Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological evaluation and in silico studies

Abstract As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a–c, 9 and 10a–e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitumor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results were obtained with N-propylindole –5-methylisatin hybrid 8a which displayed broad spectrum anti-proliferative action with efficient sub-panel GI50 (MG-MID) range from 1.33 to 4.23 µM, and promising full-panel GI50 (MG-MID) equals 3.10 µM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC50= 0.85 ± 0.03 and 0.46 ± 0.02 µM, respectively). Interestingly, molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.


Introduction
In the current medical era, cancer is considered as a major public health problem worldwide and one of the most leading causes of death throughout the world. So, development of more effective new drugs for management of different human malignancies is a major requirement. The expanded knowledge for the functional relationship between the different molecules which constitute the cell cycle and checkpoint pathways furnished novel promising strategies for the management of tumours.
Cyclin-dependent kinases (CDKs) are considered as crucial factors that affect diverse key transitions in cell cycle for the cancer cell, in addition to regulation of apoptosis, transcription and exocytosis, therefore therapeutic strategies based on inhibition of CDKs stand out as a promising opportunity for anticancer drug discovery and an efficient approach for management of different human malignancies. On the other hand, apoptosis, an automatic cancer cell death, was found as an important consequence of CDKs inhibition and can be assessed by cell cycle arrest at low concentration or even mitochondrial damage at high concentration 1 . This fact was discovered from previous studies on CDKs inhibitors as Roscovitine and Purvalanol and their effect on three different prostate cancer cell lines 2 . In addition, the link between CDKs and apoptosis was revealed in the investigational research of the effect of Ibulocydine, a prodrug CDK inhibitor, on hepatocellular carcinoma 3 .
Isatin moiety served as a contributor in various CDKs inhibitors having an apoptotic effect. For example, isatin dimers, such as Indirubin-3 0 -oxime (Figure 1), revealed a potent inhibition against CDK1, CDK2, and CDK5 with IC 50 s ¼ 180 nM, 500 nM and 250 nM, respectively 4 . In addition to its effect against CDKs, it possessed an apoptotic effect by disruption of cell cycle phases through arresting G2/M phase 5 . Indirubin-3 0 -oxime is regarded as a lead compound. It is especially known in Chinese traditional medicine, and its other derivatives as 5-Bromoindirun and Indirubin-5-sulfonate proved to be potent CKD inhibitors against CDK2 with IC 50 s ¼ 1 and 0.5 mM, respectively. Moreover, they were co-crystallized with CDK2 (2BHE and 1E9H, respectively) ( Figure 1) 6,7 . This dimeric scaffold is initiative for the design of other derivatives with a significant activity against CDKs as reported 8,9 . Other isatin derivatives decorated with 3-substitution expressed potent inhibition for CDK2 as shown in SU9516 which inhibits CDK2 with IC 50 ¼ 25 nM (Figure 1) 10 . Moreover, compound (I) is another isatin derivative with hydrazino linker at position 3 (CDK2; IC 50 ¼ 60 nM) that was co-crystallized with its target and submitted to protein data bank (1VFT). Several modifications on compound (I) were performed to get compound (II) which inhibits CDK2 in sub-nanomolar range (IC 50 ¼ 0.54 nM) (Figure 1) 11 .
In last few years, our research team has developed diverse small molecules based on the isatin scaffold as efficient anticancer agents (structures III-V 12-14 , Figure 1) with diverse cellular and enzymatic targets; for example triggering of apoptosis in several tumour cell lines 15,16 , inhibition of tumour-linked human carbonic anhydrase isoform IX 17,18 , and inhibition of VEGFR-2 kinase 19 . In 2018, we have reported the conjugation between the isatin and indole moieties via methylenehydrazono spacer (HC¼N-N¼) to develop new three different series of [(3-indolylmethylene)hydrazono]indolinone derivatives as anticancer agents with promising pro-apoptotic activity 20 . As concluded from SAR analysis for this study 20 , hybridisation of N-propyl indole moiety with N-unsubstituted isatin moiety (compound VI, Figure 1) achieved the most effective anticancer activity.
Inspired by these findings and in connection with our previous research work, it is thought advantageous to broaden our investigations to probe new N-alkylindole-isatin conjugates 7, 8a-c, 9 and 10a-e exerting promising anticancer and pro-apoptotic actions. Utilising the SAR outputs from the previous study 20 , our design here is based on appending four alkyl groups with different length (ethyl 7, n-propyl 8a-c), bulkiness (isopropyl 9) and unsaturation (allyl 10a-e) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates.
All the synthesised conjugates were examined for their cytotoxicity towards a list of three different human cancer cell lines HCT-116 (Colon), A-549 (NSCLC), and MDA-MB-231 (Breast) utilising SRB assay. Furthermore, seven hybrids (8a-c, 9, 10a, 10c and 10d) were screened for their possible in vitro anticancer action in accordance with US-NCI protocol, then, 8a was furthermore chosen for testing at the five-dose assay. Subsequently, we examined the growth inhibition mechanism of hybrid 8a in relation to cell cycle regulation as well as apoptosis induction in breast T-47D cancer cells through the DNA flow cytometry and Annexin V-FITC/ PI assays. Moreover, inhibitory actions of hybrid 8a against the cell cycle regulator protein CDK2 kinase and the anti-apoptotic protein Bcl-2 were explored. Finally, docking simulations were conducted in order to explore the behaviour of 8a within the active site of both CDK2 and Bcl-2, and to justify its activity.

