First-in-class pan caspase inhibitor developed for the treatment of liver disease

Article abstract of DOI:10.1021/jm050307e

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of α-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Full text of DOI:10.1021/jm050307e

J. Med. Chem. 2005, 48, 6779-6782
6779
First-in-Class Pan Caspase Inhibitor
Developed for the Treatment of Liver
Disease
Steven D. Linton,* Teresa Aja, Robert A. Armstrong,
Xu Bai, Long-Shiuh Chen, Ning Chen, Brett Ching,
Patricia Contreras, Jose-Luis Diaz, Craig D. Fisher,
Lawrence C. Fritz, Patricia Gladstone, Todd Groessl,
Xin Gu, Julia Herrmann, Brad P. Hirakawa,
Niel C. Hoglen, Kathy G. Jahangiri,
Vincent J. Kalish, Donald S. Karanewsky,
Lalitha Kodandapani, Joseph Krebs, Jeff McQuiston,
Steven P. Meduna, Kip Nalley, Edward D. Robinson,
Robert O. Sayers, Kristen Sebring, Alfred P. Spada,
Robert J. Ternansky, Kevin J. Tomaselli,
Figure 1.
The majority of early discovery programs targeting
caspases centered on the development of reversible
inhibitors of caspase-1.4 This caspase is not directly
involved in apoptosis per se, but is involved in the
processing of the zymogen pro-IL1â to the active cytok-
ine, IL-1â. Numerous studies have been published
targeting apoptotic mechanisms using caspase-3 selec-
tive or pan caspase inhibitors (PCIs),5 and several
reviews concerning the therapeutic utility of regulating
apoptosis via caspase inhibition have been written.6
This letter describes structure-activity relationships,
lead optimization, and selected in vivo data that led to
the identification of IDN-6556 (1). Compound 1 is the
first irreversible PCI to enter clinical development. This
compound is currently in Phase II clinical trials for the
treatment of liver disease.
Our work in developing PCIs has evolved from early
leads taken from tetrapeptide inhibitors, indolyl dipep-
tide (2)7 and oxo-azepino indole aspartyl inhibitors (3),8
to acyloxy dipeptides (4),9 and ultimately to an irrevers-
ible10 oxamyl dipeptide11 inhibitor (1, Figure 1). After
extensive evaluation, our data suggested that potent,
irreversible inhibition of caspases was very successful
in achieving effective anti-apoptotic activity.12 Although
these are irreversible inhibitors, they were shown to be
selective for caspases, demonstrating high micromolar
activity against related cysteine and serine proteases
such as calpain, cathepsin B, chymotrypsin, papain, etc.
(unpublished results). In addition to highly potent pan
caspase inhibitory activity, high potency in a functional
cell-based apoptosis assay was deemed to be critical for
compound progression. We established a variety of such
models that were useful for this purpose. In particular,
a cellular assay utilizing an anti-Fas antibody-stimu-
lated Jurkat E6.1 cell lymphoma cell line was a key part
of our flowchart13 and was useful in discriminating
among potent compounds in vitro from those that
possessed acceptable functional activity. This assay is
quite revealing in that the challenged Jurkat cells
trigger the extrinsic apoptotic pathway, exhibiting all
the standard hallmarks of apoptosis. These caspase
inhibitors prevented apoptosis in the challenged cells
and also maintained their basic cellular morphology.
Making use of the same mechanism, these data can also
be reconciled with in vivo activity observed in a murine
model of R-Fas induced liver damage,14 using ALT levels
(alanine aminotransferase) and histology as endpoints.
This model is particularly relevant in the setting of liver
Brett R. Ullman, Karen L. Valentino,
Suzanne Weeks, David Winn, Joe C. Wu,
Pauline Yeo, and Cheng-zhi Zhang
Idun Pharmaceuticals, 9380 Judicial Drive,
San Diego, California 92121
Received April 5, 2005
Abstract: A series of oxamyl dipeptides were optimized for
pan caspase inhibition, anti-apoptotic cellular activity and in
vivo efficacy. This structure-activity relationship study fo-
cused on the P4 oxamides and warhead moieties. Primarily
on the basis of in vitro data, inhibitors were selected for study
in a murine model of R-Fas-induced liver injury. IDN-6556 (1)
was further profiled in additional in vivo models and phar-
macokinetic studies. This first-in-class caspase inhibitor is now
the subject of two Phase II clinical trials, evaluating its safety
and efficacy for use in liver disease.
