Design and synthesis of carbazole–acrylamide–pyridinium hybrids as dual acetylcholinesterase/butyrylcholinesterase inhibitors and evaluation of their cytotoxic profile on SH-SY5Y and HEK293 cell lines

Abstract

A novel series of seventeen carbazole–acrylamide–pyridinium-N-phenylacetamide hybrids (12a–q) was designed and synthesized as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) for potential Alzheimer's disease therapy. Their inhibitory activity was evaluated against both enzymes using tacrine and galantamine as reference standards. Tacrine exhibited IC₅₀ values of 58.34 nM against AChE and 160.50 nM against BChE, while galantamine showed 124.72 nM and 216.23 nM, respectively. The synthesized derivatives demonstrated IC₅₀ values ranging from 6.41 to 74.93 nM against AChE and from 38.05 to 191.96 nM against BChE. All compounds were more potent than galantamine, and most outperformed tacrine as well. Compound 12i was the most potent AChE inhibitor (IC₅₀ = 6.41 nM), showing 9.1-fold and 19.5-fold higher potency than tacrine and galantamine, respectively. Compound 12g was the strongest BChE inhibitor (IC₅₀ = 38.05 nM), with 4.2-fold and 5.7-fold higher activity than tacrine and galantamine. Kinetic analysis revealed that all of the most potent compounds act as competitive inhibitors. Cytotoxicity evaluation revealed that compounds 12i and 12g exhibited lower toxicity toward HEK293 cells than sorafenib, with favorable selectivity toward SH-SY5Y cells. These results establish the carbazole–acrylamide–pyridinium scaffold as a promising lead for developing safer cholinesterase inhibitors for Alzheimer's disease.

A novel series of seventeen carbazole–acrylamide–pyridinium-N-phenylacetamide hybrids (12a–q) was designed and synthesized as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) for potential Alzheimer's disease therapy.

Introduction

Alzheimer's disease (AD) is recognized as the most prevalent neurodegenerative disorder and the leading cause of dementia, accounting for 60–80% of all dementia cases, with substantial economic and social burden on patients, families, and healthcare systems.¹ Although AD etiology is multifactorial, involving amyloid β accumulation, tau pathology, oxidative stress, neuro-inflammation, and disruptions in multiple neurotransmitter systems, the cholinergic hypothesis remains the only well-validated neurochemical basis with established pharmacological therapeutic approaches.² Specifically, the degeneration of cholinergic neurons and the resulting deficiency in acetylcholine (ACh) represents a hallmark feature contributing to cognitive decline and functional impairment.³ While current standard-of-care medications including donepezil, rivastigmine, and galantamine provide symptomatic benefit by selectively inhibiting acetylcholinesterase (AChE) to elevate synaptic ACh levels, recent pharmacological studies have demonstrated that butyrylcholinesterase (BChE), particularly in advanced stages of AD, significantly contributes to ACh degradation.⁴ Consequently, dual inhibition strategies targeting both AChE and BChE have emerged as a more comprehensive and potentially superior therapeutic approach to enhance cholinergic neurotransmission and improve cognitive outcomes in AD patients.⁵

Molecular hybridization is an important strategy in medicinal chemistry for designing new bioactive compounds.⁶ To apply this approach, effective pharmacophore fragments are first identified from the literature based on the desired therapeutic target. These fragments are then connected using appropriate linkers.⁷ This method can potentially reduce side effects and improve efficacy by linking only the key bioactive regions.

One of the most recognized pharmacophore in medicinal chemistry is carbazole.^8,9 It is a tricyclic nitrogen-containing heterocycle found in natural, semisynthetic, and synthetic biologically active compounds.¹⁰ One of the reported effects of carbazole-containing compounds is the inhibition of AChE and BChE. For example, carbazole derivatives A and B (Fig. 1) were found to be active against cholinesterase (ChE) enzymes.^11,12 Compounds B contain an acrylamide unit in their structures, which is also present in ChE inhibitors C (Fig. 1).¹³ Therefore, acrylamide can serve as an appropriate linker for the design of new ChE inhibitors. On the other hand, the pyridinium-N-phenylacetamide moiety was found in potent ChE inhibitors D (Fig. 1).¹⁴ Recently, the pyridinium moiety has gained significant attention as a suitable bioisosteric replacement for the protonatable nitrogen of donepezil, mimicking its ionic interaction with the catalytic anionic site of the enzyme.¹⁴

Fig. 1. Design strategy for carbazol–acrylamid–pyridinium-N-phenylacetamide derivatives as new potent ChE inhibitors.

Based on these observations, carbazole and pyridinium-N-phenylacetamide were selected as two pharmacophores that can confer ChE inhibitory activity. Connecting these pharmacophores with an acrylamide unit provided a rational design for a carbazole–acrylamide–pyridinium-N-phenylacetamide scaffold as a new class of ChE inhibitors (Fig. 1). Consequently, seventeen derivatives (compounds 12a–q) of this scaffold were synthesized through efficient chemical reactions and evaluated both in vitro and in silico as promising anti-AD agents.

Several studies have demonstrated that, in addition to the established roles of cholinesterase inhibitors in neurodegenerative disorders, these agents also exhibit significant antiproliferative and pro-apoptotic effects across various cancer cell lines, including neuroblastoma.^15–17 In this context, the newly designed carbazole–acrylamide–pyridinium-N-phenylacetamide hybrids were also evaluated for their potential anti-neuroblastoma activity, aiming to explore their dual therapeutic relevance in both neurodegenerative and oncological settings.

Experimental

Instrumentation

Melting points were determined using an Electrothermal-9200 melting point apparatus (Electrothermal Engineering Ltd., Essex, UK) in open glass capillaries under air. FTIR spectra were recorded on a Shimadzu IRSpirit QATR-S spectrophotometer (Shimadzu Corporation, Kyoto, Japan) in the range of 600–4000 cm⁻¹ at room temperature. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were obtained on a Bruker Avance III HD 400 spectrometer (Bruker BioSpin, Billerica, MA, USA) using CDCl₃ as solvent and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) spectra were obtained using the electrospray ionization (ESI) technique on a Thermo Fisher Scientific Q Exactive™ Hybrid Quadrupole-Orbitrap™ instrument. Elemental analyses (C, H, N) were performed using a Heraeus CHN-O-Rapid analyzer (Heraeus GmbH, Hanau, Germany).

General procedure for the synthesis of 9-benzyl-9H-carbazole (3a) and 9-ethyl-9H-carbazole (3b)

Carbazole 1 (20 mmol) was dissolved in CH₃CN for the preparation of 9-benzyl-9H-carbazole 3a, or in DMSO for the synthesis of 9-ethyl-9H-carbazole 3b. Potassium hydroxide (30 mmol) was then added, and the resulting mixture was stirred at room temperature for 15 minutes. Subsequently, ethyl iodide (24 mmol) or benzyl bromide (24 mmol) was added for the synthesis of 3a or 3b, respectively. The reaction mixture was stirred at r.t for 24 hours. After the reaction was completed (monitored by TLC), cold water was added, and the precipitate was filtered. The crude product was recrystallized with tert-butyl ether to afford the corresponding pure product. Yield: 91% (light cream solid).¹⁸

Selective formylation of 1-(9-benzyl-9H-carbazol-3-yl)ethan-1-one (5a) and 1-(9-ethyl-9H-carbazol-3-yl)ethan-1-one (5b)

Phosphorus oxychloride (12 mmol) was added dropwise to 10 mmol of DMF (4), in a cooled condition at 0–5 °C, and stirred in an ice bath for 1 hour. Then, carbazole (10 mmol) and Phosphorus oxychloride (10 ml, as solvent) was added and reacted at 70 °C for 5–6 days. The mixture was poured into 200 mL of ice water, followed by the addition of NaOH to adjust the pH to 8.0. The crude product was filtered off and washed with water and purified with ethyl acetate to give monoformaldehydation of 9-benzyl-9H-carbazole and 9-ethyl-9H-carbazole 5a and 5b, respectively. Yield 85–88% (cream solid).

