Research Article INSILICO DRUG DESIGN AND MOLECULAR DOCKING STUDIES OF SOME NOVEL BENZOTHIAZOLE DERIVATIVES AS ANTI-CANCER AND ANTI-INFLAMMATORY AGENTS DEEPTHY CHANDRAN, LEENA. K. PAPPACHEN*, MANJU PRATHAP, JINSHA.M.J, JILSHA.G. Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Kochi 682041, Kerala, India. Email: [email protected]. Received: 30 Sep 2013, Revised and Accepted: 03 Jan 2014 ABSTRACT Objective: Cancer is a disease characterized by uncontrollable, irreversible, independent, autonomous, uncoordinated and relatively unlimited and abnormal over growth of tissues. Benzothiazole is an important class of heterocyclic compound which possess interesting biological activities like anti-tumor, anti-microbial, anti-tubercular, anti-malarial, anti-convulsant, anthelmintic, analgesic and anti-inflammatory activity. The drugs containing oxadiazole groups were the first effective chemotherapeutic agents which were systematically proved for the prevention and cure of bacterial infection in human beings. The Objective of the study is too carried out the docking studies of benzothiazole derivatives containing oxadiazole groups or amino groups with known anti-cancer and anti-inflammatory targets like estrogen receptor, cox1 and cox2 by using Argus lab and Auto dock programmes. Methods: Docking studies were carried out using Argus lab and Auto dock version 4.0 for all ten ligands and docking scores were compared with the scores of standard drugs Tamoxifen and Indomethacin. Validation of ligands was carried out by using Lipinski rule of five. Results: 10 ligands show higher docking scores and shows better drug-likeness properties as compared to the reference drugs. The compounds show lowest docking energy and hydrogen bondings stabilize the interactions. Conclusion: The study concluded that all benzothiazole derivatives will be significant lead for further investigation of anti-cancer and anti- inflammatory agents. Keywords: Benzothiazole, Oxadiazole, estrogen receptor, Cox1, Cox2, Anti-cancer and Anti-inflammatory targets. INTRODUCTION Benzothiazole is a heterocyclic compound which possess various biological activities and still of great scientific interest now a days. Benzothiazoles are fused membered rings which consist of thiazole ring fused with benzene ring. They are widely found in bioorganic and medicinal chemistry with application in drug discovery [1] .Benzothiazole moieties are part of compounds showing numerous biological activities like anti-fungal [2] ,anti-cancer [3] ,anti- inflammatory [4] , anti-diabetic [5] ,anti-convulsant [6] ,anti- microbial [7] ,diuretic [8] ,anti-tubercular [9] ,schictosomicidal [10] ,anthelmintic [11] activities. They have also found application in industry as anti-oxidants and vulcanization accelerators [12] . Benzothiazole N S 1 2 3 4 5 3a 6 7a 7 Benzothiazole is used in research as a starting material for the synthesis of various bioactive structures. Its aromaticity makes its relatively stable. Due to its important pharmaceutical utilities, the synthesis of derivative compounds is of considerable interest. Benzothiazole can scavenge reactive oxygen species and suppress inflammation, edema and pain. The potential of benzothiazole for anti-inflammatory activities and related metabolic pathways have been recently reviewed. Cyclooxygenase (Cox) is the rate limiting enzyme that catalyses the conversion of arachidonic acid to important inflammatory mediators like prostaglandins, prostacyclins and thromboxanes. Cyclooxygenase exist in two distinct isoforms, Cox1and Cox2.Cox1 is involved in cytoprotection of GIT and Cox2 is responsible for inflammation. Thus inhibition of both isoforms by NSAID’s causes gastrointestinal ulceration [13] .The research project involves the Insilco designing of novel benzothiazole derivatives having good potency, less toxicity against inflammatory targets, cox1 and cox2. Along with this, anti-cancer activities of the same benzothiazole analogues were evaluated for their insilico anti-cancer activity against estrogen