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

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Exploring pyrrolidinyl-spirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/CDK2 inhibitors for halting breast cancer cells © 2024 Nafie, Al-Majid, Ali, Alayyaf, Haukka, Ashraf, Ul-Haq, El-Faham and Barakat Published version Nafie, Mohamed S.; Al-Majid, Abdullah Mohammed; Ali, M.; Alayyaf, Abdulmajeed Abdullah; Haukka, Matti; Ashraf, Sajda; Ul-Haq, Zaheer; El-Faham, Ayman; Barakat, Assem Nafie, M. S., Al-Majid, A. M., Ali, M., Alayyaf, A. A., Haukka, M., Ashraf, S., Ul-Haq, Z., El-Faham, A., & Barakat, A. (2024). Exploring pyrrolidinyl-spirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/CDK2 inhibitors for halting breast cancer cells. Frontiers in Chemistry, 12, Article 1364378. https://doi.org/10.3389/fchem.2024.1364378 2024 Exploring pyrrolidinyl-spirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/ CDK2 inhibitors for halting breast cancer cells Mohamed S. Nafie 1 , 2 , Abdullah Mohammed Al-Majid 3 , M. Ali 3 , Abdulmajeed Abdullah Alayyaf 3 , Matti Haukka 4 , Sajda Ashraf 5 , Zaheer Ul-Haq 5 , Ayman El-Faham 6 * and Assem Barakat 3 * 1 Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates, 2 Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt, 3 Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia, 4 Department of Chemistry, University of Jyväskylä, Jyväskylä, Finland, 5 Dr. Panjwani Center for Molecular medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan, 6 Department of Chemistry, Faculty of Science, Alexandria University, Alexandria, Egypt 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 threecomponent 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 (IC 50 = 3.4 and 4.12 μM, respectively) and MDA-MB-231 cells (IC 50 = 8.45 and 4.32 μM, respectively) compared to Erlotinib. Conversely, compounds 5a-f displayed promising cytotoxicity against MCF-7 cells with IC 50 values range (IC 50 = 5.87–18.5 μM) with selective activity against MDA-MB-231 cancer cells. Compound 5g demonstrated the highest cytotoxicity (IC 50 = 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 IC 50 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 IC 50 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 (IC 50 = 0.03 μM, 95.4%). Furthermore, these compounds exhibited potent CDK-2 inhibition, with IC 50 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 (IC 50 = 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 OPEN ACCESS EDITED BY Xuetao Xu, Wuyi University, China REVIEWED BY Eman Nossier, Al-Azhar University, Egypt Ibrahim F. Nassar, Ain Shams University, Egypt *CORRESPONDENCE Ayman El-Faham, [email protected] Assem Barakat, [email protected] RECEIVED 02 January 2024 ACCEPTED 15 February 2024 PUBLISHED 29 February 2024 CITATION Nafie MS, Al-Majid AM, Ali M, Alayyaf AA, Haukka M, Ashraf S, Ul-Haq Z, El-Faham A and Barakat A (2024), Exploring pyrrolidinylspirooxindole natural products as promising platforms for the synthesis of novel spirooxindoles as EGFR/CDK2 inhibitors for halting breast cancer cells. Front. Chem. 12:1364378. doi: 10.3389/fchem.2024.1364378 COPYRIGHT © 2024 Nafie, Al-Majid, Ali, Alayyaf, Haukka, Ashraf, Ul-Haq, El-Faham and Barakat. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Chemistry frontiersin.org01 TYPE Original Research PUBLISHED 29 February 2024 DOI 10.3389/fchem.2024.1364378 3, 8, and 9 with notable fold changes while decreasing the expression of the antiapoptotic 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. KEYWORDS spirooxindole, [3+2] cycloaddition, breast cancer (MCF-7 and MDA-MB-231), EGFR, CDK-2, molecular dynamics Introduction Cancer, a widespread and intricate group of diseases, presents asignificant 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. FIGURE 1 Naturally occurring spirooxindole compounds possessing anti-tumor properties. Frontiers in Chemistry frontiersin.org02 Nafieetal. 10.3389/fchem.2024.1364378 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). Figure 1 depicts diverse spirooxindole frameworks sourced from nature, showcasing potent anti-cancer activity (Yu et al., 2015). Of particular note is Spirobrassinin, an oxindole alkaloid renowned for its robust anti-tumor properties (Budovská et al., 2020), along with spindomycins A and B, identified as potential inhibitors of the tyrosine kinase Bcr-Abl (Guo et al., 2014). Spirotryprostatins A and B exhibit noteworthy inhibitory effects against mouse breast cancer, specifically targeting tsFT210 (Ding et al., 2005;Al-Rashood et al., 2020). Pteropodine and Uncarine F have shown robust inhibitory effects against CEM-C7H2 cells, whereas Mitraphylline has exhibited significant inhibitory activity against various cancer cell lines, including neuroblastoma SKN-BE, glioma GAMG, human Ewing’s sarcoma MHH-ES-1, and breast cancer MT-3 cells in a dose-dependent manner (García Giménez et al., 2010). Strychnofoline is an additional example, demonstrating efficacy against melanoma and Ehrlich tumor cells (Yu et al., 2018; Yuenyongsawad et al., 2013). Mitraphylline, Uncarine F, and Pteropodine represent natural spirooxindole alkaloids extracted from Uncaria tomentosa (Bacher et al., 2006). 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)andspiro(Al-Jassas, et al., 2023;Barakat, et al., 2023)anticancer 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 Frontiers in Chemistry frontiersin.org03 Nafieetal. 10.3389/fchem.2024.1364378 thioproline 1were reacted under refluxed conditions in MeOH for 2 h, resulting in the desired stereo-selective bi-spirooxindole- incorporated rhodanine analog 5a-o. The reaction proceeded in two steps: first, isatin derivatives 2a-e reacted with secondary amino acid (thioproline) 1to 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 FIGURE 2 Rational design inspired by both natural products and novel synthetic spirooxindoles. SCHEME 1 Synthesis of compounds 5a-o via a 32CA reaction of AY 3a-e with ethylene derivative 4a-f. Frontiers in Chemistry frontiersin.org04 Nafieetal. 10.3389/fchem.2024.1364378 C9, C12, C13 and C14 atoms. This indicated and assigned the absolute configuration of the final spiroxindoles adduct. Cytotoxic activity The cytotoxicity of the synthesized compounds was tested using the MTT assay on breast cancer cells (MCF-7 and MDAMB-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 =2.14µM),andthey exhibited potent cytotoxicity against MDA-MB-231 with IC 50 values range of 4.3–8.4 μM compared Erlotinib (IC 50 =3.25µM). 