Antitumor Agents Based on Ansamitocin P3
FULL PAPER
ture was directly purified by reversed phase HPLC (Trentec Reprosil-Pur
120 C18 AQ 5 mm, column: 250 mm ꢃ 8 mm, guard 40 mm ꢃ 8 mm; gra-
dient 0.1% in water TFA/MeCN 99:1 ! 100% MeCN in 55 min, then
5 min 100% MeCN, flow rate 2.25 ! 4 mLminꢀ1; tr =42.4 min) to yield
169.8 (s, C-25), 168.2 (s, C-1), 166.3 (s, C-34), 160.5 (s, C-42), 153.3 (s, C-
43), 151.2 (s, NHCOO), 150.7 (s, C-38), 149.4 (s, C-40), 148.4 (d, C-45),
142.0 (s, C-44), 141.0 (s, C-18), 140.8 (s, C-16), 139.4 (s, C-14), 132.4 (s, C-
19), 132.2 (d, C-12), 129.9 (d, C-23), 129.7 (d, C-21), 129.0 (d, C-36),
128.9 (d, C-17), 128.1 (d, C-11), 128.0 (s, C-41), 126.6 (d, C-20), 124.4 (d,
C-13), 124.2 (d, C-22), 121.4 (s, C-35), 111.2 (d, C-37), 88.3 (d, C-10), 80.1
(s, C-9), 75.9 (d, C-3), 73.4 (d, C-7), 65.8 (d, C-5), 60.5 (s, C-4), 56.1 (q,
10-OMe), 52.2 (d, C-33), 51.3 (d, C-29), 45.9 (t, C-39), 45.5 (t, C-15), 40.7
(t, C-24), 39.8 (t, C-28), 37.7 (d, C-6), 36.2 (q, NMe), 36.1 (t, C-8), 35.0 (t,
C-26), 33.7 (t, C-27), 33.0 (d, C-2’), 32.3 (t, C-2), 31.9 (t, C-31), 26.6 (t, C-
32), 19.8 (q, 2’-Mea), 17.8 (q, 2’-Meb), 15.3 (q, 14-Me), 14.5 (q, 6-Me),
11.5 ppm (q, 4-Me); HRMS (ESI): m/z: calcd for C59H72N11O16S2
[MꢀH]ꢀ: 1254.4600, found: 1254.4594.
the title compound 23 (2.6 mg, 1.91 mmol, 42%) as
a yellow solid.
1H NMR (500 MHz, [D6]DMSO, [D5]DMSO=2.50 ppm): d = 8.70 (m,
1H, 24-NH), 8.66 (s, 1H, 48-H), 8.32 (m, 1H, 32-NH), 8.22 (m, 1H, 36-
NH), 7.80 (d, J=7.3 Hz, 2H, 27-H), 7.65 (d, J=8.8 Hz, 2H, 39-H), 7.44
(m, 2H, 28-H), 7.30 (brs, 1H, 45-OH), 7.20 (s, 1H, 43-NHa), 7.10 (s, 1H,
43-NHb), 7.07 (s, 1H, 17-H), 6.99 (s, 1H, 38-NH), 6.90 (s, 1H, 9-NH),
6.70 (s, 1H, 21-H), 6.63 (d, J=8.8 Hz, 2H, 40-H), 6.58 (dd, J=15.3,
11.1 Hz, 1H, 12-H), 6.36 (m, 2H, 22-H, 23-H), 6.19 (d, J=11.1 Hz, 1H,
13-H), 5.40 (dd, J=15.3, 8.9 Hz, 1H, 11-H), 4.56 (dd, J=11.9, 2.8 Hz,
1H, 3-H), 4.48 (s, 2H, 42-H), 4.44 (m, 1H, 32-H), 4.29 (m, 1H, 36-H),
4.25 (m, 1H, 30-H), 4.07 (m, 3H, 7-H, 24-H), 3.88 (s, 3H, 20-OMe), 3.49
(d, J=8.9 Hz, 1H, 10-H), 3.48 (d, J=12.5 Hz, 1H, 15-Ha), 3.23 (d, J=
12.8 Hz, 1H, 15-Hb), 3.22 (s, 3H, 10-OMe), 2.92 (s, 3H, NMe), 2.90 (m,
1H, 31-Ha), 2.75 (m, 1H, 31-Hb), 2.67 (d, J=9.8 Hz, 1H, 5-H), 2.64 (sep,
