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Design, synthesis, anticancer activity, bioimaging, and molecular docking of novel fluorescent isatin derivativesTables: 3
Merve İnel1*, Ayse Yildirim2, Mustafa Yilmaz2, Bahadir Ozturk1
Manuscript body text: 2868 words
Garphical Abstract: ✓
Figures: 5
Supplementary Information: ✓
Mİ: minel2016@gmail.com https://orcid.org/0000-0001-5457-4294
AY: ayse.yldrm2020@gmail.com https://orcid.org/0000-0003-1219-5514
MY: myilmaz@selcuk.edu.tr https://orcid.org/0000-0003-2904-160X
BO: bahadirozturk@selcuk.edu.tr https://orcid.org/0000-0003-2654-7621
*Corresponding Author: minel2016@gmail.com
1 Department of Medical Biochemistry, Faculty of Medicine, Selcuk University, Selcuklu, 42031 Türkiye.
2 Discipline of Organic Chemistry, Department of Chemistry, Faculty of Science, Selcuk University, Konya, Türkiye.
Design, Synthesis, Anticancer Activity, Bioimaging, and Molecular Docking of Novel Fluorescent Isatin Derivatives
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ABSTRACT
Breast cancer remains a leading global health challenge, driving the urgent need for innovative therapeutic strategies. This study presents the initial results of the design, synthesis, characterization, and in vitro evaluation of a novel fluorescent agent for breast cancer treatment, focusing on its subcellular localization and molecular docking. Seven novel fluorescent compounds (3a-g) were synthesized via isatin derivatives and 4-bromo 1,8-naphthalimide conjugation. The compounds were spectroscopically characterized and tested in MDA-MB-231 and MCF-7 cells using viability assays, Annexin-V and propidium iodide flow cytometry to define cytotoxic mechanisms, confocal microscopy with nuclear and mitochondrial markers for subcellular localization, and molecular docking to VEGFR2. Several conjugates, particularly 3a and 3g in MCF-7 and 3c in MDA-MB-231, showed strong activity, while 3b was observed to be largely inactive. Docking indicated that 3c binds VEGFR2 through a binding mode distinct from sunitinib, supporting its promise as a lead together with 3a.
Graphical Abstract
Keywords:
Anticancer
drug development
fluorescent
isatin
colocalization
molecular docking
1. Introduction
According to the International Agency for Research on Cancer, cancer-related mortality is a global crisis with an estimated 20 million new cases and 9.7 million cancer deaths worldwide in 2022 [1, 2]. Breast cancer accounts for the highest share of all cancer types worldwide [3].
Fig. 1
The molecular target profiles of Sunitinib, Nintedanib and one of the novel isatin-based compounds 3c, demonstrate distinct pharmacological mechanisms, with Sunitinib and the novel 3c primarily targeting kinases, while Nintedanib also interacts with proteases. Today, the serious systemic side effects of current cancer treatments require understanding cancer metabolism and targeted drug development [4–8]. In this respect, isatin, an simple heterocyclic compound, and its derivatives have been used in studies and approved drugs including Sunitinib, Nintedanib [9–11].
Isatin, an endogenous compound found in mammalian tissues, exhibits low toxicity, mutagenicity, and genotoxicity in vivo [12]. Studies have shown that isatin and its derivatives generally comply with drug-likeness criteria and demonstrate low systemic toxicity in animal models [13, 14]. At physiological concentrations, isatin inhibits certain enzymes and receptors, while higher concentrations can induce apoptosis in various cell lines, including tumor cells [12]. Isatin derivatives have also shown potential as anti-proliferative cytostatic effects in cancer cell lines [14].Recent studies show that the presence of a substituent at position 5 of the isatin nucleus is beneficial for its antitumor activity [10, 15]. Compared to the Nintedanib, Sunitinib features a fluorine at position 5 of the isatin ring, rather than a methoxycarbonyl group at position 6 [16]. For this reason, in this study halogens, nitroisatin[17] and methylisatin at position 5 isatin were selected. Hovewer, there are limited reports on the creation of isatin-based fluorescent compounds for biological screening [18]. And the emission wavelengths of probes synthesized from 1,8-naphthalimide derivatives and benzoxazole, benzothiophene and oxazole [4,5-b]pyridine structures were increased to image fluctuations in mitochondrial viscosity [19].
