Molecular Docking and in vitro-Anti-Inflammatory evaluation of Novel Isochromen-1-one Analogues Frometodolac

 

Ayyakannu Arumugam Napoleon, Vijay Sharma

Pharmaceutical Chemistry Laboratory, SAS, VIT University, Vellore - 632014, Tamil Nadu, India.

 

ABSTRACT:

In the present work, the three synthesizedisochromen-1-one, their thio and N-methylated analogues derived from the anti-inflammatory drug Etodolacare assessed for its in-vitro anti-inflammatory activity and carried out molecular docking studies. Docking study result shows that compound, 5 possess good binding affinity with the COX-1 receptor showing a docking score of -8.2 and lipophilic interaction of -2.5 which is greater when compared to standard drugs such as Etodolac, Aceclofenac and Diclofenac were used. The same compound 5 also shows comparable in-vitroanti-inflammatory activity with etodolac as standard in both the methods screened.

 

 

 

INTRODUCTION:

Inflammation is a confined physical illness with engorgement, blush generally with acheand as part of the complex biological reaction of body tissues to vicious impetuses, such as pathogen or spoiled cells. In addition, it may damage to the skin, or a localized infection which persuades an inflow of phagocytic blood cells and proteins to the affected area to mending tissue injury. When this happens, the inflammation itself causes damage and responsible for the major symptoms of the disease like anaphylaxis, asthma, atherosclerosis and rheumatoid arthritis. Presently used anti-inflammatory drugs have the danger of critical myocardial and some severe coronary heart diseases for prolonged uses¹.However, plentiful Non-steroidal anti-inflammatory drugs are existing in market, it is quiettask of the scientist to improve more effective and less noxious agents to treat the signs and symptoms of inflammation.

 

Isochromen-1-ones are a class of naturally arising lactones which have a diverse range of pharmacological activities. Some of the synthetic isochromen-1-one analogues were reported to have potential therapeutic properties such as antifungal^2-3, cytotoxic^4-6, antioxidant and anti-inflammatory^7-8, anti-allergic and antimicrobial activity⁹. Etodolac, 1 is an anti-inflammatory drug used in the treatment of numerous musculoskeletal problems, arthritis, inflammations and also any pain ¹⁰.

 

By using Etodolac as starting material, we have synthesized the following compounds via2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetyl chloride,2 namely3-((1,8-diethyl -1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)methyl)-1H-isochromen-1one, 3  3-((1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)methyl)-1-H-isochromene-1-thione, 4and3- ((1,8-diethyl-1,3,4,9-tetrahydro-9-methyl pyrano[3,4-b]indol-1-yl) methyl)-1H-isochromen-1-one, 5.

 

To discover the anti-inflammatory potential of isochromen-1-ones, their thio and methyl analogues derived from etodolac and our continued interest on isochromen-1-ones, the current work is proposed on themolecular docking studies and in vitro-anti-inflammatory evaluations has been carried out and compared with that of etodolac.

 

MATERIALS AND METHODS:

In the current study we used the databases such as Drug Bank and PDB (Protein Data Bank). It contains structural evidence of the macromolecules dogged by X-ray crystallographic, NMR methods etc. The docking analysis of compounds 3, 4 and 5 and standards Etodolac, Aceclofenac, Diclofenac and ketoprofen for anti-inflammatory efficacy were carried out using AutoDock.v.1.5.6. The prostaglandin H2 synthase (COX-1) receptor (ID-1HT5) was taken from protein data bank. In the current type, both the receptor plus ligand kept fixed, and for each derivative nearly hundred confirmations have been screened at various poses within the receptor. Finally the most stable confirmations were designated and exposed to Auto Dock simulation with optimization option. The rototranstional area in to which the ligand is allowed to move freely around the active range is restricted to 10 A^º.  The derivatives of the final compounds were docked with the receptor and the interaction of ligands with the receptor were shown.

 

RESULTS AND DISCUSSION:

i.       Docking studies:

The most stable docking structure of isocoumarin derivatives complexed with PGHS receptor as presented in (Fig: 1-2) which obviously shows that the synthesized ligands are having stronger binding interactions with the receptor when related to standards.  The results (Table:1) attained from the docking studies have exhibited that compound 5have good binding affinity with the docking score of -8.2 kcal/mole which is greater than that of standard Aceclofenac (-7.9 kcal/mole) Diclofenac (-7.7 kcal/mol), Etodolac (-7.0 kcal\mol) and comparable to that of ketoprofen (-9.0 kcal/mol).

