Chemical synthesis of potential inhibitors. Reagents and solvents were from Aldrich, Alfa Aesar or Acros. Reactions were monitored by TLC, which was performed on precoated aluminum-backed plates (Merck, silica 60 F254). Melting points were determined using a Leica Galen III hot-stage melting point apparatus and microscope. Infrared spectra were recorded from Nujol mulls between sodium chloride discs, on a Bruker Tensor 27 FT-IR spectrometer. NMR spectra were acquired using a Bruker DPX500 NMR spectrometer. Chemical shifts (δ) are given in ppm, and the multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br). Coupling constants J are given in Hz (± 0.5 Hz). High resolution mass spectra (HRMS) were recorded using a Bruker MicroTOF spectrometer. The purity of all compounds synthesized were ≥95% as determined by analytical reverse-phase HPLC (Ultimate 3000). Daminozide (Alar™) and compound 28 are commercially available. The synthesis and characterisation of compounds 22 25 , 25 26 (link), 27 27 , 36 28 , 37 29 and 38 26 (link) has been reported. The synthesis of compounds 31-35 , 39-41 and 13C NMR spectra for 22 , 23 , 24 , 26 , 31-35 are given in the Supporting Information .
4-(2,2,2-Trimethylhydrazinyl)-4-oxobutanoate22 . The synthesis of compound 22 was as reported25 , thus reaction of daminozide (500mg, 3.1 mmol) with methyl iodide (700mg, 0.31 mL, 5.0 mmol) gave 22 as a white solid (75% yield), mp: 137-138 °C (lit.1 137-138.5 °C); 1H NMR (500 MHz, MeOD): δ 2.40 (t, J = 6.5 Hz, 2H), 2.51 (t, J = 6.5 Hz, 2H), 3.56 (s, 9H); 13C NMR (125 MHz, MeOD): δ 28.5, 29.1, 56.1, 170.4, 173.4; IR (neat) υ/cm−1: 3405, 3312, 1729, 1693; HRMS (m/z): [M]+ calcd. for C7H15N2O3, 175.1077; found, 175.1081.
General procedure for the coupling of hydrazine to succinic anhydride. To a stirred solution of the appropriate hydrazine (1 equiv.) in acetonitrile (5 mL) was added dropwise a solution of succinic anhydride (200 mg, 2.0 mmol, 1 equiv.) in acetonitrile (5 mL). The mixture was stirred at room temperature for 24 h, after which the solvent was evaporated in vacuo and the resulting crude purified using semipreparative reverse-phase HPLC, performed on a phenomenex C18 column (150 mm × 4.6 mm). Separation was achieved using a linear gradient of solvent A (water + 0.1% CF3CO2H) and solvent B (acetonitrile + 0.1% CF3CO2H), eluting at a flow rate of 1 mL/min and monitoring at 220 nm: 0% B to 40% B over 30 min.
4-(2-Methylhydrazinyl)-4-oxobutanoic acid23 . Compound 23 is a colourless oil (63% yield), 1H NMR (500 MHz, DMSO-d6): δ 2.35 (t, J = 7.0 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.98 (s, 3H), 4.76 (s, 1H), 7.74 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 28.3, 29.1, 170.1, 173.6; IR (neat) υ/cm−1: 33 3219, 3057, 1708, 1632; HRMS (m/z): [M+Na]+ calcd. for C5H10N2NaO3, 169.0584; found, 169.0577.
4-Hydrazinyl-4-oxobutanoic acid24 . Compound 24 is a colourless oil (87% yield), 1H NMR (500 MHz, DMSO-d6): δ 2.34 (t, J = 7.0 Hz, 2H), 2.60 (t, J = 7.0 Hz, 2H), 5.86 (s, 1H), 8.99 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 28.2, 29.1, 170.8, 173.9; IR (neat) υ/cm−1: 3303, 3290, 3199, 1712, 1624; HRMS (m/z): [M-H]- calcd. for C4H7N2O3, 131.0462; found, 131.0468.
4-Oxo-4-(1,2,2-trimethylhydrazinyl)butanoic acid26 . Compound 26 is a white solid (56% yield), mp: 97-98 °C, 1H NMR (500 MHz, DMSO-d6): δ 2.37 (t, J = 7.0 Hz, 2H), 2.66 (t, J = 7.0 Hz, 2H), 2.74 (s, 3H), 11.98 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 28.2, 29.8, 43.4, 48.7, 173.5, 175.0; IR (neat) υ/cm−1: 2958, 1723, 1615; HRMS (m/z): [M+Na]+ calcd. for C7H14N2NaO3, 197.0897; found, 197.0895.
