Synthesis and biological evaluation of biphenylsulfonamide carboxylate aggrecanase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.10.001

Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenyl

Full text of DOI:10.1016/j.bmcl.2005.10.001

Bioorganic & Medicinal Chemistry Letters 16 (2006) 311–316
Synthesis and biological evaluation
of biphenylsulfonamide carboxylate aggrecanase-1 inhibitors
Jason S. Xiang,a Yonghan Hu,a,* Thomas S. Rush,a Jennifer R. Thomason,a Manus Ipek,a
Phaik-Eng Sum,b Leila Abrous,b Joshua J. Sabatini,b Katy Georgiadis,c Erica Reifenberg,c
Manas Majumdar,c Elisabeth A. Morrisc and Steve Tama
aDepartment of Chemical and Screening Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
bDepartment of Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
cDepartment of WomenÕs Health and Bone, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
Received 24 August 2005; revised 29 September 2005; accepted 3 October 2005
Available online 3 November 2005
Abstract—Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors
may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of
biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of pro-
teoglycan degradation at 10 lg/mL and has an oral bioavailability in rat of 35%.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Osteoarthritis (OA) is a debilitating disease resulting
from the breakdown of articular cartilage and character-
ized by chronic joint pain and inflammation, which re-
sults in significant reduction in the quality of life.
Currently there is no therapy available to halt the pro-
gression of this disease.
fied enzyme. Inhibition of agg-1 may impart overall car-
tilage protection and offer a potential therapy that could
alter the progression of OA.2
Several reports in the literature described aggrecanase
inhibitors that contain hydroxamic acid zinc-chelating
groups.3,4 Modification around hydroxamic acid func-
tional group by introducing polar functionality was
reported to have a favorable effect on absorption and
clearance through steric hindrance and intramolecular
hydrogen bonding.3a The use of other zinc-chelating
groups, such as the carboxylic acid, has become a pop-
ular approach for hydroxamic acid replacement in other
matrix metalloproteinase programs.5
Aggrecan, a multidomain proteoglycan, is a major com-
ponent of cartilage and provides compressive resistance
to articular cartilage. During the early stages of osteoar-
thritis, and then throughout the disease, there is in-
creased loss of GAG (glycosaminoglycan)-rich
aggrecan fragments via proteolysis attributable to Ôagg-
recanaseÕ activity. Eventually, the cartilage is eroded
and replacement joint surgery is usually required.
In our research efforts toward agg-1 inhibitors, we
conducted high throughput screening of our corporate
library and discovered that the hydroxamate com-
pound 1a, reported by Novartis as a non-selective
stromelysin inhibitor (CGS 27023A),6 showed 92%
inhibition of agg-1 at a concentration of 25 lM.7 To
our surprise, 1b (the S-isomer of 1a) was inactive to-
ward agg-1 (Table 1).
Aggrecanase-1 (agg-1)1 is a member of ADAMTS (A
Disintegrin and Metalloprotease possessing Thrombo-
spondin domain) family of zinc containing metallopro-
teases, responsible for the cleavage of aggrecan IGD
(Interglobular domain) at the Glu373-Ala374 peptide
bond, a unique site untouched by any previously identi-
This enantiomeric preference for an R-configuration was
examined using an agg-1 homology model derived from
the venom metalloproteinase, Atrolysin C (PDB code:
1DTH).8 The catalytic domains of the two proteins
Keywords: Aggrecanase-1 inhibitor; Biphenylsulfonamide carboxylate;
Benzofuran; Osteoarthritis.
*
Corresponding author. Tel.: +1 617 665 5621; fax: +1 617 665
5682; e-mail: fhu@wyeth.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.001

312
J. S. Xiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 311–316
Table 1. High throughput screening hit
(3) entropic gains from the hydrophobic effect, as this
area is encapsulated by hydrophobic residues (e.g.,
Leu330, Met395, and Phe357).
