Introduction

AD is the most common type of dementia worldwide. It is characterized by a severe and progressive loss of memory (Craig et al., 2011; Jahn, 2013). The prevalence of AD, which increases with age, ranges from 1 to 2 % in the 65 years age group to 35 % or higher in the 85 years age group (Tayeb et al., 2012; Mayeux, 2003). General consensus supports the multifactorial nature of AD with decrease in the neurotransmitter acetylcholine (ACh), formation of amyloid β-protein (Aβ) plaques and abnormal posttranslational modifications of tau protein to yield neurofibrillary tangles. These pathological findings are considered to be closely associated with AD (Tomiyama et al., 1996; Krall and Sramek, 1999; Lahiri et al., 2003; Cummings, 2004; Bartolini et al., 2007; Gauthier and Poirier, 2008).

Most of the currently available compounds intended to treat AD do this by compensating for ACh deficiency and enhancing ACh-mediated transmission (Silman and Sussman, 2005). This is achieved by inhibition of cholinesterases (ChEs) (Giacobini, 2004), a family containing acetylcholinesterase (AChE, E.C. 3.1.1.7.) and butyrylcholinesterase (BChE, E.C. 3.1.1.8.) enzymes that cause degradation of ACh (Darvesh et al., 2003; Greig et al., 2001, 2005; Rizzo et al., 2011; Yiannopoulou and Papageorgiou, 2013). Tacrine, donepezil, rivastigmine and galantamine belong to group of ChE inhibitors (ChEIs) capable of cognitive, functional and behavioural improvement, although none of them proved to be effective against the progression of AD (Fig. 1) (Zemek et al., 2014). Tacrine (9-amino-1,2,3,4-tetrahydroacridine, THA) was the first ChEI approved for the treatment of AD. However, tacrine was withdrawn due to hepatotoxicity (Dejmek, 1990; Summers et al., 1989; Patocka et al., 2008). The 7-methoxy derivative, 9-amino-7-methoxy-1,2,3,4-tetrahydroacridine (7-MEOTA, Fig. 1), was found also to be effective and less hepatotoxic ChEI (Soukup et al., 2013; Marx, 1987; Korabecny et al., 2010, 2011; Korabecny et al., 2014).

Fig. 1
figure 1

Structures of ChEIs, derivatives of tacrine, polycyclic amines and 7-MEOTA-amantadine thioureas 4–10

Full size image

Aminoadamantanes (memantine, amantadine, Fig. 1) are used in the treatment of Parkinson’s disease (PD) and AD (Caumont and Octave, 2006; Ossola et al., 2011). Memantine is a non-competitive N-methyl-d-aspartate (NMDA) glutamate receptor antagonist (Bormann, 1989) approved for moderate to severe AD (Forest Pharmaceuticals Inc., 2003; Manning et al., 2011). Memantine (1-amino-3,5-dimethyladamantane) is used as a neuroprotective agent in the treatment of AD with different mechanism of action (NMDA receptor antagonist) (Kotermanski and Johnson, 2009; Lipton, 2006; Schmitt, 2005). Amantadine (1-aminoadamantane, adamantylamine) is currently used as an antiviral and an anti-PD drug (Blanpied et al., 2005). Amantadine is a weak NMDA receptor antagonist, which is considered to be beneficial for PD and AD patients that need the increase in dopaminergic transmission in order to compensate for the dopamine/glutamate imbalance (Page et al., 2000; Peeters et al., 2002; Magazanik et al., 1996).

Simoni et al. (2012) synthesized a novel series of compounds linking galantamine and memantine following the strategy of multi-target-directed ligands (MTDLs). These compounds seem to be effective in treating complex diseases due to their ability to interact with multiple targets responsible for the disease pathogenesis. Selected compounds from this study (1–3) were found to be capable of inhibiting rat AChE in the nanomolar range. In the MK-801 binding assay against NMDA receptor and against NR2B-containing NMDA receptor (using the ifenprodil binding assay), these dual hybrids showed activity in the micromolar range. Furthermore, neuroprotective profile against NMDA-mediated neurotoxicity was tested using SHSY-5Y cell 5Y cell viability assay and these analogues were found to inhibit NMDA-induced neurotoxicity in sub-nanomolar range (Cavalli et al., 2008; Spilovska et al., 2013; Simoni et al., 2012; Zheng et al., 2014).

Based on the MTDL strategy by Simoni et al., a series of 7-MEOTA-amantadine urea-linked derivatives were designed, synthesized and evaluated as AChE/BChE inhibitors. The design was focused on the compounds development capable of interacting with both the active site gorge and the peripheral anionic site (PAS) of the enzyme (i.e. a dual binding site inhibitors). These heterodimers were compared with 7-MEOTA-amantadine thioureas, 7-MEOTA, tacrine and galantamine-memantine dimers reported previously (Cavalli et al., 2008; Spilovska et al., 2013; Simoni et al., 2012). The synthetic approach and in vitro ChE inhibition properties are discussed along with molecular modeling studies.

