Design, synthesis and docking studies of novel 4-aminophenol-1,2,4-oxadiazole hybrids as apoptosis inducers against triple negative breast cancer cells targeting MAP kinase

Abstract In our study, a series of novel 4-aminophenol benzamide-1,2,4-oxadiazole hybrid analogues have been designed and synthesized by condensing 4-hydroxyphenyl arylamides (3a–c) and 5-chloromethyl-3-aryl-1,2,4-oxadiazoles (6a–d). The structure of the synthesised compounds was verified by various spectroscopic techniques (1H NMR, 13C NMR, IR and LC-MS). All the prepared compounds were subjected to in silico and in vitro antiproliferative study against TNBC cell lines MDA-MB-468 and MDA-MB-231. The investigations revealed that compound 7k significantly promoted apoptosis against MDA-MB-468 and MDA-MB-231 cells with IC50 values of 22.31 µM and 26.27 µM, respectively. Compound 7k interacted with crucial active sites of MAPK and exhibited the highest docking score of −7.06 kcal/mol. Docking was validated with molecular dynamic studies with simulation for 100 ns, depicting various stable interactions with MAPK. Consequently, 7k forms stable H-Bonds and π-π stacking with amino acid residues along with π-cation. Our investigations reveal that the in vitro antiproliferative study of 7k was in good correlation with the in silico studies. Hence, 7k serves as a potential novel lead for the inhibition of TNBCs by downregulating MAPK P38. Communicated by Ramaswamy H. Sarma Highlights Novel 4-aminophenol benzamide-1,2,4 oxadiazole library of small molecules displayed potent antiproliferative activity. Compound 7k induces apoptosis significantly against triple-negative breast cancer cells. Compound 7k potentiates apoptosis by targeting MAPK P38 and altering mitochondrial membrane potential. Molecular docking and molecular dynamic simulations confirm the efficient binding of compound 7k with MAPK (Docking score of –7.06 kcal/mol).


Introduction
Worldwide cancer is a major human health problem.Cancer is the second leading cause of death globally and is responsible for an estimated nearly 10 million deaths in 2020 (WHO).Detailed analyses of the structure of compounds and the interaction mechanism of molecules with cancer cells light up to develop novel anticancer/antitumor drugs with minimal side effects (Dasari & Bernard Tchounwou, 2014).The recent surveys and studies revealed that the synergistic effect of aromatic compounds significantly reduces the risk of cancer mortality (Siegel et al., 2023).FDA in 2023 approved organic molecules as anticancer drugs such as pirtobrutinib and tucatinib.Both are aromatic molecules possessing heteroatoms such as O, N and F. These are having lone pair of electrons that facilitate interaction with DNA, RNA, proteins and so forth, making the molecules biologically and pharmacologically potent.
Heterocycle scaffolds have found a prominent position in drug discovery and medicinal chemistry due to the significant chemical properties they possess.Among these, 1,2,4oxadiazoles have received tremendous attention in the pharmaceutical industry which serves as bioisosteric equivalence to heterocyclic amides and esters (Luthman et al., 1999).It is also used in peptide building blocks and the formation of dipeptidomimetics.Over the decades, the 1,2,4oxadiazole motif has received considerable attention globally from various research communities.
1,2,4-Oxadiazoles are also present in the natural products, two indole alkaloids Phidianidine A and Phidianidine B isolated from sea slug Opisthobranch, Phidiana militaris (Carbone et al., 2011), exhibit in vitro cytotoxic activity against tumor and nontumor mammalian cell lines.
In spite of being endowed with better activity for the existing drugs, its drawbacks, such as poor solubility and cytotoxicity to normal human cells is a never-ending phenomenon.Considering the aforementioned points, we designed and synthesized novel 4-aminophenol benzamide-1,2,4-oxadiazole hybrid analogues to examine the synergistic antiproliferative activity with less cytotoxicity.

