Synthesis, cytotoxicity, metabolic stability and pharmacokinetic evaluation of fluorinated docetaxel analogs

Article abstract of DOI:10.1016/j.ejmech.2011.02.027

Three novel fluorinated docetaxel analogs, along with six previous reported, were evaluated for their cytotoxicity against five tumor cell lines. The results indicated that these analogs maintained similar/more potent activity than docetaxel against these tumor cell lines. They were also evaluated for their metabolic stability and pharmacokinetics, which demonstrated that these analogs showed better profiles of metabolic stability and pharmacokinetics than that of docetaxel.

Full text of DOI:10.1016/j.ejmech.2011.02.027

European Journal of Medicinal Chemistry 46 (2011) 1743e1748
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity, metabolic stability and pharmacokinetic evaluation of
fluorinated docetaxel analogs
,
,
*
Hong-Fu Lu a, Cheng Xie a, Jun Chang a, Guo-Qiang Lin a b, Xun Sun a
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China
b Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel fluorinated docetaxel analogs, along with six previous reported, were evaluated for their
cytotoxicity against five tumor cell lines. The results indicated that these analogs maintained similar/
more potent activity than docetaxel against these tumor cell lines. They were also evaluated for their
metabolic stability and pharmacokinetics, which demonstrated that these analogs showed better profiles
of metabolic stability and pharmacokinetics than that of docetaxel.
Received 23 November 2010
Received in revised form
29 December 2010
Accepted 14 February 2011
Available online 22 February 2011
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Fluorinated docetaxel analogs
Cytotoxicity
Metabolic stability
Pharmacokinetics
1. Introduction
reported a series of taxoids in which 30-Ph was replaced by alkyl
or alkenyl substitutions, in combination with the changes of the
The naturally occurring paclitaxel (Taxol) is a diterpenoid orig-
inally isolated from the bark of Taxus brevifolia [1], which is also
prepared by semisynthesis and microorganisms now. Paclitaxel (1)
and its semi-synthetic derivative docetaxel (2) (Fig. 1), are currently
considered to be the most important and promising anticancer
agents in the treatment of refractory breast and ovarian cancers due
to their unique mechanism of action by binding tubulin and
stabilizing microtubule formation, which ultimately disrupts
mitosis and causes cell death [2]. However, some drawbacks of
paclitaxel hampered its clinical usefulness, such as poor aqueous
solubility, multi-drug resistance (MDR), and low activity for oral
administration [3]. To develop new anticancer agents, many
approaches have been applied in preparing of analogs with better
activity, conjugates or prodrugs with better bioavailability and
specificity, and new formulations with improved physical proper-
ties [4].
substitutions on the C-2 and C-10. The 30-trifluoromethyl-10-acetyl
analogs of docetaxel exhibited better cytotoxicity than paclitaxel
and possessed more than one order of magnitude higher potency
than paclitaxel and docetaxel against a drug-resistant human
breast cancer cell line. Further systematically studies [7e9]
demonstrated that incorporating fluorine at the metabolism site
in the paclitaxel molecules did indeed block some of the metabolic
pathways by enzymes of P450 (CYP) family and increased their
stability, in which no hydroxylation was observed for 2-(3-fluo-
robenzoyl) paclitaxel both at the phenyl ring of 2-(3-fluorobenzoyl)
and at the 30-phenyl ring. This remarkable effect may be contrib-
uted by the direct influence from the outlying fluorine moiety upon
enzymeesubstrate recognition and action. These findings lay the
solid foundation for this course of work that the fluorine replace-
ment on the taxoid should be a viable tool to enhance the potency,
perhaps against MDR cancer cell lines.
Extensive studies on the structureeactivity relationships of
paclitaxel and docetaxel were mainly focused on the modifications
on C-13 side chain, B and C ring [5]. The importance of C-13
substituted phenylisoserine side chain to the bioactivity of pacli-
taxel has been acknowledged for a long time. Ojima’s group [6]
Along with the pursuing of more potent docetaxel analogs as
anticancer agents, we also pay our attention to these analogs’
metabolic stability and pharmacokinetic profiles. It is believed that
only the optimized combination can lead to the success to find
desired clinical candidates. As our continuous work to design and
synthesis of fluorine-containing docetaxel analogs [10], three novel
docetaxel analogs (3aec) were synthesized here (Fig. 2). Along with
the previous six analogs (3dei), these nine analogs will be evalu-
ated for their cytotoxicity against five tumor cell lines. Also
* Corresponding author. Tel./fax: þ86 21 51980003.
E-mail address: sunxunf@shmu.edu.cn (X. Sun).
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.027

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H.-F. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 1743e1748
para-toluenesulfonate (PPTS) followed by saponification of formed
intermediate 12 afforded acid 13 in 98.6% yield.
