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

Article abstract of DOI:10.1021/jm801144h

The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of the Wnt pathway have been proposed as anabolic a

Full text of DOI:10.1021/jm801144h

J. Med. Chem. 2009, 52, 105–116
105
Modulation of Wnt Signaling Through Inhibition of Secreted Frizzled-Related Protein I
(sFRP-1) with N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides†
William J. Moore,*,‡ Jeffrey C. Kern,‡ Ramesh Bhat,| Thomas J. Commons,‡ Shoichi Fukayama,| Igor Goljer,‡
Girija Krishnamurthy,§ Ronald L. Magolda,‡ Lisa Nogle,‡ Keith Pitts,§ Barb Stauffer,| Eugene J. Trybulski,‡
Gregory S. Welmaker,‡ Matthew Wilson,‡ and Peter V. N. Bodine|
Chemical and Screening Sciences, Wyeth Research, 500 Arcola Road, CollegeVille, PennsylVania 19426, Chemical and Screening Sciences,
Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965, Women’s Health and Musculoskeletal Biology, Wyeth Research,
500 Arcola Road, CollegeVille, PennsylVania 19426
ReceiVed September 12, 2008
The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors
of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of
the Wnt pathway have been proposed as anabolic agents for the treatment of osteoporosis or other bone-
related disorders. Details of the structure-activity relationships and biological activity from the first structural
class of this scaffold will be discussed.
Introduction
The skeletal system is one of the largest organs in the human
body, and the maintenance of healthy and strong bones is critical
to good health and quality of life.1 The incidence of bone disease
and fractures continues to rise, especially in our aging popula-
tion. According to the U.S. Surgeon General, 1.5 million elderly
people will suffer an osteoporotic-related fracture annually, often
leading to an overall decline in physical and mental health.1
The occurrence of osteoporotic-related fractures is expected to
dramatically increase in the next 10 years, resulting in a greater
burden on the health care system.1 Osteoporosis is a bone disease
predominately affecting postmenopausal women. This disease
disrupts the balance in bone formation and resorption, the later
of which predominates, resulting in the catabolic loss of
trabecular bone mass and structure.1 Traditional therapies have
Figure 1. Diphenylsulfonyl sulfonamide scaffolds are inhibitors of
sFRP-1.
involved anticatabolic agents, targeting osteoclasts that are
responsible for bone resorption. These agents arrest the resorp-
skeletal development.4 The Wnt/ꢀ-Catenin canonical signaling
tive process and minimize further loss in bone mass, however,
pathway is activated by the binding of Wnt to a membrane
bone mass that has been lost is not restored. To restore
receptor complex composed of a frizzled (Fzd) GPCR and a
catabolized bone, an anabolic agent is often necessary to regain
low-density lipoprotein receptor-related protein (LRP). The
bone integrity and structure. Currently, the only anabolic agent
binding results in a signaling cascade, leading to the stabilization
of ꢀ-catenin, translocation to the nucleus, and activation of
approved by the FDA for the treatment of chronic osteoporosis
is Teriparatide, a truncated synthetic peptide of the natural
transcriptional factors.3,4 The Wnt signaling process is regulated
by many factors, including extracellular components such as
WIF-1, Dkk-1, SOST, and secreted frizzled-related protein-1
(sFRP-1 or SARP-2).3a,5 Human sFRP-1 is a 35 kD protein
consisting of 313 amino acids and containing 2 distinct structural
domains, a netrin domain, and a cysteine-rich domain (CRD).
The later domain is homologous to the Fzd receptor.6a,b sFRP-1
is thought to interact with Wnt via the CRD domain in a
competitive manner with the Fzd receptor. Thus sFRP-1 is a
negative regulator or an antagonist of the Wnt signaling
pathway.7 Increased expression of sFRP-1 in OB cells results
in suppression of Wnt signaling, leading to decreased OB
survival, activation, and differentiation.8 Conversely, deletion
of the sFRP-1 gene in mice results in increased Wnt signaling,
leading to OB activation, proliferation, and differentiation
resulting in increases in trabecular bone.9
human parathyroid hormone (hPTHa).2 The signaling processes
invoked by PTH treatment and responsible for the subsequent
anabolic effects on bone are not completely understood.
Recently, the anabolic action of PTH on bone has been linked
to the modulation of the Wnt signaling pathway in osteoblasts
(OB).3a,b The Wnt signaling pathway, activated by secreted Wnt
glycoproteins, regulate various biological processes, including
†
Dedicated to the memory of Ronald L. Magolda.
* To whom correspondence should be addressed. Phone: 484-865-7827.
Fax: 484-865-9399. E-mail: moorew2@wyeth.com.
‡
Chemical and Screening Sciences, Collegeville, PA 19426.
Chemical and Screening Sciences, Pearl River, NY.
Women’s Health and Musculoskeletal Biology, Collegeville, PA 19426.
§
|
a Abbreviations: sFRP-1, secreted-frizzled related protein-1; Fzd, frizzled;
Wnt, wingless; hPTH, human parathyroid hormone; OB, osteoblasts; GPCR,
glycoprotein coupled receptor; WIF-1, Wnt inhibitory factor-1; Dkk-1,
dickkopf-1; SOST, sclerostin; CRD, cysteine-rich domain; LRP, low-density
lipoprotein receptor-related protein; FP, fluorescent polarization; TCF, T-cell
factor; FI, fold-induction; OS, osteosarcoma; RLM, rat liver microsomes;
HLM, human liver microsomes.
The goal of our drug discovery program was to identify an
orally bioavailable small-molecule inhibitor of sFRP-1 (Figure
1), which liberates Wnt for binding to the LRP-frizzled receptor
10.1021/jm801144h CCC: $40.75
2009 American Chemical Society
Published on Web 12/12/2008

106 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Moore et al.
Scheme 1a
a Reagents and conditions: (a) benzene, AlCl3; (b) CF2Br2, Cu metal, charcoal, DMA; (c) SnCl2, MeOH/H2O; (d) HCl, AcOH; (e) NaNO2; (f) SO2(g),
CuCl2; (g) 4-amino-Boc piperidine, TEA; (h) HCl.
Scheme 2a
complex resulting in increased canonical Wnt-signaling. A
molecule with these characteristics would be a potential anabolic
agent for the treatment of bone diseases such as osteoporosis.
Initial accounts of the discovery and subsequent elaboration of
small molecule inhibitors of sFRP-1 were disclosed.10,11 The
piperidinyl diphenylsulfonyl sulfonamide 1 (WAY-316606) was
identified as an inhibitor of sFRP-1 with moderate binding
affinity, good functional activity, and robust anabolic activity
in an ex vivo calvaria tissue assay. The SAR of the diphenyl-
sulfone portion of compound 1 was explored, and substitution
ortho to the sulfonamide was found to be critical. While lower
alkyl and halogen groups were tolerated, the trifluoromethyl
group appeared to be optimal.10b N-substitution on the piperidi-
nyl group was found to accommodate a wide variety of
functionality. Thus, 1 provided an opportunity for us to capitalize
on derivatization of the piperidine group, and the focus of this
communication will highlight our efforts to discover sFRP-1
inhibitors 3-8 (Figure 1) with an improved target profile.
Chemistry
The methods used to synthesize diphenylsulfonyl sulfona-
mides 1 and 3-8 are described below. The requisite sulfonyl
chloride 2 for the synthesis of 1 was prepared in five steps
starting with the sulfonylation of benzene with commercially
available 3-nitro-4-chlorobenzene sulfonyl chloride 9 using a
Friedel-Crafts catalyst provided the sulfone 10 (75-84%),
Scheme 1. Introduction of the trifluoromethyl group was
accomplished by reaction of the sulfone intermediate using
methodology described by Clark12a,b and Burton.12c We found
that the procedure described by Burton using dibromodifluo-
romethane and copper metal in the presence of charcoal afforded
the desired product 11 in moderate yield (56%). Reduction of
the nitro group upon exposure to SnCl2 in aqueous methanol
provided the aniline 12 (95%), which was diazotized and
subsequently transformed to 2 by exposure to sulfur dioxide
gas in the presence of CuCl2 (31%). Reaction of 2 with N-Boc-
4-aminopiperidine provided the sulfonamide 13 (60%), followed
by acidic removal of the protecting group to afford 1 (97%).
R-Aminoacetamide derivatives 3a-f were prepared by one
of two synthetic routes. The first route was a two-step sequence
involving the direct coupling of chloroacetyl chloride to 1 in
the presence of a base followed by exposure of the intermediate
R-chloroacetamide 3g with an ethanolic solution containing an
appropriate amine afforded the aminomethyl 3a, pyrrolidinyl
3c, and morpholino 3d compounds shown in Scheme 2. The
free bases were converted into the hydrochloride salts upon
treatment with HCl. In the case of the dibasic piperazinyl
a Reagents and conditions: (a) 2-chloroacetyl chloride, TEA, DCM; (b)
amine, ethanol; (c) HCl, ethyl acetate; (d) for 3b: 2-(N,N-dimethylami-
no)acetyl chloride, TEA, DCM; (e) for 3f: 1H-imidazol-1-acetic acid, EDC,
DMAP, DCM.
derivative 3e, N-Boc-piperazine was reacted with the R-chlo-
roacetamide in an ethanolic solution buffered with triethylamine
(TEA) followed by removal of the protecting group under acidic
conditions. Alternatively, 1 was directly reacted with either
commercially available R-N,N-dimethylaminoacetyl chloride and
base to afford the dimethylamino analogue 3b or activation of
an R-1H-imidazol-1-acetic acid with N-(3-dimethylaminopro-
pyl)-N′-ethylcarbodiimide (EDC) in the presence of N,N-
dimethylaminopyridine (DMAP) to provide 3f.
The syntheses of the constrained R-aminoacetamides (4) were
conducted by activation of the R or S enantiomer of com-
mercially available, Boc-protected proline (X ) CH2, Scheme
3). The protected amino acids were coupled to 1 using standard
amino acid coupling procedures, affording derivatives 4a and
4b respectively in moderate yields (45-56%) as indicated in
Scheme 3. A lactam derivative of L-proline (X ) CO) was
coupled to 1 in a similar manner, affording amide 4c. The
urethanes were deprotected under acidic conditions providing
compounds 5a-c.
L-Proline amides 6a (R2 ) Ac, X ) CH2) and 8a (R2 ) Me,
X ) CH2) were also prepared from compound 1 using the
reaction sequence outlined in Scheme 3 employing either

