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H H H H
N N
N N
N 6 O O O N 6 O O
1 1
N N O P O P O P O N N O P O P O
O O O O O
O O
H H O H H
H H O P O H H
OH OH OH OH
O
Protein Kinases
Tyrosine kinases
O O
H H H H
N N
Protein Protein Protein Protein
O
ATP ADP
P
OH O OH
OH
Protein Kinases
Serine-threonine kinases
O O
H H H H
N N
Protein Protein Protein Protein
OH O
ATP ADP P
Serine O OH
OH
O
O H H
H H N
N Protein Protein
Protein Protein
H3C O
H3C OH ATP ADP
P
Threonine O OH
OH
Protein Kinases
Active Site
•ATP binding site is similar but not identical for all protein kinases
Gln-767
Hydrophobic
H2NOC
pocket
H
N
H O
HC N HBD
Leu-768 3 H H
H3C O N
Cleft
HBA
Met-769 N N
S H
N O O O
H3C
N N O P O P O P O
O O O
O O
H H
H H
OH OH
Ribose pocket
Gln-767
Ribose pocket
•Type I inhibitors bind to the ATP binding site and block access to ATP
•Type II inhibitors bind to the enzyme and stabilise the inactive conformation
Type I inhibitors
Gefitinib, erlotinib, SU11248 and seliciclib
Type II inhibitors
Imatinib, lapatinib, sorafenib and vatalanib
Gefitinib (Iressa)
Aniline
HN Cl O
4 6 O N
N3
1 Morpholine
N 7 OMe
Quinazoline
Notes
•Developed by Astra Zeneca
•Inhibits the kinase active site of the epidermal growth factor receptor
•The EGF-receptor is a tyrosine kinase receptor
•Gefitinib is a 4-anilinoquinazoline structure
Gefitinib (Iressa)
Lead compound
Secondary
amine
HN CH3 Small lipophilic group
4 6 OMe
N Electron-donating
substituents
N 7 OMe
I; IC50 5 nM
Notes
•The secondary amine, electron-donating substituents and small lipophilic group
are all important for activity
•Useful in vitro activity
•Lower in vivo activity due to rapid metabolism
•Metabolised by cytochrome P450 enzymes
Gefitinib (Iressa)
Metabolism of the lead compound
OH
Cytochrome OH
HN CH3 HN HN CH3
P450 enzymes
4 6 OMe OMe + OMe
N Oxidation N N
I; IC50 5 nM II III
Notes
•Methyl group and para-position of aromatic ring are susceptible positions
•Blocking metabolism should improve the half life of the drug
Gefitinib (Iressa)
Drug design
HN CH3 HN Cl
4 OMe
6 OMe
N N
N 7 OMe N OMe
I; IC50 5 nM IV: IC50 9 nM
Notes
•Fluoro-substituent blocks para-hydroxylation of the aromatic ring
•Fluorine is similar in size to hydrogen and has no steric effect
•Methyl group is replaced by a chloro substituent
•Chlorine and methyl group have similar sizes and lipophilicities
•Chlorine acts as a bio-isotere for the methyl group
•Chlorine is resistant to oxidation
•Compound is less active in vitro, but more active in vivo
Gefitinib (Iressa)
Drug design
F F
Morpholine
HN CH3 HN Cl HN Cl O
4 OMe 4
6 OMe 6 O N
N N N3
Spacer
1
N 7 OMe N OMe N 7 OMe
I; IC50 5 nM Gefitinib Ionisable
IV: IC50 9 nM
Notes
•Morpholine ring increases water solubility
•Morpholine nitrogen allows generation of water soluble amine salts
•Spacer allows morpholine to protrude out of the active site
•Remains solvated when the drug is bound
•Avoids a desolvation penalty
Gefitinib (Iressa)
Binding interactions
•Identified by a molecular modelling experiment
•Gefitinib is docked with a model binding site
•Binds to the ATP binding site
•Aniline ring occupies the normally vacant hydrophobic pocket opposite the ribose
binding pocket
•Quinazoline binds to the same region as the purine ring of ATP
Hydrophobic
pocket
F
H
Thr-830 N Cl O
HBA O N
OH2 N
Cleft
N OMe
HBA
Met-769
3. Gefitinib (Iressa)
Synthesis of gefitinib and analogues
Aniline substituent
Cl NHAr NHAr
OAc OAc OH
N MeOH N
SOCl2 ArNH2 N
NH4OH
N OMe N OMe N OMe
Ar
HN O
R2N(CH2)nBr O N
N
N OMe
4. Lapatinib and Etlotinib
F
O Aniline ring
Aniline ring
NH
Cl NH 4
O
N OMe
N O
HN OMe
N O
N SO2Me
Quinazoline ring
Quinazoline ring
Notes
•4-Anilinoquinazoline structures - compare gefitinib
•EGF-receptor kinase inhibitors
5. PKI 166
Me
NH
4 Pyrrole
N
OH
Pyrimidine
N N
H
HBA HBD
Notes
•Pyrrolopyrimidine structure
•EGF-receptor kinase inhibitor
•Different binding mode from ATP or anilinoquinazolines
