S. K. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 4973–4984
4981
isobutylchloroformate were added. The reaction mix-
ture was stirred for 4 h at ꢁ20 ꢁC. A solution of
167 mg (1.36 mmol) 4-aminobenzyl alcohol in dry tetra-
hydrofuran (4 mL) was added dropwise, and the reac-
tion mixture was allowed to come to room
temperature and then stirred for 16 h. Tetrahydrofuran
was evaporated, and dichloromethane was added. The
organic layer was washed with a saturated sodium bicar-
bonate solution, a 0.5 N potassium bisulfate solution,
and brine, then dried over anhydrous sodium sulfate
and evaporated. The residual crude product was purified
by column chromatography (ethyl acetate–hexane, 8:2)
purified by HPLC to give 25 mg (0.015 mmol, 65%) of
compound 2. H NMR (400 MHz, CDCl3) d 1.0 (6H,
1
m, 2 CH3-Leu), 1.21 (6H, s), 1.27 (3H, s), 1.29 (2H, m,
CH2-Lys), 1.54 (2H, m, CH2-Lys), 1.72 (3H, m), 1.75
(2H, m, CH2-Leu), 1.80 (3H, m, CH2-Lys and CH-
Leu), 1.9 (2H, m), 2.00 (2H, m), 2.02 (3H, s), 2.05
(3H, s), 2.16-2.18 (8H, m), 2.58 (2H, m, CH2-Leu),
2.78 (1H, s), 3.05 (2H, m, CH2-His), 3.38 (2H, m),
3.47 (4H, m, CH2-morpholine), 3.67 (4H, m, CH2-mor-
pholine), 4.0 (2H, m, Ha), 4.4 (2H, m), 4.5 (2H, m), 4.6
(3H, m, Ha), 4.65 (4H, m, 2CH2-Ser), 4.79 (2H, dd),
4.85 (1H, m, Ha), 4.94 (2H, dd), 5.52 (1H, s), 5.44
(2H, dd), 5.80 (1H, d), 6.10 (1H, d), 6.8 (1H, CH-His),
7.0 (1H, s), 7.12 (2H,), 7.15 (2H, d), 7.19 (9H, m), 7.24
(2H,), 7.37 (2H, d), 7.4 (2H, m), 7.44 (1H, CH-His),
7.46 (3H, m), 7.52 (7H, m), 7.84 (2H, d), 8.0 (2H, m).
MS (MALDI) m/e 1797 (M+H)+, 1818 (M+Na)+. Anal
(C89H113N13O27ÆH2O) C, H, N.
1
to yield 59 mg of the Fmoc-Gln-PABA 10 (92%): H
NMR (400 MHz, CD3OD) d 2.17 (2H, m), 2.18 (2H,
m), 4.4 (1H, m), 4.5 (1H, m), 4.79 (2H, dd), 7.17 (2H,
m), 7.2 (2H, m), 7.4 (2H, m), 7.57 (4H, m), 7.84 (2H,
d),; MS (EI) m/e 474 (M+H)+, 495 (M+Na)+. Anal.
(C27H27N3O5ÆH2O) C, H, N.
5.5. 20-Z -7-(Fmoc-Gln-PABA)-paclitaxel (11)
5.7. 20-[4-Nitrophenyl-carbonate]paclitaxel (12)
To a solution of 80 mg (0.07 mmol) 7 in dry tetrahydro-
furan-dichloromethane (2 mL, 1:1) under an argon
atmosphere were added 44 mg (0.09 mmol, 1.2 eq) 10
and N,N0-diisopropylethylamine (20 mg, 2 eq). The
reaction mixture was stirred at room temperature for
48 h. After 48 h, 25 mL of ethyl acetate was added.
The organic layer was washed with 10% citric acid,
brine, and water, then dried over anhydrous sodium sul-
fate and evaporated. The residue was subjected to col-
umn chromatography (ethyl acetate–hexane, 1:1;
chloroform–methanol, 30:1, respectively) to afford
60 mg of the desired carbonate 11 (58%); 1H NMR
(400 MHz, CDCl3) d 1.22 (6H, s), 1.28 (3H, s), 1.72
(5H, m), 2.00 (2H, m), 2.02 (3H, s), 2.05 (3H, s), 2.17
(2H, m), 2.18 (2H, m), 2.2 (1H, OH), 2.78 (1H, s),
3.38 (2H, m), 4.00 (1H, s), 4.4 (1H, m), 4.5 (2H, m),
4.79 (2H, dd), 4.94 (2H, dd), 5.52 (1H, s), 5.44 (2H,
dd), 5.80 (1H, d), 6.10 (1H, d), 7.0 (1H, s), 7.12 (2H,),
7.19 (9H, m), 7.4 (2H, m), 7.24 (2H,), 7.37 (2H, d),
7.46 (3H, m), 7.51 (5H, m), 7.84 (2H, d), 7.99 (2H, m),
8.2 (1H, NH); MS (EI) m/e 1488 (M+H)+, 1509
(M+Na)+. Anal (C83H82N4O22) C, H, N.
