Exploring pyrrolidinyl-spirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/CDK2 inhibitors for halting breast cancer cells

Cancer represents a global challenge, and the pursuit of developing new cancer treatments that are potent, safe, less prone to drug resistance, and associated with fewer side effects poses a significant challenge in cancer research and drug discovery. Drawing inspiration from pyrrolidinyl-spirooxindole natural products, a novel series of spirooxindoles has been synthesized through a one-pot three-component reaction, involving a [3 + 2] cycloaddition reaction. The cytotoxicity against breast cancer cells (MCF-7 and MDA-MB-231) and safety profile against WISH cells of the newly developed library were assessed using the MTT assay. Compounds 5l and 5o exhibited notable cytotoxicity against MCF-7 cells (IC50 = 3.4 and 4.12 μM, respectively) and MDA-MB-231 cells (IC50 = 8.45 and 4.32 μM, respectively) compared to Erlotinib. Conversely, compounds 5a-f displayed promising cytotoxicity against MCF-7 cells with IC50 values range (IC50 = 5.87–18.5 μM) with selective activity against MDA-MB-231 cancer cells. Compound 5g demonstrated the highest cytotoxicity (IC50 = 2.8 μM) among the tested compounds. Additionally, compounds 5g, 5l, and 5n were found to be safe (non-cytotoxic) against WISH cells with higher IC50 values ranging from 39.33 to 47.2 μM. Compounds 5g, 5l, and 5n underwent testing for their inhibitory effects against EGFR and CDK-2. Remarkably, they demonstrated potent EGFR inhibition, with IC50 values of 0.026, 0.067, and 0.04 μM and inhibition percentages of 92.6%, 89.8%, and 91.2%, respectively, when compared to Erlotinib (IC50 = 0.03 μM, 95.4%). Furthermore, these compounds exhibited potent CDK-2 inhibition, with IC50 values of 0.301, 0.345, and 0.557 μM and inhibition percentages of 91.9%, 89.4%, and 88.7%, respectively, in contrast to Roscovitine (IC50 = 0.556 μM, 92.1%). RT-PCR analysis was performed on both untreated and 5g-treated MCF-7 cells to confirm apoptotic cell death. Treatment with 5g increased the gene expression of pro-apoptotic genes P53, Bax, caspases 3, 8, and 9 with notable fold changes while decreasing the expression of the anti-apoptotic gene Bcl-2. Molecular docking and dynamic simulations (100 ns simulation using AMBER22) were conducted to investigate the binding mode of the most potent candidates, namely, 5g, 5l, and 5n, within the active sites of EGFR and CDK-2.


