From bench to brain: novel thieno-oxazine hybrids as potent pleiotropic anti-Alzheimer’s agents with in vivo/in vitro validation and in silico insights

Eman A Fayed; Mazin A A Najm; Khulood H Oudah; Maha A Ebrahim; Nirvana A Gohar; Karema Abu-Elfotuh; Ehsan Khedre Mohamed; Ahmed M E Hamdan; Najla A Albalawi; Shahad Faisal Alzahrani; Amira M Hamdan; Reema Almotairi; Shaimaa M Hafez; Triveena M Ramsis

doi:10.1080/14756366.2025.2598741

. 2026 Jan 2;41(1):2598741. doi: 10.1080/14756366.2025.2598741

From bench to brain: novel thieno-oxazine hybrids as potent pleiotropic anti-Alzheimer’s agents with in vivo/in vitro validation and in silico insights

Eman A Fayed ^a,^✉, Mazin A A Najm ^b, Khulood H Oudah ^b, Maha A Ebrahim ^a, Nirvana A Gohar ^c, Karema Abu-Elfotuh ^d,^e, Ehsan Khedre Mohamed ^f, Ahmed M E Hamdan ^g,^h, Najla A Albalawi ⁱ, Shahad Faisal Alzahrani ⁱ, Amira M Hamdan ^j, Reema Almotairi ^h,^k, Shaimaa M Hafez ^l, Triveena M Ramsis ^m

^{^a}Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt

^{^b}Department of Pharmacy, Mazaya University College, Nasiriyah, Iraq

^{^c}Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Cairo, Egypt

^{^d}Department of Clinical Pharmacy, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt

^{^e}College of Pharmacy, Al-Ayen Iraqi University, Thi-Qar, Iraq

^{^f}Department of Biochemistry, Egyptian Drug Authority (EDA), formerly National Organization of Drug Control and Research (NODCAR), Giza, Egypt

^{^g}Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia

^{^h}Prince Fahad Bin Sultan Chair for Biomedical Research (PFSCBR), University of Tabuk, Tabuk, Saudi Arabia

^ⁱFaculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia

^{^j}Oceanography Department, Faculty of Science, Alexandria University, Alexandria, Egypt

^{^k}Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk, Saudi Arabia

^{^l}Department of Anatomy and Embryology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

^{^m}Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sinai University, Ismailia, Egypt

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14756366.2025.2598741.

^✉

CONTACT Eman A. Fayed alfayed_e@azhar.edu.eg; alfayed_e@yahoo.com Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt

Roles

Eman A Fayed: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Mazin A A Najm: Formal analysis, Writing – original draft, Writing – review & editing

Khulood H Oudah: Formal analysis, Writing – original draft, Writing – review & editing

Maha A Ebrahim: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Nirvana A Gohar: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Karema Abu-Elfotuh: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing

Ehsan Khedre Mohamed: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Ahmed M E Hamdan: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Najla A Albalawi: Data curation, Writing – original draft, Writing – review & editing

Shahad Faisal Alzahrani: Data curation, Writing – original draft, Writing – review & editing

Amira M Hamdan: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Reema Almotairi: Formal analysis, Writing – original draft, Writing – review & editing

Shaimaa M Hafez: Formal analysis, Writing – original draft, Writing – review & editing

Triveena M Ramsis: Data curation, Formal analysis, Methodology, Software, Writing – original draft, Writing – review & editing

PMCID: PMC12777904 PMID: 41481412

Abstract

Due to their various pharmacological effects, several substituted sulphur heterocycles containing thiophene have recently attracted a great deal of attention. A novel 2,3-diaryl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (9–14) was synthesised starting from cyclohexa[b]thiophene. Compounds 9 and 10 showed the greatest gene expression downregulation of BAX by 75.1% and 79.7%, and upregulation of Bcl-2 gene expression by 8.1 folds for each. It also decreased the level of AChE by 70.2 and 75%; respectively. Compounds 9 and 10 significantly increased Wnt3a levels by 5.8 and 6.6 folds, and β-Catenin levels by 10.1 and 10.5 folds, respectively, compared to donepezil. They significantly downregulated 5-GSK3β gene expression by 77.1%, and 78.7%, respectively. Even though all compounds exhibited potent inhibition of AChE, all synthesised compounds, except for compounds 5 and 11 demonstrated higher selectivity towards BChE (SI < 1). In-silico ADMET calculations as well as molecular docking have been performed for synthetic compounds.

Keywords: Thieno-oxazines, Alzheimer’s disease, AChE and BChE inhibitors, ADMET and docking studies, multi-targets and histopathological examinations

Graphical abstract

graphic file with name IENZ_A_2598741_UF0001_C.jpg

Introduction

Mood disorders (depression and anxiety), agitation or aggression, psychosis, and impairments in emotional communication and facial emotional expressions are all linked to Alzheimer’s disease (AD). AD is a rather complex neurodegenerative disease that is attributed to a combination of multiple factors associated with cognitive decline due to cholinergic and glutamatergic deficits¹^,². According to earlier biochemical research, the main underlying causes of the illness include Aβ accumulation, hyperphosphorylated τ-protein aggregation, and oxidative stress^3–5. Reduced ACh levels in particular brain regions that regulate memory and learning processes cause an exclusive decrease in cholinergic neurons, which is the main cause of AD^6–8. Acetylcholinesterase inhibitors raise the amount of ACh in the synaptic cleft by preventing its breakdown^9–12.

The B cell leukemia-2 gene product (Bcl-2) family controls apoptosis; Bcl-2 inhibits apoptosis, while BAX promotes it¹³. The overexpression of BAX and the downregulation of Bcl-2 gene expression in AD cause a significant shift in the ratio of pro-apoptotic BAX to anti-apoptotic Bcl-2¹⁴. β-secretase, also referred to as BACE1 and γ-secretase enzymes, cleaves APP to produce Aβ^15–17. Neuroinflammation, oxidative stress, and neuronal apoptosis/necrosis are all brought on by Aβ diffusing across the brain parenchyma¹⁸^,¹⁹. When Aβ attaches itself to TLR4, it triggers an inflammatory cascade that activates the NLRP3 inflammasome multiprotein complex²⁰, that causes neurodegeneration²¹^,²². Reduced Aβ clearance causes buildup in the brain, contributing to the aetiology of Alzheimer’s disease^23–25. In AD patients, the harmful effects of Aβ can be lessened and neurogenesis can be encouraged by treatment strategies that inhibit GSK3β and/or induce Wnt/β-catenin signalling in neural cells^26–28. One crucial signal transduction system that controls many biological functions, including cell survival, is Wnt/β-catenin signalling²⁹^,³⁰. In addition to being essential for neuronal survival and neurogenesis, Wnt/β-catenin signalling also regulates synaptic plasticity and the integrity and function of the blood-brain barrier (BBB)^31–33. Furthermore, τ-protein hyperphosphorylation and amyloid-β formation in the brain are inhibited by Wnt/β-catenin signalling activity³⁴^,³⁵. Crucially, the AD brain exhibits a significant suppression of Wnt/β-catenin signalling through a variety of pathogenic pathways^36–38. Restoring Wnt/β-catenin signalling thus offers a remarkable potential for the rational development of innovative AD treatments²⁶^,³⁹.

Acetylcholinesterase inhibitor medications, such as donepezil⁴⁰, rivastigmine⁴¹, memantine⁴² and galantamine⁴³, are used to treat Alzheimer’s disease (Figure 1)⁴⁴.

Figure 1. — Structures of FDA approved anti-Alzheimer’s drugs.

Since the pathological process of AD involves several mechanisms, all of which result in increased oxidative stress and inflammation, we used a rat model to examine the effects of our newly synthesised compounds on behavioural parameters as a therapeutic strategy for slowing the progression of AD against AlCl₃-induced Alzheimer’s disease. The results of the docking experiments and ADMET properties will be useful for further lead optimisation.

More and more evidence points to the necessity of BChE for regulating ACh levels in the brain during the latter phases of Alzheimer’s disease^45–48. In severe AD, AChE levels in the brain fall to 55–67% of normal ranges, whereas BChE levels increase to 165% of normal levels⁴⁹. It is also worth noting that BChE inhibition has no adverse peripheral effects. As such, a big breakthrough has been made with the development of highly specific and potent BChE inhibitors that can raise ACh levels in the brain with a great reduction in negative effects on the peripheral nervous system^49–52.

Owing to their widespread occurrence in nature, heterocyclic molecules find extensive application in both the synthesis and biological fields⁵³. Thiophene is a heterocyclic sulphur-containing moiety that demonstrates a variety of biological properties, including antibacterial, antiviral, cytotoxic, and fungal properties^54–58.

The purpose of this article as a continuation of our prior work which was about the synthesis of bicyclic and tricyclic cyclohepta[b]thiophene derivatives is to produce novel lead compounds comprising cyclohexa-thieno-oxazine hybrids that may be employed as AChE and BChE inhibitors to treat Alzheimer’s disease⁴⁵. We developed derivatives of bicyclic and tricyclic cyclohexa[b]thiophene and tested their influence on various pathways, such as learning and memory, antioxidant activity, neuroinflammatory inhibitory activity, apoptotic biomarkers, β-amyloid deposition, and improving BDNF levels in brain tissues, as well as, BAX/Bcl-2 and Wnt/β-catenin, in order to find new potential candidates to serve as AChE and BChE inhibitors (Figure 2). Among the most potent hybrids, the mechanisms of action of AChE and BChE inhibitors were also determined. The results of the completed docking investigations and the predictions of ADMET properties could help with further lead optimisation.

Figure 2. — Reasoned analysis of the recently created synthetic materials.

Material and methods

Chemistry

General details

Solvents were purchased from local suppliers. Fine reagents were purchased from Sigma Aldrich. The melting points were recorded uncorrected using the Electrothermal LA 9000 SERIS, Digital Melting Point Apparatus. At Al-Azhar University’s Faculty of Pharmacy (Girls), IR spectra were acquired using an Agilent IR 200 FT IR Spectrophotometer and the KBr disc technique. The outcomes are shown in cm⁻¹. The ¹H NMR Spectra at 400 MHz and the ¹³C NMR Spectra at 100 MHz on the Gemini 400 Mercury were performed using the NMR Spectrometer at the Main Chemical Warfare Laboratories, Chemical Warfare Department, Ministry of Defense. Using DMSO-d₆ as the solvent, chemical changes were tracked and calculated in parts per million (ppm) in relation to TMS, which served as the internal standard. A Schimadzu GC/MS-QP5050A Spectrometer was used at the Regional Centre for Mycology and Biotechnology at Al-Azhar University to record mass spectra at a voltage of 70 eV.

The purities of the tested compounds were determined by UHPLC-PDA (Thermo Fisher Scientific) at the Regional Centre for Mycology and Biotechnology at Al-Azhar University. The separation of analytes was carried out using Acquity BEH C18 100 mm × 2.1 mm column (particle size, 1.7 μm). The solvent system consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). UHPLC-method: In a gradient run, using acetonitrile and water; the percentage of eluent (B) increased from an initial concentration of 5% at 0 min to 100% at 5.5 min, kept at 100% for 1 min, and flushed back to 5% in 0.5 min. The flow rate was set to 0.6 ml/min.

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1)

Created utilising the documented process²³^,⁵⁹.

Ethyl 2-hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2)

Compound 1 (0.005 mol) was dissolved in 30 millilitres of water and 4.5 millilitres of 37%w/v hydrochloric acid. The diazonium salt was obtained by adding a dropwise, stirring addition of a cold solution of sodium nitrite (0.005 mol) in water (1 ml) to the cooled amine solution (0–5 °C). To guarantee a full response, stirring was maintained for 15 minutes. Adding an equivalent amount of water and boiling it in a warm water bath at 50 °C for 30 min made hydrolysis simple. Once N₂ evolution stopped, the precipitate that had formed was filtered, dried, and crystallised from pure ethanol to produce the desired hydroxy product.

Ethyl 2-(aryloxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (4,5)

Standard operating procedure

A stirred solution of compound 2 (0.01 mol) in N,N-dimethyl formamide (DMF) (20 ml) with five drops of triethylamine was heated under reflux for six hours before the addition of the proper benzoyl chloride or 4-chloro benzoyl chloride (0.01 mol). Pour the mixture into ice-cold water once it has cooled. Following a water rinse and a dry filter, the solid crystallised from the ethanol.

