4.1 Chemistry
All the solvents used for the synthesis of the developed compounds were dried by distillation techniques before use. All the chemicals and reagents were obtained from the CDH chemicals (India), Qualigen (India), S.D. Fine Chemicals (India) and Finar chemicals (India). The progress of reactions was checked by thin-layer chromatography (TLC) on precoated silica gel 60 F254 (MerckKGaA) and were checked under UV light. Column chromatographic purifications were done using silica gel 60-120 mesh size (Avra synthesis, India). 1H nuclear magnetic resonance (1H-NMR) and 13C NMR spectra were measured on Bruker Advance, 500 MHz spectrometers with tetramethylsilane (TMS) as the internal standard. The NMR solvents used were CDCl3 or DMSO‑d6 as indicated. Chemical shifts were measured in ppm and coupling constants (J) were measured in Hz. The following abbreviations are used to describe peak splitting patterns when appropriate: d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, br = broad. Coupling constants J are reported in Hertz (Hz).
4.1.1. General procedure for synthesis of chalcone (3a-3c)
Into a stirring solution of 4-hydroxyacetophenone (2) (1.2 g, 8.81 mmol, 1.0 equiv.) in ethanol (20 mL), substituted benzaldehyde (1a-1c) (1.75 g, 10.57 mmol, 1.20 equiv.) were added. After 5 min, KOH (1.48 g, 26.43 mmol, 3.0 mmol) was added to the reaction mixture. The mixture was refluxed for 10-12 h. After cooling down to RT, the solution was acidified with dilute HCl and left for 2 hr. The obtained precipitate was filtered and washed with a mixture of water. The resulting yellow solid was crystalized using ethanol to yield compound 3a-3c as a solid yellow powder in good to high yield.
4.1.1.1. (E)-3-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (3a)
Compound 3a was prepared according to general procedure mentioned above. 4-hydroxyacetophenone (2) (1.2 g, 8.81 mmol, 1.0 equiv.), 3, 4-methoxybenzaldehyde (1a) (1.75 g, 10.57 mmol, 1.2 equiv.), KOH (1.48 g, 26.43 mmol, 3.0 mmol) were mixed in 20 mL ethanol, and refluxed for 10 hr. Yellow solid powder (1.242 g, 81% yield), TLC (EtOAc:Hexane 30:70 v/v), Rf = 0.60. 1H NMR (500 MHz, DMSO-d6):δ10.46 (bs, 1H, -OH), 8.09 (d, J = 8.75 Hz, 2H, Ar-H), 7.81 (d, J = 15.50 Hz, 1H, -CH=CH-), 7.66 (d, J = 15.50 Hz, 1H,-CH=CH-), 7.52 (d, J = 5.50 Hz, 1H, Ar-H), 7.34 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H,Ar-H), 6.99 (d, J = 6.50 Hz, 1H, Ar-H), 6.93 (d, J = 8.50 Hz, 2H, Ar-H), 3.86 (s, 3H, -OCH3), 3.80 (s, 3H, -OCH3).
4.1.1.2. (E)-3-(2,5-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (3b)
Compound 3b was prepared according to general procedure mentioned above. 4-hydroxyacetophenone (2) (1.2 g, 8.81 mmol, 1.0 equiv.), 2, 5-methoxybenzaldehyde (1b) (1.75g, 10.57 mmol, 1.2 equiv.), KOH (1.48 g, 26.43 mmol, 3.0 mmol) were mixed in 20 mL ethanol, and refluxed for 10 hr. Yellow solid powder (1.32 g, 78% yield), TLC (EtOAc:Hexane 35:65 v/v), Rf = 0.55. 1H NMR (500 MHz, DMSO-d6):δ10.32 (bs, 1H, -OH), 8.08 (d, J = 8.0 Hz, 2H, Ar-H), 7.99 (d, J = 16.0 Hz, 1H, -CH=CH-), 7.88 (d, J = 16.0 Hz, 1H, -CH=CH-), 7.53 (d, J = 6.0 Hz, 1H, Ar-H), 7.01-7.00 (m, 2H, Ar-H), 6.92 (d, J = 6.50 Hz, 2H, Ar-H), 3.83 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3).
