R. Echemendía et al. / Tetrahedron 70 (2014) 3297e3305
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reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and sodium
ascorbate (0.08 mmol) according to the click conjugation procedure.
Flash column chromatography purification (CH2Cl2/EtOAc 3:2)
afforded conjugate 7 (211 mg, 89%) as a white solid. Mp (AcOEt):
were reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and
sodium ascorbate (0.08 mmol) according to the click conjugation
procedure. Flash column chromatography purification (CH2Cl2/
EtOAc 3:2) afforded conjugate 14 (177 mg, 71%) as a white solid. Mp
203e204 ꢀC. 1H NMR (400 MHz, CD3OD):
d¼0.78 (d, 3H, J¼6.8 Hz);
(AcOEt): 235e236 ꢀC. 1H NMR (400 MHz, CDCl3):
d¼0.70e0.96 (m,
0.79e0.84 (m, 12H); 0.93 (d, 6H, J¼6.6 Hz); 0.98e1.53 (m, 30H);
1.86e1.63 (m, 8H); 1.98e2.10 (m, 6H); 2.39 (d,1H,1H, J¼12.3 Hz); 2.57
(td, 1H, J¼13.3/4.9 Hz); 2.93 (dd, 1H, J¼13.6/9.1 Hz); 3.07e3.12 (m,
4H); 3.73e3.95 (m, 4H); 3.99e4.10 (m, 2H); 4.26e4.31 (m, 2H);
4.34e4.40 (m, 1H); 4.45 (m, 2H); 4.61 (s, 1H); 4.72 (m, 1H); 5.05 (m,
2H); 6.05 (s, 1H); 6.20 (dd,1H, J¼10.1/1.9 Hz); 7.18 (m, 1H); 7.22e7.27
22H); 1.19e1.95 (m, 54H); 2.20 (m, 2H); 2.63 (m, 1H); 2.79 (m, 1H);
2.98 (m, 2H); 3.16 (m, 2H); 3.36 (t,1H, J¼10.8 Hz); 3.47 (m,1H); 3.53
(m, 1H); 4.17e4.325 (m, 2H); 4.46e4.36 (m, 4H); 4.62 (m, 1H); 4.84
(m, 1H); 5.05 (s, 2H); 5.08 (m, 1H); 7.13 (m, 3H); 7.17 (m, 1H);
7.33e7.39 (m, 12H); 7.85 (m, 1H). 13C NMR (100 MHz, CDCl3):
d¼11.4, 14.6, 15.2, 16.7, 16.9, 17.4, 19.4, 19.8 (CH3); 21.4 (CH2); 23.6
(m, 5H); 7.33 (m, 5H); 7.80 (s,1H).13C NMR (100 MHz, CDCl3):
d¼12.4,
(CH); 24.7, 25.0, 27.4 (CH2); 28.2 (CH3); 28.6, 29.1 (CH2); 29.4, 30.6
(CH); 31.3, 31.6, 33.4, 35.2, 34.9, 36.4 (CH2); 39.0 (C); 39.5, 40.3
(CH2); 40.6 (C); 41.6 (CH2); 42.6, 43.1(CH); 44.6 (CH2); 46.8, 47.6
(CH); 51.3 (C); 52.8, 54.6, 59.4, 60.4, 62.6 (CH); 66.2, 68.4 (CH2); 73.1
(C); 74.2, 77.6, 80.8 (CH); 81.2, 109.2 (C); 124.9, 125.8, 126.3, 127.2,
128.4, 129.0, 135.8 (CH); 136.4, 137.3 (C); 156.8, 157.7, 171.0, 171.7,
172.7, 173.1 (C]O). HRMS (ESI-FT-ICR) m/z: 1272.7623 [MþNa]þ;
calculated for C69H103N9NaO12: 1272.7664.
16.6, 16.8, 18.0, 18.4, 19.1 (CH3); 22.4, 24.6 (CH2); 25.2 (CH); 26.4, 27.2
(CH2); 28.2 (CH3); 29.6, 30.2, 30.8 (CH2); 31.6 (CH); 32.4, 34.1 (CH2);
35.6, 36.4 (CH); 38.4, 39.2, 41.6 (CH2); 42.6, 43.2 (C); 47.2, 48.4, 52.3,
52.8, 53.1 (CH); 53.8 (CH2); 55.4 (CH); 56.6, 58.3, 59.4, 66.4 (CH2); 81.2
(C); 123.4, 126.2, 127.1, 127.8, 128.3, 129.6, 130.4, 130.8 (CH); 136.2,
136.4, 138.8 (C); 155.8 (CH); 156.1,156.3 (C]O); 169.4 (C); 171.0,172.0,
172.7, 173.2, 186.3 (C]O). HRMS (ESI-FT-ICR) m/z: 1209.7042
[MþNa]þ; calculated for C66H94N10NaO10: 1209.7091.
