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T. Sambaiah et al. / European Journal of Medicinal Chemistry 37 (2002) 349–353
parts per million (ppm) and coupling constants (J) are
given in Hertz (Hz). The splitting pattern abbreviations
are as follows: s, singlet; d, doublet; t, triplet; q, quar-
tet; br, broad; m, unresolved multiplet; and dd, doublet
of doublets. Electrospray ionisation mass spectrometry
(ESI–MS) analyses were performed on a quadrupole
ion trap mass analyser fitted with an electrospray ioni-
sation source (Finnigan LCQ, Finnigan MAT, San
Jose, CA). A three-point calibration was carried out
with a standard mixture of the peptide MRFA, caf-
feine, and Ultramark. Samples were injected at 5.0 mL
min−1. Ions were produced with a spray voltage of 4.25
keV, with the heated capillary set at 200 °C. Spectra
were collected in the positive ion mode. Each spectrum
was an average of 10–20 individual scans, which were
composed of three microscans.
Purification on silica gel refers to gravity column
chromatography or preparative thin layer chromatogra-
phy on Merck silica gel. Analytical thin layer chro-
matography was performed on precoated plates
purchased from Merck (silica gel 60 F254).
2%-(Triethylsilyl)paclitaxel (2) was prepared according
to the literature method [21,22]. The immunofluores-
cence assay was performed by use of a Zeiss Axiophot
microscope.
(d, J=8.0 Hz, 2H, ArH), 8.14 (d, J=7.6 Hz, 2H,
ArH); 13C-NMR (CDCl3, 75 MHz): l 4.32, 6.47, 10.84,
14.45, 20.61, 21.34, 22.92, 26.35, 33.36, 35.42, 35.55,
39.01, 43.32, 46.77, 55.12, 55.69, 55.94, 70.32, 71.36,
74.51, 74.69, 75.12, 76.36, 78.55, 80.94, 83.98, 113.02,
126.07, 126.43, 127.01, 127.71, 127.98, 128.53, 128.68,
128.74, 129.07, 129.77, 130.17, 131.74, 133.69, 134.08,
138.24, 138.36, 140.85, 146.34, 157.75, 167.09, 168.77,
169.80, 171.59, 172.04, 201.75.
5.1.2. 7-(i-Alanyl)paclitaxel (4) [23]
A solution of tritylated paclitaxel 3 (39.8 mg, 0.0306
mmol) containing a mixture of AcOH, H2O, and THF
(6:3:1, 5.0 mL) was heated at 45–50 °C with stirring
for 24 h. The reaction mixture was cooled to r.t.,
neutralised with 10% NaHCO3 aq. solution, and ex-
tracted with EtOAc (2×30 mL). The combined organic
layers were washed with water (20 mL), saturated NaCl
aq. solution (20 mL), dried over MgSO4 (s), filtered,
and concentrated under reduced pressure. The residue
was purified by use of the preparative thin layer chro-
matography on silica gel (10% MeOH in CH2Cl2 as
eluant) to afford 4 (16.8 mg, 0.0182 mmol) as a colour-
1
less solid in 59% yield: H-NMR (CDCl3, 300 MHz): l
1.10 (s, 3H, C(17)H3), 1.17 (s, 3H, C(16)H3), 1.76 (s,
3H, C(19)H3), 1.81 (s, 3H, C(18)H3), 2.13–2.20 (m, 3H,
COCH2+C(6)Hb), 2.15 (s, 3H, C(4)OCOCH3), 2.35–
2.43 (m, 2H, C(14)H2), 2.38 (s, 3H, C(10)OCOCH3),
2.43–2.58 (m, 1H, C(6)Ha), 2.65 (br, t, J=7.3 Hz, 2H,
NCH2), 3.32 (br, s, 2H, NH2), 3.84 (d, J=6.9 Hz, 1H,
C(3)H), 4.14 (d, J=7.8 Hz, 1H, C(20)Hb), 4.27 (d,
J=7.8 Hz, 1H, C(20)Ha), 4.86 (d, J=2.8 Hz, 1H,
C(2%)H), 4.92 (d, J=8.8 Hz, 1H, C(5)H), 5.58–5.73 (m,
3H, C(2)H+C(3%)H+C(7)H), 6.09 (br, t, J=8.0 Hz,
1H, C(13)H), 6.12 (s, 1H, C(10)H), 6.85 (d, J=7.2 Hz,
1H, NH), 7.26–7.63 (m, 11H, ArH), 7.82 (d, J=7.1
Hz, 2H, ArH), 8.08 (d, J=7.68 Hz, 2H, ArH); 13C-
NMR (CDCl3, 75 MHz): l 10.83, 14.48, 20.81, 21.15,
22.53, 26.37, 31.08, 33.07, 35.58, 35.67, 43.09, 46.84,
55.58, 55.89, 71.52, 71.98, 73.87, 74.68, 75.08, 76.22,
78.58, 80.72, 83.63, 127.27, 127.34, 127.87, 128.55,
128.78, 129.12, 130.15, 131.76, 132.38, 132.94, 133.48,
138.30, 141.39, 166.79, 167.60, 170.66, 170.96, 171.04,
172.96.
