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Aug 27, 2014 - Their molecular repertoire is generated first in the liver with the production of primary bile acids, cholic acid (CA), and chenodeoxyc...
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Modification on Ursodeoxycholic Acid (UDCA) Scaffold. Discovery of Bile Acid Derivatives As Selective Agonists of Cell-Surface G‑Protein Coupled Bile Acid Receptor 1 (GP-BAR1) Valentina Sepe,† Barbara Renga,‡ Carmen Festa,† Claudio D’Amore,‡ Dario Masullo,† Sabrina Cipriani,‡ Francesco Saverio Di Leva,† Maria Chiara Monti,§ Ettore Novellino,† Vittorio Limongelli,† Angela Zampella,*,† and Stefano Fiorucci‡ †

Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy Department of Clinical and Experimental Medicine, Nuova Facoltà di Medicina, Via Gambuli 1 06132 Perugia, Italy § Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084, Fisciano (Salerno), Italy ‡

S Supporting Information *

ABSTRACT: Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of proglucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

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INTRODUCTION

Cell-surface G-protein coupled bile acid receptor 1 (GPBAR1, TGR5, M-BAR1) belongs to the rhodopsin-like superfamily of G protein coupled receptors (GPCRs). GPBAR1 activation results in the elevation of intracellular cAMP levels, which in turn activates specific intracellular signaling cascades. Similarly to FXR, GP-BAR1 is highly expressed in the liver and in the intestine, but differently from FXR it is also located in tissues and cells not participating in the enterohepatic circulation such as muscle, brain, adipose tissue, macrophages, and endothelial cells. In muscle and brown adipose tissue, GP-BAR1 activation increases energy expenditure and oxygen consumption,12 while in entero-endocrine L cells, it stimulates the secretion of glucagon-like peptide (GLP)1, an incretin that improves insulin release from pancreas, thus regulating glucose blood levels, gastrointestinal motility, and appetite.13 All these data underlay the role of GP-BAR1 agonists in preventing obesity, atherosclerosis, and hepatic steatosis in

Bile acids (BAs) are signaling molecules interacting with two types of dedicated cellular receptors, intracellular nuclear receptors, and cell-surface receptors. Nuclear receptors include farnesoid X receptor (FXR),1,2 pregnane X receptor (PXR),3 vitamin D3 receptor (VDR),4 and constitutive androstane receptor (CAR),5 with FXR identified as the endogenous bile acid sensor. Highly expressed in enterohepatic tissues (liver and intestine), FXR regulates bile acid homeostasis, preventing their accumulation and associated toxicity through the induction of gene expression for conjugating enzymes and export pumps with the final aim to provide bile acid elimination from the body. Apart from this pivotal role, FXR controls further important metabolic pathways6 regulating also lipid7 and glucose homeostasis.8 Additionally, FXR agonists were proved to exert anti-inflammatory9 and antifibrotic effects,10 making this nuclear receptor an appealing pharmacological target in the treatment of common human diseases ranging from metabolic syndrome to cancer.11 © 2014 American Chemical Society

Received: June 11, 2014 Published: August 27, 2014 7687

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about 3% of total bile acids, UDCA has been shown effective in biliary and liver diseases and is now considered as the first-line treatment for primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), intrahepatic cholestasis of pregnancy (ICP), and several less common adult and pediatric cholestatic conditions.17 UDCA is a molecule with a good safety profile and a clinical history with minimal side effects, also when used in large doses. Additionally, UDCA is endowed with several beneficial effects such as neuroprotective and antiapoptotic actions, chemopreventative effects in colon cancer with attenuation of carcinogenic signaling cascades induced by toxic bile acids.18−20 UDCA is the 7β-hydroxy epimer of CDCA and, despite its pharmacological profile resembling a modulation of FXR, UDCA is not a FXR agonist and its mechanism of action remains unclear and still subjected to intense scientific debates. In this context we have decided to manipulate UDCA chemical scaffold to obtain selective GP-BAR1 agonists. Thus, a series of side chain modified 3α,7β-dihydroxyl cholanoids (Figure 2) has been prepared and screened in their ability to modulate FXR/GP-BAR1. This research work resulted in the identification of several UDCA analogues endowed with GPBAR1 selective agonistic profile. Computational studies have been performed to elucidate their binding mechanism to GPBAR1 and the pharmacological activity of derivative 16, the most potent compound generated in this study, has been characterized through a series of in vitro and in vivo experiments.

mice on a high-fat diet, thus pointing toward GP-BAR1 as a promising target in the pharmacotherapy of enterohepatic and metabolic disorders.14−16 Thus, today, several efforts are focused toward the discovery and development of potent and selective GP-BAR1 small molecule agonists belonging to bile acid or nonbile acid classes as promising therapeutic strategy in lipid and glucose diseases such as in the treatment of nonalcoholic steatohepatitis, hypercholesterolaemia, hypertriglyceridaemia, and type 2 diabetes mellitus (T2DM).14−16 In this context, the identification of potent GP-BAR1 small molecule agonists with improved selectivity over FXR remains the aim of the medicinal chemists. From a chemical point of view, BAs are truncated cholesterol side chain derivatives (Figure 1). Their molecular repertoire is

Figure 1. Endogenus bile acids.

generated first in the liver with the production of primary bile acids, cholic acid (CA), and chenodeoxycholic acid (CDCA), successively subjected to microbial transformation in the gut, affording secondary bile acids, deoxycholic acid (DCA), and lithocholic acid (LCA) and their glycine and taurine conjugates. Interestingly, the activity toward the two BAs receptors is structure dependent, with CDCA and its conjugated forms the most potent endogenous FXR activators and LCA and taurolithocholic acid (TLCA) the strongest natural agonists of GPBAR1. Among BAs, ursodeoxycholic acid (UDCA) represents a puzzling molecule. Present in human bile at low concentrations,

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RESULTS The steroidal nucleus of both CA and CDCA adopts a bent shape due to the A/B cis ring juncture that forces ring A to lie outside of the plane of the BCD ring system. As a result, two well distinct faces are generated: (i) the hydrophobic area (βsite) of the cholanoic acid nucleus and (ii) the α-hydrophilic region defined by the two hydroxyl groups at C3 and C7. These structural features allow primary BAs (CDCA and CA) to bind the FXR ligand binding domain (FXR-LBD) and activate this nuclear receptor.21 If compared with CDCA, the reduced FXR

Figure 2. Ursodeoxycholane derivatives generated in this study. 7688

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Scheme 1. nor-Ursodeoxycholane Derivativesa

a

Reagents and conditions: (a) HCOOH, HClO4, 96%; (b) TFA, trifluoroacetic anhydride, NaNO2, 96%; (c) KOH 30% in MeOH/H2O 1:1 v/v, 97%; (d) p-TsOH, MeOH dry, 97%; (e) DMT-MM, Et3N, taurine, DMF dry, 28%; (f) LiBH4, MeOH dry, THF, 0 °C, quantitative yield; (g) Et3N· SO3, DMF, 95 °C.

