Noncovalent tripeptidyl benzyl- And cyclohexyl-amine inhibitors of the cysteine protease caspase-1

Article abstract of DOI:10.1021/jm901790w

Potent and noncovalent inhibitors of caspase-1 were produced by incorporating a secondary amine (reduced amide) isostere in place of the conventional electrophile (e.g., aldehyde) that normally confers high potency to cysteine protease inhibitors. Benzyl- or cyclohexylamines produced potent, reversible, and competitive inhibitors that were selective for caspase-1 (e.g., K_i = 47 nM) over caspases 3 and 8 with minimal cytotoxicity. Unlike most cysteine protease inhibitors, these compounds do not react covalently and indiscriminately with thiols.

Full text of DOI:10.1021/jm901790w

J. Med. Chem. 2010, 53, 2651–2655 2651
DOI: 10.1021/jm901790w
Noncovalent Tripeptidyl Benzyl- and Cyclohexyl-Amine Inhibitors of the Cysteine Protease Caspase-1
†
Reik Loser, Giovanni Abbenante, Praveen K. Madala, Maria Halili, Giang T. Le, and David P. Fairlie*
€
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072,
Australia. †Present address: Forschungszentrum Dresden-Rossendorf, Institut fu€r Radiopharmazie, 01328 Dresden, Germany.
Received December 2, 2009
Potent and noncovalent inhibitors of caspase-1 were produced by incorporating a secondary amine
(reduced amide) isostere in place of the conventional electrophile (e.g., aldehyde) that normally confers
high potency to cysteine protease inhibitors. Benzyl- or cyclohexylamines produced potent, reversible,
and competitive inhibitors that were selective for caspase-1 (e.g., Ki = 47 nM) over caspases 3 and 8 with
minimal cytotoxicity. Unlike most cysteine protease inhibitors, these compounds do not react
covalently and indiscriminately with thiols.
Introduction
Results and Discussion
A common strategy for inhibiting some classes of enzymes
istoincorporatea reactiveelectrophile(e.g., aldehyde, ketone,
nitrile, chloromethyl ketone, epoxide) in a compound to
induce covalent bonding with an enzyme active site nucleo-
phile, such as the side chain of a serine or cysteine residue.1,2
Such electrophiles can confer up to 103- to 104-fold enhance-
ments to inhibitor potency of enzyme inhibitors, but also
result in indiscriminant interactions with other nucleophiles in
vivo, leading to side effects and reduced bioavailability.1
Cysteine proteases are pivotal enzymes in tumor metastasis
(e.g., cathepsin B), osteoporosis (cathepsins K, L), athero-
sclerosis, inflammation (cathepsin S), aging, apoptosis, neu-
rodegenerative diseases (caspases 3, 8), malaria (falcipain 2
and 3), Chagas disease (cruzipain), and viral infections like the
common cold (rhinovirus 3C protease). Many potent inhibi-
tors of cysteine proteases have been developed,1,2 but few have
progressed in clinical trials and none are in the marketplace.1,2
Here, we report a series of secondary/tertiary amines with-
out electrophiles that are the first potent and stable non-
covalent inhibitors of caspase-1, an important proinflamma-
tory cysteine protease that converts interleukin-1 (IL-1) to the
inflammatory cytokine IL-1β. Caspase-1 inhibition reduces
formation of IL-1β in vitro and in vivo, with efficacy in
inflammatory diseases in animals and humans.3 No caspase-1
inhibitor has succeeded in clinical trials.1,3 A few examples of
noncovalent inhibitors of the lysosomal cysteine proteases,
cathepsins K, S, and L, have been described,4-6 but there are
no examples of stable noncovalent inhibitors for caspases,
some of which are key enzyme mediators of cell death. Indeed,
the presence of an electrophilic isostere is still thought by most
to be essential for producing potent inhibitors of caspases.1-3
Our strategy to inhibit cysteine proteases avoids covalent
bonding to the cysteine, instead obtaining affinity by targeting
a prime side binding site. This could be a valuable approach to
other inhibitors of cysteine proteases, including cathepsins
and other caspases.
Our initial lead compound was the benzylamine 3 (Table 1),
featuring a secondary amine at the C-terminus or P10 position.