Experimental section

Materials and methods

7-MEOTA was prepared at the Department of Toxicology and Military Pharmacy according to the method described earlier (Pohanka et al., 2008). All reagents were obtained from Sigma-Aldrich in reagent grade quality. All experiments were carried out under nitrogen atmosphere. Thin-layer chromatography (TLC) was performed on aluminium sheets pre-coated with silica gel 60 F254 (Merck). Column chromatography was performed at normal pressure on silica gel 100 (particle size 0.063–0.200 mm, 70–230 mesh ASTM, Fluka). Elemental analysis was measured at Perkin-Elmer CHN Analyser 2400 Serie II apparatus. Mass spectra were recorded using a combination of high-performance liquid chromatography and mass spectrometry. The HP1100 HPLC system was obtained from Agilent Technologies (Waldbronn, Germany). It consisted of a G1322A vacuum degasser, G1311A quaternary pump, G1313A autosampler and a MSD1456 VL quadrupole mass spectrometer equipped with an electrospray ionization source. Nitrogen for mass spectrometer was supplied by a Whatman 75–720 nitrogen generator. Data were collected in positive ion mode with an ESI probe voltage of 4,000 V. The pressure of nebulizer gas was set up to 35 psig. Drying gas temperature was operated at 335 °C and flow at 13 L/min. 1H-NMR and 13C-NMR spectra were recorded with a Varian S500 spectrometer operating at 500 and 125 MHz, respectively, in deuteriochloroform (CDCl3; 7.27 (D), 77.2 (C) ppm) or hexadeuteriodimethylsulfoxide (DMSO-d 6; 2.50 (D), 39.7 (C) ppm) using tetramethylsilane (TMS) as internal reference (= 0 ppm for both nuclei). Chemical shifts are reported in parts per milion (ppm, δ) relative to TMS. The assignment of chemical shifts is based on standard NMR experiments (1H, 13C, 1H-1H COSY, 1H-13C HSQC, HMBC, DEPT). Melting points were measured on a micro heating stage PHMK 05 (VEB Kombinant Nagema, Radebeul, Germany) and are uncorrected.

General synthetic procedure

In this paper, the required 1-adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)alkyl)urea 2,3-dihydroxysuccinates (11–17) were obtained via the reaction of early prepared 7-MEOTA-adamantylamine thioureas (4–10; 10 mmol) with 2,4,6-trimethylbenzonitrile-N-oxide (11 mmol) in dichloromethane (5 mL). The reaction mixture was stirred 24 h at room temperature and purified via column chromatography using chloroform/methanol (9:1) as mobile phase. The final products were converted to tartaric salts by the addition of equimolar tartaric acid and stirred in absolute ethanol (10 mL) for 24 h. The final 1-adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)alkyl)urea 2,3-dihydroxysuccinates (11–17) were obtained as white to yellow powders in good yields.

Experimental procedures and compound characterization

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl)urea 2,3-dihydroxysuccinate (11)

White powder yields 70.8 %; m.p. = 60.4–63.0 °C; 1H-NMR (DMSO-d 6) δ 7.76 (m, 1H, CH, H-5), 7.72 (m, 1H, CH, H-8), 7.45 (m, 1H, CH, H-6), 4.07 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.94 (s, 3H, OCH3), 3.81 (m, 2H, CH2, H-1′), 2.95 (m, 2H, CH2, H-4), 2.77 (m, 2H, CH2, H-1), 2.49 (m, 6H, 3 × CH2, H-2″, H-6″, H-10″), 1.97 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.82 (m, 6H, 3 × CH2, H-2, H-3, H-2′), 1.58 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″); 13C NMR (DMSO-d 6) δ 173.9 (C-1′″, C-4′″), 158.8 (C=O), 156.4 (C-7), 154.4 (C-9), 151.0 (C-4a), 134.2 (C-10a). 123.3 (C-6), 122.8 (C-5), 118.0 (C-9a), 111.9 (C-8a), 103.4 (C-8), 71.8 (C-2′″, C-3′″), 55.9 (OCH3), 50.3 (C-1′), 42.1 (C-2′), 40.2 (C-2″, C-6″, C-10″), 36.2 (C-4″, C-7″, C-9″), 29.1 (C-4), 28.3 (C-3″, C-5″, C-8″), 24.9 (C-1), 22.2, 21.0 (C-2, C-3); Elemental analysis: calculated 62.19 % C, 7.07 % H, 9.36 % N; found 62.10 % C, 6.93 % H, 9.55 % N; ESI–MS: m/z 448.2 [M]+ (calculated for: [C27H37N4O2]+ 448.3).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl)urea 2,3-dihydroxysuccinate (12)

