Synthesis and pharmacological evaluation of donepezil-based agents as new cholinesterase/monoamine oxidase inhibitors for the potential application against Alzheimer’s disease

Abstract

In a continuing effort to develop multitargeted compounds as potential treatment agents against Alzheimer's disease (AD), a series of donepezil-like compounds were designed, synthesized and evaluated. In vitro studies showed that most of the designed compounds displayed potent inhibitory activities toward AChE, BuChE, MAO-B and MAO-A. Among them, w18 was a promising agent with balanced activities, which exhibited a moderate cholinesterase inhibition (IC₅₀, 0.220 μM for eeAChE; 1.23 μM for eqBuChE; 0.454 μM for hAChE) and an acceptable inhibitory activity against monoamine oxidases (IC₅₀, 3.14 μM for MAO-B; 13.4 μM for MAO-A). Moreover, w18 could also be a metal-chelator, and able to cross the blood–brain barrier with low cell toxicity on PC12 cells. Taken together, these results suggested that w18 might be a promising multitargeted compound for AD treatment.

Keywords:

• Alzheimer’s disease
• donepezil
• cholinesterase
• monoamine oxidase
• molecular modeling

Introduction

Alzheimer’s disease (AD), the most common cause of dementia in elderly people, is a complex and progressive neurodegenerative disorder characterized by memory loss, decline in language ability and other cognitive imparment1,2. Over 100 years, the exact etiology of AD still remain elusive, multiple factors such as β-amyloid deposite, dyshomeostasis of biometal, oxidative and low levels of acetylcholine (ACh) are considered to play significant roles in the pathophysiology of AD3.

Among the multiple factors that induce AD, the cholinergic hypothesis has been proposed to explain the mechanism of AD development4. This hypothesis asserts that dysfunction of cholinergic system, mainly decline of acetylcholine (ACh) level, leads to the memory and cognitive deficits associated with AD, and inhibiting the cholinesterase (ChE) responsible for the hydrolysis of ACh is therefore supposed to be clinically beneficial to patients5,6. Two types of ChEs, acetycholinesterase (AChE) and butyrylcholinesterase (BuChE), existed in the central nervous system of human. Compared to BuChE, AChE is more active and can hydrolyze the major ACh in healthy brains7. However, in the case of AD, BuChE is a major modulator in regulating the ACh level8,9. As AD progresses, the activity of AChE is found to be decreased, and that of BuChE is significantly increased in the hippocampus and temporal cortex10,11. Consequently, both AChE and BuChE are important target, and inhibition of both of them will be more beneficial to the treatment of AD.

In addition, many studies have found that monoamine oxidase (MAO) also play a very important role in the pathogenesis of AD, as the increase of MAO in brain may result in a cascade of biochemical events leading to neuronal dysfunction12,13. MAOs are important FAD-dependent enzymes (flavoenzymes), which have two functional isozymic forms, namely MAO-A and MAO-B, identified by their different substrate and inhibitor specificity14,15. Catecholaminergic neurons predominantly contain MAO-A, while MAO-B is located in serotonergic glia and neurons16,17. The MAOs have been used as drug targets and inhibitors of these enzymes are used to treat neuropsychiatric syndromes18,19. Selective MAO-B inhibitors have been applied to treat the neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases18, while selective MAO-A inhibitors have been revealed to treat depression and anxiety19. Several lines of evidence indicate that, AD patients also commonly present depressive symptom20. Based on these aspects, simultaneous inhibition of both MAO-A and MAO-B, could provide additional benefits in AD therapy.

At present, there are three FDA-approved drugs for AD treatment, these anti-AChE agents include galanthamine, donepezil, and rivastigmine, which can only provide a temporary symptom alleviation instead of preventing or slowing the progressive neurodegeneration21–23. However, the multiple etiologies of AD make single-target strategy difficult to shed good therapeutic effect. Thus, multi-target-directed ligand (MTDL) raises as an effective strategy for the treatment of AD24,25. Attempts to combine anti-AChE and anti-MAO activities in one molecular entity have previously been reported26. For example, N-pyrimidine-4-acetylaniline derivatives possessing AChE and reversible MAO-A inhibitory activity in vitro have been reported, and our group has also reported the synthesis of tacrine–coumarin hybrids as multitargeted agents against AD27–29.

Recently, donepezil, an effective anti-AChE drug for AD treatment, has attracted considerable attention30,31. Replacing the indanone fragment of donepezil with additional bioactive molecules to produce multitargeted inhibitors is a good strategy. For example, donepezil and N-[(5-(benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine hybrids have been designed as multitargeted agents capable of inhibiting ChEs and MAOs32. On the other hand, lazabemide and its analog Ro 16–6491 are reversible MAO inhibitors with remarkably high potency and selectivity for MAO-B33,34. They could serve as adjuvants in the therapy of AD and other degenerative brain disorders35. Moreover, moclobemide, a another reversible and short-acting preferential MAO-A inhibitor, and it has been shown to have antidepressant effects on human36,37. The neurochemical and pharmacological characteristics of these carboxamide derivatives lazabemide, Ro16–6491 and moclobemide have been studied as to effect monoamine levels in human brain33–37.

Given the activities of them, and in an attempt to obtain new multi-targeted molecules with both ChEs and MAOs inhibitory activity for the treatment of AD38,39, a series of novel compounds have been designed and synthesized. The strategy is to retain the 1-benzylpiperidine fragment from donepezil with ChEs inhibition and introduce the benzamide or 2-picolinamide moiety from lazabemide, Ro16–6491 and moclobemide with MAOs inhibitory activity (Figure 1). Besides, we also introduces a 2-thiophenecarboxamide moiety for its structural similarity to benzamide. Since the length of the linker could affect the accommodation of the hybrid in AChE40, we changed the length of carbon spacer to obtain optional conformation that could make the activity of designed compounds better. Although a part of donepezil-like compounds have been known, their biological activities such as the inhibition of ChEs and MAOs have not been determined41–43. Meanwhile, the amide of 2-picolinamide moiety also has the ability to chelate metal ions44,45. In this study, we described the design, synthesis, and evaluation of series of donepezil-like compounds which were found to show potential abilities, including the inhibition of ChEs, inhibition of MAOs, metal chelation and penetration of the blood–brain barrier (BBB). The structure–activity relationships were discussed based on the pharmacological activities. Moreover, to further investigate the interaction mechanism with ChEs and MAOs, kinetic analysis and molecular modeling studies were also performed.

Figure 1. Design strategy for donepezil-like compounds.

Materials and methods

Materials

All common reagents and solvents were obtained from commercial suppliers and used without purification. Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qing-Dao, China), and the spots were detected under UV light (254 nm). Melting points were determined on an XT-4 micromelting point instrument and uncorrected. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). Column chromatography was performed on silica gel (90–150 μm; Qingdao Marine Chemical Inc.). ¹H NMR and ¹³C NMR spectra were measured on a Bruker ACF-500 spectrometer (Bruker, Karlsruhe, Germany) at 25 °C and referenced to TMS. Chemical shifts are reported in ppm (δ) using the residual solvent line as internal standard. Mass spectra were obtained on a MS Agilent 1100 (Agilent Technologies, Santa Clara, CA) Series LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectrometer (HRESIMS), respectively.

General procedures for the preparation of compounds w1–23

A solution of 1a–u (1.0 mmol) and 1, 0-carbonyldiimidazole (1.2 mmol) in 10 mL of anhydrous CH₂Cl₂ was stirred at room temperature for 1 h. The 1-benzylpiperidin-4-amine or 2-(1-benzylpiperidin-4-yl) ethanamine (1.0 mmol) was added to the solution, and stirring was continued overnight. The reaction mixture was diluted with H₂O and extracted with CH₂Cl₂. The organic extracts were combined, washed with brine, and dried with anhydrous Na₂SO₄, and the solvent was evaporated in vacuo to give the crude product, which was purified by silica gel chromatography with CH₂Cl₂:MeOH = 30:1 as an eluent to afford corresponding target compounds.

