Indole Glucocorticoid Receptor Antagonists Active in a Model of

Article pubs.acs.org/jmc

Indole Glucocorticoid Receptor Antagonists Active in a Model of Dyslipidemia Act via a Unique Association with an Agonist Binding Site. John G. Luz,‡ Matthew W. Carson,† Bradley Condon,‡ David Clawson,† Anna Pustilnik,‡ Daniel T. Kohlman,† Robert J. Barr,† James S. Bean,† M. Joelle Dill,† Dana K Sindelar,† Milan Maletic,‡ and Michael J. Coghlan*,† †

Lilly Research Laboratories, A Division of Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana 46285 United States Eli Lilly Biotechnology Center, 10300 Campus Point Drive, Suite 200, San Diego, California 92121 United States

‡

S Supporting Information *

ABSTRACT: To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (−74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts.

■

INTRODUCTION The glucocorticoid receptor (GR) is a nuclear hormone receptor (NHR) that mediates a myriad of biological processes (e.g., inflammation, gluconeogenesis, immunity, homeostasis, and development) through transcriptional and nontranscriptional mechanisms. GR positively regulates transcription via transactivation, a process by which GR, bound to an endogenous steroid ligand, translocates from the cytoplasm into the nucleus and activates gene expression by binding to glucocorticoid response elements (GREs) in susceptible promoter regions or by interacting with other transcription factors. Negative regulation of transcription, known as transrepression, occurs when the GR/ligand complex binds directly to DNA negative regulatory elements or GR tethering to DNA-bound transcription factors such AP-1 and NF-κB.1−6 The transrepression phenomenon is a widely cited mechanism inducing the potent anti-inflammatory actions of glucocorticoids. The activity of GR is modulated not only by endogenous steroid ligands but also via interaction with coactivator and corepressor proteins and by post-translational modifications such as phosphorylation and acetylation of the receptor itself.7−9 © XXXX American Chemical Society

Because of their potent anti-inflammatory properties, GR agonists have found broad application in the treatment of inflammation-related conditions such as asthma, rheumatoid arthritis, and allergy. Clinically approved steroidal GR agonists include cortisol, prednisolone, dexamethasone (dex), and fluticasone furoate, and efforts continue to identify novel nonsteroidal GR ligands seeking improved efficacy, reduced side effect profile, as well as exploration of novel indications.10−16 Recently, the potential uses of GR antagonists have been further validated. Mifepristone (MIF, Chart 1) is clinically employed as a progesterone receptor (PR) antagonist, but it also acts as a strong GR antagonist in vivo.17 GR antagonism by MIF has elicited clinical interest as a potential treatment for conditions induced by excessive glucocorticoid action, such as Cushing’s Syndrome.18 Other potential indications for GR antagonists include glaucoma,19 psychotic depression,20,21 and type 2 diabetes.22 A measure of efficacy in the treatment of some of the aforementioned disorders23,24 with MIF has been Received: May 12, 2015

A

DOI: 10.1021/acs.jmedchem.5b00736 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2-fold to accommodate different ligands.33 Antagonist conformations informed by the GR LBD/MIF crystal structures reveal dramatic rearrangements in the secondary and tertiary structure α helix 12 (h12). Specifically, the position of h12 is variable and the conformations of novel GR antagonists within the GR LBD could potentially lead to a preferred h12 conformation or a breadth of conformations which may enable varied biological outcomes. Mindful of this body of data, the crystal structure of GR LBD in complex with compound 8 was pursued in order to improve our understanding of the structural basis of GR antagonism as well as to provide a framework for further development of nonsteroidal GR antagonists.

Chart 1. Structures of Mifepristone (MIF) and Compound 8

■

RESULTS In Vitro Profile of NHR Selectivity and GR Antagonism. The 3-aryl-7-sulfonamidoindole scaffold was identified by screening the Lilly compound library for GR ligands using a conventional radioligand binding assay. Structure−activity relationships within this scaffold ultimately resulted in the identification of compound 8 prepared in Scheme 1 as a highly potent and reasonably selective nonsteroidal GR antagonist. Sonogashira coupling34 between commercially available intermediates aryl bromide 1 and alkyne 2 gave 3 in excellent yield (92%). Key intermediate 4 was prepared quantitatively via acetylation of 3 with trifluoroacetic anhydride. Aminopalladation between alkyne 4 and 5-fluoro-2-iodobenzene 5 in the presence of palladium catalyst and base afforded indole 635 in moderate yield (44%). Reduction of the nitro group provided aniline 7. The synthesis of compound 8 was then completed by sulfonylation of 7 with methanesulfonyl chloride. Supporting SAR binding data are summarized in Table 1. These indole sulfonamides rely on two key groups for potency

demonstrated, and MIF has been approved for the clinical treatment of refractory Cushing’s disease.25 Very recently, a member of the novel GR antagonist family of indole sulfonamides introduced in this work was shown to reduce the weight gain associated with atypical antipsychotics in rodents.26 The overall profile of pharmacology associated with this class of ligands led us to investigate additional novel treatment modalities and to pursue the structural basis for this activity. Crystal structures of GR LBD in complex with steroidal27,28 as well as nonsteroidal agonists29,30 and with the steroid antagonist MIF have been determined.31,32 The tertiary structure and steroid binding mode of the GR LBD were found to be similar to that of other NHR LBDs, an α-helical globular domain with a hydrophobic binding pocket toward its base where α-helices 3, 8, and 11 impinge. In GR LBD agonist crystal structures, an association with N564 is a prerequisite for agonism and the overall volume of the binding site can increase Scheme 1. Synthesis of Compound 8

B



Article

Table 1. Binding Data for the Series of Indole GR Ligandsa

binding Ki [nM] (SEM) n compd no.

R2

R3

R5

R7

GR

MR

AR

PR

6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 MIF

CH2N(Me)Bn CH2N(Me)Bn Me CH2N(Me)Bn H Me Me H CH2N(Me)Bn Bn Ph Me H H H H H

2-OMe-4-F Ph 2-OMe-4-F Ph Ph 2-OMe-4-F Ph 2-OMe-5-F Ph Ph 2-OMe-5-F Ph H 2-OMe-4-F Ph 2-OMe-4-F Ph 2-OMe-5-F Ph 2-OMe-4-F Ph 2-OMe-5-Cl Ph 3-F-4-OMe Ph 3-Cl Ph 2-OMe Ph 4-F Ph

i-Pr i-Pr H F H OMe OMe H t-Bu i-Pr H F H H H H H

NO2 NHSO2Me H NH2 NHAc NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me NHSO2Me

b 1.22 (0.299) 4 b b 654 39.8 115 50 2.67 (0.564) 2 0.543 53.7 1.32 5.45 (0.853) 2 1410 5.04 3.06 94.6 0.299(0.107) 6

NTc 1580 (435) 4 NT 2120 6530 3890 38.4 952 (214) 2 3880 4250 3920 44.7 (6.58) 2 4470 NT 2.1 27.4 225 851(318) 6

NT 232 (77.2) 4 NT 4140 6120 4060 1120 355 (158) 2 231 684 905 1130 (611) 2 697 NT 91 401 496 5.95 (2.34) 6

NT 64.2 (40.6) 4 NT 4190 688 3940 22.3 104 (4.98) 2 253 4000 44.2 20.5 (48.2) 2 233 NT 5.51 86 29 0.998(0.456) 7

a Single values are the mean of assays run in triplicate. None of these analogues were found to be ER ligands up to 10 μM. bNo binding observed at 10 μM. cNT = not tested.

In Vivo Lipid Profiling. After identifying compound 8, we explored the pharmacologic uses for GR antagonists noted from the literature.37−41 Cushing’s patients exhibit dyslipidemia, diabetes, and obesity, similar to the condition dubbed “metabolic syndrome” in the wider population. Dyslipidemia has been associated with elevated cortisol levels in patients with cardiovascular disease, diabetes, and obesity.42,43 This link between excess glucocorticoids with aberrant lipid levels is further supported by reports of 11β HSD1 inhibitors acting to improve lipid profiles in rats fed a high fat diet via reduced circulating corticosteroids.44 We surmised that the effect of GR antagonism on lipid profiles would be persuasively assessed in a conventional hamster model of dyslipidemia.45 After assessment of PK properties showed an attractive profile (Table 3),

and GR selectivity in NHR binding assays. The sulfonamide group at R7 is an essential function for GR binding (compare compounds 8, 10, and 11), and ortho-methoxy phenyl substitution at the 3 position of the indole provides additional GR affinity (compound 14 versus 22). Additional selectivity versus the remaining steroid hormone receptors occurs with the addition of bulky alkyl groups at R5 in many cases. Analogues with GR Ki values less than 10 nM were brought into functional GR assays. Table 2 summarizes antagonist data Table 2. In Vitro GR Antagonist Activity36 IC50 [nM] (SEM) n compd