With intermediate 13 in hand, the previous optimized synthetic
route was adopted for the synthesis of target compounds 3aec
(Scheme 2) [10]. First, hydroxyl groups at the C7 and C10 of the
natural product 10-deacetylbaccatin III (10-DAB) were protected
with 2,2,2-trichloroethyl chloroformate (TrocCl) by using pyridine
as the base to obtain 7,10-diTroc-10-deacetylbaccatin (15). Then, 15
was coupled with 13 to provide the corresponding intermediate 16
in 86% yield in the presence of dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP). After removing the 30-N-Boc
and acetonide-protecting group of 16 with 98% formic acid at room
temperature, the amino alcohol 17 was obtained in 55% yield. After
that, 19aec were prepared by acylation of amino group of 17 with
freshly prepared fluorine containing acyl chlorides (18aec) [15,16],
removing the 7,10-Troc protecting groups on 19aec with zinc in
acetic acid, three corresponding fluorinated docetaxel derivatives
(3aec) were synthesized in desirable yields.
Fig. 1. Structures of paclitaxel and docetaxel.
preliminary metabolic stability and pharmacokinetic study will be
executed. Based on the observation from Ojima group, it was
anticipated that fluorination(s) on docetaxel should have impact on
their metabolic stability and pharmacokinetics profiles. Herein, we
reported the syntheses, cytotoxic activity, metabolic stability and
pharmacokinetics of a series of fluorinated docetaxel analogs.
2.2. In vitro cytotoxic activity
2. Results and discussion
The invitro cytotoxicityof fluorinated docetaxel derivatives (3aei)
were evaluated against two human cancer cell lines [oral epidermoid
carcinoma (KB-0528) and human promyelocytic leukemia cells
(HL60-0607-2)], and threemultiple drugresistance(MDR) cancercell
lines (KBR-0530, KBR-0602, and HL60OR-0604-2) (Table 1).
Compared with docetaxel, except 3c which had 18-fold more potent
than docetaxel against KB-0528, all other analogs (3aeb and 3dei)
showed similar or slight better potency against both human cancer
cell lines (KB-0528 and HL60-0607-2). Interestingly, analog 3c also
exhibited 20- and 10-fold more potent than docetaxel against HL60-
0607-2 and HL60OR-0604-2, while others had similar potency as
docetaxel.
2.1. Chemistry
The synthesis of side chain [11e14] commenced from 4-fluo-
robenzaldehyde (4), a commercially available starting material, as
illustrated in Scheme 1. 4-Fluorobenzaldehyde was first trans-
formed into 4-fluoro-(E)-cinnamic acid (6) via the reaction of ethyl
bromoacetate with triphenylphosphine in the presence of catalytic
amount of titanium tetrachloride, followed by hydrolysis and
crystallization. The obtained 4-fluoro-(E)-cinnamic acid (6) was
then converted to isopropyl cinnamate (7) by using isopropanol
and thionyl chloride at 0 ꢀC, and then stirred at room temperature
overnight in 78% yield. Compound 7 was subjected to a Sharpless
asymmetric aminohydroxylation by using (DHQ)2PHAL as the
ligand, freshly prepared N-bromoacetamide as the nitrogen source,
and potassium osmate as the oxidant to give the desired amino
alcohol (8) as a single isomer in 83% yield and with >99% ee.
Compound 8 was subjected to hydrolysis in aqueous 10% HCl under
reflux to furnish the free amine which was concomitantly trans-
ferred to be the methyl ester (10). Subsequently, the amine group
was transformed into the Boc protected amino alcohol 11 by using
Boc2O in the presence of triethylamine. Cyclic protection using
methoxypropene in the presence of catalytic amount of pyridinium
2.3. In vitro metabolic stability
The metabolic study results for fluorinated docetaxel analogs
upon incubation with pooled human liver microsome are shown in
Table 2 [17e19]. Compared with Docetaxel (22.6% remaining after
60 min), all analogs (3aei) exhibited much better metabolic
stability profile. Among them, 3a and 3e were two most metabolic
stable analogs in this condition with 88.5% and 89.5% intact after
60 min. This result demonstrated that incorporating fluorine(s) at
the metabolism site(s) in the paclitaxel molecules did indeed block
Fig. 2. Fluorinated docetaxel analogs.

