Barbiturate–sulfonate hybrids as potent cholinesterase inhibitors: design, synthesis and molecular modeling studies

Abstract

Aim: Design and synthesis of a series of 5-benzylidene(thio)barbiturates 3a–r.

Methodology: Evaluation of the inhibitory activity of the new chemical entities on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) using Donepezil as the standard reference.

Results & Conclusion: Compound 3r emerged as the most potent AChE inhibitor (IC₅₀ = 9.12 μM), while compound 3q exhibited the highest inhibitory activity against BChE (IC₅₀ = 19.43 μM). Toxicological bioassays confirmed the absence of cytotoxicity for the most potent compounds at the tested doses. Molecular docking analysis demonstrated that the tested derivatives effectively bind to the active sites of both enzymes. Overall, this study sheds light on the potential of barbiturate–sulfonate conjugates as promising drug candidates.

Plain language summary

Article highlights.

• A series of 5-benzylidene(thio)barbiturates were synthesized, and evaluated for their inhibitory properties against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).

• Compounds 3r and 3q were the most potent analogs against AChE and BChE, respectively, with no cytotoxicity detected on the normal cell line.

• Antioxidant assays revealed respectable antioxidant values.

• Computational studies were utilized to validate the binding interactions.

• The study highlighted the potential of barbiturate–sulfonate hybrids as drug candidates.

1. Backround

Alzheimer's disease is a degenerative neurological disorder that impacts cognitive functions such as memory, thinking and behavior, and is the primary cause of dementia, which refers to a decline in cognitive abilities that affect daily life [1–3]. The disease results in the damage and death of brain cells, leading to a decline in cognitive function and difficulty performing basic tasks such as dressing, eating and bathing. Although the precise causes of Alzheimer's disease are not entirely clear, genetic, environmental and lifestyle factors are thought to contribute to its development [4–6].

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are all members of the serine esterase family, and their general structures are similar. Furthermore, their active sites are made up of a choline-binding pocket and three catalytic components. The proportion of AChE in the normal brain is approximately 80%, while BChE accounts for only 20%. However, as the disease continues, the concentration of BChE in the patient's brain may grow, while the concentration of AChE drops to 10–15% [7–9]. As a result, simultaneously inhibiting BChE and AChE in proper quantities is a better optimal therapeutic technique.

AChE is responsible for breaking down acetylcholine in the brain. In Alzheimer's disease, there is a decrease in the levels of acetylcholine, which is believed to contribute to cognitive impairment [10–14]. To treat Alzheimer's disease, cholinesterase inhibitors like donepezil, rivastigmine, tacrine and galantamine are commonly used (Figure 1) [15]. These drugs work by blocking the activity of both AChE and BChE, leading to an increase in the concentration of acetylcholine in the brain, which can alleviate some of the symptoms of Alzheimer's disease, such as memory loss and confusion. However, cholinesterase inhibitors do not cure Alzheimer's disease, and they do not halt its progression. Additionally, these drugs can cause side effects, including nausea, vomiting and diarrhea, and may interact with other medications [16–21]. Therefore, it is crucial to carefully monitor their use and explore other potential therapeutic options for Alzheimer's disease.

Figure 1.

Examples of drugs in the treatment of Alzheimer's disease. AChEI: Acetylcholinesterase inhibitor.

Moreover, the excessive production of reactive oxygen species results in oxidative stress, a condition often associated with the development of Alzheimer's disease (AD) [22]. Therefore, implementing strategies to reduce oxidative stress and enhance antioxidant defense is considered an effective approach to delaying the initial and advancing stages of AD and offering neuroprotective advantages [23,24].

Barbiturates and thiobarbiturates-containing scaffolds are widely used in medicinal chemistry for their therapeutic properties, including anticancer [25], anti-inflammatory [26], antioxidant [27], antibacterial [27,28], anticonvulsant [29] and antidiabetic [28,30]. However, they have been found to inhibit both AChE and BChE [31,32].

Sulfonate-containing compounds are important motifs in medicinal chemistry due to their ability to modulate the biological activity of diverse class of compounds [33–36]. Compounds containing sulfonate groups have exhibited promising abilities to suppress cholinesterase enzymes. For instance, arylsulfonates I and II, based on scopoletin, have been found to have inhibitory effects [37]. Additionally, derivative III, which is a methanesulfonate, and derivative IV, which is a sulfonate derivative of aurone, have shown significant potential in inhibiting AChE [38,39] (Supplementary Figure S1).

Donepezil molecule consists mainly of an aromatic system that can interact with the peripheral anionic domain (PAS), and a basic center that can bind to the catalytic active site (CAS) of the AChE enzyme [40]. In this study, the barbiturates/thiobarbiturates ring (a hetero aromatic ring) acts as the aromatic system to interact with PAS and alkane sulfonate moieties as a basic center to bind to CAS.

Consequently, a new scaffold has been designed to incorporate both barbiturates/thiobarbiturates that act as the aromatic system to interact with PAS and alkane sulfonate moieties as a basic center to bind to CAS (Figure 2), expected to be a potent candidate for the treatment of Alzheimer's disease through the inhibition of AChE and/or BChE [41].

Figure 2.

Design strategy of targeted compounds for the treatment of Alzheimer's disease.

AChE: Acetylcholinesterase; BChE: Butyrylcholinesterase; CAS: Catalytic active site; PAS: Peripheral anionic site.

2. Experimental

Melting points were recorded on a Stuart SMP30 melting point apparatus (Staffordshire, UK). IR spectra (KBr) were recorded on a JASCO 6100 spectrophotometer, JASCO, (PA, USA). NMR spectra were recorded on a JEOL AS 500 (¹H: 500 MHz, ¹³C: 125 MHz) (CA, USA) and spectrometer. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX (EI, 70 eV) spectrometer (Kyoto, Japan). Elemental microanalyses were recorded on a Vario El Elementar analyzer (Langenselbold, Germany). Aldehyde–sulfonate derivatives 2a–r [39] were prepared following to the previously described procedures, the spec. charts of compounds 3a–r are displayed in sup. inf. file (Supplementary Figure S2–S34).

2.1. General procedure for the synthesis of compounds 3a–r

To a solution of barbituric acid or thiobarbituric acid 1 (1.0 mmol) in ethanol (20 ml), was added, the various 4-formyl phenyl alkane sulfonate derivatives (2) namely; 4-formyl phenyl methanesulfonate, 4-formyl phenyl ethanesulfonate or 4-formyl phenyl propane-1-sulfonate (1.0 mmol). The reaction mixture was heated under reflux for 2 h before being cooled to room temperature. The precipitate obtained was filtered, washed with ethanol, dried and recrystallized from ethanol to yield compounds 3a–r.

2.1.1. 4-((2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl methanesulfonate (3a)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formylphenyl methanesulfonate (0.200 g, 1 mmol) to yield compound 3a as an off-white solid. Yield: 89%; mp 261–263°C; IR (KBr) cm^-1, ν: 3399 (NH), 3324 (NH), 1686 (C=O), 1620 (CH=C), 1391 (SO₂), 1291 (CO); ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.45 (s, 3H, CH₃), 7.43 (d, 2H, J = 9.0 Hz, H-2 and H-6), 8.17 (d, 2H, J = 9.5 Hz, H-3 and H-5), 8.28 (s, 1H, CH=C), 11.27 (s, 1H, NH), 11.42 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 37.83 (CH₃), 119.69 (CH=C), 121.62 (C-2 and C-6), 131.69 (C-4), 134.97 (C-3 and C-5), 150.15 (NH-CO-NH), 151.12 (C-1), 152.92 (C=CH), 161.50 (C=O), 163.18 (C=O); Anal. calc. for C₁₂H₁₀N₂O₆S (310.28): C, 46.45; H, 3.25; N, 9.03; Found: C, 46.34; H, 3.33; N, 9.11.

