Novel quercetin and apigenin-acetamide derivatives: Design, synthesis, characterization, biological evaluation and molecular docking studies

Article abstract of DOI:10.1039/d0ra04559d

Flavonoids exhibit essential but limited biological properties which can be enhanced through chemical modifications. In this study, we designed, synthesized, and characterized two novel flavonoid derivatives, quercetin penta-acetamide (1S3) and apigenin t

Full text of DOI:10.1039/d0ra04559d

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PAPER
Novel quercetin and apigenin-acetamide
derivatives: design, synthesis, characterization,
biological evaluation and molecular docking
studies†
Cite this: RSC Adv., 2020, 10, 25046
a
b
a
a
*
Daniel Isika, Mustafa Çesme, Francis J. Osonga and Omowunmi A. Sadik
¸
Flavonoids exhibit essential but limited biological properties which can be enhanced through chemical
modifications. In this study, we designed, synthesized, and characterized two novel flavonoid derivatives,
quercetin penta-acetamide (1S3) and apigenin tri-acetamide (2S3). These compounds were confirmed
using (1H, 13C) NMR, UV-Vis, and FT-IR characterizations. Their interaction with fish sperm DNA (FS-DNA)
at physiological pH was investigated by UV-Vis and fluorescence spectrophotometry. The binding
constant (Kb) for the UV-Vis experiment was found to be 1.43 ꢀ 0.3 ꢁ 104 Mꢂ1 for 1S3 and 2.08 ꢀ 0.2 ꢁ
104 Mꢂ1 for 2S3. The binding constants (KSV) for the fluorescence quenching experiment were 1.83 ꢁ 104
Mꢂ1 and 1.96 ꢁ 104 Mꢂ1 for 1S3 and 2S3, respectively. Based on molecular modeling and docking
studies, the binding affinities were found to be ꢂ7.9 and ꢂ9.1 kcal molꢂ1, for 1S3 and 2S3, respectively.
The compound–DNA docked model correlated with our experimental results, and they are groove
binders. Furthermore, mutagenicity potential was examined. 1S3 and its metabolites showed no
mutagenic activity for both TA98 and TA100 strains. 2S3 did not show any mutagenic activity for the
strain TA 98, while its metabolites were only active at high doses. Both 2S3 and its metabolites showed
mutagenic activity in the TA100 strain.
Received 23rd May 2020
Accepted 20th June 2020
DOI: 10.1039/d0ra04559d
rsc.li/rsc-advances
functionalization of A- and B-rings and chemical manipulation
of the phenolic hydroxyl groups.10
1. Introduction
Quercetin penta-phosphate and quercetin sulfonic acid are
water soluble and effective in the reduction of Cr4+ ions.19,20
Phosphorylated avonoids have improved aqueous solubility
and metal chelation.20,21 They are reducing, stabilizing and
capping agents in the synthesis of gold, palladium and silver
nanoparticles with enhanced antimicrobial properties.21–23
In this study, we chemically modied quercetin and apige-
nin to yield quercetin- and apigenin-acetamide derivatives. The
rationale is that the acetamide group comprises of a sp3
hybridized carbon which, when incorporated into the avonoid
molecule, will induce exibility and enhance the overall lip-
ophilicity of the resultant derivative. The amide group would
potentially increase hydrogen bonding capability and
Flavonoids are a class of polyphenolic plant metabolites struc-
turally consisting of 15-carbons where two benzene rings are
linked by a three-carbon chain to afford a C6–C3–C6 carbon
skeleton. They possess anticancer,1 antibacterial,2 antifungal3
and anti-inammatory4 properties. They exist in various classes
notably avones (such as apigenin) and avanols (such as
quercetin); structures are shown in Fig. 1. Apigenin occurs in
vegetables and fruits.5 It has anticancer,5–7 anti-inammatory8
and antibacterial9 properties. Quercetin occurs in medicinal
plants, fruits and vegetables.5 It exhibits anti-cancer,10 anti-
bacterial,11 gastroprotective,12 antiviral13 and anti-inamma-
tory14 properties.
Biological applications of avonoids are limited by low
bioavailability,15 fast body clearance,16 low aqueous solu-
bility10,17 enzyme degradation18 and fast body metabolism.10 To
address these limitations, quercetin has been modied by the
aDepartment of Chemistry and Environmental Science, Sensors Mechanisms Research
& Technology (The SMART Center), New Jersey Institute of Technology, 161 Warren
Street, University Heights, Newark, NJ 07102, USA. E-mail: sadik@njit.edu
bDepartment of Chemistry, Faculty of Art and Sciences, Kahramanmaras Sutcu Imam
University, Kahramanmaras, 46040, Turkey
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra04559d
Fig. 1 Structure of apigenin and quercetin.
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interaction sites, of the derivatives. The viability of these PG spectrophotometer (T80+, UK) equipped with 1.0 cm quartz
derivatives to applications in biological systems was evaluated cells. Fluorescence spectra were collected on a PerkinElmer
by biological and molecular docking studies. Acetamide deriv- LS55 luminescence spectrophotometer. All samples were
atives possess antimicrobial24 and anti-inammatory25 activi- prepared in spectrophotometric grade solvents and analyzed in
ties. Acetamides can inhibit hepatitis C virus26 and are possible a 1.0 cm optical path quartz cuvette. Infrared spectra were
analgesics.27 Modanil, levetiracetam, acetaminophen and recorded on a PerkinElmer spectrometer (Spectrum 400)
acetazolamide are some acetamide-based drugs.
equipped with ATR accessory. A pH meter (Thermo Scientic
Nucleic acids are powerful tools in the recognition and Orion Star A215 pH/conductivity benchtop) was used for the
monitoring of vital compounds.28–31 The monitoring of DNA–drug adjustment of pH values. The 1H and 13C NMR data were
interactions using spectroscopic methods is based on the fact recorded on a Bruker Avance III HD 400 MHz Spectrometer.
that the uorescence of free ligands interacts with DNA in the
electronic absorption spectra.32,33 The Ames test34 is used to
determine the mutations occurring by chemicals at the cell level.
