Discovery of benzochromene derivatives first example with dual cytotoxic activity against the resistant cancer cell MCF-7/ADR and inhibitory effect of the P-glycoprotein expression levels

Abstract A series of 1H-benzo[f]chromene moieties (4a–z) were synthesised under Ultrasonic irradiation and confirmed with spectral analyses. Derivative 4i solely possessed an X-ray single crystal. The anti-proliferative efficacy of the desired molecules has been explored against three cancer cells: MCF-7, HCT-116, and HepG-2 with the cytotoxically active derivatives screened against MCF-7/ADR and normal cells HFL-1 and WI-38. Furthermore, compounds 4b–d, 4k, 4n, 4q, and 4w, which possessed good potency against MCF-7/ADR, were tested as permeability glycoprotein (P-glycoprotein [P-gp]) expression inhibitors. The attained data confirmed that 4b–d, 4q, and 4w exhibited strong expression inhibition against the P-gp alongside its cytotoxic effect on MCF-7/ADR. The western blot results and Rho123 accumulation assays showed that compounds 4b–d, 4q, and 4w effectively inhibited the P-gp expression and efflux function. Meanwhile, 4b–d, 4q, and 4w induced apoptosis and accumulation of the treated MCF-7/ADR cells in the G1 phase and 4k and 4n in the S phase of the cell cycle.


Introduction
An efficient and green tool for organic reactions is to use the Ultrasound technique as a non-conventional energy source, which can provide a range of benefits, such as shorter reaction times, easier operation, and improved yields of pure products [1][2][3][4] .

Aim of the work and rationale
Herein, we report the synthesis of anti-proliferative 1H-benzo[f]chromene derivatives (a-z).In this work, we explore the potent and anti-proliferative effects of the substituents on the C-1 position of the phenyl-bearing moieties of the 1H-benzo[f]chromene derivatives.To achieve this goal, three tumour cell lines MCF-7 (breast cancer), HCT-116 (human colon cancer), and HepG-2 (hepatocellular carcinoma) were utilised to investigate the anti-proliferative activity of the desired molecules.Subsequently, the highly cytotoxically-active derivatives 4b-e, 4g, 4i, 4k, 4n, 4o, 4q, 4r, 4u, 4w, and 4z were subjected to further screenings against Adriamycin (ADR)-resistant human breast cancer cells (MCF-7/ADR) and the normal cell lines human foetal lung (HFL-1) alongside WI-38 human (diploid fibroblasts).Furthermore, compounds 4b-d, 4k, 4n, 4q, and 4w revealed good potency towards MCF-7/ADR cells and were examined for the inhibition of P-gp expression; meanwhile, 4b-d, 4q, and 4w displayed high potency against Pgp expression MDR in MCF-7/ADR and they are able to inhibit Pgp.Besides, western blot results and Rho123 accumulation assays showed that compounds 4b-d, 4q, and 4w effectively inhibited Pgp expression and efflux function, while compounds 4b-d, 4q, and 4w induced accumulation of the treated MCF-7/ADR cells in the G1 phase, and compounds 4k and 4n in the S phase of the cell cycle, as illustrated in Chart 1.
The rational design of the 9-methoxy-1H-benzo[f]chromene derivatives was based on the following considerations: (i) the 1Hbenzo[f]chromene template, (ii) the existence of the efficacious 9position substituent, (iii) the varying sorts of substituents located on the aryl moieties bound to the 9-methoxy-1H-benzo[f]chromene framework at the 1-position which emerges as a critical component in cytotoxic behaviour, and (iv) comparative analyses regarding the performances of the freshly prepared molecules and the formerly prepared molecules with a bromine atom at the 9position 24 as illustrated in Chart 2.
The final feature in this rationale study revealed that the recent molecules 4b, 4g, 4n, 4q, 4u, and 4z possessed a remarkable influence regarding their behaviours against tumour cells, which had an elevated potency in comparison with the molecules 1-4 24 .

