Recent Advances in the Medicinal Chemistry of Liver X Receptors

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Recent Advances in the Medicinal Chemistry of Liver X Receptors Miniperspective Bahaa El-Dien M. El-Gendy,*,†,‡ Shaimaa S. Goher,‡ Lamees S. Hegazy,† Mohamed M. H. Arief,‡ and Thomas P. Burris§ †

Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, United States Chemistry Department, Faculty of Science, Benha University, Benha 13518, Egypt § Center for Clinical Pharmacology, Washington University School of Medicine and St. Louis College of Pharmacy, St. Louis, Missouri 63110, United States

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‡

ABSTRACT: Nuclear hormone receptors represent a large family of ligandactivated transcription factors that include steroid receptors, thyroid/retinoid receptors, and orphan receptors. Among nuclear hormone receptors, the liver X receptors have emerged as very important drug targets. These receptors regulate some of the most important metabolic functions, and they were also identified as anti-inflammatory transcription factors and regulators of the immune system. The development of drugs targeting liver X receptors continues to be a challenge, but advances in our knowledge of receptor structure and function move us forward, toward achieving this goal. This review highlights the latest advances in the development of synthetic LXR modulators in the primary literature from 2013 to 2017. In this review, we place great emphasis on the structure and function of LXRs because of their essential role in the drug design process. The structure− activity relationships of the most active and promising synthetic modulators are discussed.

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INTRODUCTION Nuclear hormone receptors (NRs) comprise a superfamily of ligand-activated transcription factors that incorporate a group of 48 members in humans and 49 in mice.1−3 NRs are intracellular proteins, and they all share common structural features and are composed of an N-terminal domain (AF-1), a DNA-binding domain (DBD), a hinge region, a ligand-binding domain (LBD), and a C-terminal domain (AF-2) (Figure 1). Both the DNA-binding domain (DBD) and the ligand-binding domain (LBD) are highly conserved.

as master regulators of lipid and cholesterol metabolism in addition to their notable anti-inflammatory activities.7−11 LXRs belong to the thyroid/retinoid hormone receptor subfamily, and they were isolated initially as orphan receptors, as their natural ligand was not yet identified.12 Following the discovery of the endogenous oxysterols as specific ligands for LXRs, they were subsequently considered deorphanized.13−15 LXRs play a paramount role in many physiological processes. LXRs regulate different metabolic functions, such as cholesterol metabolism,16−19 lipogenesis,20,21 carbohydrate metabolism, and energy metabolism. Moreover, LXRs regulate inflammation and immune function in many cell types and have been identified as anti-inflammatory transcription factors.22,23 LXRs are also involved in the regulation of cell growth, development, reproduction, and homeostasis.24,25 Consequently, LXR ligands may have utility for the treatment of skin diseases,26,27 rheumatoid arthritis,28 antithrombotic,29 Alzheimer’s disease (AD),7,8,30 Parkinson’s disease (PD),31 dyslipidemia,32 atherosclerosis,33−36 and multiple sclerosis (MS).37 In cancer biology, LXR ligands have shown antiproliferative effects on different cancer cell types; the activation of LXRs decreases proliferation by reducing the intracellular cholesterol level necessary for lymphocytes to synthesize its cellular

Figure 1. NR domains.

NRs are classified according to the homology sequence into six subfamilies, including steroid receptors, thyroid/retinoid receptors, and orphan receptors.4,5 The natural ligands of steroid receptors and thyroid/retinoid receptors are wellknown, but those of orphan receptors have not been identified.6 Among the NR superfamily, the LXRs have emerged as very promising drug targets because of their role © XXXX American Chemical Society

Received: January 11, 2018 Published: July 13, 2018 A

DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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membrane during proliferation,38 and in glioblastomas, the activation of LXRs decreases cholesterol levels and hence promotes tumor cell death.7,39−42 Alternatively, the activation of LXRβ induces the transcription of tumoral and stromal apolipoprotein E (ApoE), which is a strong suppressor of melanoma metastasis. The administration of LXR agonists leads to the suppression of tumor growth and metastasis in a broad spectrum of melanoma lines.43 LXR/ApoE activation using a potent LXRβ agonist currently in clinical trials, RGX104, was found to reduce myeloid-derived suppressor cells (MDSCs) abundance by increasing their apoptosis. In human cancer patients with metastatic cancers, RGX-104 depleted MDSCs and activated T cells in a remarkable way. In mice, RGX-104 enhanced the efficacy of checkpoint inhibitors without noticeable signs of toxicity.44 The vast majority of research conducted to find LXR modulators of therapeutic utility has been directed toward developing LXR agonists. A major drawback of using LXR agonists as drugs is the elevation of plasma triglycerides and hepatic steatosis due to the direct regulation of the lipogenic pathway by LXR in the liver. This undesirable effect has impeded its development into commercial drugs. To overcome this hurdle, new strategies have been developed such as developing LXRβ-selective agonists and tissue-selective agonists. Antagonists have been mainly used as chemical probes to study the biology of LXRs. These antagonists block the LXR agonists from binding to the ligand binding domain and prevent them from inducing conformational changes in the receptor. Inverse agonists repress transcription by recruiting corepressor proteins when bound to the LBD of a constitutively active receptor such as LXR. Inverse agonists of LXRs are fairly new modulators that were first developed by our group and showed potential for the treatment of nonalcoholic fatty liver disease (NAFLD),45 nonalcoholic steatohepatitis (NASH),46 and different kinds of cancers without noticeable side effects in preclinical studies.21,47

cholesterol 7α-hydroxylase, carbohydrate regulatory element binding protein (ChREBP), apolipoprotein E (ApoE), cholesteryl ester transfer protein (CETP), sterol regulatory element-binding protein 1c (SREBP-1c), and ATP-binding cassette (ABC) transporters.15 LXRs regulate gene expression in different ways. In the unliganded state, the LXR/RXR heterodimer bound to LXR response element (LXRE) interacts with nuclear receptor corepressor (NCoR) or with the silencing retinoic acid and thyroid hormone receptor mediator (SMRT).56 This interaction is followed by the recruitment of histone deacetylase (HDAC) through interaction with the stress-activated MAP kinase interacting protein 3A (Sin3A) and consequent blocking of transcription.9,23,35,36,57 Upon binding with agonists, conformational changes occur and result in the dissociation of the corepressor complex associated with LXRE. The coactivator complex, such as activating signal integrator-2 (ASC2),58 receptor-integrating protein140 (RIP140),59 or steroid receptor coactivators (SRC),60 is then recruited onto helix 12 (H12) of the LBD, leading to activation of the transcription of target genes.61 Upon binding with antagonists or inverse agonists, LXR-corepressor recruitment occurs, and the expression of target genes is downregulated to below basal levels.21,45 Activation of LXRs by ligands results in the transrepression of inflammatory pathways by the inhibition of signal-dependent transcription factors such as nuclear factor κB (NF-κB). For LXRs to interact with the corepressor docked to the transcription factor, such as STAT1 complex, and to consequently inhibit the proinflammatory pathways, LXRs must first be SUMOylated. In this regulatory mechanism of SUMOylation, LXRs are conjugated to small ubiquitin-like modifier 2 or 3 (SUMO2 or SUMO3), a step required for their transrepression and consequent inhibition of the proinflammatory pathways.9,62,63 Several X-ray crystal structures of LXRs were obtained and deposited in the Protein Data Bank (PDB) in multiple forms; as a monomer, homodimer, or heterodimer (Table 1).64 The ligand-binding domain in the reported X-ray crystal structures was complexed with agonists or partial agonists, but no X-ray crystal structures have been reported for antagonists or inverse agonists. The ligand-binding pocket (LBP) of LXR is mainly hydrophobic, with a few polar amino acid residues that make hydrogen-bonding interactions with the bound ligand. We used SiteMap65 to calculate the volume of the active site in two different X-ray structures to gain knowledge about the binding cavity. It became obvious that the binding cavity can accommodate different ligand sizes.66,67 For example, in the case of small ligands such as T0901317 (1) (Figure 2), the LXRβ binding pocket volume is 266 Å3 (Figure 3A), while with larger ligands such as GW3965 (2) (Figure 2), the volume is 533 Å3 (Figure 3B). In addition, based on the bound ligand, a polar tunnel cavity that is composed of the solventexposed residues opens (Figure 3).68 Upon binding with an agonist, H12 of the LBD adopts a conformation that allows the binding of coactivator protein (Figure 4A). Studying the crystal structures of the LBDs complexed with either steroidal or nonsteroidal agonists provided important information about the most common interactions that were found to activate LXR. The hydrogen bond between the ligand and His421 in helix 10 (H10) for LXRα or with His435 for LXRβ in helix 11 (H11) is essential

