Succinic acid amides as P2-P3 replacements for inhibitors of interleukin-1β converting enzyme (ICE or caspase 1)

Article abstract of DOI:10.1016/j.bmcl.2010.07.004

Succinic acid amides have been found to be effective P2-P3 scaffold replacements for peptidic ICE inhibitors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affinities. Utilization of an acylal prodrug moiety was

Full text of DOI:10.1016/j.bmcl.2010.07.004

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5184–5190
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Succinic acid amides as P2–P3 replacements for inhibitors
of interleukin-1b converting enzyme (ICE or caspase 1)
a
a
a
a
a
Paul Galatsis a, , Bradley Caprathe , John Gilmore , Anthony Thomas , Kristin Linn , Susan Sheehan ,
William Harter a, Catherine Kostlan a, Elizabeth Lunney a, Charles Stankovic a, John Rubin a, Kenneth Brady b,
Hamish Allen b, Robert Talanian b
*
a Pfizer Global R&D, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
b BASF Bioresearch Corporation, 100 Research Drive, Worcester, MA 01605, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Succinic acid amides have been found to be effective P2–P3 scaffold replacements for peptidic ICE inhib-
itors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affin-
ities. Utilization of an acylal prodrug moiety was required to overcome biopharmaceutical issues which
led to the identification of 17f, a potential clinical candidate.
Received 26 March 2010
Revised 1 July 2010
Accepted 2 July 2010
Available online 8 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
ICE inhibitor
P2–P3 replacement
Succinic acid amides
H2N
Cl
O
O
Interleukin IL-1b (IL-1b) is a pro-inflammatory cytokine that
has been implicated in a number of pathophysiological states.1
Thus inhibitors of ICE, the key enzyme that converts the inactive
pro-IL-1b to the biologically active form, would be of pharmacolog-
ical value.2 Structurally, ICE inhibitors can be dissected into three
components; a core fragment, a P4–P2 peptidomimetic head-frag-
ment, and a prime side tail-fragment. We have recently reported3
our efforts at delineating optimal binding fragments for the prime
side. ICE has a key requirement for Asp as the core fragment4 as re-
vealed by SAR data buttressed by X-ray crystallography.5 However,
most SAR campaigns have truncated the prime side at the Asp car-
bonyl employing an acylal as a ‘prodrug’ (Fig. 1) focusing synthetic
efforts at optimizing the P2–P4 segment.
O
N
N
O
H
N
O
O
N
O
O
N
H
O
N
H
OEt
O
O
N
H
N
OR
2a R=H (VRT-18858)
2b R=Et (VX-740)
3 (VX-765)
This peptide inhibitor has been the epicenter of numerous optimi-
zation studies for more drug-like compounds. VX-740 (pralnacasan)
2b,7 prodrug of 2a, and VX-765 38 are two examples of ICE inhibi-
tors containing P2–P3 replacements that have embodied sufficient
biopharmaceutical properties to merit clinical investigation. Both
compounds leverage the requirement for aspartic acid (ethyl acylal
prodrug form) at P1 and the pyridazinodiazepine and proline cores,
respectively, of 2 and 3, to effectively mimic the b-sheet H-bonding
Tetrapeptide Ac-YVAD-CHO (1, Fig. 2)5a,6 is a potent competitive
ICE inhibitor. The P1–P4 residues of 1 represent the natural binding
amino acids exhibiting the obligatory P1 aspartic acid. This
requirement for Asp at P1 is a consequence of its interaction with
the oxy-anion hole formed by Arg341, Arg179, and Gln283. The
remaining residues interact with the S2, S3, and S4 pockets utiliz-
ing the alanine methyl, valine isopropyl, and tyrosine phenol moi-
eties, respectively.
O
O
CO2H
P4 P2
O
O
P4 P2
P4 P2
H
O
N
H
N
H
N
H
OH
OR
core (Asp)
acylal
acylal prodrug
* Corresponding author at present address: Pfizer Global R&D, MS 8220-4225,
Eastern Point Road, Groton, CT 06340, USA. Tel.: +1 860 686 9379.
E-mail address: paul.galatsis@pfizer.com (P. Galatsis).
