Benzothiazines As Novel Peptide Mimetic Calpain Inhibitors

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Discovery and development of a novel peptidomimetic for potential treatment of ischemic stroke or traumatic spinal cord injury.

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Benzothiazines As Novel Peptide Mimetic Calpain Inhibitors

  1. 1. 3,4-Dihydro-1,2-Benzothiazine-1,1-dioxides as Novel Peptide Mimetic Calpain I Inhibitors
  2. 2. Properties of Calpain <ul><li>Calcium-activated Neutral Endopeptidases with close homology to papain (CANP) </li></ul><ul><li>Large family of intracellular cysteine proteases </li></ul><ul><li>Two major forms: Calpain I (  -calpain, low [Ca +2 ] activated) and Calpain II (m-calpain, higher [Ca +2 ] activated) </li></ul><ul><li>Heterodimer – 30 kDa regulatory + 80 kDa catalytic subunits </li></ul><ul><li>Ubiquitous throughout animal kingdom and even insects </li></ul><ul><li>> Cytoplasmic (non-lysosomal) </li></ul><ul><li>> Erythrocytes contain only Calpain I </li></ul>
  3. 3. Heterodimeric Structure of Human Procalpain I Ca +2 Ca +2 Cys His I II III IV V VI 714AA (80 kDa) 268AA (30 kDa) <ul><li>Large catalytic subunit of Calpain II is distinct </li></ul><ul><li>Small subunit is identical </li></ul>Calcium-activated autoproteolysis: 80 to 76 kDa 30 to 17 kDa
  4. 4. Calpain Substrates <ul><li>Cytoskeletal Proteins – Structural Integrity </li></ul><ul><ul><li>Spectrin (fodrin): K m = 50nM </li></ul></ul><ul><ul><li>Microtubule-associated proteins: K m <50nM </li></ul></ul><ul><ul><li>Talin, actin, neutrofilament </li></ul></ul><ul><li>Membrane Proteins </li></ul><ul><ul><li>EGF receptors, integrin (gpII a III b ) </li></ul></ul><ul><li>Enzymes </li></ul><ul><ul><li>PKC, MLCK, Calcineurin, Phospholipase C </li></ul></ul><ul><li>Transcriptional Activators </li></ul><ul><ul><li>fos, jun </li></ul></ul><ul><li>Miscellaneous </li></ul><ul><ul><li>Cytokines, crystallins </li></ul></ul>
  5. 5. Involvement in Pathophysiological States <ul><li>Ischemia: Cerebral, Myocardial, Liver, etc. </li></ul><ul><li>Brain and Spinal Cord Trauma </li></ul><ul><li>Alzheimers Disease </li></ul><ul><li>Arthritis </li></ul><ul><li>Restinosis </li></ul><ul><li>Cataracts </li></ul><ul><li>Peripheral Neuropathy </li></ul>
  6. 6. Role of Calpain in Ischemic Neurodegeneration Calpain Activation AMPA NMDA Voltage- gated Ca +2 Ca +2 Hydrolysis of Cytoskeletal Proteins Neuronal Cell Death Kemp, TIPS (1994) Bigge, Ann Rep Med Chem, 29 , 13 (1994) Siman, Neurotox Excit Amino Acids , p.145 (1990) Ca +2 Ca +2 Ca +2 Ca +2 Ca +2
  7. 7. Substrate Hydrolysis Mechanism
  8. 8. Inhibitor Classes Reversible Irreversible
  9. 9. Peptide Aldehydes – Prior Art <ul><li>Leupeptin: Ac-Leu-Leu-Arg-H (Umezawa, 1969, 1972; Suzuki, 1978) </li></ul><ul><li>Ac-Phe-Gly-H - Papain Inhibitor (Westerik & Wolfenden, 1972) </li></ul><ul><li>Leupeptin as Calpain Inhibitor: Ac-Leu-Leu-Arg-H (Suzuki, 1978) </li></ul><ul><li>Chymostatin (Umezawa, 1973) </li></ul><ul><li>Leupeptin in vivo – ventricular infusion (Lee, 1991) </li></ul><ul><li>Z-Val-Phe-H in vivo – i.v. injection (Hong, 1994) </li></ul>Molt-4 Cell IC 50 = 0.8mM MDL28170 (Z-Val-Phe-H) IC 50 = 20nM
