Synthesis and cell imaging applications of fluorescent mono/di/tri-heterocyclyl-2,6-dicyanoanilines

Article abstract of DOI:10.1016/j.bmcl.2016.12.074

Synthesis of 3,4,5-triheterocyclyl-2,6-dicyanoanilines, starting from heterocyclic aldehydes and 1,2-diheterocycle-substituted ethanones, is described. 2,6-Dicyanoanilines with one or two heterocyclic substituents have also been synthesized. It was found

Full text of DOI:10.1016/j.bmcl.2016.12.074

Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cell imaging applications of fluorescent
mono/di/tri-heterocyclyl-2,6-dicyanoanilines
Mahesh M. Pisal a, Ritesh A. Annadate a, Meghana C. Athalye b, Deepak Kumar c, Subhash P. Chavan a,
Dhiman Sarkar b, Hanumant B. Borate a,
⇑
a Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pashan, Pune 411 008, India
b Combichem Bio-resource Centre, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pashan, Pune 411 008, India
c Physical Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pashan, Pune 411 008, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of 3,4,5-triheterocyclyl-2,6-dicyanoanilines, starting from heterocyclic aldehydes and 1,2-
diheterocycle-substituted ethanones, is described. 2,6-Dicyanoanilines with one or two heterocyclic
substituents have also been synthesized. It was found that some of these molecules have selective
cell-staining properties useful for cell imaging applications. The compounds 1g, 10f and 11 were found
to stain cytoplasm of the cells in contact but not the nucleus while the compound 12 showed affinity
to apoptotic cells resulting in blue fluorescence. The cell imaging results with compound 12 were similar
to Annexin V-FITC, a known reagent containing recombinant Annexin V conjugated to green-fluorescent
FITC dye, used for detection of apoptotic cells. These compounds were found to be non-cytotoxic and
have potential application as cell imaging agents.
Received 17 November 2016
Revised 8 December 2016
Accepted 28 December 2016
Available online 31 December 2016
Keywords:
Dicyanoanilines
Cell imaging
Fluorescence
Heterocyclic
Ó 2016 Elsevier Ltd. All rights reserved.
Substituted 2,6-dicyanoanilines are important compounds
studied1a as such or as intermediates in diverse fields like
optical materials, dyes, textile printing, heat resistant polymers,
chiral stationary phases in chromatography etc. They also
constitute molecular skeleton of a number of compounds, with
an array of structural variations, some of which have been studied
for potential medicinal use. The synthetic methods to prepare
these moieties and their applications have been reviewed
recently1a and the continued interest in these compounds
has resulted in further publications.1b,1c,2a–c We desired to study
the synthesis and optical/biological properties of dicyanoanilines
with one, two or three heterocyclic substituents. The literature
survey revealed that there are many references for 3-heterocyclyl-2,
describing cell imaging potential of 2,6-dicyanoanilines and these
preliminary results will lead to new applications of dicyanoanilines
for cell imaging.
Various
hitherto
unknown
3,4,5-triheterocyclyl-2,6-
dicyanoanilines were prepared from 1-benzyl-1,2,3[1H]-triazole-
4- carboxaldehyde 8, malononitrile and required 1,2-diheterocy-
clyl ethanone as exemplified by the synthesis of 2-amino-4-(1-
benzyl-1H-1,2,3-triazol-4-yl)-5-(4-oxo-6-propylthieno[2,3-d]
pyrimidin-3(4H)-yl)-6-(thiophen-2-yl)isophthalonitrile (1a)
shown in Scheme 1.
