A highly potent and selective caspase 1 inhibitor that utilizes a key 3-cyanopropanoic acid moiety

Article abstract of DOI:10.1002/cmdc.200900531

Herein, we examine the potential of a nitrile-containing propionic acid moiety as an electrophile for covalent attack by the active-site cysteine residue of caspase 1. The syntheses of several cyanopropanate-containing small molecules based on the optimiz

Full text of DOI:10.1002/cmdc.200900531

MED
DOI: 10.1002/cmdc.200900531
A Highly Potent and Selective Caspase 1 Inhibitor that
Utilizes a Key 3-Cyanopropanoic Acid Moiety
Matthew B. Boxer, Amy M. Quinn, Min Shen, Ajit Jadhav, William Leister, Anton Simeonov,
Douglas S. Auld, and Craig J. Thomas*[a]
Herein, we examine the potential of a nitrile-containing pro-
pionic acid moiety as an electrophile for covalent attack by the
active-site cysteine residue of caspase 1. The syntheses of sev-
eral cyanopropanate-containing small molecules based on the
optimized peptidic scaffold of prodrug VX-765 were accom-
plished. These compounds were found to be potent inhibitors
of caspase 1 (IC50 values ꢀ1 nm). Examination of these novel
small molecules against a caspase panel demonstrated an im-
pressive degree of selectivity for caspase 1 inhibition over
other caspase isozymes. Assessment of hydrolytic stability and
selected ADME properties highlighted these agents as poten-
tially useful tools for studying caspase 1 down-regulation in
various settings, including in vivo analyses.
Introduction
Caspases are cysteine proteases with a strict specificity for
cleaving peptide sequences C-terminal to aspartic acid resi-
dues.[1] Currently, 12 caspase isozymes have been identified in
humans with numerous reported activities.[1,2] Caspases are
often subcategorized as either pro-apoptotic or pro-inflamma-
tory enzymes. A prominent member of the pro-inflammatory
class is caspase 1 (also known as interleukin-converting
enzyme or ICE), which is responsible for the proteolytic activa-
tion of interleukin (IL)-1b and IL-18.[3] IL-1b and IL-18 are cyto-
kines that play a major role in the immune response and
within numerous autoimmune and inflammatory diseases.[4]
Caspase 1 is constitutively expressed, but can also be induced,
in immune response elements, such as T cells, macrophages
and neutrophils.[3,5] Procaspase 1 is known to associate with
several multiprotein complexes capable of responding to nu-
merous external stimuli suggesting that caspase 1 is a major
regulator of the inflammation response.[6]
pase 1 that enhance the potency of the interaction and confer
a modest degree of selectivity. Reports demonstrate that VX-
765 (1) and VRT-043198 (2b) are capable of blocking lipopoly-
saccharide-stimulated IL-1b and IL-18 release from human pe-
ripheral blood mononuclear cells (PBMC), whole blood and in
mice (both ip and po administration).[11] Additional in vivo
studies clearly showed mitigation of numerous inflammatory
markers and models.[11] In 2004, Vertex Pharmaceuticals report-
ed that VX-765 had entered a phase II clinical study targeting
psoriasis. Subsequent reports on the clinical development of
VX-765 have yet to be released.
The design of small-molecule inhibitors of cysteine proteases
relies heavily on covalent modification of the active-site cys-
teine through reaction with the highly nucleophilic thio-
late.[12–14] Electrophilic ‘warhead‘ moieties suitable for this
modification include the aforementioned aldehyde, Michael ac-
ceptors (e.g., vinyl sulfones), a-halo ketones, epoxides and ni-
triles. In particular, nitrile-based cysteine protease inhibitors
have found utility against cathepsin K,[15] TbCatB[16] and cru-
zain.[17] Covalently modifying small molecules rely upon bind-
ing that optimally aligns the reactive thiolate nucleophile and
the ‘warhead‘ electrophile. Oballa et al. recently described a
general method to gauge the electrophilic character of the
cyano functionality and reported calculated reaction energies
for diversely substituted nitriles.[18] In general, heterocyclic ni-
triles (e.g., triazine and pyridine nitriles) scored as strong elec-
Targeting proteases, and specifically caspases, via small-mol-
ecule therapeutics is an active area of research.[7–10] Small-mole-
cule inhibitors of selected proteases have entered clinical trials
and many have received approval for use in the clinic. Inhibi-
tors of caspase 1 are sought for intervention strategies within
ischemic disorders, Huntington’s disease, amyotrophic lateral
sclerosis (ALS), rheumatoid arthritis, osteoarthritis, inflammato-
ry bowel disease and sepsis. To date, at least three caspase 1
inhibitors have entered clinical evaluation including pralnaca-
san (VX-740), IDN-6556 and VX-765 (1). All three agents are
active-site inhibitors that act through reversible (pralnacasan
and 1) or irreversible (IDN-6556) covalent modification of the
catalytic cysteine residue. VX-765 (1) is a prodrug that requires
esterase cleavage of the 5-ethoxydihydrofuran-2(3H)-one
moiety to yield the aldehyde functionality of the drug VRT-
043198 (2b), which acts as a potent electrophile for attack by
the active-site cysteine thiol (Figure 1).[11] The remainder of the
VX-765 (1) molecule establishes key binding contacts with cas-
[a] Dr. M. B. Boxer, Dr. A. M. Quinn, Dr. M. Shen, Dr. A. Jadhav, Dr. W. Leister,
Dr. A. Simeonov, Dr. D. S. Auld, Dr. C. J. Thomas
National Institutes of Health, National Human Genome Research Institute
NIH Chemical Genomics Center
9800 Medical Center Drive, Rockville, Maryland 20850 (USA)
Fax: (+1)301-217-5736
E-mail: craigt@mail.nih.gov
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900531.
730
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Potent and Selective Caspase Inhibitors
Figure 1. a) The structure of VX-765 (1) and schematic representation of esterase cleavage of the 5-ethoxydihydrofuran-2(3H)-one moiety to yield the active
drug VRT-043198 (2b). b) The structure of NCGC00185682 (3) and putative esterase cleavage of the ethyl-3-cyanopropanoate moiety to yield active agent
NCGC00183434 (4).
trophiles, a-aminonitriles were modest electrophiles, and phe-
nolic and aliphatic nitriles were calculated as relatively weak
electrophiles.
are not commercially available and therefore required synthetic
elaboration. As we wanted to explore both an active and pro-
drug form of our conceived molecule, we examined alternative
protecting group strategies for the acid side chain (Scheme 1).
