Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect

Abstract In our effort to develop novel and powerful agents with anti-proliferative activity, two new series of 1H-benzo[f]chromene derivatives, 4a–h and 6a–h, were synthesised using heterocyclocondensation methodologies under microwave irradiation condition. The structures of the target compounds were established on the basis of their spectral data, IR, 1H NMR, 13 C NMR, 13 C NMR-DEPT/APT, and MS data. The new compounds have been examined for their anti-proliferative activity against three cancer cell lines, MCF-7, HCT-116, and HepG-2. Vinblastine and Doxorubicin have been used as positive controls in the viability assay. The obtained results confirmed that most of the tested molecules revealed strong and selective cytotoxic activity against the three cancer cell lines. Moreover, these molecules exhibited weak cytotoxicity on the HFL-1 line, which suggested that they might be ideal anticancer candidates. The SAR study of the new benzochromene compounds verified that the substituents on the phenyl ring of 1H-benzo[f]chromene nucleus, accompanied with the presence of bromine atom or methoxy group at the 8-position, increases the ability of these molecules against the different cell lines. Due to their high anti-proliferative activity, compounds 4c and 6e were selected to be examined their proficiency to inhibit the invasiveness of the highly sensitive and invasive breast cancer cell line, MDA-MB-231. The anti-invasion behaviour of these molecules against the highly sensitive, non-oestrogen, and progesterone MDA-MB-231 cell line gave rise to their decreasing metastatic effect compared to the reference drug. Furthermore, this report explores the apoptotic mechanistic pathway of the cytotoxicity of the target compounds and reveals that most of these compounds enhance the Caspase 3/7 activity that could be considered as potential anticancer agents.

In our efforts to be a part of the development process of novel cytotoxic agents, we have recently focussed on utilising the 1H-benzo[f]chromene scaffold with methoxy substituent at the 9-position to create various potent and high anti-proliferative compounds such as 3-amino-1-(4-chlorophenyl)-9-methoxy-1H-benzo[f]chromene-2-carbonitrile 27 (H). Since it was revealed that the position and the type of the substituent have a great effect on the cytotoxic activity of the desired compounds, the main goal of the present study is to synthesise a new family of substituted 1H-benzo[f]chromene at the 8-postion with electron-withdrawing and electron-donating groups. The rational design of the new compounds was based on the following considerations: (i) the 1Hbenzo[f]chromene scaffold itself, (ii) the presence of the effective 8-postion substitution, and (iii) the type of the aryl groups linked to chromene scaffold which seems to play a crucial role in the cytotoxic activity.
Herein, we introduced the microwave irradiation synthesis of the 3-amino-1H-benzo[f]chromene-2-carbonitrile derivatives. The target compounds were examined for their anti-proliferative activity towards three cancer cell lines, MCF-7, HCT-116, and HepG-2 and the viability assay was evaluated against two standard drugs, Vinblastine and Doxorubicin. The Structure-Activity Relationships of these molecules elucidated the effect of the substituents at the  C-1 and C-8 positions of the 1H-benzo[f]chromene moiety on their cytotoxic activity.

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). The 13 C NMR spectra were obtained using distortion less enhancement by polarisation transfer (DEPT), where the signals of CH and CH 3 carbon atoms appear normal (up), and the signals of carbon atoms in CH 2 environments appear negative (down). The 13 C N MR spectra were also obtained using attached proton test (APT), with this technique, the signals of CH and CH 3 carbon atoms appear normal (up) and the signal of CH 2 and Cq environments appears negative (down

Biological screening
2.3.1. Cell culture and cytotoxicity evaluation using viability assay Compounds 4a-h and 6a-h were initially evaluated for in vitro antitumour activity against three different human cell lines: MCF-7, HCT-116, and HepG-2, and only the representative active cytotoxic compounds were further evaluated against the normal HFL-1 cell line. The in vitro cytotoxicity evaluation was performed at the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University under different concentrations (50, 25, 12.5, 6.25, 3.125, 1.56, and 0 mg/mL); Vinblastine and Doxorubicin are used as reference cytotoxic compounds. The measurements of cell growth and the in vitro cytotoxicity evaluation were determined using viability assay as described in literature [31][32][33] and the result was cited in Table 1 and Figure 2.

