S. Kecel-Gunduz, Y. Budama-Kilinc, B. Gok et al.
Journal of Molecular Structure 1239 (2021) 130539
droxyl groups are potent electron/hydrogen donors to free radicals.
Many coumarin derivatives have special abilities to scavenge reac-
(NaHNH4(PO4.4H2O)) and 4-nitro-o-phenylenediamine (NPD) were
purchased from Sigma Aldrich. Sodium azide (SA, NaN3), dimethyl
sulfoxide (DMSO), tris base, ethylenediamintetraacetic acid (EDTA),
hydrochloric acid (HCl), sodium hydroxide (NaOH) were pur-
chased from Merck Millipore. DMEM, MTT and dexometasone
were acquired from Sigma–Aldrich. Penicillin and streptomycin
were purchased from I.E. Ulagay. DMSO, which is used for MTT
assay was obtained from Merck. RPMI-1640 Medium, FBS, Trypsin,
PBS, L-glutamine were obtained from Gibco. Absorbances were
measured with BioTek, ELx800 Microplate Reader.
Iminocoumarin is a subclass of coumarin family. The imino
group, including Schiff bases compound, is important for several
biological applications [34–39]. It is examined that Schiff bases is
related with antimicrobial, antiviral and anticancer activity. Thus,
iminocoumarins are also important structures that stand out in
biological and medical applications and have anticancer and an-
timicrobial properties [40–43]. Imino coumarin, have also anti-
cancer properties [34] on different human cancer cell lines such as
and on cervical cancer cell lines [50].
2.2. Methods
Encouraging improvements in biological activities showed by
coumarins direct the researcher’s attention to design and synthesis
of new more effective derivatives. But still there is need to develop
effective anticancer drugs.
2.2.1. Synthesis of 7-hydroxy-8-(((1-hydroxy-3-phenylpropan-2-
yl)imino)methyl)-4-(trifluoromethyl)-2H-chromen-2-one
(D3)
2-Amino-3-phenyl-1-propanol (1 mmol) and 8-formyl-7-
hydroxy-4-(trifluoromethyl)coumarin (2, 1 mmol) were dissolved
in absolute ethanol. The mixture was refluxed under inert atmo-
sphere for 4 h. The reaction was monitored by TLC. Then alcohol
was evaporated, and the crude product was purified by column
chromatography on silica gel (ethyl acetate-hexane 1:1). Yellow
solid, m.p. 193.1–196.2 °C, yield 92%. 1H NMR (500 MHz, CDCl3,
δ): 2.92 (dd, 1H, J = 13.7, 8.5 Hz, CH2Ph), 3.05 (dd, 1H, J = 13.5,
5.0 Hz, CH2Ph), 3.72 (dd, 1H, J = 11.5, 8.0 Hz, CH2OH), 3.78–3.80
(m, 1H, CH), 3.94 (apparent dd, 1H, J = 11.5, 2.5 Hz, CH2OH), 4.72
(bs, 1H, OH), 6.32 (s, 1H, =CH), 6.50 (d, 1H, J = 9.5 Hz, ArH),
7.15 (d, 2H, J = 6.5 Hz, ArH), 7.21–7.24 (m, 1H, ArH), 7.28–7.32
In developing a new drug, it is evaluated three basic crite-
ria: efficacy, quality and pharmacological safety. Regulatory bod-
ies such as the Food and Drug Administration (FDA), the Euro-
pean Medicines Agency (EMA) and the National Health Surveil-
lance Agency (Anvisa) state that preclinical trials should be the
starting point for a new drug development process [51–53]. DNA is
the target in the development of new anticancer drugs. In this con-
text, understanding the interactions of small molecules with DNA
is important for developing new and effective drugs for clinical ap-
plications. In addition, the Ames/Salmonella assay is a routine part
of preclinical trials in determining the safety of a new drugs [54].
Uncovering the DNA binding properties of coumarin com-
pounds, which have antitumor, antioxidant, antiviral, antibacterial,
antifungal properties, has an important place in the development
of new therapeutic reagents. In this study, spectroscopic (UV / VIS)
and in silico methods (molecular docking) were used to determine
the relationship of the coumarin compound with the property of
being a potential anticancer drug, and its interaction mechanism
was investigated at the atomic level.
