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Identification and In Vivo Evaluation of Liver X Receptor # (LXR#) Selective Agonists for the Potential Treatment of Alzheimer’s Disease Shawn J. Stachel, Celina Zerbinatti, Michael T Rudd, Mali Cosden, Sokreine Suon, Kausik K. Nanda, Keith Wessner, Jillian M Dimuzio, Jill Maxwell, Zhenhua Wu, Jason M. Uslaner, Maria S. Michener, Peter Szczerba, Edward Brnardic, Vanessa Rada, Yuntae Kim, Robert S. Meissner, W Peter Wuelfing, Yang Yuan, Jeanine Ballard, Marie Holahan, Daniel J. Klein, Jun Lu, Xavier Fradera, Gopal Parthasarathy, Victor N. Uebele, Zhongguo Chen, Yingjie Li, Jian Li, Andrew J. Cooke, Jonathan Bennett, Mark Bilodeau, and John Renger J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00176 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Journal of Medicinal Chemistry

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Identification and In Vivo Evaluation of Liver X Receptor β (LXRβ β) Selective Agonists for the Potential Treatment of Alzheimer’s Disease Shawn J. Stachel,*† Celina Zerbinatti,‡ Michael T. Rudd, † Mali Cosden,‡ Sokreine Suon,‡ Kausik K. Nanda,† Keith Wessner,‡ Jillian DiMuzio,‡ Jill Maxwell,‡ Zhenhua Wu,‡ Jason M. Uslaner,# Maria S. Michener,# Peter Szczerba,# Edward Brnardic,† Vanessa Rada,† Yuntae Kim,† Robert Meissner,† Peter Wuelfing,§ Yang Yuan,¶ Jeanine Ballard,¶ Marie Holahan,# Daniel J. Klein,^ Jun Lu,^ Xavier Fradera,^ Gopal Parthasarathy,^ Victor N. Uebele,‡ Zhongguo Chen,° Yingjie Li,° Jian Li,° Andrew J. Cooke,† D. Jonathan Bennett,† Mark Bilodeau,† and John Renger‡

†

Department of Medicinal Chemistry; ‡Department of Neuroscience, ¶Department of Pharmacology,

^

Department of Structural Chemistry, #Department of Imaging , §Department of Pharmaceutical

Sciences, Merck Research Laboratories, P. O. Box 4, West Point, Pennsylvania 19486; °

WuXi AppTec Co., Ltd, Shanghai 200131, PR China

Received February 2, 2016

ABSTRACT Herein we describe the development of a functionally selective LXRβ agonist series optimized for Emax selectivity, solubility and physical properties to enable efficacy and safety studies in vivo. Compound 9 showed central pharmacodynamic effects in rodent models, evidenced by statistically significant increases in apoE and ABCA1 levels in the brain, along with a greatly improved peripheral lipid safety profile when compared to full dual agonists. These findings were replicated by subchronic

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dosing studies in non-human primates, where CSF levels of apoE and Aβ peptides were increased concomitantly with an improved peripheral lipid profile relative to that of non-selective compounds. These results suggest that optimization of LXR agonists for Emax selectivity may have the potential to circumvent the adverse lipid-related effects of hepatic LXR activity. Introduction. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory and cognitive function. The presence of amyloid-β (Aβ) peptide deposits in the hippocampal and cortical regions of the brain is a major hallmark of AD pathology. Aβ peptides are released from the transmembrane amyloid precursor protein (APP) following sequential cleavage by βand γ-secretases, and oligomers of these peptides have been shown to cause toxicity to both neurons and glia in vitro and in vivo.1 The major genetic factor associated with increased risk for late-onset AD (LOAD) is the ε4 allele of the APOE gene, which encodes for apolipoprotein E (apoE), the main lipid transporter protein in the CNS.2 Three human apoE isoforms arise from polymorphisms within the APOE gene, named E2, E3 and E4. While only 15% of the normal population is APOE4 carriers, up to 70% of LOAD patients have one or two copies of the APOE4 gene. The mechanisms by which apoE4 increases the risk and accelerates the onset of AD are not clear. However, it is known from human genetic data that both healthy and LOAD individuals carrying the apoE4 isoform have increased brain amyloid burden,3,4 which raised the hypothesis of a role for apoE in the basal clearance of amyloid from the brain. Interestingly, APP transgenic (APP-Tg) mice overexpressing (human) hapoE4 also have increased brain amyloid load compared to hapoE3 mice, further corroborating the human findings. Additionally, previous studies have demonstrated that hapoE4 mice have reduced total levels of apoE in the CNS5-7 and show decreased association of astrocyte-secreted apoE with lipids when compared to hapoE3 mice.8 These differences in the ability of apoE isoforms to transport lipids may underlie the reduced association of apoE4 with Aβ, which might be crucial for its effective clearance from and within the CNS.9 Also in support of this hypothesis are recent data showing that both half-life and



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steady-state levels of soluble Aβ are significantly increased in APP-Tg hapoE4 mice compared to APPTg hapoE3 and hapoE2 mice,10 confirming a pivotal clearance role for apoE, as well as the compromised ability of the E4 isoform to perform this function.

We have previously proposed that apoE protein associates with and transports lipid particles that act as carriers for amyloid peptide transport both within and out of the CNS.9 The majority of lipids associated with brain apoE are contributed by the ATP-binding cassette protein A1 (ABCA1).11 Liver X receptors, LXRα and LXRβ, are nuclear hormone receptors that control the expression of genes involved in cholesterol and lipid metabolism. Of particular relevance to the CNS, LXRs β and α are known to regulate the expression of both apoE and ABCA1 in astrocytes12 and therefore control the overall expression and lipidation levels of apoE in the healthy brain. Recent studies have demonstrated that agonism of brain LXR induces up-regulation of apoE and ABCA1, both of which have known genetic deficiencies associated with increased brain amyloid burden in mouse models of Alzheimer's disease. Up-regulation of these genes has also been shown to promote amyloid–β peptide (Aβ) transport and clearance, and to lower brain concentrations of amyloid. As such, LXR agonism has emerged as a potential therapeutic approach for Alzheimer's disease.

In light of these findings, we envisaged a strategy of increasing apoE and apoE lipidation in the CNS with a LXRβ-selective agonist to improve Aβ clearance mechanisms and decrease amyloid burden in AD patients. This disease prevention/modifying strategy is supported by recent in vivo labeling experiments showing that while Aβ production is mostly unaffected in LOAD patients, Aβ clearance is decreased by 25-30% in comparison to normal individuals.13 In addition to improving the clearance of amyloid, CNS activation of LXR has the potential advantage of targeting other important apoE functions that may be compromised in the brain of AD patients, including cholesterol transport for synaptic plasticity, signal transduction and inflammatory response. Finally, an efficacious brain penetrant LXRβ-


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selective agonist could be combined with either symptomatic or disease-modifying targeted mechanisms currently under development to increase the overall probability of success of AD therapeutics.