H.-F. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 1743e1748
1745
Scheme 1. Synthesis of key intermediate 13. Reagents and conditions: (i) Ph3P]CHCOOEt, toluene, reflux, 3 h; rt, overnight; (ii) KOH/EtOH, rt, 3 h; (iii) i-PrOH/SOCl2, 0 ꢀC - rt,
overnight; (iv) LiOHeH2O/K2OsO2(OH)4, t-BuOHeH2O/(DHQ)2PHAL, AcNHBr, 0 ꢀC, 10 h; (v) 10% HCl aqueous, reflux, 4 h; (vi) MeOH/SOCl2, 0 ꢀC - rt, overnight; (vii) Boc2O/Et3N,
CH2Cl2, 0 ꢀC - rt, 48 h; (viii) CH2]C(OCH3)CH3, PPTs, toluene, 90 ꢀC, 3 h; (ix) LiOH, THF/MeOH/H2O, rt, 2 h.
some of the metabolic pathways by enzymes of P450 (CYP) family,
which resulted in the significant increase of their metabolic
stability. In the inactive pooled human liver microsome, the
unchanged drugs of 3aei at 60 min were 109.2%, 96.9%, 85.2%,
73.2%, 86.9%, 95.0%, 54.6%, 92.9% and 82.5%, respectively, in which
the concentration of both 3d and 3g decreased along with the
prolongation of the incubation time may due to the adsorption of
protein or self-degradation. The results of CLint (Table 2) indicated
that 3c was a low intrinsic clearance drug and had a better meta-
bolic stability, while the others (3aeb and 3dei) were medium
intrinsic clearance drugs, compared with docetaxel. In conclusion,
mono-fluorinated tert-butyl docetaxel analogs (eg. 3a, 3d and 3g)
had lower metabolic stabilities than those with di- or tri-fluori-
nated tert-butyl docetaxel analogs (eg. 3bec, 3eef, and 3hei).
total plasma clearance (CLz) and apparent volume of distribution
(Vz) decreased significantly. The AUC of tri-fluorinated tert-butyl
docetaxel analogues (3c, 3f, and 3i) was higher than those of mono,
or di-fluorinated tert-butyl docetaxel analogs, while CLz and Vz of
those (3c, 3f, and 3i) were lower. This indicated that fluorine
substitution on the side chain of tert-butyl could improve the
AUC0ꢁt and AUC0ꢁN, however, 2-(3-fluorobenzoyl) substitution
and at the 30-(4-fluorophenyl) substitution didn’t influence the
AUC0ꢁt and AUC0ꢁN. Except 3a and 3g, the other fluorinated
docetaxel analogs had a longer elimination half-life time than
docetaxel. All fluorinated derivatives had a lower total plasma
clearance than docetaxel. Interestingly, the AUC0ꢁt and AUC0ꢁN of
3h were lower than those of 3g, and the result was confirmed by
repeated experiments. Further investigation is on the way (Table 3).
2.4. In vivo pharmacokinetics
3. Conclusions
The pharmacokinetics of fluorinated docetaxel analogs were
defined after i.v. administration of 5 mg/kg in rat. The plasma was
sampled between 5 min and 24 h after dosing. Liquideliquid
extraction followed by isocratic high-performance liquid chroma-
tography (HPLC) with MSeMS was used to determine drug
concentrations in plasma. Compared with docetaxel, AUC0ꢁt and
AUC0ꢁN of fluorinated docetaxel analogs increased significantly,
especially the AUC of 3c, 3f, and 3i increased 6-fold. Meanwhile, the
Fluorinated docetaxel analogs were synthesized and evaluated
for their cytotoxicity, metabolic stability and pharmacokinetics. It
was found that some analogs showed potent cytotoxicity, good
metabolic stability, and improved pharmacokinetics. Among them,
3c was shown up as one of the most promising analogs. It was also
found that fluorinated substituted on the side chain tert-butyl
significantly improved the AUC0ꢁt and AUC0ꢁN, while 2-(3-fluo-
robenzoyl) substitution and at the 30-(4-fluorophenyl) substitution
Scheme 2. Synthesis of new fluorinated docetaxel derivatives (3aec). Reagents and conditions: (i) TrocCl, Pyridine, rt, 30 m; (ii) 13, DCC, DMAP, toluene; (iii) HCOOH, rt, 2 h; (iv)
Et3N, CH2Cl2; (v) Zn/HOAc, MeOH, 50 ꢀC.

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H.-F. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 1743e1748
Table 1
In vitro cytotoxicities (IC50 mM) of fluorinated docetaxel analogs.