Cell invasion assay
The cell invasion assay was performed by Reaction Biology Corporation, One Great Valley Parkway, PA. All compounds were dissolved in DMSO and tested for their ability to cell invasion assay. Compounds 4c, 6e, and reference compound bosutinib were purchased from Sellechem (Houston, TX). Corning Bio Coat Fluoro Blok Insert System-96-well plates were purchased from Corning (Corning Incorporated, Tewksbury, MA). Calcein AM labelling reagent was purchased from Molecular Probes (Eugene, Oregon). MDA-MB-231 cell line was purchased from American Type Culture Collection (Manassas, VA). MDA-MB-231 cells were grown in DMEM medium supplemented with 10% foetal bovine serum (FBS), 100 mg/mL of penicillin, and 100 mg/mL of streptomycin. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO 2 and 95% air-following incubation; medium was carefully removed from the apical chambers. The insert system was transferred into a second 96-square well plate containing 200 ml/well of 4 mg/mL Calcein AM in HBSS. The plates were  incubated for 1 h at 37 C, 5% CO 2 . Fluorescence of invaded cells was read at wavelengths of 492/535 nm (Ex/Em) from bottom reading using Envision 2104 Multilabel Reader (PerkinElmer, Santa Clara, CA). The IC 50 curves were plotted and IC 50 values were calculated using the Graph Pad Prism 4 program for windows (Graph Pad software, San Diego California. www.graphpad.com) based on a sigmoidal dose-response equation.

Caspase 3/7 activity assay
The Caspase-Glo 3/7 activity assay was performed by Reaction Biology Corporation, One Great Valley Parkway, PA. Examined compounds 4c, 4e, 4h, 6d, and 6e were dissolved in DMSO as 20 mM stock and tested for their ability to Caspase-Glo 3/7 activity assay. The reference compound staurosporine was purchased from Sigma-Aldrich (Saint Louis, MO) and dissolved in DMSO in 10 mM stock. Caspase-Glo 3/7 activity assay reagent was purchased from Promega (Madison, WI). MDA-MB-231 cell line was provided by ATCC. MDA-MB-231 cell line culture conditions: DMEM with 10% FBS. 100 mg/mL penicillin and 100 mg/mL streptomycin were added to the culture media. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO 2 and 95% air.

c-Src kinase assay
The effect of the synthesised compounds on the activity of c-Src kinase was determined by HTScan Src Kinase Assay Kit, catalogue number 7776 from Cell Signaling Technology (Danvers, MA); according to the manufacturer's protocol. Streptavidin-coated plates were purchased from Pierce (Rockford, IL). In brief, the kinase reaction was started with the incubation of the 12.5 mL of the reaction cocktail (0.5 ng/mL of GST-Src kinase in 1.25 mM DTT) with 12.5 mL of prediluted compounds (dissolved in 1% DMSO) for 5 min at room temperature. ATP/substrate (25 mL, 20 mM/1.5 uM) cocktail was added to the mixture. The biotinylated substrate (catalogue number 1366) contains the residues surrounding tyrosine 160 (Tyr160) of signal transduction protein and has a sequence of EGIYDVP. The reaction mixture was incubated for 30 min at room temperature. The kinase reaction was stopped with the addition of 50 mL of 50 mM EDTA (pH 8.0). The reaction solution (25 mL) was transferred into 96-well streptavidin plates (Pierce, part number 15125), diluted with 75 mL double distilled water, and incubated at room temperature for 60 min. At the end of the incubation, the wells were washed three times with 200 mL of 0.05% Tween-20 in PBS buffer (PBS/T). After that to each well was added 100 mL of phosphotyrosine antibody (P-Tyr-100) (1:1000 dilution in PBS/T with 1% BSA) and the wells were incubated for another 60 min. After washing three times with 0.05% Tween-20 in PBS/T, the wells were incubated with 100 mL secondary anti-mouse IgG antibody, which was HRP-conjugated (1:500 dilution in PBS/T with 1% BSA) for next 30 min at room temperature. The wells were washed five times with 0.05% Tween-20 in PBS and then were incubated with 100 mL of 3,3 0 ,5,5 0 -tetramethylbenzidine dihydrochloride substrate for 5 min. The reaction was stopped by adding 100 mL/well of stop solution to each well and mixed well and read the absorbance at 450 nm using a microplate reader (Molecular devices, spectra Max M2). IC 50 values of the compounds were calculated using ORIGIN 6.0 (Origin Lab Corporation, Northampton, MA) software. IC 50 is the concentration of the compound that inhibited enzyme activity by 50%. All the experiments were carried out in triplicate 24 .

