Electronic Supplementary Material

4,5-Diaryl imidazoles with hydroxamic acid appendages as anti-hepatoma agents

Pietro Di Fazioa, Susanne Lingelbacha, Rainer Schobertb, Bernhard Biersackb

aDepartment of Visceral, Thoracic and Vascular Surgery, PhilippsUniversityMarburg, 35043 Marburg, Germany

bOrganic Chemistry Laboratory, University Bayreuth, 95440 Bayreuth, Germany

TOC

General / S2
Synthesis of compounds 1 and 2 / S3
Real time cell viability of Hep3B cells / S8
1H and 13C NMR spectra of compounds 2 and 3a-c / S9

General

Melting points were recorded using a Gallenkamp apparatus and are uncorrected. IR: Perkin-Elmer Spectrum One FT-IR spectrophotometer equipped with an ATR sampling unit. NMR: Bruker Avance 300 spectrometer; chemical shifts are given in parts per million (δ) downfield from Me4Si as internal standard; coupling constants (J) are given in Hz. MS: Varian MAT 311A (EI). ESI-MS: Waters UPLC-Q-TOF.Microanalyses indicated by the symbols of the elements were within ± 0.2% of the theoretical values for all new compounds. The starting compounds and pure solvents were purchased from the usual sources and were used without further purification. Merck silica gel 60 (230-400 mesh) was used for column chromatography. The synthesis of compounds 3a-c will be reported elsewhere.

Synthesis of compounds 1 and 2

The hydroxamates 1 and 2 were prepared via a van-Leusen three-component reaction of TosMIC reagent 4 (Van Leusen AM et al. Tetrahedron Lett. 1972, 23, 2367)with t-butyl 4-formylbenzoate or t-butyl 4-formylcinnamate and methyl amine to give the respective 1-methyl-4-phenyl-5-p-benzoate or -cinnamate. These were treated with TFA to afford the corresponding carboxylic acids which in turn were reacted in the presence of EDCI with either benzyloxyamine furnishing the benzyl hydroxamates 5aand 5b. Catalytic hydrogenation of 5aor of 5bleft the hydroxamic acids 1 or 2, respectively. Attempts to remove the benzyl group of 5b without affecting the olefin by treatment with TMSBr failed and the bromide 2was isolated after catalytic hydrogenation.

Scheme S1. Reagents and conditions: (i) a) MeNH2, t-BuOH, reflux, 2h, then 4, K2CO3, t-BuOH, reflux, 3h; b) TFA, DCM, rt, 1 h; c) BnO-NH2, Et3N, EDCI, DMAP, DCM, rt, 24 h; (ii) H2, 10% Pd/C, MeOH, rt, 5 h; (iii) TMSBr, THF, rt, 1 h, then H2, 10% Pd/C, MeOH, rt, 5 h.

4.2.1. 1-Methyl-4-phenyl-5-(4’-N-benzoxyaminocarbonylphenyl)-imidazole 5a

A mixture of 4-t-butoxycarbonylbenzaldehyde (97 mg, 0.47 mmol) and 33% MeNH2/ethanol (260 µL, 2.10 mmol) in t-butanol (15 mL) was refluxed for 2 h. After cooling down to room temperature, 4(110 mg, 0.40 mmol) and K2CO3 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanol 95:5) giving the t-butoxycarbonylphenylimidazole intermediate as yellow oil. Yield: 60 mg (0.18 mmol, 45%, with regard to compound 4); Rf = 0.40 (ethyl acetate); IR (ATR): νmax 2978, 1708, 1510, 1368, 1294, 1250, 1116, 774, 722, 696 cm-1; 1H NMR (300 MHz, CDCl3):  1.59 (s, 9H), 4.45 (s, 3H), 7.3-7.5 (m, 9H); 13C NMR (75.5 MHz, CDCl3): 21.0, 32.3, 60.4, 78.2, 126.7, 126.9, 127.4, 128.2, 128.6, 128.7, 130.7, 134.0, 135.5, 138.0, 138.9. The t-butoxycarbonyl-phenylimidazole intermediate (60 mg, 0.18 mmol) was dissolved in CH2Cl2 (3 mL), treated with TFA (3 mL) and stirred at room temperature for 1 h. The solvent was evaporated, the oily residue featuring the deprotected 4-carboxyphenylimidazole intermediate was dried in vacuum and used for the next step without further purification. For this reason the deprotected intermediate was dissolved in dry CH2Cl2 and EDCI (35 mg, 0.18 mmol), DMAP (12 mg, 0.09 mmol), triethyl amine (55 µL, 0.40 mmol) and benzyl hydroxylamine (50 µL, 0.43 mmol) were added. The reaction mixture was stirred at room temperature under argon for 24 h. The solvent was evaporated and the residue was purified by column chromatography (silica gel 60, 5% methanol in ethyl acetate). Yield: 40 mg (0.10 mmol, 56%); Rf = 0.50 (ethyl acetate / methanol, 95:5); IR (ATR) νmax : 2948, 1651, 1504, 1301, 1013, 857, 774, 696 cm-1; 1H NMR (300 MHz, CDCl3):  3.41 (s, 3H), 5.03 (s, 2H), 7.0-7.2 (m, 5H), 7.2-7.7 (m, 9H), 9.75 (s, 1H); 13C NMR (75.5 MHz, CDCl3):  19.5, 28.1, 28.9, 32.1, 32.2, 73.1, 81.3, 126.2, 126.9, 127.8, 127.9, 128.0, 128.9, 129.9, 130.3, 130.5, 131.9, 134.2, 134.5, 137.3, 138.9, 165.2.

