P. C. A. Rodrigues et al. / Bioorg. Med. Chem. Lett. 13 (2003) 355–360
359
8. de Groot, F. M. H.; van Berkom, L. W. A.; Scheeren, H. W.
J. Med. Chem. 2000, 43, 3093.
9. Greenwald, R. B.; Gilbert, C. W.; Pendri, A.; Conover,
C. D.; Xia, J.; Martinez, A. J. Med. Chem. 1996, 39, 424.
10. Safavy, A.; Raisch, K. P.; Khazaeli, M. B.; Buchsbaum,
D. J.; Bonner, J. A. J. Med. Chem. 1999, 42, 4919.
75.59 (C-10), 75.11 (C-2), 72.12 (C-13), 72.11 (C-7), 58.52
(C-8), 53.20 (C-30), 45.59 (C-3), 43.19 (C-15), 35.55 (C-6),
35.54 (C-14), 26.90 (C-700), 26.83 (C-17), 22.69 (4-OCOCH3),
22.14 (C-16), 20.83 (10-OCOCH3), 14.88 (C-18), 9.61 (C-19).
MS (ESI: 4.5 kV, 250 ꢁC) on a Finnigan MAT 312 mass spec-
+
trometer: m/z=1022 (100) [M+ +Na ].
.
11. Paradis, R.; Page
´
, M. Anticancer Res. 1998, 18, 2711.
27. Data for one representative experiment is given: Prepara-
tion of 3a: 100 mg (0.1 mmol) 1, 173 mg (0.5 mmol) 3-mal-
eimidobenzoic acid hydrazide trifluoroacetate and 77 mL (1
mmol) trifluoroacetic acid were dissolved in 10 mL methanol and
stirred for a period of 14 h at room temperature in the dark. The
solvent was removed in vacuo and the residue dissolved in 1.5 mL
THF. Purification was performed by chromatography (silica gel,
12. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K.
J. Control. Rel. 2000, 65, 271.
13. Noguchi, Y.; Wu, J.; Duncan, R.; Strohalm, J.; Ulbrich,
K.; Akaike, T.; Maeda, H. Jpn. J. Cancer Res. 1998, 89, 307.
14. Ceruti, M.; Crosasso, P.; Brusa, P.; Arpicco, S.; Dosio, F.;
Cattel, L. J. Control. Rel. 2000, 63, 141.
15. Greenwald, R. B.; Conover, C. D.; Choe, Y. H. Crit. Rev.
Ther. Drug Carrier Syst. 2000, 17, 101.
16. Li, C.; Yu, D.; Inoue, T.; Yang, D. J.; Millas, L.; Hunter,
N. R.; Kim, E. E.; Wallace, S. Anti-Cancer Drugs 1996, 7, 642.
17. Wang, D.; Kopeckova, P.; Minko, T.; Nanayakkara, V.;
Kopecek, J. Biomacromolecules 2000, 3, 313.
18. Kratz, F.; Fichtner, I.; Roth, T.; Fiebig, H. H.; Unger, C.
J. Drug Targeting 2000, 8, 305.
19. Kratz, F.; Muller-Driver, R.; Hofmann, I.; Drevs, J.;
Unger, C. J. Med. Chem. 2000, 43, 1253.
20. Kratz, F.; Beyer, U.; Schumacher, P.; Kruger, M.; Zahn,
H.; Roth, T.; Fiebig, H. H.; Unger, C. Bioorg. Med. Chem.
Lett. 1997, 7, 617.
21. Beyer, U.; Roth, T.; Schumacher, P.; Maier, G.; Unold,
A.; Frahm, A. W.; Fiebig, H. H.; Unger, C.; Kratz, F. J. Med.
Chem. 1998, 41, 2701.
22. Rodrigues, P. A.; Schumacher, P.; Beyer, U.; Roth, T.;
Fiebig, H. H.; Unger, C.; Paper, D.; Messori, L.; Orioli, P.;
Mulhaupt, R.; Kratz, F. Bioorg. Med. Chem. 1999, 7, 2517.
23. Zalipsky, S. Adv. Drug Delivery Rev. 1995, 16, 157.
24. The synthesis ofmaleimide spacer derivatives 2a and 2c
were described previously. 2b was prepared according to 2a
and 2c. (see Beyer, U.; Kruger M.; Schumacher, P.; Unger, C.;
Kratz, F., Monatshefte Chemie 1997, 128, 91).