The potential therapeutic value of LXR agonists has been considered for cardiovascular disease; however, pharmacological activation of hepatic LXRα has been linked to the marked up-regulation of SREBP1, a master switch for genes involved in triglyceride synthesis. Therefore, the development of synthetic LXR ligands as therapeutic agents has been largely hampered by hepatic steatosis (fatty liver). While previous literature based on dosing LXRα/LXRβ knockout (KO) mice with non-selective agonists suggested that selective hepatic LXRβ agonism could mitigate the undesired lipogenic effects attributed to LXRα,14 the effects resulting from the selective pharmacological activation of LXRβ have not been fully assessed in wild type mice expressing both LXR isoforms in the liver. Further, the level of hepatic triglyceride accumulation upon selective pharmacological activation of LXRβ in more relevant pre-clinical species, such as non-human primates (NHP), has not yet been fully described. Here we report the development of a new agonist displaying Emax selectivity for LXRβ over LXRα, including the CNS and peripheral efficacy and safety profiles from subchronic dosing in both mice and the rhesus monkey.


Journal of Medicinal Chemistry F3C

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O

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HO2C

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Figure 1. Examples of reported LXR agonists.

Agonist Design. A large number of non-selective LXR agonists have been reported in the literature, Figure 1. Compounds 1 (TO-091317) and 2 (GW3965) are two well-studied dual LXR agonist that have been shown to reduce atherosclerotic burden in mice, but lipogenic activity and adverse events limited their development.15,16 In addition to a plethora of dual agonists, several LXRβ selective agonists (such as compound 3 and 4) have recently been reported in the literature.17,18 Compound 3 (LXR-623) in particular was shown to have an improved lipid safety profile in the non-human primate,19 but adverse CNS effects observed in clinic trials halted further development and precluded evaluation of the safety profile in humans.20

To successfully prosecute the target and differentiate from previous iterations, it was paramount that we abide by two main tenets for our target molecule design, those of brain permeability and selectivity. To achieve facilitation of Aβ clearance from and within the CNS as a result of brain LXRβ agonism, it was ACS Paragon Plus Environment

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essential that the compound possessed the appropriate physicochemical properties and lacked Pgp susceptibility.

Secondly, isoform selectivity for LXRβ over LXRα would be required, however

identification of LXR isoform-selective orthosteric agonists has been challenging as the ligand binding domains of LXRα and β are well conserved, with approximately 78% homology.21 By choosing to focus on Emax- rather than potency-based selectivity (Figure 2), we hypothesized that we could maximize the beneficial efficacy of LXRβ agonism while effectively eliminating the negative efficacy of LXRα agonism independent of the pharmacokinetic profile of the agents. With this approach, LXRα agonist activity would be independent of the peripheral exposure thereby reducing the potential for hepatosteatosis.

Figure 2. Methods for achieving isoform selectivity.

As a starting point, we succeeded in identifying compound 6, which displayed Emax selectivity for LXRβ over LXRα, from an uHTS of the MRL compound collection (Figure 3). Compound 6 displayed moderate Emax selectivity in the cofactor recruitment assay (CFR) using the ligand binding domains of LXRβ (EC50 = 0.94 µM, Emax = 106%) and LXRα (EC50 = 1.48 µM, Emax = 50% efficacy). This selectivity profile was also present in a cell-based transactivation assay with endogenous LXRβ (EC50 = 0.77 µM, Emax = 103%) and LXRα (EC50 = 0.28 µM, Emax = 40%), using compound 1 as the full agonist control (LXRβ EC50 = 0.02 µM, Emax = 100%; LXRα EC50 = 0.02 µM, Emax = 100%), however the modest potency and severely limited solubility of the compound precluded pharmacodynamic and safety studies in vivo. SAR in the system revealed that replacement of the t-butyl carbamate with a mandelate group, as depicted by compound 7, resulted in a substantial increase in ACS Paragon Plus Environment


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binding, as reflected in a significant left-shift in EC50, but also resulted in loss of Emax selectivity in both CFR assay, LXRβ (EC50 = 0.008 µM, Emax = 67%); LXRα (EC50 = 0.041 µM, Emax = 62%) and transactivation assay, LXRβ (EC50 = 0.007 µM, Emax = 64%); LXRα (EC50 = 0.045 µM, Emax = 66%), without much improvement in solubility.

In an attempt to improve solubility, the central

cyanophenyl ring was replaced with a bispiperidine system, resulting in compound 8, which exhibited markedly improved solubility properties (pH 2 solubility = 197 µM) compared to compound 7 (pH 2 solubility = 3 µM) while retaining selectivity in the transactivation assay, LXRβ (EC50 = 0.55 µM, Emax = 91%); LXRα (EC50 = 0.50 µM, Emax = 24%). Unfortunately Pgp susceptibility limited CNS penetration (MDCK cells BA:AB = 4.3 rat, 1.8 human), detracting from the utility of compound 8 as an in vivo tool. Finally, replacement of the phenyl trifluoromandalate with an isopropyl trifluoromandelate resulted in compound 9, which retained acceptable solubility properties (pH 2 solubility = 205 µM) but showed significantly reduced Pgp susceptibility (Pgp efflux ratio 0.8 rat; 0.6 human; Papp = 24 x 10-6 cm/s). Compound 9 displayed full agonist activity towards LXRβ, but only partial agonism towards LXRα in both the CFR assay, LXRβ (EC50 = 0.03 µM, Emax = 103%); LXRα (EC50 = 1.9 µM, Emax = 49%) and transactivation assay, LXRβ (EC50 = 0.42 µM, Emax = 104%); LXRα (EC50 = 0.83 µM, Emax = 31%). The selectivity profile of compound 9 was also confirmed in a gene expression assay (supplemental material).

In addition to favorable physical properties for CNS penetration (rat

brain/plasma ratio = 0.62), compound 9 also exhibited a clean ancillary profile with no significant responses < 10 µM in a counterscreen panel consisting of a variety of 36 enzymes and receptors as well as a panel of 18 nuclear hormone receptors. While the in vivo clearance rate in rat was high, (rat Cl = 87 mL/min/kg; t1/2 = 2.9 h, Vdss = 8.8 L/kg, %F = 71, rat PPB = 98.2%) we were able to achieve sufficient and sustained plasma concentrations through subcutaneous dosing for pharmacodynamic studies. Mouse PK was comparable to that of the rat with slightly higher exposures observed with similar dose levels. Clearance was lower in the rhesus (Cl = 18 mL/min/kg, t1/2 = 2.0 h Vdss = 2.7 L/kg, %F = 77%, PPB = 99.3%) and oral dosing was amenable for a multi-day dosing studies (vida infra). ACS Paragon Plus Environment

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Figure 3. Evolution of hit to lead

Chemistry: The synthesis of compound 9 began with the mono-acylation of bispiperidine 10 to produce the differentially protected amine 11 (Scheme 1). Palladium catalyzed amination of intermediate 11 with 4-bromo-2-chloro-N,N’-dimethylbenzamide resulted in the Boc-protected tricyclic compound 13 in excellent yield. Acid mediated deprotection of the Boc group followed by PyBOP mediated coupling with 3-methyl-2-oxobutanoic acid yielded the α-ketoamide 14.

