Synthesis, Biological Evaluation and Molecular Docking Study of Hydrazone-Containing Pyridinium Salts as Cholinesterase Inhibitors

Abstract

A series of pyridinium salts bearing alkylphenyl groups at 1 position and hydrazone structure at 4 position of the pyridinium ring were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. The cholinesterase (ChE) inhibitory activity studies were carried out by using the Ellman’s colorimetric method. All compounds displayed considerable AChE and BuChE inhibitory activity and some of the compounds manifested remarkable anti-AChE activity compared to the reference compound, galantamine. Among the title compounds, the series including benzofuran aromatic ring exhibited the best inhibitory activity both on AChE and BuChE enzymes. Compound 3b, 4-[2-(1-(benzofuran-2-yl)ethylidene)hydrazinyl]-1-(3-phenylpropyl)pyridinium bromide, was the most active compound with IC₅₀ value of 0.23 (0.24) µM against enantiomeric excess (ee)AChE (human (h)AChE) while compound 3a, 4-[2-(1-(benzofuran-2-yl)ethylidene)hydrazinyl]-1-phenethylpyridinium bromide, was the most active compound with IC₅₀ value of 0.95 µM against BuChE. Moreover, 3a and b exhibited higher activity than the reference compound galantamine (eeAChE (hAChE) IC₅₀ 0.43 (0.52) µM; BuChE IC₅₀ 14.92 µM). Molecular docking studies were carried out on 3b having highest inhibitory activity against AChE.

Acetylcholinesterase (AChE) is an enzyme which is responsible for the hydrolysis of acetylcholine (ACh).¹⁾ AChE inhibitors bind to the enzyme thereby increases the level of acetylcholine by inhibiting the hydrolysis of ACh to choline and acetate.²⁾ AChE inhibitors are used as therapeutic agents in the treatment of disorders related to ACh diminution such as Alzheimer disease, myasthenia gravis, glaucoma.³⁾

AChE has a deep, narrow gorge approximately 20 Å long. Catalytic active site (CAS) of the enzyme is located at the bottom of the gorge whereas peripheral anionic site (PAS) is at the entrance.⁴⁾ Although anti-AChE drugs show a wide range of chemical diversity, they generally include aromatic-heterocyclic ring systems and a nitrogen atom as chemical structure (Fig. 1). These structures have an important role for the interaction with specific amino acids in the enzyme.⁵⁾ Aromatic–heterocyclic nuclei generally have potential hydrophobic and charge-transfer interaction, while nitrogen atom and its quaternary form can make hydrogen or ionic bonds as well as dipole–dipole or ion–dipole interactions with amino acids in the enzyme gorge.^5–10) In addition, quaternary nitrogen cation displays highly positive binding stabilization due to cation–π interactions with amino acid residues in AChE enzyme.^9–12)

Fig. 1. FDA Approved AChE Inhibitors

Many reports indicated that compounds bearing aromatic/heteroaromatic rings on lateral parts of pyridinium displayed high AChE inhibitory activity and act as dual binding inhibitors.^13–16) According to the docking studies of these compounds, it was found that both terminal rings attached to the pyridinium were able to bind simultaneously to both CAS and PAS of the enzyme resulting in higher AChE inhibitory activity.^13–16) Among the reported studies, pyridinium derivatives having benzofuran ring were shown to have a significant AChE inhibitory activity and it was observed that benzofuran and phenyl moieties as terminal rings were interacted with both PAS and CAS of AChE.^15,16)

On the other hand, many of compounds bearing hydrazone-hydrazide functional group have been reported to possess ChE inhibitory activity.^17,18) The hydrazones have hydrogen donor and acceptor nitrogen atoms and ability to make hydrogen bound with amino acids in the gorge of the enzyme. Thus, these groups are preferred in the molecular structures. In our previous studies, hydrazone and pyridinium derivatives bearing oxime group were synthesized and evaluated for their inhibitory activities on ChE enzymes and amyloid-β aggregation.^19–21) Some of these compounds exhibited remarkable AChE inhibitory activity and enhanced the fibril destruction. Previous structure–activity relationship results suggested that adding a phenyl ring to propyl side chain attached to the pyridinium nitrogen noticeably enhances the activity (Fig. 2).

Fig. 2. Pyridinium Derivatives as AChE Inhibitors

Based on the above data, we synthesized a series of pyridinium derivatives bearing different alkylphenyl groups on pyridine nitrogen and hydrazone structure at 4 position of pyridine ring. For this purpose, acetophenone, p-chloroacetophenone and 2-acetylbenzofuran were selected as ketone function to form their hydrazones. Ethylene and propylene chains were selected as linkers and one of the CH₂ group in propylene linker was replaced with ether function which is isoster of methylene group to compare the effect on the interaction of oxygen atom with the enzyme.