KB-0528
KBR-0530
KBR-0602
HL60-0607-2
HL60OR-0604-2
3a
3b
3c
3d
3e
3f
3g
3h
9.146 ꢂ 10ꢁ4
3.969 ꢂ 10ꢁ4
2.589 ꢂ 10ꢁ5
5.057 ꢂ 10ꢁ4
2.820 ꢂ 10ꢁ4
8.342 ꢂ 10ꢁ5
2.476 ꢂ 10ꢁ4
7.076 ꢂ 10ꢁ5
2.101 ꢂ 10ꢁ4
4.536 ꢂ 10ꢁ4
2.318 ꢂ 10ꢁ4
3.207 ꢂ 10ꢁ4
3.318 ꢂ 10ꢁ4
0.322 ꢂ 10ꢁ4
3.321 ꢂ 10ꢁ4
6.941 ꢂ 10ꢁ4
6.695 ꢂ 10ꢁ5
1.609 ꢂ 10ꢁ4
1.076 ꢂ 10ꢁ4
1.317 ꢂ 10ꢁ4
1.945 ꢂ 10ꢁ4
8.854 ꢂ 10ꢁ4
4.323 ꢂ 10ꢁ4
7.917 ꢂ 10ꢁ4
3.693 ꢂ 10ꢁ4
6.260 ꢂ 10ꢁ4
2.049 ꢂ 10ꢁ4
1.817 ꢂ 10ꢁ4
1.492 ꢂ 10ꢁ4
2.341 ꢂ 10ꢁ4
1.399 ꢂ 10ꢁ4
1.681 ꢂ 10ꢁ5
3.583 ꢂ 10ꢁ7
1.564 ꢂ 10ꢁ4
1.762 ꢂ 10ꢁ4
2.625 ꢂ 10ꢁ4
1.110 ꢂ 10ꢁ6
1.834 ꢂ 10ꢁ4
5.468 ꢂ 10ꢁ5
1.149 ꢂ 10ꢁ5
5.100 ꢂ 10ꢁ4
2.085 ꢂ 10ꢁ3
1.545 ꢂ 10ꢁ5
9.583 ꢂ 10ꢁ4
5.539 ꢂ 10ꢁ4
4.099 ꢂ 10ꢁ4
2.973 ꢂ 10ꢁ3
5.840 ꢂ 10ꢁ4
1.909 ꢂ 10ꢁ4
1.140 ꢂ 10ꢁ4
3i
Docetaxel
didn’t significantly influence the AUC0ꢁt and AUC0ꢁN. Overall, tri-
fluorinated tert-butyl docetaxel analogs exhibited the best meta-
bolic stability and the lowest total plasma clearance.
4.2. Chemistry
4.2.1. Iso-propyl (2E)-3-(4-fluorophenyl) prop-2-enoic acid (7)
Compound 5 (16.08 g, 82.86 mmol) was treated with KOH (16 g)
in ethanol (220 mL) at room temperature for 3 h. Then the ethanol
was removed under reduced pressure. The residue was diluted with
water and extracted with CH2Cl2 twice to remove organic impuri-
ties. The obtained aqueous layer was then acidified with dilute HCl,
extracted with ether (400 mL ꢂ 2). The combined ether was dried
over anhydrous Na2SO4 and evaporated to afford the crude acid.
The crude acid was dissolved in i-PrOH (230 mL), and SOCl2
(9.05 mL, 124.3 mmol) was added dropwise at 0 ꢀC under nitrogen
atmosphere. The reaction mixture was stirred at 85 ꢀC for 4 h, and
then quenched with saturated NaHCO3 to pH ¼ 8. The solvent was
evaporated under reduced pressure, and diluted with H2O. The
water phase was extracted with EtOAc for three times. The
combined organic phases were dried over anhydrous Na2SO4 and
the solvent was removed under reduced pressure to give the
4. Experimental section
4.1. Material and physical measurements
Reagents were purchased from Aldrich and TCI Chemical
companies. All solvents are purified and dried in accordance with
standard procedures, unless otherwise indicated. Ether, tetrahy-
drofuran and toluene ware dried over natrium/benzophenone;
dichloromethane and pyridine ware distilled from calcium hydride;
and DMF was distilled from calcium hydride by reduced pressure.
Oxygen-free and water-free operations were carried out under
argon atmosphere in dried glassware unless otherwise noted. All
reactions were monitored by TLC with precoated silica gel plates
GF254, 10e40 mm (Yantai, China). The purity of the samples was by
product 7 (13.42 g, 77.9%); 1H NMR (300 MHz, CDCl3):
d 1.31
column chromatography with the silica gel H 300e400 mesh
(Yantai, China). Melting points (mp) were determined using an X-4
microscope melting point apparatus and were uncorrected. All 1H
NMR spectra were recorded on Varian Mercury 300 (300 MHz), or
Bruker DRX-400 (400 MHz) spectrometer at room temperature,
(d, J ¼ 6.3 Hz, 6H), 5.14 (m, 1H), 6.35 (d, J ¼ 15.9 Hz, 1H), 7.10
(t, J ¼ 8.7 Hz, 2H), 7.51 (dd, J ¼ 8.7, 5.4 Hz, 2H) 7.63 (d, J ¼ 15.9 Hz,
1H); 13C NMR (100 MHz, CDCl3):
d 22.1, 68.1, 116.1, 116.3, 118.8,
130.0, 131.0, 143.2, 166.6; EIMS m/z 208, 166, 149; HRMS (EI) m/z
calcd for C12H13O2F: 208.0906, found 208.0900.
