Ionic and Non-Ionic Counterparts Based on Bis(Uracilyl)Alkane Moiety with Highest Selectivity Towards Acetylcholinesterase for Protection Against Organophosphate Poisoning and Treating Alzheimer’s Disease

Abstract

A series of bisuracils, in which uracil and 3,6-dimethyluracil moieties were bridged with a polymethylene spacer, and the uracil moiety contained a pentamethylene radical with ionic and non-ionic aminobenzyl groups, were synthesised. These bisuracils have been identified as cholinesterase inhibitors with exceptional selectivity for acetylcholinesterase (AChE) over butyrylcholinesterase (BuChE). These bisuracils, which have been identified as highly effective AChE inhibitors, demonstrated activity at nano- and sub-nanomolar concentrations, with exceptional selectivity for AChE over BuChE. In kinetic studies of lead bisuracils 2b and 3c, both compounds exhibited mixed-type inhibition against AChE and BuChE. Additionally, molecular dynamic simulations demonstrated robust and stable interactions of 2b and 3c with the binding sites of their target. Bisuracil 2b showed significant potential for protection of AChE from irreversible inhibition by paraoxon; the most effective dose of 0.01 mg/kg was shown to reduce mortality in paraoxon-poisoned mice. Bisuracil 3c effectively inhibited brain AChE activity, reversing scopolamine-induced amnesia in mice at a dose of 5 mg/kg, which indicates its potential for cognitive enhancement. These findings position ionic bisuracils as promising prophylactics against organophosphate poisoning and non-ionic bisuracils as viable candidates for Alzheimer’s disease therapeutics.

1. Introduction

Cholinesterase inhibitors represent a class of compounds that interfere with the action of the enzyme cholinesterase to prevent the breakdown of acetylcholine, which is an important neurotransmitter in the nervous system. Such compounds, which interact with the enzyme as a primary target, can be used both as drugs and toxins [1]. The nature of the interaction between the inhibitor and the enzyme determines whether the inhibitor will be reversible or irreversible. Reversible inhibitors are widely used in the treatment of neurodegenerative disorders. Particular attention has been paid to currently approved cholinesterase inhibitors donepezil, rivastigmine, and galantamine in the pharmacotherapy of Alzheimer’s disease (AD) [2]. Reversible cholinesterase inhibitors competitively bind to AChE, preventing irreversible inhibition by OPs. This protective effect is most effective before OP exposure, as it temporarily shields the enzyme’s active site [3,4]. However, once OP-induced ‘aging’ occurs (irreversible phosphorylation of AChE), reversible inhibitors are ineffective, highlighting the need for prophylactic use. Reversible cholinesterase inhibitors are additionally utilised as pre-treatment for poisoning by irreversible cholinesterase inhibitors, represented by organophosphorus compounds (OPs), which are widely used as pesticides [5].

OPs have been identified as a prevalent cause of fatal poisonings, especially in rural areas of developing countries [4,6,7]. The prevailing standard of care for acute OP poisoning, which includes the administration of atropine, a muscarinic ACh receptor blocker, as well as various oxime compounds for activating acetylcholinesterase (AChE), has not met patient expectations [8]. However, if exposure to OPs is anticipated, preventive measures include the use of non-OP AChE inhibitors, which are capable of reversibly binding to the enzyme, thereby protecting its active site from irreversible inhibition by OPs [5,9].

Representing the most common cause of adult-onset dementia in people over 65, AD is characterised by progressive impairment of learning and memory functions at an early stage and ultimately resulting in death as the disease progresses [10]. The most extensively studied pathological features of AD are β-amyloid (Aβ) plaques primarily composed of aggregated forms of the Aβ peptide and neurofibrillary tangles, characterised by the presence of hyperphosphorylated tau protein associated with microtubules [11]. Current symptomatic treatments for AD include cholinesterase inhibitors (e.g., donepezil) that align with the cholinergic hypothesis, though disease-modifying therapies targeting amyloid-β and tau are under investigation. While current symptomatic treatments for AD primarily target the cholinergic hypothesis—which links cognitive decline to acetylcholine deficiency and loss of cholinergic neurons [12]—emerging evidence highlights the multifaceted role of AChE in neurodegeneration. Beyond its catalytic function, AChE accelerates amyloid-β aggregation [13] and interacts with tau pathology [14,15], suggesting broader involvement in AD progression. Notably, non-catalytic roles of AChE in neuroinflammation and synaptic plasticity [16] further complicate therapeutic targeting. By partially inhibiting these enzymes, acetylcholine levels can be increased, leading to improvements in learning and memory [17,18,19].

In a recent work, we reported a new class of selective AChE inhibitors with a bisuracil scaffold. Based on the proposed concept, bisuracils described by formula 1 were successfully designed and synthesised (Figure 1). These compounds consisted of 3,6-dimethyluracil and uracil rings bridged to each other by a polymethylene spacer, with the N1 atom of the uracil ring containing a pentyl(diethylammonium)benzyl radical. Electron-withdrawing nitro- and trifluoromethyl substituents were located in the o-position of the benzene ring of the benzyl moiety. The efficacy of these ionic bisuracils 1 as inhibitors of AChE was demonstrated at nano- and sub-nanomolar concentrations, exhibiting a highest degree of selectivity for AChE over BuChE. The lead compound in this series, namely bisuracil 1 with a hexamethylene spacer between uracil moieties and the trifluoromethyl substituent at the benzene ring, has been demonstrated to enhance the efficacy of antidotal therapy as a pre-treatment for poisoning by OPs [20].

The present study expands the concept of selective AChE inhibitors based on the bisuracil scaffold in two directions, the first consisting in the introduction of nitrile substituents into bisuracils—in particular, bisuracil 2 (Figure 2). In this instance, it is particularly important to consider the extent to which the substitution with electron-withdrawing nitrile groups in compounds with a similar structure to bisuracil 1, with electron-withdrawing nitro- and trifluoromethyl groups, could affect their activity against AChE. It has been demonstrated that nitrile groups exhibit a specific effect on biological activity [21,22]. In the present study, a series of bisuracil 2 was afforded, which exhibited the same structure as bisuracil 1, but, in contrast, carried nitrile substituents at the benzene ring. Earlier experience in studying the effect of the nitrile group on the activity of AChE inhibitors using 1,3-bis[ω-(substituted benzylethylamino)alkyl]uracils served as an example. The hypothesised nitrile-group effect indeed manifested in an outstanding affinity and selectivity against AChE exhibited by the studied compounds [23].

In the second direction of expanding the concept of bisuracil scaffold-based AChE inhibitors, we aimed to design and synthesise bisuracils that are isostructural to bisuracil 2 but have a non-ionic rather than a charged structure. Although the neutral bisuracil 3 (Figure 2) is described as having the same bisuracil scaffold as compounds 1 and 2, it contains a non-ionic pentyl(ethylamino)benzyl radical at the uracil ring. It is known that charged AChE inhibitors cannot cross the blood–brain barrier (BBB), whereas non-ionic AChE inhibitors are able to pass the BBB into the brain. AChE inhibitors that do not have charged moieties in their structure are considered potential drugs for the treatment of AD symptoms. Bisuracil 3, which can be expected to have the capability of crossing the BBB, may consequently be useful in the treatment of AD.

Herein, we describe the synthesis, in silico studies, and biological evaluation in vitro and in vivo of a series of new bisuracil scaffold-based AChE inhibitors—namely, the ionic and non-ionic counterparts 2 and 3.

2. Results and Discussion

2.1. Synthesis of Bisuracils 2 and 3

The target bisuracils from a series 2 and 3 (Figure 2) with ionic and non-ionic amino-moieties were synthesised by starting from the same α,ω-{(3,6-dimethyluracil-1-yl)-[3-(5-bromopentyl)uracil-1-yl]}alkane (4) reagent. This bromide was first prepared from the reaction of 1-(ω-bromoalkyl)-3,6-dimethyluracil (5a–d) with 2,4-bis(trimethylsilyl)uracil (6) and alkylation of the resulting bis(uracil)alkane 7a–d with 1,5-dibromopentane (Scheme 1). The steps leading from bromide 4 to charged bisuracils 2a–d included a replacement of the terminal Br atom in the bromide with a diethylamino-group and subsequent quaternisation of the N atom in amines 8a–d with o-nitrile benzyl bromide (9). To produce non-ionic bisuracils 3a–d, it is necessary to amine bromide 4 not with diethylamine as in the case of charged bisuracil 2a–d preparation, but with ethylamine, resulting in the isolation of amines 10a–d. Finally, the targeted non-ionic bisuracils 3a–d were prepared via the alkylation of the secondary N atoms in the pentamethylene chains of amines 10a–d with bromide 9 (Scheme 2).

2.2. Inhibitory Effects on Cholinesterases of Counterparts 2 and 3 In Vitro

The inhibitory activities of bisuracils 2a–d and 3a–d against human AChE and BuChE for the 50% inhibitory concentration (IC₅₀) values and their selectivity indexes for AChE over BuChE are presented in

Table 1. In vitro inhibition of recombinant human AChE and BChE from human plasma by ionic bisuracils 1 and 2a–d, varied polymethylene chains and substituents at benzene rings, and pyridostigmine bromide.

