Modulation of Wnt signaling through inhibition of secreted frizzled-related protein I (sFRP-1) with N-substituted piperidinyl diphenylsulfonyl sulfonamides: Part II

Article abstract of DOI:10.1016/j.bmc.2009.11.002

Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein. Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/β-catenin canonical signaling pathway. Details of the structure-activity relationships and biological activity of this structural class of compounds will be discussed.

Full text of DOI:10.1016/j.bmc.2009.11.002

Bioorganic & Medicinal Chemistry 18 (2010) 190–201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modulation of Wnt signaling through inhibition of secreted frizzled-related
protein I (sFRP-1) with N-substituted piperidinyl diphenylsulfonyl
sulfonamides: Part II
a
b
b
b
William J. Moore a, , Jeffrey C. Kern , Ramesh Bhat , Peter V. N. Bodine , Shoichi Fukyama ,
*
Girija Krishnamurthy a, Ronald L. Magolda a, Keith Pitts a, Barb Stauffer b, Eugene J. Trybulski a
a Chemical Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, United States
b Women’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding
affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein.
Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/b-catenin canonical signaling
pathway. Details of the structure–activity relationships and biological activity of this structural class of
compounds will be discussed.
Received 24 September 2009
Revised 29 October 2009
Accepted 2 November 2009
Available online 6 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Secreted-frizzled related protein-1 (sFRP-1)
Wnt pathway
Osteoblasts
Osteoporosis
1. Introduction
in the activation of transcriptional factors.1,2,4 There are many reg-
ulators of the Wnt signaling process, including extracellular com-
Secreted Wnt glycoproteins are responsible for activation of the
Wnt signaling pathway. This signaling process is responsible for
the regulation of various cellular developmental processes includ-
ing cell proliferation, differentiation, and apoptosis which has been
the topic of several recent reviews.1–3 The Wnt/b-catenin canonical
signaling pathway is initiated by the extracellular binding of Wnt
to a membrane receptor complex composed of a Frizzled (Fzd)
ponents such as WIF-1, Dkk-1, SOST, and secreted Frizzled-
Related Protein-1 (sFRP-1 or SARP-2).4,5 The sFRPs are one of the
largest families of Wnt antagonists and share high homology to
the membrane bound frizzled receptor. Human sFRP-1 is a 35 kD
glycosylated protein consisting of 313 amino acids and eight disul-
fide linkages, five of which are located in the cysteine rich domain
(CRD) while the remaining three disulfide bonds are located within
the netrin domain.6 sFRP-1, via the CRD domain, competes with the
Fzd receptor for Wnt. Thus sFRP-1 is a negative regulator or an
antagonist of the Wnt signaling pathway.7a Therefore, inhibitors
GPCR and
a low-density lipoprotein receptor-related protein
(LRP). The binding results in a signaling cascade leading to the sta-
bilization of b-catenin, which translocates to the nucleus, resulting
O
NH
O
N
O
S
O
S
NH
O
S
O
S
N
N
H
N
H
O
O
O
O
CF3
CF3
1 (WAY-316606)
FP Binding IC50 0.5 µM
Wnt-luc EC50 0.65 µM
2 (WAY-362692)
FP Binding IC50 0.02 µM
Wnt-luc EC50 0.03 µM
* Corresponding author. Tel.: +1 484 865 7827; fax: +1 484 865 9399.
E-mail address: moorew2@wyeth.com (W.J. Moore).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.002

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
191
O
NH
N
N
O
S
N
O
S
O
S
N
Ar
O
S
O
S
N
O
a
a or b
N
5d
N
1
O
O
H
(45 %)
O
O
H
(61 - 98 %)
CF3
CF3
3b-d, 3f
5e
4a-e
O
S
Scheme 3. Reagents and condition: (a) TMS–N3, Al(CH3)3, toluene, 80 °C.
O
S
O
S
N
Ar
O
c
N
O
O
H
(14 - 88 %)
CF3
CO2H
O
S
O
S
N
5a-e
a, b
N
1
Scheme 1. See Table 1 for aryl (Ar) definitions. Reagents: (a) ArCOCl, TEA; (b)
O
O
H
(9 - 19 %)
ArCO2H, EDC, DMAP; (c) ArSO2Cl, TEA.
CF3
6a, meta
6b, para
of sFRP-1 could be pharmacologically useful agents for bone disor-
ders like osteoporosis or fracture repair in which activation of the
Wnt signaling pathway would be beneficial.7b,c Recently, the dis-
covery of the diphenylsulfonyl sulfonamide scaffold was reported
followed by a detailed account of the piperidinyl diphenylsulfonyl
Scheme 4. Reagents and conditions: (a) 3- or 4-(tertbutoxycarbonyl)phenylboronic
acid, Cu(OAc)2, TEA; (b) HCl, EtOAc or dioxane.
sulfonamide
1 (WAY-316606) and L-proline derived urea 2
( )n
R1
(WAY-362692).8,9 Compound 2 exhibited binding affinity (IC50
20 nM) to the sFRP-1 protein and potent functional activity in both
the cell-based functional assay (EC50 30 nM) and the ex vivo mouse
calvaria tissue culture.9,11,12 During the course of the SAR investi-
gations functionality was identified that could be substituted on
the piperidinyl group to expand the SAR knowledge about the
target as well as improve the chemical properties of the series.
Herein, we highlight the synthesis and SAR of our efforts to expand
the piperidinyl diphenylsulfonyl sulfonamide scaffold as sFRP-1
inhibitors.
O
S
O
S
N
O
N
H
1
O
O
CF3
a (53 %)
7a (n = 1), R1 = NH2
7b (n = 1), 7c (n = 2); R1 = OH
b, c (57 - 77 %)
O
O
( )n
O
S
O
S
N
OH
N
O
O
H
CF3
2. Chemistry
d (32 - 77 %)
7d (n = 2), 7e (n = 3)
The synthesis of compound 1 was described, including the de-
tailed preparation of sulfonyl chloride 9.9 The synthetic routes
illustrating the preparation of N-substituted piperidinyl diph-
enylsulfonyl sulfonamides 3–8 are shown in Schemes 1–3.
Treatment of 1 with an aryl or heteroaryl acid chloride, aryl sul-
fonyl chloride or activated aryl carboxylic acid in the presence of
an amine base afforded amides 3b–d, 3f 4a–e and sulfonamides
5a–e, respectively in moderate to good yields (Scheme 1). Alterna-
tively, amide derivatives 3a, 3e or 4f–h could be derived from the
reaction of sulfonyl chloride 9 and the appropriately substituted 4-
aminopiperidinyl carboxamides (Scheme 2). The aromatic nitrile
5d was converted to the tetrazole 5e following a convenient proce-
dure described by Huff involving the reaction of an aryl nitrile with
azidotrimethylsilane in the presence of the Lewis acid trimethyl-
aluminum (Scheme 3).13
OH
R2
O
S
O
S
N
R2
N
O
O
H
CF3
a (30 %)
e (70 %)
7f, R2 = H
7g, R2 = CH3
Scheme 5. Reagents and conditions: (a) 2-bromoethanol or 2-bromoacetamide,
TEA; (b) tert-butyl 2-bromoacetate or tert-butyl 3-bromopropionate, TEA; (c) HCl,
EtOAc or dioxane; (d) succinic or glutaric anhydride, EtOH,
l wave, 150 °C; (e)
isobutylene oxide, EtOH, 75 °C.
benzoate. The tertiary butyl esters were further treated with acid
to provide hydrochloride salts of the corresponding benzoic acid
isomers.
As shown in Scheme 4, compounds 6a and 6b were furnished
albeit in modest yields via copper catalyzed N-arylation between
1 and the meta- or para-substituted boronic acids of tertiary butyl
The syntheses of derivatives 7a–g are illustrated in Scheme 5.
Alkylation of 1 at elevated temperatures with primary alkyl bro-
mides in the presence of an acid scavenger such as triethylamine
afforded compounds 7a (n = 1, R1 = NH2) and 7f (R2 = H), respec-
tively. Treatment of 1 in a similar manner, with the tertiary butyl
esters of 2-bromoacetic acid or 3-bromopropionic acid followed
by acidic hydrolysis provided the aliphatic carboxylic acids
(R1 = OH) 7b (n = 1) and 7c (n = 2). Alternatively, the tertiary alco-
hol 7g (R2 = CH3) can be prepared by alkylating 1 with the epoxide
of isobutylene. Reaction of 1 in an ethanolic solution containing
either succinic or glutaric anhydride was facilitated by employing
microwave irradiation affording the amido acids 7d (n = 2) and
7e (n = 3).
O
O
S
O
S
N
Ar
PhO 2
S
PhO 2
S
a
Cl
N
O
O
H
CF3
CF3
9
3a, 3e
4f-h
Scheme 2. Reagents and conditions: (a) (4-aminopiperidin-1-yl) arylcarbonyl, TEA,
CH2Cl2 (23–76%).

