Identification of diarylsulfone sulfonamides as secreted frizzled related protein-1 (sFRP-1) inhibitors

Article abstract of DOI:10.1021/jm801069w

Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure-activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.

Full text of DOI:10.1021/jm801069w

7670
J. Med. Chem. 2008, 51, 7670–7672
Identification of Diarylsulfone Sulfonamides
as Secreted Frizzled Related Protein-1
(sFRP-1) Inhibitors
Ariamala Gopalsamy,*,† Mengxiao Shi,† Barbara Stauffer,#
Ramesh Bahat,# Julia Billiard,# Helga Ponce-de-Leon,#
Laura Seestaller-Wehr,# Shoichi Fukayama,‡
Annamarie Mangine,# Robert Moran,#
Figure 1. SFRP-1 inhibitor hit identified from HTS.
Scheme 1a
Girija Krishnamurthy,† and Peter Bodine#
Chemical and Screening Sciences, Wyeth Research, 401 N.
Middletown Road, Pearl RiVer, New York 10965, Women’s Health
and Musculoskeletal Biology, Wyeth Research, Cambridge,
Massachusetts 02140, and Women’s Health and Musculoskeletal
Biology, Wyeth Research, CollegeVille, PennsylVania 19426
ReceiVed August 27, 2008
Abstract: Inhibitor of secreted frizzled related protein-1 (sFRP-1)
would be a novel potential osteogenic agent, since loss of sFRP-1 affects
osteoblast proliferation, differentiation, and activity, resulting in
improved bone mineral density, quality, and strength. We have identified
small molecule diarylsulfone sulfonamide derivatives as sFRP-1
inhibitors. Structure-activity relationship generated for various regions
of the scaffold was utilized to improve the biochemical profile, resulting
in the identification of potent selective analogues, such as 16 with
desirable pharmaceutical profile.
a Reagents: (a) AlCl3, RT, overnight, 92%; (b) (1) ClSO3H, neat, 50 °C,
16 h, 90%; (c) Phenethyl amine, Et3N, CH2Cl2, 37 °C, 16 h, 82%.
with sFRP-1 inhibitor could increase bone mass and decrease
the risk of osteoporotic fractures.
A cell-based high-throughput screening (HTS) assay was
developed in U2OS human osteosarcoma cells that were
transfected with human sFRP-1, Wnt-3, and a TCF-luciferase
reporter gene that measures activation of the canonical Wnt
pathway.8 One of the hits identified from this effort is the
diphenylsulfone sulfonamide9 1 (Figure 1).
Wnts are secreted glycoproteins that mediate fundamental
biological processes like embryogenesis, organogenesis, and
tumorigenesis.1 Many extracelluar and intracellular proteins
control Wnt signaling. Among the extracelluar regulators are a
variety of secreted proteins2 that include Wnt inhibitory factor,
secreted frizzled-related proteins (sFRPs),3 Cerberus, and dick-
Compound 1 showed an EC50 of 3.9 µM in the cell based
functional assay and was selective for sFRP-1 vs -2, -3, -4, and
-5. It was active across species (human, mouse, and rat sFRP-
1) and was active with Wnt-1 and Wnt-3. With fluorescence
spectroscopy techniques, KD of 0.35 µM was determined from
the changes in the endogenous tryptophan fluorescence of the
protein upon inhibitor binding, at the emission and excitation
wavelengths of 340 and 295 nm, respectively. From these
experiments the stoichiometry of binding was found to be a
1:1 ratio indicative of specific binding. The compound also
suppressed osteocytic apoptosis induced by sFRP-1. The favor-
able biochemical profile exhibited by this hit prompted us to
undertake further follow-up study. Herein, we describe our
optimization efforts on this hit to obtain the structure-activity
relationship and to improve pharmaceutical properties like
aqueous solubility and microsomal stability.
Compounds required for establishment of structure-activity
relationship were prepared as shown in Scheme 1 starting from
appropriately substituted phenylsulfonyl chlorides 2. Friedel-
Crafts sulfonylation of benzene 3 afforded the required diphe-
nylsulfone intermediate 4. The sulfone 4 underwent regiose-
lective chlorosulfonylation to give the advanced intermediate
sulfonyl chloride 5. Reaction of 5 with various amines of interest
afforded the final target sulfonamides. The reactions were carried
out in parallel, and the products were purified by preparatory
HPLC and characterized to ensure purity and integrity.
