Synthesis, Docking and Biological Activity of Various ...

RESEARCH ARTICLE

Synthesis, Docking and Biological Activity of Various Substituted Zolpidem based GABAA Inhibitors Endowed Potent Hypnotic and Sedative Activity M Vijey Aanandhi1*, Debojit Bhattacherjee1, R Kamalraj2 Abstract: Zolpidem based compounds affects GABAA regulation in locomotor activity and sleep. The study was undertaken to evaluate the effect of zolpidem based derivatives in mice. Synthesis, characterizations, biological profiling and molecular docking studies were carried out to understand the biological activity and binding selectivity of the synthesized compounds. The study indicates that substituted zolpidem based compounds were shown potent phenobarbitone induced hypnosis as well as locomotor activity throughout the study. Molecular docking study by AutoDock 4.2 MGL tools helped to understand the hypothetical selectivity of the compounds and also endowed the future prospects of de novo design for future prototype ligands. Compound 7a and 7d produced significant reduction in onset and prolongation of sleep duration induced by phenobarbitone. In the second model (locomotor activity in actophotometer) activity was found to be maximum for 7a and 7d (30 mg/kg), produced 31.2 and 33.2% decreased in locomotors activity, where standard drug phenobarbitone produced 59.37% decreased in activity. Molecular docking studies also concluded the selectivity of compound 7d was appreciable in respect to the standard. The binding energy and the bond distances of phenobarbitone and Compound 7d between the target were found to be -7.24 kcal and -6.6 kcal and 2.829 Å (PRO 85), 1.896 Å (PHE 78), (LEU 76) respectively. The study revealed the possibilities in future research of zolpidem based derivatives for establishment of new generation CNS acting agents.

INTRODUCTION Zolpidem (Figure 1) is an imidazopyridine derivative is widely prescribed in clinical practice for the treatment of sleep disorder. [1-4] Zolpidem produces sedative and hypnotic effects via interaction with GABA benzodiazepine receptor complex, with relative selectivity for the Type 1 (omega-1) benzodiazepine receptor subtype. [5-6] Due to its selective binding, Zolpidem has very weak anxiolytic, myorelaxant and anticonvulsant properties but very strong hypnotic properties. [7-9] Our present work concentrates on synthesis, characterization, sedative and hypnotic activity of the six different substituted Zolpidem derivatives. All the synthesized compounds were tested for hypnotic and sedative effect by phenobarbitone induced hypnosis and loco motor activity using actophotometer. The molecular docking study also performed to confirm the selectivity of the synthesized compounds. The comparative study also performed to ensure the efficacy of the test as well as standard compounds.

MATERIALS AND METHODS Melting points of the synthesized compounds were recorded on Gallen kamp digital melting point apparatus MFB-595-101M in open-end capillary tubes. IR, Shimadzu 435 IR spectrophotometer (Shimadzu, Tokyo, Japan), 1HNMR (400 MHz) Bruker (Avance DRX-400) NMR spectrometer in DMSO-d6 using TMS as internal standard were used. Mass spectra were recorded on JEOL SX 102/DA–6000 Mass spectrometer. Elemental analyses were performed on a Perkin Elmer EAL 240 elemental analyzer. AutoDock 4.2 MGL Tools and PyMOL visualize software were taken for the molecular docking study. Experimental Methyl acetophenone was taken as an initial compound to synthesis p-bromomethyl acetophenone [1] by reacting with bromine and acetic acid. p-bromomethyl acetophenone [1] undergoes condensation with substituted 2-amino pyridine to give 2-p-toly 1H-imidazo[1,2-a]pyridine. [2] Which upon undergoes Mannich reaction to give 2-p-toly 1H-imidazo [1,2-a]pyridine-3-yl methyl [sub]-amine. [3] Compound further converted into its cyno,[sub]-2-ptolyl-imidazole [1,2a]pyridine-3-yl methyl [sub]-amine [5] through a methyl iodide quaternary salt. cyno,[sub]-2-ptolyl-imidazole [1,2a] pyridine-3-yl methyl [sub]-amine [5] undergoes alkaline hydrolysis by treating with potassium hydroxide and ethanol reflux to yield [sub]-2-ptolyl-imidazole [1,2a] pyridine-3-yl-acetic acid. [6] Finally,the key intermediate was reacted with imidazole and anhydrous substituted amine to yield substituted zolpidem derivative, n.n-dialkyl2-(-2-p-tolyl-imidazo[1,2-2]pyridin-3-yl)-acetamide(7a-7f). These reactions are summarized in (Scheme 1).