192
W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
R1
ity were also monitored to prioritize compounds with drug-like
O
N
properties. Select compounds were further profiled in an ex vivo
mouse calvaria tissue model of bone formation.
( )n
O
S
O
S
N
Tables 1–3 summarize the in vitro binding and functional activ-
ity profiles of the prepared sFRP-1 inhibitors. As the SAR around
the piperidinyl diphenylsulfonyl sulfonamide developed, it became
apparent that optimized substitutions to the growing scaffold re-
sulted in molecules that began to resemble peptidomimetic-like
structures. Due to the protein–protein relationship between
sFRP-1 and Wnt, it was not unreasonable to expect that higher
molecular weight compounds would be required to perturb the
interaction and influence the functional activity.
a
N
1
O
O
H
(70%)
CF3
R1 = Boc, 8a-b
b (44-54%)
R1
R1 = H, 8g-h
O
N
O
S
O
N
S
a
N
An unsubstituted aryl group adjoined to the piperidine by either
carbonyl 3a (IC50 0.07
O
O
H
(45 - 55%)
lM, EC50 0.25 lM) or sulfonyl 5a (IC50
CF3
0.13 M, EC50 0.21 M) were equivalent in binding affinity and po-
l
l
R1 = Boc, 8c-f
R1 = H, 8i-l
tency (Tables 1 and 3). Chloro substitution on the phenyl at the
ortho (3b), meta (3c), or para (3d) positions resulted in derivatives
exhibiting a 2.5–5-fold loss in activity relative to the unsubstituted
derivative 3a. Additional para-substituted compounds bearing the
trifluoromethyl 3e and dimethylamino 3f were similarly character-
ized as sFRP-1 inhibitors with moderate binding affinities
b (27-44%)
Scheme 6. Reagents: (a) CDI, TEA or EDC, DMAP; (b) HCl, EtOAc or dioxane.
The synthesis of N-Boc protected and deprotected R- and S-pro-
line enantiomers 8c–d and 8i–j have been previously reported.9
The remaining heterocycloalkyl derivatives were obtained in a
similar manner starting with commercially available N-tert-butoxy
carbonyl protected piperidine-4-carboxylic acid, piperidin-4-
ylacetic acid, and the R or S-enantiomers of pyrrolidine-3-carbox-
ylic acid. As depicted in Scheme 6, the acids were activated using
standard procedures mediated by either carbonyl diimidazole
(CDI) and triethylamine or N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide (EDC) and N,N-dimethylaminopyridine (DMAP).
When the activated acids were coupled with compound 1, piperid-
inyl 8a–b or pyrrolidinyl 8c–f amides were provided. The depro-
tected analogs 8g–l were acquired in good yields upon exposure
to acid yielding the corresponding hydrochloride salts.
(>0.2 lM) and functional activity (0.2–1.9 lM) and offered no
advantages over the parent molecule 3a. Positioning of a nitrogen
atom within the aromatic ring relative to the carbonyl was most
effective at the 2- or 3-positions, while substitution at the 4-posi-
tion provide an sFRP-1 inhibitor with weaker binding and func-
tional activity. The pyridyl derivatives 4b (IC50 0.06
0.23 M) and 4c (IC50 0.08 M, EC50 0.12 M) had comparable
binding affinity and functional activity while the para-situated
compound 4a (IC50 0.20 M, EC50 0.60 M) had 2.5–4.5-fold less
lM, EC50
l
l
l
l
l
activity in either binding or functional response. Further substitu-
tion of 4c led to compounds containing chloro (4d), trifluoromethyl
(4e) and dimethylamino (4f) groups and resulted in compounds
with similar binding affinity and functional activity. It should be
noted that while compound 4f (IC50 0.04
onstrated analogous binding affinity to the initial phenyl derivative
3a (IC50 0.07 M, EC50 0.25 M) it exhibited a fourfold improve-
lM, EC50 0.07 lM) dem-
3. Results and discussion
l
l
ment in functional potency. The fold induction response (2- and
4-FI) in the functional assay provided alternate functional evidence
of these derivatives ability to modulate Wnt signaling. Four of the
3-pyridyl analogs (4c–f) invoked a fourfold induction of luciferase
Recent disclosures of the diphenylsulfonyl sulfonamide scaffold
as sFRP-1 inhibitors have been reported.8,9,11,12 Substitution on the
piperidinyl group of the 4-sulfonamidopiperidinyl derivative 1 re-
sulted in inhibitors, such as compound 2, which possessed potent
binding affinity to sFRP-1 as well as improved potency in our cell
and tissue based assays.9 These results prompted us to explore fur-
ther optimization. Expansion of the N-substituted diphenylsulfone
sulfonamide scaffold was facilitated by the use of 1 as an interme-
diate core from which several amides, sulfonamides, N-aryl and
alkylated derivatives were prepared. A competitive binding assay
as well as a cellular functional assay were devised to evaluate
the small molecule inhibitors. The binding affinity of the com-
pounds to purified sFRP-1 protein was initially determined using
a tryptophan quenching assay. A rapid and more efficient compet-
itive binding assay was developed using a fluorescein derivative of
compound 1.10,11c The cellular assay was derived from a U2-OS cell
line that was virally infected with human sFRP-1, Wnt-3, and a TCF
luciferase reporter. The cell-based activity (Wnt signaling) was
measured as a function of luciferase production (fold induction,
FI) after incubation of the cells with a test compound for 16–
18 h. The functional activity was assessed in a dose–response for-
mat (EC50). As a means of comparing the relative activity, constant
fold induction thresholds were selected and the concentration of
the sFRP-1 inhibitor was monitored. The fold induction constant
values were extrapolated from the dose–response curve. Generally,
there was good correlation between the binding affinity (IC50) and
the functional dose–response assays. In addition to the target pro-
file, solubility, cytochrome p450 inhibition, and microsomal stabil-
signal at concentrations of 0.2 lM or less. Additional amino pyrid-
inyl derivatives of 4f that reduced the number of rotatable bonds
while increasing the steric bulk were explored. A decline in binding
and functional activity was observed with 3-pyridyl derivatives in
which the dimethylamino group of 4f was replaced with heterocy-
cles such as the pyrrolidino 4g (IC50 0.87
lM, EC50 0.23 lM) or
morpholino 4h (IC50 0.13 M, EC50 0.14 M).
l
l
Summarized in Table 2 for compounds 4c–h are the calculated
properties for the ‘R’ groups as well as the in vitro solubility and
microsomal stability profiles of these compounds. The calculated
molar refractivity (cMR) for compounds 4g and 4h increases signif-
icantly relative to analogs 4d–f, which suggests that an increase in
steric bulk may partially account for the decrease in binding affin-
ity. However, a multivariate combination of molecular properties
may account for the differentiation observed between 4g and 4f.
In addition to an increase in the cMR, the c log P of the pyrrolidino
group is increased whereas the morpholino group shares the same
cMR value albeit with a c log P value analogous to compounds 4d–
f.
As demonstrated in Table 2, the pyridyl derivatives provided an
ionizable group to facilitate aqueous solubility (4c, aqueous solu-
bility 78
lM) while the substituted pyridyl targets exhibited
diminished solubility in aqueous pH 7.4 buffer (<10
lM). As antic-
ipated, the pyridyl derivatives possessed poor stability in phase I
metabolizing microsomes, with the exception of the derivatives

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
193
Table 1
Comparison of aryl/heteroaryl carbonyl/sulfonyl derivatives 3a–f and 4a–h using fluorescence polarization binding and U2-OS functional activity assays
O
O
S
O
S
N
Ar
N
H
O
O
CF3
3a-f, 4a-h
Compound
Ar = phenyl
FP bindinga
(
l
M)
Wnt-lucb
2-FIc
(lM)
IC50
Max concn
EC50
4-FIc
3a
3b
3c
3d
3e
3f
H
2-Cl
3-Cl
4-Cl
4-CF3
4-N(CH3)2
0.07
0.20
0.30
0.30
0.40
0.30
12.5
6.3
6.3
3.1
6.3
50
0.25
1.9
0.87
1.8
0.97
0.19
0.24
0.49
0.49
0.85
0.53
0.15
NAd
2.6
1.4
2.4
146
NAd
Ar = pyridine
4a
4b
4c
4d
4e
4f
4-yl
2-yl
3-yl
6-Cl-3-yl
6-(CF3)-3-yl
6-N(CH3)2-3-yl
6-(Pyrrolidino)-3-yl
6-(Morpholino)-3-yl
0.20
0.06
0.08
0.11
0.05
0.04
0.87
0.13
50
100
50
0.60
0.23
0.12
0.14
0.14
0.07
0.23
0.14
0.14
0.07
0.04
0.06
0.02
0.03
0.10
0.08
0.57
0.41
0.22
0.17
0.16
0.18
0.68
0.39
25
12.5
12.5
12.5
25
4g
4h
a
The affinity of test compounds for sFRP-1 was determined using the FP binding assay. The dose–responses were run in duplicate and IC50’s averaged. The mean confidence
value (CV) for the binding assay IC50 determinations was 15%.
b
The osteosarcoma cell line, U2-OS, was used for the functional assay.
Micromolar concentration that elicited a twofold (2-FI) or fourfold (4-FI) increase in Wnt signaling. Each point from the dose–response was measured in quadruplicate.
c
The standard deviations for the Wnt-luc assay were typically 9% of the mean or less.
d
A fourfold induction of the luciferase signal was not achieved (NA).
Table 2
Calculated R-group properties, aqueous solubility and liver microsomal stability for 3-pyridinyl derivatives
O
O
S
O
S
N
N
H
N
R
O
O
CF3
4c-h
R group propertiesa
cMR
Solubility pH 7.4b
(lM)
Liver microsome t1/2 (min)
c
Compound
R
c log P
PSA
Human
Rat
4c
4d
4e
4f
4g
4h
H
Cl
CF3
0
0
0
0
0
3
3
78
6
2
9
16
2
2
8
0.7
0.9
0.7
1.5
0.4
0.5
0.5
1.3
2.0
2.2
1.7
3.2
6.7
9.6
4.7
>30
>30
3
4
5
N(CH3)2
Pyrrolidin-1-yl
Morpholin-4-yl
12
a
b
c
Daylight method was used to calculate c log P and cMR values and PSAs were calculated from an internally developed algorithm.
Solubility was assessed in an aqueous buffer from a DMSO stock solution.
Human and rat liver microsomes were incubated with 1 lM DMSO stock solution of compounds.
containing electron withdrawing groups (t1/2 >30 min). The trends
emerging from the SAR were directing toward targets with subop-
timal physical properties (e.g., limited aqueous solubility) thus we
were prompted to examine an alternate ionizable groups to pro-
mote aqueous solubility. Functionality such as saturated heterocy-
cles (8), alcohols (7) or deprotonatable species such as carboxylic
acids (5 or 6) were investigated.
The saturated heterocyclic analogs shown in Table 3 (8a–l) are
related to compound 2. Compounds 8c and 8i were previously dis-
closed, are direct precursors to 2, and were included for SAR com-
parison. In the case of the six-membered heterocycles, removal of
the carbamate group resulted in 2.5–5-fold improved binding and
functionally potent inhibitors, 8g (IC50 0.12 M, EC50 0.15 M) and
8h (IC50 0.10 M, EC50 0.21 M). The linker, n = 0 or 1, did not have
a significant effect on target activity. The basic piperidinyl moiety
in compounds 8g and 8h was fortuitous since this functionality
l
l
l
l
provided improved aqueous solubility (>100 lM) as well as stabil-
ity in the microsomal assay for rat and human (>30 min), as indi-
cated in Table 4. Conversely, the pyrrolidine scaffold containing
the tertiary butyl carbamate appendage was better suited relative
to the basic secondary nitrogen. In the previous disclosure, we
indicated that the S-enantiomer 8c was favored, 2–3-fold, over