receptor which is present in clinical trials for the prevention of breast cancer. MATERIALS AND METHODS Group of compounds were designed by making substitutions at 2 nd , 4 th and 6 th position of benzothiazole nucleus. At 2 nd position, amine group and oxadiazole derivatives have been substituted and at 4 th and 6 th position, chlorine and fluorine group have been substituted. Ten substitutions were selected to complete the group. In this study, Tamoxifen and Indomethacin have been used as reference drugs for cancer and inflammation respectively. N S R2 R4 R6 Docking study Selection of target and lead A small molecule library of 10 compounds was docked into the enzyme and the potential leads were discovered. Targets were selected for anti-cancer and anti-inflammatory activity. Ligand Preparation The smiles formulas of the drug molecules were obtained from ChemSketch which is a chemically intelligent drawing interface freeware developed by Advanced Chemistry Department. Molecular Network software packages provide CORINA, which was used for the generation of 3D coordinates from smiles. Again using Converter of these same server 2D structures of the drugs was converted into PDB format which is an acceptable form for any standard docking software. Finally using ARGUS LAB ligand molecules were prepared by the addition of hydrogen atoms. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Suppl 2, 2014 Academic Sciences
Transcript
Research Article
INSILICO DRUG DESIGN AND MOLECULAR DOCKING STUDIES OF SOME NOVEL BENZOTHIAZOLE DERIVATIVES AS ANTI-CANCER AND ANTI-INFLAMMATORY AGENTS
DEEPTHY CHANDRAN, LEENA. K. PAPPACHEN*, MANJU PRATHAP, JINSHA.M.J, JILSHA.G.
Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Kochi 682041, Kerala, India. Email: [email protected].
Received: 30 Sep 2013, Revised and Accepted: 03 Jan 2014
ABSTRACT
Objective: Cancer is a disease characterized by uncontrollable, irreversible, independent, autonomous, uncoordinated and relatively unlimited and abnormal over growth of tissues. Benzothiazole is an important class of heterocyclic compound which possess interesting biological activities like anti-tumor, anti-microbial, anti-tubercular, anti-malarial, anti-convulsant, anthelmintic, analgesic and anti-inflammatory activity. The drugs containing oxadiazole groups were the first effective chemotherapeutic agents which were systematically proved for the prevention and cure of bacterial infection in human beings. The Objective of the study is too carried out the docking studies of benzothiazole derivatives containing oxadiazole groups or amino groups with known anti-cancer and anti-inflammatory targets like estrogen receptor, cox1 and cox2 by using Argus lab and Auto dock programmes.
Methods: Docking studies were carried out using Argus lab and Auto dock version 4.0 for all ten ligands and docking scores were compared with the scores of standard drugs Tamoxifen and Indomethacin. Validation of ligands was carried out by using Lipinski rule of five.
Results: 10 ligands show higher docking scores and shows better drug-likeness properties as compared to the reference drugs. The compounds show lowest docking energy and hydrogen bondings stabilize the interactions.
Conclusion: The study concluded that all benzothiazole derivatives will be significant lead for further investigation of anti-cancer and anti-inflammatory agents.
Benzothiazole is a heterocyclic compound which possess various biological activities and still of great scientific interest now a days. Benzothiazoles are fused membered rings which consist of thiazole ring fused with benzene ring. They are widely found in bioorganic and medicinal chemistry with application in drug discovery[1].Benzothiazole moieties are part of compounds showing numerous biological activities like anti-fungal[2],anti-cancer[3],anti-inflammatory[4], anti-diabetic[5],anti-convulsant[6],anti-microbial[7]
,anthelmintic[11] activities. They have also found application in industry as anti-oxidants and vulcanization accelerators[12].