The hit to lead optimization was initiated by obtaining
preliminary SAR for various regions of the molecule. As shown
in Table 1, introducing a small substituent like methyl or fluoro
to ring A of the diphenylsulfone scaffold was well tolerated (6
kopfs (DKKs).a 4 Inhibitors like sFRPs that bind Wnts or frizzled
,
receptors have the ability to blunt all Wnt-activated pathways,
whereas DKKs only suppress the canonical pathway.
A sFRP was isolated from human osteoblast cells and
identified as sFRP-1 (also known as SARP-2).5 It is a 35 kDa
protein consisting of 313 amino acids. This secreted protein
contains two distinct structural domains, a netrin domain and a
CRD domain, the latter of which is homologous to the frizzled
receptor. sFRP-1 is thought to interact with Wnt via the CRD
domain in a competitive manner with the frizzled receptor; thus,
sFRP-1 is an antagonist of the Wnt signaling pathway. Over-
expression of sFRP-1 in human osteoblasts in vitro accelerates
programmed cell death, while deletion of sFRP-1 in mice leads
to decreased osteoblast apoptosis and increased trabecular bone
formation.6 Loss of sFRP-1 also affects osteoblast proliferation,
differentiation, and activity and improves bone mineral density,
quality, and strength. Moreover, sFRP-/- animals do not have
significant extra skeletal defects.7 Thus, an inhibitor of sFRP-1
could have osteogenic potential, since it would prolong osteo-
blast life and allow these cells to produce more bone. Treatment
* To whom correspondence should be addressed. Phone: 845-602-2841.
Fax: 845-602-3045. E-mail: gopalsa@wyeth.com.
†
Chemical and Screening Sciences, Wyeth Research, NY.
Women’s Health and Musculoskeletal Biology, Wyeth Research, PA.
Women’s Health and Musculoskeletal Biology, Wyeth Research, MA.
#
‡
a Abbreviations: sFRP-1, secreted frizzled related protein-1; DKKs,
dickkopfs; SARP, secreted apoptosis related protein; CRD domain, cysteine-
rich domain; HTS, high throughput screen.
10.1021/jm801069w CCC: $40.75
2008 American Chemical Society
Published on Web 12/03/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7671
Table 1. sFRP-1 inhibitor activity of diphenylsulfone derivatives
Table 2. Pharmaceutical Properties of sFRP-1 inhibitor diphenylsulfone
derivatives
Properties
1
15
16
17
Cell-based TCF-reporter
3.88
1.27
9
6.97
functional assay EC50 (µM)
Aq solubility @ pH7.4 (µg/mL)
Rat microsomal stability (t1/2, min)
In Vitro microsomal
1
5
-
26
12
26.7
>100
16
-
1
1
-
Fold
Comp
R1
R2
R3
n
W
Inductiona
stability-Human (t1/2, min)
Cyp 3A4 (% Inhibition @ 3 µM)
Cyp 2D6 (% Inhibition @ 3 µM)
Cyp 2C9 (% Inhibition @ 3 µM)
70
12
41
43
9
14
78
25
25
96
11
84
1
6
7
8
H
Me
F
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
2
2
2
2
2
2
0
1
3
2
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3.3b
2.5b
2.2b
2.7b
H
H
H
Cl
H
H
H
H
H
H
<1.02
3.3b
9
Et
change brought about by a substituent in that postion is critical.
Among the substitutents explored the ethyl analogue 9 was more
active than the electron releasing methoxy analogue 10. SAR
for the linker region indicated that the sulfonamide NH is
critical, since the N-methylated analogue 14 was found to be
less active. Changing the phenethyl moiety to benzyl (12) or
anilide (11) decreased the activity significantly. Extending the
linker to phenylpropyl (13) was not advantageous as well.
However, the aromatic ring region of the phenethyl group
was found to be very flexible, and a variety of groups were
accommodated. Hence, this region was probed extensively.
