Synthesis, biological evaluation and molecular docking studies of novel 2-alkylthiopyrimidino-tacrines as anticholinesterase agents and their DFT calculations

Journal Pre-proof Synthesis, biological evaluation and molecular docking studies of novel 2alkylthiopyrimidino-Tacrines as anticholinesterase agents and their DFT calculations

Chamseddine Derabli, Houssem Boulebd, Ahmed B. Abdelwahab, Celia Boucheraine, Sarah Zerrouki, Chawki Bensouici, Gilbert Kirsch, Raouf Boulcina, Abdelmadjid Debache PII:

S0022-2860(20)30226-X

DOI:

https://doi.org/10.1016/j.molstruc.2020.127902

Reference:

MOLSTR 127902

To appear in:

Journal of Molecular Structure

Received Date:

18 January 2020

Accepted Date:

13 February 2020

Please cite this article as: Chamseddine Derabli, Houssem Boulebd, Ahmed B. Abdelwahab, Celia Boucheraine, Sarah Zerrouki, Chawki Bensouici, Gilbert Kirsch, Raouf Boulcina, Abdelmadjid Debache, Synthesis, biological evaluation and molecular docking studies of novel 2alkylthiopyrimidino-Tacrines as anticholinesterase agents and their DFT calculations, Journal of Molecular Structure (2020), https://doi.org/10.1016/j.molstruc.2020.127902

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Journal Pre-proof Graphical Abstract Synthesis, biological evaluation and molecular docking studies of novel 2alkylthiopyrimidino-Tacrines as anticholinesterase agents and their DFT calculations Chamseddine Derabli, Houssem Boulebd, Ahmed B. Abdelwahab, Celia Boucheraine, Sarah Zerrouki, Chawki Bensouici, Gilbert Kirsch, Raouf Boulcina* and Abdelmadjid Debache

Cl

Cl

NH2 CN

N S

N

N

NH2

S

Starting point Molecular docking of compound 3d

N

N

Potent AChE inhibitor (3d) IC50= 4.32 µM

Journal Pre-proof Synthesis, biological evaluation and molecular docking studies of novel 2alkylthiopyrimidino-Tacrines as anticholinesterase agents and their DFT calculations Chamseddine Derablia,b, Houssem Boulebdb, Ahmed B. Abdelwahabc, Celia Boucherainea, Sarah Zerroukia, Chawki Bensouicid, Gilbert Kirsche, Raouf Boulcinab,f * and Abdelmadjid Debacheb National Higher School of Biotechnology "Toufik Khaznadar" University city Ali Mendjeli, 25100,

a

Constantine, Algeria b

Laboratory of Synthesis of Molecules with Biological Interest, Frères Mentouri Constantine 1 University,

25000, Constantine, Algeria c

Plant Advanced Technologies (PAT), 19 avenue de la Forêt de Haye, 54500, Vandoeuvre-lès-Nancy, France

d

Biotechnology Research Center, Constantine, Algeria

e L2CM,

Lorraine University, 1 Boulevard Arago, 57070, France

f Faculty

of Technology, Batna 2 University, 05000 Batna, Algeria

*Corresponding author. E-mail: [email protected]; Phone: +213-559-110-329.

Journal Pre-proof Abstract To search for effective and selective inhibitors of cholinesterases (AChE and BuChE), a series of poly-functionalized Tacrine-derived compounds specifically 2-(alkylthio)-4-aryl-6,7,8,9tetrahydropyrimido[4,5-b]quinolin-5-amines were designed an synthesized via Friedlander reaction. The structures of the newly synthesized compounds were confirmed on the basis of their spectral data (1H NMR,

13C

NMR) and elemental analyses (CHNS). Compounds 3a-h

were evaluated for their abilities to inhibit AChE and BChE. The obtained biological results revealed that some synthesized compounds displayed higher anti-cholinesterase activity in comparison to Galantamine. Among them, compound 3d bearing S-ethyl and 4-chlorophenyl moieties showed the most potent activity against AChE/BuChE with IC50s values of 4,32 and 15,10 µM, respectively. The anti-AChE activity of 3d was 5-fold more than that of reference drug Galantamine. Moreover, molecular docking studies were performed for the most active derivatives, 3d and 3c, in which binding mode between these compounds and the receptors were determined. Density functional theory (DFT) method at B3LYP/6-311++G (d,p) level of theory was employed to gain insights into the molecular structure of the target compounds. Molecular electrostatic potential (MEP) mapping, reactivity indices such as electronegativity, electrophilic index, softness and hardness as well as frontier molecular orbitals HOMOLUMO have been investigated for 3a-3c as representative compounds. In addition, their antiradical activity has been predicted by computing bond dissociation enthalpies (BDEs).

Keywords:

Alzheimer’s

disease;

2-Alkylthiopyrimidino-Tacrines;

cholinesterases; Molecular docking; DFT calculations.