Scheme 2. Ursodeoxycholane and Bis-homo Derivativesa

Reagents and conditions: (a) LiBH4, MeOH dry, THF, 0 °C, 93%; (b) 2,6-lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2, 0 °C; (c) LiBH4, MeOH dry, THF, 0 °C, quantitative yield over two steps; (d) DMSO, oxalyl chloride, TEA dry, CH2Cl2, −78 °C, then methyl(triphenylphosphoranylidene)acetate, 76%; (e) HCl 37%, MeOH, quantitative yield; (f) NaOH 5% in MeOH/H2O 1:1 v/v, 93%; (g) DMTMM, Et3N, taurine, DMF dry, 26%; (h) H2, Pd(OH)2/C Degussa type, THF/MeOH 1:1, then HCl 37%, MeOH, quantitative yield; (i) NaOH 5% in MeOH/H2O 1:1 v/v, 60%; (j) DMT-MM, Et3N, taurine, DMF dry, 45%; (k) LiBH4, MeOH dry, THF, 0 °C, 77%. a

activation of UDCA can be explained by the lower hydrophobicity of the β-site due to the presence of the β-oriented hydroxyl group at C7. This stresses the pharmacophoric role of the configuration at C7 hydroxyl methine carbon in bile acid derivatives to achieve FXR agonism. On the other hand, according to structural studies, the BAs alkyl side chain points toward a rather large and polar cavity both in FXR and GPBAR1.21,22 Consequently, this part of the BAs can be functionalized by producing derivatives endowed with different

pharmacological profiles, ranging from full potent dual agonism22 to selective FXR or GP-BAR1 modulation.23,24 To get more insights into the structural requisites ruling the activity of bile acid derivatives on GP-BAR1 and FXR, we have developed a library of compounds introducing on the UDCA scaffold: (i) side chains of different length (C23, C24, and C26) and flexibility, (ii) side chains functionalized with a number of diverse polar functional groups such as carboxylic, alcoholic, and sulfate moieties. In fact, we have recently identified a 7689

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Scheme 3. Bis-homo Chenodeoxycholane Derivativesa

Reagents and conditions: (a) LiBH4, MeOH dry, THF, 0 °C; (b) 2,6-lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2, 0 °C; (c) LiBH4, MeOH dry, THF, 0 °C; (d) DMSO, oxalyl chloride, TEA dry, CH2Cl2, −78 °C; (e) LiOH, TEPA, THF dry, reflux; (e) HCl 37%, MeOH, 60% yield over five steps; (f) NaOH 5% in MeOH/H2O 1:1 v/v, 78% yield; (g) LiBH4, MeOH dry, THF, 0 °C, 73% yield. a

purification allowed us to obtain the corresponding tauro conjugated derivative 20. Double bond hydrogenation on 24 gave methyl ester 15, which was used as starting material in the preparation of carboxyl acid 16 and its corresponding alcohol 14, through treatment with LiOH and LiBH4, respectively. Finally, 16 was easily transformed in the corresponding tauro-conjugated (taurine, DMT-MM, 45% yield) derivative 17. In this study, several derivatives were proved selective GPBAR1 agonists (see below in In Vitro Pharmacological Evaluation), e.g., compounds 4, 14, and 16. These results represent an interesting evidence of the hierarchical role played by the length and the functionalization of the side chain rather than the configuration at C7 in BA recognition of their receptors. To give further support to this hypothesis, also bishomoCDCA (26) and corresponding alcohol 27 were prepared (Scheme 3) starting from CDCA methyl ester and following the synthetic protocol already described for the corresponding bis-homo ursodeoxycholane derivatives (Scheme 2). In Vitro Pharmacological Evaluation. Ursodeoxycholane derivatives (3−20, Figure 2) and chenodeoxycholane analogues 26 and 27 were tested in the luciferase reporter assays on HepG2 and HEK-293T cells transfected with FXR and GPBAR1, respectively. Our pharmacological data suggest that the stereochemistry at C7 is a determinant factor for the activity on FXR and not on GP-BAR1. In fact, as shown in Figures 3 and 4, all the newly synthesized UDCA derivatives, which have the 7βOH group, are inactive on FXR, while compounds like CDCA, which presents a 7α-OH group, activates this receptor. At variance with FXR, in GP-BAR1 the presence of the 7α-OH as in CDCA, the 7β-OH as in 16 and even the absence of the hydroxyl group at C7 as in TLCA are tolerated. Additionally as previously demonstrated,29 side chain elongation on CDCA scaffold produce positive effects toward FXR when an hydroxyl function is placed as end-terminus (27), whereas the corresponding carboxyl acid derivative 26 was almost inactive when tested at 10 μM (Figure 3A). Moreover, when compound 26 was tested at 50 μM in the presence of CDCA (Figure 3B), the ratio RLU/RRU was higher than that of CDCA, indicating that 26 might be an FXR modulator. Indeed, 26 was demonstrated cytotoxic on HEK-293T cells (Figure 4B) at 50 μM, thus ruling out the pharmacological potential of this compound.

potent GP-BAR1 agonist showing the CDCA scaffold with a sulfate group on the side chain.22 Thus, it is worth investigating the effect of this substituent on the GP-BAR1 activity of UDCA derivatives. nor-Ursodeoxycholane Derivatives. A key step in the synthetic protocol toward nor-ursodeoxycholane derivatives was Beckmann one-carbon degradation at C24 on the UDCA perfomate derivative 21, generated through UDCA Fisher’s esterification with formic acid and acetic anhydride,25 by treatment with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid (Scheme 1).26,27 Alkaline hydrolysis of the resulting C23-nitrile (22) furnished 11 in 97% chemical yield, which in a small aliquot was subjected to the reaction of amidation with taurine in the presence of the versatile coupling agent, 4-(4,6-dimethoxy1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM),28 giving the amide derivative as ammonium sulfate salt. Purification on RP-18 column followed by HPLC furnished 12 in pure form and as sodium salt (28% chemical yield). Methanol/p-toluenesulfonic acid treatment on 11 afforded C23 methyl ester 13 that in turn was transformed in the corresponding C23 alcohol 4 by treatment with lithium borohydride (88% chemical yield over two steps). Sulfation (Et3N·SO3 10 equiv) afforded a crude reaction product (Scheme 1) containing trisulfate derivative 10, the mixture of disulfate derivatives 8−9, and the mixture of monosulfate derivatives 5−7. RP-18 column separation with a gradient eluting system from water to methanol followed by HPLC of the enriched fractions furnished pure derivatives 5−10, differing in the sulfation pattern on nor-ursodeoxycholane scaffold. Ursodeoxycholane and Bis-homo-ursodeoxycholane Derivatives. First, treatment of ursodeoxycholic methyl ester with LiBH4 afforded triol 3 in high chemical yield (Scheme 2). A four-step reaction sequence on UDCA methyl ester, including protection of alcoholic functions at C3 and C7 (2,6lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2), reduction of the side chain methyl ester (LiBH4, MeOH/THF), and subsequent one-pot Swern oxidation/ Wittig C2 homologation gave the protected conjugated methyl ester 24 in a total 76% chemical yield. Silyl deprotection furnished pure Δ24,25 ester 18 that was subjected to methyl ester hydrolysis to give the corresponding bis-homo conjugated acid 19. Finally, amidation with taurine and RP18/HPLC 7690

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Figure 3. Transactivation assays on FXR. (A) HepG2 cells were transfected with pSG5-FXR, pSG5-RXR, pCMV-βgal, and p(hsp27)TKLUC vectors. Cells were stimulated with compounds 3−20, 26, and 27 (10 μM). CDCA (1, 10 μM) was used as a positive control, UDCA (2, 10 μM) as negative control. Results are expressed as mean ± standard error; *p < 0.05 versus not treated cells (NT). (B) HepG2 cells were transfected with pSG5-FXR, pSG5-RXR, pCMV-βgal, and p(hsp27)TKLUC vectors. Cells were stimulated with CDCA (1) 10 μM in combination with UDCA (2, 50 μM) as negative control and with 3−20, 26, and 27, 50 μM. Results are expressed as mean ± standard error. *p < 0.05 versus NT; #p < 0.05 versus CDCA; °cytotoxic at 50 μM.