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LXR STRUCTURE AND FUNCTION LXRs are known as nuclear oxysterol receptors and have two isoforms: LXRα (NR1H3), initially named OR-1, and LXRβ (NR1H2), initially named NER and UR.48−51 In humans, LXRα consists of 447 amino acids and is highly expressed in the liver, kidney, intestine, adipose tissue, and macrophages.12,50 LXRβ consists of 460 amino acids and is expressed ubiquitously in most tissues.49,52 Similar to other nuclear receptors, LXRs are composed of four functional domains (Figure 1): (i) the N-terminal domain (A/B domain); (ii) two zinc finger DNA-binding motifs with their DNA-binding domain (DBD); (iii) a hinge region; and (iv) a globular ligand-binding domain that also contains AF-2 (activating function 2), responsible for the recruitment of coactivators.7,53−55 Both the LBD and DBD of the two LXR isoforms share approximately 74−75.6% of their amino acid sequence identity and differ by only two residues in the ligandbinding pocket: V261/I277 (LXRα/LXRβ) and V295/I311 (LXRα/LXRβ).54 LXRs form a heterodimer with the 9-cis retinoic acid receptor RXR. The LXR/RXR heterodimer is activated by ligands for either LXR or RXR and is thus considered “permissive”. Many target genes are involved in the regulation of different biological processes by LXRs in both normal and pathological functions. Among those target genes are fatty acid synthase (FAS), cytochrome P450 isoform 7A1 (CYP7A1), B



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Table 1. Available LXR X-ray Structures PDB code

ligand effect

receptor crystallized form

ref

1P8D 1PQ6 1PQ9 1PQC 1UHL 1UPW 1UPV 2ACL 3FAL 3FC6 3IPS 3IPU 3IPQ 3KFC 3L0E 4DK7 4DK8 4NQA 4RAK 5AVI 5AVL 5HJP 5HJS 5I4V 5JY3 5KYA 5KYJ

agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist agonist partial agonist agonist partial agonist agonist agonist agonist agonist agonist partial agonist agonist agonist

LXRβ monomer LXRβ homodimer LXRβ homodimer LXRβ homodimer LXRα-RXRα heterodimer LXRβ monomer LXRβ monomer LXRα-RXRα heterodimer LXRα-RXRα heterodimer LXRα-RXRα heterodimer LXRα homodimer LXRα homodimer LXRα homodimer LXRβ homodimer LXRβ homodimer LXRβ homodimer LXRβ homodimer LXRβ-RXRα heterodimer on DNA LXRβ homodimer LXRα homodimer LXRα homodimer LXRβ homodimer LXRα -RXRα heterodimer LXRβ homodimer LXRβ homodimer LXRβ homodimer LXRβ-RXRβ heterodimer

69 70 70 70 61 67 67 71 72 73 74 74 74 75 76 64 64 77 78 79 79 80 80 81 82 83 83

Figure 3. Hydrophobic (yellow), donor (blue), and acceptor (red) maps for the cavities in the LBD of LXRβ. The protein is shown in ribbon representation, and the picture background is shown in black. The ligand is shown in stick representation. (A) The ligand-binding pocket of LXRβ bound to small agonist 1 (PDB code 1PQC). (B) The ligand-binding pocket of LXRβ bound to larger agonist 2 (PDB code 1PQ6), where a polar tunnel cavity opens.

The ligands that bind tightly to LXR and prevent H12 from adopting an agonist conformation may act as antagonists.84 In addition, it was observed that the larger is the size of the bound ligand, the more the antagonism or partial agonism effect was observed.76 This finding may be attributed to the perturbation of the LBD of the receptor by larger ligands. For instance, the high-affinity LXR antagonist 3 possesses a larger size, hydrophobic property, and bulky substituent than all agonists in the same sulfonamide series developed by Zuercher et al.76 It is likely that larger ligands such as 3 can perturb the binding pocket of the protein, which constitutes helix 3 (H3), H10/11, and the H12 loop and can prevent the binding of coactivator proteins, resulting in partial agonism or antagonism behavior.76 Although no X-ray crystal structures for LXR inverse agonists have been reported, molecular dynamics simulations that predict the binding mode and molecular basis of an inverse agonist 4 (27-norcholesteonic acid) were described recently.85 Upon binding with this molecule, the key interactions between His435 in H11 and Trp457 in H12 were disrupted, causing structural changes in the overall confirmation of H12 and in the H11/H12 loop. In addition, this disruption caused dynamic changes in the protein

for LXR activation, and the bond strength defines the stability of the ligand−protein complex. This interaction positions the histidine imidazole to make edge-to-face π-electron cloud interactions with the indole of Trp457. Previous studies have shown that the two conserved amino acids His435 and Trp457 interact within the ligand-binding pocket and function as an activation switch that drives the conformational rearrangement of the AF-2 domain (Figure 4B). With shorter hydrogen bond distance, the histidine imidazole is properly positioned 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 H12 and the AF-2 domain in the active agonist conformation.66,69,78,83

Figure 2. LXR Agonists 1−4. C



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Figure 4. (A) Secondary structure of LXRβ ligand-binding domain (LBD) bound with the agonist {2-[(2R)-4-[4-(hydroxymethyl)-3(methylsulfonyl)phenyl]-2-(propan-2-yl)piperazin-1-yl]-4-(trifluoromethyl)pyrimidin-5-yl}methanol (28) (green). H12 (red) exists in active conformation to allow coactivator binding (blue) (PDB code 5I4V). (B) Interactions of the ligand with key residues in the LBD. Hydrogenbonding interactions with His435 and Glu281 are shown as blue lines, and hydrophobic contacts are demonstrated with orange lines.

Figure 5. LXR pyrazole agonists 5−12.

The first modification was to replace the o-chlorine atom on the phenyl ring in 7 with the trifluoromethyl (CF3) in 8. This simple modification increased the binding selectivity of LXRβ 15-fold. Replacement of the CF3 on the pyrazole ring in 8 with the ester group in 9 decreased the stability of the compound in mouse microsomes. However, replacement of the same group with dimethyl carbinol in 10 increased the LXRβ binding selectivity 18-fold over 9. Later, the thiophene ring was replaced with a phenyl to provide the biphenyl sulfones 11 and 12 (Figure 5). This ring replacement improved the LXRβ selectivity 10- to 14-fold in 11 and 12. Both compounds displayed similar selectivity, but 11 was found to be more potent, with an EC50 of 49 nM in a cellular transactivation assay.78 Stachel et al. developed a series of agonists to selectively target LXRβ in the brain for the treatment of Alzheimer’s disease (Figure 6).80 To measure the isoform selectivity for LXRβ over LXRα, the authors focused on measuring Emaxbased selectivity instead of potency-based selectivity because the ligand-binding domains of both isoforms are highly conserved. First, the authors identified compound 13 from the HTS of their library collection to be moderately selective toward LXRβ in the cofactor recruitment assay (CFR) (Table

backbone to a larger extent than what occurred by complexation with an agonist.85

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MEDICINAL CHEMISTRY OF LXR MODULATORS LXR Agonists. Wrobel et al. developed LXR-623 (5) as a potent LXR agonist (Figure 5).86 Compound 5 showed potency toward both LXRα (179 nM) and LXRβ (24 nM), with 7-fold selectivity for LXRβ. To the best of our knowledge, 5 was the first LXR agonist with published clinical trial results from a single ascending dose trial and was tested in humans, where it showed upregulation of the LXR target genes ABCA1 and ABCG1 in a dose-dependent manner.87 Upregulation of these genes enhances the process of reverse cholesterol transport (RCT) and could inhibit or delay the progress of atherosclerosis. Although 5 showed adverse CNS effects and did not go to market; it was a hallmark in validating LXRs as an important therapeutic target. Pyrazole agonists 6 and 7 are structurally similar to 5 and were developed as highly potent LXR agonists (Figure 5).88 Pyrazole 7 was found to be a potent LXRβ agonist, with EC50 = 108 nM and 90% efficacy. To increase LXRβ selectivity, many analogs were synthesized using array synthesis and single compound synthesis. D



