Discovery of a Novel, Orally Efficacious Liver X Receptor (LXR) Î²

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Discovery of a novel, orally efficacious Liver X Receptor (LXR) # agonist Ya-Jun Zheng, Linghang Zhuang, Kristi Yi Fan, Colin M. Tice, Wei Zhao, Chengguo Dong, Stephen D Lotesta, Katerina Leftheris, Peter R Lindblom, Zhijie Liu, Jun Shimada, Paul B Noto, Shi Meng, Andrew Hardy, Lamont Howard, Paula Krosky, Joan Guo, Kerri Lipinski, Geeta Kandpal, Yuri Bukhtiyarov, Yi Zhao, Deepak Lala, Rebecca Van Orden, Jing Zhou, Guozhou Chen, Zhongren Wu, Brian M McKeever, Gerard M McGeehan, Richard E Gregg, David A. Claremon, and Suresh B Singh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b02029 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 20, 2016

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Journal of Medicinal Chemistry

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Discovery of a novel, orally efficacious Liver X Receptor (LXR) β agonist

Yajun Zheng, Linghang Zhuang, Kristi Yi Fan, Colin M. Tice, Wei Zhao, Chengguo Dong, Stephen D. Lotesta, Katerina Leftheris, Peter R. Lindblom, Zhijie Liu, Jun Shimada, Paul B. Noto, Shi Meng, Andrew Hardy, Lamont Howard, Paula Krosky, Joan Guo, Kerri Lipinski, Geeta Kandpal, Yuri Bukhtiyarov, Yi Zhao, Deepak Lala, Rebecca Van Orden, Jing Zhou, Guozhou Chen, Zhongren Wu, Brian M. McKeever, Gerard M. McGeehan, Richard E. Gregg, David A. Claremon, Suresh B. Singh*

Vitae Pharmaceuticals, Inc., 502 W. Office Center Drive, Fort Washington, PA 19034, USA

ACS Paragon Plus Environment

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Abstract This paper describes the application of Contour® to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) agonist (17). Contour® technology is a structurebased drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRβ and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour® was used to design agonists containing a piperazine core. Compound 17 binds to LXRβ with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

Introduction The LXRs play a pivotal role in lipid metabolism and have been investigated as targets for the treatment of atherosclerosis, inflammation, Alzheimer’s disease, dermatological conditions, and cancer in humans.1, 2 Two isoforms of LXR, LXRα and LXRβ, exist. LXRα is expressed in intestine, liver, adipose, and macrophages, while LXRβ is ubiquitously expressed.3, 4

LXRs function as heterodimers in complex with retinoid X receptors (RXRs).5 LXRs consist of a ligand independent N-terminal domain, a zinc finger containing DNA binding domain, a connecting hinge region, a ligand binding domain, and a C-terminal activation domain. In their



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resting state, with the ligand binding pocket unoccupied, LXR heterodimers are hypothesized to be found in the nucleus bound to the promoter regions of target genes and corepressor proteins.6-8 The binding of an agonist to the LXR ligand binding domain releases corepressor proteins, and allows the activation domain on helix 12 to adopt a conformation in which a coactivator protein can bind and allow gene transcription to occur.6, 9, 10

A number of LXR agonist chemotypes have been discovered over the past two decades.11 Oxysterols such as 1 (Figure 1) are believed to be endogenous LXR ligands.12 Synthetic LXR agonists 2 (T0901317),13 3 (GW3965),14 and 4 (WAY-252263)15 have played prominent roles in the validation of LXRs as therapeutic targets.11, 15-17 Indazole 4 is the only LXR agonist to have entered clinical trials.18 Methyl phenyl sulfones, exemplified by 5,19 constitute a large group of LXR agonists that has been explored by several research groups.20-22

Contour® technology is a structure-based drug design platform that comprises a context perceptive growth algorithm and a contact sensitive scoring function.23, 24 A flexible and programmable growth engine creates drug-like molecules25, 26 by assembling fragments in the context of protein binding pockets. The context sensitive algorithm is designed to generate novel molecules that naturally complement hydrophilic and hydrophobic features of the protein binding site through two dynamic growth features: dynamic vector selection (DVS) and dynamic fragment selection (DFS). These two features in concert generate molecules that best complement the shape and the features of protein binding sites.


