Design, Synthesis, and Potent Anticancer Activity of Novel Indole-Based Bcl-2 Inhibitors

The Bcl-2 family plays a crucial role in regulating cell apoptosis, making it an attractive target for cancer therapy. In this study, a series of indole-based compounds, U1–6, were designed, synthesized, and evaluated for their anticancer activity against Bcl-2-expressing cancer cell lines. The binding affinity, safety profile, cell cycle arrest, and apoptosis effects of the compounds were tested. The designed compounds exhibited potent inhibitory activity at sub-micromolar IC50 concentrations against MCF-7, MDA-MB-231, and A549 cell lines. Notably, U2 and U3 demonstrated the highest activity, particularly against MCF-7 cells. Respectively, both U2 and U3 showed potential BCL-2 inhibition activity with IC50 values of 1.2 ± 0.02 and 11.10 ± 0.07 µM using an ELISA binding assay compared with 0.62 ± 0.01 µM for gossypol, employed as a positive control. Molecular docking analysis suggested stable interactions of compound U2 at the Bcl-2 binding site through hydrogen bonding, pi-pi stacking, and hydrophobic interactions. Furthermore, U2 demonstrated significant induction of apoptosis and cell cycle arrest at the G1/S phase. Importantly, U2 displayed a favourable safety profile on HDF human dermal normal fibroblast cells at 10-fold greater IC50 values compared with MDA-MB-231 cells. These findings underscore the therapeutic potential of compound U2 as a Bcl-2 inhibitor and provide insights into its molecular mechanisms of action.


Introduction
The B-cell lymphoma 2 (Bcl-2) protein is a key regulator of apoptosis, a programmed cell death process required for tissue homeostasis and the elimination of damaged cells [1]. Bcl-2 is a member of the Bcl-2 protein family, which is categorized into pro-apoptotic (such as Bax and Bak) and anti-apoptotic members (such as Bcl-2, Bcl-XL, and Mcl-1) [2]. Bcl-2 is an anti-apoptotic protein that suppresses cell death by inhibiting the activation of pro-apoptotic proteins, hence favouring cell survival [3]. Bcl-2 expression or function dysregulation contributes to cancer development and progression [4,5]. Several studies have linked the overexpression levels of the anti-apoptotic Bcl-2 protein with resistance to chemotherapy, radiation treatment, and targeted therapies in many cancer types [6,7]. This resistance permits cancer cell survival, resulting in therapeutic resistance and disease progression [8]. Furthermore, multiple Bcl-2 inhibitors are being tested in clinical studies

Rational Design
The rational design of Bcl-2 inhibitors has been explored using various small-molecule inhibitors with core heterocyclic scaffolds, such as isoquinoline [2,46] and indole [39,47], along with different substitutions, to enhance their activity and physiochemical properties. We previously designed a series of indole-based compounds with triazole [48], oxadiazole [3,49], and quinoline-fused triazolothiadiazole [2] scaffolds. The inclusion of a triazole ring was beneficial in targeting anti-apoptotic proteins Bcl-2 and Mcl-1 [45]. Morpholino substitution has demonstrated efficacy in targeting Bcl-2 proteins as in the case of ABT-263 [50,51]. Additionally, acetamide derivatives have been investigated for their ability to inhibit anti-apoptotic Bcl-2 proteins [52]. Various substitutions of electron-donating or -withdrawing groups have been introduced to the aforementioned compounds to alter the electronic properties and physicochemical characteristics of the molecule, potentially impacting its interactions with the target protein or modulating its biological activity. The rational construction of a series of molecules involves incorporating various functional groups and substitution patterns, including an indole ring, a 1,2,4-triazole ring, a thioether linkage, an acetamide linker, and a morpholino moiety. The arrangement and connectivity of these structural elements can contribute to the compound's functional activity. It is worth noting that the discovery of small-molecule inhibitors targeting the Bim binding site of anti-apoptotic Bcl-2 is an active area of research ( Figure 2). The rational design of these compounds aims to optimize their potency, selectivity, and other pharmacological properties, with the ultimate goal of developing effective therapeutic agents for the treatment of specific diseases.

