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Recent Progress on Bile Acid Receptor Modulators for Treatment of Metabolic Diseases Yanping Xu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00342 • Publication Date (Web): 15 Feb 2016 Downloaded from http://pubs.acs.org on February 17, 2016

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Journal of Medicinal Chemistry

Recent Progress on Bile Acid Receptor Modulators for Treatment of Metabolic Diseases Yanping Xu* Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA

Abstract: Bile acids are steroid-derived molecules synthesized in the liver, secreted from hepatocytes into the bile canaliculi, and subsequently stored in the gall bladder. During the feeding, bile flows into the duodenum where it contributes to the solubilization and digestion of lipid-soluble nutrients. After a meal, bile-acid levels increase in the intestine, liver, and also in the systemic circulation. Therefore, serum bile-acid levels serve as an important sensing mechanism for nutrient and energy. Recent studies have described bile acids as versatile signaling molecules endowed with systemic endocrine functions. Bile acids are ligands for G‑protein-coupled receptors (GPCRs) such as TGR5 (also known as GPBAR1, M-BAR and BG37) and nuclear hormone receptors including farnesoid X receptor (FXR; also known as NR1H4). Acting through these diverse signaling pathways, bile acids regulate triglyceride, cholesterol, glucose homeostasis, and energy expenditure. These bile acid‑controlled signaling pathways have become the source of promising novel drug targets to treat common metabolic and hepatic diseases.

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Traditionally, bile acids (BAs) have been viewed as detergents that solubilize cholesterol, fatty acids, and liposoluble vitamins to facilitate the digestion, transportation, and gastrointestinal absorption of nutrients. BAs have also been shown to be involved in a large variety of cellular processes. Recent discoveries have unveiled novel actions of BAs as signaling hormones endowed with a wide array of endocrine functions.1-3 In 1999, three research groups independently identified bile acids as endogenous ligands for FXR.4-6 In early 2000, two research groups discovered that a novel Class A G-Protein Coupled Receptor (GPCR), TGR5 (also known as GPBAR1, GPCR19, GPR131, BG37, M-BAR, Rup 43), can be activated by bile acids.7,8 Highly expressed in the liver, intestine, kidney, adrenal glands, and adipose tissue, FXR is a master regulator of the synthesis and pleiotropic actions of endogenous BAs.9 Conjugated and unconjugated bile acids are the natural ligands for FXR. Activation of FXR by BAs or synthetic ligands lowers plasma triglycerides by a mechanism involving repression of hepatic sterol regulatory element binding protein-1c (SREBP-1c) expression and modulation of glucosedependent lipogenic genes.10,11 Furthermore, FXR controls lipid and glucose metabolism through regulation of gluconeogenesis and glycogenolysis in the liver and through regulation of peripheral insulin sensitivity in striated muscle and adipose tissue.12-14 Similar to effects in the liver, FXR agonists modulate lipid metabolism and promote anti-inflammatory and anti-fibrotic effects in the kidney, suggesting a potential use of FXR agonists to treat diabetic nephropathy and other fibrotic renal diseases.15

TGR5 is expressed in the brown adipose tissue, muscle, liver, intestine, gallbladder,16 and selected areas of the central nervous system.8 TGR5 activation in intestinal enteroendocrine L cells stimulates secretion of glucagon-like peptide-1 (GLP-1).17 Mice fed with a high fat diet


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demonstrated lower serum glucose and insulin levels and enhanced glucose tolerance when treated with a TGR5 agonist, oleanolic acid.15 In addition, administration of BAs to mice increased energy expenditure in brown adipose tissue, preventing obesity and insulin resistance via TGR5-mediated cAMP-dependent induction of type 2 iodothyronine deiodinase (D2), which locally stimulates thyroid hormone-mediated thermogenesis.19 In rodent intestinal enteroendocrine L (STC-1) cells, bile acids can activate the TGR5 receptor and promote GLP-1 secretion in a dose-dependent manner.20 Treatment of diet-induced obese (DIO) mice with oleanolic acid lowered serum glucose and insulin levels and enhanced glucose tolerance.21 In obese diabetic humans, rectal administration of sodium taurocholate dose-dependently stimulated a robust increase of plasma concentration of GLP-1 and PYY.22 Therefore, small molecule TGR5 agonists are targeted to promote GLP-1 secretion, increase energy expenditure, and improve insulin sensitivity in the treatment of type 2 diabetes. TGR5 is also expressed in sinusoidal endothelial cells, which are transiently exposed to high concentrations of bile acids. TGR5 regulates nitric oxide production via cyclic AMP-dependent activation of endothelial nitric oxide synthase. This mechanism may scavenge bile-acid-induced reactive oxygen species and protect the liver against lipid peroxidation and bile-acid-induced injury.23 In addition, high TGR5 expression was found in monocytes of human macrophages. Compared to normal macrophages, TGR5-/- macrophages showed increased cytokine levels. Treatment of human macrophage with TGR5 agonists led to the reduction of cytokine production,24-26 suggesting a therapeutic potential of TGR5 agonists for treatment of inflammatory diseases like atherosclerosis, colitis and nonalcoholic fatty liver disease. This perspective review will highlight the most recent publications in the field of FXR modulators27 and provide a comprehensive literature summary of TGR5 agonists.28



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1. FXR AGONISTS

Bile acids such as chenodeoxycholic acid (CDCA) have been known to be FXR ligands. Data from human and animal studies suggested a key role for FXR in lipid regulation.29,30 Early nonsteroidal FXR agonists, e.g. GW4064 (1)31, have served as useful in vitro molecules. However, several liabilities associated with 1 limited its utility as a drug to treat FXR-mediated diseases. These liabilities included poor rat PK (high clearance and low bioavailability), a potentially toxic stilbene pharmacophore, and stilbene-mediated UV light instability.

1

2

In an FXR transient transfection (TT) assay 1 demonstrated excellent potency with an EC50 of 65 nM (eff = 100%). SAR development at the 3- and 5-positions of the isoxazole ring revealed a preference for hydrophobic substituents.32 In addition, some polarity was tolerated in the tether at the 3-position of the isoxazole linked to the phenyl group, e.g. 2 (FXR TT EC50 = 89 nM, eff = 89%). A rat PK study demonstrated that 2 had an improved half-life (2 h) and clearance (CL = 20 mL/min/kg). Unfortunately, the low oral bioavailability (F = 9%) suggested the stilbene moiety could be the predominant limiting structural feature. To address this issue, a series of alternatively conformational constrained analogs were explored.33 Benzothiophene analog 3 (FXR TT EC50 = 32 nM, eff = 87%) was equipotent to 1. Indole analog 4a was slightly weaker


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(FXR TT EC50 = 210 nM, eff = 84%). In rat PK studies, compound 3 had very high clearance (CL = 66 ml/min/kg), short half-life (t1/2 = 15 min), and poor bioavailability (F = 9%).

4a: X = C 4b: X = N

3

Indole 4a had significantly lower clearance (CL = 6.7 ml/min/kg); however, the half-life was modest (t1/2 = 45 min). The oral bioavailability was also low (F = 12%). These results suggested that the poor bioavailability may be due to poor solubility. Thus a second nitrogen atom was incorporated into the indole ring to give benzimidazole 4b. Although this compound was significantly less active at FXR (FXR TT EC50 = 5 µM, eff = 40%), a two-fold improvement in bioavailability was achieved (F = 26%) with little alteration of clearance or half-life supporting the hypothesis that absorption of 1 may be dissolution limited. The co-crystal structure of 1 with FXR33 suggested the potential for favorable hydrogen bond interactions between the isoxazole 3-aryl group and several receptor residues such as Tyr373 and Ser336. Replacing the 2,6-dichloro phenyl with a 2,6-dichloro-4-pyridyl moiety maintained both FXR binding and functional activity. Combining this pyridine moiety with an N-methyl indolephenyl ring afforded 5, which demonstrated FXR binding affinity in the human SPA binding assay of 94 nM compared to 64 nM for 1.34



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5

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6

Oxidation of the pyridine to the corresponding N-oxide yielded the most potent compound in this series 6, with a FXR binding affinity of 45 nM. Additionally, 6 was the most permeable analog in the series (PAMPA, 5.87 x 10-4 cm/s). From molecular docking studies, the N-oxide oxygen most likely acted as a hydrogen bond acceptor to interact with Tyr373 on helix 7 and/or Ser336 on helix 5. As a novel class of FXR agonist, azepino[4,5-b]indole 7 (hEC50 = 600 nM, efficacy (eff) = 100%) was identified as a lead from a high throughput screening effort.35 Structure-activity relationship (SAR) studies around the azepine ring indicated that dialkyl substitution at C-1 led to a 30-fold improvement in potency. In addition, incorporation of an isopropyl ester yielded another ~3-fold boost in potency. Compound 8 represented the most potent FXR agonist within the series (hEC50 = 4 nM, eff = 149%).