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Our objective was to discover an LXRβ-selective, agonist with drug-like characteristics. It has been hypothesized that such a compound would induce reverse cholesterol transport without elevating triglyceride levels in plasma and liver.27 A publicly available X-ray structure of LXRβ complexed with a synthetic agonist was used as the starting point for the application of Contour to design novel, potent, orally bioavailable, and efficacious agonists. These efforts led to the identification of a drug-like lead compound with acceptable potency, selectivity, and in vivo efficacy for the treatment of atopic dermatitis.28

Results and discussion Design of compound 17 At the initiation of design, eleven crystal structures of LXRα and LXRβ were publicly available. Apart from the crystal structures of LXR in complex with oxysterols (1), complexes of LXRβ with 2 and 329 were also known; however, no X-ray structures with ligands from the methyl phenyl sulfone class were available at that time.

We chose to use the crystal structure of LXRβ complexed with 3, a LXRβ selective synthetic non-steroid agonist (PDB code 1PQ6), since it had the largest binding pocket of all the available complexes.29 The coordinates of the LXRβ protein from 1PQ6, without the ligand, were imported into the Contour system. At that time the most potent compound in LXRβ binding and cell assays that we were aware of was ligand 5 from a Roche patent application.19 Modeling was initiated by docking compound 5 into the ligand binding site from 1PQ6 using the QXP/Flo+ program.30, 31 In one of the high scoring docked poses (Figure 2) one of the sulfone oxygens of 5 hydrogen bonded to the backbone NH of Leu330, similar to the hydrogen bond present for the



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carboxylate group of 3 in the crystal structure. This orientation allowed us to rationalize the SAR of this class of compounds presented in Roche’s patent.19 In addition, the hydroxyl group forms a hydrogen bond with the side chain of the carboxyl group of Glu281. The phenyl ring of 5 has a stacked orientation with Phe329 (Figure 2). The proposed hydrogen bond between the phenyl sulfone and the backbone NH of Leu-330 was later confirmed by a crystal structure (3KFC).32 To generate structurally novel compounds, the bulk of ligand 5 was removed graphically. The 2-(methylsulfonyl)benzyl alcohol fragment (6) (Figure 1) resulting from 5 was used as a starting point to grow novel molecules with Contour in the context of the binding site.

Using the process described in the experimental section, Contour® generated over 2,000 structurally distinct molecules. The top scoring 200 structures were selected for graphical examination with the synthetic chemists. Of all structures viewed graphically, compound 7 was selected due the presence of binding elements similar to those in compound 5 (Figure 3), and its structural novelty compared to the other LXR agonists published in the literature. To design a synthetically more accessible molecule, compound 7 was modified graphically. In particular, the saturated ring in the tetrahydroquinoline moiety of 7 was broken and tied back to the three carbon flexible linker to generate a piperazine ring as in compound 8 (Figures 4 and 5). The weakly basic piperazine ring in 8 was introduced in an effort to impart water solubility. Compound 9 lacking the hydroxymethyl group and the methyl group on the piperazine ring, was accessible in fewer synthetic steps than 8, and was synthesized as a racemic mixture to test the Contour generated hypothesis. Fortuitously, in agreement with the model, compound 9 exhibited an LXRβ Ki value of 1275 nM (Table 1). To provide support for the binding model of compound 7, we prepared 10 which lacks the methyl sulfone moiety. In accord with the model,


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compound 10 had no detectable cellular activity underscoring the importance of methyl sulfone to the activity.

In the binding model of 9 to LXRβ, the phenyl group at the 2-position of the piperazine ring occupied an equatorial configuration and the (S) isomer was predicted to be more potent. The enantiomers of 9 were separated by chromatography on a chiral column to give 9a and 9b. One isomer, 9a, with an LXRβ Ki value of 460 nM, was shown to be at least 5x more potent than its enantiomer 9b and thus have at least 5x binding selectivity for LXRβ over LXRα. In addition, 9a was 9x more potent than 9b in the cellular assay (Table 1). Both the isomers are agonists of LXRβ. Isomer 9a was assumed to be the (S) isomer based on modeling, however the absolute configuration was not verified with an experimental structure determination.