Commercially available Fmoc protected d-isoasparagine (5) of-
fered a convenient entry point to both required building
blocks. Treatment of 5 with 2-(trimethylsilyl)ethanol, EDC and
DMAP in dichloromethane provided the TMSE-protected deriv-
ative 6 in good yield. Conversion of 6 to the corresponding ni-
trile 7 was accomplished by treatment with trifluoroacetic an-
hydride (TFAA) and diisopropylethylamine (DIPEA). A similar se-
quence was used to produce the ethyl ester 9. In addition to
Based upon successes surrounding nitrile-based cysteine
protease inhibitors, and the potential alignment of reactivities
between a nitrile-containing aspartic acid mimetic and the
active site thiol, we endeavored to examine the potential of
the 3-cyanopropanoic acid moiety within a known caspase 1
inhibitor scaffold. Fairlie and co-workers included a 3-cyano-
propanoic-acid-containing dipeptide in a study aimed at dis-
covering caspase 1 inhibitors but reported that it had no activi-
ty.[19] Several patents cover various small molecules with 3-cy-
anopropanoic acids but do not
go into detail regarding the ac-
tivities of these agents.[20–22]
From these limited studies, we
felt that the promise of cyano-
propanoates as caspase inhibi-
tors remained in question. To ex-
plore the potential of this func-
tional moiety, we took advant-
age of the peptidic scaffold of
VX-765 (1). Furthermore, we in-
corporated the ethyl-3-cyanopro-
panoate moiety to mimic the
prodrug qualities associated
with 1 (Figure 1).
Results and Discussion
Design and synthesis
Scheme 1. Conditions and reagents: a) EDC, DMAP, CH2Cl2, 6 h, 59%; b) TFAA, DIPEA, CH2Cl2, 08C, 30 min, 90%;
c) thionyl chloride (excess), EtOH, 08C, 95%; d) TFAA, DIPEA, CH2Cl2, 08C, 30 min, 86%; e) NMM, DME, then
NH4OH, 73%; f) TMSN3, Bu2SnO (0.6 equiv), toluene, microwave, 1008C, 1h, 77%; g) TFAA, DIPEA, CH2Cl2, 08C,
Appropriately substituted ethyl-
3-cyanopropanoate derivatives 30 min, 80%.
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C. J. Thomas et al.
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the ester prodrug and the active cyanopropionic acid, it was of
interest to explore carboxylic acid mimetics. As such, we un-
dertook the synthesis of a tetrazole analogue of the key ethyl-
3-cyanopropanoate moiety. Here, we used the previously re-
ported Fmoc-protected (S)-2-amino-3-cyanopropanoic acid
(10).[23] Conversion to amide 11 was required prior to forma-
tion of tetrazole 12. The amide was formed via the mixed an-
hydride followed by treatment with ammonium hydroxide. Tet-
razole formation was accomplished via microwave irradiation
of nitrile 11 and TMS-azide in the presence of dibutylstanna-
none.[24] Dehydration to nitrile 13 was accomplished in a
manner analogous to 7 and 9.
DBU in DMF gave the free amines to which 15, DIPEA and
finally HATU were sequentially added to yield the coupled
products. The generation of 4 further required TBAF-mediated
removal of the TMSE group.
In order to compare the activities of our newly synthesized
compounds to VX-765 (1) and VRT-043198 (2b) we undertook
the synthesis of these agents as well. In 2008, Magdziak and
co-workers reported a synthesis of 1 that relied upon a well-
engineered Pd-catalyzed coupling reaction between the ethox-
yfuranone vinyl bromide and the amide of a Cbz-protected
proline amide.[25] For our purposes, it was convenient to begin
with the orthogonally protected d-b-homoserine 17, which
was transformed into aldehyde 18 via the Parikh–Doering oxi-
dation in good yield (Scheme 3).[26] Conversion to the diethyl
acetal and removal of the Fmoc protecting group provided the
free amine 19 in modest yields
With the appropriately substituted/protected cyanopro-
panoate building blocks in hand, we next turned our attention
to the trimer core of VX-765 (Scheme 2). Both Fmoc-protected
over two steps. Standard cou-
pling of 19 and 15 with HATU
and DIPEA was followed by
treatment with TFA in dry di-
chloromethane to generate the
5-ethoxydihydrofuranone pres-
ent in 1. In our hands, genera-
tion of hemiketal 2a/VRT-043198
(2b) was accomplished by treat-
ing 1 with HCl in a THF/water
mixture.
In vitro pharmacology
With the desired compounds in
hand, we turned our attention
to their biochemical capabilities.
Our first evaluations were aimed
at determining the inhibition po-
tency of each agent against cas-
pase 1. For this purpose we used
a
well-established
protocol,
whereby caspase 1 activity is
measured using the enzyme in
the presence and absence of
compound and
a pro-fluores-
cence peptide substrate (Ac-
LEHD-AMC). Compounds were
Scheme 2. Conditions and reagents: a) EDC, HOBt, DMF, RT, 8 h; b) DBU, CH2Cl2, RT, 71% (two steps); c) HATU,
DIPEA, DMF, RT, 2 h; d) TFA/CH2Cl2 (1:1), RT, 4 h, 85% (two steps); e) DBU, DMF, 5 min, then 15, HATU, DIPEA, DMF,
08C, 2 h; f) TBAF, THF, 08C, 72% (two steps); g) DBU, DMF, 5 min, then 15, HATU, DIPEA, DMF, 08C, 2 h, 91%;
h) DBU, DMF, 5 min, then 15, HATU, DIPEA, DMF, 08C, 2 h, 82%.
examined across
a
titration
series (0.65 pm ! 57.5 mm) and
data were recorded via fluores-
cence detection over 20 min, fol-
l-tert-leucine and tert-butyl-l-prolinate are commercially avail-
able and were easily coupled via treatment with EDC and
HOBt. Fmoc removal was effected by treatment with DBU re-
sulting in the protected dimer 14. Coupling of 14 with
4-amino-3-chlorobenzoic acid was accomplished using HATU
and DIPEA in DMF. Trifluoroacetic acid (TFA)-mediated removal
of the tert-butyl group yielded the carboxylic acid 15. A single
pot deprotection–coupling sequence was used to generate
the desired final products. Deprotection of 7, 9 and 13 with
lowing a pre-incubation period of 15 min at room temperature
using 5 mm of peptide substrate. We examined VRT-043198
(2b), NCGC00185682 (3), NCGC00183434 (4) and the tetrazole
NCGC00183681 (16), and the results are displayed in Figure 2.
