1. LIGAND BASED DRUG DESIGN OF NOVEL
PYRIMIDINE DERIVATIVES AS
TANKYRASE INHIBITORS FOR THE
TREATMENT OF COLORECTAL CANCER
Research work carried out by-
Mr. Abhishek Patel
13MPH601
M.Pharm in Drug Discovery
Department of Pharmaceutical Chemistry
Institute of Pharmacy, Nirma University
Gujarat , India.
Guided by-
Dr. Hardik Bhatt
Co-guided by
Ms. Bhumika Patel
13-05-2015 1
2. Content
Introduction
Literature Review
Aim and Objectives
Computational Work
• Pharmacophore Modeling
• Virtual Screening
• 3D-QSAR
Designing of Molecules
Molecular Docking
In-silico ADMET studies
Experimental Work
In-vitro Cytotoxicity and Cell-Line Study
13-05-2015 2
3. Introduction
• Cancer is a term used for the diseases where abnormal cell division takes
place and also invades other tissues.
• Cancer is a leading cause of death worldwide, accounting 8.2 million
deaths in 2014 as per WHO.
• Colorectal cancer (also known as colon cancer, rectal cancer or bowel
cancer) is the development of cancer in the colon or rectum.
• Tankyrase as a drug target for cancer
• Belongs to PARP family poly(ADP-ribose) polymerase.
• Cellular processes like DNA repair and Wnt-signaling pathway are cause
for targeting this Tankyrase as target.
• An increased expression of Tankyrase has been mainly found in colon
cancer.
• Here, tankyrase-1 tends to down-regulate compared to normal tissue
whereas, tankyrase-2 tends to up-regulate expression of tissues.
http://seer.cancer.gov/statfacts/html/colorect.html Riffell et al.; Nature Reviews; 2012; 11; 923
13-05-2015 3
4. Nicotinamide site-
Nicotinamide binding site of donor NAD substrate. This
Site is conserved among Tankyrase.
Provides anchoring of many Tankyrase inhibitors:
TNKS 2 – Ser-1068 and Gly-1032
TNKS1 – Gly-1185 and Ser-1221
Potency increases mainly of hydrophobic features( Phe-1035 of TNKS2 and
Phe-1188 in TNKS1)
Adenosine site-
IWR-1 binds to adenosine (induced) pocket of TNKS.
G007-LK also binds to adenosine subsite and forms π-π stacking with His-1048
as IWR-1 (a molecule in clinical phase does).
Structure basis of Tankyrase inhibition
Zhan et al.; Mol. Biosyst.; 2014; 10; 2783
13-05-2015 4
5. Literature review
1.Schultz et al., synthesized pyrimidnone
derivatives using Lipophilic efficiency
approach.
2.Schultz et al., synthesized [1,2,4]Triazol-
3-ylamines as Novel Nictonamide Isoteres
using bioisosteric replacement
4.Hua et al., developed Novel Dual Binders
as potent, Selective, and Orally
Bioavailable Tankyrase inhibitors.
3.Threadgill et al., designed and
synthesized 2-Arylquinazolin-4-ones as
potent and selective Tankyrase inhibitors.
5.Liscio et al. discovered methoxy[1]benzothieno[2,3-c]quinolin-
6(5H)-one scaffold using scaffold hopping approach
N
NH
Br
O
N
NH
O
S O
N
H
O
CN
S
N
NN
NH
O R
O
N
NH
O
N
O
N
O
O
N
SO
O
1.Schultz et al;J.Med.Chem;2013;56;6495-6511
3.Threadgill et al.; ACS Med. Chem. Lett; 2013; 4; 1173-1177
52.Schultz et al.; J.Med.Chem; 2013; 56; 7049-7059
4. Hua et al.; J. Med. Chem.;2013; 56; 10003-10015
5.Liscio et al.; European J. Med. Chem.; 2014; 87; 611-623
6. 6.Bergman et al., designed and
synthesized Novel Oxazolidinones binding
to induced pocket of Tankyrase Inhibitors
using Structure-Based Molecular
Hybridization
7.Larson et al., identified 4-Methyl-1,2-
dihydroquinolin-2-one as high potent
fragment binding to Nicotinamide pocket
of Tankyrase
9.Huang et al., synthesized 2-
Aminopyridine Oxazolidinones as
selective Tankyrase Inhibitors using
structure based drug design.
