inhibition of the release of acetylcholine from synaptic
terminals.4
functionality, a monoanionic bidentate chelator of zinc that
has been previously used for the inhibition of metallopro-
teinases,11 (2) a para-halogenated phenyl ring was needed,12
and (3) an ortho,para-dihalogenated ring was superior to
para-substition alone10 (Figure 1). Indeed, X-ray crystal-
lographic analysis of the bound inhibitors revealed hydroxyl
coordination of the hydroxamate to the Zn(II) ion in the
active site in addition to phenyl ring interaction with a
hydrophobic portion of the enzyme.2 Addition of the ortho
chlorine substituent created an additional favorable interac-
tion with an active site arginine, making this the most potent
inhibitor reported to date.2
While our initial research has been critical to lending
credence that small nonpeptidic molecules can be tailored
to the BoNT/A protease, it is readily apparent that more
potent inhibitors will be required if a therapeutic is the final
goal. As such, we sought to synthesize a series of molecules
containing additional chemical diversity for potential interac-
tion(s) with the enzyme active site. We envisioned the
preparation of hydroxy ethyl hydroxamates (R)- and (S)-4
and -5, which would not only extend within polar space of
the enzyme’s combining site but also explore the importance
of molecular chirality and its impact on enzyme inhibition.
Current treatment for BoNT intoxication employs an
antitoxin to sequester the toxin followed by removal of the
complex from the body. However, this treatment option is
dependent upon prompt diagnosis of intoxication and must
be administered rapidly before the toxin can enter the cell.5
The antitoxin also suffers from the risk of allergic reaction,6
long-term effects of unknown origin,7 and insufficient
available quantities for treatment of a large-scale attack.8
BoNT serotype A (BoNT/A) is considered to be the most
deadly BoNT serotype based on the following: First, and
most importantly, BoNT/A has an extremely long duration
of action, i.e., weeks to months. Second, there is no antidote
for BoNT/A intoxication, with severe intoxication cases
requiring mechanical ventilation due to respiratory paralysis.9
Third, BoNT/A, together with serotype B, is responsible for
∼1000 cases of human BoNT poisoning per year.1 Finally,
BoNT/A has a high potential for bioterrorism use due to its
extreme toxicity and ease of dissemination through the food
or water supply. Thus, the inhibition of BoNT/A LC is an
attractive target for nonpeptidic small molecules that can
target the enzyme within an intoxicated cell.
Scheme 1
.
(A) Structures of Hydroxamates (R)- and (S)-4 and
-5; (B) Retrosynthetic Analysis
Figure 1
inhibitors.
. Structure and IC50 values of previously identified BoNT/A
Previous research from one of our laboratories screened a
diverse library of small molecules and discovered several
inhibitors of BoNT/A, with trans-cinnamic hydroxamate 1
being our initial lead (Figure 1). Hydroxamate 2 stemmed
from structure-activity studies, and is one of the most potent
inhibitors known, having a Ki value of 300 ( 12 nM.10 From
these studies several lessons were learned, including the
following: (1) all structures required the hydroxamate
We viewed these structures as rational choices based on
active-site molecular modeling wherein a key goal was to
displace a nacent water molecule within the enzyme active
site, which on the basis of previous protease inhibitors
targeting active site water molecules could improve Ki up
to 100-fold.13 To access such structures required an asym-
metric synthetic strategy that would construct both enantio-
mers of the final hydroxamate products from a single, readily
(3) Blasi, J.; Chapman, E. R.; Link, E.; Binz, T.; Yamasaki, S.; De.
Camilli, P.; Sudhof, T. C.; Niemann, H.; Jahn, R. Nature 1993, 365, 160–
163.
(4) Hambleton, P. J. Neurol. 1992, 239, 16–20.
(5) Capkova, K.; Salzameda, N. T.; Janda, K. D. Toxicon 2009, 54, 575–
582.
(6) Black, R. E.; Gunn, R. A. Am. J. Med 1980, 69, 567–570.
(7) Villar, R. G.; Elliott, S. P.; Davenport, K. M. Infect. Dis. Clin. North
Am. 2006, 20, 313–327.
(8) Arnon, S. S.; Schechter, R.; Inglesby, T. H.; Henderson, D. A.;
Bartlett, J. G.; Ascher, M. S.; Eitzen, E.; Fine, A. D.; Hauer, J. JAMA,
J. Am. Med. Assoc. 2001, 285, 1050–1079.
(11) (a) Skiles, J. W.; Gonnella, N. C.; Jeng, A. Y. Curr. Med. Chem.
2004, 22, 2911–2977. (b) Grant, S.; Easley, C.; Kirkpatrick, P. Nat. ReV.
Drug DiscoV. 2007, 6, 21–22.
(9) Hicks, R. P.; Hartell, M. G.; Nichols, D. A.; Bhattacharjee, A. K.;
Von Hamont, J. E.; Skillman, D. R. Curr. Med. Chem. 2005, 12, 667–690.
(10) Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Org. Lett. 2006, 8, 1729–
1732.
(12) Capkova, K.; Yoneda, Y.; Dickerson, T. J.; Janda, K. D. Bioorg.
Med. Chem. Lett. 2007, 17, 6463–6466.
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