Oxamyl dipeptide caspase inhibitors developed for the treatment of stroke

Article abstract of DOI:10.1016/j.bmcl.2003.12.106

Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein.

Full text of DOI:10.1016/j.bmcl.2003.12.106

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2685–2691
Oxamyl dipeptide caspase inhibitors developed for the
treatment of stroke
Steven D. Linton,a,* Teresa Aja,a Peter R. Allegrini,b Thomas L. Deckwerth,a
Jose-Luis Diaz,a Bastian Hengerer,b Julia Herrmann,a Kathy G. Jahangiri,a Joerg Kallen,b
Donald S. Karanewsky,a Steven P. Meduna,a Kip Nalley,a Edward D. Robinson,a
Silvio Roggo,b Giorgio Rovelli,b Andre Sauter,b Robert O. Sayers,a Albert Schmitz,b
Robert Smidt,a Robert J. Ternansky,a Kevin J. Tomaselli,a Brett R. Ullman,a
Christoph Wiessnerb and Joe C. Wua
aIdun Pharmaceuticals, Inc., 9380 Judicial Drive, San Diego, CA 92121, USA
bNovartis Pharma Ltd, Pharma Research, CH-4002 Basel, Switzerland
Received 9 May 2003; accepted 23 December 2003
Abstract—Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl
dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are
described herein.
Ó 2004 Elsevier Ltd. All rights reserved.
The caspases1 are a family of cysteine proteases with
aspartic acid specificity at P1 involved in both cytokine
maturation and apoptosis.2 Caspase 1 (interleukin-1b
converting enzyme, ICE) is involved in the induction of
inflammation by catalyzing the cleavage of the pro-form
of IL-1b. Other caspases may play a role in the regula-
tion of apoptosis, either as apoptosis signaling molecules
or as downstream effectors. Inhibition of caspases, either
broad spectrum or caspase specific, would be of thera-
peutic value3 in the treatment of inflammatory and
degenerative diseases, such as rheumatoid arthritis,
ALS, Alzheimer’s disease, Parkinson’s disease, and
stroke. Up to now there have been few reports in the
literature on caspase inhibitors, other than a broad body
of work associated with caspase 1.3
A series of acyl dipeptide aldehydes was previously
prepared4 as peptidomimetic caspase inhibitors. The
naphthyloxyacetyl dipeptide 1 showed broad spectrum
caspase inhibition5 (Table 1). Results from our modeling
studies suggest that the aryl ether oxygen of compound 1
may occupy the space occupied by the P3 amide-NH
of tetrapeptide inhibitors.6 Replacement of the ether
oxygen in compound 1 by NH (2) led to a dramatic
enhancement in potency against the caspases tested7
(Table 1).
Conversion of the methylene in the amino-amide moiety
of compound 2 to a carbonyl generated the oxamide 3.8
Of the three analogues, the oxamide 3 showed the best
enhancement in potency against caspases 1, 3, and 9.
Compound 3 was also significantly more potent than the
parent ether analogue (1) in the monocyte8;9 assay
(Table 1). Due to this improved cellular activity, along
with the observed relative chemical instability of ana-
logues such as amine 2, an SAR study was initiated on
the oxamyl dipeptide series of caspase inhibitors.
Our aim was to develop a series of novel, peptidomi-
metic, broad spectrum caspase inhibitors with low
nanomolar activity. These compounds could then be
optimized and developed as therapeutics for CNS dis-
orders, such as stroke, traumatic brain injury, and
others.