Results and Discussion

Chemistry

The synthesis of arylidenehydrazinylpyridinium derivatives was carried out according to the literature^22,23) as summarized in Chart 1. In the first step, 4-hydrazinylpyridine was obtained by nucleophilic substitution of hydrazine with 4-chloropyridine then corresponding hydrazone derivatives 1–3 were furnished by the condensation of 4-hydrazinylpyridine with aromatic ketones in ethanol under reflux. Finally, the title compounds 1a–3c were synthesized by quaternization of intermediate compounds 1–3 with alkyl halides in ethanol under reflux condition.

Chart 1. Synthesis Pathway of the Pyridinium Compounds

All title compounds were reported for the first time in this study except for 1b and 2b.²³⁾ The structures of the compounds (1a–3c) were identified by IR, ¹H-NMR, ¹³C-NMR and the purity levels of the compounds were confirmed by high resolution (HR)-MS analysis. The spectroscopic properties were in accordance with the proposed structures. In the IR spectra, N–H stretching band was observed at 3480–3375 cm⁻¹ while C=C and C=N stretching bands were detected between 1645–1443 cm⁻¹. According to the ¹H-NMR spectra of the final compounds, the aromatic and aliphatic proton signals were observed in the expected regions with expected integrations and divisions.²⁴⁾ The proton signal due to the NH group was recorded as a broad singlet between δ 11.24–12.19 ppm. The singlet signal which is seen in aliphatic region between δ 2.43–2.52 ppm belongs to the methyl proton of –NH–N=C–CH₃ group. Pyridinium protons at 2, 6 positions for the final compounds were observed between δ 8.34–8.60 ppm. The relatively higher frequency values of these chemical shifts according to 3, 5 positions (δ 7.19–7.87 ppm) can be interpreted by the withdrawing effect of the nitrogen atom. The aromatic protons of the phenyl ring attached to pyridinium nitrogen with alkyl chain were resonated in the δ 6.88–7.34 ppm region integrating for five protons as expected. On the other hand, the protons of the aromatic ring in the hydrazone moiety were observed between δ 7.39–7.95 ppm for series 1 while the aromatic protons at 2, 6 and 3, 5 positions in series 2 having chlorine substituent at para position were appeared as two doublets between δ 7.92–7.94 ppm and δ 7.48–7.52 ppm, respectively. In series 3, the furan proton of benzofuran ring was detected as a singlet at δ 7.55, δ 7.57 ppm for 3c and b, respectively.

Biological Activities

Inhibitory activities of the target compounds against AChE and BuChE enzymes were evaluated by modified Ellman’s spectrophotometric method.^9,25) Galantamine was used as reference standard. The results were summarized in Table 1 as IC₅₀ values.

Table 1. AChE/BuChE Inhibitory Activities and Selectivity of Pyridinium Compounds

	IC50±S.E.M. (µM)a)
	eeAChE	hAChE	eqBuChE	Selectivity eeAChE/eqBuChE
1a	8.89±0.13	6.30±2.33	1.91±0.04	4.65
1b	0.84±0.02	0.96±0.07	2.48±0.04	0.34
1c	8.81±0.17	10.83±1.36	3.86±0.17	2.28
2a	5.59±0.19	8.08±0.15	5.11±0.10	1.09
2b	0.74±0.03	0.56±0.02	2.0±0.04	0.37
2c	4.38±0.11	1.54±0.15	1.69±0.03	2.59
3a	0.32±0.03	0.62±0.03	0.95±0.03	0.33
3b	0.23±0.01	0.24±0.002	1.95±0.04	0.11
3c	4.24±0.59	9.96±1.89	1.47±0.05	2.88
Galantamine	0.43±0.03	0.52±0.06	14.92±0.57	0.03

a) Data are the mean±S.E.M. of triplicate independent experiments.

According to the enantiomeric excess (ee)AChE results, all compounds exhibited from good to moderate inhibitory activity in the range of 0.23 to 8.89 µM. In comparison of the nonsubtituted (1a–c) to chlorosubstituted (2a–c) phenyl derivatives, 1a–c have inhibitory potency with IC₅₀ values of 8.89, 0.84, 8.81 µM, whereas 2a–c exhibited the inhibition with IC₅₀ values of 5.59, 0.74, 4.38 µM, respectively. The compounds bearing chlorine atom on phenyl ring at para position displayed better activity than nonsubstituted phenyl derivatives. Thus, the introduction of a chlorine atom to the phenyl ring positively influences the inhibitory capacity of the target compounds.

When each series having phenylethyl (1a, 2a, 3a), phenylpropyl (1b, 2b, 3b) and phenoxyethyl (1c, 2c, 3c) side chains was evaluated among themselves, the highest activity was detected in the 3a–c containing benzofuran ring instead of phenyl ring. Among the tested compounds, compound 3a and b with benzofuran ring were found to be the most active derivatives against eeAChE enzyme. Moreover, 3a and b with IC₅₀ values of 0.32, 0.23 µM, respectively, exhibited better inhibitory activity than the reference compound galantamine (IC₅₀ 0.43 µM).

