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 involved anticatabolic agents, targeting osteoclasts that are responsible for bone resorption. These agents arrest the resorp- tive 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 (hPTH a ). 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 skeletal development. 4 The Wnt/-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 -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 † 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. J. Med. Chem. 2009, 52, 105–116 105 10.1021/jm801144h CCC: $40.75 2009 American Chemical Society Published on Web 12/12/2008
Transcript
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 inhibitorsof the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators ofthe 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 structuralclass of this scaffold will be discussed.
Introduction
The skeletal system is one of the largest organs in the humanbody, and the maintenance of healthy and strong bones is criticalto good health and quality of life.1 The incidence of bone diseaseand fractures continues to rise, especially in our aging popula-tion. According to the U.S. Surgeon General, 1.5 million elderlypeople will suffer an osteoporotic-related fracture annually, oftenleading to an overall decline in physical and mental health.1
The occurrence of osteoporotic-related fractures is expected todramatically increase in the next 10 years, resulting in a greaterburden on the health care system.1 Osteoporosis is a bone diseasepredominately affecting postmenopausal women. This diseasedisrupts the balance in bone formation and resorption, the laterof which predominates, resulting in the catabolic loss oftrabecular bone mass and structure.1 Traditional therapies haveinvolved anticatabolic agents, targeting osteoclasts that areresponsible for bone resorption. These agents arrest the resorp-tive process and minimize further loss in bone mass, however,bone mass that has been lost is not restored. To restorecatabolized bone, an anabolic agent is often necessary to regainbone integrity and structure. Currently, the only anabolic agentapproved by the FDA for the treatment of chronic osteoporosisis Teriparatide, a truncated synthetic peptide of the naturalhuman parathyroid hormone (hPTHa).2 The signaling processesinvoked by PTH treatment and responsible for the subsequentanabolic effects on bone are not completely understood.Recently, the anabolic action of PTH on bone has been linkedto the modulation of the Wnt signaling pathway in osteoblasts(OB).3a,b The Wnt signaling pathway, activated by secreted Wntglycoproteins, regulate various biological processes, including
skeletal development.4 The Wnt/�-Catenin canonical signalingpathway is activated by the binding of Wnt to a membranereceptor complex composed of a frizzled (Fzd) GPCR and alow-density lipoprotein receptor-related protein (LRP). Thebinding results in a signaling cascade, leading to the stabilizationof �-catenin, translocation to the nucleus, and activation oftranscriptional factors.3,4 The Wnt signaling process is regulatedby many factors, including extracellular components such asWIF-1, Dkk-1, SOST, and secreted frizzled-related protein-1(sFRP-1 or SARP-2).3a,5 Human sFRP-1 is a 35 kD proteinconsisting of 313 amino acids and containing 2 distinct structuraldomains, a netrin domain, and a cysteine-rich domain (CRD).The later domain is homologous to the Fzd receptor.6a,b sFRP-1is thought to interact with Wnt via the CRD domain in acompetitive manner with the Fzd receptor. Thus sFRP-1 is anegative regulator or an antagonist of the Wnt signalingpathway.7 Increased expression of sFRP-1 in OB cells resultsin suppression of Wnt signaling, leading to decreased OBsurvival, activation, and differentiation.8 Conversely, deletionof the sFRP-1 gene in mice results in increased Wnt signaling,leading to OB activation, proliferation, and differentiationresulting in increases in trabecular bone.9
The goal of our drug discovery program was to identify anorally bioavailable small-molecule inhibitor of sFRP-1 (Figure1), which liberates Wnt for binding to the LRP-frizzled receptor
† 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;
Figure 1. Diphenylsulfonyl sulfonamide scaffolds are inhibitors ofsFRP-1.
J. Med. Chem. 2009, 52, 105–116 105
10.1021/jm801144h CCC: $40.75 2009 American Chemical SocietyPublished on Web 12/12/2008
complex resulting in increased canonical Wnt-signaling. Amolecule with these characteristics would be a potential anabolicagent for the treatment of bone diseases such as osteoporosis.Initial accounts of the discovery and subsequent elaboration ofsmall molecule inhibitors of sFRP-1 were disclosed.10,11 Thepiperidinyl diphenylsulfonyl sulfonamide 1 (WAY-316606) wasidentified as an inhibitor of sFRP-1 with moderate bindingaffinity, good functional activity, and robust anabolic activityin an ex vivo calvaria tissue assay. The SAR of the diphenyl-sulfone portion of compound 1 was explored, and substitutionortho to the sulfonamide was found to be critical. While loweralkyl and halogen groups were tolerated, the trifluoromethylgroup appeared to be optimal.10b N-substitution on the piperidi-nyl group was found to accommodate a wide variety offunctionality. Thus, 1 provided an opportunity for us to capitalizeon derivatization of the piperidine group, and the focus of thiscommunication will highlight our efforts to discover sFRP-1inhibitors 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 sulfonylchloride 2 for the synthesis of 1 was prepared in five stepsstarting with the sulfonylation of benzene with commerciallyavailable 3-nitro-4-chlorobenzene sulfonyl chloride 9 using aFriedel-Crafts catalyst provided the sulfone 10 (75-84%),Scheme 1. Introduction of the trifluoromethyl group wasaccomplished by reaction of the sulfone intermediate usingmethodology described by Clark12a,b and Burton.12c We foundthat the procedure described by Burton using dibromodifluo-romethane and copper metal in the presence of charcoal affordedthe desired product 11 in moderate yield (56%). Reduction ofthe nitro group upon exposure to SnCl2 in aqueous methanolprovided the aniline 12 (95%), which was diazotized andsubsequently transformed to 2 by exposure to sulfur dioxidegas in the presence of CuCl2 (31%). Reaction of 2 with N-Boc-4-aminopiperidine provided the sulfonamide 13 (60%), followedby acidic removal of the protecting group to afford 1 (97%).
