Selective inhibition of matrix metalloproteinase isozymes and in vivo protection against emphysema by substituted γ-keto carboxylic acids

Article abstract of DOI:10.1021/jm051101g

The synthesis and matrix metalloproteinase (MMP) inhibitory activity of a series of γ-keto carboxylic acids are described. Among nine MMP isozymes tested, compound 1j displays selective inhibition of MMP-2, -9, and -12 with IC₅₀ values between

Full text of DOI:10.1021/jm051101g

456
J. Med. Chem. 2006, 49, 456-458
Selective Inhibition of Matrix Metalloproteinase
Isozymes and in Vivo Protection against
Emphysema by Substituted γ-Keto Carboxylic
Acids
Dawei Ma,*,† Yongwen Jiang,†,# Fangping Chen,‡,#
Li-kun, Gong,‡ Ke Ding,† Yong Xu,† Renxiao Wang,†
Aihua Ge,† Jin Ren,*,‡ Jingya Li,§ Jia Li,§ and Qizhuang Ye§
State Key Laboratory of Bioorganic and Natural Products
Chemistry, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Lu, Shanghai 201203, China, and
Chinese National Center for Drug Screening,
Figure 1. Design of a new class of γ-keto acids as selective MMP
inhibitors.
Shanghai Institute of Materia Medica, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences,
functional group, such as a carboxylic acid, hydroximic acid,
or sulfhydryl, capable of chelating the active-site Zn(II) ion (zinc
binding group, ZBG), at least one functional group that provides
a hydrogen bond interaction with the enzyme backbone, and
one or more side chains that undergo effective van der Waals
interactions with subsites of the enzyme. Carboxylates, repre-
sented by compound 68 (Figure 1), have been shown to be
effective MMP inhibitors. In light of these studies, we have
designed and synthesized a series of γ-keto carboxylic acids 1
in which the γ-oxo-benzenebutanoic acid skeleton was retained
to maintain the necessary Zn(II)-binding group and substituents
at either the 4-position of the benzene ring or the â-position of
the carboxyl group were introduced to provide van der Waals
interactions with the enzyme. It was hoped to identify some
structurally novel compounds with higher inhibitory activity and
selectivity.
189 Guo-Shou-Jing Road, Shanghai 201203, China
ReceiVed NoVember 1, 2005
Abstract: The synthesis and matrix metalloproteinase (MMP) inhibi-
tory activity of a series of γ-keto carboxylic acids are described. Among
nine MMP isozymes tested, compound 1j displays selective inhibition
of MMP-2, -9, and -12 with IC50 values between 0.20 and 1.51 µM,
and in male golden Syrian hamsters, it shows protection against PPE-
induced emphysema.
There is increasing evidence1-3 that some matrix metallo-
proteinases, particularly macrophage elastase (MMP-12), play
an important role in the progression of emphysema, a disease
caused by prolonged exposure to particles and gases such as
those in tobacco smoke.4 The earliest direct observation for this
hypothesis was reported by Hautamaki et al.2 who found that
wild-type mice possessing the metalloelastase gene (MMP-
12+/+) developed emphysema after chronic exposure to cigarette
smoke, while mice lacking this gene (MMP-12-/-) did not.
Recently, it was proposed that MMP-12 mediates smoke-
induced inflammation by releasing TNF-R from macrophages
with subsequent endothelial activation, neutrophil influx, and
proteolytic matrix breakdown caused by neutrophil-derived
proteases.4 Accordingly, it is expected that selective inhibitors
for MMP-12 should serve as novel therapeutic agents for
treatment of chronic obstructive pulmonary disease (COPD).
However, to the best of our knowledge, there have appeared
no reports regarding in vivo protection against emphysema by
selective MMP-12 inhibitors although much effort has in recent
years been directed to the development of selective MMP
inhibitors.5,6 In studies toward selective MMP inhibitors,7 we
have shown that some substituted γ-keto acids inhibit MMP-
12 selectively and display promising in vivo protection against
emphysema.
