5-((1H-Pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one inhibitors of ADAMTS-5

Article abstract of DOI:10.1016/j.bmcl.2006.12.020

A series of 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one inhibitors of ADAMTS-5 (aggrecanase-2) is described. These compounds show μM functional inhibition of ADAMTS-5, and represent a new class of agents with the potential of inhibiting degradation of aggrecan, a major component of cartilage which is lost in osteoporosis. Compound 12 is noteworthy in that it has an ADAMTS-5 IC₅₀: 1.1 μM and shows >40-fold functional selectivity over ADAMTS-4 (aggrecanase-1).

Full text of DOI:10.1016/j.bmcl.2006.12.020

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1189–1192
5-((1H-Pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one
inhibitors of ADAMTS-5
Adam M. Gilbert,a,* Matthew G. Bursavich,a Sabrina Lombardi,a
Katy E. Georgiadis,b Erica Reifenberg,b Carl R. Flanneryb and Elisabeth A. Morrisb
aExploratory Medicinal Chemistry, Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road,
Pearl River, NY 10965, USA
bWomen’s Health and Musculoskeletal Biology, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
Received 25 October 2006; revised 7 December 2006; accepted 8 December 2006
Available online 12 December 2006
Abstract—A series of 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one inhibitors of ADAMTS-5 (aggrecanase-2) is
described. These compounds show lM functional inhibition of ADAMTS-5, and represent a new class of agents with the potential
of inhibiting degradation of aggrecan, a major component of cartilage which is lost in osteoporosis. Compound 12 is noteworthy in
that it has an ADAMTS-5 IC50: 1.1 lM and shows >40-fold functional selectivity over ADAMTS-4 (aggrecanase-1).
Ó 2006 Elsevier Ltd. All rights reserved.
Osteoarthritis (OA), a disease where aggrecan and colla-
gen in the articular cartilage matrix are slowly degraded,
often leads to debilitating chronic joint pain and inflam-
mation. While collagen is the major structural compo-
nent of aggrecan and provides strength to the tissue,
aggrecan is responsible for the tissue’s flexible and elas-
tic nature. Aggrecan is lost in the initial phases of the
disease, while collagen is lost in the disease’s later stages.
Thus an agent that would inhibit aggrecan breakdown
could be a disease-modifying osteoarthritis treatment
as opposed to treatments that only provide symptomatic
relief (e.g., acetaminophen, COX-2 inhibitors, NSAIDs
or intraarticular steroids).1
gest that ADAMTS-5 could be the major player as
ADAMTS-5 knockout mice show reduced OA severity
after surgical induction of joint instability, whereas the
corresponding ADAMTS-4 knockouts show a normal
OA progression.3 ADAMTS-5 is also known to be the
major ADAMTS in mouse cartilage and in a mouse
model of inflammatory arthritis.4 In this letter, we re-
port our initial studies on a series of 5-((1H-pyrazol-4-
yl)methylene)-2-thioxothiazolidin-4-one
ADAMTS-5
inhibitors 1 found from high throughput screening and
show that they are useful as potentially selective
ADAMTS-5 therapeutics to treat OA.5 To our knowl-
edge, these compounds represent the first disclosure of
inhibitors of ADAMTS-5. Examples of general aggre-
canase inhibitors have been previously disclosed.6–9
Aggrecan is catabolized by the proteolytic enzymes
ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggre-
canase-2), two members of the ‘‘A Disintegrin And
O
Metalloproteinase
with
ThromboSpondinmotifs’’
(ADAMTS) family of proteins. These enzymes cleave
the aggrecan interglobular domain at the Glu373–
Ala374 peptide bond.2 Both enzymes are 1000-fold more
efficient at cleaving aggrecan than any other enzymes
although it is not known which enzyme is the primary
source of aggrecanase activity in cartilage. Studies sug-
R3
NH
S
X
N
R5
N
R1
1
Thioxothiazolidin-4-one ADAMTS-5 inhibitors 1 are
prepared according to Scheme 1.10 5-Hydroxypyrazoles
3 are synthesized by combining acetylacetic esters 2 with
hydrazines.11
Keywords: ADAMTS-5; Aggrecanase-2; Thioxothiazolidin-4-ones;
Osteoporosis.
*
Corresponding author. Tel.: +1 845 602 4865; fax: +1 845 602
5561; e-mail: gilbera@wyeth.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.020

