Download INSILICO DRUG DESIGN AND MOLECULAR DOCKING STUDIES OF SOME NOVEL

Academic Sciences
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 6, Suppl 2, 2014
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 antiinflammatory 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],antiinflammatory[4],
anti-diabetic[5],anti-convulsant[6],antimicrobial[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].
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.
Benzothiazole
R4
4
5
3
3a
N
N
2
6
7a
7
R2
S
S
R6
1
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
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.
Pappachen et al.
Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
Table 1: Substituent’s at R groups
Compound code
BT1
BT2
BT3
R2
–NH2
–NH2
Cl
R4
-Cl
-F
H
R6
-Cl
-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
N
N
NH2
BT4
O
O
CH3
N
N
BT5
O
Cl
N
N
BT6
N
N
O
BT7
BT8
-SCH3
N
Cl
N
O
O
Cl
BT9
O
N
N
Br
BT10
O
N
N
Table 2: Compounds and IUPAC names of Benzothiazole derivatives of Protein Structure
Compounds
BT1
BT2
BT3
BT4
BT5
BT6
BT7
BT8
BT9
BT10
Iupac name
4,6-dichloro-1,3-benzothiazol-2-amine
4, 6-difluoro-1, 3-benzothiazol-2-amine
2-[5-(1, 3-benzothiazol-2-yl)-1, 3, 4-oxadiazol-2-yl]-3-chloroaniline
2-[5-(3-methoxyphenyl)-1, 3, 4-oxadiazol-2-yl]-1, 3-benzothiazole
2-[5-(3-chlorophenyl)-1, 3, 4-oxadiazol-2-yl]-1, 3-benzothiazole
2-{5-[(E)-2-phenylethenyl]-1, 3, 4-oxadiazol-2-yl}-1, 3-benzothiazole
2-{5-[(2, 4-dichlorophenoxy) methyl]-1, 3, 4-oxadiazol-2-yl}-1, 3-benzothiazole
2-(methylsulfanyl)-1, 3-benzothiazole
2-(5-phenyl-1, 3, 4-oxadiazol-2-yl)-1, 3-benzothiazole
2-[5-(2-bromophenyl)-1, 3, 4-oxadiazol-2-yl]-1, 3-benzothiazole
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 6.00A 0were 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.
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Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
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
1
2
3
4
5
6
7
8
9
10
11
12
BT1
BT2
BT3
BT4
BT5
BT6
BT7
BT8
BT9
BT10
Tamoxifen
Indomethacin
Docking score(kcal/mol)
Cox1
-7.817
-6.139
-8.036
-7.567
-8.178
-8.569
-11.1120
-7.604
-8.001
-8.314
Cox2
-9.460
-6.678
-9.630
-9.377
-9.720
-11.524
-12.296
-10.O21
-9.002
-9.969
-11.089
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
ER
-9.68
-9.491
-6.45
-8.50
-9.59
-11.9651
-11.1988
-9.64
-7.96
-9.26
-9.26
-10.9612
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
1
2
3
4
5
6
7
8
9
10
11
12
BT1
BT2
BT3
BT4
BT5
BT6
BT7
BT8
BT9
BT10
Tamoxifen
Indomethacin
Docking score(kcal/mol)
COX 1
-5.78
-5.58
-8.84
-8.4
-8.73
-8.98
-9.84
-5.93
-8.42
-8.78
COX2
-6.77
-5.76
-8.82
-8.72
-11.66
-11.57
-9.45
-6.11
-8.59
-9.4
-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.
