Experimental

Materials and methods

Reagents and solvents for compound preparation were purchased from Sigma-Aldrich (St. Louis, MO, USA) and BLD Pharmatech GmbH (Reinbek, Germany). CEM Focused Microwave TM Synthesis System (Discover SP, Matthews, NC, SAD) was used for microwave synthesis. The reactions were monitored by thin-layer chromatography plates coated with silica gel (Sigma-Aldrich, St. Louis, MO, USA). TLC plates were visualized by UV irradiation (254 nm) or by iodine fumes. 1D and 2D 1H and 13C NMR spectra were recorded on a Bruker Avance III HD 400 MHz/54 mm Ascend spectrometer (Bruker Optics Inc, Billerica, MA, USA) in deuterated chloroform or methanol at 298 K. Chemical shifts are given in ppm downfield from tetramethylsilane (TMS) as an internal standard. Hydrogen and carbon atoms of the phenyl group are marked with a Ph; of the benzyl group with a Bn; of the morpholine ring with a morph; of the tert-butyl group as t-bu; of the cyclohexyl ring with a chx; of iso-butyl as i-bu; of the formaldehyde group as form; of the acetic group as Ac; and of the tosyl group as ts. Methylene groups are marked with CH2 x, where x is the mark from which the component CH2 group is formed. Note regarding the 1H and 13C spectra of compounds 1 to 10 at 298 K, some Ugi adducts may appear as a mixture of conformers (Figures S1-S21). Melting points were determined on a Melting Point B-540 apparatus (Büchi, Essen, Germany) and are uncorrected. All compounds showed purities >97 % by HPLC analyses performed on an Agilent 1260 series instrument equipped with a quaternary pump, autosampler, column compartment, and diode-array detector (DAD). HPLC conditions: Zorbax Extend-C18 column, 4.6×250 mm, 5 μm pore size; column temperature 40 °C; flow rate 1.0 mL min-1; mobile phase A: 0.1 % TFA in H2O; mobile phase B: 0.1 % TFA in CH3CN; linear gradient 10/90/90/10/10 % B in time intervals 0/10/15/20/25; the volume of injection 5 μL; UV detection at 220 nm (Figures S22-S32). HRMS analyses were carried out on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer.

Synthesis of compounds

Conventional synthesis - general procedure: Appropriate amine (1 mmol), aldehyde (1 mmol), carbocyclic acid (1 mmol), and isocyanide (1 mmol) were weighed into a round-bottom flask, and methanol (1 mL) was added. The solution was mixed on a magnetic stirrer for 24 h. Methanol was evaporated, and the reaction mixture was made alkaline with sodium hydrogen carbonate solution (pH 8). After extraction with ethyl acetate (2×20 mL), the organic extracts were dried over anhydrous sodium sulphate. Ethyl acetate was evaporated, and the residue was purified by column chromatography (silica gel, DCM : MeOH = 9 : 1).

Microwave-aided synthesis - general procedure: Appropriate amine (1 mmol), aldehyde (1 mmol), carbocyclic acid (1 mmol), and isocyanide (1 mmol) were added to a microwave vial together with methanol (0.5 mL) and a stirrer bar. After the designated time and temperature, methanol was evaporated, and the reaction mixture was made alkaline with the addition of sodium hydrogen carbonate solution (pH 8). After extraction with ethyl acetate (2×20 mL), the organic extracts were dried over anhydrous sodium sulphate. Ethyl acetate was evaporated, and the residue was purified by column chromatography (silica gel, DCM : MeOH = 9 : 1).

N-benzyl-N-{[(morpholine-4-yl)ethylaminocarbonyl]methyl} benzamide (1): yellow oil, yield = 30 %; _R_f=0.44; 1H NMR (400 MHz, CD3OD) δ/ppm: 2.39-2.52 (m, 6 H, H2, H6 morph, CH21 morph) 3.34-3.39 (m, 1 H, CH22 morph) 3.64-3.69 (m, 4 H, H3, H5 morph) 3.82 (s, 1 H, CH2 form) 4.08 (s, 1 H, CH2 form) 4.61 (s, 1 H, CH2 Bn) 4.78 (s, 1 H, CH2 Bn) 7.20 (m, 1 H, H Bn) 7.27-7.39 (m, 4 H, H Bn) 7.41-7.55 (m, 5 H, H Ph); 13C NMR (100 MHz, CD3OD) δ/ppm: 37.2 (CH22 morph) 50.4 (CH2 Bn) 50.7 (CH2 form) 52.1 (CH2 form) 54.8 (C2, C6 morph) 55.3 (CH2 Bn) 58.5 (CH21 morph) 67.7 (C3, C5 morph) 127.8; 127.9; 128.8; 128.9; 129.42; 129.73; 129.9; 131.1; 131.2; 136.7; 137.1 (C1 Bn) 137.5; 137.8 (C1 Ph) 170.3 (C=O) 174.9 (C=O); HPLC: _t_R=10.56 min, 98.8 %; HRMS/+ESI: C22H28N3O3+ calculated 382.2125, found 382.2124.