HEK cell

TAT

8 15 16 18 19 21 22 MIF

2.76(0.460) 4 4.49(0.919) 3 7.54 22 143(17.5) 2 180 235(72.7) 2 0.298(0.246) 14

10.6(4.96) 5 14.4(5.88) 6 292 63.4(32.8) 3 1250 76 1180 1.94(1.17) 64

Table 3. Rat PK for Compound 8 Values (SEM)36

for several analogues compared to MIF in HEK cells transfected with functional GR and the tyrosine amino transferase (TAT) assay run in H4−II E cells.36 The indole sulfonamides were ∼10× less potent than the reference steroid, yet most analogues were more selective than MIF. In this instance, the size of the indole R2 substituent shows a trend toward improved antagonism. (Compounds 8, 15, 16, 18, and 19 were also assayed in models of GR agonism and transrepression where they did not show significant activity36 (Supporting Information Table S1)).

dose, route of administration

1 mg/kg, iv

3 mg/kg, po

AUC (ng·h/mL) t1/2 (h) CL (mL/min/kg) Vdss (mL/kg) t max (h) Cmax (ng/mL) F (%)

315 (145) 3.96 (0.599) 58.2 (25.6) 13800 (3980)

488 (242) 3.31 (0.281)

4.00(0) 63.3(32.7) 51.9 (9.87)

groups (n = 6) of Syrian golden hamsters were maintained on a high fat, high cholesterol (HFHC) diet over 14 days as MIF and compound 8 were dosed po at 30 mg/kg qd. The resulting improvement in lipid profile is shown in Figure 1. A 74% decrease in LDL and a 73% increase of HDL levels were observed for compound 8 dosed hamsters versus untreated HFHC controls (p < 0.001 for both comparisons). A summary of the complete data set for compound 8 compared with MIF is C



Article

Figure 1. Lipid changes from GR antagonists in hamsters receiving a high fat, high cholesterol (HFHC) diet. Syrian golden hamsters (n = 6) were maintained on the HFHC diet for 14 days with and without concurrent treatment with test article (compound 8 and MIF) at 30 mg/kg po. Compound 8 substantially improves both LDL (74% decrease) and HDL (73% increase) levels versus untreated controls, and the effects are comparable to those observed with MIF (43% LDL lowering, 35% HDL increase). Data represent mean values with standard errors. (* p < 0.001).

shown in Figure 1. These results provide additional evidence for the role of GR antagonists such as compound 8 for the potential treatment of dyslipidemia mediated by disrupted homeostatic function. The recent reports of GR antagonism ameliorating the weight gain associated with antipsychotic drugs26 prompted us to evaluate compound 8 versus quetiapine in a prophylactic model. Results shown in Supplemental Figure 5a indicate that compound 8 effectively reduced weight gain over 14 days dosing. No changes in overall food intake were noted for these dose groups (Supplemental Figure 5b), leading to the conclusion that the reduced weight gain was mitigated by GR antagonism of compound 8. Structural Biology. Protein Expression and Purification. The attractive in vitro and in vivo properties of 8 led to the pursuit of a structure-based understanding of GR antagonist action. Consequently, we sought to crystallize 8 within the GR LBD in comparison with the known binding conformations of MIF and dex. Purification of stable nonaggregated GR LBD bound to 3-aryl-sulfonamidoindole antagonists required screening to identify those compounds that supported soluble expression when added to the expression media. Compound 8 was thus identified and subsequently used to generate purified nonaggregated GR LBD. Because this complex failed to crystallize, we introduced additional mutations to increase protein solubility. These mutations (L525S, L528S, L535A, V538T) were chosen because of their solvent accessibility as well as their remoteness from the binding site. A pair of additional surface entropy mutations were introduced that were described previously as stabilizing the MIF antagonist binding conformation (E684A, E688A).32 We also designed a novel corepressor peptide derived from NcoR truncated to remove disordered residues (NLGLEDIIRKALMGS). This combination of mutations, increased protein yields, and a truncated NcoR peptide led to the successful crystallization and crystal structure determination of the trimeric complex. Overall Structure. The overall crystal structure of GR LBD (Table 4) bound to compound 8 (Figure 2a, PDB accession code 4MDD) is similar to that of previously determined cocrystal structures of GR LBD/agonist complexes with an average rmsd of 1.4 Å for all equivalent Cα atoms when superimposed upon the GR LBD/dex bound structure,28 excluding the α-1 and α-12 helices. (The numbering of the

Table 4. X-ray Collection Data and Refinement Statistics for Compound 8 Data Collection space group cell dimensions (a,b,c) (Å) angles (α,β,γ) (deg) resolution (Å) completeness (%) Rsym (%) mean I/σ(I) redundancy wavelength (Å) Refinement resolution range reflections Rwork Rfree rmsd bonds lengths (Å) angles (deg) total no. residues chain A chain B chain C chain D total no. protein atoms heteroatoms total no. waters average B (Å2) all peptide chain A chain B chain C chain D ligand (chain I) water (chain W) a

P3221 72.54, 72.54, 229.5 90, 90, 120 2.3−30 (2.3−2.44)a 99.6 (100) 12.8 (72) 10.3 (3.9) 10.8 (11) 0.97931 2.35−19.8 (2.35−2.43) 29970 21.9% (23.5%) 25.8% (28.9%) 0.01 1.08 247 220 13 12 3957 72 118 53.6 52.2 54.2 63.0 82.0 34.5 49.7

(parentheses = highest resolution shell).

helices corresponds to that originally defined by the first crystal steroid NHR structure of the estrogen receptor.46) As had been observed in the GR LBD/MIF/NcoR complex, α-helix 1 forms a domain swapping interaction, in this case with the second trimer of the asymmetric unit (Supporting D



Article

Figure 2. Crystal structure of GR LBD bound to GR ligands. (a) compound 8 (PBD accession code 4MDD) (colored by atom: C, yellow; N, blue; O, red; F, light blue; S, dark yellow) is located in the steroid binding pocket at the base of the globular α-helical domain (cyan). The new NcoR peptide (orange ribbon) binds at the corepressor binding site. (b) Compound 8 is packed between helices α-3, α-6, α-7, and α-11, which is oriented with the methylsulfonamide below and the methoxyfluorophenyl above. Critical hydrogen bonds are formed between compound 8 and the N564 side chain amide, and the additional hydrogen bond to the indole N-1 of compound 8 draws the ligand deeper into the pocket. (c) GR-MIF does not hydrogen bond with N564, rather Q570, and MIF is higher in the binding pocket than compound 8. The methoxyfluorophenyl of 8 lies in approximately the same plane as the MIF A and B rings. (d) In the GR-DBX agonist structure, a hydrogen bond is observed between N564 and DBX.

Figure 3. Comparison of h12 in the crystal structures of GR LBD bound to compound 8, mifepristone, and dex. (a) The 8, antagonist, and agonist bound GR LBD forms are superimposed. The body of the domain is colored gray for all four structures. The α-12 helices for the ligand bound forms are colored as follows: magenta, MIFp; cyan, compound 8; yellow, MIFa; orange, dex. The α-12 helix of the compound 8 bound form adopts an intermediate position relative to that demonstrated by the MIFp and MIFa antagonist conformations. (b) The crystal structures showing h12 for MIFp (magenta), MIFa (orange), and GR LBD/compound 8(cyan) are shown as ribbon diagrams with the ligands removed.

base of the binding site while the methoxyfluorophenyl points upward, making contact with the α-6 helix which forms the roof of the binding site. The new NcoR derived peptide is located in the expected corepressor binding site, and its disposition is superimposable with that observed in the GR LBD/MIF/NcoR complex. The orientation of compound 8 is the same in both

Information Figure S1). Compound 8 is bound in the steroid ligand binding site occupied by known NHR ligands found at the bottom of the domain and formed by a constellation of generally hydrophobic residues primarily comprised of αhelices 3, 6, 8, and 11 (Figure 2b−d). The compound 8 sulfonamide is wedged between the α-3 and α-11 helices at the E