Concerning human (h)AChE activity results, the compounds possessed similar inhibitory activity to eeAChE. Also, compounds 3a (IC₅₀ 0.62 µM) and 3b (IC₅₀ 0.24 µM) were the most active derivatives against hAChE as well.

The AChE inhibitory activity increased when the distance between the pyridinium nitrogen and the phenyl ring was lengthened from two to three methylene groups. However, the replacement of methylene group with ether function in propyl chain leads to a remarkably diminution in AChE inhibitory potency. This result suggested that the length of the side chain and its hydrophobic feature is important for the activity and it can be speculated that a hydrophobic interaction can be formed between the propylphenyl chain and AChE enzyme.

In respect to BuChE activity results, most of the compounds displayed inhibitory activity better than the reference compound. When compared to AChE inhibitory results, as generally the derivatives bearing benzofuran ring are the most active compounds in the phenylethyl (a), phenylpropyl (b) and phenoxyethyl (c) series, no further correlations were established between side chains and BuChE inhibitory activity. Among the title compounds, 3a is the most active compound with IC₅₀ value of 0.95 µM towards BuChE.

Regarding the eeAChE/equation (eq)BuChE selectivity, all of the compounds except for 2a displayed selectivity towards AChE or BuChE enzyme. Besides, the selectivity tendency of the title compounds shifts from BuChE to AChE in b series, whereas this tendency was observed conversely in c series. Compounds 1b, 2b, 3a and b which have the highest inhibitory activity against AChE are also the most selective compounds towards AChE.

Among the tested derivatives, the series including benzofuran aromatic ring exhibited the best inhibitory activity on both AChE and BuChE enzymes. Benzofuran ring is an isoster of indane ring which is the pharmacophoric group of donepezil.

Docking Study

In this study, molecular docking study was performed using GOLD 5.2.1 to predict the interaction mode of compound 3b for AChE. The X-ray crystal structure of Torpedo californica AChE (TcAChE) and hAChE in complex with donepezil (PDB code 1EVE and 4EY7, respectively) was selected to build the starting model of AChE. Compound 3b was chosen for molecular modeling as the most active compound in the series (Table 1).

As shown in Fig. 3, compound 3b has three major binding interactions with TcAChE. Hydrazone moiety interacts with the carbonyl group of Phe330 residue via H-bond. Phe330 is located in the hydrophobic pocket near the CAS of AChE and it is known that several AChE inhibitors have binding interactions with Phe330.⁸⁾ On the other hand, pyridinium ring interacts with Tyr334 residue and the phenyl ring interacts with Trp279 residue by π–π interactions. Tyr334 and Trp279 are located in the PAS of the enzyme⁴⁾ and it can be proposed that compound 3b can interact both with the CAS and PAS of AChE therefore compound 3b can be speculated to have a dual binding potential. Regarding hAChE, hydrazone moiety of compound 3b interacts with both the hydroxyl group of Ser203 and the carbonyl group of Glu202 residue through two hydrogen bonds. Ser203 is one of the amino acid of the catalytic triad (Ser203, Glu334, His447) and these amino acid residues are located in the active site.²⁶⁾ The phenyl ring of compound 3b interacts with Tyr341, located in the peripheral anionic binding site at the mouth of the gorge by π–π interactions. Moreover, the benzofuran ring reaches to Trp84 amino acid residue located at the bottom of the enzyme gorge. To sum up the docking results, hydrazone group interacts with amino acid residues of both enzymes via hydrogen bond. While phenyl ring interacts with PAS, the other lateral part, benzofuran moiety, reaches to CAS of both enzymes. In conclusion, the docking results are consistent with the activity of compound 3b.

Fig. 3. (A) Proposed Binding Mode for Compound 3b Inside TcAChE (pdb Code 1EVE) and (B) Proposed Binding Mode for Compound 3b Inside hAChE (pdb Code 4EY7)

The active compound is represented as black sticks in TcAChE and hAChE. The most involved residues are named and represented as grey sticks for TcAChE and hAChE. Hydrogen bond interactions are represented as dashed lines.

On the other hand, when we compare the activity results of the series b with the series c (series b carry propylphenyl group as the side chain whereas series c carry phenoxyethyl group), the activity results of the series b are remarkably higher than the results of the series c. In terms of molecular modeling studies, we can speculate that the higher activity of the series b might result from the π–π interaction with Trp279 and the interaction might be lost with the series c due to the replacement of the CH₂ group with O atom causing a different orientation of the phenyl ring inside the enzyme.

