Development of 2-oindolin-3-ylidene-indole-3-carbohydrazide derivatives as novel apoptotic and anti-proliferative agents towards colorectal cancer cells

Abstract Mitochondrial anti-apoptotic Bcl2 and BclxL proteins, are overexpressed in multiple tumour types, and has been involved in the progression and survival of malignant cells. Therefore, inhibition of such proteins has become a validated and attractive target for anticancer drug discovery. In this manner, the present studies developed a series of novel isatin–indole conjugates (7a-j and 9a-e) as potential anticancer Bcl2 and BclxL inhibitors. The progression of the two examined colorectal cancer cell lines was significantly inhibited by all of the prepared compounds with IC50 ranges132–611 nM compared to IC50 = 4.6 µM for 5FU, against HT-29 and IC50 ranges 37–468 nM compared to IC50 = 1.5 µM for 5FU, against SW-620. Thereafter, compounds 7c and 7g were selected for further investigations. Interestingly, both compounds exhibited selective cytotoxicity against both cell lines with high safety to normal fibroblast (HFF-1). In addition, both compounds 7c and 7g induced apoptosis and inhibited Bcl2 and BclxL expression in a dose-dependent manner. Collectively, the high potency and selective cytotoxicity suggested that conjugates 7c and 7g could be a starting point for further optimisation to develop novel pro-apoptotic and antitumor agents towards colon cancer.


Introduction
Cancer is a group of diseases that is characterised by uncontrolled and rapid cell proliferation and differentiation mechanisms with the potential to invade or spread to other body parts 1 . Since several decades, cancer has been one of the main world health problems and is still considered a serious leading cause of death worldwide. Early strategies of cancer treatment were based on the unspecific induction of cell death mainly targeting the replication machinery [2][3][4][5] and/or the DNA synthesis [6][7][8][9] . Therefore, the traditional anticancer drugs were associated with severe adverse effects due to the unselective toxicity towards the normal cells in addition to the resistance emerged towards them 9 . Thus, the development of effective and safe new antitumour drugs with increased selectivity towards cancer cells is still an active search 10,11 . On the other hand, recent strategies of targeted therapies target specific biomarkers essential for the regulation of cancer cells proliferation and/or cell apoptosis such as deregulated, mutated, or overexpressed proteins 12 and thus, selectively affect cancer cells or their supporting environment with minimum effects on normal cells 13 . Among these targets are the pro-apoptotic and anti-apoptotic proteins that control the cellular apoptosis [14][15][16] .
Apoptosis, a form of programmed cell death takes place in multicellular organisms, is a series of biochemical events that result in characteristic cell changes and death 17 . Apoptosis could be launched through one of two pathways (intrinsic pathway and extrinsic pathway). The mitochondria-dependent apoptotic pathway (intrinsic pathway), one of the main pathways of induction of the cell apoptosis 18,19 , is controlled by the Bcl2 proteins family. The Bcl2 family members have dual functions; some are anti-apoptotic Bcl2 proteins, such as Bcl2 and BclxL that inhibit apoptosis, while others are pro-apoptotic Bcl2 proteins such as Bax and Bak that promote apoptosis 14 . In this regard, it was reported that most of cancer cells are characterised by over-expression of the anti-apoptotic Bcl2 proteins which could lead to apoptosis prevention as well as drug resistance 20,21 . Thus, the development of anti-apoptotic Bcl2 proteins inhibitors has become an important strategy for introducing potential anti-cancer agents 15,16 . In this manner, several heterocyclic scaffolds including isatin 22 were developed as candidates targeting Bcl2 proteins.
Isatin (1H-indole-2,3-dione), as a special class in drug design and discovery, represents one of the most favourable scaffolds of heterocyclic systems which possesses many interesting biological activities including anti-SARS-CoV-2 23 , antimicrobial 24 , anticonvulsant 25 and mainly anticancer [26][27][28] . Therefore, isatin nucleus was broadly used by our group for the development of diverse effective oxindole-based small molecules (structures I-III 29-31 , Figure 1) with anticancer activities that target different enzymatic and cellular targets such as inhibition of cancer-related carbonic anhydrase IX isoform [32][33] , inhibition of different kinases [34][35] , in addition to apoptosis induction in different human cancer cell lines [36][37] .
Motivated by the aforementioned findings and in continuation to our previous work, in the present study, a novel series of isatin/ indole conjugates (7a-j and 9a-e, Figure 1) were designed and synthesised. The antiproliferative effect of the new compounds against HT-29 and SW-620 colorectal cancer cell lines were examined. In addition, the levels of the mitochondria-related anti-apoptotic proteins Bcl2 and BclxL in HT-29 and SW-620 colorectal cancer cell lines after incubation with isatin derivatives 7c and 7g were determined.

