Structure-activity relationships within a series of caspase inhibitors. Part 2: Heterocyclic warheads

Article abstract of DOI:10.1016/j.bmcl.2005.05.036

Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val- Asp backbone have been prepared. A study of their structure-activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.

Full text of DOI:10.1016/j.bmcl.2005.05.036

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3632–3636
Structure–activity relationships within a series of caspase
inhibitors. Part 2: Heterocyclic warheads
Brett R. Ullman,a,* Teresa Aja,a Ning Chen,a Jose-Luis Diaz,a Xin Gu,a Julia Herrmann,a
Vincent J. Kalish,a Donald S. Karanewsky,a Lalitha Kodandapani,a Joseph J. Krebs,a
Steven D. Linton,a Steven P. Meduna,a Kip Nalley,a Edward D. Robinson,a
Silvio P. Roggo,b Robert O. Sayers,a Albert Schmitz,c Robert J. Ternansky,a
Kevin J. Tomasellia and Joe C. Wua
aIdun Pharmaceuticals, Inc., 9380 Judicial Drive, San Diego, CA 92121, USA
bDiscovery Technologies, Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002 Basel, Switzerland
cNeuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002 Basel, Switzerland
Received 8 April 2005; revised 5 May 2005; accepted 7 May 2005
Available online 17 June 2005
Abstract—Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A study
of their structure–activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell
death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.
Ó 2005 Elsevier Ltd. All rights reserved.
Apoptosis (programmed cell death) is a highly regulated
biological process involved in maintaining normal tissue
homeostasis. Caspases (cysteinyl aspartate-specific pro-
teases) play a critical role in the regulation of inflamma-
tion, apoptosis, and cytokine maturation.1,2 Caspases
can be classified into two subfamilies with distinct func-
tions: apoptosis, caspase-2, 3, -6, -7, -8, -9, -10, and -12
and cytokine activation, caspses-1, -4, and -5.3
of caspase-1 inhibitors as anti-inflammatory agents and
more recently research has focused on pan caspase inhib-
itors and small molecule inhibitors of caspase-3.6
Previously, we described use and SAR of phenoxy-
methyl ketone warheads on a naphthyloxy acyl dipep-
tide backbone.7 Herein, we describe modification and
analysis of heteroaryl hetero-methyl ketones as the war-
head functionality (the electrophilic moiety that cova-
lently interacts with the active site of the enzyme) on
the same naphthyloxy-Val-Asp backbone (Fig. 1). Cel-
lular activity in the Jurkat (JFas) assay,8 a Fas-induced
model of apoptosis, is also discussed.
Therefore, inhibitors of the caspase family of cysteine
proteases may be useful therapeutic agents for the treat-
ment of inflammatory conditions such as arthritis and
hepatitis; reperfusion injuries such as myocardial
infarction and ischemic kidney disease; neurodegenera-
tive diseases such as HuntingtonÕs, ParkinsonÕs, and Alz-
heimerÕs disease; and for the treatment of stroke.4 Such
inhibitors may also be useful for the prolonging of organ
viability for use in transplantation.5
Preparation of the acyldipeptide aryloxy or arylthio
methyl ketones is described in Scheme 1. Dipeptide 1
was prepared from 1-naphthyloxyacetic acid and L-val-
yl-L-asparyl-b-(tert-butyl ester)methyl ester 29 using
ethyl-(dimethylaminopropyl)carbodiimide (EDAC)/1-
hydroxybenzotriazole (HOBt) coupling conditions in
Inhibitors of caspases represent a class of compounds use-
ful for the control of the previously stated disease states.
Early research in this area has been focused on the use
*
Corresponding author. Tel.: +858 646 8188, fax: +1 858 625 2677;
e-mail: bullman@idun.com
Figure 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.036