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A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1189–1192
H
from nearly pure Z isomers (as drawn in Scheme 1) to
80:20 Z:E isomeric mixtures.
R3
R3
O
O
O
a
b
N
N
R3
OEt
OH
Cl
N
R1
3
N
ADAMTS-5 activity was determined using a fluores-
cence resonance energy transfer (FRET) assay using
a QF-peptide containing the aggrecanase cleavage site.
Binding to target studies were conducted using fluores-
R1
4
2
cence polarization experiments using
a xanthene-
c
e
d
ADAMTS-5 hydroxamic acid inhibitor. Several
compounds were analyzed for ADAMTS-4 activity
using the same QF-peptide used for the ADAMTS-5
activity studies.
H
H
H
R3
N
R3
R3
O
O
O
N
N
SR2
NR2
OR2
N
R1
N
N
R1
R1
Biological data of 3-CF3-1H-pyrazole thioxothiazolidin-
4-ones are shown in Table 1. When R1 = Me and
R5 = SPh (9) or SCH2Ph (10), weak ADAMTS-5 activ-
ity is seen. Replacing the Ph in 10 with the isosteric
2-thiophene in 11 produces similarly weak ADAMTS-
5 potency. However the addition of a p-Cl moiety to
11 produces 12, a compound which displays micromolar
ADAMTS-5 inhibitory activity (IC50: 1.1 lM). Move-
ment of the p-Cl moiety in 12 to the o-position affords
a compound with ꢀ20-fold less activity (13: IC50,
23.2 lM). The p-Cl moiety in 12 may be replaced by a
p-t-Bu in 14 (IC50, 2.2 lM), but not a p-OMe as in 15.
Removing the benzyl CH2 methylene in 12 gives the
p-Cl phenyl analog 16 which has reduced ADAMTS-5
inhibition. In addition to R1 = Me, R1 can be Ph (17:
IC50, 2.5 lM) and cyclohexyl (18: IC50, 4.2 lM). The
SCH2 moiety in 12 and 17 can be replaced by a pipera-
zine to give compounds with similar ADAMTS-5 activ-
ity (19: IC50, 9.0 lM and 20: IC50, 2.1 lM).
5
6
7
f
O
S
R3
NH
S
N
R5
N
R1
1
Scheme 1. Reagents and conditions: (a) R1NHNH2, H2SO4, EtOH,
reflux, 17 h; (b) POCl3, DMF, 80 °C, 12 h; (c) R2SH, Et3N, DMSO,
40 °C, 12 h; (d) R2NH, Et3N, DMF, 100 °C, 12 h; (e) t-BuOK, DMF,
23 °C, 12 h; (f) rhodanine, b-alanine, AcOH, 110 °C, 12 h.
The corresponding 4-formyl-5-chloropyrazoles 3 are
prepared using POCl3 in hot DMF.11 The 5-chloro moi-
ety can be displaced with the following nucleophiles: thi-
ols to produce 5 (Et3N, DMSO, 40 °C), amines to
produce 6 (Et3N, DMF, 100 °C), and alcohols to pro-
duce 7 (t-BuOK, DMF, 23 °C). The corresponding thi-
oxothiazolidin-4-ones 1 are produced using rhodanine
and b-alanine in hot HOAc.12 1H NMR spectroscopy
shows that thioxothiazolidin-4-ones 1 exist in a range
The 3-CF3 moiety in 9–20 can be modified to produce
active compounds if other pyrazole substituents are
modified as well (Table 2). The 1,3-diphenyl-1H-pyra-
zole analog 21 shows ADAMTS-5 inhibition. This
contrasts with the R1 = Me, R3 = 3-CF3 analog 11.
The 2-thiophene moiety in 21 may be changed to a furan
Table 1. In vitro ADAMTS-5 inhibition data for 5-((3-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-ones
O
NH
F3C
S
S
N
R5
N
R1
R1
R5
% Inhib. @ 20a (lM)
IC50 (lM)
a
Compound
9
10
11
12
13
14
15
16
17
18
19
20
Me
Me
Me
Me
Me
Me
Me
Me
Ph
SPh
SCH2Ph
28
44
40
SCH2(2-thiophene)
SCH2Ph(4-Cl)
SCH2Ph(2-Cl)
SCH2Ph(4-t-Bu)
SCH2Ph(4-OMe)
SPh(4-Cl)
1.1
23.2
2.2
53
47
SCH2Ph(4-Cl)
SCH2Ph(4-Cl)
PiperazinePh(4-Cl)
PiperazinePh(4-Cl)
2.5
4.2
9.0
2.1
CyH
Me
Ph
a Values are means of two experiments, standard deviations are 10%.

