ANTICANCER AGENTS

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ANTICANCER AGENTS PROTEIN KINASE INHIBITORS
Chapter 21
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Protein Kinases

• Enzymes that catalyse phosphorylation reactions
  on protein substrates
• 500-2000 estimated protein kinases in a cell
• Protein kinases are present in the cytoplasm
• 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

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Protein Kinases
Tyrosine kinases
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Protein Kinases
Serine-threonine kinases
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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

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1. Protein Kinases
ATP binding site
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Protein Kinases
ATP binding site

• Purine base is buried deep into the binding site
• Purine forms two hydrogen bonding interactions to
  the binding site
• Ribose sugar binds to a ribose binding pocket
• Triphosphate chain lies along a cleft towards the
  enzyme surface
• Triphosphate interacts with two metal ions and
  amino acids
• Specificity surface is an area of unoccupied
  binding site
• An empty hydrophobic pocket lies opposite the
  ribose binding pocket
• 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

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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
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Gefitinib (Iressa)

• 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

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Gefitinib (Iressa)
Lead compound
Secondary amine
Small lipophilic group
Electron-donating substituents

• 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

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Gefitinib (Iressa)
Metabolism of the lead compound

• Notes
• Methyl group and para-position of aromatic ring
  are susceptible positions
• Blocking metabolism should improve the half life
  of the drug

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Gefitinib (Iressa)
Drug design

• 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

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Gefitinib (Iressa)
Drug design
Morpholine

• 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

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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

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3. Gefitinib (Iressa)
Synthesis of gefitinib and analogues
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4. Lapatinib and Etlotinib

• Notes
• 4-Anilinoquinazoline structures - compare
  gefitinib
• EGF-receptor kinase inhibitors

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5. PKI 166

• Notes
• Pyrrolopyrimidine structure
• EGF-receptor kinase inhibitor
• Different binding mode from ATP or
  anilinoquinazolines

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5. PKI 166

• Comparison of binding interactions
• ATP and EGF-receptor kinase inhibitors all
  contain a pyrimidine ring
• Different binding modes are possible

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6. Imatinib (Glivec or Gleevec)

• 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

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6. Imatinib (Glivec or Gleevec)
Lead compound
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6. Imatinib (Glivec or Gleevec)
Drug design
Pyridine
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6. Imatinib (Glivec or Gleevec)
Drug design
Piperazine
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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

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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

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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)

Piperazinyl group
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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

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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

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6. Imatinib (Glivec or Gleevec)
Synthesis of imatinib and analogues
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7. Second Generation Bcr-Abl inhibitors
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7. Second Generation Bcr-Abl inhibitors

• Notes
• Inhibits two protein kinase targets (Abl and Src)
• Currently in clinical trials
• Less likely to fall prey to drug resistance

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7. Second Generation Bcr-Abl inhibitors
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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

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8. Inhibitors of cyclin-dependent kinases

• 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

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8. Inhibitors of cyclin-dependent kinases
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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

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9. Kinase Inhibitors of FGF-R and VEGF-R
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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

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10. Multi-tyrosine receptor kinase inhibitors

• 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

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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

Urea
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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

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10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib - variation of rings
Isoxazole
Lead compound IC50 17 mM

• Notes
• Variation of rings also carried out
  systematically
• Isoxazole ring is not good for activity
• Conventional medicinal chemistry strategies fail
  to achieve further improvement

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10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
Lead compound IC50 17 mM

• 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

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10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
Lead compound IC50 17 mM
Pyridine
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10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
Lead compound IC50 17 mM
Pyridine
V IC50 0.23 mM
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10. Multi-tyrosine receptor kinase inhibitors
Sorafenib - binding interactions

• 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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11. Inhibitors of heat shock protein 90

• Notes
• HSP 90 is a kinase protein and acts as a
  molecular chaperone
• Important to survival of cells - inhibition
  likely to lead to cell death
• HSP 90 interacts selectively with many of the
  proteins implicated in tumours
• 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
• Resistant cells likely to be more vulnerable to
  inhibitors of HSP 90

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11. Inhibitors of heat shock protein 90
Notes Inhibitors bind to the ATP binding site
Lead compound - geldanamycin
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11. Inhibitors of heat shock protein 90
Geldanamycin analogues