G.Wells | June 2011

3,4-Dihydro-1,2-Benzothiazine-1,1-dioxidesas Novel Peptide Mimetic Calpain I Inhibitors

SN

O O

O

NH

Y

O

R3

R2

R1

G.Wells | June 2011

Properties of Calpain

• Calcium-activated Neutral Endopeptidases with close homology to papain (CANP)

• Large family of intracellular cysteine proteases

• Two major forms: Calpain I (µ-calpain, low [Ca+2] activated) and Calpain II (m-calpain, higher [Ca+2] activated)

• Heterodimer – 30 kDa regulatory + 80 kDa catalytic subunits

• Ubiquitous throughout animal kingdom and even insects> Cytoplasmic (non-lysosomal)> Erythrocytes contain only Calpain I

G.Wells | June 2011

Heterodimeric Structure of Human Procalpain I

Ca+2

Ca+2

Cys His

I II III IV

V VI

714AA (80 kDa)

268AA (30 kDa)

Large catalytic subunit of Calpain II is distinct Small subunit is identical

Calcium-activated autoproteolysis: 80 to 76 kDa 30 to 17 kDa

G.Wells | June 2011

Calpain Substrates

• Cytoskeletal Proteins – Structural Integrity

– Spectrin (fodrin): Km = 50nM

– Microtubule-associated proteins: Km <50nM

– Talin, actin, neutrofilament

• Membrane Proteins

– EGF receptors, integrin (gpIIaIIIb)

• Enzymes– PKC, MLCK, Calcineurin, Phospholipase C

• Transcriptional Activators– fos, jun

• Miscellaneous

– Cytokines, crystallins

G.Wells | June 2011

Involvement in Pathophysiological States

• Ischemia: Cerebral, Myocardial, Liver, etc.• Brain and Spinal Cord Trauma• Alzheimers Disease• Arthritis• Restinosis• Cataracts• Peripheral Neuropathy

G.Wells | June 2011

CalpainActivation

Role of Calpain in Ischemic Neurodegeneration

AMPA

NMDA

Voltage-gated

Ca+2

Ca+2

Hydrolysis ofCytoskeletal

Proteins

NeuronalCell 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

G.Wells | June 2011

Substrate Hydrolysis Mechanism

R NN

NR'

O R2

H O

H

R1

O

H

R1'

O

SH

N

NH

S

R NN

NR'

O R2

H O

H

R1

O

H

R1'

ONH+

NH

H O

H

NH2

R'

R1'

O

R NN

O R2

H O

H

R1

O

S

NH

:NR NN

O R2

H O

H

R1

O

OH

SH

NH

:N

P3 P2 P1 P1'

Calpain

Calpain

Calpain

Calpain

H2O

G.Wells | June 2011

Inhibitor Classes

R NN

O R2

H O

H

R1

OH

G

S

NH

:N

R NN

O R2

H O

H

R1

O

G

SH

NH

N

R NN

O R2

H O

H

R1

O

S NH

:N

R NN

O R2

H O

H

R1

O

X

SH

NH

N

Calpain

Calpain

Calpain

Calpain

(G = H, R, CONR3R4, CO2R5, Het) (X = F, Cl, O(CO)R3, S+RRY-, OP(O)(OR4)2, OHet)

Reversible Irreversible

G.Wells | June 2011

Peptide Aldehydes – Prior Art

• Leupeptin: Ac-Leu-Leu-Arg-H (Umezawa, 1969, 1972; Suzuki, 1978)• Ac-Phe-Gly-H - Papain Inhibitor (Westerik & Wolfenden, 1972)• Leupeptin as Calpain Inhibitor: Ac-Leu-Leu-Arg-H (Suzuki, 1978)• Chymostatin (Umezawa, 1973)• Leupeptin in vivo – ventricular infusion (Lee, 1991)• Z-Val-Phe-H in vivo – i.v. injection (Hong, 1994)

H

O

N

H

O

NO

O

H

Molt-4 Cell IC50 = 0.8mM

MDL28170 (Z-Val-Phe-H) IC50 = 20nM

G.Wells | June 2011

Enzyme-Reactive Group Survey

• Aldehydes 10• a-Ketoamides/a-Ketoesters >=20• Hydroxy Cyclopropenones 200• Semicarbazones >1000• Methyl Vinyl Ethers ~1000• Ketones >10,000• Trifluoromethyl Ketones >>10,000• Vinyl Sulfones & Sulfonates >>10,000