Molecular docking
Molecular docking simulation was carried out with the program AutoDock 4.0.1 34 (GNU General Public License). The three-dimensional (3D) structure of c-Src kinase was obtained from Protein Data Bank (PDB entry no. 2src). The ligand and solvent molecules were removed from the crystal structure to obtain the docking grid and the active site was defined using AutoGrid. The grid box was centred on the centre of the ligand from the corresponding crystal structure complexes. The Lamarckian genetic algorithm issued for docking with the following settings: a maximum number of 2,500,000 energy evaluations, an initial population of 50 randomly placed individuals, a maximum number of 37,000 generations, a mutation rate of 0.02, across over rate of 0.80, and an elitism value (number of top individuals that automatically survive) of 1. The ligand was fully optimised inside the binding site during the docking simulations. Finally, the conformation with the lowest predicted binding free energy of the most occurring binding modes in c-Src active pocket was selected. Hydrogen atoms were added to the structure with the Molecular Operating Environment (MOE 2012) 35 and atomic partial charges were calculated using Auto-Dock Tools. Selected active compounds were docked into the active site of microorganisms' targets to predict compound binding modes. For flexible docking, Auto-Dock standard parameter settings were applied. High-scoring binding poses were selected on the basis of visual inspection.

Statistical analysis
All statistical calculations were done using computer programs, Microsoft excel version 10, SPSS version 20.00 (SPSS Inc., IBM in 2009, Chicago, IL) statistical program at 0.05, 0.01, and 0.001 level of probability 36 . Comparisons of inhibiting tumour growth between treatments groups or the control were done using Student's t test, one-way ANOVA, and post hoc the least significant difference tests measurement.

Chemistry
Benzochromene molecules have been synthesised using the synthetic strategy illustrated in Schemes 1 and 2. In a one-pot three component heterocyclocondensation process, 3-amino-1-aryl-8bromo-1H-benzo[f]chromene-2-carbonitrile 4a-h were obtained via the reaction of 6-bromo-2-naphthol 1 with a mixture of appropriate aromatic aldehydes 2a-h and malononitrile 3 in ethanol/ piperidine solution under microwave irradiation conditions for 2 min at 140 C.
The IR spectra of compounds 4a-h revealed the presence of three peaks at t 3477-3409, 3369-3321, 3217-3199 cm À1 , and one peak at t 2204-2181 cm À1 which are assigned to the amino and cyano groups, respectively. In the meantime, the 1 H NMR spectra of compounds 4a-h displayed a singlet signal of the 1H protons in the range of d 5.47-5.26 ppm and a broad signal of the amino group in the range of d 7.10-6.90 ppm. In addition, compounds 4a and 4e-h revealed the presence of D 2 O as a replacement to the NH 2 proton at d 7.04-6.90 ppm. Furthermore, the 1 H NMR spectra of compounds 4e and 4f-h exhibited signals of the methyl and methoxy groups at d 2.21 and 3.85-3.67 ppm, respectively. On the other hand, the 13 C NMR spectra of compounds 4a-h displayed one signal of the chiral carbon resonating at d 37.89-31.26 ppm, and compounds 4f-h exhibited signals of the methyl and methoxy groups at d 20.51 and 55.96-54.96 ppm, respectively. Also, the 13 C NMR-DEPT spectra at 45 , 90 , and 135 C as well as the 13 C NMR-APT spectra of compounds 4b, 4e-h provided additional evidence to support the proposed structures (See supplemental data).
The same methodology has been employed to prepare the second series of benzochromene molecules; compounds 6a-h, Scheme 2 illustrated the interaction of 6-methoxy-2-naphthol 5 with a mixture of appropriate aromatic aldehydes, 2a-h, and malononitrile 3 to afford the corresponding 3-amino-1-aryl-8methoxy-1H-benzo[f]chromene-2-carbonitriles, 6a-h. The maximum power of the microwave irradiation was optimised by repeating the reaction at different watt powers and periods of time. The optimum condition was obtained by using the microwave irradiations at 400 W and reaction time 2 min, which gave the highest yield of the desired compounds. It is also important to note that the 1-position of compounds 4a-h and 6a-h is a chiral centre and all the reactions were controlled using the TLC technique.
The IR spectra of the target compounds 6a-h confirmed the presence of the characteristics of the NH 2 absorption bands around t 3475-3414, 3337-3321, 3219-3192 cm À1 and the CN absorption bands around t 2203-2185 cm À1 . The 1 H NMR spectra of 6a-h showed singlet signals of 1H, NH 2 , and methyl or methoxy groups protons at d 5.46-5.21, 7.02-6.81 and 2.21, and 3.88-3.66 ppm, respectively. Furthermore, the 13 C NMR spectra of compounds 6a-h showed signals resonating in the range of d 38.15-31.12 ppm which are attributed to the chiral carbon, while the carbons of the methyl and methoxy groups appeared in the range of d 20.51 and 55.45-54.95 ppm, respectively. Besides, the 13 C NMR-DEPT spectra at 45 , 90 , and 135 C as well as the 13 C NMR-APT spectra of compounds 6f-h provided additional evidence in supporting the proposed structures (See supplemental data).