Rational Design
The rational design of Bcl-2 inhibitors has been explored using various small-molecule inhibitors with core heterocyclic scaffolds, such as isoquinoline [2,46] and indole [39,47], along with different substitutions, to enhance their activity and physiochemical properties. We previously designed a series of indole-based compounds with triazole [48], oxadiazole [3,49], and quinoline-fused triazolothiadiazole [2] scaffolds. The inclusion of a triazole ring was beneficial in targeting anti-apoptotic proteins Bcl-2 and Mcl-1 [45]. Morpholino substitution has demonstrated efficacy in targeting Bcl-2 proteins as in the case of ABT-263 [50,51]. Additionally, acetamide derivatives have been investigated for their ability to inhibit anti-apoptotic Bcl-2 proteins [52]. Various substitutions of electron-donating or -withdrawing groups have been introduced to the aforementioned compounds to alter the electronic properties and physicochemical characteristics of the molecule, potentially impacting its interactions with the target protein or modulating its biological activity. The rational construction of a series of molecules involves incorporating various functional groups and substitution patterns, including an indole ring, a 1,2,4-triazole ring, a thioether linkage, an acetamide linker, and a morpholino moiety. The arrangement and connectivity of these structural elements can contribute to the compound's functional activity. It is worth noting that the discovery of small-molecule inhibitors targeting the Bim binding site of anti-apoptotic Bcl-2 is an active area of research ( Figure 2). The rational design of these compounds aims to optimize their potency, selectivity, and other pharmacological properties, with the ultimate goal of developing effective therapeutic agents for the treatment of specific diseases.

Compounds U2 and U3 Showed Potent Inhibitory Activity towards Bcl-2-Expressing Cancer Cells
The designed compounds demonstrated inhibitory activity against Bcl-2-expressing human cell lines, including breast cancer lines MCF-7 and MDA-MB-231 and A549 lung cancer cells at sub-micromolar concentrations, indicating their potency (Table 1, Figure 3). The most potent activity was observed in U2, followed by U3, U1, and U4. Overall, the antiproliferative activity of all compounds was highest against the MCF-7 cancer cell line. Compounds U2 and U3 showed IC 50 values of 0.83 ± 0.11 and 1.17 ± 0.10 µM against MCF-7; 0.73 ± 0.07 and 2.98 ± 0.19 µM against A549; and 5.22 ± 0.55, 4.07 ± 0.35 µM against the metastatic and treatment-refractory triple-negative breast cancer cell line MDA-MB-231, respectively. The physicochemical characteristics of various substitution patterns and the potent activity of U2 and U3 led to their selection for further investigation.

ELISA Indicated the Superior Activity of Compound U2 against Bcl-2 Protein
The ability of compounds U2 and U3 to bind to the Bcl-2 binding pocket as BH3 mimetics was tested with the ELISA binding assay, as previously described by our group [48,49]. The different attributes of the substitution, such as size, shape, and electrostatic interaction, influence their effectiveness as Bcl-2-competitive inhibitors. Compound U2 exhibited an IC 50 of 1.2 ± 0.02 µM, which is two-fold less potent than gossypol (IC 50 0.62 ± 0.01 µM), while U3 exhibited lower binding affinity with an IC 50 of 11.10 ± 0.07 µM (Table 2, Figure 4). These results indicated the potential activity of compound U2 as a Bcl-2 inhibitor, warranting further investigation.

