Identification of iminooxothiazolidines as secreted frizzled related protein-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.09.085

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.

Full text of DOI:10.1016/j.bmcl.2009.09.085

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6337–6339
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of iminooxothiazolidines as secreted frizzled related
protein-1 inhibitors
b
b
b
b
Mengxiao Shi a, , Barbara Stauffer , Ramesh Bhat , Julia Billiard , Helga Ponce-de-Leon ,
Laura Seestaller-Wehr b, Shoichi Fukayama b, Annamarie Mangine b, Robert Moran b, Girija Krishnamurthy c,
Peter Bodine b, Ariamala Gopalsamy a
*
a Chemical Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Womens Health and Musculoskeletal Biology, 500 Arcola Road, Wyeth, Collegeville, PA 19426, USA
c Screening Sciences, Wyeth Research, 401 N. Middletown Road., Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2009
Revised 21 September 2009
Accepted 22 September 2009
Available online 25 September 2009
Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of
osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and
proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as
sFRP-1 inhibitors. Structure–activity relationships were established for various regions of the scaffold
along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keyword:
sFRP1 inhibitor osteoporosis
Wnts are a large family of growth factors that mediate funda-
mental biological processes like embryogenesis, organogenesis
and tumorigenesis.1–4 Recent research indicates that they also play
an important role in bone formation, and the Wnt pathway compo-
nents are being pursued as potential drug targets for osteoporosis
and other metabolic bone diseases.5–9 Secreted frizzled related
proteins, sFRPs, are Wnt antagonists.10,11 Overexpression of sFRP-
1 in human osteoblasts in vitro accelerates apoptosis, and deletion
of sFRP-1 in mice leads to osteoblast proliferation resulting in bone
formation.12,13 Moreover, sFRP-1 knockout animals do not have
significant extra skeletal defects.14 Thus, an sFRP-1 inhibitor has
the potential to be a novel osteogenic agent, since it would prolong
the life of osteoblasts and therefore allow these cells to produce
more bone.
A cell-based high throughput screening (HTS) assay for sFRP-1
inhibitors was therefore developed. This assay involves transient
transfection of U2OS human osteosarcoma cells with human
sFRP-1, Wnt-3 and a TCF-luciferase reporter gene that measures
activation of the canonical Wnt-pathway.15,16 Iminooxothiazoli-
dine 1 was identified as a hit from this screening effort (Fig. 1).17
phan fluorescence of the protein upon inhibitor binding at the
emission and excitation wavelengths of 340 and 295 nm, respec-
tively. From these experiments the stoichiometry of binding was
found to be 1:1, indicative of specific binding. The favorable bio-
chemical profile displayed by this hit prompted us to conduct a fol-
low up study around this scaffold. A structure–activity relationship
was established for this chemotype and preliminary ex vivo data
were obtained.
The compounds for exploration of SAR were synthesized by fol-
lowing synthetic routes analogous to those in Schemes 1–3. The
iminooxothiazolidine derivatives were synthesized by a modified
literature procedure.18 Ring opening of maleic anhydride 3 with
2-amino-4,5-dimethyl-thiophene-3-carboxylic acid ethyl ester 2
resulted in the intermediate 2-(3-carboxy-acryloylamino)-4,5-di-
methyl-thiophene-3-carboxylic acid ethyl ester 4, which was
further reacted with thiourea to give the 2-[2-(2-imino-4-oxo-
thiazolidin-5-yl)-acetylamino]-4,5-dimethyl-thiophene-3-carbox-
ylic acid ethyl ester 5 (Scheme 1). Following the same procedure
and using different aminothiophenes or anilines with thiourea or
Compound 1 showed an EC50 of 7.2 lM in the U2OS TCF-lucifer-
ase assay and was selective against sFRP-2 and -5. It was active
across species (human, mouse and rat sFRP-1) and was active with
COOMe
O
Wnt-3. With fluorescence spectroscopic techniques
a KD of
NH
NH
S
0.72 M was determined from changes in the endogenous trypto-
l
NH
S
O
1
* Corresponding author. Tel.: +1 8456021857; fax: +1 8456023045.
E-mail address: shim2@wyeth.com (M. Shi).
Figure 1. High throughput screen hit identified.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.085