R-Aminoacetamide derivatives 3a-f were prepared by oneof two synthetic routes. The first route was a two-step sequenceinvolving the direct coupling of chloroacetyl chloride to 1 inthe presence of a base followed by exposure of the intermediateR-chloroacetamide 3g with an ethanolic solution containing anappropriate amine afforded the aminomethyl 3a, pyrrolidinyl3c, and morpholino 3d compounds shown in Scheme 2. Thefree bases were converted into the hydrochloride salts upontreatment with HCl. In the case of the dibasic piperazinyl
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 acidicconditions. Alternatively, 1 was directly reacted with eithercommercially available R-N,N-dimethylaminoacetyl chloride andbase to afford the dimethylamino analogue 3b or activation ofan 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) wereconducted by activation of the R or S enantiomer of com-mercially available, Boc-protected proline (X ) CH2, Scheme3). The protected amino acids were coupled to 1 using standardamino acid coupling procedures, affording derivatives 4a and4b respectively in moderate yields (45-56%) as indicated inScheme 3. A lactam derivative of L-proline (X ) CO) wascoupled to 1 in a similar manner, affording amide 4c. Theurethanes were deprotected under acidic conditions providingcompounds 5a-c.
L-Proline amides 6a (R2 ) Ac, X ) CH2) and 8a (R2 ) Me,X ) CH2) were also prepared from compound 1 using thereaction sequence outlined in Scheme 3 employing either
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
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.
106 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 Moore et al.
commercially available N-acetyl-L-proline or N-methyl-L-prolineand activated by treatment with EDC in the presence of DMAP.
The synthesis of various carbonyl and alkyl derivativesstemmed from compound 5a as shown in Scheme 4. Reactionof 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 treatmentof 5a with alkylisocyanates or via the chloroformates of primaryor secondary amines as shown in Scheme 4, route ii. Alkylatedderivatives were acquired (Scheme 4, route iii) by eitherreductive amination of 5a, with the appropriate aldehyde in thepresence of sodium triacetoxyborohydride in methanol to afford8b and 8c, or direct alkylation of 5a, with benzyl bromideemploying microwave-assisted heating and buffered with tri-ethylamine to afford the benzyl derivative 8d.
Results and Discussion
During the early phase of our lead optimization and after thediscovery of 1, a fluorescent probe was identified that allowedus to develop a high-throughput fluorescent polarization (FP)binding assay using purified human sFRP-1 protein.10c As partof 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 aswell as a functional assay derived from a U2-OS cell line thatwas virally infected with human sFRP-1, Wnt-3, and a TCFluciferase reporter. The level of activity (Wnt signaling) wasmeasured as a function of luciferase production (fold-induction,FI) after incubation of the cells with a test compound for 16-18h. The activity from the functional assay was evaluated at asingle concentration or in a dose response format (EC50). Ingeneral, there was a good correlation between the bindingaffinity (IC50) and the functional dose response assays. Whilethe EC50 values could be used for comparison of functionalactivity, the concentration of the sFRP-1 inhibitor that produceda constant fold-induction was also monitored as a means oftracking potency by standardizing the efficacy in lieu of a naturalligand. This value was extrapolated from the dose responsecurve. In addition to the target profile, we also monitoredphysical and pharmaceutical properties such as solubility,cytochrome p450 inhibition, and microsomal stability. Advancedcompounds were evaluated in an ex vivo mouse calvaria assayof bone formation as well as in vivo pharmacokinetic experiments.
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 carbonylchloride, DIPEA, DCM; (d) 3,3-dimethylbutyraldehyde or c-hexane carboxaldehyde, NaBH(OAc)3, MeOH; (e) benzyl bromide, TEA, microwave, 120 °C,10 min.
Tables 1-6 represent the in vitro biological data generatedfor these compounds. The initial R-aminoacetamide derivatives3a and 3b have comparable binding and functional activity tolead compound 1, while the heterocyclic derivatives 3c-fdemonstrated a significant improvement in both binding andfunctional activity (Table 1). It is interesting to note that themost dramatic differences can be observed in the concentrationat which the compounds elicit a 2- and 4 fold-induction in Wntsignaling. Noting the improvement in activity of the R-hetero-cycle acetamides 3c-f, we then considered a constrained versionof the R-aminoacetamide functionality to reduce or minimizethe number of rotatable bonds.
Exploration of the proline derivatives provided significantimprovements in binding affinities and functional activity over1, Table 2. All of the proline derivatives 4a-c and 5a-c appearto have similar binding affinities to sFRP-1, however, the datafrom 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- andL-isomers of the proline amides (4a/4b or 5a/5b) were compared.
The observation that the carbamates were more potent thanthe deprotected N-H compounds led us to pursue carbonylanalogues that would be less prone to hydrolysis in a low pHenvironment such as gastrointestinal fluids. On the basis of thedata for 4a, the L-proline amide 5a was used as a template forfurther analoguing. The alkyl 6a-f, aryl 6g and 6h, andheteroaryl 6i derivatives demonstrated comparable bindingaffinities relative to 4a (Table 3), and the level of bindinginteraction of these compounds with sFRP-1 reached a plateau,which was not a function of limiting aqueous solubility. On
Table 1. Comparison of R-Aminoacetamides 3a-f to 1 UsingFluorescence Polarization Binding and U2-OS Functional ActivityAssays
a The affinity of test compounds for sFRP-1 was determined using theFP binding assay. b The osteosarcoma cell line, U2-OS, was used for thefunctional 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 responsewas measured in quadruplicate. The standard deviations for these assayswere typically (9% of the mean or less.
Table 2. Data from Fluorescence Polarization Binding and U2 OSFunctional Activity Assays for Compounds 4a-c and 5a-c
a The affinity of test compounds for sFRP-1 was determined using theFP binding assay. b The osteosarcoma cell line, U2-OS, was used for thefunctional assay. c Micromolar concentration that elicited a 2-fold or 4-foldincrease in Wnt signaling. Each point from the dose response was measuredin quadruplicate. The standard deviations for these assays were typically(9% of the mean or less.
