Review on Isatin and It's Anticancer Scaffolds

Isatin (1H-indole-2,3-dione) is a biologically active heterocyclic compound that serves as a key structural motif in numerous natural and synthetic molecules. Since its first synthesis in the 19th century, isatin has gained considerable attention in medicinal chemistry due to its wide spectrum of pharmacological properties, including antimicrobial, antiviral, anticonvulsant, antioxidant, and anticancer activities. ^1-5 Its unique indole-dione framework provides reactive sites suitable for diverse chemical modifications, making it an important intermediate for drug design and synthesis. ^6-8

Recent studies have shown that isatin-based compounds exhibit a wide range of biological activities, with notable effects against different cancer cell lines such as breast, lung, colon, and leukemia models.^11-14 These derivatives act through several pathways including induction of apoptosis, inhibition of cell proliferation, and interference with receptor tyrosine kinases like EGFR and VEGFR2.^15.16  Apart from their anticancer role, many isatin analogues have also shown antioxidant, antimicrobial, anticonvulsant, and enzyme inhibitory activities, indicating their broad pharmacological potential.^17,19

Chemically, isatin is a versatile scaffold that can easily undergo condensation, cyclization, and Schiff base formation, leading to a variety of heterocyclic structures with improved biological activity. The metal-chelating ability of isatin-derived Schiff bases further enhances their interaction with biomolecules, contributing to their diverse pharmacological profile.^20,21

In recent years, researchers have also emphasized more sustainable methods for synthesizing isatin derivatives, including the use of mild catalysts and natural extracts that minimize the use of toxic reagents. Such eco-friendly approaches reflect the current trend of combining medicinal chemistry with green chemistry principles to achieve safer and more efficient drug design.²²

Structure Activity Relationship (SAR) of Isatin

Figure 2. Structure Activity Relationship (SAR) of isatin

The isatin nucleus (1H-indole-2,3-dione) serves as a versatile pharmacophore exhibiting a wide spectrum of biological activities. Substitution at various positions on the indole ring (N-1, C-5, C-6, and C-7) and modifications at the C-2 and C-3 carbonyl groups significantly influence its pharmacological profile, particularly anticancer activity. The electronic nature, steric effects, and hydrogen-bonding potential of these substituents determine the interaction of isatin derivatives with biomolecular targets such as kinases, caspases, and topoisomerases.

1. Modifications at N-1 Position

• Substitution at the nitrogen atom (N-1) greatly affects lipophilicity and cellular permeability.
• Alkyl or aryl groups at this position enhance cytotoxic activity by improving membrane penetration and interaction with hydrophobic pockets of enzymes.
• N-benzyl and N-phenyl isatin derivatives have shown potent antiproliferative activity against breast and lung cancer cell lines.^3,7,31
• Example
  1. N-benzyl isatin

Figure 3. Structure of N-benzyl isatin

• 2. N-methylisatin

Figure 4. Structure of N-methyl isatin

2. Substitution at C-5, C-6, and C-7 Positions

1. Electron-donating groups (–OCH?, –OH) at C-5 or C-7 generally enhance activity due to improved hydrogen bonding and radical scavenging properties.
2. Electron-withdrawing groups (–Cl, –Br, –NO?) at C-5 or C-6 increase cytotoxicity by promoting electrophilic character and enhancing binding with enzyme receptors.
3. 5-Fluoro and 5-Chloro isatin derivatives exhibit remarkable anticancer potential by inducing apoptosis in tumor cells.^2,5,20,26
4.  Examples
   1. 5-Fluoroisatin

Figure 5. Structure of 5-Fluoro isatin

1. 2. 7-Chloroisatin

Figure 6. Structure of 7- Chloro isatin

1. 3. 5-Nitroisatin

Figure 7. Structure of 5-Nitro isatin

3.  Modifications at the C-2 and C-3 Positions

• The C-3 carbonyl group is highly reactive and can form Schiff bases, hydrazones, or oximes, leading to enhanced cytotoxic and apoptosis-inducing properties.³
• C-3 hydrazone derivatives are particularly active due to their ability to chelate metal ions and generate reactive oxygen species (ROS) in cancer cells.^14,34
• The C-2 position can accommodate bulky or heterocyclic moieties, which may improve binding affinity toward cancer-related enzymes.
•  Example