N-benzyl-N-{[(morpholine-4-yl)ethylaminocarbonyl]methyl}-3-bromobenzamide (2): yellow solid, m.p.=109.8 °C, yield=41 %; _R_f=0.46; 1H NMR (400 MHz, CD3OD) δ/ppm: 2.40-2.49 (m, 6 H, H2, H6 morph, CH21 morph) 3.37 (t, 1 H, J=6.6 Hz, 1 H, CH22 morph) 3.64-3.69 (m, 4 H, H3, H5 morph) 3.81 (s, 1 H, CH2 form) 4.09 (s, 1 H, CH2 form) 4.58 (s, 1 H, CH2 Bn) 4.76 (s, 1 H, CH2 Bn) 7.20 (m, 1 H, H Bn) 7.30-7.41 (m, 5 H, H Ph) 7.45-7.50 (m, 1 H, H Bn) 7.62-7.71 (m, 2 H, H Bn); 13C (100 MHz, CD3OD) NMR δ/ppm: 37.2 (CH22 morph) 50.6 (CH2 Bn) 52.0 (CH2 form) 54.6 (C2, C6 morph) 55.3 (CH2 Bn) 58.5 (CH21 morph) 67.8 (C3, C5 morph) 123.5 (C3 Ph) 126.6; 128.3; 128.9; 129.0; 129.5; 129.9; 130.0; 130.8; 131.0; 131.6; 131.6; 134.1; 134.2; 137.3; 137.6 (C1 Bn) 138.9; 139.2 (C1 Ph) 170.1; 170.3 (C=O) 173.0 (C=O); HPLC: _t_R=11.69 min, 99.0 %; HRMS/+ESI: C22H27N3O3Br+ calculated 462.1210, found 462.1207.

N-benzyl-N-{[(morpholine-4-yl)ethylaminocarbonyl]methyl}-3-chlorobenzamide (3): yellow solid, m.p.=99.5 °C, yield=33 %; _R_f=0.50; 1H NMR (400 MHz, CD3OD) δ/ppm: 2.41-2.49 (m, 6 H, H2, H6 morph, CH21 morph) 3.37 (t, J=6.4 Hz, 1 H, CH22 morph) 3.64-3.70 (m, 4 H, H3, H5 morph) 3.82 (s, 1 H, CH2 form) 4.09 (s, 1 H, CH2 form) 4.58 (s, 1 H, CH2 Bn) 4.76 (s, 1 H, CH2 Bn) 7.20-7.22 (m, 1 H, H Bn) 7.28-7.32 (m, 1 H, H Bn) 7.34-45 (m, 5 H, H Ph) 7.46-7.56 (m, 2 H, H Bn); 13C NMR (100 MHz, CD3OD) δ/ppm: 37.2 (CH22 morph) 49.1 (CH2 form) 50.6 (CH2 Bn) 52.0 (CH2 form) 54.6 (C2, C6 morph) 55.3 (CH2 Bn) 58.5 (CH21 morph) 67.7 (C3, C5 morph) 126.2; 127.9; 128.1; 128.3; 128.9; 129.0; 129.5; 129.9; 131.1; 131.3; 131.4; 131.5; 135.6 (C3 Ph) 137.3; 137.6 (C1 Bn) 138.7; 139.0 (C1 Ph) 170.1; 170.3 (C=O) 173.2 (C=O); HPLC: _t_R=11.51 min, 99.2 %; HRMS/+ESI: C22H27N3O3Cl+ calculated 416.1735, found 416.1736.