Initially, thienopyrimidinone was chosen as one of the hetero-
cycles because thienopyrimidinones5 with various substituents
can be synthesized easily from substituted 2-aminothiophene-3-
carboxylates which in turn can be prepared by Gewald synthesis.6
Thus, ketone 6a was prepared from 2-bromoacetylthiophene7 3a
and 6-propylthieno[2,3-d]pyrimidin-4(3H)-one 5a5 in the presence
of triethyl amine in ethyl acetate and reacted8 with 1-benzyl-1,2,3
[1H]-triazole-4-carboxaldehyde 89 and malononitrile in DMF in the
presence of morpholine to obtain the 3,4,5-triheterocyclyl-2,6-
dicyanoaniline 1a in 43% isolated yield. The 3,4,5-triheterocyclyl-
2,6-dicyanoanilines and 3,4-diheterocyclyl-5-aryl-2,6-dicyanoani-
lines 1a–j thus prepared, using aldehyde 8 and appropriate
ketones, are shown in Table 1.
6-dicyanoanilines,1a
6-dicyanoanilines2
a
few references for 3,5-diheterocyclyl-2,
and very few references for
a
4-heterocyclyl-2,6-dicyanoanilines3 but there are no references for
3,4-diheterocyclyl-2,6-dicyanoanilines or 3,4,5-triheterocyclyl-
2,6-dicyanoanilines. We report herein synthesis of various hitherto
unknown 3,4-diheterocyclyl-2,6-dicyanoanilines and 3,4,5-trihete-
rocyclyl-2,6-dicyanoanilines, their fluorescence properties and cell
imaging applications. Though there are a large number of publica-
tions about cell imaging,4 to our knowledge, this is the first report
Structures of these compounds were assigned based on 1H NMR,
13C NMR, IR and HRMS data.10 The structures of representative
⇑
Corresponding author.
E-mail address: hb.borate@ncl.res.in (H.B. Borate).
http://dx.doi.org/10.1016/j.bmcl.2016.12.074
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

980
M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
O
CH3
EtO
H2N
Bn N3
+
S
C3H7
S
O
HO
2a
4a
d
a
b
O
Bn
N
HN
S
Br
C3H7
N
S
O
N
HO
N
5a
3a
7
e
c
Bn
O
N
N
N
S
N
N
C3H7
OHC
O
S
8
6a
f
C3H7
S
N
Bn
N
N
O
N
N
S
NC
CN
NH2
1a
Scheme 1. Reagents and conditions: (a) Br2, ether, 0 °C, 1.5 h, 72%; (b) Formamide, ammonium acetate, 145 °C, 10 h, 75%; (c) Triethyl amine, acetone, 0 °C, 1 h, RT, 3 h, 86%;
(d) CuSO4ꢂ5H2O, sodium ascorbate, t-butanol-water, RT, 12 h, 94%; (e) MnO2, ethyl acetate, RT, 2 h, 92%; (f) Malononitrile, morpholine, DMF, 80 °C, 12 h, 43%.
compounds 1c and 1e were further confirmed by X-ray
1g, 10f, 11 and 12 and N,N-dimethylated compounds 13e and
14f are shown in Fig. 1A while fluorescence spectra for these com-
pounds are shown in Fig. 1B.
crystallography.10
Using
a
similar strategy, various hitherto unknown
3-heterocyclyl-2,6-dicyanoanilines 9, 10a–f, 11 and 12 were
prepared by reacting aldehyde 8 with appropriate aldehyde/ketone
(Table 2). The dicyanoanilines 1e and 1f were methylated2a by
subjecting them to NaH-MeI in THF to get the corresponding
N,N-dimethylated compounds 5-(1-benzyl-1H-1,2,3-triazol-4-yl)-
3-(dimethylamino)-20,40-difluoro-6-(1H-1,2,4-triazol-1-yl)-[1,10-
biphenyl]-2,4-dicarbonitrile (13e) and 5-(1-benzyl-1H-1,2,
3-triazol-4-yl)-3-(dimethylamino)-40-fluoro-6-(1H-1,2,4-triazol-1-
yl)-[1,10-biphenyl]-2,4-dicarbonitrile (13f).10 Similarly, compounds
10a–f were methylated to obtain the corresponding N,N-dimethy-
lated compounds 14a–f (Table 2).
