The Medicinal Chemistry of Liver X Receptor (LXR) Modulators

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The Medicinal Chemistry of Liver X Receptor (LXR) Modulators Colin Michael Tice, Paul B Noto, Kristi Yi Fan, Linghang Zhuang, Deepak S Lala, and Suresh B Singh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500442z • Publication Date (Web): 15 May 2014 Downloaded from http://pubs.acs.org on May 27, 2014

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Journal of Medicinal Chemistry

The Medicinal Chemistry of Liver X Receptor (LXR) Modulators

Colin M. Tice,* Paul B. Noto, Kristi Yi Fan, Linghang Zhuang, Deepak S. Lala and Suresh B. Singh

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

Abstract LXRs have been of interest as targets for the treatment of atherosclerosis for over a decade. In recent years, LXR modulators have also garnered interest for potential use in the treatment of inflammation, Alzheimer’s disease (AD), dermatological conditions, hepatic steatosis and oncology. To date, no LXR modulator has successfully progressed beyond phase I clinical trials. In this review, we summarize published medicinal chemistry efforts in the context of the available crystallographic data, druglikeness and isoform selectivity. In addition, we discuss the challenges to be overcome before an LXR modulator can reach clinical use.

LXRs: target genes and major therapeutic applications Nuclear receptors (NRs) comprise a group of 48 ligand-activated transcription factors in humans. Many marketed drugs are modulators of NRs.1 Liver X receptors (LXRs) are NRs that act as oxysterol (OHC) sensors, regulating genes involved in cholesterol and lipid metabolism.2 The potential utility of LXRs as drug targets, particularly for atherosclerosis,3 has been recognized for over a decade. A number of excellent scientific reviews summarize recent developments in

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our understanding of the biology of the LXRs,4,5,6,7 as well as the chemistry of LXR modulators from the recent patent literature.8 This review focuses on the biological activity, crystallographic binding modes and relevant physical chemical properties of LXR modulators reported in the published medicinal chemistry literature. Two isoforms of LXR are known, LXRα (NR1H3) and LXRβ (NR1H2). While LXRα is primarily expressed in liver, intestine, adipose tissue and macrophages, LXRβ is present in all tissues and organs.9,10 Elevated concentrations of intracellular cholesterol and OHCs lead to activation of LXRs which, as heterodimers with retinoid X receptors (RXRs), bind to LXR response elements (LXREs) present within promoters to turn on the expression of several genes via a mechanism termed transactivation. In the absence of LXR ligands, the LXR/RXR heterodimer is associated with co-repressors, such as nuclear co-repressor 1 (NCoR1), and constitutively bound to the promoter of LXR target genes.11-12 This is known as basal gene repression. The presence of agonist ligands causes dissociation of NCoR1 from the promoters of LXR target genes and induces recruitment of co-activators by LXRs,13,14 leading to initiation of gene transcription. Some of the key LXR target genes include ABC transporters expressed in multiple tissues, including ABCA1/G1 in macrophages15,16 and ABCG5/G8 in liver and intestine.17 ABCA1 mediates the transport of phospholipids and cholesterol to poorly-lipidated apolipoproteins, such as Apo-A1,18 contributing to stabilization of high-density lipoproteins (HDL) and initiating reverse cholesterol transport (RCT). By contrast, ABCG1 promotes cholesterol efflux to phospholipid-containing acceptors, such as HDL particles previously lipidated by ABCA1.19 ABCG5/G8 transporters play a crucial role in the secretion of hepatic sterols into bile, as mice lacking either transporter show increased levels of cholesterol in the liver and reduced levels of bile acids.20 An additional LXR target gene is the phospholipid


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transfer protein (PLTP).21 PLTP mediates the transfer of phospholipids and cholesterol from triglyceride (TG)-rich lipoproteins (TRL) into HDL, contributing to the formation of β-HDL particles, which are very efficient acceptors of cholesterol from peripheral cells.22 In rodents, but not in humans, LXRs have been shown to upregulate CYP7A1 in the liver. CYP7A1 is a ratelimiting enzyme in the synthesis of bile acids from cholesterol.23 LXRs also play a role in cholesterol homeostasis at the intestinal level. LXR activation has been shown to reduce the expression levels of the Niemann–Pick C1 like 1 (NPC1L1) gene both in human colon carcinoma cells (CaCo-2) and in mouse intestine.24 NPC1L1 is required for intestinal cholesterol absorption and it is primarily expressed in the brush border membrane of enterocytes in the small intestine.25 Statins, which inhibit HMG-CoA-reductase, the rate-limiting enzyme in cholesterol synthesis, are currently the primary pharmacological intervention for atherosclerosis.26 Although statins effectively lower blood-circulating cholesterol and therefore ameliorate the condition of patients with cardiovascular disorders, new drugs that actually promote cholesterol efflux from overloaded macrophages are needed in order to reduce existing atherosclerotic plaques and to decrease the probability of thrombotic events. The ability of LXRs to promote RCT via direct up regulation of the genes of several ABC transporters in macrophages and intestine, while limiting absorption of cholesterol in the small intestine, makes them an attractive therapeutic target for the treatment of atherosclerosis.27 However, upon ligand binding in liver, LXRs are also responsible for the upregulation of the sterol regulatory element-binding protein-1c (SREBP1c) gene28 and several other genes involved in lipogenesis, such as fatty acid synthase (FAS)27 and stearoyl-CoA desaturase (SCD).29 Oral