Table 3. Data Comparison of Boc-Protected Proline Derivative 4a withAcylated Compounds 6a-6i from Fluorescence Polarization Bindingand U2-OS Functional Activity Assays
a The affinity of test compounds for sFRP-1 was determined using theFP binding assay. b The osteosarcoma cell line, U2 OS, was used for thefunctional assay. c Micromolar concentration that elicited a 2-fold or 4-foldincrease in Wnt signaling. Each point from the dose response was measuredin quadruplicate. The standard deviations for these assays were typically( 9% of the mean or less.
Table 4. Comparison of Fluorescence Polarization Binding and U2-OSFunctional Activity Data for Boc Protected Proline Derivative 4a withUreas, 7a-7e
FP bindinga (µM) Wnt-lucb (µM)
compound R4 R5 IC50 solubility EC50 2-FI c 4-FI c
4a 0.06 25 0.06 0.02 0.167a Et H 0.03 100 0.09 0.04 0.107b tBu H 0.02 50 0.03 0.01 0.037c Ph H 0.02 25 0.31 0.09 0.677d Me Me 0.03 100 0.16 0.06 0.227e morpholine 0.04 50 0.28 0.11 0.52
a The affinity of test compounds for sFRP-1 was determined using theFP binding assay. b The osteosarcoma cell line, U2OS, was used for thefunctional assay. c Micromolar concentration that elicited a 2-fold or 4-foldincrease in Wnt signaling. Each point from the dose response was measuredin quadruplicate. The standard deviations for these assays were typically(9% of the mean or less.
108 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 Moore et al.
the basis of the functional activity, the cyclohexyl 6f andaromatic derivatives 6 g-i were less effective at facilitatingWnt signaling than the lower alkyl analogues 6a-d. The isostereof 4a, compound 6d, was equivalent in potency but did notsignificantly distinguish itself from the other analogues. Thedimethyl amino group in compound 6e was introduced toincorporate water-solublizing properties (6e ) 100 µM versus6d ) 25 µM). The effect of the polar group toward target affinitywas minimal when compared to the lipophilic derivatives 6b-d.This was a trend that demonstrated the tolerability of the bindingsite to a variety of functionality in this region of the scaffold.
As shown in Table 4, the isosteric urea 7b was comparableto carbamate 4a in binding affinity and potency, but perhapsmost impressive was the concentration that was necessary toevoke a 4-fold induction (0.16 µM versus 0.03 µM) for 4a and7b, respectively. There was no significant difference betweenthe mono 7a and dialkylated ureas 7d. In contrast, the phenyl7c 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. Compound8b, an isoster of the urea derivative 7b, had binding affinity(30 nM), comparable to 7b. The substitution of the ureafunctionality with a methylene group resulted in a modest dropin the total polar surface area albeit at a cost of increasing thecalculated logP. The most dramatic difference observed for thedescarbonyl compounds was the decline of functional activity;in particular, the concentration of inhibitor required to induceda 4-fold induction (g0.17 µM) of Wnt signaling. This datasuggested that the carbonyl was an important structural featurein this class of compounds.
While little structural information is known about theprotein-protein interaction between sFRP-1 and Wnt, sFRP-1is thought to engage Wnt via the CRD.13a A truncated versionof sFRP-1, in which the netrin domain is deleted, can beproduced in the U2-OS cell-based assay and the modifiedsecreted protein antagonizes Wnt signaling albeit with dimin-ished efficiency.13b The sFRP-1 inhibitors described herein haveno detectable effect on Wnt signaling when examined in thefunctional assay containing the modified protein, suggesting thatthis class of compounds disrupt the protein-protein interactionby association with the sFRP-1 netrin domain. Advanced bindingexperiments conducted with Wnt, sFRP-1 and 1, have revealed
that the small molecule does prevent the direct binding ofsFRP-1 to the Wnt protein. The details of these experimentswill be communicated in a separate publication.
Selected sFRP-1 inhibitors were evaluated in the ex vivomouse calvaria assay. During the early characterization of 1 inthe ex vivo assay, we found that the concentrations of inhibitorrequired to induce increases in total bone area were significantlylower than the observed in vitro EC50. Evidence of increasedbone formation as well as other anabolic attributes such as boneremodeling, including resorption, osteoblast activation, anddifferentiation were determined by histological assessment. Onthe basis of the potent activity observed in the functional assay,compound 7b (WAY-362692) was selected to advance into thecalvaria assay for characterization. Treatment of the calvariawith a 1 nM DMSO stock solution of compound 7b resulted ina 75% increase in total bone area relative to the control withno significant increase at a lower concentration (0.1 nM) asshown in Figure 2. However, in both treatment groups, thehistology assessment indicated activation of the osteoblast cellsand active bone remodeling. As seen in Figure 3, a picture ofthe histology slide from the treatment of the calvaria with 7b,the OB activation is noted by the rounding of the cells (a), whileactive bone remodeling can be observed by the irregular surface(b). The increase in total bone area was evident from the lightercolor, made possible by differential staining (c). Despitestimulation of the osteoblasts and a trend indicating an increasein 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 ofthe calvaria with the hormone under the same conditions willresult not only in an increase in the total bone area but also adramatic increase in the number of OB.
All sFRP-1 inhibitors were screened in a panel of profilingassays to monitor stability in liver microsomes (rat and human)as a means of identifying potential first pass metabolism. Inaddition, inhibition of cytochrome p450 isozymes (3A4, 2D6,and 2C9) was monitored. These compounds have minimalinhibition of the 2D6 and 2C9 isozymes with moderate to highinhibition of the 3A4 isozyme, as represented by the isostericderivatives 4a, 6d, 7b, and 8b, Table 6. These compounds wererepresentative 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.
