Activation of sulfonate ester based matrix metalloproteinase proinhibitors by hydrogen peroxide

Article abstract of DOI:10.1007/s00775-010-0727-x

This study details the development of matrix metalloproteinase inhibitor prodrugs (proMMPi) that are activated in the presence of reactive-oxygen species (ROS). Conventional matrix metalloproteinase inhibitors (MMPi) utilize a zinc-binding group (ZBG) that chelates to the catalytic zinc(II) ion of matrix metalloproteinases (MMPs) to inhibit their activity. To create ROS-sensitive prodrugs, sulfonate esters were used as a protecting group for the ZBG to block their metal binding ability. Surprisingly, these sulfonate esters were found to be cleaved by H₂O₂ only when the ZBG contained an N-oxide donor atom moiety. Sulfonate ester derivatives of full-length MMPi based on these ROS-triggerable systems were synthesized. It was found that proMMPi with sulfonate ester protecting groups showed relatively high rates of cleavage in the presence of H₂O₂ to release the active MMPi. In vitro MMP inhibition studies confirmed a significant increase in inhibitory activity of proMMPi upon addition of H₂O₂, demonstrating the use of sulfonate esters to act as cleavable triggers for ROS-activated prodrugs.

Full text of DOI:10.1007/s00775-010-0727-x

J Biol Inorg Chem (2011) 16:313–323
DOI 10.1007/s00775-010-0727-x
ORIGINAL PAPER
Activation of sulfonate ester based matrix metalloproteinase
proinhibitors by hydrogen peroxide
•
Kevin B. Daniel Jody L. Major Jourden
•
•
Kimberly E. Negoescu Seth M. Cohen
Received: 30 August 2010 / Accepted: 19 October 2010 / Published online: 4 November 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract This study details the development of matrix
metalloproteinase inhibitor prodrugs (proMMPi) that are
activated in the presence of reactive-oxygen species (ROS).
Conventional matrix metalloproteinase inhibitors (MMPi)
utilize a zinc-binding group (ZBG) that chelates to the cata-
lytic zinc(II) ion of matrix metalloproteinases (MMPs) to
inhibit their activity. To create ROS-sensitive prodrugs, sul-
fonate esters were used as a protecting group for the ZBG to
block their metal binding ability. Surprisingly, these sulfonate
esters were found to be cleaved by H2O2 only when the ZBG
contained an N-oxide donor atom moiety. Sulfonate ester
derivatives of full-length MMPi based on these ROS-trigg-
erable systems were synthesized. It was found that proMMPi
with sulfonate ester protecting groups showed relatively high
rates of cleavage in the presence of H2O2 to release the active
MMPi. In vitro MMP inhibition studies confirmed a signifi-
cant increase in inhibitory activity of proMMPi upon addition
of H2O2, demonstrating the use of sulfonate esters to act as
cleavable triggers for ROS-activated prodrugs.
extracellular proteins [1–3]. MMPs are secreted as zymogens
and become activated by a variety of pathways through
proteolytic cleavage of the propeptide domain by proteases,
other MMPs, and reactive-oxygen species (ROS) [1, 3].
Owing to the ability of MMPs to cleave proteins in the
extracellular matrix, overexpression and misregulation of
MMPs has been associated with a variety of pathologic
disorders, including arthritis, cancer, and cardiovascular
disease [2, 4–6]. Of particular note is MMP activation
associated with stroke, which is a leading cause of disable-
ment and death [7–10]. In stroke, the inflammatory response
induced by ischemia initiates the formation of ROS, which
activates MMPs, and can lead to the breakdown of the blood–
brain barrier, resulting in cell death and tissue damage
[7–11]. The association of ROS with MMP activation after
ischemia has generated interest in using MMP inhibitors
(MMPi) to treat reperfusion injury associated with stroke and
other cardiovascular ailments [12]. It has been shown that
MMP inhibition with broad-based inhibitors upon the onset
of stroke can reduce ischemic-related brain injury [9, 13, 14].
However, because MMPs have both beneficial and
pathogenic roles with respect to normal physiologic
function and disease progression, systemic inhibition of
MMPs can lead to side effects such as musculoskeletal
syndrome [1, 4, 5, 15–18]. Hence, there is an impetus to
control both the spatial and temporal activity of these
inhibitors [19].
Keywords Matrix metalloproteinases ꢀ Reactive-oxygen
species ꢀ Prodrugs
Introduction
Matrix metalloproteinases (MMPs) comprise a family of
Zn(II)-dependent endopeptidases involved in the cleavage of
To localize the activity of MMPi, a prodrug approach
can be employed to develop latent forms of the inhibitor
(i.e., proinhibitor) that are activated under specific, desired
conditions. Prochelators activated by biological or chemi-
cal stimuli have been explored as a means to modulate
metal ion chelation in various disease states [20–25]. By
blocking the zinc-binding group (ZBG) of the inhibitor
[which coordinates to the catalytic Zn(II) ion] with a
K. B. Daniel ꢀ J. L. Major Jourden ꢀ K. E. Negoescu ꢀ
S. M. Cohen (&)
Department of Chemistry and Biochemistry,
University of California, San Diego,
9500 Gilman Drive, La Jolla,
CA 92093-0358, USA
e-mail: scohen@ucsd.edu
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stimulus-responsive protecting group, one can greatly
attenuate or abolish the inhibitory activity of the MMPi.
Under the appropriate biological stimulus, the proinhibitor
can be activated, releasing the MMPi, thereby allowing for
localized delivery and inhibition. In general, metalloen-
zyme proinhibitors have not been widely investigated, with
only a few systems reported for MMPs [26–31]. To
establish the use of ZBG protecting groups for the devel-
opment of MMP proinhibitors, MMPi containing carbo-
hydrate protecting groups that are cleaved in the presence
of b-glucosidase were described [30]. Quotient IC50 values,
which represent the ratio of the IC50 values of the MMPi
prodrug (proMMPi) to IC50 values of the active inhibitor
[32], were excellent at greater than 1,000 with MMP-8
[30]. Similarly, proMMPi based on concepts from
prochelators and fluorescent probes sensitive to ROS [33,
34] were developed for potential use in ischemic injury
[31]. Boronic ester protecting groups appended via a self-
immolative linker to the ZBG of MMPi were reported by
our group and showed rapid activation in the presence of
H2O2, leading to the release of the free MMPi [31].
We sought to identify additional protecting groups to
develop proMMPi that could be triggered in an ischemic
setting by ROS. Fluorescent probes incorporating sulfo-
nate ester protecting groups have been shown to be
sensitive for detecting several ROS (hydrogen peroxide,
superoxide anion) [35–37]. These studies prompted the
use of sulfonate esters as selective protecting groups for
proMMPi that could be cleaved under conditions of
oxidative stress (e.g., ischemia). The work described
herein demonstrates that various ZBGs could be pro-
tected with sulfonate esters, creating molecules that can
be activated with H2O2. Four different ZBGs employing
oxygen donor atoms were selected for this study. The
ability of H2O2 to liberate the ZBGs was assessed, and
suitable candidates were developed into full-length
proMMPi that show increased potency against MMP-12
in the presence of H2O2.
of Chemistry and Biochemistry at the University of Cali-
fornia, San Diego. Elemental analyses was preformed by
NuMega Resonance Labs, San Diego.
