Synthesis, structural characterisation and biological evolution of chromanones

Article abstract of DOI:10.1016/j.molstruc.2017.05.013

Four new chromanone derivatives (4a-d) are synthesized and evaluated for their DNA binding, cleavage and antioxidant activities. The synthesized compounds are characterized by Fourier Transform Infrared (FTIR), ¹H and ¹³C nuclear magnetic resonance (NMR), mass spectrometry, UV–Visible, thermal and fluorescence spectral studies. Finally, structures of the compounds 4a and 4b are confirmed by single crystal X-ray diffraction method. The DNA binding study is carried out by electronic absorption spectroscopy and fluorescence spectroscopy. The observation from the results is that the chromanone derivatives 4a-d binds to CT-DNA in an intercalation mode. The DNA cleavage studies are investigated using supercoiled pET28a plasmid DNA by agarose gel electrophoresis. These studies reveal that the compounds act as efficient cleaving agents even in the absence of an oxidative agent H₂O₂. The antioxidant studies of these compounds in vitro are evaluated and compared with that of standard drug ascorbic acid (AA).

Full text of DOI:10.1016/j.molstruc.2017.05.013

Accepted Manuscript
Synthesis, structural characterisation and biological evolution of chromanones
Mayuri Bheemarasetti, P. Kavitha, Srinivas Basavoju, G. Bhargavi, K. Laxma
Reddy
PII:
S0022-2860(17)30596-3
10.1016/j.molstruc.2017.05.013
MOLSTR 23754
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
17 October 2016
27 April 2017
04 May 2017
Please cite this article as: Mayuri Bheemarasetti, P. Kavitha, Srinivas Basavoju, G. Bhargavi, K.
Laxma Reddy, Synthesis, structural characterisation and biological evolution of chromanones,
Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.05.013
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ACCEPTED MANUSCRIPT
Highlights



New chromanones (4a-d) were synthesized and characterized.
The X-ray crystal structures of the chromanones 4a and 4b were elucidated.
DNA binding constant Kb values indicate that chromanones bind with DNA possibly by
an intercalative mode.


All the chromanones were showed good ability to cleave the pET28a plasmid DNA.
Antioxidant activity of the chromanoes was examined.