Conclusion
In summary, we have presented data for a class of novelsmall-molecule inhibitors of sFRP-1, which appear to disruptthe 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) thatdemonstrated improved binding and potency. Optimization of4a led to a series of derivatives that included isosteric derivatives(see Table 6 for isostere comparison). All of the isosteres ofcompound 4a exhibited good binding affinity to sFRP-1 (e60nM) but varied in their functional potency (2-15-fold). Thiseffort led to the identification of compound 7b that demonstratedpotent binding affinity (IC50 20 nM) to sFRP-1 protein. In acell-based assay that detects Wnt signaling, 7b provided potentfunctional activity (EC50 30 nM). This compound increased totalbone area (75%) relative to the vehicle control in an ex vivomouse calvaria model for bone formation, wherein parametersof anabolic activity such as activation of osteoblasts and activebone remodeling were observed. In vitro metabolism studies
Table 5. Fluorescence Polarization Binding and Wnt-LuciferaseActivity from U2-OS Cells for Alkylated Proline Analogues 8a-d
a The affinity of test compounds for sFRP-1 was determined using theFP binding assay. b The osteosarcoma cell line, U2-OS, was used for thefunctional assay. c Micromolar concentration that elicited a 2-fold or 4-foldincrease in Wnt signaling. Each point from the dose response was measuredin quadruplicate. The standard deviations for these assays were typically(9% of the mean or less.
of 7b predicted significant first-pass oxidative metabolism, andthis property limited its use as an orally administered anabolicbone agent. Further studies are ongoing to examine the in vivoproperties of this class of sFRP-1 inhibitors on bone as well asother 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 andused without further purification. Reagents were purchased fromcommercial sources and used as received. 1H spectra were recordedon either a Varian Inova 400 or Bruker instrument with chemicalshifts reported in δ values (parts per million, ppm) relative to aninternal 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 onan Aglient MSD. HPLC analyses were obtained on an Agilent 1200using the following conditions: Waters Xterra RP18 HPLC column(3.5 µ, 150 mm L × 4.6 mm ID), 40 °C column temperature, 1.2mL/min flow rate, photodiode array detection (210-400 nm), linearmobile phase gradient of 15-95% B over 10 min, holding 5 minat 95% B (mobile phase A: 10 mM ammonium formate in water,pH 3.5; mobile phase B: 1:1 methanol/acetonitrile). Chiral HPLCanalyses were run on a Berger Analytical SFC (Thar Technologies,Inc., Pittsburgh, PA) using a Chiralcel OD-H column, 5 µ, 250mm L × 20 mm ID (Chiral Technologies, Inc., Exton, PA) withcolumn temperature of 35 °C, flow rate 2.0 mL/min, UV detectorat 220 nm, 100 bar outlet pressure, and carbondioxide modifier of15% MeOH with 0.2% dimethylethylamine as additive. Opticalrotations of individual enantiomers were obtained on a JASCOP-1020 polarimeter at a wavelength of 589 nm using a 1.0 cmmicrocell and methanol as the solvent.
5-(Phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonyl Chloride(2). Step 1: 1-Chloro-2-nitro-4-(phenylsulfonyl)benzene (10). To astirred slurry of aluminum chloride (15.6 g, 117 mmol) in benzene(50 mL) under nitrogen was added 4-chloro-3-nitrobenzenesulfonylchloride (97.6 mmol, 25.0 g). The resulting solution was stirred atroom temperature for 2 days, then poured over ice and extractedwith ethyl acetate twice. The combined organic layers were washedwith brine, dried over magnesium sulfate, filtered, and concentrated.Trituration of the material in diethyl ether gave 10 as a yellowsolid, mp 113-122 °C (21.8-24.5 g, 75-84%). 1H NMR (400MHz, 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 forC12H8ClNO4S, 296.98626, found (EI, M+) 296.9849. HPLC purity92.7%, 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 anhydrousdimethylacetamide (150 mL) under nitrogen was added 3 µm copperpowder (27.8 g, 437 mmol), oven-dried 100 mesh activated carbonpowder (11.0 g), and dibromodifluoromethane (20 mL, 219 mmol).The resulting solution was heated to 100 °C and for 6 h. Thesolution was allowed to cool to room temperature and filteredthrough Celite. The filtrate was concentrated to approximately one-third of the volume and partitioned between ethyl acetate andsaturated aqueous ammonium chloride solution. The aqueous phasewas extracted with additional ethyl acetate. The combined organiclayers were washed with water several times, followed by a brinewash. The organic layer was dried over magnesium sulfate, filtered,and concentrated. The crude mixture was flash column separatedusing 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 forC13H8F3NO4S, 331.01261. found (EI, M+), 331.0128. HPLC purity97.6%, RT 9.6 min.
Step 3: 5-(Phenylsulfonyl)-2-(trifluoromethyl)aniline (12). To astirred solution of 11 (13.6 g, 41.0 mmol) not totally dissolved inmethanol (300 mL) under nitrogen was added water (10 mL), andtin(II) chloride (31.13 g, 164 mmol). The resulting solution washeated to 70 °C for three days. The reaction was allowed to coolto room temperature and slowly quenched with saturated aqueoussodium bicarbonate solution (400 mL). This resulted in a thick whitemixture, which was diluted with water and extracted several timeswith ethyl acetate. The combined organic layers were washed withbrine, dried over magnesium sulfate, filtered, and concentrated togive 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 ) 1.56Hz, 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 +
Table 6. In Vitro Data Comparison for sFRP-1 Isosteric Inhibitors
CYP450b (% inhibition)
compound X Y FP binding IC50 (µM) Wnt-luc EC50 (µM) RLMa t1/2 (min) HLMa t1/2 (min) 3A4 2D6 2C9
a Rat liver (RLM) and human liver (HLM) microsomal stability was determined from a 1 µM DMSO solution of compound. b CYP450 inhibitions weredetermined 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 0.1and 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.
110 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 Moore et al.
H+ + CH3CN 343.07226, found [M + H + ACN]+) 343.0735.HPLC purity 97.1%, RT 9.0 min.