1,8-naphthalimide used as a fluorophore has a long stroke shift. Stroke shift helps to distinguish between the excitation and emission wavelengths of a fluorophore [20]. We aim to synthesize an isatin–1,8-naphthalimide conjugate with strong fluorescence and a large Stokes shift to study its anticancer effects in breast cancer cells using confocal microscopy.
Isatin and its derivatives are promising anticancer agents, particularly as tyrosine kinase inhibitors. Recent studies used computational methods to design new isatin scaffolds for BCR-ABL inhibition. These compounds showed good ADMET properties and stable enzyme binding [21]. Isatin-hydrazones strongly inhibit EGFR and VEGFR-2. Docking results suggest both ATP-competitive and non-competitive mechanisms[22]. Isatin derivatives with pyridine groups show strong cytotoxicity and kinase inhibition [23]. Structure–activity studies on drugs like Sunitinib and Nintedanib stress the role of isatin nitrogen and carbonyls at positions 1 and 2 in hydrogen bonding[16]. In this study, in silico analysis demonstrates how isatin derivatives bind to tyrosine kinase enzymes.
2. Methods
2.1. Materials and reagents
All chemicals used were of analytical grade and were purchased from Merck, Aldrich, Fluka, or Sigma. Ultrapure water was obtained using a Millipore Milli-Q Plus purification system. The 1H-NMR (400MHz, CDCl3) and 13C-NMR (101MHz, (CH₃)₂SO-d₆) spectra were recorded using a Varian spectrometer and chemical shifts are reported in ppm. IR spectra were obtained using a Perkin Elmer 1605 FTIR spectrometer equipped with an ATR probe. Fluorescence spectra were recorded on a Perkin Elmer LS55 spectrometer, and UV-visible measurements were performed using a Shimadzu 160A spectrophotometer. For cell culture experiments, MCF-7, MDA-MB-231, and HEK293 cell lines were acquired from ATCC and cultured from stocks available in the Cell Culture Laboratory of the Department of Medical Biochemistry. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco), all used under sterile conditions.
2.2. Synthesis
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As shown in the synthesis in Fig. 2; 4-bromo-1,8-naphthalic anhydride (3.6 mmol) was refluxed with 3-ethoxypropylamine (4.33 mmol) in ethanol for 24 hours. The reaction progress was monitored by TLC. The product was crystallized from ethanol-chloroform (9:1) and dried in a vacuum oven at 90°C. A yellow-white solid, designated as compound 1, was obtained. Compound 1 (5.3 mmol) was refluxed with ethylenediamine (32 mmol) in 2-methoxyethanol under nitrogen at 120°C for 30 hours. The product was dried in a vacuum oven at 70°C to obtain an orange-colored solid, referred to as compound 2. Compound 2 (0.0014 mmol) was refluxed with each of the 7 isatin derivatives (0.0017 mmol) in ethanol for 36 hours. The products were crystallized from ethanol-chloroform (9:1) and dried in a vacuum oven at 50°C, yielding range of yellow to tile red solids designated as compounds 3a-g. (FT-IR, 1H-NMR and 13C-NMR spectra in Table S1)Fig. 2
Conjugation of isatin derivatives with 4-bromo-1,8-naphthylanhydride. 4-Bromo-1,8-naphthalic anhydride was refluxed with 3-ethoxypropylamine in ethanol at 90°C for 24 hours. The resulting intermediate was then reacted with ethylenediamine in 2-methoxyethanol under nitrogen at 120°C for 30 hours. Finally, the product was refluxed with isatin derivatives in ethanol for 36 hours to obtain the target compound.
