Recent Advances in the Medicinal Chemistry of Liver X Receptors Miniperspective † ‡ ‡ † ‡ Bahaa El-Dien M

Perspective

Cite This: J. Med. Chem. 2018, 61, 10935−10956 pubs.acs.org/jmc

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

ABSTRACT: Nuclear hormone receptors represent a large family of ligand- activated 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.

■ INTRODUCTION as master regulators of lipid and cholesterol metabolism in fl 7−11 Nuclear hormone receptors (NRs) comprise a superfamily of addition to their notable anti-in ammatory activities. LXRs ligand-activated transcription factors that incorporate a group belong to the thyroid/retinoid hormone receptor subfamily, − of 48 members in humans and 49 in mice.1 3 NRs are and they were isolated initially as orphan receptors, as their fi 12 intracellular proteins, and they all share common structural natural ligand was not yet identi ed. Following the discovery of the endogenous oxysterols as specific ligands for LXRs, they features and are composed of an N-terminal domain (AF-1), a 13−15 DNA-binding domain (DBD), a hinge region, a ligand-binding were subsequently considered deorphanized.

Downloaded via UNIV OF FLORIDA on January 12, 2019 at 01:12:35 (UTC). domain (LBD), and a C-terminal domain (AF-2) (Figure 1). LXRs play a paramount role in many physiological ﬀ Both the DNA-binding domain (DBD) and the ligand-binding processes. LXRs regulate di erent metabolic functions, such 16−19 20,21 domain (LBD) are highly conserved. as cholesterol metabolism, lipogenesis, carbohydrate

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 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 Figure 1. NR domains. (MS).37 fi In cancer biology, LXR ligands have shown antiproliferative NRs are classi ed according to the homology sequence into ff ff six subfamilies, including steroid receptors, thyroid/retinoid e ects on di erent cancer cell types; the activation of LXRs receptors, and orphan receptors.4,5 The natural ligands of decreases proliferation by reducing the intracellular cholesterol steroid receptors and thyroid/retinoid receptors are well- level necessary for lymphocytes to synthesize its cellular known, but those of orphan receptors have not been identified.6 Among the NR superfamily, the LXRs have Received: January 11, 2018 emerged as very promising drug targets because of their role Published: July 13, 2018

© 2018 American Chemical Society 10935 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective membrane during proliferation,38 and in glioblastomas, the cholesterol 7α-hydroxylase, carbohydrate regulatory element activation of LXRs decreases cholesterol levels and hence binding protein (ChREBP), apolipoprotein E (ApoE), − promotes tumor cell death.7,39 42 Alternatively, the activation cholesteryl ester transfer protein (CETP), sterol regulatory of LXRβ induces the transcription of tumoral and stromal element-binding protein 1c (SREBP-1c), and ATP-binding apolipoprotein E (ApoE), which is a strong suppressor of cassette (ABC) transporters.15 melanoma metastasis. The administration of LXR agonists LXRs regulate gene expression in different ways. In the leads to the suppression of tumor growth and metastasis in a unliganded state, the LXR/RXR heterodimer bound to LXR broad spectrum of melanoma lines.43 LXR/ApoE activation response element (LXRE) interacts with nuclear receptor using a potent LXRβ agonist currently in clinical trials, RGX- corepressor (NCoR) or with the silencing retinoic acid and 104, was found to reduce myeloid-derived suppressor cells thyroid hormone receptor mediator (SMRT).56 This inter- (MDSCs) abundance by increasing their apoptosis. In human action is followed by the recruitment of histone deacetylase cancer patients with metastatic cancers, RGX-104 depleted (HDAC) through interaction with the stress-activated MAP MDSCs and activated T cells in a remarkable way. In mice, kinase interacting protein 3A (Sin3A) and consequent RGX-104 enhanced the efficacy of checkpoint inhibitors blocking of transcription.9,23,35,36,57 Upon binding with without noticeable signs of toxicity.44 agonists, conformational changes occur and result in the The vast majority of research conducted to find LXR dissociation of the corepressor complex associated with LXRE. modulators of therapeutic utility has been directed toward The coactivator complex, such as activating signal integrator-2 developing LXR agonists. A major drawback of using LXR (ASC2),58 receptor-integrating protein140 (RIP140),59 or agonists as drugs is the elevation of plasma triglycerides and steroid receptor coactivators (SRC),60 is then recruited onto hepatic steatosis due to the direct regulation of the lipogenic helix 12 (H12) of the LBD, leading to activation of the pathway by LXR in the liver. This undesirable effect has transcription of target genes.61 Upon binding with antagonists impeded its development into commercial drugs. To overcome or inverse agonists, LXR-corepressor recruitment occurs, and this hurdle, new strategies have been developed such as the expression of target genes is downregulated to below basal developing LXRβ-selective agonists and tissue-selective ago- levels.21,45 nists. Antagonists have been mainly used as chemical probes to Activation of LXRs by ligands results in the transrepression study the biology of LXRs. These antagonists block the LXR of inflammatory pathways by the inhibition of signal-depend- agonists from binding to the ligand binding domain and ent transcription factors such as nuclear factor κB (NF-κB). prevent them from inducing conformational changes in the For LXRs to interact with the corepressor docked to the receptor. Inverse agonists repress transcription by recruiting transcription factor, such as STAT1 complex, and to corepressor proteins when bound to the LBD of a consequently inhibit the proinflammatory pathways, LXRs constitutively active receptor such as LXR. Inverse agonists must first be SUMOylated. In this regulatory mechanism of of LXRs are fairly new modulators that were first developed by SUMOylation, LXRs are conjugated to small ubiquitin-like ourgroupandshowedpotentialforthetreatmentof modifier 2 or 3 (SUMO2 or SUMO3), a step required for their 45 nonalcoholic fatty liver disease (NAFLD), nonalcoholic transrepression and consequent inhibition of the proinflamma- 46 steatohepatitis (NASH), and different kinds of cancers tory pathways.9,62,63 21,47 without noticeable side effects in preclinical studies. Several X-ray crystal structures of LXRs were obtained and deposited in the Protein Data Bank (PDB) in multiple forms; ■ LXR STRUCTURE AND FUNCTION as a monomer, homodimer, or heterodimer (Table 1).64 The LXRs are known as nuclear oxysterol receptors and have two ligand-binding domain in the reported X-ray crystal structures isoforms: LXRα (NR1H3), initially named OR-1, and LXRβ was complexed with agonists or partial agonists, but no X-ray − (NR1H2), initially named NER and UR.48 51 In humans, crystal structures have been reported for antagonists or inverse LXRα consists of 447 amino acids and is highly expressed in agonists. the liver, kidney, intestine, adipose tissue, and macro- The ligand-binding pocket (LBP) of LXR is mainly phages.12,50 LXRβ consists of 460 amino acids and is expressed hydrophobic, with a few polar amino acid residues that make ubiquitously in most tissues.49,52 hydrogen-bonding interactions with the bound ligand. We Similar to other nuclear receptors, LXRs are composed of used SiteMap65 to calculate the volume of the active site in two four functional domains (Figure 1): (i) the N-terminal domain different X-ray structures to gain knowledge about the binding (A/B domain); (ii) two zinc finger DNA-binding motifs with cavity. It became obvious that the binding cavity can their DNA-binding domain (DBD); (iii) a hinge region; and accommodate different ligand sizes.66,67 For example, in the (iv) a globular ligand-binding domain that also contains AF-2 case of small ligands such as T0901317 (1)(Figure 2), the (activating function 2), responsible for the recruitment of LXRβ binding pocket volume is 266 Å3 (Figure 3A), while − coactivators.7,53 55 Both the LBD and DBD of the two LXR with larger ligands such as GW3965 (2)(Figure 2), the isoforms share approximately 74−75.6% of their amino acid volume is 533 Å3 (Figure 3B). In addition, based on the bound sequence identity and differ by only two residues in the ligand- ligand, a polar tunnel cavity that is composed of the solvent- binding pocket: V261/I277 (LXRα/LXRβ) and V295/I311 exposed residues opens (Figure 3).68 (LXRα/LXRβ).54 Upon binding with an agonist, H12 of the LBD adopts a LXRs form a heterodimer with the 9-cis retinoic acid conformation that allows the binding of coactivator protein receptor RXR. The LXR/RXR heterodimer is activated by (Figure 4A). Studying the crystal structures of the LBDs ligands for either LXR or RXR and is thus considered complexed with either steroidal or nonsteroidal agonists “permissive”. Many target genes are involved in the regulation provided important information about the most common of different biological processes by LXRs in both normal and interactions that were found to activate LXR. The hydrogen pathological functions. Among those target genes are fatty acid bond between the ligand and His421 in helix 10 (H10) for synthase (FAS), cytochrome P450 isoform 7A1 (CYP7A1), LXRα or with His435 for LXRβ in helix 11 (H11) is essential

10936 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

Table 1. Available LXR X-ray Structures

PDB code ligand effect receptor crystallized form ref 1P8D agonist LXRβ monomer 69 1PQ6 agonist LXRβ homodimer 70 1PQ9 agonist LXRβ homodimer 70 1PQC agonist LXRβ homodimer 70 1UHL agonist LXRα-RXRα heterodimer 61 1UPW agonist LXRβ monomer 67 1UPV agonist LXRβ monomer 67 2ACL agonist LXRα-RXRα heterodimer 71 3FAL agonist LXRα-RXRα heterodimer 72 3FC6 agonist LXRα-RXRα heterodimer 73 3IPS agonist LXRα homodimer 74 3IPU agonist LXRα homodimer 74 Figure 3. Hydrophobic (yellow), donor (blue), and acceptor (red) maps for the cavities in the LBD of LXRβ. The protein is shown in 3IPQ agonist LXRα homodimer 74 β ribbon representation, and the picture background is shown in black. 3KFC agonist LXR homodimer 75 The ligand is shown in stick representation. (A) The ligand-binding β 3L0E agonist LXR homodimer 76 pocket of LXRβ bound to small agonist 1 (PDB code 1PQC). (B) 4DK7 agonist LXRβ homodimer 64 The ligand-binding pocket of LXRβ bound to larger agonist 2 (PDB 4DK8 partial agonist LXRβ homodimer 64 code 1PQ6), where a polar tunnel cavity opens. 4NQA agonist LXRβ-RXRα heterodimer on DNA 77 4RAK partial agonist LXRβ homodimer 78 5AVI agonist LXRα homodimer 79 5AVL agonist LXRα homodimer 79 The ligands that bind tightly to LXR and prevent H12 from 84 5HJP agonist LXRβ homodimer 80 adopting an agonist conformation may act as antagonists. In 5HJS agonist LXRα -RXRα heterodimer 80 addition, it was observed that the larger is the size of the bound 5I4V agonist LXRβ homodimer 81 ligand, the more the antagonism or partial agonism effect was 76 5JY3 partial agonist LXRβ homodimer 82 observed. This finding may be attributed to the perturbation 5KYA agonist LXRβ homodimer 83 of the LBD of the receptor by larger ligands. For instance, the 5KYJ agonist LXRβ-RXRβ heterodimer 83 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 for LXR activation, and the bond strength defines the stability It is likely that larger ligands such as 3 can perturb the binding of the ligand−protein complex. This interaction positions the pocket of the protein, which constitutes helix 3 (H3), H10/11, histidine imidazole to make edge-to-face π-electron cloud and the H12 loop and can prevent the binding of coactivator interactions with the indole of Trp457. Previous studies have proteins, resulting in partial agonism or antagonism behavior.76 shown that the two conserved amino acids His435 and Trp457 Although no X-ray crystal structures for LXR inverse interact within the ligand-binding pocket and function as an agonists have been reported, molecular dynamics simulations activation switch that drives the conformational rearrangement that predict the binding mode and molecular basis of an of the AF-2 domain (Figure 4B). With shorter hydrogen bond inverse agonist 4 (27-norcholesteonic acid) were described distance, the histidine imidazole is properly positioned toward recently.85 Upon binding with this molecule, the key the more strongly negative π-electron cloud of the phenyl interactions between His435 in H11 and Trp457 in H12 portion of the indole ring rather than the weaker π-cloud of the were disrupted, causing structural changes in the overall pyrrole, thereby stabilizing H12 and the AF-2 domain in the confirmation of H12 and in the H11/H12 loop. In addition, active agonist conformation.66,69,78,83 this disruption caused dynamic changes in the protein

Figure 2. LXR Agonists 1−4.