N
O
OMe
HO
N
*
N
S
Compound 2 was synthesized and showed an agg-1 IC50
of 3.0 lM (Table 2).10 Biophysical studies confirmed
that 2 binds to agg-1 in 1:1 stoichiometry. A compound
with a bent biphenyl configuration (3) was inactive pre-
sumably because it does not fit into the S10 narrow
hydrophobic channel. Attempts to generate a hydrogen
bond between the enzyme and the inhibitor from the
biphenyl backbone (4–7) were not successful. It was
thought that the substituents may either disrupt the flat-
tened cylindrical shape of the molecule (4–6) or were too
congested to fit into the narrow S10 pocket (4–7), leading
to reduced activity against agg-1. A para hydroxy group
(8), which retained its flattened cylindrical shape, re-
tained activity. Therefore, this para-position provided
a good attachment point for further modifications.
H
O
O
Compound
Enantiomer
Agg-1 inhibition at 25 lM
1a
1b
R
S
92%
Not active
share ꢀ43% homology.9 The homology model was built
in Sybyl 6.7 (Tripos, Inc.) using the Composer module
for Homology Modeling. Docking of compound 1a to
the homology model placed the hydroxamic acid moiety
around the Zn, the sulfonyl group in H-bonding dis-
tance to backbone NHs of Leu330 and Gly331, the
methoxy phenyl within the S10 pocket, and the pyridine
substituent facing the solvent near the S20 area of the ac-
tive site. Only the R-enantiomer 1a can dock in this
manner, as the comparable conformation of the S-enan-
tiomer 1b would clash with Met332 and other residues
within the S20 area of the active site (the bC–bD loop)
(Fig. 1).
Another point of modification was the amino acid
headpiece. A variety of amino acids have been ex-
plored (Table 3). Compound 9 with an L-valine head-
piece had an IC50 > 100 lM toward agg-1, further
confirming the enantiomeric preference of this enzyme.
Agg-1 IC50 of the homologated acid 10 was ꢀ100 lM.
The extra carbon atom increases the spacing between
the carboxylic acid and the P10 group such that they
cannot simultaneously achieve the required interac-
tions. Most other modifications (11–14) were tolerat-
ed, suggesting that these side chains might reside in
a spacious domain. As suggested by the homology
model, there is limited space for N-substitution, and
indeed, a small N-alkyl substituent (15) yielded good
The binding model of compound 1a led us to explore
carboxylic acid derivatives with the same enantiomeric
preference. Structure-based explorations using the
homology model suggested that a biphenyl P10 ligand
(2) might be best because of the potential for: (1) enthal-
pic p-stacking interactions with the active site His
(His361), (2) enthalpic van der Waals interactions due
to the shape of the S10 pocket (flattened cylinder), and
Table 2. Modifications on biphenyl backbone
O
H
R
N
HO
S
O
O
Compound
R
Agg-1 IC50 (lM)
2
3.0
3
4
5
6
>100
ꢀ30
ꢀ50
ꢀ20
NH2
O2N
HO
OH
7
8
9.0
4.6
Figure 1. A close-up and schematic view of the predicted binding
mode of compound 1a to agg-1 homology model derived from
Atrolysin C.
OH

J. S. Xiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 311–316
313
Table 3. Modification of amino acid headpieces
Table 4. Linker modification
R
R
N
O
H
R'
S
N
O
O
HO
S
O
O
Compound
R-N-R0
Agg-1 IC50 (lM)
Compound
R
Agg-1 IC50 (lM)
0.35
O
O
O
O
O
NH
NH
2
3.0
HO
17
O
O
O
18
19
20
21
22
23
24
11.6
9
10
11
12
13
>100
ꢀ100
5.5
HO
O
O
0.086
7.5
HO
O
NH
O
NH
O
O
O
O
HO
O
1.4
O
HO
1.1
O
3.8
O
O
OH
O
4.3
HO
0.4
O
Ph
O
O
HO
0.7
H
14
15
16
2.8
1.6
12
N
O
O
The pharmacokinetics of compound 24 were examined
via two different routes of administration, intravenous
(iv) and oral (po), to male Sprague–Dawley rats. Ani-
mals received a single iv bolus of 2 mpk and a po dose
of 10 mpk. The compound exhibited low clearance
(16.1 mL/min/kg). The oral bioavailability was 35%
(T1/2 = 277 min, Cmax = 1360 ng/mL).