Compounds U2 and U3 Showed Potent Inhibitory Activity towards Bcl-2-Expressing Cancer Cells
The designed compounds demonstrated inhibitory activity against Bcl-2-expressing human cell lines, including breast cancer lines MCF-7 and MDA-MB-231 and A549 lung cancer cells at sub-micromolar concentrations, indicating their potency (Table 1, Figure 3). The most potent activity was observed in U2, followed by U3, U1, and U4. Overall, the antiproliferative activity of all compounds was highest against the MCF-7 cancer cell line. Compounds U2 and U3 showed IC50 values of 0.83 ± 0.11 and 1.17 ± 0.10 µM against MCF-7; 0.73 ± 0.07 and 2.98 ± 0.19 µM against A549; and 5.22 ± 0.55, 4.07 ± 0.35 µM against the metastatic and treatment-refractory triple-negative breast cancer cell line MDA-MB-231, respectively. The physicochemical characteristics of various substitution patterns and the potent activity of U2 and U3 led to their selection for further investigation.

ELISA Indicated the Superior Activity of Compound U2 against Bcl-2 Protein
The ability of compounds U2 and U3 to bind to the Bcl-2 binding pocket as BH3 mimetics was tested with the ELISA binding assay, as previously described by our group [48,49]. The different attributes of the substitution, such as size, shape, and electrostatic interaction, influence their effectiveness as Bcl-2-competitive inhibitors. Compound U2 exhibited an IC50 of 1.2 ± 0.02 µM, which is two-fold less potent than gossypol (IC50 0.62 ± 0.01 µM), while U3 exhibited lower binding affinity with an IC50 of 11.10 ± 0.07 µM (Table 2, Figure 4). These results indicated the potential activity of compound U2 as a Bcl-2 inhibitor, warranting further investigation. Table 2. IC50 values of the selected compounds using ELISA against Bcl-2.

Compounds U1-6 Showed an Excellent Safety Profile in Human Normal Cells
All tested compounds displayed minimal inhibitory activity against HDF human dermal normal fibroblast cells at a concentration of 50 µM (over 24 hrs), indicative of a high safety profile. In particular, compounds U1, U3, U4, and U5 showed no significant change compared with the negative control.

Compounds U1-6 Showed an Excellent Safety Profile in Human Normal Cells
All tested compounds displayed minimal inhibitory activity against HDF human dermal normal fibroblast cells at a concentration of 50 µM (over 24 hrs), indicative of a high safety profile. In particular, compounds U1, U3, U4, and U5 showed no significant change compared with the negative control. U6 and U2 exhibited limited toxicity on fibroblasts with p-values of <0.0001 and 0.01, respectively ( Figure 5). The data were analysed using GraphPad Prism and calculated with nonlinear retrogression curve analysis. DMSO was used as a negative control. The data display the mean ± standard error (SEM) of three replicates.

Compounds U1-6 Showed an Excellent Safety Profile in Human Normal Cells
All tested compounds displayed minimal inhibitory activity against HDF human dermal normal fibroblast cells at a concentration of 50 µM (over 24 hrs), indicative of a high safety profile. In particular, compounds U1, U3, U4, and U5 showed no significant change compared with the negative control. U6 and U2 exhibited limited toxicity on fibroblasts with p-values of <0.0001 and 0.01, respectively ( Figure 5).

U1
U2 . The data were analysed using one-way ANOVA and calculated with the multiple comparison test. The significance levels are indicated by asterisks (** p < 0.01; **** p < 0.0001). ns means non-significant. The data display the mean ± standard error (SEM) of three replicates.

Compound U2 Induced Apoptosis and Cell Cycle Arrest at G1/S Phase
Compound U2 effectively inhibited cell growth through apoptosis and significantly increased early apoptosis, displaying a 43-fold increase compared with control untreated cells ( Figure   . The data were analysed using one-way ANOVA and calculated with the multiple comparison test. The significance levels are indicated by asterisks (** p < 0.01; **** p < 0.0001). ns means non-significant. The data display the mean ± standard error (SEM) of three replicates.