4.1.19. Peptideesteroid conjugate 16. Peptidyl alkyne 15 (132 mg,
0.2 mmol) and azidosteroid 10 (83 mg, 0.2 mmol) in THF (5 mL)
were reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and
sodium ascorbate (0.08 mmol) according to the click conjugation
procedure. Flash column chromatography purification (CH2Cl2/
EtOAc 3:2) afforded conjugate 16 (192 mg, 90%) as a white solid. Mp
4.1.16. Peptideesteroid conjugate 9. Peptidyl alkyne 6 (165 mg,
0.2 mmol) and azidosteroid 8 (63 mg, 0.2 mmol) in THF (5 mL) were
reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and sodium
ascorbate (0.08 mmol) according to the click conjugation procedure.
Flash column chromatography purification (CH2Cl2/EtOAc 3:2)
afforded conjugate 9 (198 mg, 86%) as a white solid. Mp (AcOEt):
(AcOEt): 199e200 ꢀC. 1H NMR (400 MHz, CDCl3):
d¼0.63 (s, 3H);
197e199 ꢀC. 1H NMR (400 MHz, CD3OD):
d
¼0.78 (d, 3H, J¼6.8 Hz);
0.75 (d, 3H, J¼6.2 Hz); 0.81 (d, 3H, J¼6.6 Hz); 0.85 (d, 3H, J¼6.8 Hz);
0.86 (d, 6H, J¼6.4 Hz); 0.88 (s, 3H); 0.94e2.08 (m, 50H); 2.27 (d, 1H,
J¼13.0 Hz); 2.94 (dd, 1H, J¼13.0/8.46 Hz); 3.17 (m, 2H); 3.23 (m,
1H); 4.04 (m, 1H); 4.22 (m, 1H); 4.43 (dd, 1H, J¼15.1/5.2 Hz); 4.52
(m, 2H); 4.87 (m, 1H); 5.08 (m, 2H); 5.36 (t, 1H, J¼5.6 Hz); 5.75 (d,
1H, J¼5.0 Hz); 7.03 (m,1H); 7.12e7.17 (m, 6H); 7.33 (m, 5H); 7.43 (m,
0.82 (t, 3H, J¼7.4 Hz); 0.85 (s, 3H); 0.93 (d, 6H, J¼6.8 Hz); 0.94 (s, 3H);
1.04e2.15 (m, 38H); 2.40 (dd, 1H, J¼11.0/8.5 Hz); 2.88e2.96 (m, 1H);
3.07e3.12 (m, 3H); 3.75 (m, 1H); 3.86e3.91 (m, 2H); 4.04 (d, 1H,
J¼7.1 Hz); 4.27 (m, 1H); 4.46 (m, 2H); 4.61 (m, 1H); 4.72 (d, 1H,
J¼7.1 Hz); 5.04 (m, 2H); 7.17e7.33 (m, 10H); 7.93 (s, 1H). 13C NMR
(100 MHz, CDCl3):
d¼11.1, 12.2, 12.4, 16.2 18.2, 19.2 (CH3); 20.3, 20.8
1H); 7.58 (s, 1H). 13C NMR (100 MHz, CDCl3):
d¼11.8, 12.2, 19.2
(CH2); 21.4 (CH); 22.1, 23.3, 24.5 (CH2); 24.6 (CH); 24.7, 26.8 (CH2);
28.2 (CH3); 28.6, 28.9, 29.0, 29.2 (CH2); 31.3 (C); 32.1 (CH); 32.9 (CH2);
35.4, 35.8 (CH); 36.3, 36.8, 38.6 (CH2); 43.0 (C); 47.1, 47.8, 51.1, 52.3,
54.8(CH); 56.259.4, 66.2(CH2);71.4(CH); 80.0(C); 125.4,126.8,127.4,
128.3, 128.4, 130.0, 135.7 (CH); 131.4, 136.4, 138.2 (C); 156.6, 157.8;
158.3; 171.0, 171.4, 172.7, 173.2, 205.3 (C]O). HRMS (ESI-FT-ICR) m/z:
1171.6893 [MþNa]þ; calculated for C62H92N10NaO10: 1171.6924.
(CH3); 20.7, 22.4 (CH2); 22.5, 22.8 (CH3); 23.9, 24.1, 25.3 (CH2); 28.0
(CH); 28.2 (CH2); 28.6 (CH3); 29.4 (CH2); 30.1 (CH); 31.6, 32.7, 33.1,
35.1 (CH2); 35.3, 35.8 (CH); 36.1, 37.9, 39.5 (CH2); 39.7 (CH); 39.8,
39.9 (CH2); 42.5 (C); 53.9 (CH); 55.1 (C); 56.1, 56.2, 56.3 (CH); 66.6
(CH2); 80.2 (C); 121.6, 126.6, 128.0, 128.3, 128.5, 130.0 (CH); 136.5,
136.8, 144.1 (C); 156.4, 156.9, 170.8, 171.0, 173.2 (C]O). HRMS (ESI-
FT-ICR) m/z: 1099.7231 [MþNa]þ; calculated for C63H96N8NaO7:
1099.7298.
