Synthesis and structure-activity relationships of oxamyl dipeptide caspase inhibitors developed for the treatment of liver disease

Article abstract of DOI:10.1016/j.bmcl.2008.10.117

The P4 region of a series of oxamyl dipeptide caspase inhibitors was optimized by the combination of anti-apoptotic activity in the Jurkat/Fas (JFas) cellular assay and membrane permeability in the PAMPA assay. Two highly potent anti-apoptotic agents with moderate membrane permeability, 29 and 36, showed strong in vivo efficacy in a murine model of α-Fas-induced liver injury.

Full text of DOI:10.1016/j.bmcl.2008.10.117

Bioorganic & Medicinal Chemistry Letters 19 (2009) 199–202
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of oxamyl dipeptide caspase
inhibitors developed for the treatment of liver disease
a
a
a
a
a
Hirokazu Ueno a, , Makoto Kawai , Hirohisa Shimokawa , Masako Hirota , Masashi Ohmi , Rie Sudo ,
*
Atsuko Ohta a, Yoshimasa Arano a, Kazunari Hattori a, Takashi Ohmi b, Naoto Kato b, Midori Kojima b,
Yoshinobu Ueno b, Mitsuko Yamamoto b, Yukiko Moriguchi b, Hiroyuki Eda b, , Kazunao Masubuchi
a
*
a Discovery Chemistry Research, Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
b Discovery Biology Research, Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The P4 region of a series of oxamyl dipeptide caspase inhibitors was optimized by the combination of
anti-apoptotic activity in the Jurkat/Fas (JFas) cellular assay and membrane permeability in the PAMPA
assay. Two highly potent anti-apoptotic agents with moderate membrane permeability, 29 and 36,
Received 26 September 2008
Revised 22 October 2008
Accepted 27 October 2008
Available online 5 November 2008
showed strong in vivo efficacy in a murine model of
a
-Fas-induced liver injury.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Caspase
Apoptosis
Liver
Jurkat
a-Fas
Permeability
PAMPA
Oxamyl dipeptide
Apoptosis, or programmed cell death, is a highly regulated bio-
logical process involved in maintaining normal tissue homeosta-
sis.1–4 Caspases are a family of cysteine proteases with strict
substrate specificity requiring aspartic acid as P1 amino acid.5
Caspases can be divided into two groups. One group, represented
by caspase-1, plays an important function in cytokine maturation.6
The other group, including caspase-3, -8, and -9, plays a critical role
in apoptosis by cleaving numerous important proteins.7 Because of
the important function of caspases in both inflammation and apop-
tosis, the discovery and development of caspase inhibitors could
result in novel anti-inflammatory and anti-apoptotic drugs for
the treatment of a variety of diseases.8
maceuticals as irreversible pan-caspase inhibitors for the treat-
ment of liver diseases.12,13 They reported that the 2-substituted
phenyl or halogenated phenyl oxamides such as compounds 1, 2,
and 3 showed potent caspase inhibitory activities and anti-apopto-
tic activity in the Jurkat/Fas (JFas) cellular assay (Fig. 1).14 How-
ever, some of the oxamyl dipeptide derivatives showed only
weak in vivo efficacy in a murine model of
a-Fas-induced liver in-
jury in spite of their potent caspase inhibitory activity and cellular
P4
P3– P2
P1
Warhead
F
Many dipeptide caspase inhibitors have been designed and syn-
thesized based on substrate specificity of caspases.9–11 Among
them, oxamyl dipeptide derivatives were identified by Idun Phar-
OH
F
O
O
O
H
N
R
N
N
O
F
H
H
* Corresponding authors at present addresses. Pfizer Global Research and
Development, Groton Laboratories, Pfizer Inc., MS 8220-3034 Eastern Point Road,
Groton, CT 06340, USA. Tel.: +1 860 686 9495 (H.U.); Pfizer Global Research and
Development, St. Louis Laboratories, Pfizer Inc., 700 Chesterfield Parkway West,
Mail Zone AA3C, Chesterfield, MO 63017, USA. Tel.: +1 636 247 2166; fax: +1 636
247 5300 (H.E.).
O
O
F
1 (IDN-7314): R=2,6-di-F-Ph
2 (IDN-7568): R=2-Cl-Ph
3 (IDN-6734): R=2-CF3-Ph
29: R=2,5-di-Cl-PhCH2
36: R=2,5-di-Me-PhCH2CH2
E-mail addresses: Hirokazu.ueno@pfizer.com (H. Ueno), hiroyuki.eda@pfizer.
com (H. Eda).
Figure 1. Oxamyl dipeptide caspase inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.117