Yellow powder yields 90.1 %; m.p. = 75.2–78.1 °C; 1H-NMR (DMSO-d 6) δ 7.81 (d, 1H, CH, H-5, J = 8.1 Hz), 7.66 (m, 1H, CH, H-8), 7.45 (d, 1H, CH, H-6), 4.11 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.92 (s, 3H, OCH3), 3.69 (m, 2H, CH2, H-1′), 3.06 (m, 2H, CH2, H-3′), 3.00 (m, 2H, CH2, H-4), 2.74 (m, 2H, CH2, H-1), 1.96 (m, 6H, 3 × CH2, H-2″, H-6″, H-10″), 1.82 (m, 6H, 3 × CH2, H-2, H-3, H-2′), 1.70 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.58 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″); 13C NMR (DMSO-d 6) δ 174.0 (C-1′″, C-4′″), 158.1 (C=O), 156.5 (C-7), 154.1 (C-9), 151.0 (C-4a), 134.6 (C-10a), 123.4 (C-6), 122.8 (C-5), 118.2 (C-9a), 112.2 (C-8a), 103.0 (C-8), 72.0 (C-2′″, C-3′″), 55.9 (OCH3), 44.3 (C-1′), 42.1 (C-2′), 36.3 (C-4″, C-7″, C-9″), 35.6 (C-3′), 32.1 (C-3″, C-5″, C-8″), 29.1 (C-2″, C-6″, C-10″), 29.0 (C-4), 25.0 (C-1), 22.2, 20.9 (C-2, C-3); Elemental analysis: calculated 62.73 % C, 7.24 % H, 9.14 % N; found 62.32 % C, 7.20 % H, 9.05 % N; ESI–MS: m/z 462.2 [M]+ (calculated for: [C28H39N4O2]+ 462.3).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)butyl)urea 2,3-dihydroxysuccinate (13)

White powder yields 67.0 %; m.p. = 80.5–83.3 °C; 1H-NMR (DMSO-d 6) δ 7.79 (d, 1H, CH, H-5, J = 8.5 Hz), 7.59 (m, 1H, CH, H-8), 7.45 (m, 1H, CH, H-6), 4.03 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.89 (s, 3H, OCH3), 3.63 (m, 2H, CH2, H-1′, 2.95 (m, 2H, CH2, H-4), 2.93 (m, 2H, CH2, H-4′), 2.70 (m, 2H, CH2, H-1), 1.95 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.80 (m, 8H, 4 × CH2, H-2, H-3, H-2′, H-3′), 1.57 (m, 6H, 3 × CH2, H-2″, H-6″, H-10″), 1.37 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″); 13C NMR (DMSO-d 6) δ 174.3 (C-1′″, C-4′″), 157.3 (C = O), 156.3 (C-7), 153.2 (C-9), 151.8 (C-4a), 136.2 (C-10a), 124.1 (C-6), 122.7 (C-5), 118.7 (C-9a), 113.3 (C-8a), 103.0 (C-8), 71.9 (C-2′″, C-3′″), 55.9 (OCH3), 49.5 (C-1′), 47.0 (C-2′, C-3′), 38.6 (C-4′), 36.3 (C-4″, C-7″, C-9″), 29.8 (C-4), 29.1 (C-3″, C-5″, C-8″), 27.6 (C-2″, C-6″, C-10″), 25.0 (C-1), 22.3, 21.3 (C-2, C-3); Elemental analysis: calculated 63.24 % C, 7.40 % H, 8.94 % N; found 63.35 % C, 7.35 % H, 8.95 % N; ESI–MS: m/z 476.2 [M]+ (calculated for: [C29H41N4O2]+ 476.3).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)pentyl)urea 2,3-dihydroxysuccinate (14)