General procedure for the synthesis of (E)-3-(9-benzyl-9H-carbazol-3-yl)acrylic acid (6a) and (E)-3-(9-ethyl-9H-carbazol-3-yl)acrylic acid (6b)

A mixture of compound 5a or 5b (12.5 mmol), malonic acid (30 mmol) and β-alanine (3 mmol) dissolved in pyridine (10 mL) and stirred at 100 °C for 2 hours. After completion of the reaction (monitored by TLC), cold water was added to the reaction mixture, followed by 37% HCl to adjust the pH to 4. The resulting precipitate was collected by filtration, washed with water, and purified by ethyl acetate to afford the desired products. Yield: 90% (light yellow solid).

Procedure for the synthesis of (E)-3-(9-benzyl-9H-carbazol-3-yl)-N-(pyridin-3-ylmethyl)acrylamide (8a) and (E)-3-(9-ethyl-9H-carbazol-3-yl)-N-(pyridin-3-ylmethyl)acrylamide (8b)

Compound 6a or 6b (10 mmol, 1.0 equiv.) was dissolved in 7 mL of DMF, and triethylamine (Et₃N, 15 mmol, 1.5 equiv.) was added to the solution. The mixture was stirred in an ice bath for 15 minutes. Then, TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 12.5 mmol, 1.25 equiv.) was added, and the reaction was allowed to continue at room temperature for 1 hour. After that, pyridin-3-ylmethanamine 7 (12 mmol, 1.2 equiv.) was added, and stirring was continued. The progress of the reaction was monitored by thin-layer chromatography (TLC). After the completion of the reaction, cold water was added, and the precipitate was filtered. The crude product was purified with ethanol to afford the corresponding pure compounds. Yield: 71% (light yellow solid).¹⁹

A general procedure for the synthesis of 2-chloro-N-substituted acetamide derivatives (11a–i)

Aniline derivatives 9a–i (50 mmol) were suspended in 25 mL DMF and cooled to 0 °C. Then, chloroacethyl chloride (10, 65 mmol) was added. The reaction mixture was then stirred at room temperature for 24 h. At the end of the reaction, the reaction mixture was diluted with water, poured into crashed ice, and the obtained precipitate were filtered off, Finally, residue was washed with water to obtain 2-chloro-N-substituted acetamide derivatives 11a–i.²⁰

The general procedure for the synthesis of final products 12a–q

A mixture of 2-chloro-N-substituted acetamide derivatives 11a–i (1.2 mmol) and compound 8a or 8b (1 mmol) in acetonitrile (10 mL) was stirred under reflux for 48–72 hours. After completion of the reaction (monitored by TLC), the solvent was removed under reduced pressure. The crude product was purified by recrystallization from ethyl acetate. The resulting crystals were collected by filtration and dried at room temperature to afford the final products 12a–q.²¹

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(phenylamino)ethyl)pyridin-1-ium chloride (12a)

Yield: 83%. Light yellow solid, mp: 245–247 °C; IR (KBr, ν_max): 3302.99, 3020.33, 1683.06, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.21 (s, 1H), 9.12 (s, 1H), 9.07 (t, J = 5.9 Hz, 1H), 9.03 (d, J = 6.1 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.25–8.18 (m, 2H), 7.75–7.63 (m, 6H), 7.47 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.8 Hz, 2H), 7.30–7.23 (m, 4H), 7.19 (d, J = 6.9 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 6.79 (d, J = 15.7 Hz, 1H), 5.77 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.7 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.64, 163.66, 145.53, 145.23, 145.15, 141.49, 141.25, 141.13, 140.75, 138.84, 138.04, 129.42, 129.10, 127.83, 127.62, 127.24, 126.86, 126.50, 125.85, 124.39, 123.11, 122.65, 121.18, 121.05, 120.14, 119.63, 118.93, 110.61, 110.46, 62.72, 46.20; anal. calcd for C₃₆H₃₁ClN₄O₂: C, 73.65; H, 5.32; N, 9.54; found: C, 73.86; H, 5.48; N, 9.70.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(o-tolylamino)ethyl)pyridin-1-ium chloride (12b)

Yield: 79%. Light yellow solid, mp: 241–243 °C; IR (KBr, ν_max): 3281.47, 3004.54, 1667.28, 1619.93 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.39 (s, 1H), 9.13 (s, 1H), 9.08 (t, J = 5.9 Hz, 1H), 9.04 (d, J = 6.1 Hz, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.26–8.18 (m, 2H), 7.75–7.64 (m, 4H), 7.52–7.42 (m, 2H), 7.30–7.23 (m, 5H), 7.21–7.11 (m, 4H), 6.79 (d, J = 15.7 Hz, 1H), 5.81 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 2.31 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.63, 163.93, 145.48, 145.20, 145.13, 141.49, 141.27, 141.13, 140.74, 138.04, 135.80, 132.12, 130.97, 129.10, 127.83, 127.64, 127.24, 126.86, 126.49, 126.15, 125.85, 125.21, 123.12, 122.65, 121.19, 121.05, 120.14, 118.91, 110.61, 110.47, 62.50, 46.20, 18.50; anal. calcd for C₃₇H₃₃ClN₄O₂: C, 73.93; H, 5.53; N, 9.32; found: C, 74.10; H, 5.68; N, 9.50.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(p-tolylamino)ethyl)pyridin-1-ium chloride (12c)

Yield: 85%. Light yellow solid, mp: 244–246 °C; IR (KBr, ν_max): 3305.86, 3023.20, 1662.97, 1612.75 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.11 (s, 1H), 9.11 (s, 1H), 9.07 (t, J = 6.0 Hz, 1H), 9.02 (d, J = 6.1 Hz, 1H), 8.63 (d, J = 8.2 Hz, 1H), 8.44 (s, 1H), 8.25–8.17 (m, 2H), 7.75–7.64 (m, 4H), 7.54 (d, J = 8.2 Hz, 2H), 7.47 (t, J = 7.7 Hz, 1H), 7.32–7.21 (m, 4H), 7.21–7.18 (m, 2H), 7.14 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 15.7 Hz, 1H), 5.74 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 2.26 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.64, 163.36, 145.50, 145.20, 145.13, 141.49, 141.26, 141.13, 140.74, 138.04, 136.33, 133.36, 129.80, 129.10, 127.83, 127.61, 127.24, 126.86, 126.50, 125.85, 123.12, 122.65, 121.18, 121.05, 120.14, 119.61, 118.93, 110.61, 110.46, 62.68, 46.20, 20.93; anal. calcd for C₃₇H₃₃ClN₄O₂: C, 73.93; H, 5.53; N, 9.32; found: C, 74.11; H, 5.69; N, 9.50.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-methoxyphenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12d)

Yield: 81%. Light yellow solid, mp: 230–232 °C; IR (KBr, ν_max): 3313.03, 3017.46, 1677.32, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 9.10 (s, 1H), 9.05 (t, J = 5.9 Hz, 1H), 9.01 (d, J = 6.1 Hz, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.44 (s, 1H), 8.25–8.17 (m, 2H), 7.76–7.64 (m, 4H), 7.56 (d, J = 8.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.30–7.22 (m, 4H), 7.19 (d, J = 6.9 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 15.7 Hz, 1H), 5.72 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 3.72 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.63, 163.05, 156.06, 145.50, 145.19, 145.11, 141.49, 141.26, 141.13, 140.73, 138.04, 131.92, 129.10, 127.83, 127.61, 127.24, 126.86, 126.49, 125.85, 123.11, 122.65, 121.18, 121.13, 121.05, 120.14, 118.91, 114.51, 110.61, 110.47, 62.59, 55.63, 46.19; anal. calcd for C₃₇H₃₃ClN₄O₃: C, 72.01; H, 5.39; N, 9.08; found: C, 72.19; H, 5.57; N, 9.29.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((2-fluorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12e)

Yield: 81%. Light yellow solid, mp: 221–223 °C; IR (KBr, ν_max): 3013.15, 1667.28, 1618.49 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 9.19 (t, J = 5.9 Hz, 1H), 9.16 (s, 1H), 9.06 (d, J = 6.1 Hz, 1H), 8.65 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.26–8.18 (m, 2H), 7.95–7.86 (m, 1H), 7.75–7.69 (m, 3H), 7.67 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.34–7.18 (m, 9H), 6.83 (d, J = 15.7 Hz, 1H), 5.84 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.65, 164.40, 155.22, 152.78, 145.54, 145.30, 145.21, 141.48, 141.20, 141.12, 140.78, 138.04, 129.10, 127.82, 127.64, 127.24, 126.85, 126.53, 126.46, 126.39, 125.83, 125.79, 125.67, 125.02, 124.99, 124.34, 123.11, 122.66, 121.20, 121.05, 120.13, 119.01, 116.31, 116.12, 110.61, 110.46, 62.67, 46.20; anal. calcd for C₃₆H₃₀ClFN₄O₂: C, 71.46; H, 5.00; N, 9.26; found: C, 71.67; H, 5.22; N, 9.39.