5. PKI 166
Comparison of binding interactions
•ATP and EGF-receptor kinase inhibitors all contain a pyrimidine ring
•Different binding modes are possible
Me F
Gefitinib
PKI 166 H
NH O
N Cl
4
O N
N HBA N
OH
N N N OMe
H
HBA
HBA HBD
HBD
H H
N ATP
HBA N N
O O O
N N O P O P O P O
O O O
O
H H
H H
OH OH
6. Imatinib (Glivec or Gleevec)
N
Me
H
N N
N O
H
N
N
Me
Notes
•First protein kinase inhibitor to reach the market
•Selective inhibitor for a hybrid tyrosine kinase (Bcr-Abl)
•Bcr-Abl is active in certain tumour cells
6. Imatinib (Glivec or Gleevec)
Lead compound
•Phenylaminopyrimidine structure
•Identified by random screening of compound libraries
•Originally identified as a PKC inhibitor
•PKC is a serine-threonine kinase
6. Imatinib (Glivec or Gleevec)
Drug design
Pyridine
N N
H H H
N N 3' N N N N
N N N
Amide
I II IV N O
(IC 50 5 M) H
Inhibits tyrosine
kinases as well
6. Imatinib (Glivec or Gleevec)
Drug design
Conformational
N
blocker N
N Me
Me
H H
H N N N N
N N
N N N
N O IV N O
N O
H
H (IC 50 5 M) H
Imatinib
CGP 53716
(IC 50 0.1 M)
Piperazine
N Spacer
•Increased activity vs
tyrosine kinases
N
Me •No activity against
serine-threonine kinases
•Piperazine increases activity,
selectivity and water solubility
•Spacer inserted to avoid
aniline structure
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Identified from a crystal structure of an inhibitor-Abl kinase complex
•Amide serves as an ‘anchoring group’ and orientates the molecule
•Amide binds to Glu and Asp
•Glu and Asp are important to the catalytic mechanism
Hydrophobic region
Selectivity region 2
H
Glu
O N N Me
Hydrophobic pocket
Selectivity region 1
O
H
N N
N N O
H N
O N H O
H
O N
H Me
Met O
Asp
MeS O2C
Thr
Gatekeeper
residue
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Other interactions determine target selectivity
•A hydrogen bond to the gatekeeper Thr is essential to activity
•N-Alkylation eliminates activity
Hydrophobic region
Selectivity region 2
H
Glu
O N N Me
Hydrophobic pocket
Selectivity region 1
O
H
N N
N N O
H N
O N H O
H
O N
H Me
Met O
Asp
MeS O2C
Thr
Gatekeeper
residue
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Molecular modelling studies suggest that the piperazinyl group interacts with a
glutamate residue
•Imatinib inhibits protein kinases containing this glutamate residue (Abl, c-Kit
and PDGF-R)
Hydrophobic region
Selectivity region 2
Ionic
bond
Glu
H
Glu
O N N Me
Hydrophobic pocket
Selectivity region 1
O Piperazinyl
group
H
N N
N N O
H N
O N H O
H
O N
H Me
Met O
Asp
MeS O2C
Thr
Gatekeeper
residue
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Conformational blocker aids selectivity
•Binds to a hydrophobic pocket that is not accessible if a larger gatekeeper
residue was present
Hydrophobic region
Selectivity region 2
H
Glu
O N N Me
Hydrophobic pocket
Selectivity region 1
O
H
N N
N N O
H N
O N H O
H
O N
H Me
Met O
Asp
MeS O2C
Thr
Gatekeeper
residue Conformational
blocker
6. Imatinib (Glivec or Gleevec)
Drug resistance
•Mutation of the gatekeeper residue to isoleucine introduces resistance (T315I
mutation)
•Isoleucine unable to form an important hydrogen bond to the amine
Hydrophobic region
Selectivity region 2
H
Glu
O N N Me
Hydrophobic pocket
Selectivity region 1
O
H
N N
N N O
H N
O N H O
H
O N
H Me
Met O
Asp
MeS O2C
Thr
Gatekeeper
residue Mutation to Isoleucine
6. Imatinib (Glivec or Gleevec)
Synthesis of imatinib and analogues
N
N N Me
HC(OEt)2NMe2 Phenylguanidine H
N N
O O derivative
N
Me
II
I
NMe2 NO2
N N
Me Me
H H
H2 N N N N
ArCOCl
Pd/C
Reduction N Acylation N
NH2 N O
H
III Amide
Ar
7. Second Generation Bcr-Abl inhibitors
F3C
Glu-286 N
N
H
Me
N N
N N O
N Asp-381
Met-318
H
Thr-315 Me
Nilotinib
Me
Thr-315 N
N
N OH
Me H N N
N N
S
H
O Met-318
Cl
Dasatinib; BMS-354825
7. Second Generation Bcr-Abl inhibitors
Cl Cl
HN OMe
MeO CN
N O N
MeN
Bosutinib
Notes
•Inhibits two protein kinase targets (Abl and Src)