General procedure for the synthesis of sulfonate ester
ZBGs
The ZBG compound was dissolved in pyridine on ice. To
this was added the desired sulfonyl chloride. The reaction
flask was removed from the ice bath and left stirring
overnight under nitrogen while warming to room temper-
ature. The pyridine was removed by rotary evaporation and
the resulting oil was redissolved in dichloromethane and
washed with 1 M HCl (approximately 30 mL), water, and
brine. The organic layer was dried over MgSO4, filtered,
and then concentrated via rotary evaporation. The product
was purified on a silica gel column and eluted with 1%
MeOH in dichloromethane unless otherwise noted.
2-Oxopyridin-1(2H)-yl benzenesulfonate
2-Hydroxypyridine-1-oxide (ZBG1; 1.0 g, 9.1 mmol) was
reacted with benzenesulfonyl chloride (1.27 mL,
10.0 mmol) in 75 mL of pyridine to afford 2-oxopyridin-
1(2H)-yl benzenesulfonate (PZBG-1a) in 77% yield
(1.75 g, 7.0 mmol). 1H NMR (500 MHz, CDCl3) d = 8.02
(d, J = 8.0 Hz, 2H), 7.75 (t, J = 8.0 Hz, 1H), 7.59 (m,
3H), 7.28 (dt, J1 = 6.9 Hz, J2 = 2.3 Hz, 1H), 6.52 (d,
J = 9.8 Hz, 1H), 6.15 (t, J = 7.5 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d = 157.0, 139.7, 137.1, 136.0, 133.8,
130.0, 129.5, 123.4, 105.4. Electrospray ionization mass
spectrometry (ESI-MS)(?): m/z 252.01 [M ? H]?, 273.95
[M ? Na]?. Anal. calcd for C11H9NO4S: C, 52.58; H,
3.61; N, 5.57. Found: C, 52.21; H, 3.99; N, 5.44.
2-Oxopyridin-1(2H)-yl 4-methylbenzenesulfonate
ZBG1 (0.5 g, 4.5 mmol) was reacted with p-toluenesul-
fonyl chloride (2.57 g, 13.5 mmol) in 40 mL of pyridine to
afford 2-oxopyridin-1(2H)-yl 4-methylbenzenesulfonate
(PZBG-1b) in 89% yield (1.06 g, 4.0 mmol). 1H NMR
[500 MHz, dimethyl sulfoxide (DMSO)-d6] d = 7.82 (d,
J = 8.6 Hz, 2H), 7.75 (dd, J1 = 7.5 Hz, J2 = 1.8 Hz, 1H),
7.49 (d, J = 8 Hz, 2H), 7.41 (dt, J1 = 7.5 Hz,
J2 = 1.7 Hz, 1H), 6.48 (dd, J1 = 9.2 Hz, J2 = 1.8 Hz,
1H), 6.21 (dt, J1 = 7.5 Hz, J2 = 1.8 Hz, 1H), 2.42 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3) d = 156.6, 148.9,
141.2, 138.1, 131.1, 130.5, 130.0, 122.9, 106.1, 22.0. ESI-
MS(?): m/z 266.10, [M ? H]?, 287.99 [M ? Na]?. Anal.
calcd for C12H11NO4S: C, 54.33; H, 4.18; N, 5.28. Found:
C, 54.24; H, 4.35; N, 5.24.
Materials and methods
General
Starting materials and solvents were purchased from
commercial suppliers (Sigma–Aldrich, Alfa Aesar, Fisher,
and others) and used as received. 1H/13C NMR spectra
were recorded at ambient temperature with a 400 or
500 MHz Varian Fourier transform NMR instrument or a
500 MHz JEOL instrument, located in the Department of
Chemistry and Biochemistry at the University of Califor-
nia, San Diego. Mass spectra were obtained at the
Molecular Mass Spectrometry Facility in the Department
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315
2-Oxopyridin-1(2H)-yl 4-nitrobenzenesulfonate
2-methyl-4-oxo-4H-pyran-3-yl benzenesulfonate (PZBG-
1
2a) in 71% yield (1.50 g, 5.6 mmol). H NMR (500 MHz,
ZBG1 (0.5 g, 4.5 mmol) was reacted with 4-nitro-
benzenesulfonyl chloride (3.0 g, 13.5 mmol) in 40 mL of
pyridine to afford 2-oxopyridin-1(2H)-yl 4-nitrobenzene-
CDCl3) d = 8.12 (d, J = 8.6 Hz, 2H), 7.69 (t, J = 7.5 Hz,
1H), 7.65 (d, J = 5.8 Hz, 1H), 7.58 (t, J = 8.0 Hz, 2H),
6.33 (d, J = 5.2 Hz, 1H), 2.46 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3) d = 172.1, 163.1, 154.3, 138.4, 136.6,
134.7, 129.2, 129.0, 117.7, 16.3. ESI-MS(?): m/z 267.06
[M ? H]?, 289.03 [M ? Na]?.
1
sulfonate (PZBG-1c) in 92% yield (1.23 g, 4.2 mmol). H
NMR (500 MHz, CDCl3) d = 8.42 (d, J = 8.6 Hz, 2H),
8.23 (d, J = 9.2 Hz, 2H), 7.65 (dd, J1 = 7.5 Hz,
J2 = 1.8 Hz, 1H), 7.34 (dt, J1 = 9.2 Hz, J2 = 1.7 Hz,
1H), 6.52 (d, J = 9.2 Hz, 1H), 6.22 (t, J = 7.7 Hz, 1H).
13C NMR (100 MHz, DMSO) d = 156.6, 152.3,
141.6, 139.4, 138.3, 131.8, 125.6, 122.8, 106.4. ESI-MS(?):
m/z 297.28 [M ? H]?, 319.02 [M ? Na]?.
2-Methyl-4-oxo-4H-pyran-3-yl
4-methylbenzenesulfonate
ZBG2 (0.5 g, 4.0 mmol) was reacted with p-toluenesul-
fonyl chloride (2.27 g, 11.9 mmol) in 40 mL of pyridine to
afford 2-methyl-4-oxo-4H-pyran-3-yl 4-methylbenzene-
sulfonate (PZBG-2b) in 64% yield (0.71 g, 2.5 mmol). 1H
NMR (500 MHz, CDCl3) d = 7.99 (d, J = 8 Hz, 2H),
7.64 (d, J = 5.8 Hz, 1H), 7.37 (d, J = 8 Hz, 2H), 6.34 (d,
2-Oxopyridin-1(2H)-yl 2,4-dinitrobenzenesulfonate
ZBG1 (0.5 g, 4.5 mmol) was reacted with 2,4-dini-
trobenzenesulfonyl chloride (1.32 g, 5.0 mmol) in 40 mL
of pyridine to afford 2-oxopyridin-1(2H)-yl 2,4-dini-
trobenzenesulfonate (PZBG-1d) in 31% yield (0.48 g,
1.4 mmol). 1H NMR (500 MHz, CDCl3) d = 8.96 (d,
J = 2.3 Hz, 1H), 8.42 (dd, J1 = 9.2 Hz, J2 = 2.3 Hz, 1H),
7.68 (dd, J1 = 6.9 Hz, J2 = 1.7 Hz, 1H), 7.50 (dt,
J1 = 7.5 Hz, J2 = 2.3 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H),
6.82 (dd, J1 = 9.2 Hz, J2 = 1.8 Hz, 1H), 6.35 (dt,
J1 = 6.9 Hz, J2 = 1.8 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d = 157.0, 155.6, 140.5, 135.1, 129.6, 124.0,
122.9, 116.0, 106.6.
