Modulation of Wnt Signaling Through Inhibition of Secreted Frizzled

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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 bonerelated 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 population. 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 involved anticatabolic agents, targeting osteoclasts that are responsible for bone resorption. These agents arrest the resorptive process and minimize further loss in bone mass, however, bone mass that has been lost is not restored. To restore catabolized bone, an anabolic agent is often necessary to regain bone integrity and structure. Currently, the only anabolic agent approved by the FDA for the treatment of chronic osteoporosis is Teriparatide, a truncated synthetic peptide of the natural 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: [email protected]. ‡ 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.

Figure 1. Diphenylsulfonyl sulfonamide scaffolds are inhibitors of sFRP-1.

skeletal development.4 The Wnt/β-Catenin canonical signaling pathway is activated by the binding of Wnt to a membrane receptor complex composed of a frizzled (Fzd) GPCR and a low-density lipoprotein receptor-related protein (LRP). The binding results in a signaling cascade, leading to the stabilization of β-catenin, translocation to the nucleus, and activation of 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 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

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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.

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 diphenylsulfone 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 piperidinyl 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.

Scheme 2a

Chemistry The methods used to synthesize diphenylsulfonyl sulfonamides 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 dibromodifluoromethane 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-Boc4-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-dimethylamino)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-chloroacetamide 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-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in the presence of N,Ndimethylaminopyridine (DMAP) to provide 3f. The syntheses of the constrained R-aminoacetamides (4) were conducted by activation of the R or S enantiomer of commercially 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

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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 diisopropylethylamine 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 triethylamine to afford the benzyl derivative 8d. 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

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.

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Table 1. Comparison of R-Aminoacetamides 3a-f to 1 Using Fluorescence Polarization Binding and U2-OS Functional Activity Assays

FP bindinga(µM)

Wnt-lucb(µM)

R1

IC50

solubility

EC50

2-FIc

4-FIc

NHCH3 N(CH3)2 pyrrolidin-1-yl morpholin-1-yl 4H-piperazin-1-yl imidazol-1-yl

0.50 0.20 0.30 0.11 0.10 0.14 0.03

50 50 100 25 100 50 100

0.65 0.62 0.28 0.19 0.16 0.25 0.23

0.75 0.16 0.21 0.06 0.07 0.09 0.05

NA 0.61 NA 0.22 0.45 0.75 0.19

compound 1 3a 3b 3c 3d 3e 3f

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

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.

Table 2. Data from Fluorescence Polarization Binding and U2 OS Functional Activity Assays for Compounds 4a-c and 5a-c

FP bindinga (µM)

Wnt-lucb (µM)

compound

R3

IC50

solubility

EC50

4a 6a 6b 6c 6d 6e 6f 6g 6h 6i

Ot-Bu Me i-Pr t-Bu neopentyl CH2N(CH3)2 c-Hex Ph Ph, 4-N(CH3)2 Pyrid-4-yl

0.06 0.16 0.04 0.04 0.02 0.09 0.05 0.07 0.11 0.07

25 12.5 100 50 25 100 12.5 50 100 100

0.06 0.25 0.09 0.27 0.12 0.31 0.46 0.38 0.83 0.35

c

2-FI

0.02 0.09 0.03 0.07 0.04 0.09 0.22 0.14 0.46 0.13

c

4-FI

0.16 0.68 0.11 0.33 0.22 0.49 0.87 0.92 1.82 0.88

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 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) compound

X

R2

1 4a 4b 4c 5a 5b 5c

CH2 CH2 CO CH2 CH2 CO

L-Boc D-Boc L-Boc L-H D-H L-H

Wnt-lucb (µM)

IC50

solubility

EC50

0.50 0.06 0.12 0.13 0.11 0.20 0.09

50 25 25 50 50 25 100

0.65 0.06 0.21 0.09 0.35 0.71 0.24

2-FI

c

0.75 0.02 0.09 0.04 0.17 0.34 0.11

4-FI

c

NA 0.16 0.97 0.25 1.99 2.98 0.99

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.

