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Chemmedchem 9 10 11 12 Supporting Information 13 14 15 16 17 18 Bicyclic Imidazolium Inhibitors of Gli Transcription Factor 19 20 Activity 21 + + 22 Marisa E

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Chemmedchem 9 10 11 12 Supporting Information 13 14 15 16 17 18 Bicyclic Imidazolium Inhibitors of Gli Transcription Factor 19 20 Activity 21 + + 22 Marisa E 1 2 3 4 5 6 7 8 ChemMedChem 9 10 11 12 Supporting Information 13 14 15 16 17 18 Bicyclic Imidazolium Inhibitors of Gli Transcription Factor 19 20 Activity 21 + + 22 Marisa E. Hom , Alison E. Ondrus , Tomoyo Sakata-Kato, Paul G. Rack, and James K. Chen* 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Wiley VCH Dienstag, 09.06.2020 2012 / 164877 [S. 1050/1050] 1 Table of Contents Supplementary Figures Figure S1 ...................................................................................................................................... 2 Figure S2 ...................................................................................................................................... 3 Figure S3 ...................................................................................................................................... 4 Figure S4 ...................................................................................................................................... 5 Figure S5 ...................................................................................................................................... 6 Synthetic Procedures General methods .......................................................................................................................... 7 Instrumentation ............................................................................................................................. 7 General procedure of characterization data .............................................................................. 7-8 Characterization data Imidazolium 2 .......................................................................................................................... 8, 16 Imidazolium 3 .......................................................................................................................... 9, 17 Imidazolium 5 .......................................................................................................................... 9, 18 Imidazolium 6 ........................................................................................................................ 10, 19 Imidazolium 7 ........................................................................................................................ 10, 20 Imidazolium 8 ........................................................................................................................ 11, 21 Imidazolium 9 ........................................................................................................................ 11, 22 Imidazolium 10 ...................................................................................................................... 12, 23 Indole 13 .......................................................................................................................... 12-13, 24 Pyrazole 14 ...................................................................................................................... 14-15, 25 Biological Assays Cell culture conditions and methods ...................................................................................... 26-29 Reagents .................................................................................................................................... 30 1 SUFU-KO-LIGHT 120 Vismodegib SANT1 100 Cyclopamine 80 GANT61 60 40 20 Luciferase activity (% control) 0 10-4 10-3 10-2 10-1 100 101 102 [Inhibitor] (µM) Figure S1. SMO inhibitors do not inhibit Gli-dependent luciferase activity in SUFU-KO-LIGHT cells. Cells were incubated with the indicated inhibitors for 24 h, and the resulting luciferase reporter activities were measured. Data are the average of three biological replicates ± s.e.m. 2 FLAG-GLI1-expressing NIH-3T3 cells Whole cell DMSO 10 Cyc 10 Cyt Nuc Cyt Nuc Cyt Nuc DMSO Cyclop GLI1 Lamin A Lamin C GAPDH Figure S2. Bicyclic imidazoliums do not alter the subcellular localization of FLAG-GLI1. Western blot analyses of FLAG-GLI1-expressing NIH-3T3 cells treated with either 5 µM 10, 5 µM cyclopamine (Cyc), or DMSO vehicle for 4 h and then lysed and fractionated by centrifugation. Subcellular fractionation was confirmed using lamins and GAPDH as nuclear and cytosolic markers, respectively. A representative blot from three biological replicates is shown, and all samples were analyzed on the same blot. 3 Panel/Cell Line Log10 GI50 GI50 Less sensitive More sensitive Leukemia CCRF-CEM -6.65 HL-60(TB) -7.37 K-562 -6.76 MOLT-4 -6.53 RPMI-8226 < -8.00 SR -6.52 Non-Small Cell Lung