Apoptosis, or programmed cell death, is a highly
regulated biological process involved in maintaining
normal tissue homeostasis.1 Dysregulation of apoptosis
can lead either to pathological loss of cells (stroke,
neuronal degeneration, myocardial infarction, liver
disease) or to uncontrolled cell survival and excessive
accumulation of cells (cancer, chronic inflammatory
conditions, autoimmunity).1c,d A family of cysteine pro-
teases known as caspases2 (cysteine aspartate-specific
proteases) are responsible for the disassembly and
phagocytosis of an apoptotic cell.3 The enzymatic cas-
cade leading to apoptosis can be triggered through an
extrinsic (extracellular) pathway, or an intrinsic (intra-
cellular) pathway. The extrinsic pathway is driven by
the cytokines Fas or TNF-R, and subsequent receptor-
mediated activation of caspase-8. The intrinsic pathway
is activated during normal tissue homeostasis and is
governed at the level of the mitochondria. Internal
damage to the cell leads to loss of integrity of the
mitochondrial membrane, leakage of cytochrome C and
depletion of ATP. Cytochrome C is a critical cofactor for
the activation of caspase-9 via the apoptosome complex.
Caspases 8 and 9 are commonly referred to as initiator
or apical caspases, since these proteases activate down-
stream caspases (3, 6, and 7) that degrade structural
proteins and execute the apoptotic program.
* Corresponding author. Tel.: (858) 646-8154, Fax: (858) 625-2677,
E-mail: slinton@idun.com.
10.1021/jm050307e CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/06/2005

6780 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
Scheme 1a
Noting this tolerant P4 oxamide SAR, it was believed
that the warhead would be critical in evaluating the
overall potency and cellular activity of these inhibitors,
as previously shown in a survey of various methyl
ketone warheads on a naphthyloxy dipeptide backbone9c
and a publication involving the use of irreversible
caspase inhibitors in the context of apoptosis.12 A subset
of the P4 groups was selected for an SAR study,
comparing the more active warhead moieties based on
related SAR studies: tetrafluorophenoxy, dichloroben-
zoyloxy, and diphenylphosphoryloxy methyl ketones.
These were shown to be more potent than a standard
fluoromethyl ketone on both the acyl dipeptide back-
bone9c and the oxamyl valinyl backbone.11 Comparing
the activities of these three warhead analogues on four
oxamyl-alanyl backbones (28-35) demonstrated an
overall trend that tetrafluorophenoxy warhead generally
showed better caspase inhibitory activity, and generally
gave more potent results in the JFas cellular assay.
Activity seen in the R-Fas-induced Jurkat cellular
assay prompted in vivo screening of these PCIs in an
R-Fas-mediated liver model.14 Tetrafluorophenoxy and
diphenylphosphoryloxy methyl ketone warhead ana-
logues of selected P4-ala-asp backbones were studied.
This model of acute liver damage is associated with
marked hepatocellular apoptosis, elevated plasma ALT
activities and lethality within 6 h.15 These endpoints
were dose-dependently reduced by pan caspase inhibi-
tors as demonstrated by the ED50s shown in Table 1.
In general, the tetrafluorophenoxy methyl ketones were
consistently more potent in this model than other
classes of inhibitors. Using various routes of adminis-
tration, compound 1 was further evaluated in the R-Fas
model; ip, iv, po, and im. ED50 values of 0.08 (0.06-
0.12) mg/kg, 0.38 (0.11-1.27) mg/kg, 0.31 (0.24-0.42)
mg/kg, and 0.04 (0.02-0.07) mg/kg were observed,
respectively.15
To discriminate between warheads, compounds 1 and
28 were studied in a second and complementary model
of liver injury, a murine D-Gln/LPS model.15 After i.p.
and i.v. administration of 1, ED50 values were 0.17 and
0.09 mg/kg, respectively, as compared to less potent
results with compound 28 (0.97 and 5.03 mg/kg). Oral
administration of compound 1 in two independent
experiments provided an ED50 determined to be less
than 0.01 mg/kg.