N-(1-benzylpiperidin-4-yl)benzamide (w1)

Yield 68%, white solid, m.p. 171–173 °C; IR (KBr) ν 3303, 2916, 2792, 1632, 1548, 1381, 1341, 743, 701 cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.24 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 7.2 Hz, 2H), 7.53 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 7.4 Hz, 2H), 7.37–7.30 (m, 4H), 7.29–7.25 (m, 1H), 3.85–3.71 (m, 1H), 3.48 (s, 2H), 2.84 (d, J = 11.0 Hz, 2H), 2.04 (t, J = 11.0 Hz, 2H), 1.79 (d, J = 10.6 Hz, 2H), 1.60 (qd, J = 12.1, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 166.21, 138.96, 135.22, 131.48, 129.26, 128.63, 127.72, 127.36, 62.58, 52.70, 47.39, 31.89. ESI-MS m/z: 295.12 [M + H]⁺; HRMS: calcd for C₁₉H₂₂N₂O [M + H]⁺295.1805, found 295.1803.

N-(1-benzylpiperidin-4-yl)-4-chlorobenzamide (w2)

Yield 73%, pale white solid, m.p. 192–194 °C; IR (KBr) ν 3340, 2924, 1631, 1544, 1453, 1070, 803, 757, 706  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.32 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.38–7.30 (m, 4H), 7.27 (t, J = 6.8 Hz, 1H), 3.83–3.70 (m, 1H), 3.49 (s, 2H), 2.84 (d, J = 11.5 Hz, 2H), 2.03 (t, J = 14.4 Hz, 2H), 1.79 (d, J = 11.1 Hz, 2H), 1.59 (qd, J = 12.1, 3.2 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 165.03, 139.13, 136.28, 133.97, 129.69, 129.19, 128.69, 128.62, 127.31, 62.60, 52.69, 47.54, 31.93. ESI-MS m/z: 329.13 [M + H]⁺; HRMS: calcd for C₁₉H₂₁ClN₂O [M + H]⁺329.1415, found 329.1416.

N-(1-benzylpiperidin-4-yl)-5-chloropicolinamide (w3)

Yield 65%, yellow solid, m.p. 74–76 °C; IR (KBr) ν 3315, 2945, 2789, 1656, 1527, 1469, 1079, 771, 707, 652  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.70 (d, J = 2.2 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.13 (dd, J = 8.4, 2.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.39–7.29 (m, 4H), 7.26 (t, J = 6.8 Hz, 1H), 3.89–3.70 (m, 1H), 3.48 (s, 2H), 2.81 (d, J = 11.6 Hz, 2H), 2.07 (t, J = 11.0 Hz, 2H), 1.77 (d, J = 10.9 Hz, 2H), 1.66 (qd, J = 12.1, 3.5 Hz, 2H). 13C NMR (125 MHz, DMSO) δ 162.95, 148.94, 147.47, 138.04, 134.36, 129.96, 129.64, 128.72, 127.67, 123.89, 62.26, 52.36, 46.91, 31.22. ESI-MS m/z: 330.11 [M + H]⁺; HRMS: calcd for C₁₈H₂₁ClN₃O [M + H]⁺330.1368, found 330.1365.

N-(1-benzylpiperidin-4-yl)nicotinamide (w4)

Yield 75%, yellow solid, m.p. 133–135 °C; IR (KBr) ν 3294, 2917, 2793, 1632, 1551, 1063, 742, 701, 660  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 9.01 (s, 1H), 8.71 (d, J = 3.6 Hz, 1H), 8.46 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.50 (dd, J = 7.9, 4.8 Hz, 1H), 7.37–7.30 (m, 5H), 7.27 (t, J = 6.4 Hz, 1H), 3.81 (qd, J = 11.7, 5.7 Hz, 1H), 3.49 (s, 2H), 2.84 (d, J = 11.7 Hz, 2H), 2.05 (t, J = 10.9 Hz, 2H), 1.82 (d, J = 10.9 Hz, 2H), 1.60 (qd, J = 12.1, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 164.78, 152.16, 148.84, 139.01, 135.52, 130.67, 129.26, 128.64, 127.36, 123.86, 62.58, 52.60, 47.53, 31.84. ESI-MS m/z: 296.10 [M + H]⁺; HRMS: calcd for C₁₈H₂₂N₃O [M + H]⁺296.1757, found 296.1755.

N-(1-benzylpiperidin-4-yl)-5-chlorothiophene-2-carboxamide (w5)

Yield 78%, pale white, solid m.p. 174–176 °C; IR (KBr) ν 3299, 2924, 1613, 1554, 1454, 742, 701  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.34 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.37–7.30 (m, 4H), 7.26 (t, J = 6.8 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 3.78–3.66 (m, 1H), 3.48 (s, 2H), 2.83 (d, J = 11.4 Hz, 2H), 2.03 (t, J = 11.4 Hz, 2H), 1.79 (d, J = 10.9 Hz, 2H), 1.57 (qd, J = 12.1, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 160.12, 139.54, 138.65, 133.43, 129.38, 128.64, 128.43, 128.36, 127.42, 62.50, 52.49, 47.57, 31.70. ESI-MS m/z: 335.07 [M + H]⁺; HRMS: calcd for C₁₇H₂₀ClN₂OS [M + H]⁺335.09, found 335.0977.

N-(2-(1-benzylpiperidin-4-yl)ethyl)benzamide (w6)

Yield 58%, yellow solid, m.p. 94–96 °C; IR (KBr) ν 3314, 2924, 1631, 1535, 1490, 1435, 802, 774, 695  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.42 (t, J = 5.3 Hz, 1H), 7.85 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.47 (t, J = 7.3 Hz, 2H), 7.36–7.27 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 3.44 (s, 2H), 3.30 (dd, J = 13.3, 6.9 Hz, 2H), 2.79 (d, J = 11.3 Hz, 2H), 1.90 (t, J = 11.0 Hz, 2H), 1.68 (d, J = 12.0 Hz, 2H), 1.48 (d, J = 7.2 Hz, 2H), 1.33–1.28 (m, 1H), 1.17 (qd, J = 12.3, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 166.56, 139.17, 135.26, 131.40, 129.21, 128.67, 128.55, 127.58, 127.22, 63.02, 53.75, 37.40, 36.39, 33.52, 32.38. ESI-MS m/z: 323.19 [M + H]⁺; HRMS: calcd for C₂₁H₂₇N₂O [M + H]⁺323.2118, found 323.2116.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-2-chlorobenzamide (w7)

Yield 70%, white solid, m.p. 145–147 °C; IR (KBr) ν 3276, 2918, 1640, 1553, 1452, 763, 733, 709  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.37 (t, J = 5.2 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.46–7.42 (m, 1H), 7.39 (d, J = 4.2 Hz, 2H), 7.35–7.29 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 3.45 (s, 2H), 3.27 (dd, J = 13.0, 6.9 Hz, 2H), 2.80 (d, J = 11.3 Hz, 2H), 1.90 (t, J = 10.9 Hz, 2H), 1.67 (d, J = 12.0 Hz, 2H), 1.46 (q, J = 6.9 Hz, 2H), 1.42–1.34 (m, 1H), 1.17 (qd, J = 12.3, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 166.65, 139.10, 137.78, 131.02, 130.27, 130.00, 129.26, 129.19, 128.56, 127.54, 127.25, 63.01, 53.78, 37.06, 36.04, 33.26, 32.27. ESI-MS m/z: 357.16 [M + H]⁺; HRMS: calcd for C₂₁H₂₆ClN₂O [M + H]⁺357.1728, found 357.1727.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-2-(2-bromophenyl)acetamide (w8)

Yield 58%, yellow solid, m.p(0).83–85 °C; IR (KBr) ν 3124, 2921, 1646, 1546, 1448, 1057, 755, 660  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 7.98 (t, J = 5.1 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.37–7.28 (m, 6H), 7.26 (t, J = 6.9 Hz, 1H), 7.22–7.17 (m, 1H), 3.58 (s, 2H), 3.46 (s, 2H), 3.12 (dd, J = 12.7, 6.8 Hz, 2H), 2.80 (d, J = 11.0 Hz, 2H), 1.91 (t, J = 11.0 Hz, 2H), 1.63 (d, J = 12.2 Hz, 2H), 1.37 (dd, J = 13.6, 6.8 Hz, 2H), 1.32–1.29 (m, 1H), 1.14 (qd, J = 12.4 Hz, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 169.30, 138.53, 135.69, 132.71, 132.32, 129.44, 129.08, 128.57, 128.02, 127.37, 122.17, 62.87, 53.59, 42.88, 36.79, 36.05, 33.05, 31.99. ESI-MS m/z: 415.08 [M + H]⁺; HRMS: calcd for C₂₂H₂₇BrN₂O [M + H]⁺415.1380, found 415.1378.