7

8

A rat pharmacokinetic (PK) study showed that 8 had good oral bioavailability (F = 38%) and a long half-life (t1/2 = 24 h). Normal C57BL/6 mice dosed orally with 8 at 10 mg/kg/d for 7 days


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experienced statistically significant reductions of triglycerides (TG, 24%) and total cholesterol (22%) levels. When administered to low density lipoprotein receptor knockout (LDLR-/-) mice fed a Western diet for 8 weeks, 8 lowered both TG (19% and 39% at 1 and 3 mg/kg, respectively) and total cholesterol (23% and 50% at 1 and 3 mg/kg, respectively). However, this molecule was poorly soluble. Guided by crystallographic data, the appended morpholine analogs 9a and 9b showed dramatic 400-fold improvements in equilibrium solubility measured in 0.5% methylcellulose/2% Tween-80 in water.

( )n

9a: n = 3 9b: n = 2 Both compounds were potent at mouse FXR (9a: mEC50 = 52 nM, eff = 117%; 9b: mEC50 = 188 nM, eff = 110%) similar to compound 8 (mEC50 = 152 nM, eff = 174%). Oral administration of 9a and 9b to LDLR-/- led to a dose-dependent reduction of low density lipoprotein cholesterol (LDLc). In female rhesus monkeys, 9a dosed 60 mg/kg/d orally for 4 weeks resulted in a significant lowering of TG, very low density lipoprotein cholesterol (VLDLc), and LDLc.10 From a separate screening effort, benzimidazolyl acetamide 10 was discovered as a novel FXR agonist with binding affinity of 70 nM.11,36 Attempts to improve the physical properties of this compound by replacing the cyclohexyl groups with more polar moieties proved unsuccessful. This result was consistent with the co-crystal structure of compound 10 and hFXR, where the cyclohexyl groups were oriented within highly lipophilic pockets.



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10

11

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12a: R = H 12b: R = F

The lead molecule from this series, 11 (IC50 SPA = 13 nM), was evaluated in LDLR-/- mice. It significantly reduced total cholesterol (45%), LDL (48%), and TG (52%) when orally administered at a dose of 30 mg/kg/d for 5 days.11 The poor physiochemical properties of 11, namely high lipophilicity and low aqueous solubility, limited its potential for further development. In addition, this molecule inhibited the hERG potassium channel in vitro (IC50 = 1.6 µM). Further structural analysis revealed a more polar and yet unexplored pocket consisting of Gln267, Asn297, His298, Arg335, and three water molecules near the region where Ncyclohexyl group binding region.37 Subsequent SAR efforts to replace this cyclohexyl group with a 4-carboxyphenyl ring yielded compound 12a with no loss of receptor binding activity (FXR IC50 SPA = 50 nM), significantly improved solubility (88 µg/mL vs 20 µM).37-39 Fluoro-analog 12b showed enhanced FXR binding (IC50 SPA = 37 nM) and solubility (115 µg/mL) with no significant hERG activity (IC50 >20 µM). As a result of its good murine in vitro potency (IC50 = 290 nM, EC50 = 870 nM, eff = 38%) and mouse PK profile (CL = 10 mL/min/kg, F = 33%), 12b was evaluated in LDLR-/- mice. After five days of treatment (10 mg/kg/d, po), statistically significant decreases in plasma total cholesterol (41%), LDLc (33%), and TG (59%) were observed.37


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In an effort aimed to find a novel class of non-steroidal FXR agonists, Marinozzi et al. reported the discovery of 4-(2,4-dimethoxyphenyl-1-(2-methyphenyl)-4,8-dihydro-1H-pyrazole[3,4,e][1,4]thiazepin-7-one, 13, as a novel FXR agonist.40 Subsequent SAR led to the identification of 14 as a potent and efficacious (EC50 = 1.4 µM; eff = 130%) FXR agonist in a cofactor recruitment assay (AlphaScreen). SAR study suggested that the pyrazole C-3 alkyl group (Me or Et) seemed essential for activity. Based on a computational docking model, these alkyl groups occupy a hydrophobic cleft in the ligand binding site defined by Phe284, Thr454 and Phe461. The resultant Van der Waals interactions stabilize the orientation of the AF-2 helix (residue range 461-468) and helix 3, which are directly involved in coactivator recruitment. The SAR study also revealed that receptor recognition is highly stereoselective for this class of agonists, as only (4R,6S)-14 exhibited low micromolar potency and full efficacy compared to all other stereoisomers. O

O O S 3

N

N

13

N H

O

N

N

S 6 N H

O

14

In pursuit of non-steroidal FXR agonists with improved ADME and toxicological properties, the authors hypothesized that a partial agonist approach can mitigate various side effects of a full agonist during a long-term treatment. Merk et al. identified a class of anthranilic acid derivatives as FXR partial agonist.41 The initial lead 15 possessed partial agonistic activity at FXR with 11% activity at 30 µM in a cell-based full-length FXR transactivation assay.



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15

16

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17

SAR data suggested that all functional groups of the anthranilic acid moiety were crucial for FXR activation. Larger lipophilic tails, as exemplified in compound 16, led to improved activity (EC50 = 8.6 µM; eff = 37%). By introduction of an aromatic moiety within the acidic head group, the potency was enhanced further. Compound 17 constituted a potent partial FXR agonist with an EC50 of 1.5 µM and maximal FXR activation of 37%. Given their reasonable potencies, ADME and physiochemical properties for compounds 16 and 17 were evaluated. Both compounds were soluble in a variety of solvents including water at alkaline pH. Compound 16 had an aqueous solubility of 45 mg/L and was highly stable against metabolic degradation by liver microsomes with 92% of parent detected after a 60 min incubation. Compound 17 showed an aqueous solubility of 0.3 mg/L and moderate metabolic stability with 61% of parent detected after a 60 min incubation in liver microsomes. Neither compound showed any toxicity in the HeLa cell based reporter gene assay up to the highest concentration tested (60 µM). To further improve potency, the naphthyl ring of 17 was replaced with 4- tBu phenyl group yielded compound 18 with EC50 of 0.28 µM and partial agonist profile (eff = 9%).42 SAR of acid head group revealed that carboxylic acid functional group can be replaced by different bioisosteric moieties without losing the potency. Nevertheless 18 remained as the most desirable analog based on potency and solubility. Different substitution on the anthranilic central core resulted 4OMe analog 19 as the most potent partial agonist (EC50 = 8 nM; eff = 18%) of the series. The


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fact that the effect of 19 on FXR target genes (SHP, OSTα, BSEP, and IBABP) was not concentration dependent in the concentration range of 0.1 to 10 µM suggested that 19 is a true partial FXR agonist. Compound 19 also demonstrated good selectivity against other nuclear receptors PPARγ (375 fold), PPARα (>1000 fold), PPARδ (>1000 fold), and the membrane bile acid receptor TGR5 (>1000 fold).

19

18

With the aim of developing a potent FXR partial agonist, Merk et al. investigated a fragmentbased design approach.43 From the co-crystal structure of 1 with the FXR ligand binding domain (FXR-LBD), the 5-isopropyl-3-phenylisoxazole moiety was bound near helices 11 and 12; the space around helix 12 could accommodate a C-5 substituent large than isopropyl. The authors hypothesized that the 3,5-disubstituted isoxazole moiety played an essential role for FXR activation. The 1,2,4-oxadiazole core was designed as an attractive isoxazole replacement based on its higher polarity yet comparable size and geometry.



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20

21

22

23

Replacing the isopropyl group with a more sterically demanding phenyl ring resulted in oxadiazole analog 20, which possessed good FXR agonist activity with an EC50 of 530 nM (eff = 19%). Introduction of substituents on both phenyl rings yielded 21 with enhanced in vitro potency (EC50 = 120 nM; eff = 11%). To improve solubility, various polar functional groups were evaluated. Replacing para OMe by COOH of the 5-phenyl ring gave 22 without diminished in vitro potency (EC50 = 220 nM; eff = 16%). Moving the carboxylic acid to the meta resulted in 23, a highly potent partial FXR agonist with an EC50 of 7.2 nM (eff = 14%). Compound 23 displayed good aqueous solubility (33 mg/L) and reasonable rat liver microsomal stability with 54% of the compound remaining after 60 min incubation. As a partial agonist, compound 23 induced the expression of the FXR target genes, i.e. BSEP, SHP, OSTα, and IBABP in HepG2 and HT-29 cells. There have been numerous recent patent applications disclosing novel chemical scaffolds as FXR agonists. In a 2009 US patent application, hexahydropyrroloazepines exemplified by 24 were claimed as FXR agonists.44 Using a Gal4/hFXR fusion protein expressed in the HEK293 cell line, compound 24 showed an EC50 of 280 nM. A related tetrahydropyrroloazepine series of


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FXR agonists was disclosed in a separate US patent application.45 Representative compound 25 showed an EC50 of 3 nM in the HEK293 cell assay.