2.1.2. 4-((2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl ethanesulfonate (3b)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formylphenyl ethane sulfonate (0.214 g, 1 mmol) to yield compound 3b as an off-white solid. Yield: 92%; mp 254–256°C; IR (KBr) cm^-1, ν: 3388 (NH), 3345 (NH) 2807 (CH aliphatic), 1680 (C=O), 1600 (CH=C), 1381 (SO₂), 1288 (C-O); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.39 (t, 3H, J = 6.5 Hz, CH₃), 3.59 (q, 2H, J = 6.5 Hz, CH₂), 7.41 (d, 2H, J = 7.5 Hz, H-2 and H-6), 8.16 (d, 2H, J = 7.5 Hz, H-3 and H-5), 8.27 (s, 1H, CH=C), 11.27 (s, 1H, NH), 11.42 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.07 (CH₃), 45.08 (CH₂), 119.66 (CH=C), 121.48 (C-2 and C-6), 131.55 (C-4), 134.97 (C-3 and C-5), 150.12 (NH-CO-NH), 150.99 (C-1), 152.89 (C=CH), 161.49 (C=O), 163.18 (C=O); Anal. calc. for C₁₃H₁₂N₂O₆S (324.31): C, 48.15; H, 3.73; N, 8.64; Found: C, 48.22; H, 3.61; N, 8.59.

2.1.3. 4-((2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl propane-1-sulfonate (3c)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formylphenyl propane-1-sulfonate (0.228 g, 1 mmol) to yield compound 3c as an off-white solid. Yield: 90%; mp 221–223°C; IR (KBr) cm^-1, ν: 3369 (NH), 3255 (NH), 1688 (C=O), 1671 (C=O), 1630 (CH=C), 1370 (SO₂), 1291 (CO); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.04 (t, 3H, J = 8.0 Hz, CH₃), 1.39 (sextet, 2H, J = 7.5 Hz, CH₂), 3.57 (t, 2H, J = 8.0 Hz, CH₂), 7.40 (d, 2H, J = 9.5 Hz, H-2 and H-6), 8.16 (d, 2H, J = 9.5 Hz, H-3 and H-5), 8.28 (s, 1H, CH=C), 11.27 (s, 1H, NH), 11.42 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.31 (CH₃), 16.96 (CH₂), 51.78 (CH₂), 119.64 (CH=C), 121.50 (C-2 and C-6), 131.54 (C-4), 134.98 (C-3 and C-5), 150.12 (NH-CO-NH), 150.99 (C-1), 152.91 (C=CH), 161.50 (C=O), 163.16 (C=O); Anal. calc. for C₁₄H₁₄N₂O₆S (338.33): C, 49.70; H, 4.17; N, 8.28; Found: C, 49.61; H, 4.21; N, 8.19.

2.1.4. 2-Methoxy-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl methanesulfonate (3d)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formyl-2-methoxyphenyl methanesulfonate (0.230 g, 1 mmol) to yield compound 3d as a yellow solid. Yield: 88%; mp 238–240°C; IR (KBr) cm^-1, ν: 3410 (NH), 3210 (CH Ar), 1677 (C=O), 1670 (C=O), 1649 (CH=C), 1351 (SO₂), 1285 (CO); ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.44 (s, 3H, CH₃), 3.92 (s, 3H, OCH₃), 7.42 (br.s, 1H, H-6), 7.75 (br.s, 1H, H-5), 8.18 (s, 1H, H-3), 8.31 (s, 1H, CH=C), 11.31 (s, 1H, NH), 11.47 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 38.73 (CH₃), 56.14 (OCH₃), 117.96 (C-3), 119.65 (C-6), 123.31 (C-5), 126.79 (C-4), 132.57 (C-1), 140.21 (NH-CO-NH), 150.16 (C-2), 150.66 (C=CH), 153.35 (CH=C), 161.68 (C=O), 163.29 (C=O); Anal. calc. for C₁₃H₁₂N₂O₇S (340.31): C, 45.88; H, 3.55; N, 8.23; Found: C, 46.99; H, 3.40; N, 8.39.

2.1.5. 2-Methoxy-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl ethanesulfonate (3e)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formyl-2-methoxyphenyl ethanesulfonate (0.244 g, 1 mmol) to yield compound 3e as a yellow solid. Yield: 91%; mp 220–222°C; IR (KBr) cm^-1, ν: 3380 (NH), 3366 (NH), 2240 (CH aliphatic), 1669 (C=O), 1666 (C=O), 1655 (CH=C), 1349 (SO₂); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.39 (t, 3H, J = 7.5 Hz, CH₃), 3.54 (q, 2H, J = 7.5 Hz, CH₂), 3.87 (s, 3H, OCH₃), 7.37 (dd, 1H, J = 8.5, 4.0 Hz, H-5), 7.69 (d, 1H, J = 8.5 Hz, H-6), 8.12 (s, 1H, CH=C), 8.26 (d, 1H, J = 3.5 Hz, H-3), 11.29 (s, 1H, NH), 11.43 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.63 (CH₃), 46.45 (CH₂), 56.66 (OCH₃), 118.48 (C-3), 120.14 (C-6), 123.78 (C-5), 127.33 (C-4), 132.95 (C-1), 140.73 (NH-CO-NH), 150.66 (C-2), 151.12 (C=CH), 153.83 (CH=C), 161.22 (C=O), 163.81 (C=O); Anal. calc. for C₁₄H₁₄N₂O₇S (354.33): C, 47.46; H, 3.98; N, 7.91; Found: C, 47.56; H, 3.83; N, 7.82.

2.1.6. 2-Methoxy-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl propane-1-sulfonate (3f)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 4-formyl-2-methoxyphenyl propane-1-sulfonate (0.258 g, 1 mmol) to yield compound 3f as an orange solid. Yield: 89%; mp 196–198°C; IR (KBr) cm^-1, ν: 3338 (NH), 3110 (CH Ar), 1688 (C=O), 1670 (C=O), 1615 (CH=C), 1353 (SO₂), 1225 (C-O); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.04 (t, 3H, J = 7.5 Hz, CH₃), 1.87 (sextet, 2H, J = 7.5 Hz, CH₂), 3.53 (t, 2H, J = 7.5 Hz, CH₂), 3.87 (s, 3H, OCH₃), 7.37 (d, 1H, J = 9.0 Hz, H-6), 7.72 (d, 1H, J = 9.0 Hz, H-5), 8.12 (s, 1H, H-3), 8.27 (s, 1H, CH=C), 11.29 (s, 1H, NH), 11.43 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.35 (CH₃), 12.09 (CH₂), 51.88 (CH₂), 55.48 (OCH₃), 113.05 (C-3), 117.11 (C-6), 125.33 (C-5), 128.77 (C-4), 136.55 (C-1), 136.88 (NH-CO-NH), 150.11 (C-2), 152.99 (C=CH), 155.55 (CH=C); 162.11 (C=O), 163.49 (C=O); Anal. calc. for C₁₅H₁₆N₂O₇S (368.36): C, 48.91; H, 4.38; N, 7.61; Found: C, 48.80; H, 4.21; N, 7.50.

2.1.7. 2-Methoxy-5-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl methanesulfonate (3g)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 5-formyl-2-methoxyphenyl methanesulfonate (0.230 g, 1 mmol) to yield compound 3g as a yellow solid. Yield: 90%; mp 291–293°C; IR (KBr) cm^-1, ν: 3380 (NH), 3290 (NH), 1676 (C=O), 1629 (CH=C), 1344 (SO₂); ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.42 (s, 3H, CH₃), 3.96 (s, 3H, OCH₃), 7.33 (d, 1H, J = 9.0 Hz, H-3), 8.18 (dd, 1H, J = 9.0, 2.0 Hz, H-4), 8.25 (s, 1H, CH=C), 8.56 (d, 1H, J = 2.5 Hz, H-6), 11.25 (s, 1H, NH), 11.36 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 38.41 (CH₃), 56.55 (OCH₃), 112.99 (C-3), 117.09 (C-6), 125.24 (C-4), 128.85 (C-5), 136.50 (C-1), 137.08 (C-2), 150.11 (NH-CO-NH), 153.42 (C=CH), 155.37 (CH=C), 162.11 (C=O), 163.54 (C=O); Anal. calc. for C₁₃H₁₂N₂O₇S (340.31): C, 45.88; H, 3.55; N, 8.23; Found: C, 45.97; H, 3.46; N, 8.11.