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-heterocycle 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

FP bindinga (µM) compound 4a 7a 7b 7c 7d 7e

Wnt-lucb (µM)

R5

IC50

solubility

EC50

Et H tBu H Ph H Me Me morpholine

0.06 0.03 0.02 0.02 0.03 0.04

25 100 50 25 100 50

0.06 0.09 0.03 0.31 0.16 0.28

R4

2-FI

c

0.02 0.04 0.01 0.09 0.06 0.11

4-FI

c

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, 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.

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

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Table 5. Fluorescence Polarization Binding and Wnt-Luciferase Activity from U2-OS Cells for Alkylated Proline Analogues 8a-d

FP bindinga (µM)

Wnt-lucb (µM)

compound

R6

IC50

solubility

EC50

7b 8a 8b 8c 8d

H neopentyl c-Hex Ph

0.02 0.12 0.03 0.04 1.4

50 100 12.5 25 25

0.03 0.23 0.45 0.32 0.72

2-FI

c

0.01 0.11 0.04 0.09 0.27

4-FI

c

0.03 0.74 0.17 0.32 0.77

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 characteristics, 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. 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 diminished 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

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 probability of limited oral bioavailability due to phase I metabolism. 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

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Table 6. In Vitro Data Comparison for sFRP-1 Isosteric Inhibitors

CYP450b (% inhibition) compound 1 4a 6d 7b 8b

X CO CO CO CH2

a

a

Y

FP binding IC50 (µM)

Wnt-luc EC50 (µM)

RLM t1/2 (min)

HLM t1/2 (min)

3A4

2D6

2C9

O CH2 NH CH2

0.50 0.06 0.02 0.02 0.03

0.65 0.06 0.12 0.03 0.45

>60c 1 3 3 8

>60c 1 1 2 5

51 55 74 49 84

0 0 0 0 24

0 9 30 0 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.

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.

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 communications 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. Highresolution 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.

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 solid, mp 113-122 °C (21.8-24.5 g, 75-84%). 1H 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 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 onethird 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. 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 give 12 as an off-white solid (11.8 g, 95%), mp 135-138 °C. 1H (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 +

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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. 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. 1 H (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. 5-(Phenylsulfonyl)-N-piperidin-4-yl-2-(trifluoromethyl)benzenesulfonamide (1). Step 1: tert-Butyl 4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)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 (2.56 g, 60%). 1H (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 in ethyl acetate gave 1 as an HCl salt (3 g, 97%). 1H NMR (400

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. N-[1-(N-Methylglycyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)-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 concentrated. Flash column separation using 10-70% ethyl acetate/ hexane gradient gave N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (0.43 g, 61%). Step 2. To a stirred solution of N-[1-(chloroacetyl)piperidin-4yl]-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%). 1 H (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 hydrochloride (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-[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)-

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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%). 1 H (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%). 1 H (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. 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-4yl]-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 HCl salt (0.051 g, 58%). 1H (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. tert-Butyl (2S)-2-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]-sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxylate (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.

Moore et al.

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]D21 ) -20.3 (c 0.95, MeOH). 1H (400 MHz, DMSOd6) 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. tert-Butyl (2R)-2-{[4-({[5-(Phenylsulfonyl)-2-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxylate (4b). To a stirred solution of N-(tert-butoxycarbonyl)-Dproline (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-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1carboxylate (4c). To a stirred solution of 5-oxo-pyrrolidine-1,2dicarboxylic 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-L-prolylpiperidin-4-yl)-2-(trifluoromethyl)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, 98%). Specific optical rotation [R]D21 ) ( 27.2 (c 1.15, MeOH). 1 H (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. 5-(Phenylsulfonyl)-N-(1-D-prolylpiperidin-4-yl)-2-(trifluoromethyl)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

