In situ "click" assembly of small molecule matrix metalloprotease inhibitors containing zinc-chelating groups

Article abstract of DOI:10.1021/ol802286g

(Chemical Equation Presented) A panel of small molecule-based MMP inhibitors containing rhodanine warheads was assembled using "one-pot" click chemistry. Upon biological screening, moderate inhibitors were identified which specifically targets MMP-7 and MMP-13 over other MMPs.

Full text of DOI:10.1021/ol802286g

ORGANIC
LETTERS
2008
Vol. 10, No. 24
5529-5531
In Situ “Click” Assembly of Small
Molecule Matrix Metalloprotease
Inhibitors Containing Zinc-Chelating
Groups
Mingyu Hu, Junqi Li, and Shao Q. Yao*
Departments of Chemistry and Biological Sciences, NUS MedChem Program of the
Office of Life Sciences, National UniVersity of Singapore, Singapore 117543
chmyaosq@nus.edu.sg
Received October 2, 2008
ABSTRACT
A panel of small molecule-based MMP inhibitors containing rhodanine warheads was assembled using “one-pot” click chemistry. Upon biological
screening, moderate inhibitors were identified which specifically targets MMP-7 and MMP-13 over other MMPs.
Matrix metalloproteases (MMPs) consist of a family of zinc-
dependent endoproteinases which are involved in tissue
remodeling and degradation of the extracellular matrix
(ECM), angiogenesis, and cell motility. Currently, at least
26 human MMPs have been discovered. Abnormal MMP
activities are linked to many serious human diseases.1
Therefore, the development of potent MMP inhibitors has
received considerable interest in recent years.2 In most cases,
MMP inhibitors contain a well-known zinc-binding group
(ZBG) that chelates to the catalytic Zn2+ ion located in the
enzyme active site and a peptide or nonpeptide group that
interacts with the surrounding sites. One of the most widely
exploited ZBGs of MMP inhibitors is the hydroxamic acid
group, which has produced numerous nanomolar inhibitors
but has not been successful in clinical trials.1,2 The reason
is that inhibitors containing this ZBG normally exhibit very
potent but broad-spectrum inhibition toward most metallo-
proteases rather than MMPs alone. Furthermore, they exhibit
poor pharmacokinetic and bioavailability properties. As a
result, there has been intensive research efforts in the
discovery of new ZBGs which may be further developed
into highly potent and selective MMP inhibitors.3 Of
particular interest is the work of Cohen et al., where they
exploited the pyrone- and thiopyrone-containing moieties as
potential ZBGs and systematically evaluated their inhibitory
properties against various classes of metalloproteases includ-
ing MMPs and Anthrax Lethal Factor (ALF).3b-e Our
ongoing research interests in the use of high-throughput
amenable chemistry (such as “click chemistry” and amide-
forming reactions)4 have recently led us to the development
of “click” assembled, hydroxamate-containing peptide ana-
logues which showed micromolar inhibitory activities against
various MMPs.5 Herein, we describe a “one-pot” click
chemistry procedure for the rapid assembly of small molecule
(3) (a) Jacobsrn, F. E.; Lewis, J. A.; Cohen, S. M. ChemMedChem 2007,
2, 152–171. (b) Puerta, D. T.; Lewis, J. A.; Cohen, S. M. J. Am. Chem.
Soc. 2004, 126, 8388–8389. (c) Lewis, J. A.; Mongan, J.; McCammon,
J. A.; Cohen, S. M. ChemMedChem 2006, 1, 694–697. (d) Yan, Y.-L.;
Cohen, S. M. Org. Lett. 2007, 9, 2517–2520. (e) Agrawal, A.; Romero-
Perez, D.; Jacobsen, J. A.; Villarreal, F. J.; Cohen, S. M. ChemMedChem
2008, 3, 812–820.
(4) Kalesh, K. A.; Yang, P.-Y.; Srinivasan, R.; Yao, S. Q. QSAR Comb.
Sci. 2007, 26, 1135–1144. (b) Brik, A.; Wu, C.-Y.; Wong, C.-H. Org.
Biomol. Chem. 2006, 4, 1446–1457.
(1) Overall, C. M.; Kleifeld, O. Nat. ReV. Cancer 2006, 6, 227–239.
(2) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Chem.
ReV. 1999, 99, 2735–2776.
(5) Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S. Q. Org. Lett.
2006, 8, 3821–3824.
10.1021/ol802286g CCC: $40.75
Published on Web 11/17/2008
2008 American Chemical Society