and chemical shifts are reported in ppm (d) relative to TMS (0.00) as
internal standard; expressed in peak shape, the number of
hydrogen, and coupling constant in Hertz. 13C NMR spectra data
were recorded on Bruker DRX-400 (100 MHz), or Bruker DPX-300
(75 MHz) spectrometer at room temperature, and chemical shifts
4.2.2. Isopropyl (2R,3S)-3-acetylamino-2-hydroxy-3-(4-
fluorophenyl) propionate (8)
To a flash (100 mL) charged with aqueous solution (25 mL) of
LiOH-H2O (0.222 g, 5.27 mmol) was added K2OsO2(OH)4 (0.045 g,
0.122 mmol, 2.4 mmol%). The mixture was stirred for 30 min and
then t-BuOH (50 mL) and ligand (DHQ)2PHAL (1.58 mmol%, 0.062 g,
0.0789 mmol) were added. The resulting mixture was stirred for 1 h
at room temperature to give a clear solution. Water (25 mL) was
subsequently added and the reaction mixture was cooled to 0 ꢀC,
7 (1.04 g, 5.0 mmol) was added followed by the addition of fresh
are reported in ppm (d) relative to d-substituted solvents (d-chlo-
roform at 77.2 ppm, d6-acetone at 30.6 ppm and 206.7 ppm) as
internal standard. Mass spectra ware performed at the testing and
analysis center of Shanghai Institute of Organic Chemistry: Low-
resolution mass spectra (ESI) were performed on Shimadzu LCMS-
2010EV; High-resolution mass spectra on IonSpec 4.7 T FTMS
(MALDI) or Bruker Daltonics, Inc. APEXIII7.0 TESLA FMS (ESI).
Table 3
Table 2
Pharmacokinetics parameters in vivo of fluorinated docetaxel analogs.
T1/2 in liver microsomes and predicted CLint in vivo of fluorinated docetaxel analogs.
AUC0ꢁt
AUC0ꢁ
N
MRT t1/2
(h)
5.06 3.02
4.28 13.4 1.51
2.78 8.50 1.28
5.62 12.2 5.09
CLz
Vz
Unchanged drug
at 60 min（%）
T
1/2（min）
CLint（mL/min/kg）
（ng,h/mL） （ng,h/mL） (h)
(L/h/kg) （L/kg）
3a
3b
3c
3d
3e
3f
3g
3h
1640
3147
3874
914
2857
3859
2192
1932
3867
646
1659
3361
3979
994
2932
3960
2238
1989
3944
667
2.13
22.0
27.4
15.8
89.7
20.2
18.1
22.7
36.2
13.7
78.8
3a
3b
3c
3d
3e
3f
3g
3h
53.8
72.4
88.5
47.2
89.5
58.1
46.8
71.0
63.6
22.6
144.4
364.8
8.62
3.41
0.36
10.24
3.23
5.21
10.06
4.67
5.75
3465.7
121.6
385.1
239.0
123.8
266.6
216.6
70.0
2.76
2.32
2.30
7.69 1.79
9.91 1.26
6.80 2.26
2.40 10.15 2.55
3i
2.13
3.34
7.62 1.28
7.37 7.51
3i
Docetaxel
Docetaxel
17.78

H.-F. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 1743e1748
1747
AcNHBr (0.897 g, 6.5 mmol) in one portion. The reaction mixture
was stirred at 0 ꢀC and monitored by TLC. After completion, the
reaction mixture was treated with Na2SO3 (2 g) and stirred for
30 min at room temperature, after which EtOAc was added. The
organic layer was separated and the water layer was extracted with
EtOAc for three times. The combined organic layers were washed
with brine, dried over Na2SO4, and evaporated. The residue was
purified by silica gel column chromatography using petroleum
ether/EtOAc (3/2) to afford 8 (1.176 g, 83.0%) as a white solid. 1H
