Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones as potential cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2020.127432

The new derivatives based on (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-one scaffold was synthesized and evaluated for their in vitro cytotoxic potential against a panel of cancer cell lines, viz., A549 (human lung cancer), HCT-116 (human colorectal cancer), B16F10 (murine melanoma cancer), BT-474 (human breast cancer), and MDA-MB-231 (human triple-negative breast cancer). Among them, many of the synthesized compounds exhibited promising cytotoxic potential against the panel of tested cancer cell lines with IC₅₀ <30 μM. Based on the preliminary screening results, the structure-activity relationship (SAR) of the compounds was established. Among the synthesized compounds, 15i displayed a potential anti-proliferative activity against HCT-116 cancer cell line with an IC₅₀ value of 1.21 ± 0.14 μM. Flow cytometric analysis revealed that compound 15i arrested the G0/G1 phase of the cell cycle. Moreover, increased reactive oxygen species (ROS) generation, clonogenic assay, acridine orange staining, DAPI nuclear staining, measurement of mitochondrial membrane potential (ΔΨm), and annexin V-FITC assays revealed that compound 15i promoted cell death through apoptosis.

Full text of DOI:10.1016/j.bmcl.2020.127432

Journal Pre-proofs
Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-
yl)prop-2-en-1-ones as potential cytotoxic agents
Geeta Sai Mani, Pratibha Anchi, Satish Sunkari, Kavitha Donthiboina,
Chandraiah Godugu, Nagula Shankaraiah, Ahmed Kamal
PII:
S0960-894X(20)30543-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127432
BMCL 127432
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
27 May 2020
18 July 2020
20 July 2020
Please cite this article as: Sai Mani, G., Anchi, P., Sunkari, S., Donthiboina, K., Godugu, C., Shankaraiah, N.,
Kamal, A., Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones as potential
cytotoxic agents, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127432
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones
as potential cytotoxic agents
Geeta Sai Mani,a Pratibha Anchi,b Satish Sunkari,c Kavitha Donthiboina,a Chandraiah Godugu,b* Nagula
Shankaraiah,a* Ahmed Kamala,c,d
*
aDepartment of Medicinal Chemistry, bDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad
500037, India
cMedicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad-500007, India.
dSchool of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi 110062, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The new derivatives based on (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-
en-1-one scaffold was synthesized and evaluated for their in vitro cytotoxic potential against a
panel of cancer cell lines, viz., A549 (human lung cancer), HCT-116 (human colorectal cancer),
B16F10 (murine melanoma cancer), BT-474 (human breast cancer), and MDA-MB-231 (human
triple-negative breast cancer). Among them, many of the synthesized compounds exhibited
promising cytotoxic potential against the panel of tested cancer cell lines with IC50 <30 µM.
Based on the preliminary screening results, the structure-activity relationship (SAR) of the
compounds was established. Among the synthesized compounds, 15i displayed a potential anti-
proliferative activity against HCT-116 cancer cell line with an IC50 value of 1.21±0.14 µM.
Flow cytometric analysis revealed that compound 15i arrested the G0/G1 phase of the cell cycle.
Moreover, increased reactive oxygen species (ROS) generation, clonogenic assay, acridine
orange staining, DAPI nuclear staining, measurement of mitochondrial membrane potential
(ΔΨm), and annexin V-FITC assays revealed that compound 15i promoted cell death through
apoptosis.
Keywords:
Imidazo[1,5-a]pyridines
Cytotoxicity
Cell cycle analysis
Annexin-V
Mitochondrial membrane potential
Cancer can be considered as one of the hyper-proliferative
diseases characterized by the deregulation of cell functioning,
which eventually modifies the cell death causing apoptosis.