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susceptibility was reduced significantly, and LXRβ selectivity was preserved as measured via CFR assay (Table 2).80 This compound exhibited good CNS penetration properties and a clean ancillary profile. When compound 16 was tested in an animal model of Alzheimer’s disease, the brain ApoE and ABCA1 levels increased significantly without raising liver triglyceride levels. Additionally, when tested in a rhesus monkey model, ApoE and amyloid-β peptides changed positively, while the liver triglycerides were not elevated. In light of these findings, improving the LXRβ selectivity would help to overcome the adverse side effects resulting from the undesirable activation of LXRα in the liver. In cell-based transactivation assays, the S diastereoisomer of 16 shows similar activity against LXRα (EC50 = 2.0 μM and Emax = 31%) and much lower activity against LXRβ (EC50 = 0.84 μM and Emax = 117%). The crystal structures of compound 16 in complex with the LXRα homodimer and the LXRβ/RXR heterodimer were determined at 1.7 and 2.6 Å resolution, respectively (Figure 7). Compound 16 binds the ligand-binding domain of LXRα and LXRβ in a similar manner; it forms hydrophobic π−π and alkyl−π interactions with hydrophobic residues in the active site such as Phe329 and Met312. Compound 16 makes hydrogen-bonding interactions with Leu330, Arg319 and His435 (LXRβ numbering). No noticeable differences were observed in the interaction of compound 16 with the His435Trp457 activation switch in both LXR isoforms. The authors explained isoform selectivity as being achieved via the cumulative effect of small conformational differences of 16 when bound to LXRα and LXRβ (Figure 7). If we compare compound 15 to compound 16, then the only difference is the rigid phenyl- versus the conformationally flexible isopropyl trifluoromandelate group. Therefore, the high isoform selectivity observed in 16 is most likely due to the observed differential flexibility of the isopropyl trifluoromandelate group.80 Moreover, the S enantiomer of compound 16 was 2-fold less active against LXRβ than the R enantiomer (16) in cell-based transactivation assays, which means that the spatial arrangements of the isopropyl trifluoromandelate group play a crucial role in achieving higher isoform selectivity. Different stereochemistry around the amide carbonyl of 16 that forms a hydrogen bond with His435 would affect the strength of the

Figure 6. Piperazine agonists 13−16.

2). This hit was not suitable for in vivo studies because of insolubility and weak potency. A remarkable increase in Table 2. Emax Selectivity of 13−16 from the Cofactor Recruitment Assay (CFR) compd

LXRα EC50 (μM)

Emax (%)

LXRβ EC50 (μM)

Emax (%)

13 14 15 16 (R)

1.48 0.041 0.50 1.9

50 62 24 49

0.94 0.008 0.55 0.03

106 67 91 103

binding was achieved upon replacement of the tert-butyl carbamate in 13 with the mandelate group in 14, as shown by a significant left shift in EC50. However, the solubility did not improve, and the Emax selectivity was lost, as shown in the CFR assay (Table 2). Replacing the cyanopyridyl ring with a bispiperidine resulted in the formation of compound 15, which has much better solubility and similar selectivity compared to 14 when measured in a transactivation assay. Compound 15 suffered from low CNS penetration because of liability to Pglycoprotein (Pgp). To overcome this problem, compound 16 was synthesized with an isopropyl trifluoromandelate replacing the phenyl trifluoromandelate group. This compound was obtained as a mixture of enantiomers and was subjected to chiral separation to obtain the R enantiomer, which is the most active form. Pgp

Figure 7. Compound 16 interactions in the LBD of LXRα (green) and LXRβ (pink) (Protein Data Bank entries 5HJS and 5HJP, respectively). Residue numbering is based on LXRβ numbering. Hydrogen-bonding interactions are shown as blue lines. E



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Scheme 1. Design Strategy of Piperazine Core Agonists Developed by Zheng et al.

hydrogen bond and might slightly disrupt the His435-Trp457 switch. Zheng et al. used Contour, a structure-based drug design platform, to identify new drug-like lead molecules by assembling fragments in the protein-binding pockets that naturally complement hydrophilic and hydrophobic features of the protein-binding site.81 The algorithm of Contour uses dynamic vector selection (DVS) and dynamic fragment selection (DFS) as two methods of dynamic growth to generate novel scaffolds that best fit the binding site. They were able to develop some novel potent, bioavailable, and orally active LXRβ selective agonists. The novel agonists were designed to induce the reverse cholesterol transport (RCT) without increasing the triglyceride formation in both liver and plasma. The modeling study used the most potent agonist 17 (Scheme 1) developed by Roche and the LXRβ crystal structure (PDB code 1PQ6), which comprises the largest binding pocket.89 The 2-(methylsulfonyl)benzyl alcohol fragment 18 (Scheme 1) resulting from 17 was used as a starting point to grow novel molecules in the cavity of the ligand-binding domain using Contour. The top-scoring 200 structures generated were examined graphically; compound 19 was selected due to the presence of binding elements similar to those in compound 17 and because of its structural novelty compared to the other LXR agonists. Compound 19 was modified by introducing the weakly basic piperazine ring to enhance the water solubility. This modification led to the identification of agonist 20, which was further simplified to produce the synthetic agonist 21. Replacement of the pyridyl methyl group in 21 with pyrimidinyl piperazine group gave 22. Replacement of the

phenyl group on the piperazine ring in 22 with an isopropyl group improved the affinity and selectivity of the resulting compound 23 toward LXRβ (Table 3). Table 3. Assay Results for Piperazines 21−29 compd

LXRα Ki (nM)

LXRα EC50 (nM)

LXRβ Ki (nM)

LXRβ EC50 (nM)

α/β selectivity

21aa,b 21ba,b 22aa,b 22ba,b 23 24 25 26 27 28 29

>3300 2200 1100 1552 43 25 3 146 157 81 1027

>20000 2500 3825 >10000 257 254 92 590 763 244 1734

>2500 460 94 211 4 1 1 9 8 3 160

9200 990 1289 >10000 53 53 21 68 80 21 503

5 12 7 11 25 3 16 20 27 7

a Isomers were separated on chiral column. b The absolute configuration was not verified experimentally but assigned based on modeling only.

As compound 23 lacked metabolic stability in the rodent liver microsomes, a fluorine atom was introduced into position 4 of the phenyl ring to produce 24. The affinity of 24 improved, but the stability did not improve. In contrast, adding a chlorine atom on the pyrimidine ring in 25 improved the stability and maintained good selectivity toward LXRβ (3× over LXRα). The addition of hydroxymethyl and 2-hydroxy-2propyl onto the pyrimidine ring, i.e., 26 and 27, retained the β selectivity. Moreover, replacing the fluorine atom in 26 with F



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with Glu281, and one of the sulfone oxygens forms another hydrogen bond with Leu330. Although compound 28 is a potent LXRβ agonist, limited CNS penetration hindered its use as a probe to study the effect of LXRβ agonist on brain Aβ levels to treat Alzheimer’s disease.81 This compound was developed into a suitable probe by replacing the central piperazine core with 2,4,5,6tetrahydropyrrolo[3,4-c]pyrazole as in 30.83 Compound 30 possessed better physical properties for CNS penetration and has higher metabolic stability in liver microsomes. Since the methyl sulfone group was expected to form a key hydrogen bond with the Leu330 backbone NH based on X-ray structures of similar agonists and the computational model of 31 in the LXRβ binding site, the right-hand part must be maintained. Therefore, SAR efforts were directed toward modifying the left-hand part of compound 30 (Figure 10).83 Changing the position of one of the pyrimidine’s nitrogen in 32 led to compound 33, which has similar cellular potency. The potency of 33 was doubled by introducing a methyl group in 34 (Table 4). The introduction of a methoxy group as a replacement for the trifluormethyl group in 34 produced partial agonist 35 and full agonist 36. Compound 35 was a moderate inhibitor of CYP2C9 and was more selective toward LXRβ (17× over LXRα). Full agonist 36 has a comparable cellular potency to 34 but increased the inhibition of CYP2D6. Drug candidates that inhibit CYP2D6 are highly undesirable because they can cause serious adverse effects by increasing the plasma concentration of any drug that is metabolized by CYP2D6. Fluorinated derivative 37 has good potency but strongly inhibited CYP2C9. The X-ray crystal structures of both 31 and 37 cocrystallized with the LXRβ ligand binding domain (PDB codes 5KYA and 5KYJ, respectively) are presented in Figure 11.83 One of the sulfone oxygens of both 31 and 37 forms a hydrogen bond with the Leu330 backbone NH. The hydroxymethyl group of 31 forms a hydrogen bond with the imidazole of His435, an interaction that is absent in the case of 37. This absence is most likely the reason behind the lower agonistic activity of 37 compared to 31 toward LXRα (Table 4).

the hydroxymethyl group produced the most potent agonist 28 and its less potent S-isomer 29 (Table 3 and Figure 8).81

Figure 8. LXR agonists 24−29.

