One-pot three-component synthesis of novel spirooxindoles with potential cytotoxic activity against triple-negative breast cancer MDA-MB-231 cells

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited treatment options due to its heterogeneity and the lack of well-defined molecular targets. In our endeavour towards the development of novel anti-TNBC agents, herein we report a one-pot three-component synthesis of novel spirooxindoles 6a–p, and evaluation of their potential anti-proliferative activity towards TNBC MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i emerged as the most potent analogues with IC50 = 6.70, 6.40 and 6.70 µM, respectively. Compounds 6a and 6e induced apoptosis in MDA-MB-231 cells, as evidenced by the up-regulation of the Bax and down-regulation of the Bcl-2, besides boosting caspase-3 levels. Additionally, 6e displayed significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34 to 44%. Furthermore, spirooxindoles 6e and 6i displayed good inhibitory activity against EGFR (IC50 = 120 and 150 nM, respectively). Collectively, these data demonstrated that 6e might be a potential lead compound for the development of effective anti-TNBC agents.


Introduction
Breast cancer is the fifth most common cause of death in women world-wide. In fact, it represents about 12% of all new cancer cases and 25% of all cancers in women with nearly 1.7 million new cases diagnosed in 2012 1 . Routine breast cancer case showed an expression of three distinctive receptors which are oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 receptor (Her2). About 15-20% of the women diagnosed with breast cancer lacked in overexpression of these three receptors (ER, PR and Her2) 2 . This case is known as triplenegative breast cancer (TNBC) and it does not respond to normal protocol for the treatment of normal breast cancer. As a result, TNBC is responsible for high incident among the total percent of deaths regarded to breast cancer 3,4 . Unfortunately, TNBC patients were subjected to traditional cytotoxic therapies during their treatment protocol as till now there is no approved targeted cytotoxic drug for TNBC 5 .
Based on the aforementioned findings and as a continuation of our research program on the development of novel effective anti-TNBC candidates [12][13][14] , it was thought worthwhile to extend our investigations to probe for novel spirooxindoles possessing promising anti-proliferative activity towards TNBC. In the present work we report the synthesis of a novel series of spirooxindoles 6a-p, Figure 1, and their in vitro efficacy against the proliferation of the aggressive TNBC MDA-MB-231 cell line. In addition, spirooxindoles were further investigated regarding their potential apoptotic induction and their effects on cell cycle progression in the MDA-MB-231 cancer cells to acquire a perception for the mechanism of the antitumor activity of target spirooxindoles. Since the epidermal growth factor receptor (EGFR) is frequently overexpressed in TNBC 15,16 , the most potent spirooxindoles in this study will be assayed for their potential inhibitory activity towards EGFR.

Chemistry
Melting points were measured with a Stuart melting point apparatus and were uncorrected. Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR 8400 S spectrophotometer. Mass spectral data are given by GCMS-QP1000 EX spectrometer at 70 e.V. NMR Spectra were recorded on a Varian Mercury NMR spectrometer. 1 H spectrum was run at 400 MHz and 13 C spectrum was run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts are expressed in values (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. Some representative NMR charts are provided in the Supplementary material.

Synthesis of 3-oxo-3-phenylpropanenitriles (3)
To ethyl benzoates 2a-d (100 mmol) and acetonitrile (4.1 ml, 100 mol) in dry benzene (250 ml) and dimethylformamide (10 ml), sodium hydride (4.8 g, 60%) was added. The reaction mixture was heated under reflux for 4 h then allowed to cool to room temperature. The solid formed was collected by filtration, washed with ether and dried. This solid was dissolved in water and then neutralized with conc. HCl to pH 7. The precipitated product was collected by filtration, washed with water and dried. Recrystallization from ethanol gave compounds 3 17 .

