Isatin derivatives as potent cholinesterase inhibitors: recent developments and future challenges

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, memory impairment, and neuronal loss, primarily associated with β-amyloid plaque deposition, tau hyperphosphorylation, and cholinergic dysfunction. Since the disruption of cholinergic neurotransmission is a key pathological feature of AD, the cholinesterase (ChE) inhibitors targeting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) remain the mainstay of symptomatic treatment by enhancing synaptic acetylcholine levels. However, the currently approved small molecules, including tacrine, donepezil, rivastigmine, and galantamine, are limited by their modest efficacy, poor selectivity, and adverse effects, necessitating the development of improved therapeutic agents. Among emerging scaffolds, isatin (1H-indole-2,3-dione) has attracted considerable research attention owing to its structural versatility, synthetic accessibility, and favorable pharmacological properties. Recent studies have reported diverse isatin-based derivatives, including hydrazones, Schiff bases, thiosemicarbazones, spirooxindoles, and molecular hybrids, exhibiting potent AChE and BChE inhibitory activities, often in the submicromolar or nanomolar ranges. Structure–activity relationship studies have revealed the critical influence of electronic effects, linker optimization, N-substitution, and molecular hybridization on inhibitory potency and selectivity. This review summarizes the most promising isatin-based cholinesterase inhibitors reported from 2020 to the present, highlighting their biological activities, structure–activity relationships, and potential for the development of next-generation anti-Alzheimer therapeutics.

Structural modifications and pharmacophore orientation drive cholinesterase inhibitory activity in isatin derivatives, offering new insights for the design of next-generation anti-Alzheimer's agents.

1. Introduction

Alzheimer's disease (AD) is a chronic and irreversible neurodegenerative disease¹ characterized clinically by the progressive deterioration of cognitive function, intellectual decline, memory loss, and behavioral changes.² A central pathological feature of AD is the deterioration of cholinergic neurons within the basal forebrain, specifically those neurons projecting from the nucleus basalis of Meynert, and this leads to diffuse cortical acetylcholine (ACh) deficiency.^3,4 This reduction is accompanied by decreased choline acetyltransferase (ChAT) activity and disrupted ACh synthesis and release, and these changes are parallel to the development of cognitive impairment.⁵ The cholinergic hypothesis suggests that the symptoms of AD, such as memory impairment and executive dysfunction, are characterized by the loss of neurotransmitters.⁶ Within the synaptic cleft, acetylcholine (ACh) undergoes rapid hydrolysis catalyzed by cholinesterases (ChEs), a family of enzymes that regulate cholinergic signaling. The two principal ChEs are AChE and BChE.⁷ AChE is predominantly found in cholinergic and adrenergic nerve endings, muscle fibers, erythrocytes, and placental tissue, where it plays a central role in terminating synaptic transmission. In contrast, BChE (EC 3.1.1.8) is mainly produced in the liver and secreted into the circulatory system, serving a compensatory function⁸ as AChE activity diminishes in the course of the disease. A characteristic peculiarity of AChE is its substrate inhibition at high concentrations of the substrate due to its special catalytic process and substrate binding kinetics, a feature that is not commonly found with BChE.⁹ One of the major strategies to control AD is the suppression of the hydrolysis of ACh in order to increase its concentration in the synaptic cleft, which can be achieved by the inhibition of AChE and BChE.^10–12

Three ChE inhibitors—donepezil, rivastigmine, and galantamine—are clinically approved for the symptomatic management of AD.¹³ These agents exert their therapeutic effects by enhancing cholinergic neurotransmission; however, their efficacy is largely limited to symptomatic relief. In addition, their clinical use is frequently associated with adverse effects, including gastrointestinal disturbances, anorexia, vomiting, and bronchoconstriction.¹⁴ Given the established significance of cholinesterase inhibition in AD therapy, substantial research efforts are currently directed toward the discovery and development of novel inhibitors with improved therapeutic efficacy and reduced side-effect profiles.

Isatin (1H-indole-2,3-dione) is a natural bioactive heterocyclic framework consisting of a five-membered non-aromatic ring fused with a six-membered aromatic ring, and it possesses two carbonyl groups at positions 2 and 3 and a secondary nitrogen at position 1.^15–19 It is a highly versatile pharmacophore for the development of a wide array of biologically active compounds with anticonvulsant,²⁰ anticancer,^21–23 antidepressant,²⁴ antidiabetic,^25,26 antiulcer,²⁷ anti-HIV,²⁸ antimalarial,²⁹ antibacterial,³⁰ and antioxidant³¹ activities. The C-3 keto functionality is reactive towards nucleophilic addition, which facilitates the preparation of pharmacologically interesting derivatives, such as hydrazones, imines, oxindoles, and thiosemicarbazones.^32–34 For the treatment of AD, isatin-derived AChE inhibitors have the ability to interact with the aromatic residues of the peripheral anionic site (PAS) of AChE though π–π interactions, thereby improving their therapeutic value.³⁵ Therefore, it would be worthwhile to highlight the recently reported promising ChE inhibitors based on the isatin moiety. This may facilitate the search for novel drug candidates having improved safety and pharmacological profiles by catching the attention of the drug development community.

2. Cholinesterase inhibition mechanism

Cognitive deterioration in AD is attributed to β-amyloid (Aβ) deposition, dysfunctional cholinergic neurotransmission, and oxidative injury, which all result in neuronal dysfunction and structural impairment.³⁶ Reactive oxygen species (ROS) contribute significantly by mediating oxidative damage to neuronal lipids and proteins, which compromise cellular integrity. Of these pathological factors, cholinergic deficiency is the most enduring biochemical signature, evidenced by the diminished levels of ACh and AChE in brain tissues obtained post-mortem. AChE, a major serine hydrolase, accelerates the hydrolysis of ACh at an extremely high turnover rate of approximately 25 000 molecules per second, resulting in the swift synaptic loss of ACh, which is engaged in learning and memory.^37–39 The active site of the enzyme, consisting of anionic and esteratic subsites, has been structurally elucidated, providing insights into its catalytic activity. Based on the cholinergic hypothesis, cholinergic neuron degeneration plays a key role in age-related cognitive impairment. Therefore, AChE inhibitors have been designed to enhance cholinergic transmission by inhibiting ACh breakdown (Fig. 1). These compounds prolong the duration of ACh availability in synapses, thereby yielding relief from cognitive symptoms. Various pharmacophoric models have been utilized to design effective AChE inhibitors, and as a result, the FDA has approved drugs, such as physostigmine, rivastigmine, tacrine, galantamine and donepezil, for the symptomatic treatment of AD and other neurodegenerative disorders.⁴⁰

Fig. 1. Mechanism of the AChE inhibitors.

3. Isatin derivatives as cholinesterase inhibitors

3.1. N-substituted isatin-based derivatives

Triazole, a nitrogenous heterocyclic moiety, possesses a unique electronic structure and a rigid framework that make it a privileged scaffold in medicinal chemistry, exhibiting an impressive spectrum of pharmacological activities, including anti-inflammatory, antiproliferative, anticancer, and antimicrobial activities.⁴¹ Coumarin derivatives are also known to inhibit ChE enzymes predominantly by binding to the peripheral anionic site (PAS).⁴² In this regard, a series of coumarin-triazole isatin hybrids (8) was synthesized (Scheme 1) and evaluated for their ChE inhibitory activity.⁴³ The target hybrids (8) were synthesized through a multistep synthetic approach. Initially, isatin (1) was treated with propargyl bromide (2) to afford the corresponding N-propargyl isatin derivatives (3). In the parallel pathway, 4-hydroxycoumarin (4) reacted with dibromoalkane (5) under basic conditions to yield a bromoalkyl coumarin intermediate (6), which was subsequently converted into an azide derivative (7) using NaN₃ in DMF. Finally, the key intermediates (7 and 3) were coupled via a copper(i)-catalyzed azide–alkyne cycloaddition (CuAAC) using CuSO₄/sodium ascorbate under mild conditions, affording the desired coumarin-triazole-isatin hybrid (8) (yield: 93%).

