2
B. Yilmaz et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118904
2
2
. Experimental
149.12, 145.08, 133.45, 132.75, 131.11, 130.81, 129.92, 128.56, 128.39,
27.91, 127.69, 124.53, 110.02, 104.87, 98.29, 68.21, 44.38, 31.21,
12.61. Anal. calcd. For C140 16: C, 67.29; H, 5.81; N, 8.97; S,
.13. Found: C, 67.33; H, 5.77; N, 9.01; S, 5.19%.
1
.1. Chemistry
144 20 4
H O S N
5
1H and 13C NMR spectra were recorded on Agilent Premium Com-
pact spectrometer operating at 600 MHz. FT-IR spectra were obtained
with Bruker Vertex FT-IR spectrometer (ATR). All chemicals used were
of analytical grade, and they were used as received. HPLC grade solvents
were used for both emission and absorption spectral measurements.
The starting compounds, (Rh-A), and (1–4) were prepared by following
the procedure described elsewhere [23–27]. Targeted novel rhodamine
based calix[4]arene derivatives (5–7) were prepared for the first time as
follows.
2.2. Spectrophotometric measurements
For absorbance and emission studies, 1 mM of stock solution of (5–
7) was prepared in DMF. The solution of (5–7) was then diluted to 0.1
μM. Solutions of the metal cations (1 mM) were prepared in deionized
water (20 μM HEPES buffer, pH 7.2) for titration and/or metal selectivity
experiment. In absorption and emission experiments, the volume
of studied solutions was adjusted as 2.0 mL. The absorption spectra of
(5–7) with and without metal cations were recorded in the range of
200–600 nm. All emission spectra were obtained at room temperature
under the excitation of 520 nm. The solutions were scanned (1200
nm/min) with 400 watt of PMT voltage in a spectrofluorometer with
the range of 540–700 nm. The widths of the slit for the both excitation
and emission were adjusted at 5 nm. The best fluorescence intensity
at 580 nm was determined under the excitation at the wavelength of
520 nm.
Compounds (5): The mixture of aldehyde based calixarene com-
pound (4) (1.0 mmol) and Rhodamine amine compound (Rh-A) (5.0
mmol) in 50 mL of ethanol-chloroform solution (1:2) was refluxed for
7
2 h under dry condition. The precipitates were filtered off and washed
with excess amount cold methanol to gain a crude product. After crys-
tallization of crude product with chloroform-methanol (5:2), Schiff
base based calix[4]arene derivative (5) was obtained with 62% yields.
−
1 1
FT-IR (ATR) (ν): 1645 (C=N) cm . H NMR (600 MHz CDCl
(
3
): δ 8.54
m, 4H, Ar\\H), 7.95–7.94 (m, 4H, Ar\\H), 7.49–7.45 (m, 8H, Ar\\H),
.19 (m, 6H, Ar\\H), 7.14–7.13 (m, 4H, Ar\\H), 6.41 (m, 12H, Ar\\H),
.13–6.12 (m, 8H, Ar\\H), 3.19–3.20 (m, 40H, CH CH and ArCH Ar),
): C NMR (CDCl ): δ
7
6
1
1
1
9
7
2.3. Biology applications
2
13
3
2
13
2+
.02 (m, 48H, CH
2
-CH
3
). C NMR (DMSO‑d
67.87, 155.55, 151.93, 149.42, 146.91, 138.47, 132.15, 131.23, 131.11,
30.81, 129.92, 129.30, 130.01, 128.81, 127.29, 127.23, 114.11, 106.58,
7.77, 76.37, 47.19, 31.07, 12.88. Anal. calcd. For C144
5.50; H, 6.34; N, 9.78. Found: C, 75.48; H, 6.39; N, 9.81.
Compounds (6): To chloroform solution (50 mL) of
6
3
2.3.1. Cell applications: cytotoxicity and Hg imaging
Human epithelial cell line HEK 293 (human embryonic kidney 293)
was cultured in DMEM (4.5 g/L glucose; Gibco, Invitrogen, Karlsruhe
Germany) containing 0.01% gentamicin and 100 units/mL penicillin/
streptomycin, MCF-7 (human breast adenocarcinoma) and MIA PaCa-
2 were separately grown in RPMI (110 mg/L sodium pyruvate and 1
g/L glucose; Gibco, Invitrogen, Karlsruhe, Germany) containing 100
units/mL penicillin/streptomycin. All media supplemented with 10%
fetal bovine serum (FBS) was incubated in a humidified atmosphere
144 12
H O N16: C,
tetrachloromethylated calix[4]arene compound (2) (1.0 mmol), 4.1
mmol of Rhodamine amine compound (Rh-A) in 25 mL of chloroform
in presence of catalytic amount of triethylamine (TEA) was added grad-
ually at room temperature and reaction flask was refluxed under nitro-
gen atmosphere for 48 h. All formed precipitates were filtered off and
clear solution was evaporated to dryness. Obtained crude product was
crystallized in methanol-dichloromethane solution (1:5) and shine
crystals of compound (6) with 53% yields were obtained after overnight.