ACCEPTED MANUSCRIPT
Synthesis, structural characterisation and biological evolution of
chromanones
Mayuri Bheemarasettia, P. Kavithaa Srinivas Basavojua, G. Bhargavib, K. Laxma Reddya,*
,
aDepartment of Chemistry, National Institute of Technology, Warangal-506 004, Telangana, India.
bSchool of Chemistry, University of Hyderabad, Hyderabad, Gachibowli, 500 046, Telangana,
India.
Email: laxmareddychem12@gmail.com
Tel: +91870-2462663, Fax: +91-870 2459547
Abstract
Four new chromanone derivatives (4a-d) are synthesised and evaluated for their DNA binding,
cleavage and antioxidant activities. The synthesized compounds are characterized by Fourier
1
13
Transform Infrared (FTIR), H and C nuclear magnetic resonance (NMR), mass spectrometry,
UV-Visible, thermal and fluorescence spectral studies. Finally, structures of the compounds 4a and
4b are confirmed by single crystal X-ray diffraction method. The DNA binding study is carried out
by electronic absorption spectroscopy and fluorescence spectroscopy. The observation from the
results is that the chromanone derivatives 4a-d binds to CT-DNA in an intercalation mode. The
DNA cleavage studies are investigated using supercoiled pET28a plasmid DNA by agarose gel
electrophoresis. These studies reveal that the compounds act as efficient cleaving agents even in the
absence of an oxidative agent H2O2. The antioxidant studies of these compounds in vitro are
evaluated and compared with that of standard drug ascorbic acid (AA).
Keywords: Antioxidant activity, Chromanones, DNA binding, cleavage, Single crystal X-ray
diffraction
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1.
Introduction
Due to significant biological activities, the chromanones have attracted much attention in
current research. Most of the biologically active molecules from both synthetic and natural
compounds contain chromanone moiety as a core structure. These are also minor constituents of
human diet [1]. Several chromanone derivatives exhibit a significant biological and
pharmacological activities such as antimicrobial, antioxidant, DNA binding, cleavage, anticancer,
antiallergic, neuroprotective and pesticidal agents [2–10]. Emami et al. reported
imidazolylchromanone, (1,2,4-triazolyl)chromanone and their oxime derivatives as anticonvulsants
[11]. Agostinha et al. and Lamprontia et al., reported 2-methyl-4-chromanone with efficient activity
against in vitro cell growth of human K562 cells [12, 13]. Some of the chromanones also showed a
potential application in the treatment of status epilepticus [14]. Dawood et al. has reported
chromanone derivatives as antihuman-immunodeficiency-virus (HIV-1) [15]. 6-hydroxy-7-
methoxy-4-chromanones are evaluated for their potential antioxidant activities [16]. The derivatives
of chromanone show a significant function in biotic and abiotic stress response phenomena in plants
[17]. Consequently, chromanone chemistry extends its importance in the area of synthesis and
medicinal chemistry.
A well-known method to synthesize chromanone derivatives is the reaction of
3-formylchromone with an aromatic amine. The 3-formylchromone (4-oxo-4H-1-benzopyran-3-
carboxaldehyde) is an important precursor to synthesise the derivatives of chromones and
chromanones. According to literature, the reaction between 3-formylchromone and primary
aromatic amines leads to form various products depending on the nature of the amine and/or
experimental conditions [18–20]. Even several reports presented on the synthesis and biological
properties of chromanones in literature, a fewer reports dealt with structural determination by single
crystal X-ray diffraction. Thus, in this paper, we aim to study the Synthesis, structural
characterisation and biological evolutions of chromanones.
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2. Experimental
2.1. Chemicals and instrumentation
All the used chemicals and solvents were purchased from MERCK, SD Fine and Himedia
chemical companies. Melting points of the chromanones were determined with Stuart SMP30 and
were uncorrected. The progress of the reactions is monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 aluminium sheets (MERCK). The FTIR spectra of the compounds were
recorded on Perkin-Elmer 100S spectrometer within the range 4000-400 cm–1 using the KBr disc.
13
1H and C NMR spectra of the compounds were recorded on a Bruker-Avance III HD 400 MHz
spectrometer in CDCl3 using tetramethylsilane (TMS) as an internal standard. Mass spectra were
recorded on a Jeol JMSD-300 spectrometer. The electronic spectra of the compounds are recorded
on Evolution 300 UV-Visible spectrophotometer in the range of 200-800 nm. The fluorescence
spectra of the compounds were recorded on Horiba yvon Flurolog Spectrophotometer. The
thermogravimetric measurement was recorded on TA instruments (SDT 200) at a heating rate of
20°C and a nitrogen flow rate of 20 mL/min.
2.2.
Crystal structure determination of chromanones 4a and 4b
Yellow coloured crystals of chromanones 4a and 4b for X-ray structure determination were
obtained by slow evaporation technique. Suitable single crystals of the dimensions 0.50×0.30×0.28
mm3 of 4a and 0.50×0.34×0.30 mm3 of 4b were picked by observing under the LEICA DFC295
polarizing microscope. The single crystal X-ray data of the chromanones were collected on a
Bruker Kappa APEX-II CCD DUO X-Ray diffractometer at 23 °C using graphite-monochromated
Mo Kα radiation (λ = 0.71073 Å). No absorption correction was carried out. The total reflections of
single crystal 4a was measured 26924 in a 2θ range of 1.21 to 24.99°, and those of single crystal 4b
was measured 28332 in a 2θ range of 2.25 to 24.99°; 2687 and 2862 independent reflections were
measured for 4a and 4b, respectively. The unit cell dimensions were determined by least-squares
analysis and the reflection data was integrated using the SHELXTL program [21].The crystal
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structures were solved by direct methods using SHELXS-97, and refinement is carried out by full-
matrix least-squares technique (F2) using SHELXL-97 [22]. Anisotropic displacement parameters
are included for other than H-atoms. The N−H hydrogen atoms are located from difference Fourier
maps. All the aromatic and aliphatic C−H hydrogens were positioned geometrically and treated as
riding on their parent C-atoms with C−H distances of 0.93-0.97 Å. The software Mercury 2.3 (Build
RC4), ORTEP-3 and X-Seed was used to prepare material for publication [23].
2.3.
General procedure for the synthesis of chromanones
4a-d
3-Formylchromone was prepared by the Vilsmeier-Haack synthesis [24]. Chromanones 4a-d
were prepared by adding o-anisidine (0.11 mL, 1 mM) dropwise to the 3-formylchromone (0.174 g,
1 mM) in different solvents (methanol (a), ethanol (b), 2-propanol (c) and 1-butanol (d)) with
continuous stirring for 1 h. All the compounds (4a-d) formed are yellow in colour. The products
thus obtained were recrystallized from their corresponding solvents. All the experimental details
were given in Scheme 1.
2-methoxy-3-(((2-methoxyphenyl)amino)methylene)chroman-4-one (4a)
Yellow solid; M.P 117-119 °C; IR (KBr, cm−1): 3169 (N–H), 3058 (aromatic C–H), 2977 (aliphatic
C–H), 1748, 1649 (C=O), 1283 (C–N); 1H-NMR (400 MHz, CDCl3): δH (ppm) 3.52 (s, 3H), 3.97 (s,
13
3H), 5.70 (s, 1H), 6.93–8.03 (m, 9H), 12.20 (d, J = 12.4 Hz, 1H, NH). C-NMR (100 MHz,
CDCl3): δC (ppm) 55.3, 55.9, 101.9, 103.7, 111.2, 113.6, 117.7, 121.0, 122.0, 123.0, 124.3, 126.3,
129.1, 134.0, 142.9, 148.7, 155.6, 180.8. ESI mass spectrum (m/z): calcd. for [C15H8N2O]: 311.12,
obsd.; 334 (M+Na), 280 fragment.
2-ethoxy-3-(((2-methoxyphenyl)amino)methylene)chroman-4-one (4b)
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Yellow solid; M.P 119-120 °C; IR (KBr, cm−1): 3169 (N–H), 3058 (aromatic C–H), 2977 (aliphatic
C–H), 1748, 1649 (C=O), 1283 (C–N); 1H NMR (400 MHz, CDCl3): δH (ppm) 1.20 (t, J = 7.1 Hz,
3H), 3.71 (q, J = 7.1 Hz, 2H), 3.96 (s, 3H), 5.81 (s, 1H), 6.92-7.54 (m, 8H), 8.02 (dd, J = 7.8 Hz, J
13
=1.7 Hz, 1H), 12.18 (d, J = 12.7 Hz, 1H, NH). C NMR (100 MHz, CDCl3): δC (ppm) 15.1, 55.9,
