Synthesis of AAZTA5-SA
1,4-Dibenzyl-6-methylpentanoate-6-nitroperhydro-1,4-diazepine (1)
A mixture of 2-nitrocyclohexanone (2.00 g, 13.9 mmol), Amberlyst® A21 (1.05 g) and abs. methanol (35 mL) was stirred at 60 °C under reflux for 1 hour. N,N’-Dibenzylethylenediamine (3.36 g, 13.9 mmol) and paraformaldehyde (1.67 g, 55.5 mmol) were added to the solution. The resulting suspension was heated to 80 °C and stirred overnight. After completion of the reaction, the mixture was filtered and the filtrate was evaporated under reduced pressure. After purification via column chromatography (cyclohexane/ethylacetate 9:1, Rf = 0.27) product 1 was obtained as yellow oil (5.20 g, 11.8 mmol, 85%). 1H-NMR (CDCl3, 400 MHz, δ [ppm]): 7.29 (m, 10 H); 3.66 (s, 3 H); 3.67 (dd, J = 13.5 Hz, 4 H); 3.25 (dd, J = 14.0 Hz, 4 H); 2.63 (m, 4 H); 2.12 (m, 2 H); 1.59 (m, 2 H); 1.32 (m, 2 H); 0.78 (m, 2 H). 13C-NMR (CDCl3, 100 MHz, δ [ppm]): 173.6 (s); 139.1 (s); 129.1 (s); 128.3 (s); 127.3 (s); 94.8 (s); 64.9 (s); 61.8 (s); 58.9 (s); 51.5 (s); 36.5 (s); 33.6 (s); 24.6 (s); 22.6 (s). MS ESI+ (m/z): found 440.3 [M + H+], calculated for C25H33N3O4: 439.25
1,4-Di(tert-butylacetate)-6-methylpentanoate-6-amino-di(tert-butylacetate)-perhydro-1,4-diazepine (3)
To a mixture of 1 (1.05 g, 2.39 mmol) and Pd(OH)2/C (0.62 g, 10 wt%) in abs. ethanol (20 mL) acetic acid (411 µL, 7.19 mmol) was added and the resulting solution was stirred at room temperature overnight under an atmosphere of hydrogen. After completion of the reaction, the mixture was filtered over Celite® and the filtrate was evaporated under reduced pressure. The crude product 2 was used for the following reaction without further purification.
To a solution of 2 (2.39 mmol) and K2CO3 (1.32 g, 9.57 mmol) in abs. acetonitrile (30 mL) tert-butyl bromoacetate (1.41 mL, 9.57 mmol) and sodium iodide (0.80 g, 4.82 mmol) were added. The resulting mixture was stirred overnight at 40 °C. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was subsequently purified via column chromatography (cyclohexane/ethylacetate 7:1, Rf = 0.15) to yield product 3 as yellow oil (0.89 g, 1.30 mmol, 54%). 1H-NMR (CDCl3, 400 MHz, δ [ppm]): 3.65 (s, 4 H); 3.61 (s, 4 H); 3.22 (s, 3 H); 2.99 (d, J = 14.1 Hz, 2 H); 2.85–2.65 (m, 4 H); 2.63 (d, J = 14.1 Hz, 2 H); 2.31 (t, J = 7.4 Hz, 2 H); 1.62–1.52 (m, 4 H); 1.44 (s, 18 H); 1.43 (s, 18 H); 1.25 (m, 2 H). 13C-NMR (CDCl3, 100 MHz, δ [ppm]): 174.4 (s); 172.9 (s); 170.9 (s); 80.9 (s); 80.4 (s); 65.3 (s); 63.2 (s); 62.6 (s); 59.4 (s); 52.1 (s); 51.6 (s); 37.3 (s); 34.3 (s); 28.3 (s); 28.3 (s); 25.9 (s); 21.8 (s). MS ESI+ (m/z): found 686.5 [M + H+], 708.4 [M + Na+], calculated for C35H63N3O10: 685.45
1,4-Di(tert-butylacetate)-6-pentanoic acid-6-(amino-di(tert-butylacetate))-perhydro-1,4-diazepine (4)
To a solution of 3 (172 mg, 0.25 mmol) in 1,4-dioxane/water (2:1, 3 mL) a 1 M solution of LiOH (375 µL, 0.38 mmol) was added and the resulting mixture was stirred overnight at room temperature. After completion of the reaction the solvent was evaporated under reduced pressure and 1 M NaHCO3 (10 mL) was added to the residue. The mixture was extracted with chloroform (5 × 5 mL) and the combined organic extracts were washed with water, dried over Mg2SO4 and evaporated under reduced pressure. Product 4 was obtained as yellow oil without further purification (116 mg, 0.17 mmol, 69%). 1H-NMR (CDCl3, 400 MHz, δ [ppm]): 3.60 (s, 4 H); 3.23 (s, 4 H); 3.00-2.97 (d, J = 14.1 Hz, 2 H); 2.88–2.60 (m, 6 H); 2.36–2.32 (t, J = 7.9 Hz, 2 H); 1.64–1.52 (m, 4 H); 1.43 (s, 18 H); 1.42 (s, 18 H); 1.24 (m, 2 H). 13C-NMR (CDCl3, 100 MHz, δ [ppm]): 178.9 (s); 172.9 (s); 170.9 (s); 81.0 (s); 80.5 (s); 65.1 (s); 63.1 (s); 59.4 (s); 52.2 (s); 34.2 (s); 29.8 (s); 28.3 (s); 28.2 (s); 25.6 (s); 22.8 (s); 21.9. MS ESI+ (m/z): found 672.4 [M + H+], 694.5 [M + Na+], calculated for C34H61N3O10: 671.44