2.3 Synthesis of the compounds
To the best our knowledge, this is the rst time the synthesis
2.3.1 Triethyl
2,20,200-((2-(3,4-bis(2-ethoxy-2-oxoethoxy)
of 1S3 and 2S3 is being reported. DNA-binding of these phenyl)-4-oxo-4H-chromene-3,5,7-triyl)tris(oxy))triacetate (1S1).
compounds was investigated using uorescence quenching and This step was achieved by using a modied literature procedure
UV-Vis spectroscopy. The atomic details of these ligands–DNA by Lee et al. (2007)35 and Xia et al. (2010).36
interactions were investigated through the molecular docking
Quercetin, 1S0, (604 mg, 2 mmol, 1 eq.) was dissolved in dry
method. The results based on the in silico DNA–ligand interac- DMF (10 mL) in an oven-dried round-bottomed ask forming
tion studies show that 1S3 and 2S3 ligands are groove binders a brown solution. Anhydrous K2CO3 (2070 mg, 15 mmol, 7.5 eq.)
and that H-bond interactions play a principal role in the was added and the mixture was stirred for 1 hour at room
stability of these ligand–DNA complexes. This research exhibits temperature. Ethyl chloroacetate (1716 mg, 14 mmol, 7 eq.) was
the details of mutagenicity, the mode of binding interaction, slowly added into the reaction mixture, which was vigorously
binding affinity, main interaction forces of ligands with FS-DNA stirred at room temperature, under nitrogen gas. The progress
and structure of ligand–FS-DNA complex. The interaction of FS- of the reaction was monitored by TLC and the reaction was
DNA with the two synthesized compounds is signicant in stopped aer 37 hours. The nal reaction mixture was ltered,
pharmaceutical drug discovery. Based on the in silico molecular and the excess solvent was removed in a rotavapor. The resul-
docking studies, the binding score of the DNA interaction of the tant crude solid product was dissolved in ethyl acetate and
compounds is quite satisfactory. When the results obtained washed with 5% sodium bicarbonate, brine and nally water.
from the DNA interaction and the mutagenicity tests are eval- The nal product was puried by ash column chromatography
uated, it can be concluded that these compounds can be used in using silica gel in (hexane/ethyl acetate/acetone, 3 : 2 : 1, v/v/v)
biological applications and in vivo studies. This is aer further to afford 1S1, a light yellow solid, 73% yield, Rf ¼ 0.22. The
considering that these compounds are not toxic.
structure of this compound was veried by 1H and 13C NMR
characterizations.
1S1: d1H (400 MHz, DMSO-d6): 7.82–7.67 (2H, m), 7.14–7.02
(1H, m), 6.92–6.84 (1H, m), 6.47 (1H, d, J ¼ 2.3 Hz), 5.03–4.71
(10H, m), 4.24–4.08 (10H, m), 1.27–1.13 (15H, m). d13C (400
MHz, DMSO-d6): 171.71, 168.64, 168.57, 168.40, 168.17, 167.93,
161.68, 158.58, 157.69, 151.03, 149.30, 146.60, 138.57, 123.23,
122.47, 114.65, 113.60, 108.94, 97.83, 94.83, 67.60, 65.69, 65.65,
65.20, 65.05, 60.85, 60.73, 60.63, 60.45, 14.01, 13.99, 13.91.
2. Materials and methods
2.1 Reagents and solutions
High purity quercetin and apigenin were purchased from
Indone Chemical Company, Hillsborough, NJ, USA. Ethyl
chloroacetate, dimethylformamide, thionyl chloride, lithium
hydroxide monohydrate, anhydrous potassium carbonate,
sodium chloride, ethyl acetate, hexane, acetone, ammonia
solution, silica gel, methyl sulfoxide-d6, sodium bicarbonate,
and anhydrous sodium sulfate were purchased from Millipore
Sigma (Burlington, MA, USA). All chemicals were of analytical
grade and were used without further purication. Fish sperm
DNA (74782) and Tris–HCl (RES3098T-B7) were supplied by
Sigma-Aldrich Chemie GmbH (Germany). Ethidium bromide
(EtBr) and sodium chloride ($99.5%) were purchased from
Sigma Aldrich, USA. Other chemicals and reagents purchased
from Sigma or Merck and used in this work were all of the
analytical grades.
2.3.2 Diethyl
2,20-((2-(4-(2-ethoxy-2-oxoethoxy)phenyl)-4-
oxo-4H-chromene-5,7-diyl)bis(oxy))diacetate (2S1). Apigenin
(540 mg, 2 mmol, 1 eq.) was dissolved in dry DMF (10 mL) in
a clean oven-dried round-bottomed ask. Anhydrous potassium
carbonate (1242 mg, 9 mmol, 4.5 eq.) was added and the reac-
tion mixture was stirred at room temperature for 45 minutes.
Ethyl chloroacetate (1030 mg, 0.9 mL, 8.4 mmol, 4.2 eq.) was
added into the reaction mixture, which was stirred at room
temperature. The progress of the reaction was monitored by
TLC for 24 hours. The reaction mixture was ltered and dried;
the crude solid was re-dissolved in 10 mL of ethyl acetate and
washed with 5% sodium bicarbonate, brine and water (2 ꢁ
5 mL each). The organic layer was dried in anhydrous sodium
sulfate prior to purication in ash column chromatography
2.2 Apparatus
Absorption measurements were carried out using a double- using silica gel in (hexane/ethyl acetate, 1 : 1, v/v) to yield
beam spectrophotometer (PerkinElmer, Lambda 25, USA) and a brown solid, 992.9 mg, 94%. Rf ¼ 0.16 (hexane/ethyl acetate,
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1 : 1, v/v). The structure of the intermediate product was 4 hours. The excess solvents were removed in a rotavapor fol-
1
conrmed using H and 13C NMR characterizations.
lowed by the purication in a ash column chromatography.
2S1: d1H NMR (400 MHz, DMSO-d6) d 7.99 (d, J ¼ 9.0 Hz, 2H), The nal product was a light brown solid, 329 mg, 80% yield.
7.10 (d, J ¼ 9.0 Hz, 2H), 6.93 (d, J ¼ 2.3 Hz, 1H), 6.71 (s, 1H), 6.46 The products were conrmed by H NMR, 13C NMR and FT-IR
1
(d, J ¼ 2.3 Hz, 1H), 4.98–4.87 (m, 6H), 4.25–4.13 (m, 6H), 1.27– characterizations.
1.18 (m, 9H). d13C NMR (400 MHz, DMSO-d6) d 175.38, 168.36,
1S3: dH (400 MHz, DMSO-d6): 8.07 (1H, s), 7.88–7.65 (4H, m),
168.27, 168.00, 161.53, 160.13, 159.66, 158.83, 158.52, 127.72, 7.61 (1H, s), 7.56–7.33 (6H, m), 7.12 (1H, d, J ¼ 8.6 Hz), 6.88 (1H,
123.63, 115.04, 109.04, 106.94, 98.11, 95.24, 65.66, 64.99, 64.73, s), 6.67 (1H, s), 4.59 (8H, dd, J ¼ 16.0, 10.2 Hz), 4.33 (2H, s).
60.84, 60.74, 60.69, 14.01.
d13C (400 MHz, DMSO-d6): 172.93, 170.04, 169.94, 169.66,
2.3.3 2,20,200-((2-(3,4-Bis(carboxymethoxy)phenyl)-4-oxo-4H- 169.57, 169.52, 168.92, 162.45, 157.67, 152.63, 149.91, 147.22,
chromene-3,5,7-triyl)tris(oxy))triacetic acid (1S2). This step was 139.17, 122.97, 122.75, 114.59, 113.71, 108.43, 98.29, 94.94,
achieved by using a modied literature procedure by Lee et al. 70.24, 68.12, 67.77, 67.45, 67.03.
