Synthesis of deuterium-labelled paclitaxel and its hydroxyl metabolite

Article abstract of DOI:10.1002/jlcr.1870

This paper describes the synthesis of deuterium-labelled paclitaxel and its hydroxyl metabolite. Paclitaxel labelled with ²H was obtained in four steps using the commercially available [²H₅]benzoic chloride as the stable l

Full text of DOI:10.1002/jlcr.1870

Research Article
Received 18 May 2010,
Revised 10 November 2010,
Accepted 13 November 2010
Published online 17 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1870
Synthesis of deuterium-labelled paclitaxel
and its hydroxyl metabolite
Ã
Lei Tian,a Keying Wu,a Jie Tao,a and Liqin Chenb
This paper describes the synthesis of deuterium-labelled paclitaxel and its hydroxyl metabolite. Paclitaxel labelled with 2H
was obtained in four steps using the commercially available [2H5]benzoic chloride as the stable labelled reagent with a 40%
overall yield. The hydroxyl metabolite labelled with 2H was prepared starting from deuterium-labelled paclitaxel in six
steps with a 42% overall yield based on unrecovered starting material.
Keywords: deuterium labelled; paclitaxel; metabolite; 6a-hydroxy-paclitaxel
stirred at room temperature for 8 h. The solvent and excess
chlorotriethylsilane were removed under reduced pressure at
Introduction
Paclitaxel, a substance originally isolated from the Pacific yew tree,
has been approved for the clinical treatment of cancer patients
301C. The remaining residue was dissolved in dichloromethane
(200mL) and washed with H2O (5 ꢀ 50mL). The organic layer was
and now became one of the most significant drugs in cancer
therapy.1 This molecule exerts its special anticancer activity by
inhibiting mitosis through enhancement of the polymerization of
tubulin and consequent stabilization of microtubules.2 The
determination of paclitaxel and its metabolites in biological fluids
has been reported in the literature.3–7 6a-Hydroxy-paclitaxel was
detected to be the principal metabolite of paclitaxel in human
hepatic microsomes, human liver slices, and patient biliary
excretions.4,5 Metabolism studies revealed that paclitaxel is mainly
metabolized by hydroxylation through CYP2C8 to the still active
metabolite 6a-hydroxyl metabolite, as shown in Figure 1.7
dried over anhydrous Na2SO4, and concentrated under reduced
pressure to give a white solid, which was recrystallized from
CH2Cl2/hexanes to afford (2) as a white solid (10.01g, 83.7%).
1H-NMR(CDCl3) d ppm: 8.12(dd, 2H, o-Ph), 7.62(t, 1H, p-Ph),
7.48(m, 2H, m-Ph), 6.46 (s, 1H, 10-H), 5.65(d, 1H, J = 6.0 Hz, 2-H),
4.95(d, 1H, J = 9.0 Hz, 5-H), 4.83 (t, 1H, 13-H), 4.50(dd, 1H, J = 12.0,
6.0 Hz, 7-H), 4.31(d, 1H, J = 9.0 Hz, 20-Ha), 4.13 (d, 1H, J = 9.0 Hz,
20-Hb), 3.90(d, 1H, J = 6.0 Hz, 3-H), 2.51(m, 1H, 6-Ha), 2.29(s, 3H,
4-COCH3), 2.27(m, 2H, 14-H), 2.19(s, 3H, 10-COCH3), 2.17(s, 3H,
18-CH3), 1.88(m, 1H, 6-Hb), 1.63(s, 3H, 19-CH3), 1.20(s, 3H, 17-CH3),
1.04(s, 3H, 16-CH3), 0.93 (t, 3H, SiCH2 CH3), 0.55(m, 2H, SiCH2 CH3).
