D. Güçlü et al.
Bioorganic&MedicinalChemistryLetters28(2018)2647–2651
Table 2
Theoretically determined values of Log BB and Caco-2 permeability for imidazopyridines 5a–5i.
Tests
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.419
8340
0.794
8351
0.644
8369
0.290
8340
0.176
4718
0.284
8340
1.012
8370
0.411
8342
0.647
8347
Caco-2 Permeability (nm/s)b
a
The appropriate range for Log BB is between −3.0 and 1.2.
Higher than 500 nm/s means that the compound can be absorbed by human intestinal system.
b
IC50 of 0.6 and 7.8 μM, respectively (Fig. 1).21 Herein, we therefore
describe the discovery and development of a series of imidazopyridines
that demonstrate toxicity against cancer cell lines, LN-405. We used the
MTT test to identify the cytotoxicities of the prepared compounds in the
first stage of the cell study.22 To test the antitumor activity, we eval-
uated the activity of compounds 5a–i against a glioblastoma cell line
(LN-405); the results are summarized in Fig. 2. Following the interac-
tions between compounds and the cells, the results revealed that the
most potent molecules were 5a and 5i, which exhibited potent cyto-
toxicity activity against the LN-405 cell line. Compounds 5a and 5i
exhibited high activity at 100 µM. Conversely, compounds 5b, 5c, 5f,
5g, and 5h at 100 µM did not present any cytotoxicity in the LN-405 cell
lines. On the other hand, the cell viability with compound 5d at 100 µM
was 80%. Compounds 5a and 5i showed stronger effects with cell vi-
abilities of 6% and 32%, respectively (Fig. 2). Owing to the fact that
compound 5e was crystallized in the cell growth media, it was not
possible to determine the effect of this compound on cell viability.
Compounds 5a and 5i were thus selected as lead compounds and
their IC50 values were determined to be 10 µM for 5a and 75 µM for 5i
by screening at concentrations of 100, 75, 50, 25, 10, and 1 µM (Fig. 3).
The compounds containing phenyl (5a) and para-fluorophenyl (5i)
rings exhibited biological activity against LN-405 cell lines. We have
suggested that aromatic rings show their effects via π–π bonding.23
Even though some of the other prepared compounds, such as 5b, 5d, 5f,
and 5h, have one more phenyl ring or substituents on the para position,
they did not demonstrate a noteworthy cytotoxicity affect. For example,
para-biphenyl (5f), β-naphthyl (5h), or para-bromo phenyl (5b) sub-
stituted molecules exhibited the same results as seen in the control. It
was assumed that size of substituent at para-position of phenyl ring did
affect antiproliferation on LN-405 cell line. Relation between size and
activity can be found as H > F > OMe > Br = Ph. Additionally,
imidazopyridines derivatized with thiophene (5c and 5g) did not pre-
sent any antiproliferation effects, as seen in some of the phenyl-sub-
stituted compounds, and the aforementioned results might be caused by
a poor fit between the active site of the target and the synthesized
molecules.
To determine whether the synthesized molecules cause DNA da-
mage, the comet assay was performed only for compounds 5a and 5i.
Cell number for control, 5a, and 5i are 1026, 1180, and 1014, respec-
tively. The results showed that only compound 5a led to statistically
significantly more DNA damage compared to the control groups
(p < 0.001) in which compound 5a affected 14% of cells. Therefore,
we can predict that the toxicity of compound 5a was owing to DNA
damage and the other compounds have no DNA-damaging effects.
Comparison between 5i and control (C) did not give statically positive
result (P > 0.05). So, the comet assay results indicated that the DNA-
damaging effect of compound 5i was not noteworthy (Table 1).
Antiproliferative and DNA-damaging effects were determined for
lead compounds. It is very vital to find out the safety of lead com-
pounds. Hence, we have tested the cytotoxicity of compound 5a and 5i
on WS1 (skin fibroblast) cell line which are normal and non-cancerous
cell. We have carried out MTT method to calculate IC50 values of
compound 5a and 5i on WS1, determined as 480 and 844 µM, respec-
tively (Fig. 4). With this information, effective concentrations of lead
compounds 5a and 5i can be found as safety on normal cell when
cancer cell affects.
After carrying out the MTT and comet assays, flow cytometry was
performed to detect the effects of the synthesized compounds on the
cell cycle. These compounds were added to the cell media at the pre-
viously determined IC50 values and incubated for 72 h to determine
their real cell cycle and early and real apoptotic effects; the results are
shown in Fig. 5. The flow cytometry results show that the percentages
of the LN-405 cell population in a GO/G1 state are almost the same for
both compounds at 99.2% for 5a and 98.2% for 5i. While a very low
percentage of the cells incubated with 75 µM of 5i were able to reach
the S phase, none of the cells incubated with 10 µM of 5a were detected
in the S phase. This means that 5a and 5i effectively prevented DNA
duplication. A statistically significant difference was found between the
control and 5a, the control and 5i, and 5i and 5a groups in terms of cell
populations at cell cycles (p < 0.001). The flow cytometry results
showed that both of the lead compounds 5a and 5i might have anti-
proliferative effects on the LN-405 cell line. These antiproliferative ef-
fects could be achieved via several mechanisms, such as disruption of
the control of cell cycle regulator proteins or increased/decreased ex-
pression of tumor suppressor genes/proteins and other genetic factors
that participate in the cell proliferation process.
Glioblastoma is a type of brain cancer and any chemotherapeutic
drug that is taken orally should be absorbed in the gastrointestinal
system and pass through the blood–brain barrier. To determine these
vital parameters of the synthesized compounds theoretically, we uti-
lized Schrödinger (QikProp) software to calculate the Log BB (blood–-
brain barrier) and Caco-2 permeability (to predict human intestinal
permeability).25 According to the software data, analyzing 95% of
drugs showed that the range for Log BB is between −3.0 and 1.2.
Furthermore, if Caco-2 permeability exceeds 500 nm/s, the molecule
can easily pass through this membrane. The Log BB values for the
imidazopyridines 5a–5i were found to be between 0.284 and 1.012.
The predicted Log BB for compounds 5a and 5c, which do not contain
bromine, were 0.419 and 0.644, respectively, while similar skeletons
containing a bromine atom (5b and 5 g) gave Log BB of 0.794 and
1.012, respectively. Davis and co-workers have found that chloro- and
bromo-substituted peptides enhanced the lipophilicity and passage
across blood brain barrier.26 The Caco-2 permeability values were
found to range between 8340 and 8370, except for 5e, which has a
single phenyl ring and a predicted Caco-2 permeability of 4718. The-
oretical values indicated that synthesized compounds 5a–i should pass
through the blood–brain barrier. Furthermore, Caco-2 permeability
predicted that all of the synthesized molecules can be administered
To sum up, various imidazopyridine derivatives bearing different
functional groups have been synthesized and their cytotoxicities against
a glioblastoma cell line (LN-405) were investigated experimentally.
Two lead compounds with IC50 values of 10 and 75 µM were identified
for glioblastoma. These lead compounds were subjected to comet assay
to determine their genotoxicities; DNA damage was caused by com-
pound 5a, which possesses two unsubstituted phenyl rings. The two
lead compounds were analyzed by flow cytometry to determine their
effects on the cell cycle of LN-405 cells. It was found that the lead
compounds prevented cells from passing the synthesis phase.
Furthermore, theoretical parameters, Log BB, and Caco-2 permeability
showed that the lead compounds can be administered orally.
Pharmacomodulation of these compounds and their structure–activity
2650