Unlocking the Potential of Isatin Scaffolds in Acetylcholinesterase Inhibition

ABSTRACT

The therapeutic significance of acetylcholinesterase (AChE) inhibition is most prominently observed in the management of various neurodegenerative and neuromuscular disorders, including Alzheimer's disease (AD), and myasthenia gravis (MG). The present review examines isatin‐based compounds as a promising class of drugs for treating these conditions, focusing on their ability to inhibit AChE. While AD is characterized by a significant loss of cholinergic neurons leading to a deficiency in acetylcholine (ACh), MG is an autoimmune disease where the body produces antibodies that block or destroy ACh receptors at the neuromuscular junction. By inhibiting AChE, therapeutic agents increase the availability of ACh, thereby enhancing cholinergic neurotransmission and improving muscle strength or cognitive function. The underlying issue in MG is the reduced number of functional receptors rather than a primary decrease in AChE production. However, inhibition of the enzyme remains a vital strategy for symptom management. Accordingly, this review provides a systematic overview of the various synthetic strategies reported in the literature over the last decade for enhancing isatin‐based AChE inhibitory activity. These strategies include modifications of the isatin nitrogen, such as N‐alkylation, N‐acylation, or N‐arylation. Another strategy is aromatic ring substitution, where various substituents (e.g., halogens, nitro, amino, and alkyl groups) are introduced typically at the C₅, C₆, and/or C₇ positions. C₃‐carbonyl functionalization is also reported, exemplified by the formation of Schiff bases, hydrazones, or spirocyclic derivatives. Our findings highlight the exceptional potential and adaptability of isatin derivatives in medicinal chemistry, highlighting the major advances within the last 10 years and the future directions.

Structure–activity relationship (SAR) of isatin‐based AChEIs.

1. Introduction

Acetylcholinesterase (AChE) is a vital enzyme found mainly at the connections between nerves and muscles (neuromuscular junctions) and at nerve‐to‐nerve communication points using acetylcholine (ACh). Its main function is to stop nerve signals from being transmitted. AChE achieves this by rapidly breaking down the neurotransmitter ACh into two parts: choline and acetic acid. AChE ensures that nerve signals are precise and regulated, and thereby stops the receptors on the receiving cell from being constantly activated [1, 2, 3]. The incredibly high speed of AChE in performing this breakdown makes it one of the most rapid enzymes known, emphasizing its critical role in the nervous system. Based on subunit assembly and anchoring, AChE is classified into four symmetric, globular forms (G1–G4). These are subdivided according to the number of catalytic subunits: G1 (monomer) has one; G2 (dimer) has two. The G4 (tetramer) is the largest soluble form in the brain and most tissues, possessing four catalytic subunits [4].

Asymmetric forms (A) like A₄, A₈, and A₁₂ are those with a catalytic subunit that is tethered to it by a structural subunit, often a collagen‐like tail. These forms are important at neuromuscular junctions, anchored to the basal lamina. These variant molecular forms of AChE are characterized by specific localization and ensure the execution of specialized functions in cholinergic neurotransmission in different tissues. Different tissues, developmental stages, and even disease states can have different ratios of these subtypes.

AChE could also be classified into AChE‐T (Tailed Form), AChE‐H (Hydrophobic Form), and AChE‐R (Readthrough Form) based on differences in their C‐terminal regions and the mechanisms by which they are localized or associated with cellular structures [5, 6].

The tailed form, AChE‐T, is the dominant isoform in the central nervous system and at neuromuscular junctions. Its primary function is the rapid hydrolysis of ACh at these synaptic sites, ensuring precise and timely termination of cholinergic signaling. AChE‐T's characteristic feature is its association with cellular membranes [1]. This anchoring is facilitated by its proline‐rich attachment domain (PRAD), which interacts with proline‐rich membrane anchor (PRiMA) proteins on the neuronal and muscle cell surfaces [7].

AChE‐H is primarily found in erythrocytes (red blood cells); its membrane association in this context is achieved through a glycosylphosphatidylinositol (GPI) anchor attached to the C‐terminus of AChE‐H, conferring its hydrophobic nature. Moreover, AChE‐H exists as a dimer on the erythrocyte membrane. While the precise physiological role of AChE in erythrocytes is still under investigation, it is hypothesized to be involved in regulating local ACh levels, which may influence vascular tone and inflammatory responses [8].

Furthermore, AChE‐R arises from alternative splicing of the AChE gene, resulting in the bypass of the canonical stop codon and the addition of a unique C‐terminal sequence. This alternative splicing event leads to the production of a predominantly soluble, monomeric form of AChE that lacks the membrane‐anchoring domains present in AChE‐T and AChE‐H [9, 10].

Interestingly, the expression of AChE‐R is often upregulated under various stress conditions, including psychological stress and exposure to certain toxins, suggesting a role in the cellular stress response. AChE‐R is found in both the central nervous system and peripheral tissues as well as blood plasma [9, 11].

While the three main isoforms of AChE are all derived from the same AChE gene through alternative splicing, they exhibit differences in their overall structure due to variations in their C‐terminal regions and associated subunits. However, a crucial aspect is that they all possess the same catalytic active site (CAS), which is responsible for the hydrolysis of ACh. This active site comprises a catalytic triad of serine, histidine, and glutamate residues, along with other conserved amino acids essential for substrate binding and catalysis. Therefore, the fundamental mechanism of ACh hydrolysis is the same for all isoforms [8, 12].

AChE's active site is a deep gorge containing the CAS (Figure 1). Ser203 is the nucleophilic residue that initiates the attack on the carbonyl carbon of ACh, and His447 activates Ser203 for nucleophilic attack. Subsequently, it aids in choline departure. Glu334 orients and polarizes histidine, thereby enhancing its catalytic function [13, 14].

Figure 1.

Schematic diagram of the active site of AChE. Created by BioRender.com.

In addition, the peripheral anionic site (PAS) is located in close proximity to the CAS; it is crucial for the binding and precise orientation of the positively charged quaternary ammonium group of ACh. The key residues in PAS include Trp286, which plays an essential role in substrate binding via cation–π interactions with the quaternary ammonium group of ACh. Moreover, inhibitors that bind to the PAS can allosterically modulate the enzyme's activity or physically block the entry of ACh into the active site [8, 14]. Dual‐site inhibitors that interact with both the PAS and the CAS often exhibit enhanced potency and selectivity [13, 15, 16].

The gorge is lined with numerous aromatic amino acid residues like Phe338 and Tyr121, which are thought to guide the substrate toward the active site and participate in transient binding interactions [17]. When the enzyme AChE breaks down ACh, a temporary unstable structure is formed. This unstable structure has a negatively charged oxygen atom. The enzyme has a special pocket, called the oxyanion hole (made of specific amino acids like Gly121, Gly122, and Ala204), that grabs onto and stabilizes this negatively charged oxygen. This stabilization makes it easier for the reaction to proceed and speeds up the breakdown process [18]. Inhibitors that interact with this region can disrupt the catalytic mechanism by preventing the stabilization of the unstable intermediate [15, 16, 17].