Experimental

Chemistry Materials and Methods

Melting points were determined using a Stuart SMP30 (Staffordshire, ST15 OSA, U.K.) melting point apparatus and are not corrected. IR spectra of the compounds were recorded as potassium bromide pellets on a PerkinElmer 100 Fourier transform (FT)-IR spectrophotometer (PerkinElmer, Inc., MA, U.S.A.). ¹H- and ¹³C-NMR spectra were recorded with a Varian AS 400 Mercury Plus NMR spectrometer (Varian, Palo Alto, CA, U.S.A.) operated at 400 and 100 MHz for ¹H and ¹³C, respectively, in deutero-dimethyl sulfoxide (DMSO). Chemical shifts are given in ppm (δ) with tetramethylsilane (TMS) as an internal standard. Abbreviations for data quoted are: s (singlet), d (doublet), t (triplet), quin (quintet), m (multiplet), br s (broad singlet). HR-MS of the title compounds were recorded on a HPLC-TOF Waters Micromass LCT Premier XE (Milford, MA, U.S.A.) mass spectrometer using an electrospray ion source (ESI). All chemicals, reagents and solvents used for synthesis were high-grade commercial products and they were purchased from Sigma, Acros, Fluka, U.S.A., and Merck Companies. Reactions were checked by TLC on pre-coated silica gel aluminum plates (Kieselgel 60, F₂₅₄, E. Merck, Germany); spots were visualized by UV at 254 nm.

Synthesis of 4-Hydrazinylpyridine Hydrochloride

A solution of 4-chloropyridine (15 g; 0.1 mol) and hydrazine monohydrate (9.7 mL, 0.2 mol) in 1-propanol (30 mL) was refluxed for 18 h. The solution was cooled to 0°C and the precipitate was filtered, washed with cool 1-propanol and crystallized from ethanol.²²⁾

General Procedure for Synthesis of Compounds 1–3

4-Hydrazinylpyridine (0.8 g, 7.5 mmol) was condensated with the appropriate ketones (acetophenone, 4-chloroacetophenone and 2-acetylbenzofuran) (1.05, 1.17 mL, 1.44 g, 9 mmol) in ethanol (30 mL) for 20–38 h in order to obtain hydrazone derivatives. The precipitate was filtered and washed with cool ethanol–water (1 : 1) mixture and crystallized from ethanol. Compounds 1 and 2 were synthesized in our previous study.²³⁾

4-(2-[1-(Benzofuran-2-yl)ethylidene]hydrazinyl)pyridine (3)

Yield 65%; ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.30 (3H, s, CH₃), 7.15 (2H, dd, J=4.0/8.0 Hz, pyridinium-H), 7.33–7.43 (3H, m, benzofuran-H), 7.82 (2H, d, J=8.0 Hz, benzofuran-H), 8.23 (2H, d, J=8.0 Hz, pyridinium-H), 9.77 (1H, br s, NH).

General Procedure for Synthesis of the Final Compounds 1a–3c

A mixture of 1–3 (0.21, 0.25, 0.25 g, 1 mmol) and the corresponding alkyl halide (phenethyl bromide, 3-phenypropyl bromide, 2-phenoxyethyl bromide) (0.25, 0.27 mL, 0.36 g, 1.8 mmol) in ethanol (30 mL) were heated under reflux for 66–118 h. The mixture was cooled to room temperature and the obtained precipitate was filtered and washed with cool ethanol. The crude products were crystallized from ethanol–petroleum ether (1 : 1) mixture to give the target compounds 1a–3c. Compounds 1b and 2b were synthesized in our previous study.²³⁾

1-Phenethyl-4-[2-(1-phenylethylidene)hydrazinyl]pyridinium Bromide (1a)

Yield 65%; mp 152°C; IR (KBr) cm⁻¹: 3435, 3033, 2960, 2860, 1645, 1578, 1555, 1539, 1184, 835, 760, 746. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.44 (3H, s, CH₃), 3.16 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 4.54 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 7.20–7.32 (5H, m, Ar-H), 7.37 (1H, d, J=7.6 Hz, pyridinium-H), 7.44–7.47 (3H, m, Ar-H), 7.61 (1H, d, J=7.6 Hz, pyridinium-H), 7.89–7.93 (2H, m, Ar-H), 8.34 (2H, br s, pyridinium-H), 11.24 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.30, 36.67, 58.83, 107.81, 109.76, 126.94, 127.32, 128.94, 129.00, 129.36, 130.20, 137.07, 137.75, 143.21, 144.49, 153.30, 154.91. HR-MS (ESI⁺) Calcd for C₂₁H₂₂N₃⁺ 316.1814. Found 316.1818.

