C24-hydroxylated stigmastane derivatives as Liver X Receptor agonists

C24-hydroxylated stigmastane derivatives as Liver X Receptor agonists

Accepted Manuscript Title: C24-hydroxylated stigmastane derivatives as Liver X Receptor agonists Authors: Francisco Fermin Castro Navas, Gianluca Gior...

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Accepted Manuscript Title: C24-hydroxylated stigmastane derivatives as Liver X Receptor agonists Authors: Francisco Fermin Castro Navas, Gianluca Giorgi, Daniela Maggioni, Manuela Pacciarini, Vincenzo Russo, Maura Marinozzi PII: DOI: Reference:

S0009-3084(17)30273-6 https://doi.org/10.1016/j.chemphyslip.2018.01.005 CPL 4631

To appear in:

Chemistry and Physics of Lipids

Received date: Revised date: Accepted date:

11-10-2017 22-12-2017 15-1-2018

Please cite this article as: Navas, Francisco Fermin Castro, Giorgi, Gianluca, Maggioni, Daniela, Pacciarini, Manuela, Russo, Vincenzo, Marinozzi, Maura, C24-hydroxylated stigmastane derivatives as Liver X Receptor agonists.Chemistry and Physics of Lipids https://doi.org/10.1016/j.chemphyslip.2018.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

C24-Hydroxylated Stigmastane Derivatives as Liver X Receptor Agonists Francisco Fermin Castro Navas,a Gianluca Giorgi,b Daniela Maggioni,e Manuela Pacciarini,a

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Vincenzo Russo,c and Maura Marinozzia* [a] Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, Via del Liceo 1,

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06123 Perugia (Italy).

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[b] Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. Moro,

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53100 Siena, (Italy).

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[c] IRCCS, Istituto Scientifico Ospedale San Raffaele, Via Olgettina 58, 20132 Milano (Italy).

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*Corresponding Author: Maura Marinozzi, Dipartimento di Scienze Farmaceutiche, Università

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degli Studi di Perugia, Via del Liceo 1, 06123 Perugia (Italy); Tel. +39 075 5855159; Fax +39 075

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Highlights:

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5855161; e-mail: [email protected]

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The efficient synthesis of four stigmastane derivatives, endowed with a hydroxyl group at C24 position, is described. Thanks to X-ray crystallography the absolute configuration of the newly created chiral centers was definitively assigned for all the four compounds. The ability of the two 24(S)-epimers, 10b and 11b, to interact with LXRs is reported. The gene expression profiling of 10a and 11a demonstrated the capability of both the compounds to induce the expression of four well-known LXR target genes, such as ABCA1, SREBP1c, FASN, and SCD1 in U937 monocytic cell line

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Abstract Phytosterols are stucturally correlated to the endogenous ligands of Liver X Receptor (LXR), a ligand-activated nuclear receptor that has emerged as an attractive drug target due to its ability to integrate metabolic and inflammatory signaling. Natural and semi-synthetic phytosterol derivatives

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characterized by the presence of side-chain oxygenated functions have shown to be able to modulate LXR activity. Here, we describe the efficient synthesis of four stigmastane derivatives,

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endowed with a hydroxyl group at C24 position, namely (24R)- and (24S)-stigmasta-5,28-diene3β,24-ols (also referred to as saringosterols, 10a and 10b) and (24R)- and (24S)-stigmasta-5-ene3β,24-ols (11a and 11b), starting from the readily available stigmasterol. Thanks to X-ray

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crystallography the absolute configuration of the newly created chiral centers was definitively

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assigned for all the four compounds. The subsequent luciferase assays with GAL-4 chimeric

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receptors evidenced the ability of the two 24(S)-epimers, 10b and 11b, to interact with LXRs,

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showing the same degree of affinity as (22R)-hydroxycholesterol (1). With regard to the isoform

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selectivity both the derivatives 10b and 11b showed a preference for LXRβ, up to 4-fold in terms of efficacy for 11b. The gene expression profiling of (24S)-stigmasta-5,28-diene-3β,24-ol (10a) and

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(24S)-stigmasta-5-ene-3β,24-ol (11a) demonstrated the capability of both the compounds to induce the expression of four well-known LXR target genes, such as ABCA1, SREBP1c, FASN, and SCD1

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in U937 monocytic cell line, thus supporting the hypothesis they were LXR positive modulators.

