All reactions were performed with reagents of commercial high purity quality without ... The synthesis steps of 1-((2-methylbenzofuran-3-yl)methyl)-3-(thiophen-2-.
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SUPPORTING INFORMATION
Synthesis of S07662. All reactions were performed with reagents of commercial high purity quality without further purification unless otherwise mentioned. Reactions were monitored by thin-layer chromatography using aluminum sheets coated with silica gel 60 F245 (0.24 mm) with suitable visualization. 1H NMR and
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C NMR were recorded on a Bruker Avance AV
500 (Bruker Biospin, Switzerland) spectrometer operating on 500.1 and 125.8 MHz, respectively. CDCl3 was used as a solvent, and tetramethylsilane (TMS) was used as an internal standard. The spectra were processed from the recorded FID files with TOPSPIN 2.1 software. Chemical shifts (d) are reported in parts per million (ppm) downfield from TMS. Following abbreviations are used: s, singlet; br s, broad singlet; d, doublet; t, triplet; dd, doublet of doublets; m, multiplet. Coupling constants are reported in Hz. ESI-MS spectra were acquired using a LCQ quadrupole ion trap mass spectrometer equipped with an electrospray ionization source (Thermo LTQ, San Jose, CA, USA). Elemental analyses for C, H, and N were performed on a ThermoQuest CE Instruments EA1110 CHNS-O elemental analyzer (ThermoQuest, Italy). The analytical HPLC system consisted of a following set up: Agilent 1100 HPLC (Agilent Technologies, Germany), a C18 column (Zorbax Eclipse XDBc18, 4.6 cm x 50 mm, 1.8 µm), isocratic runs of solvent B (acetonitrile) in solvent A (H2O) with 40 : 60 proportion for 5 min with flow rate of 1.5 mL/min. The compound (6) was detected by UV at λ = 242 nm (range 190-400 nm).
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Figure S1. The synthesis steps of 1-((2-methylbenzofuran-3-yl)methyl)-3-(thiophen-2ylmethyl)ure (S07662).
Preparation of 2-methylbenzofuran-3-carbaldehyde oxime (2). 2-methylbenzofuran-3carbaldehyde (1) (1.6 g, 9.98 mmol, in 16 ml ethanol) was added to a 100 ml round bottom flask (RBF), followed by a solution of NH2OH.HCl (2.42 g, 34.93 mmol) and aq. Na2CO3 (1.85 g, 17.46 mmol) in 8 ml of water at RT. The mixture was refluxed for 16 h at 78 oC. The progress of the reaction was monitored by TLC using 20 % ethyl acetate in petroleum ether as a mobile phase. Solvents were concentrated under vacuum and the residue obtained was dissolved in 50 ml ethyl acetate and washed with water (3 x 50 ml) and 50 ml brine. The organic layer was dried over sodium sulphate, filtered and concentrated under vacuum to afford white solid compound (1.1 g, 62.9 %); 1H NMR (CDCl3, 500 MHz): δ 8.24 (s, 1H), 7.86-7.83 (m, 1H), 7.56 (br s , 1H), 7.34-7.33 (dd, J = 5.5 and 2.0 Hz, 1H), 7.22-7.17 (m, 2H), 2.48 (s, 3H).
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Preparation of (2-methylbenzofuran-3-yl) methanamine (3). 2-methylbenzofuran-3carbaldehyde oxime (2) (1.1 g, 6.27 mmol) in methanol (8.25 ml) was placed into a 250 ml two-necked RBF and sodium cyanoborohydride (3.94 g, 62.79 mmol) and ammonium acetate (5.32 g, 69.07 mmol) were added at 0-5 oC. A solution of TiCl3 (30% wt in 2N HCl, 25 ml) was added dropwise and the mixture was stirred for 1h at RT. The reaction mixture was basified with 5N NaOH (pH < 3) and diluted with 1% NH4OH (250ml). TLC checked using 40 % ethyl acetate in petroleum ether as a mobile phase. Solvents were concentrated under vacuum and the residue obtained was dissolved in 250 ml ethyl acetate and washed with water (3 x 250 ml) and 100 ml brine. The organic layer was dried over sodium sulphate, 1 filtered and concentrated under vacuum to afford light yellow solid (1.0 g, yield: 99%); H
NMR (CDCl3, 500 MHz): δ 7.47-7.45 (m, 1H), 7.36-7.32 (m, 1H), 7.22 (t, J = 4.5 Hz, 1H), 7.19-7.16 (m, 1H), 4.19 (br s , 2H), 3.90 (t, 2H), 2.43 (s, 3H).
