Design, synthesis, and evaluation of a mechanism-based inhibitor for gelatinase A

Article abstract of DOI:10.1021/jo050339+

Matrix metalloproteinases (MMPs), of which 26 are known, have been implicated in a number of pathological conditions, including tumor metastasis. We have previously described the first mechanism-based inhibitor for MMPs (J. Am. Chem. Soc. 2000, 122, 6799-

Full text of DOI:10.1021/jo050339+

and cancer, just to mention a few. Inhibition of MMPs
as a means to intervention of disease is highly sought.2
With very few exceptions, the known inhibitors of MMPs
are broad-spectrum molecules, designed to chelate the
active-site zinc ions of these enzymes. This broad breadth
of activity has been problematic in clinical trials of MMP
inhibitors, as the molecules show serious side effects.3
We have been interested in selective inhibition of
gelatinases, MMP-2 and -9 (also known as gelatinases A
and B, respectively). The excessive and unregulated
activities of these two enzymes have been indicated in a
number of cancer metastases.4 We disclosed for the first
time a novel strategy in mechanism-based inhibition of
MMP by a thiirane-containing inhibitor, where the
thiirane sulfur first coordinates with the active-site zinc
ion.5 The coordinated thiirane predisposes it to nucleo-
philic attack by the active site glutamate (Glu-404 in
MMP-2) in these enzymes, a process that leads to
covalent modification of the enzyme and the attendant
loss of activity. This process is depicted below for the
prototypic member (compound 1) of this class of inhibitors
(1 f 2).
Design, Synthesis, and Evaluation of a
Mechanism-Based Inhibitor for
Gelatinase A
Masahiro Ikejiri,† M. Margarida Bernardo,‡
Samy O. Meroueh,† Stephen Brown,† Mayland Chang,†
Rafael Fridman,‡ and Shahriar Mobashery*,†
Department of Pathology, Wayne State University School of
Medicine, Detroit, Michigan 48201, and Department of
Chemistry and Biochemistry and the Walther Cancer
Research Center, University of Notre Dame, Notre Dame,
Indiana 45665
mobashery@nd.edu
Received February 22, 2005
Matrix metalloproteinases (MMPs), of which 26 are known,
have been implicated in a number of pathological conditions,
including tumor metastasis. We have previously described
the first mechanism-based inhibitor for MMPs (J. Am. Chem.
Soc. 2000, 122, 6799-6800), which in chemistry mediated
by the active site zinc ion selectively and covalently inhibits
MMP-2, -3, and -9. Computational analyses indicated that
this selectivity in inhibition of MMPs could be improved by
design of new variants of the inhibitor class. We report
herein the syntheses of methyl 2-(4-{4-[(2-thiiranylpropyl)-
sulfonyl]phenoxy}phenyl)acetate (3) and 2-(4-{4-[(2-thiiranyl-
propyl)sulfonyl]phenoxy}phenyl)acetic acid (4), and show
that compound 3 serves as a mechanism-based inhibitor
exclusively for MMP-2. This molecule should prove useful
in delineating the functions of MMP-2 in biological systems.
Inhibitor 1 underwent the chemistry shown above with
three MMPs: MMP-2, -3, and -9.5 We have been keen
on developing inhibitors of this class that show even a
more narrow spectrum of inhibition than that exhibited
by 1. Specific interactions at the bottom of the deep S1′
pocket of MMP-2 and MMP-9 were identified that could
be exploited with a structural variant of inhibitor 1 to
enhance selectivity toward gelatinases (Figure 1). Two
Matrix metalloproteinases (MMPs) are zinc-dependent
endopeptidases with important pathological and physi-
ological functions.1 A total of 26 MMPs are known. Their
unregulated and uncontrolled activities have been as-
sociated with a number of disease processes, including
neurological disorders, arthritis, cardiovascular diseases,
(2) Cristofanilli, M.; Charnsangavej, C.; Hortobagyi, G. N. Nature
Rev. 2002, 1, 415-426. Buolamwini, J. K. Curr. Opin. Chem. Biol.
1999, 3, 500-509. Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A.
J. H. Chem. Rev. 1999, 99, 2735-2776. Skiles, J. W.; Gonnella, N. C.;
Jeng, A. Y. Curr. Med. Chem. 2001, 8, 425-474. Lee, M.; Fridman,
R.; Mobashery, S. Chem. Soc. Rev. 2004, 33, 401-409.