Biological evaluation
In vitro anti-proliferative activity assay Synthetic spirooxindoles 6a-p were tested for their anti-proliferative potency on TNBC cells (MDA-MB-231). Cells lines were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin sulfate. Cells were sub-cultured with trypsine/EDTA solution, counted with haemocytometer and plated onto 96-well plates (5000 cells/well) and left overnight to form a semi-confluent monolayer. We employed a modified method utilizing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye (Carbosynth, UK) that is based on the reduction of the dye by mitochondrial dehydrogenases of metabolically active cells to insoluble formazan crystals 18,19 . Briefly, cell monolayers were treated in quadrates with vehicle (DMSO, 0.1% v/v), test samples or Adriamycin as positive control for an exposure time of 48 h. At the end of exposure, MTT solution in PBS (5 mg/ml) was then added to all wells including no cell blank and left to incubate for 90 min 20 . The formation of formazan crystals were visually confirmed using phase contract microscopy. DMSO (100 ml/well) was added to dissolve the formazan crystals with shaking for 10 min after which the absorbance was read at 590 nm against no cell blanks on a FLuo Star Optima microplate reader (BMG Technologies, Germany). Cell proliferation was calculated comparing the OD values of the DMSO control wells and those of the samples represented as % proliferation to the control. Doseresponse experiment was performed on samples producing !50% loss of cell proliferation using five serial 2-fold dilutions (50, 25, 12.5, 6.25 and 3.125 mM) of the sample. IC 50 values (concentration of sample causing 50% loss of cell proliferation of the vehicle control) were calculated using non-linear regression curve fitting of the dose response plots on GraphPad Prism V.6.0 software (Graphpad Inc, San Diego, CA). Assessment of morphological changes of MDA-MB-231 cells following treatment with the most active hits were performed using phase contrast inverted microscope (Carl Zeiss Microscopy GmbH, Gottingen, Germany) and photomicrographs were taken using digital camera.
In vitro cytotoxic activity WI-38 cells (human lung fibroblast normal cell line) WI-38 cells (normal breast cells), were obtained from American Type Culture Collection. The cells were propagated in DMEM supplemented with 10% heat-inactivated FBS (Hyclone), 10 lg/ml of insulin (Sigma), and 1% penicillin-streptomycin. All of the other chemicals and reagents were from Sigma, or Invitrogen. Cytotoxicity was determined using MTT assay following a reported procedure 18 . The 50% inhibitory concentration (IC 50 ) was estimated from graphic plots of the dose response curve for each conc. using Graphpad Prism software (San Diego, CA).

Assay of the apoptosis markers (Bax, caspase-3 and Bcl-2) levels
The levels of the apoptotic markers (Bax, caspase-3) as well as the anti-apoptotic marker (Bcl-2) were assessed using ELISA colorimetric kits per the manufacturer's protocol and referring to reported instructions 21,22 .

Cell cycle analysis
The MDA-MB-231 cells were treated with 6.40 lM of compound 6e for 24 h. 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 0.1 mg/mL RNase, stained with 40 mg/mL PI, and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, BD, USA). The cell cycle distributions were calculated using CellQuest software (Becton Dickinson).
Annexin V-FITC apoptosis assay Phosphatidylserine externalization was measured using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions, as reported earlier 22 . MDA-MB-231cells were treated with 6e at defined concentrations for 24 h.
EGFR kinase ELISA assay EGFR enzyme inhibition was measured using a BPS Biosciences Colorimetric 96-well EGFR assay kit (catalog no. 40321), according to the manufacturer's instructions. Percent inhibition was calculated by the comparison of compounds treated to control incubations. The concentration of the test compound causing 50% inhibition (IC 50 ) was calculated from the concentration-inhibition response curve (triplicate determinations) and the data were compared with Erlotinib as standard EGFR inhibitor.  absorption bands of the (NH) groups in the region 3205-3387 cm À1 , in addition to the carbonyl bands in the region 1709-1716 cm À1 and CN bands in the region 2202-2206 cm À1 . 1 H NMR spectra of 6a-p showed two singlet D 2 O-exchangeable signals attributable to two (NH) protons at range d 10.09-10.52 and 10.45-10.89 ppm. In addition, the methoxy (-OCH 3 ) protons of compounds 6c, 6 g, 6k and 6 m-p were displayed as singlet signals in the range d 3.68-3.81 ppm, whereas, the methyl (-CH 3 ) protons of compounds 6 b, 6f, 6j and 6n appeared as singlet signals around d 2.35 ppm. Moreover, 13 C NMR spectra of spirooxindoles 6a-p showed signals resonating in the range d 177.72-178.10 ppm attributable for the carbon of the carbonyl (C¼O) groups, whereas the carbons of the methoxy (-OCH 3 ) groups of compounds 6 g, 6k and 6 m-o and carbons of the methyl (-CH 3 ) groups of compounds 6 b, 6j and 6n appeared as two signals around d 56.0 and 21.4 ppm, respectively.

Biological evaluation
In vitro anti-proliferative activity The in vitro anti-proliferative activity of the newly synthesized spirooxindoles 6a-p was examined against TNBC MDA-MB-231 cells. This assay was performed utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as described by Mosmann 18 . Adriamycin was included in this assay 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).
As shown in Figure 2, morphological assessment using bright field phase contrast microscopy revealed adverse effects on cell morphology such as monolayer disruption in addition to cell shrinkage and rounding, as indicated by arrows.