J = 5.8 Hz, 1H), 2.46 (s, 3H, CH3), 2.45 (s, 3H, CH3). 13
C
NMR (100 MHz, CDCl3) d = 127.2, 163.2, 154.2, 145.8,
138.4, 133.6, 129.8, 129.0, 117.7, 22.0, 16.3. ESI-MS(?):
m/z 281.01 [M ? H]?, 303.03 [M ? Na]?.
2-Methyl-4-oxo-4H-pyran-3-yl
4-nitrobenzenesulfonate
ZBG2 (0.5 g, 4.0 mmol) was reacted with 4-nitro-
benzenesulfonyl chloride (0.88 g, 4.0 mmol) in 15 mL of
pyridine to afford 2-methyl-4-oxo-4H-pyran-3-yl 4-nitro-
benzenesulfonate (PZBG-2c) in 58% yield (0.71 g,
2.3 mmol). 1H NMR (500 MHz, CDCl3) d = 8.42 (d,
J = 9.2 Hz, 2H), 8.31 (d, J = 9.2 Hz, 2H), 7.69 (d,
J = 5.7 Hz, 1H), 6.34 (d, J = 5.8 Hz, 1H), 2.53 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3) d = 171.8, 163.5,
154.6, 151.2, 142.3, 138.6, 130.4, 124.3, 117.6, 16.3. ESI-
MS(?): m/z 312.06 [M ? H]?.
4-(((2-Oxopyridin-1(2H)-yl)oxy)sulfonyl) benzoic acid
ZBG1 (0.21 g, 1.9 mmol) was reacted with 4-(chlorosul-
fonyl) benzoic acid (0.62 g, 2.8 mmol) in 5 mL of pyri-
dine. The solvent was evaporated, leaving a yellow oil.
Addition of 5 mL of dichloromethane followed by the
addition of 5 mL of ethyl acetate allowed for precipitation
of 4-(((2-oxopyridin-1(2H)-yl)oxy)sulfonyl)benzoic acid
1
(PZBG-1e) in 30% yield (0.17 g, 0.57 mmol). H NMR
2-Methyl-4-oxo-4H-pyran-3-yl
2,4-dinitrobenzenesulfonate
(400 MHz, DMSO) d = 8.18 (d, J = 8.8 Hz, 2H), 8.08 (d,
J = 8.4 Hz), 7.88 (dd, J1 = 7.6 Hz, J2 = 2 Hz), 7.45 (td,
J1 = 8.2 Hz, J2 = 2 Hz, 1 H), 6.50 (dd, J1 = 9.2 Hz,
J2 = 1.6 Hz, 1 H), 6.26 (td, J1 = 7 Hz, J2 = 1.6 Hz, 1 H).
13C NMR (100 MHz, CDCl3) d = 166.5, 156.6, 141.5,
138.2, 137.9, 137.3, 131.2, 130.4, 122.8, 106.2. ESI-MS(-):
m/z 294.26 [M - H]-. Anal. calcd. for C12H9NO6S: C,
48.81; H, 3.07; N, 4.74. Found: C, 48.91; H, 3.37; N 4.84.
ZBG2 (0.5 g, 4.0 mmol) was reacted with 2,4-dini-
trobenzenesulfonyl chloride (1.58 g, 5.9 mmol) in 40 mL
of pyridine to afford 2-methyl-4-oxo-4H-pyran-3-yl 2,4-
dinitrobenzenesulfonate (PZBG-2d) in 28% yield (0.39 g,
1.1 mmol). 1H NMR (500 MHz, CDCl3) d = 8.72 (d,
J = 2.3 Hz, 1H), 8.56 (dd, J1 = 9.2 Hz, J2 = 2.3 Hz, 1H),
8.44 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 5.8 Hz, 1H), 6.31
(d, J = 5.8 Hz, 1H), 2.53 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3) d = 171.6, 163.3, 154.9, 139.3, 136.4,
133.8, 126.8, 120.5, 117.5, 16.1. ESI-MS(?): m/z 357.03
[M ? H]?, 378.99 [M ? Na]?.
2-Methyl-4-oxo-4H-pyran-3-yl benzenesulfonate
3-Hydroxy-2-methyl-4H-pyran-4-one
(ZBG2;
1.0 g,
7.9 mmol) was reacted with benzenesulfonyl chloride
(3.0 mL, 23.7 mmol) in 75 mL of pyridine to afford
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1,2-Dimethyl-4-oxo-1,4-dihydropyridin-3-yl
benzenesulfonate
H), 7.82 (d, J = 7.6 Hz, 1 H), 7.03 (d, J = 9.2 Hz, 1 H),
6.25 (d, J = 7.6 Hz, 1 H), 3.68 (s, 3H), 2.31 (s, 3H). 13C
NMR (100 MHz, CDCl3) d = 169.35, 154.98, 144.00,
142.61, 141.42, 140.76, 138.54, 129.69, 122.24, 118.15,
116.78, 59.63, 13.29.
3-Hydroxy-1,2-dimethylpyridin-4(1H)-one (ZBG-3; 0.5 g,
3.6 mmol) was reacted with benzenesulfonyl chloride
(0.51 mL, 4.0 mmol) in 40 mL of pyridine to afford 1,2-
himethyl-4-oxo-1,4-dihydropyridin-3-yl benzenesulfonate
7-Oxocyclohepta-1,3,5-trien-1-yl benzenesulfonate
1
(PZBG-3a) in 43% yield (0.43 g, 1.5 mmol). H NMR
(500 MHz, CDCl3) d = 8.19 (d, J = 6.9 Hz, 2H), 7.66 (t,
J = 7.5 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.23 (d,
J = 8 Hz, 1H), 6.34 (d, J = 8.1 Hz, 1H), 3.63 (s, 3H,
NCH3), 2.49 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3)
d = 171.3, 144.8, 140.7, 139.9, 137.3, 134.3, 129.0, 128.9,
118.3, 41.8, 14.7. ESI-MS(?): m/z 280.09 [M ? H]?.
2-Hydroxycyclohepta-2,4,6-trienone
(ZBG4;
0.2 g,
1.7 mmol) was reacted with benzenesulfonyl chloride
(0.63 mL, 4.9 mmol) in 5 mL of pyridine to afford 7-
oxocyclohepta-1,3,5-trien-1-yl benzenesulfonate (PZBG-
1
4a) in 76% yield (0.33 g, 1.3 mmol). H NMR (400 MHz,
DMSO) d = 7.95 (d, J = 7.6 Hz, 2H), 7.80 (t, J = 7.6 Hz,
1H), 7.67 (t, J = 8 Hz, 2H), 7.43–7.37 (m, 2H), 7.25 (t,
J = 8.4 Hz, 1H), 7.14–7.09 (m, 2H). 13C NMR (100 MHz,
CDCl3) d = 179.24, 154.79, 141.19, 138.21, 136.37,
136.33, 135.57, 131.96, 130.92, 130.27, 128.76. ESI-
MS(?): m/z 262.97 [M ? H]?, 279.72 [M ? NH4]?,
284.99 [M ? Na]?.
