Acyl dipeptides as reversible caspase inhibitors. Part 1: Initial lead optimization

Article abstract of DOI:10.1016/S0960-894X(02)00629-7

Parallel synthesis was used to explore the SAR of a peptidomimetic caspase inhibitor. The most potent compound had nanomolar activity against caspases 1, 3, 6, 7, and 8.

Full text of DOI:10.1016/S0960-894X(02)00629-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2969–2971
Acyl Dipeptides as Reversible Caspase Inhibitors. Part 1: Initial
Lead Optimization
Steven D. Linton,* Donald S. Karanewsky, Robert J. Ternansky, Joe C. Wu,
Brian Pham, Lalitha Kodandapani, Robert Smidt, Jose-Luis Diaz,
Lawrence C. Fritz and Kevin J. Tomaselli
Idun Pharmaceuticals, Inc., 9380 Judicial Drive, San Diego, CA 92121, USA
Received 31 January 2002; accepted 1 July 2002
Abstract—Parallel synthesis was used to explore the SAR of a peptidomimetic caspase inhibitor. The most potent compound had
nanomolar activity against caspases 1, 3, 6, 7, and 8.
# 2002 Elsevier Science Ltd. All rights reserved.
Caspases1 are a family of cysteine proteases with speci-
ficity for aspartic acid at the S1 subsite of the enzyme
that are involved in both cytokine maturation and
apoptosis.2 Caspase-1 (interleukin-1b converting
enzyme, ICE) is involved in the induction of inflamma-
tion by catalyzing the cleavage of the pro-formof
IL-1b.1c Other caspases play a role in the regulation of
apoptosis, either as apoptosis signaling molecules or as
downstreameffectors. Inhibition of caspases, either
broad spectrumor caspase specific, could be of ther-
apeutic value3 in the treatment of inflammatory and
degenerative diseases, such as rheumatoid arthritis,
ALS, Alzheimer’s disease, Parkinson’s disease, stroke,
and myocardial infarction. Up to now there have been few
reports in the literature on caspase inhibitors, other than a
significant body of work associated with caspase 1.3
Table 1. Caspase activity of classical tetra- and tripeptides compared
to acylated dipeptides
Compd
Caspase activitya IC50 (mM)
mCsp-1 Csp-3 Csp-6 Csp-7 Csp-8
1
2
3
4
5
6
Ac-DEVD-H
Z-ELD-H
Z-FLD-H
Z-VD-H
0.05
0.0065 0.0023
0.043 0.137
5.85
18.82
0.0035 0.01 0.01 0.08
—
0.01 0.03
2.53 0.68 8.7
10 6.81 11.96
14.03 21.9 50
18.56 8.87 10
1.75
3.47
0.94
Z-LD-H
2-Naphthyloxy acetyl-LD-H 10
aAssay conditions are described in ref 5.
Herein we describe a series of novel, potent, broad
spectrumcaspase inhibitors. Our starting point was the
known peptide inhibitor, Ac-DEVD-H, which upon
truncation to a Z-XXD-H tripeptide4 retained potent,
broad spectrumactivity (Table 1, Fig. 1, compounds
2
and 3). However, further truncation to a Z-XD-H
dipeptide resulted in a significant loss in caspase inhibi-
tory activity. Given this, we sought to prepare other
truncated compounds containing a more active P3 sur-
rogate in the context of an acyl dipeptide inhibitor.
Using parallel synthesis, incorporating a resin bound
*Corresponding author. Fax: +1-858-625-2677; e-mail: slinton@idun.
com
Figure 1. Tetra- and tripeptide inhibitors leading to an acylated
dipeptide.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00629-7

2970
S. D. Linton et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2969–2971
Fmoc protected leucine. Upon Fmoc deprotection, the
resin-bound dipeptide aldehyde was coupled to a col-
lection of carboxylic acids. Hydrolysis of the tert-butyl
ester and subsequent cleavage4,5a fromthe resin affor-
ded the desired library of acyl dipeptide aldehyde inhi-
bitors (Table 2, Fig. 2).
Figure 2.