(E)-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-fluorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12f)

Yield: 84%. Light yellow solid, mp: 254–256 °C; IR (KBr, ν_max): 3314.47, 3018.89, 1683.06, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.38 (s, 1H), 9.12 (s, 1H), 9.08 (t, J = 6.0 Hz, 1H), 9.03 (d, J = 6.1 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.25–8.19 (m, 2H), 7.70 (dt, J = 13.0, 7.8 Hz, 6H), 7.47 (t, J = 7.7 Hz, 1H), 7.32–7.15 (m, 8H), 6.80 (d, J = 15.7 Hz, 1H), 5.77 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.64, 163.61, 159.95, 157.56, 145.52, 145.25, 145.15, 141.49, 141.25, 141.13, 140.75, 138.04, 135.29, 135.26, 129.10, 127.83, 127.62, 127.24, 126.86, 126.50, 125.85, 123.12, 122.65, 121.47, 121.39, 121.18, 121.04, 120.14, 118.94, 116.15, 115.93, 110.61, 110.46, 62.62, 46.20; anal. calcd for C₃₆H₃₀ClFN₄O₂: C, 71.46; H, 5.00; N, 9.26; found: C, 71.68; H, 5.23; N, 9.49.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-chlorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12g)

Yield: 83%. Light yellow solid, mp: 255–257 °C; IR (KBr, ν_max): 3331.69, 3017.46, 1683.06, 1612.75 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.43 (s, 1H), 9.10 (s, 1H), 9.07–9.00 (m, 2H), 8.63 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.25–8.18 (m, 2H), 7.70 (q, J = 8.6 Hz, 6H), 7.48 (t, J = 7.7 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.31–7.23 (m, 4H), 7.19 (d, J = 7.5 Hz, 2H), 6.79 (d, J = 15.8 Hz, 1H), 5.77 (s, 2H), 5.70 (s, 2H), 4.66 (d, J = 5.8 Hz, 2H), ¹³C NMR (101 MHz, DMSO-d₆) δ 166.63, 163.90, 145.53, 145.28, 145.15, 141.49, 141.26, 141.13, 140.76, 138.04, 137.82, 129.36, 129.10, 127.97, 127.83, 127.63, 127.24, 126.86, 126.49, 125.85, 123.12, 122.65, 121.21, 121.18, 121.04, 120.14, 118.91, 110.61, 110.46, 62.70, 46.20; HRMS (ESI⁺) m/z: calculated for C₃₆H₃₀Cl₂N₄O₂⁺ ([M]⁺) 585.20573, found 585.20932; calculated for C₃₆H₃₀Cl₂N₄O₂⁺ ([M + H]⁺) 586.21355, found 586.21251. Anal. calcd for C₃₆H₃₀Cl₂N₄O₂: C, 69.57; H, 4.87; N, 9.01; found: C, 69.74; H, 4.98; N, 9.15.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((2,4-dichlorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12h)

Yield: 84%. Light yellow solid, mp: 243–245 °C; IR (KBr, ν_max): 3327.38, 3070.55, 1693.11, 1618.49 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.73 (s, 1H), 9.15–9.09 (m, 2H), 9.04 (d, J = 6.1 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H), 8.44 (s, 1H), 8.26–8.18 (m, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.74–7.69 (m, 4H), 7.67 (d, J = 8.2 Hz, 1H), 7.54–7.41 (m, 2H), 7.31–7.22 (m, 4H), 7.19 (d, J = 7.5 Hz, 2H), 6.80 (d, J = 15.7 Hz, 1H), 5.83 (s, 2H), 5.70 (s, 2H), 4.66 (d, J = 5.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.64, 164.74, 145.56, 145.35, 145.21, 141.49, 141.24, 141.13, 140.77, 133.74, 130.43, 129.65, 129.10, 128.19, 127.83, 127.70, 127.65, 127.41, 127.24, 126.86, 126.49, 125.83, 123.11, 122.65, 121.19, 121.04, 120.14, 118.94, 110.61, 110.47, 62.61, 46.20; anal. calcd for C₃₆H₂₉Cl₃N₄O₂: C, 65.91; H, 4.46; N, 8.54; found: C, 66.07; H, 4.62; N, 8.76.

E-3-((3-(9-Benzyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-nitrophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12i)

Yield: 87%. Light yellow solid, mp: 243–245 °C; IR (KBr, ν_max): 3284.34, 3004.54, 1690.24, 1617.06 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 12.06 (s, 1H), 9.13 (s, 1H), 9.09–9.03 (m, 2H), 8.65 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.30–8.19 (m, 4H), 7.94 (d, J = 8.9 Hz, 2H), 7.76–7.63 (m, 4H), 7.47 (t, J = 7.7 Hz, 1H), 7.30–7.22 (m, 4H), 7.19 (d, J = 6.8 Hz, 2H), 6.80 (d, J = 15.7 Hz, 1H), 5.86 (s, 2H), 5.70 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.66, 164.95, 145.58, 145.41, 145.20, 144.96, 143.15, 141.48, 141.28, 141.12, 140.80, 138.03, 129.10, 127.83, 127.66, 127.23, 126.86, 126.49, 125.85, 125.60, 123.11, 122.65, 121.18, 121.04, 120.14, 119.53, 118.91, 110.60, 110.45, 62.90, 46.20; HRMS (ESI⁺) m/z: calculated for C₃₆H₃₀ClN₅O₄⁺ ([M]⁺) 596.22978, found 596.23363; calculated for C₃₆H₃₀ClN₅O₄⁺ ([M + H]⁺) 597.23760, found 597.23665. Anal. calcd for C₃₆H₃₀ClN₅O₄: C, 68.40; H, 4.78; N, 11.08; found: C, 68.57; H, 4.96; N, 11.32.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(phenylamino)ethyl)pyridin-1-ium chloride (12j)

Yield: 78%. Light yellow solid, mp: 208–210 °C; IR (KBr, ν_max): 3186.77, 3024.63, 1667.37, 1594.10 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.17 (s, 1H), 9.11 (s, 1H), 9.08–8.99 (m, 2H), 8.63 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.26–8.16 (m, 2H), 7.77–7.60 (m, 6H), 7.50 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 6.79 (d, J = 15.8 Hz, 1H), 5.76 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.68, 163.66, 145.53, 145.23, 145.13, 141.38, 140.85, 140.78, 140.49, 138.83, 129.43, 129.29, 127.63, 126.71, 126.09, 125.68, 124.40, 123.00, 122.57, 121.21, 121.03, 119.80, 119.63, 118.67, 110.13, 109.98, 62.71, 37.62, 14.23; anal. calcd for C₃₁H₂₈ClN₅O₄: C, 65.32; H, 4.95; N, 12.29; found: C, 65.54; H, 5.12; N, 12.50.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(o-tolylamino)ethyl)pyridin-1-ium chloride (12k)

Yield: 78%. Light yellow solid, mp: 198–200 °C; IR (KBr, ν_max): 3166.68, 3117.90, 1690.24, 1589.80 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 1H), 9.13 (s, 1H), 9.09 (t, J = 5.9 Hz, 1H), 9.04 (d, J = 6.1 Hz, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.26–8.16 (m, 2H), 7.77–7.62 (m, 4H), 7.50 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.28–7.23 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.3 Hz, 1H), 6.80 (d, J = 15.8 Hz, 1H), 5.81 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 2.31 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.67, 163.94, 145.47, 145.19, 145.11, 141.37, 140.86, 140.77, 140.49, 135.81, 132.13, 130.97, 127.64, 126.71, 126.49, 126.15, 126.10, 125.68, 125.21, 123.00, 122.57, 121.22, 121.03, 119.80, 118.67, 110.14, 109.98, 62.50, 56.49, 37.62, 18.50, 14.24: anal. calcd for C₃₂H₃₁ClN₄O₂: C, 71.30; H, 5.80; N, 10.39; found: C, 71.49; H, 5.98; N, 10.54.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-oxo-2-(p-tolylamino)ethyl)pyridin-1-ium chloride (12l)