Chemistry
The synthesis of 4-hydroxyphenyl aryl amides 3a-c was carried out as shown in Scheme 1.To a solution of 4-aminophenol 2 and aroyl chloride (1a-c) in acetonitrile at 0 � C added dry triethyl amine dropwise (Gao et al., 2018).The reaction mixture was refluxed for 3-4 h for the completion of the reaction.
The synthetic sequence for 5-(chloromethyl)-3-aryl-1,2,4oxadiazoles (6a-d) is outlined in Scheme 2. Aromatic substituted nitriles (4a-d) were treated with hydroxylamine hydrochloride and potassium carbonate in the ethanol-water mixture followed by reflux to afford substituted benzamidoximes (5a-d).Compounds (5a-d) were dissolved in toluene and stirred at ambient temperature.To this chloroacetyl chloride solution in toluene was added dropwise followed by overnight reflux to furnish (6a-d).
The novel 4-aminophenolbenzamide-1,2,4-oxadiazole hybrid analogues 7a-l (Table 1) were obtained by condensing 4-hydroxyphenyl aryl amides (3a-c) with 5-chloromethyl-3-aryl-1,2,4-oxadiazoles (6a-d) dissolved in dry acetone in presence of potassium carbonate and a catalytic amount of TBAB as depicted in the Scheme 3. The formation and structures of compounds (6a-d) were confirmed with 1 H NMR and mass spectral analysis.The characteristic data complied well with the already reported literature.In 1 H NMR, methylene proton appeared at d 4.5-4.8,aromatic protons at d 7.2-8.6,whereas methyl and methoxy protons resonated in the range d 2.4-3.9.The formation of molecular hybrid analogues 4-aminophenolbenzamide-1,3,4 oxadiazole 7a-l was confirmed by 1 H NMR, 13 C NMR and mass spectral analysis.In 1 H NMR spectra, the methylene protons appeared at d 5.2-5.5, greatly deshielded when compared with 1,2,4-oxadiazole methylene protons, which can be attributed to the greater electronegativity of oxygen atom to which methylene carbon being attached in 7a-l.The 13 C spectra showed peaks centered at d 175-177, 115-165 and 61-62 corresponding to the carbonyl carbon, aromatic carbons and methylene carbon, respectively.The aromatic methyl and methoxy carbons appeared in the range of d 21.3-21.8and 61.6-61.9,respectively.
The IR spectra of compounds 7a-l showed absorption peaks at 3420-3460, 1620-1680 and 1420-1480 cm À 1 , corresponding to the stretching vibrations of NH, C ¼ O and C ¼ N, respectively.Furthermore, mass spectral analysis displayed [M þ H] þ as the base peak, confirming the structure and formation of 7a-l.

Compound 7k inhibits the proliferation of TNBC cells
Breast tumorigenesis is a multistep complex process involving the role of growth factors, oncogenic signaling and various transduction events (Beckwith & Yee, 2014).Mammary malignancies are caused by several etiological factors mainly environmental, genetic and immunological defects (Pharoah et al., 2004).Amongst several dysregulated  signaling and growth factor pathways, the insulin-like growth factor (IGF) axis is fundamental that regulates proliferation, differentiation, migration, cell survival/apoptosis and transformation of signaling events in breast carcinogenesis (Bonneterre et al., 1987;Pollak, 2008).Both IGF1 and the receptor (IGF1R) have been reported to be strongly associated with the risk of breast cancer (Kang et al., 2014).Cancer cell sensitization by chemotherapeutic candidates mainly targets apoptosis reactivation by intervening in a variety of cellular signaling pathways (Shabbits et al., 2003).
We  2a,b).Interestingly 7k displayed less cytotoxic effect against normal kidney cell lines (HEK) (Figure 2c).Collectively these results demonstrate that compound 7k had potential antiproliferation effects on TNBC cells and a lesser effect (adversity) on noncancer cells.

Compound 7k disrupts mitochondrial membrane potential in TNBC cells
Mitochondria plays a pivotal role in energy metabolism, homeostasis and cell death.Cytochromes involved in the electron transport chain in mitochondria are crucial to trigger an apoptotic cascade.Alteration of mitochondrial membrane potential drains the cytochrome to activate the caspases and results in apoptosis of the cells.Notably, cell apoptosis is initiated through mitochondria which have a certain mitochondrial membrane potential designated as DwM or MMP (Ly et al., 2003).MMP, hence, serves as an important molecular biochemical parameter for mitochondrial activity and integrity and an indicator of normal or apoptotic cells.Therefore, normal functioning cells show a higher DwM and apoptotic cells showed a lower DwM due to JC-10 monomeric form with green fluorescence.Treatment of compound 7k to MDA-MB-468 and 231 cells displayed efficient apoptosis induction by altering the membrane potential which was unveiled by aggregation of JC-10 dye.Compound 7k displayed induction of apoptosis in a concentration-dependent manner against both the TNBC cells (Figure 3a,b).Alterations in MMP by compound 7k indicate its pro-apoptotic properties that could cause the initiation of the apoptotic cascade.

