Introduction

Alzheimer’s disease (AD), the most common form of dementia, is a complex neurodegenerative disorder occurring in the central nervous system (CNS), characterized by progressive cognitive decline, memory loss and learning and behavioral disturbances. AD primarily affects the elderly section of the population, specifically people aged 65 or older (Piazzi et al., 2003; Naj et al., 2011; Seshadri et al., 2010; Lambert et al., 2009). The significance of AD is further compounded, as the number of identified cases is estimated to quadruple by the year 2050 (Brookmeyer et al., 2007). The definite reason of the AD is still ambiguous, but in general the following hypothesis has been put forward on the basis of the various causative factors such as cholinergic, amyloid, tau, and metal hypothesis (Singh et al., 2013). The cholinergic hypothesis is one of the oldest and most popular hypothesis outlining the pathogenesis of AD. According to the cholinergic hypothesis, the main objective of current pharmacotherapy for AD are drugs intended to raise the levels of acetylcholine (ACh) by the inhibition of cholinesterase enzymes (ChEs) (Tai et al., 2012; Akasofu et al., 2008; Dumas and Newhouse, 2011). Studies have shown that AD is defined by the rapid loss of acetylcholinesterase (AChE) activity in the early stages of the disease, along with the increasing ratio of AChE as the disease progresses (Darvesh et al., 2003; Greig et al., 2005).

Two forms of the ChE enzymes, AChE and butyrylcholinesterase (BuChE), are localized within the cholinergic regions of the CNS. Both enzymes are able to hydrolyze ACh, but AChE has a 1013-fold higher hydrolytic ACh activity than does BuChE under the same conditions (Skrzypek et al., 2013). In addition, BuChE has been found capable of compensating for the missing AChE catalytic functions in the synaptic cleft and its activity increases, 30–60 %, during AD (Jhee et al., 2002; Bullock and Lane, 2007; Mesulam et al., 2002; Kamal et al., 2008). Due to the role of BuChE in hydrolysis of ACh, inhibition of both cholinesterase enzymes using a dual inhibitor should results in the increased levels of ACh in the brain that provides more successful clinical efficacy of AD (Khoobi et al., 2013; Basiri et al., 2013).

The structural character of AChE shows that the catalytic anionic site (CAS) is located at the bottom of a narrow 20-Å-deep gorge. Besides, the entrance of the gorge as a regulatory site is termed the peripheral anionic site (PAS) (Xinga et al., 2013). ChE inhibitors (ChEI) may inhibit both cholinesterase enzymes via a competitive mechanism, by interacting with the CAS, via a non-competitive mechanism, by binding with the PAS, or via mixed-type mechanisms, by exerting a dual binding ChE inhibition (Samadi et al., 2012).

Recent studies have indicated that another approach, called metal hypothesis, may contribute to AD pathology (Liu et al., 2006). The level of metal ions (Fe2+, Cu2+, Zn2+) in AD patients is 3–7 times higher than in healthy individuals (Zatta et al., 2009). The excessive accumulation of metals increases the formation of beta-amyloid fibril aggregates, which induce inflammation and activate neurotoxic pathways, leading to the dysfunction and death of brain cells (Opazo et al., 2002; Dong et al., 2003; Yan et al., 2013). Thus, metal chelation therapy aimed at precluding and scavenging the occurrence of free radical metals may be considered a promising clinical approach to AD. Heavy metals, such as Cu2+, Fe2+ and Zn2+, may contribute to the production of reactive oxygen species and oxidative stress (Huang et al., 2004). Therefore, chelators, such as clioquinol, a specific Cu2+ and Zn2+ chelators been recommended as potential therapeutic treatments for AD (Bush, 2008; Ritchie et al., 2003).

Several anti-cholinesterase agents, such as rivastigmine (Exelon), galantamine (Razadyne), and donepezil (Aricept), only offer symptomatic relief without any disease-modifying effects. Unfortunately, these inhibitors have many side effects. One in particular, tacrines been shown to have a serious hepatotoxic effect (Sugimoto, 2008; Smith et al., 1997). In the view of the above mentioned reasons, we focused on the development of more active and selective dual inhibitors which are capable of interacting with the CAS and PAS of both ChEs.

α,β-unsaturated carbonyl-based compounds were obtained and evaluated for their cholinesterase inhibition capacity. Some of these compounds exhibited a respectable activity on AChE and BuChE inhibition with the half maximal inhibitory concentration (IC50) values in the micromolar (µM) ranges (Hasan et al., 2005; Yerdelen and Gul, 2013). In this study, a new series of oxamide and fumaramide derivatives was synthesized, evaluated and molecularly modeled. The pharmacological evaluations of these new compounds included AChE and BuChE inhibition, the kinetics of enzyme inhibition and metal chelation. Finally, molecular modeling studies were performed to understand the interactions between the related enzyme and the most potent compounds.

Materials and methods

Chemistry

The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded with tetramethylsilane (TMS) as the internal standard on a Bruker FT-400(100) MHz spectrometer using CDCl3 as the solvent. Coupling constants were given in Hertz (Hz). Mass (LC-MS) spectra were recorded on Agilent 1200 mass spectrometer at 10 eV. Melting points of the compounds were obtained on Electrothermal 9100 melting-point apparatus. Benzaniline compounds were synthesized with CEM 3100 microwave oven. Reaction progress and product mixtures were routinely checked by thin-layer chromatography (TLC) on Merck SilicaGel F254 aluminum plates. Column chromatography was performed with SiO2 (70–230 mesh). The chemical reagents and solvents used in this study were purchased from Merck or Sigma Aldrich.

General synthesis procedure under solvent-free phase-transfer catalysis conditions (1a–s)

A mixture of meta-substituted aniline (10 mmol), benzyl chloride, or p-methylbenzyl chloride (5 mmol) was adsorbed on a mixture of potassium carbonate (6.25 mmol) and tetrabutylammonium bromide (1 mmol) and then the resulting fine powder was irradiated with CEM microwave (150 W, 120 °C) for 5 min. Completion of the reaction was checked by TLC and all of the compounds were purified by column chromotography on silica gel with hexane:ethyl acetate (9:1) mobile phase. Synthesis pathway is shown on Scheme 1.

Scheme 1
scheme 1

Synthesis of the target compounds 2a–s and 3a–s

Full size image

A general procedure for the synthesis of oxamide compounds (2a–s)

A mixture of the meta-substituted benzaniline (1.3 mmol), triethylamine (TEA) (1.3 mmol), and oxalyl chloride (0.66 mmol) in tetrahydrofuran (5 mL) was stirred at room temperature for 12 h. The reaction mixture was quenched with 15 mL of distilled water and the aqueous phase was extracted with two portions of CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel with hexane:ethyl acetate (7:3) mobile phase. The obtained solid was recrystallized from EtOH.

