Manish Rana1, Md Imam Faizan2, Sajad Hussain Dar1, Tanveer Ahmad2. 1. Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India. 2. Multidisciplinary Centre for Advanced Research & Studies, Jamia Millia Islamia, New Delhi 110025, India.
Abstract
To discover anticancer drugs with novel structures and expand our research scope, pyrazoline derivatives (3a-3l) were designed and synthesized through cyclization of chalcones with thiosemicarbazide/semicarbazide in CH3COOH as a solvent. All newly synthesized pyrazoline derivatives were fully characterized using several spectroscopic experiments such as 1H, 13C NMR, FT-IR spectroscopy, and mass analysis. By HPLC, the purity of all analogs was found above 95% and both lead compounds (3a and 3h) were also validated by HRMS. Anticancer activity of synthesized pyrazoline derivatives (3a-3l) was investigated by the MTT assay against the human lung cancer cell (A549), human cervical cancer cell (HeLa), and human primary normal lung cells (HFL-1). Staurosporine (STS) was used as a standard drug. The anticancer results showed that two potent analogs 3a and 3h exhibit excellent activity against A549 (IC50 = 13.49 ± 0.17 and 22.54 ± 0.25 μM) and HeLa cells (IC50 = 17.52 ± 0.09 and 24.14 ± 0.86 μM) and low toxicity against the HFL-1 (IC50 = 114.50 ± 0.01 and 173.20 ± 10 μM). The flow cytometry was further used to confirm the anticancer activity of potent derivatives against the A549 cancer cell line. DNA binding interaction of anticancer agents 3a and 3h with Ct-DNA has been carried out by absorption, fluorescence, EtBr (dye displacement assay), circular dichroism, cyclic voltammetry and time-resolved fluorescence, which showed noncovalent binding mode of interaction. Anticancer activity of both lead compounds (3a and 3h) may be attributed to DNA binding. The evaluation of the antioxidant potential of pyrazoline analogs 3a and 3h by 2,2-diphenyl-1-picrylhydrazyl free radical showed promising antioxidant activity with IC50 values of 0.132 ± 0.012 and 0.215 ± 0.025 μg/mL, respectively. In silico molecular docking of pyrazoline derivatives was also performed using autodock vina software against the DNA hexamer with PDB ID: 1Z3F and ADMET properties to explore their best hits.
To discover anticancer drugs with novel structures and expand our research scope, pyrazoline derivatives (3a-3l) were designed and synthesized through cyclization of chalcones with thiosemicarbazide/semicarbazide in CH3COOH as a solvent. All newly synthesized pyrazoline derivatives were fully characterized using several spectroscopic experiments such as 1H, 13C NMR, FT-IR spectroscopy, and mass analysis. By HPLC, the purity of all analogs was found above 95% and both lead compounds (3a and 3h) were also validated by HRMS. Anticancer activity of synthesized pyrazoline derivatives (3a-3l) was investigated by the MTT assay against the human lung cancer cell (A549), human cervical cancer cell (HeLa), and human primary normal lung cells (HFL-1). Staurosporine (STS) was used as a standard drug. The anticancer results showed that two potent analogs 3a and 3h exhibit excellent activity against A549 (IC50 = 13.49 ± 0.17 and 22.54 ± 0.25 μM) and HeLa cells (IC50 = 17.52 ± 0.09 and 24.14 ± 0.86 μM) and low toxicity against the HFL-1 (IC50 = 114.50 ± 0.01 and 173.20 ± 10 μM). The flow cytometry was further used to confirm the anticancer activity of potent derivatives against the A549 cancer cell line. DNA binding interaction of anticancer agents 3a and 3h with Ct-DNA has been carried out by absorption, fluorescence, EtBr (dye displacement assay), circular dichroism, cyclic voltammetry and time-resolved fluorescence, which showed noncovalent binding mode of interaction. Anticancer activity of both lead compounds (3a and 3h) may be attributed to DNA binding. The evaluation of the antioxidant potential of pyrazoline analogs 3a and 3h by 2,2-diphenyl-1-picrylhydrazyl free radical showed promising antioxidant activity with IC50 values of 0.132 ± 0.012 and 0.215 ± 0.025 μg/mL, respectively. In silico molecular docking of pyrazoline derivatives was also performed using autodock vina software against the DNA hexamer with PDB ID: 1Z3F and ADMET properties to explore their best hits.
Cancer
is one of the most critical health issues as well as the
leading cause of mortality in the world. Among all types of cancer,
lung and cervical cancer are the leading causes of cancer death. However,
in the twenty-first century, effective malignant tumor therapy is
a problem, and new and less hazardous anticancer medicines with a
broader range of tumor cell cytotoxicity may be necessary.[1−3] Nearly all kinds of cancer belong to a wide category of diseases
in which abnormal cells develop out of control and invade nearby organs.
The three most frequent treatments are general surgery, chemotherapy,
and radiation. However, in most malignant tumor types, there may not
be a treatment that is completely successful.[4−7] There are many drugs currently
used for cancer therapy worldwide, however, anticancer drugs have
major shortcomings such as dose-limiting side effects, induced cellular
resistance, intrinsic acquired resistance, acceptable specificity,
reduced bioavailability, severe toxicity, uncomfortable, cost-intensive
way of administration, and a spectrum of activity limited to narrow
range of tumor types. Despite such limitations, researchers took efforts
to develop anticancer agents for reducing these effects.[8,9] In the last several years, the FDA has approved a large number of
heterocyclic analogs as chemotherapeutic drugs.[10,11] Pyrazoline is a pyrazole substructure found in just a few of them.