2.3. Cell Culture Preparation
In the study, cell lines were cultured in medium containing 1% L-glutamine, 10% FBS and 1% penicillin-streptomycin at 37 ℃ and 5% CO2 and when 85–90% confluent, they were washed with Ca2+ and Mg2+ free PBS and removed with Trypsin-EDTA.
2.4. Cell Viability Assay
To determine the effect of seven different synthesis substances on MCF-7 and MDA-MB-231 cell viability, stocks were prepared as 5x103 µM by dissolving in 0.2% DMSO. It was diluted with medium and prepared in 6 different concentrations (0-150 µM). Cells were collected by trypsinizing. They were manually counted in a hemacytometer and seeded in 96-well plates at 8x103 cells/well. After 24 hours incubation in 37 ℃ and 5% CO2, 5 repeated doses were applied. After 24th and 48th hours, the medium was incubated with 0.05 g/ml MTT for 3 hours. After the medium was aspirated and treated with DMSO for 15 minutes, absorbance was determined in a microplate reader at 570 nm. Cytotoxicity assay was performed with HEK-293 at the determined IC50 doses.
2.5. Apoptotic Necrotic Cell Analysis
For apoptosis detection using the Annexin V-FITC/PI staining kit, 7.5 × 10⁴ cells were seeded in 6-well plates in triplicate, and IC₅₀ concentrations of the test compounds were administered overnight. After 24 hours of treatment, cells were washed with PBS and detached using trypsin. Following centrifugation, the cell pellets were resuspended in 100 µL of 1X binding buffer and stained with 2.5 µL each of Annexin V-FITC and Propidium Iodide. The samples were incubated in the dark for 20 minutes at room temperature. After staining, 300 µL of 1X binding buffer was added and gently mixed. Apoptotic populations were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and dot plots were obtained from the FITC channel.
2.6. Colocalization Imaging
To determine the intracellular localization, mitochondrial and nuclear staining were performed on MCF-7 cells. After a 24-hour incubation with 3 µM of the synthesized compounds, cells were treated with Mitoview 633 (5 µM) for 40 minutes. Following this, the cells were rinsed with PBS and then stained with DAPI (5 µM) in the dark for 15 minutes. After a final PBS wash, fluorescence imaging was carried out using a confocal microscope (Nikon A1R+/A1+).
2.7. Target Prediction
Small-molecule targets were predicted using SwissTargetPrediction, which employs 2D and 3D similarity to known ligands for accurate target identification of bioactive compounds under 500 g/mol [24].
2.8. In-Silico Protein Binding
The VEGFR2 crystal structure (PDB ID: 3G0E) was obtained from the RCSB Protein Data Bank (USA) [25, 26]. Protein and ligand preparation, as well as molecular docking, were performed using AutoDock Tools (ADT v1.5.7, The Scripps Research Institute, USA) and AutoDock Vina (v1.2.7, The Scripps Research Institute, USA), with the grid centered on the sunitinib binding site [27, 28]. Docking results were visualized and analyzed in PyMOL (v3.1.6.1, Schrödinger, LLC, USA), with 3c and sunitinib colored blue and green, respectively. Hydrogen bond interactions were measured in PyMOL. The active site of the receptor and ligand were visualized via BIOVIA Discovery Studio Visualizer (v4.5) [29].
3. Results and Discussion
Here, we report fluorescent isatin derivatives, evaluate their antiproliferative effects on breast cancer cell lines, and perform molecular docking, finding that compounds 3a and 3c notably reduce cell viability and increase apoptosis/necrosis in MCF-7 and MDA-MB-231 cells while showing selectivity over HEK control cells.
3.1. Cell Viability of Breast Cancer Cell Lines
All compounds dose-dependently reduced MCF-7 cell viability, with 3c showing the strongest effect (0.55–1.01% at 50–75 µM) and 3a also highly potent (6.86% at 75 µM) at 24 h, and at 48 h, 3c nearly completely inhibited viability at ≥ 5 µM and 3a reduced it to 0% at ≥ 50 µM (p < 0.0001) as shown in Table 1.