Noting the oral efficacy of 1 compared to other routes
of administration, a pharmacokinetic study was con-
ducted in rats.15 After a single bolus administration (iv,
ip, or sc) 1 had terminal half-lives of 51, 47, and 46 min,
respectively. The oral bioavailability of compound 1 (10
mg/kg, fasted rats) was low, ranging from 2.7 to 4%.
This is in contrast to the systemic bioavailability of 49
and 70% achieved following ip and sc administration,
respectively. Portal and systemic concentrations were
compared in an attempt to reconcile the low systemic
oral bioavailability of 1 in the rat with the observed
similarity in efficacy between ip and po administration,
also in the rat.15 After oral administration, AUCinf and
MRTinf of 1 in the portal vein were 5.9-5.3-fold higher,
respectively, than in the systemic compartment, sug-
gesting a marked first-pass effect.
a (a) Methyl chlorooxoacetate (1.1 equiv), triethylamine (TEA)
(1.1 equiv), 0 °C-room temperature (rt) 1 h; (b) 1 N LiOH (1.2
equiv), 1,4-dioxane, rt, 1 h; (c) bis(trimethylsilyl)-trifluoroaceta-
mide (2 equiv), rt 30 min, Z-Ala-OSu ester (1 equiv), DMF, rt, 24
h, 82%; (d) i. isobutyl chloroformate (1.5 equiv), N-methylmorpho-
line (NMM) (1.6 equiv), THF, -10 °C, 20 min; ii. CH2N2/Et2O (2.5
equiv), 0 °C, 15 min; iii. HBr (aq 48%) THF, 0 °C-rt, 30 min, 52%;
(e) 2,3,5,6-tetrafluorophenol (1.2 equiv), KF (4 equiv), DMF, rt,
16 h, used crude; (f) NaBH4 (4 equiv) EtOH, 0 °C, 1 h, 78%; (g) H2
(1 atm), 10% Pd/C, MeOH, rt, 2 h, 70%; (h) oxamic acid, 5 (1 equiv),
HOBt (1.05 equiv), EDCI (1.4 equiv), NMM (1.05 equiv), CH2Cl2;
rt, 16 h; (i) iodobenzene diacetate (3.8 equiv), TEMPO (0.2 equiv),
CH2Cl2; rt, 16 h; (j) TFA/CH2Cl2/anisole (66:33:1), rt, 1 h.
disease, where R-Fas is the major cytokine responsible
for liver damage. Studies on 1 in other animal models
will be discussed, as well as early clinical data.
The synthesis of compounds 1 and 9-23 is outlined
in Scheme 1.11 The oxamic acid (5) was prepared using
methyl chlorooxoacetate and the amine of choice, fol-
lowed by ester hydrolysis. The dipeptide fragment (6)
is prepared by coupling the carbobenzyloxy alanine
hydroxysuccinimide with the â-protected aspartic acid
using bis(trimethylsilyl)-trifluoroacetamide. Conversion
of 6 to its bromomethyl ketone derivative (7), followed
by displacement with 2,3,5,6-tetrafluorophenol provided
the tetrafluorophenoxy methyl ketone. After reduction
of the ketone to the alcohol and hydrogenolysis of the
protected amine, the requisite oxamic acid (5) is then
coupled. Oxidation of the alcohol and subsequent tert-
butyl ester hydrolysis provides the P4 oxamyl dipeptide
analogues in Table 1.