10937 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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. Hydrogen- bonding 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. backbone to a larger extent than what occurred by complex- The first modification was to replace the o-chlorine atom on 85 fl ation with an agonist. the phenyl ring in 7 with the tri uoromethyl (CF3)in8. This simple modification increased the binding selectivity of LXRβ ■ MEDICINAL CHEMISTRY OF LXR MODULATORS 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 LXR Agonists. Wrobel et al. developed LXR-623 (5)asa mouse microsomes. However, replacement of the same group potent LXR agonist (Figure 5).86 Compound 5 showed with dimethyl carbinol in 10 increased the LXRβ binding potency toward both LXRα (179 nM) and LXRβ (24 nM), 9 with 7-fold selectivity for LXRβ. To the best of our knowledge, selectivity 18-fold over . Later, the thiophene ring was fi replaced with a phenyl to provide the biphenyl sulfones 11 and 5 was the rst LXR agonist with published clinical trial results β from a single ascending dose trial and was tested in humans, 12 (Figure 5). This ring replacement improved the LXR where it showed upregulation of the LXR target genes ABCA1 selectivity 10- to 14-fold in 11 and 12. Both compounds and ABCG1 in a dose-dependent manner.87 Upregulation of displayed similar selectivity, but 11 was found to be more potent, with an EC50 of 49 nM in a cellular transactivation these genes enhances the process of reverse cholesterol 78 transport (RCT) and could inhibit or delay the progress of assay. atherosclerosis. Although 5 showed adverse CNS effects and Stachel et al. developed a series of agonists to selectively β ’ did not go to market; it was a hallmark in validating LXRs as an target LXR in the brain for the treatment of Alzheimer s 80 important therapeutic target. disease (Figure 6). To measure the isoform selectivity for β α Pyrazole agonists 6 and 7 are structurally similar to 5 and LXR over LXR , the authors focused on measuring Emax- were developed as highly potent LXR agonists (Figure 5).88 based selectivity instead of potency-based selectivity because β Pyrazole 7 was found to be a potent LXR agonist, with EC50 the ligand-binding domains of both isoforms are highly = 108 nM and 90% efficacy. To increase LXRβ selectivity, conserved. First, the authors identified compound 13 from many analogs were synthesized using array synthesis and single the HTS of their library collection to be moderately selective compound synthesis. toward LXRβ in the cofactor recruitment assay (CFR) (Table

10938 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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 α μ Figure 6. Piperazine agonists 13−16. similar activity against LXR (EC50 = 2.0 M and Emax = 31%) β μ and much lower activity against LXR (EC50 = 0.84 M and 2). This hit was not suitable for in vivo studies because of Emax = 117%). insolubility and weak potency. A remarkable increase in The crystal structures of compound 16 in complex with the LXRα homodimer and the LXRβ/RXR heterodimer were E − Table 2. max Selectivity of 13 16 from the Cofactor determined at 1.7 and 2.6 Å resolution, respectively (Figure 7). Recruitment Assay (CFR) Compound 16 binds the ligand-binding domain of LXRα and LXRβ in a similar manner; it forms hydrophobic π−π and compd LXRα EC (μM) E (%) LXRβ EC (μM) E (%) 50 max 50 max alkyl−π interactions with hydrophobic residues in the active 13 1.48 50 0.94 106 site such as Phe329 and Met312. Compound 16 makes 14 0.041 62 0.008 67 hydrogen-bonding interactions with Leu330, Arg319 and 15 0.50 24 0.55 91 His435 (LXRβ numbering). No noticeable differences were 16 (R) 1.9 49 0.03 103 observed in the interaction of compound 16 with the His435- Trp457 activation switch in both LXR isoforms. The authors explained isoform selectivity as being achieved via the binding was achieved upon replacement of the tert-butyl ff ff carbamate in 13 with the mandelate group in 14, as shown by a cumulative e ect of small conformational di erences of 16 fi when bound to LXRα and LXRβ (Figure 7). If we compare signi cant left shift in EC50. However, the solubility did not ff improve, and the E selectivity was lost, as shown in the CFR compound 15 to compound 16, then the only di erence is the max fl assay (Table 2). Replacing the cyanopyridyl ring with a rigid phenyl- versus the conformationally exible isopropyl fl bispiperidine resulted in the formation of compound 15, which tri uoromandelate group. Therefore, the high isoform has much better solubility and similar selectivity compared to selectivity observed in 16 is most likely due to the observed 14 when measured in a transactivation assay. Compound 15 differential flexibility of the isopropyl trifluoromandelate 80 suffered from low CNS penetration because of liability to P- group. Moreover, the S enantiomer of compound 16 was glycoprotein (Pgp). 2-fold less active against LXRβ than the R enantiomer (16)in To overcome this problem, compound 16 was synthesized cell-based transactivation assays, which means that the spatial with an isopropyl trifluoromandelate replacing the phenyl arrangements of the isopropyl trifluoromandelate group play a trifluoromandelate group. This compound was obtained as a crucial role in achieving higher isoform selectivity. Different mixture of enantiomers and was subjected to chiral separation stereochemistry around the amide carbonyl of 16 that forms a to obtain the R enantiomer, which is the most active form. Pgp hydrogen bond with His435 would affect the strength of the

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.

10939 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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

hydrogen bond and might slightly disrupt the His435-Trp457 phenyl group on the piperazine ring in 22 with an isopropyl switch. group improved the affinity and selectivity of the resulting Zheng et al. used Contour, a structure-based drug design compound 23 toward LXRβ (Table 3). platform, to identify new drug-like lead molecules by assembling fragments in the protein-binding pockets that Table 3. Assay Results for Piperazines 21−29 naturally complement hydrophilic and hydrophobic features of α α β β α β 81 LXR Ki LXR EC50 LXR Ki LXR EC50 / the protein-binding site. The algorithm of Contour uses compd (nM) (nM) (nM) (nM) selectivity dynamicvectorselection(DVS)anddynamicfragment a,b selection (DFS) as two methods of dynamic growth to 21a >3300 >20000 >2500 9200 21ba,b 2200 2500 460 990 5 generate novel scaffolds that best fit the binding site. They 22aa,b 1100 3825 94 1289 12 were able to develop some novel potent, bioavailable, and 22ba,b 1552 >10000 211 >10000 7 orally active LXRβ selective agonists. The novel agonists were 23 43 257 4 53 11 designed to induce the reverse cholesterol transport (RCT) 24 25 254 1 53 25 without increasing the triglyceride formation in both liver and 25 3 92 1 21 3 plasma. The modeling study used the most potent agonist 17 β 26 146 590 9 68 16 (Scheme 1) developed by Roche and the LXR crystal 27 157 763 8 80 20 structure (PDB code 1PQ6), which comprises the largest 28 81 244 3 21 27 89 binding pocket. 29 1027 1734 160 503 7 The 2-(methylsulfonyl)benzyl alcohol fragment 18 (Scheme aIsomers were separated on chiral column. bThe absolute 1) resulting from 17 was used as a starting point to grow novel configuration was not verified experimentally but assigned based on molecules in the cavity of the ligand-binding domain using modeling only. Contour. The top-scoring 200 structures generated were examined graphically; compound 19 was selected due to the As compound 23 lacked metabolic stability in the rodent presence of binding elements similar to those in compound 17 liver microsomes, a fluorine atom was introduced into position and because of its structural novelty compared to the other 4 of the phenyl ring to produce 24. The affinity of 24 LXR agonists. Compound 19 was modified by introducing the improved, but the stability did not improve. In contrast, adding weakly basic piperazine ring to enhance the water solubility. a chlorine atom on the pyrimidine ring in 25 improved the This modification led to the identification of agonist 20, which stability and maintained good selectivity toward LXRβ (3× was further simplified to produce the synthetic agonist 21. over LXRα). The addition of hydroxymethyl and 2-hydroxy-2- Replacement of the pyridyl methyl group in 21 with propyl onto the pyrimidine ring, i.e., 26 and 27, retained the β pyrimidinyl piperazine group gave 22. Replacement of the selectivity. Moreover, replacing the fluorine atom in 26 with

10940 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective the hydroxymethyl group produced the most potent agonist 28 with Glu281, and one of the sulfone oxygens forms another and its less potent S-isomer 29 (Table 3 and Figure 8).81 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 2,4,5,6- tetrahydropyrrolo[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 Figure 8. LXR agonists 24−29. bond with the Leu330 backbone NH based on X-ray structures of similar agonists and the computational model of 31 in the β Surprisingly, 28 (VTP-766) (Figure 8) exhibited remarkable LXR binding site, the right-hand part must be maintained. β × Therefore, SAR efforts were directed toward modifying the potency and selectivity toward LXR . Compound 28 was 27 83 more selective toward LXRβ than LXRα (K was obtained from left-hand part of compound 30 (Figure 10). i ’ radioligand binding assay with LXRα and LXRβ ligand-binding Changing the position of one of the pyrimidine s nitrogen in domains). It also showed significant inhibition activity against 32 led to compound 33, which has similar cellular potency. rCYP2C9, with an IC50 value of 610 nM. Moreover, The potency of 33 was doubled by introducing a methyl group compound 28 was found to regulate the ABCA1 and in 34 (Table 4). The introduction of a methoxy group as a fl SREBP-1c LXR target genes in THP1 (EC50 = 4.5 nM) and replacement for the tri uormethyl group in 34 produced HepG2 cells (EC50 = 6.3 nM), respectively. partial agonist 35 and full agonist 36. Compound 35 was a Recently, the crystal structure of 28 complexed with LXRβ moderate inhibitor of CYP2C9 and was more selective toward coordinates was deposited in the Protein Data Bank (PDB LXRβ (17× over LXRα). Full agonist 36 has a comparable code 5I4V).81 The structure shown in Figure 9 has the cellular potency to 34 but increased the inhibition of CYP2D6. pyrimidine rotamer that forms hydrogen bond interactions Drug candidates that inhibit CYP2D6 are highly undesirable with His435 in accordance with its agonist activity. The agonist because they can cause serious adverse effects by increasing the response observed for the compound is rationalized based on plasma concentration of any drug that is metabolized by the hydrogen bond present between the hydroxyl group on the CYP2D6. Fluorinated derivative 37 has good potency but pyrimidine ring and the imidazole ring of His435. This strongly inhibited CYP2C9. interaction helps to orient and stabilize the imidazole side The X-ray crystal structures of both 31 and 37 cocrystallized chain edge-to-face interaction of His435 with the indole side with the LXRβ ligand binding domain (PDB codes 5KYA and chain of Trp457 located on H12, which helps maintain the 5KYJ, respectively) are presented in Figure 11.83 One of the agonist conformation of the LBD.28,36,37 Although there is no sulfone oxygens of both 31 and 37 forms a hydrogen bond significant difference in the binding affinity and agonistic with the Leu330 backbone NH. The hydroxymethyl group of activity of 23 and 28, this hydrogen bond is likely responsible 31 forms a hydrogen bond with the imidazole of His435, an for the remarkable selectivity of 28 against other nuclear interaction that is absent in the case of 37. This absence is hormone receptors (>3000×). In addition, the hydroxyl most likely the reason behind the lower agonistic activity of 37 methyl group on the phenyl ring forms a hydrogen bond compared to 31 toward LXRα (Table 4).

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

10941 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

Figure 10. LXR agonists 30−39.

Table 4. LXR Bioassay Data of Agonists 31−39 pyrazole ring with an imidazole heterocycle. Imidazole 40 α β (Figure 12) induced the ATP-binding transporters ABCA1 and compd LXR EC50 (nM) Emax (%) LXR EC50 (nM) Emax (%) μ ABGC1 in human blood (EC50 = 1.2 M). This compound 31 249 67 46 43 displayed remarkable pharmacokinetic effects in mice as it 32 111 104 28 91 induced peripheral ABCA1 at 3 mg/kg and 10 mg/kg doses 33 199 88 35 81 without increasing either plasma or hepatic triglycerides.78 34 72 95 18 78 When 40 was orally dosed in male cynomolgus monkeys (1 35 271 5 16 29 mg/kg), the t for 40 and gene induction was 2 h with a C 36 57 91 18 81 max max of 2227 nM and a maximal ABCG1 induction of 4.7-fold. By 37 491 45 15 29 24 h, the levels of ABCG1 mRNA were close to baseline; by 48 38 189 72 45 67 90 39 166 83 38 77 h, the plasma concentration of 40 was 154 nM. Moreover, compound 40 displayed similar potency in vivo, Compound 39 was synthesized based on a computational inducing LXR target genes in cynomolgus monkeys with an model that showed that the replacement of the isopropyl group EC50 of 610 nM. However, this compound was less potent on the pyrrolidine ring in 38 with tert-butyl group could than 1, increasing plasma triglycerides and LDL cholesterol by slightly enhance the activity (Table 4). When this compound 29- and 12-fold, respectively. Testing this compound in fi was dosed orally to wild-type C57BL mice at 1 mg/kg or 3 primates showed an improvement in the lipid pro le compared mg/kg, it exhibited the highest level of ABCA1 mRNA to full agonists, which suggests that limiting LXRα activity and induction in the rat brain among all the compounds in this increasing LXRβ selectivity can improve the therapeutic series but did not significantly change the levels of Aβ1−40 or window of LXR.90 Aβ1−42 in the rat cerebrum or CFS.83 Recently, compound 41 (BMS-852927) was developed as a Kick et al. have synthesized a series of imidazole partial selective LXRβ agonist and has a desirable profile in animal agonists selective for LXRβ. These imidazoles were derived models and a wide therapeutic index in both non-human from pyrazole agonists 6−12 (Figure 5) by replacing the primates and mice. This compound selectively activates LXRβ

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.