VRT-043198 (2b) was confirmed as a potent caspase 1 inhibitor
with an IC50 value of 11.5 nm. NCGC00183434 (4), containing
the key cyanopropanoate moiety, was found to inhibit cas-
pase 1 with an impressive IC50 value of 0.316 nm. We were fur-
ther gratified to find that the ethyl ester 3 and tetrazole 16 re-
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Potent and Selective Caspase Inhibitors
Having established that the ni-
trile-containing compound 4 is a
potent inhibitor of caspase 1, we
next turned our attention to the
selectivity of these agents.
Randle and co-workers have re-
ported the Ki values of 2b
against caspases 1, 3–4 and 6–9,
and against granzyme B, cathep-
sin B and trypsin.[11] This report
presents evidence that 2b is
nearly equipotent against cas-
pases 1 and 4 (Ki =<1 nm) and
modestly potent against caspas-
es 6, 8 and 9 (100 nm, 560 nm
and 1030 nm, respectively), while
possessing little activity against
the remaining targets. We sub-
mitted compounds 2b, 3, 4 and
16 for evaluation against a com-
Scheme 3. Conditions and reagents: a) SO3–pyridine, DIPEA, CH2Cl2, DMSO, 75%; b) (EtO)3CH, PPTS, EtOH, 508C,
24 h; c) DBU, CH2Cl2, 63% (two steps); d) 19, DMF, 5 min, then 15, HATU, DIPEA, DMF, 08C, 1 h; e) TFA/CH2Cl2 (1:1),
1 h, 72% (two steps); f) HCl, THF/H2O, quantitative.
mercial panel of caspases offered by Reaction Biology Corpora-
tion (RBC).[27] The results, shown in Table 1, confirmed the
potent inhibition of caspase 1 by 2b (IC50 =0.204 nm), howev-
er, the IC50 values found against caspase 4 (IC50 =14.5 nm) and
caspase 8 (IC50 =3.3 nm) differed slightly from the reported Ki
values.[11] The results against caspase 6 (IC50 =>10000 nm) and
caspase 9 (IC50 =5.07 nm) were significantly different from
those reported by Randle and co-workers.[11] The RBC panel
also included caspase 5 (IC50 =10.6 nm), caspase 10 (IC50 =
66.5 nm) and caspase 14 (IC50 =58.5 nm), and the data present-
ed here represents the first disclosure of the IC50 values for 2b
against these targets.
The results for 4 demonstrated an impressive potency of the
compound against caspase 1 (IC50 =0.023 nm), and a similar se-
lectivity profile as compound 2b. The only prominent diver-
gence between the selectivity profiles of 4 and 2b was a sig-
nificant difference in their ability to inhibit caspase 14 (IC50 =
801 nm and IC50 =58.5 nm, respectively). The caspase 1 inhibi-
tion data generated in this panel for 3 and 16 were similar to
the data generated in our caspase 1 assay with reported IC50
values of 43.4 nm and 2.58 nm, respectively. A particularly inter-
esting aspect of these molecules was the high selectivity for
Figure 2. Concentration response curves for VRT-043198 (2b; ^;
IC50 =11.5 nm), NCGC00185682 (3; ~; IC50 =144.7 nm), NCGC00183434 (4;
^; IC50 =0.316 nm)and the tetrazole NCGC00183681 (16; &; IC50 =20.4 nm).
tained impressive potencies against caspase 1 (IC50 =144.7 nm
and IC50 =20.4 nm, respectively). The Ki value of 4 was estimat-
ed to be 0.4 nm for caspase 1 using a competitive inhibition
model (see Supporting Information).
Table 1. IC50 values for selected compounds versus caspase panel.[a]
Compound
IC50 [nm]
caspase 1
caspase 3
caspase 4 caspase 5 caspase 6
caspase 7
caspase 8
caspase 9 caspase 10 caspase 14
5.07 66.5 58.5
VRT-043198 (2b) (Drug)
3 (Nitrile ester)
4 (Nitrile acid)
16 (Nitrile tetrazole)
20 YVAD-CN
Ac-LEHD-CHO (standard)
Ac-DEVD-CHO (standard)
0.204
43.4
0.023
2.58
2.16
15.0
ND
>10000
>10000
>10000
>10000
>10000
ND
14.5
>10000
13.8
1380
114
81.7
ND
10.6
1570
3.60
1300
29.0
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
3.3
>10000
25.2
>10000
726
1610 >10000 >10000
2.17 89.7
91.5 >10000
297 187
40.4
ND
801
>10000
116
21.3
ND
ND
122
ND
3.82
ND
49.2
ND
134
ND
3.04
3.54
[a] Data was generated by Reaction Biology (http://www.reactionbiology.com/). Data is presented as an IC50 value using a (Z-LEHD)2-R110 tetrapeptide sub-
strate for caspase 1, 4, 5, 8, 9, 10, 14, and a (Z-DEVD)2-R110 tetrapeptide substrate for caspases 3, 6 and 7. Data represents the results from three separate
experiments. ND=not determined.
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C. J. Thomas et al.
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caspase 1. NCGC00183681 (16) exhibited an IC50 value of
91.5 nm against caspase 9. All other activities were above the
1 mm threshold.
In addition to the primary compounds of this study, we
were interested in establishing cyanopropanoates as general
caspase directing ‘warhead‘ for future utility in the search for
other potent and selective small-molecule inhibitors of caspas-
es. As such, we included the general nitrile–aspartic acid direct-
ing group into the common peptide caspase inhibitor YVAD.
The resulting agent YVAD-CN (20) was profiled and the results
clearly demonstrate that cyanopropanoates represent a gener-
al moiety capable of reversible, covalent modification of cas-
pases.