Compound 7k efficiently induces apoptosis in TNBC cells
Anticancer drugs impede the proliferation of cancer cells and induce apoptosis by targeting various regulatory proteins involved in various mechanisms.Next, we performed experiments to study the mechanisms by which compound 7k induced cell death in TNBC cells.Induction of apoptosis in cancer cells by treatment with compound 7k was assessed using Hoechst/PI dual staining (Figure 4).Compound 7k treated cells displayed uptake of propidium iodide into cells which unveil that the cells underwent apoptosis due to a lack of membrane potential in dead cells.Compound 7k treated cells show induction of cell death in comparison to DMSO-treated cells which served as negative control (Figure 4a,b).Collectively the results from cell proliferation and apoptosis studies suggest that compound 7k induced mitochondrial intrinsic apoptosis in TNBC cells in a mitochondria-dependent mechanism.These observations add that apoptosis induction is one of the key mechanisms behind the antiproliferation effects of compound 7k on TNBC cells.

Gaussian-based QSAR model generation
The study focused on analysing the relationship between the structure of 7a-l with MAPK.The researchers developed QSAR models to predict the activity and evaluated their Scheme 3. Synthesis of 4-aminophenolbenzamide-1,2,4-oxadiazole hybrid analogues 7a-l.
performance using several parameters.These parameters included the squared correlation co-efficient (r 2 ), which measures the accuracy of the model's predictions compared to the actual values.They also used the cross-validated correlation coefficient (q 2 training) to assess the model's predictive ability on new data.To determine the statistical significance of the model, the researchers employed Fisher's test, which calculates the variance ratio.The p-value, derived from Fisher's test, indicated the level of significance with smaller values indicating a higher degree of significance.The stability of the models was evaluated to gauge their consistency when subjected to small changes in the dataset or model parameters.Additional parameters considered were the standard deviation of the regression, which quantified the spread of predicted values around the regression line, and the root-mean-square error test, which measured the average difference between predicted and actual values in the test set.The Pearson-r coefficient was used to determine the correlation between observed and predicted activities in the test set (Table 2, Figure 5a).According to the findings of this model, the hydrophobic contribution was determined to be 0.982, while the electron-withdrawing contribution was found to be 0.015.These results indicate that, in protein-ligand interactions, the hydrophobic field has a greater significance compared to the electrostatic groups.More detailed information regarding the hydrophobic field is depicted in Figure 5b.

Molecular docking
The investigations of molecular docking reveal that compound 7k possessing strong electronegative group has a better binding with MAPK than those analogues with different functional groups.Ligand 7k containing 1,2,4-oxadiazole ring and 4-aminophenol benzamide along with p-chloro    substituent at both the ends representing higher electronegativity present at para position exhibit strong binding with the MAPK.The docking score of ligand 7k is found to be À 7.06 kcal/mol, which possesses high hydrogen bonding energy (-1.0 kcal/mol) (Table 3).Based on the docking result, it suggests that the ligands 7b, 7f and 7k with 124 oxadiazole ring and 4-aminophenol benzamide moieties possess good binding (Table 3), and interestingly ligand 7f has no hydrogen bonding interactions.Hence to verify the docking result, all sets of ligands were processed for MM/GBSA (Assaran Darban et al., 2017).