Compound U2 Induced Apoptosis and Cell Cycle Arrest at G1/S Phase
Compound U2 effectively inhibited cell growth through apoptosis and significantly increased early apoptosis, displaying a 43-fold increase compared with control untreated cells ( Figure 6A-C), while inducing a remarkable 111-fold increase in late apoptosis ( Figure 6A-C). Additionally, the compound caused cell cycle arrest at the G1/S phase, as indicated in Figure 7D-F. These findings highlight the ability of compound U2 to induce programmed cell death and disrupt cell cycle progression.    (Table 3, Figure 7C). The collective interactions resulted in an overall glide score of −5.8 kcal/mole, suggesting a strong binding affinity. Figure 7D-F show the 2D interaction maps of compounds U2, U3, and U1, respectively, within the Bcl-2 binding site.

Compound U2 Showed Improved Properties Compared with Small-Molecule Inhibitor ABT263
The bioavailability radars (Swiss-ADME) of the newly designed and highly potent compound U2 were compared with those of the ABT-263 compound (Figure 8). The pink areas in the figure represent the optimal range of six properties, namely, unsaturation, insolubility, lipophilicity, flexibility, polarity, and size. It was observed that the U2 compound fell within the desired range and exhibited acceptable parameters when compared with ABT-236. This suggests that U2 exhibited favourable bioavailability characteristics, making it a promising candidate for further development.

Discussion
Venetoclax, an FDA-approved Bcl-2 inhibitor, is quickly evolving as the standard of care for acute myeloid leukaemia and chronic lymphocytic leukaemia. Because of the concomitant upregulation of anti-apoptotic proteins, particularly MCL-1, following prolonged exposure to venetoclax [53], which provides an escape route leading to venetoclax resistance, its use is limited, and hence, it has become part of a combination therapy [54]. To overcome this limitation, researchers have studied the dual inhibition of MCL-1 and Bcl-2 [55,56]. This includes the use of an indole-based lead compound that exhibits potent MCL-1 inhibition and moderate Bcl-2 inhibition [57].
Indole-based compounds showed potent anticancer activity through the dual inhibition activity of both Bcl-2/Mcl-1 proteins [58]. Indole-based derivatives designed by incorporating amide, biphenyl, and sulphonamide pharmacophoric moieties aimed to enhance inhibitory activity against Bcl-2 and Mcl-1 [59], potentially leading to more effective dual inhibitors [58]. Furthermore, indole-based coumarin compounds have been reported to be potent anticancer agents by targeting Bcl-2, and halogen substitution has shown the highest activity [60]. We previously designed and synthesized a series of indole-based compounds by incorporating various heterocyclic rings, such as oxadiazole [61], triazole [62], thiazole [2,63], quinoline [2], and a fused triazolo-thiazole ring [64]. Our previous studies highlight the chloro-substitution of indole-based oxadiazole amine as a promising inhibitor of Bcl-2 with a similar binding affinity to gossypol; triazolo-thiazole and the dimethoxy derivatives showed two-fold less potent binding affinity to Bcl-2 compared with gossypol, employed as a positive control [64].
The structure modifications aimed to optimize the binding interactions between the inhibitor and the protein. The extension to occupy more of the binding pocket of Bcl-2 played a vital role in enhancing the inhibitory activity against both proteins. The design of new molecular features that enhance potency and specificity was achieved by studying the pharmacophoric features of potent inhibitors and structural characteristics to make favourable interactions. A molecular docking study indicated the binding affinity of the designed compounds with Bcl-2 active site key amino acid residues due to the amide carbonyl moiety incorporated with H-bond interactions. A structure-activity relationship

