼ 9.4, 1H),
7.23e7.12 (m, 5H), 7.09e7.02 (m, 2H), 6.88 (d, J ¼ 1.5, 2H), 4.42 (br s,
1H), 3.92 (d, J ¼ 5.3, 2H), 3.83 (s, 3H), 3.75 (s, 3H); 13C NMR (75 MHz,
CDCl3)
d 154.3, 150.2, 140.0, 139.0, 134.0, 128.4, 128.2, 128.0, 127.4,
126.9, 124.0, 122.6, 118.0, 115.6, 113.6, 106.6, 57.2, 55.9, 52.4; HRMS
(m/z) calculated for C22H21BrN3O2, (M þ H), 388.0817, found
438.0806.
NMR (300 MHz, DMSO) d 12.28 (s,1H), 9.65 (s,1H), 9.02 (s,1H), 8.03
(d, J ¼ 8.7, 1H), 7.84 (d, J ¼ 9.2, 1H), 7.50 (d, J ¼ 8.1, 1H), 7.42 (s, 1H),
4.1.1.5. 2-(-3,4-bis(Benzyloxy)phenyl)-6-bromo-N-cyclohexylimidazo
[1,2-a]pyridine-3-amine 5. 2-Amino-5-bromopyridine (0.200 g,
1.156 mmol) was dissolved in dioxane (5 mL). 3,4-
Dibenzyloxybenzaldehyde (0.0405 g, 1.27 mmol), K10 (0.210 g)
and cyclohexyl isocyanide (0.129 g, 1.171 mmol) was added to the
mixture, together with more dioxane (3 mL). The mixture was
stirred at reflux for 6 h and then at rt for 18 h. The product 5
crystallized in the reaction flash and was recrystallised from EtOAc
to afford 5 (0.295 g) as a yellow solid in a yield of 44%.
Mp ¼ 188e190 ꢀC; IR ¼ nmax (cmꢂ1) ¼ 2920, 1580, 1504, 1436, 1403,
6.97 (d, J ¼ 6.2, 2H), 6.77 (t, J ¼ 6.9, 1H), 6.30 (s, 1H), 6.15 (d, J ¼ 7.1,
1H), 1.93 (s, 6H); 13C NMR (75 MHz, DMSO)
d 159.1, 158.3, 139.6,
138.4, 137.0, 134.9, 129.5, 128.6, 126.6, 122.7, 122.1, 121.3, 118.7, 115.4,
107.4, 106.8, 103.0, 18.3; HRMS (m/z) calculated for C21H19N4O4,
(M þ H), 391.1406, found 391.1389.
4.1.1.2. 4-[6-Bromo-3-(2,6-dimethyl-phenylamino)-imidazo[1,2-a]pyr-
idin-2-yl]-benzene-1,3-diol 2. 2-Amino-5-bromopyridine (0.2895 g,
1.67 mmol) was dissolved in 1,4-dioxane (5 mL) in a small round-
bottomed flask. 2,4-dihydroxybenzaldehyde (0.2708 g, 1.96 mmol),
2,6-dimethylphenyl isocyanide (0.2782 g, 2.12 mmol), K10 (0.3365 g)
was added to the flask with further 1,4-dioxane (5 mL). The reaction
was refluxed for 80 h at 110 ꢀC and then worked up as previously
described to afford a yellow-brown solid. The solid was dissolved in
methanol and the yellow solid of 2 that crystallized to the bottom of
the flask was collected (0.5258 g, 74%). Mp ¼ 265e270 ꢀC; IR ¼ nmax
(cmꢂ1) ¼ 3593, 3337, 2920, 1595, 1500, 1473, 1452, 1403, 1329; 1H
1318; 1H NMR (300 MHz, CDCl3)
d
8.24 (s, 1H), 7.72 (d, J ¼ 1.7, 1H),
7.57e7.42 (m, 6H), 7.42e7.28 (m, 6H), 7.18 (dd, J ¼ 9.4, 1.7, 1H), 7.00
(d, J ¼ 8.4, 1H), 5.28 (s, 2H), 5.20 (s, 2H), 3.31 (br s, 1H), 2.91 (s, 1H),
1.81e1.53 (m, 5H), 1.27e1.08 (m, 5H); 13C NMR (75 MHz, CDCl3)
d
149.3, 148.9, 139.6, 137.5, 137.4, 137.1, 128.6, 128.6, 127.9, 127.5,
124.7, 123.0, 120.5, 117.7, 115.1, 114.0, 106.8, 71.4, 71.4, 56.9, 34.2,
25.8, 24.9; HRMS (m/z) calculated for C33H33BrN3O2, (M þ H),
582.1756, found 582.1730.
