Design and synthesis of a potent and selective peptidomimetic inhibitor of caspase-3

Article abstract of DOI:10.1021/jm049248f

In this paper we report the synthesis and characterization of a novel potent and selective inhibitor of caspase-3, a member of the caspase family of cysteine proteases which plays an important role in many human disorders. This molecule represents 3(S)-acetylamino-N-{1-[(((3S)-2-hydroxy-5-oxo- tetrahydrofuran-3-yl)carbamoyl)methyl]-2-oxo-5-phenyl-2,3-dihydro-1H/-benzo[e] [1,4]diazepin-3-yl}succinamic acid, a monocyclic conformationally constrained form of the tetrapeptide Ac-DEVD-H, in which a 1,4-benzodiazepine nucleus is introduced internally to the peptidic sequence.

Full text of DOI:10.1021/jm049248f

J. Med. Chem. 2004, 47, 6455-6458
6455
Chart 1
Design and Synthesis of a Potent and
Selective Peptidomimetic Inhibitor of
Caspase-3
Nicola Micale,*,† Rajendran Vairagoundar,‡
Alexander G. Yakovlev,§ and Alan P. Kozikowski†
Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois at Chicago, 833 South Wood Street,
Illinois 60612, ChemBridge Research Laboratories,
16981 Via Tazon, San Diego, California 92127,
and Georgetown University Medical Center,
3970 Reservoir Road, Washington, D.C. 20057
Received September 15, 2004
Abstract: In this paper we report the synthesis and charac-
terization of a novel potent and selective inhibitor of caspase-
3, a member of the caspase family of cysteine proteases which
plays an important role in many human disorders. This
molecule represents 3(S)-acetylamino-N-{1-[(((3S)-2-hydroxy-
5-oxo-tetrahydrofuran-3-yl)carbamoyl)methyl]-2-oxo-5-phenyl-
2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl}succinamic acid, a
monocyclic conformationally constrained form of the tetrapep-
tide Ac-DEVD-H, in which a 1,4-benzodiazepine nucleus is
introduced internally to the peptidic sequence.
P3 amido hydrogens, which are best retained for high-
affinity binding.7
Furthermore, one of the most striking features in the
design of peptidomimetics is the incorporation of non-
peptidic scaffolds into the amino acidic sequences with
the perspective to force peptides into bioactive confor-
mations, enhance stability toward degradation by en-
zymes, and improve biological selectivity.9
The caspase (cysteinyl-aspartate protease) family
represents a class of intracellular proteases playing a
critical role in apoptotic cell death pathways and activa-
tion of pro-inflammatory cytokines.1 Their enzymatic
properties are governed by a nearly absolute specificity
for substrates containing aspartic acid at the P1 site and
by the use of a cysteine side-chain for peptide-bond
hydrolysis.2
Taking into account these considerations, we synthe-
sized a monocyclic conformationally constrained ana-
logue of Ac-DEVD-H in which the P2 amide nitrogen
has been “tied back” to the P3 side chain with the
purpose to improve the selectivity for caspase-3, analo-
gously to what was observed by other authors.10 In this