In MDA-MB-231 cells, 3a was the most potent compound at both 24 and 48 h, 3c, 3d, 3e, and 3g showed strong cytotoxicity at higher doses, while 3b and especially 3f were less effective as shown in Table 2.
3.2. Apoptotic Necrotic Cell Analysis
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As in shown Table 3 and Table S2-4, In MCF-7 cells, 3c induced both early apoptosis (42.75%) and necrosis (35.93%), 3a and 3g caused predominantly necrotic cell death, and 3a displayed selective toxicity as HEK cells maintained > 87% viability (TableS5). In MDA-MB-231 cells, 3a and 3c were most effective with high late and early apoptosis. Unlike prior studies [10, 15], the unsubstituted 5-position isatin showed the strongest anticancer effect. These compounds may induce cell death via caspase-mediated apoptosis and mitochondrial dysfunction. [30, 31]. 3c caused high early apoptosis, suggesting intrinsic apoptosis through mitochondrial disruption. In contrast, 3g showed high necrosis, indicating alternative pathways like ferroptosis. [32]. Apoptosis-inducing compounds act through DNA damage, p53 activation, and Bcl-2/Bax balance. Our findings suggest these derivatives target similar pathways. Other compounds showed limited apoptosis, possibly due to active anti-apoptotic proteins like Bcl-2. [33, 34].Fig. 3
Light microscopy images of MCF-7 cell line before (a), 8 hours (b), 24 hours (c) and 48 hours (d) after IC50 dose application with 3a.
Light microscopy images of the synthesized isatin-derived fluorescent substances in MCF-7 cancer cells showed time-dependent cytotoxic effect (Fig. 3).
3.3. Colocalization Assay
Confocal microscopy showed 3a strongly colocalized with mitochondria (Pearson’s:0.916, Fig. 4). The compounds preferentially accumulated in mitochondria, suggesting potential disruption of membrane potential and induction of apoptosis. Despite lacking a cationic structure, their mitochondrial targeting is notable, as cancer cell mitochondria have higher membrane potential and metabolic activity than normal cells. [35]. Similar mitochondrial targeting by lipophilic cationic compounds has been reported to trigger apoptosis in cancer cells [36]. Although the compounds share the same core structure and differ only in the R group, cytotoxicity and apoptotic effects varied between cell lines, highlighting complex structure–activity relationships and “activity cliffs” [37].
Fig. 4
Intracellular localization images of 3a in MCF-7 cell line: A. Fluorescence emission collected from nucleus stain (blue) B. Fluorescence emission collected from mitochondria stain (red), C. Fluorescence emission collected from 3a(green), D. Merge E. Pearson's correlation graphic of red versus green. 3a showed a high overlap with the mitochondrial dye, indicating a particularly mitochondrial uptake within the cell (Pearson's correlation = 0.916).
3.4. Molecular Docking with Sunitinib
Sunitinib is an oral tyrosine kinase inhibitor that targets multiple receptors exhibiting potent antiangiogenic and antitumor activity [38] as shown in Fig. 1. Molecular docking and dynamics studies have been conducted to investigate the interactions between VEGFR-2 and various tyrosine kinase inhibitors with sunitinib. These studies reveal that sunitinib forms hydrophobic interactions and hydrogen bonds with specific residues in the VEGFR-2 binding pocket, contributing to its biological activity [39]. Comparative analysis of sunitinib, lenvatinib, and sorafenib binding to VEGFR-2 highlights the importance of van der Waals interactions and key residues in drug binding [40]. A 5-methoxy-substituted sunitinib analog has been synthesized and radiolabeled, showing similar binding orientation to VEGFR-2 as sunitinib [41]. Docking studies of sunitinib and other anticancer drugs with VEGF demonstrate their ability to selectively inhibit the protein [42]. Among VEGFR2 the 3G0E structure, is used as a model for VEGFR2 its widespread use in many studies allows for reliable inference of inhibitor binding modes [25, 26, 43].
