Bio-evaluation of the 2-nitrochalcones as potential anti-lung cancer agents, inducers of apoptosis and inhibitors of protein kinase (VEGFR-2)

A series of the 2-nitrochalcones 3a–3k was synthesized and evaluated for cytotoxicity against the human lung adenocarcinoma (A549) and human embryonic kidney (HEK293-T) cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The 3-(4-fluorophenyl) 3c and the 3-(4-(1,1,2,2-tetrafluoroethoxy)phenyl derivative 3k induced early (25–29%) and late (48–60%) apoptosis of A549 cells as determined by the Annexin V-FITC/PI method. The 3-(4-fluorophenyl) 3c, 3-(4-methoxyphenyl) 3h, 3-(2,3,4-trimethoxyphenyl) 3j and the 3-(4-(1,1,2,2-tetrafluoroethoxy)phenyl derivative 3k were also found to exhibit significant inhibitory activity against vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase compared to staurosporine (0.035 ± 0.002 µM) or nintedanib (IC50 = 0.021 ± 0.001 µM) with IC50 values of 31.49 ± 0.02, 39.95 ± 0.17, 36.90 ± 0.16 and 29.10 ± 0.16 µM, respectively. Molecular docking studies were also conducted on 3c and 3k as representative models to recognize the hypothetical binding motif of the title compounds within the active site of VEGFR-2.


Introduction
Lung cancer related mortality rate accounts for 25% of cancer deaths worldwide [1], with nearly 80-90% of lung cancer cases being non-small cell lung cancer (NSCLC) [2].The problem with cancer is that the affected cells tend to proliferate uncontrollably, avoid apoptosis, and readily metastasize [3].Angiogenesis, a process of formation of new blood vessels from pre-existing ones plays a significant role in tumor growth as the blood vessels are essential for the increase of tumor size [4].The vascular endothelial growth factor (VEGF) is the main mediator of angiogenesis and the vascular endothelial growth factor receptor-2 (VEGFR-2) is over-expressed in NSCLC patients [5,6].Inhibition of VEGFR-2 tyrosine kinase activity affects the blood supply to tumour cells, in turn, reduces angiogenesis and suppresses tumour growth and metastasis [6].VEGFR-2 has the strongest pro-angiogenic activity and as a result, inhibition of VEGFR-2 tyrosine kinase activity has become a central focus of molecularly targeted cancer therapy [7].Moreover, dysregulation of VEGFR-2 tyrosine kinase activity is also associated with HIV infection and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [8].A wide range of VEGFR-2 kinase inhibitors with antitumor properties such as sorafenib, regorafenib, tivozanib, vandetanib, sunitinib, pazopanib, cabozantinib, lenvatinib, axitinib and apatinib have been developed [9].However, clinical results demonstrate resistance to the anti-angiogenic drugs in certain types of cancers, and this is one of the major obstacles to current anti-angiogenic therapy.Despite tremendous progress made to-date in the development of effective drugs for the treatment of cancers, there is still a need to design and synthesize more cost-effective and potent antitumor agents with reduced or no toxicity.To this end, research focusing on the syntheses and antitumor activity studies of chalcones is justified because these easily accessible compounds exhibit no adverse side effects and tend to reduce the clinical signs and symptoms with decent bioavailability [10].Chalcones and their derivatives have demonstrated potential in vitro and in vivo activity against various cancers via multiple mechanisms, including induction of apoptosis, DNA and mitochondrial damage, inhibition of angiogenesis, tubulin inhibition, kinase inhibition, and drug efflux protein activities, or a combination of some of these mechanisms [10,11].4-Hydroxychalcone A shown in Fig. 1, for example, has been found to display inhibitory effect against VEGFR-2 tyrosine kinase which is an essential factor in angiogenesis [12].The anticancer or cancer chemopreventive activities of chalcones and their derivatives form part of several review articles [13,14], thus showing the potential of chalcone derivatives to be considered as the new chemical space in drug discovery.Previous studies on the structure-activity relationship (SAR) of synthetic chalcones emphasized the importance of the ketoethylinic/ketovinyl (-COCH = CH-) moiety and the electronic properties of the substituents on the A and/or