A comprehensive review and recent advances on isatin-based compounds as a versatile framework for anticancer therapeutics (2020–2025)

Abstract

Isatin (1H-indole-2,3-dione) is a privileged nitrogen-containing heterocyclic framework that has received considerable attention in anticancer drug discovery owing to its general biological behavior and structural diversity. This review focuses on isatin–heterocyclic hybrids as a valuable model in the development of new anti-cancer drugs that may reduce side effects and help overcome drug resistance, discussing their synthetic approaches and mechanism of action as apoptosis induction through kinase inhibition. With various chemical modifications, isatin had an excellent ability to build powerful isatin hybrids and conjugates targeting multiple oncogenic pathways. It is worth mentioning that isatin-hybrids exhibited anticancer activity against various cancer cell lines, such as breast, liver, colon, lung, and multidrug-resistant carcinomas. Their mechanisms include mitochondrial-mediated apoptosis, caspase activation, tubulin polymerization inhibition, and kinase modulation, particularly VEGFR-2, EGFR, CDK2, and STAT-3. Numerous synthesized isatin-based compounds have shown superior cytotoxicity compared to established chemotherapeutics, with favorable IC₅₀ values and minimal toxicity toward normal cells. In addition, this review summarizes more recent synthetic innovations, e.g., microwave-assisted and multi-component techniques, which offer improved pharmacological profiles of these isatin–heterocyclic hybrids with improved cytotoxicity and target signaling pathways. Overall, these results underscore the value of isatin as a flexible scaffold for the rational design of new anticancer agents. To increase bioavailability and targeted delivery, especially in solid tumors, and to lead to the creation of novel, potent anticancer therapies, nano-formulation drug delivery systems with revolutionary drug signaling pathways will be further advocated in the future.

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1. Introduction

Cancer remains one of the most formidable challenges in modern medicine, not only for its complexity and adaptability but also for the collateral damage inflicted by its treatment. Chemotherapy, a cornerstone of cancer therapy, is designed to eradicate rapidly dividing cancer cells. However, its lack of absolute specificity often results in significant toxicity to normal, healthy cells, particularly those in the bone marrow, digestive tract, and hair follicles, which limits the therapeutic window and diminishes patients' quality of life.^1,2 This dual-edged nature of chemotherapy underscores a central dilemma: maximizing the destruction of malignant cells while preserving the integrity and function of normal tissues. Therefore, it is essential to create novel anti-cancer medications that are highly selective and effective against drug-sensitive and drug-resistant tumors.^3,4

In medicinal chemistry, heterocyclic compounds are beneficial molecules.⁵ They display various pharmacological and biological activities.^6,7 As a fundamental building block of a vast heterocyclic library, nitrogen-containing heterocyclic congeners are widely employed in many scientific fields.^8,9 Moreover, nitrogen-containing heterocycles have remarkable structural properties and are commonly found in a variety of herbal components, including alkaloids and vitamins.¹⁰ Isatin is a nitrogen-containing single scaffold. Many bioactive natural compounds have the isatin skeleton, e.g., chitosenine, horsfiline, javaniside, spirotryprostatin A, spirotryprostatin B, and strychnofoline (Fig. 1).¹¹

Fig. 1 Bioactive natural products containing isatin.

Isatin's structure allows the insertion of various substituents into almost any position of the moiety. Numerous derivatives with enhanced biological activity have been produced as a result of structural modifications to the isatin ring.^12,13 Additionally, numerous cancer forms, even those that are resistant to treatment, may be treated by specific anti-cancer treatments that contain isatin, indicating the possibility of creating new anti-cancer drugs.¹⁴ Fig. 2 represents currently marketed isatin-based anti-cancer drugs.¹⁵

Fig. 2 Isatin-based marketed anti-cancer drugs.

Isatin is an indole derivative that is produced by oxidizing indigo dye. Its chemical name is 1H-indole-2,3-dione. First synthesized by Erdman and Laurent in 1841,¹⁶ isatin has since become a pivotal entity in organic synthesis, attributable to its structural intricacy and chemical reactivity. Isatin is a naturally occurring substance that can be found in plants like Calanthe discolor and Couroupita guianensis. It has also been found within the secretion of the parotid gland of Bufo frogs and human metabolic pathways stemming from adrenaline. Additionally, various plant species include substituted isatins, such as the melastatin alkaloids obtained from the Caribbean tumorigenic plant Melochia tomentosa.^14,15

2. Chemical properties

Isatin is an endogenous compound with the molecular formula C₆H₅NO₂. According to Fig. 3, it contains two carbonyl groups at positions two and three and a nitrogen atom at position one. It consists of two cyclic rings, one with five members and the other with six. The two rings are flat. Whereas the 5-membered ring has an anti-aromatic property, the 6-membered ring has an aromatic one.¹⁷

Fig. 3 Structure of isatin.

Isatin can undergo chemical reactions in three different places: N-alkylation, aromatic ring substitution, and carbonyl reaction at its C2 and/or C3 carbonyl functionalities (Fig. 4).¹⁸

Fig. 4 Possible substitution on the isatin scaffold.¹⁹

Derivatives of isatin exhibit improved anticancer properties through strategic hybridization. Imidazole–isatin hybrids inhibit COX-2 and PI3K enzymes, key targets in inflammation and breast cancer. Isatin-hydrazones suppress Bcl-2, activate caspases, and induce ROS-mediated apoptosis in breast cancer cells. Triazole-tethered isatin–coumarin hybrids inhibit tubulin polymerization (IC₅₀ ≈ 1–5 μM) and overcome multidrug resistance in prostate and breast cancers. Brominated isatin derivatives from marine organisms target CDK2, a kinase critical for cell cycle progression.^20,21

Due to these remarkable and fantastic properties and conversions (Fig. 5), the generation of macrocyclic complexes with isatin or its derivatives is being explored more.²²

Fig. 5 Tautomerism in isatin. (A) Lactam form, (B) lactim form and (C) imide form.

3. Molecular targeting of isatin-based derivatives as anti-cancer agents

The apoptotic (programmed cell death) effects of isatin-based derivatives on several types of cancer cells make them promising candidates for use as anticancer agents. The apoptotic (programmed cell death) effects of isatin-based derivatives on several types of cancer cells make them promising contenders for anticancer drug development. Drugs that inhibit CDK2, receptor tyrosine kinases, and histone deacetylases can be designed using the isatin scaffold. These drugs affect cell cycle progression, mitosis, and epigenetic control in tumor cells, as summarized in Fig. 6.

Fig. 6 The signaling therapeutic pathways of isatin-based derivatives as anti-cancer agents. This figure is partially generated by Biorender and reproduced from our previously published work.³⁴

3.1. Molecular mechanisms of isatin-induced apoptosis

3.1.1. Kinase-mediated apoptosis. Reducing tumor angiogenesis and metastasis, isatin-based derivatives inhibit vascular endothelial growth factor receptor 2 (VEGFR2) and epidermal growth factor receptor (EGFR). By inhibiting receptors for the MAPK and PI3K/AKT signaling pathways, which are frequently dysregulated in cancers, the compounds reduce the availability of survival signals for cancer cells.²³ This promotes cell survival and proliferation. Anticancer treatments can be more effective if specific pathways are targeted. Many malignancies have dysregulated signaling pathways that allow cells to survive and proliferate, such as RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR.²⁴

Also, isatin-based derivatives have a significant impact on cell cycle regulation. By blocking the G1-S phase transition and consequently inhibiting cell growth, these compounds have shown promise as cyclin-dependent kinase 2 (CDK2) inhibitors. Moreover, these chemicals cause cell cycle arrest by inhibiting histone deacetylase (HDAC), which in turn induces chromatin remodeling and suppresses oncogenic transcription.²⁵

There have been other investigations into isatin-based compounds that target HER-2. On the other hand, they are known to suppress the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR signaling pathways, which are involved in cancer cell survival and metastasis. Thus, blocking these pathways may slow cancer progression and improve therapy options.²⁶

3.1.2. Mitochondrial pathway activation. Isatin derivatives effectively decrease the expression of anti-apoptotic Bcl-2 protein while maintaining Bax (pro-apoptotic) expression levels, significantly reducing the Bcl-2/Bax ratio. This altered ratio is a critical regulatory step that sets the threshold for apoptosis susceptibility in the mitochondrial pathway. The disruption of mitochondrial integrity represents one of the earliest events in isatin-induced apoptosis. Treatment with isatin markedly decreases the mitochondrial transmembrane potential in cancer cells, indicating mitochondrial dysfunction. This destabilization leads to elevated cytochrome c release into the cytosol, a universal feature of the apoptotic process.^27–31

3.1.3. Caspase cascade activation. Upon cytochrome c release, isatin initiates the apoptotic program by triggering a caspase cascade. Isatin stimulates caspase-9 and caspase-3, according to studies. The dicarbonyl functionality is crucial to isatin's activity because it activates caspases. By interacting with the nucleophilic cysteine thiolate functionality and the electrophilic C-3 carbonyl carbon of isatin, this group forms a thiohemiketal that binds to the cysteine residue at the active site of activated caspases. In the next step, caspase-3 cleaves the ICAD inhibitor, which activates caspase-activated DNase (CAD) and causes DNA fragmentation within nuclear internucleosomes. The hallmarks of cell death, such as DNA fragmentation and chromatin condensation, have been seen in cancer cells treated with isatin.^32,33

4. Isatin–hybrids as anti-cancer agents

Drug hybridization is a beneficial strategy in pharmaceutical development, as such compounds can enhance efficacy, improve target specificity, and combat resistance.³⁵ The isatin scaffold serves as a valuable model in the development of new anti-cancer drugs. By merging the isatin core with other anti-cancer pharmacophores (Fig. 7), it is possible to design hybrid molecules that may reduce side effects and help overcome drug resistance. These isatin-based hybrids represent promising candidates for novel cancer therapies.³⁶ This section displays recent advances in isatin-based hybrid compounds as potential anti-cancer agents. It includes isatin scaffold with quinazoline, indole,1,2,3-triazole, 1,2,4-triazole, pyrazole, sulphonamide, hydrazone, benzofuran, thiazolidine, thiazole, and thiadiazole hybrids, respectively.

Fig. 7 Hybridization of isatin core with other anti-cancer pharmacophores.

4.1. Isatin–quinazoline hybrids

Kandeel et al. reported synthesizing indolinone-based derivatives as cytotoxic kinase inhibitors.³⁷ The synthetic routes employed to synthesize the target derivatives (7 and 12) are represented in Schemes 1 and 2, respectively. In Scheme 1, anthranilic acid 1 was heated with formamide to afford 4-quinazolinone 2, which was nitrated with a nitrating mixture to give 6-nitroquinazolinone 3. Also, 6-nitroquinazolinone 3 can be obtained through the cyclization of 2-amino-5-nitro-benzonitrile 4 with formic acid. Furthermore, 6-nitroquinazolinone 3 was then reduced using iron and ammonium chloride in isopropanol, producing 6-aminoquinazolinone 5. Finally, 6-aminoquinazolinone 5 was allowed to react with 4-chloroisatin 6 in EtOH and in the presence of catalytic acetic acid under reflux to furnish the target 6-(indolylidonamino)quinazolinone 7 (yield: 59%).

Scheme 1 Synthesis of isatin–quinazoline 7. Reagents and conditions: (i) HCONH₂, reflux, 6 h; (ii) HNO₃, H₂SO₄, 90 °C, 30 min; (iii) HCOOH, H₂SO₄, reflux, 1 h; (iv) Fe, NH₄C1, iPrOH, reflux, 1.5 h; (v) EtOH, gl. AcOH (cat.), reflux, 4–6 h.

Scheme 2 Synthesis of isatin–quinazoline hybrid 12. Reagents and conditions: (i) CuC1₂·2H₂O, EtOH, reflux, 16 h; (ii) Fe, HC1; (iii) EtOH, gl. AcOH (cat.) reflux, 6 h.

On the other hand, Scheme 2, synthesis was started via heating a mixture of anthranilamide 8 with p-nitrobenzaldehyde 9 and copper(II) chloride in EtOH, which yielded the 2-(nitro-phenyl)quinazolinone 10. The latter compound 10 was reduced with iron and hydrochloric acid afforded 2-(4-aminophenyl)quinazolinone 11. Finally, compound 11 was condensed with 4-chloroisatin 6 in EtOH and in the presence of catalytic acetic acid under reflux for 6 h to furnish the target 12 (yield: 61%).