MMP inhibitors containing novel rhodanine ZBGs (Fig-
ure 1).
triazole ring is formed regioselectively by reacting a terminal
alkyne with an azide in the presence of a Cu(I) catalyst. In
recent years, interests have been drawn toward the “one-
pot” click chemistry approaches using in situ generated
alkynes or azides.8 In our case, we found the two TMS-free
rhodanine warheads A and B (Scheme 1) rapidly decomposed
during column purification and could not be isolated in
acceptable purity. We therefore developed a “one-pot”
approach by using the TMS-protected rhodanine warheads
instead (D and E; see also Figure 1) during click chemistry.
Synthesis of the TMS-protected rhodanines involved three
steps. Propargyl alcohol 1 was TMS-protected to give 2,
which was subsequently oxidized to give aldehyde 3. The
base-catalyzed Knoevenagel condensation between 3 and 4/5
then afforded the TMS-protected, alkyne-containing D and
E. A total of 28 aromatic azides, C1-C28, were synthesized
from the corresponding arylamines using standard azidation
methods (Scheme 1).
Figure 1. General structures of MMP inhibitors containing a
rhodanine warhead and the “one-pot” click chemistry used in our
approach. The rhodanine ZBG is highlighted (in red). Inset: known
ALF inhibitors.
For the “one-pot” click chemistry between the TMS-
protected D and E and the azides C (Scheme 2), a variety
The design principle of our inhibitors was based on a
recent report that rhodanine-based molecules (inset in Figure
1), identified from high-throughput screening (HTS), are
efficient micromolar inhibitors of ALF.6 Crystallographic
analysis of the inhibitor-ALF complex further confirmed
that the rhodanine ring is capable of interacting with Zn2+
ion via the thiazolidine sulfur atom. However, MMP inhibi-
tors based on rhodanine ZBGs have yet to be reported in
the literature. Our aims in this project are therefore to
evaluate (1) whether rhodanine-containing small molecules
constitute a novel class of potential MMP inhibitors and (2)
whether our previously reported “click” approach could be
used to rapidly assemble these inhibitors.5 The Cu(I)-
catalyzed 1,3-dipolar cycloaddition between an alkyne and
azide is the prime example of “click chemistry” popularized
by Sharpless.7 In most cases, the 1,4-disubstituted-1,2,3-
Scheme 2. “One-Pot” Click Chemistry of Rhodanine Inhibitors
of conditions were explored to ensure efficient TMS cleavage
while maintaining minimum interference of the subsequent
“click” assembly of the product followed by direct in situ
Scheme 1. (Left) Synthetic Routes of Alkyne Building Blocks A and B. (Right) Synthesis and List of Aromatic Azides Used
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Org. Lett., Vol. 10, No. 24, 2008