1. Isatin-3-hydrazone derivative

Figure 8. Structure of Isatin-3-hydrazone

2. Isatin-thiosemicarbazones derivative

Figure 9. Structure of Isatin thiosemicarbazone derivative

4. N-1 and C-3 Dual Substitution

• Simultaneous modification at N-1 and C-3 positions significantly improves selectivity and potency.
• N-1 aryl–C-3 hydrazone derivatives exhibit synergistic effects due to combined lipophilicity and enhanced receptor binding.^7,10,13,28
• These dual-substituted compounds have demonstrated broad anticancer efficacy against leukemia, colon, and ovarian cancer lines.^3,14,28
•  Example

N-phenyl isatin-3-thiosemicarbazone derivative [7]

Figure 10. Structure of N-phenyl isatin-3-thiosemicarbazone derivative

5. Role of Heterocyclic Fusion

• Incorporation of heterocyclic rings (e.g., quinoline, pyridine, thiazole) into the isatin scaffold expands conjugation and improves enzyme inhibition (like EGFR, Bcl-2, and caspase-3).^12,13
• Spiro-isatin derivatives and isatin-quinoline hybrids have been reported to display high selectivity toward tumor cells while minimizing normal cell toxicity.^12,13,28
• Example

Spiro[indole-3,4′-oxan]-2(1H)-one derivate ¹

Figure 11.  Structure of spiro[indole-3,4′-oxan]-2(1H)-one derivate

Methods of synthesis of Isatin:

1. Sandmeyer method

The Sandmeyer reaction offers a well-established method for the synthesis of isatin from aniline derivatives. The process begins with the formation of an N-aryl oxime intermediate through the reaction of aniline with trichloroacetaldehyde (chloral hydrate) and hydroxylamine hydrochloride under acidic conditions. Subsequent acid-catalyzed cyclization of the oxime intermediate in the presence of sulfuric acid and water leads to the formation of isatin (1H-indole-2,3-dione). This reaction pathway efficiently constructs the isatin core and allows for various substitutions on the aromatic ring via the aniline precursor, making it a versatile approach for functionalized isatin synthesis.^6,33

Scheme 1. Sandmeyer Synthesis

2. Stolle’s method

The Stolle synthesis is a widely recognized method for preparing N-substituted isatins from anilines. Initially, the aniline reacts with oxalyl chloride, leading to the formation of a chlorooxalylanilide intermediate with the elimination of hydrogen chloride. This intermediate subsequently undergoes intramolecular cyclization in the presence of a Lewis acid catalyst, such as aluminium chloride (alcl3), yielding the corresponding isatin derivative. The reaction conditions are typically mild, and the method provides access to a variety of N-substituted isatins by simply varying the substituents on the starting aniline.^6,25

Scheme 2. Stolle’s Synthesis method

3. Gassman method

The Gassman reaction offers a route starting from N-substituted anilines, which react with trichloro acetyl chloride followed by oxidation to produce isatin derivatives. The advantage of this method is the ability to introduce substituents directly on the nitrogen atom, enabling the synthesis of various N-substituted isatins useful for biological screening.⁶

Scheme 3. Gassman Synthesis method

Most Potent Anticancer Isatin Derivatives :

1.  Bis (indolin-2,3-dione) Hybrid

Figure 12. Structure of Compound 29

Althagafi et al. Synthesized a series of bis-(indoline-2,3-dione) via reactions of isatin with 1,3-dibromopropane with subsequent condensation reaction with hydrazine derivatives. The antiproliferative activity of the synthesized hybrids was assayed on MCF-7 cells and revealed some compounds exhibiting IC50s ranging from 0.0028 to 0.028 µm, with Compound 29 being the most potent, having an IC50 of 0.0028 µm on MCF-7 cancer cells.⁴²