  10. 10. Enzyme-Reactive Group Survey <ul><li>Aldehydes 10 </li></ul><ul><li>a-Ketoamides/a-Ketoesters >=20 </li></ul><ul><li>Hydroxy Cyclopropenones 200 </li></ul><ul><li>Semicarbazones >1000 </li></ul><ul><li>Methyl Vinyl Ethers ~1000 </li></ul><ul><li>Ketones >10,000 </li></ul><ul><li>Trifluoromethyl Ketones >>10,000 </li></ul><ul><li>Vinyl Sulfones & Sulfonates >>10,000 </li></ul>Group Calpain IC 50 (nM) > 50 Inactivators – leaving groups (up to 300,000 M -1 s -1 ) Wells, Bihovsky Exp. Opin.Ther. Patents 1998, 1707
  11. 11. Cephalon Irreversible Inhibitors Chatterjee, Wells, et al., JMC; 1997, 3820 Tao, Wells, et al., JMC; 1998, 3912 <ul><li>Primarily di- or tri-peptidyl mimics containing a readily displaced leaving group </li></ul><ul><li>Classified on the basis of time-dependent, second-order inhibition kinetics (M -1 /s -1 ) </li></ul><ul><li>Best-in-class included various peptidyl fluoromethyl and phosphorous-oxymethyl ketones </li></ul><ul><li>Class dropped due to issues of stability, selectivity, poor brain penetration in-vivo </li></ul>
  12. 12. Cephalon Side-chain Optimization Summary of SAR from >100 L,L-Dipeptidyl aldehydes P 2 : Specific Leu > Val ~ Nle > Ile Capping Group: Tolerant Z ~ 4-Nitro-Z > Ts ~ (+)-Menthyloxy-CO ~ FMOC >> Ac P 1 : Tolerant Val > Cha ~ Leu > Nle His ~ Phe ~ Met > Arg Most potent inhibitor IC 50 = 4nM Iqbal, et al., Bioorg. Med. Chem. Lett. 1997, 7, 539
  13. 13. P 2 -D-Amino Acid Analogs Calpain I IC 50 = 11nM Chatterjee, Bihovsky JMC , 1998, 41, 2663 Molt-4 IC 50 = 1.4  M 10 130 Calpain I IC 50 (nM) Molt-4 IC 50 (  M) X 0.9 ND H
  14. 14. Intact-Cell Calpain Inhibitor Assay <ul><li>Molt-4 Cells: Human leukemic T-lymphocytes </li></ul><ul><ul><li>Calpain I predominates </li></ul></ul><ul><ul><li>Stimulate with Ionomycin (Calcium ionophore) </li></ul></ul><ul><ul><li>Measure Spectrin BDP; Western Blot w/specific polyclonal AB </li></ul></ul><ul><ul><li>Good measure of cell permeability, solubility </li></ul></ul><ul><li>Earlier use of Cortical neuronal cell line </li></ul><ul><li>discontinued due to poor reproducibility, viability </li></ul>
  15. 15. P 2 -Achiral, P ’ -Extended a-Ketoamides Chatterjee, Bihovsky, Wells BMCL , 1999, 2371 K i (nM) R K i (nM) R 62 PhCH(CH 3 )(CH 2 ) 2 - 26 2,6-Dichlorophenyl 1100 Ph(CH 2 ) 3 - 49 2,5-Dichlorophenyl 21 (CH 3 ) 2 CHCH 2 CH 2 - 46 2-Chloro-5-methoxyphenyl 420 (CH 3 ) 2 CHCH 2 - 130 2,6-Dimethylphenyl 1100 (CH 3 ) 2 CH- 430 3,5-Bis(trifluoromethyl)phenyl 57 2,6-Dichloronicotinyl >1000 3,4-Methylenedioxyphenyl 14 2,6-Difluorophenyl 570 Phenyl
  16. 16. P 2 -Achiral, P’-Extended a-Ketoamides X K i (nM) 14 21 8 59 71 15
  17. 17. Hypothesis Overlap of P 2 -Phe/P 3 -N-SO 2 Ph Groups gives a Novel 1,2-Benzothiazine Peptide Mimetic <ul><li>X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature </li></ul>IC 50 = 11nM
  18. 18. Hypothesis Overlap of P 2 -Phe/P 3 -N-SO 2 Ph Groups gives a Novel 1,2-Benzothiazine Peptide Mimetic <ul><li>X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature </li></ul>IC 50 = 11nM IC 50 = 20nM
  19. 19. Hypothesis Overlap of P 2 -Phe/P 3 -N-SO 2 Ph Groups gives a Novel 1,2-Benzothiazine Peptide Mimetic <ul><li>X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature </li></ul>IC 50 = 11nM IC 50 = 20nM
  20. 20. Dreiding Model Analysis <ul><li>No obvious discouraging intermolecular interactions </li></ul><ul><li>Literature search uncovered no competing IP </li></ul>LET’S GO FOR IT!
