Tethering identifies fragment that yields potent inhibitors of human caspase-1

Article abstract of DOI:10.1016/j.bmcl.2005.10.048

Disulfide Tethering was applied to the active site of human caspase-1, resulting in the discovery of a novel, tricyclic molecular fragment that selectively binds in S4. This fragment was developed into a class of potent inhibitors of human caspase-1. Seve

Full text of DOI:10.1016/j.bmcl.2005.10.048

Bioorganic & Medicinal Chemistry Letters 16 (2006) 559–562
Tethering identifies fragment that yields potent inhibitors
of human caspase-1q
Bruce T. Fahr,a,* Tom OÕBrien,b Phuongly Pham,a Nathan D. Waal,a
Subramanian Baskaran,a Brian C. Raimundo,a Joni W. Lam,b Michelle M. Sopko,b
Hans E. Purkeya and Michael J. Romanowskic
aDepartment of Chemistry, Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA
bDepartment of Biology, Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA
cDepartment of Structural Biology, Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA
Received 12 September 2005; revised 14 October 2005; accepted 17 October 2005
Available online 4 November 2005
Abstract—Disulfide TetheringÒ was applied to the active site of human caspase-1, resulting in the discovery of a novel, tricyclic
molecular fragment that selectively binds in S4. This fragment was developed into a class of potent inhibitors of human caspase-
1. Several key analogues determined the optimal distance of the tricycle from the catalytic residues, the relative importance of var-
ious features of the tricycle, and the importance of the linker.
Ó 2005 Elsevier Ltd. All rights reserved.
The caspases (cysteinyl aspartate-specific proteases) are
a family of endopeptidases that share a preference for
an aspartic acid in the substrate P1 position.2,3 Cas-
pase-1 (interleukin-1b converting enzyme, ICE) carries
out cytokine processing that results in inflammatory
responses, and this processing is believed to play a key
biological role for the enzyme.4,5 There are two known
endogenous substrates for caspase-1: pro-interleukin-
1b (pro-IL-1b) and pro-interleukin-18 (pro-IL-18). IL-
1b is a proinflammatory cytokine that is released by
peripheral blood mononuclear cells in response to
inflammatory stimuli. This cellular release of IL-1b re-
quires processing of pro-IL-1b by caspase-1.6 Inhibitors
of caspase-1 might therefore attenuate the inflammatory
processes that occur in various diseases.
O
O
N
N
Fragment identified
by Tethering
N
H
N
H
O
H
N
N
N
N
H
H
O
N
H
CO2H
1c Casp-1 Ki=0.19 µM
Figure 1. Initial hit derived from Tethering.
itor, we herein report some of our efforts to develop a
more advanced lead compound.
Our initial hit 1c was assembled by linking the tricyclic
fragment to a methylene scaffold bearing an aspartyl
Tethering identifies small molecular fragments that bind
to a specific site on a protein surface and can form disul-
fide bonds with a cysteine residue near that site.7,8 Using
Tethering, we discovered a tricyclic molecular fragment
that binds in the S4 region of caspase-1 (Fig. 1).9 Having
quickly converted this fragment into a promising inhib-
aldehyde, a well-known P1 moiety for caspase
inhibitors.10
The length of the tether used in the disulfide formation
led us to expect that the optimal length of our inhibitors
would require a 6-aminohexanoic acid linker between
the tricyclic fragment and the aspartyl aldehyde.9 This
length corresponds to a direct atom-for-atom replace-
ment of the Tethering disulfide linkage with a methylene
chain. Indeed, a study of unsubstituted, flexible linkers
Keywords: Caspase; Interleukin-1b; Tethering; ICE.
q
See Ref. 1.
*
Corresponding author. Tel.: +1 650 266 3627; fax: +1 650 266
3501; e-mail: bfahr@sunesis.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.048