63.6, 100.7, 103.9, 111.2, 113.7, 117.7, 121.0, 121.9, 123.1, 124.2, 126.4, 129.3, 134.0, 142.8,
148.7, 155.8, 181.0. ESI mass spectrum (m/z): calcd. for [C15H8N2O]: 325.13, obsd.: 326 (M+1),
280 (base peak).
2-isopropoxy-3-(((2-methoxyphenyl)amino)methylene)chroman-4-one (4c)
Yellow solid; M.P 127-128 °C; IR (KBr, cm−1): 3164 (N–H), 2936 (aliphatic C–H), 1642 (C=O),
1
1282 (C–N); H NMR (400 MHz, CDCl3): δH (ppm) 1.16 (d, J = 6.2 Hz, 3H), 1.26 (d, J = 6.2 Hz,
3H), 3.97 (s, 3H), 4.20 (hept, J = 6.2 Hz, 1H,), 5.90 (s, 1H), 6.95–7.51 (m, 8H), 8.02 (dd, J= 7.8 Hz,
J =1.7 Hz, 1H,), 12.17 (d, J = 12.7 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3): δC (ppm) 22.0, 23.3,
55.9, 70.0, 99.2, 104.2, 111.2, 113.7, 117.7, 121.0, 121.8, 123.1, 124.1, 126.4, 129.3, 133.9, 142.5,
148.7, 155.9, 181.2. ESI mass spectrum (m/z): calcd. for [C20H21NO4]: 339.15, obsd: 340.10 [M+1],
280.05 (base peak).
2-butoxy-3-(((2-methoxyphenyl)amino)methylene)chroman-4-one (4d)
Yellow solid; M.P 123-125 °C; IR (KBr, cm−1): 3167 (N–H), 2997 (aromatic C–H), 2937 (aliphatic
1
C–H), 1648 (C=O), 1282 (C–N); H NMR (400 MHz, CDCl3): δH (ppm) 0.86 (t, J = 7.4 Hz, 3H),
1.29 (sext, J = 7.4 Hz, 2H), 1.55 (quint, J = 7.4 Hz, 2H), 3.75 (t, J = 7.4 Hz, 2H), 3.97 (s, 3H), 5.79
(s, 1H), 6.93–7.11 (m, 5H), 7.20 (d, J = 11.6 Hz, 1H,), 7.42–7.46 (m, 1H), 7.52 (d, J = 12.8 Hz,
13
1H), 8.02 (dd, J= 7.8 Hz, J =1.7 Hz, 1H,) 12.18 (d, J = 12.7 Hz, 1H, NH). C NMR (100 MHz,
CDCl3): δC (ppm) 13.8, 19.2, 31.5, 55.9, 67.9, 100.9, 104.0, 111.2, 113.7, 117.7, 121.0, 121.9,
123.1, 124.2, 126.3, 129.3, 134.0, 142.7, 148.7, 155.8, 181.1. ESI mass spectrum (m/z): calcd. for
[C21H23NO4]: 353.16, obsd: 354 [M+1], 280.05 (base peak).
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2.4.
DNA binding studies
2.4.1. Absorbance spectroscopic studies
Stock solution of CT-DNA was prepared in 5 mM Tris–HCl/50 mM NaCl in nuclease free
water at pH = 7.2 and are kept it overnight at –20 °C. The concentration of CT-DNA stock solution
is determined at 260 nm by UV absorbance intensity using the molar absorption coefficient as 6600
M−1 cm−1. Solution of CT-DNA in 5 mM Tris-HCl/50 mM NaCl (pH = 7.2) gave a ratio of UV
absorption at 260 nm and 280 nm A260/A280 of 1.82, indicating that the DNA is sufficiently free
from protein [25]. Stock solutions were stored at 4 °C and used within four days. The electronic
absorption spectra were recorded using Evolution 300 UV-Visible spectrophotometer. Electronic
absorption titrations were performed at constant concentration of the compounds (80 μM) and by
increasing the concentration of CT-DNA from 0.026×10-4 M to 0.261×10-4 M. For the compounds
4a-d, from the absorption titration data, the binding constants (Kb) were determined using the
following equation [26].
[DNA] / (εa–εf) = [DNA] / (εb–εf) + 1 / Kb (εb–εf)
Where εa is apparent extinction coefficient and it is obtained by calculating Aobsd/ [Compound]. The
expressions εf correspond to the extinction coefficients of free (unbound) and εb correspond to the
extinction coefficients of fully bound complexes. A plot of [DNA]/(εa–εf) vs [DNA] will give a
slope of 1/(εb–εf) and an intercept of 1/Kb (εb–εf). The binding constant (Kb) was obtained by the
ratio of the slope and intercept.
2.4.2. Fluorescence spectroscopic studies
Quantitatively to compare the binding affinity of the chromanones 4a-d to CT-DNA, the intrinsic
binding constant K of the compounds were obtained by the fluorescence spectroscopic titrations.
The compounds (4a-d) with 10 µM concentrations were titrated against the CT-DNA with
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increasing concentrations from 0.15 to 0.6 μM. All the compounds were excited at 358nm and
emission was recorded at 415-650 nm in 5 mM Tris–HCl/50 mM NaCl buffer. The concentration of
the bound compound was calculated by using the following formula [27]
Cb= Ct [(F - F0) / (Fmax– F0)]
Where, Ct is the total compound concentration, F is the observed fluorescence emission intensity at
given DNA concentration, F0 is the intensity in the absence of DNA, and Fmax is the fluorescence of
the totally bound compound. Binding constant K value was obtained from the Scathchard plot [28]
of r/Cf versus r, where r is the binding ratio Cb/[DNA] and Cf is the free ligand concentration.
EB displacement studies:
Further support for compounds binding to DNA by intercalation mode was given through the
emission quenching experiment. EB is a common fluorescent probe for DNA structure and has been
selected for the examinations of the mode and process of compound binding to DNA [29]. A 3ml
solution of 1.5μM DNA and 1 x 10-5M EB was titrated by 1.5 to 6.0 μM compound (λex = 520 nm,
λex = 550.0–750.0 nm). According to the classical Stern–Volmer equation [30]:
F0/F = Kq [Q] + 1
Where F0 is the emission intensity in the absence of quencher, F is the emission intensity in the
presence of quencher, Kq is the quenching constant, and [Q] is the quencher concentration. Plots of
F0/F versus [Q] appear to be linear and Kq depends on temperature.
2.5.
DNA cleavage activity
The DNA cleavage experiments of supercoiled pET28a plasmid DNA was determined by agarose
gel electrophoresis. Chromanone stock samples (1 mg/mL) were prepared using DMSO solvent.
From stock solution, 100 µg/mLsolutions were prepared by using Tris-HCl buffer, from which 15
µL is added to the isolated pET28a plasmid DNA. The samples were incubated at 37 °C for 2 hrs
and then 30 µL of DNA sample along with loading dye was directly loaded on 0.8% agarose gel for
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analysis by gel electrophoresis at 70 V for 2 hrs. After electrophoresis, the gel was stained with
ethidium bromide (10.0 mg/mL) and the gel images were captured by Mediccare GELSTAN gel
documentation system and the extent of DNA cleavage was quantitatively calculated by labImage
1D software.
2.6.
Antioxidant activity
The antioxidant activity of the chromanones 4a-d was determined by using 2,2-diphenyl-1-
picrylhydrazyl (DPPH) as per the literature [31, 32] and ascorbic acid used as standard. 0.004%
DPPH stock solutionin methanol was prepared. The chromanones 4a-d and ascorbic acid were
dissolved in methanol 10 mg/10 mL and they were used as stock solution. Different concentrations
of test compounds (0.025-0.100 mg/mL) were prepared from the stock solutions, by using
methanol. For each 1 mL test compound of different concentrations, 3 mL of methanolic solution of
0.004% DPPH was added. The test compounds were incubated at room temperature for 30 min.
After 30 minutes, the absorbance of the test compounds was recorded at 517 nm using UV-Visible
spectrophotometer, which was compared with the corresponding absorbance of standard ascorbic
acid (0.025-0.100 mg/mL). Methanol was used as blank and DPPH solution was used as control.
The scavenging activity of DPPH in percentage was calculated by using the formula as given below
(
)
퐴표 ‒ 퐴푖
퐴표
푆푐푎푣푒푛푔푖푛푔푎푐푡푖푣푖푡푦(%) =
× 100
[
]
Where Ao is the absorbance of the control and Ai is the absorbance of the sample. IC50 values of
chromanones 4a-d were calculated.
3. Results and discussion
Four chromanone derivatives (4a-d) were synthesised by the reaction of 3-formylchromone
with o-anisidine in different solvents like methanol, ethanol, 2-propanol and 1-butanol. As it is
evidenced from the literature [19], the solvent molecules (alcohols) underwent Michael addition
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with compound 2 at position-2. Subsequently, 2-methoxyaniline (3) reacts with 3-formylchromone
(2)
and
obtained
the
derivatives
of
chromanone
(2-methoxy-3-(((2-
(2-ethoxy-3-(((2-
2-isopropoxy-3-(((2-
2-butoxy-3-(((2-
methoxyphenyl)amino)methylene)chroman-4-one)
methoxyphenyl)amino)methylene)chroman-4-one)
methoxyphenyl)amino)methylene)chroman-4-one,
4a,
4b,
4c,
methoxyphenyl)amino)methylene)chroman-4-one, 4d. Ligands 4a and 4b were obtained as yellow
crystals from the slow evaporation of the solvents methanol and ethanol respectively in 7-days
period of time and they were stable at room temperature. The presence of the alkoxy groups which