(2007)35 and Xia et al. (2010).36
FT-IR: (ATR, cmꢂ1): 3411 n(N–H), 3310 n(N–H), 1682 n(C]
1S1 (732 mg, 1 mmol, 1 eq.) was dissolved in THF/H2O (1 : 2). O)ketone, 1601 n(C]O)amide UV-Vis, lmax , (3 mꢂ1 cmꢂ1) (DI
nm
Into the resultant reaction mixture, LiOH$H2O (419.6 mg, water as solvent): 255 (1.80 ꢁ 104), 300 (9.2 ꢁ 103), 345 (1.71 ꢁ
10 mmol, 10 eq.) was added. The reaction mixture was stirred at 104). Conductivity: 11.3 mS.
room temperature and the progress of the reaction was moni-
2.3.6 2,20-((2-(4-(2-Amino-2-oxoethoxy)phenyl)-4-oxo-4H-
tored by TLC for 24 hours. The excess lithium hydroxide was chromene-5,7-diyl)bis(oxy))diacetamide (2S3). 2S2 (311 mg,
neutralized with 1 N HCl to afford a light-yellow precipitate 0.7 mmol, 1 eq.) was suspended in excess thionyl chloride in
which was ltered off to obtain a yellow residue. The nal a round-bottomed ask; the reaction mixture was reuxed for
product was concentrated in vacuo and le in a line vacuum 12 hours and concentrated in vacuo to achieve a yellow powder;
overnight to achieve 1S2 as a ne light-yellow solid, 515 mg, the acyl chloride intermediate. The intermediate was added into
87% yield. The structure was conrmed using 1H NMR an ice-cooled ammonium hydroxide. The reaction was stirred at
characterization.
room temperature and was monitored by TLC for 4 hours. The
1S2: dH (400 MHz, DMSO-d6): 13.07 (5H, s), 7.81 (1H, d, J ¼ nal product was puried in ash column chromatography to
2.1 Hz), 7.74 (1H, dd, J ¼ 8.6, 2.1 Hz), 7.06 (1H, d, J ¼ 8.8 Hz), afford an orange-brown solid, 250 mg, 81% yield. The structure
6.86 (1H, d, J ¼ 2.2 Hz), 6.45 (1H, d, J ¼ 2.3 Hz), 4.87–4.80 (6H, of the product was conrmed using 1H NMR, 13C NMR and FT-
m), 4.79 (2H, s), 4.70 (2H, s).
IR characterizations.
2.3.4 2,20-((2-(4-(Carboxymethoxy)phenyl)-4-oxo-4H-
2S3: 1H NMR (400 MHz, DMSO-d6) d 8.25 (1H, s), 8.02 (2H, d,
chromene-5,7-diyl)bis(oxy))diacetic acid (2S2). 2S1 (528 mg, J ¼ 8.4 Hz), 7.68 (1H, s), 7.60 (2H, s), 7.45 (2H, d, J ¼ 17.4 Hz),
1 mmol, 1 eq.) was dissolved in H2O/THF, 2 : 1. LiOH$H2O 7.12 (2H, d, J ¼ 8.6 Hz), 6.92 (1H, s), 6.76 (1H, s), 6.66 (1H, d, J ¼
(252 mg, 6 mmol, 6 eq.) was slowly added into the reaction 2.1 Hz), 4.64–4.51 (6H, m). 13C NMR (400 MHz, DMSO-d6)
mixture at room temperature, the reaction was monitored by d 176.93, 170.30, 169.88, 169.46, 162.79, 161.06, 160.97, 159.12,
TLC for 14 hours. The reaction mixture was neutralized with 1 M 158.13, 128.33, 123.84, 115.75, 108.91, 107.30, 99.17, 95.74,
HCl to form a light yellow precipitate. The precipitate was 68.39, 67.48, 67.20.
ltered off and puried by ash column chromatography on
FT-IR: (ATR, cmꢂ1): 3391 n(N–H), 3311 n(N–H), 1674 n(C]
silica gel (methanol/water, 9 : 1, v/v). The nal product was O)ketone, 1587 n(C]O)amide UV-Vis lmax , (3 mꢂ1 cmꢂ1) (DMSO
nm
concentrated en vacuo and le in a line vacuum overnight to as solvent): 260 (1.8 ꢁ 104), 290 (1.59 ꢁ 104), 357 (2.36 ꢁ 104).
achieve 2S2, as a ne light-yellow solid, 395.4 mg, 89% yield. Conductivity: 7.9 mS.
The structure of the product was conrmed using 1H
characterization.
2S2: dH (400 MHz, DMSO-d6): 13.15 (3H, s), 8.06–7.97 (2H,
2.4 DNA interaction study
m), 7.13–7.05 (2H, m), 6.93 (1H, d, J ¼ 2.3 Hz), 6.75 (1H, s), 6.49
(1H, d, J ¼ 2.3 Hz), 4.87–4.79 (6H, m).
The stock solution of FS-DNA was prepared in a Tris–HCl buffer
with a pH of 7.4 and a concentration of 50 mM then stored in
2.3.5 2,20,200-((2-(3,4-bis(2-amino-2-oxoethoxy)phenyl)-4-
oxo-4H-chromene-3,5,7-triyl)tris(oxy))triacetamide (1S3). This the refrigerator at 4 ꢃC for a maximum of 72 hours. In all
step was achieved by using a modied literature procedure by Li experiments, the FS-DNA concentration was determined using
et al. (2012).37
the absorption band at 260 nm, and the molar absorption
1S2 (414.7 mg, 0.7 mmol, 1 eq.) was mixed with excess thi- coefficient reported as 6600 L molꢂ1 cmꢂ1 the relevant calcu-
onyl chloride in an oven-dried round-bottomed ask. The lations were made based on this concentration.38 To determine
reaction mixture was vigorously stirred under reux conditions; that FS-DNA did not contain protein impurity; the purity of the
the reaction was monitored by TLC and was stopped aer 13 prepared stock FS-DNA solution was conrmed by the UV
hours. The reaction mixture was concentrated under reduced absorption rate (A260/A280 > 1.8) as a result of measurements
pressure to form a yellow powder, the acyl chloride intermediate obtained at 260 and 280 nm.39 Similarly, stock solutions of the
product. This product was slowly transferred into ice-cold ligands were prepared in deionized water and then diluted to
aqueous ammonium hydroxide. The reaction mixture was stir- desired concentrations by Tris–HCl buffer. The EtBr used in
red at this temperature for 2 hours and at room temperature for photoluminescence studies was prepared at a concentration of
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3.0 ꢁ 10ꢂ3 mol Lꢂ1 in ethanol and used for the experiment where I0 and I are the emission intensities in the absence and
immediately.
presence of the quencher, KSV is the quenching constant, and
2.4.1 UV-Visible spectroscopy. Electronic absorption spec- [Q] is the ligand (quencher) concentration. The KSV values have
troscopy is one of the simplest and easiest methods for studying been derived from the slopes of the plots of I0/I versus [Q].
binding affinity in the interaction of biomolecules with ligands.
