New and potential boron-containing compounds for treatment of Alzheimer's disease and cancers

ABSTRACT

Aim

In this study, new boron-containing carbamate compounds were synthesized and evaluated as potential acetylcholinesterase (AChE) inhibitors by in vitro and in silico analyses.

Materials & methods

The structures were characterized by spectroscopic analysis including ¹H NMR, ¹³C NMR, ¹¹B NMR, and MS. The purities of the compounds were determined by HPLC analysis. In vitro and in silico analyses were performed.

Results

Based on our findings, compounds (1–4) demonstrated more potent AChE inhibitory activity compared to tacrine, which is an FDA-approved AChE inhibitor. Compound 4 had the highest inhibitory activity with an IC₅₀ of 37.87 ± 0.96 nM and was more effective than tacrine (74.23 ± 0.83 nM). Compounds 1, 2, and 3, respectively, showed 1.78-, 1.73-, and 1.58-fold more potent enzyme inhibition activity compared to tacrine. The strong interactions with critical residues in the binding pocket of AChE were identified between protein and the compounds. Furthermore, compound 4 exerted an antiproliferative activity against various human cancer cell lines (32.91 ± 4.92 µM in HT29 and 42.38 ± 2.73 µM in MCF-7).

Conclusion

Our study indicates the discovery of new boron-containing AChE inhibitors as potential candidates for the treatment of Alzheimer’s disease and cancer.

GRAPHICAL ABSTRACT

1. Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by cognitive decline, memory loss, and behavioral changes [1]. The main pathological features of AD are characterized by the accumulation of amyloid-β (Aβ) plaques and hyperphosphorylated tau proteins, leading to synaptic dysfunction and neuronal loss [2]. Thus, the pathological hallmarks of AD include neuronal loss, the accumulation of misfolded β-amyloid peptides (Aβ), and tau protein aggregation [3]. However, the pathophysiological process of AD is not limited to Aβ and tau accumulations alone. Neuroinflammation also has an important effect on this process [4]. The recent studies suggest that neuroinflammation contributes to the progression of AD and synaptic dysfunction [5]. These changes lead to cerebral atrophy and the formation of amyloid plaques and neurofibrillary tangles in the brain [6]. Current treatments for AD are limited in their effectiveness [7]. Cholinesterase inhibitors, which increase acetylcholine levels in the brain, are among the few drugs that have shown clinical benefits in managing AD symptoms [7]. Up to date, various potential cholinesterase inhibitors such as donepezil, tacrine, rivastigmine, and memantine have been reported [8–12]. The urgent need for new inhibitors and targeted therapeutics in AD is underscored by the progressive nature of the condition and the lack of disease-modifying treatments [13]. The studies have highlighted the involvement of various factors such as zinc status, neuroinflammation, and genetic biomarkers in progression of AD [13].

Acetylcholinesterase (AChE) is a key enzyme in AD due to its role in breaking down acetylcholine (ACh), a neurotransmitter critical for memory and learning. The loss of cholinergic neurons in AD leads to reduced ACh levels, which is strongly associated with cognitive decline, as described by the cholinergic hypothesis [14,15]. The changes in AChE activity, observed in plasma and cerebrospinal fluid (CSF) of AD patients, also correlate with disease progression. The certain AChE isoforms interact with amyloid-beta (Aβ) peptides, forming complexes that exacerbate neurotoxicity and contribute to the hallmark pathology of AD [16,17]. Current treatments include AChE inhibitors like donepezil, rivastigmine, and galantamine, which improve cholinergic transmission by preventing ACh breakdown, offering symptomatic relief and modestly slowing disease progression. However, these treatments are limited by side effects, such as gastrointestinal issues, which restrict dosing [15]. To overcome these limitations, ongoing research explores novel inhibitors, including synthetic compounds and natural products, aiming to enhance efficacy and reduce toxicity [18,19]. In summary, AChE is central to the pathophysiology and treatment of AD with its inhibition remaining a primary therapeutic strategy. Advances in this area hold promises for developing more effective and tolerable treatments to manage the disease.

AD and cancer are two prevalent and debilitating conditions that have been the focus of extensive research due to their impact on global health. While traditionally viewed as distinct diseases affecting different systems in the body, emerging evidence suggests a potential relationship between AD and cancer as some AChE inhibitors exerted antiproliferative effects in cancer models [20,21]. The studies have shown intriguing connections between AD and cancer at the molecular level. For instance, phosphorylation events in specific sites of one of the tumor suppressors such as WWOX have been implicated in both cancer progression and neurodegeneration [22]. Additionally, the role of exosomes in facilitating cancer cell metastasis and the downregulation of cancer cell PTEN expression in cancer cells have been linked to neurodegenerative diseases like AD [20]. Furthermore, higher plasma levels of amyloid-β peptides and characteristic of AD have been associated with an increased risk of cancer, suggesting a potential biological link between the two conditions [23]. The inverse relationship between AD and cancer has also been explored with the studies indicating a negative association between the two diseases [24–26]. Biological mechanisms underlying this inverse correlation have been proposed with cancer characterized by excessive cell proliferation and neurodegeneration marked by premature cell death [27]. Moreover, increase of AChE activity in cancer cells has been suggested to contribute to cancer cell proliferation, further highlighting the role AChE inhibitors play in treating both of AD and cancer [25,28].