N-benzyl-N-{[(morpholine-4-yl)ethylaminocarbonyl]methyl}-3-nitrobenzamide (4): yellow oil, yield=28 %; _R_f=0.48; 1H NMR (400 MHz, CD3OD) δ/ppm: 2.38-2.50 (m, 6 H, H2, H6 morph, CH21 morph) 3.39 (t, J=6.4 Hz, 1 H, CH22 morph) 3.64-3.68 (m, 4 H, H3, H5 morph) 3.84 (s, 1 H, CH2 form) 4.15 (s, 1 H, CH2 form) 4.60 (s, 1 H, CH2 Bn) 4.79 (s, 1 H, CH2 Bn) 7.20-7.22 (m, 1 H, H Bn) 7.28-7.39 (m, 4 H, H Ph) 7.70 (q, J=7.9 Hz, 1 H, H Bn) 7.90-7.92 (m, 1 H, H Ph) 7.32-7.41 (m, 2H, H Bn); 13C (100 MHz, CD3OD) NMR δ/ppm: 37.2 (CH22 morph) 49.3 (CH2 form) 50.8 (CH2 Bn) 52.0 (CH2 form) 54.6 (C2, C6 morph) 55.3 (CH2 Bn) 58.5 (CH21 morph) 67.7 (C3, C5 morph) 123.0; 123.2; 125.7; 128.3; 128.9; 129.0; 129.6; 129.9; 130.2; 131.3; 133.9; 134.2; 137.2; 137.5 (C1 Ph) 138.4; 138.7 (C1 Bn) 149.5 (C3 Bn) 170.0; 170.3 (C=O) 172.3 (C=O); HPLC: _t_R=10.78 min, 99.3 %; HRMS/+ESI: C22H27N4O5+ calculated 427.1976, found 427.1977.

N-benzyl-N-[(tert-butylamino)carbonylmethyl]benzamide (5): white solid, m.p. = 133.7-134.8 °C, yield=85 %; _R_f=0.85; 1H NMR (400 MHz, CDCl3) δ/ppm: 1.34 (s, 9 H, CH3 t-bu) 3.73 (s, 0.6 H, CH2 form) 3.97 (s, 1.4 H, CH2 form) 4.64 (s, 1.4 H, CH2 Bn) 4.81 (s, 0.6 H, CH2 Bn) 5.24 (s, 0.3 H, NH) 6.15 (s, 0.6 H, NH) 7.18 (s, 1 H, H4 Ph) 7.27-7.51 (m, 9 H, H2,H3,H4,H5,H6 Bn, H2,H3,H5, H6 Ph); 13C NMR (100 MHz, CDCl3) δ/ppm: 28.8 (CH3 t-bu) 35.7 (C(CH3)3 t-bu) 50.2, 51.4 (CH2 form) 53.0, 54.2 (CH2 Bn) 126.9 (C4 Bn) 127.3 (C4 Ph) 128.0 (C2, C6 Bn) 128.7 (C2, C6 Ph) 129.0 (C3, C5 Bn) 130.2 (C3, C5 Ph) 135.4 (C1 Bn) 136.2 (C1 Ph) 167.8 (C=O) 172.8 (C=O); HPLC: _t_R=13.14 min, 97.8 %; HRMS/+ESI: C20H25N2O2+ calculated 325.1911, found 325.1909.

N-isobutyl-N-[(cyclohexylamino)carbonylmethyl]benzamide (6): white solid, m.p. = 139.2-140.1 °C, yield=64 %; _R_f=0.83; 1H NMR (400 MHz, CDCl3) δ/ppm: 0.75 (bs, 4.7 H, CH3 i-bu) 0.99 (bs, 1.3 H, CH3 i-bu) 1.11-1.29 (m, 3 H, H2, H4, H6, CH2 chx) 1.31-1.45 (m, 2 H, H3, H5, CH2 chx) 1.53-1.64 (m, 1H, H4, CH2 chx) 1.66-1.78 (m, 2 H, H3, H5, CH2 chx) 1.84-1.95 (m, 2 H, H2, H6 CH2 chx) 2.00 (bs, 1 H, CH i-bu) 3.09-3.26 (m, 1.5 H, CH2 form) 3.28-3.49 (m, 0.4 H, CH2 form) 3.74-3.83 (m, 1 H, H1 chx) 3.85-4.00 (m, 0.4 H, CH2 i-bu) 4.11 (s, 1.6 H, CH2 i-bu) 5.67 (s, 0.2 H, NH) 6.98 (s, 0.7 H, NH) 7.31-7.48 (m, 5 H, H2, H3, H4, H5, H6 Ph); 13C NMR (100 MHz, CDCl3) δ/ppm: 19.9 (CH3 i-bu) 24.8 (C3, C5 chx) 25.6 (C4 chx) 27.1 (CH i-bu) 33.0 (C2, C6 chx) 48.1 (C1 chx) 51.9, 53.6 (CH2 form) 59.1 (CH2 i-bu) 127.1 (C4 Ph) 128.7 (C2, C6 Ph) 129.8 (C3, C5 Ph) 135.9 (C1 Ph) 168.7 (C=O) 173.5 (C=O); HPLC: _t_R=14.66 min, 97.8 %; HRMS/+ESI: C19H29N2O2+ calculated 317.2224 found 317.2223.