Chemistry
Synthetic routes herein proposed in order to get the targeted conjugates (7, 8a-c, 9 and 10a-e) have been illustrated in Schemes 1 and 2. With regard to Scheme 1, preparation of 1Hindole-3-carbaldehyde 2 was achieved through Vilsmeier formylation of indole 1 by the use of N, N-dimethylformamide (DMF) and phosphorus oxychloride (POCl 3 ), then aldehyde 2 was undergone to N-alkylation using different alkyl halides 3a-d in DMF with the aid of sodium hydride base to get N-substituted indole-3-carbaldehyde intermediates 4a-d. The different N-substituted indole-3-carbaldehyde derivatives 4a-d were condensed with hydrazine hydrate via heating under reflux temperature in ethyl alcohol in order to produce the key intermediates N-substituted-3-(hydrazonomethyl)-1H-indoles 5a-d. In Scheme 2, the key intermediates 5a-d were reacted with different isatin derivatives 6a-g in absolute ethyl alcohol with the aid of catalytic amount of glacial acetic acid to furnish targeted [(3-indolylmethylene)hydrazono]indolinones 7, 8a-c, 9 and 10a-e.
Postulated structures for the newly prepared key intermediates and target compounds 7, 8a-c, 9 and 10a-e have been in full consistent with the data of both spectral and elemental analyses.

Biological evaluation
2.2.1. Antiproliferative activities towards A-549, MDA-MB-231 and HCT-116 cancer cell lines All the newly prepared target conjugates (7, 8a-c, 9 and 10a-e) were investigated for the prospective growth inhibitory actions against three different cancer cell lines viz.; A-549, MDA-MB-231, and HCT-116, using the sulforhodamine B colorimetric (SRB) assay 21 . Staurosporine, a clinically used antitumor drug, was coassayed as a reference agent for the experiment. The results have been conveyed as IC 50 values, and listed in Table 1.

Nci-USA cancer cell lines screening
Seven hybrids (8a-c, 9, 10a, 10c and 10d) have been selected and screened by USA-National Cancer Institute (NCI-DTP; www. dtp.nci.nih.gov) for their possible in vitro anti-proliferative actions towards a panel of 59 human cancer cell lines representing breast, ovarian, CNS, colon, NSCLC, leukaemia, melanoma, renal and prostate cancers, according to the NCI, Bethesda, Drug Evaluation Branch protocol 22-24 .
2.2.2.1. Preliminary single (10 mM) dose screening. Antitumor activities of here reported conjugates 8a-c, 9, 10a, 10c and 10d were first screened in an initial single dose (10 mM) screening, utilising the SRB assay to estimate cells viability and growth 21 . The obtained results were presented as percent growth inhibition (GI %) for the examined conjugates towards the different treated tumour cell lines, as presented in Table 2.
Exploring the attained GI% values ( Table 2), disclosed that hybrid 8a stood out as the most efficient anti-proliferative agent in the herein reported NCI screening exhibiting mean GI % equals to 59, with broad-spectrum action towards all human cancer cell lines in all the herein examined cancer subpanels, except towards Renal cancer TK-10 cell line.