Yield: 80%. Light yellow solid, mp: 247–249 °C; IR (KBr, ν_max): 3285.77, 3040.42, 1687.37, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.04 (s, 1H), 9.10 (s, 1H), 9.07–8.98 (m, 2H), 8.63 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.25–8.16 (m, 2H), 7.77–7.62 (m, 4H), 7.56–7.46 (m, 3H), 7.25 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 8.1 Hz, 2H), 6.79 (d, J = 15.7 Hz, 1H), 5.73 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 2.26 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.67, 163.36, 145.51, 145.19, 145.10, 141.38, 140.85, 140.76, 140.49, 136.31, 133.38, 129.81, 127.61, 126.71, 126.09, 125.68, 123.00, 122.57, 121.21, 121.02, 119.80, 119.60, 118.66, 110.13, 109.98, 62.67, 37.62, 20.92, 14.24; anal. calcd for C₃₂H₃₁ClN₄O₂: C, 71.30; H, 5.80; N, 10.39; found: C, 71.49; H, 5.97; N, 10.63.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((2-fluorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12m)

Yield: 81%. Light yellow solid, mp: 233–235 °C; IR (KBr, ν_max): 3173.86, 3004.54, 1690.24, 1591.23 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.83 (s, 1H), 9.17–9.12 (m, 2H), 9.04 (d, J = 6.1 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.26–8.16 (m, 2H), 7.95–7.86 (m, 1H), 7.77–7.61 (m, 4H), 7.50 (t, J = 7.7 Hz, 1H), 7.35–7.29 (m, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.22–7.16 (m, 2H), 6.82 (d, J = 15.7 Hz, 1H), 5.82 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.69, 164.40, 155.19, 152.75, 145.54, 145.30, 145.18, 141.33, 140.85, 140.80, 140.48, 127.64, 126.70, 126.46, 126.38, 126.11, 125.78, 125.66, 125.03, 125.00, 124.29, 123.00, 122.57, 121.22, 121.02, 119.80, 118.73, 116.31, 116.12, 110.13, 109.97, 62.67, 37.62, 14.23; anal. calcd for C₃₁H₂₈ClFN₄O₂: C, 68.57; H, 5.20; N, 10.32; found: C, 68.75; H, 5.41; N, 10.49.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-fluorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12n)

Yield: 83%. Light yellow solid, mp: 238–240 °C; IR (KBr, ν_max): 3265.68, 3040.42, 1680.19, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.52 (s, 1H), 9.20–9.13 (m, 2H), 9.05 (d, J = 6.0 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.26–8.16 (m, 2H), 7.76–7.69 (m, 4H), 7.64 (t, J = 8.7 Hz, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 8.7 Hz, 2H), 6.83 (d, J = 15.7 Hz, 1H), 5.80 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.70, 163.61, 159.94, 157.55, 145.49, 145.19 (d, J = 6.9 Hz), 141.33, 140.81 (d, J = 5.6 Hz), 140.47, 135.32 (d, J = 2.6 Hz), 127.62, 126.69, 126.13, 125.66, 123.00, 122.58, 121.48, 121.40, 121.23, 121.02, 119.79, 118.75, 116.11, 115.89, 110.11, 109.95, 62.64, 37.62, 14.22; anal. calcd for C₃₁H₂₈Cl₂N₄O₂: C, 68.57; H, 5.20; N, 10.32; found: C, 68.78; H, 5.38; N, 10.54.

(E)-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-chlorophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12o)

Yield: 82%. Light yellow solid, mp: 255–257 °C; IR (KBr, ν_max): 3252.77, 3034.68, 1665.84, 1615.62 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 11.49 (s, 1H), 9.11 (s, 1H), 9.06 (t, J = 6.0 Hz, 1H), 9.02 (d, J = 6.1 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.77–7.61 (m, 6H), 7.50 (t, J = 7.7 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.25 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 15.7 Hz, 1H), 5.78 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.67, 163.90, 145.53, 145.27, 145.14, 141.35, 140.85, 140.78, 140.48, 137.84, 129.35, 127.95, 127.62, 126.70, 126.10, 125.67, 123.00, 122.57, 121.20, 121.02, 119.80, 118.69, 110.13, 109.97, 62.71, 56.49, 37.62, 14.23; anal. calcd for C₃₁H₂₈Cl₂N₄O₂: C, 66.55; H, 5.04; N, 10.01; found: C, 66.73; H, 5.20; N, 10.27.

(E)-1-(2-((2,4-Dichlorophenyl)amino)-2-oxoethyl)-3-((3-(9-ethyl-9H-carbazol-3-yl)acrylamido)methyl)pyridin-1-ium chloride (12p)

Yield: 83%. Light yellow solid, mp: 233–235 °C; IR (KBr, ν_max): 3295.82, 2977.28, 1694.54, 1618.49 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.71 (s, 1H), 9.14 (s, 1H), 9.10 (t, J = 6.0 Hz, 1H), 9.04 (d, J = 6.1 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.26–8.16 (m, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.77–7.62 (m, 5H), 7.50 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 6.80 (d, J = 15.7 Hz, 1H), 5.83 (s, 2H), 4.66 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.68, 164.74, 145.55, 145.34, 145.20, 141.35, 140.86, 140.80, 140.49, 133.74, 130.43, 129.65, 128.19, 127.68, 127.65, 127.39, 126.71, 126.09, 125.66, 123.00, 122.57, 121.21, 121.02, 119.80, 118.69, 110.13, 109.98, 62.61, 37.62, 14.24; anal. calcd for C₃₁H₂₇Cl₃N₄O₂: C, 62.69; H, 4.58; N, 9.43; found: C, 62.87; H, 4.74; N, 9.59.

E-3-((3-(9-Ethyl-9H-carbazol-3-yl)acrylamido)methyl)-1-(2-((4-nitrophenyl)amino)-2-oxoethyl)pyridin-1-ium chloride (12q)

Yield: 85%. Light yellow solid, mp: 258–260 °C; IR (KBr, ν_max): 3371.86, 2919.89, 1706.02, 1596.97 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 12.02 (s, 1H), 9.12 (s, 1H), 9.03 (dd, J = 6.2, 3.7 Hz, 2H), 8.65 (d, J = 8.1 Hz, 1H), 8.41 (s, 1H), 8.27 (d, J = 9.1 Hz, 2H), 8.22 (d, J = 6.2 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.9 Hz, 2H), 7.77–7.61 (m, 4H), 7.50 (t, J = 7.7 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 15.7 Hz, 1H), 5.85 (s, 2H), 4.67 (d, J = 5.8 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.68, 164.96, 145.59, 145.40, 145.17, 144.96, 143.15, 141.38, 140.85, 140.83, 140.48, 127.66, 126.71, 126.08, 125.67, 125.61, 123.00, 122.57, 121.20, 121.02, 119.80, 119.52, 118.66, 110.13, 109.98, 62.91, 37.62, 14.23; anal. calcd for C₃₁H₂₈ClN₅O₄: C, 65.32; H, 4.95; N, 12.29; found: C, 65.50; H, 5.12; N, 12.51.