In the field of drug design, pyrazoline (4,5-dihydropyrazoles) is
one of the most prominent instances of a physiologically active five-membered
ring. The N–N bond in the pyrazoline ring and its biological
applications appear to be one of the most important factors. Natural
compounds have fewer N–N bonds than living creatures because
they are more difficult to form. Heterocyclic analogs such as 1H-pyrazole-1-carbothioamide/carboxamide are often used to
design and develop physiologically active novel medications.[12−16] The pyrazoline core showed the potential anticancer activity[17−21], and 1H-pyrazole-1-carbothioamide
types also exhibited biological activities such as antibacterial,[22] antifungal,[23] antiviral,[24] antimalarial,[25] antioxidant,[26] anti-inflammatory,[27,28], and analgesic effects.[29−31] Anticancer drugs have
DNA as their primary intracellular target. In the present era, researchers
are learning about the interaction of medications with DNA, and they
believe that this interaction is responsible for DNA damage produced
by malignant tumor cells’ inability to proliferate quickly.[32,33] Medication and micro-molecules commonly employ noncovalent, intercalation,
groove binding, and electrostatic binding to interact with DNA. In
today’s market, there are numerous different substances with
pyrazoline rings that have a variety of actions,[34,35] that is, examples of adducible medicines include antipyrine, metamizole,
propyphenazone, and ramifenazone (Figure ). The carbothioamide/carboxamide-based pyrazoline
analog (1) displayed potent cytotoxic activity against breast cancer
cell line (MCF-7) with an IC50 value of 0.08 μM.[36] Moreover, the pyrazoline derivatives (2 and
3) exhibited promising cytotoxicity against HeLa with IC50 values of 0.21 and 0.25 μM, respectively[37] (Figure ). For our present research work, the design strategy for the synthesis
of pyrazoline derivatives is depicted in Figure . A549 cells are lung adenocarcinoma cells,
and thus, an ideal cancer cell line to test our synthetic drug products.
As lung cancer is one of the leading causes of mortality worldwide,
we believe that our lead compounds 3a and 3h will be an ideal candidate for the treatment of lung cancer. To
expand the anticancer activity of the drug products, we further chose
Hela cells, which are well-established cell lines for testing the
anticancer drugs and other therapeutic products. For us, the rationale
to use Hela cells was to validate the findings of A549 in another
cell line, and of a tissue of different origin besides lungs. Therefore,
using more than one cell line and of different tumor origin provides
a more comprehensive evaluation of the screened drug products. The
apoptosis, DNA binding, molecular docking, ADMET assay, and antioxidant
assay of the lead analogs were carried out.
Figure 1
Some available bioactive
drugs of pyrazoline containing scaffold.
Some available bioactive
drugs of pyrazoline containing scaffold.Reported
carbothioamide/carboxamide-based pyrazoline derivatives.Design strategy of new pyrazoline compounds.
Experimental Section
Materials and Methods
All reagents
and solvents, TLC (precoated 60 F254Al sheets, Merck),
melting point, IR, 1H and 13C NMR spectra, chemical
shift values in ppm, mass spectral analysis and UV–visible,
fluorescence, time-resolved fluorescence, dye displacement assay,
and cyclic voltammetry to examine the DNA-drug interaction performed
as described previously.[38]
Synthesis of Carbothioamide/Carboxamide Pyrazoline
Derivatives (3a–3l)
As reported
earlier,[39] chalcone analogs (2a–2l) were synthesized in ethanol as solvent by
the reaction of substituted aldehydes with acetophenone derivatives
in the presence of a base (NaOH). 1 mmol chalcone derivatives (2a–2f, 2j, and 3l) were stirred with 1 mmol thiosemicarbazide in glacial acetic acid
(5 mL) and heated under reflux conditions for 4–6 h. The same
procedure was followed for chalcone derivatives (2g–2i and 2k) with 1 mmol semicarbazide in acetic
acid. The precipitates (3a–3l) were
filtered, washed with water, dried, and recrystallized in chloroform.
The synthesis of pyrazoline analogs is illustrated in Scheme .
Scheme 1
Preparation of Pyrazoline
Derivatives (3a–3l)
(i)
NaOH (50%), absolute ethanol,
stir; (ii) thiosemicarbazide/semicarbazide, reflux for 4–6
h.
The American type culture collection
(ATCC, USA) provided human lung
cancer cell line (A549), cervical cancer cell line (HeLa), and human
primary normal lung cells (HFL-1) for this investigation. In a water-jacketed
cell culture incubator, all of these cell types were cultured according
to conventional culture conditions at 37 °C with 5% CO2. These cells were cultured in appropriate cell culture media. A549
cells were grown in DMEM media with 2 mM glutamine, HeLa cells were
grown in Eagle’s minimum essential medium (EMEM) as recommended,
whereas HFL-1 cells were also grown in F-12K. Fetal bovine serum 10%
(FBS) was added to the entire medium. At a rate of 8000 cells were
seeded in triplicate in a single 96-well plate for the MTT technique
of cell death investigation. The plate was treated with an increasing
concentration of pyrazoline derivatives (5, 10, 15, 20, 25, and 50
μM) and Staurosporine dissolved in DMSO. The MTT assay was used
to determine whether the test substances have any effect on cell death
after 24 h of treatment. After the treatment period was finished,
the media was removed, and each well was gently washed three times
with PBS to clear any remaining residue just before the experiment.