194
W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
Table 3
bias, albeit in favor of the R-isomer. The assignment of stereochem-
istry based on commercially available chiral intermediates and the
procedures used to couple the two entities would not be expected
to invert the established stereochemistry. Also consistent with our
earlier disclosure, the carbamates or acylated derivatives had lim-
ited aqueous solubility. The carbamate also rendered these deriva-
tives with lower stability in liver microsomes. Alternatively, the
deprotected derivatives 8g–l generally demonstrated moderate to
Comparison of piperidinyl/pyrrolidinyl carbonyl derivatives 8a–l using fluorescence
polarization binding and U2-OS functional activity assays
R1
O
N
O
R1
( )n
N
O
S
O
S
N
N
N
N
O
O
H
H
CF3
8a-b, 8g-h
8c-f, 8i-l
good aqueous solubility (77 to >100 lM) and like the piperidinyl
analogs were stable in the presence of liver microsomes. Interest-
ingly, the regioisomers from the 3-substituted pyrrolidine moiety
(8k, 8l) were more stable (t1/2 >30 min) in the microsomal stability
assay than the 2-substituted isomers (8i, 8j).
Substituted aryl sulfonamides and a direct linked N-aryl group
containing a carboxyl group at either the meta (5b, 6a) or para
(5c, 6b) positions were evaluated and the data is summarized
in Table 5. The unsubstituted aryl sulfonamide 5a exhibited rela-
tively good binding and functional activity. Addition of a carbox-
ylic acid at the meta 5b or para 5c positions was well tolerated
and the two sulfonyl benzoic acid derivatives demonstrated
Compound
n
R1
Isomer
FP bindinga
(
lM)
Wnt-lucb
(lM)
IC50
Max concn EC50 2-FIc 4-FIc
8a
8b
8c
8d
8e
8f
8g
8h
8i
0
1
Boc
Boc
0.30
25
0.42
0.40
0.06
0.21
0.15
0.08
0.15
0.21
0.35
0.71
0.70
0.38
0.28
0.16
0.02
0.09
0.06
0.03
0.16
0.06
0.17
0.34
0.22
0.12
NA
0.49 12.5
0.06 25
0.12 25
0.52
0.16
0.97
0.26
0.14
NA
0.25
2.0
3.0
Boc 2S
Boc 2R
Boc 3S
Boc 3R
H
0.07
0.05
25
12.5
0
1
0.12 100
0.10 100
0.11 50
H
H
H
H
H
2S
2R
3S
3R
8j
8k
8l
0.20
0.04
0.06
25
25
12.5
2.1
1.6
equivalent binding affinity (IC50 0.02
compound 5a (IC50 0.13 M, EC50 0.21
fold separation differentiated the functional activity (EC50 0.19
and 0.11 M, respectively). Comparison of compounds 5b or 5c,
lM), 6.5-fold better than
l
lM) while less than a two-
a–c
See Table 1.
l
to the direct linked or nonsulfonyl benzoic acids, 6a and 6b, dem-
onstrated a 5–6-fold decrease in binding with only a slight de-
cline in potency. The functional efficacy for acid derivatives 5b
and 6b resulted in a fourfold induction at concentrations that
were representative of the functional potency. Replacement of
the carboxylic acid with the tetrazole moiety provided compound
5e. The bioisostere had respectable binding affinity (IC50
Table 4
Aqueous solubility and microsomal stability for heterocyclic derivatives
Solubility pH 7.4a
(lM)
Liver microsome t1/2 (min)
b
Compound
Human
Rat
8g
8h
8i
8j
8k
8l
>100
>100
>100
>100
77
>30
>30
19
15
>30
NA
>30
>30
24
9
>30
>30
0.03
1.2
l
M), but significantly decreased functional potency (EC50
l
M). The nitrile 5d (IC50 0.09 M, EC50 0.17 M) possessed
l
l
binding and functional activity that was comparable to the parent
sulfonamide 5a or the acids (5b and 5c), the distinction was more
pronounced in the concentration required to induce a fourfold
70
a,b
induction (>0.5 lM).
See Table 2.
The realization that a group such as a carboxylic acid was toler-
ated led to the design of targets intended to provide aqueous solu-
bility while reducing the molecular weight. Table 6 summarizes
the aliphatic N-alkyl derivatives as well as the N-acyl derivatives.
The acids 7b, 7d, and 7e exhibited moderate binding (IC50 0.2–
the R-enantiomer but not to the extent that was traditionally ob-
served in a eutomer/distomer relationship of a targeted receptor.
We also described substitutions that were tolerated on this group
that resulted in the identification of 2. The binding and functional
data for stereo isomers 8e and 8f, indicated that a modest chiral
0.5
lM) to sFRP-1 and functional potency (EC50 0.4–0.57 lM) that
was more analogous to compound 1. The drop in activity for com-
Table 5
Comparison of aryl/heteroaryl carbonyl/sulfonyl derivatives 3a–f and 4a–i using fluorescence polarization binding and U2-OS functional activity assays
X
O
S
O
S
N
Ar
N
O
O
H
CF3
5a-e, 6a-b
Compound
X
Ar = phenyl
FP bindinga
(
l
M)
Wnt-lucb
2-FIc
(lM)
IC50
Max concn
EC50
4-FIc
5a
5bd
5c
5d
5e
6a
6b
SO2
SO2
SO2
SO2
SO2
Bond
Bond
H
0.13
0.02
0.02
0.09
0.03
0.10
0.12
25
0.21
0.19
0.11
0.17
1.2
0.12
0.083
0.10
0.096
0.39
0.27
NAe
0.18
NAe
0.57
2.9
3-CO2H
4-CO2H
3-CN
3-(Tetrazol-5-yl)
3-CO2H
4-CO2H
12.5
12.5
6.3
100
100
25
0.35
0.23
0.41
0.22
0.061
a–c
See Table 1.
d
Average from 13 independent dose response determinations.
NA, data was not available.
e

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
195
Table 6
Comparison of alkyl/acylated carboxylic acids and alcohols 7a–g using fluorescence polarization binding and U2-OS functional activity assays
X
R1
( )n
O
S
O
S
N
N
O
O
H
CF3
7a-g
Compound
n
R1
X
FP bindinga
(
l
M)
Wnt-lucb
2-FIc
(lM)
IC50
Max concn
EC50
4-FIc
7a
7b
7c
7d
7e
7f
0
0
1
2
3
1
1
CONH2
CO2H
CO2H
CO2H
CO2H
CH2
CH2
CH2
CO
CO
CH2
CH2
0.06
0.19
0.9
0.27
0.50
0.50
0.20
100
100
100
25
50
100
50
0.31
0.57
1.2
0.40
0.57
0.85
0.63
0.18
0.41
0.99
0.35
0.33
0.12
0.15
NA
NA
NAd
NA
NA
0.52
0.64
CH2OH
C(CH3)2OH
7g
a–c
See Table 1.
d
Maximum fold induction achieved was 3-FI at 19 lM.
(P90 lM). The acids were associated with good stability in hu-
Table 7
man and rat liver microsomes. The alcohols, 7f and 7g, provided
improved aqueous solubility but little improvement in micro-
somal stability.
Aqueous solubility and microsomal stability for carboxylic acids and alcohols
b
Compound
Solubility pH 7.4a
(lM)
Liver microsome t1/2 (min)
Human
Rat
Select sFRP-1 inhibitors based on the potent activity observed
in the functional assay, compounds 4c, 4g, 5b, 5c, and 8h were
evaluated in the ex vivo mouse calvaria assay.14 As described in
our previous disclosure, screening concentrations in the ex vivo
assay were significantly lower than the observed in vitro potency.
Evidence of anabolic activity such as increased bone formation
and remodeling, osteoblast activation and differentiation, and in-
creases in the osteoblast population were determined by histo-
logical assessment. Treatment of the calvaria with either 100 or
1 nM DMSO stock solutions of the sFRP-1 inhibitors resulted in
significant increases in total bone area relative to the controls
as shown in Table 8. For the three basic molecules 4c, 4g, and
8h significant increases in osteoblast population were observed,
whereas for the two aryl carboxylic acids, 5b and 5c, no increase
in the osteoblast numbers was observed. In each treatment group
the qualitative histology assessment indicated activation of the
osteoblast cells and active bone remodeling. The significance of
increasing osteoblast numbers in the ex vivo calvaria model
and the relationship to bone related disease has not been rigor-
ously established. However, typical treatment of the calvaria with
hPTH(1–34) under the same conditions results not only in an in-
crease in the total bone area, but also a dramatic increase in the
number of osteoblast cells. Data from a representative experi-
ment conducted with hPTH(1-34) are shown in Table 8 for
comparison.
5b
6a
7b
7d
7f
>100
>100
90
95
>100
>100
>30
>30
>30
>30
3
23
17
>30
>30
7
7g
<1
3
a
Solubility was assessed in an aqueous buffer from a DMSO stock solution.
Human and rat liver microsomes were incubated with
solution of compounds.
b
1 lM DMSO stock
Table 8
Anabolic activity of sFRP-1 inhibitors demonstrated in an ex vivo calvaria tissue
culture assay
Compound
Concentration (
lM)
% Increase relative to control
TBA (SE)
OB #s (SE)
hPTH (1–34)
4c
4f
5b
5c
8h
0.001
0.1
0.001
0.001
0.001
0.001
48 (19)b
65 (7)a,d
135 (21)a,d
56 (11)a,d,f
49 (8)a,e
87 (11)a
36 (8)b
78 (15)b
0
0
47 (9)b,e
27 (5)c
a
b
c
d
e
f
p <0.0001.
p <0.01.
p <0.05.
Number of calvaria = 4.
Number of calvaria = 5.
Average of five experiments.
4. Conclusion
We have presented an expanded SAR disclosure of the pipe-
ridinyl diphenylsulfonyl sulfonamide scaffold. The in vitro and
ex vivo data provided evidence of sFRP-1 inhibition, which mod-
ulated Wnt signaling through perturbation of the protein–pro-
tein interaction between Wnt and sFRP-1. Compound 1 (IC50
pound 7c (IC50 0.9 lM, EC50 1.2 lM) relative to the corresponding
derivatives was attributed to the proximity of the piperidinyl nitro-
gen to the acid. The two groups are situated to form a spirocyclic
hydrogen bond, and a zwitterionic species that diminishes the
interaction with the protein. Binding and functional activity were
restored to levels comparable to 7b when the acidity of the pro-
ton was reduced in the form of either a primary amide 7a or as
the primary or tertiary alcohols, 7f and 7g. The presence of a car-
boxylic acid group was desired to provide an opportunity to im-
prove aqueous solubility and Table 7 highlights some selected
acids and alcohols that were previously described. As expected,
the aqueous solubility of the acid derivatives was generally good
0.5 lM, EC50 0.65 lM) allowed us the opportunity to probe var-
ious functionality on the novel diphenylsulfonyl sulfonamide
scaffold to improve the drug-like properties. Several compounds
described herein have demonstrated potent binding affinity
(IC50 6 100 nM) to the sFRP-1 protein. In a cell-based Wnt sig-
naling assay many of the molecules demonstrated good binding
affinity and potent functional activity (EC50 70–150 nM). Five
sFRP-1 inhibitors increased total bone area (47–135%) relative