1,2-Dimethyl-4-oxo-1,4-dihydropyridin-3-yl
4-methylbenzenesulfonate
ZBG-3 (0.2 g, 1.4 mmol) was reacted with p-toluenesul-
fonyl chloride (0.82 g, 4.3 mmol) in 10 mL of pyridine
to afford 1,2-dimethyl-4-oxo-1,4-dihydropyridin-3-yl 4-
methylbenzenesulfonate (PZBG-3b) in 86% yield (0.35 g,
1.2 mmol). 1H NMR (500 MHz, CDCl3) d = 7.99 (d,
J = 8.4 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.33 (d,
J = 8 Hz, 2H), 6.40 (d, J = 7.2 Hz, 1H), 3.64 (s, 3H), 2.42
(s, 3H), 2.41 (s, 3H). 13C NMR (100 MHz, CDCl3)
d = 170.71, 145.67, 145.54, 141.48, 134.07, 129.72,
128.96, 126.07, 117.53, 42.25, 22.00, 14.7. ESI-MS(?):
m/z 294.05 [M ? H]?, 315.97 [M ? Na]?.
7-Oxocyclohepta-1,3,5-trien-1-yl
4-methylbenzenesulfonate
ZBG4 (0.2 g, 1.7 mmol) was reacted with p-toluenesul-
fonyl chloride (0.41 g, 2.0 mmol) in 10 mL of pyridine to
afford 7-oxocyclohepta-1,3,5-trien-1-yl 4-methylbenzene-
sulfonate (PZBG-4b) in 63% yield (0.04 g, 0.1 mmol). 1H
NMR (400 MHz, CDCl3) d = 7.92 (d, J = 8.4 Hz, 2H),
7.46 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H),
7.26–7.16 (m, 2H), 7.13–7.06 (m, 1H), 6.98 (t, J = 10 Hz,
1H), 2.45 (s, 3H). 13C (125 MHz, CDCl3) d = 179.41,
155.15, 145.50, 141.23, 136.32, 134.61, 133.41, 130.81,
130.00, 129.60, 128.59, 21.78. ESI-MS(?): m/z 277.21
[M ? H]?, 293.99 [M ? NH4]?.
1,2-Dimethyl-4-oxo-1,4-dihydropyridin-3-yl
4-nitrobenzenesulfonate
ZBG-3 (0.2 g, 1.5 mmol) was reacted with 4-nitro-
benzenesulfonyl chloride (0.488 g, 2.2 mmol) in 10 mL of
pyridine to afford 1,2-dimethyl-4-oxo-1,4-dihydropyridin-
3-yl 4-nitrobenzenesulfonate (PZBG-3c) in 50% yield
(0.23 g, 0.7 mmol). 1H NMR (400 MHz, CDCl3)
d = 8.41–8.34 (m, 4H), 7.26 (d, J = 7.6 Hz, 1H), 6.33 (d,
J = 7.6 Hz, 1H), 3.67 (s, 3H), 2.53 (s, 3H). 13C NMR
(100 MHz, CDCl3) d = 170.9, 150.9, 144.9, 143.2,
140.9, 140.1, 130.4, 124.0, 118.4, 41.9, 14.6. ESI-MS(?):
m/z 325.11 [M ? H]?, 346.96 [M ? Na]?.
7-Oxocyclohepta-1,3,5-trien-1-yl
4-nitrobenzenesulfonate
ZBG4 (0.2 g, 1.7 mmol) was reacted with 4-nitro-
benzenesulfonyl chloride (1.1 g, 4.9 mmol) in 5 mL of
pyridine. Addition of 10 mL of water allowed for precip-
itation of 7-oxocyclohepta-1,3,5-trien-1-yl 4-nitroben-
zenesulfonate (PZBG-4c) in 71% yield (0.38 g, 1.2 mmol)
without the need for further purification. 1H NMR
(400 MHz, DMSO) d = 8.45 (d, J = 8.8 Hz, 2H), 8.22 (d,
J = 8.8 Hz, 2H), 7.52 (d, J = 9.2 Hz, 1H), 7,46 (dd,
J1 = 10.2 Hz, J2 = 3.6 Hz, 1 H), 7.31 (dd, J1 = 9.8 Hz,
J2 = 2 Hz, 1 H), 7.15 (dd, J1 = 10.6 Hz, J2 = 3.6 Hz, 1
H). 13C (100 MHz, CDCl3) d = 179.09, 154.86, 151.50,
141.95, 141.27, 138.59, 136.87, 132.06, 131.48, 130.42,
125.42. ESI-MS(?): m/z 308.01 [M ? H]?, 324.73
[M ? NH4]?.
1,2-Dimethyl-4-oxo-1,4-dihydropyridin-3-yl
2,4-dinitrobenzenesulfonate
ZBG-3 (0.10 g, 0.73 mmol) was reacted with 2,4-dini-
trobenzenesulfonyl chloride (0.3 g, 1.1 mmol) in 10 mL of
pyridine to afford 1,2-dimethyl-4-oxo-1,4-dihydropyridin-
3-yl 2,4-dinitrobenzenesulfonate (PZBG-3d) in 23% yield
(0.07 g, 0.2 mmol). 1H NMR (400 MHz, CDCl3) d = 8.83
(d, J = 2.8 Hz 1H), 8.37 (dd, J1 = 9.6 Hz, J2 = 2.8 Hz, 1
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7-Oxocyclohepta-1,3,5-trien-1-yl
2,4-dinitrobenzenesulfonate
(2H)-yl benzenesulfonate (1a) in 67% yield (0.05 g,
0.1 mmol). 1H NMR (400 MHz, CDCl3) d = 8.01 (d,
J = 7.6 Hz, 2H), 7.76 (t, J = 7.6 Hz, 1 H), 7.61 (m, 6H), 7.47
(m, 4H), 7.38–7.31 (m, 2H), 6.62 (d, J = 8.0 Hz, 1H), 6.57 (d,
J = 6.4 Hz, 1H), 4.63 (d, J = 5.2 Hz, 2H). 13C (100 MHz,
CDCl3) d = 159.2, 157.0, 142.7, 141.2, 140.8, 138.9, 136.0,
134.5, 129.9, 129.5, 129.1, 128.9, 127.8, 127.7, 127.3, 125.2,
107.6, 44.5. ESI-MS(?): m/z 461.13 [M ? H]?, 483.13
[M ? Na]?. Anal. calcd for C25H20N2O5Sꢀ0.5 H2O: C, 63.95;
H, 4.51; N, 5.99. Found: C, 63.68; H, 5.14; N, 5.99.