Docking studies showed that the 3c and sunitinib bind differently to the VEGFR2 structure with PDB code 3G0E. 3c forms two hydrogen bonds with the amino acid Leu595 at distances of 2.3 angstroms and 4.5 angstroms. Sunitinib as shown as light green molecule [25], on the other hand, forms hydrogen bonds with other residues at distances of 3.8, 2.8, 2.0, 1.9, and 1.7 angstroms (Fig. 5).
Fig. 5
Visual representation of the VEGFR2 (3G0E) active site, showing the hydrogen bond (dashed lines), distancesbetween 3c (cyan blue) or sunitinib (light green) and key residues in the VEGFR2 (3G0E) binding pocket. 3c uniquely formed two hydrogen bonds with Leu595, with bond distances of 2.3 Å and 4.5 Å, and an additional hydrogen bond with another receptor residue at 1.9 Å. In contrast, sunitinib established hydrogen bonds with a different set of residues, with bond distances of 3.8, 2.8, 2.0, 1.9, and 1.7 Å.
In the analysis of possible bonds between receptor and both ligands that containing isatin substitute, which is emphasized for its increased anticancer activity [10], Sunitinib demonstrated strong binding affinity to the receptor, primarily stabilized through halogen bonding between its F substitue of isatin and Asp810, chain of VEGFR2, Phe811 contributed to the stability of the complex (FigS1). Hydrophobic interactions with key residues like Cys809, Val654, and Tyr672 further reinforced the ligand’s binding. Conventional hydrogen bonds between the carbonyl oxygen of the isatin group and Cys673, as well as between the nitrogen of the isatin ring and Glu671, provided additional stabilization. Furthermore, a p-sigma bond with Leu595 was observed, enhancing the overall binding affinity of sunitinib to the receptor.
In comparison, 3c formed a halogen bond with Trp557, a critical residue within the active site of VEGFR-2, and a conventional hydrogen bond between the carbonyl group of isatin and Glu671 and Cys673, similar to sunitinib (FigS2). Additionally, alkyl interaction between naphtylamides and Leu799, Cys674; the ligand’s propylamine group and Leu595 was identified, which contributed to the ligand’s hydrophobic complementarity with the receptor. These findings suggest that, while both compounds share some common interaction patterns like Glu671 and Cys673 hydrogene bond that involved in main interaction[10], 3c exhibits distinct binding characteristics, particularly in its interactions with Trp557 and Leu595 which involved in the ATP recognition at the catalytic site [44].
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Docking results for 3c indicated that the best binding mode had a predicted affinity of − 6.2 kcal/mol. The top ten docking poses showed affinities ranging from − 6.2 to − 5.7 kcal/mol, with root-mean-square deviation (RMSD) values from the best mode ranging from 0.000 to 7.363 Å (Table S6). The relatively small differences in binding affinity among the top poses, combined with RMSD values below 2 Å for several modes, suggest that the novel compound can adopt multiple, closely related binding orientations within the VEGFR2 pocket. The most favorable pose, which engages Trp577 and Leu595 through hydrogen bonding, is likely to represent the biologically relevant binding mode (Fig. 5). Higher RMSD values observed in some poses indicate alternative orientations that are less similar to the best mode and may be less favorable energetically or sterically.4. Conclusion
3c strongly induces early apoptosis, 3a causes potent necrosis, and small structural differences lead to varied effects across cell lines, influenced by both molecular properties and cell-specific factors; computational analysis shows the novel compound binds VEGFR2 differently from sunitinib, highlighting its potential as a selective anticancer agent and the need for further in silico, in vitro, and in vivo studies.
Electronic Supplementary Material
Below is the link to the electronic supplementary material
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Data Availability
All data supporting the findings of this study are available within the paper and its Supplementary Information.
References
1.