Chemistry
The synthetic strategies deliberate for the development of the final compounds (7a-j and 9a-e) were illustrated in Schemes 1-2. In Scheme 1, indole 1 was subjugated to formylation via Vilsmeier haack reaction to produce 1H-indole-3-carbaldehyde 2, in which the CHO functionality was oxidised by KMnO 4 in acetone to furnish 1H-indole-3-carboxylic acid 3. Then, the acid analogue 3 was subjected to esterification through refluxing in dry methanol to get the carboxylate analogue 4, where the ester group reacted with hydrazine hydrate in methyl alcohol to produce the key intermediate 1H-indole-3-carbohydrazide 5. Finally, the key intermediate 5 was condensed with different isatin derivatives 6a-j in glacial acetic acid to give the final targeted compounds 7a-j.
On the other hand, in Scheme 2, three isatins 6a, 6c and 6d were alkylated with methyl iodide, propyl bromide and benzyl bromide in DMF with the presence of K 2 CO 3 and catalytic amount of KI to give the N-substituted isatin derivatives 8a-e, which heated under reflux with the carbohydrazide 5 in acetic acid to furnish the final compounds 9a-e, respectively. The structure of the synthesised 2-oxindolin-3-ylidene-indole-3-carbohydrazide was confirmed under the basis of spectral and elemental analyses which were in full agreement with the proposed structures.

Anti-proliferative activity against HT-29 and SW-620 colorectal cancer cell lines
The anti-proliferative potential of the final compounds (7a-j and 9a-e) was examined against two human colorectal cancer HT-29 and SW-620 cell lines. While, HT-29 is an adenocarcinoma cell line, SW-620 represents metastatic cancer cell line. These effects were compared with known anti-cancer drug, 5-Fluorouracil (5FU) commonly used in colorectal cancer treatment. All of the compounds were found to inhibit the cell viability of cancer cells with varied sub-micro-molar efficacy. The IC 50 values ranged from 105 to 611 nM for all compounds were calculated using Graph Pad prism 8 (Table 1).
Regarding the activity towards HT-29 cell line, all compounds showed superior activity to the reference drug (5FU) with IC 50 ranged from 132-611 nM compared to IC 50 ¼4.6 mM for 5FU. Compound 7h was the most active against HT-29 cell line with IC 50 ¼132 nM that is approximately 35-fold more than 5FU. Also, the results revealed that the N-alkylation of isatin moiety (series 9a-e) resulted in diversified effect on the potency according to the alkyl group added and/or the substitution on indole moiety. For the unsubstituted indole (7a, IC 50 ¼408 nM), while N-alkylation with CH 3 -group significantly increase the potency (9a, IC 50 ¼176 nM) and N-alkylation with benzyl-group slightly increase the potency (9c, IC 50 ¼405 nM), the N-alkylation with propyl group dramatically decrease the potency (9b, IC 50 ¼525 nM). Furthermore, N-propylation of both chloro-(7c, IC 50 ¼206 nM) and bromo-(7d, IC 50 ¼320 nM) derivatives significantly increase the potency (9d, IC 50 ¼166 nM) and (9e, IC 50 ¼290 nM), respectively. Similarly, the activity towards SW-620 cell line of all compounds was more that 5FU with IC 50 ranged from 37-468 nM compared to IC 50 ¼1.5 mM for 5FU. Compound 7f was the most active against SW-620 cell line with IC 50 ¼37 nM that is approximately 32 folds more than 5FU. However, N-alkylation of isatin moiety resulted in reduction of potency for all series except for compounds 9b (IC 50 ¼105 nM) compared to compound 7a (IC 50 ¼145 nM) ( Table 1). Figure 2 illustrated that treatment of HT-29 and SW-620 cell lines with different concentrations of compounds 7c and 7g resulted in a dose-dependent inhibition of cell viability. Compound 7c was found to inhibit HT-29 and SW-620 with IC 50 ¼188 nM and 206 nM, respectively ( Figure 2(A,B)), whereas compound 7g was found to exhibit IC 50 of 279 nM against HT-29 and 299 nM against SW-620 cell lines (Figure 2(C,D)), compared to the IC 50 for 5FU that was found to be 1.5 mM against HT-29 and 4.6 mM against SW-620 cell lines.
Furthermore, to investigate the selective cytotoxicity of the tested compounds, the effect of compounds 7c and 7g was studied on normal human skin fibroblast (HFF-1). Compounds 7c and 7g were found to have no/or little effect on cell viability of HFF-1 (Figure 3(A,B)). This finding indicates that compounds 7c and 7g inhibited cell viability of cancer cells without affecting normal fibroblast.