Anti-ChE assays

The inhibitory activities of all synthesized compounds (12a–q) against AChE (E.C. 3.1.1.7, from electric eel) and BChE (E.C. 3.1.1.8, from equine serum) were evaluated using the Ellman colorimetric assay, as previously described.²² Acetylthiocholine iodide (AChI) and butyrylthiocholine iodide (BTC) were employed as substrates for AChE and BChE, respectively. The enzyme activity was quantified by measuring the change in absorbance with 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB). For the assay, 50 μL of AChE/BChE solutions (5.32 × 10⁻³ EU) were mixed with 100 μL of buffer solution (pH 8.0, Tris/HCl, 1.0 M) and varying amounts (50–200 μL) of sample solutions, which were dissolved in deionized water. The mixture was incubated at 20 °C for 10 minutes. Following incubation, 50 μL of DTNB (0.5 mM in 25 mL) and AChI/BTC were added to the reaction mixture, with 50 μL of AChI/BTC initiating the enzymatic reaction. The enzyme activity was then measured spectrophotometrically at 412 nm. Tacrine and galantamine were used as reference inhibitors in the cholinesterase inhibition assays. Tacrine was included as a historically well-established benchmark compound widely used in anti-cholinesterase research, whereas galantamine served as a clinically approved anti-Alzheimer drug currently used in the symptomatic treatment of AD. The concentration-response curve was analyzed by nonlinear regression to determine the IC₅₀ values, corresponding to the concentration required to inhibit 50% of enzyme activity. The data are expressed as means ± standard deviation (SD).

Molecular docking and molecular dynamics

Molecular docking and molecular dynamics (MD) simulations were performed using the Schrödinger Molecular Modeling Software (2025-1) with the Maestro interface (v14.3) and Desmond. All protein and ligand preparations were carried out following established protocols from our group, including proper protonation states, bond orders, and minimization of steric clashes.^23–25 The high-resolution 3D structures of AChE (PDB ID: 4EY7) and BChE (PDB ID: 5NN0) were retrieved from the Protein Data Bank and further optimized using the Schrödinger's Protein Preparation Wizard, which included assignment of protonation states at physiological pH and addition of missing side chains and hydrogen atoms. Docking studies were conducted using Glide XP and the induced fit docking (IFD) protocol to account for receptor flexibility and achieve a more realistic representation of ligand binding. For each ligand, twenty distinct binding poses were generated, and the top-ranked poses were selected based on IFD scores, considering both docking score and the complementarity of ligand–protein interactions. To quantify binding affinity, prime MM-GBSA calculations were performed using the VSGB solvation model, providing estimates of the free energy of binding for each ligand–protein complex.^26–28 MD simulations were conducted using Desmond (release 2024-3). Each protein–ligand complex was placed in an orthorhombic simulation box and solvated with TIP4P water molecules, with counter ions added to neutralize the system and 0.15 M NaCl to mimic physiological ionic strength. The OPLS4 force field was applied to describe bonded and non-bonded interactions. Following energy minimization to remove steric clashes, the systems were equilibrated under NVT and NPT ensembles. Production MD simulations were then performed for 500 ns at 300 K and 1.01325 bar, using the Nosé–Hoover thermostat and Martyna–Tobias–Klein barostat to maintain temperature and pressure. Trajectory analysis was conducted to monitor stability and conformational changes in the complexes, including calculations of root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) for the protein backbone and ligand atoms. These simulations provided detailed insights into the dynamic behavior of ligands within the active sites of AChE and BChE, complementing the docking results and allowing evaluation of interaction persistence, flexibility, and overall complex stability.^29–32

Cytotoxicity assay

The human neuroblastoma cell line SH-SY5Y (ATCC® CRL-2266™) and the human embryonic kidney cell line HEK293 (ATCC® CRL-1573™) were cultured in DMEM/F-12 supplemented with 10% heat-inactivated FBS and 1% penicillin–streptomycin solution. Cells were maintained at 37 °C in a humidified incubator under 5% CO₂ atmosphere. SH-SY5Y and HEK293 cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and allowed to adhere overnight in a humidified incubator maintained at 37 °C with 5% CO₂. After cell attachment, eight serially diluted concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM) of the target compounds were applied for 24 hours. Each concentration was tested in triplicate. Following exposure, 10 μL of MTT solution (5 mg mL⁻¹ in PBS) was added to each well and incubated for an additional 4 hours under the same conditions. Subsequently, the medium was carefully removed, and the resulting formazan crystals were dissolved in 100 μL of DMSO per well. Absorbance was measured at 570 nm using a microplate reader. The net absorbance values (after blank correction) were used to determine the percentage of cell viability relative to untreated control cells, which were defined as 100%. Data are presented as the mean ± standard deviation (SD) of three independent experiments.^33,34

Results and discussion

Chemistry

The carbazol–acrylamid–pyridinium-N-phenylacetamide derivatives 12a–q were synthesized according to the pathway illustrated in Scheme 1. N-Alkylation of carbazole (1) was achieved under phase-transfer conditions using benzyl bromide (2a) or ethyl iodide (2b) in the presence of KOH, yielding the benzyl–carbazole derivative 3a or the ethyl–carbazole derivative 3b. Subsequent Vilsmeier formylation selectively functionalized the carbazole core at the C3 position, employing POCl₃/DMF to afford aldehydes 5a–b. These aldehydes were condensed with malonic acid via Knoevenagel condensation catalyzed by β-alanine/pyridine, generating acrylic acid derivatives 6a–b. After that, the latter derivatives 6a–b reacted with pyridin-3-ylmethanamine (7) in the presence of TBTU/NEt₃ to give carbazol-acrylamid-pyridine derivatives 8a–b. On the other hand, 2-chloro-N-substituted acetamides 11a–i were prepared by reaction of aniline derivatives 9a–i with chloroacetyl chloride (10). Final compounds 12a–q were obtained through SN₂ alkylation of carbazol–acrylamid–pyridine derivatives 8a–b with chloroacetamide derivatives 11a–i in acetonitrile under reflux.

Scheme 1. Synthesis of carbazol–acrylamid–pyridinium-N-phenylacetamide derivatives 12a–q.

Evaluation of target compounds 12a–q against AChE and BChE

The synthesized derivatives 12a–q were evaluated for their inhibitory activity against AChE and BChE, using tacrine and galantamine as reference inhibitors. Tacrine inhibited AChE and BChE with IC₅₀ values of 58.34 nM and 160.50 nM, respectively, while galantamine displayed corresponding IC₅₀ values of 124.72 nM and 216.23 nM. Notably, most of the synthesized compounds demonstrated superior potency compared to tacrine, and all derivatives were more potent than galantamine. The IC₅₀ values ranged from 6.41 to 74.93 nM against AChE and from 38.05 to 191.96 nM against BChE. Among the series, compound 12i emerged as the most potent AChE inhibitor, exhibiting approximately 19.5-fold and 9.1-fold higher potency than galantamine and tacrine, respectively. In terms of BChE inhibition, compound 12g displayed the strongest activity, with approximately 5.7-fold and 4.2-fold greater potency than galantamine and tacrine, respectively.

In term of structural perspective, new derivatives 12a–q are divided into two categories: benzyl derivatives (compounds 12a–i) and ethyl derivatives (compounds 12j–q). In each series, structural optimization was performed by alteration of substituents on the phenyl ring of the N-phenacylamide moiety to enhance anti-ChE activity. The SAR analysis on these two series that was schematically showed in Fig. 2, reveals critical insights into how structural modifications influence potency. Given that all synthesized derivatives outperformed galantamine against both AChE and BChE, we used tacrine as the primary reference standard in our SAR discussion.

Fig. 2. SARs in the new compounds 12a–q in anti-AChE assay.

The benzyl derivatives 12a–i generally exhibited high inhibitory activity against AChE, with IC₅₀ values ranging from 6.41 to 71.47 nM. The type and position of substituents on the phenyl ring of the N-phenacylamide moiety were decisive for anti-AChE activity (Table 1 and Fig. 2). The un-substituted compound 12a showed moderate activity (IC₅₀ = 40.90 nM). Introduction of methyl substituents at the 2-position (compound 12b, IC₅₀ = 63.26 nM) or 4-position (compound 12c, IC₅₀ = 31.43 nM) did not significantly improve the enzymatic inhibitory activity, and in the case of the 2-position, it even reduced potency. Similarly, the 4-methoxy derivative (compound 12d, IC₅₀ = 71.47 nM) showed diminished activity, likely due to the electron-donating nature and steric hindrance of the substituent. In contrast, electron-withdrawing substituents such as halogen atoms or nitro group markedly enhanced activity. In this regards, the most active compound of this series was the 4-nitro derivative 12i (IC₅₀ = 6.41 nM). Other the most potent compounds were 4-fluoro derivative 12f (IC₅₀ = 9.02 nM) and 4-chloro derivative 12g (IC₅₀ = 9.94 nM). Moreover, the 2-fluoro (compound 12e, IC₅₀ = 28.61 nM) and 2,4-di-Cl derivative (compound 12h, IC₅₀ = 20.71 nM) also demonstrated improved activity in comparison to un-substituted derivative 12a or derivatives 12b–d with electron donating groups (Fig. 2).