Ferlay J, Colombet M, Soerjomataram I et al (2021) Cancer statistics for the year 2020: An overview. Int J Cancer 149:778–789. https://doi.org/https://doi.org/10.1002/ijc.33588
2.
WHO (2022) Global Cancer Observatory 2022. In: https://gco.iarc.fr/en
3.
Sağlık Bakanlığı TC (2020) 2020 Yılı Türkiye Kanser İstatistikleri. In: https://hsgm.saglik.gov.tr/depo/birimler/kanser-db/Dokumanlar/Istatistikler/Turkiye_Kanser_Istatistikleri_2020.pdf
4.
Fu X, Shi Y, Qi T et al (2020) Precise design strategies of nanomedicine for improving cancer therapeutic efficacy using subcellular targeting. Signal Transduct Target Ther 5:262. https://doi.org/10.1038/s41392-020-00342-0
5.
Orbay S, Sanyal R, Sanyal A (2023) Porous Microgels for Delivery of Curcumin: Microfluidics-Based Fabrication and Cytotoxicity Evaluation. Micromachines (Basel) 14:. https://doi.org/10.3390/mi14101969
6.
Orer HS, Nomak G, Oksuzoglu B et al (2022) 1200TiP Phase Ia/Ib study of RS-0139, a novel tumor-targeted delivery of docetaxel, in patients with recurrent, locally advanced, or metastatic non-small cell lung cancer (NSCLC). Ann Oncol 33:S1097. https://doi.org/10.1016/j.annonc.2022.07.1876
7.
Bagci-Onder T, Kutuk O, Chonghaile TN, Knippschild U (2022) Editorial: Cell Death and Targeted Cancer Therapies. Front Cell Dev Biol 10:967720. https://doi.org/10.3389/fcell.2022.967720
8.
Oh S, Kim BJ, Singh NP et al (2009) Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide. Cancer Lett 274:33–39. https://doi.org/https://doi.org/10.1016/j.canlet.2008.08.031
9.
Ding Z, Zhou M, Zeng C (2020) Recent advances in isatin hybrids as potential anticancer agents. Arch Pharm (Weinheim) 353:1900367. https://doi.org/https://doi.org/10.1002/ardp.201900367
10.
Meleddu R, Petrikaite V, Distinto S et al (2019) Investigating the Anticancer Activity of Isatin/Dihydropyrazole Hybrids. ACS Med Chem Lett 10:571–576. https://doi.org/10.1021/acsmedchemlett.8b00596
11.
Vine KL, Matesic L, Locke JM et al (2009) Cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from 2000–2008. Anticancer Agents Med Chem 9 4:397–414. https://doi.org/10.2174/1871520610909040397
12.
Medvedev A, Buneeva O, Gnedenko O et al (2018) Isatin, an endogenous nonpeptide biofactor: A review of its molecular targets, mechanisms of actions, and their biomedical implications. BioFactors 44:95–108. https://doi.org/https://doi.org/10.1002/biof.1408
13.
Nalini CN, Deepthi S, Baid C (2011) Toxicity Risk Assesment Of Isatins. Rasayan J Chem 4(4):829–833
14.
Ramshid PK, Jagadeeshan S, Krishnan A et al (2010) Synthesis and in vitro evaluation of some isatin-thiazolidinone hybrid analogues as anti-proliferative agents. Med Chem 6 5:306–312. https://doi.org/10.2174/157340610793358909
15.
Oguz A, Saglik BN, Oguz M et al (2024) Novel mitochondrial and DNA damaging fluorescent Calix[4]arenes bearing isatin groups as aromatase inhibitors: Design, synthesis and anticancer activity. Bioorg Med Chem 98:117586. https://doi.org/https://doi.org/10.1016/j.bmc.2023.117586
16.
Roth GJ, Binder R, Colbatzky F et al (2015) Nintedanib: From Discovery to the Clinic. J Med Chem 58:1053–1063. https://doi.org/10.1021/jm501562a
17.