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administration of a synthetic LXR agonist, T0901317 (1, Fig. 1), to mice and hamsters leads to increased plasma and hepatic triglyceride levels.30 Control of lipogenesis is not restricted to plasma and liver, as LXR activation also promotes lipid accumulation in human mature adipocytes.31 Additionally, LXRs positively regulate the expression of cholesteryl ester transfer protein (CETP), which mediates a bidirectional exchange of cholesteryl esters and triglycerides between lipoproteins, such as HDL and low-density lipoproteins (LDL).32,33 Importantly, plasma CETP activity is not present in all species; rats and mice do not express the CETP gene. Induction of CETP in humans represents a major liability in the pharmacological activation of LXRs, as increased CETP activity is associated with an enhanced atherogenic lipoprotein profile.34 Administration of two synthetic non-steroidal LXR agonists, 3 and SB247881 (4), to CETPexpressing species, such as hamsters and cynomolgus monkeys, caused a significant increase of both very low density lipoprotein (VLDL) and LDL cholesterol levels in plasma.35 LXRs have been shown to act as negative regulators of key inflammatory genes, such as tumornecrosis factor alpha (TNFα), interleukins (IL-1β, IL-6), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB).36 Additionally, in murine peritoneal macrophages, ligand-activated LXRs can counteract the LPS-induced expression of matrix metalloproteinase 9 (MMP-9), which is involved in degradation of extracellular matrix (ECM) components during normal and pathogenic tissue remodeling.37 The mechanism by which LXRs exert anti-inflammatory properties is less well understood, but may, at least in part, rely upon association with co-repressor complexes that prevent the recruitment of the transcriptional machinery onto the promoters of pro-inflammatory genes.38 This mechanism is known as transrepression and does not rely on the presence of LXREs. While less well


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understood compared to transactivation, transrepression appears to involve at least in part, ligand-dependent ubiquitin-like modifications of the lysine residues in the LBDs of LXRs with small ubiquitin related modifier (SUMO) proteins. The SUMOylated LXRs are recruited to the promoter regions of certain constitutively repressed inflammatory genes preventing the clearance of the co-repressor (NCoR1) complex in response to transcriptional stimuli, maintaining them in a repressed state. The anti-inflammatory effect of LXR agonists also appears to play an important role in their anti-atherosclerotic activity.39 Since the early characterization of LXR ligands, such as OHCs and 1, a substantial effort has been dedicated to the identification of LXR ligands capable of turning on ABC transporter genes as well as reducing inflammation, without affecting SREBP1c gene levels. Given that in liver LXRα expression is higher than LXRβ expression,10 several research groups have directed their efforts towards the discovery of LXRβ-selective modulators, in an effort to avoid activation of LXRα, which seems to play a more significant role in SREBP1c gene regulation in the liver.40 Studies with LXR knockout mice provided support for this hypothesis.41 However, more recently, LXR knockout experiments in murine models of atherosclerosis (LDLR-/- and ApoE-/-) have shown the requirement for LXRα, but not LXRβ, in macrophages in order to achieve robust reduction of atherosclerotic plaques and efficient cholesterol efflux to ApoA1.42,43 Consistent with this, increased expression of LXRα in the intestine of LDLR-/- mice, obtained via knock in of a VP16-LXRα construct, led to improved RCT and decreased severity of aortic lesions.44 Evidence also supports a significant role for LXRs in innate immunity. In response to intracellular bacteria, induction of LXRα expression in murine macrophages leads to increased survival, as well as decreased apoptosis, and LXR-mediated gene upregulation of the Scavenger Receptor Cystine-Rich Repeat Protein (Spα),45 which appears to have a critical role in the



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clearance of bacterial pathogens.46,47 Consistent with this observation, LXR activation has been shown to potentiate the LPS response in human macrophages, by inducing the expression of the TLR-4 gene.48 This might suggest a dual role for LXRs, as they initially prepare macrophages to elicit an antibacterial response, and then, once the inflammatory stimulus is present, exert antiinflammatory actions to restore normal cell conditions. Nonetheless, regulation of TLR-4 is known to be species-specific, since LXRs do not induce this gene in mice. The antiinflammatory effects of LXRs are not limited to macrophages. For instance, LXRs can also suppress the hepatic expression of the C-reactive protein (CRP), a typical human acute phase protein. This has been demonstrated in human hepatocytes, showing that LXRs can maintain the CRP gene in a repressed state by preventing the cytokine-induced clearance of nuclear receptor/co-repressor complexes.49 Recently, modulation of LXRs has been proposed for the treatment of (AD).50,51 Currently, no therapy exists to block or reverse the progression of this debilitating neurological disease. Recent studies have demonstrated that activation of LXRs results in increased apolipoprotein E (apoE) levels in murine and human macrophages52,53 and in rat brain, in which higher levels of lipidated apoE positively correlate with amyloid Aβ clearance.54 LXRβ expression and activation has also been shown to protect dopaminergic neurons in a mouse model of Parkinson’s disease (PD) by modulating the microglia-mediated neuronal toxicity.55 Taken together with the anti-inflammatory properties of LXRs, development of central nervous system (CNS)-penetrant LXR modulators might prove beneficial in the setting of neurological disorders, such as AD and PD, either alone or in combination with other agents. The LXRβ-mediated upregulation of ApoE has also been associated with a reduction of melanoma growth in both human in vitro systems and rodent in vivo models, where increased


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levels of ApoE effectively suppressed metastatic invasion and angiogenesis leading to prolonged animal survival.56 Anti-proliferative and pro-apoptotic effects were also reported in the context of hormonally regulated cancers, such as those of breast and prostate. Indeed, the tumor suppressive properties of LXR have been shown via the LXR-mediated increase of ApoE, leading to growth suppression of the MCF-7 human line,57 and the suppression of gene networks involved in tumor progression, such as those regulated by E2F genes, upon activation of LXRs with the synthetic ligand GW3965.58 Similarly, LXR knock-out studies have shown the protective role of LXRs against the development and progression of prostate cancer.59 In addition, activation of LXRs via treatment of human prostate cancer cells, in both culture and xenografted nude mice, with T0901317 has been shown to induce apoptosis and blockage of tumor progression.60 Pharmacological modulation of LXRs has also been proposed for the treatment of skin disorders, such as atopic dermatitis. In mouse skin, LXR activation with both natural and synthetic ligands, such as OHCs (e.g., 2) and GW3965 (3), results in reduced expression of pro-inflammatory cytokines while preventing keratinocyte differentiation and promoting epidermal development, by increasing lipid production and thereby improving barrier function.61,62 Treatment of human airway smooth muscle cells (hASM) with the synthetic agonist 1 reduced levels of a series of cytokines and pro-inflammatory factors, suggesting an additional application for LXR modulation in the context of airway inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD).63 LXRs may also play a therapeutic role in stroke. In rodent models of experimental stroke, treatment with two synthetic LXR agonists, 1 and 3, diminished the levels of several ischemia-



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related inflammatory markers, such as iNOS, MMP-9, COX-2 and TNFα.64 Therefore, in cases of cerebral ischemia, LXR modulators may offer neuroprotection by reducing brain inflammation and neurological deficits.65 Suppression of LXR activity in liver has been proposed for the treatment of hepatic diseases, such as fatty liver, cirrhosis and non-alcoholic hepatosteatosis (NASH).30,66 A number of groups have identified LXR antagonists;67,68,69,70 one group has demonstrated the capacity, both in vitro and in vivo, of synthetic LXRα/β antagonists to down regulate lipogenic genes in liver, limiting hepatic accumulation of lipids, and reducing plasma cholesterol levels in a mouse model of NASH.70 Therefore, development of liver-specific LXR antagonists may prove very useful in the treatment of metabolic disorders associated with fat accumulation in the liver.