Compounds 7 and 12 were examined against two human tumor cancer cell lines (HepG-2 and MCF-7), using indirubin as the positive control. They displayed the highest cytotoxicity against the HepG2 (IC₅₀ = 2.53 and 3.08 μM) and MCF-7 (IC₅₀ = 7.54 and 5.28 μM) cell lines, respectively, compared to indirubin (IC₅₀ = 6.92 and 6.12 μM). Compound 7 demonstrated potent inhibition against VEGFR2 and CDK-2 (IC₅₀ = 56.74 and 9.39 nM), respectively. Compound 7 was around five times more potent than indirubin when inhibiting CDK-2. On the other hand, compound 12 demonstrated potent inhibition of both EGFR and VEGFR-2 with IC₅₀ values of 14.31 nM and 32.65 nM, respectively. Molecular docking studies supported the potential binding modes and interactions 7 with CDK-2 and 12 with VEGFR-2.³⁷

4.2. Isatin–indole hybrids

Al-Wabli et al. synthesized a new isatin–indole conjugate.³⁸ The synthesis started with the esterification of indole-2-carboxylic acid 13 to give the methyl ester 14. Next, compound 14 was allowed to react with N₂H₄·H₂O, forming the acid hydrazide 15. The target conjugate 17 was prepared by reacting the acid hydrazide 15 with isatin derivative 16 in EtOH-containing drops of acetic acid (yield: 54%), Scheme 3.

Scheme 3 Synthesis of isatin–indole hybrid 17. Reagents and conditions: (i) MeOH, H₂SO₄ (cat.), reflux, 4 h; (ii) N₂H₄·H₂O, MeOH, reflux, 2 h; (iii) EtOH, gl. AcOH (cat.), reflux, 4 h.

Using human breast (ZR-75), colon (HT-29), and lung (A-549) carcinoma cells, the antiproliferative efficacy of compound 17 was assessed. With IC₅₀ values of 0.74, 2.02, and 0.76 μM, respectively, it exhibited the most potent anticancer activity when contrasted with the standard drug sunitinib, which had IC₅₀ values of 8.31, 10.14, and 5.87 μM, respectively.³⁸

A novel isatin–indole conjugate was synthesized by Eldehna et al.³⁹ The synthetic strategy used for the preparation of the target compound 23 was illustrated in Scheme 4. First, 1H-indole 18 was formylated via the Vilsmeier–Haack reaction to produce 1H-indole-3-carbaldehyde 19, in which the CHO functionality was then oxidized by KMnO₄ in acetone to furnish 1H-indole-3-carboxylic acid 20. Furthermore, the acid 20 was esterified through refluxing in dry methanol (MeOH) containing a catalytic dehydrating agent, H₂SO_4, to get carboxylate 21, where the ester group reacted with N₂H₄·H₂O in MeOH to produce the intermediate 1H-indole-3-carbohydrazide 22. Finally, the intermediate 22 was condensed with 5-chloroisatin 6 in glacial acetic acid to give the targeted compound 23 (yield: 75%).

Scheme 4 Synthesis of isatin–indole hybrid 23. Reagents and conditions: (i) DMF, POC1₃, reflux 8 h, (ii) KMnO₄, acetone, stirring, r.t., 12 h, (iii) MeOH, H₂SO₄ (cat.), reflux, 7 h, (iv) N₂H₄·H₂O, MeOH, reflux, 4 h, (v) gl. AcOH, reflux, 5–7 h.

Compound 23 was examined against two human cell lines, colorectal cancer HT-29 and SW-620. In comparison to standard 5-FU, which had IC₅₀ values of 4600 and 1500 μM, respectively, it exhibited effective and selective cytotoxicity at 206 and 188 nM, respectively.³⁹

Based on the structural analysis of the reported CDK2 inhibitor, a new compound with 3-hydrazonoindolin-2-one scaffold 29 was developed by Al-Sanea et al.⁴⁰ The target compound 29 was prepared through a four-step reaction. First is the coupling dehydration of glycolic acid 24 with indole 18 to give indol-3yl-acetic acid 25, which is esterified with EtOH in an acidic medium to afford indole acetic acid ester 26. The ester 26 reacted with N₂H₄·H₂O to give hydrazide 27. 5-Methoxyindole 2,3-dione 28 was condensed with 27 in EtOH and in the presence of catalytic glacial acetic acid under reflux for 4 h to produce the target 29 (yield: 62%), Scheme 5.

Scheme 5 Synthesis of isatin–indole hybrid 29. Reagents and conditions: (i) KOH, HC1, H₂O; (ii) EtOH, H⁺, reflux, 10 h; (iii) N₂H₄·H₂O, EtOH, reflux, 2 h; (iv) EtOH, gl. AcOH (cat.), reflux, 4 h.

Metastatic cancer (MCF-7, MDA-MB-231) and ovarian cancer (NCI-ADR) cell lines were used to test compound 29's antiproliferative activity. Compared to Dox, which inhibits the proliferation of the breast cancer cell line MCF-7 with an IC₅₀ value of 6.81 ± 0.22 μM, compound 29's antiproliferative action is four times more potent, with a value of 1.15 ± 0.04 μM, while it was found to be equipotent with Dox in inhibiting the proliferation of the breast cancer cell line MDA-MB-231. Furthermore, it has demonstrated antiproliferative activity against ovarian cancer cells. NCI-ADR. 29 exhibited pronounced CDK2 inhibitory activity with IC₅₀ value of 6.32 μM. Additionally, docking studies have shown that it can interact with CDK2.⁴⁰

Al-Warhi et al. reported synthesizing and biologically evaluating certain oxindole–indole conjugates as anticancer CDK inhibitors.⁴¹ The targeted conjugate 32 was prepared, as shown in Scheme 6. The first step involved the esterification of indole 2-carboxylic acid 13 through refluxing in EtOH in the presence of thionyl chloride to get ethyl-1H-indole-2-carboxylate 30. Subsequently, the ester 30 was treated with N₂H₄·H₂O in boiling EtOH to get intermediate 15. Final target 32 was prepared by condensing hydrazide 15 with 5-methylisatin 31 in refluxing glacial acetic acid (yield: 69%).

Scheme 6 Synthesis of isatin–indole hybrid 32. Reagents and conditions: (i) EtOH, SOC1₂, reflux, 6 h; (ii) N₂H₄·H₂O, EtOH, reflux, 2 h; (iii) gl. AcOH, reflux, 4 h.

The antiproliferative activity of compound 32 was evaluated in vitro against breast cancer MCF-7 and MDA-MB-231 cell lines. It showed more cytotoxic activity with (IC₅₀ = 0.39 μM) against MCF-7 than MDA-MB-231 cell line compared to the reference drug staurosporine with (IC₅₀ = 6.81 μM). It displayed good CDK4 inhibitory activity with an IC₅₀ equal to 1.26 μM. The ability of 32 to interact with CDK4 was also confirmed by a docking study.⁴¹

A novel set of N-alkylindole-isatin conjugates is developed by Al-Warhi et al. to prepare more efficient isatin-based anticancer candidates.⁴² Synthetic routes proposed to get the targeted conjugate 36 have been illustrated in Scheme 7. In the first step, Vilsmeier formylates indole 18 using DMF and phosphorus oxychloride to form 1H-indole-3-carbaldehyde 19. Then, aldehyde 19 underwent N-alkylation with propyl bromide 33 in DMF and sodium hydride base to get intermediate 34. The latter compound 34 was condensed with N₂H₄·H₂O under reflux in EtOH to furnish hydrazide 35. Finally, the key intermediate 35 was reacted with 5-methylisatin 31 in EtOH in the presence of catalytic glacial acetic acid to afford the target product 36 (yield: 74%).

Scheme 7 Synthesis of isatin–indole hybrid 36. Reagents and conditions: (i) DMF, POC1₃, reflux, 8 h; (ii) DMF, NaH, stirring, r.t., 24 h; (iii) N₂H₄·H₂O, EtOH, reflux, 2 h; (iv) EtOH, gl. AcOH (cat.), reflux, 3 h.

A-549, MDA-MB-231, and HCT-116 cell lines were all significantly inhibited by compound 36, with IC₅₀ values of 7.3, 4.7, and 2.6 μM, respectively, indicating its potent antiproliferative effect. With an IC₅₀ value of 2.6 ± 0.17 μM, it was found to be the most powerful analog, surpassing the reference drug DOX (IC₅₀ = 3.7 ± 0.24 μM). It exhibited good inhibitory action against CDK2 with IC₅₀ values equal to 0.85 ± 0.03 μM. Results from docking experiments showed that 36 bound firmly to the active sites of CDK-2 and formed a stable complex.⁴²

4.3. Isatin-1,2,3-triazole hybrids

Mohite et al. reported the synthesis of isatin-1,2,3-triazole hybrids as anticancer agents.⁴³ The synthetic strategy for preparing the target compound 44 is outlined in Scheme 8. The 2-azido-N-(p-tolyl)acetamide 40 was produced by the reaction of 4-methyl aniline 37 with chloroacetyl chloride 38 in acetone, followed by the reaction of compound 39 with sodium azide in DMF. The N-alkylation of isatin 41 with propargyl bromide 42 was performed using K₂CO₃ to give the N-terminal alkyne 43. Lastly, the synthesis of a triazole–isatin hybrid 44 by combining azide 40 with the terminal alkyne 43 in a solution of CuSO₄·5H₂O and L-sodium ascorbate in n-butanol/H₂O (1 : 1 v/v) at room temperature afforded the target 44 (yield: 88%).

Scheme 8 Synthesis of isatin-1,2,3-triazole hybrid 44. Reagents and conditions: (i) acetone, r.t., 2–3 h; (ii) NaN₃, DMF, r.t., 24 h; (iii) K₂CO₃, DMF, r.t., 24 h; (iv) CuSO₄·5H₂O, sodium ascorbate, n-BuOH/H₂O (1 : 1 v/v), DMF, 24 h.

Compound 44 demonstrated potent activity, displaying a submicromolar IC₅₀ value against MCF-7 (IC₅₀ = 0.67 ± 0.12 μM) and HCC1937 (IC₅₀ = 0.53 ± 0.11 μM) cell lines. It was evaluated for its potential PARP-1 inhibitory activity, utilizing olaparib as a reference PARP-1 inhibitor. 44 as the most effective PARP-1 inhibitors showed IC₅₀ value of 13.65 ± 1.42 nM. A molecular docking study revealed excellent binding strength in the active site vicinity of PARP-1.⁴³

Preeti et al. have reported the synthesis and apoptotic assessment of triazole–isatin hybrids.⁴⁴ The synthetic methodology for the synthesis of the target hybrid 51 is outlined in Scheme 9. Base-promoted alkylation of 5,7-dibromoisatin 45 with dibromopropane 46 yielded 47, which was followed by subsequent treatment with sodium azide, resulting in the desired azide 48. The preparation of spirocyclopropyl oxindole 49 from isatin 41 was done by treating it with trimethylsulfoxonium iodide (TMSI) in the presence of base NaH in dry DMF through Domino Corey–Chaykovsky reaction. Subsequent treatment of 49 with propargyl bromide 42 resulted in the formation of alkyne 50. By applying Cu-promoted azide–alkyne cycloaddition, the precursors 48 and 50 were used to synthesize the desired isatin hybrid 51 in DMSO in a microwave reactor at 80 °C for 10 min (yield: 77%).

Scheme 9 Synthesis of isatin-1,2,3-triazole hybrid 51. Reagents and conditions: (i) K₂CO₃, DMF, 100 °C, 10 min, MW; (ii) NaN₃, DMF, 120 °C, 20 min, MW; (iii) TMSI, NaH, DMSO, r.t.; (iv) K₂CO₃, DMF, 60 °C, 2 h; (v) DMSO, CuI, DIPEA, 80 °C, 10 min, MW.

Compound 51 was screened for its anticancer activity against the MDAMB-231 cell line. It displayed the best IC₅₀ value of 0.73 μM compared to tamoxifen citrate, with the best IC₅₀ value of 12.88 μM. Docking studies revealed a stable complex and strong binding affinity of 51 to the active sites of EGFR.⁴⁴

Utilizing the molecular hybridization approach, Seliem et al. reported the synthesis of sets of triazole–isatin hybrids.⁴⁵ The synthetic protocol developed for the preparation of the targeted hybrid 56 is adopted in Scheme 10. 5-Methylisatin 31 was treated with propargyl bromide 42 in the presence of K₂CO₃ in DMF at room temperature to obtain alkyne 52. The synthesized alkyne 52 was coupled with 1-azido-2-methoxybenzene 53 using a click chemistry approach in the presence of CuSO₄·5H₂O and sodium D-isoascorbate in t-butanol/water mixture under microwave irradiation for 2 h at 100 °C to furnish the desired triazole 54. Finally, the reaction of triazole 54 with compound 55 in EtOH at room temperature for 2 h gave the desired isatin 56 (yield: 84%).