Group Calpain IC50 (nM)

> 50 Inactivators – leaving groups (up to 300,000 M-1s-1)

Wells, Bihovsky Exp. Opin.Ther. Patents 1998, 1707

P2 NH

O

ERG

P1

G.Wells | June 2011

Cephalon Irreversible Inhibitors

N NH

NH

F

O

O

O

Ph

O NH

NH

O

O

O

O

R'

Ph

P(OR)2

(k = 276,000 M-1s-1) (k = 100-365 x 103 M-1s-1)

(O)0-1

Chatterjee, Wells, et al., JMC; 1997, 3820 Tao, Wells, et al., JMC; 1998, 3912

Primarily di- or tri-peptidyl mimics containing a readily displaced leaving group Classified on the basis of time-dependent, second-order inhibition kinetics (M-1/s-1) Best-in-class included various peptidyl fluoromethyl and phosphorous-oxymethyl ketones

Class dropped due to issues of stability, selectivity, poor brain penetration in-vivo

G.Wells | June 2011

Cephalon Side-chain Optimization

Summary of SAR from >100 L,L-Dipeptidyl aldehydes

P2: SpecificLeu > Val ~ Nle > Ile

Capping Group: TolerantZ ~ 4-Nitro-Z > Ts ~(+)-Menthyloxy-CO ~FMOC >> Ac

P1: TolerantVal > Cha ~ Leu > NleHis ~ Phe ~ Met > Arg

Most potent inhibitor IC50 = 4nM

Iqbal, et al., Bioorg. Med. Chem. Lett. 1997, 7, 539

R H

H

HR2

R1

O

O

O

G.Wells | June 2011

P2-D-Amino Acid Analogs

O NH

O

H

ONHS

O O

Ph

O NH

O

ONHS

O O

Ph

NH

O

X(D) (D)

10

130

Calpain I

IC50 (nM)

Molt-4

IC50 (µM)X

0.9

NDH

S NNHSO2

Calpain I IC50 = 11nM

Chatterjee, Bihovsky JMC, 1998, 41, 2663

Molt-4 IC50 = 1.4µM

G.Wells | June 2011

Intact-Cell Calpain Inhibitor Assay

• Molt-4 Cells: Human leukemic T-lymphocytes– Calpain I predominates– Stimulate with Ionomycin (Calcium ionophore)– Measure Spectrin BDP; Western Blot w/specific

polyclonal AB– Good measure of cell permeability, solubility

• Earlier use of Cortical neuronal cell line

discontinued due to poor reproducibility, viability

G.Wells | June 2011

P2-Achiral, P’-Extended a-Ketoamides

Ki (nM)RKi (nM)R

62PhCH(CH3)(CH2)2-262,6-Dichlorophenyl

1100Ph(CH2)3-492,5-Dichlorophenyl

21(CH3)2CHCH2CH2-462-Chloro-5-methoxyphenyl

420(CH3)2CHCH2-1302,6-Dimethylphenyl

1100(CH3)2CH-4303,5-Bis(trifluoromethyl)phenyl

572,6-Dichloronicotinyl>10003,4-Methylenedioxyphenyl

142,6-Difluorophenyl570Phenyl

Chatterjee, Bihovsky, Wells BMCL, 1999, 2371

R NH

NH

NHSO2

O

O

O

Ph

S N

G.Wells | June 2011

P2-Achiral, P’-Extended a-Ketoamides

NH

NH

NHSO2-X

O

O

O

PhCl

Cl

S

O

S

NHAc

S

N O

X Ki (nM)

14

21

8

59

71

15

S

CN

N

N N

N

NAc

G.Wells | June 2011

Hypothesis

Overlap of P2-Phe/P3-N-SO2Ph Groupsgives a Novel 1,2-Benzothiazine Peptide Mimetic

X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature

O

NH

NH

O

H

O

Ph

S

OO

IC50 = 11nM

G.Wells | June 2011

Hypothesis

Overlap of P2-Phe/P3-N-SO2Ph Groupsgives a Novel 1,2-Benzothiazine Peptide Mimetic

X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature

NH

NH

O

H

O

Ph

S

OO

O

NH

NH

O

H

O

Ph

S

OO

IC50 = 11nM IC50 = 20nM

G.Wells | June 2011

Hypothesis

Overlap of P2-Phe/P3-N-SO2Ph Groupsgives a Novel 1,2-Benzothiazine Peptide Mimetic

X-Ray Crystal Structure of Calpain-ligand active site unavailable due to autolytic nature

NH

NH

O

H

O

Ph

S

OO

O

NH

NH

O

H

O

Ph

S

OO

SNH

NH

O

H

O

O O

Ph

IC50 = 11nM IC50 = 20nM

G.Wells | June 2011

Dreiding Model Analysis

No obvious discouraging intermolecular interactions Literature search uncovered no competing IP

LET’S GO FOR IT!