Interestingly, also 16 and the corresponding Δ 24,25 conjugated analogue 19 were demonstrated inducers of cAMP-luciferase reporter gene, with 16 showing a potency similar to that of TLCA, the most potent endogenous GPBAR1 agonist (Figure 4). This result demonstrates that side chain elongation on the ursodeoxycholane scaffold produce positive effects toward GP-BAR1 activation also when a carboxyl function is placed as end-terminus. The above activity declines in the corresponding tauro-conjugates (compare 16 with 17 and 19 with 20 in Figure 4), thus demonstrating that further elongation of UDCA side chain is not tolerated by the

None of the tested compounds turned out to be FXR antagonist (Figure 3B). Results of transactivations of CREB-responsive elements in HEK-293T cells transiently transfected with the membrane bile acid receptor GP-BAR1 (Figure 4) revealed that all ursodeoxycholane alcohols generated in this study, compounds 3, 4, and 14, transactivate GP-BAR1, thus demonstrating that the introduction of an hydroxyl group on the side chain of ursodeoxycholane scaffold produces, independently by side chain length (C23 in 4, C24 in 3 and C26 in 14), derivatives endowed with membrane bile acid receptor agonism. 7691

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Figure 4. Transactivation assays on GP-BAR1. (A) HEK-293T cells were cotransfected with GP-BAR1 and a reporter gene containing a cAMP responsive element in front of the luciferase gene. Then 24 h post transfection, cells were stimulated with CDCA (1), UDCA (2), and compounds 3−20, 26, and 27 (10 μM). Luciferase activity served as a measure of the rise in intracellular cAMP following activation of GP-BAR1. TLCA (10 μM) was used as a positive control. Results are expressed as mean ± standard error. *p < 0.05 versus NT cells. (B) Antagonistic activity of compounds 1−27 on transactivation of cAMP responsive element induced by TLCA in HEK-293T cells transfected with GP-BAR1. Experimental conditions were the same described in (A). None of the compound had an antagonistic activity. Concentrations used were: TLCA 10 μM, while all the other compounds were tested at the concentration of 50 μM. Results are expressed as mean ± standard error. *p < 0.05 versus NT cells; °cytotoxic at 50 μM.

receptor. Of interest is also the behavior of chenodeoxycholane derivatives 26 and 27, with the alcohol 27 almost inactive and the carboxylic acid 26 less potent than the corresponding UDCA derivative 16. Notably, 3, 4, 14, 16, and 19 are selective GP-BAR1 agonists devoid of any activity toward FXR (Figure 3). Moreover, none of tested compounds was able to induce cAMP-luciferase reporter gene in absence of GP-BAR1 (data not shown), thus indicating that the observed cAMP induction is GP-BAR1 mediated. Among all tested compounds, our attention was focused on 4 and 16, both selective GP-BAR1 agonists, but quite different in their chemical scaffold. As shown in Figure 5, compounds 4 and 16 exerted a concentration-dependent effect on activation of

cAMP responsive element in HEK-293T cells transfected with GP-BAR1 with an EC50 of 24.4 and 17.2 μM, respectively. Computational Studies. To elucidate the structural requisites of the activity of the UDCA derivatives on GPBAR1, we performed docking simulations on the most active compounds of the series (3, 4, 14, and 16). In these calculations, we used the tridimensional structure of GPBAR1 that we have very recently reported22 (see Experimental Section for details). The best-scored docking solution shows the most potent derivative of the series, compound 16, Hbonding with the Glu169 and Asn93 side chains through the 3α- and 7β-hydroxyl groups, respectively (Figure 6). On the other side of the molecule, the flexible side chain points toward the transmembrane (TM) helices 1, 2, and 7, where the ligand carboxylate group engages H-bonds with the 7692

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Figure 5. Concentration−response curves on GP-BAR1 activation by compounds 4 and 16. HEK-293T cells were transfected with GP-BAR1 and stimulated with increasing concentrations of 4 and 16 (0.1, 1, and 10 μM). Results are mean ± standard error of at least three determination in duplicate.

Figure 6. Binding mode of 16 (upper left), 14 (upper right), 3 (lower right), and 4 (lower left) in the GP-BAR1 homology model. Ligands are represented as cyan (16), green (14), orange (3), and yellow (4) sticks. The receptor is shown as gray cartoons and sticks. Extracellular loops and nonpolar hydrogens are omitted for clarity.

substantially alter the ligand binding mode. In fact, the alcohol derivative 14 shows a binding mode very similar to that of 16 (Figure 6). In particular, the steroidal scaffold of 14 engages the same interactions established by 16, while its C26 side chain points toward the polar region formed by Ser21, Ser267, and Ser270, where the hydroxyl group H-bonds with the Ser270 side chain. Our results show that in this pocket the presence of a negatively charged group, as in compound 16, is desirable but not necessary, while polar groups, able to form H-bond interactions, are important for GP-BAR1 activation. Our in silico predictions explain the pharmacological data, which show

side chains of Ser21 and Ser270. This ligand binding mode is further stabilized by a series of hydrophobic interactions engaged by the steroidal scaffold with residues such as Leu71, Phe83, Leu174, and Trp237. It is interesting to note that the computed binding mode of 16 is very similar to that of other potent GP-BAR1 agonists previously reported by our group.22 Furthermore, mutagenesis data showed a reduced activity of bile acid derivatives in the Asn93Ala, Glu169Ala, and Ser270Ala GP-BAR1 mutants if compared to the wild-type receptor.30 The involvement of these residues in the binding mode of 16 is in line with these experimental observations. The substitution of the carboxylate group with a hydroxyl one does not 7693

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Figure 7. In vivo evaluation on compound 16. In vivo administration of 16 has no effect on expression of FXR target genes in the liver but increases the expression of pro-glucagon 1, a GP-BAR1 regulated gene. Livers were collected 6 h after administration of 16 or UDCA (2) and 24 h after administration of 2; *p < 0.05 versus control mice.

Figure 8. Effect of UDCA and 16 in a rodent model of cholestasis. In vivo administration of UDCA (2) and 16 alters the bile acid pool. Cholestasis was induced by feeding mice with ANIT alone or in combination with 2, 15 mg/kg, or 16, 16 mg/kg, for 7 days. Blood was collected at the end of experiments and bile acids concentrations measured as described in the Experimental Section. (A) Analysis of body weight loss in control mice, mice administered ANIT, mice administered ANIT plus 2, and mice administered ANIT plus 16. (B) Blood concentrations of total conjugated bile acids. (C) Administration of 16 caused a robust increase in tauro-muricholic acid (tMU) and tauro-cholic acid (tCA). *p < 0.05 versus control mice; #p < 0.05 versus ANIT treated mice. 7694

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effect became apparent only 24 h after its administration (p < 0.05 versus control mice). In contrast, expression of pro-glucagon-1 gene, a GP-BAR1 regulated target, in the intestine was increased by 2-fold in response to 6 h treatment with compound 16 (Figure 7). In summary, compound 16 is an effective and selective GP-BAR1 ligand in vivo and in vitro, and its activity is different from UDCA, its parent compound. To further confirm the role of compound 16 as a GP-BAR1 agonist, we also performed a cholestatic model by administering mice with α-naphthylisothiocyanate (ANIT). As shown in Figure 8, exposure to ANIT caused cholestasis as confirmed by the analysis of plasma bile acid concentration in ANIT treated animals which revealed that these animals had an increase in the total conjugated bile acids in comparison with not treated animals. In addition, ANIT treated animals had a significant lost weight in comparison with not treated animals, while no significant differences among the groups ANIT and ANIT plus UDCA was observed in terms of both weight loss and bile acid concentration. By the contrast, administration of compound 16 significantly increased total amount of plasma bile acids in comparison with ANIT treated mice (p < 0.05 versus ANIT treated mice). It is noteworthy that mice administered with compound 16 had a significant increase in plasma levels of tauro-muricholic acid (tMU) and tauro-cholic acid (tCA) compared with ANIT treated mice (p < 0.05 versus ANIT treated mice).