It was observed that there was a red shift in absorption maxi-
mum of the amino group after methylation due to enhanced elec-
tron-donating ability e.g. the kmax value for amino compound 1e
was 367 nm while that for its corresponding N,N-dimethylated
derivative 13e was 395 nm. It was observed that the Stokes shift
for the amino compound 1e was 47 nm while the stokes shift for
the corresponding N,N-dimethylated derivative 13e was 74 nm
indicating that the Stokes shift is increased after N,N-dimethylation
of amino group. There was change in color of fluorescence in ace-
tonitrile solutions of same concentration of these compounds. The
compounds studied in the present work are soluble in DMSO, DMF,
THF, chloroform, dichloromethane, ethyl acetate, acetone, acetoni-
trile, methanol and ethanol while they are insoluble in water and
pet ether. The HOMO-LUMO gaps estimated from DFT calcula-
tions11 were found to be in good agreement with the experimental
values derived from uv-visible spectra (Please see Supplementary
data).
There is a possibility to modify the structures of the compounds
in the present work, in order to tune the optical properties, as there
is a lot of flexibility in the synthetic schemes described.
These compounds were studied for their optical properties as
they were colored in solution (Please see Supplementary data
Table S2).
The UV–visible absorption spectra, of 2.5 ꢀ 10ꢁ5 M solutions in
acetonitrile, for representative 2,6-dicyanoaniline compounds 1e,
The fluorescent molecules have a wide application in biological
research where they have been developed as valuable tools to

M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
981
study basic physiological processes within biological systems and
there are a number of reports wherein they have been explored
for cell imaging studies.4
Apoptosis is a process of programmed cell death in which the
cell shrinks, nuclear envelope disassembles and DNA breaks into
fragments.12 The cell surface is altered which serves as a signal
for the neighboring macrophages to phagocytose it.13,14 Phos-
phatidylserine (PS) which is actively held on the interior (cytosolic)
side of the lipid bilayer is externalized which serves as a cell sur-
face signal for apoptosis.15,16 Annexin V is a Ca2+ dependent
Table 1
The 3,4,5-triheterocyclyl-2,6-dicyanoanilines and 3,4-diheterocyclyl-5-aryl-2,6-dicyanoanilines prepared.
Entry no
1
Ketone used
Product obtained
Yield %
43
C3H7
S
N
O
S
S
N
C3H7
N
NC
N
S
S
O
H2N
Bn
S
N
6a
O
O
N
CN
N
1a
1b
2
45
C6H13
S
N
S
N
C6H13
N
NC
N
O
O
H2N
Bn
N
6b
N
CN
N
3
44
N
S
N
N
N
NC
N
N
S
H2N
Bn
O
N
N
N
CN
1c
N
6c
4
38
N
N
Bn
N
N
N
N
O
O
O
NC
CN
6d
NH2
1d
5
49
N
N
Bn
N
N
N
N
F
F
N
N
N
N
N
F
O
F
CN
NC
6e
NH2
1e
6
52
N
N
Bn
N
N
N
F
F
N
N
O
CN
NC
6f
NH2
1f
7
31
F
C5H11
S
N
O
S
N
F
C5H11
N
F
N
O
NC
H2N
Bn
N
O
F
N
CN
N
6g
1g
(continued on next page)

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M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
Table 1 (continued)
Entry no
8
Ketone used
Product obtained
Yield %
37
F
CH3
S
N
O
S
N
O
F
CH3
N
F
F
N
O
NC
H2N
Bn
N
N
CN
F
N
6h
1h
9
43
N
N
Bn
N
N
F
N
N
N
N
F
O
CN
F
NC
6i
6j
NH2
1i
10
47
N
N
Bn
N
F
F
N
N
N
O
CN
NC
NH2
1j
Table 2
The novel 3-heterocyclyl-2,6-dicyanoanilines and 3,4-diheterocyclyl-2,6-dicyanoanilines prepared.