Scheme 10 Synthesis of isatin-1,2,3-triazole hybrid 56. Reagents and conditions: (i) K₂CO₃, DMF, r.t., 4 h; (ii) sodium D-isoascorbate, CuSO₄·5H₂O, t-butanol/H₂O, MW, 100 °C, 2 h; (iii) EtOH, r.t., 2 h.

Compound 56 was screened for antiproliferation properties against breast cancer MCF7, HCT116 (colon), and PaCa2 (pancreatic) cell lines. It showed a higher potency of cytotoxicity against MCF7 (IC₅₀ = 5.361 μM) than the standard reference sunitinib (IC₅₀ = 11.304 μM). It also displayed higher antiproliferation properties against HCT116 (IC₅₀ = 12.50 μM) than 5-FU (IC₅₀ = 20.43 μM). It revealed high VEGFR2 inhibition properties (% inhibition = 77.6) comparable to that of sunitinib (% inhibition = 67.1).⁴⁵

As a potential dual VEGFR2/STAT-3 inhibitor, Elsebaie et al. reported the synthesis of isatin-incorporated phenyl-1,2,3-triazole derivatives.⁴⁶ Scheme 11 shows the synthesis route of the target isatin 63. First, 1-azido-4 methoxybenzene 57 was allowed to react with ethyl acetoacetate 58 at 90 °C in diethylamine and dimethyl sulfoxide to afford triazole intermediate 59. Then, the synthesis of carbohydrazide 60 was achieved by the reaction of compound 59 with N₂H₄·H₂O (99%) in absolute EtOH under reflux. Isatin 41 underwent an N-alkylation via reacting with benzyl bromide 61 in the presence of KI and K₂CO₃ using acetonitrile to afford compound 62. Target compound 63 has been synthesized by reacting hydrazide 60 with N-alkylated isatin 62 in absolute EtOH and catalytic glacial acetic acid under reflux (yield: 81%).

Scheme 11 Synthesis of isatin-1,2,3-triazole hybrid 63. Reagents and conditions: (i) Et₂NH, DMSO, reflux, 6 h; (ii) N₂H₄·H₂O, EtOH, reflux, 4 h; (iii) K₂CO₃, KI, MeCN, reflux, 6 h; (iv) EtOH, gl. AcOH (cat.), reflux, 12 h.

Prostate cancer (PC3) and pancreatic cancer (PANC1) cells were used to study compound 63's anti-proliferative activity. It showed more effective cytotoxicity against PANC1 (IC₅₀ = 0.13 μM) and PC3 (IC₅₀ = 0.10 μM) compared to DOX (IC₅₀ = 0.45 μM and 0.24 μM, respectively) and sunitinib (IC₅₀ = 1.49 μM and 0.60 μM, respectively). Its IC₅₀ value of 26.3 nM showed an effective suppression of VEGFR2, in contrast to sunitinib's IC₅₀ value of 30.7 nM. The STAT-3 inhibitory potential of compound 63 was also investigated. Its IC₅₀ value of 5.63 nM demonstrated its efficacy in inhibiting STAT-3. Molecular docking analyses showed that the powerful 63 binds to the VEGFR2 and STAT-3 active sites in a significant way.⁴⁶

A series of novel indole-2-one derivatives based on 1,2,3-triazole scaffolds were synthesized by Wang et al.⁴⁷ The synthetic route adopted to synthesize the target indole-2-one-1,2,3-triazole derivative 70 is depicted in Scheme 12. First, the preparation of 1-azido-4-methylbenzene 67 involved diazo-reaction and displacement reaction with sodium azide. Then, click chemistry via Cu(I)-catalyzed azide–alkyne-type cycloaddition between aryl-azide 67 and 4-ethynylbenzaldehyde 68 in the presence of sodium ascorbate and CuSO₄·5H₂O as a catalyst in DMF and water mixture afforded a 1,2,3-triazole 69. Isatin 41 was prepared from aniline 64 via Sandmeyer's method. Moreover, compound 66 was prepared from isatin 41 by reacting with N₂H₄·H₂O. Finally, the preparation of the title compound 70 was accomplished by employing Claisen–Schmidt condensation between indolin-2-one 66 and 1,2,3-triazole aromatic aldehyde 69 with a catalytic amount of piperidine as a base (yield: 51.64%).

Scheme 12 Synthesis of isatin-1,2,3-triazole hybrid 70. Reagents and conditions: (i) chloral hydrate, Na₂SO₄, NH₂OH·HCL, HC1, H₂O, 85 °C, 3 h; (ii) conc. H₂SO₄, 60 °C, 0.5 h, 90 °C, 1.5 h; (iii) N₂H₄·H₂O, EtOH, H₂O, 100 °C, 10 h; (iv) NaNO₂, HC1, NaN₃, DCM, H₂O, 0–5 °C, 3–5 h; (v) CuSO₄·5H₂O, ascorbic acid, KI, DMF, H₂O, 50 °C, 6–10 h; (vi) EtOH, piperidine, 80 °C, 4–8 h.

Human colon cancer (HT-29), gastric cancer (MKN-45), and umbilical vein endothelial cells (HUVECs) cell lines were used to test compound 70's antiproliferative activities. In comparison to the positive control, sunitinib, which had IC₅₀ values of 10.34 and 9.25 μM, respectively, it effectively inhibited cell viability for HT-29 and MKN-45 cells, with IC₅₀ values of 1.61 and 1.92 μM, respectively. Furthermore, it had lower toxicity to HUVECs than of sunitinib. It exhibited excellent inhibitory activity against VEGFR2 with an IC₅₀ value of 26.3 nM compared to sunitinib with an IC₅₀ value of 83.2 nM. The results of docking experiments and molecular dynamics simulations showed that 70 bound firmly to the active sites of VEGFR2, forming a stable complex.⁴⁷

Nazari et al. reported the synthesis of a distinctive family of isatin derivatives. The synthesis of the most active compound 74 is depicted in Scheme 13. First, the nucleophilic reaction of isatin 41 and propargyl bromide 42 in DMF in the presence of anhydrous K₂CO₃ gave N-propargyl isatin 71. Second, Cu-catalyzed click reaction of 71 and azide 43 under ultrasonic irradiation in t-BuOH–H₂O for 1 h was used to give the target triazole 72. Finally, the 1,2,3-triazol-linked oxindole–thiosemicarbazone conjugate 74 was prepared by the reaction of 72 and thiosemicarbazide 73 in isopropyl alcohol under ultrasonic irradiation (yield: 80%).⁴⁸

Scheme 13 Synthesis of isatin-1,2,3-triazole hybrid 74. Reagents and conditions: (i) K₂CO₃, DMF, r.t., 3 h; (ii) CuSO₄·5H₂O, sodium ascorbate, t-BuOH, H₂O, r.t., sonication, 1 h; (iii) iPrOH, 65 °C, sonication, 1 h.

Compound 74 showed encouraging cytotoxicity against a variety of cell types, including A375, MDA-MB-231, PC3, and LNCaP. In terms of cytotoxic activity, it was most effective against the A375, MDA-MB-231, PC3, and LNCaP cell lines, with IC₅₀ values of 25.91 μM, 18.42 μM, 15.32 μM, and 29.23 μM, respectively, compared to etoposide (IC₅₀ = 24.46, 31.02, 30, and 31.21 μM).⁴⁸

4.4. Isatin-1,2,4-triazole hybrids

Elsawi et al. developed 1,2,4-triazole-tethered indolinones as new cancer-fighting small molecules targeting VEGFR2.⁴⁹ The preparation of the targeted hybrid 86 is illustrated in Scheme 14. 4-Aminohippuric acid 78 was synthesized by acylating the amino group of glycine amino acid 76 using p-nitrobenzoyl chloride 75 in an aqueous NaOH solution. Subsequently, the nitro group was reduced to the required amino group using Pd/C. To obtain compound 80, 4-aminohippuric acid 78 was heated with acetic anhydride 79, resulting in the acylation of two distinct functional groups. The active methylene of compound 80 was then coupled through the Kuskov-like reaction with freshly prepared diazonium salt 82 derived from 4-fluoroaniline 81 using sodium acetate salt to give hydrazone linker-tethered compound 83. Next, the azalactone ring of compound 83 was opened and underwent Sawdey rearrangement via refluxing in EtOH with N₂H₄·H₂O, ultimately forming hydrazide 84. Finally, hydrazide 84 underwent condensation with 5-bromoisatin 85 under reflux in absolute EtOH in the presence of glacial acetic acid as a catalyst to furnish hybrid 86 (yield: 44.3%).

Scheme 14 Synthesis of isatin-1,2,4-triazole hybrid 86. Reagents and conditions: (i) NaOH (aq.), r.t., 1 h; (ii) MeOH, Pd/C, r.t., 3 h; (iii) heating, 75 °C, 40 min; (iv) HC1, NaNO₂, 0–5 °C, 20 min; (v) AcONa, 0–10 °C, 3 h; (vi) EtOH, N₂H₄·H₂O, reflux, 1 h; (vii) EtOH, AcOH, reflux, 2 h.

Two cell lines, PANC1 and HepG2, were used to evaluate compound 86. In comparison to the reference medication DOX, which had IC₅₀ values of 0.19 and 0.43 μM, respectively, it demonstrated cytotoxic activity with an IC₅₀ value of 1.16 and 0.73 μM. With IC₅₀ values of 8.35 ± 0.62 μM, it exhibited minimal toxicity to normal vero cells as well. In contrast to Sorafenib, which exhibited weak VEGFR2 inhibitory action, it exhibited robust activity, with an IC₅₀ value of 16.3 nM. The most potent inhibitor of VEGFR2 thus far, 86, was simulated using molecular docking, and the results showed a robust binding to the essential amino acid residues of the VEGFR2 ATP binding site.⁴⁹

Utilizing a hybrid pharmacophore approach, Rasgania et al. reported the synthesis of triazole-functionalized isatin hybrids with potent antiproliferative activity.⁵⁰ The synthetic procedures adopted for the synthesis of target 94 is outlined in Scheme 15. First, reactive chloroacetyl isatin 93 was obtained by refluxing isatin 41 with chloroacetyl chloride 38. Secondly, the triazole derivative 92 was synthesized by a four-step reaction starting with the esterification of o-chlorobenzoic acid 87 with H₂SO₄ in MeOH. Subsequently, 88 reacted with N₂H₄·H₂O to generate 89. Compound 91 was obtained by heating 89 with ammonium thiocyanate 90 in the presence of conc. HCl as a catalyst. Finally, triazole 92 was efficiently synthesized by refluxing compound 91 with sodium hydroxide in H₂O.⁵¹ The target product 94 has been synthesized by condensation of chloroacetyl isatin 93 and 5-(2-chlorophenyl)-4H-1,2,4-triazole-3-thiol 92 in EtOH under reflux and in the presence of sodium carbonate. The nucleophilic attack of the thiol of the triazole moiety on the carbonyl carbon of chloroacetyl isatin leads to the desired novel 94 (yield: 85%).

Scheme 15 Synthesis of isatin-1,2,4-triazole hybrid 94. Reagents and conditions: (i) MeOH, H₂SO₄, reflux, 8 h; (ii) N₂H₄·H₂O, MeOH, r.t., 4 h; (iii) EtOH, conc. HC1, reflux, 6 h; (iv) NaOH, H₂O, reflux, 4 h; (v) reflux, 140 °C, 5 h, r.t., overnight; (vi) EtOH, K₂CO₃, reflux, 4 h.