4.1.1.3. (E)-1-(4-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3c)
Compound 3c was prepared according to general procedure mentioned above. 4-hydroxyacetophenone (2) (1.2 g, 8.81 mmol, 1.0 equiv.), 4-methoxybenzaldehyde (1c) (1.42 g, 10.57 mmol, 1.2 equiv.), KOH (1.48 g, 26.43 mmol, 3.0 mmol) were mixed in 20 mL ethanol, and refluxed for 10 hr. Yellow solid (1.10 g, 76% yield), TLC (EtOAc:Hexane40:60 v/v), Rf = 0.50. 1H NMR (500 MHz, DMSO-d6):δ10.24 (bs, 1H, -OH), 8.01 (d, J = 7.5 Hz, 2H, Ar-H), 7.82 (d, J = 15.75 Hz, 1H, -CH=CH-), 7.78 (d, J = 15.75 Hz, 1H, -CH=CH-), 7.53 (d, J = 8.5 Hz, 2H, Ar-H), 7.11-7.07 (m, 2H, Ar-H), 6.98 (d, J = 8.5 Hz, 2H, Ar-H), 3.68 (s, 3H, -OCH3).
4.1.2. General procedure for synthesis of substituted 2-hydrazinobenzothaizole
To a 60 mL seal tube equipped with magnetic stirrer, 4-methylbenzo[d]thiazol-2-amine (1.64g, 10 mmol, 1.0 equiv.), ethylene glycol (30 ml), and hydrazine hydrate (1.6 mL, 50 mmol, 5 equiv.) were added. The reaction mixture was then heated at 150 oC for 10 hr. The progress of reaction was monitored by TLC followed by the addition of water (20 ml). The intermediates were then filter washed with water to get aforementioned product.
4.1.2.1. 2-hydrazinyl-6-methylbenzo[d]thiazole (6a)
Compound was prepared according to general procedure mentioned above. 6-methylbenzo[d]thiazol-2-amine (1.64g, 10 mmol, 1.0 equiv.), ethylene glycol (30 ml), and hydrazine hydrate (1.6 mL, 50mmol, 5 equiv.) were mixed and refluxed for 10 hr. White solid powder (1.72, 78% yield), TLC (EtOAc:Hexane 50:50 v/v), Rf = 0.55. 1H NMR (500 MHz, DMSO-d6):δ9.01 (bs, 1H, -NH), 7.49 (dd, J1 = 7.5 Hz, J2 = 2.5 Hz, 1H, Ar-H), 7.01 (d, J = 8.0 Hz, 1H, Ar-H), 6.88 (t, J = 6.5 Hz, 1H, Ar-H), 4.99 (s, 2H, -NH2), 2.51 (s, 3H, -CH3).
4.1.2.2. 2-hydrazinyl-4-methylbenzo[d]thiazole (6b)
Compound was prepared according to general procedure mentioned above. 4-methylbenzo[d]thiazol-2-amine (1.64 g, 10 mmol, 1.0 equiv.), ethylene glycol (30 ml), and hydrazine hydrate (1.6 mL, 50 mmol, 5 equiv.) were mixed and refluxed for 10 hr. White solid crystalline powder (1.8 g, 81% yield), TLC (EtOAc:Hexane 50:50 v/v), Rf = 0.60. 1H NMR (500 MHz, DMSO-d6): δ 8.86 (bs, 1H, -NH), 7.47 (s, 1H, Ar-H), 7.20 (d, J = 7.5 Hz, 1H, Ar-H), 7.01 (dd, J1 = 8.5 Hz, J2 = 2.0 Hz, 1H, Ar-H), 4.96 (s, 2H, -NH2), 2.50 (s, 3H, -CH3).
4.1.3. General procedure for synthesis of pyrazolo-benzothiazole derivatives
To a 30 mL seal tube equipped with magnetic stirrer, compound 3a-3c (1.0 equiv.), and compound 6a-6b (1.2 equiv.) were refluxed in n-butanol for 10-12h. The progress of reaction was monitored by TLC (EtOAc:Hexane, 1:1). The crude residues were then filter and subjected to column chromatography to get aforementioned product.