Article

defined by the steroid C and D rings of MIF while the benzylamine moiety, extending from the 2 position of compound 8, juts across the α-11 helix and sterically blocks adoption of the agonist h12 helix conformation, occupying a position analogous to that of the MIF dimethylaniline at C11. The isopropyl group extending from position 5 of the compound 8 core indole overlaps with the propynyl group of the MIF D ring. A pair of hydrogen bonds between the C3 carbonyl oxygen of the MIF A ring and GR LBD side chains (Q570 and R611) are formed and conserved in the various steroid GR structures. An additional hydrogen bond between the MIF D ring 17βhydroxyl and the Q642 side chain amide occurs in MIFp but not in MIFa. Compound 8 does not form such hydrogen bonds with any of these residues. Interestingly, the critical hydrogen bonds between compound 8 and GR are formed with the N564 side chain in a mode more similar to that seen for GR agonists (Figure 2b,d). At the apex of the binding cavity, an unconventional interaction is formed between the fluorine of the compound 8 methoxyfluorophenyl and the backbone carbonyl of M604. The benzylamine at R2 of 8 directs h12 into an antagonist conformation. This observation supports the observed SAR trend where larger R2 groups act as better functional antagonists (Table 2). Edge-to-face π-stacking is observed between the F623 side chain phenyl and the compound 8 methoxyfluorophenyl, which lies in approximately the same plane as the B ring of MIF. Side chain reorganization that cannot be attributed to gross structural changes in the disposition or ordering of helices is apparent in residue M560, which must adopt a different conformer to accommodate the compound 8 methylsulfonamide. Relative to MIF, compound 8 extends deeper into and occupies a greater volume at the base of the ligand binding site (Figure 3b,c). Comparison with GR LBD Bound to Agonists. In the GR LBD−dex structure, the N-terminus of h12 is pressed against the α-3 helix. This contrasts starkly with h12 location in the GR LBD/compound 8/NcoR structure in which its N-terminus has swung away from the main body of the domain and the Cα carbon of the first residue of h12 (M752) is shifted by ∼17 Å relative to the same atom in the GR LBD−dex crystal structure. The GR LBD engages compound 8 through a series of hydrogen bonds found between the N564 side chain and the ligand (Figure 2b). The N564 side chain oxygen forms two hydrogen bonds with 8: one with the indole NH, and the other with the sulfonamide nitrogen. To form the hydrogen bond between the N564 side chain oxygen and the indole nitrogen, the ligand must move deeper into the base of the pocket relative to the recently reported nonsteroidal dibenzoxapane agonist (DBX) (Figure 2b,c).36 In contrast to the GR LBD/ DBX agonist crystal structure, no hydrogen bond is formed with the T736 side chain. On the basis of previous structural data, such hydrogen bonding to the N564 side chain might be expected to potentiate agonism because both the nonsteroidal trifluoromethyl carbinol and steroidal agonists also form hydrogen bonds with N564. The intricate network of stabilizing hydrogen bonds between compound 8 and N564 would appear to test the hypothesis that hydrogen bonding between N564 and a ligand is a sufficient structural prerequisite for agonism and transrepression. Perhaps this hypothesis can be modified to acknowledge that such hydrogen bonding may be required but alone it is not sufficient for agonist function.

copies of the trimeric complex in the asymmetric unit, and the molecular contacts between the GR LBD and compound 8 are the same for both complexes with the exception of F737, where in one trimer, the F737 side chain rotates to form an edge-toface π-stacking interaction with the compound 8 phenyl. For the purposes of this discussion, the analysis of all GR crystal structures will refer to the most ordered copy in the unit cell unless otherwise specified. Comparison with GR LBD Bound to MIF. The crystal structures of GR LBD bound to MIF exhibit significant differences in the relative orientations of α-helix 12 (h12).31,33 For one crystal structure with MIF, the orientation of h12 varies considerably between monomers in the tetrameric asymmetric unit. Moreover, the residues that contact the ligand exhibit alternate conformations in the different GR/MIF structures. In contrast, h12 in the GR LBD compound 8 crystal structure occupies an intermediate position relative to the orientations in the published MIF/GR structures (Figure 3). The phenyl moiety of compound 8 is not sterically compatible with the orientation of h12 observed in the MIF binding conformation associated with active antagonism (MIFa, PDB code 3H52) in which the helix is found packed closely against the main body of the LBD, more reminiscent of the h12 position displayed by GR LBD agonist crystal structures. However, h12 in the GR LBD complex crystal structures juts out less than in the MIF binding conformation associated with passive antagonism (MIFp, PDB code 1NHZ (Figure 3)). Notably h12 appears to be completely disordered in chain B of the GR LBD crystal structure which may be an indication of the inherent flexibility of this structural element when bound to antagonists. Although the phenyl in compound 8 protrudes into the space between the α-3 and α-11 helices (Supporting Information Figure S3) as observed in the NHR antagonist complexes of mifepristone, raloxifene, and bicalutamide, it is found in closer proximity to the α-11 helix relative to the functionally similar moieties of the other GR antagonists (Figure S3). The structural divergence between the GR LBD antagonist structures becomes apparent in the residues preceding those encoding h12. After approximately residue V729, these structures exhibit a remarkable degree of plasticity. The trajectory of the α-11 in MIFa diverges from that of α-11 in the GR LBD/compound 8 complex, with the base of α-11 packed more closely into the ligand binding site in the latter (Figure 3b). In the MIFp complex, the α-helical content dissipates after L733 with a random coil observed between residues 733 and 739. A short accessory α-helix is then formed from residues 739−746. This combination of structural elements forms a flexible tether which allows h12 in MIFp to project away from the LBD domain such that none of the intradomain contacts between the main body of the LBD and h12 are preserved. Despite the discrepancies between the MIF bound structures, the overall orientation of the ligand and its effect on h12 remains the same, thereby providing a reasonable basis for comparison with the binding mode of compound 8. Compound 8 is oriented with the fluorine of the methoxyfluorophenyl pointing upward in the binding site and occupying roughly the same space as the A and B rings of MIF (compare Figure 2b,c). The compound 8 methylsulfonamide lies at the base of the cavity, extending below the 17β-hydroxyl of the steroid D ring in MIF with much of the bulk of compound 8 in sites associated with other/GR complexes. The indole core of compound 8 angles forward and out of the plane F



Article

Figure 4. 2D representations of contacts between GR and ligands. (a) dexamethasone, (b) mifepristone, (c) compound 8. (d) 2D representation of potential contacts for GR LBD ligands: Green asterisk, critical hydrogen bond contact common to dex and 8. Red asterisk, critical hydrogen bond contacts common to dex and MIF. Hydrogen bonding to N564, generally associated with agonism, may be a significant contact for the development of novel antagonists. Adding critical hydrogen bonding contacts common to both dex and MIF to the 2D contact map of 8 in (d) provides a schematic view of how unique receptor-based pharmacophores can be designed to model binding to the GR LBD.

■

DISCUSSION AND CONCLUSION

crystal structure of GR LBD bound to compound 8 confirms previous findings that reordering and repositioning of h12 is critical to the differentiation of the biological outcomes of agonism and antagonism. In this context, the GR LBD crystal structure complements the accepted paradigm defined by preexisting structural information associated with the positioning of h12. In addition, the location of the compound 8 phenyl moiety between the α-3 and α-11 helices is compatible with the expected binding conformation of NHR antagonists originally defined by the ER/raloxifene complex. However, in several

In this study, the novel GR antagonist, compound 8, was identified and characterized as a GR ligand in vitro and in vivo. Structural biology also provides the binding mode of 8 within the GR LBD. Compound 8 binds to GR (Ki = 1.2 nM) with high affinity, and it was determined to be a potent nonsteroidal antagonist which is more selective than MIF. Notably this represents the second of a series of indole sulfonamide GR antagonists showing interesting GR-mediated physiology. The G