The synthetic pathways to the target molecules 1 are shown
in Scheme 1. The key intermediates 4 were obtained by two
routes. One was direct esterification of 2, a Friedel-Crafts
reaction product of bromobenzene with succinic anhydride, to
afford 4g. The other is alkylation of aryl ketones 3 with ethyl
bromoacetate mediated by LDA. The ketones 3a-c and 3f were
prepared by a direct Friedel-Crafts reaction of bromobenzene
with a suitable acyl chloride, while 3d and 3e were prepared
by a Wittig reaction of a 2,4′-dibromoacetophenone-derived
ylide with an aromatic aldehyde followed by hydrogenation.
Sonogashira reaction of esters 4 with different 1-alkynes
catalyzed by Pd/CuI gave the coupling products 5. Upon
treatment with aqueous NaOH in methanol, these esters were
hydrolyzed to furnish the target compounds 1.
The synthetic γ-keto carboxylic acids 1 were evaluated as
MMP inhibitors using galardin as a standard as described in
our previous paper.7 As summarized in Table 1, when R was
an aliphatic substituent, the resultant compounds 1a-d showed
weak inhibition of MMP-2 and MMP-12. Introduction of an
alkyl group at the R-position of the ketone moiety slightly
enhanced the potency (compare 1c with 1d). The potency was
further increased by about 10-fold by switching the alkyl
substituents of the alkyne moiety to aryl (compare 1d to 1e).
Better selectivity for MMP-12 over MMP-2 was seen when R′
was a larger group (compare 1e with 1f-h). It is noteworthy
that 1j showed the highest potency toward MMP-12, indicating
that replacing the phenyl group in 1g with a 4-chlorophenyl
group led to increased inhibitory activity. Although this
compound had considerable potency for MMP-2 and MMP-9,
it did not show activity against MMP-1, MMP-7, MMP-14,
During the past decade, a variety of different structural classes
of MMP inhibitors have been discovered using different methods
including structure-based design and combinatorial chemistry.5a
From these results, it can be seen that the requirements for a
molecule to be an effective inhibitor of MMPs include a
* To whom correspondence should be addressed. For D. Ma: phone
86-21-54925130; fax 86-21-64166128; e-mail madw@mail.sioc.ac.cn. For
J. Ren: phone 86-21-50806031; fax 86-21-50807088; e-mail:
jren@mail.shcnc.ac.cn.
† Shanghai Institute of Organic Chemistry.
‡ Shanghai Institute of Meteria Medica.
§ Chinese National Center for Drug Screening.
# These authors contributed equally to this work.
10.1021/jm051101g CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2005

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 457
Scheme 1
Table 1. IC50 Values for Inhibition of MMPs by Compounds 1 (µM)
compound
MMP-1
MMP-2
MMP-7
MMP-9
MMP-12
MMP-14
MMP-15
MMP-16
MMP-26
1a (R ) CH2OH, R′ ) H)
1b (R ) n-C8H17, R′ ) H)
1c (R ) CH2OBn, R′ ) H)
1d (R ) CH2OBn, R′ ) Et)
1e (R ) Ph, R′ ) Et)
a
a
a
a
a
15.37
3.07
23.10
5.74
0.44
1.66
1.12
2.11
3.43
0.55
0.007
a
a
a
a
a
a
24.69
12.48
25.74
7.56
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
N.A.
a
18.07
3.60
5.41
3.39
9.29
10.03
1.51
0.015
0.48
30.60
13.94
32.04
1f (R ) Ph, R′ ) n-C6H13)
1g (R ) Ph, R′ ) c-pentylmethyl)
1h (R ) Ph, R′ ) Bn)
0.45
0.32
0.60
1i (R ) Ph, R′ ) 4-FC6H4CH2)
1j (R ) 4-ClC6H4, R′ ) n-C6H13)
galardin
1.14
a
a
0.20
a
a
a
a
0.006
0.013
0.023
0.006
0.008
0.017
a No activity up to 50 µM.
MMP-15, MMP-16, and MMP-26 and could be considered as
a relatively selective MMP inhibitor.