Chemistry
A series of substituted fused heterocyclic derivatives (4a-z) were synthesised using 7-methoxynaphthalen-2-ol (1) as a starting material.The attained molecules 4e, 4f, 4j, and 4r-u are described herein for the first time, while previous reported compounds 4a-d, 4g-i, 4k-q, and 4v-z have been acquired employing an ultrasound as a new synthetic strategy.The interaction of 1 with a number of aldehydes derivatives (2a-z) in the presence of and malononitrile (3) and absolute ethanol/piperidine solution has been achieved utilising 60 W of ultrasonic irradiation conditions at ambient temperature and allowed for the formation of b-enamionitriles containing 9-methoxy-1H-benzo[f]chromene motifs (4a-z) as shown in Scheme 1.
The 1-position of compounds 4a-z is a chiral centre and all the reactions were controlled using TLC technique.The optical execution of 4a-z was detected utilising using a Carl Zeiss polarimeter and displayed zero rotation (i.e.optically inactive) due to their occurrence as a racemic (±) mixture 52,53 , as shown in Scheme 1.
The structure's identity of the novel molecules 4e, 4f, 4j, and 4r-u was substantiated via their IR, 1 H NMR, 13 C NMR, 13 C NMR-APT, and MS data.Supporting evidences for the suggested structures comes from their infra-red spectra which exhibited at t 3448-3402, 3338-3291, 3261-3200 cm À1 for an NH 2 group and at t 2204-2167 cm À1 for the CN group of compounds 4e, 4f, 4j, and 4r-u.The 1 H NMR data of 4e, 4f, 4j, and 4r-u demonstrated singlet signals at d 7.17-6.93ppm that is corresponding to the amino protons, while signals at d 6.06-5.26are attributable for the methine protons.Moreover, the 13 C NMR-APT of compound 4s as well as the MS spectrum, the single crystal X-ray analysis of compounds 4d, 4 g, 9,54 and 4i provided a conclusive verification for the desired molecules (see Supplementary Materials 1S-100S).