Antitumour assay
The in vitro cytotoxic evaluation of the prepared compounds 4a-h and 6a-h was performed against MCF-7 (breast cancer), HCT-116 (human colon cancer), HepG-2 (liver cancer), and HFL-1(normal fibroblast lung) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay as described in literature [31][32][33] . Doxorubicin, one of the most effective anticancer agents, and the cancer drug Vinblastine was included in the experiment as positive controls. The results were expressed as growth inhibitory concentration (IC 50 ) values, which represent the compounds' concentrations required to produce a 50% inhibition of the cell growth after 24 h of incubation, compared to the untreated controls as shown in Table 1 and Figure 2.
According to the obtained results, it was obvious that most of the synthesised compounds displayed excellent anti-proliferative activity against the tested cancer cell lines. Investigations of the cytotoxic activity against the three cancer cell lines, MCF-7, HCT-116, and HepG-2, indicated that the HepG-2 was the most sensitive cell line to the influence of the target compounds. In the meantime, the treatment of the MCF-7 cancer cell lines was very successful using the first series compounds, 4a-h which displayed IC 50 values in the range of 0.5-5.2 mg/mL. The same effect was obtained using compounds 6a-f of the second series which exhibited IC 50 values between 0.3 and 4.4 mg/mL, Table 1. These values were found to be more potent and efficacious against MCF-7 cancer cell lines than Vinblastine (IC 50 ¼ 6.1 mg/mL) while compound 6e (IC 50 ¼ 0.3 mg/mL) was found to be the most potent derivative against MCF-7 as compared to Doxorubicin (IC 50  range of 0.8-2.5 mg/mL displayed significant growth inhibitory activity in comparison to Vinblastine (IC 50 ¼ 2.6 mg/mL). In particular, compound 4d (IC 50 ¼ 0.5 mg/mL) was equipotent as Doxorubicin (IC 50 ¼ 0.5 mg/mL). The same effect has also been achieved using compounds 4c, 4e, 6d, and 6e with IC 50 between 0.7 and 0.9 mg/mL. On the other hand, the cytotoxic evaluation against HepG-2 cell lines revealed that the second series elicited considerable anti-proliferative activity than the first series. For instance, seven members of the first series 4a-g with IC 50 values in the range of 0.5-4.4 mg/mL and four members of the second series 6b-e with IC 50 values between 0.3 and 0.7 mg/mL were more potent and efficient than Vinblastine (IC 50 ¼ 4.6 mg/mL) while compounds 6b-e, 4a, and 4b with IC 50 ranging from 0.3 to 0.8 mg/mL displayed significant growth inhibitory activity in comparison to Doxorubicin (IC 50 ¼ 0.9 mg/mL). Compound 4e (IC 50 ¼ 0.9 mg/mL) was equipotent as Doxorubicin. In addition, the potent compounds 4c, 4e, 4h, 6d, and 6e were evaluated for their cytotoxic effect against normal fibroblast cell line. The obtained data revealed that all compounds displayed weak cytotoxicity (IC 50 ¼ 13-35 mg/mL) compared to their anticancer effect, proving the selectivity behaviour. Finally, the remaining compounds exhibited equipotent or moderate to fair cytotoxic activities against the three tumour cell types compared to Vinblastine and Doxorubicin, and the highly active ones could be considered as selective anticancer agents.