Table 1. Structures and anti-ChE activities of new compounds 12a–q.

Compound	R1	R2	AChE (IC50, nM)	BChE (IC50, nM)
12a	Benzyl	H	40.90 ± 0.61	144.03 ± 0.84
12b	Benzyl	2-Me	63.26 ± 0.83	180.62 ± 0.75
12c	Benzyl	4-Me	31.43 ± 0.24	138.65 ± 0.62
12d	Benzyl	4-OMe	71.47 ± 0.81	176.27 ± 0.91
12e	Benzyl	2-F	28.61 ± 0.21	81.97 ± 0.82
12f	Benzyl	4-F	9.02 ± 0.09	43.80 ± 0.25
12g	Benzyl	4-Cl	9.94 ± 0.18	38.05 ± 0.27
12h	Benzyl	2,4-di-Cl	20.71 ± 0.61	72.44 ± 0.37
12i	Benzyl	4-NO2	6.41 ± 0.11	47.89 ± 0.28
12j	Et	H	59.56 ± 0.46	169.02 ± 0.53
12k	Et	2-Me	56.58 ± 0.51	191.96 ± 0.39
12l	Et	4-Me	74.93 ± 0.47	130.52 ± 0.46
12m	Et	2-F	23.75 ± 0.39	95.21 ± 0.74
12n	Et	4-F	11.62 ± 0.24	51.76 ± 0.53
12o	Et	4-Cl	6.78 ± 0.13	60.73 ± 0.65
12p	Et	2,4-di-Cl	19.24 ± 0.07	90.84 ± 0.55
12q	Et	4-NO2	14.10 ± 0.18	46.06 ± 0.26
Tacrine	—	—	58.34 ± 0.21	160.50 ± 0.71
Galantamine	—	—	124.72 ± 0.06	216.23 ± 0.50

The ethyl-substituted series 12j–q also displayed considerable inhibitory potency, with IC₅₀ values ranging from 6.78 to 74.93 nM. The un-substituted derivative 12j (IC₅₀ = 59.56 nM) showed comparable activity to the standard reference drug tacrine (IC₅₀ = 58.34 nM). Introduction of 2-methyl substituent (compound 12k, IC₅₀ = 56.58 nM) had little improvement effect on un-substituted compound (compounds 12j) but introduction of the 4-methyl substituent (compound 12l, IC₅₀ = 74.93 nM) led to reduced activity. Similar to benzyl derivatives, halogen substituents once again exerted a beneficial influence on anti-AChE activity. In this regard, compounds 12m (2-F, IC₅₀ = 23.75 nM), 12n (4-F, IC₅₀ = 11.62 nM), 12o (4-Cl, IC₅₀ = 6.78 nM), and 12p (2,4-di-Cl, IC₅₀ = 19.24 nM) were dramatically more potent than tacrine. In addition, the 4-nitro analog 12q (IC₅₀ = 14.10 nM) also showed considerable inhibitory activity, consistent with the favorable role of electron-withdrawing substituents.

General comparison of benzyl and ethyl derivatives demonstrated that the benzyl series showed a slightly broader activity range (IC₅₀ range = 6.41–71.47 nM) compared to the ethyl series (IC₅₀ range = 6.78–74.93 nM). Further details of comparison of benzyl derivatives 12a–i with their corresponding analogs of ethyl series are illustrated in Fig. 2.

The SAR for BChE inhibition reveals distinct trends influenced by the benzyl or ethyl scaffold and substituent effects on the phenyl ring of N-phenylacetamide moiety. Compared to tacrine (IC₅₀ = 160.50 nM), most derivatives exhibited superior BChE inhibition, with potency enhancements driven by electronic and steric factors. SAR analysis of the title compounds was schematically showed in Fig. 3.

Fig. 3. SARs in the new compounds 12a–q in anti-BChE assay.

SAR survey on benzyl derivatives 12a–i revealed that electron withdrawing groups at the 4-position of phenyl ring significantly boosted BChE inhibition (Fig. 3). In this regard, the best BChE inhibitors among the all synthesized compounds were benzyl derivatives 12g (IC₅₀ = 38.06 nM) and 12f (IC₅₀ = 43.80 nM) with 4-Cl and 4-F substituents, respectively. Moreover, 4-NO₂ derivative (compound 12i, IC₅₀ = 47.89 nM) in this series was a strong BChE inhibitor. This compound was slightly weaker than 4-halogented derivatives 12g and 12f with a substituent, and was significantly more potent than 2,4-dichloro derivative 12h (IC₅₀ = 72.441 nM), possibly due to excessive polarity or steric constraints. It seems that steric hindrance has a negative impact in the observed activities because 2-F derivative 12e (IC₅₀ = 81.97 nM) and 2-methyl derivative 12b (IC₅₀ = 180.62 nM) were the less potent compounds among the electron withdrawing and donating derivatives, respectively. In the latter compound changing the position of substituent of 2-position to 4-position led to a significant increase in anti-BChE activity as observe in 4-fluoro derivative 12f (IC₅₀ = 43.80 nM) and 4-methyl derivative 12c (IC₅₀ = 138.65 nM).

In the ethyl derivatives 12j–q, 4-NO₂ derivative 12q (IC₅₀ = 46.06 nM), 4-F derivative 12n (IC₅₀ = 51.76 nM), 4-Cl derivative 12o (IC₅₀ = 60.74 nM), and 2,4-dichloro derivative 12p (IC₅₀ = 90.845 nM) displayed the highest potency. Similar to benzyl derivatives, 2-substituented derivatives exhibited the less potency in comparison to the corresponding analogs with same substituent on 4-position (2-F derivative 12mvs. 4-F derivative 12n and 2-methyl derivative 12kvs. 4-methyl derivative 12l). Un-substituted derivative 12j (IC₅₀ = 169.02 nM) showed minimal improvement over tacrine.

As can be seen in Table 1 and Fig. 3, with the exception of one case (4-nitro derivatives), benzyl derivatives generally were more potent than ethyl analogs, emphasizing the role of aromatic stacking in BChE inhibition.

Kinetic studies of the most potent compounds against AChE and BChE

To further validate the inhibition mechanisms and strengthen the SAR analysis, kinetic studies were performed on the most potent compounds identified against AChE and BChE. Based on the in vitro inhibition results, compounds 12i, 12o, and 12f were selected for AChE, whereas compounds 12g, 12f, and 12q were selected for BChE. The inhibition types were determined using Lineweaver-Burk plots and the corresponding secondary plots, as presented in Fig. 4 and 5. As shown in the kinetic analyses, increasing concentrations of the tested compounds did not significantly affect V_max values, whereas apparent K_m values increased. These findings indicate that the selected compounds inhibit both AChE and BChE through a competitive inhibition mechanism. The corresponding K_i values were calculated from secondary plots and are summarized in Table 2.

Fig. 4. Kinetic analysis of AChE inhibition by compounds 12i, 12o, and 12f.

Fig. 5. Kinetic analysis of BChE inhibition by compounds 12g, 12f, and 12q.

Table 2. K _i values and inhibition types of the most potent compounds against AChE and BChE.