196
W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
to the vehicle control in an ex vivo mouse calvaria model,
wherein parameters of anabolic activity such as activation of
osteoblasts and active bone remodeling were observed. In partic-
organic layers were washed with brine several times, dried
(Na2SO4), filtered and concentrated. Flash column separation
using hexane/ethyl acetate (30–100% gradient provided the de-
sired amide.
ular, compound 4f (IC50 0.04 lM, EC50 0.07 lM) demonstrated
robust activity in the ex vivo model, not only increased total
bone area (135%) but also promoted an increase in the popula-
tion of bone forming osteoblasts (78%). Other aspects of anabolic
activity such as osteoblast activation and active remodeling were
also coupled with this activity. While compound 4f had a good
in vitro and ex vivo profile, it demonstrated low stability in
the human and rat liver microsomal assays (t1/2 3 and 2 min,
respectively) and would be predicted to have limited oral bio-
availability due to first pass metabolism. Compound 8h (IC50
5.1.3. General procedure for the EDC coupling of an acid to 1
using triethylamine
To a stirred solution of 5-oxo-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester (0.075 g, 0.32 mmol) in methylene chloride
(3 mL) was added DMAP (0.04 g, 0.32 mmol) and EDC (0.07 g,
0.035 mmol). The resulting solution was stirred at room tempera-
ture for 20 min at which time 1 (0.12 g, 0.27 mmol) was added and
stirred overnight at room temperature. The reaction was washed
with saturated aqueous ammonium chloride solution and concen-
trated. Flash column separation using 0–8% methanol/methylene
chloride gradient gave 4c (0.10 g, 56%).
0.10 lM, EC50 0.21 lM) demonstrated robust activity in the
ex vivo model, also regulating bone formation (47%) as well as
increasing OB numbers (27%). This piperidinyl derivative also
demonstrated good aqueous solubility (>100
in the human and rat liver microsomal assays (t1/2 >30 min).
The benzoic acid derivative, 5b (IC50 0.02 M, EC50 0.19 M)
lM) and stability
5.1.3.1. N-(1-Benzoylpiperidin-4-yl)-5-(phenylsulfonyl)-2-trifluo-
romethyl)benzenesulfonamide (3a). 1H NMR (400 MHz, DMSO-
d6) d 8.61 (d, J = 6.50 Hz, 1H), 8.58 (d, J = 1.56 Hz, 1H), 8.41 (dd,
J = 1.04, 8.32 Hz, 1H), 8.23 (d, J = 8.32 Hz, 1H), 8.00–8.05 (m, 2H),
7.59–7.72 (m, 3H), 7.43–7.50 (m, 3H), 7.32–7.38 (m, 2H), 4.20 (br
s, 1H), 3.27–3.48 (m, 2H), 2.73–3.05 (m, 2H), 1.21–1.70 (m, 4H);
MS (ES) m/z 553.1; HRMS: calculated for C25H23F3N2O5S2 + H+,
553.10732; found (ESI, [M+H]+), 553.1075; HPLC purity 98.6%, RT
9.4 min.
l
l
was also very active in the calvaria assay, but was ineffective
at increasing the number of osteoblasts. Appending an acid
group to the aryl sulfonamides also resulted in increased aque-
ous solubility (>100 lM) as well as improved microsomal stabil-
ity affording extended half-lives in the rat (23 min) and human
(>30 min) liver microsomal assays.15 Future communications
we will detail additional SAR studies of related scaffolds.
5.1.3.2. N-[1-(2-Chlorobenzoyl)piperidin-4-yl]-5-(phenylsulfo-
nyl)-2-(trifluoromethyl)benzenesulfonamide (3b). 1H NMR
(400 MHz, DMSO-d6) d 8.61 (dd, J = 7.54, 11.70 Hz, 1H), 8.57 (d,
J = 1.56 Hz, 1H), 8.41 (dd, J = 1.17, 8.19 Hz, 1H), 8.23 (d, J = 8.58 Hz,
1H), 8.00–8.05 (m, 2H), 7.60–7.71 (m, 3H), 7.50–7.58 (m, 1H),
7.34–7.49 (m, 2H), 7.30 (dd, J = 1.82, 7.28 Hz, 1H), 4.18–4.32 (m,
1H), 3.29–3.42 (m, 1H), 3.02–3.15 (m, 1H), 2.76–2.98 (m, 2H),
1.57–1.66 (m, 1H), 1.19–1.49 (m, 3H); MS (ES) m/z 587.0; HRMS: cal-
culated for C25H22ClF3N2O5S2 + H+, 587.06835; found (ESI, [M+H]+),
587.0666; HPLC purity 93.6%, RT 9.6 min.
5. Experimental procedures
5.1. Chemistry
Solvents were purchased as anhydrous grade and used without
further purification. Reagents were purchased from commercial
sources and used as received. 1H NMR spectra were recorded on
a Varian Inova 400 instrument with chemical shifts reported in d
values (parts per million, ppm) relative to an internal standard
(tetramethylsilane). High-resolution mass spectra were obtained
on an Agilent 6210 TOF. Low-resolution electrospray (ESI) mass
spectra were recorded on an Aglient MSD. HPLC analyses were ob-
tained on an Agilent 1200 using the following conditions: Waters
5.1.3.3. N-[1-(3-Chlorobenzoyl)piperidin-4-yl]-5-(phenylsulfonyl)-
2-(trifluoromethyl)benzenesulfonamide (3c). 1H NMR (400 MHz,
DMSO-d6) d 8.56–8.64 (m, 2H), 8.38–8.45 (m, 1H), 8.23 (d, J = 8.32
Hz, 1H), 8.00–8.07 (m, 2H), 7.61–7.73 (m, 3H), 7.52–7.56 (m, 1H),
7.47–7.52 (m, 1H), 7.42 (t, J = 1.56 Hz, 1H), 7.31 (dt, J = 1.36, 7.41 Hz,
1H), 4.09–4.27 (m, 1H), 3.24–3.41 (m, 2H), 2.74–3.06 (m, 2H), 1.42–
1.70 (m, 2H), 1.22–1.41 (m, 2H); MS (ES) m/z 587.0; HRMS: calculated
for C25H22ClF3N2O5S2 + H+, 587.06835; found (ESI, [M+H]+), 587.0674;
HPLC purity 98.4%, RT 9.9 min.
Xterra RP18 HPLC column (3.5
l
, 150 mm L ꢀ 4.6 mm ID), 40 °C
column temperature, 1.2 mL/min flow rate, photodiode array
detection (210–370 nm), linear mobile phase gradient of 15–95%
B over 10 min, holding 5 min at 95% B (mobile phase A: 10 mM
ammonium formate in water, pH 3.5/mobile phase B: 1:1 metha-
nol/acetonitrile).
5.1.1. General procedure for the coupling of an acid or sulfonyl
chloride to 1 using triethylamine
5.1.3.4. N-[1-(4-Chlorobenzoyl)piperidin-4-yl]-5-(phenylsulfonyl)-
2-(trifluoromethyl)benzenesulfonamide (3d). 1H NMR (400 MHz,
DMSO-d6) d 8.61 (d, J = 6.76 Hz, 1H), 8.58 (d, J = 1.82 Hz, 1H), 8.41 (dd,
J = 1.04, 8.32 Hz, 1H), 8.23 (d, J = 8.32 Hz, 1H), 8.00–8.06 (m, 2H),
7.68–7.73 (m, 1H), 7.62–7.67 (m, 2H), 7.51–7.55 (m, 2H), 7.37–7.41
(m, 2H), 4.17 (br s, 1H), 3.26–3.49 (m, 2H), 2.72–3.08 (m, 2H), 1.43–
1.70 (m, 2H), 1.22–1.41 (m, 2H); MS (ES) m/z 587.0; HRMS: calculated
for C25H22ClF3N2O5S2 + H+, 587.06835; found (ESI, [M+H]+), 587.0698;
HPLC purity 98.2%, RT 9.9 min.
To a stirred solution of 1 (0.22 mmol) in methylene chloride
(3 mL) with triethylamine (0.70 mmol) was added ArCOCl or Ar-
SO2Cl (0.22 mmol) and the resulting solution was stirred overnight
at room temperature. The reaction mixture was washed with water
and concentrated. Product was purified using flash column separa-
tion using either a methylene chloride/methanol (1–10%) or hex-
ane/ethyl acetate (5–50%) gradient.
5.1.2. General procedure for the carbonyldiimidazole coupling
of an acid to 1 using triethylamine
The acid (0.70 mmol) was activated in the presence of CDI
(0.68 mmol) and triethylamine (0.14 mmol) in DMF (2 mL) for
30 min followed by the addition of 1 (0.45 mmol). The resulting
solution was heated to 100 °C for 15 min. The mixture was
diluted with a saturated aqueous ammonium chloride solution
and extracted several times with ethyl acetate. The combined
5.1.3.5. 5-(Phenylsulfonyl)-2-(trifluoromethyl)-N-{1-[4-(trifluo-
romethyl)benzoyl]piperidin-4-yl}benzenesulfonamide (3e). 1H
NMR (400 MHz, DMSO-d6) d 8.62 (br s, 1H), 8.58 (d, J = 1.82 Hz, 1H),
8.41(dd, J = 1.17, 8.45 Hz, 1H), 8.23 (d, J = 8.32 Hz, 1H), 7.99–8.07(m,
2H), 7.84 (d, J = 8.06 Hz, 2H), 7.61–7.73 (m, 3H), 7.58 (d, J = 7.80 Hz,
2H), 4.11–4.29 (m, 1H), 3.25–3.42 (m, 2H), 2.76–3.07 (m, 2H),