LXR Structure and Function

Full length LXRα and β consist of 44771 and 46172 amino acids, respectively. In common with other nuclear receptors, the proteins include the following domains: an N-terminal ligandindependent activation domain, a zinc finger DNA binding domain (DBD), a hinge region, the ligand binding domain (LBD) and a C-terminal domain.73 The DBD and LBD regions of LXRα and LXRβ have 75.6% and 74% sequence identity, respectively.74 The three-layered α-helical sandwich structure of the LBD is well conserved across the nuclear receptor family. In the case of LXR, the LBD contains 10 α-helices; helix 2 is absent and helices 10 and 11 are merged (Fig 2.).75 In vivo, LXRs form heterodimers with RXRs. In the unliganded state, the LXR/RXR heterodimer is present in the nucleus bound to LXREs and to corepressor proteins.13 Upon binding of an agonist ligand to LXR, corepressors are released and helix 12, also known as


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activation function 2 (AF2), adopts a conformation that closes the ligand binding pocket and forms a groove to which coactivator proteins can bind, allowing gene transcription to occur.76 By analogy with the estrogen receptor (ER), it can be anticipated that ligands that bind tightly to LXR and prevent helix 12 from adopting an agonist conformation will function as active antagonists. Alternatively, ligands that bind tightly to the receptor but fail to stabilize the agonist conformation of helix 12 may function as passive antagonists.77

X-ray Crystal Structures Seven LXRα and eleven LXRβ LBD X-ray crystal structures have been deposited into the Brookhaven Protein Databank (PDB) since 2003, with resolution in the range from 2.1 to 3.1 Å.69,75,76,78,79,80,81,82,83,84,85, All the structures include an agonist complexed to the ligand binding domain. The ligands range from epoxycholesterol 2, a weak affinity endogenous LXR activator,78 to various synthetic non-steroid agonists including 184 and 3.76 The crystal structures include monomers, (PDB IDs: 1P8D, 1UPV, 1UPW, 3L0E), homodimers (PDB IDs: 3IPQ, 3IPS, 3IPU, 1PQ6, 1PQ9, 1PQC, 3KFC, 4DK7, 4DK8) and heterodimers with RXR (PDB IDs: 1UHL, 2ACL, 3FC6, 3FAL, 4NQA). The last of these structures (PDB ID: 4NQA) includes the DBDs of both LXRβ and RXRα in addition to the two LBDs.86

In all of the reported structures, the ligand occupies the ligand binding site behind helix 3, and therefore allows helix 12 to adopt an agonist conformation and pack against the LBD, facilitating coactivator binding. A schematic diagram of the LXR LBD based on LXRβ complexed to a 4(3-aryloxyaryl) quinoline sulfone (PDB ID: 3KFC) is shown in Fig. 2.82 Examination of the available LXR X-ray crystal structures reveals that the ligand binding site stretches from helix 12



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to either the β-sheet regions when a small ligand such as 1 is bound (PDB ID: 1PQC), or to the polar sub-cavity formed by Ser242, Glu281, Arg319, and Tyr335 when a larger ligand is bound (PDB IDs: 3KFC and 1PQ6). The ligand binding site is hydrophobic in nature, but contains polar residues that can form interactions with the ligand. The most important interaction for receptor activation is hydrogen bonding between the ligand and His421/His435 (LXRα /LXRβ) in helix 10/11. This interaction stabilizes the stacking interaction between His421/His435 (LXRα /LXRβ) and Trp443/Trp457 LXRα/LXRβ) in the C-terminal helix 12.78 This interaction is believed to promote the association of co-activators with the LXR receptors. In addition, a hydrogen bond between the ligand and the backbone NH of Leu330 was observed for several LXRβ complex structures.69,82,76

The X-ray structure of the human RXRα:LXRβ heterodimer complexed with DNA (PDB ID: 4NQA) has recently been resolved at 3.1 Å resolution.86 The binding sites of both proteins are occupied by agonist ligands; LXRβ by 3 and RXRα by retinoic acid. The structure shows an Xshaped arrangement of the two domains of LXRβ and RXRα criss-crossing each other. This configuration has the LXRβ DBD docked onto the 3’ site half of the DR-4 oligonucleotide and RXRα DBD toward the 5’ site half respectively (Fig. 3). In addition, a typical zinc-finger organization was observed for both NR DBDs, having the expected contacts with DR-4. The extended X-shaped arrangement differs from the parallel domain arrangement previously reported for PPARγ:RXRα heterodimer bound to DR-1.87 The researchers suggested that the observed arrangement of the RXRα:LXRβ heterodimer on DNA places the LXRβ LBD for optimal exposure of the AF-2 surfaces and co-activator recruitment.86


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As expected, based on the mLXRα/hRXRα LBD crystal structures (PDB IDs: 2ACL, 3FAL, and 3FC6), helices 10 and 11 of the two proteins form the heterodimer interface. Four hydrogen bonding interactions are observed between the two proteins. As shown in Fig. 4, the interacting residue pairs are LXRβ R429(h11)/ RXRα S427(h11), LXRβ E393(h9)/ RXRα K356(h7), LXRβ R420(h10)/ RXRα E394(h9), and LXRβ D414 (loop between h9 and h10)/ RXRα S222(loop toward DBD). Overall, the LBDs of both receptors in the complex of 4NQA demonstrate a typical active agonist bound conformation with SRC-2 co-activator docked against AF2.