Benzothiazole
N
S1
2
34
5 3a
6 7a
7
Benzothiazole is used in research as a starting material for the synthesis of various bioactive structures. Its aromaticity makes its relatively stable. Due to its important pharmaceutical utilities, the synthesis of derivative compounds is of considerable interest. Benzothiazole can scavenge reactive oxygen species and suppress inflammation, edema and pain. The potential of benzothiazole for anti-inflammatory activities and related metabolic pathways have been recently reviewed. Cyclooxygenase (Cox) is the rate limiting enzyme that catalyses the conversion of arachidonic acid to important inflammatory mediators like prostaglandins, prostacyclins and thromboxanes. Cyclooxygenase exist in two distinct isoforms, Cox1and Cox2.Cox1 is involved in cytoprotection of GIT and Cox2 is responsible for inflammation. Thus inhibition of both isoforms by NSAID’s causes gastrointestinal ulceration [13].The research project involves the Insilco designing of novel benzothiazole derivatives having good potency, less toxicity against inflammatory targets, cox1 and cox2. Along with this, anti-cancer
activities of the same benzothiazole analogues were evaluated for their insilico anti-cancer activity against estrogen receptor which is present in clinical trials for the prevention of breast cancer.
MATERIALS AND METHODS
Group of compounds were designed by making substitutions at 2nd, 4th and 6th position of benzothiazole nucleus. At 2nd position, amine group and oxadiazole derivatives have been substituted and at 4th and 6th position, chlorine and fluorine group have been substituted. Ten substitutions were selected to complete the group. In this study, Tamoxifen and Indomethacin have been used as reference drugs for cancer and inflammation respectively.
N
S
R2
R4
R6
Docking study
Selection of target and lead
A small molecule library of 10 compounds was docked into the enzyme and the potential leads were discovered. Targets were selected for anti-cancer and anti-inflammatory activity.
Ligand Preparation
The smiles formulas of the drug molecules were obtained from ChemSketch which is a chemically intelligent drawing interface freeware developed by Advanced Chemistry Department.
Molecular Network software packages provide CORINA, which was used for the generation of 3D coordinates from smiles. Again using Converter of these same server 2D structures of the drugs was converted into PDB format which is an acceptable form for any standard docking software. Finally using ARGUS LAB ligand molecules were prepared by the addition of hydrogen atoms.
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Suppl 2, 2014
Academic Sciences
Pappachen et al. Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
204
Table 1: Substituent’s at R groups
Compound code R2 R4 R6
BT1 –NH2 -Cl -Cl
BT2 –NH2 - F - F
BT3
O
N N
Cl
NH2
H H
BT4
O
N N
OCH3
H H
BT5
O
N N
Cl
H H
BT6
O
NN
H H
BT7 -SCH3 H H
BT8
O
N N
O
Cl
Cl
H H
BT9
O
N N
H H
BT10
O
N N
Br
H H
Table 2: Compounds and IUPAC names of Benzothiazole derivatives of Protein Structure
Crystallographic structures of the targets (6cox, 1cqe, 1qkm) was obtained from PBD (Protein Data bank) and saved in standard 3D coordinate format.
Active Site Identification the targets were possessing natural ligand and so active site residue identification was carried out taking advantage of the same.
The protein was loaded in SWISS PDB viewer. Protein which had many chains was cleaned and a single chain of interest was selected.
Using the control panel of this software, natural ligand molecules were selected. All the residues surrounding this ligand which comes in 6.00A0were identified and selected. These molecules were checked in previous literature to confirm the selection and also their hydrophobic properties were checked to confirm its
presence in the binding pocket. The same procedure was carried for all targets.
Preparation of Active Site
Explicit Hydrogen atoms missing in the PDB structure were added using Argus Lab, docking software. Furthermore, the atom list of the molecules were prepared, which represents numbers of all the atoms of the active site residues involved.
Energy Minimization
Hydrogen added clean files of the proteins were reloaded in the Swiss PDB Viewer. The conformations and energy states of the newly added hydrogen were fixed and corrected by minimizing the energy. New energy levels were checked for the RMSD deviation with its actual PDB structures.
Pappachen et al. Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
205
Molecular Docking
Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. The docking was done by using two softwares, Argus lab and Autodock. Preliminary docking was performed by using Argus lab and it was faster than Autodock. The preliminary screening helps to compare the docking score of standard drugs against ligands. Then the pre-screened ligands were validated using Autodock version 4.0 which is more efficient. Accuracy is more for the score obtained from the Autodock if it takes more time than the Argus lab. In Autodock, the proteins were refined by removing water molecules and polar hydrogen’s and kollmann charges were added. Grid box
for docking simulations were constructed with 60 points in x, y, and z direction to be centered in the active site using Autogrid utility of the Autodock programme. The target ligand complex was subjected to 2.5 million evaluations. The binding energies are compared with the docking score of the standard ligands, Tamoxifen and Indomethacin.