N 1,N 2-dibenzyl-N 1,N 2-di-m-tolyl-oxamide (2a)

Intermediate 3-methyl-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2a as a white solid with a yield of 45 %; mp 152–155 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.28 (s, 6H, 2× CH3), 4.80 (s, 4H, 2× CH2-N), 6.78–6.76 (d, 2H, J = 8.0 Hz), 6.95 (s, 2H), 7.06–7.04 (d, 4H, J = 7.7 Hz), 7.11–7.09 (m, 2H), 7.18–7.14 (m, 6H), 7.36–7.34 (d, 2H, J = 7.9 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 20.32 (Ph-CH3), 53.24 (CH2-N), 124.28, 128.48, 128.49, 128.55, 129.16, 129.87, 131.35, 136.22, 138.75, 140.42, 158.01 (C=O). ESI-MS m/z [M + H] = 449.5.

N 1,N 2-bis-(4-methyl-benzyl)-N 1,N 2-di-m-tolyl-oxamide (2b)

Intermediate 3-methyl-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2b as a white solid with a yield of 40 %; mp 148–152 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.23 (s, 6H), 2.28 (s, 6H), 4.80 (s, 4H, 2× CH2-N), 6.72–6.70 (d, 2H, J = 7.9 Hz), 6.85 (s, 2H), 7.09 (bs, 8H), 7.15–7.13 (d, 2H, J = 7.8 Hz), 7.19–7.15 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.17 (p-CH3), 22.31 (m-CH3), 53.13 (CH2-N), 124.98, 127.67, 129.56, 130.37, 133.26, 134.49, 137.22, 140.29, 140.56, 159.37 (C=O). ESI-MS m/z [M + H] = 477.6.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-trifluoromethyl-phenyl)-oxamide (2c)

Intermediate 3-trifluoromethyl-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2c as a white solid with a yield of 57 %; mp 130–134 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.89 (s, 4H, 2× CH2-N), 7.09–7.06 (m, 6H), 7.23–7.20 (m, 2H), 7.28–7.24 (m, 6H), 7.38–7.36 (d, 2H, J = 7.7 Hz), 7.58 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 51.25 (CH2-N), 122.66, 126.12, 127.23, 129.31, 130.28, 132.02, 135.44, 139.65, 157.20 (C=O). ESI-MS m/z [M + H] = 557.5.

N 1,N 2-bis-(4-methyl-benzyl)-N 1,N 2-bis-(3-trifluoromethyl-phenyl)-oxamide (2d)

Intermediate 3-trifluoromethyl-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2d as a white solid with a yield of 42 %; mp 114–117 °C. 1H NMR (400 MHz, CDCl3): δ ppm 3.92 (s, 6H, 2× Ph-CH3), 4.83 (s, 4H, 2× CH2-N), 7.11–6.98 (d, 2H, J = 7.9 Hz), 7.01–6.99 (d, 4H, J = 8.0 Hz), 7.10–7.08 (d, 4H, J = 7.7 Hz), 7.24–7.22 (m, 2H), 7.34–7.32 (d, 2H, J = 7.6 Hz), 7.79 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.13 (Ph-CH3), 52.56 (CH2-N), 124.09, 128.63, 129.95, 130.48, 131.18, 135.36, 136.57, 139.92, 158.25 (C=O). ESI-MS m/z [M + H] = 585.2.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-ethyl-phenyl)-oxamide (2e)

Intermediate 3-ethyl-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2e as a yellow oil with a yield of 82 %. 1H NMR (400 MHz, CDCl3): δ ppm 1.13–1.11 (t, 6H, 2× CH2 CH 3 ), 2.42–2.39 (q, 4H, 2× CH 2 CH3), 4.80 (s, 4H, 2× CH2-N), 6.88–6.85 (d, 2H, J = 7.6 Hz), 7.05–7.01 (m, 6H), 7.18–7.14 (m, 6H), 7.21 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 18.45 (CH2–CH 3 ), 27.38 (CH 2 –CH3), 52.64 (CH2-N), 124.37, 126.99, 127.09, 128.24, 128.57, 131.23, 135.34, 140.78, 159.91 (C=O). ESI-MS m/z [M + H] = 477.6.

N 1,N 2-bis-(3-ethyl-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2f)

Intermediate 3-ethyl-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2f as a yellow oil with a yield of 59 %. 1H NMR (400 MHz, CDCl3): δ ppm 1.20–1.16 (t, 6H, 2× CH2 CH 3 ), 2.28 (s, 6H, 2× CH3-Ph), 2.63–2.58 (q, 4H, 2× CH 2 CH3), 4.84 (s, 4H, 2× CH2-N), 6.78–6.76 (bs, 4H), 7.03 (bs, 8H), 7.15–7.13 (d, 2H, J = 7.69 Hz), 7.26–7.21 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 13.33 (CH2-CH 3 ), 20.42 (Ph-CH3), 27.57 (CH 2 -CH3), 52.28 (CH2-N), 125.03, 126.78, 127.02, 127.97, 128.69, 130.34, 133.51, 135.49, 139.32, 143.00, 160.32 (C=O). ESI-MS m/z [M + H] = 505.6.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-methoxy-phenyl)-oxamide (2g)

Intermediate 3-methoxy-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2g as a white solid with a yield of 48 %; mp 152–155 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.28 (s, 6H, 2× OCH3), 4.83 (s, 4H, 2× CH2-N), 6.73–6.72 (d, 2H, J = 7.6 Hz), 6.90 (s, 2H), 7.11–7.09 (d, 4H, J = 7.8 Hz), 7.12–7.09 (m, 2H), 7.20–7.16 (m, 6H), 7.25–7.24 (d, 2H, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 20.19 (OCH3), 52.67 (N-CH2), 125.08, 126.25, 127.19, 128.11, 128.71, 131.37, 135.66, 137.84, 140.19, 161.23 (C=O). ESI-MS m/z [M + H] = 481.5.