N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 107
Scheme 3a
a Reagents and conditions: (a) CDI, TEA, DCM; (b) EDC, DMAP, DCM; (c) HCl, ethyl acetate.
Scheme 4a
a Reagents and conditions: (a) R3COCl, DIPEA, DCM; (b) ethyl or t-butylisocyanate, DIPEA, DCM; (c) phenyl, dimethylamino, or morpholino carbonyl
chloride, DIPEA, DCM; (d) 3,3-dimethylbutyraldehyde or c-hexane carboxaldehyde, NaBH(OAc)3, MeOH; (e) benzyl bromide, TEA, microwave, 120 °C,
10 min.
commercially available N-acetyl-L-proline or N-methyl-L-proline
and activated by treatment with EDC in the presence of DMAP.
The synthesis of various carbonyl and alkyl derivatives
stemmed from compound 5a as shown in Scheme 4. Reaction
of 5a with acid chlorides and a base such as diisopropylethy-
lamine in dichloromethane afforded amides 6b-i (Scheme 4,
route i). Urea derivatives 7a-e were prepared by the treatment
of 5a with alkylisocyanates or via the chloroformates of primary
or secondary amines as shown in Scheme 4, route ii. Alkylated
derivatives were acquired (Scheme 4, route iii) by either
reductive amination of 5a, with the appropriate aldehyde in the
presence of sodium triacetoxyborohydride in methanol to afford
8b and 8c, or direct alkylation of 5a, with benzyl bromide
employing microwave-assisted heating and buffered with tri-
ethylamine to afford the benzyl derivative 8d.
molecules in the aqueous assay medium was conducted.
Compounds were screened in the competitive binding assay as
well as a functional assay derived from a U2-OS cell line that
was virally infected with human sFRP-1, Wnt-3, and a TCF
luciferase reporter. The level of 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 activity from the functional assay was evaluated at a
single concentration or in a dose response format (EC50). In
general, there was a good correlation between the binding
affinity (IC50) and the functional dose response assays. While
the EC50 values could be used for comparison of functional
activity, the concentration of the sFRP-1 inhibitor that produced
a constant fold-induction was also monitored as a means of
tracking potency by standardizing the efficacy in lieu of a natural
ligand. This value was extrapolated from the dose response
curve. In addition to the target profile, we also monitored
physical and pharmaceutical properties such as solubility,
cytochrome p450 inhibition, and microsomal stability. Advanced
compounds were evaluated in an ex vivo mouse calvaria assay
of bone formation as well as in vivo pharmacokinetic experiments.
Results and Discussion
During the early phase of our lead optimization and after the
discovery of 1, a fluorescent probe was identified that allowed
us to develop a high-throughput fluorescent polarization (FP)
binding assay using purified human sFRP-1 protein.10c As part
of the FP binding assay, an assessment of the solubility of our

108 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Moore et al.
Table 1. Comparison of R-Aminoacetamides 3a-f to 1 Using
Fluorescence Polarization Binding and U2-OS Functional Activity
Assays
Table 3. Data Comparison of Boc-Protected Proline Derivative 4a with
Acylated Compounds 6a-6i from Fluorescence Polarization Binding
and U2-OS Functional Activity Assays
FP bindinga(µM)
IC50 solubility EC50 2-FIc 4-FIc
Wnt-lucb(µM)
FP bindinga (µM)
Wnt-lucb (µM)
compound
R1
NHCH3
N(CH3)2
pyrrolidin-1-yl
morpholin-1-yl
c
c
compound
R3
IC50
solubility EC50 2-FI
4-FI
1
0.50
0.20
0.30
0.11
0.10
50
50
100
25
100
50
100
0.65 0.75 NA
0.62 0.16 0.61
0.28 0.21 NA
0.19 0.06 0.22
0.16 0.07 0.45
0.25 0.09 0.75
0.23 0.05 0.19
3a
3b
3c
3d
3e
3f
4a
6a
6b
6c
6d
6e
6f
6g
6h
6i
Ot-Bu
Me
0.06
0.16
0.04
0.04
0.02
0.09
0.05
0.07
25
12.5
100
50
25
100
12.5
50
100
100
0.06 0.02
0.25 0.09
0.09 0.03
0.27 0.07
0.12 0.04
0.31 0.09
0.46 0.22
0.38 0.14
0.83 0.46
0.35 0.13
0.16
0.68
0.11
0.33
0.22
0.49
0.87
0.92
1.82
0.88
i-Pr
t-Bu
4H-piperazin-1-yl 0.14
imidazol-1-yl 0.03
neopentyl
CH2N(CH3)2
c-Hex
a The affinity of test compounds for sFRP-1 was determined using the
FP binding assay. b The osteosarcoma cell line, U2-OS, was used for the
functional assay. c Micromolar concentration that elicited a 2-fold (2-FI)
or 4-fold (4-FI) increase in Wnt signaling. Each point from the dose response
was measured in quadruplicate. The standard deviations for these assays
were typically (9% of the mean or less.
Ph
Ph, 4-N(CH3)2 0.11
Pyrid-4-yl 0.07
a The affinity of test compounds for sFRP-1 was determined using the
FP binding assay. b The osteosarcoma cell line, U2 OS, was used for the
functional assay. c Micromolar concentration that elicited a 2-fold or 4-fold
increase in Wnt signaling. Each point from the dose response was measured
in quadruplicate. The standard deviations for these assays were typically
( 9% of the mean or less.
Table 2. Data from Fluorescence Polarization Binding and U2 OS
Functional Activity Assays for Compounds 4a-c and 5a-c
Table 4. Comparison of Fluorescence Polarization Binding and U2-OS
Functional Activity Data for Boc Protected Proline Derivative 4a with
Ureas, 7a-7e
FP bindinga (µM)
Wnt-lucb (µM)
c
c
compound
X
R2
IC50
solubility EC50 2-FI
4-FI
1
0.50
50
25
25
50
50
0.65
0.06
0.21
0.09
0.35
0.71
0.24
0.75 NA
4a
4b
4c
5a
5b
5c
CH2 L-Boc 0.06
CH2 D-Boc 0.12
CO L-Boc 0.13
0.02
0.09
0.04
0.17
0.34
0.11
0.16
0.97
0.25
1.99
2.98
0.99
FP bindinga (µM)
Wnt-lucb (µM)
CH2 L-H
CH2 D-H
CO L-H
0.11
0.20
0.09
c
c
compound
R4
Et
tBu
Ph
R5
IC50
solubility EC50 2-FI
4-FI
25
100
4a
7a
7b
7c
7d
7e
0.06
0.03
0.02
0.02
0.03
0.04
25
100
50
0.06
0.09
0.03
0.31
0.16
0.28
0.02
0.04
0.01
0.09
0.06
0.11
0.16
0.10
0.03
0.67
0.22
0.52
a The affinity of test compounds for sFRP-1 was determined using the
FP binding assay. b The osteosarcoma cell line, U2-OS, was used for the
functional assay. c Micromolar concentration that elicited a 2-fold or 4-fold
increase in Wnt signaling. Each point from the dose response was measured
in quadruplicate. The standard deviations for these assays were typically
(9% of the mean or less.
H
H
H
Me
25
Me
morpholine
100
50
a The affinity of test compounds for sFRP-1 was determined using the
FP binding assay. b The osteosarcoma cell line, U2OS, was used for the
functional assay. c Micromolar concentration that elicited a 2-fold or 4-fold
increase in Wnt signaling. Each point from the dose response was measured
in quadruplicate. The standard deviations for these assays were typically
(9% of the mean or less.
Tables 1-6 represent the in vitro biological data generated
for these compounds. The initial R-aminoacetamide derivatives
3a and 3b have comparable binding and functional activity to
lead compound 1, while the heterocyclic derivatives 3c-f
demonstrated a significant improvement in both binding and
functional activity (Table 1). It is interesting to note that the
most dramatic differences can be observed in the concentration
at which the compounds elicit a 2- and 4 fold-induction in Wnt
signaling. Noting the improvement in activity of the R-hetero-
cycle acetamides 3c-f, we then considered a constrained version
of the R-aminoacetamide functionality to reduce or minimize
the number of rotatable bonds.
Exploration of the proline derivatives provided significant
improvements in binding affinities and functional activity over
1, Table 2. All of the proline derivatives 4a-c and 5a-c appear
to have similar binding affinities to sFRP-1, however, the data
from the functional assay indicates greater activity for the
carbamates (4a-c) over the unsubstituted compounds (5a-c).
There was minimal stereochemical preference when the D- and
L-isomers of the proline amides (4a/4b or 5a/5b) were compared.
The observation that the carbamates were more potent than
the deprotected N-H compounds led us to pursue carbonyl
analogues that would be less prone to hydrolysis in a low pH
environment such as gastrointestinal fluids. On the basis of the
data for 4a, the L-proline amide 5a was used as a template for
further analoguing. The alkyl 6a-f, aryl 6g and 6h, and
heteroaryl 6i derivatives demonstrated comparable binding
affinities relative to 4a (Table 3), and the level of binding
interaction of these compounds with sFRP-1 reached a plateau,
which was not a function of limiting aqueous solubility. On