LXRα and LXRβ have 74% amino acid identity in the LBD, and differ by only two residues in the ligand binding site: V261/I277 (LXRα /LXRβ) and V295/I311/ (LXRα//LXRβ). The high structural similarity between LXRα and LXRβ in the ligand binding site has made the design of isoform selective compounds challenging. Details of the interactions of specific compounds with the ligand binding site will be discussed in the medicinal chemistry section below. The volume of the ligand binding site of LXR varies substantially depending on the size and shape of the bound ligand. For instance, the ligand binding site of LXRβ bound to 1, has a volume of 466 Å3 (PDB ID: 1PQ9),84,88 while the complex with 3 has a volume of 831 Å3 (PDB ID: 1PQC).76,88 The major conformational change responsible for the enlargement of the ligand binding pocket in 1PQC is the rotation of the Phe329 side chain. This rearrangement of the Phe329 side chain was also observed for two other LXRβ structural entries (3KFC and 3L0E). The diphenylmethyl group of 3 promotes movement of the side chains of Phe271 and Phe340, in addition to Phe329. Furthermore, the helix 2/β-sheet region of the ligand binding site displays a high degree of flexibility, while the region near the His435 (helix 11)/Trp457 (helix 12) switch is more rigid. In summary, the available co-crystal structures and molecular dynamic simulation



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studies88-90 suggest that the receptor adopts a multitude of conformations and thus can accommodate ligands with varied shapes and dimensions.

Medicinal Chemistry

Steroidal Ligands Naturally occurring oxysterols, such as 24(S),25-epoxycholesterol 2 (Fig. 1), are endogenous LXR ligands.91,92 The earliest reported medicinal chemistry efforts directed towards LXR explored natural and synthetic oxysterols.2,93 The importance of a hydrogen bond acceptor on the steroid side chain was established in these studies. Subsequently an X-ray crystal structure of 2 bound to the LXRβ LBD (PDB ID: 1P8D) revealed a hydrogen bond between the epoxide oxygen and NHε of His435.78 The hydroxyl group of 2 is oriented towards a polar sub-cavity near helix 1 to form a hydrogen bond with the side chain carbonyl of Glu281. Very modest isoform selectivity was identified among these early steroidal analogs. For example, diepoxide 5 (Fig. 5) has a Ki values of 390 and 1700 nM against LXRα and β respectively. Furthermore, this compound activates LXRα with an EC50 of 7 µM but has little effect on LXRβ up to 40 µM. N,N-dimethylamide 6 was one of the more potent compounds identified in early studies, with LXRα and β binding Ki values of 130 and 100 nM. Incubation of HepG2 cells with 10 µM of 5 resulted in an approximately 3-fold induction of ABCA1 mRNA compared to vehicle treated cells, similar to the effect seen with 1 (Table 1).94 However, the SREBP1c mRNA induction in this cell line with 6 was much reduced compared to 1 (3.3 vs 26). A similar experiment in THP1 cells also showed reduced SREBP1c induction compared to 1 (5.4 vs 14.7). The most dramatic effects were observed in murine J774 cells where 6 effected a greater induction of ABCA1 than


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1 (10.3 vs 4.8) combined with a reduction in SREBP1c mRNA below vehicle control. Concentration response experiments in HepG2 cells demonstrated that 6 is less potent than 1 as an inducer of both ABCA1 and SREBP1c; however, the effects on activation level diverge. In particular, 6 shows a much reduced activation level (33%) against SREBP1c. In chow-fed mice, 6 dosed intraperitoneally (i.p.) at 50 mg/kg/day for 6 days gave a similar induction of ABCA1 in liver compared to 1 administered orally at the same dose (1.8 vs 2.2), combined with much less induction of SREBP1c (1.5 vs 12.3). Consistent with this result, circulating and liver triglycerides were unchanged in mice treated with 50 mg/kg/day of 6.

Table 1. Regulation of ABCA1 and SREBP1c by 1 and 5. Cpd

1 6 a

HepG2 ABCA1 FIa 3.2 3.4

HepG2 SREBP1c FIa 26 3.3

THP-1 ABCA1 FIa 12.2 10.9

THP-1 SREBP1c FIa 14.7 5.4

J774 ABCA1 FIa 4.8 10.3

J774 SREBP1c FIa 2.6 0.4

HepG2 ABCA1 EC50(act)b 0.04 (100) 1.8 (139)

HepG2 SREBP1c EC50(act)b 0.01 (100) 0.58 (33)

fold induction at 10 µM. b EC50 in µM, activation level expressed as %, with 1 set at 100%.

Based on its promising profile, the impact of compound 6 on atherogenesis was further studied in apoE -/- mice.95 Mice were fed a Western diet ± 6 such that they received an 8/mg/kg/day dose for 11 weeks. Both male and female mice in the treated group exhibited >45% reductions in lesion areas in aortic valve and >25% reductions in lesion areas in en face aorta preparations. At this dose no increases in liver triglycerides or total cholesterol were observed. Compound 6 was also found to inhibit 3β-hydroxysterol-∆24-reductase, the enzyme that catalyzes the final step in cholesterol biosynthesis.96 This second site of action is responsible for an increase in desmosterol in plasma and feces.