Identification and quantification of drugs and metabolites by
LC/MS relies very much on stable isotope-labelled analogues.8,9
A renewed interest has been recently raised to develop a robust
and validated LC/MS method to determine paclitaxel and its
metabolite in biological fluids. The preparation of the stable
labelled versions of the title compounds with M15 was
13-((4S, 5R)-3-(tert-butoxycarbonyl)-2-(4-methoxyl-benzyl)-
4-phenyl-5-oxazolidinecarboxylic formyl)-7-trithylsilyl bac-
catin III (4)
To a stirred solution of (2) (5.00 g, 7.1 mmol) and (4S, 5R)-3-(tert-
butoxycarbonyl)-2-(4-methoxyl-benzyl)-4-phenyl-5-oxazolidine-
carboxylic acid (3) (8.55 g, 21.4 mmol) in dry toluene (214 mL)
was added DCC (4.30 g, 20.8 mmol) and DMAP (0.44 g,
3.6 mmol). The reaction was allowed to proceed at 801C for
1 h. The reaction mixture was cooled to room temperature and
stirred for 1 h, followed by filtering the resulting dicyclohexylur-
ea. The filter cake was rinsed thoroughly with toluene (50 mL)
and the combined organic layer was concentrated under
3
requested. Although H-paclitaxel and 14C-paclitaxel have been
prepared for pharmacological studies,10,11 the synthesis of its
stable labelled internal standards has not been disclosed in
detail.12 In this paper, the synthetic routes to [2H5]paclitaxel and
its 6a-hydroxyl metabolite are described in detail.
Experimental
General
aCollege of Material Science and Technology, Nanjing University of Aeronautics
and Astronautics, 29 Yudao Street, Nanjing, Jiangsu 210016, People’s Republic of
China
All reagents were obtained from Sigma-Aldrich and CDN Isotope.
Mass spectra were recorded using a Quattro micro API mass
spectrometer. 1H NMR spectra were recorded on a Bruker
300MHz instrument. Chemical purities were determined by an bLiqin Chen, Hi-Tech Research Institute and State Key Laboratory of Materials-
Oriented Chemical Engineering, Nanjing University of Technology, 5 Xinmofan
Road, Nanjing, Jiangsu 210009, People’s Republic of China
Agilent 1200 HPLC with a XDB-C18 column, 5 mm, 4.6 ꢀ 150 mm.
7-Triethylsilyl baccatin III (2)
*Correspondence to: Liqin Chen, Hi-Tech Research Institute and State Key
Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of
Technology, 5 Xinmofan Road, Nanjing, Jiangsu 210009, People’s Republic of China.
E-mail: liqin_chen@hotmail.com
A solution of Baccatin III (10 g, 17mmol) in dry pyridine (250mL)
was treated with chlorotriethylsilane (69.3 mL, 341 mmol) and
J. Label Compd. Radiopharm 2011, 54 318–323
Copyright r 2011 John Wiley & Sons, Ltd.

L. Tian et al.
20-Ha), 4.06(d, 1H, J = 9.0 Hz, 20-Hb), 3.60(d, 1H, J = 6.0 Hz, 3-H),
2.40(m, 1H, 6-Ha), 2.18(s, 3H, 4-COCH3), 2.16(m, 2H, 14-H), 2.14(s,
3H, 10-COCH3), 1.78(s, 3H, 18-CH3), 1.78(s, 3H, 19-CH3), 1.68(s,
1H, 6-Hb), 1.59(s, 3H, 17-CH3), 1.11(s, 3H, 16-CH3).
[2H5]Paclitaxel (6)
The solution of [2H5]benzoyl chloride (0.47 g, 3.23 mmol) in
CH2Cl2 (5 mL) was added slowly to a solution of (5) (2.19 g,
2.92 mmol) in CH2Cl2 (29.2 mL) containing Et3N (0.55 mL,
3.96 mmol) while cooling in an ice-bath. The reaction mixture
was stirred at room temperature for half an hour and the solvent
was removed under reduced pressure to give a white solid. The
crude product was purified by flash chromatography on a silica
gel column, eluted with CH2Cl2/MeOH (99:1) to afford (6) as a
white solid (2.01 g, 80.4%).