4.1.3.1.4-(5-(3,4-dimethoxyphenyl)-1-(6-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4a)
Compound was prepared according to general procedure mentioned above. (E)-3-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3a) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-6-methylbenzo[d]thiazole (6a) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid crystalline powder (0.289 mg, 80% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.60. 1H-NMR (500 MHz, DMSO) δ ppm: 7.60 (m, 3H, Ar-H), 7.06 (d, J = 7.3 Hz, 1H, Ar-H), 7.01 – 6.95 (m, 2H, Ar-H), 6.87 – 6.80 (m, 3H, Ar-H), 6.67 (d, J = 3.0 Hz, 1H, Ar-H), 5.84 (dd, J = 11.8, 5.3 Hz, 1H, -CH), 3.93 (q, 1H, -CH2), 3.78 (s, 3H, -OCH3), 3.62 (s, 4H, -OCH3, methylene -H), 2.31 (s, 3H, -CH3). 13C-NMR (126 MHz, DMSO) δ ppm: 161.98, 154.87, 153.51, 151.75, 151.17, 130.95,128.73, 128.62,126.82, 121.82, 119.06, 116.28, 114.72, 114.30,113.36, 113.03, 59.48, 56.82, 55.74, 18.11.
4.1.3.2.4-(5-(3,4-dimethoxyphenyl)-1-(4-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4b)
Compound was prepared according to general procedure mentioned above. (E)-3-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3a) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-4-methylbenzo[d]thiazole (6b) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid crystalline powder (0.25 mg, 75% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.60. 1H NMR (500 MHz, DMSO) δppm: 7.60 (dd, J = 17.7, 8.1 Hz, 3H, Ar-H), 7.06 (d, J = 7.3 Hz, 1H, Ar-H), 7.06 – 6.90 (m, 2H, Ar-H), 6.93 – 6.75 (m, 3H, Ar-H), 6.67 (d, J = 3.0 Hz, 1H), 5.84 (dd, J = 11.8, 5.3 Hz, 1H), 3.94 (d, J = 11.8 Hz, 2H, -CH2), 3.84 (d, J = 6.5 Hz, 3H, -OCH3), 3.62 (s, 3H, -OCH3), 2.31 (s, 3H, -CH3).13C NMR (126 MHz, CDCl3) δppm: 163.14, 161.19, 147.41, 146.74, 143.04, 130.69, 130.63, 127.80, 121.88, 115.28, 115.11, 114.80, 114.40, 109.92, 62.03, 55.98, 31.93, 29.70.
4.1.3.3. 4-(5-(2,5-dimethoxyphenyl)-1-(6-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4c)
Compound was prepared according to general procedure mentioned above. (E)-3-(2,5-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3b) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-6-methylbenzo[d]thiazole (6a) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid powder (0.26 mg, 76% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.50. 1H-NMR (500 MHz, DMSO) δ ppm: 7.69 (d, J = 7.5 Hz, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 7.01 (d, J = 7.5 Hz, 2H, Ar-H), 6.92 (s, 1H, Ar-H), 6.68– 6.62 (m, 2H, Ar-H), 6.57 (d, J = 3.0 Hz, 1H, Ar-H), 5.75 (dd, J = 8.5, 3.5 Hz, 1H, -CH), 4.01 (t, J = 6.5, 1H, -CH2), 3.63 (s, 3H, -OCH3), 3.51 (s, 4H, -OCH3, methylene -H), 2.39 (s, 3H, -CH3). 13C-NMR (126 MHz, DMSO) δ ppm: 160.63, 151.36, 149.68, 148.61, 147.38, 145.36,131.31, 125.18,122.63, 121.45, 118.74, 115.64, 112.31, 111.27,110.32, 110.17, 58.62, 57.21, 54.32, 19.64.
