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HPLC analysis: Analytical HPLC was performed on a HP Series
1
050 system equipped with a Vydac C18 reverse-phase column
(
218TP, 250ꢁ4.6 mm, 5 mm). Separation was achieved with a flow
À1
rate of 1 mLmin and the following mobile phase: 5% MeOH+
0
.1% formic acid in H O (A) and 0.1% formic acid in MeOH (B).
2
Starting with 95% A and 5% B, an isocratic gradient was run for
5 min to a final solvent mixture of 5% A and 95% B, which was
1
held for 5 min before ramping back down to 95% A and 5% B
over 2 min, and holding for an additional 4 min. Compounds were
prepared in HEPES buffer (50 mm, pH 7.5) at a concentration of
1
mm. Retention times of compounds PY-2, and 1,2-HOPO-2 were
determined under identical HPLC conditions prior to evaluation of
esterase cleavage of the protected compounds.
To determine the efficiency of esterase cleavage for the matrix
metalloproteinase proinhibitors (proMMPI), a 1 mm solution of test
compound (1 mL) in HEPES buffer (50 mm, pH 7.5) was prepared
and treated with PLE (50 U). The sample was incubated at 258C for
Figure 3. Inhibition assay results for compounds 7–9, 11 and 12 against
MMP-8 and MMP-12 and in the absence or presence of porcine liver esterase
1
h prior to analysis (Figures S8 and S9 in the Supporting Informa-
(PLE).
tion).
To evaluate the hydrolytic stability of the proMMPI, a 1 mm solu-
tion of test compound (1.0 mL) in HEPES buffer (50 mm, pH 7.4)
was prepared and a HPLC trace was obtained immediately. The
sample was then incubated in the buffer solution for 24 h at 378C
before a second HPLC trace was obtained. The stability of each
sample was determined based on the area under the curve (Figur-
es S11–S14 in the Supporting Information).
benzyl ether linkage (approaches 2 and 3) is superior to the
conventional direct linkage of the acetate protecting group
(approach 1) with respect to kinetics and aqueous stability.
Testing of these compounds in a biochemical assay shows no
inhibition by the proinhibitors against either MMP-8 or MMP-
1
2. Upon treatment with esterase, the promoieties effectively
MMP inhibition assays: Inhibition values of compounds 7–9, and
cleave to generate the active MMPI, which inhibits the targets
as expected. We hope that the superior reaction-based strat-
egies presented here will serve as a platform for esterase-re-
sponsive prodrug design.
1
1–12 were determined using a previously described commercially
[15]
available fluorescent-based assay kit. MMP activity was measured
in 96-well plates using a Bio-Tek Flx800 fluorescent plate reader.
Each test compound was dissolved in DMSO to a concentration of
1
mm and diluted in HEPES buffer (50 mm, pH 7.5) to a concentra-
tion of 50 mm. Each sample was then treated with PLE such that
0 U of protein was present. This mixture was incubated for 1 h at
Experimental Section
5
room temperature. The esterase was removed via micro-centrifuga-
tion (258C, 5 min, 13000 rpm) using 10 kDa molecular weight cut-
off filters. The filtered esterase-treated compounds were then
added to appropriate wells at their respective IC50 values. Each well
contained 20 mL of MMP-8 or MMP-12 (1.82 UmL or 0.35 UmL ,
respectively), 60 mL MMP assay buffer (50 mm HEPES, 10 mm CaCl2,
Synthesis and characterization: The detailed synthesis and char-
acterization of compounds 1–12 are provided in the Supporting In-
formation. All chemicals were purchased from commercial suppli-
ers (Sigma–Aldrich, Acros Organics, TCI America) and were used
without further purification. Chromatography was performed using
a CombiFlash Rf 200 automated system from TeledyneISCO (Lin-
coln, USA). NMR spectra were recorded on a Varian FT 400 NMR in-
strument. Mass spectrometry (MS) was performed at the Molecular
Mass Spectrometry Facility (MMSF) in the Department of Chemistry
À1
À1
0
.10% Brij-35, pH 7.5), and the esterase-treated MMPI (10 mL). After
a 30 min incubation at 378C, a reaction was initiated with the addi-
tion of 10 mL (40 mm) of the fluorescent substrate (Mca-Pro-Leu-
Gly-Leu-Dpa-Ala-Arg-NH ) where Mca=(7-methoxycoumarin-4-yl)-
2
&
Biochemistry at the University of California, San Diego.
acetyl and Dpa=N-3-(2,4-dinitrophenyl)-l-a-b-diaminopropiony-
l)),and the kinetic activity was monitored every 40 s for 30 min
with excitation and emission wavelengths at 335 nm and 405 nm,
respectively. Enzymatic activity and thus inhibition was calculated
with respect to the control experiment (no inhibitor present).
Measurements were performed in duplicate in two independent
experiments.
UV–vis spectroscopy: Absorption spectra of compounds 1–6, 11,
and 12 were collected on a PerkinElmer Lambda 25 UV–vis spec-
trophotometer. A 0.05 mm solution of test compound (1.0 mL) in
HEPES buffer (50 mm, pH 7.5) was treated with porcine liver ester-
ase (PLE) (3.57 U), and the absorption was monitored over time at
room temperature (Figure S1–S7 in the Supporting Information).
Calculation of kinetic rate constant: Pseudo-first-order rate con-
stants (kobs) were calculated by monitoring absorption over time in
the presence of PLE. A 50 mm solution of test compound (1.0 mL)
in HEPES buffer (50 mm, pH 7.5) was treated with PLE (0.178 U),
and the absorption was monitored over 10–20 min at room tem-
perature; at least 100 spectra were recorded for each sample. The
change in absorption was monitored at 274 nm for the maltol
Acknowledgements
The authors thank Dr. Yongxuan Su (Molecular Mass Spectrome-
try Facility (MMSF) at University of California, San Diego, USA)
for performing mass spectrometry experiments. The authors ac-
knowledge the California Alliance for Minority Participation (C.P.).
This work was funded by a grant from the US National Institutes
series (1–3) and at 338 nm for the PY-2 series (7–9), we term Amax
.
The rate constant (kobs) was determined by monitoring the appear-
ance of the absorption peak by plotting the linear slope of ln-
[
(AmaxÀA)/(Amax)].
ꢀ
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1662 – 1667 1666
Perez, Christian