24

25

A series of benzofuran/benzothiophene/benzothiazole derivatives was disclosed as FXR modulators. Representative compound 26 showed an EC50 of 1.95 µM in a hFXR transactivation assay in CV-1 cells 46,47

26

27

In a 2009 patent application from GSK, a biaryl carboxylate scaffold led to novel FXR agonists.48 Compound 27 was exemplified with an EC50 60 min.


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52

53

54

55

56

57

Using surface plasmon resonance (SPR)-based binding assay, the same research group identified 57 as an FXR ligand which bound to FXRα-LBD with a Kd of 8.3 µM.70 In HepG2 cells under luciferase reporter assay format, 57 antagonized either CDCA (50 nM)- or 1 (50 nM)-activated transactivation activity with IC50’s of 4.5 or 2.6 µM, respectively. Compound 57 was a selective FXR antagonist with no effects on the transactivation activities of other nuclear receptors tested, LXRα/β, MR, RXRα, PPARγ, PR, and ERα/β. From the crystal structure of the FXRα-LBD-57 complex, 57 induced rearrangements of helices 11 and 12 by forming a homodimer of the FXRα-LBD. Ligand 57 competitively occupied the same binding pocket as compound 1, causing the hydrophobic part of helix 11 to bend to the ligand binding pocket and stabilize the inactive conformation of the LBD. As a result, helix 12 blocked co-activators binding. In mouse primary hepatocytes, 57 effectively reversed the 1-induced stimulation of BSEP and SHP mRNA expression. Upon 4-week treatment of db/db mice at 24 mg/kg/d, 57 significantly downregulated gluconeogenesis-related genes; including phosphoenolpyruvate carboxykinase, glucose 6phosphatatse, BSEP, and SHP. This result shed light on the pharmacological potential of an FXR antagonist as an antidiabetic agent.



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While exploring a collection of natural products, Zhou et al. reported that 14-aryl ether andrographolide derivatives were potent FXR antagonists. As an example, compound 58 had an IC50 < 5 µM in a luciferase reporter assay.71

21 26

3

59

58

As a part of research effort directed toward the discovery of marine natural nuclear receptor ligands, Sepe et al. investigated a library of sulfated sterols isolated from marine echinoderms.72 Compounds were evaluated in an hepatocarcinoma cell line (HepG2 cells) transfected with FXR, RXR, and β-galactosidase vectors and with p(hsp27)TKLUC reporter vector containing the promoter of the FXR target gene heat shock protein 27 (hsp27). At 10 µM, compound 59 exhibited potent antagonist activity resulting in 80% inhibition of FXR transactivation induced by CDCA at 100 µM, the inhibition was nearly complete. From a molecular docking simulation, both sulfate groups at C-3 and C-21 make hydrogen bond interactions with key amino acid residues of the receptor (namely, Tyr358 in helix7, His444 in helix10/11, Trp466 in helix 12, Arg328 in helix 5, and Met262 in helix 2). In addition, the presence of the C-26 hydroxyl group appeared to be critical for FXR antagonist activity, mainly through a hydrogen bond with Thr267 in helix 2. This research group also isolated a rare class of 4-methylenesteroids from Theonella sponges exemplified by 60 and 61.73 Using a luciferase reporter assay in FXR transfected HepG2 cell,


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both inhibited CDCA induced FXR activation with IC50’s of 40µM and 35 µM, respectively. These results can be rationalized at the molecular level by docking calculations using X-ray structure of the human FXR-LBD (pdb code: 1OSV). Pi interactions of the A-ring double bonds with Tyr358, His444, and Trp451 were deemed the main driving force to form stable and efficient sterol-receptor complexes.

1 2

3

4

60

61

Unlike previously reported steroidal FXR antagonist 39, compound 60 is a selective antagonist for FXR.74 This selectivity was confirmed by transactivation and microarray experiments in HepG2 cells. While 60 antagonizes the transactivation of FXR by it natural ligand CDCA, it did not modulate the expression of a wide array of mammalian nuclear receptors or activate PPARγ, LXR, PXR and VDR in transactivation assays. In a bile duct ligation model (BDL mice), mice were administered compound 60 at 10 mg/kg or FXR agonist 6-ECDCA at 30 mg/kg for 3 days. Compound 60 effectively reduced intrahepatic bile duct pressure as measured by the size of the common bile duct. It also attenuated the extent of liver injury assessed by plasma AST levels and liver histopathology. Furthermore, analysis of expression of MRP-4 in the liver of BDL mice confirmed that pharmacological antagonism of FXR increased MRP-4 gene expression. This result was opposite to the observation in mice administered a potent FXR agonist. In 2013, the same research group reported the discovery of suvanine 62, a furano sesterterpene sulfate as a novel FXR antagonist with an IC50 of 24 µM.75



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62

63

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64

Toward novel FXR ligand design, computational studies were performed on the FXR complex with agonist 6-ECDCA and antagonist 62. For the first time, molecular dynamic simulation was used to elucidate the conformational changes leading to FXR inactivation. In the 6-ECDCA/FXR complex, a strong cation-pi interaction between His444 and Trp466 stabilized the helix 12 in a conformation enabling coactivator binding. Compound 62 formed hydrogen bonds with the side chains of these residues to break the cation-pi interaction. The subsequent shift of His444 in the LBD changed the interaction patterns of residues on helix 12. As a consequence, helix 12 underwent a conformational rearrangement unsuitable for coactivator binding. Assisted by the computational modeling, a series of analogs were designed to replace the exocyclic enol-sulfate moiety with chemically more stable groups. From this effort, compound 63 was identified as an antagonist with IC50 of 25µM. And compound 64 was found as a moderate FXR agonist with EC50 of 30 µM (eff = 21%).

3. TGR5 AGONISTS

3.1 Non-bile acid agonists A high-throughput screen using a BacMam transduced human osteosarcoma cell line (U2-OS) led to the discovery of isoxazole 65 as a TGR5 agonist with a pEC50 of 5.3 and 100% maximum


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response.76 SAR optimization on the amide phenyl ring, exemplified by compound 66 (pEC50 = 7.5), suggested that para-substitution was preferred.77 In melanophore cells, compound 66 was equipotent at both human (pEC50 = 7.5) and canine receptors (pEC50 = 7.2). In a conscious dog model, intrajejeunal injection of glucose (0.125 g/kg) with co-administration of 66 at a dose of 1 mg/kg afforded a significant improvement in portal vein GLP-1 secretion and glucose reduction compared to vehicle.76

65

66

67

68

Compound 66 showed high in vivo clearance (CL = 85 mL/min/kg) in rats and high intrinsic clearance (CLint = 48 mL/min/g) in rat liver microsomes suggesting potential challenges to the developability of this series. In addition, 66 had measurable activity against two cytochrome P450 (CYP450) isoforms including 2C19 (pIC50 = 6.5) and 3A4 (pIC50 = 5.9). SAR development at the 5-position of the isoxazole revealed that some increased steric bulk was well tolerated, exemplified by 67. Compound 67 showed improved in vitro potency (pEC50 = 8.4) and reduced in vitro clearance in rat (CLint = 10 mL/min/g). Interestingly, replacement of the isoxazole with a 1,2,3-triazole, 68, afforded further reduction of intrinsic clearance (CLint = 6.5



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mL/min/g) as well as an improved CYP450 profile (pIC50 > 10 µM). Linker homologation to the phenethylamine and bromine substitution on the phenyl ring led to compound 70a with a significant improvement in potency at both human (hEC50 = 65 nM) and mouse (mEC50 = 3.2 µM) receptors. Phenol deprotection gave analog 70b, which was considerably more active at the mouse receptor (mEC50 = 0.28 µM) but much less active at the human receptor (hEC50 = 5.1 µM). To test the hypothesis that activation of TGR5 stimulates GLP-1 release, 70b (30 mg/kg) was orally administered to DIO mice. Following an oral glucose challenge, a statistically significant increase in plasma GLP-1 levels was observed. Acute administration of 70b (30 mg/kg po) to DIO mice prior to an oral glucose tolerance test also resulted in significantly enhanced glucose clearance. In a separate study, treatment of C57BL/6 DIO mice with 70b for two weeks at doses of 3, 30 and 100 mg/kg bid reduced fasting glucose, post prandial TG, and HDL levels.79