Annexin V-FITC apoptosis assay
Induction of apoptosis is mainly considered as an important strategy for the development of new anti-proliferative agents 38 . Compounds 7c and 7g were further investigated for their potential role of apoptosis induction in the colorectal SW-620 cancer cell line (SW-620 cell line was selected because most of the new compounds showed significant potency more than HT-29 cell  line). Exposure of SW-620 cells to compounds 7c and 7g resulted in the induction of apoptosis in a dose dependent manner ( Figure  4). As shown in the results, compound 7c at concentrations of 250 nM and 500 nM was able to induce approximately 5.2-and 10.66-fold, respectively, total apoptosis increase compared to the control for SW-620 cell line (Figure 4(A)). Similarly, compound 7g induced approximately 7.3 and 9.6 fold total apoptosis increase compared to the control when incubated with SW-620 cell line at concentration of 250 nM and 500 nM, respectively (Figure 4(B)). These findings encouraged us to further investigate the effect of compounds 7c and 7g towards the anti-apoptotic mitochondrial markers Bcl2 and BclxL.

Impact of compounds 7c and 7g on the anti-apoptotic (Bcl2, and BclxL) markers levels
Bcl2 and BclxL, as anti-apoptotic proteins, are known to be overexpressed in diverse tumours causing cancer cell survival and drug resistance 14 . Inhibition of these proteins expression resulted in cancer cell death and has been exploited as a strategy for anticancer drug discovery [15][16] . Treatment of SW-620 with compound 7c resulted in a dose-dependent inhibition of Bcl2 and BclxL protein expression ( Figure 5(A)). Compound 7g was also found to inhibit Bcl2 and BclxL expression in SW-620 cells ( Figure 5(B)). These findings thus indicate that compound 7c and 7g inhibited cell viability by inhibiting Bcl2 and BclxL resulting in the apoptosis.