N
N
HO
O
N
HO
Compound 24 was tested for its ability to inhibit MMP-
1, MMP-2, MMP-13, and MMP-14. This compound is
selective against MMP-1 and -14, but is a more potent
inhibitor of MMP-2 and MMP-13 (Table 5). Since
MMP-13 plays an important role in cartilage degrada-
tion, inhibition of MMP-13 is also beneficial to prevent-
ing osteoarthritis.2,14
activity, while a bigger group (16) led to diminished
activity. For simplicity, we kept the D-valine headpiece
for further modifications.
Since the homology between agg-1 and Atrolysin C is
much lower in the depths of the S10 pocket, our model
was less useful in predicting substitutions beyond the
biphenyl P10 group. Table 4 shows the SAR on further
modification of the para-position of the biphenyl back-
bone. A benzofuran ester P10 ligand (17) improved
agg-1 activity by 10-fold. The smaller furan analog
(18) resulted in decreased activity. A C3-methyl group
(19) on the benzofuran further improved agg-1 activity
by 4-fold (IC50 = 86 nM). Other linkages intended to im-
prove metabolic stability while mimicking ester binding,
including amide (20), ether (21), and ketone (22), gave
reduced activity. A trans double bond configuration
(23) retained activity; however, this group is prone to
metabolism. The C3-methyl ether 24 is 2-fold more ac-
tive than ether 21.11
Compound 24 demonstrated excellent cartilage penetra-
tion properties.12 In an interleukin-1 stimulated bovine
cartilage explant assay,13 it gave 89% inhibition of pro-
teoglycan inhibition at 10 lg/mL after a 3-day incuba-
tion (Fig. 2). According to early work published by
Pratta2 and Little,14 the aggrecan is degraded early, dur-
ing the first week of culture (day 3–7), whereas the col-
Table 5. Inhibitory activities (IC50, nM) of compound 24
MMP-1
MMP-2
28
MMP-13
4.4
MMP-14
3000
>100,000

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J. S. Xiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 311–316
NO2
Cl
NO2
100.00
80.00
60.00
40.00
20.00
0.00
.HCl
O
O
O
a
OO S
NH
OO S
tBuO
NH2
tBuO
b
NH
tBuO
29
30
31
a
d, e
O
O
4
OO S
NH
Br
OO S
NH
R
c, e
10 g/mL
1
g/mL
0.1 g/mL
tBuO
HO
Figure 2. Proteoglycan inhibition of compound 24.
OH
32
OH
R:
O2N
HO
lagen is not rapidly degraded until later in the culture
period (day 8–14). Therefore, the activity in this 3-day
assay is indicative of aggrecanase inhibition. There is
no significant involvement of MMPs during this early
stage of proteoglycan degradation.2,14
5
6
7
8
Scheme 3. Reagents and conditions: (a) DIEA, DCM, 3 h (91–95%);
(b) PhB(OH)2, Pd(PPh3)2Cl2, CsF, NMP, 100 ꢁC, 18 h (55%); (c) for 5,
boronic acid, Pd(OAc)2, PPh3, Et3N, DMF, 100 ꢁC, 2 h (43%). For 6–
7, boronic acid, Pd(PPh3)4, K2CO3, DME/H2O, 80 ꢁC, 12 h (71–83%);
(d) Pd/C, H2, THF, 12 h (100%); (e) TFA, DCM, 3 h (100%).
The synthesis of compounds 2–24 is shown in Schemes
1–3. Due to the presence of a chiral center sensitive to
racemization, the synthesis of these compounds was car-
ried out in the absence of strong basic media.15
Suzuki coupling was used to construct the second ben-
zene ring of the biphenyl backbone, leading to com-
pounds 31, 5–8 (Scheme 3), 33 and 34 (Scheme 4).
Hydrogenation of 31, followed by hydrolysis, gave 4.
As shown in Scheme 1, sulfonylation of the amino ester
25 gave 26, which upon hydrolysis formed compounds 2
and 9 in excellent yield. Alkylation of the sulfonamide
nitrogen atom and hydrolysis led to compounds 15
and 16. Sulfonamide formation was performed on b-
amino acid 28 using transient silylation condition16 to
give 10. Compounds 11–14 were synthesized from Wang
resin through Fmoc deprotection, sulfonamide forma-
tion, and TFA cleavage, in excellent yields (Scheme 2).
Esterification (17–19) or alkylation (21, 24) of the OH
group on compound 33 led to compounds with P10 ester
or ether linkages. Heck coupling on 34 gave compound
23. 2-Acylbenzofuran 36 was synthesized according to
the literature procedure.17 Stille coupling of tributyltin
compound 35 (synthesized from 29) with 32 gave 22.