Finally, formation of the α-

trifluoromethyl alcohol through the influence of Rupert’s reagent21 followed by chiral separation of the enantiomers produced the desired target 9.

The absolute stereochemistry of compound 9 was

determined to be the (R)-enantiomer using Vibrational Circular Dichroism (VCD).22 The S-diastereomer was approximately 2-fold less active against LXRβ in the transactivation with similar activity against LXRα (LXRβ EC50 = 0.84 µM, Emax = 117%; LXRα EC50 = 2.0 µM, Emax = 31%), as such the R enantiomer was selected for further study.



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Scheme 1: Synthetic route to compound 9.

Previous work has demonstrated that LXR agonists, including the oxysterol natural ligands, induce conformational rearrangements of helix 12 and the AF2 domain required for receptor activation.23 Two conserved amino acids, His435 and Trp457 (LXRβ numbering), interact within the ligand-binding pocket and have been suggested to function as an activation switch that drives the conformational rearrangement of the AF2 domain. In particular, formation of a hydrogen bond between LXR agonists and the imidazole of His435 orients that latter to make edge-to-face π-electron cloud interactions with the indole of Trp457, and these interactions are associated with the activated state of the receptor. Past studies have also suggested that shorter hydrogen bond distances between the agonist and His435 position the latter’s imidazole toward the more strongly negative π-electron cloud of the phenyl portion of the indole ring rather than the weaker π-cloud of the pyrrole, thereby stabilizing helix 12 and the AF2 domain in the active conformation. In this regard, the imidazole of His435 can act as either a hydrogen


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bond donor, as in the case of the native oxysterols, or as a hydrogen bond acceptor, as observed with compound 1.

To better understand the structural basis for the observed isoform selectivity of 9, we determined cocrystal structures of 9 in complex with the LXRα homodimer and the LXRβ/RXR heterodimer, at 1.7 and 2.6 Å resolution respectively.

Compound 9 binds the two orthosteric pockets of the LXRα

homodimer in a similar overall conformation (Figure 4A). However, close inspection reveals subtle differences for the isopropyl trifluoromandelate group consistent with its weaker electron density and higher crystallographic B-factors (Figure 4A), suggesting that this group retains some conformational flexibility in the LXRα-bound state. The amide carbonyl of 9 is positioned at distances of 2.8 and 3.0 Å from the imidazole of His435 for the two copies of the LXRα dimer. By comparison, the crystal structure of 9 bound to the LXRβ-RXR heterodimer demonstrates that the amide carbonyl of 9 interacts with the His435-Trp457 activation switch via hydrogen bonding distances that are essentially indistinguishable from that observed for LXRα. Therefore, the crystal structures do not implicate differential interactions with the activation switch in the final agonist-bound state as the key mechanism underlying isoform selectivity. Rather, our structures are more consistent with isoform selectivity being achieved via the cumulative effect of small conformational differences in 9 when bound to LXRα and LXRβ (Figure 4B), such as the observed differential flexibility of the isopropyl trifluoromandelate group.



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Figure 4. Compound 9 bound to the active sites of LXRα and LXRβ (PDB codes: 5HJS and 5HJP respectively)

CNS Efficacy and Liver Lipid Analysis in Rodents. With the successful identification of a moderately selective LXRβ agonist with acceptable potency, brain penetration and pharmacokinetic properties we moved to evaluate the effects of compound 9 in pre-clinical models. While our transactivation assays are human derived cell-lines, neuroglioma H4 for LXRβ; HepG2 for LXRα, the species homology for the LXR paralogs is reported to be highly conserved in the DNA- and ligand-binding domains24 between rat and human leading us to infer that similar levels of Emax and functional selectivity would be operant.

However, it should be noted that due to

differences in LXR signaling pathways between rodents and humans additional studies would be needed. As such, the rodent studies mainly served to build confidence for efficacy and safety moving toward more resource intensive non-human primate studies. We began with a preliminary assessment of compound 9 in rats with 4-day subcutaneous dosing that resulted in a favorable pharmacodynamic response as measured by increased levels of apoE and Aβ measured in CSF (supplemental material). We did not observe significant changes in either liver or plasma triglycerides following 4-day treatment with compound 9, confirming an improved peripheral lipid safety margin compared with non-selective LXR agonists. However, while these results were encouraging, we determined that a longer sub-chronic dosing paradigm would be needed to fully assess the lipogenic potential of compound 9. For this study ACS Paragon Plus Environment

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we employed Tg2576 transgenic mice that overproduce Aβ peptides and develop amyloid plaques similar to those observed in AD patients. For the study design, 12-month old Tg2576 mice were dosed subcutaneously for 3 three weeks with either compound 1 (10 mg/kg) or compound 9 (50 mg/kg). CNS efficacy of compound 9 was confirmed by significant increases in the protein level of the LXR-target gene ABCA1 as well as significant increases in soluble apoE protein levels in the brain (Fig. 5).25 In addition, a reduction in the levels of DEA-soluble Aβ was detected in the brain that, while trending downward, did not achieve levels of statistical significance by ANOVA analysis due to variability. More importantly, compound 9 did not cause any statistically significant changes in plasma or liver triglycerides when compared to the vehicle group, whereas both plasma and liver triglyceride levels were significantly increased in mice treated with the non-selective agonist 1 (Figure 6).

(a)

(b)

(c)

Figure 5. Mouse efficacy: Brain levels of LXRβ biomarkers. (a) increases in brain ABCA1 levels (b) fold-increase in soluble apoE protein in mouse brain, dashed line represents vehicle control levels. (c) decreases in DEA-soluble brain Aβ levels.



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Liver Triglyceride

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Figure 6. Mouse lipids: Liver lipid biomarkers.

In addition to the analysis of key biochemical pharmacodynamic readouts in these mice, we also investigated potential behavioral effects of the treatment regimen. It has been reported that Tg2576 mice display increased locomotor activity when compared to the wild type (WT) controls and this phenotype is correlated with increased brain amyloid burden.26 As such, the animals from the above three week study were evaluated for locomotor activity in the open field assay after 16 days of dosing. Compound 9 was able to rescue the locomotor behavioral phenotype when dosed (50 mg/kg) subcutaneously (Figure 7).

Reversal of hyperlocomotor behavior showed amyloid dependence, as

compound 9 did not significantly alter locomotor behavior in wild type mice. Furthermore, compound effects were likely not due to off-target activity since administration of a single dose of compound 9 4 hours prior to the test did not result in the reversal of the Tg2576 hyperlocomotor phenotype (data not shown).

Figure 7. Locomotor activity in the Open Field Test (first 10 min).