Article

chromatography (4:1 to 7:3 to 6:4 hexanes:EtOAc) gave 6.9 g (92%) of the titled compound as a dark oil. 1H NMR(DMSO-d6) δ 7.84 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.22−7.32 (m, 5H), 6.98 (br s, 2H), 3.61 (s, 2H), 3.57 (s, 2H), 2.83 (sept, J = 6.8 Hz, 1H), 2.26 (s, 3H), 1.15 (d, J = 6.8 Hz, 6H). 13C NMR(DMSO-d6) δ 144.3, 138.9, 138.1, 136.0, 131.3, 129.2, 128.7, 127.5, 123.2, 112.2, 93.4, 80.2, 60.1, 46.2, 41.8, 32.4, 23.8. HRMS (ESI) m/z calcd for C20H23N3O2 [M + H]+ 338.1870, found 338.1860. 2-[3-(Benzyl(methyl)amino)prop-1-ynyl]-4-isopropyl-6-nitro-aniline (4). Trifluoroacetic anhydride (4.2 mL, 29.9 mmol) was added dropwise via a syringe to a solution of 2-[3-(benzyl(methyl)amino)prop-1-ynyl]-4-isopropyl-6-nitro-aniline 3 (6.9 g, 20.1 mmol) and pyridine (4.7 mL, 4.8 mmol) in dichloromethane (265 mL) at 0 °C under nitrogen. The solution was allowed to warm to room temperature over a 6 h period. The solvent was then removed under reduced pressure, and the crude residue was triturated with diethyl ether. The solid was filtered and dried to give the title compound (11.0 g, 99%). 1H NMR(DMSO-d6) δ 7.94 (d, J = 2.0 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.24−7.33 (m, 5H), 3.54 (s, 2H), 3.14 (s, 2H), 3.04 (sept, J = 6.8 Hz, 1H), 2.23 (s, 3H), 1.22 (d, J = 6.8 Hz, 6H). 13C NMR(DMSO-d6) δ 155.9 (q, J = 38 Hz), 150.6, 147.0, 138.7, 137.2, 135.7, 129.2, 128.7, 127.6, 124.4, 123.3, 116.2 (q, J = 292 Hz), 92.4, 80.5, 59.7, 49.0, 45.7, 33.3, 23.5. HRMS (ESI) m/z calcd for C22H22F3N3O3 [M + H]+ 434.1693, found 434.1692. N-Benzyl-1-[3-(4-fluoro-2-methoxyphenyl)-5-isopropyl-7-nitro1H-indol-2-yl]-N-methyl-methanamine (6). A mixture of 2-[3(benzyl(methyl)amino)prop-1-ynyl]-4-isopropyl-6-nitro-aniline 4 (5.50 g, 12.7 mmol), 4-fluoro-2-iodoanisole 5 (3.50 g, 13.9 mmol), and cesium carbonate (12.6 g, 38.2 mmol) in acetonitrile (40 mL) was bubbled with nitrogen in a 150 mL pressure flask. Pd(PPh3)4 (1.15 g, 1.00 mmol) was added, the flask was sealed, and the mixture was heated at 100 °C for 3 h. The mixture was allowed to cool, and 250 mL of EtOAc was added. The mixture was washed with water and then brine (60 mL each). The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude oil was purified by flash chromatography (8:2 to 7:3 hexanes:EtOAc) to afford 2.6 g (44%) of the titled compound as a yellow solid. 1H NMR(DMSO-d6) δ 11.31 (s, 1H), 7.93 (d, J = 1.0 Hz, 1H), 7.48 (d, J = 1.0 Hz, 1H), 7.11−7.46 (m, 6H), 7.02 (dd, J = 11.6, 2.4 Hz, 1H), 6.85 (td, J = 8.4, 2.4 Hz, 1H), 3.70 (s, 2H), 3.68 (s, 3H), 3.66 (s, 2H), 3.03 (sept, J = 6.8 Hz, 1H), 1.97 (s, 3H), 1.22 (d, J = 6.8 Hz, 6H). 13C NMR(DMSOd6) δ 164,2, 161.8, 158.8 (d, J = 9.9 Hz), 147.0, 140.0, 139.1, 138.0, 132.4, 132.3, 129.0, 128.4, 127.2, 127.0, 118.5 (d, J = 3.0 Hz), 116.8, 112.2, 107.1 (d, J = 20.3 Hz), 100.3 (d, J = 25.7 Hz), 61.1, 56.0, 52.6, 41.7, 33.4, 24.6. HRMS (ESI) m/z calcd for C27H28FN3O3 [M + H]+ 462.2195, found 462.2188. 2-[(Benzyl(methyl)amino)methyl]-3-(4-fluoro-2-methoxyphenyl)5-isopropyl-1H-indol-7-amine (7). N-Benzyl-1-[3-(4-fluoro-2-methoxyphenyl)-5-isopropyl-7-nitro-1H-indol-2-yl]-N-methyl-methanamine 6 (2.60 g, 5.63 mmol), Raney nickel (3 mL), THF (30 mL), and ethanol (30 mL) were added to a 150 mL pressure flask. The system was evacuated with nitrogen/vacuum (3×), and the dark mixture was then stirred rapidly under 50 psi hydrogen for 6 h. Additional Raney nickel (3 mL) and the mixture was stirred for 18 h and then filtered through Celite. The filtrate concentrated under reduced pressure, diluted with EtOAc (250 mL), and then washed successively with water and brine (50 mL each). The organic layer was dried over MgSO4 and concentrated under reduced pressure to give the titled compound (2.38 g, 98%), which was used in the next step without further purification: LC/MS [M + H]+ = 432 (72% purity). N-[2-[(Benzyl(methyl)amino)methyl]-3-(4-fluoro-2-methoxyphenyl)-5-isopropyl-1H-indol-7-yl]methanesulfonamide (8). Methanesulfonyl chloride (0.49 mL, 6.33 mmol) was added dropwise via syringe to a solution of 2-[(benzyl(methyl)amino)methyl]-3-(4-fluoro2-methoxyphenyl)-5-isopropyl-1H-indol-7-amine 7 and pyridine (1.3 mmol, 16 mmol) in dichloromethane (55 mL) at 0 °C. The solution was allowed to warm to room temperature and stirred for 18 h. The solution was concentrated and purified via column chromatography (50:1 to 25:1 dichloromethane:methanol). A second purification was conducted via reverse phase (methanol:water) to give the titled

respects, this GR LBD crystal structure represents a novel finding. Hydrogen/deuterium exchange mass spectrometry and the MIFa structure demonstrated that, in the corepressor-bound antagonist conformation, h12 adopts a conformation similar to that observed in the agonist-bound form.28 In contrast, h12 of the GR LBD/compound 8/NcoR complex is displaced relative to its position in agonist complexes albeit not to the extent of the MIFp structure. Perhaps most importantly the extensive hydrogen bonding between the compound 8 sulfonamide and the N564 side chain is strongly reminiscent of GR agonist binding modes. In summary, this study identifies and characterizes a novel class of indole sulfonamide GR antagonists. Compound 8 represents the first nonsteroidal GR antagonist to be crystallized within the GR LBD, thereby providing additional support for the structural conformation of antagonist action. The significance of the findings described herein suggests that protein expression and engineering strategies can be successfully utilized in the prosecution of the X-ray crystallography of GR bound to diverse scaffolds, that GR antagonists can share key binding interactions with agonists, and that although the position of h12 can vary, it is more likely to adopt the passive antagonist form. The structure also reveals how unconventional interactions, such as those observed between the fluorine of compound 8, the backbone carbonyl of the M604 and the association of the sulfonamide with N564, might be exploited in molecular design. Figure 4 depicts a two-dimensional rendering of the residues involved in the breadth of binding modes of dex, MIF and 8 along with their associated residues within the GR LBD which could be used to initiate structure-based design. Taken together, these observations can assist in the understanding of the structural basis of GR ligand function, leading to development of new agents targeted toward the amelioration of disorders related to GR pharmacology.

■

EXPERIMENTAL SECTION

Chemical Synthesis. All reactions, unless specified, were carried under an atmosphere of nitrogen in oven-dried glassware. Reagents and solvents were obtained from Aldrich Chemical or Acros Organics and used without further purification. Catalysts were purchased from Strem. 2-Bromo-4-isopropyl-6-nitro-aniline and 4-fluoro-2-iodoanisole were obtained from Oakwood Products, Inc. TLC was performed on 0.25 mm E. Merck silica gel 60 F254 plates and visualized under UV light. Flash chromatography was performed using Agilent Super Flash silica cartridges. Reverse-phase purification was carried out on a Waters X-Bridge Prep C18, OBD 19 mm × 100 mm with a gradient of 5−95% acetonitrile/water (with 0.1% TFA) over 8 min at 20 mL/min. LCMS analysis was carried out on an Agilent 1100 series LC/MSD using 5− 100% acetonitrile/0.1% formic acid over 7.0 min. NMR spectra were recorded on a Varian 400, and chemical shifts are expressed in ppm relative to solvent signals: CDCl3 (1H 7.26; 13C 77.0 ppm) or DMSOd6 (1H 2.50; 13C 39.5 ppm). High resolution mass spectra were obtained electrospray ionization mass spectrometry. Test articles were refined to >95% purity using the chromatography methods noted above. 2-[3-(Benzyl(methyl)amino)prop-1-ynyl]-4-isopropyl-6-nitro-aniline (3). A solution of 2-bromo-4-isopropyl-6-nitro-aniline 1 (6.82 g, 22.3 mmol) and N-benzyl-N-methyl-prop-2-yn-1-amine 2 (4.00 g, 25.2 mmol) in THF (40 mL) was degassed for 10 min with nitrogen in a 500 mL round-bottomed flask prior to the addition of CuI (0.26 g, 1.3 mmol), PdCl2(PPh3)2 (0.48 g, 0.68 mmol), and triethylamine (43.0 mL). The resultant mixture was stirred at 25 °C for 1 h prior to diluting with EtOAc (500 mL) The mixture was then washed with saturated aqueous ammonium chloride, water, and brine (100 mL each). The organic layer was dried over Na2SO4 and concentrated under reduced pressure, and the crude residue was purified via flash H