B ring on biological activity [15][16][17][18][19].The α,β-unsaturated carbonyl framework serves as a Michael acceptor to facilitate the interaction of chalcones with sulfhydryl groups in cysteine residues of the targeted proteins or with thiol groups resulting in a broad spectrum of biological properties of this class of compounds [16].Moreover, the chalcone structure comprises a flat framework for possible π-π stacking interaction and a carbonyl group as hydrogen-bond acceptor, which are pharmacophoric features common in various VEGFR-2 inhibitors [20].The biological activity of chalcones is also correlated with the electron-donor and/or electron-acceptor properties of the substituents on both the aryl groups.As a result, chemical modifications of the A-and/or B-ring of the chalcone scaffold continue to preoccupy the attention of medicinal chemists with the intent to develop more potent and efficient anticancer, anti-inflammatory, antidiabetic, antimicrobial, anti-oxidant, antiparasitic, psychoactive, and neuroprotective agents [15,19].
The A-ring nitro substituted chalcones have attracted the interest of medicinal chemists among the synthetic chalcones because of their wide applications as potential antimicrobial, antihyperglycemic, antinociceptive and antitumor agents [21].Although this group is prone to metabolic reduction into reactive nitroso and hydroxylamine derivatives leading to increased toxicity [22], several nitro substituted compounds are in the market as drugs for the treatment of various diseases with no safety risks [23].The strong electron-withdrawing inductive and resonance effect of this group cause changes in the polarity of the molecules which, in turn, favours interaction of the drug molecules with nucleophilic sites of protein structures to inhibit enzyme activity [24].The nitro group represents a masked electrophile in the design of covalent inhibitors and it targets the binding pockets of enzymes or receptors with appropriately placed cysteine and acid residues [25].The A-ring nitro substituted chalcone derivative, (E)-3-(3,4,5-trimethoxyphenyl)-1-(4-nitrophenyl)prop-2-en-1-one B, for example, was identified as a lead compound with promising anticancer, anti-inflammatory and antioxidant properties with no toxicity [26].Both oral and intraperitoneal administration of the A-ring ortho-, metaor para-nitro substituted chalcones C into Wistar rats resulted in strongest antiinflammatory protective effect of the ortho-nitrochalcone compared to the other isomers [27].It was concluded based on the results of the carrageenan-induced rat hind paw edema model that a nitro group located at the ortho position of chalcone framework plays an important role in both the antiinflammatory protective effect and the bioavailability.The 4'-(2,5-dichlorobenzene)sulfonamido appended 3-nitrochalcone hybrid (CL185) D, on the other hand, was found to stimulate inflammation and angiogenic activity, and also to upregulate VEGF in vivo [28].Inflammation itself is a critical component of tumour progression and many cancers arise from sites of infection, chronic irritation and inflammation [29].It has also been observed that in some cancers, inflammatory conditions aid proliferation and survival of the tumor cells, while in the other types of cancers, inflammation is induced by the oncogenic changes in tumor microenvironment [29,30].
Within the framework of our research on chalcone derivatives as candidate anticancer agents, we became interested in the evaluation of the 2-nitrochalcones bearing different substituents on the B-ring for cytotoxicity in vitro against the human lung adenocarcinoma (A549) cell line.Their toxicity against the human embryonic kidney (HEK293-T) cells has also been evaluated to assess their selectivity and safety profile at least in vitro.Since cytotoxicity does not define a specific cellular death mechanism, we also evaluated whether these α,β-unsaturated ketones could induce cell death in the A549 cancer cells.Additionally, their potential to inhibit VEGFR-2 tyrosine kinase activity has been evaluated experimentally through enzymatic assay (in vitro) and theoretically through molecular docking to rationalize their possible mode of interaction with amino acid residues in the active sites of this enzyme.