Graphical visualization of compound 9 suggested that the left hand pyridylmethyl group could be replaced with a directly attached aryl or heteroaryl group e.g. pyrimidinyl piperazine 11. The pyrimidine ring in 11 was modeled in a coplanar conformation with the plane of the piperazine ring (small molecule X-rays in Cambridge Structural Database (CSD) show a ±30o variation around coplanarity), while, in contrast to the model of 9 discussed above, the phenyl substituent on the piperazine ring of 11 was modeled in an axial configuration, consistent with the X-ray structure of a small molecule in the CSD (CSD code POGPEI).33, 34 Based on this model, the (R) isomer was predicted to be more potent than the (S) isomer of compound 11. A racemic mixture of pyrimidinyl piperazine 11 was synthesized, and the two enantiomers 11a and 11b were separated chromatographically. Again, one enantiomer, 11a, was more potent than 11b in the binding assay and was an agonist in the cellular assay. The absolute configuration of 11a was



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not verified with experimental structure determination, but was assumed to be (R) based on the model.

Further graphical examination of the binding model of the (R) isomer of 11, and the shape of the pocket, guided the replacement of the phenyl ring with an isopropyl group. This led to the synthesis of (R) isopropyl piperazine derivative 12 (Table 2) from commercially available monoBoc protected 2-()-isopropyl piperazine. Compound 12 has substantially improved affinity towards LXRβ and agonist activity in the cellular assay. As in the case of 11, our model of 12 bound to LXRβ places the isopropyl group in an axial configuration. The axial configuration for isopropyl group is preferred over the equatorial orientation by 9.1 kcal/mol, based on computation with Jaguar using B3LYP/6-31G** level of theory.35 This is also consistent with Brameld et al.’s observation that the alkyl substituent adjacent to the sp2 nitrogen in the piperidine ring is axial (CSD codes REYPUG, BOAYPI).36

However, compound 12 lacked sufficient metabolic stability in rodent liver microsomes. The 4position of the phenyl ring and 5-position of the pyrimidine ring were hypothesized to be likely sites of oxidative metabolism based on our empirical knowledge. In an effort to improve metabolic stability, these positions were substituted with functional groups empirically known to mitigate oxidative metabolism. Introduction of a fluorine atom at the 4-position of the phenyl ring (13) failed to improve stability in rat and liver microsomes. When a chlorine atom was appended to the pyrimidine ring of 13 to give 14, an improved stability in rat liver microsomes was observed. Compound 14 (cLog D = 5.3) showed full agonist activity with a 3x separation between LXRα and LXRβ.


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The hydroxyl methyl group, as initially modeled in compound 8, was reintroduced at this stage of lead optimization. Introduction of hydroxyl containing substituent at the site of oxidation was anticipated to improve the physical properties as well as reduce the metabolic liability of the compound. Addition of hydroxymethyl (15, clogD = 4.0) and 2-hydroxy-2-propyl (16, clogD = 4.6) substituents onto the pyrimidine ring restored >10x isoform selectivity, albeit with diminished potency in the cell based assay. In addition, tertiary alcohol 16 exhibited substantially improved metabolic stability in human and rodent liver microsomes. Unfortunately, the tertiary alcohol functionality of 16 proved to be acid labile, readily eliminating water to afford a propenyl group. This led us to focus on modifications to primary alcohol 15; replacement of the fluorine substituent with a hydroxymethyl group provided diol 17 (clog D = 3.3). Compound 18, (S) isomer of 17, was shown to be much less potent than 17 (Table 2), confirming that our modeling had predicted the disparity in activity for the two enantiomers. The preferred (R) stereochemistry of the isopropyl group was further verified experimentally by the 2.61Å crystal structure of 17 bound to LXRβ.

Compound 17 combined a number of desirable properties: it binds potently to LXRβ with a Ki value of 3 nM and is 27x selective over LXRα (Table 3). It is an agonist of LXRβ, has moderate (HLM, MLM) to good metabolic stability (RLM). It also exhibits excellent selectivity against other nuclear hormone receptors (>3000X). It weakly inhibits rCYP2D6 and rCYP3A4 (IC50 values > 25 μM)), but is a more potent inhibitor of rCYP2C9 (IC50 = 610 nM). Compound 17 showed dose-dependent upregulation of two important LXR target genes - ABCA1 in THP1 cells and SREBP1c in HepG2 cells (Figure 6).