4.2.3.3. N-De-t-butoxycarbonyl-N-[2-(1,1,1-trifluoro-2-methyl)propy-
loxycarbonyl]-7,10-di(2,2,2-trichloroethyloxycarbonyl)-30-dephenyl-30-
(4-fluorophenyl)docetaxel (19c). Yield 50.0%; mp 170e172 ꢀC; 1H
NMR (300 MHz, CDCl3):
d 1.21 (s, 3H), 1.27 (s, 3H), 1.54 and 1.58 (2s,
6H), 1.86 (s, 3H), 1.93 (s, 3H), 2.31 (m, 2H), 2.36 (s, 3H), 2.07 and 2.63
(2m, 2H), 3.90 (d, J ¼ 6.6 Hz, 1H), 4.18 and 4.34 (2d, J ¼ 8.7 Hz, 2H),
4.60 and 4.92 (2d, J ¼ 12.0 Hz, 2H), 4.62 (s, 1H), 4.78 (s, 2H), 4.96
(d, J ¼ 10.2 Hz, 1H), 5.25 (br d, J ¼ 9.0 Hz, 1H), 5.53 (dd, J ¼ 10.8,
7.5 Hz, 1H), 5.68 (m, 1H), 5.69 (d, J ¼ 6.3 Hz, 1H), 6.24 (s, 1H), 6.24
(t, J ¼ 9.3 Hz, 1H), 7.11 (t, J ¼ 8.7 Hz, 2H), 7.40 (dd, J ¼ 8.4, 5.4 Hz, 2H),
7.51 (t, J ¼ 7.5 Hz, 2H), 7.64 (t, J ¼ 7.5 Hz, 1H), 8.10 (d, J ¼ 7.5 Hz, 2H);
NMR (300 MHz, CDCl3):
d
1.28 (d, J ¼ 6.6 Hz, 3H), 1.32 (d, J ¼ 6.3 Hz,
3H), 2.00 (s, 3H), 3.23 (br s, 1H), 4.44 (d, J ¼ 2.1 Hz, 1H), 5.11 (m, 1H),
5.53 (dd, J ¼ 9.0, 1.5 Hz, 1H), 6.41 (d, J ¼ 9.3 Hz, 1H), 7.03 (t,
J ¼ 8.7 Hz, 2H), 7.38 (dd, J ¼ 8.7, 5.1 Hz, 2H); 13C NMR (100 MHz,
13C NMR (100 MHz, CDCl3):
d 10.7, 14.7, 19.4, 19.6, 20.7, 22.5, 26.4,
33.3, 35.2, 43.1, 46.9, 55.6, 56.3, 72.3, 73.2, 74.0, 76.4, 77.1, 77.4, 78.6,
79.1, 81.0, 83.6, 94.1, 115.8, 128.6, 128.7, 128.9, 130.1, 132.3, 133.9,
142.0, 153.2, 153.3, 154.8, 163.7, 166.9, 170.4, 172.1, 200.6; MALDIMS
m/z 1266.2 [M þ K]þ; HRMS (ESI) m/z calcd for C49H51NO18F4Cl6Naþ:
1250.1078 [M þ Na]þ, found 1250.1125.
CDCl3):
d 21.7, 21.7, 23.3, 58.6, 69.9, 73.7, 115.5, 115.7, 128.9, 135.1,
163.7, 172.5; ESIMS m/z 306.0 [M þ Na]þ; HRMS (ESI) m/z calcd for
C14H19NO4Fþ: 284.1301 [M þ H]þ, found 284.12926.
4.2.3. General procedure for the preparation of 19aec
To a stirred solution of 17 (500 mg, 0.46 mmol) in anhydrous
THF (10 mL) was dropwise added excessive 18 at 0 ꢀC, followed by
a solution of triethylamine (0.13 mL, 0.93 mmol) in anhydrous THF
(5 mL). The resulting mixture was stirred at room temperature for
1 h, and then extracted with ethyl acetate (20 mL ꢂ 3). The
combined organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The obtained residue was
purified by silica gel flash chromatography column (petroleum
ether/ethyl acetate: 2/1) to afford 19aec as white solid.
4.2.4. General procedure for the preparation of 3aec
To a solution of 19aec (0.19 mmol) in methanol (10 mL) were
added glacial acetic acid (4.60 mL) and zinc powder (0.46 g,
7.08 mmol). The resulting mixture was stirred at 50 ꢀC for 1 h. The
reaction mixture was filtered to remove the zinc and solid formed.
Removal of the solvent by distillation gave a white solid. The
obtained solid was then dissolved in ethyl acetate (60 mL), which
was washed with saturated NaHCO3 and brine, dried over Na2SO4,
and concentrated in vacuo. The obtained residue was purified by
silica gel flash chromatography column (petroleum ether/Acetone:
2/1) to give 3aec as a white solid.