N 1,N 2-bis-(3-methoxy-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2h)

Intermediate 3-methoxy-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2h as a yellow oil with a yield of 75 %. 1H NMR (400 MHz, CDCl3): δ ppm 2.21 (s, 6H, 2× Ph-CH3), 3.68 (s, 6H, 2× OCH3), 4.82 (s, 4H, 2× CH2-N), 6.76–6.74 (d, 2H, J = 7.7 Hz), 6.91 (s, 2H), 7.10 (bs, 8H), 7.22–7.20 (d, 2H, J = 7.6 Hz), 7.23–7.20 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.12 (Ph-CH3), 52.22 (OCH3), 55.75 (N-CH2), 111.65, 112.47, 119.49, 128.30, 130.11, 132.83, 135.73, 140.42, 153.92, 159.22 (C=O). ESI-MS m/z [M + H] = 509.6.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-ethoxy-phenyl)-oxamide (2i)

Intermediate 3-ethoxy-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2i as a yellow solid with a yield of 77 %; mp 94–98 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.33–1.30 (t, 6H, 2× CH2 CH 3 ), 3.85–3.82 (q, 4H, 2× CH 2 CH 3 ), 4.80 (s, 4H, 2× CH2-N), 6.53–6.51 (d, 2H, J = 7.8 Hz), 6.90 (s, 2H), 7.08-7.05 (d, 4H, J = 7.8 Hz), 7.12–7.09 (m, 2H), 7.14–7.11 (m, 6H), 7.20–7.18 (d, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 15.03 (OCH2 CH 3 ), 52.57 (OCH 2 CH3), 60.11 (N-CH2), 111.03, 119.89, 122.34, 125.43, 128.31, 130.99, 134.21, 139.31, 155.22, 159.28 (C=O). ESI-MS m/z [M + H] = 509.6.

N 1,N 2-bis-(3-ethoxy-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2j)

Intermediate 3-ethoxy-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2j as a orange solid with a yield of 87 %; mp 78–81 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.38–1.35 (t, 6H, 2× OCH2 CH 3 ), 2.20 (s, 6H, 2× CH3), 3.86–3.83 (q, 4H, 2× OCH 2 CH3), 4.85 (s, 4H, 2× CH2-N), 6.68 (s, 2H), 6.73–6.72 (d, 2H, J = 8.1 Hz), 6.81–6.78 (d, 2H, J = 8.0 Hz), 7.00–6.94 (bs, 8H), 7.23–7.19 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 15.23 (OCH2 CH 3 ), 20.11 (Ph-CH3), 53.09 (OCH 2 CH3), 61.59 (N-CH2), 112.89, 113.96, 119.22, 125.18, 128.35, 130.09, 131.57, 135.92, 140.01, 152.74, 159.94 (C=O). ESI-MS m/z [M + H] = 537.6.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-fluoro-phenyl)-oxamide (2k)

Intermediate 3-fluoro-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2k as a white solid with a yield of 59 %; mp 155–159 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.82 (s, 4H, 2× CH2-N), 6.88–6.86 (d, 2H, J = 7.6 Hz), 7.03–7.00 (m, 2H), 7.15–7.13 (d, 4H, J = 7.8 Hz), 7.25–7.22 (m, 6H), 7.30–7.28 (d, 2H, J = 7.9 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 51.22 (N-CH2), 112.11, 113.57, 121.00, 125.23, 128.61, 129.15, 131.62, 134.23, 140.21, 157.98, 159.11 (C=O). ESI-MS m/z [M + H] = 457.4.

N 1,N 2-bis-(3-fluoro-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2l)

Intermediate 3-fluoro-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2l as a white solid with a yield of 55 %; mp 112–115 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.21 (s, 6H, 2× CH3), 4.81 (s, 4H, 2× CH2-N), 6.78–6.70 (m, 4H), 7.01–6.98 (d, 4H, J = 7.8 Hz), 7.06–7.04 (d, 4H, J = 8.0 Hz), 7.28–7.25 (d, 2H, J = 8.0 Hz), 7.32–7.29 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.01 (Ph-CH3), 52.24 (N-CH2), 111.09, 112.47, 121.04, 125.35, 128.57, 129.90, 131.26, 135.21, 139.82, 157.25, 160.91 (C=O). ESI-MS m/z [M + H] = 485.6.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-chloro-phenyl)-oxamide (2m)

Intermediate 3-chloro-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2m as a white solid with a yield of 44 %; mp 128–130 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.82 (s, 4H, 2× CH2-N), 6.81–6.80 (d, 2H, J = 7.9 Hz), 7.00 (s, 2H), 7.25–7.24 (d, 4H, J = 8.0 Hz), 7.27–7.24 (m, 2H), 7.28–7.26 (m, 6H), 7.32–7.30 (d, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 52.45 (N-CH2), 123.31, 125.64, 126.11, 127.25, 127.98, 129.14, 130.72, 131.67, 132.42, 138.38, 160.59 (C=O). ESI-MS m/z [M + H] = 490.3.

N 1,N 2-bis-(3-chloro-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2n)

Intermediate 3-chloro-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2n as a white solid with a yield of 57 %; mp 144–148 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.21 (s, 6H, 2× CH3), 4.83 (s, 4H, 2× CH2-N), 6.80 (s, 2H), 7.05 (bs, 8H), 7.06–7.04 (d, 2H, J = 8.0 Hz), 7.24–7.20 (m, 2H), 7.30–7.28 (d, 2H, J = 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 20.34 (Ph-CH3), 53.02 (N-CH2), 113.28, 114.58, 121.15, 126.49, 128.21, 129.01, 130.59, 131.08, 135.22, 140.55, 158.23 (C=O). ESI-MS m/z [M + H] = 518.4.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-bromo-phenyl)-oxamide (2o)

Intermediate 3-bromo-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2o as a white solid with a yield of 45 %; mp 114–119 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.85 (s, 4H, 2× CH2-N), 6.83–6.81 (d, 2H, J = 7.7 Hz), 7.00 (s, 2H), 7.11–7.08 (d, 4H, J = 7.8 Hz), 7.25–7.24 (m, 2H), 7.30–7.27 (m, 6H), 7.36–7.34 (d, 2H, J = 7.9 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 51.54 (N-CH2), 121.10, 123.20, 125.54, 126.47, 129.11, 130.11, 130.91, 131.12, 133.27, 139.47, 158.77 (C=O). ESI-MS m/z [M + H] = 579.2.

N 1,N 2-bis-(3-bromo-phenyl)-N 1,N 2-bis-(4-methyl-benzyl)-oxamide (2p)

Intermediate 3-bromo-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2p as a white solid with a yield of 45 %; mp 146–149 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.31 (s, 6H, 2× CH3), 4.81 (s, 4H, 2× CH2-N), 6.85–6.83 (d, 2H, J = 7.8 Hz), 7.01–7.00 (d, 4H, J = 8.1 Hz), 7.04–7.01 (d, 4H, J = 7.9 Hz), 7.28 (s, 2H), 7.32–7.30 (d, 2H, J = 8.1 Hz), 7.44–7.41 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.44 (Ph-CH3), 51.13 (N-CH2), 120.24, 125.13, 127.45, 128.85, 130.13, 130.92, 131.21, 132.99, 133.42, 139.51, 159.46 (C=O). ESI-MS m/z [M + H] = 607.4.