4.2.2.-4.2.3 1-Methyl-4-phenyl-5-(4’-benzoxyamincarbonylethenylphenyl)-imidazole 5b

A mixture of 4-formyl-t-butylcinnamate (97 mg, 0.42 mmol) and 33% MeNH2/ethanol (260 µL, 2.10 mmol) in t-butanol (15 mL) was refluxed for 2 h. After cooling down to room temperature, 4(110 mg, 0.40 mmol) and K2CO3 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanol 95:5) giving the t-butylcinnamoylimidazole intermediate as yellow oil. Yield: 90 mg (0.25 mmol, 63%, with regard to compound 4); Rf = 0.76 (ethyl acetate / methanol, 9:1); IR (ATR): νmax 2982, 1706, 1634, 1601, 1512, 1481, 1456, 1443, 1367, 1321, 1285, 1249, 1206, 1143, 1123, 1069, 1041, 1014, 983, 973, 952, 915, 873, 831, 814, 778, 766, 720, 698 cm-1; 1H NMR (300 MHz, CDCl3):  1.53 (s, 9H), 3.50 (s, 3H), 6.40 (d, J = 16.0 Hz, 1H,), 7.1-7.2 (m, 3 H), 7.22 (d, J = 16.0 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.4-7.6 (m, 5H); 13C NMR (75.5 MHz, CDCl3):  28.1, 32.2, 80.6, 121.0, 126.5, 126.7, 128.1, 128.4, 130.9, 132.0, 134.5, 134.7, 137.8, 142.5, 143.5, 166.0. The intermediate was dissolved in CH2Cl2 (3 mL), treated with TFA (2 mL) and stirred at room temperature for 1 h. The solvent was evaporated, the residue was dried in vacuum and used for the next step without further purification. It was dissolved in dry CH2Cl2 and EDCI (126 mg, 0.65 mmol), DMAP (24 mg, 0.18 mmol), triethyl amine (150 µL, 0.71 mmol) and benzyl hydroxylamine (92 µL, 0.78 mmol) were added. After stirring at room temperature for 24 h, the solvent was evaporated and the residue was purified by column chromatography (silica gel 60, ethyl acetate / methanol, 9:1).

Yield: 90 mg (0.25 mmol, 100%); Rf = 0.20 (ethyl acetate); IR (ATR): νmax 3119, 2972, 2944, 1661, 1626, 1603, 1512, 1498, 1454, 1444, 1367, 1331, 1248, 1207, 1195, 1050, 1036, 1028, 979, 953, 907, 831, 774, 729, 696 cm-1; 1H NMR (300 MHz, CDCl3):  3.43 (s, 3H), 4.97 (s, 2H), 7.0-7.2 (m, 5H), 7.3-7.5 (m, 11), 7.68 (d, J = 15.8 Hz, 1H); 13C NMR (75.5 MHz, CDCl3):  32.3, 64.7, 78.1, 117.9, 124.9, 126.4, 126.8, 128.1, 128.5, 129.2, 130.8, 131.6, 134.1, 134.9, 135.5, 137.8, 138.6, 140.4, 164.9; MS (EI) m/z 409 (93) [M+], 350 (12), 303 (92), 287 (100), 91 (26).