2-benzoyl-N-(2-(1-benzylpiperidin-4-yl)ethyl)benzamide (w9)

Yield 65%, white solid, m.p. 144–146 °C; IR (KBr) ν 3294, 2963, 1678, 1261, 800, 698 cm⁻¹; 1H NMR (500 MHz, DMSO) δ 7.72 (d, J = 7.0 Hz, 1H), 7.59–7.49 (m, 2H), 7.39–7.29 (m, 7H), 7.29–7.21 (m, 4H), 3.41 (s, 2H), 3.40–3.35 (m, 2H), 2.71 (d, J = 2.4 Hz, 2H), 1.84 (dd, J = 19.7, 9.2 Hz, 2H), 1.60 (d, J = 12.4 Hz, 1H), 1.48 (d, J = 12.4 Hz, 1H), 1.44–1.32 (m, 1H), 1.32–1.21 (m, 1H), 1.15 (dd, J = 6.9, 3.6 Hz, 1H), 1.00 (qd, J = 11.7, 3.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 167.11, 150.09, 140.72, 138.85, 132.84, 131.15, 129.61, 129.29, 128.87, 128.56, 128.51, 127.29, 126.32, 123.16, 122.80, 62.88, 53.52, 37.08, 35.26, 33.44, 32.16. ESI-MS m/z: 427.20 [M + H]⁺; HRMS: calcd for C₂₈H₃₁N₂O₂ [M + H]⁺427.2380, found 427.2379.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-3-nitrobenzamide (w10)

Yield 75%, yellow solid, m.p. 125–127 °C; IR (KBr) ν 3353, 2923, 1638, 1530, 1350, 821, 724  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.81 (t, J = 5.3 Hz, 1H), 8.68 (s, 1H), 8.39 (dd, J = 8.0, 1.7 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.36–7.27 (m, 4H), 7.25 (t, J = 7.0 Hz, 1H), 3.45 (s, 2H), 3.35 (dd, J = 11.8, 4.5 Hz, 2H), 2.80 (d, J = 11.0 Hz, 2H), 1.91 (t, J = 11.0 Hz, 2H), 1.67 (t, J = 15.3 Hz, 2H), 1.51 (dd, J = 14.2, 7.0 Hz, 2H), 1.36–1.32 (m, 1H), 1.19 (qd, J =12.2, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 164.40, 148.28, 139.53, 136.53, 134.05, 130.54 (s), 129.22, 128.55, 127.24, 126.14, 122.32, 62.97, 53.70, 37.65, 36.17, 33.44, 32.31. ESI-MS m/z: 368.19 [M + H]⁺; HRMS: calcd for C₂₁H₂₆N₃O₃ [M + H]⁺368.1969, found 368.1970.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-nitrobenzamide (w11)

Yield 73%, yellow solid, m.p. 113–115 °C; IR (KBr) ν 3330, 2928, 1644, 1597, 1542, 1518, 741, 726, 698  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.76 (t, J = 5.2 Hz, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H), 7.35–7.27 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 3.45 (s, 2H), 3.34 (d, J = 11.5 Hz, 2H), 2.80 (d, J = 11.0 Hz, 2H), 1.91 (t, J = 11.0 Hz, 2H), 1.68 (d, J = 12.0 Hz, 2H), 1.50 (dd, J = 14.1, 7.0 Hz, 2H), 1.34–1.31 (m, 1H), 1.18 (qd, J = 12.2, 3.5 Hz, 2H). ¹³C NMR (126 MHz, DMSO) δ 164.99, 149.40, 140.81, 139.06, 129.24, 129.09, 128.55, 127.24, 123.95, 62.98, 53.69, 37.65, 36.13, 33.46, 32.28. ESI-MS m/z: 368.18 [M + H]⁺; HRMS: calcd for C₂₁H₂₆N₃O₃ [M + H]⁺368.1969, found 368.1968.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-methoxybenzamide (w12)

Yield 75%, yellow solid, m.p. 125–127 °C; IR (KBr) ν 3293, 2918, 1627, 1558, 1509, 1255, 843, 736, 673  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.26 (t, J = 5.3 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.35–7.28 (m, 5H), 7.25 (t, J = 6.9 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.44 (s, 2H), 3.28 (dd, J = 13.4, 6.6 Hz, 2H), 2.79 (d, J = 11.3 Hz, 2H), 1.90 (t, J = 10.9 Hz, 2H), 1.68 (d, J = 11.7 Hz, 2H), 1.47 (dd, J = 14.1, 6.9 Hz, 2H), 1.32–1.28 (m, 1H), 1.17 (qd, J = 12.3, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 166.13, 161.90, 139.04, 129.38, 129.27, 128.56, 127.43, 127.26, 113.91, 62.98, 55.80, 53.72, 37.33, 36.43, 33.49, 32.31. ESI-MS m/z: 353.20 [M + H]⁺; HRMS: calcd for C₂₂H₂₉N₂O [M + H]⁺353.2224, found 353.2222.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-methylbenzamide (w13)

Yield 77%, yellow solid m.p. 85–92 °C; IR (KBr) ν 3321, 2925, 1633, 1550, 1453, 1311, 730, 697  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.31 (t, J = 5.4 Hz, 1H), 7.75 (d, J = 8.1 Hz, 2H), 7.35–7.29 (m, 4H), 7.25–7.23 (m, 3H), 3.45 (s, 2H), 3.29 (dd, J = 13.4, 6.9 Hz, 2H), 2.79 (d, J = 11.0 Hz, 2H), 2.36 (s, 3H), 1.92 (t, J = 11.0 Hz, 2H), 1.68 (d, J = 11.9 Hz, 2H), 1.47 (dd, J = 14.2, 6.9 Hz, 2H), 1.33–1.29 (m, 1H), 1.17 (qd, J = 12.2, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 166.40, 141.21, 139.07, 132.44, 129.23, 129.18, 128.55, 127.59, 127.25, 62.95, 53.72, 37.32, 36.40, 33.49, 32.33, 21.38. ESI-MS m/z: 337.21 [M + H]⁺; HRMS: calcd for C₂₂H₂₉N₂O [M + H]⁺337.2274, found 337.2273.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-chlorobenzamide (w14)

Yield 88%, yellow solid, m.p. 122–124 °C; IR (KBr) ν 3305, 2914, 1632, 1543, 845, 743, 701  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.49 (t, J = 5.3 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.37–7.27 (m, 4H), 7.25 (t, J = 6.7 Hz, 1H), 3.44 (s, 2H), 3.30 (dd, J = 13.4, 6.7 Hz, 2H), 2.79 (d, J = 11.0 Hz, 2H), 1.91 (t, J = 11.0 Hz, 2H), 1.68 (d, J = 11.9 Hz, 2H), 1.48 (dd, J = 14.1, 7.0 Hz, 2H), 1.32–1.29 (m, 1H), 1.17 (qd, J = 12.2, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 165.59, 136.32, 135.07, 133.89, 129.53, 129.41, 128.80, 128.61, 127.43, 62.77, 53.59, 37.44, 36.19, 33.31, 32.08. ESI-MS m/z: 357.16 [M + H]⁺; HRMS: calcd for C₂₁H₂₆ClN₂O [M + H]⁺357.1728, found 357.1727.

N-(2-(1-benzylpiperidin-4-yl)ethyl)quinoline-2-carboxamide (w15)

Yield 85%, yellow solid, m.p. 95–97 °C; IR (KBr) ν 3382, 2938, 1667, 1522, 1496, 781, 737, 697  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.90 (t, J = 5.9 Hz, 1H), 8.57 (d, J = 8.5 Hz, 1H), 8.18–8.14 (m, 2H), 8.10 (d, J = 8.0 Hz, 1H), 7.89 (dd, J = 11.2, 4.1 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.35–7.27 (m, 4H), 7.25 (t, J = 6.8 Hz, 1H), 3.42 (dd, J = 15.6, 8.2 Hz, 4H), 2.80 (d, J = 11.0 Hz, 2H), 1.91 (t, J = 11.0 Hz, 2H), 1.72 (d, J = 12.1 Hz, 2H), 1.55 (dd, J = 14.2, 7.0 Hz, 2H), 1.37–1.29 (m, 1H), 1.17 (qd, J = 12.1, 3.3 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 164.40, 150.78, 146.48, 138.28, 130.94, 129.94, 129.65, 129.33, 129.23, 128.58, 128.53, 128.46, 127.35, 119.09, 62.85, 53.62, 37.20, 36.45, 33.42, 32.18. ESI-MS m/z: 374.21 [M + H]⁺; HRMS: calcd for C₂₄H₂₈N₃O [M + H]⁺374.2227, found 374.2226.