In UV-Vis measurements, the absorption spectra of compounds
were recorded in the 200–800 nm wavelength range. For the
study of the interaction between FS-DNA and ligands, the
spectra obtained by adding certain amounts of FS-DNA (0–20
mM) by titration method were recorded for each ligand solution
(10 mM). All measurements were in triplicate and before the
measurements of the corresponding spectrum of the mixture
were taken for each sample, falcon tubes were slightly shaken
and incubated for 15 minutes. The absorption spectrum ob-
tained with the added FS-DNA was subtracted from the spec-
trum in the DNA–ligand measurement (the same amount of FS-
DNA added) and exact spectra were gained. The intrinsic
binding constant Kb was determined from the plot of [DNA]/(3a
ꢂ 3f) vs. [DNA] graph, where [DNA] is the concentration of DNA,
and the apparent absorption coefficient 3a, 3f, 3b correspond to
Aobs/[Ligand], the extinction coefficient for the free ligand
compounds (1S3 and 2S3) and the extinction coefficient of the
ligand compounds in the totally bound form, respectively.40 The
observed values were appointed to eqn (1), with a slope equal to
1/(3b ꢂ 3f) and the intercept equal to 1/[Kb(3b ꢂ 3f)]. Kb was
acquired from the ratio of the slope to the intercept.39
2.5 Mutagenicity assay
2.5.1 Bacterial strains. Histidine decient (hisꢂ) tester
strains TA 98 and TA 100 of Salmonella typhimurium were kindly
provided by J. L. Swezey, Curator, ARS Patent Culture Collection,
Microbial Genomics, and Bioprocessing Research Unit, North
University Street, Peoria, Illinois 61604, USA. The TA98 strain
was used for the detection of frameshi mutagens while TA100
strain was used for the detection of base-pair substitution
mutagens. Before their use for this assay, each strain was
checked for the presence of strain-specic marker as described
by Maron and Ames.42
2.5.2 Salmonella/microsome test (Ames). The standard
plate-incorporation assay was examined with Salmonella typhi-
murium TA98 and TA100 strains both in the presence and
absence of S9 mixture, according to Maron and Ames.42 0.5 mL
of S9 mixture containing 50 mL of S9 factor per plate was used
for the assay. Compounds 1S3 and 2S3 were each dissolved in
distilled water to attain the concentrations of 0.40, 0.20, 0.10,
0.05 and 0.02 mg per plate. In this study, 2-AF (2-aminoourene)
was used as a positive mutagen (20 mg per plate) for both TA98
and TA100 strains in the presence of S9 mix. 4-NPD (4-nitro-
phenylene diamine) was used as a positive mutagen (200 mg per
plate) for TA98 and TA100, per sample. The study was done with
ve replicate plates and all experiments were performed twice.
2.5.3 Preparation of S9. The male albino rats (Rattus nor-
vegicus var. albinos) weighing 200 g were pre-treated with 80 mg
kgꢂ1 concentration of 3-methylcolanthrene (Oekanal, cat. no:
45794) (dissolved in sunower seed oil) for 5 days and the S9
fraction and S9 mixture were prepared following the procedure
by Maron and Ames.42 The S9 tablets were purchased from
Roche (cat. no: 1.745.425). The freshly prepared S9 fraction was
distributed in 1 mL portions in small plastic tubes, which were
[DNA]/(3a ꢂ 3f) ¼ [DNA]/(3b ꢂ 3f) + 1/[Kb(3b ꢂ 3f)]
(1)
2.4.2 Fluorescence spectroscopy. Fluorescence spectros-
copy is a highly sensitive and effective method for investigating
the binding of ligands, particularly drugs and drug candidates,
to biomacromolecules. The uorescence quenching experiment
is signicant in the determination of the number of binding
sites, binding constants, binding mode, and intermolecular
distances of compounds with interacting potential. The inter-
actions of DNA with the synthesized ligands were studied using
a uorescence quenching experiment. Excitation and emission
slits were set at 5 and 10 nm, respectively, using a quartz cuvette
with an optical path length of 1 cm. In order to investigate the
binding of ligands with DNA, the FS-DNA solution was carefully
mixed with ethidium bromide (EtBr), which was prepared just
before the experiment, with a concentration ratio of DNA : EtBr
(10 : 1) for 15 minutes at 25 ꢃC. The binding of EtBr molecules
to the double helix structure of DNA by intercalation mode
causes a signicant increase in the uorescence intensity of EB.
Subsequently, various amounts of ligand solutions (0–70 mM)
were added to this mixture, allowing the nal mixture to incu-
bate for 15 minutes. Fluorescence spectra of the resulting nal
solutions were displayed in the range of 500–700 nm with an
ꢃ
frozen immediately and stored at ꢂ35 C. The S9 mixture was
prepared fresh for each mutagenicity assay. Each tablet was
dissolved in 18 mL distilled water supplemented with 2 mL of
S9 fraction.
2.5.4 Statistical signicance. The statistical signicance
between control revertants and revertants of the treated groups
was determined using the t-test. Between the averages of the
data obtained, considering differences in the level (p < 0.05)
were identied. The experiments were carried out in two
groups, with and without S9. The average of the test results was
compared with the positive and negative control groups and
evaluated.
excitation wavelength of 520 nm. Fluorescence quenching effi- 2.6 Docking and molecular modelling studies
ciency was evaluated according to the following equation
(Stern–Volmer equation, eqn (2)).41
The synthesized ligands, 1S3 and 2S3, were sketched in drawing
BIOVIA Draw 2019 soware and their energies minimized by
Chem3D 19. Molecular modeling studies of these ligands were
realized using HyperChem 8.03.43 The molecular docking
analysis was operated on the ligands against 1BNA by utilizing
I0/I ¼ KSV[Q] + 1
(2)
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40, 5 and 7. It has three rings: two benzene rings (ring A and B)
and a pyran ring, ring C. Quercetin (2-(3,4-dihydroxyphenyl)-
3,5,7-trihydroxy-4H-chromen-4-one) structure consist of 2-
phenylchromen-4-one with ve hydroxyl groups at positions 3,
30, 40, 5 and 7. It has three rings: two benzene rings (ring A and
B) and a pyran ring, ring C; Fig. 1.
Quercetin and apigenin-acetamide derivatives were synthe-
sized and characterized, starting from the avonoid molecules.