NMR (300 MHz, MeOD)
d
7.95 (d, J ¼ 1.0, 1H), 7.65 (d, J ¼ 8.6,1H), 7.49
(d, J ¼ 9.4, 1H), 7.36 (dd, J ¼ 9.4, 1.8, 1H), 6.94 (d, J ¼ 7.4, 2H), 6.75 (t,
J ¼ 7.5, 1H), 6.32 (d, J ¼ 2.4, 1H), 6.18 (dd, J ¼ 8.6, 2.4, 1H), 1.94 (s, 6H);
4.1.1.6. [6-Bromo-2-(3,4-dimethoxy-phenyl)-imidazo[1,2-a]pyridin-
3-yl]-(2,6-dimethylphenyl)-amine
(0.3900 g, 2.25 mmol) was dissolved in 1,4-dioxane (3 mL) in
a small round-bottomed flask. 3,4-dimethoxybenzaldehyde
6. 2-Amino-5-bromopyridine
13C NMR (75 MHz, MeOD)
d 159.9, 159.7, 141.5, 138.8, 138.2, 130.7,
129.9, 129.0, 127.2, 123.7, 122.2, 121.8, 117.6, 110.0, 108.3, 107.8, 104.0,
18.7, 18.7; HRMS (m/z) calculated for C21H19BrN3O2, (M þ H),
424.0661, found 424.0645.
(0.3927 g, 2.36 mmol), 2,6-dimethylphenyl isocyanide (0.3108 g,
2.37 mmol), K10 (0.3707 g) were added to the flask with further
1,4-dioxane (3 mL). The reaction was refluxed for 6 h at 110 ꢀC and
stirred for a further 92 h at rt. After work-up of the reaction, as
described above, a brown solid was obtained. The solid was dis-
solved in methanol and on cooling yielded a yellow solid which
was filtered and collected. The product 6 a yellow, crystalline
material was obtained by re-crystallization (CH2Cl2/hexane) in
a yield of 67% (0.6944 g). Mp ¼ 208e210 ꢀC (CH2Cl2/hexane); IR
nmax (cmꢂ1) ¼ 3361, 2960, 1687, 1623, 1584, 1505, 1469, 1411, 1356,
4.1.1.3. [2-(3,4-Dimethoxy-phenyl)-6-methyl-imidazo[1,2-a]pyridin-
3-yl]-(2,6-dimethylphenyl)-amine
(0.2630 g, 2.23 mmol) was dissolved in 1,4-dioxane (3 mL) in
small round-bottomed flask. 3,4-dimethoxybenzaldehyde
3. 2-Amino-5-methylpyridine
a
(0.5555 g, 3.34 mmol), 2,6-dimethylphenyl isocyanide (0.3544 g,
2.70 mmol), K10 (0.2609 g) were added to the flask with further
1,4-dioxane (3 mL). The reaction was refluxed for 8 h at 110 ꢀC and
stirred for a further 88 h at rt, the reaction was then stopped and
worked-up as previously described to yield a brown oil. The oil was
purified by silica gel column chromatography (80% EtOAc/hexane)
to afford an off-white/pink powder of 3 in 42% yield (0.3620 g).