J=7.0 Hz, 1H, 2’-H), 2.48 (m, 1H, 2-Ha), 2.25 (m, 2H, 34-H), 2.02 (m,
2H, 2-Hb, 35-Ha), 1.91 (m, 1H, 35-Hb), 1.62 (s, 3H, 14-Me), 1.60 (m, 3H,
30-Me), 1.43 (m, 1H, 6-H, 8-Ha), 1.31 (m, 1H, 8-Hb), 1.16 (d, J=7.0 Hz,
3H, 2’-Mea), 1.12 (d, J=6.3 Hz, 3H, 6-Me), 1.10 (d, J=7.0 Hz, 3H, 2’-
Meb), 0.81 ppm (s, 3H, 4-Me); 13C NMR (125 MHz, [D6]DMSO,
[D6]DMSO=39.52 ppm): d = 175.4 (s, C-1’), 173.8 (s, 36-CO2H), 171.9 (s,
32-CO2H), 171.8 (s, C-33), 168.2 (s, C-1), 166.3 (s, C-37), 165.8 (s, C-25),
160.7 (s, C-45), 157.9 (s, C-20), 153.5 (s, C-46), 151.2 (s, NHCOO), 150.7
(s, C-41), 148.9 (s, C-43), 148.4 (d, C-48), 144.9 (s, C-29), 142.2 (s, C-47),
142.2 (s, C-18), 140.9 (s, C-16), 139.2 (s, C-14), 133.7 (s, C-26), 132.2 (d,
C-12), 132.1 (d, C-23), 129.0 (d, C-39), 128.1 (d, C-11), 127.9 (s, C-44),
127.4 (d, C-28), 127.3 (d, C-27), 124.4 (d, C-13), 121.3 (s, C-38), 121.2 (d,
C-22), 120.9 (s, C-19), 120.1 (d, C-21), 112.6 (d, C-17), 111.1 (d, C-40),
88.2 (d, C-10), 80.0 (s, C-9), 76.1 (d, C-3), 73.3 (d, C-7), 65.8 (d, C-5), 60.5
(s, C-4), 56.1 (q, 10-OMe), 55.9 (q, 20-OMe), 52.1 (d, C-36), 51.2 (d, C-
32), 48.6 (d, C-30), 45.9 (t, C-42), 45.8 (t, C-15), 42.2 (t, C-24), 39.9 (t, C-
31), 37.7 (d, C-6), 36.0 (t, C-8), 35.8 (q, NMe), 33.0 (d, C-2’), 32.3 (t, C-2),
31.9 (t, C-34), 26.6 (t, C-35), 20.1 (q, 2’-Mea), 19.8 (q, 30-Me), 17.7 (q, 2’-
Meb), 15.3 (q, 14-Me), 14.5 (q, 6-Me), 11.6 ppm (q, 4-Me); HRMS (ESI):
m/z: calcd for C66H78N11O17S2 [MꢀH]ꢀ: 1360.5019, found: 1360.4988.
Folic acid–ansamitocin conjugate 24: Pte-l-Glu-l-Cys 21 (1.6 mg,
2.94 mmol, 1.0 equiv) was suspended in water (600 mL), the mixture was
deaerated by passing nitrogen through and adjusted to pH 7 by adding a
solution of 0.1m NaHCO3. After addition of disulfide 18 (2.4 mg,
2.94 mmol, 1.0 equiv), dissolved in MeCN (600 mL), stirring was continued
until complete conversion was detected by UPLC-MS. The reaction mix-
ture was directly purified by reversed phase HPLC (Trentec Reprosil-Pur
120 C18 AQ 5 mm, column: 250 mm ꢃ 8 mm, guard 40 mm ꢃ 8 mm; gra-
dient 0.1% in water TFA/MeCN 99:1 ! 100% MeCN in 55 min, then
5 min 100% MeCN, flow rate 2.25 ! 4 mLminꢀ1; tr =36.2 min) to yield
the title compound 24 (2.8 mg, 2.23 mmol, 76%) as a yellow solid.