Rasheed HAM, Al-Majidi SMH (2024) Synthesis of Novel 1,2,4-Triazole Derivatives of N-Benzyl-5-nitroisatin: Anticancer Activity, Anti-oxidant Activity, and Molecular Design. Org Prep Proced Int 1–13. https://doi.org/10.1080/00304948.2024.2365517
18.
Kundu A, Pathak S, Pramanik A (2013) Synthesis and Fluorescence Properties of Isatin-Based Spiro Compounds: Switch off Chemosensing of Copper(II) Ions. Asian J Org Chem 2:869–876. https://doi.org/https://doi.org/10.1002/ajoc.201300153
19.
Wei Y-F, Zhang X-Q, Sun R et al (2021) Fluorescent probes based 1,8-naphthalimide-nitrogen heterocyclic for monitoring the fluctuation of mitochondrial viscosity. Dyes Pigm 194:109559. https://doi.org/https://doi.org/10.1016/j.dyepig.2021.109559
20.
Yilmaz MD, Altves S, Erbas-Cakmak S (2024) Naturally Occurring Flavonol, Quercetagetin 5,6,7,3′,4′-Pentamethyl Ether (Marionol), as a Nontoxic Plant-Based Fluorescent Probe for Rapid, Sensitive, and Selective Detection of Cu2 + in Water. ACS Omega 9:47342–47349. https://doi.org/10.1021/acsomega.4c09069
21.
Mchichi LEL, Alaqarbeh M, Lakhlıfı T, Bouachrıne M (2024) Discovery of a New Isatin Scaffold for BCR-ABL Tyrosine Kinase Inhibitors Using a Comprehensive Computational Approach. ChemistrySelect 9:e202402053. https://doi.org/10.1002/slct.202402053
22.
Al-Salem HSA, Arifuzzaman M, Issa IS, Rahman AFMM (2021) Isatin-Hydrazones with Multiple Receptor Tyrosine Kinases (RTKs) Inhibitory Activity and In-Silico Binding Mechanism. Appl Sci 11:3746. https://doi.org/10.3390/app11093746
23.
Ibrahim NW, Mahdi M, Raauf AMR (2022) Design, Molecular Docking, Synthesis and Evaluation of New Isatin Derivatives Bearing Pyridine Moiety as Potential Tyrosine Kinase Inhibitors. Egypt J Chem 65:9–18. https://doi.org/10.21608/ejchem.2021.72747.3607
24.
Gfeller D, Grosdidier A, Wirth M et al (2014) SwissTargetPrediction: a web server for target prediction of bioactive small molecules. Nucleic Acids Res 42:W32–W38. https://doi.org/10.1093/nar/gku293
25.
Gajiwala KS, Wu JC, Christensen J et al (2009) KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients. Proceedings of the National Academy of Sciences 106:1542–1547. https://doi.org/10.1073/pnas.0812413106
26.
Laderoute K, Calaoagan JM, Madrid PB et al (2010) SU11248 (sunitinib) directly inhibits the activity of mammalian 5’-AMP-activated protein kinase (AMPK). Cancer Biol Ther 10:68–76. https://doi.org/10.4161/cbt.10.1.12162
27.
Eberhardt J, Santos-Martins D, Tillack AF, Forli S AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and, Bindings P (2021) J Chem Inf Model 61:3891–3898. https://doi.org/10.1021/acs.jcim.1c00203
28.
Trott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461. https://doi.org/https://doi.org/10.1002/jcc.21334
29.
Muhammed TM, Akı-yalcın E (2021) Pharmacophore Modeling in Drug Discovery: Methodology and Current Status. Journal of the Turkish Chemical Society, Section A: Chemistry 8:749–762. https://doi.org/0000-0003-0050-5271
30.
Kaufmann SH, Earnshaw WC (2000) Induction of Apoptosis by Cancer Chemotherapy. Exp Cell Res 256:42–49. https://doi.org/https://doi.org/10.1006/excr.2000.4838
31.