(m, 3H, ArH), 8.50 (s, 1H, CH N); 13C NMR (125 MHz, CDCl3, δ):
=
37.9 (CH2Ph), 64.4 (CH), 68.9 (OCH2), 100.7 (C-3), 104.1 (CaroH,
C-7), 108.1 (Caro, C-5), 120.0 (CaroH), 122.4 (CaroH), 127.2 (CaroH),
128.9 (CaroH), 129.2 (Caro), 130.1 (Caro), 135.8 (Caro), 141.9 (Cq,
–
JCF=130 Hz, CF3), 157.0 (C-4), 158.6 (Caro and C OH), 160.0 (C=N),
176.2 (C=O) ppm. FTIR (ATR): ν = 3551, 3419, 3061, 3028, 2946,
2855, 1723, 1629, 1575, 1496, 1381, 1284 cm−1; LC-MS (ESI-QTOF):
m/z [M-H]+ calcd for C20H16 F3NO4, 390.0953; found, 390.0977.
To investigate the mutagenic effects and antioxidant activity of
D3, Ames/Salmonella assay and diphenyl-2-picrylhydrazyl (DPPH)
radical scavenger test were performed. The cytotoxic activity of D3
was also studied via MTT assay using human breast cancer (MCF-
7), human epithelial cervical cancer (HeLa) and rat prostate cancer
(MAT-Lylu).
2.2.2. DNA binding assay
DNA binding assay was performed using the UV–vis absorption
titration method in Tris-HCl/NaCl buffer (pH 7.2) at room temper-
ature. The purity of the CT-DNA solution in Tris-HCl / NaCl buffer
was confirmed as 1.9 ratio which is the ratio of UV absorbance val-
ues at 260 and 280 nm wavelengths. This ratio indicates that the
CT-DNA is free of protein impurities [57,58]. The experiment was
carried out by keeping the D3 concentration (30 μM) constant in
the buffer and adding increasing concentrations (0–270 μM) of CT-
DNA. The solutions were incubated for 5 min at room temperature,
and then spectra were recorded. The percentage ratio of changes in
the absorbance intensity of D3 was calculated by the following for-
2. Materials and methods
2.1. Materials and equipments
Reagent quality solvents were used without further purifica-
tion. Column chromatography was conducted on silica gel 60 (40–
63 μM) (Merck). TLC was carried out on aluminum sheets pre-
coated with silica gel 60F254 (Merck). IR spectra were determined
on a Thermo Scientific NICOLET IS10 spectrometer. NMR spectra
were recorded on Bruker Avance III 500 MHz spectrometer. Chem-
ical shifts, δ are reported in ppm with TMS as internal standard
and the solvents are CDCl3. LC-MS (QTOF) spectra were obtained
on Agilent G6530B model TOF/Q-TOF Mass Spectrometer. The syn-
thesis of compounds 1 and 2 were carried out according to the
literature procedure [55,56].
%H = A − A / A × 100
[(
s) ( i)]
(1)
i
in this equation, Ai indicates the free absorbance intensity of the
compound, and as indicates the absorbance intensity of the com-
pound after adding DNA at the maximum concentration.
The intrinsic binding constant (Kb) used to investigate the bind-
ing strength of the D3 molecule to DNA was calculated using
Eq. (2). In the formula, εA; damping coefficient in the measured
concentross, εb; damping coefficient after binding of all com-
pounds to DNA and εf; damping coefficient of the free compound.
In the [DNA]/(εA − εf) versus [DNA] plot, the ratio of the slope to
the point of intersection gives Kb [57].
Calf thymus DNA (CT-DNA), 1,1-Diphenyl-2-picril-hydrazine
(DPPH), ethanol, magnesium sulfate heptahydrate (MgSO4.7H2O),
citric acid monohydrate (C6H8O7.H2O), potassium phos-
phate (K2HPO4), sodium dihydrogen phosphate monohydrate
(NaH2PO4.H2O), sodium hydroxide (NaOH), sodium chloride
(NaCl), disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O),
potassium chloride (KCl) agar (Difco), nutrient broth (Oxoid no: 2)
L-histidine, D-biotin, sodium ammonium phosphate tetrahydrate
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
DNA / ε − εf
=
DNA / ε − εf + 1 / K εB − εf
(2)
[
]
[
]
A
B
b
2