Step 4: 5-(Phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonylchloride (2). To a stirred solution of 12 (13.2 g, 43.8 mmol) inacetonitrile (310 mL) under nitrogen at 0 °C was added glacialacetic acid (25 mL) followed by concentrated hydrochloric acid(25 mL). This results in a thick white mixture, which is difficult tostir. 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. Theresulting solution was stirred 20 min, during which time the whiteprecipitate disappeared and the solution became homogeneous.Sulfur dioxide was bubbled into the solution over a period of 20min, immediately followed by the addition of an aqueous solutionof 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 roomtemperature and stirred overnight. The solution was then partitionedbetween ethyl acetate and water. The aqueous layer was extractedwith ethyl acetate. The combined organic layers were washed witha saturated aqueous ammonium chloride solution once, then withwater several times, and once with brine. The organic layer wasdried over magnesium sulfate, filtered, and concentrated. The crudemixture 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.
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 (3.3mL, 23.6 mmol) in dichloromethane (25 mL) under nitrogen wasadded a solution of 1-Boc-4-aminopiperidine (1.87 g, 9.36 mmol)dissolved in dichloromethane (5 mL) slowly over two minutes. Theresulting solution was stirred at room temperature for 1 h, and thenwashed with a saturated aqueous ammonium chloride solution. Theorganic phase was dried over magnesium sulfate, filtered andconcentrated. The crude mixture was flash column separated using0-30% 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 forC23H27F3N2O6S2 + Na+, 571.11548, found (ESI, [M + Na]+
)benzene-sulfonamide (1). To a stirred solution of 13 (3.8 g, 6.93mmol) in 1,4-dioxane (40 mL) under nitrogen was added a 4N HCldioxane solution (13 mL, 52 mmol). The resulting solution wasstirred overnight at room temperature, and concentrated. Triturationin ethyl acetate gave 1 as an HCl salt (3 g, 97%). 1H NMR (400
N-[1-(N-Methylglycyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-fluoromethyl)-benzenesulfonamide (3a). Step 1. To a stirredsolution of 1 (0.60 g, 1.34 mmol) in methylene chloride (15 mL)with triethylamine (0.4 mL, 2.87 mmol) was added chloroacetylchloride (0.15 g, 1.34 mol), and the resulting solution was stirredovernight at room temperature. The reaction mixture was washedwith 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 (0.08g, 0.15mmol) in ethanol (1 mL) was added 33% methylamine inethanol (0.1 mL) and the resulting solution was stirred overnightat room temperature. The mixture was concentrated and flashcolumn separated using 0-10% methanol/methylene chloridegradient gave 3a, which was dissolved in ethyl acetate, treated withHCl 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 purity98.4%, RT 6.9 min.
N-[1-(N,N-Dimethylglycyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoro-methyl)benzenesulfonamide (3b). To a stirred solution of1 (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 stirredfor overnight at room temperature. The reaction mixture was washedwith water and concentrated. Flash column separation using 0-8%methanol/methylene chloride gradient gave 3b, which was dissolvedin ethyl acetate, treated with HCl gas and concentrated to provide theHCl 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 mannerto 3a, step 2, N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-
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 onbone.
N-[1-(Morpholin-4-ylacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (3d). In an analogous mannerto 3a, step 2, N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide and morpholine were usedto prepare 3d, which was dissolved in ethyl acetate, treated withHCl 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.
5-(Phenylsulfonyl)-N-[1-(piperazin-1-ylacetyl)piperidin-4-yl]-2-(trifluoro-methyl)benzenesulfonamide (3e). In an analogous mannerto 3a, step 2, a stirred solution of N-[1-(chloroacetyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(trifluoromethyl)benzenesulfonamide (0.80g, 0.15 mmol) in ethanol was treated with piperazine-1-carboxylicacid tert-butyl ester (0.06 g, 0.32 mmol) and the resulting solutionwas stirred overnight at room temperature. The reaction mixturewas washed with water and concentrated. The resulting solid wasdissolved in ethyl acetate (2 mL) and HCl gas was bubbled in.The solution was stirred overnight and filtered to give 3e as anHCl 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 ) 11.02Hz, 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 ofimidazol-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 temperaturefor 20 min. To this was added 1 (0.10 g, 0.22 mmol) and the resultingsolution was stirred for 3 days. The mixture was washed with asaturated aqueous sodium bicarbonate solution at which time aprecipitate formed. The solid was filtered, then triturated in methylenechloride to give 3f, which was dissolved in ethyl acetate, treated withHCl 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 ) 13.32Hz, 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)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, 0.67mmol) and the resulting solution was heated to 100 °C for 15 min.
The mixture was diluted with a saturated aqueous ammoniumchloride solution and extracted several times with ethyl acetate.The combined organic layers were washed with brine several timesand concentrated. Flash column separation using 30-100% ethylacetate/hexane gradient gave 4a (0.12 g, 56%). Specific opticalrotation [R]D
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) andtriethylamine (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 aqueousammonium chloride solution and extracted several times with ethylacetate. The combined organic layers were washed with brineseveral times and concentrated. Flash column separation using30-100% ethyl acetate/hexane gradient gave 4b (0.13 g, 45%).Specific optical rotation [R]D
yl)benzenesulfonamide (5a). 4a (0.09 g, 0.14 mmol) was dissolvedin ethyl acetate (2 mL) and HCl gas was bubbled in. The solutionwas stirred overnight and filtered to give 5a as an HCl salt (0.08 g,98%). Specific optical rotation [R]D
5-(Phenylsulfonyl)-N-(1-D-prolylpiperidin-4-yl)-2-(trifluorometh-yl)benzenesulfonamide (5b). 4b (0.096 g, 0.15 mmol) was dissolvedin ethyl acetate (2 mL) and HCl gas was bubbled in. The solution
112 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 Moore et al.
was stirred overnight and filtered to give 5b as an HCl salt (0.078g, 90%). Specific optical rotation [R]D
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)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) inmethylene chloride (2 mL) was added diisopropylethylamine (0.1mL, 0.58 mmol) and ethylisocyanate (0.009 g, 0.126 mmol) andthe resulting solution was stirred at room temperature for severalhours. The reaction was washed with saturated aqueous ammoniumchloride solution and concentrated. Flash column separation using30-100% 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.
