Nicotinyl aspartyl ketones as inhibitors of caspase-3

Article abstract of DOI:10.1016/S0960-894X(03)00390-1

Caspase-3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. Since P₁ aspartic acid is a required element of recognition for this enzyme, a

Full text of DOI:10.1016/S0960-894X(03)00390-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2137–2140
Nicotinyl Aspartyl Ketones as Inhibitors of Caspase-3
Elise Isabel,a,* W. Cameron Black,a Christopher I. Bayly,a Erich L. Grimm,a
Marc K. Janes,a Daniel J. McKay,a Donald W. Nicholson,a Dita M. Rasper,a
Johanne Renaud,a Sophie Roy,a John Tam,a Nancy A. Thornberry,b
John P. Vaillancourt,a Steven Xanthoudakisa and Robert Zambonia
aMerck Frosst Centre for Therapeutic Research, Merck Frosst Canada & Co., PO Box 1005, Pointe-Claire-Dorval,
Quebec, Canada H9R 4P8
bMerck Research Laboratories, Rahway, NJ 07065, USA
Received 15 May 2002; accepted 8 April 2003
Abstract—Caspase-3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the
treatment of several degenerative diseases. Since P1 aspartic acid is a required element of recognition for this enzyme, a library of
capped aspartyl aldehydes was synthesized using solid-phase chemistry. The 5-bromonicotinamide derivative of the aspartic acid
aldehyde was identified to be an inhibitor of caspase-3. Substitution at the 5-position of the pyridine ring and conversion of the
aldehyde to ketones led to a series of potent inhibitors of caspase-3.
# 2003 Elsevier Science Ltd. All rights reserved.
The human caspases are a family of at least 11 cysteinyl-
aspartate-specific proteinases that are central compo-
nents in the molecular pathways that result in the
apoptosis of cells.1 These enzymes are divided into three
groups. Group I caspases (1, 4, and 5) mediate cytokine
maturation and are implicated in the inflammatory
response. Group II caspases (2, 3 and 7) are the major
effectors of cell death. Group III caspases (6, 8, 9, and
10) are upstream activator enzymes of the group II cas-
pases. Caspase-14 is a heratinocyte specific caspase that
has not yet been classified. Caspase-3 appears to be a
critical participant in apoptosis in neurons. Prototype
peptidyl inhibitors of caspase-3 have shown efficacy in
models such as stroke, traumatic brain/spinal cord
injury, hypoxic brain damage, and cardiac ischemia/
reperfusion injury.2 DEVD-CHO is a tetrapeptide inhi-
bitor based on the preferred amino acid sequence
recognized by caspase-3. The aspartic acid is an essen-
tial element of recognition of the enzyme and was used
as the basis to develop new inhibitors. Here we report
nicotinyl aspartyl ketones as potent and selective
inhibitors of caspase-3.
In order to generate libraries of aspartyl aldehydes,
rapid analogues synthesis was performed using solid-
phase chemistry (Scheme 1). Fmoc-aspartyl aldehyde 1
was reacted with a semi-carbazone linked resin 33.3,4
The Fmoc protecting group was then cleaved under
standard conditions to afford the polymer 2. The free
amine was submitted to peptidic couplings with a wide
variety of carboxylic acids. Reactions were routinely
conducted on a 100-mg scale of resin. Subsequent
treatment with 9:1 TFA/H2O led to cleavage from the
resin with concomitant t-butyl ester removal to give
final compounds with a good level of purity. Two alde-
hyde replacements were considered: alkyl and thioether
ketones. Alkyl ketones were prepared from Fmoc
aspartate alcohol 9 (Scheme 2), which were submitted to
Swern oxidation followed by an in situ addition of
Grignard reagents. Resulting alcohols were oxidized to
ketones using the Dess–Martin periodinane. These
ketones were then attached to resin 33. The Fmoc pro-
tecting group was cleaved and the free amine was cou-
pled to the 5-bromonicotinic acid. Cleavage from the
resin with wet TFA afforded ketones such as 10–12.
Thioether ketones were synthesized using the Fmoc-
aspartic acid b-t-butyl ester 13 (Scheme 3). After for-
mation of the mixed anhydride, treatment with CH2N2
and addition of a 1:1 mixture of aqueous 48% HBr/
CH3COOH, the a-bromoketone 14 was obtained. As in
*Corresponding author. Tel.: +1-514-428-3655; fax: +1-514-428-
4939; e-mail: elise_isabel@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00390-1