1326; 1H NMR (300 MHz, CDCl3)
J ¼ 8.4, 1.9, 1H), 7.48 (d, J ¼ 9.4, 1H), 7.44 (d, J ¼ 1.8, 1H), 7.21 (dd,
J ¼ 9.4, 1.7, 1H), 6.98 (d, J ¼ 7.5, 2H), 6.85e6.75 (m, 2H), 5.50 (br s,
1H), 3.88 (s, 3H), 3.72 (s, 3H), 2.00 (s, 6H); 13C NMR (75 MHz,
d
7.94 (d, J ¼ 0.9, 1H), 7.58 (dd,

N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
4581
CDCl3):
d
149.0, 149.0, 140.5, 139.7, 139.0, 130.3, 128.0, 125.8, 124.8,
4.1.1.10. 4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridine-2-yl)benzene-
1,3-diol 10. Aminopyridine (0.4812 g, 5.113 mmol), 2,4-
dihydroxybenzaldehyde (0.7768 g, 5.624 mmol), cyclohexyl
isocyanide (0.6194 g, 5.622 mmol) and K 10 (0.48 g) was dissolved in
1,4-dioxane (20 mL). The reaction mixture was refluxed for 30 h at
105 ꢀC. The reaction mixture was worked-up as previously described
and was subjected column chromatography on silica gel (50% EtOAc/
hexane) to give the desired product 10 (0.2480 g, 15%) as a yellow
solid. Mp ¼ 195e200 ꢀC; IR nmax (cmꢂ1) ¼ 3040, 2929, 2853, 2625,
1618, 1589, 1497, 1449, 1385, 1357, 1328, 1315; 1H NMR (300 MHz,
122.6, 121.1, 120.0, 119.9, 117.8, 111.0, 110.0, 107.3, 55.9, 55.7, 18.7,
18.6; HRMS (m/z) calculated for C23H22BrN3O2, (M þ H), 452.0974,
found 452.0975.
4.1.1.7. 2-(2,4-bis(Benzyloxy)phenyl)-3-(cyclohexylamino)imidazo[1,
2-a]pyridine-6-carboxamide 7. 6-Aminonicotinamide (0.500 g,
3.64 mmol), 2,4-bis(benzyloxy)benzaldehyde (1.160 g, 3.64 mmol),
cyclohexyl isocyanide (0.398 g, 3.64 mmol) and K10 (0.50 g) was
dissolved in 1,4-dioxane (20 mL) at rt. The reaction mixture was
refluxed for 40 h at 110 ꢀC while monitoring the reaction progress
with TLC. After work-up of the reaction as described previously, the
residue obtained was subjected column chromatography on silica
gel (ethyl acetate) to furnish a yellow compound which was crys-
tallized from hexane to give pure desired product 2-(2,4-
bis(benzyloxy)phenyl)-3-(cyclohexylamino)imidazo[1,2-a]pyri-
MeOD)
d
8.29 (d, J ¼ 6.8,1H), 7.81 (d, J ¼ 8.5, 1H), 7.49 (d, J ¼ 9.0,1H),
7.35e7.26 (m, 1H), 6.97 (t, J ¼ 6.8, 1H), 6.44 (dd, J ¼ 8.5, 2.4, 1H), 6.39
(d, J ¼ 2.4, 1H), 2.94e2.82 (m, 1H), 1.81e1.49 (m, 6H), 1.29e1.10 (m,
5H); 13C NMR (75 MHz, MeOD)
d 160.1, 157.9, 142.3, 140.0, 136.4,
131.1, 126.5, 124.1, 116.6, 113.6, 112.2, 108.6, 104.6, 58.1, 34.4, 26.8,
25.9; (ESI negative) HRMS (m/z) calculated for C19H20N3O2, (M ꢂ H),
322.1556, found 322.1556.
dine-6-carboxamide
7
(1.70 g, 85%) as
a
yellow solid.