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
197
1.44–1.70 (m, 2H), 1.22–1.43 (m, 2H); MS (ES) m/z 621.1; HRMS: cal-
culated for C26H22F6N2O5S2 + H+, 621.09471; found (ESI, [M+H]+),
621.0933; HPLC purity 100%, RT 10.0 min.
1H), 1.48–1.59(m, 1H), 1.30–1.47(m, 2H);MS(ES) m/z621.9;HRMS:
calculated for C25H21F6N3O5S2 + H+, 622.08996; found (ESI, [M+H]+),
622.0897; HPLC purity 100.0%, RT 9.5 min.
5.1.3.6. N-{1-[4-(Dimethylamino)benzoyl]piperidin-4-yl}-5-
(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (3f).
1H NMR (400 MHz, DMSO-d6) d 8.55–8.64 (m, 2H), 8.37–8.45
(m, 1H), 8.23 (d, J = 8.46 Hz, 1H), 7.99–8.07 (m, 2H), 7.60–7.73
(m, 3H), 7.20–7.28 (m, 2H), 6.76 (d, J = 8.46 Hz, 2H), 3.73–4.04
(m, 2H), 3.26–3.39 (m, 1H), 2.96 (s, 6H), 2.80–2.92 (m, 2H),
1.45–1.57 (m, 2H), 1.20–1.36 (m, 2H); MS (ES) m/z 595.8; HRMS:
calculated for C27H28F3N3O5S2 + H+, 596.14952; found (ESI,
[M+H]+), 596.1509; HPLC purity 95.8%, RT 9.7 min.
5.1.3.12. N-(1-{[6-(Dimethylamino)pyridin-3-yl]carbonyl}pipe-
ridin-4-yl)-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfo-
namide (4f). 1H NMR (400 MHz, DMSO-d6) d 8.65 (d, J = 7.17 Hz,
1H), 8.59 (d, J = 1.79 Hz, 1H), 8.42 (dd, J = 1.02, 8.20 Hz, 1H), 8.24
(d, J = 8.46 Hz, 1H), 8.01–8.11 (m, 3H), 7.71–7.79 (m, 2H), 7.63–
7.71 (m, 2H), 6.97 (br s, 1H), 3.69–3.98 (m, 2H), 3.27–3.43 (m,
1H), 3.16 (s, 6H), 2.93 (m, 2H), 1.52–1.65 (m, 2H), 1.28–1.45 (m,
2H); MS (ES) m/z 597.0; HRMS: calculated for C26H27F3N4O5S2 + H+,
597.14477; found (ESI, [M+H]+), 597.1453; HPLC purity 100%, RT
8.8 min.
5.1.3.7. 5-(Phenylsulfonyl)-N-[1-(pyridin-2-ylcarbonyl)piperidin-
4-yl]-2-(trifluoromethyl)benzenesulfonamide (4a). 1H NMR
(400 MHz, DMSO-d6) d 8.64 (d, J = 7.43 Hz, 1H), 8.61 (ddd, J = 1.02,
1.79, 4.87 Hz, 1H), 8.59 (d, J = 1.79 Hz, 1H), 8.39–8.45 (m, 1H), 8.24
(d, J = 8.20 Hz, 1H), 8.01–8.06 (m, 2H), 7.95 (td, J = 1.79, 7.69 Hz, 1H),
7.67–7.73 (m, 1H), 7.61–7.67 (m, 2H), 7.53–7.57 (m, 1H), 7.50 (ddd,
J = 1.28, 4.87, 7.69 Hz, 1H), 4.17–4.28 (m, 1H), 3.45–3.59 (m, 1H),
3.31–3.44 (m, 1H), 2.90–3.04 (m, 1H), 2.76–2.87 (m, 1H), 1.59–1.68
(m, 1H), 1.42–1.52 (m, 1H), 1.29–1.42 (m, 2H); MS (ES) m/z 554.0;
HRMS: calculated for C24H22F3N3O5S2 + H+, 554.10257; found (ESI,
[M+H]+), 554.1008; HPLC purity 94.8%, RT 8.6 min.
5.1.3.13. 5-(Phenylsulfonyl)-N-{1-[(6-pyrrolidin-1-ylpyridin-3-
yl)carbonyl]piperidin-4-yl}-2-(trifluoromethyl)benzenesulfon-
amide (4g). 1H NMR (400 MHz, DMSO-d6) d 8.65 (d, J = 7.17 Hz,
1H), 8.59 (d, J = 1.54 Hz, 1H), 8.41 (d, J = 9.74 Hz, 1H), 8.24 (d,
J = 8.46 Hz, 1H), 8.01–8.07 (m, 3H), 7.76 (br s, 1H), 7.73–7.79 (m,
1H), 7.64–7.71 (m, 2H), 6.89 (br s, 1H), 3.29–3.79 (m, 8H), 2.96
(br s, 1H), 1.99–2.05 (m, 4H), 1.55–1.66 (m, 2H), 1.31–1.44 (m,
2H); MS (ES) m/z 623.0; HRMS: calculated for C28H29F3N4O5S2 + H+,
623.16042; found (ESI, [M+H]+), 623.1609; HPLC purity 92.8%, RT
8.9 min.
5.1.3.8. 5-(Phenylsulfonyl)-N-[1-(pyridin-3-ylcarbonyl)piperidin-
4-yl]-2-(trifluoromethyl)benzenesulfonamide (4b). 1H NMR (400
MHz, DMSO-d6) d 8.76 (dd, J = 1.67, 5.00 Hz, 1H), 8.71 (dd, J = 0.64,
2.18 Hz, 1H), 8.67 (d, J = 7.17 Hz, 1H), 8.59 (d, J = 1.54 Hz, 1H),
8.39–8.44 (m, 1H), 8.24 (d, J = 8.46 Hz, 1H), 8.00–8.07 (m, 3H),
7.63–7.74 (m, 4H), 4.12–4.28 (m, 1H), 3.28–3.46 (m, 2H), 2.95–
3.12 (m, 1H), 2.79–2.92 (m, 1H), 1.59–1.73 (m, 1H), 1.46–1.58 (m,
1H), 1.30–1.46 (m, 2H); MS (ES) m/z 554.0; HRMS: calculated for
C24H22F3N3O5S2 + H+, 554.10257; found (ESI, [M+H]+), 554.1022;
HPLC purity 93.8%, RT 8.4 min.
5.1.3.14. N-{1-[(6-Morpholin-4-ylpyridin-3-yl)carbonyl]piperi-
din-4-yl}-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfon-
amide (4h). 1H NMR (400 MHz, DMSO-d6) d 8.63 (d, J = 7.43 Hz,
1H), 8.59 (d, J = 1.54 Hz, 1H), 8.39–8.44 (m, 1H), 8.24 (d,
J = 8.46 Hz, 1H), 8.16 (d, J = 1.79 Hz, 1H), 8.00–8.07 (m, 2H), 7.70–
7.76 (m, 1H), 7.61–7.69 (m, 3H), 6.95 (d, J = 8.71 Hz, 1H), 4.49 (br
s, 4H), 3.65–3.75 (m, 4H), 3.52–3.61 (m, 4H), 2.83–3.00 (m, 1H),
1.50–1.61 (m, 2H), 1.25–1.41 (m, 2H); MS (ES) m/z 639.1; HRMS:
calculated for C28H29F3N4O6S2 + H+, 639.15534; found (ESI,
[M+H]+), 639.1536; HPLC purity 98.3%, RT 9.1 min.
5.1.3.9. N-(1-Isonicotinoylpiperidin-4-yl)-5-(phenylsulfonyl)-2-
(trifluoromethyl)benzenesulfonamide (4c). 1H NMR (400 MHz,
DMSO-d6) d 8.66–8.70 (m, 2H), 8.62 (d, J = 7.02 Hz, 1H), 8.58 (d,
J = 1.56 Hz, 1H), 8.41 (dd, J = 1.17, 8.19 Hz, 1H), 8.23 (d, J = 8.32 Hz,
1H), 8.01–8.06 (m, 2H), 7.67–7.73 (m, 1H), 7.62–7.67 (m, 2H),
7.31–7.38 (m, 2H), 4.19 (d, J = 11.44 Hz, 1H), 3.22–3.41 (m, 2H),
2.92–3.05 (m, 1H), 2.78–2.90 (m, 1H), 1.57–1.69 (m, 1H), 1.44–
1.55 (m, 1H), 1.22–1.43 (m, 2H); MS (ES) m/z 554.1; HRMS: calcu-
lated for C24H22F3N3O5S2 + H+, 554.10257; found (ESI, [M+H]+),
554.1038; HPLC purity 99.4%, RT 8.3 min.
5.1.3.15. 5-(Phenylsulfonyl)-N-[1-(phenylsulfonyl)piperidin-4-
yl]-2-(trifluoromethyl)benzenesulfonamide (5a). 1H NMR (400
MHz, DMSO-d6) d 8.54 (d, J = 7.17 Hz, 1H), 8.50 (d, J = 1.54 Hz,
1H), 8.36–8.42 (m, 1H), 8.20 (d, J = 8.20 Hz, 1H), 7.97–8.04 (m,
2H), 7.70–7.80 (m, 4H), 7.59–7.69 (m, 4H), 3.29–3.40 (m, 2H),
2.95–3.13 (m, 1H), 2.25–2.38 (m, 2H), 1.52–1.64 (m, 2H), 1.33–
1.48 (m, 2H); MS (ES) m/z 588.5; HRMS: calculated for
C24H23F3N2O6S3 + H+, 589.07431; found (ESI, [M+H]+), 589.0731;
HPLC purity 98.0%, RT 9.8 min.
5.1.3.16. 3-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl
]sulfonyl}amino)piperidin-1-yl]sulfonyl}benzoic acid (5b). 1H
NMR (400 MHz, DMSO-d6) d 13.59 (br s, 1H), 8.53 (d, J = 7.17 Hz,
1H), 8.49 (d, J = 1.54 Hz, 1H), 8.36–8.41 (m, 1H), 8.23–8.28 (m,
1H), 8.20 (d, J = 8.46 Hz, 1H), 8.16 (t, J = 1.67 Hz, 1H), 7.99–8.03
(m, 2H), 7.96 (dt, J = 1.41, 8.20 Hz, 1H), 7.73–7.84 (m, 2H), 7.60–
7.66 (m, 2H), 3.32–3.42 (m, 2H), 3.00–3.17 (m, 1H), 2.28–2.41
(m, 2H), 1.53–1.64 (m, 2H), 1.33–1.46 (m, 2H); HRMS: calculated
for C25H23F3N2O8S3 + H+, 633.06414; found (ESI, [M+H]+),
633.0627; HPLC purity 100%, RT 9.2 min.
5.1.3.10. N-{1-[(6-Chloropyridin-3-yl)carbonyl]piperidin-4-yl}-
5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (4d).
1H NMR (400 MHz, DMSO-d6) d 8.63 (br s, 1H), 8.59 (d,
J = 1.79 Hz, 1H), 8.44 (d, J = 1.79 Hz, 1H), 8.41 (d, J = 8.20 Hz, 1H),
8.23 (d, J = 8.20 Hz, 1H), 8.01–8.06 (m, 2H), 7.88 (dd, J = 2.31,
8.20 Hz, 1H), 7.70–7.75 (m, 1H), 7.64–7.69 (m, 2H), 7.62 (dd,
J = 0.77, 8.20 Hz, 1H), 4.09–4.27 (m, 1H), 3.26–3.47 (m, 2H), 2.78–
3.12 (m, 2H), 1.47–1.72 (m, 2H), 1.27–1.44 (m, 2H); MS (ES) m/z
588.0; HRMS: calculated for C24H21ClF3N3O5S2 + H+, 588.06360;
found (ESI, [M+H]+), 588.0640; HPLC purity 100%, RT 9.1 min.
5.1.3.17. 4-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl
]sulfonyl}amino)piperidin-1-yl]sulfonyl}benzoic acid (5c). 1H
NMR (400 MHz, DMSO-d6) d 13.51 (br s, 1H), 8.54 (d, J = 7.43 Hz,
1H), 8.49 (d, J = 1.79 Hz, 1H), 8.36–8.42 (m, 1H), 8.20 (d,
J = 8.46 Hz, 1H), 8.14–8.18 (m, 2H), 7.97–8.03 (m, 2H), 7.82 (d,
J = 8.71 Hz, 2H), 7.73–7.79 (m, 1H), 7.60–7.67 (m, 2H), 3.33–3.45
(m, 2H), 3.01–3.16 (m, 1H), 2.31–2.43 (m, 2H), 1.52–1.65 (m,
2H), 1.30–1.47 (m, 2H); MS (ES) m/z 632.5; HRMS: calculated for
5.1.3.11. 5-(Phenylsulfonyl)-2-(trifluoromethyl)-N-(1-{[6-(tri-
fluoromethyl)pyridin-3-yl]carbonyl}piperidin-4-yl)benzenesul-
fonamide (4e). 1H NMR (400 MHz, DMSO-d6) d 8.78 (d, J = 1.79 Hz,
1H), 8.65 (d, J = 6.66 Hz, 1H), 8.59 (d, J = 1.54 Hz, 1H), 8.39–8.43 (m,
1H), 8.24 (d, J = 8.20 Hz, 1H), 8.08–8.12 (m, 1H), 7.99–8.06 (m, 3H),
7.68–7.74 (m, 1H), 7.63–7.68 (m, 2H), 4.14–4.33 (m, 1H), 3.26–
3.49 (m, 2H), 2.98–3.13 (m, 1H), 2.83–2.95 (m, 1H), 1.60–1.72 (m,

