Discovery of Potent Indolyl-Hydrazones as Kinase Inhibitors for Breast Cancer: Synthesis, X-ray Single-Crystal Analysis, and In Vitro and In Vivo Anti-Cancer Activity Evaluation

According to data provided by the World Health Organization (WHO), a total of 2.3 million women across the globe received a diagnosis of breast cancer in the year 2020, and among these cases, 685,000 resulted in fatalities. As the incidence of breast cancer statistics continues to rise, it is imperative to explore new avenues in the ongoing battle against this disease. Therefore, a number of new indolyl-hydrazones were synthesized by reacting the ethyl 3-formyl-1H-indole-2-carboxylate 1 with thiosemicarbazide, semicarbazide.HCl, 4-nitrophenyl hydrazine, 2,4-dinitrophenyl hydrazine, and 4-amino-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione to afford the new hit compounds, which were assigned chemical structures as thiosemicarbazone 3, bis(hydrazine derivative) 5, semicarbzone 6, Schiff base 8, and the corresponding hydrazones 10 and 12 by NMR, elemental analysis, and X-ray single-crystal analysis. The MTT assay was employed to investigate the compounds’ cytotoxicity against breast cancer cells (MCF-7). Cytotoxicity results disclosed potent IC50 values against MCF-7, especially compounds 5, 8, and 12, with IC50 values of 2.73 ± 0.14, 4.38 ± 0.23, and 7.03 ± 0.37 μM, respectively, compared to staurosproine (IC50 = 8.32 ± 0.43 μM). Consequently, the activities of compounds 5, 8, and 12 in relation to cell migration were investigated using the wound-healing test. The findings revealed notable wound-healing efficacy, with respective percentages of wound closure measured at 48.8%, 60.7%, and 51.8%. The impact of the hit compounds on cell proliferation was assessed by examining their apoptosis-inducing properties. Intriguingly, compound 5 exhibited a significant enhancement in cell death within MCF-7 cells, registering a notable increase of 39.26% in comparison to the untreated control group, which demonstrated only 1.27% cell death. Furthermore, the mechanism of action of compound 5 was scrutinized through testing against kinase receptors. The results revealed significant kinase inhibition, particularly against PI3K-α, PI3K-β, PI3K-δ, CDK2, AKT-1, and EGFR, showcasing promising activity, compared to standard drugs targeting these receptors. In the conclusive phase, through in vivo assay, compound 5 demonstrated a substantial reduction in tumor volume, decreasing from 106 mm³ in the untreated control to 56.4 mm³. Moreover, it significantly attenuated tumor proliferation by 46.9%. In view of these findings, the identified leads exhibit promises for potential development into future medications for the treatment of breast cancer, as they effectively hinder both cell migration and proliferation.


Introduction
Globally, cancer stands as the foremost contributor to mortality, with breast cancer ranking among the leading causes of death in women.The complexity of this ailment poses a significant challenge to medical therapy.Despite their capacity to eliminate cancer cells, conventional anticancer treatments often induce a multitude of adverse effects, proving detrimental to healthy tissues.Traditional antineoplastic drugs, aimed at inhibiting specific molecules fostering tumor growth, commonly result in side effects.In response, scientists are actively exploring novel anticancer drugs, engaging in the design and discovery of new compounds tailored for treating various cancer types.This pursuit aims to identify targeted therapies that can potentially offer more effective and less harmful treatment options [1,2].Kinases, constituting the sixth largest class of proteins in the human body [3][4][5], play a crucial role in cellular function.Inhibitors of kinases are indispensable for maintaining proper cellular activities, as they modulate kinase dysregulation associated with various diseases and disorders, including cancer, inflammatory conditions, and responses to external stimuli.Through the regulation of protein kinases, these inhibitors effectively impede the growth of their substrates, thereby exerting control over the viability and proliferation of cells [6][7][8][9].
Various therapeutic targets for kinase inhibitors exist, encompassing EGFR, CDK, AKT, PI3K, and other specific targets [10][11][12].Indole-containing compounds have garnered prolonged attention from researchers and evolved into a dynamic field of study.The indole moiety demonstrates a high affinity for binding to several receptors, paving the way for the development of new bioactive medications.Its widespread utilization in target-based discovery and the design of anticancer drugs is well-documented [13][14][15][16][17][18].
Building upon the findings from the aforementioned studies, the conceptualization of synthesizing novel compounds that incorporate ester and azomethine groups, along with an indole scaffold in a single compound, is expected to yield potent anticancer medicines.This anticipation stems from the potential of these compounds to function as both hydrogen bond donors and/or acceptors upon interaction with receptors.Building upon the findings from the aforementioned studies, the conceptualization of synthesizing novel compounds that incorporate ester and azomethine groups, along with an indole scaffold in a single compound, is expected to yield potent anticancer medicines.This anticipation stems from the potential of these compounds to function as both hydrogen bond donors and/or acceptors upon interaction with receptors.