Compound	IC50 [nM]		AChE Selectivity 1
	AChE	BuChE
1a 2	0.4 ± 0.03	40,000 ± 380	112,676
1b 2	0.1 ± 0.01	6000 ± 430	60,000
1c 2	2.0 ± 0.2	440,000 ± 10,000	220,000
1d 2	1.0 ± 0.1	200,000 ± 1500	200,000
1e 2	0.5 ± 0.03	60,000 ± 1470	120,000
1f 2	0.2 ± 0.01	40,000 ± 1600	200,000
2a	0.3 ± 0.03	370,000 ± 27,000	1,258,503
2b	0.06 ± 0.003	210,00 ± 50,000	3,374,422
2c	0.2 ± 0.06	70,000 ± 100	388,888
2d	0.5 ± 0.16	30,000 ± 500	57,034
pyridostigmine bromide	350 ± 20	1000 ± 130	2.9

¹ (IC₅₀ BuChE)/(IC₅₀ AChE); ² for bisuracils 1a–f, the data given in reference [20] were used.

and

Table 2. In vitro inhibition of human AChE and BuChE by non-ionic bisuracils 3a–d with varied polymethylene chains, in comparison to donepezil hydrochloride.

Compound	IC50 [nM]		AChE Selectivity 1
	AChE	BuChE
3a	6.1 ± 0.7	69,100 ± 1000	11,290
3b	7.9 ± 0.25	48,300 ± 3000	6090
3c	2.8 ± 0.5	28,000 ± 1000	10,071
3d	5.4 ± 0.7	95,000 ± 1100	17,639
Donepezil	11.3 ± 0.1	5260 ± 270	465.5

¹ (IC₅₀ BuChE)/(IC₅₀ AChE).

, respectively. In order to facilitate comparison of the data obtained for the new nitrile bisuracils 2a–d with those for bisuracils with trifluoromethyl-l and nitro-groups, Table 1 provides a summary of the data published in an earlier report on the most active inhibitors from the series of ionic bisuracils 1a–f [20]. The myasthenia drug pyridostigmine bromide for the charged AChE inhibitors in Table 1 and the AD drug donepezil for the non-ionic AChE inhibitors in Table 2 were used as reference drugs.

Ionic bisuracils 1a–f have been shown to be highly effective and selective inhibitors of AChE, with the ability to inhibit the enzyme at picomolar or nanomolar concentrations. Additionally, these compounds have been observed to exhibit increased selectivity towards AChE compared to BuChE by more than 200,000 times. Bisuracils with CF₃-substituents at benzene rings demonstrate greater selectivity towards AChE than bisuracils with NO₂ substituents. In addition to the extreme selectivity towards AChE of bisuracils with CF₃ and NO₂ substituents, the introduction of nitrile substituents into the benzyl moiety further reduces IC₅₀ values towards AChE and increases the selectivity for AChE vs. BChE. Thus, bisuracils 2a–d with nitrile groups inhibit AChE at picomolar concentrations with selectivity for AChE over BuChE exceeding that of bisuracils with CF₃ and NO₂ groups by more than an order of magnitude. The reference drug inhibits AChE at a concentration hundreds of times higher than that of bisuracils and with a selectivity for AChE vs. BuChE of about three. The following can be observed in the change in inhibitory activity against AChE in the series of bisuracils 2a–d. When the number of methylene groups in the spacer between 3,6-dimethyluracil moieties increased from three to four, the inhibitory activity against AChE and selectivity of AChE vs. BuChE increased. However, a lengthening of the spacer from four to five methylene groups, and further from five to six methylene groups, led to a decrease in inhibitory activity and selectivity. The bisuracils 2a,b with three and four methylene groups in the spacer between 3,6-dimethyl and uracil moieties are unprecedentedly selective inhibitors of AChE vs. BuChE, with a selectivity of more than one million for bisuracil 2a and more than three million for bisuracil 2b (Table 1). To our knowledge, no AChE inhibitors with such extraordinary selectivity for the enzyme have previously been discovered.

Non-ionic bisuracils 3a–d exhibit an inhibitory effect against AChE, with an IC₅₀ more than an order of magnitude higher than that of ionic counterparts 2a–d (Table 2). Meanwhile, their selectivity for AChE vs. BuChE is one to two orders of magnitude lower than the selectivity of bisuracils 2a–d. Like bisuracils 2a–d, as well as inhibiting AChE at nanomolar concentrations, the reference drug donepezil demonstrates more than an order of magnitude lower selectivity for AChE. The moderate 50-fold selectivity of galantamine for AChE over BuChE [24] and the non-selective inhibition profile of rivastigmine [25] sharply contrasts with the exceptional more than 10,000-fold AChE selectivity exhibited by bisuracils 3c,d. This pronounced selectivity may significantly reduce off-target effects compared to conventional cholinesterase inhibitors.

In the series of non-ionic AChE inhibitors 3a–d, there is no clear relationship between the number of methylene groups in the bridge between the uracil moieties and the IC₅₀ or the selectivity with respect to AChE. For bisuracils 3a–d, with a number of methylene groups in the bridge from three to six, IC₅₀ changes in the range of 2.8–7.9 nM, while the selectivity against AChE vs. BChE changes from 6090 to 17,639 (Table 2).

Based on the data from the study of inhibitory activity in a series of ionic and non-ionic AChE inhibitors, bisuracils 2b and 3c were selected as lead compounds. Exhibiting the lowest values of IC₅₀ and the highest selectivity for AChE in comparison with BuChE, this series of compounds also offers better prospects for AChE inhibition in vivo.

In order to study the mechanism of AChE and BuChE inhibition by bisuracils 2b and 3c, the inhibition constants (K_i) were determined. Experiments were performed using four different concentrations of ATCh as a substrate for AChE, and with BuTCh as a substrate for BuChE. According to the analysis of Dixon and Cornish–Bowden plots for bisuracil 2b, AChE and BuChE inhibition is of a mixed type characterised both by competitive inhibition (K_ci) and uncompetitive inhibition (K_ui) components. For AChE, K_ci was determined to be 55.7 ± 8.1 pM while K_ui = 156 ± 18 pM (Figure 3A,B), and K_ci = 0.13 ± 0.02 mM while K_ui = 0.7 ± 0.06 mM for BuChE (Figure 3C,D). Inhibition of AChE and BuChE by bisuracil 3c was of a mixed type. For the AChE constant, K_ci was 0.7 ± 0.04 nM while K_ui 4.45 ± 1.76 nM (Figure 4A,B); for the BuChE constant, K_ci = 0.04 ± 0.007 мM while K_ui = 0.68 ± 0.08 мM (Figure 4C,D).

2.3. In Vivo Biological Assays

2.3.1. Acute Toxicity

The median lethal doses (value of LD₅₀) for mice of the lead compounds were calculated to be 5.16 mg/kg (95% confidence limits: 3.68–6.94 mg/kg) for bisuracil 2b and 22.8 mg/kg (95% confidence limits: 15.04–28.73 mg/kg) for bisuracil 3c.

2.3.2. Pre-Treatment of Poisoning with OPs

The concept of using reversible AChE inhibition as a preventive strategy to enhance the efficacy of traditional antidotes in OP poisoning is a well-established principle [5,26]. This study explores the potential of bisuracil 2b as a preventive agent to protect AChE from irreversible inhibition caused by paraoxon (POX), a model OP. Mice were pre-treated with bisuracil 2b via intraperitoneal (i.p.) injection at different doses 30 min before being exposed to 2xLD₅₀ POX (0.45 mg/kg). Following POX administration, the standard antidote atropine was given at a dose of 15 mg/kg within one minute. The protective effects of bisuracil 2b are summarised in

Table 3. Selection of bisuracil 2b dose for pre-treatment of mice poisoned with 2×LD50 of POX.

Group	n/N *
POX	0/12
POX + Atropine	6/12
Bisuracil 2b - 1 mg/kg + POX + Atropine	4/12
Bisuracil 2b - 0.5 mg/kg + POX + Atropine	4/12
Bisuracil 2b - 0.1 mg/kg + POX + Atropine	5/12
Bisuracil 2b - 0.05 mg/kg + POX + Atropine	7/12
Bisuracil 2b - 0.01 mg/kg + POX + Atropine	10/12
Bisuracil 2b - 0.005 mg/kg + POX + Atropine	8/12

* n—number of mice surviving 5 days after POX poisoning; N—total number of mice in the group. POX—0.42 mg/kg; IP; bisuracil 2b was i.p. administrated 30 min before POX; atropine at the dose 15 mg/kg was i.p. administrated 1 min after POX.

. The most effective protective effect of bisuracil 2b was observed with a dose of 0.01 mg/kg at which 10 out of 12 mice survived. Increasing the dose of bisuracil 2b reduced the protective effect in POX poisoned animals (Table 3).

We also estimated the relative risk of death (RRD) as a function of precocity (from min to hours) over a period of 10 h by Cox survival analysis [27]. Statistical differences in the number of surviving animals between experimental groups were tested using the Cox–Mantel multisample logarithmic rank criterion [27,28]. It was shown that, in mice exposed to 0.42 mg/kg of POX, RRD = 1 (Figure 5). However, in animals that received atropine within 1 min after poisoning, POX-induced mortality was lower (RRD = 0.5; Figure 5).