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5α,6α-Epoxycholesterol (7, Fig. 5 is an oxysterol found in processed foods. It was shown to displace tritiated 1 from LXRα with an IC50 of 76 nM, however it was unable to displace 3H-1 from LXRβ.97 Nonetheless, at a concentration of 10 µM, compound 7 was able to recruit certain coregulator peptides to both LXRα and β. Some differences in the coregulator recruitment profile of 7 vs 1 and 2 were observed. Compound 7 was demonstrated to act as an antagonist of several LXR mediated genes in Huh7 and A549 cells; however in THP-1 cells, it functioned as a partial agonist of ABCA1 expression. Inspired by the structure of 1, the closely related compounds ATI-829 (8) and ATI-111 (9), bearing hexafluoroisopropanol groups on the steroid side-chain, were synthesized.98 Compound 8 is a more potent agonist of LXRα in a luciferase reporter gene assay in HEK293 cells than 2, and almost as potent as 1. In a similar assay against LXRβ, 8 is approximately equipotent to 2 and distinctly less potent than 1.98 In HepG2 cells at 1 µM, 8 induced a 2 fold increase in SREBP-1c mRNA vs 12-fold for 1; however, in THP-1 cells, 8 and 1 induced 8- and 11-fold increases in ABCA1 mRNA respectively. Compound 8 was demonstrated to be orally bioavailable in mice when delivered as a microemulsion, with a half-life of about 7 h. The livers of LDLR-/- mice which had been gavaged with 10 mg/kg/day of 8 for 2 weeks showed no significant change in the expression of LXR target genes, including SREBP-1c and other lipogenic genes, and no increase in liver triglycerides. At the same dose of 8, statistically significant reductions in atherosclerotic lesion area in the innominate artery and the aortic route were measured. Introduction of unsaturation into the side chain of 8 afforded 9.99 In a reporter gene assay, 9 proved to be more potent against LXRα than 1 with an EC50 of 60 nM; however, against LXRβ, its EC50 was 700 nM. Treatment of LDLR-/- mice fed an atherogenic diet with 5 mg/kg/day by


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mouth (p.o.) of 9 for 8 weeks gave a 1.5-fold increase in ABCA1 mRNA in peritoneal macrophages and a ~2-fold increase in SREBP-1c expression in liver. Despite the increase in SREBP-1c mRNA, no accumulation of cholesterol or triglycerides in liver was observed. This may be a result of inhibition of SREBP-1c protein processing by 9. At the same dose, atherosclerotic plaque size in the innominate artery, ascending aorta and aortic root were reduced by 78%, 67% and 54% respectively. In terms of isoform selectivity, the steroidal LXR ligands discussed above range from nonselective to modestly α-selective. Their alogP values exceed 4.5. As steroid analogs, they may be prone to off-target pharmacology through cross reactivity with other nuclear receptors or interaction with steroid processing enzymes e.g. inhibition of 3β-hydroxysterol-∆24-reductase by 6.

Natural Products and Derivatives A number of non-steroidal natural products and have been described as LXR modulators, 67,100, 101, 102

however, few of these have spawned medicinal chemistry efforts. Apparent LXR binding

in a sample of podocarpic acid was traced to contamination with 10 (Fig. 6), the anhydride of podocarpic acid which had binding IC50s of 2 and 1 nM against LXRα and β, respectively (Table 1).103 It also functioned as a potent agonist of both isoforms in Gal4 assays. Replacement of the anhydride with an imide afforded 11, which was slightly less potent in Gal4 assays than 10. Imide 11 had 22% oral bioavailability in rat. Administration of 10 mg/kg twice daily (b.i.d.) for 8 days to hamsters and mice increased HDL cholesterol levels by 30 and 19% respectively. Plasma triglycerides were increased by 51 and 36% in the two species. Compound 12, the bis(acetate) of 10, was also potent and was demonstrated to elevate ABCA1 mRNA expression



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in THP-1 cells to a much greater extent than the natural oxysterol 22-(R)-hydroxycholesterol.104 The compound also stimulated cholesterol efflux from cholesterol loaded human fibroblasts with an apparent EC50 value of ~1 nM. A library of podocarpic acid amides was prepared in an attempt to replace one of the podocarpate moieties in 11 with a simpler group. The compounds prepared were less potent in binding e.g. 13.105 In an HTRF assay, 14 recruited the coactivator peptide SRC1 to LXRα with an EC50 of 50 nM, but was ineffective at recruiting this cofactor to LXRβ. Nonetheless, in Gal4 assays, 13 functioned as an agonist on both isoforms. These compounds have very high alogP values and show little evidence of any isoform selectivity.

Table 2. Potency of podocarpic acid derivatives. LXRα Binding LXRβ Binding LXRα Gal4EC50a LXRβ Gal4EC50a IC50a IC50a (FIb) (FIb) 2 1 1 (50x) 1 (85x) 10 1 1 50 µM against LXRα and β respectively.41 Furthermore 34 is a full agonist of LXRα with an EC50 of 1.2 µM, while displaying little activation of LXRβ at 50 µM. Subsequently X-ray structures of 32 (PDB ID: 3IPS) and 34 (PDB ID: 3IPU) bound to LXRα were reported.80 The former structure has a resolution of 3.2 Å, with missing residues in H1 (Gln222 and above), while the latter structure is better resolved at 2.7 Å. In both cases the benzisoxazole nitrogen and oxygen atoms lie in the vicinity of His421, although not within hydrogen bond distance. However, thioether 32 is is about 4 Å shorter than urea 34, and the aryl(alkyl)carboxylic acid portions of the two ligands induce different conformational changes in the protein, resulting in distinct ligand protein interactions. In the complex of 32, the terminal carboxylic acid interacts with either the backbone NH of Leu316 in the A chain or Arg305 in the B chain. In either case, the observed hydrogen bond interaction was solvent exposed. Although the urea moiety of compound 34 binds to a similar position as the thioether in 32, it forms novel water-mediated hydrogen bonds to the Leu260 backbone carbonyl oxygen and to the Arg232 Nɛ atom. This is presumably a strong network since it is buried in a hydrophobic pocket. The terminal acid group of 34 is located further outside of the polar sub-cavity near helix 1, where it may interact with Asn225 and Lys317.


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All three examples of this chemotype have MW < 500 and 33 and 34 have alogP < 5. The carboxylic acid can be expected to impart some water solubility. Although starting compound 32 has no isoform selectivity, 34 is remarkably LXRα-selective.