Article

compound (1.2 g, 43%). 1H NMR(DMSO-d6) δ 10.68 (s, 1H), 9.49 (s, 1H), 7.19−7.27 (m, 6H), 6.96−7.00 (m, 2H), 6.80−6.85 (m, 2H), 3.68 (s, 3H), 3.40 (s, 2H), 3.30 (s, 2H), 2.99 (s, 3H), 2.85 (sept, J = 6.8 Hz, 1H), 2.05 (s, 3H), 1.15 (d, J = 6.8 Hz, 6H). 13C NMR(DMSOd6) δ 163.8, 161.4, 158.6 (d, J = 10.1 Hz), 140.2, 138.8, 135.1, 132.9 (d, 9.9 Hz), 129.8, 129.2, 128.5, 127.3, 127.0, 122.2, 119.6 (d, J = 3.1 Hz), 112.6, 110.7, 107.4 (d, J = 21.1 Hz), 100.2 (d, J = 25.3 Hz), 61.2, 61.1, 55.9, 53.3, 42.3, 34.0, 25.0. HRMS (ESI) m/z calcd for C27H28FN3O3 [M + H]+ 510.2239, found 510.2232. 2-Methyl-3-phenyl-1H-indole (9) and N-[1H-Indol-7-yl]methanesulfonamide (14). 14 was prepared using literature methods.47,48 The following compounds were prepared using the general method described for the synthesis of compound 8. N-[2-[(Benzyl(methyl)amino)methyl]-3-(4-fluoro-2-methoxyphenyl)-5-fluoro-7-amino Indole (10). 1H NMR (DMSO): δ 10.66 (s, 1H), 7.10−7.31 (m, 6H), 6.95 (dd, J = 8.4, 2.1 Hz, 1H), 6.80 (dt, J = 8.4, 1.5 Hz, 1H), 6.08 (dd, J = 8.4, 1.8 Hz, 1H), 6.05 (dd, J = 8.2, 1.8 Hz, 1H), 5.55 (bs, 2H), 3.68 (s, 3H), 3.39 (s, 2H), 3.30 (s, 2H), 2.04 (s, 3H). MS m/z 408 (M+ + H) observed in positive mode LC/MS. N-[3-(5-Fluoro-2-methoxyphenyl)-7-indolyl]acetamide (11). 1H NMR (DMSO): δ 10.14 (bs, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.46 (bs, 1H), 7.32 (dd, J = 9.2, 2.6 Hz, 1H), 7.06 (t, J = 7.7 Hz, 1H), 6.95−6.15 (m, 2H), 6.74 (d, J = 7.4 Hz, 1H), 3.81 (3, 3H), 2.31 (s, 3H). MS m/z 299 (M + + H). HRMS calcd for ([C17H15FN2O2]+H)+ 298.1118, found 298.1118. N-[2-Methyl-3-phenyl-5-methoxy-1H-indol-7-yl]methanesulfonamide (12). 1H NMR (DMSO): δ 8.89 (bs, 1H), 7.43−7.51 (m, 4H), 7.29−7.35 (m, 1H), 7.06 (s, 1H), 6.54 (s, 1H), 6.40 (bs, 1H), 3.80 (s, 3H), 3.05 (s, 3H), 2.49 (s, 3H). MS m/z 331 (M+ + H). HRMS calcd for ([C17H18FN2O3S])+ 330.1038, found 330.1022. N-[3-(3-Fluoro-4-methoxyphenyl)-1H-indol-7-yl]methanesulfonamide (13). 1H NMR (DMSO): δ 9.33 (bs, 1H), 7.82 (m, 1H), 7.30−7.39 (m, 3H), 7.15 (t, J = 7.58 Hz), 6.95 (m, 1H), 6.39 (bs, 1H), 3.95 (s, 3H), 3.03 (s, 3H). MS m/z 335 (M+ + H). HRMS calcd for (C16H15FN2O3S)+ 334.0787, found 334.0780. N-[2-[(Benzyl(methyl)amino)methyl]-3-(4-fluoro-2-methoxyphenyl)-5-t-butyl-1H-indol-7-yl]methanesulfonamide (15). 1H NMR (DMSO): δ 9.94 (bs, 1H), 7.24−7.37 (m, 8H), 7.07 (bs, 1H), 6.74−6.82 (m, 2H), 3.54−3.89 (m, 4H), 3.74 (s, 3H), 3.09 (s, 3H), 2.27 (s, 3H), 1.32 (s, 9H). MS m/z 524 (M+ + H). HRMS calcd for ([C29H34FN3O3S])+ 523.2305, found 523.2302. N-[2-Benzyl-3-(4-fluoro-2-methoxyphenyl)-5-cyclopropyl-1Hindol-7-yl]methanesulfonamide (16). 1H NMR (DMSO): δ10.60 (s, 1H), 9.19 (s, 1H), 7.12−7.26 (m, 4H), 7.08 (d, J = 7.5 Hz, 2H), 6.99 (dd, J = 11.5, 2.4 Hz, 1H), 6.82 (dt, J = 8.2, 2.5 Hz, 1H), 6.78 (bs, 2H), 6.70 (bs, 2H), 3.95 (bs, 2H), 3.67 (s, 3H), 2.96 (s, 3H), 1.91 (m, 1H), 0.84 (m, 2H), 0.51 (m, 2H). MS m/z 463 (M+ - H) in negative mode. HRMS calcd for (C26H25FN2O3S)+ 464.1570, found 464.1572. N-[2-Phenyl-3-(5-fluoro-2-methoxyphenyl)-1H-indol-7-yl]methanesulfonamide (17). 1H NMR (DMSO): δ 9.47 (bs, 1H), 7.39−7.44 (m, 2H), 7.27−7.35 (m, 2H), 7.25−7.26 (m, 4H), 6.98− 7.10 (m, 2H), 6.85−6.94 (m, 1H), 6.49 (bs, 1H), 3.49 (s, 3H), 3.07 (s, 3H). MS m/z 411 (M+ + H). HRMS calcd for ([C22H19FN2O3S])+ 410.1100, found 410.1102. N-[2-Methyl-3-(4-fluoro-2-methoxyphenyl)-5-fluoro-1H-indol-7yl]methanesulfonamide (18). 1H NMR (DMSO): δ 9.01 (bs, 1H), 7.22 (dd, J = 8.5, 6.6, 1H), 6.96 (dd, J = 9.2, 1.9 Hz, 1H), 6.73−6.79 (m, 2H), 6.65 (dd, J = 9.6, 1.8 Hz, 1H), 6.47 (bs, 1H), 3.79 (s, 3H), 3.07 (s, 3H), 2.36 (s, 3H). MS m/z 367 (M+ + H). HRMS calcd for ([C17H16F2N2O3S])+ 366.0850, found 366.0856. N-[3-(5-Chloro-2-methoxyphenyl)-1H-indol-7-yl]methanesulfonamide (19). 1H NMR (DMSO): δ 9.14 (bs, 1H), 7.56 (d, J = 2.75 Hz, 1H), 7.54 (d, 2.75 Hz, 1H), 7.22−7.26 (m, 2H), 7.12 (t, J = 7.91 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.86 (t, J = 8.1 Hz, 1H), 6.53 (bs, 1H), 3.84 (s, 3H), 3.03 (s, 3H). MS m/z 351 (M+ + H). HRMS calcd for ([C16H15ClN2O3S])+ 350.0492, found 350.0487. N-[3-(3-Fluoro-4-methoxyphenyl)-1H-indol-7-yl]methanesulfonamide (20). 1H NMR (DMSO): δ 9.01 (bs, 1H), 7.22 (dd, J = 8.5, 6.6, 1H), 6.96 (dd, J = 9.2, 1.9 Hz, 1H), 6.73−6.79 (m,