(3) Egeblad, M.; Werb, Z. Nature Rev. Cancer 2002, 2, 161-174.
Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002, 295,
2387-2392.
* Address correspondence to this author. Phone: 574-631-2933.
Fax: 574-631-6652.
† University of Notre Dame.
‡ Wayne State University School of Medicine.
(4) Bartsch, J. E.; Staren, E. D.; Appert, H. E. J. Surg. Res. 2003,
110, 383-392. Nakopoulou, L.; Tsirmpa, I.; Alexandrou, P.; Louvrou,
A.; Ampela, C.; Markaki, S.; Davaris, P. S. Breast Cancer Res. Treat.
2003, 77, 145-155. Mook, O. R.; Frederiks, W. M.; Van Noorden, C. J.
Biochim. Biophys. Acta 2004, 1705, 69-89. Aglund, K.; Rauvala, M.;
Puistola, U.; Angstrom, T.; Turpeenniemi-Hujanen, T.; Zackrisson, B.;
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Oncol. 2004, 30, 560-4. Fundyler, O.; Khanna, M.; Smoller, B. R. Mod.
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Metalloproteases: Structures, Evolution and Diversification. FASEB
J. 1998, 12, 1075. Forget, M.-A.; Desrosier, R. R.; Be´liveau, R. Can.
J. Physiol. Pharmacol. 1999, 77, 465-480. Nelson, A. R.; Fingleton,
B.; Rothenberg, M. L.; Matrisian, L. M. J. Clin. Oncol. 2000, 18,
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563-572. Kerbel, R.; Folkman, J. Nature Rev. 2002, 2, 727-739. Fidler,
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Fridman, R.; Mobashery, S. J. Am. Chem. Soc. 2000, 122, 6799-
6800.
10.1021/jo050339+ CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/09/2005
J. Org. Chem. 2005, 70, 5709-5712
5709

hydrolyzed on C18-reverse phase silica gel to afford the
desired carboxylic acid 4 in 65% yield (with an attendant
12% recovery of 3).
The synthetic route to compounds 3 and 4 is different
than those reported for inhibitor 1.5,9 Syntheses of 1
began from the commercially available diphenyl ethers
11 or 12. The synthetic route of Scheme 1 allows more
flexibility in creating structural diversity in the two-ring
systems and should find more general applicability for
entries into this molecular class of versatile enzyme
inhibitors.
FIGURE 1. Stereoview of compound 3 bound to the active
site of MMP-2. A Connolly solvent-accessible surface is con-
structed in the active site (shown in green), while the protein
is rendered in purple. Compound 3 along with the active site
Glu-404 and the three histidines that are coordinated to the
catalytic zinc are shown in capped-stick representation, colored
according to atom types (yellow, red, blue, and white for S, O,
N, and C, respectively). The zinc ion is shown as an orange
sphere. The white arrow points to the S1′ pocket. The side
chain hydroxyl of Thr428 is expected to hydrogen bond (2.8
Å) to the ester carbonyl of compound 3. The methyl group of
compound 4 is located near Leu399, Leu420, and Phe431
resulting in favorable hydrophobic interactions that would
likely contribute to the overall binding affinity.
Compounds 3 and 4 were evaluated with a representa-
tive set of MMPs. As shown in Table 1, compound 4
inhibits MMP-2 with a Ki of 460 nM. Whereas compound
3 exhibits dissociation constants for MMP-2 and -9 in the
low nanomolar levels, it behaves as a slow-binding
inhibitor that leads to mechanism-based inhibitor only
with MMP-2. Therefore, compound 3 is a selective and
potent mechanism-based inhibitor of MMP-2 (gelatinase
A). Both compounds are merely poor competitive inhibi-
tors (micromolar) of the other MMP that were tested.
Hence, high selectivity in inhibition of MMP-2 has been
achieved.
Superimposition of the X-ray structures for MMP-2 and
MMP-9 reveals some important differences.10 Midway
through the S1′ loop, an arginine residue is present in
MMP-9, as opposed to a threonine in MMP-2. The pocket
of MMP-9 appears to be more constricted than that of
MMP-2, as the backbone of the S1′ loop of MMP-9 is
about 2-3 Å inward. This could potentially lead to
unfavorable steric interaction for the methyl moiety of
compound 3 in MMP-9.
such molecules, 3 and 4, were designed by computational
analyses. We report herein the syntheses of these mol-
ecules. Furthermore, we document that compound 3
shows the pattern of slow-binding inhibition that leads
to covalent chemistry only with MMP-2.