Basic heterocycles were introduced as water solubilizing groups
in an effort to improve the pharmaceutical properties of the
molecule. As seen in the Figure 2, the SAR preference observed
for the R2 substituents (i.e., Et > Me > OMe > H) using the
phenethyl group was valid for the other R4 modifications as
well, using groups A-K. Observed SAR preference within the
groups A-K was minimal; however, the tolerance of group B
is noteworthy because it shows that substitution off the ethyl
linker is accommodated. However, a ring constrained group at
R4 like K did not provide any improvement over the phenethyl
moiety in the lead.
10
11
12
13
14
15
16
17
OMe
Me
Me
Me
Me
Et
1.5b
1.0c
<1.0c
1.3b
<1.0b
C (Figure 2)
A (Figure 2)
G (Figure 2)
Et
Et
4.2b
3.0b
a Fold induction was determined by adding. b 15 µM of inhibitor (or).
c 30 µM of inhibitor. The luciferase signal observed in absence of inhibitor
was set at 1. Fold induction was measured in quadruplicate.
Some of the modifications carried out during this optimization
process resulted in improved pharmaceutical properties like
aqueous solubility and microsomal stability in analogues like
15 (R2 ) Et; R4 ) C) and 16 (R2 ) Et; R4 ) A) compared to
the HTS hit 1 as shown in Table 2.
To further establish that this class of sFRP-1 inhibitors
can indeed act as anabolic agents, 17 (R2 ) Et; R4 ) G) was
evaluated in ex vivo mouse calvaria assay (Figure 3).10
Treatment of calvaria with 17 at 5 µM resulted in a 50%
increase in total bone area compared to the vehicle control.
Significant increase in the number of osteoblasts was also
observed.
Figure 2. Structure activity relationship for R2 and R4 substituents of
diphenyl sulfone sulfonamides. Fold Induction determined using 15
µM of the inhibitors when R4 ) Et, Me, OMe and 30 µM of the
inhibitors when R4 ) H.
and 7). However, in ring B, a substituent adjacent to the
sulfonamide group was necessary and removal of the methyl
group rendered 8 less active. Probably the conformational
Figure 3. Evaluation of compound 17 in ex-vivo mouse calvaria assay
at 5 µM. Each bar gives the Mean ( SE for 5 calvaria per treat-
ment.

7672 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Letters
(5) (a) Rattner, A.; Hsieh, J. C.; Smallwood, P. M.; Gilbert, D. J.;
Copeland, N. G.; Jenkins, N. A.; Nathans, J. A family of secreted
proteins contains homology to the cysteine-rich ligand-binding domain
of frizzled receptors. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2859–
2863. (b) Chong, J. M.; Uren, A.; Rubin, J. S.; Speicher, D. W.
Disulfide bond assignments of secreted frizzled-related protein-1
provide insights about frizzled homology and netrin modules. J. Biol.
Chem. 2002, 277, 5134–5144.
(6) (a) Bodine, P. V.; Zhao, W.; Kharode, Y. P.; Bex, F. J.; Lambert,
A. J.; Goad, M. B.; Gaur, T.; Stein, G. S.; Lian, J. B.; Komm, B. S.
The Wnt antagonist secreted frizzled-related protein-1 is a negative
regulator of trabecular bone formation in adult mice. Mol. Endocrinol.
2004, 18, 1222–1237. (b) Bodine, P. V. N.; Billiard, J.; Moran, R. A.;
Ponce-de-Leon, H.; McLarney, S.; Mangine, A.; Scrimo, M. J.; Bhat,
R. A.; Stauffer, B.; Green, J.; Stein, G. S.; Lian, J. B.; Komm, B. S.
The Wnt antagonist secreted frizzled-related protein-1 controls os-
teoblast and osteocyte apoptosis. J. Cell. Biochem. 2005, 96, 1212–
1230.
In summary, we have identified diphenylsulfone sulfona-
mide derivatives as sFRP-1 inhibitors. The hit to lead
optimization afforded SAR for various regions of the
molecule, resulting in inhibitors with improved physical
properties. A representative compound from this class induced
significant increase in total bone area in a murine calvarial
organ culture assay.