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Br N H

N H MeO

N

N

RO

S

S

70a: R = Me 70b: R = H

69

O O

O

O N

N N

O

N N

N

O

N

O

Cl

N

N Cl

N

O Cl

O

N N

Cl F3 C

Cl

N

Cl O

71

72

73

N H

N H

74

In a search for small molecule TGR5 agonists, nicotinamide 71 was identified from a highthroughput screening with EC50’s of 0.396 µM and 2.02 µM in human and murine TGR5 receptors, respectively.80 However, the physiochemical properties of this compound were poor, i.e. low aqueous solubility, high metabolic clearance, and some degree of CYP inhibition. At the onset of SAR exploration, Martin et al. investigated the scope of the substitution pattern of phenoxy side chain and demonstrated that 2,5-dichloro analog 72 had fully potent agonist activity against human TGR5 receptor (102% measured relative to lithocholic acid (LCA)) with an EC50 of 10 nM but with a mouse EC50 of only 2.56 µM. Similar to compound 71, the physiochemical properties of 72 remained as an issue with clogP of 5.2, aqueous solubility < 1 µg/mL, and high clearance in both human (CL = 1418 ml/min/g) and mouse (2134 ml/min/g) microsome. From microsomal metID studies, the tetrahydroquinoline moiety is the primary site of oxidative metabolism for both human and mouse. Three metabolic pathways were identified:



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1) oxidation of the alkyl bridge of the tetrahydroquinoline ([M+O]); 2) oxidation of the alkyl bridge followed by elimination ([M-2H]); 3) hydroxylation of the phenyl ring para to the amide nitrogen ([M+O]). Attempts to replace the tetrahydroquinoline with other heteroaryl groups (i.e. tetrahydroimidazo[1,2-a]pyrimidine, indole, benzoimidazole) failed to maintain the desirable potency. Substituting the phenyl ring with F or Cl to block the potential metabolic site yielded analogs with reasonable cellular potency, but did not improve in vitro metabolic stability. The most important finding was introducing a N atom at benzylic position to afford compound 73, which maintains good in vitro potency (EC50 = 14 nM) with modest improvement of aqueous solubility (14 µg /mL). Aiming to further improve physiochemical properties of the scaffold, SAR effort focused on the identification of positions that would allow an attachment of polar and charged side chains (under physiological conditions) such as a carboxylic acid. Assisted by computational modeling, an alignment was generated by docking bile acids such as DCA, LCA or taurolithocholic acid (TLCA), and nicotinamides into the hydrophilic binding cavity of TGR5 three dimensional homology model. According to the predicted binding mode, the tetrahydroquinoline of compound 72 was placed into the hydrophobic cavity and thus overlaid with the A and B rings of bile acids. Based on this docking hypothesis, the optimal position to incorporate polar side chains was at the 2,5-dichlorophenyl ring para to the O linker to match the hydrophilic side of bile acids. Among the newly synthesized analogs, compound 74 demonstrated good potency with EC50 of 76 nM and 350 nM for human and mouse, respectively. This compound also had much improved aqueous solubility (535 µg /mL) and acceptable microsomal clearance in human (CL = 56 µl/min/mg protein) and mouse (CL = 56 µl/min/mg protein).


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O

O N

N

N

N

N

O

HO

N

Cl Cl

N

O Cl

Cl

75

76

Given the high microsomal clearance of compound 75, Park et al. hypothesized that replacement of the pyridine core with pyrimidine would reduce the clogP and improve overall physiochemical properties.81 Compound 76 was identified with comparable TGR5 agonist potency (EC50 = 3.9 nM) compared to compound 75 (EC50 = 6.1 nM). With lower clogP (2.9 versus 5.0), compound 76 showed moderate in vivo clearance (58 mL/min/kg) and bioavailability 14% in a mouse pharmacokinetic study. In a patent application from Takeda,82 oxazepinone 77 (30 µM) was reported to stimulate GLP-1 secretion by 249% in NCI-H716 cells and by 360% in a rat bowel primary culture cell. To evaluate in vivo GLP-1 secretion and glucose-dependent insulin secretion, 77 was administered orally to male F344 rats at doses of 30 and 100 mg/kg. Animals treated with 100 mg/kg of 77 showed significant GLP-1 and insulin secretion after a glucose challenge compared to the vehicle group.



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77

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78

80

79

81

Toward novel chemical scaffolds, a Chinese research group used compound 77 (EC50 = 19 nM in HEK293 cell) as a starting point.83 Breaking the oxazepine ring gave acetamide 78 with moderate potency (EC50 = 910 nM; eff = 117%) and a moderate ligand efficiency (LE) of 0.26. Removing the carbonyl group and introducing a 2-F substituent in the phenyl ring afforded compound 79 with modestly improved potency (EC50 = 458 nM; eff = 81%) and LE (0.29). Interestingly, with a CN substituent at the benzylic position, 80 resulted in a greater than 10-fold improvement in potency with EC50 of 35 nM (eff = 101%). A similar improvement was noted when the benzylic methylene was replaced by a sulfonyl group to give compound 81 with an EC50 of 63 nM (eff = 102%). Compound 80 possessed more favorable mTGR5 activity with an EC50 of 150 nM (eff = 121%) compared to 81 (EC50 = 820 nM; eff = 148%). With a favorable rodent activity profile, the acute effect of 80 on blood glucose was assessed in ICR mice using an oral glucose tolerance test (OGTT). Unfortunately due to poor pharmacokinetic properties (high


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clearance, low plasma exposure), compound 80 did not reduce the glucose excursion compared with the vehicle control group when dosed at 50 mg/kg.

82

83

84

85

Zhu et al. proposed a ligand-based pharmacophore model by thorough analysis of structurally diverse structures of TGR5 agonists in the public domain.84 The authors hypothesized that hydrophilic aromatic rings in compound 77 formed a triangular backbone to anchor the TGR5 receptor through pi-pi stacking or hydrophobic interactions. For novel ligand design, the same ligand conformation was targeted by removing the oxazepine ring and replacing the amide group with a five-membered heterocycle. Among the various heterocycles studied, the 4,5-dihydro1,2,4-oxadiazole was most effective, leading to 82 with cellular EC50 of 79 nM (eff = 93%). Introducing additional substituents in the newly introduced phenyl ring resulted in a significant reduction of in vitro potency. These results indicated that the binding pocket of TGR5 in this region is relatively narrow with insufficient space to accommodate larger substituents. With the



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less steric bulk, 2-furanyl analog 83 displayed improved potency with an EC50 of 15 nM (eff = 102%). Focusing on the 4-phenylpyridine moiety, ortho substitution of the phenyl ring was desirable regardless electronic characteristics. Relocating the substituents to the meta or para position on the phenyl ring only resulted in a loss of potency. Therefore, the activity is likely determined by the dihedral angle between the phenyl and pyridine rings; the orientation of the phenyl ring impacts binding significantly. Additionally, removing or changing the position of the pyridine N led to a dramatic decrease in activity, suggesting the N atom may engage in a key hydrogen bond with the TGR5 receptor. Finally, SAR study around the bis-trifluoromethyl phenyl ring suggested that all positions favored electron-withdrawing and lipophilic substituents. Attempts to increase the electron density of the phenyl ring only resulted in significantly reduced in vitro potency. Adding a 2-Cl substituent afforded compound 84 with enhanced in vitro potency (EC50 = 5.6 nM; eff = 90%). Finally the corresponding 2,6-difluoro phenyl analog was most potent compound of the series. Isolated by chiral chromatography, enantiomer 85 showed an EC50 of 1.4 nM (eff = 96%). The absolute stereochemistry was determined by single-crystal X-ray diffraction and quantum chemical solid-state TDDFT-ECD methods.