Surprisingly, 28 (VTP-766) (Figure 8) exhibited remarkable potency and selectivity toward LXRβ. Compound 28 was 27× more selective toward LXRβ than LXRα (Ki was obtained from radioligand binding assay with LXRα and LXRβ ligand-binding domains). It also showed significant inhibition activity against rCYP2C9, with an IC50 value of 610 nM. Moreover, compound 28 was found to regulate the ABCA1 and SREBP-1c LXR target genes in THP1 (EC50 = 4.5 nM) and HepG2 cells (EC50 = 6.3 nM), respectively. Recently, the crystal structure of 28 complexed with LXRβ coordinates was deposited in the Protein Data Bank (PDB code 5I4V).81 The structure shown in Figure 9 has the pyrimidine rotamer that forms hydrogen bond interactions with His435 in accordance with its agonist activity. The agonist response observed for the compound is rationalized based on the hydrogen bond present between the hydroxyl group on the pyrimidine ring and the imidazole ring of His435. This interaction helps to orient and stabilize the imidazole side chain edge-to-face interaction of His435 with the indole side chain of Trp457 located on H12, which helps maintain the agonist conformation of the LBD.28,36,37 Although there is no significant difference in the binding affinity and agonistic activity of 23 and 28, this hydrogen bond is likely responsible for the remarkable selectivity of 28 against other nuclear hormone receptors (>3000×). In addition, the hydroxyl methyl group on the phenyl ring forms a hydrogen bond

Figure 9. X-ray structure of compound 28 bound to LXRβ (PDB code 5I4V). Hydrogen-bonding interactions are shown as blue lines. G



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Figure 10. LXR agonists 30−39.

pyrazole ring with an imidazole heterocycle. Imidazole 40 (Figure 12) induced the ATP-binding transporters ABCA1 and ABGC1 in human blood (EC50 = 1.2 μM). This compound displayed remarkable pharmacokinetic effects in mice as it induced peripheral ABCA1 at 3 mg/kg and 10 mg/kg doses without increasing either plasma or hepatic triglycerides.78 When 40 was orally dosed in male cynomolgus monkeys (1 mg/kg), the tmax for 40 and gene induction was 2 h with a Cmax of 2227 nM and a maximal ABCG1 induction of 4.7-fold. By 24 h, the levels of ABCG1 mRNA were close to baseline; by 48 h, the plasma concentration of 40 was 154 nM.90 Moreover, compound 40 displayed similar potency in vivo, inducing LXR target genes in cynomolgus monkeys with an EC50 of 610 nM. However, this compound was less potent than 1, increasing plasma triglycerides and LDL cholesterol by 29- and 12-fold, respectively. Testing this compound in primates showed an improvement in the lipid profile compared to full agonists, which suggests that limiting LXRα activity and increasing LXRβ selectivity can improve the therapeutic window of LXR.90 Recently, compound 41 (BMS-852927) was developed as a selective LXRβ agonist and has a desirable profile in animal models and a wide therapeutic index in both non-human primates and mice. This compound selectively activates LXRβ

Table 4. LXR Bioassay Data of Agonists 31−39 compd

LXRα EC50 (nM)

Emax (%)

LXRβ EC50 (nM)

Emax (%)

31 32 33 34 35 36 37 38 39

249 111 199 72 271 57 491 189 166

67 104 88 95 5 91 45 72 83

46 28 35 18 16 18 15 45 38

43 91 81 78 29 81 29 67 77

Compound 39 was synthesized based on a computational model that showed that the replacement of the isopropyl group on the pyrrolidine ring in 38 with tert-butyl group could slightly enhance the activity (Table 4). When this compound was dosed orally to wild-type C57BL mice at 1 mg/kg or 3 mg/kg, it exhibited the highest level of ABCA1 mRNA induction in the rat brain among all the compounds in this series but did not significantly change the levels of Aβ1−40 or Aβ1−42 in the rat cerebrum or CFS.83 Kick et al. have synthesized a series of imidazole partial agonists selective for LXRβ. These imidazoles were derived from pyrazole agonists 6−12 (Figure 5) by replacing the

Figure 11. X-ray crystal structure of compound 31 (A) and 37 (B) bound to LXRβ (PDB codes 5KYA and 5KYJ). Hydrogen bonds are shown as blue lines. H



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Figure 12. LXR agonists 40 and 41.

Figure 13. Imidazole partial agonists complexed to the active site of LXRβ. (A) Compound 40 bound to LXRβ (PDB code 4RAK), (B) Compound 41 bound to LXRβ (PDB code 5JY3). Hydrogen bonds are shown as blue lines. (C) Overall structure of LXRβ LBD (PDB code 4RAK) homodimer in complex with compound 40, where the secondary structure is depicted in ribbon representation. Chain B (right) is in active conformation where H12 (red) adapts a close and active conformation. However, H12 on chain A (left) adapts an open and inactive conformation. Similar behavior was observed with the X-ray structure 5JY3.

undesirable effect of 41 was the unexpected decrease in the circulating neutrophil counts in healthy subjects.91 When 41 was dosed at 15 mg, there was a 47% decrease in the neutrophil counts and the treatment was discontinued for two subjects when the neutrophil counts decreased to 5.52 1.01 >5.32 4.89 6.02 3.48 4.29 3 6.25 10.4 3.09

ia = inactive at 10 μM.

a

23α expression and increased Mertk expression in a dosedependent manner, causing a reduction in neutrophil production and stimulating its clearance pathway. Both 40 and 41 show similarities in their binding mode, with three hydrogen bonds in their X-ray crystal structure (Figure 13). However, both compounds lack interactions with His435 that are known to activate the His435-Trp457 switch, which was observed in the potent full pan agonists.91 The fluorine atom on the hydroxymethyl phenyl group of 41 improved the shape complementarity to the pocket near this group, most likely providing enhanced potency. In addition, a π-stacking interaction between the benzylic phenyl ring in 41 and Phe340 was observed, while the second chlorine allows the benzylic methyl to rotate and improve the molecular shape related to the LXR binding pocket.82 Both X-ray structures (4RAK and 5JY3) are composed of homodimers, where H12 adopted two different conformational states. In one monomer, H12 exists in

an active agonist conformation, while in the other monomer, it exists in a nonactive conformation (Figure 13). Compound 42 (Figure 14) was developed as a selective LXRβ agonist to overcome the side effects normally associated with LXR activation, such as elevated triglycerides in the liver and plasma. Indeed, the oxochromene 42 has shown a lowering effect on lipids accumulated in the aortic arch in hamster and a rise in the HDL-C levels.92 When tested via GAL4-LXR luciferase assay, compound 42 was inactive against LXRα at 10 μM, but it was active against LXRβ with EC50 = 0.96 μM and 8% efficacy (this is the ratio between the maximal fold induction for the test compound and the fold induction for 1 at 10 μM). A head-to-tail molecular design approach was used to improve the selectivity and potency of 42 and led to the identification of compound 43 (Figure 14 and Table 5). Compound 43 exhibited a lower Emax for LXRα (26%) and higher Emax for LXRβ (146%). Increasing the LXRβ selectivity led to significant lipid accumulation suppression and decreased J



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Scheme 2. Molecular Design of the Head Structure in 48 by Modifying the Head Structure in 44

However, enantiomer (−)-64 showed agonistic activity for both LXRα and LXRβ (EC50 for LXRα = 0.20 μM, Emax = 164%, EC50 for LXRβ = 0.007 μM, Emax = 255%, and EC50 α/β = 28.5). Moreover, this compound has a good pharmacokinetic profile and has increased HDL-C levels. Additionally, this compound decreased the accumulation of lipids in high-fat and cholesterol-fed LDL receptor knockout mice.94 Two more LXRβ agonists were discovered recently. The first agonist is the benzofuran-2-carboxylate 65 (E17110) (Figure 15), which showed significant efficacy, with an EC50 value of