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 39.33–47.2 μM. EGFR/CDK-2 inhibition Inhibitory activities of 5g, 5l, and 5n were tested against EGFR and CDK-2. Interestingly, as seen in Table 2, they exhibited potent EGFR inhibition, with IC 50 values of 0.026, 0.067, and 0.04 μM with percentages of inhibition of 92.6%, 89.8%, 91.2% compared to Erlotinib (IC 50 = 0.03 μM, 95.4%). Additionally, they exhibited potent CDK-2 inhibition, with IC 50 values of 0.301, 0.345, and 0.557 μM with percentages of inhibition of 91.9%, 89.4%, 88.7% compared to Roscovitine (IC 50 = 0.556 μM, 92.1%). These findings highlight the promising EGFR/CDK-2 enzyme inhibition. 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. FIGURE 3 ORTEP for compound 5e. Frontiers in Chemistry frontiersin.org05 Nafieetal. 10.3389/fchem.2024.1364378 TABLE 1 Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay. Chemical structures IC 50 ±SD[μM] MCF-7 MDA-MB-231 WISH 5.87 ± 0.5 29.5 ± 1.3 NT 7.8 ± 0.32 32.5 ± 0.42 NT 15.8 ± 0.4 24.2 ± 1.1 NT 19.4 ± 0.4 9.8 ± 0.35 NT (Continued on following page) Frontiers in Chemistry frontiersin.org06 Nafieetal. 10.3389/fchem.2024.1364378 TABLE 1 (Continued) Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay. Chemical structures IC 50 ±SD[μM] MCF-7 MDA-MB-231 WISH 9.8 ± 0.7 5.4 ± 0.6 NT 18.5 ± 0.6 35.5 ± 1.1 NT 2.8 ± 0.4 23.5 ± 0.9 39.33 ± 1.8 24.3 ± 0.9 31.5 ± 1.0 NT (Continued on following page) Frontiers in Chemistry frontiersin.org07 Nafieetal. 10.3389/fchem.2024.1364378 TABLE 1 (Continued) Cytotoxicity of the tested compounds against MCF-7 and MDA-MB-231 breast cancer cells using the MTT assay. Chemical structures IC 50 ±SD[μM] MCF-7 MDA-MB-231 WISH 28.7 ± 0.8 35.3 ± 1.2 NT 23.4 ± 1.2 21.4 ± 0.8 NT 21.4 ± 0.9 19.5 ± 0.7 NT 3.4 ± 0.5 8.43 ± 0.6 43.5 ± 2.0 (Continued on following page) Frontiers in Chemistry frontiersin.org08 Nafieetal. 10.3389/fchem.2024.1364378 135.57, 133.42, 131.58, 129.60, 125.81, 123.36, 92.33, 79.20, 76.28, 75.75, 59.09, 58.38, 55.59, 52.35, 52.30, 46.85, 40.80, 40.68, 40.59, 40.48, 40.38, 40.27, 40.17, 40.06, 39.85, 39.72, 39.64, 39.43, 31.40, 31.30, 31.20, 31.10; Chemical Formula: C 29 H 28 BrFN 4 O 6 S 2 ;LCMS(m/z): 692.59 [M + H] + , Elemental Analysis: [Calculated: C, 50.37; H, 4.08; N, 8.10; S, 9.27; Found: C, 50.36; H, 4.09; N, 8.07; S, 9.29]. FIGURE 9 RMSD, RMSF and RoG of the CDK2 systems calculated as a function of time. FIGURE 10 RMSD, RMSF and RoG of the EGFR systems calculated as a function of time Based on the visual examination of simulation trajectories, it can be inferred that compound 5n achieves stability within the cavity of CDK2 and EGFR by effectively facilitating hydrophobic and hydrophilic contacts with the active site residue. The examination of time-varying paths of 5n reveals intriguing findings. The polar atoms of the dioxothiazolidin moiety maintain contact with the protein through polar interactions with Glu12 and Lys89, with occupancies of 65% and 55%, respectively. Frontiers in Chemistry frontiersin.org15 Nafieetal. 10.3389/fchem.2024.1364378 Methyl (2-((3S,6′S,7′S,7a′S)-6-chloro-7’-(4- chlorophenyl)-2,2″,4″-trioxo-7′,7a′- dihydro-1′H,3′H-dispiro [indoline-3,5′- pyrrolo [1,2-c]thiazole-6′,5″-thiazolidin]- 3″-yl)acetyl)alaninate 5n The 6-chloro-isatin 2b (90.5 mg) and 4d (221.5 mg) were utilized according to the general method, and the spirocompound 5n was obtained in 84% yield. 