Our first approach was to prepare various irreversible
warhead analogues using both valine and leucine at
P2,10 based on our previous work with the acyl dipeptide
series5;7;11 (Scheme 1). The term ‘warhead’ is used here to
describe the electrophilic moiety that covalently interacts
* Corresponding author. Fax: +1-858-625-2677; e-mail: slinton@
idun.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.106

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S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
Table 1. Transition from acyl dipeptide to oxamyl dipeptide
Compd
Enzyme assaysa IC50 (lM)
Monocyte assay
mCsp-1b
Csp-3
Csp-6
Csp-8
Csp-9
% inhib @ 10 lM
1
2
3
0.570
0.033
0.027
0.135
0.013
0.010
0.940
0.037
1.50
0.770
0.007
0.179
2.72
0.160
0.230
17%
18%
79%c
aAssay conditions are described in Refs. 8 and 14.
bMurine caspase-1 enzyme. Other enzyme and cellular data are from human.
cIC50 ¼ 3:08 lM.
Scheme 1. Reagents and conditions: (a) Methyl chlorooxoacetate (1.1 equiv), triethylamine (TEA) (1.1 equiv), 0 °C to room temperature (rt), 1 h;
(b) 1 N LiOH (1.2 equiv), 1,4-dioxane, rt, 1 h, 78% overall; (c) 4 (1 equiv), HATU (1 equiv), DIEA (3 equiv), N-methylpyrolidinone (NMP)/CH2Cl2
(1:1), rt, 2 h, 73%; (d) 1 N LiOH (1.02 equiv), 1,4-dioxane, rt, 1 h; (e) isobutyl chloroformate (1.1 equiv), N–Me morpholine (NMM) (1 equiv), THF,
ꢀ10 °C, 30 min; (f) 1.5 equiv CH2N2/Et2O, 0 °C, 1 h, 62%; (g) HBr (aq 48%) THF, 0 °C, 15 min; (h) Nucleophile (X), KF (3 equiv), DMF, rt 16 h;
(i) TFA/CH2Cl2/anisole, (1:1:0.5), rt, 1 h.
with the active site of the enzyme and its prime side
substitution. These analogues were prepared8 by acyla-
tion of 1-aminonaphthalene with methyl chlorooxoace-
tate, then basic hydrolysis to yield the oxamic acid, 4.
Coupling to the p-tosylate salt of amine 58 using HATU
followed by basic hydrolysis gave the oxamyl dipeptide 6.
Three-step conversion to the bromomethyl ketone
employing diazomethane12 afforded the key intermediate
7. This compound could then be reacted with the
nucleophile of choice employing modified Finklestein
conditions,13 followed by acidic hydrolysis of the tert-
butyl ester to yield the inhibitors (Tables 2 and 3).
Table 2. Warhead SAR, valine versus leucine
Compd
X
P2
Cellular assaysa IC50 (lM)
Csp-3 k3/Ki
Csp-8 k3/Ki
(Mꢀ1 sꢀ1
)
(Mꢀ1 sꢀ1
)
Con A
Jurkat
8
OCO(2,6-Cl2-Ph)
OCO(2,6-Cl2-Ph)
O(2,3,5,6-F4-Ph)
O(2,3,5,6-F4-Ph)
O(2,4,6-F3-Ph)
O(2,4,6-F3-Ph)
Leu
Val
Leu
Val
Leu
Val
282,834
740,466
180,453
288,748
85,300
112,166
119,197
177,219
447,761
145,011
189,358
0.50
0.35
2.58
0.55
4.61
0.30
1.3
0.0043
2.5
9
10
11
12
13
0.0061
0.535
0.0052
340,398
a Assay conditions are described in Refs. 8 and 14.