N 1,N 2-dibenzyl-N 1,N 2-bis-(3-iodo-phenyl)-oxamide (2r)

Intermediate 3-iodo-N-(benzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2r as a white solid with a yield of 46 %; mp 138–142 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.87 (s, 4H, 2× CH2-N), 6.91–6.89 (d, 2H, J = 7.9 Hz), 7.08–7.04 (m, 2H), 7.15–7.13 (d, 4H, J = 7.6 Hz), 7.24 (bs, 6H), 7.49 (s, 2H), 7.68–7.66 (d, 2H, J = 7.6 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 52.55 (N-CH2), 124.72, 125.27, 126.66, 129.12, 129.87, 134.40, 135.35, 136.85, 140.30, 159.68 (C=O). ESI-MS m/z [M + H] = 673.2.

N 1,N 2-bis(3-iodophenyl)-N 1,N 2-bis(4-methylbenzyl)-oxamide (2s)

Intermediate 3-iodo-N-(4-methylbenzyl)aniline was reacted with oxalyl chloride following the general procedure to give the product 2s as a white solid with a yield of 90 %; mp 143–147 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.32 (s, 6H, 2× CH3), 4.86 (s, 4H, 2× CH2-N), 6.85–6.83 (d, 2H, J = 8.0 Hz), 7.03–7.00 (d, 4H, J = 8.1 Hz), 7.04–7.02 (d, 4H, J = 8.1 Hz), 7.28 (s, 2H), 7.32–7.31 (d, 2H, J = 7.9 Hz), 7.46–7.41 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 20.03 (Ph-CH3), 50.53 (N-CH2), 122.46, 126.64, 127.85, 128.56, 130.73, 135.84, 136.02, 138.21, 140.02, 160.15 (C=O). ESI-MS m/z [M + H] = 701.3.

A general procedure for the synthesis of fumaramide compounds (3a–s)

A mixture of meta-substituted benzaniline compound (1.3 mmol), TEA (1.3 mmol), and dry ethylacetate (5 mL). The mixture was cooled with an ice bath to 0–5 °C and fumaryl chloride (0.65 mmol) in 5 mL dry ethylacetate was added dropwise by syringe over 30 min. The reaction mixture was stirred at room temperature for over night. The reaction mixture was quenched with 50 mL of distilled water and the aqueous phase was extracted with two portions of CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated by rotary evaporation. The target compounds were recrystallized from ethanol.

N 1,N 4-dibenzyl-N 1,N 4-di-m-tolyl-fumaramide (3a)

Intermediate 3-methyl-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3a as a white solid with a yield of 61 %; mp 180–184 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.31 (s, 6H, 2× CH3), 4.88 (s, 4H, 2× CH2-N), 6.75–6.74 (d, 2H, J = 7.60 Hz), 6.81 (s, 2H, fumaryl CH=CH), 6.89 (s, 2H), 7.13–7.11 (d, 4H, J = 7.60 Hz), 7.16–7.14 (m, 2H), 7.22–7.18 (m, 6H), 7.26–7.24 (d, 2H, J = 7.6 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 21.29 (Ph-CH3), 53.58 (CH2-N), 125.28, 127.40 (CH=CH), 128.35, 128.49, 128.63, 129.04, 129.16, 132.07, 137.07, 139.75, 141.22, 164.43 (C=O). ESI-MS m/z [M + H] = 475.1.

N 1,N 4-bis(4-methylbenzyl)-N 1,N 4-di-m-tolyl-fumaramide (3b)

Intermediate 3-methyl-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3b as a white solid with a yield of 55 %; mp 202–204 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.29 (s, 6H), 2.32 (s, 6H), 4.83 (s, 4H, 2× CH2-N), 6.74–6.72 (d, 2H, J = 7.7 Hz), 6.81 (s, 2H, ethylene protons CH=CH), 6.88 (s, 2H), 7.00 (bs, 8H), 7.12–7.10 (d, 2H, J = 7.8 Hz), 7.22–7.18 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 21.09 (p-CH3), 21.29 (m-CH3), 53.33 (CH2-N), 125.33, 128.50 (CH=CH), 128.61, 129.01, 129.37, 132.07, 134.06, 137.00, 139.69, 141.30, 164.38 (C=O). ESI-MS m/z [M + H] = 503.1.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-(trifluoromethyl)phenyl)-fumaramide (3c)

Intermediate 3-trifluoromethyl-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3c as a white solid with a yield of 52 %; mp 161–165 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.92 (s, 4H, 2× CH2-N), 6.83 (s, 2H, ethylene protons CH=CH), 7.12–7.10 (m, 6H), 7.26–7.24 (m, 2H), 7.29–7.26 (m, 6H), 7.50–7.48 (d, 2H, J = 7.8 Hz), 7.60 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 53.49 (CH2-N), 124.87, 125.20 (CH=CH), 127.85, 128.63, 130.41, 131.82, 132.29, 136.20, 141.65, 163.94 (C=O). ESI-MS m/z [M + H] = 583.4.

N 1,N 4-bis(4-methylbenzyl)-N 1,N 4-bis(3-(trifluoromethyl)phenyl)-fumaramide (3d)

Intermediate 3-trifluoromethyl-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3d as a white solid with a yield of 46 %; mp 176–180 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.29 (s, 6H, 2× Ph-CH3), 4.87 (s, 4H, 2× CH2-N), 6.80 (s, 2H, ethylene protons CH=CH), 7.00–6.90 (d, 2H, J = 7.9 Hz), 7.05–7.03 (d, 4H, J = 8.6 Hz), 7.11–7.09 (d, 4H, J = 7.8 Hz), 7.27–7.26 (m, 2H), 7.49–7.47 (d, 2H, J = 7.4 Hz), 7.59 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 21.08 (Ph-CH3), 53.25 (CH2-N), 125.11, 128.62 (CH=CH), 129.26, 130.35, 131.88, 132.28, 133.17, 137.57, 141.72, 163.90 (C=O). ESI-MS m/z [M + H] = 611.2.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-ethylphenyl)-fumaramide (3e)

Intermediate 3-ethyl-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3e as a white solid with a yield of 16 %; mp 138–140 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.19–1.15 (t, 6H, 2× CH2 CH 3 ), 2.62–2.57 (q, 4H, 2× CH 2 CH3), 4.88 (s, 4H, 2× CH2-N), 6.90 (s, 2H, ethylene protons CH=CH), 6.78–6.77 (d, 2H, J = 7.9 Hz), 7.15–7.13 (m, 6H), 7.24–7.22 (m, 6H), 7.26 (s, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 15.21 (CH2-CH 3 ), 28.53 (CH 2 -CH3), 53.50 (CH2-N), 125.37, 127.39, 127.79, 128.34, 128.69, 129.49 (CH=CH), 132.12, 137.12, 141.24, 164.47 (C=O). ESI-MS m/z [M + H] = 503.8.