Mp ¼ 112e115 ꢀC; IR nmax (cmꢂ1) ¼ 2924, 1667, 1607, 1504, 1452,
1366; 1H NMR (300 MHz, CDCl3)
d
8.75 (s, 1H), 7.80 (d, J ¼ 9.1, 1H),
4.1.1.11. 2-(2,4-Dimethoxyphenyl)-N-(2,6-dimethylphenyl)-6-nitroimi-
dazo[1,2-a]pyridine-3-amine 11. 2-Amino-5-nitropyridine (0.3010 g,
2.1637 mmol), 2,4-dimethoxybenzaldehyde (0.3955 g, 2.380 mmol),
2,6-dimethylphenyl isocyanide (0.3148 g, 2.3815 mmol) and K 10
(0.30 g) was dissolved in 1,4-dioxane (20 mL) at room temperature.
The reaction mixture was refluxed for 24 h at 110 ꢀC. After work-up
as described previously the residue was subjected column chroma-
tography on silica gel (30% EtOAc/hexane) to give pure desired
7.49e7.27 (m, 12H), 6.81e6.72 (m, 2H), 6.52 (br s, 1H), 5.08 (s, 2H),
5.05 (s, 2H), 3.88 (d, J ¼ 8.1, 1H), 2.71e2.55 (m, 1H), 1.68e1.40 (m,
5H), 1.13e0.83 (m, 5H); 13C NMR (75 MHz, CDCl3)
d 167.8, 159.9,
156.2, 141.7, 136.7, 136.0, 135.0, 132.6, 128.8, 128.7, 128.5, 128.2,
127.8, 127.7, 124.9, 121.6, 118.3, 117.2, 116.4, 107.4, 101.6, 71.7, 70.3,
56.6, 34.0, 25.7, 24.8; HRMS (m/z) calculated for C34H35N4O3,
(M þ H), 547.2709, found 547.2707.
product,
2-(2,4-dimethoxyphenyl)-N-(2,6-dimethylphenyl)-6-
4.1.1.8. 2-(3,4-bis(Benzyloxy)phenyl)-N-cyclohexyl-6-nitroimidazo[1,
nitroimidazo[1,2-a]pyridine-3-amine 11 (0.5832 g, 68%) as a yellow
solid. Mp ¼ 172e176 ꢀC; IR nmax (cmꢂ1) ¼ 2926, 2847, 2660, 1636,
1615, 1575, 1535, 1451, 1429, 1387, 1348; 1H NMR (300 MHz, CDCl3)
2-a]prridin-3-amine
2.700 mmol),
8. Amino-5-nitropyridine
2,4-dibenzyloxybenzaldehyde
(0.3714
(0.8628
g,
g,
2.710 mmol), cyclohexyl isocyanide (0. 2988 g, 2.712 mmol) and
K10 (0.37 g) was dissolved in 1,4-dioxane (18 mL). The reaction
mixture was refluxed for 25 h at 105 ꢀC. The reaction mixture was
worked-up as previously described and was subjected column
chromatography on silica gel (30% EtOAc/hexane) to give the
d
8.61 (s,1H), 7.93 (s,1H), 7.86 (dd, J ¼ 9.7,1.8,1H), 7.71 (d, J ¼ 8.6,1H),
6.97 (d, J ¼ 7.4, 2H), 6.88 (dd, J ¼ 8.3, 6.5, 1H), 6.62 (dd, J ¼ 8.6, 2.2,
1H), 6.52 (d, J ¼ 2.2, 1H), 6.47 (s, 1H), 3.93 (s, 3H), 3.85 (s, 3H), 1.97 (s,
6H); 13C NMR (75 MHz, CDCl3)
d 161.7, 157.5, 140.1, 138.1, 137.0, 135.1,
135.1, 132.0, 129.7, 128.4, 126.0, 123.2, 122.3, 116.9, 116.8, 114.8, 106.0,
99.2, 99.2, 56.2, 55.7, 18.5, 18.5; HRMS (m/z) calculated for
C23H23N4O4, (M þ H), 419.1719, found 419.1736.
desired product 10 (0.6298 g, 43%) as
a orange solid.