Discussion
Venetoclax, an FDA-approved Bcl-2 inhibitor, is quickly evolving as the standard of care for acute myeloid leukaemia and chronic lymphocytic leukaemia. Because of the concomitant upregulation of anti-apoptotic proteins, particularly MCL-1, following prolonged exposure to venetoclax [53], which provides an escape route leading to venetoclax resistance, its use is limited, and hence, it has become part of a combination therapy [54]. To overcome this limitation, researchers have studied the dual inhibition of MCL-1 and Bcl-2 [55,56]. This includes the use of an indole-based lead compound that exhibits potent MCL-1 inhibition and moderate Bcl-2 inhibition [57].
Indole-based compounds showed potent anticancer activity through the dual inhibition activity of both Bcl-2/Mcl-1 proteins [58]. Indole-based derivatives designed by incorporating amide, biphenyl, and sulphonamide pharmacophoric moieties aimed to enhance inhibitory activity against Bcl-2 and Mcl-1 [59], potentially leading to more effective dual inhibitors [58]. Furthermore, indole-based coumarin compounds have been reported to be potent anticancer agents by targeting Bcl-2, and halogen substitution has shown the highest activity [60]. We previously designed and synthesized a series of indole-based compounds by incorporating various heterocyclic rings, such as oxadiazole [61], triazole [62], thiazole [2,63], quinoline [2], and a fused triazolo-thiazole ring [64]. Our previous studies highlight the chloro-substitution of indole-based oxadiazole amine as a promising inhibitor of Bcl-2 with a similar binding affinity to gossypol; triazolo-thiazole and the dimethoxy derivatives showed two-fold less potent binding affinity to Bcl-2 compared with gossypol, employed as a positive control [64].
The structure modifications aimed to optimize the binding interactions between the inhibitor and the protein. The extension to occupy more of the binding pocket of Bcl-2 played a vital role in enhancing the inhibitory activity against both proteins. The design of new molecular features that enhance potency and specificity was achieved by studying the pharmacophoric features of potent inhibitors and structural characteristics to make favourable interactions. A molecular docking study indicated the binding affinity of the designed compounds with Bcl-2 active site key amino acid residues due to the amide carbonyl moiety incorporated with H-bond interactions. A structure-activity relationship study indicated that the substitution of electron-donating groups at the para-position, such as p-methoxy U2 and p-methyl substituted U3, showed the highest activity, followed by electron-withdrawing functional groups such as p-fluoro-substituted U1. These modifications can impact the compound's pharmacokinetic profile and potentially improve its efficacy and safety. A future priority will be to test the activity of the U2 compound on anti-apoptotic members such as BCL-XL and MCL-1.
The U2 compound showed cytotoxic effects mainly by inducing apoptosis and G1/S cell cycle arrest. This inference is consistent with earlier research highlighting the involvement of Bcl-2 inhibitors in cell cycle arrest, specifically in the G0/G1 and S phases. For example, TW-37, a small-molecule Bcl-2 inhibitor, induces S-phase arrest in tumours [65]. Obatoclax inhibited the G0/G1 cell cycle in human oesophageal cancer cells [66]. Other studies have found that Bcl-2 has the most pronounced effect on the cell cycle by delaying passage from the G0/G1 phase to the S phase [67] and that direct inhibition of Bcl-2 caused by obatoclax improves the G1/G0 to S phase transition rather than causing G1/G0 phase arrest [68]. Gossypol also limited cell cycle progression by triggering S phase arrest [69]. These findings contribute to continuing attempts to find more effective Bcl-2 inhibitors, perhaps leading to new therapeutic alternatives for cancer treatment. A future study will be carried out to compare the activity of our designed compound compared with further positive control compounds in the cell cycle and apoptosis assays. More study and optimization of these compounds are required to increase their potency and selectivity.

Chemistry
The synthesised compounds were monitored with thin-layer chromatography (TLC) using pre-coated silica gel plates (Kieselgel 60F254, BDH, Taufkirchen, Germany) and visualised using UV light at 254 nm. A Gallenkamp melting point apparatus (London, UK) was used to measure the melting points (mps). 1 H NMR spectra were acquired at 500 MHz with a Bruker spectrometer. Chemical shifts were indicated in parts per million (ppm) relative to TMS; coupling constant (J) values were expressed in hertz (Hz); and the signals were labelled as s (singlet), d (doublet), t (triplet), and m (multiplet). Positive mode electrospray ionization (ESI) mass spectroscopy (Bruker Daltonics mass spectrometer, Bremen, Germany) was used to confirm molecular mass and formula.