1H NMR (500 MHz, [D6]DMSO, [D5]DMSO=2.50 ppm): d = 8.68 (s,
1H, 45-H), 8.34 (d, J=8.1 Hz, 1H, 33-NH), 8.21 (m, 2H, 24-NH, 29-
NH), 7.67 (d, J=9.0 Hz, 2H, 36-H), 7.63 (m, 1H, 20-H), 7.36 (brs, 1H,
42-OH), 7.32 (d, J=8.1 Hz, 1H, 21-H), 7.19 (s, 1H, 43-NHa), 7.09 (s, 1H,
43-NHb), 7.05 (s, 1H, 17-H), 6.99 (s, 1H, 38-NH), 6.91 (s, 1H, 9-NH),
6.65 (d, J=9.0 Hz, 2H, 37-H), 6.57 (dd, J=15.9, 11.7 Hz, 1H, 12-H), 6.35
(d, J=15.9 Hz, 1H, 22-H), 6.24 (ddd, J=15.9, 6.4, 5.8 Hz, 1H, 23-H),
6.19 (d, J=11.7 Hz, 1H, 13-H), 5.39 (dd, J=15.9, 9.0 Hz, 1H, 11-H), 4.54
(dd, J=12.3, 2.8 Hz, 1H, 3-H), 4.51 (s, 2H, 39-H), 4.46 (m, 1H, 33-H),
4.29 (m, 1H, 29-H), 4.08 (ddd, J=12.0, 11.2, 1.4 Hz, 1H, 7-H), 3.85 (m,
2H, 24-H), 3.48 (d, J=9.0 Hz, 1H, 10-H), 3.47 (d, J=14.5 Hz, 1H, 15-
Ha), 3.25 (d, J=14.5 Hz, 1H, 15-Hb), 3.23 (s, 3H, 10-OMe), 3.10 (dd, J=
13.7, 4.6 Hz, 1H, 28-Ha), 2.98 (s, 3H, NMe), 2.96 (brs, 1H, OH), 2.89 (m,
3H, 27-H, 28-Hb), 2.66 (d, J=9.9 Hz, 1H, 5-H), 2.63 (sep, J=7.0 Hz, 1H,
2’-H), 2.47 (m, 3H, 31-H, 2-Ha), 2.24 (m, 2H, 26-H), 2.05 (m, 1H, 32-Ha),
2.02 (dd, J=13.9, 2.8 Hz, 1H, 2-Hb), 1.91 (m, 1H, 32-Hb), 1.59 (s, 3H, 14-
Me), 1.43 (m, 2H, 6-H, 8-Ha), 1.31 (m, 1H, 8-Hb), 1.17 (d, J=7.0 Hz, 3H,
2’-Mea), 1.10 (2x d, J=7.0 Hz, 6H, 6-Me, 2’-Meb), 0.75 ppm (s, 3H, 4-
Me); 13C NMR (125 MHz, [D6]DMSO, [D6]DMSO=39.52 ppm): d =
175.4 (s, C-1’), 173.8 (s, 33-CO2H), 172.0 (s, 29-CO2H), 171.9 (s, C-30),
Folic acid–ansamitocin conjugate 25: Pte-l-Glu-l-Cys 21 (2.5 mg,
4.59 mmol, 1.0 equiv) was suspended in water (900 mL), the mixture was
deaerated by passing nitrogen through and adjusted to pH 7 by adding a
solution of 0.1m NaHCO3. After addition of disulfide 19 (3.9 mg, 4.59
mmol, 1.0 equiv), dissolved in MeCN (900 mL), stirring was continued
until complete conversion was detected by UPLC-MS. The reaction mix-
ture was directly purified by reversed phase HPLC (Trentec Reprosil-Pur
120 C18 AQ 5 mm, column: 250 mm ꢃ 8 mm, guard 40 mm ꢃ 8 mm; gra-
dient 0.1% in water TFA/MeCN 99:1 ! 100% MeCN in 55 min, then
5 min 100% MeCN, flow rate 2.25 ! 4 mLminꢀ1; tr =32.9 min) to yield
the title compound 25 (3.5 mg, 2.72 mmol, 59%) as
a yellow solid.