Mp ¼ 175e178 ꢀC; IR nmax (cmꢂ1) ¼ 2924, 2852, 1631, 1584, 1537,
1508, 1435, 1347, 1320; 1H NMR (300 MHz, CDCl3)
d
9.21 (d, J ¼ 1.8,
1H), 7.89 (dd, J ¼ 9.8, 2.2, 1H), 7.73 (d, J ¼ 1.8, 1H), 7.64e7.43 (m,
6H), 7.42e7.29 (m, 6H), 7.01 (d, J ¼ 8.4, 1H), 5.28 (s, 2H), 5.21 (s, 2H),
3.48 (br s, 1H), 3.03e2.90 (m, 1H), 1.80e1.55 (m, 5H), 1.29e1.07 (m,
4.1.1.12. 6-Bromo-2(2,4-dimethoxyphenyl)-N-(2,6-dimethylphenyl)
imidazo[1,2-a]pyridine-3-amine 12. 2-Amino-5-bromopyridine
(0.5031 g, 2.88 mmol), 2,4-dimethoxybenzaldehyde (0.4802 g,
2.88 mmol), 2,6-dimethylphenyl isocyanide (0.3790 g,
2.88 mmol) and K10 (0.50 g) was dissolved in 1,4-dioxane (20 mL)
at room temperature. The reaction mixture was refluxed for 30 h
at 110 ꢀC. After work-up as described previously, the residue was
subjected column chromatography on silica gel (30% EtOAc/
hexane) that afforded a light brown/yellow compound which was
crystallized from hexane to give pure desired product 12 (1.104 g,
84%) as a yellow solid. Mp ¼ 159e163 ꢀC; IR nmax (cmꢂ1) ¼ 3288,
1655, 1613, 1568, 1518, 1473, 1439, 1404, 1326, 1302; 1H NMR
5H); 13C NMR (75 MHz, CDCl3)
d 149.5, 149.3, 141.0, 139.5, 137.3,
137.1, 128.7, 128.7, 128.0, 127.5, 127.5, 126.4, 126.2, 123.1, 120.7, 118.6,
116.4, 115.0, 114.1, 71.5, 71.3, 57.2, 34.2, 25.6, 24.9; HRMS (m/z)
calculated for C33H33N4O4, (M þ H), 549.2502, found 549.2506.
4.1.1.9. 4-(3-(2,6-Dimethyl-phenylamino)imidazo[1,2-a]pyridine-2-yl)
benzene-1,3-diol 9. Aminopyridine (0.4270 g, 4.537 mmol), 2,4-
dihydroxybenzaldehyde (0.62811 g, 4.548 mmol), 2,6-dime-
thylphenyl isocyanide (0.60110 g, 4.547 mmol) and K 10 (0.42 g)
was dissolved in 1,4-dioxane (15 mL) at room temperature. The
reaction mixture was refluxed for 10 h at 110 ꢀC. The mixture was
worked up as previously described to afford residue was subjected to
silica gel column chromatography (30% EtOAc/hexane) which affor-
ded a light brown/yellow compound which was crystallized from
hexane to give pure desired product 9 (0.2977 g, 19%) as a yellow
solid. Mp ¼ 245e250 ꢀC; IR nmax (cmꢂ1) ¼ 3348, 2917, 1596, 1524,
(300 MHz, CDCl3)
d
7.56 (d, J ¼ 8.4, 2H), 7.44 (d, J ¼ 9.4, 1H), 7.10
(dd, J ¼ 9.4, 1.7, 1H), 6.90 (d, J ¼ 7.4, 2H), 6.80e6.71 (m, 1H), 6.58
(dd, J ¼ 8.5, 2.2, 1H), 6.49 (d, J ¼ 2.1, 1H), 5.96 (s, 1H), 3.84 (s, 3H),
3.81 (s, 3H), 1.89 (s, 6H); 13C NMR (75 MHz, CDCl3)
d 161.1, 157.4,
139.2, 139.1, 134.3, 131.9, 129.4, 127.0, 126.0, 123.7, 122.0, 121.7,
118.1, 115.9, 106.6, 105.6, 99.0, 99.0, 55.9, 55.5, 18.3, 18.3; HRMS
(m/z) calculated for C23H23BrN3O2, (M þ H), 452.0974, found
452.0963.