N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 109
Table 5. Fluorescence Polarization Binding and Wnt-Luciferase
Activity from U2-OS Cells for Alkylated Proline Analogues 8a-d
that the small molecule does prevent the direct binding of
sFRP-1 to the Wnt protein. The details of these experiments
will be communicated in a separate publication.
Selected sFRP-1 inhibitors were evaluated in the ex vivo
mouse calvaria assay. During the early characterization of 1 in
the ex vivo assay, we found that the concentrations of inhibitor
required to induce increases in total bone area were significantly
lower than the observed in vitro EC50. Evidence of increased
bone formation as well as other anabolic attributes such as bone
remodeling, including resorption, osteoblast activation, and
differentiation were determined by histological assessment. On
the basis of the potent activity observed in the functional assay,
compound 7b (WAY-362692) was selected to advance into the
calvaria assay for characterization. Treatment of the calvaria
with a 1 nM DMSO stock solution of compound 7b resulted in
a 75% increase in total bone area relative to the control with
no significant increase at a lower concentration (0.1 nM) as
shown in Figure 2. However, in both treatment groups, the
histology assessment indicated activation of the osteoblast cells
and active bone remodeling. As seen in Figure 3, a picture of
the histology slide from the treatment of the calvaria with 7b,
the OB activation is noted by the rounding of the cells (a), while
active bone remodeling can be observed by the irregular surface
(b). The increase in total bone area was evident from the lighter
color, made possible by differential staining (c). Despite
stimulation of the osteoblasts and a trend indicating an increase
in the OB number, the increase was not statistically significant.
This observation is in contrast to the ex vivo profile of hPTH
(1-34) in this assay, data not shown. Typically, treatment of
the calvaria with the hormone under the same conditions will
result not only in an increase in the total bone area but also a
dramatic increase in the number of OB.
All sFRP-1 inhibitors were screened in a panel of profiling
assays to monitor stability in liver microsomes (rat and human)
as a means of identifying potential first pass metabolism. In
addition, inhibition of cytochrome p450 isozymes (3A4, 2D6,
and 2C9) was monitored. These compounds have minimal
inhibition of the 2D6 and 2C9 isozymes with moderate to high
inhibition of the 3A4 isozyme, as represented by the isosteric
derivatives 4a, 6d, 7b, and 8b, Table 6. These compounds were
representative of the majority of this class of compounds and,
when compared to compound 1, exhibited short half-lives (t1/2)
in the microsomal stability assays, indicating a greater prob-
ability of limited oral bioavailability due to phase I metabolism.
FP bindinga (µM)
Wnt-lucb (µM)
c
c
compound
R6
IC50
solubility
EC50 2-FI
4-FI
7b
8a
8b
8c
8d
0.02
0.12
0.03
0.04
1.4
50
100
12.5
25
0.03
0.23
0.45
0.32
0.72
0.01
0.11
0.04
0.09
0.27
0.03
0.74
0.17
0.32
0.77
H
neopentyl
c-Hex
Ph
25
a The affinity of test compounds for sFRP-1 was determined using the
FP binding assay. b The osteosarcoma cell line, U2-OS, was used for the
functional assay. c Micromolar concentration that elicited a 2-fold or 4-fold
increase in Wnt signaling. Each point from the dose response was measured
in quadruplicate. The standard deviations for these assays were typically
(9% of the mean or less.
the basis of the functional activity, the cyclohexyl 6f and
aromatic derivatives 6 g-i were less effective at facilitating
Wnt signaling than the lower alkyl analogues 6a-d. The isostere
of 4a, compound 6d, was equivalent in potency but did not
significantly distinguish itself from the other analogues. The
dimethyl amino group in compound 6e was introduced to
incorporate water-solublizing properties (6e ) 100 µM versus
6d ) 25 µM). The effect of the polar group toward target affinity
was minimal when compared to the lipophilic derivatives 6b-d.
This was a trend that demonstrated the tolerability of the binding
site to a variety of functionality in this region of the scaffold.
As shown in Table 4, the isosteric urea 7b was comparable
to carbamate 4a in binding affinity and potency, but perhaps
most impressive was the concentration that was necessary to
evoke a 4-fold induction (0.16 µM versus 0.03 µM) for 4a and
7b, respectively. There was no significant difference between
the mono 7a and dialkylated ureas 7d. In contrast, the phenyl
7c and morpholino 7e ureas were significantly weaker.
Evaluation of noncarbonyl substitution was also explored
(Table 5). These derivatives maintain good binding character-
istics, with the exception of the benzyl derivative 8d. Compound
8b, an isoster of the urea derivative 7b, had binding affinity
(30 nM), comparable to 7b. The substitution of the urea
functionality with a methylene group resulted in a modest drop
in the total polar surface area albeit at a cost of increasing the
calculated logP. The most dramatic difference observed for the
descarbonyl compounds was the decline of functional activity;
in particular, the concentration of inhibitor required to induced
a 4-fold induction (g0.17 µM) of Wnt signaling. This data
suggested that the carbonyl was an important structural feature
in this class of compounds.
Conclusion
In summary, we have presented data for a class of novel
small-molecule inhibitors of sFRP-1, which appear to disrupt
the protein-protein interaction between Wnt and sFRP-1.
Derivatization of compound 1 (IC50 0.5 µM, EC50 0.65 µM)
led to compound 4a (IC50 0.06 µM, EC50 0.06 µM) that
demonstrated improved binding and potency. Optimization of
4a led to a series of derivatives that included isosteric derivatives
(see Table 6 for isostere comparison). All of the isosteres of
compound 4a exhibited good binding affinity to sFRP-1 (e60
nM) but varied in their functional potency (2-15-fold). This
effort led to the identification of compound 7b that demonstrated
potent binding affinity (IC50 20 nM) to sFRP-1 protein. In a
cell-based assay that detects Wnt signaling, 7b provided potent
functional activity (EC50 30 nM). This compound increased total
bone area (75%) relative to the vehicle control in an ex vivo
mouse calvaria model for bone formation, wherein parameters
of anabolic activity such as activation of osteoblasts and active
bone remodeling were observed. In vitro metabolism studies
While little structural information is known about the
protein-protein interaction between sFRP-1 and Wnt, sFRP-1
is thought to engage Wnt via the CRD.13a A truncated version
of sFRP-1, in which the netrin domain is deleted, can be
produced in the U2-OS cell-based assay and the modified
secreted protein antagonizes Wnt signaling albeit with dimin-
ished efficiency.13b The sFRP-1 inhibitors described herein have
no detectable effect on Wnt signaling when examined in the
functional assay containing the modified protein, suggesting that
this class of compounds disrupt the protein-protein interaction
by association with the sFRP-1 netrin domain. Advanced binding
experiments conducted with Wnt, sFRP-1 and 1, have revealed

110 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Table 6. In Vitro Data Comparison for sFRP-1 Isosteric Inhibitors
Moore et al.
CYP450b (% inhibition)
compound
X
Y
FP binding IC50 (µM)
Wnt-luc EC50 (µM)
RLMa t1/2 (min)
HLMa t1/2 (min)
3A4
2D6
2C9
1
0.50
0.06
0.02
0.02
0.03
0.65
0.06
0.12
0.03
0.45
>60c
>60c
51
55
74
49
84
0
0
0
0
24
0
9
30
0
4a
6d
7b
8b
CO
CO
CO
CH2
O
CH2
NH
1
3
3
8
1
1
2
5
CH2
0
a Rat liver (RLM) and human liver (HLM) microsomal stability was determined from a 1 µM DMSO solution of compound. b CYP450 inhibitions were
determined from a 3 µM DMSO solution of compound. c Advanced microsomal stability performed in the presence of NADPH with and without UDPGA.
5-(Phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonyl Chloride
(2). Step 1: 1-Chloro-2-nitro-4-(phenylsulfonyl)benzene (10). To a
stirred slurry of aluminum chloride (15.6 g, 117 mmol) in benzene
(50 mL) under nitrogen was added 4-chloro-3-nitrobenzenesulfonyl
chloride (97.6 mmol, 25.0 g). The resulting solution was stirred at
room temperature for 2 days, then poured over ice and extracted
with ethyl acetate twice. The combined organic layers were washed
with brine, dried over magnesium sulfate, filtered, and concentrated.
Trituration of the material in diethyl ether gave 10 as a yellow
1
solid, mp 113-122 °C (21.8-24.5 g, 75-84%). H NMR (400
MHz, DMSO-d6) δ 8.68 (d, J ) 2.08 Hz, 1H), 8.25 (dd, J ) 2.08,
8.58 Hz, 1H), 8.00-8.09 (m, 3H), 7.72-7.80 (m, 1H), 7.62-7.71
(m, 2H). MS (ESI, [M + H]+, m/z) 296.9. HRMS: calculated for
C12H8ClNO4S, 296.98626, found (EI, M+) 296.9849. HPLC purity
Figure 2. Evaluation of 7b in the ex vivo mouse calvaria assay at 0.1
and 1 nM. Each bar gives the mean ( SE for 5 (control) cultures.
Control values were 0.0045244 ( 0.0002607 mm2 (total bone area)
and 87 ( 8.2 (number of osteoblasts). ** P < 0.01.
92.7%, RT 9.3 min.
Step 2: 2-Nitro-4-(phenylsulfonyl)-1-(trifluoromethyl)benzene
(11). To a stirred solution of 10 (21.7 g, 72.9 mmol) in anhydrous
dimethylacetamide (150 mL) under nitrogen was added 3 µm copper
powder (27.8 g, 437 mmol), oven-dried 100 mesh activated carbon
powder (11.0 g), and dibromodifluoromethane (20 mL, 219 mmol).
The resulting solution was heated to 100 °C and for 6 h. The
solution was allowed to cool to room temperature and filtered
through Celite. The filtrate was concentrated to approximately one-
third of the volume and partitioned between ethyl acetate and
saturated aqueous ammonium chloride solution. The aqueous phase
was extracted with additional ethyl acetate. The combined organic
layers were washed with water several times, followed by a brine
wash. The organic layer was dried over magnesium sulfate, filtered,
and concentrated. The crude mixture was flash column separated
using 0-20% ethyl acetate/hexane to give 11 as an off-white solid
(13.6 g, 56%), mp 97-99 °C. 1H (400 MHz, DMSO-d6) δ 8.76 (d,
J ) 1.82 Hz, 1H), 8.47 (dd, J ) 1.04, 8.32 Hz, 1H), 8.28 (d, J )
8.32 Hz, 1H), 8.05-8.14 (m, 2H), 7.74-7.84 (m, 1H), 7.64-7.73
(m, 2H). MS (ESI, [M + H]+, m/z) 331. HRMS: calculated for
C13H8F3NO4S, 331.01261. found (EI, M+), 331.0128. HPLC purity
97.6%, RT 9.6 min.
of 7b predicted significant first-pass oxidative metabolism, and
this property limited its use as an orally administered anabolic
bone agent. Further studies are ongoing to examine the in vivo
properties of this class of sFRP-1 inhibitors on bone as well as
other potential therapeutic applications.14-16 Future communica-
tions will detail additional SAR studies of related scaffolds.
Experimental Procedures
Chemistry. Solvents were purchased as anhydrous grade and
used without further purification. Reagents were purchased from
commercial sources and used as received. 1H spectra were recorded
on either a Varian Inova 400 or Bruker instrument with chemical
shifts reported in δ values (parts per million, ppm) relative to an
internal standard tetramethylsilane in CDCl3 or DMSO-d6. 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 obtained on an Agilent 1200
using the following conditions: Waters Xterra RP18 HPLC column
(3.5 µ, 150 mm L × 4.6 mm ID), 40 °C column temperature, 1.2
mL/min flow rate, photodiode array detection (210-400 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 methanol/acetonitrile). Chiral HPLC
analyses were run on a Berger Analytical SFC (Thar Technologies,
Inc., Pittsburgh, PA) using a Chiralcel OD-H column, 5 µ, 250
mm L × 20 mm ID (Chiral Technologies, Inc., Exton, PA) with
column temperature of 35 °C, flow rate 2.0 mL/min, UV detector
at 220 nm, 100 bar outlet pressure, and carbondioxide modifier of
15% MeOH with 0.2% dimethylethylamine as additive. Optical
rotations of individual enantiomers were obtained on a JASCO
P-1020 polarimeter at a wavelength of 589 nm using a 1.0 cm
microcell and methanol as the solvent.
Step 3: 5-(Phenylsulfonyl)-2-(trifluoromethyl)aniline (12). To a
stirred solution of 11 (13.6 g, 41.0 mmol) not totally dissolved in
methanol (300 mL) under nitrogen was added water (10 mL), and
tin(II) chloride (31.13 g, 164 mmol). The resulting solution was
heated to 70 °C for three days. The reaction was allowed to cool
to room temperature and slowly quenched with saturated aqueous
sodium bicarbonate solution (400 mL). This resulted in a thick white
mixture, which was diluted with water and extracted several times
with ethyl acetate. The combined organic layers were washed with
brine, dried over magnesium sulfate, filtered, and concentrated to
1
give 12 as an off-white solid (11.8 g, 95%), mp 135-138 °C. H
(400 MHz, DMSO-d6) δ 7.86-7.96 (m, 2H), 7.70-7.79 (m, 1H),
7.62-7.70 (m, 2H), 7.54 (d, J ) 8.32 Hz, 1H), 7.44 (d, J ) 1.56
Hz, 1H), 7.06 (dd, J ) 1.04, 8.32 Hz, 1H), 6.21 (s, 2H). MS (ESI,
[M + H]+, m/z) 302.1. HRMS: calculated for C13H10F3NO2S +