Visual Inspection
The solution structures of each drug candidates against all the targets were visualized and inspected for their goodness of fit and orientation inside the active site. This was done with PYMOL, standalone visualization software. Also the conformation and contacts with all amino acids were checked manually.
Table 3: The docking scores obtained from the preliminary docking programme by using Argus lab were listed below:
Sl no. Compound code Docking score(kcal/mol)
Cox1 Cox2 ER
1 BT1 -7.817 -9.460 -9.68
2 BT2 -6.139 -6.678 -9.491
3 BT3 -8.036 -9.630 -6.45
4 BT4 -7.567 -9.377 -8.50
5 BT5 -8.178 -9.720 -9.59
6 BT6 -8.569 -11.524 -11.9651
7 BT7 -11.1120 -12.296 -11.1988
8 BT8 -7.604 -10.O21 -9.64
9 BT9 -8.001 -9.002 -7.96
10 BT10 -8.314 -9.969 -9.26
11 Tamoxifen -9.26
12 Indomethacin -11.089 -10.9612
RESULTS AND DISCUSSION
Docking Analysis
The docking scores were obtained from the analogues against Cox1, Cox2 and ER receptors. The output of all ligands were given by energy values in kcal/mol as shown in Table 3.
All the compounds shows good docking scores when compared to standard drugs. Docking score of the compounds targeted estrogen receptor was compared with the score of the drug Tamoxifen which is used as a potent drug for the treatment of breast cancer and docking score of the compounds targeted Cox1 and Cox2 receptor was compared with the score of the drug Indomethacin which is
used as NSAID to reduce fever, pain and swelling. In Argus lab, BT7 shows the highest docking score than the standard drugs. Then BT6 shows higher docking score against the receptor ER and Cox2 than the standard drugs, Tamoxifen and Indomethacin. Next comes, BT8 with high docking score against the receptor Cox2.
Benzothiazole derivatives were docked with the crystallographic structures of the targets by Autodock version 4.0 screening programme as shown in Table 4.The analogues were examined for their binding energies and hydrogen bonding. The conformations with highest binding energies and greater number of hydrogen bonds of all the ligands were taken in consideration for ranking the analogues.
Table 4: Autodock Docking scores
S. No. Compound code Docking score(kcal/mol)
COX 1 COX2 COX3
1 BT1 -5.78 -6.77 -6.29
2 BT2 -5.58 -5.76 -5.25
3 BT3 -8.84 -8.82 -7.19
4 BT4 -8.4 -8.72 -7.48
5 BT5 -8.73 -11.66 -7.45
6 BT6 -8.98 -11.57 -7.68
7 BT7 -9.84 -9.45 -8.86
8 BT8 -5.93 -6.11 -5.35
9 BT9 -8.42 -8.59 -7.05
10 BT10 -8.78 -9.4 -7.55
11 Tamoxifen -3.86
12 Indomethacin -9.87 -8.97
All the analogues show higher docking scores when compared to standard drugs. Autodock shows lowest docking scores than Argus lab but have more accuracy.BT7 shows higher docking scores with receptor ER and BT6 shows higher docking score with receptor Cox2.Studies have proved that compounds showing good Cox2 inhibition can also be considered as good agents for anti cancer therapy. The interactions were stronger (energetically lesser) for all the ligands which are used for docking simulation.
Potential Binding Site in Receptors
Autodock version 4.0 screening programme is also used for the identification of most potential active site where the ligand can bind and interact with the target protein, Cox1, Cox2 and ER. Residues ARG 374,ARG 928,ARG 926,ASN 375,ARG 376,ARG 346,THR 206,TYR 385,HIS 386,THR 212,TRP 345,VAL 280,PRO 278 were predicted as active site in the target proteins,Cox1,Cox2 and ER as shown in Table 2.Number of
Pappachen et al. Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
206
hydrogen bonding will considerably increase the affinity of ligand-target interaction. Autodock results shows that most of the benzothiazole derivatives shows higher hydrogen bonding between the ligand-target interactions.