2. AChE‐Related Diseases

Disturbed levels of AChE are linked to several diseases [19]. The most important diseases caused by AChE disturbance are presented below.

2.1. Alzheimer's Disease (AD)

The primary pathology of AD involves amyloid plaques and neurofibrillary tangles. There is also a significant loss of cholinergic neurons in brain regions crucial for memory and cognition, particularly the basal forebrain. This neuronal loss leads to decreased ACh synthesis and release. ACh is vital for various cognitive functions, including memory, attention, learning, and arousal. The deficiency in this neurotransmitter is strongly linked to the cognitive symptoms of AD. Based on the cholinergic hypothesis, a primary class of drugs used to manage the symptoms of AD is acetylcholinesterase inhibitors (AChEIs). These medications work by inhibiting the activity of AChE, thereby slowing down the breakdown of ACh. The result is a temporary increase in the concentration of ACh in the brain, which can help to improve communication between the remaining cholinergic neurons and provide modest symptomatic relief in cognitive functions like memory and thinking [3, 20, 21, 22, 23, 24].

2.2. Parkinson's Disease (PD)

PD is one of the most prevalent neurodegenerative disorders globally. It is pathologically characterized by the accumulation of intracellular Lewy bodies and Lewy neurites (composed primarily of α‐synuclein) within the substantia nigra. This leads to the progressive loss of dopaminergic neurons; clinical motor symptoms typically manifest once approximately 50%–70% of these neurons have deteriorated. Beyond dopaminergic loss, the cholinergic system is significantly compromised in PD, particularly as the disease progresses toward Parkinson's disease dementia (PDD). Cortical cholinergic loss is linked to the degeneration of the nucleus basalis of Meynert (NBM) [25], contributing heavily to cognitive decline. Furthermore, dysfunction of the pedunculopontine nucleus (PPN) results in reduced cholinergic input to the thalamus [26]. This specific pathway is clinically associated with postural instability, gait disturbances, and an increased risk of falls. While the use of AChEIs requires careful management in PD due to the potential for exacerbating motor tremors, they remain a primary therapeutic strategy for addressing the cognitive impairments and visual hallucinations associated with PDD; for example, rivastigmine is FDA‐approved specifically for the treatment of cognitive decline in PDD, where the benefit to cognition often outweighs the potential risk of motor exacerbation [27, 28, 29].

2.3. Myasthenia Gravis (MG)

MG is an autoimmune disease where the body produces antibodies that block or destroy ACh receptors at the neuromuscular junction. Interestingly, drugs used to treat MG are often AChEIs. By inhibiting AChE, these drugs increase the amount of ACh available to bind to the remaining functional receptors, thus improving muscle strength. However, the underlying issue in MG is the reduced number of functional receptors, not necessarily a primary decrease in AChE production by the body itself [30].

2.4. Organophosphate Poisoning

Beyond common diseases, organophosphate pesticides act as irreversible inhibitors of AChE. This results in an overwhelming accumulation of ACh, leading to a cholinergic crisis. This induces many symptoms such as paralysis of muscles, cessation of breathing, convulsions, and even death. This illustrates the drastic effects of sharply decreased AChE activity [31].

3. AChEIs

The inhibition of AChE is an important and complex process involving specific molecular interactions within the enzyme's active site. A primary class of drugs used to manage the symptoms of ACh deficiency are AChEIs, such as donepezil, physostigmine, and tacrine (Figure 2). These drugs act by inhibiting the remaining AChE activity, thereby increasing the availability of ACh at the synapses and temporarily improving cholinergic neurotransmission. Moreover, the search for effective therapeutic agents to combat neurodegenerative diseases (NDDs) has long been a paramount focus in medicinal chemistry. Among the many promising candidates, isatin derivatives have distinguished themselves as a particularly potent and versatile class of compounds, garnering significant attention as powerful AChEIs [32, 33, 34].

Figure 2.

AChEIs drugs.

Based on their chemical structure, AChEIs can be categorized into several key groups [35].

3.1. Organophosphates

Echothiophate (Figure 2), a therapeutic agent used in the management of glaucoma, serves as a prominent clinical example of this class. Its mechanism involves the formation of a stable, often irreversible, covalent bond with the active site of AChE [36].

3.2. Carbamates

Examples of this class include physostigmine, rivastigmine, and neostigmine (used in AD) (Figure 2). Carbamates inhibit AChE through a process of carbamoylation, which is reversible, though the recovery of enzyme activity is slower compared to simple reversible inhibitors [37].

3.3. Piperidines

A prominent example of piperidines is donepezil (Figure 2), a widely used reversible AChEI for AD [38].

Donepezil is a leading medication for AD symptoms, which works by inhibiting the AChE enzyme. This slows the degradation of ACh, thereby improving nerve cell function. The manner in which donepezil binds to the AChE active site is a sophisticated interaction. Basically, it acts like a bridge that blocks the long gorge of the AChE active site [39]. Donepezil is long enough to simultaneously bind to two important regions, the CAS deep within the gorge, and the PAS near the gorge's entrance. Moreover, there are aromatic stacking π–π interactions. The binding is primarily stabilized by stacking interactions between the flat aromatic rings of donepezil and the aromatic side chains of amino acids in the enzyme, particularly Tyr70, Trp84, and Trp279. Donepezil does not interact directly with the enzyme's catalytic triad (Ser203, His447, and Glu334). Instead, it simply obstructs the pathway, preventing ACh from reaching the site where it would be broken down [40, 41] (Figure 3).

Figure 3.

The binding mode of donepezil to the AChE active site.

3.4. Isatin‐Containing Compounds

Isatin scaffold serves as a highly adaptable platform for designing potent AChEIs through diverse synthetic strategies. Key approaches include N ₁‐substitution strategies like N‐alkylation, N‐acylation, or N‐arylation to modify the indole nitrogen [42]. Another strategy is aromatic ring substitution by the introduction of various substituents (e.g., halogens, nitro, amino, alkyl groups) typically at the C₅, C_6, and/or C₇ positions. The third strategy is C₃‐carbonyl functionalization, such as the formation of Schiff bases, hydrazones [43], or spirocyclic derivatives, which often link isatin to other pharmacophoric fragments. These distinct derivatization strategies can be systematically classified and evaluated based on structure–activity relationship (SAR) studies, which correlate the specific structural modifications achieved through each approach with the resulting AChE inhibitory potency [42, 44, 45].