N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 111
Figure 3. Histological Evaluation of Calvaria after treatment with 0.1 and 1.0 nM of Compound 7b. (a) Activated osteoblasts. (b) Irregular surface.
(c) Formation of new bone (light pink). All characteristics are indications of active bone remodeling and are associated with anabolic action on
bone.
H+ + CH3CN 343.07226, found [M + H + ACN]+) 343.0735.
HPLC purity 97.1%, RT 9.0 min.
MHz, DMSO-d6) δ 8.82 (br s, 1H), 8.60 (d, J ) 1.79 Hz, 1H),
8.57 (br s, 1H), 8.42 (dd, J ) 1.15, 8.33 Hz, 1H), 8.25 (d, J )
8.20 Hz, 1H), 8.02-8.08 (m, 2H), 7.76-7.83 (m, 1H), 7.67-7.74
(m, 2H), 3.35-3.48 (m, 1H), 3.11-3.22 (m, 2H), 2.77-2.90 (m,
2H), 1.57-1.78 (m, 4H). MS (ESI, [M + H]+, m/z) 449.1. HRMS:
calculated for C18H19F3N2O4S2 + H+ 449.08111, found (ESI, [M
+ H]+) 449.083. HPLC purity 98.0%, RT 6.8 min.
Step 4: 5-(Phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonyl
chloride (2). To a stirred solution of 12 (13.2 g, 43.8 mmol) in
acetonitrile (310 mL) under nitrogen at 0 °C was added glacial
acetic acid (25 mL) followed by concentrated hydrochloric acid
(25 mL). This results in a thick white mixture, which is difficult to
stir. To this stirred mixture was added a solution of sodium nitrite
(3.63 g, 52.6 mmol) in water (5 mL) dropwise over 10 min. The
resulting solution was stirred 20 min, during which time the white
precipitate disappeared and the solution became homogeneous.
Sulfur dioxide was bubbled into the solution over a period of 20
min, immediately followed by the addition of an aqueous solution
of copper(II) chloride dihydrate (7.5 g, 44.0 mmol in 7 mL H2O)
in one portion, which resulted in the vigorous evolution of gas.
The resulting solution was slowly allowed to warm to room
temperature and stirred overnight. The solution was then partitioned
between ethyl acetate and water. The aqueous layer was extracted
with ethyl acetate. The combined organic layers were washed with
a saturated aqueous ammonium chloride solution once, then with
water several times, and once with brine. The organic layer was
dried over magnesium sulfate, filtered, and concentrated. The crude
mixture was flash column separated using 0%-20% ethyl acetate/
hexane to give 2 as a white solid (5.2 g, 31%), mp 104-105 °C.
1H (400 MHz, CDCl3) δ 8.84 (d, J ) 1.54 Hz, 1H), 8.41 (dd, J )
0.77, 8.20 Hz, 1H), 8.13 (d, J ) 8.20 Hz, 1H), 7.96-8.06 (m, 2H),
7.66-7.76 (m, 1H), 7.54-7.66 (m, 2H). MS (ESI, [M + H]+, m/z)
384.9.
N-[1-(N-Methylglycyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-
fluoromethyl)-benzenesulfonamide (3a). Step 1. To a stirred
solution of 1 (0.60 g, 1.34 mmol) in methylene chloride (15 mL)
with triethylamine (0.4 mL, 2.87 mmol) was added chloroacetyl
chloride (0.15 g, 1.34 mol), and the resulting solution was stirred
overnight at room temperature. The reaction mixture was washed
with a saturated aqueous ammonium chloride solution and con-
centrated. Flash column separation using 10-70% ethyl acetate/
hexane gradient gave N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenyl-
sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (0.43 g, 61%).
Step 2. To a stirred solution of N-[1-(chloroacetyl)piperidin-4-
yl]-5-(phenylsulfonyl)-2- (trifluoromethyl)benzenesulfonamide (0.08
g, 0.15mmol) in ethanol (1 mL) was added 33% methylamine in
ethanol (0.1 mL) and the resulting solution was stirred overnight
at room temperature. The mixture was concentrated and flash
column separated using 0-10% methanol/methylene chloride
gradient gave 3a, which was dissolved in ethyl acetate, treated with
HCl gas and concentrated to provide the HCl salt (0.05 g, 61%).
1H (400 MHz, DMSO-d6) δ 8.59 (d, J ) 1.79 Hz, 1H), 8.42 (dd,
J ) 1.28, 8.20 Hz, 1H), 8.24 (d, J ) 8.46 Hz, 1H), 8.00-8.08 (m,
2H), 7.75-7.83 (m, 1H), 7.66-7.73 (m, 2H), 4.05-4.18 (m, 1H),
3.50-3.75 (m, 4H), 2.86-3.03 (m, 1H), 2.65 (t, 1H), 2.41 (s, 3H),
1.49-1.67 (m, 2H), 1.30-1.45 (m, 1H), 1.12-1.27 (m, 1H). MS
(ESI, [M + H]+, m/z) 520.0. HRMS: calculated for C21H24F3N3O5S2
+ H+ 520.11822, found (ESI, [M + H]+) 520.1174. HPLC purity
98.4%, RT 6.9 min.
N-[1-(N,N-Dimethylglycyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-
(trifluoro-methyl)benzenesulfonamide (3b). To a stirred solution of
1 (0.10 g, 0.22 mmol) in methylene chloride (3 mL) with triethylamine
(0.1 mL, 0.70 mmol) was added dimethylaminoaceyl chloride hydro-
chloride (0.035 g, 0.22 mmol) and the resulting solution was stirred
for overnight at room temperature. The reaction mixture was washed
with water and concentrated. Flash column separation using 0-8%
methanol/methylene chloride gradient gave 3b, which was dissolved
in ethyl acetate, treated with HCl gas and concentrated to provide the
HCl salt (0.055 g, 46%). 1H (400 MHz, DMSO-d6) δ 9.50 (br s, 1H),
8.71 (d, J ) 7.17 Hz, 1H), 8.60 (d, J ) 1.79 Hz, 1H), 8.42 (dd, J )
1.15, 8.07 Hz, 1H), 8.25 (d, J ) 8.46 Hz, 1H), 8.00-8.11 (m, 2H),
7.75-7.85 (m, 1H), 7.64-7.75 (m, 2H), 4.16-4.38 (m, 2H), 4.05-4.15
(m, 1H), 3.43-3.53 (m, 1H), 2.95-3.09 (m, 1H), 2.62-2.87 (m, 7H),
1.55-1.76 (m, 2H), 1.39-1.50 (m, 1H), 1.19-1.33 (m, 1H). HRMS:
calculated for C22H26F3N3O5S2 + H+ 534.13387, found (ESI, [M +
H]+) 534.1341. MS (ESI, [M + H]+, m/z) 533.8. HPLC purity 93.9%,
RT 7.1 min.
5-(Phenylsulfonyl)-N-piperidin-4-yl-2-(trifluoromethyl)benzene-
sulfonamide (1). Step 1: tert-Butyl 4-({[5-(Phenylsulfonyl)-2-(trif-
luoromethyl)phenyl]sulfonyl}-amino)piperidine-1-carboxylate (13).
To a stirred solution of 2 (3.0 g, 7.8 mmol) and triethylamine (3.3
mL, 23.6 mmol) in dichloromethane (25 mL) under nitrogen was
added a solution of 1-Boc-4-aminopiperidine (1.87 g, 9.36 mmol)
dissolved in dichloromethane (5 mL) slowly over two minutes. The
resulting solution was stirred at room temperature for 1 h, and then
washed with a saturated aqueous ammonium chloride solution. The
organic phase was dried over magnesium sulfate, filtered and
concentrated. The crude mixture was flash column separated using
0-30% ethyl acetate/hexane gradient to give 13 as a white solid
1
(2.56 g, 60%). H (400 MHz, DMSO-d6) δ 8.58 (d, J ) 1.56 Hz,
1H), 8.55 (d, J ) 7.54 Hz, 1H), 8.42 (dd, J ) 1.17, 8.19 Hz, 1H),
8.24 (d, J ) 8.32 Hz, 1H), 8.01-8.07 (m, 2H), 7.74-7.81 (m,
1H), 7.65-7.73 (m, 2H), 3.71 (d, J ) 13.26 Hz, 2H), 3.14-3.28
(m, 1H), 2.56-2.77 (m, 2H), 1.47 (dd, J ) 3.12, 13.00 Hz, 2H),
1.40 (s, 9H), 1.16-1.27 (m, 2H). HRMS: calculated for
C23H27F3N2O6S2 + Na+, 571.11548, found (ESI, [M + Na]+
observed) 571.1151. HPLC purity 97.4%, RT 10.1 min.
Step 2: 5-(Phenylsulfonyl)-N-piperidin-4-yl-2-(trifluoromethyl-
)benzene-sulfonamide (1). To a stirred solution of 13 (3.8 g, 6.93
mmol) in 1,4-dioxane (40 mL) under nitrogen was added a 4N HCl
dioxane solution (13 mL, 52 mmol). The resulting solution was
stirred overnight at room temperature, and concentrated. Trituration
5-(Phenylsulfonyl)-N-[1-(pyrrolidin-1-ylacetyl)piperidin-4-yl]-2-
(trifluoro-methyl)benzenesulfonamide (3c). In an analogous manner
to 3a, step 2, N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-
1
in ethyl acetate gave 1 as an HCl salt (3 g, 97%). H NMR (400