4.2.3.1. N-De-t-butoxycarbonyl-N-[2-(1-fluoro-2-methyl)propylox-
ycarbonyl]-7,10-di-(2,2,2-trichloroethyloxycarbonyl)-30-dephenyl-30-
(4-fluorophenyl)docetaxel (19a). Yield 55.8%; mp 156e158 ꢀC; 1H
4.2.4.1. N-De-t-butoxycarbonyl-N-[2-(1-fluoro-2-methyl)propyloxycar-
bonyl]-30-dephenyl-30-(4-fluorophenyl)docetaxel (3a). Yield 57.5%; mp
NMR (300 MHz, CDCl3):
d 1.21 (s, 3H), 1.27 (s, 3H), 1.35 (s, 6H), 1.87
(s, 3H), 1.96 (s, 3H), 2.32 (m, 2H), 2.39 (s, 3H), 2.07 and 2.63 (2m,
2H), 3.91 (d, J ¼ 6.9 Hz, 1H), 4.19 and 4.34 (2d, J ¼ 8.1 Hz, 2H), 4.32
(m, 2H), 4.61 and 4.91 (2d, J ¼ 11.7 Hz, 2H), 4.62 (m,1H), 4.78 (s, 2H),
4.96 (d, J ¼ 8.4 Hz, 1H), 5.27 (m, J ¼ 9.6 Hz, 1H), 5.53 (d, J ¼ 9.9 Hz,
1H), 5.54 (m, 1H), 5.70 (d, J ¼ 6.9 Hz, 1H), 6.24 (s, 1H), 6.24 (t,
J ¼ 8.1 Hz, 1H), 7.10 (t, J ¼ 8.7 Hz, 2H), 7.38 (dd, J ¼ 8.7, 5.1 Hz, 2H),
7.51 (t, J ¼ 7.5 Hz, 2H), 7.63 (t, J ¼ 7.5 Hz, 1H), 8.11 (d, J ¼ 7.2 Hz, 2H);
168e170 ꢀC; [
CD3COCD3):
a
]20D ꢁ9.8ꢀ (c 0.50 MeOH); 1H NMR (300 MHz,
d
1.16 (s, 3H), 1.21 (s, 3H), 1.36 (s, 6H), 1.73 (s, 3H), 1.91 (s,
3H), 2.31 (m, 2H), 2.42 (s, 3H),1.84 and 2.46 (2m, 2H), 3.77 (s, 1H), 3.94
(d, J ¼ 7.2 Hz, 1H), 4.17 (s, 2H), 4.24 (m, 1H), 4.32 (m, 1H), 4.35 (s, 1H),
4.33 and 4.50 (2m, 2H), 4.66 (m, 1H), 4.91 (d, J ¼ 6.6 Hz, 1H), 4.97 (d,
J ¼ 7.8 Hz, 1H), 5.24 (s, 1H), 5.24 (m, 1H), 5.69 (d, J ¼ 7.5 Hz,1H), 6.20 (t,
J ¼ 8.4 Hz, 1H), 6.82 (d, J ¼ 9.3 Hz, 1H), 7.17 (t, J ¼ 8.7 Hz, 2H), 7.54 (m,
2H), 7.56 (t, J ¼ 7.8 Hz, 2H), 7.66 (t, J ¼ 7.5 Hz, 1H), 8.10 (d, J ¼ 7.2 Hz,
13C NMR (100 MHz, CDCl3):
d 10.7, 14.7, 20.9, 22.2, 22.5, 26.3, 33.3,
35.3, 43.2, 46.9, 53.4, 56.3, 72.3, 73.3, 74.1, 76.4, 77.1, 78.6, 79.1, 79.9,
80.9, 83.6, 86.8, 94.2, 115.8, 126.7, 128.6, 128.7, 128.8, 129.0, 130.2,
132.1,133.8,138.0,142.3,153.2,153.2,154.8,161.1,166.8,170.4,172.4,
200.7; MALDIMS m/z 1192.0 [M þ H]þ; HRMS (ESI) m/z calcd for
C49H53NO18F2Cl6Naþ: 1214.1267 [M þ Na]þ, found 1214.1258.
2H); 13C NMR (100 MHz, CDCl3):
d 9.4, 13.5, 20.4, 21.6, 21.7, 22.1, 26.1,
36.0, 36.7, 43.3, 46.6, 57.7, 71.4, 71.6, 74.0, 74.3, 75.0, 76.0, 77.7, 79.9,
80.9, 84.1, 86.0, 87.7, 115.0, 128.5, 129.2, 129.9, 130.4, 133.1, 136.7, 137.5,
155.0, 165.7, 170.0, 210.6; ESIMS m/z 866.5 [M þ Na]þ; HRMS (MALDI)
m/z calcd for C43H51NO14F2Naþ: 866.3154 [M
866.31699.