Wound-Healing Activity
As shown in Table 3 and Figure 3, the wounded area between cell layers following a scratch was partially filled by migrating MCF-7 control cells (94.07%wound closure), while treatments of compounds 5, 8, and 12 significantly inhibited wound-healing activity, with percentages of wound closure of 48.88, 60.74, and 51.85%, respectively, compared to control.

Wound-Healing Activity
As shown in Table 3 and Figure 3, the wounded area between cell layers following a scratch was partially filled by migrating MCF-7 control cells (94.07%wound closure), while treatments of compounds 5, 8, and 12 significantly inhibited wound-healing activity, with percentages of wound closure of 48.88, 60.74, and 51.85%, respectively, compared to control.

Apoptotic Induction Activity
To investigate the apoptotic activity of compounds 5, 8, and 12, flow cytometric evaluation of Annexin V/PI staining was utilized to examine apoptotic cell death in untreated and treated MCF-7 cells.Table 4 shows that compound 5 dramatically increased cell death in MCF-7 cells by 39.26% (29.35% for apoptosis and 9.91% for necrosis), compared to the untreated control group, which increased it by 1.27% (0.4% for apoptosis and 0.87% for

Apoptotic Induction Activity
To investigate the apoptotic activity of compounds 5, 8, and 12, flow cytometric evaluation of Annexin V/PI staining was utilized to examine apoptotic cell death in untreated and treated MCF-7 cells.Table 4 shows that compound 5 dramatically increased cell death in MCF-7 cells by 39.26% (29.35% for apoptosis and 9.91% for necrosis), compared to the untreated control group, which increased it by 1.27% (0.4% for apoptosis and 0.87% for necrosis).Additionally, compounds 8 and 12 caused total cell death by 24.4% and 37%, with apoptosis ratios of 15.72% and 21.0%, respectively.After being treated with a cytotoxic chemical, the cell population in each cell phase was then ascertained by DNA flow cytometry.Compound 5 increased the S-phase cell population by 56.2%, compared to control, which increased it by 41.33%, as Figure 4 illustrates, whereas cells in other phases decreased negligibly.Consequently, compound 5 stopped MCF-7 cells from proliferating at the S-phase by inducing apoptosis.

Kinase-Inhibition Activity
To highlight their effective molecular target, the most cytotoxic and apoptotic compound 5 was screened for its activity towards a panel of kinase activities, including PI3K-

In Vivo (SEC-Bearing Mice)
A solid Ehrlich carcinoma cell was implanted, and 5 was injected intraperitoneally (IP) throughout the experiment to confirm its anticancer efficacy, as shown in Figure 5, which summarizes the main findings of the antitumor activity experiments.As a result, tumor proliferation revealed an increase in solid tumor mass of approximately 398.1 mg, which is related to tumor proliferation.Following treatment with 5, the solid tumor mass decreased to 126.5 mg, compared to 110 mg in the 5-FU treatment.As a result, treatments with 5 considerably reduced tumor volume from 10 6 mm 3 in the untreated control to 56.4 mm 3 and significantly decreased tumor proliferation by 46.9%, while 5-FU reduced tumor volume to 43.7 mm 3 and inhibited tumor development by 58.8%.