N R1

CH3

N O R2

Figure 1: Chemical Structure of Zolpidem

1Department of Pharmaceutical Chemistry, School of Pharmaceutical sciences, Vels University (VISTAS), Chennai-600117, India. E-mail: [email protected] *Corresponding author

Preparation of P-Bromomethyl Acetophenone [1] To a pre cooled solution of methanol and 4-methyl acetophenone, aluminum chloride was added under stirring at 0-5°C. To this reaction mixture bromine was added slowly at 0-5°C and stirred for 30 minutes.

2Department of Pharmaceutical Chemistry, Saastra College of Pharmaceutical Education and Research, Nellore-524001, Andra Pradesh, India.

Inventi Rapid: Med Chem Vol. 2014, Issue 2 [ISSN 0976-3821]

1

2014 pmc 305, CCC: $10 © Inventi Journals (P) Ltd Published on Web 15/02/2014, www.inventi.in

RESEARCH ARTICLE Scheme Step 1 H2N N 2-amino pyridine

Br

Br/CH3COOH 5-00C

O

O

p-methyl bromo acetophenone (1)

p- methyl aceto phenone

N N

2-p-tolylH-imidazo[1,2-a]pyridine

(2)

Step 2 H N

/ Acetic acid

O

N

R2

formalin

N

N

N

2-p-tolylH-imidazo[1,2-a]pyridine

(2)

R1

2-p-tolylH imidazo[1,2a]pyridine-3yl methyl)[sub]-amine (3)

Step 3

I

R2

Acetone/CH3I/Reflux N

R2 N

R1

N

R1

NaCN,C2H5OH

N

R2

Intermediate (4)

N N

R1

cyano,[sub]-2-ptolylH-imidazo [1,2a]pyridine-3-yl methyl)[sub]-amine (5) Step 4 R2

O

HO N N

N

KOH/ethenol/reflux R1

R1

N

[sub]2-p-tolylH-imidazo[1, 2-a] pyridin-3-yl)-acetic acid (6)

cyano,[sub]-2-ptolylH-imidazo [1,2a]pyridine-3-yl methyl)[sub]-amine (5)

Step 5

HO

HN

O

N

N N N

R1

[sub]2-p-tolylH-imidazo[1, 2-a] pyridin-3-yl)-acetic acid (6)

N

imidazole

R2

R1, R2

O

N, N-Dialkyl-2-(-2-p-tolylH-imidazo [1, 2-a]pyridin-3-yl)-acetamide (7a-7f)

: 7a. R1 =3-CH3, R2 = Dimethylamine, 7b. R1 =3-CH3 , R2 = Dimethylamine, 7c. R1= 5-CH3, R2 = Dimethylamine, 7d. R1 =3-CH3 , R2= Diisopropylamine, 7e. R1 =4-CH3 , R2= Diisopropylamine, 7f. R1 = 5-CH3 , R2= Diisopropylamine,

Scheme 1: Synthesis based on mannich base reaction


2


RESEARCH ARTICLE protons appeared at 3.38 ppm and 1.71 ppm respectively. The pyridine ring protons were observed in the region 6.55-8.11 ppm. The other aromatic protons were observed at expected regions. The 13CNMR also performed and the data showed satisfactory results. In the mass spectra of all compounds (7a-7f), M+1 peak was observed. All compounds gave satisfactory elemental analysis.