Scheme 1. Synthesis of the coumarin-triazole isatin hybrid (8). Reagents and conditions: (i) K₂CO₃, DMF, 4 h, and rt, (ii) K₂CO₃, DMF, 6 h, and rt, (iii) DMF, 4 h, and rt, and (iv) CuSO₄, sodium ascorbate, DMF, distilled water, 0.5–2 h, and rt.

In vitro evaluation demonstrated that most of the derivatives (8) exhibited good to excellent inhibition for BChE, with IC₅₀ values ranging from 1.74 ± 0.29 µM to 14.84 ± 1.44 µM, while showing comparatively weak activity for AChE. Structure–activity relationship (SAR) studies revealed that minimal or no substitution at the C-5 position of isatin improved enzyme binding by reducing steric hindrance. Substitution with fluorine yielded moderate activity, whereas relatively bulky halogens, nitro, and methyl groups lowered potency. Short linkers restricted flexibility, hindering AChE interactions. Compound 8, bearing (R H) substituents (Table 1), exhibited significant BChE inhibitory activity (IC₅₀ = 1.74 ± 0.29 µM), surpassing the reference drug, donepezil (IC₅₀ = l5.24 ± 1.16 µM).

Table 1. ChE inhibitory activity of the N-substituted isatin-based derivatives^a.

Code	Structure	AChE		BChE		Ref.
		IC50 (µM)	Standard IC50 (µM)	IC50 (µM)	Standard IC50 (µM)
8		15.28 ± 1.22	Donepezil (0.08 ± 0.02)	1.74 ± 0.29	Donepezil (5.24 ± 1.16)	43
13		155 ± 1.65*	Donepezil (42 ± 0.78*)	—	—	44
19		18.2 ± 1.2	Galantamine (275 ± 25)	—	—	45
23		0.52 ± 0.073**	Donepezil (8.234 ± 0.628**)	—	—	46
31		0.71 ± 0.04	0.23 ± 0.4	1.08 ± 0.02	0.31 ± 0.03	47
35		24.7	Pyridostigmine bromide (21.7)	—	—	50
37		82.1 ± 8.1	Rivastigmine 275 ± 25	3.77 ± 0.45	Rivastigmine 1.14 ± 0.13	51
41		—	—	4.49 ± 0.43	Rivastigmine (1.14 ± 0.13)	52

^a* Units of IC₅₀ value = nM and ** units of IC₅₀ value = µg mL⁻¹.

Furthermore, Bhagat and coworkers⁴⁴ reported the synthesis and evaluation of isatin triazole hybrids (13) as potent AChE inhibitors. The target molecule (13) was synthesized (Scheme 2) by first preparing N-propargylated isatin (10) by the reaction of 5-fluoroisatin (1) with propargyl bromide (9) in DMF, using potassium carbonate as a base. Meanwhile, phenacyl azide (12) was formed from 4-chlorophenacyl bromide (11) and NaN₃ in DMF. Subsequently, the coupling reaction of N-propargylated isatin (10) and phenacyl azide (12) was conducted through the CuAAC click reaction using DMF with catalytic CuSO₄ and sodium ascorbate, affording the desired product (13) (yield: 82%).

Scheme 2. Synthesis of the isatin triazole hybrid (13). Reagents and conditions: (i) K₂CO₃, DMF, and rt, (ii) DMF, NaN₃, and rt, and (iii) DMF, sodium ascorbate, CuSO₄, and rt.

Results revealed that most of the compounds exhibited comparable inhibitory activities, with IC₅₀ values ranging between 155 ± 1.65 nM and 852 ± 7.17 nM (Table 1). SAR analysis also identified that the electronegative substituents (–F and –Cl) on both the isatin and benzoyl phenyl rings showed the highest AChE inhibitory activity, while –CH₃ and –OCH₃ were least favorable. Overall, isatin ring substitutions followed this activity trend: F > Cl > H > CH₃ > OCH₃. Compound 13, having (R F and R₁ Cl) substituents, demonstrated the best AChE inhibitory activity, with an IC₅₀ value of 155 ± 1.65 nM, as compared to the reference drug, donepezil (IC₅₀ = 42 ± 0.78 nM).

Afterward, the synthesis and biological evaluation of a series of isatin-linked thiazole hybrids (19) were reported. The synthesis (Scheme 3) was initiated with the N-alkylation of isatin (14) using 1,3-dibromopropane (15), affording the corresponding N-alkylated isatin intermediate (16). Meanwhile, the thiazolidine-2-thione heterocycle (18) was synthesized from 1,1-dimethylpropargylamine (17) and CS₂ in the presence of a Pd catalyst. Subsequently, the N-alkylated isatin intermediate (16) was coupled with the prepared thiazolidine-2-thione derivative (18) in the presence of K₂CO₃, furnishing the desired hybrid compound (19) (yield: 42%).

Scheme 3. Synthesis of the isatin-linked thiazole hybrid (19). Reagents and conditions: (i) K₂CO₃ and DMF, (ii) Pd (PPh₃)₄ and toluene and (iii) K₂CO₃ and MeCN.

All the synthesized compounds demonstrated excellent to moderate AChE inhibitory activity.⁴⁵ Out of them, compound 19 (n = 3) was a hit derivative, exhibiting superior AChE inhibition (IC₅₀ = 18.2 ± 1.2 µM) compared to the reference drug, galantamine (IC₅₀ = 275 ± 25 µM) (Table 1). SAR studies have shown that inhibitory activity is influenced by the chain length of the linker between the isatin and thiazole moieties. Optimal activity was observed in derivatives bearing a linker of three or four carbon atoms, while deviation from it (either shorter or longer) resulted in a considerable reduction in inhibitory activity.

Similarly, considering the pivotal role of Isatin derivatives in the development of bioactive compounds, Mehreen et al. synthesized and explored the in vitro AChE inhibitory activity of a library of phenoxy pendant isatin (23).⁴⁶ The synthetic strategy was initiated by treating 4-fluorophenol (20) with a large excess of 1,2-dibromoethane (21) at 50 °C in a CH₃CN : DMF (9 : 1) mixture, affording the desired intermediate (22). The intermediate (22) was then reacted with isatin (14) in DMF in the presence of K₂CO₃ as a base at 60 °C to furnish a phenoxy pendant isatin (23) (yield: 83%) (Scheme 4).

Scheme 4. Synthesis of the phenoxy pendant isatin derivative (23). Reagents and conditions: (i) K₂CO₃/CH₃CN : DMF (9 : 1)/50 °C–90 °C and (ii) K₂CO₃/DMF/60 °C.

Biological evaluation demonstrated that most of the synthesized compounds exhibited superior inhibitory activities against AChE, with IC₅₀ values ranging from 0.52 ± 0.073 µg mL⁻¹ to 1.31 ± 0.083 µg mL⁻¹ in comparison to donepezil. The SAR analysis of the phenoxy pendant isatin revealed that halogen substitution at position 5 of the isatin core and on the phenoxy pendant tended to decrease the AChE inhibitory activity. Among halogens, fluorine resulted in the smallest loss of activity, probably because of its small size and high electronegativity, which influenced electronic properties but had little steric effect. Among them, the substituted compound (23), with R₁ H and R₂ H, emerged as a lead candidate, showcasing excellent AChE inhibition, with an IC₅₀ of 0.52 ± 0.073 µg mL⁻¹ compared to donepezil (IC₅₀ = 8.234 ± 0.628 µg mL⁻¹) (Table 1).

A later study by Macha and coworkers⁴⁷ established the potential derivatives of cycloalkyl-fused quinoline–isatin Schiff base hybrids (31) and investigated their biological properties against ChE. As shown in Scheme 5, the synthesis of the hybrid molecule (31) was carried out via a four-step process. First, 2-aminobenzonitrile (24) was heated under reflux in dry toluene in the presence of BF₃·Et₂O and cycloheptanone (25) for 24 hours to obtain the cycloalkanoquinoline amine (26). This amine (26) was then reacted with 3-chloropropanoyl chloride (27) in dry dichloromethane, along with triethylamine, from 0 °C to room temperature to give rise to the cycloalkanoquinolinyl-N-chloroacetamide compound (28). In a parallel step, isatin (14) was reacted with aniline (29) by refluxing for 3–5 hours in the presence of absolute ethanol containing glacial acetic acid to give the isatin Schiff base compound (30). Lastly, these isatin Schiff bases (30) were reacted with a cycloalkanoquinolinyl-N-chloroacetamide compound (28) in dry dimethylformamide (DMF), along with K₂CO₃, yielding the desired derivative (31) (yield: 49%).