In mice pre-treated with bisuracil 2b at a dose of 0.01 mg/kg, the RRD significantly decreased as compared to the poisoned group treated only with atropine (p = 0.02). However, further increases in the dose of bisuracil 2b did not result in a reduction in the RRD (Figure 5). Therefore, it can be concluded that bisuracil 2b significantly reduced mortality in mice at the dose of 0.01 mg/kg.

2.3.3. Inhibition of Brain AChE

Bisuracil 3c was identified as the most promising for the inhibition of brain AChE in vivo. To evaluate its inhibitory potential, we measured AChE activity in mouse brain homogenates at 30 min after i.p. injection of 3c at doses of 0.5 LD₅₀ (10 mg/kg) and 0.25 LD₅₀ (5 mg/kg). Bisuracil 3c was observed to inhibit AChE activity in the brain by 54.2 ± 6.6% at a dose of 10 mg/kg and 14.9 ± 3.8% at a dose of 5 mg/kg as compared to the mean AChE activity in the brains of control mice. Consequently, 3c has been demonstrated to effectively inhibit brain AChE, rendering it a promising candidate for the treatment of memory impairment associated with AD.

2.3.4. Behavioural Test

This study explored the ability of bisuracil 3c to counteract scopolamine-induced amnesia in mice, utilising behavioural assessments conducted through the novel object recognition test. The test operates on the principle that animals demonstrating a significant preference for exploring new objects, as opposed to familiar ones, can be considered evidence of intact recognition memory [29]. The induction of amnesia was facilitated by the i.p. injection of scopolamine (1.5 mg/kg) 50 min prior to testing. Subsequently, donepezil (1 mg/kg, i.p.) or bisuracil 3c (5 or 2 mg/kg, i.p.) was administered 30 min prior to testing. An equivalent amount of the vehicle was administered to a control group of mice.

It was shown that the mice in the control group preferred a novel object over a familiar object with a probability of 66.0 ± 2.9%. Administration of scopolamine at a dose of 1.5 mg/kg significantly reduced the preference for the novel object by 34.1% as compared to the control group (p = 0.0001). In contrast, the administration of bisuracil 3c at a dose of 5 mg/kg served to prevent scopolamine-induced memory impairment. It was demonstrated that the administration of bisuracil 3c resulted in a significant 30.5% increase in the preference for the novel object compared to the scopolamine-treated group (p = 0.01) (Figure 6). Concurrently, the values did not deviate from those of the control group of animals (p = 0.975). The administration of donepezil at a dose of 1 mg/kg similarly resulted in a significant 32.2% (p = 0.03) increase in the preference for the novel object over the familiar one as compared to the group receiving scopolamine. Notably, bisuracil 3c at a dose of 2 mg/kg had no significant effect on the preference index (Figure 6).

Thus, memory deficits were improved when either compound bisuracil 3c at a dose of 5 mg/kg or donepezil at a dose of 1 mg/kg was administered.

2.3.5. Effect on the Level of Locomotor Activity in Mice

The ability of effective doses of bisuracil 2b (0.01 mg/kg) and bisuracil 3c (5 mg/kg) to induce incapacitation was evaluated. Locomotor activity, measured as the total distance travelled, and exploratory behaviour, assessed by the number of rearings (head inclinations) and head dips, were evaluated using the open-field test. Additionally, motor coordination was analysed using the rotarod test, where the ability of mice to stay on the rotating rod was recorded. The compounds were administered i.p. 30 min before testing, while the control group received an equivalent volume of the vehicle.

The results showed that both groups of 2b- and 3c-treated mice explored the open field without any reduction in locomotor activity. As a result, the distance travelled by mice in both experimental groups was comparable to that of the control group, showing no significant differences (Figure 7). Furthermore, this study revealed that neither of the tested compounds significantly influenced the number of rearings or head dips (Figure 7B,C). The rotarod test results indicated that mice treated with bisuracils 2b and 3c exhibited no signs of motor impairment. The mean fall time of mice receiving 2b (0.01 mg/kg, i.p.) was 124 ± 20 s, which was not significantly different from the control group, whose mean fall time was 173 ± 21 s (p = 0.125) (Figure 7D). A similar outcome was observed in mice administered 3c at the maximum dose (5 mg/kg, i.p.), whose mean fall time was 170 ± 19 s, which was not significantly different from the control group, whose mean fall time was 154 ± 20 s (p = 0.137) (Figure 7D).

Consequently, the open-field and rotarod tests showed no impairments in locomotor or exploratory activity in mice following exposure to both bisuracils 2b and 3c. The results indicated that the tested compounds did not induce negative effects on movement coordination and stereotyped exploratory behaviour in mice.

2.4. Computational Modelling

2.4.1. Molecular Docking and Molecular Dynamics

Molecular docking and molecular dynamics (MD) simulations were performed to compare docking scores and ligand–receptor interactions [30,31]. This analysis aims to provide deeper insights into the structure–activity relationships of bisuracils, thereby guiding subsequent structural optimisation efforts and facilitating the identification of superior lead compounds. For this reason, we used MOE for molecular docking simulation of the lead compounds 2b and 3c. The docking scoring results are shown in

Table 4. Docking score of compounds and human AChE or BuChE (kcal/mol).

Compound	AChE (PDB: 7E3H)	BuChE (PDB: 6ESY)
Donepezil	−8.2999	−5.8870
2b	−10.5200	−6.5570
3c	−10.0700	−7.1540

.

To evaluate the binding mode and interaction between bisuracils 2b, 3c, and AChE, the title compounds were docked into the crystal structure of hAChE complexed with donepezil (PDB: 7E3H). The detailed interactions between ligands and receptors are shown in

Table 5. Interactions between bisuracils 2b and 3c and human acetylcholinesterase by molecular docking of the bisuracils into the crystal structure of hAChE complexed with donepezil (PDB: 7E3H).

Compound	Interactions	hAChE Residues	Distance (Å)	E (kcal/mol)
	H-donor	Ser 293	3.40	−0.4
	H-acceptor	Val 294	3.30	−0.3
	H-acceptor	Phe 295	3.08	−2.4
	H-pi	Trp 286	3.94	−0.2
	H-pi	Trp 286	3.69	−0.3
	H-pi	Tyr 341	3.93	−0.5
	H-pi	Tyr 337	4.27	−0.2
	pi-H	Tyr 341	3.92	−0.2
	pi-H	His 447	3.46	−0.5
	H-donor	Asp 74	3.38	−0.7
	H-acceptor	Tyr 133	2.79	−2.1
	H-pi	Tyr 337	4.29	−0.2
	H-pi	Tyr 337	4.78	−0.2
	H-pi	Trp 286	3.95	−0.3
	pi-H	Trp 86	4.12	−0.5
	pi-H	Trp 86	3.73	−0.2
	pi-pi	Tyr 341	3.48	0

. Figure 8A shows a possible binding mode and binding site for bisuracil 2b (IC₅₀ = 64.9 pM), while Figure 8B indicates the interaction between bisuracil 2b and receptor residues. Bisuracil 2b forms close interactions with residues Ser293, Val294, and Phe295 through three hydrogen bonds; C5′H acts as a hydrogen bond donor forming a hydrogen bond with Ser293 (distance = 3.40 Å), while carbonyl oxygen in C4′=O of pyrimidine ring acts as a hydrogen bond acceptor interacting with Val294 and Phe295 (distances = 3.30 Å, 3.08 Å), respectively. Bisuracil 2b also has extensive H-pi interactions between C10H, C10′H, and the ethyl group at quaternised N with residues Trp286, Tyr341, and Tyr337, while C10H interacts twice with Trp286 (distance = 3.94 Å, 3.69 Å, 3.93 Å, and 4.27 Å, respectively), and uracil and benzene rings interact with Tyr341 and His447 by pi-H interaction (distance = 3.92 Å, 3.46 Å). The 3,6-dimethyluracil cycle of 2b extends into the solvent space outside the binding pocket to interact with the solvent. Moreover, bisuracil 2b, which occupies PAS, has strong interactions with key residues Trp286, Tyr341, and Phe295. Furthermore, 2b interacts with His447 of the CAS catalytic triad, as well as residues of the anion-binding hydrophobic sites Tyr341 and Tyr337. These unique interactions play a crucial role in its excellent activity.

In comparison with charged bisuracil 2b, Figure 8C demonstrates a possible binding mode and binding site for non-ionic bisuracil 3c (IC₅₀ = 2.78 nM), while Figure 8D indicates the interaction between 3c and receptor amino acid residues. Bisuracil 3c forms interactions with residues Asp74 and Tyr133 through hydrogen bonds. C7′H acts as a hydrogen bond donor forming a hydrogen bond with Asp74 (distance = 3.38 Å), while C≡N acts as a hydrogen bond acceptor interacting with Tyr133 (distances = 2.79Å). In addition, bisuracil 3c also has extensive H-pi interactions of C5′H, C9′H, and C7H with residues Tyr337, Tyr341, and Trp286 (distance = 4.29 Å, 4.78 Å, and 3.95 Å, respectively); the benzene ring interacts twice with Trp86 by pi-H interaction (distance = 3.73 Å, 4.12 Å) and with residues Tyr341 by pi-pi interaction (distance = 3.48 Å). The 3,6-dimethyluracil cycle of 3c extends into the solvent space outside the binding pocket and interacts with the solvent.