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Keywords: Liver X Receptor; LXR; Phytosterols; Oxysterols; Natural products; Stigmastane derivatives; Saringosterol.

1. Introduction The beneficial effects of dietary phytosterols on human health, in particular their cholesterollowering properties, are well-known and have been mainly, but not exclusively, ascribed to their 2

ability to reduce intestinal cholesterol absorption. (Racette et al., 2015; Brauner et al., 2012; Plat et al., 2015; Ling & Jones, 1995) However, despite the vast literature devoted to this matter the mechanisms underlying the phytosterols health effects have been so far only marginally understood. (Plat et al., 2015) The α and β liver X receptors (LXR α/β) are members of the nuclear receptor superfamily of ligand-activated transcription factors. They are able to play pivotal roles in the

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mammalian lipid homeostasis by controlling the expression of a set of genes involved in cholesterol, fatty acids and phospholipids metabolism through direct binding to LXR response

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elements (LXREs) in their target promoters. (Song et al., 1994; Shinar et al., 1994; Apfel et al., 1994; Teboul et al., 1995; Willy et al., 1995) The ability of LXRs to stimulate the reverse cholesterol transport through the up-regulation of ABC transporter genes has made them an

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attractive therapeutic target for the treatment of atherosclerosis. However, the concomitant observed

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lipogenic effects has so far prevented the clinical application of LXR agonists in this therapeutic

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area. (Kirchgessner et al., 2016) Being LXRs also key regulators of cholesterol homeostasis and

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inflammatory processes in the central nervous system, they have been recently emerged as potential

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drug target for different neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. (Courtney & Landreth, 2016) Moreover, a number of studies has

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evidenced the ability of LXR activation to inhibit tumorigenesis by modulating metabolism, microenvironment and cell cycle allowing to hypothesize LXR modulation as novel anticancer co-

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adjuvant strategy. (Bovenga et al., 2015). The observation that phytosterols share a close structural similarity with the LXR α/β endogenous

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ligands, namely oxysterols 1-3 (Figure 1), has stimulated investigation into the potential activity of natural and semi-synthetic phytosterols at these rceptors. (Kaneko et al., 2003; Premalatha et al., 2014); The ability of the derivatives 4 and 5 to interact with LXRs and therefore modulate the expression of specific genes involved in the cholesterol metabolism pathways has been thus demonstrated. (Minh-Hien et al., 2012; Plat et al., 2005)

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Figure 1. Main endogenous ligands of LXRs.

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Figure 2. Selected examples of phytosterol derivatives acting as LXRs modulators.

In the frame of a vast research project aimed at the synthesis of phytosterol derivatives bearing oxygenated side-chains, we have recently reported the preparation and the LXR activity of two series of C22 and/or C23-oxygenated stigmasterol and ergosterol derivatives. (Marinozzi et al., 2017) Among them, PFM002 (6), PFM006 (7) and PFM005 (8) were identified as LXRα-selective 4

agonists, whereas PFM018 (9) as a LXRβ-selective ligand. Interestingly, most of the derivatives, in particular PFM018 (9) and PFM009 (10), showed to be LXR target gene-selective modulators, by strongly inducing the expression of ABCA1, while poorly or not activating the lipogenic genes SREBP1 and SCD1, or FASN. (Marinozzi et al., 2017) With the aim to continue the side-chain functionalization we turned our attention toward some

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naturally occurring C24-hydroxystigmastane derivatives, such as 24(S)-stigmast-5-ene-3β,24-diol (11b), isolated from the methanolic extract of Ficus pumila L. fruit, (Kitajima et al., 1998) as well