Synthesis:

Scheme 4. Synthesis of Compound 29

2.  N-Benzyl 5-Aryl Isatin

• Structure:

Figure 13. Structure of compound 51

Zhang et al. Synthesized a series of N-1 benzyl substituted 5-arylisatins and evaluated Their antiproliferative activities on human leukemia K562 and liver cancer hepg2 cells using An MTT assay. The presence of N-benzyl and C-5 phenyl substituents along with an intact Carbonyl functionality at the C-3 position proved to be important for good antiproliferative Activities. Compound 51 proved to be the most potent among the series, exhibiting the Highest antiproliferative activity against K562 cells, with an IC50 of 0.03 µm, while an IC50 Of 0.05 µm was observed on hepg2 cells (Figure 8). The morphological results and flow Cytometry analysis revealed that the cell cycle arrest resulted in a time- and dose-dependent Manner. Additionally, the results of cell tube formation have shown the inhibition of the Angiogenesis of HUVEC cells by 51 via a dose-dependent inhibitory effect.¹¹

• Synthesis:

Scheme 5. Synthesis of compound 51

3, Indolin-2-one Thiadiazole Hybrid

Figure 14. Structure of compound 197

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 mm In comparison to DOX, which had IC50 values of 3.46, 1.15, and 0.98 mm, respectively. It showed IC50 values of 2.0, 10, and 40 mm, in contrast to DOX’s IC50 values of 3.46, 1.15, and 0.98 mm, 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 197.¹²

• Synthesis

Scheme 6. Synthesis of compound 197

4.  Isatin–thiazole-based hybrid

Figure 15. Structure of Compound 14

MTT-based screening assays against two cancer Cell lines MCF-7 (breast cancer) and HT-29 (colorectal Cancer) revealed some important structure activity trends. Compounds with phthalimide ring, thiadiazole moiety and 4-nitrophenyl group (attached to diazole ring) displayed Higher activity than the compounds with alternative structure features (viz. Maleimide ring, oxadiazoles moiety and 4-hydroxyphenyl group). Compound C14 (2-(4-chloro-3-((5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)diazenyl)phenyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione) displayed Significant anticancer activity against both cell lines with IC50 values of 0.09 ± 0.02µm (MCF-7) and 0.11 ± 0.03µm (HT-29).

Compound C16 displayed highest anticancer activity against MCF-7 cell line with IC50 = 0.07 ± 0.02 µm. Compounds with thiadiazoles moiety (C5 to C16) showed higher cytotoxicity than their respective oxygen analogues.^24,32

Figure 16. Structure of compound 16

• Synthesis of  compound 14 and 16 :

Scheme 7. Synthesis of compound 14 and 16

5. N-Substituted Isatin Thiosemicarbazone Metal Complexes   

Figure 18. Structure of compound 40a

The group further extended the approach and synthesized a series of nickel(II) complexes [Ni(L)2] of N-substituted isatin thiosemicarbazones. In vitro cytotoxicity evaluation Revealed substantial activities on human breast (MCF7) and lung (A549) cancer cells, with The best outcomes being recorded for complexes 40a and 40b, exhibiting IC50 < 0.1 µm. Complexes 40a and 40b showed a stronger DNA binding affinity than the other Complexes, which could be attributed to the presence of benzyl and allyl groups.^14,34

Synthesis:

Scheme 8. Synthesis of compound 40a

6. Isatin–α, β-Unsaturated Ketone Hybrids

Figure 20. Structure of compound 5b

 Compounds 5b and 5g were the most potent with IC50 values 0.38 ± 0.08 µm And 0.57 ± 0.05 µm, which are comparable with the positive control staurosporine. To Assess the safety of the synthesized compounds, we screened the cytotoxicity of the most Potent compound 5b on the normal peripheral blood mononuclear cells (PBMC).