G.Wells | June 2011

General Synthesis

Popel Pharmazie,1980, 266

G.Wells | June 2011

SY

R6

R7

NH

O

H

O

R1

O O

3

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

G.Wells | June 2011

Excellent selectivity compared to dipeptide aldehydes

O NH

H

O

ONHMeSO2

Ph

NH

H

O

ONHMeSO2

Ph

NH

H

O

O

Ph

NH

O

O

Ph

O

O SN

O O

NH

O

H

Ph

O

(Z-Val-Phe-H)

CEP-3501

Structure

>>1000

(44%@1µM)7

8211

520

3811

Cathepsin B

IC50 (nM)

Calpain I

IC50 (nM)

G.Wells | June 2011

Synthesis of Lead Molecule and Identification of Absolute Configuration from L-DOPA

NH2

OH

OH

CO2H

NHCbz

O

O

CO2Me

O

O SN

CO2H

O O

O

O SN

O O

NH

O

H

Ph

O

NH2

OH

Ph

L-DOPA

1. MeOH, SOCl22. Cbz-OSu

3. BrCH2CH2Br, K2CO3

1. ClSO3H; then Et3N, DMAP

2. EtI, K2CO3, DMF

3. 2M NaOH, MeOH

1.

2. Dess-Martin oxidation

BOP, HOBt, NMM

CEP-3501

3

Calpain I IC50 = 7nM

Molt 4 Cell IC50 = 0.50µM

Confirms (S)-configuration at C-3 68% Enantiomeric purity

G.Wells | June 2011

Unsaturated Analogs

SN

R3O O

R1

R2 CO2Me

SN

R3O O

R1

R2

R4

CO2Me

SN

MeO O

R1

R2

OH

CO2Me

SN

R3O O

R1

R2

R4

NH

OPh

CHO

(as previously)

NBS, (BzO)2 MeI, K2CO3

(Piroxicam precursor)

3.2

2.1

0.9

2.9

Molt-4 Cell

IC50 (µM)

37OMeMeHH

8HCH2CH3OCH2CH2O

6HMeOCH2CH2O

15HMeClCl

Calpain I IC50 (nM)

R4R3R2R1

G.Wells | June 2011

Benzothiadiazine Analogs

NH2

SO2NH2

R2

R1

NH

SO2NH2

R2

R1

COCO2Et

SNH

N

O O

CO2EtR2

R1

SNH

N

O O

R2

R1

NH

OPh

CHO

ClCOCO2Et

Et3N

NaOEt

(as previously)

28OCH2CH2O

83HH

Calpain I

IC50 (nM)

R2R1

G.Wells | June 2011

Isoquinoline Analogs

NH

O

CO2H

N

O

CO2H

Me

N

O

NH

O

R

CHO

Ph

1. MeI, AgO

2. NaOH

(as previously)

~100085Me

--~5000H

Isomer 2Isomer 1R

Calpain I IC50 (nM)

G.Wells | June 2011

3,4-Dihydro-1,2-benzothiazine a-KetoamidesR’-Alkyl Groups

Et EtEt Bu (diastereomers)H BuEt CH2CH2OCH3

Et CH(CH3)2

Et CH2-c-propaneEt (CH2)4CH3

Et CH2PhEt CH2CH2PhEt CH2CH=CH2

Et (CH2)3-(imidazol-1-yl)Et (CH2)3-(2-ketopyrrolidin-1-yl)Et (CH2)3-(morpholin-4-yl)Et CH2-(pyridin-2-yl)Et CH2-(pyridin-4-yl)

34050; 300

2002002052861508163

200~5000

500195170240

R R’ IC50 (nM)

O

O SN

NH

NH

O

O

O

O O

R

R'