According to the results of molecular docking, the flexible polymethylene chain allows the title bisuracils to bind both key sites CAS and PAS of AChE. The aromatic ring mainly interacts with the CAS site, while the uracil ring and pentamethylene chain favour interactions with the key residues of the PAS site. The 3,6-dimethyluracil cycle extends outward to interact with solvents. This unique binding mode enables this kind of bisuracil to exhibit excellent AChE inhibitory activity.

Mixed-type inhibition suggests simultaneous binding at CAS and PAS, corroborated by docking (Figure 8A–D). This dual binding may sterically hinder substrate access, enhancing inhibition.

In order to explore the details of the 2b and 3c title compounds binding to cholinesterases and further structural modifications, two series of representative bisuracils 2b and 3c were subjected to 100 ns MD simulations with AChE and BuChE, respectively. The root-mean-square deviations (RMSDs) of the bisuracils 2b and 3c were calculated to evaluate the stability and convergence of the titled bisuracil AChE and BuChE complex during the MD simulations (Figure 9 and Figure 10).

Figure 10 illustrates the critical role played by the quaternary ammonium moiety and terminal 3,6-dimethyluracil cycle in the binding process; the carbonyl group on the central uracil ring also significantly contributes to the interaction. The RMSD analysis from MD simulations provided insights into the stability of bisuracils 2b and 3c binding to AChE and BuChE. For both compounds, the trajectory fluctuations were notably smaller when binding to AChE than those observed for BuChE (Figure 9 and Figure 10). This characteristic allowed them to more rapidly reach a steady state, as well as maintaining a more stable binding mode when interacting with AChE. Notably, bisuracil 2b exhibits superior stability compared to compound 3c when bound to AChE. The smoother trajectory of 2b assumes a more consistent and durable interaction within the enzyme’s active site. This enhanced stability was corroborated by its excellent performance in molecular docking studies, which further rationalised its superior activity relative to bisuracil 3c. The significant difference in the binding stability between AChE and BuChE for 2b and 3c underscored the structural distinctions between these enzymes. Bisuracil 2b’s pronounced selectivity for AChE over BuChE can be attributed to its optimal fit within the active site of AChE, resulting in higher affinity and lower IC₅₀ values.

2.4.2. ADMET Prediction

ADMET refers to the Absorption, Distribution, Metabolism, Excretion, and Toxicity of compounds in the human body. The ADMET prediction results (

Table 6. Prediction of ADMET properties of synthesised compounds.

Compd.	logP a	Solubility b	pKa c	BBB d	T1/2 e	Acute Oral Toxicity on Rodents f
2a	2.37	−4.15	8.80	0.59	0.12	0.26
2b	2.76	−4.41	8.82	0.59	0.11	0.32
2c	3.15	−4.67	8.84	0.59	0.11	0.35
2d	3.54	−4.93	8.84	0.59	0.11	0.34
3a	1.83	−3.71	7.77	0.72	0.32	0.14
3b	2.22	−3.97	7.71	0.72	0.34	0.17
3c	2.61	−4.23	7.59	0.72	0.37	0.21
3d	3.00	−4.49	7.47	0.72	0.40	0.27

^a LogP value is the ratio of the concentration of a compound in n-octanol and water at equilibrium, and logP is the logarithm (base 10) of the calculated coefficient. It is a measure of a compound’s hydrophilicity. ^b Water solubility logarithm (base 10) of the molar solubility of a compound in water (mol/L). The larger the number, the higher the water solubility. When the number is less than −6, it can be assumed that the compound has poor water solubility. ^c Acid dissociation constant (pK_a) represents the ability of an acid to dissociate hydrogen ions. The smaller the pK_a of an acid, the more acidic it is and the more likely it is to be able to grow in complete dissociation in water. ^d Blood–brain-barrier permeant (BBB) determines whether a compound can enter the brain. The result is a real number between 0 and 1. The closer the number is to 1, the higher the probability of the compound passing through the blood–brain barrier. ^e The half-life (T_1/2) of a compound in the body is the time it takes for its concentration to fall to 1/2 of its initial value, with a short half-life (< 3 h) generally indicating a shorter therapeutic time window, higher toxicity, and more frequent dosing. The result is a real number of 0–1. The closer the number is to 1, the higher the probability that the half-life of the compound is greater than 3 h. ^f LD₅₀: Dose that causes half of the animals to die in acute oral toxicity tests on rodents within a certain period of time. The result is a real number between 0 and 1. The closer the number is to 1, the higher the probability that the compound will induce an acute toxic reaction in rodents. If the compound’s LD₅₀ for rodents is less than 500 mg/kg, it is recorded as 1; otherwise, it is recorded as 0.

) indicated that synthesised bisuracils 2a–d and 3a–d have relatively good safety profiles, with a low probability of acute oral toxicity, and possess good lipophilicity and transmembrane ability, which enables them to penetrate the blood–brain barrier effectively and exert their effects in the brain.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

The NMR experiments were carried out on Bruker spectrometers AVANCE-400 (400.1 MHz (¹H), 100.6 MHz (¹³C)). Chemical shifts (δ in ppm) were referenced to the solvents DMSO-d₆ (δ = 2.50 ppm for ¹H and 40.0 ppm for ¹³C NMR) and CDCl₃ (δ = 7.26 ppm for ¹H and 77.0 ppm for ¹³C NMR). MALDI-TOF mass spectra were recorded on a Bruker ULTRAFLEX III mass spectrometer using p-nitroaniline or 2,5-dihydroxybenzoic acid as a matrix, and conditions of mass spectrum recording: Nd:YAG laser, λ = 355 nm, repetition rate 100 Hz, and linear mode with the registration of positively or negatively charged ions. Microanalyses of C, H, and N were performed with a CHNS analyser Vario Macro cube (Elementar Analysensysteme GmbH, Germany); they were within ±0.3% of theoretical values for C, H, and N. Thin-layer chromatography was performed on Silufol-254 plates (the solvent system is diethyl ether or an ethylacetate–methanol mixture); visualisation of spots was carried out under UV light (λ = 254 nm). For column chromatography, silica gel of 60 mesh from Fluka was used. All solvents were dried according to standard protocols.

Procedures for the preparation of α,ω-{(3,6-dimethyluracil-1-yl)-[3-(5-bromopentyl)uracil-1-yl]}alkanes (4a–d) and α,ω-{(3,6-dimethyluracil-1-yl)-[3-(5-diethylaminopentyl)uracil-1-yl]}alkanes (8a–d) from 4a–d were as described previously [20].

3.1.2. Synthesis of Charged Bisuracils with Nitrile Substituent

General Procedure to Obtain Bisuracils 2a–d

A solution of amine 8a–d (1.0 mmol) and 1.1-fold excess o-nitrilebenzyl bromide 9 in MeCN (30 mL) was refluxed for 16 h at 60–65 °C. The solvent was distilled off and the residue was triturated in anhydrous diethyl ether (2 × 30 mL), each time decanted after settling of the compound. Water (70 mL) and activated carbon (0.1–0.15 g, powder) were added to the residue. The suspension was stirred for 1 h at 35–40 °C and filtered after cooling to room temperature. The solvent was evaporated, and the residue was dissolved in CHCl3 (200 mL) and dried with MgSO₄ to afford the titled bisuracil as a hygroscopic light brown powder with an unclear melting point in the temperature range of 40–60 °C.

1,3-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(diethyl)ammonio)pentyl)-uracil-1-yl]}propane bromide (2a): 0.42 g, 67%; ¹H NMR (CDCl₃, 400 MHz) δ 8.32 (d, J = 7.6 Hz, 1H, C3′′H), 7.83–7.79 (m, 2H, C4′′H, C5′′H), 7.68–7.65 (m, 1H, C6′′H), 7.38 (d, J = 7.6 Hz,1H, C6′H), 5.72 (d, J = 7.6 Hz, 1H, C5′H), 5.63 (s, 1H, C5H), 5.17 (br. s, 2H, NCH₂Ph), 3.94–3.86 (m, 6H, C7H₂, C9H₂, C7′CH₂), 3.72–3.70 (m, 4H, 2NCH₂), 3.55 (m, 2H, C11′H₂), 3.29 (s, 3H, N3CH₃), 2.25 (s, 3H, C6CH₃), 2.09 (m, 2H, C8H₂), 1.85 (m, 2H, C10′H₂), 1.69 (m, 2H, C8′H₂), 1.25–1.22 (m, 8H, C9′H₂, 2CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 163.0 (C′4), 162.2 (C4), 152.7 (C6′), 152.4 (C6), 151.4 (C2), 150.8 (C2′), 142.7 (C2′′), 135.5 (C5′′), 134.3 (C1′′), 133.9 (C6′′), 131.5 (C4′′), 130.9 (C3′′), 101.9, 101.7 (C5′, C5), 61.2 (C11′), 59.2 (NCH₂Ph), 55.2 (NCH₂CH₃), 47.5 (C9), 42.3 (C7), 40.0 (C7′), 28.5 (C10′), 27.9 (N3CH₃), 26.6 (C8′), 23.4 (C8), 22.2 (C9′), 19.9 (C6CH₃), 9.0 (NCH₂CH₃); MALDI-MS [M − H]⁺, [M − Br + H]⁺ calcd for C₃₀H₄₁BrN₆O₄ m/z 627.2, 549.3, respectively. Found: 627.2, 549.3. Anal. calcd for C₃₀H₄₁BrN₆O₄: C, 57.23; H, 6.56; Br, 12.69; N, 13.35. Found: C, 57.31; H, 6.46; Br, 12.81; N, 13.28 (Figures S1–S3).