Entry no
1
Aldehyde/ketone used
Product obtained
Isolated Yield %
50
Corresponding methylated product prepared
ND
Isolated Yield %
–
Acetaldehyde + 8
Bn
N
N
NC
N
CN
H2N
9
2
3
4
Propionaldehyde + 8
Butyraldehyde + 8
Valeraldehyde + 8
61
64
59
75
73
70
Bn
Bn
N
N
CH3
CH3
N
N
N
N
NC
NC
NC
CN
CN
CN
NC
NC
NC
CN
NH2
10a
N(CH3)2
14a
Bn
N
Bn
N
N
N
C2H5
C2H5
N
N
CN
NH2
10b
N(CH3)2
14b
Bn
N
Bn
N
N
N
C3H7
C3H7
N
N
CN
NH2
10c
N(CH3)2
14c

M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
983
Table 2 (continued)
Entry no
5
Aldehyde/ketone used
Product obtained
Isolated Yield %
60
Corresponding methylated product prepared
Isolated Yield %
Hexanal + 8
Heptanal + 8
Octanal + 8
72
69
77
Bn
N
Bn
N
N
N
N
C4H9
C4H9
N
NC
CN
NC
CN
NH2
10d
N(CH3)2
14d
6
61
Bn
N
Bn
N
N
N
C5H11
C5H11
N
N
NC
CN
NC
CN
NH2
10e
N(CH3)2
14e
7
63
Bn
N
Bn
N
N
N
C6H13
C6H13
N
N
NC
CN
CN
NC
F
N(CH3)2
NH2
14f
10f
8
55
ND
–
Bn
N
F
F
N
CH3
+ 8
N
O
F
NC
CN
S
NH2
11
9
65
ND
–
S
N
C4H9
C4H9
N
+ 8
N
N
O
O
CHO
Bn
NC
N
N
N
H2N
CN
12
1e
1g
10f
11
12
13e
14f
1e
1g
10f
11
12
13e
14f
1.5
1.0
0.5
1.0
0.8
0.6
0.4
0.2
0.0
A
B
0.0
200
300
Wavelength [nm]
400
350
400
450
500
550
600
650
Wavelength [nm]
Fig. 1. (A) UV–visible absorption and (B) fluorescence spectra of compounds 1e, 1g, 10f, 11, 12, 13e and 14f.
phospholipid-binding protein with high affinity for PS. Annexin V
conjugated with a fluorophore, fluorescein isothiocyanate (FITC),
is widely used to detect this PS externalization which indicates
apoptotic cells in a cell population. In some biological studies it
becomes necessary to identify such cell populations in order to
interpret cell death inducing mechanisms if necessary. Propidium
iodide is another widely used dye which intercalates with the
apoptosis.17 This has
a profound application in identifying

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M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
Fig. 2. Cell images captured after staining THP-1 cells with compounds 1g, 10f, 11 and 12 (blue fluorescence). Imaging assay buffer: Pre-warmed (37 °C) phosphate-buffered
saline (PBS, pH 7.2) containing compounds at 30 g/mL final concentrations. Incubation time: 20 min, Temperature: 37 °C in CO2 incubator.
l
Fig. 3. MCF-7 cells stained with the compound 1g, SYTO 9 and Nile red. The cells were stained with compound 1g (100 lg/mL, 0.16 mM, blue fluorescence), SYTO 9 which
stains nucleus (green fluorescence) and Nile red which stains cell membrane (red fluorescence) observed at 60X magnification. (A) Cells seen in bright field; (B) Overlay image
of blue and green channels, Blue – compound 1g, Green – SYTO 9 (stains nucleus); (C) Overlay image of green and red channels: Red – Nile red (stains cell membrane),
Green – SYTO 9 (stains nucleus); (D) Overlay image of (B) and (C). Scale on image measures 50
lm. Imaging assay buffer: Pre-warmed (37 °C) phosphate-buffered saline (PBS,
pH 7.2) containing compound 1g (100 g/mL, 0.16 mM), SYTO 9 and Nile red. Incubation time: 20 min, Temperature: 37 °C in CO2 incubator.