Apoptosis is an important physiological process involved in
tissue homeostasis and its inappropriate regulation or disruption
leads to numerous diseases including cancer.1 Various
transcription factors are involved in the apoptotic pathway and
therefore, anticipated as effective anticancer drug targets.2 Thus,
compounds are designed to induce apoptosis in cancer cells by
targeting these specific factors involved in apoptosis.3,4 In this
regard, many drugs are available based on anti-proliferative
activity. However, most of the currently available drugs usually
act on rapidly dividing metabolically active cells and suffer poor
selectivity between normal and cancer cells. Moreover, high
toxicity and poor tolerance are the major problems associated
with currently available anticancer drugs. Therefore, the
discovery and development of new chemical entities which are
more selective towards cancerous cells that particularly inhibit
key mechanisms involved in the progression and development of
tumor.5 In this regard, various small synthetic molecules are
currently being investigated as newer anticancer agents.6
approved drugs contain a nitrogen-bridged fused heterocycles.7
Among these heterocycles, imidazoles represent a significant
class of heterocyclic molecules with a promising biological and
pharmaceutical importance. They are found to be common
moieties in highly important biomolecules such as histamine,
biotin, and in numerous alkaloids that displays a wide spectrum
of biological properties. One of the interesting and most widely
used heterocyclic motifs from this category is imidazopyridine,8 a
bicyclic N-fused imidazole which was known to exhibit a broad
spectrum of biological activities for various targets such as
positive inotropic agents,9 thromboxane synthase inhibitors,10
antiviral,11 antibacterial,12 estrogen production suppressors,13
anxioselective and hypnoselective agents.14 Moreover,
imidazopyridines display cytotoxic activity through various
molecular mechanisms, for instance through the inhibition of
vascular endothelial growth factor (VEGF)-receptor KDF
(Kinase insert domain receptor) and through the initiation of
apoptosis.15 Recently, we have reported the synthesis of
imidazopyridine-linked oxadiazoles (1),16 imidazopyridine
oxindoles (2),17 imidazopyridine-linked benzimidazoles (3),18
bisindole-imidazopyridine derivatives19 as potential cytotoxic
agents as shown in Figure 1.
Next, nitrogen-containing heterocycles are the most important
structural components in many pharmacologically active
molecules. According to the U.S.FDA database 59% of the
Moreover,
numerous
small
molecules
containing
arylaminoprop-2-en-1-ones are reported to possess anticancer
activity as shown in Figure 1. In continuation to our earlier
efforts, we have designed and synthesized a series of (Z)-
(diphenyl pyrazolyl)-2-propenones (5),20 (Z)-(arylamino)-
Authors to whom correspondence should be addressed. Corresponding
authors; Dr. Ahmed Kamal, E-mail: ahmedkamal@iict.res.in;
Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in;
Dr. Chandraiah Godugu, E-mail.: chandra.niperhyd@gov.in
pyrazolyl/isoxazolyl-2-propenones,21
benzo[d]imidazo[2,1-
1

b]thiazole-propenones
(6)22
and
2-anilinopyridine-
in a sequential step manner, the first step involves a nucleophilic
amination of 2-pyridyl-methanamine (7) on substituted benzoyl
chlorides 8a-d in the presence of triethylamine to deliver N1-(2-
pyridylmethyl)-substituted benzamides 9a-d which were cyclized
into the corresponding imidazo[1,5-a]pyridines 10a-d by
refluxing in POCl3. The imidazo[1,5-a]pyridine derivatives 10a-
d were formylated under Vilsmeier-Haack conditions with POCl3
in dry DMF provided 3-(substituted phenyl)imidazo-[1,5-
a]pyridine-1-carbaldehyde intermediates 11a-d.18 Later, these
aldehydes were treated with ethynyl magnesium bromide to
afford 1-aryl-2-propyn-1-ols 12a-d. Next, oxidation of 12a-d
with 2-iodoxybenzoic acid (IBX) in the presence of dimethyl
sulfoxide (DMSO) gave 1-aryl-2-propyn-1-ones 13a-d.20 Finally,
condensation of 13a-d with different aryl amines 14a-g in
ethanol at room temperature provides (Z)-3-(arylamino)-1-(3-
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones 15a-r in
good to excellent yields (68-85%, Scheme 1).
arylpropenones23 as effective apoptosis-inducing and potential
cytotoxic agents. Furthermore, these studies also revealed that the
inclusion of propen-1-one moiety into the molecules has resulted
in compounds with promising cytotoxic activity. For instance,
(Z)-1-aryl-3-arylamino-2-propen-1-ones (4)24 exhibited effective
cytotoxicity towards various tested cancer cell lines with anti-
tubulin activity by arresting the G2/M phase of the cell cycle.