N-isobutyl-N-[(tert-butylamino)carbonylmethyl]benzamide (7): white solid, m.p.=114.8-116.5 °C, yield=27 %; _R_f=0.57; 1H NMR (400 MHz, CDCl3) δ/ppm: 0.76 (s, 4.6 H, CH3 i-bu) 0.98 (s, 1 H, CH3 i-bu) 1.37 (s, 9 H, CH3 t-bu) 2.00 (s, 1 H, CH i-bu) 3.20 (s, 1.6 H, CH2 form) 3.39 (s, 0.4 H, CH2 form) 3.84 (s, 0.3 H, CH2 i-bu) 4.04 (s, 1.7 H, CH2 i-bu) 5.41 (s, 0.1 H, NH) 6.87 (s, 0.7 H, NH) 7.32-7.48 (m, 5H, H2, H3, H4, H5, H6 Ph); 13C NMR (100 MHz, CDCl3) δ/ppm: 20.0 (CH3 i-bu) 27.2 (CH i-bu) 28.8 (CH3 t-bu) 52.8 (CH2 form) 59.1 (CH2 i-bu) 127.1 (C4 Ph) 128.6 (C2,C6 Ph) 129.8 (C3,C5 Ph) 135.9 (C1 Ph) 168.8 (C=O) 173.3 (C=O); HPLC: _t_R=13.05 min, 100.0 %; HRMS/+ESI: C17H27N2O2+ calculated 291.2068 found 291.2065.

N-isobutyl-N-[(tosylmethylamino)carbonylmethyl]benzamide (8): white solid, m.p.=80.1-80.3 °C, yield=34 %; _R_f=0.44; 1H NMR (400 MHz, CDCl3) δ/ppm: 0.56-1.04 (m, 6 H, CH3 i-bu) 1.84 (bs, 1 H, CH i-bu) 2.42 (s, 3 H, CH3 ts) 3.08 (s, 2 H, CH2 form) 4.09 (s, 2 H, CH2 i-bu) 4.68 (s, 2 H, CH2 ts) 7.24-7.48 (m, 7 H, H3, H5 ts, H2, H3, H4, H5, H6 Ph) 7.66-7.86 (m, 3 H, NH, H2, H6 ts); 13C NMR (100 MHz, CDCl3) δ/ppm: 19.9 20.7 (CH3 i-bu) 21.8 22.0 (CH3 ts) 27.0 (CH i-bu) 50.1 (CH2 form) 58.3 60.4 (CH2 i-bu) 61.2 63.9 (CH2 ts) 127.2 (C4 Ph) 128.7 (C2, C6 Ph) 128.9 (C2, C6 ts) 129.6 130.0 (C3, C5 Ph) 130.1 130.5 (C3, C5 ts) 134.3 (C1 Ph, C4 ts) 145.4 (C1 ts) 169.1 (C=O) 173.7 (C=O Ts); HPLC: _t_R=13.84 min, 99.6 %; HRMS/+ESI: C21H27N2O4S+ calculated 403.1687 found 403.1686.