We used 10 μL of MTT reagent (Sigma, USA, cat. no. 11465007001)
in each well with a working stock concentration of 5 mg/mL, as previously
described.[40,41] The plates were incubated at
37 °C for 4 h after the MTT was added. We used DMSO to remove
crystals after the incubation period was completed. A hybrid multi-mode
plate reader (BioTek, USA) with a microplate reader was used to measure
absorbance at 570 nm. The percentage inhibition was calculated using eq :where ODtreated is the mean optical
density (OD) of the treated cells and ODcontrol is the OD of the vehicle
control cells (negative control).With all of the experimental
drugs, we conducted the MTT assay three times. Prism8 software (GraphPad)
was used to determine the IC50 values of the various drugs,
which were then expressed as a drug concentration (μM).
Apoptosis Studies
We used flow cytometry
analysis on A549 and HeLa cells for the quantitative apoptosis assay.
As previously stated, cells were cultivated at 1.0 × 105 cells/ml and plated in 24-well culture plates (Corning) for 24 h.
Cells were cultured for further 24 h after being treated with 4 μm
of target drugs 3a and 3h. For analysis,
the medium was discarded and cells were washed briefly in PBS, and
then, the trypsinization protocol was carried out. Furthermore, cells
were suspended in PBS and stained with annexin in V-FITC/PI according
to the supplier technique (Sigma, USA, catalogue number: APOAF-20TST).
After adding the reagents, the cells were allowed to incubate for
30 min at room temperature before being analyzed in PBS. The analysis
was conducted on a BD FACS Accuri (BD Biosciences, USA), and the results
were analyzed using FlowJo software (BD Biosciences, USA).[42]
Molecular Docking Study
Investigators
used molecular docking to determine which drugs exhibit anticancer
effects, including the target area’s binding domain. Crystal
structure B-DNA was downloaded from Protein Data Bank (http://www.pdb.org/, ID: 1Z3F). PDB file of drug
structures 3a–3l (as a flexible ligand)
was obtained via Mervin software. The hexamer B-DNA was docked with
pyrazoline analogs using the autodock vina software 4.0 version. Autodock
was used to eliminate heteroatoms and water molecules around the duplex.
Introduced to the specific receptor are polar hydrogen atoms, Kollman
unified atom type charges, and gasteiger partial charges. For BNA,
a grid box with 74 × 64 × 117 Å point spacing of 0.375
Å was used to reformate structure files into PDBQT formats. The
number of generated algorithm runs and assessments were both limited
to 100. To compare structural similarities, the most optimized model
with the minimum binding energy was chosen (RMSD = 0.0). The Discovery
studio visualizer was used to examine the docked DNA-drug complex
image including hydrogen bonds, π–π stacking, and
bond lengths.[43,44]
In Silico
ADMET Assay
The drug’s
likeness features of all heterocyclic analogs (3a–3l) were screened out to determine the drug-like behavior.
The ADMET characteristics of proposed chemical compounds were computed
using the free online Swiss ADMET program after 2D structures were
converted to canonical SMILES representation. It is important in the
drug development process because it provides free access to reliable
models for physicochemical pharmacokinetics and drug-likeness property
prediction.[45,46]
DNA Binding
Studies
Absorption Titration
The DNA interaction
affinity of the test compound is determined using electronic absorption
titration. First, in Tris-buffer solutions, the absorbance of pure
DNA, compounds, and λmax has been calculated. The
absorbance proportion at fixed wavelengths 260 and 280 nm was 1.9:1,
indicating that DNA was free from protein contamination. The intrinsic
binding constant (Kb) was utilized to
evaluate pyrazoline DNA binding affinity by using eq . 2 mL of Tris-HCl/NaCl buffer (pH
= 7.4) was used to dilute a 5 μL stock solution of pyrazoline
derivatives (5 mg/2 mL in DMSO). The molar absorption coefficient
(ε260) 6600 L mol–1 cm–1 was used to evaluate the concentration of DNA in the stock solution.
The UV–visible spectrum of the compound–DNA mixture
solution was obtained when an increasing concentration of Ct-DNA solution
(10–100 μM) was added to the solution of the test compound
(10 μM) at 25 °C. The mixture solution was allowed to incubate
for 3 min before observations. Titration experiments were repeated
three times (n = 3) to ensure consistent results.
The binding affinity (Kb) was calculated
using eq :[47−49]where Aobsd/[compound],
the extinction coefficient of the complex in the bound form, and the
extinction coefficient for the complex are represented by absorption
coefficients, εa, εb, and εf, respectively.
Fluorescence Measurements
Drug
interactions with Ct-DNA have been studied using fluorescence assays.
The experiments were achieved by varying drug concentration from 10
to 80 μM while keeping the concentration of DNA (10 μM)
constant. Before making emission observation, the synthesized compound
was incubated for 3 min with mixing DNA, after which emission titration
was collected in Tris-buffer (pH 7.4) excitation at wavelength 260
nm, and the quenching results were analyzed using the Stern–Volmer
equation (eq ):Slit width has been kept constant at 10 nm
during emission and excitation. The titration experiments were repeated
three times (n = 3) to ensure consistent results.[50−52]
Competitive Fluorescent Displacement Assays
Competitive fluorescence binding experiments utilizing ethidium
bromide, a standard intercalation indicator, were used to evaluate
the degree of molecular binding interaction mechanism of an active
drug with Ct-DNA. The presence of compounds, which is highly effective
in replacing EB from the binding site, might quench the rising fluorescence
intensity. The observable changes in the EB–DNA adduct’s
emission spectrum as a function of quencher addition are particularly
valuable for researching compound–DNA interactions. Fluorescence
measurements were done in Tris-buffer at a 515 nm excitation wavelength
in the range of 530–700 nm. Slit widths for both emission and
excitation were set at about 10 nm.[54,69]
Circular Dichroism
This experimental
technique is a simple and effective way to observe structural changes
that take place as a result of DNA and drug interactions. These studies
were carried out in the free DNA and presence of the compounds (10
μM) at room temperature in Tris buffer (pH = 7.4) in the wavelength
range from 200 to 320 nm.[55] At 50 nm/min
scan rate, the spectrum was generated by averaged triple scans and
eliminating buffer background.