Cl
H
F
O
N
CF3
N
NH
O
N
N
O
NH
O
NH
O
O
N
N
N
N
OH
N
F
Br
F
O
O
O
1
2
3
4
O
GI50 = 1.30 µM
GI50 = 3.46 µM
GI50 = 1.37 µM
IC50 = 0.06±0.05 µM
(Prostate cancer cell line)
NH
(CNS cancer cell line) (Lung cancer cell line) (Breast cancer cell line)
R2
R
F
O
O
S
H
O
NH2
N
N
O
O
N
N
Cl
N
DMF, POCl3
N
N
N
POCl3
7
OH
N
O
NH
0 oC to 100 oC, 3 h
(76-83%)
HN
R
TEA, dry THF,
0 oC to rt, 3 h
(76-82%)
reflux, 2 h
(76-80%)
N
8a
; R = CF3
; R = F
; R = H
N
NH
N
8b
8c
8d
R
HN
10a-d
11a-d
R1
R
; R = OCH3
5
6
9a-d
15a-r
ethynylmagnesium bromide
THF, 0 oC to rt, 8-9 h
(60-70%)
IC50 = 1.6±0.1µM
(Cervical cancer cell line)
IC50 = 1.5±0.45 µM
(Lung cancer cell line)
R1
O
O
OH
HN
R1-NH2
14a-g
Figure 1. Chemical structures and anticancer activities of imidazopyridines
1–3, aminoprop-2-en-1-one derivatives 4–6 and (Z)-3-(arylamino)-1-(3-
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones 15a–r.
N
N
N
2-iodoxybenzoic acid
N
N
N
DMSO, rt, 5 h
(70-72%)
ethanol, rt, 4 h
(68-85%)
15a-r
R
R
12a-d
13a-d
R
The development of small molecules through molecular
hybridization from known structural motifs is one of the current
trends in drug discovery. It is anticipated that the improved
cytotoxicity may be obtained by the structural conjugation of two
potent pharmacophoric units. Hence, herein we have designed
; R = F, R1 = 2-methyl 5-indolyl
; R = CF3, R1 = 4F-C6H4
15j
15a
15b
15c
15d
15e
15f
; R = F, R1 = 4OCH3-C6H4
; R = CF3, R1 = 4Cl-C6H4
; R = CF3, R1 = 5-indolyl
; R = CF3, R1 = 6-indolyl
; R = CF3, R1 = 3-quinoline
15k
; R = F, R1 = 3,4-dimethoxy-C6H3
15l
; R = H, R1 = 3-quinoline
15m
15n
; R = H, R1 = 4OCH3-C6H4
; R = OCH3, R1 = 4F-C6H4
; R = OCH3, R1 = 4Cl-C6H4
; R = OCH3, R1 = 5-indolyl
; R = CF3, R1 = 4OCH3-C6H4
; R = CF3, R1 = 3,4-dimethoxy-C6H3
; R = F, R1 = 4F-C6H4
15o
15p
15q
15r
and synthesized
a
new series of imidazopyridyl-linked
15g
15h
15i
arylaminoprop-2-en-1-ones by replacing oxadiazole group of
imidazopyridine-linked oxadiazole derivatives16 (1) with 3-
arylamino-2-propen-1-one group of (Z)-1-aryl-3-arylamino-2-
propen-1-ones24 as shown in Figure 2. Thus, a new series of
compounds related to this template with diverse substituents on
both biologically active pharmacophoric subunits have been
synthesized.
; R = OCH3, R1 = 4OCH3-C6H4
; R = F, R1 = 4Cl-C6H4
Scheme 1. Schematic representation for the synthesis of imidazopyridine-
linked 3-arylaminoprop-2-en-1-ones 15a-r.
All the synthesized compounds 15a-r were assessed for their
in vitro cytotoxic potential against a panel of selected cancer cell
lines, viz., A549 (human lung cancer cell line), HCT-116 (human
colorectal cancer cell line), B16F10 (murine melanoma cancer
cell line), BT-474 (human breast cancer), and MDA-MB-231
(human triple-negative breast cancer cell line) using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.25 The obtained values were compared to the standard drug
nocodazole as shown in Table 1 and expressed in IC50 values.
Interestingly, the screening results revealed that the compounds
15b, 15c, 15f, 15h, 15i, 15k, 15n, and 15q displayed a potential
cytotoxic activity (IC50 <30 µM) against all the tested cell lines.