To guarantee a comprehensive coverage of the literature regarding isatin scaffolds as AChEIs, a literature search was performed across electronic databases including PubMed, Scopus, and ScienceDirect. Search keywords were combinations of terms including “Acetylcholinesterase inhibition, Alzheimer's disease, Parkinson's disease, Isatin, and Structure modification.” Inclusion criteria were restricted to peer‐reviewed original research articles and short communications published primarily within the last 10 years, focusing on structural modifications and biological evaluation, such as IC₅₀ values of new isatin derivatives as AChEIs. Non‐English literature and patents were excluded.

This systematic review details recent synthetic advancements aimed at optimizing isatin‐based AChE inhibitors. It categorizes these developments into N‐substitution, aromatic ring modification (notably at C₅, C₆, and C₇), and C₃‐carbonyl functionalization. The findings emphasize the scaffold's versatile role in AChE inhibition over the last decade while outlining prospective research directions.

3.4.1. N₁‐Substitution of the Isatin Ring

Ozgun et al. [42] investigated the effect of N ₁‐Mannich base isatin derivatives bearing different amines (piperidinyl, morpholinyl, or N‐methylpiperazinyl) on the inhibitory activity of AChE (Figure 4). The findings revealed that all three compounds 1a–c exhibited significant inhibitory effects against AChE. Compounds 1a–c can be considered as promising leads for the development of new AChEIs, as they demonstrated 2.2–5.9 times greater inhibitory activity against this enzyme compared to tacrine. AChE was effectively inhibited by compounds 1a–c with IC₅₀ values in the range 1.66–2.34 nM, compared to 4.36 nM for tacrine.

Figure 4.

The design strategy of compounds 1a–c and 2.

While N‐alkyl isatin derivatives are known for their various biological activities, a comprehensive study specifically on the effect of N‐alkylation on their AChE inhibitory activity was lacking. Alcorn et al. [44] examined the impact of various N‐alkyl isatin derivatives on Electrophorus electricus (eeAChE) inhibition. Their findings demonstrated that the introduction of an N‐alkyl chain significantly augmented the inhibitory potency compared to the unsubstituted isatin core. Compound 2 (N‐dodecyl derivative) exhibited outstanding potency (IC₅₀ = 62.2 μM) compared to rivastigmine (IC₅₀ = 275 μM). Molecular docking results revealed that compound 2 attached to the AChE active site through a parallel π–π stacking interaction between its isatin moiety and Trp84. Moreover, a hydrogen bond was formed between the compound's 2‐oxo group and the Ser122 side‐chain hydroxyl group.

Zhou et al. [46] prepared and evaluated a new series of isatin derivatives for their ability to inhibit AChE (Figure 5). Previous research had shown that compounds like isatin can maintain important cellular proteins and stop the clumping of amyloid‐beta (Aβ), which is linked to AD [47]. The biological testing demonstrated that several of these compounds exhibited potent AChE inhibitory activity, with IC₅₀ values in the nanomolar range. Notably, the N‐benzoyl derivative 3 showed an IC₅₀ of 47.86 nM, which was comparable to the established drug donepezil with an IC₅₀ of 50.12 nM. Molecular docking predictions indicated that compound 3 would bind to the AChE active site through a parallel π–π stacking interaction between its isatin moiety and Tyr337. Additionally, a hydrogen bond was expected to form between the 2‐oxo group of the isatin and Tyr341.

Figure 5.

The design strategies applied for the preparation of compounds 3–5.

Lan research team [48] synthesized three sets of isatin derivatives bearing 1‐benzyl‐1H‐1,2,3‐triazole group to target AChE inhibition, inspired by the structural characteristics of donepezil (Figure 5). It was proposed that the inclusion of the triazole moiety would preserve a nitrogen atom similar to that in donepezil and introduce a heterocycle with more possible interactions with the enzyme binding site. Among the synthesized compounds, compound 4 exhibited the strongest AChE inhibitory activity, showing a 50% inhibition of the enzyme relative to donepezil when tested at a concentration of 100 μM. Therefore, a molecular docking simulation was used to gain more perception about SAR of compound 4 through the binding domain of AChE. The isatin heterocyclic system of 4 showed interactions with the residues in the PAS and acyl pocket of AChE. Also, the triazole ring of compound 4 displayed massive hydrophobic interactions with Phe295 and Tyr341, alike the interactions afforded by the piperazine moiety of donepezil. Furthermore, strong H‐bond and van der Waals interactions were developed between the 1‐benzyl‐1H‐1,2,3‐triazole moiety and Tyr124.

In 2021, isatin‐linked 4,4‐dimethyl‐5‐methylene‐4,5‐dihydro‐thiazole‐2‐thiols were specifically designed by Davis and Eckroat [49] to block AChE. While 2‐thiazoline hadn't been previously used as an AChEI, it was believed that chemically attaching it to isatin could lead to a more potent, multi‐functional AChEI. This was due to 2‐thiazoline's several heteroatoms that could interact with the PAS, and its suitability for further chemical modification. The aim was for this new compound to simultaneously engage with both the CAS and the PAS. The most effective compounds in this series were compounds 5a,b (with IC₅₀ values of 18.2 and 27.5 μM, respectively). Compounds 5a,b were more potent than rivastigmine with an IC₅₀ of 275 μM and showed almost similar potency to galantamine. Further studies on compound 5a revealed that it inhibited AChE in a non‐competitive manner. Its isatin moiety was located within the CAS, while the alkyl chain stretched into the gorge, placing the 2‐thiazoline moiety near the PAS. In the CAS, the isatin group formed crucial π–π stacking interactions with Trp84 and Phe330, a common binding mode for aromatic compounds. At the PAS, the isatin's 2‐oxo group formed a hydrogen bond with Ser122, and the 2‐thiazoline's nitrogen formed a hydrogen bond with Tyr121.

A series of quercetin‐1,2,3‐triazole hybrid molecules were synthesized and evaluated for their ability to act as antioxidants, to protect cells against oxidative stress caused by hydrogen peroxide, and to inhibit the activity of AChE and butyrylcholinesterase (BChE). Molecules with the 1,2,3‐triazole heterocyclic are known for their broad pharmacological properties, including AChE inhibitory activity [50] (Figure 6). To enhance quercetin's AChE inhibitory activity, it was combined with the 1,2,3‐triazole heterocycle and isatin molecule using a molecular hybridization approach. This combination also appeared to offer enhanced enzyme inhibitory potential. Several compounds, specifically 6, demonstrated inhibitory activity against eeAChE, with 24.31% inhibition compared to quercetin, which had 8.18% inhibition at a concentration of 100 µM [51].

Figure 6.

Molecular hybridization of quercetin, 1,2,3‐triazole heterocycle and isatin.