A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1189–1192
1191
Table 2. In vitro aggrecanase-2 inhibition data for 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-ones
O
R3
NH
S
S
N
R5
N
R1
a
Compound
R1
R3
R5
% Inhib. @ 20a (lM)
IC50 (lM)
21
22
23
24
25
26
27
28
29
30
31
Ph
Ph
Ph
Ph
SCH2(2-thiophene)
SCH2(2-furan)
SCH2(2-furan)
SCH2(2-furan)
SCH2Ph
3.7
6.7
4.6
CyH
Ph
Ph
CF3
CF3
Ph
42
Ph
16.9
14.1
8.3
Me
Me
CyH
CyH
Ph
SCH2(2-furan)
SPh(4-Me)
Ph
Ph
Piperazine(2-pyrimidine)
Piperazine(2-pyrimidine)
Piperazine(Me)
8.0
CF3
Ph
11.7
4.6
Ph
CF3
Piperazine(Me)
45
a Values are means of two experiments, standard deviations are 10%.
22 with little change in activity, while the corresponding
compound where R1 = CyH 23 has similar ADAMTS-5
potency. Interestingly, 24 where R3 = CF3 does not
show good ADAMTS-5 activity (compare with 22),
but 25 shows moderate activity where R5 = SCH2Ph
(compare with 10). The R1 = Me and R3 = Ph substitu-
tion pattern gives moderately active compounds (26 and
27) and piperazinyl moieties can be incorporated at R5
(28–31) as previously shown above (Table 1).
O
S
O
EtO2C
Me
CO2Et
Me
NH
S
NH
F3C
F3C
N
H
S
S
N
N
R5
R5
N
N
toluene, reflux
17: R5 = SCH2Ph(4-Cl)
24:R5 = SCH2(2-furan)
31: R5 = piperazine(Me)
36: ADAMTS-5 IC50: 5.8 M
37: ADAMTS-5: 42% @ 20 M
38: ADAMTS-5: < 40% @ 20 M
The sulfur at the R5 position of the pyrazole can be
substituted with an oxygen substituent provided that
an extra CH2 group is added to the linker (Table 3).
Thus, 32 possesses ADAMTS-5 activity (IC50, 4.2 lM;
compare with 17). The corresponding p-F phenyl com-
pound 33 also shows ADAMTS-5 inhibition (IC50,
7.1 lM) as does the 2-thiophene analog 34 (IC50,
9.1 lM). Similarly to 15, the p-OMe-phenyl analog 35
shows reduced ADAMTS-5 activity again showing the
importance of the p-substituent on phenyl group of the
R5 side chain (Table 4).
Reduction of the double bond attached to the thioxo-
thiazolidin-4-one headpiece using the Hantzsch ester13
produces compounds with reduced ADAMTS-5 activity.
Thus while 17 only loses 2-fold ADAMTS-5 potency on
double bond reduction (36: IC50, 5.8 lM), the case is
worse for 24 and 31; both compounds lose substantial
potency when converted to their single bond analogs 37
and 38.
Given the previously cited suggestive mouse ADAMTS-5
knockout data,3 we have begun to look at ADAMTS-5/
ADAMTS-4 selectivity for a few of the thioxothiazoli-
din-4-ones presented in this manuscript using an
ADAMTS-4 FRET assay. Two of the more potent
ADAMTS-5 inhibitors show selectivity over ADAM-
TS-4. The p-Cl-Ph analog 12 shows good functional
selectivity over ADAMTS-4 (ADAMTS-5 IC50,
1.1 lM; ADAMTS-4 IC50, 44 lM), something that is
not surprising since ADAMTS-5 has approximately
40% sequence homology to ADAMTS-4 overall and
Table 3. In vitro ADAMTS-5 inhibition data for 5-((5-alkoxy-1H-
pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-ones
O
NH
F3C
S
S
N
R5
N
Compound R5
% Inhib.
@ 20a (lM)
IC50 (lM)
a
Table 4. In vitro ADAMTS-5 and ADAMTS-4 inhibition data for
selected 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-ones
32
33
34
35
O(CH2)2Ph(4-Cl)
O(CH2)2Ph(4-F)
4.2
7.1
9.1
a
a
Compound ADAMTS-5 IC50 (lM) ADAMTS-4 IC50 (lM)
O(CH2)2(2-thiophene)
O(CH2)2Ph(4-OMe)
12
14
1.1
2.2
44
7.5
31
a Values are means of two experiments, standard deviations are 10%.
a Values are means of two experiments, standard deviations are 10%.