N-benzyl-N-{[(morpholine-4-yl)ethylaminocarbonyl]methyl} acetamide (9): yellow oil, yield=26 %; _R_f=0.44; 1H NMR (400 MHz, CD3OD) δ/ppm: 2.16-2.21 (m, 3 H, CH3 Ac) 2.42-2.50 (m, 6 H, H2, H6 morph, CH21 morph), 3.32-3.35 (m, 1 H, CH22 morph), 3.66-3.69 (m, 4 H, H3, H5 morph), 3.97 (s, 2 H, CH2 form), 4.60 (s, 1 H, CH2 Bn), 4.67 (s, 1 H, CH2 Bn), 7.24-7.38 (m, 5 H, H Bn); 13C NMR (100 MHz, CD3OD) δ/ppm: 21.5; 21.6 (CH3 Ac) 37.1; 37.2 (CH22), 49.8 (CH2 form), 50.7 (CH2 Bn), 51.6 (CH2 form), 54.4 (CH2 Bn), 54.6 (C2, C6 morph), 58.5 (CH21), 67.7; 67.8 (C3, C5 morph) 128.0; 128.6; 128.9; 129.3; 129.7; 130.0 (Ar C) 137.7 (C1 Bn) 138.1 (C1 Bn) 170.4 (C=O) 171.0 (C=O) 174.4 (C=O); HPLC: _t_R=8.22 min, 98.6 %; HRMS/+ESI: C21H27N2O4S+ calculated 320.1969 found 320.1967.

N-benzyl-N-[(tert-butylamino)carbonylmethyl]acetamide (10): white solid, m.p.=115.2-115.4 °C, yield=28 %; _R_f=0.80; 1H NMR (400 MHz, CDCl3) δ/ppm: 1.16 (s, 3 H, CH3 t-bu) 1.32 (s, 6 H, CH3 t-bu) 2.12 (s, 1 H, CH3 Ac) 2.21 (s, 2 H, CH3 Ac) 3.85 (s, 0.7 H, CH2 form) 3.86 (s, 1.3 H, CH2 form) 4.61 (s, 0.7 H, CH2 Bn) 4.66 (s, 1.3 H, CH2 Bn) 5.26 (s, 0.3 H, NH) 6.06 (s, 0.6 H, NH) 7.19 (d, J=7.0 Hz, 1 H, H4 Bn) 7.28-7.42 (m, 4 H, H2, H3, H5, H6 Bn); 13C NMR (100 MHz, CDCl3) δ/ppm: 21.6 21.9 (CH3 Ac) 28.5 28.9 (CH3 t-bu) 51.2 51.3 (CH2 form) 53.5 53.6 (CH2 Bn) 126.8 128.0 (C4 Bn) 128.2 129.0 (C2, C6 Bn) 129.1 129.3 (C3, C5 Bn) 136.1 137.3 (C1 Bn) 167.2 168.2 (C=O) 171.6 171.9 (C=O); HPLC: _t_R=11.96 min, 96.2 %; HRMS/+ESI: C15H23N2O4+ calculated 263.1755 found 263.1754.

N-benzyl-N-[2-(tert-butylamino)carbonylprop-2-yl]acetamide (11): white solid, m.p.=94.1-95.2 °C, yield=44 %; _R_f=0.75; 1H NMR (400 MHz, CDCl3) δ/ppm: 1.34 (s, 9 H, CH3 t-bu) 1.41 (s, 6 H, (CH3)2C) 2.13 (s, 3 H, CH3 Ac) 4.64 (s, 2 H, CH2 Bn) 5.54 (s, 1 H, NH) 7.27-7.32 (m, 1 H, H4 Bn) 7.35-7.42 (m, 4H, H2, H3, H5, H6 Bn); 13C NMR (100 MHz, CDCl3) δ/ppm: 23.4 ((CH3)2C) 24.9 (CH3 Ac) 28.7 (CH3 t-bu) 48.6 51.1 (CH2 Bn) 63.0 ((CH3)2C) 126.2 (C4 Bn) 127.5 (C2, C6 Bn) 129.1 (C3, C5 Bn) 139.0 (C1 Bn) 171.9 (C=O), 174.1 (C=O); HPLC: _t_R=13.27 min, 99.6 %; HRMS/+ESI: C17H27N2O4+: calculated 291.2064 found 291.2068.

Kinetic studies

Sources of AChE and BChE were native human erythrocytes and native human plasma, respectively, from two healthy donors at the Institute for Medical Research and Occupational Health, Croatia, following approval by the Ethics Committee of the Institute. Enzyme substrate acetylthiocholine iodide (ATCh) was purchased from Sigma-Aldrich, Steinheim, Germany, and thiol reagent 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) from Sigma-Aldrich, St. Louis, USA, ATCh and DTNB were dissolved in 0.1 M sodium phosphate buffer (pH 7.4).