Ma Z, Hou L, Jiang Y et al (2014) The endogenous oxindole isatin induces apoptosis of MCF-7 breast cancer cells through a mitochondrial pathway. Oncol Rep 32:2111–2117. https://doi.org/10.3892/or.2014.3426
32.
Dixon SJ, Lemberg KM, Lamprecht MR et al (2012) Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell 149:1060–1072. https://doi.org/10.1016/j.cell.2012.03.042
33.
Fulda S (2009) Tumor resistance to apoptosis. Int J Cancer 124:511–515. https://doi.org/https://doi.org/10.1002/ijc.24064
34.
Hanahan D, Weinberg RA (2011) Hallmarks of Cancer: The Next Generation. Cell 144:646–674. https://doi.org/10.1016/j.cell.2011.02.013
35.
Wallace DC (2012) Mitochondria and cancer. Nat Rev Cancer 12:685–698. https://doi.org/10.1038/nrc3365
36.
İnel M, Yildirim A, Ozturk B, Yilmaz M (2025) Recent Advances in the Application of Mitochondria-Targeted Fluorescent Probes. Curr Med Chem 32:9653–9678. https://doi.org/10.2174/0109298673368188250613112621
37.
Maggiora GM (2006) On Outliers and Activity CliffsWhy QSAR Often Disappoints. J Chem Inf Model 46:1535. https://doi.org/10.1021/ci060117s
38.
Chow LQM, Eckhardt SG (2007) Sunitinib: from rational design to clinical efficacy. J Clin Oncol 25 7:884–896. https://doi.org/10.1200/JCO.2006.06.3602
39.
Malińska M, Jarzembska KN, Góral A et al (2014) Sunitinib: from charge-density studies to interaction with proteins. Acta Crystallogr D Biol Crystallogr 70 Pt 5:1257–1270. https://doi.org/10.1107/S1399004714002351
40.
Wang Y, Peng C, Wang G et al (2019) Exploring binding mechanisms of VEGFR2 with three drugs lenvatinib, sorafenib, and sunitinib by molecular dynamics simulation and free energy calculation. Chem Biol Drug Des 93:934–948. https://doi.org/10.1111/cbdd.13493
41.
Caballero J, Muñoz C, Alzate-Morales JH et al (2012) Synthesis, in silico, in vitro, and in vivo investigation of 5-[11C]methoxy-substituted sunitinib, a tyrosine kinase inhibitor of VEGFR-2. Eur J Med Chem 58:272–280. https://doi.org/10.1016/j.ejmech.2012.10.020
42.
Igwe KK, Otuokere IE, Ikpeazu OV (2017) Probing the binding sites, global energy and molecular interactions between ponatinib, cabozantinib and sunitinib anticancer drugs with vascular endothelial growth factor. J Drugs Pharm Sci 1(1):9–15. https://doi.org/10.31248/JDPS2017.007
43.
Baker SD, Zimmerman EI, Wang Y-D et al (2013) Emergence of Polyclonal FLT3 Tyrosine Kinase Domain Mutations during Sequential Therapy with Sorafenib and Sunitinib in FLT3-ITD–Positive Acute Myeloid Leukemia. Clin Cancer Res 19:5758–5768. https://doi.org/10.1158/1078-0432.CCR-13-1323
44.
Chaudhari P, Bari S (2016) In silico exploration of c-KIT inhibitors by pharmaco-informatics methodology: pharmacophore modeling, 3D QSAR, docking studies, and virtual screening. Mol Divers 20:41–53. https://doi.org/10.1007/s11030-015-9635-x
Statements & Declarations
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Funding
This work was supported by Scientific Research Foundation of Selçuk University (24202058) as M.İ.’s master thesis. Author M.İ. has received research support from Türkiye Scientific and Technological Research Institution (TÜBİTAK-BİDB-2210).
Competing Interests
The authors have no relevant financial or non-financial interests to disclose
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Author Contribution
This work does not involve the use of humans or animals.
Ethical statement
This work does not involve the use of humans or animals.