Synthesis and characterization
The 2-nitrochalcones 3a-3k were synthesized via base (5% NaOH (aq)) mediated Claisen-Schmidt condensation of 2-nitroacetophenone with benzaldehyde derivatives in ethanol at room temperature as shown in Scheme 1. Aqueous work-up and acidification with hydrochloric acid followed by filtration and recrystallization afforded pure compounds characterised using a combination of NMR, IR and mass spectroscopic techniques.The corresponding copies of NMR spectra have been included as Fig. S1 in the Supplementary Materials.The designation of Ar and the substitution pattern on this ring are indicated in Table 1 below.Their 1 H-NMR spectra revealed the presence of the olefinic proton signals of the α,β-unsaturated carbonyl framework of compounds, which resonate as two sets of doublets in the aromatic region with vicinal coupling constant (J αβ ) value of 15.0-16.5 Hz.This value is consistent with the trans geometry about the ethylene linkage for the analogous compounds and the geometry previously observed in the crystal structure of these compounds [31].The other protons exhibited additional resonance signals typically present on the scaffold of the respective chalcone derivative.These structurally simple chalcone derivatives have previously been found to undergo cyanide anion triggered Michael addition followed by a cascade cyclization mechanistically related to the Baeyer-Drewson reaction to afford the 2-(3oxoindolin-2-ylidene) acetonitriles [32].
Our interest in chalcone derivatives as candidate anticancer agents encouraged us to evaluate the 2-nitrochalcone derivatives 3 for cytotoxicity in vitro against the A549 cancer cell line using the MTT assay.The compounds were also evaluated for toxicity against the human embryonic kidney (HEK293-T) cells.

Biological studies
The compounds were assayed at concentrations ranging from 0.5 to 100 µM with DMSO as the negative control, and doxorubicin and gefitinib reference standards for the assays.Gefitinib has previously been found to enhance apoptosis and suppress growth of lung cancer (A549) cells [33].The IC 50 values (lethal concentration at which 50% of the cancer cells are killed) of compounds 3a-k averaged from three independent experiments against doxorubicin, nitendanib and gefitinib are represented in Table 2.The dose-response curve used to calculate IC 50 values of the test compounds and the percentage cell viability against the A549 and HEK293-T cell lines are listed as Fig. S2 and Tables S1 and S2  3-position of the conjugated framework.Among the 3-(halogenophenyl) substituted derivatives 3b-e, compound 3c substituted with a moderately π-electron delocalizing fluorine atom at the para position of the 3-phenyl ring exhibited higher activity against the A549 cells with an IC 50 value of 7.75 ± 0.73 µM.This compound exhibited higher activity compared to the 3-phenyl substituted derivative 3a (IC 50 = 16.94 ± 2.00 µM).A halogen atom confers aromatic rings with an electron-withdrawing inductive effect and an electron-donating resonance effect.A moderately π-electron delocalizing 4-fluorophenyl group of 3c would probably result in increased hydrophobic (eg, π-π stacking, π-π T-shaped, πalkyl, alkyl) interactions with the protein residues in the receptors and therefore enhance the biological profile of this compound.The strong electron-withdrawing effect of this halogen atom, on the other hand, would help in forming hydrophilic (halogen and/or hydrogen bonds) interactions that help to increase the number of interactions of drug molecules with their protein targets, thereby enhancing biological activity [34].The presence of fluorine in a ring system may increase biological half-life of molecules by delaying oxidative biotransformation and can also increase bio-absorption by lipophilic effects [35].For the chlorinated derivatives, significant cytotoxicity was observed for 3d (IC 50 = 13.35 ± 0.62 μM) substituted with a chlorine atom in position 2 of the B ring than 3e (IC 50 = 26.16± 1.25 μM) with the same atom at the para position.The presence of a chlorine atom on a phenyl ring has been found to lead to increased lipophilicity of the drug molecule and also to polarize the phenyl ring to result in increased non-bonding interactions with protein groups in the binding site of the receptor [36].Steric halogen effect is more pronounced at the 2-and 6-position of the phenyl group [37].