1H-NMR (300 MHz, CDCl3) d ppm: 8.14(dd, 2H, o-Ph), 7.61(dd,
2H, o-Ph), 7.53(t, 1H, p-Ph), 7.48(m, 2H, m- Ph), 7.36–7.43(m, 3H,
Ph), 6.98(d, 1H, J = 9.0 Hz, NH), 6.28(s, 1H, 10-H), 6.23 (t, 1H,
J = 9.0 Hz, 13-H), 5.80(dd, J = 9.0, 3.0 Hz, 1H), 5.68(d, 1H, J = 7.0 Hz,
2-H), 4.97(d, 1H, J = 9.0 Hz, 5-H), 4.80(d, 1H, J = 3.0 Hz, 20-H),
4.40(dd, 1H, J = 12.0, 6.0 Hz, 7-H), 4.29(d, 1H, J = 9.0 Hz, 20-Ha),
4.20(d, 1H, J = 9.0 Hz, 20- Hb), 3.80(d, 1H, J = 6.0 Hz, 3-H), 2.54(m,
1H, 6-Ha), 2.38(s, 3H, 4-COCH3), 2.30(m, 1H, 14-H), 2.23(s, 3H,
10-COCH3), 1.88(m, 2H, 6-Hb,14-H), 1.80(s, 3H, 18-CH3), 1.69(s, 3H,
19-CH3), 1.24(s, 3H, 17-CH3), 1.15(s, 3H, 16-CH3). HPLC (XDB-C18,
CH3CN/H2O = 53/47, 1.0 mL/min): tR 5.96 min (499.3%). MS-EI
(m/z): 860.1(20), 881.3(MNa1, 30), 932.4(100), 933.4(55).
Figure 1. Paclitaxel is mainly metabolized by CYP2C8 through hydroxylation at
a-C-6 to provide 6a-hydroxy-paclitaxel, which is still active.
[2H5]20-O-(t-Butyldimethylsilyl)-paclitaxel (7)
reduced pressure to give a yellow solid. The crude product was
purified on a silica gel column, eluted with hexanes/ethyl
acetate (7:3) to afford (4) as a light yellow solid (6.42 g, 82.9%).
1H-NMR (300 MHz, CDCl3) d ppm: 8.03(dd, 2H, o-Ph), 7.61(t, 1H,
p-Ph), 7.50(m, 2H, m-Ph), 7.47(m, 2H, Ph), 7.42(m, 7H, Ph),
6.35(s, 1H, 10-H), 6.08(t, 1H, J = 9.0 Hz, 13-H), 5.65(d, 1H,
J = 6.0 Hz, 2-H), 5.04(m, 1H, 40-H), 4.87(dd, 1H, J = 9.0, 3.0 Hz,
5-H), 4.46(d, 1H, J = 6.0 Hz, 50-H), 4.45(dd, 1H, J = 9.0, 6.0 Hz, 7-H),
4.25(d, 1H, J = 9.0 Hz, 20-Ha), 4.10(d, 1H, J = 9.0 Hz, 20-Hb),
3.81(s, 3H, OCH3), 3.80(d, 1H, J = 6.0 Hz, 3-H), 2.51(m, 1H, 6-Ha),
2.18(s, 3H, 4-COCH3), 2.17(s, 3H, 10-COCH3), 2.16(m, 2H, 14-H),
2.05(s, 1H, 18-CH3), 1.82(m, 1H, 6-Hb), 1.66(s, 1H, 19-CH3), 1.22(s,
1H, 17-CH3), 1.21(s, 1H, 16-CH3), 1.10(brs, 9H, Boc), 0.92(t, 3H,
SiCH2CH3), 0.56(m, 2H, Si CH2 CH3).
To a solution of [2H5]paclitaxel (2.5 g, 2.91 mmol) in dry
DMF(25 mL) was added imdazole (0.9908 g, 14.55 mmol) and
t-butyldimethylchlorosilane (1.0982 g, 7.28 mmol). The mixture
was stirred at 701C for 8 h. The reaction mixture was diluted with
ethyl acetate (50 mL) and washed with H2O (50 mL), 2% HCl
solution (50 mL), NaHCO3 solution (50 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure to give a
white solid. The crude product was purified by column
chromatography on a silica gel column, eluted with hexanes/
ethyl acetate (6:4) to afford (7) as a white solid (2.2 g, 77.7%).