The structures of the intermediates and the products are shown in
Fig. 2 and Table 1. The NMR characterization data conformed
with the structures of both the key intermediates and the nal
products. The modication process was designed based on the
chemistry of the phenol hydroxyl groups. These hydroxyl groups
were sequentially derivatized into the desired acetamide group
through an ester, carboxylic acid and acyl chloride intermediates.
The hydroxyl groups in the quercetin and apigenin mole-
cules, S0, were converted into ester intermediates, S1, through
an ether-bond formation. This step was achieved through the
Williamson ether synthesis. In step 2, the ester intermediate
(S1) was converted into a carboxylic acid intermediate (S2) in an
ester-hydrolysis reaction. In the nal step, step 3, the carboxylic
acid derivative was converted into an acyl chloride, which was
subsequently amidated to achieve the acetamide derivative, S3.
The general steps followed are shown in Scheme 1.
Fig. 2 General structure of the flavonoids, the intermediates and the
final products. R3, R0 ; R0 ; R5 and R7 variables are shown in Table 1.
3
4
the PyRx docking soware (academic licensed version 0.9.8).44
To evaluate the binding and interaction of the two compounds
to DNA, we employed the Dickerson–Drew dodecamer (DDD)
with the sequence d(CGCGAATTCGCG)2 as a model system.45
The high-resolution structure of the free DNA was downloaded
from the PDB database (PDB code: 1BNA) (http://www.rcsb.org/
pdb). The heteroatoms were removed from 1BNA.pdb, to
remove any free ligand from the complex receptor. DNA was
prepared by the addition of Kollman united atom charges, polar
hydrogens, and solvation parameters. The default exhaustive-
ness value for both positions was set to 8 to achieve the highest
level of accuracy for binding conformational analysis. Ligands
were arranged by combining non-polar hydrogens and assign-
ing the Gasteiger charge. In order to dock the synthesized
ligands to DNA, a blind docking (placement) was conducted
with 128 lattice points throughout the X, Y, and Z axes to nd
the binding site of complexes on DNA with a grid point range of
Step 1 was achieved through the Williamson ether synthesis
reaction. Hypothetically, this reaction proceeds through an SN2
reaction pathway. Anhydrous potassium carbonate was used to
deprotonate the avonoid hydroxyl groups yielding poly phen-
oxide ions. These anions served as the nucleophile, whereas
ethyl chloroacetate served as the substrate for the reaction.
Excess potassium carbonate was used to ensure optimum
deprotonation of the avonoid hydroxyl groups into the corre-
sponding nucleophiles.
˚
0.375 A. In the next step, the center of the grid box was located at
the binding site, and the second docking was performed using
a cubic box with 60 ꢁ 60 ꢁ 60 dimensions. The search for best
conformers was done using a Lamarckian genetic algorithm
(LGA).46 The docked complexes (1S3–DNA and 2S3–DNA) were
assessed on the lowest binding energy (kcal molꢂ1) values. The
graphical depictions and related calculations of all the docking
complexes were carried out and monitored using Discovery
Studio 2020.
1
The S1 derivatives were conrmed using H and 13C NMR
characterizations. In 1S1, 1H NMR conrmed 5 aromatic, 10
methylene (ether), 10 methylene (ester), and 15 methyl (ester)
protons, by integration (Fig. S1†). 2S1 1H NMR integration
conrmed 7 aromatic, 6 methylene (ether), 6 methylene (ester)
and 9 methyl (ester) protons (Fig. S2†).
In step 2, the avonoid-ester intermediate (S1) was converted
3. Results and discussion
3.1 Synthesis and NMR study of the compounds
Apigenin (40,5,7-trihydroxyavone) structure consists of 2-
into
a avonoid-carboxylic acid derivative (S2) through
a saponication reaction. The ethoxy (–OCH2CH3) group of the
avonoid ester derivative was cleaved in an ester hydrolysis
phenylchromen-4-one with three hydroxyl groups at positions reaction. The formation of the S2 derivatives was conrmed
Table 1 R3, R0 ; R0 ; R5 and R7 variables in Fig. 2
3
4
R3
OH
R30
R40
R5
R7
1S0
1S1
1S2
1S3
2S0
2S1
2S2
2S3
OH
OH
OH
OH
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
H
H
H
H
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
H
H
H
H
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
OH
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
OH
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
OH
OCH2COOCH2CH3
OCH2COOH
OCH2CONH2
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Scheme 1 Steps in the synthesis of 1S3 & 2S3 derivatives. Reagents and reaction conditions: step 1 (S0 / S1) ethyl chloroacetate, K2CO3 (excess),
DMF, rt. Step 2 (S1 / S2) LiOH$H2O, THF/H2O, rt. Step 3 (S2 / S3) SOCl2 (excess), reflux, 75 ꢃC; NH4OH (excess), 0 ꢃC – rt.
using 1H NMR characterization. The 1H NMR integration conrmed 6-CONH2, 7 aromatic and 6 methylene protons,
conrmed 5-COOH protons, 5 aromatic protons and 10 meth- Fig. S7.†
ylene protons in 1S2, Fig. S3.† In 2S2, 3-COOH protons, 7
3.1.1 UV-Visible spectroscopy. The preliminary optical
aromatic and 6 methylene protons were conrmed by the studies were examined by using UV-Vis spectrophotometer.
1
integration of the H NMR, Fig. S4.†
Fig. 3 shows the UV-Vis absorption spectrum of 1S3 and 2S3.
Step 3 involved the conversion of the S2 intermediate into Both 1S3 and 2S3 have two distinct peaks and a shoulder
the desired avonoid acetamide derivative (S3). This was done (Fig. 3). The common characteristic absorption peaks were
by rst converting the –COOH group into an acyl chloride observed in two curves at lmax 255 and 345 nm for 1S3. These
(–COCl) by reuxing the carboxylic acid derivative with thionyl peaks were detected at 260 and 335 nm for 2S3. These sharp
chloride. The acyl chloride intermediate, with a better leaving peaks were attributed to p–p* transition within the B ring of
group, was nally converted into S3 product using an aqueous cinnamoyl structure (the band I); the second peak (band II) may
ammonium solution. The S3 product was characterized using be appointed to transitions in the ring A of benzoyl structure. As
1H & 13C NMR. The 1H NMR conrmed the amide, aromatic and we have previously reported, these characteristic peaks in the
the methylene protons.