Some of the derivatives showing oxadiazole moieties show more than two hydrogen bonds commonly ARG 374, ASN 375, ARG 376.This hydrogen bonding interactions helps to increase the binding energy of ligand-protein interactions.
Table 5: Autodock screening programme of derivatives having hydrogen bonding interactions
S. No. Compound code Hydrogen bonding interactions (kcal/mol)
COX 1 COX2 COX3
1 BT1 ARG 374 THR 206
TYR 385
NIL
2 BT2 ARG 928
ARG 374
ARG 926
THR 206 ARG 346 HIS386
3 BT3 ARG 374
ASN 375
ARG 376
TYR 385
THR 212
TRP 345
ARG346
4 BT4 ARG 926
ASN 927 ARG 928
ALA 378 NIL
5 BT5 ARG 376
ARG 374 ASN 375
ALA 378 NIL
6 BT6 ARG 926
ARG 928
NIL VAL 280
7 BT7 ARG 374 NIL NIL
8 BT8 ASN 375
ARG 926
NIL NIL
9 BT9 ARG 926 ASN 927
ARG 928
TYR 385 NIL
10 BT10 ASN 375 ARG 926
NIL PRO 278
Of the high docking scores ligand-target complexes with different hydrogen bonding interactions generated by Autodock 4.0 programme were shown below
QSAR and toxicity studies was performed to obtain the molecular properties of all ligands as shown in Table 6.QSAR studies reveals that all ligands was passed and acted as a drug molecule by their adherence to the properties such as Absorption, Distribution, Metabolism and Excretion (ADME) as per the Lipinski Rule Of 5[14].The results shows that all the values of analogues were relays within the optimal range.
Also the compounds have molecular weight less than 500 Daltons and number of hydrogen bond donors and hydrogen bond acceptors of all the analogues is below 5 and 10 respectively.
All the values of partition coefficient and number of rotatable bonds were coming under the limit of 5.All these data indicates that the analogues shows no more violations likely to be an orally active drug.
Table 6: Analysis of Lipinski rule of 5 for the novel proposed analogues.
S. No. Compound
Code
Molecular
Weight
No. Of
Hba
No. Of
Hbd
C log p No. Of
Rot.b
n violation
1 BT1 219.O96 2 2 3.238 0 0
2 BT2 186.186 2 2 2.209 0 0
3 BT3 328.784 2 5 4.348 2 0
4 BT4 309.35 0 5 4.316 3 0
5 BT5 313.769 0 4 4.937 2 0
6 BT6 305.362 0 4 4.296 3 0
7 BT7 378.24 0 5 4.52 4 0
8 BT8 181.285 0 1 3.092 1 0
9 BT9 279.324 0 4 4.283 2 0
10 BT10 388.22 4 4 4.22 0 0
CONCLUSION
Flexible docking of ligand to receptor molecules is an emerging approach and is extensively used to reduce cost and time in drug discovery. In this study the approach utilized is successful in finding potent inhibitors against Cox1, Cox2 and ER receptors. All the compounds show lowest docked energy and hydrogen bonding stabilizes the interactions. The analogues of benzothiazole at second position showed good receptor binding with the selected targets. Heterocyclic oxadiazole ring is having anti-cancer, anti-inflammatory and anti-microbial activity. Substitution on 5th position of oxadiazole ring with aromatic group also increases the activity. Introduction of an ethylene bridge between oxadiazole and
aromatic substitution resulted in an analogue with best binding potency.
The final assessment of drug-likeness and its related parameters helps to confirm the oral activity of compounds. The study concluded that all benzothiazole derivatives will be significant lead for further investigation of anti-cancer and anti-inflammatory agents.
ACKNOWLEDGEMENT
We would wish to express our sincere gratitude to Amrita School of Pharmacy, AIMS Health Sciences Campus, Kochi, Kerala, for providing us the facilities for carrying out this research work.