Ethyl 2-(benzoyloxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (4)

In the same manner as in the previous method, compound 2 was combined with benzoyl chloride to form compound 4.

Yield: 90%; m.p.: 170–172 °C; IR (KBr, cm⁻¹)= 3075 (CH aromatic), 2928, 2870 (CH aliphatic), 1654 (2 C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.30 (t, 3H, CH₃CH₂), 1.70 (m, 4H, 2CH₂ at position 5, 6), 2.71 (m, 2H, CH₂ at position 4), 2.87 (m, 2H, CH₂ at position 7), 4.27 (q, 2H, CH₃CH₂), 7.02 (t, 1H, ArH), 7.22 (t, 1H, Ar-H), 7.48 (d, 1H, J = 7.6 Hz, Ar-H), 7.56 (d, 1H, J = 7.2 Hz, Ar-H), 7.88 (t, 1H, ArH); ¹³C NMR (100 MHz, DMSO–d₆): δ = 14 (CH₂CH₃), 20, 21, 22, 23 (cyclohexa-C_4,5,6,7), 60.9 (CH₂CH₃), 113.5 (thiophene C₃), 127, 128 (cyclohexa-C_4a, C_7a), 129.72, 130.2, 131,132, 133, 134 (ArCs), 151 (C₂), 161 (COOC₂H₅), 165.89 (COO-phenyl); MS (EI, 70 eV): m/z (%)= 330.03 [M^{. +}] (5.97%), 104.99 (100%); Anal. Calc. for C₁₈H₁₈O₄S (330.40): C, 65.44; H, 5.49; S, 9.70; Found: C, 65.47; H, 5.51; S, 9.68.

Ethyl 2-((4-chlorobenzoyl)oxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5)

Yield: 92%; m.p.: 167–168 °C; IR (KBr, cm⁻¹)= 3090 (CH aromatic), 2980, 2860 (CH aliphatic), 1677 (2 C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.30 (t, 3H, CH₃CH₂), 1.70 (m, 4H, 2CH₂ at position 5, 6), 2.65 (m, 2H, CH₂ at position 4), 2.86 (m, 2H, CH₂ at position 7), 4.27 (q, 2H, CH₃CH₂), 7.46 (d, 1H, J = 8.4 Hz, Ar-H), 7.51 (d, 1H, J = 8.8 Hz, Ar-H), 7.67 (d, 1H, J = 8.4 Hz, Ar-H), 7.89 (d, 1H, J = 8.8 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 14 (CH₃CH₂), 20.5, 22, 23, 24 (cyclohexa-C_4,5,6,7), 60.1 (CH₃CH₂), 113 (thiophene C₃), 129.12, 129.21 (cyclohexa-C_4a, C_7a), 130.109, 131.56, 131.59, 131.68, 138.23 (4-chlorophenyl Cs), 152 (C₂), 161 (COOC₂H₅), 166.91 (COO phenyl); MS (EI, 70 eV): m/z (%)= 366.09 [M + 2] (17.21%), 364.87 [M^{. +}] (22.69), 290.35 (100%); Anal. Calc. for C₁₈H₁₇ClO₄S (364.84): C, 59.26; H, 4.70; S, 8.79. Found: C, 59.25; H, 4.73; S, 8.80.

2,3-Diaryl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (9–14)

Standard operational policy.

The appropriate amine (0.01 mol) and ketoester 4, 5 (0.01 mol) were heated under reflux for 10 h in 10 millilitres of glacial acetic acid. The reaction mixture was cooled and concentrated under reduced pressure before being triturated with diethyl ether. The separated substance was filtered and crystallised using the proper solvent.

2,3-Diphenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (9)

As previously indicated, compound 9 was created by combining aniline with compound 4.

Crystallised from ethanol; Yield: 79%; m.p.: 205- 206 °C; IR (KBr, cm⁻¹)= 3030 (CH aromatic), 2929 (CH aliphatic), 1655 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 2.01 (s, 4H, 2CH₂ at position 5, 6), 2.71 (m, 2H, CH₂ at position 4), 2.86 (m, 2H, CH₂ at position 7), 6.97 (t, 2H, Ar-H), 7.23 (t, 2H, Ar-H), 7.48 (s, 1H, oxazine-H), 7.54 (d, 2H, J = 8 Hz, Ar-H), 7.60 (m, 2H, Ar-H), 7.92 (d, 2H, J = 8.4 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 22, 23, 24.45, 24.39 (cyclohexa-thiophene C_4,5,6,7), 75, 119.37 (oxazine Cs), 119.47, 120 (cyclohexa-thiophene C_4a, C_7a), 123.38, 123.43, 129.03, 129.11, 129.13, 129.14, 139.74 (phenyl Cs), 167 (CO), 168.73 (oxazine Cs); MS (EI, 70 eV): m/z (%)= 361 [M^{. +}] (10.54), 299 (100%); Anal. Calc. for C₂₂H₁₉NO₂S (361.46): C, 73.10; H, 5.30; N, 3.88; S, 8.87; Found: C, 73.11; H, 5.32; N, 3.84; S, 8.85.

3-(4-Chlorophenyl)-2-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (10)

As previously indicated, compound 10 was created by combining 4-chloroaniline with compound 4. Crystallised formed from acetic acid as crystals; Yield: 85%; m.p.: 218–220 °C; IR (KBr, cm⁻¹)= 3080 (CH aromatic), 2910 (CH aliphatic), 1655 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.44 (s, 4H, 2CH₂ at position 5, 6), 2.01 (m, 2H, CH₂ at position 4), 2.21 (m, 2H, CH₂ at position 7), 7.18 (s, 1H, oxazine-H), 7.31 (d, 2H, J = 8.4 Hz, Ar-H), 7.44 (t, 1H, Ar-H), 7.51 (d, 2H, J = 8.8 Hz, Ar-H), 7.64 (t, 1H, Ar-H), 7.97 (m, 1H, Ar-H), 8.24 (d, 2H, J = 7.6 Hz, Ar-H); MS (EI, 70 eV): m/z (%)= 397 [M + 2] (0.77), 395 [M^{. +}], 40 (100%); Anal. Calc. for C₂₂H₁₈ClNO₂S (395.90): C, 66.74; H, 4.58; N, 3.54; S, 8.10; Found: C, 66.75; H, 4.57; N, 3.56; S, 8.12.

3-(4-Methoxyphenyl)-2-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazi-n-4-one (11)

As previously indicated, compound 11 was created by combining 4-methoxyaniline with compound 4, formed a crystal out of ethanol; Yield: 79%; m.p.: 260–261 °C; IR (KBr, cm⁻¹) = 3060 (CH aromatic), 2927 (CH aliphatic), 1655 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.89 (s, 4H, 2CH₂ at position 5, 6), 1.97 (s, 4H, 2CH₂ at position 4, 7), 3.68 (s, 3H, OCH₃), 6.83 (d, 3H, J = 8.8 Hz, Ar-H), 6.89 (s, 1H, oxazine-H), 7.43 (m, 5H, J = 12 Hz, Ar-H), 7.50 (d, 2H, J = 7.6 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 22, 23, 24.19, 24.27 (cyclohexa-thiophene C_4,5,6,7), 55.65 (OCH₃), 75, 114.17 (oxazine Cs), 120.87, 121.05 (cyclohexa-thiophene C_4a, C_7a), 127, 128, 129, 132.96, 133.9, 140, 155 (phenyl Cs), 166 (CO), 168.69 (oxazine Cs); MS (EI, 70 eV): m/z (%)= 391 [M^{. +}] (8.70), 52 (100%); Anal. Calc. for C₂₃H₂₁NO₃S (391.49): C, 70.57; H, 5.41; N, 3.58; S, 8.19; Found: C, 70.55; H, 5.42; N, 3.59; S, 8.21.

2-(4-Chlorophenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (12)

As previously indicated, compound 12 was created by combining aniline and compound 5, formed from ethanol as crystals; Yield: 83%; m.p.: 271–273 °C; IR (KBr, cm⁻¹) = 3070 (CH aromatic), 2927, 2854 (CH aliphatic), 1655 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.72 (s, 2H, CH₂ at position 5), 2.01 (s, 2H, CH₂ at position 4,6), 2.62 (s, 2H, CH₂ at position 7), 6.97 (t, 1H, Ar-H), 7.18 (s, 1H, oxazine-H), 7.24 (t, 2H, Ar-H), 7.55 (d, 4H, J = 8 Hz, Ar-H), 7.92 (d, 2H, J = 8.4 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 20, 21, 22, 23 (cyclohexa-thiophene C_4,5,6,7), 75.47, 119 (oxazine Cs), 123, 124 (cyclohex-athiophene C_4a, C_7a), 129.14, 129.24, 130.086, 131.604, 138.23, 139 (phenyl Cs), 166.90 (CO), 168.69 (oxazine Cs); MS (EI, 70 eV): m/z (%) = 397 [M + 2] (8.24%), 395 [M^{. +}] (61.81), 382 (100%); Anal. Calc. for C₂₂H₁₈ClNO₂S (395.90): C, 66.74; H, 4.58; N, 3.54; S, 8.10; Found: C, 66.77; H, 4.59; N, 3.50; S, 8.08.

2,3-Bis(4-chlorophenyl)-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (13)

As previously indicated, compound 13 was created by combining 4-chloroaniline and compound 5. Crystallised from ethanol; Yield: 89%; m.p.:180–181 °C; IR (KBr, cm⁻¹)= 3060 (CH aromatic), 2934, 2870 (CH aliphatic), 1597 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.72 (s, 2H, CH₂ at position 5), 2.02 (s, 2H, CH₂ at position 6), 2.62 (s, 2H, CH₂ at position 4), 2.72 (s, 2H, CH₂ at position 7), 7.31 (d, 2H, J = 8.8 Hz, Ar-H), 7.53 (s, 1H, oxazine-H), 7.55 (d, 2H, J = 12 Hz, Ar-H), 7.69 (d, 2H, J = 8.8 Hz, Ar-H), 7.92 (d, 2H, J = 8.4 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 22.5, 23, 24.38, 24.46 (cyclohexa-thiophene C_4,5,6,7), 85, 112.4 (oxazine Cs), 120.83, 120.96 (cyclohexa-thiophene C_4a, C_7a), 126.91, 128.92, 129.08, 129.13, 129.24, 130.08, 131.25, 131.57, 131.61, 138.23, 138.71 (4-chlorophenyl Cs), 166.90 (CO), 168.87 (oxazine Cs); MS (EI, 70 eV): m/z (%)= 433 [M + 4] (0.83%), 431 [M + 2] (4.85%), 429 [M^{. +}] (3.17%), 138 (100%); Anal. Calc. for C₂₂H₁₇Cl₂NO₂S (429.34): C, 61.40, H, 3.98; N, 3.25; S, 7.45; Found: C, 61.45, H, 3.97; N, 3.23; S, 7.44.

2-(4-Chlorophenyl)-3-(4-methoxyphenyl)-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (14)

As previously indicated, compound 14 was created by combining 4-methoxyaniline and compound 5. Crystallised from ethanol; Yield: 90%; m.p.: 229–230 °C; IR (KBr, cm⁻¹)= 3068 (CH aromatic), 2930, 2850 (CH aliphatic), 1647 (C = O); ¹H NMR (400 MHz, DMSO–d₆): δ = 1.72 (s, 2H, CH₂ at position 5), 1.97 (s, 2H, CH₂ at position 6), 2.62 (s, 2H, CH₂ at position 4), 2.72 (s, 2H, CH₂ at position 7), 3.68 (s, 3H, OCH₃), 6.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.44 (d, 2H, J = 8.8 Hz, Ar-H), 7.54 (missing signal, d,? H, J = 8.8,? ?), 7.68 (d, 2H, J = 8.8 Hz, Ar-H), 7.89 (s, 1H, oxazine-H), 7.92 (d, 2H, J = 8.8 Hz, Ar-H); ¹³C NMR (100 MHz, DMSO–d₆): δ = 23.5, 23.9, 24.17, 24.28 (cyclohexa-thiophene C_4,5,6,7), 55.46 (CH₃), 86, 112 (oxazine Cs), 114.14, 114.30 (phenyl Cs), 120.84, 121.08 (cyclohexa-thiophene C_4a, C_7a), 129.1, 129.2, 130.09, 131.57, 131.61, 132.97, 138.23, 155.45 (phenyl Cs), 166.90 (CO), 168.14 (oxazine Cs); MS (EI, 70 eV): m/z (%)= 427 [M + 2] (17.35%), 425 [M^{. +}] (11.12%), 362 (100%); Anal. Calc. for C₂₃H₂₀ClNO₃S (425.93): C, 64.86; H, 4.73; N, 3.29; S, 7.53; Found: C, 64.87; H, 4.75; N, 3.31; S, 7.51.