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as 24(R)- and 24(S)-stigmast-5,28-diene-3β,24-diols (saringosterols, 10a and 10b), identified in different seaweed members of the genus Sargassum (Figure 3). (Ikekawa et al., 1966; Catalan et al., 1983) Although the biological activity of 11b has been so far not yet investigated, Chen et al. have

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recently described 10b as a selective LXRβ agonist, able to induce the expression of LXR target

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genes in multiple cell lines. (Chen et al., 2014)

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The procedures for the isolation and purification of 11b and 10a,b from the respective natural

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sources proved to be tedious and laborious affording only tiny amounts of pure compounds, thus

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constituting a major problem for their complete biological characterization, as well as for their use

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as starting materials for the preparation of analogs.

Figure 3. 24(R)- and 24(S)-saringosterols (10a and 10b), and 24(R)- and 24(S)-stigmast-5-ene3β,24-diols (11a and 11b).

Therefore, by deeming of interest to evaluate the potential activity of 11b and its 24-epimer 11a and to explore the biological profile of 10a,b on our models, we report herein the efficient synthesis, the 5

unambiguous configuration assignment and the preliminary biological evaluation of 10a,b and 11a,b. 2. Results and Discussion 2.1. Chemistry The aldehyde 12, prepared from i-stigmasterol methyl ether by ozonolysis, (Foley et al., 2010) was

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reacted with 3-methyl-1-(triphenylphosphoranylidene)-2-butanone to give the corresponding 22trans-6β-methoxy-3α,5-cyclo-5α-cholest-22-en-24-one, then submitted as a crude product, to the

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subsequent double-bond catalytic hydrogenation step, thus obtaining 6β-methoxy-3α,5-cyclo-5αcholestan-24-one (13) in 60% overall yield (Scheme 1). The reaction of 13 with vinyl magnesium bromide in tetrahydrofuran afforded in good yield the 1:1 mixture of the corresponding tertiary

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alcohols 24(R)-14a and 24(S)-14b, isolated both as pure single diastereoisomers by medium

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pressure chromatography. The final acidic treatment of the single isomers 14a and 14b afforded

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24(R)- and 24(S)-saringosterols (10a and 10b) in 82% and 76% yield, respectively. On the other

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side, the catalytic hydrogenation of the vinyl moiety of 24(R)-14a led to the corresponding (24R)-

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ethyl derivative, finally converted into 24(R)-stigmasta-5-ene-3β,24-ol (11a) in 66% overall yield. An analogous sequence starting from 24(S)-14b gave (24S)-stigmasta-5-ene-3β,24-ol (11b) in 76%

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overall yield.

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Scheme 1. Synthesis of 24(R)-and 24(S)-saringosterols (10a and 10b), and 24(R)-and 24(S)-

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stigmast-5-ene-3β,24-diols (11a and 11b). Reagent and conditions: (a) i. Ph3P=CHC(O)CH(CH3)2,

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toluene, 92 h, reflux; ii. H2, 40 psi, 10% Pd/C, NaHCO3, ethyl acetate/tetrahydrofuran, 30 h, room temperature; 60% yield. (b) 1M CH2=CHMgBr in tetrahydrofuran, diethyl ether, 2 h, reflux; ii.

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medium pressure chromatography, 79% yield. (c) pTosOH·H2O, 75% aqueous dioxane, 75 °C, 1 h;

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(d) H2, 40 psi, 10% Pd/C, NaHCO3, ethyl acetate/tetrahydrofuran, 12 h, room temperature.

2.2. Absolute Configuration Assignment

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Another issue, which was an important concern for us, was the unambiguous assignment of the absolute configuration at C24 for the four compounds prepared. Catalan et al., indeed, had supposed the configuration of 10a and 10b and their reduction derivatives 11a and 11b by correlation with fucosterol and isofucosterol 24(28)-epoxides, whose stereochemistry had been in turn tentatively assigned by a series of chemical transformations. (Catalan et al., 1983; Fujimoto et al., 1980)

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After several attempts we were successful in obtaining from 11b a crystal suitable for X-ray analysis (Figure 4); thus, 11b was unambiguously characterized as 24(S)-stigmasta-5,28-diene3β,24-ol and consequently the same spatial arrangement of the atoms at C24 had to be owned by its corresponding formal starting product 10b. Thus, the less polar saringosterol 10a was endowed with

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24(R)-configuration as well as its corresponding reduced derivative 11a.