UV spectra showed absorption bands at about 257 and 374 nm
In 1S3, 10 amide (–CONH2) protons, 5 aromatic protons and for the quercetin which is the precursor of the 1S3 compound,
10 methylene protons were positively conrmed by the inte- at about 269 and 340 nm for the apigenin, the precursor of the
gration of the 1H NMR, Fig. S5.† The additional carbons the CH2 2S3.19,47
and the amide carbons were conrmed from the 13C NMR
spectrum, as shown in Fig. S6.† The 2S3 H NMR integration peaks in the FT-IR spectra of the 1S3 and 2S3 derivatives are due
to the amide N–H stretch. Primary amides are characterized by
3.1.2 FTIR spectroscopy. The prominent characteristic
1
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To examine the interaction between the ligands with FS-
DNA, 1S3 and 2S3 UV-Vis absorption spectra were recorded in
the absence and presence of different DNA concentrations. The
UV-Vis spectra of 1S3 and 2S3 in the absence and presence of FS-
DNA are given in Fig. 4. In the absence of DNA, 1S3 showed two
different absorbance peaks at 255 and 335 nm, respectively. As
the absorption in the 255 nm band increased with the addition
of DNA, the absorption at 335 nm decreased and a partial red
shi was observed. Similarly, the absorption of the band at
260 nm in 2S3 increased with the addition of DNA, while the
absorption of the band at 345 nm decreased and a slight
redshi was observed. This can be explained by a decrease in
the number of chromophores in the solution medium and
conformational changes in the DNA as a result of DNA–ligand
interaction. Furthermore, as a result of interaction with DNA in
both compounds, approximately 300 nm isosbestic point
occurred. This situation is a strong evidence48 of the formation
of a new DNA–ligand complex on the interaction of compounds
and DNA affinity. Based on variations in the absorbance bands,
the binding constant (Kb) for the ligand–DNA was calculated
using the Benesi–Hildebrand equation (eqn (1)) and the rele-
vant values are summarized in Table 2. The binding constant
was found to be 1.43 ꢀ 0.3 ꢁ 104 Mꢂ1 for 1S3 and 2.08 ꢀ 0.2 ꢁ
104 Mꢂ1 for 2S3. When these values are compared with the
classical intercalation agent ethidium bromide, which is known
Fig. 3 Absorption spectra of 10 mmol Lꢂ1 1S3 and 2S3 in Tris–HCl
buffer (0.1 mol Lꢂ1, pH 7.4).
two N–H absorptions. In 1S3, the amide N–H absorptions were
recorded at 3411 and 3310 cmꢂ1. The corresponding N–H
absorptions in 2S3 were recorded at 3391 and 3311 cmꢂ1
.
Additionally, the amide and ketone (C-4) C]O stretches were
relatively identied in the FR-IR spectra. The ketone C]O
stretching absorptions were recorded at 1682 and 1674 cmꢂ1 for
1S3 and 2S3, respectively. The amide C]O stretching absorp-
tions were recorded at 1601 and 1587 cmꢂ1 for 1S3 and 2S3,
respectively. The key FT-IR spectra peaks are shown in
Table S1.† The FT-IR spectra of the two compounds are shown
in Fig. S8.†
3.1.3 Molecular modeling studies. Geometric model
structures of 1S3 and 2S3 were obtained by optimization of
bond lengths, dihedral angles, and bond angles. Structures
were optimized with minimum energies obtained by applying
quantum chemical calculations. As shown in Fig. S9,† the
ligands examined have a planar structure. The bond lengths
between atoms and the bond angles are presented in detail in
Tables S2 and S3.†
3.2 Compound–DNA interaction study
3.2.1 Absorption spectroscopy studies. Electronic absorp-
tion spectroscopy, one of the most employed test techniques, is
extensively used in the examination of the interaction of DNA
with small molecules. Commonly, the mechanism in binding
small molecules to DNA through intercalative binding mode is
elucidated, resulting in hypochromism and bathochromism of
the resulting absorption bands.48 When occurring electrostatic
interaction between DNA and compounds, the hyperchromic
effect can be seen, in which changes occur in the conformation
of DNA. However, in the event of groove binding mode between
DNA and compounds, the hypochromic effect can be recog-
nized with the position of the absorption band substantially
unaltered, which can be associated with the superimposing of
the electronic states of the chromophore in the DNA grooves of
the complex with the nitrogenous bases.49,50
Fig. 4 Absorption spectra of 10 mmol Lꢂ1 1S3 (a) and 10 mmol Lꢂ1 2S3
(b) in the presence of FS-DNA at different concentrations (a: 0.0, b:
1.94, c: 3.89 d: 5.84, e: 7.78, f: 9.73, g: 11.68, h: 13.63, i: 15.58, and j:
17.52 mmol Lꢂ1 in Tris–HCl buffer (0.1 mol Lꢂ1, pH 7.4)). The inset
showing the plot of [DNA/(3a ꢂ 3f)] versus [DNA].
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Table 2 Data obtained from spectro-analytical methods and calcu-
lations for the interaction of compounds with DNA
Property
1S3
2S3
Absorbance (nm)
335
345
Kb (Mꢂ1
)
1.43 ꢀ 03 ꢁ 104
2.08 ꢀ 02 ꢁ 104
Hypochromism (%)
Isosbestic point (nm)
Shi (nm)
22.54
300
3
20.91
301
3
to interact with DNA (although the EtBr–DNA (Kb ¼ 1.4 ꢁ 106)
complex is signicantly lower than binding) it can be said that it
falls within the range of groove binding51 constant from binding
modes when DNA interacts with a small molecule.
3.2.2 Fluorescence quenching studies. In order to further
analyze the interaction mode of 1S3 and 2S3 with DNA, a uo-
rescence quenching experiment was conducted. To study this
interaction, EtBr, a powerful intercalation agent well known to
interact with DNA, was selected as a uorescence indicator. EtBr
has a weak uorescence alone; however, in the presence of DNA,
it shows intense uorescence at 597 nm in stimulation at
280 nm due to its robust placement between adjacent DNA base
pairs (intercalation). Therefore, the interaction of the
compounds with FS-DNA can be examined in terms of changes
of this characteristic peak. In the literature, it has been proven
that the increased uorescence of DNA–EtBr can be quenched
by adding a second molecule through a competition between
EtBr and the second molecule to bind to DNA.31 While a signif-
icant increase in the uorescence density of EtBr is observed
upon the addition of DNA, it shows a weak uorescence emis-
sion in solution due to the transfer of hydrogen into solution as
a result of non-radiative decay from one of the amino groups of
EtBr.52 Thus, these ligands can replace EtBr by altering DNA
conformation. As a result, DNA-bound EtBr molecules are
transformed into their free forms in solution, quenching the
uorescence.
Fig. 5 Quenched Fluorescence spectra of FS-DNA bound to the EtBr
system with the addition of compounds 1S3 (a) and 2S3 (b). Inset: I0/I
versus [DNA] (mM).
3.3 Mutagenicity properties of the compounds
The uorescence spectra of 1S3 and 2S3 in the absence and
presence of FS-DNA were shown in Fig. 5. As 1S3 and 2S3 are
gradually titrated into the EtBr–DNA complex, the maximum
emission of the solution is reduced. This data has been vali-
dated by the interaction of the ligands with FS-DNA, which
causes the formation of non-uorescent complexes in both
compounds and quenches the endogenous uorescence.