AChE plays a crucial role in modulating the levels of ACh in the tumor microenvironment, influencing cancer progression and treatment outcomes across various malignancies. The recent studies have highlighted the ACh’s involvement in tumorigenesis and the therapeutic potential of targeting cholinergic signaling pathways. ACh is not limited to neuromodulation; it also has significant non-neuronal functions, including the promotion of tumor growth and drug resistance. In the context of non-small cell lung cancer (NSCLC), ACh signaling can influence drug tolerance mechanisms, contributing to tumor relapse following treatment with epidermal growth factor receptor (EGFR) inhibitors [29]. These findings indicate the role of ACh in enhancing survival and proliferation in various cancer cell types, suggesting that it may function as a growth factor [30]. The impact of AChE is critical as it directly regulates ACh levels by hydrolyzing this neurotransmitter, thus modulating signaling pathways that promote tumor progression. Altered expression or elevated activity of AChE during tumor development can lead to increased bioavailability of ACh, which engages cholinergic receptors and stimulates tumor growth [28,31]. Notably, cholinergic signaling via specific muscarinic receptors, such as M3, has been implicated in driving cancer progression across multiple tumor types, including colorectal and gastrointestinal cancers [32,33]. This emphasizes the therapeutic implications of inhibiting AChE or modulating cholinergic receptor signaling as potential strategies in cancer treatment. Emerging data suggest the presence of cholinergic CD4+ T-cells that influence tumor immunogenicity and response. In hepatocellular carcinoma, these cholinergic immune cells have been observed to support favorable antitumor immune responses, indicating that the cholinergic system may mediate not just tumor growth, but also immune evasion [34]. Furthermore, the link between the peripheral nervous system and tumor biology illustrates the complex interactions whereby neurotransmitters like ACh could initiate cascades involving signaling pathways conducive to cancer metastasis and aggressiveness [35]. Moreover, the modulation of cholinergic systems in the tumor microenvironment suggests a potential avenue for therapy [35].

Boron-containing compounds have garnered attention in medicinal chemistry due to their diverse applications and potential therapeutic benefit [36]. Thus, the boron containing compounds have a significant potential in drug development and pharmaceutical chemistry due to their unique properties that can enhance the efficacy and specificity of medications [36]. The incorporation of boron atoms into pharmaceuticals can increase binding affinity with enzymes, facilitating interactions through dative bonds and enhancing drug–target interactions [37]. Boronic acids and boronate esters serve as indispensable building blocks in transition-metal-catalyzed cross-coupling reactions, allowing for the formation of diverse carbon–carbon and carbon-heteroatom bonds [38]. The ease of handling, stability, and functional group tolerance of aryl boronic acids make them valuable tools in organic synthesis and pharmaceutical chemistry [39]. One notable example of a boron-containing drug is bortezomib, a boronic acid derivative, which has potent anticancer activity by selectively inhibiting the proteasome, a cellular proteolytic complex involved in protein degradation, a chemotherapeutic agent used in the treatment of multiple myeloma and mantle cell lymphoma [40,41]. Boron-based drugs have also been studied for their potential in boron neutron capture therapy, a targeted radiation treatment for brain cancers and other malignancies [42]. The chemical structures of some FDA-approved boron-containing drugs such as Bortezomib are given in Figure 1.

Figure 1.

Some FDA-approved boron-containing drugs and candidates [43,44]. (a) Bortezomib, (b) ixazomib, (c) ixazomib citrate, (d) tavaborole, (e) crisaborole, (f) vaborbactam, (g) 4-(3,9-Dihydroxybenzo[f] [1,2]benzoxaborinin-2-yl)benzene-1,2-diol, (h) taniborbactam, (i) talabostat.

In drug design, computational methods such as molecular docking have been pivotal in identifying boron-containing AChE inhibitors [45]. The studies emphasize that while AChE inhibitors are critical for managing AD symptoms, their side effects remain a significant concern, necessitating a careful evaluation of newer compounds [46]. Furthermore, it was reported that the necessity of identifying boron-based compounds capable of penetrating the blood–brain barrier, which could enhance the treatment landscape for neurodegenerative diseases such as AD [47]. A novel boron-based hybrid containing an antioxidant moiety inhibited AChE activity, increased cellular viability under conditions mimicking AD pathology, thereby highlighting the therapeutic potential of boron-containing compounds [48]. Moreover, exploration into computational modeling studies has facilitated the design of boron-containing hybrids with enhanced targeting of AChE, amplifying their inhibitory effects and offering novel therapeutic avenues for AD treatment [49].