Preparation of Sub-2-Ptolyl-Imidazol [1-2a] Pyridine [2] A mixture p-bromomethyl acetophenone [1] undergoes condensation by treating with water, sodium carbonate and substituted 2-amino pyridine at 25-30°C and stirred for reaction completion. Preparation of [Sub]-2-Ptolyl-Imidazole [1, 2a] Pyridine-3-Yl Methyl [Sub]-Amine [3] To a mixture of sub-2-ptolyl-imidazol[1-2a] pyridine [2] in acetic acid, aqueous dimethyl amine solution and formalin solution were added slowly and stirred at 25-30°C for reaction completion. The reaction mass was cooled to 010°C and pH was adjusted to 8-9 using 20% aqueous sodium hydroxide.

1. N, N, 5-Trimethyl-2-Phenyl-2, 3-Dihydroimidazo [1, 2a] Pyridine-3-Carboxamide (7a) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 6.12-7.39 (m, 5H ArH), 6.558.11 (m, 3h, pyridine).13CNMR (TMS, δ ppm): δ 164(C of 1imine,1-C=C), δ 122.6-133.0(CH of 1-ethylene,s,1-C=C),δ 162.8(C of 1-ethylene,1-C=C,1-C,1-N), δ 170.4(C of 1-amide, 1 -C-C, 2 -C from N-amide),δ 140.7(C of 1-benzene, 1 -C-C),δ 127.8-128.7(CH of 1-benzene,1-C-C),δ 20-37.1(CH3 of – C=C,-C=N,-C,-C(=O)-N,-N).Ms [m+1]: m/z 84.84.

Preparation of Methyl 1, [Sub]-2-Ptolyl-Imidazole [1, 2a] Pyridine-3-Yl Methyl) [Sub]-Amine [4] To a mixture of [sub]-2-ptolyl-imidazole [1,2a] pyridine-3yl methyl [sub]-amine [3] and acetone, precooled methyl iodide was added under stirring. The reaction mass was stirred at 20-30°C for 8 hours and filtered.

2. N, N, 6-Trimethyl-2-Phenyl-2, 3-Dihydroimidazo [1, 2a] Pyridine-3-Carboxamide (7b) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 6.12-7.39 (m, 5H ArH), 6.558.11 (m, 3h, pyridine). 13CNMR (TMS, δ ppm): δ 164(C of 1imine,1-C=C), δ 122.6-137.9 (CH of 1-ethylene,s,1-C=C),δ 120.0(C of 1-ethylene,1-C=C,1-C,1-N), δ 170.4(C of 1-amide, 1 -C-C, 2 -C from N-amide),δ 140.7(C of 1-benzene, 1 -C-C),δ 127.8-128.7(CH of 1-benzene,1-C-C),δ 19.8-37.1(CH3 of – C=C,-C=N,-C,-C(=O)-N,-N).Ms [m+1]: m/z 84.32.

Preparation of Cyano [Sub]-2-Ptolyl-Imidazole [1, 2a] Pyridine-3-Yl Methyl [Sub]-Amine [5] A mixture of methyl 1, [sub]-2-ptolyl-imidazole [1, 2a] pyridine-3-yl methyl) [sub]-amine [4] and sodium cyanide were added to water and stirred at 80-95°C until reaction completion. The reaction mass was cooled to room temperature and washed with toluene. The aqueous layer pH was adjusted to 5.0-6.0 with acetic acid. The precipitate compound was filtered, washed, dried at 85°C and recrystallized from methanol to yield an off-white solid. Preparation of [Sub]-2-Ptolyl-Imidazole [1, 2a] Pyridine-3-Yl-Acetic Acid [6] A mixture cyno,[sub]-2-ptolyl-imidazole [1,2a]pyridine-3yl methyl [sub]-amine [5] and potassium hydroxide were added and refluxed for 4 hours using methanol and cooled to room temperature. Preparation of N, N-Dialkyl-2-(-2-P-Tolyl-Imidazo [1, 22] Pyridin-3-Yl)-Acetamide (7a-7f) A mixture of [sub]-2-ptolyl-imidazole [1, 2a] pyridine-3-ylacetic acid, [6] carbonyl diimidazole and followed by addition of dimethyl amine were added and stirred for 8090°C until reaction completion. The reaction mass was cooled to room temperature, washed and dried.

