3,4-Disubstituted benzofuran P1′ MMP-13 inhibitors: Optimization of selectivity and reduction of protein binding

Article abstract of DOI:10.1016/j.bmcl.2009.07.008

Potent 3,4-disubstituted benzofuran P1′ MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1′ moiety of the molecule. By replacing a backbone benzene with a pyridine and va

Full text of DOI:10.1016/j.bmcl.2009.07.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4546–4550
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3,4-Disubstituted benzofuran P10 MMP-13 inhibitors: Optimization
of selectivity and reduction of protein binding q
a
a
a
b
Wei Li a, Yonghan Hu a, , Jianchang Li , Jennifer R. Thomason , Dianne DeVincentis , Xuemei Du ,
Junjun Wu a, Rajeev Hotchandani a, Thomas S. Rush III a, , Jerauld S. Skotnicki b, Steve Tam a,
Priya S. Chockalingam c, Elisabeth A. Morris c, Jeremy I. Levin b
*
a Department of Chemical Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
b Department of Chemical Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
c Department of Tissue Repair, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
a r t i c l e i n f o
a b s t r a c t
Potent 3,4-disubstituted benzofuran P10 MMP-13 inhibitors have been prepared. Selectivity over MMP-2
was achieved through a substituent at the C4 position of the benzofuran P10 moiety of the molecule. By
replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with
greatly reduced plasma protein binding were also obtained.
Article history:
Received 26 May 2009
Revised 22 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MMP
Osteoarthritis
Plasma protein binding
MMP-13 (matrix metalloproteinase-13, collagenase-3) is a mem-
ber of a family of zinc-containing enzymes that are involved in the
degradation and remodeling of extracellular matrix proteins.1,2
Exploration of the therapeutic potential for small molecule inhibi-
tors of these enzymes to treat various pathologies has been contin-
uing for more than two decades, and many drug candidates
generated from this approach have been advanced to clinical trials.3
Human MMP-13 was first discovered and cloned in human breast
carcinoma and was later found expressed in primary and trans-
formed chondrocytes derived from joint tissue. It is inducible by
IL-1. The expression of MMP-13 in osteoarthritic cartilage and its
activity against type II collagen suggest that this enzyme plays a sig-
nificant role in cartilage collagen degradation.
In clinical trials, non-selective MMP inhibitors have not shown
efficacy, despite significant activity in pre-clinical models of osteo-
arthritis and cancer. One possible explanation for these failures
may be dose-limiting toxicity in the form of musculoskeletal side
effects. This toxicity has been proposed to be caused by the non-
selective inhibition of MMPs.4 Therefore, selective, efficacious
MMP-13 inhibitors that avoid this side effect are highly desirable.5
Biphenylsulfonamide carboxylates have been reported by us6 as
well as others7–9 as selective MMP-13 inhibitors. In compounds I
and II we have used a benzofuran P10 moiety to gain MMP-13 po-
tency and varied the substitution at the C3, C4, and C5 positions to
optimize selectivity against the highly homologous MMP-2 (Fig. 1).
The loop region of MMP-2 that forms its S10 pocket is two amino
acids shorter than this region of MMP-13, presenting the possibil-
ity that MMP-13 will tolerate larger P10 moieties that cannot be
accommodated by MMP-2. Furthermore, we hoped that in addition
to the selectivity gained over MMP-2 in this manner, these analogs
would be selective over a wide variety of other MMPs with lower
sequence homology.
We have previously reported6b that the 4-methoxy substituent
of compound II clashes with Thr229 of MMP-2 in the S10 pocket
while the bromine atom at C5 helps constrain the mobility of the
methoxy group. No such steric clash exists for these substituents
within the larger S10 pocket of MMP-13. In this Letter, we report
that bulky or rigid functional groups at the C4-position, in the ab-
sence of a C5 group, afford analogs with good selectivity over
MMP-2 while retaining excellent MMP-13 activity. We also report
that the plasma protein binding of these selective inhibitors, as
gauged by the enzyme IC50 shift in the presence of plasma, can
be significantly reduced by modifying the amino acid or biphenyl
portions of the inhibitor.
q
Portions of this work were presented at 7th World Congress of Inflammation,
Melbourne, Australia, 2005, Aug. 20.
* Corresponding author. Tel.: +1 617 665 5621; fax: +1 617665 5682.
E-mail address: fhu@wyeth.com (Y. Hu).