Introduction
Cancer, a widespread and intricate group of diseases, presents a significant challenge in global healthcare, impacting millions of lives (Sung et al., 2021).Characterized by uncontrolled cell growth, it forms malignant tumors and persists as a major public health concern despite medical advancements.The multifaceted nature of cancer involves diverse forms and complex interactions of genetic, environmental, and lifestyle factors.The problem's gravity is highlighted by its global prevalence and substantial emotional, economic, and healthcare burdens on individuals and communities.Urgency is fueled by rising incidence rates and the ongoing search for effective treatments and prevention.The introduction emphasizes the need to understand cancer's intricacies for developing innovative therapies and prevention strategies, setting the stage for exploring its multifaceted aspects and addressing the challenges it poses to public health improvement.
Among women globally, breast cancer stood out as the predominant form of cancer, comprising 30% of the total newly diagnosed cases in the year 2021 (Sung et al., 2021).Breast cancer cell lines, such as MCF-7 and MDA-MB-231, play a crucial role in cancer research.Derived from breast cancer tumors, these cell lines serve as invaluable tools for studying the disease's biology, testing treatments and understanding molecular mechanisms.They are cultured in laboratories, allowing researchers to investigate various aspects of breast cancer, including genetic makeup, treatment responses, and drug resistance.These cell lines are vital for the preclinical testing of new therapies, contributing to developing more effective treatments for breast cancer patients.
The distinct structural framework of these compounds, characterized by a spiro ring fusion at position-3 of the oxindole, gives rise to their diverse biological activities.This arrangement enables the oxindole moiety to serve as either a hydrogen bond donor or acceptor, thereby augmenting its interactions with diverse biological targets.Furthermore, their adaptability in forming combinations with various bioactive cycloalkyl or heterocyclic motifs substantially boosts their effectiveness across various applications (Zhou et al., 2020).
Spirotryprostatin A (Cui, et al., 1996;Edmondson et al., 1999) is an example of a neutrally occurring spirooxindole scaffold targeting breast cancer cells.Derived from the tryprostatin alkaloid family (Islam et al., 2023;Marti and Carreira, 2003), this compound exhibits notable inhibitory effects against breast cancer cell lines.The unique structural features of the spirooxindole scaffold, including a spiro ring fusion at position-3 of the oxindole, contribute to its ability to interact with biological targets in breast cancer cells.Studies suggest that Spirotryprostatin A hinders breast cancer cell proliferation and induces apoptosis, making it a potential candidate for further exploration in the development of targeted breast cancer therapies.The compound exemplifies the potential of spirooxindole scaffolds in the quest for innovative and effective treatments for breast cancer.
Cyclin-Dependent Kinase 2 (CDK2) (Tadesse, et al., 2018;Golsteyn, 2005) is a compelling target in cancer therapy due to its crucial role in regulating the cell cycle, particularly the transition from G1 to S phase.Aberrant activation of CDK2 is associated with uncontrolled cell proliferation in various cancers.Inhibitors designed to selectively target CDK2 have shown promise in preclinical and clinical settings by inducing cell cycle arrest and triggering apoptosis in cancer cells.Targeting CDK2 offers a strategic approach to impede cancer cell division, and ongoing research aims to optimize CDK2 inhibitors for enhanced efficacy and reduced side effects, highlighting its potential as an innovative avenue in cancer therapy.We built upon the groundwork established by benchmark oxindolebased CDK2 inhibitors (I) (Luk, 2004;Venkanna, et al., 2020;Bramson, et al., 2001) and spiro (Al-Jassas, et al., 2023;Barakat, et al., 2023) anti-cancer agents recognized for their kinase inhibition, specifically those aimed at CDK2.This rational study involved a detailed exploration of the CDK2 inhibitory potential within the investigated series, as depicted in Figure 2. Al-Jassas (Al-Jassas, et al., 2023) designed, synthesized, and assessed a novel spirooxindole scaffold for its dual inhibitory properties against CDK2 and EGFR.Compound II exhibited notable inhibition, with IC 50 values of 0.189 ± 0.01 µM (MCF-7) and 1.04 ± 0.21 µM (HepG2).Additionally, it demonstrated potent CDK-2 inhibition (34.98 nM) and an IC 50 of 96.6 nM for EGFR inhibition.Compound II also effectively modulated the expression of pro-apoptotic genes (P53, Bax, caspases-3, 8, and 9) while downregulating the anti-apoptotic gene Bcl-2.
Barakat research group has reported a combinatorial stereoselective synthesis of rationally designed spiroindeno [1,2-b] quinoxaline-based CDK2 inhibitors III for non-small cell lung cancer (NSCLC) therapy (Barakat, et al., 2023).Among the derivatives tested, hit III emerged as the most promising, exhibiting potent inhibitory effects against A549 cells and normal lung fibroblasts Wi-38, with an IC 50 value of 54 nM and a selectivity index (SI) of 6.64.
Biao Wang et al. (Wang, et al., 2020) identified the THN-fused spirooxindole derivative, IV, as a potent inhibitor using a rational drug design approach, complemented by the asymmetric synthesis of the designed compounds.Notably, IV exhibited robust inhibitory effects on both MDM2 and CDK4 in glioblastoma cells expressing either wild-type or mutant P53.Molecular dynamics simulations suggested a tight binding affinity of IV to both MDM2 and CDK4.Furthermore, IV demonstrated the ability to induce substantial apoptosis and G1 phase cell cycle arrest.
Based on the aforementioned findings, this study explores the realm of pyrrolidinyl-spirooxindole natural products, drawing inspiration from their distinctive chemical structures for the potential development of therapeutic agents.These naturally occurring compounds serve as intriguing templates, providing valuable insights into the design of novel medications.The investigation aims to unveil the therapeutic potential inherent in pyrrolidinyl-spirooxindoles, with the goal of developing innovative and effective therapeutic agents for diverse medical applications (Galliford and Scheidt., 2007).The study involves the synthesis and evaluation of a new set of spirooxindoles against breast cancer cells, along with an assessment of their inhibitory activities against CDK2 and EGFR.Additionally, the study explores apoptotic cell death, pro-apoptotic genes, and anti-apoptotic gene assays.Finally, molecular docking and dynamic simulations are employed to investigate the binding modes of the most potent candidates within the active sites of EGFR and CDK-2.