were obtained from solvent molecules (alcohols) was confirmed by all the spectral methods (1H
NMR, 13C NMR, mass, FTIR and X-ray crystallography).
3.1.
Spectral analysis
In all the compounds (4a-d) IR stretching frequencies found in the range of 3169-3164 cm‒1
indicate the presence of N‒H group. Similarly, the stretching frequencies around 1282 cm‒1
corroborate the presence of C‒N group in 4a-d. The presence of these N‒H and C‒N stretching
frequencies indicate that the imine (CH=N) group existing in its tautomeric structure (=C‒NH).
This was further confirmed by single crystal X-ray diffraction.
1
In the H NMR spectrum, the methoxy (–OCH3) group which was attached at 2-position of the
chromanone 4a gave a singlet at δ 3.52 indicating that the methanol underwent Michael addition
with compound 2. Similarly the quartet at δ value 3.71 (2H) and a triplet at δ 1.20 (3H) with same
coupling constant values (J = 7.1 Hz) in 4b indicate the presence of ethoxy group which has come
from ethanol. In compound 4c, a septet at a δ value 4.20 and two doublets at δ values 1.16 and 1.26
with same coupling constant values (J = 6.2 Hz) confirms the presence of an isopropoxy group. The
presence of n-butoxy group at 2-position of the chromanone moiety was confirmed by the presence
of a triplet at a δ value 3.75 (2H), 1.55 quintet (2H), 1.29 sextet (2H) and a triplet at 0.86 (3H) with
same coupling constant values (J = 7.4 Hz) in 4d. However, the confirmation of the Michael
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addition of solvent molecules (alcohols) at the 2-position of the parent compound 3-
formylchromone (2) is the hydrogen atom at the 2-position. The proton at 2-position in 3-
formylchromone shows a singlet at δ 8.56 i.e., in the aromatic region [33], where as in the case of
compounds 4a, 4b, 4c and 4d, the proton at 2-position gave a singlet at δ values 5.70, 5.81, 5.90 and
5.79 respectively i.e., the δ values were shifted to upfield region. This is due to the Michael addition
of alcohols (solvents) at 2-position.
13
The C NMR spectrum of compound 4a show two peaks at δ 55.3 and 55.9 confirms the
presence of –OCH3 group at 2-position of chromanone moiety and the aromatic –OCH3 group
respectively. Similarly, δ 15.1 and 55.9 in 4b indicates the presence of -OCH2-CH3. The aromatic –
OCH3 appears at δ 63.6. The aliphatic peaks at δ 22.0, 23.3 and 55.9 corroborate the presence of
isopropoxy group in compound 4c. The aromatic –OCH3 group appears at δ 70.0 in 4c. In the
compound 4d the δ valuesat13.8, 19.2, 31.5 and 55.9 indicate the presence of -O-CH2-CH2-CH2-
CH3 on chromanone moiety at 2-position. The aromatic –OCH3 group appears at a δ value of 67.9.
However, in the parent compound 3-formylchromone (2) the C-atom at 2-position shows a peak at a
δ value of 172.5 [34]. But in case of compounds 4a, 4b, 4c and 4d, the peak for C-atom at 2-
position appeared at δ 101.9, 100.7, 99.2 and 100.9 respectively. The δ values are shifted to upfield
in the final compounds compared to 3-formychromone. This is due to the Michael addition of
alcohols at 2-position.
The mass spectral fragmentation analysis of all the compounds 4a-d, was observed that the
presence of a fragment with m/z value 280 confirming the cleavage of alkoxy group from the 2-
position of chromanone moiety, which was attached from the respective solvents during the
synthesis.
3.2.
Crystal Structure Analysis
Chromanones 4a and 4b crystallize in the centrosymmetric orthorhombic Pbcn space group
with one molecule in the asymmetric unit [Fig. 1 and 4]. The unit cell values show that the two
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crystal structures of 4a and 4b are isomorphous and isostructural. It was found that in 4a both (R)-
and (S)-chromanone stereoisomers were crystallized in the crystal structure (racemate). The crystal
structure analysis of chromanone 4a reveals that the molecules form ‘V’-shaped 1D-ribbon like
structure C–H···O hydrogen bonds. Both the chromanone moiety and methoxyphenyl moiety both
are almost coplanar with each other due to intramolecular N–H···O hydrogen bonding. This
intramolecular hydrogen bonding is due to the interaction of N–H group of tautomeric form of
imine group with the oxygen atom of chromanone moiety carbonyl group and oxygen atom of
methoxy phenyl group [N1–H1A···O2; D = 2.6843(18) Å, d = 1.91 Å, θ = 134º; N1–H1A···O4; D
= 2.5980(17) Å, d = 2.19 Å, θ = 102º]. The hydrogen atom of the methoxyphenyl group of (R)-
enantiomer interacts with translation related chromanone carbonyl oxygen of another (R)-isomer
via C–H···O (C18–H18C···O2; D = 3.484(2) Å, d = 2.53 Å, θ = 146º) hydrogen bond; and the
hydrogen atom of the phenyl group interacts with translation related chromanone methoxy group O-
atom via C–H···O (C7–H7···O3; D = 3.535(2) Å, d = 2.55 Å, θ = 151º) hydrogen bonds. These
interactions are extended along the crystallographic b-axis and form a 1D-ribbon like structure [Fig.
2]. Another 1D-ribbon like structure is formed perpendicular to the above structure by (S)-
enantiomers with the same hydrogen bonds forming a 'V'-shaped pattern. These 'V'-shaped structure
is interacting with adjacent inverted 'V'-shaped structure via C–H···O hydrogen bonds [Fig. 3].
Further, the 1D-ribbon like structures are stabilized by weak N–H···π (N1–H1A···πC4-C5-C6-C7-C8-C9
;
d = 3.664 Å, 102.94º) and C–H···π [C7–H7···πC2; D = 3.568 Å; d = 3.192 Å, θ = 106.35º; C18–
H18B···πC16-C17; d = 3.131 Å, θ = 150.51º] interactions.
The crystal structure analysis of chromanone 4b reveals that the molecules form ‘V’-shaped
1D-ribbon like structure with C–H···O hydrogen bonds. In this structure also both the chromanone
moiety and methoxyphenyl moiety both are almost coplanar with each other due to intramolecular
N–H···O hydrogen bonding. This intramolecular hydrogen bonding is due to the interaction of N–H
group of tautomeric form of imine group with the oxygen atom of chromanone moiety carbonyl
group and oxygen atom of methoxy phenyl group [N1–H1A···O2; D = 2.682(2) Å, d = 1.92 Å, θ =
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130º; N1–H1A···O4; D = 2.598(3) Å, d = 2.18 Å, θ = 103º]. The mode of interactions in this
crystal structure is also very similar to the compound 4a. The methyl hydrogen atom of
methoxyphenyl group of (R)-enantiomer interacts with translation related chromanone carbonyl
oxygen of another (R)-isomer via C–H···O (C19–H19C···O2; D = 3.431(3) Å, d = 2.49 Å, θ =
144º) hydrogen bond; and the hydrogen atom of the phenyl group interacts with translation related
chromanone ethoxy group O-atom via C–H···O (C7–H7···O3; D = 3.431(3) Å, d = 2.46 Å, θ =
148º) hydrogen bonds. These interactions are extended along the crystallographic b-axis and form a
1D-ribbon like structure as in 4a [Fig. 5]. Another 1D-ribbon like structure is formed perpendicular
to the above structure by (S)-enantiomers with the same hydrogen bonds forming a 'V'-shaped
pattern. These 'V'-shaped structure is interacting with adjacent inverted 'V'-shaped structure via C–
H···O hydrogen bonds and extended along the crystallographic b-axis [Fig. 6]. Further, the 1D-
ribbon like structures are stabilized by weak N–H···π (N1–H1A···πC4-C5-C6-C7-C8-C9; d = 3.596 Å,
104.67º) and C–H···π [C7–H7···πC2; D = 3.517 Å; d = 3.180 Å, θ = 103.64º; C18–H18···πC4-C5; d =
3.126 Å, θ = 160.02º] interactions. All the pertinent crystallographic data and geometrical
parameters of hydrogen bonds are given in Tables 1 and 2.
3.3.
Fluorescence studies
The emission spectra of all the compounds were recorded in the solid state and shown in Fig. 7.
The chromanones 4a-d were characterised by emission bands at 504, 507, 512 and 492 nm upon
photo excitation at 400 nm. All the four compounds show the emission bands in the green light
region. Chromanone-4d shows more hyperchromic and hypsochromic shift when compared to the
remaining chromanones (4a-c). The intensity of the emission bands of the chromanones increases
with increase in the number of carbons of alkoxy group which are attached at 2-position of
chromanone ring except in the case of 4c.
3.4.
Thermal study
12

