Investigation of self-immolative linkers in the design of hydrogen peroxide activated metalloprotein inhibitors

Article abstract of DOI:10.1039/c1cc12526e

A series of self-immolative boronic ester protected methyl salicylates and metal-binding groups with various linking strategies have been investigated for their use in the design of matrix metalloproteinase proinhibitors.

Full text of DOI:10.1039/c1cc12526e

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COMMUNICATION
Investigation of self-immolative linkers in the design of hydrogen
peroxide activated metalloprotein inhibitorsw
Jody L. Major Jourden, Kevin B. Daniel and Seth M. Cohen
Received 29th April 2011, Accepted 31st May 2011
DOI: 10.1039/c1cc12526e
A series of self-immolative boronic ester protected methyl
salicylates and metal-binding groups with various linking stra-
tegies have been investigated for their use in the design of matrix
metalloproteinase proinhibitors.
over the more commonly used carbonate ester linkage
(compare compound 1 vs. 2 in Fig. 1) due to better synthetic
accessibility, superior hydrolytic stability, and comparably fast
cleavage kinetics. Recently, this ether linkage was utilized in
studies on ROS-sensitive luciferase probes15 and protease-
sensitive fluorophores.13 Nonetheless, there are essentially no
studies on the generality and utility of this promising linking
strategy.
Prodrugs are a class of therapeutics that can be activated
in vivo to generate an active drug, providing targeted inhibitory
activity and thereby reducing side effects.1,2 Approximately
6% of all drugs approved worldwide can be classified as
prodrugs, including a handful of metalloprotein inhibitors.2,3
Recent efforts in prodrug strategies for metalloprotein inhibitors
have focused largely on redox mechanisms for activation.4,5
However, general approaches for developing prodrugs that
target metalloproteins has not been widely investigated.
In an effort to develop broadly applicable methods to
metalloprotein prodrugs, we have focused on developing
‘proinhibitors’ of the zinc(II)-dependent matrix metalloproteinases
(MMPs), a canonical metalloprotein target of medicinal
interest.6,7 MMP inhibitors (MMPi), like most metalloprotein
inhibitors, generally employ a metal-binding group (MBG),
which if blocked abolishes inhibitory activity. Prodrug matrix
metalloproteinase inhibitors (‘proMMPi’) have been developed
that are activated enzymatically or by reactive oxygen species
(ROS).8–10
This report investigates the scope of this ether-connected,
self-immolative proinhibitor strategy with a variety of functional
groups and MBGs. The behavior of different activation strategies
is explored using related, but distinct self-immolative linkers
for coupling to the MBGs. All of the strategies studied here
use boronic ester protecting groups that can be selectively
removed by H2O2. A series of methyl salicylate derivatives
containing phenol, thiophenol, aniline, and benzylamine
leaving groups were investigated using either an ether linkage,
a carbonate/carbamate ester linker, or no linker to the boronic
In the development of ROS-activated proMMPi, we employed
a relatively underutilized self-immolative protecting group
with several apparent advantages over previously described
systems. The use of self-immolative linkers has become
increasingly popular in drug development, molecular sensors,
and polymeric delivery systems.1,11,12 Linkers that undergo
self-immolative elimination upon removal of the protecting
group can release an active species through a 1,6-benzyl
elimination (Fig. 1). This reaction is thermodynamically
driven by the release of CO2 when a carbonate or carbamate
ester linkage is employed.12–14 However, in the development of
proMMPi, it was found that the use of an ether linkage
between the activating group and the inhibitor was preferred
Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, California, 92093-0358, USA.
E-mail: scohen@ucsd.edu; Fax: +1 858-822-5598;
Fig. 1 Three approaches to the development of ROS-activated
boronic ester proMMPi demonstrated with the methyl salicylate
derivatives 1–3 using either an ether or ester linked self-immolative
linker or through direct linkage of the protecting group.
Tel: +1 858-822-5596
w Electronic supplementary information (ESI) available: Synthetic
details, characterization of all compounds and details of hydrogen
peroxide activation. Fig. S1–S19. See DOI: 10.1039/c1cc12526e
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7968 Chem. Commun., 2011, 47, 7968–7970
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ester protecting group (Fig. 1 and Fig. 2). In addition, we
looked at a variety of MBGs protected with a boronic ester
self-immolative leaving group to expand our inventory of
MBGs for use in novel metalloprotein prodrugs.
Compounds 1–3 were designed to release methyl salicylate
in the presence of H2O2 using a self-immolative ether linkage
(1), a carbonate ester linkage (2), or no self-immolative linker
(3) to directly compare three possible designs of a prodrug
scaffold. The syntheses of these compounds are described in
the Supporting Information. Compounds 1–3 were first
examined for activation in the presence of H2O2 using UV-Vis
spectroscopy. To a solution of the boronic ester derivative in
HEPES buffer (50 mM, pH 7.5) was added H2O2 and the
change in absorbance was monitored over time. As shown in
Fig. 3 for compound 1, the absorbance over time shows an
increase at 302 nm indicative of the emergence of methyl
salicylate with a clear isobestic point at 293 nm. Similar results
were obtained with compounds 2 and 3. While compounds 1
and 2 achieved 490% cleavage within 45 min using an 18-fold
excess of H2O2 (Fig. S1–S2w), deprotection of compound 3
required a 180-fold excess of H2O2 to realize cleavage in a