In conclusion, the ability of boron atoms underscores their significance in drug design and development, particularly in combating diseases characterized by uncontrolled proliferation. Herein, this study focused on the discovery of boron containing compounds with synthesis, in vitro, and in silico analysis to be used in AD and cancer.

2. Experimental

2.1. General

Sigma-Aldrich, Merck and VWR were used in this study. To monitor the reactions, Merck 60 F254 aluminum sheets 20 × 20 cm were used for thin layer chromatography (TLC), and the spots were visualized under CAMAG UV light. Melting point was determined with the X-4 Melting Point Apparatus. For the FT-IR analysis, a Perkin Elmer Spectrum 100 FTIR spectrophotometer was used using the ATR technique. Agilent Premium Compact 600 MHz and JEOL and Varian 400 MHz were used for ¹H NMR, ¹³C NMR, and ¹¹B NMR analyses. Mass spectrometry (MS) (Q-TOF and LC-MS/MS) was performed. Coupling constants (J values) and chemical shifts (expressed in parts per million, ppm) were given in NMR spectra.

2.2. General procedure for the synthesis of boron-containing compounds

2.2.1. Synthesis of some boron-containing benzyl carbamate derivatives

2.2.1.1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzylchloroformate

Phosgene solution in toluene 15% (6 eq.) is added dropwise to the mixture of 4-(hydroxymethyl) phenylboronic acid pinacol ester (2 eq.) in tetrahydrofuran (THF) (2–3 mL). The reaction mixture is stirred under Argon atmosphere at room temperature for 24 h. The excess solvent and phosgene are evaporated at reduced pressure, dried in vacuum and stored at + 4°C [50]. Colorless viscous product (Yield: 95%).

2.2.2. General method for synthesis of the compounds (1–4)

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzylchloroformate in CH₂Cl₂ was added to the solution of the amine derivative (1 eq.) and Et₃N (1.1 eq.) in CH₂Cl₂ at 0°C and stirred for 30 min and continued at rt up to 24 h. The solvent was evaporated, and the residue was dissolved in ethyl acetate (EtOAc) and washed with saturated brine and dried in Na₂SO₄ [51]. The product was dried in vacuum and stored at + 4°C.

2.2.2.1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl (2-hydroxyethyl)carbamate (compound 1)

Viscous (Yield: 60%). LC-MS/MS m/z = [M+H]⁺ = 322.10 (100.00%). HRMS (Q-TOF) m/z calcd. for C₁₆H₂₄BNO₅ [M+H]⁺ 322.18303; [M+Na]⁺ 344.16344 (Figure S1). ¹H NMR (400 MHz, Chloroform-d, (CDCl₃), ppm): δ 7.76 (d, J = 7.8 Hz, 2H, Ar-H), 7.31 (d, J = 7.8 Hz, 2H, Ar-H), 5.31 (s, 1H, NH), 5.08 (s, 2H, CH₂O), 4.11–4.06 (q, 1H, OH), 3.68–3.65 (m, 2H, CH₂-CH₂), 3.31 (q, J = 6.1 Hz, 2H, CH₂-CH₂), 1.32 (s, CH_3, 12H) (Figure S2). ¹³C NMR (100 MHz, CDCl₃, ppm): δ 157.14, 139.51, 135.05, 127.20, 83.94, 76.81, 66.79, 62.22, 60.51, 43.56, 24.91 (Figure S3). ¹¹B NMR (CDCl₃, 128.27 MHz, 25°C, δ ppm): 29 (Figure S4).

2.2.2.2. tert-Butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) ethane-1,2-diyldicarbamate (compound 2)

White crystals (Yield: 95%; m.p.: 132–134°C). LC-MS/MS m/z [M+H]⁺ = 421.20 (100.00%), 422.20 (30.95%). HRMS (Q-TOF): m/z [M+Na]⁺ calcd. for C₂₁H₃₃BN₂O₆ = 443.23161 (Figure S5). ¹H NMR (600 MHz, Chloroform-d, (CDCl₃), ppm): δ 7.78 (d, J = 7.6 Hz, 2H, Ar-H), 7.73 (d, J = 7.6 Hz, 2H, Ar-H), 5.18 (s, 1H, NH), 5.09 (s, 2H, CH₂O), 4.84 (s, 1H, NH), 3.28 (t, J = 5.7 Hz, 2H, CH₂-CH₂), 3.27–3.22 (m, 2H, CH₂-CH₂), 1.41 (s, 12H, CH₃), 1.32 (s, 9H, CH₃) (Figure S6). ¹³C NMR (150 MHz, CDCl₃, ppm): δ 156.69, 156.42, 139.46, 134.96, 127.11, 83.83, 79.62, 66.59, 41.49, 40.55, 28.34, 24.84 (Figure S7). ¹¹B NMR (CDCl₃, 128.27 MHz, 25°C, δ ppm): 29 (Figure S8).