Results and discussion
Scheme 1 illustrates the efficient and highly selective synthesis of the targeted bi-spirooxindole-incorporated rhodanine analog.The starting material chalcones based rhodanine motif 4a-f, was synthesized following a literature-reported method (Barakat et al., 2021).Employing a one-pot multicomponent 32CA reaction, the arylidene rhodanine analogue 4a-f, isatin derivatives 2a-e, and thioproline 1 were reacted under refluxed conditions in MeOH for 2 h, resulting in the desired stereo-selective bi-spirooxindoleincorporated rhodanine analog 5a-o.The reaction proceeded in two steps: first, isatin derivatives 2a-e reacted with secondary amino acid (thioproline) 1 to generate the azomethine ylide (AY).In the second step, the generated azomethine ylide (AY 3) reacted with arylidene rhodanine analog 4a-f through completely ortho regioselective and exo stereoiselective.Spectral data analysis and elucidation confirmed the proposed structure, and single crystal X-ray diffraction analysis further validated the chemical structure.

Crystal structure description
The X-ray structure of the studied compound 5e (Figure 3) revealed the formation of the target organic hybrid, which crystallized with one molecule of methanol as a crystal solvent.It crystallized in monoclinic crystal system and P2 1 /n as a space group.The unit cell parameters are a = 11.5475(3), b = 15.7550(4) c = 14.6493 (3) Å and β = 104.655(2)˚.There is one molecule as asymmetric formula while z = 4.It is evident from the reported X-ray structure the presence of four stereogenic centers located at

Cytotoxic activity
The cytotoxicity of the synthesized compounds was tested using the MTT assay on breast cancer cells (MCF-7 and MDA-MB-231).As seen in Table 1, compounds 5l-5o showed potent cytotoxicity against MCF-7 cells with IC 50 values range of 3.4-4.5 μM compared to Erlotinib (IC 50 = 2.14 µM), and they exhibited potent cytotoxicity against MDA-MB-231 with IC 50 values range of 4.3-8.4μM compared Erlotinib (IC 50 = 3.25 µM).Compounds 5a-f showed promising cytotoxicity against MCF-7 cells with IC 50 values range of 5.87-18.5 μM, with selective cytotoxicity against MDA-MB-231 cancer cells with higher IC 50 values.Interestingly, compound 5g had the highest cytotoxicity among the tested compounds, with IC 50 value of 2.8 μM.Furthermore, potent compounds 5g, 5l, and 5n were safe (non-cytotoxic) against the WISH cells with higher IC 50 values with an IC 50 value range of 39.33-47.2μM.

Apoptotic investigation Annexin V/PI staining with cell cycle analysis
The apoptotic activity of compounds 5g was determined by flow cytometric analysis of Annexin V/PI staining of untreated and treated MCF-7 cells.Figure 4A) showed that compounds 5g significantly activated apoptotic cell death, increasing the cell population in total apoptosis by 31.9% (10.15% late and 21.87% early apoptosis) compared to the untreated control group (1.98%).Additionally, they induced necrotic cell death by 5.43% compared to 2.12% in the untreated control.Hence, compound 5g-treatment induced apoptosis more than necrotic cell death.
Additionally, As can be shown in Figure 4B), the cell population in the G0-G1-phase was considerably raised by 39.8% after treatment with compound 5g, compared to the control 31%, whereas the cell population in the S-phase was significantly increased by 45.2% after treatment compared to the control 32.1%, hence, in contrast, cells population at G2/M phase were decreased upon treatment.

RT-PCR gene expression of apoptosisrelated genes
Both the untreated and treated MCF-7 cells were subjected to RT-PCR to confirm apoptotic cell death (Figure 5).The expression of pro-apoptotic genes P53, Bax, caspases 3, 8, and 9 was upregulated by 5g treatment, with corresponding fold changes of 4. 1, 6.26, 9.2, 1.7, and 6.13, respectively.Concurrently, it resulted in a 0.39-fold reduction in the expression of the anti-apoptotic gene Bcl-2.These findings are in line with the possibility of triggering cell death by blocking enzymes.Activation of the intrinsic apoptotic pathway leads to mitochondrial potential loss and cytochrome c release.When the ratio of proteins that promote cell death to those that prevent it rises, a cascade reaction involving caspases 3 and 9 is set in motion, leading to cell death by caspase-dependent apoptosis.Structure-Activity Relationship (SAR) analysis is pivotal in understanding the correlation between the structural features of the synthesized spirooxindoles and their biological activities.In the case of these compounds inspired by pyrrolidinyl-spirooxindole natural products, SAR exploration involves assessing how variations in the molecular structure impact cytotoxicity against specific cancer cell lines, inhibitory activities against enzymes like EGFR and CDK-2, and safety profiles.The goal is to identify key structural elements that contribute to therapeutic efficacy, guiding further optimization for the development of more effective compounds.The results indicate that among the synthesized library of inspired spiroxindoles, compound 5g stands out as the most active.Notably, it features a p-Cl-substituted benzene, 5-Clsubstituted oxindole, and N-substituted acid in its chemical structure (Figure 6).