(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 (0.076,85%). Specific optical rotation [R]D
N-[1-(1-Methyl-L-prolyl)piperidin-4-yl]-5-(phenylsulfonyl)-2-(tri-fluoromethyl)benzenesulfonamide (8a). To a stirred solution ofN-methyl-L-proline (0.035 g, 0.27 mmol) in methylene chloride (3mL) was added DMAP (0.033 g, 0.27 mmol) and EDC (0.056 g,0.027 mmol). The resulting solution was stirred at room temperaturefor 20 min, at which time 1 (0.10 g, 0.22 mmol) was added andstirred overnight at room temperature. The reaction was washedwith water and concentrated. Flash column separation using 0-10%methanol/methylene chloride gradient gave 8a, which was dissolvedin ethyl acetate, treated with HCl gas, and concentrated to formthe 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-(phenyl-sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (8b). To a stirredsolution 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) andthe 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 temperatureseveral more hours and concentrated. Flash column separation using0-10% methanol/methylene chloride gradient gave 8b, which wasdissolved in ethyl acetate, treated with HCl gas, and concentratedto 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-(phenyl-sulfonyl)-2-(trifluoromethyl)benzenesulfonamide (8c). In an analo-gous manner to 8b, 5a, cyclohexanecarboxaldehyde, and sodiumtriacetoxyborohydride were used to prepare 8c, which was dissolvedin ethyl acetate, treated with HCl gas, and concentrated to formthe 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-(tri-fluoromethyl)benzenesulfonamide (8d). To a stirred solution of 5a(0.09 g, 0.15 mmol) in THF (2 mL) was added triethylamine (0.1mL, 0.70 mmol) and benzyl bromide (0.03 mmol, 0.15 mmol) andthe resulting solution was microwave irradiated to 120 °C for 10min. The resulting mixture was partitioned between ethyl acetateand a saturated sodium bicarbonate solution. The organic layer wasconcentrated and flash column separated using 0-10% methanol/methylene chloride gradient gave 8d, which was dissolved in ethylacetate, treated with HCl gas, and concentrated to form the HClsalt (0.042 g, 43%). 1H (400 MHz, DMSO-d6) δ ) 9.62 (br. s., 1H), 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
114 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 Moore et al.
(m, 5 H), 1.17-1.39 (m, 3 H). HRMS: calculated forC30H32F3N3O5S2 + 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 testcompounds for sFRP-1 was determined using a fluorescencepolarization binding assay. According to the assay design, a probecompound was bound to sFRP-1. The fluorescence anisotropy valueof the probe compound increased upon binding to sFRP-1. Uponthe addition of a test compound, the fluorescence anisotropy valuefor the probe compound decreased due to competitive displacementof the probe by the test compound. The decrease in anisotropy asa function of increasing concentration of the test compound provideda direct measure of the test compound’s binding affinity forsFRP-1.
To determine IC50 values, fluorescence polarization experimentswere conducted in a 384-well format according to the followingprocedures. A 20 mM stock solution of the probe compound wasprepared in 100% DMSO and dispensed in 10 µL aliquots for long-term storage at -20 °C. The binding assay buffer was prepared bycombining stock solutions of Tris-Cl, NaCl, glycerol, and NP40 atfinal concentrations of 25 mM Tris-Cl pH 7.4, 0.5 M NaCl, 5%glycerol, and 0.002% NP40. Master stock solutions of the testcompounds were prepared in 100% DMSO at final concentrationsof 20 mM. Typically the working stock solutions of the testcompounds were prepared by serially diluting the 20 mM masterstock solution to 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125mM, 0.156 mM, 78 µM, 39 µM, 19.5 µM, 9.8 µM, 4.9 µM, 2.44µM, 1.22 µM, 0.31 µM, 76 nM, and 19 nM in DMSO. The workingstock solutions of the test compounds were further diluted bycombining 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 ofthe 20 mM fluorescence probe compound was diluted 1000-foldin 100% DMSO to a final concentration of 20 µM. Then 6 µL ofthe 20 µM probe was combined with 5.4 mL of the assay buffer,mixed well, and 18 µL of the resulting solution was dispensed into384-well plates.
sFRP-1/probe complex was prepared by combining 11 µL of 20µM probe compound with 9.9 mL of the assay buffer and sFRP-1stock solution to final concentrations of 22 nM probe compoundand 50 nM sFRP-1. Then 18 µL of the sFRP-1/probe complex wasdispensed into the 384-well plates.
Aliquots of the test compounds from the 10× working stocksolutions (2 µL) were removed and dispensed into the platecontaining the sFRP-1/probe complex and the resultant solutionswere mixed by pipetting 10 µL up and down twice. The finalconcentrations of sFRP-1 and probe in the assay solutions were 45and 20 nM, respectively. In a typical experiment, each plate wasused to test 14 compounds.
The plate was incubated in the dark for 30 min. The fluorescenceof the sFRP-1/probe complexes was read in the Tecan Ultra platereader at excitation and emission maxima of 485 and 535 nm.
Fluorescence anisotropy results from the emission of polarizedlight in the parallel and perpendicular directions when a fluorophoreis excited with vertically polarized light. The anisotropy of the probein the free and bound state was determined using the followingequation: r ) [I(|) - I(⊥ )] ÷ [I(|) + 2I(⊥ )], where I(|) and I(⊥ )are the parallel and perpendicular emission intensities, respectively.
Monitoring the anisotropy changes of the probe compoundrevealed that it bound saturably to sFRP-1 with a KD of 20-30nM. The binding affinity was independently verified using atryptophan fluorescence-quenching assay.