MM/GBSA
The MM/GBSA method was employed to calculate the binding energies of the ligand-protein complexes, specifically for ligands 7a-l.These binding energies are significant as they provide insights into the thermodynamics and desolvation parameters associated with the interactions.Among these parameters, the most favourable energies observed during ligand binding are related to van der Waals and nonpolar solvation terms.Conversely, the binding process is counteracted by polar solvation, which opposes the ligand's interaction with the macromolecule.The MM/GBSA scoring, which evaluates the ligand's affinity for the target macromolecule independently, often correlates well with biological activity data across a diverse set of compounds (Lyne et al., 2006).The computed results for the ligand-protein interaction include various components such as DG binding energy, DG (Coulomb) , DG (Covalent energy) , DG (Hbond) , DG (Lipo) and DG (vdW) .These energy terms contribute to our understanding of the interaction between the ligand and the protein (Table 3).Among the calculated energies, ligand 7k is the most active compound with highest DG bind value of À 65.90 kcal/mol (Table 3).Hence, ligand 7k was further subjected to molecular dynamic simulation, under the protein and ligand dynamics in a complex with respect to time.

Molecular dynamics simulations
MDS provide the most important data to support the docking prediction.Desmond helps to study the dynamics of stable protein-ligand complex conformation and analyses the simulation along with the stability of the complex which can be effectively examined.Therefore, in this study, we conducted MDS for a docked complex consisting of MAPK and ligand 7k.The docked complex of MAPK with ligand 7k was stable throughout the simulation upto 100 ns, which is given by the protein and ligand RMSD (Figure 6a) and the protein root-mean-square fluctuation plot shows the multiple ligand contact point with the amino acid residues of MAPK (Figure 6b).The 2D interaction plot of ligand 7k with MAPK is depicted with the various interactions which are hydrogen bond with His107, Thr106 and Glu71, p-p stacking with Phe169 along with p-cation with Lys53 (Figure 7a).Ligand RMSF shows that the pharmacophore which actually involved in the interaction, this data also provides detailed atoms of the ligand which are involved in strong binding (Figure 7b).The secondary structural element (SSE) of the protein MAPK when bound to ligand 7k, the resulted % of secondary conformation changes of protein, i.e., % of a-helix and b-strand changes from the unbound protein to bound protein is found to a total of 43.77% (Figure 7c).Ligand 7k  interacting with MAPK near the active site pocket is given by the normalized stacked bar chat, which provides the type of interactions possessed by the individual amino acids throughout the MDS study (Figure 7d) and how the interaction is very close to the amino acid throughout the time frame of molecular simulation is provided in a timeline chart with the number of contact point along with strength (Figure 7e) (Malek-Esfandiari et al., 2023).

In silico ADMET
The major descriptors and physiochemical parameters of pharmacokinetic ADMET (Absorption, Distribution, Metabolism Excretion and Toxicity) properties were calculated using the prediction tool QikProp.For newly synthesized compounds, Qikprop modules predict a variety of molecular attributes so that they can be compared to those of 95% of known drugs.Each ligand complies with Lipinski's rules, and each ligand also meets the requirements for the partition coefficients of octanol/gas (QPlogPoct), water/gas (QPlogPw), brain/blood (QPlogBB), skin permeability (QPlogKp) and aqueous solubility (QPlogS) with the predicted values falling within the acceptable range.All twelve produced compounds demonstrated high oral absorption, as was anticipated by the Qualitative Model for Human Oral Absorption (Table 4).

Conclusion
A series of novel 4-aminophenol benzamide-1,2,4-oxadiazole hybrid analogues 7a-k were synthesized and the structures were identified by spectral analysis.2d).Compound 7k displayed a potent cytotoxic effect against the screened cells by targeting the MAP-kinase pathway protein.MAP-kinase has proven to be an attractive target to design novel molecules, which can be attributed to its role in aberrant activation of cell proliferation, expression of pro-inflammatory cytokines and regulation in protein synthesis by phosphorylation of various substrate proteins.We utilized in silico techniques such as 3D-QSAR, molecular docking, MM/GBSA, ADMET, and molecular dynamics simulations to explore the potential of compound 7k as a potent anticancer molecule.3D-QSAR model revealed the significant structural factors that influence the anticancer activity of compound 7k.The structure-activity relationship indicates that the presence of electron-withdrawing groups in compound 7k has increased the activity greatly when compared with electron-donating groups.Interestingly existence of an electronwithdrawing group at the para position of both the phenyl rings in 4-aminophenol benzamide and 1,2,4-oxadiazole pharmacophore is greatly responsible when compared with the presence of other electron donating groups.Compound 7k possessing electron withdrawing group, -Cl group at the para position of both the phenyl rings has displayed significant antiproliferative activity when compared with the rest of the molecular hybrid analogues synthesized.
The electron-withdrawing group at the para position of both the phenyl ring bearing 4-aminophenol benzamide and 1,2,4oxadiazole pharmacophore is a prime requirement for the synergistic antiproliferative activity.Consequently, 7k serves as a promising lead candidate to combat TNBCs, which can be further modified to increase the efficacy of the molecule.
Molecular dynamic studies reveal that the ligand 7k plays a significant role in downregulating the MAPK and the protein-ligand complex is stable throughout the MDS, it forms various interactions which are hydrogen bonds with His107, Thr106 and Glu71, p-p stacking with Phe169 along with p-cation with Lys53.Hence, based on in silico studies the targeted protein for the 7k is MAPK.