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Figure 4. X-ray structure of 11b (ellipsoids enclose 50% probability). The structure crystallizes

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with three independent molecules and a water molecule in the asymmetric unit. Only one molecule

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is reported for clarity.

2.3. LXRs Binding

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The four synthesized compounds were tested for their ability to activate LXRs by using luciferase assays with GAL-4 chimeric receptors, as described in the experimental section. Briefly, we co-

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transfected plasmids encoding hLXRα- and β-binding domains fused to GAL-4, with the respective responsive element conjugated with the luciferase reporter gene into the human embryonic kidney

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293 cells. The results of the assays are listed in Table 1 and showed in Supplementary Figures 1 A and B.

Table 1. LXR Agonist Profile of Compounds 10a,b and 11a,b

LXRα

LXRβ

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22R-HC (1) 10a 10b

Efficacy (%) ± SD

EC50 (μM)a ± SD (95% C.I.)b 3.12 ± 0.78 (1.06 - 8.5) 3.28 ± 0.32 (2.51 - 4.89) 1.69 ± 0.22 (0.66 - 5.13)

100 12.85 ± 4.45 43.44 ± 17.65

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NAd

NAd

NAd

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3.72 ± 0.5 (1.85 - 6.49)

11.04 ± 4.08

3.21 ± 0.6 (0.89 - 8.64)

Efficacy (%)c± SD

100 28.72 ± 7.8 61.04 ± 17.36 NAd

48.37 ± 5.0

Fifty% maximal activation (EC50) ± standard deviations (SD) was determined by dose-response

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4.52 ± 0.08 (2.42 - 8.4) 4.05 ± 0.07 (3.13 - 5.75) 2.92 ± 0.05 (1.03 - 7.07)

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Compd

EC50 (μM)a ± SD (95% C.I.)b

curve of titrating concentrations of compounds 10a,b-11a,b (32, 16, 8, 4, 2 and 1 μM) tested by luciferase assays. The results were mean of four independent experiments; b95% Confidence

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interval; cEfficacy: % of compound effect ± SD versus 8 μM of 22R-HC (1); dNA: not active.

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Among the four compounds the two 24(S)-epimers, 10b and 11b, exhibited low micromolar EC50

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retaining or, in some cases, improving that of the endogenous ligand 22R-HC (1). However, none of

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the tested compounds showed higher LXR efficacy than the reference 1. Among the series, derivative 10b was the best of the series due to its lower EC50 value and higher efficacy with respect

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to 11b. However, when comparing the efficacy for the two isoforms, 11b resulted to be the ligand

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endowed with the highest preference for LXR whereas 10b showed a  efficacy ratio of only 1.4, confirming the data published by Chen et al., who, however, defined this derivative as a

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selective LXRagonist. (Chen et al., 2014) The strength of the stigmastane scaffold as a starting point for obtaining LXRβ modulators, already evidenced by our derivative PFM018 (9), was thus confirmed. (Marinozzi et al., 2017). Furthermore, the inactivity or the scarce efficacy of the 24(R)-

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epimers, 10b and 10a, respectively, corroborated the evidence that the spatial disposition of the oxygenated side-chain moiety is a crucial parameter for the biological activity.

2.4. Gene expression profile

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LXR agonists are able to induce the expression of target genes, which are involved in cholesterol homeostasis, particularly in the reverse cholesterol transport pathway, such as ABCA1. (Hong & Tontonoz, 2014) Furthermore, LXR activation promotes the de novo lipogenesis by inducing the expression of the master regulator of hepatic lipogenesis sterol-regulatory element-binding protein 1C (SREBP-1c), as well as several downstream genes in the SREBP-1c pathway, including steroyl

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CoA desaturase 1 (SCD1) and fatty acid synthase (FASN). (Hong & Tontonoz, 2014) Accordingly, we investigated by quantitative PCR (qPCR) the ability of 10b and 11b to induce the expression of

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these four genes, by using RNA from monocytic U937 cells and 22R-HC (1), as a positive control.