10.Huang et al., Discovered
a class of Tankyrase inhibitors
that bind to Both Nicotinamide
pocket and Induced pocket
through structure-based
virtual screening.
8.Narwal et al., discovered Flavones
inhibiting Tankyrase with increased
potency and isoenzyme selectivity
NH
H
N
OCl
O
O
O
OH
O
O
N N
NH
O O
F
CN
O
N
N
N
N
NH2
O
6
6.Bregman et al.; J. Med. Chem.; 2013; 56; 4320-4342
7.Larson et al.; J. Med. Chem.; 2013; 56; 4497-4508
8.Narwal et al. ; J. Med. Chem.; 2013; 56; 7880-7889
9.Huang et al. ; ACS Med. Chem. Lett.; 2013; 4; 1218-1223
10.Huang et al.; J. Med. Chem.; 2013; 56; 1341-1345
N
NH
O
O
O
O
7. Rationale
13-05-2015 7
Tankyrase
• Role in cellular processes like cell proliferation, telomere homeostasis
etc.
• Expression increases in various cancer especially Colorectal cancer
Medicinal
chemistry
strategies
• Bioisosteric replacement, Scaffold hopping, Fragment based drug
discovery.
• To find potent and selective Tankyrase inhibitors
New Target
• Drugs are under clinical trials.
• Extensive bio-clinical utility makes it as a favorable target.
8. Aim -
The aim of research work is to design and synthesize novel Tankyrase
inhibitors for the treatment of Colorectal Cancer.
Objectives-
Literature Review.
Pharmacophore Modeling.
Virtual Screening.
Quantitative Structure Activity Relationship (3D-QSAR).
Designing of Molecules.
Molecular Docking.
In-silico ADMET Studies.
Synthesis of Designed Molecules.
Spectral Analysis of Synthesized Molecules using FTIR, 1HNMR,
13C-NMR and mass spectroscopy.
In-vitro Cytotoxicity Studies.
13-05-2015 8
9. Pharmacophore modeling
Model no Feat. Hits Specificity Energy
17 5 5 3.9860 10.56
9 5 3 3.560 4.56
7 diverse structure were taken targeting
on Tankyrase enzyme.
GALAHAD Module
20 models were generated
Model no 17 was found to best
Total number of compounds in database 200
Total number of actives 21
Total hits (Ht) 25
Active hits (Ha) 19
% yield of actives [(Ha /Ht )* 100] 76%
% ratio of actives [(Ha /A )* 100] 90.47%
Enrichment factor, EF [(Ha*D)/(Ht *A)] 7.23
Goodness of Hit (GH) score 0.76
AUC 0.832
Sensitivity 0.90
Specificity 0.97
Pharmacophore validation by GH score and ROC curve method
13-05-2015 9
10. NCI database
search 2 lakhs
molecules
Remove
counter ions
17,490
molecules
Remove
duplicate
structures and
bad fragments
9,246 molecules
Analyzing
Lipinski rule
of five 6,565
molecules
N
O
O
N
N N
H
H
N O
NCI ID NO: 91556
Qfit Value: 83.50
NCI ID NO: 696228
Qfit Value: 85.65
Virtual screening
13-05-2015 10
11. 3D- QSAR
3D-QSAR (CoMFA and CoMSIA) studies was carried out by using series of 41
Pyrimidinone derivatives acting on Tankyrase enzyme.
Contour maps were generated.
Training set- 32 Molecules (80%)
Test set – 9 molecules (20%)
Core Structure Alignment Structure
N
NH
O
Linear Regression Analysis Graph
y= 0.961x+ 0.263
R²=0.984
0
2
4
6
8
10
4 5 6 7 8 9 10
PredictedPIC50
ExpeerimentalPIC50
CoMFAMODEL
Linear()
Linear()
y= 0.870x+ 0.849
R²=0.965
4
5
6
7
8
9
10
4 5 6 7 8 9 10
PredictedPIC50
ExperimentalPIC50
CoMSIAMODEL
Linear()
Statistical
parameters
Distill (Rigid body)
alignment
CoMFA CoMSIA
q2 0.648 0.749
r2
ncv 0.977 0.979
r2
cv 0.605 0.744
r2
bs 0.988 0.990
N 6 6
Ftest 177.452 191.744
SEE 0.175 0.169
R2
pred 0.990 0.969
Field
contribution:-
Steric 0.774 0.173
Electrostatic 0.226 0.116
Hydrophobic - 0.321
HBD - 0.090
HBA - 0.300
13-05-2015 11
12. CoMFA Contour Maps
CoMFA (STERIC) CoMFA (ELECTROSTATIC)
CoMSIA Contour Maps
CoMSIA
(DONOR)
CoMSIA
(ACCEPTOR)
CoMSIA
(HYDROPHOBIC)
N
NH
CF3
O
S
Hydrophobic
favorable
Steric and
Hydrophobic
Favorable
13-05-2015 12
13. Designing of Molecules
• Using Ligand-based Pharmacophore Features like Acceptor, Donor and
Hydrophobic was obtained.