4.1.3.4. 4-(5-(2,5-dimethoxyphenyl)-1-(4-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4d)
Compound was prepared according to general procedure mentioned above. (E)-3-(2,5-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3b) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-4-methylbenzo[d]thiazole (6b) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid powder (0.275 mg, 80% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.60. 1H-NMR (500 MHz, DMSO) δ ppm: 7.64 (d, J = 8.5 Hz, 2H, Ar-H), 7.50 (s, 1H, Ar-H), 7.08 (s, 1H, Ar-H), 6.99 (m, 3H, Ar-H), 6.71 (d, J = 8.5, 2H, Ar-H), 6.60 (d, J = 7.5 Hz, 1H, Ar-H), 5.76 (d, J = 8.5, 1H, -CH), 3.94 (d, J = 7.5, 2H, -CH2), 3.70 (s, 3H, -OCH3), 3.44 (s, 3H, -OCH3), 2.56 (s, 3H, -CH3). 13C-NMR (126 MHz, DMSO) δ ppm: 163.23, 155.36, 154.12, 149.62, 148.14, 147.65,141.74, 141.02,136.21, 127.32, 119.82, 116.84, 117.62, 114.61,109.51, 108.62, 60.32, 59.65, 55.41, 20.14.
4.1.3.5.4-(5-(4-methoxyphenyl)-1-(6-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4e)
Compound was prepared according to general procedure mentioned above. (E)-3-(4-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3c) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-6-methylbenzo[d]thiazole (6a) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid crystalline powder (0.245 mg, 75% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.60. 1H-NMR (500 MHz, DMSO) δ ppm: 7.54 (d, J = 8.5 Hz, 2H, Ar-H), 7.44 (d, J = 7.5 Hz, 2H, Ar-H), 7.05 (d, J = 7.5, 2H, Ar-H), 6.94 (s, 1H, Ar-H), 6.66 (d, J = 7.5, 2H, Ar-H), 6.47 (d, J = 7.5 Hz, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 5.80 (d, J = 6.5, 1H, -CH), 4.12 (d, J = 6.5, 2H, -CH2), 3.61 (s, 3H, -OCH3), 3.51 (s, 3H, -OCH3), 2.41 (s, 3H, -CH3). 13C-NMR (126 MHz, DMSO) δ ppm: 161.32, 158.14, 151.32, 147.52, 144.65, 143.84,140.66, 137.63,135.41, 125.62, 118.14, 115.11, 114.21, 113.21, 106.44, 57.32, 56.44, 53.94, 18.65.
4.1.3.6.4-(5-(4-methoxyphenyl)-1-(4-methylbenzo[d]thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (4f)
Compound was prepared according to general procedure mentioned above. (E)-3-(4-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one(3c) (0.2 g, 0.703 mmol, 1.0 equiv.) and 2-hydrazinyl-4-methylbenzo[d]thiazole (6b) (0.126 g, 0.703 mmol, 1.0 equiv.) were refluxed in n-butanol for 10-12h. White solid powder (0.29 mg, 82% yield), TLC (EtOAc:Hexane 1:1 v/v), Rf = 0.55. 1H-NMR (500 MHz, DMSO) δ ppm: 7.61 (d, J = 7.5 Hz, 2H, Ar-H), 7.56 (d, J = 6.5 Hz, 2H, Ar-H), 6.99 (d, J = 8.5, 2H, Ar-H), 6.91 (m, 3H, Ar-H), 6.40 (d, J = 8.5 Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 5.84 (d, J = 7.5, 1H, -CH), 4.21 (d, J = 7.0, 2H, -CH2), 3.66 (s, 3H, -OCH3), 3.55 (s, 3H, -OCH3), 2.44 (s, 3H, -CH3). 13C-NMR (126 MHz, DMSO) δ ppm: 160.32, 159.74, 154.12, 150.21, 147.63, 147.15,138.15, 133.12,131.50, 124.32, 116.50, 111.16, 110.74, 109.84, 108.63, 60.84, 58.62, 55.12, 20.32.