Compound 94 was screened for its anticancer activity against the MDAMB-231 and MCF-7 cell lines. It has shown the inhibition of MDAMB-231 and MCF-7 with GI₅₀ values of 0.003 and 2.00 × 10⁻⁴, respectively, compared to adriamycin with GI₅₀ values of 2.00 × 10⁻⁷ and 2.00 × 10⁻⁸. Molecular docking studies supported the potential binding modes and interactions of compound 94 with the VEGFR-2 active site.⁵⁰

4.5. Isatin–pyrazole hybrids

Shreedhar Reddy et al. developed a one-pot synthesis of isatin–pyrazole hybrids as VEGFR2 inhibitors.⁵² The synthesis of targeted isatin–pyrazole hybrid 99 was achieved in two main steps, Scheme 16. The first step involved the condensation reaction between 1-methylisatin 95 and propargyl amine 96 in MeOH at 60 °C for 9 h. Later, the acyl-Sonogashira coupling of 1-methyl-3-(prop-2-yn-1-ylimino)isatin 97 with 4-nitrobenzoyl chloride 75 in the presence of sodium lauryl sulphate and K₂CO₃ in water at 65 °C for 7 h gave the corresponding in situ α,β-unsaturated ynone 98, that subsequently treated with N₂H₄·H₂O at 65 °C for 12 h to provide the desired product 99 (yield: 78%).

Scheme 16 Synthesis of isatin–pyrazole hybrid 99. Reagents and conditions: (i) MeOH, 60 °C, 9 h; (ii) PdC1₂(PPh₃)₂, CuI, sodium laurylsulfate, K₂CO₃, water, 65 °C, 7 h; (iii) N₂H₄·H₂O, 65 °C, 12 h.

The synthesized compound 99 was evaluated for its potential to inhibit the proliferation of TNBC cell lines MDA-MB-231 and MDA-MB-468. It showed the most potent cytotoxicity with IC₅₀ values of 10.24 ± 1.27 and 8.23 ± 1.87 μM against MDA-MB-468 and MDA-MB-231 cancer cells, respectively, compared to both TAM and 5-fluorouracil with IC₅₀ values of 15.29 μM and 12.4 ± 1.3 μM against MDA-MB-468, respectively, and 23.05 μM and 10.5 ± 1.2 μM against MDA-MB-231 cancer cells, respectively. Docking studies revealed a stable complex and strong binding affinity of 99 to the active sites of EGFR.⁵²

Emami et al. reported the preparation of novel isatin–pyrazole hybrids as a new class of antiproliferative agents.⁵³ The synthetic route of target compound 102 is described in Scheme 17. The preparation was achieved by the reaction of 5-chloroisatin 6 with K₂CO₃ as a base in acetonitrile, followed by N-benzylation with 3-chlorobenzyl chloride 100 at 80 °C, which afforded the intermediate 101. Then, condensation of 101 with 4-aminoantipyrine (ampyrone) 55 in absolute EtOH in the presence of a catalytic amount of glacial acetic acid under reflux for 24 h gave the desired product 102 (yield: 92%).

Scheme 17 Synthesis of isatin–pyrazole hybrid 102. Reagents and conditions: (i) K₂CO₃, TBAB, MeCN, reflux, 24 h; (ii) EtOH, AcOH (cat.), 50–60 °C, 24 h.

In comparison to cisplatin, which served as a positive control, compound 102 exhibited superior activity against MCF-7, A549, and SCOV3, with IC₅₀ values of 5.12, 25.5, and 12.9 μM, respectively. Evidence from docking and MD simulations suggests that 102 binds most strongly to the VEGFR and JNK3 MAP kinase receptors.⁵³

4.6. Isatin–sulphonamide hybrids

As potential anti-cancer agents, Demirel et al. reported the synthesis of novel sulfonamide derivatives of isatin Schiff bases.⁵⁴ The synthesis of target compound 111 is shown in Scheme 18. The starting material, 5-nitroisatin 103 was treated with 2,2-dimethylpropane-1,3 diol 104 with catalytic PTSA. Then, the nitro group in compound 105 was reduced by using 1 atm H₂/Pd–C (%10) in MeOH to yield the amine 106. The resulting amine 106 was allowed to react with p-toluene sulphonyl chloride 107 in DCM in the presence of pyridine to afford sulfonamide 108. Finally, after deprotection of the third position with a mixture of glacial acetic acid and HCl, Schiff base of sulfonamide 111 was obtained by reaction of sulfonamide 109 with 4-chloroaniline 110 in MeOH with catalytic PTSA (yield: 51%).

Scheme 18 Synthesis of isatin–sulphonamide hybrid 111. Reagents and conditions: (i) PTSA, cyclohexane, reflux, 24 h; (ii) Pd/C, H2, MeOH, r.t., 24 h; (iii) pyridine, DCM, r.t., 24 h; (iv) gl. AcOH, HC1, 30 °C, overnight; (v) PTSA, MeOH, 80 °C, 8 h.

The novel synthesized compound 111 was investigated in vitro to determine its cytotoxicity against four cancer cell lines (PC-3, HepG2, SH-SY5Y, and A549) by using an MTT assay. HepG2 cell were more sensitive to cytotoxic activity among the other studied cell lines. 111 induced the potential inhibition of cellular proliferation activity against HepG2 cells with IC₅₀ value of 37.81 μM, which was more potent than a standard drug, DOX with IC₅₀ value of 51.15 μM. A selectivity index of 111 was found to be 8.57, so it might be safe for treatment.⁵⁴

Saied et al. reported the synthesis and biological assessment of a series of novel indolinone-based benzenesulfonamides.⁵⁵ The synthesis of the target isatin-based benzenesulfonamide 118 is described in Scheme 19. The synthesis started with acetylating 4-aminobenzenesulfonamide 112 with 2-bromopropionyl bromide 113 in dioxane and TEA, which afforded compound 114. Then, the produced amide 114 was alkylated with ethyl nipecotate 115 in refluxing acetone with dry K₂CO₃ and catalytic KI, which afforded compound 116. Moreover, hydrazinolysis of 116 under reflux with N₂H₄·H₂O in EtOH afforded hydrazide; compound 117. Finally, hydrazide 117 was condensed with isatin 41 in EtOH and glacial acetic acid to afford the target compound 118 (yield: 75%).

Scheme 19 Synthesis of isatin–sulphonamide hybrid 118. Reagents and conditions: (i) dioxane, Et₃N, stirring, r.t., 20 h; (ii) acetone, K₂CO₃, KI, stirring, r.t., 2 h; (iii) N₂H₄·H₂O, EtOH, reflux, 4 h; (iv) EtOH, gl. AcOH (cat.), reflux, 6 h.

The in vitro antiproliferative effect of compound 118 against the MDA-MB-231 and MCF-7 breast cancer cell lines was investigated. When tested against MDA-MB-231 cell lines, it showed a more substantial growth inhibitory effect with an IC₅₀ value of 4.083 μM, compared to the standard drug (5-FU; IC₅₀ = 8.704 μM). When tested against MCF-7 cell lines, it showed an IC₅₀ value of 9.997 μM, which was similar to the standard drug (5-FU; IC₅₀ = 5.167 μM). It seems to be the most effective VEGFR2 inhibitor, with an IC₅₀ of 204 nM, which was on par with the gold standard (sorafenib; IC₅₀ = 41 nM). Analysis of molecular docking data showed that the potent molecule 118 bound to the VEGFR2 active site in a significant way.⁵⁵

The development of sulfonamide-tethered isatin derivatives as novel anticancer agents and VEGFR2 inhibitors was discovered by Shaldam et al.⁵⁶ Preparation procedures used in synthesizing the designed compound 123 are shown in Scheme 20. The first step in synthesis was performing chlorosulfonation of compound 119 using thionyl chloride and chlorosulfonic acid, which afforded benzenesulfonyl chloride 120. Then, the reaction of compound 120 with ammonia using EtOH as solvent afforded benzenesulfonamide 121. Moreover, the synthesis of hydrazone 122 was accomplished by refluxing compound 121 with N₂H₄·H₂O for 4 h in EtOH and in the presence of catalytic glacial acetic acid. N-benzylated isatin 16 was synthesized by reacting 5-chloroisatin 6 with benzyl bromide 61 in acetonitrile and K₂CO₃ under reflux. Finally, compound 16 was allowed to react with hydrazone 122 in the presence of a catalytic amount of glacial acetic acid and under reflux (yield: 82%).

Scheme 20 Synthesis of isatin–sulphonamide hybrid 123. Reagents and conditions: (i) HOSO₂C1, SOC1₂, 0 °C, 30 min, r.t., 26 h; (ii) EtOH, ammonia, r.t.; (iii) N₂H₄·H₂O, EtOH, gl. AcOH (cat.) reflux, 4 h; (iv) K₂CO₃, MeCN, reflux, 5 h; (v) EtOH, gl. AcOH (cat.), reflux, 4 h.

Compound 123 was evaluated in vitro against T47D breast cancer cell line. It demonstrated cytotoxic activity (IC₅₀ = 3.59 ± 0.16 μM) compared to DOX (IC₅₀ of 2.26 μM). It demonstrated good VEGFR2 inhibition with an IC₅₀ of 23.10 nM, compared to sorafenib (IC₅₀ = 29.70 nM). Docking studies and molecular dynamic simulations revealed a stable complex and strong binding affinity of 123 to the active sites of VEGFR2.⁵⁶

The cytotoxic effect of novel synthesized isatin sulfonamide-molecular hybrid derivatives targeting EGFRs have been investigated by Eldeeb et al.⁵⁷ The synthesis of the target compound 126 was done by the reaction of the parent molecule 5-(piperidin-1-ylsulfonyl) indoline-2,3-dione 124 with 1-(p-tolyl)ethenone 125 in the presence of ethylamine in MeOH, Scheme 21.

Scheme 21 Synthesis of isatin–sulphonamide hybrid 126. Reagents and conditions: (i) EtNH₂, MeOH.

The antiproliferative effects of compound 126 were tested against two human hepatocellular carcinoma cell lines, HepG2 and Huh7. The IC₅₀ value of 16.80 ± 1.44 μM, which was lower than the DOX IC₅₀ value of 21.60 ± 0.81 μM, demonstrated a higher selectivity to HepG2 than Huh7. Additionally, it demonstrated a lack of cytotoxicity when tested on the RPE1 cell line, which is not malignant, indicating a promising safety profile as a selective anticancer drug. With EGFR levels reduced to 42 ± 2.3 pmol per mg protein, it demonstrated a notable decrease. Results from docking experiments showed a stable compound 126 with an affinity for the 126 active sites on EGFR.⁵⁷

4.7. Isatin–hydrazone hybrids

A novel poly(ADP-ribose) polymerase inhibitor, El Hassab et al. have reported the synthesis of a novel series of isatin–hydrazone hybrids.⁵⁸ The general strategy for preparing the targeted molecule 133 is presented in Scheme 22. First, ethyl 4-aminobenzoate 127 was heated under reflux with N₂H₄·H₂O in EtOH to afford 4-aminobenzohydrazide 128. Furthermore, 5-chloroisatin 6 was allowed to react with 1-iodo-2-methylpropane 129 in DMF at 100 °C to furnish 5-chloro-1-isobutylisatin 130. Through condensation of 4-aminobenzohydrazide 128 with 5-chloro-1-isobutylisatin 130 in refluxing absolute EtOH in the presence of catalytic glacial acetic acid, hydrazide 131 was formed. Thereafter, the latter compound 131 reacted with phthalic anhydride 132 via heating in glacial acetic acid using anhydrous sodium acetate to afford the target molecule 133 (yield: 89%).

Scheme 22 Synthesis of isatin–hydrazone hybrid 133. Reagents and conditions: (i) N₂H₄·H₂O, EtOH, reflux, 6 h; (ii) K₂CO₃, DMF, 100 °C, 9 h; (iii) EtOH, gl. AcOH (cat.), reflux, 7 h; (iv) gl. AcOH, AcONa, reflux, 6 h.

Compound 133 was evaluated for its in vitro cytotoxicity against three human cancer cell lines, A549, PC3, and MCF-7. It displayed the highest cytotoxic activity with IC₅₀ values of 5.32, 35.1, and 4.86 μM against A549, PC3, and MCF-7 cells, respectively, compared to 5-FU with IC₅₀ values of 12.3, 68.4, and 13.15 μM. It displayed double inhibitory activity with an IC₅₀ value of 16.28 ± 1.21 nM compared to sorafenib, which has shown inhibitory activity with an IC₅₀ value of 35.62 ± 1.52 nM. Molecular docking studies of compound 133 towards human VEGFR2 kinase have shown good binding interactions with the target protein.⁵⁸

Al-Rasheed et al. demonstrated an efficient strategy for merging s-triazine and isatin via a hydrazone linkage as new potential anticancer derivatives.⁵⁹ The new target s-triazine–isatin hydrazone 140 was synthesized following the procedures in Scheme 23. The initial step involved the nucleophilic substitution of the chlorine atom of cyanuric chloride 134 by piperidine 135 at 0–5 °C to afford the 2,4-dichloro-6-(piperidin-1-yl)-1,3,5-triazine 136. The second step involved the replacement of the second chlorine atom by 4-chloroaniline 110 at room temperature, yielding compound 137. The compound 137 was reacted with N₂H₄·H₂O (80%) under reflux in EtOH for 8–12 h to afford 138. Finally, compound 138 was condensed with 5-fluoroisatin 139 in EtOH and in the presence of catalytic acetic acid to afford the target product 140 (yield: 93%).

