Medicinal Chemistry

Lectures Note 12:

ANTICANCER AGENTS:
PROTEIN KINASE INHIBITORS
1. Protein Kinases
•Enzymes that catalyse phosphorylation reactions on protein substrates

•Protein kinases are present in the cytoplasm

•500-2000 estimated protein kinases in a cell

•Protein kinase receptors - dual role as receptor and enzyme

•Overexpression can result in cancer

•Tyrosine kinases, serine-threonine kinases and histidine kinases

•ATP used as enzyme cofactor - phosphorylating agent

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 H H
OH OH O P O OH OH
O
1. Protein Kinases
Tyrosine kinases

O O
H H H H
N N
Protein Protein Protein Protein

O
ATP ADP
P
OH O OH
OH
1. 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
1. Protein Kinases
Active Site

•Contains the binding site for the protein substrate

•Contains the binding site for the ATP cofactor

•Clinically useful inhibitors target the ATP binding site

•ATP binding site is similar but not identical for all protein kinases

•Allows selectivity of inhibitor action

1. Protein Kinases
ATP binding site

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
N O O O
S H
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

1. Protein Kinases H2NOC

H
N
Hydrophobic
pocket

ATP binding site HC

H N
O
HBD
Leu-768 3
Specificity… H3C O
H
N
H

HBA Cleft
Met-769 N N
N O O O
S H
H 3C
N N O P O P O P O
O O O
O O
H H
H H
OH OH

Ribose pocket

•An empty hydrophobic pocket lies opposite the ribose binding pocket

•Specificity surface is an area of unoccupied binding site

•The gatekeeper residue is an amino acid situated at the entrance to the

hydrophobic pocket

•The size of the gatekeeper residue is important in drug design

•The nature of amino acids in the binding pockets is important to drug design
2. Protein Kinase Inhibitors
Notes

•Type I inhibitors act on the active conformation of the enzyme

•Type I inhibitors bind to the ATP binding site and block access to ATP

•Type II inhibitors act on the inactive conformation of the enzyme

•Type II inhibitors bind to the enzyme and stabilise the inactive conformation

•Type II inhibitors are likely to be more selective

Type I inhibitors
Gefitinib, erlotinib, SU11248 and seliciclib

Type II inhibitors
Imatinib, lapatinib, sorafenib and vatalanib
3. 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

3. Gefitinib (Iressa)
Lead compound
Secondary
amine
HN CH3 Small lipophilic group
4 OMe
6
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

3. Gefitinib (Iressa)
Metabolism of the lead compound

OH
Cytochrome
OH
HN CH3
P450 HN HN CH3
enzymes
4
6 OMe OMe + OMe
N Oxidation N N

N 7 OMe N OMe N OMe

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

3. 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

•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

3. 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

•Spacer allows morpholine to protrude out of the active site

3. Gefitinib (Iressa)
Binding interactions
•Identified by a molecular modelling experiment

•Gefitinib is docked with a model binding site

•Binds to the ATP binding site

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

O O Phenol O Protecting group

OMe OH
HN Methionine OAc
HN Pyridine
HN
MeSO3H
Ac2O
N OMe N OMe N OMe

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
Quinazoline ring
ring

Lapatinib Erlotinib (Tarceva)

IC50 2 nM

Notes
•4-Anilinoquinazoline structures - compare gefitinib

•EGF-receptor kinase inhibitors

•The acetylene group acts on the hydrophobic group guarded by Thr

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
N Cl O
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

Pyrimidine H Anilino substituent

N N

•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 N O
IV
(IC50 5 M) H

Increased inhibition of PKC

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 (IC50 5 M) H

Imatinib
CGP 53716
(IC50 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

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

Me N
I II
NO2
NMe2

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
More active against tumour-resistant cells (excpet T315I-mutant strains)
F3C

Glu-286 N
N
H
Me
N N
N N O
N Asp-381
Met-318
H
Thr-315 Me

Nilotinib
Nilotinib 20-30 fold increase in activity
Me

Thr-315 N
N
N OH
Me H N N

N N
S
H
O Met-318
Cl
Dasatinib
Dasatinib; BMS-354825 Best activity
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 •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

•Inhibited by cyclin-dependent kinase inhibitors

•Synthetic inhibitors bind to the ATP binding site

 8. Inhibitors of cyclin-dependent kinases
HBD HBA
OH O
Benzopyran
Cl

HO O
OH

Piperidine Phenyl
N ring

Me

Flavopiridol

•Benzopyran binds to the adenine binding region

•Piperidine binds to the region occupied by the first phosphate of ATP

•Phenyl lies over the ribose binding pocket

•Undergoing clinical trials (has antiangiogenic effect and can induce

apoptosis)
 8. Inhibitors of cyclin-dependent kinases
H
HBD HBA N
R O
OH O 7
Benzopyran
Cl

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

Flavopiridol Staurosporine; R=H R-Roscovitine

7-Hydroxystaurosporin; R=OH (seliciclib)

7-Hydroxystaurosporin Shows selectivity for

is undergoing clinical CDK2
trials (more selective) Undergoing clinical
trials (induce apoptosis)
9. Kinase Inhibitors of FGF-R and VEGF-R
FGF-R and VEGF-R

•FGF-R = fibroblast growth factor receptor

•VEGF-R = vascular endothelial growth factor receptor

•Associated with angiogenesis

•Inhibitors bind to the ATP binding site

•Currently undergoing clinical trials

•These agents are not guaranteed to inhibits agiongenesis since other

pathways can be followed.
9. Kinase Inhibitors of FGF-R and VEGF-R
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

•SU 5416 in clinical trials Phase III clinical trials in 2006

for treatment of colorectal
cancer
•Oxindole binds to same
region as adenine of ATP

These agents are not guaranteed to inhibits agiongenesis since other

pathways can be followed.
10. Multi-tyrosine receptor kinase inhibitors
Notes

•Designed to be selective against a range of tyrosine receptor kinases

implicated in tumours

•Drug resistance unlikely to occur for all kinase targets

•Equivalent of combination therapy (poly-pharmacology)

•Sometimes called ‘dirty drugs’

•Promising agents against tumours that are driven by several

abnormalities
 10. Multi-tyrosine receptor kinase inhibitors
H
H
N N
H N
N O
H3C O Cl O NEt2
O CF3 Me
N
H
Sorafenib IC50 12 nM

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

MeO2C MeO2C MeO2C

H H H H H H
N N N N N N
S S S
O O O
H Me O

Lead compound II; IC50 1.7 M III; Poor activity

IC50 17 M

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

Lead compound VI; Poor activity

IC50 17 M

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
new lead compound
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
 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
N •Ring variation
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

1000-fold increase in activity

10. Multi-tyrosine receptor kinase inhibitors
Sorafenib - binding interactions

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

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