1H-NMR (300 MHz, CDCl3) d ppm: 8.14(dd, 2H, o-Ph), 7.60(dd,
2H, o-Ph),7.55(t, 1H, p-Ph), 7.32–7.43(m, 5H, Ph), 7.05(d, 1H,
J = 9.0 Hz, NH), 6.30(s, 1H, 10-H), 6.28(t, 1H, J = 9.0 Hz, 13-H),
5.73(dd, 1H, J = 9.0, 6.0 Hz, 30-H), 5.68(d, 1H, J = 6.0 Hz, 2-H),
4.98(d, 1H, J = 9.0 Hz, 5-H), 4.66(d, 1H, J = 3.0 Hz, 20-H), 4.42(dd,
1H, J = 12.0, 6.0 Hz, 7-H), 4.29(d, 1H, J = 9.0 Hz, 20-Ha), 4.20(d, 1H,
J = 9.0 Hz, 20-Hb), 4.12(m, H, OH), 3.82(d, 1H, J = 6.0 Hz, 3-H),
2.57(s, 3H, 4-COCH3), 2.45(m, 1H, 6-Ha), 2.36(m, 1H, 14-H), 2.23(s,
3H, 10-COCH3), 2.12(m, 2H, 6-Hb, 14-H), 1.90(s, 3H, 18-CH3),
1.80(s, 3H, 19-CH3), 1.24(s, 3H, 17-CH3), 1.14(s, 3H, 16-CH3), 0.80
(s, 9H, C(CH3)3), ꢁ0.04(t, 3H, SiCH3), 0.29(s, 3H, SiCH3).
13-(O-2R-Hydroxy-3S-amine-phenylpropionyl)-baccatin III (5)
A solution of (4) (8.47 g, 7.83 mmol) in formic acid (176.1 mL) was
stirred at room temperature for 2 h. The mixture was concen-
trated to about 20 mL, diluted with CH2Cl2 (50 mL), neutralized
with saturated NaHCO3 solution and solid Na2CO3 to pH 8. The
organic layer was separated and the aqueous layer was
extracted with CH2Cl2/methanol 95:5 (6 ꢀ 50 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to afford a yellow solid. The
crude product was purified by flash chromatography on a silica
[2H5]20-O-(t-Butyldimethylsilyl)-7b-O-trifluoromethanesulfo-
nylpaclitaxel (8)
gel column, eluted with CH2Cl2/MeOH (95:5) to afford (5) as a To a stirred solution of silyl ether (7) (2.66g, 2.73mmol) in dry
brown solid (3.3 g, 56.2%). trichloromethane (26.6 mL) was added DMAP (0.5009 g, 4.1 mmol)
1H-NMR(CDCl3)d ppm: 7.80(dd, 2H, o-Ph), 7.66(m, 1H, p-Ph), and trifluoromethanesulfonyl chloride (0.5527g, 3.28mmol).
7.62(m, 2H, o-Ph), 7.53(m, 2H, m-Ph), 7.36(m, 3H, Ph), 6.32(s, 1H, The mixture was kept stirring at room temperature for 2.5 h.
10-H), 5.98(t, 1H, J = 9.0 Hz, 13-H), 5.53(d, 1H, J = 6.0 Hz, 2-H), The reaction mixture was diluted with trichloromethane (150mL)
4.93(d, 1H, J = 9.0 Hz, 30-H), 4.86(d, 1H, J = 9.0 Hz, 5-H), 4.78(d, 1H, and washed with H2O (2 ꢀ 100 mL), 1% HCl solution (100mL),
J = 9.0 Hz, 20-H), 4.27(brt, 1H, J = 6.0 Hz, 7-H), 4.18(d, 1H, J = 9.0 Hz, H2O (100mL), saturated NaHCO3 solution (100mL), dried over
J. Label Compd. Radiopharm 2011, 54 318–323
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

L. Tian et al.
anhydrous Na2SO4, and concentrated under reduced pressure to J = 9.0 Hz, NH), 6.83(s, 1H, 10-H), 6.29(t, 1H, J = 9.0 Hz, H-13),
give a white solid. The crude product was purified by column 5.77(dd, 1H, J = 9.0, 3.0 Hz, 30-H), 5.74(d, 1H, J = 6.0 Hz, 2-H),
chromatography on a silica gel column, eluted with hexanes/ethyl 4.67(d, 1H, J = 3.0 Hz, 5-H), 4.66(brs, 2H, 20-H), 4.37(s, 1H, 20-H),
acetate (3:1) to afford (8) as a white solid (2.75 g, 91%).