To rationalize the structure-activity relationship of our
compounds, we have analyzed the three-dimensional structures
of MMPs that are available in the Protein Data Bank.9 Regarding
the formation and the shape of their S1′ pockets, MMPs can be
roughly clustered into three groups. As illustrated in Figure 2,
the S1′ pockets on MMP-1 and MMP-7 are short. In contrast,
the S1′ pockets on MMP-2, MMP-9, and MMP-12 are relatively
long and wide. The characteristics of the S1′ pockets on MMP-
14 and MMP-16 are more elusive since they could be very
restricted (as on MMP-16) or a bit more open (as on MMP-
14). In fact, MMP-14, MMP-15, and MMP-16 share a high
sequence homology and form a subfamily of membrane-type
matrix metalloproteinases (MT-MMPs). The orientation of one
flexible glutamine residue, which locates at the middle of the
S1′ pocket, largely determines the openness of this pocket. But
in general, the S1′ pockets on MMP-14 and MMP-16 can be
considered to be quite distinctive from their counterparts on
MMP-2, MMP-9, and MMP-12. So, if there is a large alkynyl
group at the 4-position of the phenyl ring (Table 1), such a
compound can only be well-accommodated by MMP-2, MMP-
9, and MMP-12. This may explain the high selectivity of this
series of compounds to MMP-2, MMP-9, and MMP-12 over
other subtypes. In addition, we found in our molecular docking
results that the alkynyl groups at the 4-position on the phenyl
ring can extend into the bottom of the S1′ pocket, forming
favorable hydrophobic contacts with residues Ala216, Val217,
Ala234, Val235, Met236, Phe237, Pro238, Tyr240, Tyr242,
Val243, and Phe248 (as numbered on MMP-12, PDB entry
1JK3). This may explain the difference in inhibitory activity
exhibited by different R groups. The most potent compound in
this series, that is, compound 1j, has a chlorine atom at the
very end of the R group (Table 1). Our molecular docking results
suggested that its potency may be due to the extra polar
interaction formed between this chlorine atom and one polar
residue at the bottom of S1′ pocket (Lys241, Thr227, and
Arg424 in MMP-12, MMP-2, and MMP-9, respectively).
Figure 2. S1′ pocket of MMPs generated by the MOLCAD program
encoded in Sybyl 6.8.8 Key residues determining the size and the shape
of the binding pocket are labeled. The structures of MMPs were
retrieved from the RCSB Protein Data Bank.9 The PDB entries used
for making these figures are 1HFC (MMP-1), 1QIB (MMP-2), 1MMQ
(MMP-7), 1GKC (MMP-9), 1JK3 (MMP-12), 1BUV (MMP-14), and
1RM8 (MMP-16).

458 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
Table 2. Effect of 1j on Wet Weight of Left Lung, VL, LV and RVHR
in Hamster
of the compounds 1j. This material is available free of charge via
the Internet at http://pubs.acs.org/.
weight of
left lung (g)
group
n
VL (mL)
LV (mL/g)
RVHR
References
saline
PPE
1j/PPE
1j/saline
5
5
5
5
0.188 ( 0.011
0.240 ( 0.030b
1.45 ( 0.78
2.28 ( 0.21b
1.20 ( 0.48
2.39 ( 0.45b
233.31 ( 25.39
302.49 ( 10.73b
(1) For a review, see: Belvisi, M. G.; Bottomley, K. M. The Role of
Matrix Metallopropeinases (MMPs) in the Pathophysiology of
Chronic Obstructive Pulmonary Diease (COPD): a Therapeutic Role
for Inhibitors of MMPs. Inflammation Res. 2003, 52, 95-100.
(2) Hautamaki, R. D.; Kobayashi, D. M.; Senior, R. M.; Shapiro, S. D.
Requirement for Macrophage Elastase for Cigarette Smoke-Induced
Emphysema in Mice. Science 1997, 277, 2002-2004.
0.203 ( 0.018a,c 1.96 ( 0.57b,c 1.82 ( 0.38a,c 255.00 ( 22.73a,c
0.196 ( 0.014c
1.07 ( 0.74c
1.14 ( 0.50c
237.10 ( 30.22c
a p < 0.05. b p < 0.01 vs Saline group. c p < 0.01 vs PPE groups.