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The thermal stability of chromanone 4a was studied in the range of 25-800 °C in air
atmosphere. The thermograph of chromone derivative is presented in Fig. 8. The TG curve shows
three decomposition steps. The chromone derivative is stable up to 130 °C. The first decomposition
step observed in temperature range of 130-200 °C, may be due to the loss of solvent molecule. The
second and third steps are observed in the range of 201-340 °C and 341-670 °C may be due to the
loss of chromone and remaining organic part present in the analysed compound.
3.5.
DNA binding studies
3.5.1. Absorbance spectroscopic studies
The DNA interaction properties of the chromanones on CT-DNA were studied by using
electronic absorption titration technique. The experiments were carried out by maintaining a
constant concentration of the chromanone and varying the concentration of CT-DNA. The
electronic absorption spectra of the chromanone (4a) in the absence as well as presence of CT-DNA
were shown in Fig. 9. The results show decrease in intensity with increasing concentration of the
CT-DNA. This hypochromic effect is observed in all the chromanones. It indicates an intercalative
mode of binding between an aromatic chromophore and the base pairs of DNA [34]. The
hypochromism is commonly extent with the strength of DNA interaction. As it is evidenced from
the crystal structures of the chromanones 4a and 4b which exhibit the C–H···O and C–H···π
interactions, these results indicate that the planar aromatic rings of the chromanone molecules (4a
and 4b) bind to DNA helical structure in an intercalation mode via hydrogen bonding and π···π
stacking interactions [35]. The intrinsic binding constant (Kb) for chromanones 4a-d was found to
be 1.33×104M–1, 2.66×104 M–1, 3.11×104 M–1 and 4.70×104 M–1, respectively. From the observed
Kb values, it was evident that chromanone 4d binds more strongly to CT-DNA when compared to
remaining chromanones. Further, binding affinity increases with rise in the number of carbons of
alkoxy group which were attached at 2-position of chromanone ring.
13