The Compound showed weak cytotoxicity on normal cells with an IC50 value of 14.17 µm, and Selectivity index (SI) of 37.2. These results demonstrated that the synthesized compounds Have selective cytotoxic activity against cancer cells, without affecting normal body cells. Therefore, the target compounds are promising anticancer lead compounds.

Figure 21. Structure of compound 5g

Synthesis:

Scheme 9. Synthesis of compound 5b and 5g.[39]

7. 5-Substituted Isatin-Capped Hydroxamic Acid Derivatives (HDAC Inhibitors)           

Figure 22. Structure of compound 15

In a recent study, 5-substituted isatin capped hydroxamic Acid derivatives (15) have been reported as HDAC inhibitors and their anti-proliferative activity against cervical tumor cell lines Has been evaluated. SAR showed that halogenation of the parent Compound (15a) produced the most active compounds of the Series (15c, 15d, and 15e). Out of the halogenated compounds the 5-chloro-substituted derivative (15d) was found to be the Most potent, with IC50 = 0.97 ± 0.26 μm. ²⁰

8. Isatin Indole Hybrid

Figure 23. Structure of compound 12c

Indoles constitute an important subunit for the discovery of new drug candidates. It is widely distributed in natural Products and bioactive molecules and is responsible for the faecal smell in human feces, scents of flowers, and the flowery Smell of perfumes [84–86]. The indole moiety is a versatile Molecule with several biological properties such as antifungal, antimicrobial, antiviral, and antitubercular properties.

The hybrid 12c showed potent antiproliferative activity with an IC50 value of 1.17 µm which was approximately sevenfold greater than Sunitinib, a well-known anticancer Medication. The SAR studies revealed that hybrids bearing N-benzyl moiety on isatin were more active with better antiproliferative activity.²³

9. Isatin Azole Hybrid

Figure 24. Structure of compound 2b

Azole, a privileged scaffold of choice when designing novel Therapeutic agents, is mainly found as core structure in several natural products and synthesized compounds that are used by pharmaceutical or agrochemical industries. Most azole compounds are used as antifungal drugs and some of its derivatives possess a variety of biological properties such as anticancer , antibacterial, and antitubercular properties. Several isatin-Azole hybrids have been synthesized and reported to possess diverse pharmacological properties. The hybrid 2b was identified as the most active analogue portraying broad-spectrum Activity against breast, colon, and lung human cancer cell Lines with an average IC50 value of 2.14 μm. SAR studies Revealed that the 5-pyrazolyl moiety was crucial and played An important role in the enhanced activity of this compound.¹⁴

10. N-Benzyl and C-5 Phenyl–Substituted Isatin Derivatives

Figure 25. Structure of compound 2m

In conclusion, a series of novel N-1 benzyl and C-5 phenyl Substituted isatin derivatives were synthesized and tested for their In vitro antitumor activity against two strains of cancer cell lines (human leukemia K562 cells and liver cancer hepg2 cells). Among Them, compounds 2m exhibited excellent cancer inhibitory activity In vitro against K562 cell lines (IC50 ¼ 0.03 mm) and hepg2 cell lines (IC50 ¼ 0.05 mm). SAR analysis showed that the carbonyl group of the isatin, N-benzyl and C-5 phenyl substituted pattern, the para-methoxyl group of the benzyl played a significant role in the anti-tumor activity. Both morphological results and flow cytometry analysis showed that compound 2m induce apoptosis and cause cell cycle arrest in a time-dependent and dose-dependent manner. Furthermore, wound healing and trans well experiments showed that compound 2m could inhibit the migration and repair ability of hepg2 cells. In Addition, the results of cell tube formation showed that the compound 2m could inhibit the angiogenesis of HUVEC cells and has a Dose-dependent inhibitory effect. The effects of indole derivative compound 2m on tumor cell growth, migration and angiogenesis were studied, which provide a Strong foundation for further study on the antitumor activity and mechanism of indole compounds.¹¹

• Synthesis:

Scheme 10. Synthesis of compound 2m