a decreased activity of the hydroxyl compound 14 if compared with 16, while the more hydrophobic ester derivative 15 shows an even lower activity. To provide further structural insights on the binding mechanism to GP-BAR1 of this series of compounds, we studied also the binding mode of the alcohol derivatives 3 and 4. These ligands bear a shorter side chain with respect to 14 and 16, specifically 3 and 4 are C24 and C23 derivatives, respectively. Docking calculations show that these compounds occupy the binding site similarly to 14 and 16, demonstrating that shorter side chains are allowed if they present H-bonding groups which can interact with the neighboring serine residues through direct or water mediated H-bonds (Figure 6). This finding is in agreement with the pharmacological data showing that 3 and 4 are both able to activate GP-BAR1 in transactivation assays. It is important to stress that our simulations highlight the high conformational flexibility of the ligand side chain, which allows the proper binding of the ligand in the binding pocket and the formation of the interactions with the receptor. Compounds with a more rigid side chain, such as the Δ24,25 compound 19 (see Supporting Information, Figure S1), show indeed a lower activity. A reduced activity was observed also for the tauro-conjugated compounds 12 and 20, where the presence of the amide group decreases the conformational flexibility of the ligand side chain. Finally, the presence of bulky groups on rings A and B, like sulfate in compounds 6−10, hampers the proper orientation of the ligand in the binding site to interact with residues such as Asn93 and Glu169, causing a drop in the activity. On the basis of our pharmacological assays, all the UDCA derivatives are inactive on FXR. These data prompted us to investigate the binding mechanism of UDCA to FXR in the attempt to elucidate the structural bases of its inactivity toward this receptor. Docking calculations on UDCA in the FXR-LBD indicates that it is able to form a salt-bridge with the Arg328 side chain through its C24 carboxylate group and two hydrogen bonds with the Tyr358 and His444 side chains through its 3αOH (see Supporting Information, Figure S2). Similar interactions are engaged by the potent FXR agonist 6ECDCA,21 however, at variance with 6-ECDCA, UDCA is not able to form hydrogen bonds with the Tyr366 and Ser329 side chains due to the presence of the β-oriented 7-hydroxyl group (see Supporting Information, Figure S2). The lack of these interactions might explain the inactivity of UDCA toward FXR as also previously reported by Fujino et al.31 This conclusion is further supported by experimental data, showing that the interaction with Tyr366 and Ser329 is determinant for the agonistic activity on FXR. In fact, compounds such as 3deoxyCDCA, which interacts with Tyr366 and Ser329 and not with His444 and Trp466, have an agonist profile in FXR transactivation assays.21 In Vivo Evaluation on Compound 16. Compound 16, the most potent GP-BAR1 agonist generated in this study, was further investigated in vivo and its effects on FXR and GPBAR1 target genes in the liver and intestine assessed by RTPCR. In these experiments CD1, 11 week old male mice were orally administered with compound 16 (64 mg/kg) or its parent compound UDCA (60 mg/kg) and liver, intestine, and blood collected after 3, 6, and 24 h (n = 3). As illustrated in Figure 7, compound 16 failed to induce the expression of OST and BSEP mRNA in the liver at 6 and 24 h (data not shown) after administration. UDCA increased the expression of these two FXR-target genes although that the

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CONCLUSION In conclusion, in this study we demonstrated that manipulating UDCA side chain represents a promising strategy to generate bile acid derivatives as selective GP-BAR1 agonists. Pharmacological evaluation on 16 demonstrated this compound as a promising lead for the treatment of diabetes. Administration of compound 16 selectively increase the expression of proglucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. This study provides new opportunities for the treatment of enterohepatic and metabolic disorders.

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EXPERIMENTAL SECTION

Chemistry. General. Specific rotations were measured on a Jasco P-2000 polarimeter. High-resolution ESI-MS spectra were performed with a Micromass Q-TOF mass spectrometer. NMR spectra were obtained on Varian Inova 400 and Varian Inova 500 NMR spectrometers (1H at 400 and 500 MHz, 13C at 100 and 125 MHz) equipped with Sun hardware and recorded in CDCl3 (δH = 7.26 and δC = 77.0 ppm) and CD3OD (δH = 3.30 and δC = 49.0 ppm). J are in hertz, and chemical shifts (δ) are reported in ppm and referred to CHCl3 and CHD2OD as internal standards. HPLC was performed using a Waters model 510 pump equipped with Waters Rheodine injector and a differential refractometer, model 401. Reaction progress was monitored via thin-layer chromatography (TLC) on Alugram silica gel G/UV254 plates. Silica gel MN Kieselgel 60 (70−230 mesh) from Macherey-Nagel Company was used for column chromatography. All chemicals were obtained from Sigma-Aldrich, Inc. Solvents and reagents were used as supplied from commercial sources with the following exceptions. Tetrahydrofuran, dichloromethane, diisopropylamine, and triethylamine were distilled from calcium hydride immediately prior to use. Methanol was dried from magnesium methoxide as follows. Magnesium turnings (5 g) and iodine (0.5 g) were refluxed in a small (50−100 mL) quantity of methanol until all of 7695

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Journal of Medicinal Chemistry

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mm i.d. × 250 mm) with MeOH/H2O (60:40) as eluent (flow rate 1 mL/min) to give 7 mg (28%) of compound 12 (tR = 12.4 min); [α]25D = −4.8 (c 0.15, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.58 (2H, t, J = 6.6 Hz), 3.48 (2H, m), 2.95 (2H, t, J = 6.6 Hz), 0.97 (3H, d, ovl), 0.96 (3H, s), 0.71 (3H, s). HR ESIMS m/z 484.2735 [M − Na]−, C25H42NO6S requires 484.2732. Methyl 3α,7β-Dihydroxy-24-nor-5β-cholan-23-oate (13). Compound 11 (100 mg, 0.26 mmol) was dissolved in 30 mL of dry methanol and treated with p-toluenesulfonic acid (251 mg, 1.3 mmol). The solution was left to stand at room temperature for 5 h. The mixture was quenched by addition until neutrality of NaHCO3 saturated solution. Most of the solvent was evaporated, and the residue was extracted with EtOAc. The combined extract was washed with brine, dried with Na2SO4, and evaporated to give 13 as amorphous solid (100 mg, 97% yield). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (9:1) as eluent (flow rate 1 mL/min) to give compound 13 (tR = 9.5 min); [α]25D = +19.8 (c 0.22, CHCl3). Selected 1H NMR (400 MHz CDCl3): δ 3.60 (3H, s), 3.51 (2H, m), 0.92 (3H, d, J = 6.3 Hz), 0.88 (3H, s), 0.65 (3H, s). 13C NMR (100 MHz CDCl3): δ 173.8, 71.0, 70.9, 55.7, 54.9, 51.3, 43.6, 43.4, 42.4, 41.3, 39.9, 39.1, 37.1, 37.0, 34.8, 33.9, 33.6, 30.0, 28.6, 26.7, 23.3, 21.0, 19.5, 12.0. HRMS-ESI m/z 393.3009 [M + H]+, C24H41O4 requires 393.3005. 3α,7β-Dihydroxy-24-nor-5β-cholan-23-ol (4). To a solution of 13 (90 mg, 0.23 mmol) in dry THF (5 mL) at 0 °C under argon were added LiBH4 (0.8 mL, 2 M in THF, 1.6 mmol) and dry methanol (65 μL, 1.6 mmol), and the resulting mixture was stirred for 3 h at 0 °C. The mixture was quenched by addition of NaOH (1 M, 1.4 mL) and then allowed to warm to room temperature. Ethyl acetate was added, and the separated aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (CH2Cl2/ MeOH 97:3) gave compound 4 as a colorless oil (85 mg, quantitative yield). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (8:2) as eluent (flow rate 1 mL/min) to give compound 4 (tR = 17.8 min); [α]25 D = +23.7 (c 0.18, CH3OH). 1H NMR (400 MHz CD3OD): δ 3.60 (1H, m ovl), 3.51 (1H, m), 3.50 (2H, m), 0.97 (3H, d, ovl), 0.96 (3H, s), 0.72 (3H, s). 13C NMR (100 MHz CD3OD): δ 72.1, 71.9, 60.8, 57.5, 57.1, 44.8, 44.5, 44.0, 41.6, 40.7, 39.9, 38.6, 38.0, 36.1, 35.2, 34.1, 31.0, 29.8, 27.9, 23.9, 22.4, 19.5, 12.6. HRMS-ESI m/z 365.3053 [M + H]+, C23H41O3 requires 365.3056. 3α,7β-Dihydroxy-24-nor-5β-cholan-23-yl-23-sodium Sulfate (5), 7β,23-Dihydroxy-24-nor-5β-cholan-3α-yl-3-sodium Sulfate (6), 3α,23-Dihydroxy-24-nor-5β-cholan-7β-yl-7-sodium Sulfate (7), 7βHydroxy-24-nor-5β-cholan-3α,23-diyl-3,23-sodium Disulfate (8), 3α-Hydroxy-24-nor-5β-cholan-7β,23-diyl-7,23-sodium Disulfate (9), and 24-nor-5β-Cholan-3α,7β,23-tryl-3,7,23-sodium Trisulfate (10). The triethylamine−sulfur trioxide complex (253 mg, 1.4 mmol) was added to a solution of compound 4 (50 mg, 0.14 mmol) in DMF dry (5 mL) under an argon atmosphere, and the mixture was stirred at 95 °C for 24 h. The solution was then concentrated under vacuum. To the solid dissolved in methanol (15 mL) was added three drops of HCl 37% v/v, and the mixture was stirred for 3 h at room temperature. At the end of reaction, silver carbonate was added to precipitate chloride. Then the reaction mixture was centrifuged, and the supernatant was concentrated in vacuo. The residue was poured over a RP18 column. Fraction eluted with H2O/MeOH 99:1 gave a mixture that was further purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (35:65) as eluent (flow rate 1 mL/ min) to give 3.7 mg (4%) of compound 10 (tR = 5 min); fraction eluted with H2O/MeOH 95:5 and with H2O/MeOH 9:1 gave a mixture that was purified as previously to afford respectively 8.7 mg (11%) of compound 8 (tR = 15 min) and 7 mg (9%) of compound 9 (tR = 14 min). Fraction eluted with H2O/MeOH 75:25 gave a mixture that was further purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (65:35) as eluent (flow rate 1 mL/min), to give 15 mg (23%) of compound 7 (tR = 7 min). Fraction eluted with H2O/MeOH 7:3 gave a mixture that was further