was stirred overnight and filtered to give 5b as an HCl salt (0.078 g, 90%). Specific optical rotation [R]D21 ) +19.8 (c 0.8, MeOH). 1 H (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-(5-oxo-L-Prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)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, 88%). 1H (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-Acetyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6a). In an analogous manner to 4c, 1 and N-acetyl-L-proline were used to prepare 6a (0.102 g, 78%). 1 H (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-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 gradient gave 6b (0.064 g, 75%). 1H (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-(2,2-Dimethylpropanoyl)-L-prolyl]piperidin-4-yl}-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6c). In an analogous manner to 6b, 5a, diisopropylethylamine, and 2,2dimethyl-propionyl chloride were used to prepare 6c (0.068 g, 78%). 1 H (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-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6d). In an analogous manner to 6b, 5a, diisopropylethylamine, and 3,3dimethyl-butyryl chloride were used to prepare 6d (0.075 g, 85%). 1 H (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

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 113

(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. N-[1-(N,N-Dimethylglycyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-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 salt (0.049 g, 56%). 1H (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. N-{1-[1-(Cyclohexylcarbonyl)-L-prolyl]piperidin-4-yl}-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6f). In an analogous manner to 6b, 5a, diisopropylethylamine, and cyclohexanecarbonyl 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-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-[4-(Dimethylamino)benzoyl]-L-prolyl}piperidin-4-yl)-5(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (6h). In an analogous manner to 6b, 5a, diisopropylethylamine, and 4-dimethylaminobenzoyl chloride were used to prepare 6h, which was dissolved in ethyl acetate, treated with HCl gas, and concentrated to form the HCl salt (0.041 g, 43%). 1H (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-Isonicotinoyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)2-(trifluoromethyl)benzenesulfonamide (6i). In an analogous manner 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%). 1 H (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

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. (2S)-N-Ethyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxamide (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%). 1 H (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. (2S)-N-(tert-Butyl)-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxamide (7b). In an analogous manner to 7a, 5a, diisopropylethylamine, and tert-butylisocyanate were used to prepare 7b (0.076, 85%). Specific optical rotation [R]D21 ) -1.2 (c 1.3, MeOH). 1H (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. (2S)-N-Phenyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxamide (7c). In an analogous manner to 7a, 5a, diisopropylethylamine, 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. (2S)-N,N-Dimethyl-2-{[4-({[5-(phenylsulfonyl)-2-(trifluoromethyl)phenyl]sulfonyl}amino)piperidin-1-yl]carbonyl}pyrrolidine-1-carboxamide (7d). In an analogous manner to 7a, 5a, diisopropylethylamine, 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. 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, 61%). 1H (400 MHz, DMSO-d6) δ ) 8.49-8.74 (m, 2 H), 8.42 (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

Moore et al.

(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. N-[1-(1-Methyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)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 the HCl salt (0.055 g, 44%). 1H (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. N-{1-[1-(3,3-Dimethylbutyl)-L-prolyl]piperidin-4-yl}-5-(phenylsulfonyl)-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 to form the HCl salt (0.027 g, 28%). 1H (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. N-{1-[1-(Cyclohexylmethyl)-L-prolyl]piperidin-4-yl}-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (8c). In an analogous 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 the HCl salt (0.024 g, 25%). 1H (400 MHz, DMSO-d6) δ ) 8.96 (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-(trifluoromethyl)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 salt (0.042 g, 43%). 1H (400 MHz, DMSO-d6) δ ) 9.62 (br. s., 1 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, 5 H), 1.17-1.39 (m, 3 H). HRMS: calculated for 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. 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 longterm 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. 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-LogIC50)) × Hillslope, where “X” is the logarithm of concentration, “Y” is the anisotropy, and “Bottom” and “Top” correspond to the anisotropy values of the free and sFRP1-bound probe prior to the addition of the test compound, respectively.

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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. 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 Ad5sFRP-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) penicillinstreptomycin] 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) penicillinstreptomycin. 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. After organ culture, calvaria were fixed in 10% neutral phosphatebuffered 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

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by one-way ANOVA using the Dunnett’s test with JMP software (SAS Institute, Cary, NC).

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.

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Supporting Information Available: Analytical HPLC chromatograms 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 temperature experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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