2.2.2.3. (4-(((4-(tert-Butyl-1,2-ethylenediamine-1,4-carbonyl)oxy)methyl)phenyl)boronic acid (compound 3)

White crystals (Yield: 60%; m.p.: 187–189.5°C). LC-MS/MS m/z [M+H]⁺ = 339.15 (100.00%), 340.20 (17.29%); HRMS (Q-TOF): m/z [M+Na]⁺ calcd. for C₁₅H₂₃BN₂O₆ = 361.15323 (Figure S9). ¹H NMR (400 MHz, Chloroform-d, (CDCl₃), ppm) δ 8.19 (s, 1H, OH), 7.73 (s, 1H, OH), 7.42 (d, 4H, Ar-H), 5.15 (d, J = 25.6 Hz, 2H, NH), 4.84 (s, 2H, CH₂O), 3.28 (s, 4H, CH₂-CH₂), 1.43 (s, 9H, CH₃) (Figure S10). ¹³C APT (100 MHz, DMSO-d₆, CDCl₃, ppm): δ 156.73, 156.00, 138.75, 134.44, 126.90, 78.55, 66.11, 41.14, 40.57, 28.51. ¹³C NMR (100 MHz, DMSO-d₆, CDCl₃, ppm): δ 156.57, 156.03, 139.32, 134.56, 134.35, 130.35, 126.97, 115.43, 78.07, 65.61, 62.45, 40.71, 26.07 (Figure S11). ¹¹B NMR (DMSO-d6, 128.27 MHz, 25°C, δ ppm): 29 (Figure S12).

2.2.2.4. 1-(tert-Butyl) 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) piperazine-1,4-dicarboxylate (compound 4)

It was synthesized according to the method for compound 5. White crystals (Yield: 75%). LC-MS/MS m/z [M+H]⁺ = 447.20 (27.58%), 448.25 (5.97%); HRMS (Q-TOF): m/z [M+Na]⁺ calcd. for C₂₃H₃₅BN₂O₆ = 469.24754 (Figure S13). ¹H NMR (400 MHz, Chloroform-d, (CDCl₃), ppm): δ 7.82–7.76 (m, 2H, Ar-H), 7.37–7.30 (d, J = 8.1 Hz, 2H, Ar-H), 5.13 (s, 2H, CH₂O), 4.83 (b, 2H, CH₂), 3.45 (dd, J = 7.0, 3.8 Hz, 4 H, CH₂), 3.39 (b, 2H, CH₂), 1.45 (s, 9H, CH₃), 1.32 (s, 12H, CH₃) (Figure S14). ¹³C NMR (100 MHz, CDCl₃, ppm): δ 155.24, 154.71, 139.56, 135.09, 127.26, 83.97, 80.31, 67.34, 59.60, 43.83, 38.22, 31.34, 28.47, 24.94 (Figure S15). ¹¹B NMR (CDCl₃, 128.27 MHz, 25°C, δ ppm): 29.53 (Figure S16).

2.2.2.5. Synthesis of bis(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) piperazine-1,4-dicarboxylate (compound 5)

Starting compound (2.02 eq.) was dissolved in CH₂Cl₂ was added to the solution of the amine derivative (1 eq.) and Et₃N (2.2 eq.) in CH₂Cl₂ at 0°C. It was stirred at 0°C for 30 min and treated for 24 h at rt. The solvent was evaporated, and the residue was dissolved in EtOAc, washed with saturated brine and dried in Na₂SO₄ [52]. After the solvent was evaporated, and the resulting product was dried in vacuum and stored at + 4°C. (Yield: 80%; m.p.: 147–150°C). LC-MS/MS m/z [M+H]⁺ calcd. for C₃₂H₄₄B₂N₂O₈ = 607.35 (16.05%) (Figure S17). ¹H NMR (400 MHz, Chloroform-d, (CDCl₃), ppm): δ 7.80–7.78 (d, J = 7.8 Hz, 4H, Ar-H), 7.34–7.32 (d, J = 7.8 Hz, 4H, Ar-H), 5.12 (s, 4H, CH₂O), 3.48–3.44 (s, 8H, piperazine), 1.32 (s, 24H, CH₃) (Figure S18). ¹³C NMR (100 MHz, CDCl₃, ppm): δ 155.18, 139.48, 135.08, 127.23, 83.93, 77.32, 67.39, 43.69, 24.92 (Figure S19). ¹¹B NMR (CDCl₃, 128.27 MHz, 25°C, δ ppm): 29.09 (Figure S20).

HPLC spectra for the synthesized compounds were given in Figures S21-S25.