White–yellow powder yield 87.9 %; m.p. = 83.8–86.2 °C; 1H-NMR (DMSO-d 6) δ 7.79 (d, 1H, CH, H-5, J = 9.2 Hz), 7.60 (m, 1H, CH, H-8), 7.43 (dd, 1H, CH, H-6, J = 9.2, 2.1 Hz), 4.08 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.89 (s, 3H, OCH3), 3.67 (m, 2H, CH2, H-1′), 2.96 (m, 2H, CH2, H-4), 2.89 (m, 2H, CH2, H-5′), 2.70 (m, 2H, CH2, H-1), 1.95 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.80 (m, 8H, 4 × CH2, H-2, H-3, H-3′ H-4′), 1.65 (m, 2H, CH2, H-2′), 1.56 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″), 1.30 (m, 6H, 3 × CH2, H-2″, H-6″, H-10″); 13C NMR (DMSO-d 6) δ 174.0 (C-1′″, C-4′″), 157.3 (C=O), 156.4 (C-7), 153.8 (C-9), 151.3 (C-4a), 135.2 (C-10a), 123.3 (C-6), 123.1 (C-5), 118.3 (C-9a), 112.7 (C-8a), 103.3 (C-8), 71.9 (C-2′″, C-3′″), 56.0 (OCH3), 47.2 (C-1′), 42.2 (C-3′, C-4′), 38.8 (C-5′), 36.3 (C-4″, C-7″, C-9″), 30.3 (C-2′), 29.3 (C-4), 29.1 (C-3″, C-5″, C-8″), 24.9 (C-1), 23.8 (C-2″, C-6″, C-10″), 22.1, 21.0 (C-2, C-3); Elemental analysis: calculated 63.73 % C, 7.55 % H, 8.74 % N; found 63.60 % C, 7.60 % H, 8.55 % N; ESI–MS: m/z 490.2 [M]+ (calculated for: [C30H43N4O2]+ 490.3).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)hexyl)urea 2,3-dihydroxysuccinate (15)

Yellow powder yields 67.0 %; m.p. = 84.0–86.8 °C; 1H-NMR (DMSO-d 6) δ 7.80 (d, 1H, CH, H-5, J = 9.1 Hz), 7.60 (m, 1H, CH, H-8), 7.42 (dd, 1H, CH, H-6, J = 9.1, 2.2 Hz), 4.07 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.89 (s, 3H, OCH3), 3.66 (t, 2H, CH2, H-1′, J = 6.1 Hz), 2.96 (m, 2H, CH2, H-4), 2.87 (m, 2H, CH2, H-6′), 2.70 (m, 2H, CH2, H-1), 1.95 (m, 3H, 3 × CH, H-3″, H-5″ H-8″), 1.81 (m, 8H, 4 × CH2, H-2, H-3, H-4′, H-5′), 1.63 (m, 2H, CH2, H-2′), 1.56 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″), 1.31 (m, 8H, 4 × CH2, H-3′, H-2″, H-6″, H-10″); 13C NMR (DMSO-d 6) δ 174.0 (C-1′″, C-4′″), 157.3 (C=O), 156.4 (C-7), 153.7 (C-9), 151.4 (C-4a), 135.4 (C-10a), 123.5 (C-6), 123.0 (C-5), 118.4 (C-9a), 112.8 (C-8a), 103.2 (C-8), 71.9 (C-2′″, C-3′″), 55.9 (OCH3), 47.0 (C-1′), 42.2 (C-4′, C-5′), 38.7 (C-6′), 36.3 (C-4″, C-7″, C-9″), 30.6 (C-2′), 30.2 (C-3′), 29.4 (C-4), 29.1 (C-3″, C-5″, C-8″), 26.1 (C-2″, C-6″, C-10″), 25.0 (C-1), 22.2, 21.1 (C-2, C-3); Elemental analysis: calculated 64.20 % C, 7.70 % H, 8.56 % N; found 64.15 % C, 7.83 % H, 8.50 % N; ESI–MS: m/z 504.3 [M]+ (calculated for: [C31H45N4O2]+ 504.4).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)heptyl)urea 2,3-dihydroxysuccinate (16)

Orange powder yields 81.5 %; m.p. = 65.2–68.2 °C; 1H-NMR (DMSO-d 6) δ 7.80 (d, 1H, CH, H-5, J = 9.1 Hz), 7.62 (m, 1H, CH, H-8), 7.44 (m, 1H, CH, H-6), 4.10 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.89 (s, 3H, OCH3), 3.68 (t, 2H, CH2, H-1′, J = 6.5 Hz), 2.96 (m, 2H, CH2, H-4), 2.86 (m, 2H, CH2, H-7′), 2.69 (m, 2H, CH2, H-1), 1.95 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.81 (m, 8H, 4 × CH2, H-2, H-3, H-5′ H-6′, 1.65 (m, 2H, CH2, H-2′, 1.56 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″), 1.23 (m, 8H, 4 × CH2, H-3′, H-2″, H-6″, H-10″); 13C NMR (DMSO-d 6) δ 173.9 (C-1′″, C-4′″), 157.3 (C=O), 156.4 (C-7), 153.9 (C-9), 151.1 (C-4a), 135.0 (C-10a), 123.2 (C-6), 123.1 (C-5), 118.2 (C-9a), 112.6 (C-8a), 103.3 (C-8), 72.0 (C-2′″, C-3′″), 56.0 (OCH3), 47.1 (C-1′), 42.2 (C-5′, C-6′), 38.9 (C-7′), 36.3 (C-4″, C-7″, C-9″), 30.5 (C-2′), 30.1 (C-3′), 29.4 (C-4), 29.1 (C-3″, C-5″, C-8″), 28.6 (C-4′), 26.4 (C-2″, C-6″, C-10″), 24.9 (C-1), 22.1, 21.0 (C-2, C-3); Elemental analysis: calculated 64.65 % C, 7.84 % H, 8.38 % N; found 64.58 % C, 7.83 % H, 8.40 % N; ESI–MS: m/z 518.4 [M]+ (calculated for: [C32H47N4O2]+ 518.5).