LDL-C without causing an elevation in the level of TG in plasma high-fat-and-cholesterol-fed Bio F1B hamster. To improve the interaction between the LXRβ receptor and the ligand, the structure−activity relationship efforts focused on the head moiety because the headgroup can modulate the activation of the His435-Trp457 switch.93 The headgroup in compound 43 was developed through a series of SAR modifications following the strategy depicted in Scheme 2 to give the desired bis-n-propyl phenyl structure. In brief, the sixmembered ring was removed, and the trifluoromethyl group was maintained because it is a key factor in the head structure for LXR activation. In addition, another trifluoromethyl group and n-propyl group were incorporated to establish the head structure in 48 (Scheme 2).93 To develop 43 into a treatment of atherosclerosis, the potency, selectivity, and pharmacokinetic profile must be improved. Therefore, the SAR of the linker moiety (butylene unit) between the head (1,1-bistrifluoromethylcarbinol) and the tail (imidazolidine-2,4-dione) was studied (Table 5). It was clear from the LXR transactivation assay that the aromatic linkers are superior to aliphatic linkers (Table 5). The introduction of a methyl substituent to the m-position in the benzene ring (56) enhanced LXRβ selectivity but increased the ClogP value to 8.12. To overcome this problem, polar groups such as the methoxy group (57) and the hydroxyl group (58) were used to replace the methyl group. Interestingly, agonistic activity toward LXRβ was enhanced significantly in 57, and ClogP was decreased slightly (=7.50). The metabolically labile benzylic position was then modified by introducing a hydroxyl group (59), which diminished activity. The conversion of hydroxyl into carbonyl (60) improved both the potency and lipophilicity (ClogP = 6.54). Among the series of aromatic linkers, compounds substituted at the 2-position of acetophenone (61, 62, and 63) gave the best LXRβ selectivity, with EC50 α/β of 6.25, 10.4, 3.09 for 61, 62, and 63, respectively. These results could not be improved further to suppress the activity of LXRα, and attention was directed toward modifying the tail part. Therefore, compound 64, bearing the pyridylhydantoin moiety, was prepared as a more potent and selective LXRβ agonist (EC50 for LXRα = 0.33 μM, Emax = 68%, EC50 for LXRβ = 0.058 μM, Emax = 329%, and EC50 α/β = 5.69). Both enantiomers of 64 were tested in Gal4-LXRα and LXRβ luciferase assays. Enantiomer (+)-64 showed no LXRα agonistic activity and very low LXRβ agonistic activity.

Figure 15. LXRβ selective agonists 64, 65 (E17110), and 66 (IMB808).

0.72 μM. As with other LXR agonists, 65 increased the ABCA1 and ABCG1 target genes and enhanced the cholesterol efflux in RAW264.7 macrophages in vitro, suggesting that it may have antiatherosclerotic activity.95 The second compound, 66 (IMB-808) (Figure 15), was identified as a promising antiatherosclerotic agent.96 When tested in the LXR-GAL4 luciferase reporter assay, this compound exerted a dual LXR partial agonistic activity in vitro, with an EC50 value of 0.15 μM and 0.53 μM toward LXRα and LXRβ, respectively. Compound 66 increased the RCT and cholesterol metabolism target genes in murine and human macrophages. There was a significant increase in the levels of both mRNA and protein of ABCA1 and ABCG1 in RAW264.7 and THP-1 macrophage cells. However, mRNA and protein levels of ApoE were only slightly increased compared to 1. Compound 66 enhanced cholesterol efflux from macrophages and reduced cellular lipid accumulation in assays of foam cells that were carried in RAW264.7 cells. K



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Figure 16. LXR agonists identified through two-step virtual screening protocol.

strong metal chelator that can bind to a wide range of biological targets with high affinities.100 Two new liver X receptor agonists were discovered using pharmacophore modeling and shape-based virtual screening. This study applied a virtual screening workflow to identify potential selective LXRβ agonists from a 3D compound database. A library of 14 compounds was selected based on their LXR selectivity. Discovery Studio was used to create a 3D multiconformational library for the selected hits. These compounds were then screened against six pharmacophore models developed by Schuster and co-workers to account for receptor flexibility.97 Moreover, a shape-based rapid overlay of chemical structures (ROCS) screening was performed using a selective LXRβ compound to increase the probability of finding an LXRβ-selective hit. Molecules that were found to fit the query shape were selected for further evaluation via LXR luciferase reporter gene assay. Compounds 70 and 71 (Figure 17) were found to activate both LXR isoforms, and compound 70 was found to be slightly selective to LXRβ (1.8-fold over LXRα).101

Interestingly, 66 did not induce the expression of lipogenic genes such as FAS, SREBP-1c, and SCD-1. Compound 1 induces these lipogenic genes 4-fold at 1 μM compared to 66. To better understand the difference between 1 and 66 in terms of gene regulations, the authors used docking to determine the key amino acid residues involved in the binding of 1 and 66 in both isoforms. The amino acids that bind to 1 were different from the ones that bind to 66. The amino acids H421 and W443 in LXRα (H435 and W457 in LXRβ) were the most important for binding to 1 but interacted at a moderate level with 66. Phe257 and Arg305 in LXRα (Phe271, Met312, and Thr316 in LXRβ) form the most important interactions with 66. These key amino acids for 66 binding were replaced by site-directed mutagenesis, and the luciferase activity was measured for the different mutants. There was a distinct difference in the agonistic activity of the wild type and various mutants. Two mutants, LXRα-R305G and LXRβ-F271A, were selected to study the effect of mutation on cofactor recruitment by TR-FRET. While the activity of 1 in co-regulator recruitment was not affected, the activity of 66 was affected significantly. Compound 66 was weak in recruiting coactivator TRAP220 (∼18% compared to 1) and moderate in replacing the corepressor NCoR (∼31% compared to 1) in LXRα-R305G. For LXRβ-F271A, 66 was weak in recruiting coactivator D22 (∼13% compared to 1) and in displacing the corepressor SMRT (∼23% compared to 1). These results indicate that compound 66 has a different mechanism in activating LXR than the full agonist 1, and it acts as a partial dual agonist of LXRα/β. Since 66 reduces lipogenesis, it has a good potential to be advanced as a treatment for atherosclerosis.96 A two-step virtual screening protocol was applied to identify active compounds that included 3D-pharmacophore filters and rescoring by shape alignment.97 LigandScout 2.3 was used for generating multiple pharmacophores based on multiple X-ray structures, thereby accounting for different binding modes related to receptor flexibility. The second step of virtual screening was a reranking of the screening hits with the TanimotoCombo scoring function of ROCS, a method for fast alignment and comparison to the bioactive ligand conformation as a query molecule. Eighteen virtual hits were tested in vitro using a reporter gene assay, and compounds 67−69 were found to activate LXR with low micromolar EC50; thus, they can be used for future lead optimization (Figure 16).97 However, these compounds are considered PAINS (pan assay interference compounds) and have the potential for cross reactivity with many targets.98 For example, compound 67 might expel the diethylamine moiety via a retro-Mannich reaction and might generate metabolically reactive quinone methide. The formation of quinone methides can lead to hepatotoxicity or idiosyncratic toxicity, and one must be cautious when using this molecule as a lead compound.99 Both 67 and 68 have 8-hydroxyquinoline (8-HQ) moiety, which is a

Figure 17. LXR agonists 70 and 71.

Steroid Agonists and Antagonists. Sterol-based agonists were found to have anti-inflammatory effects without inducing liver lipid accumulation as with 1. For example, N,N-dimethyl3-hydroxycholenamide (DMHCA) (72), a gene-selective LXR synthetic agonist, exhibited anti-inflammatory activity in leukocytes without affecting liver lipid accumulation. Additionally, this compound potently induced the expression of the target gene ABCA1 in the liver, small intestine, and peritoneal macrophages and stimulated cholesterol transport. This compound showed less potency at increasing the hepatic SREBP-1c mRNA and did not alter the circulating plasma TG in vitro or in vivo. Although 72 is a less potent LXR activator, it significantly enhanced the cholesterol efflux in macrophages compared to nonsteroidal agonists.102 3β-Hydroxycholenamide (MePipHCA) (73) is another sterol-based LXR agonist derived from 72 via replacement of the N,N-dimethyl group in 72 with the 4-methoxypiperidine group. Compound 73 has slightly better potency and efficacy than 72 (for compound 72, EC50 = 422 nM and % of 1 = 45; for compound 73, EC50 = 230 nM and % of 1 = 58). Compound 73 selectively improved both cellular and in vivo L



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Figure 18. Steroidal LXR modulators 72−77.