2.3. In vitro AChE inhibition activity

The assay was carried out with slight modifications based on the Ellman method. The AChE enzyme activity (EU) (Electrophorus electricus (electric eel)) was defined as the hydrolysis of 1.0 μmol of acetylcholine (ACh) to choline and acetate per minute at pH 8.0 and 37°C. Tacrine was used as a reference inhibitor. The absorbances were measured at 405 nm. All measurements were performed in triplicate [52–55].

2.4. MTT assay

To screen the cytotoxic effects of the synthesized compounds on breast and colon cancer cell lines, MTT assay was conducted according to our reported study [9]. The cells were cultured in DMEM/F12 supplemented with fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS 1%), under 5% CO₂ in an incubator. The absorbances were measured at 570 nm. All measurements were performed in duplicate.

2.5. Statistical analysis

Welch test was performed by using GraphPad Prism p < 0.05 values that are statistically significant and are indicated with an asterisk.

2.6. Molecular docking

A molecular docking study, Glide/SP method was carried out to evaluate the binding affinities of boron-containing compounds against AChE [56]. The crystal structure of the target protein was obtained from the Protein Data Bank (PDB code 4EY7). For the molecular docking study, water molecules were removed, and missing hydrogen atoms were added to the protein, which was prepared at a physiological pH of 7.4. Protein Preparation Wizard [57] and LigPrep (LigPrep, Schrödinger, LLC, New York, NY, 2024) were used. The OPLS4 force field was used [58,59], and binding energies were calculated in kcal/mol. The intermolecular interactions between the ligands and protein were analyzed in detail using 2D ligand interaction diagrams.

2.7. Molecular dynamics (MD) simulations

Molecular dynamics (MD) simulations were performed by subjecting the top docking poses of protein-ligand complexes to energy minimization and equilibration using standard protocols [60]. MD simulations were carried out under physiological conditions with the system set at a temperature of 300 K and a pressure of 1 atm. The simulation time for each complex was set to 100 ns. The TIP3P water model was used to solve the systems, and counterions were added to neutralize the system’s charge. The stability of the protein-ligand complexes was evaluated by analyzing the root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) of the protein backbone and ligand over the simulation period. Additionally, molecular interactions were assessed to identify key residues that contribute to binding stability [54,61].

2.8. Molecular mechanics with generalized born and surface area solvation (MM/GBSA) analysis

To calculate the binding free energies (ΔG_bind) as kcal/mol, Prime method of Maestro was performed utilizing each pose of compounds and reference drugs after 100 ns MD simulations [62].

3. Results and discussion

3.1. Chemistry

The reaction conditions were given in Figure 2 [63]. The starting compound, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate, was synthesized by the reaction of commercially available 4-(hydroxymethyl)phenylboronic acid pinacol ester and 15% phosgene solution in toluene in tetrahydrofuran (THF) under Argon atmosphere and at a room temperature (rt) for 24 h. The pure product was obtained with a high yield of 90%, and it was used as a starting compound in the second step for the synthesis of new compounds (1–5). Then, substituted amide compounds were successfully obtained in the presence of triethylamine (Et₃N) and dichloromethane (CH₂Cl₂) at 0°C to rt. The reactions were successfully synthesized with high yields. Throughout the spectroscopic analysis, the characterization of the synthesized compounds was performed with the help of ¹H NMR and ¹³C NMR analysis. MS analysis (Q-TOF HRMS or LC-MS/MS) was performed to determine their molecular ion peaks. In ¹¹B NMR spectra, the signals were observed between 29.53 and 27.16 ppm. The expected characteristic peaks of the compounds were observed in all spectra (Figures S1-S20). All compounds were determined with the purities of 100% by HPLC analysis (Figures S21-S25). In ¹H NMR spectra, characteristic aromatic protons appeared approximately between 7.80 and 7.30 ppm. Two protons belonging to methylene (-OCH₂-) characterized at 5.05–5.13 ppm. In ¹³C NMR spectra, the chemical shifts were obtained between 157 and 24 ppm. [M+H]⁺ was detected with LC-MS/MS and/or Q-TOF HRMS analysis. The synthesis of compound 2 has been recently reported by a different method [64].

Figure 2.

Synthesis of the compounds (1–5). Reagents and conditions. i. Phosgene solution in toluene 15%, THF, under Ar, r.t., 24 h. ii. Et₃N, CH₂Cl₂, 0 °C, r.t. 5 h. iii. NH₄OAc, NaIO₄, acetone:H₂O (1:1), r.t., reduction step for compound 3 [63].