4-((Dimethylamino)oxy)-4-oxobutanoic acid29 . N,N-Dimethylhydroxylamine (39 mg, 0.63 mmol, 1.1 equiv. ) was added to a solution of 4-(tert-butoxy)-4-oxobutanoic acid (100 mg, 0.57 mmol, 1 equiv.), hydroxybenzotriazole (100 mg, 0.74 mmol, 1.3 equiv.), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( 140 mg, 0.74 mmol, 1.3 equiv.) and diisopropylethylamine ( 0.2 mL, 1.14 mmol, 2.0 equiv.) in CH2Cl2 (10 mL). The reaction was stirred at room temperature overnight, washed with water, HCl 1N, brine, dried on MgSO4. The organic phase was evaporated in vacuo and purified by chromatography (MeOH/CH2Cl2 0.5/9.5) to obtain 110 mg of tert-butyl 4((dimethylamino)oxy)-4-oxobutanoate (90% yield). CF3CO2H (0.04 ml, 0.37 mmol, 4 equiv.) was added to a solution of tert-butyl 4((dimethylamino)oxy)-4-oxobutanoate (20 mg, 0.09 mmol, 1 equiv.) in CH2Cl2 (1.5 ml). The reaction was stirred at room temperature for 4h and evaporated in vacuo to give 14 mg of 29 (yield 95%). 1H NMR (500 MHz, CD3OD) δ 2.59 (s, 6H), 2.57 (s, 4H); 13C NMR (500 MHz, CD3OD) δ 176.2, 172.0, 48.5, 29.9; IR (neat) 3341, 2485,1717, 1120, 1026, 975 cm−1; HRMS (m/z):[M+]calcd. for C6H11NO4 161.0688; found 161.0923.
N‘1, N‘1, N‘4, N‘4-Tetramethylsuccinohydrazide30 . A solution of succinic acid (100 mg, 0.85 mmol, 1 equiv.), hydroxybenzotriazole (350 mg, 2.11 mmol, 2.3 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (421 mg, 2.11 mmol, 2.3 equiv.), diisopropylethylamine (0.6 mL, 3.4 mmol, 4 equiv.) and 1,1-dimethylhydrazine (0.16 mL, 2.04 mmol, 2.2 equiv.) in CH2Cl2 (20 mL) was stirred at room temperature overnight. CH2Cl2 (10 mL) was added and the reaction mixture was washed with water, a saturated solution of NaHCO3, brine and dried on MgSO4. The organic phase was evaporated in vacuo and purified by chromatography (MeOH/ CH2Cl2 1/9) to give 77 mg of 30 (45% yield). 1H NMR (500 MHz, CD3OD) δ 2.87 (s, 6H), 2.65 (s, 2H); 13C NMR (500 MHz, CD3OD) δ 178.2, 43.8, 27.6; IR (neat) 3356, 2485, 2071, 1695, 1120, 1027, 974 cm−1; HRMS (m/z):[(M-2CH3)−]calcd. for C6H14N4O2, 174.1117; found, 174.1022.
4-(2,2,2-Trimethylhydrazinyl)-4-oxobutanoate
General procedure for the coupling of hydrazine to succinic anhydride. To a stirred solution of the appropriate hydrazine (1 equiv.) in acetonitrile (5 mL) was added dropwise a solution of succinic anhydride (200 mg, 2.0 mmol, 1 equiv.) in acetonitrile (5 mL). The mixture was stirred at room temperature for 24 h, after which the solvent was evaporated in vacuo and the resulting crude purified using semipreparative reverse-phase HPLC, performed on a phenomenex C18 column (150 mm × 4.6 mm). Separation was achieved using a linear gradient of solvent A (water + 0.1% CF3CO2H) and solvent B (acetonitrile + 0.1% CF3CO2H), eluting at a flow rate of 1 mL/min and monitoring at 220 nm: 0% B to 40% B over 30 min.
4-(2-Methylhydrazinyl)-4-oxobutanoic acid
4-Hydrazinyl-4-oxobutanoic acid
4-Oxo-4-(1,2,2-trimethylhydrazinyl)butanoic acid
4-((Dimethylamino)oxy)-4-oxobutanoic acid
N‘1, N‘1, N‘4, N‘4-Tetramethylsuccinohydrazide