Figure 19. LXR modulators 78−80.

Figure 20. LXR antagonists 81−83.

77 complex showed a stable agonist conformation with small RMSD (root-mean-square deviation) for His435 and slightly larger RMSD for Trp457. In contrast, 76 showed conformational differences compared to 2 and 77, as the Trp457 side chain rotated in a way that exposed the indole nitrogen atom to form a strong hydrogen bond with the carboxylate group, which interrupted the agonist conformation.105 These results suggest that the negatively charged carboxylate in 75 and 76 is required to disrupt the His435-Trp457 aromatic−aromatic interaction and gives rise to the inverse agonist activity. Novel amide derivatives of the Fernholtz acid (78) (Figure 19) have been synthesized and evaluated in both in silico and in vitro studies because the stereochemistry and functionality of carbon number 22 in cholesterol play a vital role in LXR target gene expression. Compound 79 (22-ketocholesterol) selectively upregulated the ATP-binding cassette transporter ABCA1 in skeletal muscle cells, with no obvious effect on lipogenesis and little effect on FAS and SCD1. This kind of selectivity makes 79 a good candidate for further studies as an antiatherosclerotic drug. In contrast, the Fernholtz cyclohexylamide (80) downregulated the de novo lipogenesis in a dose-dependent manner and counteracted the effect of 1 on LXR.106 Recently, Astrand et al. developed a series of LXRβ antagonists using a molecular modeling approach. The

potency and reduced the inflammation in dextran sulfate sodium (DSS)-induced colitis and traumatic brain injury without causing lipid accumulation or liver injury (Figure 18).103 Compound 74, a 22SHC (22-S-hydroxycholesterol) mimic, has the same agonistic effect of 1. This compound significantly upregulates the gene expression of SCD1 and increases lipogenesis in myotubes in vitro. Additionally, this compound increased lipogenesis when combined with 1 more than 1 did alone.104 The two fluorinated oxysterols 76 and 77 were developed based on the inverse agonist 75 (Figure 18). The replacement of the methylene group hydrogens in the inverse agonist 75 with fluorine atoms altered both the polarity and lipophilicity of the side chains. The difluoroacid 76 significantly reduced the basal levels of luciferase activity, acting as an inverse agonist, while the difluoro alcohol 77 showed agonist activity.105 Molecular dynamic simulations of both 76 and 77 in comparison to that of the 2 were studied to explore the molecular basis of their interactions.105 The fluorine atoms in 2 and 77 actively participate in the interaction with several residues in the ligand-binding pocket. These interactions led to stabilization of the active agonist conformation, which was confirmed by the reporter gene assay. Interestingly, the LXRβ/ M



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Figure 21. LXRs antagonists of fenofibrate family 85−88.

structures of these antagonists were based on the selective antagonist 22SHC (81). The authors synthesized the best novel compounds that resulted from the molecular docking and tested them in vitro in myotubes and HepG2 cells.107 Compounds 82 and 83 (Figure 20) were the most potent antagonists and produced results similar to those of 81 in regulating lipogenic genes, reducing lipogenesis, and abolishing the effect of known LXR agonist 1. Conversely, the plasma concentration of 82 was remarkably low. Therefore, the structure of this compound needs to be optimized to enhance the bioavailability before performing more in vivo studies.107 Synthetic Antagonists. Fenofibrate (84), a drug marketed as Tricor, was developed as a peroxisome proliferatoractivated receptor α (PPARα) modulator and used successfully in treating hypertriglyceridemia and mixed hyperlipidemia,108 early diabetic retinopathy in type 2 diabetes mellitus patients, and related cardiovascular disorders.107,109−112 Interestingly, fenofibrate esters (85−88) were found to inhibit lipogeneses by binding with LXRs and repressing SREBP1 and FAS mRNA genes involved in lipogeneses in the liver. Similarly, other fenofibrate esters bind directly to LXRs and function as LXR antagonists (Figure 21).113 The affinity and specificity of these fibrate esters to LXR depend mainly on the presence of the ester group. While the fibrate esters bound only to LXRs, the corresponding fibric acids bound only to PPARα. Moreover, the fibrate esters reduced the transcriptional activity induced by LXR agonists, whereas the carboxylic acids did not affect the transcriptional activity induced by the same agonists. There is a large degree of similarity in the primary amino acid sequence and ligandbinding domains between PPARα and LXRs, and it is possible to have structurally similar ligands that can modulate both receptors. Jiao and his co-workers114 discovered a new series of LXR antagonists using a structure-based approach. This new series was based on the well-known agonist 1, which is a dual LXRα/ β agonist. Structural modification of 1 led to the identification of compound 94 (Figure 22), which was the first LXR antagonist suitable for in vivo studies in rodents. This compound has good antagonistic activity, as shown in the LXRβ SPA binding assay (IC50 = 0.5 μM) and the LXRβ GAL4 assay (IC50 = 2 μM). Moreover, 95 has high microsomal stability, as >95% remained in rat liver microsomes after incubation at 1 μM for 30 min. This compound has the best pharmacokinetic profile among all the synthesized compounds; it showed both good oral availability and good exposure following oral dosing in rats. Compound 94 displayed lower


clearance (CL = 1.1 L h−1 kg−1) and 8-fold higher area under the curve (AUC = 885 μg h/L) compared to 95 (CL = 4.8 L h−1 kg−1 and AUC = 102 μg h/L) following iv dosing in male Sprague-Dawley rats. Following oral dosing in the same rats (5 mg/kg), 94 displayed good oral availability (F = 31%) and exposure (AUC = 1380 μg h/L), suggesting that 94 could be used as a good in vivo compound. Careful examination of the structure−activity relationship of developed compounds revealed the following important observations: (i) replacement of the trifluoromethyl group in 1 with alkyne substituents improved the binding affinity (for 89, IC50 = 0.6 μM in LXRβ SPA binding assay); (ii) using larger trifluoroethyl (e.g., 1) or isobutyl groups (e.g., 89−93) instead of N-methylsulfonamide groups enhanced the antagonistic activity (for 90, IC50 = 0.3 μM in LXRβ SPA binding assay); (iii) substitution at the meta position of the benzenesulfonamide with small groups such as CN enhanced the binding affinity slightly over the unsubstituted derivative (e.g., 89 vs 90); (iv) substituting phenylacetylene with 3methylsulfonyl or 4-methylsulfonyl with or without CN on benzenesulfonamide produced compounds with good binding affinity and strong antagonistic activity (e.g., 91, 92, and 93).114 The tricyclic 5,11-dihydro-5-methyl-11-methylene-6Hdibenz[b,e]azepin-6-one (96) (Figure 23) was selected as the lead compound to design novel compounds with LXR antagonistic activity.110 The structural modification of 96 was based on weakening or abolishing the hydrogen bond interaction between 96 and His421 on H11 in the LXRα ligand-binding domain, which is responsible for its transactivational agonistic activity (Figure 23).110 Substituting hexafluoropropanol moiety with alkyl groups proved to be a successful strategy. The alky groups were unable to form the hydrogen bond with His421 in LXRs and therefore prevented the proper N