Present address: Drug design and optimization, Merck Research Laboratories, 33
Avenue Louis Pasteur, Boston, MA 02115, USA.
The synthesis of biphenylsulfonamide analogs disubstituted at
C3 and C4 of the benzofuran is shown in Schemes 1–3. Carboxylic
esters 3 were synthesized via nitro reduction of compound 1 to
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.008

W. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4546–4550
4547
2
The synthesis of compounds with a pyridin-2-ylphenylsulfona-
mide backbone is shown in Scheme 2. The valine or O-tert-butyl
threonine methyl esters 5 were coupled to 4-bromobenzenesulfo-
nyl chloride to give compounds 6 in good yield. Palladium cata-
lyzed reaction of 6 with bis(pinacolato)diborane then afforded
boronic ester 7 which next underwent Suzuki coupling with 2-bro-
mo-5-nitropyridine to give pyridin-2-ylphenylsulfonamides 8.
Reduction of the nitro group and subsequent amide coupling was
followed by ester hydrolysis to give final compounds 11.
The synthesis routes to the requisite benzofuran-2-carboxylic
acids used in Schemes 1 and 2 are shown in Scheme 3. Thus, 4-hy-
droxy-3-methylbenzofuran-2-carboxylate (12) was treated with
triflic anhydride to give 13 which was then used for installing a
variety of functionalities at the C4 position of the benzofuran in
compounds 15–20. Alkylation of 12 with an alkyl halide gave com-
pound 14. Suzuki coupling of 13 with a boronic acid or boronic
1
3
R
O
HN S
O
R
3
4
R
5
O
NH
O
O
HO
MMP-13
IC50 (nM)
MMP-2
IC50 (nM)
MMP-2/
MMP-13
R1
H
R2
H
R3
H
I
2.4
5.0
2
II
Me
OMe
Br
2.3
1700
739
Figure 1. Biphenylsulfonamide carboxylate MMP-13 inhibitors.
give aniline 2, followed by amide coupling with the desired benzo-
furan-2-carboxylic acid. Saponification of 3 with lithium hydroxide
gave the biphenylsulfonamide carboxylates 4 without racemiza-
tion of the valine chiral center (Scheme 1).
O
O
O O
S
NH2
O O
S
NO2
NH
NH
a
O
O
2
1
R
O
R
HO
O
O O
S
NH
O
O
NH
O
O
b
3
R
O
S
O O
NH
NH
HO
O
O
c
4
Scheme 1. Reagents and conditions: (a) H2, Pd/C, 50 psi, 5 h (95%); (b) BOP, DIEA, DMF, 18 h (40–80%); (c) 1 N LiOH/THF/MeOH (1:3:1), 18 h (40–70%).
O
O
O
O
O
O O
S
Br
O
O S
Cl
O
B
B
Br
NH
.HCl
NH2
R1
O
O
O
a
b
R1
5
6
O
O
S
O
N
O O
S
B
O O
NO2
N
Br
NO2
O
NH
NH
O
c
R1
R1
8
7
R2
O
N
HO
O O
S
NH2
O
O
NH
e
O
d
R1
9
R2
O
N
O O
S
NH
NH
f
O
O
O
R1
10
R2
O
S
N
O O
NH
O
NH
HO
O
R3
11
Scheme 2. Reagents and conditions: R1 = CH3 or OtBu, R3 = CH3 or OH (a) DIEA, DCM, 0 °C–rt, 1 h (88–95%); (b) PdCl2(dppf)ꢀCH2Cl2, KOAc, DMSO, 80 °C, 18 h (69–77%); (c)
Pd(PPh3)4, K2CO3, DME, H2O, 90 °C, 3 h (61–81%); (d) H2, Pd/C, THF, 2d (50–90%); (e) BOP, DIEA, DMF, 18 h (57–65%); (f) when R1 = OtBu; (i) TFA, DCM, 3 h (100%); (ii) 1 N
LiOH/THF/MeOH (1:3:1), 18 h (40–70%). When R1 = Me, 1 N LiOH/THF/MeOH (1:3:1), 18 h (40–70%).