3. N, N, 7-Trimethyl-2-Phenyl-2, 3-Dihydroimidazo [1, 2a] Pyridine-3-Carboxamide (7c) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 6.12-7.39 (m, 5H ArH), 6.558.11 (m, 3h, pyridine). 13CNMR (TMS, δ ppm): δ 164(C of 1imine,1-C=C), δ 122.9 (CH of 1-ethylene,s,1-C=C),δ 114.1147.5(CH of 1-ethylene,s,1-C=C,1-N),δ 147.7(C of 1ethylene,1-C=C,1-C), δ 170.4(C of 1-amide, 1 -C-C, 2 -C from N-amide),δ 140.7(C of 1-benzene, 1 -C-C),δ 127.8-128.7(CH of 1-benzene,1-C-C),δ 22.2-37.1(CH3 of –C=C,-C=N,-C,C(=O)-N,-N). Ms [m+1]: m/z 84.28.

Characterization of the Compounds The structures of the compounds (7a-7f) were confirmed by IR, 1H-NMR and mass spectral data and Elemental analyses. In the IR spectra of all compounds (7a-7f), all derivatives have a strong, characteristic band in the region 1685–1675 cm−1 due to the C=O stretching vibration. The bands due to C=C and C=N stretching vibrations were observed in the region 1620–1400 cm−1. The C-N stretching vibrations bands were observed in the region 14451455cm−1. In the 1H NMR spectra of all compounds (7a-7f) the signals due to the CH of acetate and CH of methyl

4. 5-Hydroxy-N, N-Diisopropyl-2-Phenyl-2, 3Dihydroimidazo [1, 2a] Pyridine-3 Carboxamide (7d) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 1.25(m,12 H of CH of iso propyl), 6.12-7.39 (m, 5H ArH), 6.55-8.11 (m, 3h, pyridine). 13CNMR (TMS, δ ppm): δ 164(C of 1-imine,1C=C), δ 122.6-133.0 (CH of 1-ethylene,s,1-C=C),δ 150.5(C of 1-ethylene, 1 -C=C, 1 –O, 1 -N),δ127.8-128.7(CH of 1benzene, 1-C-C),δ 21.4(CH3 of –C=C,-C=N,-C,-C(=O)-N,-N),δ


3


RESEARCH ARTICLE 169.2(C of 1-amide,1-C-C,2-C(C)-C from N-amide).Ms [m+1]: m/z 83.92.

group using the formula (Percent decrease in activity = (1Wa/Wb) ×100).Where Wa and Wb are average activity scores after and before drug administration respectively and average decrease in activity was calculated for all groups. [12] Table 2 showed the activity data.

5. N, N – Diisopropyl - 6 – Methyl - 2- Phenyl - 2, 3Dihydroimidazo[1,2a]Pyridine-3-Carboxamide (7e) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 1.25(m,12 H of CH of isopropyl), 6.12-7.39 (m, 5H ArH), 6.55-8.11 (m, 3h, pyridine). 13CNMR (TMS, δ ppm): δ 164 (C of 1-imine,1-C=C), δ 122.6-137.9 (CH of 1-ethylene,s,1-C=C), δ 150.5(C of 1-ethylene, 1 -C=C, 1 –O, 1 -N), δ 169.2(C of 1-amide,1-C-C,2-C(C)-C from Namide), δ127.8-128.7(CH of 1-benzene, 1-C-C), δ 21.4(CH3 of –C=C,-C=N,-C,-C(=O)-N,-N),δ 19.8(CH3 of 1-C=C,1-N,1 C=N,1-C) . Ms [m+1]: m/z 84.84.