2
containing 5% CO at 37 °C for 24 h. Alamar Blue® assay was used for
the determination of cytotoxicity of studied compounds on epithelial
and cancer cell lines [28]. This assay measures cell viability by determin-
ing the mitochondrial activity of the cells, therefore it shows the relative
toxicity of the test compound. Briefly, after seeding and incubating of
cell lines (MCF-7, MIA PaCa-2 or HEK 293) on 96 well plates (10,000
−
1 1
FT-IR (ATR) (ν): 3310 (R-NH) cm
.91–7.87 (m, 4H, Ar\\H), 7.58–7.55 (m, 4H, Ar\\H), 7.18 (m, 4H,
Ar\\H), 7.12 (m, 4H, Ar\\H), 6.77 (s, 8H, Ar\\H), 6.54–6.48 (m, 8H,
Ar\\H), 6.34–6.23 (m, 16H, Ar\\H), 3.97 (s, 8H, CH NH), 3.44–3.37
m, 32H, CH CH ), 3.35–3.27 (m, 4H, ArCH Ar), 3.05–2.92 (m, 4H,
ArCH Ar), 1.15 (m, 48H, CH
): δ 161.27, 152.77, 151.22, 149.52, 139.47, 135.08, 132.65,
3
. H NMR (600 MHz CDCl ): δ
7
2
cells/well) at 37 °C and 5% CO for 6 h, each well was treated with differ-
ent concentrations of (5, 6 and 7) (20, 40, 60, 80 and 100 μM). At the end
of incubation for 18 h, Alamar Blue solution was added into the each
well (1:10, v/v) and cell lines were incubated for an additional 4 h,
then spectrophotometric measurements were carried out at 570 nm
and 600 nm. Cell viabilities of each sample were calculated and control
wells was taken as 100% and the percent viability in the presence of
tested compounds was expressed as a percent of control. For the cell
imagine experiments, MCF7 (for (5) and (7)) and MIA PaCa-2 (for
(7)) were used as model cell lines. Cell lines were incubated in a 10
μM of (5) or (7) solution in the culture medium at 37 °C for 1 h. After
washing the cells with phosphate-buffered saline (PBS), 5 μM of Hg2
was added to the medium. After incubation, Bright-field and fluores-
cence images of MCF-7 or MIA PaCa-2 cells were taken by a fluorescence
microscope (Leica™ DM3000) with an objective lens of 100×
magnification.
2
(
2
3
2
1
3
13
2
2 3 6
-CH ). C NMR (DMSO‑d ): C NMR
(
CDCl
3
1
1
C
31.75, 131.11, 130.81, 129.92, 129.30, 128.91, 128.56, 127.69, 126.53,
14.15, 106.52, 98.71, 73.77, 54.51, 47.12, 31.33, 12.90. Anal. calcd. For
144
152 12
H O N16: C, 75.24; H, 6.66; N, 9.75. Found: C, 75.29; H, 6.69;
N, 9.71.
Compound (7): 15 mL of dry THF solution of excess amount of Rho-
damine amine compound (Rh-A) was added dropwise to 30 mL of dry
THF solution of chlorosulfonated calix[4]arene compound (3) (1.5
mmol) in the presence of catalytic amount of triethylamine at 0 °C;
the reaction mixture was stirred under nitrogen atmosphere at room
temperature for 3 h. After completion of the reaction, excess amount
of solvent and volatile reactant was removed under reduced pressure
+
and the residue was treated with CH
After cooling the solution, organic phase was slowly washed three
times with water (3 × 20 mL) and dried over Na SO . Crude sulfon-
amide based calix[4]are derivative (7) was purified by column chroma-
tography (SiO , CH Cl -MeOH, 7:2). FT-IR (ATR) (ν): 1139 (SO NH)
cm . H NMR (600 MHz CDCl ): δ 7.96–7.90 (m, 4H, Ar\\H), 7.59
m, 4H, Ar\\H), 7.43 (s, 8H, Ar\\H), 7.12 (m, 4H, Ar\\H), 7.02 (m, 4H,
Ar\\H), 6.41–6.38 (m, 8H, Ar\\H), 6.30–6.26 (m, 16H, Ar\\H),
.34–3.33 (m, 40H, CH -CH and ArCH Ar), 1.16 (m, 48H, CH -CH ).
): C NMR (CDCl ): δ 164.32, 156.97, 151.22,
2
Cl
2
(80 mL) and refluxed for 3 h.
3. Results and discussion
2
4
3.1. Synthesis and characterization of chemosensor (5–7)
2
2
2
2
Rhodamine based calixarene molecules (5–7) were synthesized by
the coupling of Rhodamine amine compound (Rh-A) with tetra-
aldehyde-derivatized calixarene (4), tetra-chloro-methylated calix[4]
arene compound (2), and tetra-chloro-sulfonated calix[4]arene com-
pound (3) for targeted chemosensor molecules (5), (6) and (7), respec-
tively (Fig. 1). In Fourier transform infrared (FTIR-ATR) spectra of
−
1 1
3
(
3
2
3
2
2
3
13
13
C NMR (DMSO‑d
6
3