Based on the acquired cytotoxicity assay results on different
cancer cell lines, a structure-activity relationship (SAR) is
established. The effect of different groups on the imidazo[1,5-
a]pyridine ring as well as aryl/heteroaryl amine-linked to prop-2-
en-1-one was demonstrated by synthesizing the compounds
containing both electron-withdrawing and electron-donating
substituents. Compounds containing H, F atoms, and CF3, OMe
groups on the phenyl group linked to imidazo[1,5-a]pyridin-1-yl
residue showed superior cytotoxic activity. On the other hand,
arylamine residue containing compounds such as 15a, 15b, 15f,
15g, 15h, 15i, 15k, 15l, 15n, 15o, 15p, and 15r (Table 1)
displayed good anticancer effects against the selected cancer cell
lines compared to heteroaryl amines except the compounds
containing 5-indolylaryl group (15c and 15q). The activity of the
Cl
F
N
N
O
R1
HN
N
N
O
1
N
N
Conjugation
O
O
+
O
O
NH
O
Apoptosis
R
O
O
New conjugates 15ar
OH
Br
4
O
Figure 2. The conjugation of scaffolds 1 and 4 leads to new series of (Z)-3-
(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones 15a–r.
The imidazopyridine-linked 3-arylaminoprop-2-en-1-one
conjugates 15a–r were synthesized by employing a general
synthetic approach as illustrated in Scheme 1. The target
compounds were synthesized from various substituted 1-(3-
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-yn-1-ones 13a-d and
different substituted anilines 14a-g. The key intermediates such
as imidazo[1,5-a]pyridinyl propynones 13a-d were synthesized
2

compounds containing 3-quinolinyl, 6-indolyl aryl groups
showed decreased activity against all the tested cancer cell lines.
The introduction of the methyl group on 2-position of the
indolylaryl group (15j) showed decreased cytotoxicity. It was
noted that the introduction of the chloro group on the aryl amine-
linked to prop-2-en-1-one (15b, 15i, and 15p) displayed potent
cytotoxic activity against all the tested cancer cell lines followed
by a methoxy group (15f, 15n, and 15r). However, the
introduction of an additional OMe group on the meta position of
aryl amine-linked to prop-2-en-1-one (15l) showed a significant
reduction in potency. Moreover, compounds bearing the fluoro
group on para-position of arylamino prop-2-en-1-one display
reduced cytotoxicity. Among all the synthesized compounds 15c,
15i, and 15q exhibited potent cytotoxic activity against all the
tested cancer cell lines. Based on these observations, structure-
activity relationship (SAR) of (Z)-3-(arylamino)-1-(3-
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones was derived
and depicted as shown in Figure 3.
Based on acquired cytotoxicity results, the most potent
compound 15i was selected as a model substrate against HCT-
116 cell line to carry further experiments to identify the
mechanism of cancer cell growth inhibition
Many cytotoxic compounds displayed their cytotoxicity by
arresting at a particular checkpoint of the cell cycle or through
the induction of apoptosis or by a collective effect of both cell
cycle arrest and apoptosis.26 In vitro screening results revealed
that compound 15i exhibited significant cytotoxic activity against
human colon cancer cell line (HCT-116). Hence, it was
considered of interest to know whether this cell growth inhibition
was due to cell cycle arrest or not. In the present study, HCT-116
cells were treated with the potential conjugate 15i at a
concentration of 0.5, 1.0, 2.5 µM for 48 h. Flow cytometric
analysis revealed that compound 15i arrested G0/G1 phase of the
cell cycle in a dose-dependent manner i.e. the ratio of HCT-116
cells increased in G0/G1 phase from 28.2% at control, 31.5% at
0.5 µM, 38.7% at 1 µM and 41.9% at 2.5 µM as shown in Figure
4. Therefore, the acquired results concluded that compound 15i
produces cytotoxicity by inducing G0/G1 phase of cell cycle
arrest.
Table 1. In vitro cytotoxicity (IC50 in µM) for the compounds 15a–r
on different cancer linesa
Compound A549b
HCT-
116c
B16F10d
BT-474e
MDA-
MB-231f
15a
>30
>30
>30
>30
>30
15b
15c
18.48±2.74
5.19±0.30
>30
11.52±0.50
27.14±1.45
>30
>30
3.82±0.93
5.19±0.06
>30
3.84±0.72
1.18±0.066
>30
18.20±0.96
>30
15d
15e
>30
>30
>30
>30
>30
15f
21.83±0.14
>30
7.72±1.25
>30
>30
>30
>30
15g
>30
>30
>30
15h
15i
13.18±0.39
5.09±1.32
>30
>30
>30
16.94±1.64
7.03±1.73
>30
25.11±1.58
4.25±0.37
>30
1.21±0.14
>30
11.00±1.74
>30
15j
15k
15l
19.15±4.41
>30
>30
2.56±0.08
>30
15.67±5.25
>30
>30
>30
>30
15m
15n
15o
>30
>30
3.78±0.13
1.79±0.39
>30
19.70±1.05
12.80±0.33
>30
12.28±0.49
28.25±5.86
>30
Figure 4. Cell cycle analysis of HCT-116 cells upon treatment with
compound 15i by flow cytometry. DNA histogram depicts various stages of
cell cycle i.e., SubG1, G0/G1, S, G2/M. Each phase of treated cell cycle
results was evaluated with DMSO control cells, cell cycle analysis. 10,000
events were examined for the results.