To a solution of 200 mg (0.23 mmol) paclitaxel in dry
dichloromethane under an argon atmosphere was added
pyridine (4 drops). At ꢁ50 ꢁC, 275 mg (6.0 eq) of
4-nitrophenyl chloroformate dissolved in dry dichloro-
methane was added. The reaction mixture was stirred
at ꢁ50 ꢁC, and after 4 h, 4-nitrophenyl chloroformate
(4.2 eq) was added. After 1 h, the mixture was diluted
with dichloromethane, washed with 0.5 N potassium
bisulfate and brine, and dried over anhydrous sodium
sulfate. After evaporation of the solvents, the residual
yellow film was purified by means of column chroma-
tography (ethyl acetate–hexane, 1:1) to yield 125 mg of
activated paclitaxel 7 (65%): 1H NMR (400 MHz,
CDCl3) d 1.17 (s, 3H), 1.24 (s, 3H), 1.69 (s, 3H), 1.92
(s, 3H), 2.25 (s, 3H), 2.49 (s, 3H), 2.55 (m, 1H), 3.82
(d, 1H), 4.24 (d, 1H), 4.38 (d, 1H), 4.42 (m, 1H), 4.96
(bd, 1H), 5.53 (d, 1H), 5.70 (d, 1H), 6.10 (dd, 1H),
6.29 (s, 1H), 6.34 (m, 1H), 6.90 (d, 1H), 7.34 (d, 2H),
7.37–7.65 (m, 11H), 7.75 (d, 2H), 8.15 (d, 2H), 8.26 (d,
2H); MS (FAB) m/e 1020 (M+H)+, 1042 (M+Na)+.
Anal. (C54H54N2O18) C, H, N.
5.8. 20-Fmoc-Gln-PABA-paclitaxel (13)
5.6. 7-[Mu-His-Ser-Ser-Lys-Leu-Gln-PABA]paclitaxel
(2)
To a solution of 100 mg (0.09 mmol) 12 in dry tetrahy-
drofuran-dichloromethane (5 mL, 1:1) under an argon
atmosphere were added 48 mg (0.09 mmol) 10 and
N,N0-diisopropylethylamine (25 mg, 2 equiv). The reac-
tion mixture was stirred at room temperature for 48 h.
After 48 h, ethyl acetate was added. The organic layer
was washed with 10% citric acid, brine, and water, dried
over anhydrous sodium sulfate, and evaporated. The
residue was purified by means of column chromatogra-
phy (ethyl acetate–hexane, 1:1; chloroform–methanol,
30:1, respectively) to afford 106 mg of the desired car-
To a solution of 50 mg (0.033 mmol) of compound 11 in
ethyl acetate was added a catalytic amount of 10% Pd-C.
The mixture was shaken for 3 h under an H2 atmo-
sphere. The Pd-C was removed by filtering through Cel-
ite. The solvent was evaporated to give 40 mg
(0.029 mmol, 89%) of 7-(Fmoc-Gln-PABA)-paclitaxel.
After addition of 20% piperidine in DMF, the mixture
was stirred for 30 min to give 7-(Gln-PABA)-paclitaxel,
which was further dried in vacuo for 2 h and utilized for
the following coupling reaction without further purifica-
tion: To a cooled solution of Mu-His-Ser-Ser-Lys(F-
moc)-Leu-OH (22 mg, 0.024 mmol), HBTU (9.2 mg,
0.024 mmol), and the above 7-(Gln-PABA)-paclitaxel
in 0.5 mL DMF, DIEPA (8 lL, 0.046 mmol) were added
and stirred for 6 h. To this mixture, acetonitrile and
water (1:1) (10 mL) was added, and the product was
1
bonate 13 (79%); H NMR (400 MHz, CDCl3) d 1.21
(6H, s), 1.29 (3H, s), 1.75 (5H, m), 2.0 (2H, m), 2.12
(3H, s), 2.15 (3H, s), 2.17 (4H, m), 2.2 (1H, OH), 2.78
(1H, s), 3.38 (2H, m), 4.00 (1H, s), 4.4 (1H, m), 4.5
(2H, m), 4.79 (2H, dd), 4.94 (2H, dd), 5.42 (1H, s), 5.7
(2H, dd), 5.80 (1H, d), 6.10 (1H, d), 7.0 (1H, s), 7.12
(2H,), 7.19 (4H, m), 7.4 (2H, m), 7.24 (2H,), 7.37 (2H,