The syntheses of compounds 3 and 4 were accom-
plished according to Scheme 1. An N,N-dimethyl glycine-
promoted Ullmann coupling reaction6 between the com-
mercially available aryl bromide 5 and phenol 7,7 which
was in turn prepared from 4-hydroxythiophenol (6) by
chemoselective allylation, proceeded smoothly to give 8
in 65% yield. Oxidation of the sulfur and olefin moieties
in 8 was achieved by the use of an excess of m-CPBA (12
equiv) to afford oxirane 9 in 92% yield, which was treated
with thiourea to provide thiirane 3 in 77% yield. At-
tempts at hydrolysis of the methyl ester of 3 to the
corresponding carboxylic acid 4 under various basic
conditions were unsuccessful, probably due to the depro-
tonation of the acidic R-position to the sulfonyl moiety,
followed by â-elimination of the thiolate. The method of
Mascaretti with the use of (Bu3Sn)2O in toluene at 80
°C8 was found to be most effective for this conversion.
Under these conditions, the methyl ester 3 was converted
to the corresponding tin ester 10, which was subsequently
Experimental Section
Methods. Enzyme kinetics were performed as described
earlier.5
4-(Allylthio)phenol (7). To a stirred solution of 4-hydroxy-
thiophenol (6) (4.30 g, 34.1 mmol) in DMF (25 mL) were added
K2CO3 (4.71 g, 34.1 mmol) and allyl bromide (3.09 mL, 34.1
mmol) at ice-water temperature, and the mixture was stirred
for 15 min, prior to stirring overnight at room temperature. After
the addition of 1 M aqueous HCl, the mixture was extracted with
ether (3×). The combined organic layer was washed with water
and brine, dried over MgSO4, and concentrated under reduced
pressure. The resultant residue was purified by silica gel column
chromatography (ethyl acetate/hexane ) 1/10 to 1/6) to give 7
(5.74 g, 70%) as a white semisolid. The 1H and 13C NMR spectra
and mass spectrum were identical with the reported values.7
Methyl 2-{4-[4-(Allylthio)phenoxy]phenyl}acetate (8). A
mixture of 5 (1.51 g, 6.59 mmol), 7 (1.64 g, 9.88 mmol), Cs2CO3
(4.30 g, 13.2 mmol), N,N-dimethylglycine hydrochloride salt (276
mg, 1.98 mmol), CuI (125 mg, 0.659 mmol), and degassed 1,4-
dioxane (14 mL) was heated at 90 °C for 22 h under a nitrogen
atmosphere. After dilution with water, the mixture was ex-
tracted with ethyl acetate. The combined organic layer was
(6) Ma, D.; Cai, Q. Org. Lett. 2003, 5, 3799-3802.
(7) Goux, C.; Lhoste, P.; Sinou, D. Tetrahedron 1994, 50, 10321-
10330.
(8) Mata, E. G.; Mascaretti, O. A. Tetrahedron Lett. 1988, 29, 6893-
6896. Salomon, C. J.; Mata, E. G.; Mascaretti, O. A. J. Org. Chem.
1994, 59, 7259-7266.
(9) Lim, I. T.; Brown, S.; Mobashery, S. J. Org. Chem. 2004, 69,
3572-3573.
(10) Park, H. I.; Jin, Y.; Hurst, D. R.; Monroe, C. A.; Lee, S.;
Schwartz, M. A.; Sang, Q.-X. A. J. Biol. Chem. 2003, 278, 51646-
51653.