Reduction of a peptide bond to a secondary amine at a pepti-
dase cleavage site was successfully used in the design and
development ofaspartic proteaseinhibitors7 and hasalso been
applied to inhibitor development for a cysteine protease from
Leishmania mexicana.8 Replacement of a peptide moiety, at
the prime side binding sites of inhibitors containing secondary
amines, with an aryl residue has been suggested to lead to
inhibitors of thiol-dependent cathepsins.4
The peptidic moiety in 3 (2-Nap-Val-Ala-Asp) was pre-
viously identified for selective inhibition of caspase-1,9 and we
have made no attempt in this paper to improve this segment or
to make it nonpeptidic. Instead we have focused on investi-
gating alternatives to electrophilic isosteres appended to the
C-terminal asparate residue. N-Capped tripeptidyl fragments
such as 2Nap-Val-Ala-Asp were prepared (Scheme 1) by
starting with Z-Asp(OtBu)-ol 1.10 Deprotection with hydro-
gen, catalyzed by Pd/C, and reaction of the resultant free
amine with Z-Ala-OH, using HBTUa for coupling, gave di-
peptide alcohol Z-Ala-Asp(OtBu)-ol. Sequential deprotec-
tion, followed by coupling of Z-Val-OH then 2-naphthoic
acid, yielded alcohol 2. Oxidation with Dess-Martin reagent,
reductive amination with various benzylamines or cyclohexyl-
amine, and deprotection with TFA yielded target compounds
3-12 (Scheme 1, Table 1).
With the GOLD program, 3 was docked into the substrate-
binding site of caspase-1 (Figure 1). The model predicted that
the benzylamine portion of 3 would project into the prime side
S10 binding site of the enzyme. In this binding region, the side
chains of enzyme residues Asp288 and His248 were identified
as potential partners for polar interactions with inhibitors. It
wasenvisagedthatthose residuescouldpotentially betargeted
aAbbreviations: RT, room temperature; 2-Nap, 2-naphthyl; IL, interleukin;
TCP, tritylchloride polystyrene; HBTU, O-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluoroborate; DIPEA, N,N-diisopro-
pylethylamine; SPPS, solid phase peptide synthesis; AMC, 7-amino-
4-methylcoumarine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]
1-propanesulfonate; MTT, methylthiazoletetrazolium.
*To whom correspondence should be addressed. Phone: þ61 7 33462989.
Fax: þ61 7 33462990. E-mail: d.fairlie@imb.uq.edu.au.
r
2010 American Chemical Society
Published on Web 02/19/2010
pubs.acs.org/jmc

2652 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Table 1. Benzylamine Inhibitors of Caspase-1
Lo€ser et al.
Figure 1. Modeled conformation of 4 docked in the active site of
caspase-1 (PDB: 2HBQ). The gray (hydrophobic), blue (basic), and
red (acidic) surface represents the active site of the enzyme. The
green stick diagram represents ligand 4. The proposed hydrogen
bond is indicated by a dashed line.
Scheme 1. Synthesis of Tripeptide-Derived Amines 3-12a
by substituents at the ortho or para positions, respectively,
of the benzyl moiety. A hydroxyl substituent was chosen for
this task, potentially making a hydrogen bond from 4 and
7 to Asp288 or His248, respectively. These compounds were
synthesized and examined for inhibition of human caspase-1.
Compound 4 proved to be >10-fold more potent as an
inhibitor of caspase-1 than its parent compound 3. To esta-
blish the reason for this increase in potency, the o-hydroxy
group in 4 was replaced by an o-fluorine substitutent (5) or an
o-methoxy group (6), resulting in 25- and 20-fold respective
decreases in inhibitory potency versus 4.
a (a) H2/Pd/C, 1% TFA/EtOAc, RT; (b) Z-Ala-OH, HBTU, DIPEA,
DMF, RT; (c) Z-Val-OH, HBTU, DIPEA, DMF, RT; (d) 2-naphthoic
acid, HBTU, DIPEA, DMF, RT; (e) Dess-Martin reagent, CH2Cl2,
RT; (f) RNHR0, NaBH(OAc)3, CH2Cl2, RT; (g) TFA/CH2Cl2 1:1, RT.