Scheme 23 Synthesis of isatin–hydrazone hybrid 140. Reagents and conditions: (i) NaHCO₃, 0 °C, 1–2 h; (ii) acetone, H₂O, NaHCO₃, 0 °C to r.t., overnight; (iii) N₂H₄·H₂O, EtOH, reflux, 6–12 h; (iv) EtOH, AcOH (cat.), reflux, 6–8 h.

Compound 140 was evaluated for its antiproliferative activity against the lung cancer cell line (A549). It showed cytotoxicity with an IC₅₀ value of 0.114 μM compared to sorafenib IC₅₀ value of 0.195 μM. Compound 140 exhibited promising anti-trypsin effects at its anticancer IC₅₀ value (75.123 ± 4.32 μM) when compared to the inhibitory effect of rivaroxaban (53.223 ± 0.98 μM). It exhibited potentially greater potency as EGF inhibitors (65.34 ± 5.42 μM) compared to the IC₅₀ of sorafenib (68.25 ± 5.93 μM). Docking studies supported the obtained results and demonstrated the ability of these derivatives to interact with EGFR active sites, as well as broad-spectrum anti-trypsin activity.⁵⁹

M. M. Alanazi and A. S. Alanazi have reported the synthesis of novel isatin–hydrazone hybrid compounds as protein kinase inhibitors.⁶⁰ Initially, the ethyl 4-aminobenzoate 127 was added to a 4-chloro-7H-pyrrolo[2,3-d]pyrimidine 141 solution in absolute EtOH under reflux for 7 h. Then, ethyl-4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoate 142 was refluxed in excess of N₂H₄·H₂O for 5 h afforded hydrazide 143. In the final step, the hydrazide 143 and 5-methoxyisatin 28 were mixed in absolute EtOH and glacial acetic acid under reflux to furnish the final target 144 (yield: 92.26%), Scheme 24.

Scheme 24 Synthesis of isatin–hydrazone hybrid 144. Reagents and conditions: (i) EtOH, reflux, 7 h; (ii) N₂H₄·H₂O, EtOH, reflux, 5 h; (iii) EtOH, AcOH, reflux, 6–9 h.

The antiproliferative activity of compound 144 was evaluated in vitro against four human cancer cell lines: hepatocellular carcinoma (HepG2), mammary gland cancer (MCF-7), breast cancer (MDA-MB-231), and epithelioid cervix carcinoma (HeLa), using DOX and sunitinib as reference drugs. It had a potent antiproliferative activity with IC₅₀ values of 6.11, 5.93, 2.48, and 1.98 μM against HepG2, MCF-7, MDA-MB-231, and HeLa cell lines, respectively. It exhibited multi-kinase inhibition and exhibited inhibitory activities against EGFR, HER2, VEGFR2, and CDK2 with IC₅₀ values of 0.103, 0.081, 0.178, and 0.131 μM, respectively, comparable to reference drugs, ribociclib, erlotinib, lapatinib, and sorafenib. A molecular docking study revealed a stable binding interaction in the active site of the selected protein kinase enzymes.⁶⁰

Based on a molecular hybridization strategy, Alanazi et al. reported the synthesis of novel isatin–hydrazone hybrids.⁶¹ The synthesis started with refluxing 6-chloro-9H-purine 145 in excess of N₂H₄·H₂O for 1 h to furnish 6-hydrazinyl-9H-purine 146. Synthesis of the final compound 147 started with mixing 6-hydrazinyl-9H-purine 146, 5-methoxyisatin 28, and glacial acetic acid in absolute EtOH under reflux (yield: 95.19%), Scheme 25.

Scheme 25 Synthesis of isatin–hydrazone hybrid 147. Reagents and conditions: (i) N₂H₄·H₂O, reflux, 1 h; (ii) EtOH, gl. AcOH (cat.), reflux, 3–7 h.

The antiproliferative activity of compound 147 was evaluated in vitro against four human cancer cell lines: hepatocellular carcinoma (HepG2), mammary gland cancer (MCF-7), breast cancer (MDA-MB-231), and epithelioid cervix carcinoma (HeLa), using sunitinib as a reference drug. It demonstrated cytotoxic activity comparable to the reference drug sunitinib against the HepG2, MCF-7, MDA-MB-231, and HeLa cell lines, with IC₅₀ values of 9.61, 10.78, 14.89, and 8.93 μM, respectively. It exhibited inhibitory activities against EGFR, HER2, VEGFR2, and CDK2 with IC₅₀ values of 0.143, 0.15, 0.192, and 0.534 μM, respectively. A molecular docking study revealed a stable binding interaction in the active site of the investigated protein kinase enzymes.⁶¹

The discovery of new isatin hybrids as novel CDK2 inhibitors with potent in vitro antiproliferative activity was reported by Eldehna et al.⁶² The synthetic strategy deliberated for the synthesis of the target hybrid 153 is illustrated in Scheme 26. 1H-benzimidazole-2-thiol 148 was reacted with ethyl 2-chloro-3-oxobutanoate 149 in absolute EtOH to furnish intermediate 150, which then heterocyclized to compound 151 via heating in acetic anhydride 79. The ester analog 151 was subjected to hydrazinolysis to produce hydrazide 152, condensed with 5-methoxyisatin 28 in glacial acetic acid to give the targeted hybrid 153 (yield: 84%).

Scheme 26 Synthesis of isatin–hydrazone hybrid 153. Reagents and conditions: (i) EtOH, TEA, reflux, 6 h; (ii) reflux, 5 h; (iii) N₂H₄·H₂O, iPrOH, reflux, 6 h; (iv) gl. AcOH, reflux, 3–5 h.

The antiproliferative activity of compound 153 was screened in vitro towards MDA-MB-231 and MCF-7 breast cancer cell lines; staurosporine, an anticancer agent, was used as a positive control drug. It was the most active derivative, with an IC₅₀ value of 3.30 ± 0.21 μM against MDA-MB-231 compared to staurosporine, which displayed an IC₅₀ value equal to 4.29 ± 0.72 μM. It showed inhibitory activity against the cell cycle regulator CDK2 protein kinase with an IC₅₀ value of 26.24 nM, superior to staurosporine, which exhibited an IC₅₀ value of 38.5 nM. Molecular docking revealed that the compound achieved the best binding score (−11.2 kcal per mole) and formed the most stable complex with the CDK2 enzyme.⁶²

Al-Salem et al. synthesized a series of novel isatin-hydrazones in excellent yields.^63,64 The synthesis of target compound 156 was straightforward, as illustrated in Scheme 27. First, isatin 41 was refluxed with N₂H₄·H₂O in EtOH to afford isatin monohydrazone 154. Next, the isatin monohydrazone 154 was refluxed with 2,6-dichlorobenzaldehyde 155 in EtOH and in the presence of a catalytic glacial acetic acid to afford the target compound 156 (yield: 98%).

Scheme 27 Synthesis of isatin–hydrazone hybrid 156. Reagents and conditions: (i) N₂H₄·H₂O, MeOH, reflux, 1 h; (ii) EtOH, gl. AcOH, reflux, 4 h.

Compound 156 was tested for its cytotoxicity against human breast adenocarcinoma (MCF7) and human ovarian adenocarcinoma (A2780) cell lines. It (IC₅₀ = 1.51 ± 0.09 μM) showed excellent activity against MCF-7 compared to A2780 (IC₅₀ = 26 ± 2.24 μM) and to DOX (IC₅₀ = 3.10 ± 0.29 μM and 0.20 ± 0.03 μM, respectively). Compound 156 (IC₅₀ = 0.245 μM) exhibited good inhibitory activity against the cell cycle regulator CDK2 protein kinase compared to imatinib (IC₅₀ = 0.131 μM). Its ability to interact with CDK2 was also confirmed by a docking study.⁶³

As potential VEGFR2 inhibitors, Eldehna et al. reported the preparation of novel N′-(2-oxoindolin-3-ylidene) piperidine-4-carbohydrazide derivatives.⁶⁵ The targeted molecule 161 was synthesized via straightforward methodologies outlined in Scheme 28. Firstly, piperidine carboxylate ester 158 was allowed to react with 4-methoxyphenyl isocyanate 157 in toluene at 90 °C for 2 h to give compound 159. In the next step, the ester 159 reacted with an excess of N₂H₄·H₂O to give the desired hydrazide 160. After that, the hydrazide 160 was condensed with 5-bromoisatin 85 in glacial acetic acid under reflux for 4 h to afford the target compound 161 (yield: 85%).

Scheme 28 Synthesis of isatin–hydrazone hybrid 161. Reagents and conditions: (i) toluene, stirring, 90 °C, 2 h; (ii) N₂H₄·H₂O, EtOH, reflux, 4 h; (iii) gl. AcOH, reflux, 4 h.

Compound 161 was tested for its cytotoxicity using the MDA-MB-231 and MCF-7 breast cancer cell lines. Cytotoxic activity was shown with IC₅₀ values of 1.03 and 8.00 μM, respectively. It was investigated for its inhibitory effects on VEGFR2 using sorafenib as the reference medication. It displayed the most promising inhibitory efficacy with an IC₅₀ value of 45.9 nM, compared to sorafenib, with an IC₅₀ value of 48.6 nM. Within the VEGFR2 active region, molecular docking and dynamic simulations uncovered 161 important binding interactions.⁶⁵

The design and synthesis of CDK2 inhibitors using an isatin-based scaffold have been developed by Espinosa-Rodriguez et al.⁶⁶ The synthesis began with the reaction of isatin 41 with N₂H₄·H₂O in MeOH under reflux, which afforded hydrazone 154. The latter hydrazone 154 was allowed to react with isatin 41 in MeOH under reflux, which yielded the desired product 162 (yield: 95%), Scheme 29.

Scheme 29 Synthesis of isatin–hydrazone hybrid 162. Reagents and conditions: (i) N₂H₄·H₂O, EtOH, reflux, 1 h; (ii) EtOH, gl. AcOH (cat.), reflux, 3 h.

The cytotoxicity of compound 162 was examined by subjecting it to tests on MCF-7 and PC-3 cell lines. In comparison to the positive control lapatinib, which exhibited cytotoxic activity with IC₅₀ values of 50.61 μM and 32.4 μM in MCF-7 and PC-3 cells, respectively, it exhibited cytotoxic activity with IC₅₀ values of 19.07 μM and 41.17 μM. Additionally, docking tests demonstrated that it might bind to CDK2 active sites and so suppress their activity.⁶⁶

The in vitro antiproliferative activities of novel synthesized fluorinated isatin–hydrazones were investigated by Başaran et al.⁶⁷ As shown in Scheme 30, a mixture of 5-fluoroisatin 139 and N₂H₄·H₂O in EtOH was refluxed for 1 h to give compound 163. Then, compound 163 was allowed to react with 4-nitobenzaldehyde 9 in absolute EtOH using catalytic drops of glacial acetic acid under reflux for 3 h, affording the desired hydrazone 164 (yield: 65%).

Scheme 30 Synthesis of isatin–hydrazone hybrid 164. Reagents and conditions: (i) N₂H₄·H₂O, EtOH, reflux, 1 h; (ii) EtOH, gl. AcOH (cat.), reflux, 3 h.

In vitro tests were conducted on Compound 164 using the lung cancer cell line and the liver cancer cell line. The IC₅₀ value was 42.43 μM, indicating a significant suppression of lung cell development, while the IC₅₀ value was 48.43 μM, indicating cytotoxicity, when tested on the HepG2 cell line. Further evidence of its capacity to bind to and inhibit the function of EGFR and VEGFR2 was provided by docking studies.⁶⁷

Munir et al. reported the synthesis of novel N-benzylisatin-based hydrazones.⁶⁸ N-Benzylation of isatin was done via a reaction of isatin 41 with benzyl bromide 61 in acetonitrile and in the presence of K₂CO₃ and KI to yield 62. Then, the desired hydrazone 166 was synthesized by refluxing the equimolar ratio of N-benzylisatin 62 and 4-nitrobenzohydrazide 165 in EtOH and a catalytic amount of acetic acid (yield: 85%), Scheme 31.