Synthesis of 2-Chloro-N-(4-nitrophenyl)acetamide (2)
Chloro-acetyl chloride (1 mL, 10 mmol) was added dropwise to a suspension of p-nitro aniline (1.4 g, 10 mmol) and potassium carbonate (20 mmol) in DCM (20 mL). The reaction mixture was refluxed for 4 h. The aqueous layer was extracted with dichloromethane, dried over anhydrous sodium sulphate, and evaporated in vacuo until dry. The crude product was recrystallized from ethanol. Yield

Synthesis of N-(4-Amino-phenyl)-2-morpholin-4-yl-acetamide (4)
2-Morpholin-4-yl-N-(4-nitrophenyl)acetamide (3, 2.7 mL, 10 mmol) in 75% ethanol (30 mL) was added to a solution of reduced iron 5 gm in water (10 mL) and concentrated hydrochloric acid (0.5 mL) solution. The reaction mixture was heated to reflux for 1 h. Iron was removed through filtration, and the filtrate was rinsed three times with ethanol. The filtrate was then run through a silica pad to eliminate any leftover iron residues. The filtrate was evaporated in vacuo until dry and purified using preparative TLC. Yield

General Procedure for Triazole Thiol (7a-f) Preparation
A solution of previously prepared indolyl-3-carbonyl-N-substituted phenyl thiosemicarbazides (6a-f, 10 mmol) in 2N NaOH (4 mL) was refluxed for 3 h [49]. Water was added, and the solution was carefully neutralized with diluted HCl. The corresponding precipitate was filtered, dried, and recrystallized from ethanol.

Cytotoxicity Assay
The antiproliferative activity of compounds U1-6 was assessed using the 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, as previously described [40,64]. Cancer cell lines were seeded in 96-well flatbottom plates at a density of 10 4 cells/well for 24 h. The cells were subsequently treated with U1-6 compounds at a screening concentration of 50 µM and incubated at 37 • C for 48 h in a humidified incubator containing 5% CO 2 . Serial dilution concentrations ranged from 1, 10, 25, 50, to 100 µM and were used for IC 50 calculations of each compound for the three cancer cell lines. DMSO was used as a negative vehicle control (1% concentration). The culture media were then removed and incubated for 2 h with fresh 200 µL culture media containing 5 mg/mL MTT. After removing the culture media and dissolving the produced formazan crystals in 100 µL DMSO, they were incubated for another 30 min at 37 • C. The produced colour was measured at 570 nm using a microplate reader (Thermo-Scientific, Vantaa, Finland). The GraphPad Prism version 9.1 software was used to generate plots of absorbance versus the compound's concentration. Three independent repeat experiments were performed for each concentration to ensure reproducibility. IC 50 values were obtained from nonlinear regression plots of absorbance versus the log concentration of the tested compounds using the GraphPad Prism software version 5.0 (San Diego, CA, USA).

Cell Cycle Assay
Most active compound U2 was applied to MCF7 cells for 24 h at its IC 50 concentration. DMSO vehicle was used as a negative control (1% concentration). The cells were collected, centrifuged, and fixed in 70% ethanol on ice for 20 min. The fixed cells were incubated for 1 h at room temperature with staining solution (50 mg/mL propidium iodide PI, 0.05% Triton X-100, 0.1 mg/mL RNaseA). A Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) was used to measure the cell cycle proportion [70].

Apoptosis Assay
The Annexin-V-FITC apoptosis detection kit (Catalog # k101-25, Biovision, Mountain View, USA) was used for the apoptosis experiment according to previously published data. MCF-7 cells were treated with U2 compound for 24 h. The negative control was 0.1% DMSO. The cells were collected via centrifugation and resuspended in 500 µL of 1X Binding Buffer. Treated and control cells were stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI 50 mg/mL) and then incubated at room temperature for 5 min in the dark. Fluorescence-activated cell sorting (FACS) was quantified via flow cytometry. Flow cytometry (excision at 488 nm; emission at 530 nm) was used to examine Annexin V-FITC binding and PI staining using the FITC signal detector and a phycoerythrin emission signal detector [71].