Preparation of 1-((2-methylbenzofuran-3-yl) methyl)-3-(thiophen-2-ylmethyl) urea (6 = S07662). Thiophen-2-ylmethanamine (4) (500 mg, 4.41 mmol) in THF (3.5 ml) was placed into a 100 ml RBF and carbonyldiimidazole (750 mg, 4.63 mmol) was added. The mixture was stirred for 1 h at 78 oC. A solution of (2-methylbenzofuran-3-yl) methanamine (3) (640 mg) in THF was added to the mixture at RT and refluxed for 1 h at 78 oC. The progress of the reaction was monitored by TLC using 40 % ethyl acetate in petroleum ether as a mobile phase. Solvent was concentrated under vacuum to afford off-white solid which was stirred in ethyl acetate: methanol (9 : 1) to afford 700 mg. The 700 mg crude compound was purified by HPLC (Shimadzu LC-10Avp) using reversed phase column (Kromasil 100 C8 5 µm, 150 x 20 mm) and so lvent system of MeOH : H2O (0.001% acetic acid) with 50 : 50 proportion used as an eluent with a flow rate of 20 mL/min. The compound (6) was detected by UV at λ
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= 254 nm. The product was extracted from the collected fractions with ethyl acetate and evaporation of solvent afforded white solid compound (350 mg, yield: 26%); 98.82% purity by HPLC (Rf = 1.67); 1H NMR (CDCl3, 500 MHz): δ 7.47 (d, J = 7.75 Hz, 1H), 7.36 (d, J = 7.75 Hz, 1H), 7.20-7.15 (m, 3H), 6.90 (t, J = 4.21 Hz, 2H), 4.61 (br s, 1H), 4.54 (d, J = 5.55 Hz, 2H), 4.51 (br s, 1H), 4.44 (d, J = 5.55 Hz, 2H), 2.42 (s, 3H); 13C NMR (CDCl3): δ 157.4, 153.9, 152.6, 142.1, 128.4, 126.8, 125.4, 124.9, 123.5, 122.5, 118.9, 111.9, 110.6, 39.5, 34.3, 11.9; Anal. Calcd for C16H16N2O2S: C, 63.98; H, 5.37; N, 9.33%. Found: C, 62.62; H, 5.40; N, 8.45%.
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Screening of the Maybridge compounds. 35 compounds purchased from Maybridge were screened for human CAR activity with the mammalian 1-hybrid assay. The normalized reporter activity data for each compound is standardized to the reference compound clotrimazole and vehicle control DMSO reporter activities using the equation below. The results of the screening are presented in Table S1. The average –fold activation by clotrimazole was 3.69 ± 0.41 (mean ± SD) which was similar to our previous results (Küblbeck et al., 2008). This screening assay performed adequately as judged from the values of Z’ (0.41) and signal window (2.69) factors (Iversen et al., 2006).
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Table S1. Screening of the Maybridge compounds.
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Compound
Act. ± SEM
Assignment
BTB01778
262.2 ± 3.8
agonist
BTB07658
27.9 ± 0.9
BTB08903
206.3 ± 57.7
agonist
BTB10119
135.2 ± 47.4
agonist
CD12037
20.9 ± 0.8
HAN00020
-46.8 ± -4.3
inverse agonist
HTS00509
215.9 ± 15.5
agonist
HTS02622
677.6 ± 126.8
agonist
HTS04556
26.2 ± 1.4
HTS06274
35.7 ± 1.9
HTS07948
-72.9 ± -3.4
HTS09666
-2.7 ± -0.7
inverse agonist
Structure
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Compound
Act. ± SEM
Assigment
HTS12762
25.3 ± 1.9
JFD03557
22.2 ± 0.9
KM01623
20.8 ± 3.9
RH00070
89.7 ± 8.6
agonist
RJC02543
122.4 ± 10.3
agonist
S01399
12.2 ± 0.1
S06212
24.6 ± 2.9
S06275
110.5 ± 13.4
agonist
S07662
-108.5 ± -5.3
inverse agonist
S15114
47.4 ± 10.8
S15423
5.6 ± 1.0
SCR00064
7.3 ± 0.4
Structure
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Compound
Act. ± SEM
Assignment
SCR00560
90.0 ± 3.0
agonist
SCR00954
29.0 ± 1.2
SEW01741
64.3 ± 2.1
agonist
SEW04160
64.4 ± 4.1
agonist
SEW04239
31.0 ± 2.6
SEW04440
-14.2 ± -3.0
SP01153
3.6 ± 0.1
SP01155
27.0 ± 2.3
SPB02065
291.6 ± 5.6
agonist
SPB04331
157.0 ± 17.5
agonist
SPB03188
93.2 ± 17.3
agonist
Structure
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Molecular modeling. The calculated root mean square deviations (RMSD) of the alpha carbon atoms of CAR show that the structures were stable during the MD runs (Figure S2). The change seen in the apo structure is caused by the opening of the loop between helices 2 and 3 and has been discussed in the main text (tai jotain). The atomic positional fluctuation (APF) analysis of the CAR backbone atoms show the flexible regions of the LBD in the MDs.
Figure S2. Root mean square deviations (RMSD) of CAR LDB structures during 10 ns MD. Apo (grey), CITCO (green), PK11195 (blue), S07662 (red).
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Figure S3. Atomic positional fluctuation of the CAR LPD backbone atoms during 10 ns MD simulation. Secondary structure elements are indicated below x-axis (numbers indicate helices and β indicates beta-sheet).
REFERENCES Iversen, P.W.; Eastwood, B.J.; Sittampalam, G.S.; Cox, K.L. A comparison of assay performance measures in screening assays: signal window, Z' factor, and assay variability ratio. J. Biomol. Screen. 2006, 11, (3), 247–52 .