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F3 C

F3 C

N

N

N

N

N

N

H N

N

N

N

H N

O

H N

O

O

Cl

CN

86

CN

87

88 CN

H N

F3 C

N

F3 C

N

H N

N

N

O

N

N

N

H N F3 C

N

O

N

O N

CN

N

N

N

CN OH O

89

90

91

From an HTS effort, TGR5 agonist nipecotamide 86 was identified as an interesting starting point due to its good human (EC50 = 0.88 µM) and mouse (EC50 = 1.29 µM) potency.85 At the start of the hit-to-lead effort, a clear preference for the R over the S-enantiomer was demonstrated. Subsequent SAR study in the pyrimidine ring region revealed that (pyrimidin-4yl)piperidine-3-carboxamide 88 (hEC50 = 140 nM, mEC50 =23 nM) was more selective for mTGR5 than the (pyrimidin-2-yl)piperidine-3-carboxamide 87 (hEC50 = 17 nM, mEC50 = 130 nM). When the Cl on the distal phenyl ring of the original hit 86 was replaced by a CN group, the in vitro potency was maintained while the clogP was lowered. Adding a piperazine group onto the pyrimidine ring gave 89, the most potent and efficacious TGR5 agonist of the series for both human (EC50 = 3 nM) and mouse (EC50 = 1 nM). In a murine GLUTag cell line, compound 89 stimulated GLP-1 secretion with an EC50 of 256 nM. However, strong CYP3A4 (IC50 < 1



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µM) and hERG inhibition (IC50 = 3.2 µM) were liabilities. Molecular docking studies with a model of CYP3A4 suggested that compound 89 is anchored into the active site via a hydrogen bond between the amide carbonyl and S119 and by Van der Walls interaction between the CF3 group and a hydrophobic pocket formed by F108, F213, F220, and F241. Also the piperazine nitrogen forms a salt bridge with E308, explaining why the corresponding des-methyl piperazine analog is a more potent CYP3A4 inhibitor. To reduce CYP inhibition, structure modifications were targeted to disrupt the aforementioned favorable ligand and enzyme interaction by replacing the piperidine-3-carboxamide with smaller and less lipophilic rings. As the result, azetidine 90 showed enhanced TGR5 potency (hEC50 = 19 nM; mEC50 = 4 nM) and much reduced CYP3A4 (IC50 = 7.8 µM) and hERG (IC50 > 30 µM) inhibition. While this strategy proved to be effective to identify compounds with good in vitro potency and selectivity, these compounds suffered from poor microsomal stability and poor in vivo plasma exposure. Several functional groups were considered to replace the N-alkylpiperazine group to improve metabolic stability. Carboxylic acid substituents are polar and ionized at physiological pH and generally more metabolically stable than their basic and neutral analogs. Moreover, these acidic compounds are less prone to be substrates for the hERG channel. SAR studies demonstrated that the methyl piperazine can be replaced with 4-(4-trans-carboxycyclohexyl)-phenyl to afford compound 91, which maintained good in vitro potency (hEC50 = 15 nM; mEC50 = 1 nM). 91 was potent in the GLP-1 (EC50 = 46 nM) and PBMC (EC50 = 215 nM) functional assays without inhibiting CYP (IC50 > 25µM) or hERG (IC50 > 30 µM). In a selectivity panel screen of 20 receptors, 91 showed only moderate inhibition of human phosphodiesterase type 4D (hPDE4D; IC50 = 4.6 µM), while all other receptors tested had IC50 values over 30 µM. In an in vivo GLP-1 secretion study, a single 3 mg/kg oral administration of 91 in C57BL/6 mice resulted in an


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increase of plasma total GLP-1 by 310% versus vehicle control. To determine if chronic treatment of a TGR5 agonist would improve glucose disposal, 91 was dosed in ob/ob mice for 5 weeks. For this study, a 30 mg/kg dose of 91 was chosen to maximize the coverage of the compound during the course of the study. Interestingly, an OGTT at day 1 demonstrated efficacy nearly equivalent to a DPP4 inhibitor, 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)pyrrolipyrrolidine (PKF275). 86 However, after 26 days of q.d. dosing with 91 the OGTT efficacy was ablated even though compound plasma exposure was maintained constant over the course of the study. The authors concluded that the lack of efficacy upon chronic dosing was likely due to receptor desensitization or tachyphylaxis.

R

N

N

OH

OH N

OH N

O

N

92a: R = H 92b: R = Me

OH N

OH N O

N

93

94

Oxime 92a was identified in a high-throughput screening campaign as a full agonist with EC50 values of 45 nM and 2.0 µM at recombinant (CHO-expressed) human and mouse TGR5, respectively.87 Initial SAR data around 92a revealed that replacing the 4-pyridyl head group by 2- or 3-pyridyl, phenyl, or 4-fluorophenyl was not tolerated. o-Methylation was also found less favorable. Introduction of an ortho methyl group on the unsubstituted phenyl ring afforded compound 92b with moderately improved hTGR5 potency (hTGR5 EC50 = 12 nM; eff = 124%). However, 92b was a potent inhibitor of CYP3A4. That is consistent with its overall high



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lipophilicity and an ortho-unsubstituted pyridine group. CYP3A4 inhibition can be minimized by introducing a methyl group next to the pyridine nitrogen, and the lipophilicity can be reduced by replacing the N,N-dimethyl group with a 4-carboxyphenyl as in compound 93. After chiral chromatography, the R enantiomer was found to be about 5 fold more potent than the S enantiomer with a hTGR5 EC50 of 20 nM (eff = 81%) and a mTGR5 EC50 of 93 nM (eff = 123%) without CYP3A4 inhibition (IC50 > 50 µM). This compound was also found inactive in a FXR transactivation assay. In an in vivo PYY secretion assay, 93 significantly induced PYY secretion at dose of 50 or 100 mg/kg. The high dose requirement is consistent with the high protein binding of 93 (plasma free fraction about 0.2%). To reduce plasma protein binding, the 4carboxyphenyl group was replaced by a piperidine-carboxylic acid to give compound 94 as the most potent compound in this series with an EC50 of 4 nM (eff = 102%) and 28 nM (eff = 163%) for human and mouse receptors, respectively. Importantly this compound had moderate lipophilicity (logD7.4 = 1.5) and a plasma free fraction 10-fold higher than 93. Compounds 93 (100 mg/kg) and 94 (10 and 30 mg/kg) were selected for an oral glucose tolerance test (OGTT) in hTGR5-KI mice. Both compounds produced an approximately 30% decrease in postprandial glucose excursion. Plasma GLP-1 and PYY levels were markedly increased in all treatment groups, with 94 dosed 10 mg/kg being as effective as 93 dosed 100 mg/kg. In order to effectively evaluate TGR5 agonism, the Pfizer research team developed two independent in vitro assays; one with a high level of receptor expression to provide increased sensitivity and a large dynamic range for screening compounds, and another lower-expression system to provide a more conservative estimate of agonist potency and intrinsic activity. Highthroughput screening of the Pfizer compound collection yielded racemic hit 95a with an EC50 of 120 nM and 5780 nM in the induced- and uninduced cAMP assays, respectively.88 Given modest


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in vitro potency and high liver microsomal intrinsic clearance (CLint > 320 µL/min/mg), the initial SAR efforts centered on improving lipophilic efficiency (LipE = -logEC50 uninduced – logD).

95a: Y = F 95b: Y = Cl

96

97

SAR exploration the phenyl ring suggested lipophilic substituents were favorable. 2,3-dichloro analog 95b showed an approximate 25-fold improvement in potency with an EC50 of 231 nM in the uninduced-cAMP assay. Further in vitro potency enhancement was noted upon modifying the pyridinyl piperidine amide region of the molecule. Ring-opened analog 96 demonstrated an EC50 of 105 nM with much improved LipE relative to early lead 95a (3.5 vs 2.0). To reduce the clearance, various heterocycles to replace the pyridine were explored. 5-thiazolyl analog 97 was identified with good in vitro potency, an EC50 of 60 nM in the uninduced-cAMP assay. However, high intrinsic human liver clearance persisted among all active analogs; for 97 (CLint = 76 µL/min/mg) only a modest improvement was noted relative to 95a. Although LipE was improved during the medicinal chemistry effort, potent analogs with lower logD values could not be found. Metabolic instability hindered further development of this series. In a 2007 patent application, a series of bis-phenyl sulfonamide TGR5 agonists was disclosed.89 In hTGR5 transfected melanophore cells, compound 98 showed agonist activity with a pEC50



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between 6.0 and 6.9. Anesthetized CD rats administered 98 by intracolonic injection at a dose of 2.5 mg/kg showed a significant increase of plasma GLP-1 levels (measured by both active and total GLP-1). In a 16 day chronic study, conscious Goto-Kakizaki rats were dosed intracolonically with 98 (0.3 mg/kg q.d.). On day 16, an intravenous glucose tolerance test was performed. Significant glucose reduction was achieved in treated animals compared to the vehicle control group.

98

99

100

101

Two separate publications described different types of arylpyridines as TGR5 agonists.90,91 In HEK293 cells expressing hTGR5, compounds 99-101 increased cAMP production with EC50 values less than 10 µM. Two patent applications from Kalypsys disclosed different classes of pteridinone compounds as potent TGR5 agonists.92,93 Representative compounds 102 and 103 stimulated cAMP production with EC50 values less than 10 µM in HEK293 cells expressing hTGR5.