5.1.1. 2%-(Triethylsilyl)-7-[N-(4-methoxytrityl)-
i-alanyl]paclitaxel (3)
To a solution of 2%-(triethylsilyl)paclitaxel (2) (18.3
mg, 0.0189 mmol, 1.0 equiv.) and N-(4-methoxytrityl)-
b-alanine (7.7 mg, 0.022 mmol, 1.2 equiv.) in CH2Cl2
(5.0 mL) were added DCC (20.1 mg, 0.0974 mmol, 5.2
equiv.) and DMAP (3.10 mg, 0.0254 mmol, 1.3 equiv.).
The reaction mixture was stirred at room temperature
(r.t.) under nitrogen atmosphere for 36 h. After filtra-
tion, the filtrate was washed with water (2×10 mL),
saturated NaCl aq. solution (5.0 mL), dried over
MgSO4 (s), filtered, and concentrated under reduced
pressure. The residue was purified by column chro-
matography on silica gel (20% EtOAc in hexanes as
eluant) to give 3 (16.1 mg, 0.0124 mmol) as a colourless
1
solid in 66% yield: H-NMR (CDCl3, 300 MHz): l 0.45
(q, J=8.0 Hz, 6H, 3×SiCH2), 0.83 (t, J=8.0 Hz, 9H,
3×CCH3), 1.16 (s, 3H, C(17)H3), 1.21 (s, 3H,
C(16)H3), 1.79 (s, 3H, C(19)H3), 1.96 (s, 3H, C(18)H3),
2.10 (s, 3H, C(4)OCOCH3), 2.16–2.22 (m, 3H,
COCH2+C(6)Hb), 2.35–2.50 (m, 4H, NCH2+
C(14)H2), 2.54 (s, 3H, C(10)OCOCH3), 2.58–2.62 (m,
1H, C(6)Ha), 3.76 (s, 3H, OCH3), 3.92 (d, J=7.3 Hz,
1H, C(3)H), 4.22 (d, J=8.1 Hz, 1H, C(20)Hb), 4.34 (d,
J=8.1 Hz, 1H, C(20)Ha), 4.70 (d, J=1.8 Hz, 1H,
C(2%)H), 4.96 (d, J=8.1 Hz, 1H, C(5)H), 5.58 (dd,
J=9.5, 6.2 Hz, 1H, C(7)H), 5.70 (m, 2H, C(2)H+
C(3%)H), 6.23 (br, t, J=8.3 Hz, 1H, C(13)H), 6.25 (s,
1H, C(10)H), 6.79 (d, J=8.9 Hz, 2H, ArH), 6.84 (d,
J=7.8 Hz, 1H, NH), 7.08–7.62 (m, 23H, ArH), 7.75
5.1.3. 7-(5%-Biotinylamidopropanoyl)paclitaxel (5)
A solution of biotinyl-N-hydroxysuccinimide (15.1
mg, 0.0442 mmol, 2.0 equiv.) in dry DMF was added to
a stirred solution of 7-(b-alanyl)paclitaxel (4) (20.4 mg,
0.0221 mmol, 1.0 equiv.) and N-ethylmorpholine (4.40
mg, 0.038 mmol, 1.7 equiv.) in CH2Cl2 (5.0 mL) at
0 °C. After the solution was stirred at r.t. for 10 h, the
solvent was removed under reduced pressure. The
residue was dissolved in EtOAc (50 mL), washed with
10% citric acid solution (10 mL), water (10 mL), dried
over MgSO4 (s), and concentrated under reduced pres-