Enzyme	Compound	K i value	Inhibition type
AChE	12i	5.03 ± 0.37	Competitive
AChE	12o	5.61 ± 0.45	Competitive
AChE	12f	6.93 ± 0.60	Competitive
AChE	Tacrine	50.35 ± 7.10	Competitive
AChE	Galantamine	97.28 ± 9.34	Competitive
BChE	12g	31.48 ± 4.83	Competitive
BChE	12f	35.70 ± 6.20	Competitive
BChE	12q	41.22 ± 5.01	Competitive
BChE	Tacrine	126.08 ± 7.41	Competitive
BChE	Galantamine	173.56 ± 9.98	Competitive

Molecular docking studies

Molecular docking is widely used to analyze ligand–protein interactions and binding affinities. The induced fit docking (IFD) method increases accuracy by allowing protein flexibility, while MM-GBSA calculations provide quantitative estimates of binding free energies. In this study, the most active compounds against AChE and BChE were selected to investigate their binding mechanisms. Docking was carried out using the IFD protocol, and MM-GBSA ΔG binding energies were calculated. The results are summarized in Table 3. The docking and MM-GBSA analyses revealed that the investigated compounds exhibit stronger interactions with both AChE and BChE compared to the reference drug tacrine. Compound 12i displayed the most favorable binding affinity toward AChE, with an IFD docking score of −16.755 and a ΔG binding energy of −100.88 kcal mol⁻¹, which are markedly more negative than those obtained for tacrine (−10.530 and −55.36 kcal mol⁻¹, respectively). This suggests that 12i establishes more extensive or stable noncovalent polar and nonpolar interactions within the AChE active site. Similarly, compound 12g demonstrated potent binding to BChE (IFD score: –14.808; ΔG: −99.88 kcal mol⁻¹), surpassing tacrine (−7.500; −46.87 kcal mol⁻¹) by a considerable margin, which highlights its potential selectivity and strength against BChE.

Table 3. Docking scores and MM-GBSA ΔG binding free energies of the most active compounds and reference drug tacrine against AChE and BChE.

Complexes	IFD docking scores (kcal mol−1)	MM-GBSA ΔG bind (kcal mol−1)
12i–AChE	−16.755	−100.88
12g–BChE	−14.808	−99.88
Tacrine–AChE	−10.530	−55.36
Tacrine–BChE	−7.500	−46.87

The 2D and 3D ligand–protein interactions of 12i–AChE and 12g–BChE are presented in Fig. 6 and 7, respectively. Fig. 6a shows that the 12i molecule establishes multiple and diverse polar and nonpolar interactions within the AChE active site. The pyridinium ring acts as a key pharmacophore in the compound. The pyridinium nitrogen forms both a π–cation interaction (red line) and a π–π stacking interaction (green line) with Trp-286. The α,β-unsaturated carbonyl oxygen engages in two hydrogen bonds (purple arrows) with Arg-296 and Phe-295, which are key residues for AChE inhibition. Additionally, the amide nitrogen of Tyr-72 forms another hydrogen bond, while the carbazole ring participates in multiple π–π stacking interactions with Phe-338, Tyr-337, His-447, and Tyr-124. Collectively, these interactions contribute to the stable binding of 12i within the AChE active site, forming a well-defined and energetically favorable complex.

Fig. 6. Molecular docking interactions of compound 12i within the AChE active site: (a) 2D interaction diagram and (b) 3D binding pose showing key protein–ligand interactions.

Fig. 7. Binding interactions of 12g with BChE: (a) 2D interaction diagram and (b) 3D binding pose.

Fig. 6b represents the 3D binding pose: yellow dashed lines indicate hydrogen bonds, turquoise lines show π–π stacking, and red lines represent π–cation interactions. The blue surface depicts the ligand's binding surface, while the gray surface represents the protein's binding surface. The greater the overlap between these surfaces, the better the ligand fits into the binding site. In this case, only the nitro group extends beyond the binding surface, likely due to the molecule's size and linear shape.

The 2D interactions of 12g with BChE are shown in Fig. 7a. Similar to 12i, the pyridinium ring stands out as the key pharmacophore. The pyridinium ring forms a salt bridge with Glu-197, two π–cation interactions with Trp-82, and two π–π stacking interactions also with Trp-82. The amide nitrogen atoms form hydrogen bonds with Asp-70 and His-438, while the chlorobenzene ring engages in π–π stacking with Trp-231 and Phe-329.

The 3D binding interactions are presented in Fig. 7b, showing a complete overlap between the protein and ligand binding surfaces, indicating an excellent fit. Unlike in the 12i–AChE complex, the carbazole ring of 12g does not participate in any interactions within the BChE active site.

Overall, 12i and 12g show strong binding to AChE and BChE, respectively, surpassing tacrine. Key interactions include 12i's pyridinium ring with Trp-286, carbonyl hydrogen bonds with Arg-296 and Phe-295, and carbazole π–π stacking with Phe-338, Tyr-337, His-447, and Tyr-124, while 12g forms a salt bridge with Glu-197, π–cation and π–π interactions with Trp-82, amide hydrogen bonds with Asp-70 and His-438, and chlorobenzene π–π stacking with Trp-231 and Phe-329, stabilizing both complexes.

Molecular dynamics simulations

To further validate the docking results and evaluate the dynamic stability of the ligand–protein complexes, molecular dynamics (MD) simulations were performed. Both the 12i–AChE and 12g–BChE complexes were subjected to 500 ns MD simulations. The results of the MD analyses are presented in Fig. 8 and 9.

Fig. 8. Molecular dynamics simulation results of the 12i–AChE complex over 500 ns. (a) 2D ligand–protein interaction profile showing key residues and their interaction fractions during the simulation. (b) RMSD plots: Cα RMSD of the protein backbone (pale blue), ligand RMSD with respect to the protein (red), and ligand RMSD with respect to itself (pink). (c) RMSF plot highlighting residue flexibility and interaction regions. (d) Histogram of fractional interactions indicating hydrogen bonds (green), water bridges (blue), and hydrophobic contacts (purple).

Fig. 9. Molecular dynamics simulation results of the 12g–BChE complex over 500 ns. (a) 2D ligand–protein interaction profile showing key residues and their interaction fractions during the simulation. (b) RMSD plots: Cα RMSD of the protein backbone (pale blue), ligand RMSD with respect to the protein (red), and ligand RMSD with respect to itself (pink). (c) RMSF plot highlighting residue flexibility and interaction regions. (d) Histogram of fractional interactions indicating hydrogen bonds (green), water bridges (blue), and hydrophobic contacts (purple).

Fig. 8a illustrates the 2D ligand–protein interactions of compound 12i with AChE during the 500 ns MD simulation, along with the percentage of the simulation period in which each interaction was maintained. 12i exhibited excellent binding stability within the active site of AChE, with several key interactions persisting throughout nearly the entire trajectory, indicating the formation of a highly stable ligand–protein complex. The carbonyl oxygen of the α,β-unsaturated amide group formed a hydrogen bond with Phe-295 for 99% of the simulation, while the amide nitrogen established another hydrogen bond with Tyr-341 for 71% of the simulation. The pyridinium ring also emerged as a crucial pharmacophoric moiety: the pyridinium nitrogen engaged in a π–cation interaction with Trp-286 (99% of the simulation), and the ring itself maintained a π–π stacking interaction with the same residue for 99%. An additional π–cation interaction with Tyr-72 was observed for 35% of the trajectory. The carbazole ring contributed further through multiple π–π stacking interactions with Trp-86 (64% of the simulation time), Tyr-337 (32% and 38% of the sim.), Tyr-124 (47% of the sim), and Tyr-341 (64% of the sim). Collectively, these interactions highlight the significance of both the α,β-unsaturated system and the pyridinium ring as essential contributors to ligand binding and complex stability.

Fig. 8b presents the RMSD profiles. The 12i–AChE complex demonstrated superior structural stability with an average Cα RMSD of 1.20 Å (pale blue), an average ligand RMSD relative to the protein of 1.5 Å (red), and an average ligand RMSD relative to itself of only 0.75 Å (pink). These consistently low RMSD values strongly support the stability of the complex.

Fig. 8c depicts the RMSF values. The average Cα RMSF was approximately 0.6 Å, further confirming the rigidity of the system. Green bars indicate amino acid residues in contact with the ligand, showing that around 20 residues maintained direct interactions with 12i throughout the simulation. Finally, Fig. 8d displays the histogram of fractional interactions observed throughout the simulation. In this plot, green bars represent direct hydrogen bonds, blue bars indicate water-bridged hydrogen bonds, and purple bars correspond to hydrophobic interactions. According to the histogram, the most persistent and dominant contributors were Trp-286 and Tyr-341, both of which played central roles in stabilizing the 12i–AChE complex. Additional key residues included Phe-295, Tyr-337, and Trp-86, each providing significant hydrophobic and stacking contributions.