Figure 1. General structural features of an ICE inhibitor.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.004

P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
5185
We now report the identification of succinic acid amides 4 as
suitable replacements for the P2–P3 fragment. Figure 3 provides
a simplistic view toward the origins for these compounds. Upon vi-
sual comparison of the P2–P3 residues of 1 with the succinic acid
moiety of 4, our proposed scaffold replacement, the analogy be-
comes apparent. The P2 methyl group in 1 is now the isopropyl
moiety of 4.9 Moreover, the P3 residue of 1 is directly related to
the distal amide nitrogen, relative to the P1 aspartic acid, providing
a synthetically amenable method for probing this chemical space.
Finally, the R-group of 4 aligns with the P4 residue found in 1.
Thus, in a compact and more easily accessible synthetic frame-
work, one can design and optimize ICE inhibitors with greater po-
tential for clinically viability.
The desired target compounds 910 could be obtained using a
synthetic route starting with the known oxazolidinone 5 (Scheme
1).11 Treating the enolate of 511 with benzyl bromoacetate fol-
lowed by removal of the chiral auxiliary afforded 6. Diimide cou-
pling of b-t-butyl ester alcohol of aspartic acid produced the
corresponding amide alcohol 7. Hydrogenolysis of the benzyl ester
moiety followed by amide coupling installed the P4 substituent
present in 8. The synthetic protocol was completed by IBX oxida-
tion of the alcohol to the corresponding aldehyde and deprotection
of the t-Bu ester to generate the required P4 substituted carboxy
aldehydes 9.
Figure 2. Binding pose of Ac-YVAD-CHO 1 with ICE.
HO
P2
P2
P4
P1
P1
O
H
N
O
H
N
O
O
H
N
O
R
N
H
N
H
N
H
P3
O
O
O
P4
CO2H
CO2H
P3
With compound 9 in hand, their affinity for ICE was evaluated.
Table 1 presents the results of the incorporation of substituted
phenpropyl fragments at the P4 position. Overlays, as exemplified
in Figure 3, indicated this was the best arrangement to access the
region of P4 occupied by the phenoxy fragment of tyrosine. Data
for 1 and 2a have been included to serve as a benchmark for the
novel compounds now being disclosed. Compound 9a clearly indi-
cated that potent ICE inhibitors could be obtained with our succi-
nic acid amide P2–P3 scaffold replacement linked to a minimally
elaborated P4 moiety. With this reference point, one could now
systematically probe the SAR space for this template. Compound
9b showed that substitution on the phenyl ring could be tolerated.
While the oxo modification to the linker, present in 9c, was toler-
ated, a sevenfold increase in potency was observed with an oxa-
modification found in 9d. The overlays (Fig. 3) also indicated that
one could reach out to the S3 pocket by substituting off the distal
amide (relative to 1) nitrogen. The natural residue for P3 is valine,
thus 9e introduces an isopropyl group to this position which re-
sulted in a sixfold loss in potency. However, it had been deter-
mined that the most potent arrangement of amino acid residues
for the P1–P4 segment was WEHD and not the natural YVAD.4
Compound 9f introduces a propionic acid moiety at this position
to mimic the glutamate found in this substrate. About a twofold in-
crease in potency relative to 9d was observed. Thus, we concluded
that substitution at this position had minimal effects on potency
but at the cost of physical–chemical properties.
1
4
Figure 3. Top panel shows a comparison of P2–P4 residues. Bottom panel shows an
overlay of 1 (gray) with 4 (green) truncated at P4.
O
O
O
a, b, c
d
OH
O
N
BnO
O
5
6
Ph
O
H
N
O
H
N
e, f
R
BnO
OH
N
OH
Compound 9g attempted to restrict the bond rotation of the lin-
ker and was found to be tolerated. Relative to 9d, compound 9h
was 18-fold more potent. This compound took advantage of the
knowledge that phenyl substitution was tolerated but now intro-
duced a nitrogen atom in the linker in exchange for the oxygen
atom. Interestingly, rigidifying the linker, as exemplified by 9i, re-
sulted in a dramatic loss in potency. Presumably, the linear confor-
mation produced in this example indicated this was not the
bioactive conformation. This observation was reinforced with 9j.
The compound maintains the core fragment found in 9a but puts
a kink in the linker with the introduction of the fused phenyl ring.
It was observed from the crystal structure of 1 (Fig. 1) that the
tyrosine at P4 must bend back toward the protein and 9j repro-
duces this conformational requirement, such that, it exhibited a
200-fold improvement in potency, relative to 9a.