In vivo assessment

Animals and induction of AD rat models

The Faculty of Pharmacy’s Research Ethics Committee of Sinai University’s Kantara Branch, Kantara East, Ismailia, Egypt, approved all animal treatment practices and procedures (SU.REC.2024 (18 A)). All procedures and experiments were conducted in compliance with the applicable National Institutes of Health standards and regulations for the Care and Use of Laboratory Animals. In this experiment, adult male Dawley rats in excellent condition weighing between 300 and 320 g were used. The animals were acquired from the Nile Company for Pharmaceuticals and Chemical Industries (Cairo, Egypt), licence number is USER-219. They were placed in a controlled laboratory setting with unrestricted access to water, a temperature range of 24 to 26 °C, and 12-h light-dark cycles. Three rats per cage were housed in polycarbonate cages that were individually packed with paper and encased with stainless steel wire⁶⁰.

Experimental design

The rats were randomly assigned into 11 groups with 10 rats each as follows. Group 1: Rats were administered saline (i.p., daily for 5 weeks) and considered as a vehicle control. Group 2: AlCl₃-induced AD (AD) by administering rats with 70 mg/kg AlCl₃, i.p., daily for 5 weeks) as reported⁶⁰. Groups 3–11: AD + either donepezil, compound 4, compound 5, compound 9, compound 10, compound 11, compound 12, compound 13, or compound 14 (orally, 30 mg/kg)⁶¹. At the end of the study, after five weeks, behavioural tests were used to evaluate the extent of spatial recognition and memory impairment^62–64.

Animal justification

We made statistical justification using the G*Power tool to ensure that the sample size is sufficient to detect a true effect with a desired power (0.95) and statistical significance (alpha level). We obtained 300 rats as total sample size.

However, regarding the ethical considerations and practical needs, such as accounting for animal loss and the cost of experimentation, we use the final number (110 rats, 10 rats per group; 6 rats for biochemical tests and 4 rats for histopathological tests) to observe the differences between the intervention and control groups and to be robust to achieve scientific objectives while minimising adverse effects and improving welfare as a neutral balancing of benefits and harms.

In addition, a strong justification can be based on the number of animals used in prior, comparable studies where the desired statistical significance was achieved.

Behavioural tests

Morris water maze test (MWM)

Spatial learning and memory were investigated using the Morris water maze test⁶⁵. A circular water tank, 150 cm in diameter and 60 cm in height, was filled with tap water to a depth of 30 cm (25 ± 2 ◦C). To make the water translucent, non-toxic white paint was applied. The east, west, north, and south quadrants of the pool were essentially separated into four equal sections. At a specific spot in the middle of one quadrant, an escape platform with a diameter of 10 cm was buried 2 cm below the water’s surface. The platform stood in the exact same quadrant throughout the testing. The rats’ swimming route was recorded by a video tracking camera situated above the pool. From a designated spot in each quadrant, each rat was submerged in the water with its back turned to the pool wall, and it was then left to swim to the platform. The rats received training sessions every day for three days in a row, with four trials conducted in each session. Before the next trial began, the animals were given a maximum of 60 s to explore the hidden platform. After that, they were permitted to relax on it for 20 s. The rat was positioned gently on the platform and allowed 20 s to rest if it took longer than 60 s to locate it. The time it took to find the platform, or the escape latency, was recorded. In order to do a probing test on the fourth day, the platform was removed away, and the rats were given 60 s to swim freely. We used a specially designed room with an indirect light in order to avoid interference with the camera and the tracking software. A Microsoft Surface Pro 4 Tablet with its built-in-camera was used in order to record the rat videos for behavioural analysis. The videos were acquired in the MP4 format with a resolution of 1920 × 1080 pixels at 30 frames per second. The video files are processed by a MATLAB script (ver R2018b) downloaded from the following website: https://mathworks.com/downloads. The amount of time spent in the assigned quadrant was measured⁶⁵.

Y-maze spontaneous alternation (SAB) test

One kind of short-term memory that SAB can represent is spatial working memory. Three arms, designated A, B, or C were employed in a black hardwood Y-maze with a symmetrical triangular core space. Rats were briefly positioned at the edge of one arm and given 8 min to freely navigate around the maze. When the rat’s rear paws were fully inside the arm, the entries were counted. The following formula was used to determine SAB based on the total number of arm entries and alternations: SAB (%) = [number of alternations/(total arm entries-2)] × 100 ⁶⁶.

Tissue sampling and preparation

Cervical dislocation was performed under thiopental sodium anaesthesia (50 mg/kg, i.p.). The brain tissues of the rats were removed and rigorously washed in isotonic saline 24 h after they were euthanized following the last behavioural test. For histological analysis, four brains per group were preserved for the whole night in 10% neutral buffered formalin. The remaining six brains were split into two sections each. In order to create a 10% homogenate (w/v), the first component was instantaneously homogenised using an ice-cold solution that included 300 mM sucrose and 50 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 1800× g for 10 min at 4 ◦C for biochemical tests, and the supernatant was thereafter kept at −20 ◦C. The second portion was set aside for use in real-time PCR analysis at −80 °C⁶⁶.

Biochemical measurements

Fluorometric technique

Brain monoamine levels were promptly assessed following the rats’ euthanasia to prevent changes in the substance’s concentration. Dopamine (DA), norepinephrine (NE), and serotonin (5-HT) fluorometric tests were determined in brain tissue homogenate using the Ciarlone method. The process involved oxidising monoamines to their adrenochromes, rearranging them to their adrenolutins, and then fluorometrically detecting them using samples at λ_ex/λ_em 320/385 nm, 385/485 nm, and 360/470 nm for DA, NE, and 5-HT, respectively. The concentrations were expressed as nanograms per gram of fresh tissue using the fluorescence of reference solutions⁶⁷.

Colorimetric technique

Malondialdehyde (MDA) was evaluated using the thiobarbituric acid method at a wavelength of 532 nm in order to colorimetrically detect the degree of lipid peroxidation in brain homogenate. The superoxide dismutase (SOD) enzyme activity was measured using the Marklund et al. method, which is based on the enzyme’s capacity to decrease the nitro blue tetrazolium dye at a wavelength of 540 nm⁶⁸. To evaluate the total antioxidant capacity (TAC), a colorimetric approach was employed to estimate the amount of hydrogen peroxide that remains after 3,5-dichloro-2-hydroxybenzene sulphonate is converted to a coloured product at 660 nm⁶⁸. Additionally, a colorimetric measurement was made at 412 nm to assess the amount of Acetylcholinesterase (AChE) in the brain tissue homogenate⁶⁹.

ELISA technique

Brain tissue homogenate was used to test the levels of interleukin-1β (IL-1β) and tumour necrosis factor-alpha (TNF-α) using ELISA kits (Ray Biotech, Inc. Cat No: IQR-IL1b and My BioSource, Inc., San Diego, CA, USA Cat No: MBS175904, respectively). Brain-derived neurotrophic factor (BDNF) and β-amyloid were among the biomarkers of cognition and the degree of neurodegeneration that were estimated using ELISA kits (MyBioSource, Inc., San Diego, CA, USA). Furthermore, ELISA kits from MyBioSource, Inc., San Diego, CA, USA, were used to quantify the concentration of the β-secretase enzyme (BACE1). Following the manufacturer’s instructions, the brain concentrations of Wnt Family Member 3 A (Wnt3a) (orb555678, Biorbyt Ltd., Cambridge, UK) and Rat β-catenin ELISA Kit (K3383, Biovision Inc.) were assessed. Every step of the quantitative sandwich ELISA procedure was carried out according to the manufacturer’s instructions⁷⁰^,⁷¹.

Real-time quantitative polymerase chain reaction

Using the Applied Biosystems Step One Plus device, real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the mRNA levels of GSK-3β, BAX, and Bcl-2. Total RNA was isolated using the Qiagen tissue extraction kit (Qiagen, Germantown, MD, USA) in accordance with the manufacturer’s instructions. The Maxima SYBR Green qPCR kit (Fermentas, Hanover, MD, USA) was used to amplify the extracted mRNA after it had been reverse-transcribed using a Sense Rapid cDNA Synthesis kit (CAT No. BIO-65053) (Table 1). The ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used to quantify the mRNA levels. In order to determine the relative expression of the target genes, the findings were normalised to β-actin expression using the 2 − ΔΔCT technique⁶³.

Table 1.

The sequences of primers employed in real-time RT-PCR analysis.

Target gene	Primer sequence	Accession number
BAX	F: 5′-CACGTCTGCGGGGAGTCA-3′ R: 5′-TAGGAAAGGAGGCCATCCCA-3′	NM_017059
Bcl-2	F: 5′-CATCTCATGCCAAGGGGGAA-3′ R: 5′-TATCCCACTCGTAGCCCCTC-3′	NM_016993
GSK-3β	F: 5′-AGCCTATATCCATTCCTTGG-3′ R: 5′-CCTCGGACCAGCTGCTTT-3′	NM_032080
β-actin	F: 5′-CCGTAAAGACCTCTATGCCA-3′ R: 5′-AAGAAAGGGTGTAAAACGCA-3′	NM_031144

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AChE inhibition assay

All newly created compounds were subjected to an acetylcholinesterase enzyme inhibitory screen. This was done with the Colorimetric Acetylcholinesterase Inhibitor Screening Kit (BioVision Catalogue # K197-100). The 96-well assay kit provides a rapid, simple, and reliable way to screen for AChE inhibitors in large quantities. The candidate inhibitors were dissolved at 100X or higher and then further diluted to a concentration of 20X using the AChE test buffer. A completely transparent 96-well plate with a flat bottom was filled with 10 µl of test inhibitors that were diluted (20X). AChE Assay Buffer was used to dilute 2 μL of the stock (10 mM) solution to 100 μL in order to create the inhibitor control (donepezil). AChE Assay Buffer was then used to further dilute the donepezil solution to 40 μL. Ten microliters of the 20 μM donepezil working solution were added to the chosen well. After being reconstituted, acetylcholinesterase was diluted 25 times. 10 μL of diluted acetylcholinesterase was applied to each well that contained sample compounds, solvent control, and inhibitor control. A 12-fold dilution of the AChE substrate was made. 25 μL of AChE assay buffer, 5 μL of probe mix, and 10 μL of diluted AChE substrate were mixed to create the reaction mix. The reaction mix was mixed with the sample compound, enzyme control, and inhibitor control. After 40 min at room temperature in kinetic mode, the absorbance (OD) at a wavelength of 412 nm was measured using a temperature-controlled plate reader. In a control test, DMSO was utilised as the solvent and was anticipated to have 100% enzyme activity. The IC₅₀ was calculated using nonlinear regression analysis using the response-concentration (log) curve⁷².

BChE inhibition assay

Using an Invitrogen BChE test kit (Catalogue #EIABCHEF), the assay was performed in compliance with the supplier’s instructions. The study was conducted using black 96-well plates. 100 μL of each diluted tested chemical or tacrine was added to the 96-well plate. Each well was then filled with 50 μL of the reaction mixture, which consisted of fluorescence detection reagent and butyrylthiocholine iodide in assay buffer. The plate was then left to incubate for 20 min at room temperature. Finally, excitation at 390 nm was used to observe emission at 510 nm using a microplate reader. Background fluorescence was further eliminated for each data point. In a single run, the assay was conducted in triplicate⁷³^,⁷⁴.