Materials and methods
All starting materials and solvents used were AR grade and purchased from Spectrochem, Avra, Alfa Aesar, Thermo Fisher Scientific, Gibco and Sigma-Aldrich.The 1 H NMR and 13 C NMR spectra were recorded on a Bruker 400 MHz and a Bruker 600 MHz spectrometer and the chemical shifts have been reported in d (ppm) using CDCl 3 or DMSO-d 6 as a solvent and TMS as internal standard.Coupling constants (J) of ligands are reported in Hertz (Hz).Proton signal splitting patterns are indicated as dd: doublet of doublet, m: multiplet, q: quartet, t: triplet, d: doublet, s: singlet.Xevo G2-XS QTof with an ESI interface were used to record mass spectra.Perkin Elmer FTIR spectrophotometer were used to record IR spectra (cm À 1 ) using KBr method.Silica gel column chromatography was performed to purify the compounds using Merck silica gel (100-200 mesh).Thin Layer Chromatography was performed using Merck pre-coated silica GF254 plates to monitor the progress of the reactions.

General procedure for the synthesis of N-(4-hydroxyphenyl)arylamide (3a-c)
The solution of aroyl chloride (1a-c) (0.5 mmol) in dry acetonitrile were added slowly to the stirred solution containing 4-aminophenol (0.5 mmol) and dry triethyl amine (1 mmol) at 0 � C. The reaction mixture was refluxed for 2-4 h, cooled and checked for the completion of the reaction through TLC.The crude reaction mixture was concentrated under reduced pressure, dissolved in 10% NaOH solution and filtered.The filtrate was neutralized and adjusted the pH to 3-4 using 20% HCl to obtain the precipitate.The crude solid was filtered, and recrystallized from methanol to obtain the desired Table 4. Computer-aided ADMET screening of N-(4-((3-aryl-1,2,4-oxadiazol-5-yl)methoxy)phenyl)benzamide (7a-l).

General procedure for the synthesis of 5-(chloromethyl)-3aryl-1,2,4-oxadiazole (6a-d)
The solution of substituted benzonitriles (4a-d) (0.048 mol) in ethanol (50 mL) were added hydroxylamine hydrochloride (0.121 mol) in portions and sodium carbonate (0.077 mol) dissolved in water.The reaction mixture was heated to reflux for 8 h.The progress of the reaction was monitored through TLC and cooled to room temperature.The solvent was removed through rotaory evaporator and extracted with ethyl acetate.
The combined organic layers were washed with water, brine and dried over sodium sulfate.The organic layer was concentrated under reduced pressure to obtain a crude solid which was recrystallized using aqueous ethanol to obtain substituted benzamidoximes (5a-d) in good yield.
A solution of chloroacetyl chloride in toluene was added dropwise to the solution of substituted benzamidoximes (5a-d) in toluene at 0 � C. The reaction mixture was allowed to reflux overnight and checked for the completion of the reaction through TLC.Toluene was removed under reduced pressure to obtain a residue, which was extracted with ethyl acetate.The organic layers were combined, washed with water, 20% sodium bicarbonate solution, brine, dried over sodium sulfate and filtered.Solvent was removed in vacuo and the residue was recrystallized from ethanol to obtain 5-(Chloromethyl)-3-aryl-1,2,4-oxadiazole (6a-d) in good yield.The synthesized compounds were confirmed with 1 H NMR, and mass spectral analysis which complies well with the reported literature.