3.4.2. C₃‐Carbonyl Functionalization

In 2015, 3‐(2‐(4‐benzoylpiperazin‐1‐yl)ethylimino)indolin‐2‐one derivatives were designed and synthesized to explore their AChE inhibitory activity (Figure 7). The design was inspired by previous research showing that compounds with phenylpiperazine, benzylpiperidine, or anilino moieties like compound 7 were effective enzyme inhibitors, and that aromatic rings, such as the indoline substructure, improved the binding to the enzyme's PAS section [52]. By incorporating the key pharmacophoric elements of donepezil, the researchers synthesized these new donepezil‐like compounds. The inhibitory efficacy of these compounds against eeAChE was then quantitatively evaluated using Ellman's assay, with donepezil as a reference standard. The results were remarkable, with most of the new compounds demonstrating superior AChE inhibitory potency. Notably, compound 8 was the most potent inhibitor, with an IC₅₀ of 0.01 nM, vastly outperforming donepezil [53].

Figure 7.

The design strategy of compound 8.

Tripathi et al. [54] designed a collection of semicarbazone derivatives from 3,4‐(methylenedioxy)aniline 10a,b as promising AChEIs inspired by the structure of the previously reported compound 9 [55] (Figure 8). The semicarbazone chemical structure includes amino and carbamoyl ends that can be easily modified to produce new versions with enhanced pharmacodynamic or pharmacokinetic profiles. Previous research revealed that semicarbazones derived from aryl and heteroaryl compounds were potent inhibitors of the AChE enzyme. The synthesized compounds were assessed for their potential to inhibit AChE. Most of the new compounds selectively inhibited AChE that was isolated from rat brain, displaying IC₅₀ values in either the micro‐ or nanomolar range. Compounds 10a,b exhibited a promising inhibitory profile against AChE (IC₅₀ = 3.26 ± 0.087 and 0.307 ± 0.003 μM, respectively) compared to donepezil IC₅₀ = 0.021 μM, showing no toxicity to neurons. Molecular docking studies revealed that compound 10a formed hydrogen bonds with Asp74, Tyr124, and Tyr133, and engaged in π–π interactions with Tyr341. Additionally, Compound 10b formed hydrogen bonds with Asp74, Tyr124, Ser125, and Phe295, and engaged in π–π interactions with Trp86 and Tyr341.

Figure 8.

Design of semicarbazone derivatives as AChEIs.

Kaya et al. [43] synthesized three novel palladium (II) complexes 12a–c (Figure 9) utilizing asymmetric isatin bisthiocarbohydrazone ligands and PdCl₂(PPh₃)₂. In the context of designing AChEIs, palladium (II) complexes are of particular interest due to their known ability to act as enzyme inhibitors [43]. Starting from the previously reported compounds 11a–c [56], Pd(II) complexes 12a–c were designed for AChE inhibition and further enhanced by coordination with phosphine ligands, such as triphenylphosphine (PPh₃) [43]. This strategic combination of a Pd(II) core with a bulky phosphine ligand presented a promising approach for developing new and effective AChEIs. These complexes were diamagnetic and possessed a square planar geometry. The asymmetric isatin bisthiocarbohydrazone ligands coordinated to the Pd (II) ion in a tridentate manner, engaging through the phenolic oxygen, imine nitrogen, and thiol sulfur atoms, thereby forming five‐ and six‐membered chelate rings within their structures. The fourth coordination site in these complexes was occupied by triphenylphosphine (PPh₃). The synthesized Pd(II) complexes were evaluated for their inhibitory activity against AChE. All complexes exhibited highly potent inhibition of AChE, with IC₅₀ values ranging from 3.6 to 8.6 nM compared to an IC₅₀ of 33.5 nM for donepezil. The findings demonstrated the effective AChE inhibitory properties of both the synthesized ligands and their palladium (II) complexes [43].

Figure 9.

Synthesized novel palladium (II) complexes of isatin derivatives.

In 2022, isatin‐thiosemicarbazone hybrids were designed and synthesized as AChEIs (Figure 10). While some research existed on isatin compounds as carbonic anhydrase inhibitors and anticancer agents [57, 58, 59], this study was the first to investigate the combined anticancer, antimicrobial, and enzyme inhibition properties of isatin‐thiosemicarbazone hybrids. In the enzymatic assays, compound 13 emerged as the most potent inhibitor of AChE with an IC₅₀ of 1.23 μM. Notably, nearly all the tested compounds displayed superior inhibitory profiles compared to tacrine's IC₅₀ of 11.14 μM. Molecular docking estimated that compound 13 would bind to the AChE active site through a hydrogen bond between the 2‐oxo group of the isatin and Leu236 [45].

Figure 10.

Isatin‐thiosemicarbazone hybrids.

Mavroidi et al. [60] synthesized two novel isatin‐thiosemicarbazone derivatives as AChEIs, inspired by the structural characteristics of donepezil (Figure 10). It was proposed that the inclusion of thiosemicarbazone can improve the enzyme inhibitory activity. The compounds demonstrated a moderate ability to inhibit the AChE enzyme. Specifically, compound 14 had an IC₅₀ value of 54 μM, which was superior to the reference drug tacrine.

Khan et al. [61] synthesized a series of 16 isatin/bis‐Schiff base compounds incorporating a thiocarbohydrazone (Figure 11). The strategy of this study was the synthesis of a hybrid of an isatin molecule with Schiff base derivatives to afford new promising AChEIs. All of the synthesized analogs demonstrated significant inhibitory activity against AChE. Compound 15 exhibited outstanding inhibitory potency (IC₅₀ = 0.2 ± 0.05 μM), compared to the reference drug donepezil (IC₅₀ = 2.16 ± 0.12 μM). Concerning molecular docking, compound 15 was expected to bind to the active site of AChE with π–π stacking between the isatin moiety and Trp59, in addition to a H‐bonding interaction between the 2‐oxo group and Gln306, and H‐bonding interactions between NO₂ groups and Gln 63 His101, in addition to different amino acid interactions through Van der Wall forces.

Figure 11.

The design strategy of isatin/bis‐Schiff base compounds incorporating a thiocarbohydrazone.

In 2022, isatin thiosemicarbazone (ITSC) derivatives were recognized for their significant ability to inhibit many enzymes like AChE, valuable neuroprotective properties, and robust anti‐Aβ aggregation activity [60]. Building on these findings, this study aimed to optimize isatin's diverse properties, especially for dual anti‐Aβ aggregation and AChE inhibitory activity. Compound 16 was identified as a moderate in vitro AChEI with an IC₅₀ of 44 µM. Subsequently, Hassan et al. [62] were inspired by the potential of compound 16 as an AChEI [60] and synthesized a new series of isatin‐based Schiff base derivatives through reacting 3‐hydrazino‐isatin with various aldehydes (Figure 12). These compounds were then tested in vitro for their antioxidant, anti‐diabetic, and anti‐AD properties. Derivatives (17a–c) displayed the most promising activity across these tests. Among these potent compounds, compound 17a exhibited AChE inhibitory activity of 36.38% ± 0.08% at a concentration of 100 µM. Molecular docking predictions indicated that compound 17a displayed the strongest binding to the AChE active site. Its binding involved a hydrogen bond between Glu202 and the isatin's NH, complemented by π–π interactions between the pyrazole core and Tyr341, and the N‐phenyl of pyrazole with Trp286. Furthermore, compound 17b also showed notable activity, primarily through multiple π–π interactions. Compound 17b interacted with Tyr124 and Tyr341 via the isatin's phenyl, and with Trp286 via the pyrazole's N‐phenyl. Compound 17c engaged Trp286, Tyr337, and Tyr341 with the isatin's NH, pyrazole's N‐phenyl, and pyrazole core, respectively [60].