25. Paclitaxel was purchased from Hande Tech USA, Inc.
Preparation of 1: To 6 g (36.6 mmol) 4-acetylbenzoic acid dis-
solved in 250 mL toluene were added 80 mL (0.11 mol) thionyl
chloride, and the mixture was refluxed for 1 h. Thionyl chlo-
ride as well as toluene were removed in vacuo, and the residue
crystallized in 100 mL diethylether to yield the acid chloride as
yellow crystals.
˚
0.6–1.0 A, in THF/iso-hexane=2:1) to yield 3a as a colorless
solid. Analytical data: 3a: yield 54 mg (0.045 mmol): Rf=0.38
1
(THF/iso-hexane=2:1); H NMR (DMSO-d6/TMS): d=11.69/
10.99 (s, 1H, NH-N), 9.32 (d, 1H, NH), 8.12–7.28 (m, 23 arom.
H), 7.22 (s, 2H, 1000-H/1100-H), 6.31 (s, 1H, 10-H), 5.85 (t, 1H,
13-H), 5.77/5.57 (d, 1H, 30-H), 5.38 (d, 1H, 20-H), 5.36 (d, 1H,
2-H), 4.94 (dd, 1H, 5-H), 4.92 (s, 1H, 1-OH), 4.70 (s, 1H, 7-OH),
4.13 (dd, 1H, 7-H), 4.03/3.80 (dd, 2H, 20a-H/20b-H), 3.63 (d, 1H,
3-H), 2.43 (s, 3H, 700-H), 2.35 (m, 1H, 6a-H), 2.30 (s, 3H,
4-OCOCH3), 2.11 (s, 3H, 10-OCOCH3), 1.94/1.76 (m, 2H, 14-H),
1.85 (s, 3H, 18-H), 1.68 (m, 1H, 6b-H), 1.51 (s, 3H, 19-H), 1.04 (s,
3H, 16-H), 1.01 (s, 3H, 17-H); 13C NMR (75.4 MHz, DMSO-d6/
TMS): d=202.22 (C-9), 169.73 (C-900/C-1200), 169.62 (C-10),
168.66 (10-OCOCH3), 168.65 (4-OCOCH3), 166.69 (30-
NHCOPh), 166.68 (20-OCOPh), 165.09 (C-800), 164.82 (C-100),
150.15/146.25 (C-600), 143.10 (C-12), 139.14 (C-11), 134.94 (C-1000/
C-1100),
136.94/134.31/133.40/131.42/
129.83/129.48/129.42/
128.67/128.57/128.31/127.69/127.32/126.73 (C-arom.), 83.52 (C-5),
80.18 (C-4), 76.65 (C-1), 75.44 (C-20), 74.41 (C-20), 71.07 (C-10),
70.32 (C-2), 66.51 (C-13), 66.50 (C-7), 57.31 (C-8), 53.73 (C-30),
46.01 (C-3), 42.88 (C-15), 36.32 (C-6), 34.25 (C-14), 26.24 (C-17),
23.46 (4-OCOCH3), 22.45 (C-16), 20.56 (10-OCOCH3), 13.89 (C-
18), 13.84/13.47 (C-700), 9.69 (C-19); MS (ESI: 4.5 kV, 250 ꢁC): m/z
=1235 (100) [M+ꢄ +Na+], 1267 (40) [(Mꢀ1)+Na++CH3OH],
1022 (30) [(Mꢀ1)+Na+ꢀC11H7N2O3]. Anal. (C67H64N4O18)
M=1213.24 g/mol.
28. Benassi, R.; Taddei, F. Biochemistry 1985, 17, 1499.
29. HPLC studies were performed on a reversed-phase RP 18
(Lichrosorb, RT 250-4, Merck); mobile phase: acetonitrile/
0.004 M sodium phosphate (pH 6.0)=60/40; Kontron 422
pump (flow 1 mL/min); UV/VIS detector Kontron 535 and
integrator (at l=254 and 230 nm); Auto sampler Merck/
Hitachi AS4000, injection volume 50 mL PEEK-Loop.
26. To 700 mg (0.85 mmol) paclitaxel and 240 mL (1.7 mmol)
triethylamine dissolved in 50 mL CH2Cl2 were added dropwise
193 mg (1.06 mmol) 4-acetylbenzoic acid chloride dissolved in
10 mL CH2Cl2 over a period of30 min. The mixture was stir-
red for 12 h in the dark. The solvent was removed in vacuo
and the residue purified through chromatography on silica gel
30. e-values in THF: e280 (paclitaxel)=1180ꢂ90 Mꢀ1 cmꢀ1
,
e280 (1)=2880ꢂ120 Mꢀ1 cmꢀ1, e309 (3a)=18,400ꢂ250 Mꢀ1
cmꢀ1, e309 (3b)=18,930ꢂ250 Mꢀ1 cmꢀ1, e303 (3a)=21,940ꢂ
240 Mꢀ1 cmꢀ1
.
˚
(0.4–0.6 A//CH2Cl2/methanol=97:3) to yield 1 as a colorless
31. Data for one representative experiment is given: Prepara-
tion of PEG20000-(3a)2: 8.2 mg (7 mmol) of 3a were dissolved
in 1 mL dimethylformamide and added to 40 mg (2 mmol)
PEG-20000(SH)2 dissolved in 1 mL buffer (0.004 M sodium
phosphate, 0.15 M NaCl, pH 7.4). The mixture was homo-
genized and stirred at 30 ꢁC for 30 min. The solvent was
removed in vacuo, the residue dissolved in 2 mL methanol and
chromatographed over Sephadex1 LH20 Gel (100 mmꢅ10
mm, loop size: 2 mL, flow: 1.0 mL/min, retention time: 15–16
min, eluent: 100% methanol HPLC grade, l=280 nm).
Methanol was removed in vacuo, the residue dissolved in 200
mL CH2Cl2 and the product was precipitated with diethylether.
The colorless solid was dried in vacuo. The purity ofthe
samples was analyzed through a semi-analytical FPLC-col-
umn (Sephadex1 LH 20, flow: 0.1 mL/min of100% Methanol
HPLC-grade, retention time: 15–17 min, l=280 nm).
solid. Analytical data: 1: yield 781 mg (0.78 mmol); Rf 0.7
(THF: iso/hexane=2:1); 1H NMR data: 300 MHz (CDCl3) f or
1: d=8.12 (dd, 2H, arom. H: O–Bz), 8.02/8.03 (dd, 4H, arom.
H: 300-H/400-H), 7.75 (dd, 2H, arom. H: N–Bz), 7.62–7.3 (m,
11H, arom. H), 7.05 (d, 1H, NH), 6.3 (s, 1H, 10-H), 6.26 (t,
1H, 13-H), 6.07 (dd, 1H, 30-H), 5.71 (d, 1H, 20-H), 5.67 (d, 1H,
2-H), 5.30 (s, 1H, 1-OH), 5.15 (s, 1H, 7-OH), 4.97 (dd, 1H,
5-H), 4.45 (dd, 1H, 7-H), 4.31/4.18 (dd, 2H, 20a-H/20b-H),
3.82 (d, 1H, 3-H), 2.64 (s, 3H, 700-H), 2.54 (m, 1H, 6a-H), 2.45
(s, 3H, 4-OCOCH3), 2.34/2.14 (m, 2H, 14-H), 2.22 (s, 3H,
10-OCOCH3), 1.97 (s, 3H, 18-H), 1.85 (m, 1H, 6b-H), 1.67 (s,
3H, 19-H), 1.23 (s, 3H, 16-H), 1.13 (s, 3H, 17-H). 13C NMR-data:
(75.4 MHz, CDCl3): d=203.80 (C-9), 197.30 (C-600), 171.28 (C-10),
169.80 (10-OCOCH3), 167.99 (4-OCOCH3), 167.21 (30-
NHCOPh), 167.03 (20-OCOPh), 164.89 (C-100), 142.66 (C-12),
140.96
130.13/129.22/128.81/128.75/128.46/127.07/126.67
84.46 (C-5), 81.09 (C-4), 79.19 (C-1), 77.21 (C-20), 76.44 (C-20),
(C-11),
136.71/133.67/132.90/132.23/132.12/130.22/
32. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.
33. FPLC studies: Studies at pH 4.0 and pH 7.4 were per-
formed with the PEG paclitaxel conjugates. 50 mL ofthe stock
(C-arom.)