the magnesium had reacted. The mixture was diluted (up to 1 L) with reagent grade methanol, refluxed for 2−3 h, then distilled under nitrogen. All reactions were carried out under argon atmosphere using flame-dried glassware. The purity of tested compounds was determined to be always greater than 95% by analytical HPLC analysis using a Nucleodur 100− 5 C18 (5 μm; 4.6 mm i.d. × 250 mm) column eluting with the solvent system (flow rate 1 mL/min) reported below in the section corresponding to each individual compound. 3α,7β-Diformyloxy-5β-cholan-24-oic Acid (21). A solution of ursodeoxycholic acid 2 (550 mg, 1.3 mmol) in 10 mL of 90% formic acid containing 25 μL of 70% perchloric acid was stirred at 47−50 °C for 12 h. The temperature of the heating bath was lowered to 40 °C, then 5 mL of acetic anhydride was added over 10 min and the mixture was stirred for 10 min more. The solution was cooled to room temperature, poured into 50 mL of water, and extracted with diethyl ether. The organic layers were washed with water to neutrality, dried over Na2SO4, and evaporated to give 570 mg of 21 (96%). An analytic sample was obtained by silica gel chromatography eluting with CH2Cl2/MeOH 9:1. Selected 1H NMR (400 MHz CD3OD): δ 8.03 (1H, s), 7.98 (1H, s), 4.89 (1H, m), 4.80 (1H, m), 0.98 (3H, s), 0.93 (3H, d, J = 6.5 Hz), 0.69 (3H, s). 13C NMR (100 MHz CD3OD): δ 178.1, 163.0, 162.5, 74.9, 74.8, 56.6, 56.4, 44.8, 43.5, 41.3, 41.2, 40.7, 36.6, 35.4 (2C), 35.1, 34.1, 34.0, 32.3, 32.0, 29.5, 27.6, 23.6, 22.3, 18.9, 12.5. HRMS-ESI m/z 449.2907 [M + H]+, C26H41O6 requires 449.2903. 3α,7β-Diformyloxy-24-nor-5β-cholan-23-nitrile (22). Crude 21 (500 mg, 1.1 mmol), 1.7 mL of cold trifluoroacetic acid, and 466 μL (3.3 mmol) of trifluoroacetic anhydride were stirred at 0−5 °C until dissolution. Sodium nitrite (83 mg, 1.21 mmol) was added in small portions. After the addition was complete, the reaction mixture was stirred first at 0−5 °C for 1 h, then at 38−40 °C for 8 h. On completion, the reaction was neutralized with NaOH 2 N, then the product was extracted with 50 mL of diethyl ether (3 × 50 mL), followed by washing with brine and dried over anhydrous Na2SO4. The ether was removed under reduced pressure to afford 440 mg of 22 (96%) that was subjected to next step without any purification. An analytic sample was obtained by silica gel chromatography eluting with n-hexane/ethyl acetate 8:2. Selected 1H NMR (400 MHz CD3OD): δ 8.04 (1H, s), 7.99 (1H, s), 4.87 (1H, m), 4.73 (1H, m), 1.12 (3H, d, J = 6.5 Hz), 0.98 (3H, s), 0.71 (3H, s). 13C NMR (100 MHz CD3OD): δ 163.1, 162.6, 120.3, 74.8 (2C), 56.4, 55.5, 44.8, 43.2, 41.2, 40.9, 40.6, 35.4, 35.1, 34.6, 34.1, 34.0, 30.9, 29.3, 27.6, 25.1, 23.6, 22.3, 19.8, 12.5. HRMS-ESI m/z 416.2807 [M + H]+, C25H38NO4 requires 416.2801. 24-nor-Ursodeoxycholic Acid (11). Crude compound 22 (400 mg, 0.96 mmol) was refluxed in ca. 50 mL of methanol/water 1:1 with 30% KOH. After stirring for 48 h, the basic aqueous solution was neutralized with HCl 6 N. Then methanol was evaporated, and the residue was extracted with AcOEt (3 × 50 mL). The combined organic layers were washed with brine, dried, and evaporated to dryness to give white solid residue, which was purified by silica gel chromatography, eluting with CH2Cl2/MeOH 9:1 (355 mg, 97%). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (70:30) as eluent (flow rate 1 mL/min), to give compound 11 (tR = 20.7 min); [α]25D = +11.0 (c 0.41, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.44 (2H, m), 0.98 (3H, d, J = 6.5 Hz), 0.93 (3H, s), 0.71 (3H, s). 13C NMR (100 MHz CD3OD): δ 177.3, 72.1, 71.9, 57.5, 56.6, 44.8, 44.5, 44.0, 42.5, 41.4, 40.7, 38.6, 38.0, 36.1, 35.2, 34.9, 31.0, 29.7, 27.9, 23.9, 22.4, 20.2, 12.7. HRMS-ESI m/z 379.2844 [M + H]+, C23H39O4 requires 379.2848. 3α,7β-Dihydroxy-24-nor-5β-cholan-23-oyl Taurine Sodium Salt (12). Carboxylic acid 11 (20 mg, 0.05 mmol) in DMF dry (2 mL) was treated with DMT-MM (41 mg, 0.15 mmol) and triethylamine (174 μL, 1.25 mmol), and the mixture was stirred at room temperature for 10 min. Then to the mixture was added taurine (37 mg, 0.3 mmol). After 1 h, the reaction mixture was concentrated under vacuo and dissolved in water (5 mL). The solution was poured over a C18 silica gel column. Fraction eluted with H2O/MeOH 99:1 gave a mixture that was further purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 7696