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The calculated physical properties of 17 (MW = 489, clogD = 3.2, tPSA = 115 Å2, 2 aromatic rings) compare favorably with those of many LXR modulators reported in the literature2 and with those of compound 5 (MW = 596, clogD = 5.5, tPSA = 137 Å2) used as the starting point for design.

X-ray structure of 17 The heterodimer of the ligand-binding domains (LBD) of human LXRβ and human RXRβ was co-crystallized with compound 17 and the structure solved to a resolution of 2.61 Å. Both receptors assume the canonical “activated” conformation with helix 12 tucked against the body of the LBD and the tethered SRC2-2 co-activator peptide sitting above helix12 in the grove created by helices 3, 5 and 12.37 While the ligand-binding site of the RXRβ-LBD is empty, but a strong positive density is observed in the Fo-Fc map for compound 17 in the LXRβ-LBD binding site to allow for the fitting of the ligand and refinement of the complex crystal structure.

The X-ray structure reveals some disorder with a portion of the ligand 17. The density for the 4(trifluoromethyl)-5-(hydroxymethyl)pyrimidine is somewhat smeared and ambiguous. Initial attempts to model this with a single conformation of compound 17 resulted in the distortion of the ligand’s molecular geometry. Allowing for compound 17 to be modeled as two conformations, where the torsion angle around N1-C5 could be either 0 or 180 degrees, reduced the distortion and did help partly explain the smeared electron density. The two conformations of compound 17 modeled in each LXRβ-LBD monomer has their occupancy set to 0.50 in the structure deposited into the PDB.


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The structure shown in Figure 7 has the pyrimidine rotamer that forms hydrogen bond interaction with His435 in accordance with its agonist activity. The compound also forms hydrogen bonds with Glu281 and Leu330. The agonist response observed for the compound is rationalized based on the hydrogen bond present between the hydroxyl group on the pyrimidine ring and the imidazole ring of His435. This interaction helps orient and stabilize the imidazole side chain, edge-to-face interaction of His435 with indole side chain of Trp457 located on helix 12, which helps maintain the agonist conformation of the ligand-binding domain29, 38, 39

The experimental details for the expression, purification, crystallization and structure solution of the LXRβ-LBD/RXRβ-LBD/17 complex are described in Section S3 of the Supporting Information.

Chemistry Detailed experimental procedures for all compounds tested, except for 17, are provided in the Supporting Information, Section S4. The synthesis and characterization of compound are shown in the experimental section. In general, analog synthesis began with commercially available monoprotected 2-phenyl and 2-isopropyl piperazines, as illustrated for key compound 17 (Scheme 1). Palladium catalyzed reaction of monoBoc protected piperazine 19 with bromobenzene 20 provided 21. Removal of the Boc group was followed by SNAr reaction with ethyl 2-chloro-4-(trifluoromethyl)pyrimidine-5-carboxylate to give 23. Finally, reduction of the two esters with DiBAl afforded the diol 17.



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Conclusions We present the rational design of 17, a novel LXRβ partial agonist starting from a phenylsulfone fragment and growing molecules using Contour® structure-based design technology. This effort led to a potent, selective, orally bioavailable, compound that exhibits efficacy in vitro and in vivo.28 The X-ray structure of 17 shows that the compound binds to the ligand binding domain of LXRβ in the predicted pose. The further optimization of 17, led to the candidate currently in human clinical trials for treating atopic dermatitis.