Physical properties
Based upon the data provided in this panel, it was clear that
these agents represent important new tools for caspase 1 in-
hibition. However, the contributing functional groups for these
agents (i.e., ethyl acetals, aldehydes, nitriles and esters) are all
subject to hydrolysis under various conditions. To appreciate
the utility of these compounds as molecular probes or even
clinically useful agents, it was essential to fully understand
their stability profile. Therefore, we examined derivatives 1, 2b,
3, 4 and 16 within an aqueous degradation study under neu-
tral (pH 7), acidic (pH 2), and basic (pH 8) conditions. The study
was conducted by monitoring the degradation of each agent
by LC/MS analysis at various time points over 96 h (Figure 3).
Prodrug 1 showed moderate degradation in water with over
50% of the compound decomposed after 48 h. This degrada-
tion was amplified under both basic and acidic conditions.
Conversely, the active agent 2b was very stable under both
neutral and acidic conditions, and its degradation at pH 8 was
moderate. The potent analogue 4 was exceedingly stable
under basic conditions, and its stability in neutral and acidic
conditions was moderate-to-good (degradation of 50% under
both conditions after 72 h). The ethyl ester 3 was exceptionally
stable under neutral and acidic conditions (no degradation
noted), however, it was fully degraded under basic conditions
after 22 h (presumably due to saponification of the ester). Fi-
nally, the tetrazole 16 was found to be resistant to degradation
under all conditions tested. Interestingly, these data suggest
Figure 3. Aqueous stability of a) prodrugs VX-765 (1; ^) and
NCGC00185682 (3; ꢁ) and b) drugs VRT-043198 (2b, ~), NCGC00183434 (4;
&) and NCGC00183681 (16; *) under neutral (pH 7, c black), acidic
(pH 2, c red), and basic (pH 8, c blue) conditions.
levels reported for 1 and 3 strongly suggested an active trans-
port mechanism, and a control experiment with verapamil con-
firmed that these agents are substrates for Pgp efflux. Unsur-
prisingly, free acids 2b and 4 and tetrazole 16 had significantly
higher free fractions in both human and rat protein binding
assays relative to the more hydrophobic prodrug 1 and ethyl
1
ester 3. The clearance rates (CLint) and t for 2b, 3, 4 and 16
2
were all moderate. Ester 3 was noted to possess a slight
degree of degradation in liver microsomes without NADPH as
a cofactor suggesting a nonenzymatic-related degradation
mechanism. Prodrug 1 was only minimally metabolized by
1
that 1 may have a short half-life (t ) as an oral agent due to its
2
1
2
instability under acidic conditions, such as those found in the
liver microsomes, with a t of >9400 min. It is unknown how
gastric environment (40% degradation after 3.5 h at pH 2). In
contrast, these data strongly suggest that 3 and 16 will be
suitable reagents for all manner of examinations (cell based
and in vivo studies), and even the highly active derivative 4
persists beyond 24 h.
this extended stability affects the toxicity profile of this agent.
Modeling
Finally, we examined the binding mechanism of these agents
through molecular modeling. Several crystal structures of cas-
pase 1 exist including structures with reversible and nonrever-
sible inhibitors (PDB codes: 1BMQ, 1IBC, 1ICE, 1RWK, 1RWM,
1RWN, 1RWO, 1RWP, 1RWV, 1RWW, 1RWX, 1SC1, 1SC3, 1SC4,
2FQQ, 2H48, 2HBQ, 2HBR, 2HBY, 2HBZ, 2FQR, 2FQS, 2FQU,
2FQZ).[29–32] We identified crystal structure 2HBQ as the best
template for 4 binding to caspase 1 (2HBQ is a co-crystal struc-
ture of Z-VAD-FMK bound to caspase 1). When building a
Given the aqueous stability of these new agents, it was of
interest to examine selected ADME properties for chosen com-
pounds. As such, derivatives 1, 2b, 3, 4 and 16 were submitted
to Cyprotex[28] for a profile of bi-directional Caco-2 permeabili-
ty, plasma protein binding (both human and rat) and microso-
mal stability (both human and rat) studies (Table 2). All agents
possessed relatively low A!B permeability; however, prodrug
1 and ester 3 had moderately better levels. The high B!A
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Potent and Selective Caspase Inhibitors
drolysis during proteolysis.[34]
This may have consequences for
both the binding affinity of ni-
trile-based cysteine proteases in-
hibitors and their ultimate reso-
lution through hydrolysis of the
thioimidate intermediate.
Table 2. In vitro ADME properties for selected compounds.[a]
Compound
Caco Papp[b] [ꢁ10ꢁ6 cm sꢁ1
]
Protein Binding[c]
fraction unbound CLint [mL minꢁ1 mgꢁ1 protein] t [min]
Microsomal Stability[d]
1
2
A!B
B!A
VX-765 (2a) (Prodrug)
VRT-043198 (2b) (Drug)
3 (Nitrile ester)
4 (Nitrile acid)
18 (Nitrile tetrazole)
0.797
ND
0.445
0.144
0.130
32.7
0.173
9.59
0.060
0.193
0.006
0.420
0.071
0.431
0.243
0.147
6.72
27.4
10.3
9.38
9430
206
50.7
134
148
[a] Data was generated by Cyprotex (http://www.cyprotex.com/home/). ND=not determined. [b] Caco-2 per-
meability (Papp) assay over three separate experiments with two separate internal control groups. Agents
were also profiled in the presence of the known Pgp substrate verapamil and data from these experiments
highly suggested that 2a and 3 were substrates for efflux by Pgp. [c] Plasma protein binding assays were per-
formed using an equilibrium dialysis method in 100% plasma (profiles versus human and rat plasma were ob-
tained: see the Supporting Information section for rat plasma binding data). [d] Microsomal stability (intrinsic
clearance (CLint) and half-life) was profiled alongside internal control compounds, minus NADPH and minus
compound (profiles versus human and rat plasma were obtained).
