Novel Thiazolidinone/Thiazolo[3,2-a]Benzimidazolone-Isatin Conjugates as Apoptotic Anti-proliferative Agents Towards Breast Cancer: One-Pot Synthesis and In Vitro Biological Evaluation

In connection with our research program on the development of new isatin-based anticancer candidates, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates (4a–n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a–d), and in vitro evaluation of their antiproliferative activity towards two breast cancer cell lines; triple negative MDA-MB-231, and MCF-7. Compounds 4m and 7b emerged as the most active congeners against MDA-MB-231 cells (IC50 = 7.6 ± 0.5 and 13.2 ± 1.1 µM, respectively). Compounds 4m and 7b were able to provoke apoptosis in MDA-MB-231 cells, evidenced by the up-regulation of Bax and down-regulation of Bcl-2, besides boosting caspase-3 levels. Hybrid 4m induced a fourfold increase in the percentage of cells at Sub-G1, with concurrent arrest in G2-M phase by 2.5-folds. Furthermore, hybrid 4m resulted in a sixfold increase in the percentage of annexin V-FITC positive apoptotic MDA-MB-231 cells as compared with the control. Moreover, the cytotoxic activities of the active conjugates were assessed towards two nontumorigenic cell lines (breast MCF-10A and lung WI-38) where both conjugates 4m and 7b displayed mean tumor selectivity index: 9.6 and 13.9, respectively. Finally, several ADME descriptors were predicted for the active conjugates via a theoretical kinetic study.


Introduction
Breast cancer has become the most frequently diagnosed malignancy among women, and the second leading cause of cancer-related deaths in women [1][2][3]. The deficiency in the maintenance of

Chemistry
The target conjugates 4a-n and 7a-d were synthesized adopting the chemical pathways outlined in Schemes 1 and 2. In Scheme 1, preparation of conjugates 4a-n was accomplished via two synthetic routes. The first route comprised a one-pot three-component synthesis, involving a Knoevenagel condensation, followed by cyclization among isatins 1a-g, utilising an equimolar amount of N,N′diphenyl/p-tolyl thiourea 2a,b and bromoacetic acid 3 in refluxing glacial acetic acid in the presence of sodium acetate to furnish conjugates 4a-n. This route proved successful to prepare conjugates 4an in high yields; 70-86% (Scheme 1). Alternatively, N,N′-diphenyl/p-tolyl thiourea 2a,b reacted with bromoacetic acid 3 to furnish compounds 5a,b, which subsequently condensed with isatins 1a-g in acetic acid in the presence of sodium acetate to afford the target conjugates 4a-n, with overall 55-68% Sunitinib (Sutent ® ) II (Figure 1), an isatin-based multitarget tyrosine kinase inhibitor, was granted its FDA approval in 2006 for the treatment of imatinib-resistant gastrointestinal stromal tumors (GIST) and advanced metastatic renal cell carcinoma (RCC) [15]. By 2014, Nintedanib (III, Ofev ® , Figure 1), an orally available isatin-based triple-angiokinase inhibitor, was approved by the FDA for the management of idiopathic pulmonary fibrosis [16]. One year later, the European Medicines Agency approved Nintedanib, under a trade name Vargatef ® , as a second-line treatment in combination with docetaxel for patients with advanced non-small cell lung cancer of adenocarcinoma who have progressed to first-line chemotherapy [17].
Taking the above into account, herein we adopted the hybrid pharmacophore approach to design and synthesize two novel series of thiazolidinone-isatin (4a-n) and thiazolo [3,2-a]benzimidazoloneisatin conjugates (7a-d) (Figure 1), with the goal of developing potent antiproliferative agents toward TNBC MDA-MB-231, and MCF-7 breast cancer cell lines. Furthermore, the most potent antiproliferative congeners were evaluated for their apoptosis induction potential in MDA-MB-231 cells, to gain insight into the mechanism of the anticancer activity for the prepared hybrids. Finally, the cytotoxic activity of the active conjugates was tested towards nontumorigenic human normal lung fibroblast cell line  to investigate the safety of the newly prepared conjugates.