Inhibitors

of

Journal Pre-proof 1. Introduction The human brain is so complex that scientists are constantly researching its anatomy, physiology and dysfunctions at the root of the various diseases that can destabilize it, the most important of which are neurodegenerative diseases. In view of the gravity and extent of the progression of Alzheimer's disease (AD), scientists are interested in this pathology, which affects about 60 to 70% of elderly people with dementia [1, 2]. AD is a significant problem for old people worldwide, it is one of the neurodegenerative diseases that affects the elderly by inducing irreversible memory loss [3]. This disease characterized by neurofibrillary degeneration with a continuous, progressive and irreversible decline in cognitive function until the ability to perform daily tasks is completely lost [4, 5]. From a pathophysiological point of view, β-amyloid plaques and neurofibrillary tangles are the major pathological signs in the brain of AD patients [6]. Also, there is no cure for AD, only four drugs have been approved for AD therapy: Memantine, a N-methyl-D-aspartate receptor antagonist [7, 8]; three of them are acetylcholinesterase inhibitors (AChEIs) including Donepezil, Rivastigmine, and Galantamine [9], able to restore the neurotransmitter acetylcholine levels. (Figure 1) Tacrine, the first marketed AChEI which significantly improved certain functions of recognition, reasoning, perception, attention, decision-making and many others. O NH2

R2

NH2

N

N

O H

N A

R1

N

S

HO

3a-h

Tacrine

Galantamine

O N

O

NH2 O

N

O O

Donepezil

N

Rivastigmine

N Memantine

Figure 1. Drugs for Alzheimer’s disease and the structure of compounds 3a-h described in this work. Indeed, it restarted brain chemistry by restoring neurotransmission at the synapses by protecting acetylcholine and butyrylcholine neurotransmitters. Despite the effectiveness of the treatment, the patients who took it quickly developed rather serious side effects:

Journal Pre-proof hepatotoxicity, heart problems, neuropsychic disorders, digestive disorders, etc [10, 11], and since then many researchers have been interested in the development of Tacrine analogues by minimizing adverse reactions as much as possible [12, 17]. In this context, we have recently reported the synthesis and pharmacological studies of a series

of

readily

available

Tacrine-pyranopyrazole

[18]

and

alkylbis(4-amino-5-

cyanopyrimidine) derivatives [19], as promising agents for AD therapy. In our efforts to contribute to the development of new compounds that may be useful in the treatment of AD, we are focusing on the synthesis of new Tacrine analogs which result from exchanging the benzene ring (A) with a potentially active pharmacophore: 2-alkylthiopyrimidine. In fact, pyrimidine derivatives and their analog exhibit a variety of pharmacological activities, including antitumoral properties [20]. They can be used as anticancer [21], antimicrobial [22], antitubercular agents [23] and so forth. As our continuous interest in the synthesis of the biologically important heterocycles, herein we describe the synthesis of a novel series of highly

functionalized

2-(alkylthio)-4-aryl-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-

amines as cholinesterase inhibitors. In addition, molecular docking analysis was also performed to disclose the binding interaction template of the most active inhibitors to the amino acid residues composing active site of the AChE and BChE enzymes and the findings are presented below. Furthermore, optimized molecular geometry, frontier molecular orbitals HOMO-LUMO, molecular electrostatic potential (MEP) mapping and reactivity indices (electronegativity, electrophilic index, softness and hardness) have been investigated for 3a3c as representative compounds using DFT/B3LYP method. At the same level of theory, the antiradical properties of 3a-3c have been also examined by computing bond dissociation enthalpies (BDEs). 2. Experimental section 2.1.

Materials and equipments

All chemicals were purchased from the Sigma-Aldrich and used without further purification. All solvents used for spectroscopic and synthesis studies were reagent grade and further purified by literature methods. Melting points were determined on an Electrothermal capillary fine control apparatus. 1H and

13C

NMR spectra were recorded on a Bruker Avance 400

instrument at 400 and 100 MHz respectively, in CDCl3 or DMSO–d6. Chemical shifts (δ) are given in part per million downfield from TMS as an internal standard for 1H and 13C NMR. Coupling constants (J) values were indicated in Hz. The chemicals were used as obtained

Journal Pre-proof commercially. Elemental analyses were carried out on a Microanalyzer Flash EA1112 CHNS/O Thermo Electron. 2.2.