2.1.8. 2-Methoxy-5-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl ethanesulfonate (3h)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 5-formyl-2-methoxyphenyl ethanesulfonate (0.244 g, 1 mmol) to yield compound 3h as a yellow solid. Yield: 92%; mp 258–260°C; IR (KBr) cm^-1, ν: 3320 (NH), 3258 (CH Ar), 1666 (C=O), 1660 (C=O), 1613 (CH=C), 1361 (SO₂); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.42 (t, 3H, J = 7.5 Hz, CH₃), 3.55 (q, 2H, J = 7.5 Hz, CH₂), 3.95 (s, 3H, OCH₃), 7.32 (dd, 1H, J = 8.5, 2.0 Hz, H-4), 8.16 (d, 1H, J = 8.5 Hz, H-3), 8.24 (s, 1H, CH=C), 8.58 (d, 1H, J = 2.5 Hz, H-6), 11.24 (s, 1H, NH), 11.35 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.09 (CH₃), 45.59 (CH₂), 56.54 (OCH₃), 112.92 (C-3), 117.05 (C-6), 125.24 (C-4), 128.82 (C-5), 136.56 (C-1), 137.02 (C-2), 150.10 (NH-CO-NH), 153.42 (C=CH), 155.41 (CH=C), 162.14 (C=O), 163.56 (C=O); Anal. calc. for C₁₄H₁₄N₂O₇S (354.33): C, 47.46; H, 3.98; N, 7.91; Found: C, 47.55; H, 3.81; N, 7.80.

2.1.9. 2-Methoxy-5-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl propane-1-sulfonate (3i)

It is obtained from the reaction of barbituric acid (0.128 g, 1 mmol) with 5-formyl-2-methoxyphenyl propane-1-sulfonate (0.258 g, 1 mmol) to yield compound 3i as a yellow solid. Yield: 93%; mp 243–245°C; IR (KBr) cm^-1, ν: 3409 (NH), 3308 (NH), 2278 (CH aliphatic), 1672 (C=O), 1666 (C=O), 1629 (CH=C), 1332 (SO₂); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.05 (t, 3H, J = 7.5 Hz, CH₃), 1.88 (sextet, 2H, J = 7.5 Hz, CH₂), 3.53 (t, 2H, J = 7.5 Hz, CH₂), 3.95 (s, 3H, OCH₃), 7.31 (d, 1H, J = 9.5 Hz, H-4), 8.16 (d, 1H, J = 9.0 Hz, H-3), 8.24 (s, 1H, CH=C), 8.57 (s, 1H, H-6), 11.24 (s, 1H, NH), 11.35 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.37 (CH₃), 17.05 (CH₂), 52.30 (CH₂), 56.53 (OCH₃), 112.91 (C-3), 117.05 (C-6), 125.22 (C-4), 128.82 (C-5), 136.48 (C-1), 136.99 (C-2), 150.09 (NH-CO-NH), 153.39 (C=CH), 155.38 (CH=C), 162.09 (C=O), 163.53 (C=O); Anal. calc. for C₁₅H₁₆N₂O₇S (368.36): C, 48.91; H, 4.38; N, 7.61; Found: C, 48.80; H, 4.21; N, 7.50.

2.1.10. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl methanesulfonate (3j)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formylphenyl methanesulfonate (0.200 g, 1 mmol) to yield compound 3j as a yellow solid. Yield: 89%; mp 278–280°C; IR (KBr) cm^-1, ν: 3410 (NH), 3391 (NH), 1679 (C=O), 1605 (CH=C), 1350 (SO₂), 1233 (C-O), 1101 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.31 (s, 3H, CH₃), 7.44 (d, 2H, J = 9.0 Hz, H-2 and H-6), 8.21 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.29 (s, 1H, CH=C), 12.37 (s, 1H, NH), 12.48 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 37.88 (CH₃), 121.69 (CH=C), 122.93 (C-2 and C-6), 131.55 (C=CH), 135.31 (C-3 and C-5), 151.42 (C-4), 153.13 (C-1), 161.50 (C=O), 163.18 (C=O), 178.61 (C=S); Anal. calc. for C₁₂H₁₀N₂O₅S₂ (326.24): C, 44.17; H, 3.09; N, 8.58; Found: C, 44.28; H, 3.15; N, 8.69.

2.1.11. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl ethanesulfonate (3k)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formylphenyl ethanesulfonate (0.214 g, 1 mmol) to yield compound 3k as a yellow solid. Yield: 91%; mp 268–270°C; IR (KBr) cm^-1, ν: 3391 (NH), 3385 (NH), 2270 (CH aliphatic), 1675 (C=O), 1636 (CH=C), 1360 (SO₂) 1150 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.39 (t, 3H, J = 8.0 Hz, CH₃), 3.60 (q, 2H, J = 8.0 Hz, CH₂), 7.42 (dd, 2H, J = 9.5, 0.5 Hz, H-2 and H-6), 8.21 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.29 (s, 1H, CH=C), 12.37 (s, 1H, NH), 12.48 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.02 (CH₃), 45.19 (CH₂), 121.56 (CH=C), 122.79 (C-2 and C-6), 131.53 (C=CH), 135.32 (C-3 and C-5), 151.32 (C-4), 153.00 (C-1), 161.50 (C=O), 163.18 (C=O), 178.60 (C=S); Anal. calc. for C₁₃H₁₂N₂O₅S₂ (340.37): C, 45.87; H, 3.55; N, 8.23; Found: C, 45.94; H, 3.43; N, 8.34.

2.1.12. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl propane-1-sulfonate (3l)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formylphenyl propane-1-sulfonate (0.228 g, 1 mmol) to yield compound 3l as a yellow solid. Yield: 88%; mp 256–258°C; IR (KBr) cm^-1, ν: 3288 (NH), 3100 (CH Ar), 1660 (C=O), 1643 (CH=C), 1361 (SO₂), 955 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.04 (t, 3H, J = 7.5 Hz, CH₃), 1.39 (sextet, 2H, J = 7.5 Hz, CH₂), 3.58 (t, 2H, J = 8.0 Hz, CH₂), 7.42 (d, 2H, J = 9.0 Hz, H-2 and H-6), 8.21 (d, 2H, J = 9.0 Hz, H-3 and H-5), 8.28 (s, 1H, CH=C), 12.37 (s, 1H, NH), 12.48 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.30 (CH₃), 16.96 (CH₂), 51.81 (CH₂), 121.55 (C-2 and C-6), 122.78 (CH=C), 131.50 (C=CH), 135.31 (C-3 and C-5), 151.30 (C-4), 153.75 (C-1), 159.35 (C=O), 161.44 (C=O), 178.57 (C=S); Anal. calc. for C₁₄H₁₄N₂O₅S₂ (354.40): C, 47.45; H, 3.98; N, 7.90; Found: C, 47.53; H, 3.85; N, 7.81.

2.1.13. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl methanesulfonate (3m)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formyl-2-methoxyphenyl methanesulfonate (0.230 g, 1 mmol) to yield compound 3m as an orange solid. Yield: 92%; mp 252–254°C; IR (KBr) cm^-1, ν: 3365 (NH), 3241 (NH), 1692 (C=O), 1666 (C=O), 1630 (CH=C), 1361 (SO₂), 899 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.43 (s, 3H, CH₃), 3.88 (s, 3H, OCH₃), 7.41 (d, 1H, J = 8.5 Hz, H-6), 7.78 (dd, 1H, J = 9.0, 1.5 Hz, H-5), 8.15 (d, 1H, J = 1.5 Hz, H-3), 8.28 (s, 1H, CH=C), 12.38 (s, 1H, NH), 12.49 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) 37.35 (CH₃), 55.63 (OCH₃), 113.11 (C-3), 117.88 (C-6), 127.43 (C-5), 132.50 (CH=C), 140.35 (C-4), 150.62 (C-1), 152.11 (C-2), 154.44 (C=CH), 160.34 (C=O), 161.55 (C=O), 179.13 (C=S); Anal. calc. for C₁₃H₁₂N₂O₆S₂ (356.37): C, 43.82; H, 3.39; N, 7.86; Found: C, 43.94; H, 3.48; N, 7.97.