Ph

Wells, Bihovsky BMCL, 2004, 1035

G.Wells | June 2011

3,4-Dihydro-1,2-benzothiazine a-KetoamidesR’-Sulfonamides

Et CH2CH2NHSO2CH3

Et CH2CH2NHSO2(4-NO2-Ph)Et CH2CH2NHSO2(3,4-Cl2-Ph)Et CH2CH2NHSO2PhH CH2CH2NHSO2PhEt CH2CH2NHSO2(4-F-Ph)Et CH2CH2NHSO2(5-(2-pyridyl)thiophen-2-yl)Et (CH2)3NHSO2PhEt (CH2)3NHSO2(4-F-Ph)Et (CH2)3NHSO2(4-NO2-Ph)Et (CH2)3NHSO2(3,4-Cl2-Ph)

O

O SN

NH

NH

O

O

O

O O

R

R'

Ph8947564076292035505056

R R’ IC50 (nM)

Wells, Bihovsky BMCL, 2004, 1035

G.Wells | June 2011

General Synthesis of α-Ketoamides

O

O

SN

CO2H

O OO

O

SN

O O

NH

OPh

CHO

O

O

SN

O O

O

NH

X

O

OH

Ph

O

O

SN

O O

NH

O

O

X

OPh

(a), (b) (c)

(for R' = Bu)

(d)

(b)

X = OCH3

X = NHR'

(e)

(a) H2NCH(CH2Ph)CH2OH, HOBt, BOP, NMM, DMF; (b) Dess-Martin, DCM; (c) BuNC, TiCl4, DCM;(d) HCl-H2NCH(CH2Ph)CH(OH)CO2CH3, HOBt, BOP, NMM, DMF; (e) R'NH2, neat, rt.

G.Wells | June 2011

General Synthesis of α-Ketoamide R’-Sulfonamides

O

O

SN

CO2H

O O O

O

SN

O O

NH

O

O

NH(CH2)nNHSO2R'

OPh

(a), (b)

(a) HCl-H2NCH(CH2Ph)CH(OH)CONH(CH2)nNHSO2R', HOBt, BOP, NMM, DMF; (b) Dess-Martin, DCM, t-BuOH

Chatterjee, Bihovsky, Wells BMCL, 1999, 2371

G.Wells | June 2011

CEP-3501 PK

G.Wells | June 2011

Profile of CEP-3501

Calpain I IC50 7 nMMolt-4 IC50 0.5 nMBrain Concentration 1.6 µM (infused @ 22 mg/kg/h)Clearance 144 mL/mg/kgSolubility 0.22 mg/mLGerbil global ischemia 85% reduction in spectrin breakdownMini-Ames Test Negative

O

O SN

O O

NH

O

H

Ph

O

(S)

(S)

(Mol. Wt. = 444)

G.Wells | June 2011

Synthesis and study of bisulfite addition product

O

O SN

O O

NH

O

H

Ph

O

O

O SN

O O

NH

O

SO3Na

Ph

OH

NaHSO3, EtOAc, H2O

(87%)

(MW 444) (MW 548)

IC50 = 7nM IC50 = 8nM

Molt-4 IC50 = 0.5µM Molt-4 IC50 = 1.2µM

• Superior water solubility of bisulfite product v. aldehyde (>>1mg/mL v. 0.2mg/mL, resp.)

• Comparable in-vitro potency but weaker Molt-4 cellular potency

• Inhibits spectrin breakdown in gerbil forebrain global ischemia model (BCAO) by 88% (100mg/kg bolus + 24h infusion at 30mg/kg/h)

• Not neuroprotective four days after ischemia dosed at this regimen, despite evidence of robust inhibition of spectrin breakdown following autopsy

G.Wells | June 2011

Acknowledgements

Medicinal Chemistry

Ron BihovskySankar ChatterjeeManoj DasBruce DembofskyDerek DunnBethany FreedZi-Qiang GuMohamed IqbalKurt JosefJames KauerJohn MallamoPatricia MessinaMing TaoRabindranath TripathyGregory Wells

Biochemistry

Mark AtorWilliam BiazzoDonna Bozyczko-CoyneSatish MallyaBeth McKennaTerry O’KaneShobha Senadhi

Molecular Biology

Diane LangSheryl MeyerChrysanthe Spais

Pharmacology

Lisa AimoneRichard DiRoccoBruce JonesVal MarcyMatthew MillerJeffrey SkellJeffry Vaught

SmithKline Beecham

Wayne BowenGregory GallagherJohn GleasonJackie HunterWilliam KingsburyGordon MooreIsrail Pendrak