Conclusions
In the present work, a series of novel isatin-indole conjugates (7aj and 9a-e) was designed and synthesised as potential antiproliferative agents towards colon cancer cells with promising inhibitory activity against the anti-apoptotic Bcl2 and BclxL proteins. The cell growth of two examined colorectal cancer (HT-29 and SW-620) cell lines was significantly inhibited by all of the prepared compounds with IC 50 ranges 132-611 nM against HT-29 and IC 50 ranges 37-468 nM against SW-620 as compared to IC 50 ¼4.6 and 1.5 mM for 5FU, respectively. For further mechanistic and selective cytotoxicity studies, compounds 7c and 7g were examined and proved to exhibit selective cytotoxicity against both cancer cell lines with high safety profile to normal fibroblast (HFF-1). In addition, towards SW-620 cell line, both candidates inducted apoptosis and inhibited anti-apoptotic Bcl2 and BclxL proteins in a dose dependent manner. Collectively, the high potency and selective cytotoxicity suggested that conjugates 7c and 7g could serve as lead compounds for further optimisation to develop novel antitumor agents and Bcl2/BclxL inhibitors.

General
The NMR spectra have been recorded by Bruker spectrometer at 400 MHz. 13 C NMR spectra were run at 100 MHz in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (d H ) are reported relative to the solvent (DMSO-d 6 ). Infra-red spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer. Elemental analyses have been performed at the Regional Centre for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.

Synthesis of the target compounds 7a-j and 9a-e
To hot stirred solution of 1H-indole-3-carbohydrazide 5 (0.3 g, 1.7 mmole) in 15 ml of glacial acetic acid, an equivalent amount of appropriate isatin derivatives 6a-j and 8a-e was added. The reaction mixture was heated under reflux for (5-7) h, and then was cooled to room temperature. The formed precipitate was collected by filtration, washed with cold water, hexane and recrystallized from DMF-MeOH mixture to furnish the targeted novel compounds 7a-j and 9a-e, respectively. Representative NMR spectra charts were provided in the Supplementary data.

Cell viability assay
The cytotoxicity effect of compounds on the colorectal cancer cell lines, HT29 and SW620 in addition to the normal human fibroblasts was measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) as previously described 39 . Briefly, cells were seeded in 96 well culture plates at 5000/well for HT29 and 10,000/well for SW620 for 24 h. Cells were then incubated with different compounds from WAG1 series (1-15) 24 h at 37 C and humidified 5% CO 2 incubator. Freshly prepared 10 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT 5 mM) solution were added to the cells and further incubated for 2 h. Thereafter, 100 ml of dimethyl sulphoxide (DMSO) were added in each well and the crystals were dissolved through careful pipetting. In certain experiment, cells were treated different concentration of 5FU for 72 h. The absorbance of the product was measured at 540 nm using a Synergy TM 2 multi-mode microplate reader (Biotech, VA, USA). The experiments were performed in triplicate for each condition.

Measurement of apoptosis by annexin V-FITC/PI assay:
Induction of apoptosis was measured by Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (Thermofischer scientific, OR, USA) according to the manufacturer's instruction. As described previously (39), cells were seeded in a 6-well plate (3 Â 10 5 cells per well) and treated with the various compound for 24 h. Both floating and adherent cells were harvested, pooled together, and incubated with Annexin V-FITC and PI for 15 min on ice in dark. The cells were analysed by BD FACSCalibur TM cell analyser (BD Biosciences, CA, USA) at an emission of 530 nm (FL1 channel) and >575 nm (FL3).

Western blot analysis:
All cells were seeded in a 100 mm dish (1 Â 10 6 cells per dish) in 5% CO 2 at 37 C in the appropriate culture medium. The cells with around 50% confluency were treated with compounds for 24 h. At the experiment day, cells were washed with 1x PBS, harvested and lysed in RIPA lysis buffer, combined with protease inhibitors, (Sigma-Aldrich, St. Louis, MO, USA) as described previously 39 ). The total protein concentration was evaluated by the colorimetric Bradford protein assay (BIO-RAD inc, CA, USA) at 595 nm absorbance. Lysates were loaded in equal concentration and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane by semi-dry. Blocking of the membrane was done by 5% non-fat dried milk for one hour, incubated with the primary antibodies. The primary antibodies used were Bcl2 (cat. no. sc-7382), BclxL (cat. no. sc-8392) and b Actin (cat. no. sc-69879) from (Santa Cruz