10942 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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. α ff (Emax = 88%) over LXR (Emax = 20%). This compound undesirable e ect of 41 was the unexpected decrease in the showed potency in the in vitro human whole blood target gene circulating neutrophil counts in healthy subjects.91 When 41 ffi 82,91 assay (WBA), with an EC50 of 9 nM and 26% e cacy. was dosed at 15 mg, there was a 47% decrease in the Compound 41 caused both positive and adverse effects in neutrophil counts and the treatment was discontinued for two multiple ascending dose (MAD) clinical studies indicating that subjects when the neutrophil counts decreased to <1000 cells/ preclinical studies predict only therapeutic effects and not μL. The decrease in neutrophil counts was not predicted in adverse ones. In animal models, 41 decreased the lipogenic rats or in cynomolgus monkey models but was predicted in activity in primates and inhibited atherosclerosis in mice. mice. Treatment of mouse peritoneal macrophages with 41 Likewise, this compound increased hepatic and plasma TG as caused a dose-dependent decrease in circulating neutrophils. well as plasma LDL-C but decreased HDL-C. An important This response likely occurred because 41 downregulated IL-

10943 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

Figure 14. LXRβ-selective agonists developed through a head-to-tail molecular design.

a Table 5. LXR Activity of Phenylhydantoin Agonists 49−63

α μ β μ α β compd RXLXR EC50 ( M) Emax (%) LXR EC50 ( M) Emax (%) EC50 ratio for LXR /LXR 43 1.12 26 1.15 146 0.97 49 ia 0 ia 1 50 1.23 40 0.8 182 1.54 51 0.86 27 0.61 149 1.41

52 CH2 1.96 53 0.5 429 3.92

53 HCH2 ia 0 1.81 22 >5.52

54 (CH2)2 2.89 23 2.85 126 1.01

55 H (CH2)2 ia 1 1.88 223 >5.32

56 CH3 (CH2)2 3.14 53 0.7 615 4.89

57 OCH3 (CH2)2 ia 0 1.66 534 6.02

58 OH (CH2)2 ia 1 2.87 284 3.48

59 H CH(OH)CH2 ia 5 2.33 176 4.29

60 H COCH2 0.36 46 0.12 236 3

61 CH3 COCH2 0.4 71 0.064 264 6.25

62 OCH3 COCH2 3.74 38 0.36 306 10.4

63 OH COCH2 0.34 55 0.11 348 3.09 aia = inactive at 10 μM.

23α expression and increased Mertk expression in a dose- an active agonist conformation, while in the other monomer, it dependent manner, causing a reductioninneutrophil exists in a nonactive conformation (Figure 13). production and stimulating its clearance pathway. Compound 42 (Figure 14) was developed as a selective Both 40 and 41 show similarities in their binding mode, with LXRβ agonist to overcome the side effects normally associated three hydrogen bonds in their X-ray crystal structure (Figure with LXR activation, such as elevated triglycerides in the liver 13). However, both compounds lack interactions with His435 and plasma. Indeed, the oxochromene 42 has shown a lowering effect on lipids accumulated in the aortic arch in hamster and a that are known to activate the His435-Trp457 switch, which 92 was observed in the potent full pan agonists.91 The fluorine rise in the HDL-C levels. When tested via GAL4-LXR luciferase assay, compound 42 was inactive against LXRα at 10 atom on the hydroxymethyl phenyl group of 41 improved the μ β μ M, but it was active against LXR with EC50 = 0.96 M and shape complementarity to the pocket near this group, most ffi π 8% e cacy (this is the ratio between the maximal fold likely providing enhanced potency. In addition, a -stacking induction for the test compound and the fold induction for 1 at interaction between the benzylic phenyl ring in 41 and Phe340 10 μM). A head-to-tail molecular design approach was used to was observed, while the second chlorine allows the benzylic improve the selectivity and potency of 42 and led to the methyl to rotate and improve the molecular shape related to identification of compound 43 (Figure 14 and Table 5). 82 α the LXR binding pocket. Both X-ray structures (4RAK and Compound 43 exhibited a lower Emax for LXR (26%) and β β 5JY3) are composed of homodimers, where H12 adopted two higher Emax for LXR (146%). Increasing the LXR selectivity different conformational states. In one monomer, H12 exists in led to significant lipid accumulation suppression and decreased

10944 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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

LDL-C without causing an elevation in the level of TG in However, enantiomer (−)-64 showed agonistic activity for α β α μ plasma high-fat-and-cholesterol-fed Bio F1B hamster. both LXR and LXR (EC50 for LXR = 0.20 M, Emax = β β μ α β To improve the interaction between the LXR receptor and 164%, EC50 for LXR = 0.007 M, Emax = 255%, and EC50 / the ligand, the structure−activity relationship efforts focused = 28.5). Moreover, this compound has a good pharmacokinetic on the head moiety because the headgroup can modulate the profile and has increased HDL-C levels. Additionally, this activation of the His435-Trp457 switch.93 The headgroup in compound decreased the accumulation of lipids in high-fat and compound 43 was developed through a series of SAR cholesterol-fed LDL receptor knockout mice.94 modifications following the strategy depicted in Scheme 2 to Two more LXRβ agonists were discovered recently. The first give the desired bis-n-propyl phenyl structure. In brief, the six- agonist is the benzofuran-2-carboxylate 65 (E17110) (Figure fi ffi membered ring was removed, and the trifluoromethyl group 15), which showed signi cant e cacy, with an EC50 value of 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 β groups such as the methoxy group (57) and the hydroxyl Figure 15. LXR selective agonists 64, 65 (E17110), and 66 (IMB- group (58) were used to replace the methyl group. 808). Interestingly, agonistic activity toward LXRβ was enhanced fi signi cantly in 57, and ClogP was decreased slightly (=7.50). 0.72 μM. As with other LXR agonists, 65 increased the ABCA1 fi The metabolically labile benzylic position was then modi ed and ABCG1 target genes and enhanced the cholesterol efflux by introducing a hydroxyl group (59), which diminished in RAW264.7 macrophages in vitro, suggesting that it may activity. The conversion of hydroxyl into carbonyl (60) have antiatherosclerotic activity.95 improved both the potency and lipophilicity (ClogP = 6.54). The second compound, 66 (IMB-808) (Figure 15), was Among the series of aromatic linkers, compounds substituted identified as a promising antiatherosclerotic agent.96 When at the 2-position of acetophenone (61, 62, and 63) gave the tested in the LXR-GAL4 luciferase reporter assay, this β α β best LXR selectivity, with EC50 / of 6.25, 10.4, 3.09 for 61, compound exerted a dual LXR partial agonistic activity in μ μ 62, and 63, respectively. These results could not be improved vitro, with an EC50 value of 0.15 M and 0.53 M toward further to suppress the activity of LXRα, and attention was LXRα and LXRβ, respectively. Compound 66 increased the directed toward modifying the tail part. RCT and cholesterol metabolism target genes in murine and Therefore, compound 64, bearing the pyridylhydantoin human macrophages. There was a significant increase in the moiety, was prepared as a more potent and selective LXRβ levels of both mRNA and protein of ABCA1 and ABCG1 in α μ agonist (EC50 for LXR = 0.33 M, Emax = 68%, EC50 for RAW264.7 and THP-1 macrophage cells. However, mRNA β μ α β LXR = 0.058 M, Emax = 329%, and EC50 / = 5.69). Both and protein levels of ApoE were only slightly increased enantiomers of 64 were tested in Gal4-LXRα and LXRβ compared to 1. Compound 66 enhanced cholesterol efflux luciferase assays. Enantiomer (+)-64 showed no LXRα from macrophages and reduced cellular lipid accumulation in agonistic activity and very low LXRβ agonistic activity. assays of foam cells that were carried in RAW264.7 cells.

10945 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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

Interestingly, 66 did not induce the expression of lipogenic strong metal chelator that can bind to a wide range of genes such as FAS, SREBP-1c, and SCD-1. Compound 1 biological targets with high affinities.100 induces these lipogenic genes 4-fold at 1 μM compared to 66. Two new liver X receptor agonists were discovered using To better understand the difference between 1 and 66 in pharmacophore modeling and shape-based virtual screening. terms of gene regulations, the authors used docking to This study applied a virtual screening workflow to identify determine the key amino acid residues involved in the binding potential selective LXRβ agonists from a 3D compound of 1 and 66 in both isoforms. The amino acids that bind to 1 database. A library of 14 compounds was selected based on were different from the ones that bind to 66. The amino acids their LXR selectivity. Discovery Studio was used to create a 3D H421 and W443 in LXRα (H435 and W457 in LXRβ) were multiconformational library for the selected hits. These the most important for binding to 1 but interacted at a compounds were then screened against six pharmacophore moderate level with 66. Phe257 and Arg305 in LXRα (Phe271, models developed by Schuster and co-workers to account for Met312, and Thr316 in LXRβ) form the most important receptor flexibility.97 Moreover, a shape-based rapid overlay of interactions with 66. These key amino acids for 66 binding chemical structures (ROCS) screening was performed using a were replaced by site-directed mutagenesis, and the luciferase selective LXRβ compound to increase the probability of activity was measured for the different mutants. finding an LXRβ-selective hit. Molecules that were found to fit There was a distinct difference in the agonistic activity of the the query shape were selected for further evaluation via LXR wild type and various mutants. Two mutants, LXRα-R305G luciferase reporter gene assay. Compounds 70 and 71 (Figure and LXRβ-F271A, were selected to study the effect of mutation 17) were found to activate both LXR isoforms, and compound on cofactor recruitment by TR-FRET. While the activity of 1 70 was found to be slightly selective to LXRβ (1.8-fold over 101 in co-regulator recruitment was not affected, the activity of 66 LXRα). 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 96 treatment for atherosclerosis. Figure 17. LXR agonists 70 and 71. A two-step virtual screening protocol was applied to identify active compounds that included 3D-pharmacophore filters and 97 Steroid Agonists and Antagonists. Sterol-based agonists rescoring by shape alignment. LigandScout 2.3 was used for were found to have anti-inflammatory effects without inducing generating multiple pharmacophores based on multiple X-ray liver lipid accumulation as with 1. For example, N,N-dimethyl- structures, thereby accounting for different binding modes fl 3-hydroxycholenamide (DMHCA) (72), a gene-selective LXR related to receptor exibility. The second step of virtual synthetic agonist, exhibited anti-inflammatory activity in screening was a reranking of the screening hits with the leukocytes without affecting liver lipid accumulation. Addi- TanimotoCombo scoring function of ROCS, a method for fast tionally, this compound potently induced the expression of the alignment and comparison to the bioactive ligand conforma- target gene ABCA1 in the liver, small intestine, and peritoneal tion as a query molecule. Eighteen virtual hits were tested in macrophages and stimulated cholesterol transport. This − vitro using a reporter gene assay, and compounds 67 69 were compound showed less potency at increasing the hepatic found to activate LXR with low micromolar EC50; thus, they SREBP-1c mRNA and did not alter the circulating plasma TG 97 can be used for future lead optimization (Figure 16). in vitro or in vivo. Although 72 is a less potent LXR activator, However, these compounds are considered PAINS (pan assay it significantly enhanced the cholesterol efflux in macrophages interference compounds) and have the potential for cross compared to nonsteroidal agonists.102 98 reactivity with many targets. For example, compound 67 3β-Hydroxycholenamide (MePipHCA) (73)isanother might expel the diethylamine moiety via a retro-Mannich sterol-based LXR agonist derived from 72 via replacement of reaction and might generate metabolically reactive quinone the N,N-dimethyl group in 72 with the 4-methoxypiperidine methide. The formation of quinone methides can lead to group. Compound 73 has slightly better potency and efficacy hepatotoxicity or idiosyncratic toxicity, and one must be than 72 (for compound 72,EC50 = 422 nM and % of 1 = 45; 99 cautious when using this molecule as a lead compound. Both for compound 73,EC50 = 230 nM and % of 1 = 58). 67 and 68 have 8-hydroxyquinoline (8-HQ) moiety, which is a Compound 73 selectively improved both cellular and in vivo

10946 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

Figure 18. Steroidal LXR modulators 72−77.

Figure 19. LXR modulators 78−80.

