Selektivní kinázové inhibitory na bázi pyrazolo [1,5-a]p yrimidinu

1. Selektivní kinázové inhibitory na bázi pyrazolo[1,5-a]pyrimidinu Dr. Kamil Paruch Schering-Plough Research Institute, Kenilworth, NJ, USA Ústav chemie, Masarykova univerzita, Brno • CDK inhibitor SCH 727965 (dinaciclib) • CHK1 inhibitor SCH 900776

2. CDKs participate in cell cycle progression and cellular transcription Sausville, EA. Trends Molec Med 8: S32, 2002 Sausville, EA. Trends Molec Med 8: S32, 2002 1

3. Targeting the cell cycle and CDKs • Inhibition of CDK activity is an attractive therapeutic concept • Anticipated effects, based on mechanism, include: • Tumor cell-specific apoptosis • Reversible inhibition of proliferation in normal cells • Phenotypes confirmed by extensive experimental validation • (siRNA, dominant negatives and reference inhibitors) • Activity versus CDK2 and CDK1 shown to be essential for efficacy 2

4. Target candidate profile • Potent, selective inhibitor of CDK2 and CDK1 • Broad spectrum of activity (in vitro and in vivo) • Differential effects on tumor versus normal cells • Apoptosis in tumors • Transient arrest in normal tissues • Non-genotoxic MOA • Acceptable therapeutic index • Superior to competition and standards of care • Suitable for iv formulation and infusion • Reversible and manageable side-effects 3

5. Selectivity issue CDK2 vs. GSK3b 4

6. Compound A CDK2 IC50 = 0.50 uM Compound B CDK2 IC50 = 0.8 uM Lead finding • Early effort: synthesis based on molecular modeling: chemotypes capable of binding to the protein backbone (e.g. indazoles) - acceptable in vitro activity (< 50 nM), poor potency in the cell. • Variety of compound libraries purchased to improve hit rate inkinase inhibitor programs. • CDK2 actives found in Biofocus kinase-directed libraries. Pyrazolo[1,5-a]pyrimidines Imidazo[1,2-a]pyrazines 5

7. Proposed binding modes Crystal structure of A in CDK2 Purvalanol B CDK2 IC50 = 0.009 uM Compound A CDK2 IC50 = 0.50 uM • Initial SAR Plan: Increase potency by incorporating 3-substitution. 6

8. 3-Substitution improves potency Compound A CDK2 IC50 = 0.50 uM Thym. IC50 = 21 uM Compound A1 CDK2 IC50 = 0.003 uM Thym. IC50 = 0.48 uM Follow-up SAR plan: evaluate similar heterocyclic cores: 7

9. Synthesis of pyrazolo[1,5-a]pyrimidines Bioorg. Med. Chem. Lett. 2007, 17, 6216. 8

10. Synthesis of pyrazolo[1,5-a]pyridines Bioorg. Med. Chem. Lett. 2007, 17, 6216. 9

11. Synthesis of imidazo[1,2-a]pyrazines Bioorg. Med. Chem. Lett. 2007, 17, 6216. 10

12. Synthesis of imidazo[1,2-a]pyridines Bioorg. Med. Chem. Lett. 2007, 17, 6216. 11

13. CDK2 activity in various heterocyclic cores • focus on SAR of pyrazolo[1,5-a]pyrimidines 12

14. Validating the concept Does the lead series give the desired activity in cell culture? Can we derive a therapeutic index? Can the desired activity be demonstrated in vivo? 13