4548
W. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4546–4550
O
N
OH
N
tBu
O R
O
O
O
Et
O
O
O
O
O
12
16
17
a
e
d
b
R2
R1
OTf
O
O
O
R
O
O
tBu
O
Et
f
O
O
O
O
R = tBu or Et
14
13
18
O
R4
O
O
S
R3
c
NH2
g
h
H2N
O
O
R4
O
S
Ar
R3
NH
NH
O
tBu
O
Et
O
O
Et
O
O
O
O
O
15
19
20
Scheme 3. Reagents and conditions: (a) Tf2O, DIEA, DCM, ꢂ10 °C, 2 h (95–100%); (b) R1–X, K2CO3, DMF, 12 h (70–90%); (c) ArB(OH)2, Pd(PPh3)4, DME, satd NaHCO3, 85 °C,
18 h (58–75%); when Ar = vinyl, vinyltributyltin, Pd(PPh3)4, LiCl, DMF, 90 °C, 3 h (75%); (d) Zn(CN)2, Pd(PPh3)4, DMF, 90 °C, 18 h (86%); (e) morpholine, K3PO4, Pd(OAc)2,
dioxane, 85 °C, 18 h (63%); (f) for reaction with R2CHCH:PdCl2(PPh3)2, Et3N, DMF, 90 °C (38–52%); for reaction with CH3CHCHSnBu3:Pd(PPh3)4, toluene, reflux, 18 h (15%); for
reaction with cyclopropylCCHSi(CH3)3:PdCl2(PPh3)2, Et3N, TBAF, 70 °C, 24 h (7%); (g) Pd2(dba)3, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, Cs2CO3, dioxane, reflux,
3d (90–100%); (h) Pd2(dba)3, biphenyl-2-yl-di-tert-butyl-phosphane, K3PO4, toluene, reflux, 3 h (22–67%).
Table 2
ester provided 15. Coupling of triflate 13 with Zn(CN)2 or alkynes
Selectivity of 3,4-disubstituted benzofurans: C-linker
afforded compounds 16 and 18, respectively. Palladium catalyzed
1
amination of 13 gave morpholine 17. Finally, Buchwald coupling
was used to prepare amides 19 or sulfonamides 20.
R
O
HN S
O
O
NH
O
The MMP-13 activity, and selectivity over MMP-2, for biphenyl-
sulfonamide carboxylates with various functionality at the C4 po-
sition of the benzofuran moiety is shown in Tables 1 and 2.
Compounds 21–40 are all very potent inhibitors of MMP-13. As ex-
pected, various functional groups at C4 of the benzofuran are toler-
ated since the relatively spacious S10 pocket of MMP-13 can
accommodate a large group at this position, although the largest
substituents do diminish MMP-13 activity (e.g., compounds 24,
25, 29, 30 and 31). Importantly, analogs 21–40 are from 50 to
O
HO
IC (nM)
50
Entry
R1
MMP-13
MMP-2
MMP-2/MMP-13
32
33
34
35
36
37
38
39
40
Me
Et
4.8
2.3
7.1
1150
1410
4410
ꢁ4000
581
287
613
621
364
157
98
1579
243
322
(CH2)3OH
(CH2)3OCH3
CN
11
3.7
6.1
9.5
15
6.4
CCH
CCCH3
Ph
595
15,000
3650
2060
3-Furyl
Table 1
Selectivity of 3,4-disubstituted benzofurans: O- & N-linkers
1
R
O
HN S
O
greater than 1000-fold selective for MMP-13 over MMP-2, presum-
ably due to a clash of the C4 moiety with Thr229 in the smaller S10
pocket of MMP-2.