N-(2-(1-benzylpiperidin-4-yl)ethyl)nicotinamide (w16)

Yield 65%, yellow solid, m.p. 64–66 °C; IR (KBr) ν 3306, 2925, 1636, 1548, 1311, 731, 707, 663  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 9.00 (s, 1H), 8.71 (dd, J = 4.8, 1.4 Hz, 1H), 8.61 (t, J = 5.2 Hz, 1H), 8.18 (dt, J = 7.9, 1.8 Hz, 1H), 7.51 (dd, J = 7.9, 4.8 Hz, 1H), 7.35–7.28 (m, 5H), 7.25 (t, J = 6.9 Hz, 1H), 3.44 (s, 2H), 3.31 (d, J = 6.9 Hz, 2H), 2.79 (d, J = 11.9 Hz, 2H), 1.91 (t, J = 10.8 Hz, 2H), 1.68 (d, J = 11.9 Hz, 2H), 1.49 (dd, J = 14.2, 7.0 Hz, 2H), 1.35–1.30 (m, 1H), 1.18 (qd, J = 12.2, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 165.14, 152.13, 148.73, 135.34, 129.28, 128.56, 127.29, 123.89, 62.92, 53.67, 37.42, 36.20, 33.42, 32.24. ESI-MS m/z: 324.18 [M + H]⁺; HRMS: calcd for C₂₀H₂₆N₃O [M + H]⁺324.2070, found 324.2068.

N-(2-(1-benzylpiperidin-4-yl)ethyl)isonicotinamide (w17)

Yield 78%, yellow oil; IR (KBr) ν 3294, 2925, 1648, 1551, 1451, 1309, 1065, 742, 701, 660  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.73 (d, J = 5.8 Hz, 1H), 7.75 (d, J = 5.8 Hz, 1H), 7.35–7.27 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 7.03 (s, 1H), 3.44 (s, 2H), 3.33–3.29 (m, 2H), 2.79 (d, J = 11.3 Hz, 2H), 1.96–1.86 (m, 2H), 1.66 (t, J = 14.0 Hz, 1H), 1.49 (dd, J = 14.0, 6.9 Hz, 1H), 1.34–1.28 (m, 1H), 1.18 (qd, J = 12.1, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 165.04, 150.63, 142.07, 138.88, 129.29, 128.57, 127.30, 121.65, 62.91, 53.64, 37.50, 36.09, 33.40, 32.20. ESI-MS m/z: 324.18 [M + H]⁺; HRMS: calcd for C₂₀H₂₆N₃O [M + H]⁺324.2070, found 324.2071.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-chloropicolinamide (w18)

Yield 66%, yellow solid, m.p. 65–69 °C; IR (KBr) ν 3415, 3301, 2930, 2855, 1663, 1533, 1455, 1110, 790, 732, 684  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.80 (t, J = 5.8 Hz, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.13 (dd, J = 8.4, 2.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.38–7.28 (m, 4H), 7.26 (t, J = 6.9 Hz, 1H), 3.47 (s, 2H), 3.34–3.30 (m, 2H), 2.80 (d, J = 8.7 Hz, 2H), 1.92 (s, 2H), 1.69 (d, J = 12.3 Hz, 2H), 1.49 (dd, J = 14.0, 6.9 Hz, 2H), 1.33–1.28 (m, 1H), 1.17 (qd, J = 12.3, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 163.38, 149.15, 147.47, 138.84, 138.84, 137.98, 134.24, 129.30, 128.56, 127.30, 123.80, 62.89, 53.64, 37.10, 36.29, 33.36, 32.18. ESI-MS m/z: 358.15 [M + H]⁺; HRMS: calcd for C₂₀H₂₅ClN₃O [M + H]⁺358.1681, found 358.1680.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-fluoropicolinamide (w19)

Yield 55%, yellow solid, m.p. 81–83 °C; IR (KBr) ν 3306, 2928, 1661, 1530, 1469, 1227, 732, 682  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.71 (t, J = 5.7 Hz, 1H), 8.64 (d, J = 2.7 Hz, 1H), 8.11 (dd, J = 8.7, 4.7 Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.34–7.28 (m, 4H), 7.24 (t, J = 6.9 Hz, 1H), 3.44 (s, 2H), 3.38–3.33 (m, 2H), 2.78 (d, J = 11.2 Hz, 2H), 1.89 (t, J = 11.2 Hz, 2H), 1.68 (d, J = 11.2 Hz, 2H), 1.49 (dd, J = 14.0, 6.9 Hz, 2H), 1.31–1.26 (m, 1H), 1.16 (qd, J = 12.2, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 163.27, 160.04, 147.37, 139.03, 137.15, 129.24, 128.54, 127.24, 124.85, 124.43, 62.96, 53.68, 37.08, 36.36, 33.42, 32.26. ESI-MS m/z: 342.18 [M + H]⁺; HRMS: calcd for C₂₀H₂₅FN₃O [M + H]⁺342.1976, found 342.1974.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-6-methylpicolinamide (w20)

Yield 65%, yellow oil; IR (KBr) ν 3389, 2923, 1673, 1593, 1526, 1452, 740, 699  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.58 (t, J = 5.8 Hz, 1H), 7.90–7.80 (m, 2H), 7.45 (d, J = 7.3 Hz, 1H), 7.36–7.27 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 3.43 (s, 2H), 3.35 (d, J = 13.7 Hz, 2H), 2.78 (t, J = 11.3 Hz, 2H), 2.57 (s, 3H), 1.90 (t, J = 11.3 Hz, 2H), 1.69 (d, J = 11.9 Hz, 2H), 1.49 (dd, J = 14.2, 6.9 Hz, 2H), 1.31–1.23 (m, 1H), 1.17 (qd, J = 12.1, 3.3 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 164.36, 157.57, 149.84, 138.32, 129.34, 128.58, 127.31, 126.37, 119.36, 62.93, 53.64, 36.97, 36.45, 33.42, 32.16, 24.32. ESI-MS m/z: 338.22 [M + H]⁺; HRMS: calcd for C₂₁H₂₈N₃O [M + H]⁺338.2227, found 338.2226.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-chlorothiophene-2-carboxamide (w21)

Yield 80%, yellow solid, m.p. 115–117 °C; IR (KBr) ν 3299, 2929, 1661, 1531, 1453, 1108, 730, 682  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.53 (t, J = 5.4 Hz, 1H), 7.63 (d, J = 4.0 Hz, 1H), 7.35–7.28 (m, 4H), 7.25 (dd, J = 9.3, 4.4 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 3.47 (s, 2H), 3.26 (dd, J = 13.4, 6.7 Hz, 2H), 2.80 (d, J = 11.1 Hz, 2H), 1.93 (t, J = 11.1 Hz, 2H), 1.67 (d, J = 12.1 Hz, 2H), 1.46 (dd, J = 14.2, 7.0 Hz, 2H), 1.31–1.29 (m, 1H), 1.17 (qd, J = 12.3, 3.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 160.40, 139.90, 133.07, 129.27, 128.57, 128.43, 128.08, 127.31, 62.87, 53.64, 37.30, 36.28, 33.36, 32.21. ESI-MS m/z: 362.12 [M + H]⁺; HRMS: calcd for C₁₉H₂₄ClN₂OS [M + H]⁺363.1292, found 363.1293.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-bromothiophene-2-carboxamide (w22)

Yield 59%, yellow solid, m.p. 124–126 °C; IR (KBr) ν 3279, 2924, 1617, 1564, 1421, 1304, 736, 694  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.50 (t, J = 5.4 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.35–7.28 (m, 4H), 7.28 (d, J = 4.0 Hz, 1H), 7.25 (t, J = 6.9 Hz, 1H), 3.44 (s, 2H), 3.25 (dd, J = 13.5, 6.6 Hz, 2H), 2.79 (d, J = 10.9 Hz, 2H), 1.90 (t, J = 10.9 Hz, 2H), 1.66 (d, J = 11.7 Hz, 2H), 1.46 (dd, J = 14.2, 6.9 Hz, 2H), 1.36–1.29 (m, 1H), 1.17 (qd, J =12.3, 3.3 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 160.40, 142.53, 139.03, 131.86, 129.24, 128.91, 128.55, 127.25, 116.81, 62.96, 53.67, 37.30, 36.27, 33.41, 32.26. ESI-MS m/z: 407.07 [M + H]⁺; HRMS: calcd for C₁₉H₂₄BrN₂OS [M + H]⁺407.0787, found 407.0786.