The ortho-chlorophenyl group is expected to be oriented on the conjugated framework in such a way to avoid steric interactions between Cl and the β-hydrogen atom and/or the nitro group.Such an orientation probably resulted in a conformation that embedded or anchored the 2-chlorophenyl group of 3d into the binding pocket of the receptor to interact with several amino acid residues resulting in increased biological activity compared to isomer 3e.The presence of a moderate electron donating methyl group on the para position of the 3-phenyl ring of 3f (IC 50 = 16.10 ± 1.70 μM) resulted in a slightly higher activity compared to the parent compound 3a.Derivative 3g substituted with an electron donating methoxy group at the meta position of the 3-phenyl ring exhibited slightly higher cytotoxicity (IC 50 = 15.18 ± 1.01 µM) compared to 3a and 3f.
Previous studies on methoxylated chalcones suggested that the number and the position of strong π-electron delocalizing methoxy groups on the aromatic ring/s are critical for their cytotoxicity [35].Derivative 3h containing a methoxy group in the para position or 3i substituted at the meta positions with two such groups were found to exhibit comparable cytotoxicity (IC 50 = 14.20 ± 0.63 μM and 14.67 ± 0.71 μM, respectively).
The presence of an additional methoxy group at the meta position of 3i resulted in increased electron density of the ring and slightly increased biological activity compared to 3g substituted with only one such group on the 3-phenyl ring.Compound 3j substituted with three (3) methoxy groups on the B-ring was found to be the least active among the methoxy substituted derivatives (IC 50 = 31.35± 0.20 µM) and to exhibit significantly reduced cytotoxicity among the test compounds.Significantly reduced activity of 3j is probably due to steric effects of the bulky 2,3,4-trimethoxyohenyl.Compound 3k substituted with a 4-(1,1,2,2-tetrafluoroethoxy)phenyl group at the 3-position of the conjugated scaffold, on the other hand, exhibited significant cytotoxicity (IC 50 = 10.74 ± 0.74 µM) compared to the alkoxy analogues 3h-j substituted with the strongly π-electron delocalizing methoxy group/s on the 3-phenyl ring.
The treatment of cancer requires the drug used to specifically inhibit proliferation of cancer cells by interfering with molecular target/s in cancer cells with high selectivity index against healthy or non-cancerous cells [38].The test compounds were also evaluated for toxicity against the human embryonic kidney 293-T (HEK293-T) cell line which has been found to be highly sensitivity to free doxorubicin [39].Selectivity index (SI = IC 50 of non-cancerous cells/IC 50 of cancerous cells) refers to the ability of a drug to efficiently kill cancer cells with minimal effect to the noncancerous cells.A compound with a higher SI value is said to be more selective to kill a cancer cell and hence less toxic to normal ones.SI values of 1 or less relate to lack of selective cytotoxicity of a compound to kill a cancer cell whereas the high values (SI > 1) suggest that the cancer cells will be killed at a higher rate than normal ones [40].Cytotoxicity against the human embryonic kidney cell line HEK-293-T was also evaluated and the selectivity was thus assessed.The parent compound 3a with an SI value of 0.82 will kill both A549 and non-cancerous cells.The B-ring substituted derivatives 3b, 3f, 3g and 3h with moderate cytotoxicity against the A549 cell line (SI = 1.22,1.85, 1.88 and 1.65, respectively) and derivatives 3c and 3k with significant activity against this cancer cell line (SI = 1.86 and 1.02, respectively) were also be slightly toxic to the HEK293-T cells.Chalcone derivative 3d substituted with a -C 6 H 4 (2-Cl) group at the 3-position of the conjugated framework with an IC 50 value of 13.35 ± 0.62 µM against the A549 and IC 50 value of 29.86 ± 0.80 µM against the HEK293-T cell