The AChE and BChE activity was measured by the Ellman spectrophotometric method [32] at different substrate concentrations (s; 0.050 to 0.50 mM) in the absence (_v_0) and presence (_v_i) of a given compound concentration (i) selected to inhibit the enzymes for 20 to 80 %. At least three inhibitor concentrations for each substrate concentration were used in at least two experiments. The apparent inhibition constant (_K_i,app) was calculated using the Hunter-Downs equation (1) and the linear regression analysis:

where the y-intercept determines the enzyme-inhibitor dissociation constants (_K_i), while the x-intercept determines the enzyme-substrate dissociation constant, K(S). The equation was used with the assumption that, due to the low substrate concentrations used in the experiments, the substrate binds only to the catalytic site, whereas the inhibitor can bind to both sites, the catalytic and the peripheral site.

No side interactions of the tested compounds with ATCh or DTNB were detected. Measurements were done at 25 °C on a Tecan Infinite M200Pro plate reader (Austria).

Determination of IC50 values

eqBChE (EC, 3.1.1.8), type IV-S lyophilized powder from horse serum (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used without further purification. The activity of the enzyme was measured using Ellman’s method. [32] ATCh was used as a substrate (2.2 mM), and the reaction was monitored spectrophotometrically at 412 nm using a Molecular Devices SpectraMax iD3 Multi-Mode Microplate Reader. Assays were performed in phosphate buffer (0.1 mmol dm-3, pH 7.4) at 25 °C, with a total reaction volume of 0.3 mL in 96-well plates. Residual activity of the enzyme was measured in the presence of six to eight different concentrations of inhibitor [I], selected to inhibit the enzyme 10-90 %. Reagents were added in the following order: buffer, eqBChE, inhibitor (1 to 11), DTNB, and ATCh. Inhibitor concentrations (log [I]) were plotted against the percentage of enzyme inhibition, % (Inhibition = [1 - (rate of reaction with I) / (rate of control reaction)]×100), with 50 % inhibition identified by the linear regression method.

Principal component analysis

Multivariate analyses of inhibition of human AChE and BChE by tested compounds were conducted using the 2nd-order tensor decomposition tool principal component analysis (PCA) [33-34]. Data matrix X (2), which has a rank r, is numerically decomposed into the sum of matrices tipiτ (each one of rank 1):

where ti is a score vector, whereas pi is a loading vector. PCA provides the best linear projection of multidimensional data by minimizing the least squares objective function. Score values determine the locations of original samples in the coordinate system defined by the calculated principal component axes, while loadings describe the variability in the data. Experimentally determined inhibition data were written in the data matrix X and PCA was performed on the data covariance matrix using our parallelized code for multi- and univariate analysis [35-38]. Eigenvector extraction was performed with the non-linear iterative partial least squares (NIPALS) algorithm [39].

Quantum chemical docking

A comprehensive search of the configurational space of small molecules within the BChE active site was performed using quantum-chemical docking. A parallelized Monte Carlo sampling algorithm was used for the structure generation of configurations of molecules docked into the BChE’s active site (107 structures) [40]. A semi-flexible quantum-mechanical approach to molecular docking was employed, accounting for all translational, rotational, and torsional degrees of freedom of the selected α-acylaminoacetamides, and configurations with atomic overlaps were discarded. Initially, the binding energies within the active site were estimated using single‑point calculations with the PM7 Hamiltonian [41]. The top 1000 local minima, sorted by calculated enthalpies, were further refined by geometry optimization, clustered, and ranked by estimated binding enthalpies. The lowest-energy structures obtained were further analysed and compared with experimental crystal structures.

Single-point quantum-chemical calculations using the PM7 method were performed to estimate binding energies at the active site. For the top 1000 local minima, additional full geometry optimizations were performed. These refined structures with optimized geometries were clustered by similarity and ranked based on the estimated binding energy. The results were analysed using automated molecular interactions search subroutine and further visually inspected. The lowest-energy structures obtained were further analysed and compared with experimental crystal structures of hBChE-tacrine complex (PDB 4BDS) [42].