Cell invasion assay
Tumour invasion is a common feature of triple-negative breast cancers. Triple-negative breast cancers (12-24% of breast cancers) 37,38 which are characterised by the absence of ER, progesterone receptors, and HER-2 expression, resulted in high morbidity and mortality owing to their rapid growth rate, invasive potential, and frequently acquired treatment resistance. The invasion process has become an important prerequisite for cancer progression and metastasis. Therefore, therapeutic strategies for preventing or suppressing cancer invasion and metastasis can significantly improve the survival of triple-negative breast cancer patients. The two most active compounds 4c and 6e were examined for their ability to inhibit the invasiveness of the highly sensitive and invasive breast cancer cell line, MDA-MB-231. A bosutinib drug was used as a control ant-invasive agent. As shown in Figure 3, 4c and 6e derivatives have markedly suppressed the invasion of the MDA-MB-231 cell up to the highest percentage. As shown in Table 2, they showed comparable potent ant-invasion activity with IC 50 of 10.5 and 16.5 mM, respectively. The obtained data might confirm that the cytotoxicity of novel molecules led to the death of the cancer cells and stopped the metastatic processes.

Caspase 3/7 activity assay
Caspases are ATP-dependent enzymes that promote apoptosis. Caspases are vital biochemical features of apoptotic signalling. The apoptotic response of MDA-MB-231 cells to potent anti-proliferative compounds was examined via quantifying the activity of caspases 3/7. To determine whether the cell growth inhibition in response to the benzochromene derivatives was mediated by apoptosis and was caspase dependent, MDA-MB-231 cells were treated with 10 different concentrations of the target compounds for 24 h. As shown in Figure 4, caspase-3/7 activity was activated in a dose-dependent manner. The target benzochromene compounds significantly increased caspase-3/7 activity to a 10-fold at a certain concentration and time point, Table 3 and Figure 4.

c-Src kinase assay
As a biological step for the discovery of their anti-proliferative mechanism, further c-Src kinase inhibitory activity of the potent compounds 4c, 4e, 4h, 6d, and 6e were performed, Table 4. It is clearly presented that their inhibitory activity is potent and the IC 50 values ranged between 9 and 329 nM, which mostly exhibited higher activity than the reference drug. For instance, compound 4e, with the 4-methylphenyl substitution, exhibited higher c-Src kinase inhibitory activity (IC 50 ¼ 9 nM) than the rest. Similarly, compound 6e, with the 4-methylphenyl substitution, was the most effective compound in the 8-methoxy series with an IC 50 value of 9 nM. Additionally, 3,4-dimethoxyphenyl and 4bromophenyl substituted compounds 4 h and 6d were among the moderately effective inhibitors in both the 8-bromo and the 8-methoxy series of compounds with IC 50 ranging from 214 to 329 nM. Lastly, the substitution of the aryl terminal moiety with the electron-donating group (methyl group) in compounds 4e and 6e led to increase in inhibitory activity, suggesting that substitution at position 4 of the phenyl ring is detrimental on the c-Src kinase inhibition.