line and SI value of 2.23 will kill cancer cells with minimal effect to the normal cells.Compound 3i (Ar = -C 6 H 3 (3,5-OMe)) with an SI value of 2.66 will also be more selective against the A549 cell line compared to the other derivatives.The known anticancer drugs, nintendanib and getifinib exhibited increased activity against A549 cell line (IC 50 = 0.46 ± 0.06 and 0.57 ± 0.03, respectively) and also against the HEK293-T cell line (IC 50 = 0.98 ± 0.08 µM and 1.60 ± 0.15 µM, respectively) with SI values of 2.13 and 2.80, respectively.The SI values of 3d and 3i, which are higher than 2 in our view suggest relatively good cytocompatibility for these 2-nitrochalcone derivatives.Although compounds 3c and 3k substituted with a fluorine atom or fluorine-containing group at the 4-position of the 3-phenyl ring exhibited significant activity against the A549 cell line, these compounds will be less selective against the normal cells.However, further cytotoxicity tests against different normal cell lines need to be conducted to confirm the safety and/or cytotoxicity of these compounds.
The mode of action of many anticancer drugs is to induce cell death through apoptosis which is characterised by biochemical and morphological changes in cells [41].The changes may involve cellular shrinkage and cell membrane blebbing, chromatin condensation and/or extensive doublestranded DNA fragmentation [41,42].Literature precedents revealed chalcones as powerful inducers of apoptosis in different tumor cells, however, their mechanisms of cancer cell death vary [43].Since the test compounds were relatively non-toxic to the normal (HEK293-T) cells compared to doxorubicin, we have investigated their mode of cancer cell death against the A549 cell line using the Annexin V-fluorescein isothiocyanate (FITC) propidium iodide (PI) assay.The population of apoptotic cells was determined using flow cytometry (raw data enclosed as Table S3 in SI), which is one of the tools used for the investigation of molecular and morphological events occurring during cell death and cell proliferation.The presence of positive cells staining with propidium iodide, a compound permanently staining the DNA of dead cells may suggest that apoptosis is only one of the processes that cells undergo under the influence of the tested compounds.As shown in Fig. 2, treatment of A549 cells with the test compounds for 24 h showed cell increment in early and late apoptosis with percentage ranges of 24-41% and 15-60%, respectively.Treatment of the A549 cells with doxorubicin on the other hand, resulted in 30% and 43% of the early and late apoptotic cells after 24 h, respectively.This preliminary assay prompted us to deduce that doxorubicin was the strongest inducer of cell apoptosis compared to the test compounds.The most cytotoxic 2-nitrochalcone derivatives against the A549 cell line, namely, compounds 3c and 3k, exhibited less percentages of early apoptosis (25-29%) and high late apoptosis (48-60%) with approximately (15-22%) viable cell percentage observed.The high late apoptosis is presumably associated with DNA fragmentation, which occurs during later stages of the apoptotic process.However, it would be conclusive to complement these preliminary results using additional methods for detecting DNA fragmentation such as DNA ladder assay, comet assay, and/or TUNEL assay [6].Previous studies have also shown that chalcones play an anticancer role through tumor cell apoptosis induction, anti-angiogenesis, microtubule polymerization, anti-inflammatory and (MDR) inhibition [11,43].
Inhibition of VEGFR-2 tyrosine kinase activity has become a central focus of molecularly targeted cancer therapy [7].We took this consideration in mind and the fact that the effects of 2-nitrochalcones on angiogenesis have not been studied before and then evaluated compounds 3a-k for potential to inhibit VEGFR-2 activity.