• QSAR contour maps suggested Steric and Hydrophobic Features will
Increase the potency of designed Tankyrase inhibitors.
• Steric group (R) - aromatic ring with Halogen group, Aromatic amines may
increase the activity
13-05-2015 13
N
NH2N NH-R
NH-R - Substituted
amines
14. Docking of Designed
Molecules
Designed Molecules Docking Total Score No of Hydrogen
Interactions
Standard(XAV-939) 7.96 Gly-1032
HA-1(3a) 6.34 Gly-1032,Tyr-1071
HA-2(3b) 6.77 His-1031,His-1048
HA-3(3c) 5.88 Gly-1032,Tyr-1071
HA-4(3d) 6.74 Ser-1068,Tyr-
1060,Ala-1049
HA-5(3e) 6.49 Gly-1032, Ser-1068
HA-6(3f) 4.86 Gly-1032
HA-7(3g) 5.94 Gly-1032, Ser-1068
HA-8(3h) 5.55 Gly-1032, Ser-1068
TAE13-05-2015
14
PDB ID- 3KR8, SurFlex-dock module
Standard XAV-939
3d
15. In-Silico ADMET studies
Parameters (3a) (3b) (3c) (3d) (3e) (3f) (3g) (3h)
Mutagenicity No No No No No No No No
Tumorigenicity No No No No No No No No
Irritant No No No No No No No No
Reproductive
effects
No No No No No No No No
cLog P 1.94 4.88 2.15 3.78 4.8 2.36 3.19 2.89
Solubility -4.88 -5.39 -3.35 -4.56 -4.76 -4.06 -3.87 -4.15
Molecular
weight
234.07 230.7 252.63 279.01 214.75 241.51 254.08 200.63
Drug likeness 1.0 2.31 0.86 0.87 0.56 0.45 0.36 0.41
Drug score 0.24 0.82 0.44 0.23 0.36 0.15 0.57 0.63
13-05-2015 15
16. Experimental Work
70-80 C˚
Conc. HCl
3 h
IPA
13-05-2015 16
N
N
N
HH
Cl
N
N
HN
Cl
Cl
NH2
N
N
Cl NH2
N
N
N
H
NH2
R
4-chloro-6-methylpyrimidin-2-amine
Ar-NH2
20. In-Vitro Cytotoxicity study
13-05-2015 20
COMPOUND NO VERO (CC50 ) μM MCF-7 (IC50 )μM
3A >100 >100
3B >100 22.49
3C >100 NC
3D >100 23.97
3E >100 47.19
3F NC >100
3G NC >100
3H NC > 100
Standard (Paclitaxel) >100 4.31
NC – not covered
21. Conclusion
• Using Ligand based drug design (pharmacophore modeling) three features
were obtained by GALAHAD module three hydrophobic, one hydrogen
bond acceptor and one hydrogen bond donor .
• Contour Maps of CoMFA and CoMSIA generated using 3D-QSAR was
very effective and played a very important role in drug design . Steric and
hydrophobic features were found to be important for the selectivity and
potency of the compound
• Two Ligand based drug designing techniques were compared and
concluded that hydrophobic feature was important for the potency of the
compound.
• Tankyrase enzyme has a hydrophobic binding pocket and compound was
designed according to pharmacophore modeling and 3D-QSAR.
• Designed drug also had good docking score using SurFlex-module of sybyl
and designed compounds were synthesized.
• In-Vitro cytotoxic activity will be performed .
13-05-2015 21