Experimental Section Contour growth calculations The 4- position of the fragment 6 was marked for initiation of growth. Then Contour® growth was initiated by assembling fragments from the fragment library. The fragment library consists of monocyclic rings (5 and 6 membered rings), fused bicyclic rings, flexible linkers, functional groups, and atoms.23 Contour carries out the following steps for each molecule that gets grown: (i) in the first step, Contour was instructed to start from the 4-position of the fragment 6. The binding site from this point of attachment was mapped out using the dynamic fragment selection (DFS) protocol. The properties of the binding site features mapped out by DFS guided the selection of fragments. A subset of fragments from the fragment library (1% of 10,000 fragments) that best complement the shape and features of the binding site was selected to attach to the 4-position of the starting fragment. (ii) in the second step, conformational sampling around the bond between fragment 6 and the attached fragment was carried out. Each rotamer was optimized with a numerical gradient based method to minimize steric repulsion and form hydrogen bond interactions in the binding site. The fragments with better than 85% steric


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tolerance and acceptable hydrogen bonds, where possible, were accepted and saved for the next step.;23 (iii) in the third step, Contour used a dynamic vector selection (DVS) feature to identify the best attachment sites (atoms containing hydrogen atoms) on the accepted fragments from the second step. After the identification of attachment site(s), the iterative process of identifying fragments, attaching them, and optimizing them continued with the procedure described above. This process of growth was used to generate molecules containing fragments ranging from 3-6 fragments, with a molecular weight limit of 500, and a minimum Contour score of 4 (~10 µM). More detailed description of the Contour growth process is provided in papers describing the design of inhibitors of renin and 11β HSD-1.23, 40 The technical details of the Contour growth algorithm is provided in the Supporting Information, Section S1.

Docking procedure Molecular docking was performed using the QXP program, a part of the Flo+ software.30,

31

The MCDOCK+ procedure in QXP was used to dock ligand 5. The ligand was placed in the binding pocket with an initial 100 Monte Carlo steps. Then the ligand was subjected to 10,000 cycles of Monte Carlo to generate multiple orientations and conformations followed by energy minimization with AMBER 3.0 force field. Each docked orientation resulting from this process was scored to yield an ensemble of 25 top scoring poses.

QM optimization The axial and equatorial configurations of the isopropyl group in 12 were built using Maestro modeling software and energy minimized in the gas phase with Jaguar using the B3LYP/631G** level of theory.35



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Chemistry Starting reagents were purchased from commercial suppliers and were used without further purification unless otherwise specified. Normal phase column chromatography was carried out in the indicated solvent system (in the percentage of volume) using pre-packed silica gel cartridges for use on the Isco CombiFlash® Companion®. Analytical thin layer chromatography (TLC) visualization was performed using 254 nm wavelength ultraviolet light. Preparative HPLC purifications (reverse-phase) were performed using a Gilson 215 Liquid Handler with Unipoint software, typically with a SunFireTM Prep C18 OBDTM 5µm 19 × 50 mm column. A 10-minute run (20 mL/min, 10% MeCN/H2O, 0.1%CF3CO2H to 90% MeCN/H2O, 0.1% CF3CO2H) with UV detection at 254 nm was typically used. SFC separations were carried out on a Berger MultiGramTM SFC, Mettler Toledo Co, Ltd using an OJ 250 mm*30 mm, 20 um column. The mobile phases were A: supercritical CO2, B: EtOH or MeOH, A:B =60:40 at 80 ml/min, the column temperature 38 oC, the nozzle pressure 100 Bar, the nozzle temperature 60 oC, the evaporator temperature 20 oC and the trimmer temperature25 oC. UV detection was at 220 nm. Nuclear Magnetic Resonance spectra were recorded on a Varian Mercury 400 spectrometer operating at 399.932 MHz for 1H NMR, at 376.312 MHz for 19F NMR and at 100.623 MHz for 13

C NMR. Spectra were taken in the indicated solvent at ambient temperature, and the chemical

shifts are reported in parts per million (ppm (δ)) relative to the lock of the solvent used. Resonance patterns are recorded with the following notations: br (broad), s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). High resolution mass spectra (HRMS) were obtained on an Agilent 6210 Series time of flight LC/MS using electrospray ionization in positive mode.


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The purity of final compounds was determined based on LC-MS data acquired on a Water Micromass SQD system UV detector set to at 220 nm. Samples were injected (2 µL of 10 µg/mL in MeCN) onto an ACQUITY CSH C18 1.7 µM, 2.1 x 50mm column maintained at 40 °C. A linear gradient from 5% to 95% B (MeCN + 0.01% CF3CO2H) in 1.9 min was followed by pumping 95% B for another 0.3 minutes with A being H2O + 0.01% CF3CO2H. The flow rate was 0.65 mL/min. The mass spectral (MS) data were acquired in positive ion mode using an electrospray ionization (ESI) source. All final compounds were >95% pure by this method.