Statistical analysis

The Tukey-Kramer test was used for posthoc analysis after the one-way ANOVA was used for multiple comparisons. The findings are shown as mean ± SEM, and a p values of less than 0.05 was deemed statistically significant. The GraphPad Prism software (version 8, ISI^®, San Diego, CA, USA) was used in order to perform statistical analysis and to create the graphs.

Histopathological examination of brain tissue

Brain tissue samples were fixed in 10% formalin for 24 h, washed with water, and then serially diluted with alcohol to induce dehydration. After being submerged in paraffin, the specimens were cut into segments that were 4 µm thick using a microtome. After being collected on glass slides, the tissue samples were deparaffinized and stained with haematoxylin and eosin in order to conduct a standard histological examination under a light microscope¹⁸.

In silico ADME and toxicity prediction

All synthetic compounds in this study, as well as the conventional reference pharmaceuticals Tacrine and Donepezil, were examined via the Swiss ADME web interface (http://www.sib.swiss)⁷⁵. The drug-likeness and characteristics of the in silico ADME were investigated. The molecular structures were transformed into SMILES databases using Chemdraw 19.0. Using these SMILES as input, the SwissADME website was used to assess the physicochemical characteristics, pharmacokinetic properties, lipophilicity, ADME parameters, and medicinal chemistry compatibility⁷⁶^,⁷⁷. BBB permeability was predicted using ADMETLab3.0 webtool (https://admetlab3.scbdd.com/)⁷⁸^,⁷⁹.

Toxicity of the most active hits, 9, 11, and 12, together with two reference anti-Alzheimer’s medications, Tacrine and Donepezil, based on web tools: ProTox-II (https://tox-new.charite.de/protox_II) and pkCSM (http://biosig.unimelb.edu.au/pkcsm/prediction)⁸⁰^,⁸¹.

Molecular docking

The docking simulation utilised the 3D crystal structures of AChE in complex with Donepezil (PDB ID: 4EY7, resolution: 2.35 Å) and BChE in complex with Tacrine (PDB ID: 4BDS, resolution: 2.10 Å) retrieved from the RCSB PDB (https://www.rscb.org) in PDB format^82–85. The protein crystal structure was imported in Molecular Operating Environment (Montreal, QC, Canada) (MOE 2014.09)⁸⁶. The MMFF94x forcefield was set before the simulation study. The imported proteins were prepared by rectifying usual errors, such as adding missing H atoms and minimising energy^87–90. All water molecules were eliminated from the structure^91–94. The structures of the synthesised compounds were sketched in ChemBioDraw Ultra 19.0, saved as .mol files, and imported into a database. The triangle matcher was utilised for placement, while London dG was employed as a scoring function throughout the docking procedure. The Rigid Receptor approach was utilised for refining^95–97.

Results and discussion

Chemistry

The target hybrids were created using the procedures indicated in Scheme 1 and Scheme 2. Compound 1 was created by applying the Gewald reaction²³. 2-Hydroxy-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl ester 2 was prepared from 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl ester 1 as reported⁶³.

The esters 4 and 5 are prepared from the reaction of benzoyl chloride and 4-chlorobenzene chloride with 2-hydroxy thiophene derivative 2, respectively⁹⁸^,⁹⁹.

The ¹H NMR spectrum of hybrid 4 represented three triplets and two doublet signals at δ 7.02 –7.88 ppm corresponding to the phenyl five protons, in addition to the ¹³C NMR spectrum which showed a signal at δ 165.89 ppm due to COO-phenyl. In addition, signals at δ 129.72, 130.2, 131,132, 133, 134 ppm due to phenyl C_1-6 carbons.

The ¹H NMR spectrum of derivative 5 revealed four doublets at δ 7.45, 7.50, 7.66, and 7.87 ppm due to the para-substituted chlorophenyl protons. Moreover, the ¹³C NMR spectrum showed peaks at 130.10, 131.56, 131.59, 131.68, and 138.23 corresponding to 4-chlorophenyl C₁-₆ carbons, in addition to a peak at δ 166.91 ppm due to COO phenyl.

Different aniline derivatives were reacted with the thiophene esters 4 and 5 to afford 1,3-oxazine 4-one derivatives 9–14¹⁰⁰^,¹⁰¹. The ¹H NMR spectra of compound 9 revealed a singlet signal at δ 6.97 ppm corresponding to the N-CH proton. In addition, the multiplet signals at δ 6.97–7.92 ppm is due to the two phenyl protons.

Moreover, the ¹³C NMR spectrum of compound 9 showed a signal at δ 75 ppm corresponding to oxazine C₂ and signals at 123.38, 123.43, 129.03, 129.11, 129.13, 129.14, and 139.74 due to the two phenyl rings carbons.

The ¹H NMR spectrum of compound 10 revealed a singlet signal at δ 7.18 ppm due to oxazine-H, in addition to two doublets at δ 7.56 and 8.24 ppm corresponding to 4-chlorophenyl protons, respectively.

The structure of compound 11 was evidenced by the presence of two singlet signals at δ 3.68 ppm and 6.89 ppm in the ¹H NMR spectrum characteristic for OCH₃ and oxazine-H protons, respectively. Moreover, the ¹³C NMR spectrum for the same structure showed signals at δ 55.65 and 75 ppm due to OCH₃ and oxazine-H carbons.

Furthermore, the ¹H NMR spectrum of compound 12 revealed a singlet signal at δ 7.18 ppm due to oxazine-H proton and two doublets at 7.54 and 7.91 due to 4-chlorophenyl-Hs). Furthermore, the ¹³C NMR spectrum of compound 12 showed three signals at δ 75.47, 131.60, and 138.23 ppm, corresponding to oxazine C₂, 4-chlorophenyl C_3,5 and 4-chlorophenyl C_{2, 6}).

The ¹H NMR spectrum of compound 13 revealed a singlet signal at δ 7.55 ppm due to oxazine-H proton, while the ¹³C NMR spectrum revealed a signal at δ 85.00 ppm due to oxazine C₂ carbon in addition to signals δ 129.24, 130.08, 131.25, 131.5 ppm due to N-4-chlorophenyl carbons. Moreover, the mass spectrum of compound 13 showed three peaks at m/z 433 [M + 4], 431 [M + 2] and 429 [M^{. +}].

The ¹H NMR spectrum of compound 14 revealed a singlet signal at δ 3.86 ppm due to OCH₃ protons, in addition to singlet signal at δ 7.89 ppm and four doublets at δ 6.82, 7.43, 7.67, 7.91 ppm due to Ar-H protons. While the ¹³C NMR spectrum revealed two singlet signals at δ 55.46 and 86 ppm due to OCH₃ and oxazine C₂ carbons, respectively. Mass spectrum of compound 14 showed two peaks at 427 and 425 due to [M^.+2] and [M^. +], respectively.

In vivo assessment

Effects on behavioural tests in AlCl₃-induced AD

All the tests were done using donepezil as a control drug. Compound 9 demonstrated substantial reduction in escape latency by 77.4%, surpassing that of donepezil, whereas 11 and 13 resulted in 69% and 66.8% reduction in escape latency, which is relatively equal to donepezil. Hybrids 12 and 14 demonstrated moderate escape latency reduction by 53.3% and 56.7%, respectively. However, compound 4 showed the mild reduction in escape latency by 35.1% (Figure 3(A-D)).

Figure 3. — Effects on Behavioural Tests in AlCl₃-induced AD: (A) The escape latency during Probe/retention trial on day 4. (B) Time spent in target quadrant in the MWM test. (C) SAB (%) in Y-Maze test. The data is presented as means ± SE (n = 6).

However, the administration of compounds 9 and 10 increased the residence time by nearly 5.9 and 5.5 times, which are higher than donepezil, while 11 and 12 showed a relatively equal activity to donepezil by 5.3 and 5.4 times, respectively.

Compounds 9 and 11 administrations showed an increase in SAB% of about 64.5% for both, confirming the ability to donepezil. In addition, 10 and 13 showed relatively high increase in SAB% by 51.8% and 58.9%, respectively. Compounds 5 and 14 demonstrated moderate increase in SAB% by 41.7% and 41.4%, respectively. A mild increase in SAB% was exhibited by compounds 4 and 12 by values of 21.5 5% and 38.8%, respectively (Tables 2 and 3).

Table 2.

The effect of compounds 4 and 5 on escape latency (sec) and time spent in the target quadrant (sec) as tested by MWM and SAB% as tested by Y-maze.


Cpd. No.	R¹	R²	MWM		Y-maze
Cpd. No.	R¹	R²	Escape latency (sec)	Time spent in the target quadrant (sec)	SAB%
Control	–	–	6.667 ± 0.31	54.0 ± 1.21	93.5 ± 0.85
AD	–	–	53.17 ± 1.05^a	7.83 ± 0.70^a	50 ± 2.89^a
Donepezil	–	–	14.78 ± 0.83^ab	42.67 ± 0.61^ab	85 ± 0.58^ab
4	H	–	36.83 ± 0.97^abc	24.5 ± 1.15^abc	62.17 ± 0.99^abc
5	Cl	–	34.5 ± 1.18^abc	26.67 ± 1.36^ab	72.5 ± 0.33^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 3.

The effect of compounds 9–14 on escape latency (sec) and time spent in the target quadrant (sec) as tested by MWM and SAB% as tested by Y-maze.


Cpd. No.	R¹	R²	MWM		Y-maze
Cpd. No.	R¹	R²	Escape latency (sec)	Time spent in the target quadrant (sec)	SAB%
Control	–	–	6.667 ± 0.31	54.0 ± 1.21	93.5 ± 0.85
AD	–	–	53.17 ± 1.05^a	7.83 ± 0.70^a	50 ± 2.89^a
Donepezil	–	–	14.78 ± 0.83^ab	42.67 ± 0.61^ab	85 ± 0.58^ab
9	H	H	12.0 ± 0.58^ab	46.0 ± 1.16^ab	84.17 ± 0.92^ab
10	H	Cl	19.5 ± 0.67^abc	42.83 ± 0.87^ab	77.67 ± 0.88^abc
11	H	OCH₃	16.5 ± 1.07^ab	41.83 ± 0.48^abc	84.17 ± 1.24^ab
12	Cl	H	24.83 ± 0.33^abc	34.17 ± 1.25^abc	71.0 ± 0.92^abc
13	Cl	Cl	17.67 ± 0.6^ab	36.0 ± 1.07^abc	81.33 ± 0.88^ab
14	Cl	OCH₃	23.0 ± 0.88^abc	37.83 ± 0.87^abc	72.33 ± 0.48^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effect on BACE1 and amyloid-β

Treatment with compounds 9, 10, and 11 diminished β-amyloid levels significantly by 75.4%, 82.6%, and 70.7%; respectively, confirming superiority to Donepezil (reduction by 68.8%). Compounds 13 and 14 showed a high decrease in β-amyloid by 62.4%, 50.9%; respectively. Compound 12 only showed moderate decrease in β-amyloid by 43.3%, whereas compounds 4 and 5 exhibited minor reduction of β-amyloid by 29.4% and 36.4%; respectively in comparison to donepezil (Figure 4).

Figure 4. — Effects on BACE1 and amyloid- β biomarker in AlCl₃-induced AD. The data is presented as means ± SE (n = 6).

In parallel, compounds 9, 10, and 11 demonstrated a significant decrease in the levels of BACE1 by 73.4%, 78.2% and 68%, respectively. Treatment with compounds 13 and 14 revealed a quite good reduction in BACE1 levels by 63.4% and 57.5%, whereas treatment with compounds 4, 5 and 12 revealed a minimal reduction of BACE1 levels by 21.6%, 33.9% and 44.5%, respectively (Tables 4 and 5).

Table 4.

Effects of 4 and 5 on BACE1 (ng/mL) and amyloid-β (ng/g tissue).


Cpd. No.	R¹	R²	BACE1 (ng/mL)	Amyloid-β (ng/g tissue)
Control	–	–	1.012 ± 0.01	1.592 ± 0.03
AD	–	–	29.55 ± 0.61^a	26.06 ± 0.57^a
Donepezil	–	–	4.93 ± 0.04^ab	8.1 ± 1.71^ab
4	H	–	23.16 ± 0.26^abc	18.39 ± 0.30^abc
5	Cl	–	19.53 ± 0.45^abc	16.57 ± 0.17^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c) relative to donepezil. Significance: p < 0.05.