Inhibition of VEGFR-2 by compounds 3a-k
The compounds were evaluated for inhibitory effect against VEGFR-2 tyrosine kinase activity in vitro at concentrations ranging from 0.1 to 100 µM with DMSO as the negative control with staurosporine and nintedanib as reference standards for the assay (Table 3).Staurosporine has previously been found to lead to a reduction or a lack of proliferation of the A549 cells in a concentration-dependent manner [44] and this drug is a broadly used selective and potent protein kinase inhibitor with submicromolar binding to the vast majority of the protein kinases [45].Moreover, this microbial alkaloid has previously been used several times as a reference standard for the inhibition of VEGFR-2 activity.Nintedanib, on the other hand, is an orally available potent and selective triple angiokinase inhibitor that blocks the activities of VEGFR, plateletderived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) and was approved as a second-line treatment of NSCLC [46].The results obtained from the VEGF-2 inhibitory assay showed that derivatives 3c, 3h, 3j and 3k exhibit moderate inhibitory effects compared to gefitinib (0.025 ± 0.007 µM), staurosporine (0.035 ± 0.002 µM) or nintedanib (IC 50 = 0.021 ± 0.001 µM) with IC 50 values of 31.49± 0.02, 39.95 ± 0.17, 36.90 ± 0.16 and 29.10 ± 0.16 µM, respectively.Compounds 3d, 3f, 3g and 3i, on the other hand, exhibited weak activity, while compounds 3a, 3b and 3e demonstrated lowest enzyme inhibitory activity against VEGF-2 with IC 50 values in the range of 55.31-57.88µM.These preliminary results indicate that chalcones 3c and 3k exhibit the highest inhibitory activity among the test compounds against VEGFR-2 activity consistent with their cytotoxicity and potential to induce apoptosis of A549 cells.

Molecular docking into VGEFR-2 active site
The test compounds consist of a planar conjugated framework for possible π-π interaction with the carbonyl and nitro group as hydrogen bond acceptors, which are some of the prerequisites for interaction with hinge region of VEGFR-2 [20].A molecular docking study was performed on the most active derivatives against this enzyme from the series, namely 3c and 3k, to predict their interactions with the active site of VEGFR-2 (PDB: 2OH4 and 4ASD) (Fig. 3).The control docking of the crystal ligand in 2OH4 and 4ASD has the root mean square deviation (RMSD) value of 0.67 Å and 0.57 Å, respectively, thus showed the appropriateness of the used docking parameters.Same binding free energy value (−8.2 kcal/mol) was calculated for the docking of the two compounds into 2OH4.Compounds 3c and 3k are predicted to have comparable free binding energy, −8.82 and −8.37 kcal/mol with 4ASD, respectively.The nitrobenzene region of both compounds forms at least two hydrogen bonds with 2OH4/4ASD.Compound 3c forms the same number of hydrogen bonds (three hydrogen bonding) with 2OH4 (Fig. 3a) and 4ASD (Fig. 3b), while 3k forms three and five hydrogen bonds with 2OH4 and 4ASD, respectively.These compounds form three and six hydrophobic interactions with 2OH4, respectively.Six (3c) and five (3k) hydrophobic interactions are predicted with 4ASD.The benzene rings of these two chalcone derivatives also form π-π interaction with Phe916/1045 of 2OH4 or Phe918/1047 of 4ASD, and these could be the key for the recognition between the ligand-protein that might be useful for possible future compound optimization.

Drug-likeness Estimation of Chalcones 3a-k
Early predictions of the pharmacokinetic behaviour of the promising anticancer compounds based on their structure could help to find safer and more effective leads for future preclinical testing.The drug-likeness features play a key role in assessing the quality of compounds whereby it can help to narrow down the list of candidates and identify those that behave similar to the target drug that is suitable as anti-cancer candidates.In order to assess the drug-likeness of the synthesized compounds, absorption, distribution, metabolism, and  The results represented in Table 4 show that the test compounds possess drug-likeness features except the 3-(3-methoxyphenyl) substituted derivative 3g with one violation on the Lipinski's Rule of five.