The fluorine substituent in 5 retains the electronegative
character and steric demand of the hydroxyl group in 4 but
removes the H-bond donating property. The methoxy group
in 6 removes the H-bond donor but maintains the H-bond
accepting oxygen at the ortho position. These assay results
indicate that the o-hydroxy group acts as a hydrogen bond
donor, as predicted from modeling of 4 in the enzyme active
site (Figure 1). The model also supported an extended con-
formation11 for the inhibitor, orienting the 2-naphthyl residue
into the S4 subsite of the enzyme, with Val, Ala, and Asp
residues in the S3, S2, and S1 subsites, respectively. The
2-hydroxybenzylamine portion of the molecule was oriented
into the S10 binding pocket. The protonated amine nitrogen
atom was found to position between the catalytic residues Cys
because of a longer distance between the hydroxyl group and
the His side chain. Therefore, the hydroxyl substituent in 7
was replaced by a primary amide in 8 to achieve better
interactions with the His side chain. If the imidazole of
His237 is protonated, it may hydrogen-bond to the primary
amide that can serve as an H-bond acceptor or as well a H-
bond donor if the His is unprotonated. However, 8 was even
less active than 7 as an inhibitor of caspase-1 (Table 1). Thus,
His248 seemed to be difficult to target by H-bond forming
functional groups.
The docking of 3 in the enzyme had suggested that there is
more space in the prime side binding site that could potentially
be occupied by extension of the benzyl residue. It was thus
decided to prepare 9, bearing a piperonyl substituent at the
amine nitrogen. This residue is a privileged structure in
numerous bioactive natural and synthetic products. Indeed,
9 was a 3-fold more potent inhibitor than 3. Modeling
had predicted that the bicyclic benzodioxole component of
9 would fill the P10 side pocket of caspase-1 very well
˚
˚
285 (N S = 3.74 A) and His 237 (N N = 2.81 A), with a
3 3 3
3 3 3
predicted hydrogen-bonding interaction with His237. The
2-hydroxybenzyl group fills the S10 subsite, with the 2-OH
function directed toward Asp288 and potentially forming a
hydrogen bond with the carboxylic acid side chain of this
˚
residue (O O = 2.71 A).
3 3 3
Compound 7 was synthesized with the intention of target-
ing His248 through polar interactions exerted by the hydroxy
group in the para position. In comparison to unsubstituted
3, 7 proved to be a less potent caspase-1 inhibitor probably

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2653
Scheme 2. Synthesis of Carba Analogue 15a
a (a) EtOAc, reflux; (b) H2 (40 psi), Pd/C, 3% TFA/ethyl acetate, RT; (c) TFA, RT; (d) FmocOSu, NaHCO3, THF/H2O 1:1, 4 °C f RT; (e) TCP resin,
DMF, RT.
(Supporting Information). The CH-Cipso-CH2-NH dihe-
dral angle in the piperonyl derivative 9 suggests that the
piperonyl ring is oriented perpendicularly to the plane of the
benzene ring of 4. This leads to the conclusion that it may be
difficult to structurally combine the space-filling properties of
bicyclic moieties with an H-bond donor that targets Asp 288.
Thus, when the aromatic benzyl moiety was replaced by
cyclohexyl, as in 10, there was a slight decrease in inhibitory
potency compared to 3.
Attention was next focused on the potential impact of the
basic nitrogen on the inhibitory potency. The tertiary methyl-
amine 11 proved to be 3 times less potent as an inhibitor than
the corresponding secondary amine 3. In the case of the
cyclohexylamines 10 and 12, the tertiary methylamine 12 was
more active than its secondary amine counterpart 10. The
importance of the amino functionality to the potency of these
inhibitors is highlighted by the carba analogue 15, which was
synthesized from the aldehyde Z-Asp(OtBu)-H10 (Scheme 2).
A Wittig reaction of this aldehyde with 1-phenyl-2-(triphenyl-
λ5-phosphanylidene)ethanone,12 a stabilized phosphorus ylide,
gave intermediate enone 13 that could be completely reduced
and N-deprotected with hydrogen catalyzed by Pd/C. Deprotection
with TFA and introduction of the Fmoc protecting group via
Fmoc-OSu gave 14. Compound 14 was attached to TCP resin
and the target compound 15 assembled via standard solid phase
peptide synthesis (Supporting Information). Carba analogue
15 was a very poor inhibitor of caspase-1, suggesting that the
amino group of these inhibitors forms crucial polar contacts
with the enzyme. None of the compounds described herein
showed any detectable cytotoxicity, at the concentrations tested
(e200 μM), in an MTT assay performed with HT29 human
cells (see Experimental Methods).