N 1,N 4-bis(3-ethylphenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3f)

Intermediate 3-ethyl-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3f as a yellow solid with a yield of 50 %; mp 171–173 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.20–1.16 (t, 6H, 2× CH2 CH 3 ), 2.28 (s, 6H, 2× CH3-Ph), 2.63–2.58 (q, 4H, 2× CH 2 CH3), 4.84 (s, 4H, 2× CH2-N), 6.78–6.76 (bs, 4H), 6.88 (s, 2H, ethylene protons CH=CH), 7.03 (bs, 8H), 7.15–7.13 (d, 2H, J = 7.69 Hz), 7.26–7.21 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 15.21 (CH2-CH 3 ), 21.08 (Ph-CH3), 28.53 (CH 2 -CH3), 53.26 (CH2-N), 125.43, 127.41, 127.72, 128.67, 129.00, 129.44 (CH=CH), 132.12, 134.11, 137.00, 141.32, 145.93, 164.42 (C=O). ESI-MS m/z [M + H] = 531.6.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-methoxyphenyl)-fumaramide (3g)

Intermediate 3-methoxy-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3g as a white solid with a yield of 76 %; mp 180–182 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.31 (s, 6H, 2× OCH3), 4.89 (s, 4H, 2× CH2-N), 6.75–6.73 (d, 2H, J = 7.60 Hz), 6.81 (s, 2H, ethylene protons CH=CH), 6.90 (s, 2H), 7.13–7.11 (d, 4H, J = 7.3 Hz), 7.16–7.14 (m, 2H), 7.22–7.18 (m, 6H), 7.26–7.24 (d, 2H, J = 7.6 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 21.09 (OCH3), 53.57 (N-CH2), 125.28, 127.39, 128.49, 128.63 (CH=CH), 129.05, 129.41, 132.07, 137.07, 139.74, 141.22, 164.43 (C=O). ESI-MS m/z [M + H] = 507.7.

N 1,N 4-bis(3-methoxyphenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3h)

Intermediate 3-methoxy-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3h as a white solid with a yield of 37 %; mp 192–194 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.28 (s, 6H, 2× Ph-CH3), 3.73 (s, 6H, 2× OCH3), 4.84 (s, 4H, 2×CH2-N), 6.45 (s, 2H, ethylene protons CH=CH), 6.56–6.54 (d, 2H, J = 7.6 Hz), 6.90 (s, 2H), 7.04 (bs, 8H), 7.23–7.21 (d, 2H, J = 8.0 Hz), 7.26–7.25 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 21.09 (Ph-CH3), 53.18 (OCH3), 55.33 (N-CH2), 113.70, 113.93, 120.52, 128.66 (CH=CH), 130.30, 132.11, 134.03, 137.08, 142.42, 160.35, 164.32 (C=O). ESI-MS m/z [M + H] = 535.6.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-ethoxyphenyl)-fumaramide (3i)

Intermediate 3-ethoxy-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3i as a brown solid with a yield of 55 %; mp 184–188 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.39–1.36 (t, 6H, 2× CH2 CH 3 ), 3.93–3.91 (q, 4H, 2× CH 2 CH 3 ), 4.88 (s, 4H, 2× CH2-N), 6.50 (s, 2H, ethylene protons CH=CH), 6.55–6.53 (d, 2H, J = 7.60 Hz), 6.92 (s, 2H), 7.17–7.15 (d, 4H, J = 7.6 Hz), 7.19–7.16 (m, 2H), 7.23–7.21 (m, 6H), 7.26–7.24 (d, 2H, J = 8.4 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 14.66 (OCH2 CH 3 ), 53.45 (OCH 2 CH3), 63.59 (N-CH2), 114.34, 120.29, 127.43, 128.39 (CH=CH), 128.66, 130.31, 132.11, 137.08, 142.31, 159.77, 164.39 (C=O). ESI-MS m/z [M + H] = 536.8.

N 1,N 4-bis(3-ethoxyphenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3j)

Intermediate 3-ethoxy-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3j as a white solid with a yield of 52 %; mp 178–182 °C. 1H NMR (400 MHz, CDCl3): δ ppm 1.40–1.37 (t, 6H, 2× OCH2 CH 3 ), 2.28 (s, 6H, 2× CH3), 3.96–3.91 (q, 4H, 2× OCH 2 CH3), 4.83 (s, 4H, 2× CH2-N), 6.50 (s, 2H), 6.54–6.52 (d, 2H, J = 8.05 Hz), 6.84–6.82 (d, 2H, J = 8.4 Hz), 6.90 (s, 2H, ethylene protons CH=CH), 7.06–7.04 (bs, 8H), 7.26–7.19 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 14.66 (OCH2 CH 3 ), 21.08 (Ph-CH3), 53.19 (OCH 2 CH3), 63.59 (N-CH2), 114.27, 114.36, 120.35, 128.64 (CH=CH), 129.04, 130.26, 132.11, 134.06, 137.03, 142.39, 159.74, 164.34 (C=O). ESI-MS m/z [M + H] = 563.1.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-fluorophenyl)-fumaramide (3k)

Intermediate 3-fluoro-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3k as a brown solid with a yield of 73 %; mp 162–165 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.90 (s, 4H, 2× CH2-N), 6.73 (s, 2H, ethylene protons CH=CH), 6.78–6.76 (d, 2H, J = 7.9 Hz), 7.07–7.05 (m, 2H), 7.16–7.30 (d, 4H, J = 7.4 Hz), 7.30–7.25 (m, 6H), 7.33–7.30 (d, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 53.45 (N-CH2), 115.56, 115.67, 124.11, 127.70 (CH=CH), 128.56, 130.85, 130.95, 132.18, 136.50, 142.66, 161.67, 164.07 (C=O). ESI-MS m/z [M + H] = 483.9.

N 1,N 4-bis(3-fluorophenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3l)

Intermediate 3-fluoro-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3l as a brown solid with a yield of 34 %; mp 189–191 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.29 (s, 6H, 2× CH3), 4.85 (s, 4H, 2× CH2-N), 6.78–6.70 (m, 4H), 6.87 (s, 2H, ethylene protons CH=CH), 7.03–7.01 (d, 4H, J = 8.3 Hz), 7.06–7.04 (d, 4H, J = 8.3 Hz), 7.30–7.28 (d, 2H, J = 8.0 Hz), 7.34–7.32 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 21.11 (Ph-CH3), 53.19 (N-CH2), 115.38, 115.60, 124.15, 128.57 (CH=CH), 129.21, 130.80, 130.90, 132.18, 137.38, 142.73, 161.65, 164.13 (C=O). ESI-MS m/z [M + H] = 511.0.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-chlorophenyl)-fumaramide (3m)

Intermediate 3-chloro-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3m as a brown solid with a yield of 44 %; mp 178–180 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.89 (s, 4H, 2× CH2-N), 6.84–6.83 (d, 2H, J = 7.7 Hz), 6.86 (s, 2H, ethylene protons CH=CH), 7.03 (s, 2H), 7.15–7.13 (d, 4H, J = 8.1 Hz), 7.25–7.24 (m, 2H), 7.29–7.26 (m, 6H), 7.34–7.32 (d, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 53.51 (N-CH2), 126.68, 127.73, 128.21, 128.50 (CH=CH), 128.60, 128.70, 130.65, 132.20, 135.26, 136.42, 142.32, 164.04 (C=O). ESI-MS m/z [M + H] = 516.9.