1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)octyl)urea 2,3-dihydroxysuccinate (17)

Yellow–brown powder yield 60.1 %; m.p. = 57.3–60.0 °C; 1H-NMR (DMSO-d 6) δ 7.81 (d, 1H, CH, H-5, J = 9.1 Hz), 7.61 (m, 1H, CH, H-8), 7.44 (m, 1H, CH, H-6), 4.10 (m, 2H, 2 × CH, H-2′″, H-3′″), 3.89 (s, 3H, OCH3), 3.68 (t, 2H, CH2, H-1′ J = 6.8 Hz), 2.96 (m, 2H, CH2, H-4), 2.86 (m, 2H, CH2, H-8′), 2.69 (m, 2H, CH2, H-1), 1.95 (m, 3H, 3 × CH, H-3″, H-5″, H-8″), 1.81 (m, 8H, 4 × CH2, H-2, H-3, H-6′, H-7′), 1.65 (m, 2H, CH2, H-2′), 1.56 (m, 6H, 3 × CH2, H-4″, H-7″, H-9″), 1.23 (m, 10H, 5 × CH2, H-3′, H-4′, H-2″, H-6″, H-10″); 13C NMR (DMSO-d 6) δ 174.0 (C-1′″, C-4′″), 157.3 (C=O), 156.4 (C-7), 153.9 (C-9), 151.2 (C-4a), 135.0 (C-10a), 123.2 (C-6), 123.1 (C-5), 118.2 (C-9a), 112.6 (C-8a), 103.3 (C-8), 72.0 (C-2′″, C-3′″), 56.0 (OCH3), 47.1 (C-1′), 42.2 (C-6′, C-7′), 38.9 (C-8′), 36.3 (C-4″, C-7″, C-9″), 30.5 (C-2′), 30.2 (C-3′), 29.4 (C-4), 29.1 (C-3″, C-5″, C-8″), 28.8 (C-4′), 26.4 (C-2″, C-6″, C-10″), 24.9 (C-1), 22.1, 21.0 (C-2, C-3); Elemental analysis: calculated 65.08 % C, 7.97 % H, 8.20 % N; found 65.01 % C, 8.10 % H, 8.35 % N; ESI–MS: m/z 532.4 [M]+ (calculated for: [C33H49N4O2]+ 532.5).

In vitro testing

A sunrise multichannel spectrophotometer (Tecan, Salzburg, Austria) was used for all cholinesterase activity measurements. A previously optimized Ellman procedure was slightly modified in order to estimate anticholinergic properties (Bielavsky, 1977). 96-well photometric microplates made from polystyrene (Nunc, Rockilde, Denmark) were used for measuring purposes. Human recombinant AChE or human plasmatic BChE (Aldrich; commercially purified by affinity chromatography) was suspended into phosphate buffer (pH 7.4) up to final activity 0.002 U/μL. Cholinesterase (5 μL), freshly mixed solution of 0.4 mg/mL 5,5′-dithio-bis(2-nitrobenzoic) acid (40 μL), 1 mM acetylthiocholine chloride in phosphate buffer (20 μL) and appropriate concentration of inhibitor (1 mM–0.1 nM; 5 μL), were injected per well. Absorbance was measured at 412 nm after 5-min incubation using automatic shaking of the microplate. The obtained data were used to compute percentage of inhibition (I; Eq. 1):

$$ I = 1 - \frac{{\Delta A_{i} }}{{\Delta A_{0} }} \left[ \% \right] $$
(1)

where ΔA i indicates absorbance change provided by cholinesterase exposed to hAChE inhibitors, and ΔA 0 indicates absorbance change caused by intact cholinesterase (phosphate buffer was applied instead of hAChE inhibitor). IC50 values were calculated using Origin 6.1 (Northampton, MA, USA). Percentage of inhibition for the given anticholinergic compound was overlaid by proper curve chosen according to optimal correlation coefficient. IC50 as well as upper limit of inhibition (maximal inhibition provided by given compound) was computed.