2.2.2.2.
In vitro full NCI panel five dose assay. The obtained data from the preliminary single dose (10 mM) screening disclosed that hybrid 8a (NSC: D-771612/1) is the most efficient anticancer agent here in this study, displaying promising effectiveness against numerous cancer cell lines from different subpanels (Table 2, Figure 2). Subsequently, hybrid 8a was chosen to carry out further biological screening at a (0.01-100 mM) five-dose assay. Three response parameters (GI 50 , TGI and LC 50 ) were evaluated for hybrid 8a towards each herein examined cancer cell line, and listed in Table 3. GI 50 values reflect the level for growth inhibitory effect, whereas TGI represents cytostatic impact. In addition, LC 50 parameter is considered to be the cytotoxicity parameter for the examined hybrid. Moreover, both full panel and subpanel mean graph midpoints (MG-MID), for full panel and individual subpanels cell lines, were calculated on the level of GI 50 parameter, providing an average potency parameter for the tested hybrid 8a (Table 4). As shown in Table 3, hybrid 8a displayed potent anti-proliferative action at a single-digit micromolar concentration against all herein examined human cancer cell lines with GI 50 values range: 1.17-8.41 mM, except for Melanoma (UACC-257), Renal (TK-10) and Breast (HS 578 T) cell lines which possessed GI 50 values equal 12.30, 11.10 and 11.30 mM, respectively (Table 3). Interestingly, hybrid 8a showed superior sub-micromolar activity towards leukaemia (MOLT-4 and SR), Ovarian cancer (OVCAR-4) and Breast cancer (T-47D) cells (GI 50 ¼ 0.68, 0.52, 0.93 and 0.41 mM, respectively).
With regards to the sensitivity for diverse tumour cell lines, hybrid 8a possessed relatively homologous growth inhibitory action for the whole of NCI panel, with efficient sub-panel GI 50 (MG-MID) range of 1.33-4.23 mM, and promising full-panel GI 50 (MG-MID) ¼ 3.10 mM. leukaemia, prostate cancer, colon cancer, and breast cancer subpanels were the most vulnerable herein examined cancer subpanels to the impact of hybrid 8a [GI 50 (MG-MID) ¼ 1.33, 2.47, 2.85 and 3.09 mM, respectively] (Table 4).
Furthermore, dividing the full-panel MG-MID (mM) for the examined derivative by its individual subpanel MG-MID (mM) provides an index which is regarded as a measure for its selectivity. A value between three and six refers to moderate selectivity against the corresponding cancer cell line, ratio more than six indicates high selectivity, whereas if the compound does not meet any of such criteria is considered as non-selective 25 . In this regards, hybrid 8a was found to be a non-selective anticancer agent that exhibits broad-spectrum potency towards all cancer subpanels herein examined at the GI 50 level, with selectivity ratios spanning in the range from 0.73 to 2.33 (Table 4).  -62  60  11  --25  15  19  Ovarian cancer  IGROV1  61  ---28  31  23  OVCAR-3 98

Cell cycle analysis
The superior sub-micromolar anti-proliferative activity of conjugate 8a on breast cancer T-47D cells (GI 50 ¼ 0.41 mM, Table 3) prompted us to further investigate about the growth inhibitory mechanism of the target conjugates. Both regulation of cell cycle progression and apoptosis induction have been considered as significant strategies to control the proliferation of different cancer cells, accordingly, we primarily examined the growth inhibition mechanism of hybrid 8 in relation to cell cycle progression and regulation in human breast T-47D cancer cells.
The impact on cell cycle distribution was assessed by a DNA flow cytometry analysis, upon incubation of T-47D cells with conjugate 8a at its GI 50 concentration (0.41 mM) for 24 h (Figure 3). From the obtained results it was found that T-47D cells exposed to hybrid 8 significantly arrested at the G2/M phase of the cell cycle with an escalation in G2/M phase fraction from 19.19% (in control cells) to 34.05% (in 8a-treated T-47D cells). Furthermore, the cell population in sub-G1 phase was drastically augmented from 1.25% (in control cells) to 17.73% (in 8a-treated T-47D cells).
Generally the upsurge of populations in the sub-G1 phase indicates the induction of apoptotic cell death. So, a subsequent study will be conducted to reveal whether the G2/M phase cell cycle arrest afforded by conjugate 8a was accompanied by apoptosis.