1192
A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1189–1192
50% active site sequence similarity.14 The closely
related t-Bu-Ph analog 14 is less functionally selective
(ADAMTS-5 IC50, 2.2 lM; ADAMTS-4 IC50,
7.5 lM). In addition, both 12 and 14 successfully dis-
placed a xanthene-ADAMTS-5 hydroxamic acid inhibi-
tor from the ADAMTS-5 protein showing that their
inhibitory activity involved binding to ADAMTS-5.15
5. For an accompanying letter describing a series of related
aryl thioxothiazolidin-4-one ADAMTS-5 inhibitors, see:
Bursavich, M. G.; Gilbert, A.M.; Lombardi, S.; Georgi-
adis, K.E.; Reifenberg, E.; Flannery, C.R.; Morris, E.A.
Bioorg. Med. Chem. Lett. 2007, 17, in press.
6. Xiang, J. S.; Hu, Y.; Rush, T. S.; Thomason, J. R.; Ipek,
M.; Sum, P.-E.; Abrous, L.; Sabatini, J. J.; Georgiadis, K.;
Reifenberg, E.; Majumdar, M.; Morris, E. A.; Tam, S.
Bioorg. Med. Chem. Lett. 2006, 16, 311.
Thus, we have presented a series of 5-((1H-pyrazol-4-
yl)methylene)-2-thioxothiazolidin-4-one
7. Cherney, R. J.; Mo, R.; Meyer, D. T.; Wang, L.; Yao, W.;
Wasserman, Z. R.; Liu, R.-Q.; Covington, M. B.; Torto-
rella, M. D.; Arner, E. C.; Qian, M.; Christ, D. D.;
Trzaskos, J. M.; Newton, R. C.; Magolda, R. L.; Decicco,
C. P. Bioorg. Med. Chem. Lett. 2003, 13, 1297.
8. 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. 2001, 12, 101.
9. 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.
ADAMTS-5
inhibitors. This series of compounds has tractable
SAR and several analogs show modest functional selec-
tivity for ADAMTS-5 over ADAMTS-4. The continued
development of selective ADAMTS-5 inhibitors is cur-
rently ongoing and will be reported in due course.
Acknowledgments
We thank Dr. John Ellingboe for support of this work.
We also thank Dr. Kristina Cunningham for performing
fluorescence polarization binding to target studies.
10. All newly prepared compounds were characterized by
reversed phases-HPLC/MS spectroscopy. A selected num-
ber of compounds were also characterized by H NMR.
1
11. Lee, L. F.; Schleppnik, F. M.; Schneider, R. W.; Camp-
bell, D. H. J. Heterocycl. Chem. 1990, 27, 243.
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