Molecular Docking
To illustrate the virtual mechanism of binding towards the EGFR, PI3K, and CDK2 binding sites, molecular docking research was carried out.As seen in Figure 6, compound 5 was properly docked inside the protein active sites of EGFR (A), PI3K (B), and CDK2 (C), with binding energies of −23.15, −21.32, and −23.44 Kcal/mol, and it formed good binding interactions with their active sites.Compound 5 exhibited strong binding interactions with the amino acids Lys721, Cys773, and Leu694 inside EGFR.It formed two H-bond interactions with Val882 inside the PI3K active site, and it formed two arene-cation interactions with Lys89 inside the CDK2 active site like the co-crystallized ligands.These outcomes corroborated the kinase inhibition experiment findings.Previous literature reported the downstream inhibition pathway of EGFR/PI3K/AKT, which is linked to CDK2 inhibition, as a promising target for inducing apoptosis in cancer cells [32].(IP) throughout the experiment to confirm its anticancer efficacy, as shown in Figure 5, which summarizes the main findings of the antitumor activity experiments.As a result, tumor proliferation revealed an increase in solid tumor mass of approximately 398.1 mg, which is related to tumor proliferation.Following treatment with 5, the solid tumor mass decreased to 126.5 mg, compared to 110 mg in the 5-FU treatment.As a result, treatments with 5 considerably reduced tumor volume from 10 6 mm 3 in the untreated control to 56.4 mm 3 and significantly decreased tumor proliferation by 46.9%, while 5-FU reduced tumor volume to 43.7 mm 3 and inhibited tumor development by 58.8%.

Molecular Docking
To illustrate the virtual mechanism of binding towards the EGFR, PI3K, and CDK2 binding sites, molecular docking research was carried out.As seen in Figure 6, compound 5 was properly docked inside the protein active sites of EGFR (A), PI3K (B), and CDK2 (C), with binding energies of −23.15, −21.32, and −23.44 Kcal/mol, and it formed good binding interactions with their active sites.Compound 5 exhibited strong binding interactions with the amino acids Lys721, Cys773, and Leu694 inside EGFR.It formed two H-bond interactions with Val882 inside the PI3K active site, and it formed two arene-cation interactions with Lys89 inside the CDK2 active site like the co-crystallized ligands.These outcomes corroborated the kinase inhibition experiment findings.Previous literature reported the downstream inhibition pathway of EGFR/PI3K/AKT, which is linked to CDK2 inhibition, as a promising target for inducing apoptosis in cancer cells [32].As summarized in Figure 7, compound 5, as an indolyl-hydrazone derivative, induced potent cytotoxicity against MCF-7 as an apoptosis inducer through the downstreaming pathway of EGFR/PI3K/AKT and CDK2 inhibition.The effective pathway induced cell cycle arrest at the S-phase, and it led to apoptosis in the MCF-7 cells.EGFR, and its downstreaming pathway is considered one of the promising effective pathways As summarized in Figure 7, compound 5, as an indolyl-hydrazone derivative, induced potent cytotoxicity against MCF-7 as an apoptosis inducer through the downstreaming pathway of EGFR/PI3K/AKT and CDK2 inhibition.The effective pathway induced cell cycle arrest at the S-phase, and it led to apoptosis in the MCF-7 cells.EGFR, and its downstreaming pathway is considered one of the promising effective pathways for cancer treatment, and our results agreed with previous reported studies for the same compounds' scaffold affecting cytotoxic activities through apoptosis [33][34][35].for cancer treatment, and our results agreed with previous reported studies for the same compounds' scaffold affecting cytotoxic activities through apoptosis [33][34][35].

