Post on 10-Jul-2015
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