N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-methylthiophene-2-carboxamide (w23)

Yield 66%, yellow solid, m.p. 107–110 °C; IR (KBr) ν 3281, 2923, 1616, 1562, 1449, 1308, 736, 694  cm⁻¹; ¹H NMR (500 MHz, DMSO) δ 8.27 (t, J = 5.4 Hz, 1H), 7.53 (d, J = 3.5 Hz, 1H), 7.37–7.28 (m, 4H), 7.25 (t, J = 6.9 Hz, 1H), 6.83 (d, J = 3.5 Hz, 1H), 3.45 (s, 2H), 3.24 (dd, J = 13.4, 6.6 Hz, 2H), 2.79 (d, J = 11.0 Hz, 2H), 2.46 (s, 3H), 1.91 (t, J = 11.0 Hz, 2H), 1.67 (d, J = 11.9 Hz, 2H), 1.45 (dd, J = 14.2, 6.9 Hz, 2H), 1.33–1.28 (m, 1H), 1.17 (qd, J = 12.2, 3.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 161.42, 144.60, 139.07, 138.21, 129.22, 128.55, 128.36, 127.24, 126.68, 62.96, 53.70, 37.19, 36.46, 33.44, 32.32, 15.56. ESI-MS m/z: 343.17 [M + H]⁺; HRMS: calcd for C₂₀H₂₇N₂OS [M + H]⁺343.1839, found 343.1838.

Biological activity

Inhibitory activity against AChE and BuChE

Acetylcholinesterase (eeAChE, E.C. 3.1.1.7, from electric eel and hAChE, EC 3.1.1.7, from human erythrocyes), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), S-butyrylthiocholine iodide (BTCI), acetylthiocholine iodide (ATCI), and donepezil hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). The capacity of the test compounds (w1–23) to inhibit AChE and BuChE activities were assessed by Ellman’s method. Stock solution of test compounds was dissolved in a minimum volume of DMSO (1%) and was diluted using the buffer solution (50 mM Tris-HCl, pH= 8.0, 0.1 M NaCl, 0.02 M MgCl₂·6H₂O). In 96-well plates, 160 μL of 1.5 mM DTNB, 50 μL of AChE (0.22 U/mL prepared in 50 mM Tris-HCl, pH =8.0, 0.1% w/v bovine serum albumin, BSA) or 50 μL of BuChE (0.12 U/mL prepared in 50 mM Tris-HCl, pH = 8.0, 0.1% w/v BSA) were incubated with 10 μL of various concentrations of test compounds (0.001–100 μM) at 37 °C for 6 min followed by the addition of the substrates (30 μL) acetylthiocholine iodide (15 mM) or S-butyrylthiocholine iodide (15 mM) and the absorbance was measured at different time intervals (0, 60, 120, and 180 s) at a wavelength of 405 nm. The concentration of compound producing 50% of enzyme activity inhibition (IC₅₀) was calculated by nonlinear regression analysis of the response-concentration (log) curve, using the Graph-Pad Prism program package (Graph Pad Software, San Diego, CA). Results are expressed as the mean ± SD of at least three different experiments performed in triplicate.

Inhibitory activity against hMAO-A and hMAO-B

Monoamine Oxidases (hMAO-A, hMAO-B, E.C. 1.4.3.4), p-tyramine, Amplex Red and horseradish peroxidase (E.C. 1.11.1.7) were purchased from Sigma-Aldrich. Firstly, MAOs activity were adjusted to obtain in our experimental conditions the same reaction velocity in the presence of both isoforms (i.e., to oxidize (in the control group) the same concentration of substrate: 165 pmol of p-tyramine/min(hMAO-A: 1.1 μg protein; specific activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 μg protein; specific activity: 22 nmol of p-tyramine transformed/min/mg protein). Then compounds were dissolved in DMSO (10 mM) and diluted in 0.05 M KH₂PO₄/K₂HPO₄ buffer (pH = 7.4) to the desired final concentration. All the compounds are soluble at the tested concentration. Test drugs (20 μL) and MAO (80 μL) were incubated at 37 °C for 15 min in a flat-black-bottom 96-well microtest plate in dark. The reaction was started by adding 200 μM Amplex Red reagent, 2 U/mL horseradish peroxidase, and 2 mM p-tyramine for hMAO at 37 °C for 20 min. The production of H₂O₂ and consequently, of resorufin, was quantified at 37 °C in a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA) multi-mode detection platform reader based on the fluorescence generated (excitation, 545 nm; emission, 590 nm). The specific fluorescence emission was calculated after subtraction of the background activity. The background activity was determined from wells containing all components except the MAO isoforms, which were replaced by a sodium phosphate buffer solution (0.05 mM, pH 7.4). The percent inhibition was calculated by the following expression: (1 − IF_i/IF_c) × 100 in which IF_i and IF_c are the fluorescence intensities obtained for hMAO in the presence and absence of inhibitors after subtracting the respective background.

Kinetic study of AChE inbition

To obtain of the mechanism of action w18, reciprocal plots of 1/velocity versus 1/substrate were constructed at different concentrations of the substrate thiocholine iodide 0.05–0.5 mM by using Ellman’s method. Three concentrations of w18 were selected for the studies: 0.440, 0.220 and 0.110 μM for the kinetic analysis of AChE inhibition. The plots were assessed by a weighted least-squares analysis that assumed the variance of velocity (v) to be a constant percentage of v for the entire data set. Slopes of these reciprocal plots were then plotted against the concentration of w18 in a weighted analysis and K_i was determined as the intercept on the negative x-axis. Data analysis was performed with GraphPad Prism 4.03 software (GraphPad Software Inc., San Diego, CA).

Reversibility and kinetic studies of hMAO-B inhibition

To determine whether the inhibition of hMAO-B by the donepezil-like compounds were reversible or irreversible, the time-dependence of inhibition of the selected inhibitor w18 and reference compound pargyline were examined. Compounds were allowed to pre-incubate with recombinant human hMAO-B for various periods of time (0, 15, 30, 60 min) at 37 °C in potassium phosphate buffer (0.05 mM, pH 7.4). The concentrations of the compounds were about twofold the measured IC₅₀ values for the inhibition of hMAO-B. The reactions were subsequently diluted two-fold to yield a final enzyme concentration of 0.015 mg mL⁻¹ and concentrations of the inhibitors that are about equal to the IC₅₀ values. The reactions were incubated at 37 °C for a further 15 min. All measurements were carried out in triplicate and are expressed as mean ± SD.

Then, the type of hMAO-B inhibition was determined by constructing a set of Lineweaver–Burk plots. Six different concentrations of the substrate p-tyramine (0.05, 0.1, 0.25, 0.33, 0.5, and 1.0 mM) was applied, and the initial catalytic rates of hMAO-B were measured in the absence and in the presence of three different concentrations (6.28, 3.14, and 1.57 μM) of compound w18. The assay conditions and measurements were similar to the IC₅₀ determination. The plots were assessed by a weighted leastsquares analysis that assumed the variance of velocity (v) to be a constant percentage of v for the entire data set. Slopes of these reciprocal plots were then plotted against the concentration of w18 in a weighted analysis. Data analysis was performed with GraphPad Prism 4.03 software (San Diego, CA).