SAR
The structure-reactivity relationship of the synthesized compounds is summarized as follows in Figure 8: The hydrazone derivative 10, featuring a p-nitro group-substituted benzene ring, exhibited the lowest reactivity, with an IC50 value of 25.4 ± 1.54 µM.In contrast, the aldehyde-based indole derivative 1, the starting material, demonstrated better reactivity, with an IC50 value of 19.7 ± 2.31 µM.The thiosemicarbazide 3 and its isosteric semicarbazide 6 enhanced reactivity, with IC50 values of 9.42 ± 0.57 and 10.2 ± 0.53 µM, respectively.The presence of two nitro groups on the substituted benzene ring of hydrazone 12 significantly improved reactivity, with an IC50 value of 7.03 ± 0.37 µM, due to the high electron-withdrawing group effect.The introduction of the thio-triazole indolebased Schiff base 8 significantly increased activity (IC50 = 4.38 ± 0.23 µM) up to 1.9-fold higher than the reference drug, while the symmetrical bis-esters azine 5 emerged as the most potent compound in inhibiting breast cancer cells, with an IC50 of 2.73 ± 0.14 µM, 3fold more potent than the standard drug staurosporine (IC50 = 8.32 ± 0.43 µM).

SAR
The structure-reactivity relationship of the synthesized compounds is summarized as follows in Figure 8: The hydrazone derivative 10, featuring a p-nitro group-substituted benzene ring, exhibited the lowest reactivity, with an IC 50 value of 25.4 ± 1.54 µM.In contrast, the aldehyde-based indole derivative 1, the starting material, demonstrated better reactivity, with an IC 50 value of 19.7 ± 2.31 µM.The thiosemicarbazide 3 and its isosteric semicarbazide 6 enhanced reactivity, with IC 50 values of 9.42 ± 0.57 and 10.2 ± 0.53 µM, respectively.The presence of two nitro groups on the substituted benzene ring of hydrazone 12 significantly improved reactivity, with an IC 50 value of 7.03 ± 0.37 µM, due to the high electron-withdrawing group effect.The introduction of the thio-triazole indole-based Schiff base 8 significantly increased activity (IC 50 = 4.38 ± 0.23 µM) up to 1.9-fold higher than the reference drug, while the symmetrical bis-esters azine 5 emerged as the most potent compound in inhibiting breast cancer cells, with an IC 50 of 2.73 ± 0.14 µM, 3-fold more potent than the standard drug staurosporine (IC 50 = 8.32 ± 0.43 µM).

General
The values for the melting points were uncorrected and were determined in open capillaries using a Temp-melt II melting point equipment.On silica gel 60 (230-400 mesh ASTM), flash chromatography was carried out.On silica gel 60 F254 aluminum plates (E.Merck, layer thickness 0.2 mm), thin-layer chromatography (TLC) was performed.The spots were found using a UV lamp.Using DMSO-d6 and CDCl3 as solvents, the 1 H and 13 C-NMR spectra were captured on Bruker instruments at 400 MHz for 1 H NMR and 101 MHz for 13 C NMR, respectively.Using KBr and a PerkinElmer 1430 ratio-recording infrared spectrophotometer, Bruker's Fourier-transform infrared (FT-IR) spectrophotometry was used to record the IR spectra.

Synthesis
A mixture of 1 (1.0 mmol, 0.22 g), thiosemicarbazide, and semicarbazide.HCl (1.1 mmol, 0.1 g, and 0.12 g respectively) was grinded and fused on a hotplate for 5 min until all reactants turned to products.The products were purified by recrystallization from DMF/EtOH to 3 and 5, respectively.