Figure 5. Regulation of ABCA1 (A), SREBP1c (B), FASN (C), and SCD1 (D) genes by the title compounds assessed by qPCR. U937 cells differentiated with PMA for 72 hours were treated with

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22R-HC (1) or with the tested compound (10 μM). The results show mean ± SD of three biological samples. (n = 3/group). ns= not significant, **p < 0.01, ***p < 0.001.

As shown in Figure 5 the two compounds were able to induce ABCA1 expression (Fig. 5A), little less than 1. Similar results were obtained with SREBP1c, though the activation induced by 11b was

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not significant (Fig. 5A). Of note, 10b induced the activation of FASN (Fig. 5C) suggesting a possible lipogenic effect mediated by this compound, while both compounds failed to activate

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SCD1 (Fig. 5D).

3. Conclusions

In this work, we reported an efficient synthetic approach for the preparation of naturally occurring

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24(S)-saringosterol (10b) and 24(S)-stigmast-5-ene-3β,24-ol (11b) and their 24-epimers, 10a and

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11a, respectively. Moreover, for the first time the C-24 absolute configuration of these compounds

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was unambiguously assigned. The evaluation of the activity towards LXRs confirmed the ability of

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24(S) saringosterol (10b) to activate these receptors although with scarce isoform preference; the

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selectivity towards LXR was more pronounced in the case of 11b. The LXR activation by 10b and 11b was supported by gene expression studies, which evidenced a similar profile between both our

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compounds and 22R-HC (1), though 10b was the only inducing the expression of FASN target gene.

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In conclusion, this study reiterated the importance of the stigmastane nucleus as a promising scaffold for the discovery of LXR modulators. Certainly the derivative prepared here in such

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efficient way could be used as starting material for further chemical manipulation.

4. Experimental section 4.1. Chemistry All reactions involving moisture- or air-sensitive reagents were performed in oven-dried glassware under inert atmosphere. Melting points were determined by the capillary method on a Büchi 535 electrothermal apparatus and are uncorrected. 1H- and 13C NMR spectra were taken on Bruker AC 11

400 spectrometers as solutions in CDCl3 unless otherwise indicated. The spin multiplicities are indicated by the symbols s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and bs (broad). Flash chromatography was performed on Merck silica gel (0.040-0.063 mm). Medium pressure chromatography (mpc) was performed on Merck LiChroprep Si 60 Lobar columns. 4.1.1. 6β-Methoxy-3α,5-cyclo-5α-cholestan-24-one (13). 3-Methyl-1-triphenylphosphoranylidene-2-

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butanone (11.8 g, 34.0 mmol) was added to a stirred solution of the aldehyde 12 (3.9 g, 11.3 mmol) in dry toluene (140 mL), kept under argon atmosphere at room temperature. The resulting mixture

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was refluxed for 92 h and then allowed to reach room temperature. The solid in the meantime formed was removed by filtration and the resulting solution was concentrated to give a residue (3.9 g), which was directly submitted to catalytic hydrogenation. After its solubilisation in 80:20 ethyl

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acetate/tetrahydrofuran mixture (40 mL), the resulting solution was hydrogenated at 40 psi in the

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presence of sodium hydrogen carbonate (1.9 g, 22.7 mmol) and 10% Pd/C (0.125 g). After 30 h the

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reaction mixture was filtered through a Celite pad. The solvent was then removed in vacuo to

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furnish a solid, which was purified by flash chromatography. Elution with light petroleum/ethyl

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acetate (80:15) gave 2.85 g (60%) of 13 as a white solid. mp 92.5-94.6 °C; 1H NMR (400 MHz, CDCl3): δ 3.32 (s, 3H), 2.78 (s, 1H), 1.73 (m, 3H), 1.08 (m, 4H), 1.02 (s, 4H), 0.92 (m, 2H), 0.84 (s

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3H); 13C NMR (100 MHz, CDCl3): δ 215.5, 82.3, 56.5, 56.4, 56.0, 47.9, 43.3, 42.7, 40.8, 40.2, 37.2, 35.4, 35.2, 34.9, 33.3, 30.4, 29.8, 28.2, 24.9, 24.1, 22.7, 21.4, 19.2, 18.4, 18.3, 13.0, 12.2.