Synthesis and characterization

2.2.1. Synthesis of 2-(Alkylthio)-4-aryl-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3): In a 100 mL round bottom flask, a mixture of 3-amino-2-cyanopyrimidine (1, 1 mmol), aluminum chloride (2 mmol) and cyclohexanone (2, 2 mmol) was added into adequate volume of dry 1,2-dichloroethane (DCE) and stirred at reflux for 24 hours. (Monitored by TLC) After completion, water was added and the mixture was basified with 10% sodium hydroxide solution to pH = 8-9. After stirring for 30 min, the precipitate was filtered and washed with water. Purification by recrystallization in ethyl acetate afforded the title compounds. 2.2.2. Spectral characterization 4-(4-Chlorophenyl)-2-(methylthio)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3a) Compound 3a was obtained as a yellow solid (41%). M.p: 251–254 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.44 (m, 4H), 4.49 (br s, 2H, NH2), 2.97 (t, J=6.0 Hz, 2H), 2.64 (s, 3H, SCH3), 2.32 (t, J=6.0 Hz, 2H), 1.86-1.81 (m, 4H).13C NMR (100 MHz, CDCl3) δ (ppm) 170.3, 165.8, 165.6, 158.3, 149.1, 137.4, 136.5, 130.0, 129.8, 129.6, 129.4, 111.9, 103.7, 34.5, 23.5, 22.4, 14.4. Anal. calcd for C18H17ClN4S (4%H2O): C, 58.15; H, 5.05; N, 15.00; S, 8,62; Found: C, 58.53; H, 4.65; N, 14.50, S, 8.36. 4-(4-Methoxyphenyl)-2-(methylthio)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5amine (3b): Compound 3b was obtained as a yellow solid (58%). M.p: 176-178 °C. 1H NMR (400 MHz, CDCl3) δ(ppm): 7.43 (dd, J=6.8, 2.4 Hz, 2H), 6.96 (dd, J=6.8, 2.0 Hz, 2H), 4.58 (br s, 2H, NH2), 3.81 (s, 3H, O-CH3), 2.97 (t, J=6.0 Hz, 2H), 2.64 (s, 3H, S-CH3), 2.32 (t, J=6.4 Hz, 2H), 1.86-1.81 (m, 4H).13C NMR (100 MHz, CDCl3): δ (ppm): 170.3, 166.6, 165.4, 161.1, 158.4, 149.5, 132.6, 131.2, 130.2, 114.9, 111.6, 103.8, 55.5, 34.4, 26.8, 23.5, 22.5, 22.4, 14.4. Anal. calcd for C19H20N4OS: C, 64,75; H, 5,72; N, 15,90; S, 9.10 ; Found: C, 63,75; H, 5,39; N, 13,20, S, 7.64.

Journal Pre-proof 2-(Methylthio)-4-(3-nitrophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3c): Compound 3c was obtained as a orange brown solid (55%). M.p: 210-216 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.39 (s, 1H), 8.32(dd, J= 8.0, 1.6 Hz, 1H), 7.88 (dd, J= 7.6, 1.2 Hz, 1H), 7.67 (t, J= 8.0 Hz, 1H), 4.38 (br s, 2H, NH2), 3.0 (t, J= 5.6 Hz, 2H), 2.63 (s, 3H), 2.36 (t, J= 6.0 Hz, 2H), 1.87-1.82 (m, 4H).

13C

NMR (100 MHz, CDCl3): δ (ppm): 170.5,166.5,

163.9, 158.3, 148.6, 148.4, 140.5, 134.8, 130.2, 124.9, 124.1, 112.4, 103.6, 34.5, 23.5, 22.3, 14.5. Anal. calcd for C18H17N5 O2S: C, 58.84; H, 4.66; N, 19.06; S, 8.73 ; Found: C, 45.68; H, 4.02; N, 12.89, S, 5.61. 4-(4-Chlorophenyl)-2-(ethylthio)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3d) Compound 3d was obtained as an orange brown (57%). M.p 216-219 °C.1H NMR (400 MHz, CDCl3) δ (ppm) 7.44 (m, 4H), 4.47 (br s, 2H, NH2), 3.28 (q, J=7.2 Hz, 2H), 2.97 (t, J=6.0 Hz, 2H), 2.32 (t, J=6.0 Hz, 2H), 1.85-1.81 (m, 4H), 1.36 (t, J= 7.2 Hz, 3H).13C NMR (100 MHz, CDCl3) δ (ppm) 169.9, 165.8, 165.6, 158.3, 149.0, 137.5, 136.5, 130.0, 129.4, 111.9, 103.7, 34.5, 25.3, 23.5, 22.4, 22.3, 14.4. Anal. calcd for C19H19ClN4S (2% H2O): C, 60.29; H, 5.29; N, 14.80; S, 8.47; Found: C, 60.85; H, 5.09; N, 14.36, S, 8.23. 2-(Butylthio)-4-(4-chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3e) : Compound 3e was obtained as a yellow solid (58%). M.p: 230-232 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.53 (m, 4H), 4.56 (br s, 2H, NH2), 3.39 (t, J=7.2 Hz, 2H), 3.06 (t, J=6.4 Hz, 2H), 2.41 (t, J=6.0 Hz, 2H), 1.95-1.90 (m, 4H), 1.77 (qt, J=7.2 Hz, 2H), 1.52 (qt, J=7.6 Hz, 2H), 0.96 (t, J= 7.2 Hz, 3H).

13C

NMR (100 MHz, CDCl3): δ (ppm): 170.2, 165.8, 165.5,

158.3, 149.0, 137.5, 136.5, 130.1, 129.4, 111.8, 103.7, 34.5, 30.9, 23.5, 22.4, 22.3, 22.1, 13.8. Anal. calcd for C21H23ClN4S (9%H2O): C, 57.53; H, 6.29; N, 12.77; S, 7.31; Found: C, 57.65; H, 5.37; N, 12.21, S, 6.95. 2-(Butylthio)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3f) : Compound 3f was obtained as a yellow solid (39%). M.p: 188-190°C. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.53 (dd, J=6.8, 2.0 Hz, 2H), 7.05 (dd, J=6.8, 2.0 Hz, 2H), 4.81 (br s, 2H, NH2), 3.90 (s, 3H, O-CH3), 3.37 (t, J=7.2 Hz, 2H), 3.07 (t, J=6.0 Hz, 2H), 2.43 (t, J=6.0 Hz,