H
O
O
7
CO2tBu
CO2tBu
8
O
H
N
O
g, h
R
N
H
O
CO2H
9
Scheme 1. Reagents: (a) NaHMDS; (b) BrCH2CO2Bn; (c) LiOH, H2O2; (d)
H2NCH(CO2tBu)CH2OH, EDCI, HOBt, NMM; (e) H2, 20% Pd/C; (f) ArCH2CH2NH2,
EDCI, HOBt, NMM; (g) IBX; (h) TFA.
interactions observed from X-ray crystal structure data of
1
(Fig. 2),5a while removing several of the metabolically labile amide
bonds.

5186
P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
Table 1
Table 2
ICE enzyme inhibition dataa
ICE enzyme inhibition dataa
Entry Structure
Ki (nM) IC50 (nM)
Entry
Structure
Ki (nM)
IC50 (nM)
23.1
HO
O
H
N
O
9k
5.02
N
H
N
H
O
H
N
O
H
N
O
1
0.70
0.60
17
O
CO2H
N
H
N
H
O
O
F
CO2H
O
O
O
H
N
O
9l
7.9
37
N
N
N
H
CO2H
N
H
O
N
2a
3.4
O
O
N
CO2H
O
O
O
N
H
H
O
O
H
N
O
H
N
9m
9n
9o
1.29
0.90
4.03
18
N
H
N
H
N
H
9a
9b
9c
9d
9e
115
70.9
118
16.9
100
444
410
398
539
584
Me
O
CO2H
O
CO2H
O
H
N
O
O
H
N
N
H
42.5
45.3
N
N
H
O
Me
MeO
CO2H
O
Me
CO2H
O
O
O
O
O
H
N
O
N
H
O
H
N
O
O
O
N
CO2H
O
N
H
H
N
O
CO2H
O
O
O
N
H
a
For a description of the assay see Ref. 12. Values are the geometric mean of two
or more experiments.
CO2H
O
H
N
benzimidazoles, and quinolines. The modulation of affinity, one
could explore with these scaffolds, could be conducted in a bifur-
cated manner. First, the presumed bioactive conformation would
be maintained in a gross manner, but these systems introduce sub-
tle variations that could be exploited. Secondly, the introduction of
heteroatoms provides the ability to control physical–chemical
properties that may lead to improved ADME profiles.
Table 2 presents the results obtained from incorporation of
substituted indole fragments. Compound 9k represents the indole
equivalent to the naphthalene found in 9j and was found to be
about ninefold less potent. As was observed previously, substitu-
tion of the phenyl moiety was tolerated as exemplified by the 5-
fluoro in 9l. The 2-methyl substituent of 9m afforded a fourfold in-
crease in potency. An additional 1.4-fold potency increase was
found with 9n by the addition of a second methyl group to the
1-position of the indole ring. Switching the point of attachment
of the fused bicyclic heterocycle, as presented in 9o, was tolerated
and did not have a dramatic effect.
The benzimidazole SAR is summarized in Table 3. Compound 9p
is the benzimidazole equivalent to 9j or 9k and was found to be
about fourfold less potent than 9k. As was observed in the previous
series, substitution of the phenyl or imidazole ring was tolerated.
Indeed, compounds 9q–9u indicated that substitution of the phe-
nyl or imidazole ring could recover this activity and 9t indicated
that fairly large groups could be accommodated at the imidazole
2-position without a dramatic loss in potency. While, in general,
the SAR for these substitutions was fairly flat as most groups re-
sulted in equipotent compounds, substitution on both rings, as
shown in 9u, provided the optimal substitution pattern and re-
sulted in a compound of comparable potency to 9j and 9m.
The subtle nuances seen in this SAR provided the desire for a
greater understanding of the binding interactions for this series
of compounds. This greater insight was partially achieved when
N
N
O
CO2H
O
H
N
9f
9.24
21.5
0.95
114
170
11.2
O
O
CO2H
O
CO2H
O
H
N
O
N
9g
9h
CO2H
O
H
N
O
H
N
N
H
O
CO2H
Cl
O
H
N
O
N
9i
9j
1020
0.58
7810
5.7
N
O
CO2H
O
H
N
O
N
H
O
CO2H
a
For a description of the assay see Ref. 12. Values are the geometric mean of two
or more experiments.