112 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Moore et al.
2-(trifluoromethyl)benzenesulfonamide and pyrrolidine were used
to prepare 3c, which was dissolved in ethyl acetate, treated with
HCl gas and concentrated to provide the HCl salt (0.03 g, 35%).
1H (400 MHz, DMSO-d6) δ 9.79 (br s, 1H), 8.71 (d, J ) 7.43 Hz,
1H), 8.60 (d, J ) 1.79 Hz, 1H), 8.38-8.47 (m, 1H), 8.25 (d, J )
8.20 Hz, 1H), 8.00-8.09 (m, 2H), 7.75-7.85 (m, 1H), 7.65-7.74
(m, 2H), 4.24-4.47 (m, 2H), 4.10 (d, J ) 13.32 Hz, 1H), 3.43-3.62
(m, 3H), 3.02 (t, J ) 11.02 Hz, 4H), 2.68-2.80 (m, 1H), 1.81-2.07
(m, 4H), 1.65-1.74 (m, 1H), 1.55-1.65 (m, 1H), 1.39-1.53 (m,
1H), 1.21-1.33 (m, 1H). HRMS: calculated for C24H28F3N3O5S2
+ H+ 560.14952, found (ESI, [M + H]+ observed) 560.1497. MS
(ESI, [M + H]+, m/z) 560.0. HPLC purity 98%, RT 7.2 min.
N-[1-(Morpholin-4-ylacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-
(trifluoromethyl)benzenesulfonamide (3d). In an analogous manner
to 3a, step 2, N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-
2-(trifluoromethyl)benzenesulfonamide and morpholine were used
to prepare 3d, which was dissolved in ethyl acetate, treated with
HCl gas, and concentrated to provide the HCl salt (0.077 g, 88%).
1H (400 MHz, DMSO-d6) δ 8.55-8.63 (m, 2H), 8.38-8.45 (m,
1H), 8.24 (d, J ) 8.46 Hz, 1H), 8.01-8.08 (m, 2H), 7.74-7.83
(m, 1H), 7.65-7.72 (m, 2H), 4.02-4.11 (m, 1H), 3.78-3.92 (m,
1H), 3.57 (br s, 4H), 3.15-3.25 (m, 1H), 2.85-3.06 (m, 2H),
2.55-2.61 (m, 1H), 2.35 (br s, 4H), 1.33-1.67 (m, 3H), 1.12-1.26
(m, 1H). MS (ESI, [M + H]+, m/z) 576.0. HPLC purity 98.2%, RT
7.1 min. HRMS: calculated for C24H28F3N3O6S2 + H+ 576.14444,
found (ESI, [M + H]+) 576.1428.
The mixture was diluted with a saturated aqueous ammonium
chloride solution and extracted several times with ethyl acetate.
The combined organic layers were washed with brine several times
and concentrated. Flash column separation using 30-100% ethyl
acetate/hexane gradient gave 4a (0.12 g, 56%). Specific optical
rotation [R]D ) -20.3 (c 0.95, MeOH). H (400 MHz, DMSO-
d6) δ 8.49 - 8.74 (m, 2H), 8.42 (d, J ) 8.46 Hz, 1H), 8.24 (d, J
) 8.46 Hz, 1H), 8.00-8.08 (m, 2H), 7.74-7.83 (m, 1H), 7.64-7.73
(m, 2H), 4.55 (d, J ) 6.92 Hz, 2H), 4.14 (d, J ) 11.27 Hz, 1H),
3.78 (d, J ) 11.02 Hz, 1H), 2.96-3.08 (m, 1H), 2.85-2.96 (m,
2H), 2.06-2.24 (m, 1H), 1.48-1.87 (m, 8H), 1.32-1.44 (m, 9H).
MS (ESI, [M - H]-, m/z) 644.2. HRMS: calculated for
C28H34F3N3O7S2 + H+ 646.18630, found (ESI, [M + H]+) 646.188.
HPLC purity 100%, RT 9.5 min.
21
1
tert-Butyl (2R)-2-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)-
phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-car-
boxylate (4b). To a stirred solution of N-(tert-butoxycarbonyl)-D-
proline (0.15 g, 0.70 mmol) and CDI (0.11 g, 0.68 mmol) and
triethylamine (0.2 mL, 0.14 mmol) in DMF (2 mL) was added 1
(0.20 g, 0.45 mmol) and 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 organic layers were washed with brine
several times and concentrated. Flash column separation using
30-100% ethyl acetate/hexane gradient gave 4b (0.13 g, 45%).
Specific optical rotation [R]D21 ) +21.8 (c 0.65, MeOH). 1H (400
MHz, DMSO-d6) δ 8.50-8.74 (m, 2H), 8.42 (d, J ) 9.48 Hz, 1H),
8.24 (d, J ) 8.46 Hz, 1H), 7.99-8.08 (m, 2H), 7.73-7.84 (m,
1H), 7.63-7.74 (m, 2H), 4.48-4.65 (m, 1H), 4.08-4.19 (m, 1H),
3.72-3.86 (m, 1H), 2.85-3.06 (m, 2H), 2.05-2.23 (m, 2H),
1.51-1.86 (m, 8H), 1.32-1.44 (m, 9H). MS (ESI, [M - H]-, m/z)
644.2. HRMS: calculated for C28H32F3N3O8S2 + Na+ 682.14751,
found (ESI, [M + Na]+ observed) 682.1473. HPLC purity 97.0%,
RT 9.5 min.
tert-Butyl(5S)-2-oxo-5-{[4-({[5-(Phenylsulfonyl)-2-(trifluorome-
thyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-
carboxylate (4c). 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 temperature 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 concentrated. Flash column separation using 0-8% methanol/
methylene chloride gradient gave 4c (0.10 g, 56%). 1H (400 MHz,
DMSO-d6) δ 8.52-8.76 (m, 2 H), 8.39-8.45 (m, 1 H), 8.24 (d, J
) 8.5 Hz, 1 H), 8.01-8.08 (m, 2 H), 7.75-7.82 (m, 1 H),
7.66-7.72 (m, 2 H), 4.96-5.08 (m, 1 H), 3.99-4.16 (m, 1 H),
3.69-3.83 (m, 1 H), 2.91-3.14 (m, 1 H), 2.56-2.77 (m, 1 H),
2.14-2.44 (m, 4 H), 1.61-1.73 (m, 2 H), 1.49-1.60 (m, 1 H),
1.32-1.46 (m, 9 H), 1.15-1.29 (m, 1 H). MS (ESI, [M + H]+,
m/z) 658.0. HRMS: calculated for C28H32F3N3O8S2 + H+ 660.16557,
found (ESI, [M + H-tboc]+) 560.1172. HPLC purity 95.7%, RT
8.9 min.
5-(Phenylsulfonyl)-N-[1-(piperazin-1-ylacetyl)piperidin-4-yl]-2-
(trifluoro-methyl)benzenesulfonamide (3e). In an analogous manner
to 3a, step 2, a stirred solution of N-[1-(chloroacetyl)piperidin-4-
yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (0.80
g, 0.15 mmol) in ethanol was treated with piperazine-1-carboxylic
acid tert-butyl ester (0.06 g, 0.32 mmol) and the resulting solution
was stirred overnight at room temperature. The reaction mixture
was washed with water and concentrated. The resulting solid was
dissolved in ethyl acetate (2 mL) and HCl gas was bubbled in.
The solution was stirred overnight and filtered to give 3e as an
1
HCl salt (0.051 g, 58%). H (400 MHz, DMSO-d6) δ 9.49 (br s,
1H), 8.71 (d, J ) 7.69 Hz, 1H), 8.61 (d, J ) 1.54 Hz, 1H),
8.38-8.45 (m, 1H), 8.25 (d, J ) 8.20 Hz, 1H), 8.00-8.09 (m,
2H), 7.75-7.85 (m, 1H), 7.65-7.74 (m, 2H), 4.09 (d, J ) 11.02
Hz, 1H), 3.59 (br s, 3H), 2.93-3.09 (m, 1H), 2.64-2.78 (m, 1H),
1.65-1.76 (m, 1H), 1.55-1.65 (m, 1H), 1.40-1.54 (m, 1H),
1.20-1.32 (m, 1H). HRMS: calculated for C24H29F3N4O5S2 + H+
575.16042, found (ESI, [M + H]+ observed) 575.1606. MS (ESI,
[M - H]-, m/z) 572.9. HPLC purity 98%, RT 7.1 min.
N-[1-(1H-Imidazol-1-ylacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-
2-(trifluoromethyl)benzenesulfonamide (3f). To a stirred solution of
imidazol-1-yl-acetic acid (0.035 g, 0.27 mmol) and DMAP (0.03 g,
0.27 mmol) in methylene chloride (2 mL) was added EDC (0.06 g,
0.03 mmol) and the resulting solution was stirred room temperature
for 20 min. To this was added 1 (0.10 g, 0.22 mmol) and the resulting
solution was stirred for 3 days. The mixture was washed with a
saturated aqueous sodium bicarbonate solution at which time a
precipitate formed. The solid was filtered, then triturated in methylene
chloride to give 3f, which was dissolved in ethyl acetate, treated with
HCl gas, and concentrated to provide the HCl salt (0.068 g, 55%). 1H
(400 MHz, DMSO-d6) δ 8.59 (d, J ) 2.05 Hz, 1H), 8.28 (br s, 1H),
8.13 (br. s., 1H), 8.01 (d, J ) 7.43 Hz, 2H), 7.73-7.82 (m, 1H),
7.64-7.73 (m, 2H), 7.51 (s, 1H), 7.00-7.07 (m, 1H), 6.85 (s, 1H),
4.85-5.08 (m, 2H), 3.97 (d, J ) 8.97 Hz, 1H), 3.68 (d, J ) 13.32
Hz, 1H), 3.18 (br s, 1H), 3.00 (t, J ) 10.50 Hz, 1H), 2.63-2.77 (m,
1H), 1.54-1.67 (m, 1H), 1.43-1.53 (m, 1H), 1.29-1.43 (m, 1H),
1.10-1.25 (m, 1H). HRMS: calculated for C23H23F3N4O5S2 + H+
557.11347, found (ESI, [M + H]+ observed) 557.1134. MS (ESI, [M
+ H]+, m/z) 556.8. HPLC purity 99.2%, RT 7.3 min.
5-(Phenylsulfonyl)-N-(1-L-prolylpiperidin-4-yl)-2-(trifluorometh-
yl)benzenesulfonamide (5a). 4a (0.09 g, 0.14 mmol) was dissolved
in ethyl acetate (2 mL) and HCl gas was bubbled in. The solution
was stirred overnight and filtered to give 5a as an HCl salt (0.08 g,
21
98%). Specific optical rotation [R]D ) ( 27.2 (c 1.15, MeOH).
1H (400 MHz, DMSO-d6) δ 9.53 (br. s., 1 H), 8.62-8.75 (m, 1 H),
8.59-8.62 (m, 1 H), 8.36-8.51 (m, 2 H), 8.25 (d, J ) 8.5 Hz, 1
H), 8.02-8.08 (m, 2 H), 7.76-7.82 (m, 1 H), 7.66-7.74 (m, 2 H),
4.45-4.64 (m, 1 H), 4.03-4.15 (m, 1 H), 3.65-3.75 (m, 1 H),
3.01-3.28 (m, 4 H), 2.71-2.85 (m, 1 H), 2.29-2.44 (m, 1 H),
1.58-1.97 (m, 6 H), 1.21-1.49 (m, 2 H). HRMS: calculated for
C23H26F3N3O5S2 + H+ 546.13387, found (ESI, [M + H]+)
546.1358. MS (ESI, [M + H]+, m/z) 546.1. HPLC purity 100%,
RT 7.2 min.
tert-Butyl (2S)-2-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phe-
nyl]-sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carbox-
ylate (4a). To a stirred solution of N-(tert-butoxycarbonyl)-L-proline
(0.15 g, 0.70 mmol) and CDI (0.11 g, 0.68 mmol) and triethylamine
(0.1 mL, 0.7 mmol) in DMF (2 mL) was added 1 (0.30 g, 0.67
mmol) and the resulting solution was heated to 100 °C for 15 min.
5-(Phenylsulfonyl)-N-(1-D-prolylpiperidin-4-yl)-2-(trifluorometh-
yl)benzenesulfonamide (5b). 4b (0.096 g, 0.15 mmol) was dissolved
in ethyl acetate (2 mL) and HCl gas was bubbled in. The solution

