Characterization of a selective inhibitor for matrix metalloproteinase-8 (MMP-8)

Article abstract of DOI:10.1039/c4md00172a

MMP-8 has been implicated in various diseases. Selective MMP-8 inhibitors are needed to ascertain the role of this enzyme. We synthesized two inhibitors reported previously as selective for MMP-8. Compound 1 selectively inhibited MMP-8 and MMP-13; compoun

Full text of DOI:10.1039/c4md00172a

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Characterization of a selective inhibitor for matrix
metalloproteinase-8 (MMP-8)†
Cite this: Med. Chem. Commun., 2014,
, 1381
5
Derong Ding, Katerina Lichtenwalter, Hualiang Pi, Shahriar Mobashery*
and Mayland Chang*
Received 16th April 2014
Accepted 17th June 2014
MMP-8 has been implicated in various diseases. Selective MMP-8 inhibitors are needed to ascertain the role
of this enzyme. We synthesized two inhibitors reported previously as selective for MMP-8. Compound 1
selectively inhibited MMP-8 and MMP-13; compound 2 was a potent broad-spectrum inhibitor,
notwithstanding that it is used as a selective one.
DOI: 10.1039/c4md00172a
www.rsc.org/medchemcomm
Matrix metalloproteinases (MMPs) constitute a family of 26 ablation of a particular MMP might result in compensatory
enzymes that perform important functions in restructuring the increases in other MMPs, which might exhibit some overlap in
1
extracellular matrix environment. The roles of these enzymes their activities.
in the course of many physiological processes are well docu-
Hence, small-molecule inhibitors that exhibit high selec-
2
mented. However, one or more of the MMPs might also be tivity in the inhibition of a targeted MMP are keenly needed for
3
–6
expressed in the course of various pathological events.
Indeed, the expression of MMPs in either physiological or
pathological conditions is highly regulated. These enzymes are progression, and wound repair.
use in wild-type organisms.
14
MMP-8 has been implicated in inammation, cancer
15
16,17
In this vein, we have had a
expressed as zymogens, which need to be activated by proteol- need for a selective MMP-8 inhibitor for use in our investiga-
7
ysis. The active enzymes are further regulated by their complex tions of these mechanisms. A search of the literature identied
formation with tissue inhibitors of metalloproteinases (TIMPs), two inhibitors, compounds 1 and 2, which have been reported
an association that inhibits the activity of the enzymes. It is the and used as selective towards inhibition of MMP-8. We
uncomplexed activated MMPs that can play a biological role in synthesized these compounds by a variation of methods that
the living organism.
have been reported for the preparation of each, one in six steps
As regards the pathological conditions, these enzymes are and the other in four steps. The breadth of activity of these two
worthy targets for inhibition by small-molecule inhibitors. inhibitors was evaluated in vitro in order to explore the degree of
However, broad-spectrum inhibition of MMPs, as explored in selectivity of each in the inhibition of representative MMPs, in
investigations of cancer, has not borne fruit. Aside from the an assessment of the usefulness of the inhibitors for in vivo
multiplicity of these enzymes, the situation is complicated by work in animal models.
the fact that some MMPs might play benecial (repair) roles in
the given disease, whereas others might play detrimental
(pathological) roles. When multiple forms of these enzymes are
expressed, the study becomes complicated.
We have used selective inhibitors of MMPs to great advan-
tage in elucidation of the roles of specic MMPs in various
8–13
diseases.
These selective agents behave as “surgical” tools
The synthesis of compound 1 has been reported using a
that arrest the functions of specic enzymes at the precise time
points in the progression of the disease. A comparable experi-
mental control is not possible with the use of MMP-knockout
animals, as the knockout organism will lack the given MMP
from the moment of the fertilization of the egg. Moreover, gene
linear sequence of seven steps from the commercially available
18
ethyl 2-oxocyclohexylacetate (3) in an overall yield of 6%. Our
initial attempts to synthesize ethyl 2-amino-4-ethoxy-
carbonylmethyl-4,5,6,7-tetrahydrobenz[b]thiophene-3-carboxy-
18
late (4) followed the reported two-step sequence. This process
did not work well in our hands as the conditions were harsh and
led to the formation of side products, which proved difficult to
separate from the desired product. Hence the yield was low. In
order to overcome these difficulties, we devised a one-step
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
Indiana, USA. E-mail: mobashery@nd.edu; mchang@nd.edu; Fax: +574 631–6652;
Tel: +574 631-2933; +574 631-2965
†
Electronic supplementary information (ESI) available: syntheses of compounds transformation of 3 to 4, which furnished the product in 57%
and 2 and enzyme inhibition. See DOI: 10.1039/c4md00172a
1
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Table 1 Kinetic Parameters for Inhibition of MMPs
Compound 1
Compound 2
MMP
K
i
(nM)
K
i
(nM)
a
MMP-1
24000 Æ 3000
3000 Æ 200
17000 Æ 2000
53000 Æ 6000
25 Æ 2
6.8 Æ 0.3
Scheme 1 Synthetic scheme for the preparation of compound 1.
MMP-2a
0.10 Æ 0.01
1.2 Æ 0.3
MMP-3
MMP-7a
MMP-8a
MMP-9
135 Æ 26
0.6 Æ 0.1
76000 Æ 8000
0.53 Æ 0.30
45000 Æ 4000
0.37 Æ 0.02
0.023 Æ 0.008
0.22 Æ 0.04
yield. As depicted in Scheme 1, compound 3 was treated with
ethyl cyanoacetate and sulfur in pyridine in the presence of
diethylamine at room temperature. Compound 4 was obtained
in 57% yield aer column purication. With this intermediate
in hand, the target molecule was then synthesized following the
MMP-13a
MMP-14
a
Catalytic domain.
18
earlier procedure in ve additional steps. This synthesis was
accomplished in six steps with an overall yield of 10%.
The synthesis of compound 2 has been given only as a
scheme in the literature and the detailed procedures were not
provided. Based on the reported synthetic route, we devised a
slight modication of it to arrive at the target molecule. As
outlined in Scheme 2, the synthesis started from (R)-phenyl-
alanine (5).
<
25 nM for MMP-13, >2500 nM for MMP-3, and >10000 nM for
18
19
19
MMP-1, -2, -7, -9, and -14.
On the other hand, compound 2 strongly inhibited all the
tested MMPs in a competitive manner, with K values in the
i
nanomolar range for MMP-1, -3, and -7 and in the picomolar
range for MMP-2, -8, -9, -13, and À14 (Table 1), for which it is a
tight-binding inhibitor. The enzyme kinetics for compound 2
has not been reported previously according to a search of the
literature, except for that of MMP-8 for which an IC50 value of 4
nM was indicated. We have since learned that compound 2 is
being marketed by several commercial entities as a selective
MMP-8 inhibitor. According to our results, although this
compound is exceptionally potent, it is not selective for any
MMP.
Compound 6, which was prepared according to the litera-
20
ture, was used for the next step directly to afford compound 8
21
by the literature method. With 8 in hand, the initial attempts
to prepare compound 2 followed a two-step procedure involving
activation of the acid by thionyl chloride and subsequent
treatment with hydroxylamine hydrochloride. However, TLC
analysis indicated the presence of a complex mixture from
which it was difficult to purify the desired product. We resorted
to a different method. Compound 8 was used to form a mixed
anhydride by the reaction with ethyl chloroformate in the
presence of NMM, which was allowed to react with hydroxyl-
amine hydrochloride using triethylamine as base to afford the
19
Conclusions