This last observation was the genesis of implementing the use
of fused-bicyclic heteroaromatic ring systems for the P4 substitu-
ent. To this end, three related frameworks were examined, indoles,

P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
5187
Table 3
Table 4
ICE enzyme inhibition dataa
ICE enzyme inhibition dataa
Entry
Structure
Ki (nM)
IC50 (nM)
33.7
Entry
Structure
Ki (nM)
IC50 (nM)
O
H
N
O
N
O
H
N
O
9p
N
N
20
9v
1.31
4.77
3.61
5.81
N
H
N
H
O
CO2H
O
O
CO2H
O
O
H
N
N
N
O
H
N
O
9q
9r
4.85
2.81
15.3
14.5
N
N
9w
9x
3
N
H
N
H
O
O
CO2H
CO2H
N
O
H
N
O
H
N
O
1.81
N
N
N
H
N
H
O
O
CO2H
CO2H
N
O
O
H
O
9s
9t
N
N
N
4.68
3.85
23.8
26.5
N
N
H
9y
9z
16.7
102
O
O
H
N
O
CO2H
N
H
O
H
N
O
O
CO2H
N
N
N
H
O
CO2H
O
H
N
O
N
4.3
12
N
N
H
O
CO2H
O
H
N
O
9u
1.26
9.18
a
For a description of the assay see Ref. 12. Values are the geometric mean of two
N
N
N
H
or more experiments.
O
CO2H
a
For a description of the assay see Ref. 12. Values are the geometric mean of two
or more experiments.
Table 5
Comparison of in vitro and in vivo data
Entry
muKi (nM)
muKi/huKi
PBMC (IC50
l
M)
huSCID (%)
1
9.0
7.0
13.4
18.7
14.4
12.8
11.7
10.3
3.98
8.0
1.2
69
62
54
68
52
2a
9m
9s
9x
0.38
1.50
2.90
0.65
hole, as previously observed. The isopropyl substituent of the suc-
cinic acid amide P2–P3 scaffold replacement was found interacting
with the S2 pocket, defined by Trp340 and Val338. While it appears
the second methyl group of the isopropyl side chain was not mak-
ing any apparent contacts, the ethyl variation of the succinic acid
amide was found to be a less effective ICE inhibitor (data not
shown).9 As predicted, the succinic acid amide scaffold does not
provide for any S3 interactions, and from this pose one would ex-
pect the amide nitrogen substituents to be pointing out into sol-
vent. This is most likely the case for the N-iPr substituent of 9e,
however, for 9f, which incorporated an N-propionic acid substitu-
ent, the extended nature of this fragment presumably allowed it to
make minimal contacts in S3. Finally, the benzimidazole moiety at
P4 appears to be interacting with the Arg383 and confirms the
kinked bioactive conformation, in that the interaction with S4 ap-
pears to be with the phenyl fragment.
Figure 4. Docking pose for X-ray crystal structure of 9s (3NS7).
The last P4 scaffold investigated was based on a quinoline group
and is presented in Table 4. In this series, the SAR appears to be
more tolerant of substitution and of comparable potency to the
most potent indoles, 9n, and benzimidazoles, 9u. Compounds 9v,
an X-ray crystal structure of 9s (3NS7) bound in the active site of
ICE was determined (Fig. 4). As can be discerned from the binding
pose, the Asp residue was found to be binding in the oxy-anion

5188
P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
O
a 4-quinolinyl-analog, and 9x, a 5-quinolinyl-analog, are equipo-
tent. An obvious exception was 9y which exhibited a 12.7-fold loss
in potency by the incorporation of a dimethylamino-ethoxy substi-
tuent at the 2-position of the quinoline ring.
O
O
OH
OH
H2O
H2O
OH
O
R
R
R
N
H
N
H
N
H
OH
OH
O
Having identified potent compounds in each P4 variant, one
compound from each series was selected for additional profiling.
Table 5 presents the differences inherent in these compounds.
The first point of differentiation was related to species specificity
and the ability to use in vivo models. Since the compounds were
optimized for human potency, the differences between the human
and mouse isoforms of ICE could be determined and were found to
be significant (4- to 10-fold weaker) which resulted in the neces-
sity of using the in vivo huSCID model.13 The second point of differ-
entiation was related to a dramatic loss of potency observed for
these compounds in going from the enzyme assay to cell-based as-
say formats (e.g., PBMC14), which was not unique to our com-
pounds. Presumably, ionization of the carboxylic acid reduces the
permeability giving rise to the observed potencies. Despite this dis-
connect, significant IL-1b reduction was observed in the in vivo
huSCID model.13 In addition to the species selectivity that was ob-
served, we also determined that these compounds showed selec-
tivity across several members of the caspase enzyme family.