Scheme 31 Synthesis of isatin–hydrazone hybrid 166. Reagents and conditions: (i) KI, K₂CO₃, MeCN, reflux, 4 h; (ii) EtOH, AcOH (cat.), reflux, 4–6 h.

Both the MDA-MB-231 breast cancer cell line and the MCF-10A breast epithelial cell line were tested in vitro to determine the potential of compound 166. When examined in vitro using the triple-negative MDA-MB-231 breast cancer cell line. The antiproliferative activities against the MDA-MB-231 were encouraging, with an IC₅₀ value of 15.8 ± 0.6 μM. Furthermore, docking studies confirmed that it suppressed EGFR activity by interacting with its active regions.⁶⁸

Abu-Hashem and Al-Hussain reported the design and synthesis of compound 172.⁶⁹ The synthesis started with a one-pot synthesis using the Mannich reaction by stirring a mixture of isatin 41 with freshly distilled isonicotinaldehyde 167 and morpholine 168 in sodium ethoxide solution to compound 169. Moreover, the latter compound 169 was refluxed with thiosemicarbazide 73 in glacial acetic acid to produce the target 170. Finally, the nucleophilic aromatic substitution reaction of compound 170 with 2,3-dichloro-quinoxaline 171 under reflux in absolute EtOH and in the presence of catalytic triethylamine (TEA) gave 1-(morpholino(pyridin-4-yl)methyl)-3-((2-thioxo-2,3-dihydro-1H-imidazo[4,5-b]quinoxalin-1-yl)imino)indolin-2-one 172 in 74% yield, Scheme 32.

Scheme 32 Synthesis of isatin–hydrazone hybrid 172. Reagents and conditions: (i) EtONa, stirring, r.t., 8–10 h; (ii) gl. AcOH, reflux, 5–7 h; (iii) EtOH, TEA, reflux, 22–25 h.

Using 5-FU as a reference medication, compound 172's antiproliferative activity was tested in vitro against four human cancer cell lines: gastric carcinoma cells (MGC-803), breast adenocarcinoma cells (MCF-7), nasopharyngeal carcinoma cells (CNE2), and oral carcinoma cells (KB). Compared to 5-FU, the cytotoxic action was demonstrated with IC₅₀ values of 9.7, 9.6, 9.5, and 9.4 μM, respectively, compared to 10.7, 10.5, 10.3, and 10.1 μM for 5-FU.⁶⁹

4.8. Isatin–benzofuran hybrid

Eldehna et al. reported the preparation of novel isatin–benzofuran hybrids.⁷⁰ The strategy designed for the preparation of the target compound 177 is illustrated in Scheme 33. First, the reaction of ethyl bromoacetate 174 with 5-bromosalicylaldehyde 173 in acetonitrile to furnish ethyl 5-bromobenzofuran-2-carboxylate 175. Thereafter, hydrazinolysis of the ester 175 via refluxing with N₂H₄·H₂O gave the intermediate hydrazide 176. Finally, key intermediate 176 was condensed with 5-bromoisatin 85 in absolute EtOH with catalytic drops of glacial acetic acid to get the final compound 177 (yield: 87%).

Scheme 33 Synthesis of isatin–benzofuran hybrid 177. Reagents and conditions: (i) MeCN, K₂CO₃, reflux, 4 h; (ii) N₂H₄·H₂O, MeOH, reflux, 3 h; (iii) EtOH, gl. AcOH (cat.), reflux, 4–7 h.

T-47D and MCF-7 breast cancer cell lines were used to study compound 177's antiproliferative effects. Relative to the standard staurosporine, which exhibited IC₅₀ values of 4.34 and 4.81 μM, respectively, it had a potent cytotoxic effect at concentrations of 3.82 and 3.41 μM. It showed the most potent inhibitory activity on CDK-2 and GSK-3β with IC₅₀ of 37.77 and 32.09 nM, comparable to staurosporine IC₅₀ of 38.5 and 43.38 nM. Molecular docking studies revealed important binding interactions of potent compound 177 with the CDK-2 and GSK-3β active sites.⁷⁰

4.9. Isatin–thiazolidine hybrid

As potential VEGFR2 inhibitors, Taghour et al. developed new thiazoldine–isatin hybrids.⁷¹ For preparing the target hybrid 181, the synthetic route is clarified in Scheme 34. Primarily, compound 179 was prepared by the reaction of isatin 41 with thiazolidine-2,4-dione 178 under reflux in glacial acetic acid with catalytic sodium acetate for 5 h. Heating a mixture of compound 179 with 2-chloro-N-(2,6-dichlorophenyl) acetamide 180 in dry DMF/KI yielded the corresponding target compound 181 (yield: 89%).

Scheme 34 Synthesis of isatin–thiazolidine hybrid 181. Reagents and conditions: (i) gl. AcOH, AcONa, reflux, 5 h; (ii) DMF, K₂CO₃, KI, reflux, 6 h.

Compound 181 was tested for its cytotoxic activity against the MCF-7 cell line. It exhibited cytotoxicity with an IC₅₀ value of 12.47 μM compared to 5-FU as a reference drug. A molecular docking study was conducted against the Hsp90 protein and obtained crucial molecular interactions.⁷¹

Yousef et al. reported the synthesis of novel isatin-based derivatives as potential anticancer agents.⁷² The target molecule 187 was synthesized utilizing a three-step reaction, Scheme 35. The first step involved the reaction of 5-nitroisatin 103 with propylamine 182 to furnish N-propyl intermediate 183. In the following step, refluxing 5-nitro-N-propylisatin 183 with 4-methyl-3-thiosemicarbazide 184 in EtOH in the presence of a catalytic amount of glacial acetic acid afforded isatin-3-(Z)-thiosemicarbazone 185. The final step was the cyclization of 185 with chloroacetic acid 186 in refluxing EtOH in the presence of catalytic anhydrous sodium acetate, which gave the target compound 187 (yield: 69%).

Scheme 35 Synthesis of isatin–thiazolidine hybrid 187. Reagents and conditions: (i) K₂CO₃, DMF, 80 °C, 45 min; (ii) EtOH, gl. AcOH (cat.), reflux, 2 h; (iii) EtOH, AcONa (cat.), reflux, 24 h.

The in vitro cytotoxicity of compound 187 was evaluated against three cell lines: human liver cancer cells (HepG2), breast cancer cells (MCF-7), and human colon cancer cells (HT-29), using DOX as a reference. It exhibited cytotoxic activity with IC₅₀ values of 4.97, 5.33, and 3.29 μM, respectively, compared to DOX (IC₅₀ = 4.50, 4.17, and 4.01 μM, respectively). The inhibitory effect of 187 against CDK1 was also significant, with IC₅₀ = 0.38 μM, compared to reference DOX (IC₅₀ = 0.42 μM). A docking study also confirmed the ability of 187 to interact with CDK1.⁷²

Novel azine-linked hybrids of isatin and thiazolodinone scaffolds as CDK2 inhibitors were reported by Qayed et al.⁷³ The designed compound 189 was synthesized as illustrated in Scheme 36. The route started with the synthesis of N-benzyl-5-chloroisatin 16 via the reaction of 5-chloroisatin 6 with benzyl bromide 61 in DMF in the presence of K₂CO₃ and KI at 80 °C. Next, refluxing N-benzyl-5-chloroisatin 16 with 4-methyl-3-thiosemicarbazide 184 in EtOH in the presence of catalytic glacial acetic acid afforded (Z)-thiosemicarbazone 188. Finally, the target hybrid 189 was produced from the cyclization of compound 188 by reacting with chloroacetic acid 186 in EtOH in the presence of a catalytic amount of anhydrous sodium acetate (yield: 37%).

Scheme 36 Synthesis of isatin–thiazolidine hybrid 189. Reagents and conditions: (i) K₂CO₃, DMF, KI, 80 °C, 45 min; (ii) EtOH, gl. AcOH (cat.), reflux, 3 h; (iii) EtOH, AcONa (cat.), reflux, 24 h.

Using Dox as a reference medicine, compound 189 showed antiproliferative efficacy against HepG2, MCF7, and HCT-29 cell lines, which are human liver, breast, and colon, respectively. It showed cytotoxic activity with IC₅₀ values of 3.0, 5.19, and 3.10 μM, respectively, compared to Dox with IC₅₀ values of 4.15, 4.61, and 4.65 μM. The inhibitory effect of 189 against CDK2 was also significant, with IC₅₀ = 27.42 nM, compared to the reference drug sunitinib (IC₅₀ = 23.8 nM). The molecular dynamics simulations and docking studies showed that there is a stable complex with a strong binding affinity of 189 to the active regions of CDK2.⁷³

As possible VEGFR2 inhibitors, Mallikarjuna Rao et al. reported the synthesis of some isatin-thiazolidine-2,4-dione-pyrazoles.⁷⁴ The synthetic path followed to get the designed compound 194 is shown in Scheme 37. Initially, Knoevenagel condensation between 1-methylindoline-2,3-dione 95 and thiazolidine-2,4-dione 178 under piperidine catalyst in EtOH under reflux for 24 h afforded compound 190. Later, treatment of compound 190 with propargyl bromide 42 in acetonitrile at 80 °C for 10 h gave the terminal alkyne 194 (yield: 67%).

Scheme 37 Synthesis of isatin–thiazolidine hybrid 194. Reagents and conditions: (i) EtOH, piperidine, reflux, 24 h; (ii) Cs₂CO₃, MeCN, 80 °C, 10 h; (iii) PdC1₂(PPh₃)₂, Cul, K₂CO₃, sodium lauryl sulfate, H₂O, 65 °C, 8 h; (iv) N₂H₄·H₂O, 65 °C, 12 h.

Human cancer cell lines HepG2, Caco-2, and MDA-MB231 were used to study compound 194's antiproliferative properties. It displayed remarkable activity (HepG2; IC₅₀ = 2.4 μM, Caco-2; IC₅₀ = 6.2 μM and MDA-MB231; IC₅₀ = 7.5 μM) against all cancer cell lines and this was higher than the standard drug DOX (HepG2; IC₅₀ = 2.9 μM, Caco-2; IC₅₀ = 8.3 μM and MDA-MB231; IC₅₀ = 9.2 μM). The results showed that 194 inhibited VEGFR2 more effectively than sorafenib (IC₅₀ = 51.3 nM vs. 53.8 nM). Through molecular docking studies, it was found to bind extensively to the VEGFR2 active site.⁷⁴

Taghour et al. reported the synthesis of thiazolidine-2,4-diones hybrids as potential VEGFR-2 inhibitors.⁷⁵ The sequence of chemical synthesis is clarified in Scheme 38. The synthesis started with the preparation of thiazolidine-2,4-dione 178 through the reaction of thiourea 195 with chloroacetic acid 186 under reflux in conc. HCl. Moreover, condensation of isatin 41 with thiazolidine-2,4-dione 178 was done with sodium acetate in acetic acid to give the isatin derivative 179. The treatment of compound 179 with the alcoholic solution of KOH provided its potassium salt 196. Then, heating a mixture of compound 196 with 2-chloro-N-(p-tolyl)acetamide 39 in dry DMF and KI afforded the target compound 197 (yield: 79%).

Scheme 38 Synthesis of isatin–thiazolidine hybrids 197 and 199. Reagents and conditions: (i) (a) H₂O, 0–5 °C, stirring, 15 min; (b) conc. HC1, reflux, 10 h; (ii) AcOH, AcONa, reflux, 6 h; (iii) KOH, EtOH, reflux; (iv) DMF, K₂CO₃, KI, reflux, 6 h.