5710 J. Org. Chem., Vol. 70, No. 14, 2005

SCHEME 1
TABLE 1. Kinetic Parameters for Competitive
Inhibition of MMPs by the Synthetic Inhibitors
was washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (ethyl acetate/hexane )
2/5) to give 3 (400 mg, 77%) as a colorless oil. 1H NMR (500 MHz,
CDCl3) δ 2.15 (dd, 1H, J ) 5.5, 2.0 Hz), 2.53 (dd, 1H, J ) 6.5,
2.0 Hz), 3.05 (m, 1H), 3.17 (dd, 1H, J ) 14.5, 7.0 Hz), 3.51 (dd,
1H, J ) 14.5, 5.5 Hz), 3.65 (s, 2H), 3.72 (s, 3H), 7.03-7.05 (m,
2H), 7.08-7.10 (m, 2H), 7.33-7.34 (m, 2H), 7.85-7.86 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 24.2, 26.0, 40.3, 52.1, 62.6, 117.7,
120.5, 130.7, 130.9, 131.1, 131.9, 153.8, 162.8, 171.8; HRMS
(FAB) calcd for C18H19O5S2 (M + H+) 379.0674, found 379.0645.
Ki (nM)
3
4
MMP-2
MMP-9
MMP-14cat
MMP-1
MMP-3
MMP-7
50 ( 14a
460 ( 30
40 ( 2
(4.1 ( 0.2) × 103
(5.3 ( 0.3) × 104
(4.5 ( 0.9) × 103
(5.4 ( 1.0) × 105
(2.5 ( 0.1) × 105
590 ( 70
(1.1 ( 0.2) × 104
(8.7 ( 0.5) × 103
1.3 × 104
2-(4-{4-[(2-Thiiranylpropyl)sulfonyl]phenoxy}phenyl)-
acetic Acid (4). To a stirred solution of 3 (312 mg, 0.83 mmol)
in toluene (11 mL) was added bis(tributyltin)oxide (1.05 mL, 2.06
mmol) at room temperature, and the mixture was stirred at 80
°C for 12 h. The solution was cooled to room temperature and
concentrated to dryness under reduced pressure. The residue
was dissolved in acetonitrile, and the solution was washed with
hexane (3×) and concentrated under reduced pressure to leave
the crude tin ester 10 (532 mg) as a pale-yellow oil. Subse-
quently, 10 was passed through a C18-reverse phase silica gel
pad (ODS silica gel 20 g, washed with water, 1:2 water/
acetonitrile and acetonitrile) to afford a mixture of 3 and 4, which
was purified by silica gel column chromatography (chloroform/
methanol ) 30/1 to 10/1) to give 4 (195 mg, 65%) as a white
solid with the recovery of some of 3 (38 mg, 12%). Compound
10: 1H NMR (300 MHz, CDCl3) δ 0.90 (t, 9H, J ) 7.2 Hz), 1.23-
1.38 (m, 12H), 1.54-1.64 (m, 6H), 2.16 (dd, 1H, J ) 5.4, 1.8 Hz),
2.54 (dd, 1H, J ) 6.0, 1.8 Hz), 3.07 (m, 1H), 3.15 (dd, 1H, J )
13.8, 7.8 Hz), 3.54 (dd, 1H, J ) 13.8, 5.1 Hz), 3.64 (s, 2H), 7.03
(m, 2H), 7.08 (m, 2H), 7.35 (m, 2H), 7.86 (m, 2H); mass (FAB)
m/z 655 (M + H+); Rf 0.3 (chloroform/methanol ) 10/1).
Compound 4: mp 133-134 °C; 1H NMR (500 MHz, CDCl3) δ
2.16 (d, 1H, J ) 4.0 Hz), 2.54 (d, 1H, J ) 5.5 Hz), 3.06 (m, 1H),
3.19 (dd, 1H, J ) 14.0, 8.0 Hz), 3.52 (dd, 1H, J ) 14.0, 6.0 Hz),
3.68 (s, 2H), 7.05 (br d, 2H, J ) 8.5 Hz), 7.10 (br d, 2H, J ) 8.5
a Parameters for the slow-binding component of inhibition: kon
) (1.2 ( 0.3) × 104 M-1 s-1, koff ) (6.2 ( 0.7) × 10-4 s-1
.
washed with water and brine, dried over Na2SO4, and concen-
trated under reduced pressure. The resultant residue was
purified by silica gel column chromatography (ethyl acetate/
hexane ) 1/12) to give 8 (1.35 g, 65%) as a pale yellow semisolid.
1H NMR (300 MHz, CDCl3) δ 3.49 (d, 2H, J ) 7.2 Hz), 3.61 (s,
2H), 3.71 (s, 3H), 5.03-5.10 (m, 2H), 5.86 (m, 1H), 6.91-6.97
(m, 4H), 7.23-7.26 (m, 2H), 7.32-7.35 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 38.5, 40.3, 52.1, 117.5, 119.0, 119.1, 129.0, 129.3,
130.6, 132.9, 133.7, 156.0, 156.3, 172.0; HRMS (FAB) calcd for
C
18H18O3S (M+) 314.0977, found 314.0986.