Molecular modelling studies

Docking calculations were performed using AutoDock Vina (Yan and Wang, 2012). The molecular models were built and minimized with UCSF chimera 1.3 (Amber Force Filed) (Trott and Olson, 2010). The structure of both enzymes, human AChE (hAChE, PDB ID: 1B41) and human BChE (hBChE, PDB ID: 1P0I), was prepared using PyMol 1.1 from crystal structures (Pettersen et al., 2004; Kryger et al., 2000). Compounds used in this study and both enzymes were prepared using AutoDock Tools 1.5.2. in charged form (The Pymol Molecular Graphics System, Morris et al., 2009). Molecules of water with other nonenzymatic molecules were removed (withdrawing the fasciculin 2 from hAChE and molecules of water from both enzymes), and hydrogens were added (Harel et al., 1993). The 3D affinity grid box in the x-, y- and z-axes was 66, 66 and 66 with spacing 0.253 Å for hAChE, within the hBChE grid box dimensions were set to x = 46, y = 60 and z = 46 with spacing 0.375 Å. For the hAChE docking, the grid for energy was set in the coordinates x = 119.775, y = 117.597 and z = −128.964, within hBChE the coordinates were adjusted to x = 137.871, y = 115.156 and z = 38.652. The hAChE residues Trp86, Tyr72, Trp286, Asp74, Tyr341 and Phe297 were set to be flexible by AutoDock Tools 1.5.2, for hBChE amino acid residues Glu325, His438, Trp82, Asp70 and Tyr332 were selected as flexible. Flexible ligand docking was performed for the selected compound 14 and compared to previously reported compound 7. The docking calculations were made on Mac Pro 4.1 Quad-Core Intel Xeon 2.93 GHz. At the end of calculation, AutoDock Vina performed cluster analysis. The visualization was carried out in PyMol 1.1. Hydrogens were finally removed to improve figures clarity.

Results and discussion

Chemistry

The synthesis of prepared 7-MEOTA-amantadine thioureas 2,3-dihydroxysuccinates 4–10 was described in previous work (Spilovska et al., 2013). The preparation of 1-adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)octyl)ureas 2,3-dihydroxysuccinates 11–17 was accomplished in two steps. The 7-MEOTA-amantadine thioureas were transferred to 7-MEOTA-amantadine ureas using 2,4,6-trimethylbenzonitril-N-oxide (MNO) as oxidative agent. The reaction was performed in dichloromethane for 24 h at room temperature. The formation of urea moiety was confirmed by the 13C NMR signal in the range 156–159 ppm for the carbonyl carbon. The final heterodimers were converted to tartaric salts by addition of equimolar amount of tartaric acid. The conversion to tartaric salt was necessary for better solubility for in vitro assessment. 7-MEOTA-amantadine ureas 2,3-dihydroxysuccinates were acquired in yields ranging from 60 to 90 %. The synthetic route leading to novel heterodimers 11–17 is shown in Scheme 1.

Scheme 1
scheme 1

Synthesis of 7-MEOTA-amantadine ureas derivatives

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Biological assay

The AChE and BChE inhibitory activity of all 14 7-MEOTA-amantadine derivatives were determined by the spectroscopic method described by Ellman et al. using human AChE and BChE (Ellman et al., 1961; Pohanka et al., 2008). Amantadine, THA and 7-MEOTA were used as standards. The results were expressed as IC50 values for hAChE/hBChE, and they are summarized in Table 1. The novel urea heterodimers seem to have a favourable effect on AChE and BChE inhibition. Compared to THA, which had IC50 0.5 µM for hAChE and IC50 0.023 µM for hBChE, the other two reference standards (7-MEOTA and amantadine) were less potent inhibitors.

Table 1 IC50 values of reference standards and tested heterodimers and logP values
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7-MEOTA and amantadine exhibited two orders of magnitude decrease in hAChE inhibitory activity in comparison with THA. Moreover, THA was stronger hBChE inhibitor compared to 7-MEOTA and amantadine. All new hybrids were more potent hAChE and hBChE inhibitors than 7-MEOTA with IC50 values ranging from 5.02 to 0.47 µM for thioureas (4–10) and from 4.98 to 0.69 µM for ureas (11–17). In the 7-MEOTA-amantadine thioureas, series five compounds had IC50 values in sub-micromolar range for hAChE. Only two derivatives (5 and 7) exhibited inhibition potency in sub-micromolar range for hAChE. However, compounds 4, 7, 8, 9 and 10 showed sub-micromolar inhibition potency for hBChE. The best inhibitory activity from 4 to 10 had compound 7, bearing five methylene groups in the linker. This derivative displayed potency in the same order of magnitude to THA for hAChE. The selectivity index (SI) was calculated for all newly evaluated compounds. All novel compounds (except analogues 5, 12 and 17) have lower SI values compared to 7-MEOTA or amantadine and can be considered as agents more selective for hBChE.