nal product in good yield. In summary, this four-step sequence
Of the two compounds reported as selective MMP-8 inhibitors,
only compound 1 exhibits selectivity in inhibition of MMP-8
and MMP-13. This compound is not commercially available.
However, compound 2 is currently commercially available and
is marketed as a selective MMP-8 inhibitor. Compound 2 has
been used in several studies to ascertain the role of MMP-8 in
the pathology of diseases. Using this inhibitor, MMP-8 was
indicated to play a role in bacterial meningitis by preventing
detachment of infected human brain microvascular endothelial
furnished the desired compound 2 with an overall yield of 27%.
Compound 1 was a selective competitive inhibitor of MMP-8
and MMP-13, with K
respectively (Table 1). Inhibition of MMP-1, -2, -3, -7, -9, and -14
i
values of 25 Æ 2 nM and 0.54 Æ 0.30 nM,
was poor, with dissociation constants (K ) in the micromolar
i
range and at least 120-fold greater than that of MMP-8. The K
i
values for compound 1 had not been reported previously;
however, the reported IC50 values were 7.4 nM for MMP-8,
22
cells and restoring blood–brain barrier permeability.
Compound 2 was evaluated in mice with experimental auto-
immune encephalomyelitis, an animal model for multiple
23
sclerosis, and found to reduce the severity of the disease. The
use of inhibitor 2 was instrumental in implicating MMP-8 in
24