Scheme 5. Synthesis of the cycloalkyl-fused quinoline–isatin Schiff base hybrid (31). Reagents and conditions: (i) BF₃, reflux, and 24 h, (ii) DCM, 0 °C, and 4 h, and (iii) acetic acid, EtOH, reflux, and 6 h.

Biological evaluation demonstrated that all the synthesized compounds exhibited comparable inhibitory activities, with IC₅₀ values ranging from 0.71 ± 0.04 µM to 3.05 ± 0.03 µM for AChE and from 1.08 ± 0.02 to 4.21 ± 0.14 µM (Table 1) for BChE. Out of them, compound 31, possessing (n = 3, n′ = 2, and R H) substituents, showed the best activity, with IC₅₀ values of 0.71 ± 0.04 µM (AChE) and 1.08 ± 0.02 µM (BChE), in comparison to the reference drug, tacrine. SAR studies showed that increasing the cycloalkane ring size from five to seven members and extending the linker length enhanced AChE inhibition. Additionally, substitution at the para-position of the phenyl ring also influenced activity, with fluorine generally reducing BChE inhibition, while chlorine improved AChE potency in certain analogs.

On the other hand, oximes, being hydroxy-imine derivatives, exhibit a broad spectrum of pharmacological activities, including antifungal, antibacterial, antioxidant, and anticancer effects. Remarkably, these compounds serve as effective antidotes to nerve agents by reactivating inhibited AChE.^48,49 In light of their therapeutic relevance, a series of isatin–pyridine oxime hybrids (35) has been explored for their AChE inhibition potential.⁵⁰ Isatin (14) was reacted with 1,5-dibromopentane (32) in dry DMF at 80 °C in the presence of K₂CO₃ as a base to afford the N-alkylated isatin intermediate (33). This intermediate (33) was subsequently treated with pyridine-4-aldoxime (34) in dry acetonitrile with a catalytic amount of KI. The reaction was carried out at 80 °C, yielding the corresponding isatin–oxime hybrid (35) (Scheme 6). In vitro analysis showed that compound 35 (n = 5 and oxime position = 4) (Table 1) behaved as a competitor inhibitor of AChE, exhibiting a promising inhibitory activity, with IC₅₀ = 24.7 µM, as compared to the reference drug, pyridostigmine bromide (IC₅₀ = 21.7 µM). SAR studies revealed the critical effect of the linker length between the isatin and pyridine-oxime moieties, with the most potent inhibition observed for a linker encompassing five methylene units.

Scheme 6. Synthesis of the isatin–pyridine oxime hybrid (35). Reagents and conditions: (i) K₂CO₃, DMF, and 80 °C and (ii) CH₃CN, KI cat. and 80 °C.

Moreover, building on the excellent inhibitory activity of previously reported N-alkyl-isatin hybrids, a library of N-alkyl isatin derivatives (37) was recently prepared and evaluated for their AChE and BChE inhibitory activities.⁵¹ The target compound (37) was synthesized by the reaction of isatin (14) with 1-bromononane (36), using K₂CO₃ as a base in DMF at room temperature, affording the target compound (37) (yield: 42%) (Scheme 7).

Scheme 7. Synthesis of the N-alkyl isatin derivative (37). Reagents and conditions: (i) K₂CO₃, DMF, and rt.

Results showed that all the compounds proved to exhibit moderate to high AChE inhibition (IC₅₀ = 62.2 ± 8.5 to 647 ± 57 µM), along with BChE inhibition (IC₅₀ = 3.77 ± 0.45 µM to >1000 µM) (Table 1). SAR analysis indicated no definite relationship between AChE inhibition and the length of the N-alkyl chain, although compounds with more elongated chains had increased activities. Conversely, BChE inhibition was consistently enhanced with chain length, likely due to indirect hydrophobic and steric interactions suggested by molecular docking. Out of them, compound 37, bearing an alkyl chain of n = 8, was a potent one, showing inhibitory activity for AChE (IC₅₀ = 82.1 ± 8.1 µM) and BChE (IC₅₀ = 3.77 ± 0.45 µM), as compared to the reference drug, rivastigmine (AChE: IC₅₀ = 275 ± 25 µM and BChE: IC₅₀ = 1.14 ± 0.13 µM).

In a recent report, a set of 3-indolyl-3-hydroxy-2-oxindole dimers (41) was synthesized and tested for their cholinesterase inhibitory activity.⁵² Target 41 was synthesized through straightforward one- and two-step procedures. The synthesis of an isatin dimer (39) was achieved through the N-alkylation of isatin (14), with 1,5-dibromopentane (38) as an alkylating agent in DMF, using K₂CO₃. The further reaction of the isatin dimer (39) with indole (40) in KOH resulted in the formation of the desired product (41) (Scheme 8).

Scheme 8. Synthesis of the 3-indolyl-3-hydroxy-2-oxindole dimer (41). Reagents and conditions: (i) KOH and MeOH, (ii) K₂CO₃ and DMF, and (iii) KOH and MeOH.

Although none of the compounds exhibited marked AChE inhibition, several of them had moderate to effective BChE activity, with IC₅₀ values ranging from 3.20 ± 0.20 µM to 357 ± 6 µM (Table 1). SAR analysis showed that isatin dimerization improved BChE inhibition compared to the parent isatin and that the length of the linkers was critical, with the intermediate-sized linkers in most cases showing the highest activity. Compound 41 was the most effective in both series, exhibiting similar BChE inhibition, with IC₅₀ values of 4.49 ± 0.43 µM, as compared to the standard drug, rivastigmine (IC₅₀ = 1.14 ± 0.13 µM).

3.2. Isatin-derived Schiff bases (imines)

Hydrazone derivatives, possessing a biologically active azomethine moiety, have gained significant interest in medicinal and pharmaceutical chemistry.⁵³ In this context, Arshad and colleagues developed a series of hydrazone conjugates by coupling heteroaromatic aldehydes with N-alkyl-5-chloroisatins (47)⁵⁴ and screened for in vitro ChE inhibitory activity. The synthesis involved carrying out the N-alkylation reaction on 5-chloroisatin (42) with 2-bromoethylbenzene (43), using K₂CO₃ as the base in DMF at 45 °C–50 °C for 3 h, giving compound N-(2-phenethyl)-5-chloroisatin (44). On the other hand, the reaction of tetrahydrothiophene-2-carbaldehyde (45) with hydrazine hydrate (NH₂NH₂·H₂O) in a MeOH solvent, with Et₃N as the catalyst at reflux temperature for 2 h, produced compound 46. Finally, the condensation of 44 with 46 in isopropanol (i-PrOH) and acetic acid (AcOH) under reflux for 3 h furnished the target thioether-linked isatin hydrazone derivative (47) (yield: 87%) (Scheme 9).

Scheme 9. Synthesis of the phenoxy pendant isatin (47). Reagents and conditions: (i) K₂CO₃, DMF, 45 °C-50 °C, and 3 h, (ii) NH₂NH₂·H₂O, MeOH, Et₃N, reflux, and 2 h, and (iii) i-PrOH, AcOH, reflux, and 3 h.