Figure 20. LXR antagonists 81−83. potency and reduced the inflammation in dextran sulfate 77 complex showed a stable agonist conformation with small sodium (DSS)-induced colitis and traumatic brain injury RMSD (root-mean-square deviation) for His435 and slightly without causing lipid accumulation or liver injury (Figure larger RMSD for Trp457. In contrast, 76 showed conforma- 18).103 tional differences compared to 2 and 77, as the Trp457 side Compound 74, a 22SHC (22-S-hydroxycholesterol) mimic, chain rotated in a way that exposed the indole nitrogen atom has the same agonistic effect of 1. This compound significantly to form a strong hydrogen bond with the carboxylate group, upregulates the gene expression of SCD1 and increases which interrupted the agonist conformation.105 These results lipogenesis in myotubes in vitro. Additionally, this compound suggest that the negatively charged carboxylate in 75 and 76 is increased lipogenesis when combined with 1 more than 1 did required to disrupt the His435-Trp457 aromatic−aromatic alone.104 interaction and gives rise to the inverse agonist activity. The two fluorinated oxysterols 76 and 77 were developed Novel amide derivatives of the Fernholtz acid (78)(Figure based on the inverse agonist 75 (Figure 18). The replacement 19) have been synthesized and evaluated in both in silico and of the methylene group hydrogens in the inverse agonist 75 in vitro studies because the stereochemistry and functionality with fluorine atoms altered both the polarity and lipophilicity of carbon number 22 in cholesterol play a vital role in LXR of the side chains. The difluoroacid 76 significantly reduced target gene expression. Compound 79 (22-ketocholesterol) the basal levels of luciferase activity, acting as an inverse selectively upregulated the ATP-binding cassette transporter agonist, while the difluoro alcohol 77 showed agonist ABCA1 in skeletal muscle cells, with no obvious effect on activity.105 lipogenesis and little effect on FAS and SCD1. This kind of Molecular dynamic simulations of both 76 and 77 in selectivity makes 79 a good candidate for further studies as an comparison to that of the 2 were studied to explore the antiatherosclerotic drug. In contrast, the Fernholtz cyclo- molecular basis of their interactions.105 The fluorine atoms in 2 hexylamide (80) downregulated the de novo lipogenesis in a and 77 actively participate in the interaction with several dose-dependent manner and counteracted the effect of 1 on residues in the ligand-binding pocket. These interactions led to LXR.106 stabilization of the active agonist conformation, which was Recently, Astrand et al. developed a series of LXRβ confirmed by the reporter gene assay. Interestingly, the LXRβ/ antagonists using a molecular modeling approach. The

10947 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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 Figure 22. LXR antagonists 89−95. Synthetic Antagonists. Fenofibrate (84), a drug mar- keted as Tricor, was developed as a peroxisome proliferator- activated receptor α (PPARα) modulator and used successfully clearance (CL = 1.1 L h−1 kg−1) and 8-fold higher area under 108 in treating hypertriglyceridemia and mixed hyperlipidemia, the curve (AUC = 885 μg h/L) compared to 95 (CL = 4.8 L early diabetic retinopathy in type 2 diabetes mellitus patients, h−1 kg−1 and AUC = 102 μg h/L) following iv dosing in male 107,109−112 and related cardiovascular disorders. Interestingly, Sprague-Dawley rats. Following oral dosing in the same rats (5 fenofibrate esters (85−88) were found to inhibit lipogeneses mg/kg), 94 displayed good oral availability (F = 31%) and by binding with LXRs and repressing SREBP1 and FAS mRNA exposure (AUC = 1380 μg h/L), suggesting that 94 could be genes involved in lipogeneses in the liver. Similarly, other used as a good in vivo compound. fenofibrate esters bind directly to LXRs and function as LXR Careful examination of the structure−activity relationship of 113 antagonists (Figure 21). developed compounds revealed the following important The affinity and specificity of these fibrate esters to LXR observations: (i) replacement of the trifluoromethyl group in depend mainly on the presence of the ester group. While the 1 with alkyne substituents improved the binding affinity (for fi fi μ β brate esters bound only to LXRs, the corresponding bric 89,IC50 = 0.6 M in LXR SPA binding assay); (ii) using acids bound only to PPARα. Moreover, the fibrate esters larger trifluoroethyl (e.g., 1) or isobutyl groups (e.g., 89−93) reduced the transcriptional activity induced by LXR agonists, instead of N-methylsulfonamide groups enhanced the antag- ff μ β whereas the carboxylic acids did not a ect the transcriptional onistic activity (for 90,IC50 = 0.3 M in LXR SPA binding activity induced by the same agonists. There is a large degree assay); (iii) substitution at the meta position of the of similarity in the primary amino acid sequence and ligand- benzenesulfonamide with small groups such as CN enhanced binding domains between PPARα and LXRs, and it is possible the binding affinity slightly over the unsubstituted derivative to have structurally similar ligands that can modulate both (e.g., 89 vs 90); (iv) substituting phenylacetylene with 3- receptors. methylsulfonyl or 4-methylsulfonyl with or without CN on Jiao and his co-workers114 discovered a new series of LXR benzenesulfonamide produced compounds with good binding antagonists using a structure-based approach. This new series affinity and strong antagonistic activity (e.g., 91, 92, and was based on the well-known agonist 1, which is a dual LXRα/ 93).114 β agonist. Structural modification of 1 led to the identification The tricyclic 5,11-dihydro-5-methyl-11-methylene-6H- of compound 94 (Figure 22), which was the first LXR dibenz[b,e]azepin-6-one (96)(Figure 23) was selected as the antagonist suitable for in vivo studies in rodents. This lead compound to design novel compounds with LXR compound has good antagonistic activity, as shown in the antagonistic activity.110 The structural modification of 96 was β μ β LXR SPA binding assay (IC50 = 0.5 M) and the LXR based on weakening or abolishing the hydrogen bond μ α GAL4 assay (IC50 =2 M). Moreover, 95 has high microsomal interaction between 96 and His421 on H11 in the LXR stability, as >95% remained in rat liver microsomes after ligand-binding domain, which is responsible for its transactiva- incubation at 1 μM for 30 min. This compound has the best tional agonistic activity (Figure 23).110 Substituting hexafluor- pharmacokinetic profile among all the synthesized compounds; opropanol moiety with alkyl groups proved to be a successful it showed both good oral availability and good exposure strategy. The alky groups were unable to form the hydrogen following oral dosing in rats. Compound 94 displayed lower bond with His421 in LXRs and therefore prevented the proper

10948 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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). 3,4- Dimethoxy 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 117 Figure 23. Design strategy for LXR antagonists 97−99. analogue (102). Compound 103 showed the most potent LXR antagonistic μ α β folding of H12, which is required to induce the transactiva- activity, with an IC50 of 3.3 and 4.3 M for LXR and LXR , tional agonistic activity (Figure 23).110 respectively. Moreover, this compound possesses high A series of substituted azepin-6-one derivatives that exhibit antagonistic selectivity, as it did not show agonistic or inverse potent and promising selective antagonistic activity were agonistic activity toward LXR at 30 μM. In addition, 103 did developed, and compounds 97 and 98 were selected as not increase the levels of ABCA1 or SREBP-1c mRNA second-generation lead compounds.113 These two compounds expression in THP-1 cells. Therefore, this compound is a good stabilized binding of corepressors NCoR and SMRT in a dose- lead compound because it is not expected to increase the blood dependent manner and did not recruit coactivators SRC1 and triglycerides in addition to its interesting anti-inflammatory in DRIP205. Moreover, compounds 97 and 98 were selective for vitro activity.117 LXR over closely related farnesoid X receptor (FXR), PPARγ, The binding of 103 to LXRβ was studied using the TR- and retinoid X receptor α (RXRα). The low antagonistic FRET assay. Compound 103 inhibited the binding of activity of compounds 97 and 98 compared to 96 was coactivators with LXR in a dose-dependent manner and was ffi β μ 117 attributed to its lower binding a nity. Therefore, compound found to bind directly to LXR , with an IC50 of 1.8 M. 99, which possesses a hydroxyl group, was developed as a more Molecular docking of 103 into the cocrystal structure of LXRα μ potent and selective antagonist (IC50 = 3.5 M) through LBD complexed with 2 showed that 103 binds at the binding fi further structural modi cation. Compound 99 shows high site of LXRα. The docking model predicts hydrogen-bonding metabolic stability in human liver microsomes and exhibits interaction of the carbonyl group on 103 and the hydroxyl good in vitro ADME properties. Therefore, this compound has group on the Thr302 of LXRα. Unlike 1, no hydrogen-bonding potential for further development. interaction with His421 was observed. Since this interaction is Ishikawa and his co-workers115 developed 101 as a selective α μ α μ necessary for inducing the proper folding of H12 and LXR antagonist (IC50 = 0.2 M for LXR and >30 M for β recruiting the coactivators, this might be the reason behind LXR )(Figure 24). This antagonist was developed from a 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 β 76 an IC50 of 31.8 nM toward LXR . 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 ffi mandatory for high a nity, 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. fi − The rst selective synthetic LXR inverse Figure 24. LXR antagonists 101 104. agonist, SR9238 (105), is a tertiary sulfonamide that was developed by our group (Figure 25).45 In a cell-based thalidomide-related phthalimide derivative, PP2P (100), which cotransfection assay, this compound exhibits high potency was identified previously as an α-glucosidase inhibitor with toward both LXR isoforms but shows greater selectivity for α β μ α β α dual LXR / antagonistic activity (IC50 = 9.8 M for LXR LXR (IC50 = 43 nM) over LXR (IC50 = 214 nM). and 44 μM for LXRβ)(Figure 24).116 In a cell-based screening Compound 105 enhanced the interaction of corepressors of antagonistic activity, compound 100 reduced the NCoR ID1 and NCoR ID2 with both LXR isoforms in a dose- interleukin-6 (IL-6) level by 31% at 10 μM and was selected dependent manner.

10949 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

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 Figure 25. First potent LXR inverse agonists 105 and 106. (SMRT) and does not enhance coactivator recruitment; it therefore inhibits LXR activation. Accordingly, glycolytic and lipogenic gene expression is reduced, leading to inhibition of ff Compound 105 was liver specificandreducedthe the Warburg e ect (aerobic glycolysis) and lipogenesis in ffi expression of lipogenic genes in the liver and spared LXR cancer cells. The e cacy of 106 as an anticancer agent was ff target genes outside the liver to avoid adverse effects. When assessed in di erent cancer cell types. The cancer cell viability 105 was injected into mice (30 mg/kg, ip), it was not detected in an MTT reduction assay of prostate, colorectal, and lung cell in the plasma, but it was detected in the liver 2 h after the lineswasreducedatnanomolar concentrations. Most injection. In another experiment, when mice were injected for importantly, 106 is nontoxic to nonmalignant cells and induces 3 days with 105, high levels of 105 were detected in the liver. cancer cell death without noticeable side effects both in vitro The compound was also detected in the intestine to a lower and in vivo. In vitro, 106 was selective in suppressing the extent but not in the plasma, skeletal muscle, or brain. This elevated glycolytic output in cancer cells without disrupting result is mostly because the ester group is rapidly metabolized glycolytic genes in normal cells. Glycolysis is required in to the corresponding carboxylic acid by plasma lipases. The normal cells for energy production, but glycolytic enzyme acid analogue of 105 displays no LXR activity in the cell-based inhibitors usually disrupt the activity of glycolysis enzymes and cotransfection assay. Compound 105 displayed high selectivity cause undesirable side effects. When immune competent for LXR, and when screened in a nuclear receptor specificity (C57BL6J) tumor-bearing mice were treated with 106 at a 60 panel, it showed no activity at any of the tested nuclear mg/kg dose, 106 was shown to inhibit Lewis lung carcinoma receptors. Moreover, 105 suppresses the hepatic steatosis and (LLC1) tumor growth and increase Tnfα levels within tumors, inflammation that are present in a mouse model of without increasing the expression levels of other cytokines in nonalcoholic hepatosteatosis and hence shows great potential the liver. Moreover, 106 did not promote weight loss after 14 to treat nonalcoholic fatty liver disease (NAFLD). Interest- days of treatment. There is no available X-ray structure for ingly, 105 can play an important role in cholesterol reduction LXR bound with inverse agonists. We performed modeling and since it was found to reduce the levels of total plasma energy minimization for both 105 and 106 complexed with cholesterol, plasma LDL, and plasma HDL in diet-induced LXRβ using MacroModel.112 obesity mice.45 In an animal model of nonalcoholic The X-ray structure 3L0E of agonist GSK1305158 bound to steatohepatitis (NASH), 105 was very potent in treating LXRβ and TIF2 peptide was used as a reference for ligand mice with severe hepatic inflammation, hepatic steatosis, and positioning and modeling.76 The energy of both structures was fibrosis.46 When mice were treated with 105 (30 mg kg−1 minimized using the Polak−Ribiere conjugate gradient method day−1 ip) for 30 days while being maintained on the NASH (PRCG) and OPLS3 force field111 with a gradient convergence diet, the expression of hepatic inflammatory markers (e.g., IL- threshold value of 0.05. Both compounds were predicted to 6, IL-1β, and IL-12) was reduced. Moreover, the hepatic make mainly intermolecular hydrophobic interactions with

