Acyl dipeptides as reversible caspase inhibitors. Part 2: Further optimization

Article abstract of DOI:10.1016/S0960-894X(02)00630-3

A new structural class of broad spectrum caspase inhibitors was optimized for its activity against caspases 1, 3, 6, 7, and 8. The most potent compound had low nanomolar broad spectrum activity, in particular, single digit nanomolar inhibitory activity ag

Full text of DOI:10.1016/S0960-894X(02)00630-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2973–2975
Acyl Dipeptides as Reversible Caspase Inhibitors.
Part 2: Further Optimization
Steven D. Linton,a,* Donald S. Karanewsky,a Robert J. Ternansky,a Ning Chen,a
Xian Guo,a Kathy G. Jahangiri,a Vincent J. Kalish,a Steven P. Meduna,a
Edward D. Robinson,a Brett R. Ullman,a Joe C. Wu,a Brian Pham,a
Lalitha Kodandapani,a Robert Smidt,a Jose-Luis Diaz,a Lawrence C. Fritz,a
U. von Krosigk,b Silvio Roggo,b Albert Schmitzb and Kevin J. Tomasellia
aIdun Pharmaceuticals, Inc., 9380 Judicial Drive, San Diego, CA 92121, USA
bNovartis Pharma Ltd., Pharma Research, CH-4002 Basel, Switzerland
Received 31 January 2002; accepted 1 July 2002
Abstract—A new structural class of broad spectrum caspase inhibitors was optimized for its activity against caspases 1, 3, 6, 7, and
8. The most potent compound had low nanomolar broad spectrum activity, in particular, single digit nanomolar inhibitory activity
against caspase 8.
# 2002 Elsevier Science Ltd. All rights reserved.
The caspase family1 of cysteine proteases play a key role
in both cytokine maturation and apoptosis2 (pro-
grammed cell death). These are a unique set of proteases
having a aspartic acid specificity at P1. Caspase inhibi-
tors could be of therapeutic value3 in the treatment of
inflammatory and degenerative diseases, such as rheu-
matoid arthritis, ALS, Alzheimer’s disease, Parkinson’s
disease, stroke, and myocardial infarction. Other than
publications on caspase-1 inhibitors,3 there have been
few reports in the literature on caspase inhibitors, broad
spectrum or specific.
As discussed in our previous publication, truncation of
the known peptide inhibitor, Ac-DEVD-H (1) resulted
in a substantial loss of inhibitory activity (Table 1, Fig.
1).4 Truncated compounds containing a more active P3
surrogate in the context of an acyl dipeptide inhibitor
were then prepared by parallel synthesis, utilizing a
resin bound Fmoc-Leu-Asp(OtBu)-semicarbazone.5
Compound 5 was identified as a broad spectrum caspase
inhibitor. Herein we describe further optimization of
this structural class of caspase inhibitors in order to
obtain analogues with potent, broad spectrum activity.
*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)00630-3