brocyte migration. Compound 2 was found to decrease
25
ventilator-induced lung injury in mice, which led to the
conclusion that MMP-8 promotes acute inammation aer
ventilator-induced lung injury. In the light of our results indi-
cating the broad spectrum of activity for inhibition of MMPs by
compound 2, we regretfully conclude that the mechanistic role
of MMP-8 in the pathology of these diseases might not have
been established.
Scheme 2 Synthetic scheme for the preparation of compound 2.
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5 A. Gutierrez-Fernandez, A. Fueyo, A. R. Folgueras,
C. Garabaya, C. J. Pennington, S. Pilgrim, D. R. Edwards,
D. L. Holliday, J. L. Jones, P. N. Span, F. C. Sweep,
X. S. Puente and C. Lopez-Otin, Cancer Res., 2008, 68,
2755–2763.
Notes and references
1
A. Page-McCaw, A. J. Ewald and Z. Werb, Nat. Rev. Mol. Cell
Biol., 2007, 8, 221–233.
2
3
T. H. Vu and Z. Werb, Genes Dev., 2000, 14, 2123–2133.
E. Dejonckheere, R. E. Vandenbroucke and C. Libert, Drug 16 E. Pirila, J. T. Korpi, T. Korkiamaki, T. Jahkola, A. Gutierrez-
Discovery Today, 2011, 16, 762–778.
C. Gialeli, A. D. Theocharis and N. K. Karamanos, FEBS J.,
Fernandez, C. Lopez-Otin, U. Saarialho-Kere, T. Salo and
T. Sorsa, Wound Repair Regen., 2007, 15, 47–57.
17 M. Gooyit, Z. Peng, W. R. Wolter, H. Pi, D. Ding, D. Hesek,
M. Lee, B. Boggess, M. M. Champion, M. A. Suckow,
S. Mobashery and M. Chang, ACS Chem. Biol., 2014, 9,
505–510.
4
5
6
2011, 278, 16–27.
W. C. Parks, C. L. Wilson and Y. S. Lopez-Boado, Nat. Rev.
Immunol., 2004, 4, 617–629.
V. W. Yong, S. M. Agrawal and D. P. Stirling,
Neurotherapeutics, 2007, 4, 580–589.
18 G. Pochetti, R. Montanari, C. Gege, C. Chevrier, A. G. Taveras
and F. Mazza, J. Med. Chem., 2009, 52, 1040–1049.
7
8
R. Visse and H. Nagase, Circ. Res., 2003, 92, 827–839.
R. D. Bonl, A. Sabbota, S. Nabha, M. M. Bernardo, Z. Dong, 19 H. Matter, M. Schudok, W. Schwab, W. Thorwart, D. Barbier,
H. Meng, H. Yamamoto, S. R. Chinni, I. T. Lim, M. Chang,
L. C. Filetti, S. Mobashery, M. L. Cher and R. Fridman, Int.
J. Cancer, 2006, 118, 2721–2726.
J. Cui, S. Chen, C. Zhang, F. Meng, W. Wu, R. Hu, O. Hadass,
T. Lehmidi, G. J. Blair, M. Lee, M. Chang, S. Mobashery,
G. Y. Sun and Z. Gu, Mol Neurodegen, 2012, 7, 21.
G. Billen, B. Haase, B. Neises, K. Weithmann and
T. Wollmann, Bioorg. Med. Chem., 2002, 10, 3529–3544.
20 A. P. Kozikowski, D. Ma, Y. P. Pang, P. Shum, V. Likic,
P. K. Mishra, S. Macura, A. Basu, J. S. Lazo and R. G. Ball,
J. Am. Chem. Soc., 1993, 115, 3957–3965.
9
21 H. Matter, M. Knauf, W. Schwab and E. F. Paulus, J. Am.
Chem. Soc., 1998, 120, 11512–11513.
1
1
1
1
0 Z. Gu, J. Cui, S. Brown, R. Fridman, S. Mobashery,
A. Y. Strongin and S. A. Lipton, J. Neurosci., 2005, 25, 6401– 22 A. Schubert-Unkmeir, C. Konrad, H. Slanina, F. Czapek,
408.
S. Hebling and M. Frosch, PLoS Pathog., 2010, 6, e1000874.
1 D. H. Kim, C. Lilliehook, B. Roides, Z. Chen, M. Chang, 23 A. R. Folgueras, A. Fueyo, O. Garcia-Suarez, J. Cox,
6
S. Mobashery and S. A. Goldman, J. Neurosci., 2008, 28,
08–216.
2 A. Kruger, M. J. Arlt, M. Gerg, C. Kopitz, M. M. Bernardo,
A. Astudillo, P. Tortorella, C. Campestre, A. Gutierrez-
Fernandez, M. Fanjul-Fernandez, C. J. Pennington,
D. R. Edwards, C. M. Overall and C. Lopez-Otin, J. Biol.
Chem., 2008, 283, 9465–9474.
2
M. Chang, S. Mobashery and R. Fridman, Cancer Res.,
2
005, 65, 3523–3526.
24 C. Garcia-de-Alba, C. Becerril, V. Ruiz, Y. Gonzalez, S. Reyes,
J. Garcia-Alvarez, M. Selman and A. Pardo, Am. J. Respir. Crit.
Care Med., 2010, 182, 1144–1152.
3 R. Ota, C. Kurihara, T. L. Tsou, W. L. Young,
Y. Yeghiazarians, M. Chang, S. Mobashery, A. Sakamoto
and T. Hashimoto, J. Cereb. Blood Flow Metab., 2009, 29, 25 G. M. Albaiceta, A. Gutierrez-Fernandez, E. Garcia-Prieto,
1
547–1558.
4 P. Van Lint and C. Libert, Cytokine Growth Factor Rev., 2006,
7, 217–223.
X. S. Puente, D. Parra, A. Astudillo, C. Campestre,
S. Cabrera, A. Gonzalez-Lopez, A. Fueyo, F. Taboada and
C. Lopez-Otin, Am. J. Respir. Cell Mol. Biol., 2010, 43, 555–
1
1
563.
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