Table 2 lists a set of diverse analogues designed to probe
the SAR in the S3 caspase subsite. Naphthoyl (14 and
15) and naphthyl acetyl (16 and 17) analogues showed
no significant improvement in potency over analogue 6.
A notable improvement in broad spectrum caspase
inhibitory activity was seen with compound 18, imply-
ing that orientation of the naphthyl ring is critical to
binding with respect to compound 6. The carbon iso-
stere of 18 (19) showed a 3-fold or greater loss of activ-
ity across the panel of casapase isozymes. Addition of a
methyl group in the alpha position of the (aryloxy)acetyl
moiety as in 21 also resulted in a loss in caspase inhibitory
activity. The sulfur isosteres of analogues 18 and 6 (23 and
24) were interesting in that 23 showed caspase 3 selectivity,
while the naphthyl positional isomer showed improved
Fmoc-Leu-Asp(OtBu)-semicarbazone,5 we identified
compound 6 as the basis for a more directed library of
analogues.
In order to prepare a directed library of aspartyl alde-
hyde inhibitors, we employed a strategy utilizing an
aldehyde linked to solid phase via a semicarbazone was
employed (Scheme 1).5a,b Commercially available Fmoc-
Asp (OtBu)-OH was converted to the corresponding
aldehyde via the Weinreb amide, then condensed with
an aminoethylphenoxyacetic acid semicarbazide linker.5a
The Fmoc protected aspartyl semicarbazone b-t-butyl
ester could now be coupled to aminomethylpolystyrene
resin,5b the amine deprotected, and then coupled to
Table 2. Caspase activity of inhibitors froma directed parallel solid-phase synthesis
Compd
R
Caspase activity IC50 (mM)
mCsp-1
Csp-3
5.06
>10
6.74
Csp-6
Csp-7
Csp-8
>10
>10
>10
13
14
15
16
17
18
6
4-Biphenyl acetyl
1-Naphthoyl
2-Naphthoyl
1-Naphthyl acetyl
2-Naphthyl acetyl
7.43
2.30
1.74
3.24
>10
>10
>10
—
>10
>10
>10
7.27
>10
1.81
8.87
8.77
9.72
1.32
32.4
1.74
2.52
18.2
11.3
7.43
1.37
9.65
3.60
5.89
4.16
4.08
3.38
3.61
34.7
4.57
3.85
3.48
1.51
>10
6.04
>10
2.02
3.75
12.0
3.36
4.41
6.82
2.20
7.25
1.49
5.23
2.79
8.24
2.74
2.31
0.853
0.849
0.135
0.944
1.59
1.39
0.376
4.81
0.195
0.269
2.76
0.967
1.42
0.059
0.910
0.412
0.837
0.375
0.263
0.231
0.357
1.57
0.232
0.412
0.407
0.147
3.4
—
3.14
11.11
0.940
18.56
4.18
5.05
1.28
13.8
1.43
3.16
14.5
11.8
10.3
0.305
5.90
2.72
1.62
32.4
22.6
12.0
21.4
>50
>50
>50
42.7
12.5
>50
>50
>50
0.717
14.5
20.5
10.1
18.9
21.2
9.75
33.4
3.37
51.2
5.86
32.5
11.8
28.6
12.7
0.770
>10
12.2
13.4
2.43
29.1
7.42
11.0
>50
11.2
5.23
9.81
15.2
16.3
15.0
4.14
1.45
1.69
3.04
>50
>50
4.02
3.64
2.24
>10
>10
>10
1.71
3.86
7.53
6.05
4.72
9.28
4.34
8.60
1.86
5.90
3.87
4.35
1.75
4.95
1-Naphthyloxy acetyl
2-Naphthyloxy acetyl
0.570
10
1.86
0.650
3.99
6.84
2.75
0.792
1.80
0.408
0.543
0.686
1.32
0.563
0.611
0.843
0.831
0.429
0.141
44.2
35.3
5.25
5.14