According to the eqn (2), the KSV values of the compounds have
been calculated to be 1.83 ꢁ 104 Mꢂ1 and 1.96 ꢁ 104 Mꢂ1 for
1S3 and 2S3, respectively. KSV values calculated from the Stern–
Volmer equation have lower than the values of ethidium
bromide, acridine orange, and methylene blue, which are well
known to have intercalation interaction with DNA.53 Therefore,
for both 1S3 and 2S3 compounds, it cannot be said that it
interacts with DNA by intercalation and displaces EtBr.54 The
decrease in the uorescence intensity of EtBr–DNA is an indi-
cation that these compounds may be partially intercalated, but
this interaction is also unstable in line with the KSV values ob-
tained and the uorescence intensity of magnitude.55
Salmonella, a microsome test system, is used to detect point
mutations in DNA. In this test, TA98 and TA100 strains were
obtained from the LT2 ancestral strain of Salmonella typhimu-
rium by a mutation in vitro.56 This test was developed by Ames in
1971.34,42,56–58 Among these strains, TA100 had base change
mutation, and TA98 had frameshi mutation. Due to these
mutations, these strains are unable to synthesize histidine
(hisꢂ). The principle of the experiment is based on the correc-
tion of these mutations by treating these mutated strains (TA98
and TA100) with various chemicals, i.e., calculating the histi-
dine expression frequencies of the strains (his+). Point mutation
and frameshi mutations are the basis of many genetic diseases
observed in humans. There is strong evidence that it induces
tumor formation in humans and animals due to base change
mutations in tumor suppressor genes in both oncogenes and
somatic cells.34 Frameshi mutations occur as a result of
adding/removing the base as much as 1, 2, and its multiples
occurring in DNA. The mRNA synthesized from this mutated
DNA molecule and the protein chain synthesized then make
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Fig. 6 Comparison graphs showing the changes in revertant colony percentages in the presence (+S9) and absence (ꢂS9) of the metabolic
activation system for TA98 and TA100 strains of compounds 1S3 and 2S3.
major changes in the amino acid sequence (in the entire chain). mutations occur as a result of the structural change of one of
Therefore, if a chemical frame-shi mutation is induced, major the bases in the DNA molecule, much fewer changes occur in
changes occur in the protein as a result. However, since point the mRNA synthesized from this DNA molecule and
Table 3 The mutagenicity of compounds 1S3 and 2S3 on S. Typhimurium TA98 and TA100 strains in the presence or absence of S9 mixturea
TA98
TA100
Concentration,
mg per plate
Test substances
ꢂS9
+S9
ꢂS9
+S9
Spontaneous control
—
16.20 ꢀ 1.77
4260 ꢀ 291.75
—
14.20 ꢀ 2.26
—
162.60 ꢀ 12.23
—
—
135.80 ꢀ 7.35
—
NPD
2-AF
SA
200 mg mLꢂ1
20 mg mLꢂ1
1 mg mLꢂ1
0.40
2504.60 ꢀ 397.74
3207.40 ꢀ 385.93
—
—
1497.2 ꢀ 230.28
88.80 ꢀ 14.07 a2b3
80.20 ꢀ 10.65 a2b3
85.00 ꢀ 7.17 a3b3
94.00 ꢀ 8.43 a3b3
112.40 ꢀ 3.28 a3b3
136.80 ꢀ 5.95
—
—
1S3
11.60 ꢀ 1.63 a1b3
13.20 ꢀ 1.01 a1b3
12.20 ꢀ 0.66 a2b3
14.40 ꢀ 2.20 b3
12.00 ꢀ 1.41 a1b3
16.20 ꢀ 1.77
3808.40 ꢀ 221.78
—
7.20 ꢀ 1.68 a1b3
11.20 ꢀ 2.97 b3
14.00 ꢀ 2.60 b3
7.80 ꢀ 1.11 a2b3
9.00 ꢀ 0.44 a3b3
14.20 ꢀ 2.81
—
82.20 ꢀ 5.37 a3b3
47.00 ꢀ 6.73 a3b3
62.00 ꢀ 4.95 a3b3
48.80 ꢀ 12.85 a2b3
47.00 ꢀ 7.16 a3b3
133.20 ꢀ 4.56
—
0.20
0.10
0.05
0.02
Spontaneous control
NPD
2-AF
SA
4135.80 ꢀ 181.49
—
3518.40 ꢀ 385.76
—
—
1506.40 ꢀ 114.82
171.40 ꢀ 8.55 a1b3
200.00 ꢀ 8.44 a2b3
185.60 ꢀ 7.20 a2b3
195.40 ꢀ 6.96 a3b3
182.40 ꢀ 9.15 a2b3
—
2S3
0.40
0.20
0.10
0.05
0.02
19.20 ꢀ 2.08 b3
16.60 ꢀ 1.80 b3
20.00 ꢀ 2.25 b3
17.40 ꢀ 1.12 b3
17.00 ꢀ 1.00 b3
24.60 ꢀ 2.11 a2b3
22.00 ꢀ 1.37 a2b3
25.40 ꢀ 1.83 a2b3
18.40 ꢀ 1.74 b3
15.60 ꢀ 2.52 b3
210.40 ꢀ 2.31 a3b3
183.60 ꢀ 9.35 a2b3
196.60 ꢀ 15.75 a2b3
190.80 ꢀ 11.16 a2b3
187.40 ꢀ 18.93 a1b3
a
(a) Signicant from control. (b) Signicant from positive control, (1) P # 0.05; (2) P # 0.01; (3) P # 0.001, NPD: 4-nitro-o-phenylenediamine, 2-AF: 2-
aminoourene, SA: sodium azide.
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system.34 In addition, 2S3 signicantly increased the number of
revertant colonies for TA100 strain both in the presence and the
absence of the activation system. This shows that both the 2S3
and its metabolites are potentially mutagenic for the TA100
strain at high doses. It is evident that strain TA98 shows
frameshi mutation and strain TA100 shows base change
mutation.58 In summary, 1S3 does not have any mutagenic
potential in both TA98 and TA100 strains. Compound 2S3 does
not show any mutagenic activity in the TA98 strain; however, its
metabolites have a mutagenic potential at high doses. In addi-
tion, both the 2S3 itself and its metabolites have mutagenic
activity in the TA100 strain. The values of both compounds are
given in detail in Fig. 6 and Table 3. According to the data that
we obtained from this study, we can conclude that compound
1S3 by itself and its metabolites are not mutagenic because they
do not cause frameshis and point mutations. On the other
hand, compound 2S3 by itself does not induce the frame-shi
mutation, but it induces metabolites at high doses. It further
induces the point mutation of both 2S3 itself and its metabo-
lites. Therefore, we can conclude that it has mutagenic
potential.