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Cl

O N N

F

O N

N N

N

Cl

N N

F

102

103

Br

O N

Cl

H N

CF3

H N

O H

N

N

CF3

N F

104

105

Compound 104 was reported to represent a novel class of quinazolinone-based TGR5 agonists.94 In HEK293 cells expressing hTGR5, 104 stimulated cAMP production with an EC50 value less than 10 µM. In an oral glucose tolerance test, mice dosed orally with 104 at 30 mg/kg demonstrated a 52% reduction in glucose compared to the vehicle control group. Concomitant increases in insulin (130%) and GLP-1 (70%) levels compared to vehicle-treated animals were noted. In a 2007 patent application, a family of diazepine derivatives was claimed to be TGR5 agonists.95 Representative compound 105 stimulated cAMP secretion in a HEK293 cell line expressing hTGR5 with an EC50 value less than 10 µM. Takeda reported a series of oxazepine compounds as TGR5 agonists.96,97 In CHO cells expressing hTGR5, compound 106 at 1 µM stimulated cAMP production by 100%. In NCI-



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H716 cells, a 100% increase in cAMP production was observed in the presence of compound 107 at 10 µM. In the same cell line, compound 107 (5 µM) increased GLP-1 secretion by 157%.

106

107

108

109

A set of heteroarylacetamide TGR5 agonists was claimed in a 2006 patent application.98 Among these, dihydroquinoxaline 108 at 30 µM elicited 251% GLP-1 secretion in rat bowel primary culture cells. In a rat intestine perfusion model, 108 at 10 µM gave a significant increase in portal vein GLP-1 concentration. In 2010, a series of aryl amide TGR5 agonists was discovered.99 In CHO cells expressing hTGR5, compound 109 increased cAMP production with an EC50 of 7 nM. Research from Novartis resulted in two patent applications published in 2007. One disclosed a series of heterocyclic amides exemplified by compound 110 as TGR5 agonists.100 Another patent


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application described a class of pyridazine/pyridine/pyran derivatives as TGR5 agonists.101 Compound 111 and 112 are representative structures. Biological data were not reported.

110

111

113

112

114

A series of triazole and imidazole TGR5 agonists exemplified by 113 and 114 were reported by Exelixis.102 In a hTGR5/CRE-luciferase assay, both compounds showed receptor activation with EC50 values less than 100 nM. In mouse STC-1 cells under high glucose conditions, 113 effectively stimulated GLP-1 secretion with an EC50 of 17 nM. In vivo, a two-fold increase in GLP-1 secretion was achieved when fasted C57BL/6 mice were treated with 113 at an oral dose of 30 mg/kg. In a 2010 patent application, a series of isoquinoline-based TGR5 agonists was claimed.103 Representative compound 115 stimulated cAMP production in HEK293 cells expressing hTGR5 with an EC50 of 7.37 µM. Another patent application from this group claimed a series of



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isoquinolinyloxy-methyl heteroaryl analogs exemplified by 116.104 In the same HEK293 cellular assay, compound 116 stimulated cAMP production with an EC50 of 229 nM. F

F N Cl

Cl N

N

O N

O CN

F

N

F

115

O

O

116

H N

OH S O

OH

Cl

O N N

O

F Cl

Cl

S

N

S O O

H N

N O

O

O

O

117

118

In 2012 Bristol-Myers Squibb published a patent application claiming a series of bicyclic nitrogen-containing heterocycles, tetrahydrobenzoquinoline and dihydrobenzothiazine, as TGR5 agonists.105 In CHOK1 cells expressing hTGR5, representative compounds 117 and 118 demonstrated potent TGR5 activity with EC50 values of 24 nM and 23 nM, respectively. In a 2012 patent application, a series of 3-aminopyridines was claimed as potent TGR5 agonists exemplified by 119.106 In hTGR5 expressed CHO cells, compound 119 dose-dependently stimulated cAMP secretion with an EC50 of 9 nM.


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119

120

121

A series of 1-hydroxyimino-3-phenyl propanes exemplified by 120 was claimed as potent TGR5 agonists in a 2012 US patent.107 In hTGR5 expressed CHO cells, compound 120 dosedependently stimulated cAMP secretion at EC50 of 1 nM. In a 2012 patent application, 4-acyl pyrazoles were disclosed to be TGR5 agonists.108 In human T lymphocytes, compound 121 showed dose-dependent stimulation of cAMP with an EC50 of 7 nM. Several patents from Cadila claimed different classes of heterocyclic TGR5 agonists. In a hTGR5 CHO cell CRE-Luciferase assay, compound 122 and 123 at 100 nM showed 147%109 and 97%,110 respectively.



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122

123

124

125

In another application using the same assay,111 compound 124 demonstrated a 129% response at 100 nM compared to the control. In C57/Bl6 mice, upon oral administration at 30 mg/kg, 124 stimulated GLP-1 secretion up to 320% compared to the vehicle control group. In 2013, a series of Erythrina alkaloid TGR5 agonists was claimed and exemplified by compound 125.112 However, no biological data was disclosed. In a 2013 patent,113 substituted imidazopyrimidines were claimed as TGR5 agonists. In hTGR5 transfected CHO cells, compound 126 stimulated cAMP secretion with an EC50 of 90 nM. Incubating 126 in a human enteroendocrine NCI-H716 cell line gave a 3.7 fold in GLP-1 release with respect to vehicle control (the compound concentration was not given).


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126

127

128

129

During an oral glucose tolerance test (OGTT) in a Streptozotocin-induced diabetic hamster model, compound 126 at 38 mg/kg significantly decreased glucose excursion by 39%. In a 2week hamster study, treatment with 126 at 20 mg/kg bid increased oxygen consumption by 7.9%, reduced body weight by 5.2%, and enhanced insulin secretion in response to an oral glucose bolus by 80%. Also, plasma TG levels decreased by 43%, and the HDL:LDL ratio improved by 24%; specifically, HDL increased by 7% and non HDL and LDL decreased by 24% and 14%, respectively. Hoffmann-La Roche published a patent application in 2013 claiming 1-heterocycylhydroxyimino-3-phenyl-propane analogs as TGR5 agonists.114 In a hTGR5 expressed CHO cell, compound 127 dose dependently stimulated cAMP production with an EC50 of 29 nM. In another patent from this group,115 1-pyridazinyl-hydroxyimino-3-phenyl-propanes were claimed



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as TGR5 agonists. In hTGR5 CHO cells, compound 128 proved to be a potent TGR5 agonist with an EC50 of 6 nM. In a third 2013 patent application,116 pyridazine benzamides were reported as TGR5 modulators. Compound 129, the most potent analog reported, activated the TGR5 receptor in hTGR5 CHO cells with an EC50 of 75 nM. In a 2013 patent application,117 a class of thiazolotriazoles and thiazoloimidazoles exemplified by 130 was reported to have TGR5 agonist activity. In a hTGR5 HEK293 cell line, 130 stimulated cAMP secretion with an EC50 < 0.5 µM.

130

131

132

A 2013 patent application from the Chinese Academy of Science disclosed dihydroquinoxalines as TGR5 agonists.118 In a hTGR5 HEK293/CRE-Luciferase assay, compound 131 showed potent agonist activity with an EC50 of 0.43 nM. From the same research group, dihydro-1,2,4oxadiazoles were reported as TGR5 agonists in a 2014 patent application.119 In a hTGR5 HEK293/CRE-Luciferase assay, compound 132 demonstrated agonist activity with an EC50 < 100 nM.


