Med Chem Res
Calcd. for C15H22N6O6S: C, 43.47; H, 5.35; N, 20.28; O,
23.16; S, 7.74. Found: C, 43.51; H, 5.31; N, 20.22; O,
23.19; S, 7.77. 1H-NMR (CDCl3) d/ppm: 8.58 (s, 4H,
NH2), 8.05 (s, H, aNH), 8.0 (s, H, NH), 7.44 (m, 2H, Ar),
7.4 (m, 3H, Ar), 7.19 (s, 2H, NH2), 5.11 (s, 2H, PhCH2),
4.57 (m, H, CH), 4.12 (s, 2H, CH2), 3.41 (m, 2H, cCH2),
2.37 (m, 2H, bCH2); 13C-NMR (CDCl3) d/ppm: 172.8,
169.9, 158.4, 155.9, 136.4, 128.5, 127.8, 127.4, 66.7, 56.7,
53.4, 42.5, 15.5; 11b: MS-ES, m/z: 401 [M?] (400.41);
Anal. Calcd. for C14H20N6O6S: C, 41.99; H, 5.03; N,
20.99; O, 23.97; S, 8.01. Found: C, 41.95; H, 5.05; N,
21.01; O, 23.92; S, 8.07. 1H-NMR (CDCl3) d/ppm: 8.55 (s,
4H, NH2), 8.11 (H, aNH), 8.05 (s, H, NH), 7.5 (m, 2H, Ar),
7.37 (m, 3H, Ar), 7.18 (s, 2H, NH2), 5.12 (s, 2H, PhCH2),
4.52 (m, H, CH), 4.15 (t, 2H, CH2), 4.01, 3.82 (m, 2H,
bCH2); 13C-NMR (CDCl3) d/ppm: 172.1, 169.6, 158.7,
156.3, 136.5, 129.3, 127.7, 126.8, 66.9, 61.04, 52.3, 42.1.
(660.70);Anal. Calcd. forC28H36N8O9S:C, 50.90;H,5.49;N,
16.96; O, 21.79; S, 4.85. Found: C, 50.85; H, 5.52; N, 16.93;
O,21.78;S,4.95. 1H-NMR(CDCl3)d/ppm:8.57(s, 4H, NH2),
8.07(s, 3H, aNH), 7.45(m, 2H, Ar),7.34(m, 3H, Ar), 7.22(m,
2H, Ar), 7.12 (s, 2H, NH2), 6.74 (m, 2 J, Ar), 5.30 (s, H, OH),
5.06 (s, 2H, PhCH2), 4.99 (m, H, CH), 4.57 (m, H, CH), 4.44
(m, H, CH), 4.1 (m, 2H, CH2), 4.05, 3.77 (m, 2H, bCH2), 3.54,
3.47 (m, 2H, CH2), 3.41, 3.2 (m, 2H, CH2), 2.3, 2.1 (m, 2H,
CH2), 2.01, 1.88 (m, 2H, CH2); 13C-NMR (CDCl3) d/ppm:
172.1, 171.7, 170.6, 169.7, 158.5, 156.2, 155.4, 136.3, 130.4,
129.1, 128.7, 127.9, 127.3, 67.9, 66.5, 61.07, 56.7, 50.8, 49.3,
42.5, 37.1, 29.1, 24.0.
Synthesis of Pro-sArg-Gly-NH2, N-(1-((2-amino-2-
oxoethyl)amino)-4-(N-(diaminomethylene)sulfamoyl)-1-
oxobutan-2-yl)pyrrolidine-2-carboxamide, (8a), Pro-
NsArg-Gly-NH2, N-(1-((2-amino-2-oxoethyl)amino)-3-(N-
(diaminomethylene)sulfamoyl)-1-oxopropan-2-yl)
pyrrolidine-2-carboxamide, (8b), sArg-Gly-NH2, 2-amino-
N-(2-amino-2-oxoethyl)-4-(N-(diaminomethylene)
sulfamoyl)butanamide, (12a), NsArg-Gly-NH2,
2-amino-N-(2-amino-2-oxoethyl)-3-(N-(diaminomethylene)
sulfamoyl)propanamide, (12b), Tyr-Pro-sArg-Gly-NH2,
N-(1-((2-amino-2-oxoethyl)amino)-4-(N-
Synthesis of Z-Tyr-Pro-sArg-Gly-NH2, benzyl (1-(2-((1-((2-
amino-2-oxoethyl)amino)-4-(N-(diaminomethylene)
sulfamoyl)-1-oxobutan-2-yl)carbamoyl)pyrrolidin-1-yl)-3-
(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate, 13a
(Z-Tyr-Pro-NsArg-Gly-NH2, benzyl (1-(2-((1-((2-amino-2-
oxoethyl)amino)-3-(N-(diaminomethylene)sulfamoyl)-1-
oxopropan-2-yl)carbamoyl)pyrrolidin-1-yl)-3-
(diaminomethylene)sulfamoyl)-1-oxobutan-2-yl)-1-(2-
amino-3-(4-hydroxyphenyl)propanoyl)pyrrolidine-2-
carboxamide, (14a), and Tyr-Pro-NsArg-Gly-NH2, N-
(1-((2-amino-2-oxoethyl)amino)-3-(N-(diaminomethylene)