4.12(m, 1H, 6-H), 3.87(d, 1H, J = 7.0 Hz, H-3), 3.71(dd, 1H, J = 12.0,
1H-NMR(300 MHz, CDCl3) d ppm: 8.13(dd, 2H, o-Ph), 7.61(d, 6.0 Hz, 7-H), 2.90(d, 1H, J = 9.0 Hz, OH), 2.68(s, 3H, 4-COCH3),
2H, o-Ph), 7.53(t, 1H, p-Ph), 7.33–7.42 (m, 5H, Ph), 7.06(d, 1H, 2.33(m, 1H, 14-Ha´), 2.20(s, 3H, 10-COCH3), 2.13(m, 1H, 14-Hb),
J = 9.0 Hz, NH), 6.62(s, 1H, 10-H), 6.25(t, 1H, J = 9.0 Hz, 13-H), 1.91(s, 3H, 18-CH3), 1.60(s, 3H, 19-CH3), 1.24(s, 3H, 17-CH3),
5.75(dd,2H, J = 9.0, 3.0 Hz, 2-H), 5.48(dd, 1H, J = 12.0, 6.0 Hz, 7-H), 1.14(s, 3H, 16-CH3), 0.78(s, 9H, C(CH3)3), ꢁ0.04(t, 3H, SiCH3),
4.96(d, 1H, J = 9.0 Hz, 5-H), 4.64(d, 1H, J = 3.0 Hz, 20-H), 4.36(d, 1H, ꢁ0.30(s, 3H).
J = 9.0 Hz, 20-Ha), 4.22(d, 1H, J = 9.0 Hz, 20-Hb), 4.14(m, 1H, OH),
4.12(m, 1H, OH), 3.96(d, 1H, J = 6.0 Hz, 3-H), 2.86(m, 1H, 6-Ha), [2H5]20-O-(t-Butyldimethylsilyl)-6a-hydroxy-paclitaxel (11)
2.60(s, 3H, 4-COCH3), 2.39(m, 1H,14-H), 2.20(s, 3H, 10-COCH3),
To a solution of (10) (1.95 g, 1.971 mmol) in dry toluene(19.5 mL)
2.17(m, 1H, 6-Hb), 2.17(m, 1H, 14-H), 2.06(s, 3H, 18-CH3), 1.90(s,
was added 1, 8-diazobicyclo [5, 4, 0] udec-7-ene (DBU, 0.6 g,
3H, 19-CH3), 1.22(s, 3H, 17-CH3), 1.19(s, 3H, 16-CH3), 0.80(s, 9H,
3.94 mmol) and the mixture was stirred at 801C for 1 h. The
C(CH3)3), ꢁ0.02(s, 3H, SiCH3), ꢁ0.29(s, 3H, SiCH3).
reaction mixture was concentrated under reduced pressure to
give a brown solid. The crude product was purified by flash-
[2H5]20-O-(t-Butyldimethylsilyl)-6,7-dehydropaclitaxel (9)
chromatograph on a silica gel, eluted with hexanes/ethyl
acetate (7:3) to afford (11) as a white solid (0.15 g, 7.67%) and
(10) as a white solid too (1.6 g, 82%).
To a stirred solution of (8) (1.75 g, 1.58 mmol) in CH2Cl2 (17.5 mL)
was added 1, 8- diazabicyclo [5, 4, 0] undec-7-ene (2.4104 g,
15.8 mmol). The mixture was kept stirring at 401C for 8 h. The
solvent was removed under reduced pressure to afford a white
solid. The crude product was purified by flash chromatography
on a silica gel column, eluted with hexanes/ethyl acetate (7:3) to
afford (9) as a white solid (1.54 g, 98.7%).