O
NH
O
O
HO
IC (nM)
50
When compared to tri-substituted benzofuran II, compound 22
has lower selectivity over MMP-2 (110-fold vs 739-fold for II) be-
cause it lacks a C5 substituent to constrain the mobility of the
methoxy group. The smaller C4 hydroxy analog, 21, is almost two-
fold less selective than 22, while the larger ethoxy derivative 23 is
almost 500-fold selective for MMP-13 over MMP-2, even without
any C5 substituent. Increasing steric bulk at C4 further in the ether
series, as in 24 (248-fold selective) and phenoxy derivative 25
(263-fold selective) does not provide additional improvement in
selectivity. The same trend is observed for the C4 amine derivatives
26–31. The N,N-dimethyl group (28) is over 750-fold selective but
the primary amine and mono-substituted amine analogs are
Entry
R1
MMP-13
MMP-2
MMP-2/MMP-13
21
22
23
24
25
26
27
28
29
30
31
OH
OMe
OEt
OCMe2COOMe
OPh
NH2
NHCH3
N(CH3)2
NHCOCH3
NHSO2CH3
N-Morpholine
6.1
9.5
7.4
25
379
63
110
485
248
263
75
108
786
152
165
51
1050
3590
6200
ꢁ5000
323
19
4.3
8.0
6.5
25
13
32
886
5110
3810
2150
1630

W. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4546–4550
4549
Table 3
MMP-13 and MMP-2 IC50 with and/or without addition of rabbit serum albumin
R1
O
X
O O
S
NH
O
NH
HO
O
Y
Entry
X
Y
R1
cLog P
MMP-13 (nM)
MMP-2 (nM)
MMP-2/MMP-13
(+RSA) MMP-13a (nM)
(+RSA)/(-RSA) MMP-13b
36
30
41
42
43
44
CH
CH
N
N
CH
N
Me
Me
Me
Me
OH
OH
CN
NHSO2Me
CN
NHSO2Me
NHSO2Me
NHSO2Me
5.1
4.3
4.6
4.0
2.5
2.2
4.0
13
14
40
13
581
2150
2840
7500
2470
8990
145
165
203
188
190
257
13,100
9040
5250
8950
406
3257
695
375
224
31
35
307
9
a
MMP-13 activity assayed in the presence of 25 mg/mL rabbit serum albumin (RSA).
Fold difference with/without RSA.
b
considerably less selective. Morpholine analog 31 is the least selec-
tive C4-heteroatom linked analog, presumably due to constraint of
the amine substituents into a ring.
show significant IC50 shifts (695 and 3275-fold, respectively),
indicative of high serum binding for these two compounds (Table
3). As it has been reported that introducing heteroatoms into drug
molecules can reduce plasma binding,12,13 we then explored mod-
ifying the biphenyl backbone and the amino acid of the inhibitor to
decrease cLog P and reduce protein binding. A pyridine ring was
therefore introduced to form compounds 41, 42, and 44 by replac-
ing one benzene ring of the biphenylsulfonamide carboxylate
inhibitors and a threonine was used in compounds 43 and 44 to re-
place the valine moiety. The introduction of a pyridyl nitrogen (41,
42, and 43) appears to reduce MMP-13 activity by threefold (c.f. 36,
30 and 43, respectively). By comparing the MMP-13 IC50 shift in
the presence of RSA for two pairs of compounds (36:41, 30:42),
it was found that the benzene to pyridine switch reduced the
IC50 shift significantly, up to 10-fold in the case of the 36:41 pair.
A more dramatic change, over 20-fold, was apparent for the threo-
nine-based inhibitors as compared to the corresponding valine
analogs (30:43, 42:44). In the case of compound 44, where both
the pyridine and threonine were introduced, the MMP-13 IC50 shift
is only ninefold. The lower lipophilicity (cLog P = 2.2) of 44 is likely
one of the factors involved in the observed lowering of plasma pro-
tein binding.
The selectivity of analogs bearing carbon-linked substituents at
the benzofuran C4-position is shown in Table 2. A C4-methyl group
(32) gave selectivity of 287-fold. The larger ethyl group (33) gave
higher selectivity (613-fold) than methyl derivative 32 or the isos-
teric C4 methoxy compound 22. Further lengthening of the C4-
group, as for compounds 34 and 35 did not enhance selectivity rel-
ative to 33. Small linear functionality such as a nitrile (36) or ter-
minal alkyne (37) provides only moderate MMP-2 selectivity.
Interestingly, the propynyl derivative 38 afforded the best selectiv-
ity of any of the analogs prepared, 1579-fold, possibly because of
the steric clash of this longer propynyl group with MMP-2 than ni-
trile and terminal alkyne. Aromatic groups at C4 (39 and 40) also
retained MMP-13 activity and had good selectivity over MMP-2.