Acknowledgment. We thank Dr. John Ellingboe for support
of this work. We also thank Wyeth Dicovery Analytical
Department for compound characterization; in particular, we
thank Dr. Xidong Feng for HRMS data and Dr. Li Di for
solubility determination under assay conditions.
Supporting Information Available: Experimental details and
spectral data for the compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(7) Gaur, T.; Rich, L.; Lengner, C. J.; Hussain, S.; Trevant, B.; Ayers,
D.; Stein, J. L.; Bodine, P. V. N.; Komm, B. S.; Stein, G. S.; Lian,
J. B. Secreted frizzled-related protein-1 regulates Wnt signaling for
BMP2 induced chondrocyte differentiation. J. Cell. Physiol. 2006, 208,
87–96.
References
(8) (a) Bhat, R. A.; Stauffer, B.; Komm, B. S.; Bodine, P. V. Regulated
expression of sFRP-1 protein by the GeneSwitch system. Protein
Expression Purif. 2004, 37, 327–335. (b) Bhat, R. A.; Stauffer, B.;
Komm, B. S.; Bodine, P. V. N. Structure-function analysis of secreted
frizzled-related protein-1 for its Wnt antagonist function. J. Cell.
Biochem. 2007, 103, 1519–1528.
(9) Gopalsamy, A.; Moore, W. J.; Kern, J. C.; Molinari, A. J.; Shi, M.;
Welmaker, G. S.; Wilson, M. A.; Krishnamurthy, G.; Commons, T. J.;
Webb, M. B.; Woodworth, R. P. Preparation of Diarylsulfone
Sulfonamides and Their Use as Secreted Frizzled Related Protein-1
Modulators for Bone Disorders Such as Osteoporosis. U.S. 2006/
0276464, 2006.
(10) (a) Mundy, G.; Garrett, R.; Harris, S.; Chan, J.; Chen, D.; Rossini,
G.; Boyce, B.; Zhao, M.; Gutierrez, G. Stimulation of bone formation
in vitro and in rodents by statins. Science 1999, 286, 1946–1949. (b)
Traianedes, K.; Dallas, M. R.; Garrett, I. R.; Mundy, G. R.; Bonewald,
L. F. 5-Lipoxygenase metabolites inhibit bone formation in vitro.
Endocrinology 1998, 139, 3178–3184.
(1) (a) Wodarz, A.; Nusse, R. Mechanisms of Wnt signaling in develop-
ment. Annu. ReV. Cell DeV. Biol. 1998, 14, 59–88. (b) Miller, J. R.
The Wnts. Genome Biol. 2002, 3, 3001. (c) Moon, R. T.; Kohn, A. D.;
De Ferrari, G. V.; Kaykas, A. WNT and beta-catenin signalling:
diseases and therapies. Nat. ReV. Genet. 2004, 5, 691–701. (d) Logan,
C. Y.; Nusse, R. The Wnt signaling pathway in development and
disease. Annu. ReV. Cell DeV. Biol. 2004, 20, 781–810.
(2) Kawano, Y.; Kypta, R. Secreted antagonists of the Wnt signalling
pathway. J. Cell Sci. 2003, 116 (13), 2627–2634.
(3) Melkonyan, H. S.; Chang, W. C.; Shapiro, J. P.; Mahadevappa, M.;
Fitzpatrick, P. A.; Kiefer, M. C.; Tomei, L. D.; Umansky, S. R. SARPs:
a family of secreted apoptosis-related proteins. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 13636–13641.
(4) (a) Morvan, F.; Boulukos, K.; Clement-Lacroix, P.; Roman-Roman,
S.; Suc-Royer, I.; Vayssiere, B. Deletion of a single allele of the Dkk1
gene leads to an increase in bone formation and bone mass. J. Bone
Miner. Res. 2006, 21, 934–945. (b) Li, X.; Ominsky, M. S.; Niu, Q.-
T.; Sun, N.; Daugherty, B.; D’Agostin, D. Targeted deletion of the
sclerostin gene in mice results in increased bone formation and bone
strength. J. Bone Miner. Res. 2008, 23, 860–869.
JM801069W