A panel of three cancer cell lines, namely colon (Caco-2), hepatocellular (HepG2), and breast (MDA-MB-231) cancer cell lines, was used to evaluate the potential anti-proliferative effects of compound 197. It displayed IC₅₀ values of 2.0, 10, and 40 μM in comparison to DOX, which had IC₅₀ values of 3.46, 1.15, and 0.98 μM, respectively. It showed IC₅₀ values of 2.0, 10, and 40 μM, in contrast to DOX's IC₅₀ values of 3.46, 1.15, and 0.98 μM, respectively. The molecular dynamics and docking studies have revealed the presence of a stable complex that binds to the active areas of VEGFR2 with a high affinity of 197.⁷⁵

Elkaeed et al. also reported the synthesis of thiazolidine-2,4-diones hybrid as an apoptotic VEGFR2 inhibitor. The synthetic procedure was the same as adopted for the synthesis of 197 in Scheme 38, except in the final step. 2-Chloro-N-phenylacetamide 198 was heated with compound 196 in dry DMF, and KI gave the target compound 199 (yield: 87%).⁷⁶

Four cancer cell lines were tested for compound 199's antiproliferative effects: A549, Caco-2 (colon cancer), HepG2 (hepatocellular cancer), and MDA-MB-231 (breast cancer). The compound demonstrated superior cytotoxic effects compared to DOX, with IC₅₀ values of 49.5, 9.3, and 28 μM against A549, Caco-2, and MDA-MB-231, respectively. The IC₅₀ value was 69.11 nM, indicating a substantial inhibitory action against VEGFR2. This compound's molecular docking investigations on the target VEGFR2 protein demonstrated its binding capabilities.⁷⁶

4.10. Isatin–thiazole hybrid

An optimization strategy was adopted for synthesizing a new series of 2-oxindole conjugates by Ismail et al.⁷⁷ The synthetic route adopted for the preparation of the target 3-(methylene)-indol-2-one 206 is depicted in Scheme 39. The synthesis started with the preparation of urea intermediate 202 by the reaction of mono-BOC protected phenylenediamine 200 with 2-isocyanatothiazole 201. Then, BOC removal with trifluoroacetic acid (TFA) gave the required compound 203.⁷⁷ Furthermore, condensation of the active methylene group of 2-oxindole 66 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) 204 to afford the N-methylene intermediate 205. Finally, the latter compound 205 reacted with the prepared amine 203 in glacial acetic acid to give the target compound 206 (yield: 84%).

Scheme 39 Synthesis of isatin–thiazole hybrid 206. Reagents and conditions: (i) DCM, −25 °C to r.t.; (ii) TFA, DCM, 0 °C; (iii) toluene, reflux, 2 h; (iv) AcOH, reflux, 4 h.

Compound 206 was screened in vitro for its cytotoxicity towards human MCT-7 (Breast), DU-145 (Prostate), and HCT-116 (Colon) cancer cell lines. It showed distinct potent and broad antiproliferative activity with IC₅₀ values of 4.39, 1.06, and 0.34 nM, respectively, against MCT-7, DU 145, and HCT-116 cell lines. It showed the most active inhibition activity against FGFR, VEGFR2, and RET kinases, showing IC₅₀ values of 1.28, 0.117, and 1.18 μM, respectively. Molecular docking studies, which demonstrated its ability to achieve essential interactions, are crucial for inhibiting FGFR, VEGFR-2, and RET kinases.⁷⁷

4.11. Isatin–thiadiazole hybrid

A series of 1-(2-((aryl-1,3,4-thiadiazol-2-yl)amino)acetyl)indoline-2,3-diones as anti-breast cancer leads was produced by Rasgania et al.⁷⁸ The synthesis of the target compound 208 is outlined in Scheme 40. First, the synthesis of compound 207 via a four-step method. o-Chlorobenzoic acid 87 was refluxed in H₂SO₄ containing MeOH for 16 h to get the benzoate 88. By refluxing the benzoate with N₂H₄·H₂O for 4 h, the hydrazide 89 was obtained, which was further converted into its respective thiosemicarbazide 91 by refluxing with KSCN in acidic media (HCl/H₂O) for 5 h. 2-Amino-5-(2-chlorophenyl)-1,3,4-thiadiazole 207 was obtained by stirring thiosemicarbazides in H₂SO₄ for 6 h, followed by neutralization with an ammonia solution. Second, isatin 41 was vigorously refluxed with chloroacetyl chloride for 5 h, followed by an overnight stir at room temperature, affording 1-(2-chloroacetyl)indoline-2,3-dione 93 as yellow crystals. The target molecule 208 was efficiently synthesized by the thermal integration of the synthesized thiadiazole 207 with chloroacetyl isatin 93 under reflux in EtOH for 4 h (yield: 91%).

Scheme 40 Synthesis of isatin–thiadiazole hybrid 208. Reagents and conditions: (i) H₂SO₄, MeOH, reflux, 16 h; (ii) N₂H₄·H₂O, reflux, 4 h; (iii) KSCN, HC1, H₂O, reflux, 5 h; (iv) H₂SO₄, stirring, r.t., 6 h; (v) (a) reflux, 5 h; (b) stirring, r.t., 24 h; (vi) EtOH, K₂CO₃, reflux, 4 h.

The antiproliferative activity of compound 208 was evaluated in vitro against a triple-negative breast cancer (MDA-MB-231) cell line. It showed the highest level of potency when tested in vitro, with an IC₅₀ value of 57.79 μg ml⁻¹, when compared to tamoxifen citrate as the positive control. Additionally, docking tests verified that it could bind to EGFR active sites and so decrease its activity, suggesting that it acted as an EGFR inhibitor.⁷⁸

Table 1 summarizes the biological findings of some recently synthesized isatin hybrids as anti-cancer agents, highlighting the effective molecular targets, melting points, and cytotoxicity values.

Table 1 Cytotoxicity of isatin-based derivatives with highlighted molecular targets

Feature	Scheme	Structure	m.p. (°C)	Kinase inhibition activity			Anticancer activity			Ref.
Isatin–quinazoline hybrids	1		292–294	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		37
					Cpd 7	Indirubin		Cpd 7	Indirubin
				VEGFR2	56.74 ± 4.3	126.42 ± 20	HepG2	2.53 ± 0.11	6.92 ± 0.65
				EGFR	87.48 ± 6.71	175.46 ± 18.33
				CDK-2	9.39 ± 0.51	45.60 ± 2.24	MCF-7	7.54 ± 0.71	6.12 ± 0.35
				CDK-4	36.39 ± 4.52	23.64 ± 2.15
	2		>300	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		37
					Cpd 12	Indirubin		Cpd 12	Indirubin
				VEGFR2	14.31 ± 2.70	126.42 ± 20	HepG2	3.08 ± 0.35	6.92 ± 0.65
				EGFR	32.65 ± 1.61	175.46 ± 18.33
				CDK-2	225.32 ± 12.56	45.60 ± 2.24	MCF-7	5.28 ± 0.22	6.12 ± 0.35
				CDK-4	244.32 ± 12.21	23.64 ± 2.15

---

Isatin–indole hybrids	3		268–270	Enzymes	—	Cell lines	IC50 [μM]		38
							Cpd 17	Sunitinib
				—	—	HT-29	2.02 ± 0.36	10.14 ± 0.8
						ZR-75	0.74 ± 0.88	8.31 ± 2.4
						A-549	0.76 ± 0.12	5.87 ± 0.3
	4		>300	Enzymes	—	Cell lines	IC50 [μM]		39
							Cpd 23	5-FU
				—	—	HT-29	206	4600
						SW-620	188	1500
	5		232.5–234	Enzymes	IC50 [μM]	Cell lines	IC50 [μM]		40
					Cpd 29		Cpd 29	DOX
				CDK2	6.32	MCF-7	1.15 ± 0.04	6.81 ± 0.22
						MDA-MB-231	10.54 ± 0.43	10.29 ± 0.72
						NCI-ADR	9.17	—

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Isatin–indole hybrids	6		> 300	Enzymes	% inhibition		Cell lines	IC50 [μM]		41
					Cpd 32	Staurosporine		Cpd 32	Staurosporine
				CDK4	1.26	0.017	MCF-7	0.39 ± 0.05	6.81 ± 0.22
							MDA-MB-231	22.54 ± 1.67	10.29 ± 0.72
	7		195–197	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		42
					Cpd 36	Roscovitine		Cpd 36	DOX
				CDK2	0.85 ± 0.03	0.1 ± 0.01	A-549	7.3 ± 0.42	2.3 ± 0.17
							MDA-MB-231	4.7 ± 0.28	4.5 ± 0.29
							HCT-116	2.6 ± 0.17	3.7 ± 0.24

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Isatin–1,2,3-triazole hybrids	8		ND	Enzymes	Docking study	Cell lines	%Growth inhibition (PGI)/lethality	43
					Cpd 44		Cpd 44
				CDK2	Compound 44 displayed the best docking score of −8.89	Panel of cell lines	Ranging from 3%–98%

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Isatin–1,2,3-triazole hybrids	9		110–113	Enzymes	Docking study	Cell lines	IC50 [μM]			44
					Cpd 51		Cpd 51	5-FU	Tamoxifen
				EGFR	Compound 51 displayed the best docking score of −7.33	MDA-MB-231	8.23 ± 1.87	10.5 ± 1.2	23.05
						MDA-MB-468	10.24 ± 1.27	12.4 ± 1.3	15.29

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Isatin–1,2,3-triazole hybrids	10		252–254	Enzymes	% Inhibition		Cell lines	IC50 [μM]			45
					Cpd 56	Sunitinib		Cpd 56	Sunitinib	5-FU
				VEGFR-2	77.6%	67.1%	MCF7	5.361 ± 0.31	11.304 ± 0.28	—
							HCT116	12.50 ± 0.88	9.67 ± 0.84	20.43 ± 1.99
							PaCa-2	12.128 ± 0.79	6.596 ± 0.43	—
	11		228	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]			46
					Cpd 63	Sunitinib		Cpd 63	Sunitinib	Dox
				VEGFR2	26.3 ± 0.38	30.70 ± 0.17	PANC1	0.13 ± 0.01	1.49 ± 0.04	0.45 ± 0.01
							PC3	0.10 ± 0.01	0.60 ± 0.02	0.24 ± 0.02
				STAT-3	5.63 ± 0.34	—	WPMY-1 (normal cell)	>10.0	>10.0	>3.0
							Selectivity index	>100	>16	>12

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Isatin–1,2,3-triazole hybrids	12		248	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		47
					Cpd 70	Sunitinib		Cpd 70	Sunitinib
				VEGFR2	26.38 ± 1.09	83.20 ± 1.36	HT-29	1.61 ± 0.45	10.34 ± 0.96
							MKN-45	1.92 ± 0.37	9.25 ± 0.77
							HUVECs	7.94 ± 0.36	6.37 ± 0.59

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Isatin–1,2,3-triazole hybrids	13		190–192	Enzymes	—	Cell lines	IC50 [μM]		48
							Cpd 74	Etoposide
				—	—	A375	25.91 ± 0.005	24.46 ± 0.019
						MDA-MB231	18.42 ± 0.002	31.02 ± 0.051
						PC-3	15.32 ± 0.002	30 ± 0.037
						LNCaP	29.23 ± 0.003	31.21 ± 0.005
						HDF (normal cell)	>100	>100

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Isatin–1,2,4-triazole hybrids	14		>300	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		49
					Cpd 86	Sorafenib		Cpd 86	DOX
				VEGFR2	16.3 ± 0.42	29.7 ± 0.39	PANC1	1.16 ± 0.02	0.19 ± 0.01
							HepG2	0.73 ± 0.02	0.43 ± 0.02

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Isatin–1,2,4-triazole hybrids	15		101–102	Enzymes	Docking study	Cell lines	IC50 [μM]		50,51
					Cpd 94		Cpd 94	Tamoxifen citrate
				EGFR	Compound 94 displayed the best docking score of −9.2	MDA-MB-231	0.73	12.88

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Isatin–pyrazole hybrids	16		ND	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		52
					Cpd 99	Sorafenib		Cpd 99	5-FU
				VEGFR-2	16.28 ± 1.21	35.62 ± 1.52	A549	5.32 ± 0.78	12.30 ± 0.48
							PC3	35.10 ± 1.54	68.48 ± 1.42
							MCF-7	4.86 ± 0.48	13.15 ± 1.02

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Isatin–pyrazole hybrids	17		205–209	Enzymes	Docking study	Cell lines	IC50 [μM]		53
					Cpd 102		Cpd 102	Cisplatin
				VEGFR	Compound 102 displayed the best docking score of −9.9	MCF-7	5.12 ± 1.34	14.9 ± 2.1
						A549	25.5 ± 1.8	12 ± 1.9
				JNK3	Compound 102 displayed the best docking score of −9.4	SCOV3	12.9 ± 2.9	18.7 ± 0.58
						MCF-10A (normal cell)	32.6 ± 3.1	42.5 ± 1.7
Isatin–sulphonamide hybrids	18		125–127	Enzymes	—	Cell lines	IC50 [μM]		54
							Cpd 111	DOX
				—	—	HepG2	37.81 ± 5.05	51.15 ± 9.9
						NIH/3T3 (normal cell)	324.2 ± 20.51	53.8 ± 6.8