B. R. Ullman et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3632–3636
3633
Scheme 1. Reagents and conditions: (a) EDAC (1.5 equiv.), HOBt (1.05 equiv.), N-methylmorpholine (NMM) (1 equiv.), CH2Cl2, 0 °C 2 h, 20 h,
83%; (b) LiOH (1 equiv.), dioxane/H2O (3:1), 1 h, 99%; (c) (i) Isobutyl-chloroformate (1.05 equiv.), NMM (1.5 equiv.), THF, ꢀ20 °C, 20 min, (ii)
CH2N2, and (iii) HBr (50%, H2O), Et2O, 0 °C, 30 min, 71%; (d) KF (2.5 equiv.), NaI (0.1 equiv.), (1 equiv.), DMF, 40–80%; (e) TFA/CH2Cl2/anisole
(4/3/1), 99%.
Table 1. Warhead analysis of five-membered heterocycles
Compound
R
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
)
Cellular assay IC50 (lM)
mCsp-1b
Csp-3
Csp-6
Csp-8
2,266
Jurkat
4
5
6
7,572
71,138
23,909
28
46
9,396
1,445
35,478
5,674
509
295
0c
1,141
>200
7
8
3,671
42,463
173
21,844
143,850
850
220
5,804
22
279
6,344
0c
>200
71
9
>200
>200
10
1,201
11,546
570
212
11
199
1,391
22
0c
>200
>200
12
13
186
217
2,295
2,731
228
231
0c
0c
>200
(continued on next page)

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B. R. Ullman et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3632–3636
Table 1 (continued)
Compound
R
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
Csp-3 Csp-6
)
Cellular assay IC50 (lM)
mCsp-1b
Csp-8
218
Jurkat
14
200
2,010
0c
>200
15
16
17
18
19
20
21
22
23
24
25
26
1,191
280
38,119
4,798
401
975
110
17
3,059
276
0
102
150
115
>200
>200
190
180
2,259
5,239
19,186
34,433
221
83
293
5,477
400
0c
224
2,150
0c
0c
153
3,629
6506
>200
>200
>200
>200
>200
>200
0c
0c
0c
168
1,769
3,569
25
10
202
0c
0c
41
958
0c
2
28,324
121,491
8,534
14,518
a Assay conditions are described in Ref. 13.
b Murine caspase 1.
c Indicates reversible inhibition.
methylene chloride followed by ester hydrolysis with
lithium hydroxide in dioxane/water (3:1). Bromomethyl
ketone 3 was obtained by treatment of the mixed anhy-
dride of 2 with diazomethane10 followed by reaction of
the diazo ketone with hydrogen bromide. Warheads
were attached by treating 3 with the appropriate alcohol
or thiol, sodium iodide, and potassium fluoride in DMF
under the modified Finkelstein11 conditions. Removal of
the t-butyl ester protecting group with trifluoroacetic
acid produced the target inhibitors, after trituration in
diethyl ether.
In our continuing focus on warhead optimization, we
synthesized various heterocyclic hetero-methyl ketones.

B. R. Ullman et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3632–3636
Table 2. Warhead analysis six-membered heterocycles
3635
Compound
R
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
)
Cellular assay IC50 (lM)
mCsp-1b
Csp-3
Csp-6
Csp-8
Jurkat
>200
ND
27
28
29
138
0c
733
5
8
348
0c
0c
0c
0c
0c
0c
>200
30
31
32
33
34
35
36
37
38
39
40
41
1,326
10
7,484
283
903
367
>200
ND
0c
0c
950
24,834
479
0c
7,130
656
5,147
156
0c
120
3,199
0c
>200
37
113,766
1,752
>200
>200
>200
>200
>200
>200
>200
>200
0c
0c
0c
0c
0c
0c
152
67
1,763
8
0c
979
4,359
3,350
343
0c
9
1,261
693
83
140
96
39
8
0c
4
42
43
0c
0c
0c
0c
166
360
1,307
462
177
106
(continued on next page)