560
B. T. Fahr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 559–562
Table 1. P2 groups and length dependence
Table 2. Variation of linker
Compound
Casp-1 Ki (lM)
O
R
O
H
N
N
N
6
0.12
0.10
2.10
1.95
0.035
0.005
0.26
0.12
1.0
N
H
n
H
O
7
N
CO2H
8
H
9
10
Compound
n
R
Ki (lM)
11
12
1a
1b
1c
1d
2
1
2
3
4
3
3
3
3
H
H
H
H
5.2
2.8
13
14
0.190
2.6
15
Z-VAD-CHO
0.004
0.008
2-Thienyl
Ethyl
Phenyl
0.005
0.016
0.005
0.008
3
4
5
2-Fluorophenyl
most potent non-peptide. Pyridyl and benzenoid linkers,
similar to those in 7 and 11, had previously been
incorporated into a series of reversible caspase inhibitors
by Cytovia11 and into a series of irreversible inhibitors
by Vertex.12 The thiophene-linked compound 10 was
9-fold less potent at 35 nM.
(compounds 1a–d, Table 1) demonstrated that the Ki
of the inhibitors was strongly dependent on length,
with 6-aminohexanoic acid giving the most potent inhib-
itor (1c).
We had previously found that caspase-3 inhibitors bear-
ing a hydrophobic P2 substituent often displayed good
potency against caspase-1.10 We therefore attached our
P4 tricycle to a set of flexible linkers with various hydro-
phobic P2 groups (Table 1). As anticipated, the presence
of a P2 group greatly improved potency. Although
several compounds displayed low nanomolar potency,
concerns about the number of rotatable bonds led us
to examine a series of more constrained linkers (Chart
1, Table 2). The synthesis of these linkers has been
reported elsewhere,8 except for the peptidyl analogue
15, which was assembled using standard solid-phase
peptide techniques. Several linkers produced high-
affinity inhibitors, with the pyridyl-linked 11 being the
We next turned our attention to the tricycle in order to
establish which elements are important for inhibition
and to improve the physical properties of the inhibitors
(Chart 2, Table 3) For this study, we chose two linkers,
6-aminohexanoic acid and the thiophene-substituted
linker in compound 2. Truncating the quinoxaline
system (16a,b) reduced the potency, which could be
partially restored by placing a chlorine at the pyrazine
6-position (17b). Replacing the bridging NH with an
oxygen moderately reduced the potency (18a,b). Methyl
groups are tolerated at several positions with only mod-
est changes in potency (19a,b and 20b). The reverse
amide (21a) analogue of our initial lead (1a) had an
approximately 10-fold reduced potency.
The syntheses of the P4 groups are summarized below
(Scheme 2). With the exception of the reverse amide
21a, all P4 analogues were synthesized as carboxylic
acids and then elaborated into the full inhibitors using
the resin chemistry shown in Scheme 1. Similar resin
chemistry to prepare aldehydes has been previously
N
Ar
=
N
N
H
O
O
O
H
N
N
S
Ar
N
O
H
Ar
N
H
H
N
H
O
H
CO2H
6
O
R1
O
11
CO2H
H
O
N
R2
H
H
Ar
N
S
O
Ar
N
H
O
H
N
H
N
O
CO2H
O
H
H
O
O
R2=
O
O
O
O
7
N
N
N
N
12
13
CO2H
CO2H
N
H
N
H
S
O
O
N
O
Cl
N
H
H
N
H
N
H
N
H
Ar
Ar
17b: R1=2-thienyl
H
O
H
16a: R1=H
16b: R1=2-thienyl
Ar
N
H
O
O
CO2H
O
CO2H
8
O
O
N
N
N
H
N
H
N
N
Ar
Ar
N
O
N
H
O
H
N
H
N
N
H
N
N
O
H
H
Me
H
18a: R1=H
18b: R1
19a: R1=H
19b: R1=2-thienyl
9
O
O
O
O
14
CO2H
=2-thienyl
CO2H
O
O
H
O
O
N
N
Me
N
H
N
H
N
H
N
H
N
N
Ar
H
N
H
H
S
H
O
N
N
N
N
O
O
H
H
CO2H
10
CO2H
20b: R1=2-thienyl
15
21a: R1=H
Chart 1. Rigid linkers.
Chart 2. Variation of P4 group.