 

Table: 1 Molecular docking studies for anti-inflammatory efficacy of synthesized  isochromen-1-one derivatives and standards

Ligand	AutoDock Score [grid]	H bond	Lipo philicity	RMSD	Torsion
3	-7.8	-3.4	-1.4	90.83	4
4	-7.6	-2.7	-1.2	83.27	2
5	-8.4	-3.8	-2.5	92.19	2
Std a	-7.0	-2.3	-1.9	81.54	3
Std b	-7.9	-3.1	-2.1	88.26	2
Std c	-7.7	-2.9	-1.0	85.67	3
Std d	-9.0	-4.2	-1.6	94.18	3

a = Etodolac; b = Aceclofanec; c = Diclofanac; d = Ketoprofen.

 

Prostglandin H2 synthase (PGHS) is a dimeric enzyme normally catalyses prostaglandin synthesis step selectively. These are lipid mediators play a vital role in various physiological processes includes inflammation, platelet aggregation modification of vascular tone. Therefore activity of PGHS is significant during the treatment of inflammation, heart disease, fever, in the stoppage of Alzheimer’s disease and colon cancer.  The synthesis of prostaglandin H2 carried out in two steps at separate active sites namely cyclooxygenase (COX), peroxidise site by PGHS. During first step at the COX site arachidonic acid is oxygenated to produce hydroperoxide prostaglandin G2 (PGG2) which is subsequently reduced at the peroxidise site to corresponding alcohol PGH2. These two steps are linked together by catalytic residue Tyr 385. Finally the peroxidase active site changes this residue to tyrosyl radical species which is further necessary for the activation of COX reaction cycle. Currently inhibition of COX reaction is restricted to pharmacological alteration of PGHS activity. Presence of isocoumarin moiety along with the anti-inflammatory drug etodolac, the combined synergertic efficacy resulted in the high binding affinity with the receptor PGHS which is quite higher when compared to etodolac alone.

 

Fig.1; The binding affinity of synthesized isochromen derivatives with the PGHS receptor [A] The interaction of compound 3 [B] The hydrogen bonding interaction of compound 3 [C] The hydrophobic interaction of compound 3 receptor [A] The interaction of compound 4 [B] The hydrogen bonding interaction of compound 4 [C] The hydrophobic interaction of compound 4. [A] The interaction of compound 5 [B] The hydrogen bonding interaction of compound 5 [C] The hydrophobic interactions of compound 5.

 

Fig.2;The binding affinity of  various standards with the PGHS receptor [A] The interaction Aceclofenac[B] The interaction of Diclofenac [C] The interaction of ketoprofen [D] The interaction of etodolac.

 

ii.      In-vitro Anti-inflammatory Studies:

In the present work in-vitro anti-inflammatory studies were also carried out by two methods for example membrane stabilization test and proteinase inhibitory test. RBCs membrane was studied since they resemble to lysosomal membrane¹¹. Inhibition of heat induced haemolysis will probably inhibit the release of lysosomal content of the neutrophils at the site of inflammation. These neutrophil lysosomal components include bactericidal enzymes and protease, which upon extracellular release gives further tissue inflammation and damage. Proteinase has been associated in arthritic reaction. Neutrophils are identified to be a good source of proteinase which carries numerous serine proteinases in their lysosomal granules. Leukocytes proteinase show an important role in the progress of tissue injury during inflammatory reactions and substantial level of protection was delivered by proteinase inhibitors.

 

1.      Membrane stabilization test:

Preparation of red blood cells (RBCs) suspension:

Fresh human blood (10 mL) was taken from a healthy volunteer and transferred to heparinized centrifuged tubes. Tubes were centrifuged at 3000 rpm for 10 minutes. Then washed 3 times with equal volume of saline solution. The volume of blood was measured and reconstituted to 10 % with normal saline.

 

Heat induced haemolysis:

Equal volume of test samples (3, 4 and 5) of various concentrations (50, 75 and 100 µg/ml) and 10% v/v RBC suspension¹² (2 ml) were transferred to centrifuged tubes. In control test tube instead of drug only saline was added and using Etodolac as standard drug. All centrifuge tubes were incubated in water bath at 56 ºC for half an hour. Reaction mixture was then centrifuged at 2500 rpm for 5 minutes.  Absorbance of the supernatant was taken at 560 nm. The test was performed in triplicates and the percentage membrane stabilization activity was calculated by:

 

(% Inhibition) = [(A_b-A_s)/A_b] × 100

Where A_b= absorption of blank sample`

A_s= absorption of sample

 

Table: 2Membrane stabilization activity of etodolac derivatives, 3, 4, 5 and standard

a = Etodolac

 

2.      Proteinase inhibitory activity:

1ml of 20 mMTrisHCl buffer with the pH of 7.4is added to the 2 ml of the reaction mixture having 0.06 mg trypsin. Then 1 ml test sample of different concentrations were added. Etodolac was taken as standard drug. The mixture was incubated at 37ºC for 5 min and then 1 ml of 0.8% (w/v) casein was added. Incubated for an additional 20 min. andto terminate the reaction, 2 ml of 70 % perchloric acid was added. Cloudy suspension obtained was centrifuged. Absorbance of the supernatant was measured at 210 nm against buffer as blank. This test was performed in triplicate. Proteinase inhibitory activity in terms of percentage inhibition was calculated by:

 

(% Inhibition) = [(A_b-A_s)/A_b] × 100

Where A_b= absorption of blank sample

A_s= absorption of sample

 

Table: 3 Proteinase inhibitory activity of etodolac derivative, 3, 4, 5 and standard

a = Etodolac

 

 

Fig. 3; Anti-inflammatory efficacy of isochromen derivatives and standard [A] Membrane stabilization method [B] Proteinase inhibitory method.