The 7-MEOTA-amantadine ureas series 11–17 were synthesized and biologically evaluated to determine different binding affinities towards both human cholinesterases. These heterodimers demonstrated potent inhibitory activity against hAChE and hBChE with IC50 values ranging from the micromolar to sub-micromolar. In particular, compound 14 represented by 7-MEOTA and amantadine linked with five carbon chain exhibited the highest inhibition activity of hAChE and hBChE. However, this derivative resulted in a 1.4-fold decreased activity for hAChE and 9.6-fold decreased inhibition potency for hBChE relative to THA. Furthermore, compound 14 was ascertained to be 15.2-fold more potent inhibitor of hAChE than 7-MEOTA. None of the novel compounds presented significant activity in comparison with nanomolar galantamine-memantine dimers (1–3, Table 1).

Considering differences between 7-MEOTA-amantadine thioureas and their urea analogues in relation to the linker length, thioureas derivatives 4–7 and 10 had very similar IC50 values as their urea analogues 11–14 and 17 for hAChE. By other two thiourea (8, 9) and urea derivatives (15, 16), this trend in inhibition ability was not observed. Essentially, compound 8 and 9 exhibited one order of magnitude higher inhibitory activity than heterodimers 15 and 16. Moreover, five derivatives of urea (11, 12, 13, 15, 16) proved enhanced inhibitory capability for hAChE in comparison with their thiourea counterparts. Regarding the inhibitory activity of tested ureas for hBChE (apart from the analogue 14), these hybrids had lower inhibition ability than original thioureas. However, the difference in the IC50 values was statistically insignificant. Regarding selectivity index results, 7-MEOTA was found more selective towards hAChE, whereas heterodimers 7 and 14 were predominantly selective towards hBChE. As shown in Table 1, from both series of thiourea and urea, derivatives are the best cholinesterase inhibitors compounds 7 and 14 bearing five methylene groups in the linker. This may be due the fact that so long linker is optimal to interact with the gorge of the enzyme in comparison with other thiourea and urea heterodimers. Futhermore, IC50 values of these two inhibitors 7 and 14 are very similar. Enzyme activities for reference standards THA and 7-MEOTA and of tested urea 14 for hAChE/hBChE are displayed in Figs. 2 and 3.

Fig. 2
figure 2

hAChE activity for THA, 7-MEOTA and 14

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Fig. 3
figure 3

hBChE activity for THA, 7-MEOTA and 14

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Based on the fact that studying lipophilicity is important in the evaluation of anti-AD drugs, the logP values were calculated. The logP (partition coefficient) is one of the important coefficients, which was well-defined by Lipinski in the “rule of five” for “drug-like” molecules (Lipinski et al., 2001). According to Lipinski, logP value for chemical compounds to be centrally active should be less than five. The logP values for 7-MEOTA-amantadine thiourea and urea derivatives are displayed in Table 1. All of the compounds showed logP values more than five proposing their highly lipophilic character. In comparison with thiourea subset, urea derivatives demonstrated lower logP values, plausibly due to their ability to easier formation of hydrogen bonds with amino acid residues within ChE active site. Even though newly synthesized inhibitors possess logP values far from the optimal, their effect to cross-biological membranes needs to be determined in vivo.

Based on in vitro results, it is not clear whether the most active urea analogue manages higher potency towards both binding sites (active and peripheral) of hAChE as thiourea analogue with the same spacer length. Therefore, only the most promising inhibitors (7 and 14) were subjected to kinetic analysis of the hAChE inhibition and molecular modelling studies to clarify the differences in their binding.

The inhibition mechanism of compounds 7 and 14 was investigated using steady-state inhibition of acetylthiocholine (ATCh) hydrolysis. The inhibition type was elucidated from the nonlinear regression analysis using GraphPad Prism 5.02 software (GraphPad Software, San Diego, CA, USA). Results for each inhibition model (competitive, noncompetitive, uncompetitive and mixed) were compared with sum of squares F test. Analysis confirmed competitive type of inhibition (p < 0.05) for both compounds. Figure 4 shows Lineweaver–Burk reciprocal plots of measured data. A K i value 0.409 ± 0.157 µM for inhibitor 7 and 0.163 ± 0.057 µM for 14 was estimated by the nonlinear regression analysis.