198
W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
C25H23F3N2O8S3 + H+, 633.06414; found (ESI, [M+H]+), 633.0644;
HPLC purity 100%, RT 9.2 min.
in 4 N HCl in dioxane solution (1 mL) and stirred overnight. The
solution was concentrated and trituration with ethyl acetate gave
7b (0.09 g, 59%). 1H NMR (400 MHz, DMSO-d6)
d 8.84 (d,
J = 6.92 Hz, 1H), 8.59 (d, J = 1.54 Hz, 1H), 8.41 (dd, J = 1.28,
8.20 Hz, 1H), 8.24 (d, J = 8.46 Hz, 1H), 8.00–8.10 (m, 2H), 7.75–
7.84 (m, 1H), 7.65–7.74 (m, 2H), 4.00 (br s, 2H), 3.34 (br s, 4H),
3.02 (br s, 1H), 1.81 (br s, 4H); HRMS: calculated for
C20H21F3N2O6S2 + H+, 507.08659; found (ESI, [M+H]+), 507.0854;
HPLC purity 100%, RT 6.8 min.
5.1.3.18. N-{1-[(3-Cyanophenyl)sulfonyl]piperidin-4-yl}-5-(phen-
ylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (5d). 1H
NMR (400 MHz, DMSO-d6) d 8.54–8.61 (m, 1H), 8.50 (d, J =
2.05 Hz, 1H), 8.39 (d, J = 9.48 Hz, 1H), 8.18–8.24 (m, 3H), 7.98–
8.06 (m, 3H), 7.83–7.89 (m, 1H), 7.76–7.81 (m, 1H), 7.62–7.69
(m, 2H), 3.36–3.46 (m, 2H), 3.03–3.18 (m, 1H), 2.36–2.46 (m,
2H), 1.54–1.65 (m, 2H), 1.33–1.47 (m, 2H); MS (ES) m/z 613.9;
HPLC purity 98.4%, RT 9.7 min.
5.1.3.24. 3-[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]-
sulfonyl}amino)piperidin-1-yl]propanoic acid (7c). 1H NMR
(400 MHz, DMSO-d6) d 8.78–8.90 (m, 1H), 8.58 (d, J = 1.79 Hz,
1H), 8.42 (dd, J = 1.15, 8.07 Hz, 1H), 8.25 (d, J = 8.20 Hz, 1H),
8.02–8.08 (m, 2H), 7.77–7.83 (m, 1H), 7.68–7.75 (m, 2H), 3.24–
3.46 (m, 4H), 3.18 (br s, 2H), 2.89 (br s, 1H), 2.76 (t, J = 7.43 Hz,
2H), 1.77 (br s, 4H); HRMS: calculated for C21H23F3N2O6S2 + H+,
521.10224; found (ESI, [M+H]+), 521.1035; HPLC purity 97.6%, RT
6.8 min.
5.1.3.19. 5-(Phenylsulfonyl)-N-(1-{[3-(2H-tetrazol-5-yl)phenyl]-
sulfonyl}piperidin-4-yl)-2-(trifluoromethyl)benzenesulfonamide
(5e). To a stirred solution of trimethylaluminum 2 M in toluene
(0.16 mL, 0.32 mmol) and trimethylsilylazide (0.04 g, 0.32 mmol)
in toluene (1 mL) at 0 °C was added 5d (0.15 g, 0.24 mmol). The
resulting solution was heated to 80 °C overnight, cooled to 0 °C,
quenched with 6 N HCl solution and extracted with ethyl acetate.
The organic layer was concentrated and the resulting solid was
flash column separated using a 0–10% MeOH/CH2Cl2 gradient to
5e (0.072 g, 45%). 1H NMR (400 MHz, DMSO-d6) d 8.55 (d,
J = 7.17 Hz, 1H), 8.49 (d, J = 2.05 Hz, 1H), 8.32–8.40 (m, 3H), 8.19
(d, J = 8.46 Hz, 1H), 7.96–8.01 (m, 2H), 7.87–7.93 (m, 2H), 7.69–
7.75 (m, 1H), 7.57–7.65 (m, 2H), 3.36–3.46 (m, 2H), 3.03–3.15 (m,
1H), 2.36–2.44 (m, 2H), 1.55–1.65 (m, 2H), 1.37–1.48 (m, 2H); MS
(ES) m/z 657.0; HRMS: calculated for C25H23F3N6O6S3 + H+,
657.08660; found (ESI, [M+H]+), 657.0853; HPLC purity 93.4%, RT
8.6 min.
5.1.3.25. 4-Oxo-4-[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)-
phenyl]sulfonyl}amino)piperidin-1-yl]butanoic acid (7d).
A
stirred solution of 5-(phenylsulfonyl)-N-piperidin-4-yl-2-(trifluo-
romethyl)benzenesulfonamide (0.10 g, 0.22 mmol) and succinic
anhydride (0.023 g, 0.23 mmol) in EtOH (1 mL) was microwave
irradiated for 10 min at 150 °C. The resulting solution was concen-
trated and flash column separation using 0–10% methanol/methy-
lene chloride gradient afforded 7d (0.04 g, 33%). 1H NMR (400 MHz,
DMSO-d6) d 8.45–8.70 (br s, 1H), 8.58 (d, J = 1.79 Hz, 1H), 8.42 (d,
J = 9.48 Hz, 1H), 8.24 (d, J = 8.20 Hz, 1H), 8.01–8.07 (m, 2H), 7.75–
7.82 (m, 1H), 7.66–7.73 (m, 2H), 4.01–4.13 (m, 1H), 3.62–3.74
(m, 1H), 2.87–2.99 (m, 1H), 2.35–2.43 (m, 2H), 1.52–1.61 (m,
1H), 1.42–1.51 (m, 1H), 1.23–1.38 (m, 1H), 1.09–1.21 (m, 1H);
MS (ESI) m/z 549; HRMS: calculated for C22H23F3N2O7S2 + H+,
549.09715; found (ESI, [M+H]+), 549.095; HPLC purity 100%, RT
8.1 min.
5.1.3.20. 3-[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]
sulfonyl}amino)piperidin-1-yl]benzoic acid (6a). 1H NMR (400
MHz, DMSO-d6) d 8.56–8.64 (m, 2H), 8.40–8.46 (m, 1H), 8.25 (d,
J = 8.46 Hz, 1H), 8.01–8.09 (m, 2H), 7.74–7.81 (m, 1H), 7.66–7.73
(m, 2H), 7.50 (br s, 1H), 7.32–7.44 (m, 2H), 7.19–7.28 (m, 1H),
3.49–3.64 (m, 2H), 3.19–3.34 (m, 1H), 2.68–2.86 (m, 2H), 1.43–
1.69 (m, 4H); HRMS: calculated for C25H23F3N2O6S2 + H+,
569.10224; found (ESI, [M+H]+), 569.1022; HPLC purity 100%, RT
9.7 min.
5.1.3.26. 5-Oxo-5-[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)-
phenyl]sulfonyl}amino)piperidin-1-yl]pentanoic acid (7e). 1H
NMR (400 MHz, DMSO-d6) d 12.01 (br s, 1H), 8.54–8.61 (m, 2H),
8.42 (dd, J = 1.02, 8.20 Hz, 1H), 8.24 (d, J = 8.20 Hz, 1H), 8.01–8.07
(m, 2H), 7.75–7.82 (m, 1H), 7.66–7.73 (m, 2H), 4.04–4.15 (m, 1H),
3.60–3.71 (m, 1H), 3.21–3.29 (m, 1H), 2.85–2.98 (m, 1H), 2.16–
2.35 (m, 4H), 1.63–1.75 (m, 2H), 1.43–1.59 (m, 2H), 1.11–1.34 (m,
2H); HRMS: calculated for C23H25F3N2O7S2 + H+, 563.11280; found
(ESI, [M+H]+), 563.1128; HPLC purity 100%, RT 8.2 min.
5.1.3.21. 4-[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]-
sulfonyl}amino)piperidin-1-yl]benzoic acid (6b). 1H NMR
(400 MHz, DMSO-d6) d 8.50–8.64 (m, 2H), 8.37–8.46 (m, 1H),
8.25 (d, J = 8.20 Hz, 1H), 8.01–8.08 (m, 2H), 7.73–7.82 (m, 3H),
7.64–7.72 (m, 2H), 6.92 (d, J = 9.22 Hz, 2H), 3.64–3.80 (m, 2H),
3.20–3.38 (m, 1H), 2.72–2.86 (m, 2H), 1.52–1.64 (m, 2H), 1.35–
1.48 (m, 2H); HRMS: calculated for C25H23F3N2O6S2 + H+,
569.10224; found (ESI, [M+H]+), 569.0995.
5.1.3.27. N-[1-(2-Hydroxyethyl)piperidin-4-yl]-5-(phenylsulfo-
nyl)-2-(trifluoromethyl)benzenesulfonamide (7f). To a stirred
solution of 1 (0.09 g, 0.20 mmol) in 1,4-dioxane (1 mL) was added
2-bromoethanol (0.05, 0.40 mmol) and triethylamine (0.1 mL,
0.7 mmol). The resulting solution was heated 150 °C in a micro-
wave for 10 min and concentrated. Flash column separation using
0–5% methanol/methylene chloride gradient gave 7f (0.03 g, 30%).
1H NMR (400 MHz, DMSO-d6) d 8.57 (d, J = 1.54 Hz, 1H), 8.39–8.44
(m, 1H), 8.24 (d, J = 8.46 Hz, 1H), 8.01–8.06 (m, 2H), 7.76–7.82 (m,
1H), 7.67–7.73 (m, 2H), 3.53 (br s, 4H), 2.81–3.17 (m, 6H), 1.30–
1.97 (m, 4H); MS (ES) m/z 493.1; HRMS: calculated for
C20H23F3N2O5S2 + H+, 493.10732; found (ESI, [M+H]+), 493.1083;
HPLC purity 98.3%, RT 6.8 min.
5.1.3.22. 2-[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]-
sulfonyl}amino)piperidin-1-yl]acetamide (7a). 1H NMR (400
MHz, DMSO-d6) d 8.51–8.61 (m, 2H), 8.41 (dd, J = 1.03, 8.20 Hz,
1H), 8.23 (d, J = 8.46 Hz, 1H), 7.99–8.07 (m, 2H), 7.74–7.82 (m,
1H), 7.64–7.72 (m, 2H), 7.10 (br s, 2H), 2.89–3.02 (m, 1H), 2.77 (s,
2H), 2.62 (d, J = 12.04 Hz, 2H), 1.83–1.98 (m, 2H), 1.37–1.50 (m,
4H); HRMS: calculated for C20H22F3N3O5S2 + H+, 506.10257; found
(ESI, [M+H]+), 506.103; HPLC purity 98.6%, RT 6.8 min.
5.1.3.23. [4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]-
sulfonyl}amino)piperidin-1-yl]acetic acid (7b). A stirred solu-
tion of 1 (0.14 g, 0.30 mmol) and tert-butylbromoacetate (0.06 g,
0.30 mmol) and triethylamine (0.1 mL, 0.7 mmol) in THF (1 mL)
was microwave irradiated for 10 min at 120 °C. The resulting solu-
tion was concentrated and flash column separation using 0–50%
ethyl acetate/hexane gradient gave a solid. The solid was dissolved
5.1.3.28. N-[1-(2-Hydroxy-2-methylpropyl)piperidin-4-yl]-5-
(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (7g).
To a stirred mixture of 1 (0.075 g, 0.167 mmol) in ethanol
(2 mL) was added isobutylene oxide (0.05 g, 0.69 mmol) and the
resulting mixture was heated to reflux overnight. The crude