1H-NMR (300 MHz, CDCl3) d ppm: 8.10(dd, 2H, o-Ph), 7.50(d,
2H, o-Ph), 7.38(t, 1H, p-Ph), 7.33–7.42(m, 5H, Ph), 7.06(d, 1H,
J = 9.0 Hz, NH), 6.25(t, 1H, J = 9.0 Hz, 13-H), 6.24(s, 1H, 10-H),
6.10(dd, 1H, J = 9.0, 6.0 Hz, 6-H), 5.88(d, 1H, J = 9.0 Hz, 7-H),
5.86(d, 1H, J = 6.0 Hz, 2-H), 5.55(d, 1H, J = 9.0 Hz, 30-H), 4.67(d, 1H,
J = 3.0 Hz, 20-H), 4.20(d, 1H, J = 9.0 Hz, 20-Ha), 4.12(d, 1H,
J = 9.0 Hz, 20-Hb), 4.03(d, 1H, J = 6.0 Hz, 3-H), 2.58(s, 3H,
4-COCH3), 2.40(m, 1H, 14-Ha), 2.22(m, 2H, 6-H, 14-Hb), 2.11(s,
3H, 10-COCH3), 1.88(s, 3H, 18-CH3), 1.82(s, 3H, 19-CH3), 1.26(s,
3H, 17-CH3), 1.15(s, 3H, 16-CH3), 0.79(s, 9H, C(CH3)3), ꢁ0.05(s, 3H,
SiCH3), ꢁ0.31(s, 3H, SiCH3).
1H-NMR (300 MHz, CDCl3) d ppm: 8.14(dd, 2H, o-Ph), 7.60(d,
2H, o-Ph), 7.53(t, 1H, p-Ph), 7.32–7.43 (m, 5H, Ph), 7.06(d, 1H,
J = 9.0 Hz, NH), 6.29(s, 1H, 10-H), 6.29(m, 1H, J = 9.0 Hz, H-13),
5.77(d, 1H, J = 9.0, 3.0 Hz, 30-H), 5.74(d, 1H, J = 9.0 Hz, 2-H), 4.86
(d, 1H, 5-H), 4.66(d, 1H, J = 3.0 Hz, 20-H), 4.34(d, 1H, J = 9.0 Hz, 20-
Ha), 4.24(m, 1H, 7-H), 4.23(d, 1H, J = 9.0 Hz, 20-Hb) 3.96(m, 2H, 6-H
and 3-H), 2.59(s, 3H, 4-COCH3), 2.42(m, 1H, 14-Ha), 2.26(s, 3H,
10-COCH3), 2.14(m, 1H, 14-Hb), 1.91(s, 3H, 18-CH3), 1.64(s, 3H,
19-CH3), 1.20(s, 3H, 17-CH3), 1.14(s, 3H, 16-CH3), 0.78(s, 9H,
C(CH3)3), ꢁ0.05(t, 3H, SiCH3), ꢁ0.30(s, 3H, SiCH3).
[2H5]6a-Hydroxy-paclitaxel (12)
The reaction mixture of (11) (0.23 g, 0.233 mmol), acetic acid
(0.112 g, 0.106 mL, 1.86 mmol), and tetrabutyl ammonium
fluoride hydrate (0.1822 g, 0.69 mmol) was stirred in THF(3.0 mL)
at room temperature for 4 h. It was diluted with EtOAc
(10 mL). The excess TBAF was quenched with 2% HCl solution
(10 mL) and the mixture was extracted with ethyl acetate
(5 ꢀ 10 mL). The combined organic extracts were washed with
saturated NaHCO3 solution (2 ꢀ 10 mL), dried over Na2SO4, and
concentrated under reduced pressure to afford the desired
product (12) as a white solid (0.18 g, 79.1%).
1H-NMR (300 MHz, CDCl3) d ppm: 8.14(dd, 2H, o-Ph), 7.62(t, 1H,
p-Ph), 7.48(m, 2H, o-Ph), 7.35–7.42(m, 5H), 6.96(d, 1H, J = 9.0 Hz,
NH), 6.26(s, 1H, 10-H), 6.25(t, J = 9.0 Hz, 13-H), 5.78(dd, 1H, J = 9.0,
3.0 Hz, 30-H), 5.66(d, 1H, J = 6.0 Hz, 2-H), 4.82 (s, 1H, 5-H), 4.79(dd,
1H, J = 9.0, 3.0 Hz, 20-H), 4.30(d, 1H, J = 9.0 Hz, 20-Ha), 4.21(d, 1H,
J = 9.0 Hz, 20-Hb), 4.20(d, 1H, J = 9.0 Hz, 7-H), 3.94 (m, 2H, 6-H
and 3-H), 2.39(s, 3H, 4-COCH3), 2.33(m, 2H, 14-H), 2.24(s, 3H,
10-COCH3), 1.90(brs, 1H, OH), 1.82(s, 3H, 18-CH3), 1.65(s, 3H,
19-CH3), 1.25(s, 3H, 17-CH3), 1.12(s, 3H, 16-CH3). MS-EI (m/z):
242.2(100), 243.2(18), 897.2(MNa1, 25), 898.2(17), HPLC (XDB-
C18, CH3CN/H2O = 47/53, 1.0 mL/min): tR 6.05 min (498.9%).