4.1.17. Peptideesteroid conjugate 11. Peptidyl alkyne 6 (165 mg,
0.2 mmol) and azidosteroid 10 (83 mg, 0.2 mmol) in THF (5 mL)
were reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and
sodium ascorbate (0.08 mmol) according to the click conjugation
procedure. Flash column chromatography purification (CH2Cl2/
EtOAc 3:2) afforded conjugate 11 (204 mg, 82%) as a white solid. Mp
4.1.20. Peptideesteroid conjugate 19. Peptidyl alkyne 17 (68 mg,
0.2 mmol) and azidosteroid 18 (83 mg, 0.2 mmol) in THF (5 mL)
were reacted in the presence of Cu(OAc)2$H2O (0.04 mmol) and
sodium ascorbate (0.08 mmol) according to the click conjugation
procedure. Flash column chromatography purification (CH2Cl2/
EtOAc 3:2) afforded conjugate 19 (140 mg, 93%) as a white solid. Mp
(AcOEt): 212e214 ꢀC. 1H NMR (400 MHz, CD3OD):
d
¼0.67 (s, 3H);
0.78 (d, 3H, J¼6.4 Hz); 0.82 (d, 3H, J¼7.2 Hz); 0.87 (d, 6H, J¼6.8 Hz);
0.91 (s, 3H); 089e0.94 (m, 12H); 1.00e2.11 (m, 52H); 2.28e2.33 (m,
2H); 2.95 (m, 1H); 3.10 (m, 3H); 3.80 (m 1H); 3.88 (m, 1H); 3.92 (m,
1H); 4.03 (m, 1H); 4.27 (m, 1H); 4.45e4.50 (m, 2H); 4.61 (m, 2H);
5.05 (s, 2H); 7.23 (m, 5H); 7.32 (m, 5H); 7.93 (s, 1H). 13C NMR
(AcOEt): 188e189 ꢀC. 1H NMR (400 MHz, CDCl3):
d
¼0.65 (s, 3H);
0.85e0.89 (d, 6H); 0.92 (d, 3H, J¼6.2 Hz); 1.01 (s, 3H); 1.17e1.47 (m,
14H); 1.14 (d, 6H, J¼6.6 Hz); 1.37 (d, 3H, J¼7.1 Hz); 1.43 (s, 9H);
1.54e1.60 (m, 2H); 1.74e1.98 (m, 10H); 2.17e2.25 (dd, 2H); 2.36 (m,
1H); 3.67 (s, 3H); 3.84 (m, 1H); 3.94 (t, 1H, J¼7.0 Hz); 4.53 (m, 1H);
5.18 (d, 1H, J¼7.6 Hz); 6.82 (d, 1H, J¼7.5 Hz); 7.34 (br s, 1H); 7.58 (s,
(100 MHz, CDCl3):
d
¼11.4, 12.3, 14.6, 15.8, 16.5, 16.8, 17.8, 18.6, 19.1
(CH3); 19.6 (CH); 20.4, 23.5, 23.8, 24.1 (CH2); 24.8 (CH); 25.2, 28.1,
28.3 (CH2); 28.6 (CH3); 29.1, 29.4, 30.2, 30.6 (CH2); 31.8, 32.6 (CH);
33.6 (C); 34.2 (CH); 35.4, 35.6, 35.8, 38.6, 39.2, 39.7, 41.5 (CH2); 42.4
(C); 42.8, 47.4, 48.1, 48.6, 52.4 (CH); 53.6 (CH2); 55.9, 56.2 (CH); 56.8,
59.4 (CH2); 71.2 (CH); 80.1 (C); 124.2, 125.8, 126.4, 127.2, 128.6,
129.2, 130.0 (CH); 136.4 (C); 146.2, 148.6 (C); 156.4, 156.8, 158.3,
171.2, 171.4, 172.7, 173.2 (C]O). HRMS (ESI-FT-ICR) m/z: 1269.8352
[MþNa]þ; calculated for C71H110N10NaO9: 1269.8394.
1H). 13C NMR (100 MHz, CDCl3):
d
¼11.1, 11.7, 12.2, 15.6, 18.2 (CH3);
20.6 (CH2); 23.4 (CH3); 23.5 (CH); 24.1, 24.7, 26.3, 27.1, 27.9 (CH2);
28.3 (CH3); 30.9, 31.0, 33.9 (CH2); 34.7 (C); 35.1 (CH2); 35.3, 35.8,
37.1 (CH); 39.9 (CH2); 40.5, 42.1, 42.7 (CH); 43.6 (C); 48.7 (CH2); 51.5
(CH3); 55.8, 56.2, 61.0 (CH); 80.1 (C); 120.2 (CH); 136.1 (C); 156.9,
171.4, 172.3, 174.9 (C]O). HRMS (ESI-FT-ICR) m/z: 777.5254
[MþNa]þ; calculated for C42H70N6NaO6: 777.5321.
4.1.21. Cyclopeptideesteroid conjugate 22. Azidosteroid 5 (166 mg,
0.3 mmol) was dissolved in CH2Cl2 (7 mL) and treated with tri-
fluoracetic acid (TFA, 3 mL) at 0 ꢀC. The reaction mixture was
4.1.18. Peptideesteroid conjugate 14. Peptidyl alkyne 12 (155 mg,
0.2 mmol) and azidosteroid 13 (95 mg, 0.2 mmol) in THF (5 mL)