1 Supplemental data A Supplemental Figure 1: Alignment of potential ERRE1 and 2 in human, mouse and rat PEPCK promoter.
2 A B C Supplemental Figure 2: Molecular structures of 4-T analogs. a-b, GSK5182 (A), 4-T (B) and D4 (C) in ligand binding pocket of ERRγ. GSK5182 improves the specificity for ERRγ over ERα through additional non-covalent interactions with Y326 and N346 32. These structures were taken from the Brookhaven Protein Data Bank, Accession No. 2EWP.
3 30 pfr-luc 25 Fold activity 20 15 10 5 0 Gal4ERRγ - + + + + PGC-1α - - + + + GSK5182(1uM) - - - + - D4(1uM) - - - - + Supplemental Figure 3: GSK5182 decreases PGC-1α-dependent ERRγ transcriptional activity. Experiments was performed in HepG2 cells using transient transfection, and followed by treatment of GSK5182 and D4 for final 24 h. Error bars in show ± s.e.m.
4 Supplemental Methods D4 Synthesis General Techniques: All reactions were performed either in oven-dried glassware or microwave vessel under dry argon atmosphere. Tetrahydrofuran (THF) was dried by distillation from sodium benzophenone immediately prior to use. Dichloromethane (DCM) was dried by distillation from CaH 2. ther solvents and organic reagents were purchased from commercial venders and used without further purification unless otherwise mentioned. Zinc powder, titanium(iv) chloride, imidazole, 2,6-lutidine, dimethylaminopyridine (DMAP), trifluoroacetic anhydride, palladium (II) acetate, 1,3- bis(diphenylphosphino)propane (dppp), tetrabutylammonium fluoride (TBAF) solution (1.0 M in THF), 2-(dimethylamino)ethyl chloride hydrochloride and cesium carbonate were purchased from Sigma-Aldrich (St. Louis, M, USA). 4,4 - Dihydroxybenzophenone, triisopropylsilyl chloride (TIPSCl), triethylamine (TEA) and propiophenone were purchased from TCI (Tokyo chemical industry Co., Ltd.,Japan). The 1 H and 13 C NMR spectra were recorded on a Varian Inova-500 (Varian Assoc., Palo Alto, USA), and chemical shifts were measured in ppm downfield from internal tetramethylsilane (TMS) standard. Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); br s (broad singlet), etc. Coupling constants were reported in Hz. The identity of final compounds was confirmed using mass spectrometry (MS) analysis which was performed with Micromass & Waters Q- TF Ultima Global Mass Spectrometer at the Mass spectrometry facility of the National Center for Inter-university Research Facilities, Seoul National University. Microwave reaction was performed using CEM Discover Benchmate in described condition. Reverse phase HPLC analysis was performed on a VPDS C-18 column (150 x 4.6 mm) at a flow rate of 1.0 ml/min for analysis, and PRC-DS C-18 column (250 x 20 mm) at a flow rate of 10.0 ml/min for preparation, Shimadzu LC-6AD pump,
5 SPD-10A detector (Japan). HPLC solvents consist of water containing 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B). Preparation of 4,4'-(2-phenylbut-1-ene-1,1-diyl)diphenol (1): H H 1 Compound 1 was prepared by performing McMurry coupling reaction of 4,4 - dihydroxybenzophenone with propiophenone. To a suspension of zinc powder (4.12 g, 63.0 mmol) in THF (30 ml) stirred at 0, added titanium(iv) chloride (3.08 ml, 28.01 mmol) in dropwise over 15-min period, and the reaction mixture was refluxed for 3 h. 4,4 -Dihydroxybenzophenone (1 g, 4.57 mmol) and propiophenone (1.86 ml, 14.0 mmol) in anhydrous THF (10 ml) were added to the previously prepared solution of TiCl 4 and Zn at 0. The reaction mixture was refluxed again for 2 h, and then quenched with 10% K 2 C 3 (aq) for 30 min. The resulting dark slurry was filtered by celite-packed glass filter, and the filtrate was treated with brine, and extracted twice with ethyl acetate (EtAc). The combined organic layer was dried over anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo and purified with silica-gel flash column chromatography (1:5 = ethylacetate:hexanes, v/v) to provide 1 (1.4 g). R f = 0.45 (1:2 = EtAc:n-hexanes, v/v); 1 H NMR (500 MHz, CD 3 D) δ 7.20 6.94 (m, 7H), 6.76 (d, J = 8.31 Hz, 2H), 6.65 (d, J = 8.56 Hz, 2H), 6.40 (d, J = 8.56 Hz, 2H), 2.48 (q, J = 7.42 Hz, 2H), 0.90 (t, J = 7.46 Hz, 3H); 13 C NMR (125 MHz, CD 3 D) δ; 157.32, 156.42, 144.39, 141.61, 140.11, 136.67, 136.30, 133.20, 131.73, 131.04, 128.96, 136.99, 115.92, 115.17, 30.01, 14.11.