N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 113
was stirred overnight and filtered to give 5b as an HCl salt (0.078
g, 90%). Specific optical rotation [R]D ) +19.8 (c 0.8, MeOH).
(m, 1 H), 7.66-7.73 (m, 2 H), 4.69-4.89 (m, 1 H), 3.76-3.89
(m, 1 H), 3.43-3.61 (m, 2 H), 2.89-3.11 (m, 1 H), 2.03-2.25
(m, 3 H), 1.40-1.92 (m, 5 H), 0.94-1.06 (m, 9 H). HRMS:
calculated for C29H36F3N3O6S2 + H+ 644.20704, found (ESI, [M
+ H]+) 644.205. MS (ESI, [M + H]+, m/z) 643.9. HPLC purity
95.9%, RT 9.6 min.
21
1H (400 MHz, DMSO-d6) δ 9.67 (br s, 1H), 8.63-8.76 (m, 1H),
8.60 (s, 1H), 8.42 (d, J ) 8.46 Hz, 2H), 8.25 (d, J ) 8.20 Hz, 1H),
8.06 (d, J ) 7.69 Hz, 2H), 7.76-7.84 (m, 1H), 7.66-7.74 (m,
2H), 4.45-4.64 (m, 1H), 4.01-4.16 (m, 1H), 3.65-3.77 (m, 1H),
2.99-3.28 (m, 3H), 2.64-2.88 (m, 1H), 2.26-2.43 (m, 1H),
1.78-2.01 (m, 2H), 1.55-1.77 (m, 4H), 1.20-1.52 (m, 2H).
HRMS: calculated for C23H26F3N3O5S2 + H+ 546.13387, found
(ESI, [M + H]+) 546.1329. MS (ESI, [M + H]+, m/z) 546.1. HPLC
purity 97.7, RT 7.2 min.
N-[1-(N,N-Dimethylglycyl-L-prolyl)piperidin-4-yl]-5-(phenylsul-
fonyl)-2-(trifluoromethyl)benzenesulfonamide (6e). In an analogous
manner to 6b, 5a, diisopropylethylamine, and dimethylamino-acetyl
chloride were used to prepare 6e, which was dissolved in ethyl
acetate, treated with HCl gas, and concentrated to form the HCl
N-[1-(5-oxo-L-Prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluo-
romethyl)benzenesulfonamide (5c). 4c (0.064 g, 0.10 mmol) was
dissolved in ethyl acetate (2 mL) and HCl gas was bubbled in.
The solution was stirred overnight and filtered to give 5c (0.049 g,
1
salt (0.049 g, 56%). H (400 MHz, DMSO-d6) δ 9.73 (br s, 1 H),
8.55-8.80 (m, 2 H), 8.42 (d, J ) 8.5 Hz, 1 H), 8.25 (d, J ) 8.2
Hz, 1 H), 8.01-8.09 (m, 2 H), 7.75-7.84 (m, 1 H), 7.66-7.74
(m, 2 H), 4.77-4.92 (m, 1 H), 4.17-4.29 (m, 1 H), 3.78-3.93
(m, 1 H), 3.43-3.55 (m, 2 H), 3.18-3.42 (m, 2 H), 2.94-3.17
(m, 1 H), 2.72-2.90 (m, 5 H), 2.56-2.71 (m, 1 H), 2.06-2.29
(m, 1 H), 1.79-1.98 (m, 2 H), 1.13-1.79 (m, 5 H). HRMS:
calculated for C27H33F3N4O6S2 + H+ 631.18664, found (ESI, [M
+ H]+ observed) 631.1862. MS (ESI, [M + H]+, m/z) 630.9. HPLC
purity 87.6%, RT 7.4 min.
1
88%). H (400 MHz, DMSO-d6) δ 8.54-8.69 (m, 2H), 8.42 (d, J
) 8.71 Hz, 1H), 8.24 (d, J ) 8.46 Hz, 1H), 7.99-8.09 (m, 2H),
7.75-7.83 (m, 1H), 7.63-7.73 (m, 2H), 4.49 (d, J ) 6.92 Hz,
1H), 4.02-4.13 (m, 1H), 3.63-3.77 (m, 1H), 2.88-3.08 (m, 1H),
2.56-2.72 (m, 1H), 2.21-2.35 (m, 1H), 2.01-2.16 (m, 3H),
1.69-1.86 (m, 1H), 1.49-1.67 (m, 2H), 1.14-1.42 (m, 2H).
HRMS: calculated for C23H24F3N3O6S2 + H+ 560.11314, found
(ESI, [M + H]+) 560.1150. MS (ESI, [M + H]+, m/z) 559.8. HPLC
purity 93.8%, RT 7.6 min.
N-{1-[1-(Cyclohexylcarbonyl)-L-prolyl]piperidin-4-yl}-5-(phenyl-
sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6f). In an analo-
gous manner to 6b, 5a, diisopropylethylamine, and cyclohexane-
carbonyl chloride were used to prepare 6f (0.073 g, 81%). 1H (400
MHz, DMSO-d6) δ ) 8.49-8.80 (m, 2 H), 8.38-8.46 (m, 1 H),
8.20-8.29 (m, 1 H), 8.01-8.09 (m, 2 H), 7.74-7.83 (m, 1 H),
7.65-7.74 (m, 2 H), 4.63-5.00 (m, 1 H), 3.76-4.24 (m, 2 H),
3.46-3.66 (m, 2 H), 2.83-3.09 (m, 1 H), 1.81-1.97 (m, 2 H),
1.38-1.80 (m, 10 H), 1.04-1.37 (m, 7 H). HRMS: calculated for
C30H36F3N3O6S2 + H+ 656.20704, found (ESI, [M + H]+ observed)
656.2069. MS (ESI, [M + H]+, m/z) 655.9. HPLC purity 96.2%,
RT 9.8 min.
N-[1-(1-Acetyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-
fluoromethyl)benzenesulfonamide (6a). In an analogous manner to
4c, 1 and N-acetyl-L-proline were used to prepare 6a (0.102 g, 78%).
1H (400 MHz, DMSO-d6) δ 8.49-8.71 (m, 2 H), 8.42 (d, J ) 9.0
Hz, 1 H), 8.24 (d, J ) 8.2 Hz, 1 H), 8.02-8.07 (m, 2 H), 7.75-7.83
(m, 1 H), 7.66-7.74 (m, 2 H), 4.66-4.93 (m, 1 H), 3.97-4.18
(m, 1 H), 3.72-3.89 (m, 1 H), 3.46-3.57 (m, 1 H), 3.24-3.42
(m, 1 H), 2.89-3.15 (m, 1 H), 1.83-2.00 (m, 3 H), 1.42-1.81
(m,
5 H), 1.10-1.28 (m, 1 H). HRMS: calculated for
C25H28F3N3O6S2 + H+ 588.14444, found (ESI, [M + H]+),
588.1462. MS (ESI, [M + H]+, m/z) 587.9. HPLC purity 99.5%,
RT 5.9 min.
N-[1-(1-Benzoyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-
(trifluoromethyl)benzenesulfonamide (6g). In an analogous manner
to 6b, 5a, diisopropylethylamine, and benzoyl chloride were used
to prepare 6g (0.078 g, 88%). 1H (400 MHz, DMSO-d6) δ )
8.50-8.76 (m, 2 H), 8.42 (d, J ) 8.5 Hz, 1 H), 8.24 (d, J ) 8.7
Hz, 1 H), 7.99-8.10 (m, 2 H), 7.75-7.85 (m, 1 H), 7.64-7.74
(m, 2 H), 7.41-7.56 (m, 4 H), 7.22-7.37 (m, 1 H), 4.62-5.04
(m, 1 H), 3.85-4.17 (m, 2 H), 3.33-3.70 (m, 3 H), 2.93-3.23
(m, 1 H), 2.56-2.79 (m, 1 H), 2.16-2.37 (m, 1 H), 1.12-1.96
(m, 7 H). HRMS: calculated for C30H30F3N3O6S2 + H+ 650.16009,
found (ESI, [M + H]+) 650.1616. MS (ESI, [M + H]+, m/z) 649.8.
HPLC purity 94.7%, RT 9.2 min.
N-[1-(1-Isobutyryl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-
(trifluoromethyl)benzenesulfonamide (6b). To a stirred solution of
5a (0.08 g, 0.13 mmol) in methylene chloride (2 mL) was added
diisopropylethylamine (0.1 mL, 0.58 mmol) and isobutyryl chloride
(0.015 g, 0.13 mmol) and the resulting solution was stirred at room
temperature for several hours. The reaction was washed with
saturated aqueous ammonium chloride solution and concentrated.
Flash column separation using 30-100% ethyl acetate/hexane
1
gradient gave 6b (0.064 g, 75%). H (400 MHz, DMSO-d6) δ
8.50-8.73 (m, 2H), 8.42 (d, J ) 8.71 Hz, 1H), 8.21-8.27 (m,
1H), 8.01-8.08 (m, 2H), 7.74-7.82 (m, 1H), 7.66-7.73 (m, 2H),
4.65-4.80 (m, 1H), 3.95-4.09 (m, 1H), 3.76-3.89 (m, 1H),
3.46-3.63 (m, 2H), 3.33-3.42 (m, 1H), 2.91-3.06 (m, 1H),
2.63-2.76 (m, 1H), 1.09-1.94 (m, 9H), 0.82-1.05 (m, 6H).
HRMS: calculated for C27H32F3N3O6S2 + H+ 616.17574, found
(ESI, [M + H]+) 616.1791. MS (ESI, [M + H]+, m/z) 615.8. HPLC
purity 100.0%, RT 9.0 min.
N-(1-{1-[4-(Dimethylamino)benzoyl]-L-prolyl}piperidin-4-yl)-5-
(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6h). In an
analogous manner to 6b, 5a, diisopropylethylamine, and 4-dim-
ethylaminobenzoyl chloride were used to prepare 6h, which was
dissolved in ethyl acetate, treated with HCl gas, and concentrated
1
to form the HCl salt (0.041 g, 43%). H (400 MHz, DMSO-d6) δ
) 8.50-8.77 (m, 2 H), 8.42 (d, J ) 9.0 Hz, 1 H), 8.24 (d, J ) 8.5
Hz, 1 H), 7.98-8.10 (m, 2 H), 7.61-7.85 (m, 3 H), 7.18-7.50
(m, 2 H), 6.63-6.86 (m, 2 H), 5.10-5.82 (m, 3 H), 4.71-5.04
(m, 1 H), 3.85-4.19 (m, 2 H), 3.52-3.73 (m, 2 H), 2.09-2.30
(m, 1 H), 1.42-1.96 (m, 5 H), 1.11-1.42 (m, 2 H). HRMS:
calculated for C32H35F3N4O6S2 + H+ 693.20229, found (ESI, [M
+ H]+) 693.2034. MS (ESI, [M + H]+, m/z) 692.9. HPLC purity
94.2%, RT 9.6 min.
N-{1-[1-(2,2-Dimethylpropanoyl)-L-prolyl]piperidin-4-yl}-5-(phe-
nylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6c). In an
analogous manner to 6b, 5a, diisopropylethylamine, and 2,2-
dimethyl-propionyl chloride were used to prepare 6c (0.068 g, 78%).
1H (400 MHz, DMSO-d6) δ 8.48-8.76 (m, 2 H), 8.41 (d, J ) 7.7
Hz, 1 H), 8.24 (d, J ) 8.2 Hz, 1 H), 8.01-8.08 (m, 2 H), 7.74-7.83
(m, 1 H), 7.65-7.74 (m, 2 H), 4.69-4.85 (m, 1 H), 3.95-4.11
(m, 1 H), 3.67-3.91 (m, 2 H), 3.51-3.65 (m, 1 H), 2.86-3.08
(m, 1 H), 1.77-2.14 (m, 3 H), 1.40-1.70 (m, 4 H), 1.06-1.26
(m, 11 H). HRMS: calculated for C28H34F3N3O6S2 + H+ 630.19139,
found (ESI, [M + H]+) 630.1919. MS (ESI, [M + H]+, m/z) 629.8.
HPLC purity 95.8%, RT 9.4 min.
N-{1-[1-(3,3-Dimethylbutanoyl)-L-prolyl]piperidin-4-yl}-5-(phe-
nylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6d). In an
analogous manner to 6b, 5a, diisopropylethylamine, and 3,3-
dimethyl-butyryl chloride were used to prepare 6d (0.075 g, 85%).
1H (400 MHz, DMSO-d6) δ 8.49-8.75 (m, 2 H), 8.42 (d, J ) 9.0
Hz, 1 H), 8.24 (d, J ) 7.9 Hz, 1 H), 8.01-8.08 (m, 2 H), 7.74-7.83
N-[1-(1-Isonicotinoyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-
2-(trifluoromethyl)benzenesulfonamide (6i). In an analogous man-
ner to 6b, 5a, diisopropylethylamine, and 4-isonicotinoyl chloride
were used to prepare 6i, which was dissolved in ethyl acetate, treated
with HCl gas, and concentrated to form the HCl salt (0.068 g, 76%).
1H (400 MHz, DMSO-d6) δ ) 8.53-8.85 (m, 4 H), 8.38-8.47
(m, 1 H), 8.20-8.29 (m, 1 H), 8.00-8.09 (m, 2 H), 7.76-7.83
(m, 1 H), 7.42-7.74 (m, 4 H), 4.38-5.47 (m, 2 H), 3.83-4.17
(m, 2 H), 2.97-3.69 (m, 3 H), 2.58-2.74 (m, 1 H), 2.14-2.40
(m,
1 H), 1.08-2.05 (m, 7 H). HRMS: calculated for