200
H. Ueno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 199–202
activity.15 Therefore, further optimization at the P4 region of the
oxamyl dipeptides has been conducted in order to improve
in vivo efficacy of caspase inhibitors.
Synthesis of the various P4 derivatives of the oxamyl dipeptides
was carried out as outlined in Scheme 1. Commercially available
methyl chlorooxoacetate was converted to the tert-butyl methyl
oxalate 4 by treatment with tert-BuOH in the presence of pyridine.
in caspase inhibitory activity (k3/Ki values: 10,000–1,000,000)
was observed for the compounds exhibiting potent JFas cellular
activities (<20 nM). In the case of these derivatives, the potency
of the cellular activity tended to depend on inhibitory activities
against other caspases in addition to caspase-8. Ultimately the
derivatives with a broad spectrum of caspases inhibition against
caspase-3, caspase-8, and caspase-9 turned out to have potent cel-
lular activity (Table 1). Notably the substituted benzyl derivative
11 and the phenethyl derivative 12 showed potent JFas cellular
activity comparable to 1 (IDN-7314) (see Table 2).
Hydrolysis of the methyl ester
hydroxide followed by coupling with H-Ala-OBzlÁHCl using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
4 with aqueous potassium
fluorophosphate (HATU) gave the compound 5. Removal of the
benzyl group of 5 by catalytic hydrogenation yielded the P2–P3
segment 6. The P1-warhead segment 8 was prepared from the
bromomethylketone 716 by treatment with potassium 2,3,5,6-tet-
rafluorophenolate12 followed by removal of the Boc group. The
carboxylic acid 6 was coupled with compound 8 using HATU
followed by removal of the tert-butyl group affording the key
intermediate 9. Preparation of the P4 derivatives was achieved
by coupling of compound 9 with various amines followed by
removal of the benzyl group.
The in vivo activity of compounds 11 and 12 was evaluated in a
murine model of
a
-Fas-induced liver injury15,17 (single dose oral
administration) and the ED50 values were 0.7 and 0.2 mg/kg,
respectively. Although the cellular activities of the compounds 14
and 16 were weaker than those of 11 and 12, these compounds
were more efficacious than compound 1 by comparing their ED50
values. In order to explain these results, key factors such as cell
permeability that have an effect on the in vivo efficacy have been
investigated. Using a parallel artificial membrane permeability as-
say (PAMPA18), effective permeability (Pe) of 11 and 12 was found
to be low (Pe: <1.7 Â 10À6 cm/s). In contrast, compounds 14 and 16
had moderate permeability (Pe: >3.1 Â 10À6 cm/s). These results
suggested that the in vivo activity of compounds could be pre-
dicted to some extent by both the cellular activity and permeabil-
The relevance of the caspase inhibitory activities and the JFas
cellular activity of the P4 derivatives were initially analyzed. The
results revealed that the cellular activity was correlated with the
caspase-8 inhibitory activity (Fig. 2). However, a large variation
O
O
O
O
a
b, c
H
OMe
OMe
N
OBn
Cl
O
O
O
O
O
4
5
O
O
d
H
N
OH
O
O
6
OBn
O
OBn
F
F
F
e, f
O
HCl
H2N
F
Boc-HN
Br
O
O
O
7
8
OBn
O
F
F
F
g, h
i, j
O
O
H
6
N
O
F
HO
N
H
O
F
O
9
OH
F
O
O
O
H
N
R
F
N
H
O
N
H
F
O
O
10
Scheme 1. Synthetic route of the P4 derivatives of the oxamyl dipeptides. Reagents and conditions: (a) t-BuOH, pyridine, Et2O, rt (70%); (b) KOH, CH3CN, H2O, rt; (c) H-Ala-
OBz HCl, HATU, DIEA, CH2Cl2, DMF, rt (85%, 2 steps); (d) H2, 10% Pd-C, AcOEt, rt (92%); (e) potassium 2,3,5,6-tetrafluorophenolate, NaI, acetone, rt (90%); (f) 4 N HCl-AcOEt, rt
(95%); (g) 8, HATU, DIEA, CH2Cl2, DMF, rt (50%); (h) TFA, CH2Cl2, rt (90%); (i) R-NH2, HATU, DIEA, DMA, rt; (j) H2, 10% Pd-C, THF, 50 °C or HCO2NH4, 10% Pd-C, MeOH, THF, 40 °C.