SAR studies
The preliminary SAR study has focussed on the effect of the presence of the phenyl ring on the antitumour activities of the synthesised compounds. The influence of replacing the H atom at the 4-position in the phenyl ring of the 1H-benzo[f]chromene moiety with the electron-withdrawing (F, Cl, Br) or electron-donating (Me, MeO) groups, and the presence of a bromine atom (electron-withdrawing) or methoxy group (electron-donating) at the 8-position has also been discussed. In a comparison of the cytotoxic activities of the two series 4a-h and 6a-h against MCF-7 breast cancer, we found that the highest growth inhibitory effect of the first series 4a-h was associated with the presence of 4-chlorophenyl 4c, 3,4dimethoxyphenyl 4h, unsubstituted phenyl 4a, 4-bromophenyl 4d, 4-methylphenyl 4e, 2,4-dimethoxyphenyl 4g, 4-methoxyphenyl 4f, and 4-fluorophenyl 4b, respectively. As previously mentioned, these molecules showed IC 50 values in the range of 0.5-5.2 mg/mL against MCF-7, which are considered excellent activities relative to Vinblastine (IC 50 ¼ 6.1 mg/mL), Table 5. On the other hand, the inhibitory effect of the second series 6a-h was more potent and efficacious than Vinblastine (IC 50 ¼ 6.1 mg/mL). The IC 50 values of these molecules were between 0.3 and 4.4 mg/mL against MCF-7, and their effect was as follows: 4-methylphenyl 6e, 4-methoxyphenyl 6f, 4-bromophenyl 6d, un-substituted phenyl 6a, 4-chlorophenyl 6c, and 4-fluorophenyl 6b, respectively, Table 5. According to the obtained data and regarding the first series, it was found that the replacement of the H atom by the chlorine atom (electron-withdrawing group) at the 4-position in the phenyl ring of the 1H-benzo[f]chromene moiety was significant for its activity against the MCF-7 compared to the dimethoxy (electron-donating group) at 3,4-positions on the same ring with bromine atom at the 8-position. However, the introduction of a methyl or methoxy group at 4-position (electron-donating group) on the phenyl ring, at position 1 of the second series is more beneficial than an electron-withdrawing substituent like Br, Cl, or F atoms, taking into consideration that the second series 6a-h (more electron-donating group) exhibited significant growth inhibitory activity against MCF-7 which is more effective than the first series 4a-h (more electron-withdrawing group). In general, the SAR image of the novel potent derivatives indicates that the presence of different pharmacophoric functionalities with small hydrophobic moieties as halogens or slightly hydrophobic fragments as methoxy play an important role in the cytotoxic activity.
Investigations of the cytotoxic activity against HCT-116, human colon cancer, indicated that compounds 4a-e of the first series with IC 50 ¼ 0.5-1.2 mg/mL and compounds 6a,c-e of the second series with IC 50 ¼ 0.8-2.5 mg/mL displayed a good inhibitory activity compared to Vinblastine (IC 50 ¼ 2.6 mg/mL), Table 6. These results are intimate that the grafting of a lipophilic electron-withdrawing substituent (halogen atoms) is more beneficial than an electron-donating substituent (methyl or unsubstituted phenyl group) for the activity of the bromine atom or the methoxy group at the 8-position.    Moreover, the first series 4a-h (electron-withdrawing group) revealed significant growth inhibitory activity against HCT-116 in comparison to the second series 6a-h (electron-donating group).
Regarding the activity against the HepG-2 cell line (liver cancer), the order of the antitumour activity of the first series was found to be 4a > 4b > 4e > 4c > 4d > 4g > 4f (IC 50 ¼ 0.7-4.4 mg/mL) and for the second series 6c > 6b > 6e > 6d, with IC 50 ¼ 0.3-0.7 mg/mL, Table 7. These results suggested that the un-substituted phenyl (electron-donating group) of the first series is preferred as an antitumor agent in comparison to the substituted phenyl (electron-withdrawing group) with bromine atom at the 8-position; however, the electron-withdrawing substituent is preferred compared to electrondonating substituent for the second series with methoxy group at the 8-position, keeping in mind that the second series, 6a-h (electron-withdrawing and electron-donating groups), is more effective towards the HepG-2 than the first series, 4a-h (electrondonating and electron-withdrawing groups).
On the other hand, the rest of the compounds showed almost equipotent or moderate to fair cytotoxic activities against the three tumour cell lines compared to Vinblastine. While compound 6e (IC 50 ¼ 0.3 mg/mL) was found to be the most potent derivative against MCF-7 compared to Doxorubicin (IC 50 ¼ 0.4 mg/mL) as shown in Table 8. Compound 4c (IC 50 ¼ 0.5 mg/mL) was almost as equipotent as Doxorubicin, and compound 4d (IC 50 ¼ 0.5 mg/mL) was equipotent as Doxorubicin against HCT-116. Furthermore, compounds 6c, 6b, 6e, 4a, and 4b with IC 50 ¼ 0.3-0.8 mg/mL displayed significant growth inhibitory activity against HepG-2 in comparison to Doxorubicin (IC 50 ¼ 0.9 mg/mL) as shown in Table 7, and compound 4e (IC 50 ¼ 0.9 mg/mL) was equipotent as Doxorubicin indicating that the electron-withdrawing substituent is preferred for antitumour activity compared to the electrondonating substituent.
Finally, we can deduce that the substitution pattern of the phenyl group at the 1-position of the 1H-benzo[f]chromene moiety played a vital role in its antitumour activity. The incorporation of halogen atoms (electron-withdrawing group) has also greatly enhanced the activity of the desired compounds in comparison to the electrondonating groups as methyl or methoxy groups with bromine atom or methoxy group at the 8-position. Overall, the growth inhibitory activity of the second series exceeded the first one.