The decrease in the anisotropy of the probe upon addition ofthe competing test compound was fitted to a sigmoidal doseresponse curve of the equation shown below: Y ) Bottom + ((Top- Bottom))/((1 + 10X-LogIC50)) × Hillslope, where “X” is thelogarithm 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.
For automated IC50 determinations, the equation shown above wasused in the program GraphPad Prism. The “Hillslope” was keptconstant at 1. The value for “Bottom” was fixed but was determinedby the blank (probe-only) wells in the plate. The values for “Top”and “IC50” were determined by the data fit. The value for “Top” wastypically close to 120, equivalent to approximately 50% bound probe,and the value for “Bottom” was around 30 due to free probe. If thetest compound interfered with the probe in the fluorescence assay athigh concentrations, the range for the fitted data was limited to thelower concentration range.
Cell-Based, TCF-Reporter Functional Assay. U2OS bone cellswere infected with recombinant adenovirus 5 (Ad5)-WNT3 at amultiplicity of infection (MOI) of 2, followed by infection with Ad5-sFRP-1 and Ad5-16xTCF-luciferase, each at an MOI of 10. Four hoursafter infection, the cells were frozen in sterile cryogenic vials at a celldensity of 9 × 106 cells/mL and stored in a -150 °C freezer. For theassay, a vial of frozen cells was thawed, and the cells were resuspendedin plating medium [phenol red-free RPMI 1640 medium containing5% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-streptomycin] to a final cell density of 1.5 × 105 cells/mL. Theresuspended cells were then plated in 96-well tissue culture treatedplates 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 andstarted to spread. Prior to the addition of test compounds, the mediumwas replaced with 50 µL/well of phenol red-free RPMI 1640 containing10% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-streptomycin. Test compounds, or vehicle (typically DMSO), dilutedin phenol red-free RPMI 1640 containing 2 mM GlutaMAX-l, and l% (v/v) penicillin-streptomycin were then added to the wells inreplicates of 4 wells/dilution and the plates were incubated at 37 °Covernight. Dose-response experiments were performed with thecompounds in 2-fold serial dilutions from 10000-4.9 nM. After theovernight incubation, the cells were washed twice with 150 uL/wellof PBS w/o calcium or magnesium and lysed with 50 µL/well of 1×cell culture lysis reagent (Promega Corporation) on a shaker at roomtemperature for 30 min. Aliquots of the cell lysates (30 µL) weretransferred to 96-well luminometer plates, and the luciferase activitywas 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 measuredfor 10 s after a 1.6 s delay. The luciferase activity data was transferredfrom the luminometer to a PC and analyzed using the SAS/Excelprogram to determine EC50 values. The luciferase data was analyzedusing the SAS/Excel program. EC50 determinations for dose responsecurves were determined using the SAS/Excel program.
Ex Vivo Bone Formation Assay. Neonatal mouse calvaria wereprepared from 4-day-old pups as described previously.17 Briefly,calvaria were excised and cut in half along the sagittal suture. Calvariawere incubated overnight in serum-free BGJ medium containing 0.1%(w/v) bovine serum albumin (BSA). Each half-calvaria was placedwith the concave surface downward on a stainless steel grid (SmallParts Inc., Miami, FL) in a 12-well tissue culture dish (BectonDickinson, Oxnard, CA). Each well contained 1.0 mL of BGJ mediumwith 1.0% (v/v) fetal bovine serum (FBS). Calvaria were incubatedfor 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 orcompounds added.
After organ culture, calvaria were fixed in 10% neutral phosphate-buffered formaldehyde at room temperature for at least 72 h, thendecalcified for 6 h in 10% EDTA in phosphate buffered saline (PBS).Calvaria were embedded in parallel in the same paraffin block, and 4µm sections were stained with hematoxylin-eosin. Consistent boneareas (200 µm away from the frontal sutures) were selected forhistomorphometric analysis. A 200 µm square grid was placed on eachcalvarium, and total bone area, as well as the number of osteoblastswithin the grid, was determined with the Osteomeasure System(Osteometrics Inc., Atlanta, GA). All cells on the bone surface werecounted as osteoblasts. Data were analyzed for statistical significance
by one-way ANOVA using the Dunnett’s test with JMP software (SASInstitute, Cary, NC).
Acknowledgment. We thank the Departments of AnalyticalChemistry and Chemical Technologies, Wyeth, for analytical andprofile data. We also wish to thank our management, Drs. MagidAbou-Gharbia and Len Freedman, for their support of this work.
Supporting Information Available: Analytical HPLC chromato-grams for target compounds 3a-f, 4a-c, 5a-c, 6a-i, 7a-e, and8a-d. Analytical chiral HPLC analyses of compounds 4a-b, 5a-b,and 7b. Full NMR characterization of compound 7b for protonassignment including multinuclear 19F, 13C, 15N, and variable temper-ature experiments. This material is available free of charge via theInternet at http://pubs.acs.org.
References(1) Carmona, R. H. Bone Health and Osteoporosis: A Report of the
Surgeon General U.S. Department of Health and Human Services,Public Health Service; Office of the Surgeon General: Rockville,MD,October 14, 2004. Gass, M.; Dawson-Hughes, B. Preventingosteoporosis-related fractures: an overview. Am. J. Med. 2006, 119(4 Suppl 1), S3-S11.
(2) Neer, R. M.; Arnaud, C. D.; Zanchetta, J. R.; Prince, R.; Gaich, G. A.;Reginster, J-Y.; Hodsman, A. B.; Eriksen, E. F.; Ish-Shalom, S.;Genant, H. K.; Wang, O.; Mitlak, B. H.; Mellstrom, D.; Oefjord, E. S.;Marcinowska-Suchowierska, E,; Salmi, J.; Mulder, H.; Halse, J.;Sawicki, A. Z. Effect of Parathyroid Hormone (1-34) on Fracturesand Bone Mineral Density in Postmenopausal Women with Os-teoporosis. N. Engl. J. Med. 2001, 344, 1434–1441.