Electrochemical
Measurements
Using
this experiment, a drug–DNA binding interaction has also been
investigated. The solution’s pH was maintained before analysis,
and the entire analytical equipment was built at 25 °C with a
constant potential of 100 mV s–1 scan rate and a
voltage range of −1 to +1 V. The spectra of a test compound
(60 μL) were recorded using a screen-printed electrode (SPE).
These experiments were carried out in a 1:1 ferri/ferrocyanide solution
ratio. Various concentrations of Ct-DNA (Tris-buffer) were used to
further evaluate test compounds 3a and 3h. The working electrode is made of the test compound, whereas the
counter and reference electrodes are made of gold-coated copper. Binding
constants were obtained using eq :where I0 and I are the
peak currents of compound and compound–DNA
complexes, respectively.[56]
Time-Resolved Fluorescence
A study
was carried out using TRF spectroscopy to decide the lifetime decay
measurement of free compound (excited at 280 nm) and when DNA is presented.
The investigations were carried out at room temperature with a quartz
cuvette with an optical path of 10 mm. Using a dilute suspension of
colloidal silica to scatter the excitation beam, the instrument response
function may be determined. Reduced statistic and residual distributions
were used to test the fit’s adequacy.[57−59]
Antioxidant Assay
The antioxidant
activity of the 3a and 3h was determined
using the DPPH (2,2-diphenyl-2-picrylhydrazyl) radical scavenging
technique, as reported.[60,73] In methanol, the DPPH
radical has a purple color and has a strong absorption band with a
maximum of 517 nm. Compound concentrations ranging from 1 to 4 μM
were mixed with DPPH at a concentration of 10 μM. The mixtures
were properly mixed and then incubated at room temperature for 1 h
in the dark. The standard reference was ascorbic acid. As a control,
DPPH in methanol was utilized. The % of DPPH radical scavenged was
used to calculate the antioxidant activity of the compounds, which
was calculated using eq :where Acontrol = DPPH absorbance; Asample = sample
with DPPH absorbance.
Results
and Discussion
Chemistry
The
nucleophilic addition
procedure was used to treat appropriate chalcone analogs (2a–2l) with thiosemicarbazide/semicarbazide in
glacial acetic acid. The pyrazoline ring is synthesized by 1,4 nucleophilic
attack of thiosemicarbazide/semicarbazide on chalcone derivatives,
followed by cyclization and finally, dehydration. In IR spectra of
pyrazoline analogs (3a–3l), the vibrational
bands at 3122–3491, 1557–1591, and 1322–1364
cm–1 correspond to the NH2, nitrile,
and C=S functional groups, respectively. The 1H
NMR spectra of analogs show an ABX pattern among −CH2 protons at 3.02–3.25 ppm (HA) and 3.73–4.12
ppm (HB) as a doublet of doublet confirmed the creation
of the pyrazoline ring. When a 2J coupling occurs
with a proton from the −CH2 group that is not magnetically
equivalent, the −CH proton exhibits a doublet of doublet and
resonates at 5.31–5.97 ppm (HX). At 6.50–9.29
ppm, aromatic proton signals were detected. Carbon signals for −CH2 and methyne were detected in the range of 40.33–45.54
ppm using 13C NMR spectroscopy. The other signal, which
ranges from 154.85 to 178.58 ppm and is attributed to −C=O/–C=S,
supports the carboxamide/carbothioamide assessment. The 1H NMR spectra of lead compounds 3a and 3h are given in Figure and HRMS spectra of both lead compounds are given in Figure S5, and the remaining 1H and 13C NMR, IR, mass, and HPLC spectral analyses were also supported
by the structure of pyrazoline analogs (3a–3l) (Figures S1–S4).
Figure 4
1H NMR spectra of compounds 3a and 3h.
1H NMR spectra of compounds 3a and 3h.
Cytotoxicity
Cytotoxicity
of pyrazoline
derivatives 3a–3l against human lung
cancer (A549), cervical cancer cell line (HeLa), and human primary normal lung cell (HFL-1) were assessed
in vitro using the MTT assay.[62] Staurosporine
(STS) was used as a standard drug. It is extensively used to induce
apoptosis in cell culture systems, including a large number of cancer
cells. Furthermore, staurosporine is well established in cell culture
as a positive control to induce cell death. Considering that our study
is evaluating the anticancer activity of the synthesized drug products,
which act by inducing the apoptosis, we thus preferred to use staurosporine
as a control drug. The results in terms of IC50 values
are given in Table , and dose–response curve is also shown in Figure . The IC50 values
demonstrate that mostly pyrazoline derivatives have moderate to good
cytotoxicity against A549 and HeLa cell lines, and extremely low toxicity
against HFL-1. Compounds 3a, 3d, 3e, 3h, 3k, and 3l showed remarkable
cytotoxicity as compared to the reference drug (staurosporine). Analogs p-CH3 substituent 3e showed IC50 values of 37.07 ± 0.14 and 14.05 ± 0.40 μM
against A549 and HeLa cell lines, respectively.