2.1.14. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl ethanesulfonate (3n)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formyl-2-methoxyphenyl ethanesulfonate (0.244 g, 1 mmol) to yield compound 3n as an orange solid. Yield: 87%; mp 241–243°C; IR (KBr) cm^-1, ν: 3389 (NH), 3191 (CH Ar), 1662 (C=O), 1651 (CH=C), 1330 (SO₂), 1100 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.40 (t, 3H, J = 8.0 Hz, CH₃), 3.56 (q, 2H, J = 7.5 Hz, CH₂), 3.87 (s, 3H, OCH₃), 7.39 (d, 1H, J = 9.0 Hz, H-6), 7.77 (d, 1H, J = 9.0 Hz, H-5), 8.15 (s, 1H, H-3), 8.28 (s, 1H, CH=C), 12.38 (s, 1H, NH), 12.49 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.08 (CH₃), 46.04 (CH₂), 56.16 (OCH₃), 112.78 (C-3), 118.14 (C-6), 127.14 (C-5), 132.39 (C=CH), 140.50 (C-4), 150.63 (C-1), 152.08 (C-2), 154.16 (CH=C), 159.50 (C=O), 161.58 (C=O), 178.52 (C=S); Anal. calc. for C₁₄H₁₄N₂O₆S₂ (370.39): C, 45.40; H, 3.81; N, 7.56; Found: C, 45.33; H, 3.70; N, 7.66.

2.1.15. 4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl propane-1-sulfonate (3o)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 4-formyl-2-methoxyphenyl propane-1-sulfonate (0.258 g, 1 mmol) to yield compound 3o as a yellow solid. Yield: 81%; mp 213–215°C; IR (KBr) cm^-1, ν: 3465 (NH), 3255 (NH), 2215 (CH aliphatic), 1681 (C=O), 1611 (CH=C), 1340 (SO₂), 1266 (C-O), 1105 (C=S) ; ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.04 (t, 3H, J = 7.5 Hz, CH₃), 1.90 (sextet, 2H, J = 7.5 Hz, CH₂), 3.53 (t, 2H, J = 7.5 Hz, CH₂), 3.88 (s, 3H, OCH₃), 7.39 (d, 1H, J = 8.5 Hz, H-6), 7.78 (dd, 1H, J = 8.5, 2.0 Hz, H-5), 8.15 (d, 1H, J = 2.0 Hz, H-3), 8.28 (s, 1H, CH=C), 12.38 (s, 1H, NH), 12.49 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.34 (CH₃), 17.11 (CH₂), 51.90 (CH₂), 55.35 (OCH₃), 113.15 (C-3), 117.53 (C-6), 127.11 (C-5), 132.44 (C=CH), 140.22 (C-4), 150.60 (C-1), 162.43 (C-2), 154.11 (CH=C), 160.13 (C=O), 161.35 (C=O), 179.55 (C=S); Anal. calc. for C₁₅H₁₆N₂O₆S₂ (384.42): C, 46.87; H, 4.20; N, 7.29; Found: C, 46.91; H, 4.10; N, 7.35.

2.1.16. 5-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl methanesulfonate (3p)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 5-formyl-2-methoxyphenyl methanesulfonate (0.230 g, 1 mmol) to yield compound 3p as a yellow solid. Yield: 90%; mp 275–277°C; IR (KBr) cm^-1, ν: 3375 (NH), 3245 (NH), 1688 (C=O), 1659 (C=O), 1610 (CH=C), 1350 (SO₂), 966 (C=S) ; ¹H NMR (500 MHz, DMSO-d₆) δ_H 3.36 (s, 3H, CH₃), 3.97 (s, 3H, OCH₃), 7.34 (d, 1H, J = 10.0 Hz, H-3), 8.23 (d, 1H, J = 9.5 Hz, H-4), 8.26 (s, 1H, H-6), 8.61 (s, 1H, CH=C), 12.34 (s, 1H, NH), 12.43 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 38.45 (CH₃), 56.63 (OCH₃), 113.09 (C-3), 117.11 (C-6), 123.91 (C-4), 129.16 (C=CH), 137.13 (C-5), 154.40 (C-1), 155.82 (C-2), 156.49 (CH=C), 159.90 (C=O), 161.84 (C=O), 178.33 (C=S); Anal. calc. for C₁₃H₁₂N₂O₆S₂ (356.37): C, 43.82; H, 3.39; N, 7.86; Found: C, 43.77; H, 3.22; N, 7.77.

2.1.17. 5-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl ethanesulfonate (3q)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 5-formyl-2-methoxyphenyl ethanesulfonate (0.244 g, 1 mmol) to yield compound 3q as a yellow solid. Yield: 92%; mp 250–252°C; IR (KBr) cm^-1, ν: 3388 (NH), 2275 (CH aliphatic), 1677 (C=O), 1616 (CH=C), 1365 (SO₂), 1277 (C-O), 1100 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.41 (t, 3H, J = 7.5 Hz, CH₃), 3.56 (q, 2H, J = 7.5 Hz, CH₂), 3.96 (s, 3H, OCH₃), 7.33 (d, 1H, J = 9.5 Hz, H-3), 8.22 (dd, 1H, J = 9.5, 2.0 Hz, H-4), 8.26 (s, 1H, CH=C), 8.62 (d, 1H, J = 2.5 Hz, H-6), 12.34 (s, 1H, NH), 12.43 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 8.10 (CH₃), 45.65 (CH₂), 56.63 (OCH₃), 113.02 (C-3), 117.06 (C-6), 125.32 (C-4), 129.12 (C=CH), 137.05 (C-5), 154.40 (C-1), 155.85 (C-2), 159.94 (CH=C), 161.85 (C=O), 161.88 (C=O), 178.32 (C=S); Anal. calc. for C₁₄H₁₄N₂O₆S₂ (370.39): C, 45.40; H, 3.81; N, 7.56; Found: C, 45.55; H, 3.77; N, 7.44.

2.1.18. 5-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2-methoxyphenyl propane-1-sulfonate (3r)

It is obtained from the reaction of 2-thiobarbituric acid (0.144 g, 1 mmol) with 5-formyl-2-methoxyphenyl propane-1-sulfonate (0.258 g, 1 mmol) to yield compound 3r as a yellow solid. Yield: 91%; mp 227–229°C; IR (KBr) cm^-1, ν: 3403 (NH), 3343 (NH), 2100 (CH aliphatic), 1683 (C=O), 1675 (C=O), 1615 (CH=C), 1345 (SO₂), 1200 (C=S); ¹H NMR (500 MHz, DMSO-d₆) δ_H 1.06 (t, 3H, J = 8.5 Hz, CH₃), 1.90 (sextet, 2H, J = 8.0 Hz, CH₂), 3.53 (t, 2H, J = 8.0 Hz, CH₂), 3.97 (s, 3H, OCH₃), 7.34 (d, 1H, J = 8.5 Hz, H-3), 8.21 (d, 1H, J = 8.6 Hz, H-4), 8.26 (s, 1H, H-6), 8.61 (s, 1H, CH=C), 12.34 (s, 1H, NH), 12.42 ppm (s, 1H, NH); ¹³C NMR (125 MHz; DMSO-d₆) δ_C 12.36 (CH₃), 17.04 (CH₂), 52.33 (CH₂), 56.60 (OCH₃), 113.00 (C-3), 117.11 (C-6), 125.30 (C-4), 129.08 (C=CH), 136.95 (C-5), 154.35 (C-1), 155.82 (CH=C), 156.39 (C-2), 159.86 (C=O), 161.81 (C=O), 178.30 (C=S); Anal. calc. for C₁₅H₁₆N₂O₆S₂ (384.42): C, 46.87; H, 4.20; N, 7.29; Found: C, 46.91; H, 4.11; N, 7.15.