Figure 2. Cornish-Bowden plot showing competitive inhibition of
caspase-1 by 4. The steady-state rates for caspase-1 catalyzed
hydrolysis of Ac-Trp-Glu-His-Asp-AMC were measured in the
presence of different concentrations of inhibitor 4 ([4] = 0, 50, 80,
100, 200, 300, 400, 500 nM). The substrate concentrations were
8 μM (squares), 20 μM (triangles), and 30 μM (lozenges).
varying substrate concentrations. Data were analyzed using a
Cornish-Bowden plot,13 which revealed a set of approxi-
mately parallel lines (Figure 2). This confirms a competitive
binding and inhibition mode, as expected for such substrate-
like inhibitors.13
The tripeptide-derived amines are slow-binding inhibitors for
caspase-1. All Ki values were determined in preincubation assays
(Supporting Information). As an example, 4 was also evaluated
in an assay in which the reaction was started by adding enzyme to
follow the binding kinetics (Supporting Information). This led to
confirmation of a one-step mechanism for the enzyme-inhibitor
interaction, with a second-order rate rate constant for formation
of the enzyme-inhibitor complex of kon = 1389 M-1 s-1 and a
first-order rate constant describing the decay of the complex
koff = 6.5 ꢀ 10-5 s-1 14
. Compound 4 was also evaluated for
All of the structure-activity relationships interpreted
above are based on the assumption that every compound
binds in the substrate-binding active site of the enzyme. To
establish that the benzylamine compounds were indeed direc-
ted toward the active site, we needed to demonstrate that
inhibition was competitive with substrate. To facilitate this
analysis, the inhibition by 4 was investigated in more detail at
caspase selectivity by testing against caspases 3 and 8 (Figure S2
of Supporting Information). No inhibition of caspase-3 was
observed up to micromolar concentrations, while some inhibi-
tion of caspase-8 was detected at high micromolar concentra-
tions. This selectivity for caspase-1 is undoubtedly a result of the
naphthyl group being at P4 in 4 and has little to do with the
benzylamine isostere.

2654 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Lo€ser et al.
Chart 1. Aniline Derivative 16 and Corresponding Aldehyde 17
serine protease inhibitors and has since translated effectively
into clinical trials.1
Experimental Methods
Synthesis of Tripeptidylamine-Based Inhibitors of Caspase-1.
The synthesis of 4 only is described here, while the synthesis of
all other compounds is reported in the Supporting Information.
(S)-3-((S)-2-((S)-2-(2-Naphthamido)-3-methylbutanamido)-
propanamido)-4-(2-hydroxy)benzylaminobutanoic Acid, 4. This
is also a general procedure used for the syntheses of tripeptidyl-
amines 3-12. The tetrapeptide aldehyde (0.05 g, 0.1 mmol)
was dissolved in dichloromethane (5 mL) and benzylamine
(0.013 mL, 0.12 mmol) (or an equivalent amount of substituted
benzylamine or cyclohexylamine) was added followed by solid
sodium triacetoxyborohydride (0.11 g, 0.5 mmol). After the
mixture was stirred at room temperature for 2 h, the solvent was
removed in vacuo and the residue suspended in 1 N NaOH and
extracted with ethyl acetate (3 ꢀ 20 mL). The combined organic
layers were washed with saturated NaCl (10 mL), dried over
MgSO4, and evaporated to yield an oily colorless residue which
was taken up in CH2Cl2/TFA (1:1, 10 mL). After the mixture
was stirred for 2 h, the volatiles were removed in the N2 stream.
The oily material was subjected to purification by preparative
HPLC. The product-containing fractions (monitored by ESMS)
were combined and lyophilized to give the pure final product 4,
as the trifluoroacetate salt, in 30% overall yield. 1H NMR (600
MHz, DMSO-d6) δ in ppm = 0.95 (3J = 6.5 Hz, 3H, Val-CH3);
0.96 (3J = 6.5 Hz, 3H, Val-CH3); 1.23 (d, 3J = 7.0 Hz, 3H, Ala-
CH3); 2.10-2.19 (m, 1H, Val-CβH); 2.53 (d, 3J = 6.4 Hz, Asp-
CH2); 3.08-3.15 (m, 2H, CHCH2NH); 4.06-4.15 (m, 2H,
PhCH2NH); 4.19-4.25 (m, 1H, Ala-CH); 4.26-4.37 (m, 2H,
Asp-CH, Val-CRH); 6.80-6.85 (m, 1H, Ph-5-H); 6.90 (br d,
3J = 8.1 Hz, 1H, Ph-3-H); 7.20-7.25 (m, 1H, Ph-4-H); 7.31 (br
d, 1H, 7.5 Hz, Ph-6-H); 7.57-7.64 (m, 2H, Nap-6-H, 7-H);
7.92-8.00 (m, 4H, Nap-3-H, 4-H, 5-H, Asp-NH); 8.03 (br d,
3J = 7.9 Hz, 1H, Nap-8-H); 8.25 (d, 3J = 6.8 Hz, 1H, Ala-NH);
8.42 (d, 3J = 8.2 Hz, Val-NH); 8.50 (br s, 1H, Nap-1-H); 10.21
(br s, OH). 13C NMR (150 MHz, DMSO-d6) δ in ppm = 17.63
(Ala-CH3); 18.95 (Val-CH3); 19.32 (Val-CH3); 30.14 ((CH3)2-
CH); 36.44 (CH2COOH); 43.45 (Asp-CH); 46.00 (PhCH2NH);
48.61 (Ala-CH); 49.14 (CHCH2NH); 59.14 (Val-CH); 115.29
(Ph-3-C); 117.88 (Ph-1-C); 119.08 (Ph-5-C); 124.47, 126.72,
127.61, 127.64, 127.75, 127.78, 128.85, 130.55, 131.43 (CHarom);
131.50, 132.08, 134.19 (Carom); 156.06 (Ph-2-C); 166.75
(Nap-CO); 171.04 (Val-CO); 171.66 (COOH); 172.46 (Ala-CO).
HR-ESMS calcd for C30H37N4O6 [M þ H]þ = 549.2713, found
549.2701. Analytical HPLC 100% A to 100% B in 20 min: %
purity = 98.3%, tR = 13.73 min.
With regard to inhibitor stability, the benzylamines described
here represent an important advance over covalent and non-
covalent inhibitors previously described for cysteine proteases.
Covalent inhibitors that possess a reactive functional group for
interaction with the active site of the enzyme are generally prone
to racemization. For example, the well-known aldehyde inhibi-
tor of caspase-1, Ac-Tyr-Val-Ala-Asp-H, readily racemizes in
solution at room temperature.9 Noncovalent arylamines have
been reported as inhibitors of cathepsins K and S,4 but we have
found that such compounds are highly susceptible to oxidation,
forming traces of aldehyde intermediates. The presence of even
trace amounts of aldehyde in noncovalent arylamines can confer
the impression of potent inhibition and thus seriously compro-
mise their assessment as potent and noncovalent inhibitors, as
found for azide inhibitors of cysteine proteases.15 For example,
when 16 (Chart 1) was allowed to stand for a few weeks at room
temperature, its apparent potency against caspase-1 increased
dramatically. This is attributed to the presence of trace amounts
(1-2%) of the potent aldehyde inhibitor 17 (IC50 = 0.2 nM)
detected after aging the samples for 2 weeks (Supporting
Information). The aldehyde was produced by oxidation of the
aromatic amine of 16.16 A series of arylethylamine inhibitors of
caspase-1 were similarly all found to increase in inhibitory
potency over time, and all showed the formation of traces of
17 in aged samples when analyzed by LCMS (data not shown).
By contrast, no aldehyde was detected after storing 4for <1 year
at room temperature.
Conclusion
Covalent interactions made between cysteine proteases and
their prospective inhibitors have previously been identified by
some as a major impediment to successful clinical develop-
ment of cysteine protease inhibitors.1,17 However, progress
toward the development of noncovalent inhibitors of cysteine
proteases, which by contrast do not indiscriminately interact
with numerous nucleophiles in vivo, has been very slow. The
present study has contributed one possible solution to this
problem. By incorporating a secondary amine (reduced
amide) isostere into putative inhibitors, with additional focus
on making key interactions between inhibitor and the prime
side binding sites of the enzyme, this study has produced
potent, reversible, and noncovalent inhibition of caspase-1.
This strategy of avoiding electrophilic isosteres could be
applied to developing inhibitors of other important cysteine
proteases, withthecaveatthat somearylamines, vide infra, are
unstable and convert to potent aldehyde contaminants that
can compromise enzyme inhibition results. We believe that
this field should progress from cysteine protease inhibitors
bearing electrophilic warheads to the design of noncovalent
inhibitors, a step that occurred successfully some years ago for
Methods for Molecular Docking. The crystal structure co-
ordinates of caspase-1 were obtained from PDB entry 2HBQ.18
Coordinates for peptidic ligands were developed using the Sybyl
sketch module and further refined by minimization using a
conjugate gradient method19 and molecular dynamics using
the Tripos force field.20 Docking of ligands was performed using
Gold, version 3.2,21 with default docking settings. The active site
of the enzyme was defined by taking Ser339 at the center of
˚
the active site with a 12 A radius. Ligands were docked in
30 independent experiments (each involving 100 structures and
up to 100 000 operations). Mutations, migration, and operator
weights for crossover were set to 95, 10, and 95, respectively. To
˚
allow poor nonbonded contacts, a maximum distance of 5 A was
set between hydrogen donor and fitting points. To speed up
˚
calculations, docking was stopped when rmsd = 1.5 A between
the top three solutions. Docking was done without any bonding
constraints between the protein and the ligand. The best-docked
conformation of the ligand was identified manually by considering
the highly conserved orientation with maximum number of inter-
actions within the active site.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2655
Inhibition Experiments and Inhibition Kinetics of Compound 4.
Recombinant human caspase-1 (Calbiochem) was diluted with
700 μL of buffer (identical to the assay medium except that it
lacks DMSO), divided into 70 μL aliquots, and stored at -80 °C.
A 1:20 dilution of these aliquots was used in the assays. Enzyme
activities were calculated from kinetic measurements by fluori-
metric detection of the product 7-amino-4-methylcoumarine
(AMC) at 30 °C in a plate reader (Polarstar BMG Labtech)
using black 96-well plates (final volume of 200 μL). The wave-
lengths for excitation and emission were 380 and 460 nm, respec-
tively. A 2 mM stock solution of the fluorogenic substrate Ac-Trp-
Glu-His-Asp-AMC (Calbiochem) was prepared in DMSO. The
final concentration was 5 μM. The assay medium consisted of
50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT, 1 mM
EDTA, 10% glycerol, 0.1% CHAPS, 1% DMSO. Stock solutions
of the inhibitors (each 5 mM) were prepared in DMSO.
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Inhibition by 4 of caspases 3 and 8 is described in the
Supporting Information (Figure S2).
MTT Cytotoxicity Assay. The cytotoxicity of the caspase-1
inhibitors was tested against HT29 colon cancer cells. Cells were
plated in a 96-well plate at 1 ꢀ 105 cells/well, using RPMI 1640
medium, and left to adhere overnight at 37 °C. The following day,
cells were treated with various drug concentrations and left to
incubate for 24 h at 37 °C. MTT dissolved in RPMI 1640 media
at 1 μg/mL was added to the cells (100 μL/well) and incubated for 1 h
at 37 °C to allow for MTT to be metabolized. Cells were then lysed
and crystals dissolved using isopropanol. Measurements were taken
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Acknowledgment. We thank the Australian Research
Council for a Federation Fellowship to D.P.F. and both
ARC and the National Health and Medical Research Council
of Australia for research support. R.L. thanks the German
Research Society (DFG) for a postdoctoral scholarship
(Grant LO1165/2-1) and Alun Jones for recording HRMS
spectra.
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by proteolytic enzymes. J. Mol. Recognit. 1999, 12, 363–370.
(12) Babu, K. S.; Li, X.-C.; Jacob, M. R.; Zhang, Q.; Khan, S. I.;
Ferreira, D.; Clark, A. M. Synthesis, antifungal activity, and
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J. Med. Chem. 2006, 49, 7877–7886.
Supporting Information Available: Synthetic procedures and
characterization data of all compounds, docking images for 3
and 9, inhibition kinetics of 4 and demonstration of selectivity
over caspases 3 and 8, and experimental evidence for the
autoxidation of 16. This material is available free of charge via
the Internet at http://pubs.acs.org.
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