Tertiary Amine Carboxylic Acids A group at GlaxoSmithKline (GSK) employed parallel solid phase synthesis to optimize the cell potency of a screening hit to give tertiary amino acid 3 and the corresponding carboxamide GW6340 (35, Fig 12).126,127 Compound 3 has an EC50 of 190 nM (67%) against LXRα in a Gal4 assay. X-ray structures of 3 bound to both LXRα (PDB ID: 3IPQ)80 and LXRβ (PDB IDs: 1PQ6, 4NQA)76,86 are publicly available. The similarity between the three bound structures provides an indication of the challenge of designing isoform selective compounds. An overlay of the LBDs of the full length heterodimer (4NQA) and hLXRβ homodimer (1PQ6) structures revealed almost identical binding pockets with little side chain movement. The RMSD of Cα atoms between the corresponding residues in the two structures is only 0.96 Å. In all three structures, the chloro(trifluoromethyl)benzyl moiety forms hydrophobic interactions with the pocket near the His421/Trp443 (LXRα) switch of helix11/12, while the terminal acetic acid moiety of 3 interacts with Arg305, Glu267 and the backbone nitrogen of Leu316. Compound 3 is selective against a panel of 12 nuclear receptors but does activate LXRβ and PXR. The compound has 70% oral bioavailability in mice when dosed at 10 mg/kg p.o and when administered b.i.d. at this dose it induced increased expression of ABCA1 by 8x in intestine and 7x in peripheral macrophages. Plasma levels of HDLc increased by 30% after 3 days and remained at this level until the end of the study at 14 days.



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The analogous carboxamide 35 has an EC50 of 425 nM against LXRα in a Gal4 assay. This compound was shown in a mouse study to activate LXR dependent genes, such as ABCA1, in intestine but not in liver.128 Despite the prominent role tertiary amine 335, 61, 126,129,130 has played in development of the biology of LXR agonists, only limited additional work on this series has been reported. Introduction of a (R)-methyl group on the propylene linker gave 4 which was ~2x more potent than 3 against both LXR isoforms (Table 4) in a FRET assay measuring recruitment of SRC1 and in a functional assay measuring cholesterol efflux from mouse 264.7 macrophages.127 Further methylation adjacent to the carboxylic acid gave 36 which had EC50s of 9 and 11 nM against LXRα and β and was fully efficacious. In the mouse macrophage cholesterol efflux assay 36 was at least 10x more potent than 3. To better fill the binding pocket the phenyl acetic acid moiety in 3 was replaced with various heterocycles, including an indole and indole acetic acid affording 37 and 38.81 These compounds were full agonists with little isoform selectivity. Compound 38 was 45% orally bioavailable in rat. An X-ray structure of 38 bound to LXRα (PDB ID: 3FC6) was solved. One of the carboxylate oxygens of 38 forms a hydrogen bond with the backbone NH of Leu314, while the other forms a salt bridge interaction with Arg303 and Asn223.


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Table 4. Potency of GSK LXR modulators

Cpd

LXRα LXRβ Cholesterol efflux a a EC50 (act) EC50 (act) EC50b 63 (110) 40 (110) 29 1 200 (100) 40 (100) 29 3 74(100) 25 (89) 17 35 9 (100) 11 (100) 550 and alogP > 5 and potency appears to be driven by hydrophobic interactions. In addition, the molecules are flexible with >10 rotatable bonds each. Nonetheless, the amine and carboxylic acid moieties presumably impart some water solubility to the molecules and oral bioavailability has been demonstrated. Most of the compounds show weak selectivity towards LXRβ.

Quinoline Carboxylic Acids Structure based optimization of a screening hit by a group at Wyeth led to the discovery of quinoline carboxylic acids 39 (WAY-254011) and 40 (Fig. 13).131 Compound 39 binds potently to both LXR isoforms with 4.5x selectivity for LXRβ (Table 5). In Gal4 assays, isoform selectivity was reduced to 2.5x and EC50 values were more than 25x lower than the corresponding binding IC50 values. In a cellular assay against full length human LXRβ (hLXRβ), 39 was a full agonist with an EC50 of 70 nM. Compound 40, in which the ether linkage was replaced by an amino group, had similar binding IC50 values, but was up to 2x more



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active when tested in cellular assays. Both compounds were potent in functional assays measuring ABCA1 expression in THP-1 macrophages and SREBP-1c expression in Huh7 cells, but neither exhibited useful gene selectivity. Both 39 and 40 were agonists for all three isoforms of PPAR with EC50s ranging from 320 to 1320 nM. The oral bioavailability of 39 was 67% in mouse. Oral administration of 10 mg/kg/day of 39 for 8 weeks to LDLR knockout mice raised on a high fat diet resulted in a 45% reduction in atherosclerotic lesion burden vs control. An X-ray structure of 39 bound to hLXRβ indicated that the quinoline nitrogen interacted with His435 while the acetic acid moiety formed a network of hydrogen bonds with the backbone NH of Leu330 and the side chain of Arg319. The benzyl group occupied a hydrophobic pocket formed by Phe340, Phe349 and Phe271. The binding pose of 40 bound to hLXRβ was identical to that of 39.132 Modifications to the acetic acid region of 39 were undertaken to take advantage of the residue difference between LXRα (Val263) and β (Ile277) in this region.133 Thus, replacement of the phenyl acetic acid with a naphthalene acetic acid gave 41 which had improved binding selectivity for LXRβ over LXRα; however, isoform selectivity in a Gal4 assay was not improved. Furthermore, an increase PPARγ potency was observed: EC50 84 nM (54%). Efforts were made to overcome the PPAR liability by replacement of the acetic acid moiety. Morpholine benzamide 42 retained comparable potency to 39 in binding and Gal4 assays but improved potency in the macrophage ABCA1 assay.134 An X-ray structure of compound 42 bound to hLXRβ reveals that the amide carbonyl oxygen forms a hydrogen bond to the backbone NH of Leu330. The acetic acid moiety could also be replaced with a 2-hydroxy-2-propyl group (43) with retention of binding potency and improved macrophage ABCA1 potency, but reduced isoform selectivity.135


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Replacement of the quinoline with a cinnoline and incorporation of an indole ring gave 44 which had >70x selectivity for binding to LXRβ vs LXRα.136 In Gal4 assays, EC50 selectivity was reduced to 8x, although the activation level was also lower for LXRα (23%) than for LXRβ (67%). Compound 44 was free of PPAR cross reactivity. Unfortunately, the half-life of 44 in mouse liver microsomes was < 5 min, and no plasma exposure was detectable in mice after oral administration of 10 mg/kg. The quinoline carboxylic acids (39-41) and related compounds (42-44) have MW values >500, alogP values >7 and aromatic ring counts of 5 or 6. However, compounds 41 and 44 are of interest because of the >20x selectivity for binding to LXRβ that they exhibit (Table 5). Pyridone carboxylic acid 45 was disclosed as a pan full agonist of both LXR isoforms with EC50s in the 1 – 2 µM range and was used as an assay standard.137