1,4-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(diethyl)ammonio)pentyl)-uracil-1-yl]}butane bromide (2b): 0.46 g, 71%; ¹H NMR (CDCl₃, 400 MHz) δ 8.17–8.14 (m, 1H, C3′′H), 7.83–7.80 (m, 2H, C4′′H, C5′′H), 7.70–7.67 (m, 1H, C6′′H), 7.39 (d, J = 6.0 Hz,1H, C6′H), 5.68 (d, J = 7.6 Hz, 1H, C5′H), 5.58 (s, 1H, C5H), 5.17 (br. s, 2H, NCH₂Ph), 3.89–3.82 (m, 6H, C7H₂, C10H₂, C7′CH₂), 3.65 (m, 4H, 2NCH₂), 3.46 (m, 2H, C11′H₂), 3.27 (s, 3H, N3CH₃), 2.26 (s, 3H, C6CH₃), 1.87 (m, 2H, C10′H₂), 1.75–1.68 (m, 6H, C8H₂, C9H₂, C8′H₂), 1.48–1.41 (m, 8H, C9′H₂, 2CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.8 (C′4), 160.0 (C4), 152.0 (C6′), 151.1 (C6), 150.9 (C2), 150.8 (C2′), 142.8 (C2′′), 134.8 (C5′′), 134.0 (C1′′), 133.8 (C6′′), 131.3 (C4′′), 130.0 (C3′′), 101.3, 101.2 (C5′, C5), 60.5 (C11′), 58.7 (NCH₂Ph), 54.8 (NCH₂CH₃), 46.6 (C10), 44.1 (C7), 39.7 (C7′), 27.5 (C10′), 25.8 (N3CH₃), 26.6 (C8′), 23.6, 23.2 (C8, C9), 22.3 (C9′), 19.6 (C6CH₃), 8.7, 8.5 (NCH₂CH₃); MALDI-MS [M − H]⁺, [M − Br + H]⁺ calcd for C₃₁H₄₃BrN₆O₄ m/z 641.3, 563.3, respectively. Found: 641.3, 563.4. Anal. calcd for C₃₁H₄₃BrN₆O₄: C, 57.85; H, 6.73; Br, 12.41; N, 13.06. Found: C, 57.71; H, 6.70; Br, 12.31; N, 13.18 (Figures S4–S6).

1,5-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(diethyl)ammonio)pentyl)-uracil-1-yl]}pentane bromide (2c): 0.55 g, 84%; ¹H NMR (CDCl₃, 400 MHz) δ 8.26 (m, 1H, C3′′H), 7.78–7.75 (m, 2H, C4′′H, C5′′H), 7.65–7.62 (m, 1H, C6′′H), 7.20 (d, J = 6.4 Hz,1H, C6′H), 5.67 (d, J = 7.4 Hz, 1H, C5′H), 5.57 (s, 1H, C5H), 5.14 (br. s, 2H, NCH₂Ph), 3.88 (m, 2H, C7′H₂), 3.77–3.69 (m, 8H, C11H₂, C7CH₂, 2NCH₂), 3.48 (m, 2H, C11′H₂), 3.26 (s, 3H, N3CH₃), 2.23 (s, 3H, C6CH₃), 1.81 (m, 2H, C10′H₂), 1.67 (m, 6H, C8H₂, C10H₂, C8′H₂), 1.48–1.41 (m, 10H, C9H₂, C9′H₂, 2CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 163.1 (C′4), 162.3 (C4), 152.2 (C6′), 151.3 (C6), 151.1 (C2, C2′), 142.6 (C2′′), 135.5 (C5′′), 134.2 (C1′′), 131.4 (C6′′), 130.9 (C4′′, C3′′), 101.4 (C5′, C5), 61.1 (C11′), 59.1 (NCH₂Ph), 55.2 (NCH₂CH₃), 49.4 (C11), 44.7 (C7), 39.9 (C7′), 28.4 (C10′), 28.2, 27.7 (C8, C8′), 26.6 (C10, N3CH₃), 23.5 (C9′), 22.2 (C9), 19.8 (C6CH₃), 9.0 (NCH₂CH₃); MALDI-MS [M − Br]⁺ calcd for C₃₂H₄₅BrN₆O₄ m/z 577.4, respectively. Found: 577.4. Anal. calcd for C₃₂H₄₅BrN₆O₄: C, 58.44; H, 6.90; Br, 12.15; N, 12.78. Found: C, 58.35; H, 7.00; Br, 12.06; N, 12.83 (Figures S7–S9).

1,6-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(diethyl)ammonio)pentyl)-uracil-1-yl]}hexane bromide (2d): 0.60 g, 90%; ¹H NMR (CDCl₃, 400 MHz) δ 8.37–8.35 (d, J = 7.6 Hz, 1H, C3′′H), 7.83–7.78 (m, 2H, C4′′H, C5′′H), 7.66–7.63 (m, 1H, C6′′H), 7.16–7.14 (d, J = 8.4 Hz,1H, C6′H), 5.72–5.69 (d, J = 7.6 Hz, 1H, C5′H), 5.62 (s, 1H, C5H), 5.18 (br. s, 2H, NCH₂Ph), 3.92 (m, 2H, C7′H₂), 3.82–3.78 (m, 2H, C12H₂), 3.73 (m, 6H, C7CH₂, 2NCH₂), 3.54 (m, 2H, C11′H₂), 3.31 (s, 3H, N3CH₃), 2.24 (s, 3H, C6CH₃), 1.82 (m, 2H, C10′H₂), 1.70 (m, 6H, C8H₂, C11H₂, C8′H₂), 1.48–1.41 (m, 12H, C9H₂, C10H₂, C9′H₂, 2CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.9 (C′4), 162.4 (C4), 152.3 (C6′), 151.3 (C6), 150.9 (C2, C2′′), 142.1 (C2′), 137.7 (C5′′), 134.4 (C1′′), 132.2 (C6′′), 125.8 (C4′′), 122.1 (C3′′), 101.7, 101.6 (C5′, C5), 59.0 (C11′), 58.7 (NCH₂Ph), 55.3 (NCH₂CH₃), 49.6 (C7), 46.6, 45.0 (C12, C7′), 28.9 (C10′), 27.8 (N3CH₃), 27.1 (C8′), 26.4 (C9′), 26.3, 26.0 (C8, C11), 23.0, 22.9 (C9, C10), 19.8 (C6CH₃), 9.0, 8.6 (NCH₂CH₃); MALDI-MS [M − H]⁺, [M − Br + H]⁺ calcd for C₃₃H₄₇BrN₆O₄ m/z 669.3, 591.4, respectively. Found: 669.3, 591.4. Anal. calcd for C₃₃H₄₇BrN₆O₄: C, 59.01; H, 7.05; Br, 11.90; N, 12.51. Found: C, 58.95; H, 7.12; Br, 12.02; N, 12.60 (Figures S10–S12).

3.1.3. Synthesis of Non-Ionic Bisuracils with Nitrile Substituent

General Procedure to Replace Atom of Br in Bromides 4a–d with Ethylamino-Group

A 20% EtNH₂ solution in i-PrOH (2.5 mL, ca. 11.0 mmol) was added to the mixture of bromide 4a–d (2.0 mmol) and potassium carbonate (0.28 g, 2.0 mmol) in MeCN (50 mL), and the mixture was stirred at 35–40 °C for 8 h. A precipitate was filtered out, and the solvent was evaporated in vacuo to afford the targeted amine as a clear light yellow oil.

1,3-{(3,6-Dimethyluracil-1-yl)-[3-(5-ethylaminopentyl)uracil-1-yl]}propane (10a): 0.77 g, 95%; ¹H NMR (CDCl₃, 400 MHz) δ 7.20 (d, J = 6.4 Hz, 1H, C6′H), 5.70 (d, J = 7.2 Hz, 1H, C5′H), 5.58 (s, 1H, C5H), 3.89–3.84 (m, 6H, C7′H₂, C9H₂), 3.80–3.77 (m, 2H, C7H₂), 3.27 (s, 3H, N3CH₃), 2.63–2.56 (m, 4H, C11′H₂, NCH₂), 2.24 (s, 1H, NH), 2.21 (s, 3H, C6CH₃), 2.07–2.01 (m, 2H, C8H₂), 1.62–1.56 (m, 2H, C8′H₂); 1.54–1.48 (m, 2H, C10′H₂), 1.37–1.30 (m, 2H, C9′H₂), 1.09–1.06 (t, J = 6.0 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.7 (C′4), 162.0 (C4), 154.0 (C6′), 152.3 (C6), 150.4 (C2), 141.9 (C2′), 102.0 (C5′, C5), 49.3 (C11′), 47.3 (NCH₂CH₃), 43.9, 42.1 (C7, C9), 41.1 (C7′), 29.4 (C10′), 28.5 (C8′), 27.8 (N3CH₃), 27.3 (C8), 24.6 (C9′), 19.5 (C6CH₃), 14.9 (NCH₂CH₃); ESI-MS [M + H]⁺ calcd for C₂₀H₃₁N₅O₄ m/z 406.3. Found: 406.3. Anal. calcd for C₂₀H₃₁N₅O₄: C, 59.24; H, 7.71; N, 17.27. Found: C, 59.14; H, 7.62; N, 17.21 ((Figures S13–S15).