(3) (a) Kulkarni, N. H.; Halladay, D. L.; Miles, R. R.; Gilbert, L. M.;Frolik, C. A.; Galvin, R. J. S.; Martin, T. J.; Gillespie, M. T.; Onyia,J. E. Effects of parathyroid hormone on Wnt signaling pathway inbone. J. Cell. Biochem. 2005, 95, 1178–1190. (b) Bodine, P. V. N.;Seestaller-Wehr, L.; Kharode, Y. P.; Bex, F. J.; Komm, B. S. Boneanabolic effects of parathyroid hormone are blunted by deletion ofthe Wnt antagonist secreted frizzled-related protein-1. J. Cell. Phys.2007, 210 (2), 352–357.
(4) (a) Wodarz, A; Nusse, R. Mechanisms of Wnt signaling in develop-ment. Ann. ReV. Cell DeV. Biol. 1998, 14, 59–88. (b) Baron, R.;Rawadi, G.; Roman-Roman, S. Wnt signaling: a key regulator of bonemass. Curr. Top. DeV. Biol. 2006, 76, 103–127.
(5) Kawano, Y.; Kypta, R. Secreted antagonists of the Wnt signallingpathway. J. Cell Sci. 2003, 116 (13), 2627–2634.
(6) (a) Rattner, A.; Hsieh, J. C.; Smallwood, P. M.; Gilbert, D. J.;Copeland, N. G. Jenkins, N. A.; Nathans, J. A family of secretedproteins contains homology to the cysteine-rich ligand-binding domainof frizzled receptors. Proc. Natl. Acad. Sci. 1997, 94, 2859–2863. (b)Jones, S. E.; Jomary, C. Secreted frizzled-related proteins: searchingfor relationships and patterns. BioEssays 2002, 24, 811–820. (c) Chong,J. M.; Uren, A.; Rubin, J. S.; Speicher, D. W. Disulfide bondassignments of secreted Frizzled-related protein-1 provide insightsabout frizzled homology and netrin modules. J. Biol. Chem. 2002,277 (7), 5134–5144.
(7) Roman-Roman, S.; Shi, D.-L.; Stiot, V.; Hay, E.; Vayssiere, B.; Garcia,T.; Baron, R.; Rawadi, G. Murine Frizzled-1 Behaves as an Antagonistof the Canonical Wnt/b-Catenin Signaling. J. Biol. Chem. 2004, 279(7), 5725–5733.
(8) (a) Bodine, P. V. N.; Billiard, J.; Moran, R. A.; Ponce-de-Leon, H.;McLarney, S.; Mangine, A.; Bhat, R., A.; Stauffer, B.; Green, J.; Stein,G. S.; Lian, J. B.; Komm, B. S. The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis. J. Cell.Biochem. 2005, 96, 1212–1230. (b) Bodine, P. V. N.; Robinson, J. A.;
Bhat, R. A.; Billiard, J.; Bex, F. J.; Komm, B. S. The role of Wntsignaling in bone and mineral metabolism. Clin. ReV. Bone Miner.Metabol. 2006, 4, 73–96.
(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 negativeregulator of trabecular bone formation in adult mice. Mol. Endocrinol.2004, 18, 1222–1237.
(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 secretedfrizzled-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 sulfonamidesas secreted frizzled-related protein-1 (sFRP-1) antagonists: SAR andoptimization. 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 abinding assay for the identification of secreted frizzled-related protein-1(sFRP-1) antagonists. Abstracts of Papers, 234th ACS NationalMeeting, Boston, MA, August 19-23, 2007, American ChemicalSociety: 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,J. H.; McClinton, M. A.; Blade, R. J. J. Chem. Soc., Chem. Commun.1988, 10, 638–639. (c) Wiemers, D. M.; Burton, D. J. J. Am. Chem.Soc. 1986, 108, 832–834.
(13) (a) Lin, K; Wang, S; Julius, M A; Kitajewski, J; Moos, M, Jr; Luyten,F P. The cysteine-rich frizzled domain of Frzb-1 is required andsufficient for modulation of Wnt signaling. Proc. Natl. Acad. Sci.U.S.A. 1997, 94 (21), 11196–11200. (b) Bhat, R. A.; Stauffer, B.;Komm, B. S.; Bodine, P. V. N. Structure-function analysis of secretedfrizzled-related protein-1 for its Wnt antagonist function. J. Cell. Biol.2007, 102 (6), 1519–1528.
(14) Li, C. H.; Amar, S. Role of secreted frizzled-related protein 1 (SFRP1)in wound healing. J. Dent. Res. 2006, 85, 374–378.
(15) Kumar, S.; Leontovich, A.; Coenen, M. J.; Bahn, R. S. Gene expressionprofiling of orbital adipose tissue from patients with Graves’ ophthal-mopathy: A potential role for secreted frizzled-related protein-1 inorbital adipogenesis. J. Clin. Endocr. Metab. 2005, 90, 4730–4735.
(16) Wang, W.-H.; McNatt, L. G.; Pang, I.-H.; Millar, J. C.; Hellberg, P. E.;Hellberg, M. H.; Steely, H. T.; Rubin, J. S.; Fingert, J. H.; Sheffield,V. C.; Stone, E. M.; Clark, A. F. Increased expression of the WNTantagonist sFRP-1 in glaucoma elevates intraocular pressure. J. Clin.InVest. 2008, 118, 1056–1064.
(17) (a) Mundy, G.; Garrett, R.; Harris, S.; Chan, J.; Chen, D.; Rossini,G.; Boyce, B.; Zhao, M.; Gutierrez, G. Stimulation of bone formationin vitro and in rodents by statins. Science 1999, 286, 1946–1949. (b)Traianedes, K.; Dallas, M. R.; Garrett, I. R.; Mundy, G. R.; Bonewald,L. F. 5-Lipoxygenase metabolites inhibit bone formation in vitro.Endocrinology 1998, 139, 3178–3184.
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