Analog 3l containing para N,N-diethyl group of one benzene ring and other benzene-containing
−Br at para position exhibited IC50 values of 43.93
± 0.13 and 17.65 ± 0.42 μM against A549 and HeLa cell lines, respectively. Analog ortho-Cl substituent 3d showed an IC50 value 15.0
± 0.46 μM against the HeLa cell line. Analog 3k showed an IC50 value of 52.76 ± 0.28 μM
against human lung cancer cell (A549). The compounds 3a and 3h demonstrate magnificent cytotoxicity
IC50 values of 13.49 ± 0.17 and 22.54 ± 0.25 μM against A549 and 17.52 ± 0.09 and 24.14 ± 0.86 μM against the HeLa cancer cell lines.
Table 1
Selectivity Index (SI) Shows That
Pyrazoline Derivatives,3a and 3h, Were More
Selective for Both Cancer Cell Lines as Compared to the Reference
Drugs
Figure 5
Dose–response curve of compounds 3a–3l concentration vs % cell survival,
and obtained results
are given as mean ± SEM. *** indicates P less
than 0.001. Non-parametric t-test was used for calculating
statistical values by using GraphPad prism.
Dose–response curve of compounds 3a–3l concentration vs % cell survival,
and obtained results
are given as mean ± SEM. *** indicates P less
than 0.001. Non-parametric t-test was used for calculating
statistical values by using GraphPad prism.
Apoptosis
We employed two lead compounds 3a and 3h for flow cytometry analysis to validate
the MTT findings of cell death and provide a quantitative evaluation
of cell death.[63] The % of cells that are
positive in each of the three sub-regions was used to calculate cell
death; PI-positive cells (Q1), AV + PI positive cells (Q2), AV positive
cells(Q3), and live cells(Q4). In the case of staurosporine-treated
cells, there was an increase in signal from Q1 to Q3, as well as a
decrease in Q4. Control cells, on the other hand, saw a high % of
cells in Q4, confirming the assay’s accuracy in detecting cell
death. Similarly, active analogs 3a and 3h were analyzed with the same number of cells. An increased signal
in all quadrants Q1, Q2, and Q3 was observed in cells treated with
the drug, indicating enhanced cell death. Compound 3a causes 25.86% (12.23 + 11.69 + 1.94) cell death, while 3h causes 20.60% (13.48 + 6.02 + 1.08), compared
to 11.41% in the control (9.72 + 1.25 + 0.43). These
findings indicate that both active analogs cause considerable cell
death, as compared to staurosporine (STA)-induced cell death (Figure ). Overall, these
findings support those of the MTT assay, implying that both potential
derivatives could be used as anti-cancer drugs.
Figure 6
Graphical dot plots of
apoptotic A549 cells following 24 h of treatment
with 4 μM concentrations of compounds 3a, 3h, control, and reference drug. Flow cytometry was used to
evaluate the cells after they were extracted and labeled with Annexin-V
(AV) and PI. For the analysis of statistical significance between
drug and control, the histogram investigation of drug treatment has
been used. The results are shown in terms of mean ± SEM. * indicates P less than 0.05 and *** indicates P less
than 0.001 with n = 4 for each analysis. Non-parametric t-test was used for calculating statistical values by using
the GraphPad prism.
Graphical dot plots of
apoptotic A549 cells following 24 h of treatment
with 4 μM concentrations of compounds 3a, 3h, control, and reference drug. Flow cytometry was used to
evaluate the cells after they were extracted and labeled with Annexin-V
(AV) and PI. For the analysis of statistical significance between
drug and control, the histogram investigation of drug treatment has
been used. The results are shown in terms of mean ± SEM. * indicates P less than 0.05 and *** indicates P less
than 0.001 with n = 4 for each analysis. Non-parametric t-test was used for calculating statistical values by using
the GraphPad prism.
Molecular
Docking
The anticancer
agent ellipticine binds DNA via intercalative binding based on stacking
interactions along the major base-pair axis in the PDB ID: 1Z3F structure, but other
binding modes have been proposed, particularly for ellipticine derivatives
between base pairs and inhibition of topoisomerase II enzyme, a DNA
replication enzyme, blocking it and providing potent antitumor action.
Therefore, we have done molecular docking of all the synthesized derivatives
with 1Z3F and
also used it to confirm the experimental results and determine the
binding manner of both lead compounds with DNA. All the compounds 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, and 3l binding
energies were determined to be −7.1, −6.4, −6.6,
−6.3, −6.7, −6.5, −6.6, −6.9, −6.7,
−7.6, −7.8, and −6.3 kcal/mol. Docked models
of both compounds were prepared by using the discovery studio visualizer.
A DNA duplex of sequence hexamers d(CGATCG)2 was used as
a target for molecular docking of the pyrazoline analogs (3a–3l). Compounds 3a and 3h have a significant binding affinity (−7.1 and −6.9
kcal/mol, respectively), indicating that the B-DNA results were of
excellent quality.[64,65] According to the docking experiments,
the analogs 3a and 3h interacted with DNA
via an intercalative binding mode, as illustrated in Figure .
Figure 7
Intercalation of lead
compounds 3a and 3h with DNA base pairs
(PDB ID: 1Z3F).
Intercalation of lead
compounds 3a and 3h with DNA base pairs
(PDB ID: 1Z3F).