Chemistry
The target conjugates 4a-n and 7a-d were synthesized adopting the chemical pathways outlined in Schemes 1 and 2. In Scheme 1, preparation of conjugates 4a-n was accomplished via two synthetic routes. The first route comprised a one-pot three-component synthesis, involving a Knoevenagel condensation, followed by cyclization among isatins 1a-g, utilising an equimolar amount of N,N -diphenyl/p-tolyl thiourea 2a,b and bromoacetic acid 3 in refluxing glacial acetic acid in the presence of sodium acetate to furnish conjugates 4a-n. This route proved successful to prepare conjugates 4a-n in high yields; 70-86% (Scheme 1). Alternatively, N,N -diphenyl/p-tolyl thiourea 2a,b reacted with bromoacetic acid 3 to furnish compounds 5a,b, which subsequently condensed with isatins 1a-g in acetic acid in the presence of sodium acetate to afford the target conjugates 4a-n, with overall 55-68% yield over the two steps (Scheme 1).  (II & III), some reported isatin-based hybrids with potent antiproliferative activity against breast cancer (VII-IX), and the target conjugates (4a-n and 7a-d).

Chemistry
The target conjugates 4a-n and 7a-d were synthesized adopting the chemical pathways outlined in Schemes 1 and 2. In Scheme 1, preparation of conjugates 4a-n was accomplished via two synthetic routes. The first route comprised a one-pot three-component synthesis, involving a Knoevenagel condensation, followed by cyclization among isatins 1a-g, utilising an equimolar amount of N,N′diphenyl/p-tolyl thiourea 2a,b and bromoacetic acid 3 in refluxing glacial acetic acid in the presence of sodium acetate to furnish conjugates 4a-n. This route proved successful to prepare conjugates 4an in high yields; 70-86% (Scheme 1). Alternatively, N,N′-diphenyl/p-tolyl thiourea 2a,b reacted with bromoacetic acid 3 to furnish compounds 5a,b, which subsequently condensed with isatins 1a-g in acetic acid in the presence of sodium acetate to afford the target conjugates 4a-n, with overall 55-68% yield over the two steps (Scheme 1).
Postulated structures of the newly synthesized conjugates 4a-n and 7a-d were in full agreement with their spectral and elemental analyses data.

In Vitro Antiproliferative Activity
The in vitro antiproliferative activity of the newly synthesized conjugates 4a-n and 7a-d was evaluated against breast cancer MDA-MB-231 and MCF-7 cell lines, following the Crystal Violet (CV) cell cytotoxicity assay [36]. Doxorubicin was included in this assay as a positive control, and Sunitinib was used as a reference drug. The results were expressed as median growth inhibitory concentration (IC50) values that represent the compounds concentrations required to afford a 50% inhibition of cell growth after 48 h of incubation, compared to untreated controls (Table 1).
Breast cancer is composed of multiple subtypes with distinct morphologies and clinical implications. In this study, the sensitivity of two different breast cancer subtypes towards the target conjugates was tested to examine if the target compounds possess selective growth inhibitory activity toward certain subtype. MCF-7 is a Luminal A breast cancer cells which is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive), HER2 negative, and has low levels of the protein Ki-67, which helps control how fast cancer cells grow. Luminal A cancers are low-grade, tend to grow slowly and have the best prognosis. Whereas, MDA-MB-231 is a triple-negative/basal-like breast cancer cell line, which is hormone-receptor negative (estrogen-receptor and progesteronereceptor negative) and HER2 negative. This type of cancer is more common in women with BRCA1 gene mutations. Similar to Scheme 1, target conjugates 7a-d were prepared in a one-pot three-component reaction of isatins 1c, e-g with equimolar amounts of 2-mercaptobenzimidazole 6 and bromoacetic acid 3 in refluxing glacial acetic acid in the presence of sodium acetate to produce conjugates 7a-d, in good yields; 80-88% (Scheme 2). The latter conjugates prepared through cyclization of 2-((benzimidazol-2-yl)thio)acetic acid 8 into compound 9, with subsequent condensation with isatins 1c, e-g, with low overall yield; 45-61%.
Postulated structures of the newly synthesized conjugates 4a-n and 7a-d were in full agreement with their spectral and elemental analyses data.