In addition to excellent enzyme activity, in order for MMP-13
inhibitors to modulate cartilage degradation they need to distrib-
ute to the synovial fluid of the arthritic joint and then diffuse into
the cartilage. Plasma protein binding limits free drug concentration
and is expected to hamper drug penetration into the final target
tissue. Unfortunately, many of the biphenylsulfonamide carboxyl-
ates in Tables 1 and 2 display high plasma protein binding. Also,
both compounds I and II were >98% protein bound in an equilib-
rium dialysis assay.10 Plasma protein binding has been assessed
by measuring in vitro enzymatic and cellular IC50 in the presence
and absence of serum protein.11 The shift in potency due to the
addition of serum proteins can be used as a rough guidance for
the amount of plasma protein binding. While this method may sac-
rifice some accuracy, it has an operational advantage of being eas-
ily used in very high throughput fashion. This can then be used to
further identify suitable compounds for more detailed study of
plasma protein binding. Therefore, we evaluated the ratio of the
MMP-13 IC50 values of the inhibitor in the presence and absence
of 25 mg/mL of rabbit serum albumin (RSA) as an indication of
plasma protein binding. Rabbit serum albumin was used since
we intended to use a rabbit surgical efficacy model to ultimately
assess compounds in vivo. In this assay, compounds 30 and 36 both
Compounds 30 and 36 were also evaluated for selectivity
against MMP-9, MMP-14, and aggrecanse-1, and their ability to in-
hibit degradation of articular cartilage in vitro (Table 4). Nitrile 36
is more than 145-fold selective over the MMPs screened and is ac-
tive in the cartilage degradation assay down to 0.1 lM. Methyl sul-
fonamide 30 is somewhat more selective, with 200-fold selectivity
over all of the MMPs screened and provides 51% inhibition in the
cartilage degradation assay at 0.01 l
M.14
In summary, we have successfully synthesized a series of potent
MMP-13 inhibitors with excellent selectivity over MMP-2 by intro-
ducing rigid or bulky functional groups at C4 of the benzofuran P10
moiety. Extra size and flexibility of the S10 pocket allow MMP-13 to
accommodate a larger group at the C4-position of the benzofuran
moiety. Since MMP-2 is one of the most homologous enzyme to
MMP-13, compounds with selectivity over MMP-2 might achieve
broader selectivities over other MMPs. These broader selectivities
Table 4
Inhibitory activities of MMPs and degradation of extracellular matrix molecules
IC50 (nM)
Cartilage degradation modela % inhibition
Entry
MMP-1
MMP-2
MMP-9
MMP-14
AGG-1
1
l
M
0.1
lM
0.01 lM
30
36
12,000
24,000
2150
581
>10,000
>10,000
11,200
5450
2200
NT
96%
100%
69%
87%
51%
23%
a
In vitro articular cartilage model for inhibition of degradation of extracellular matrix molecules; NT = not tested.

4550
W. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4546–4550
Schmid, J.; Skotnicki, J. S.; Tam, S.; Thomason, J. R.; Wang, Q.; Levin, J. I. Bioorg.
Med. Chem. 2005, 13, 6629.
are exemplified by selected compounds (30 and 36). Many of these
biphenylsulfonamide carboxylates have high plasma protein bind-
ing that could be attenuated by structural changes that lowered
the cLog P of the inhibitor, providing attractive leads for in vivo ac-
tive compounds. Selected compounds also had good activity in an
in vitro cartilage degradation assay.
7. (a) Natchus, M. G.; Bookland, R. C.; Laufersweiler, M. J.; Pikul, S.; Almstead, N.
G.; De, B.; Janusz, M. J.; Hsieh, L. C.; Gu, F.; Pokross, M. E.; Patel, V. S.; Garver, S.
M.; Peng, S. X.; Branch, T. M.; King, S. L.; Baker, T. R.; Foltz, D. J.; Mieling, G. E. J.
Med. Chem. 2001, 44, 1060; (b) Pikul, S.; Phler, N. E.; Ciszewski, G.;
Laufersweiler, M. C.; Almstead, N. G.; De, B.; Natchus, M. G.; Hsieh, L. C.;
Janusz, M. J.; Peng, S. X.; Branch, T. M.; King, S. L.; Taiwo, Y. O.; Mieling, G. E. J.
Med. Chem. 2001, 44, 2499; (c) Tullis, J. S.; Laufersweiler, M. J.; VanRens, J. C.;
Natchus, M. G.; Bookland, R. G.; Almstead, N. G.; Pikul, S.; De, B.; Hsieh, L. C.;
Janusz, M. J.; Branch, T. M.; Peng, S. X.; Jin, Y. Y.; Hudlicky, T.; Oppong, K. Bioorg.
Med. Chem. Lett. 2001, 11, 1975.