>30
7.01±0.42
>30
>30
15p
15q
15r
ND
>30
1.24±0.17
5.12±0.33
2.12±0.22
3.12±0.77
37.32±0.89
12.98±0.25
15.42±0.10
2.72±1.57
>30
4.80±1.03
ND
17.4±1.09
>30
28.69±0.59
>30
The proliferative potential and ability of adhered cells to form
colonies from a single cell to multicellular solid tumors can be
evaluated using the colony formation assay. Therefore, it is the
method to determine the effectiveness of cytotoxic molecules
based on cell reproductive death and cell survival. In this assay,
the colony-forming ability of HCT-116 cells was measured by
treating media with various concentrations (0.25, 0.5, and 1 μM)
of compound 15i over for 10 days to form colonies. Cells were
then stained with crystal violet to visualize the colonies. The
Nocodazole
3.63±0.24
2.82±1.32
1.83±0.40
a50% inhibitory concentration after 48 h of drug treatment and the values are
averages of three individual experiments, bhuman lung cancer cell line,
chuman colorectal cancer cell line, dmurine melanoma cancer cell line,
ehuman breast cancer cell line, human triple-negative breast cancer cell line.
f
ND: Not Determined.
O
R1
HN
results concluded that there is
a concentration-dependent
N
inhibition of colony formation by 15i on HCT-116 cells. Hence,
this study confirmed that compound 15i was efficient in
inhibiting the colony formation even at a low concentration and
maximum colonies disappeared at 1 µM concentration as shown
in Figure 5.
Cytotoxicity: Aryl>Heteroaryl
N
Aryl: Cl>OMe>H>F
Heteroaryl: 5-indolyl>3-Quinoline>6-
indolyl
better activity when
R = CF3, F, H and OMe
R
Figure 3. SAR of (Z)-3-(arylamino)-1-(3-phenylimidazo[1,5-a]pyridin-1-
yl)prop-2-en-1-ones.
3

Figure 5. Effect of conjugate 15i on the colony-forming capability of HCT-
116 cells. (i) a) Control cells (HCT-116), b) 15i (0.25 µM), c) 15i (0.5 µM),
d) 15i (1 µM), (ii) Bar graph displaying the number of colonies remained
following treatment of the cells through increasing concentration of the
conjugate 15i.
To examine whether the treatment with compound 15i could
lead to loss of cell viability and stimulate apoptosis, HCT-116
cells were treated with 0.5, 1, and 2.5 µM concentrations of
compound 15i. Upon 48 h post-treatment, cells were observed
through
a
phase-contrast microscope and representative
Figure 7. Acridine orange (AO) staining in HCT-116 cell line displaying
apoptosis. HCT-116 cells were treated with increased concentrations of
compound 15i and stained with acridine orange. Reduced viable cells and
apoptotic features were noticed at 200× magnification.
photographs were taken as shown in Figure 6. Results revealed
that compound 15i treated cells show a visible difference in
morphology and viability. Based on the results, we concluded
that as a concentration of the compound increased, there is a
distinct decrease in cell viability with significant morphological
changes such as cell shrinkage and detachment of cells from the
substratum.
DAPI is a blue coloured fluorescent dye that stains ds DNA
by strongly binding to A-T rich sequence of nucleic acid in a
rigid manner. Nuclear modifications that were formed during
apoptosis such as condensed nuclei, nuclear-fragmented bodies
can be visualized through DAPI staining.29 To investigate the
compound 15i induced nuclear changes in the HCT-116 cells, the
DAPI staining was exploited. HCT-116 cells were treated with
various concentrations of 15i (0.5, 1.0, and 2.5 µM) for 48 h and
stained with DAPI. The results from Figure 8 concluded that 15i
treated HCT-116 cells displayed condensed and micronuclei
formation along with the loose cell structure in a dose-dependent
manner. Whereas, untreated control cells displayed normal intact
nucleus which implies that conjugate 15i has the potential to
induce apoptosis in HCT-116 cells.