Cell lines and culture condition
The

MTT cell proliferation assay
MTT assay was performed as described previously (Girish et al., 2022;Sreenatha et al., 2022).Briefly, the cells were initially seeded and then treated with varying concentrations of compound 7k.After 48 h of incubation, the cells were subjected to the MTT assay, which involved incubating them for an additional 4 h.The resulting insoluble formazan crystals were dissolved using DMSO, and the absorbance was measured at 570 nm using a Microplate Reader from Tecan Instruments in Switzerland.To determine the percent cell count, the mean ± standard deviation (SD) was calculated.
The experiment was repeated independently a minimum of three times, and the data were presented in the form of a histogram.

Mitochondrial membrane potential assay
Mitochondrial membrane potential assay was performed as described previously (Girish et al., 2022).Cells were treated with compound 7k and incubated for 24 h.Cells were collected and washed by using PBS and subjected to MMP assay as per the manufacturer's protocol (MAK159, Sigma Aldrich).Cells were centrifuged at 800 rpm and imaged using an inverted fluorescence microscope (CKX53; Olympus, USA).

Apoptotic analysis by Hoechst/PI staining
Hoechst/PI double staining assay was performed as described (Uttarkar et al., 2023).Briefly, MDA-MB-231 and MDA-MB-468 cells were seeded into 6 well plates and incubated overnight.
Then treated with an increasing concentration of compound 7k and incubated for 48h.Cells were collected, washed with PBS and subjected to Apoptotic analysis by Hoechst/PI double staining and imaged.

In silico studies
Gaussian-based 3D QSAR Dataset collection.The structures of molecules 7a-l were drawn using a 2D Skecher and imported into the workspace.
Experimental IC 50 values were assigned to each ligand.The 2D structure of ligands was optimized using minimization tools of the Macromodel module using a force field of OPLS_ 2005.To linearize the data, the IC 50 values of the data set were converted to negative logarithmic forms (Eq.( 1)).
In this work, pIC 50 values were employed as a dependent variable for the creation of 3D-QSAR models.Using the random selection process used in 3D-Gaussian based QSAR, the dataset was split into training and test (validation) sets (70:30) (Table 2).The most important need for effective model development is the accurate alignment of optimized conformers from a chosen dataset in a defined lattice.It relies on how accurately the molecules from the dataset are arranged relative to one another.Utilizing the structural alignment tools (flexible shape-based alignment, common scaffold alignment, maximum common substructure, smarts), the molecules in this study were positioned correctly (Singh et al., 2022(Singh et al., , 2023)).

Generation of 3D QSAR model
The models were constructed using Maestro 12.9, with a grid spacing of 0.1 Å and an extension of 3.0 Å beyond the boundaries of the training set.Within a range of 2.0 Å, the force field settings were ignored.The default value for the shortened steric and electrostatic force field was set to 30 kcal mol À 1 .During cross-validation, one ligand was omitted and variables with a standard deviation less than 0.01 were eliminated.The model incorporated five features: steric, electrostatic, H-bond acceptor, H-bond donor and hydrophobic field.The 3D-QSAR model was built using partial least squares (PLS).To validate the models, several statistical parameters were considered, including standard deviation (SD), cross-correlation validation (rCV 2 ), r 2 scramble, Fischer's test (F-test), variance ratio (p-value), root mean squared error (RMSE), Pearson-r values, leave-one-out (LOO) cross-validated correlation coefficient (q 2 ) and Pearson-r.Multiple statistical benchmarks were employed to evaluate the predictive capability of the QSAR models, using Gaussian intensities as descriptors (independent variables) and pIC 50 values as dependent factors (Morris et al., 2009;Singh et al., 2023).