S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
2687
Table 3. Warhead SAR, P2¼valine
Compd
X
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
)
Cellular assaysa IC50 (lM)
mCsp-1b
Csp-3
Csp-6
Csp-8
119,197
THP-1
Con A
9
OCO(2,6-Cl2-Ph)
O(2,3,5,6-F4-Ph)
O(2,4,6-F3-Ph)
OPOPh2
1,052,775
892,596
879,183
2,950,000
1,404,853
1,295,045
1,069,270
532,101
2969
740,466
288,748
340,398
876,478
529,376
492,401
293,818
264,194
342
234,318
142,903
37,438
222,420
165,406
49,970
13,349
12,805
459
0.52
0.770
0.40
0.65
0.09
0.48
0.25
0.28
8.30
1.93
3.06
1.12
0.35
0.55
0.30
0.65
2.00
0.45
2.00
9.00
11
13
14
15
16
17
18
19
20
21
22
447,761
189,358
2,447,090
847,197
311,465
60,347
88,785
1
OPO(CH3)Ph
O(2,6-F2-Ph)
O(2-CF3-4-pyrimidyl)
O(5-CO2CH3-3-isoxazyl)
OSOPh
>50
O(2-naphthyl)
O(1-naphthyl)
O-Ph(4-Ph)
155,894
378,200
58,032
9455
11,653
1659
31
268
20
12
7.30
2.70
0
0
>50
a Assay conditions are described in Refs. 8 and 14.
b Murine caspase-1 enzyme. Other enzyme data are from recombinant human.
Given our experience with irreversible inhibitors and
their ability to block cell death in cellular models of
apoptosis,11;14 we focused primarily on warheads that
were potentially irreversible in nature.11;12 The results of
this SAR (Table 2) indicated that valine at P2 was better
than leucine, especially with respect to cellular activity.
Some of these derivatives exhibited extremely potent
activities for inhibition of cell death in the Jurkat cel-
lular assay.9 Additional warhead analogues were then
prepared, using only valine at P2 (Table 3).
Additional P2 analogues of the tetrafluorophenoxy
warhead were prepared as shown in Scheme 2.8 Com-
mercially available Z-Asp(O-tBu)-OH was converted to
the bromomethyl ketone.12 Displacement with the
potassium salt of 2,3,5,6-tetrafluorophenol gave com-
pound 24. Reduction of the ketone and hydrogenolysis
afforded the key intermediate 25, which could then be
coupled to the protected amino acid of choice. Depro-
tection of the amine and coupling to the oxamic acid 4
yields an oxamyl dipeptide alcohol. Dess–Martin oxi-
dation and subsequent hydrolysis of the tert-butyl ester
gave the series of analogues in Table 4. The SAR of this
set of analogues was evaluated as before by comparing
broad spectrum caspase inhibition and activity in cel-
lular models. The valine derivative was optimal when
considering cellular activity. From the perspective of
broad spectrum caspase inhibition and cellular activity,
The dichlorobenzoyloxy (9),12b polyfluorophenoxy (11,
13), and diphenylphosphinyl (14)15 warhead analogues
in particular exhibited excellent broad spectrum enzy-
matic and cellular activities (see Table 3). From these
analogues, the 2,3,5,6-tetrafluorophenoxy warhead (11)
was selected for further study.
Scheme 2. Reagents and conditions: (a) (i) isobutyl chloroformate (1.2 equiv), NMM (1.1 equiv), THF ꢀ10 °C, 20 min; (ii) 3 equiv CH2N1/Et2O, 0 °C,
15 min; (iii) HBr (aq 48%) THF, 0 °C, rt, 30 min, used crude; (b) 2,3,5,6-tetrafluorophenol, (1.14 equiv), KF (3.2 equiv), DMF, rt, 16 h, 95%;
(c) NaBH4 (0.74 equiv) EtOH, 0 °C, 1 h, 94%; (d) H2 (1 atm), 10% Pd/C, MeOH, rt, 4 h, 93%; (e) Fmoc or Cbz amino acid, HATU (1.2 equiv), DIEA
(2 equiv), CH2Cl2, rt, 16 h; (f) 10% piperidine/DMF, rt, 1 h or H2 (1 atm), 10% Pd/C, MeOH, rt, 4 h; (g) 4 (1.5 equiv), HATU (1.2 equiv), DIEA
(2 equiv), CH2Cl2, rt, 16 h; (h) Dess–Martin periodinane (2.8 equiv), CH2Cl2, rt, 30 min; (i) TFA/CH2Cl2/anisole (1:1:0.2), rt, 30 min.