B. T. Fahr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 559–562
561
CO2t-Bu
CO2t-Bu
6-methyl quinoxaline 28 was prepared from the chloro-
quinoxaline 27 by treating 27 with 4-aminobenzoic acid
in acidic methanol to give the methyl ester of 28, which
was then hydrolyzed to provide the acid 28. The synthe-
sis of the reverse amide 21a was accomplished by first
forming the tricyclic amine, followed by reaction with
adipic anhydride, then coupling of the resulting carbox-
ylic acid onto the resin-supported aldehyde.
O
FmocHN
H
H
N
A
N
FmocHN
HN
HN
H
N
O
HN
HN
a, b
c, d,
e, f
O
CO2H
O
Aldehyde Resin
H
R
N
H
A
N
H
Unfortunately, none of the P4 analogues in Table 3
showed potency greater than that of the original tricycle.
Nevertheless, the apparent specificity of the original
tricycle for the S4 site demonstrates the power of
Tethering to discover molecular fragments that form a
tight and selective fit with a protein surface.
O
O
Scheme 1. Inhibitor syntheses. Reagents: (a) 20% piperidine/DMF; (b)
FMOC-NH-A-CO2H; (c) 20% piperidine/DMF; (d) RCO2H, PyBoP,
DIEA; (e) TFA/AcOH/TFA, MeCHO; (f) CH2Cl2, TFA.
N
N
CO2H
N
N
CO2H
a
+
Some of our more potent analogues showed low micro-
molar cell activity in a peripheral blood mononuclear
cell (PBMC) assay measuring the release of IL-1b (Table
3).15 Compound 1c, having a completely unsubstituted
and flexible linker, in fact displayed the best cell activity
in the series at 4.2 lM. This activity was approximately
equal to that of the peptide aldehyde Z-YVAD-CHO we
used as a standard. As a control, for compounds tested
in the cell assay we also measured IL-6 production and
found in all cases that it was unaffected at inhibitor con-
centrations up to 200 lM.
N
Cl
Cl
H2N
H
22
O
N
CO2H
N
N
b,c
Ot-Bu
+
Cl
N
H
Cl
N
Cl
H2N
H
23
24
N
CO2Me
CO2H
N
CO2H
CO2H
d,e
+
N
Cl HO
N
O
25
N
N
N
N
Crystal structures of 11 and 18b bound to caspase-1
showed the tricycle in two different binding modes
(Fig. 2). The tricycle of compound 11 sits in a lower
groove in the S4 pocket, whereas the tricycle in
compound 18b sits in an upper groove. The fact that
compound 11 was more potent suggests that the lower
binding mode is more energetically preferred. This
hypothesis is further supported by crystal structures
we recently disclosed in another publication, in which
the highest potency analogues all displayed the lower
binding mode.9
f
+
Cl H2N
N
H
Me
Me
26
Me
NH2
Me
N
Me
N
g
h
NH2
N
H
O
N
Cl
27
CO2H
Me
N
CO2H
i, j
+
27
H2N
N
N
H
28
Space-filling models show a remarkably tight filling of
the space within S4. The guanidine of Arg383 sits over
the phenyl ring of the tricycle, possibly forming a
p-charge interaction. Compound 11 forms a hydrogen
bond between the pyridyl nitrogen and the backbone
nitrogen of Arg341. This hydrogen bond presumably
contributes to the high potency of compound 11.
H
NHBoc
N
N
CO2H
n=3
k, l, m
O
H2N
N
N
H
resin chemistry
21a
Scheme 2. Syntheses of P4 groups. Reagents and conditions: (a) HCl,
H2O, 100 °C, 1.5 h, 8%; (b) NaH, DMSO, rt, 9 days, 22%; (c) HCl,
dioxane, rt, 44%; (d) NaH, DMSO, 80 °C, 16 h, 85%; (e) LiOH, H2O,
dioxane, 93%; (f) HCl, H2O, 100 °C, 1 h, 56%; (g) glyoxylic acid(aq),
MeOH, rt, 42%; (h) POCl3, PCl5, 110 °C, 2.5 h,89%; (i) HCl, MeOH,
90 °C, 46%; (j) LiOH, H2O, dioxane, 60%; (k) 17, EtOH, D; (l) TFA;
(m) adipic anhydride.
Table 3. Variation of P4 group
Compound
R1
Casp-1 Ki (lM) IL-1b EC50 (lM)
1c
H
0.19
0.007
4.2
20.5
2
16a
2-Thienyl
H
37
>200
16b
17b
2-Thienyl
2-Thienyl
H
0.10
0.054
2.4
20.0
n.d.
27.0
12.4
7.7
reported by groups at Corvas,13 Idun,14 and Sunesis.8
The pyrazine 22 was prepared by condensing 4-amino-
benzoic acid with chloropyrazine. The 6-chloro ana-
logue thereof (24) was prepared as the hydrochloride
salt, using an SNAr reaction of 23 with 2,6-dichloropyr-
azine, followed by cleavage of the tert-butyl ester. The
oxygen-bridged tricyclic analogue 25 was prepared
by reacting methyl 4-hydroxybenzoate with 2-chloro-
quinoxaline, followed by hydrolysis of the ester. The
18a
18b
2-Thienyl
H
0.043
0.32
0.004
0.005
2.1
19a
19b
2-Thienyl
2-Thienyl
H
7.7
20b
21a
n.d.
n.d.
3.9
Z-YVAD-CHO
—
0.18
n.d., not determined.