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Rhesus Monkey Efficacy and Lipid Safety Study. While the efficacy and peripheral safety margin findings in the rodent models were compelling, one must be cautious with the interpretation of these results due to the well-known differences in lipid profiles across species. For example, CETP, a protein that facilitates the transport of cholesteryl esters and triglycerides between lipoproteins, is not expressed in mice and rats, but plays an important role in the distribution of lipids between HDL and LDL in humans. Because CETP expression is also directly regulated by LXRs,27 the translational value of the non-CETP rodents for evaluation of lipid changes upon LXR ligand treatment is highly questionable. To gain a more accurate view of the potential safety margin in higher species, we turned to the non-human primate to assess both CNS efficacy and peripheral lipid effects. The study was designed to be non-terminal with CNS efficacy being evaluated by measurement of the LXR target apoE in CSF. Compound effects on peripheral lipids were assessed by measurement of serum triglyceride levels and magnetic resonance spectroscopy (MRS) as a noninvasive method to assess changes in total liver fat content. The study intent was to compare compound 9 to that of the non-selective agonists 1 and 5 (ORG693),28 a peripherally restricted full dual agonist that significantly increased liver TG content in the cynomolgus monkey at doses ≥ 3 mg/kg over a period of 2 weeks in internal studies (unpublished data).

A 2-week daily oral dosing study was performed using compound 9 at doses of 10 and 20 mg/kg, compound 1 at 10 mg/kg and compound 5 at 5 mg/kg dose. Three baselines serum samples, to evaluate lipid profiles and clinical chemistries for each monkey, were obtained over a period of 2 weeks prior to the beginning of the study. Baseline liver fat content, measured by MRS, and CSF samples, via lumbar puncture, were also obtained for each monkey. Monkeys were randomized so that there were no statistically significant differences for all serum lipids, liver enzymes and liver fat content baseline averages between the groups. Serum samples, liver fat content and CSF were evaluated at trough following the last day of dosing. As shown in Figure 8, 2 weeks treatment with compound 9 at both the 10 and 20 mg/kg dose showed a strong pharmacodynamic effect, producing a significant 3-fold increase ACS Paragon Plus Environment


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in baseline levels of CSF apoE. It should be noted that while substantial accumulation of compound 9 was observed with dosing at 20 mg/kg, leading to an estimated Cmax of 18 µM, this effect was not observed at the 10 mg/kg dose, which resulted in a Cmax of approximately 1µM throughout the 2-week study. As such, our results suggest that a 3-4 fold increase in CSF apoE levels may be the maximal response expected from CNS LXR agonism as similar levels of apoE increases were observed for both groups dosed with compound 9. In support of that assertion, two week treatment with the brain penetrant full dual agonist 1 also resulted in only an approximate 4-fold elevation of CSF apoE. In contrast, compound 5, which is a peripherally restricted compound due to a high Pgp efflux ratio, did not increase CSF apoE despite a significant increase observed in plasma apoE levels. These findings suggest that CNS target engagement was entirely responsible for the elevations of apoE levels observed in the CSF with compounds 9 and 1, as opposed to potential transport of apoE from peripheral compartments. Treatment with brain penetrant compounds 9 and 1 also resulted in a significant 25% increase in both Aβ40 and Aβ42 in the rhesus CSF (Figure 9), whereas no increase in CSF Aβ levels was observed with the peripherally restricted agonist 5, further corroborating the central compartment effects. An increase in CSF Aβ levels likely reflects increased transport of Aβ out of the brain parenchyma and ultimately a decrease in total brain Aβ levels. However, since this was not a terminal study we were unable to quantify total brain Aβ levels as we did in mice where brain Aβ levels were observed to decrease. More importantly, the positive changes in the primary efficacy biomarkers observed for compound 9 were not accompanied by alteration in fat liver content, as measure by MRS (Figure 10) indicating a potential advantage of targeting selective LXRβ agonism. The percentage fat liver levels following treatment at both 10 and 20 mg/kg doses of compound 9 were similar to vehicle. In contrast, monkeys receiving full dual agonists 1 or 5 showed significant 4-fold increase in hepatic fat content as measured by MSR imaging. It should be noted however that while compound 9 did not significantly alter overall serum or liver lipids, significant elevations in LDL cholesterol levels and corresponding decrease in the ratio HDL/LDL cholesterol at plasma concentrations above the in vitro ACS Paragon Plus Environment

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LXRα Emax were observed. These changes in cholesterol between the HDL and LDL fractions can be attributed to the upregulation of CETP protein levels (2.8-fold increase observed at the 20 mg/kg dose). Therefore, while these initial results are encouraging, identification of compounds with even greater levels of selectivity will be needed to fully address the lipogenic potential associated with activation of

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Figure 8. CSF apoE level in 2 week rhesus monkey study



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Figure 10. Liver fat content in 2-week rhesus monkey study.

Conclusion. We succeeded in identifying a new series of functionally selective LXRβ agonists. This series was optimized for Emax selectivity, solubility and physical properties to enable in vivo studies. Compound 9 displayed Emax selectivity for LXRβ, favorable physical properties for CNS penetration and a clean ancillary profile thereby enabling in vivo proof of concept studies. Compound 9 produced statistically significant increases in brain apoE and ABCA1 levels without effecting liver triglyceride levels thereby providing an improved lipid safety profile in rats and mice compared with full dual agonists. These results translated favorable into the rhesus monkey model, where positive changes in primary efficacy biomarkers were accompanied by lack of alterations in liver fat content, as measured by ACS Paragon Plus Environment

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MRS. Progression of this series with emphasis on further improving selectivity by elimination of residual LXRα activity, including in vivo efficacy and lipid ramifications will be reported in a future publication.

EXPERIMENTAL SECTION General Chemistry Information: 1H NMR spectra were recorded on a Varian Inova 400 (400 MHz) spectrometer. Chemical shifts are reported in ppm from tetramethylsilane with tetramethylsilane as the internal standard. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants (Hz), integration. Low resolution mass spectra were recorded on a Bruker Daltonics 3T Fourier transform ion cyclotron resonance mass spectrometer (FT/ICR) with electrospray ionization.

Analytical thin layer chromatography was

performed on EM Reagent 0.25 mm silica gel 60-F plates. Automated flash chromatography was performed on an ISCO Combiflash Sg 100c with Biotage Flash 40 cartridges. Preparative reverse phase chromatography was performed using a Gilson 215 liquid handler and a YMC CombiPrep Pro C18 column (50 mm x 20 mm i.d.) with a linear gradient over 20 minutes (95:5 to 5:95 water/acetonitrile, containing 0.1% TFA) with collection triggered by UV detection at 220 or 254 nm. Compound purity was determined to be >95% by analytical HPLC analysis on an Agilent 1090 HPLC with binary pump and diode array detector with area quantification performed at 214 nM (Method 1: Zorbax RX-C18, 75 mm x 4.6 mm, 3.5 µM, 98% A/2% B to 100% B over 5.5 min then 100% B to 6.0 min (A = 0.1% H3P04/water v/v; B = acetonitrile). Method 2: Luna C8(2), 75 mm x 4.6 mm, 3 µM 98% A/2% B to 100% B over 5.5 min then 100B to 6.0 min (A = 0.1% H3PO4/water v/v; B = acetonitrile). Solvents for extraction and chromatography were HPLC grade. Unless otherwise noted all reactions were conducted in oven (80oC) or flame-dried glassware with magnetic stirring under an inert atmosphere of dry nitrogen.