l
Fig. 4. Differentiated THP-1 cells (A) In bright field; (B) Stained with compound 12; (C) Stained with Annexin V-FITC. Imaging assay buffer: 10 mM HEPES, 140 mM NaCl and
2.5 mM CaCl2, pH 7.4, compound 12 (final concentration 100 lg/mL, 0.2 mM), Annexin V-FITC conjugate (5 lL v/v). Incubation time: 15 min, Temperature: 37 °C in CO2
incubator
DNA. It is specifically used to detect dead cell population in
research18 and is often used along with Annexin-V-FITC.
compounds 1g, 10f, 11 and 12 are shown in Fig. 2. Differentiated
THP-1 cells were observed to retain these fluorescent compounds.
The compounds 1g, 10f and 11 were found to stain all the cells
in contact while compound 12 was found to stain only some cells
out of total cells. The compounds 1g, 10f, 11 and 12 were studied
further for staining MCF-7 and MDA-MB-231 cells. The representa-
tive cell-imaging results for the compound 1g for MCF-7 cells are
shown in Fig. 3.
The detailed cell images for staining of differentiated THP-1,
MCF-7 and MDA-MB-231 cell lines with the compounds 1g, 10f
and 11 are given in the Supplementary Information. The com-
pounds 1g, 10f and 11 were found to specifically stain only the
cytoplasm of the cells but not the nucleus. This was verified by
using known counter stains like SYTO 9 (for nucleus) which gives
green fluorescence and Nile red (lipid molecules and cell mem-
brane) which gives red fluorescence.
The fluorescent molecules can be tagged or conjugated to speci-
fic moieties and generate fluorescent signals which thus serve as
detectable or measurable signals. These have applications in
microscopy, fluorimetry, flow cytometry, etc. The molecules syn-
thesized in the present work show fluorescent properties which
were explored for cell imaging studies. Cell imaging analysis was
carried out on fluorescent compounds prepared in the present
work and 4 compounds (1g, 10f, 11 and 12) were found to stain
eukaryotic cells. Three cell lines (differentiated THP-1, MCF-7 and
MDA-MB-231) were tested for cell staining using known staining
agents [SYTO 9 which stains nucleus (green fluorescence), Nile
red which stains cell membrane (red fluorescence), Annexin-V-
FITC which detects apoptotic cells (green fluorescence) and Propid-
ium iodide which stains dead cells (red fluorescence)].
Cell images captured, using High Content Screening System
(ArrayScanXTI, Thermo Scientific) at 20X magnification, after
staining differentiated THP-1 cells with 30 lg/mL solutions of
However, compound 12 was found to stain selectively some
cells out of total cells and the patterns were similar to Annexin
V-FITC as shown in Fig. 4.

M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
985
Fig. 5. Differentiated THP-1 cells stained with compound 12, Annexin-V-FITC and propidium iodide. The cells were stained with compound 12 (100
lg/mL, 0.2 mM, blue
fluorescence), Annexin-V-FITC which detects apoptotic cells (green fluorescence) and propidium iodide (PI) which stains dead cells (red fluorescence) observed at 60X
magnification. (A) Cells seen in bright field; (B) Overlay image of green and red channels: Green – Annexin-V-FITC (apoptosis), Red – PI (dead); (C) Overlay image of blue and
red channels: Blue – compound 12, Red – PI (dead); (D) Overlay image of bright field with blue channel – Blue - compound 12 (E) - Overlay image of (A), (B) and (C); (F) -
Overlay image of (B) and (C). Imaging assay buffer: 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4, Annexin V-FITC conjugate (5
compound 12 at final concentration of 100 g/mL, 0.2 mM. Incubation time: 15 min, Temperature: 37 °C in CO2 incubator.