1,4-{(3,6-Dimethyluracil-1-yl)-[3-(5-ethylaminopentyl)uracil-1-yl]}butane (10b): 0.81 g, 96%; ¹H NMR (CDCl₃, 400 MHz) δ 7.13 (d, J = 6.4 Hz, 1H, C6′H), 5.67 (d, J = 6.0 Hz, 1H, C5′H), 5.57 (s, 1H, C5H), 3.88–3.85 (t, J = 6.0 Hz, 2H, C7′H₂), 3.84–3.81 (t, J = 6.0 Hz, 1H, C10H₂), 3.76–3.73 (t, J = 5.6 Hz, 2H, C7H₂), 3.26 (s, 3H, N3CH₃), 2.71–2.59 (m, 5H, C11′H₂, NCH₂, NH), 2.21 (s, 1H, NH), 2.21 (s, 3H, C6CH₃), 1.75–1.52 (m, 8H, C8H₂, C8′H₂, C9H₂, C10′H₂), 1.36–1.30 (m, 2H, C9′H₂), 1.11–1.08 (t, J = 5.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.9 (C′4), 162.2 (C4), 152.3 (C6′), 151.3 (C6), 150.7 (C2), 142.7 (C2′), 101.8 (C5′, C5), 49.0 (C11′), 44.2 (NCH₂CH₃), 43.7, 41.0 (C7, C10, C7′), 28.9 (C10′), 27.7 (C8′), 27.2 (N3CH₃), 26.0, 25.7 (C8, C9), 24.5 (C9′), 19.6 (C6CH₃), 14.4 (NCH₂CH₃); MALDI-MS [M + H]⁺ calcd for C₂₁H₃₃N₅O₄ m/z 420.3. Found: 420.1. Anal. calcd for C₂₁H₃₃N₅O₄: C, 60.12; H, 7.93; N, 16.69. Found: C, 60.18; H, 8.00; N, 16.58 (Figures S16–S18).

1,5-{(3,6-Dimethyluracil-1-yl)-[3-(5-ethylaminopentyl)uracil-1-yl]}pentane (10c): 0.80 g, 92%; ¹H NMR (CDCl₃, 400 MHz) δ 7.06 (d, J = 6.0 Hz, 1H, C6′H), 5.65 (d, J = 6.4 Hz, 1H, C5′H), 5.53 (s, 1H, C5H), 3.87–3.84 (t, J = 6.0 Hz, 2H, C7′H₂), 3.76–3.73 (t, J = 6.4 Hz, 1H, C11H₂), 3.70–3.67 (t, J = 6.0 Hz, 2H, C7H₂), 3.24 (s, 3H, N3CH₃), 2.61–2.53 (m, 4H, C11′H₂, NCH₂), 2.21 (s, 3H, C6CH₃), 1.92 (s, 1H, NH), 1.70–1.63 (m, 4H, C8H₂, C10H₂), 1.59–1.55, 1.49–1,46 (both m, 2H each, C8′H₂, C10′H₂), 1.37–1.30 (m, 4H, C9H₂, C9′H₂), 1.06–1.03 (t, J = 5.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.8 (C′4), 162.2 (C4), 152.1 (C6′), 151.2 (C6), 150.7 (C2), 141.8 (C2′), 101.6 (C5′, C5), 49.4 (C11′), 49.2 (NCH₂CH₃), 44.6, 43.9, 41.0 (C7, C11, C7′), 29.5 (C10′), 28.4 (C8′), 28.1 (N3CH₃), 27.7, 27.3 (C8, C10), 24.6, 23.3 (C9, C9′), 19.5 (C6CH₃), 15.0 (NCH₂CH₃); MALDI-MS [M + H]⁺ calcd for C₂₂H₃₅N₅O₄ m/z 434.3. Found: 434.4. Anal. calcd for C₂₂H₃₅N₅O₄: C, 60.95; H, 8.14; N, 16.15. Found: C, 61.04; H, 8.03; N, 16.23 (Figures S19 and S20).

1,6-{(3,6-Dimethyluracil-1-yl)-[3-(5-ethylaminopentyl)uracil-1-yl]}hexane (10d): 0.86 g, 96%; ¹H NMR (CDCl₃, 400 MHz) δ 7.05 (d, J = 7.6 Hz, 1H, C6′H), 5.63 (d, J = 8.0 Hz, 1H, C5′H), 5.52 (s, 1H, C5H), 3.87–3.83 (t, J = 5.2 Hz, 2H, C7′H₂), 3.75–3.72 (t, J = 6.0 Hz, 1H, C12H₂), 3.67–3.64 (t, J = 6.4 Hz, 2H, C7H₂), 3.24 (s, 3H, N3CH₃), 2.60–2.53 (m, 4H, C11′H₂, NCH₂), 2.43 (s, 1H, NH), 2.18 (s, 3H, C6CH₃), 1.66–1.46 (m, 8H, C8H₂, C11H₂, C8′H₂, C10′H₂), 1.36–1.30 (m, 6H, C9H₂, C10H₂, C9′H₂), 1.07–1.04 (t, J = 7.2 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.9 (C′4), 162.2 (C4), 152.1 (C6′), 151.2 (C6), 150.8 (C2), 142.0 (C2′), 101.4 (C5′, C5), 49.4 (C11′), 48.7 (NCH₂CH₃), 44.8, 43.4, 40.9 (C7, C12, C7′), 28.7 (C10′), 28.5 (C8′), 28.4 (N3CH₃), 27.6, 27.1 (C8, C11), 26.1, 25.8, 24.4 (C9, C10, C9′), 19.5 (C6CH₃), 14.0 (NCH₂CH₃); MALDI-MS [M + H]⁺ calcd for C₂₃H₃₇N₅O₄ m/z 448.3. Found: 448.3. Anal. calcd for C₂₃H₃₇N₅O₄: C, 61.72; H, 8.33; N, 15.65. Found: C, 61.64; H, 8.43; N, 15.73 (Figures S21–S23).

Synthesis of Non-Ionic Cholinesterase Inhibitors Based on Bisuracils 3a–d: General Procedure

A mixture of amine 10a–d (1.0 mmol), ortho-nitrilebenzyl bromide 9 (0.2 g, 1.0 mmol), and potassium carbonate (0.14 g, 1.0 mmol) was stirred in MeCN (30 mL) at 45–50 °C for 8 h. The precipitate was filtered out. The solution was concentrated to 10–15 mL and transferred to a column with SiO₂. The column was successively washed with petroleum ether, ethyl acetate, and 10:1 ethyl acetate diethylamine mixture. The target bisuracils 3a–d were isolated from the 10:1 ethyl acetate diethylamine mixture fractions as clear light brown oil.

1,3-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(ethyl)amino)pentyl)-uracil-1-yl]}propane (3a): 0.42 g, 80%; ¹H NMR (CDCl₃, 400 MHz) δ 7.56–7.51 (m, 2H, C3′′H, C4′′H), 7.48–7.45 (m, 1H, C5′′H), 7.26–7.23 (m, 1H, C6′′H), 7.21 (d, J = 8.0 Hz,1H, C6′H), 5.64 (d, J = 8.0 Hz, 1H, C5′H), 5.53 (s, 1H, C5H), 3.83–3.74 (m, 6H, C7H₂, C9H₂, C7′CH₂), 3.66 (br. s, 2H, NCH₂Ph), 3.21 (s, 3H, N3CH₃), 2.49–2.44, 2.40–2.37 (both m, 2H each, NCH₂, C11′H₂), 2.16 (s, 3H, C6CH₃), 2.01–1.98 (m, 2H, C8H₂), 1.54–1.48 (m, 2H, C10′H₂), 1.46–1.40 (m, 2H, C8′H₂), 1.26–1.20 (m, 2H, C9′H₂), 0.97–0.94 (t, J = 6.8 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.6 (C′4), 162.0 (C4), 152.1 (C6′), 151.2 (C6), 150.5 (C2, C2′), 141.9 (C2′′), 132.5, 132.4 (C1′′, C5′′), 129.6 (C3′′, C4′′, C6′′), 101.7, 101.6 (C5′, C5), 55.9 (C11′), 52.9 (NCH₂Ph), 47.2 (NCH₂CH₃), 42.0, 40.9 (C7, C7′, C9), 28.3 (C10′), 27.6 (N3CH₃), 27.1 (C8′), 26.3 (C8), 24.4 (C9′), 19.4 (C6CH₃), 11.3 (NCH₂CH₃); MALDI-MS [M − Et − CN + 2H]⁺, [M − H]⁺ calcd for C₂₈H₃₆N₆O₄ m/z 468.3, 520,3, respectively. Found: 468.2, 520.2. Anal. calcd for C₂₈H₃₆N₆O₄: C, 64.60; H, 6.97; N, 16.14. Found: C, 64.71; H, 7.06; Br, 12.81; N, 16.08 (Figures S24–S26).