 

In-vitro anti-inflammatory efficacy:

The three etodolac analogues made were successfully explored for their in-vitro anti-inflammatory activities. The present study revealed that compound3 and 4 showed  moderate anti-inflammatory activity in both the membrane stabilization test and proteinase inhibitory activity test whereas compound 5 showed anti-inflammatory activity which is comparable to Etodolac (Fig.3) (Table:2-3).

 

CONCLUSION:

In conclusion the isochromen-1-one, their thio and N-methylated analogues derived from etodolac through molecular docking studies clearly revealed that these are having good anti-inflammatory efficacy and their interaction with PGHS receptor. In this modern era in order to treat the signs and symptoms of both chronic and acute inflammatory diseases there is a need to develop effective therapeutic agents with less side effects. Hence in the present work novel isochromen-1-one ring containing compounds have been shown their synergistic anti-inflammatory efficacy which is explored successfully.

 

ACKNOWLEDGEMENTS:

The authors desire to express their gratitude to the management of VIT University, Vellore for their support and all the necessary facilities facilities for carrying out this study.

REFERENCES:

1.       Hiller PL, Hodd KO, Wilson RL, Anti-inflammatory drugs: protection of a bacterial virus as an in vitro biological measure of free radical activity. Chem. Biol. Interact, 1983; 47: 293– 305

2.       Meepagala K, Sturtz G, Wedge D, Antifungal constituents of the essential oil fraction of Artemisia dracunculus L. Var. dracunculus,J. Agric. Food Chem., 2002; 50: 6989–6992.

3.       Nozawa, K, Yamada M, Tsuda Y, Kawai K, Nakajima S, Syntheses of antifungal isocoumarins. II. Synthesis and antifungal activity of 3-substituted isocoumarins

4.       Chem. Pharm. Bull. 1981, 29, 2491-2495

5.       Whyte C, Gloer JB, Scott JA, Malloch D, Cercophorins AC, Cercophorins A-C: novel antifungal and cytotoxic metabolites from the coprophilous fungus Cercophora areolata, J. Nat. Prod., 1996; 59: 765–769.

6.       Cai Y, Kleiner H, Johnston D, Dubowski A, Botic S, Ivie W, Giovanni JD, Effect of naturally occurring coumarins on the formation ofepidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in SENCAR mice, Carcinogenesis., 1997; 18: 1521–1527.

7.       Ai-Yun Peng and Yi-Xiang Ding, The Synthesis of Phosphaisocoumarins by Cu (I)-catalyzed intramolecular Cyclization of Ethynylphenylphosphonic Acid Monoesters,  J. Am. chem. soc. 2003, 125, 15006-15007.

8.       Napoleon AA, Bhagwat S, Nawaz khan F, Pharmacological evaluation of isochromen-1-ones and their thioanalogues derived from anti-inflammatory drug ketoprofen, res chem intermed., 2013; 39: 1343-1351.

9.       Qadeer G,  Hasan Rama N, Gardun Ramırez ML,  Synthesis and anti-inflammatory activity of fluorinated isocoumarins and 3,4-dihydroisocoumarins,  Journal of Fluorine Chemistry, 128 (2007) 641–646.

10.     Matsuda H, Shimoda H, Yoshikawa M, Structure-Requirements of isocoumarins, phthalides, and stilbenes from hydrangeaedulcis folium for inhibitory activity on histamine release from rat peritoneal mast cells, Bioorg. Med. Chem. 1999; 7: 1445– 1450.

11.     Pareek A, Chandurkar N, Tenpe CR, Yeole PG, Payghan R, International Journal of Pharmacology and Therapeutics., 2011, 1: 1-6.

12.     Uma Singh,  Tabibian J, Senthil K. Venugopal, Sridevi D, and Ishwarlal J, Development of an In Vitro Screening Assay to Test the Anti-inflammatory Properties of Dietary Supplements and Pharmacologic Agents, Clin. Chem. 2005; 51: 2252–2256.

13.     T. L. Lenike, D.A. Williams, V.F. Roche, S.W. Zito, Foye’s Principles of Medicinal Chemistry, W. Williams and Wilkins, Philadelphia, 2008.

 

 

 

Accepted on 05.09.2017        © RJPT All right reserved