6338
M. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6337–6339
Table 1
O
COOEt
sFRP-1 inhibitor activity of iminooxothiazolidine derivatives
R1
R2
COOEt
NH2
a
O
+
NH COOH
S
R3
NH
S
O
O
2
4
O
3
NH
S
NH
COOEt
S
O
b
O
NH
NH
S
Compd
R1
R2
R3
Fold induction (@15 lM)
NH
S
O
1
5
7
Me
Me
Me
Me
H
H
H
Me
Me
Me
Br
H
Et
COOMe
COOEt
COOEt
COOEt
COOEt
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
CONH2
CN
1.9
2.3
2.6
2.0
1.7
1.2
0.9
0.8
1.8
2.5
1.5
1.9
1.6
1.1
1.2
5
15
16
17
18
19
20
10
21
22
23
24
25
Scheme 1. Reagents and conditions: (a) Et2O, rt 10 h, 72%; (b) HOBT, EDCI, thiourea,
DMF, 60 °C, 12 h, 52%.
H
Ph
Ph
Me
Br
COOEt
COOEt
Ph
CH2NHPh
–CH2CH2CH2–
–CH2(CH2)3CH2–
–CH2(CH2)2CH2–
CH2(CH2)2CH2–
CH2(CH2)2CH2–
a
O
O
Br
NH
NH
NH
S
NH
S
NH
NH
S
O
S
O
7
6
Br
Br
COOEt
NH2
COOEt
b
+
O
O
NH
NH
NH
S
NH
S
phene scaffold provides the best activity of the functional groups
surveyed. Replacing the ester with an amide (compound 24) or ni-
trile (compound 25) decreases the activity in the case of a bicyclic
scaffold. From the analogs synthesized it was clear that a range of
substituents like methyl (compounds 7 and 15), phenyl (com-
pound 20) or anilinomethyl (compound 10) were tolerated in the
4-position of the thiophene ring. However the 5-position showed
less tolerance for steric bulk. The 5-phenyl analogs 18 and 19 were
much less active compared to compounds with smaller substitu-
ents like methyl or ethyl at the 5-position (compounds 5 and
16). However, a bromo group in this position, as in analogs 7 and
10, was well accommodated indicating that the region was sensi-
tive to the stereoelectronics of the substituent. Replacing the thio-
phene scaffold with cycloalkyl fused thiophenes (compounds 21–
23) was also tolerated.
NH
NH
S
9
O
S
O
8
6
c
HN
Br
COOEt
O
NH
NH
S
NH
S
O
10
Scheme 2. Reagents and conditions: (a) NBS, chloroform, 0 °C 1 h, 67%; (b)
bromine, chloroform, 6 h, 70%; (c) K2CO3, DMF, rt, 12 h, 25%.
EtOOC
H2N
Scaffold hopping was attempted next using substituted phenyl
(compounds 26–30) and benzyl derivatives (compound 31) in the
place of the thiophene, as shown in Table 2. Overall this effort
did not improve the activity for the series. Variations on the imi-
nooxothiazolidine region were also explored and the results are
shown in Table 3. Thus, the unsubstituted iminooxothiazolidine
was found to be critical for activity. Replacing it with a 2,4-di-
oxo-thiazolidine (14) or alkylation of the iminooxothiazolidine
nitrogen (32–34) decreases activity significantly, irrespective of
the scaffold employed.
O
O
O
Cl
a
COOH
HN
HN
+
S
S
S
O
O
12
13
11
H
COOEt
N
O
O
b
S
NH
S
O
14
Scheme 3. Reagents and conditions: (a) oxalyl chloride, DMF (cat.), CH2Cl2 30 min;
(b) pyridine, 1 h. 30%.
Table 2
SAR for central ring scaffold replacement
substituted thioureas, another series of iminooxothiazolidine ana-
logs were synthesized for SAR studies. Compounds 7 and 8 were
made by treating compound 6 with different brominating agents
such as NBS or bromine. Compound 8 was further reacted with ani-
line to form the 5-bromo-2-[2-(2-imino-4-oxo-thiazolidin-5-yl)-
acetylamino]-4-phenylamino methyl-thiophene-3-carboxylic acid
ethyl ester 10 (Scheme 2). The 2,4-dioxo-thiazolidine 14 was
synthesized from commercial acid 11 through a typical amide
formation procedure (Scheme 3).
H
N
O
X
NH
NH
R4
S
O
Compd
R4
X
Fold induction (@30 lM)
26
27
28
29
30
31
3-Cl
Bond
Bond
Bond
Bond
Bond
CH2
1.7
1.5
1.1a
1.1
1.0
1.0
3-COOEt
2-COOEt
H
2,-3 DiCl
3-Cl
The hit to lead optimization was focused on exploring various
regions of the hit molecule 1 to obtain preliminary SAR. As shown
in Table 1, a methyl or ethyl ester at the 3-position of the thio-
a
Fold induction was determined at 15 lM inhibitor concentration.

M. Shi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6337–6339
6339
Table 3
In summary, a series of iminooxothiazolidines identified by
high throughput screening was investigated to understand the
SAR requirements of the scaffold. These compounds represent a
novel sFRP-1 inhibitor scaffold with an interesting in vitro and
ex vivo profile.
SAR for iminooxothiazolidine replacement
R1
COOEt
R6
N
O
R2
NH
S
R5
S
Acknowledgements
O
Compd
R1
R2
R5
R6
Fold induction (@15 lM)
The authors thank Dr. John Ellingboe for his support of this
work. We also thank Discovery Analytical Chemistry group for ana-
lytical data.
5
Me
Me
Me
Me
Me
Me
Me
Me
NH
NMe
NEt
NMe
NH
O
H
H
H
Me
H
2.3
1.2
1.2
1.2
1.6
1.0
32
33
34
23
14
References and notes
–CH2(CH2)2CH2–
CH2(CH2)2CH2–
H
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