General
The values for the melting points were uncorrected and were determined in open capillaries using a Temp-melt II melting point equipment.On silica gel 60 (230-400 mesh ASTM), flash chromatography was carried out.On silica gel 60 F254 aluminum plates (E.Merck, layer thickness 0.2 mm), thin-layer chromatography (TLC) was performed.The spots were found using a UV lamp.Using DMSO-d 6 and CDCl 3 as solvents, the 1 H and 13 C-NMR spectra were captured on Bruker instruments at 400 MHz for 1 H NMR and 101 MHz for 13 C NMR, respectively.Using KBr and a PerkinElmer 1430 ratio-recording infrared spectrophotometer, Bruker's Fourier-transform infrared (FT-IR) spectrophotometry was used to record the IR spectra.

Synthesis
A mixture of 1 (1.0 mmol, 0.22 g), thiosemicarbazide, and semicarbazide.HCl (1.1 mmol, 0.1 g, and 0.12 g respectively) was grinded and fused on a hotplate for 5 min until all reactants turned to products.The products were purified by recrystallization from DMF/EtOH to 3 and 5, respectively.

X-ray Structure Determination
The general protocol for the collection of crystalline compounds 3 and 5 is provided in the supporting materials [36][37][38].
All cells were cultured, following routine tissue culture work, in 5% CO 2 humidified at 37 • C. Cells were exposed to compounds at concentrations of 0.01, 0.1, 1, 10, and 100 µM on the second day of culturing.After 48 h, cell viability was evaluated using the MTT solution (Promega, Madison, WI, USA) [38].MTT dye (20 µL) was placed into each well, and the plate was then incubated for three hours.Absorbance was subsequently measured at 570 nm using the ELISA microplate reader (BIO-RAD, model iMark, Tokyo, Japan), and the percentage of cell viability was calculated, compared to control, as (mean absorbance of tested compound)/(mean absorbance in control) × 100.Finally, IC 50 values were found using the nonlinear dose-response sigmoidal curve in GraphPad Prism 7 [39].

Investigation of Apoptosis
Annexin V/PI staining and cell cycle analysis 3-10 5 MCF-7 cells were added to 6well culture plates, which were then placed in the incubator for the night.Following that, cells were treated for 48 h to compound 5 at its IC 50 levels.Following that, PBS was rinsed with ice-cold water before cells and media supernatants were gathered.The cells were then treated with "Annexin V-FITC solution (1:100) and propidium iodide (PI)" at a concentration of 10 g/mL for 30 min in the dark after being suspended in 100 L of annexin-binding buffer solution, which was composed of 25 mM CaCl 2 , 1.4 M NaCl, and 0.1 M Hepes/NaOH, pH 7.4.Then, labeled cells were collected using the Cytoflex FACS system.The data were assessed using the cytExpert program [39].

Wound-Healing Assay (Scratch Assay)
The wound-healing test was mentioned in previous research [40,41].Six-well plates containing starvation media were filled with four 105 MCF-7 cells per well, and the plates were subsequently incubated at 37 • C for the whole night.A sterile 1 mL pipette tip was used to generate a scratch of the cell monolayer once it was established the following day that the cells had adhered to the well and that cell confluence had reached 90%.Starvation media was used to clean the cells before they were removed from the plates.For 48 h, the cells were cultivated in a CO 2 incubator with the IC 50 of compounds 5, 8, and 12 in the full medium.After 48 h, the medium was immediately changed to PBS, the wound gap was examined, and cells-both control and treated-were captured on camera with a digital camera attached to an Olympus microscope.The region where the wound closes was measured [42,43].

Kinase Inhibitory Assays
EGFR (catalog #40321), CDK2 (catalog #79599), AKT (catalog #78038), PI3K-α (catalog #40639), β (catalog #79802), and δ (catalog #40628) kinase inhibitions were conducted using an ELISA kit in accordance with the manufacturer instructions from Bioscience, USA.To assess the inhibitory potency of compound 5 against the kinase activity, kinase inhibitory tests were carried out.The following calculation was used to compute the proportion that chemicals inhibited autophosphorylation: 100 − [(A Control )/(A Treated ) − A Control ].Using the GraphPad prism7 program, the IC 50 was calculated using the curves of percentage inhibition of five concentrations of each chemical [44].