Figure 6. Phase-contrast images of HCT-116 cells treated with 0.5, 1, and 2.5
µM concentration of compound 15i and observed for the compound induced
morphological changes at 200× magnification.
Acridine orange (AO) is a fluorescent cationic dye that allows
identifying the apoptotic cells. It can easily traverse through the
intact cell membrane and intercalates into double-stranded DNA,
releases green colour fluorescence upon excitation at 480-490
nm.28 HCT-116 cells were incubated with the compound 15i at
0.5, 1.0, 2.5 µM concentrations for 24 h and stained with acridine
orange. After executing this assay, it was observed that the
untreated control cells displayed normal homogenous
morphology, and cells appeared to be a bright green colouration,
indicating healthy and viable cells. However, upon treatment
with compound 15i, we observed irregular distribution of
chromatin, membrane blebbing, and also green fluorescence
diminished gradually as shown in Figure 7 in a concentration-
dependent manner indicating DNA break down, which is a
typical hallmark of apoptosis.
Figure 8. DAPI stain for nuclear morphology in HCT-116 cancer cell lines
following 48 h post-treatment with conjugate 15i. The images were captured
with a fluorescence microscope using a DAPI filter at 200× magnification.
An increased level of reactive oxygen species (ROS) causes
oxidative damage to mitochondrial permeability transition pore
causing depletion of mitochondrial membrane potential and
further it leads to the initiation of the intrinsic apoptotic
4

cascade.30 As compound 15i induce apoptosis in HCT-116 cells,
it was considered of interest to identify the effect of 15i on the
ROS generation. Hence, cells were incubated with 15i for 24 h at
0.5, 1, 2.5 µM concentration, and the intracellular ROS
Maintenance of mitochondrial membrane potential (ΔΨm) is
essential for bio-energetic function and mitochondrial integrity.
Alteration of ΔΨm is an indication of early events that occur
during apoptosis and chemical-hypoxia induced necrosis.31
Mitochondrial damage induced by 15i was estimated by
identifying the drop in mitochondrial membrane potential. After
48 h of drug treatment with the compound 15i at 0.5, 1.0, 2.5 µM
concentrations, it was identified that ΔΨm of HCT-116 cells was
reduced as shown in Figure 10, measured by JC-1 staining. JC-1
is a cationic lipophilic dye employed to stain the mitochondria.
generation
was
observed
by
employing
2'-7'-
Dichlorodihydrofluorescein diacetate (DCFDA) staining using
fluorescent microscopy. DCFDA is a non-fluorescent dye which
is altered into a fluorescent green DCF via intracellular esterases.
The fluorescence intensity of DCF corresponds to the quantity of
ROS generated. As shown in Figure 9, there is a considerable
enhancement of DCF cell population by 15i as compared to
JC-1 binds dominantly to
J monomers formed during
control which indicates that it induces apoptosis in
a
hyperpolarisation in cells which emits fluorescence at 590±17.5
nm. On the other hand, healthy normal cells contain more J
aggregates and fewer monomers due to which JC-1 dye could not
bind and emits fluorescence at 530±15 nm. Flow cytometric
analysis of HCT-116 cells after treatment with compound 15i
revealed that the compound has disturbed the ΔΨm in contrast to
control. Based on the acquired results, we have identified an
increase of J-monomers (disrupted mitochondrial cells) formed
from normal polarised J aggregate. In the present study, the
analysis was symbolized as a P1 (normal ΔΨm) and P2
populations (altered ΔΨm).
concentration-dependent manner through intracellular ROS
generation in HCT-116 cells. This was also confirmed with the
observed fluorescence emission where a considerable increase
was observed dose-dependently. Overall, these results conclude
that compound 15i induced apoptosis via the mitochondrial
pathway.
Figure 9. DCFDA images illustrating the effect of compound 15i on the
generation of Reactive Oxygen Species (ROS) in arbitrary units at
concentrations 0.5, 1, and 2.5 µM on HCT-116 cancer cell lines. The
corresponding graph represents fluorescence emitted at excited and emission
wavelengths 480/535 nm in comparison to control at various doses.