work, by using a 3-amino-2,3-dihydro-2-oxo-5-phenyl-
1H-benzo[e][1,4]diazepin-1-acetic acid moiety11 as a P3-
P2 dipeptide replacement, a novel, potent, and specific
inhibitor 16 of caspase-3 has been discovered (Chart 1).
The strategy adopted by us to obtain this compound
required the construction of the peptidomimetic frame
containing the diazepine ring (8), and subsequent in situ
coupling with the aspartyl aldehyde building block (13),
followed by coupling with the N-Ac-aspartic acid at the
P4 site.
To date, 11 human caspases have been identified.3
Among them, caspase-3 plays a role of the major
executioner in cell death triggered by a variety of pro-
apoptotic stimuli.1c,4
Since activation of caspase-3-dependent apoptotic cell
death has been implicated in the etiology of many
harmful human disorders, such as immunodeficiency,
Alzheimer’s, Parkinson’s, and Huntington’s diseases, as
well as ischemia, brain trauma, and amyotrophic lateral
sclerosis, inhibition of this caspase is believed to be a
valuable therapeutic approach.3d,5
Previous peptidomimetic studies6 resulted in identi-
fication of a semispecific inhibitor of caspase-3. This
inhibitor is the tetrapeptide Ac-Asp-Glu-Val-Asp-CHO3a
(Ac-DEVD-H, Chart 1) with an aldehyde functional
group that reversibly forms a covalent bond with the
thiol of the active-site cysteine, thereby inactivating it.7
While the Asp residue at P1 is strictly required for
activity, the Asp at P4 represents the most critical
determinant of the inhibitor’s specificity.3a Liberal
substitution is tolerated at P2 without loss of potency.
The P2 amide nitrogen is not utilized in a hydrogen
bonding interaction with the enzyme,8 unlike the P1 and
The synthesis of the 5-phenyl-1,4-benzodiazepine ring
system was accomplished according to a well-known
sequence of reactions shown in Scheme 1 by condensa-
tion of 2-aminobenzophenone (1) and N-(benzyloxycar-
bonyl)-2-(propylthio)glycine (2),12 a protected R-ami-
noglycine equivalent, which in turn was obtained in two
steps from glyoxylic acid, benzyl carbamate, and 1-pro-
panethiol.13
The building block 2 thus formed was converted to
its mixed anhydride with isobutyl chloroformate in the
presence of N-methylmorpholine and reacted in situ
with 2-aminobenzophenone (1) to give the amide 3 in
good yield after extractive workup. In the critical step,
the (propylthio)glycinamide 3 was dissolved in dry
tetrahydrofuran, and the solution was cooled to 0 °C
and saturated with ammonia. Mercuric chloride was
then added in one portion to the stirred mixture, while
a continuous stream of ammonia gas was bubbled
* To whom correspondence should be addressed. Present address:
Dipartimento Farmaco-Chimico, Viale Annunziata, 98168, Messina,
Italy. Tel: +39 090 6766466. Fax: +39 090 355613. E-mail: nmicale@
pharma.unime.it.
† University of Illinois at Chicago.
‡ ChemBridge Research Laboratories.
§ Georgetown University Medical Center.
10.1021/jm049248f CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/18/2004

6456 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Letters
Scheme 1a
Scheme 2a
a Reagents: (a) i-BuOCOCl, N-methylmorpholine, CH2Cl2, 0 °C,
15 min, then 0 °C - rt, 12 h. (b) NH3 (g), THF, 0 °C - rt, 3 h. (c)
NH4OAc, HOAc, rt, 12 h. (d) Et3SiH, Pd(OAc)2, CH2Cl2, rt, 3 h.
(e) (Boc)2O, CH2Cl2, rt, 2 h. (f) NaH, DMF, 0 °C, 30 min, then
BrCH2COOEt, 0 °C - rt, 4 h. (g) 1 N LiOH, MeOH, 0 °C - rt, 6
h.
into it. The intermediate R-aminoglycinamide 4 was
obtained in essentially quantitative yield and immedi-
ately treated with ammonium acetate in glacial acetic
acid with stirring at room-temperature overnight to
allow the cyclization. In this way, 5 was obtained as a
crystalline solid in 70-75% yield. Deprotection of the
NH-Cbz group of 5 was carried out with triethylsilane
and palladium acetate as catalyst.14 Quenching with a
solution of aqueous ammonium chloride and brine
followed by extraction of the reaction mixture removed
the excess of silane reagent and provided the crude
amine which was reacted with 1.25 equiv of (Boc)2O
without further purification to give 6 in good yield. The
side chain at N-1 was introduced by treatment with
sodium hydride and ethyl bromoacetate in dimethylfor-
mamide at ambient temperature to provide 7 after
chromatographic purification. Saponification of the ester
group with 1 N LiOH in methanol afforded the dipep-
tidic N-protected intermediate 8 which is equivalent to
the P3-P2 sites of Ac-DEVD-H.
The building block 12 was synthesized as described
in Scheme 2. First, commercially available aspartic acid
â-tert-butyl ester 9 was N-protected with allyl chloro-
formate in the presence of sodium bicarbonate. The
product 10 was then converted to a mixed anhydride
with isobutyl chloroformate and N-methylmorpholine,
which was reduced to the corresponding alcohol 11 with
sodium borohydride. The alcohol 11 was oxidized to the
aldehyde under Swern conditions,15 and the latter
immediately protected with benzyl alcohol and p-tolu-
enesulfonic acid in the presence of 3 Å molecular sieves.
Trifluoroacetic acid (TFA)-promoted deprotection of the
tert-butyl ester moiety was accompanied by cyclization
a Reagents: (a) Allyl chloroformate, aq NaHCO3/THF (8/2), rt,
12 h; (b) i-BuOCOCl, N-methylmorpholine, CH2Cl2, 0 °C, 30 min;
(c) NaBH4, MeOH/THF (5/1), -78 °C, 4 h; (d) (COCl)2, DMSO,
i-Pr2NEt, -65 °C, 1 h; (e) BnOH, TsOH, 3 Å mol. sieves, CH2Cl2,
rt, 16 h; (f) CF3CO2H, CH2Cl2, rt, 1 h; (g) PdCl2(PPh3)2, n-Bu3SnH,
CH2Cl2, 0 °C, 30 min; (h) EDCI, HOBt, CH2Cl2, rt, 15 h; (i)
CF3CO2H, CH2Cl2, rt, 4 h; (j) Ac-Asp(OBn)-OH, EDCI, HOBt,
CH2Cl2, rt, 15 h; (k) H2, Pd(OH)2/C, EtOH, 6 h.
to the desired acylal 12, which was isolated as a 1:1
mixture of diastereomers.
Cleavage of the N-allyloxycarbonyl (Alloc) group16
gave the intermediate 13 which, being volatile and
sensitive to acid and base,17 was not isolated and was
in situ coupled to 8 for an efficient peptide elongation.
To incorporate the last aspartate residue at the P4
position, the Boc group in 14 was first removed by TFA,
and the resulting amine was coupled to N-acetylaspartic
acid â-benzyl ester by the procedure reported above.
Hydrogenolysis of the tetrapeptide 15 using Pd(OH)2/C
as catalyst in ethanol provided the final 3(S)-acety-
lamino-N-{1-[(((3S)-2-hydroxy-5-oxo-tetrahydrofuran-3-
yl)carbamoyl)methyl]-2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diazepin-3-yl}succinamic acid 16 in a
reasonable yield.
Potency of the synthesized compound in the inhibition
of caspase-3 was tested in vitro using the pure recom-
binant active human enzyme expressed in E. coli.
Briefly, 2 Units of caspase-3 was added to the reaction