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133

134

135

Based on preclinical in vivo efficacy data, TGR5 represents an attractive molecular target for the treatment of T2D. However, mechanism-mediated side effects have also been reported with systemically exposed TGR5 agonists. Using high-throughput screening, Piotrowski et al. from Pfizer identified compound 133 as a potent TGR5 agonist (induced hTGR5 EC50 = 3.6 nM; uninduced hTGR5 EC50 = 158 nM; dTGR5 EC50 = 213 nM).120 Early assessment showed that compound 133 had very high intrinsic clearance in human (HLM CLint > 317 µL/min/mg) and rat liver microsomes (RLM CLint > 564 µL/min/mg). Initial structure optimization was designed to improve clearance by reducing lipophilicity, to minimize metabolic soft spots, and to maximize TGR5 potency. Aromatic moieties linked by an ethylene spacer to the amide carbonyl were highly preferred. Changing the linking ethylene group (truncation, heteroatom interruption, and alkyl substitution) resulted in compounds with diminished potency. A significant boost in potency was noted by increasing the size of the para-substituent on the terminal phenyl ring from H to iPr (133) or OCF3 (134). Introducing a N atom into the phenyl ring led to 135 with a significant reduction in logD (1.4 compared to 3.0 for 133) and an improvement in microsomal clearance (HLM CLint = 18 µL/min/mg). However, this improvement came at the expense of a



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20-40 fold decrease in potency for 135 (induced hTGR5 EC50 = 43 nM; uninduced hTGR5 EC50 = 2990 nM) compared to 134 (induced hTGR5 EC50 = 2.3 nM; uninduced hTGR5 EC50 = 75 nM; dTGR5 EC50 = 135 nM). Given its good potency and reasonable metabolic profile, compound 134 was further evaluated in human NCI-716 cells (EC50 = 145 nM) and in peripheral blood mononuclear cells isolated from whole human blood (EC50 = 110 nM). In a 7-day dog toxicology study of compound 134, dose-dependent changes in heart rate and blood pressure were noted without treatment-related changes to electrocardiogram waveform parameters. These data suggest involvement of a mechanism-based toxicity associated with the systemic exposure of this compound. Toward a structurally distinct series, scientists from the same research group selected compound 136a as the starting point for SAR exploration.121 In a high TGR5 expression cell line, 136a exhibited modest TGR5 activity with an EC50 of 53 nM. In human and dog liver microsomes, 136a showed high clearances of > 320 µL/min/mg and 630 µL/min/mg, respectively. Introduction of a 3-methoxy group afforded compound 136b with a significant boost in potency (EC50 = 2.1 nM), however no appreciable improvement on clearance was noted.

136a: X = H 136b: X = OMe

137


138

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The team postulated that incorporating an electron-withdrawing group and /or increasing polarity in the aniline region of the molecule might lower the CYP-mediated oxidative metabolism by reducing the overall lipophilicity and oxidative liability of these molecules. Replacing the chlorophenyl group with a 2-trifluoromethyl pyridyl group and changing the 5-ethyl group on the 1,2,3-triazole ring to a 5-cyclopropyl group yielded compound 137 with the lowest clearance (HLM CLint = 30 µL/min/kg) in this series, albeit at the expense of potency (EC50 = 160 nM). Cyano analogue 138 demonstrated reduced clearance (HLM CLint = 98 µL/min/mg compared to that of 136a) while maintaining excellent potency (EC50 = 5.5 nM). In a dog PK study, compound 138 showed moderate clearance (CL = 22 mL/min/kg) and bioavailability (F = 17%). In a human ex vivo whole blood assay, 138 caused a dose dependent suppression of TNFα release after treating human whole blood with LPS. In a panel of 67 cross reactivity assays, 138 showed 7 in a human TGR5 stably transfected HEK293 cell. In C57BL/6 mice incretin secretion assay, compound 146 dosed 30 mg/kg strongly stimulated total GLP-1 (~2-3 fold over vehicle control) and PYY (~2 fold over vehicle control) secretion throughout a 16 h period. At the same dose in CD-1 mice, compound 146 has no significant effect on gallbladder weight.



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146 In 2014, two patent applications from Exelixis claimed a class of imidazole and triazole containing TGR5 agonists. 126,127 As examples, compounds 147 and 148 were designed as GI restricted agents by containing quarternary ammonium salt groups. However, no biological data was disclosed.

Cl-

Cl-

147

148 `

3.2

Bile acid derivatives

In 2009, a semisynthetic cholic acid (CA) derivative, 6α-ethyl-23(S)-methyl-CA (EMCA), was reported to be a selective TGR5 agonist.128 Initial SAR studies unveiled that incorporating a methyl group at C-23 on the side chain afforded the selective, albeit not very potent, TGR5 agonist 149 with an EC50 of 3.58 µM (FXR EC50 > 100 µM). The S-configuration at C-23 was critical for TGR5 potency.


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23

TGR5 EC50

FXR EC50

3.58 µM

>100 µM

150: R = Et 0.095 µM

11.8 µM

149: R = H 6

An improvement in TGR5 and FXR in vitro potency was noted by introducing a small alkyl substituent at C-6 as in compound 150. Unfortunately, this compound suffered from poor physical properties, namely low solubility and high serum albumin binding. Structurally, CA (151) differs from CDCA (152) at C-12 by having an additional α-hydroxyl group oriented on the polar side of the molecule. This “minor” structural change accounts for the markedly different solubilities between these bile acids.

Water Solubility albumin in 0.1 HCl binding (%)

12

151: R = OH 270 µM

93

152: R = H

54

30 µM

Moreover, 151 is devoid of FXR activity (EC50 > 100 µM) while maintaining good TGR5 activity (EC50 = 13.6 µM). Introducing a C-12 α-hydroxyl group into compound 150 afforded compound 153 (EMCA, INT-777), which showed potent TGR5 activity (EC50 = 0.8 µM, eff = 166%) and excellent selectivity over FXR (EC50 > 100 µM). Compound 153 appeared to be stable to human stool broth culture, with more than 95% of compound unmodified after a 12 h incubation. The 6α-ethyl group was believed to provide steric hindrance against the bacterial 7α-dehydroxylation process. 153 was resistant to conjugation since more than 90% of parent compound was secreted into the bile unchanged. The α-methyl group at C-23 was thought to



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prevent carboxyl CoA activation and subsequent conjugation, thereby favoring the cholehepatic shunt pathway with ductular absorption and a potent choleretic effect. O

OH

OH

HO

H

OH

153 DIO C57BL/6 mice treated with compound 153 for 10 weeks at 30 mg/kg/d as a diet admixture showed a significant increase in energy expenditure as determined by increases in O2 consumption, CO2 production, and respiratory quotient. In addition, liver function was improved as evidenced by a reduction in liver steatosis. Significant reductions in plasma TG and nonesterified fatty acids were also observed. Treating these mice with 153 for 3 weeks at 30 mg/kg/d admixed with diet significantly improved glucose tolerance and insulin sensitivity.129 Based on these results, the team continued to investigate the therapeutic potential of naturally occurring BAs from 677 vertebrate species.130 Of particular interest were C24 BAs bearing a hydroxyl group at the C-16 α-position, detected in the biliary composition of birds and snakes. Polar groups at this position were expected to form favorable interactions with the TGR5 receptor as predicted by the QSAR model.


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16

16

6

154a: 16α OH 154b: 16β OH

155

Compound 154a showed only marginal potency as TGR5 agonist with an EC50 of 160 µM. Interestingly, the C16 epimer 154b was nearly an order of magnitude more potent with an EC50 of 25 µM. This data suggested the preference of the hydroxyl group to form hydrogen bond interactions when placed in the equatorial position on the steroidal nucleus. Introduction of 6αethyl group yielded compound 155 with a marked increase of TGR5 potency (EC50 = 650 nM; eff = 120%). Being detergent-like molecules, the tendency to form micelles is a major concern for BAs. Values for the critical micellar concentration (CMC) and the surface tension at CMC (STCMC) are correlated with the cytotoxicity potential of BAs. The relatively higher CMC for 155 (5.9 mM) versus 153 (2 mM) suggested a lower micellar aggregation number and weaker detergency that may lead to lower toxicity for 155. In mouse liver, compound 155 is fully conjugated with taurine, while 153 is only partially conjugated with taurine. The taurine conjugate of compound 155 might also be absorbed by the terminal ileum through an active mechanism leading to increased bioavailability and bio-distribution relative to 153. A 2014 patent claimed a class of bile acid analogs exemplified by compound 156 as potent TGR5 agonist. 131 In hTGR5 HEK293 cells, 156 stimulated intracellular cAMP with an EC50 of 220 nM. This compound proved to be a selective TGR5 agonist with no activity in a FXR functional assay. Computational modeling studies suggested that the stereochemistry at C-23 is



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important for TGR5 activity. With the 23-S configuration, compound 156 can dock easily into the TGR5 ligand binding domain (LBD) while the corresponding R epimer suffers a steric clash with the receptor backbone. O 23

OH

HO

H

156 4

FXR/TGR5 DUAL AGONISTS

In 2010, 157 (INT-767) was reported to be a FXR/TGR5 dual agonist.132 Using an AlphaScreen coactivator recruitment assay, the potency of 157 at FXR was 30 nM. In NCI-H716 cells, 157 stimulated intracellular cAMP secretion with an EC50 of 0.63 µM, similar to selective TGR5 agonist 153 (EC50 = 0.8 µM). Compound 157 also induced a dose-dependent increase of GLP-1 secretion from NCI-H716 cells.