Crystal data
Table 1 illustrates the crystallographic analysis and the refinement information of molecule 4i with the chemical formula of C 22 H 18 N 2 O 2 .The asymmetric unit of compound 4i is containing one molecule as shown in Figure 1.The length of all bonds and the angles are in normal ranges 55 .In the crystal packing, Figure 1, molecules of compound 4i were linked via two intermolecular hydrogen bonds (Table 2).
The central pyran ring (O1-C1-C10-C11-C12-C13) is almost planar with the largest deviation from the mean plan of À0.013(2) at C10 atom, which connected to naphthalene ring and to a tolyl ring (Figure 1).The dihedral angle between the main core ring and the tolyl is 73.51 (7) .The mean plan through the naphthalene ring (C8-C13) is nearly orthogonal to that of the tolyl ring as signified by the dihedral angle between them of 144.44 (4) .Also, there are two intermolecular hydrogen bonds interactions, N2-H2N1N1, given in Table 2.
Crystal explorer17 56 was used to investigate the Hirschfield-surface "HS" with their 2D-fingerprint surfaces "2D-FS" analysis, which displayed the intermolecular interactions in the crystal packing.HS was mapped over d norm for 4i (Figure 1).The HS-volume "HSV" and the surface area "HSA" are 433.89Å 3 and 382.56 Å 2 , respectively, which represented in red(Àd norm ), blue(þd norm ), and white (normal d norm ) colour schemes.Negative, positive, and normal d norm , are shorter, longer and equal contacts than van-der-Waalsradii, respectively.The decomposed 2D-HS in (Figure 1) showed the chief contribution in C-H and H-H contacts, which contribute the most (30.9%) to the total HSA.The lowest contributions are from C-C, C-N contacts, while C-O, O-H, and N-H displayed contributions at 2.7, 6.9, and 14.1 contacts.The strong intermolecular interactions appear as distinct spikes in the fingerprint plots.
Shape index (SI) and curvedness (CS) were used to study the curvature of the surface (Figure 1).The SI sheds light on the stacking arrangement for 4i, which was used to identify the complementary cavities (red) and bumps (blue) where two molecular HS close to each other.The blue and red coloured triangular-shaped patterns represented the particular stacking arrangement of 4i.Molecule 4i displayed no significant triangular-patterns on SI map, which suggested the absence of the p-p interaction.The CS was represented by the function of the r.m.s.curvature.CS mapped was found to be in range of (À4.0-4.0) which categorised by bulky green-coloured regions and detached by deep blue boundaries.Since there is no flat surface seen on the CS plot, there is no planar stacking between the molecules.
The B3LYP/6-311G(d,p) function was employed for simulating the intermolecular interaction energies in the crystal to examine their stabilisation degree of the crystal lattice (Table 3).Figure 1 represents their energy framework as a cylinder-shaped, which displayed the relative-strength for the interaction energies and also give a clear view for their role in the stabilisation of the crystal packing.According to Table 3, it is clear that the dispersion force (À233.3kJ/mol) plays a central role, which having a maximum energy of À233.3 kJ/mol, among the total interaction energy which is À244.7 kJ/mol.
The effect of compounds 4b-d, 4k, and 4w on the inhibition of P-gp expression in MCF-7/ADR was also confirmed using western blot analysis as shown in Figure 4(B).Compounds 4b-d, 4k, 4q, and 4w have strong cytotoxic effects on MCF-7/ADR cells; they also displayed excellent inhibitory performance on the P-gp content (Figure 4(A)) with the exception of compound 4k.These results demonstrated that only compounds 4b-d and 4w possessed high inhibitory effect on the expression of P-gp which subsequently have a great potency in reversal the MDR in MCF-7/ ADR, similar results have been reported previously 60,61 .Furthermore, the reversion of P-gp mediated multidrug resistance may be achieved by down-regulation of P-gp expression and or inhibition of P-gp efflux function 62 .For this reason, the effect of     our synthesised drugs was tested for inhibitory potential of P-gp activity using a Rhodamine 123 Accumulation Assay (Rhodamine Competitive ELISA Kit) (Table 6).
The data in Table 6 portrayed the (IC 50 ) of Rhodamine 123 for evaluating the P-gp functional inhibition of compounds 4b-d, and 4w which ranging from 10.6 to 25.6 mM compared to 14.3 mM of the reference drug Verapamil.Compounds 4b-d and 4w had an impact on the restoration of the sensitivity to MCF-7/ADR cells by reducing not only the P-gp expression but also its function.On the other hand, compounds 4k and 4q exhibited cytotoxic activity against MDR which may be associated with different types of mechanisms such as genetic factors, growth factors, increased DNA repair capacity 63 .

Cell cycle arrest in treated MCF-7/ADR cancer cells
Development of anticancer agents targeting cell cycle arrest represents an important therapeutic intervention in treating diseases like cancer.Cancer cells undergo unscheduled cell divisions by the down regulation of the four cell cycle stages (G1, S, G2, and M) 64,65 .P-gp breast cancer resistant proteins (BCRP) normally prevent intercellular drug accumulation, affecting the anticancer agent's impact on the cell cycle arrest 66 .Therefore, the effect of the most potent newly synthesised compounds 4b-d, 4k, 4n, 4q, and 4w on regulating cell cycle progression of MCF-7/ADR cancer cells cycle was analysed by the flow cytometry, exploiting the FACS Calibres (Becton Dickinson).The distribution of cells along the G1 (2n), G2/M (4n), and S (2n-4n) phases of the cycle was exhibited in the representative cell cycle distribution histogram of the stained DNA in Figure 5(a).The MCF-7/ADR cancer cells were remedied with each derivative at its IC 50 values for 24h, a controlled experiment with no treatment done.
The outcomes of the cell cycle progression showed that all the tested compounds have expressed a significant increased percentage of 10-15% (60-65%) of cells at the G1 phase in comparison to the (55%) control cells expect for compounds 4k and 4n which show increased percentages 40% and 43%, respectively, in S phase compared to the control (30%).In addition, these results were accompanied by a considerable decrease in percentage at the G2/M phases compared to the untreated control cells (Figure 5(b)).The cell cycle evaluation presented that the tested derivatives significantly arrested the cells' progression by restricting the G1 and S phases.