4-[2-(1-Phenylethylidene)hydrazinyl]-1-(3-phenylpropyl)pyridinium Bromide (1b)

Yield 60%; mp 106°C; IR (KBr) cm⁻¹: 3480, 3010, 2990, 2860, 1644, 1538, 1445, 840, 754, 692. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.17 (2H, quin, J=7.4 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 2.47 (3H, s, CH₃), 2.64 (2H, t, J=7.4 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 4.33 (2H, t, J=7.4 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 7.20–7.33 (5H, m, Ar-H), 7.45–7.49 (4H, m, pyridinium-1H, Ar-3H), 7.69 (1H, d, J=7.6 Hz, pyridinium-H), 7.93–7.95 (2H, m, Ar-H), 8.48 (2H, dd, J=3.7/7.7 Hz, pyridinium-H), 11.30 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.24, 32.02, 32.33, 57.79, 108.09, 110.02, 126.50, 126.92, 128.66, 128.86, 128.96, 130.20, 137.80, 140.94, 143.26, 144.53, 153.22, 154.98. HR-MS (ESI⁺) Calcd for C₂₂H₂₄N₃⁺ 330.1970. Found 330.1983.

1-(2-Phenoxyethyl)-4-[2-(1-phenylethylidene)hydrazinyl]pyridinium Bromide (1c)

Yield 77%; mp 203°C; IR (KBr) cm⁻¹: 3470, 3070, 2917, 1644, 1539, 1490, 1443, 1189, 1050, 842, 764. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.50 (3H, s, CH₃), 4.36 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 4.65 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 6.88–6.92 (3H, m, Ar-H), 7.23 (2H, t, J=8.0 Hz, Ar-H), 7.39–7.41 (3H, m, Ar-H), 7.61 (1H, d, J=8.0 Hz, pyridinium-H), 7.85–7.87 (3H, m, pyridinium-1H, Ar-2H), 8.49 (2H, dd, J=3.7/8.0 Hz, pyridinium-H), 12.19 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.79, 57.13, 66.84, 107.72, 109.87, 115.05, 121.71, 126.93, 128.89, 130.00, 130.13, 137.92, 143.48, 144.94, 153.63, 155.46, 158.11. HR-MS (ESI⁺) Calcd for C₂₁H₂₂N₃O⁺ 332.1763. Found 332.1765.

4-[2-(1-(4-Chlorophenyl)ethylidene)hydrazinyl]-1-phenethylpyridinium Bromide (2a)

Yield 75%; mp 225°C; IR (KBr) cm⁻¹: 3399, 3055, 2902, 2855, 1645, 1582, 1553, 1539, 1489, 1203, 1176, 1093, 847, 729. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.43 (3H, s, CH₃), 3.16 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 4.54 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 7.20–7.32 (5H, m, Ar-H), 7.38 (1H, br s, pyridinium-H), 7.51 (2H, d, J=8.4 Hz, Ar-H), 7.62 (1H, d, J=7.2 Hz, pyridinium-H), 7.94 (2H, d, J=8.4 Hz, Ar-H), 8.34 (2H, br s, pyridinium-H), 11.27 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.22, 36.67, 58.85, 107.91, 109.84, 127.31, 128.72, 128.91, 128.99, 129.36, 134.87, 136.54, 137.04, 143.26, 144.51, 152.03, 154.88. HR-MS (ESI⁺) Calcd for C₂₁H₂₁ClN₃⁺ 350.1424. Found 350.1431.

4-[2-(1-(4-Chlorophenyl)ethylidene)hydrazinyl]-1-(3-phenylpropyl)pyridinium Bromide (2b)

Yield 63%; mp 115°C; IR (KBr) cm⁻¹: 3425, 3020, 2951, 2832, 1643, 1583, 1538, 1513, 1482, 823, 763, 680. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.15 (2H, quin, J=8.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 2.44 (3H, s, CH₃), 2.61 (2H, t, J=8.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 4.32 (2H, t, J=8.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 7.18–7.31 (5H, m, Ar-H), 7.45 (1H, d, J=8.0 Hz, pyridinium-H), 7.52 (2H, d, J=8.0 Hz, Ar-H), 7.68 (1H, d, J=8.0 Hz, pyridinium-H), 7.94 (2H, d, J=8.0 Hz, Ar-H), 8.48 (2H, dd, J=4.0/8.0 Hz, pyridinium-H), 11.31 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.22, 32.02, 32.36, 57.82, 108.19, 110.11, 126.49, 128.65, 128.70, 128.85, 128.93, 134.86, 136.60, 140.93, 143.31, 144.55, 151.94, 154.96. HR-MS (ESI⁺) Calcd for C₂₂H₂₃ClN₃⁺ 364.1581. Found 364.1585.