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S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
Table 4. P2 SAR
Compd
Amino acid
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
)
Cellular assaysa IC50 (lM)
mCsp-1b
Csp-3
Csp-6
Csp-8
447,761
THP-1
Con A
11
10
26
27
28
29
30
31
32
33
34
35
Valine
Leucine
892,596
253,514
35,955
288,748
180,453
9849
142,903
83,651
11,557
0.77
2.13
0.55
2.58
16
177,219
19,481
Homoproline
Cyclohexyl glycine
Phenylalanine
tert-Butyl glycine
(4,5-Benzo)proline
Phenyl glycine
norLeucine
1,220,081
1,285,268
2,323,255
912
153,367
42,326
98,551
511
170,466
136,846
160,076
5583
388,557
1,025,631
456,491
1136
1.44
1.26
0.27
22.14
2.77
1.49
7.53
0.84
0.93
1.5
2.7
2.9
50
1,075,916
2,571,212
2,680,884
1,537,859
767,316
275,734
148,881
62,940
96,254
70,631
229,873
174,831
1,082,958
160,779
8227
1305,449
1,834,206
1,826,721
234,028
51,317
0.5
1
homoPhenylalanine
tBu-alanine
3
2
2.5
Aminocyclopentane
carboxylic acid
a Assay conditions are described in Refs. 8 and 14.
b Murine caspase-1 enzyme. Other enzyme data are from human.
the P2 tert-butyl and phenyl glycine analogues (29 and
31) were quite potent. Other bulky P2 amino acids, such
as phenylalanine and homophenylalanine (28 and 33)
showed potent caspase inhibition but limited cellular
activity (Table 4).
tion with the potassium salt of 2,3,5,6-tetrafluorophenol.
After reduction of the ketone and deprotection of the
amine, the requisite oxamic acid is then coupled. Dess–
Martin oxidation of the alcohol and subsequent tert-
butyl ester hydrolysis yields the oxamyl dipeptide.
The synthesis of compound 78 is outlined in Scheme 3 as
an example of how the analogues in Table 5 were pre-
pared.8 This involves preparation of the oxamic acid,
using methyl chlorooxoacetate and the amine of choice,
followed by ester hydrolysis. The valinyl-aspartyl tetra-
fluorophenoxy methyl ketone portion of the inhibitor is
prepared by converting Z-Val-Asp(OtBu)-OH to its
bromomethyl ketone derivative, followed by substitu-
This SAR study involved an extensive investigation of
the oxamyl terminal group effect on in vitro activity. The
results of this P4 SAR are summarized in Table 5. As
can be seen from the table, several analogues exhibited
potent in vitro activities and were considered candidates
for in vivo analysis. Alkyl oxamides showed modest
activity; the t-octyl and adamantyl analogues (38 and
40) indicated a preference for bulk, although the methyl
Scheme 3. Reagents and conditions: (a) methyl chlorooxoacetate (1.1 equiv), (TEA) (1.1 equiv), 0 °C, rt, 1 h; (b) 1 N LiOH (1.2 equiv), 1,4-dioxane,
rt, 1 h, (65%, two steps); (c) isobutyl chloroformate (1.1 equiv), (NMM) (1 equiv), THF, ꢀ10 °C, 30 min; (d) 1.5 equiv CH2N2/Et2O, 0 °C, 1 h; (e) HBr
(aq 48%) THF, 0 °C, rt, 15 min (87% three steps); (f) KO(2,3,5,6-Fr)Ph, NaI, acetone, rt; (g) NaBH4; MeOH/THF, 0 °C; (h) H2 (1 atm), 10% Pd/C,
MeOH, rt, 4 h; (i) (2,3,5,6-Cl4-Ph)NHCOCOOH (1.5 equiv), HATU (1.2 equiv), DIEA (2 equiv), CH2Cl2, rt, 2 h; (j) Dess–Martin (1.25 equiv),
CH2Cl2, rt, 15 min; (k) TFA/CH2Cl2/anisole, (1:1:0.5), rt, 1 h.