sulfamoyl)-1-oxopropan-2-yl)-1-(2-amino-3-
(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate, 13b)
A solution of Z-Tyr-Pro-OH (0.5 g, 1.2 mM) and NMM
(0.13 ml, 1.2 mM) in 10 ml DMF was cooled down to
-10 °C and Piv-Cl (0.15 ml, 1.2 mM) was added drop
wise. 10 min later a solution of HBr.sArg-Gly-NH2
(HBr.NsArg-Gly-NH2) (1.2 mM) and Et3N (0.17 ml,
1.2 mM) in 10 ml DMF was added. The reaction mixture was
stirred at room temperature for 24 h. 20 ml water was added
and the mixture was extracted with CHCl3 (3 9 20 ml). The
organic layer was washed consequently with 5 % NaHCO3
(3 9 20 ml), 5 % NaHSO4 (3 9 20 ml), and water, and then
dried over anhydrous Na2SO4, and the solvent was evapo-
rated. The products obtained 13a (0.38 g, 48 %) and 13b
(0.41 g, 51 %) were used without further purification. 13a:
MS-ES, m/z: 675 [M?] (674.73); Anal. Calcd. for
C29H38N8O9S: C, 51.62; H, 5.68; N, 16.61; O, 21.34; S, 4.75.
(4-hydroxyphenyl)propanoyl)pyrrolidine-2-carboxamide,
(14b)
The N-Protected peptide analogue (0.1 mM) was dissolved
in 1 ml AcOH, and HBr/AcOH (0.5 ml) was added. A
deprotection was carried out for 1 h at room temperature.
The solvent was removed and the crude product was
treated three times with MeOH (3 9 20 ml), which was
also evaporated. The HBr-salts of the kyotorphin analogues
obtained were transformed to free bases with Et3N in
CH2Cl2. The CH2Cl2 was evaporated. The final peptides
were obtained after column purification (Silicagel 60,
CH3CN: H2O, 4:1). 8a (0.03 g, 78 %): MS-ES, m/z: 378
[M?] (377.42); Anal. Calcd. for C12H23N7O5S: C, 38.19;
H, 6.14; N, 25.98; O, 21.20; S, 8.50. Found: C, 38.22; H,
6.10; N, 25.95; O, 21.21; S, 8.52. 1H-NMR (CDCl3) d/ppm:
8.50(s, 4H, NH2), 8.1 (s, 2H, aNH), 7.10 (s, 2H, NH2), 4.50
(t, H, CH), 4.12 (s, 2H, CH2), 3.69 (m, H, CH), 3.4(m, 2H,
cCH2), 2.31 (m, 2H, bCH2), 2.80, 2.70 (m, 2H, CH2), 2.04
(m, H, NH), 1.95, 1.73 (m, 2H, CH2), 1.64, 1.52 (m, 2H,
CH2); 13C-NMR (CDCl3) d/ppm: 172.5, 171.07, 170.4,
158.8, 62.7, 55.8, 53.2, 45.6, 42.5, 30.8, 24.0, 15.1; 8b
(0.028 g, 77 %): MS-ES, m/z: 364 [M?] (363.39); Anal.
Calcd. for C11H21N7O5S: C, 36.36; H, 5.82; N, 26.98; O,
1
Found: C, 51.57; H, 5.71; N, 16.62; O, 21.28; S, 4.82. H-
NMR (CDCl3) d/ppm: 8.54(s, 4H, NH2), 8.04 (s, 3H, aNH),
7.48(m, 2H, Ar), 7.39(m, 3H, Ar), 7.18 (s, 2H, NH2), 7.12(m,
2H, Ar), 6.71 (m, 2H, Ar), 5.32 (s, H, OH), 5.11 (s, 2H,
PhCH2), 4.92(t, H, CH), 4.55(t, H, CH), 4.40(m, H, CH, Pro),
4.12 (s, 2H, CH2), 3.51, 3.41 (m, 2H, CH2), 3.44, 3.19 (m, 2H,
CH2), 3.42(m, 2H, cCH2), 2.34 (m, 2H, bCH2), 2.31, 2.09 (m,
2H, CH2), 2.0, 1.90 (m, 2H, CH2); 13C-NMR (CDCl3) d/ppm:
172.9, 171.04, 170.5, 169.4, 158.7, 155.9, 155.4, 136.5, 130.1,
129.5, 128.4, 127.2, 126.7, 115.5, 67.9, 66.5, 55.9, 55.1, 53.6,
49.1, 37.4, 29.5, 24.6, 15.3; 13b: MS-ES, m/z: 661 [M?]
123