comparable time frame. Release of methyl salicylate for all
three compounds was confirmed by HPLC (Fig. S3–S5w).
The rates of conversion to methyl salicylate were determined
by monitoring the change in absorbance under pseudo first-order
reaction conditions with an excess of H2O2. The calculated
rate constants are presented in Table 1. Consistent with earlier
reports, the carbonate ester derivative 2 displayed the fastest
rate of conversion, but 2 also underwent spontaneous
hydrolytic cleavage in buffer, whereas compound 1 was stable
in buffer over a 4 h period (data not shown). Introduction of
the carbonate group into the self-immolative linker of 2 leads
to hydrolytic instability facilitated by nucleophilic attack of
water at the carbonyl position which is not possible in
compound 1.16 Interestingly, while the hydrolytic stability
of 3 was comparable to the ether linkage used in 1, the rate
of conversion for 3 was about two orders of magnitude slower
Fig. 3 Absorption spectra of 1 (50 mM in HEPES buffer, pH 7.5) in
the presence of H2O2 (18 eq.) monitored every 2 min over 60 min. The
dashed line is the starting spectrum and the bold solid line is the final
spectrum. A sample of methyl salicylate is shown as a dotted line.
The arrow represents the change in absorption over time.
Table 1 Pseudo first-order rate constants calculated with an excess of
H2O2
Compound
k (MÀ1 sÀ1
)
Compound
k (MÀ1 sÀ1
)
1
2
3
10
1.12 Æ 0.04
2.7 Æ 0.1
11
12
13
14
5.9 Æ 0.2
3.5 Æ 0.3
2.9 Æ 0.1
4.1 Æ 0.2
0.031 Æ 0.002
3.1 Æ 0.5
than either 1 or 2, suggesting that use of self-immolative linker
facilitates conversion to the desired active compound.
Based on the behavior of compounds 1–3, the most promising
linking strategy is the benzyl ether linkage seen in compound
1. The benzyl ether linkage shows excellent stability in buffer
while maintaining rapid cleavage kinetics upon activation.
Therefore, we investigated the use of this motif with other
leaving groups. Compounds 4–7 were synthesized to study the
effects of using sulfur (4), aniline (5–6), or benzyl amine (7)
leaving groups (Fig. 2). Evaluation with UV-Vis absorption
spectroscopy of 4–7 in the presence of H2O2 showed no
cleavage of the protecting group suggesting that this strategy
may only be relevant for oxygen-derived leaving groups
(Fig. S6–S9w). Further evaluation with LC-MS showed that
the boronic ester of compound 6 was cleaved to the phenol
group, but that the cascade reaction did not proceed as
expected to release the aniline group (Fig. S10w). This is
reflective of the general robustness of these amine derivatives
and their inability to become easily ionized.17 Compounds 8
and 9 were then evaluated to investigate the use of a carbamate
ester linkage (Fig. 2). Unlike compound 7, the carbamate self-
immolative linkers in 8 and 9 showed that the desired benzyl
amine is released in the presence of H2O2, thermodynamically
driven by the release of CO2 (Fig. S11 and S12w). This suggests
that for the release of nitrogen-derived leaving groups,
the carbamate linkage may still be preferable for prodrug
development.1
To validate our observations in the context of MBGs, a
series of activatable MBGs (prochelators)18 were synthesized
Fig. 2 Methyl salilcylate derivatives 1–9 investigated in this study
with varying leaving groups and linkage strategies.
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A thorough investigation of boronic ester prochelators
shows that the use of benzyl ether self-immolative linkers
provides a superior platform for the development of metallo-
protein proinhibitors with oxygen-based leaving groups. These
compounds show excellent hydrolytic stability as well as fast
rates of cleavage to the active compounds in the presence of
H2O2. The use of boronic acids (instead of esters) results in
even faster cleavage and better aqueous solubility with no loss
in hydroylic stability. These findings are significant in the
development of triggered metalloprotein proinhibitors,
H2O2-activated prodrugs, and further examination of the
benzyl-ether self-immolative strategy with other triggering
groups is currently under way to obtain proinhibitors sensitive
to a variety of chemical and biological stimuli.
We thank Dr. Y. Su for performing mass spectrometry
experiments. This work was supported by a grant from the
National Institutes of Health (R21 HL094571). J.L.M.J. is
supported by an American Heart Association postdoctoral
fellowship and K.B.D. is supported by an NIH/NIDDK
Training Grant (5T32-DK007233).
Fig. 4 Protected MBGs (prochelators) designed with a benzyl-ether
self-immolative linker.
(compounds 10–14, Fig. 4) and evaluated. Compounds 10 and 11
were designed with a boronic acid protecting group to improve
water solubility of the protected MBGs.10 Several other
protected MBGs were examined including the oxygen-binding
3-hydroxy-1,2-dimethylpyridin-4(1H)-one (12), tropolone (13),
and 8-hydroxyquinoline (14). Compounds 10–14 showed
rapid cleavage to the desired MBG in the presence of H2O2,
as determined by absorption spectroscopy (Table 1), thus
confirming the broader utility of the benzyl ether self-
immolative strategy for designing metalloprotein proinhibitors
(Fig. S13–S17w). Additionally, use of the boronic acid derivative
in 10 and 11 shows both improved solubility and an increase in
the rate of cleavage when compared to their boronic ester
counterparts.10 The pinacol boronic ester analog of 10 had a
Notes and references
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evaluated. In the presence of H2O2 compound 16 showed no
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group, but no further cascade reaction occurs to release the
hydroxamic acid (vide supra, Fig. S19w).
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7970 Chem. Commun., 2011, 47, 7968–7970
This journal is The Royal Society of Chemistry 2011