Fig. 4
figure 4

Steady-state inhibition of hAChE hydrolysis substrate ATCh by compounds 7 and 14. Lineweaver–Burk reciprocal plots of initial velocity and substrate concentrations (0.781–6.25 nM) for different concentrations of tested inhibitors. Lines were derived from a weighted least-squares analysis of data

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Molecular docking simulations for derivative 14 into hAChE active site were performed using the AutoDock Tools 1.5.2 software. We used the X-ray structure of the hAChE-fasciculin-2 complex (PDB ID: 1B41), as the enzyme of same origin was used in the in vitro biochemical assay (Fig. 5). Moreover, hAChE obtained from PDB is available in high resolution and represents a good tool for molecular modeling. Acquired docking simulations showed dual binding site character; 7-MEOTA moiety was observed to enter the catalytic anionic site (CAS), while adamantyl cage is located at the peripheral anionic site (PAS) (Fig. 5). 7-MEOTA moiety provides parallel π–π stacking with Trp86 (3.4 Å) and cation-π contact with Tyr337 (3.4 Å). Tyr337 could provide additional hydrogen bonding between the heterocyclic tacrine nitrogen and the Tyr337 hydroxyl group. Adamantyl scaffold could have aliphatic-π contact with both Trp286 (3.6 Å), and Phe297 (4.0 Å) and several weak Van der Waals interactions (Val294—4.1 Å, Leu289—4.1 Å). Spacer has two sections: urea group interacts via hydrogen bonds to Tyr124 (2.3 Å, 2.4 Å) and Tyr341 (3.6 Å), while methylene bridge is constricted to aromatic amino acid residues (Phe338—3.6 Å; Phe297—3.7 Å) through hydrophobic interactions. The binding energy for 14 (−12.0 kcal/mol) is similar to that found for the most potent derivative 7 (−11.1 kcal/mol) in the 7-MEOTA-amantadine thiourea class. Similar spatial orientation of 7 and 14 within hAChE active site can explain similar in vitro activity (Table 1). Only His438 (3.9 Å), from the catalytic triad seems to provide weak hydrophobic interaction with cyclohexyl moiety of 7-MEOTA.

Fig. 5
figure 5

Top-scored docking poses of the compounds 14- and 7-hAChE complex. The binding pattern of compound 14 is shown in green, previously reported analogue 7 is depicted in yellow and important amino acid residues are highlighted in pale-brown (Color figure online)

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The flexible docking procedure was applied to hBChE (PDB ID: 1P01) to determine possible differences of binding modes concerning compounds 14 and 7. Since no crystal structure of tacrine and tacrine derivatives within hBChE active site is available the choice of hBChE crystal structure from PDB was based on a good resolution of the enzyme. In addition, the enzyme of the same origin was used in the in vitro assay. The docking simulations confirmed predicted orientations for both derivatives 14 (−10.4 kcal/mol) and 7 (−10.3 kcal/mol) with minor changes in their spatial arrangements (Fig. 6). Evaluation of 14 revealed hydrogen bond interactions between heterocyclic nitrogen of 7-MEOTA moiety and amino acid residues of catalytic triad, Ser198 (3.9 Å), His438 (4.2 Å), while Glu325 is not affected. His438 (3.7 Å) also provides T-shaped π–π interaction. Besides the three ring core of 14, it is also involved in T-shaped π–π interaction with Phe329 (4.3 Å) and parallel π–π stacking with Trp231 (4.0 Å). Furthermore, the most energetically favoured binding mode of 14 places the adamantyl skeleton to vicinity of Trp82 (3.6 Å), Tyr440 (4.1 Å) and Trp440 (4.9 Å) displaying aliphatic-π contact. In this orientation, urea moiety in the linker established hydrogen bonding to Tyr332 (2.7 Å) and Asp70 (3.4 Å).

Fig. 6
figure 6

Two major binding modes of 14 (green) and 7 (yellow) hBChE. Both compounds as well as attached amino acid residues (pale-brown) are rendered as sticks (Color figure online)

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Conclusion

In conclusion, we have reported the synthesis and pharmacological evaluation of a new series of multi-target-directed ligands derived from 7-MEOTA and amantadine for the treatment of AD. Our study extended the previously reported series of biologically active 7-MEOTA-amantadine thiourea derivatives to 7-MEOTA-amantadine ureas. These urea analogues were evaluated as potential inhibitors of AChE and BChE, but expected rise in cholinesterase inhibition potency was not observed. The Lineweaver–Burk plot revealed that the best thiourea derivate 7 and best urea analogue 14 inhibited hAChE competitively. Further investigations of AD therapeutic candidates (anti-amyloid aggregation, channel activity of NMDA receptor) based on these results are in progress.