Patrick: An Introduction to Medicinal Chemistry 5e Chapter 21 ANTICANCER AGENTS PROTEIN KINASE INHIBITORS modified

1. 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

1. Protein Kinases Tyrosine kinases ATP ADP Wiggly lines removed

1. Protein Kinases Serine-threonine kinases ATP ADP ATP ADP Wiggly lines removed

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 N O H 2 C 3 S Gln-767 Met-769 Leu-768 Ribose pocket Hydrophobic pocket Cleft HBD HBA Atoms modified

1. Protein Kinases ATP binding site H 2 C 3 S Gln-767 Met-769 Leu-768 HBD HBA Ribose pocket Hydrophobic pocket Cleft 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

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, but there is a greater risk that random mutation of the target will weaken binding interactions and lead to drug resistance Type III inhibitors are currently being studied Type III inhibitors bind to regions of the active site not occupied by ATP

2. Protein Kinase Inhibitors Examples of type I and type II inhibitors Type I inhibitors Gefitinib Erlotinib SU11248 Seliciclib Type II inhibitors Imatinib Nilotinib Sorafenib Vatalanib

3. 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

3. Gefitinib (Iressa) Lead compound Notes 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

3. Gefitinib (Iressa) Metabolism of the lead compound + Notes Cytochrome P450 enzymes Oxidation Notes Methyl group and para-position of aromatic ring are susceptible positions Blocking metabolism should improve the half life of the drug arrow

3. 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 Fluoro substiutent

3. Gefitinib (Iressa) Drug design Notes Morpholine Spacer Ionisable 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

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 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 O H 2 Hydrophobic pocket Cleft Met T h r HBA

3. Gefitinib (Iressa) Synthesis of gefitinib and analogues Methionine Pyridine A c 2 O A r N H 2 S O C l 2 N H 4 O M e R 2 N ( C H ) n B r

4. Lapatinib and Etlotinib Erlotinib (Tarceva) IC50 2 nM Quinazoline ring Aniline ring Quinazoline ring Aniline ring Notes 4-Anilinoquinazoline structures - compare gefitinib EGF-receptor kinase inhibitors

5. PKI 166 Notes Pyrrolopyrimidine structure Pyrrole Pyrimidine HBA HBD Notes Pyrrolopyrimidine structure EGF-receptor kinase inhibitor Different binding mode from ATP or anilinoquinazolines May need updated

5. PKI 166 Comparison of binding interactions ATP and EGF-receptor kinase inhibitors all contain a pyrimidine ring Different binding modes are possible HBA PKI 166 Gefitinib ATP PKI 166 Gefitinib ATP HBA HBD HBD HBA

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

6. Imatinib (Glivec or Gleevec) Lead compound Pyrimidine Anilino substituent 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 Amide Increased inhibition of PKC Inhibits tyrosine kinases as well

6. Imatinib (Glivec or Gleevec) Drug design Conformational blocker Imatinib Piperazine Spacer Increased activity vs tyrosine kinases 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 Wiggly lines removed

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 Wiggly lines removed

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) Glu Ionic bond Piperazinyl group Wiggly lines removed

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 Wiggly lines removed 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 Wiggly lines removed Mutation to Isoleucine

6. Imatinib (Glivec or Gleevec) Synthesis of imatinib and analogues

7. Second-generation Bcr-Abl inhibitors Met-318 Thr-315 Asp-381 Glu-286 Second generation Dotted lines

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 Second-generation

7. Second-generation Bcr-Abl inhibitors Notes Allosteric inhibitor of Bcr-Abl Does not bind to ATP binding site Stabilises inactive form of the enzyme Binds to an autoregulatory cleft Potential agent for treating leukaemia Notes Binds to the protein substrate site Currently under study Second-generation

8. Inhibitors of 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 HBA HBD Benzopyran Piperidine Phenyl ring 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

8. Inhibitors of cyclin-dependent kinases HBA HBD R-Roscovitine (seliciclib) Benzopyran Piperidine Phenyl ring 7-Hydroxystaurosporin is undergoing clinical trials Shows selectivity for CDK2 Undergoing clinical trials

9. Kinase Inhibitors of 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

9. Kinase Inhibitors of FGF-R and VEGF-R Anilino substituent Phthalazine Pyridine Pyrrole Oxindole HBA HBD Phase III clinical trials in 2006 SU 5416 in clinical trials for treatment of colorectal cancer Oxindole binds to same region as adenine of ATP

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 ‘promiscuous drugs’ Promising agents against tumours that are driven by several abnormalities promiscuous

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 Pazopanib approved in 2009

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 Lead compound; IC50 17 mM

10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib - variation of substituents Lead compound IC50 17 mM II; IC50 1.7 mM III; Poor activity Notes Methyl substituent in structure II 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 Isoxazole VI; Poor activity 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

10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Isoxazole Phenoxy group IV; IC50 1.1 mM IV; IC50 1.1 mM 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

10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Isoxazole Phenoxy group IV; IC50 1.1 mM V; IC50 0.23 mM Lead compound IC50 17 mM Pyridine Ring variation 5-fold increase in activity Increase in aqueous solubility and cLogP

10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Isoxazole Phenoxy group IV; IC50 1.1 mM Lead compound IC50 17 mM Ring variation Substituent Sorafenib IC50 12 nM Pyridine Sorafenib IC50 12 nM V; IC50 0.23 mM 1000-fold increase in activity

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

11. Inhibitors of heat shock protein 90 Notes Inhibitors bind to the ATP binding site Lead compound - geldanamycin Natural product Potent inhibitor Urethane group is crucial to activity Binds to region occupied by adenine Poor solubility Reactive quinone moiety Quinone Urethane

11. Inhibitors of heat shock protein 90 Geldanamycin analogues Alvespimycin Tanespimycin IPI 504