Drug
Likeness
Oral bioavailability
is a critical component in the development of bioactive compounds
as medicinal treatments. Lipinski’s rule of five (RO5) is a
useful technique for evaluating a molecule’s drug-likeness
features.[66] The value ranges of numerous
main variables for all of the compounds are given in Table . The molecular weight range
was 338.37–431.39 g/mol, and the evaluated numbers of H-bond
donors (HBD) and H-bond acceptors (HBA) to/from H2O molecules
in aqueous solution were 0.0–2.0 and 1.0–4.0, respectively.
The predicted octanol/water partition coefficients (QPlogPo/w) were 1.89–4.25, and the predicted aqueous solubility (QPlogS)
values were −4.04 to −6.41. The predicted numbers of
rotatable bonds (RB) were in the range of 4–6, and the apparent
Caco-2 cell permeability (QPPCaco) values were 20.25–41.09
nm/s. The apparent MDCK cell permeability (QPPMDCK) values
were 0.09–133.47 nm/s, and the predicted skin permeability
(QPlogKp) values were −6.65 to −5.52. The
human GI absorption values were high. The topological polar surface
area (TPSA) values were in the range of 61.93–122.77, and the
values of the logarithm of the partition coefficient (IlogP) were
0.00–3.78. The molar refractivity (MR) values were 104.54–124.24,
and the fractions of sp3-hybridized carbon (fraction Csp3) were 0.18–0.26. For every compound, the number of
Lipinski violations was zero. In Figure , the red lines which represent compounds 3a and 3h are incorporated in the pink area predicting
good oral bioavailability. The results were established to be within
an allowable extent when compared to the pyrazoline derivatives’
drug-likeness features, implying that they have the potential to be
druggable compounds.
Table 2
Compounds (3a–3l)
Drug-likeness Propertiesa
compounds
MW
RB
fraction
Csp3
HBA
HBD
TPSA
IlogP
GI absorption
QPlogPo/w
QPlogKp
LV
MR
QPPCaco (nm/s)
QPPMDCK (nm/s)
QPlogS
3a
358.89
4
0.22
1
1
76.95
2.86
high
3.24
–6.02
0
111.74
32.72
49.43
–4.36
3b
402.35
4
0.26
1
1
73.71
3.27
high
4.25
–5.52
0
114.80
35.72
0.09
–5.31
3c
369.44
5
0.22
3
1
122.77
1.99
high
1.90
–6.65
0
115.55
20.25
1.56
–4.11
3d
358.89
4
0.22
1
1
76.95
2.56
high
3.18
–6.02
0
111.74
32.72
12.81
–5.36
3e
338.47
4
0.26
1
1
76.95
2.87
high
3.04
–6.08
0
111.70
26.75
60.12
–5.14
3f
403.34
4
0.22
1
1
76.95
2.94
high
3.32
–6.25
0
114.43
35.43
0.10
–5.56
3g
342.82
4
0.22
2
1
61.93
2.95
high
2.81
–6.35
0
104.54
23.38
133.47
–5.17
3h
387.27
4
0.22
2
1
61.93
3.08
high
2.90
–6.57
0
107.23
23.93
0.51
–5.37
3i
338.37
4
0.22
4
0
81.73
2.07
high
1.89
–6.47
0
105.68
20.84
4.61
–4.04
3j
374.50
4
0.18
1
1
76.95
0.00
high
3.01
–5.67
0
124.24
27.01
28.75
–6.41
3k
358.44
4
0.18
2
1
61.93
3.05
high
3.18
–6.00
0
117.03
22.07
66.38
–6.21
3l
431.39
6
0.18
1
1
76.95
3.78
high
4.04
–5.89
0
124.04
41.09
0.15
–6.34
STA
466.53
2
0.32
4
2
69.45
3.19
high
3.17
–6.85
0
139.39
48.76
153.09
–7.59
Abbreviations representing physicochemical
attributes for “drug likeness” analyzed from SWISS ADMET:
MW = molecular weight, RB = number of rotatable bonds, HBA = number
of hydrogen-bond acceptors, HBD = number of hydrogen-bond donors,
TPSA = topological polar surface area, IlogP = logarithm of the partition
coefficient, GI = gastrointestinal, QPlogPo/w = predicted
octanol/water partition coefficient, QPlogKp = predicted
skin permeability, LV = number of Lipinski violations, MR = molar
refractivity, QPPCaco = apparent Caco-2 cell permeability,
QPPMDCK = apparent MDCK cell permeability, QPlogS = predicted
aqueous solubility.
Figure 8
Radar charts for an indicator of oral bioavailability
arising by
Swiss ADME web tool; pink zone shows ideal values for oral bioavailability
and red zone shows drugs.
Radar charts for an indicator of oral bioavailability
arising by
Swiss ADME web tool; pink zone shows ideal values for oral bioavailability
and red zone shows drugs.Abbreviations representing physicochemical
attributes for “drug likeness” analyzed from SWISS ADMET:
MW = molecular weight, RB = number of rotatable bonds, HBA = number
of hydrogen-bond acceptors, HBD = number of hydrogen-bond donors,
TPSA = topological polar surface area, IlogP = logarithm of the partition
coefficient, GI = gastrointestinal, QPlogPo/w = predicted
octanol/water partition coefficient, QPlogKp = predicted
skin permeability, LV = number of Lipinski violations, MR = molar
refractivity, QPPCaco = apparent Caco-2 cell permeability,
QPPMDCK = apparent MDCK cell permeability, QPlogS = predicted
aqueous solubility.