Methyl 2-(4-{4-[(2-Oxiranylpropyl)sulfonyl]phenoxy}-
phenyl)acetate (9). To a stirred solution of 8 (500 mg, 1.59
mmol) in CH2Cl2 (20 mL) was added m-CPBA (ca. 70%, 4.7 g,
19.1 mmol) at ice-water temperature, and the mixture was
subsequently stirred at room temperature for 8 days. With ice-
cooling, the reaction was quenched with a saturated Na2S2O3
solution, followed by saturated NaHCO3 solution, and the
mixture was extracted with ethyl acetate (3×). The combined
organic layer was washed with saturated Na2S2O3 solution,
saturated NaHCO3 solution, water, and brine, dried over Na2-
SO4, and concentrated under reduced pressure. The resultant
residue was purified by silica gel column chromatography (ethyl
acetate/hexane ) 1/2 to 2/3) to give 9 (528 mg, 92%) as a color-
less oil. 1H NMR (500 MHz, CDCl3) δ 2.47 (dd, 1H, J ) 5.0,
2.0 Hz), 2.82 (m, 1H), 3.26-3.33 (m, 3H), 3.65 (s, 2H), 3.72 (s,
3H), 7.03-7.05 (m, 2H), 7.08-7.10 (m, 2H), 7.32-7.34 (m, 2H),
7.86-7.88 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 40.3, 45.8,
52.1, 59.6, 117.6, 120.6, 130.5, 130.9, 131.1, 132.4, 153.8, 162.8,
171.8; HRMS (FAB) calcd for C18H18O6S (M+) 362.0824, found
362.0829.
Hz), 7.35 (br d, 2H, J ) 8.5 Hz), 7.86 (br d, 2H, J ) 8.5 Hz); 13
C
NMR (125 MHz, CDCl3) δ 24.2, 26.0, 40.2, 62.6, 117.8, 120.5,
130.2, 130.7, 131.3, 132.0, 154.1, 162.7, 177.1; HRMS (FAB) calcd
for C17H17O5S2 (M + H+) 365.0517, found 365.0495; Rf 0.2
(chloroform/methanol ) 10/1).
Computational Procedures. The X-ray structure of MMP-2
provided the Cartesian coordinates for the molecular docking
study (RCSB code 1QIB). The Sybyl program (Tripos Inc., St.
Louis, MO) was used for the manipulation and visualization of
all structures and for the protonation of the bound ligand. AM1-
BCC charges were computed for the ligand by using the
antechamber module from the AMBER 7 suite of programs.11
The ligand was docked into the active site of MMP-2 with
use of a Lamarckian genetic algorithm as implemented in
Methyl 2-(4-{4-[(2-Thiiranylpropyl)sulfonyl]phenoxy}-
phenyl)acetate (3). To a stirred solution of 9 (500 mg, 1.38
mmol) in MeOH-CH2Cl2 (10:1, 11 mL) was added thiourea (262
mg, 3.45 mmol) at room temperature, and the mixture was
stirred overnight. After concentration under reduced pressure,
the residue was dissolved in ethyl ether. The ethyl ether solution
J. Org. Chem, Vol. 70, No. 14, 2005 5711

the AutoDock 3.04 program.12 Parameters for the docking
runs were similar to those used previously,12 except for the
following differences: the quaternion step, the translation
step, and the torsion step were set to 0.2, 5, and 5, respectively.
The number of evaluations was increased to 2.5 × 107 from
250 000 and the ligand was fully flexible during the docking
runs.
Acknowledgment. This work was supported by the
National Institutes of Health. S.O.M. is the recipient
of a Walther Cancer Institute Postdoctoral Fellowship.
(11) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T.
E., III; Wang, J.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz,
K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsui,
V.; Gohlke, H.; Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I.; Seibel,
G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A.; AMBER, 7th ed.;
University of California: San Francisco, CA, 2002.
(12) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639-
1662.
Supporting Information Available: NMR spectra for the
synthetic molecules. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO050339+
5712 J. Org. Chem., Vol. 70, No. 14, 2005