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

10950 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective active site hydrophobic residues Phe329, Phe340, and Met312 the therapeutic treatment of cancer, fatty liver diseases, Alzheimer and hydrogen-bonding interactions with Leu330 or Ser278 disease, and atherosclerosis. Recently, he was awarded The Egyptian (Figure 26). Unlike the structurally similar GSK1305158, State Prize in Chemical Science and the Presidential Medal of interactions of sulfonamide oxygen with His435 in H12 is lost. Excellence (First Class) for outstanding contribution from the Therefore, the His435-Trp457 switch responsible for agonist President of Egypt. conformation is disrupted. The methyl sulfone oxygen on the Shaimaa Goher received her B.Sc. degree in Chemistry from the opposite end of the ligand forms the second hydrogen bond 85,110 Faculty of Science, Benha University (Egypt) in 2013. She is currently with Leu330 or Ser278 (Figure 26). working toward her M.Sc. degree under the supervision of Prof. Bahaa ■ CONCLUSIONS El-Gendy. Her research project focuses on the development of novel liver X receptor modulators as antihepatitis C virus. Her research The LXRs play a major role in regulating lipid and cholesterol interests include medicinal chemistry, computational chemistry, metabolism in addition to their anti-inflammatory activities. organic chemistry, and drug design. She was selected among the These activities have led to expanded interest in developing 400 most qualified young scientists to attend the Lindau Nobel new small molecule modulators for these receptors. Cardio- meeting in Lindau, Germany, 2017. vascular diseases were the main driving force behind the development of early LXR modulators, but these compounds Lamees Hegazy is a Research Assistant Professor at the Department suffered from undesirable side effects such as the induction of of Pharmacology and Physiology at Saint Louis University School of hepatic steatosis. Elegant work by many research groups led to Medicine. She has a doctoral degree in Computational Biochemistry a better understanding of the structure and function of LXRs from the University of Florida. Dr. Hegazy is an expert in employing and allowed scientists to decipher novel mechanisms and molecular modeling and computational chemistry methods to study pathways that may lead to the discovery of new therapeutics. the dynamics and function of biological macromolecules. Her current Therefore, we placed substantial emphasis on the structure research focus is the use of molecular dynamics simulations, enhanced and function of LXRs because of their essential role in the drug sampling simulations, and free energy-based lead optimization to study the conformational behavior of nuclear receptors and design design process. We aimed to provide deeper insight into the ff important structural features of LXRs, and we discussed the modulators that target di erent conformational states. structure−activity relationships of most active and promising Mohamed Arief has been a Professor of Organic Chemistry since synthetic modulators in the past few years. 1999 in the Department of Chemistry, Faculty of Science, Benha The field is growing rapidly, and the focus is directed toward University, Egypt. He was awarded both a M.Sc. and a Ph.D. in developing selective liver X receptor modulators to avoid the Organic Chemistry from Ain Shams University, Egypt. From 1983 to undesirable side effects caused by the first generation of LXR 1984, he worked as a postdoctoral fellow at Institut für Organische modulators. Currently, there are three drugs in clinical trials at Chemie (Austria), with Professor Sauter. He worked as an Assistant different phases of study. RGX-104-001 is a drug used for the Professor starting from 1987 at Benha University, where he focused treatment of patients with advanced solid tumors and on efficient microwave-assisted solvent-free synthesis and molecular lymphoma, and it is in phase 1. VTP-38543 and ALX-101 docking studies, synthesis and reactions of some heterocyclic are in phase 2 clinical trials for the treatment of atopic compounds of expected biological activities, spectroscopic studies, dermatitis. The structures of these molecules have not been medicinal and pharmaceutical chemistry, and natural product reported in peer-reviewed manuscripts; thus, they were not chemistry. part of this review. Thomas P. Burris is Alumni Endowed Professor of Pharmacology in fi The discovery of the rst LXR inverse agonists 105 and 106 the Center for Clinical Pharmacology at Washington University that showed therapeutic potential in nonalcoholic fatty liver School of Medicine and St. Louis College of Pharmacy. Prior to his disease, nonalcoholic steatohepatitis, and cancer without current position he was the William Beaumont, M.D. Endowed ff noticeable side e ects in animal models is an area deserving Professor of Physiology and Chairman of the Department of of further exploration by medicinal chemists. Pharmacology & Physiology at Saint Louis University School of Medicine (2013−2018) and Professor at The Scripps Research ■ AUTHOR INFORMATION Institute (2007−2013). He held drug discovery research positions at Corresponding Author Johnson & Johnson and Eli Lilly for a decade before returning to *Phone: +1-3149775171. Fax: +1-3149776411. E-mail: academia. Dr. Burris is an expert in chemical biology and [email protected]. pharmacology of nuclear hormone receptors and has considerable ORCID experience developing drugs that target this class of drug target. Bahaa El-Dien M. El-Gendy: 0000-0003-4800-7976 Thomas P. Burris: 0000-0003-2922-4449 ■ ACKNOWLEDGMENTS Notes Bahaa El-Gendy thanks the Science and Technology Develop- The authors declare no competing financial interest. ment Fund (Egypt) for financial support (STDF-STF Grant Biographies 11969). Bahaa El-Dien El-Gendy is an Assistant Professor at the Department of Pharmacology and Physiology in the Saint Louis University School ■ ABBREVIATIONS USED of Medicine and Associate Professor of Bio-Organic Chemistry in NR, nuclear hormone receptor; AF-1, activation function 1; Benha University, Egypt. He received his Ph.D. in Chemistry from DBD, DNA-binding domain; LBD, ligand-binding domain; University of Florida under the supervision of Prof. Alan R. Katritzky AF2, activation function 2 (C-terminal domain); ER, estrogen and completed postdoctoral training at the Scripps Research Institute. receptor; AR, androgen receptor; TR, thyroid hormone The focus of Dr. El-Gendy’s research group is the development of receptor; RAR, vitamin A receptor (retinoic acid receptor); small molecule modulators for various nuclear hormone receptors for VDR, vitamin D receptor; PR, progesterone receptor; PPAR,

10951 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective peroxisome proliferator-activated receptor; FXR, farnesoid X and Evolution from the Rat Genome. Genome Res. 2004, 14, 580− receptor; PXR, pregnane X receptor; CAR, constitutive 590. androstane receptor; LXR, liver X receptor; AD, Alzheimer’s (3) Gronemeyer, H.; Gustafsson, J. Å.; Laudet, V. Principles for ’ Modulation of the Nuclear Receptor Superfamily. Nat. Rev. Drug disease; PD, Parkinson s disease; MS, multiple sclerosis; RXR, − retinoic acid receptor; LXRE, liver X receptor response Discovery 2004, 3 (11), 950 964. element; FAS, fatty acid synthase; CYP7A1, cytochrome (4) Laudet, V. Evolution of the Nuclear Receptor Superfamily: Early α Diversification from an Ancestral Orphan Receptor. J. Mol. Endocrinol. P450 isoform 7A1; ChREBP, cholesterol 7 -hydroxylase, 1997, 19 (3), 207−226. carbohydrate regulatory element binding protein; ApoE, (5) Laudet, V.; Auwerx, J.; Gustafsson, J. Å.; Wahli, W. A Unified apolipoprotein E; CETP, cholesteryl ester transfer protein; Nomenclature System for the Nuclear Receptor Superfamily. Cell ABC, ATP binding cassette; NCoR, nuclear corepressor; 1999, 97 (100), 161−163. SMRT, silencing retinoic acid and thyroid hormone receptor (6) Sladek, F. M. Nuclear Receptors as Drug Targets: New mediator; HDAC, histone deacetylase enzyme; Sin3A, stress Developments in Coregulators, Orphan Receptors and Major activated MAP kinase interacting protein 3A; ASC2, activating Therapeutic Areas. Expert Opin. Ther. Targets 2003, 7 (5), 679−684. signal integrator-2; RIP140, receptor-integrating protein140; (7) Komati, R.; Spadoni, D.; Zheng, S.; Sridhar, J.; Riley, K.; Wang, SRC, steroid receptor coactivator; NF κB, nuclear factor κB G. Ligands of Therapeutic Utility for the Liver X Receptors. Molecules (transcription factor); STAT1, signal transducer and activator 2017, 22 (1), 88. of transcription 1; SUMO2 or SUMO3, small ubiquitin-like (8) Hong, C.; Tontonoz, P. Liver X Receptors in Lipid Metabolism: fi Opportunities for Drug Discovery. Nat. Rev. Drug Discovery 2014, 13 modi er 2 or 3; PDB, Protein Data Bank; ABCA1, ATP- − binding cassette transporter A1; ABCG1, ATP-binding cassette (6), 433 444. (9) Ducheix, S.; Montagner, A.; Theodorou, V.; Ferrier, L.; Guillou, subfamily G member 1; CNS, central nervous system; HTS, H. The Liver X Receptor: A Master Regulator of the Gut-Liver Axis high throughput screening; CFR, Code of Federal Regulations; and a Target for Non Alcoholic Fatty Liver Disease. Biochem. Pgp, P-glycoprotein; Boc2O, di-tert-butyl dicarbonate; RCT, Pharmacol. 2013, 86 (1), 96−105. reverse cholesterol transport; rCYP2C9, recombinant cyto- (10) Zhao, C.; Dahlman-Wright, K. Liver X Receptor in Cholesterol chrome P450 2C9; SREBP1c, sterol regulatory element- Metabolism. J. Endocrinol. 2010, 204 (3), 233−240. binding transcription factor 1; THP1, human leukemic (11) Zelcer, N.; Tontonoz, P. Liver X Receptors as Integrators of monocyte cell line; HepG2, hepatocellular carcinoma cell Metabolic and Inflammatory Signaling. J. Clin. Invest. 2006, 116 (3), line; DIBAL, diisobutyl aluminum hydride, a strong and bulky 607−614. reducing agent; Aβ,amyloidβ;SAR,structure−activity (12) Willy, P. J.; Umesono, K.; Ong, E. S.; Evans, R. M.; Heyman, R. relationship; CFS, chronic fatigue syndrome; SUMO, small A.; Mangelsdorf, D. J. LXR, a Nuclear Receptor That Defines a − ubiquitin related modifier; TG, triglyceride; LDL, low-density Distinct Retinoid Response Pathway. Genes Dev. 1995, 9 (9), 1033 lipoprotein; MAD, multiple ascending dose; DMHCA, N, N- 1045. dimethyl-3-hydroxycholenamide; DSS, dextran sulfate sodium; (13) Janowski, B. A.; Willy, P. J.; Devi, T. R.; Falck, J. R.; Mangelsdorf, D. J. An Oxysterol Signalling Pathway Mediated by the SCD1, stearoyl-CoA desaturase-1; RMSD, root-mean-square Nuclear Receptor LXR Alpha. Nature 1996, 383 (6602), 728−731. deviation of atomic positions; 22KC, 22-ketocholesterol; LDL- (14) Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver, T. C, low-density lipoprotein C; HDL-C, high-density lipoprotein A.; Oliver, B. B.; Su, J.; Sundseth, S. S.; Winegar, D. A.; Blanchard, D. C; PK, pharmacokinetics; Gal4, DNA-binding yeast tran- E.; Spencer, T. A.; Willson, T. M. Activation of the Nuclear Receptor scription factor; ClogP, the logarithm of partition coefficient LXR by Oxysterols Defines a New Hormone Response Pathway. J. between n-octanol and water log(Coctanol/Cwater); RAW264.7, a Biol. Chem. 1997, 272 (6), 3137−3140. macrophage-like, Abelson leukemia virus-transformed cell line (15) Edwards, P. A.; Kennedy, M. A.; Mak, P. A. LXRs; Oxysterol derived from BALB/c mice; QSAR, quantitative structure− Activated Nuclear Receptors That Regulate Genes Controlling Lipid activity relationship; ZINC database, database for the Homeostasis. Vasc. Pharmacol. 2002, 38 (4), 249−256. commercially available chemical compounds for virtual screen- (16) Tobin, K. A. R.; Ulven, S. M.; Schuster, G. U.; Steineger, H. H.; ing; ROCS, software for virtual screening; 22SHC, 22-S- Andresen, S. M.; Gustafsson, J. Å.; Nebb, H. I. Liver X Receptors as Insulin-Mediating Factors in Fatty Acid and Cholesterol Biosynthesis. hydroxycholesterol; TBS, tert-butyldimethylsilyl group; DMP, − Dess−Martin periodinate; ADME, pharmacokinetic properties J. Biol. Chem. 2002, 277 (12), 10691 10697. absorption, distribution, metabolism, and excretion; IL-6, (17) Whitney, K. D.; Watson, M. A.; Collins, J. L.; Benson, W. G.; fl Stone, T. M.; Numerick, M. J.; Tippin, T. K.; Wilson, J. G.; Winegar, interleukin-6; TR-FRET, time-resolved uorescence resonance D. A.; Kliewer, S. A. Regulation of Cholesterol Homeostasis by the energy transfer assay; NAFLD, nonalcoholic fatty liver disease; Liver X Receptors in the Central Nervous System. Mol. Endocrinol. NASH, nonalcoholic steatohepatitis; MTT, a colorimetric 2002, 16 (6), 1378−1385. assay for cell metabolic activity; DCM, dichloromethane; (18) Wójcicka, G.; Jamroz-wisniewska,́ A.; Horoszewicz, K.; PRCG, Polak−Ribiere conjugate gradient method Beltowski, J. Liver X Receptors (LXRs). Part I: Structure, Function, Regulation of Activity, and Role in Lipid Metabolism. Postep. Hig Med. ■ REFERENCES Dosw 2007, 61, 736−759. (1) Robinson-Rechavi, M.; Carpentier, A. S.; Duffraisse, M.; Laudet, (19) Courtney, R.; Landreth, G. E. LXR Regulation of Brain V.; Escriva, H.; Al, E.; Gronemeyer, H.; Laudet, V.; Kliewer, S. A.; Al, Cholesterol: From Development to Disease. Trends Endocrinol. Metab. E.; Adams, M. D.; Al, E.; Sluder, A. E.; Maina, C. V.; Committee, N. 2016, 27 (6), 404−414. R. N.; Consortium, I. H. G. S.; Venter, J. C.; Al, E.; Graveley, B. R.; (20) Cha, J. Y.; Repa, J. J. The Liver X Receptor (LXR) and Hepatic Black, D. L.; Shoemaker, D. D.; Al, E.; Kuiper, G. G.; Al, E.; Altschul, Lipogenesis: The Carbohydrate-Response Element-Binding Protein Is S. F.; Al, E.; Burge, C.; Karlin, S.; Galtier, N.; Al, E.; Saitou, N.; Nei, a Target Gene of LXR. J. Biol. Chem. 2007, 282 (1), 743−751. M.; Zanaria, E.; Al, E.; Seol, W.; Al, E.; Stassen, M. J.; Al, E. How (21) Flaveny, C. A.; Griffett, K.; El-Gendy, B.-E. M.; Kazantzis, M.; Many Nuclear Hormone Receptors Are There in the Human Sengupta, M.; Amelio, A. L.; Chatterjee, A.; Walker, J.; Solt, L. A.; Genome? Trends Genet. 2001, 17 (10), 554−556. Kamenecka, T. M.; Burris, T. P. Broad Anti-Tumor Activity of a Small (2) Zhang, Z.; Burch, P.; Cooney, A. Genomic Analysis of the Molecule That Selectively Targets the Warburg Effect and Lipo- Nuclear Receptor Family: New Insights into Structure, Regulation, genesis. Cancer Cell 2015, 28 (1), 42−56.