DNA Binding
The capability
of Ct-DNA to interact with active molecules 3a and 3h was investigated using UV–visible spectroscopy to
investigate the binding mechanism of DNA. During compound absorption
titrations, two traits emerge: hypochromic and hyperchromic. Hypochromic
refers to a drop in absorbance caused by a strong stacking interaction
between DNA base pairs and the aromatic chromophore of the compounds,
which is thought to be a unique property of the intercalative form
of binding.[67]Figure illustrates absorption spectra of small
molecules 3a and 3h without and with increasing
concentration of DNA. In the absence of DNA, 3a displayed
three absorbance peaks at 220, 260, and 350 nm, and 3h showed 215, 255, and 300 nm. With the addition of DNA, the absorbance
at all three bands decreased. For quantitative investigation of the
binding strength of compounds to DNA, intrinsic binding constants Kb of compounds with Ct-DNA were determined by
utilizing eq . Intercept
plots of [DNA]/(εa – εf)
vs [DNA] determined the intrinsic binding constants (Kb) for compounds 3a and 3h to
be 3.8 × 104 and 1.3 × 104 M–1, respectively.
Figure 9
UV spectrum and inset plot between [DNA]/[εa –
εf] vs [DNA] of 3a and 3h (10 μM) with increasing amount of Ct-DNA (10–90 μM).
UV spectrum and inset plot between [DNA]/[εa –
εf] vs [DNA] of 3a and 3h (10 μM) with increasing amount of Ct-DNA (10–90 μM).
Emission Titration
Emission titrations
were used to explore the binding properties of compounds 3a and 3h with DNA as a suitable addition to the previous
experiments. Ct-DNA emission titrations were performed in the increasing
concentration of both the compounds. Emission intensity decreases
dramatically, with the addition of compounds aliquot, as seen in Figure . The quenching
of emission of the compounds is caused by the transfer of a photoelectron
from DNA’s guanine nitrogenous base to excited states of compounds.[68] The fraction of quenching may be calculated
experimentally using the value of KSV obtained
from the SV plot shown in Figure , and the Stern–Volmer constant (KSV) is the slope of F0/F versus [Q] found to be 5.80 × 103 and
4.28 × 103 M–1 for the compounds 3a and 3h, respectively.
Figure 10
Emission titration spectra
of a constant concentration of DNA (10
μM) with varying compounds 3a and 3h concentration (10–80 μM). The Stern–Volmer plot
of compounds 3a and 3h.
Emission titration spectra
of a constant concentration of DNA (10
μM) with varying compounds 3a and 3h concentration (10–80 μM). The Stern–Volmer plot
of compounds 3a and 3h.
Dye Displacement Assay
The low
fluorescence intensity of EtBr offers information regarding DNA-free
EtBr, which is utilized to determine the mechanism of DNA binding
interaction. When ethidium bromide (EtBr) is intercalated into DNA,
the emission peak is significantly increased. Fluorescence quenching
was seen after the test chemical was continuously added to the EB-DNA
complex. The dye displacement assay shows that compound 3c interacts with DNA in an intercalative manner. As the concentration
of both test compounds increases, the Stern–Volmer eq was employed to quantify
the degree of fluorescence quenching of the EB-DNA system. The value
of KSV obtained from the SV plot may be
used to experimentally calculate the fraction of quenching and so
predict the optimal DNA binding interaction mode. A competitive fluorescence
displacement test is commonly used in the general approach to DNA
binding research.[69] The quenching suggests
that compounds 3a and 3h cause the release
of ethidium bromide from DNA helix, implying that they bind to DNA
in an intercalative manner. KSV values
for the fluorescence quenching depth of EB were found to apply the
Stern–Volmer plot. Stern–Volmer constant (KSV) is the slope of Fo/F vs [Q] found to be 1.84 × 104 and 1.69
× 104 M–1 for the compounds 3a and 3h, respectively. The other parameters
of the dye displacement measurement are mentioned in Figure .
Figure 11
Displacement of ethidium
bromide from Ct-DNA by 3a and 3h.
Displacement of ethidium
bromide from Ct-DNA by 3a and 3h.The circular
dichroism technique is widely employed to detect conformational changes
in Ct-DNA and proteins, as a result of their interactions with ligand
molecules. To explore the structural modification found throughout
compound–DNA binding interaction, a CD spectrum of Ct-DNA was
used without and with active compounds (3a, 3h). Generally, native Ct-DNA shows two distinct peaks, a positive
peak at 275 nm due to DNA helicity and a negative peak at 245 nm due
to π–π base stacking.[70]Figure shows
that compounds binding to DNA have only a significant result on a
negative peak location of 245 nm and also a slight shift at 275 nm.
The results demonstrate that the compounds 3a and 3h interact with Ct-DNA by intercalative mode, which in turn
causes conformational alterations in the DNA structure.
Figure 12
CD spectra
of Ct-DNA (50 μM) without and with active analogs 3a and 3h (50 μM).
CD spectra
of Ct-DNA (50 μM) without and with active analogs 3a and 3h (50 μM).
Cyclic Voltammetry
The manners
of compound–DNA binding interaction were investigated using
an electrochemical method. The change in peak potential of the cyclic
voltammogram provides important information about the compound’s
binding mode with DNA. Figure shows a cyclic voltammogram of compounds 3a and 3h in the absence and presence of DNA. The peak
current was reduced when Ct-DNA was added to the compounds, as shown
in the graph. As a result, the development of a compound–DNA
complex formation may occur. This may be due to the complex creation
of compounds with bulky Ct-DNA that diffuses gradually toward the
working electrode. The greater oxidation peak currents of pyrazoline
analogs (3a, 3h) than their DNA adduct correspond
to easier mobilities and faster responses toward working electrodes
due to faster diffusion. The plot between log(I/I0 – I) versus log(1/[DNA])
was utilized to calculate binding constant values K for compounds 3a and 3h, which are found
to be 1.25 × 104 and 7.1 × 103, respectively,
as depicted in ref (71).