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
199
mixture was concentrated and purified using flash column chro-
matography employing 50–100% ethyl acetate/hexane gradient
and afforded 7g (0.06 g, 70%). 1H NMR (400 MHz, DMSO-d6) d
8.57 (d, J = 1.82 Hz, 1H), 8.48 (d, J = 7.28 Hz, 1H), 8.41 (dd,
J = 1.17, 8.19 Hz, 1H), 8.23 (d, J = 8.32 Hz, 1H), 8.01–8.06 (m, 2H),
7.74–7.80 (m, 1H), 7.66–7.72 (m, 2H), 4.02 (s, 1H), 2.85–2.98 (m,
1H), 2.69–2.78 (m, 2H), 2.09 (s, 2H), 1.88–1.98 (m, 2H), 1.35–
1.45 (m, 4H), 1.05 (s, 6H); MS (ES) m/z 519.0; HRMS: calculated
for C22H27F3N2O5S2 + H+, 521.13862; found (ESI, [M+H]+),
521.1403; HPLC purity 96.8%, RT 7.1 min.
was bubbled HCl gas. The resulting solution was stirred at room
temperature overnight, and concentrated. The resulting solid was
triturated with diethyl ether and provided 8g as a white solid
(0.085 g, 85%). 1H NMR (400 MHz, DMSO-d6) d 8.83 (br s, 1H),
8.63 (d, J = 7.17 Hz, 1H), 8.60 (d, J = 1.79 Hz, 1H), 8.46–8.57 (m,
1H), 8.40–8.44 (m, 1H), 8.24 (d, J = 8.20 Hz, 1H), 8.03–8.07 (m,
2H), 7.76–7.81 (m, 1H), 7.67–7.73 (m, 2H), 4.04–4.16 (m, 1H),
3.77–3.88 (m, 1H), 3.20–3.36 (m, 4H), 2.97–3.06 (m, 1H), 2.85–
2.97 (m, 3H), 2.54–2.63 (m, 1H), 1.63–1.80 (m, 5H), 1.49–1.56
(m, 1H), 1.29–1.40 (m, 1H), 1.14–1.26 (m, 1H); MS (ES) m/z
559.8; HRMS: calculated for C24H28F3N3O5S2 + H+, 560.14952;
found (ESI, [M+H]+), 560.1478; HPLC purity 100%, RT 7.2 min.
5.1.3.29. tert-Butyl 4-{[4-({[5-(phenylsulfonyl)-2-(trifluoro-
methyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}piper-
idine-1-carboxylate (8a). 1H NMR (400 MHz, DMSO-d6) d 8.59 (d,
J = 1.54 Hz, 1H), 8.57 (d, J = 7.18 Hz, 1H), 8.42 (dd, J = 1.28, 8.20 Hz,
1H), 8.24 (d, J = 8.46 Hz, 1H), 8.02–8.08 (m, 2H), 7.75–7.81 (m, 1H),
7.66–7.73 (m, 2H), 4.05–4.16 (m, 1H), 3.86–3.99 (m, 2H), 3.75–3.85
(m, 1H), 2.91–3.05 (m, 1H), 2.69–2.87 (m, 4H), 2.52–2.62 (m, 1H),
1.45–1.68 (m, 4H), 1.39 (s, 9H), 1.26–1.37 (m, 3H), 1.10–1.22 (m,
1H); MS (ES) m/z 657.8; HRMS: calculated for C29H36F3N3O7S2 + H+,
660.20195; found (ESI, [M+H]+), 660.2032; HPLC purity 100%, RT
9.8 min.
5.1.4.2. 5-(Phenylsulfonyl)-N-[1-(piperidin-4-ylacetyl)piperidin-
4-yl]-2-(trifluoromethyl)benzenesulfonamide (8h). 1H NMR
(400 MHz, DMSO-d6) d 8.55–8.65 (m, 3H), 8.30–8.46 (m, 2H), 8.24
(d, J = 8.20 Hz, 1H), 8.02–8.07 (m, 2H), 7.75–7.82 (m, 1H), 7.66–
7.73 (m, 2H), 3.66–4.19 (m, 1H), 3.17–3.35 (m, 4H), 2.77–3.01 (m,
4H), 2.54–2.63 (m, 1H), 2.26 (d, J = 6.66 Hz, 2H), 1.88–2.02 (m,
1H), 1.74–1.84 (m, 2H), 1.45–1.68 (m, 1H), 1.10–1.40 (m, 4H); MS
(ES) m/z 573.9; HRMS: calculated for C25H30F3N3O5S2 + H+,
574.16517; found (ESI, [M+H]+), 574.1636; HPLC purity 98.0%, RT
7.3 min.
5.1.3.30. tert-Butyl 4-{2-oxo-2-[4-({[5-(phenylsulfonyl)-2-(tri-
fluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]ethyl}piper-
idine-1-carboxylate (8b). 1H NMR (400 MHz, DMSO-d6) d 8.58
(d, J = 1.79 Hz, 1H), 8.56 (br s, 1H), 8.41 (dd, J = 1.02, 7.94 Hz, 1H),
8.24 (d, J = 8.46 Hz, 1H), 8.02–8.07 (m, 2H), 7.75–7.81 (m, 1H),
7.66–7.73 (m, 2H), 4.07–4.16 (m, 1H), 3.84–3.95 (m, 2H), 3.67–
3.77 (m, 1H), 3.24–3.30 (m, 1H), 2.88–3.00 (m, 1H), 2.61–2.81
(m, 2H), 2.21 (d, J = 6.15 Hz, 2H), 1.77–1.89 (m, 1H), 1.54–1.67
(m, 4H), 1.44–1.52 (m, 1H), 1.38 (s, 9H), 1.25–1.34 (m, 1H), 1.13–
1.21 (m, 1H), 0.95–1.06 (m, 2H); HRMS: calculated for
C30H38F3N3O7S2 + H+, 674.21760; found (ESI, [M+H]+), 674.2167;
HPLC purity 97.7%, RT 10.0 min.
5.1.4.3. 5-(Phenylsulfonyl)-N-{1-[(3S)-pyrrolidin-3-ylcarbonyl]-
piperidin-4-yl}-2-(trifluoromethyl)benzenesulfonamide (8k).
1H NMR (400 MHz, DMSO-d6) d 9.19 (br s, 1H), 9.00 (br s, 1H),
8.65 (dd, J = 7.30, 17.30 Hz, 1H), 8.60 (d, J = 1.54 Hz, 1H), 8.42 (d,
J = 7.94 Hz, 1H), 8.25 (d, J = 8.20 Hz, 1H), 8.02–8.09 (m, 2H), 7.76–
7.83 (m, 1H), 7.67–7.74 (m, 2H), 4.05–4.19 (m, 1H), 3.73–3.82
(m, 1H), 3.42–3.53 (m, 1H), 3.10–3.39 (m, 4H), 2.96–3.08 (m,
1H), 2.60–2.71 (m, 1H), 2.11–2.25 (m, 1H), 1.76–1.90 (m, 1H),
1.60–1.73 (m, 2H), 1.50–1.60 (m, 1H), 1.31–1.42 (m, 1H), 1.18–
1.29 (m, 1H); HPLC purity 90.4%, RT 7.3 min.; HRMS: calculated
for C23H26F3N3O5S2 + H+, 546.13387; found (ESI, [M+H]+),
546.1302.
5.1.3.31. tert-Butyl (3S)-3-{[4-({[5-(phenylsulfonyl)-2-(trifluo-
romethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyr-
rolidine-1-carboxylate (8e). 1H NMR (400 MHz, DMSO-d6) d
8.53–8.61 (m, 2H), 8.42 (dd, J = 1.15, 8.33 Hz, 1H), 8.24 (d,
J = 8.46 Hz, 1H), 8.01–8.09 (m, 2H), 7.75–7.82 (m, 1H), 7.65–7.73
(m, 2H), 4.05–4.16 (m, 1H), 3.74–3.86 (m, 1H), 3.17–3.47 (m,
5H), 2.92–3.06 (m, 1H), 2.54–2.64 (m, 1H), 1.76–2.03 (m, 3H),
1.57–1.66 (m, 1H), 1.45–1.56 (m, 1H), 1.36–1.43 (m, 9H), 1.28–
1.35 (m, 1H), 1.15–1.23 (m, 1H); HRMS: calculated for
C28H34F3N3O7S2 + H+, 646.18630; found (ESI, [M+H]+), 646.1891;
HPLC purity 100.0%, RT 9.8 min.
5.1.4.4. 5-(Phenylsulfonyl)-N-{1-[(3R)-pyrrolidin-3-ylcarbonyl]-
piperidin-4-yl}-2-(trifluoromethyl)benzenesulfonamide (8l).
1H NMR (400 MHz, DMSO-d6) d 9.00 (br s, 2H), 8.56–8.70 (m, 2H),
8.42 (d, J = 8.71 Hz, 1H), 8.25 (d, J = 8.20 Hz, 1H), 8.01–8.08 (m, 2H),
7.75–7.84 (m, 1H), 7.65–7.74 (m, 2H), 4.03–4.16 (m, 1H), 3.72–3.82
(m, 1H), 3.43–3.53 (m, 1H), 3.10–3.41 (m, 4H), 2.96–3.08 (m, 1H),
2.62–2.72 (m, 1H), 2.10–2.24 (m, 1H), 1.75–1.90 (m, 1H), 1.61–
1.73 (m, 2H), 1.52–1.60 (m, 1H), 1.30–1.44 (m, 1H), 1.17–1.29 (m,
1H); HRMS: calculated for C23H26F3N3O5S2 H+, 546.13387; found
(ESI, [M+H]+), 546.1336; HPLC purity 95.7%, RT 7.3 min.
5.1.3.32. tert-Butyl (3R)-3-{[4-({[5-(phenylsulfonyl)-2-(trifluo-
romethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyr-
rolidine-1-carboxylate (8f). 1H NMR (400 MHz, DMSO-d6) d
8.53–8.61 (m, 2H), 8.42 (d, J = 8.71 Hz, 1H), 8.24 (d, J = 8.46 Hz,
1H), 8.01–8.08 (m, 2H), 7.75–7.82 (m, 1H), 7.63–7.74 (m, 2H),
4.06–4.15 (m, 1H), 3.73–3.87 (m, 1H), 3.17–3.47 (m, 5H), 2.92–
3.04 (m, 1H), 2.54–2.63 (m, 1H), 1.74–2.01 (m, 3H), 1.57–1.67
(m, 1H), 1.47–1.56 (m, 1H), 1.37–1.42 (m, 9H), 1.26–1.36 (m,
1H), 1.11–1.24 (m, 1H); HRMS: calculated for C28H34F3N3O7S2 + H+,
646.18630; found (ESI, [M+H]+), 646.189; HPLC purity 100.0%, RT
9.8 min.
6. sFRP-1 In vitro characterization assays
6.1. Fluorescence polarization binding assay
The affinity of test compounds for sFRP-1 was determined using
a fluorescence polarization binding assay. According to the assay
design, a probe compound was bound to sFRP-1. The fluorescence
anisotropy value of the probe compound increased upon binding to
sFRP-1. Upon the addition of a test compound, the fluorescence
anisotropy value for the probe compound decreased due to com-
petitive displacement of the probe by the test compound. The de-
crease in anisotropy as a function of increasing concentration of
the test compound provided a direct measure of the test com-
pound’s binding affinity for sFRP-1.
5.1.4. General procedure for the acid catalyzed hydrolysis of
tertiary butyl esters
5.1.4.1. 5-(Phenylsulfonyl)-N-[1-(piperidin-4-ylcarbonyl)piperi-
To determine IC50 values, fluorescence polarization experi-
ments were conducted in a 384-well format according to the
din-4-yl]-2-(trifluoromethyl)benzenesulfonamide (8g). To
a
stirred solution of 8a (0.10 g, 0.15 mmol) in ethyl acetate (2 mL)
following procedures.
A 20 mM stock solution of the probe