N 1,N 4-bis(3-chlorophenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3n)

Intermediate 3-chloro-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3n as a brown solid with a yield of 36 %; mp 197–199 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.29 (s, 6H, 2× CH3), 4.84 (s, 4H, 2× CH2-N), 6.81 (s, 2H, ethylene protons CH=CH), 6.84 (s, 2H), 7.02 (bs, 8H), 7.06–7.04 (d, 2H, J = 8.2 Hz), 7.28–7.24 (m, 2H), 7.32–7.30 (d, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 21.11 (Ph-CH3), 53.19 (N-CH2), 115.38, 115.60, 124.15, 128.57 (CH=CH), 128.69, 129.21, 130.80, 130.90, 132.18, 137.38, 142.73, 164.13 (C=O). ESI-MS m/z [M + H] = 544.5.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-bromophenyl)-fumaramide (3o)

Intermediate 3-bromo-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3o as a brown solid with a yield of 56 %; mp 174–178 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.88 (s, 4H, 2× CH2-N); 6.85 (s, 2H, ethylene protons CH=CH); 6.88 (bs, 2H); 7.14 (m, 4H); 7.21–7.20 (m, 4H, Ha); 7.24 (bs, 6H); 7.48–7.46 (d, 2H, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ ppm 53.54 (N-CH2), 123.10, 127.20, 127.74, 128.50, 128.63 (CH=CH), 130.91, 131.06, 131.61, 132.20, 136.40, 142.42, 164.02 (C=O). ESI-MS m/z [M + H] = 605.5

N 1,N 4-bis(3-bromophenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3p)

Intermediate 3-bromo-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3p as a white solid with a yield of 59 %; mp 196–198 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.29 (s, 6H, 2× CH3), 4.84 (s, 4H, 2× CH2-N), 6.83 (s, 2H, ethylene protons CH=CH), 6.87–6.85 (d, 2H, J = 8.0 Hz), 7.03–7.00 (d, 4H, J = 8.0 Hz), 7.06–7.00 (d, 4H, J = 8.0 Hz), 7.18 (s, 2H), 7.22–7.20 (d, 2H, J = 8.0 Hz), 7.48–7.46 (m, 2H). 13C NMR (100 MHz, CDCl3): δ ppm 21.10 (Ph-CH3), 53.29 (N-CH2), 123.05, 127.24, 128.61, 129.21 (CH=CH), 130.85, 131.06, 131.52, 132.21, 133.37, 137.40, 142.51, 163.98 (C=O). ESI-MS m/z [M + H] = 634.3.

N 1,N 4-dibenzyl-N 1,N 4-bis(3-iodophenyl)-fumaramide (3r)

Intermediate 3-iodo-N-(benzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3r as a white solid with a yield of 50 %; mp 210–212 °C. 1H NMR (400 MHz, CDCl3): δ ppm 4.87 (s, 4H, 2× CH2-N), 6.84 (s, 2H, ethylene protons CH=CH), 6.91–6.89 (d, 2H, J = 7.6 Hz), 7.08–7.04 (m, 2H), 7.15–7.13 (d, 4H, J = 7.3 Hz), 7.24 (bs, 6H), 7.49 (s, 2H), 7.68–7.66 (d, 2H, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): 53.55 (N-CH2), 127.72, 127.89, 128.55 (CH=CH), 128.66, 131.03, 132.21, 136.40, 136.84, 137.49, 142.30, 164.01 (C=O). ESI-MS m/z [M + H] = 699.7.

N 1,N 4-bis(3-iodophenyl)-N 1,N 4-bis(4-methylbenzyl)-fumaramide (3s)

Intermediate 3-iodo-N-(4-methylbenzyl)aniline was reacted with fumaryl chloride following the general procedure to give the product 3s as a white solid with a yield of 39 %; mp 142–147 °C. 1H NMR (400 MHz, CDCl3): δ ppm 2.30 (s, 6H, 2× CH3), 4.60 (s, 4H, 2× CH2-N), 6.66 (s, 2H, ethylene protons CH=CH), 6.97–6.95 (d, 2H, J = 7.9 Hz), 7.05–7.01 (bs, 8H,), 7.08 (s, 2H); 7.13–7.12 (m, 2H); 7.69–7.67 (d, 2H). 13C NMR (100 MHz, CDCl3): 21.15 (Ph-CH3), 51.58 (N-CH2), 128.20, 128.30, 129.26 (CH=CH), 129.45, 130.41, 132.73, 137.11, 137.37, 137.54, 140.35, 163.85 (C=O). ESI-MS m/z [M + H] = 727.5.

Inhibition studies on AChE and BuChE

AChE (E.C. 3.1.1.7, from electric eel), BuChE (E.C. 3.1.1.8, from equine serum), 5,5-dithiobis-(2-nitrobenzoic acid) DTNB, acetylthiocholine iodide (ATCI), and butyrylthiocholine iodide (BTCI) were purchased from Sigma Aldrich. Inhibitory activities of AChE and BuChE of the test compounds were evaluated by colorimetric Ellman’s method (Ellman et al., 1961) with some modifications using commercially available neostigmine bromide (Skrzypek et al., 2013), galantamine bromide (Singh et al., 2013), and ambenonium dichloride (Musileka et al., 2011) as the reference compounds. The test compounds were dissolved in dimethylsulphoxide and then diluted in 50 mM Tris buffer (pH 8.0) to provide a final concentration range. In a 96-well plate, the assay medium in each well consisted of 50 µL of a Tris buffer, 125 µL of 3 mM DTNB (Ellman’s reagent), 25 µL of 0.2 U/mL enzyme (AChE or BuChE) and 15 mM substrate (ATCI or BTCI). The assay mixture containing enzyme, buffer, DTNB and 25 µL of inhibitor compound was preincubated for 15 min at 37 °C, before the substrate was added to begin the reaction. Galantamine bromide, neostigmine bromide, ambenonium dichloride, and all test compounds were prepared at different concentrations such as 0.195, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µg/mL. The absorbance of the reaction mixture was then measured three times at 412 nm every 45 s using a microplate reader (Bio-Tek ELx800, USA). Results are presented as means ± standard errors of the experiment. The IC50 values of the compounds showing percentage inhibition, the measurements and calculations were evaluated by nonlinear regression analysis using GraphPad Prism software.