10952 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

(22) Bensinger, S. J.; Bradley, M. N.; Joseph, S. B.; Zelcer, N.; Cells by Activating the LXRs/SREBP-1c/FAS Signaling Cascade. Janssen, E. M.; Hausner, M. A.; Shih, R.; Parks, J. S.; Edwards, P. A.; Cancer Res. 2016, 76 (16), 4696−4707. Jamieson, B. D.; Tontonoz, P. LXR Signaling Couples Sterol (41) Lin, C. Y.; Gustafsson, J. Å. Targeting Liver X Receptors in Metabolism to Proliferation in the Acquired Immune Response. Cell Cancer Therapeutics. Nat. Rev. Cancer 2015, 15 (4), 216−224. 2008, 134 (1), 97−111. (42) El Roz, A.; Bard, J. M.; Valin, S.; Huvelin, J. M.; Nazih, H. (23) A-Gonzalez,́ N.; Castrillo, A. Liver X Receptors as Regulators of Macrophage Apolipoprotein E and Proliferation of MCF-7 Breast Macrophage Inflammatory and Metabolic Pathways. Biochim. Biophys. Cancer Cells: Role of LXR. Anticancer Res. 2013, 33 (9), 3783−3790. Acta, Mol. Basis Dis. 2011, 1812 (8), 982−994. (43) Pencheva, N.; Buss, C. G.; Posada, J.; Merghoub, T.; Tavazoie, (24) Bonet, M. L.; Ribot, J.; Felipe, F.; Palou, A. Vitamin A and the S. F. Broad-Spectrum Therapeutic Suppression of Metastatic Regulation of Fat Reserves. Cell. Mol. Life Sci. 2003, 60 (7), 1311− Melanoma through Nuclear Hormone Receptor Activation. Cell 1321. 2014, 156 (5), 986−1001. (25) Novac, N.; Heinzel, T. Nuclear Receptors: Overview and (44) Tavazoie, M. F.; Pollack, I.; Tanqueco, R.; Ostendorf, B. N.; Classification. Curr. Drug Targets: Inflammation Allergy 2004, 3 (4), Reis, B. S.; Gonsalves, F. C.; Kurth, I.; Andreu-Agullo, C.; Derbyshire, 335−346. M. L.; Posada, J.; Takeda, S.; Tafreshian, K. N.; Rowinsky, E.; Szarek, (26) Yin, K.; Smith, A. G. Nuclear Receptor Function in Skin Health M.; Waltzman, R. J.; Mcmillan, E. A.; Zhao, C.; Mita, M.; Mita, A.; and Disease: Therapeutic Opportunities in the Orphan and Adopted Chmielowski, B.; Postow, M. A.; Ribas, A.; Mucida, D.; Tavazoie, S. F. − Receptor Classes. Cell. Mol. Life Sci. 2016, 73 (20), 3789 3800. LXR/ApoE Activation Restricts Innate Immune Suppression in (27) Bedewi, A. El; Awad, M.; Nada, E. Role of Tissue Liver X- Cancer. Cell 2018, 172 (4), 825−840. Receptors Level in Skin Diseases. Int. J. Pept. Res. Ther. 2013, 19 (4), (45) Griffett, K.; Solt, L. A.; El-Gendy, B.-E. M.; Kamenecka, T. M.; − 275 279. Burris, T. P. A Liver-Selective LXR Inverse Agonist That Suppresses (28) Chintalacharuvu, S. R.; Sandusky, G. E.; Burris, T. P.; Burmer, Hepatic Steatosis. ACS Chem. Biol. 2013, 8 (3), 559−567. G. C.; Nagpal, S. Liver X Receptor Is a Therapeutic Target in (46) Griffett, K.; Welch, R. D.; Flaveny, C. A.; Kolar, G. R.; − Collagen-Induced Arthritis. Arthritis Rheum. 2007, 56 (4), 1365 Neuschwander-Tetri, B. A.; Burris, T. P. The LXR Inverse Agonist 1367. SR9238 Suppresses Fibrosis in a Model of Non-Alcoholic (29) Spyridon, M.; Moraes, L. A.; Jones, C. I.; Sage, T.; Sasikumar, Steatohepatitis. Mol. Metab. 2015, 4 (4), 353−357. P.; Bucci, G.; Gibbins, J. M. LXR as a Novel Antithrombotic Target. − (47) Steffensen, K. R. Are Synthetic Compounds That Silence the Blood 2011, 117 (21), 5751 5761. Liver-X-Receptor the Next Generation of Anti-Cancer Drugs? (30) Wang, Y.; Rogers, P. M.; Stayrook, K. R.; Su, C.; Varga, G.; ’ Altering the Course of Small Cell Lung Cancer: Targeting Cancer Shen, Q.; Nagpal, S.; Burris, T. P. The Selective Alzheimer s Disease Stem Cells via LSD1 Inhibition. Cancer Cell 2015, 28 (1), 3−4. Indicator-1 Gene (Seladin-1/DHCR24) is a Liver X Receptor Target (48) Teboul, M.; Enmark, E.; Li, Q.; Wikstrom, A. C.; Pelto-Huikko, Gene. Mol. Pharmacol. 2008, 74 (6), 1716−1721. M.; Gustafsson, J. Å. OR-1, a Member of the Nuclear Receptor (31) Dai, Y. B.; Tan, X. J.; Wu, W. F.; Warner, M.; Gustafsson, J. Å. Superfamily That Interacts with the 9-Cis-Retinoic Acid Receptor. Liver X Receptor β Protects Dopaminergic Neurons in a Mouse Proc. Natl. Acad. Sci. U. S. A. 1995, 92 (6), 2096−2100. Model of Parkinson Disease. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (49) Shinar, D. M.; Endo, N.; Rutledge, S. J.; Vogel, R.; Rodan, G. (32), 13112−13117. A.; Schmidt, A. NER, a New Member of the Gene Family Encoding (32) Bełtowski, J. Liver X Receptors (LXR) as Therapeutic Targets − the Human Steroid Hormone Nuclear Receptor. Gene 1994, 147 (2), in Dyslipidemia. Cardiovasc. Ther. 2008, 26 (4), 297 316. − (33) Varin, A.; Thomas, C.; Ishibashi, M.; Menégaut,́ L.; Gautier, T.; 273 276. Trousson, A.; Bergas, V.; De Barros, J. P. P.; Narce, M.; Lobaccaro, J. (50) Apfel, R.; Benbrook, D.; Lernhardt, E.; Ortiz, M. A.; Salbert, G.; M. A.; Lagrost, L.; Masson, D. Liver X Receptor Activation Promotes Pfahl, M. A Novel Orphan Receptor Specific for a Subset of Thyroid Polyunsaturated Fatty Acid Synthesis in Macrophages: Relevance in Hormone-Responsive Elements and Its Interaction with the Retinoid/ Thyroid Hormone Receptor Subfamily. Mol. Cell. Biol. 1994, 14 (10), the Context of Atherosclerosis. Arterioscler., Thromb., Vasc. Biol. 2015, − 35 (6), 1357−1365. 7025 7035. (34) Kappus, M. S.; Murphy, A. J.; Abramowicz, S.; Ntonga, V.; (51) Song, C.; Kokontis, J. M.; Hiipakka, R. A.; Liao, S. Ubiquitous Welch, C. L.; Tall, A. R.; Westerterp, M. Activation of Liver X Receptor: A Receptor That Modulates Gene Activation by Retinoic Receptor Decreases Atherosclerosis in Ldlr Mice in the Absence of Acid and Thyroid Hormone Receptors. Proc. Natl. Acad. Sci. U. S. A. − ATP-Binding Cassette Transporters A1 and G1 in Myeloid Cells. 1994, 91 (23), 10809 10813. Arterioscler., Thromb., Vasc. Biol. 2014, 34 (2), 279−284. (52) Song, C.; Hiipakka, R. A.; Kokontis, J. M.; Liao, S. Ubiquitous (35) Fievet,́ C.; Staels, B. Liver X Receptor Modulators: Effects on Receptor: Structures, Immunocytochemical Localization, and Modu- Lipid Metabolism and Potential Use in the Treatment of lation of Gene Activation by Receptors for Retinoic Acids and − Atherosclerosis. Biochem. Pharmacol. 2009, 77 (8), 1316−1327. Thyroid Hormones. Ann. N. Y. Acad. Sci. 1995, 761,38 49. (36) Zhu, Y.; Li, Y. Liver X Receptors as Potential Therapeutic (53) Robinson-Rechavi, M.; Escriva, H.; Laudet, V. The Nuclear − Targets in Atherosclerosis. Clin. Invest. Med. 2009, 32 (5), E383− Receptor Superfamily. J. Cell Sci. 2003, 116 (4), 585 586. E394. (54) Tice, C. M.; Noto, P. B.; Fan, K. Y.; Zhuang, L.; Lala, D. S.; (37) Wang, Z.; Sadovnick, A. D.; Traboulsee, A. L.; Ross, J. P.; Singh, S. B. The Medicinal Chemistry of Liver x Receptor (LXR) Bernales, C. Q.; Encarnacion, M.; Yee, I. M.; De Lemos, M.; Modulators. J. Med. Chem. 2014, 57 (17), 7182−7205. Greenwood, T.; Lee, J. D.; Wright, G.; Ross, C. J.; Zhang, S.; Song, (55) Viennois, E.; Mouzat, K.; Dufour, J.; Morel, L.; Lobaccaro, J.; W.; Vilarino-Guell, C. Nuclear Receptor NR1H3 in Familial Multiple Baron, S. Selective Liver X Receptor Modulators (SLiMs): What Use Sclerosis. Neuron 2016, 90 (5), 948−954. in Human Health? Mol. Cell. Endocrinol. 2012, 351 (2), 129−141. (38) Zhang, R.; Liu, Z.; Li, Y.; Wu, B. LXR Agonist Regulates the (56) Hu, X.; Li, S.; Wu, J.; Xia, C.; Lala, D. S. Liver X Receptors Proliferation and Apoptosis of Human T-Cell Acute Lymphoblastic Interact with Corepressors to Regulate Gene Expression. Mol. Leukemia Cells via the SOCS3 Pathway. Int. J. Biochem. Cell Biol. Endocrinol. 2003, 17 (6), 1019−1026. 2016, 78, 180−185. (57) Jones, P. L.; Sachs, L. M.; Rouse, N.; Wade, P. A.; Shi, Y. B. (39) Derangere,̀ V.; Rebé,́ C. LXRβ Subcellular Localization: A New Multiple N-CoR Complexes Contain Distinct Histone Deacetylases. J. Tool to Investigate Cancer Cell Response to LXR Ligand-Induced Biol. Chem. 2001, 276 (12), 8807−8811. Cytotoxicity? Cancer Cell Microenviron. 2016, 3 (1), 1−4. (58) Lee, S.; Lee, J.; Lee, S.-K.; Lee, J. W. Activating Signal (40) Zhao, Y.; Li, H.; Zhang, Y.; Li, L.; Fang, R.; Li, Y.; Liu, Q.; Cointegrator-2 Is an Essential Adaptor to Recruit Histone H3 Lysine Zhang, W.; Qiu, L.; Liu, F.; Zhang, X.; Ye, L. Oncoprotein HBXIP 4 Methyltransferases MLL3 and MLL4 to the Liver X Receptors. Mol. Modulates Abnormal Lipid Metabolism and Growth of Breast Cancer Endocrinol. 2008, 22 (6), 1312−1319.