Figure 12.

Design of isatin‐based Schiff base derivatives as AChE.

In 2025, two series of isatin hydrazones, including either morpholine or piperazine units, were reported, and their inhibitory activity against human recombinant (hAChE) enzyme was evaluated (Figure 13). Recent reports suggested isatin‐based hydrazones as promising scaffolds for developing AChEIs, especially when various pharmacophores are attached at the C₃ position [63]. Based on this finding, a series of novel isatin derivatives were synthesized featuring a hydrazone linker that connected either a 4‐(4‐methylpiperazin‐1‐yl)benzylidene or morpholinobenzylidene group to the C₃ position of isatin. The results showed that compound 18a was particularly effective at inhibiting AChE by 74%, with an IC₅₀ of 1.60 ± 0.51 μM, compared to donepezil. A clear advantage was observed in this series: incorporation of electron‐withdrawing groups like chlorine (Cl) and bromine (Br) at the C₅ position of the isatin core significantly boosted AChE inhibition, chlorine substituted derivative proved more effectiveness than the bromine derivative. More broadly, it was found that morpholine derivatives consistently outperformed their corresponding piperazine derivatives as AChEIs. The docking simulation with AChE revealed that the isatin moiety's carbonyl group in compound 18a formed a hydrogen bond with Phe295, while its NH group also hydrogen‐bonded with Ser293. The aromatic benzene ring of the morpholine benzaldehyde component engaged in a π–π stacking interaction with Phe338. Compound 18a also exhibited extensive hydrophobic interactions with several key residues, including Tyr124, Phe297, Val294, Tyr337, Tyr341, Trp286, and Trp86. Furthermore, it made polar contact with Ser203 and His447. These critical interactions collectively explained the inhibitory effect of 18a against AChE. Notably, 18a displayed similar hydrophobic interaction patterns within the AChE binding site when compared to the reference ligand, donepezil [64].

Figure 13.

Design of isatin hydrazones as AChEIs.

3.4.3. Spirocyclic Derivatives

Barakat et al. [65] synthesized a new class of spirooxindole‐benzo[b]thiophene analogs (Figure 14) and tested their ability to inhibit AChE. The broad‐spectrum biological activity of both structural classes made them highly valuable for developing novel scaffolds inspired by previously reported compound 19, which had an IC₅₀ value of 3.33 µM [66]. All of the synthesized compounds showed AChE inhibitory activity, with IC₅₀ values ranging from 20.8 to 121.6 μM. Compound 20a emerged as the most effective inhibitor, exhibiting an IC₅₀ value of 20.8 μM. Its high inhibitory activity was linked to the presence of a 5‐chloro‐substituted oxindole core combined with a benzo[b]thiophene moiety. The compound achieved strong dual‐site binding at both the CAS and PAS of the protein, stabilized by a network of π‐stacking interactions and hydrophobic interactions with key aromatic residues, including Tyr72, Trp86, and Trp286. This extensive network of interactions suggested that the compound's multiple aromatic cores contributed significantly to its high activity. Further stabilization was provided by a hydrophilic interaction through a hydrogen bond formed between Ser125 and the carbonyl group of 20a. A notable halogen bond interaction between the 5‐Cl atom and Asp74 also contributed to its stable binding. Several other analogs containing octahydroindole moieties 20b–f also displayed acceptable inhibitory activity, with IC₅₀ values ranging from 34.0 to 41.5 μM, which were compared to the positive control, galantamine.

Figure 14.

Design of new class of spirooxindole analogs as AChEIs.

In 2021, compound 19 was further modified to afford a new series of spirooxindole analogs, containing a pyrazole scaffold [67]. The compounds were synthesized using a [3 + 2] cycloaddition reaction between a chalcone‐based pyrazole and isatin derivatives (Figure 14). This research aimed to combine the structural advantages of pyrazole and oxindole scaffolds. The synthesized spirooxindole analogs were then evaluated for their neuroprotective potential by assessing their ability to inhibit the AChE enzyme using Ellman's method. Compound 21 demonstrated moderate AChE inhibition, with an IC₅₀ value of 5.7 μM compared to the standard drug galantamine's IC₅₀ value of 1.2 μM. Moreover, compound 21 exclusively interacted with amino acid residues located within the PAS, specifically with Tyr124, Tyr72, Ser293, His287, and notably, Tyr341 and Trp286. The latter two residues (Tyr341 and Trp286) are known to enlarge the gorge, facilitating inhibitor binding and stabilizing the inhibitor‐ligand complex through π–π stacking interactions. This binding mode suggested that compound 21 inhibited the enzyme primarily by engaging the PAS, rather than the CAS, which aligned with recent findings on structurally similar AChEIs [67].

The active site of the AChE enzyme is primarily hydrophobic. In contrast, BChE has a similar overall structure to AChE, but its active site contains more non‐aromatic residues like valine and leucine. This allows BChE to accommodate larger molecules. Both of these enzymes are key targets for treating AD, and previous studies have shown that spiropyrrolidines can inhibit them [68]. Inspired by these findings, Basiri et al. [69] used molecular modeling to design new piperidone‐spiropyrrolidine hybrids that could effectively inhibit both AChE and BChE. The results showed that compound 22 (Figure 15) was particularly effective, inhibiting AChE with an IC₅₀ value of 1.37 μM compared to galantamine with an IC₅₀ value of 2.9 μM. The most significant finding of this study demonstrates that substitution of the isatin scaffold at the C₆ position is highly beneficial for maximizing AChE inhibition. Specifically, the introduction of a NO₂ group represents the optimal modification, extremely enhancing inhibitory potency. Further analysis through molecular docking simulations revealed that compound 22 fit well into the active site of the AChE enzyme. It formed hydrophobic interactions with residues at both the entrance of the gorge, specifically Trp279, Tyr121, and Tyr334 (part of the PAS), and with key residues deeper within the channel, including Trp84 (involved in choline binding) and His440 (part of the catalytic triad). This dual‐site interaction engaging residues at both the gorge entrance and the active site likely explained the observed high efficiency of compound 22 in blocking the enzyme's channel, thereby preventing substrate entry and hydrolysis, which accounted for its remarkable inhibitory activity.

