Inhibitors of Na-acetyl-L-ornithine deacetylase
1163
conditions to yield the corresponding isocyanate (Tsai et al.
2002). After refluxing in pyridine with a series of alcohols
determination. Only five Orn derivatives (1a, 1f–1i) were
weak inhibitors of B. subtilis-168 in the range 250–500 lM
for CFUs amounting to millions of bacteria. As in the drop
diffusion test, no MIC activity of Orn derivatives toward E.
coli was found.
´ˇ
(Hlavacek et al. 1976), the corresponding Orn derivatives
of general formulae Na–R–O–CO–Nd–Z–Orn–OMe were
obtained. Hydrogenation on Pd/C, followed by saponifi-
cation with NaOH yielded 2a–2e, which was desalted using
ion exchange chromatography on Dowex 50 and purified
by HPLC (Table 1). Triphosgene in DCM was found to
exhibit excellent reactivity by modifying only the a-amino
group under mild alkaline conditions. Interestingly, no
cyclization or lactam formation was observed until after
Nd–Z deprotection by hydrogenolysis of the corresponding
Na–R–O–CO–Nd–Z–Orn–OMe derivative. Finally, (CH3)3
C–O–CO–Orn–OH (2f) was prepared by direct reaction of
H–Nd–Z–Orn–OMe with (Boc)2O in a dioxane–water
mixture at pH 8 adjusted with NaHCO3 (Moroder et al.
1976), followed by deprotection of the d-amino and car-
boxyl groups, desalting and purification as previously
described.
Probably, the weak activity of Orn derivatives toward B.
subtilis might correspond to a decrease or elimination of
the hydrolytic activity of ArgE via inhibition of the amide
bond cleavage in Na-acetylornithine. Above data correlate
well with the IC50 values determined for ArgE, suggesting
an indirect evidence that Orn derivatives are capable of
getting across the cell membrane and that ArgE is the
likely bacterial enzymatic target.
In conclusion, the new Orn based compounds described
herein represent a new class of inhibitors for ArgE
enzymes. Interestingly, several of these compounds exhibit
weak antibacterial activity toward B. subtilis. Therefore,
ornithine provides a realistic structural platform for the
design of new inhibitors for ArgE enzymes that may pos-
sess antimicrobial activity. In general, small molecules that
are non-degradable substrate analogues should provide
effective inhibitors of bacterial enzymes and represent a
reasonable approach toward the development of a previ-
ously undescribed antimicrobial agent. Ultimately, refining
the structural aspects of these substrate analog inhibitors in
order to match inhibitor structural features to those of the
enzyme, including metal binding interactions, will be
crucial to the discovery of a clinically relevant antimicro-
bial agent.
All of the Orn derivatives synthesized were examined
for their ability to inhibit the catalytic activity of ArgE. Of
the 18 compounds synthesized, nearly all inhibited ArgE
activity as evidenced by IC50 values in the lM range.
These data indicate that all of the ornithine derivatives
prepared in this study are moderately strong inhibitors of
ArgE. The Orn derivative that provided the best inhibitory
response was Na-chloroacetyl-L-ornithine (1g), which
exhibited an IC50 value of 85 lM. Similarly, the trifluo-
roacetyl-(1f) and ethoxycarbonyl-(2b) ornithine derivatives
provided IC50 values of 200 and 250 lM, respectively. It
should be noted that the Na-dichloroacetyl-L-ornithine (1h),
the Na-trichloroacetyl-D-ornithine (1j), the Na-butyryl-L-
ornithine (1d), and the Na-acetyl-D-ornithine (1a) deriva-
tives all exhibited moderate inhibition of ArgE providing
IC50 values in the range 320–450 lM (Table 1). Inspection
of these inhibitory data provides some clues into the
active site of ArgE enzymes. The best inhibitors have small
R groups with the relative inhibitory order CH2Cl ꢀ
CF3 [ CHCl2 [ CH3 with the D and L derivatives of
CHCl2 inhibiting ArgE equally. This trend suggests that
this R group must recognize specific binding pocket near to
the dinuclear Zn(II) active site of ArgE.
Acknowledgments This research was supported by the Research
Project Z4 055 0506, the Grant Agency of the Czech Academy of
Sciences (No. IAA400550614) and the National Science Foundation
(CHE-0652981, RCH).
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In order to determine if Orn derivatives are potential
antibiotic leads their antimicrobial activity was examined
against B. subtilis-168 and E. coli B (Table 1). A majority
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Hlavacek