1 H NMR (400 MHz, DMSO-d 6 )δ11.18 (d, J= 5.0 Hz, 1H), 8.63 (t, J= 6.8 Hz, 1H), 7.72–7.59 (m, 1H), 7.47 (d, J= 8.1 Hz, 2H), 7.41 (d, J= 8.2 Hz, 2H), 7.25 (t, J= 8.9 Hz, 1H), 7.05 (t, J= 9.4 Hz, 1H), 6.90 (s, 1H), 4.82 (q, J= 7.9, 7.4 Hz, 1H), 4.35–4.23 (m, 1H), 4.21–4.12 (m, 1H), 4.10–3.91 (m, 2H), 3.82 (d, J= 6.0 Hz, 1H), 3.64 (d, J= 6.7 Hz, 3H), 3.49–3.41 (m, 1H), 3.01 (t, J= 7.7 Hz, 1H), 1.28 (q, J= 7.1 Hz, 3H); 13 C NMR (101 MHz, DMSO-d 6 )δ181.16, 176.20, 174.37, 172.16, 169.71, 167.74, 164.67, 151.40, 143.74, 135.98, 135.63, 135.05, 133.41, 132.31, 131.02, 126.13, 125.81, 123.05, 112.07, 94.30, 84.51, 76.27, 75.58, 59.25, 54.52, 52.58, 48.11, 46.69, 40.71, 40.63, 40.51, 40.30, 40.09, 39.88, 39.67, 39.46, 17.74; Chemical Formula: C 27 H 24 Cl 2 N 4 O 6 S 2 ; LCMS (m/z): 636.59 [M + H] + , Elemental Analysis: [Calculated: C, 51.03; H, 3.81; N, 8.82; S, 10.09; Found: C, 51.03; H, 3.81; N, 8.82; S, 10.09]. Methyl 4-(2-((3S,6′S,7′S,7a′S)-7’-(4- bromophenyl)-6-chloro-2,2″,4″-trioxo- 7′,7a′-dihydro-1′H,3′H-dispiro [indoline- 3,5′-pyrrolo [1,2-c]thiazole-6′,5″- thiazolidin]-3″-yl)acetamido)butanoate 5o The 6-chloro-isatin 2b (90.5 mg) and 4e (228.5 mg) were utilized according to the general method, and the spirocompound 5o was obtained in 81% yield. 1 H NMR (400 MHz, DMSO-d 6 )δ11.18 (s, 1H), 8.16 (t, J= 5.6 Hz, 1H), 7.68 (d, J= 8.6 Hz, 0H), 7.63 (d, J= 8.7 Hz, 0H), 7.52–7.38 (m, 5H), 7.26 (d, J= 8.5 Hz, 1H), 7.06 (dd, J= 8.4, 2.3 Hz, 1H), 6.90 (d, J= 2.1 Hz, 1H), 4.82 (q, J= 7.7, 7.2 Hz, 1H), 4.29–4.12 (m, 2H), 4.07–3.77 (m, 3H), 3.45 (d, J= 6.1 Hz, 1H), 3.13–3.00 (m, 3H), 3.05–2.96 (m, 1H), 2.77 (dd, J= 9.8, 7.6 Hz, 1H), 1.72–1.57 (m, 3H); 13 C NMR (101 MHz, DMSO-d 6 )δ176.22, 174.78, 173.62, 173.08, 168.74, 167.22, 165.52, 164.72, 145.38, 136.00, 135.64, 133.40, 132.33, 129.46, 129.42, 123.04, 121.61, 112.52, 112.22, 76.19, 75.48, 70.60, 63.64, 56.11, 51.86, 50.22, 48.78, 47.03, 43.86, 40.71, 40.63, 40.50, 40.47, 40.29, 40.09, 39.88, 39.67, 39.54, 39.46, 38.62, 32.26, 31.11, 24.92; Chemical Formula: C 28 H 26 BrClN 4 O 6 S 2 ; LCMS (m/z): 695.02 [M + H] + , Elemental Analysis: [Calculated: C, 48.46; H, 3.78; N, 8.07; S, 9.24; Found: C, 48.49; H, 3.80; N, 8.11; S, 9.25]. 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. Biological investigations The methods for the Cytotoxic activity (Mosmann, 1983;Nafie et al., 2020a); EGFR/CDK-2 enzyme inhibition (Nafie et al., 2022a); Flow cytometry using Annexin V/PI staining; Gene expression analysis using RT-PCR (Nafie et al., 2022b); are amended 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. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. Author contributions MN: Formal Analysis, Methodology, Software, Writing–review and editing. AA-M: Supervision, Visualization, Writing–review and editing. MA: Investigation, Methodology, Writing–review and editing. AA: Investigation, Methodology, Writing–review and editing. MH: Data curation, Formal Analysis, Software, Writing–review and editing. SA: Data curation, Investigation, Software, Validation, Writing–review and editing. ZU-H: Data curation,Investigation,Software, Validation, Writing–review and editing. AE-F: Methodology, Supervision, Writing–review and editing. AB: Funding acquisition, Methodology, Project administration, Writing–original draft, Writing–review and editing. Frontiers in Chemistry frontiersin.org16 Nafieetal. 10.3389/fchem.2024.1364378 Funding The author(s) declare financial support was received for the research, authorship, publication of this article. The authors would like to extend their sincere appreciation to the Researchers Supporting Project (RSP 2024R64), King Saud University, Riyadh, Saudi Arabia. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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