Molecular modeling studies of w18 with ChEs and hMAO-B

Molecular modeling calculations and docking studies were performed using Molecular Operating Environment (MOE) software version 2008.10 (Chemical Computing Group, Montreal, Canada). The X-ray crystallographic structure of AChE in complexed with donepezil (PDB code 1EVE), hBuChE (PDB code 1P0I) and human MAO-B in complexed with 7-(3-chlorobenzyloxy)-4-formylcoumarin (PDB code 2V60) were obtained from the Protein Data Bank. All water molecules in PDB files were removed and hydrogen atoms were subsequently added to the protein. The compound w18 was built using the builder interface of the MOE program and energy minimized using MMFF94x forcefield. Then the w18 was docked into the active site of the protein by the “Triangle Matcher” method, which generated poses by aligning the ligand triplet of atoms with the triplet of alpha spheres in cavities of tight atomic packing. The Dock scoring in MOE software was done using ASE scoring function and forcefield was selected as the refinement method. The best 10 poses of molecules were retained and scored. After docking, the geometry of resulting complex was studied using the MOE’s pose viewer utility.

Spectrophotometric measurement of complex with Cu²⁺

The study of metal chelation was performed in methanol at 298 K using UV–Vis spectrophotometer (SHIMADZU UV-2450PC) with wavelength ranging from 200 to 500 nm. The difference UV–Vis spectra due to complex formation was obtained by numerical subtraction of the spectra of the metal ions alone and the compound alone (at the same concentration used in the mixture) from the spectra of the mixture. A fixed amount of w18 (50 μM) was mixed with growing amounts of metal ions (10–80 μM).

In vitro BBB permeation assay

Brain penetration of compounds was evaluated using a parallel artificial membrane permeation assay (PAMPA). Commercial drugs were purchased from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and the acceptor microplate were both from Millipore (St. Charles, MO). The 96-well UV plate (COSTAR^@) was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 μL of PBS:EtOH (7:3), and the filter membrane was impregnated with 4 μL of PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to achieve a concentration of 100 mg/mL, 200 μL of which was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 16 h at 25 °C. After incubation, the donor plate was carefully removed and the concentration of compound in the acceptor wells was determined using a UV plate reader (Flexsta-tion^@ 3). Every sample was analyzed at five wavelengths, in four wells, in at least three independent runs, and the results are given as the mean ± standard deviation. In each experiment, 9 quality control standards of known BBB permeability were included to validate the analysis set.

Rat pheochromocytoma (PC12) cell toxicity

The toxicity effect of compounds on the rat pheochromocytoma (PC12) cells was examined. The PC12 cells were routinely grown at 37 °C in a humidified incubator with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, 100 units/mL penicillin, and 100 units/mL of streptomycin. Cells were subcultured in 96-well plates at a seeding density of 10 000 cells per well and allowed to adhere and grow. When cells reached the required confluence, they were placed into serum-free medium and treated with compound w18. Twenty-four hours later the survival of cells was determined by MTT assay. Briefly, after incubation with 20 μL of MTT at 37 °C for 4 h, living cells containing MTT formazan crystals were solubilized in 200 μL DMSO. The absorbance of each well was measured using a microculture plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. Results are expressed as the mean ± SD of three independent experiments.

Results and discussion

Chemistry

The synthetic route for target compounds is shown in Scheme 1. Activation of different carboxylic acid compounds with 1,1′-carbonyldiimidazole (CDI) and subsequent coupling to 1-benzylpiperidin-4-amine or 2-(1-benzylpiperidin-4-yl) ethanamine afforded target compounds w1–23 in good yields46–48. Structures of all synthesized compounds were characterized by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy (see Supplementary data).

Scheme 1. Reagents and conditions: (i) 1,1′-carbonyldiimidazole (CDI), CH₂Cl₂, r.t. overnight.

Inhibitory activity against AChE and BuChE

To determine the potential interest of the new donepezil-like compounds for the treatment of AD, the ChEs inhibitory activities were assayed by the method of Ellmam et al. using donepezil and galanthamine as reference compounds49. The AChE inhibitory was tested against the Electrophorus electricus enzyme (eeAChE), and the inhibition of BuChE was carried out using the equine serum enzyme (eqBuChE). The IC₅₀ values of all tested compounds and their slectivity index for AChE over BuChE are summarized in Table 1.

Table 1. Cholinesterases and human recombinant MAO isoforms inhibitory activities of tested compounds and reference compounds.

			IC50^a (μM)		Selectivity index	IC50^a (μM)		Selectivity index
Compound	R	n	eeAChE	eqBuChE	eqBuChE/eeAChE	hMAO-A	hMAO-B	hMAO-A/hMAO-B
w1	Ph	0	76.6 ± 4.5	70.4 ± 3.2	0.92	N	N	–
w2	3-Cl Ph	0	80.7 ± 4.0	N^b	>1.24	N	N	–
w3	3-Cl-2-pyridine	0	9.95 ± 0.12	58.7 ± 2.7	5.90	N	N	–
w4	3-Pyridine	0	54.2 ± 2.9	N	>1.85	N	N	–
w5	5-Cl-2-thiophene	0	23.6 ± 1.5	N	>4.34	N	N	–
w6	Ph	2	1.51 ± 0.22	1.82 ± 0.08	1.21	N	N	–
w7	2-Cl Ph	2	1.86 ± 0.09	2.49 ± 0.14	1.34	N	N	–
w8	2-Br PhCH2	2	8.23 ± 0.34	0.323 ± 0.033	0.0392	N	46.8 ± 2.2	>2.14
w9	2-(PhCO) Ph	2	52.2 ± 1.3	3.57 ± 0.14	0.0684	N	N	–
w10	3-NO2 Ph	2	0.298 ± 0.031	0.918 ± 0.080	3.08	92.4 ± 7.1	26.2 ± 1.5	>3.53
w11	4-NO2 Ph	2	0.123 ± 0.022	0.570 ± 0.035	4.63	22.6 ± 1.8	68.3 ± 4.0	0.331
w12	4-OCH3 Ph	2	0.662 ± 0.029	1.26 ± 0.11	2.03	N	N	–
w13	4-CH3 Ph	2	1.45 ± 0.07	1.65 ± 0.08	1.14	N	94.1 ± 4.9	1.06
w14	4-Cl Ph	2	0.378 ± 0.015	0.620 ± 0.07	1.64	23.2 ± 1.8	9.27 ± 1.3	2.5
w15	2-quinoline	2	0.174 ± 0.009	0.601 ± 0.06	3.54	24.5 ± 2.3	48.6 ± 3.3	0.504
w16	3-pyridine	2	0.791 ± 0.045	13.6 ± 0.6	17.2	N	N	–
w17	4-pyridine	2	0.340 ± 0.031	5.40 ± 0.27	15.9	N	N	–
w18	5-Cl-2-pyridine	2	0.220 ± 0.006	1.23 ± 0.10	5.6	13.4 ± 0.9	3.14 ± 027	4.27
w19	5-F-2-pyridine	2	0.502 ± 0.011	3.85 ± 0.27	15.3	N	N	–
w20	6-CH3-2-pyridine	2	0.434 ± 0.019	3.64 ± 0.18	8.39	N	2.53 ± 1.4	>39.5
w21	5-Cl-2-thiophene	2	0.269 ± 0.017	2.75 ± 0.13	10.2	76.4 ± 5.4	11.5 ± 0.9	6.64
w22	5-Br-2-thiophene	2	0.768 ± 0.037	0.208 ± 0.009	0.271	96.2 ± 4.8	9.47 ± 0.54	10.2
w23	5-CH3-2-thiophene	2	1.55 ± 0.013	0.370 ± 0.021	0.239	N	N	–
Donepezil	–	–	0.035 ± 0.003	2.32 ± 0.10		nt	nt	–
Galantamine	–	–	2.67 ± 0.18	12.7 ± 0.3		nt	nt	–
Lazabemide	–	–	nt^c	nt	–	nt	0.105 ± 0.008	–
Iproniazide	–	–	nt	nt	–	6.7 ± 0.4	7.69 ± 0.23	–

^aIC₅₀: 50% inhibitory concentration (means ± SD of three experiments).

^b Inactive at 100 μM (highest concentration tested), at higher concentrations the compounds precipitate.

^c nt = not tested.