15. Initial lead compound In Vitro IC50’s (uM) CDK2/E = 0.031 CDK2/A = 0.030 CDK1 = 0.045 CDK4 = 0.045 MAPK = 1.2 GSK3b = 0.15 3A4, 2D6, 2C9, 2C19 > 30 uM In-Cell, thy uptake, A2780 IC50 (10% FBS) = 0.16 uM IC90 (10% FBS) = 0.29 uM hERG Rb:17% @ 1.5 uM Rb: 44% @ 5 uM VC: IC50 = 3.2 uM Compound 27 Mouse PK (IV/PO, 5 mpk) AUC = 8.4 uM.hr Cmax = 5.8 uM IV t1/2 = 2.4 hrs (NOEL) Rat PK (IV/PO, 5/10 mpk) PO AUC = 34.5 uM.hr Cmax = 4.4 uM IV t1/2 = 5.9 hrs clearance = 18 mL/min/kg (IV, PO NOEL) Monkey PK (IV/PO, 1/5 mpk) PO AUC = 29.9 uM.hr Cmax = 2.5 uM IV t1/2 = 11.0 hrs (IV: NOEL; PO: 2/3 diarrhea) • Efficacy: A2780 (ovarian): 96% I @ 40 mpk, qd; MiaPaCa (pancreatic): 77% I @ 40 mpk, qd. • Increasing exposure with increasing dose; physical properties appear to limit Cmax. • Poor solubility has precluded development of an IV formulation. 14

16. A2780 Apoptosis WS1 Cell cycle delay/arrest In-cell activity/therapeutic index Compound 27 Continuous Exposure Assays A2780 (Tumor) WS1 (Normal) Compound 27 Pulsed Exposure Assays • Expose cells to vehicle, IC50, IC90, 2 x IC90 for 24 hrs • Wash out compound, follow recovery of culture • Support of efficacy (putative trough/exposures) • Cell cultures show very distinct responses 15

17. Program progression Is the current lead appropriate to progress for further evaluation? What are the key issues? What approach do we take to resolve them? 16

18. Improving the potency of the lead compound • 3-Position: • Tight SAR. • Br, Cl, ethyl optimal. • Kinase selectivity domain. • 5-Position: • Variety of polar/nonpolar functionality tolerated. • Greatest potential for increases in potency. • High affinity for –OH substitution. • Substitution influences selectivity. • 7-Position: • Solvent exposed; wide variety of functionality tolerated. • Potency improvements with modulation of physical characteristics. • Substitution influences selectivity. 17

19. 3-Substitution • Tight SAR; only small hydrophobic substituents tolerated. • 3-Ethyl similar to 3-Br. Bioorg. Med. Chem. Lett.2007,17, 6220. 18

20. 7-Substitution • Solvent exposed; wide variety of functionality tolerated. • Potency improvements with modulation of physical characteristics. Bioorg. Med. Chem. Lett.2007,17, 6220. 19

21. 5-Substitution: aryl • Variety of polar/nonpolar functionality tolerated. • Greatest potential for increases in potency. 20 Bioorg. Med. Chem. Lett.2007,17, 6220.

22. 5-Substitution: alkyl 21

23. Compounds with improved potency/PK Compound 27 CDK2/A IC50 = 0.030 uM Thy. IC50 = 0.16 uM Rat PK (IV/PO, 5/10 mpk) PO AUC = 34.5 uM.hr IV t½ = 5.9 hrs Monkey PK (IV/PO, 1/5 mpk) PO AUC = 29.9 uM.hr IV t1/2 = 11.0 hrs Compound 28 CDK2/A IC50 = 0.016 uM Thy. IC50 = 0.16 uM Rat PK (IV/PO, 5/10 mpk) AUC = 28 uM.hr IV t½ = 2.1 hr Monkey PK (IV/PO, 2/10 mpk) AUC = 71 uM.hr t½ = 12 hrs Compound 31 CDK2/A IC50 = 0.018 uM Thy. IC50 = 0.025 uM Rat PK (IV/PO, 2/10 mpk) AUC = 15 uM.hr IV t½ = 3.6 hr Monkey PK (IV/PO, 0.5/1 mpk) AUC = 10 uM.hr t½ = 12 hrs 22

24. 5-Position: alcohol substitution Compound 70 CDK2 IC50 = 0.011 uM Thy. IC50 = 0.48 uM Compound 71 CDK2 IC50 = 0.063 uM Thy. IC50 = 2.5 uM Compound 72 CDK2 IC50 = 0.027 uM Thy. IC50 = 0.26 uM Compound 73 CDK2 IC50 = 0.0004 uM Thy. IC50 = 0.006 uM Compound 74 CDK2 IC50 = 0.063 uM Thy. IC50 = 1.1 uM Compound 75 CDK2 IC50 = 0.018 uM Thy. IC50 = 0.85 uM Compound 76 CDK2 IC50 = 0.006 uM Thy. IC50 = 0.2 uM Compound 77 CDK2 IC50 = 0.0003 uM Thy. IC50 = 0.002 uM • Alcohol substitution improves potency and solubility, but increases rate of clearance and decreases exposure 23