Apoptosis induction in MCF-7/ADR cancer cells
Several lines of evidence indicate that many anticancer compounds exerted their effects by blocking the cell cycle progression, by inducing apoptosis, or the combined effect of both 67 .Also, P-gp inhibits apoptosis by preventing the release of cytochrome c which is mediated by the intrinsic mitochondrial pathway 68 .To further assess the pivotal relation of the newly synthesised MCF-7/ADR anticancer compounds and apoptosis, phosphatidylserine (PS) translocation to the cell membrane as a marker for apoptosis was measured by the means of the Annexin V/PI double staining flow cytometric assay 69 .The representative dot plots of the double-stained MCF-7/ADR cells after treatment with the diverse examined compounds were displayed in Figure 6(a).
Unlike necrosis, which was not observed in all the results, all treated cells illustrated up to 40% in total apoptosis in evaluation against 30% and 2% of the Doxorubicin and untreated cells respectively.Moreover, all the tested compounds showed early (Annexin V positive, PI negative) as well as late (Annexin V positive, PI positive) apoptosis for all the treated cells (Figure 6(b)).Our results proposed that the induction of MCF-7/ADR cytotoxicity occurs via mechanisms associated with apoptosis with no obvious negative effects of the P-gp.

Structure-activity relationship SAR
The initial SAR investigations performed were centred on the impact of substituting hydrogen atoms on the phenyl group at 1-position of the 1H-benzo[f]chromene platform by halogen atoms, methyl or methoxy groups and methoxy group at 9-position as illustrate in Scheme 2. The implementation of the halogen or methoxy monosubstituted for the first series (4a-j) reduced their activities against MCF-7 cell line (IC 50 in the range of 0.6-69.2mM) as compared with Vinblastine.This behaviour could be attributed to the effect of the grafting of a lipophilic electron withdrawing or electron donating groups on the phenyl group at 1-position of the 1H-benzo[f]chromene moiety.Meanwhile, the second series with disubstituted halogens atoms or methoxy groups (4k-w) revealed a fluctuation in their antiproliferative activities with a simultaneous variation in the position and size of the disubstituted halogens or methoxy.Among the third series with the trisubstituents, compounds (4x-z), only compound (4z) possessed adequate activity in assessment with Vinblastine.
Regarding the influence of the substituent's groups of the desired molecules from the first series on their potency against HCT-116 cell lines, the halogenated or methylated monosubstituent (4a-j) exhibited diminished IC 50 in the range of 1.9-27.7 mM values, while for compounds (4k-w) the activities were in the range of 1.3-96.5 mM.Furthermore, through the trisubstituents of the third series (4x-z), only compound (4z) exhibited activity approximate to Vinblastine, while the other derivatives were inactive, intimating that the disubstituent demonstrated superior potency in evaluation with the mono-and tri-substituent analogues.
In addition, selected derivatives of the three series demonstrated superior potency against HepG-2 cell lines.For instance, compounds 4q, 4g, 4z, 4e, and 4j had IC 50 values in the range of 1.5-5.3mM in appraisal with Vinblastine and Doxorubicin.Also, compounds 4b-d, 4k, 4n, 4q, and 4w possessed good potency against resistant cell strains (MCF-7/ADR) with IC 50 in the range of 10.9-15.5 lM.Such results tentatively suggested that grafting a lipophilic electron withdrawing with moderate size (difluoro or dichloro substituents) is more beneficial than other substituents for the activity.Furthermore, compounds 4a-e, 4g, 4i, 4k, 4n, 4q, 4r, 4u, 4w, 4 y, and 4z have been screened against two normal cell lines, HFL-1 and WI-38 and displayed IC 50 ranging from 29.9 to 83.0 mM, which confirm their inadequate performance against these control cell lines.Finally, we can deduce that the position and the type of the substituent on the phenyl group at the 1-position of the 1H-benzo[f]chromene moiety played a vital role in its antitumor activity.