Molecular docking study
Using the MOE docking suite, the most active compound of the series and the reference anti-cancer drugs roscovitine and erlotinib were docked into the active sites of the target proteins CDK2 (PDB ID 6Q4G) (Wood et al., 2019) and EGFR (PDB ID 1M17) (Stamos et al., 2002), respectively, to explore the anti-cancer potential of spirooxindole engrafted rhodanine derivatives.The binding modes of the most active compounds, 5g, 5l, and 5n, were established using MOE with binding energies ranging from −5.3 to −7.6 kcal mol-1.Figure 7A depicts the binding pose of compound 5g, where the oxygen of the dioxothiazolidin ring is involved in a hydrogen bond interaction with the nitrogen of the side chain of Lys9 at a distance of 3.2Å.The proteinligand interactions are further stabilized by hydrophobic interactions between the ligand and Ile10, Lys88, and Val163 of the CDK2 protein.
In the case of compound 5l, a strong hydrogen bond interaction is observed between the oxygen of the dioxothiazolidin ring and the side chain of Glu12 at a distance of 2.1Å (Figure 7B).Meanwhile, in the case of compound 5n, which is the most active compound of the series, two hydrogen bond interactions are exhibited with the main chain of Glu12 at distances of 1.9 and 3.4Å (Figure 7C).Another hydrogen bond is observed between the oxygen of the dioxothiazolidin ring and the side chain of Lys89 at a distance of 3.4Å.Apart from the H-bond interactions, an oxygen atom of compounds 5l and 5n participates in a salt bridge interaction with the positively charged Lys33 residue.These compounds also exhibit hydrophobic interactions with Val18, Gln131, and Val163.
In EGFR docking, the conformation exhibiting the most favorable binding energy (approximately −7.4 to −8.3 kcal mol-1) substantiates the notion that the compounds are effectively incorporated within the binding pocket.The binding patterns are also slightly different, which may be responsible for the variations in activity.It is important to mention that the reference inhibitor forms a hydrogen bond with the backbone NH of Met769 in the Hinge region, while the compound is deeply embedded into the EGFR active site via hydrophobic interactions that are conserved in the majority of the structures.In the case of compound 5g, in addition to hydrophobic interactions, a salt bridge interaction is also observed with Lys721 (Figure 8A).The plausible binding mode of compound 5l is depicted in Figure 8B.The nitrogen of the thiazole and indolin ring is involved in a hydrogen bond interaction with the oxygen of Leu694 and Arg817 at distances of 3.3 and 3.5 Å, respectively.Another hydrogen bond interaction is observed between the oxygen of the dioxothiazolidin ring and the side chain of Gly772 at a distance of 3.3 Å. Figure 8C presents the binding mode of compound 5n.This compound also exhibits three hydrogen bond interactions with Leu694, Gly772, and Arg817 at distances of 3.4, 3.3, and 2.5 Å, respectively.Further, anchorage is provided by hydrophobic interactions with Leu694, Val702, Leu768, and Leu820.
A 100 ns simulation using AMBER22 was conducted to understand the dynamic behavior of the active chemical.An evaluation of the system's overall stability and simulation quality was conducted using the RMSD, RMSF, and RG (radius of gyration) as metrics for quantitative analysis (RoG).
The RMSD of the heavy atoms in the main chain of the proteins was computed using the "rms" tool in CPPTRAJ.Figures 9,10; Supplementary Figure S10 illustrate the RMSD of the heavy atoms in the protein backbone.Figures 7, 8 clearly demonstrate the system's stability, as indicated by an average RMSD value of 2.7 Å in the case of CDK2.In the case of EGFR, higher RMSD is observed due to flexibility in its domain.This observation was further corroborated by examining the RoG, indicating that the systems were tightly compressed (Figures 7, 8).In addition, to comprehend the behavior of the side chains of residues, the RMSF of the protein was computed over time (Figures 9,10).The results indicated that the amino acid residues in the protein-ligand complex remained stable upon interaction with the active chemical of the series.
Regarding EGFR, a visual examination of the paths shows that 5n engages in hydrophobic interactions with Leu694, Val702, Leu768, and Leu820.Compound 5n demonstrates a significant affinity for the "hinge region key residue," Met769, of the EGFR target.This residue is essential for the active site and is occupied 68% of the time.Throughout the simulation, the majority of the protein-ligand interactions were observed to align with the docking position.