In summary, we described herein the design, synthesis,
and biological evaluation of biphenylsulfonamide car-
.HCl
O
NH2
R1
25
O
O
H
b
tBuO
N S R
O
tBuO
2, 9
R1
a
c, h
17-19
26
O
d
OO
a
S
OH
d, h
NH
tBuO
21, 24
R2
b
O
O
33
15, 16
N S R
O
tBuO
R1
27
O
O
HO
NH2
c
OO S
NH
Br
OO S
NH
e, h
a
10
tBuO
tBuO
23
O
28
34
32
Scheme 1. Reagents and conditions: (a) RSO2Cl, DIEA, DCM, 3 h
(93–100%); (b) TFA, DCM, 3 h (95–100%); (c) (i)—Me3SiCl, DCM,
reflux, 5 h; (ii)— Et3N, Ph-PhSO2Cl (33%); (d) R2X, K2CO3, CH3CN,
reflux, 5 h (45–77%).
Si
O
f
b
Br
O
OO S
SnBu3
O
O
NH
36
g, h
tBuO
22
O
H
O
N
a, b
35
FMoc
HN
S
O
O
O
R
Scheme 4. Reagents and conditions: (a) boronic acid, Pd(PPh3)4,
K2CO3, DME/H2O, 80 ꢁC, 12 h (80–82%); (b) (Bu3Sn)2, Pd(PPh3)4,
toluene, reflux, 12 h (56%); (c) carboxylic acid, DCC, DMAP, DCM,
3.5 h (31–71%); (d) benzofuran-2-CH2Br, K2CO3, DMF, 90 ꢁC, 18ꢁh
R
O
(Wang resin)
(35–48%); (e) 2-bromobenzofuran, Pd(dba)3, [tBu3PH]+BF4
,
c
11-14
Cy2NMe, dioxane, microwave, 180 ꢁC, 1 h (25%); (f) 2-(4-bromophe-
nyl)acetyl chloride, TiCl4, DCM, ꢁ78 ꢁC, 20 min (17%); (g) Pd(PPh3)4,
toluene, reflux, 12 h (20%); (h) TFA, DCM, 3 h (95–100%).
Scheme 2. Reagents and conditions: (a) piperidine, DMF, 1 h; (b)
biphenyl-4-sulfonyl chloride, iPr2NEt, CH2Cl2; (c) TFA.

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
1. Tortorella, M. D.; Burn, T. C.; Pratta, M. A.; Abbaszade,
I.; Hollis, J. M.; Liu, R.; Rosenfeld, S. A.; Copeland, R.
A.; Decicco, C. P.; Wynn, R.; Rockwell, A.; Yang, F.;
Duke, J. L.; Solomon, K.; George, H.; Bruckner, R.;
Nagase, H.; Itoh, Y.; Ellis, D. M.; Ross, H.; Wiswall, B.
H.; Murphy, K.; Hillman, M. C., Jr.; Hollis, G. F.;
Newton, R. C.; Magolda, R. L.; Trzaskos, J. M.; Arner, E.
C. Science 1999, 284, 1664.
2. Pratta, M. A.; Yao, W.; Decicco, C.; Tortorella, M. D.;
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.
Chem. Lett. 2005, 15, 2808; (c) Noe, M. C.; Snow, S. L.;
Wolf-Gouveia, L. A.; Mitchell, P. G.; Lopresti-Morrow,
L.; Reeves, L. M.; Yocum, S. A.; Liras, J. L.; Vaughn, M.
Bioorg. Med. Chem. Lett. 2004, 14, 4727.
4. (a) Yao, W.; Wasserman, Z. R.; Chao, M.; Reddy, G.; Shi,
E.; Liu, R.-Q.; Covington, M. B.; Arner, E. C.; Pratta, M.
A.; Tortorella, M.; Magolda, R. L.; Newton, R.; Qian,
M.; Ribadeneira, M. D.; Christ, D.; Wexler, R. R.;
Decicco, C. P. J. Med. Chem. 2001, 44, 3347; (b) Yao, W.;
Chao, M.; Wasserman, Z. R.; Liu, R.-Q.; Covington, M.