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3.5.2. Fluorescence spectroscopic studies
The enhancements in the emission intensity of the chromanone 4d with increasing CT-DNA
concentration are shown in Fig. 10. In the absence of DNA, chromanones 4a, 4b, 4c and 4d emit
weak fluorescence in Tris buffer at ambient temperature with maximum emission wavelengths 451,
457, 458 and 457 nm, respectively. In the presence of DNA,the emission intensity of chromanones
(4a-d) increases with respect to DNA concentration. However, the corresponding 3, 7, 5 and 7 nm
blue shifts were observed with the increase of emission intensities. This phenomenon is related to
the extent to which the compounds penetrate into the hydrophobic environment inside the DNA,
thereby avoiding the quenching effect of solvent water molecules. The binding of compounds to
CT-DNA leads to a marked increase in the emission intensity, which also agrees with those
observed for other intercalators [28-30]. According to the Scathchard equation, a plot of r/Cf versus
r gave the binding constants of 2.56×104, 3.29×104, 4.12×104 and 6.07×104 M–1 from the
fluorescence data for the compounds 4a, 4b, 4c and 4d, respectively. These results show that the
chromanone 4d binds more strongly compared to the remaining chromanones.
Ethidium bromide (EB) displacement studies
The mode of interaction of the compounds (4a-d) with DNA was further investigated by the
competitive binding experiment using EB. Even though, EB is a weak fluorescent compound, its
fluorescent intensity can be greatly enhanced in the presence of DNA due to its strong intercalation
between the adjacent DNA base pairs. A compound’s competitive binding to EB-bound DNA can
reduce the emission intensity due to displacement of bound EB from DNA [36]. The fluorescence
quenching spectrum of DNA–EB system by the 4d compound is shown in Fig. 11.The fluorescence
emission intensity of the DNA–EB system decreased with the increase of the compound
concentration. The resulting decrease in fluorescence was caused by EB changing from a
14