Conclusion
In this study, we set out to ex-
amine the potential of nitriles as
a ‘warhead‘ functionality for re-
versible, covalent modification of
the active-site cysteine residue
of caspases. The synthesis of a
cyanopropanoate derivative of
the clinically used caspase 1 in-
model, the mechanism-of-inhibition for the binding of 4 to
caspase 1 was presumed to be via a covalent, reversible inter-
action. The nitrile carbon was therefore held at a proximal dis-
tance (2.6 ꢂ) from the catalytic cysteine residue (C285) by con-
straint docking, and flexibility was granted to the remainder of
the small molecule to achieve an optimal binding pose using
FRED.[33] The results, shown in Figure 4, demonstrate comple-
mentarity between the peptidic fragment of 4 and the peptide
binding domain of caspase 1. Key interactions were noted for
the acid moiety and arginine residues 341 and 179 in similar
fashion to other aspartic acid-containing, small-molecule cas-
pase 1 inhibitors. While direct interrogation of a covalent inter-
action between the nitrile and C285 was not pursued in our
model, this representation does illustrate the open binding
cavity that accommodates the tetrahedral intermediate that
forms as a result of covalent binding with aldehyde-based in-
hibitors (a mimetic of the hemithiolacetal intermediate associ-
ated with transition state 1 (TS1) during proteolysis). In con-
trast, covalent interactions between a thiol and a nitrile form a
thioimidate intermediate that mimics transition state 2 (TS2) of
an enzymatic proteolytic event between a cysteine proteases
and a substrate. Mꢃnard and co-workers examined aldehyde
and nitrile inhibitors of papain and found that the thioimidate
intermediate engages the oxyanion hole interaction in a
manner that more closely mimics the natural process of hy-
hibitor VRT-043198 (2b) was accomplished. Several related ni-
trile-containing agents were examined and found to be highly
potent and selective caspase 1 inhibitors. Interestingly, the car-
boxylic acid moiety of aspartic acid was found to confer a sig-
nificant increase in potency to NCGC00183434 (4), however,
ester and tetrazole analogues of this compound were also
found to be potent and selective caspase 1 inhibitors. Aqueous
solubility studies and profiles of selected ADME properties also
suggest that these novel agents possess appropriate proper-
ties to be utilized as molecular probes of caspase 1 and, poten-
tially, within in vivo settings.
Experimental Section
Caspase assay
Caspase 1 was dispensed (3 mL) into a 1536-well black solid-
bottom microplate (Greiner Bio-One, Monroe, USA) using a Bio-
RAPTR (Beckman Coulter, Fullerton, USA) nanoliter dispenser for
automated addition of reagents, to give a final enzyme concentra-
tion of 2 nm. Assay buffer was 50 mm HEPES, pH 7.4, containing
1m sodium citrate, 100 mm NaCl, 0.1 mm EDTA, 10 mm DTT, 0.01%
CHAPS, 1% DMSO, and 0.1 mgmLꢁ1 bovine serum albumin (BSA).
Aliquots of test compound (23 nL) from a DMSO stock solution
were transferred to the enzyme-containing wells using a pintool
transfer station (Kalypsys Inc., San Diego, USA), and the plates
were incubated for 15 min at room temperature. Each compound
was added as a 12-point titration
in duplicate. Substrate was dis-
pensed (1 mL) to give a final pro-
fluorescence peptide substrate
(Ac-LEHD-AMC) concentration of
5 mm, and the initial reaction rate
was monitored over 20 min at
room temperature. Free AMC was
measured by fluorescence detec-
tion (lex =340 nm, lem =450 nm)
with
a ViewLux charge-coupled
device-based imager (PerkinElmer,
Waltham, USA). Percent inhibition
was calculated from the median
values of the uninhibited and the
Figure 4. Molecular model [a) ribbon and b) space filling] of NCGC00183434 (4) bound to caspase 1.
ChemMedChem 2010, 5, 730 – 738
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
735

C. J. Thomas et al.
MED
uncatalyzed controls, and IC50 values were determined by fitting
the concentration–response data with a four-parameter Hill equa-
tion[35] as previously described (http://www.ncgc.nih.gov/pub/
openhts/curvefit/).
with DIPEA (4.17 mL, 23.89 mmol, 3 equiv), followed by the drop-
wise addition of TFAA (2.25 mL, 15.93 mmol, 2 equiv). The yellow
solution was stirred until all starting material was consumed (con-
firmed by TLC; 30 min). The reaction was quenched with aq
NaHCO3. The reaction was washed with aq NaHCO3 (ꢁ2) and brine
(ꢁ1), dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by column chromatography (hexane/EtOAc, 9:1!1:1) gave 7
as an off-white powder (3.13 g, 90%): [a]2D0 =ꢁ26 (c=1.0 in
Chemistry
General Methods: All air- or moisture-sensitive reactions were per-
formed under a positive pressure of N2 with oven-dried glassware.
Anhydrous solvents, such as THF, toluene, CH2Cl2, DMF, MeCN,
MeOH and Et3N, were purchased from Sigma–Aldrich.
1
MeOH); H NMR (400 MHz, [D6]DMSO): d=8.23 (d, J=7.6 Hz, 1H),
7.88 (d, J=7.4 Hz, 2H), 7.68 (d, J=7.4 Hz, 2H), 7.37–7.45 (m, 2H),
7.32 (t, J=7.4 Hz, 2H), 4.76 (q, J=7.6 Hz, 1H), 4.35–4.46 (m, 2H),
4.24 (t, J=6.5 Hz, 1H), 4.02–4.21 (m, 2H), 2.77–3.00 (m, 2H), 0.85–
1.05 (m, 2H), 0.00 ppm (s, 9H); 13C NMR (100 MHz, [D6]DMSO): d=
170.0, 156.8, 145.1, 142.3, 129.2, 128.6, 126.6, 121.7, 120.4, 67.6,
64.4, 50.2, 48.1, 37.8, 18.3, 0.0 ppm.
Preparative purification was performed on a Waters semi-prepara-
tive HPLC using a Phenomenex Luna C18 column (5 mm, 30ꢁ
75 mm) at a flow rate of 45 mLminꢁ1. The mobile phase consisted
of MeCN and water (each containing 0.1% TFA). A gradient of 10%
to 50% MeCN over 8 min was used during the purification. Frac-
tion collection was triggered by UV detection (220 nm). Analytical
analysis was performed on an Agilent LC/MS (Agilent Technologies,
Santa Clara, USA).