lL v/v) and PI (10 lg/mL) along with
l
Fig. 6. Differentiated THP-1 cells with induced apoptosis using Paclitaxel, stained with the compound 12, Annexin-V-FITC and propidium iodide. Differentiated THP-1
(macrophage) cells with induced apoptosis using Paclitaxel IC90 concentration, were stained with the compound 12 (100 g/mL, 0.2 mM, blue fluorescence), Annexin-V-FITC
l
(green fluorescence for apoptotic cells) and propidium iodide (PI) (red fluorescence for dead cells) observed at 60X magnification. (A) Cells seen in bright field; (B) Overlay
image of green and red channels, Green – Annexin-V-FITC (stains apoptotic cells), Red – PI (stains dead cells); (C) Overlay image of blue and red channels: Blue – compound
12, Red – PI (stains dead cells); (D) Overlay image of bright field with blue channel– compound 12; (E) Blue – compound 12; (F) Overlay image of (A) and (C). Imaging assay
buffer: 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4, Annexin V-FITC conjugate (5
100 g/mL, 0.2 mM. Incubation time: 15 min, Temperature: 37 °C in CO2 incubator.
lL v/v) and PI (10 lg/mL) along with compound 12 at final concentration of
l
This differential staining was studied further using known
markers and inducers. In order to study the differential staining
property of 12, the cells were seeded and apoptosis / cell death
was induced using known anti-cancer, cytotoxic compounds
namely Paclitaxel and Doxorubicin at their IC50 and IC90 concen-
trations. Known markers like Annexin V-FITC for apoptosis and

986
M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
Fig. 7. MCF-7 cells stained with the compound 12, Annexin-V-FITC (green fluorescence) and propidium iodide. The MCF-7 cells were stained with the compound 12
(100 g/mL, 0.2 mM, blue fluorescence), Annexin-V-FITC (green fluorescence) and propidium iodide (red fluorescence) observed at 60X magnification. (A) Cells seen in bright
field; (B) Overlay image of green and red channels: Green – Annexin-V-FITC (apoptosis), Red – PI (dead cells); (C) Overlay image of blue and red channels: Blue – the
compound 12, Red – PI (dead cells); (D) Blue – the compound 12; (E) - Overlay image of (A) and (B); (F) Overlay image of (A) and (C). Scale on image measures 50 m. Imaging
assay buffer: 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4, Annexin V-FITC conjugate (5 L v/v) and PI (10 g/mL) along with compound 12 at final concentration of
100 g/mL, 0.2 mM. Incubation time: 15 min, Temperature: 37 °C in CO2 incubator.
l
l
l
l
l
(D)
(C)
(A)
(B)
Fig. 8. MCF-7 cells with induced apoptosis using Doxorubicin, stained with the compound 12, Annexin-V-FITC and propidium iodide. MCF-7 cells with induced apoptosis
using Doxorubicin IC50 concentration were stained with the compound 12 (100 g/mL, 0.2 mM, blue fluorescence), Annexin-V-FITC (green fluorescence) and propidium
iodide (red fluorescence) observed at 60X magnification. (A) Cells seen in bright field; (B) Blue – compound 12 (C) Overlay image of green, red and blue channels: Green –
Annexin-V-FITC (apoptosis), Red – (dead cells) and Blue – compound 12; (D) - Overlay image of (A) and (B). Scale on image measures 50 m. Imaging assay buffer: 10 mM
g/mL) along with compound 12 at final concentration of 100 g/mL, 0.2 mM.
l
l
HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4, Annexin V-FITC conjugate (5
lL v/v) and PI (10
l
l
Incubation time: 15 min, Temperature: 37 °C in CO2 incubator.
propidium iodide for dead cells were used along with the com-
pound 12. Standard staining protocol for apoptosis/necrosis using
Annexin V-FITC/PI was carried out with addition of this new com-
pound 12. Healthy cells were used as negative control. The cell
images for differentiated THP-1 (macrophage) cells stained with
compound 12 is similar to Annexin-V-FITC (which shows green
fluorescence for apoptotic cells).
Further, apoptosis was induced in MCF-7 cells by treating them
with Doxorubicin and the cells were stained with the compound
12, Annexin-V-FITC and Propidium iodide and the results are
shown in Fig. 8.
the compound 12 (100 lg/mL, 0.2 mM, blue fluorescence),
Annexin-V-FITC which detects apoptotic cells (green fluorescence)
and Propidium iodide (PI) which stains dead cells (red fluores-
cence) are shown in Fig. 5.