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hydrophobic environment to an aqueous environment [37]. Such a characteristic change is often
observed in intercalative DNA interactions [38-41]. The quenching plots illustrate that the
quenching of EB bound to DNA by the compounds 4a-d was in good agreement with the linear
Stern-Volmer equation. In the plots of F0/F versus [Q],Kq is given by the ratio of the slope to the
intercept. The Kqvalues for the compounds 4a, 4b, 4c and 4d are 1.95×103, 2.88×103, 5.97×103 and
7.09×103 respectively. This data suggests that the interaction of the compound 4d with CT-DNA is
stronger than that of the remaining compounds, which is consistent with the above absorption
spectral results.
3.6.
DNA cleavage activity
The chromanone derivatives (4a-d) were investigated for their DNA cleavage ability by agarose gel
electrophoresis method using pET28a plasmid DNA in the absence as well as presence of an
oxidizing agent H2O2 [Fig.12 and 13]. The cleavage activity of chromanones 4a-d towards SC
pET28a DNA has been investigated by measuring the extent of formation of nicked circular (NC)
DNA. In the presence of oxidative agent H2O2, all chromanones show more cleavage activity than
in the absence of H2O2. The DNA cleavage activity of the chromanones (4a-d) increases with
increasing the carbon chain in the chromanones (4a < 4b < 4c < 4d).
3.7.
Antioxidant activity
Synthesized chromanones (4a-d) possess moderate scavenging activity. The scavenging activity
of chromanones 4a-d was increased with the increasing concentration of the compound [Fig. 14].
Ascorbic acid (AA) was used as the standard (IC50 value 0.052 mg/mL). The IC50 values of the
compounds were 0.677 (4a), 0.654 (4b), 0.402 (4c) and 0.445 (4d) mg/mL. Chromonone 4c
exhibited high activity compared to the remaining chromonones but less activity compared to the
standard drug AA.
4. Conclusion
15