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tert-Butyl

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4-{5-[3-chloro-4-(dimethylcarbamoyl)phenyl]-3-cyanopyridin-2-yl}piperazine-1-

carboxylate (6). To a solution of tert-butyl piperazine-1-carboxylate (9.4 g, 51 mmol, 1.1 eq) and 5bromo-2-chloropyridine-3-carbonitrile (10 g, 46 mmol, 1.0 eq) in acetonitrile (200 mL) was added N,Ndiisopropylethylamine (9.6 mL, 55 mmol, 1.2 eq) and the resulting solution was heated at reflux for 16 hours. The reaction mixture was concentrated and the residue was dissolved in dichloromethane (200 mL). The organic layer was washed with 1 N aqueous HCl solution (50 mL) followed by 10% aqueous sodium bicarbonate solution (50 mL) then dried over sodium sulfate and concentrated to yield tert-butyl 4-(5-bromo-3-cyanopyridin-2-yl)piperazine-1-carboxylate (16 g, 94%) as a tan solid.

1

H NMR (400

MHz, CDCl3): δ 8.35 (d, J = 2.5 Hz, 1 H); 7.85 (d, J = 2.5 Hz, 1 H); 3.71-3.65 (m, 4 H); 3.59-3.54 (m, 4 H); 1.48 (s, 9 H). LRMS m/z (M+H) 367.1 found, 367.1 required. A mixture of tert-butyl 4-(5-bromo-3-cyanopyridin-2-yl)piperazine-1-carboxylate (8.0 g, 22 mmol, 1.0 eq), 3-chloro-4-(dimethylcarbamoyl)phenyl boronic acid (6.0 g, 26 mmol, 1.2 eq) and 2M sodium carbonate solution (11 mL, 22 mmol, 1.0 eq) in acetonitrile (100 mL) was degassed with nitrogen for 15 minutes prior to the addition of bis(triphenylphosphine) palladium (II) chloride (0.76 g, 1.1 mmol, 0.050 eq). The resulting reaction mixture was heated at reflux under nitrogen for 16 hours. The reaction mixture was concentrated and the residue was partitioned between dichloromethane (200 mL) and water (100 ml). The organic layer was separated, dried over sodium sulfate and concentrated and the resulting residue was purified by silica gel chromatography (0-100% ethyl acetate/hexanes) to yield

compound

6,

tert-butyl

4-{5-[3-chloro-4-(dimethylcarbamoyl)phenyl]-3-cyanopyridin-2-

yl}piperazine-1-carboxylate, (6.3 g, 62%) as a light yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.52 (d, J = 2.6 Hz, 1 H); 7.93 (d, J = 2.6 Hz, 1 H); 7.49 (s, 1 H); 7.41-7.35 (m, 2 H); 3.78-3.72 (m, 4 H); 3.61-3.55 (m, 4 H); 3.13 (s, 3 H); 2.89 (s, 3 H); 1.46 (s, 9 H). LRMS m/z (M+H) 470.2 found, 470.2 required.

2-Chloro-4-(5-cyano-6-{4-[(2R)-3,3,3-trifluoro-2-hydroxy-2-phenylpropanoyl]piperazin-1yl}pyridin-3-yl)-N,N-dimethylbenzamide (7). To a solution of compound 6, tert-butyl 4-{5-[3-chloroACS Paragon Plus Environment

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4-(dimethylcarbamoyl)phenyl]-3-cyanopyridin-2-yl}piperazine-1-carboxylate, (6.3 g, 13 mmol, 1.0 eq) in methanol (100 mL) was added 2 M hydrochloric acid in diethyl ether (100 mL, 100 mmol, 7.5 eq) and the resulting reaction mixture was stirred at 23°C for 16 hours. The reaction mixture was concentrated under reduced pressure to give the bis-HCl salt of 2-chloro-4-[5-cyano-6-(piperazin-1-yl)pyridin-3-yl]N,N-dimethylbenzamide (5.9 g, 100%) as light yellow solid. 1H NMR (400 MHz, D2O): δ 8.51 (d, J = 2.5 Hz, 1 H); 8.22 (d, J = 2.5 Hz, 1 H); 7.64 (s, 1 H); 7.50 (d, J = 8.0 Hz, 1 H); 7.33-7.31 (m, 1 H); 3.78-3.73 (m, 4 H); 3.33-3.28 (m, 4 H); 2.99 (s, 3 H); 2.80 (s, 3 H). LRMS m/z (M+H) 370.1 found, 370.1 required To a solution of the above bis-hydrochloride salt of 2-chloro-4-[5-cyano-6-(piperazin-1yl)pyridin-3-yl]-N,N-dimethylbenzamide (1.7 g, 3.8 mmol, 1.0 eq) and N,N-diisopropylethylamine (2.7 mL, 15 mmol, 4.0 eq) in dichloromethane (30 mL) was added (2R)-3,3,3-trifluoro-2-hydroxy-2phenylpropanoic acid (0.93 g, 4.2 mmol, 1.1 eq) followed by HBTU (2.2 g, 5.7 mmol, 1.5 eq) and the resulting reaction was stirred at 23°C for 16 hours. The reaction mixture was purified by silica gel chromatography (0-100% ethyl acetate/hexanes) to yield compound 7, 2-chloro-4-(5-cyano-6-{4-[(2R)3,3,3-trifluoro-2-hydroxy-2-phenylpropanoyl]piperazin-1-yl}pyridin-3-yl)-N,N-dimethylbenzamide (1.2 g, 54%). 1H NMR (400 MHz, DMSO): δ 8.74 (d, J = 2.5 Hz, 1 H); 8.47 (d, J = 2.5 Hz, 1 H); 8.16 (s, 1 H); 7.87 (d, J = 1.7 Hz, 1 H); 7.71 (dd, J = 8.0, 1.8 Hz, 1 H); 7.53-7.35 (m, 5 H); 3.75-3.41 (m, 8 H); 2.98 (s, 3 H); 2.76 (s, 3 H). LRMS m/z (M+H) 572.3 found, 572.2 required.