10953 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

(59) Herzog, B.; Hallberg, M.; Seth, A.; Woods, A.; White, R.; Evans, C.; Jaye, M.; Thompson, S. K. Synthesis and SAR of Potent Parker, M. G. The Nuclear Receptor Cofactor, Receptor-Interacting LXR Agonists Containing an Indole Pharmacophore. Bioorg. Med. Protein 140, Is Required for the Regulation of Hepatic Lipid and Chem. Lett. 2009, 19 (4), 1097−1100. Glucose Metabolism by Liver X Receptor. Mol. Endocrinol. 2007, 21 (74) Fradera, X.; Vu, D.; Nimz, O.; Skene, R.; Hosfield, D.; (11), 2687−2697. Wynands, R.; Cooke, A. J.; Haunsø, A.; King, A.; Bennett, D. J.; (60) Huuskonen, J.; Fielding, P. E.; Fielding, C. J. Role of P160 Mcguire, R.; Uitdehaag, J. C. M. X-Ray Structures of the LXRα LBD Coactivator Complex in the Activation of Liver X Receptor. in Its Homodimeric Form and Implications for Heterodimer Arterioscler., Thromb., Vasc. Biol. 2004, 24 (4), 703−708. 2010 − ̈ Signaling. J. Mol. Biol. , 399 (1), 120 132. (61) Svensson, S.; Ostberg, T.; Jacobsson, M.; Norström, C.; (75) Bernotas, R. C.; Singhaus, R. R.; Kaufman, D. H.; Travins, J. Stefansson, K.; Hallen,́ D.; Johansson, I. C.; Zachrisson, K.; Ogg, D.; M.; Ullrich, J. W.; Unwalla, R.; Quinet, E.; Evans, M.; Nambi, P.; Jendeberg, L. Crystal Structure of the Heterodimeric Complex of Olland, A.; Kauppi, B.; Wilhelmsson, A.; Goos-Nilsson, A.; Wrobel, J. LXRα and RXRβ Ligand-Binding Domains in a Fully Agonistic 4-(3-Aryloxyaryl)quinoline Sulfones Are Potent Liver X Receptor Conformation. EMBO J. 2003, 22 (18), 4625−4633. Agonists. Bioorg. Med. Chem. Lett. 2010, 20 (1), 209−212. (62) Ghisletti, S.; Huang, W.; Ogawa, S.; Pascual, G.; Lin, M. E.; (76) Zuercher, W. J.; Buckholz, R. G.; Campobasso, N.; Collins, J. Willson, T. M.; Rosenfeld, M. G.; Glass, C. K. Parallel SUMOylation- L.; Galardi, C. M.; Gampe, R. T.; Hyatt, S. M.; Merrihew, S. L.; Dependent Pathways Mediate Gene- and Signal-Specific Trans- Moore, J. T.; Oplinger, J. A.; Reid, P. R.; Spearing, P. K.; Stanley, T. γ − repression by LXRs and PPAR . Mol. Cell 2007, 25 (1), 57 70. B.; Stewart, E. L.; Willson, T. M. Discovery of Tertiary Sulfonamides (63) Lee, J. H.; Park, S. M.; Kim, O. S.; Lee, C. S.; Woo, J. H.; Park, as Potent Liver X Receptor Antagonists. J. Med. Chem. 2010, 53 (8), α β S. J.; Joe, E.-h.; Jou, I. Differential SUMOylation of LXR And LXR 3412−3416. Mediates Transrepression of STAT1 Inflammatory Signaling in IFN- (77) Lou, X.; Toresson, G.; Benod, C.; Suh, J. H.; Philips, K. J.; γ − -Stimulated Brain Astrocytes. Mol. Cell 2009, 35 (6), 806 817. Webb, P.; Gustafsson, J.-A. Structure of the Retinoid X Receptor α- (64) Kopecky, D. J.; Jiao, X. Y.; Fisher, B.; McKendry, S.; Labelle, Liver X Receptor β (RXRα−LXRβ) Heterodimer on DNA. Nat. M.; Piper, D. E.; Coward, P.; Shiau, A. K.; Escaron, P.; Danao, J.; Struct. Mol. Biol. 2014, 21 (3), 277−281. Chai, A.; Jaen, J.; Kayser, F. Discovery of a New Binding Mode for a (78) Kick, E.; Martin, R.; Xie, Y.; Flatt, B.; Schweiger, E.; Wang, T. Series of Liver X Receptor Agonists. Bioorg. Med. Chem. Lett. 2012, 22 − L.; Busch, B.; Nyman, M.; Gu, X. H.; Yan, G.; Wagner, B.; Nanao, M.; (7), 2407 2410. Nguyen, L.; Stout, T.; Plonowski, A.; Schulman, I.; Ostrowski, J.; (65) Halgren, T. A. Identifying and Characterizing Binding Sites and − Kirchgessner, T.; Wexler, R.; Mohan, R. Liver X Receptor (LXR) Assessing Druggability. J. Chem. Inf. Model. 2009, 49 (2), 377 389. Partial Agonists: Biaryl Pyrazoles and Imidazoles Displaying a (66) Beautrait, A.; Karaboga, A. S.; Souchet, M.; Maigret, B. Induced Preference for LXRβ. Bioorg. Med. Chem. Lett. 2015, 25 (2), 372−377. Fit in Liver X Receptor Beta: A Molecular Dynamics-Based − (79) Matsui, Y.; Yamaguchi, T.; Yamazaki, T.; Yoshida, M.; Arai, M.; Investigation. Proteins: Struct., Funct., Genet. 2008, 72 (3), 873 882. Terasaka, N.; Honzumi, S.; Wakabayashi, K.; Hayashi, S.; Nakai, D.; (67) Hoerer, S.; Schmid, A.; Heckel, A.; Budzinski, R.; Nar, H. Hanzawa, H.; Tamaki, K. Discovery and Structure-Guided Opti- Crystal Structure of the Human Liver X Receptor β Ligand-Binding mization of tert-Butyl 6-(Phenoxymethyl)-3-(Trifluoromethyl)-Ben- Domain in Complex with a Synthetic Agonist. J. Mol. Biol. 2003, 334, zoates as Liver X Receptor Agonists. Bioorg. Med. Chem. Lett. 2015, 25 853−861. (18), 3914−3920. (68) Chen, M.; Yang, F.; Kang, J.; Yang, X.; Lai, X.; Gao, Y. Multi- (80) Stachel, S. J.; Zerbinatti, C.; Rudd, M. T.; Cosden, M.; Suon, S.; Layer Identification of Highly-Potent ABCA1 up-Regulators Target- Nanda, K. K.; Wessner, K.; Dimuzio, J.; Maxwell, J.; Wu, Z.; Uslaner, ing LXRβ Using Multiple QSAR Modeling, Structural Similarity J. M.; Michener, M. S.; Szczerba, P.; Brnardic, E.; Rada, V.; Kim, Y.; Analysis, and Molecular Docking. Molecules 2016, 21 (12), 1639. (69) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert, Meissner, R.; Wuelfing, P.; Yuan, Y.; Ballard, J.; Holahan, M.; Klein, M. H.; Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.; D. J.; Lu, J.; Fradera, X.; Parthasarathy, G.; Uebele, V. N.; Chen, Z.; Li, Y.; Li, J.; Cooke, A. J.; Bennett, D. J.; Bilodeau, M. T.; Renger, J. Parks, D.; Watson, M.; Wisely, B.; Willson, T. M. X-Ray Crystal β Structure of the Liver X Receptor β Ligand Binding Domain: Identification and in Vivo Evaluation of Liver X Receptor -Selective Agonists for the Potential Treatment of Alzheimer’s Disease. J. Med. Regulation by a Histidine-Tryptophan Switch. J. Biol. Chem. 2003, − 278 (29), 27138−27143. Chem. 2016, 59 (7), 3489 3498. (70) Farnegä rdh,̊ M.; Bonn, T.; Sun, S.; Ljunggren, J.; Ahola, H.; (81) Zheng, Y.; Zhuang, L.; Fan, K. Y.; Tice, C. M.; Zhao, W.; Dong, Wilhelmsson, A.; Gustafsson, J. Å.; Carlquist, M. The Three- C.; Lotesta, S. D.; Leftheris, K.; Lindblom, P. R.; Liu, Z.; Shimada, J.; Dimensional Structure of the Liver X Receptor β Reveals a Flexible Noto, P. B.; Meng, S.; Hardy, A.; Howard, L.; Krosky, P.; Guo, J.; Ligand-Binding Pocket That Can Accommodate Fundamentally Lipinski, K.; Kandpal, G.; Bukhtiyarov, Y.; Zhao, Y.; Lala, D.; Van Different Ligands. J. Biol. Chem. 2003, 278 (40), 38821−38828. Orden, R.; Zhou, J.; Chen, G.; Wu, Z.; McKeever, B. M.; McGeehan, (71) Jaye, M. C.; Krawiec, J. A.; Campobasso, N.; Smallwood, A.; G. M.; Gregg, R. E.; Claremon, D. A.; Singh, S. B. Discovery of a β Qiu, C.; Lu, Q.; Kerrigan, J. J.; De Los Frailes Alvaro, M. D. L. F.; Novel, Orally Efficacious Liver X Receptor (LXR) Agonist. J. Med. − Laffitte, B.; Liu, W.; Marino, J. P.; Meyer, C. R.; Nichols, J. A.; Parks, Chem. 2016, 59 (7), 3264 3271. D. J.; Perez, P.; Sarov-blat, L.; Seepersaud, S. D.; Steplewski, K. M.; (82) Kick, E. K.; Busch, B. B.; Martin, R.; Stevens, W. C.; Bollu, V.; Thompson, S. K.; Wang, P.; Watson, M. A.; Webb, C. L.; Haigh, D.; Xie, Y.; Boren, B. C.; Nyman, M. C.; Nanao, M. H.; Nguyen, L.; Caravella, J. A.; Macphee, C. H.; Willson, T. M.; Collins, J. L. Plonowski, A.; Schulman, I. G.; Yan, G.; Zhang, H.; Hou, X.; Valente, Discovery of Substituted Maleimides as Liver X Receptor Agonists M. N.; Narayanan, R.; Behnia, K.; Rodrigues, A. D.; Brock, B.; and Determination of a Ligand-Bound Crystal Structure. J. Med. Smalley, J.; Cantor, G. H.; Lupisella, J.; Sleph, P.; Grimm, D.; Chem. 2005, 48, 5419−5422. Ostrowski, J.; Wexler, R. R.; Kirchgessner, T.; Mohan, R. Discovery of (72) Chao, E. Y.; Caravella, J. A.; Watson, M. A.; Campobasso, N.; Highly Potent Liver X Receptor β Agonists. ACS Med. Chem. Lett. Ghisletti, S.; Billin, A. N.; Galardi, C.; Wang, P.; Laffitte, B. A.; 2016, 7 (12), 1207−1212. Iannone, M. A.; Goodwin, B. J.; Nichols, J. A.; Parks, D. J.; Stewart, (83) Tice, C. M.; Noto, P. B.; Fan, K. Y.; Zhao, W.; Lotesta, S. D.; E.; Wiethe, R. W.; Williams, S. P.; Smallwood, A.; Pearce, K. H.; Dong, C.; Marcus, A. P.; Zheng, Y. J.; Chen, G.; Wu, Z.; Van Orden, Glass, C. K.; Willson, T. M.; Zuercher, W. J.; Collins, P. J. Structure- R.; Zhou, J.; Bukhtiyarov, Y.; Zhao, Y.; Lipinski, K.; Howard, L.; Guo, Guided Design of N-Phenyl Tertiary Amines as Transrepression- J.; Kandpal, G.; Meng, S.; Hardy, A.; Krosky, P.; Gregg, R. E.; Selective Liver X Receptor Modulators with Anti-Inflammatory Leftheris, K.; McKeever, B. M.; Singh, S. B.; Lala, D.; McGeehan, G. Activity. J. Med. Chem. 2008, 51 (18), 5758−5765. M.; Zhuang, L.; Claremon, D. A. Brain Penetrant Liver X Receptor (73) Washburn, D. G.; Hoang, T. H.; Campobasso, N.; Smallwood, (LXR) Modulators Based on a 2,4,5,6-Tetrahydropyrrolo[3,4-c]- A.; Parks, D. J.; Webb, C. L.; Frank, K. A.; Nord, M.; Duraiswami, C.; Pyrazole Core. Bioorg. Med. Chem. Lett. 2016, 26 (20), 5044−5050.