1503, 1472, 1447, 1390, 1343, 1301; 1H NMR (300 MHz, MeOD)
d 7.82
(d, J ¼ 6.9,1H), 7.76 (d, J ¼ 8.6, 1H), 7.54 (d, J ¼ 9.0, 1H), 7.34e7.24 (m,
1H), 6.89 (dd, J ¼ 13.0, 7.1, 3H), 6.71 (t, J ¼ 7.5,1H), 6.34 (d, J ¼ 2.4,1H),
6.21 (dd, J ¼ 8.6, 2.4, 1H), 1.94 (s, 6H); 13C NMR (75 MHz, MeOD)
4.1.1.13. 2-(2,4-Dimethoxyphenyl)-N-(2,6-dimethlyphenyl)-6-nitroimi-
dazo[1,2-a]pyridine-3-amine 13. 2-Amino-5-methylpyridine (0.500 g,
4.62 mmol), 2,4-dimethoxybenzaldehyde (0.768 g, 4.62 mmol), 2,6-
dimethylphenyl isocyanide (0.606 g, 4.62 mmol) and K10 (0.5 g) was
dissolved in 1,4-dioxane (20 mL) at room temperature. The reaction
mixture was refluxed for 48 h at 110 ꢀC. The residue after work-up was
d
159.7, 159.7, 142.0, 140.4, 137.5, 130.7, 129.9, 126.9, 126.2, 123.6,
121.8, 121.3, 116.8, 113.8, 110.4, 107.7, 104.0, 18.8, 18.7; (ESI negative)
HRMS (m/z) calculated for C21H18N3O2, (M ꢂ H), 344.1399, found
344.1403.

4582
N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
subjected column chromatography on silica gel (50% EtOAc/hexane)
affording a brown pasty compound which was crystallized from
hexane to give pure desired product 13 as a light brown solid (1.502 g,
84%). Mp ¼ 137e140 ꢀC; IR nmax (cmꢂ1) ¼ 3201, 3001, 2922, 2833,
1596, 1582, 1532, 1467, 1449, 1436, 1411, 1395; 1H NMR (300 MHz,
4.5. Assessment of apoptosis
4.5.1. Annexin V
One of the most important stages of apoptosis involves the
attainment of surface changes by dying cells that would eventually
result in phagocytosis. Many studies have shown that cells under-
going apoptosis lose the phospholipid asymmetry of their plasma
membrane and expose PS, which is translocated to the outer layer
of the membrane. Annexin V is a useful tool in detecting apoptotic
cells since it binds preferentially to negatively charged phospho-
lipids such as PS. The translocation of PS occurs in both necrosis and
apoptosis; hence Annexin V is combined with propidium iodide
(PI). The cell staining was assessed using fluorescein isothiocyanate
(FITC)-labelled Annexin V (green fluorescence) as well as dye
exclusion of PI (negative for red fluorescence). It is possible to
detect and quantitate the apoptotic cells on a single-cell basis by
flow cytometry and to identify the intact cells (FITC-PI-), early
apoptotic (FITCþPI-), late apoptotic or necrotic cells (FITC þ PI) [21].
The Annexin V-FITC apoptosis detection kit (BD Biosciences) was
used to determine whether apoptosis induction had occurred. The
cells were analysed by FACS flow cytometry (BD LSR II flow
cytometer) and the data displayed as a two-colour dot plot with
FITC-Annexin V (green fluorescence, X axis) vs. PI (red fluorescence,
Y axis).
CDCl3)
d
7.57 (d, J ¼ 8.4, 1H), 7.45 (d, J ¼ 9.1,1H), 6.88 (dd, J ¼ 15.5, 4.4,
3H), 6.74e6.65 (m, 1H), 6.56 (dd, J ¼ 8.5, 2.2, 1H), 6.47 (d, J ¼ 2.2, 1H),
5.88(s,1H), 3.80(s,3H), 3.78(s,3H), 2.15(d, J¼ 6.7, 3H),1.87(s,6H); 13C
NMR (75 MHz, CDCl3)
d 160.8, 157.4, 140.1, 139.9, 133.6, 131.8, 129.3,
126.4, 126.0, 122.8, 121.3, 120.9, 119.7, 116.8, 116.5, 105.4, 98.9, 55.8,
55.5, 18.5, 18.3, 18.3; HRMS (m/z) calculated for C24H26N3O2, (M þ H),
388.2025, found 388.2025.