2H), 6.65 (dd, J = 9.6, 1.8 Hz, 1H), 6.47 (bs, 1H), 3.79 (s, 3H), 3.07 (s, 3H), 2.36 (s, 3H). MS m/z 367 (M+ + H). HRMS calcd for ([C17H16F2N2O3S])+ 366.0850, found 366.0856. N-[3-(3-Chlorophenyl)-1H-indol-7-yl]methanesulfonamide (21). 1 H NMR (DMSO): δ 11.25 (bs, 1H), 9.37 (s, 1H), 7.79 (d, J = 2.75 Hz, 1H), 7.70 (dd, J = 7.8, 1.4 Hz, 1H), 7.67 (m, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.27 (d, J = 8.2 Hz, 1H), 7.12 (m, 2H), 2.99 (s, 3H). MS m/z 321.4 (M+ + H). N-[3-(2-Methoxyphenyl)-1H-indol-7-yl]methanesulfonamide (22). 1H NMR (DMSO): δ 11.01 (s, 1H), 9.38 (s, 1H), 7.52, (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 7.3, 1.3 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.25 (dt, J = 7.6, 1.7 Hz, 1H), 7.10 (m, 1H), 7.08 (m, 1H), 7.02 (d, J = 7.7 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 3.77 (s, 3H), 2.99 (s, 3H). MS m/z 317.0 (M+ + H). N-[3-(4-Fluoro-phenyl)-1H-indol-7-yl]methanesulfonamide (23). 1 H NMR (DMSO): δ 11.1 (s, 1H), 9.36 (s, 1H), 7.66 (m, 4H), 7.25 (m, 2H), 7.09 (m, 2H), 2.99 (s, 3H). MS m/z 305.0 (M+ + H). In Vitro Assays. Steroid receptor competition binding assays using lysates from human embryonic kidney HEK293 cells overexpressing human MR, GR, AR, or PR were run in a buffer containing 20 mM HEPES buffer (pH = 7.6), 0.2 mM EDTA, 75 mM NaCl, 1.5 mM MgCl2, 20% glycerol, 20 mM sodium molybdate, 0.2 mM DTT, 20 μg/mL aprotinin, and 20 μg/mL leupeptin (assay buffer). The binding assays included radiolabeled ligands such as 0.25 nM [3H]-aldosterone for MR binding, 0.3 nM [3H]-dexamethasone for GR binding, 0.36 nM [3H]-methyltrienolone for AR binding, and 0.29 nM [3H]methyltrienolone for PR binding, and either 20 μg of 293-MR lysate, 20 μg of 293-GR lysate, 22 μg of 293-AR lysate, or 40 μg of 293-PR lysate per well in 96-well format. Competing test compounds were added at various concentrations ranging from 0.01 nM to 10 μM. Nonspecific binding was determined in the presence of 0.5 μM aldosterone for MR binding, 0.5 μM dexamethasone for GR binding, or 0.5 μM methyltrienolone for AR and PR binding. The binding reactions (140 μL) were incubated overnight at 4 °C, then 70 μL of cold charcoal−dextran buffer (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) was added to each reaction. Plates were mixed for 8 min on an orbital shaker at 4 °C, followed by centrifugation at 3000 rpm at 4 °C for 10 min. An aliquot of 120 μL of the binding reaction mixture was transferred to another 96-well plate, and 175 μL of Wallac Optiphase Hisafe 3 scintillation fluid was added to each well. Plates were sealed and shaken vigorously on an orbital shaker. After an incubation of 2 h, plates were read in a Wallac MICROBETA counter. The data were used to calculate an estimated IC50 and percentage inhibition at 10 μM. The IC50 values for compounds are converted to Ki using the Cheng−Prusoff equation. The Kd for [3H]-aldosterone for MR binding, [3H]-dexamethasone for GR binding, [3H]-methyltrienolone for AR binding, or [3H]methyltrienolone for PR binding was determined by saturation binding. Antagonist (GRAnt) activity was determined by measuring intrinsic agonist activity in HEK293 cells transiently transfected with GREs controlling a luciferase (luc) reporter gene. The tyrosine aminotransferase (TAT) enzyme assay was used to determine whether these compounds act as GR agonists or GR antagonists.36 The full GR agonist, dexamethasone, induced GR transactivation, resulting in the synthesis of tyrosine aminotransferase in hepatoma tissue culture (HTC) cells. As an indicator of functional activity of test compound on the GR, the tyrosine aminotransferase enzyme (TAT) activity assay was performed in agonist and antagonist modes. Briefly, rat hepatoma cells H4IIEC3 (ATCC no. CRL-1600) were grown in T150 cm2 flasks in DMEM media with 10% FBS. The cells were trypsinized and replated in 96-well plates at 100000 cells per well and incubated overnight at 37 °C under 5% CO2. After this incubation, the media was replaced with DMEM/F12 containing 5% CS-FBS and incubated for another 24 h at 37 °C under 5% CO2. To test for agonist activity, cells were exposed to various concentrations of test compounds ranging from about 0.09 nM to 25 μM and incubated for an additional 24 h at 37 °C under 5% CO2. To test for antagonist activity, cells were treated with various concentrations of test compounds ranging from 0.09 nM to 25 μM for 1 h prior to the I



Article

addition of 1 nM dexamethasone and incubated for an additional 24 h at 37 °C under 5% CO2. Following incubation with test compounds, the cells were washed in PBS, lysed with solubilization buffer at pH 7.6 containing 125 mM potassium phosphate, 1 mM EDTA, 1 mM DTT, and 0.5% nonyl phenoxypolyethoxylethanol (NP-40), and frozen at −80 °C. Upon thawing the plates, TAT substrate solution at pH 7.6 containing 125 mM potassium phosphate, 4.74 mM L-tyrosine, 13.3 mM α-ketoglutarate, and 0.07 mM pyridoxyl phosphate was added to the lysates. The reaction was allowed to develop for 2 h at 37 °C under 5% CO2. After 2 h, the p-hydroxyphenylpyruvate produced in the reaction was converted to p-hydroxybenzaldehyde with the addition of 10 N potassium hydroxide and incubation for 30 min. Absorbance at 340 nM was read for each well using a spectrophotometer. The phydroxybenzaldehyde concentration of samples was calculated based on a standard curve, and the resulting data were fit to a four parameterfit logistics to determine EC50 values for agonist activity and IC50 values for antagonist activity. In Vivo Plasma Lipid Model. The procedure of Bravo45,49 was used for live phase assessment of HDL and LDL in Syrian golden hamsters. Analysis of plasma lipids was done using the method of Kieft and co-workers.50 In Vivo Model for Prevention of Atypical Antipsychotic Associated Weight Gain. A modification of the procedure of Gehlert et al. was used.26 In this instance, a prophylactic protocol was employed whereby a dose response of compound 8 and a fixed dose (10 mg/kg) of quetiapine were administered po to groups (n = 9−10) of lean female Wistar rats concurrently for 14 days. Food intake, body weight, and changes in body composition were recorded over the course of the study. Protein Expression Purification and Crystallization. A gene encoding the GR LBD (amino acid residues 522−777) with the following mutations, L525S, L528S, L535A, V538T, F602Y, C638D, E684A, E688A, and W712S, was expressed in Escherichia coli as an Nterminally his-tagged SMT fusion protein and cofermented with 50 μM of compound 8. Cell pellets were stored at −80 °C and subsequently lysed by sonication in cold buffer containing 0.02 M TrisHCl, pH 8.0, 0.5 M NaCl, 10% glycerol, 0.025 M imidazole, 5 mM BME, 0.2% CHAPS, 50 μM compound 8, benzonase, and protease inhibitor (Roche complete EDTA-free, catalogue no. 13317600). Cell lysates were clarified by centrifugation (JLA-16.25 at 16K RPM at 4 °C), and the his-tagged recombinant protein was purified using NiNTA beads (Qiagen) in batch mode. The beads were collected in a drip column after incubation with the decanted supernatant at 4 °C with stirring and then washed with 20 bed volumes of cold lysis buffer. Recombinant GR LBD was eluted with lysis buffer containing 250 mM imidazole without compound 8. Then 0.5% ULP1 protease was added to the eluted sample followed by dialyzing overnight at 4 °C against 0.02 M Tris, pH 8.0, 0.5 M NaCl, 10% glycerol, 0.2% CHAPS, and 5 mM βME. The sample was reapplied to Ni-NTA (His-TRAP HP, GE Healthcare), and the flow through was collected and concentrated for further purification after the addition of 50 μM compound 8. The sample was applied to an s200 Superdex (GE Healthcare) gel filtration column equilibrated with 0.02 M Tris-HCl, pH 8.0, 0.1 M NaCl, 10% glycerol, 0.2% CHAPS, and 2 mM TCEP. GR LBD containing fractions were pooled, and equimolar compound 8, a 6-fold molar excess of modified NcoR peptide (NLGLEDIIRKALMGS), and an additional 0.2 M NaCl were added. The sample was incubated overnight on ice and then concentrated to 6 mg/mL. Crystals in the space group P3221 (a,b = 72.5; c = 229.5; α,β = 90°; γ = 120°) were grown by sitting drop vapor diffusion at 21 °C using a 1:1 drop ratio with a drop volume of 1.5 μL against a mother liquor containing 0.1 M Tris, pH 7.0, 24% PEG 8K, and 6% 1−6 hexanediol. Crystals were cryocooled by immersion in N2 (l) using 20% glycerol as the cryoprotectant. Data Collection and Refinement. X-ray diffraction data (Table 1) were collected at the LRL-CAT beamline at the Advance Photon Source (Chicago, Illinois) (λ = 0.97931 Å). The data were processed using XDS. The crystal structure was determined by molecular replacement (two copies of the trimeric complex/a.u.) using GR bound to the DBX agonist (4LSJ) as the search model (PHASER)51,52

and refined (BUSTER) at 2.35 Å resolution, after several rounds of model building (COOT)53 to an Rwork of 21.9% and an Rfree of 25.8% (Table 1). The first copy of the trimer in the asymmetric unit consists of residues 530−776 of GR LBD (chain A), residues 2−14 of the NcoR peptide, and one copy of compound 8. The second copy of the trimer in the asymmetric unit consists of residues 531−776 of GR_LBD (chain B), residues 3−14 of the NcoR peptide, and one copy of compound 8. In chain B, residues 742−767 are not included in the model due to lack of representative electron density. Side chains for the following residues are truncated due to incomplete electron density: R633, Y640 (chain A); T531, I769, K770 (chain B). There are 118 water molecules modeled into the structure. There are no residues in the disallowed region of the Ramachandran plot. Chain B of the GR LBD superimposes upon chain A with an rmsd of 0.7 Å for all equivalent Cα atoms. Molecular graphics were rendered, and atomic distances were measured, using PYMOL.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00736. Additional data regarding the X-ray conformation of 8/ GR LBD/cofactor complex including electron density maps; data showing changes in lipid profile using MIF (PDF) Chemical formula strings (CSV) Accession Codes

The coordinates for the structure of 8within the GR LBD are available in the Protein Data Bank under the accession code 4MDD.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 317 651 1620. E-mail: [email protected]. Address: Mail Drop Code 0528, Lilly Research Laboratories, A Division of Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana 46285 United States. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the staff at the LRL-CAT beamline at APS, Stephen Wasserman, Sonali Sojitra, John Koss, and Laura Morisco, for data collection. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We are also indebted to Dr. Mathew Schiffler for providing valuable editing.