114 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Moore et al.
C29H29F3N4O6S2 + H+ 651.15534, found (ESI, [M + H]+)
651.1552. MS (ESI, [M + H]+, m/z) 650.8. HPLC purity 94.6%,
RT 8.4 min.
(m, 3 H), 2.07-2.24 (m, 1 H), 1.38-1.94 (m, 7 H), 1.09-1.33
(m, 2 H). HRMS: calculated for C28H33F3N4O7S2 + H+ 659.18155,
found (ESI, [M + H]+ observed) 659.1810. MS (ESI, [M + H]+,
m/z) 658.8. HPLC purity 100.0%, RT 8.6 min.
(2S)-N-Ethyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)ph-
enyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carbox-
amide (7a). To a stirred solution of 5a (0.075 g, 0.129 mmol) in
methylene chloride (2 mL) was added diisopropylethylamine (0.1
mL, 0.58 mmol) and ethylisocyanate (0.009 g, 0.126 mmol) and
the resulting solution was stirred at room temperature for several
hours. The reaction was washed with saturated aqueous ammonium
chloride solution and concentrated. Flash column separation using
30-100% ethyl acetate/hexane gradient gave 7a (0.040 g, 50%).
1H (400 MHz, DMSO-d6) δ ) 8.48-8.73 (m, 2 H), 8.39-8.45
(m, 1 H), 8.24 (d, J ) 8.5 Hz, 1 H), 8.01-8.08 (m, 2 H), 7.76-7.83
(m, 1 H), 7.67-7.73 (m, 2 H), 6.02-6.11 (m, 1 H), 4.60-4.73
(m, 1 H), 3.98-4.09 (m, 1 H), 3.74-3.87 (m, 1 H), 3.22-3.31
(m, 3 H), 2.88-3.11 (m, 3 H), 2.01-2.13 (m, 1 H), 1.78-1.89
(m, 2 H), 1.40-1.66 (m, 4 H), 1.10-1.33 (m, 2 H), 0.93-1.05
(m, 3 H). HRMS: calculated for C26H31F3N4O6S2 + H+ 617.17099,
found (ESI, [M + H]+) 617.1716. MS (ESI, [M + H]+, m/z) 616.8.
HPLC purity 99.1%, RT 8.6 min.
N-[1-(1-Methyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-
fluoromethyl)benzenesulfonamide (8a). To a stirred solution of
N-methyl-L-proline (0.035 g, 0.27 mmol) in methylene chloride (3
mL) was added DMAP (0.033 g, 0.27 mmol) and EDC (0.056 g,
0.027 mmol). The resulting solution was stirred at room temperature
for 20 min, at which time 1 (0.10 g, 0.22 mmol) was added and
stirred overnight at room temperature. The reaction was washed
with water and concentrated. Flash column separation using 0-10%
methanol/methylene chloride gradient gave 8a, which was dissolved
in ethyl acetate, treated with HCl gas, and concentrated to form
1
the HCl salt (0.055 g, 44%). H (400 MHz, DMSO-d6) δ ) 9.54
(br s, 1 H), 8.57-8.76 (m, 2 H), 8.42 (d, J ) 8.2 Hz, 1 H), 8.25 (d,
J ) 8.5 Hz, 1 H), 8.02-8.10 (m, 2 H), 7.76-7.83 (m, 1 H),
7.66-7.74 (m, 2 H), 4.46-4.65 (m, 1 H), 4.03-4.17 (m, 1 H),
3.51-3.65 (m, 2 H), 3.35-3.44 (m, 2 H), 3.00-3.17 (m, 2 H),
2.73-2.87 (m, 4 H), 2.02-2.16 (m, 1 H), 1.59-1.96 (m, 4 H),
1.24-1.55 (m, 2 H). HRMS: calculated for C24H28F3N3O5S2 + H+
560.14952, found (ESI, [M + H]+) 560.1518. MS (ESI, [M + H]+,
m/z) 560.2. HPLC purity 100.0%, RT 7.3 min.
(2S)-N-(tert-Butyl)-2-{[4-({[5-(phenylsulfonyl)-2-(trifluorometh-
yl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-car-
boxamide (7b). In an analogous manner to 7a, 5a, diisopropyleth-
ylamine, and tert-butylisocyanate were used to prepare 7b (0.076,
N-{1-[1-(3,3-Dimethylbutyl)-L-prolyl]piperidin-4-yl}-5-(phenyl-
sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (8b). To a stirred
solution of 5a (0.09 g, 0.15 mmol) in anhydrous methanol (1 mL)
was added 3,3-dimethyl-butyraldehyde (0.016 g, 0.16 mmol) and
the resulting solution was stirred at room temperature several hours.
To this mixture was added sodium triacetoxyborohydride (0.05 g,
0.22 mmol) and the reaction was allowed to stir at room temperature
several more hours and concentrated. Flash column separation using
0-10% methanol/methylene chloride gradient gave 8b, which was
dissolved in ethyl acetate, treated with HCl gas, and concentrated
21
1
85%). Specific optical rotation [R]D ) -1.2 (c 1.3, MeOH). H
(400 MHz, DMSO-d6) δ ) 8.48-8.76 (m, 2 H), 8.41 (d, J ) 7.9
Hz, 1 H), 8.24 (d, J ) 8.2 Hz, 1 H), 8.01-8.09 (m, 2 H), 7.75-7.83
(m, 1 H), 7.65-7.74 (m, 2 H), 5.19 (s, 1 H), 4.59-4.78 (m, 1 H),
3.76-4.13 (m, 2 H), 3.19-3.43 (m, 2 H), 2.85-3.08 (m, 1 H),
1.95-2.13 (m, 1 H), 1.73-1.94 (m, 2 H), 1.38-1.67 (m, 4 H),
1.06-1.36 (m, 11 H). HRMS: calculated for C28H35F3N4O6S2 +
H+ 645.20229, found (ESI, [M + H]+) 645.2024. MS (ESI, [M +
H]+, m/z) 644.9. HPLC purity 100.0%, RT 9.3 min.
1
to form the HCl salt (0.027 g, 28%). H (400 MHz, DMSO-d6) δ
) 9.31 (br s, 1 H), 8.56-8.78 (m, 2 H), 8.42 (dd, J ) 1.3, 8.2 Hz,
1 H), 8.25 (d, J ) 8.5 Hz, 1 H), 8.02-8.10 (m, 2 H), 7.76-7.84
(m, 1 H), 7.67-7.74 (m, 2 H), 4.51-4.75 (m, 1 H), 4.05-4.19
(m, 1 H), 3.56-3.72 (m, 2 H), 2.74-3.18 (m, 5 H), 1.99-2.15
(m, 1 H), 1.57-1.87 (m, 3 H), 1.20-1.57 (m, 4 H), 0.83-0.96
(m, 9 H). HRMS: calculated for C29H38F3N3O5S2 + H+ 630.22777,
found (ESI, [M + H]+) 630.2308. MS (ESI, [M + H]+, m/z) 630.0.
HPLC purity 100.0%, RT 8.8 min.
(2S)-N-Phenyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)-
phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-car-
boxamide (7c). In an analogous manner to 7a, 5a, diisopropyleth-
ylamine, and phenylisocyanate were used to prepare 7c (0.074 g,
81%). 1H (400 MHz, DMSO-d6) δ ) 8.49-8.73 (m, 2 H), 8.42 (d,
J ) 8.2 Hz, 1 H), 8.24 (d, J ) 8.2 Hz, 1 H), 8.16 (d, J ) 5.6 Hz,
1 H), 8.05 (d, J ) 7.7 Hz, 2 H), 7.75-7.83 (m, 1 H), 7.66-7.73
(m, 2 H), 7.42-7.56 (m, 2 H), 7.15-7.27 (m, 2 H), 6.86-6.97
(m, 1 H), 4.73-4.88 (m, 1 H), 3.98-4.10 (m, 1 H), 3.79-3.90
(m, 1 H), 3.45-3.61 (m, 2 H), 2.91-3.11 (m, 1 H), 2.07-2.23
(m, 1 H), 1.83-1.98 (m, 3 H), 1.43-1.71 (m, 4 H), 1.10-1.37
(m, 2 H). HRMS: calculated for C30H31F3N4O6S2 + H+ 665.17099,
found (ESI, [M + H]+ observed) 665.1703. MS (ESI, [M + H]+,
m/z) 664.8. HPLC purity 100.0%, RT 9.4 min.
N-{1-[1-(Cyclohexylmethyl)-L-prolyl]piperidin-4-yl}-5-(phenyl-
sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (8c). In an analo-
gous manner to 8b, 5a, cyclohexanecarboxaldehyde, and sodium
triacetoxyborohydride were used to prepare 8c, which was dissolved
in ethyl acetate, treated with HCl gas, and concentrated to form
1
the HCl salt (0.024 g, 25%). H (400 MHz, DMSO-d6) δ ) 8.96
(2S)-N,N-Dimethyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluorometh-
yl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-car-
boxamide (7d). In an analogous manner to 7a, 5a, diisopropyl-
ethylamine, and dimethylaminocarbonyl chloride were used to
prepare 7d (0.045 g, 57%). 1H (400 MHz, DMSO-d6) δ )
8.50-8.71 (m, 2 H), 8.42 (dd, J ) 1.0, 8.2 Hz, 1 H), 8.24 (d, J )
8.5 Hz, 1 H), 8.00-8.08 (m, 2 H), 7.75-7.83 (m, 1 H), 7.66-7.74
(m, 2 H), 4.64-4.80 (m, 1 H), 4.00-4.11 (m, 1 H), 3.82-3.93
(m, 1 H), 3.38 (dd, J ) 4.7, 8.3 Hz, 2 H), 2.89-3.06 (m, 1 H),
2.68-2.77 (m, 6 H), 2.52-2.60 (m, 1 H), 2.03-2.22 (m, 1 H),
1.81-1.93 (m, 1 H), 1.39-1.77 (m, 5 H), 1.09-1.38 (m, 2 H).
HRMS: calculated for C26H31F3N4O6S2 + H+ 617.17099, found
(ESI, [M + H]+) 617.1701. MS (ESI, [M + H]+, m/z) 616.8. HPLC
purity 99.2%, RT 8.7 min.
(br s, 1 H), 8.57-8.76 (m, 2 H), 8.42 (d, J ) 8.2 Hz, 1 H), 8.25 (d,
J ) 8.2 Hz, 1 H), 8.02-8.08 (m, 2 H), 7.76-7.83 (m, 1 H),
7.66-7.74 (m, 2 H), 4.51-4.76 (m, 1 H), 4.03-4.19 (m, 1 H),
3.56-3.82 (m, 3 H), 3.02-3.20 (m, 3 H), 2.73-2.99 (m, 3 H),
1.85-2.16 (m, 3 H), 1.53-1.82 (m, 9 H), 1.07-1.41 (m, 4 H),
0.86-1.02 (m, 2 H). HRMS: calculated for C30H38F3N3O5S2 + H+
642.22777, found (ESI, [M + H]+) 642.2297. MS (ESI, [M + H]+,
m/z) 641.9. HPLC purity 100.0%, RT 8.9 min.
N-[1-(1-Benzyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-
fluoromethyl)benzenesulfonamide (8d). To a stirred solution of 5a
(0.09 g, 0.15 mmol) in THF (2 mL) was added triethylamine (0.1
mL, 0.70 mmol) and benzyl bromide (0.03 mmol, 0.15 mmol) and
the resulting solution was microwave irradiated to 120 °C for 10
min. The resulting mixture was partitioned between ethyl acetate
and a saturated sodium bicarbonate solution. The organic layer was
concentrated and flash column separated using 0-10% methanol/
methylene chloride gradient gave 8d, which was dissolved in ethyl
acetate, treated with HCl gas, and concentrated to form the HCl
N-{1-[1-(Morpholin-4-ylcarbonyl)-L-prolyl]piperidin-4-yl}-
5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (7e).
In an analogous manner to 7a, 5a, diisopropylethylamine, and
morpholine-4-carbonyl chloride were used to prepare 7e (0.055 g,
1
1
61%). H (400 MHz, DMSO-d6) δ ) 8.49-8.74 (m, 2 H), 8.42
salt (0.042 g, 43%). H (400 MHz, DMSO-d6) δ ) 9.62 (br. s., 1
(dd, J ) 1.3, 7.9 Hz, 1 H), 8.24 (d, J ) 8.5 Hz, 1 H), 8.02-8.08
(m, 2 H), 7.76-7.83 (m, 1 H), 7.67-7.73 (m, 2 H), 4.64-4.82
(m, 1 H), 3.99-4.13 (m, 1 H), 3.81-3.94 (m, 1 H), 3.49-3.66
(m, 4 H), 3.33-3.46 (m, 2 H), 3.13-3.26 (m, 2 H), 2.90-3.11
H), 8.52-8.78 (m, 2 H), 8.40-8.45 (m, 1 H), 8.25 (d, J ) 8.5 Hz,
1 H), 8.05 (d, J ) 7.9 Hz, 2 H), 7.75-7.84 (m, 1 H), 7.65-7.73
(m, 2 H), 7.19-7.59 (m, 5 H), 4.36-4.78 (m, 2 H), 3.49-4.33
(m, 4 H), 2.77-3.06 (m, 3 H), 2.02-2.20 (m, 1 H), 1.44-1.85