4-[2-(1-(4-Chlorophenyl)ethylidene)hydrazinyl]-1-(2-phenoxyethyl)pyridinium Bromide (2c)

Yield 82%; mp 235°C; IR (KBr) cm⁻¹: 3375, 3060, 2904, 1643, 1541, 1488, 1242, 1189, 1089, 832, 789, 760. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.43 (3H, s, CH₃), 4.39 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 4.68 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 6.91–6.96 (3H, m, Ar-H), 7.27 (2H, t, J=8.0 Hz, Ar-H), 7.48 (2H, d, J=8.0 Hz, Ar-H), 7.67 (1H, d, J=8.0 Hz, pyridinium-H), 7.79 (1H, d, J=8.0 Hz, pyridinium-H), 7.92 (2H, d, J=8.0 Hz, Ar-H), 8.50 (1H, d, J=8.0 Hz, pyridinium-H), 8.53 (1H, d, J=8.0 Hz, pyridinium-H), 12.09 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 15.52, 57.21, 66.81, 107.87, 109.97, 115.05, 121.73, 128.72, 128.90, 130.01, 134.87, 136.73, 143.61, 144.99, 152.39, 155.41, 158.10. HR-MS (ESI⁺) Calcd for C₂₁H₂₁ClN₃O⁺ 366.1373. Found 366.1382.

4-[2-(1-(Benzofuran-2-yl)ethylidene)hydrazinyl]-1-phenethylpyridinium Bromide (3a)

Yield 64%; mp 130°C; IR (KBr) cm⁻¹: 3408, 3028, 2904, 1639, 1579, 1532, 1448, 1181, 836, 742. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.50 (3H, s, CH₃), 3.20 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 4.59 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–Ph), 7.23–7.34 (6H, m, pyridinium-1H, Ar-5H), 7.42 (1H, td, J=1.2/8.0 Hz, benzofuran-H), 7.55–7.58 (3H, m, pyridinium-1H, benzofuran-2H), 7.66 (1H, d, J=8.0 Hz, benzofuran-H), 7.72 (1H, d, J=8.0 Hz, benzofuran-H), 8.47 (2H, br s, pyridinium-H), 11.49 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 14.91, 36.64, 59.02, 107.94, 108.97, 110.03, 111.83, 122.32, 123.93, 126.59, 127.34, 128.35, 129.01, 129.36, 137.02, 143.36, 144.67, 144.70, 153.39, 154.68, 155.19. HR-MS (ESI⁺) Calcd for C₂₃H₂₂N₃O⁺ 356.1763. Found 356.1772.

4-[2-(1-(Benzofuran-2-yl)ethylidene)hydrazinyl]-1-(3-phenylpropyl)pyridinium Bromide (3b)

Yield 60%; mp 77°C; IR (KBr) cm⁻¹: 3396, 3026, 2991, 2933, 1641, 1582, 1560, 1533, 1450, 1186, 838, 745, 735. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.18 (2H, quin, J=7.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 2.52 (3H, s, CH₃), 2.64 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 4.36 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–CH₂–Ph), 7.19–7.33 (6H, m, pyridinium-1H, Ar-5H), 7.42 (1H, td, J=1.2/8.0 Hz, benzofuran-H), 7.57 (1H, s, benzofuran-H), 7.72–7.74 (4H, m, pyridinium-1H, benzofuran-3H), 8.55 (1H, br s, pyridinium-H), 8.60 (1H, br s, pyridinium-H), 11.52 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 14.93, 32.03, 32.29, 57.96, 108.25, 108.96, 110.30, 111.83, 122.32, 123.93, 126.50, 126.59, 128.36, 128.66, 128.85, 140.91, 143.44, 144.61, 144.70, 153.42, 154.75, 155.20. HR-MS (ESI⁺) Calcd for C₂₄H₂₄N₃O⁺ 370.1919. Found 370.1906.

4-[2-(1-(Benzofuran-2-yl)ethylidene)hydrazinyl]-1-(2-phenoxyethyl)pyridinium Bromide (3c)

Yield 73%; mp 172°C; IR (KBr) cm⁻¹: 3400, 3035, 2929, 1637, 1597, 1587, 1530, 1495, 1246, 1184, 1117, 1048, 841, 744, 739. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 2.50 (3H, s, CH₃), 4.39 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 4.68 (2H, t, J=7.0 Hz, N⁺–CH₂–CH₂–O–Ph), 6.92–6.96 (3H, m, Ar-H), 7.26–7.31 (3H, m, pyridinium-1H, Ar-2H), 7.40 (1H, t, J=8.0 Hz, benzofuran-H), 7.55 (1H, s, benzofuran-H), 7.63–7.71 (4H, m, pyridinium-1H, benzofuran-3H), 8.50 (1H, br s, pyridinium-H), 8.56 (1H, br s, pyridinium-H), 11.92 (1H, br s, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ ppm: 14.94, 57.38, 66.78, 107.94, 108.98, 110.15, 111.82, 115.05, 121.77, 122.34, 123.96, 126.62, 128.38, 130.01, 143.80, 144.97, 145.28, 153.51, 155.13, 155.21, 158.10. HR-MS (ESI⁺) Calcd for C₂₃H₂₂N₃O₂⁺ 372.1712. Found 372.1710.