(3) For a review, see: Buhl, R.; Farmer, S. G. Current and Future
Pharmacologic Therapy of Exacerbations in Chronic Obstructive
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136-142.
It is known that emphysema can be induced in hamsters by
the intratracheal instillation of porcine pancreatic elastase
(PPE).10 This model was therefore used to examine the in vivo
activity of our selective MMP-12 inhibitor 1j. Forty male golden
Syrian hamsters were divided into four groups, which were
treated with saline, PPE (400 IU/kg), PPE/1j (400 IU/kg and 5
mg/(kg‚day)) and saline/1j (5 mg/(kg‚day)), respectively (1j was
injected intraperitoneally for 28 days after saline or PPE was
instilled intratracheally). After 28 days, the hamsters were killed
and their lungs were examined by measuring the wet weight of
left lung, lung volumes (VL), relative lung volumes (LV, LV )
VL/body weight × 102), and right ventricular hypertrophy (RVH,
RVH ) right ventricle/(left ventricle + interventricular septum)
× 103). As indicated in Table 2, all these parameters increased
significantly for the PPE-treated group in comparison with the
parameters from the saline-treated control hamsters. This is
consistent with those results reported previously and clearly
shows emphysema to have been produced in PPE-treated
hamsters. This trend was inhibited greatly by 1j, as evidenced
by the lower wet weight of left lung, lung volumes, relative
lung volumes, and right ventricular hypertrophy observed in
PPE/1j-treated hamsters when compared with PPE-treated
hamsters. It should be noted that the data from 1j/saline treated
hamsters are close to those from saline-treated control hamsters.
Furthermore, morphometric results in the PPE/1j-treated group
imply that 1j tends to protect the integrity of the alveolar septal
walls and protect elastic fibers from cleavage. Histologic data
in the hamster revealed that 1j significantly protects against lung
hemorrhage on the first day after PPE is injected intratracheally.
From these results, it is concluded that 1j provides in vivo
protection against emphysema.
(4) Churg, A.; Wang, R. D.; Tai, H.; Wang, X.; Xie, C.; Dai, J.; Shapiro,
S. D.; Wright, J. L. Macrophage Metalloelastase Mediates Acute
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Release. Am. J. Respir. Crit. Care Med. 2003, 167, 1083-1089.
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Thomason, J. R.; Xiang, J. S.; Skotnicki, J. S.; Tam, S.; Cunningham,
K. M.; Chockalingam, P. S.; Morris, E. A.; Levin, J. I. Synthesis
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A.; Blais, S.; Cluzeau, P.; Courte´, K.; Hamon, J.; Moreau, F.; Prunet,
M.-L.; Tertre, A. Structure-Based Design and Synthesis of Novel
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K. J.; Barbosa, J.; McDonnell, P. A.; Metzler, W. J.; Rankin, B. M.;
Liu, R.; Vaccaro, W.; Pitts, W. Structure-Based Design of Potent
and Selective Inhibitors of Collagenase-3 (MMP-13). Bioorg. Med.
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Cuniasse, P.; Dive, V.; Yiotakis, A. Evaluation of P1′-Diversified
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In conclusion, we have found that some γ-keto carboxylic
acids are relatively selective inhibiters of MMP-12. The most
potent compound in this series also displayed promising in vivo
protection against PPE-induced emphysema in male golden
Syrian hamsters. This result provides additional evidence for
involvement of MMP-12 in development of emphysema and
should stimulate further exploration of MMP-12 as a target for
treatment of emphysema.
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BMC Physiol. 2005, 5, No 6.
Acknowledgment. The authors are grateful to the Chinese
Academy of Sciences, National Natural Science Foundation of
China (Grants 20321202 and 20132030), and Science and
Technology Commission of Shanghai Municipality (Grants
02JC14032 and 05DZ19334) for their financial support.
Supporting Information Available: Experimental procedure
for the preparation of compounds 1 and pharmacological studies
JM051101G