Cancer A549/ATCC -6.29 EKVX -6.82 HOP-62 -5.89 HOP-92 -6.73 NCI-H226 -6.11 * NCI-H23 -6.99 NCI-H322M -5.74 NCI-H460 -6.41 * NCI-H522 -6.77 Colon Cancer COLO 205 -6.63 HCC-2998 -6.50 HCT-116 -6.64 HCT-15 -5.36 HT29 -6.55 KM12 -6.54 SW-620 -6.23 CNS Cancer SF-268 -6.37 SF-295 -6.44 SF-539 -6.46 SNB-19 -6.58 SNB-75 -6.49 U251 -6.42 Melanoma LOX IMVI -6.49 MALME-3M -6.80 M14 -6.35 MDA-MB-435 -6.57 SK-MEL-2 -6.63 SK-MEL-28 -6.42 SK-MEL-5 -6.78 UACC-257 -6.50 UACC-62 -6.80 Ovarian Cancer IGROV1 -6.21 OVCAR-3 -6.63 OVCAR-4 -6.94 OVCAR-5 -6.18 OVCAR-8 -6.28 NCI/ADR-RES -4.70 SK-OV-3 -6.43 Renal Cancer 786-0 -5.88 A498 -6.21 ACHN -5.61 CAKI-1 -4.94 RXF 393 -6.06 SN12C -6.54 TK-10 -6.20 UO-31 -5.66 Prostate Cancer PC-3 -6.64 DU-145 -6.35 Breast Cancer MCF7 -6.64 MDA-MB-231/ATCC -6.56 HS 578T -5.97 BT-549 -6.82 T-47D -7.11 MDA-MB-468 < -8.00 Figure S3. Activity profile of imidazolium 5 against the NCI60 cancer cell lines. Compound concentrations required for 50% of maximal inhibition (GI50s) are shown. The H23 and H522 lines (asterisks) express high levels of GLI1. 4 DMSO 1 μM Oligomycin A 5 μM Cyclopamine 100 nM 10 1 μM 10 5 μM 10 Figure S4. Bicyclic imidazoliums can disrupt mitochondrial membrane potential. NIH-3T3 cells were treated with the designated compounds for 24 h and then stained for 30 min with 500 nM tetramethylrhodamine (TMRM), which selectively labels mitochondria with a membrane potential. Scale bar: 100 µm. 5 125 100 75 50 25 OCR (% control) 0 0 5 2.5 10 1.25 [Cyclopamine] (μM) Figure S5. Hh pathway inhibition does not alter mitochondrial respiration. Oxygen consumption rates (OCRs) of NIH-3T3 cells treated with varying doses of cyclopamine, as measured by the Seahorse Mito Stress assay. The SMO antagonist did not significantly inhibit OXPHOS as determined by one-way ANOVA against DMSO. Data are the average of three biological replicates ± s.e.m. 6 General Methods. All reactions were performed in thick-walled pressure vessels under a positive pressure of nitrogen. Stainless steel syringes or cannulae were used to transfer air- and moisture sensitive liquids. Organic solutions were concentrated on Büchi R-200 rotary evaporators at ~20 Torr (house vacuum) at 25–35 °C, then at ~1 Torr (vacuum pump) unless otherwise indicated. Column chromatography was performed using 200−300 mesh silica gel. Commercial reagents and solvents were used as received unless otherwise noted. Compounds 1, 4, 11, and 12 were commercially available; the synthesis of 2, 3, 6, and 8 was contracted to Chao Che (Peking University); and the synthesis of 13 and 14 was contracted to Kemio Solutions (Bangalore, India). Compounds 5, 7, 9, and 10 were synthesized in-house. Instrumentation. Proton nuclear magnetic resonance (1H NMR) spectra were recorded with a Bruker 300, Bruker 500, or Varian 500 INOVA spectrometer. High resolution mass spectra (HRMS) were obtained on a quadrupole time-of-flight (QTOF) mass spectrometer. Chemical shifts are reported in parts per million from internal tetramethylsilane on the δ scale and are referenced from the residual protium in the NMR solvent (CHCl3: δ 7.27 ppm, CD2HOD: δ3.31 ppm, CD2HCN: δ1.94 ppm). General Procedure for the Synthesis of Bicyclic Imidazolium Compounds (D): NH2 Br R2 R1 Br Br A O C HO H R2 R2 MeO N N N neat, 85 °C THF N N Ac2O N N 6 days R1 78 °C, 16 h 120 °C, 5 h R1 R1 n n n n B E F D 7 A mixture of aniline A (1.0 equiv) and azepine B (1.0 equiv) was heated to 85 ºC and stirred under a nitrogen atmosphere for 6 days. The resulting precipitate was collected by vacuum filtration and washed with 3×3 mL Et2O to yield a 7-amino-azepine intermediate E. 2-Bromoketone C (1.0 equiv) was added to a pressure vessel charged with a suspension of the corresponding 7-amino- azepine intermediate E (1.0 equiv) in THF (to 2.0 M) and the vessel was sealed and placed on an oil bath at 78 ºC. The reaction mixture was stirred at 78 ºC for 16 h, then allowed to cool to room temperature and the solution was concentrated under reduced pressure. The crude hydrate F was dissolved in acetic anhydride (to 1.5 M) in a pressure vessel, and the vessel was sealed and maintained on an oil bath at 120 ºC for 5 h. The vessel was then allowed to cool to room temperature and the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (MeOH:CH2Cl2, 1:20) to give bicyclic imidazolium compound D. Characterization data Me Me N Me N Me O Br NaBH4, BF3Et2O Br THF N N EtO N 0 ➝ 23 °C EtO N S1 2 Bicyclic imidazolium compound S1 was prepared according to the General Procedure with A = 4- ethoxyaniline, B = 7-methoxy-3,4,5,6-tetrahydro-2H-azepine, and C = N-(4-(2- bromoacetyl)phenyl)-N-methylpropionamide, and used directly in the next step. To the solution of (S1) (30 mg) in 5 mL of anhydrous THF was added NaBH4 (40 mg) and BF3OEt2 (0.2 mL) at 0 ºC. The reaction mixture was stirred at 0 ºC for 1 h and at room temperature overnight. The reaction was then quenched
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