2.2. Estimation of the inhibitory activity against acetylcholine esterase & butyrylcholine esterase enzymes

The assays were conducted using a 96-well plate, as reported previously [42–44]. A microplate reader (Tecan, Männedorf, Switzerland) was used. Bovine serum albumin was purchased from Sigma-Aldrich (MO, USA). AChE from electric eel (type VI-S lyophilized powder, EC 3.1.1.7) and horse BChE (EC 3.1.1.8) enzymes were purchased from Sigma-Aldrich. Butyrylthiocholine iodide and acetylthiocholine were utilized as substrates in BChE and AChE assays, respectively and purchased from Sigma-Aldrich. Dithio-bis-(2-nitrobenzoic acid) (DTNB) was served as an indicator and purchased from Sigma-Aldrich. Buffers and other chemicals were of extra pure analytical grade. Briefly, 170 μl of Tris-HCl buffer (200 mM, pH 7.5) was added followed by 20 μl at different concentrations of the tested compounds (125–1.953 μg ml^-1) and then 20 μl of the enzyme solution (0.1 U ml^-1). After an incubation period of 10 min at 25°C, 40 μl of DTNB and then 20 μl of the substrate (1.11 mM) were added. All samples were dissolved in MeOH. The intensity of the developed color was measured at 405 nm using a microplate reader (reading A) and control without the inhibitor was measured (reading B). Blank assays were performed by replacing the enzyme (20 μl) with buffer and their absorbances were recorded for correction of the spontaneous lysis of the indicator or inherent color of the inhibitor. All the reactions were performed in triplicate. Linear regression was performed for the calculation of the IC₅₀ (50% inhibitory concentration). Microsoft Excel 2010 (WA, USA) and GraphPad Instat 6.0 (Graphpad Software Inc, CA, USA) software were used for the data analysis where the % of inhibition was calculated according to the following equation.

$$\%\text{ Inhibition}=\left(1-\frac{\mathrm{reading}A}{\mathrm{reading}B}\right)\times 100$$

The study was approved by the Medical Research Ethical Committee of the National Research Centre (04411023).

2.3. Enzyme kinetics & mode of inhibition

Lineweaver-Burk plot analysis was utilized to determine the mode of inhibition of compound 3r against the AChE enzyme. Plots were constructed at inhibitor concentration levels (3.04, 9.12 and 27.3 μM) over concentrations of substrate (1.11, 0.56, 0.27, 0.14 μM) [42]. K_i was calculated using GraphPad Prism software utilizing the equation adopted for the uncompetitive inhibition type of inhibition [45,46].

$${\text{V}}_{max}\text{App}=\frac{{\text{V}}_{max}}{1+\frac{\text{I}}{{\text{AlphaK}}_{\text{i}}}}$$

$${\text{K}}_m\text{App}=\frac{{\text{K}}_m}{1+\frac{\text{I}}{{\text{AlphaK}}_{\text{i}}}}$$

$$\text{Y}={\text{V}}_{max}\text{App}\times \frac{\text{X}}{{\text{K}}_{\text{m}}\text{App}+\text{X}}$$

Parameter I is the concentration of inhibitor.

The parameters V_max, K_m and K_i were determined by Prism which fits one best-fit value for the entire set of data.

2.4. Molecular docking

In this molecular docking study, we investigated the potential inhibitory activity of a series of new barbiturate–sulfonate hybrids against cholinesterase enzymes (AChE and BChE). The synthesized barbiturate derivatives 3a–r were assessed for their binding poses and affinities toward AChE and BChE enzymes. Initial x-ray structures of the AChE and BChE enzymes were retrieved from the Protein Data Bank (PDB) website (https://www.rcsb.org) using PDB codes 4EY7 and 1P0P, respectively. The AChE enzyme was bound to the inhibitor Donepezil, while the BChE enzyme was in complex with the substrate analog (BCH). For the docking process, we prepared the receptor by removing unnecessary chains and water molecules, adding polar hydrogens and fixing partial charges, creating suitable receptor models for docking simulations using Biovia Discovery Studio. Autodock 4 software was utilized for the docking studies. Initially, we docked the co-crystallized native ligands (Donepezil and BCH) into the active sites of the AChE and BChE enzymes, respectively, to validate the docking protocol [47].

The docking procedure involved preparing coordinate files for both the docking molecule and the target molecule. An affinity grid was calculated for the target molecule based on the position of the co-crystallized ligand. We selected a grid box of size 50 × 50 × 50 with a grid point spacing of 0.375 Å. The Lamarckian Genetic Algorithm (LGA) was employed for conformation searches, executing the Genetic Algorithm (GA) in 10 sets with a maximum of 27,000 generations per run. The gene mutation rate was set to 0.02, and the crossover rate was set to 0.8. AutoDock 4.2 was run multiple times to generate several docked conformations, and the best solution was identified by analyzing the predicted energy and the consistency of the results.

2.5. Molecular dynamics simulation

Executing molecular dynamics simulations over 100 ns utilized the Desmond software package developed by Schrödinger LLC [48]. The computational resources for these simulations were hosted on the Smart Encoders website [49]. Initial configurations of protein-ligand complexes were derived from preceding docking studies, providing predictions of ligand binding behavior under static conditions. Subsequent simulations aimed to forecast how ligand binding would unfold within a physiological context. The Protein Preparation Wizard or Maestro was employed for the initial processing of protein–ligand complexes, incorporating optimization and minimization steps for complex refinement. The System Builder tool facilitated the assembly of all systems. The solvent model chosen featured an orthorhombic box, specifically the TIP3P (Transferable Intermolecular Interaction Potential 3 Points) model. The OPLS_2005 force field governed the ensuing simulations [50], and neutralization of models was achieved by introducing counter ions as needed. To emulate physiological conditions, a salt concentration of 0.15 M (NaCl) was introduced. The NPT ensemble (N for moles, P for pressure, and T for temperature conservation) was selected for the complete simulation, maintaining a temperature of 300 K and a pressure of 1 atm. Prior to initiating the simulation, models underwent relaxation. Trajectories were saved at 50 ps intervals for subsequent analysis, and the stability of the simulations was evaluated by calculating the root mean square deviation (RMSD) of both the protein and ligand over time.

2.6. Physicochemical properties prediction

Physicochemical properties prediction is founded on a diverse array of computational methods and empirical models. SWISSADME utilizes a combination of established algorithms and databases to forecast properties crucial for drug discovery. These include molecular weight, which influences pharmacokinetics and dosing; lipophilicity (logP), which indicates a compound's affinity for lipid environments and its potential to penetrate cell membranes; water solubility, a key determinant of a drug's dissolution and bioavailability; and gastrointestinal absorption, which evaluates the likelihood of a compound being absorbed into the bloodstream after oral administration. SWISSADME integrates these predictions with other parameters like drug-likeness and medicinal chemistry friendliness, which are essential for identifying molecules with desirable characteristics for further development [51].

The accuracy of SWISSADME predictions stems from its reliance on validated datasets and robust computational methodologies. While no prediction tool can guarantee absolute accuracy, SWISSADME has been extensively validated against experimental data, demonstrating reliable performance across various chemical classes and molecular structures. Additionally, continuous updates and improvements ensure that the server remains aligned with the latest advancements in predictive modeling and drug discovery research. Researchers can thus have confidence in the predictions generated by SWISSADME, using them as a guide to prioritize compounds for synthesis and experimental evaluation, thereby enhancing the efficiency of the drug development process [52,53].

2.7. Cytotoxic effect on hTERT-immortalized retinal pigment epithelial cell line (RPE-1)

Cell viability was assessed by the mitochondrial dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan. Procedure: All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, ME, USA). Cells were suspended in DMEM-F12 medium, 1% antibiotic-antimycotic mixture (10,000 U/ml Potassium Penicillin, 10,000 μg/ml Streptomycin Sulfate and 25 μg/ml Amphotericin B) and 1% L-glutamine at 37°C under 5% CO₂. Cells were batch cultured for 10 days, then seeded at concentration of 10 × 10³ cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37°C for 24 h under 5% CO₂ using a water jacketed Carbon dioxide incubator (Sheldon, TC2323, OR, USA). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100-50-25-12.5-6.25-3.125-0.78 and 1.56 μg/ml). After 48 h of incubation, medium was aspirated, 40 μl MTT salt (2.5 μg/ml) were added to each well and incubated for further four hours at 37°C under 5% CO₂. To stop the reaction and dissolving the formed crystals, 200 μl of 10% Sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37°C. DOX were used as positive control at 100 μg/ml gives 100% lethality under the same conditions. The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc, model 3350, CA, USA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. DMSO is the vehicle used for dissolution of plant extracts and its final concentration on the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula: ([Reading of extract/Reading of negative control]-1) × 100. A probit analysis was carried for IC₅₀ and IC₉₀ determination using SPSS 11 program [54,55].