25. What is the optimal profile for a CDK inhibitor? • Series with distinct properties were identified: • Compound 7: average potency, good PK • Compound 31: improved in-cell potency, excellent PK • Compound 73A: high potency, rapid clearance • How do they compare to known CDK inhibitors? • BMS-387032: average potency, rapid clearance • Flavopiridol: average potency, rapid clearance • How do we identify the optimal profile (potency, PK)? BMS-387032 Compound 73A Compound 31 24

26. In vivo screening paradigm • Goal: identification of optimal profile • Readout: efficacy below MTD in mouse 25

27. Initially evaluated compounds • *MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy); **TI (therapeutic index) = MTD/MED; MTD: dose (ip, qdx7) giving 20% BW loss. • Amino alcohol series (high potency, short t½) gives best therapeutic index. • High potency, short t½ contrary to conventional wisdom • What is the rationale for the activity seen with compound 73A? 26

28. Identification of SCH 727965 *MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy) vs. • in vivo differentiation of compounds • with nearly identical in vitro profile ACS Med. Chem. Lett. 2010, 1, 204. 27

29. SCH 727965: in vitroproperties In Vitro A/CDK2 IC50 = 0.001 uM B/CDK1 IC50 = 0.004 uM p35/CDK5 IC50 = 0.001 uM D/CDK4 IC50 = 0.1 uM H/CDK7 IC50 = 0.07 uM GSK3b IC50 = 0.78 uM MAPK IC50 = 4.1 uM UBI Kinase Profiler – No issues PanLabs – No issues GPCR – No issues CYP – No issues hERG (VC) – 5% @ 1 uM Invest. CV – No issues up to ~0.37 uM SCH 727965 In Cell IC50 (A2780) = 0.004 uM Marker IC50 (phospho-Rb) = ~0.01 uM 2 hour apoptosis = 0.025 uM (BMS-387032 = 1 uM) Cell Line Survey (106 cell lines, NCI-60 plus others) Multiple assays (FACS, markers, viability, apoptosis) 100% arrest; >85% apoptosis. Predominant tumor cell response - apoptosis Normal cell response (WS-1) - cell cycle arrest, little apoptosis CDK2/cyclin A + SCH 727965 Mol.CancerTher. 2010, 9, 2344. 28

30. In vivoefficacy: SCH 727965 is active < MTD A2780 (ovarian cancer) RPMI 8226 (multiple myeloma) • same model used for preclinical studies of Velcade • complete cures in 20 mpk group (no regrowth of tumor) A549 (lung cancer) • complete cures in 20 mpk group • (no regrowth of tumor) • no effect on RBC, platelets • mild reversible myelosupression 29

31. CHK1 inhibitors template-based approach Antimetabolites induce activation (and phosphorylation) of CHK1 kinase CHK1 inhibition + HU (or gemcitabine or cytarabine) induce DNA damage and cell death 30

32. 31

33. Bioorg. Med. Chem. Lett. 2011, 21, 471. 32

34. CHK1 inhibitors template-based approach SCH 900776 + HU (or Gem or Ara-C) induce DNA damage and cell death in vivo Mol.CancerTher. 2011, doi:10.1158/1535-7163.MCT-10-0324. 33

35. CHK1 small molecule inhibitor + HU (or Gem or Ara-C) induce DNA damage and cell death • gemcitabine • inhibits RNR (as triphosphate) • gets incorporated into DNA • relatively toxic • limited stability • gem analogs (WO 2009/061781 A1) • gem-like phenotype in combination with CHK1 inhibitor • no DNA incorporation • current effort • compounds with targeted biological activity • synthetic lethal treatment CHK1 + DNA polymerase-a inhibition induce DNA damage and cell death Taricani, L.; Shanahan, F.; Parry, D. Cell Cycle2009, 8, 482. 34

36. The End