From the table, it can be seen that most of our target compounds showed potent inhibitory activity to both ChEs with IC₅₀ values ranging from micromolar to nanomolar. The length of linker of the new compounds has great influences on the inhibitory activities. From the IC₅₀ values, compounds w6–23 with N-ethylcarboxamide linkage exhibited a higher activity than those w1–5 with carboxamide linkage. This suggested that the suitable linker length seemed to be 2 carbon atoms for ChEs inhibition. Then, the introduction of substituents with different sizes to phenyl ring and pyridine ring were planned. Among compounds w6–20, w11 (IC₅₀=0.123 μM) showed the highest inhibitory activity against AChE, which was 20 times stronger than that of reference compound galanthamine (IC₅₀=2.67 μM), and 3 times less than that of donepzil (IC₅₀=0.035 μM). Compound w8 exhibited strongest inhibition against BuChE with IC₅₀ value of 0.323 μM, which was 7 times more potent than that of donepezil (IC₅₀=2.32 μM), and showed the highest selectivity with a selectivity index of 0.0392. Compared with the unsubstituted compound w6 (IC₅₀=1.51 μM for AChE; IC₅₀=1.82 μM for BuChE), introduction of Cl or NO₂ group on the 4-position of phenyl ring (w11, w14), showed inhibitory activities for both ChEs better than those of compound w6. On the contrary, incorporating the benzoyl group in the 2-position of phenyl ring (w9) showed a decreased ChEs inhibitory activities (IC₅₀=52.2 μM for AChE; IC₅₀=3.57 μM for BuChE). This might be attributed to the steric hindrance of the benzoyl group. Moreover, different substituents were introduced to the 5- or 6- position of pyridine ring, most of them showed good inhibitory activity against AChE. Especially, the inhibitory activities of w18 against AChE and BuChE (IC₅₀=0.220 μM for AChE; IC₅₀=1.23 μM for BuChE) were 12-fold and 10-fold more potent, respectively, than those of reference compound galanthamine (IC₅₀=2.67 μM for AChE; IC₅₀=12.7 μM for BuChE). To extend the series of our compounds, compounds w21–23 with thiophene moiety were synthesized. As the trend with w21–23 for AChE inhibition, w21–23 were also sensitive to the size of substituents at 5-position of thiophene ring. For example, the AChE inhibitory activity of w21 (IC₅₀=0.269 μM) for was 6-fold more potent than that of w23 (IC₅₀=1.55 μM). However, BuChE inhibitory activities of w21–23 seemed to be the opposite trend compared with the AChE inhibitory activities, inhibitory activity of w22 for BuChE (IC₅₀=0.208 μM) was 13-fold more potent than that of w21 (IC₅₀=2.75 μM). This suggested the thiophene ring also might be favorable for ChEs inhibition.

Inhibitory activity against MAOs

For all target compounds, the MAO inhibitory activities were measured, and lazabemine and iproniazide were used as reference compounds. The corresponding IC₅₀ values and MAO selectivity ratios are also shown in Table 1. Based on the screening data, it could be seen that only a part of the tested compounds could effectively inhibit MAO-A or MAO-B. Among the synthesized compounds, w20 was the most potent and selective inhibitor against MAO-B (IC₅₀=2.53 μM, SI > 39.5), which is nearly 3 times stronger than that of iproniazide. Compound w18 (IC₅₀=13.4 μM for MAO-A; IC₅₀=3.14 μM for MAO-B) with 5-Cl substituent at pyridine ring exhibited the most potent MAO-A and MAO-B. From the IC₅₀ values, compounds w1–5 with carboxamide linkage showed no activity, which suggested that the suitable linker length seemed to be 2 carbon atoms for MAOs inhibition.

Compared to no substituted compound w6 (no activity at 100 μM), introduction of different sizes to the 2-position of phenyl ring also showed no activity at 100 μM with exception of w8 (IC₅₀=46.8 μM for MAO-B). Furthermore, among w6–14, compounds with 3- and 4-position electron-withdrawing substitutions of phenyl ring were more potent inhibition for MAOs than those with 2-position substitution. Replacement of Cl group (w18) with F group (w19) in 5-position of pyridine ring presented a total loss of inhibitory activity for both MAO-A and B. In addition, the electronic properties of substitutions at 5-position of thiophene ring also affected the MAOs inhibitory activity. Compared to w21 and w22, compound w21 (IC₅₀=76.4 μM for MAO-A; IC₅₀=11.5 μM for MAO-B, SI = 6.64) and w22 (IC₅₀=96 μM for MAO-A; IC₅₀=9.47 μM for MAO-B, SI = 10.2) were potent for MAO-A and MAO-B, and good selective inhibitors toward MAO-B. Finally, we found that, no matter introducing the Cl group to 4-position of phenyl ring, 5-position of pyridine or thiophene ring, respectively, all of them showed good inhibition for both MAO-A and B.

Inhibitory activity against hAChE

Based on the results of ChEs and MAOs inhibitory activity, compounds w10, 11, 14, 15, 18, 21, 22 showed good inhibition. However, their ChE inhibitory activity was initially tested on enzymes of animal origin due to the lower cost. To better evaluate them, their inhibitory activity was retested on human AChE and the results are summarized in Table 2. Most of the tested compounds gave IC₅₀ values in nanomolar, which were slightly less potent inhibition for hAChE than for eeAChE. However, compound w22 exhibited the inhibitory activity for hAChE (IC₅₀=0.363 μM) was 2 times stronger than for eeAChE (IC₅₀=0.768 μM). Among them, we chose compound w18, which showed balanced potential to inhibit ChEs (eeAChE: IC₅₀=0.220 μM; eqBuChE: IC₅₀=1.23 μM; hAChE: IC₅₀=0.454 μM) and MAOs (MAO-A: IC₅₀=13.4 μM; MAO-B: IC₅₀=3.14 μM) as a promising multi-targeted inhibitor for further study.

Table 2. Inhibition of human AChE activity.

Compound	R	n	IC50^a (μM) hAChE^b
w10	3-NO2 Ph	2	0.544 ± 0.023
w11	4-NO2 Ph	2	0.325 ± 0.009
w14	4-Cl Ph	2	0.621 ± 0.036
w15	2-quinoline	2	0.527 ± 0.010
w18	5-Cl-2-pyridine	2	0.454 ± 0.023
w21	5-Cl-2-thiophene	2	0.412 ± 0.017
w22	5-Br-2-thiophene	2	0.363 ± 0.027
Donepezil	–	–	0.029 ± 0.002

^aIC₅₀: 50% inhibitory concentration (means ± SD of three experiments).

^b AChE from human erythrocytes.

Kinetic study of AChE

Kinetic study of compound w18 was further examined to investigate the AChE inhibitory mechanism. Graphical analysis of the Lineweaver–Burk reciprocal plots (Figure 2) indicated that both increasing slopes and intercepts with increasing inhibitor concentrations. This pattern suggested w18 is a mixed-type of inhibition and this revealed that it could interact simultaneously with dual sites (PAS and CAS) of AChE. Replots of the slope versus concentration of w18 gave an estimate of competitive inhibition constant, K_i, of 0.220 μM.

Figure 2. Kinetic study on the mechanism of eeAChE inhibition by compound w18. Overlaid Lineweaver–Burk reciprocal plots of AChE initial velocity at increasing substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the presence of w18 are shown. Lines were derived from a weighted least-squares analysis of the data points.

Kinetic study for MAO-B

From the point of view of AD treatment, reversible inhibitors of MAO-B have significant advantages over the irreversible inhibitors. Therefore, to examine whether w18 was reversible or irreversible MAO-B inhibitor, the time dependencies of inhibition were evaluated with an irreversible inhibitor, pargyline, as reference compound50. Compound w18 was preincubated for various time periods (0–60 min) with human MAO-B at a concentration of 6.28 μM. This concentration of the inhibitor are twofold the measured IC₅₀ value for the inhibition of MAO-B. As shown in Figure 3, we could observe that w18 was reversible MAO-B inhibitors as evidenced by the time-dependent decrease of their inhibitory activity. In contrast, after treatment of MAO-B with pargyline, the enzyme inhibitory activity was increased.

Figure 3. Reversibility studies of hMAO-B inhibition by compound w18. Compound w18 and pargyline were preincubated for various periods of time (0–60 min) with hMAO-B at concentrations equal to twofold the IC₅₀ values for the inhibition of the enzyme. After dilution to concentrations of w18 and pargyline equal to IC₅₀, the inhibitory rates were recorded.