Along with caspase 1, caspase 3, 4, 6, and 8 activity was deter-
mined. While 2a showed a minimum of 53-fold selectivity over
these enzymes, our compounds exhibited greater than 200-fold
selectivity for these caspases, thereby providing a wider therapeu-
tic index against these targets.
In following up with PK studies, it was observed that these com-
pounds did not all behave in a similar manner. Figure 5 clearly
shows that 9x has comparable exposure to 2a and better exposure
than 9s, which essentially had no oral bioavailability. In the cases
of 2b and 3, there were clear benefits, from a bioavailability per-
spective, for advancing the acylal prodrug version of the inhibitor.
Based on this and other data, a systematic exploration of prodrugs
for these compounds was pursued.
We began our prodrug approach by initiating studies focused on
gaining an understanding into the various forms of the acylal pro-
drugs. A simple 1H NMR experiment was conducted to chemically
assess the potential species that this moiety could participate
(Fig. 6). Taking Asp-CHO 10 in water or methanol, the chemical shift
11
12
10
~5.2-5.5 ppm
~4.5 ppm
MeOH
O
O
O
OH
OMe
OH
OMe
H+
MeOH
O
R
R
R
N
N
H
N
H
H
OMe
OMe
OH
13
14
15
~5.2-5.5 ppm
~4.5 ppm
~4.5 ppm
Figure 6. Summary of 1H NMR experiments looking to decipher the various
prodrug acylal species that could exist in solution.
O
H
N
OR
O
O
a, b, c
OH
BnO
BnO
g, h
O
O
16
6
O
O
H
N
OR
O
Ar
N
H
O
17
O
Scheme 2. Reagents: (a) H2NCH(CO2tBu)CH2OH, EDCI, HOBt, NMM; (b) IBX; (c)
TFA, ROH with chromatography to separate diastereomers; (d) H2, 20% Pd/C; (f)
ArCH2CH2NH2, EDCI, HOBt, NMM.
for the acetal methane hydrogen was determined and correlated
with structural substitution features. From this analysis, the pre-
dominant isomer in methanol-d4 was the acyclic hemi-acetal 13
and not 11 nor was it methyl acylal 14. What was observed was a
mix of diastereomers with two acetal methine doublets at
ꢀ4.5 ppm. Basedonthechemicalshifts of cyclicacylal intermediates
Figure 5. Bioavailability for selected compounds. Wistar rats were dosed 25 mg/kg po using a vehicle of 10% DMSO, 11.25% Trappsol, D5W and the compounds were detected
by LC/MS.

P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
5189
Table 6
Table 8
Comparison of in vivo half-lives of selected compounds
Comparison of compound bioavailability data
Entry
Structure
t
(h)
Entry
Structure
%F
½
O
O
O
O
N
N
N
N
O
O
O
O
O
O
N
2b
0.87
2b
N
H
N
H
O
O
N
H
O
N
H
N
OEt
OEt
N
N
N
N
H
N
OEt
O
O
H
N
OEt
O
N
H
N
17a
17b
17c
1.27
1.60
0.87
17d
17f
17g
17j
25
N
H
N
H
O
O
O
O
O
O
O
O
H
N
OEt
O
O
O
O
H
N
OnPr
N
9
N
H
N
H
O
O
O
O
O
N
O
H
N
OEt
O
H
N
OnPr
35
N
H
N
H
O
O
O
H
N
O
O
5
N
H
Table 7
O
Effect of compound stereochemistry on plasma stability
N
determined to have similar stabilities in vivo. More likely, this is
a reflection of the stability of acylal moiety under the conditions
found in vivo and indicates one may be able to modulate pharma-
cokinetic properties by suitable modification of the acylal group.
With this data in hand, we next focused our attention to under-
standing the effect of the prodrug substituent on plasma stability.
Table 7 presents the results of these studies. For each acylal pro-
drug, two possible stereoisomers are possible; cis and trans relative
to the lactone ring fragment. The first general trend observed was
that the trans isomer appeared to be more stable in plasma com-
pared to the cis stereoisomer. The second trend observed was re-
lated to the steric bulk of the prodrug substituent. As this group
increased in size, irrespective of the stereochemistry, the stability
of the prodrug increased. Thus one could use this SAR to modulate
the biopharmaceutical properties of the drug to fit the desired indi-
cation, that is, short or long plasma half-life.