Sulfones A substantial proportion of recently described LXR modulators incorporate a methyl phenyl sulfone moiety. X-ray structures of representative compounds (PDB IDs: 3KFC and 3L0E) indicate that the sulfone occupies a similar region of the binding site to the carboxylic acid function in the compounds discussed above and one of the sulfone oxygens forms a hydrogen bond with the backbone NH of Leu 330 (LXRβ), while the methyl group occupies a small hydrophobic pocket. Early examples of this chemotype, illustrated by 46 and 47, were discovered by workers at XCeptor and Exelixis.138-139 Comparison of the structure of 46 to that of 45 strongly suggests that the methyl sulfone was discovered in an effort to replace the acetic acid moiety. Further development of this chemotype at Exelixis and BMS led to analogs such as 48 which is reported



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to be a partial agonist of LXRα with an EC50 of 1000 68 3.0

9 2.1 1.9 5 1.9 3.3 14 0.92 2.8 49 7 2.7 16 5 4 41 53 4.4 2.6

IC50 in nM.

macrophages.

b

LXRα Gal4 EC50 (act)b

LXRβ Gal4 EC50 (act)b

240 (90) 160 (82) 685 (57) 345 (90)

90 (63) 87 (70) 355 (48) 138 (80)

8320(23)

2540 (25)

1160 (67) 11 (78) 214 (44) 840 (37)

3000 (57)

630 (72)

Macrophage ABCA1 EC50 (act)b 44 (100)c 84 (155)c 33 (151)c 280 (75)c 34 (96)d 12 (122)d 68 (109)d 5 (108)d 70 (87)d 125 (99)c 425 (126)c 425 (104)c 440 (107)c

>1000 (3.1) 525 (73)

85 (94)d 1900 (91)d 1090 (79)c 77 (94)d

>1000 (1)

EC50 in nM, activation level expressed as % with 1 set as 100%. d

c

Human THP-1

Mouse J774 macrophages.

In the course of the Wyeth group’s effort to replace the carboxylic acid in their LXR modulators (vide supra), they also discovered promising compounds incorporating a methyl sulfone.82 For example, 56 (Fig 16) which is closely related to earlier compounds such as 39-44, was very potent in the binding assay but had little isoform selectivity (Table 5). It was also a potent agonist in the Gal4 LXRβ assay and induced ABCA1 expression in a J774 mouse macrophage cell line. However, in rat and human liver microsomes its half-lives were 16 and 12 min. Deletion of the benzyl group gave 57 which retained good binding potency against LXRβ with much improved isoform selectivity. It displayed moderate cellular agonism and metabolic stability.82 An X-ray crystal structure of 57 bound to hLXRβ (PDB ID: 3KFC, Fig. 2) reveals the expected interaction of the quinoline nitrogen with His 435 seen previously in the complex of


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56 with LXRβ In addition, a hydrogen bond is observed between one of the sulfone oxygens and the backbone NH of Leu 330. The quinoline ring system in 57 was replaced with imidazopyridine (58)144 and benzimidazole (59,60)145 rings in an effort to discover more polar, water soluble analogs. Compound 58 bound almost 50x more weakly to LXRβ than 57, although it has substantially improved water solubility of >100 µg/mL. Less binding potency was sacrificed with benzimidazole analogs 59 and 60, however, EC50s for ABCA1 induction in THP-1cells were >400 nM, while EC50s for triglyceride accumulation in HepG2 cells were 271 and 218 nM respectively. Introduction of a polar carboxamide substituent onto the 3-position of the 8-chloroquinoline analog of 57 afforded 61.146 This compound had an LXRβ IC50 of 16 nM and 34x selectivity over LXRα; however, as observed with earlier analogs, the selectivity in Gal4 assays was much reduced, to 5x in this case. Compound 61 was shown to be orally bioavailable and did not penetrate the brain. It effectively raised ABCA1 levels and stimulated cholesterol efflux from THP-1 cells with EC50s of 440 nM (107%) and 10 nM (71%) respectively. Its EC50 for triglyceride accumulation in HepG2 cells was 1250 nM (68%). By contrast, 1 raised ABCA1 levels and stimulated cholesterol efflux from THP-1 cells with EC50s of 44 nM (100%) and 3 nM (100%) and its EC50 for triglyceride accumulation in HepG2 cells was 137 nM (100%). Despite the improved LXRβ selectivity of 61 vs 1, it did not demonstrate an improvement in selectivity for ABCA1 induction vs triglyceride accumulation and it was deemed unlikely to possess a favorable triglyceride profile in vivo. Another avenue for the introduction of polar substituent was explored in the context of a quinazoline series of which 62 is a prototype.147 This compound gains 30x in LXRβ binding potency from the chlorine substituent and has good isoform selectivity. Additional polar groups



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were introduced by modifying the methyl sulfone into a sulfonamide e.g. 63 or by replacing the methyl group with alkylene chains bearing polar substituents e.g. 64.148 While the Nmethylsulfonamide in 63 retained comparable binding to 62, 64 suffered an 8x loss in potency vs. 62; however, 64 was shown to have much reduced brain penetration compared to close analogs of 62. Further efforts by the Wyeth group led to analogs in which the biaryl ether was replaced by a biaryl system. Quinoxaline WYE-672 (65) has an LXRβ binding IC50 of 53 nM and ≥20x isoform selectivity.149 It showed negligible activation of either isoform in Gal4 assays run in Huh-7 cells but was an agonist against both isoforms when the Gal4 assay was performed in HEK-293 cells with EC50 values and activation levels of 1700 nM (7%) against LXRα and 580 nM (45%) against LXRβ. The standard LXR agonist 1 was equipotent in both cell lines. In THP-1 cells, 65 induced ABCA1 with an EC50 of 1090 nM (79%) but it had very little effect on lipid levels in HepG2 cells. In a coregulator peptide recruitment assay against LXRβ, 65 had a very similar profile to 1 and 61; however, it did not effectively recruit any of the peptides tested to LXRα. In an accelerated atherosclerosis lesion study, conducted in high fat/high cholesterol fed mice, 65 dosed in the feed at 3 mg/kg/day for 8 weeks resulted in a 51% reduction in lesion burden. Plasma cholesterol and triglyceride levels were unaffected. In the duodenum, the compound induced ABCA1, ABCG5 and ABCG8 expression, while having little effect on SREBP1c. Thus 65 demonstrated isoform and tissue selectivity. Biaryl quinoline 66 bound to LXRβ with an IC50 of 4.4 nM and was ~15x more potent on this isoform than LXRα.150 Interestingly, isoform selectivity was lost when small alkyl substituents were introduced at the 3-position of the quinoline. In a Gal4 assay against LXRβ, 66 had an EC50 of 525 nM with an activation level of 73%. It was metabolically stable with t1/2 > 30 min in