S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
2689
Table 5. P4 SAR
Compd
R
Enzyme assaysa K3/Ki (Mꢀ1 sꢀ1
)
Cellular assays IC50 (lM)
mCsp-1b
Csp-3
Csp-6
Csp-8
Jurkat
THP-1
Con A
SKW 6.4
36
37
38
39
40
41
42
43
44
45
46
11
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
Me
n-Heptyl
t-Octyl
16,846
87,791
149,284
47,953
126,292
52,600
21,165
21,407
643,095
92,584
154,580
281,943
124,16
71,761
37,831
206,906
0.11
1.28
0.013
0.06
1.76
0.43
0.0771
1.55
0.52
4.7
1.23
7.71
1.51
0.72
1.79
0.07
0.57
3.5
7
1.04
7.80
0.41
1.78
0.72
0.94
0.91
18.33
1.59
0.61
7
241,885
126,294
171,800
158,722
259,386
314,557
166,660
345,243
441,760
892,596
625,087
46,041
243,190
164,873
68,859
444,855
142,818
128,669
274,616
218,579
119,655
35,050
2
Cyclohexyl
Adamantyl
Ph
6.12
3.5
1.5
1.5
9
327,155
388,350
156,637
55,558
PhCH2
3,4,5-Trimethoxybenzyl
1-Naphthyl-CH2
PhCH2CH2
Ph2CH
0.37
0.08
0.17
0.77
0.7
0.8
3.5
7.3
0.5
3.28
2.4
2
243,319
67,743
317,122
54,920
119,087 >200
1-Naphthyl
2-Naphthyl
1,2,3,4-H4-1-naphthyl
5,6,7,8-H4-1-naphthyl
(1-Naph)(Me)
4-Cl-1-naphthyl
1-Anthryl
288,748
395,393
20,525
142,903
483,945
21,079
447,761
588,771
18,719
0.0061
1.61
0.38
0.86
1.07
0.0007
0.37
0.175
2.91
1.10
904,928
236
18,802
138,864
2
4282
132,427
78
2319
1,150,059
0
9825
>100
>50
>100
0.03
0.01
0.15
0.2
0.61
0.22
0.66
0.16
1.77
1.85
0.4
0.75
0.85
0.55
2.5
2.5
1.1
2.5
2
2.45
1.96
0.75
0.48
1.78
2.00
1.20
1.56
2.24
2.01
9.90
17,606,236
43,267
260,083
141,709
158,011
78,594
263,666
191,060
335,251
202,138
185,911
98,510
1,917,858
39,064
2-Anthryl
(2-Ph)-Ph
2,593,143
1,502,281
190,759
369,375
1,156,360
1,234,278
5,594,712
59,480
1,269,323
933,186
82,742
2-Phenoxy-Ph
3-Phenoxy-Ph
2-Methoxy-Ph
2-Benzyl-Ph
Ph2-N
0.66
1.15
0.31
0.0090
0.19
0.07
2.35
244,710
100,239
187,179
168,506
104,026
113,104
78,333
441,478
935,882
666,672
526,289
70,373
213,969
130,873
123,918
143,499
66,233
0.18
0.78
0.63
6.58
4.87
2.3
1-Ph,1-Bz-N
Ph-NH
PhCH2O
20
36,278
46,344
30.8
>32
15
1
5-Indanyl
2-tBu-Ph
289,114
955,812
210,683
11,732
189,287
268,006
104,370
22,681
146,052
424,103
308,387
18,592
305,616
2,302,210
1,338,965
18,657
0.041
0.0022
0.017
0.27
1.28
1.85
2.64
6.6
0.27
0.34
1.87
13.00
0.23
0.50
0.92
0.11
1.27
1.54
1.63
0.15
1.19
0.58
14.02
0.41
30.10
2.20
19.09
5.68
2,5-di-tBu-Ph
4-n-Heptyl-Ph
2-CF3-Ph
2
>50
1,942,965
194,786
106,344
2,019,148
7,157,535
1,980,934
277,593
>10,000,000
266,379
415,922
236,329
3,908,091
13,844
842,242
221,052
8,161,648
1,173,738
1,274,281
563,535
322,524
206,962
113,021
207,576
141,958
279,263
54,288
745,467
191,812
247,449
654,890
504,107
373,103
280,613
132,506
62,630
1,626,372
247,890
179,386
2,137,136
2,346,190
2,328,063
396,449
83,952
0.0017
0.097
0.019
0.016
0.356
0.081
0.136
0.01