Figure 15.

Piperidone‐grafted spiropyrrolidine as AChEIs.

Mohamed Yusoff et al. [70] synthesized a new scaffold of dispiropyrrolidine compounds through [3 + 2] cycloaddition reactions (Figure 15). Dispiropyrrolidine compounds demonstrated a wide range of promising biological activities, including anticancer [57, 58, 59], anti‐AD, and antidiabetic effects. The research team focused on creating new spiro heterocyclic compounds that combine both oxindole and pyrrolidine structures based on their previous work on this series [69]. The compounds were then tested in vitro using Ellman's assay to determine their ability to inhibit AChE. The results indicated that these compounds generally displayed better inhibitory activity against BChE compared to AChE. Compound 23 emerged as an adequate AChEI, exhibiting inhibition of 43.50% ± 5.70% at 10 μM in comparison to physostigmine (tested at 0.2 μM), which displayed 70.13% ± 2.37% inhibition.

In 2023, spiro indolin‐1,2‐diazepine compounds were reported as AChEIs (Figure 16). The compounds were designed by structure modification of compound 24 (IC₅₀ = 0.44 nM) into spiro indolin‐1,2‐diazepine [52]. The newly synthesized compounds were then evaluated for their ability to inhibit the enzymes BChE and AChE. The results showed that compound 25 was a potent and selective AChEI, with an IC₅₀ value of 3.98 ± 1.07 μM compared to donepezil with an IC₅₀ value of 0.079 ± 0.05 μM. Docking simulations of compound 25 with AChE revealed a multi‐point binding interaction. The 5‐nitroindolinone moiety specifically engaged with Trp86 in the anionic site and formed a hydrogen bond with Tyr124. Additionally, the indolinone ring established two interactions with His447 within the CAS pocket. Moving to the other end of the molecule, the hydroxyphenyl amine portion formed two hydrogen bonds with Tyr72 and Asp74, both located near the PAS pocket [71].

Figure 16.

A new series of spiro indolin‐1,2‐diazepine compounds.

In a study reported by Pourtaher et al. [72], a novel scaffold of isatin derivatives was synthesized and screened for dual inhibitory activity against AChE and BChE (Figure 17). The authors also utilized a highly efficient, catalyst‐free, one‐pot synthetic method to simplify the production of these new compounds. The synthesized compounds 26a–d demonstrated percentage inhibition at 50 μM ranging from 63.13% ± 4.32% to 75.47% ± 3.68%, with an IC₅₀ value range of 6.02 ± 2.59–42.55 ± 4.11 μM for AChE compared to the donepezil IC₅₀ value of 0.079 ± 0.05 μM. Moreover, compounds 26a–d showed percentage inhibition at 50 μM ranging from 13.94% ± 3.68% to 38.94% ± 7.36% for BChE. Compound 26d, an enantiomeric mixture, emerged as the leading candidate, prompting a detailed investigation into its R and S isomers' binding within the AChE active site. Molecular docking assessed both enantiomers, revealing that the S enantiomer of 26d was optimally oriented in the active site; the indolinone moiety formed a hydrogen bond with Tyr124 and a π–π stacking interaction with Trp86. Furthermore, the pyrazole ring made two hydrogen bonds with Asp74 and Tyr337, and a π–π stacking interaction with Tyr337, and the nitro group exhibited a π‐cation interaction with Tyr341. The 3‐Cl substituent formed a halogen bond with Phe295. Conversely, the R isomer displayed halogen interaction with Phe295, a hydrogen bond with Ser203, π–π stacking interaction with Tyr341, and both π‐cation and π–π stacking interactions with His447.

Figure 17.

A novel scaffold of spiroindolinone‐pyrazole derivatives.

3.4.4. Miscellaneous Core Modification Strategies

Based on earlier discoveries that a type of isatin derivative could prevent Aβ aggregation, researchers identified compound 28 (a 5‐methoxyisatin derivative) as a multi‐target inhibitor in 2017 [73] (Figure 18). This compound not only interfered with Aβ aggregation but also inhibited AChE with an IC₅₀ value of 10.4 ± 0.9 μM. To improve its water solubility, a Mannich‐base derivative, compound 29, was synthesized. It showed a remarkable increase in solubility at an acidic pH and proved to be more stable than 28. It also inhibited eeAChE with an IC₅₀ value of 10.4 ± 0.9 μM, compared to galantamine with an IC₅₀ of 0.51 ± 0.10 μM. Interestingly, compound 29 was metabolized rapidly in human serum to afford compound 28. In cell studies, compound 29 was found to be highly neuroprotective. On the other hand, the eeAChE inhibition tests revealed that both compounds were less potent than the reference drug, galantamine [74].

Figure 18.

Modification of 5‐methoxyisatin derivatives into AChEIs.

Negi et al. [75] performed a study combining piperidine and isatin scaffolds to create a series of novel benzylpiperidine and isatin hybrids (Figure 19). Piperidine and isatin are valuable scaffolds in drug design because they can easily cross the blood‐brain barrier and can be derivatized. The goal was to develop efficient inhibitors for AChE, the key enzyme implicated in NDDs, taking compound 10b as a lead [64]. The synthesized hybrids were evaluated for their inhibitory effects on the AChE enzyme. Compound 30 was considered a potent lead compound, effectively inhibiting eeAChE (IC₅₀ = 0.034 μM), even outperforming the reference drug donepezil (IC₅₀ = 0.084 ± 0.002 μM) against AChE. Compound 30 effectively fit within the AChE gorge, interacting with both the CAS and PAS. Docking simulations indicated that its benzylpiperidine moiety was preferably situated within the PAS, while the substituted isatin moiety occupied the CAS, with a flexible hydrazino linker connecting them. The stability of this enzyme‐ligand complex was attributed to hydrogen bonding, π–π interactions, and van der Waals forces. Specifically, three hydrogen bonds were observed with Tyr124, one from the isatin's carbonyl oxygen, and two from the NH hydrogens within the hydrazino linker. Furthermore, a π–σ interaction occurred between the phenyl ring of Trp86 and the chlorine atom of the isatin. Two π–π stacking interactions were identified, one between the isatin's phenyl ring and Tyr337, and another between the benzylpiperidine's phenyl ring and Trp286. Additional π–alkyl interactions were noted with Phe295, Phe297, Tyr337, Phe338, His447, and Tyr449, complemented by various van der Waals interactions with Tyr72, Gly121, Gly122, Ser203, Ala204, Leu289, Tyr341, and Gly448.

Figure 19.

Design a series of novel benzylpiperidine and isatin hybrids.