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both rat and human microsomes and had ~100% oral bioavailability in mice. In vivo, 66 decreased lesion progression by 59% at 10 mg/kg/day but also raised triglyceride levels. The Wyeth group also described analogs which incorporated an ester linkage for dermatological uses.151 The ester functionality allow for rapid metabolic clearance, minimizing systemic exposure. One example, compound 67, had excellent binding potency, with no isoform selectivity. This compound induced ABCA1 expression in human foreskin fibroblasts and KERTr cells with EC50 values of 1 and 2.8 nM, respectively. Workers at Vitae reported methyl sulfone LXR modulators incorporating fused indole and benzimidazole ring systems (Fig. 17).152, 153 These compounds incorporate the vicinal methylsulfonyl and hydroxymethyl substituents present in 50, 51 and 55. Benzimidazole 68 had an LXRβ binding Ki of 20 nM, with 16x isoform selectivity.152 In a Gal4 assay, the LXRα and LXRβ EC50 values were 340 nM and 13 nM, respectively. Indole 69 had an LXRβ binding Ki of 2 nM with 12x isoform selectivity.153 In a Gal 4 assay, the LXRα and LXRβ EC50 values were 88 nM and 10 nM respectively. The sulfone chemotype encompasses a broad range of structures and calculated properties. MW values range from 427 to 690, while alogP values lie between 3.5 and 7.5. Compounds that combine favorable MW and alogP values include 50, 61 and 68. Furthermore, these three compounds are reported to bind LXRβ with IC50s of 20 nM or better and the latter two display >10x isoform β selectivity. Nonetheless, in functional assays 61 did not show any evidence of improved selectivity over 1. Tissue-selective compound 65 is also promising in vivo although its binding potency is unimpressive.

Indazoles



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Page 34 of 101

A team at Wyeth identified indazole 70 as a promising, low MW lead (Fig. 18).154 The compound exhibits ~7x binding selectivity for LXRβ vs LXRα. In Gal4 assays, its EC50s were 4270 nM (31%) and 3260 nM (53%) against the α and β isoforms, respectively (Table 6). The lower potency and efficacy against the α isoform were regarded as attractive; however, in mice the compound was rapidly cleared by oxidative metabolism of the phenyl rings. Introduction of halogens on the phenyl rings gave 71 (WAY-214950) and 72 (WAY-252623, LXR623) which retained similar potency and selectivity levels in the binding and Gal4 assays combined with improved metabolic stability. Both 71 and 72 were shown to upregulate ABCA1 mRNA expression in THP-1 cells with EC50s ~ 550 nM and to upregulate SREBP1c mRNA in HepG2 cells with EC50s of 1222 nM and 2114 nM. When dosed p.o. at 10 mg/kg 71 had excellent oral bioavailability and moderate clearance. An X-ray structure of 72 bound to hLXRβ revealed that indazole N1 formed an H-bond with His 435 NHε, while the CF3 group experienced attractive electrostatic interactions with the same residue. Some reorganization of side chains of the protein, compared to other X-ray structures, optimized hydrophobic interactions with the ligand.

Table 6. Potency and PK parameters of Wyeth Indazole LXR modulators Cpd 1 3 70 71 72 a

LXRα IC50a 13 100 279 248 179

IC50 in nM.

b

LXRβ IC50a 9 12 41 33 24

LXRα Gal4 EC50 (act)b 140 (100) 660 (58) 4270 (31) 6100 (35) 6660 (53)

LXRβ Gal4 EC50 (act)b 170 (100) 310 (77) 3260 (53) 4150 (64) 3670 (73)

i.v. t1/2

%F (mouse)

1.9 7.9

16 106

EC50 in nM, activation level expressed as % with 1 set as 100%.

Compound 72 was extensively characterized in animal studies. Treatment of LDLR-/- mice fed high fat, high cholesterol diets with 15 mg/kg/day of 72 reduced lesion area by 37% compared to


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vehicle.155 The compound was also administered by gavage to Golden Syrian hamsters, a CETP expressing species, at doses up to 120 mg/kg/day. No effect was observed on serum cholesterol or triglycerides, or on liver weight, cholesterol or triglycerides. In cynomolgus monkeys, 72 dosed for 28 days at 50 mg/kg/day p.o. caused a 45-57% reduction in serum total cholesterol. A statistically significant reduction in LDL cholesterol was observed at this dose while HDL showed a trend towards lower levels. Microarray analysis of tissues from this study revealed differential induction of LXR target genes in duodenum vs liver. For example SREBP1 mRNA expression in duodenum was increased by ~3x, while in liver it was reduced by ~60%. An MRI imaging study of atherosclerotic plaques in New Zealand White Rabbits treated with 1.5 or 5mg/kg/day of 72 for 6 months showed reduced plaque progression, compared to placebo, of a magnitude similar to that produced by simvastatin at 5 mg/kg/day.156 Compound 72 was advanced into a single ascending dose study with healthy participants. Doses ranged from 12.5 to 300 mg. Cmax and AUC were dose proportional. Cmax was achieved after 2 h and the terminal half life was calculated as ~42 h. Activation of ABCA1 and ABCG1 expression was observed at doses of 75 mg and above. At the top two doses (300 and 150 mg), CNS related adverse effects were observed and further development was discontinued. It was not determined whether the CNS symptoms were LXR-mediated or off target effects.157 The indazoles are low MW (

The Medicinal Chemistry of Liver X Receptor (LXR) Modulators

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