Apoptosis assay
In an attempt to further investigate whether the antiproliferative activity for conjugate 8a is harmonious with the apoptosis induction within T-47D cells pointed out by the increased cell population in sub-G1 phase in 8a-treated T-47D cells (Figure 3), Annexin V-FITC/PI dual staining analysis was used for the apoptosis assay ( Figure 4).
The outcomes of the Annexin V-FITC/PI assay suggested that treatment of T-47D cells with conjugate 8a led to an early and late cellular apoptosis, which proved through the significant increase for the apoptotic cells percentage in both the early apoptotic phase (from 0.26% to 4.82%) and the late apoptotic phase (from 0.47% to 14.62%) that signifies about 26-fold increase in total apoptosis, when compared to the untreated control ( Figure 4).

Cdk2 and Bcl-2 inhibitory activity
The promising anti-proliferative impact of conjugate 8a, in addition to its cell cycle disruption and pro-apoptotic effects, provoked a further exploration for their possible inhibitory activities against the cell cycle regulator CDK2 protein kinase and the anti-   Table 5. Results in Table 5 showed that the tested hybrid 8a exhibited good inhibitory action against CDK2 and Bcl-2 with IC 50 values equal 0.85 ± 0.03 and 0.46 ± 0.02 mM, respectively.

Molecular docking studies
The docking simulation studies were conducted to investigate the behaviour of compound 8a in the active site of both CDK2 and Bcl-2 (pdb code 2BHE 7 and 4 26 ). From several X-ray structures for CDK2 enzyme, as mentioned before in the introduction, it was observed that the active site should be filled by planar molecules [8][9][10][11] . In the docking simulations 8a has taken the planar orientation, although it has a certain flexibility, with some little deviation at the indole moiety to fill the part of the corresponding hydrophobic pocket by hydrophobic contact with Lys89 as shown in (Figure 4(b)), whereas the N-propyl group interacted with the part of the terminal hydrophobic pocket by alkyl hydrophobic contact with Lys20. Moreover, the Molecular docking simulations showed an important hydrogen bond interaction with Leu83, via C¼O of isatin, which is a key interaction with distance 2.5 Å. Also the docking poses of hybrid 8a showed that 5-methyl group, decorated on the benzenoid part of the isatin moiety, made an anchoring hydrophobic mixed with p interactions with Val18 and Leu134 from both sides as shown in Figure 4(a).
On the other hand, the molecular docking studies explored the binding pattern of hybrid 8a within the active site of Bcl-2 to justify its apoptotic effect. As reported, the pro-apoptotic effect of the small molecules could be done at the molecular level by mimicking the BH3 a-helix and then binding to the hydrophobic groove of anti-apoptotic Bcl-2 proteins preventing their heterodimerization, which eventually leads to apoptosis 27 . The attitude of a-helical BH3 domains complexed with Bcl-2 family besides the docking poses of other small molecules as Bcl-2 inhibitors were discussed as a guide for our docking study [28][29][30][31] .   Molecular docking of compound 8a, as a small molecule inhibitor, inside the hydrophobic groove of Bcl-2 showed significant interactions at the front of this groove ( Figure 5(a)). The (NH) functionality of isatin moiety can donate a hydrogen bond (2.00 Å) to the key amino acid Asn102 at the margin of the hydrophobic groove. This anchoring hydrogen bond was supported by the p-stack of isatin ring with Arg105 ( Figure 5(a)). On the other side of the groove, N-propyl group attached to indole ring was embedded in the hydrophobic pocket formed of three amino acids; Phe112, Phe71 and Met74. The p-stack interaction of indole ring with Leu96 and Ala108 would act as a middle support for the whole compound inside the opening of the hydrophobic groove ( Figure 5(b)).