In 2019, three series of isatin derivatives incorporating a 1‐benzyl‐1H‐1,2,3‐triazole moiety were synthesized (Figure 20). The design of these compounds was inspired by the structural features of donepezil to create potent AChEIs. By incorporating a triazole group, a key nitrogen atom was maintained, similar to the one found in donepezil, while also introducing a heterocyclic ring that allowed for more favorable interactions within the enzyme's active site. Among the synthesized compounds, derivative 31f demonstrated AChE inhibitory activity, achieving inhibition percentages of 51% at a concentration of 100 μM. These findings indicated that, although the synthesized compounds were not yet potent AChEIs, they showed some ability to inhibit the enzyme. This suggested that further modifications of the benzyl group or changes to the substitution patterns at positions 3 or 5 on the 2‐oxoindoline core could lead to the development of new and more powerful AChEIs. Moreover, docking studies indicated that compound 31f displayed optimal interactions within both the PAS and the acyl pocket of AChE. It achieved multiple stacking interactions with Trp286 and established three hydrogen bonds with Phe295, Arg296, and Tyr341. Furthermore, the triazole ring of 31f engaged in hydrophobic interactions with Phe295 and Tyr341, mirroring the interactions observed with donepezil's piperazine moiety. Strong hydrogen bond and van der Waals interactions were also noted between the 1‐benzyl‐1H‐1,2,3‐triazole portion and Tyr124 [48].

Figure 20.

Design of isatin derivatives 31a–f bearing 1‐benzyl‐1H‐1,2,3‐triazole moiety.

Riazimontazer et al. [76] synthesized a new scaffold of hybrid molecules, combining the structural features of tacrine and isatin via N‐hexyl group linkage (Figure 21). Biological evaluation demonstrated that most of these compounds potently inhibited eeAChE. The majority of the synthesized compounds inhibited eeAChE with IC₅₀ values ranging from 0.42 to 79.66 nM. Notably, compounds 32a–c, all featuring a six‐carbon linker between the tacrine and isatin Schiff base moieties, displayed the most potent eeAChE inhibitory activity, with IC₅₀ values of 0.42, 0.62, and 0.95 nM, respectively. These compounds were found to be 92, 62, and 41 times more active against eeAChE than tacrine, with an IC₅₀ value of 38.72 nM. In addition, compounds 32a and 32b were identified as effective inhibitors of AChE‐induced Aβ aggregation. Compounds 32a–c demonstrated dual‐site binding as AChEIs, engaging the CAS, PAS, and mid‐gorge according to the docking studies. The tacrine moiety generally settled in the CAS. Here, the tacrine ring and its protonated nitrogen formed π–π stacking with Trp84 and a cation–π interaction with Phe330. The protonated tacrine nitrogen was hydrogen‐bonded with His440. Separately, the Schiff base's protonated nitrogen could form a cation–π interaction with Trp279 and π–π stacking with its own ring in the PAS. In compound 32c, the linker nitrogen of tacrine showed a cation–π interaction with Trp84 in the CAS, while the isatin's protonated nitrogen formed a cation–π interaction with Tyr334 in the PAS.

Figure 21.

Hybrid molecules combining the structural features of tacrine and isatin.

Conversely, in 32a,b, the tacrine ring exhibited a reversed orientation, π–π stacking against Trp279 in the PAS, while the Schiff base ring formed a π–π interaction with Trp84 in the CAS. Additionally, Tyr334 and Phe330 formed cation–π interactions with the protonated isatin nitrogen and Schiff base nitrogen in the CAS. Tyr121 and Tyr334 were involved in interactions with the mid‐gorge. These molecular docking results confirmed that compounds 32a,b simultaneously bind to the CAS, PAS, and mid‐gorge of AChE, consistent with their design as dual‐site inhibitors and their kinetic study findings.

Further optimization of compound 32a–c was reported in 2020, aiming to develop new molecules by altering the size of the six‐membered cycloalkane ring of tacrine. The authors designed and synthesized conjugates that feature five‐, six‐, and seven‐membered “C” rings (n = 1–3), as depicted in the general structure. A promising approach involved binding tacrine derivatives to isatin Schiff bases using an amide linker; the study identified several compounds with potent inhibitory activity against AChE and BChE collected from mouse brain, particularly those with a propionyl and butyroyl linker (Figure 21). Compound 33 appeared as the most effective cholinesterase inhibitor, demonstrating remarkable potency with an IC₅₀ value of 0.71 ± 0.004 μM against AChE and 1.08 ± 0.02 μM against BChE. Compound 33 exhibited significant interactions within the AChE binding pocket. A key hydrogen bond was formed, with the compound's carbonyl group accepting a hydrogen bond with Ser198. Its hydrazone phenyl group and the isatin scaffold were positioned within a hydrophobic pocket defined by Ile69, Trp82, and Tyr128. Concurrently, the central cycloheptane‐fused quinoline ring was situated in a separate hydrophobic pocket, surrounded by Phe229, Leu289, and Phe398 [77].

The multi‐target directed ligand (MTDL) approach involves designing a single molecule to act on multiple biological targets simultaneously, offering superior activity than conventional single‐target molecules. In this context, the MTDL approach was adopted to design compounds having a trifunctional profile, such as AChE inhibition, antioxidant activity, and biometal chelation, via creating hybrid compounds from triazine 34, isatin, and aniline derivative 8 (Figure 22).

Figure 22.

Design of novel hybrid compounds from triazine, isatin, and aniline derivatives.

The biological evaluation showed that these new hybrids were exceptionally potent AChEIs. The most effective compounds, 35a and 35b, demonstrated outstanding inhibitory activity against eeAChE with IC₅₀ values of 0.7 ± 0.03 and 0.2 ± 0.08 nM, compared to donepezil with an IC₅₀ value of 22.4 ± 2.1 nM, respectively. Additionally, these molecules exhibited strong inhibitory effects on BChE with IC₅₀ values of 0.09 ± 0.003 and 0.03 ± 0.03 μM, respectively. Furthermore, the inclusion of a hydroxyl group on the aniline moiety endowed some of the compounds with good antioxidant activity. Compounds 35a,b demonstrated consistent interactions with the key AChE residues: Tyr124 and Trp286 in the PAS, Trp86 in the CAS, and Tyr341 in the mid‐gorge. This pervasive binding pattern likely explained the non‐competitive inhibition observed for compound 35b. In detail, N‐benzyl‐substituted isatin derivatives showed interactions with Tyr72 in the PAS and Tyr337 in the CAS, as well as a hydrophobic interaction between their benzyl moiety and Leu289 in the PAS, potentially increasing their potency. As designed, compound 35b exhibited a binding profile related to donepezil, with its isatin portion engaging multiple residues in the PAS (hydrophobic contacts with Leu289, Trp286, Tyr341, π‐stacking with Trp286, hydrogen bonding with Phe295). Its aniline moiety significantly interacted with the CAS, where the aniline methoxy group formed hydrogen bonds with Ser203 and His447, and the aniline aromatic ring had a hydrophobic interaction with Trp86. The triazine group and its methoxy substituent formed π‐stacking and hydrogen bonds with Tyr337 (CAS) and Tyr341 (mid‐gorge), respectively. Additionally, Tyr124 formed a hydrogen bond with the hydrazone nitrogen [78].