Notably, results demonstrated promising inhibitory activity for all synthesized compounds against AChE, with IC₅₀ values ranging between 16.22 ± 0.18 and 20.21 ± 0.06 µM, and BChE, with IC₅₀ values ranging from 20.11 ± 0.12 to 28.01 ± 0.01 µM, as compared to the reference drug, galantamine. SAR studies through the docking analysis of compounds exhibited strong BChE binding due to the hydrogen and arene-H interactions of the thiophene ring with Phe278 and Phe295, along with electrostatic interactions involving the carbonyl group. Compound 47, with R CH₂–C₆H₅ (Table 2), exhibited significant dose-dependent inhibition of AChE, with IC₅₀ values of 16.22 ± 0.18 µM, and BChE, with IC₅₀ values of 20.11 ± 0.12 µM, in comparison to the reference drug, galantamine (AChE: IC₅₀ = 10.13 ± 0.01 µM; and BChE: IC₅₀ = 22.00 ± 0.01 µM).

Table 2. ChE inhibitory activity of the isatin-derived Schiff bases (imines).

Code	Compound	AChE		BChE		Ref.
		IC50 (µM)	Standard IC50 (µM)	IC50 (µM)	Standard IC50 (µM)
47		16.22 ± 0.18	Galantamine (10.13 ± 0.01)	20.11 ± 0.12	Galantamine (22.00 ± 0.01)	54
50		1.60 ± 0.51	Donepezil (0.016 ± 0.002)	—	—	55
57		0.86 ± 0.03	Donepezil (22.4 ± 2.1)	0.66 ± 0.05	Donepezil (0.21 ± 0.04)	57
64		0.2 ± 0.08	Donepezil (22.4 ± 2.1)	0.03 ± 0.03	Donepezil (0.21 ± 0.04)	58
70		0.034 ± 0.002	Donepezil (0.084 ± 0.002)	—	—	59
74		1.23	Tacrine (11.14)	—	—	61
78		0.20 ± 0.05	Donepezil (2.16 ± 0.12)	0.90 ± 0.20	Donepezil (4.5 ± 0.11)	62
81(a-b)		(81a) 0.20 ± 0.05	Donepezil (2.16 ± 0.12)	0.90 ± 0.20	Donepezil (4.5 ± 0.11)	65
		—	—	(81a) 0.008 ± 0.002	Donepezil (8.234 ± 0.628)

Building upon this concept, a novel study was undertaken to synthesize a set of isatin-linked piperazine/morpholine derivatives (50) and evaluate their cholinesterase inhibition potential.⁵⁵ The target compound (50) was synthesized in two steps. First, isatin hydrazone (48) was prepared by refluxing 5-chloroisatin (42) with hydrazine hydrate in methanol in the presence of glacial acetic acid for 3 h. Subsequently, the obtained isatin hydrazone (48) was condensed with morpholine benzaldehyde (49) in methanol, using glacial acetic acid as a catalyst under reflux at 160 °C for 72 h to afford the final product (50) (Scheme 10).

Scheme 10. Synthesis of the phenoxy pendant isatin (50). Reagents and conditions: (i) EtOH, glacial acetic acid, reflux and 3 h and (ii) EtOH, glacial acetic acid, reflux, 160 °C and 72 h.

Interestingly, most of the compounds selectively displayed moderate to significant AChE inhibition, with an IC₅₀ value ranging between 1.60 ± 0.51 µM and 5.73 ± 0.97 µM. SAR studies showed that the derivatives of the morpholine series bearing electron-withdrawing groups on the C-5 position of the isatin moiety exhibited more potent human cholinesterase (hAChE) inhibition, following the order Cl > Br > F > CH₃. In contrast, the use of electron-donating substituents, such as methyl or methoxy, led to diminished inhibition. In addition, morpholine-based halogenated compounds generally surpassed their piperazine counterparts. Notably, compound 50, having (R Cl and R₁ 4-(4-morpholinyl) (Table 2), showed notable hAChE inhibition, with an IC₅₀ value of 1.60 ± 0.51 µM, in comparison to the reference drug, donepezil (IC₅₀ = 0.016 ± 0.002 µM).

Similarly, the 1,3,5-triazine scaffold is seen in several bioactive compounds showing anticancer, antimicrobial, anti-inflammatory, and AD activities.⁵⁶ Following these observations, Abibigloo et al. designed and synthesized a series of isatin 1,3,5-triazine benzylamine derivatives (57) as multi-target-directed ligands (MTDLs) for AD.⁵⁷ The 2,4,6-trichloro-1,3,5-triazine compound (51) was initially methoxylated through the reaction of methanol in the presence of NaHCO₃ in an ice bath to give rise to 4,6-dichloro-2-methoxy-1,3,5-triazine (52). Then, the obtained intermediate (52) was subjected to reaction with benzylamine (53) in the presence of 1,4-dioxane/acetone, with NaHCO₃ as the base, at temperatures ranging between 0 °C and 5 °C to obtain the benzylamino triazine intermediate (54). The obtained intermediate (54) was reacted with hydrazine hydrate in 1,4-dioxane, yielding an intermediate (55). Finally, the obtained intermediate (55) was subjected to a condensation reaction with an appropriate isatin derivative (56) in ethanol under reflux, yielding the desired derivative (57) (yield: 65%) (Scheme 11).

Scheme 11. Synthesis of the isatin 1,3,5-triazine benzylamine derivatives (57). Reagents and conditions: (i) CH₃OH, NaHCO₃, and 0 °C, (ii) NaHCO₃, 0 °C, and 1,4-dioxane, (iii) NH₂NH₂·H₂O, rt, and 1,4-dioxane and (iv) glacial acetic acid, ethanol, and reflux.

Remarkably, these compounds were found to possess strong in vitro inhibitory activities towards AChE, with IC₅₀ values in the range of 0.86–36.30 µM, and BChE, with IC₅₀ values ranging between 0.66 µM and 3.88 µM, in comparison with the reference drug, donepezil (eeAChE: IC₅₀ = 22.4 ± 2.1 µM and eqBChE: IC₅₀ = 0.21 ± 0.04 µM) (Table 2). SAR studies indicated that N-benzyl isatin hybrids improved AChE inhibition via hydrophobic interactions. Furthermore, replacing aniline with benzylamine improved activity in isatin and N-benzyl analogs but reduced potency in N-methyl derivatives. Additionally, 4-OCH₃ groups lowered AChE activity, and benzylamine substitution decreased BChE inhibition. Among them, hybrid 57, having (R₁ R₂ R₃ H and R₄ CH₂Ph) substituents, exhibited marvelous inhibitory activity against both electrophorus electricus acetylcholinesterase (eeAChE) (IC₅₀ = 0.86 ± 0.03 µM) and equine butyrylcholinesterase (eqBChE) (IC₅₀ = 0.66 ± 0.05 µM), outperforming the standard reference drug, donepezil.

In the same way, a library of compounds of s-triazine, isatin, and aniline derivatives (64) was synthesized and assessed as anti-ChE. The target compound (64) was prepared in multiple steps from aniline (58), which was first treated with chloral hydrate and hydroxylamine hydrochloride in the presence of Na₂SO₄ and H₂SO₄ to produce isonitrosoacetanilide (59). Thereafter, cyclization was carried out under concentrated H₂SO₄, affording isatin (14). The isatin (14) was then reacted with benzyl bromide (43) in the presence of K₂CO₃ in DMF to form an intermediate (60). On the other hand, cyanuric chloride (51) was subjected to methoxylation with methanol at 0 °C to produce an intermediate (52), which was then reacted with o-anisidine (61) in a NaHCO₃ aqueous solution to yield an intermediate (62). The intermediate (62) was further treated with hydrazine hydrate in acetone at room temperature to produce a hydrazinyl-s-triazine intermediate (63). The condensation of intermediates 59 and 63 in ethanol containing acetic acid gave the desired product (64) (yield: 74%) (Scheme 12).

Scheme 12. Synthesis of s-triazine, isatin, and aniline derivatives (64). Reagents and conditions: (i) chloral hydrate, hydroxylamine, and Na₂SO₄, (ii) H₂SO₄ and heat, (iii) K₂CO₃ and DMF, (iv) CH₃OH, NaHCO₃, and 0 °C, (v) NaHCO₃, 0 °C, and acetone, (vi) NH₂NH₂·H₂O, r.t, and 1,4-dioxane and (vii) glacial acetic acid, ethanol, and reflux.