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4.1.2. 24(R)- and 24(S)-6β-Methoxy-3α,5-cyclo-5α-stigmast-28-ene-24-ols (14a and 14b). 1 M Vinyl magnesium bromide in tetrahydrofuran (7.6 mL, 7.6 mmol) was added to a stirred solution of

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13 (0.4 g, 1.1 mmol) in dry diethyl ether (10 mL), kept under argon atmosphere at room temperature. The resulting mixture was refluxed for 2 h, and then allowed to reach room temperature. After ice cooling, NH4Cl solution (10 mL) was dropwise added. The mixture was extracted with diethyl ether (3 x 15 mL) and the combined organic layers were washed with water (10 mL) and then dried over Na2SO4. Evaporation of the solvent in vacuo furnished a yellow oil which was purified by medium pressure chromatography. Elution with light petroleum/ethyl acetate 12

(85:20) afforded pure samples of the desired compounds in 85% total yield. Compound 14a, colorless oil, 1H NMR (400 MHz, CDCl3): δ 0.33 (m, 1H), 0.55 (m, 1H), 0.61 (s, 3H), 0.92 (s, 3H), 2.67 (s, 1H), 3.23 (s, 3H), 5.04 (d, 1H, J = 10.0 Hz), 5.10 (d, 1H, J = 16.7 Hz), 5.71 (dd, 1H, J = 10.0 and 16.7 Hz); 13C NMR (100 MHz, CDCl3): δ 12.18, 13.00, 16.40, 17.51, 18.71, 19.24, 21.42, 22.70, 24.09, 24.90, 28.21, 28.95, 30.40, 33.27, 34.75, 34.96, 35.19, 35.83, 36.07, 40.17, 42.69,

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43.30, 47.91, 55.93, 56.40, 56.49, 77.64, 82.34, 112.89, 142.39. Compound 14b, colorless oil; 1H NMR (400 MHz, CDCl3): δ 0.30 (m, 1H), 0.53 (m, 1H), 0.91 (s, 3H), 2.66 (s, 1H), 3.21 (s, 3H),

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5.02 (d, 1H, J = 10.9 Hz), 5.08 (d, 1H, J = 17.3 Hz), 5.69 (dd, 1H, J = 10.9 and 17.3 Hz); 13C NMR (100 MHz, CDCl3): δ 12.17, 13.00, 16.41, 17.48, 18.70, 19.23, 21.41, 22.70, 24.09, 24.90, 28.25, 28.98, 30.39, 33.27, 34.53, 34.95, 35.18, 35.88, 36.00, 40.17, 42.69, 43.29, 47.92, 55.89, 56.42,

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56.48, 77.62, 82.33, 112.80, 142.46.

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4.1.3. 24(R)-Stigmasta-5,28-diene-3β,24-ol (10a). p-Toluenesulfonic acid monohydrate (0.0039 g,

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0.021 mmol) was added to a stirred solution of 14a (0.095 g, 0.215 mmol) in 25% aqueous dioxane

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(7.7 mL) and the resulting mixture was heated at 75 °C for 1 h, then allowed to reach room

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temperature. After the addition of water (10 mL), the mixture was extracted with ethyl acetate (3 x 10 mL) and the combined organic layers were washed with brine (10 mL) and dried over Na2SO4.