4.1.1.14. [2-(3,4-Dimethoxy-phenyl)-6-phenyl-imidazo[1,2-a]pyridin-
3-yl]-(2,6-dimethyl-phenyl)-amine 14. 6-Bromo-2-(3,4-dimethoxy-
phenyl)-N-(2,6-dimethylphenyl)imidazo[1,2-a]pyridin-3-amine
6
(0.25 g, 0.55 mmol), phenylboronic acid (0.101 g, 0.82 mmol),
Pd(PPh3)4 (0.064 g, 10 mol%) and CsF (0.209 g, 1.38 mmol) was
dissolved in DME (3 mL) in microwave tube under argon. The
reaction mixture was irradiated in a Discovery microwave at 150 ꢀC
and 150 W for 20 min. The reaction was concentrated and subjected
to column chromatography on silica gel (30e50% EtOAc/hexane) to
give desired product 14 (0.188 g, 95%) as a crimson red solid.
Mp ¼ 158e160 ꢀC (methanol); IR nmax (cmꢂ1) ¼ 2929, 1636, 1587,
1509, 1470, 1408; 1H NMR (300 MHz, CDCl3)
d 7.86 (s, 1H), 7.60 (dd,
J ¼ 18.7, 8.6, 2H), 7.42 (d, J ¼ 23.3, 6H), 6.96 (d, J ¼ 7.1, 2H), 6.76 (d,
4.5.2. Colorimetric assay for caspase detection
J ¼ 8.0, 2H), 5.50 (s, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 2.01 (s, 6H); 13C
The activation of caspases initiates apoptosis in mammalian
cells. Caspase 3 and caspase 8 activity were detected using the
CPP32 and FLICE colorimetric assay kits, respectively. Treated cells
were lysed, and the cell lysate was then tested for caspase 3 and
caspase 8 activity by the addition of a caspase-specific peptide
conjugated to the chromophore p-nitroaniline (pNA). Time-
dependent studies were performed for protease activity by
obtaining results 2, 4, 6, 12, and 24 h after treatment with the
compounds.
NMR (75 MHz, CDCl3) d 148.8, 148.6, 140.9, 140.7, 138.7, 137.3, 130.1,
129.1, 127.7, 126.7, 126.5, 126.3, 124.9, 124.7, 120.8, 120.1, 119.7, 119.4,
116.9, 110.9, 109.9, 55.7, 55.6, 18.6, 18.6; HRMS (m/z) calculated for
C29H28N3O2, (M þ H), 450.2182, found 450.2185.
4.2. Biological cell lines
The study made use of two colonic carcinoma cell lines, namely
the HT-29 and Caco-2 (Highveld Biological, South Africa). The cells
were maintained in Dulbecco’s Modified Eagles Medium (DMEM)
(Highveld Biological, South Africa) supplemented with 5% heat-
inactivated foetal bovine serum (FBS), and incubated at 37 ꢀC in
a CO2 humidified incubator.
4.5.3. Mitochondrial membrane potential
The fluorescent mitochondrial-specific cationic dye JC-1 was
used according to the manufacturer’s specification to measure the
collapse of the electrochemical gradient across the mitochondrial
membrane.
Camptothecin is
a S-phase-specific anticancer agent that
inhibits the activity of the enzyme DNA topoisomerase 1. It was
used as a positive control in the study as several studies have shown
the effectivity of camptothecin to induce apoptosis in a dose-
dependent manner in vitro [23].
The cells were analysed by flow cytometry using excitation/
emission filters of 485/540 nm (green/FL-1); 540/590 nm
(red/FL-2).