2138
E. Isabelet a.l/ Bioorg. Med. Chem. Lett. 13 (2003) 2137–2140
Scheme 1. Synthesis of libraries of capped aldehydes: (a) 33, AcOH, THF; (b) 20% piperidine/DMF; (c) RCOOH, HATU, DMF, (iPr)2NEt; (d) 9:1
TFA/H2O.
Scheme 2. Synthesis of alkyl ketones: (a) DMSO, CH2Cl2, (COCl)2, (iPr)2NEt, RMgBr; (b) Dess–Martin periodinane, CH2Cl2; (c) 33, AcOH, THF;
(d) 20% piperidine/DMF; (e) 5-bromonicotinic acid, HATU, DMF, (iPr)2NEt; (f) 9:1 TFA/H2O.
Scheme 3. Synthesis of thioether ketones: (a) i-BuOCOCl, NMM, CH2Cl2 (b) CH2N2, Et2O; (c) HBr/AcOH; (d) 33, AcOH, THF; (e) RSH, DMF,
(iPr)2NEt; (f) 20% piperidine/DMF; (g) 5-bromonicotinic acid, HATU, DMF, (iPr)2NEt; (h) 9:1 TFA/H2O.
the case of alkyl ketones, the a-bromoketone 14 was
attached to the resin 33. The bromide was displaced by
a variety of thiols. The Fmoc group was removed and
the free amine was coupled to the 5-bromonicotinic
acid. Compounds such as 15–17 were cleaved from the
resin when treated with wet TFA. Modifications to the
pyridine ring were carried out as follows. Pyridine 5-
carboxamides like 20–23 were readily available by first
treating 18 with 3,5-pyridinecarboxylic acid under stan-
dard conditions to afford 19 (Scheme 4). This acid was
used to synthesize a variety of amides under standard
conditions. Sulfonyl chloride 26 was synthesized in six
steps starting from 5-bromonicotinic acid (Scheme 5).
Esterification followed by bromide displacement by
NaSMe5 afforded thioether 24. Oxidation to the sulfox-
ide using MMPP, and Pummerer rearrangement with
TFAA6 provided thiol 25 after treatment with Et3Nin
MeOH. The crude material was treated with chlorine in
AcOH7 for a few min to afford the sulfonyl chloride 26.
It was reacted with different amines to obtain a wide
range of sulfonamides. Methyl esters were cleaved to the
corresponding carboxylic acids and were coupled to the
intermediate 18 under standard conditions. Final com-
pounds8 such as 27–32 were cleaved from the resin as
previously described.
Tetrapeptide inhibitors of caspase-3 show poor cell
penetration. The purpose of this work was to identify a
cell-penetrant family of inhibitors. The first library
revealed that introduction of a nicotinic acid (6) was
offering a 2-fold improvement over a benzoic acid deri-
vative (4) when tested against caspase-39 (Table 1).
Others isomers of the nicotinic acid were also synthe-
sized and were shown to be less potent. Introduction of
a bromine atom at the 5-position provided a promising
lead for the replacement of the tetrapeptide (8). It was
found later by X-ray crystallography that the 3-pyridyl
nitrogen interacts st0rongly with the enzyme. To take
advantage of the P1 subsite and to increase inhibitor
stability, aldehydes were replaced by a variety of
Scheme 4. Synthesis of 5-amido-substituted pyridines: (a) 3,5-Pyridinecarboxylic acid, HATU, DMF, (iPr)2NEt; (b) R1R2NH, HATU, DMF,
(iPr)2NEt; (c) 9:1 TFA/H2O.