General
Melting points were measured with a Stuart melting point apparatus, and are uncorrected. Nuclear magnetic resonance; 1 H NMR and 13 C NMR spectra were recorded using a Bruker NMR spectrometer (400/100 MHz), in deuterated dimethylsulphoxide (DMSOd 6 ). Chemical shifts (d H ) were reported relative to TMS as an internal standard. The coupling constant values (J) were given in hertz. Chemical shifts (d C ) were reported as follows: s, singlet; d, doublet; m, multiplet. HRMS spectra were performed on a Bruker MicroTOF spectrometer. Infra-red (IR) Spectra are obtained by the use of Schimadzu FT-IR 8400S spectrophotometer as KBr discs and expressed in wave number (cm À1 ). Compounds 2 and 4a-d were prepared as reported previously 32 .

4.1.3.
General procedures for preparation of target compounds 7, 8a-c, 9 and 10a-e To a hot stirred solution of isatin derivatives 6a-g (1 mmol) in absolute ethyl alcohol (5 ml) and catalytic drops of acetic acid, the appropriate N-substituted-3-(hydrazonomethyl)-1H-indole derivative 5a-d (1 mmol) was added. The resulting mixture was stirred at reflux for 3 h, and then filtrated. The collected precipitate was dried and then recrystallized from isopropyl alcohol to afford target hybrids 7, 8a-c, 9 and 10a-e, respectively.
Full characterisation for the key intermediates (5a, 5c and 5d) as well as the target conjugates (7, 8a-c, 9 and 10a-e) have been presented in the Supporting Information.

Biological evaluation
Experimental procedures for biological evaluations for the herein reported conjugates (7, 8a-c, 9 and 10a-e) were provided in the Supplementary material (Supplementary materials can be found at www.mdpi.com/xxx/s1).

Antiproliferative action against A-549, MDAMB-231 and HCT-116 cancer cell lines
The three examined cancer cell lines (non-small cell lung A-549, Breast MDA-MB-231 and colon HCT-116 cells) were obtained from American Type Culture Collection (ATCC). Examination of the cytotoxic activity for the target conjugates was carried out in accordance with the SRB colorimetric assay protocol 21 , as described previously 33 .

Cell cycle analysis
Flow cytometric analysis (FACS) was carried out to examine the cell cycle distributions in breast T-47D cancer cells upon treatment with conjugate 8a, by BD FACS Calibur flow cytometer, as described earlier 36 .

Apoptosis assay
Phosphatidylserine externalisation effect of conjugate 8a over breast T-47D cancer cell line was investigated using Annexin-V-FITC Apoptosis Detection Kit by flow cytometry, as described earlier 37 .

Cdk2 and bcl-2 inhibitory activity
These assays were carried out as reported earlier 38,39 .

Molecular docking studies
The target proteins, CDK2 (2BHE) and BCl-2 (4AQ3), were downloaded from protein databank and prepared by the default protocol in Discovery Studio 4. All other steps including preparation of small molecules, determination of the active site and docking protocol were followed as reported 40 .

Conclusions
To conclude, a novel series of N-alkylindole-isatin conjugates (7, 8a-c, 9 and 10a-e) was designed and synthesised, utilising the SAR outputs from the previous study. The design is based on appending four alkyl groups with different length (ethyl, n-propyl), bulkiness (isopropyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. All conjugates were screened for their potential cytotoxicity towards a panel of three different human cancer cell lines HCT-116 (Colon), A-549 (NSCLC), and MDA-MB-231 (Breast) utilising SRB assay. Conjugate 8a, superiorly, displayed the best anti-proliferative activity against the three examined cell lines (IC 50 ¼ 7.3 ± 0.42, 4.7 ± 0.28 and 2.6 ± 0.17 mM, respectively). Furthermore, 8a displayed broad spectrum anti-proliferative action with efficient subpanel GI 50 (MG-MID) range of 1.33-4.23 mM, and promising full panel GI 50 (MG-MID) ¼ 3.10 mM, at the NCI fivedose assay. In particular, hybrid 8a showed superior sub-micromolar activity towards leukaemia (MOLT-4 and SR), ovarian cancer (OVCAR-4) and breast cancer (T-47D) cell lines (GI 50 ¼ 0.68, 0.52, 0.93 and 0.41 mM, respectively). Conjugate 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Moreover, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC 50 ¼ 0.85 ± 0.03 and 0.46 ± 0.02 mM, respectively). The molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.

Disclosure statement
No potential conflict of interest was reported by the author(s).