Journal Pre-proof 2H), 1.95-1.92 (m, 4H),1.76 (qt, J=7.2 Hz, 2H), 1.50 (qt, J=7.2 Hz, 2H), 0.95 (t, J= 7.2 Hz, 3H).13C NMR (100 MHz, CDCl3): δ (ppm): 170.3, 166.6, 164.9, 161.2, 158.2, 149.7, 131.1, 130.3, 114.6, 111.6, 104.5, 103.8, 55.5, 34.2, 30.9, 30.8, 23.5, 22.4, 22.3, 22.1, 19.7, 13.8. Anal. calcd for C22H26N4OS: C, 66,97; H, 6,64; N, 14,20; S, 8.13; Found: C, 66,93 ; H, 6,57; N, 14,20, S, 7.40. 2-(Butylthio)-4-(3-nitrophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3g) Compound 3g was obtained as a yellow solid (62%). M.p: >260 °C.1H NMR (400 MHz, CDCl3) δ (ppm) 8.48 (d, J= 2.0 Hz, 1H), 8.41 (dd, J= 8.4, 1.2 Hz, 1H), 7.96 (d, J= 7.6 Hz 1H), 7.75 (t, J= 6.0 Hz, 1H), 4.45 (br s, 2H, NH2), 3.39 (t, J= 7.2 Hz, 2H), 3.08 (t, J= 6.0 Hz, 2H), 2.44 (t, J= 6.0 Hz, 2H), 1.95-1.88 (m, 4H), 1.68 (qt, J=7.2 Hz, 2H), 1.27 (qt, J=7.2 Hz, 2H), 0.99 (t, J= 7.2 Hz, 3H).

13C

NMR (400 MHz, CDCl3) δ (ppm) 170.3, 166.4, 163.9,

158.3, 148.5, 148.3, 140.5, 134.9, 130.1, 124.8, 124.1, 112.3, 103.6, 34.5, 30.8, 23.5, 22.3, 14.2. Anal.calcd for C22H26N4OS: C, 60,58; H, 4,80; N, 15,70; S, 8,99; Found: C, 31,69; H, 4,20; N, 6,27, S, 2,95. 4-(4-Chlorophenyl)-2-(octylthio)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-5-amine (3h): Compound 3h was obtained as a yellow solid (51%). M.p: 164-167 °C. 1H NMR (400 MHz, CDCl3) δ(ppm7.44 (m, 4H), 4.46 (br s, 2H, NH2), 3.29 (t, J= 7.2 Hz, 2H), 2.97 (t, J=6.0 Hz, 2H),2.32 (t, J= 6.0 Hz, 2H), 1.86-1.81 (m, 4H), 1.68 (qt, J=7.6 Hz, 2H), 1.40 (qt, J=7.6 Hz, 2H),1.24-1.18 (m, 8H), 0.80 (t, J= 6.8 Hz, 3H).13C NMR (400 MHz, CDCl3): δ(ppm): 170.3, 165.8, 165.5, 158.3, 149.0, 137.5, 136.5, 130.1, 129.4, 111.8, 103.8, 34.5, 31.8, 31.1, 29.2, 29.0, 28.8, 23.5, 22.7, 22.4, 22.3, 14.1. Anal. calcd for C25H31ClN4S (14% H2O): C, 56.74; H, 7.41 ; N, 10.58; S, 6.06 ; Found: C, 56.95; H, 5.99; N, 9.81, S, 5.67. 2.3. Method for Cholinesterase Activity Assay Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity was measured, by the spectrophotometric method developed by Ellman, G.L., et al. [24] Briefly, 150 µl of 100 mM sodium phosphate buffer (pH 8.0), 10 µl of sample solution dissolved in methanol at different concentrations and 20 µl AChE (5.32 x10-3 U) or BChE (6.85 x10-3U) solution were mixed and incubated for 15 min at 25ºC, and 10 µl of 0.5 mM DTNB[5,5´dithio-bis(2-nitrobenzoic) acid] were added. The reaction was then initiated by the addition of 10 µl of acetylthiocholine iodide (0.71 mM) or butyrylthiocholine chloride (0.2 mM). The

Journal Pre-proof hydrolysis of these substrates were monitored spectrophotometrically by the formation of yellow 5-thio-2-nitrobenzoate anion, as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively, at a wavelength of 412 nm, every 5 min for 15 min, utilizing a 96-well microplate reader (Perkin Elmer Multimode Plate Reader EnSpire, USA) in triplicate experiments. Galanthamine was used as reference compound. The results were given as 50% inhibition concentration (IC50) and the percentage of inhibition of AChE or BChE was determined by comparison of reaction rates of samples relative to blank sample (methanol in phosphate buffer, pH 8) using the formula: Inhibition of AChE or BChE (%) =

E―S × 100 E

Where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample. 2.4.