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S. D. Linton et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2973–2975
Table 1. Caspase activity of classical tetra- and tripeptides compared
to acylated dipeptides
Starting from an aspartyl aldehyde intermediate 8 ori-
ginally reported by Graybill et al.,6 either (N-benzyl-
oxycarbonyl)leucine N-hydroxysuccinimide ester or
(N-benzyloxycarbonyl)valine could be coupled5 (see
Scheme 1) to give the protected dipeptides 9ab. After
hydrogenolysis, the carboxylic acid of interest could be
coupled7À11 to give protected acyl dipeptides 10ab.
Hydrolysis of the semicarbazone5,6 and t-butyl esters
yielded the analogues listed in Table 2.
Compd
Caspase activitya IC50(mM)
mCsp-1 Csp-3 Csp-6 Csp-7 Csp-8
1
2
3
4
5
6
7
Ac-DEVD-H
Z-ELD-H
Z-FLD-H
0.05
0.0065 0.0023
0.043 0.137
0.0035 0.01
0.01 0.08
0.01 0.03
0.68 8.7
8.87 10
—
2.53
2-Naphthyloxy acetyl-LD-H 10
1-Naphthyloxy acetyl-LD-H 0.570 0.135
0.94
18.56
0.940 1.81 0.770
10 6.81 11.96
14.03 21.9 50
Z-VD-H
Z-LD-H
5.85
18.82
1.75
3.47
Table 2 examines the data for these optimized analo-
gues. Extension of the naphthyloxy moiety deeper into
the S4 pocket (12) did not improve potency. Analogues
incorporating a P3 glutamic acid mimetic (13 and 14)
showed a significant increase in activity against caspases
3, 6, 7, and 8 (the natural S isomer being the more
potent), but a 5- to 10-fold loss of potency against cas-
pase 1. Compounds 15, 16, and 17 show that caspase 3
and 8 activity can also be significantly increased by the
proper placement of a carboxyl group on the naphthylene
aAssay conditions are described in ref 5.
Figure 2.
Table 2. Caspase activity of optimized acyl dipeptide inhibitors
Compd
R1
X
n
R2
P2
Caspase activity IC50 (mM)
Csp-3 Csp-6 Csp-7
mCsp-1
0.135
0.336
0.25
Csp-8
5
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
H
H
Leu0.570
Val
Val
Leu2.55
Leu4.86
Val
Val
Val
Val
Val
Leu
Leu0.087
Val
Leu0.790
Leu0.330
0.940
1.81
0.770
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0.355
4.4
>10
2.10
37.03
1.20
19.59
H
R-(CH2)2CO2H
S-(CH2)2CO2H
4.6
0.021
0.0038
0.015
0.0035
0.587
0.130
0.012
0.031
1-CO2H-2-naphthyl
1-Naphthyl-2-CO2H
2-Naphthyl-3-CO2H
2-Naphthyl-3-NHSO2CF3
1-Naphthyl-5-NHSO2CF3
1-Naphthyl
H
H
H
H
H
H
CH3
H
H
H
2.96
0.401
0.054
0.731
1.92
0.207
0.013
3.61
1.43
1.90
3.03
1.01
0.037
10.9
1.65
17.0
43.66
11.0
1.32
0.733
0.048
0.200
1.42
0.615
0.0076
0.385
1.89
2.18
0.127
0.033
1-Naphthyl
1-Naphthyl
1,2-Diphenyl-ethyl
3-Phenoxy-phenyl
0.512
0.015
0.310
7.24
0.017
1.55
7.0
6.14
0.450
0.320
0.230
0.589
0.308
2.92
2.67
0.990
0.670
Scheme 1.5ꢀ Reagents and conditions: (a) Val: Cbz-Val-OH (1 equiv), EDAC (1.5 equiv), HOBt (1.05 equiv), N-methylmorpholine (NMM) (1 equiv),
.
CH2Cl2, 0 C 2 h, 20 h, 93%; Leu: Cbz-Leu-OSu (1 equiv), TsOH H-Asp(OtBu)Sc (1.3 equiv), DIEA (1.4 equiv), CH2Cl2, 16 h; (b) H2 (1 ATM), Pd/
C 10%, EtOH, 1.5 h; (c) carboxylic acid (0.9–1 equiv), EDAC (1.5 equiv), HOBt (1.4 equiv), NMM (1.2 equiv), NMP/CH2Cl2 (1:1); (d) TFA,
CH2Cl2, anisole 4/3/1, 4.5 h; (e) 37% aq HCHO, AcOH, THF, TFA 1/1//5/0.025, 4–16 h.