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
3-(1-Naphthyl)propionyl
3-(1-Naphthyl)acryloyl
2-(1-Naphthyloxy)propionyl
2-(6-Bromo-1-naphthyloxy)-propionyl
1-Naphthylmercapto acetyl
2-Naphthylmercapto acetyl
4(2-Naphthyl) butyryl
3-(1-Naphthoyl)propionyl
3-(2-Napthoyl)propionyl
3-(1-Naphthyloxy)propionyl
3-(2-Naphthyloxy)propionyl
3-(1-Naphthylmercapto)propionyl
3-(2-Naphthylmercapto)propionyl
2-Methyl-1-naphthyoxy acetyl
4-Methoxy-1-naphthyloxy acetyl
4-Chloro-1-naphthyloxy acetyl
2,4-Dichloro-1-naphthyloxyacetyl
1-Isoquinolinyloxy acetyl
4-Quinolinyloxy acetyl
5-Quinolinyloxy acetyl
5-Isoquinolinyloxy acetyl
8-Quinolinyloxy acetyl
Phenyl
3-Phenoxy propionyl
Phenoxy acetyl
2-Biphenoxy acetyl
3-Biphenoxy acetyl
4-Biphenoxy acetyl
13.7
>10
9.42
>10
0.636
1.10
0.419
3.40
0.095
0.311
0.763
0.490
0.346
0.545
0.222
0.57
0.197
0.319
0.474
0.528
0.324
0.162
1.90
(2-Benzyl)phenoxy acetyl
(4-Benzyl)phenoxy acetyl
(4-Phenoxy)phenoxy acetyl
(2-Benzyloxy)phenoxy acetyl
(4-Benzyloxy)phenoxy acetyl
(2-Cyclopentyl)phenoxy acetyl
(4-Cyclopentyl)phenoxy acetyl
[2-(1-Adamantyl)-4-methyl]phenoxy acetyl
4-(1-Adamantyl)-phenoxy acetyl
5,6,7,8-Tetrahydro-1-naphthyloxy acetyl
5,6,7,8-Tetrahydro-2-naphthyloxy acetyl
0.521
1.80
2.21
2.40
2.51
0.538
2.20
1.43
1.83
1.81
2.57

S. D. Linton et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2969–2971
2971
and retain nanomolar, broad spectrum caspase inhib-
itory activity. Further work has been done on this new
structural class of caspase inhibitors and will be the
subject of future publications.
Acknowledgements
The authors wish to thank Alfred Spada, Judy Dryden,
and Silvio Roggo (Novartis Pharma, Ltd.) for their
contributions in preparing this manuscript.
Scheme 1. Reagents and conditions: (a) N,O-dimethylhydroxyl-
amine HCl (1.2 equiv), HOBt (1.2 equiv), EDAC (1.2 equiv), N-methyl-
References and Notes
.
morpholine (1.2 equiv), THF, 0 ꢀC, 2 h, 16 h. 98%; (b) 1.0M LAH in
ether (0.5 equiv), ether, 0 ꢀC, 1 h; (c) 9 (1.03 equiv), NaOAc (1.3 equiv),
ethanol, 0 ꢀC 3 h, 16 h, 69%; (d) aminomethylpolystyrene resin, pyBOP
(1.5 equiv), DIEA (diisopropylethylamine) (3 equiv), THF/N-methyl-
pyrolidinone (NMP) (1:1), 3 h; (e) piperidine, DMF (1:4), 1 h; (f) Fmoc-
Leu-OH (2 equiv), pyBOP (3 equiv), DIEA (6 equiv), THF/NMP (1:1),
2.5 h; (g) carboxylic acid (3.75 equiv), pyBOP (3 equiv 0.25 M pyBOP in
NMP), DIEA (6.25 equiv 0.5 M DIEA in NMP), 16 h; (h) TFA, CH2Cl2,
anisole 4/3/1, 6 h; (i) 37% aq HCHO, AcOH, THF, TFA 1/1/5/0.025, 4 h,
30–88%.
1. (a) For nomenclature of the caspase family, see: Alnemri,
E. S.; Livingston, D. J.; Nicholson, D. W.; Salvesen, G.;
Thornberry, N. A.; Wong, W. W.; Yuan, J.-Y. Cell 1996, 87,
171. (b) Thornberry, N. A. Chem. Biol. 1998, 5, R97. (c)
Black, R. A.; Kronheim, S. R.; Sleath, P. R. FEBS Lett. 1989,
247, 386.