Biological Activity Assays Materials and Methods

AChE (E.C. 3.1.1.7., Type VI-S, from electric eel and recombinant human enzyme) and BuChE (E.C. 3.1.1.8, from equine serum) were purchased from Sigma-Aldrich (Steinheim, Germany). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) acetylthiocholine iodide (ATC) and butyrylthiocholine iodide (BTC) used as substrates were obtained from Fluka. Buffer compounds (potassium dihydrogen phosphate, potassium hydroxide) and sodium hydrogen carbonate were purchased from Merck. Spectrofotometric measurements were performed on a Shimadzu 160-A UV-Vis spectrophotometer.

Acetylcholinesterase/Butyrylcholinesterase Activity Assay

The inhibitory effects of the synthesized compounds on AChE and BuChE were evaluated using a slightly modified colorimetric method of Ellman et al., with galantamine as the positive control.^9,25) Prior to use, all solutions were adjusted to 20°C. Enzyme solution (100 µL) and inhibitor solution (100 µL) were added into a cuvette containing the phosphate buffer (3.0 mL, 0.1 M; pH 8.0). After 5 min incubation, required aliquots of the DTNB solution (100 µL) and of the ATC/BTC (20 µL) were added. After rapid and immediate mixing the absorption was measured at 412 nm by UV spectroscopy. As a reference, an identical solution of the enzyme without the inhibitor is processed following the same protocol. The blank reading contained 3.0 mL buffer, 200 µL water, 100 µL DTNB, and 20 µL substrate. The enzyme activity was determined in the presence of at least five different concentrations of an inhibitor, generally between 10⁻³ and 10⁻⁸, in order to obtain inhibition of AChE or BuChE activity between 0 and 100%. Each concentration was assayed in triplicate. The samples were investigated immediately after preperation. The AChE/BuChE inhibitory activities and selectivity of pyridinium compounds results are summarized in Table 1.

Molecular Docking Study

The crystal structures of donepezil in complex with AChE (pdb codes: 1EVE and 4EY7 resolved at 2.5 and 2.35 Å, respectively) were taken from the Protein Data Bank. Heteroatoms and water molecules in the PDB file were removed and hydrogen atoms were added to the protein by using MOE 2014.09.1.²⁷⁾ Prior to the docking calculations, an energy minimization using the AMBER99 force field was performed on the enzyme. Compound 3b was built and protonated using the protonate 3D protocol and energy minimized using the MMFF94 force field via MOE 2014.09.1. Docking of the ligand was carried out using the GOLD 5.2.1 program with default settings.^28,29) A sphere of 22 Å around the carbonyl group of Glu199 (Glu202 in hAChE) was defined as the binding site for the ligand docking and 250 confirmations was allowed. Chemscore and Goldscore standard precision (sp) were calculated and analyzed. The putative binding mode was carried out through visual inspection (Fig. 3).

Conclusion

A series of hydrazone-containing pyridinium salts were synthesized and assayed for their cholinesterase inhibitory activity. All compounds exhibited from good to moderate inhibitory activity on both AChE and BuChE enzymes. For the title compounds, the length of the side chain and its hydrophobic feature seems to be important for the AChE activity. Among tested derivatives, the series including benzofuran aromatic ring exhibited the best inhibitory activity against cholinesterases. Benzofuran-pyridiniumhydrazone scaffold can take place as a core structure for further investigations.

Acknowledgments

This study was supported by Research Grants from Ege University (Project Number: 13/Ecz/026). The authors would like to thank the Pharmaceutical Sciences Research Centre (FABAL) at Ege University Faculty of Pharmacy for spectral analyses of the compounds.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains Supplementary materials.