Results showed that all the synthesized compounds exhibited excellent inhibitory activity, with IC₅₀ values ranging from 0.2 µM to 734.5 µM for AChE and 0.02 µM to 1.92 µM for BChE⁵⁸ (Table 2). SAR analysis showed that ChE inhibition was strongly influenced by substituents at both the aniline (R₁) and the nitrogen atom of the isatin (R₂) moieties. Specifically, AChE inhibition was improved by introducing ortho-substituted polar groups (2-OH and 2-Ome) on the aniline moiety, whereas para-substituted polar groups (4-Ome, 4-OH) led to reduced AChE inhibition, likely due to positional and electronic effects. Moreover, the incorporation of a lipophilic bulky group (CH₂-Ph) at the nitrogen atom of the isatin scaffold considerably improved both AChE and BChE inhibition through hydrophobic or π–π interactions. Consequently, the most potent derivative (64), having (R₁ CH₂-Ph and R₂ 2-OCH₃), displayed IC₅₀ values of 0.2 ± 0.08 µM for AChE and 0.03 ± 0.03 µM for BChE, as compared to the standard, donepezil (IC₅₀ = 22.4 ± 2.1 µM for AChE and 0.21 ± 0.04 µM for BChE).

Recently, a library of benzylpiperidine-based hydrazones of isatin (70)⁵⁹ was evaluated for AChE inhibition. The desired hydrazone compound (70) was prepared in three steps. The first step involved reacting 4-amino-1-benzylpiperidine (65) with ethyl bromoacetate (66) in dry tetrahydrofuran (THF) in the presence of TEA at 60 °C for 6 hours to obtain the acetate ester compound (67). Compound 67 was then converted into an acyl hydrazide compound (68) by treatment with hydrazine hydrate in ethanol at reflux for 8 hours. The condensation of compound 68 with N-propargyl-5-chloroisatin (69) in glacial acetic acid at reflux in ethanol yielded compound 70 (yield: 75%) (Scheme 13) after 10–20 hours.

Scheme 13. Synthesis of the benzylpiperidine-based hydrazones of isatin (70). Reagents and conditions: (i) TEA, dry THF, stirring, 60 °C, and 6 h, (ii) NH₂NH₂·H₂O, EtOH, reflux, and 8 h and (iii) EtOH, glacial acetic acid, reflux, and 10–20 h.

Notably, results showed magnificent inhibitory activity, with the IC₅₀ values in the range of 0.034 ± 0.002–10.49 ± 0.28 µM. SAR studies displayed that substitution at R₁ (H, benzyl, allyl, and propargyl) and R₂ (H, Cl, and Br) on the isatin template significantly influenced AChE inhibitory activities. Propargyl substitution at R₁ exhibited the highest potency, particularly with a chlorine atom at R₂. In contrast, benzyl-substituted compounds were the least active. Out of the R₂ substituents, chlorine improved activity the most, followed by hydrogen, while bromine led to reduced potency attributed to steric hindrance. Overall, the observed activity trend for R₁ was as follows: propargyl > H > allyl > benzyl. Among the synthesized hybrids, compound 70 was a lead compound, which showed strong inhibition for AChE (IC₅₀ = 0.034 ± 0.002 µM) (Table 2), being more potent than the reference drug, donepezil (IC₅₀ = 0.084 ± 0.002 µM).

Likewise, thiosemicarbazones possess various biological activities, such as antibacterial, antioxidant, antimicrobial, antituberculosis, anti-inflammatory, anticancer, and enzyme inhibition effects.⁶⁰ Considering this, recently, Koçyiğit et al.⁶¹ Prepared isatin–thiosemicarbazone derivatives (74) and assayed their dual inhibition. The target compound (74) was synthesized (Scheme 7) via a two-step procedure. Thiosemicarbazide (72) was first obtained by reacting isothiocyanate (71) with hydrazine monohydrate in ethanol under cooling conditions. The resulting thiosemicarbazide (72) was then condensed with 5-methoxyisatin in aqueous ethanol containing catalytic HCl under reflux for 5 h to afford the desired thiosemicarbazone derivative (74) (yield: 63%) (Scheme 14). In vitro screening showed the superior inhibitory activity of all the synthesized compounds against AChE, with IC₅₀ values ranging from 1.23 µM to 5.43 µM and Ki values between 0.94 ± 0.13 µM and 5.47 ± 1.14 µM. Out of them, compound 17 was the most effective inhibitor of AChE, with an IC₅₀ value of 1.23 µM and Ki values of 0.94 ± 0.13–5.47 µM, in comparison to the reference drug, tacrine (IC₅₀ = 11.14 µM and K_i = 9.33 ± 1.45 µM) (Table 2).

Scheme 14. Synthesis of the isatin–thiosemicarbazone derivative (74). Reagents and conditions: (i) NH₂NH₂·H₂O, EtOH, and 0 °C and (ii) EtOH, HCl, 5 h, and reflux.

Similarly, isatin/bis-Schiff base-based thiosemicarbazones⁶² (78) were synthesized, and their ChE inhibition profiles were evaluated. The target compound (78) was synthesized in two steps. First, hydrazine hydrate was reacted with carbon disulfide (75) in ethanol under reflux for 4 h to obtain the thiosemicarbazide intermediate (76). Subsequently, the intermediate (76) was condensed with 5-nitroisatin (77) in methanol in the presence of catalytic acetic acid to afford the desired target molecule (78) (yield: 77%) (Scheme 15).

Scheme 15. Synthesis of the isatin/bis-Schiff base-based thiosemicarbazones (78). Reagents and conditions: (i) NH₂NH₂·H₂O, EtOH, reflux, and 4 h and (ii) MeOH, CH₃COOH, 4 h, and reflux.

The results showed superior inhibitory activity against AChE, with IC₅₀ values ranging from 0.20 ± 0.05 µM to 7.40 ± 0.20 µM, and against BChE, with IC₅₀ values ranging from 0.50 ± 0.05 µM to 9.40 ± 0.10 µM. SAR analysis revealed that ChE inhibition was significantly influenced by substitution on the isatin. The incorporation of nitro and di-chloro group (especially at 5-isatin) substituents on isatin enhanced activity through hydrogen bonding and electron-withdrawing effects, whereas bulky groups, such as bromo or ortho-substituted nitro group, reduced potency due to steric hindrance or internal interactions. Among them, compound 78, possessing (R₁ 5-NO₂) substituents (Table 2), displayed marvelous inhibitory activity against both AChE (IC₅₀ = 0.20 ± 0.05 µM) and BChE (IC₅₀ = 0.90 ± 0.20 µM), surpassing the reference drug, donepezil (IC₅₀ = 2.16 ± 0.12 µM and 4.5 ± 0.11 µM, respectively).

Furthermore, several thiosemicarbazones bearing a d-glucose moiety, along with an isatin ring, were reported to exhibit a variety of biological activities, including antibacterial, antifungal, antioxidant, and anti-MRSA activities.^63,64 Considering these outcomes, Nguyen and the group developed a new set of galactose-conjugated isatin β-thiosemicarbazones (81a-b)⁶⁵ and screened them against AChE and BChE. For their synthesis, N-Alkylated isatin derivatives (79) were synthesized by reacting isatin (14) with the appropriate alkyl halide in anhydrous DMF, using K₂CO₃ and KI, under microwave irradiation (400 W, 10–15 min). The resulting intermediates (79) were then condensed with N-(tetra-O-acetyl-β-d-glucopyranosyl) thiosemicarbazide (80) in triethylammonium acetate under microwave-assisted conditions to afford the desired d-glucose-bearing isatin-thiosemicarbazones (80) (Scheme 16).

Scheme 16. Synthesis of isatin-thiosemicarbazones bearing a d-glucose moiety (81a-b). Reagents and conditions: (i) RBr, anhyd. K₂CO₃, KI, anhyd. DMF, microwave irradiation at 400 W, and 10–15 min and (ii) ionic liquid triethylammonium acetate and MW-assisted conditions.