200
W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
compound was prepared in 100% DMSO and dispensed in 10
lL
6.2. Cell-based, TCF-reporter functional assay
aliquots for long-term storage at ꢁ20 °C. The binding assay buffer
was prepared by combining stock solutions of Tris–Cl, NaCl, glyc-
erol, and NP40 at final concentrations of 25 mM Tris–Cl pH 7.4,
0.5 M NaCl, 5% glycerol and 0.002% NP40. Master stock solutions
of the test compounds were prepared in 100% DMSO at final con-
centrations of 20 mM. Typically the working stock solutions of the
test compounds were prepared by serially diluting the 20 mM
master stock solution to 5 mM, 2.5 mM, 1.25 mM, 0.625 mM,
U2-OS bone cells are infected with recombinant adenovirus 5
(Ad5)-WNT3 at a multiplicity of infection (MOI) of 2, followed by
infection with Ad5-sFRP-1 and Ad5-16xTCF-luciferase, each at an
MOI of 10. Four hours after infection, the cells are frozen in sterile
cryogenic vials at a cell density of 9E+6 cells/mL and stored in a
ꢁ150 °C freezer. For the assay, a vial of frozen cells is thawed,
and the cells are resuspended in plating medium [phenol red-free
RPMI 1640 medium containing 5% fetal calf serum, 2 mM Gluta-
MAX-l, and 1% (v/v) penicillin–streptomycin] to a final cell density
of 1.5E+5 cells/mL. The resuspended cells are then plated in 96-
0.3125 mM, 0.156 mM, 78
2.44 M, 1.22 M, 0.31 M, 76 nM, and 19 nM in DMSO. The
working stock solutions of the test compounds were further di-
luted by combining 6 L of the solutions with 24 L of Milli-Q
lM, 39 lM, 19.5 lM, 9.8 lM, 4.9 lM,
l
l
l
l
l
well tissue culture treated plates at a volume of 100 lL of cell sus-
purity water, resulting in working stock solutions (10ꢀ compound
pension/well (i.e., 1.5E+4 cells/well). The plates are incubated at
37 °C inside a 5% CO2/95% humidified air incubator for 5 h or until
the cells have attached and started to spread. Prior to the addition
stocks) in 20% DMSO.
The assay controls were prepared as follows. A 2
the 20 mM fluorescence probe compound was diluted 1000-fold
in 100% DMSO to a final concentration of 20 M. Six microliters
of the 20 M probe were combined with 5.4 mL of the assay buffer,
lL aliquot of
of test compounds, the medium is replaced with 50 lL/well of phe-
l
nol red-free RPMI 1640 containing 10% fetal calf serum, 2 mM
GlutaMAX-l, and 1% (v/v) penicillin–streptomycin. Test com-
pounds, or vehicle (typically DMSO), diluted in phenol red-free
RPMI 1640 containing 2 mM GlutaMAX-l, and l% (v/v) penicillin–
streptomycin are then added to the wells in replicates of 4 wells/
dilution and the plates are incubated at 37 °C overnight. Dose–re-
sponse experiments are performed with the compounds in twofold
serial dilutions from 10,000 to 4.9 nM. After the overnight incuba-
l
mixed well, and 18
lL of the resulting solution was dispensed into
384-well plates.
sFRP-1/probe complex was prepared by combining 11
lL of
20
lM probe compound with 9.9 mL of the assay buffer and
sFRP-1 stock solution to final concentrations of 22 nM probe com-
pound and 50 nM sFRP-1. Eighteen microliters of the sFRP-1/probe
complex were dispensed into the 384-well plates.
tion, the cells are washed twice with 150
cium or magnesium and lysed with 50
L/well of 1ꢀ cell culture
lysis reagent (Promega Corporation) on a shaker at room tempera-
ture for 30 min. Aliquots of the cell lysates (30 L) are transferred
lL/well of PBS w/o cal-
Aliquots of the test compounds from the 10x working stock
l
solutions (2
taining the sFRP-1/probe complex and the resultant solutions were
mixed by pipetting 10 L up and down twice. The final concentra-
lL) were removed and dispensed into the plate con-
l
l
to 96-well luminometer plates, and the luciferase activity is mea-
tions of sFRP-1 and probe in the assay solutions were 45 nM and
20 nM, respectively. In a typical experiment, each plate was used
to test 14 compounds.
The plate was incubated in the dark for 30 min. The fluores-
cence of the sFRP-1/probe complexes was read in the Tecan Ultra
plate reader at excitation and emission maxima of 485 and
535 nm.
Fluorescence anisotropy results from the emission of polarized
light in the parallel and perpendicular directions when a fluoro-
phore is excited with vertically polarized light. The anisotropy of
the probe in the free and bound state was determined using the
following equation: r ¼ ½IðIIÞ ꢁ Ið?Þꢂ ꢃ ½IðIIÞ þ 2Ið?Þꢂ, where I(II)
and I(?) are the parallel and perpendicular emission intensities,
respectively.
Monitoring the anisotropy changes of the probe compound re-
vealed that it bound saturably to sFRP-1 with a KD of 20–30 nM.
The binding affinity was independently verified using a tryptophan
fluorescence-quenching assay.
sured in a MicroLumat PLUS luminometer (EG&G Berthold) using
100 lL/well of luciferase substrate (Promega Corporation).
Following the injection of substrate, luciferase activity is mea-
sured for 10 s after a 1.6 s delay. The luciferase activity data is
transferred from the luminometer to a PC and analyzed using the
SAS/EXCEL program to determine EC50 values. The luciferase data is
analyzed using the SAS/EXCEL program. EC50 determinations for
dose–response curves are determined using the SAS/EXCEL program.
7. Ex vivo bone formation assay
Neonatal mouse calvaria were prepared from 4-day-old pups as
described previously.14 Briefly, calvaria were excised and cut in
half along the sagittal suture. Calvaria were incubated overnight
in serum-free BGJ medium containing 0.1% (w/v) bovine serum
albumin (BSA). Each half calvaria was placed with the concave sur-
face downward on a stainless steel grid (Small Parts Inc, Miami, FL)
in a 12-well tissue culture dish (Becton Dickinson, Oxnard, CA).
Each well contained 1.0 mL of BGJ medium with 1.0% (v/v) fetal bo-
vine serum (FBS). Calvaria were incubated for seven days with 0.1%
(v/v) dimethyl sulfoxide (DMSO, vehicle control) or with com-
pounds in a humidified atmosphere of 95% air and 5% CO2, and
the medium was changed on day 4 with fresh DMSO or compounds
added.
The decrease in the anisotropy of the probe upon addition of
the competing test compound was fitted to a sigmoidal dose–
rðeTsoppꢁoBnotstoemÞ curve of the equation shown below: Y ¼ Bottomþ
Þ ꢄ Hillslope, where ‘X’ is the logarithm of concentration,
IC
ð1þ10XꢁLog
50
‘Y’ is the anisotropy, and ‘Bottom’ and ‘Top’ correspond to the
anisotropy values of the free and sFRP-1-bound probe prior to
the addition of the test compound, respectively.
After organ culture, calvaria were fixed in 10% neutral phos-
phate-buffered formaldehyde at room temperature for at least
72 h, then decalcified for 6 h in 10% EDTA in phosphate-buffered
saline (PBS). Calvaria were embedded in parallel in the same paraf-
For automated IC50 determinations, the equation shown above
was used in the program GraphPad Prism. The ‘Hillslope’ was kept
constant at 1. The value for ‘Bottom’ was fixed, but was determined
by the blank (probe-only) wells in the plate. The values for ‘Top’
and ‘IC50’ were determined by the data fit. The value for ‘Top’
was typically close to 120, equivalent to approximately 50% bound
probe, and the value for ‘Bottom’ was around 30, due to free probe.
If the test compound interfered with the probe in the fluorescence
assay at high concentrations, the range for the fitted data was lim-
ited to the lower concentration range.
fin block, and 4
Consistent bone areas (200
were selected for histomorphometric analysis. A 200
l
m sections were stained with hematoxylin-eosin.
m away from the frontal sutures)
m square
l
l
grid was placed on each calvarium, and total bone area, as well
as the number of osteoblasts within the grid, was determined with
the Osteomeasure System (Osteometrics Inc, Atlanta, GA). All cells
on the bone surface were counted as osteoblasts. Data were

W. J. Moore et al. / Bioorg. Med. Chem. 18 (2010) 190–201
201
8. Gopalsamy, A.; Shi, M.; Stauffer, B.; Bahat, R.; Billiard, J.; Ponce-de-Leon, H.;
Seestaller-Wehr, L.; Fukayama, S.; Mangine, A.; Moran, R.; Krishnamurthy, G.;
Bodine, P. J. Med. Chem. 2008, 51, 7670.
analyzed for statistical significance by one-way ANOVA using the
Dunnett’s test with JMP software (SAS Institute, Cary, NC).
9. Moore, W. J.; Kern, J. C.; Bhat, R.; Commons, T. J.; Fukayama, S.; Goljer, I.;
Krishnamurthy, G.; Magolda, R. L.; Nogle, L.; Pitts, K.; Stauffer, B.; Trybulski,
E. J.; Welmaker, G. S.; Wilson, M.; Bodine, P. V. N. J. Med. Chem. 2009, 52,
105.
10. Zhong, X.; Desilva, T.; Lin, L.; Bodine, P.; Bhat, R. A.; Presman, E.; Pocas, J.; Stahl,
M.; Kriz, R. J. Biol. Chem. 2007, 282, 20523.
11. (a) Moore, W. J.; Kern, J. C.; Commons, T. J.; Wilson, M. A.; Welmaker, G. S.;
Trybulski, E. J.; Pitts, K.; Krishnamurthy, G.; Stauffer, B.; Bhat, R.; Fukayama, S.;
Upthagrove, A. L.; Bodine, P. V. N. Abstracts of Papers, 234th National Meeting of
the American Chemical Society, Boston, MA, August 19–23, 2007; American
Chemical Society: Washington, DC; MEDI-385.; (b) Kern, J. C.; Moore, W. J.;
Commons, T. J.; Woodworth, R. P.; Wilson, M. A.; Welmaker, G. S.; Trybulski, E.
J.; Pitts, K.; Krishnamurthy, G.; Stauffer, B.; Bhat, R.; Bodine, P. V. N. Abstracts of
Papers, 234th National Meeting of the American Chemical Society, Boston, MA,
August 19–23, 2007; American Chemical Society: Washington, DC; MEDI-387.;
(c) Welmaker, G. S.; Wilson, M. A.; Moore, W. J.; Kern, J. C.; Trybulski, E. J.;
Magolda, R. L.; Pitts, K.; Krishnamurthy, G.; Stauffer, B.; Bhat, R.; Bodine, P. V. N.
Abstracts of Papers, 234th National Meeting of the American Chemical Society,
Boston, MA, August 19–23, 2007; American Chemical Society: Washington, DC;
MEDI-389.
Acknowledgments
The authors wish to thank the Departments of Analytical Chem-
istry and Chemical Technologies, Wyeth, for analytical and profile
data. We also wish to thank our management, Drs. M. Abou-Ghar-
bia, T. Mansour and L. Freedman, for their support of this work.
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