In Vivo (SEC-Bearing Model)
The Suez Canal University Research Ethics Committee gave the experimental procedure their seal of approval (approval number REC219/2023, Faculty of Science, Suez Canal University) [45,46].The full, detailed methodology is supported in the Supplementary Materials.

Figure 1 .
Figure 1.Selected indole structures and drugs that possess anticancer activity.

Figure 1 .
Figure 1.Selected indole structures and drugs that possess anticancer activity.

Figure 3 .
Figure 3. Migration of MCF-7 cells treated with compound 5 for 72 h observed under a light microscope as detected by the wound-healing assay.

Figure 3 .
Figure 3. Migration of MCF-7 cells treated with compound 5 for 72 h observed under a light microscope as detected by the wound-healing assay.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 8 of 17 illustrates, whereas cells in other phases decreased negligibly.Consequently, compound 5 stopped MCF-7 cells from proliferating at the S-phase by inducing apoptosis.

Figure 4 .
Figure 4. Analysis using flow cytometry.Upper panel (A): Annexin V/PI staining for evaluating necrosis and apoptosis, Q1;eEarly apoptosis is Q4, and late apoptosis is Q2.The DNA content histograms of untreated and 5-treated MCF-7 cells at each phase, "Pre-G, G1, G2/M, S" phases, with an IC50 value of 2.73 µM, 48 h, are displayed in the lower panel (B).

Figure 4 .
Figure 4. Analysis using flow cytometry.Upper panel (A): Annexin V/PI staining for evaluating necrosis and apoptosis, Q1;eEarly apoptosis is Q4, and late apoptosis is Q2.The DNA content histograms of untreated and 5-treated MCF-7 cells at each phase, "Pre-G, G1, G2/M, S" phases, with an IC 50 value of 2.73 µM, 48 h, are displayed in the lower panel (B).

Figure 5 .
Figure 5. Measurements of antitumor potentiality in the SEC-bearing mice treated with compound 5 and 5-FU."Mean ± SD values of mice in each group (n = 6)"."** Values are highly significantly different (p ≤ 0.01) between treated and SEC control", while " # values are significantly different (p ≤ 0.05) between treated SEC and SEC control mice using the un-paired test in GraphPad prism".TIR% = C − T/C × 100.

Figure 5 .Figure 6 .
Figure 5. Measurements of antitumor potentiality in the SEC-bearing mice treated with compound 5 and 5-FU."Mean ± SD values of mice in each group (n = 6)"."** Values are highly significantly different (p ≤ 0.01) between treated and SEC control", while " # values are significantly different (p ≤ 0.05) between treated SEC and SEC control mice using the un-paired test in GraphPad prism".TIR% = C − T/C × 100.Pharmaceuticals 2023, 16, x FOR PEER REVIEW 10 of 17

Figure 8 .
Figure 8. SAR for the synthesized compounds.

Figure 8 .
Figure 8. SAR for the synthesized compounds.

Table 2 .
Cytotoxicities of the investigated compounds against MCF-7 cells using the MTT assay.
*.Values are expressed as "Mean ± SD". # Significance level (p < 0.05) indicates a significant difference (unpaired Student's t-test) from the untreated control group.Data for length of migration (mm) and area are supported in the Supplementary Materials.

Table 4 .
Flow cytometry results of the three promising cytotoxic agents using Annexin V/PI and DNA-aided flow cytometry.

Table 5 .
IC 50 values of kinase activities of the tested compounds.
* "Values are expressed as an average of three independent replicates"."IC 50 values were calculated using a sigmoidal non-linear regression curve fit of percentage inhibition against five concentrations of each compound".NT = Not tested.