6 Preparation of (E/Z)-methyl 4-(2-phenyl-1-(4-(triisopropylsilyloxy)phenyl)but-1- enyl)benzoate (2): TIPS H 1 2 Compound 2 was prepared by mono-protection of 1 with TIPSCl followed by the triflation of phenolic hydroxyl group, and subsequent C insertion. To a solution of 1 (1.5 g, 4.74 mmol) and imidazole (484.1 mg, 7.11 mmol) in dichloromethane (DCM, 37 ml) and THF (5 ml) stirred at 0, added a triisopropylsilyl chloride (TIPSCl, 0.913 ml, 4.27 mmol) in dropwise over 1-h period, and the reaction mixture was stirred at room temperature for 6 h. All solvents were removed under reduced pressure. The resulting residue was treated with brine, and extracted twice with ethyl acetate. The combined organic layer was dried over anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo and purified with silica-gel flash column chromatography (1:20 = EtAc:n-hexanes, v/v) to provide a mono-silylated product as an oil (1.13 g). Fraction of resulting product (841 mg, 1.78 mmol), dimethylaminopyridine (DMAP, 43.5 mg, 0.356 mmol), and 2,6-lutidine (0.309 ml, 2.67 mmol) were dissolved in DCM (20 ml) and stirred. After 15 min, trifluoroacetic anhydride (0.449 ml, 2.67 mmol) was added in dropwise at 0. Then, the reaction temperature was warmed up to room temperature and stirred for additional 3 h. The reaction mixture was quenched with brine, and extracted twice with EtAc. The combined organic layer was dried over anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo. The resulting crude product, Pd(Ac) 2, 1,3-bis(diphenylphosphino)propane, and triethylamine (TEA) were placed in the bomb reactor with dimethylformamide (DMF, 10 ml) and Me (5mL). Then, the bomb reactor was filled with C gas at 5 atm and heated up to 70 at oil bath. The
7 resulting mixture was filtered by celite-packed glass filter, and the filtrate was treated with brine, and extracted with EtAc for 3 times. The combined organic layer was dried over anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo and purified with silica-gel flash column chromatography (1:20 ~ 1:8 = EtAc:n-hexanes, v/v) to give 57:43 mixture of (E) and (Z)-2 (631 mg). R f = 0.45 (1:10 = EtAc:n-hexanes, v/v); 1 H NMR (500 MHz, CDCl 3 ) δ 8.02 (d, J = 8.31 Hz, 0.57 2H), 7.67 (d, J = 8.56 Hz, 0.43 2H), 7.32 (d, J = 8.31 Hz, 0.57 2H), 7.19 7.04 (m, 0.43 2H, 5H), 6.94 (d, J = 8.56 Hz, 0.43 2H), 6.87 (d, J = 8.56 Hz, 0.43 2H), 6.68 (d, J = 8.56 Hz, 0.57 2H), 6.54 (d, J = 8.56 Hz, 0.57 2H), 3.93 (s, 0.57 3H), 3.82 (s, 0.43 2H), 2.53 (q, J = 7.50 Hz, 0.43 2H), 2.46 (q, J = 7.50 Hz, 0.57 2H), 1.33 1.23 (m, 0.43 3H), 1.21 1.13 (m, 0.57 3H), 1.12 (d, J = 7.34 Hz, 0.43 18H), 1.02 (d, J = 7.34 Hz, 0.57 18H), 0.97 0.91 (m, 3H). Preparation of (E/Z)-methyl 4-(1-(4-hydroxyphenyl)-2-phenylbut-1-enyl)benzoate (3): TIPS 2 3 Compound 3 was prepared from 2 by