Molecular modeling

AutodockVina 1.1.1 [25] was the software of choice to generate the binding modes of the redocked ligands and the tested compounds.The 2D structure was illustrated by ChemBioDraw Ultra 14.0. ChemBio3D Ultra 14.0from the same package was the tool of obtaining the 3D configuration. The most stable conformers were proposed by energy minimization algorithm MOPAC which operates inside VEGA ZZ 3.1.1.42enviroment [26]. All the crystal structures were downloaded in PDB format from the Protein Data Bank web site [27]. The proteins were prepared for docking by deletion of the unwanted molecules and the water using VEGA ZZ 3.1.1.42.The energy minimization of proteins were performed by YASARA server [28]. The residues of the binding sites in the active site of acetylcholinesterase were identified as Trp86, Tyr124, Ser203, Glu202, Tyr337, Phe338, His447, and Tyr449.The backbone of the binding cavity of butyrylcholinesterase has the following flexible amino acids, Asn68, Ile69, Asp70, Trp82, Gln119, Thr120, Ser198, Trp231, leu286, Ser287, Val288, phe329, Tyr332, phe398, Trp430, Met437, His438 and Tyr440. The flexible residues were allowed to move freely during the processed flexible docking. The docking grid was sized to enclose the cocrystallized molecule and the flexible residues. For acetylcholinesterase (4ey6) [29] the center of the grid box was: X= 8.2, Y = 61.5, Z = -23.8 and the size was 17 *17 *17 Å. Crystal structure AchE (4bdt) [30] has the following spatial center of the docking box X = -2.22, Y = 34.6, Z = -52.3 and the size was 17*20*18 Å. For butyrylcholinesterase (5dyw) [31] the grid

Journal Pre-proof box size was 28 *22*22 Å and the center of box: X = 137.1 Y = 114.3, Z = 39.9. Pymol software was the visualization tool for the ligands/protein complex [32]. 2.5.

Computational details

Density functional theory (DFT) calculations have been carried out using Gaussian09software [33]. The B3LYP functional [34, 35] and the 6-311++G(d,p) basis set have been used for all calculations. All the ground states were confirmed by vibrational frequency analysis (no imaginary frequency). Electronegativity, chemical softness, chemical hardness and electrophilic indexwere calculated based on HOMO and LUMO energiesas reported in the literature [36-40]. Thebond dissociation enthalpy (BDE) have been calculated as reported in our previous works [41, 42]. 3. Results and Discussion 3.1.

Chemistry

The target compounds 3a-h were synthesized via Friedlander reaction as shown in Scheme 1. First,

the

straightforward

condensation

between

2-alkylthiouronium

halides

and

arylidenemalononitriles in the presence of potassium carbonate afforded the corresponding alkylthiopyrimidine derivatives 1a-h, as previously investigated in our laboratory [43]. Condensation of the starting pyrimidines 1a-h with cyclohexanone 2 in the presence of AlCl3 in dry 1,2 dichloromethane gave eight new compounds 3a-h, with moderate to good yields (39-62%). These compounds were purified by recrystallization in ethyl acetate and characterized by 1H NMR, 13C NMR, and elemental analyses (CHNS). R2

R2

O

NH2 N R1

S

2

CN N

NH2

AlCl3, DCE reflux, 24h

1a-h

N R1

S

N

N

3a-h

3a (R1 = CH3, R2 = Cl) (41%)

3e (R1 = C4H9, R2 = Cl) (58%)

3b (R1 = CH3, R2 = OCH3) (58%)

3f (R1 = C4H9, R2 = OCH3) (39%)

3c (R1 = CH3, R2 = NO2) (55%)

3g (R1 = C4H9, R2 = NO2) (62%)

3d (R1 = C2H5, R2 = Cl) (57%)

3h (R1 = C8H17, R2 = Cl) (51%)

Scheme 1. The structures of reported Tacrine-derived AChE/BChE inhibitors.

Journal Pre-proof 3.2.

Biological evaluation

The inhibitory proprieties of target compounds 3a-h were performed by the spectroscopic method described by Ellman’s [24] using Galantamine as the standard. Most of the compounds showed potent inhibitory activity against AChE with IC50s values ranging from 4,32 to 69,19 µM. Compound 3d with IC50 value of 4.32 µM was found to be the most potent compound against AChE, this compound was 5-fold more potent than reference drug Galantamine with IC50 value of 21.81µM (Table 1). For comparison purposes, among compounds 3a, 3b and 3c having all the S-methyl group, with IC50s values of 8,76, 5,85 and 4,68µM respectively, molecule 3c with a 4-nitro group on the benzene ring is more active than the other two compounds 3a and 3b with 4-chloro and 4-methoxy groups on the benzene ring respectively. On the other hand, compounds 3e, 3f and 3g with IC50s values of 13,16, 4,94 and 69,19µM respectively which have the S-ethyl group, it was noticed that compound 3f with a 4-methoxy group on the benzene ring is more active than compounds 3e and 3g with 4-chloro and 4-nitro substituents respectively. In addition, compound 3d was more potent than 3h with IC50 value of 48,94 µM by 11-fold improvement. The difference in efficacy is due probably to the length of S-alkyl chain attached to the thiopyrimidine motiey. The BChE results revealed IC50 values ranging from 2.74 to 151.38 µM. The most active compounds are 3f and 3b with IC50s of 2.74µM and 6.78µM respectively which are more potent than Galanthamine with value of 40.72µM. Both compounds have a 4-methoxy group linked to the phenyl, which leads to deduce that it is responsible for improving BChE's inhibitory activity. By comparing compounds having the same S-alkyl chain, 3a, 3b and 3c with IC50s values of 19,97, 6,78 and 21,71µM respectively, are molecule 3b with a 4-methoxy group is more effective than the other two compounds 3a and 3c with 4-chloro and 4-nitro groups respectively. The calculation of the selectivity index allows the evaluation of the degree of affinity that the synthesized compounds have with AChE and BChE. Experimental results revealed that all compounds showed relatively more inhibitory activity to AChE as compared to BuChE with a factor ranging from 1.16 to 8.80 except 3c which on the contrary has an affinity for BChE.