2. (a) Ellis, R. E.; Yuan, J.; Horvitz, H. R. Annu. Rev. Cell
Biol. 1991, 7, 663. (b) Reed, J. C.; Tomaselli, K. J. Curr. Opin.
Biotec. 2000, 11, 586.
3. (a) Talanian, R. V.; Brady, K. D.; Cryns, V. L. J. Med.
Chem. 2000, 43, 3351. (b) Lee, D.; Long, S. A.; Murray, J. H.;
Adams, J. L.; Nuttall, M. E.; Nadeau, D. P.; Kikly, K.;
Winkler, J. D.; Sung, C.-M.; Ryan, M. D.; Levy, M. A.;
Keller, P. M.; DeWolf, W. E. J. Med. Chem. 2001, 44, 2015.
(c) Guo, Z.-M.; Xian, M.; Zhang, W.; McGill, A.; Wang, P. G.
Bioorg. Med. Chem. 2001, 9, 99. (d) Shahripour, A. B.;
Plummer, M. S.; Lunney, E. A.; Sawyer, T. K.; Stankovic,
C. J.; Connolly, M. K.; Rubin, J. R.; Walker, N. P. C.; Brady,
K. D.; Allen, H. J.; Talanian, R. V.; Wong, W. W.; Humblet,
C. Bioorg. Med. Chem. Lett. 2001, 11, 2779. (e) Shahripour,
A. B.; Plummer, M. S.; Lunney, E. A.; Albrecht, H. P.; Hays,
S. J.; Kostlan, C. R.; Sawyer, T. K.; Walker, N. P. C.; Brady,
K. D.; Allen, H. J.; Talanian, R. V.; Wong, W. W.; Humblet,
C. Bioorg. Med. Chem. Lett. 2002, 10, 31. (f) For a review of
the caspase inhibitor patent literature, see: Ashwell, S. Expert
Opin. Ther. Pat. 2001, 11, 1593.
4. These inhibitors were prepared and assayed internally. For
preparative route see: Graybill, T. L.; Dolle, R. E.; Helaszek,
C. T.; Miller, R. E.; Ator, M. A. Int. J. Peptide Protein Res.
1994, 44, 173.
5. (a) Karanewsky, D. S.; Kalish, V. J.; Robinson, E. D.;
Ullman, B. R. US Patent 6,242,422 B1, 2001. (b) A similar
approach has been used by workers at Corvas for the solid
phase synthesis of C-terminal argininal-containing peptides.
See: Murphy, A. M.; Dagnino, R.; 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.
caspase 1 activity (24). However, neither analogue had
potencies against caspases 6 or 8 comparable to that of
18. Further chain extension to analogue 25 and its
naphthoyl derivatives (26 and 27) did not improve
broad spectrumactivity, although good caspase 1 inhib-
itory activity was seen with 26 and 27. The homologues
of 18 and 6 (28 and 29) have almost the same respective
activities, except that 28 is more potent than 18 against
caspase 3, but not as potent as 18 against caspase 8. The
sulfur isosteres of 28 and 29 (30 and 31) had similar
activity as 28 and 29, except 30 was less potent against
caspase 6 and 31 was more potent against caspase 1.
Substituted naphthyloxy acetyl analogues showed good
activity against caspases 1 and 3, but were not broad
spectruminhibitors. Quinolinyloxy and isoquinoliny-
loxy analogues were inactive as broad spectruminhib-
itors, but showed caspase 3 selectivity. Some phenoxy
and 5,6,7,8-tetrahydro-naphthyloxy acetyl analogues fol-
lowed this same trend. Among this series of analogues,
the (1-naphthyloxy)acetic acid, 18, is the only inhibitor
to have nanomolar activity against caspases 1, 3, 6 and 8.
It has been shown that the peptide AcDEVD-H inhib-
itor can be truncated to a novel aryloxyacetyl dipeptide,