Molecular docking and ADME/Tox
The coordinates for MAP Kinase P38, VEGFR-1, COX-2, COX-1 and Epidermal growth factor receptors were obtained from the Brookhaven Protein Data Bank.The structures of molecules 7a-l and standard inhibitors were drawn using a 2D sketcher and then energetically optimized using the OPLS_ 2005 force field.To prepare the protein structures, a protein preparation wizard was utilized.This involved correcting any missing loops in the protein structures.The significance of water molecules in facilitating receptor interactions was taken into account during the optimization process.The protein structures were processed to assign necessary bonds, bond orders, hybridization, explicit hydrogens and charges.The OPLS_2005 force field was applied to the proteins, and a rootmean-square deviation (RMSD) of 0.30 Å was set as the criterion for the convergence of heavy atoms during preprocessing.
For the docking procedure, extra-precision docking was employed with a flexible ligand sampling approach.This allowed for a more detailed exploration of ligand conformations and interactions with the protein targets.The receptor grid was mapped by using amino acids present at the active site (Kameshwar et al., 2017;Shwetha et al., 2021).Every ligand was subjected to docking into its corresponding receptor grid, with a radius of 35 Å and specific x, y and z coordinates.The success of the docking calculation was evaluated based on the docking score (Basappa et al., 2020;Pallavi et al., 2022aPallavi et al., , 2022b)).ADME/Tox properties were calculated with ADME/Tox prediction program QikProp (Kumara et al., 2021).

Molecular mechanics-generalized born surface area
The docked position of the ligand was re-evaluated using the molecular mechanics-generalized born surface area (MM/GBSA) approach.This method allowed for the calculation of binding free energies of the protein-ligand complex, which was found to be more accurate than the initial molecular docking.The energy of optimized free receptors, a free ligand and a combination of the ligand and a receptor are all calculated by prime MM/GBSA (Pattar et al., 2020).The MM/GBSA approach utilizes several components, including the OPLS_ 2005 force field, VSGB solvent model and rotamer search algorithms.This methodology employs a surface-generalized born model, which replaces the van der Waals surface with a Gaussian surface to provide a more accurate representation of the solvent-accessible surface area.By incorporating these elements, the MM/GBSA approach improves the accuracy of calculating binding free energies in protein-ligand complexes (Suryakoppa et al., 2022).

Molecular dynamic simulations
Molecular dynamics simulations were performed in this study using the Desmond software on a 64-bit Ubuntu 20.04 platform.The aim was to investigate the behaviour of the potent ligand 7k when interacting with the MAP Kinase P38 protein.
The simulation system was set up by incorporating the SPC water model into the protein-ligand complex using the system-builder option.A solvated condition was achieved by placing the complex in an orthorhombic periodic boundary box.To ensure system neutrality, Na þ ions (61.954 mM) and Cl -ions (51.112 mM) were added.The simulations were conducted under a periodic boundary condition in the NPT ensemble, with a temperature of 310 K and atmospheric pressure.The system was relaxed using the default relaxation protocol integrated into the Desmond program.The simulations were carried out for a duration of 100 ns to study the dynamics of the system (Aamir et al., 2018).

ADMET analysis
The structures of newly synthesized ligands were drawn using 2D sketcher and energy minimization was carried out using the OPLS_2005 force field.All molecular modeling was carried out on a 64 bits Linux workstation powered by an Intel Xenon Gold processor.A set of ADMET-related properties were calculated by using the QikProp program running in normal mode.The obtained values were compared with 95% of known drugs (Raghavendra et al., 2016).

Statistical analysis
IC 50 values of the synthesized molecules were elucidated by analysing the data with the help of GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).The experiments were conducted in triplicate, with each experiment repeated three times to ensure reliability.Error bars were included in the results to represent the standard error of the mean (SEM).The statistical significance was determined by calculating the p value, which involved comparing the mean of the control group with the mean of the group treated with compound 7k.The p value obtained was found to be less than .05,indicating statistical significance.
The formation of N-(4-hydroxyphenyl)arylamide (3a-c) was confirmed by mass, 1 H NMR and 13 C NMR spectral analysis.Aromatic protons resonated at d 7-8.8, whereas phenolic -OH and amide protons appeared at d 9.03-10.1.The aromatic methyl protons appeared at d 2.38.In 13 C NMR, amide carbonyl appeared in the range at d 163-166, aromatic carbons at d 110-156, whereas methyl carbon at d 21.44.Further, the mass spectra of 3a-c shows [M þ H] þ as the base peak.
have worked on breast cancer cell lines to assess the effectiveness of 4-aminophenolbenzamide-1,2,4-oxadiazole hybrid analogues 7a-l.MDA-MB-468 cells are metastatic adenocarcinoma of the breast.These cells have extended applications in breast cancer research.Whereas MDA-MB-231 cells are breast adenocarcinoma cells.MDA-MB-468 is a basal cell line while MDA-MB-231 is a claudin-low cell line and typically with low Ki67 and E-cadherin.MDA-MB-231 cells show enrichment for markers associated with EMT and the expression of features associated with mammary cancer stem cells, so they can be different in molecular and phenotypic behavior.Many studies on potentially active agents for triple negative breast cancer disease have been conducted using indifferently MDA-MB-231 or MDA-MD-468 cells.To investigate the effect of Compound 7k on the growth of TNBC cells, MDA-MB-468 and MDA-MB-231 cells were treated and assayed the cell viability.Compound 7k mitigated cell proliferation and viability of the cancer cells.Compound 7k diminished the proliferation of TNBC cells MDA-MB-468 and MDA-MB-231 with IC 50 values of 22.31 mM and 26.27 mM, respectively (Figure