ZBG4 (0.2 g, 1.7 mmol) was reacted with 2,4-dini-
trobenzenesulfonyl chloride (0.54 g, 2.0 mmol) in 10 mL
of pyridine to afford 7-oxocyclohepta-1,3,5-trien-1-yl 2,4-
dinitrobenzenesulfonate (PZBG-4d) in 6% yield (0.04 g,
0.1 mmol). 1H NMR (400 MHz, DMSO) d = 9.00 (d,
J = 2.4 Hz 1H), 8.66 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1 H),
8.38 (d, J = 8.8 Hz 1H), 7.66 (d, J = 9.6 Hz 1H), 7.50 (td,
J1 = 8.4 Hz, J2 = 3.6 Hz, J3 = 1.2 Hz, 1 H), 7.35 (td,
J1 = 8.4 Hz, J2 = 2.4 Hz, 1 H), 7.20–7.15 (m, 2H). 13C
(125 MHz, DMSO) d = 178.71, 155.30, 151.20, 147.90,
140.96, 138.83, 137.10, 134.58, 133.22, 131.93, 131.55,
127.92, 121.11. ESI-MS(?): m/z 353.15 [M ? H]?, 375.11
[M ? Na]?.
4-(((6-(([1,10-Biphenyl]-4-ylmethyl)carbamoyl)-2-
oxopyridin-1(2H)-yl)oxy)sulfonyl) benzoic acid
1,2-HOPO-2 (0.20 g, 0.6 mmol) was dissolved in 5 mL of
pyridine. To this was added 4-(chlorosulfonyl) benzoic
acid (0.21 g, 1.0 mmol). The reaction was allowed to
proceed overnight at room temperature. The solvent was
evaporated and to the remaining oil was added 5 mL of
dichloromethane followed by 5 mL of ethyl acetate,
allowing for precipitation. The solid white product was
filtered off and collected to afford 4-(((6-(([1,10-biphenyl]-
4-ylmethyl)carbamoyl)-2-oxopyridin-1(2H)-yl)oxy)sulfo-
nyl) benzoic acid (1b) in 14% yield (0.04 g, 0.08 mmol).
1H NMR (400 MHz, DMSO) d = 9.36 (t, J = 5.6 Hz, 1H,
NH), 8.18 (d, J = 8.8 Hz, 2H), 8.06 (d, J = 8.8 Hz, 2H),
7.66–7.61 (m, 4H), 7.53 (dd, J1 = 7.8 Hz, J2 = 2.8 Hz,
1H), 7.45 (t, J = 7.6 Hz, 2H), 7.38–7.33 (m, 3H), 6.67 (dd,
J1 = 9.2 Hz, J2 = 1.6 Hz, 1H), 6.47 (dd, J1 = 6.4 Hz,
J2 = 1.2 Hz, 1H), 4.27 (d, J = 5.6 Hz, 2H). 13C NMR
(100 MHz, DMSO) d = 166.55, 159.45, 156.86, 143.21,
140.61, 140.54, 139.68, 138.26, 138.02, 137.61, 130.97,
6-(([1,10-Biphenyl]-4-ylmethyl)carbamoyl)-2-
oxopyridin-1(2H)-yl benzenesulfonate
1,2-HOPO-2 (Scheme 1) was prepared as previously
reported [38]. In a 10-mL round-bottom flask was dissolved
0.05 g (0.16 mmol) of 1,2-HOPO-2 in 3 mL of pyridine. To
this was added 60 lL (0.5 mmol) of benzenesulfonyl chlo-
ride. The reaction mixture was left stirring under nitrogen at
room temperature overnight. After 16 h, the solvent was
evaporated to leave an orange oil which was dissolved in
dichloromethane and washed once with 1 M HCl then brine.
The organic layer was dried over MgSO4, filtered, and
concentrated. The product was purified ona silica gel column
and eluted with 1% MeOH in dichloromethane to afford
6-(([1,10-biphenyl]-4-ylmethyl)carbamoyl)-2-oxopyridin-1
Scheme 1 Activation of
proinhibitor 1a or 1b with H2O2
results in generation of the
inhibitor 1,2-HOPO-2. In
contrast, treatment of
proinhibitor 2a with H2O2 did
not result in production of the
inhibitor PY-2
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J Biol Inorg Chem (2011) 16:313–323
130.09, 129.62, 128.73, 128.08, 127.33, 127.28, 124.19,
107.11, 43.05. ESI-MS(-): m/z 502.89 [M - H]-. Anal.
calcd for C26H20N2O7Sꢀ0.25 HCl: C, 60.80; H, 3.97; N,
5.45. Found: C, 60.92; H, 4.30; N, 5.73.
(kobs) was found from the linear slope of ln[(A - AZBG)/
(Ao - AZBG)] versus time, where AZBG is the absorbance of a
50 lM sample of the ZBG or full-length inhibitor and Ao is
the initial absorbance of PZBG-1a, PZBG-1b, PZBG-1e,
1a, and 1b. The rate of conversion was determined from the
slope of the line of kobs versus H2O2 concentration.
2-(([1,10-Biphenyl]-4-ylmethyl)carbamoyl)-4-oxo-4H-
pyran-3-yl benzenesulfonate
High-performance liquid chromatography
PY-2 (Scheme 1) was prepared as previously reported
[38]. In a 50-mL round-bottom flask was dissolved 0.20 g
(0.6 mmol) of PY-2 in 15 mL of pyridine. To this was
added 240 lL (1.8 mmol) of benzenesulfonyl chloride.
The reaction mixture was left stirring under nitrogen at
room temperature overnight. After 16 h, the solvent was
evaporated to leave a red oil which was dissolved in
dichloromethane and washed once with 1 M HCl then
brine. The organic layer was dried over MgSO4, filtered,
and concentrated. The product was precipitated from
MeOH to afford 2-(([1,10-biphenyl]-4-ylmethyl)carbam-
oyl)-4-oxo-4H-pyran-3-yl benzenesulfonate (2a) in 12%
Analytical High-performance liquid chromatography
(HPLC) was performed with a HP series 1050 system
equipped with a VydacÒ C18 reverse-phase column
(218TP, 250 mm 9 4.6 mm, 5 lm). Separation was
achieved with a flow rate of 1 mL min-1 and the following
solvents: solvent A was 5% MeOH and 0.1% formic acid in
H2O and solvent B was 0.1% formic acid in MeOH.
Starting with 95% solvent A and 5% solvent B, we ran an
isocratic gradient for 15 min to a final solvent mixture of
5% solvent A and 95% solvent B, which was held for
5 min before ramping back down to 95% solvent A and 5%
solvent B in 2 min and holding for an additional 4 min.
ZBG-1a and PZBG-1a were prepared in HEPES buffer
(50 mM, pH 7.5) at a concentration of 1 mM and retention
times were determined. To evaluate cleavage by H2O2, a
1 mM solution of PZBG-1a in HEPES buffer was reacted
with a 20-fold excess of H2O2 before analysis under
identical HPLC conditions as before.
1
yield (0.03 g, 0.07 mmol). H NMR (400 MHz, CDCl3)
d = 8.10 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 5.6 Hz, 1 H),
7.71 (t, J = 7.6 Hz, 1H), 7.61 (m, 6H), 7.47–7.36 (m, 6H),
6.47 (d, J = 6.0 Hz, 1H), 4.67 (d, J = 6.0 Hz, 2H). 13C
NMR (100 MHz, CDCl3) d = 172.7, 157.3, 154.8, 151.3,
141.2, 140.8, 139.2, 136.0, 135.8, 135.2, 129.3, 129.2,
129.1, 128.9, 127.8, 127.7, 127.3, 118.4, 44.3. ESI-MS(?):
m/z 461.98 [M ? H]?, 484.02 [M ? Na]?.