Table 5.

Effects of compounds 9–14 on BACE1 (ng/mL) and amyloid-β (ng/g tissue).


Cpd. No.	R¹	R²	BACE1 (ng/mL)	Amyloid-β (ng/g tissue)
Control	–	–	1.012 ± 0.01	1.592 ± 0.03
AD	–	–	29.55 ± 0.61^a	26.06 ± 0.57^a
Donepezil	–	–	4.93 ± 0.04^ab	8.1 ± 1.71^ab
9	H	H	7.87 ± 0.07^abc	6.405 ± 0.1^abc
10	H	Cl	6.437 ± 0.11^abc	4.533 ± 0.1^abc
11	H	OCH₃	9.463 ± 0.13^abc	7.625 ± 0.11^bc
12	Cl	H	16.12 ± 0.38^abc	14.78 ± 0.12^abc
13	Cl	Cl	10.81 ± 0.13^abc	9.797 ± 0.12^bc
14	Cl	OCH₃	12.5 ± 0.2^abc	12.8 ± 0.12^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effects on brain neurotransmitters levels in AlCl₃-induced AD

Administration of compounds 9, 10, 11, and 13 caused high elevation in DA levels by 4.6, 4.5, 4.4, 4.1 folds, while compounds 4, 5, 12 and 14 moderately increased the AD levels by 2.1, 2.6, 3.2 and 3.8 folds in comparison to donepezil (Figure 5).

Figure 5. — Effects on brain neurotransmitters in AlCl₃-induced AD: (A) DA, 5-HT and AChE (B) NE (C) BDNF. The data is presented as means ± SE (n = 6).

Moreover, compounds 9, 10 and 11 effectively increased the level of NE by 2.5, 2.6 and 2.3, respectively, while compounds 4, 5, 12, 13 and 14 exhibited moderate increase in the level of NE by 1.2, 1.4,1.7, 2, and 1.9 folds, respectively in comparison to the donepezil.

Furthermore, compounds 9, 10 and 11 greatly increased the 5-HT levels by 4.8, 5.6, and 4.4folds, respectively, while compounds 4, 5, 12, 13 and 14 exhibited a moderate increase in the 5-HT levels 1.7, 2.3, 2.7, 3.9, and 3.6 folds, respectively, in comparison with donepezil.

Compounds 9 and 10 showed elevation in BDNF levels by 3, 3.2 more than donepezil, compounds 11, 12, 13 and 14 relatively increased BDNF levels by 2.7, 2, 2.5, 2.2 folds, respectively, while 4 and 5 showed moderate increase by 1.5 and 1.8 folds, respectively in comparison to donepezil.

Additionally, administration of compounds 12, 13 and 14 displayed a high decrease in AChE by 70.2%, 75%, and 71.2%, respectively. Compounds 12, 13, and 14 moderately decreased AChE by 47%, 63.8%, and 61.8%, respectively. In comparison to donepezil, compounds 4 and 5 exhibited a mild reduction in AChE levels of 23.7% and 34.8%, respectively (Tables 6 and 7).

Table 6.

Effect of compounds 4 and 5 on brain monoamines: DA (ng/g tissue), 5-HT (ng/g tissue) and NE (nmol/g tissue), AChE (ng/g tissue) and BDNF (ng/mL) levels in brain tissues in comparison with Donepezil and negative control.


Cpd. No.	R¹	R²	DA (ng/g tissue)	5-HT (ng/g tissue)	NE (nmol/g tissue)	AChE (ng/g tissue)	BDNF (ng/mL)
Control	–	–	64.73 ± 0.4	13.59 ± 0.03	730.6 ± 0.36	11.93 ± 0.22	165.5 ± 0.25
AD	–	–	11.84 ± 0.09^a	1.887 ± 0.07^a	195.8 ± 0.86^a	67.34 ± 0.49^a	42.56 ± 0.48^a
Donepezil	–	–	58.12 ± 0.97^ab	11.23 ± 0.35^ab	635.4 ± 14.01^ab	13.91 ± 0.41^ab	122.4 ± 0.87^ab
4	H	–	24.71 ± 0.74^abc	3.155 ± 0.07^abc	241.2 ± 0.91^abc	51.41 ± 0.51^abc	64.95 ± 1.27^abc
5	Cl	–	30.69 ± 0.45^abc	4.292 ± 0.08^abc	270.9 ± 0.97^abc	43.89 ± 0.64^abc	75.75 ± 0.65^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 7.

Effect of compounds 9–14 on brain monoamines: DA (ng/g tissue), 5-HT (ng/g tissue), NE (nmol/g tissue) and AChE (ng/g tissue), as well as BDNF (ng/mL) levels in brain tissues in comparison with Donepezil and negative control.


Cpd. No.	R¹	R²	DA (ng/g tissue)	5-HT (ng/g tissue)	NE (nmol/g tissue)	AChE (ng/g tissue)	BDNF (ng/mL)
Control	–	–	64.73 ± 0.4	13.59 ± 0.03	730.6 ± 0.36	11.93 ± 0.22	165.5 ± 0.25
AD	–	–	11.84 ± 0.09^a	1.887 ± 0.07^a	195.8 ± 0.86^a	67.34 ± 0.49^a	42.56 ± 0.48^a
Donepezil	–	–	58.12 ± 0.97^ab	11.23 ± 0.35^ab	635.4 ± 14.01^ab	13.91 ± 0.41^ab	122.4 ± 0.87^ab
9	H	H	54.63 ± 0.36^abc	9.133 ± 0.11^abc	493.2 ± 1.25^abc	20.09 ± 0.63^abc	127.3 ± 0.96^abc
10	H	Cl	52.96 ± 1.12^abc	10.65 ± 0.11^ab	509.3 ± 2.97^abc	16.83 ± 0.10^abc	134.8 ± 0.67^abc
11	H	OCH₃	51.88 ± 0.38^abc	8.222 ± 0.04^abc	448.6 ± 0.54^abc	19.4 ± 0.36^abc	115.8 ± 0.91^abc
12	Cl	H	52.58 ± 0.37^abc	5.07 ± 0.04^abc	327.9 ± 0.8^abc	35.66 ± 0.70^abc	83.62 ± 0.92^abc
13	Cl	Cl	63.68 ± 0.25^abc	7.387 ± 0.07^abc	391.6 ± 1.1^abc	24.35 ± 0.72^abc	104.9 ± 0.94^abc
14	Cl	OCH₃	44.92 ± 0.43^abc	6.77 ± 0.10^abc	374.4 ± 1.69^abc	25.73 ± 0.79^abc	94.48 ± 1.03^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effects on neuroinflammatory biomarkers (IL-β and TNF-α) in AlCl₃-induced AD

Studying the effects of the synthesised compounds on amyloid-β, IL-1β and TNF-α proved that treatment with compounds 9, 10, 11, and 13 diminished IL-1β levels significantly by 65.3%, 69.5%, 60.4%, and 56.9%; respectively, which are higher than donepezil inhibition, compounds 12 and 14 showed moderate activity by 39.4% and 45.6%, but compounds 4 and 5 exhibited only mild inhibition activity by 22.3% and 34.3%; respectively in comparison to donepezil (Figure 6).

Figure 6. — Effects on Neuroinflammatory Biomarkers in AlCl₃-induced AD: (A) IL-1β and (B) TNF-α. The data is presented as means ± SE (n = 6).

In parallel, the administration of compounds 9, 10, and 11 decreased TNF-α levels by 73.3%, 76.7%, and 69.6%, respectively, which is higher than the donepezil inhibition effect. Compounds 12, 13, and 14 exhibited a high decrease in TNF-α levels by 57.6%, 60.4%, and 63.5%, relatively equal to donepezil, but compounds 4 and 5 showed a low decrease by 44.9% and 52%, respectively (Tables 8 and 9).

Table 8.

Effects of 4 and 5 on the neuroinflammatory biomarkers IL-β (pg/g tissue) and TNF-α (pg/g tissue).


Cpd. No.	R¹	R²	IL-β (pg/g tissue)	TNF-α (pg/g tissue)
Control	–	–	31.43 ± 0.26	26.9 ± 0.19
AD	–	–	119.4 ± 0.29^a	174.2 ± 0.58^a
Donepezil	–	–	58.75 ± 0.99^ab	61.7 ± 1.12^ab
4	H	–	92.77 ± 0.70^abc	96.07 ± 0.37^abc
5	Cl	–	78.52 ± 0.53^abc	83.54 ± 0.65^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 9.

Effects of compounds 9–14 on the neuroinflammatory biomarkers IL-β (pg/g tissue) and TNF-α (pg/g tissue).


Cpd. No.	R¹	R²	IL-β (pg/g tissue)	TNF-α (pg/g tissue)
Control	–	–	31.43 ± 0.26	26.9 ± 0.19
AD	–	–	119.4 ± 0.29^a	174.2 ± 0.58^a
Donepezil	–	–	58.75 ± 0.99^ab	61.7 ± 1.12^ab
9	H	H	41.47 ± 0.25^abc	46.49 ± 0.41^abc
10	H	Cl	36.45 ± 0.61^abc	40.66 ± 0.38^abc
11	H	OCH₃	47.33 ± 0.74^abc	52.96 ± 0.53^abc
12	Cl	H	72.42 ± 0.58^abc	73.88 ± 0.86^abc
13	Cl	Cl	51.45 ± 0.42^abc	68.98 ± 0.52^abc
14	Cl	OCH₃	65.0 ± 0.65^abc	63.55 ± 1.31^ab

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effects on BAX and bcl-2

Interestingly, compound 10 downregulated the gene expression of BAX by 79.7%, which is higher than the downregulation effect demonstrated by donepezil. Compounds 9 and 11 showed a relatively equal inhibition activity to donepezil by 75.1% and 68.5%, respectively. Compounds 13 and 14 showed a moderate effect of 58.7% and 49.5%, respectively, but compounds 4 and 5 showed a mild downregulation effect of 23.1% and 31.7%, respectively, in comparison to donepezil (Figure 7).

Figure 7. — Effects on apoptosis Biomarkers in AlCl3-induced AD Bcl2 and BAX. The data is presented as means ± SE (n = 6).

In contrast, compounds 9, 10, and 11 upregulated the gene expression of Bcl-2 by 8.1, 8.1, and 7.9 folds, respectively, which is relatively equal to donepezil upregulation. Compounds 12, 13, and 14 exhibited high upregulation by 5.4, 7, and 6 folds, whereas compounds 4 and 5 displayed moderate upregulation by 3.1 and 4.1 folds in comparison to donepezil. BAX/Bcl-2 ratio calculated for all synthesised compounds demonstrated their ability to shift the ratio to lower values, confirming their ability to improve brain cell proliferation. The BAX/Bcl-2 ratio of compound 9 is comparable to that of donepezil, while that of compound 10, is superior to that of donepezil (Tables 10 and 11).

Table 10.

Effects of 4 and 5 on the gene expression of apoptotic biomarkers Bcl-2 and BAX.


Cpd. No.	R¹	R²	Bcl-2	BAX	BAX/Bcl-2 ratio
Control	–	–	1.079 ± 0.006	1.013 ± 0.004	0.94
AD	–	–	0.120 ± 0.002^a	9.192 ± 0.16^a	76.28
Donepezil	–	–	0.980 ± 0.002^ab	2.253 ± 0.058^ab	2.30
4	H	–	0.369 ± 0.007^abc	7.070 ± 0.073^abc	19.14
5	Cl	–	0.492 ± 0.01^abc	6.283 ± 0.05^abc	12.75

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 11.

Effects of compounds 9–14 on the gene expression of apoptotic biomarkers Bcl-2 and BAX.