(S)-Ethyl 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-amino-
4-oxobutanoate (8): A suspension of 5 (10 g, 28.2 mmol, 1 equiv)
in EtOH (100 mL) was cooled in an ice bath and treated dropwise
with thionyl chloride (20.6 mL, 282 mmol, 10 equiv). The reaction
was stirred at RT until the suspension became a clear solution
(2 h). The solvents were removed in vacuo and the residue was pu-
rified by column chromatography (hexane/EtOAc, 9:1!1:9) to give
8 as a white powder (10.25 g, 95%): [a]2D0 =ꢁ7 (c=1.0 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=7.86 (d, J=7.6 Hz, 2H), 7.67 (d,
J=7.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 1H), 7.38 (t, J=7.4 Hz, 2H), 7.29
(t, J=7.5 Hz, 2H), 7.08 (br s, 2H), 4.13–4.42 (m, 4H), 3.93–4.11 (m,
2H), 2.70 (dd, J=16.0, 5.3 Hz, 1H), 2.49–2.59 (m, 1H), 1.03–
1.29 ppm (m, 3H); 13C NMR (100 MHz, [D6]DMSO): d=172.8, 170.7,
156.2, 144.2, 141.1, 128.1, 127.5, 125.7, 120.5, 66.1, 60.5, 51.6, 47.1,
36.8, 14.5 ppm.
Method 1: 7 min gradient of 4% to 100% MeCN (containing
0.025% TFA) in water (containing 0.05% TFA); 8 min run time; flow
rate=1 mLminꢁ1
; Phenomenex Luna C18 column (3 mm, 3ꢁ
75 mm); temperature=508C.
Method 2: 3 min gradient of 4% to 100% MeCN (containing
0.025% TFA) in water (containing 0.05% TFA); 4.5 min run time;
flow rate=1 mLminꢁ1; Phenomenex Gemini phenyl column (3 mm,
3ꢁ100 mm); temperature=508C.
Purity determination was performed using an Agilent Diode Array
Detector. Mass determination was performed using an Agilent
6130 mass spectrometer with electrospray ionization (ESI) in the
positive mode. 1H NMR and 13C NMR spectra were recorded on
Varian 400 spectrometers. Chemical Shifts (d) are reported in ppm
with TMS as internal standard (0 ppm) for CDCl3 solutions, or non-
deuterated solvent (DMSO at 2.49 ppm) for [D6]DMSO solutions.
High-resolution mass spectrometry was recorded on an Agilent
6210 Time-of-Flight LC/MS system. Confirmation of molecular for-
mula was accomplished using ESI in the positive mode with the
Agilent Masshunter software (version B.02). All analogues assayed
have purity greater than 95% based on both analytical methods.
(S)-Ethyl 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-cyano-
propanoate (9): A solution of 8 (2.58 g, 6.75 mmol, 1.0 equiv) in
CH2Cl2 (40 mL) was cooled in an ice bath and treated with DIPEA
(3.53 mL, 20.24 mmol, 3 equiv), followed by the dropwise addition
of TFAA (1.43 mL, 10.12 mmol, 2 equiv). The yellow solution was
stirred until all starting material was consumed (confirmed by TLC;
30 min). The reaction was quenched with aq NaHCO3. The reaction
was washed with aq NaHCO3 (ꢁ2) and brine (ꢁ1), dried (Na2SO4),
filtered and concentrated in vacuo. Purification by column chroma-
tography (hexane/EtOAc, 9:1!1:1) gave 9 as an off-white powder
(2.11 g, 86%): [a]2D0 =ꢁ24 (c=0.5 in MeOH/CH2Cl2, 1:1); 1H NMR
(400 MHz, [D6]DMSO): d=8.19 (d, J=7.6 Hz, 1H), 7.86 (d, J=7.6 Hz,
2H), 7.65 (d, J=7.4 Hz, 2H), 7.39 (t, J=7.3 Hz, 2H), 7.30 (t, J=
7.4 Hz, 2H), 4.73 (q, J=7.6 Hz, 1H), 4.39 (d, J=6.5 Hz, 2H), 4.22 (d,
J=6.3 Hz, 1H), 4.07 (q, J=7.0 Hz, 2H), 2.88 (m, 2H), 1.15 ppm (t,
J=7.1 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO): d=168.9, 155.7,
144.1, 141.2, 128.1, 127.5, 125.5, 120.6, 119.3, 66.5, 61.2, 46.9, 39.4,
36.5, 14.4 ppm.
(S)-2-(Trimethylsilyl)ethyl 3-(((9H-fluoren-9-yl)methoxy)carbonyl-
amino)-4-amino-4-oxobutanoate (6): A suspension of (S)-3-(((9H-
fluoren-9-yl)methoxy)carbonylamino)-4-amino-4-oxobutanoic acid
(5) (17.29 g, 48.8 mmol, 1 equiv) and EDC (12.16 g, 63.4 mmol,
1.3 equiv) in CH2Cl2 (250 mL) was treated with 2-(trimethylsilyl)-
ethanol (8.39 mL, 58.6 mmol, 1.2 equiv) followed by DMAP (8.35 g,
68.3 mmol, 1.4 equiv). The suspension was stirred for 6 h at RT
then quenched with saturated aq NaHCO3. The reaction was
washed with aq NaHCO3 (ꢁ2) and brine (ꢁ1), dried (Na2SO4), fil-
tered and concentrated in vacuo. Purification by column chroma-
tography (hexane/EtOAc, 9:1!1:9) gave 6 as a white powder
(13.09 g, 59%): [a]2D0 =ꢁ5 (c=1.0 in MeOH); 1H NMR (400 MHz,
CDCl3): d=7.72 (d, J=7.4 Hz, 2H), 7.54 (d, J=7.4 Hz, 2H), 7.36 (t,
J=7.4 Hz, 2H), 7.27 (t, J=7.4 Hz, 2H), 6.38 (br s., 1H), 6.03 (br s,
1H), 5.80 (br s, 1H), 4.50–4.61 (m, 1H), 4.35–4.50 (m, 2H), 4.05–4.25
(m, 3H), 2.92 (m, 1H), 2.49–2.73 (m, 1H), 0.85–1.03 (m, 2H),
0.04 ppm (s, 9H); 13C NMR (100 MHz, [D6]DMSO): d=173.9, 171.9,
157.3, 145.3, 142.2, 129.2, 128.6, 126.8, 121.6, 67.3, 63.6, 52.7, 48.1,
38.0, 18.3, 0.00 ppm.
(S)-(9H-Fluoren-9-yl)methyl 1-amino-3-cyano-1-oxopropan-2-yl-
carbamate (11): A suspension of 10 (5 g, 14.87 mmol, 1 equiv) in
DME (50 mL) was cooled in an ice bath and treated with N-methyl-
morpholine (1.63 mL, 14.87 mmol, 1 equiv). The cooled suspension
was stirred for 10 min before isobutyl chloroformate (1.95 mL,
14.87 mmol, 1 equiv) was added dropwise and stirring was contin-
ued for an additional 10 min. Ammonium hydroxide (5.79 mL,
149 mmol, 10 equiv) was then added and the reaction was re-
moved from the ice bath and allowed to warm to RT and stirred an
additional 4 h. The reaction was diluted with EtOAc (50 mL) and
water (75 mL), and the two-phase mixture was stirred for 15 min.