S. D. Linton et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2973–2975
2975
moiety of 5. Among the carboxy-substituted naphthoxy
acetyl derivatives studied, the 2-CO2H, 1-naphthoxy
acetyl analogue 16 showed the largest improvement in
caspase 3 and 8 inhibitory activity. Use of a tri-
fluoromethyl sulfonamide as a carboxylic acid isostere
(18) gave a compound (17) with a caspase inhibitory
profile similar to its carboxylic acid counterpart. This
substituent in the 5-position gained some broad spec-
trum activity, but was not as potent as compound 16.
The aminonaphthalene analogue of compound 5 (20)
showed the best overall broad spectrum activity, with
excellent potency against caspase 8. The valine analo-
gue, 22, showed caspase 1 and 8 selectivity, more than
was seen with the Leuand Val analogues of the nap-
thyloxy series, 5 and 11. Other phenyl based derivatives
showed good broad spectrum activity, but were not as
potent as 20. Results from our modeling studies suggest
that the aryl ether oxygen of compound 5 may occupy the
space occupied by the P3 amide-NH of tetrapeptide
inhibitors. An X-ray structure of Ac-DVAD-fmk bound
to caspase 3 reveals that this N–H forms a hydrogen
bond with the carbonyl oxygen of Arg-207.12 Therefore,
the enhanced potency of compound 20 could be due to
increased binding of the inhibitor due to this additional
hydrogen bonding interaction (Fig. 2).
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. Linton, S. D.; Karanewsky, D. S.; Ternansky, R. J.; Wu,
J. C.; Pham, B.; Kodandapani, L.; Smidt, R.; Diaz, J.-L.;
Fritz, L. C.; Tomaselli, K. J. Bioorg. Med. Chem. Lett. 2002,
12.
5. Karanewsky, D. S.; Kalish, V. J.; Robinson, E. D.; Ullman,
B. R. US Patent 6,242,422 B1, 2001.
6. Graybill, T. L.; Dolle, R. E.; Helaszek, C. T.; Miller, R. E.;
Ator, M. A. Int. J. Peptide Protein Res. 1994, 44, 173.
7. Compound 12 was prepared on solid phase employing the
same method in Scheme 1, using Fmoc-Valine and 4-(1-naph-
thyloxy)butyric acid.5
8. The carboxylic acid used to make compound 13 was pre-
pared from d-glutamic acid in the following manner:5 (a) KBr
(3 equiv), 2.5 N H2SO4, NaNO2 (1.7 equiv), 0 ꢀC 1 h, 45 min;
(b) CH2N2 (excess generated from 1-methyl-3-nitro-1-nitroso-
guanidine and 40% KOH), Et2O, 0 ꢀC, 51% over 2 steps; (c)
1-naphthol (1.1 equiv), K2CO3 (1.5 equiv), DMF, 3.5 h 92%;
(d) H2 (1 atm), Pd/C 10%, methanol, 1.5 h; (e) 2,4,6-tri-
chlorobenzoyl chloride (1.3 equiv), triethylamine (TEA) (1.6
equiv), THF, 18 h; (f) t-butanol, dimethylaminopyridine
(DMAP), CH2Cl2, 3.5 h, 82% overall; (g) 1 N LiOH (1.3
equiv), 3/1 1,4-dioxane/H2O, 0 ꢀC 30 min, 1.25 h, used as crude
material in coupling. The carboxylic acid used to make com-
pound 14 was prepared from l-glutamic acid following the
preceding method.
It has been shown that using solid-phase, parallel
synthesis4 followed by a focused SAR study that
AcDEVD-H can be truncated to an arylamino-acyl
dipeptide and retain good broad spectrum caspase inhi-
bitory activity. Further work in this area will be the
subject of future publications.
9. The carboxylic acids used to make compounds 15, 16, and
17 were prepared from 1-carbomethoxy-2-naphthol, 2-carbo-
methoxy-1-naphthol,
and
3-carbomethoxy-2-naphthol,
respectively, in the following manner:5 (a) t-butyl bromo-
acetate (1 equiv), K2CO3 (3 equiv), DMF, 18 h; (b) TFA/H2O
(9/1), anisole, CH2Cl2, 16 h. The carbomethoxy groups were
hydrolyzed (1 N LiOH (1.1 equiv) in 3/1 1,4-dioxane/ H2O,
4 h) after the peptide coupling and before the last 2 deprotec-
tion steps.
Acknowledgements
The authors wish to thank Alfred Spada and Judy Dry-
den for their contributions in preparing this manuscript.
10. The carboxylic acids used to make compounds 18 and 19
were prepared from 3-amino-2-naphthol and 5-amino-1-
naphthol, respectively, in the following manner:5 Compound
18: (a) t-butyl bromoacetate (1 equiv), K2CO3 (3 equiv),
DMF, 18 h, used crude; (b) trifluoromethanesulfonic anhy-
dride (1.2 equiv), TEA (1.2 equiv), CH2Cl2, À78 ꢀC 30 min,
1 h, 72% overall; (c) TFA/H2O (9/1), anisole, CH2Cl2, 16 h,
92%. Compound 19: (a) methyl bromoacetate (1.2 equiv),
K2CO3 (3 equiv), DMF, 18 h, used crude; (b) tri-
fluoromethanesulfonic anhydride (1.2 equiv), TEA (1.2 equiv),
CH2Cl2, À78 ꢀC 30 min, 1 h, used crude; (c) 1 N LiOH (2.2
equiv), 16 h, 73% overall.
11. The carboxylic acids used to make compounds 20 and 21
were prepared in the following manner:5 (a) Compound 20:
1-aminonaphthalene, methylbromoacetate (1.6 equiv), TEA
(1.1 equiv), DMF, 60 h, used crude; Compound 21: 1-amino-
naphthalene, ethyl 2-bromopropionoate (1.1 equiv), TEA (1.1
equiv), DMF, 60 ꢀC 18 h, used crude; (b) 1 N LiOH (1–1.2
equiv), 1,4-dioxane, 1.5 h. Overall yields: compound 20: 51%,
compound 21: 70%.
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