Molecular docking
c-Src kinase is a non-receptor tyrosine kinase which plays essential roles in controlling a myriad of fundamental cellular processes, including cell proliferation, adhesion, migration, and invasion 39,40 . c-Src kinase is overexpressed and/or is aberrantly activated in a variety of human tumours, including breast, colon, pancreatic, bladder, ovarian, and brain carcinomas 41,42 . Notably, increased c-Src activity is believed to correlate strongly with the metastatic potential and the poor prognosis in breast cancers 41,43,44 . Thus, c-Src kinase has been recognised as an attractive molecular target for cancer therapy, and the development of c-Src inhibitors for cancer treatment is an increasing interest. In this report, we docked the potent active anti-proliferative derivatives 4c, 4e, 4h, 6d, and 6e in the ATP-binding pocket of the target enzyme. The placement was performed based upon the position of the cocrystallised ligand ANP. A root mean square deviation (rmsd) of 0.29 and binding energies data were resulted, suggesting a high similarity of interaction to the referenced ligand. Investigation of the docking results reported different binding modes of the target compounds. These compounds consistently showed a stable hydrogen-bonding interaction with the NH of Met 341 residue as a donor with all nitrile fragments of the structures. However, in some cases, the interaction was supported with extra hydrogen bonds with the carbonyl group of Glu 339 as an acceptor through the NH 2 fragment. Moreover, Asp 404 and Lys 295 exhibited additional hydrogen bonds through the OCH 3 substituents. In addition, a hydrophobic interaction was found between the naphthalene substructure of compounds within the hydrophobic pockets in the active site, Figure 5 and Table 9. Finally, the hydrogenbonding donor-acceptor motif which was proposed as a crucial key for the kinase inhibitory activity was accomplished by the active compounds fragments with the corresponding Met 341 or Glu 399 residues 45 . This behaviour towards c-Src kinase might confirm the cytotoxic effect of the studied compounds is mediated through the inhibition of such promising target.

Prediction of drug-likeness and ADME properties
In a way to design a drug with a good oral bioavailability profile, certain parameters are considered to play an important role 46,47 . About one-third of the invented drugs fail during development processes due to their unreasonable pharmacokinetic profiles 48 . Therefore, a computational study for the prediction of the ADME properties of the synthesised molecules was performed by the calculation of lipophilicity, topological polar surface area (TPSA), absorption (% ABS), and simple molecular descriptors used by Lipinski in formulating his "rule of five" 49 . Calculations were performed using the Molinspiration online property calculation toolkit 50 . Table 10 represents a calculated percentage of the absorption (% ABS), TPSA, and Lipinski parameters of the compounds. A percentage of absorption (% ABS) was estimated using the equation: % ABS ¼109À(0.345 Â TPSA), according to Zhao  the Lipinski profile of the synthesised compounds indicated that most of the synthesised derivatives are considered to be drug-like candidates for the novel cytotoxic agents with a slight increase in the log p values for two bromobenzochromene compounds.

Conclusions
Herein, we present the synthesis of 16 compounds of 3-amino-1aryl-8-bromo/methoxy-1H-benzo[f]chromene-2-carbonitrile 4a-h and 6a-h. The new compounds have been evaluated for their cytotoxic activities against three cancer cell lines, MCF-7, HCT-116, and HepG-2. The obtained results claimed that most of the synthesised compounds displayed strong and selective cytotoxic activity against the three cell lines. Moreover, the potent compounds have been examined for their cytotoxic effect against normal fibroblast cell line and revealed weak cytotoxicity compared to their anticancer effect. These results confirm the selectivity behaviour of the target compounds. On the other hand, the SAR study has been performed on the desired compounds and elucidated that the introduction of halogens substituent (electron-withdrawing group) at the 4-position of the phenyl group at position 1 of the 1H-benzo[f]chromene moiety enhanced their antitumour activities than the electron-donating substituent like methyl or methoxy groups. In addition, the presence of the lipophilic electron-donating, methoxy group at the 8-position was essential for the activity than the electron-withdrawing substituent like a bromine atom. Caspase 3/7 intrinsic apoptotic assay has been used to explore the demise of the three cell lines upon their treatment with the target compounds. Meanwhile, the interaction behaviour of selected active benzochromene derivatives was examined computationally via their docking simulation within the c-Src kinase target, which resulted in a good interaction profile compared to the reference ligand. This behaviour classifies these molecules as potential c-Src kinase candidates with potent cytotoxic activity. The data reported in the table are extracted from MOE program showing the corresponding amino acids residues in enzyme pocket, corresponding fragments of ligands, interaction distances, types of interaction, and their binding energy to some selected drugs. Conserved amino acids are bold highlighted.