(R)-(2-(4-(4-(Hydroxymethyl)-3-(methylsulfonyl)phenyl)-2-isopropylpiperazin-1-yl)-4(trifluoromethyl)pyrimidin-5-yl)methanol (17)

To a solution of 23 (1.39 g, 2.5 mmol) in dry toluene (45 mL) at 0 °C was added diisobutylaluminium hydride (1.0 M in toluene, 15 mL, 15 mmol) slowly. After addition, the mixture was stirred at 0 oC for 2 h and quenched with aq NH4Cl solution (5 mL). The reaction mixture was poured into a vigorously stirred solution of potassium sodium tartrate (1.0 M, 40 mL) and stirred vigorously until two phases clearly separated. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 x 30 mL). The organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (EtOAc/hexanes = 60/40) afford 17 (960 mg, 79%). 1

H NMR (CD3OD, 400 MHz): δ 8.61 (s, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.27 (dd, J1 = 2.4 Hz J2 = 8.4 Hz, 1H), 4.91 (s, 2H), 4.86-4.84 (m, 1H), 4.70 (d, J = 10.4 Hz, 1H), 4.61 (s, 2H), 3.97 (d, J = 12.4 Hz, 1H), 3.75 (d, J = 10.4 Hz, 1H), 3.40-3.33 (m, 1H), 3.22 (s, 3H), 2.94-2.83 (m, 2H), 2.50-2.41 (m, 1H), 1.13 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H).



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LC-MS Method A tR = 1.49 min, m/z = 489. HRMS calcd for C21H27F3N4O4S: 488.1705107; found: 489.1774.

Tert-Butyl (R)-2-isopropyl-4-(4-(methoxycarbonyl)-3-(methylsulfonyl)phenyl)piperazine-1carboxylate (21)

To a mixture of tert-butyl (R)-2-isopropylpiperazine-1-carboxylate (19, 740 mg, 3.0 mmol), methyl 4-bromo-2-(methylsulfonyl)benzoate (20, 1.05 g, 3.6 mmol), Xphos (160, 0.33 mmol) and cesium carbonate (2.93 g, 9.0 mmol) in toluene (7 mL) was added Pd2(dba)3 (140 mg, 0.15 mmol). The mixture was purged with nitrogen and the tube was sealed. It was heated in an oil bath at 100°C for 10 h. After cooling to rt, the reaction mixture was filtered and concentrated. The residue was purified by silica gel chromatography (EtOAc/hexanes = 40/60) to afford 21 (1.17 g, 86% yield). LC-MS Method A tR = 1.66, m/z = 463 [M + 23]+.

Methyl (R)-4-(3-isopropylpiperazin-1-yl)-2-(methylsulfonyl)benzoate (22)

To a solution of 8 (1.17 g, 2.65 mmol) in dichloromethane (10 mL) at 0 °C was added TFA (2 mL). The reaction mixture was stirred at rt for 1 h. After completion of the reaction, the reaction mixture was neutralized with satd aq NaHCO3 and extracted with EtOAc (3 x 25 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to afford crude piperazine 22, which was used directly in the next step without further purification. LC-MS Method A tR = 0.65 min, m/z = 341.2 [M + H]+.

Ethyl (R)-2-(2-isopropyl-4-(4-(methoxycarbonyl)-3-(methylsulfonyl)phenyl)piperazin-1-yl)4-(trifluoromethyl)pyrimidine-5-carboxylate (23)


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To a solution of crude 22 (2.65 mmol) in DMSO (5.5 mL) were added ethyl 2-chloro-4(trifluoromethyl)pyrimidine-5-carboxylate (1.35 g, 5.3 mmol) and i-Pr2NEt (1.4 mL, 7.95 mmol). The mixture was allowed to stir at 60 °C for 2 h. After the reaction was complete, the mixture was diluted with H2O (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/hexanes = 30/70) to give 23 (1.39 g, 94%). LC-MS Method A tR = 1.88 min, m/z = 559.3 [M + H]+.