■

REFERENCES

(1) Vanden Berghe, W.; Francesconi, E.; De Bosscher, K.; RescheRigon, M.; Haegeman, G. Dissociated glucocorticoids with antiinflammatory potential repress interleukin-6 gene expression by a nuclear factor-.kappa.B-dependent mechanism. Mol. Pharmacol. 1999, 56, 797−806. (2) De Bosscher, K.; Beck, I. M.; Dejager, L.; Bougarne, N.; Gaigneaux, A.; Chateauvieux, S.; Ratman, D.; Bracke, M.; Tavernier, J.; Vanden Berghe, W.; Libert, C.; Diederich, M.; Haegeman, G. Selective modulation of the glucocorticoid receptor can distinguish between transrepression of NF-κB and AP-1. Cell. Mol. Life Sci. 2014, 71, 143. (3) McKay, L. I.; Cidlowski, J. A. Molecular control of immune/ inflammatory responses: interactions between nuclear factor-.kappa.B

J



Article

and steroid receptor-signaling pathways. Endocr. Rev. 1999, 20, 435− 459. (4) McKay, L. I.; Cidlowski, J. A. CBP (CREB binding protein) integrates NF-kB (nuclear factor-kB) and glucocorticoid receptor physical interactions and antagonism. Mol. Endocrinol. 2000, 14, 1222−1234. (5) Clark, A. R.; Belvisi, M. G. Maps and legends: The quest for dissociated ligands of the glucocorticoid receptor. Pharmacol. Ther. 2012, 134, 54−67. (6) Tuckermann, J. P.; Reichardt, H. M.; Arribas, R.; Richter, K. H.; Schutz, G.; Angel, P. The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin. J. Cell Biol. 1999, 147, 1365−1370. (7) Shipp, L. E.; Lee, J. V.; Yu, C.-Y.; Pufall, M.; Zhang, P.; Scott, D. K.; Wang, J.-C. Transcriptional regulation of human dual specificity protein phosphatase 1 (DUSP1) gene by glucocorticoids. PLoS One 2010, 5, e13754. (8) Kim, Y.; Sun, Y.; Chow, C.; Pommier, Y. G.; Simons, S. S., Jr. Effects of acetylation, polymerase phosphorylation, and DNA unwinding in glucocorticoid receptor transactivation. J. Steroid Biochem. Mol. Biol. 2006, 100, 3−17. (9) Kagoshima, M.; Wilcke, T.; Ito, K.; Tsaprouni, L.; Barnes, P. J.; Punchard, N.; Adcock, I. M. Glucocorticoid-mediated transrepression is regulated by histone acetylation and DNA methylation. Eur. J. Pharmacol. 2001, 429, 327−334. (10) Edman, K.; Ahlgren, R.; Bengtsson, M.; Bladh, H.; Backstrom, S.; Dahmen, J.; Henriksson, K.; Hillertz, P.; Hulikal, V.; Jerre, A.; Kinchin, L.; Kase, C.; Lepisto, M.; Mile, I.; Nilsson, S.; Smailagic, A.; Taylor, J.; Tjornebo, A.; Wissler, L.; Hansson, T. The discovery of potent and selective non-steroidal glucocorticoid receptor modulators, suitable for inhalation. Bioorg. Med. Chem. Lett. 2014, 24, 2571−2577. (11) Harcken, C.; Riether, D.; Kuzmich, D.; Liu, P.; Betageri, R.; Ralph, M.; Emmanuel, M.; Reeves, J. T.; Berry, A.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra, T. N.; Zuvela-Jelaska, L.; Dinallo, R.; Bentzien, J.; Nabozny, G. H.; Thomson, D. S. Identification of Highly Efficacious Glucocorticoid Receptor Agonists with a Potential for Reduced Clinical Bone Side Effects. J. Med. Chem. 2014, 57, 1583− 1598. (12) Harcken, C.; Riether, D.; Liu, P.; Razavi, H.; Patel, U.; Lee, T.; Bosanac, T.; Ward, Y.; Ralph, M.; Chen, Z.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra-Khan, T. N.; Zuvela-Jelaska, L.; Patel, M.; Thomson, D. S.; Nabozny, G. H. Optimization of Drug-Like Properties of Nonsteroidal Glucocorticoid Mimetics and Identification of a Clinical Candidate. ACS Med. Chem. Lett. 2014, 5, 1318−1323. (13) Hu, X.; Du, S.; Tunca, C.; Braden, T.; Long, K. R.; Lee, J.; Webb, E. G.; Dietz, J. D.; Hummert, S.; Rouw, S.; Hegde, S. G.; Webber, R. K.; Obukowicz, M. G. The antagonists but not partial agonists of glucocorticoid receptor ligands show substantial side effect dissociation. Endocrinology 2011, 152, 3123−3134. (14) Kuzmich, D.; Bentzien, J.; Betageri, R.; DiSalvo, D.; Fadra-Khan, T.; Harcken, C.; Kukulka, A.; Nabozny, G.; Nelson, R.; Pack, E.; Souza, D.; Thomson, D. Function-regulating pharmacophores in a sulfonamide class of glucocorticoid receptor agonists. Bioorg. Med. Chem. Lett. 2013, 23, 6640−6644. (15) Razavi, H.; Riether, D.; Harcken, C.; Bentzien, J.; Dinallo, R. M.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra-Khan, T. N.; Pack, E. J.; Zuvela-Jelaska, L.; Pelletier, J.; Panzenbeck, M.; Torcellini, C. A.; Proudfoot, J. R.; Nabozny, G. H.; Thomson, D. S. Discovery of a potent and dissociated non-steroidal glucocorticoid receptor agonist containing an alkyl carbinol pharmacophore. Bioorg. Med. Chem. Lett. 2014, 24, 1934−1940. (16) Strand, V.; Buttgereit, F.; McCabe, D.; Kolluri, S.; Tammara, B.; Rojo, R.; Hey-Hadavi, J. A PHASE 2, Randomized, Double-Blind Comparison of the Efficacy and Safety of PF-04171327 (1, 5, 10, 15 mg QD) Vs 5 and 10 Mg Prednisone QD or Placebo in Subjects with Rheumatoid Arthritis (RA) over 8 Weeks Followed By a 4-Week Taper of Study Drug. Arthritis Rheumatol. 2014, 66, 3531−3532. (17) Jung-Testas, I.; Baulieu, E. E. Inhibition of glucocorticosteroid action in cultured L-929 mouse fibroblasts by RU 486, a new anti-