Molecular docking
To account for the most profiling compounds 4b-d, 4q, and 4w against P-gp, molecular docking was conducted to investigate their possible interactions (PDB code 3G60) 70 and theoretical model was utilised in all docking experiment.I-TASSER 71 was used to generate Human P-gp, and then used AMBER force field to optimise their model.Ramachandran plots were obtained which showed that model is similar to that one gained from the experimental mouse structure, which reported in the protein data bank.We also predicted the crystal structure of mouse P-gp (code 3G60) by I-TASSER.In this docking analysis we used the P-gp structure and translocation of the substrate pore as a rigid object in the docking procedure.However, since the ligand-binding pose of Pgp (a facing inward closed contact to apo conformation) was employed, the docking data give insight in the suitable complex geometry to ATP hydrolysis.To validate the accuracy of the docking analysis we redocked the reference cyclic peptide "QZ59-RRR" bound (PDB code 3G60), and compared with original geometry for QZ59-RRR.The original QZ59-RRR was docked into the experimentally determined structure of mouse P-gp with a high accuracy with a RMSD value of 1.78 Å.To gain further validation for the docking experiment, the inhibition constant (Ki) and bioactivity factor also examined as ligand efficiency (LE) were calculated (Table 7).All investigated compounds and reference molecules appeared in acceptable range as listed in Table 7.
The docking experiment has been accomplished by Glide's module V R .The binding free energies DG are listed in Table 7.The original-inhibitors have been suitably fitted into their own binding sites for their crystal structures.The QZ59-RRR capped the 3G60 binding pocket through bounded with and formed the same H-bond with Gn721 and Ser725.All compounds 4b-d, 4q, and 4w and reference molecule (Doxorubicin) docked fruitfully into active sites in the same manner as original inhibitor through formation H-bond with Gln721 (Figure 7).From Table 7, the most potential antiproliferative compounds 4b-d, 4k, 4q, and 4w showed high promising binding affinity compared to QZ59-RRR for the investigated P-gp (PDB: 3G60).
Compound 4d showed the highest binding affinity DG ¼ À11.313 kcal/mol against all investigated compounds and reference inhibitor, which explain the highest P-gp-inhibition activity (IC 50 ¼ 13.5 lM), through blocked active site by the formation of the H-bond with vital Gln 721.Besides, the other compounds 4b, 4c, 4k, 4q, and 4w showed higher DG than QZ59-RRR, which arranged in decreasing order 4c< 4b < 4q < 4w< 4k < QZ59-RRR (Table 7).Compounds 4b, 4c, 4k, and 4q were occupied the binding pocket with the same behaviour of QZ59-RRR through engaged Gln721, only compound 4w showed high stability in binding pocket through the interaction with important Ser725 (Figure 7).

Conclusions
In continuation with our preceding endeavours in cultivating novel efficacious molecules, this report explores the design and synthesis of a series of oxygen-incorporating heterocyclic derivatives (4a-z) with the integration of a benzochromene moiety.Upon the evaluation of these derivatives for their cytotoxic behaviours against MCF-7, HCT-116, HepG-2, and MCF-7/ADR tumour cell lines in comparison with the standard reference drugs Vinblastine, and Doxorubicin, only molecules exhibiting cytotoxically active characteristics were selected for additional assessment against the representative tumour cell line ADR-resistant human breast cancer cells (MCF-7/ADR)) and two healthy cell lines (HFL-1 and human diploid fibroblasts (WI-38).Additionally, molecules 4b-d, 4k, 4n, 4q, and 4w which possessed good potency against MCF-7/ADR cell comparable to that of Doxorubicin were tested to show the possible inhibitory effect on P-gp expression and the obtained data concluded that 4b-d, 4q, and 4w possessed good inhibitory potency against P-gp expression in MCF-7/ADR cells.Furthermore, the western blot results and the Rh123 accumulation assays showed that compounds 4b-d, and 4w effectively inhibited P-gp expression and efflux function while, compounds 4b-d, 4k, 4n, 4q, and 4w induced accumulation of the treated MCF-7/ ADR cells in the G1 phase and 4k and 4n additionally in the S phase of the cell cycle.Lastly, the SARs investigations of the molecules corroborated that the variation within the substitution on the phenyl ring of the 1H-benzo[f]chromene scaffold alongside the incidence of the methoxy moiety at the 9-position elevates molecular capabilities against these diverse cell lines.