In Vitro Antiproliferative Activity
The in vitro antiproliferative activity of the newly synthesized conjugates 4a-n and 7a-d was evaluated against breast cancer MDA-MB-231 and MCF-7 cell lines, following the Crystal Violet (CV) cell cytotoxicity assay [41]. Doxorubicin was included in this assay as a positive control, and Sunitinib was used as a reference drug. The results were expressed as median growth inhibitory concentration (IC 50 ) values that represent the compounds concentrations required to afford a 50% inhibition of cell growth after 48 h of incubation, compared to untreated controls (Table 1).
Breast cancer is composed of multiple subtypes with distinct morphologies and clinical implications. In this study, the sensitivity of two different breast cancer subtypes towards the target conjugates was tested to examine if the target compounds possess selective growth inhibitory activity toward certain subtype. MCF-7 is a Luminal A breast cancer cells which is hormone-receptor positive (estrogen-receptor and/or progesterone-receptor positive), HER2 negative, and has low levels of the protein Ki-67, which helps control how fast cancer cells grow. Luminal A cancers are low-grade, tend to grow slowly and have the best prognosis. Whereas, MDA-MB-231 is a triple-negative/basal-like breast cancer cell line, which is hormone-receptor negative (estrogen-receptor and progesterone-receptor negative) and HER2 negative. This type of cancer is more common in women with BRCA1 gene mutations.  From the obtained results, it was obvious that many of the target conjugates 4a-n and 7a-d have excellent to modest growth inhibitory activity against the tested breast cancer cell lines; MDA-MB-231 and MCF-7. Also, it was noted that MDA-MB-231 cells is more sensitive to the influence of the target conjugates than MCF-7 cells, especially for the thiazolobenzimidazole conjugates 7a-d.
On the other hand, examination of the antiproliferative activity in MCF-7 cells elucidated that conjugate 4m had the best growth inhibitory activity (IC50 = 8.4 ± 0.5), with 2.2-fold decreased activity than doxorubicin (IC50 = 3.80 ± 0.4 μM). Furthermore, hybrid 4g showed good antiproliferative activity against MCF-7 cancer cell line (IC50 = 17.1 ± 1.1). Also, hybrids 4c, 4f, 4n, 7b and 7d were moderately active towards MCF-7 cells with IC50 values of 29.2 ± 2.5, 22.8 ± 2.0, 23.7 ± 1.9, 21.7 ± 1.5 and 27.6 ± 2.4, respectively. From the obtained results, it was obvious that many of the target conjugates 4a-n and 7a-d have excellent to modest growth inhibitory activity against the tested breast cancer cell lines; MDA-MB-231 and MCF-7. Also, it was noted that MDA-MB-231 cells is more sensitive to the influence of the target conjugates than MCF-7 cells, especially for the thiazolobenzimidazole conjugates 7a-d.

In Vitro Cytotoxic Activity towards Nontumorigenic Human WI-38 and MCF-10A Cells
The cytotoxic activity of the active conjugates towards MDA-MB-231 cells (4c-e, 4g, 4l-n and 7a-d) was examined against nontumorigenic human lung fibroblast cell line (WI-38) and human breast epithelial cell line (MCF-10A) to investigate the potential safety of the newly prepared conjugates towards the normal cells. Cultures derived from human fibrocystic mammary tissue (MCF-10A) are nontumorigenic and possess the features of primary cultures of breast tissue including dome formation [42]. The results were expressed as IC 50 values, and selectivity index was calculated ( Table 2). Most of the tested hybrids (4c-e, 4g, 4l-n and 7a-d) exhibited non-significant cytotoxic impact towards WI-38 and MCF-10A cells with IC 50 range 49.1 ± 1.9-353.0 ± 17.1 and 73.1 ± 2.7-250.3 ± 9.8 µM, respectively. Compounds 4m and 7a-d displayed good selectivity index range 6.0-13.9, thereby providing a good safety profile as anticancer agents.

Apoptosis Induction in TNBC MDA-MB-231 Cells
In the current medical era, induction of apoptosis in cancer cells has emerged as one of the most successful strategies for the development of cancer therapies [43][44][45]. As mentioned before, 4m and 7b emerged as the most active hybrids towards MDA-MB-231 cells. Consequently, we examined the ability of conjugates 4m and 7b to provoke apoptosis in MDA-MB-231 cells to determine the principle mechanism for their antiproliferative activity.