Journal Pre-proof Table 1. Cholinesterase inhibition efficacy of compounds 3a-h. Compound 3a 3b 3c 3d 3e 3f 3g 3h Galantamineb

IC50 (µM) ± SD / % inhibitiona AChE BuChE 8,76 ±0,22 19,97±0,86 5,85±0.28 6,78±0,51 4,68±0,73 21,71±0,41 4,32±0,87 15,10±0,19 13,16±0,14 115,82±5,95 4,94±0,42 2,74±0,74 69,19±4,99 121,24±1,40 48,94±4,05 151,38±22,2 21,81±1,15 40,72±2,85

Selectivity index AChEc 2,28 1,16 4,64 3,49 8,80 0,55 1,75 3,09 1,87

BuChEd 0,44 0,86 0,21 0,29 0,11 1,80 0,57 0,32 0,53

a

IC50 values represent the means ± SD of three parallel measurements (p<0.05).

b

Reference compound.

c

Selectivity for AChE is defined as IC50 BuChE (µM)/IC50 AChE (µM).

d

Selectivity for BuChE is defined as IC50 AChE (µM)/IC50 BuChE (µM)

3.3.

In silico studies

The suggested binding modes of the active candidates were proposed by molecular docking study. The protein data bank was accessed for obtaining the 3D structure of the enzymes [27]. Two proteins of human acetylcholinesterase were downloaded for the purpose of achieving a comparative study. One of them (pdb code: 4ey6) [29] has Galantamine as a native ligand which was the reference for the biological study. The other one (pdb code: 4bdt) [30] was cocrystallized with the Tacrine like compound (huprine w), so it could offer a model for protein interaction with our compounds which have Tacrine core in their structures. As a consequence we can identify the residues which may be involved in the stabilization of the tested compounds. The spatial distribution of the amino acids within the active sites was different between the two proteins. It was evident that the amino acids move freely to give a maximum fitting of the ligands. Energy minimization of the protein to restore the original distribution of the amino acids in the binding site to their supposed natural orientation was essential. After the energy minimization of the two proteins, the active sites showed a good matching of the orientation inside the pocket between each other. The noticed difference in the orientation of the amino acids in the binding pocket which provoked by the interaction with the bound structure implied the importance of the allowance the free rotation of the flexible amino acids inside the protein gorge. Henceforth the flexible docking was the technique of choice to calibrate the process and for the docking of the tested

Journal Pre-proof compounds. It was postulated that the resulted orientation of the ligand and the surrounding environment of the flexible amino acids residues may superimposed with that of the nonminimized protein. The validation of the docking process was conducted by drawing the native ligand using in 2D form. The 2D structure was transformed into 3D and the best conformer was picked up. The re-docking of the native molecules for both examples was performed by AutodockVina [25]. The output of the theoretical interaction between Galantamine and the protein showed a pattern similarity to that of the crystal structure. The ligand was superimposed over the cocrystallized one as well as the surrounding amino acids with a narrow margin of deviation (Figure 2 a). For huprine w, although the orientation of the ligand and the flexible amino acid backbone of the pocket wasn’t superimposed, we could notice that the spatial interaction between the ligand and the amino acids was preserved. The hydrophobic interactions between the benzene ring of the pyrimidine scaffold and Tyrosine 337 and the fused alicyclic ring with the tryptophan 86 were maintained while the hydrogen bond interaction was shifted from Ser203 to Trp86 (Figure 2 b). a

b

Figure 2. The molecular docking study of AChE a)The re-docking result of the native ligand (inside the blue mesh) with crystallized Galantamine (salmon color) is showing the near positioning between the generated orientation of the flexible amino acids residues (blue sticks) and the crystallized one (salmon sticks) of AchE (Pdb code: 4ey6) b) in silico proposed binding mode of huprine w and with the free rotated amino acids of the gorge relatively to the crystallized structure (salmon color) of AchE pdb code: 4bdt.

Journal Pre-proof The same docking parameter was applied for the assayed compound. Rigid and flexible dockings were performed where the flexible docking introduced more reliable results than the rigid and more compatible with biological study. The orientation of Tacrine core of all compounds (with exception of 3c and 3g) was found to be well oriented to give a good hydrophobic field toward Trp86 in addition to hydrogen bonds with Tyr124 (Figure 3 c). The presence of the nitro group on 3c and 3g may lead to an inverse orientation in which the nitro group made hydrogen bonds with Gly121. Trp86 was parallel in this case to the phenyl ring which offered a reduced surface area for the hydrophobic interaction which might be compensated by the hydrogen bond interaction from the nitro group and pi-interaction. This effect is suggested to potentiate the efficacy of 3c while it might have a negative impact on 3g. The decrease in the inhibitory action could be attributed to the hindrance between the long side chain of the compound and the inner wall of the pocket. This Impact could be seen also in case of 3h which showed the lowest affinity in both rigid and flexible models. The length of side chain seemed to have a key role in the interaction which may be repulsive in case of 3g and 3h while might be an additive hydrophobic site in case of 3f (Figure 3 d). The pocket of BuChE (Pdb code: 5dyw) [31] is larger than that of AChE, and the docking was a challenge. The rigid docking modeling provided results not correlated to the biological studies. The flexible docking still the better choice in this case. The binding affinity provided by flexible docking was more reasonable in a relative to the biological study comparing to the rigid one. The interesting result obtained from the calibration of the docking tool was satisfactory to consider the docking results as consistent. Figure 4 c