Kinetic study

Compounds 3o, 3p, and 2h were selected for kinetic measurements because they were found to have the highest inhibitory activity against AChE and BuChE, respectively. The test was carried out without the inhibitor and in 0.02, 0.03, and 0.04 µM concentrations of the inhibitors 3o and 3p, 0.01, 0.02, and 0.03 µM concentrations for the inhibitor 2h. Substrate concentrations were varied from 0.1 to 1.5 mM. The obtained data were used to create substrate-velocity curves, which were transformed in GraphPad Prism to Lineweaver–Burk plots.

Spectrophotometric measurement of complex with Cu2+ and Fe2+

The study of metal chelation was performed in ethanol at room temperature using UV–Vis spectrophotometer (Thermo Electron Heλios) with wavelength ranging from 200 to 500 nm. The UV absorption of the test compounds 3o and 2h, in the absence or presence of with CuSO4 and FeSO4, was recorded in a 1 cm quartz cuvette after 20 min at room temperature. The final volume of reaction mixture was 3 mL, and final concentrations of the test compounds and metals were 100 μM. All reaction mixtures were repeated for at least three times.

Molecular modeling

The docking study was performed using Surflex-Dock in Sybyl-X 2.0 by Tripos Associates. 3D structures of the compounds 2h, 3o, and 3p were constructed using the Sybyl sketcher module. The structures were minimized using the steepest descent conjugated gradient method until the gradient was 0.001 kcal/mol, max iterations: 5000 with the Tripos force field with the Gasteiger Huckel charge. The simulation system was built on the crystal structures of 1ACJ and 1P0I which were obtained from the Protein Data Bank. At the commencement of docking, all the water and ligands were removed and the random hydrogen atoms were added. Docking calculations using Surflex-Dock for 1ACJ and 1P0I were performed through protomol generation by ligand. The parameters used were threshold 0.5 and bloat 0.

Result and discussion

Chemistry

In the first step, starting benzanilines (1a–s) were prepared using a microwave heating method under solvent-free, phase-transfer catalysis conditions. N 1,N 2-diaryl-N 1,N 2-bis-(3-substituted-phenyl)-oxamides (2a–s) were synthesized by the reaction of oxalyl chloride with meta-substituted benzanilines as key intermediates in tetrahydrofuran in the presence of TEA with moderate to good yields (40–90 %). The other target compounds, N 1,N 4-diaryl-N 1,N 4-bis-(3-substituted-phenyl)-fumaramides (3a–s), were obtained in moderate yields (16–76 %) by following the same procedure described in the previously reported literature (Yerdelen, 2012). The obtained spectroscopic data are in accordance with the predicted structures. The synthesis scheme of the compounds is presented in Scheme 1. In the proton nuclear magnetic resonance (1H-NMR) spectra, the resonance signals of ethylene bridge protons are usually registered as singlets in the range of 6.45–6.90 ppm and signals for aromatic protons showed between δ 6.51 and 7.68 ppm. The N-CH2 protons appeared at δ 4.80–4.92 ppm. The carbon nuclear magnetic resonance (13C-NMR) spectrum shows characteristic carbon resonance frequencies of carbonyl atoms in the range of 157.20–161.23 ppm for the compounds 2a–s and 164.47–163.85 ppm for the compounds 3a–s. The aromatic carbons appeared at δ 113.70–161.67 ppm and N-CH2 groups appeared at δ 51.58–63.59 ppm. In the positive electron impact (EI) mass spectra, the molecular ion peaks [M + H] show that the predicted compound has formed.

Cholinesterase inhibitory activity

The inhibitory activity of the compounds 2a–s and 3a–s against AChE (from electric eel) and BuChE (from equine serum) was measured according to the colorimetric assay of Ellman et al. (1961). Neostigmine, ambenonium, and galantamine were used as the reference compounds. The IC50 values of all tested compounds and their selectivity index for AChE over BuChE are summarized in Table 1. As seen in Table 1, all new fumaramide compounds 3a–s showed good inhibitory activity to both ChEs with micromolar concentrations. Among the target compounds, 3o (IC50 = 0.03 µM) showed the most potent inhibitory activity for AChE, being 2.33-, 35-, and 63-fold stronger than the reference compounds neostigmine (IC50 = 0.07 µM), galantamine (IC50 = 1.05 µM), and ambenonium (IC50 = 1.89 µM), respectively. In contrast, 3p exhibited the strongest inhibition to BuChE with an IC50 value of 0.03 µM, which was 3-, 154-, and 441-fold more potent than those of neostigmine (IC50 = 0.09 µM), ambenonium (IC50 = 4.61 µM), and galantamine (IC50 = 13.22 µM).

Table 1 In vitro inhibition IC50 (µM) and selectivity of target compounds 2a–s and 3a–s on AChE and BuChE
Full size table

The oxamide derivatives (2a–s) did not exhibit inhibitory activity against AChE (Table 1). Compounds 2b and 2g were found to have a moderate inhibitory activity on BuChE, with an IC50 of 42.78 and 87.22 µM, respectively. Compounds 2a and 2h exhibited the strongest inhibition to BuChE with IC50 values of 0.51 and 0.02 µM, respectively. The other compounds in the series were considered to be inactive, since their IC50 values were more than 100 µM.

The target compounds (2a–s and 3a–s) were synthesized using different meta-substituted anilines with varying electronic properties. In general, the target compounds 3k–s were found the most effective inhibitors against AChE and BuChE, which have various electron-drawing halogen groups (F, Cl, Br, I) at the meta-position of the phenyl ring. To support this approach, as shown in Table 1, 3a–b possessing an electron-donating group (–CH3) at the meta-position of the aniline ring moiety showed a decreased AChE inhibitory activity, while the corresponding compounds 3c–d possessing an electron-drawing group (–CF3) showed an increased AChE inhibitory activity. However compounds 3b (–CH3) and 3i (–OC2H5), which have electron-donating groups at the meta-position, were the most inactive in the series for AChE inhibition. In addition, 3e and 3f, possessing an electron-donating group (–C2H5) at the meta-position, showed the most decreased BuChE inhibitory activity. These results imply that an electron-drawing group at the meta-position of the aniline ring is beneficial to AChE and BuChE inhibitory activity. By contrast, especially in the series 2a–s, electron-donating groups showed an increase in both ChEs inhibition. It was also observed that within both series of meta-halogenated compounds cholinesterase inhibition activity increases with increasing electron-withdrawing effect.