Acknowledgments
8. O’Brien, P. M.; Ortwine, D. F.; Pavlovsky, A. G.; Picard, J. A.; Sliskovic, D. R.; Roth,
B. D.; Dyer, R. D.; Johnson, L. L.; Man, C. F.; Hallak, H. J. Med. Chem. 2000, 43, 156.
9. (a) Tamura, Y.; Watanabe, F.; Nakatani, T.; Yasui, K.; Fuji, M.; Komurasaki, T.;
Tsuzuki, H.; Maekawa, R.; Yoshioka, T.; Kawada, K.; Sugita, K.; Ohtani, M. J. Med.
Chem. 1998, 41, 640; (b) Kiyama, R.; Tamura, Y.; Watanabe, F.; Tsuzuki, H.;
Ohtani, M.; Yodo, M. J. Med. Chem. 1999, 42, 1723.
The authors thank Dr. Tarek Mansour for his generous help in
the MMP-13 program, Dr. Joe Zaccardi for equilibrium dialysis
analysis, Dr. Nelson Huang and Ms. Ning Pan for LC-MS measure-
ments, Dr. Walter Massefski for NMR measurements, Ms. Xiufen
Yang for cartilage degradation assays, Dr. Catherine Fiorilla for
MMP-1 and MMP-14 assays of compound 30, Ms. Shihua Yao for
MMP-2 assay of compound 30, Mr. James Larocque for MMP-9 as-
say of compound 30 and 36, and Ms. Erica Reifenberg for AGG-1 as-
say of compound 30.
10. Percent bound in rabbit serum albumin using equilibrium dialysis assay: I:
98.5%; II: >99%.
11. (a) Rowley, M.; Kulagowski, J. J.; Watt, A. P.; Rathbone, D.; Stevenson, G. I.;
Carling, R. W.; Baker, R.; Marshall, G. R.; Kemp, J. A.; Foster, A. C.; Grimwood, S.;
Hargreaves, R.; Hurley, C.; Saywell, K. L.; Tricklebank, M. D.; Leeson, P. D. J. Med.
Chem. 1997, 40, 4053; (b) Rusnak, D. W.; Lai, Z.; Lansing, T. J.; Rhodes, N.;
Gilmer, T. M.; Copeland, R. A. Bioorg. Med. Chem. Lett. 2004, 14, 2309; (c) Nugiel,
D. A.; Vidwans, A.; Etzkorn, A.-M.; Rossi, K. A.; Benfield, P. A.; Burton, C. R.; Cox,
S.; Doleniak, D.; Seitz, S. P. J. Med. Chem. 2002, 45, 5224.
References and notes
12. For
a reference using hydroxyls to reduce plasma protein binding, see:
1. Skotnicki, J. S.; Levin, J. I.; Zask, A.; Killar, L. M. Matrix Metalloproteinase
Inhibitors. In Metalloproteinases as Target for Anti-inflammatory Drugs;
Bottomley, K. M. K., Bradshaw, D., Nixon, J. S., Eds.; Birkhauser: Verlag Basel/
Switzerland, 1999; pp 17–57.
2. Mitchell, P. G.; Magna, H. A.; Reeves, L. M.; Lopresti-Morrow, L. L.; Yocum, S. A.;
Rosner, P. J.; Geoghegan, K. F.; Hambor, J. E. J. Clin. Invest. 1996, 97, 761.
3. (a) Skotnicki, J. S.; DiGrandi, M. J.; Levin, J. I. Curr. Opin. Drug Disc. Dev. 2003, 6,
742; (b) Rao, B. G. Curr. Pharm. Des. 2005, 11, 295; (c) Vihinen, P.; Ala-aho, R.;
Kahari, V.-M. Curr. Cancer Drug Target 2005, 5, 203; (d) Hu, J.; Van den Steen, P.
E.; Sang, Q.-X. A.; Opdenakker, G. Nat. Rev. Drug Disc. 2007, 6, 480.
4. (a) Peterson, J. T. Cardiovasc. Res. 2006, 69, 677; (b) Peterson, J. T. Heart Failure
Rev. 2004, 9, 63; (c) Renkiewicz, R.; Qiu, L.; Lesch, C.; Sun, X.; Devalaraja, R.;
Cody, T.; Kaldjian, E.; Welgus, H.; Baragi, V. Arthritis Rheumat. 2003, 48, 1742.
