Case Study Medicinal Chemistry

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Case StudyMedicinal Chemistry

• By John Crowe, Toshi Ghosh, Janelle Slattery

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Main Ideas

• Understanding specific biochemical reactions is
  essential to designing new medicines
• A tremendous amount of testing is necessary
  before drugs can be used
• Slight modifications to molecular structure can
  have a significant effect on the effectiveness of
  a drug

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L1 Epidermal Growth Factor Receptor

• Tyrosine kinase receptors both a receptor and an
  enzyme
• Resting state Active site closed
• Receptor activation Cell signaling cascade
  leading to gene activation
• Specific TKR Epidermal growth factor
• Causes growth and division of cells
• Gene overexpression can lead to cancer
• Goal Design inhibitors of the receptor kinase

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EGF Receptor

• Located in cell membrane
• Bound by Epidermal Growth Factor (protein)
• Catalyzes phosphorylation of Tyrosine residues
• Adenosine Triphosphate (ATP) acts as the
  phosphorylating agent
• Reaction ATP transfers a phosphate group to
  tyrosine residue
• Goal create inhibitors that bind instead of ATP

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L2 Testing Procedures

• Enzyme Assay required to test compounds ability
  to cross cell membrane and inhibit kinase enzyme.
• Cell Assays developed to test inhibitors ability
  to work on whole cells crossing of cell membrane
  and prevention of signal transduction.
• In vivo Assays established if the compounds had
  satisfactory pharmacokinetic properties and any
  toxic effects.
• Selectivity Assays were vital due to the multiple
  tyrosine kinases present in the cell.

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L3 From Lead Compounds to Dianilino-phthalimides

• Lead compound- a compound that demonstrates
  useful biological activity, or has affinity for a
  target receptor or enzyme
• Staurosporine- a microbial metabolite that acts
  as a highly potent protein kinase inhibitor,
  competitive with ATP on EGF receptor kinase
• Method of action for staurosporine too broad-
  also inhibits serine-threonine kinases

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Simplification Strategies

• Simplification Finding an easier compound to
  synthesize, while retaining usefulness
• Staurosporine Simplifications
• Arcyriaflavin A selective for protein kinase C
• Bisindolyl-maleimides selective for protein
  kinase C
• Dianilino-phthalimides selective for EGF,
  inactive against other kinases
• Why do they produce different results?
• Studied with x-ray crystallography

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Kinase Inhibitor Conformations
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Structure-activity Relationships

• Over 250 analogs synthesized and tested
• By comparing similar compounds, it was possible
  to determine what structures are necessary
• CGP52411 parent structure chosen for preclinical
  trials
• Target EGF receptor kinase in psoriasis patients

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Metabolism Studies andFurther Development

• Metabolism studies
• Reveal absorptive capacity
• Reveal method and speed of metabolism/excretion
• Fluorine compounds used to block metabolism
• Activity improvement strategies
• Chain extension
• Ring expansion
• Further simplification

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L4 Model Binding Site

• An enzymes active site is modeled by
• Crystallizing the enzyme
• X-ray crystallography
• Inhibitors position ? active sites location
• Overall structure can be configured
• Molecular modeling computer software reads the
  protein-inhibitor complex
• Observes how the inhibitor is bound to the active
  site
• Amino acids
• Interactions
• Drug extensions researched
• www.chem.ed.ac.uk/chapman/p450.html

Active Site of P450-BM3 Palmitate bound to heme
face
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EGF-receptor Model

• Membrane-bound EGF-receptors cannot be
  crystallized
• ATP binding site modeled on the x-ray crystal
  structure of cyclic AMP-dependent protein kinase
• The EGF-receptor kinases amino acids were
  inserted in place of those for cyclic
  AMP-dependent protein kinase
• Hydrogen bonds between the adenine and the
  peptide bonds between Gln-767, Leu-768, and
  Met-769
• Ribose ring inserted itself into a ribose
  pocket of the binding site
• The binding site has a large, empty pocket

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Binding Model
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Binding Model of Dianilino-phthalimides

• ATP removed from the binding site by molecular
  modeling and replaced by CGP52411
• Imide moeity of dianilino-phthalimides imitates
  ATPs pyrimidine ring
• Three hydrogen bonding interactions bind the ATP
  binding site
• NH group donates a hydrogen bond
• Carbonyl oxygen accepts a hydrogen bond
• A small ribose pocket encompasses one of the
  aniline aromatic rings
• Bisindolyl-maleimides also bind via their imide
  group
• Not to the ribose pocket
• The ring expansion strategy successfully induced
  the hydrazone (R2CNNR2 ) ring that involves the
  three hydrogen bond interactions for CGP52411

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Selectivity of Action

• Ribose pockets cysteine residue selectively
  disfavors the EGF-receptor kinase
• Intensifies the pockets hydrophobic nature
• Cysteine (hydrophobic) and arginine replace the
  two glutamic acids in the EGF receptor kinase
• Binds the inhibitors aromatic ring
• Cysteines sulfur favorably interacts with
  aromatic rings, allowing the ring to fit and be
  stabilized within a ribose pocket
• www.3dchem.com/molecules.asp?ID38

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Pharmacophore

• The pharmacophore for EGF-receptor kinase
  inhibitors involves
• Hydrogen bond donor
• Hydrogen bond acceptor
• Spatially specific aromatic ring
• A bidentate hydrogen bond occurs where the
  inhibitor provided a hydrogen bond donor and a
  hydrogen bond acceptor
• The aromatic ring was to fit into the ribose
  pocket and interact with cysteine

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L5 Designing New Inhibitors

• Molecular modeling helps design new inhibitors
  that interact with the binding site, even if they
  are not structurally related to the lead compound
  (ATP)
• Target structures are created and tested to fit
  into the binding site
• Synthesized if successful

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4-(Phenylamino)-pyrrolopyrimidines

• 4-(Phenylamino)-pyrrolopyrimidine can bind in two
  ways to inhibit the EGF-receptor kinase
• Mode 1 Pyrimidine ring reacts with the binding
  site
• Mode 2 Aromatic ring fits into the ribose
  pocket along with bidentate hydrogen bonding
• The extra interaction with the aromatic ring
  makes this mode more probable

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Binding Modes
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L6 Pyrazolopyrimidines

• Two chemicals revealed that act as kinase
  inhibitors
• Docking experiments required for ribose site
  binding and bidentate hydrogen bonding abilities

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• Structure I successful binding of pyrimidine
  ring
• Structure II Stronger interaction due to lack
  ribose site occupation

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Drug Design

• Second binding mode favoured oprimary amino
  group no longer involved
• Simplification of II led to analog Iv

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• Ribose pocket must be empty when structure II or
  IV bound
• SAR study carried out activity improved with
  addition of an OH or NH2 group at meta or para
  positions

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• Chain extensions and contradictions reveal
  activity did not change
• Pocket had large capacity and activity fell with
  addition of extremely large substituents.