Effects on Mitochondrial Apoptosis Pathway Proteins Bcl-2 and Bax
Several members of the human Bcl-2 family of apoptosis-regulating proteins have been discovered, including antiapoptotic proteins such as Bcl-2 protein, and structurally similar pro-apoptotic proteins such as Bax protein, the first anti-death gene identified [44][45][46]. Bcl-2-family proteins regulate all major types of cell death, including apoptosis, necrosis and autophagy, therefore operating as nodal points at the convergence of diverse pathways with broad relevance to oncology.
In our study, we investigated the effect of conjugates 4m and 7b towards the level of the antiapoptotic Bcl2 and the level of the pro-apoptotic Bax, (Table 3, Figure 2). As displayed in Table 2, hybrid 4m induced the protein expression of Bax with 11.1 folds of the control while 7.3 folds were evaluated for hybrid 7b. On the other hand, treatment of MDA-MB-231 cancer cells with conjugates 4m and 7b significantly downregulated the expression levels of the antiapoptotic protein Bcl-2 by 66.6 and 55.1%, respectively, compared to the control. The ratio between Bax (apoptosis inducer) and Bcl-2 (apoptosis suppressor) is an important parameter and gave more profound insight into the apoptotic activity of the compounds as a determining factor for cell fate regulation, and a key indicator of therapeutic response to chemotherapy [40]. Calculating the values for Bax/Bcl2 ratio indicated that conjugates 4m and 7b boosted the Bax/Bcl-2 ratio by 33-and 16-folds, respectively, compared to the control. Conclusively, the ability of conjugates 4m and 7b to up-regulate Bax level, down-regulate Bcl2 level while significantly boosting the Bax/Bcl2 ratio proves their effectiveness as apoptosis inducers.

Effects on the Levels of Active Caspase-3 (Key Executor of Apoptosis)
The down-regulation of the antiapoptotic Bcl-2 leads to an increase in the levels of free proapoptotic Bax which in turn accumulates at the inner mitochondrial membrane forming channels, altering membrane permeability. Accordingly, the apoptotic factors can leak into the cytoplasm resulting in the caspases cascade activation. As a key executioner protease, caspase-3 is activated by upstream initiator caspases as caspase-9 [41]. Thence, the elevated Bax/Bcl-2 ratios obtained herein triggered the examination of the protein expression levels of active caspases-3. Treatment of TNBC MDA-MB-231 cells with conjugates 4m and 7b led to a significant increase in the active caspase-3 levels by 6.4 and 4.9 folds, respectively, compared to control (Table 3, Figure 3).  The ratio between Bax (apoptosis inducer) and Bcl-2 (apoptosis suppressor) is an important parameter and gave more profound insight into the apoptotic activity of the compounds as a determining factor for cell fate regulation, and a key indicator of therapeutic response to chemotherapy [47]. Calculating the values for Bax/Bcl2 ratio indicated that conjugates 4m and 7b boosted the Bax/Bcl-2 ratio by 33-and 16-folds, respectively, compared to the control. Conclusively, the ability of conjugates 4m and 7b to up-regulate Bax level, down-regulate Bcl2 level while significantly boosting the Bax/Bcl2 ratio proves their effectiveness as apoptosis inducers.

Effects on the Levels of Active Caspase-3 (Key Executor of Apoptosis)
The down-regulation of the antiapoptotic Bcl-2 leads to an increase in the levels of free pro-apoptotic Bax which in turn accumulates at the inner mitochondrial membrane forming channels, altering membrane permeability. Accordingly, the apoptotic factors can leak into the cytoplasm resulting in the caspases cascade activation. As a key executioner protease, caspase-3 is activated by upstream initiator caspases as caspase-9 [48]. Thence, the elevated Bax/Bcl-2 ratios obtained herein triggered the examination of the protein expression levels of active caspases-3. Treatment of TNBC MDA-MB-231 cells with conjugates 4m and 7b led to a significant increase in the active caspase-3 levels by 6.4 and 4.9 folds, respectively, compared to control (Table 3, Figure 3).    As displayed in Figure 4, exposure of MDA-MB-231 cells to hybrid 4m resulted in a significant rise in the percentage of cells at Sub-G 1 by 4-folds, with concurrent significant arrest in the G 2 -M phase by 2.5-folds compared to control. Arrest of G 2 -M phase and alteration of the Sub-G 1 phase were significant remarks for hybrid 4m to prompt apoptosis in TNBC MDA-MB-231 cells.