The organic layer was separated and washed with aq NaHCO3 (ꢁ2)
and brine (ꢁ1), dried (Na2SO4), filtered and concentrated in vacuo.
Purification by column chromatography (hexane/EtOAc, 9:1!1:99)
(S)-2-(Trimethylsilyl)ethyl 3-(((9H-fluoren-9-yl)methoxy)carbonyl-
amino)-3-cyanopropanoate (7): A solution of 6 (3.62 g, 7.96 mmol,
1.0 equiv) in CH2Cl2 (50 mL) was cooled in an ice bath and treated
736
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 730 – 738

Potent and Selective Caspase Inhibitors
gave 11 as a white solid (3.64 g, 73%): [a]2D0 =ꢁ10 (c=1.0 in
1H), 3.69–3.80 (m, 1H), 3.60 (m, 1H), 2.79 (dd, J=6.7, 3.1 Hz, 2H),
1.99–2.11 (m, 1H), 1.63–1.96 (m, 4H), 0.99 ppm (s, 9H); 13C NMR
(100 MHz, [D6]DMSO): d=172.2, 170.6, 169.9, 165.8, 148.0, 129.4,
128.2, 122.1, 119.1, 116.4, 114.4, 59.7, 57.8, 48.2, 37.4, 36.6, 35.3,
29.5, 27.0, 25.1 ppm; LC/MS: Method 1, tR: 4.595 min; Method 2, tR:
3.591 min; HRMS: m/z [M+H]+ calcd for C22H28N5O5Cl: 477.1779,
found: 477.1784.
1
MeOH); H NMR (400 MHz, [D6]DMSO): d=7.78–7.93 (m, 2H), 7.56–
7.76 (m, 2H), 7.47 (br s, 1H), 7.39 (t, J=7.4 Hz, 2H), 7.30 (t, J=
7.4 Hz, 2H), 4.20–4.39 (m, 3H), 2.81–2.96 (m, 1H), 2.57–2.81 (m,
1H), 1.11–1.32 ppm (m, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
171.0, 156.2, 144.2, 141.1, 128.1, 127.5, 125.7, 120.6, 118.7, 66.4,
51.1, 47.0, 20.9 ppm.
(S)-Ethyl 3-((S)-1-((S)-2-(4-amino-3-chlorobenzamido)-3,3-dime-
thylbutanoyl)pyrrolidine-2-carboxamido)-3-cyanopropanoate (3):
A solution of 9 (0.437 g, 1.200 mmol) in DMF (5 mL) at RT was
treated with DBU (0.158 mL, 1.050 mmol) and stirred for 5 min,
until LC/MS confirmed cleavage of Fmoc group. (S)-1-((S)-2-(4-
Amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2-car-
boxylic acid (0.382 g, 1 mmol) in DIPEA (0.262 mL, 1.500 mmol) and
HATU (0.456 g, 1.200 mmol) were added sequentially. The solution
was stirred at 08C for 2 h, diluted with EtOAc (30 mL) and
quenched with saturated aq NaHCO3. The organic phase was
washed with NaHCO3 (ꢁ2) and brine (ꢁ1), dried (Na2SO4), filtered
and concentrated in vacuo. Purification by reverse-phase chroma-
tography (see General Methods) gave 3 as a white powder
(460 mg, 91%): [a]2D0 =ꢁ35 (c=0.6 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.61–8.74 (m, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.64 (d,
J=8.8 Hz, 1H), 7.56 (dd, J=8.6, 2.0 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H),
4.92 (q, J=7.0 Hz, 1H), 4.65 (d, J=8.8 Hz, 1H), 4.24 (dd, J=8.2,
5.7 Hz, 1H), 4.08 (q, J=7.2 Hz, 2H), 3.69–3.80 (m, 1H), 3.55–3.66
(m, 1H), 2.85–2.93 (m, 2H), 1.97–2.10 (m, 1H), 1.64–1.95 (m, 3H),
1.17 (t, J=7.6 Hz, 3H), 0.99 ppm (s, 9H); 13C NMR (100 MHz,
[D6]DMSO): d=171.2, 170.1, 170.0, 164.9, 147.7, 129.4, 128.1, 122.1,
118.7, 116.9, 113.4, 61.1, 60.4, 56.8, 45.1, 37.1, 36.0, 35.1, 29.3, 28.4,
22.1, 15.6 ppm; LC/MS: Method 1, tR: 5.011 min; Method 2, tR:
3.981 min; HRMS: m/z [M+H]+ calcd for C24H32N5O5Cl: 505.2092,
found: 505.2100.
(S)-(9H-Fluoren-9-yl)methyl 1-amino-1-oxo-3-(1H-tetrazol-5-yl)-
propan-2-ylcarbamate (12): Compound 11 (1 g, 2.98 mmol) and
dibutylstannanone (0.445 g, 1.789 mmol) were added to a 20 mL
Biotage microwave vial and suspended in toluene (20 mL). TMS-
azide (0.910 mL, 6.86 mmol) was added and the flask was sealed
with a Teflon cap. The sealed flask was submerged in an oil bath at
1008C and stirred for 12 h. The suspension was transferred to a
round bottom flask and the toluene was removed in vacuo. Re-
verse-phase chromotography (see General Methods) gave 12 as a
white fluffy powder (870 mg, 77%): [a]2D0 =ꢁ10 (c=0.3 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=11.83–12.07 (br s, 1H), 7.87 (d,
J=7.4 Hz, 2H), 7.57–7.71 (m, 4H), 7.40 (t, J=7.2 Hz, 2H), 7.30 (t,
J=7.2 Hz, 2H), 7.19 (br s, 1H), 4.34–4.53 (m, 1H), 4.11–4.30 (m,
2H), 3.23–3.40 (m, 2H), 3.07–3.19 ppm (m, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=172.3, 156.1, 144.2, 141.1, 128.1, 127.5, 125.8, 125.6,
120.5, 66.2, 53.3, 47.0, 26.2 ppm.