MS-EI analysis of the compound(12) have the same deuterium
enrichment as compound(6).
[2H5]20-O-(t-Butyldimethylsilyl)-6a-hydroxy-7-epi-paclitaxel (10)
To a solution of (9) (1.42 g, 1.49 mmol) in THF (14.2 mL) was
added N-Methylmoprpholine N-oxide (NMO, 0.26 g, 2.2 mmol) in
H2O (4.3 mL) and OsO4 solution (4 wt. 4% solution in H2O,
0.0945 mL, 0.015 mmol) under nitrogen atmosphere. The mix-
ture was stirred at room temperature for 4 h. Additional OsO4
solution (0.2835 mL, 0.045 mmol) and NMO (0.086 g, 0.73 mmol)
was added and the mixture was stirred for 5 h. TLC analysis
showed that about 20% product was formed. This process was
repeated several times till more than 90% product was formed.
The reaction mixture was diluted with CH2Cl2 (12 mL) and
sodium metabisulfite (0.572 g) was added while cooled in ice-
bath and stirred for 1 h. Anhydrous Na2SO4 (4 g) was added and
the mixture was stirred at room temperature for another 1 h. The
suspension was filtered and the solid residue was rinsed
thoroughly with THF (3 ꢀ 8 mL). The filtrate was concentrated
to give a brown paste, which was re-dissolved in EtOAc (60 mL)
and washed with water (2 ꢀ 40 mL), 0.25 M H2SO4 (2 ꢀ 40 mL),
brine (2 ꢀ 40 mL). The organic layer was dried with Na2SO4 and
concentrated under reduced pressure to give a light green semi-
solid. The crude product was purified by flash-chromatography
on a silica gel column, eluted with hexanes/ethyl acetate (7:3) to
afford (10) as a white solid (1.16 g, 78.9%).
Results and discussion
The synthesis of unlabelled and radioactive isotope (14C, 3H)
labelled paclitaxel has been reported in detail.10–16 The chiral
side chain of taxol contains phenylisoserine ester and b-lactam.
For our synthesis, the [2H5]benzoyl group was introduced
1H-NMR (300 MHz, CDCl3) d ppm: 8.16(dd, 2H, o-Ph), 7.59(d,
2H, o-Ph), 7.52(t, 1H, p-Ph), 7.32–7.43(m, 5H, Ph), 7.06(d, 1H,
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L. Tian et al.
through N-esterification of [2H5]benzoyl chloride with an amine. (1) with triethylsilyl chloride in anhydrous pyridine produced
Initially, we choose the synthesis of [2H5]azetidinone. Based on 7-triethylsilyl baccatin III (2),19 which was condensed with the
protocols from the literature,12 [2H5]azetidinone was synthesized excess chiral oxazolidine(3) in the presence of dicyclohexyl-
in three steps starting from b-lactam. The deuterium-labelled carbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP)
reagent was introduced in the second step. The coupling of in anhydrous toluene at 801C to afford 13-O-oxazolidinyl
the lithium alkoxide of [2H5]azetidinone with 7-triethylsilyl derivative(4).17,18,20 Hydrolysis of (4) with formic acid yielded
baccatin III in THF gave [2H5]20, 7-bis(triethylsilyl)paclitaxel. 13-(O-2R-hydroxy-3S-amine-phenylpropionyl)-baccatin III(5). Yields
Removal of the carefully chosen protecting groups in [2H5]20, for this reaction were found to be highly dependent on careful
7-bis(triethylsilyl)paclitaxel could be cleanly accomplished with temperature control (if the reaction temperature was allowed to
6 M aqueous HCl in acetonitrile at ꢁ51C to give [2H5]paclitaxel. rise above 301C, yields decreased significantly). The resulting
However, the esterization of [2H5]benzoyl chloride with 13-(O- amine (5), after treatment with [2H5]benzoyl chloride and Et3N in
2R-hydroxy-3S-amine-phenylpropionyl)-baccatin III(5) to give dry CH2Cl2, gave [2H5]paclitaxel (6) in 80.4% yield. After
paclitaxel(6) is much more economic. The total yield is much purification by flash-chromatography, the desired product (6)
higher. In addition, the conditions of the whole process were was obtained with over 99% chemical purity. The 1H NMR
milder. In order to obtain 13-(O-2R-hydroxy-3S-amine-phenyl- spectrum, Rf, and chromatographic behaviour of compound (6)
propionyl)-baccatin III(5), we selected baccatin III(1) and were consistent with that of natural paclitaxel.21 Mass spectro-
phenylisoserine ester derivative(chiral oxazolidine) (3)17,18 as metry analysis of (6) revealed that the compound has more than
starting materials (Scheme 1). Selective protection of baccatin III 99% deuterium enrichment.