Materials and equipments
All chemicals were purchased from Sigma-Aldrich Chemical Co.(Sigma-Aldrich Corp., St. Louis, MO).All melting points were measured with a Stuart Scientific Co. Ltd apparatus are uncorrected.The IR spectra were recorded on a KBr disc on a Jasco FT/IR 460 plus spectrophotometer.The 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were measured on BRUKER AV 500 MHz spectrometer in DMSO-d 6 as a solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts were expressed as d (ppm). 13C-NMR spectra were obtained using distortion-free enhancement by polarisation transfer (DEPT) and the attached proton test (APT).The Ultrasonic apparatus used is Fisher Scientific UL TRASONIC CLEANER FS220.The mass spectra were determined on a Shimadzu GC/MS-QP5050A spectrometer.Elemental analysis was carried out at the Regional Centre for Mycology and Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt, and the results were within ± 0.25%.Reaction courses and product mixtures were routinely monitored by thin-layer chromatography (TLC) on silica gel precoated F 254 Merck plates.

X-ray crystallography analysis
The compounds 4i was obtained as single crystals by slow evaporation from ethanol solution of the pure compound at room temperature.

Cell culture
The tumour cell lines MCF-7, HepG-2, and PC-3 were obtained from the American Type Culture Collection (ATCC, Rockville, MD).The cells were grown on RPMI-1640 medium supplemented with 10% inactivated foetal calf serum and 50 mg/mL gentamycin.The cells were maintained at 37 C in a humidified atmosphere with 5% CO 2 and were subculture two to three times a week.

Analysis of P-glycoprotein
The content of P-gp in the MCF-7/ADR cell lysates after incubation with varying conc.(12.5-100 mM) of tested compounds 4b, 4c, 4d, 4q, and 4w following exposure for 48 h. was determined using commercial human P-gp ELISA Kit (MBS2506188, MyBioSource Inc., San Diego, CA).Absorption was recorded at 450 nm with a Spectramax Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) 76 .
Rhodamine 123 accumulation assay P-gp activity was determined by measuring intracellular accumulation of Rhodamine 123 in MCF-7/ADR cells in the absence or presence of compounds 4b-4d, 4k, and 4w, according to commercial Rhodamine Competitive ELISA Kit (AKR-5142, Cell Biolabs Inc., San Diego, CA) which provides a convenient method for the detection of total Rhodamine in extracts from cells 77 .Briefly, resistant cancer cells were harvested, washed twice, and counted.After dilution to 1 Â 10 6 cells/mL in 6 well plate each well, 1 mL of fresh media containing different concentrations of compounds 4b-4d, 4k, and 4w were added and incubated at 37 C for 4 h in an atmosphere containing 5% CO 2 .Subsequently, 5.25 lM of Rho123 was added to each well and the wells were incubated for another 30 min at 37 C. Finally, cells were washed, lysed as described before and intracellular quantification levels of rhodamine 123 were further analysed according to ELISA protocol Kit.Absorbance at 450 nm of each well was measured using Spectramax Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA).The total content of Rhodamine in each sample was determined by comparison with a Rhodamine standard curve.

Western blot analysis
Cellular protein extracts of cell lysates and western blotting were prepared after treatment of MCF-7/ADR cells with different compounds 4b-4d and 4w conc.(0.1, 1.0, and 10 lM) for 48 h as already described 78 .Cells were washed twice with ice-cold phosphate-buffered saline and total cell lysates were collected in sodium dodecyl sulphate (SDS) sample buffer.Cell lysates, containing equal amounts of protein, were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Biosciences, NJ).After being blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (pH 7.6), membranes were incubated with the appropriate primary antibodies at 4 C, overnight, and exposed to the appropriate secondary antibody for 3 h at room temperature.Finally, enzyme-linked chemiluminescence was visualised according to the ECL kit (Thermal Fisher, Waltham, MA) protocol.b-Actin was used to confirm equal loading in each lane in the samples prepared from cell lysates.