5. For an example of a selective MMP-13 inhibitor that does not cause fibroplasia
in a rat model, see: (a) Johnson, A. R.; Pavlovsky, A. G.; Ortwine, D. F.; Prior, F.;
Man, C.-F.; Bornemeier, D. A.; Banotai, C. A.; Mueller, W. T.; McConnell, P.; Yan,
C.; Baragi, V.; Lesch, C.; Roark, W. H.; Wilson, M.; Datta, K.; Guzman, R.; Han, H.-
K.; Dyer, R. D. J. Biol. Chem. 2007, 282, 27781; (b) Reiter, L. A.; Freeman-Cook, K.
D.; Jones, C. S.; Martinelli, G. J.; Antipas, A. S.; Berliner, M. A.; Datta, K.; Downs, J.
T.; Eskra, J. D.; Forman, M. D.; Greer, E. M.; Guzman, R.; Hardink, J. R.; Janat, F.;
Keene, N. F.; Laird, E. R.; Liras, J. L.; Lopresti-Morrow, L. L.; Mitchell, P. G.;
Pandit, J.; Robertson, D.; Sperger, D.; Vaughn-Bowser, Ml. L.; Waller, D. M.;
Yocum, S. A. Bioorg. Med. Chem. Lett. 2006, 16, 5822.
6. (a) Wu, J.; Rush, T. S.; Hotchandani, R.; Du, X.; Geck, M.; Collins, E.; Xu, Z.-B.;
Skotnicki, J.; Levin, J. I.; Lovering, F. L. Bioorg. Med. Chem. Lett. 2005, 15, 4105;
(b) Li, J.; Rush, T. S.; Li, W.; DeVincentis, D.; Du, X.; Hu, Y.; Thomason, J. R.;
Xiang, J. S.; Skotnicki, J. S.; Tam, S.; Cunningham, K. M.; Chockalingham, P. S.;
Morris, E. A.; Levin, J. I. Bioorg. Med. Chem. Lett. 2005, 15, 4961; (c) Hu, Y.; Xiang,
J. S.; DiGrandi, M. J.; Du, X.; Ipek, M.; Laakso, L. M.; Li, J.; Li, W.; Rush, T. S.;
Zartman, A. E.; Duong, L. T.; Fernandez-Metzler, C.; Hartman, G. D.; Leu, C.-T.;
Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan, M. E.; Meissner, R. S.
Bioorg. Med. Chem. Lett. 2005, 15, 1647.
13. For a reference using nitrogen to reduce plasma protein binding, see: Stauffer,
K. J.; Williams, P. D.; Selnick, H. G.; Nantermet, P. G.; Newton, C. L.; Homnick, C.
F.; Zrada, M. M.; Lewis, S. D.; Lucas, B. J.; Krueger, J. A.; Pietrak, B. L.; Lyle, E. A.;
Singh, R.; Miller-Stein, C.; White, R. B.; Wong, B.; Wallace, A. A.; Sitko, G. R.;
Cook, J. J.; Holahan, M. A.; Stranieri-Michener, M.; Leonard, Y. M.; Lynch, J. J.;
McMaster, D. R.; Yan, Y. J. Med. Chem. 2005, 48, 2282.
14. Bovine articular cartilage explants were harvested from carpal joints up to 4 h
post mortem. The explants were cultured in Dulbecco’s modified medium
(DMEM) (JRH Biosciences, Lenexa, KS) containing 50
(Wako, Osaka, Japan), 10 mM HEPES, pH 7.0 (Mediatech, Herndon, VA), 2 mM
-glutamine (Mediatech), 100 U/mL antibiotic-antimycotic solution
lg/mL ascorbic acid
L
(Mediatech) in the presence or absence of 5 ng IL-1/mL (SIGMA, St. Louis,
MO) to induce synthesis and activation of proteolytic enzymes including MMP-
13. Various concentrations of small molecule inhibitors were added to the
culture wells. Culture medium was collected after 3, 7, 10, 14 and 17 days in
culture and replaced with identical cytokine and inhibitor concentrations.
After day 17, cartilage explants were collected and total collagen content in
media and remaining in the explant were quantitated by biochemical analysis
of hydroxyproline content using
a modification of previously described
methods.1,2 Percent inhibition of collagen degradation by a small molecule
inhibitor was calculated as percent reduction in collagen release over the
17 day experiment compared to collagen release with IL-1 alone and no
inhibitor. For references, see (a) Bergman, I. And Loxley, R. Two improved and
simplified methods for the spectrophotometric determination of
hydroxyproline. Anal. Chem. 1963, 35, 1961. (b) Brown, S. W.; Worsfold, M.;
Sharp, C. Bio Techniques 2001, 30, 38.