COX3
-6.29
-5.25
-7.19
-7.48
-7.45
-7.68
-8.86
-5.35
-7.05
-7.55
-3.86
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
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Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
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
1
BT1
2
BT2
3
BT3
4
BT4
5
BT5
6
BT6
7
8
BT7
BT8
9
BT9
10
BT10
Hydrogen bonding interactions (kcal/mol)
COX 1
ARG 374
ARG 928
ARG 374
ARG 926
ARG 374
ASN 375
ARG 376
ARG 926
ASN 927
ARG 928
ARG 376
ARG 374
ASN 375
ARG 926
ARG 928
ARG 374
ASN 375
ARG 926
ARG 926
ASN 927
ARG 928
ASN 375
ARG 926
COX2
THR 206
TYR 385
THR 206
COX3
NIL
TYR 385
THR 212
TRP 345
ARG346
ALA 378
NIL
ALA 378
NIL
NIL
VAL 280
NIL
NIL
NIL
NIL
TYR 385
NIL
NIL
PRO 278
ARG 346 HIS386
Of the high docking scores ligand-target complexes with different hydrogen bonding interactions generated by Autodock 4.0 programme were
shown below
Fig. 1: BT2 –Cox1 (1 cqe) hydrogen bonding interactions (ARG 928,
ARG 374, ARG 926) (-8.84 kcal/mol).
Fig. 2: BT3 -Cox1 (1cqe) hydrogen bonding interactions (ARG 374,
ASN 375, ARG 376) (-8.4 Kcal/mol).
Fig. 3: BT4 -Cox 1 (1cqe) hydrogen bonding interactions (ARG 928,
ASN 927, ARG 926) (-8.73 Kcal/mol).
Fig. 4: BT5-Cox1 (1cqe) hydrogen bonding interactions (ARG 374,
ASN 375, ARG 376) (-8.98 Kcal/mol).
Fig. 5: BT6-Cox1 (1cqe) hydrogen bonding interactions (ARG 926,
ARG 928) (-9.84 Kcal/mol).
Fig. 6: BT8-Cox1 (1cqe) hydrogen bonding interactions (ASN 375,
ARG 926) (-5.93 kcal/mol).
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Int J Pharm Pharm Sci, Vol 6, Suppl 2, 203-208
Fig. 7: Cox2 hydrogen bonding interactions (THR 206, TYR 385) (6.77 Kcal/mol)
Fig. 8: BT2-Cox2 (6cox) hydrogen bonding interactions (THR 206,
HIS 387) (-8.82kcal/mol Fig 8: BT2-Cox2 (6cox) hydrogen bonding
interactions (THR 206, HIS 387) (-8.82kcal/mol)
Fig. 9: BT3-ER (1QKM) hydrogen bonding interactions (TRP 345, ARG 346)(-7.48Kcal/mol)
Validation of Ligands
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.
1
2
3
4
5
6
7
8
9
10
Compound
Code
BT1
BT2
BT3
BT4
BT5
BT6
BT7
BT8
BT9
BT10
Molecular
Weight
219.O96
186.186
328.784
309.35
313.769
305.362
378.24
181.285
279.324
388.22
No. Of
Hba
2
2
2
0
0
0
0
0
0
4
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, antiinflammatory 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
No. Of
Hbd
2
2
5
5
4
4
5
1
4
4
C log p
3.238
2.209
4.348
4.316
4.937
4.296
4.52
3.092
4.283
4.22
No. Of
Rot.b
0
0
2
3
2
3
4
1
2
0
n violation
0
0
0
0
0
0
0
0
0
0
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.
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REFERENCES
1.
2.
3.
4.
5.
6.
7.
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.
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.
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.
Venktesh P, Pandeya S N: Synthesis, characterization and antiinflammatory activity of some 2-amino benzothiazole
derivatives. International Journal of Chem. Tech research
2009; 1 Suppl 4:1354-1358.
Pattan SR, Suresh CH, Pujar VD, Reddy VVK, Rasal VP and Koti
BC: Synthesis and antidiabetic activity of 2-amino 5’(4sulphonylbenzylidine)-2,4-thiazolidinedione]-7-chloro-6fluorobenzothiazole.Indian Journal of Chemistry 2005;44
Suppl B:2404-2408.
MahfuzA, Nadeem S: Synthesis of new benzothiazole
incorporated sulphonamides as potential anticonvulsants.
Indian Journal of Heterocyclic Chemistry 2004; 6:361-4.
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.
9.
10.
11.
12.
13.
14.
15.
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.
Sunder S, Praveen S, Akash Y, Muttu CT, Ranjeeta V: Microwave
assisted synthesis of
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.
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.
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.
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.
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.
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
208