3. Results & discussion

3.1. Chemistry

A new series of 5-benzylidene(thio)barbiturates have been designed and synthesized using the well-established Knoevenegal condensation method [41]. Various aromatic aldehydes bearing (at its para position) various alkane sulfonate moieties such as methane sulfonate, ethane sulfonate and/or propane sulfonate 2a–r were heated under reflux in ethanol with barbituric or thiobarbituric acid affording 5-benzylidenebarbiturate or 5-benzylidenethiobarbiturate 3a–r in good yields, respectively, Figure 3. Different spectroscopic techniques including IR, ¹H, ¹³C-NMR and elemental analysis were employed to elucidate the chemical structure of the newly synthesized analogs.

Figure 3.

Synthetic route toward barbiturates–sulfonate hybrids derivatives 3a–r.

In the IR spectrum, a strong stretching band indicating NH was observed at 3410–3241 cm^-1, while C=C and C=O were detected at 1600–1660 cm^-1 and 1640–1690 cm^-1, respectively. Additionally, the SO₂ band appeared in the range of 1335–1372 cm^-1. The ¹H NMR spectrum displayed singlet peaks between δ_H = 8.12–8.61 ppm, indicating the presence of characteristic methine protons. The ¹³C NMR spectrum revealed the characteristic carbons of the methine group (CH=C) in the range δ_C = 153–155 ppm. The spectral data for other aromatic and aliphatic protons aligned with the expected compound structures (please see the experimental section).

The chemical structure of derivative 3a as the representative example of the synthesized analogs 3a–r has been proved according to the spectroscopic techniques, IR (KBr) of 3a showed strong stretching vibrational bands at 3399 and 3324 cm^-1 representing NH groups, as well as stretching bands perceived at 1686 cm^-1 signifying C=O groups, while the band for SO₂ has appeared at 1391 cm^-1. ¹H NMR of 3a revealed a singlet peak for methyl moiety at the upfield zone δ_H = 3.45 ppm, whereas the characteristic methine proton of C=CH was observed at δ_H = 8.28 ppm, the NH protons appeared as a singlet peak at δ_H = 11.27 and 11.42 ppm, respectively. ¹³C NMR spectrum, disclosed a characteristic peak attributable to the methane sulfonate group at δ_C = 8.17 ppm, while the peak that showed at δ_C = 152.92 ppm represented the methine carbon (C=CH) whereas the barbiturate’ carbonyls (C=O) were depicted at δ_C = 150.15, 161.50, 163.18 ppm, respectively.

3.2. Anti-cholinesterase activity

The inhibitory activities of all synthesized barbiturates–sulfonate analogs (3a–r) on AChE and BChE were evaluated using Donepezil, a US FDA-approved drug, as the reference standard. The obtained results revealed that all the tested compounds demonstrated moderate to weak inhibitory properties on both AChE and BChE enzymes, however 3r was the most potent analog on AChE with an IC₅₀ value of 9.12 μM, while against BChE, compound 3q was the most potent among the tested series with an IC₅₀ value of 19.43 μM, compared with IC₅₀ = 0.59 μM and 0.77 μM for AChE and BChE, respectively of Donepezil. Furthermore, compounds 3c, 3e, 3f, 3j, 3k and 3o demonstrated moderate inhibitory efficacy whereas derivatives 3a, 3b, 3d, 3g, 3h, 3i, 3l, 3m, 3n, 3p and 3q showed weak activity toward AChE. On the other hand, compounds 3c, 3d, 3f, 3h, 3o and 3p were the moderated hits against BChE, while derivatives 3a, 3b, 3e, 3g, 3i, 3j, 3k, 3l, 3m, 3n and 3r demonstrated week potency (Table 1 & Supplementary Figure S36).

Table 1.

IC₅₀ of the new Benzylidene(thio)barbiturates derivatives 3a-r against AChE and BChE.

Comp. No.	AChE IC50 Average ± SD (μM)	BChE IC50 Average ± SD (μM)	SI IC50 (AChE)/IC50 (BChE)	SI IC50 (BChE)/IC50 (AChE)
3a	114.78 ± 3.48	69.96 ± 0.08	1.64	0.61
3b	16.98 ± 0.30	90.09 ± 0.015	0.18	5.30
3c	22.00 ± 0.30	29.04 ± 2.54	0.75	1.32
3d	62.93 ± 0.18	25.50 ± 0.38	2.46	0.40
3e	21.79 ± 0.40	54.53 ± 1.84	0.39	2.50
3f	26.49 ± 2.61	33.01 ± 0.21	0.80	1.24
3g	30.41 ± 0.015	112.27 ± 0.87	0.27	3.69
3h	54.89 ± 2.53	28.56 ± 2.62	1.92	0.52
3i	50.36 ± 2.06	51.26 ± 2.5	0.98	1.01
3j	24.74 ± 0.37	52.65 ± 1.35	0.47	2.12
3k	25.20 ± 3.48	65.59 ± 0.47	0.38	2.60
3l	34.94 ± 0.94	106.33 ± 1.53	0.32	3.04
3m	13.65 ± 0.47	45.86 ± 2.058	0.29	3.35
3n	35.26 ± 0.11	54.46 ± 0.80	0.64	1.54
3o	23.46 ± 0.17	34.19 ± 1.23	0.68	1.45
3p	19.88 ± 0.67	43.24 ± 0.46	0.45	2.17
3q	18.27 ± 0.53	19.43 ± 0.35	0.94	1.06
3r	9.12 ± 0.61	19.64 ± 0.16	0.46	2.15
Donepezil	0.59 ± 0.083	0.77 ± 0.0088	0.76	1.30

Values represent mean ± SD (standard deviation) of triplicate readings, n = 3.

AChE: Acetylcholinesterase.

BChE: Butyrylcholinesterase.

SI: Selectivity index.

3.3. Structure–activity relationship

Based on the obtained bio-results, two primary factors appear to play a crucial role in managing the inhibitory properties. These factors include the presence of oxygen/sulfur substituents at position-2 of barbiturate/thiobarbiturate rings and the specific type of alkanesulfonate derivative positioned at the para position of the aryl ring. Regarding the first parameter, which is the presence of oxygen/sulfur substituents (barbiturate/thiobarbiturate), it is observed that compounds containing sulfur atoms (thiobarbiturate derivatives) 3j–r exhibit superior bioactivity against both enzymes (AChE and BChE) in comparison to the corresponding oxygen-containing (barbiturate) derivatives 3a–i.

During the tested series conducted, it was observed that derivatives containing methane sulfonate (OSO₂Me) groups, specifically compounds 3a, 3d, 3g, 3j, 3m and 3p, exhibited good to moderate inhibitory potency against both enzymes. Similarly, compounds containing propane sulfonate (OSO₂Pr) derivatives (3c, 3f, 3l, 3o and 3r) demonstrated reasonable efficacy compared with their methane and/or ethane counterparts, except compound 3i. On the other hand, derivatives containing ethane sulfonate (OSO₂Et) group, such as 3e, 3h, 3k and 3n, showed lower efficacy compared with the other derivatives, except for compound 3q (with IC₅₀ values of 18.27 and 19.43 μM for AChE and BChE, respectively).

As a result, it can be concluded that the presence of a propane sulfonate moiety at position 4 of the aryl group enhances the inhibitory properties against AChE, surpassing the effects of the corresponding methane sulfonate and ethane sulfonate groups. Conversely, the corresponding propane sulfonate demonstrated reasonable potency against BChE compared with methane sulfonate or ethane sulfonate groups. Additionally, it is important to note that the nature of the X substituent, whether it is oxygen or sulfur, plays a significant role in influencing the inhibitory properties.