N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides
(m, H), 1.17-1.39 (m, H). HRMS: calculated for
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 115
5
3
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 limited to the
lower concentration range.
C30H32F3N3O5S2 + H+ 636.18082, found (ESI, [M + H]+ observed)
636.1806. MS (ES) m/z 635.9. HPLC purity 100.0%, RT 8.1 min.
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 competitive displacement
of the probe by the test compound. The decrease in anisotropy as
a function of increasing concentration of the test compound provided
a direct measure of the test compound’s binding affinity for
sFRP-1.
Cell-Based, TCF-Reporter Functional Assay. U2OS bone cells
were 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 were frozen in sterile cryogenic vials at a cell
density of 9 × 106 cells/mL and stored in a -150 °C freezer. For the
assay, a vial of frozen cells was thawed, and the cells were resuspended
in plating medium [phenol red-free RPMI 1640 medium containing
5% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-
streptomycin] to a final cell density of 1.5 × 105 cells/mL. The
resuspended cells were then plated in 96-well tissue culture treated
plates at a volume of 100 µL of cell suspension/well (i.e., 1.5 ×104
cells/well). The plates were 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 of test compounds, the medium
was replaced with 50 µL/well of phenol red-free RPMI 1640 containing
10% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-
streptomycin. Test compounds, or vehicle (typically DMSO), diluted
in phenol red-free RPMI 1640 containing 2 mM GlutaMAX-l, and l
% (v/v) penicillin-streptomycin were then added to the wells in
replicates of 4 wells/dilution and the plates were incubated at 37 °C
overnight. Dose-response experiments were performed with the
compounds in 2-fold serial dilutions from 10000-4.9 nM. After the
overnight incubation, the cells were washed twice with 150 uL/well
of PBS w/o calcium or magnesium and lysed with 50 µL/well of 1×
cell culture lysis reagent (Promega Corporation) on a shaker at room
temperature for 30 min. Aliquots of the cell lysates (30 µL) were
transferred to 96-well luminometer plates, and the luciferase activity
was measured in a MicroLumat PLUS luminometer (EG&G Berthold)
using 100 µL/well of luciferase substrate (Promega Corporation).
Following the injection of substrate, luciferase activity was measured
for 10 s after a 1.6 s delay. The luciferase activity data was transferred
from the luminometer to a PC and analyzed using the SAS/Excel
program to determine EC50 values. The luciferase data was analyzed
using the SAS/Excel program. EC50 determinations for dose response
curves were determined using the SAS/Excel program.
Ex Vivo Bone Formation Assay. Neonatal mouse calvaria were
prepared from 4-day-old pups as described previously.17 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 surface 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 bovine serum (FBS). Calvaria were incubated
for 7 days with 0.1% (v/v) dimethyl sulfoxide (DMSO, vehicle control)
or with compounds in a humidified atmosphere of 95% air and 5%
CO2, and the medium was changed on day 4 with fresh DMSO or
compounds added.
To determine IC50 values, fluorescence polarization experiments
were conducted in a 384-well format according to the following
procedures. A 20 mM stock solution of the probe compound was
prepared in 100% DMSO and dispensed in 10 µL aliquots for long-
term storage at -20 °C. The binding assay buffer was prepared by
combining stock solutions of Tris-Cl, NaCl, glycerol, 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 concentrations
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, 0.3125
mM, 0.156 mM, 78 µM, 39 µM, 19.5 µM, 9.8 µM, 4.9 µM, 2.44
µM, 1.22 µM, 0.31 µM, 76 nM, and 19 nM in DMSO. The working
stock solutions of the test compounds were further diluted by
combining 6 µL of the solutions with 24 µL of Milli-Q purity water,
resulting in working stock solutions (10× compound stocks) in 20%
DMSO.
The assay controls were prepared as follows. A 2 µL aliquot of
the 20 mM fluorescence probe compound was diluted 1000-fold
in 100% DMSO to a final concentration of 20 µM. Then 6 µL of
the 20 µM probe was combined with 5.4 mL of the assay buffer,
mixed well, and 18 µL of the resulting solution was dispensed into
384-well plates.
sFRP-1/probe complex was prepared by combining 11 µL of 20
µM probe compound with 9.9 mL of the assay buffer and sFRP-1
stock solution to final concentrations of 22 nM probe compound
and 50 nM sFRP-1. Then 18 µL of the sFRP-1/probe complex was
dispensed into the 384-well plates.
Aliquots of the test compounds from the 10× working stock
solutions (2 µL) were removed and dispensed into the plate
containing the sFRP-1/probe complex and the resultant solutions
were mixed by pipetting 10 µL up and down twice. The final
concentrations of sFRP-1 and probe in the assay solutions were 45
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 fluorescence
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 fluorophore
is excited with vertically polarized light. The anisotropy of the probe
in the free and bound state was determined using the following
equation: r ) [I(|) - I( )] ÷ [I(|) + 2I( )], where I(|) and I( )
are the parallel and perpendicular emission intensities, respectively.
Monitoring the anisotropy changes of the probe compound
revealed 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.
After organ culture, calvaria were fixed in 10% neutral phosphate-
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 paraffin block, and 4
µm sections were stained with hematoxylin-eosin. Consistent bone
areas (200 µm away from the frontal sutures) were selected for
histomorphometric analysis. A 200 µm square 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 analyzed for statistical significance
The decrease in the anisotropy of the probe upon addition of
the competing test compound was fitted to a sigmoidal dose
response curve of the equation shown below: Y ) Bottom + ((Top
- Bottom))/((1 + 10X-LogIC )) × Hillslope, where “X” is the
50
logarithm of concentration, “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.

116 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Moore et al.
Bhat, R. A.; Billiard, J.; Bex, F. J.; Komm, B. S. The role of Wnt
signaling in bone and mineral metabolism. Clin. ReV. Bone Miner.
Metabol. 2006, 4, 73–96.
by one-way ANOVA using the Dunnett’s test with JMP software (SAS
Institute, Cary, NC).
(9) Bodine, P. V. N.; 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.
Acknowledgment. We thank the Departments of Analytical
Chemistry and Chemical Technologies, Wyeth, for analytical and
profile data. We also wish to thank our management, Drs. Magid
Abou-Gharbia and Len Freedman, for their support of this work.
(10) (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. Anabolic activity and pharmacokinetic profiles of secreted
frizzled-related protein-1 (sFRP-1) antagonists. Abstracts of Papers,
234th ACS National Meeting, Boston, MA, August 19-23, 2007,
American Chemical Society: Washington, DC, 2007; 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. Diaryl sulfone sulfonamides
as secreted frizzled-related protein-1 (sFRP-1) antagonists: SAR and
optimization. Abstracts of Papers, 234th ACS National Meeting,
Boston, MA, August 19-23, 2007, American Chemical Society:
Washington, DC, 2007; 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.
Design and synthesis of fluorescent probes for the development of a
binding assay for the identification of secreted frizzled-related protein-1
(sFRP-1) antagonists. Abstracts of Papers, 234th ACS National
Meeting, Boston, MA, August 19-23, 2007, American Chemical
Society: Washington, DC, 2007; MEDI-389.
(11) 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 sul-
fonamides and their use as secreted frizzled related protein-1 modula-
tors for bone disorders such as osteoporosis. U.S. Patent 2006/0276464,
2006.
(12) (a) Clark, J. H.; McClinton, M. A.; Jones, C. W.; Landon, P.; Bishop,
D.; Blade, R. J. Tetrahedron Lett. 1989, 30, 2133–2136. (b) Clark,
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1988, 10, 638–639. (c) Wiemers, D. M.; Burton, D. J. J. Am. Chem.
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Supporting Information Available: Analytical HPLC chromato-
grams for target compounds 3a-f, 4a-c, 5a-c, 6a-i, 7a-e, and
8a-d. Analytical chiral HPLC analyses of compounds 4a-b, 5a-b,
and 7b. Full NMR characterization of compound 7b for proton
assignment including multinuclear 19F, 13C, 15N, and variable temper-
ature experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
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