0.82
0.68
1.21
0.16
0.54
1.5
0.65
2-F-Ph
4-F-Ph
0.97
2
2-Cl-Ph
2-Br-Ph
0.3
0.4
1
2-I-Ph
2,6-di-F-Ph
2-F;4-I-Ph
0.38
0.41
0.57
0.27
0.47
1.05
5.67
1.43
1.44
1
0.7
3.5
2.1
2.9
0.66
2,3,4,5-F4-Ph
2,3,4,6-F4-Ph
2,3,4,5,6-F5-Ph
2,3,5,6-Cl4-Ph
4-Pyridyl (TFA salt)
2-Pyridyl (TFA salt)
2-Pyrazyl (TFA salt)
58,875
125,846
66,174
0.04
0.11
23,953
104,258
98,718
42,761
0.39
0.22
375,045
23,128
42,243
15.8
30
22,030
27,578
117,608
149,427
64,055
124,444
0.14
0.17
9.4
5.9
50,371
a For assay conditions, see Refs. 8 and 14.
b Murine caspase-1 enzyme. Other enzyme data are from human.
oxamide showed reasonable cellular activity. In another
set of analogues ranging from phenyl, benzyl, phenethyl
to tetrahydronaphthyl derivatives, the 1- and 2-naphthyl
(11 and 47), 1- and 2-anthryl (52 and 53), and the
5,6,7,8-H4-1-naphthyl (49) stood out as particularly
active compounds. This is consistent with what was
learned in the acyl dipeptide caspase inhibitor SAR.7 In
general, 2-substituted phenyl oxamides show the best
overall activity, as well as other halogenated phenyl
oxamides. Modeling of some these caspase inhibitors in
caspase 1, as well as crystal structures of various
inhibitors bound to caspase 3 (unpublished results),
show that the key interactions in the P3/P4 region are
primarily between Arg 207 and the oxamide function-
ality itself. More specifically, in a crystal structure of an
ꢀ
inhibitor bound to caspase 3, a 2.8 A interaction

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S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
between the carbonyl of Arg 207 and the distal oxamyl
ꢀ
NH was noted, as well as a 2.7 A interaction between the
References and notes
1. For nomenclature of the caspase family, see: (a) 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. Ann. Rev. Cell
Biol. 1991, 7, 663; (b) Reed, J. C.; Tomaselli, K. J. Curr.
Opin. Biotech. 2000, 11, 586.
amide hydrogen of Arg 207 and the proximal oxamyl
carbonyl. It was difficult to propose defined interactions
between the phenyl ring of substituted phenyl oxamide
analogs and residues in the P4 pocket, such as His 342
and Arg 383. This is not surprising given that the P4
pocket of caspase 1 is not a very well defined binding
pocket, generally picking up hydrophobic interactions.
This lack of defined phenyl interactions is borne out in
the methyl oxamide (36). Although not as potent as the
phenyl oxamide (41), or as the benzyl oxamide (58), the
methyl oxamide is in the same range and possesses
similar cellular activity. This is further reinforced by the
loss of activity seen with the methylated 1-naphthyl
oxamide (50). New analogues with more diverse sub-
stitution of the phenyl ring should prove interesting.