Preparation of the warhead analogues (28-35) is
outlined in Scheme 2. Coupling of the requisite oxamic
acid to the p-tosylate salt of amine (24), followed by
basic hydrolysis, gave the oxamyl dipeptide (25). Three-
step conversion to the bromomethyl ketone employing
diazomethane afforded a key intermediate, compound
26. This bromomethyl ketone could then be reacted with
the nucleophile of choice in the presence of potassium
fluoride, followed by acidic hydrolysis of the aspartyl
tert-butyl ester to yield the inhibitors of the general
structure 27 found in Scheme 2.
The P4 oxamide SAR observed with alanine at P2 is
very similar to that seen with valine,15 in that the P4
oxamide region has a very tolerant SAR with respect
to caspase activity (Table 1). However, when evaluating
both enzyme and cell data together, namely following
potency in the Jurkat/Fas (JFas) assay, 2-substituted
phenyl oxamides in general show the best overall
activity (1, 14, 17, and 19).15 Other examples include
use of basic functionalities, such as the 2-pyridyl oxa-
mide (11) having moderate JFas activity, and the
2-pyrrole phenyl oxamide (18) being among the more
potent analogues tested in this assay.
Compound 1 was studied in the bile duct ligated
(BDL) mouse, a standard model of cholestatic liver

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6781
Table 1. P4 Oxamide and Warhead SAR
enzyme assaysa k3/Ki (M-1 s-1
)
cellular assaysa IC50 (µM)
compd
R
X
mCsp-1b
Csp-3
19000
Csp-6
Csp-8
JFas
THP-1
a-Fas liver, IP, ED50 (mg/kg)
2
Figure 1
Figure 1
cyclohexyl.
phenyl
2-pyridyl
benzyl
2-F Ph
2-Br Ph
2-CF3 Ph
2-pyrrole Ph
2-Bn Ph
_
_
10100
12000000 960000
98000
25000
268
31700
23700
37100
19800
25200
45600
7750 1200000
33600
5900
18500
31900
58700 2940000
52100 2010000
34400
98000
2800
65300
201000
32483
114000
542000
332000
1.3
10
0.3
0.5
0.14
0.2
_
3
0.51
0.15
0.67
1.04
0.3
_
9
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
390000
127000
468000
674000
1140000
1340000 186000
9950000 309000
1520000
1970000
960000
1700000
270000
689000
2158000
21000
25400
44000
35500
70000
_
10
11
12
13
15
16
18
20
21
22
23
1
_
_
0.16
0.16
0.05
0.19
>32
0.54
0.8
0.06
0.12
0.27
9.09
11.8
1.84
5.87
3.13
0.54
1.04
0.32
0.5
_
0.16
0.075
0.061
0.056
0.01
0.53
0.086
0.2
0.025
0.081
0.143
0.062
0.109
1.38
0.064
0.068
1.17
0.13
3.94
0.39
_
_
_
12000
44400
51000
57000
42000
75700
99100
_
617000
154000
460000
204000
_
2-phenoxyy Ph O(2,3,5,6-F4-Ph)
_
1-naphthyl
5-indanyl
2-tBu Ph
2-tBu Ph
2-tBu Ph
2,5 ditBu Ph
2,5 ditBu Ph
2,5 ditBu Ph
2-Cl Ph
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
OPOPh2
OCO(2,6-Cl2-Ph)
O(2,3,5,6-F4-Ph) 16300000
OPOPh2
OCO(2,6-Cl2-Ph)
O(2,3,5,6-F4-Ph) 13400000 219000
OPOPh2
_
_
0.08 (0.06-0.12)d
28
29
17
30
31
14
32
33
19
34
35
0.14
45.8
8330000 236000 105000
4300
365000
970000
68
38200
18300
30000
15400
7640
6400
26000
37100 1410000
44000
5900
0.05 (0.02-0.11)
2710000
4025000
0.5
_
228000
0.05 (0.02-0.16)
2-Cl Ph
3960000 211000
8312000 256000
323000
5000000
7280000
0.04 (0.03-0.05)
2-Cl Ph
OCO(2,6-Cl2-Ph)
O(2,3,5,6-F4-Ph)
OPOPh2
109
154000
_
2-Ph Ph
3400
28400
29000
0.14 (0.08-0.23)
2-Ph Ph
22700 1570000
61000
4.12
4.36
0.44
_
2-Ph Ph
OCO(2,6-Cl2-Ph)
342000
a Assay conditions are described in ref 12. b Murine caspase-1 enzyme. Other enzyme data are from recombinant human. c Assay
conditions are described in ref 15. d Confidence interval.