H. Ueno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 199–202
201
Table 2
1,000,000
100,000
10,000
Results of JFas cellular assay, in vivo assay and PAMPA.
OH
F
F
O
O
O
H
R
N
N
N
O
F
H
H
O
O
F
Compound
R
Enzyme
assay
JFas
In vivo
PAMPA18
cellular assaya,15,17,* Pe
caspase-
8 k3/Ki
assay
IC50
(nM)
ED50 (mg/
kg)
(Â10À6 cm/
s)
(MÀ1 sÀ1
)
3
k
1 (IDN-
7314)
11
12
14
2,6-di-F-Ph
100,000
6.3
0.7
<0.9
4-Cl-PhCH2
PhCH2CH2
Cyclohexylmethyl
(1-Phenylcyclo
-propyl)methyl
34,300
79,900
23,000
50,700
8.4
5.0
19
0.7
0.2
0.1
0.3
<1.7
<1.0
3.1
16
27
3.7
a
Single dose, p.o. administration.
All experimental procedures used in this study were approved by the local
*
ethics committee (ACUP) based on international guidelines (IACUC) and adherence
to the Pfizer policy.
Table 3
Results of JFas cellular assay and PAMPA.
OH
F
F
1,000
F
O
O
O
1
10
100
1,000
H
R
N
JFas cellular activity: IC50 (nM)
N
N
O
F
H
H
O
O
Figure 2. Scatter plot of caspase-8 inhibition and JFas cellular activity.
Compound
R
JFas cellular assay
IC50 (nM)
PAMPA Pe
(Â10À6 cm/s)
1 (IDN-7314)
2,6-di-F-Ph
2-Me-PhCH2
3-Me-PhCH2
4-Me-PhCH2
2-F-PhCH2
3-F-PhCH2
4-F-PhCH2
2,3-di-Me-PhCH2
2,4-di-Me-PhCH2
2,5-di-Me-PhCH2
3,4-di-Me-PhCH2
2,3-di-Cl-PhCH2
2,4-di-Cl-PhCH2
2,5-di-Cl-PhCH2
2,6-di-Cl-PhCH2
3,5-di-Cl-PhCH2
3,5-di-F-PhCH2
2-Me-PhCH2CH2
3-Me-PhCH2CH2
6.3
52
127
50
<0.9
1.3
1.2
1.1
0.3
0.3
0.4
3.0
4.0
4.2
3.7
3.1
2.7
3.2
4.6
2.6
<0.3
1.6
1.7
7.4
4.3
6.2
3.9
3.1
Table 1
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
13
14
Pan-caspase inhibitory activity and JFas cellular activity.
OH
F
F
F
207
106
197
102
>10,000
11
>10,000
76
165
4.3
110
23
16
44
40
O
O
O
H
R
N
N
N
O
F
H
H
O
O
Compound
R
Enzyme assays14 k3/Ki (MÀ1 sÀ1
)
JFas cellular
assay14
Caspase-3 Caspase-8 Caspase-9 IC50 (nM)
1 (IDN-
7314)
11
12
13
2,6-di-F-Ph
37,400
100,000
264,000
6.3
4-Cl-PhCH2
PhCH2CH2
2-Cyclopentylethyl
Cyclohexylmethyl
2-(Dimethylamino)-
3-pyridinyl
12,200
18,600
9470
9870
4880
34,300
79,900
38,100
23,000
60,200
117,000
205,000
122,000
133,000
98,600
8.4
5.0
11
19
27
14
15
2,4-di-Me-PhCH2CH2
2,5-di-Me-PhCH2CH2
3,4-di-Me-PhCH2CH2
2-Cyclopentylethyl
Cyclohexylmethyl
84
7.1
63
11
16
(1-Phenylcyclo-
propyl)methyl
9720
50,700
125,000
27
19
ity. Therefore, further optimization around the benzyl, phenethyl
and cycloalkylmethyl derivatives was carried out to confirm this
hypothesis.
As shown in Table 3, the di-substituted benzyl derivatives 23–
31 and the di-substituted phenethyl derivatives 35–37 showed
moderate permeability (Pe: 2.6–7.4 Â 10À6 cm/s) except for the
di-fluoro derivative 32. In contrast, the permeability of the
mono-substituted benzyl derivatives 17–22 or phenethyl deriva-
tives 33 and 34 was relatively low. In terms of the cellular activity,
the substituent effect appears prominent in the di-substituted ben-
zyl and di-substituted phenethyl derivatives. Namely, the 2,5-di-
substituted benzyl (25 and 29), 3,5-di-substituted benzyl (31 and
32) and 2,5-dimethyl-phenethyl derivative 36 showed significantly
improved cellular activity compared with the other di-substituted
derivatives. Among these derivatives, the compounds 25, 29, and
36 showed potent JFas cellular activity (IC50: < 11 nM) with mod-
erate permeability (Pe: 3.2–4.3 Â 10À6 cm/s). In addition, the 2-
cyclopentylethyl derivative 13 also had a good profile comparable