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Isatin–sulphonamide hybrids	19		228–230	Enzymes	IC50 [μM]		Cell lines	IC50 [μM]		55
					Cpd 118	Sorafenib		Cpd 118	5-FU
				VEGFR2	204 ± 9	41 ± 2	MDA-MB-231	4.083 ± 0.175	8.704 ± 0.372
							MCF-7	9.997 ± 0.364	5.167 ± 0.188
	20		245–247	Enzymes	IC50 [μM]		Cell lines	IC50 [μM]		56
					Cpd 123	Sorafenib		Cpd 123	DOX
				VEGFR2	23.10 ± 0.41	29.70 ± 0.17	T47D	3.59 ± 0.16	2.26 ± 0.10

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Isatin–sulphonamide hybrids	21		238–240	Enzymes	Docking study	Cell lines	IC50 [μM]		57
					Cpd 126		Cpd 126	DOX
				EGFR	Compound 126 displayed the best docking score of −21.74	HepG2	16.80 ± 1.44	21.60 ± 0.81
						Huh7	40.00 ± 2.20	11.60 ± 0.90
						RPE1 (normal cell)	>100	—

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Isatin–hydrazone hybrids	22		270	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		58
					Cpd 133	Olaparib		Cpd 133	Olaparib
				PARP-1	13.65 ± 1.42	5.32 ± 0.78	MCF-7	0.67 ± 0.12	32.81 ± 2.26
							HCC1937	0.53 ± 0.11	>100

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Isatin–hydrazone hybrids	23		276–278	Enzymes	IC50 [μM]			Cell lines	IC50 [μM]		59
					Cpd 140	Sorafenib	Rivaroxaban		Cpd 140	Sorafenib
				EGFR	65.34 ± 5.42	68.25 ± 5.93	—	A549	0.114 ± 0.01	0.195 ± 0.02
				Trypsin	75.12 ± 4.32	—	53.22 ± 0.98	WI-38 (normal cell)	1.458 ± 0.09	2.15 ± 0.01
								Selectivity index	12.785 ± 1.05	11.025 ± 0.93

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Isatin–hydrazone hybrids

24

>300

Enzymes

IC50 [μM]

Cell lines

IC50 [μM]

60

Cpd 144

Ribocicli

Erlotinib

Lapatinib

Sorafenib

Cpd 144

DOX

Sunitinib

CDK2

0.131 ± 0.007

0.063 ± 0.003

—

—

—

HepG2

6.11 ± 0.4

4.50 ± 0.2

6.82 ± 0.5

EGFR

0.103 ± 0.006

—

0.041 ± 0.003

—

—

MCF-7

5.93 ± 0.3

4.17 ± 0.2

5.19 ± 0.4

Her2

0.081 ± 0.002

—

—

0.06 ± 0.006

—

MDA-MB-231

2.48 ± 0.1

3.18 ± 0.1

8.41 ± 0.7

VEGFR2

0.178 ± 0.009

—

—

—

0.045 ± 0.002

HeLa

1.98 ± 0.1

5.57 ± 0.4

7.48 ± 0.6

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Isatin–hydrazone hybrids	25		>300	Enzymes	IC50 [μM]					Cell lines	IC50 [μM]		61
					Cpd 147	Roscovitine	Erlotinib	Lapatinib	Sorafenib		Cpd 147	Sunitinib
				CDK2	0.534	0.143	—	—	—	HepG2	9.61 ± 0.8	6.82 ± 0.5
				EGFR	0.143	—	0.041	—	—	MCF-7	10.78 ± 0.9	5.19 ± 0.4
				Her2	0.15	—	—	0.051	—	MDA-MB-231	14.89 ± 1.2	8.41 ± 0.7
				VEGFR2	0.192	—	—	—	0.049	HeLa	8.93 ± 0.8	7.48 ± 0.6

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Isatin–hydrazone hybrids	26		>300	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		62
					Cpd 153	Staurosporine		Cpd 153	Staurosporine
				CDK2	26.24 ± 1.4	38.5 ± 2.1	MDA-MB-231	3.30 ± 0.21	4.29 ± 0.72
							MCF-7	5.82 ± 0.32	3.81 ± 0.22
	27		286–287	Enzymes	IC50 [μM]		Cell lines	IC50 [μM]		63 and 64
					Cpd 156	Imatinib		Cpd 156	DOX
				CDK2	0.2456	0.1312	MCF7	1.51 ± 0.09	3.10 ± 0.29
							A2780	26 ± 2.24	0.20 ± 0.03

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Isatin–hydrazone hybrids

28

>300

Enzymes

IC50 [nM]

Cell lines

IC50 [μM]

65

Cpd 161

Sorafenib

Cpd 161

Tamoxifen

Sorafenib

Staurosporine

VEGFR2

45.9

48.6

MCF-7

8.00 ± 0.76

8.36 ± 0.90

4.75 ± 0.56

—

MDA-MB-468

1.03 ± 0.03

—

—

5.93 ± 0.16

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Isatin–hydrazone hybrids	29		ND	Enzymes	Docking study	Cell lines	IC50 [μM]		66
					Cpd 162		Cpd 162	Lapatinib
				CDK2	Compound 162 displayed the best docking score of −9.5	MCF-7	19.07 ± 4.02	50.61 ± 12.83
						PC-3	41.17 ± 4.52	32.39 ± 2.13
	30		249–251	Enzymes	Docking study	Cell lines	IC50 [μM]		67
					Cpd 164		Cpd 164	Cisplatin
				VEGFR2	Compound 164 displayed the best docking score of −9.722	A549	42.43	4.19
				EGFR	Compound 164 displayed the best docking score of −7.17	HepG2	48.43	—
						HEK293T (normal cell)	6.04	7.48

---

Isatin–hydrazone hybrids	31		174–176	Enzymes	Docking study	Cell lines	IC50 [μM]	68
					Cpd 166		Cpd 166
				EGFR	Compound 166 displayed the best docking score of −7.561	MDA-MB231	15.8 ± 0.6
						MCF-10A	>50

---

Isatin–hydrazone hybrids	32		>350	Enzymes	—	Cell lines	IC50 [μM]		69
							Cpd 172	5-FU
				—	—	MGC-803	9.7 ± 1.1	10.7 ± 1.2
						MCF-7	9.6 ± 1.2	10.5 ± 1.1
						CNE2	9.5 ± 1.1	10.3 ± 1.3
						KB	9.4 ± 1.2	10.1 ± 1.1

---

Isatin–benzofuran hybrid	33		>300	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		70
					Cpd 177	Staurosporine		Cpd 177	Staurosporine
				CDK2	37.77 ± 2.1	38.5 ± 2.1	MCF-7	3.41 ± 0.10	4.81 ± 0.14
				GSK-3β	32.09 ± 1.7	43.38 ± 2.4	T-47D	3.82 ± 0.12	4.34 ± 0.14

---

Isatin–thiazolidine hybrids

34

166–168

Enzymes

IC50 [nM]

Cell lines

IC50 [μM]

Selectivity index

71

Cpd 181

Sorafenib

Cpd 181

DOX

181

DOX

VEGFR2

76.64

53.65

A549

5.40 ± 0.14

0.70 ± 0.22

0.07

3.57

Caco2

0.58 ± 0.01

0.82 ± 0.21

0.65

3.04

HepG2

14.45 ± 0.07

0.28 ± 0.07

0.02

8.92

MDA

0.94 ± 0.05

0.90 ± 0.08

0.4

2.77

Vero (normal cell)

0.38 ± 0.03

2.50 ± 0.14

—

—

WI-83 (normal cell)

0.90 ± 0.07

0.25 ± 0.04

—

—

---

Isatin–thiazolidine hybrids	35		193–196	Enzymes	IC50 [μM]		Cell lines	IC50 [μM]		72
					Cpd 187	DOX		Cpd 187	DOX
				CDK1	0.04 ± 0.38	0.07 ± 0.42	HepG2	4.97 ± 0.3	4.50 ± 0.2
							MCF7	5.33 ± 0.4	4.17 ± 0.2
							HCT-29	3.29 ± 0.2	4.01 ± 0.4
	36		258–260	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		73
					Cpd 189	Sunitinib		Cpd 189	DOX
				CDK2	27.42	23.8	HepG2	3.0 ± 0.92	4.15 ± 0.5
							MCF7	5.19 ± 1.15	4.61 ± 1.1
							HCT-29	3.10 ± 0.96	4.65 ± 0.6
							WI-38 (normal cell)	67.01 ± 5.78	5.05 ± 0.7
							Selectivity index	17.8	1.2
	37		222–224	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		74
					Cpd 194	Sorafenib		Cpd 194	DOX
				VEGFR2	51.3	53.8	HepG2	2.4	2.9
							Caco-2	6.2	8.3
							MDA-MB231	7.5	9.2
							Vero (normal cell)	27.5	25.2

---

Isatin–thiazolidine hybrids	38		271–272	Enzymes	IC50 [nM]		Cell lines	IC50 [μM]		Selectivity index	75
					Cpd 197	Sorafenib		Cpd 197	DOX	197
				VEGFR2	116.3	53.65	Caco-2	2.0 ± 0.005	3.46 ± 0.003	365
							HepG2	10 ± 0.001	1.15 ± 0.02	73.00
							MDA-MB-231	40 ± 0.002	0.98 ± 0.01	18.25
							Vero (normal cell)	730 ± 0.015	—	—

---

Isatin–thiazolidine hybrids

38

ND

Enzymes

IC50 [nM]

Cell lines

IC50 [μM]

Selectivity index

76

Cpd 199

Sorafenib

Cpd 199

DOX

199

DOX

VEGFR2

69.11

53.65

A549

49.5 ± 0.70

7 ± 0.22

0.54

3.57

Caco-2

9.3 ± 0.421

8.2 ± 0.21

2.85

3.05

HepG-2

149 ± 9.80

2.8 ± 0.07

0.18

8.93

MDA-MB231

28 ± 0.50

9 ± 0.77

0.95

2.78

Vero (normal cell)

26.5 ± 1.71

25 ± 1.41

—

—

---

Isatin–thiazole hybrid	39		>300	Enzymes	IC50 [μM]	Cell lines	IC50 [μM]	77
					Cpd 206		Cpd 206
				FGFR 1	1.287	MCF-7	4.39
				VEGFR 2 (KDR)	0.117	DU-145	1.06
				RET	1.185	HCT-116	0.34

---

Isatin–thiadiazole hybrid	40		136–137	Enzymes	Docking study	Cell lines	IC50 (μM)		78
					Cpd 208		Cpd 208	Tamoxifen citrate
				EGFR	Compound 208 displayed the best docking score of −9.8	MDA-MB231	57.79	7.26

---

5. Conclusion and expert opinion

In medicinal chemistry, isatin has recently come to light as an incredibly adaptable scaffold that provides a one-of-a-kind platform for the design of novel bioactive agents. It is highly favored in the field of drug development due to its structural simplicity, ease of functionalization, and wide range of biological activities. Hybrid compounds, such as isatin–heterocycles, are routinely generated using this scaffold. These molecules frequently exhibit improved selectivity and efficacy through specific target signaling pathways. Furthermore, it is critical to reveal innovative methods for producing this scaffold, to analyze the different strengths of that heterocycle, and to investigate potent applications for isatin.

Isatin can promote cell death in different types of cells and alter the expression of genes associated with cell death since it is a potent inhibitor of many enzymes and receptors. Because of the advantages of hybrid compounds in terms of efficiency, selectivity, and resistance to drug resistance, hybridization is an attractive strategy for drug discovery. The chemical and pharmacological characteristics of isatin–heterocycle hybrids are attracting attention towards novel chemotherapeutics. Various isatin analogs have been synthesized, and their bioactivities have been evaluated as lead drugs. As a future perspective, nano-formulations, drug delivery systems with innovative drug signaling pathways, will be further recommended to improve bioavailability and targeted delivery, particularly in solid tumors, to lead to the development of novel, potent anticancer medicines.

Conflicts of interest

The authors declare that they have no financial or personal interests.

Data availability

No primary research results, and no new data were generated or analysed as part of this review.

Acknowledgements

Dr Mohamed S. Nafie appreciates the Seed Research Project No. (24021440154) funded by the Research and Graduate Studies at the University of Sharjah, United Arab Emirates. Additionally, he acknowledges the electronic library sources at the University of Sharjah, which provide full access to published papers and enable searching on Chemistry databases.

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Footnote

† Both authors shared equally in this manuscript with shared first authorship. This study is a part of M. A. Alshams's Master of Science degree in Chemistry under the main supervision of M. S. Nafie.

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