Table 4 Binding affinity energies and docking parameters of the
ligand's interaction with DNA
Binding affinity
Inhibition constant
at 298.15 K (mM)
Ligand
(kcal molꢂ1
)
Kb (Mꢂ1
)
1S3
2S3
ꢂ7.9
ꢂ9.1
1.74 ꢁ 104
2.23 ꢁ 104
1.92
2.34
subsequently the synthesized protein molecule. Compound 1S3
did not cause any detectable increase in the number of revertant
colonies in both TA98 and TA100 strains, both in the presence
(+S9 mixture) and the absence of the metabolic activation
system (ꢂS9 mixture). This shows that both the 1S3 and its
metabolites do not have any mutagenic activity.
Our study reveals that compound 2S3 did not cause an
increase in the number of revertant colonies in the absence of
a metabolic activation system for the TA98 strain. However, this
compound caused a statistically signicant increase in the
number of revertant colonies in high doses (0.40, 0.20 and
0.10 mg per plate) in the presence of metabolic activation
Fig. 7 Molecular docked structure of 1S3 (a) and 2S3 (b) complexed with DNA. Surface representation showing the binding of 1S3 and 2S3 with
the dodecamer duplex sequence, d(CGCGAATTCGCG)2 (PDB ID: 1BNA).
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Fig. 8 2D plot of interaction between the 1S3 (a) and 2S3 (b) with the dodecamer duplex sequence, d(CGCGAATTCGCG)2 (PDB ID: 1BNA).
location of ligands on DNA. In general, the more negative the
binding energy resulting from the interaction, the more stable the
small molecule and the complex formed by the DNA, the stronger
the interaction between the small molecule and the DNA.30 The
docking energy values and inhibition constants of the ligands
during their interaction with DNA are given in Table 4.
According to the docking models, 1S3 binds to the major
groove of DNA, while 2S3 binds to the minor groove. The binding
affinities were found to be ꢂ7.9 and ꢂ9.1 kcal molꢂ1 according to
the best pose conditions-for the 1S3 and 2S3, respectively. These
scores relate to the minimum binding energy and the maximum
3.4 Molecular docking studies
Molecular docking studies have gained an increasing interest in
the research of the binding interaction mechanisms of biological
macromolecules with small molecules. Molecular docking is
a valuable technique in rational drug design to predict the most
stable structure, binding tendency, and binding mode of the
receptor–ligand complex so that interaction details can be better
recognized and examined during new drug discovery and devel-
opment process. This method is oen used as virtual search tools
in crucial steps of drug design and development. Molecular
docking studies have been conducted to nd the preferred
Table 5 Interactions types and distances of 1S3 and 2S3 with DNA complexes
Interactions
Distance
Bonding types
From
To
1S3–DNA complex
A:DG10:H3 – :1S3:O35
A:DG12:H22 – :1S3:O30
B:DG14:H3 – :1S3:O30
B:DG16:H22 – :1S3:O39
B:DA17:H3 – :1S3:O39
:1S3:H57 – :1S3:O25
:1S3:H60 – :1S3:O26
:1S3:H47 – A:DG12:OP1
:1S3:H48 – :1S3:O11
2.98657
2.21828
2.32444
2.77904
1.84249
2.51626
2.439
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
A:DG10:H3
A:DG12:H22
B:DG14:H3
B:DG16:H22
B:DA17:H3
:1S3:H57
:1S3:H60
:1S3:H47
:1S3:H48
:1S3:O35
:1S3:O30
:1S3:O30
:1S3:O39
:1S3:O39
:1S3:O25
:1S3:O26
A:DG12:OP1
:1S3:O11
2.37572
2.24041
Interactions
Distance
Types
From
To
2S3–DNA complex
:2S3:H42 – A:DG4:O40
:2S3:H50 – A:DT7:O2
:2S3:H50 – B:DT19:O2
:2S3:H51 – B:DT19:O2
:2S3:H51 – B:DT20:O2
:2S3:H46 – B:DG22:OP1
:2S3:H47 – :2S3:O11
:2S3:H47 – B:DC21:O30
A:DG4:H22 – :2S3:O22
A:DG4:H3 –:2S3:O22
A:DA5:H3 – :2S3:O7
A:DA6:H3 – :2S3:O28
B:DG22:H22 – :2S3:O22
A:DT8:C50 – :2S3:O32
A:DG4:H22 – :2S3
2.15093
2.71946
2.59064
2.40101
2.73073
2.49963
1.92188
2.90098
2.92642
2.4405
2.92123
2.16167
2.32328
3.25681
2.75747
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Conventional H bond
Carbon–H bond
:2S3:H42
:2S3:H50
:2S3:H50
:2S3:H51
:2S3:H51
:2S3:H46
:2S3:H47
:2S3:H47
A:DG4:H22
A:DG4:H3
A:DA5:H3
A:DA6:H3
B:DG22:H22
A:DT8:C50
A:DG4:H22
A:DG4:O40
A:DT7:O2
B:DT19:O2
B:DT19:O2
B:DT20:O2
B:DG22:OP1
:2S3:O11
B:DC21:O30
:2S3:O22
:2S3:O22
:2S3:O7
:2S3:O28
:2S3:O22
:2S3:O32
:2S3
Pi-donor-H bond
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docking cluster. These results are compatible with the spectro-
scopic results obtained. In addition, docking studies have shown
that H-bond interactions play the main role in the stability of
ligand–DNA complexes. The energetically most convenient
conformation of the docked pose of DNA–ligand complexes is
depicted in Fig. 7 as a 3D. When the 2D plot of the interaction
between DNA with 1S3 and 2S3 is examined (Fig. 8), for 1S3, DNA
appears to have 6 different H-bond interactions with both A and B
chain. Considering the nucleotide-binding and the molecular
structure of 1S3, it is seen that the hydrogen bonds formed in the
1S3–DNA complex occur via the nucleotides of the guanine and
adenine and the oxygen in the amide groups of the compound.
For 2S3, a signicant change has occurred in the conformation of
the molecule to exhibit the ability to bind along the minor groove,
and this conformational change is closely related to the structure
of the DNA minor groove. Besides, this situation can be described
as an important indication that the DNA–2S3 complex is formed.
The interaction between the ligand and DNA for the DNA–2S3
complex occurred via amide groups attached to the aromatic ring,
as in 1S3. However, apart from the conventional hydrogen bond,
carbon–H bond and Pi-donor-H bond interactions were observed.
For the 1S3–DNA and 2S3–DNA complexes, the data showing the
bonds, bond types, and bond lengths formed between the
nucleotides and ligands are summarized in Table 5. For the 1S3–
DNA and 2S3–DNA complexes, the docked model illustrations are
given in detail in the ESI, Fig. S10–S19.†
Acknowledgements
The authors acknowledge the National Science Foundation
Grant # IOS-1543944 and the Bill & Melinda Gates Foundation
for funding.
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Conflicts of interest
The authors declare no conicts of interest.
This journal is © The Royal Society of Chemistry 2020
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