Acknowledgment We thank Robert Fay for managing Vitae’s chemical and biological data system.

X-ray crystal structure The coordinates of the LXRβ complex containing the partial agonist 17 have been deposited into the Protein data bank (PDB code 5I4V). Authors will release the atomic coordinates and experimental data upon publication.

Supporting Information. A descripton of Contour ®, bioassay protocols, crystallographic data for 17, synthetic procedures and NMR, MS, and HPLC data on new compounds.

Abbreviations: ABCA1, ATP binding cassette A1; CSD, Cambridge Structural Database; DFS, dynamic fragment selection; DVS, dynamic vector selection; Gal4, DNA-binding yeast transcription factor; HEK, human embryonic kidney; HepG2, a hepatocellular carcinoma cell line;LBD,



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ligand binding domain; MR, mineralocorticoid receptor; PXR, pregnane X receptor; QXP, Flo+; ROR, retinoic acid receptor related orphan receptor; RXR, retinoid X receptor; SREBP1c, sterol regulatory element-binding protein-1c; THP-1, human monocytic cell line

Corresponding author: Suresh B. Singh 502 W. Office Center Drive Fort Washington, PA 19034 Phone: 215-461-2048 email: [email protected]

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Selected LXR agonists and fragment 6. The receptor binding affinity from the binding assay and the EC50 value from cell-based Gal4 assay for each compound is shown for LXRα and LXRβ receptors.12−15, 19


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


W457

E281

H435 M312

F268 F329

L330

F340

Figure 2. Docked model of compound 5 in LXRβ. Key residues in the vicinity of the ligand binding site are shown. The other residues within contact distance have not been displayed for clarity. Compound 5 was docked into the ligand binding site using the QXP/Flo+ program.30, 31 In one of the high scoring docked poses, one of the sulfone oxygens of 5 hydrogen bonded to the backbone NH of Leu330, similar to the hydrogen bond present for the carboxylate group of 3 in the crystal structure. This orientation allowed us to rationalize the SAR of this class of compounds presented in the patent.19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

W457 E281 M31 H435

L330 F268

F329 F340

Figure 3a. Contour® constructed model of compound 7 in LXRβ.

Compound 5 Compound 7

Figure 3b. Comparison of binding models of 5 and 7. The receptor is not displayed for clarity.


Page 26 of 34

Page 27 of 34

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Figure 4. Design of piperazine-based LXRβ agonists. Details of the growth protocol are given in the experimental section.



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Page 28 of 34

Compound 7 Compound 8

Figure 5. Binding models of compounds 7 and 8 in LXRβ. The receptor is not displayed for clarity. To design a synthetically more accessible molecule, Compound 7 was modified graphically. In particular, the saturated ring in the tetrahydroquinoline moiety of 7 was broken and tied back to the three carbon flexible linker to generate a piperazine ring as in compound 8


Page 29 of 34

Thp1 EC50 = 4.5nM Efficacy = 101%

10 ABCA1

Relative expression of

12

8 6 3.72

4 2.43

4.30

5.45

5.38

500

1uM 2

4.47

4.82

500

1uM 2

4.87

2.86

1.62

2

1.00

0 0

1

2

5 16 50 Compound 17, nM

158

HepG2 12 10 SREBP1c

Relative expression of

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EC50 = 6.3nM Efficacy = 91%

8 6 3.50

3.78

2 6 19 56 Compound 17, nM

167

4 2

2.67 1.00

1.32

0

1

3.05

1.87

0

Figure 6. LXR target gene activation by 17 vs 2 in THP1 and HepG2 cells. The units for compound 17 are in nM and for compound 2 are in µM



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

W457 H435

Page 30 of 34

E281 M312

L330 F268 F329 F340 Figure 7. X-ray structure of compound 17 bound to LXRβ. The X-ray structure reveals some disorder with a portion of the ligand 17. The density for the 4-(trifluoromethyl)-5(hydroxymethyl)pyrimidine is somewhat smeared and ambiguous. Allowing for compound 17 to be modeled as two conformations, where the torsion angle around N1-C5 could be either 0 or 180 degrees, reduced the distortion and did help partly explain the smeared electron density. One of the two rotamers of 17 consistent with its agonist response is shown here.


Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a Pd2(dba)3, X-phos, Cs2CO3, PhMe, 100 °C; b CF3CO2H, CH2Cl2, rt; c ethyl 2-chloro-4(trifluoromethyl)pyrimidine-5-carboxylate, i-Pr2NEt, DMSO, rt; d DiBAl, PhMe, 0 °C Scheme 1. Synthesis of LXR modulator 17.



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Page 32 of 34

Table 1. Assay Results for Phenyl Piperazinesa LXRα

LXRα

LXRβ

LXRβ

LXRβ

α/β

Ki

EC50

Ki

EC50

Efff

selectivity

(nM)

(nM)

(nM)

(nM)

(%)

4c

179

6600

53

24

3670

73

7

5d

20

205

51

1.3

30

48

20

9

>3,300

5375

17

1,275

2135

28

>2

9ae

2,200

2,500

19

460

990

28

5

9be

>3,300

>20,000

>2,500

9,200

12

-

10

g

>20,000

g

>20,000

11ae

1100

3825

35

94

1289

48

12

11be

1552

>10,000

-

211

>10,000

-

7

Cpd. No.

b

LXRα Eff (%)

-

a Binding potencies of LXR ligands were assessed in a competition assay, where compounds were incubated with LXR-α and LXR-β LBDs in the presence of radioactive probe [3H] 2. EC50s were measured after twenty hours post transfection. Cells containing expression vectors pG5-luc (Promega) and pGal4-LXRα LBD or pGal4-LXRβ LBD were treated with compounds for 16 hrs before measuring luciferase activity for the agonist activity. Assay results are the average of at least two independent determinations. b The structures of 4 and 5 are shown in Figure 1. The structures of 9-11 are shown in Figure 2. c Literature data.15 d Literature LXRα IC50 = 34 nM, LXRβ IC50 = 0.6 nM. e Isomers were separated by chromatography on a chiral column. f Efficacy is expressed as a percentage of the receptor activation by compound 2. g Not tested.


Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Cpd. 16 17 18No. 19 20 21 22 23 24 2512 26 27 13 28 29 3014 31 3215 33 34 3516 36 3717 38 3918 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Assay Results for Isopropyl Piperazinesa

*

X

Y

LXRα

LXRβ

α/β

H/R/MLMb

selectivity Ki

EC50

Eff

Ki

EC50

Eff

(nM)

(nM)

(%)

(nM)

(nM)

(%)

t1/2 (min)

R

H

H

43

257

85

4

53

68

11

-/11/8

R

F

H

25

254

92

1

53

68

25

-/5/11

R

F

Cl

3

92

113

1

21

114

3

19/42/-

R

F

CH2OH

146

590

57

9

68

40

16

22/36/38

R

F

CMe2OH

157

763

37

8

80

24

20

56/51/72

R

CH2OH

CH2OH

81

244

60

3

21

56

27

43/70/40

S

CH2OH

CH2OH

1072

1734

95

160

503

73

7

-/25/-

a Assay results are the average of at least two independent determinations. b H/R/MLM = human/rat/mouse liver microsomes.



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Page 34 of 34

Table 3. In vitro Profile of compound 17

LXRα

LXRβ

α/β

81

3

27

Binding Ki (nM)

Gal4 agonist

EC50 (nM)

%Eff

244

60

EC50 (nM)

%Eff

21

9

56

Cellular Efficacy Assays

EC50 (nM)

%Eff

ABCA1

4

101

SREBP1c

6

91

MR

RXR

ROR-α

PXR

>10,000

>10,000

>10,000

>10,000

Mouse

Rat

Cyno

Human

40

70

10

43

Nuclear Hormone Receptors IC50 (nM)

Microsome t1/2 (min.)

CYPs

2C9

2D6

3A4

rCYPs IC50 (nM)

610

>30,000

29,430

HLM CYPs IC50 (nM)

2200

-

6600


Page 35 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of content graphic

H435

W457 M312

F268

F271

Q281

L330 F340

X-ray structure of an LXRβ agonist


Discovery of a Novel, Orally Efficacious Liver X Receptor (LXR) Î²

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