dx.doi.org/10.1021/jm500889f | J. Med. Chem. 2014, 57, 7687−7701

Journal of Medicinal Chemistry

Article

Methyl 3α,7β-Di(tert-butyldimethylsilyloxy)-25,26-bis-homo-5βchol-24-en-26-oate (24). DMSO (2 mL, 28 mmol) was added dropwise for 15 min to a solution of oxalyl chloride (7 mL, 14 mmol) in dry dichloromethane (50 mL) at −78 °C under argon atmosphere. After 30 min, a solution of alcohol 23 (1.2 g, 2 mmol) in dry CH2Cl2 was added via cannula and the mixture was stirred at −78 °C for 30 min. Et3N (2.8 mL, 20 mmol) was added dropwise. After 1 h, methyl(triphenylphosphoranylidene)acetate (2.1 g, 3 mmol) was added and the mixture was allowed to warm to room temperature. NaCl saturated solution was added, and the aqueous phase was extracted with diethyl ether (3 × 100 mL). The combined organic phases were washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (hexane/ethyl acetate 95:5 and 0.5% TEA) gave compound 24 as a colorless oil (1 g, 76%); [α]25D = +45.0 (c 0.70, CH3OH). 1H NMR (400 MHz CDCl3): δ 6.94 (1H, dt, J = 7.3, 15.5 Hz), 5.78 (1H, d, J = 15.5 Hz), 3.66 (3H, s), 3.64 (2H, m), 0.92 (3H, d, J = 6.5 Hz), 0.90 (3H, s), 0.86 (18H, s), 0.62 (3H, s), 0.04 (6H, s), 0.03 (6H, s). 13C NMR (100 MHz CDCl3): δ 167.0, 150.0, 120.9, 72.7, 72.5, 55.5, 54.9, 51.3, 43.9, 43.7, 42.8, 39.9, 38.8, 37.9, 37.8, 35.3 (2C), 35.1, 34.3, 34.1, 30.9 (2C), 30.8, 28.9, 27.3, 26.4 (3C), 25.9 (3C), 23.5, 21.2, 18.6, 12.1, −2.8, −3.4, −4.6 (2C). HRMS-ESI m/z 661.5050 [M + H]+, C39H73O4Si2 requires 661.5047. Methyl 3α,7β-Dihydroxy-25,26-bis-homo-5β-cholan-26-oate (15). A solution of compound 24 (500 mg, 0.76 mmol) in THF dry/EtOH dry (5 mL/5 mL, v/v) was hydrogenated in the presence of Pd(OH)2 5 wt % on activated carbon Degussa type (5 mg). The flask was evacuated and flushed first with argon and then with hydrogen. After 12 h, the reaction was complete. The catalyst was filtered through Celite, and the recovered filtrate was concentrated under vacuum to give the methyl ester, which was dissolved in methanol (40 mL). At the solution was added 1 mL of HCl 37% v/v. After 1 h, silver carbonate was added to the solution to precipitate chloride. Then the reaction mixture was centrifuged, and the supernatant was concentrated in vacuo to give compound 15 as colorless amorphous solids (330 mg, quantitative yield). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (92:8) as eluent (flow rate 1 mL/min) to give compound 15 (tR = 13 min); [α]25D = +41.8 (c 0.15, CH3OH). Selected 1H NMR (400 MHz CDCl3): δ 3.67 (3H, s), 3.59 (2H, m), 2.30 (2H, dt, J = 2.5, 7.5 Hz), 0.95 (3H, s), 0.91 (3H, d, J = 6.5 Hz), 0.67 (3H, s). 13C NMR (100 MHz CDCl3): 167.1, 71.3 (2C), 55.7, 55.1, 51.4, 43.8, 43.7, 42.4, 40.1, 39.2, 37.3, 36.8, 35.5 (2C), 34.9, 34.2, 34.1, 30.4, 28.7, 26.9, 25.7, 25.4, 23.4, 21.2, 18.7, 12.1. HRMS-ESI m/z 435.3479 [M + H]+, C27H47O4 requires 435.3474. 3α,7β-Dihydroxy-25,26-bis-homo-5β-cholan-26-oic Acid (16). A portion of compound 15 (200 mg, 0.46 mmol) was hydrolyzed with NaOH (184 mg, 4.6 mmol) in a solution of MeOH/H2O 1:1 v/v (20 mL). The mixture was stirred for 4 h at reflux. The resulting solution was then acidified with HCl 6 N and extracted with ethyl acetate (3 × 50 mL). The collected organic phases were washed with brine, dried over Na2SO4 anhydrous, and evaporated under reduced pressure to give compound 16 (115 mg, 60%). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (95:5) as eluent (flow rate 1 mL/min), to give compound 16 (tR = 5 min); [α]25D = −9.0 (c 0.04, CH3OH). Selected 1 H NMR (500 MHz CD3OD): δ 3.47 (2H, m), 2.27 (2H, dt, J = 3.4, 7.4 Hz), 0.96 (3H, s), 0.94 (3H, d, J = 6.5 Hz), 0.70 (3H, s). 13C NMR (125 MHz CD3OD): δ 178.2, 72.1, 71.9, 57.6, 56.7, 44.8, 44.5, 44.0, 41.6, 40.7, 38.6, 38.0, 36.9, 36.8, 36.1, 35.3, 35.2, 30.9, 29.8, 27.9, 26.7, 26.6, 23.9, 22.4, 19.3, 12.7. HRMS-ESI m/z 421.3315 [M + H]+, C26H45O4 requires 421.3318. 3α,7β-Dihydroxy-25,26-bis-homo-5β-cholan-26-oyl Taurine Sodium Salt (17). Carboxylic acid 16 (20 mg, 0.047 mmol) in DMF dry (5 mL) was treated with DMT-MM (39 mg, 0.14 mmol) and triethylamine (164 μL, 1.18 mmol), and the mixture was stirred at room temperature for 10 min. Then to the mixture was added taurine (35 mg, 0.28 mmol). After 3 h, the reaction mixture was concentrated under vacuo and dissolved in water (5 mL). The solution was poured over a C18 silica gel column. Fraction eluted with H2O/MeOH 95:5 gave a mixture that was further purified by HPLC on a Nucleodur

purified as previously to give 5 mg (8%) of compound 5 (tR = 8.4 min) and 4 mg (6%) of compound 6 (tR = 6.4 min). 3α,7β-Dihydroxy-24-nor-5β-cholan-23-yl-23-sodium Sulfate (5). [α]25D = +35.8 (c 0.69, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.04 (2H, m), 3.48 (2H, m), 1.00 (3H, d, J = 6.5 Hz), 0.97 (3H, s), 0.72 (3H, s). HR ESIMS m/z 443.2469 [M − Na]−, C23H39O6S requires 443.2467. 7β,23-Dihydroxy-24-nor-5β-cholan-3α-yl-3-sodium Sulfate (6). [α]25D = +11.4 (c 0.04, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.23 (1H, m), 3.48 (3H, m), 0.98 (3H, d, J = 6.4 Hz), 0.97 (3H, s), 0.72 (3H, s). HR ESIMS m/z 443.2462 [M − Na]−, C23H39O6S requires 443.2467. 3α,23-Dihydroxy-24-nor-5β-cholan-7β-yl-7-sodium Sulfate (7). [α]25D = +7.8 (c 0.19, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.29 (1H, m), 3.61 (1H, m), 3.51 (2H, m), 0.97 (3H, s), 0.96 (3H, d, ovl), 0.70 (3H, s). HR ESIMS m/z 443.2465 [M − Na]−, C23H39O6S requires 443.2467. 7β-Hydroxy-24-nor-5β-cholan-3α,23-diyl-3,23-sodium Disulfate (8). [α]25D = +0.3 (c 0.23, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.23 (1H, m), 4.04 (2H, m), 3.48 (1H, m), 1.00 (3H, d, J = 6.6 Hz), 0.97 (3H, s), 0.72 (3H, s). HR ESIMS m/z 545.1859 [M − Na]−, C23H38NaO9S2 requires 545.1855. 3α-Hydroxy-24-nor-5β-cholan-7β,23-diyl-7,23-sodium Disulfate (9). [α]25D = +0.5 (c 0.15, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.29 (1H, m), 4.05 (2H, m), 3.50 (1H, m), 0.99 (3H, d, J = 6.5 Hz), 0.97 (3H, s), 0.71 (3H, s). HR ESIMS m/z 545.1857 [M − Na]−, C23H38NaO9S2 requires 545.1855. 24-nor-5β-Cholan-3α,7β,23-tryl-3,7,23-sodium Trisulfate (10). [α]25D = +13.3 (c 0.03, CH3OH). 1H NMR (400 MHz CD3OD): δ 4.31 (1H, m), 4.23 (1H, m), 4.04 (2H, m), 1.00 (3H, d, J = 6.4 Hz), 0.98 (3H, s), 0.71 (3H, s). HR ESIMS m/z 647.1247 [M − Na], C23H37Na2O12S3 requires 647.1243. 3α,7β-Dihydroxy-5β-cholan-24-ol (3). At a solution of methyl 3α,7β-dihydroxy-5β-cholan-24-oate (50 mg, 0.12 mmol) in THF dry (10 mL) were added at 0 °C dry methanol (51 μL, 1.255 mmol) and LiBH4 (300 μL, 2 M in THF, 1.25 mmol). After 1 h, the mixture was quenched by addition of NaOH (1 M, 240 μL) and then allowed to warm to room temperature. Ethyl acetate was added, and the separated aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (CH2Cl2/methanol 9:1) gave compound 3 as colorless oil (42 mg, 93%). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (85:15) as eluent (flow rate 1 mL/ min), to give compound 3 (tR = 5.8 min); [α]25D = +54.7 (c 0.36, CHCl3). Selected 1H NMR (400 MHz CD3OD): δ 3.51 (4H, m), 0.97 (3H, d, ovl), 0.97 (3H, s), 0.72 (3H, s). 13C NMR (100 MHz CD3OD): δ 72.1, 71.9, 63.6, 57.6, 56.8, 44.8, 44.5, 44.0, 41.6, 40.7, 38.6, 38.0, 37.0, 36.1, 35.2, 33.3, 31.0, 30.3, 29.7, 27.9, 23.9, 22.4, 19.4, 12.6. HRMS-ESI m/z 379.3215 [M + H]+, C24H43O3 requires 379.3212. 3α,7β-Di(tert-butyldimethylsilyloxy)-5β-cholan-24-ol (23). 2,6Lutidine (2.8 mL, 20 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (1.7 mL, 6 mmol) were added at 0 °C to a solution of methyl 3α,7β-dihydroxy-5β-cholan-24-oate (800 mg, 2 mmol) in 30 mL of CH2Cl2. After 2 h stirring at 0 °C, the reaction was quenched by addition of aqueous NaHSO4 (1 M, 100 mL). The layers were separated, and the aqueous phase was extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with NaHSO4, water, saturated aqueous NaHCO3, and brine and evaporated in vacuo to give 1.3 g of methyl 3α,7β-di(tert-butyldimethylsilyloxy)-5β-cholan-24oate in the form of colorless needles that was subjected to next step without any purification. To a solution of methyl ester (1 g, 1.6 mmol) in dry THF (30 mL), at 0 °C dry methanol (453 μL, 11.2 mmol) and LiBH4 (5.6 mL, 2 M in THF, 11.2 mmol) were added. The resulting mixture was stirred for 2 h at 0 °C. The mixture was quenched by addition of 1 M NaOH (3.2 mL) and then ethyl acetate. The organic phase was washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (hexane/ethyl acetate 99:1 and 0.5% TEA) gave 23 as a white solid (1.2 g, quantitative yield over two steps). 7697