  21. 21. General Synthesis Popel Pharmazie, 1980, 266
  22. 22. a Mixture of (3R)- and (3S)- diastereomers; b Isomer 1 is the (3S)- diastereomer . Wells, Bihovsky, Tao, Mallamo JMC , 2001, 44, 3488 3,4-Dihydro-1,2-benzothiazine 1,1-dioxide peptidomimetic aldehydes
  23. 23. Excellent selectivity compared to dipeptide aldehydes Structure >>1000 (44%@1  M) 7 82 11 5 20 38 11 Cathepsin B IC 50 (nM) Calpain I IC 50 (nM)
  24. 24. Synthesis of Lead Molecule and Identification of Absolute Configuration from L-DOPA Calpain I IC 50 = 7nM Molt 4 Cell IC 50 = 0.50  M <ul><li>Confirms (S)-configuration at C-3 </li></ul><ul><li>68% Enantiomeric purity </li></ul>
  25. 25. Unsaturated Analogs 3.2 2.1 0.9 2.9 Molt-4 Cell IC 50 (  M) 37 OMe Me H H 8 H CH 2 CH 3 OCH 2 CH 2 O 6 H Me OCH 2 CH 2 O 15 H Me Cl Cl Calpain I IC 50 (nM) R4 R3 R2 R1
  26. 26. Benzothiadiazine Analogs 28 OCH 2 CH 2 O 83 H H Calpain I IC 50 (nM) R 2 R 1
  27. 27. Isoquinoline Analogs ~1000 85 Me -- ~5000 H Isomer 2 Isomer 1 R Calpain I IC 50 (nM)
  28. 28. 3,4-Dihydro-1,2-benzothiazine a-Ketoamides R’-Alkyl Groups Et Et Et Bu (diastereomers) H Bu Et CH 2 CH 2 OCH 3 Et CH(CH 3 ) 2 Et CH 2 -c-propane Et (CH 2 ) 4 CH 3 Et CH 2 Ph Et CH 2 CH 2 Ph Et CH 2 CH=CH 2 Et (CH 2 ) 3 -(imidazol-1-yl) Et (CH 2 ) 3 -(2-ketopyrrolidin-1-yl) Et (CH 2 ) 3 -(morpholin-4-yl) Et CH 2 -(pyridin-2-yl) Et CH 2 -(pyridin-4-yl) 340 50; 300 200 200 205 286 150 81 63 200 ~5000 500 195 170 240 R R’ IC 50 (nM) Wells, Bihovsky BMCL, 2004, 1035
  29. 29. 3,4-Dihydro-1,2-benzothiazine a-Ketoamides R’-Sulfonamides Et CH 2 CH 2 NHSO 2 CH 3 Et CH 2 CH 2 NHSO 2 (4-NO 2 -Ph) Et CH 2 CH 2 NHSO 2 (3,4-Cl 2 -Ph) Et CH 2 CH 2 NHSO 2 Ph H CH 2 CH 2 NHSO 2 Ph Et CH 2 CH 2 NHSO 2 (4-F-Ph) Et CH 2 CH 2 NHSO 2 (5-(2-pyridyl)thiophen-2-yl) Et (CH 2 ) 3 NHSO 2 Ph Et (CH 2 ) 3 NHSO 2 (4-F-Ph) Et (CH 2 ) 3 NHSO 2 (4-NO 2 -Ph) Et (CH 2 ) 3 NHSO 2 (3,4-Cl 2 -Ph) 89 47 56 40 76 29 20 35 50 50 56 R R’ IC 50 (nM) Wells, Bihovsky BMCL, 2004, 1035
  30. 30. General Synthesis of  -Ketoamides
  31. 31. General Synthesis of  -Ketoamide R’-Sulfonamides Chatterjee, Bihovsky, Wells BMCL , 1999, 2371
  32. 32. CEP-3501 PK
  33. 33. Profile of CEP-3501 Calpain I IC 50 7 nM Molt-4 IC 50 0.5 nM Brain Concentration 1.6  M (infused @ 22 mg/kg/h) Clearance 144 mL/mg/kg Solubility 0.22 mg/mL Gerbil global ischemia 85% reduction in spectrin breakdown Mini-Ames Test Negative (Mol. Wt. = 444)
  34. 34. Synthesis and study of bisulfite addition product IC 50 = 7nM IC 50 = 8nM Molt-4 IC 50 = 0.5  M Molt-4 IC 50 = 1.2  M <ul><li>Superior water solubility of bisulfite product v. aldehyde (>>1mg/mL v. 0.2mg/mL, resp.) </li></ul><ul><li>Comparable in-vitro potency but weaker Molt-4 cellular potency </li></ul><ul><li>Inhibits spectrin breakdown in gerbil forebrain global ischemia model (BCAO) by 88% </li></ul><ul><li>(100mg/kg bolus + 24h infusion at 30mg/kg/h) </li></ul><ul><li>Not neuroprotective four days after ischemia dosed at this regimen, despite evidence of robust </li></ul><ul><li>inhibition of spectrin breakdown following autopsy </li></ul>
  35. 35. Acknowledgements Medicinal Chemistry Ron Bihovsky Sankar Chatterjee Manoj Das Bruce Dembofsky Derek Dunn Bethany Freed Zi-Qiang Gu Mohamed Iqbal Kurt Josef James Kauer John Mallamo Patricia Messina Ming Tao Rabindranath Tripathy Gregory Wells Biochemistry Mark Ator William Biazzo Donna Bozyczko-Coyne Satish Mallya Beth McKenna Terry O’Kane Shobha Senadhi Molecular Biology Diane Lang Sheryl Meyer Chrysanthe Spais Pharmacology Lisa Aimone Richard DiRocco Bruce Jones Val Marcy Matthew Miller Jeffrey Skell Jeffry Vaught SmithKline Beecham Wayne Bowen Gregory Gallagher John Gleason Jackie Hunter William Kingsbury Gordon Moore Israil Pendrak

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