1,4-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(ethyl)amino)pentyl)-uracil-1-yl]}butane (3b): 0.43 g, 81%; ¹H NMR (CDCl₃, 400 MHz) δ 7.57–7.51 (m, 2H, C3′′H, C4′′H), 7.49–7.46 (m, 1H, C5′′H), 7.24–7.22 (m, 1H, C6′′H), 7.12 (d, J = 6.4 Hz,1H, C6′H), 5.62 (d, J = 6.0 Hz, 1H, C5′H), 5.51 (s, 1H, C5H), 3.81–3.77 (m, 4H, C10H₂, C7′CH₂), 3.73–3.70 (m, 2H, C7H₂), 3.68 (br. s, 2H, NCH₂Ph), 3.21 (s, 3H, N3CH₃), 2.49–2.46, 2.41–2.38 (both m, 2H each, NCH₂, C11′H₂), 2.17 (s, 3H, C6CH₃), 1.70–1.64 (both m, 2H each, C8H₂, C9H₂), 1.54–1.49, 1.45–1.41 (both m, 2H each, C8′H₂, C10′H₂), 1.26–1.20 (m, 2H, C9′H₂), 0.98–0.95 (t, J = 6.0 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.6 (C′4), 162.0 (C4), 152.1 (C6′), 151.2 (C6), 150.6 (C2, C2′), 142.0 (C2′′), 132.5, 132.4 (C1′′, C5′′), 129.7, 127.1 (C3′′, C4′′, C6′′), 101.5, 101.4 (C5′, C5), 55.9 (C11′), 52.9 (NCH₂Ph), 48.7 (NCH₂CH₃), 47.2, 44.1, 40.8 (C7, C7′, C10), 27.6 (C10′), 27.1 (N3CH₃), 26.2, 25.9, 25.6, 24.4 (C8, C9, C8′, C9′), 19.5 (C6CH₃), 11.2 (NCH₂CH₃); MALDI-MS [M − H]⁺, [M − CN − 2H]⁺, [M − CH₂PhCN + 2H]⁺, calcd for C₂₉H₃₈N₆O₄ m/z 533.3, 507.3, 420.3, respectively. Found: 533.4, 507.3, 420.2. Anal. calcd for C₂₉H₃₈N₆O₄: C, 65.15; H, 7.16; N, 15.72. Found: C, 65.11; H, 7.09; N, 15.58 (Figures S27–S29).

1,5-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(ethyl)amino)pentyl)-uracil-1-yl]}pentane (3c): 0.43 g, 78%; ¹H NMR (CDCl₃, 400 MHz) δ 7.62–7.58 (m, 2H, C3′′H, C4′′H), 7.55–7.51 (m, 1H, C5′′H), 7.32–7.28 (m, 1H, C6′′H), 7.08 (d, J = 7.6 Hz,1H, C6′H), 5.70 (d, J = 7.6 Hz, 1H, C5′H), 5.58 (s, 1H, C5H), 3.91–3.87, 3.82–3.78 (both m, 2H each, C11H₂, C7′CH₂), 3.75–3.71 (m, 4H, C7H₂, NCH₂Ph), 3.30 (s, 3H, N3CH₃), 2.56–2.51 (q, J = 7.2 Hz, 2H, NCH₂), 2.47–2.43 (q, J = 7.2 Hz, 2H, C11′H₂), 2.23 (s, 3H, C6CH₃), 1.78–1.67 (both m, 2H each, C8H₂, C10H₂), 1.60–1.55, 1.51–1.46, 1.43–1.37, 1.35–1.29 (all m, 2H each, C8′H₂, C10′H₂, C9H₂, C9′H₂), 1.04–1.00 (t, J = 7.2 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.8 (C′4), 162.2 (C4), 152.2 (C6′), 151.3 (C6), 150.7 (C2, C2′), 141.9 (C2′′), 132.6, 129.8, 127.2 (C1′′, C5′′, C3′′, C4′′, C6′′), 101.5 (C5′, C5), 56.0 (C11′), 53.0 (NCH₂Ph), 49.2 (NCH₂CH₃), 47.4, 44.7, 41.0 (C7, C7′, C11), 28.4 (C10′), 28.2 (N3CH₃), 27.7, 27.3, 24.6, 23.4 (C8, C9, C10, C8′, C9′), 19.5 (C6CH₃), 11.2 (NCH₂CH₃); MALDI-MS [M − H]⁺ calcd for C₃₀H₄₆N₆O₄ m/z 547.3, respectively. Found: 547.3. Anal. calcd for C₃₀H₄₆N₆O₄: C, 65.67; H, 7.35; N, 15.32. Found: C, 65.58; H, 7.39; N, 15.18 (Figures S30–S32).

1,6-{(3,6-Dimethyluracil-1-yl)-[3-(5-o-nitrilebenzyl(ethyl)amino)pentyl)-uracil-1-yl]}hexane (3d): 0.45 g, 80%; ¹H NMR (CDCl₃, 400 MHz) δ 7.59–7.53 (m, 3H, C3′′H, C4′′H, C5′′H), 7.30 (m, 1H, C6′′H), 7.07 (d, J = 6.0 Hz,1H, C6′H), 5.67 (d, J = 6.0 Hz, 1H, C5′H), 5.56 (s, 1H, C5H), 3.89–3.86 (m, 2H, C7′CH₂), 3.78–3.67 (m, 6H, C12H₂, C7H₂, NCH₂Ph), 3.28 (s, 3H, N3CH₃), 2.52–2.44 (m, 4H, NCH₂, C11′H₂), 2.21 (s, 3H, C6CH₃), 1.63–1.50 (m, 8H, C8H₂, C11H₂, C8′H₂, C10′H₂), 1.37–1.26 (m, 6H, C9H₂, C10H₂, C9′H₂), 1.02 (br. m, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 162.9 (C′4), 162.3 (C4), 152.2 (C6′), 151.3 (C6), 150.7 (C2, C2′), 141.9 (C2′′), 132.6, 129.7, 127.1 (C1′′, C5′′, C3′′, C4′′, C6′′), 101.6 (C5′, C5), 56.0 (C11′), 53.1 (NCH₂Ph), 49.5 (NCH₂CH₃), 47.4, 44.9, 41.0 (C7, C7′, C12), 28.8 (C10′), 28.6 (N3CH₃), 27.7, 27.3, 26.2, 25.9, 24.6 (C8, C9, C10, C11, C8′, C9′), 19.6 (C6CH₃), 11.5 (NCH₂CH₃); MALDI-MS [M + Na]⁺, [M − H]⁺, [M − CH₂PhCN + 2H]⁺, calcd for C₃₁H₄₂N₆O₄ m/z 585.3, 561.3, 448.3, respectively. Found: 585.3, 561.4, 448.2. Anal. calcd for C₃₁H₄₂N₆O₄: C, 66.17; H, 7.52; N, 14.94. Found: C, 66.00; H, 7.59; N, 15.03 (Figures S33–S35).

3.2. Biological Studies

3.2.1. General Information

Paraoxon, atropine, scopolamine, acetylthiocholine iodide, butyrylthiocholine iodide, recombinant human acetylcholinesterase, butyrylcholiesterase from human plasma, and 5,5′–dithiobis–(2–nitrobenzoic) acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals and solvents were of biochemical grade.

3.2.2. Cholinesterases Inhibition Assay

In Vitro Cholinesterase Inhibition

Inhibitory activities of the tested compounds against AChE and BChE were measured according to Ellman et al. [32]. Enzyme-catalysed hydrolysis of ATCHh was carried out at 25 °C in 0.1 M phosphate buffer (pH 8.0) containing 0.25 U AChE, 1 mM ATCHh as substrates, and 0.1 mM of DTNB. In experiments with BuChE, 0.1 M phosphate buffer (pH 7.0) and BuTChh as a substrate were used. The tested compounds were pre-incubated with the enzyme for two minutes prior to the addition of the substrate and the subsequent recording of hydrolysis kinetics. The rate of substrate hydrolysis was measured for two minutes by a Shimadzu UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) at a wavelength of 412 nm. A sample without an inhibitor was used as a control (100% cholinesterase activity). A sample without a substrate was used as a blank. Experiments were performed in triplicate. IC₅₀ values (inhibitor concentration required to inhibit enzyme activity by 50%) were determined using Origin 8.5 from the Hill equation (1):

$${\displaystyle \frac{E}{E_{max}}}={\displaystyle \frac{{\left[I\right]}^n}{{IC}_{50}^n+{\left[I\right]}^n}}$$

(1)

where E—enzyme activity in the presence of the tested compound; [I]—compound concentration.

The inhibition constants (K_i) for human AChE and BuChE were determined by Ellman’s method [32] at 412 nm and 25 °C in 0.1 M sodium phosphate buffer (pH = 8.0 for AChE and pH = 7.0 for BuChE) with ATCHh or BuTCh concentrations in the range of 0.1–1.0 mM. A UV-1800 Shimadzu (Shimadzu Co., Kyoto, Japan) spectrophotometer was utilised to conduct the measurements. The final enzyme concentration in the assays was in the picomolar range. In order to ascertain the levels of spontaneous hydrolysis of the enzyme and substrate in the absence of an inhibitor, controls were performed for each substrate concentration. The change in absorption was then recorded over a period of three minutes. The inhibition constants were determined from a Dixon plot and Cornish–Bowden transformation [33].