Biological evaluation revealed that most of the synthesized compounds exhibited satisfactory inhibitory activity, with IC₅₀ values ranging from 0.029 ± 0.008 µM to 1.691 ± 0.032 µM for AChE and between 0.008 ± 0.0027 µM and 416 ± 0.342 µM for BChE (Table 2). The SAR study disclosed that the electron-withdrawing groups (e.g., nitro and halogens) on the isatin ring enhanced AChE and BChE inhibition, though bulky groups may reduce activity. Alkyl, aryl, or alkenyl substitution at position 1, especially with long, aromatic chains or branched ones, enhances inhibition through increased lipophilicity and enzyme binding. Branched and even-numbered alkyl chains were especially potent for AChE. Overall, ChE inhibition potency and selectivity were greatly influenced by electronic effects, chain length, branching, and hydrophobic interactions. From the library, derivative 81a, comprising R₁ R₂ H and R₃ Bn, displayed the highest AChE inhibitory potential, with an IC₅₀ of 0.029 ± 0.008 µM, compared to the reference drug, donepezil (IC₅₀ = 0.038 ± 0.007 µM). However, compound 81b demonstrated several-fold superior inhibitory activity for BChE, with an IC₅₀ value of 0.008 ± 0.002 µM, in comparison to donepezil (IC₅₀ = 8.234 ± 0.628 µM).

3.3. Isatin-based spiro derivatives

Spirooxindole scaffolds are widely represented in many naturally occurring molecules and exhibit a wide range of biological activities, including anticancer, anti-Alzheimer, antidiabetic, antimicrobial, antituberculosis, antiviral, antileukemic, anticonvulsant, and anesthetic activities.⁶⁶ One recent study reported synthesizing eight dispiropyrrolidines (87) through a one-pot, three-component [3 + 2] cycloaddition approach, followed by the assessment of their inhibitory activity against cholinesterase. The target dispiropyrrolidine compounds (87) were synthesised by a 1,3-dipolar cycloaddition reaction. The synthesis was initiated by treating phenylethyl-4-piperidone (82) and benzaldehyde (83) in ethanol using NaOH, affording a bis(arylidene)piperidone intermediate (84). The intermediate (84) was then reacted with 5-bromoisatin (85) and sarcosine (86) in refluxing methanol for 4–7 h via a 1,3-dipolar cycloaddition reaction to yield the target compound (87) (Scheme 17).

Scheme 17. Synthesis of dispiropyrrolidine (87). Reagents and conditions: (i) NaOH, EtOH, and 2–6 h and (ii) EtOH, reflux, and 4–7 h.

The majority of compounds exhibited weak to moderate activity; however, compound 87 emerged with more than 50% inhibition against BChE at 10 µM but with very little inhibition of AChE, showing its BChE selectivity.⁶⁷ SAR and docking analysis showed that the isomer (87) achieved seven hydrophobic interactions with the active site of BChE. Importantly, the oxindole NH and pyrrolidine CH groups provide stabilizing interactions with amino acid side chains, while the bromine substituent of the oxindole ring also improves binding through hydrophobic contacts. These interactions, especially among the three phenyl groups with active site residues, are responsible for the observed selectivity and moderate activity (IC₅₀ = 12.78 ± 1.52 µM), in comparison to the standard drug, physostigmine (IC₅₀ = 0.16 µM) (Table 3).

Table 3. ChE inhibitory activity of the isatin-based spiro derivatives.

Code	Compound	AChE		BChE		Ref.
		IC50 (µM)	Standard IC50 (µM)	IC50 (µM)	Standard (IC50 µM)
87		—	—	12.78 ± 1.52	Physostigmine (0.16)	67
92		24.1	Galantamine (1.2)	—	—	68
96		>100	—	1.8 ± 0.2	Galantamine (3.9)	69
100		3.98 ± 1.07	Donepezil (0.079 ± 0.05)	—	—	70
105		1.89 ± 0.28	Donepezil (0.59 ± 0.083)	7.24 ± 0.55	Donepezil (0.77 ± 0.0088)	71
110(a-b)		(110a) 6.02 ± 2.59	Donepezil 0.079 ± 0.05	—	-	72
		—	—	(110b) 9.13 ± 1.82	Donepezil 10.6 ± 2.1
117		—	—	1.6 ± 0.2	Galantamine (3.9 ± 0.3)	73
120		—	—	0.46	Donepezil 10.00	74
122		0.023 ± 0.003	Donepezil (0.021 ± 0.001)	—	—	75

In a recent study, a library of spirooxindole analogs (93) that incorporate indole and pyrazole moieties was prepared through a three-component [3 + 2] cycloaddition (32CA) reaction.⁶⁸ Phenylhydrazine (88) reacted with acetylacetone (89) in the presence of DMF-DMA at 80 °C for 3 h to afford the pyrazole derivative (90). Subsequently, a Knoevenagel condensation between pyrazole (90) and 5-bromoisatin (85) was carried out in ethanol using piperidine as a catalyst under reflux for 72 h, yielding the corresponding 3-(pyrazolylmethylene) indolin-2-one intermediate (91). Finally, a three-component 1,3-dipolar cycloaddition reaction of the intermediate (91), 5-nitroisatin (71), and thiazolidine-4-carboxylic acid in methanol under reflux for 2–24 h generated azomethine ylide in situ, which underwent cycloaddition to furnish the desired spirooxindole analog (92) (yield: 49%) (Scheme 18).

Scheme 18. Synthesis of the spirooxindole analog (92). Reagents and conditions: (i) DMF-DMA, 80 °C, and 3 h, (ii) piperidine, ethanol, reflux, and 72 h, and (iii) MeOH, reflux and 2–24 h.

The activities of these compounds towards AChE were determined by Ellman's assay (Table 3). Some derivatives exhibited moderate activities (IC₅₀ ≤ 50 µM), while others showed weak or no activity (IC₅₀ > 65 µM). Nevertheless, no inherent SAR pattern could be deduced in the series. Out of all the synthesized derivatives, compound 92 (X = Br, X₁ = S, Y = 5-NO₂, and R H) showed the highest inhibition, with an IC₅₀ value of 24.1 µM, as compared to the reference drug, galantamine.

Moreover, the derivatives of oxindole-based β- and γ-lactam (23), synthesized through a 4-center 3-component Ugi reaction (U4c3CR) using isatin, have been found to exhibit profound inhibition against ChE.⁶⁹ The target compound (23) was synthesized by a one-pot three-component reaction of isatin (34), isocyanide (35), and glycine (36) in 2,2,2-trifluoroethanol (TFE) at room temperature. The reaction mixture was stirred for 12–48 h, generating an azomethine ylide in situ, which underwent cycloaddition with the isocyanide intermediate, affording the corresponding derivative (23). Interestingly, all the compounds exhibited selective and promising BChE activity, with IC₅₀ values ranging from 1.8 ± 0.2 µM to >100 µM. SAR analysis demonstrated that γ-lactam analogs were always more active than β-lactams. Moreover, alkyl isocyanides, especially t-octyl, increased activity, while benzyl isocyanide decreased it. Additionally, N-phenyl and N-benzyl substitution increased potency, while methyl and propargyl resulted in inactivity caused by probable synergism with the γ-lactam ring. In contrast, aromatic substitutions at the oxindole core generally led to decreased activity (Scheme 19). Notably, compound 23 had highly potent mixed-type BChE inhibition (IC₅₀ = 1.8 ± 0.2 µM), which was more effective than that of galantamine (IC₅₀ = 3.9 µM) (Table 3).

Scheme 19. Synthesis of dispiropyrrolidines (96). Reagents and conditions: (i) TFE, rt, and 12–48 h.

Recently, an efficient, green, and high-yield cascade strategy has been reported to prepare spiroindolin-1,2-diazepine derivatives (100).⁷⁰ Compound 100 is prepared by a convergent synthesis strategy in ethanol solution under ambient conditions. On the one hand, a nitroketene analogue (97) reacted with hydrazine to give an intermediate (98). On the other hand, compound 77 and malononitrile underwent condensation to provide an intermediate (99). After that, intermediates 98 and 99 were condensed, affording a spiro-oxindole framework via a [3 + 3] formal cyclization, furnishing compound 100 (yield: 86%) (Scheme 20).