simple desilylation. To a solution of 2 (300 mg, 0.583 mmol) in THF (6 ml) stirred at room temperature, added a tetrabutylammonium fluoride (TBAF) solution (1.0 M in THF, 1.17 ml, 1.17 mmol) in dropwise. The reaction mixture was stirred at room temperature for 30 min. All solvents were evaporated under reduced pressure, and the resulting residue was treated with brine, and extracted twice with EtAc. The combined organic layer was dried over anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo and purified with silica-gel flash
8 column chromatography (1:10 = EtAc:n-hexanes, v/v) to give 55:45 mixture of (E) and (Z)-3 (207 mg). R f = 0.1 (1:10 = EtAc:n-hexanes, v/v); 1 H NMR (500 MHz, CDCl 3 ) δ 8.03 (d, J = 8.07 Hz, 0.45 2H), 7.67 (d, J = 8.07 Hz, 0.55 2H), 7.32 (d, J = 8.07 Hz, 0.45 2H), 7.22 7.03 (m, 0.55 2H, 5H), 6.94 (d, J = 8.07 Hz, 0.55 2H), 6.83 (d, J = 8.31 Hz, 0.55 2H), 6.71 (d, J = 8.56 Hz, 0.45 2H), 6.49 (d, J = 8.80 Hz, 0.45 2H), 3.93 (s, 0.45 3H), 3.83 (s, 0.55 2H), 2.52 (q, J = 7.58 Hz, 0.55 2H), 2.44 (q, J = 7.34 Hz, 0.45 2H), 0.97 0.90 (m, 3H); 13 C NMR (125 MHz, CDCl 3 ) δ 167.49, 167.48, 154.85, 153.99, 149.00, 148.71, 143.92, 142.65, 142.11, 141.99, 137.73, 137.56, 135.48, 132.29, 131.05, 131.03, 129.80, 129.75, 128.91, 128.46, 128.19, 128.16, 127.32, 126.69, 126.52, 115.37, 114.68, 52.38, 52.20, 29.32, 29.21, 13.73, 13.72. Preparation of (Z)-methyl 4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenylbut- 1-enyl)benzoate (D4): N 3 D4 Compound D4 was prepared by basic side-chain installation to 3 under given condition as E/Z stereoisomers. The final compound D4 was obtained through the purification using reverse phase HPLC equipped with C18 column as a TFA salt form. To a solution of 3 (100 mg, 0.28 mmol) and Cs 2 C 3 in anhydrous DMF (6 ml) stirred at room temperature, irradiated microwave (50W, 100, 7 min), then added a Me 2 NCH 2 CH 2 Cl HCl (79.8 mg, 0.7 mmol). Then, the resulting mixture was irradiated again with microwave (50W, 100, 30 min). The reaction mixture was diluted with water, and extracted twice with EtAc. The combined organic layer was dried over
9 anhydrous Na 2 S 4 (s). The filtrate was concentrated in vacuo and purified with reverse phase HPLC (4:1 ~ 1:4 = water:acetonitrile, 25 min; all solvents contain 0.1% TFA) to provide D4 (39 mg). R f = 0.25 (1:10 = Me:DCM, v/v); The structure was confirmed by nuclear verhauser effect (ne) analysis using 1 H NMR spectroscopy (data not shown); 1 H NMR (500 MHz, CDCl 3 ) δ; 8.02 (d, J = 7.58 Hz, 2H), 7.30 (d, J = 7.58 Hz, 2H), 7.23 7.04 (m, 5H), 6.78 (d, J = 7.58 Hz, 2H), 6.53 (d, J = 7.58 Hz, 2H), 4.22 (br s, 2H), 3.92 (s, 3H), 3.44 (br s, 2H), 2.91 (br s, 6H), 2.43 (q, J = 7.09 Hz, 2H), 0.93 (t, J = 6.97 Hz, 3H). MS (ESI+) m/z calcd for C 28 H 32 N 3 [M+H] + 430, found m/z 430.