Cpd. No.	R¹	R²	Bcl-2	BAX	BAX/Bcl-2 ratio
Control	–	–	1.079 ± 0.006	1.013 ± 0.004	0.94
AD	–	–	0.120 ± 0.002^a	9.192 ± 0.16^a	76.28
Donepezil	–	–	0.980 ± 0.002^ab	2.253 ± 0.058^ab	2.30
9	H	H	0.978 ± 0.003^ab	2.292 ± 0.08^ab	2.34
10	H	Cl	0.978 ± 0.01^ab	1.870 ± 0.07^abc	1.91
11	H	OCH₃	0.954 ± 0.01^ab	2.900 ± 0.06^abc	3.04
12	Cl	H	0.653 ± 0.022^abc	5.567 ± 0.1^abc	8.52
13	Cl	Cl	0.837 ± 0.01^abc	3.800 ± 0.03^abc	4.54
14	Cl	OCH₃	0.726 ± 0.01^abc	4.638 ± 0.09^abc	6.39

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effect on WNT signalling pathway

Studying the effects on WNT Signalling Pathway, compounds 9 and 10 exhibited high elevation in Wnt3a levels by 5.8 and 6.6 folds, respectively, whereas compounds 11, 13, and 14 showed moderate elevation by 4.9, 4.4, and 4 folds, respectively. Compounds 4, 5, and 12 exhibited mild elevation in Wnt3a levels by 2.2, 2.9, and 3.3 folds, respectively (Figure 8).

Figure 8. — Effects on Wnt3/β-Catenin/GSK3β signalling pathway in AlCl3-induced AD. The data is presented as means ± SE (n = 6).

Furthermore, compounds 9 and 10 significantly increased the level of β-Catenin by 10.1 and 10.5 folds, respectively, superior to that shown by donepezil. Compound 11 exhibited quite a high elevation of β-Catenin levels by 9.3 folds. Furthermore, compounds 12, 13, and 14 showed moderate elevation in the level of β-Catenin by 5.5, 7.9, and 6.7 folds, respectively, while compounds 4 and 5 exhibited mild increase in the level of β-Catenin by 3.3 and 4.3 folds, respectively.

On the other hand, compounds 9, 10, 11 and 13 greatly downregulated the gene expression of the 5- GSK3β by %, 77.1%, 78.7%, 69.3% and 64.4%; respectively. Compounds 12 and 14 revealed moderate downregulation to the gene expression of the 5- GSK3β by 47.5% and 53.8%; respectively, while compounds 4 and 5 exhibited the least down-regulation effect by 13.8%, 32.7%; respectively (Tables 12 and 13).

Table 12.

Effects of 4 and 5 on Wnt3/β-catenin/GSK-3β signalling.


Cpd. no.	R¹	R²	Wnt3a (ng/g tissue)	GSK3β	β-catenine (ng/L)
Control	–	–	1.012 ± 0.003	1.013 ± 0.004	17.58 ± 0.27
AD	–	–	0.129 ± 0.002^a	10.2 ± 0.029^a	1.338 ± 0.02^a
Donepezil	–	–	0.9803 ± 0.005^ab	2.045 ± 0.068^ab	12.92 ± 0.48^ab
4	H	–	0.2807 ± 0.011^abc	8.795 ± 0.116^abc	4.47 ± 0.32^abc
5	Cl	–	0.375 ± 0.01^abc	6.862 ± 0.150^abc	5.808 ± 0.10^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 13.

Effects of compounds 9–14 on Wnt3/β-catenin/GSK-3β signalling.


Cpd. No.	R¹	R²	Wnt3a (ng/g tissue)	GSK3β	β-Catenine (ng/L)
Control	–	–	1.012 ± 0.003	1.013 ± 0.004	17.58 ± 0.27
AD	–	–	0.129 ± 0.002^a	10.2 ± 0.029^a	1.338 ± 0.02^a
Donepezil	–	–	0.9803 ± 0.005^ab	2.045 ± 0.068^ab	12.92 ± 0.48^ab
9	H	H	0.7483 ± 0.01^abc	2.338 ± 0.06^ab	13.47 ± 0.11^ab
10	H	Cl	0.845 ± 0.01^abc	2.175 ± 0.1^ab	14.1 ± 0.22^abc
11	H	OCH₃	0.6283 ± 0.01^abc	3.13 ± 0.07^abc	12.37 ± 0.22^ab
12	Cl	H	0.4267 ± 0.01^abc	5.355 ± 0.07^abc	7.33 ± 0.13^abc
13	Cl	Cl	0.5733 ± 0.01^abc	3.63 ± 0.04^abc	10.53 ± 0.1^abc
14	Cl	OCH₃	0.5217 ± 0.003^abc	4.717 ± 0.05^abc	8.922 ± 0.04^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Effects on oxidative stress in brain tissues in AlCl₃-induced AD

Compounds 9, 10 and 11 exhibited improvements in TAC level by 4, 4.4, 3.9 folds, respectively, surpassing that of donepezil. Compounds 12, 13, 14 demonstrated by a 3.1, 3.4 and 3.3-fold, a comparable to that of donepezil, while compounds 4 and 5 exhibited the mild improvement of TAC by 1.9, 2.7 folds, respectively (Figure 9).

Figure 9. — Effects on oxidative stress in AlCl₃-induced AD: (A) TAC (B) SOD (C) MDA. The data is presented as means ± SE (n = 6).

Furthermore, compounds 9, 10, 11, 13, and 14 treatments demonstrated a substantial improvement in the levels of SOD activity by 6.5, 7.7, 6.3, 6.7- and 6.7 folds; respectively, while compounds 4, 5, 12 showed a mild increase in the levels of the SOD activity by 1.8, 4.6 and 5.6 folds, respectively.

Moreover, in contrast, an outstanding decrease in the MDA brain content was achieved by compounds 9, 10 and 14 by values of 68.4%, 72.8% and 79.4%, respectively. Moderate reduction of MDA levels was noted upon treatment with compounds 12 and 13 by 54.8% and 45.3%, respectively (Tables 14 and 15).

Table 14.

Effect of compounds 4 and 5 on oxidative stress represented by TAC (µmol/g tissue), SOD (U/g) and MDA (nmol/g tissue) levels in brain tissues in comparison with Donepezil and negative control.


Cpd. No.	R¹	R²	TAC (µmol/g tissue)	SOD (U/g)	MDA (nmol/g tissue)
Control	–	–	46.68 ± 0.26	5.783 ± 0.048	7.105 ± 0.05
AD	–	–	9.553 ± 0.16^a	0.38 ± 0.01^a	116.3 ± 0.30^a
Donepezil	–	–	34.88 ± 0.42^ab	3.052 ± 0.01^ab	25.25 ± 1.51^ab
4	H	–	18.26 ± 0.26^abc	0.683 ± 0.05^abc	92.07 ± 0.42^abc
5	Cl	–	25.87 ± 0.07^abc	1.747 ± 0.01^abc	77.14 ± 0.88^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Table 15.

Effect of compounds 9–14 on oxidative stress represented by TAC (µmol/g tissue), SOD (U/g) and MDA (nmol/g tissue) levels in brain tissues in comparison with Donepezil and negative control.


Cpd. No.	R¹	R²	TAC (µmol/g tissue)	SOD (U/g)	MDA (nmol/g tissue)
Control	–	–	46.68 ± 0.26	5.783 ± 0.048	7.105 ± 0.05
AD	–	–	9.553 ± 0.16^a	0.38 ± 0.01^a	116.3 ± 0.30^a
Donepezil	–	–	34.88 ± 0.42^ab	3.052 ± 0.01^ab	25.25 ± 1.51^ab
9	H	H	38.23 ± 0.23^abc	2.455 ± 0.04^abc	36.75 ± 0.85^abc
10	H	Cl	42.4 ± 0.61^abc	2.917 ± 0.08^bc	31.63 ± 0.36^abc
11	H	OCH₃	36.96 ± 0.36^abc	2.383 ± 0.03^abc	72.02 ± 0.68^abc
12	Cl	H	29.33 ± 0.18^abc	2.145 ± 0.05^abc	52.58 ± 0.37^abc
13	Cl	Cl	32.9 ± 0.53^abc	2.528 ± 0.08^abc	63.68 ± 0.53^abc
14	Cl	OCH₃	31.67 ± 0.58^abc	2.555 ± 0.11^abc	23.92 ± 0.97^abc

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Significance (^a): relative to the control group. Significance (^b): relative to the AD group. Significance (^c): relative to donepezil. Significance: p < 0.05.

Histopathological examination

As shown in Figure 10(A-D), the control group shows normal histological structure of neurons in cerebral cortex, striatum, fascia dentata and subiculum. Induction of AD using AlCl₃ resulted in high number of shrunken and degenerated neurons with sever nuclear pyknnosis in cerebral cortex, nuclear pyknosis and red neurons (necrotic neurons) in striatum, sever nuclear pyknosis in neurons of fascia dentata, presence of red neurons and neurons with sever nuclear pyknosis (Figure 10(E-H)).

Rats given donepezil demonstrated moderate nuclear pyknosis in the cerebral cortex, striatum, fascia dentata, and subiculum neurons in brain tissues (Figure 10(I-L)). Similarly, rats given either compound 4 or compound 5 displayed considerable nuclear pyknosis in the cerebral cortex, fascia dentata, striatum, and subiculum neurons (Figure 10(M-P) and (Q-T)). Like hybrids 4 and 5, hybrid 9 (Figure 11(A’-D’)) showed histologically similar neuronal structures in the cerebral cortex, fascia dentata, striatum, and subiculum. In the micrographs of rat brain sections supplied with derivative 10, the cerebral cortex showed mild nuclear pyknosis, while the striatum, fascia dentata, and subiculum neurons showed moderate pyknosis (Figure 11(E’-H’)).

Treatment with hybrids 11–14 resulted in normal histological structure of neurons in the cerebral cortex (Figure 11(I’), (M’), (Q’) and (U’)), as well as normal histological structure of fascia dentata, except for compound 11 (Figure 11(K’), (O’), (S’) and (W’)). Moderate pyknosis was observed in neurons of the striatum and subiculum upon treatment with compound 11 (Figure 11(J’) and (L’)). Mild pyknosis was seen in case of compounds 12 and 13, in striatum (Figure 11(N’) and (R’)) and subiculum neurons (Figure 11(P’) and (T’)). Treatment with hybrid 14 resulted in severe pyknosis in the histological structure of neurons in striatum and subiculum (Figure 11(V’) and (X’)).

AChE and BChE inhibition

Using donepezil and tacrine as reference medications, respectively, all synthesised hybrids were assessed as possible inhibitors of AChE and BChE. The development of very potent and selective BChE inhibitors that can raise ACh levels in the brain with few adverse effects on the periphery has advanced significantly. With IC₅₀ values less than 15 µM, all drugs demonstrated inhibition of AChE and BChE (Figure 12). The most powerful AChE inhibitors were discovered to be compounds 9, 11, and 12, with respective IC₅₀ values of 0.73 µM, 0.43 µM, and 0.92 µM. With IC₅₀ values of 2.49 µM and 2.39 µM, respectively, compounds 4 and 14 showed promising inhibitory efficacy against AChE. Additionally, compounds 5 and 10 showed modest AChE inhibitory action, with corresponding IC₅₀ values of 6.32 µM and 6.43 µM. Furthermore, compound 12 demonstrated remarkable BChE inhibitory efficacy on par with tacrine. Moreover, compounds 9 and 14 had exceptional BChE inhibitory activity, with IC₅₀ values of 0.26 µM and 0.37 µM, respectively. Additionally, compounds 4, 10, and 11 demonstrated excellent BChE inhibition, with IC₅₀ values of 0.79 µM, 0.73 µM, and 0.54 µM, respectively.

Figure 12. — IC₅₀ values of compounds 4–5 and 9–14 against AChE and BChE.

Selectivity Index (SI) is calculated as a ratio of IC₅₀ for BChE and IC₅₀ for AChE. Preferential inhibition of BChE is indicated when the value is less than 1. Even though all compounds exhibited strong inhibition of AChE, all created hybrids, except for compounds 5 and 11 demonstrated higher selectivity towards BChE (Tables 16 and 17).

Table 16.

IC₅₀ values and selectivity index (SI) of 4 and 5 against AChE and BChE vs. Donepezil and Tacrine as reference drugs.


Cpd. No.	R¹	R²	IC₅₀ (µM)		Selectivity index BChE/AChE
Cpd. No.	R¹	R²	AChE	BChE	Selectivity index BChE/AChE
4	H	-	2.49 ± 0.081	0.79 ± 0.027	0.32
5	Cl	-	6.32 ± 0.208	14.38 ± 0.475	2.28
Donepezil	–	–	0.25 ± 0.008	–	NA
Tacrine	–	–	–	0.15 ± 0.005	NA

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Table 17.