dx.doi.org/10.1021/jm500889f | J. Med. Chem. 2014, 57, 7687−7701

Journal of Medicinal Chemistry

Article

100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (45:55) as eluent (flow rate 1 mL/min) to give 12 mg (45%) of compound 17 (tR = 12.4 min); [α]25D = +108.3 (c 0.06, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.59 (2H, t, J = 6.5 Hz), 3.47 (2H, m), 2.96 (2H, t, J = 6.5 Hz), 2.18 (2H, t, J = 7.4 Hz), 0.96 (3H, s), 0.94 (3H, d, J = 6.2 Hz), 0.71 (3H, s). HRMS-ESI m/z 526.3200 [M − Na]−, C28H48NO6S requires 526.3202. 3α,7β-Dihydroxy-25, 26-bis-homo-5β-cholan-26-ol (14). At a solution of compound 15 (50 mg, 0.11 mmol) in THF dry (10 mL) were added at 0 °C dry methanol (31 μL, 0.77 mmol) and LiBH4 (382 μL, 2 M in THF, 0.77 mmol). After 1 h, the mixture was quenched by addition of NaOH (1 M, 220 μL) and then allowed to warm to room temperature. Ethyl acetate was added, and the separated aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (CH2Cl2/methanol 9:1) gave compound 14 as a colorless oil (35 mg, 77%). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (85:15) as eluent (flow rate 1 mL/ min), to give compound 14 (tR = 9 min); [α]25D = +41.8 (c 0.21, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.53 (2H, t, J = 6.5 Hz), 3.48 (2H, m), 0.95 (3H, s), 0.94 (3H, d, ovl), 0.70 (3H, s). 13 C NMR (100 MHz CD3OD): δ 72.1, 71.9, 63.0, 57.5, 56.7, 44.7, 44.4, 44.0, 41.6, 40.7, 38.5, 37.9, 37.2, 37.0, 36.1, 35.2, 33.7, 30.9, 29.8, 27.9, 27.4, 27.1, 23.9, 22.4, 19.4, 12.7. HRMS-ESI m/z 407.3520 [M + H]+, C26H47O3 requires 407.3525. Methyl 3α,7β-Dihydroxy-25,26-bis-homo-5β-chol-24-en-26-oate (18). To the compound 24 (180 mg, 0.27 mmol) dissolved in methanol (30 mL) was added 1 mL of HCl 37% v/v, and the mixture was stirred for 2 h at room temperature. At the end of reaction, silver carbonate was added to precipitate chloride. Then the reaction mixture was centrifuged and the supernatant was concentrated in vacuo to give 120 mg (quantitative yield) of the desired compound 18 as colorless amorphous solids. An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/ H2O (92:8) as eluent (flow rate 1 mL/min), to give compound 18 (tR = 14 min); [α]25D = +45.0 (c 0.70, CH3OH). 1H NMR (400 MHz CDCl3): δ 6.93 (1H, dt, J = 6.6, 15.7 Hz), 5.79 (1H, d, J = 15.7 Hz), 3.64 (3H, s), 3.58 (2H, m), 0.92 (3H, s), 0.91 (3H, d, J = 6.4 Hz), 0.65 (3H, s). 13C NMR (100 MHz CDCl3): δ 167.2, 150.1, 120.4, 71.1, 71.0, 55.9, 55.1, 51.4, 43.7, 43.6, 42.5, 40.2, 39.3, 37.3, 37.1, 35.4, 34.9, 34.3, 34.0, 30.2, 29.0, 28.7, 26.9, 23.4, 21.2, 18.5, 12.2. HRMS-ESI m/z 433.3323 [M + H]+, C27H45O4 requires 433.3318. 3α,7β-Dihydroxy-25,26-bis-homo-5β-chol-24-en-26-oic Acid (19). A portion of compound 18 (100 mg, 0.23 mmol) was hydrolyzed with NaOH (93 mg, 2.31 mmol) in a solution of MeOH/H2O 1:1 v/v (20 mL). The mixture was stirred for 1 h at reflux. The resulting solution was then acidified with HCl 6 N and extracted with ethyl acetate (3 × 50 mL). The collected organic phases were washed with brine, dried over Na2SO4 anhydrous, and evaporated under reduced pressure to give compound 19 (89 mg, 93% yield). An analytic sample was purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (95:5) as eluent (flow rate 1 mL/min) to give compound 19 (tR = 5 min); [α]25D = +203.0 (c 0.39, CH3OH). Selected 1H NMR (400 MHz CDCl3): δ 7.07 (1H, dt, J = 7.0, 15.0 Hz), 5.81 (1H, d, J = 15.0 Hz), 3.60 (2H, m), 0.94 (3H, s), 0.94 (3H, d, ovl), 0.67 (3H, s). 13C NMR (100 MHz CD3OD): δ 170.7, 151.6, 122.7, 72.1, 71.9, 57.5, 56.6, 44.8, 44.5, 44.0, 41.6, 40.7, 38.6, 38.0, 36.8, 36.1, 35.6, 35.2, 31.0, 29.9, 29.7, 27.9, 23.9, 22.4, 19.1, 12.7. HRMS-ESI m/z 419.3163 [M + H]+, C26H43O4 requires 419.3161. 3α,7β-Dihydroxy-25,26-bis-homo-5β-chol-24-en-26-oyl Taurine Sodium Salt (20). Carboxylic acid 19 (10 mg, 0.024 mmol) in DMF dry (2 mL) was treated with DMT-MM (20 mg, 0.075 mmol) and triethylamine (87 μL, 0.63 mmol), and the mixture was stirred at room temperature for 10 min. Then to the mixture was added taurine (18 mg, 0.14 mmol). After 2 h, the reaction mixture was concentrated under vacuo and dissolved in water (5 mL). The solution was poured over a C18 silica gel column. Fraction eluted with H2O/MeOH 95:5 gave a mixture that was further purified by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (45:55)