202
H. Ueno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 199–202
Table 4
Results of JFas cellular assay and PAMPA for representative derivatives.
OH
F
F
F
O
O
O
H
R
N
H
N
N
O
F
H
O
O
Compound
R
Enzyme assays k3/Ki (MÀ1 sÀ1
)
JFas celluar assay
IC50 (nM)
In vivo assay
ED50 (mg/kg)
PAMPA Pe
(Â10À6 cm/s)
Caspase-3
Caspase-8
Caspase-9
1 (IDN-7314)
2,6-di-F-Ph
37,400
26,800
20,600
9470
100,000
190,000
112,000
38,100
264,000
380,000
184,000
122,000
6.3
8.4
5.0
11
0.7
<0.01
0.02
0.01
<0.9
3.2
4.3
3.9
29
36
13
2,5-di-Cl-PhCH2
2,5-di-Me-PhCH2 CH2
2-Cyclopentylethyl
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representative pan-caspase inhibitors 29, 36, and 13 was evaluated
and, as expected, these compounds showed potent in vivo activity
(ED50: <0.01–0.02 mg/kg) in a murine model (Table 4).
In conclusion, the P4 region of the oxamyl dipeptide caspase
inhibitors was optimized by the combination of anti-apoptotic
activity in the JFas cell and membrane permeability. Among the
P4 derivatives, the compounds which showed pan-caspase inhibi-
tory activity turned out to have potent cellular activity. Ultimately
highly potent anti-apoptotic compounds with improved mem-
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pounds showed strong in vivo efficacy in a murine model of
a-
Fas-induced liver injury as predicted. Thus these compounds are
expected to be useful for the treatment of liver disease. We intend
to apply this study results to the SAR study for oxamyl dipeptide
with valine at P2 region as oxamyl dipeptides with valine at P2 re-
gion have already shown potent activities in cellular models of
apoptosis.12
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X.; Herrmann, J.; Hirakawa, B. P.; Hoglen, N. C.; Jahangiri, K. G.; Kalish, V. J.;
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Nalley, K.; Robinson, E. D.; Sayers, R. O.; Sebring, K.; Spada, A. P.; Ternansky, R.
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Acknowledgments
We greatly appreciate Steve Linton and Joe C. Wu for their great
support on the research at Idun Pharmaceuticals. We thank Rich-
ard Butt and Andrew Derrick for providing the intermediates.
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