Annexin V-FITC Apoptosis Assay
Annexin V-based flow cytometry assay elucidates either that cell death is achieved via programmed apoptosis or nonspecific necrosis [49]. The apoptotic impact of conjugate 4m was further proved by Annexin V-FITC/PI (AV/PI) dual staining analysis to examine the occurrence of phosphatidylserine externalization ( Figure 5). Flow cytometric analysis revealed that treatment of TNBC MDA-MB-231 cells with conjugate 4m resulted in a significant elevation in the percent of annexin V-FITC-positive apoptotic cells, including both the early and late apoptotic phases (UR + LR), from 1.92% to 11.43% which represents about 6-folds increase as compared with the control. Annexin V-based flow cytometry assay elucidates either that cell death is achieved via programmed apoptosis or nonspecific necrosis [49]. The apoptotic impact of conjugate 4m was further proved by Annexin V-FITC/PI (AV/PI) dual staining analysis to examine the occurrence of phosphatidylserine externalization ( Figure 5). Flow cytometric analysis revealed that treatment of TNBC MDA-MB-231 cells with conjugate 4m resulted in a significant elevation in the percent of annexin V-FITC-positive apoptotic cells, including both the early and late apoptotic phases (UR + LR), from 1.92% to 11.43% which represents about 6-folds increase as compared with the control.
In conclusion, enhanced expression of the pro-apoptotic protein Bax in addition to the reduced expression of the antiapoptotic protein Bcl-2, as well as the up-regulated active caspase-3 levels together with a harmonized increase in the Bax/Bcl-2 ratio, suggests that the antiproliferative effect of the target conjugates might be attributed, at least in part, to the induction of the intrinsic apoptotic mitochondrial pathway.
Collectively, these data have highlighted conjugate 4m as an ideal lead compound for further optimization and development as an effective anti-breast cancer therapy.

In Silico ADME Profiling
In order to examine drug-like physicochemical and pharmacokinetics properties of the target conjugates, certain ADME descriptors for the prepared conjugates (4a-n and 7a-d) were assessed through a computer-aided theoretical kinetic study using Discovery Studio software (Accelrys, San Diego, CA, USA), [50,51] Table 4.
The tested conjugates exhibited low aqueous solubility levels, except compounds 7b and 7c, which showed better solubility levels. The tested conjugates displayed good levels of human intestinal absorption, except compounds 4i-k, 4m and 4n, which showed moderate intestinal In conclusion, enhanced expression of the pro-apoptotic protein Bax in addition to the reduced expression of the antiapoptotic protein Bcl-2, as well as the up-regulated active caspase-3 levels together with a harmonized increase in the Bax/Bcl-2 ratio, suggests that the antiproliferative effect of the target conjugates might be attributed, at least in part, to the induction of the intrinsic apoptotic mitochondrial pathway.
Collectively, these data have highlighted conjugate 4m as an ideal lead compound for further optimization and development as an effective anti-breast cancer therapy.

In Silico ADME Profiling
In order to examine drug-like physicochemical and pharmacokinetics properties of the target conjugates, certain ADME descriptors for the prepared conjugates (4a-n and 7a-d) were assessed through a computer-aided theoretical kinetic study using Discovery Studio software (Accelrys, San Diego, CA, USA), [50,51] Table 4.  The tested conjugates exhibited low aqueous solubility levels, except compounds 7b and 7c, which showed better solubility levels. The tested conjugates displayed good levels of human intestinal absorption, except compounds 4i-k, 4m and 4n, which showed moderate intestinal absorption levels. On the other hand, the investigated conjugates were predicted to be CYP2D6 non-inhibitors, Table 4.