as eluent (flow rate 1 mL/min), to give 3.5 mg (26%) of compound 20 (tR = 15.8 min); [α]25D = +52.1 (c 0.08, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 6.73 (1H, dt, J = 6.5, 14.0 Hz), 5.89 (1H, d, J = 14.0 Hz), 3.65 (2H, t, J = 7.0 Hz), 3.47 (2H, m), 2.98 (2H, t, J = 7.0 Hz), 0.97 (3H, d, ovl), 0.96 (3H, s), 0.71 (3H, s). HRMS-ESI m/z 524.3043 [M − Na]−, C28H46NO6S requires 524.3046. Ethyl 25,26-Bis-homo-3α,7α-dihydroxy-5β-cholan-26-oate (25). Compound 25 (67 mg, 60% over five steps) was synthesized, starting from methyl 3α,7α-dihydroxy-5β-cholan-24-oate (100 mg, 0.25 mmol), as previously reported;22 [α]25D = +6.72 (c 0.25, CHCl3). Selected 1H NMR (400 MHz CDCl3): δ 4.14 (2H, q, J = 7.0 Hz), 3.80 (1H, br s), 3.38 (1H, m), 2.29 (2H, t, J = 6.7 Hz), 1.24 (3H, t, J = 7.0 Hz), 0.94 (3H, d, J = 6.6 Hz), 0.92 (3H, s), 0.68 (3H, s). 13C NMR (100 MHz CDCl3): δ 175.4, 72.8, 69.0, 61.4, 57.4, 51.5, 43.6, 43.1, 41.0, 40.7, 40.3, 37.0, 36.7, 36.5, 35.8, 35.2, 34.8, 34.0, 31.3, 29.3, 26.6, 26.5, 24.6, 23.5, 21.8, 19.3, 14.6, 12.3. HRMS-ESI m/z 449.3635 [M + H]+, C28H49O4 requires 449.3631. 25,26-Bis-homo-3α,7α-dihydroxy-5β-cholan-26-oic Acid (26). A portion of compound 25 (15 mg, 0.033 mmol) was hydrolyzed with NaOH (13.2 mg, 0.33 mmol) in a solution of MeOH/H2O 1:1 v/v (10 mL). The mixture was stirred for 2 h at reflux. The resulting solution was then acidified with HCl 6 N and extracted with ethyl acetate (3 × 50 mL). The collected organic phases were washed with brine, dried over Na2SO4 anhydrous, and evaporated under reduced pressure to give compound 26 (10.8 mg, 78%); [α]25D = +6.6 (c 0.38, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.79 (1H, br s), 3.40 (1H, m), 2.25 (2H, t, J = 7.5 Hz), 0.94 (3H, d, J = 6.7 Hz), 0.92 (3H, s), 0.68(3H, s). 13C NMR (100 MHz CD3OD): δ 179.6, 72.8, 68.9, 57.5, 51.5, 43.6, 43.1, 41.0, 40.7, 40.4, 37.0, 36.8, 36.6, 36.2, 35.9 (2C), 34.0, 31.3, 29.4, 26.8 (2C), 24.6, 23.5, 21.8, 19.3, 12.3. HRMSESI m/z 421.3315 [M + H]+, C26H45O4 requires 421.3318. 3α,7α-Dihydroxy-25,26-bis-homo-5β-cholan-26-ol (27). Methyl ester 25 (20 mg, 0.044 mmol) was reduced with LiBH4 (2 M in THF dry, 156 μL, 0.31 mmol) and MeOH dry (12 μL, 0.31 mmol) in THF dry at 0 °C for 5 h. The mixture was quenched by addition of 1 M NaOH solution (100 μL), and then ethyl acetate was added. The separated aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, dried (Na2SO4), and concentrated. Purification by silica gel (CH2Cl2/ MeOH 9:1) gave compound 27 as a white solid (13 mg, 73%); [α]25D = +15.0 (c 0.32, CH3OH). Selected 1H NMR (400 MHz CD3OD): δ 3.82 (1H, br s), 3.61 (2H, t, J = 6.6 Hz), 3.43 (1H, m), 0.89 (3H, d, J = 6.0 Hz), 0.88 (3H, s), 0.64 (3H, s). 13C NMR (100 MHz CDCl3): δ 71.8, 68.4, 62.8, 56.0, 50.4, 42.5, 41.4, 39.6 (2C), 39.4, 35.8, 35.6, 35.2, 35.0, 34.5, 32.8 (2C), 30.5, 28.2, 26.1, 25.8, 23.6, 22.7, 20.5, 18.6, 11.6. HRMS-ESI m/z 407.3523 [M + H]+, C26H47O4 requires 407.3525. Animal Studies. All animal experimental procedures were approved by the Ethics Committee of the University of Perugia and by the Italian Health Ministry, according to the Italian guideline for care and use of laboratory animals. The ID for this project is no. 245/ 2013-B. The authorization was released to Prof. Stefano Fiorucci, as a principal investigator, on October 10, 2013. The 6−8 week old CD1 male mice were obtained from Harlan Laboratories Srl (San Pietro al Natisone, Udine, Italy). Mice were maintained in a temperature controlled facility with a 12 h light/dark cycle and were given free access to food and water. Mice (n = 5, 6) were orally administered for 5 days with 30 mg/kg dose of α-naphthylisothiocyanate (ANIT) in olive oil, a chemical agent that causes specific damage to biliary epithelial cells leading to bile duct injury and cell proliferation, ultimately causing intrahepatic cholestasis.32,33 Animals were administered with UDCA (15 mg/kg) or 16 (16 mg/kg). At the end of the experiment, serum and liver samples were collected. During the experiment, mice were daily weight. In another experimental section, mice (n = 3, 4) were treated 6 (or 24) hours with UDCA (60 mg/kg, os) or with 16 (64 mg/kg, os). At the end of the treatments, mice were sacrificed and liver and ileum samples were collected to perform real-time PCR analysis. Transactivation Assay. For FXR mediated transactivation, HepG2 cells were plated in a 24 well-plate and transfected with 100 ng of pSG5-FXR, 100 ng of pSG5-RXR, 200 ng of the reporter vector 7698

dx.doi.org/10.1021/jm500889f | J. Med. Chem. 2014, 57, 7687−7701

Journal of Medicinal Chemistry

Article

Molecular Docking. The AutoDock4.2 software package36 was used to perform molecular docking calculations in the threedimensional model of hGP-BAR1.22 Ligands tridimensional structures were generated with the Maestro Build Panel.37 For each ligand, an extensive ring conformational sampling was performed with the OPLSAA force field38 and a 2.0 Å rmsd cutoff using MacroModel (version 9.9)39 as implemented in Maestro 9.3.37 All conformers were then refined using LigPrep40 as implemented in Maestro 9.3.37 Protonation states at pH 7.0 were assigned using Epik.41 Protein structure was prepared through the Protein Preparation Wizard through the graphical user interface of Maestro 9.3.37 Water molecules were removed, and hydrogen atoms were added and minimized using the OPLS-2005 force field.38 Ligands and receptor structures were converted to AD4 format files using ADT, and the Gesteiger−Marsili partial charges were then assigned. Grid points of 65 × 80 × 55 with a 0.375 Å spacing were calculated around the ligand binding site of GPBAR1 using AutoGrid4.2. Thus, 100 separate docking calculations were performed for each run. Each docking run consisted of 10 million energy evaluations using the Lamarckian genetic algorithm local search (GALS) method, otherwise, default docking parameters were applied. Docking conformations were clustered on the basis of their rmsd (tolerance = 2.0 Å) and were ranked based on the AutoDock scoring function.36 All the residue labels were taken from the wild-type amino acidic sequence of human GP-BAR1. Docking calculations of UDCA in the FXR-LBD were performed using the previously reported protocol.42 All figures were rendered using PyMOL (http://www.pymol.org). Statistical Analysis. All values are expressed as mean ± SD of n values per group. Comparisons of more than two groups were made with a one-way ANOVA with posthoc Tukey’s test. Comparison of two groups was made using Student’s t test for unpaired data when appropriate. Differences were considered statistically significant if p was