References and Notes

• 1) Barnard E. A., “The Peripheral Nervous System,” ed. by Hubbard J. I., Plenum Press, New York, U.S.A., 1974, pp. 201–224.
• 2) Shen T., Tai K., Henchman R. H., McCammon J. A., Acc. Chem. Res., 35, 332–340 (2002).
• 3) Colović M. B., Krstić D. Z., Lazarević-Pašti T. D., Bondžić A. M., Vasić V. M., Curr. Neuropharmacol., 11, 315–335 (2013).
• 4) Sussman J. L., Harel M., Frolow F., Oefner C., Goldman A., Toker L., Silman I., Science, 253, 872–879 (1991).
• 5) Holzgrabe U., Kapkova P., Alptuzun V., Scheiber J., Kugelmann E., Expert Opin. Ther. Targets, 11, 161–179 (2007).
• 6) Alpan A. S., Parlar S., Carlino L., Tarikogullari A. H., Alptüzün V., Güneş H. S., Bioorg. Med. Chem., 21, 4928–4937 (2013).
• 7) Dvir H., Silman I., Harel M., Rosenberry T. L., Sussman J. L., Chem. Biol. Interact., 187, 10–22 (2010).
• 8) Galdeano C., Viayna E., Arroyo P., Bidon-Chanal A., Blas J. R., Muñoz-Torrero D., Luque F. J., Curr. Pharm. Des., 16, 2818–2836 (2010).
• 9) Kapková P., Stiefl N., Sürig U., Engels B., Baumann K., Holzgrabe U., Arch. Pharm. Pharm. Med. Chem, 336, 523–540 (2003).
• 10) Conejo-García A., Pisani L., Núñez M. C., Catto M., Nicolotti O., Leonetti F., Campos J. M., Gallo M. A., Espinosa A., Carotti A., J. Med. Chem., 54, 2627–2645 (2011).
• 11) Silman I., Sussman J. L., Curr. Opin. Pharmacol., 5, 293–302 (2005).
• 12) Sepsova V., Krusek J., Zdarova-Karasova J., Zemek F., Musilek K., Kuca K., Soukup O., Physiol. Res., 63, 771–777 (2014).
• 13) Akrami H., Mirjalili B. F., Khoobi M., Nadri H., Moradi A., Sakhteman A., Emami S., Foroumadi A., Shafiee A., Eur. J. Med. Chem., 84, 375–381 (2014).
• 14) Alipour M., Khoobi M., Foroumadi A., Nadri H., Moradi A., Sakhteman A., Ghandi M., Shafiee A., Bioorg. Med. Chem., 20, 7214–7222 (2012).
• 15) Mostofi M., Mohammadi Ziarani G., Mahdavi M., Moradi A., Nadri H., Emami S., Alinezhad S., Foroumadi A., Shafiee A., Eur. J. Med. Chem., 103, 361–369 (2015).
• 16) Baharloo F., Moslemin M. H., Nadri H., Asadipour A., Mahdavi M., Emami S., Firoozpour L., Mohebat R., Shafiee A., Foroumadi A., Eur. J. Med. Chem., 93, 196–201 (2015).
• 17) Khalid H., Ur-Rehman A., Abbasi M. A., Hussain R., Mohammad-Khan K., Ashraf M., Ejaz S. A., Fatmi M. Q., Turk. J. Chem., 38, 189–201 (2014).
• 18) Utku S., Gökçe M., Orhan İ., Şahin M. F., Arzneimittelforschung, 61, 1–7 (2011).
• 19) Prinz M., Parlar S., Bayraktar G., Alptuzun V., Erciyas E., Fallarero A., Karlsson D., Vuorela P., Burek M., Forster C., Turunc E., Armagan G., Yalcin A., Schiller C., Leuner K., Krug M., Sotriffer C. A., Holzgrabe U., Eur. J. Pharm. Sci., 49, 603–613 (2013).
• 20) Alptüzün V., Kapková P., Baumann K., Erciyas E., Holzgrabe U., J. Pharm. Pharmacol., 55, 1397–1404 (2003).
• 21) Kapková P., Alptüzün V., Frey P., Erciyas E., Holzgrabe U., Bioorg. Med. Chem., 14, 472–478 (2006).
• 22) Douglas A. W., Fisher M. H., Fishinger J. J., Gund P., Haris E. E., Olson G., Patchett A. A., Ruyle W. V., J. Med. Chem., 20, 939–943 (1977).
• 23) Alptuzun V., Cakiroglu G., Limoncu M. E., Erac B., Hosgor-Limoncu M., Erciyas E., J. Enzyme Inhib. Med. Chem., 28, 960–967 (2013).
• 24) Hesse M., Meier H., Zeeh B., “Spectroscopic Methods in Organic Chemistry,” 2nd ed., ed. by Enders D., Noyori R., Trost B. M., Georg Thieme Verlag Stuttgart, New York, U.S.A., 1997.
• 25) Ellman G. L., Courtney K. D., Andres V. Jr., Feather-Stone R. M., Biochem. Pharmacol., 7, 88–95 (1961).
• 26) Cheung J., Rudolph M. J., Burshteyn F., Cassidy M. S., Gary E. N., Love J., Franklin M. C., Height J. J., J. Med. Chem., 55, 10282–10286 (2012).
• 27) Molecular Operating Environment, (2014.09.1) Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 91, Montreal H3A 2R7, Canada.
• 28) Jones G., Willett P., Glen R. C., Leach A. R., Taylor R., J. Mol. Biol., 267, 727–748 (1997).
• 29) Friesner R. A., Banks J. L., Murphy R. B., Halgren T. A., Klicic J. J., Mainz D. T., Repasky M. P., Knoll E. H., Shelley M., Perry J. K., Shaw D. E., Francis P., Shenkin P. S., J. Med. Chem., 47, 1739–1749 (2004).