IC₅₀ values and selectivity index (SI) of compounds 9–14 against AChE and BChE vs. donepezil and tacrine as reference drugs.


Cpd. No.	R¹	R²	IC₅₀ (µM)		Selectivity index BChE/AChE
Cpd. No.	R¹	R²	AChE	BChE	Selectivity index BChE/AChE
9	H	H	0.73 ± 0.025	0.26 ± 0.008	0.36
10	H	Cl	6.43 ± 0.212	0.73 ± 0.025	0.11
11	H	OCH₃	0.43 ± 0.015	0.54 ± 0.018	1.27
12	Cl	H	0.92 ± 0.03	0.14 ± 0.005	0.15
13	Cl	Cl	14.21 ± 0.467	4.88 ± 0.16	0.34
14	Cl	OCH₃	2.39 ± 0.08	0.37 ± 0.011	0.16
Donepezil	–	–	0.25 ± 0.008	–	NA
Tacrine	–	–	–	0.15 ± 0.005	NA

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SAR study

In order to comprehend the structure-activity relationship, the new compounds’ biological activities were utilised in this investigation. The tricyclic ring system is generally better than the bicyclic ring system, as demonstrated by the outstanding learning, memory, and SAB% scores of compounds 9–14 on behavioural tests. Regardless of the N-phenyl ring being substituted, compounds 9 and 11 showed the best performance in behavioural tests when the benzene ring on the C2 of oxazine was unsubstituted. The satisfactory, but small, performance shown by compounds 4 and 5 in behavioural tests has demonstrated that bicyclic compounds are practically less active than tricyclic compounds. It has been confirmed that adding a para-chloro-substitution to the N-phenyl ring improves behavioural performance in Alzheimer’s disease. Tricyclic compounds 10 and 13, which have one para-chloro substitution on the N-phenyl ring, performed well in behavioural tests, albeit marginally worse than compounds 9 and 11.

Moreover, the effect of compounds 9–14 on the inhibition of AChE expression levels confirmed that the tricyclic ring system is generally preferable to the bicyclic ring system, like behavioural performance. Maximal inhibition of AChE expression levels was achieved by compounds 10 and 11, confirming the importance of para-substituent on N-phenyl ring, whether electron-donating or electron-withdrawing group.

Additionally, the superiority of the tricyclic compounds (compounds 9–14) to bicyclic compounds (compounds 4 and 5), in minimising the levels inflammatory biomarkers confirm the essential requirement of a tricyclic ring system. Maximal anti-inflammatory activity demonstrated by tricyclic compounds 9, 10 and 11, confirm that the presence of unsubstituted 2-phenyl ring is responsible for better activity.

Compounds 4 and 5′s inhibitory effects against AChE show a SAR, highlighting the significance of the unsubstituted benzene ring’s presence in improving the activity of bicyclic compounds. Hybrids 9, 11, and 12’s inhibitory actions against AChE support a similar pattern of SAR in tricyclic compounds, which highlights the significance of having at least one unsubstituted phenyl ring. It was discovered that compound 13′s double para-chloro group substitution was detrimental to anti-AChE action, as evidenced by IC₅₀ values greater than 14 µM.

Based on the inhibitory activities of 9 and 12 against BChE, SAR demonstrated the significance of having unsubstituted N-phenyl ring which results maximal BChE inhibitory activity. The presence of an additional para-methoxy group (compounds 11 and 14) resulted in a slight reduction in BChE inhibitory activity. The N-phenyl ring’s para-chloro substitution presence in either bicyclic or tricyclic substances resulted in moderate to notable decrease in anti-BChE.

In silico ADME studies

In silico physicochemical, pharmacokinetic/ADME, and drug likeliness were done for all synthesised compounds as well as the usual reference medicines, Donepezil and Tacrine. Different physicochemical parameters like rotating contacts and lipophilicity were recorded (Table 18). None of the synthesised compounds contained H-bond donor’s groups, as observed in Donepezil, however all synthesised compounds contained two to four H-bond acceptor groups. Crossing the BBB is an essential pharmacokinetic parameter for compounds targeting AD. In order to easily cross the BBB, compounds should have log P = 2–5 and PSA ≤ 70 Å²¹⁰²^,¹⁰³. All synthesised compounds demonstrated MlogP values above 3.5. Compounds 10, 12 and 13 exhibited MlogP value above 5. High values of MlogP confirm the potential of the synthesised compounds to cross the BBB. TPSA values are presented in Table 19. Swiss ADME’s Pan Assay Interference Compounds (PAINS) discovered that all the hits had zero alerts. Though PAINS are critical characteristics to consider when developing medications to minimise false-positive outcomes, overestimation and blind application of these filters may result in the exclusion of prospective successes based on phantom PAINS¹⁰⁴. All the analogs had synthetic accessibility scores superior to that of donepezil and tacrine, with values between 3.50 and 4.14, indicating that they can be easily synthesised on a wide scale (Table 18).

Table 18.

Physicochemical properties based on Lipinskis’ rule, Veber’s rule, rotatable bonds and MlogP of synthesised compounds, donepezil and tacrine.

Molecule	#H-bond acceptors	#H-bond donors	MLogP	MW	Bioavailability score	Synthetic accessibility
4	4	0	3.82	344.42	0.55	3.51
5	4	0	4.31	378.87	0.55	3.50
9	2	0	4.86	375.48	0.55	4.00
10	2	0	5.07	409.93	0.55	3.98
11	3	0	4.22	405.51	0.55	4.14
12	2	0	5.07	409.93	0.55	3.98
13	2	0	5.54	444.37	0.55	3.99
14	3	0	4.69	439.95	0.55	4.12
Donepezil	4	0	3.06	379.49	0.55	3.36
Tacrine	1	1	2.33	198.26	0.55	2.08

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Table 19.

TPSA and calculated % ABS of synthesised compounds in comparison with Donepezil and Tacrine.

Cpd. no.	TPSA	%ABS
4	80.84	81.11
5	80.84	81.11
9	57.78	89.06
10	57.78	89.06
11	67.01	85.88
12	57.78	89.06
13	57.78	89.06
14	67.01	85.88
Donepezil	38.77	95.62
Tacrine	38.91	95.58

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All synthesised compounds have exhibited topological polar surface areas (TPSA) that are less than 200 Å². Also, the absorption (percent ABS) was determined using the equation% ABS = 109 – (0.345 × TPSA)¹⁰⁵, and the calculated percent ABS of all hits ranged from 81.11 to 89.066%, which is close to that of Donepezil and Tacrine, indicating that these derivatives may possess the needed cell membrane permeability and bioavailability (Table 19).

The forecasted pharmacokinetic/ADME attributes of the derivatives investigated are given in Table 20. The data reveals that all the tested compounds demonstrated high gastrointestinal (GI) absorption and are P-gp (p-glycoprotein) non-inhibitors except molecules 4 and 5. One crucial pharmacokinetic factor needed for anti-Alzheimer’s action is crossing the blood-brain barrier. The BBB penetration rate is expressed in cm/s. It was determined that BBB+ molecules (Category 1) had logBBB > −1, while BBB- molecules (Category 0) possessed logBBB ≤ −1. The output result indicates the likelihood that an input is BBB+ within the range of 0 to 1. Compound 4 was projected to have superior BBB permeability, while compounds 5, 11, and 14 showed a substantial likelihood of being BBB permeant, like donepezil. The remaining compounds exhibited moderate BBB permeability. Furthermore, the newly synthesised oxazines inhibit all the Cytochrome P450 isomers studied. All the analysed compounds do not inhibit CYP2D6 and CYP3A4 except 9, 10, 11 and 14, which showed CYP3A4 inhibition. The coefficient values for skin permeability (log K_p; with K_p in cm/s) of the assessed compounds were low (Table 20).

Table 20.

Pharmacokinetic/ADME properties of synthesised compounds.

Cpd. no	GI Abs ^a	BBB perm-eant^b	P-gp substrate^c	CYP1A2 inhibitor^d	CYP2C19 inhibitor^e	CYP2C9 inhibitor^f	CYP2D6 inhibitor^g	CYP3A4 inhibitor^h	Log K_pⁱ
4	High	0.138	No	Yes	Yes	Yes	No	No	−4.91
5	High	0.335	No	Yes	Yes	Yes	No	No	−4.68
9	High	0.871	Yes	Yes	Yes	Yes	No	No	−4.33
10	High	0.989	Yes	Yes	Yes	Yes	No	Yes	−4.53
11	High	0.410	Yes	Yes	Yes	Yes	No	No	−4.09
12	High	0.904	Yes	Yes	Yes	Yes	No	Yes	−4.77
13	High	0.993	Yes	Yes	Yes	Yes	No	Yes	−4.56
14	High	0.508	Yes	Yes	Yes	Yes	No	Yes	−4.33
Donepezil	High	0.353	Yes	No	No	No	Yes	Yes	−5.58
Tacrine	High	0.977	Yes	Yes	No	No	No	Yes	−5.59

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^aGastro-intestinal absorption, ^bBlood Brain Barrier permeant, ^cP-glycoprotein substrate, ^dCYP1A2: Cytochrome P450 family 1 subfamily A member 2 (PDB:2HI4), ^eCYP2C19: Cytochrome P450 family 2 subfamily C member 19 (PDB:4GQS), ^fCYP2C9: Cytochrome P450 family 2 subfamily C member 9 (PDB:1OG2), ^gCYP2D6: Cytochrome P450 family 2 subfamily D member 6 (PDB:5TFT), ^hCYP3A4: Cytochrome P450 family 3 subfamily A member 4 (PDB:4K9T), ^ISkin permeation in cm/s.

The assorted drug-likeness rules, i.e. Lipinski¹⁰⁶, Ghose¹⁰⁷, Veber¹⁰⁸, Egan¹⁰⁹ and Muegge¹¹⁰ were enforced to appraise the molecule to be an influential drug candidate. The number of violations of the formerly named rules, along with their bioavailability scores, are present in (Table 21). The Lipinski (Pfizer) strain is the vogue setter rule-of-five (RO5), and according to this rule, all the examined compounds are drug-like except 9, 10, 12, and 13. The predictions in terms of the Ghose and Egan rule flaunted that all the compounds sustain the rule and are drug candidates except 13. In the appraisal process via the Veber rule, all the derivatives exhibited drug-likeness, while only compound 4 exhibited drug-likeness according to the Muegge rule with zero violation. All the investigated molecules manifested a bioavailability score of 0.55 (Table 21).

Table 21.

Drug-likeness predictions of the synthesised compounds.

Cpd. no.	Lipinski violations	Ghose violations	Veber violations	Egan violations	Muegge violations	Bioavailability score
4	Yes	Yes	Yes	Yes	Yes	0.55
5	Yes	Yes	Yes	Yes	No	0.55
9	No	Yes	Yes	Yes	No	0.55
10	No	Yes	Yes	Yes	No	0.55
11	Yes	Yes	Yes	Yes	No	0.55
12	No	Yes	Yes	Yes	No	0.55
13	No	No	Yes	No	No	0.55
14	Yes	Yes	Yes	Yes	No	0.55
Donepezil	Yes	Yes	Yes	Yes	Yes	0.55
Tacrine	Yes	Yes	Yes	Yes	No	0.55

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In the bioavailability radar, the pink surface best represents any of the following properties: lipophilicity, XLOGP₃ ranging from 4.79 to + 6.81, MW: 330–430 g/mol MW, solubility, log S not exceeding 7, saturation, sp³ hybridisation fraction not below 0.23, and flexibility—not exceeding 6 rotatable bonds. Because it is too flexible and polar in this situation, the molecules are highly expected to be bioavailable when taken orally (Figure 13).

Figure 13. — Bioavailability radar of the tested compounds versus Donepezil and Tacrine.