3.2. AChE inhibitory activity

In the current study, AChE inhibitor activities of the newly synthesized boron-containing compounds have been examined. Based on the findings, the IC₅₀ values reveal distinct differences in the inhibitory potency of the tested compounds (Table 1, Figure S26). Among them, compound 4 had the highest inhibitory activity with an IC₅₀ of 37.87 ± 0.96 nM, more effective than tacrine (74.23 ± 0.83 nM). Compound 4 showed 1.96-fold more enzyme inhibition effect than tacrine. In contrast, compounds 1 and 2 demonstrated strong AChE inhibitor potency with an IC₅₀ value of 41.74 ± 0.63 nM and 42.93 ± 0.34 nM, respectively, compared to tacrine. Compound 3 exhibited good AChE inhibitory activity with IC₅₀ values of 46.87 ± 0.52 nM, respectively, among the synthesized compounds. Compounds 1, 2, and 3, respectively, showed 1.78-, 1.73-, and 1.58-fold more enzyme inhibition effect than tacrine. In contrast, compound 5 also showed a lower inhibitory potency against AChE with an IC₅₀ of 86.15 ± 0.41 nM.

Table 1.

The AChE inhibition (IC50) of the synthesized boron-containing compounds.

Compounds	AChE (IC50 nM) ± SD	Compounds	AChE (IC50 nM) ± SD
1	41.74 ± 0.63	4	37.87 ± 0.96
2	42.93 ± 0.34	5	86.15 ± 0.41
3	46.87 ± 0.52	Tacrine	74.23 ± 0.83

± indicates standard deviation (SD).

In the reported studies, AChE inhibition was reported with moderate activity (IC₅₀ value as 115.63 ± 1.16 µg/mL) for a newly synthesized boron-based compound derived from quercetin [65]. Then, according to a combined study including synthesis, in silico, and in vitro, AChE inhibition (%) was indicated for two quercetin-boron hybrid compounds, and this study indicated that pi–pi interactions with Trp286, Tyr337, and Trp86 residues were observed for one of the synthesized compounds [65]. The cytotoxicity with these compounds was observed with IC₅₀ value 200 ug/mL in MCF-7 and HT29 cell lines [66]. In our previously reported study, the IC₅₀ value of tacrine for AChE was found as 77.41 ± 3.12 nM [9]. Herein, we have reported the IC₅₀ value for tacrine 74.23 ± 0.83 nM. The IC₅₀ value of donepezil was reported as 0.021 ± 0.001 µM [66]. The IC₅₀ value of donepezil was also reported as 54 nM [67]. As a result of this study, newly synthesized boron-containing compounds have been reported as potential AChE inhibitor candidates.

3.3. In vitro anticancer activity

To understand the cytotoxicity of the synthesized boron-containing compounds in various human cancer cell lines, MTT assay was conducted to measure the cytotoxic activity of the newly synthesized boron-containing compounds in four different cancer cell lines including human colorectal and breast cancers for 72 h treatments. Based on the findings, compound 4 showed anticancer activity in HT29 and MCF-7 cell lines at the concentrations of 32.91 ± 4.92 µM and 42.38 ± 2.73 µM, respectively (Figure S27A,B). Moreover, compounds 1 and 3 had their IC₅₀ values above 50 µM in BT20 cell lines. Compound 2 did not show cytotoxicity up to 75 µM. We reported doxorubicin (Dox) which is a chemotherapeutic agent, showed cytotoxicity at 100 nM in MDA-MB-231 cell line [68].

The recent studies have underscored the therapeutic potential of boron-containing compounds in various biomedical applications, particularly as AChE inhibitors for neurodegenerative disorders like AD and as anticancer agents [69]. These compounds exhibit diverse mechanisms of action including the inhibition of amyloid-beta (Aβ) aggregation and the reduction of neuroinflammation associated with AD, alongside antiproliferative effects against cancer cell lines [70]. For instance, Ritacca et al. demonstrated that boron compounds could modulate multiple AD-related pathways with computational analyses suggesting their dual functionality as both neuroprotective and anticancer agents [71]. Maiti et al. observed significant reductions in amyloid accumulation and neuroinflammation in both in vitro and in vivo models, providing compelling evidence for their therapeutic promise [72]. Additionally, Cacciatore et al. highlighted the potential of boronic derivatives as innovative scaffolds for developing treatments targeting both neurodegenerative diseases and cancer [73]. Carfilzomib, a boron-containing drug, has shown efficacy in treating multiple myeloma and other hematologic malignancies by interacting with the proteasome active site [74]. Ixazomib, a boron-based proteasome inhibitor, has demonstrated activity against medullary thyroid cancer and osteosarcoma in preclinical studies by binding to the active site serine of the proteasome [75]. Molecular modeling studies have been crucial in understanding the mechanisms of action and resistance to boron-containing drugs like bortezomib, guiding the design of novel compounds with improved efficacy and reduced side effects [42]. The medicinal importance of boronic acid and boronic acid pinacol ester functional groups lies in their versatility in drug discovery and development. Bortezomib and other boron drugs show great potential in oncology and other therapeutic areas. Molecular modeling studies contribute to advance aiding in the development of innovative treatments for various diseases.

3.4. Molecular docking

Throughout the molecular docking studies, synthesized new compounds showed their binding scores between −8.837 kcal/mol and −5.579 kcal/mol against AChE. The highest docking score was obtained for compound 4 (−8.837 kcal/mol) compared to docking score of tacrine (−8.934 kcal/mol). The interactions including H bond, C–H bond, pi–pi stacked, pi–alkyl, alkyl, and pi–sigma with the key residues between the target protein and ligand were observed (Figure S28). For compound 1, the key residues, His447, Trp286, Tyr124, and Val294, were determined. An alkyl bond was observed with Trp296 and Val294 (Figure S28A). The interactions of compound 4 with the key residues including Leu76, Trp286, Trp86, His447, Tyr337, Phe338, and Tyr341 have been observed, and a boronic acid group of compound 4 facilitated to form of Leu76 (Figure S28B). In addition, compound 4 interacted with Leu76 (Figure S28B). Also, compound 3 showed the interaction with these key residues, and a boronic acid group of compound 3 was observed to interact with H-bonds interactions with Tyr341. The compound 4 and donepezil were incorporated in the binding pocket of AChE (Figure S29A,B).

Furthermore, in this study, the binding abilities and the interactions of the synthesized compounds were compared with known inhibitors of AChE such as tacrine and donepezil. Tacrine and compounds 3 and 4 interacted with the same key residues of the target enzyme. In the reported studies, the interactions such as H bond, pi–pi, and pi–alkyl between the known inhibitor donepezil and key residues of AChE were reported [76–87]. In this study, likewise, these key residues such as Tyr341, His447, Phe338, Trp286, Tyr72, and Phe295 were observed with donepezil. In our previously reported study, tacrine was used as a reference drug against AChE to compare with synthesized compounds and reported pi-pi interaction with Trp82 [9]. The docking scores of tacrine that is known as a potent inhibitor for both cholinesterase enzymes have been reported between approximately −8 kcal/mol for AChE [9,72,88–90]. In a recent study, boroxazolidones have been reported for their in silico evaluation, and in vitro acetylcholinesterase inhibition activity compared to known AChE inhibitors such as galantamine and rivastigmine. The calculations were indicated between −12.18 kcal/mol and −10.46 kcal/mol for the synthesized five boroxazolidone compounds [91]. In a conducted study, it was reported that hydroxyl groups on boron atoms interact with Tyr residue of AChE [92].

3.5. Molecular dynamics (MD) simulation

The specific interactions with key residues in the binding site of AChE were observed with compound 4 during 100 ns MD simulations. For compound 4, pi–pi stacking was obtained between Tyr341 and Trp286, and its phenyl ring. His447, Phe295, and Arg286 were interacted with a carbonyl group on carbamate via water bridges to form H-bonds (Figure 3(a)).

Figure 3.

The interactions of compounds 4 (a) and 1 (b) during 100 ns MD simulations.

Throughout MD simulations, the top docking poses of potential inhibitor candidates were analyzed. Based on our findings, the average root mean square deviation (RMSD) values of protein were found between approximately 1.31 Å and 1.71 Å (Figure 4(a)). As given in RMSD plot (Figure S8A), the average RMSD values were found to be 1.71 ± 0.24 Å, 1.71 ± 0.21 Å, 1.28 ± 0.14 Å, 1.56 ± 0.17 Å, 1.64 ± 0.22 Å, 1.75 ± 0.28 Å, and 1.31 ± 0.11 Å for compounds (1–5), donepezil, and tacrine, respectively. The fluctuations were observed in the RMSD plot of tacrine between 55 and 100 ns simulations. The average RMSD value of compound 4 increased to 4 Å after 42 ns MD simulation compared to reference drug donepezil (Figure 4(a)). Furthermore, the RMSF plot for compounds and known inhibitors exhibited the flexibility of residues. The RMSF values of the complexes were below 2.5 Å; however, it was seen that some fluctuations were obtained for four residues up to 4 Å, 7 Å, and 8 Å (Figure 4(b)).

Figure 4.

RMSD and RMSF plots for protein-ligand complexes for 100 ns MD simulations.

3.6. MM/GBSA calculations

To determine binding free energies of the most potential compounds 1 and 4, and reference drugs, MM/GBSA calculations were carried out. The binding energy (ΔG_bind) of compound 4 was calculated as −58.87 ± 6.91 kcal/mol. The energy value was found to be −49.12 ± 3.15 kcal/mol for tacrine. Donepezil was calculated with the highest binding free energy (−80.30 ± 2.70 kcal/mol). Herein, these results were correlated with docking scores and in vitro IC₅₀ values. The binding energies (ΔG_bind) for compounds 1–3, and 5, respectively, were calculated as −53.53 ± 8.37 kcal/mol, −66.85 ± 6.46 kcal/mol, −61.28 ± 8.40 kcal/mol, and −49.80 ± 5.88 kcal/mol. In the literature, the MM/GBSA method was used to determine binding free energy for one of boron-based synthesized compounds, and it was reported as −56.92 kcal/mol [65].