d

Figure 3. The molecular docking study of AChE c) 3c binding mode (blue) within the flexible amino acid residues of the binding site (salmon) d) 3d (blue) interaction with the flexible amino acids backbone of the pocket (salmon color).

Journal Pre-proof The native ligand oriented in a similar binding mode displayed by the non-minimized crystal structure of the enzyme. Some displacement in the position of Trp82 could be noticed which possibly occurred to maintain the hydrophobic and the pi-interaction toward the lateral benzene ring. Similarly Tyr440 was moved in to adjust the new position of the same benzene ring which could result in preserving the hydrophobic interaction. The hydrogen bonding interaction between the sulfite moiety and Thr120 was identical in both crystallized and redocked models. One binding mode from the docking of the compounds under investigation was proposed. This pose was selected as it afforded the maximum number of hydrogen bonds between the most active inhibitor 3f and the protein. On the other hand it was the most frequent binding mode between all poses generated by the automated flexible docking. In the selected binding mode the phenyl ring directed toward the outside of the binding cavity and pi-interaction with the phenyl ring of Tyr332 represented a possibility. Another stabilization could be provided by hydrogen bond interaction with Asp70. The hydrophobic interaction of the compounds with the protein seemed to be more limited in comparison with the native ligand which may describe the high IC50 of most of them obtained from the in vitro assay.

Figure 4. Docking results of BuChE (5dyw) a) Re-docked native ligand (blue) and the flexible amino acid in comparison with the original orientation (salmon color). b) Docking result of 3f showing the interaction with the movable amino acid residues of the active site (salmon stick). 3.4.

Theoretical investigations

After having successfully synthesized and evaluated in vitro the biological activity of the target Tacrine analogues, we next envisaged to gain insights about the reactive nature and reactivity insights of tacrines 3a-3c, as representative compounds, by using density functional theory (DFT) calculations. Optimized molecular geometries obtained by theoretical methods

Journal Pre-proof are useful to explain the three-dimensional structures of the investigated compounds. The molecular structures of 3a, 3b and 3c have been optimized at B3LYP/6-311G++(d,p) level of theory. The obtained structures are shown in Figure 5. As can be seen, the structures of 3a-3c are non-planar due to the presence of the NH2 group of the pyridine ring. The substituted benzenes are inclined by 53.67°, 46.45°and 50.85° for 3a, 3b and 3c, respectively. The fused pyridine and pyrimidine rings adopt quasi-planar geometries for all the studied compounds.

Figure 5. Optimized geometries of 3a-3c obtained at B3LYP/6-311G++(d,p) level of theory. Reactivity indices such as electronegativity, electrophilic index, softness and hardness are excellent tools that characterize the reactivity of the target compounds [44, 45]. Electronegativity is a measure of the tendency to attract electrons in a chemical bond. Electrophilicity index is related to the energy lowering associated with a maximum amount of electron flow between a donor and an acceptor [39, 46]. The chemical softness and hardness are measures of resistance to charge transfer. The reactivity indices of compounds 3a-3c were calculated in the gas-phase and are given in Table 2. Table 2. Reactivity indices of 3a-3c obtained at B3LYP/6-311++G(d,p)level of theory. Reactivity indices

3a

3b

3c

Electronegativity

4.20

3.98

4.91

Softness

0.27

0.25

0.38

Hardness

1.87

1.97

1.31

Electrophilic index

4.72

4.04

9.24

An examination of the obtained results reveals that 3a-3c have comparable values of electronegativity, softness and hardness in the range of 3.98-4.91, 1.31-1.97 and 0.25-0.38,

Journal Pre-proof respectively. 3c has the highest electrophilicity index value (9.24), whereas 3a and 3b have comparable values (4.04 and 4.72, respectively).These observations clearly shown that all the studied compounds prefer to act as electron donor rather than electron acceptor. In terms of electrophilic index, the least reactive compound as electron donor is 3c, whereas the other compounds have a comparable reactivity. Molecular electrostatic potential (MEP) mapping is a very useful approach to explore the reactivity of the investigated compounds. MEP mapping results for 3a, 3b and 3c are illustrated in Figure 6. The nucleophilic and electrophilic sites are expressed in term of different color codes; a deep red color indicates an electron-rich site, whereas deep blue indicates an electron-deficient site. As shown in Figure 6, the most electron-rich sites of 3a3c are located around the two adjacent nitrogen atoms of the pyridine and pyrimidine rings, where as the most electron-deficient sites are one the NH2 groups. These results suggest that these sites are the preferred sites for electrophilic and nucleophilic attacks, respectively.