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6457
Figure 3. Protective effect of 16 in a model of etoposide-
induced apoptosis in SH-SY5Y cells. Apoptosis was induced
by addition of 10 µM etoposide to cell cultures. (A) After 24 h
of treatment in the absence or presence of indicated concentra-
tions of caspase inhibitors, cells were stained with 5 µM calcein
AM, which is deesterified into a fluorescent product by viable
cells. Data are represented as the percent-to-control average
number of fluorescent cells + STDEV (n ) 4). (B) Caspase-3-
like activity was measured in situ after 6 h of treatment with
etoposide. Cleavage of Ac-DEVD-AMC was assayed fluoro-
metrically by measuring the accumulation of free AMC.
Figure 1. Determination of the inhibitory potency of com-
pound 16 against recombinant human caspase-3. Dissociation
constant (Ki) was calculated using the Graphpad Prism 4
computer program.
caspases-1-9 and the specific substrates for each indi-
vidual caspase. In these experiments we used 210 nM
of the inhibitor, a concentration approximately 6 times
higher than the estimated Ki value for caspase-3 inhibi-
tion. Control reactions included caspases with their
specific substrates in the absence of 16. Results that
are summarized in Figure 2 demonstrate that 16 most
potently inhibits caspase-3. Activity of this caspase in
the presence of 16 was reduced to 9 ( 2% of the control
level, whereas activity of other 8 caspases remained
essentially higher, from 58 ( 2% for caspase-6 to 95 (
4% for caspase-4. Overall, these data demonstrate
marked selectivity of the developed compound against
human caspase-3.
Relatively poor membrane permeability of the de-
scribed earlier caspase inhibitors limits possibility of
their use as anti-apoptotic drugs. Therefore, our es-
sential goal was to develop a cell permeable compound.
Cell permeability of 16 was indirectly assessed by
measuring caspase-3 activity and viability of SH-SY5Y
human neuroblastoma cells treated with 10 µM etopo-
side. Z-DEVD-FMK was used as a positive control in
parallel to 16. Results indicate that both compounds
demonstrate similar levels of protection, as shown at
the level of caspase-3 activity and cell viability (Figure
3). A fact that compound 16 is 10 times less potent than
Z-DEVD-FMK and represents a reversible inhibitor,
while Z-DEVD-FMK is irreversible, may indicate that
compound 16 demonstrates higher cell permeability.
Taken together, we synthesized a conformationally
constrained surrogate of the caspase-3 cleavage site
DEVD, in which the dipeptide Val-Glu was replaced
with a 1,4-benzodiazepine moiety. The results obtained
demonstrate that, while the inhibitory activity (Ki) of
this new compound is somewhat lower than that of the
commonly used tetrapeptide, its selectivity for caspase-3
and the ability to inhibit apoptosis in live cells make it
an attractive target for further development.
Figure 2. Determination of the inhibitory specificity of
compound 16. Two Units of each recombinant caspase were
incubated with their specific substrates in the absence or
presence of 210 nM compound 16. Ten µM concentrations of
each Ac-YVAD-AMC for caspase-1, Ac-VDVAD-AMC for
caspase-2, Ac-DEVD-AMC for caspases-3 and -7, Ac-LEVD-
AMC for caspase-4, Ac-WEHD-AMC for caspase-5, Ac-VEID-
AMC for caspase-6, Ac-IETD-AMC for caspase-8, and Ac-
LEHD-AMC for caspase-9 were used to analyze activity of the
caspases in the fluorometric assay by measuring the ac-
cumulation of free AMC. Caspase activities in the presence of
compound 16 are expressed as percent of those in the absence
of the inhibitor.
mixture containing increasing from 0 to 3 µM concen-
trations of a semispecific Z-DEVD-AMC substrate and
increasing from 0 to 3.3 µM concentrations of compound
16. Caspase-3 activity results in the cleavage of Z-
DEVD-AMC after Asp at P1 site, thus releasing the
fluorescent AMC group. Hence, fluorescence of AMC in
a sample reflects actual activity of the enzyme. We
performed measurements of AMC liberation as a func-
tion of time using a fluorescent plate reader as describe
earlier.3a Results were expressed as mean values of
three independent measurements per each experimental
point. A value for the dissociation constant Ki was
computed by fit of the reaction rates to the Michaelis-
Menten equation. Thus, Ki for 16 was estimated to be
as low as 36(9 nM at 95% confidence intervals (Figure
1). Parallel experiments demonstrated that the Ki for
Ac-DEVD-CHO, a common tetrapeptide inhibitor of
caspases-3 and -7, was equal to 3.2 ( 6 nM under the
same experimental conditions (data not shown).
Acknowledgment. The authors wish to thank Guy
S. Salvesen and Annamarie Price (The Burnham Insti-
tute, La Jolla, CA) for the biological testing of compound
16 and Werner Tueckmantel for his editorial comments
on the manuscript. Financial support from MURST is
gratefully acknowledged.
Specificity of compound 16 was estimated in the
reactions with a panel of active recombinant human

6458 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Letters
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791-800.
Supporting Information Available: Experimental pro-
cedures, characterization of new compounds, and references
to known procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
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