1,4-Di(tert-butylacetate)-6-((5-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1yl)aminoethyl)amino)-5-oxopentyl)-6-(amino-di(tert-butylacetate))-perhydro-1,4-diazepine (5)
To a solution of 4 (75 mg, 0.11 mmol) in abs. acetonitrile (1 mL) HBTU (42 mg, 0.11 mmol), HOBt (45 mg, 0.33 mmol) and DIPEA (58 µL, 0.33 mmol) were added and the resulting mixture was stirred for 1 h at room temperature. N-Boc-1,2-diaminoethane (35 µL, 0.22 mmol) was added and stirring was continued overnight. After completion of the reaction, the solvent was evaporated under reduced pressure and the residue was purified via column chromatography (cyclohexane/ethylacetate 1:1, Rf = 0.13). Product 5 was obtained as yellow oil (74.2 mg, 91 µmol, 82%). 1H-NMR (DMSO, 400 MHz, δ [ppm]): 6.34 (br, 1 H); 5.26 (br, 1 H); 3.60 (s, 4 H); 3.38–3.34 (m, 2 H); 3.26–3.24 (m, 2 H); 3.21 (s, 4 H); 2.96 (d, J = 14.1 Hz, 2 H); 2.75–2.73 (m, 2 H); 2.66–2.63 (m, 2 H); 2.59 (d, J = 14.1 Hz, 2 H); 2.19 (t, 2 H); 1.62–1.53 (m, 4 H); 1.43 (s, 18 H); 1.42 (s, 27 H); 1.28–1.20 (m, 2 H). 13C-NMR (CDCl3, 100 MHz, δ [ppm]): 174.4 (s); 173.3 (s); 172.8 (s); 165.9 (s); 82.9 (s); 82.8 (s); 63.4 (s); 62.5 (s); 62.1 (s); 55.5 (s); 54.5 (s); 47.1 (s); 40.8 (s); 39.9 (s); 35.6 (s); 29.8 (s); 28.5 (s); 28.3 (s); 28.1 (s); 27.9 (s); 26.2 (s); 23.4 (s). MS ESI+ (m/z): found 814.6 [M + H+], 836.5 [M + Na+], calculated for C41H75N5O11: 813.55
1,4-Di(acetate)-6-((5-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1yl)-aminoethyl)amino)-5-oxopentyl)-6-(amino-di(acetate))-perhydro-1,4-diazepine (6)
A solution of 5 (74.2 mg, 91 µmol) in dichloromethane/trifluoroacetic acid (1:1, 2 mL) was stirred for 3 h at room temperature. After complete deprotection, the solvent was evaporated under reduced pressure and the residue was dissolved in 0.5 M phosphate buffer (pH = 7, 4 mL). To the resulting solution 3,4-diethoxycyclobut-3-ene-1,2-dione (39 µL, 264 µmol) was added. The pH was adjusted to 7 with 1 M NaOH solution before stirring overnight at room temperature. After completion of the reaction, the product was purified via HPLC (column: Phenomenex Luna C18 semipreparative (250 × 10 mm) 10 µ, flow rate: 5 mL/min, 10% MeCN + 0.1% TFA, tR = 12.5 min) yielding 6 as colorless solid (16.2 mg, 26 µmol, 29%). 1H-NMR (D2O, 400 MHz, δ [ppm]): 4.75–4.67 (m, 2 H); 3.88 (s, 2 H); 3.76–3.66 (m, 8 H); 3.59–3.44 (m, 8 H); 3.40–3.38 (m, 2 H); 2.20 (t, 2 H); 1.52–1.45 (m, 4 H); 1.43 (t, 3 H); 1.30–1.21 (m; 2 H). 13C-NMR (D2O, 100 MHz, δ [ppm]): 176.60 (s); 176.06 (s); 175.97 (s); 173.82 (s); 170.67 (s); 70.70 (s); 70.55 (s); 62.81 (s); 59.41 (s); 58.63 (s); 52.59 (s); 52.20 (s); 43.93 (s); 39.56 (s); 39.24 (s); 35.31 (s); 33.76 (s); 25.73 (s); 22.26 (s); 15.07 (s). MS ESI+ (m/z): found 614.3 [M + H+], 636.3 [M + Na+], calculated for C26H39N5O12: 613.26
A solution of tri-tert-butyl 2,2′,2”-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclodo-decane-1,4,7-triyl)triacetate (50 mg, 81 µmol) in dichloromethane/trifluoroacetic acid (1:1, 200 µL) was stirred overnight at room temperature. After complete deprotection the solvent was evaporated under reduced pressure and the residue was dissolved in 0.5 M phosphate buffer (pH = 7, 1 mL). The pH was adjusted to 7 with 1 M NaOH solution. 3,4-diethoxycyclobut-3-ene-1,2-dione (36 µL, 244 µmol) was added and the resulting mixture was stirred overnight at room temperature. After completion of the reaction the product was purified via HPLC (column: Phenomenex Luna C18 semipreparative (250 × 10 mm) 10 µ, flow rate: 5 mL/min, 6% to 8% MeCN + 0.1% TFA in 20 min, tR = 9.5 min) yielding 7 as colorless solid (19.1 mg, 34 µmol, 41%). 1H-NMR (D2O, 600 MHz, δ [ppm]): 4.64–4.53 (dq, 2 H); 3.93–2.89 (m, 28 H); 1.41–1.33 (m, 3 H). MS ESI+ (m/z): found 571.3 [M + H+], 593.3 [M + Na+], calculated for C24H38N6O10: 570.26