Compound w18 was also used to further investigate the mode of MAO-B inhibition. The type of MAO-B inhibition was determined by the Michaelis–Menten kinetic experiments51. In this study, four different concentrations of w18 (0, 1.57, 3.14 and 6.28 μM) were selected and five different concentrations of p-tyramine (0.05–1 mM) were used as substrate. The overlaid reciprocal Lineweaver–Burk plots (Figure 4) showed that all plots for different concentrations of w18 were linear and intersected at the y-axis. This behavior indicated that compound w18 acted as a competitive MAO-B inhibitor, and this result further proved that w18 was reversible MAO-B inhibitor.

Figure 4. Kinetic study on the mechanism of hMAO-B inhibition by w18. Overlaid Lineweaver–Burk reciprocal plots of hMAO-B initial velocity at increasing p-tyramine concentration (0.05–1 mM) in the absence of inhibitor and in the presence of w18 are shown. Lines were derived from a weighted least-squares analysis of the data points.

Molecular modeling studies of ChEs

To further study the interaction mode of compound w18 for ChEs, molecular docking study was performed using software package MOE 2008.10. The X-ray crystal structure of the TcAChE complex with donepezil (PDB code: 1EVE) was applied to build the starting model of AChE. As shown in Figure 5(A) and (C), the N-benzylpiperidine moiety of w18 was oriented towards the CAS of AChE, via π-cation interaction with the quaternary nitrogen of piperidine ring from Tyr341 with the distance of 4.26 Å. Besides, its benzene ring could interact with Tyr337 via π–π stacking interaction with the distance of 4.08 Å. The 5-chloropicolinamide moiety interacted with the pridine ring from Trp286 of the PAS via π–π stacking interaction with the distance of 4.35 Å. All these results indicated that compound w18 was a dual binding site (DBS) AChE inhibitor in agreement with the kinetic study, which demonstrated the rationality of our molecular design.

Figure 5. (A) 3D docking model of compound w18 with TcAChE. (B) 3D docking model of compound w18 with hBuChE. (C) 2D schematic diagram of docking model of compound w18 with TcAChE. (D) 2D schematic diagram of docking model of compound w18 with hBuChE. The figure was prepared using the ligand interactions application in MOE.

Since the crystal structure of BuChE from equine serum has not been reported and the sequence of equine BuChE is highly similar to human BuChE, the crystal structure of hBuChE (PDB code: 1P0I) was used in the docking study. As shown in Figure 5(B) and (D), the pyridine ring of w18 stacked against the Trp82 through a π–π interaction with the distance of 2.76 Å at the CAS.

Molecular modeling studies of MAO-B

To evaluate the binding mode of compound w18 with MAO-B, docking studies were employed with MOE 2008.10, based on the protein crystal structure of MAO-B (2V60). The 3D and 2D images of binding are illustrated in Figure 6. It can be seen from Figure 6(A) and (B), the N-benzylpiperidine moiety of w18 was located within the substrate cavity of the enzyme, in close proximity of the flavin adenine dinucleotide (FAD) cofactor, and the π–π stacking interaction was seen between its benzene moiety with Tyr398 with the distance of 4.59 Å. Besides, the N-benzylpiperidine moiety via π–cation interaction with the quaternary nitrogen of piperidine ring from Gln206 had the distance of 1.75 Å. Finally, the 5-chloropicolinamide moiety of w18 occupied the hydrophobic pocket in the entrance cavity, formed by Pro104, Ser200, Leu171, Tyr326, Phe103, Pro102, Ile199 and Ile316.

Figure 6. Molecule docking of compound w18 with hMAO-B generated with MOE: (A) The 2D picture of binding was depicted; (B) The 3D picture of binding was depicted.

Metal chelating effect

The complexation ability of compound w18 for Cu²⁺in methanol was studies by using UV–Vis spectrometry with wavelength ranging from 200 to 500 nm52,53. In Figure 7(A), UV–Vis spectra of w18 at increasing Cu²⁺concentration were shown. The decrease in absorbance (at about 375 nm peak in Figure 7B), which could be better estimated by an inspection of the differential spectra, indicated that there was an interaction between Cu²⁺and compound w18. These observations indicated that our compounds could effectively chelate Cu²⁺, and thereby could serve as metal chelators in treating AD.

Figure 7. (A) UV–Vis (200–500 nm) absorption spectra of compound w18 (50 μM) in methanol after addition of ascending amounts of CuCl₂ (10–80 μM). (B) The differential spectra due to w18-Cu²⁺complex formation obtained by numerical subtraction from the above spectra of those of Cu²⁺and w18 at the corresponding concentrations.

In vitro BBB permeation assay

Because the first requirement for successful CNS drugs is to reach their therapeutic targets in brain, screening for the BBB penetration is of particular importance54. To determine whether the present compounds could penetrate into the brain, we used a parallel artificial membrane permeation assay for BBB (PAMPA-BBB), which was described by Di et al.55. Assay validation was performed by comparing experimental permeability of 9 commercial drugs with reported values (Table 3). A plot of experimental data versus bibliographic values gave a good linear correlation, P_e (exp) = 0.85 P_e (bibl.) − 0.13 (R²=0.98). From this equation-and taking into account the limits established by Di et al. for BBB permeation, we established that molecules with permeability values over 3.3 × 10⁻⁶cm s⁻¹ would be able to cross the BBB. Three compounds (w14, w18 and w21) that exhibited good activities against ChEs and MAOs were chosen as the tested compounds. The results summarized in Table 4 indicated that all of them showed higher P_e values than 3.3, which suggested they were able to cross the BBB and target the enzyme in the central nervous system.

Table 3. Permeability (P_e × 10 ^− 6cm s ^− 1) in the PAMPA-BBB assay for 9 commercial drugs, used in the experiment validation.

Commercial drugs	Bibliography^a	Experiment^b
Testosterone	17	15.6 ± 1.03
Verapamil	16	13.2 ± 0.85
β-Estradiol	12	8.90 ± 0.64
Progesterone	9.3	6.62 ± 0.43
Clonidine	5.3	4.74 ± 0.26
Corticosterone	5.1	4.92 ± 0.42
Piroxicam	2.5	1.91 ± 0.17
Hydrocortisone	1.9	1.57 ± 0.12
Dopamine	0.2	0.15 ± 0.01

^aTaken from Ref55.

^b Data are the mean ± SD of three independent experiments.

Table 4. Permeability results (P_e× 10 ^− 6cm s ^− 1) from the PAMPA-BBB assay for selected compounds with their predicted penetration into the CNS.

Compounds	Permeability (Pe × 10^−6 cm s^−1)^a	Prediction
w14	8.81 ± 1.4	CNS+
w18	10.4 ± 0.9	CNS+
w21	6.57 ± 0.7	CNS+

^aData are the mean ± SD of three independent experiments.

Rat pheochromocytoma (PC12) cell toxicity

The compound w18 was selected as the candidate to further study the potential toxicity effect on the rat pheochromocytoma (PC12) cells. After incubating the cells to compound w18 for 24 h, the cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. As indicated in Figure 8, w18 at 1–100 μM did not show significant effect on cell viability. This suggested that compound w18 was nontoxic to PC12 cells and might be a suitable drug candidate for treating AD.

Figure 8. The Cell viability of compound w18 on PC12 cells at 1–100 μM.

Conclusion

In summary, a series of donepezil-based compounds have been designed, synthesized and evaluated as multi-functional anti-AD agents with cholinesterase and MAOs inhibitory activities. Most of them displayed potent inhibitory activities toward AChE, BuChE, MAO-B and MAO-A. Among these compounds, some of them may be more potential in some ways, such as w11, w15 were stronger to inhibit ChEs than w18, w20 is more activity to interact with MAOs. But w18 was the most attractive compound with balanced bioactivity, which is able to inhibit ChEs (eeAChE: IC₅₀=0.220 μM; eqBuChE: IC₅₀=1.23 μM; hAChE: IC₅₀=0.454 μM) and MAOs (MAO-B: IC₅₀=3.14 μM; MAO-A: IC₅₀=13.4 μM). Meanwhile, compound w18 could penetrate the BBB and showed low cell toxicity on rat pheochromocytoma (PC12) cells in vitro. Altogether, the multifunctional ligand w18 endowed with balanced ChEs and MAOs inhibiting activities might be a promising anti-AD candidate for further research.

Declaration of interest

The authors declare no conflicts of interest.

We gratefully acknowledge the financial support by the National Natural Science Foundation of China (81573313), the program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Supplementary material available online