4.5.4. Detection of the release of cytochrome c determination
A quantitative concentration of cytochrome c in the cell lysates
was determined by a human cytochrome c Titerzyme Enzyme
Immunometric Assay. The kit uses a monoclonal antibody specific
for cytochrome c, a biotinylated detecting antibody and alkaline
phosphatase-conjugated streptavidin to provide a colorimetric
detection that enables a quantitative determination of cytochrome
c in the cell lysates. The treated HT-29 and Caco-2 cells were har-
vested and rinsed with ice-cold phosphate buffered saline (3.2 mM
Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl and 135 mM NaCl, pH 7.4).
The cytosolic and mitochondrial fractions were isolated according
to the protocol provided by the Mitochondria isolation kit (Assay
Designs, Inc.)
4.3. Determining cell viability via the MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay [24] was used to determine cell viability of the
two cell lines on exposure to the derivatives for 24 h. The cells were
seeded at a density of 30 000 cells/well. Formazan crystals were
dissolved in 200
using the Absorbance Labsystems Multiskan MS, version 2.4.
Compounds showing cytotoxicity at a concentration of 100
ml DMSO. The absorbance was read at 540 nm
mM
were further diluted to obtain an IC50 concentration. All tests were
performed in triplicate.
4.4. Collection of whole blood and isolation of white blood cells
from humans
4.5.5. Statistics
Significant differences between the control and experimental
samples were determined using the Prism3 Instat package, using
ANOVA, Student-t test. The values are expressed as
mean ꢁ standard error of the mean. Significance was set at
a confidence interval of 95% (p < 0.05).
The isolation of human peripheral white blood cells (WBC) was
adapted from the Versagene Blood DNA Kit according to the
manufacturer’s protocol (Gentra Systems). Ethics clearance no:
M070519, University of the Witwatersrand.

N. Dahan-Farkas et al. / European Journal of Medicinal Chemistry 46 (2011) 4573e4583
4583
[11] (a) A. Doming, I. Ugi, Multicomponent reactions with isocyanides, Angew.
Chem. Int. Ed. 39 (2000) 3168e3210;
Acknowledgements
(b) J. Zhu, H. Bienaymé (Eds.), Multicomponent Reactions, Wiley-VCH,
Weinheim, 2005.
[12] For a short review see C. Enguehard-Gueiffier, A. Gueiffier, Recent progress in
the pharmacology of imidazo[1,2-a]pyridines, Mini-Rev. Med. Chem. 7 (2007)
888e899.
[13] A.R. Katritzky, Y.-J. Xu, H. Tu, Regiospecific synthesis of 3-substituted imidazo
[1,2-a]pyridines, imidazo[1,2-a]pyrimidines, and imidazo[1,2-c]pyrimidine,
J. Org. Chem. 68 (2003) 4935e4937 and references therein.
[14] A.L. Rousseau, P. Matlaba, C.J. Parkinson, Multicomponent synthesis of imi-
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4079e4082.
This work was supported by the National Research Foundation,
Pretoria, (NRF, GUN 2053652 and IRDP of the NRF (South Africa) for
financial support provided by the Research Niche Areas pro-
gramme), the University of the Witwatersrand (Science Faculty
Research Council) and the Mellon Foundation. The sponsors played
no role in study design; in the collection, analysis and interpreta-
tion of data; in the writing of the report; and in the decision to
submit the paper for publication. CL thanks for the Mellon Foun-
[15] K.S. Gudmundssen, B.A. Johns, Imidazo[1,2-a]pyridines with potent activity
against herpesviruses, Bioorg. Med. Chem. Lett. 17 (2007) 2735e2739.
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class of drug-like Mycobacterium tuberculosis glutamine synthetase inhibitors,
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dation for a postgraduate Mellon Fellowship. Mr
R Mampa
(University of the Witwatersrand) and Mr B Moolman and Dr M
Stander (University of Stellenbosch) are thanked for providing the
NMR and HRMS MS spectroscopy services, respectively.
[17] R.B. Lacerda, C.K. de Lima, L.L. de Silva, N.C. Romeiron, A.L. Miranda,
E.J. Barreiro, C.A. Frago, Discovery of novel analgesic and anti-inflammatory 3-
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