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.; Nutall, 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. 2000, 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)
Choong, I. C.; Lew, W.; Lee, D.; Phuongly, P.; Burdett,
M. T.; Lam, J. W.; Wiesmann, C.; Luong, T. N.; Fahr, B.;
DeLano, W. L.; McDowell, R. S.; Allen, D. A.; Erlanson,
D. A.; Gordon, E. M.; O’Brien, T. J. Med. Chem. 2002,
45, 5005; (g) Han, B. H.; Xu, D.; Choi, J.; Han, Y.;
Xanthoudakis, S.; Roy, S.; Tam, J.; Vaillancourt, J.;
Colucci, J.; Siman, R.; Giroux, A.; Robertson, G. S.;
Zamboni, R.; Nicholson, D. W.; Holtzman, D. M. J. Biol.
Chem. 2002, 277, 30128; (h) For a review of the caspase
inhibitor patent literature, see: Ashwell, S. Exp. Opin.
Ther. Patents 2001, 11, 1593.
It was noted that the potency of caspase inhibition and
cellular activity do not correlate well. Lack of potency in
these cellular assays with analogues having good caspase
activity could be attributable to a number of factors,
ranging from protein binding, cell permeability to the
compounds stability in a 24 h assay run at 37 °C. Other
reasons for the poor correlation between overall caspase
inhibition and the cell assays are that some caspases are
more relevant to particular cell assays than others. For
example, the monocyte and THP-1 assays are solely
dependent on caspase 1. On the other hand, although
caspase 8 is highly relevant to the Jurkat assay, it is not
the only caspase involved. When evaluating in vitro data
of this type, it is best to look at general trends.
Based on in vitro activities (namely cellular data) ana-
logue 78 was one of several inhibitors selected for testing
in vivo. This inhibitor showed an 18% reduction in
infarct size when dosed iv at 30 mg kgꢀ1 in a rat per-
manent MCAO (Middle Cerebral Artery Occlusion)
model.16
4. Karanewsky, D. S.; Kalish, V. J.; Robinson, E. D.;
Ullman, B. R. U.S. Patent 6,242,422 B1, 2001.
5. 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, 2969.
A new, novel class of oxamyl dipeptide caspase inhibi-
tors were prepared. The SAR reveals that the activity of
this series can be optimized using either polyfluoro-
phenoxy, dichlorobenzoyloxy, or diphenylphosphinyl
as the warhead, valine, or tert-butyl valine at P2, and
2-substituted phenyl oxamides or halogenated phenyl
oxamides at P4. These analogues are potent, broad
spectrum caspase inhibitors (low nanomolar activity)
that are active in cellular models of apoptosis and
show promise in vivo. The nature of the SAR and the
limited diversity of the analogues discussed here show
that much work remains in exploring this series of
inhibitors.
6. Mittl, P. R. E.; DiMarco, S.; Krebs, J. F.; Bai, X.;
Karanewsky, D. S.; Priestle, J. P.; Tomaselli, K. J.;
Grutter, M. K. J. Biol. Chem. 1997, 272, 6539.
7. Linton, S. D.; Karanewsky, D. S.; Ternansky, R. J.; Chen,
N.; Guo, X.; Jahangiri, K. G.; Kalish, V. J.; Meduna, S.
P.; Robinson, E. D.; Ullman, B. R.; Wu, J. C.; Pham, B.;
Kodandapani, L.; Smidt, R.; Diaz, J. L.; Fritz, L. C.;
Krosigk, U.; Roggo, S.; Schmitz, A.; Tomaselli, K. J.
Bioorg. Med. Chem. Lett. 2002, 12, 2973.
8. (a) Karanewsky, D. S.; Ternansky, R. J. U.S. Patent
6,197,750, 2001; (b) Karanewsky, D. S.; Ternansky, R. J.;
Linton, S. D.; Dinh, T. PCT Patent Application WO 02/
057298, 2002.