Scheme 2a
clinically relevant improvements in ALTs and ASTs
were seen in those patients with mild liver impairment.
Compound 1 demonstrated dose-proportional pharma-
cokinetics, having a terminal half-life of 1.7-3.1 h.
There was no evidence of accumulated drug over the
7-day dosing period following multiple i.v. doses. In
general, its pharmacokinetic profile demonstrated it was
quickly cleared from the venous circulation after infu-
sion.
Given the clinical observations stated above, a 2-week
oral dosing ranging study was initiated in a population
of HCV patients refractory to currently approved HCV
treatments (interferon-R and ribavirin). This study24
demonstrated that compound 1 was well-tolerated,
normalized liver enzymes (ALT/AST), and importantly
had no adverse effect on viral load.
In summary, compound 1 has been demonstrated to
be a very potent, irreversible pan caspase inhibitor,
having potent anti-apoptotic activity in the Jurkat-fas
cellular assay. This potency translates to its in vivo
activity in murine models of liver injury and fibrosis.
The oral efficacy of this compound is very intriguing
when one considers that a cursory evaluation would
characterize it as having poor oral pharmacokinetics.
Comparisons of portal and systemic concentrations
suggest a marked first-pass effect, targeting oral ad-
ministration of the drug to the liver. Although reversible
aldehyde and ketone inhibitors were examined early in
this program, none were capable of fully inhibiting
caspases in order to affect the relevant apoptotic path-
way. The selective nature of these pan caspase inhibi-
tors has enabled them not only to be highly potent and
efficacious in both acute and chronic animal models, but
a (a) 5 (1 equiv), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetram-
ethyluronium hexafluorophosphate (HATU) (1.05 equiv), DIEA (3
equiv), N-methylpyrolidinone (NMP)/CH2Cl2 (1:1); rt, 2 h; (b) 1 N
LiOH (1.02 equiv), 1,4-dioxane, rt, 1 h; (c) i. isobutyl chloroformate
(1.1 equiv), NMM (1 equiv), THF, -10 °C, 30 min; ii. CH2N2/Et2O
(2.5 equiv), 0 °C, 15 min; iii. HBr (aq 48%) THF, 0 °C, 15 min; (d)
HX (1.2 equiv), KF (2 equiv), DMF, rt, 16 h; (e) TFA/CH2Cl2/
anisole, (66:33:1), rt, 1 h.
disease.21 The results demonstrated that a pan caspase
inhibitor reduces hepatocyte apoptosis, assessed by the
terminal deoxynucleotidyl transferase dUTP nick-end
labeling assay (TUNEL) and immunofluorescence of
active caspases 3 and 7. A reduction in liver injury was
observed by histopathology and the lowering of serum
alanine aminotransferase levels. Further, it was noted
that 1 reduced mRNA expression for markers of HSC
activation, as well as a reduction of collagen expression
and deposition.
Phase I clinical data evaluating compound 1 showed
the drug to be safe and well tolerated in a clinical
study involving 76 normal adults and patients with
mild liver impairment, including individuals with
stable HCV infection.17 Statistically significant and

6782 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
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more importantly demonstrate safety in a clinical set-
ting. Further successful clinical trials with compound
1 will open the door to the use of caspase inhibitors for
chronic liver disease and prompt research for their use
in other indications.
Acknowledgment. We thank Stuart Dimock, Yueqi
Lu, Jennifer Godwin, Michael Sertic and Asal Shoush-
tari for synthesis of intermediates, and we thank Tom
Hayes, Jay Winship, and David Shapiro for helpful
discussions.
Supporting Information Available: Experimental pro-
cedures for enzyme and cellular screening, murine R-Fas liver
model, and synthesis of compound 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
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