Inhibition assays
UV–vis spectroscopy
MMP-12 (catalytic domain, human recombinant) was
purchased from Enzo Life Sciences. The assays were car-
ried out in a 96-well plate using a Bio-Tex Flx 800 plate
reader. The activity of MMP-12 was evaluated after a 30-
min incubation in the presence of H2O2 and proMMPi. The
concentration of proMMPi used was selected to be close
to the IC50 value of the parent full-length inhibitors,
1,2-HOPO-2 and PY-2 [38]. In each well, 1 lL of proin-
hibitors 1a, 1b, and 2a and the inhibitors 1,2-HOPO-2 and
PY-2 in DMSO (5 lM) were incubated for 30 min at
37 °C with 20 lL of MMP-12 (0.35 U mL-1), 10 lL
H2O2 (1 mM in HEPES buffer, pH 7.5), and MMP assay
buffer (50 mM HEPES, 10 mM CaCl2, 0.10% Brij-35, pH
7.5) for a total volume of 99 lL. A control sample con-
taining 10 lL H2O2 (1 mM in HEPES buffer, pH 7.5) in
MMP assay buffer was also prepared to confirm that H2O2
did not inhibit MMP-12. The reaction was initiated by the
addition of 1 lL (400 lM) of the fluorescent substrate
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 [where Mca is
(7-methoxycoumarin-4-yl)acetyl and Dpa is N-3-(2,4-
dinitrophenyl)-L-a,b-diaminopropionyl)] after the 30-min
incubation period and kinetic activity was measured every
minute for 20 min with excitation and emission
Absorption spectra of compounds were taken with a
PerkinElmer Lambda 25 UV–vis spectrophotometer. To a
1.0 mL solution at 0.05 mM concentration of each com-
pound in N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic
acid (HEPES) buffer (50 mM, pH 7.5) was added H2O2
(10 lL, 0.09 M in HEPES) and absorption spectra were
monitored over time at room temperature. Hydrolytic sta-
bility was measured by monitoring each sample in HEPES
buffer over a 24-h period.
Calculation of rate constant
The pseudo-first-order rate constant was calculated follow-
ing a literature procedure [20]. To a 1.0 mL solution of
PZBG-1a, PZBG-1b, PZBG-1e, 1a, and 1b in HEPES
buffer at 50 lM was added H2O2 to final concentrations of
150, 250, 500, 750, and 900 lM. Spectra were monitored
over 15–30 min at room temperature, with at least 50 spectra
recorded at every concentration. The change in absorbance at
298 nm was monitored for PZBG-1a and PZBG-1b,
whereas the change in absorbance at 288 nm was recorded
for PZBG-1e, and at 310 nm for 1a and 1b. The rate constant
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J Biol Inorg Chem (2011) 16:313–323
319
wavelengths at 335 and 405 nm, respectively. Enzyme
activity with the inhibitor was calculated with respect to
the control experiment––no inhibitor present. Measure-
ments were performed in duplicate in two independent
experiments.
absorbance (corresponding to the formation of the free
ZBG) upon exposure to H2O2. The fact that only the five
PZBG-1 derivatives (out of 17 total combinations) were
cleaved in the presence of H2O2 strongly suggests that
the N–O group is essential for the observed reactivity.
Figure 2 shows representative absorption spectra of
PZBG-1a in the presence of H2O2. A decrease in
absorbance at 298 nm over time is noted, representing
the disappearance of PZBG-1a and a gradual increase in
absorbance at 312 nm is observed, indicating the emer-
gence of ZBG-1. In addition, analytical HPLC was used
to confirm that ZBG-1 was the product after reaction
with H2O2 (Fig. 2). Upon the addition of H2O2 to
PZBG-1a for 60 min, a peak with a retention time of
5.0 min was observed, which is identical to an authentic
sample of ZBG-1. Similarly, treatment of PZBG-1b with
H2O2 resulted in nearly identical spectra as found with
PZBG-1a, whereas PZBG-1c showed rapid hydrolysis
upon the addition of H2O2 (data not shown). It should be
noted that the absorption spectra of PZBG-1d were not
readily interpreted, owing to the overlapping of absorp-
tion profiles of the protecting group and the free ZBG;
however, thin-layer chromatography showed the emer-
gence of the free ZBG, demonstrating rapid hydrolytic
cleavage of the protecting group (even in the absence of
H2O2). These findings taken together prompted the syn-
thesis of PZBG-1e, a more water-soluble alternative to
PZBG-1c, with a carboxylic acid attached to the para
position of the sulfonate ester. A substantial increase in
solubility in buffered solution was noted, and the
cleavage behavior was similar to that of the other
compounds reported (see below), making PZBG-1e an
attractive candidate for development into a full-length
proMMPi. As mentioned above, sulfonate ester deriva-
tives of ZBG-2a–ZBG-2d, ZBG-3a–ZBG-3d, and ZBG-
4a–ZBG-4d did not show any change in absorbance over
a period of 1 h with an 18 M excess of H2O2. These
findings suggest that the N–O bond in ZBG-1 is required
for facile cleavage of the sulfonate ester group in this
series of ligands, although this will require verification
by additional studies. It is interesting to note that the
compounds tested that did not contain the N–O moiety
(those based on PZBG-2, PZBG-3, and PZBG-4)
appeared to be stable in aqueous buffer (over at least a
1-h period).
Results and discussion
Assessment of sulfonate esters as suitable protecting
groups for ZBGs
Previous studies utilizing fluorescent probes have shown
sulfonate esters to be suitable protecting groups of hydro-
xyl groups that show a turn-on response upon exposure to
ROS, including H2O2 and superoxide anion [35–37]. To
investigate the use of sulfonate ester protecting groups for
the development of ROS-activated proMMPi, a small
library of compounds was synthesized. As shown in Fig. 1,
sulfonate esters with different substituents were appended
to ZBG1-4 to evaluate which protected ZBGs (PZBGs)
provided efficient activation in the presence of H2O2.
These PZBGs were readily prepared by combining a ZBG
with the appropriate sulfonyl chloride in pyridine. In total,
17 PZBGs (PZBG-1a–PZBG-1e, PZBG-2a–PZBG-2d,
PZBG-3a–PZBG-3d, and PZBG-4a–PZBG-4d) were
prepared and tested for cleavage in the presence of H2O2.