Conclusions
The 2-nitrochalcone derivatives prepared in this study were found to generally exhibit moderate to significant cytotoxicity against the A549 cells compared to doxorubicin.The hallmark of treating cancer is to inhibit cancer cells with reduced or no effect on the normal somatic cells.Compounds 3d (Ar = -C 6 H 4 (2-Cl)) and 3i (Ar = -C 6 H 3 (3,5-OMe)) with SI values > 2 in our view may be considered as better candidates for growth suppression of the A549 cancer cells with minimal effect on the normal cells.However, further cytotoxicity tests against different cell lines need to be conducted to confirm the safety and/or cytotoxicity of these compounds.These preliminary results, nevertheless, provided evidence that the 2-nitrochalcones induced apoptosis in A549 cells presumably through DNA fragmentation pathway.Due to the complexity and number of cellular processes involved more studies should be conducted to fully understand how this class of chalcones may be used to prevent the development of cancer.The planar molecular construct of the test compounds and the presence of potential hydrogenbond accepting ketone group would probably facilitate intercalation with DNA and/or impair the function of DNA-processing enzymes.Additional methods for detecting DNA fragmentation such as DNA ladder assay, comet assay, and/or TUNEL assay require to be conducted to be conclusive.The cytotoxicity of the tested chalcones 3a-3k correlated to the VEGFR-2 enzyme inhibition presented 3c and 3k as the most inhibiting 2-nitrochalcone derivatives against this enzyme's activity.The results from molecular docking into the active sites of VEGFR-2 were found to be consistent with the in vitro results.The presence of the nitro group and the α,β-unsaturated carbonyl framework resulted in increased hydrophilic (hydrogen bonding) and hydrophobic (eg, π-π stacking) interactions with the protein residues in the active site of VEGFR-2 due consistent with the design strategy.

General
The chemicals used in this study were obtained from Merck (Modderfontein, Johannesburg, South Africa) and were used without further purification.The melting point (mp.) values of the prepared compounds were recorded on a Stuart SMP10 melting point apparatus (Coleparmer, Stone, Staffordshire, UK) and are uncorrected.The infrared (IR) spectra were recorded using the thin-film method on a Bruker VERTEX 70 FT-IR Spectrometer (Bruker Optics, Billerica, MA, USA) equipped with an ATR (diamond attenuated total reflectance) accessory.The 1 H-and 13 C nuclear magnetic resonance (NMR) spectra were acquired at 300 K on an Agilent 500 MHz NMR spectrometer (Agilent Technologies, Oxford, UK), and the chemical shift values are reported in parts per million.The 1 H-NMR and 13 C-NMR spectra were referenced to the central residual proton peak or to the central carbon peak of deuterated chloroform (CDCl 3 ), δ = 7.25 ppm and 77.0 ppm, respectively.HR-MS analysis was performed on AB SCIEX X500 QTOF system (SCIEX, Massachusetts, USA) equipped with an ESI source.The ion source temperature and the IS voltage were set to 550 °C and 5500 V, respectively.Typical procedure for the synthesis of the 2-nitrochalcones (3a-k) A stirred mixture of 1 (1 equiv.)and benzaldehyde derivative 2 (1.2 equiv.) in ethanol (30 mL/mmol of 1) was treated with 10% aqueous solution of NaOH (15 mL/mmol of 1).The mixture was stirred at room temperature (RT) for 2 h and then treated dropwise with concentrated hydrochloric acid.The resultant precipitate was filtered on a sintered funnel and washed with cold water and then recrystallized from ethanol.The crystals thus obtained were dried at 70 °C and properly stored at room temperature prior their physicochemical and spectroscopic characterizations.