E. Isabelet a.l/ Bioorg. Med. Chem. Lett. 13 (2003) 2137–2140
2139
Scheme 5. Synthesis of 5-sulfonamido-substituted pyridines: (a) MeOH, H2SO4 (cat.); (b) MeSNa, DMF, 80 C; (c) MMPP, CH2Cl2, MeOH; (d)
TFAA, CH2Cl2; (e) Et3N, MeOH; (f) Cl2, AcOH; (g) R1R2NH, CH2Cl2; (h) LiOH, MeOH, H2O; (i) 18, HATU, DMF, (iPr)2NEt; (j) 9:1 TFA/H2O.
Table 1. Enzyme inhibition of aldehydes and ketones
Compd
R
Rh-Caspase-3
IC50 (mM)
Compd
R
Rh-Caspase-3
IC50 (mM)
3
4
5
6
7
8
AcDEV
Ph
2-Pyridyl
0.027
23
55
10
>200
6
10
11
12
15
16
17
CH2Ph
(CH2)3Ph
(CH2)5Ph
63.7
13.7
96.9
11.7
1.1
3-Pyridyl
4-Pyridyl
5-Bromo-3-pyridyl
CH2SPh
CH2SCH2Ph
CH2SCH2-Ph-4-F
1.2
ketones. As shown in Table 1, a three-carbon chain
length connecting the carbonyl and an aromatic ring
was optimal. Introduction of a sulfur atom to replace
the central carbon in the chain gave a compound with
an IC50 of 1.1 mM (16). Substitution around the phenyl
ring did not provide any improvement in potency.
Having in hand a suitable ketone warhead, introduction
of an amide group at the 5-position on the pyridine ring
allowed us to obtain compounds with sub-micromolar
activity. SAR of this series suggested that lipophilic, dis-
ubstituted amides at the 5-position gave a boost in
potency relative to the bromide (Table 2). Compound 23
has an IC50 of 130 nM against caspase-3. Compounds
having an intrinsic activity better than 1 mM were also
tested in the NT2 whole cell assay.10,11 In that assay too,
lipophilic groups seemed to be favored. However there
was a large shift between the IC50 value in the enzyme
assay and the whole cell assay. Postulating that this shift
may be due to the presence of the amide bond, we next
examined sulfonamides in this position. The SAR of the
sulfonamide family is comparable to that of the amides
with the added advantage of improved whole cell
potency (Table 2). Lipophilic groups increased the
potency. Surprisingly, the best compound in the whole
cell assay (30) incorporated a small cyclopropylamine.
Using this optimized piece on the left side, the ketone
moiety was revisited (Table 3). Both aldehyde 30 and
aminoketone 34 showed a large shift in the NT2 cell
assay. However, the phenolic ether 35 demonstrated an
improved potency in this assay. Finally, the acyox-
ymethyl ketone 36, which gives an irreversible inhibitor
against caspase-3, had an IC50 of 1.3 mM in the cell assay.
Libraries of capped aspartic acid aldehydes allowed the
identification of the 5-bromonicotinic acid as a lead for
a non-peptidic caspase-3 inhibitor. Optimization of the
P10 substituent with thioethers afforded a suitable
replacement for the original aldehyde. Substitution of
the pyridine ring at the 5-position with amides and
sulfonamides improved the potency in the intrinsic
Table 2. Biological activity of amides and sulfonamides
Compd
R
Rh-Caspase-3
IC50 (mM)
NT2 cell
IC50 (mM)
Compd
R
Rh-Caspase-3
IC50 (mM)
NT2 cell
IC50 (mM)
3
AcDEVCHO
Et2NCO
Pr2NCO
(iPr)EtNCO
(iBu)2NCO
Me2NSO2
0.027
0.62
0.41
0.25
0.13
0.30
>100
>100
>100
16
28
29
30
31
32
Et2NSO2
(n-Pr)2NSO2
c-PrNSO2
c-PrMeNSO2
BnNSO2
0.17
0.17
0.40
0.27
0.34
12
25
8
22
59
20
21
22
23
27
39
21

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E. Isabelet a.l/ Bioorg. Med. Chem. Lett. 13 (2003) 2137–2140
Table 3. Biological activity of ketones
References and Notes
1. Nicholson, D. W. Cell Death Diff. 1999, 6, 1028.
2. Nicholson, D. W. Nature 2000, 407, 810 and references
therein.
3. Grimm, E. L. Unpublished results.
Compd
R
H
Rh-Caspase-3
IC50, (mM)
NT2 cell
IC50, (mM)
4. Murphy, A. M.; Dagnino, R., Jr.; Vallar, P. L.; Trippe,
A. J.; Sherman, S. L.; Lumpkin, R. H.; Tamura, S. Y.; Webb,
T. R. J. Am. Chem. Soc. 1992, 114, 3156.
5. Boussad, N.; Trefouel, T.; Dupas, G.; Bourguignon, J.;
Queguiner, G. Phosphorus Sulfur 1992, 66, 127.
6. Young, R. N.; Gauthier, J.-Y.; Coombs, W. Tetrahedron
Lett. 1984, 25, 1753.
7. Kharasch, N.; Bruice, T. C. J. Am. Chem. Soc. 1951, 73,
3240.
8. All final compounds were purified by flash chromatography
and characterized by 1H NMR.
9. Roy, S.; Bayly, C. I.; Gareau, Y.; Houtzager, V. M.; Kar-
gan, S.; Keen, S. L.; Rowland, K.; Seiden, I. M.; Thornberry,
N. A.; Nicholson, D. W. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 6132.
10. Tawa, P.; Tam, J.; Cassady, R.; Nicholson, D. W.; Xan-
thoudakis, S. Cell Death Diff. 2001, 8, 30.
30
0.4
8
33
34
35
36
1.07
0.52
0.39
0.13
57
53
4.3
1.3
11. NT2 cells were plated in 96-well plates. The following day,
camptothecin 5 mg/mL andꢀdifferent concentrations of the
inhibitors were added for 5 h. Cells were then harvested, lysed
and analyzed by cell death ELISA following manufacturer
kit’s instructions (Roche Molecular Biochemicals) measuring
DNA fragmentation.
assay. Sulfonamides proved to be superior for the cell
assay. Introduction of an acyloxymethyl ketone at the
P10 position gave a potent irreversible inhibitor, with a
potency of 1.3 mM in the NT2 cell assay.