To evaluate cleavage of the compounds by ROS, a
sample of each PZBG in HEPES buffer (50 mM, pH
7.5) was activated with excess H2O2 (0.9 mM, 18 equiv)
and the change in absorbance was monitored over time
via electronic spectroscopy. Surprisingly, only com-
pounds derived from ZBG-1 showed a change in
One key factor for any prodrug approach is the stability
of the protecting group in the absence of the triggering
stimuli. To test the stability of the sulfonate esters in buffer,
absorption spectra for PZBG-1a, PZBG-1b, and PZBG-1e
were collected over 24 h. These stability studies showed
approximately 50% cleavage of PZBG-1a and PZBG-1e
in 6 h, whereas PZBG-1b was approximately 30% cleaved
in 24 h. The rates of conversion of PZBG-1a, PZBG-1b,
Fig. 1 Zinc-binding groups (ZBGs) and their sulfonate ester deriv-
atives (protected ZBGs; PZBGs) examined in this study
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J Biol Inorg Chem (2011) 16:313–323
Fig. 2 Analysis of PZBGs in the presence of H2O2. a Absorption
spectra of PZBG-1a [0.05 mM, 50 mM N-(2-hydroxyethyl)pipera-
zine-N0-ethanesulfonic acid (HEPES) buffer, pH 7.5] in the presence
of H2O2 (0.9 mM, 18 equiv) monitored every 5 min for 60 min. The
dashed line represents the initial spectrum, and an authentic sample of
ZBG-1 is shown in red. The arrows indicate changes in spectra over
time. b High performance liquid chromatography chromatograms of
PZBG-1a, PZBG-1a ? H2O2, and ZBG-1. The retention times are
11.5 min for PZBG-1a and 5.0 min for ZBG-1. c–e Absorption
spectra of PZBG-2a, PZBG-3a, and PZBG-4a, respectively
(0.05 mM, 50 mM HEPES buffer, pH 7.5), in the presence of H2O2
(0.9 mM, 18 equiv) monitored every 5 min for 60 min. The
overlapping spectra indicate that no cleavage of the protecting group
is occurring in the presence of H2O2
and PZBG-1e were determined by monitoring the change
in absorbance using pseudo-first-order reaction conditions
with an excess of H2O2 as previously reported [20]. The
calculated rate constants indicate that PZBG-1e had the
fastest rate constant at 1.3 M-1 s-1, whereas rate constants
of 0.7 and 0.3 M-1 s-1 were determined for PZBG-1a and
PZBG-1b, respectively. It should be noted that the rate
constants determined do take into account background
hydrolysis and reactivity with H2O2; however, all kinetic
measurements were taken over a 15–30-min period, which
is before a measurable amount of hydrolysis was observed.
Experiments with PZBG-1c and PZBG-1d showed the
fastest cleavage kinetics upon exposure to H2O2, with
complete dissociation achieved in less than 3 min (no rate
constants determined). The rates of conversion for the
PZBG-1 compounds are consistent with the nature of the
respective substituents on the leaving groups. When sub-
stituents are varied on an aromatic ring, the change in free
energy of activation for a given reaction is proportional to
the change in Gibbs free energy, as summarized by the
Hammett equation [39]. Of the molecules for which rate
constants were obtained, PZBG-1e, which contains an
electron-withdrawing group (–CO2H) in the para position,
dissociates the fastest. PZBG-1b, on the other hand, with
an electron-donating group (–CH3) in the para position,
had the slowest rate of the compounds tested. PZBG-1a,
with no substituents, had a cleavage rate falling between
the others, consistent with the Hammett relationship. The
results with PZBG-1c and PZBG-1d are also consistent
with this relationship, with the strongly electron with-
drawing nitro groups producing the fastest rates, resulting
in the inability to acquire precise values.
Development of full-length proMMPi
Having demonstrated the ability of sulfonate esters to act as
cleavable protecting groups for ZBG-1, we incorporated this
chelator into a full-length proMMPi. The corresponding full-
length inhibitor of ZBG-1 with a hydrophobic biphenyl
backbone, 1,2-HOPO-2, has been previously prepared and
studied [38]. 1,2-HOPO-2 is an effective inhibitor of MMP-
3, MMP-8, and MMP-12, with IC50 values under 100 nM
[38]. Two full-length MMPi (1,2-HOPO-2 and PY-2) were
prepared by previously reported procedures and then
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321
MMP inhibition studies
protected in pyridine with an excess of the appropriate sul-
fonyl chloride to generate proMMPi 1a, 1b, and 2a
(Scheme 1). The proMMPi 1a and 1b were evaluated for
activation by H2O2 via electronic spectroscopy in the same
manner as with the PZBG compounds. As shown in
Scheme 1, the activation of 1a and 1b to the known MMPi
1,2-HOPO-2 is achieved upon the addition of H2O2. Fig-
ure 3 shows the absorption spectra of 1b, for which a
decrease at 310 nm over time is observed, indicating the
disappearance of the protected MMPi, and a gradual increase
in absorbance at 350 nm is observed, demonstrating the
emergence of 1,2-HOPO-2. Pseudo-first-order rate con-
stants were determined with an excess of H2O2 as described
earlier. Rate constants of 0.3 and 1.1 M-1 s-1 were obtained
for 1a and 1b, respectively, which are in good agreement
with the rate constants determined for the protected chelators
PZBG-1a and PZBG-1e. To evaluate the stability of the
sulfonate esters, absorption spectra of 1a and 1b were col-
lected in buffer alone. Compounds 1a and 1b showed
approximately 50% hydrolysis after 9 and 3 h, respectively.
To further confirm that the behavior of the protected
chelators (PZBGs) was readily translated to a complete
proMMPi, a proinhibitor based on PZBG-2a was synthe-
sized. Compound 2a, which contains a biphenyl backbone
like 1a, was prepared. Upon cleavage of the protecting
group, 2a should produce PY-2, a known inhibitor of
several MMPs [38]. Treatment of 2a with excess H2O2
over the course of 60 min did not result in cleavage of the
sulfonate ester (Scheme 1), as evidenced by absorption
spectroscopy (data not shown). This negative result is
consistent with all of the findings described above, showing
that the cleavage behavior of the PZBG is retained in its
full-length proMMPi.
To monitor the ability of the protected compounds to
inhibit MMP-12 in the presence of H2O2, a fluorescence-
based assay was used [40]. Compounds 1a, 1b, and 2a
were tested at concentrations close to the IC50 values of
their active parent molecules, 1,2-HOPO-2 and PY-2,
against MMP-12. The percent inhibition of proinhibitors
1a, 1b, and 2a at 50 nM was evaluated after 30 min of
incubation with and without H2O2 (Fig. 4). Before treat-
ment with H2O2, 1a and 1b were shown to exhibit
approximately 20% and 30% inhibition of MMP-12,
respectively. This significant level of inhibition is likely
due to the hydrolysis of these compounds to the active
MMPi during the incubation period, as described earlier for
the stability studies. In addition, the inhibition assays were
performed at a higher temperature than our kinetic exper-
iments, which may result in an increase in the rate of
hydrolysis and therefore lead to higher inhibition than
might otherwise be expected. After treatment with 100 lM
H2O2, the percent inhibition by proinhibitors 1a and 1b
increased to approximately 30% and 50%, respectively,
indicative of activation to 1,2-HOPO-2. Proinhibitor 2a
was used as a negative control, and as expected, displayed
no significant change in inhibitory activity upon exposure
to H2O2.