1H NMR (500 MHz, [D6]DMSO, [D5]DMSO=2.50 ppm): d = 8.69 (s,
1H, 45-H), 8.33 (d, J=8.1 Hz, 1H, 33-NH), 8.22 (m, 2H, 24-NH, 29-
NH), 7.67 (d, J=9.0 Hz, 2H, 36-H), 7.36 (brs, 1H, 42-OH), 7.20 (s, 1H,
43-NHa), 7.10 (s, 1H, 43-NHb), 7.06 (s, 1H, 17-H), 6.99 (s, 1H, 38-NH),
6.90 (s, 1H, 9-NH), 6.69 (s, 1H, 21-H), 6.64 (d, J=9.0 Hz, 2H, 37-H),
6.58 (dd, J=15.9, 11.7 Hz, 1H, 12-H), 6.26 (m, 2H, 22-H, 23-H), 6.19 (d,
J=11.7 Hz, 1H, 13-H), 5.39 (dd, J=15.9, 9.0 Hz, 1H, 11-H), 4.57 (dd, J=
12.3, 2.8 Hz, 1H, 3-H), 4.51 (s, 2H, 39-H), 4.46 (m, 1H, 33-H), 4.29 (m,
1H, 29-H), 4.08 (ddd, J=12.0, 11.2, 1.4 Hz, 1H, 7-H), 3.89 (s, 3H, 20-
OMe), 3.80 (m, 2H, 24-H), 3.48 (d, J=9.0 Hz, 1H, 10-H), 3.47 (d, J=
14.5 Hz, 1H, 15-Ha), 3.25 (d, J=14.5 Hz, 1H, 15-Hb), 3.23 (s, 3H, 10-
OMe), 3.10 (dd, J=13.7, 4.6 Hz, 1H, 28-Ha), 2.92 (s, 3H, NMe), 2.89 (m,
3H, 27-H, 28-Hb), 2.68 (d, J=9.9 Hz, 1H, 5-H), 2.62 (sep, J=7.0 Hz, 1H,
2’-H), 2.47 (m, 3H, 31-H, 2-Ha), 2.24 (m, 2H, 26-H), 2.04 (m, 1H, 32-Ha),
2.02 (dd, J=13.9, 2.8 Hz, 1H, 2-Hb), 1.91 (m, 1H, 32-Hb), 1.62 (s, 3H, 14-
Me), 1.43 (m, 2H, 6-H, 8-Ha), 1.34 (m, 1H, 8-Hb), 1.16 (d, J=7.0 Hz, 3H,
2’-Mea), 1.10 (2x d, J=7.0 Hz, 6H, 6-Me, 2’-Meb), 0.80 ppm (s, 3H, 4-
Me); 13C NMR (125 MHz, [D6]DMSO, [D6]DMSO=39.52 ppm): d =
175.4 (s, C-1’), 173.8 (s, 33-CO2H), 172.0 (s, 29-CO2H), 171.7 (s, C-30),
171.4 (s, C-25), 169.7 (s, C-1), 168.2 (s, C-34), 160.4 (s, C-42), 157.9 (s, C-
20), 153.3 (s, C-43), 151.2 (s, NHCOO), 150.6 (s, C-38), 149.5 (s, C-40),
148.3 (d, C-45), 142.2 (s, C-44), 142.2 (s, C-18), 140.9 (s, C-16), 139.2 (s,
C-14), 132.2 (d, C-12), 132.0 (d, C-23), 129.0 (d, C-36), 128.1 (d, C-11),
128.0 (s, C-41), 124.4 (d, C-13), 121.4 (s, C-35), 120.90 (s, C-22), 120.88 (d,
C-19), 120.1 (d, C-21), 112.6 (d, C-17), 111.1 (d, C-37), 88.2 (d, C-10),
80.0 (s, C-9), 76.1 (d, C-3), 73.3 (d, C-7), 65.8 (d, C-5), 60.5 (s, C-4), 56.1
(q, 10-OMe), 55.8 (q, 20-OMe), 52.1 (d, C-33), 51.2 (d, C-29), 45.9 (t, C-
39), 45.8 (t, C-15), 41.5 (t, C-24), 39.9 (t, C-28), 37.7 (d, C-6), 36.0 (q,
NMe), 35.8 (t, C-8), 34.9 (t, C-26), 33.6 (t, C-27), 33.0 (d, C-2’), 32.3 (t, C-
2), 31.8 (t, C-31), 26.5 (t, C-32), 19.8 (q, 2’-Mea), 17.7 (q, 2’-Meb), 15.3 (q,
14-Me), 14.5 (q, 6-Me), 11.5 ppm (q, 4-Me); HRMS (ESI): m/z: calcd for
C60H74N11O17S2 [M>M->H]ꢀ: 1284.4706, found: 1284.4694.
Descriptions of all experimental procedures and analytical characteriza-
tion can be found in the Supporting information.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft
(grant Ki 397/13-1). We thank Dr. J. Fohrer and Dr. E. Hofer for expert
NMR support.
Chem. Eur. J. 2012, 18, 880 – 886
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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