Structure activity relationship (SAR)
Based on the aforementioned results of the biological anti-proliferative activity assay, important structure activity relationships could be deduced. Firstly, we investigated the impact of the C-5 substitution of the indoline moiety. The abolished activity of 5methoxyindoline derivatives 6n-p along with the decreased IC 50 value of 6m, with incorporated unsubstituted phenyl group, (17.50 mM) than that of its corresponding members 6a, 6e and 6i, with unsubstituted, 5-chloro substituted and 5-bromo substituted indoline moiety, (6.70, 6.40 and 6.70 mM, respectively) indicated that unsubstitution or C-5 substitution of the indoline moiety with electron withdrawing groups (EWGs), as 5-Cl or 5-Br, is more beneficial than incorporation of electron-donating substituents, as 5-OCH 3 , to the growth inhibitory activity against MDA-MB-231 cells.
Finally, we can deduce that the substitution pattern over the synthesized spirooxindoles is a crucial element for the anti-proliferative activity towards TNBC MDA-MB-231 cell line. Incorporation of unsubstituted or 5-EWG-substituted indoline moiety along with unsubstituted phenyl group at C-6 of pyrazolo [3,4-b]pyridine moiety greatly enhances the growth inhibitory activity of the target spirooxindoles 6a-p.
In vitro cytotoxicity towards human normal WI-38 cells Three potent compounds 6a, 6e and 6i were evaluated for their ability to induce cytotoxic effect against human normal lung fibroblast cell line (WI-38 cells), to investigate their safety adopting the MTT assay procedures 18 . The results, expressed as (IC 50 ) values, and the calculated selectivity index were displayed in Table 2.
The tested spirooxindoles 6a, 6e and 6i showed non-significant cytotoxic effect with IC 50 values of 78.1, 43.2 and 39.3 mM, respectively, thereby providing a high-safety profile as anti-proliferative agents with good selectivity index rang (11.7, 6.8 and 5.9 respectively).