biological screening. We eventually found that D or E (1
equiv) and C1-C28 (1.1 equiv) with K2CO3 (1 equiv; used
for in situ deprotection of TMS) in the presence of CuSO4
(0.1 equiv) and sodium ascorbate (0.4 equiv) in a solvent
mixture of t-BuOH/H2O (1:1) afforded the 56-membered,
rhodanine-containing small molecule inhibitors in nearly
quantitative yields with minimum side products, as judged
by LCMS and NMR characterizations (see the Supporting
Information). We further confirmed that these compounds
were indeed suitable for direct in situ screening without
further purifications.
We next obtained the inhibitor fingerprints of the 56-
membered rhodanines against different MMPs (Supporting
Information). A standard microplate-based enzymatic assay
was adopted as previously described.5 Representatives from
different classes of MMPs, including collagenases (MMP-8
and MMP-13), matrilysins (MMP-7), gelatinases (MMP-9),
and membrane-type MMPs (MMP-14), were tested. At an
inhibitor concentration of 20 µM, most compounds showed
good inhibition toward MMP-13 while displaying very weak/
no inhibition toward MMP-7/-8 and MMP-9/-14, respectively
(see the Supporting Information). The most potent inhibitors
appeared to be those containing alkoxyl substituents at the
aromatic ring (Table 1). These compounds were resynthe-
and 20.9 µM against MMP-7 and MMP-13, respectively, and
appeared to show some degrees of selectivity over other
MMPs tested. These values are moderate at best but well
expected if one considers that the parental inhibitor (inset
in Figure 1) showed micromolar inhibition against ALF6 and
the previously reported “click-based” peptide hydroxamates
were also micromolar MMP inhibitors.5 Nevertheless, our
results unambiguously establish the feasibility of using
rhodanines as novel ZBGs of MMP inhibitors.
Molecular modeling was carried out for A25 with MMP-7
and MMP-13. Compared to MMP-7 (which has a shallow
S1′ pocket), MMP-13 has a relatively deep S1′ pocket. As
shown in Figure 2, A25 fit nicely into the active site of both
Figure 2. Molecular modeling of A25 in the active site of MMP-7
(left) and MMP-13 (right). The zinc ion in the active site is shown
as a white sphere.
Table 1. Inhibition of the Three Selected Inhibitors
enzymes, with the two sulfur atoms from the rhodanine
warhead chelating to the zinc ion. In addition, the aromatic
moiety in A25 appeared to extend snugly into the S1′ binding
site of both enzymes. The docking results thus appeared to
indicate that rhodanines are likely a novel class of ZBGs of
MMP inhibitors.
Several key findings arose from our current work. First,
we have been able to show rhodanines as a novel class of
ZBGs of MMP inhibitors for the first time. Second, we have
demonstrated the compatibility of these ZBGs with “click
chemistry” for rapid assembly and direct in situ screening
of potential MMP inhibitors. Finally, we have developed a
“one-pot” click chemistry protocol for above applications.
Our best inhibitors possess moderate inhibitory activities
against MMPs currently, but upon further optimizations, they
may emerge as a new class of potent small molecule-based
MMP inhibitors.
sized, purified, characterized (LCMS and NMR), and further
studied in detail (to obtain their IC50/Ki); as shown in Table
1, A25, the most potent inhibitor, displayed Ki values of 14.1
Acknowledgment. Funding support was provided by the
Ministry of Education (R143-000-280-112) and the Agency
for Science, Technology, and Research (A*Star) of Singapore
(R-154-000-274-305).
(6) Forino, M.; Sherida, J.; Wong, T. Y.; Rozanov, D. V.; Savinov, A. Y.;
Li, W.; Fattorusso, R.; Becattini, B.; Orry, A. J.; Jung, D.; Abagyan, R. A.;
Smith, J. W.; Alibek, K.; Liddington, R. C.; Strongin, A. Y.; Pellecchia,
M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9499–9504.
(7) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery Today 2003, 8, 1128–
1137.
(8) (a) Aucagne, V.; Leigh, D. A. Org. Lett. 2006, 8, 4505–4507. (b)
Appukkuttan, P.; Dehaen, W.; Fokin, V. V.; Van der Eycken, E. Org. Lett.
2004, 6, 4223–4225. (c) Hansen, T. V.; Wu, P.; Fokin, V. V. J. Org. Chem.
2005, 70, 7761–7764. (d) Beckmann, H. S. G.; Wittmann, V. Org. Lett.
2007, 9, 1–4. (e) Barral, K.; Moorhouse, A. D.; Moses, J. E. Org. Lett.
2007, 9, 1809–1811.
Supporting Information Available: Experimental details
and characterization of compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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