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In this paper, the four chromonones 4a-d have been synthesised by the reaction of 3-
1
formylchromone and o-anisidine in the presence of different solvents and characterised by H
13
NMR, C NMR, mass, FTIR, UV-Visible, fluorescence spectroscopy, and X-ray crystallography.
Compounds 4a and 4b crystallise in centrosymmetric orthorhombic Pbcn space group and it was
found that they were isomorphous. The molecules were connected by C–H···O hydrogen bonds and
form 1D-ribbon like structures parallel to the crystallographic b-axis. The DNA binding properties
of the compounds 4a-d were investigated by absorbance and fluorescence titration methods. The
DNA binding results showed that the chromanones bind to CT-DNA via an intercalative mode with
hydrogen bonding and π···π interactions. The binding affinity of chromanones increases with
increase in the carbon chain attached at 2-position of the chromanone moieties. The chromanones
displayed more DNA cleavage in the presence of oxidative agent H2O2. From the antioxidant
activity results chromanone 4c has shown good scavenging activity when compared to the
remaining chromanones, but less when compared to the standard drug ascorbic acid (AA). It has
also been established that these chromanones show efficient fluorescence in the green light region.
Acknowledgements
The authors wish to thank Dr. Ch. Subramanyam, IIT Hyderabad, Hyderabad, for providing TG
facility. Mayuri is thankful to the Ministry of Human Resource Development (MHRD), India for
research fellowship.
Supplementary data
CCDC reference numbers 1423043 and 1423042 contains the crystallographic data for the
chromanones 4a and 4b. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html.
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OCH3
H2N
O
O
O
O
O
OCH3
H
C
NH
CH3
OH
Anhydrous DMF/POCl3
H
3
O
OR
R-OH
1
2
4a-d
4a
4b
: R = -CH3
: R = -CH2-CH3
: R = CH(CH3)2
4c
4d
: R = -CH2(CH2)2CH3
Chromanone
Solvent
Methanol
Ethanol
R
4a
4b
4c
4d
-CH3
-CH2CH3
-CH(CH3)2
2-Propanol
1-Butanol
-CH2(CH2)2CH3
Scheme 1. Synthesis of chromanones 4a-d.

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Fig. 1. ORTEP representation of chromanone 4a. The thermal ellipsoids are drawn at 50%
probability level.

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Fig. 2. The one-dimensional ribbon-like structure of chromanone 4a along crystallographic b-
axis.

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Fig. 3. Perpendicularly arranged 1D-ribbon like structure connected via C–H···O hydrogen bonds
along the crystallographic b-axis

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Fig. 4. ORTEP representation of chromanone 4b. The thermal ellipsoids are drawn at 50%
probability level.

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Fig. 5. Molecules of chromanone 4b form one-dimensional ribbon like structure along the
crystallographic b-axis.

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Fig. 6. One-dimensional ribbons interact with perpendicularly arranged another layer of 1D-
ribbons with again C–H···O hydrogen bonds.

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Ex - 400 nm
500000
400000
300000
200000
100000
0
4d
4b
4c
4a
450
500
550
600
650
700
750
Wavelength (nm)
Fig. 7. Fluorescence spectrum of chromanones 4a-d excitation at 400 nm

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Fig. 8. TG-DTG graph of chromanone 4a.