158

157

In DBA/2J mice, a streptozotocin-induced type 1 diabetes model, plasma cholesterol levels are significantly higher in mice fed a western diet (WD) compared with those fed under standard


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chow. A 3-week treatment of these mice with 157 admixed at doses of 10 or 30 mg/kg/day in the WD resulted in a significant dose-dependent decrease of plasma total cholesterol levels and a significant decrease of TG levels only at the 30 mg/kg/d dose. The marked inhibition of total cholesterol induced by compound 157 treatment was correlated with normalization of LDL cholesterol levels; HDL cholesterol levels were not affected. In db/db mice, a model of type 2 diabetes, intraperitoneal administration of 157 for 2 weeks at doses of 10 and 30 mg/kg/day significantly and dose-dependently decreased plasma total cholesterol and TG levels. Using compound 157 as a template for dual agonism, D’Amore et al. described additional SAR studies that resulted in the enhanced dual agonist potency.133 Representing the newly designed analogs, compound 158 transactivated FXR with an EC50 of 1 µM in a luciferase reporter assay on HepG2 cells transfected with human FXR. In HEK-293T cells transfected with human TGR5, this compound induced TGR5 activation with an EC50 of 0.2 µM. This compound is a potent dual agonist with efficacies at 852% and 112% versus CDCA and TLCA, respectively. As a potent FXR agonist, compound 158 increased FXR targeted gene expression (SHP, OSTα and BSEP) in HepG2 cells. In GLUTag cells, this compound at 10 µM effectively increased intracellular cAMP concentration that resulted in a robust increase in GLP-1 concentrations (8 fold over control) in cell supernatants. The binding modes of 158 for both FXR and TGR5 receptors were elucidated through a series of computational studies. These simulations revealed the molecular basis to achieve potent FXR/TGR5 dual agonism profiles.

5

CLINICAL STUDIES AND FUTURE PERSPECTIVES



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To date, only a limited numbers of bile acid receptor agonists have been studied in humans. In 2009, a phase II trial result was reported on a FXR agonist, (3α,5β,6α,7α)-6-ethyl-3,7dihydroxycholan-24-oic acid (obeticholic acid; INT-747),134 in type 2 diabetes patients with comorbid fatty acid disease.135 From this double-blind placebo controlled study of 64 patients, obeticholic acid (25 mg and 50 mg for 6 weeks) significantly improved insulin sensitivity, induced weight loss, and reduced liver damage. In 2014, the Farnesoid X receptor LIgand obeticholic acid in Non-alcoholic steatohepatitis (NASH) Treatment (FLINT) trial was published.136 After 72 weeks of treatment at 25 mg q.d., patients showed improvement in the histological features of non-alcoholic steatohepatitis, including hepatic steatosis, inflammation, hepatocyte ballooning, and fibrosis. However, these improvements did not lead to disease regression. In 2013, a result of a phase I single ascending dose study of another FXR agonist, Px102 (30), was published.137 In the healthy volunteers, 30 demonstrated good pharmacokinetic profiles. In 2014, its active enantiomer (Px-104, absolute stereochemistry not disclosed)52 was advanced into a phase 2 safety pilot study in Non-alcoholic Fatty Liver Disease (NAFLD) patients. Then in late 2015, the program was terminated for the reasons undisclosed.138 In 2010, a phase II study of TGR5 agonist SB-756050 (structure undisclosed) for the treatment of type 2 diabetes was completed.139 The development of this compound was discontinued after the highest dose failed to meet the predetermined efficacy threshold. In 2012, ten obese diabetic humans were administered sodium taurocholate intrarectally at doses of 0.66, 2, 6.66 or 20 mmol (each in 20 mL of vehicle). Taurocholate dose-dependently increased GLP-1, PYY and insulin compared to placebo, with 20 mmol dose resulting in peak concentration 7.2-, 4.2- and 2.6-fold higher, respectively.22 In a separate study, a double-blind and randomized study was carried out in ten healthy men.140 After an overnight fasting, a jejunal catheter was positioned and a balloon


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inflated 30 cm beyond the pylorus with aspiration of endogenous bile. Two grams TCA in saline or saline control was infused beyond the balloon over 30 min, followed by 2 g TCA or control, together with 60 g glucose, over the next 120 minutes. In this study, TCA potently reduced the glycemic excursion in response to a small intestinal glucose infusion associated with an increase in GLP-1 and C-peptide/glucose ratio. Given this promising clinical outcome, a 16-week Phase IIa,b study of taurocholic acid using suppositories in obese type 2 diabetes was conducted. The data is being analyzed at this time.141 There have been some challenges for the clinical development of FXR and TGR5 agonists. After a 72 week clinical study with obeticholic acid, an increase of insulin resistance, higher concentrations of total serum cholesterol and LDL cholesterol, and a decrease in HDL cholesterol were noted.136 These changes could signal an increased risk of atherogenesis. Further studies of FXR agonists will need to closely monitor these biomarkers and address the consequences of these types of changes on cardiovascular outcomes. The development of TGR5 agonist has been hampered by the gallbladder distension findings as a result of receptor activation in this tissue. As these bile acid receptors are ubiquitously expressed in human body and can affect numerous genes and cell signaling pathways, the development of tissue –selective agents could be critical to mitigate undesirable side effects. In a recent publication,142 Fang et al. investigated the effects of oral treatment with fexaramine, an FXR agonist that is poorly absorbed by the intestine. In DIO mice, fexaramine demonstrated substantial metabolic benefits including increased thermogenic activity of BAT and reduced weight gain and systemic inflammation. Another publication on vertical sleeve gastrectomy143 suggested that intestinalbiased FXR modulator may provide therapeutic benefits while avoiding systemic toxicity. To avoid gallbladder distension, the concept of using GI-restricted TGR5 agonists was proposed.



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Although there were some promising results from animal studies, no GI-restricted TGR5 agonist has been advanced into the clinic. Because of the limited clinical data, the full therapeutic potential of molecules that modulate bile acid receptors has yet to be realized. Based on the prolific patent literature around these targets, it is likely that additional molecules will advance into the clinic to test their therapeutic potential over the next few years. The understanding of the role bile acids in human health and diseases will definitely benefit from the future clinical studies.

AUTHOR INFORMATION Phone: 317-433-1113. Email: [email protected] Biography Yanping Xu received a B.S. degree in chemistry in 1986 from Fudan University. He completed his Ph.D. in organic chemistry in 1996 under the supervision of Professor Carl R. Johnson at Wayne State University. From 1996 to 1998, he was a postdoctoral fellow under Professor Marvin J. Miller at Notre Dame, studying mycobactin and mycobactin analogs total syntheses. He has worked at Eli Lilly and Company since 1998 where he focuses on medicinal chemistry research in metabolic and cardiovascular diseases. ACKNOWLEDGEMENT The author would like to thank Dr. Alan Warshawsky for critical review of the manuscript.

ABBREVIATION USED ADME, absorption, distribution, metabolism and excretion; AUC, area under the curve; b.i.d., twice a day; BA, bile acids; BAT, brown adipose tissue; cAMP, 3',5'-cyclic adenosine monophosphate; clogP, calculated logP; CoA, coenzyme A; CYP, cytochrome P; EC50, half


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maximal effective concentration; GI, gastrointestinal; GLP-1, glucagon-like peptide-1; GR, glucocorticoid receptor; HTRF, homogenous time-resolved fluorescence; IC50, half-maximum inhibitory concentration; LXR, liver X receptor; LBD, ligand binding domain; metID, metabolite identification; po, oral administration; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; PXR, pregnane X receptor; q.d., once a day; QSAR, quantitative structure-activity relationship; RXR, retinoid X receptor; SAR, structure-activity relationship; t1/2, half-time; VDR, vitamin D receptor. REFERENCES (1) Kuipers, F.; Bloks, V. W.; Groen, A. K. Beyond intestinal soap-bile acids in metabolic control. Nat. Rev. Endocrinol. 2014, 10, 488-498. (2) Schaap, F. G.; Trauner, M.; Jansen, P. L. M. Bile acid receptors as targets for drug development. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 55-67. (3) Thomas, C.; Pellicciari, R.; Pruzanski, M.; Auwerx, J.; Schoonjans, K.. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discovery 2008, 7, 678-693. (4) Wang, H.; Chen, J.; Hollister, K.; Sowers, L. C.; Forman, B. M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol. Cell 1999, 3, 543-553. (5) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R. M.; Luk, A.; Hull, M. V.; Lusting, K. D.; Mangelsdorf, D. J.; Shan, B. Identification of a nuclear receptor for bile acids. Science 1999, 284, 1362-1365. (6) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.; Consler, T. G.; Kliewer, S. A.; Stimmel, J. B.; Wilson, T. M.; Zavacki, A. M.; Moore, D. D.; Lehmann, J. M. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999, 284, 1365-1368.



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