4.2. Biological evaluation
4.2.1. Determination of IC50 values
Cholinesterase inhibitory activity of developed molecules was assessed calorimetrically using modified Ellmann method.[28] ChE selectively catalyzes the breakage of acetylthiocholine to thiocholine and acetic acid, which reduces the 5,5-dithio-bis-(2- nitrobenzoic acid) (DTNB) to produces yellow color intermediate that can be spotted colorimetrically at 413 nm. Human acetylcholinesterase (hAChE) (CAS No. 9000-81-1), butyrylcholinesterase (eqBChE) (CAS NO. 9001-08-5), 5,5-dithiobis-2-nitrobenzoic acid (DTNB, CAS No. 69-78-3), acetylthiocholine iodide (ATCI, CAS No. 1866-15-5) and butyrylthiocholine iodide (BTCI, CAS No. 1866-16-6) werebrought from Sigma Aldrich, and Spectrochem chemicals respectively. All the experiments were performed in 50 mM phosphate buffer at pH 7.4.
Briefly, 50 mL of AChE (0.22 U/mL, initial concentration) and 10 μL of the test or standard compound were incubated in 96-well plates at room temperature for 30 min. Furthermore, 30 μL of the substrate viz. ATCI (1.5 mM) was added. After 30 minutes, 160 μL of DTNB (0.15 mM) was added to it and then absorbance was measured at 413 nm wavelength using microplate reader. Each assay was performed in triplicate. The blank contained all components except enzyme. The inhibition percent was calculated by the following expression: [(AcAi)/Ac] X100, where Ai and Ac are the absorbance obtained for BChE in the presence and absence of inhibitors.
The in-vitro BChE inhibition experiment was performed using the same procedure as described above. Briefly, 50 μL of BChE (0.06 U/mL) and 10 μL of the test or reference compound were incubated in 96-well plates at RT for 30 min. Thereafter, 30 μL of the substrate viz. BTCI (15 mM) was added and the solution. After 30 minutes, 160 μL of DTNB (1.5 mM) was added to it and the absorbance was measured at 413 nm wavelength using a 96-well microplate reader. The inhibition percent was calculated by the following expression: [(AcAi)/Ac] X100, where Ai and Ac are the absorbance obtained for BChE in the presence and absence of inhibitors.
4.2.2. Kinetic characterization of AChE and BChE inhibition
In order to evaluate the mechanism of action of 4a, reciprocal plots of 1/[V] versus 1/[S] were constructed using six different concentrations of the substrate ATCI (from 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM for hAChE) by using the modified Ellman method.[28] Briefly, compound 4a (10μL) at different concentrations (5μM, 10μM and 20 μM) was pre-incubated with hAChE (50 μLof 0.22 U/mL at RT for 30 minutes, followed by the addition of 30 μL of the substrate at different concentrations. The kinetic characterization of the hydrolysis of ATCI catalyzed by AChE is done spectrometrically using a 96-well microplate reader at 413 nm.
4.2.3. DPPH radical-scavenging potency
The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging method is an simple and most convenient antioxidant assay based on reduction of DPPH radical. DPPH was purchased for Sigma-Aldrich (Merck, CAS No. 1898-66-4). All the experiments were carried out in biology grade methanol (for poor solubility ethanol can be used). Eight different concentrations 1, 10, 20, 40, 80, 100, 160, and 200 μM of test sample were used. In brief, 75 μL of different concentrations of the test sample were added to a 96-well plate. Then, 75 μL of DPPH (200 μM) solution was added to it. Finally, a 96-well microplate was allowed to stand at RT for 30 minutes, followed by absorbance at 520 nm using microplate reader. The radical scavenging capacity was determined using the equation % radical scavenging activity = [(absorbance of control-absorbance of the test)/absorbance of control] X 100. All the experiments were performed in triplicate.
4.2.4. PAMPA-BBB Assay
Brain permeability prediction of the developed compound was assessed by in-vitro PAMPA-BBB assay reported method from Di et al..[29] The protocol involved the coating of porcine brain lipid solution dissolved in dodecane (5 μL) on filter membrane of donar microplate. The 500 μM concentration of test compound was prepared in phosphate buffer (pH = 7.4). The donor and acceptor microplates were filled with 500 μM (200 μL) of compound and 300 μL of phosphate buffer respectively. The assembly of acceptor and donor was sandwiched with each other and incubated at RT for 16 h). After incubation, diffusion of the test compounds in the acceptor plate was calculated by determining the absorbance spectrophotometrically. The experiment was conducted in triplicate.