Scheme 1.
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L. Tian et al.
The synthesis of unlabelled 6a-hydroxy-paclitaxel have been starting material, Scheme 2 presents the synthesis of [2H5]
described in the literature, but lacks experimental details.22–24 6a-hydroxyl paclitaxel. Selective protection of [2H5]paclitaxel (6)
Those general procedures were adopted in the synthesis of with t-butyldimethylchlorosilane in dry DMF produced
[2H5]6a-hydroxyl paclitaxel (12). With [2H5]paclitaxel (6) as the [2H5]20-O- (t-butyldimethylchlorosilyl) palictaxel (7). Reaction
Scheme 2.
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J. Label Compd. Radiopharm 2011, 54 318–323

L. Tian et al.
of (7) with trifluoromethanesulfonyl chloride and DMAP in
dry CHCl3 furnished [2H5]20-O-(t-butyldimethylchlorosilyl)-7b-O-
trifluromethanesulfonylpaclitaxel (8). The boiling point of
trifluoromethanesulfonyl chloride is 291C, so the reaction
temperature was kept below 301C to ensure good yield.
Treatment of compound (8) with 1, 8-diazabicyclo [5, 4, 0]
undec-7-ene (DBU) at 401C in dry CH2Cl2 gave the key
intermediate [2H5]20-O-(t-butyldimethylsilyl)-6, 7-dehydropacli-
taxel (9). The Rf of the starting material and product was
very close, therefore 10 equivalents of DBU were used to ensure
that the reaction was complete. The oxidation of (9) with
osmium tetroxide and N-Methylmoprpholine N-oxide (NMO) in
H2O/THF produced [2H5]20-O-(t-butyldimethylsilyl)-6a-hydroxy-7-
epi-paclitaxel (10).25 The high yield was achieved by
repeatedly adding osmium tetroxide and NMO. Treatment
of (10) with 1,8-Diazobicyclo [5, 4, 0]udec-7-ene(DBU) in
G. W. Kirby, R. E. Moore, et al.), 1993, Springer, Wien & New York,
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anhydrous toluene at 801C afforded
a single product
which was isolated and fully characterized as [2H5]20-O-(t-
butyldimethylsilyl)-6a-hydroxyl paclitaxel (11) in 7% yield,
along with 80% unreacted starting material. Since the epimer-
ization was essentially an equilibrium reaction,26 reaction time
cannot be longer than 1.5 h. Otherwise it will lead to the
formation of other impurities. Deprotection of (11) with
tetrabutylammonium fluoride and acetic acid at ambient
temperature gave the major metabolite [2H5]6a-hydroxy-pacli-
taxel (12) in 79.1% yield. It has over 98% chemical purity
and isotopic enrichment. The 1H NMR spectrum of (12) was
identical to that reported in the literature.5,6 Other spectra,
including TOCSY, HMQC, and NOESY, were consistent with the
assigned structure.5,6
In conclusion, five deuterium-labelled paclitaxel and its
hydroxyl metabolite have been successfully synthesized and
characterized. It provides important internal standards for the
clinical studies of paclitaxel.
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J. Label Compd. Radiopharm 2011, 54 318–323
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