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resonance energy transfer (FRET) substrate.39,40 The proMMPi
demonstrated essentially no inhibition against MMP-2 (o5%,
Fig. S22, ESI†). In contrast, inhibition of MMP-2 after treatment
of proMMPi with 20 equiv. of H2O2 was increased to B37%,
close to the expected value of 50% and demonstrating that the
inhibitory prolife was readily restored. The data confirm that
the proMMPi is inactive against its target (MMP-2), but regains
near full activity after being treated with H2O2.
Based on our proposed scheme (Fig. 1), a thiazolidinone mole-
cule is liberated as the prodrug becomes activated by H2O2. There-
fore, we conducted a cell viability assay to determine if the
thiazolidinone moiety was cytotoxic. An MTS assay utilizing NIH
3T3 cells was performed in which the cytotoxicity of promoiety B was
Table 2 Stability of compounds. All values represent percent of parent
compound (designated in left column) remaining in solution
Buffera + glutathione
(20 equiv.) (%)
Compound
Buffera (%)
1
2
3
4
5
6
7
8
495
495
40
50
495
74
495
495
10
78
65
67
11
o5
10
o5
a
Buffer = 100 mM Tris-Cl, pH 7.4 (37 1C).
The success of compounds 2 and 6, both of which utilize determined. Compound B demonstrated no significant cytotoxicity
promoiety B, prompted the investigation of the scope of this up to a concentration of 100 mM (Fig. S26, ESI†).
prodrug approach with an FDA approved drug, ibuprofen, and a
In summary, our findings demonstrate the development of a
well-studied matrix metalloproteinase inhibitor (Fig. 3).38 Both prodrug strategy for the H2O2-dependent release of carboxylic
prodrugs (proIBU and proMMPi, Fig. 3) were readily synthesized† acids. A study using model compounds revealed the thiazolidinone
and evaluated for ROS activation. ProMMPi activation by two ROS promoiety (B, Fig. 2) to possess high stability and was not prone to
species was evaluated. Two independent experiments were carried attack by common biological nucleophiles. Prodrugs based on the
out in which a proMMPi solution (100 mM) was added to 20 equiv. thiazolidinone B displayed little to no activity against their targets,
of H2O2 or NaClO. Activation by H2O2 to the corresponding MMPi but in the presence of H2O2 activity was restored for an MMP
was observed (Fig. S17, ESI†); however, exposure of proMMPi to inhibitor. Overall, these findings indicate that a thiazolidinone (B)
NaClO lead to degradation to unidentified side products (Fig. S18, prodrug strategy is viable for derivatizing carboxylic acid-based
ESI†). In order to determine the sensitivity of proMMPi in a therapeutics for H2O2-targeted release. Considering the widespread
quantitative fashion, pseudo-first-order kinetic measurements were use of ester-based prodrugs, such as ACE inhibitors, it is expected
performed using analytical HPLC (Fig. S17, ESI†). The observed rate that the strategy here will have applicability to produce H2O2-
constant for conversion to the desired MMPi upon exposure to activated, disease-targeted analogs of various valuable bioactive
H2O2 was kobs = 1.1 Â 10À4
s
À1. The hydrolytic stability under compounds.
We thank the UCSD Small Molecule Mass Spectrometry and
simulated physiological conditions was also determined.
The compounds were incubated in DMSO and buffer (100 mM X-ray diffraction facilities for assistance with experiments. This
Tris-Cl, pH 7.4) for 24 h at 37 1C. Additionally, each prodrug was work was supported by a grant from the National Institutes of
incubated under the same conditions with the addition of Health, National Institute of General Medical Sciences (R01
20 equiv. of lysine, serine, or glutathione, in order to demon- GM098435).
strate that the promoiety is not readily cleaved in the presence of
biologically relevant nucleophiles. Both proIBU and proMMPi
displayed remarkable stability of 495% under all of the condi-
Notes and references
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¨
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Fig. 3 Prodrug version of ibuprofen and an MMP inhibitor.
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