Molecular Docking Study To understand the biologically activity of a drug molecule as prototype therapeutic agent, the knowledge of binding selectivity towards the protein environment is very essential. Docking study was performed to understand and correlate their biological efficacy towards the selected binding domain of the protein. We used the AutoDock 4.2MGL Tools version 1.5.6 software packages for the molecular docking experiment and Pymol 1.3 Software package to analyze the results. [13] The crystal structure of the protein was extracted from the Protein Data Bank (PDB), identifier (2R2Q). Polar hydrogen atoms were added to the protein. The deletion of the both water molecule and the inorganic charges were done to avoid error. Gasteiger Charge of the macromolecule was added. Ligand docking was carried out applying the Lamarckian genetic algorithm (LGA) implemented in AutoDock 4.2. The grid size was set to 30, 30 and 30 along the X-, Y- and Zaxis to recognize the binding site. Spacing was set as 0.421 Å. The lowest binding energy conformers were selected out of 20 different conformers for each docking simulation and resultant data was further analyzed. Other miscellaneous parameters were assigned to the default values obtained from the AutoDock 4.2 program. The standard Phenobarbitone was also taken and maintained the same procedure for the docking study to compare the value of both test as well as standard.

6. N, N – Diisopropyl - 7 – Methyl – 2 – Phenyl - 2, 3Dihydroimidazo[1,2a] Pyridine-3-Carboxamide (7f) IR (KBR) νmax ∕ (cm−1):3060.6(Ar-H), 2868.3 (CH),1675.0(C=O),1599.5(C=N), 1453.5(C=C), 1445.5(C-N); 1H NMR (DMSO, δ ppm): δ 3.38 (s, 2H, CH Of Acetate), 1.71(m, 2H, CH of methyl), 1.25(m,12 H of CH of isopropyl), 6.12-7.39 (m, 5H ArH), 6.55-8.11 (m, 3h, pyridine). ). 13CNMR (TMS, δ ppm): δ 164(C of 1-imine, 1-C=C), δ 114.1147.5 (CH of 1-ethylene, s, 1-C=C, 1-N), δ127.8-128.7(CH of 1-benzene, 1-C-C), δ21.4 (CH3 of –C=C,-C=N,-C,-C (=O)-N,N). Ms [m+1]: m/z 84.56. Phenobarbitone Induced Hypnosis In this method, mice of either sex were taken randomly and divided into control, standard and test group, where each group contain six animals. Group I served as control and treated with normal saline (10 ml/kg, oral.), group II (standard) treated with standard drug Phenobarbitone (30mg/kg, oral) 15 min before the administration of test drug (30mg/kg, oral.). Test group III-VIII were treated with Compound (7a-f) (30 mg/kg) respectively. Phenobarbitone (30 mg/kg, orally.) was administered 30 min later. Onset of sleep and duration of sleep measured for the entire group. Onset of action was recorded by noting the time of loss of reflex for three consecutive trials, duration of sleep recorded by time difference between loss of righting reflex and recovery time (10-11) Table 1 shows the activity data.

Pocket Validation All receptors are having their own active site or binding domain, where the Ligands are supposed to fit. The active sites are contains a branch of amino acids. Grid generation is helped to recognize that binding region of the receptor. CastP server (http://sts.bioengr.uic.edu/castp/) has been used to validate the receptor pocket. [14] Determination of Ligand Binding Site All possible binding sites of the GABA-A receptor were determined by the open source server called FTsite server (http://ftsite.bu.edu/cite). The PDB: 2R2Q was uploaded in to the server and also created the job name for identification of the result. [15, 16]

Locomotor Activity Using Actophotometer The CNS depressant activity of the substituted Zolpidem derivative was evaluated by studying loco motor activity of mice using actophotometer Albino mice of either sex (20 25 g) were randomly divided into eight groups of six animals. The mice were placed individually inside the chamber of actophotometer for 10 min and basal activity score was noted. Group I was treated with vehicle (1% sod. CMC) and standard drug Phenobarbitone (30 mg/kg, oral.) administered to group II. The animals of the group III-VIII were treated with Compound (7a-f) (30 mg/kg) respectively and after 30 min of mice were placed again in actophotometer for 10 min and the activity was monitored. Percent decreases in activities were calculated for each


Statistical Analysis Results were expressed as Mean + SEM and ANOVA followed by Dunnet’s multiple comparison tests were applied for analysis of data. P value