2-Chloro-4-(1'-(2-hydroxy-3,3-dimethyl-2-(trifluoromethyl)butanoyl)-[4,4'-bipiperidin]-1-yl)-N,Ndimethylbenzamide (8). To a solution of compound 13 (6.1 g, 13 mmol) in MeOH (50 mL) is added a solution of HCl in dioxane (4 N, 30 mL). The reaction mixture was then at room temperature for 3 h after which the solution was concentrated to give 4-([4,4’-bipiperidin]-1-yl)-2-chloro-N,N’dimethylbenzamide dihydrochloride that was used without further purification [MS [M+H]+ 350.5 found, 350.2 required]. To a solution of this bis-hydrochloride salt of 4-([4,4’-bipiperidin]-1-yl)-2chloro-N,N’-dimethylbenzamide dihydrochloride (2.1 g, 5.0 mmol, 1.0 eq) in dichloromethane (100 ACS Paragon Plus Environment


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mL) was added N,N-diisopropylethylamine (5.2 mL, 30 mmol, 6.0 eq) followed by (2R)-3,3,3-trifluoro2-hydroxy-2-phenylpropanoic acid (1.3 g, 5.9 mmol, 1.2 eq) and HBTU (2.5 g, 6.5 mmol, 1.3 eq) sequentially. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by silica gel chromatography (0-100% ethyl acetate/hexanes) to yield compound 8, 2Chloro-4-(1'-(2-hydroxy-3,3-dimethyl-2-(trifluoromethyl)butanoyl)-[4,4'-bipiperidin]-1-yl)-N,Ndimethylbenzamide (1.5 g, 55%).

1

H NMR (400 MHz, CDCl3): δ 7.40 (m, 5H), 7.06 (d, J = 8.5 Hz,

1H), 6.80 (s, 1H), 6.71 (d, J = 8.4 Hz, 1H), 3.68-3.62 (m, 4H), 3.09 (S, 3H), 2.86 (s, 3H), 2.65-2.61 (m, 4H), 1.63-1.54 (m, 8H), 1.26-1.22 (m, 2H). LRMS m/z (M+) 552.05 found, 552.03 required.

2-Chloro-4-(1'-(2-hydroxy-3,3-dimethyl-2-(trifluoromethyl)butanoyl)-[4,4'-bipiperidin]-1-yl)-N,Ndimethylbenzamide (9). To a solution of compound 14 (60 mg, 0.13 mmol) in THF (2 mL) was added CF3TMS (36.9 mg, 0.26 mmol) followed by TBAF (1 M in THF, 0.13 mL). The reaction mixture was stirred at room temperature for 1 h afterwhich another equivalent of TBAF was added. The reaction mixture was stirred an additional 45 min and then concentrated in vacuo. The crude mixture was purified by silica gel flash chromatography using a linear gradient of 5 to 100% EtOAc in hexanes to give racemic 2-chloro-4-(1'-(2-hydroxy-3,3-dimethyl-2-(trifluoromethyl) butanoyl)-[4,4'-bipiperidin]-1yl)-N,N-dimethylbenzamide as a (54 mg, 79%) white solid. The racemic mixture was then resolved by chiral chromatography (IC column, 3 x 25 cm) using 3:2 (EtOH:heptane with 0.1% TFA). Compound 9 is the first peak to elute (elution time: 9.35 min); HRMS [M+H]+ 532.2540, 532.2554 required. Enantiomer 2 (elution time: 10.47 min); HRMS [M+H]+ 532.2543, 532.2554 required. 1H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 8.5 Hz, 1H), 6.85 (s, 1 H), 6.81 (d, J = 8.55, 1H), 5.69 (b, 1H), 4.78 (b, 1H), 4.33 (b, 1H), 3.74 (d, J = 13 Hz, 2H), 3.11 (s, 3H), 2.89 (s, 3H), 2.71 (t, J = 11 Hz, 2H), 2.44 (b, 1H), 1.87-1.78 (m, 4H), 1.39-1.21 (m, 8H), 1.12 (d, J = 6.3 Hz, 3H), 0.84 (b, 3H). HRMS [M+H]+ 532.2552 found, 532.2554 required.


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tert-Butyl 4,4'-bipiperidine-1-carboxylate (11). To a solution of 4,4'-bipiperidine dihydrochloride, compound 10, (10 g, 41.5 mmol, 1.0 eq) in 20:1 ethanol:water (420 mL) was added and a solution of 5N sodium hydroxide to adjust the pH to 8~9. Di-tert-butyl dicarbonate (9.06 g, 42.3 mmol, 1.0 eq) in ethanol (20 mL) was added and the mixture was stirred at room temperature for 12 hr. The solvent was removed in vacuo and 50 mL of water was added. The white solid (undesired di-Boc product) was removed by filteration and washed with water (3 x 20mL). The filtrate was basified by the addition of 1 N aq. NaOH to ~pH=10 and extracted with ethyl acetate (3 x 200 mL). The combined organic phase was dried over sodium sulfate and concentrated in vacuo to give compound 11, tert-butyl 4,4'bipiperidine-1-carboxylate (6.68 g, 60%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 4.17-4.11 (m, 2H), 3.46-3.44 (m, 2H), 2.96-2.92 (m, 2H), 2.67-2.61 (m, 2H), 1.88 (d, J = 16 Hz, 2H), 1.69-1.63 (m, 4H), 1.45 (s, 9H), 1.35-1.25 (m, 2H), 1.17-1.13 (m, 2H). LRMS m/z (M+H) 269.2 found, 269.2 required.

tert-Butyl 1'-(3-chloro-4-(dimethylcarbamoyl)phenyl)-[4,4'-bipiperidine]-1-carboxylate (13). To a mixture of compound 11 (6 g, 22.4 mmol), DavePhos (2.64 g, 6.71 mmol), Pd(OAc)2 (502 mg, 2.24 mmol) and sodium-t-butoxide (2.58 g, 26.8 mmol), under nitrogen, was added toluene (70 mL) followed by a solution of 4-bromo-2-chloro-N,N-dimethylbenzamide, compound 12, (5.87 g, 22.4 mmol) in toluene (30 mL). The reaction mixture was heated to 80°C for 5 h afterwhich the mixture was cooled to room temperature and partition between water and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude mixture was purified by silica gel flash chromatography using a linear gradient of 5 to 100% EtOAc in hexanes to give compound 13,

tert-butyl 1'-(3-chloro-4-

(dimethylcarbamoyl)phenyl)-[4,4'-bipiperidine]-1-carboxylate (6.1 g, 60%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 8.6 Hz, 1H), 6.81 (s, 1H), 6.78 (d, J = 8.4 Hz, 1H), 4.11-4.05 (m, 2H), 3.68-3.62 (m, 2H), 3.07 (s, 3H), 2.85 (s, 3H), 2.68-2.55 (m, 4H), 1.78-1.61 (m, 4H), 1.42 (s, 9H), 1.381.15 (m, 6H). MS [M]+ 449.99 found, 450.3 required. ACS Paragon Plus Environment


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2-Chloro-4-(1'-(3,3-dimethyl-2-oxobutanoyl)-[4,4'-bipiperidin]-1-yl)-N,N-dimethylbenzamide (14). To a solution of 4-([4,4’-bipiperidin]-1-yl)-2-chloro-N,N’-dimethylbenzamide dihydrochloride, prepared above, (40 mg, 0.095 mmol) and PyBOP (59.1 mg, 0.114 mmol) in DMF (0.5 mL) are added trimethylpyruvic acid (24.6 mg, 0.189 mmol) and DIEA (98 mg, 0.76 mmol). The reaction mixture was stirred at room temperature for 10 min. The reaction mixture was purified by reverse phase HPLC (Sunfire C18 30 x 150 mm column) using 20 to 95% MeCN in water (0.1% TFA modifier for the solvents) over 15 min at 35 mL flow rate to give compound 14, 2-chloro-4-(1'-(3,3-dimethyl-2oxobutanoyl)-[4,4'-bipiperidin]-1-yl)-N,N-dimethylbenzamide (38 mg, 88%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.14 (d, J = 8.5 Hz, 1H), 6.85 (s, 1H), 6.81 (d, J = 8.5, 1H), 4.58 (d, J = 13 Hz, 1H), 3.74 (d, J = 13 Hz, 2H), 3.66 (d, J = 13 Hz, 1H), 3.19-3.16 (m, 1H), 3.11 (s, 3H), 3.04-2.98 (m, 1H), 2.89 (s, 3H), 2.75-2.64 (m, 2H), 1.86-1.77 (m, 4H), 1.49-1.21 (m, 7H), 1.17 (d, J = 6.6 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H). LRMS m/z (M+H) 462.6 found, 462.3 required.