B.; Newton, R.; Christ, D.; Wexler, R. R.; Decicco, C. P.
Bioorg. Med. Chem. Lett. 2002, 12, 101; (c) Cherney, R. J.;
Mo, R.; Meyer, D. T.; Wang, L.; Yao, W.; Wasserman, Z.
R.; Liu, R.-Q.; Covington, M. B.; Tortorella, M. D.;
Arner, E. C.; Qian, A.; Christ, D. D.; Trzaskos, J. M.;
Newton, R. C.; Magolda, R. L.; Decicco, C. P. Bioorg.
Med. Chem. Lett. 2003, 13, 1297.
8. Botos, I.; Scapozza, L.; Zhang, D.; Liotta, L. A.; Meyer,
E. F. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2749.
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%

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J. S. Xiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 311–316
B for 2 min before each injection. The flow rate was
for 3 days in the presence or absence of recombinant
human IL-1a (rhIL-1, 5 ng/ml, Sigma) and presence or
absence of small molecule compound. Media were
replaced every day. The proteoglycan content in the
medium was measured as sulfated glucosaminoglycan
(GAG) by a colorimetric assay using dimethylmethylene
blue (DMMB) and chondroitin sulfate C from shark
cartilage (Sigma) as a standard. Measured proteoglycan
was expressed per weight of cartilage. Treatment of
cartilage with IL-1 results in the induction of catabolic
enzymes including aggrecanases that degrade cartilage
matrix proteoglycan. The cleaved proteoglycan is released
from the matrix into the media. Addition of the com-
pound together with IL-1 to the cartilage results in the
decrease of proteoglycan release, indicating the inhibition
of proteoglycan degradation. During this early phase of
proteoglycan degradation, aggrecanases are the predom-
inant catabolic enzymes that cleave aggrecan with no
significant role of other MMPs.2,14
0.2 mL/min and the injection volume was 10 lL. Mass
spectrometry was performed on a PE SCIEX API 4000
triple quadrupole mass spectrometer (SCIEX, Concord,
Ontario, Canada) using TurboIon Spray source operated
under negative electrospray ionization mode. Ion source
temperature was 400 ꢁC; spray voltage, ꢁ 4500 V; MRM,
m/z 499.1 P 266.7 (M-369) and m/z 519.3 P 403.8 (M-
048, IS). Unit mass resolution was utilized for Q1 and low
resolution was used for Q3 quadrupoles. The dwell time
was 400 ms for each selected reaction monitoring (SRM)
channel with a 5-ms pause between the scans. All ion and
tandem MS instrument parameters were optimized for
high sensitivity by infusing a standard solution of 1000 ng/
mL at a flow rate of 10 lL/min using an infusion pump
(Harvard Apparatus, South Natick, MA).
13. Bovine carpal joints were obtained from young (1- to 2-
week-old) animals. Full-depth articular cartilage plugs
were harvested using a cork borer and then sliced on a
custom die to generate individual disks ꢀ6 mm wide,
1 mm thick, and 30 mg in weight. Cartilage explants were
cultured at 37 ꢁC for 5 days in a humidified atmosphere of
5% CO2 in air in cartilage explant media (CEM) consisting
of DulbeccoÕs modified EagleÕs medium containing 1%
antimycotic/antibiotic, 2 mM glutamine, 10 mM HEPES,
and 50 lg/ml of ascorbate (all from Sigma, St. Louis,
MO). The explants were washed with CEM and 1 weighed
disk per well was placed in a 96-well culture dish with
0.2 ml media and 6–8 replicates per treatment and cultured
14. Little, C.; Hughes, C.; Curtis, C.; Janusz, M.; Bohme, R.;
Wang-Weigand, S.; Taiwo, Y.; Mitchell, P.; Otterness, I.;
Flannery, C.; Caterson, B. Matrix Biol. 2002, 21, 271.
15. Chiral purities (HPLC) were determined for selected
compounds 24 and 33 to look for possible racemization
during Suzuki coupling and base-assisted alkylation con-
ditions. No racemization was observed.
16. Varray, S.; Gauzy, C.; Lamaty, F.; Lazaro, R.; Martinez,
J. J. Org. Chem. 2000, 65, 6787.
17. Gill, M. Tetrahedron 1984, 40, 621.