Scheme 20. Synthesis of the phenoxy pendant isatin (24). Reagents and conditions: (i) EtOH and rt, (ii) EtOH and rt and (iii) EtOH and rt.

All the synthesized compounds were evaluated for AChE inhibitory activity, and most of the compounds showed selective inhibition of AChE, with IC₅₀ values ranging from 3.98 ± 1.07 µM to 22.38 ± 2.11 µM. SAR studies revealed that the electron-withdrawing groups (hydroxyl, methoxy) at the aniline scaffold (R₂) increased AChE inhibition, particularly when combined with the electron-withdrawing groups (5-NO₂, 5,7-diBr) at the isatin moiety (R₁). In contrast, the electron-withdrawing groups at the aniline scaffold (R₂) decreased activity. Amongst all, compound 100 (R 5-NO₂ and R₂ 4-OH) (Table 3) was found to be the most selective and potent AChE inhibitor (IC₅₀ = 3.98 ± 1.07 µM), although it was weaker than the standard drug, donepezil (IC₅₀ = 0.079 ± 0.05 µM). Kinetic studies on the most active compound (100) at different concentrations of acetylthiocholine (0.1–1 mM) by Ellman's assay demonstrated a mixed-mode inhibition of AChE, with a K_i value of 0.044 µM.

Furthermore, a series of dispiropyrrolodinyl oxindoles⁷¹ (105) exhibited notable ChE inhibitory activity, with IC₅₀ values ranging between 1.89 ± 0.28 µM and 159.26 ± 5.18 µM for AChE, while from 6.63 ± 0.43 µM to 578.11 ± 13.44 µM for BChE, as compared to the reference drug, donepezil (AChE: IC₅₀ = 0.59 ± 0.083 µM and BChE: IC₅₀ = 0.77 ± 0.0088 µM). Among them, compound 105 (R C₃H₅ and R₁ H₃COC₆H₄) (Table 3) demonstrated marvelous inhibitory activity against AChE (IC₅₀ = 1.89 ± 0.28 µM) and BChE (IC₅₀ = 7.24 ± 0.55 µM). Most hybrids were more selective for AChE compared to BChE. SAR analysis showed that the increase in the chain length at the indolyl N-1 position (ethyl/allyl > methyl) improved inhibitory activity. Para-substituted aryls bearing an electron-donating group (OCH₃/CH₃) showed superior inhibition compared with electron-withdrawing para-substituted aryls (F/Cl), except bromine. Furthermore, the replacement of aryls with five-membered heterocycles slightly enhanced potency, where thiophene analogs were more potent than furan analogs.

Dispiropyrrolodinyl oxindole (105) was obtained by employing a series of multicomponent reactions. First, compound 101 was treated with sarcosine (102) in an ethanol solution to produce the corresponding azomethine ylide (103) after undergoing decarboxylation. Second, the dipole that had been generated in situ was subjected to regioselective [3 + 2] cycloaddition with the exocyclic olefin present in the dienone derivative (104) in an ethanol solution at room temperature to afford the dispiropyrrolodinyl oxindole (105) (yield: 72%) (Scheme 21).

Scheme 21. Synthesis of dispiropyrrolodinyl oxindole (105). Reagents and conditions: (i) EtOH and reflux, (ii) EtOH and rt, and (iii) EtOH and reflux.

Similarly, spiroindolinone pyrazole derivatives (110a-b) were synthesized through a four-component condensation, and their assessment was performed against AChE and BChE as AD targets. 1,1-Bis(methylthio)-2-nitroethylene (107) and the appropriate arylamine (106) were refluxed in ethanol to afford N-aryl-1-(methylthio)-2-nitroethenamine (108) after 12 h. Subsequently, hydrazine hydrate and isatin derivatives (109) were added to this intermediate (108), and the reaction mixture was further stirred to furnish the corresponding target compounds (110a-b). Compound 110a (R₁ H and R₂ 3-Cl) had strong AChE activity (IC₅₀ = 6.02 ± 2.59 µM) (Table 3) with no notable BChE activity, whereas compound 110b (R₁ 5-Cl and R₂ 2-CH₃) possessed high BChE activity (IC₅₀ = 9.13 ± 1.82 µM), as compared to the reference drug, donepezil (Scheme 22). SAR analysis showed that AChE inhibition was enhanced with electron-withdrawing groups at the isatin ring (R₁) and electron-donating groups at the aniline scaffold (R₂), whereas BChE activity preferred lipophilic substituents at the aniline scaffold (R₂) and was, in general, decreased by halogens.⁷²

Scheme 22. Synthesis of spiroindolinone pyrazole (110a-b). Reagents and conditions: (i) EtOH, reflux, and 12 h and (ii) EtOH and reflux.

Within this framework, a concise library of isatin-based oxindole hybrids (117) was synthesized, and their ChE inhibitory activity was investigated by Marques and coworkers.⁷³ A brominated acetophenone derivative (111) was coupled with a glycine/amine derivative (112) in the presence of Cu(OAc)₂ and K₂CO₃ in DMF at 140 °C (overnight) to obtain an intermediate (113). The iodination reaction was performed on intermediate 113 in the presence of I₂ and TBHP in DMSO at 80 °C to form an intermediate (114). Then, this product (114) was converted into an intermediate (115) using trimethyl orthoformate (TMOF) and p-TsOH in methanol at reflux temperature (overnight). Finally, intermediate 115 was coupled with an amine derivative (116) in the presence of Pd₂(dba)₃ to afford the target molecule (117) (Scheme 23). Despite this, none of the compounds showed significant inhibitory activity for AChE; however, compound 27, bearing R₁ Bn, R₂ Bn, and R₃ propargyl substituents (Table 3), exhibited potent inhibitory activity for BChE, with an IC₅₀ value of 1.6 ± 0.2 µM, surpassing the reference drug, galantamine (IC₅₀ = 3.9 ± 0.3 µM).

Scheme 23. Synthesis of the isatin-based oxindole hybrid (117). Reagents and conditions: (i) Cu(OAc)₂, K₂CO₃, DMF, 140 °C, and overnight, (ii) I₂, TBHP, DMSO, 80 °C, and 2 d, (iii) TMOF, pTsOH, MeOH, reflux, and overnight, and (iv) Pd₂(dba)₃, DavePhos, NH₂ + NaOtBu, toluene, 90 °C, and overnight.

In the same way, Marques et al.⁷⁴ established a library of C-3-arylated isatin–triazole derivatives (120). The N-triazole–isatin derivative (118) was reacted with phenylboronic acid (119) in toluene in the presence of [Rh(C₂H₄)₂Cl]₂, (R)-BINAP, and K₂CO₃ under a nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 24 h, affording the corresponding C-3-arylated isatin–triazole derivative (120) upon work-up and purification (Scheme 24).

Scheme 24. Synthesis of the isatin-based oxindole hybrids (120). Reagents and conditions: (i) [Rh(C₂H₄)₂Cl]₂, (R)-BINAP, PhB(OH)₂, K₂CO₃, toluene, 60 °C, and 24 h.

The synthesized compounds (120) exhibited excellent BChE inhibitory activity, with IC₅₀ values ranging between 0.46 µM and 15.7 µM (Table 1). SAR analysis revealed that the incorporation of a bulky, hydrophobic, and chiral menthol moiety in the phenyl ring of analogs enhanced BChE inhibition by improving hydrophobic interactions and steric complementarity within the enzyme's active site, thereby increasing binding affinity and selectivity. Among them, the most potent compound (120), bearing the menthol scaffold, exhibited superior BChE inhibitory activity, with an IC₅₀ of 0.46 µM, outperforming the reference drug, donepezil (IC₅₀ = 10.00 µM).