Conclusions
In summary, we have demonstrated an alternative method
to prepare proMMPi that are activated in the presence of
ROS. The addition of sulfonate ester protecting groups to
Fig. 3 Absorption spectra of 1b
(0.05 mM, 50 mM HEPES
buffer, pH 7.5) in the presence
of H2O2 (0.9 mM, 18 equiv
excess) monitored every 5 min
for 60 min. The dashed line
represents the initial spectrum,
and an authentic sample of 1,2-
HOPO-2 is shown in red. The
arrows indicate changes in
spectra over time
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J Biol Inorg Chem (2011) 16:313–323
Fig. 4 Percent inhibition of
MMP-12 with proinhibitors 1a,
1b, and 2a tested at 50 nM in
the absence (gray bars) and
presence (black bars) of H2O2
after 30 min of activation
the ZBG blocks the chelating ability of the inhibitors,
thereby attenuating their activity. The sulfonate ester
proMMPi are easy to synthesize, have tunable rates of
cleavage (modulated by appropriate substituents on the
leaving group), and can be made reasonably water soluble
by addition of substituents, such as carboxylic acids, on the
aromatic ring of the protecting group. Unfortunately, the
limited hydrolytic stability of the sulfonate ester protected
ZBGs is a significant drawback. Nearly complete hydro-
lysis of the sulfonate ester proMMPi was observed after
24 h. In contrast, self-immolative boronic ester derivatized
proMMPi were entirely stable under identical conditions
[31]. In addition, the sulfonate esters were only cleaved
from one (ZBG-1) of four different chelators tested, sug-
gesting that the present strategy may not be readily applied
to inhibitors with other ZBGs. Nonetheless, complete
proMMPi based on these sulfonate ester protected chelators
were activated after treatment with H2O2 at relatively fast
rates, resulting in an increase in inhibition of MMP-12. It is
expected that combining sulfonate esters with a self-imm-
olative strategy may produce more stable, and more rapidly
cleavable proinhibitors [31, 41, 42]. These and related
experiments are under way.
References
1. Whittaker M, Flyod CD, Brown P, Gearing AJH (1999) Chem
Rev 99:2735–2776
2. Coussens L, Fingelton B, Matrisian LM (2002) Science
295:2387–2392
3. Jacobsen JA, Major Jourden JL, Miller MT, Cohen SM (2010)
Biochim Biophys Acta 1803:72–94
4. Fingleton B (2008) Semin Cell Dev Biol 19:61–68
5. Renkiewicz R, Qiu L, Lesch C, Sun X, Devalaraja R, Cody T,
Kaldjian E, Welgus H, Baragi V (2003) Arthritis Rheum
48:1742–1749
6. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM,
Podhajcer OL, Chenard MP, Rio MC, Chambon P (1990) Nature
348:699–704
7. Wang J, Tsirka SE (2005) Brain 128:1622–1633
8. Wang Q, Tang XN, Yenari MA (2007) J Neuroimmunol
184:53–68
9. Rosenberg GA, Cunningham LA, Wallace J, Alexander S,
Estrada EY, Grossetete M, Razhagi A, Miller K, Gearing A
(2001) Brain Res 893:104–112
10. Mun-Bryce S, Rosenberg GA (1998) J Cereb Blood Flow Metab
18:1163–1172
11. Fishman R (1975) N Engl J Med 293:706–711
12. Haorah J, Ramirez SH, Schall K, Smith D, Pandya R, Persidsky Y
(2007) J Neurochem 101:566–576
13. Copin J-C, Merlani P, Sugawara T, Chan PH, Gasche Y (2008)
Exp Neurol 213:196–201
14. Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA
(2007) J Cereb Blood Flow Metab 27:697–709
15. Overall CM, Kleifeld O (2006) Br J Cancer 94:941–946
16. Nagase H, Woessner JF Jr (1999) J Biol Chem 274:21491–21494
17. Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM (2000) J
Clin Oncol 18:1135–1149
18. Peterson JT (2006) Cardiovasc Res 69:677–687
19. Jin R, Yang G, Li G (2010) Neurobiol Dis 38:376–385
20. Dickens MG, Franz KJ (2010) ChemBioChem 11:59–62
21. Perez LR, Franz KJ (2010) Dalton Trans (39):2177–2187
22. Schugar H, Green DE, Bowen ML, Scott LE, Storr T, Bohmerle
K, Thomas F, Allen DD, Lockman PR, Merkel M, Thompson
KH, Orvig C (2007) Angew Chem Int Ed 46:1716–1718
Acknowledgments We thank Y. Su for performing mass spec-
trometry experiments. This work was supported by the National
Institutes of Health (R01 HL00049-01) and the American Heart
Association (0970028N). J.L.M.J. is supported by an American Heart
Association postdoctoral fellowship and K.B.D. is supported by a
National Institutes of Health Training Grant (5T32DK007233-34).
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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J Biol Inorg Chem (2011) 16:313–323
323
23. Storr T, Merkel M, Song-Zhao GX, Scott LE, Green DE, Bowen
ML, Thompson KH, Patrick BO, Schugar H, Orvig C (2007) J
Am Chem Soc 129:7453–7463
24. Wei Y, Guo M (2007) Angew Chem Int Ed 46:4722–4725
25. Wei Y, Zhang Y, Liu Z, Guo M (2010) Chem Commun
46:4472–4474
26. Failes TW, Cullinane C, Diakos N, Yamamoto N, Lyons JG,
Hambley TW (2007) Chem Eur J 13:2974–2982
27. Failes TW, Hambley TW (2006) Dalton Trans (15):1895–1901
28. Failes TW, Hambley TW (2007) J Inorg Biochem 101:396–403
29. Mitchell MB, Whitcombe IWA (2000) Tetrahedron Lett
41:8829–8834
34. Miller EW, Albers AE, Pralle A, Isacoff EY, Chang CJ (2005) J
Am Chem Soc 127:16652–16659
35. Maeda H, Fukuyasu Y, Yoshida S, Fukuda M, Saeki K, Matsuno
H, Yamauchi Y, Yoshida K, Hirata K, Miyamoto K (2004) An-
gew Chem Int Ed 43:2389–2391
36. Maeda H, Yamamoto K, Nomura Y, Kohno I, Hafsi L, Ueda N,
Yoshida S, Fukuda M, Fukuyasu Y, Yamauchi Y, Itoh N (2005) J
Am Chem Soc 127:68–69
37. Xu K, Tang B, Huang H, Yang G, Chen Z, Li P, An L (2005)
Chem Commun (48):5974–5976
38. Agrawal A, Romero-Perez D, Jacobsen JA, Villarreal FJ, Cohen
SM (2008) ChemMedChem 3:812–820
30. Major Jourden JL, Cohen SM (2010) Chem Commun
46:1241–1243
31. Major Jourden JL, Cohen SM (2010) Angew Chem Int Ed
49:6795–6797
32. Tietze LF, Feuerstein T (2003) Aust J Chem 56:841–854
33. Miller EW, Tulyathan O, Isacoff EY, Chang CJ (2007) Nat Chem
Biol 3:263–267
39. Hammett LP (1937) J Am Chem Soc 59:96–103
40. Knight CG, Willenbrock F, Murphy G (1992) FEBS Lett
296:263–266
41. Haba K, Papkov M, Shamis M, Lerner RA, Barbas CF III, Shabat
D (2005) Angew Chem Int Ed 44:716–720
42. Weinstain R, Baran PS, Shabat D (2009) Bioconjug Chem
20:1783–1791
123