•Currently in clinical trials
•Less likely to fall prey to drug resistance
7. Second Generation Bcr-Abl inhibitors
O Me
NH2 HN CO2H
MeO
OMe
O O
MeO S
N
N N MeO OMe
H
GNF-2 ON012380
Notes Notes
•Allosteric inhibitor of Bcr-Abl •Binds to the protein substrate site
•Does not bind to ATP binding site •Currently under study
•Stabilises inactive form of the enzyme
•Binds to an autoregulatory cleft
•Potential agent for treating leukaemia
8. Inhibitors of cyclin-dependent kinases
Cyclin-dependent kinases
•CDKs are involved in control of the cell cycle and are overexpressed in many cancer cells
•Serine-threonine kinases
•Activated by cyclins
HO O
OH
Piperidine Phenyl
N ring
Me
Flavopiridol
HN
HO O
N N
Me N
OH O N
Me
Piperidine Phenyl HO
N N N
N ring
MeO H
Me
Me NHMe Me
Cl
Me Anilino
R
substituent
HN
Pyrrole
Oxindole N Me N
H Phthalazine
N
O HBA
N
H HBD
SU 5416, R=H Pyridine
SU 6668, R=CH2CH2CO2H N
PTK 787 / ZK 222584
N Me
F H
NH
O
N N
N
H
N
Sunitinib
Cl
Vatalanib
Notes
•Sorafenib approved as a VEGF-R kinase inhibitor
•Sunitinib approved in 2006 - inhibits VEGF-R, PDGF-R and KIT receptor kinases
•Vatalanib undergoing clinical trials
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
•Lead compound found by high throughput screening
•200 000 compounds tested
•Tested against recombinant Raf-1 kinase
MeO2C Urea
H H
N N
S
O
H
Lead compound;
IC50 17 M
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib - variation of substituents
Notes
•Methyl substituent is optimum for activity
•10-fold increase in activity
•Phenoxy group is bad for activity
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib - variation of rings
MeO2C H H
H H Isoxazole N N
N N N
S O
O O
H
Notes
•Variation of rings also carried out systematically
•Isoxazole ring is not good for activity
•Conventional medicinal chemistry strategies fail to achieve further improvement
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C Isoxazole
H H H H
N N N N N
S O
O O
H O
Phenoxy
group
Lead compound IV; IC50 1.1 M
IC50 17 M
Notes
•Parallel synthesis - 1000 analogues synthesised with all possible
combinations of rings and substituents
•Structure IV has slightly increased activity - contradicts results from
conventional studies
•Isoxazole ring and phenoxy substituent are good for activity when combined
in the same structure - synergistic effect
•Structure IV taken as new lead compound
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C Isoxazole
H H H H
N N N N N
S O
O O
H O
Phenoxy
group
Lead compound IV; IC50 1.1 M
IC50 17 M
Pyridine
H H
N N N •Ring variation
N
O
O
•5-fold increase in activity
O •Increase in aqueous
solubility and cLogP
V; IC50 0.23 M
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C Isoxazole
H H H H
N N N N N
S O
O O
H O
Phenoxy
group
Lead compound IV; IC50 1.1 M
IC50 17 M
Ring H
Pyridine Substituent
H H variation H
N N N N N variation
N N H
O
O O N
O Cl O CH3
Substituent CF O
3
variation
V; IC50 0.23 M Sorafenib IC50 12 nM
HBD
H
H
N N HBA HBD
N H
O N
Cl HBA O CH3
CF3 O
Notes
•Urea functional group acts as a binding anchor (compare imatinib)
•Hydrogen bonds are formed to catalytic Asp and Glu
•Binding orientates the molecule
•Positions each half into two selectivity regions
11. Inhibitors of heat shock protein 90
Notes
•Targeting HSP 90 may be effective against tumour cells resistant against other
drugs
•Resistant cells contain mutated proteins - rely more on HSP 90 during the
folding process
Me
O •Natural product
Quinone
HN O
•Potent inhibitor
Urethane O
OMe •Urethane group is crucial to activity
H2N
Me •Binds to region occupied by adenine
OH O OMe
O •Poor solubility
•Reactive quinone moiety
Me OMe Me
11. Inhibitors of heat shock protein 90
Geldanamycin analogues
Me Me
O O
HN O HN O
OMe OMe
O O
Me Me NMe
H2N H2N OH O N
OH O N H
H O
O
Me OMe Me Me OMe Me
Alvespimycin
Tanespimycin Me
O
HN OH
OMe
O
Me
H2N OH HO N
H
O
Me OMe Me
IPI 504