In vitro cytotoxicity against A549 and HEK293-T cancer cell lines
Cytotoxicity was measured in adenocarcinomic alveolar epithelial (A549) and human embryonic kidney (HEK293-T) cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay against doxorubicin, nintendanib and gefitinib as positive controls.Cells were cultured in sterile Dulbecco's Minimal Essential Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution.After 24 h incubation at 37 °C in 5% CO 2 when the cells reached 80% confluency, they were harvested using 2% Trypsin-EDTA solution, centrifuged for 5 min at 3000 rpm and re-suspended in DMEM.Cell counting was done using handheld automated cell counter (Scepter 3.0™, Merck, Burlington, MA, USA) and seeded (1 × 10 4 cells/well) into a 96 well plate.After 24 h, the cells were treated with different concentration range (0.5, 1, 2.5, 5, 10, 25, 50, 100 µM) of the test compounds 3a-3k and doxorubicin prepared in DMEM as positive control.The MTT solution (20 μL) prepared in PBS (5 mg/mL) was added to all the wells and the plates were incubated for 4 h, followed by 1 h incubation with 100 µL of dimethyl sulphoxide (DMSO) to dissolve the formazan crystals.Untreated cells and 5% DMSO were included in the experiment as negative controls.All experiments were performed in triplicate and the plates were read at 570 nm and a reference wavelength of 630 nm using an ELISA plate reader (Varioskan Flash, ThermoFisher Scientific, Vantaa, Finland).The percentage of cell viability was calculated using the following formula.
where, At represents the absorbance value of test compound, and Ab the Absorbance value of blank The IC 50 values, i.e., the concentration of the drug that inhibits 50% of the cells were obtained from dose-response curves and comparisons were assessed by a one-way ANOVA.
Apoptosis assessment of 3a-3k against A549 Cells by Fluor ® 488 annexin-V/PI Staining Assay Cell apoptosis was investigated using an Alexa Fluor ® annexin V/propidium iodide (PI) apoptosis detection kit (Invitrogen).The A549 cells (2 × 10 5 ) were seeded in 6 welled plates and incubated at 37 °C in 5% CO 2 for 24 h.Cells were treated with respective 1 x IC 50 values obtained from section 2.3.The untreated and doxorubicin treated cells were included as controls.After cells were incubated for 24 h, cells were then collected by centrifugation at 1500 × g for 5 min at 4 °C.The medium was aspirated, and the cell pellets were resuspended three times in 1 mL PBS.Then, the cells were resuspended in 100 µL of annexinbinding buffer and stained with 5 µL of Fluor ® 488 annexin V/1 µL PI dyes and incubated for 15 min.at RT in the dark.The cells were diluted with 400 µL of annexin-binding buffer and then evaluated by Flow cytometer (BD FAC-SAria™ III Cell Sorter).

Inhibition of VGEFR-2 tyrosine phosphorylation by 3a-k
The effect of the compounds 3a-k on VEGFR-2 activity was carried out according to the manufacturer instructions employing the Human VEGFR-2 (KDR) kinase assay kit (BPS Bioscience Corporation catalog #40325).Compounds 3a-k and nintedanib and staurosporine as reference standards were added to the micro ELISA plate (provided in this kit) wells at concentrations 12.5-100 μM and combined with VEGFR-2 enzyme (1 ng/μL) and incubated at 30 °C for 45 min.After incubation, 50 μL kinase detection reagent (Kinase-Glo MAX, Promega) was added to each well and further incubated for 15 min at room temperature in the dark.A luminescence reading was detected and quantified by an ELISA plate reader (Varioskan Flash, ThermoFisher Scientific, Vantaa, Finland).The IC 50 values were

Fig. 2
Fig. 2 Measurement of apoptosis using flow cytometry with Annexin V-FITC staining in A549 cells exposed for 24 h to the IC 50 concentrations of compounds 3a-k and untreated cells

Table 3
Inhibitory concentrations of the tested chalcone compounds