3636
B. R. Ullman et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3632–3636
Table 2 (continued)
Compound
R
Enzyme assaysa k3/Ki (Mꢀ1 sꢀ1
)
Cellular assay IC50 (lM)
mCsp-1b
45,379
Csp-3
Csp-6
Csp-8
Jurkat
44
45
168,736
44,007
18,230
137
161
273
906
143
107
a Assay conditions are described in Ref. 13.
b Murine caspase 1.
c Indicates reversible inhibition.
Starting with the known ((1-phenyl-3-(trifluoromethyl)-
pyrazol-5-yl)oxy) (or PTP) warhead (4),12 we first inves-
tigated five-membered aromatic heterocycles, as well as
benzofused five-membered heterocycles (Table 1).
Changing the regiochemistry and substituent (phenyl
to methyl, compounds 4 and 5, respectively) of the pyr-
azole ring maintained activity in caspase-1 and -3, but
resulted in reduced activity in caspase-6 and -8.
structure to the pyrazole (4). The use of benzotriazole
and benzotriazinone (26 and 44, respectively) also
proved to be effective. In general, these inhibitors were
found to be somewhat selective for caspase-3 in nature.
Unfortunately, the cellular efficacy of the inhibitors was
moderate. We also found that some of the heterocyclic
warheads investigated proved to be reversible inhibitors
of some or all of the enzymes studied. The use and opti-
mization of these warheads will be the topic of future
publications.
Interestingly, the isoxazoles (entries 6, 7, and 8) showed
good enzyme activity with a preference for ether substi-
tution at the 3-position, which mimics the positioning of
5. Compound 8 was highly active in the enzyme assays.
However, there was little or no cellular potency associ-
ated with this series.
References and notes
1. Yuan, J.; Shaham, J.; Ledoux, S.; Ellis, H. M.; Horvitz, H.
R. Cell 1993, 75, 641.
2. Miura, M.; Zhu, H.; Rotello, R.; Hartwieg, E. A.; Yuan,
J. Cell 1993, 75, 653.
3. Fuentes-Prior, P.; Salvesen, G. S. Biochem. J. 2004, 384,
201.
4. (a) Nett-Giordalisi, M. A.; Berson, D. R.; Chaplin, D. D.
J. Cell. Biochem. B 1993, 17, 117; (b) Marx, J.; Baringa,
M. Science 1993, 259, 760.
5. Natori, S.; Higuchi, H.; Contreras, P.; Gores, G. J. Liver
Transplant. 2003, 9, 278–284.
6. For reviews see (a) Talanian, R. V.; Brady, K. D.; Cryns,
V. L. J. Med. Chem. 2000, 43, 3351; (b) Ashwell, S. Expert
Opin. Ther. Patients 2001, 11, 1593; (c) OÕBrien, T.; Lee,
D. Mini Rev. Med. Chem. 2004, 4, 153.
Thioethers (compounds 9–24) exhibited some activity in
caspase-3. The N-substituted heterocycles showed better
activity than their unsubstituted analogues (10 vs 9, 18
vs 17); this may be due to the charge on the ring. The
positioning of the heteroatoms in the oxadiazole deriva-
tives (15, 16) exhibited a large effect on potency. The tet-
razoles (20, 21) exhibited good enzyme activity which
was better than that of the triazole (18). The benzofused
analogues exhibited very little activity with the exception
of the benzotriazole (26), which was very active in the
enzymes studied but inactive in the JFas model.
7. Ullman, B. R.; Aja, T.; Deckwerth, T. L.; Diaz, J.-L.;
Herrmann, J.; Kalish, V. J.; Karanewsky, D. S.; Meduna,
S. 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.
8. 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. Biol. Chem. 1996, 271,
16850.
9. Karanewsky, D. S.; Kalish, V. J.; Robinson, E. D.;
Ullman, B. R. U.S. Patent 6,242,422 B1, 2001.
10. Arndt, F. Org. Synth. 1943, 2, 165 (Coll. Vol.).
11. March, J. In Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, 4th ed.; John Wiley & Sons:
New York, 1992, pp 430–431, and references therein.
12. Dolle, R. E.; Singh, J.; Rinker, J.; Hoyer, D.; Prasad,
C. V. C.; Graybill, T. L.; Salvino, J. M.; Helaszek, C.
T.; Miller, R. E.; Ator, M. A. J. Med. Chem. 1994, 37,
3863.
Next, we focused on the use of six-membered ring het-
erocycles (Table 2). The two pyridyl derivatives (27,
28) were inactive. However, the pyrimidine series (com-
pounds 29–36) proved to be most interesting. These war-
heads not only preferred an electron withdrawing
(trifluoromethyl) moiety, but also the appropriate regio-
chemistry to properly line up in the P0 pocket for activ-
ity. Compound (33) closely mimics that of 4 and yielded
better enzyme activity with similar cellular activity. The
benzofused analogues (38–45) of the six-membered het-
erocycle series showed very little activity, with the excep-
tion of the benzotriazinone (44) which corresponds with
the results seen for the five-membered ring series and the
benzotriazole (26).
In conclusion, we found mimicking the PTP moiety (4)
led to several new active warheads. The most notable
of these new warheads were the oxazoles (7, 8) and the
2-trifluoromethyl pyrimidine (33), which are similar in
13. Wu, J. C.; Fritz, L. C. Methods: A Companion to Methods
in Enzymology 1999, 17, 320.