Fig. 9a illustrates the 2D ligand–protein interactions observed throughout the 500 ns MD simulation of 12g–BChE complex. The pyridinium nitrogen of compound 12g formed a persistent π–cation interaction with Trp-82 that was maintained for 99% of the simulation time. The same residue also established a π–π stacking interaction with the pyridinium ring, likewise present for 99% of the trajectory, highlighting Trp-82 as a key anchoring residue. In addition, Glu-197 formed a stable salt bridge with the pyridinium nitrogen for 99% of the simulation, confirming its crucial role in ligand stabilization. The acrylamide group contributed further stability, with one amide nitrogen forming a water-bridged hydrogen bond with Ala-328 (30%) and the other forming a direct hydrogen bond with Ser-198. Furthermore, the chlorophenyl ring participated in a π–π stacking interaction with Trp-231 (44%). Similar to 12i–AChE, the pyridinium moiety again emerged as the dominant pharmacophore in the 12g–BChE complex.

Fig. 9b shows the RMSD analysis of protein and ligand atoms. The average Cα RMSD of the protein remained stable at 1.2 Å (pale blue), indicating structural stability of BChE. The ligand RMSD (ligand fit on protein, red) remained around 1.2 Å up to 300 ns but then shifted to 2.4 Å, suggesting either the disruption of a specific interaction or a conformational rearrangement of the ligand. A similar trend was observed for the ligand RMSD (ligand fit on ligand, pink), which increased from 0.8 Å to 2.0 Å after 300 ns. Fig. 9c presents the RMSF values of protein residues, which were consistently low (1.0 Å), confirming minimal flexibility and stable binding site dynamics.

Finally, Fig. 9d depicts the fractional interaction histogram. In addition to other interactions ionic interactions are highlighted in red. Trp-82 was identified as the most critical residue for 12g binding, followed by Thr-120, Glu-197, Ser-198, and Tyr-332, all of which contribute significantly to the stability of the 12g–BChE complex.

As conclusion, 500 ns MD simulations provided strong validation of the docking results and highlighted the dynamic stability of both 12i–AChE and 12g–BChE complexes. 12i maintained exceptionally stable interactions with AChE, driven primarily by the α,β-unsaturated amide group and the pyridinium ring, which engaged in persistent hydrogen bonding, π–cation, and π–π stacking interactions with key residues such as Trp-286, Tyr-341, and Phe-295. RMSD and RMSF analyses further confirmed the overall rigidity and structural integrity of the 12i–AChE complex. Similarly, 12g exhibited robust binding within the BChE active site, with the pyridinium moiety playing a central role through persistent interactions with Trp-82 and a stable salt bridge with Glu-197. Additional contributions from Ala-328, Ser-198, and Trp-231 reinforced the stability of the complex. Although a modest ligand RMSD shift was observed after 300 ns, the protein backbone remained stable, indicating sustained overall binding.

Cytotoxicity

The cytotoxic effects of the synthesized compounds were evaluated using SH-SY5Y neuroblastoma and HEK293 embryonic kidney cell lines. SH-SY5Y cells, serving as a neuronal model relevant to neurodegenerative diseases, were used to assess neurotoxicity or neuroprotection, while HEK293 cells acted as a non-neuronal control to determine general cytotoxicity and selectivity. Each compound was tested at eight serial concentrations, and the results are summarized in Table 4. According to the results, the IC₅₀ values ranged from 7.11 to 21.35 μM for SH-SY5Y and 12.34 to 50.21 μM for HEK293, indicating that most compounds exhibited moderate to strong cytotoxic activity toward neuroblastoma cells with lower toxicity toward normal cells. Among all derivatives, compound 12i (R₂ = 4-NO₂) displayed the highest potency (IC₅₀ = 7.11 μM) against SH-SY5Y cells and the greatest selectivity index (7.0), followed by 12g (R₂ = 4-Cl, IC₅₀ = 8.60 μM, SI = 5.8). These two compounds demonstrated superior selectivity compared to the reference drug sorafenib (SI = 4.2), suggesting a favorable therapeutic window. Additionally, fluorinated analogs (12e and 12f) showed noteworthy cytotoxic profiles (IC₅₀ = 21.35 μM and 10.88 μM, respectively), indicating that electron-withdrawing halogen substituents enhance anticancer activity to a certain extent. Compounds such as 12a, 12b, 12c, 12e, 12k, and 12l showed relatively weaker effects, indicating lower potency compared to the most active members of the series.

Table 4. Cell viability of SH-SY5Y and HEK293 cells treated with the compounds for 24 h. Data are mean ± SD (n = 3).

Compounds	SH-SY5Y (IC50, μM)	HEK293 (IC50, μM)	Selectivity
12a	16.14 ± 0.11	30.18 ± 0.11	1.8
12b	19.90 ± 0.19	32.34 ± 0.09	1.6
12c	18.00 ± 0.16	39.19 ± 0.14	2.2
12d	13.13 ± 0.14	35.17 ± 0.07	2.6
12e	21.35 ± 0.07	50.21 ± 0.09	2.3
12f	10.88 ± 0.07	47.36 ± 0.10	4.3
12g	8.60 ± 0.06	49.83 ± 0.12	5.8
12h	12.46 ± 0.18	40.60 ± 0.17	3.3
12i	7.11 ± 0.14	49.98 ± 0.08	7.0
12j	9.50 ± 0.16	35.22 ± 0.13	3.7
12k	18.53 ± 0.05	28.99 ± 0.14	1.5
12l	14.97 ± 0.20	25.24 ± 0.06	1.7
12m	12.94 ± 0.17	32.55 ± 0.14	2.5
12n	12.53 ± 0.08	34.91 ± 0.08	2.8
12o	9.66 ± 0.08	21.94 ± 0.06	2.2
12p	17.75 ± 0.08	33.55 ± 0.19	1.9
12q	9.34 ± 0.10	12.34 ± 0.19	1.3
Sorafenib	3.81 ± 0.12	16.55 ± 0.14	4.2

The SAR analysis reveals that the nature and position of substituents (R₂) on the phenylacetamide moiety critically influence cytotoxic potency and selectivity. Electron-withdrawing groups (–NO₂, –Cl, –F) generally enhanced the antiproliferative activity, particularly when placed at the para-position (e.g., 12i, 12g, 12f). Electron-donating groups (−Me, –OMe) led to reduced activity (12b–d, 12l), indicating that increased electron density on the aromatic ring may decrease interaction efficiency with the biological target. Changing the R₁ substituent from benzyl to ethyl slightly reduced activity overall, suggesting that the benzyl group contributes to improved lipophilicity and potential π–π stacking with hydrophobic residues at the enzyme or receptor binding site. Overall, the benzyl-substituted, para-electron-withdrawing derivatives (especially 12i and 12g) emerged as the most promising compounds, combining potent anti-neuroblastoma activity with low cytotoxicity toward non-cancerous cells.

Conclusion

In summary, this study successfully developed a new class of carbazole–acrylamide–pyridinium-N-phenylacetamide hybrids (12a–q) as potent ChE inhibitors, evaluated against the reference standards tacrine and galantamine. Notably, all synthesized derivatives outperformed galantamine against both AChE and BChE, and most also showed greater potency than tacrine, further validating the success of our molecular hybridization strategy. Compounds 12i (AChE) and 12g (BChE) were identified as the most promising candidates, both acting via competitive inhibition with significantly higher potency than both reference drugs. A strong correlation between experimental and computational data confirmed stable and favorable enzyme-ligand interactions. Importantly, cytotoxicity evaluations on SH-SY5Y neuroblastoma and HEK293 normal cells revealed that most derivatives exhibited moderate to strong activity against cancer cells while maintaining low toxicity toward normal cells, indicating a favorable therapeutic window. Collectively, our findings introduce the carbazole–acrylamide–pyridinium scaffold as a valuable framework for further optimization and development of safer anti-Alzheimer drug candidates, though in vivo and BBB permeability studies are still lacking.

Conflicts of interest

The authors declare that they have no competing interests related to the publication of this work.

Data availability

The data supporting this article have been included as part of the supplementary information (SI).

Supplementary information is available. See DOI: https://doi.org/10.1039/d5md01075f.