Molecular docking and synthesis of 1, 2, 4 - triazin analogue of diclofenac as potential ligand for parkinson’s

 

Sudhakar P^1*, Poorana Pushkalai S², Sabarinath C¹, Priyadharshini S¹, Haripriya S³

¹Department of Pharmacology and Toxicology, Swamy Vivekanandha College of Pharmacy, Tiruchengode,
Tamil Nadu- 637 205, India

²Department of Pharmacology, Vinayaka Missions College of Pharmacy, Salem, Tamil Nadu- 636 008, India

³Research Students, Department of Pharmacology, Swamy Vivekanandha College of Pharmacy, Tiruchengode, Tamil Nadu- 637 205, India

 

ABSTRACT:

Parkinson’s disease is one of the most frequent neurodegenerative disorders, characterized by the progressive loss of dopamine neurons in the substania nigra pars compacta. Studies have suggested that NSAIDs may modify the risk of developing PD. Among that diclofenac is one of the most commonly used drugs associated with severe gastric toxicity. In this present study our aim was to design, molecular docking and synthesis of 1, 2, 4 - triazin analogue of diclofenac as potential ligand against PD by replacing - COOH group. The targets used in this study are Dopamine receptor D₃ protein, Dopa decarboxylase, Adenosine A2 receptor, and P38 map kinase, MOA-B was taken from protein data bank. The structure of the ligand was drawn in ACD/ChemSketch 2012. Lamarckian genetic algorithm methodology was employed for docking implemented in AutoDock k4. At the end of the docking, the best poses were analysed and calculated by using Discovery studio 4.1. Results revealed that best fit of ligand against active site and the docking scores are D₃ protein - 6.35, DDC -6.86, AA2AR - 6.11, P38 map kinase-8.67 and MAO-B -10.25. The synthesis 1, 2, 4 - triazin analogue of diclofenac showed improvement in binding affinity and potent protective effect against the risk of PD.

 

 

 

INTRODUCTION:

Parkinson’s disease (PD) is a progressive neurodegenerative brain disorder. It is characterized by diminished facial expression, stooped posture, slowness of voluntary movement, festinating gait (progressively short- ended, accelerated steps), rigidity, and a “pill- rolling” tremor but also of many other central and peripheral neuronal systems^{1, 2}.

 

The exact aetiology of PD is unidentified but the proposed mechanisms including environmental toxins, oxidative stress and neuro inflammation³. A pathological classification of PD includes the loss of pigmented dopaminergic neurons and the presence of Lewy bodies. Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which project to the nigrostriatal pathway (the striatum), results in the loss of dopaminergic functions in individuals with PD⁴. In Dopamine loss in the basal ganglia triggers prominent secondary morphological changes and its depletion also triggers changes in the density and sensitivity of dopamine receptors⁵. The involvement of such dopamine and non-dopaminergic systems is responsible for the occurrence of the motor and non-motor Parkinson’s symptoms.

 

Neuro-inflammation is an important mechanism in defense responses to pathogenic events of PD, but it is also a key component in the progression of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurological disorders. The over-expression of COX-1 and COX-2 has been found in the central nervous system of patients with traumatic brain injury, ischemic and that with neurodegenerative diseases such as AD and PD, which indicates that COX mediated neuroinflammation, is a critical component in neuronal degeneration^{6, 7}.

 

In this condition Non steroidal anti- inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs in modern medicine⁸. NSAIDs readily cross blood brain barriers and inhibits microgial released cyclo-oxygenases and other cytokines. Among that diclofenac significantly inhibits inflammatory cytokines like TNF-α, interleukins and promote the release of IL -10.  Diclofenac is more potent in inhibiting COX-2 than COX-1 isoenzymes. Studies report that diclofenac is effective in animal models and in patients with early stage of PD⁹. It has a complex mechanism of action that involves uncompetitive, low affinity NMDA receptor open-channel blocking. Additionally, diclofenac exhibits anti radical and immunuotropic effects¹⁰.

 

Several previous studies have suggested that diclofenac may elicit appreciable GI irritation, bleeding and ulceration produced. Synthetic approaches based upon chemical modification of diclofenac have been taken with the aim of improving safety profile and in turn therapeutic window. Carboxylic group is a major reason for the GI toxicity of diclofenac. Structural replacement of carboxyl group may reduce the GI toxicity. Previous study reported that 1, 2, 4-triazin derivative may possess appropriate action on Parkinson’s action. 1, 2, 4-triazine derivative as antagonists of Adenosine A₂ receptor is expressed in the basal ganglia where it functionally opposes the actions of the dopamine D₂ receptor. i.e., inhibition of the A₂ receptor leads to enhancement of D₂ receptor function^{11, 12,}.

 

Nowadays, the discovery of new drugs to treat chronic diseases without adverse effects is one of the major challenges for pharmaceutical industry. Among the strategies useful to discovering new drugs and the molecular modification is very promising strategy. Based on above fact this study was aimed for replacement of carboxylic group with 2-Chloroacetamide to produce the 1, 2, 4 –triazin derivative of diclofenac as potential ligand for PD.