Figure 13
Cyclic voltammogram of 1.0 × 10–5 M of analogs 3a and 3h in Tris-buffer, at 100 mV s–1 scan rate free compound (black), in the presence of DNA (10–40
μM). The plot between log(I/I0 – I) vs log(1/[DNA]) for finding binding constant
value (a,b) for 3a and 3h, respectively.
Cyclic voltammogram of 1.0 × 10–5 M of analogs 3a and 3h in Tris-buffer, at 100 mV s–1 scan rate free compound (black), in the presence of DNA (10–40
μM). The plot between log(I/I0 – I) vs log(1/[DNA]) for finding binding constant
value (a,b) for 3a and 3h, respectively.Emission
decay of lead compounds 3a and 3h with and
without DNA is shown in Figure and determined parameters are shown in Table ; decay curve can be best fitted
with a tri-exponential decay model. It was observed that the relative
amplitude of the lifetime of active analogs moderately increases with
the addition of DNA. Without Ct-DNA, compounds 3a and 3h show lifetimes τ1, τ2, and τ3 of 0.90, 6.60, 0.12 and 1.26, 7.46, 0.18
ns, respectively, but in the presence of DNA small changes in all
lifetimes occur 1.0, 6.73, 0.21 and 1.38, 7.23, 0.29, respectively.
In the case of 3a and 3h, we found a slight
change in all amplitude τ1 time components from 27.81–28.57,
36.5–35.34%, τ2 time amplitude from 33.73–32.78,
17.41–19.64%, and τ3 time amplitude from 38.46–38.65,
46.09–45.05%, respectively, for upon addition of DNA. The plot
of compounds 3a and 3h in the absence and
presence of DNA demonstrate significant changes in an average lifetime
because viscosity difference of compounds solution in the presence
of DNA occurs. Results suggest that compound 3a has more
binding affinity as compared to compound 3h.[72]
Figure 14
Time-resolved fluorescence graph of active compounds 3a and 3h without and with Ct-DNA and standard
IRF.
Table 3
Lifetime Fluorescence
Spectral Details
Feature Compounds 3a and 3h
Analogs
a1
a2
a3
τ1
τ2
τ3
⟨t⟩
χ2
3a
0.10
0.02
0.88
1.26
7.46
0.18
0.43
1.08
3a–DNA
0.13
0.03
0.84
1.38
7.23
0.29
0.64
1.13
3h
0.1
0.01
0.89
0.90
6.60
0.12
0.25
1.13
3h–DNA
0.14
0.01
0.85
1.0
6.73
0.21
0.39
1.04
Time-resolved fluorescence graph of active compounds 3a and 3h without and with Ct-DNA and standard
IRF.The data were fitted using triexponential
function equation given
below:The average lifetime was determined by applying the equation
given
below:The free radical
reduced which is present on DPPH accepts an electron or an H-atom
from a molecule. The color change from violet to yellow was detected
after 1 h of incubation. The UV–visible spectrophotometer absorbance
decreases (516 nm wavelength) after the addition of compounds 3a and 3h due to pairing of its lone-pair electron
with electron of an antioxidant to form reduced DPPH. The compounds 3a and 3h show a strong antioxidant activity
with IC50 values of 0.132 ± 0.012 and 0.215 ±
0.025 μg/mL.[73] A lower IC50 value implies higher antioxidant activity. Higher antioxidant activity
of 3a as compared to 3h might be due to
the presence of nitrogen, chlorine atom, and carbothioamide group
in their structure, which donate the electron density easily. The
antioxidant activity of both the compounds was significant, as illustrated
in Figure .
Figure 15
Antioxidant
bar graph of active analogs 3a and 3h.
Antioxidant
bar graph of active analogs 3a and 3h.
Conclusions
We demonstrated
the synthesis of a series of carbothioamide/carboxamide-based
pyrazoline derivatives and characterized these by using different
spectroscopic methods. All the analogs have shown moderate to excellent
cytotoxicity against A549 and HeLa cancer cell lines. The lead compounds, 3a and 3b exhibited significant inhibitory activity
toward the A549 than the HeLa cancer cell lines. Anticancer activity
of target compounds also has been validated by apoptosis. The binding
affinity of both the active analogue with DNA was carried out by using
UV–visible, fluorescence, competitive assay method using ethidium
bromide, circular dichroism, cyclic voltammetry, and time-resolved
fluorescence. The molecular docking analysis of derivatives 3a and 3h with the DNA hexamer shows the noncovalent
intercalation binding interactions which also verify the experimental
results. The drug-likeness property of all derivatives was determined
via ADMET assay. Thus, the anticancer mechanism of action of these
compounds could be attributed to the DNA intercalation binding manner.
Authors: Irina Yu Strobykina; Alexandra D Voloshina; Olga V Andreeva; Anastasiia S Sapunova; Anna P Lyubina; Syumbelya K Amerhanova; Mayya G Belenok; Liliya F Saifina; Vyacheslav E Semenov; Vladimir E Kataev Journal: Bioorg Chem Date: 2021-09-03 Impact factor: 5.275
Authors: Dmytro Havrylyuk; Borys Zimenkovsky; Olexandr Vasylenko; Craig W Day; Donald F Smee; Philippe Grellier; Roman Lesyk Journal: Eur J Med Chem Date: 2013-06-06 Impact factor: 6.514
Figure 1
Figure 2
Figure 3
Scheme 1
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15