Apoptosis induction in TNBC MDA-MB-231 cells
Apoptosis is a programed routine that proves to be an essential physiological process for tissue development, immune response, redundant cells clearance and homeostasis by which cells signal their own termination. Accordingly, cellular integrity is conserved by this finely tuned, self-automated death 23 . Consequently, the success of cancer cells to proliferate unconditionally is allied to its ability to halt apoptosis. Thus, targeting apoptosis induction is a successful strategy for combating tumour progression.
To further elucidate the mechanism of cell death induced by the target spirooxindoles and as a part of our ongoing efforts to develop novel pro-apoptotic agents 21,22,[24][25][26] , we evaluated ability of compounds 6a and 6e to provoke apoptosis in MDA-MB-231 cells through determination of the hallmark parameters of apoptosis.
Effects on the levels of active caspase-3 Caspases, a family of cysteineaspartic proteases, are the crucial apoptosis mediators that provide essential links in cell regulatory networks controlling cell death. Caspase-3 is the key executioner caspase which modifies proteins ultimately responsible for apoptosis 27 . Accordingly, the effect of spirooxindoles 6a and 6e on the level of caspase 3 was evaluated, to give insight to the pro-apoptotic effect of the prepared spirooxindoles (Table 3).
Results in Table 3 showed that treatment of MDA-MB-231 cells with compounds 6a and 6e resulted in a significant elevation in the level of active caspase-3 by about 31.5 and 36.5 folds, respectively, compared to control.
Effects on mitochondrial apoptosis pathway (Bcl-2 family) proteins Bcl-2 family comprises a group of crucial regulatory factors in apoptosis that finely tune the apoptotic switch on/off mechanism. Based on their functional and structural criteria, the members are divided into two major classes; group I proteins that are antiapoptotic and group II proteins that are apoptotic. Group I antiapoptotic proteins exert their function by inhibiting group II apoptotic proteins through simply binding to them. Group I proteins bind selectively to the active conformations of group II proteins to prevent them from being inserted into the mitochondria and thus cease the release of pro-apoptotic factors such as cytochrome c, ultimately aborting apoptosis [25]. Thus, the inhibition of group I proteins and/or the activation of group II proteins can successfully induce apoptosis. Herein, we evaluated the impact of compounds 6a and 6e on the level of Bcl2, as a representative group I member, and the level of Bax, as a representative group II member ( Table 3).
As presented in Table 3 compound 6a induced the protein expression of Bax with 506.8 folds of the control while 442.1 folds were recorded with compound 6e. Parallel to this, the protein expression of the antiapoptotic marker Bcl-2 was down-regulated to 14.7% compared to that of the basal level in the control by compound 6a while compound 6e produced down-Cell-cycle analysis Antiproliferative agents abort cell growth by arresting its proliferation at certain well-known checkpoints. Distinguish cells in various phases of cell cycle can be detected upon treatment of cancer cells with anticancer agents 28 . Compound 6e was investigated for its activity to disrupt the cell cycle of MDA-MB-231 cancer cell lines. This effect was illustrated by DNA flow cytometric analysis which MDA-MB-231 cells was treated with compound 6e at concentration equals to the IC 50 for 24 h. Figure 3 showed that compound 6e expressed significant decrease in the G0-G1 phase by approximately 0.5 folds related to the control. Compound 6e displayed no significant change in the S phase while G2-M phase was arrested by 2.65 folds with 19.1% with respect to control (7.2%). Alteration of the Pre-G phase and arrest of G2-M phase were significant remarks for compound 6e to induce apoptosis.
Annexin V-FITC apoptosis assay Externalization of the phospholipid phosphatidylserine at the cell membrane is a one of the well-recognized hallmarks of cells going into apoptosis 17,29 . In our study, the apoptotic effect of compound 6e was further assessed by Annexin V-FITC/PI (AV/PI) dual staining assay to examine the occurrence of phosphatidylserine externalization and also to comprehend whether cell death is due to physiological apoptosis or nonspecific necrosis (Figure 4).
Flow cytometric analysis revealed that MDA-MB-231 cells treated with compound 6e displayed a significant increase in the percent of annexin V-FITC positive apoptotic cells (UR þ LR) from 1.34% to 44% which comprises about 32.8 folds compared to control.
In conclusion, the enhanced expression of the pro-apoptotic protein Bax and the reduced expression of the anti-apoptotic protein Bcl-2 as well as the up-regulated active caspase-3 level together with a harmonized increase in the Bax/Bcl-2 ratio, highlighted that the anti-proliferative activity of the target spirooxindoles 6 might be attributed, at least in part, to the induction of the intrinsic apoptotic mitochondrial pathway.
In vitro EGFR kinase ELISA assay On account of its overexpression in a significant number of TNBC, epidermal growth factor receptor (EGFR) emerged as an attractive target for developing effective therapeutic strategies for treatment of TNBC patients [28][29][30] . In this study the most potent anti-proliferative spirooxindoles 6a, 6d, 6e and 6i-k were selected to evaluate their potential inhibitory activity against EGFR by use of a colorimetric Enzyme-Linked Immunosorbent Assay (ELISA). Erlotinib, a clinically used EGFR inhibitor, was taken as the reference drug. The results were reported as a 50% inhibition concentration values (IC 50 ) which determined as triplicate determinations from the standard curve and summarized in Table 4.
Results revealed that the tested compounds exhibited EGFR inhibitory activity with IC 50 values ranging from 0.12 to 0.51 lM. Compound 6i emerged as the most potent EGFR inhibitor in this study that showed comparable potency (IC 50 ¼ 0.12 ± 0.01 lM) to the reference drug Erlotinib (IC 50 ¼ 0.11 ± 0.01 lM). Besides, compound 6e displayed good activity (IC 50 ¼ 0.15 ± 0.02 lM).

Conclusions
In summary, we have synthesized a novel series of sixteen spirooxindoles 6a-p through a one-pot three-component reaction, with the prime aim of developing potent anti-TNBC agents. All the newly synthesized spirooxindoles 6a-p was evaluated for their in vitro anti-proliferative activity towards TNBC MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i were the most potent members against MDA-MB-231 cells with IC 50 ¼ 6.70, 6.40 and 6.70 mM, respectively. Besides, compounds 6d, 6f, 6h, 6j, 6k and 6m were moderately active against MDA-MB-231 cells with IC 50 rang (12.00 -18.20 mM). Moreover, the cytotoxicity of the active counterparts 6a, 6e and 6i was examined against normal human cell line (WI-38 lung fibroblast) where none of them displayed significant cytotoxic effect, thereby providing a good safety profile. Subsequently, 6a and 6e were further estimated for their apoptosis induction potential. Both proved to induce apoptosis, which evidenced via the reduced expression of the anti-apoptotic protein Bcl-2 in addition to the enhanced expression of the pro-apoptotic protein Bax as well as the up-regulated active caspase-3 level. Moreover, 6e displayed a significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34% to 44% which comprises about 32.8 folds compared to control. As the EGFR is frequently overexpressed in TNBC, six potent spirooxindoles was assayed for their potential inhibitory activity towards EGFR. Compounds 6e and 6i displayed potent inhibitory activity against EGFR with IC 50 values of 120 and 150 nM, respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. RG-1436-038.