Cell cycle assay
Cell cycle arrest and distribution were done using Propidium Iodide Flow Cytometry Kit (ab139418, Abcam, Cambridge, UK) as previously described 79 .Briefly, MCF-7/ADR cancer cells at 1 Â 104 cells were cultured in 60-mm dishes in the presence of various tested compounds with a concentration equal to the IC 50 value for 24 h.Cells were collected and washed with PBS, fixed with precooled 70% ethanol at 4 C.Staining went along in PBS containing 40 lg/ml RNase A and 10 lg/mL propidium iodide (PI) in the dark for 15 min.The DNA content in each cell nucleus was determined by a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).Finally, Cell cycle phase distribution was analysed using Cell Quest Pro software (BD Biosciences) showing collected PI fluorescence intensity on FL2.

Annexin V-FITC apoptosis assay
Apoptosis assay was performed with an Annexin V-FITC/PI double staining apoptosis detection kit (K101, Biovison, Milpitas, CA) using a flow cytometer 80 .MCF-7/ADR cancer cells treated with different newly synthesised compounds (IC 50 value) were harvested by trypsinization, washed twice with 4 C PBS, and re-suspended in binding buffer.Annexin V-FITC and PI solutions were then added to stain the cells before analysis by flow cytometry A minimum of 10,000 cells per sample were acquired.Annexin V-FITC binding (FL1) and PI (FL2) were analysed using Cell Quest Pro software (BD Biosciences).

Statistics
All data were expressed as the means ± standard deviation (SD), from at least three independent experiments with similar results.Statistical analysis and figures were performed by Graph Pad Prism version 5.01 (Graph Pad software, San Diego, CA).

Molecule docking
The docking experiment was employed according to previous work 81 , the 3D model of P-gp was obtained from Swiss-Model 82 .Finally, the outcomes for docking were achieved by PyMol software 83 .
funding this work through General Research Project under Grant Number [RGP.1/245/43] and also, to Malak T. Mahmoud for editing and revising the manuscript.

Chart 2 .
Rationale for designing target compounds.
50 values expressed in mM as the mean values of triplicate wells from at least three experiments and are reported as the mean ± standard error, A ¼ Vinblastine and B ¼ Doxorubicin.

Figure 4 .
Figure 4. A. Inhibition of P-gp content in the lysate of MCF-7/ADR cell using varying conc.(12.5-100 mM) of tested compounds 4b-4d, 4k, 4q, and 4w follows exposure 48 h as determined by ELISA.B. Western blot analysis of P-gp expression in MCF-7/ADR cells after treatment with 0.1, 1.0, and 10.0 mM of compounds 4b-4d, and 4w for 48 h, the b-actin was used as a control.

Figure 5 .
Figure 5. Effects of compounds 4 b-d, 4k, 4n, 4q, and 4w on the cell cycle phases of MCF-7/ADR cells.(a) Representative histograms of the DNA content distribution of cells were incubated with IC 50 values for 24 h and stained with propidium iodide (PI).Their DNA content was analysed by the fluorescence flow cytometry.(b) The percentage of MCF-7/ADR cells in the G1, S, and G2/M phases after incubation with tested compounds (IC 50 value) for 24 h.The data are expressed as the mean ± SD of three independent experiments in triplicate.

Figure 6 .
Figure 6.Apoptosis of MCF-7/ADR cells treated with compounds 4b-d, 4k, 4n, 4q, and 4w.(a) The dot plot of the Annexin V/PI stained cells, treated with the indicated drugs.(b) The apoptosis percentage of MCF-7/ADR cells after incubation with tested compounds (IC 50 value) for 24 h.The data are expressed as the mean ± SD of three independent experiments in triplicate.

Table 1 .
X-Ray experimental details for compounds 4i.

Table 7 .
The binding affinity for compounds 4a-z and QZ59-RRR in (kcal/mol) against P-gp.
least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F. CCDC 2132096 contain the Supplementary crystallographic data for this compound can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
73,74were collected on a Bruker APEX-II D8 Venture area diffractometre, equipped with graphite monochromatic Mo Ka and Cu Ka radiations at 293 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT.SHELXTL-2018/373,74was used to solve structure.The final refinement was carried out by full-matrix