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as a lead compound for further SAR studies. IL-6 is one of the proinflammatory mediators that can be repressed by LXR antagonists to induce anti-inflammatory effects. Therefore, a series of styrylphenylphthalimides were synthesized and evaluated by means of LXR reporter gene assay for their LXRs’ antagonistic activity.117 These compounds are cyclic amides and are prone to ring opening by nucleophiles which might lead to potential toxicity. The IL-6 inhibitory activity was enhanced by replacing the ethyl linker in 102 with the ethylene linker (e.g., 103). 3,4Dimethoxy substituents on the phenyl ring of phenethyl moiety gave the highest inhibitory activity against IL-6 (78% inhibition at 10 μM 103). Moreover, the E-isomer (103) was superior in potency to both the Z-isomer (104) and the ethyl analogue (102).117 Compound 103 showed the most potent LXR antagonistic activity, with an IC50 of 3.3 and 4.3 μM for LXRα and LXRβ, respectively. Moreover, this compound possesses high antagonistic selectivity, as it did not show agonistic or inverse agonistic activity toward LXR at 30 μM. In addition, 103 did not increase the levels of ABCA1 or SREBP-1c mRNA expression in THP-1 cells. Therefore, this compound is a good lead compound because it is not expected to increase the blood triglycerides in addition to its interesting anti-inflammatory in vitro activity.117 The binding of 103 to LXRβ was studied using the TRFRET assay. Compound 103 inhibited the binding of coactivators with LXR in a dose-dependent manner and was found to bind directly to LXRβ, with an IC50 of 1.8 μM.117 Molecular docking of 103 into the cocrystal structure of LXRα LBD complexed with 2 showed that 103 binds at the binding site of LXRα. The docking model predicts hydrogen-bonding interaction of the carbonyl group on 103 and the hydroxyl group on the Thr302 of LXRα. Unlike 1, no hydrogen-bonding interaction with His421 was observed. Since this interaction is necessary for inducing the proper folding of H12 and recruiting the coactivators, this might be the reason behind the antagonistic activity of 103. GSK2033 (3) (Figure 2) was identified by Zuercher and his co-workers as the first potent cell-active LXR antagonist, with an IC50 of 31.8 nM toward LXRβ.76 Compound 3 antagonized the expression of ABCA1 in THP-1 cells and SREBP-1c in HepG2 cells, with an IC50 less than 100 nM. This compound has been used ever since as one of the main chemical probes to explore the biology of LXR. The iterative analogue synthesis and structure−activity relationships of this compound and its analogs revealed that the presence of a sulfonamide group is mandatory for high affinity, and a 3-MeSO2 substituent at the biaryl moiety is important to boost the antagonism activity. Compound 3 lacks LXR specificity, and a recent study by Burris et al. showed that this compound targets other nuclear receptors such as the GCR, PXR, and FXR and that it could potentially alter hepatic gene expression.109 Inverse Agonists. The first selective synthetic LXR inverse agonist, SR9238 (105), is a tertiary sulfonamide that was developed by our group (Figure 25).45 In a cell-based cotransfection assay, this compound exhibits high potency toward both LXR isoforms but shows greater selectivity for LXRβ (IC50 = 43 nM) over LXRα (IC50 = 214 nM). Compound 105 enhanced the interaction of corepressors NCoR ID1 and NCoR ID2 with both LXR isoforms in a dosedependent manner.

Figure 23. Design strategy for LXR antagonists 97−99.

folding of H12, which is required to induce the transactivational agonistic activity (Figure 23).110 A series of substituted azepin-6-one derivatives that exhibit potent and promising selective antagonistic activity were developed, and compounds 97 and 98 were selected as second-generation lead compounds.113 These two compounds stabilized binding of corepressors NCoR and SMRT in a dosedependent manner and did not recruit coactivators SRC1 and DRIP205. Moreover, compounds 97 and 98 were selective for LXR over closely related farnesoid X receptor (FXR), PPARγ, and retinoid X receptor α (RXRα). The low antagonistic activity of compounds 97 and 98 compared to 96 was attributed to its lower binding affinity. Therefore, compound 99, which possesses a hydroxyl group, was developed as a more potent and selective antagonist (IC50 = 3.5 μM) through further structural modification. Compound 99 shows high metabolic stability in human liver microsomes and exhibits good in vitro ADME properties. Therefore, this compound has potential for further development. Ishikawa and his co-workers115 developed 101 as a selective LXRα antagonist (IC50 = 0.2 μM for LXRα and >30 μM for LXRβ) (Figure 24). This antagonist was developed from a


thalidomide-related phthalimide derivative, PP2P (100), which was identified previously as an α-glucosidase inhibitor with dual LXRα/β antagonistic activity (IC50 = 9.8 μM for LXRα and 44 μM for LXRβ) (Figure 24).116 In a cell-based screening of antagonistic activity, compound 100 reduced the interleukin-6 (IL-6) level by 31% at 10 μM and was selected O



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collagen deposition was reduced significantly (almost 90%), which indicates a reduction in the hepatic fibrosis. In contrast to 105, which was liver specific due to its readily metabolized ester group, SR9243 (106) was developed as an inverse agonist that can provide systemic exposure. 21 Compound 106 was highly specific for LXR, as it did not alter the activity of any other nuclear receptor when screened in a nuclear receptor specificity panel. Compound 106 enhances the LXR-corepressor recruitment of nuclear receptor corepressor 1 (NCOR1) and nuclear receptor corepressor 2 (SMRT) and does not enhance coactivator recruitment; it therefore inhibits LXR activation. Accordingly, glycolytic and lipogenic gene expression is reduced, leading to inhibition of the Warburg effect (aerobic glycolysis) and lipogenesis in cancer cells. The efficacy of 106 as an anticancer agent was assessed in different cancer cell types. The cancer cell viability in an MTT reduction assay of prostate, colorectal, and lung cell lines was reduced at nanomolar concentrations. Most importantly, 106 is nontoxic to nonmalignant cells and induces cancer cell death without noticeable side effects both in vitro and in vivo. In vitro, 106 was selective in suppressing the elevated glycolytic output in cancer cells without disrupting glycolytic genes in normal cells. Glycolysis is required in normal cells for energy production, but glycolytic enzyme inhibitors usually disrupt the activity of glycolysis enzymes and cause undesirable side effects. When immune competent (C57BL6J) tumor-bearing mice were treated with 106 at a 60 mg/kg dose, 106 was shown to inhibit Lewis lung carcinoma (LLC1) tumor growth and increase Tnfα levels within tumors, without increasing the expression levels of other cytokines in the liver. Moreover, 106 did not promote weight loss after 14 days of treatment. There is no available X-ray structure for LXR bound with inverse agonists. We performed modeling and energy minimization for both 105 and 106 complexed with LXRβ using MacroModel.112 The X-ray structure 3L0E of agonist GSK1305158 bound to LXRβ and TIF2 peptide was used as a reference for ligand positioning and modeling.76 The energy of both structures was minimized using the Polak−Ribiere conjugate gradient method (PRCG) and OPLS3 force field111 with a gradient convergence threshold value of 0.05. Both compounds were predicted to make mainly intermolecular hydrophobic interactions with

Figure 25. First potent LXR inverse agonists 105 and 106.

Compound 105 was liver specific and reduced the expression of lipogenic genes in the liver and spared LXR target genes outside the liver to avoid adverse effects. When 105 was injected into mice (30 mg/kg, ip), it was not detected in the plasma, but it was detected in the liver 2 h after the injection. In another experiment, when mice were injected for 3 days with 105, high levels of 105 were detected in the liver. The compound was also detected in the intestine to a lower extent but not in the plasma, skeletal muscle, or brain. This result is mostly because the ester group is rapidly metabolized to the corresponding carboxylic acid by plasma lipases. The acid analogue of 105 displays no LXR activity in the cell-based cotransfection assay. Compound 105 displayed high selectivity for LXR, and when screened in a nuclear receptor specificity panel, it showed no activity at any of the tested nuclear receptors. Moreover, 105 suppresses the hepatic steatosis and inflammation that are present in a mouse model of nonalcoholic hepatosteatosis and hence shows great potential to treat nonalcoholic fatty liver disease (NAFLD). Interestingly, 105 can play an important role in cholesterol reduction since it was found to reduce the levels of total plasma cholesterol, plasma LDL, and plasma HDL in diet-induced obesity mice. 45 In an animal model of nonalcoholic steatohepatitis (NASH), 105 was very potent in treating mice with severe hepatic inflammation, hepatic steatosis, and fibrosis.46 When mice were treated with 105 (30 mg kg−1 day−1 ip) for 30 days while being maintained on the NASH diet, the expression of hepatic inflammatory markers (e.g., IL6, IL-1β, and IL-12) was reduced. Moreover, the hepatic

Figure 26. Inverse agonists modeled in the active site of LXRβ. (A) Inverse agonist 105. (B) Inverse agonist 106. P



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active site hydrophobic residues Phe329, Phe340, and Met312 and hydrogen-bonding interactions with Leu330 or Ser278 (Figure 26). Unlike the structurally similar GSK1305158, interactions of sulfonamide oxygen with His435 in H12 is lost. Therefore, the His435-Trp457 switch responsible for agonist conformation is disrupted. The methyl sulfone oxygen on the opposite end of the ligand forms the second hydrogen bond with Leu330 or Ser278 (Figure 26).85,110

the therapeutic treatment of cancer, fatty liver diseases, Alzheimer disease, and atherosclerosis. Recently, he was awarded The Egyptian State Prize in Chemical Science and the Presidential Medal of Excellence (First Class) for outstanding contribution from the President of Egypt. Shaimaa Goher received her B.Sc. degree in Chemistry from the Faculty of Science, Benha University (Egypt) in 2013. She is currently working toward her M.Sc. degree under the supervision of Prof. Bahaa El-Gendy. Her research project focuses on the development of novel liver X receptor modulators as antihepatitis C virus. Her research interests include medicinal chemistry, computational chemistry, organic chemistry, and drug design. She was selected among the 400 most qualified young scientists to attend the Lindau Nobel meeting in Lindau, Germany, 2017.