Figure 6. Lowest unoccupied molecular orbital (a), highest occupied molecular orbital (b) and molecular electrostatic potential (c) of 3a-3c calculated at B3LYP/6-311G++(d,p) level of theory.

Journal Pre-proof Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), can provide some useful information for the reactivity of molecules as well as electronic transitions within molecules [47, 48]. In terms of antiradical activity, the energy and distribution of HOMO are important parameters. The shape of the HOMO determines the sites for free radical attack, whereas, its energy is related to the ability of donating electrons. Molecules with lower HOMO energy are less likely to donate electrons [44]. The calculated frontier molecular orbitals energies and distributions in the gas phase for 3a, 3b and 3c are presented in Figure 6. The results showed that the LUMOs of 3a and 3b are distributed nearly on the entire skeleton including the pyridine, pyrimidine and benzene rigs, while that of 3c is only localized on the benzene ring. HOMOs of all the investigated compounds are located on the pyridine, pyrimidineas well as NH2 and SMe groups. From these results, it is clear that the NH2 groups of 3a-3c are potential sites for free radical attack. By comparing the HOMOs energies, theelectron donating ability of the studied compound can be ranged in the following order: 3b>3a>3c. The antiradical potential of the investigated tacrines could also be estimated by computing the bond dissociation enthalpy (BDE) of the respective NH bond. BDE corresponds to the ability of an antioxidant to donate its hydrogen atom and consequently form a radical [49]. Lower BDE values indicate weaker N-H bonds, from which the hydrogen is more easily extracted [50]. The calculated BDE values at B3LYP/6-311++G(d,p)level of theory in the gas-phase for 3a, 3b and 3c, as representative compounds, were found to be, 92.16 kcal/mol 91.65 kcal/moland 93.23 kcal/mol, respectively. These values are comparable to that of the aniline (90.83 kcal/mol) calculated at the same level of theory. These results indicate that the studied Tacrine analogues have a comparable antiradical potential than that of the aniline. This latter isconsidered as a privileged scaffold for the antiradical activity [51, 52]. Based on BDE values the sequence of the antiradical potential can be ranged in the following order 3b>3a>3c. This order is in line with HOMOs results. 4. Conclusion In the present study, we report the development of a novel series of substituted 2alkylthiopyrimidino-Tacrines 3a-h carried out by applying Friedlander reaction of 3-amino-2cyanopyrimidines 1a-h with cyclohexanone in the presence of AlCl3 in 1,2 dichloroethane. All compounds were evaluated for their potential to inhibit commercially available electric eel. acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE). The

Journal Pre-proof experimental results showed that most compounds were highly active towards AChE (IC50s ranging from 4.32 to 69.19µM) and few towards BChE with IC50s in the range of 2.74151.38µM. Also, molecular docking studies was in parallel to the in vitro results, for the purpose of assisted in explaining the structure-activity relationships of this type of compounds. Finally, theoretical calculations by means of DFT/B3LYP method have been carried out in order to gain insights about the molecular structure and reactivity insights of tacrines 3a-3c as representative compounds. Acknowledgments We thank MESRS (Ministère de l’Enseignement Supérieur et de la Recherche Scientifique) and DGRST (Direction Générale de la Recherche Scientifique et Technologique), Algeria for financial support. References [1] C. Patterson, World Alzheimer Report 2018-The state of the art of dementia research: New frontiers, Alzheimer’s Disease International, London, UK, 2018. [2] R.J. Castellani, R.K. Rolston, M.A. Smith, Alzheimer disease, Dis. Mon. 56 (2010) 484546. [3] P. Mishra, A. Kumar, G. Panda, Anti-cholinesterase hybrids as multi-target-directed ligands against Alzheimer’s disease (1998-2018), Bioorg. Med. Chem. 27 (2019) 895930. [4] P. Thomas, A. Pesce, J.P. Cassuto, maladie d’Alzheimer, 01 ed. PARIS: MASSON, 1989. [5] H. Hippius, G. Neundörfer, The discovery of Alzheimer’s disease, Dialogues in clinical neuroscience. 5 (2003) 101-108. [6] D.J. Selkoe, M.B. Podlisny, Deciphering the genetic basis of Alzheimer's disease, Annu. Rev. Genomics Hum. Genet. 3 (2002) 67-99. [7] M. Racchi, M. Mazzucchelli, E. Porrello, C. Lanni, S. Govoni, Acetylcholinesterase inhibitors: novel activities of old molecules, Pharmacol. Res. 50 (2004) 441-451. [8] K. Spilovska, F. Zemek, J. Korabecny, E. Nepovimova, O. Soukup, M. Windisch, K. Kamil, Adamantane - a Lead Structure for Drugs in Clinical Practice, Curr. Med. Chem. 23 (2016) 3245-3266.

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Journal Pre-proof Highlights 

Synthesis of new, AChE inhibitors containing Tacrine and thiopyrimidine moiety.



Their inhibitory activities against ChE were investigated and IC50 values of inhibitor compounds were determined.



All compounds showed activities comparable to or significantly higher than Galantamine.



In silico physicochemical characters assessing and DFT calculations proved compounds as promising leads.

Journal Pre-proof

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: