2412
Chen and Gabathuler
2.84–3.05 (m, 3H), 3.10–3.20 (m, 3H), 3.35–3.55 (m, 4H), 3.95 (m, 4H),
4.05 (m, 1H), 4.16 (q, J ¼ 6.6 Hz, 1H), 4.57 (s, 2H), 4.85 (m, 1H), 5.21
(s, 1H), 5.45 (br, 1H), 7.40 (d, J ¼ 8.0 Hz, 1H), 7.62 (dd, J ¼ 3.3, 3.4 Hz,
1H), 7.85 (m, 2H), 13.25 (br, 1H), 13.99 (s, 1H) ppm. 13CNMR (75 MHz,
DMSO-d6) d 17.4, 18.6, 28.8, 28.9, 29.7, 33.8, 35.7, 43.8, 44.4, 45.1, 48.9,
56.9, 64.0, 67.1, 68.6, 70.3, 75.3, 100.8. 100.9, 111.1, 119.3, 120.1, 120.3,
134.9, 135.8, 136.5, 154.8, 156.4, 161.1, 171.0, 174.6, 175.7, 177.1, 186.7,
.
186.8, 214.1 ppm. Anal. Calcd for C49H65N3O6S 3H2O: C, 56.69; H,
6.89; N, 4.05. Found: C, 56.62; H, 6.86; N, 4.17.
Dox–SMCC–S-acetic acid 5. A sample of the dox–SMCC–S-acetic
acid ammonium salt (4, 242 mg) was dissolved in MeOH (2 mL). The solution
was loaded onto Dowex weakly acidic ion-exchange resin (8.34 g), pre-
packed in a column (1.6 ꢀ 32 cm2) and eluted with water. The red aqueous
solution was extracted with CH2C12 (3 ꢀ 60 mL). The extracts were com-
bined and dried over Na2SO4. The solids were vacuum filtered, and the filter
cake was washed with CH2Cl2 (3 ꢀ 10 mL). The filtrate and washings
were combined and the solvent was completely removed under vacuum to
afford 150 mg (71%) of the free acid as a red solid: m.p. l43–1508C;
[a]2D5 þ 202.508 (c, 0.12, MeOH/CH2C12, 1/1, v/v); ESI MS m/z ¼ 877
[C41H46N2O6S þ Na]; Rf 0.29 (88/10/2 CH2Cl2/MeOH/AcOH); UV
1
(MeOH/CH2CI2, 1/1, v/v) lmax 203, 234, 252, 286, 478, 495, 529 nm; H
NMR (300 MHz, DMSO-d6) d ¼ 0.86 (m, 2H), 1.18 (m, 5H), 1.35–1.65
(m, 1H), 1.61–1.67 (m, 3H), 1.85 (m, 1H), 2.00–2.25 (m, 3H), 2.50
(m, 2H), 2.90 (q, J ¼ 12.5 Hz, 2H), 3.17–3.20 (m, 3H), 3.38–3.46 (m, 3H),
3.62 (d, J ¼ 15 Hz, 1H), 3.96 (s, 4H), 4.05 (s, 1H), 4.14 (s, 1H), 4.58
(s, 2H), 4.62 (s, 1H), 4.75 (s, 1H), 4.91 (s, 1H), 5.21 (s, 1H), 5.35 (s, 1H),
7.28 (d, J ¼ 10 Hz, 1H), 7.58 (d, J ¼ 5 Hz, 3.4 Hz, 1H), 7.85 (m, 2H), 12.69
(br, 1H), 13.19 (s, 1H), 13.90 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6)
d ¼ 16.9, 28.4 28.5, 29.2, 32.6, 35.2, 35.3, 43.4, 44.0, 44.7, 56.5, 63.6,
66.7, 68.1, 69.8, 74.9, 100.3, 110.5, 110.6, 119.6, 119.9, 133.9, 134.5, 135.4,
154.4, 155.9, 160.7, 170.5, 174.2, 175.0, 176.4, 186.2, 186.3, 213.4 ppm;
Anal. Calcd for C41H46N2O16S: C, 57.60; H, 5.42; N, 3.28. Found: C, 57.87;
H. 5.59; N, 2.97.
Synthesis of Dox-p97 Conjugate 6
A mixture of dox–SMCC–S-acetic acid 5 (6.9 mg, 7.826 mmol, 50 equiv.
of p97), BTTU (6.3 mg, 0.01956 mmol, 2.50 equiv. of 5), triethylamine
(0.04 mL), DMF (1 mL) was stirred at room temperature for 60 min. TLC
confirmed the reaction completed (MeOH/CH2Cl2, v/v, 5/95). The solution
is saved for the following reaction.