AUTHOR INFORMATION

Corresponding Author *Tel: 215-652-2273. E-mail: [email protected].

ACKNOWLEDGMENTS We thank the Shanghai Synchrotron Radiation Facility (SSRF) for collection of the diffraction data.

ABBREVIATIONS USED Aβ, amyloid β peptide; ABCA1, ATP-binding cassette transporter; AD, Alzheimer’s Disease; ANOVA, analysis of variance; apoE, apolipoprotein E; APP, amyloid precursor protein; Boc, tert-butoxycarbonyl; CETP, cholesterylester transfer protein; CFR, co-factor recruitment; CL, clearance; CNS, central nervous system; CSF, cerebrospinal fluid; EC50, half maximal effective concentration; Emax, maximal ACS Paragon Plus Environment

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efficacy; %F, percent bioavailable; HDL, high-density lipoprotein; His, histidine; LDL, low-density lipoprotein; LOAD, late-onset Alzheimer’s Disease; LXR, liver x receptor; KO, knockout; MDCK, Madin-Darby Canine Kidney Cells; MRL, Merck Research Labs; MRS, magnetic resonance spectroscopy; NHP, non-human primate; Papp, apparent permeability; Pgp, P-glycoprotein; PK, pharmacokinetic;

PPB,

plasma

protein

binding;

PyBOP,

benzotriazol-1-yl-

oxytripyrrolidinophosphonium hexafluorophosphate; RXR, retinoid x receptor; SAR, structure-activity relationship; SREBP1, sterol regulatory element-binding protein 1; t1/2, half-life; Trp, tryptophan; uHTS, ultra high throughput screening; VCD, vibrational circular dichroism; Vdss, volume of distribution; WT, wild type

Supporting Information Available: Experimental procedures and compound characterization data.

References (1) Selkoe, D. J. The Molecular Pathology of Alzheimer's Disease. Neuron 1991, 6, 487-498. (2) Strittmatter, W. J.; Saunders, A. M.; Schmechel, D.; Pericak-Vance, M.; Enghild, J.; Salvesen, G. S.; Roses, A. D. Apolipoprotein E: High-Avidity Binding to Beta-Amyloid and Increased Frequency of Type 4 Allele in Late-Onset Familial Alzheimer Disease. Proc. Natl. Acad. Sci. USA 1993, 90, 19771981. (3) Walker, L. C.; Pahnke, J.; Madauss, M.; Vogelgesang, S.; Pahnke, A.; Herbst, E. W.; Stausske, D.; Walther, R.; Kessler, C.; Warzok, R. W. Apolipoprotein E4 Promotes the Early Deposition of Abeta42 and then Abeta40 in the Elderly. Acta Neuropathol. 2000, 100, 36-42. (4) Reiman, E. M.; Chen, K.; Liu, X.; Bandy, D.; Yu, M.; Lee, W.; Ayutyanont, N.; Keppler, J.; Reeder, S. A.; Langbaum, J. B.; Alexander, G. E.; Klunk, W. E.; Mathis, C. A.; Price, J. C.; Aizenstein, H. J.; DeKosky, S. T.; Caselli, R. J. Fibrillar Amyloid-Beta Burden in Cognitively Normal People at 3 Levels of Genetic Risk for Alzheimer's Disease. Proc. Natl. Acad. Sci. USA 2009, 106, 6820-6825.



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(5) Ramaswamy, G.; Xu, Q.; Huang, Y.; Weisgraber, K. H. Effect of Domain Interaction on Apolipoprotein E Levels in Mouse Brain. J. Neurosci. 2005, 25, 10658-10663. (6) Riddell, D. R.; Zhou, H.; Atchison, K.; Warwick, H. K.; Atkinson, P. J.; Jefferson, J.; Xu, L.; Aschmies, S.; Kirksey, Y.; Hu, Y.; Wagner, E.; Parratt, A.; Xu, J.; Li, Z.; Zaleska, M. M.; Jacobsen, J. S.; Pangalos, M. N.; Reinhart, P. H. Impact of Apolipoprotein E (ApoE) Polymorphism on Brain ApoE Levels. J. Neurosci. 2008, 28, 11445-11453. (7) Bales, K. R.; Liu, F.; Wu, S.; Lin, S.; Koger, D.; DeLong, C.; Hansen, J. C.; Sullivan, P. M.; Paul, S. M. Human APOE Isoform-Dependent Effects on Brain Beta-Amyloid Levels in PDAPP Transgenic Mice. J. Neurosci. 2009, 29, 6771-6779. (8) Bu, G. Apolipoprotein E and its Receptors in Alzheimer's Disease: Pathways, Pathogenesis and Therapy. Nat. Rev. Neurosci. 2009, 10, 333-344. (9) Suon, S.; Zhao, J; Villarreal, S A.; Anumula, N.; Liu, M.; Carangia, L. M.; Renger, J. J.; Zerbinatti, C. V. Systemic Treatment with Liver X Receptor Agonists Raises Apolipoprotein E, Cholesterol, and Amyloid-β Peptides in the Cerebral Spinal Fluid of Rats. Mol. Neurodegener. 2010, 5, 44. (10) Castellano, J. M.; Kim, J.; Stewart, F. R.; Jiang, H.; DeMattos, R. B.; Patterson, B. W.; Fagan, A. M.; Morris, J. C.; Mawuenyega, K. G.; Cruchaga, C.; Goate, A. M.; Bales, K. R.; Paul, S. M.; Bateman, R. J.; Holtzman, D. M. Human ApoE Isoforms Differentially Regulate Brain Amyloid-β Peptide Clearance. Sci. Transl. Med. 2011, 89, 1-11. (11) Wahrle, S. E.; Jiang, H.; Parsadanian, M.; Legleiter, J.; Han, X.; Fryer, J. D.; Kowalewski, T.; Holtzman, D. M. ABCA1 is Required for Normal Central Nervous System ApoE Levels and for Lipidation of Astrocyte-Secreted ApoE. J. Biol. Chem. 2004, 279, 40987-40993. (12) Liang, Y.; Lin, S.; Beyer, T. P.; Zhang, Y.; Wu, X.; Bales, K. R.; DeMattos, R. B.; May, P. C.; Li, S. D.; Jiang, X. C.; Eacho, P. I.; Cao, G.; Paul, S. M. A Liver X Receptor and Retinoid X Receptor Heterodimer Mediates Apolipoprotein E Expression, Secretion and Cholesterol Homeostasis in Astrocytes. J. Neurochem. 2004, 88, 623-634.