562
B. T. Fahr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 559–562
Figure 2. (a) Crystal structure of compound 11 (PDB ID code: 1rww). In yellow are hydrogen bonds. (b) Crystal structure of 18b (PDB ID code:
˚
1rwx). A 2Fo À Fc electron density map within a 3.5-A distance of each inhibitor, contoured at the 1r level, is shown as a blue mesh.
6. Thornberry, N. A.; Bull, H. G.; Calaycay, J. R.; Chap-
man, K. T.; Howard, A. D.; Kostura, M. J.; Miller, D. K.;
Molineaux, S. M.; Weidner, J. R.; Aunins, J.; Elliston, K.
O.; Ayala, J. M.; Casano, F. J.; Chin, J.; Ding, G. J.-F.;
Egger, L. A.; Gaffney, E. P.; Limjuco, G.; Palyha, O. C.;
Raju, S. M.; Rolando, A. M.; Salley, J. P.; Yamin, T.-T.;
Lee, T. D.; Shively, J. E.; Maccross, M.; Mumford, R. A.;
Schmidt, J. A.; Tocci, M. J. Nature 1992, 356, 768.
7. Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal,
M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 9367.
In conclusion, we have used a novel tricyclic molecular
fragment, provided by Tethering, to develop a series of
potent caspase-1 inhibitors. Analogue studies of the tri-
cycle demonstrated the importance of its structural fea-
tures and multiple co-crystal structures showed the
importance of the linker structure.
Acknowledgments
8. Choong, I. C.; Lew, W.; Lee, D.; Pham, P.; Burdett, M. T.;
Lam, J. W.; Wiesmann, C.; Luong, T. N.; Fahr, B.; DeLano,
W. L.; McDowell, R. S.; Allen, D. A.; Erlanson, D. A.;
Gordon, E. M.; OÕBrien, T. J. Med. Chem. 2002, 45, 5005.
9. OÕBrien, T.; Fahr, B. T.; Sopko, M. M.; Lam, J. W.; Waal,
N. D.; Raimundo, B. C.; Purkey, H. E.; Pham, P.;
Romanowski, M. J. Acta Crystallogr. F 2005, 61, 451.
10. Allen, D. A.; Pham, P.; Choong, I. C.; Fahr, B.; Burdett,
M. T.; Lew, W.; DeLano, W. L.; Gordon, E. M.; Lam, J.
W.; OÕBrien, T.; Lee, D. Bioorg. Med. Chem. Lett. 2003,
13, 3651.
The authors thank Dan Erlanson for preparing the
covalent alkylator used in the Tethering study. We
acknowledge Ingrid Choong, Darin Allen, and Matt
Burdett for preparing some of the linkers, and Robert
McDowell for proofreading the manuscript. We further
acknowledge the helpful scientific discussions and
encouragement of Andrew Braisted.
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