3.7. In silico ADME prediction

SwissADME (http://www.swissadme.ch/) was used for the prediction of pharmacokinetic properties of the synthesized compounds (1–5). Gastrointestinal absorption (GI), cytochrome P450 (Cyp) inhibitors, blood–brain barrier (BBB), Lipinski and Veber rule, and solubility value were determined [61]. Synthesized boron-containing compounds have a good and moderate solubility except compound 5. Compound 4 may be a CYP2D6 inhibitor, and all compounds have no BBB permeability (Table 2).

Table 2.

SwissADME results for the synthesized compounds (1–5).

Compounds
	1	2	3	4	5
LogS	−2.46	−3.84	−2.06	−4.28	−6.14
GI absorption	High	High	High	High	High
BBB permeant	No	No	No	No	No
CYP1A2 inhibitor	No	No	No	No	No
CYP2C19 inhibitor	No	No	No	No	Yes
CYP2C9 inhibitor	No	No	No	No	No
CYP2D6 inhibitor	No	No	No	Yes	No
Lipinski-Veber	Yes	Yes-No	Yes	Yes	Yes
Molecular weight (g/mol)	323.38	420.31	338.16	446.34	606.32
Number H-bond acceptors	5	6	6	6	8
Number H-bond donors	2	2	4	0	0
Log Po/w	2.13	1.76	0.07	1.77	2.30

3.8. Structure–activity relationship (SAR)

In this study, we evaluated AChE inhibitory activity of the newly synthesized boron-containing carbamate compounds for their therapeutic potentials (Figure 5). Notably, the potent compounds 1-4 achieved indicating a stronger inhibitory effect than tacrine. These findings for reference drugs align with existing literature [71], supporting the potential of synthesized boron-containing compounds as AChE inhibitors and suggesting a promising direction for further optimization and development. Furthermore, an MTT assay conducted to assess anticancer activity revealed that compound 4 significantly reduced cell viability to 50% in HT29 and MCF-7 cell lines. These findings highlight the dual potential of boron-containing compounds as both AChE inhibitors and anticancer agents, underscoring their therapeutic value in the treatment of neurodegenerative diseases and cancer. The potential boron-containing benzyl carbamate compound 4 linked to tert-butyl piperazine-1-carboxylate. Piperazine moiety contributes to anti-acetylcholinesterase and anticancer activity and piperazine compounds were designed and synthesized to determine their cholinesterase activities [54,83]. Piperazine scaffold has been widely used in place of piperidine of donepezil. For instance, it was reported that a piperazine-containing compound, FK960, displayed useful effects on memory loss in AD rats and monkeys [93,94]. A piperazine containing coumarin-based drug, ensaculin is used in clinics for the treatment of dementia [93].

Figure 5.

A schematic SAR of potential AChE inhibitors.

Although compounds (2 and 3) contain tert-butyl carbamate group, 2-hydroxyethylcarbamate substituted compound 1 showed a more potent inhibition effect. It can be said that the free hydroxyl group has more contribution tert-butyl group for synthesized boron-containing carbamate compounds (2 and 3). Overall, among the synthesized compounds, piperazine-containing compound 4 more contributed to AChE inhibition activity.

4. Conclusions

In this study, new boron-containing carbamates as AChE inhibitors for the treatment of AD were investigated, and cytotoxic effects were evaluated. As a result of in vitro enzyme activity analyses, especially the strong AChE inhibition was detected for compounds 1–4 compared to tacrine. In the light of these results, compounds 1–4 have approximately 1.5- and 2-fold higher activity than tacrine. Based on our findings, molecular modeling studies revealed that the compounds exhibited strong interactions with critical accumulations in the binding site of AChE. In addition, compound 4 showed significant cytotoxic effects in colon (HT29) and breast (MCF-7) cancer cell lines. These findings indicate that new boron-containing carbamate compounds may be promising candidates for both AD and cancer treatments. Future in vitro/in vivo studies and detailed investigations will contribute to a better understanding of the therapeutic potential of these compounds. Then, new insights may be needed for the synthesized boron-containing compounds to be used in therapeutic applications and/or design of new derivatives to be used in clinic.

Funding Statement

The author(s) reported that there is no funding associated with the work featured in this article.

Article highlights

• Boron-containing carbamate compounds (1-5) were synthesized and characterized using ¹H NMR, ¹³C NMR, ¹¹B NMR, and MS techniques.

• Compounds (1-4) exhibited more potent AChE inhibitory activity compared to tacrine.

• Furthermore, compound 4 had an antiproliferative activity against various human cancer cell lines.

• The interactions with key residues in the binding pocket of AChE were identified between protein and the compounds.

• This study highlights the discovery of boron-containing compounds for the treatment of Alzheimer disease and cancer.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Data availability statement

Data available within the article or its supplementary materials

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2525065