In Vivo Cholinesterase Inhibition

In vivo experiments involving animals were carried out in accordance with the Directive of the Council of the European Union 2010/63/EU. The protocol of experiments was approved by the Animal Care and Use Committee of the FRC Kazan Scientific Center of RAS (protocol No. 2 from 9 June 2024). CD-1 mice (weighing 25–30 g, 6 weeks old) were purchased from the Laboratory Animal Breeding Facility (Branch of Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Puschino, Moscow Region, Russia) and were allowed to acclimate to their environment in vivarium for at least 1 week prior to the experiments. Animals were kept in sawdust-lined plastic cages in a well-ventilated room at 20–22 °C in a 12 h light/dark cycle, with 60–70% relative humidity, and given ad libitum access to a standard diet and water. Experiments were carried out between 8 a.m. and 12 a.m.

Toxicological experiments were performed using a single i.p. injection of the tested compounds in CD-1 mice weighing 20–25 g. Stock solutions of the tested compounds were diluted in ethanol or water. The final ethanol dose was 100 mg/kg. Nine different doses of the tested compounds were used with 12 mice per dose. Animals were observed 7 days after injection, and symptoms of toxicity were recorded. The LD₅₀ dose causing lethal effects in 50% of the animals was taken as a criterion of toxicity. LD₅₀ was determined by the method of Weiss with 95% confidence limits [34].

For the brain AChE inhibition assay, the brains of mice were removed 30 min after i.p. injection of the tested compound (experimental group, n = 6 mice) or following i.p. vehicle injection (control group, n = 6 mice). The brains were subsequently frozen in liquid nitrogen. Whole-brain homogenates were prepared in a Potter homogeniser with 0.05 M Tris–HCl, 1% Triton X–100, 1M NaCl, and 2 mM ethylenediaminetetraacetic acid (pH 7.0; 4 °C; 1 part brain to 2 parts of buffer). The homogenate was centrifuged (14,000× g rpm; t = 4 °C) for 10 min using an Eppendorf 5430R centrifuge with an FA–45–30–11 rotor (Eppendorf AG, Hamburg, Germany). AChE activity in brain homogenates was measured using the method described by Dingova et al. [35] with modification. Briefly, a 50 µL aliquot of supernatant was incubated for 30 min with 5 µL of 0.5 mM tetra–isopropyl pyrophosphoramide (iso–OMPA) as a specific BChE inhibitor. Next, the enzyme-catalysed reaction was initiated by adding 10 µL of ATCH (final concentration 1.5 mM) as a substrate. Following 10, 20, or 30 min of incubation with the substrate at 25 °C, the reaction was stopped by adding neostigmine (final concentration 0.01 M). After diluting samples 25 times in a 50 mM phosphate buffer (pH 8.0), DTNB (0.1 mM) was added. The production of yellow 5–thio–2–nitro–benzoate anion resulting from the reduction of DTNB by thiocholine (the product of enzymatic hydrolysis of ATCH) was measured spectrophotometrically using a Shimadzu UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) according to Ellman’s method [32]. The rate of thiocholine production over 20 min (from 10 to 30 min) was calculated. Brain samples of the control group were used as a control (100% of cholinesterase activity). The sample without substrate was used as a blank. All measurements for each brain sample were performed in triplicate. AChE activity was expressed in relation to the amount of total protein, which was determined by the Bradford method [36].

3.2.3. Protection Against POX Toxicity

For protection of mice against 2 × LD₅₀ POX, the experimental scheme developed by Lenina et al. was used [37]. Briefly, for the POX toxicity shift assay, atropine (15 mg/kg, i.p) was administered 1 min after i.p. injection of 2 × LD₅₀ POX. The tested compound was administered via i.p. before POX injection. The ratio of the number of mice that did not survive after challenge with 2 × LD₅₀ POX to the total number of challenged mice was used as a criterion of toxicity shift.

3.2.4. Novel Object Recognition Test

The experiments were carried out on CD1 mice of both sexes weighing 24–25 g. The novel object recognition test was conducted over a period of three days [29]. On the first day, the mice were placed individually into the square testing arena with black walls (50 cm in length, 50 cm in width, 38 cm in height) for 5 min without any objects. On the second day, two identical objects were placed in the central part of the arena, and the mice were allowed to explore the objects for 10 min. Test compounds were administered prior to testing on the second day. The induction of amnesia was facilitated by i.p. injection of scopolamine (1.5 mg/kg) 50 min prior to testing. Tested compounds were administered i.p. 30 min prior to testing. The control group of animals was administered with an equivalent amount of vehicle. On the third day, the mice were presented with a familiar and a novel object for 10 min. The time of exploration of each object by the mice was recorded using a digital camera. After each test, the arena was cleaned with a solution of 70% ethanol. At the end of the test, the preference index (exploration of novel object/total exploration time × 100) was calculated.

3.2.5. Effect on Motor Function

To analyse motor function, rotarod and open-field tests were carried out. Mice were randomly divided into 3 groups (n = 12): a control group of mice and groups of mice treated with tested compounds. Tests were conducted 1 h after compound exposure. To assess motor function, mice were first placed on a rotarod treadmill (Ugo Basile, Italy) [38]. Rotation speed was fixed at 15 rpm, and the time that each mouse remained on the rod was recorded as the score, with a maximum duration of 300 s per trial. Each mouse underwent 3 trials. After recording the length of time that mice managed to remain on the rotarod (latency to fall), the average of three trials was used for further analysis. Following the rotarod test, motor function was studied in an open field apparatus (Open Science, Krasnogorsk, Russia), as described in [39]. The numbers of rearing and head dips were estimated as indexes of exploratory activity. To assess motor function, the distance travelled in centimetres in the arena was measured, using VideoMot2 software (Version 8.02 (131) 03-07-17 (TSE Systems, Bad Homburg, Germany).

3.2.6. Statistics

All data processing was performed using OriginPro 8.5. The results are expressed as the mean ± standard deviation. ANOVA statistics with Tukey’s post hoc test were used to analyse the results of the novel object recognition test. The Mann–Whitney test was used to analyse the locomotor activity. Significance was tested at the 0.05 level of probability (p). Relative risk of death was investigated using Cox analysis.

3.3. Molecular Modelling

3.3.1. Molecular Docking

The crystal structure of human butyrylcholinesterase (hBuChE) complexed with the ligand (PDB ID: 6ESY) was selected as the template, while the crystal structure of human acetylcholinesterase (hAChE) (PDB ID: 7E3H) was used for comparison. Protein alignment and superposition were performed using the Protein Align/Superpose function in the Molecular Operating Environment (MOE) to evaluate structural overlap and similarity. This analysis facilitated the investigation of the active site architecture and binding pockets of both proteins.

3.3.2. Molecular Dynamics Simulation

Molecular dynamics (MD) simulations were performed using the Desmond simulation package (Schrödinger Release 13.5). The simulation systems were prepared by embedding the structures of compounds 2b and 3c (based on PDB IDs: 7E3H and 6ESY, respectively) into a cubic simulation box with a 10 Å buffer distance to establish a hydrated environment. Periodic boundary conditions were applied, and the box edge length was adjusted to accommodate the protein–ligand complex plus buffer. To neutralise the system and achieve physiological ionic strength (150 mM), Na⁺ and Cl⁻ ions were added using the Autoionize tool in Desmond, with a tolerance of ±0.1 charges to ensure neutrality. The systems were parameterised using the OPLS4 force field, which is a widely used force field for simulating biomolecular systems. This force field provides a balanced description of protein, lipid, nucleic acid, and small-molecule interactions. The SPC (Simple Point Charge) water model was used to represent the solvent, as it is a commonly used model that provides a good balance between computational efficiency and accuracy.

The NPT ensemble in the Desmond package was applied for system minimisation and equilibration. MD simulations were conducted for 100 ns for both compounds, with trajectory data recorded every 200 ps. Throughout the simulations, the temperature and pressure were maintained at 300 K and 1.01325 bar, respectively. Post-simulation analyses were conducted using the Simulation Interaction Diagram tool in Desmond to evaluate ligand–protein interactions. The stability of the MD simulations was assessed by monitoring the root mean square deviation (RMSD) of both ligand and protein atom positions over time. Convergence criteria were based on the RMSD values reaching a plateau, indicating the system had achieved thermodynamic equilibrium, with minimal structural drift and consistent sampling of the conformational space.

3.3.3. ADMET Predictions

The Wenxin bio-computing large model developed by Baidu and the Baidu intelligent cloud high-performance computing cluster were used to build a bio-computing high-performance computing platform PaddleHelix to predict the ADMET properties of compounds (https://paddlehelix.baidu.com/news/achieve, accessed on 8 April 2025). The ADMET prediction model is based on Baidu deep learning technology, predicting the basic physical and chemical properties of a given compound, ADMET properties, biochemistry, and another 50+ indicators. The prediction process was performed using the default settings.

4. Conclusions

In summary, bisuracils 2b and 3c exhibit potent inhibitory effects and exceptional selectivity for AChE over BuChE. Bisuracil 2b demonstrated significant prophylactic potential against OP poisoning by protecting AChE from irreversible inhibition by POX, while bisuracil 3c effectively inhibited brain AChE activity in vivo to improve memory deficits in a scopolamine-induced amnesia model in mice. Importantly, neither compound showed adverse effects on locomotor or exploratory activity, as confirmed by behavioural tests. These findings highlight the potential of bisuracils 2b and 3c as promising candidates for further development in the treatment of neurodegenerative disorders and as protective agents against OP poisoning.