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The solvent was removed in vacuo and the residue purified by flash chromatography. Elution with light petroleum/ethyl acetate (75:25) afforded 0.060 g (82%) of 10a as a white solid. mp 161-163

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°C; 1H NMR (400 MHz, CDCl3): δ 0.58 (s, 3H), 0.91 (s, 3H), 1.90-2.0 (m, 2H), 2.10-2.25 (m, 2H), 3.42 (m, 1H), 5.04 (d, 1H, J = 10.9 Hz), 5.10 (d, 1H, J = 17.3 Hz), 5.72 (dd, 1H, J = 10.9 and 17.3

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Hz); 13C NMR (100 MHz, CDCl3): δ 11.79, 16.40, 17.50, 18.74, 19.33, 21.013, 24.20, 28.10, 28.99, 31.58, 31.83, 34.71, 35.84, 36.02, 36.43, 37.20, 39.68, 42.24, 50.05, 55.77, 56.67, 71.70, 77.63, 112.88, 121.60, 140.71, 142.40. 4.1.4. 24(S)-Stigmasta-5,28-diene-3β,24-ol (10b). The derivative 14b was treated as reported for 14a to furnish 10b in 76 % yield as a white solid. mp 168-169 °C; 1H NMR (400 MHz, CDCl3): δ da rifare 13C NMR (100 MHz, CDCl3 + CD3OD): δ 11.71, 16.33, 17.40, 18.64, 19.26, 20.93, 24.15, 13

28.09, 28.92, 31.25, 31.75, 34.39, 35.80, 35.88, 36.37, 37.12, 39.61, 41.92, 42.18, 49.97, 55.66, 56.61, 71.39, 77.66, 112.77, 121.50, 140.69, 142.28. 4.1.5. 24(R)-Stigmast-5-ene-3β,24-ol (11a). A solution of 14a (0.102 g, 0.23 mmol) in 80:20 ethyl acetate/tetrahydrofuran (2.3 mL) was hydrogenated at 40 psi in the presence of sodium hydrogen carbonate (0.046 mg, 0.54 mmol) and 10% Pd/C (0.005 mg). After 12 h, the reaction mixture was

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filtered through a Celite pad. The compound (0.099 g) obtained after the removal of the solvent in vacuo was dissolved in 25% aqueous dioxane (7.8 mL), p-toluenesulfonic acid monohydrate (0.003

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g, 0.021 mmol) was added and the resulting mixture was heated at 75 °C for 1 h, then allowed to reach room temperature. After the addition of water (10 mL), the mixture was extracted with ethyl acetate (3 x 10 mL) and the combined organic layers were washed with brine (10 mL) and dried

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over Na2SO4. The solvent was removed in vacuo and the residue purified by flash chromatography.

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Elution with light petroleum/ethyl acetate (75:25) afforded 0.056 g (66%) of 11a as a white solid.

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mp 159-160 °C; 1H NMR (400 MHz, CDCl3 + CD3OD): δ 0.53 (s, 3H), 0.85 (s, 3H), 1.60-1.80 (m,

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4H), 1.80-1.90 (m, 2H), 2.0-2.20 (m, 2H), 3.34 (m, 1H), 5.20 (s, 1H); 13C NMR (400 MHz, CDCl3

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+ CD3OD): δ 7.42, 11.66, 16.59, 16.64, 18.64, 19.22, 20.90, 24.14, 28.00, 28.14, 28.84, 31.14, 31.60, 31.72, 33.70, 36.21, 36.34, 37.10, 39.58, 41.83, 42.17, 55.74, 56.58, 71.27, 76.03, 121.42,

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140.69.

4.1.6. 24(S)-Stigmast-5-ene-3β,24-ol (11b). The derivative 14b was treated as reported for 14a to

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furnish 11b in 76 % yield as white solid. mp 172-174 °C; 1H NMR (400 MHz, CDCl3 + CD3OD): δ 0.64 (s, 3H), 0.96 (s, 3H), 1.70-1.90 (m, 4H), 1.90-2.00 (m, 2H), 2.10-2.30 (m, 2H), 3.45 (m, 1H), 13

C NMR (400 MHz, CDCl3 + CD3OD): δ 7.55, 11.70, 16.50, 16.66, 18.63, 19.25,

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5.31 (s, 1H);