9. Assay definitions: (a) Monocyte: Primary monocytes
isolated from peripheral human blood by Dextran sedi-
mentation and Ficoll. Cells were stimulated overnight with
staph A. Inhibition of IL-1b production evaluated with
human IL-1b ELISA assay (R&D Systems). See: Bleeker,
M. W. P.; Netea, M. G.; Kullberg, B. J.; van der Ven-
Jongekrijgg, J.; van der Meer, J. W. Immunology 1997, 91,
548; Hamon, Y.; Luciani, M.-F.; Becq, F.; Verrier, B.;
Rubartelli, A.; Chimini, G. Blood 1997, 90, 2911; (b) Con
A: Rat embryonic cortical neurons exposed to 100 nM
concanaval A for 24 h; survival measured by MTS
Acknowledgements
The authors wish to thank Alfred Spada, Tom Hayes,
and Judy Dryden for their contributions in preparing
this manuscript.

S. D. Linton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2685–2691
2691
reduction. See: Deckwerth, T. L.; Adams, L. M.; Wiess-
ner, C.; Allegrini, P. R.; Rudin, M.; Sauter, A.; Hengerer,
B.; Sayers, R. O.; Rovelli, G.; Aja, T.; May, R.; Nalley,
K.; Linton, S.; Karanewsky, D. S.; Wu, J. C.; Roggo, S.;
Schmitz, A.; Contreras, P. C.; Tomaselli, K. T. Drug Dev.
Res. 2001, 52, 579; (c) Jurkat clone E6-1 cells exposed to
50 ng mLꢀ1 anti-fas (CH-11) for 24 h. Viability evaluated
with MTT. See: Armstrong, R. C.; Aja, T.; Xiang, J.;
Gaur, S.; Krebs, J. F.; Hoang, K.; Bai, X.; Korsmeyer, S.
J.; Karanewsky, D. S.; Fritz, L. C.; Tomaselli, K. J.
J. Biological. Chem. 1996, 271, 16850; (d) THP-1: Human
monocytic cell line primed with interferon-c. Cells were
stimulated overnight with LPS (lipopolysaccharide). Inhi-
bition of IL-1b production evaluated with human I ELISA
assay (R&D Systems). See: Humphreys, B. D.; Dubyak,
G. R. J. Immunol. 1996, 5627, For more detailed assay
conditions, see Ref. 8.
P.; Nalley, K.; Robinson, E. D.; Roggo, S. P.; Sayers, R.
O.; Schmitz, A.; Ternansky, R. J.; Tomaselli, K. J.; Wu, J.
C. Bioorg. Med. Chem. Lett. 2003, 13, 3623.
12. (a) Krantz, A.; Copp, L. J.; Coles, P. J.; Smith, R. A.;
Heard, S. B. Biochemistry 1991, 30, 4678; (b) Dolle, R. E.;
Hoyer, D.; Prasad, C. V. C.; Schmidt, S. J.; Helaszek,
C. T.; Miller, R. E.; Ator, M. A. J. Med. Chem. 1994, 37,
563.
13. March, J. In Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, 4th ed.; John Wiley: New
York, 1992; pp 430–431, and references cited therein.
14. Wu, J. C.; Fritz, L. C. Methods 1999, 17, 320.
15. Dolle, R. E.; Singh, J.; Whipple, D. I.; Osifo, K.;
Speier, G.; Graybill, T. L.; Gregory, J. S.; Harris, A. L.;
Helaszek, C. T.; Ator, M. A. J. Med. Chem. 1995, 38,
220.
16. Analogue 78 was administered iv @ 1, 3, and 5 h post-
occlusion to male Fischer 344 rats. Vehicle used was 5%
Tween 80. Infarct volume was measured at 24 h using T2-
weighted MRI. Reduction in infarct size was statistically
significant, p < 0:05.
10. Scheme 1 describes the synthetic path using valine; similar
chemistry was used for the leucine analogues.
11. Ullman, B. R.; Aja, T.; Deckwerth, T. L.; Diaz, J. L.;
Herrman, J.; Kalish, V. J.; Karanewsky, D. S.; Meduna, S.