J. S. Xiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 311–316
315
5. For a review of MMP inhibitors, see: (a) Skiles, J. W.;
Gonnella, N. C.; Jeng, A. Y. Curr. Med. Chem. 2004, 11,
2911; (b) Whittaker, M.; Floyd, C. D.; Brown, P.;
Gearing, A. J. H. Chem. Rev. 1999, 99, 2735; (c) Zask,
A.; Levin, J. I.; Killar, L. M.; Skotnicki, J. S. Curr. Pharm.
Design 1996, 2, 624.
boxylic acids as aggrecanase-1 inhibitors. The design
was based on HTS results and a homology model de-
rived from Atrolysin C. This limited SAR study suggest-
ed that biphenylsulfonamide carboxylic acids provided a
good starting point and the benzofuran moiety can im-
prove agg-1 inhibition. Compounds such as 24 show
good oral bioavailability and inhibition of proteoglycan
degradation in a cell-based assay. Compound 24 also
shows inhibitory activity against MMP-13. Detailed
study of this dual Agg-1/MMP-13 inhibition will be dis-
closed in due course.
6. MacPherson, L. J.; Bayburt, E. K.; Capparelli, M. P.;
Carroll, B. J.; Goldstein, R.; Justice, M. R.; Zhu, L.; Hu,
S.-I.; Melton, R. A.; Fryer, L.; Goldberg, R. L.; Doughty,
J. R.; Spirito, S.; Blancuzzi, V.; Wilson, D.; OÕByrne, E.
M.; Ganu, V.; Parker, D. T. J. Med. Chem. 1997, 40, 2525.
7. Compounds are assessed by their ability to inhibit
cleavage of a fluorescent peptide substrate (Abz-TEG-
ARGSVI-Dap(Dnp)) (Abz, o-aminobenzoyl; Dnp, 2,4
dinitrophenyl) (Anaspec Inc.). The peptide sequence
TEGARGSVI is based on the amino acid sequence of
the Glu373-Ala374 cleavage site of aggrecan in osteoar-
thritis. Inhibitors are pre-incubated with purified full-
length human recombinant agg-1 for 10 min followed by
the addition of substrate, at temperatures ranging from 25
to 37 ꢁC, typically at 30 ꢁC. Cleavage of the Glu-Ala bond
releases the fluorophore from internal quenching. This
results in an increase in fluorescence monitored at kex
340 nm and kex 420 nm over a period of 40 min. The initial
rate (v) at each concentration of the substrate is fit to the
following equation V ¼ V max ꢂ Sh=ðS0h:5 þ ShÞ, where h is
the Hill constant and S0.5 is the substrate concentration at
half the Vmax. The percentage activity remaining in the
presence of inhibitor is plotted as a function of inhibitor
concentration and the IC50 value is determined by fitting
the data to the following equation: % activity = 100 IC50/
(Io + IC50).
Acknowledgments
The authors thank Dr. Tarek Mansour for helpful dis-
cussions, Dr. Junjun Wu for the synthesis of com-
pound 9, Dr. Nelson Huang and Ms. Ning Pan for
LC–MS measurement, Dr. Walter Massefski for
NMR measurements, Dr. Qin Wang for PK analysis,
Dr. Sonya Glasson for cartilage penetration studies,
and Drs. Neal Green and Katherine Lee for a critical
review of the manuscript.
References and notes
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C. Science 1999, 284, 1664.
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Liu, R.-Q.; Copeland, R. A.; Magolda, R.; Newton, R. C.;
Trzaskos, J. M.; Arner, E. C. J. Biol. Chem. 2003, 278,
45539.
3. Dual MMP-13 and aggrecanase-1 inhibitors: (a) Noe, M.
C.; Natarajan, V.; Snow, S. L.; Wolf-Gouveia, L. A.;
Mitchell, P. G.; Lopresti-Morrow, L.; Reeves, L. M.;
Yocum, S. A.; Otterness, I.; Bliven, M. A.; Carty, T. J.;
Barberia, J. T.; Sweeney, F. J.; Liras, J. L.; Vaughn, M.
Bioorg. Med. Chem. 2005, 15, 3385; (b) Noe, M. C.;
Natarajan, V.; Snow, S. L.; Mitchell, P. G.; Lopresti-
Morrow, L.; Reeves, L. M.; Yocum, S. A.; Carty, T. J.;
Barberia, J. A.; Sweeney, F. J.; Liras, J. L.; Vaughn, M.;
Hardink, J. R.; Hawkins, J. M.; Tokar, C. Bioorg. Med.
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A.; Tortorella, M.; Magolda, R. L.; Newton, R.; Qian,
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9. The most significant primary sequence differences around
the active site were in the loop that defines the S20 area
(bC–bD loop; 4 residue insertion for agg-1), and in the
loop that defines the shape of the deep end of the S10
pocket (the metalloprotease ÔspecificityÕ loop; 5 residue
insertion for agg-1). Nevertheless, most residues close to
the active site Zn were conserved and thus we felt
confident in using this model for atom-based simulations.
1
10. All final compounds were characterized by H NMR and
either HRMS, LC/MS or CHN.
11. Compound 19 has an IC50 of 86 nM against aggrecanase-
1, as compared to single digit nM activity of hydroxamate
aggrecanase-1 inhibitors reported.2–4
12. Bovine articular cartilage was cultured in the absence and
presence of rabbit serum albumin for 3 days. Conditioned
media were collected and cartilage was stored at ꢁ20 ꢁC.
Cartilage was milled in the presence of liquid nitrogen.
Twenty-five milligram of wet control cartilage was
extracted with 1.5-mL acetonitrile twice, dried under
nitrogen, and reconstituted in 100 lL acetonitrile. Carti-
lage standards (0.1, 0.5, 5, 10, 50, 100, 500, and 1000 ng/
mL) and quality control samples (0.5, 50, and 500 ng/mL)
were prepared by adding appropriate amounts of M-369
stock solution to the control samples. M-048 was used as
the internal standard (IS) and was added to each cartilage
sample or standard prior to the extraction. HPLC was
performed on a Perkin Elmer Series 200 HPLC system
(Perkin Elmer, Norwalk, CT) using XTerra MS C18,
2.1 · 20 mm, 2.5 mm (Waters, Milford, MA). The mobile
phase was: solvent A = 0.1% HCOOH in H2O and solvent
B = 0.1% HCOOH in acetonitrile. The elution gradient
was isocratic at 0% B for 1 min, followed by a linear
gradient to 100% B over 4 min for the quantitative
analysis and over 14 min for the identification of the
metabolites. The column was allowed to equilibrate at 0%