General notes
"Isatin derivatives 2a-e and thioproline 1 are commercially available. 1H-NMR and 13 C-NMR are recorded in DMSO-d 6 (JEOL Spectrometer (400 MHz).The X-ray diffraction data was collected on a Rigaku Oxford Diffraction Supernova diffractometer using Cu Kα radiation.The desired starting material 4a-f was synthesized according to reported literature Abd Alhameed et al., 2020).The desired sprio-compounds derived rhodanine amino-acids 5h-k was synthesized according to previous reported literature (Barakat et al., 2021)".

General procedure for the synthesis of spiro compounds analogues 5a-o
A mixture of three components reaction including substituted isatin 2a-e (0.5 mmol), L-thioproline 1 (66.5 mg, 0.5 mmol), and compounds 4a-f (0.5 mmol) were refluxed on oil bath for 2 h.After completion of the reaction, as evident from (TLC Eluent: Ethyl acetate: n-Hexane 40%), Without additional purification, the reaction mixture was left at room temperature overnight to slowly evaporate.The solid crystalline components were filtered out to give compounds 5a-o, which were solid compounds with a light faint yellow color and an 80%-90% chemical yield.

Crystal structure determination
The technical protocol and data manipulation software details (Rikagu Oxford Diffraction CrysAlisPro, 2020;Sheldrick. 2015;Hübschle et al., 2011) are available in the Supplementary Material S1.

Molecular docking and molecular dynamic simulation
The protcol for the Molecular docking and Molecular dynamic simulation are provided in in the Supplementary Material S1 ( (Wood et al., 2019;Stamos et al., 2002;Chemical Computing Group, 2013;Case et al., 2023;Khalil et al., 2019;Roe and Cheatham, 2013).

Conclusion
In conclusion, the synthesized compounds, particularly 5g, 5l, and 5n, exhibited remarkable cytotoxicity against cancer cells, with noteworthy potency against both MCF-7 and MDA-MB-231 cells.Additionally, these compounds demonstrated promising inhibitory activities against EGFR and CDK-2, showcasing their potential as dual inhibitors.The RT-PCR results further confirmed their impact on promoting apoptotic cell death by modulating the expression of key pro-apoptotic and anti-apoptotic genes.Molecular docking and dynamic simulations provided insights into the binding modes of these compounds within the active sites of EGFR and CDK-2, reinforcing their potential as therapeutic agents.Overall, this comprehensive study underscores the multifaceted potential of these compounds in cancer treatment, warranting further investigation and development.

FIGURE 2
FIGURE 2Rational design inspired by both natural products and novel synthetic spirooxindoles.

SCHEME 1
SCHEME 1Synthesis of compounds 5a-o via a 32CA reaction of AY 3a-e with ethylene derivative 4a-f.
FIGURE 4 (A) Bar representation of the percentage of apoptosis (early and late apoptotic cell population) using Annexin V/Propidium Iodide staining.(B) Bar representation of the percentage of percentage of cell population at each cell phase in untreated and 5g-treated MCF-7 cells, at the IC 50 value at 48 h "Data shown are the average of three independent experimental runs (Mean ± SD). *p ≤ 0.05 and **p < 0.001 compared to untreated control".

1 H
NMR (400 MHz, DMSO-d 6 ) δ 11.06 (s, 1H), 8.53 (t, J = 9.0 Hz, FIGURE 7 (A) Binding pose of the 5g within the active site of CDK2 protein.(B) Binding pose of the 5l within the active site of CDK2 protein.(C) Binding pose of the 5n within the active site of CDK2 protein.Interactions between hydrogen bonds are shown by the dashed black lines.

FIGURE 6
FIGURE 6Structure reactivity relationship (SAR) of the synthesized compounds.

FIGURE 8 (
FIGURE 8 (A) Binding pose of the 5g within the active site of EGFR protein.(B) Binding pose of the 5l within the active site of EGFR protein.(C) Binding pose of the 5n within the active site of EGFR protein.The dashed black line depicts hydrogen bond interaction.

FIGURE 9 RMSD
FIGURE 9RMSD, RMSF and RoG of the CDK2 systems calculated as a function of time.

TABLE 1
Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay.

TABLE 1 (
Continued) Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay.

TABLE 1 (
Continued) Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay.

TABLE 1 (
Continued) Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay.

TABLE 2
IC 50 values of EGFR and CDK-2 kinase activities of the tested compounds. ;