Bim Enzyme-Linked Immunosorbent Assay (ELISA)
A Bim-binding assay (Thermo-Scientific, Catalogue No # BMS244-3, Vienna, Austria) was performed according to previously published data [48,49]. PBS solution containing 0.05% Tween-20 was used to wash a streptavidin-coated 96-well plate. For immobilisation, biotinylated Bim peptide (residues 81-106) was diluted in SuperBlock blocking solution and applied to each well. After incubation, the plate was washed with 0.5% BSA in PBS with Tween-20 solution. In PBS, test compounds were treated with His-tagged Bcl-2 protein and incubated for 1 h and then transferred to the wells containing the immobilised Bim peptide. After another wash, each well received anti-His antibody with horseradish peroxidase enzyme. Following incubation and washing, O-phenylenediamine and hydrogen peroxide solution were added for colour production. A plate reader (Thermo-Scientific, Vantaa, Finland) was used to measure the optical density at 450 nm. The reduction in Bim affinity was measured using a nonlinear regression curve (GraphPad Prism. Version 5.0), which was also utilized to generate the IC 50 value of Bcl-2 inhibition.

Computational Modelling
All computational work was carried out using Schrödinger suite 12.7, available at www.schrödinger.com, accessed on 5 April 2023, and using the Maestro graphical user interface software.

Protein and Ligand Preparation
The protein data bank (https://www.rcsb.org, accessed on 1 April 2023), was used to obtain the 3D crystal structure of human Bcl-2 (PDB ID: 4AQ3). The protein was prepared and refined using the Protein Preparation Wizard Maestro. Water molecules that were crystallographically larger than 5 A • were eliminated. At pH 7.3, all the missing hydrogen atoms were added to the protein for correct ionization using the EPIK module [72,73]. The tautomerization state and bond order of amino acid residues were assigned. Water molecules with three hydrogen bonds to non-waters were eliminated. Finally, to alleviate steric conflicts, energy was minimized using OPLS4 [74,75]. Ligand compounds were built and energy minimized using the LigPrep tool [76,77], and the ligand 2D structure was transformed into a 3D structure [78].

Grid Generation and Molecular Docking
In grid generation, a ligand with the crystal structure of human Bcl-2 (PDB ID: 4AQ3) was utilized. For docking investigations, a grid box was constructed at the active site's centroid, and the active site was defined around the native ligand of the Bcl-2 (PDB ID: 4AQ3) crystal structure. The prepared ligands were docked within the grid-generated Bcl-2 (PDB: 4AQ3) binding site using the standard precision (SP) mode of Glide without any limitations [79,80]. The visual inspection of the interaction indicated the affinity of the docked ligands to the binding site.

Statistical Analysis
GraphPad Prism (version 9.1.0) was used for data analysis using one-way analysis of variance (ANOVA) and the multiple comparison test. The data were represented by the mean SEM of three independent replicates. p < 0.05 was used as the statistical significance level: * represents a p-value of <0.05, ** represents a p-value of <0.01, *** represents a p-value of <0.001, **** represents a p-value of <0.0001.

Conclusions
This study investigated the effects of designed indole-based derivatives U1-6 on Bcl2expressing cancer cell growth, apoptosis, and cell cycle progression. Out of the designed compounds, compound U2, with a 4-methoxy substitution, demonstrated potent inhibitory effects against cell growth through the induction of apoptosis. In comparison with untreated control cells, the U2 compound exhibited a significant 43-fold increase in early apoptosis and a remarkable 111-fold increase in late apoptosis. Moreover, the compound induced cell cycle arrest at the G1/S phase. These findings highlight the promising anticancer activity of U2. Further research and development of U2 may lead to novel strategies for combating cell-proliferation-related diseases.