4.2.4. Inhibition of Aβ Aggregation.
Aβ peptide (CAS No. 107761-42-2), molecular biology grade DMSO (CAS No. 67-68-5), Phosphate buffer saline (PBS) was procured from Sigma-Aldrich and HiMedia respectively. Aβ peptide 0.1 mg was dissolved in 100 μL of molecular biology grade DMSO, and aliquot in five different vials. The test compounds were prepared in molecular biology grade DMSO and PBS pH 7.4 (DMSO ≤ 1% w/v final concentration). Two different ratios of Aβ peptide and 4a were evaluated (10:10 μM, and 10:20 μM respectively).
For self-induced anti-Aβ aggregation assay, the mixture of Aβ peptide (20 μM) Aβ peptide (40 μM) in PBS pH 7.4 in the presence or absence of inhibitor (20 μM, and 40 μM) was incubated (37 °C, 48 h) followed by addition of 100 μM of thioflavin T (10μM). The fluorescence intensity was measured at excitation (λex = 450 nm) and emission (λemission = 485 nm) wavelengths. The anti-Aβ aggregatory potential was calculated as percentage inhibition following an expression: [100 − (Fi/Fo × 100)]; and NFI = Fi/Fo. The Fi and Fo are the fluorescence intensities in the presence or absence of inhibitor, respectively.
4.2.5. Molecular docking
The 3D crystal structure of hAChE in complex with DPZ (PDB ID- 4EY7) was retrieved from the Brookhaven protein data bank.[30, 31] Protein Preparation Wizard of Schrodinger software package (Schrodinger, LLC, New York, NY) was used to prepare proteins. This step includes removal of water beyond 5 Å from the HET group, addition of missing hydrogen, optimization of orientations of hydroxyl and amino groups, assignment of right bond orders, and the determination of ionization of amino acids using ProtAssign utility. The resulting structures were further subjected to restrained minimization with cutoff root mean square deviation (RMSD) of 0.3 Å. Finally, the prepared complexes were further used for molecular docking and MD simulation study. All the small molecules were drawn using 2D sketcher and were subjected to ligand preparation using the LigPrep module of Schrodinger software package (Schrodinger, LLC, New York, NY). The different possible ionization states for ligands were generated at the physiological pH (7.0 ± 2), and OPLS4 force field was used to minimize the ligands. Finally, docking of all ligands was performed by the Glide module of the Schrodinger software package (Schrodinger, LLC, New York, NY) using standard operating procedures with the extra precision (XP) protocol.[32]
4.2.6 Molecular Dynamics Simulation
All-atom MD simulations were performed using the desmond-v6.6 module of Schrödinger Software Package (Schrödinger, LLC, New York, NY).[33] The system builder panel was used to prepare the initial systems for MD simulations. The apo-AChE and both docked complexes (AChE-DPZ, an AChE-4a) were placed in a cubic box of 1.0 nm size. The boxes were solvated with TIP3P water models and charged systems were neutralized using counter ions (Na+ or Cl- ions).[34] An ionic strength of 0.15 M was maintained by adding Na+ and Cl- ions to all the systems. Further, the solvated systems were minimized and equilibrated under NPT ensemble using the default protocol of Desmond. It includes a total of nine stages, among which there are two minimization and four short simulations (equilibration phase) steps.[35] All minimized and equilibrated systems were subjected to MD run with periodic boundary conditions in NPT ensemble using OPLS4 force field parameter for 100 ns.[36] During the simulation, the pressure (1 atm) and temperature (300 K) of the systems were maintained by Martyna–Tobias–Klein barostat and Nose–Hoover Chain thermostat, respectively.[37-40] The binding energy between the AChE and ligands (DPZ & 4a) was calculated using the inbuilt script thermal_mmgbsa.py.[41, 42] The binding energy was calculated from the last 25 ns of trajectory at an interval of 50 ps for both systems ((AChE-DPZ, an AChE-4a).