Conclusions
In summary, herein we report the synthesis of two novel series of thiazolidinone-isatin conjugates (4a-n) and thiazolo[3,2-a]benzimidazolone-isatin conjugates (7a-d). All the prepared conjugates were evaluated for their antiproliferative activity towards breast cancer cell lines MDA-MB-231, and MCF-7. Conjugates 4m and 7b were the most active members towards MDA-MB-231 (IC 50 = 7.6 ± 0.51 and 13.2 ± 1.07 µM, respectively). Conjugates 4d and 4m emerged as the most active derivatives against MCF-7 cells with IC 50 values of 17.1 ± 1.14 and 8.4 ± 0.47 µM, respectively. Conjugates 4m and 7b induced apoptosis in MDA-MB-231 cells, confirmed by the up-regulation of the Bax, downregulation of the Bcl-2 and boosting caspase-3 levels. Compound 4m arrested the G 2 -M phase by 2.5-folds with concurrent significant increase in the percentage of cells at Sub-G 1 by 4-folds, compared to control. Also, 4m showed significant increase in the percentage of annexin V-FITC positive apoptotic cells from 1.92% to 11.43%, approximately a 6-folds increase as compared with the control. Finally, the cytotoxic activity of the active conjugates (4c-e, 4g, 4l-n and 7a-d) were tested towards nontumorigenic human lung fibroblast WI-38 cell line and breast MCF-10A cell line, and compounds 4m and 7a-d displayed good selectivity index range 6.0-13.9, thereby providing a good safety profile as anticancer agents.

General
Using a Stuart melting point apparatus, melting points were measured and uncorrected. Using Schimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan), Infrared (IR) Spectra were recorded as KBr disks. NMR Spectra were recorded on a Bruker spectrophotometer (Bruker, Karlsruhe, Germany). 1 H spectrum was run at 400 MHz and 13 C spectrum was run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d 6 ). All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were done at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Egypt. Mass spectral data were given by a GCMS-QP1000 EX spectrometer (Shimadzu, Kyoto, Japan) at 70 e.V. High-resolution mass spectra were obtained using a Bruker MicroTOF spectrometer (Bruker Daltonics, Bremen, Germany). Compounds 5a,b [52], 8 [53], and 9 [54] were previously prepared.

Route A:
A mixture of isatins 1a-g (1 mmoL) with an equimolar amount of N,N -diphenyl/(p-tolyl)thiourea 2a,b (1 mmoL) and bromoacetic acid 3 (0.14 g, 1 mmoL) in glacial acetic acid (10 mL) in the presence of sodium acetate (0.16 gm, 2 mmoL), was heated under reflux for 3 h. The formed solid was filtered off while hot, washed with hot ethanol, dried and recrystallized from DMF to furnish the target hybrids 4a-n.

Route B:
To a hot solution of N,N -diphenyl/(p-tolyl)thiourea 2a,b (1 mmoL) and sodium acetate (0.16 gm, 2 mmoL) in glacial acetic acid (10 mL), bromoacetic acid 3 (0.14 gm, 1 mmoL) was added. The reaction mixture was refluxed for 4 h then allowed to cool to room temperature. The obtained solid was filtered off, washed with water and dried to give intermediates 3a,b. The later intermediates were added, without further purification, to a stirred solution of the appropriate isatin derivative 1a-g (1 mmoL) and sodium acetate (0.16 gm, 2 mmoL) in glacial acetic acid (5 mL) then the reaction mixture was refluxed for 3 h. The formed solid was filtered off, washed with ethanol and recrystallized from DMF to give compounds 4a-n.

Antiproliferative Activity Against Breast Cancer Cell Lines
Breast cancer cell lines MDA-MB-231 and MCF-7 cells were obtained from VACSERA Tissue Culture Unit. The cells were propagated in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine (2.5 mM), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (10 mM) and 50 µg/mL gentamycin. All cells were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and were sub-cultured two times a week. Cytotoxicity was determined following the Crystal Violet (CV) cell cytotoxicity assay [41].
Test samples were dissolved in DMSO and kept at a stock concentration of 100 mM. Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeding was done at a density of 3000 cells/well in 96-well plates. After 24 h, the monolayer cultured cells were washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 mL from 10 different dilutions of the test compounds as well as Doxorubicin was used as a standard compound in fresh maintenance medium and incubated at 37 • C for 24 h. Control wells were exposed to the same concentration of vehicle, DMSO (1%) used in the highest concentration of the test samples. Cell viability was >99% compared to untreated cells. The number of the surviving cells was determined by staining the cells with crystal violet followed by cell lysing using 33% glacial acetic acid. The absorbance was measured at 490 nm using ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA) after gentle shaking. The absorbance values from untreated cells were considered as 100% proliferation.
The number of viable cells was determined using ELISA reader as previously mentioned and the percentage of viability was calculated as [1−(ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the test sample and ODc is the mean optical density of untreated cells. The 50% inhibitory concentration (IC 50 ) was estimated from graphic plots of the dose response curve for each conc. using GraphPad Prism software, Version 5 (San Diego, CA, USA). The data presented are the mean of at least three separate experiments.