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CONCLUSIONS The LXRs play a major role in regulating lipid and cholesterol metabolism in addition to their anti-inflammatory activities. These activities have led to expanded interest in developing new small molecule modulators for these receptors. Cardiovascular diseases were the main driving force behind the development of early LXR modulators, but these compounds suffered from undesirable side effects such as the induction of hepatic steatosis. Elegant work by many research groups led to a better understanding of the structure and function of LXRs and allowed scientists to decipher novel mechanisms and pathways that may lead to the discovery of new therapeutics. Therefore, we placed substantial emphasis on the structure and function of LXRs because of their essential role in the drug design process. We aimed to provide deeper insight into the important structural features of LXRs, and we discussed the structure−activity relationships of most active and promising synthetic modulators in the past few years. The field is growing rapidly, and the focus is directed toward developing selective liver X receptor modulators to avoid the undesirable side effects caused by the first generation of LXR modulators. Currently, there are three drugs in clinical trials at different phases of study. RGX-104-001 is a drug used for the treatment of patients with advanced solid tumors and lymphoma, and it is in phase 1. VTP-38543 and ALX-101 are in phase 2 clinical trials for the treatment of atopic dermatitis. The structures of these molecules have not been reported in peer-reviewed manuscripts; thus, they were not part of this review. The discovery of the first LXR inverse agonists 105 and 106 that showed therapeutic potential in nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cancer without noticeable side effects in animal models is an area deserving of further exploration by medicinal chemists.

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Lamees Hegazy is a Research Assistant Professor at the Department of Pharmacology and Physiology at Saint Louis University School of Medicine. She has a doctoral degree in Computational Biochemistry from the University of Florida. Dr. Hegazy is an expert in employing molecular modeling and computational chemistry methods to study the dynamics and function of biological macromolecules. Her current research focus is the use of molecular dynamics simulations, enhanced sampling simulations, and free energy-based lead optimization to study the conformational behavior of nuclear receptors and design modulators that target different conformational states. Mohamed Arief has been a Professor of Organic Chemistry since 1999 in the Department of Chemistry, Faculty of Science, Benha University, Egypt. He was awarded both a M.Sc. and a Ph.D. in Organic Chemistry from Ain Shams University, Egypt. From 1983 to 1984, he worked as a postdoctoral fellow at Institut für Organische Chemie (Austria), with Professor Sauter. He worked as an Assistant Professor starting from 1987 at Benha University, where he focused on efficient microwave-assisted solvent-free synthesis and molecular docking studies, synthesis and reactions of some heterocyclic compounds of expected biological activities, spectroscopic studies, medicinal and pharmaceutical chemistry, and natural product chemistry. Thomas P. Burris is Alumni Endowed Professor of Pharmacology in the Center for Clinical Pharmacology at Washington University School of Medicine and St. Louis College of Pharmacy. Prior to his current position he was the William Beaumont, M.D. Endowed Professor of Physiology and Chairman of the Department of Pharmacology & Physiology at Saint Louis University School of Medicine (2013−2018) and Professor at The Scripps Research Institute (2007−2013). He held drug discovery research positions at Johnson & Johnson and Eli Lilly for a decade before returning to academia. Dr. Burris is an expert in chemical biology and pharmacology of nuclear hormone receptors and has considerable experience developing drugs that target this class of drug target.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1-3149775171. Fax: +1-3149776411. E-mail: [email protected]. ORCID

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Bahaa El-Dien M. El-Gendy: 0000-0003-4800-7976 Thomas P. Burris: 0000-0003-2922-4449 Biographies

ACKNOWLEDGMENTS Bahaa El-Gendy thanks the Science and Technology Development Fund (Egypt) for financial support (STDF-STF Grant 11969).

Bahaa El-Dien El-Gendy is an Assistant Professor at the Department of Pharmacology and Physiology in the Saint Louis University School of Medicine and Associate Professor of Bio-Organic Chemistry in Benha University, Egypt. He received his Ph.D. in Chemistry from University of Florida under the supervision of Prof. Alan R. Katritzky and completed postdoctoral training at the Scripps Research Institute. The focus of Dr. El-Gendy’s research group is the development of small molecule modulators for various nuclear hormone receptors for

ABBREVIATIONS USED NR, nuclear hormone receptor; AF-1, activation function 1; DBD, DNA-binding domain; LBD, ligand-binding domain; AF2, activation function 2 (C-terminal domain); ER, estrogen receptor; AR, androgen receptor; TR, thyroid hormone receptor; RAR, vitamin A receptor (retinoic acid receptor); VDR, vitamin D receptor; PR, progesterone receptor; PPAR,

Notes

The authors declare no competing financial interest.

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peroxisome proliferator-activated receptor; FXR, farnesoid X receptor; PXR, pregnane X receptor; CAR, constitutive androstane receptor; LXR, liver X receptor; AD, Alzheimer’s disease; PD, Parkinson’s disease; MS, multiple sclerosis; RXR, retinoic acid receptor; LXRE, liver X receptor response element; FAS, fatty acid synthase; CYP7A1, cytochrome P450 isoform 7A1; ChREBP, cholesterol 7α-hydroxylase, carbohydrate regulatory element binding protein; ApoE, apolipoprotein E; CETP, cholesteryl ester transfer protein; ABC, ATP binding cassette; NCoR, nuclear corepressor; SMRT, silencing retinoic acid and thyroid hormone receptor mediator; HDAC, histone deacetylase enzyme; Sin3A, stress activated MAP kinase interacting protein 3A; ASC2, activating signal integrator-2; RIP140, receptor-integrating protein140; SRC, steroid receptor coactivator; NF κB, nuclear factor κB (transcription factor); STAT1, signal transducer and activator of transcription 1; SUMO2 or SUMO3, small ubiquitin-like modifier 2 or 3; PDB, Protein Data Bank; ABCA1, ATPbinding cassette transporter A1; ABCG1, ATP-binding cassette subfamily G member 1; CNS, central nervous system; HTS, high throughput screening; CFR, Code of Federal Regulations; Pgp, P-glycoprotein; Boc2O, di-tert-butyl dicarbonate; RCT, reverse cholesterol transport; rCYP2C9, recombinant cytochrome P450 2C9; SREBP1c, sterol regulatory elementbinding transcription factor 1; THP1, human leukemic monocyte cell line; HepG2, hepatocellular carcinoma cell line; DIBAL, diisobutyl aluminum hydride, a strong and bulky reducing agent; Aβ, amyloid β; SAR, structure−activity relationship; CFS, chronic fatigue syndrome; SUMO, small ubiquitin related modifier; TG, triglyceride; LDL, low-density lipoprotein; MAD, multiple ascending dose; DMHCA, N, Ndimethyl-3-hydroxycholenamide; DSS, dextran sulfate sodium; SCD1, stearoyl-CoA desaturase-1; RMSD, root-mean-square deviation of atomic positions; 22KC, 22-ketocholesterol; LDLC, low-density lipoprotein C; HDL-C, high-density lipoprotein C; PK, pharmacokinetics; Gal4, DNA-binding yeast transcription factor; ClogP, the logarithm of partition coefficient between n-octanol and water log(Coctanol/Cwater); RAW264.7, a macrophage-like, Abelson leukemia virus-transformed cell line derived from BALB/c mice; QSAR, quantitative structure− activity relationship; ZINC database, database for the commercially available chemical compounds for virtual screening; ROCS, software for virtual screening; 22SHC, 22-Shydroxycholesterol; TBS, tert-butyldimethylsilyl group; DMP, Dess−Martin periodinate; ADME, pharmacokinetic properties absorption, distribution, metabolism, and excretion; IL-6, interleukin-6; TR-FRET, time-resolved fluorescence resonance energy transfer assay; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; MTT, a colorimetric assay for cell metabolic activity; DCM, dichloromethane; PRCG, Polak−Ribiere conjugate gradient method

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