O
H
N
OR
N
H
O
O
O
Entry
R group
Et
Stereochem
t (h)
½
17d
17e
17f
17g
17h
17i
cis
trans
cis
trans
cis
trans
cis
0.87
4.3
2.0
10.0
3.9
7.7
nPr
(S)-sBu
Cyclopentyl
17j
17k
5.1
15.7
trans
Combining all this data, we prepared several compounds within
this series and determined the bioavailability as part of a decision-
making process for moving a compound forward into clinical devel-
opment (Table 8). While most of the compounds examined had very
poor oral bioavailability, we found that 17f was comparable to 2b, a
compound Vertex had moved into clinical development.
In summary, we have identified a P2–P3 replacement fragment
that retains high in vitro potency but in a readily synthesizable
succinic acid moiety. This scaffold was also found to inhibit the
production of IL-1b in an in vivo model. While it was determined
that the parent compound did not have biopharmaceutical proper-
ties sufficient for progression into development, a prodrug ap-
proach was pursued as a solution to this issue. It was determined
that 17f had properties comparable to 2b and was identified as a
potential candidate for clinical development.
like 14 (ꢀ5.2–5.5 ppm) and of the acyclic acetal 15 (ꢀ4.5 ppm), it ap-
pears clear that the species seen for the parent prodrug in methanol-
d4 was the hemi-acetal 13.
We were able to access a variety of the acylal prodrugs by mod-
ifying the original synthetic protocol to that presented in Scheme
2. One could readily separate the diastereomers, by chromatogra-
phy, thus not only could the effect of substituents be investigated
for these prodrugs but also stereochemical implications could be
probed.
Following the examples of 2b and 3, we synthesized the ethyl
acylal prodrugs of our representative examples with Table 6 pre-
senting the human plasma stability of these compounds. The
immediate observation one can make is that plasma stability of
the prodrug is not dependent upon the nature of the P4 substituent
or the P2–P3 scaffold, since both 2b and 17a, 17b, and 17c were

5190
P. Galatsis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5184–5190
7. (a) Rudolphi, K.; Gerwin, N.; Verzijl, N.; van der Kraan, P.; van den Berg, W.
References and notes
Osteoarthritis Cartilage 2003, 11, 738; (b) Bauer, C.; Loher, F.; Dauer, M.; Mayer,
C.; Lehr, H. A.; Schonharting, M.; Hallwachs, R.; Endres, S.; Eigler, A. Dig. Dis. Sci.
2007, 52, 1642.
1. (a) Thornberry, N. A.; Bull, H. G.; Calaycay, J. R.; Chapman, K. T.; Howard, A. D.;
Kostura, M. J.; Miller, D. K.; Molineaux, S. M.; Weidner, J. R.; Aunin, 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; (b) Thornberry, N. A. Br. Med. Bull. 1996, 53, 478; (c)
Talanian, R. V.; Brady, K. D.; Cryns, V. L. J. Med. Chem 2000, 43, 3351; (d) Bird, S.;
Zou, J.; Wang, T.; Munday, B.; Cunningham, C.; Secombers, C. J. Cytokine Growth
Factor Rev. 2002, 13, 483; (e) Joosten, L. A. B.; van den Berg, W. B. Drug Discovery
Today 2006, 3, 169.
2. (a) Ator, M. A.; Dolle, R. E. Curr. Pharm. Des. 1995, 1, 191; (b) Giegel, D. A.;
Kostlan, C. R. Annu. Rep. Med. Chem. 1998, 33, 183; (c) Talanian, R. V.; Brady, K.
D.; Cryns, V. L. J. Med. Chem. 2000, 43, 3351; (d) Ashwell, S. Expert Opin. Ther.
Pat. 2001, 11, 1593; (e) Randle, J. C. R.; Harding, M. W. H.; Ku, G.; Schonharting,
M.; Kurrle, R. Expert Opin. Invest. Drugs 2001, 10, 1207; (f) Braddock, M.; Quinn,
A. Nat. Rev. Drug Disc. 2004, 3, 1; (g) Dinarello, C. A. Curr. Opin. Pharmacol. 2004,
4, 378; (h) Le, G. T. E.; Abbenante, G. Curr. Med. Chem. 2005, 12, 2963; (i)
Fischer, U.; Schulze-Osthoff, K. Pharmacol. Rev. 2005, 57, 187.