(S)-(9H-Fluoren-9-yl)methyl
1-cyano-2-(1H-tetrazol-5-yl)ethyl-
carbamate (13): A solution of 12 (0.75 g, 1.982 mmol) in CH2Cl2
(10 mL) was cooled in an ice bath and treated with DIPEA
(1.731 mL, 9.91 mmol), followed by the dropwise addition of TFAA
(0.700 mL, 4.96 mmol). The yellow solution was stirred until all
starting material was consumed (confirmed by TLC; 30 min). The
reaction was quenched with 5% HCl, the phases separated and
dried (Na2SO4), filtered and concentrated in vacuo. Purification by
reverse-phase chromatography (see General Methods) gave 13 as
a white powder (714 mg, 80%): [a]2D0 =ꢁ11 (c=0.5 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=8.35 (d, J=7.8 Hz, 1H), 7.78–7.95
(m, 2H), 7.54–7.74 (m, 2H), 7.34–7.46 (m, 2H), 7.30 (t, J=7.3 Hz,
2H), 6.33–6.83 (m, 1H), 4.99 (q, J=7.5 Hz, 1H), 4.38 (d, J=6.7 Hz,
1H), 4.13–4.28 (m, 2H), 3.46 (m, 1H), 1.18 ppm (dd, J=11.4, 7.6 Hz,
1H); 13C NMR (100 MHz, [D6]DMSO): d=157.0, 155.7, 144.4, 141.2,
128.0, 127.5, 125.6, 120.6, 120.5, 65.4, 47.2, 46.9, 41.2 ppm.
(S)-1-((S)-2-(4-Amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)-
N-((S)-1-cyano-2-(1H-tetrazol-5-yl)ethyl)pyrrolidine-2-carboxa-
mide (16): A solution of 13 (0.727 g, 2.016 mmol) in DMF (9 mL)
was cooled to 08C, treated with DBU (0.276 mL, 1.833 mmol) and
stirred for 5 min. (S)-1-((S)-2-(4-Amino-3-chlorobenzamido)-3,3-di-
methylbutanoyl)pyrrolidine-2-carboxylic acid (.7 g, 1.833 mmol),
DIPEA (0.480 mL, 2.75 mmol) and HATU (0.836 g, 2.200 mmol) were
then added sequentially to the solution. The solution was stirred at
08C for 2 h, diluted with EtOAc (30 mL) and quenched with satu-
rated aq NaHCO3. The organic phase was washed with NaHCO3 (ꢁ
2) and brine (ꢁ1), dried (Na2SO4), filtered and concentrated in
vacuo. Purification by reverse-phase chromatography (see General
Methods) gave 16 as a white powder (755 mg, 82%): [a]2D0 =ꢁ23
(c=0.5 in MeOH); 1H NMR (400 MHz, [D6]DMSO): d=8.87 (d, J=
7.6 Hz, 1H), 7.73–7.86 (m, 1H), 7.61–7.73 (m, 1H), 7.56 (dd, J=8.5,
1.9 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 5.15 (q, J=7.4 Hz, 1H), 4.64 (d,
J=8.6 Hz, 1H), 4.12–4.26 (m, 1H), 3.93–4.05 (m, 2H), 3.68–3.80 (m,
1H), 3.53–3.67 (m, 2H), 3.34–3.53 (m, 2H), 1.97–2.10 (m, 1H), 1.86–
1.97 (m, 1H), 1.75–1.86 (m, 1H), 1.57–1.75 (m, 1H), 0.99 ppm (s,
9H); 13C NMR (100 MHz, [D6]DMSO): d=172.3, 170.0, 165.9, 148.0,
129.4, 128.2, 122.1, 116.4, 114.4, 59.9, 57.8, 35.3, 29.5, 27.0,
25.1 ppm; LC/MS: Method 1, tR: 4.645 min; Method 2, tR: 3.596 min;
HRMS: m/z [M+H]+ calcd for C22H28N9O3Cl: 501.2004, found:
501.2004.
(S)-3-((S)-1-((S)-2-(4-Amino-3-chlorobenzamido)-3,3-dimethylbu-
tanoyl)pyrrolidine-2-carboxamido)-3-cyanopropanoic acid (4): A
solution of 7 (1.200 g, 2.75 mmol) in DMF (10 mL) was cooled to
08C and treated with DBU (0.414 mL, 2.75 mmol). The solution was
stirred for 5 min, and then (S)-1-((S)-2-(4-amino-3-chlorobenzami-
do)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxylic
acid
(1 g,
2.62 mmol), DIPEA (0.595 mL, 3.40 mmol) and HATU (1.195 g,
3.14 mmol) were added sequentially to the cold solution. The reac-
tion was stirred for 2 h at 08C, diluted with EtOAc (30 mL) and
quenched with saturated aq NaHCO3. The organic phase was
washed with NaHCO3 (ꢁ2) and brine (ꢁ1), dried (Na2SO4), filtered
and concentrated in vacuo. Purification by column chromatogra-
phy (CH2Cl2/MeOH; 95:5) gave the TMSE protected acid as a white
powder. A solution of this ester in THF (0.3m) was treated with a
1.0m THF solution of TBAF (2 equiv) and the reaction was stirred
for 1 h at RT. The reaction was diluted with EtOAc, washed with
water, dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by reverse-phase chromatography (see General Methods) gave
4 as a white powder (902 mg, 72% two steps): [a]2D0 =ꢁ65 (c=1.0
in MeOH); 1H NMR (400 MHz, [D6]DMSO): d=8.68 (d, J=7.4 Hz,
1H), 7.79 (d, J=1.9 Hz, 1H), 7.64 (d, J=9.0 Hz, 1H), 7.56 (dd, J=
8.6, 1.9 Hz, 1H), 6.73 (d, J=8.6 Hz, 1H), 5.95 (br s, 2H), 4.83 (q, J=
7.4 Hz, 1H), 4.64 (d, J=9.0 Hz, 1H), 4.20–4.30 (dd, J=8.2, 5.6 Hz
Acknowledgements
We thank Ms. Allison Mandich for critical reading of this manu-
script and Mr. Paul Shinn for assistance with compound manage-
ChemMedChem 2010, 5, 730 – 738
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
737

C. J. Thomas et al.
MED
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Keywords: caspases · covalent modifiers · cysteine proteases ·
inhibitors · VRT-043198 · VX-765
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