P. Jaishankar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 624–628
627
binding affinities (Ki as low as 19 nM) but significantly
reduced rate constants of inactivation (kinact). Appar-
ently, while a lipophilic P3 substituent can confer stron-
ger binding through enhanced interactions with S3, the
vinyl sulfone moiety may, as a result, be nudged into a
less favorable orientation for efficient reaction. Interest-
ingly, analogs 8a–d bearing a more hydrophilic P3 group
(pyridyl) display Ki and kinact values that are more com-
parable to the parent piperazine analogs.
non-piperazine analogs examined, only the P3 pyridyl
analog 8a was as effective as K-777. While it is tempting
to attribute this effect to the presence of a basic nitrogen
in 8a, the 2-pyridyl ring in 8a is unlikely to be signifi-
cantly protonated, even at lysosomal pH. Instead, the
activity of 8a may derive from improved permeability
(or active transport) into the parasite and/or sequestra-
tion to high concentration within particular compart-
ments. Certainly, the superior efficacy of 8a as
compared to 6a–c and 7a and 7b cannot be explained so-
lely on the basis of relative enzymatic activities.
In some cases the combination of P2 and P3 modifica-
tion confers a noteworthy synergistic effect. For exam-
ple, the combination of a 3-Me Phe at P2 with either
3,5-difluorophenylamide or benzodioxane moieties at
P3 produces analogs (6b and 9b) with noteworthy selec-
tivity for rhodesain over cruzain (up to eighteenfold).
The combination of a benzodioxane P3 substituent with
4-Me Phe at P2 affords 9d, a rhodesain-selective analog
that is fourfold more potent than K-777. Finally, the
combination of a pyridyl P3 group with a 4-Me Phe
P2 substituent affords 8d, the most highly rhodesain-
selective (twentyfold) analog described in this study.
Interestingly, in each of these cases (6b, 9b, 9d, 8d) rho-
desain selectivity derives not from increased binding
affinity (Ki) relative to cruzain but from faster rate con-
stants of inactivation (kinact).
In summary, we have conducted a systematic explora-
tion of P2 and P3 substitution in a series of vinyl sul-
fone-based cysteine protease inhibitors. Our results
suggest that the introduction of small substituents on
the phenyl ring of the P2 side chain represents a viable
strategy for generating more rhodesain-selective inhibi-
tors. We continue to explore P2/P3 modification in this
context and plan a more extensive biological evaluation
of these analogs, particularly with respect to activity
against T. brucei parasites.
Acknowledgments
Research at the SCBRPD was supported by the Sandler
Family Supporting Foundation and NIAID Grant
AI35707. The SMDC has received generous research
support from the Sandler Family Supporting Founda-
tion, the UCSF School of Medicine, the Cardiovascular
Research Institute at UCSF, and the California Institute
for Quantitative Biology (QB3). We thank Drs. James
Palmer and Mark Burlingame for helpful discussions
and Ymain Shew for technical assistance with the T. cru-
zi-infected macrophage assay.
Next, we examined selected analogs for their ability to
prolong survival of T. cruzi-infected J774 macrophages
using an established protocol1 (Table 2). This assay pro-
vides information about antiparasitic efficacy and also
provides a qualitative measure of compound toxicity
to the macrophage itself. The experiment was carried
out over 42 days and a number of the new P2/P3-mod-
ified analogs were found to exert an antitrypanosomal
effect (Table 2). The 3-methyl and 3-trifluoromethyl ana-
logs 4b and 4c were as effective as K-777, although the
latter showed some toxicity. A number of P3 benzoyl
amide analogs were also examined in this assay and
showed varying degrees of efficacy but were generally
less effective than the P3 piperazine analogs. Of the
References and notes
1. Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J.
Exp. Med. 1998, 188, 725.
2. Engel, J. C.; Doyle, P. S.; Palmer, J.; Hsieh, I.; Bainton, D.
F.; McKerrow, J. H. J. Cell Sci. 1998, 111(Pt. 5), 597.
3. Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J.
Med. Chem. 1995, 38, 3193.
Table 2. Efficacy of vinyl sulfones in
macrophage assay (10 lM compound)
a T. cruzi-infected J774
4. Abdulla, M. H.; Lim, K. C.; Sajid, M.; McKerrow, J. H.;
Caffrey, C. R. PLoS Med. 2007, 4, e14.
R
5. Bromme, D.; Klaus, J. L.; Okamoto, K.; Rasnick, D.;
Palmer, J. T. Biochem. J. 1996, 315(Pt. 1), 85.
6. Gillmor, S. A.; Craik, C. S.; Fletterick, R. J. Protein Sci.
1997, 6, 1603.
O
H
N
SO2Ph
Ph
N
H
Ar
O
7. Mackey, Z. B.; O’Brien, T. C.; Greenbaum, D. C.; Blank,
R. B.; McKerrow, J. H. J. Biol. Chem. 2004, 279, 48426.
8. McGrath, M. E.; Eakin, A. E.; Engel, J. C.; McKerrow, J.
H.; Craik, C. S.; Fletterick, R. J. J. Mol. Biol. 1995, 247,
251.
9. Brinen, L. S.; Hansell, E.; Cheng, J.; Roush, W. R.;
McKerrow, J. H.; Fletterick, R. J. Structure 2000, 8, 831.
10. Falgueyret, J. P.; Desmarais, S.; Oballa, R.; Black, W. C.;
Cromlish, W.; Khougaz, K.; Lamontagne, S.; Masse, F.;
Riendeau, D.; Toulmond, S.; Percival, M. D. J. Med.
Chem. 2005, 48, 7535.
Compound
R
Ar
J774 macrophage Macrophage
survival (days)
toxicity
No drug
K-777 (4a)
4b
—
H
—
N-MePip 42
5
—
No
No
Yes
3-Me N-MePip 42
3-CF3 N-MePip 42
4c
6a
6b
6c
7a
7b
8a
H
3,5-DiFPh 12
Yes
Yes
No
No
Yes
No
3-Me 3,5-DiFPh 12
3-CF3 3,5-DiFPh 23
H
3-Me 4-CF3Ph
4-CF3Ph
8
8
11. Somoza, J. R.; Zhan, H.; Bowman, K. K.; Yu, L.;
Mortara, K. D.; Palmer, J. T.; Clark, J. M.; McGrath, M.
E. Biochemistry 2000, 39, 12543.
H
2-Pyridyl 42