Pappachen et al. Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
208
REFERENCES
1. Sukhbir.L.Khokra, Kanika Arora, Heena Mehta, Ajay Aggarwal, Manish Yadav: Common Methods to synthesize Benzothiazole derivatives and their medical significance. International Journal of Pharmaceutical sciences and research 2011; 2 Suppl 6:1356-1377.
2. Jian-Quan Weng, Xing-Hai Liu, Hua Huang, Cheng-XiaTan, Jie Chen: Synthesis, Structure and Antifungal Activity of New 3-[(5-Aryl-1, 3, 4-oxadiazol-2-yl) methyl]benzo[d]thiazol-2(3H)-ones. Molecules 2012; 17 Suppl 2:989-1001.
3. Shi D,Bradshaw T, Wrigley S, McCall C,Lalieveld P, Fichhtner I and Stevens M: Synthesis of 2-(4-aminophenyl) benzothiazoles and evaluation of their activities against breast cancer cell lines in vitro and in vivo. J. Med. Chem 1996; 6:3375-3384.
4. Venktesh P, Pandeya S N: Synthesis, characterization and anti-inflammatory activity of some 2-amino benzothiazole derivatives. International Journal of Chem. Tech research 2009; 1 Suppl 4:1354-1358.
5. Pattan SR, Suresh CH, Pujar VD, Reddy VVK, Rasal VP and Koti BC: Synthesis and antidiabetic activity of 2-amino 5’(4-sulphonylbenzylidine)-2,4-thiazolidinedione]-7-chloro-6-fluorobenzothiazole.Indian Journal of Chemistry 2005;44 Suppl B:2404-2408.
6. MahfuzA, Nadeem S: Synthesis of new benzothiazole incorporated sulphonamides as potential anticonvulsants. Indian Journal of Heterocyclic Chemistry 2004; 6:361-4.
7. Rajeeva B, Srinivasulu N, Shantakumar SM: Synthesis and Antimicrobial Activity of some New 2‐Substituted Benzothiazole Derivatives. E. J.Chem 2009; 2:775‐779.
8. Shahar Yar M, Ansari ZH: Synthesis and in vivo diuretic activity of biphenyl benzothiazole-2-carboxamide derivatives. Acta Poloniac Pharmaceutical-Drug Research 2009; 66 Suppl 4:387-392.
9. Sunder S, Praveen S, Akash Y, Muttu CT, Ranjeeta V: Microwave assisted synthesis of
10. Fluoro, Chloro, 2-N (substituted schiff’s bases) amino benzothiazoles as potential antimicrobial and antitubercular agents. The Pharma Research 2009; 1 Suppl 4:345-356.
11. Maharan M, William S, Ramzy F and Sembel A: Synthesis and in vitro evaluation of new benzothiazole derivatives as Schistosomicidal agents. Molecules 2007; 12 Suppl 5:622-623.
12. Sreenivasa GM, Jayachandran E, Shivakumar B, Jayaraj KK, Vijay K: Synthesis of bioactive molecule fluoro benzothiazole comprising potent heterocyclic moieties for anthelmentic activity. Arch Pharma Sci Res 2009 October; 1 Suppl 2:150-7.
13. P S Yadav, Devprakash, Senthilkumar G P: Benzothiazole: Different Methods of Synthesis and Diverse Biological Activities. International Journal of Pharmaceutical Sciences and Drug Research 2011; 3 Suppl 1:01-07.
14. Gupta.A and Rawat.S: Synthesis and anti-inflammatory study of novel fluoro Benzothiazole derivatives. Journal of Chemical and Pharmaceutical Research 2010; 2 Suppl 5:244-258.
15. Lipinski C A, Franco L, Dominy B W, Feeney P J: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv.Drug Delivery Rev.2001; 46 Suppl 2:3-26
![Pyrazole-oxadiazole Conjugates: Synthesis, …Pyrazole-oxadiazole Conjugates: Synthesis, Antiproliferative Activity and Inhibition of Tubulin Polymerization Ahmed Kamal, *[a,d] Anver](https://static.documents.pub/doc/80x56/5e8c65afba3d737ddc66773e/pyrazole-oxadiazole-conjugates-synthesis-pyrazole-oxadiazole-conjugates-synthesis.jpg)