glucocorticosteroid of high affinity for the glucocorticosteroid receptor. Exp. Cell Res. 1983, 147, 177−182. (18) Nieman, L. K.; Chrousos, G. P.; Kellner, C.; Spitz, I. M.; Nisula, B. C.; Cutler, G. B.; Merriam, G. R.; Bardin, C. W.; Loriaux, D. L. Successful treatment of Cushing’s syndrome with the glucocorticoid antagonist RU 486. J. Clin. Endocrinol. Metab. 1985, 61, 536−540. (19) Phillips, C. I.; Green, K.; Gore, S. M.; Cullen, P. M.; Campbell, M. Eye drops of RU 486−6, a peripheral steroid blocker, lower intraocular pressure in rabbits. Lancet 1984, 323, 767−768. (20) Belanoff, J. K.; Flores, B. H.; Kalezhan, M.; Sund, B.; Schatzberg, A. F. Rapid reversal of psychotic depression using mifepristone. J. Clin. Psychopharmacol. 2001, 21, 516−521. (21) DeBattista, C.; Belanoff, J.; Glass, S.; Khan, A.; Horne, R. L.; Blasey, C.; Carpenter, L. L.; Alva, G. Mifepristone versus Placebo in the Treatment of Psychosis in Patients with Psychotic Major Depression. Biol. Psychiatry 2006, 60, 1343−1349. (22) Liu, Y.; Nakagawa, Y.; Wang, Y.; Sakurai, R.; Tripathi, P. V.; Lutfy, K.; Friedman, T. C. Increased glucocorticoid receptor and 11bhydroxysteroid dehydrogenase type 1 expression in hepatocytes may contribute to the phenotype of type 2 diabetes in db/db mice. Diabetes 2005, 54, 32−40. (23) Chu, J. W.; Matthias, D. F.; Belanoff, J.; Schatzberg, A.; Hoffman, A. R.; Feldman, D. Successful long-term treatment of refractory Cushing’s disease with high-dose mifepristone (RU 486). J. Clin. Endocrinol. Metab. 2001, 86, 3568−3573. (24) Fleseriu, M.; Biller, B. M.; Findling, J. W.; Molitch, M. E.; Schteingart, D. E.; Gross, C.; et al. Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing’s syndrome. J. Clin. Endocrinol. Metab. 2012, 97, 2039−2049. (25) Korlym (Mifepristone) 300 mg Tablets; Corcept Therapeutics: Menlo Park, CA, November 11, 2013; http://www.corcept.com/ korlym. (26) Sindelar, D. K.; Carson, M. W.; Morin, M.; Shaw, J.; Barr, R. J.; Need, A.; Alexander-Chacko, J.; Coghlan, M.; Gehlert, D. R. LLY2707, A Novel Nonsteroidal Glucocorticoid Antagonist That Reduces Atypical Antipsychotic-Associated Weight Gain in Rats. J. Pharmacol. Exp. Ther. 2013, 348, 192−201. (27) Biggadike, K.; Bledsoe, R. K.; Hassell, A. M.; Kirk, B. E.; McLay, I. M.; Shewchuk, L. M.; Stewart, E. L. X-ray Crystal Structure of the Novel Enhanced-Affinity Glucocorticoid Agonist Fluticasone Furoate in the Glucocorticoid Receptor-Ligand Binding Domain. J. Med. Chem. 2008, 51, 3349−3352. (28) Bledsoe, R. K.; Montana, V. G.; Stanley, T. B.; Delves, C. J.; Apolito, C. J.; McKee, D. D.; Consler, T. G.; Parks, D. J.; Stewart, E. L.; Willson, T. M.; Lambert, M. H.; Moore, J. T.; Pearce, K. H.; Xu, H. E. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 2002, 110, 93−105. (29) Biggadike, K.; Bledsoe, R. K.; Coe, D. M.; Cooper, T. W. J.; House, D.; Iannone, M. A.; MacDonald, S. J. F.; Madauss, K. P.; McLay, I. M.; Shipley, T. J.; Taylor, S. J.; Tran, T. B.; Uings, I. J.; Weller, V.; Williams, S. P. Design and x-ray crystal structures of highpotency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18114−18119. (30) Madauss, K. P.; Bledsoe, R. K.; McLay, I.; Stewart, E. L.; Uings, I. J.; Weingarten, G.; Williams, S. P. The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist. Bioorg. Med. Chem. Lett. 2008, 18, 6097−6099. (31) Kauppi, B.; Jakob, C.; Farnegardh, M.; Yang, J.; Ahola, H.; Alarcon, M.; Calles, K.; Engstrom, O.; Harlan, J.; Muchmore, S.; Ramqvist, A.-K.; Thorell, S.; Ohman, L.; Greer, J.; Gustafsson, J.-A.; Carlstedt-Duke, J.; Carlquist, M. The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligandbinding domain: RU-486 induces a transconformation that leads to active antagonism. J. Biol. Chem. 2003, 278, 22748−22754. (32) Schoch, G. A.; D'Arcy, B.; Stihle, M.; Burger, D.; Bar, D.; Benz, J.; Thoma, R.; Ruf, A. Molecular Switch in the Glucocorticoid K



Article

Receptor: Active and Passive Antagonist Conformations. J. Mol. Biol. 2010, 395, 568−577. (33) Suino-Powell, K.; Xu, Y.; Zhang, C.; Tao, Y.-g.; Tolbert, W. D.; Simons, S. S., Jr.; Xu, H. E. Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol. Mol. Cell. Biol. 2008, 28, 1915−1923. (34) Chinchilla, R.; Najera, C. Recent advances in Sonogashira reactions. Chem. Soc. Rev. 2011, 40, 5084−5121. (35) Cacchi, S.; Fabrizi, G.; Parisi, L. M. Preparation of indoles from o-alkynyltrifluoroacetanilides through the aminopalladation-reductive elimination process. Synthesis 2004, 2004, 1889−1894. (36) Carson, M. W.; Luz, J. G.; Suen, C.; Montrose, C.; Zink, R.; Ruan, X.; Cheng, C.; Cole, H.; Adrian, M. D.; Kohlman, D. T.; Mabry, T.; Snyder, N.; Condon, B.; Maletic, M.; Clawson, D.; Pustilnik, A.; Coghlan, M. J. Glucocorticoid Receptor Modulators Informed by Crystallography Lead to a New Rationale for Receptor Selectivity, Function and Implications for Structure-based Design. J. Med. Chem. 2014, 57, 849−860. (37) Andrews, J. S. Glucocorticoid antagonists and depression. Techniques in the Behavioral and Neural Sciences 2005, 15, 437−450. (38) Clark, R. D. Glucocorticoid receptor antagonists. Curr. Top. Med. Chem. (Sharjah, United Arab Emirates) 2008, 8, 813−838. (39) Dhikav, V.; Anand, K. S. Glucocorticoids may initiate Alzheimer’s disease: A Potential therapeutic role for mifepristone (RU-486). Med. Hypotheses 2007, 68, 1088−1092. (40) Gruenfeld, J. P.; Eloy, L. Glucocorticoid antagonists as probes to study the vascular effects of glucocorticoids. Kidney Int., Suppl. 1988, 34, S24−S26. (41) Nihalani, N. D.; Schwartz, T. L. Drug evaluation: mifepristone, a glucocorticoid antagonist for the potential treatment of psychotic major depression. Curr. Opin. Investig. Drugs 2007, 8, 563−569. (42) Brindley, D. N. Role of glucocorticoids and fatty acids in the impairment of lipid metabolism observed in the metabolic syndrome. Int. J. Obes. Relat. Metab. Disord. 1995, 19 (Suppl 1), S69−S75. (43) Brindley, D. N.; Wang, C.-N. Role of fatty acids, glucocorticoids and insulin resistance in the dyslipidemia of the metabolic syndrome. Prog. Obes. Res. 1996, 7, 505−510. (44) Hu, G.-X.; Lin, H.; Lian, Q.-Q.; Zhou, S.-H.; Guo, J.; Zhou, H.Y.; Chu, Y.; Ge, R.-S. Curcumin as a potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase 1: improving lipid profiles in highfat-diet-treated rats. PLoS One 2013, 8, e49976. (45) Bravo, E.; Cantafora, A.; Calcabrini, A.; Ortu, G. Why prefer the golden Syrian hamster (Mesocricetus auratus) to the Wistar rat in experimental studies on plasma lipoprotein metabolism? Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 1994, 107, 347−355. (46) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.; Carlquist, M. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 1997, 389, 753−758. (47) Tan, Y.; Hartwig, J. F. Palladium-Catalyzed Amination of Aromatic C-H Bonds with Oxime Esters. J. Am. Chem. Soc. 2010, 132, 3676−3677. (48) Bell, M. G.; Gernert, D. L.; Grese, T. A.; Belvo, M. D.; Borromeo, P. S.; Kelley, S. A.; Kennedy, J. H.; Kolis, S. P.; Lander, P. A.; Richey, R.; Sharp, V. S.; Stephenson, G. A.; Williams, J. D.; Yu, H.; Zimmerman, K. M.; Steinberg, M. I.; Jadhav, P. K. (S)-N-{3-[1Cyclopropyl-1-(2,4-difluoro-phenyl)-ethyl]-1H-indol-7-yl}-methanesulfonamide: A Potent, Nonsteroidal, Functional Antagonist of the Mineralocorticoid Receptor. J. Med. Chem. 2007, 50, 6443−6445. (49) Bravo, E.; Botham, K. M.; Mindham, M. A.; Mayes, P. A.; Marinelli, T.; Cantafora, A. Evaluation in vivo of the differential uptake and processing of high-density lipoprotein unesterified cholesterol and cholesteryl ester in the rat. Biochim. Biophys. Acta, Lipids Lipid Metab. 1994, 1215, 93−102. (50) Kieft, K. A.; Bocan, T. M. A.; Krause, B. R. Rapid on-line determination of cholesterol distribution among plasma lipoproteins after high-performance gel filtration chromatography. J. Lipid Res. 1991, 32, 859−866.

(51) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235−242. (52) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658−674. (53) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132.

L


Indole Glucocorticoid Receptor Antagonists Active in a Model of

Recommend Documents