 

MATERIAL AND METHODS:

Molecular Docking:

Preparation of ligand structure:

Structure of the 1, 2, 4 - triazine derivatives were retrieved. The structure of the ligands was drawn in ACD / ChemSketch freeware. Finally, compounds were saved in PDB format for further docking studies.   

 

Preparation of target (Protein structure):

The structure of the protein-ligand complexes for the Parkinsonism Disease associated targets were used for the molecular docking studies. They were downloaded from the Research Collaborator for Structural Bioinformatics (RCSB) Protein data bank (PDB; http://www.rcsb.org/pdb/home.do). For each crystal structure, the crystallographic water molecule were removed and edited by removing the hetero atoms^{13, 14} (Table 1).

 

Table 1. Parkinson’s enzyme targets.

 

Docking simulation:

Lamarckian genetic algorithm methodology was employed for docking simulations implemented in AutoDock k4. The standard docking procedure was used for a rigid protein and flexible ligand whose torsion angles were identified. A grid of 60, 60 and 60 points in x, y, and z directions was built with grid spacing of 0.375 Å. The default settings were used for all other parameters.

 

Analysis and visualization of docking simulation results:

At the end of the docking, the best poses were analyzed for hydrogen bonding and calculation using Discovery studio 4.1 and python software was used to view the structure. Molecular docking study estimated by molecular docking score^{15, 16}.

 

Synthesis of 1, 2, 4 - triazin derivative of diclofenac:

The chemicals used in the study were of analytical grade. Melting points were determined in open capillary tubes and were uncorrected. The FT-IR spectra of powered compounds were recorded on Perkin - Elmer KBr spectrometer. Wave values were expressed in cm^-1.

 

Scheme of synthesis:

Step I: Hydrolysis of Diclofenac sodium to 2-[(2, 6-dichloroanilino) phenyl] acetic acid

Diclofenac sodium (0.101 mol) was dissolved in ethanol (2.5mol). To this solution conc. H₂SO₄ was added drop wise to hydrolyse the salt to acid. The acid obtained was filtered, dried. Yield: 95.34 %.

 

Step II: Synthesis of ethyl-[2-(2, 6-dichloroanilino) phenyl] acetate

0.05 mol of 2-[(2, 6-dichloroanilino) phenyl] acetic acid (Step I) was dissolved in absolute ethanol (10ml), conc. H₂SO₄ (1ml) was added and the reaction mixture was refluxed for 22 hrs. Reaction mixture gave on processing ethyl ester. The solid obtained was washed with 50 ml of sodium bicarbonate solution (10%) and recrystallized from methanol. Yield: 94.07 %.

 

Step III: Synthesis of [2-(2, 6-dichloroanilino) phenyl] acetic acid hydrazide

0.01 mol of Synthesis of ethyl-[2-(2, 6-dichloroanilino) phenyl] acetate (Step II) and hydrazine hydrate (0.02 mol) were refluxed in absolute ethanol (50ml) for 20hrs. The mixture was concentrated, cooled and poured in ice cold water. The solid thus precipitated out was filtered, dried and recrystallized from ethanol. Yield: 83.78 %.

 

Step IV: Synthesis of 3-{2-[(2, 6-dichlorophenyl) amino] benzyl}-1, 6-dihydro -1, 2, 4-triazin-5(2H)-one

0.001 mol of Synthesis of [2-(2,6-dichloroanilino) phenyl] acetic acid hydrazide (Step III),                         2-chloroacetamide (0.001 mol) and dimethylformamide (80 ml) were added and the reaction mixture was refluxed for 30 hrs. It was then concentrated and cooled, whereupon the solid precipitated was filtered, washed with ethanol and recrystallized from DMF. Yield:     75.56 % ¹⁷.

 

RESULT:

Molecular Docking studies of 1, 2, 4-triazin derivative of diclofenac:

Structure of 1, 2, 4- triazin derivative of diclofenac was drawn by using chemoffice 2004 software and docking simulation was carried out against the Parkinson’s enzyme targets like Dopamine receptor D3 protein (3PBL), (Figure 1A) Dopa decarboxylase-DDC (1JS3), (Figure 1B) Adenosine A2 receptor-AA2AR (3EML), (Figure 1C) P38 map kinase (2ZAZ), (Figure 1D) Monoamino oxidase-B-MAO-B (2V5Z) (Figure 1E) enzymes target with the help of Autodock k4 program. The binding scores of designed ligand was 1-10 scores with D₃ protein, DDC, AA_2AR, MAPK and MAO-B enzymes ranging from -6.35 to -5.64 Kcal/mol, -6.86 to -5.81 Kcal/mol, -6.11 to -5.02, -8.67 to -4.63 Kcal/mol, and -10.25 to -6.76 Kcal/mol respectively (Table 2).