The other physicochemical parameter, lipophilicity, is a property used in drug designing and drug discovery that has a major effect on absorption, distribution, metabolism, excretion and toxicity properties as well as pharmacological activity. The blood–brain barrier (BBB) represents a major obstacle to the delivery of drugs to the CNS. Particularly, CNS drugs cross BBB via the passive transport, which strongly depends on their lipophilicity.

From the IC50 values, the fumaramide derivatives seemed to be more effective than the oxamide derivatives 2a–s on AChE inhibition due to the ethylene bridge for the linker between the two carbonyl groups in the 3a–s series. A similar situation was seen with BuChE inhibition, (except for 2a and 2h). It can be stated that compounds in series 3a–s with higher lipophilicity exhibited higher cholinesterase inhibition than the series 2a–s. And these findings suggest us the anticholinesterase activity is strongly dependent on the presence of ethylene moiety in the inhibitors. In our previously reported study, the results of anticholinesterase activity showed that α,β-unsaturated moiety contributed to the interaction with cholinesterase enzymes (Yerdelen and Gul, 2013). The results of this study confirmed this approach.

Kinetic study

For the kinetic studies, the potent AChE inhibitor 3o and BuChE inhibitors 3p and 2h were chosen. The mechanism of cholinesterase inhibition was investigated with Ellman’s test (Ellman et al., 1961). The type of inhibition was illuminated from the analysis of Lineweaver–Burk plots, which were reciprocal rates versus reciprocal substrate concentrations for the different inhibitor concentrations resulting from the substrate-velocity curves for ChE. The plots showed that for AChE both increased slopes and intercepts at increasing concentration of the inhibitor 3o, indicating a mixed-type inhibition (Fig. 1a). Thus, compound 3o can bind to the CAS and the PAS of AChE. In Fig. 1b and c, the lines crossed the x axis in the same point (unchanged K m) and decreased V max with increasing inhibitor concentrations. This is a typical trend of non-competitive inhibition. Docking simulations showed that compounds 3p and 2h bound mainly to the CAS and PAS of BuChE, respectively.

Fig. 1
figure 1

The Lineweaver–Burk plots for the inhibition of cholinesterase enzymes by compounds 3o (a), 3p (b), and 2h (c)

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Molecular modeling studies

Molecular modeling studies were performed to investigate possible interactions between the most active compounds, 3o with AChE, and 3p and 2h with BuChE. The molecular modeling was performed with the docking program SYBYL X 2.0 (Surflex-Dock software). The structures of the enzymes were obtained from the Protein Data Bank. The docking results showed that the compound 3o displayed multiple binding patterns with Torpedo californica (TcAChE-1ACJ), as shown in Fig. 2a. In the 1ACJ-3o complex, oxygen atom from the carbonyl group created a hydrogen bond with OH of Ser122 (2.0 Å) of PAS. And the oxygen atom of the other carbonyl group formed an interaction: H-bond with NH group of Gly118 (2.5 Å) at the CAS of the enzyme. The results indicate that the binding of 3o to the CAS and PAS gorges of TcAChE explains the promising potency for AChE inhibition and reveals a mixed-type inhibition of the compound.

Fig. 2
figure 2

Docking models of compound–enzyme complex. Compound 3o (a) interacts with residues in the binding sites of TcAChE (a). Compounds 3p (b) and 2h (c) interacts with HuBuChE

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The most potent BuChE inhibitor, 3p, showed interactions with Thr120 and Gly116 residues of human butyryl cholinesterase enzyme (HuBuChE-1P0I) (Fig. 2b). Hydrogen bond interactions between the carbonyl group and OH group of Thr120 (2.7 Å) at the CAS of HuBuChE was occured. In HuBuChE, the oxyanion hole consists of highly conserved N–H dipoles, derived from amino acids of the main chain: Gly116, Gly117, and Ala119. And the other carbonyl group showed an hydrogen bond interaction with NH group of Gly116 (1.9 Å). We also performed a docking simulation between 2h and HuBuChE (1P0I) using same program. As shown in Fig. 2c, the 3-OCH3 phenyl ring entered the PAS of BuChE and interacted with three hydrogen bonds with residues OH group of Tyr440 (1.9 Å), NH group of Trp82 (2.4 Å), and NH group of Trp430 (2.6 Å). The results of the interaction displayed non-competitive inhibition by the compounds 3p and 2h.

Metal chelating effect

The chelating effect of the most potent compounds, 3o and 2h, for the metals such as Cu2+ and Fe2+ in ethanol was studied by using UV–Visible spectrophotometry with wavelength ranging from 200 to 500 nm (Huang et al., 2010; Joseph et al., 2008). The UV absorption of the compounds 3o and 2h in ethanol changed with the titration of Cu2+ and Fe2+. An increase in absorbance can be observed after adding CuSO4 (shown in Fig. 3a), indicating the formation of complex 3o–Cu2+. Similar behavior was also observed for the compound 3o when using Fe2+. These observations indicate that 3o could effectively chelate Cu2+, Fe2+, and, thereby, could serve as a metal chelator in treating AD. Besides this, an increasing absorbance was obtained with the titration of Fe2+ and 2h. This result indicates the formation of 2h–Fe2+ metal complex. The absorbance intensity decreased with the titration of 2h–Cu2+, as shown Fig. 3b.

Fig. 3
figure 3

UV–Vis (200–500 nm) absorption spectra of compound 3o (a) and 2h (b) alone or in the presence of CuSO4 and FeSO4. The final concentration of the compounds and metals were 100 μM

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Conclusion

In summary, a series of new oxamide and fumaramide analogs was synthesized and evaluated as AChE and BuChE inhibitors. According to the inhibition data, the fumaramides generally showed moderate to high anticholinesterase activity in which compounds 3o and 3p were the most potent inhibitors acting in low micromolar concentrations toward AChE and HuBuChE, respectively. Compound 3o (IC50 = 0.03 µM) was found the most potent AChE inhibitor in its series. Compound 3o also showed strong affinity (IC50 = 0.56 µM) for BuChE but 3p was found the most effective compound (IC50 = 0.03 µM) in the series. In series 2a–s, only four compounds (2a, 2b, 2g, and 2h) showed inhibitory activity exclusively for HuBuChE. Compound 2h showed promising inhibitory ability (IC50 = 0.02 µM) toward HuBuChE better to standards galantamine, neostigmine, and ambenonium with high selectivity. The kinetic studies suggested that the inhibition mechanisms of the investigated compounds could be different. The docking simulation showed that modeled derivative 3o created many interactions with the PAS and CAS gorges of TcAChE, confirming its high inhibitor potency and revealing a mixed-type inhibition. In addition, kinetic studies indicated that compounds 2h and 3p exhibited non-competitive inhibition against the HuBuChE enzyme. Consequently, the findings imply that the presence of an ethylene bridge in the fumaramide inhibitors had more influence than the oxamide derivatives on their inhibition of AChE and BuChE.