3.5. Overview of SAR of Isatin Derivatives

Molecular docking studies consistently highlight a common SAR for AChE inhibition of isatin‐derived compounds, emphasizing the versatility of the isatin scaffold for multi‐site interactions. Generally, the isatin core is crucial, often engaging the CAS. To design a potent AChEI, the isatin scaffold can be optimized via diverse strategies (Figure 23). First, the modification of the N₁‐position of the isatin scaffold serves as a significant strategy for optimizing AChE inhibitory potency. The efficacy of these modifications is governed by three primary factors: the length of the linker, the steric nature of the substituent, and the type of functional group. The distance between the isatin core, which typically binds to the CAS, and the substituent is dominant, three to four carbons linker consistently yields the highest potency. This specific length allows the isatin ring to occupy the CAS while positioning the distal group to interact with the PAS near the gorge entrance. Short‐chain spacers of one or two carbons typically exhibit insufficient length or excessive conformational rigidity to effectively span the distance between the two binding domains, resulting in a noticeable reduction of the enzyme inhibitory potency [42, 44, 48]. On the other hand, although linkers exceeding 8 carbons often cause molecular misalignment, simple alkyl chains that lack distal pharmacophores can successfully restore activity at lengths of 7–10 carbons. This phenomenon is attributed to non‐specific hydrophobic stabilization within the enzyme's binding gorge. Regarding the substituent steric effect, nitrogen‐containing rings like morpholine, piperidine, or N‐methyl piperazine are the most effective distal substituents. Their small size allows them to fit into the limited space of the AChE gorge without steric hindrance, often resulting in nanomolar‐range inhibitory activity [42, 44, 46, 47, 48, 49]. On the other hand, bulky and rigid structures, such as quercetin attached to the N₁ linker, are too huge to fit into the narrow AChE active site, leading to diminished AChE inhibitory activity [51]. However, this same steric bulk frequently shifts selectivity toward BChE, which possesses a wider active site gorge capable of accommodating larger molecules [79]. Another important point to consider is the attachment of a heterocyclic ring. Attaching a 1,2,3‐triazole ring to the linker imitates the binding features of the benzylpiperidine of donepezil. This ring can participate in extra π–π stacking or hydrogen bonding interactions within the gorge [48, 50].

Figure 23.

Structure–activity relationship (SAR) map of isatin‐based AChEIs.

Second, the C₃‐carbonyl oxygen of isatin is inadequate for effective binding. It must be derivatized into a Schiff base, hydrazone, or thiosemicarbazone to form a flexible linker capable of bridging the distance between the enzyme's CAS and the PAS. The success of modification depends mainly on two essential factors: the linker type and the terminal group. Effective bridging is achieved using flexible ethylimine linkers, semicarbazone or thiosemicarbazone moieties [43, 45, 53, 56]. The efficacy of the C₃‐extension is strictly governed by the nature of the group at the far end of the linker. Aliphatic, non‐planar lipophilic groups such as cyclohexyl or morpholine are often more favorable than planar aromatic rings, as they fit better into the hydrophobic pockets without steric hindrance. Furthermore, when the terminal group is aromatic, potency is also enhanced by electron‐withdrawing groups. Substituents like fluoro or chloro significantly enhance binding affinity via stronger interactions with the PAS. On the other hand, electron‐donating groups like methoxy at the distal end reduce potency [54, 60, 61, 62, 63, 64].

Strategic modification at the C₅ position is essential for optimizing the electronic and steric fit of isatin derivatives. While small electron‐withdrawing groups like fluoro and chloro substituents enhance CAS binding when combined with N₁ or C₃ functionalization, electron‐donating groups or larger atoms (e.g., bromine) generally reduce potency, effectively overriding other structural optimizations [48, 61, 75, 80]. Moreover, functionalizing the C₆ position of the isatin scaffold is highly advantageous for maximizing AChE inhibition. In particular, the introduction of a NO₂ group emerges as the premier modification, extremely boosting inhibitory potency [69].

Finally, the transformation of the isatin core into a rigid, three‐dimensional spirooxindole scaffold at the C₃‐position is one of the most critical strategies for achieving high inhibitory activity. This spiro junction acts as a bridge that provides the unique orientation necessary to accommodate the narrow and deep active site gorge of the AChE enzyme by projecting a tethered heterocyclic scaffold, such as pyrrolidine, pyrazole, diazepine, or benzo[b]thiophene, in different directions. This scaffold allows the molecule to function as a dual‐site inhibitor. It simultaneously binds to the CAS at the bottom of the gorge and the PAS at the entrance [52, 65, 67, 68, 69, 70, 71, 72, 81, 82].

4. Conclusion and Future Persepectives

Based on comprehensive structural analysis, the isatin core serves as a highly adaptable scaffold capable of engaging both the CAS and the PAS. This dual‐site binding effectively bridges the enzyme's narrow active site gorge, serving as high‐potency AChEIs. This could be achieved through various structure modification strategies such as optimizing the N₁ linker and distal groups, transforming the C₃‐carbonyl into a flexible or rigid spirooxindole scaffold, and utilizing C₅ electron‐withdrawing groups. Optimized derivatives have already demonstrated extraordinary AChE inhibitory potency, reaching the nanomolar range (e.g., compound 8 displayed an IC₅₀ of 0.01 nM), which firmly establishes isatin as a focal point for the next generation of AChEIs.

Future research should focus on the most significant directions derived from these structural insights. Researchers can use the recognized criteria such as the three‐ to four‐carbon linker at the N₁ position combined with small distal nitrogen‐containing rings as a base to design novel derivatives that perfectly fit into the active site gorge without creating steric hindrance. On the other hand, transforming isatin into a rigid spirooxindole framework highlights a critical possibility for future design, as projecting new heterocyclic motifs in multiple directions from this rigid core can efficiently lock the molecules into both the CAS and PAS for enhanced interaction. Although modifications like converting the C₃‐carbonyl into flexible Schiff bases, hydrazones, or thiosemicarbazones enhance enzyme interaction, incorporating bulky groups (such as quercetin) shifts the therapeutic profile toward BChE selectivity rather than AChE inhibition.

Moreover, electronic and steric properties have to be balanced. Specifically, research should focus on pairing small electron‐withdrawing groups (like fluorine or chlorine) at the C₅‐position or a NO₂ group at the C₆‐position of isatin with optimized N₁ or C₃ modifications to maximize synergistic binding affinity, while avoiding bulky or electron‐donating groups that diminish AChE inhibitory activity.

Funding

The authors have nothing to report.

Data Availability Statement

Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study.