The imaging properties of the compound 12 were further studied
with MDA-MB-231 cells. The apoptosis was induced with Doxoru-
bicin and the cells were stained with the compound 12 (100 lg/
It was observed that the staining of the differentiated THP-1
(macrophage) cells with the compound 12 corresponds to that
with Annexin-V-FITC which is known to stain the apoptotic cells.
The compound 12 does not stain dead cells. The results were fur-
ther confirmed by inducing apoptosis in the THP-1 (macrophage)
cells with Paclitaxel and repeating the study (Fig. 6).
The results of imaging in case of MCF-7 cells stained with the
compound 12, Annexin-V-FITC and Propidium iodide are shown
in Fig. 7 which also shows that the staining pattern of the
mL, 0.2 mM, blue fluorescence), Annexin-V-FITC (green fluores-
cence) and Propidium iodide (PI) (red fluorescence) as before (Fig. 9).
All these results indicated that the staining patterns of the com-
pound 12 (which shows blue fluorescence) are similar to Annexin-
V-FITC which shows green fluorescence for apoptotic cells and the
compound 12 has potential as staining agent for imaging of apop-
totic cells.
Toxicity of the compounds 1g, 10f, 11 and 12 against THP-1
cells was studied19 and the data are given in Supplementary data.

M.M. Pisal et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 979–988
987
Fig. 9. MDA-MB-231 cells with induced apoptosis using Doxorubicin, stained with the compound 12, Annexin-V-FITC and propidium iodide. MDA-MB-231 cells with induced
apoptosis using Doxorubicin IC90 concentration, were stained with the compound 12 (100 g/mL, 0.2 mM blue fluorescence), Annexin-V-FITC (green fluorescence) and
l
propidium iodide (red fluorescence) observed at 60X magnification. (A) Cells seen in bright field; (B) Green – Annexin-V-FITC (apoptosis); (C) Overlay image of green and red
channels: Green – Annexin-V-FITC (apoptosis), Red – PI (dead cells); (D) - Overlay image of (A) and (B); (E) Bright field with blue – compound 12 and red – PI (dead cells); (F)-
Overlay image of (A) and (E). Scale on image measures 50
lm. Imaging assay buffer: 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4, Annexin V-FITC conjugate (5 lL v/
v) and PI (10 g/mL) along with compound 12 at final concentration of 100
l
l
g/mL, 0.2 mM. Incubation time: 15 min, Temperature: 37 °C in CO2 incubator.
Since the IC50 values of compounds are >100
pounds can be considered as non-cytotoxic.
l
g/mL, these com-
detailed cell imaging data for compounds 1g, 10f, 11 and 12. Sup-
plementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.074.
In conclusion, synthesis of 3,4,5-triheterocyclyl/3,4-diheterocy-
clyl/3-heterocyclyl-2,6-dicyanoanilines, starting from 1-benzyl-
1,2,3[1H]-triazole-4- carboxaldehyde 8, malononitrile and
required aldehydes/ketones was achieved. The optical properties
of the synthesized compounds were studied which indicated that
fluorescence properties of this class of compounds can be modified.
The compounds were further studied from the point of their appli-
cation for cell imaging wherein it was found that compounds 1g,
10f and 11 stain cytoplasm of the cells in contact but not the
nucleus while the compound 12 showed selective affinity to apop-
totic cells resulting in blue fluorescence. The cell imaging results
with compound 12 were compared with Annexin V-FITC, a known
reagent containing recombinant Annexin V conjugated to green-
fluorescent FITC dye, used for detection of apoptotic cells. These
encouraging results indicate the significant potential of these com-
pounds as valuable tools for cell imaging.
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The authors thank CSIR, New Delhi, India, for financial support
as part of 12th Five Year Plan Program under title ACT (CSC-0301).
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