In-silico toxicity studies

Table 22, displays the predicted toxicity of the most active hits, 9, 11, and 12, along with two reference anti-Alzheimer’s drugs, tacrine and donepezil, based on web tools: ProTox-II (https://tox-new.charite.de/protox_II) and pkCSM (http://biosig.unimelb.edu.au/pkcsm/prediction) [82, 83]. The pkCSM web tools demonstrate that only compound 11 is safer than tacrine, showing no signs of AMES toxicity, which is analogous to donepezil. Furthermore, the most active hits 9, 11, and 12 indicated a maximum tolerated human dose value superior to that of donepezil (0.329, 0.245 and 0.218 log mg/kg/day, respectively). Oral Rat Acute Toxicity (LD₅₀) values of all hits have been found to be superior to that of donepezil and tacrine. Moreover, compound 9 demonstrated Oral Rat Chronic Toxicity (LOAEL) value superior to those of donepezil and tacrine. The normal repolarization of the cardiac action potential is controlled by the potassium channels expressed by the human Ether-à-go-go Related Gene (hERG). Any blockage or impairment of these channels in heart cells can make cardiovascular diseases potentially fatal. The pharmaceutical sector has therefore shown serious concern regarding potassium channel blockage caused by drugs. While all active hits were expected to inhibit hERG-II, it was shown that none possessed any effect on hERG-I. The pkCSM server was employed to explore hepatotoxicity, where all active hits were projected to be hepatotoxic. Skin sensitivity was not observed in any of the hits.

Table 22.

Toxicity of the most active compounds 9, 11 and 12 in comparison with Donepezil and Tacrine.

	Most active compounds
	pkCSM
	9	11	12	Donepezil	Tacrine
Test	ProTox-II prediction
AMES toxicity	Yes	No	Yes	No	Yes
Max. tolerated dose (human)	0.329	0.245	0.218	−0.217	0.55
hERG I inhibitor	No	No	No	No	No
hERG II inhibitor	Yes	Yes	Yes	Yes	No
Oral Rat Acute Toxicity (LD₅₀)	2.95	2.932	3.02	2.753	2.33
Oral Rat Chronic Toxicity (LOAEL)	1.726	0.756	0.558	0.991	1.204
Hepatotoxicity	Yes	Yes	Yes	Yes	Yes
Skin Sensitisation	No	No	No	No	No
T. Pyriformis toxicity	0.313	0.29	0.314	0.804	0.642
Minnow toxicity	−1.716	−2.385	−2.221	−2.011	0.206
LD₅₀ (mg/kg)	2000	500	500	505	40
Toxicity Class	IV	IV	IV	IV	II
Immunotoxicity	Inactive (0.99)	Inactive (0.92)	Inactive (0.98)	Active (0.95)	Inactive (0.98)
Mutagenicity	Inactive (0.61)	Inactive (0.54)	Inactive (0.63)	Inactive (0.53)	Active (0.91)
Cytotoxicity	Inactive (0.68)	Inactive (0.55)	Inactive (0.66)	Active (0.63)	Inactive (0.72)
Phosphoprotein (Tumour Suppressor) p53	Inactive (0.81)	Inactive (0.75)	Inactive (0.78)	Inactive (0.94)	Inactive (0.95)

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All active hits, as well as donepezil, were predicted to fall into class four by the ProTox-II detection webtool, however tacrine was predicted to fall into class two (GHS) (Table 22). The LD₅₀ value of compound 9 was confirmed to be superior to those of Donepezil and Tacrine. In contrast to donepezil, all active hits were anticipated to be non-cytotoxic and non-immunotoxic. Furthermore, in contrast to Tacrine, all active hits were anticipated to be non-mutagenic.

Molecular docking

The correctness of the docking methods utilised in this docking simulation was verified by self-docking the original ligands (tacrine and donepezil, respectively) in the binding pocket for both the AChE (PDB ID: 4EY7) and BChE (PDB ID: 4BDS) docking protocols. The marginal RMSD values between the native ligands and the re-docked posture were 0.117 Å and 0.589 Å, respectively, and the energy scores (S) were −17.71 kcal/mol and −10.08 kcal/mol, respectively, indicating that the validation was effective. The ability of the re-docked posture to superimpose the native ligand and occupy important contacts made by the co-crystallised ligand (Supplementary Table 1) with the active site of both AChE and BChE, as shown in Figure 14, further confirms the validity of the validation.

Figure 14. — (A) Overlay between co-crystallised ligand (green) and re-docked pose (orange) of Donepezil (RMSD= 0.117 Å). (B) Ligand Interactions between the complex overlay and AChE pocket (PDB ID: 4EY7). (C) Overlay between co-crystallised ligand (green) and re-docked pose (orange) of Tacrine (RMSD= 0.589 Å). (D) Ligand Interactions between the complex overlay and BChE pocket (PDB ID: 4BDS).

The interactions of donepezil with the AChE pocket included one H-bond involving Phe295 (PDB: 4EY7). Additionally, donepezil was found to have three H-arene interactions with the residues Trp286, Tyr341, and Tyr 337 in AChE. It was also demonstrated that Trp286 had a single arene-arene interaction. Tacrine was discovered to create an H-bond with a His438 residue to interact with the BChE pocket (PDB: 4BDS). One H-arene interaction between Tacrine and His438 was noted, in addition to one arene-arene contact with the Trp82 residue (Supplementary Table 1).

The most active compounds, 9, 11, and 12, were investigated for their binding energies and interactions with both AChE and BChE. Values of −13.96, −15.50, and −15.46 kCal/mol, respectively, indicated that the binding energies were near the binding-energy of-donepezil with-AChE. Likewise, binding energies with BChE were found to be relatively close to those of tacrine, with values of −11.97, −12.43, and −13.43 kCal/mol, respectively. The most three active hybrids were found to engage with the same-amino acid residues—Phe295, Tyr341, and Trp286—that are involved in the binding of donepezil to the binding-site as they interact with the AChE pocket (Figure 15). Compound 12 exhibited an extra H-arene interaction with the Asp74 residue of AChE. The three most active hits also showed binding interactions with BChE including His438 and Trp82, two amino-acid-residues that are also involved in the binding of tacrine (Figure 16). The binding of compound 12 revealed an extra H-bond with Gly439 (Supplementary Table 2).

Figure 15. — 2D ligand interactions and 3D binding modes of the most active compounds (A) 9 (B) 11 (C) 12, with AChE (PDB ID: 4EY7).

Figure 16. — 2D ligand interactions & 3D binding modes of the most active compounds (A) 9 (B) 11 (C) 12, with BChE (PDB ID: 4BDS).

Conclusion

The anti-Alzheimer’s properties of a novel set of tricyclic and bicyclic cyclohexa[b]thiophene hybrids were assessed in this work by comparing them to donepezil and tacrine, respectively, as inhibitors of AChE and BChE. Every conjugate showed AChE and BChE inhibition with IC₅₀ values below 15 µM. It was discovered that compounds 9, 11 and 12 were the most efficient as AChE and BChE-inhibitors. All synthesised compounds, except for compounds 5 and 11 demonstrated higher selectivity towards BChE. Along with other biomarkers, TAC, MDA, SOD, BDNF, IL-β, and TNF-α were also evaluated in the brains of the rat. Our compounds were shown to lower inflammatory indicators and increase antioxidant biomarkers. All the substances demonstrated their capacity to decrease brain cell death by lowering BAX levels and raising Bcl-2 levels. All the compounds demonstrated their capacity to improve Wnt pathway activity, which in turn improved brain cell growth. Future research will be directed to experimental ADME and toxicity studies. According to these findings, the compounds are multipotent lead molecules that could be utilised in the creation of new anti-Alzheimer’s medications.

Supplementary Material

Animal Application Protocol Review Form.pdf

IENZ_A_2598741_SM3817.pdf^{(690.6KB, pdf)}

Ethical Approval.pdf

IENZ_A_2598741_SM3816.pdf^{(308.8KB, pdf)}

Book1 days.xlsx

IENZ_A_2598741_SM3815.xlsx^{(19.1KB, xlsx)}

--Supplementary_Materials_R2.docx

IENZ_A_2598741_SM3814.docx^{(36.4MB, docx)}

Funding Statement

The authors acknowledge that the funding obtained from the Research, Development, and Innovation Authority (RDIA), Saudi Arabia, Riyadh, Reactivating & Rebuilding of Existing Labs Initiative, Number (13262-Tabuk-2023-UT-R-3–1-HW-), supporting the generation of these data and publication.

Ethical approval

The minimum number of animals required for statistical significance was utilised. Rats were maintained in conditions of light/dark cycles, 50% relative humidity, and a regulated temperature of 22 ± 1 °C, with full access to water and food. After completion of experiment, rats were terminated via cervical dislocation. The current research protocol adheres to the “ARRIVE” requirements for the use and care of laboratory animals and has been authorised by the research ethics-committee at Faculty of Pharmacy, Sinai University - Kantara Branch, Kantara East, Ismailia Egypt (Permit number: SU.REC.2024 (18 A). Approval date: 10 October 2024–10 October, 2025.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data is provided within the manuscript or supplementary information files.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Animal Application Protocol Review Form.pdf

IENZ_A_2598741_SM3817.pdf^{(690.6KB, pdf)}

Ethical Approval.pdf

IENZ_A_2598741_SM3816.pdf^{(308.8KB, pdf)}

Book1 days.xlsx

IENZ_A_2598741_SM3815.xlsx^{(19.1KB, xlsx)}

--Supplementary_Materials_R2.docx

IENZ_A_2598741_SM3814.docx^{(36.4MB, docx)}

Data Availability Statement

Data is provided within the manuscript or supplementary information files.

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PERMALINK

From bench to brain: novel thieno-oxazine hybrids as potent pleiotropic anti-Alzheimer’s agents with in vivo/in vitro validation and in silico insights

Eman A Fayed

Mazin A A Najm

Khulood H Oudah

Maha A Ebrahim

Nirvana A Gohar

Karema Abu-Elfotuh

Ehsan Khedre Mohamed

Ahmed M E Hamdan

Najla A Albalawi

Shahad Faisal Alzahrani

Amira M Hamdan

Reema Almotairi

Shaimaa M Hafez

Triveena M Ramsis

Roles

Abstract

Graphical abstract

Introduction

Figure 1.

Figure 2.

Material and methods

Chemistry

General details

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1)

Ethyl 2-hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2)

Ethyl 2-(aryloxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (4,5)

Standard operating procedure

Ethyl 2-(benzoyloxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (4)

Ethyl 2-((4-chlorobenzoyl)oxy)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5)

2,3-Diaryl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (9–14)

2,3-Diphenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (9)

3-(4-Chlorophenyl)-2-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (10)

3-(4-Methoxyphenyl)-2-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazi-n-4-one (11)

2-(4-Chlorophenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (12)

2,3-Bis(4-chlorophenyl)-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (13)

2-(4-Chlorophenyl)-3-(4-methoxyphenyl)-2,3,5,6,7,8-hexahydro-4H-benzo[4,5]thieno[3,2-e][1,3]oxazin-4-one (14)

In vivo assessment

Animals and induction of AD rat models

Experimental design

Animal justification

Behavioural tests

Morris water maze test (MWM)

Y-maze spontaneous alternation (SAB) test

Tissue sampling and preparation

Biochemical measurements

Fluorometric technique

Colorimetric technique

ELISA technique

Real-time quantitative polymerase chain reaction

Table 1.

AChE inhibition assay

BChE inhibition assay

Statistical analysis

Histopathological examination of brain tissue

In silico ADME and toxicity prediction

Molecular docking

Results and discussion

Chemistry

Scheme 1.

Scheme 2.

In vivo assessment

Effects on behavioural tests in AlCl3-induced AD

Figure 3.

Table 2.

Table 3.

Effect on BACE1 and amyloid-β

Figure 4.

Table 4.

Table 5.

Effects on brain neurotransmitters levels in AlCl3-induced AD

Figure 5.

Table 6.

Table 7.

Effects on neuroinflammatory biomarkers (IL-β and TNF-α) in AlCl3-induced AD

Figure 6.

Table 8.

Table 9.

Effects on BAX and bcl-2

Effects on behavioural tests in AlCl₃-induced AD

Effects on brain neurotransmitters levels in AlCl₃-induced AD

Effects on neuroinflammatory biomarkers (IL-β and TNF-α) in AlCl₃-induced AD

Effects on oxidative stress in brain tissues in AlCl₃-induced AD