3.4. Miscellaneous isatin derivatives

Molecular hybridization involves the integration of two or more individual bioactive scaffolds into a single molecular framework, aiming to enhance the biological activities of individual pharmacophores for improved or synergistic effects.⁷⁶ Considering this, recently, a library of indolo [2,3-b] quinoxaline derivatives (122) was synthesized using indole and quinoxaline privileged structures as AChE inhibitors, aiming to develop new entities for AD. The synthesis (Scheme 25) was initiated by the N-benzylation of isatin (14) with benzyl bromide (43) in DMF in the presence of K₂CO₃, affording the corresponding N-benzyl isatin intermediate (60). Subsequently, the obtained intermediate (60) was reacted with 5-nitro-1,2-phenylenediamine (121) in acetic acid and subjected to microwave irradiation at 500 W for 5 min, leading to the formation of the desired product (122) (yield: 87%).

Scheme 25. Synthesis of the indolo [2,3-b] quinoxaline derivative (122). Reagents and conditions: (i) DMF and K₂CO₃ and (ii) 500 MW and 5 min.

The results showed that many of the synthesized compounds exhibited good inhibitory activities against AChE, with a range of IC₅₀ values from 0.023 ± 0.003 µM to 19.85 ± 0.002 µM, as compared to the reference drug, donepezil.⁷⁵ SAR investigations indicated that the unsubstituted quinoxaline derivative exhibited moderate AChE inhibitory activity, whereas the phenyl-substituted compounds had the lowest activity, which might be attributed to steric hindrance. The introduction of weak electron-donating (methyl) or weak electron-withdrawing (chloro) groups further decreased the activity. In contrast, derivatives containing electron-withdrawing groups of high strength, such as carboxylic acid, nitro, or benzoyl, showed substantially increased inhibitory activities, emphasizing the influence of electronic factors on AChE inhibition. Out of them, compound 122 (R 5-NO₂) was a hit compound with potent inhibitory activity against AChE, with IC₅₀ = 0.023 ± 0.003 µM, in comparison to donepezil (IC₅₀ = 0.021 ± 0.001 µM) (Table 3).

3.5. Structure–activity relationships and pharmacophoric features

Collectively, the preceding studies highlight several structural features that govern ChE inhibition, with the effects of substitution, N-functionalization, linker length, hydrophobicity, and molecular conformation varying across isatin-based scaffolds. In the coumarin–triazole-isatin series (8), the unsubstituted isatin derivative (R H) showed the strongest BChE inhibition. However, fluorine provided moderate activity, and relatively bulky halogen, nitro, and methyl substituents were less favorable, suggesting that steric compatibility within the binding site can outweigh the intrinsic electronic effects of substituents.⁴³ In contrast, the isatin-triazole series (13) showed a different trend, with C-5 fluoro and chloro substitution enhancing AChE inhibition relative to methyl and methoxy analogues, indicating a favorable contribution from electron-withdrawing substituents in this scaffold.⁴⁴ Similarly, hydrazone derivatives (19), bearing 5-chloroisatin, displayed potent AChE inhibition, supporting the beneficial role of C-5 halogenation in that series.⁴⁵ However, this relationship is not universal: in the phenoxy–isatin derivatives (23), halogen substitution reduced AChE inhibition, demonstrating that the effect of C-5 substitution depends on substituent identity, position, steric demand, and the surrounding pharmacophore.⁴⁶ Additional evidence from compounds 50 and 78 supports the contribution of electron-withdrawing groups, particularly chloro, bromo, and nitro substituents, although the magnitude and direction of these effects vary among chemical series.^55,62 Thus, rather than defining a universal electronic preference, the available data indicate that C-5 substitution should be optimized in conjunction with the complete molecular framework (Scheme 25).

N-functionalization and linker architecture provide additional determinants of activity. N-substitution can modulate lipophilicity, molecular shape, and hydrophobic recognition within the ChE active site. In the N-substituted series (37), increasing the N-alkyl chain length improved BChE inhibition, although a comparable systematic trend was not observed for AChE, highlighting the different steric environments of the two enzymes.⁵¹N-benzyl substitution was also favorable in compounds 57 and 64, where the appended hydrophobic group contributed to improved enzyme recognition.⁵⁷ Linker length is particularly important in molecular hybrids because it determines the relative orientation and conformational freedom of the interacting pharmacophores. For the coumarin-triazole-isatin hybrids (8), the three-carbon linker afforded the most favorable BChE inhibition, whereas short linkers were less effective.⁴³ Similarly, the isatin–thiazole derivatives (19) showed improved AChE inhibition with optimized three- to four-carbon linkers,⁴⁵ while the isatin-pyridine oxime system (35) displayed optimal activity, with an intermediate five-methylene spacer.⁵⁰ The isatin dimer (41) further demonstrated that intermediate linker lengths can provide a favorable balance between conformational flexibility and appropriate positioning of the two pharmacophoric units.⁵² Heterocyclic incorporation, including triazole, thiazole, quinoline, pyridine and triazine-containing systems, can additionally introduce π-interactions, hydrogen bonds and electrostatic contacts. Collectively, these findings indicate that N-substitution and linker optimization should be considered together rather than as independent structural variables.

Molecular hybridization, hydrophobicity and conformational rigidity further influence AChE/BChE selectivity and potency. The generally larger and more permissive BChE active site can accommodate bulky hydrophobic substituents, as illustrated by 37, 41, and the dispiropyrrolidine (87), which showed preferential BChE inhibition and extensive hydrophobic contacts within the enzyme active site.⁶⁷ In contrast, the narrow AChE active site may favor more compact architectures, although appropriately oriented aromatic and heterocyclic groups can engage the peripheral anionic site through π-interactions. Spiro and fused systems can restrict conformational freedom and thereby preorganize pharmacophores, but the available data do not support a universal relationship between increased rigidity and potency. For example, compound 87 benefited from hydrophobic interactions involving its phenyl groups and brominated oxindole moieties, whereas the spirooxindole series (92) did not show a consistent SAR trend.^67,68 The broad set of derivatives, including 100, 105, and 110a-b, further demonstrates that substituent effects are dependent on the specific molecular framework and enzyme isoform.^70–72 Overall, the emerging pharmacophore comprises a planar aromatic surface derived from the isatin and/or appended aryl/heteroaryl groups; hydrogen-bonding functionalities associated with carbonyl, imine, hydrazone, oxime or related groups; and an appropriately tuned linker or hydrophobic domain. Therefore, the combined evidence favors the balanced optimization of electronic properties, steric complementarity, hydrophobic interactions and conformational flexibility, rather than maximizing any single structural feature. Given the substantial differences in chemical series and assay conditions, these relationships should be regarded as guiding SAR principles rather than universal rules.

4. Conclusions and future outlook

The isatin scaffold has emerged as a versatile and privileged platform for the development of ChE inhibitors, offering significant promise for the treatment of AD. A wide range of structurally diverse isatin derivatives have demonstrated potent inhibitory activity against AChE and BChE, with several compounds exhibiting superior in vitro efficacy compared with clinically used agents, such as donepezil, rivastigmine, and galantamine. Structure–activity relationship studies have revealed the pivotal roles of substituent type and position, linker design, N-substitution, and molecular hybridization in modulating enzyme inhibition, selectivity, and overall biological performance.

Collectively, the evidence summarized in this review highlights the isatin scaffold as an attractive framework for the rational design of next-generation ChE inhibitors. The remarkable chemical diversity and favorable pharmacological properties of isatin-based derivatives provide valuable opportunities for the identification of novel lead compounds with enhanced therapeutic potential. Given the projected rise in the global prevalence of AD over the coming decades, the development of more effective and safer therapeutic agents remains an urgent priority. Therefore, future research should focus on improving cholinesterase isoform selectivity, minimizing off-target toxicity, enhancing blood–brain barrier permeability, and optimizing pharmacokinetic profiles. Addressing these challenges will be crucial for translating promising isatin-based ChE inhibitors into clinically viable candidates and advancing the development of effective therapies for AD and related neurodegenerative disorders.

5. Declaration

The authors used ChatGPT (OpenAI, GPT-5.5) to assist with language editing and improving the clarity and readability of the manuscript. The authors reviewed, revised, and took full responsibility for the final content of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included, and no new data were generated or analysed as part of this review.