In Vitro Cytotoxic Activity towards Nontumorigenic Human WI-38 and MCF-10A Cells
The cytotoxic activity of the active conjugates towards nontumorigenic human lung fibroblast cell line (WI-38) and human breast epithelial cell line (MCF-10A) was determined using Crystal violet (CV) cell cytotoxicity assay according to the previously published procedures [42,55].

ELISA Immunoassay
The levels of the apoptotic markers (Bax, caspase-3) as well as the antiapoptotic marker Bcl-2 were determined using ELISA colorimetric kits per the manufacturer's instructions, as reported earlier [41]. MDA-MB-231 cells were cultured as a monolayer in T-25 flasks and were seeded to attain 30% confluency prior to treatment. Cells were then treated separately with compounds 4m and 7b at their IC 50 concentrations for 48 h. At the end of treatment, cells were collected via trypsinization and centrifuged at 10,000 rpm. The pellet was then rinsed with PBS and lysed in RIPA lysis buffer at 4 • C for 45 min, then centrifuged at 14,000 rpm for 20 min to remove the cellular debris. Lysates were then collected and stored at −80 • C for later protein determination using Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer's recommendations.
The cell lysate was diluted 10 times, and 100 µL (50 mg protein) was added to the wells of four separate microtiter plates for the four ELISA kits that were pre-coated with primary antibodies specific to Bax, Bcl-2 and caspase-3 proteins, respectively. A secondary biotin-linked antibody specific to the protein captured by the primary antibody was further added to bind the captured protein, forming a "sandwich" of specific antibodies around the desired protein in the cell lysate. The streptavidin-HRP complex was then used to bind the biotin-linked secondary antibody through its streptavidin portion. The HRP domain reacted with the added TMB substrate to form a colored product that measured at 450 nm by a plate reader (ChroMate-4300, Orlando, FL, USA) after the reaction was terminated via the addition of stop solution.

Cell Cycle Analysis
TNBC MDA-MB-231 cells were treated with compound 4m for 24 h (at its IC 50 concentration), and then cells were washed twice with ice-cold phosphate buffered saline (PBS). Subsequently, the treated cells were collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with 100 µg/mL RNase, stained with 40 µg/mL PI, and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, BD, Franklin Lakes, NJ, USA). The cell cycle distributions were calculated using CellQuest software 5.1 (Becton Dickinson) [35,49].

Annexin V-FITC Apoptosis Assay
Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions, as reported earlier [35,49]. MDA-MB-231 cells were cultured to a monolayer then treated with conjugate 4m at its IC 50 concentration as described earlier. Cells were then harvested via trypsinization, and rinsed twice in PBS followed by binding buffer. Moreover, cells were re-suspended in 100 µL of binding buffer with the addition of 1 µL of FITC-Annexin V (Becton Dickinson BD Pharmingen TM , Heidelberg, Germany) followed by an incubation period of 30 min at 4 • C. Cells were then rinsed in binding buffer and resuspended in 150 µL of binding buffer with the addition of 1 µL of DAPI (1 µg/µL in PBS) (Invitrogen, Life Technologies, Darmstadt, Germany). Cells were then analyzed using the flow cytometer BD FACS Canto II (BD Biosciences, San Jose, CA, USA) and the results were interpreted with FlowJo7.6.4 software (Tree Star, Ashland, OR, USA).

Statistical Analysis
Data are presented as means ± S.D. Individual groups were compared using the two-tailed independent Student's t-test. Multiple group comparisons were carried out using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for post-hoc analysis. Statistical significance was accepted at a level of p < 0.05. All statistical analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc., La Jolla, CA, USA). Graphs were sketched using GraphPad Prism software, version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA).
Author Contributions: Wagdy M. Eldehna and Hatem A. Abdel-Aziz have formulated the research idea; Wagdy M. Eldehna, Mohamed H. El-Naggar, Hadia Almahli and Mohaned Fares carried out the experimental, interpreted the data and discussion, and prepared the manuscript; Mahmoud M. Elaasser performed the cytotoxicity assay; Amr Elgez carried out the biological work and discussed its results. All the authors reviewed and approved the manuscript.
Funding: This research received no external funding.