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(13) Mawuenyega, K. G.; Sigurdson, W.; Ovid, V.; Munshell, L.; Kasten, T.; Morris, J. C.; Yarasheski, K. E.; Bateman, R. J. Decreased Clearance of CNS β-Amyloid in Alzheimer’s Disease. Science 2010, 330, 1774. (14) Lund, E. G.; Peterson, L. B.; Adams, A. D.; Lam, M. H.; Burton, C. A.; Chin, J.; Guo, Q.; Huang, S.; Latham, M.; Lopez, J. C.; Menke, J. G.; Milot, D. P.; Mitnaul, L. J.; Rex-Rabe, S. E.; Rosa, R. L.; Tian, J. Y.; Wright, S. D.; Sparrow, C. P. Different Roles of Liver X Receptor Alpha and Beta in Lipid Metabolism: Effects on an Alpha-Selective and a Dual Agonist in Mice Deficient in each Subtype Biochem. Pharmacol. 2006, 71, 453-463. (15) Schultz, J. R.; Tu, H.; Luk, A.; Repa, J. J.; Medina, J. C.; Li, L. P.; Schwendner, S.; Wang, S.; Thoolen, M.; Mangelsdorf, D. J.; Lustig, K. D.; Shan, B. Role of LXRs in Control of Lipogenesis. Genes Dev. 2000, 14, 2831-2838. (16) Collins, J. l.; Fivush, A. M.; Watson, M. A.; Galardi, C. M.; Lewis, M. C.; Moore, L. B.; Parks, D. J.; Wilson, J. G.; Tippen, T. K.; Binz, J. G.; Plunket, K. D.; Morgan, D. G.; Beaudet, E. J.; Whitney, K. D.; Kliewer, S. A.; Willson, T. M. Identification of a Nonsteroidal Liver X Receptor Agonist through Partial Array Synthesis of Tertiary Amines. J. Med. Chem. 2002, 45, 1963-1966. (17) Wrobel, J.; Steffan, R.; Bowen, M.; Magolda, R.; Matelan, E.; Unwalla, R.; Basso, M.; Clerin, V.; Gardell, S. J.; Nambi, P.; Quinet, E.; Reminick, J. I.; Vlasuk, G. P.; Wang, S.; Wilhelmsson, A.; Zamaratski, E.; Evans, M. J. Indazole-based Liver X Receptor (LXR) Modulators with Maintained Atherosclerotic Lesion Reduction Activity but Diminished Stimulation of Hepatic Tryiglyceride Synthesis. J. Med. Chem. 2008, 51, 7161-7168. (18) Kick, E.; Martin, R.; Xie, Y.; Flatt, B.; Schweiger, E.; Wang, T.L.; Busch, B.; Nyman, M.; Gu, X.H.; Yan, G.; Wagner, B.; Nanao, M.; Nguyen, L.; Stout, T.; Plonowski, A.; Schulman, I.; Ostrowski, J.; Kirchgessner, T.; Wexler, R.; Mohan, R. Liver X Receptor (LXR) Partial Agonists: Biaryl Pyrazoles and Imidazoles Displaying a Preference for LXRβ. Bioorg. Med. Chem. Lett. 2015, 25, 372-377. (19) Quinet, E. M.; Basso, M. D.; Halpern, A. R.; Yates, D. W.; Steffan, R. J.; Clerin, V.; Resmini, C.; Keith, J. C.; Berrodin, T. J.; Feingold, I.; Zhong, W.; Hartman, H. B.; Evans, M. J.; Gardell, S. J.; ACS Paragon Plus Environment


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DiBlasio-Smith, E.; Mounts, W. M.; LaVallie, E. R.; Wrobel, J.; Nambi, P.; Vlasuk, G. P. LXR Ligand Lowers LDL Cholesterol in Primates, is Lipid Neutral in Hamster, and Reduces Atherosclerosis in Mouse. J. Lipid Res. 2009, 50, 2358-2370. (20) Katz, A.; Udata, C.; Ott, E.; Hickey, L.; Burczynski, W. E.; Burghart, P.; Vesterqvist, O.; Meng, X. Safety, Pharmacokinetics, and Pharmacodynamics of Single Doses of LXR-623, a Novel Liver XReceptor Agonist, in Healthy Participants. J. Clin. Pharm. 2009, 49, 643-649. (21) Singh, R. P.; Kirchmeier, R. L.; Shreeve, J. M. TBAF-Catalyzed Direct Nucleophilic Trifluoromethylation of α-Keto Amides with Trimethyl(trifluoromethyl)silane. J. Org. Chem. 1999, 64, 2579-2581. (22) VCD Spectral analysis of compound 9 and its enantiomer in supplemental material. Reference for VCD: Freedman, T. B.; Cao, X.; Dukor, R. K.; Nafie, L. A. Absolute Configuration Determination of Chiral Molecules in the Solution State Using Vibrational Circular Dichroism. Chirality 2003, 15, 743758. (23) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert, M. H.; Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.; Parks, D.; Watson, M.; Wisley, B.; Willson, T. M. X-ray Crystal Structure of the Liver X Receptor β Ligand Binding Domain. J. Bio. Chem. 2003, 278, 27138-27143. (24) Zhao, C.; Dahlman-Wright, K. Liver X Receptor in Cholesterol Metabolism. J. Endocrinol. 2010, 204, 233-240. (25) 1 way ANOVA (Bartlett’s test) was used and three stars is p < 0.0005. 1 star = p < 0.05 and 2 stars = p < 0.005. (26) (a) Gil-Bea, F. J.; Aisa, B. Increase of Locomotor Activity Underlying the Behavioral Disinhibition in Tg2576 Mice. Behav. Neurosci. 2007, 121, 340-344. (b) King, D. L.; Arendash, G. W. Behavioral Characteristics of the Tg2576 Transgenic Model of Alzheimer’ Disease through 19 Months. Physiol. Behav. 2002, 75, 627-642. (27) Honzumi, S.; Shima, A.; Hiroshima, A.; Koieyama, T.; Ubukata, N.; Terasaka, N. LXRalpha Regulates Human CETP Expression in Vitro and in Transgenic Mice. Atherosclerosis 2012, 212, 139ACS Paragon Plus Environment

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145. (28) Cooke, A. J.; Edwards, A. S.; Andrews, F. E.; Bennett, D. J.; Nimz, O.; Carswell, E. L. Preparation of Piperizine Derivatives as Liver X Receptor Agonists. WO 2009138438 A1 2009.

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Identification and in Vivo Evaluation of Liver X ... - ACS Publications

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