Figure 11. Annexin V-FITC/propidium iodide dual staining assay. HCT-116
cells were treated with compound 15i and labeled with Annexin V-FITC/PI
and analyzed for apoptosis using a flow-cytometer. Cells in the lower left
quadrant (Q1-LL: AV-/PI-): live cells; lower right quadrant (Q2-LR: AV-/PI-):
early apoptotic cells; upper right quadrant (Q3-UR: AV+/PI+): late apoptotic
cells and upper left quadrant (Q4-UL: AV−/PI+): necrotic cells.
The apoptotic effect of compound 15i was further analyzed
by annexin V FITC/PI (AV/PI) dual staining assay to observe the
incidence of phosphatidylserine externalization and also to
identify whether it is due to apoptosis or necrosis.32 This study
assists the detection of live cells (Q1-LL; AV−/PI−), early
apoptotic cells (Q2-LR; AV+/PI−), late apoptotic cells (Q3-UR;
AV+/PI+) and necrotic cells (Q4-UL; AV−/PI+). In the present
study, HCT-116 cells were treated with compound 15i for 48 h at
0.5, 1, 2.5 µM concentrations to identify the apoptotic effect as
shown in Figure 11. This assay reveals that compound 15i has
exhibited apoptosis in a dose-dependent manner with an increase
in late apoptotic cells i.e. control (1.48%), at 0.5 μM (5.70%), at
1 μM (6.94%) and 2.5 μM (6.97%). Besides, at 1 μM (3.49%)
Figure 10. Effect of compound 15i on mitochondrial membrane potential
(ΔΨm) in HCT-116 cancer cells upon treatment with 0.5, 1, and 2.5 µM
concentration. Cells were incubated with JC-1 dye and analyzed by flow
cytometer (BD FACSVerse™, USA). Values in the figures correspond to the
J
aggregates and
J monomers as P1 and P2 monomers populations
respectively.
5

2015;25(20):4580-4586. (j) Mullagiri K, Nayak VL, Sunkari S, Mani
GS, Guggilapu SD, Nagaraju B, Alarifi A, Kamal A. Med. Chem.
Commun. 2018;9(2):275-281.Zhang HZ, Kasibhatla S, Kuemmerle J,
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and 2.5 µM (13.30%) concentrations, the compound 15i
exhibited an increase in the necrotic cell population.
In summary, we detailed the design, synthesis, and cytotoxic
potential
of
a
series
of
(Z)-3-(phenylamino)-1-(3-
against
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-en-1-ones
selected cancer cell lines. The cytotoxic data revealed that
compound 15i displayed an effective cytotoxic potential against
the human colorectal cancer cell line (HCT-116). Moreover,
detailed biological studies such as cell cycle analysis revealed
that 15i arrest HCT-116 cells in the G0/G1 phase of cell cycle
and induced cell death by apoptosis. It was additionally
confirmed by subsequent cell viability studies such as clonogenic
assay, acridine orange staining, DAPI nuclear staining,
generation of ROS, changes in mitochondrial membrane
potential, and annexin V–FITC/PI assays. Based on the acquired
results, compound 15i can be considered as a potential lead
molecule in the designed chemical library that would become a
potential drug for the treatment of colorectal cancer.
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Acknowledgments
G. S. M. is thankful to the Department of Pharmaceuticals, the
Ministry of Chemicals and Fertilizers Govt. of India, New Delhi
for the award of a Research Fellowships. NIPER Research
Communication No.: NIPER-H/2020/M021.
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relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
Declaration of interests
☐ The authors declare that they have no known
competing financial interests or personal
6

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Graphical Abstract
Synthesis
of
(Z)-3-(arylamino)-1-(3-
phenylimidazo[1,5-a]pyridin-1-yl)prop-2-
en-1-ones as potential cytotoxic agents
Geeta Sai Mani,a Pratibha Anchi,b Satish Sunkari,c
Kavitha Donthiboina,a Chandraiah Godugu,b*
Nagula Shankaraiah,a* Ahmed Kamala,c,d
*
aDepartment of Medicinal Chemistry, bDepartment of Regulatory Toxicology,
National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad 500037, India
cMedicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of
Chemical Technology (IICT), Hyderabad-500007, India.
dSchool of Pharmaceutical Education and Research, Jamia Hamdard
University, New Delhi 110062, India
7