3.4. Cytotoxic properties

The MTT assay was utilized to evaluate the safety of the most potent cholinesterase inhibitors 3b, 3m, 3p, 3q and 3r on the hTERT-immortalized retinal pigment epithelial cell line (RPE-1). Notably, the results from this study demonstrated that these compounds exhibited no cytotoxicity toward RPE-1 cells, presuming their safety only once the appropriate in vivo studies are accomplished (Table 2 & Supplementary Table S1 & Supplementary Figure S35). These findings offer significant insights into the safety profile of these cholinesterase inhibitors, thereby informing future research endeavors and potential clinical applications.

Table 2.

Percentage cytotoxicity of compounds 3b, 3m, 3p, 3q and 3r at 100 μM on hTERT (RPE-1).

Compd. No.	The percentage cytotoxicity at 100 μM†
3b	8.25 ± 4.42
3m	17.66 ± 0.51
3p	17.66 ± 6.38
3q	16.47 ± 2.15
3r	6.53 ± 9.23

^†Percentage cytotoxicity of the compounds tested at 100 μM on hTERT-immortalized retinal pigment epithelial cell line (RPE-1).

3.5. Enzyme kinetics & mode of inhibition

This study focuses on the analysis of ligand–enzyme interactions through kinetic patterns. To investigate the inhibition mechanism of the AChE enzyme, compound 3r, the most promising derivative of Barbiturate-sulfonate, was subjected to an enzyme kinetic study alongside the reference drug Donepezil. The study calculated and presented V_max, K_m and K_i values in Supplementary Table S2. The results indicate that compound 3r is the most potent inhibitor, with the lowest IC₅₀ value (9.12 ± 0.61 μM) against AChE. Furthermore, further analysis of the compound revealed that it acts as an uncompetitive inhibitor (as shown in Supplementary Table S2). This mode of action is highly desirable for drug design as the inhibitors only bind to the enzyme when it is active and the substrate is present. Uncompetitive inhibitors decrease substrate Km and Vmax and exhibit higher inhibition with increasing substrate concentration [50]. The inhibitory constants (Ki) for compound 3r were calculated as 88.34 μM (binding to the enzyme only when a substrate is bound to the enzyme) using secondary plots (Supplementary Figure S37). The plot of three inhibition times versus the percent inhibition at the calculated IC₅₀ value of compound 3r showed that the inhibition by 3r is time-progressive indicating that the inhibitory effect of 3r on acetylcholinesterase was irreversible (Supplementary Figure S38).

3.6. Antioxidant activity

The antioxidant activity of six derivatives 3b, 3m, 3p, 3q and 3r was evaluated by measuring their ability to scavenge the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) at a concentration of 100 μg/ml, with vitamin C serving as the standard antioxidant. The results showed that all the tested candidates displayed significant antioxidant activity with reasonable IC₅₀, LC₉₀ values and percent inhibition (at 100 μg/ml) compared with the standard reference. Derivative 3q was found to be the best antioxidant agent among the tested analogs with IC₅₀ = 59.4 μg/ml, LC₉₀ = 103.4 μg/ml and percent inhibition 77% at 100 μg/ml, Supplementary Table S3.

3.7. Docking studies on AChE & BChE enzymes

The expected binding interactions of the synthesized barbiturate–sulfonate hybrids 3a–r were investigated through molecular docking studies within the binding pockets of AChE and BChE enzymes. Donepezil, renowned for its potent inhibitory role against both AChE and BChE, was chosen as the standard compound for benchmarking. Donepezil operates by inhibiting these cholinesterase enzymes, leading to increased acetylcholine levels in the brain. This rise in acetylcholine levels holds the potential to enhance cognitive function and alleviate specific symptoms associated with Alzheimer's disease. The x-ray structure of the AChE enzyme in complex with Donepezil (PDB code: 4EY7) and the x-ray structure of the BChE enzyme in complex with its substrate analog butyrylthiocholine (PDB: 1P0P) was obtained from the Protein Data Bank website.

The initial phase involved the preparation of the receptor for docking using Biovia Discovery Studio. This process included the elimination of unnecessary chains and water molecules from the receptor structure. Subsequently, polar hydrogens were incorporated, and partial charges were adjusted to ensure an accurate representation of the receptor for subsequent docking simulations. Autodock 4.2 software was employed to predict the binding interactions between the synthesized barbiturate derivatives 3a–r and the target enzymes (AChE and BChE). The validation step of the docking performance was done as we previously reported [42]. This will simplify the calculation of binding energies and allow us to visualize the ligand's position within the enzyme's binding site, thereby improving our understanding of the binding mechanisms. Initially, to verify the docking procedure, the co-crystallized native ligands (Donepezil for AChE and BCH for BChE) were re-docked into the enzymes' active sites. The resulting poses of these ligands closely matched those of the original ligands, exhibiting binding affinities of -12.34 and -12.88 kcal/mol for AChE and BChE, respectively, along with root mean square deviations (RMSD) of 0.65 and 0.74. This similarity in conformation between the docked and native ligands suggests a successful overlap and validates the docking protocol.

In this series, several compounds demonstrated significant inhibitory activity against AChE and BChE enzymes, with IC₅₀ values below 20 μM for both enzymes. Notably, compound 3r emerged as the most potent inhibitor for both AChE and BChE, with IC₅₀ values of 9.12 ± 0.61 μM and 19.64 ± 0.16 μM, respectively. Molecular docking analyses revealed that compound 3r formed favorable interactions within the AChE active site. Specifically, the carbonyl group in the thiobarbiturate ring established a crucial hydrogen bond with PHE295, resembling the interactions observed for Donepezil in the reported x-ray crystal structure (PDB ID: 4EY7). The free NH group in the thiobarbiturate ring also formed a hydrogen bond with the Tyr341 residue (Figure 4).

Figure 4.

Proposed binding interactions (3D and 2D) of compound 3r into the AChE enzyme active site. Carbon atoms of 3r are colored in orange.

Furthermore, the sulphonate group exhibited several intriguing interactions. The sulfur atom engaged in a Pi–Sulfur interaction with His447, and the S=O formed a hydrogen bond with the Gly121 residue. Additionally, the phenyl and thiobarbiturate rings participated in Pi–Pi stacking interactions with the key amino acids Trp286 and Tyr124. The diverse range of interactions, including H-bonding, Pi–Pi stacking, Pi–Sulfur and carbon–hydrogen interactions of compound 3r within the AChE active site, contributed to its potent AChE inhibition (Figure 4).

Moreover, we conducted a detailed examination of the molecular interactions of compound 3r within the BChE active site. Consistent with our observations in interactions with AChE, the thiobarbiturate ring engaged in several crucial interactions with cholinesterases. Specifically, the thio, carbonyl and amino functional groups within the thiobarbiturate ring formed three significant hydrogen bonds with Ile69, Tyr332 and Ser287 residues, respectively (Supplementary Figure S39). Additionally, the S=O bond of the sulphonate group established another hydrogen bond with the His438 residue.

Alkyl substituents displayed hydrophobic interactions within the BChE enzyme active site. The propyl substituent engaged in Pi–sigma and Pi–alkyl interactions with Trp82 and His438 residues. Additionally, the CH₃ of the methoxy substituent formed Pi–alkyl and carbon–hydrogen bonds with Ala328, Trp430 and Tyr332 residues. Variations in interactions among the compounds can be attributed to the length of the alkyl group attached to the sulfonate group and the substitution position. These factors influence the distance to adjacent amino acids, thus affecting interactions within the BChE pocket. Nonetheless, consistent interactions with His438 and Phe329 were observed across different barbiturate and thiobarbiturate derivatives.