 

 

Table 2. Docking scores for 1, 2, 4-triazin derivative of diclofenac against the Parkinson’s enzyme targets

  

Fig; 1 Docking study of 1, 2, 4-triazin derivative of diclofenac against 1(A) 3PBL, 1(B) 1JS3, 1(C) 3EML, 1(D) 2ZAZ, 1(E) 2V5Z targets

 

 

Synthesis and IR spectral details of Designed Compounds

2-[(2, 6-dichloroanilino) phenyl] acetic acid (a) was prepared from Diclofenac sodium by hydrolysis in the presence of conc.H₂SO₄ and ethanol. Compound a: IR (KBr), v, cm-1: 3100-3000 (CH), 1795.60 (C=O), 3469.70 (O-H), 1419.51 (C-O-H), 3095.54 (N-H), 1367.44 (C-N), 2509.22 (CH₂). Ethyl - [2-(2, 6-dichloroanilino) phenyl] acetate (b) was prepared from Compound a by etherification in the presence of conc. H₂SO₄ and ethanol. Compound b: IR (KBr), v, cm-1: 3307.69 (CH), 1720.39 (C=O), 1238.21 (C-O-H), 3417.63 (N-H), 1326.93 (C-N), 2981.74 (CH₂), 482.17 (C-Cl).  [2-(2, 6-dichloroanilino) phenyl] acetic acid hydrazide (c) was prepared from Compound b by Treatment with Hydrazine Hydrate in Absolute Ethanol. Compound c: IR (KBr), v, cm-1: 3056.96 (CH), 1488.94 (C=Cl), 1731.96 (C=O), 3450.41 (NH), 1240 (C-N), 3417.63 (N-H), 1326.93 (C-N), 1488.94 (CH₂), 455.03 (C-Cl).  The reaction of Compound c was refluxed with 2-Chloroacetamide and Dimethylformamide to yield 3-{2-[(2, 6-dichlorophenyl) amino] benzyl}-1, 6-dihydro-1, 2, 4-triazin-5(2H)-one (d). Compound d: IR (KBr), v, cm-1: 3056.96 (CH), 1488.94 (C=Cl), 1731.96 (C=O), 3450.41 (NH), 1240 (C-N), 3417.63 (N-H), 1326.93 (C-N), 1488.94 (CH₂), 455.03 (C-Cl) (Figure 2).

 

 

Fig; 2 Synthesis scheme of 1, 2, 4-triazin analogue of diclofenac

DISCUSSION:

NSAIDs are the most common medication used in the neuro inflammatory disorders. Among that diclofenac is one of the prominent drugs which used in Parkinson’s. Diclofenac elicit appreciable GI irritation, bleeding and ulceration produced. Synthetic approaches based upon chemical modification of Diclofenac have been taken with the aim of improving safety profile and in turn therapeutic window. Carboxylic group is a major reason for the GI toxicity of Diclofenac, so the structural replacement of carboxyl group may reduce the GI toxicity. Studies reported that 1, 2, 4-triazin derivative may possess appropriate Anti Parkinson’s action by antagonist the Adenosine A₂ receptor is expressed in the basal ganglia where it functionally opposes the actions of the dopamine D2 receptor. i.e., inhibition of the A₂ receptor leads to enhancement of D2 receptor function^{11, 18}. Hence in this study carboxyl group of diclofenac was replaced by addition of 2- chloroacetamide and produced 1, 2, 4-triazin derivative of diclofenac.

 

In this the intermediate and the final 1, 2, 4-triazin derivative of diclofenac was confirmed by IR spectra region. The compounds showed the possible peak of IR (KBr), which conforms the presence of the functional groups i.e. CH, C=O, C-O-H, N-H, C-N, CH₂, C-Cl.

 

Molecular docking of 1, 2, 4-triazin derivative of diclofenac was analysed against 5 major targets like 1) Dopamine receptor D3 protein (3PBL),2) Dopa decarboxylase-DDC (1JS3), 3) Adenosine A2 receptor-AA_2AR (3EML), 4) P38 map kinase (2ZAZ), 5) Monoamino oxidase-B-MAO-B (2V5Z) enzymes target with the help of Autodock k4 program. In this study, MAO-B , MAPK shows high affinity involved in this target and D₃ protein, DDC, AA2AR, shows slightly low binding affinity when compare to the MAO-B, MAPK.

 

CONCLUSSION:

In conclusion molecular docking study of 1, 2, 4-triazin Analogue of diclofenac shows greater interaction against MAO-B and MAPK as compared to the D3 protein, DDC and AA_2AR targets. The minimization of GI toxicity and potentiation of antiparkinson activity of diclofenac was done by replacing the carboxyl group with 2-chloroacetamide to produce a 1, 2, 4-triazin derivative of diclofenac. Further clinical data are required to explore this synthesized Analogue of Diclofenac as Potential Ligand for improving the status of PD patients.

 

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Received on 25.01.2018          Modified on 11.02.2018

Accepted on 12.03.2018       ©A&V Publications All right reserved