10954 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

(84) Shiau, A. K.; Barstad, D.; Radek, J. T.; Meyers, M. J.; Nettles, K. (97) Von Grafenstein, S.; Mihaly-Bison, J.; Wolber, G.; Bochkov, V. W.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A.; Agard, D. A.; N.; Liedl, K. R.; Schuster, D. Identification of Novel Liver X Receptor Greene, G. L. Structural Characterization of a Subtype-Selective Activators by Structure-Based Modeling. J. Chem. Inf. Model. 2012, 52 Ligand Reveals a Novel Mode of Estrogen Receptor Antagonism. Nat. (5), 1391−1400. Struct. Biol. 2002, 9 (5), 359−364. (98) Baell, J. B.; Holloway, G. A. New Substructure Filters for ́ (85) Alvarez, L. D.; Dansey, M. V.; Grinman, D. Y.; Navalesi, D.; Removal of Pan Assay Interference Compounds (PAINS) from Samaja, G. A.; Del Fueyo, M. C.; Bastiaensen, N.; Houtman, R.; Screening Libraries and for Their Exclusion in Bioassays. J. Med. Estrin, D. A.; Veleiro, A. S.; Pecci, A.; Burton, G. Destabilization of Chem. 2010, 53 (7), 2719−2740. the Torsioned Conformation of a Ligand Side Chain Inverts the (99) Bolton, J. L. Quinone Methide Bioactivation Pathway: LXRβ Activity. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2015, Contribution to Toxicity and/or Cytoprotection? Curr. Org. Chem. 1851 (12), 1577−1586. 2014, 18 (1), 61−69. (86) Wrobel, J.; Steffan, R.; Bowen, S. M.; Magolda, R.; Matelan, E.; (100)Song,Y.;Xu,H.;Chen,W.;Zhan,P.;Liu,X.8- Unwalla, R.; Basso, M.; Clerin, V.; Gardell, S. J.; Nambi, P.; Quinet, Hydroxyquinoline: A Privileged Structure with a Broad-Ranging E.; Reminick, J. I.; Vlasuk, G. P.; Wang, S.; Feingold, I.; Huselton, C.; Pharmacological Potential. MedChemComm 2015, 6 (1), 61−74. Bonn, T.; Farnegardh, M.; Hansson, T.; Nilsson, A. G.; Wilhelmsson, (101) Temml, V.; Voss, C. V.; Dirsch, V. M.; Schuster, D. Discovery A.; Zamaratski, E.; Evans, M. J. Indazole-Based Liver X Receptor of New Liver X Receptor Agonists by Pharmacophore Modeling and (LXR) Modulators with Maintained Atherosclerotic Lesion Reduc- Shape-Based Virtual Screening. J. Chem. Inf. Model. 2014, 54 (2), tion Activity but Diminished Stimulation of Hepatic Triglyceride 367−371. Synthesis. J. Med. Chem. 2008, 51 (22), 7161−7168. (102) Quinet, E. M.; Savio, D. A.; Halpern, A. R.; Chen, L.; Miller, (87) Katz, A.; Udata, C.; Ott, E.; Hickey, L.; Burczynski, M. E.; C. P.; Nambi, P. Gene-Selective Modulation by a Synthetic Oxysterol Burghart, P.; Vesterqvist, O.; Meng, X. Safety, Pharmacokinetics, and Ligand of the Liver X Receptor. J. Lipid Res. 2004, 45 (10), 1929− Pharmacodynamics of Single Doses of LXR-623, a Novel Liver X- 1942. Receptor Agonist, in Healthy Participants. J. Clin. Pharmacol. 2009, 49 (103) Yu, S.; Li, S.; Henke, A.; Muse, E. D.; Cheng, B.; Welzel, G.; (6), 643−649. Chatterjee, A. K.; Wang, D.; Roland, J.; Glass, C. K.; Tremblay, M. (88) Bayne, C. D.; Johnson, A. T.; Lu, S.; Mohan, R.; Nyman, M. C.; Dissociated Sterol-Based Liver X Receptor Agonists as Therapeutics Schweiger, E. J.; Stevens, W. C.; Wang, H.; Xie, Y. Modulators of for Chronic Inflammatory Diseases. FASEB J. 2016, 30 (7), 2570− LXR. U.S. Patent 20050080111, Apr 14, 2005. 2579. (89) Dehmlow, H.; Obst Sander, U.; Schulz-Gasch, T.; Wright, M. (104) Åstrand, O. A. H.; Sandberg, M.; Sylte, I.; Görbitz, C. H.; Preparation of Biaryl Sulfonamide Derivatives as LXRα and LXRβ Thoresen, G. H.; Kase, E. T.; Rongved, P. Synthesis and Initial Receptor Ligands for Treatment of Dyslipidemia, Diabetes, and Other Biological Evaluation of New Mimics of the LXR-Modulator 22(S)- Diseases. WO 2009/040289, Dec 17, 2009.. Hydroxycholesterol. Bioorg. Med. Chem. 2014, 22 (1), 643−650. (90) Kirchgessner, T. G.; Martin, R.; Sleph, P.; Grimm, D.; Liu, X.; (105) Rodriguez, C. R.; Alvarez, L. D.; Dansey, M. V.; Paolo, L. S.; Lupisella, J.; Smalley, J.; Narayanan, R.; Xie, Y.; Ostrowski, J.; Cantor, Veleiro, A. S.; Pecci, A.; Burton, G. Fluorinated Oxysterol Analogues: G. H.; Mohan, R.; Kick, E. Pharmacological Characterization of a Synthesis, Molecular Modelling and LXRβ Activity. J. Steroid Biochem. Novel Liver X Receptor Agonist with Partial LXRα Activity and a Mol. Biol. 2017, 165, 268−276. ̈ Favorable Window in Nonhuman Primates. J. Pharmacol. Exp. Ther. (106) Viktorsson, E. O.; Gabrielsen, M.; Kumarachandran, N.; Sylte, 2015, 352 (2), 305−314. I.; Rongved, P.; Åstrand, O. A. H.; Kase, E. T. Regulation of Liver X (91) Kirchgessner, T. G.; Sleph, P.; Ostrowski, J.; Lupisella, J.; Ryan, Receptor Target Genes by 22-Functionalized Oxysterols. Synthesis, in C. S.; Liu, X.; Fernando, G.; Grimm, D.; Shipkova, P.; Zhang, R.; Silico and in Vitro Evaluations. Steroids 2017, 118, 119−127. ̈ Garcia, R.; Zhu, J.; He, A.; Malone, H.; Martin, R.; Behnia, K.; Wang, (107) Åstrand, O. A. H.; Viktorsson, E. O.; Kristensen, A. L.; Z.; Barrett, Y. C.; Garmise, R. J.; Yuan, L.; Zhang, J.; Gandhi, M. D.; Ekeberg, D.; Røberg-Larsen, H.; Wilson, S. R.; Gabrielsen, M.; Sylte, Wastall, P.; Li, T.; Du, S.; Salvador, L.; Mohan, R.; Cantor, G. H.; I.; Rustan, A. C.; Thoresen, G. H.; Rongved, P.; Kase, E. T. Synthesis, Kick, E.; Lee, J.; Frost, R. J. A. Beneficial and Adverse Effects of an in Vitro and in Vivo Biological Evaluation of New Oxysterols as LXR Agonist on Human Lipid and Lipoprotein Metabolism and Modulators of the Liver X Receptors. J. Steroid Biochem. Mol. Biol. Circulating Neutrophils. Cell Metab. 2016, 24 (2), 223−233. 2017, 165, 323−330. (92) Matsuda, T.; Okuda, A.; Watanabe, Y.; Miura, T.; Ozawa, H.; (108) Clouet, P.; Henninger, C.; Niot, I.; Boichot, J.; Bezard, J. Tosaka, A.; Yamazaki, K.; Yamaguchi, Y.; Kurobuchi, S.; Koura, M.; Short Term Treatment by Fenofibrate Enhances Oxidative Activities Shibuya, K. Design and Discovery of 2-Oxochromene Derivatives as towards Longchain Fatty Acids in the Liver of Lean Zucker Rats. Liver X Receptor β-Selective Agonists. Bioorg. Med. Chem. Lett. 2015, Biochem. Pharmacol. 1990, 40 (9), 2137−2143. 25 (6), 1274−1278. (109) Griffett, K.; Burris, T. P. Promiscuous Activity of the LXR (93) Koura, M.; Matsuda, T.; Okuda, A.; Watanabe, Y.; Yamaguchi, Antagonist GSK2033 in a Mouse Model of Fatty Liver Disease. Y.; Kurobuchi, S.; Matsumoto, Y.; Shibuya, K. Design, Synthesis and Biochem. Biophys. Res. Commun. 2016, 479 (3), 424−428. Pharmacology of 1,1-Bistrifluoromethylcarbinol Derivatives as Liver X (110) Aoyama, A.; Endo-Umeda, K.; Kishida, K.; Ohgane, K.; Receptor β-Selective Agonists. Bioorg. Med. Chem. Lett. 2015, 25 (13), Noguchi-Yachide, T.; Aoyama, H.; Ishikawa, M.; Miyachi, H.; 2668−2674. Makishima, M.; Hashimoto, Y. Design, Synthesis, and Biological (94) Koura, M.; Yamaguchi, Y.; Kurobuchi, S.; Sumida, H.; Evaluation of Novel Transrepression-Selective Liver X Receptor Watanabe, Y.; Enomoto, T.; Matsuda, T.; Okuda, A.; Koshizawa, (LXR) Ligands with 5,11-Dihydro-5-methyl-11-methylene-6H- T.; Matsumoto, Y.; Shibuya, K. Discovery of a 2-Hydroxyacetophe- dibenz[b,e]azepin-6-One Skeleton. J. Med. Chem. 2012, 55 (17), none Derivative as an Outstanding Linker to Enhance Potency and β- 7360−7377. Selectivity of Liver X Receptor Agonist. Bioorg. Med. Chem. 2016, 24 (111) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, (16), 3436−3446. J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. (95) Li, N.; Wang, X.; Liu, P.; Lu, D.; Jiang, W.; Xu, Y.; Si, S. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. E17110 Promotes Reverse Cholesterol Transport with Liver X A. OPLS3: A Force Field Providing Broad Coverage of Drug-like Receptor Beta Agonist Activity in Vitro. Acta Pharm. Sin. B 2016, 6 Small Molecules and Proteins. J. Chem. Theory Comput. 2016, 12 (1), (3), 198−204. 281−296. (96) Li, N.; Wang, X.; Xu, Y.; Lin, Y.; Zhu, N.; Liu, P.; Lu, D.; Si, S. (112) Schrödinger Release 2017-4: MacroModel; Schrödinger, LLC: Identification of a Novel Liver X Receptor Agonist That Regulates the New York, NY, 2017. Expression of Key Cholesterol Homeostasis Genes with Distinct (113) Thomas, J.; Bramlett, K. S.; Montrose, C.; Foxworthy, P.; Pharmacological Characteristics. Mol. Pharmacol. 2017, 91, 264−276. Eacho, P. I.; McCann, D.; Cao, G.; Kiefer, A.; McCowan, J.; Yu, K. L.;

10955 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956 Journal of Medicinal Chemistry Perspective

Grese, T.; Chin, W. W.; Burris, T. P.; Michael, L. F. A Chemical Switch Regulates Fibrate Specificity for Peroxisome Proliferator- Activated Receptor α (PPARγ) Versus Liver X Receptor. J. Biol. Chem. 2003, 278 (4), 2403−2410. (114) Jiao, X.; Kopecky, D. J.; Fisher, B.; Piper, D. E.; Labelle, M.; McKendry, S.; Harrison, M.; Jones, S.; Jaen, J.; Shiau, A. K.; Escaron, P.; Danao, J.; Chai, A.; Coward, P.; Kayser, F. Discovery and Optimization of a Series of Liver X Receptor Antagonists. Bioorg. Med. Chem. Lett. 2012, 22 (18), 5966−5970. (115) Motoshima, K.; Noguchi-Yachide, T.; Sugita, K.; Hashimoto, Y.; Ishikawa, M. Separation of α-Glucosidase-Inhibitory and Liver X Receptor-Antagonistic Activities of Phenethylphenyl Phthalimide Analogs and Generation of LXRα-Selective Antagonists. Bioorg. Med. Chem. 2009, 17 (14), 5001−5014. (116) Noguchi-Yachide, T.; Aoyama, A.; Makishima, M.; Miyachi, H.; Hashimoto, Y. Liver X Receptor Antagonists with a Phthalimide Skeleton Derived from Thalidomide-Related Glucosidase Inhibitors. Bioorg. Med. Chem. Lett. 2007, 17 (14), 3957−3961. (117) Nomura, S.; Endo-Umeda, K.; Aoyama, A.; Makishima, M.; Hashimoto, Y.; Ishikawa, M. Styrylphenylphthalimides as Novel Transrepression-Selective Liver X Receptor (LXR) Modulators. ACS Med. Chem. Lett. 2015, 6 (8), 902−907.

10956 DOI: 10.1021/acs.jmedchem.8b00045 J. Med. Chem. 2018, 61, 10935−10956