3.8. Molecular dynamics simulations

Utilizing molecular dynamics simulations, we delved deeper into examining the behavior of compound 3r within the binding pocket of the AChE enzyme. The aim was to anticipate its performance in a biological context. The analysis of Desmond simulation trajectories encompassed the computation of Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and the assessment of protein–ligand contacts throughout the molecular dynamics trajectory. Supplementary Figure S6 illustrates the temporal evolution of RMSD values concerning the backbone atoms of the protein with the bound ligand. Notably, no significant alterations were observed in the alignment of the ligand concerning the protein throughout the entire simulation.

Throughout the simulation, the RMSD plot for the complex suggests the achievement of stability approximately at the 10 ns mark, with an average RMSD value persisting at 2.0 Å until 100 ns. Moreover, the variations in RMSD values consistently remain within the acceptable range of 1.9 Å, typically considered suitable for small predicted proteins. Additionally, fluctuations within 3.6 Å are observed in the RMSD values representing the ligand's alignment to the protein after stability is reached. These findings strongly imply a steady and enduring binding of the ligand to the binding site of the receptor throughout the entirety of the simulation.

The Residue-wise RMSF illustrates the computed values for specific residues of the AChE enzyme in the presence of compound 3r (Figure 5). Residues exhibiting notable peaks correlate with the N- and C-terminal segments or are commonly associated with loop regions, as discerned from the scrutiny of molecular dynamics trajectories (refer to Supplementary Figure S40). Conversely, lower RMSF values observed for residues within the binding site indicate a stable and securely established interaction between the ligand and the protein. Additionally, Supplementary Figure S41 displays the RMSD of the backbone atoms of AChE and compound 3r over time. The left Y-axis denotes the fluctuation in protein RMSD during the simulation, while the right Y-axis represents the variation in ligand RMSD throughout the duration of the simulation. Also, Supplementary Figure S42 depicts a heatmap illustrating the interactions between the protein and ligand across the entire trajectory (3r_4EY7).

Figure 5.

Root Mean Square Fluctuation (RMSF) of AChE complexed with the barbiturate–sulfonate hybrids 3r.

Various crucial interactions between ligands and proteins take center stage, emphasizing hydrogen bonds and hydrophobic interactions. Supplementary Figure S9 vividly portrays this intricate interplay. Notably, Phe295 and Arg296 play pivotal roles, especially concerning hydrogen bonds. Additionally, compound 3r exhibits diverse interactions with residues Glu81, Tyr124, Trp286, Tyr337, Phe338 and Tyr341. These interactions encompass hydrogen bonding, hydrophobic interactions, ionic bonding and water bridges. These findings align with those obtained from the molecular docking analysis of compound 3r within the active site of the AChE enzyme (see Figure 4).

To enhance the understanding of these interactions, stacked bar charts have been utilized, providing a normalized overview throughout the entire trajectory. In this context, a value of 1.0 signifies that the specific interaction persisted throughout 100% of the simulation duration. It is crucial to note that values exceeding 1.0 are also plausible, indicating scenarios where a protein residue may participate in multiple interactions of the same subtype with the ligand, introducing an intriguing layer of complexity to the molecular dynamics profile.

3.9. In-silico pharmacokinetic analysis

The physicochemical properties and drug-likeness of the recently synthesized compounds 3a–r were evaluated using the SwissADME server. These compounds exhibited promising expected drug bioavailability (Table 3). For instance, compound 3r exhibited physicochemical properties conducive to oral availability, closely resembling those of Donepezil (Figure 6). Additional figures depicting the physicochemical space for the remaining compounds are provided in the Supporting Information file (Supplementary Figures S43 & S44). Interestingly, all compounds exhibited a promising calculated topological polar surface area (TPSA) higher than the reference compound Donepezil, signifying high polarity. This aligns with their anticipated favorable gastrointestinal absorption for most of the compounds. The average molecular weight of our compounds is approximately 350 Da, providing an opportunity for further lead optimization while keeping their molecular weight within acceptable ranges.

Table 3.

In silico pharmacokinetic properties of compounds 3a-r.

No.	MW (g/mol)	#H-bond acceptor	#H-bond donors	#Rotatable bonds	TPSA	Consensus Log P	GI absorption	P-gp substrate	Lipinski #violations	PAINS #alerts
3a	310.28	6	2	3	127.02	0.27	High	No	0	1
3b	324.31	6	2	4	127.02	0.59	High	No	0	1
3c	338.34	6	2	5	127.02	0.94	High	No	0	1
3d	340.31	7	2	4	136.25	0.29	High	No	0	1
3e	354.34	7	2	5	136.25	0.62	High	No	0	1
3f	368.36	7	2	6	136.25	0.96	High	No	0	1
3g	340.31	7	2	4	136.25	0.31	High	No	0	1
3h	354.34	7	2	5	136.25	0.60	High	No	0	1
3i	368.36	7	2	6	136.25	0.94	High	No	0	1
3j	326.35	5	2	3	142.04	0.77	Low	No	0	1
3k	340.37	5	2	4	142.04	1.09	Low	No	0	1
3l	354.40	5	2	5	142.04	1.43	High	No	0	1
3m	356.37	6	2	4	151.27	0.77	Low	No	0	1
3n	370.40	6	2	5	151.27	1.11	Low	No	0	1
3o	384.43	6	2	6	151.27	1.46	Low	No	0	1
3p	356.37	6	2	4	151.27	0.79	Low	No	0	1
3q	370.40	6	2	5	151.27	1.11	Low	No	0	1
3r	384.43	6	2	6	151.27	1.45	Low	No	0	1
Donepezil	379.49	4	0	6	38.77	4.00	High	Yes	0	0

GI: Gastrointestinal; PAINS: Pan-Assay INterference CompoundS; MW: Molecular weight; TPSA: Topological polar surface area.

Figure 6.

Physicochemical space for oral availability of compound 3r and Donepezil. LIPO (Lipophilicity), POLAR (Polarity), INSOLU (Insolubility), INSATU (Instauration), FLEX (Flexibility). The colored zone in pink is the suitable physicochemical space for oral availability.

Our compounds, featuring barbiturates or thiobarbiturates as the main scaffold, are anticipated to exhibit low lipophilicity (hydrophilic) due to their relatively low molecular weight and having only one phenyl ring, resulting in consensus Log P values around one. Additionally, some compounds demonstrated high gastrointestinal (GI) absorption, and most of them are not likely to be effluxed out of cells by P-glycoprotein (P-gp), allowing them to exert their therapeutic effects. Furthermore, the compounds adhered to Lipinski's rule for drug-likeness without any violations; however, they did exhibit a single violation of PAINS (Pan-Assay INterference CompoundS), attributed to the reactivity of the exocyclic alkene in alkylidene barbiturates, which might act as a Michael Acceptor. Detailed in silico pharmacokinetic properties are provided in Table 3. The SwissADME server, while useful for predicting the pharmacokinetic properties of small molecules, has limitations including variable prediction accuracy and a narrow scope of properties covered. Additionally, it may not be suitable for high-throughput screening and is dependent on input quality and data availability for reliable predictions [51,56].

4. Conclusion

In conclusion, the synthesis and evaluation of a series of 5-Benzylidene(thio)barbiturates as inhibitors of AChE and BChE have been described. The results demonstrated that the synthesized analogs exhibited varying degrees of inhibitory activity, with compounds 3b, 3m, 3p, 3q and 3r showing the most potent effects. Compound 3r displayed the highest inhibitory activity against AChE, while compound 3q showed the best inhibitory activity against BChE. Although the inhibitory properties of the synthesized compounds were lower than that of the reference standard Donepezil, they still showed promising potential for further development as drug candidates. Furthermore, toxicological bioassays conducted on a retinal pigment epithelial cell line indicated no cytotoxicity of the most potent compounds. Antioxidant assays revealed reasonable antioxidant values for all the compounds, particularly compound 3q. Molecular docking studies provided insights into the binding interactions between the synthesized compounds and the active sites of AChE and BChE, suggesting that they fit well in the enzyme's active sites. Overall, this study contributes valuable information regarding the potential of barbiturate–sulfonate hybrids as drug candidates, highlighting their inhibitory activity, safety profile, antioxidant properties and binding interactions with the target enzymes. Further investigations and optimization of these compounds are warranted to maximize their therapeutic potential.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2366158

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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