8. Wannamaker, W.; Davies, R.; Namchuk, M.; Pollard, J.; Ford, P.; Ku, G.; Decker,
C.; Charifson, P.; Weber, P.; Germann, U. A.; Kuida, K.; Randle, J. C. R. J.
Pharmacol. Exp. Ther. 2007, 321, 509.
9. Other substituents were investigated (data not shown) and the isopropyl was
determined to be optimal. For example, cyclopentyl and cyclohexyl afforded a
fivefold loss in potency. A free hydroxyl at this position lost activity, while
ethers and esters regained some of the activity (ꢀ500 nM) and amides and
carbamates also showed a dramatic loss of potency.
10. All compounds were characterized by 1H NMR, MS, and were >98% pure by
either CHN analysis or analytical HPLC.
11. Evans, D. A.; Britton, T. C.; Dorow, R. L.; Delaria, J. F., Jr. Tetrahedron 1988, 44,
5525. This compound is also commercially available.
12. Brady, K. D. Biochemistry 1998, 37, 8508.
13. Since compounds were optimized for human ICE and were found to be less
potent in mice, the huSCID assay was developed to understand in vivo activity.
Severe combined immuno-deficient (SCID) mice were injected with human
PMBC cells 15 min before the start of the experiment. At time zero, the mice
were treated with 5 mg/kg of LPS iv followed by the compound of interest
dosed at 3 mg/kg iv 15 min before bleeding at the 2 h time point. The blood
taken was analyzed for various parameters. These included measuring the
levels of human cytokines, mouse cytokines and for the compound itself.
14. Human peripheral blood mononuclear cells (PBMC) were isolated from
heparinized blood by centrifugation over a Ficoll cushion and then washed
three times with PBS. The PBMC’s were suspended in a medium containing
RPMI 1640 with glutamine, penicillin, streptomycin, and 2% human AB serum
and then plated at 106 cells per well in a 96-well flat bottom plate. PBMC’s
were stimulated overnight with 10 ng/mL of LPS in the presence or absence of
inhibitor. The medium was harvested and the level of mature IL-1b was
determined using an ELISA kit from R&D Systems. Compound inhibition (IC50
values) was assessed by determining the concentration of agent which reduced
IL-1b levels by 50%.
3. Galatsis, P.; Caprathe, B.; Downing, D.; Gilmore, J.; Harter, W.; Hays, S.; Kostlan,
C.; Linn, K.; Lunney, E.; Para, K.; Thomas, A.; Warmus, J.; Allen, H.; Brady, K. D.;
Walker, N. Bioorg. Med. Chem. Lett. 2010, 20, 5089.
4. (a) Thornberry, N. A.; Molineaux, S. M. Protein Sci. 1994, 4, 3; (b) Rano, T. A.;
Timkey, T.; Peterson, E. P.; Rotonda, J.; Nicholson, D. W.; Becker, J. W.;
Champman, K. T.; Thornberry, N. A. Chem. Biol. 1997, 4, 149; (c) Chereau, D.;
Kodandapani, L.; Tomaselli, K. J.; Spada, A. P.; Wu, J. C. Biochemistry 2003, 42, 4151.
5. (a) Wilson, K. P.; Black, J. F.; Thomson, J. A.; Kim, E. E.; Griffith, J. P.; Navia, M. A.;
Murcko, M. A.; Chambers, S. P.; Aldape, J. P.; Raybuck, S. A.; Livingston, D. J.
Nature 1994, 370, 270; (b) Walker, N. P. C.; Talanian, R. V.; Brady, K. D.; Dang, L.
C.; Bump, N. J.; Ferenz, C. R.; Franklin, S.; Ghayur, T.; Hackett, M. C.; Hammill, L.
D.; Herzog, L.; Hugunin, M.; Houy, W.; Mankovick, J. A.; McGuiness, L.;
Orlewicz, E.; Paskind, M.; Pratt, C. A.; Reis, P.; Summani, A.; Terranova, M.;
Welch, J. P.; Moller, A.; Tracey, D. E.; Kamen, R.; Wong, W. W. Cell 1994, 78, 343.
6. (a) Chapman, K. T. Bioorg. Med. Chem. Lett. 1992, 2, 613; (b) Boost, K. A.; Hoegel,
S.; Hofstetter, C.; Flondor, M.; Stegewerth, K.; Platacis, I.; Pfeilschifter, J.; Muhl,
H.; Zwissler, B. Intensive Care Med. 2007, 33, 863.