Chemistry
Synthesis of 1
A solution of 4-aminobenzonitrile (2.36 g, 20 mmol) and maleic anhydride (5.88 g, 60 mmol) in 1,4-dioxane (30 mL) was stirred at 100°C for 1 h. To the reaction mixture, ammonium persulfate (9.12 g, 40 mmol) and dimethyl sulfoxide (DMSO) (2.84 mL) were added and the reaction mixture was heated to 100°C for 3 h. Thereafter, the reaction mixture was filtered and dioxane was removed under vacuo. The residue was dissolved in ethyl acetate and washed with diluted HCl, saturated aqueous NaHCO3, and brine. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo to afford 3.38 g of 1 (yield: 85%). 1H NMR (400 MHz, DMSO-d6) δ 7.26 (s, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 109.88, 118.49, 126.86, 133.06, 135.02, 135.88, 169.34.
Synthesis of 2
A solution of 1 (3.31 g, 16.7 mmol) in acetonitrile (10 mL) was mixed with Furan (5 mL) and stirred at 60°C for 8 h. The solvent was evaporated and ethyl acetate was added, then a solid precipitated out. The material was filtered and washed with ethyl acetate. The filtrated solution was concentrated to afford more of the product, which was also filtered and washed with ethyl acetate. The combined solid portions were dried under high vacuum at room temperature overnight to afford 2.67 g of 2 (yield: 60%). 1H NMR (400 MHz, DMSO-d6) δ 3.13 (s, 2H), 5.26 (s, 2H), 6.62 (s, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 47.71, 80.91, 110.98, 118.29, 127.55, 133.19, 136.05, 136.72, 175.27. HR-MS (FAB, pos) m/z calcd. for C15H11N2O3+, 267.0770; found 267.0768 [M + H]+.
Synthesis of 3
A solution of hydroxylamine hydrochloride (521 mg, 7.5 mmol) and triethylamine (1.04 mL, 7.5 mmol) in N,N-dimethylformamide (DMF) (5 mL) and H2O (2 mL) was added to a solution of 2 (1.33 g, 5 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 3 h. Then, H2O was added to the solution and diluted with ethyl acetate. The organic layer was dried over Na2SO4 and the solvent was evaporated under vacuo. The residue was recrystallized with chloroform and dried under high vacuum at room temperature overnight to afford 287 mg of 3 (yield: 19%). 1H NMR (400 MHz, DMSO-d6) δ 3.08 (s, 2H), 5.24 (s, 2H), 5.88 (s, 2H), 6.60 (s, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 9.74 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 47.51, 80.80, 126.00, 126.46, 132.41, 133.41, 136.66, 150.27, 175.68. HR-MS (FAB, pos) m/z calculated for C15H14N3O4+, 300.0984; found 300.0985 [M + H]+.
Synthesis of 4 (Ham-Mal)
A solution of 3 (299 mg, 1 mmol) in DMF (6 mL) and toluene (18 mL) was stirred at 115 ºC for 2 h. After evaporation of the solvent, the residue was suspended in H2O and then filtered. The solid portion was dried under high vacuum at room temperature overnight to afford 231 mg of 4 (yield: 74%). 1H NMR (400 MHz, DMSO-d6) δ 5.87 (s, 2H), 7.20 (s, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 9.72 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ 112.50, 113.05, 120.46, 126.33, 128.99, 149.57, 151.41. HR-MS (ESI, pos) m/z calculated for C11H10N3O3+, 232.0722; found 232.0725 [M + H]+.
Synthesis of 5 (Ham-Cys)
A solution of L-Cysteine (10 mg, 80 µmol) in H2O (100 µL) was adjusted to pH 7–8 by 0.1 N NaOH and then added to a solution of 4 (10 mg, 40 µmol) in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column (Nacalai Tesque, Kyoto, Japan, diameter: 10 mm, length: 250 mm, particle size: 5 µm, pore size: 120 Å) using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10] at a flow rate of 4.0 mL/min to give 3.5 mg of 5 (yield: 23%). 1H NMR (400 MHz, D2O) δ 3.07–3.14 (m, 2H), 3.24–3.34 (m, 2H), 4.08–4.18 (m, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H). ESI-HRMS m/z calculated for C14H17N4O5S+, 353.0920; found, 353.0919 [M + H]+.
Synthesis of 6 (Ham- RGD)
A solution of c(RGDfC) (PEPTIDE INSTITUTE, INC., Osaka, Japan, 2.0 mg, 4 µmol) in H2O (200 µL) was added to a solution of 4 (0.8 mg, 4 µmol) in DMF (500 µL). The reaction mixture was stirred at room temperature for 1 h. Then, the solvent was evaporated and the residue was purified by RP-HPLC on a Cosmosil 5C18-AR-II column (Nacalai Tesque, diameter: 10 mm, length: 250 mm, particle size: 5 µm, pore size: 120 Å) using the mobile phase [H2O (0.1% TFA)/acetonitrile (0.1% TFA) = 90/10 (0 min) to 70/30 (30 min)] at a flow rate of 4.0 mL/min to give 1.8 mg of 6 (64% yield). HR-MS (ESI, pos) m/z calcd. 810.2993, found 810.2988 [M + H]+.