4.2.6. 1-Methyl-4-phenyl-5-(4’-N-hydroxyaminocarbonylphenyl)-imidazole 1

Compound 5a (40 mg, 0.10 mmol) was dissolved in methanol (10 mL) and set under argon. 10 % Pd/C (150 mg) was added, the reaction mixture was evacuated, flushed with hydrogen gas and stirred under hydrogen atmosphere (1 atm) for 5 h. After filtration over celite the solvent was evaporated and the residue crystallized from ethanol/n-hexane. Yield: 20 mg (0.068 mmol, 68%); off-white gum; IR (ATR): νmax 3179, 3047, 1637, 1603, 1551, 1509, 1444, 1374, 1320, 1310, 1251, 1195, 1157, 1122, 1072, 1040, 1013, 960, 894, 857, 774, 720, 695 cm-1; 1H NMR (300 MHz, MeOD):  3.58 (s, 3H), 7.1-7.2 (m, 3H), 7.3-7.4 (m, 2H), 7.4-7.5 (m, 2H), 7.79 (s, 1H), 7.8-7.9 (m, 2H); 13C NMR (75.5 MHz, MeOD):  33.0, 128.1, 128.5, 128.9, 129.4, 129.7, 132.1, 132.2, 134.2, 134.9, 135.5, 139.8, 167.5.m/z (EI) 293 (20) [M+], 278 (100). Anal. Calcd for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found: C, 69.48; H, 5.26; N, 14.19.

4.2.7. 1-Methyl-4-phenyl-5-(4’-N-hydroxyaminocarbonylethylphenyl)-imidazole x HBr 2

Compound 5b (40 mg, 0.12 mmol) was dissolved in dry CH2Cl2 (10 mL) and TMSBr (17 µL, 0.13 mmol) was added. The reaction mixture was stirred for 30 min under argon. The reaction was terminated with methanol (20 mL) and the solvent evaporated giving the the hydrobromide salt of 5b, which was redissolved in methanol (10 mL) and set under argon. Then, 10 % Pd/C (150 mg) was added, the reaction mixture was evacuated, flushed with hydrogen gas and stirred under hydrogen atmosphere (1 atm) for 5 h. After filtration over celite the solvent was evaporated and the residue crystallized from ethanol/n-hexane. Yield: 30 mg (0.075 mmol, 60%); off-white gum; IR (ATR): νmax 3341, 3130, 3022, 2865, 1637, 1551, 1520, 1497, 1444, 1405, 1325, 1154, 1107, 1066, 1015, 998, 828, 771, 723, 694 cm-1; 1H NMR (300 MHz, DMSO-d6):  2.32 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H), 3.63 (s, 3H), 7.3-7.4 (m, 9H), 9.11 (s, 1H), 10.42 (s, 1H); 13C NMR (75.5 MHz, DMSO-d6):  32.4, 34.9, 35.3, 125.4, 128.7, 128.9, 130.2, 130.5, 130.7, 131.9, 132.3, 137.0, 144.8, 171.7; MS (ESI) 324.1774 (51), 323.1633 (19), 322.1573 (82), 321.1596 (100) [M+]. Anal. Calcd for C19H20BrN3O2: C, 56.73; H, 5.01; N, 10.45. Found: C, 56.59; H, 4.91; N, 10.29.

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Fig. S1. Real time cell viability of Hep3B cells after treatment with imidazoles.Hep3B cells were cultured on E-plates and simultaneously treated with 0.01-10 µM 1 (A), 2 (C), 3a (E), 3b (B) or 3c (D) for approximately 24 hours. Panobinostat (0.1 μM) was used as a positive control. The cell index was normalized with respect to the moment of treatment with the respective compound. The cell index was determined continuously for additional 80 hours. The results are means ± SD obtained from measurements in triplicate.

Compound 2: 1H NMR spectrum in DMSO-d6

Compound 2: 13C NMR spectrum in DMSO-d6

Compound 3a: 1H NMR spectrum in DMSO-d6

Compound 3a: 13C NMR spectrum in DMSO-d6

Compound 3b: 1H NMR spectrum in DMSO-d6

Compound 3b: 13C NMR spectrum in DMSO-d6

Compound 3c: 1H NMR spectrum in DMSO-d6

Compound 3c: 13C NMR spectrum in DMSO-d6

S1