20.92, 24.16, 28.15, 28.14, 28.94, 31.21, 31.52, 31.74, 33.71, 36.28, 36.36, 37.11, 39.61, 41.88, 42.19, 49.96, 55.76, 56.60, 71.33, 76.08, 121.47, 140.70. 4.2. Crystal structure of 11b. A single crystal of 11b was submitted to X-ray data collection on an Rigaku AFC12 FRE-VHF diffractometer with a graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 100 K. The 14

structure was solved by direct methods implemented in SHELXS program (version 2013/1). (Sheldrick, 2008) The refinement was carried out by full-matrix anisotropic least-squares on F2 for all reflections for non-H atoms by means of the SHELXL program (version 2013/4). (Sheldrick, 2008) Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 1548892. Copies of the data can be obtained, free of

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charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; (fax: + 44 (0) 1223 336 033; or e-mail: [email protected]).

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4.3. Cell Culture and Co-transfection Assays.

Human embryonic Kidney 293 cells (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle’s medium containing 10% of fetal bovine serum at 37°C in humidified

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atmosphere of 5% CO2. We transiently transfected HEK293 cells (4x104 cells per well) in 48 well

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plate with the reporter plasmids pMH100X4-TK-luc (100 ng/well), Renilla (22 ng/well) together

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with 100 ng/well of pCMX-Gal4-LXR-α or pCMX-Gal4-LXR-β plasmids using X-tremeGENE 9

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DNA Transfection Reagent (Roche). Six hours after transfection, we treated the cells with the

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appropriate compound for 24 hours. We analyzed luciferase activities by luciferase Dual Reporter Assay Systems (Promega) according to the manufacturer’s protocol. GAL4-LXRs were previously

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described. (Villablanca et al., 2010) 4.4. Quantitative Real-Time-PCR

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U937 cell line was differentiated in foam macrophages with phorbol 12-myristate 13-acetate (PMA) 10 ng/ml (Sigma) for 72 hours at 37°C in 10 mm dish at the concentration of 3x10 6 cells in 10 ml

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RPMI 10% FBS. At day 3 nuclear receptor ligands were added for 6 hours. Total RNA was purified by TRIZOL (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed incubating 2 μg of total RNA 1 hour at 42 °C with MLV-reverse transcriptase (Promega). Quantitative PCR was performed using Sybr Green Master Mix (Applied Biosystems) and real-time PCR (Viia 7 Real Time PCR System, Applied Biosystems). All PCR reactions were done in triplicate. The

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comparative Ct method was used to quantify transcripts that were normalized for human GAPDH.

GAPDH-F

ACA TCA TCC CTG CCT CTA CTG

GAPDH-R

ACC ACC TGG TGC TCA GTG TA

ABCA1-F

CCA GGC CAG TAC GGA ATT C

ABCA1-R

CCT CGC CAA ACC AGT AGG A

SREBP-1c-F

GGC GGG CGC AGA TC

SREBP-1c-R

TTG TTG ATA AGC TGA AGC ATG TCT

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We used the following primer pairs:

ACA GCG GGG AAT GGG TAC T

FAS-R

GAC TGG TAC AAC GAG CGG AT

SCD1-F

TTC AGA AAC ACA TGC TGA TCC TCA TAA TTC

SCD1-R

ATT AAG CAC CAC AGC ATA TCG CAA GAA AGT

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FAS-F

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4.5. Statistical analysis. Data are expressed as mean ± SEM and were analyzed for significance by

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ANOVA with Dunnet’s multiple comparison tests. The analysis was performed with Prism

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software. Data in Table 1 are expressed as EC50 ± SD. In particular, the standard deviations were obtained by calculating the mean of the EC50 of each experiment (three to five independent

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experiments). The efficacy (%) of the compounds was calculated as the percentage of the compound effect, in terms of LXR or  activation, versus 8 M of 22R-HC ± SD. The analyses

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were performed with Prism software.

Acknowledgments

We thank Dr. Mark E. Light, Faculty of Natural & Environmental Sciences, University of

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Southampton (UK) for X-ray data collection.

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