Figure 2 .
Figure 2. Effect of compound 7k on cell proliferation and cell viability: the study involved treating cells with varying concentrations of a compound and assessing their effects using the MTT assay.The vehicle control group was treated with DMSO.The experiments were repeated at least three times for each cell line: MDA-MB-468 (a), MDA-MB-231 (b) and HeK (c).The IC 50 values were calculated specifically for MDA-MB-468 and MDA-MB-231 cells in the presence of compound 7k (d) with concentrations ranging from 0 to 40 mM.The error bars on the graph represent the standard error of the mean and the p-value indicates that there was a statistically significant difference (p < .05) between the control group and the treated group.

Figure 3 .
Figure 3.Effect of compound 7k on mitochondrial membrane potential: the cells treated with compound 7k underwent MMP assay following the protocol provided by the manufacturer (a) MDA-MB-468 and; (b) MDA-MB-231 cells.The DMSO-treated cells were used as the vehicle control group in this experiment.Compound 7k effectively induced apoptosis in a concentration-dependent manner.Each experiment was repeated thrice.

Figure 4 .
Figure 4. Induction of apoptosis by compound 7k treatment.Compound 7k treated were subjected to Hoechst/PI dual staining in (a) MDA-MB-468 and (b) MDA-MB-231 cells.The DMSO-treated cells were used as the vehicle control group in this experiment.Each experiment has been repeated thrice.

Figure 5 .
Figure 5. Correlation analysis of predicted pIC 50 values by QSAR model and experimental pIC 50 values (a).Hydrophobic contour map for 3D-QSAR, blue represents positive coefficients and red negative coefficients (b).

Figure 6 .
Figure6.The study involved graphically representing the Root Mean Square Deviation (RMSD) of ligand 7k in relation to the active site residues of MAPK throughout molecular dynamics simulations conducted for up to 100 ns.This representation is shown in (a).Furthermore, a protein root mean square fluctuation plot was generated to illustrate the contact points between the ligand and the amino acid residues of MAPK.In this plot, the contact points are depicted as vertical green lines, and their occurrence is represented by the corresponding green colour.Several contact points can be observed in (b).

Figure 7 .
Figure 7. Putative binding site of ligand 7k through a two-dimensional interaction diagram with MAPK.The diagram (a) illustrates the interactions between the amino acids at the active site and ligand 7k showcasing the different types of interactions along with their respective strengths.Additionally, the Ligand Root mean Square Fluctuation (L-RMSF) plot (b) reveals the presence of pharmacophore sites within the ligand indicating the various bonds formed with MAPK.The simulation also monitored the protein's Secondary Structure Elements (SSE), such as a-helices and b-strands.This information is depicted in the plot above (c), which demonstrates the distribution of SSEs throughout the protein structure.The plot below summarizes the composition of SSEs for each frame of the trajectory during the simulation, while the bottom plot tracks the SSE assignment of each residue over time.To further analyze the interactions, a normalized stacked bar chart (d) displays ligand 7k's interactions with MAPK near the active site pocket highlighting the presence of hydrogen bonds, hydrophobic interactions and water bridges.Furthermore, the timeline chart (e) represents the number of contact points formed between ligand 7k and the protein over the course of the simulation.

Table 1 .
Structures of N-