þ
Na]þ, found
4.2.3.2. N-De-t-butoxycarbonyl-N-[2-(1,1-difluoro-2-methyl)propy-
loxycarbonyl]-7,10-di(2,2,2-trichloroethyloxycarbonyl)-30-dephenyl-
30-(4-fluorophenyl)docetaxel (19b). Yield 58.1%; mp 172e174 ꢀC; 1H
4.2.4.2. N-De-t-butoxycarbonyl-N-[2-(1,1-difluoro-2-methyl)propy-
loxycarbonyl]-30-dephenyl-30-(4-fluorophenyl)docetaxel (3b). Yield
NMR (300 MHz, CDCl3):
d
1.21 (s, 3H), 1.27 (s, 3H),1.39 (s, 6H), 1.86 (s,
60.4%; mp 180e182 ꢀC; [
(300 MHz, CD3COCD3): d 1.16 (s, 3H), 1.21 (s, 3H), 1.39 (s, 6H), 1.73
a
]20D ꢁ16.8ꢀ (c 0.43 MeOH); 1H NMR
3H), 1.94 (s, 3H), 2.31 (d, J ¼ 8.7 Hz, 2H), 2.37 (s, 3H), 2.07 and 2.63
(2m, 2H), 3.90 (d, J ¼ 6.6 Hz, 1H), 4.18 and 4.34 (2d, J ¼ 8.7 Hz, 2H),
4.60 and 4.92 (2d, J ¼ 12.0 Hz, 2H), 4.62 (m, 1H), 4.78 (s, 2H), 4.95
(d, J ¼ 11.4 Hz, 1H), 5.25 (d, J ¼ 8.7 Hz, 1H), 5.54 (dd, J ¼ 10.8, 7.2 Hz,
1H), 5.63 (d, J ¼ 9.9 Hz,1H), 5.69 (d, J ¼ 6.6 Hz,1H), 6.00 (t, J ¼ 5.7 Hz,
1H), 6.24 (s, 1H), 6.24 (t, J ¼ 7.5 Hz, 1H), 7.11 (t, J ¼ 8.7 Hz, 2H), 7.38
(dd, J ¼ 8.7, 5.1 Hz, 2H), 7.51 (t, J ¼ 7.5 Hz, 2H), 7.64 (t, J ¼ 7.5 Hz, 1H),
(s, 3H), 1.90 (s, 3H), 2.31 (m, 2H), 2.41 (s, 3H), 1.84 and 2.46 (2m,
2H), 3.78 (s, 1H), 3.94 (d, J ¼ 7.2 Hz, 1H), 4.17 (s, 2H), 4.24 (m, 1H),
4.30 (m, 1H), 4.35 (s, 1H), 4.67 (m, 1H), 4.95 (s, 1H), 4.96 (d,
J ¼ 7.5 Hz,1H), 5.22 (m, 1H), 5.24 (s, 1H), 5.69 (d, J ¼ 7.5 Hz, 1H), 6.14
(t, J ¼ 57.0 Hz, 1H), 6.20 (t, J ¼ 9.0 Hz, 1H), 7.04 (d, J ¼ 9.6 Hz, 1H),
7.18 (t, J ¼ 8.7 Hz, 2H), 7.55 (m, 2H), 7.56 (t, J ¼ 7.8 Hz, 2H), 7.67 (t,
J ¼ 7.5 Hz, 1H), 8.10 (d, J ¼ 7.8 Hz, 2H); 13C NMR (100 MHz, CDCl3):
8.10 (d, J ¼ 7.5 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d 10.7, 14.7, 19.8,
20.8, 22.5, 26.3, 33.3, 35.2, 43.1, 46.9, 55.5, 56.3, 72.3, 73.2, 74.1, 76.4,
77.1, 77.4, 78.6, 79.1, 79.9, 80.9, 83.6, 94.1, 114.8, 115.7, 115.9, 128.5,
128.6, 128.7, 128.9, 129.8, 130.1, 132.2, 133.9, 142.1, 153.2, 153.2, 154.2,
161.3, 163.8, 166.8, 170.4, 172.2, 200.6; MALDIMS m/z 1232.2
[M þ Na]þ; HRMS (ESI) m/z calcd for C49H52NO18F3Cl6Naþ: 1232.1173
[M þ Na]þ, found 1232.1168.
d 9.4, 13.5, 19.1, 19.3, 20.4, 22.1, 26.1, 36.0, 36.7, 43.3, 46.6, 56.7, 57.7,
71.4, 71.6, 73.9, 74.3, 75.0, 76.0, 77.7, 79.9, 80.9, 84.1, 115.04, 115.4,
128.5, 129.2, 129.3, 129.9, 130.4, 133.2, 136.7, 137.4, 154.5, 162.2,
165.7, 170.0, 172.5, 210.6; ESIMS m/z 884.4 [M þ Na]þ; HRMS
(MALDI) m/z calcd for C43H50NO14F3Naþ: 884.3071 [M þ Na]þ,
found 884.30756.

1748
H.-F. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 1743e1748
4.2.4.3. N-De-t-butoxycarbonyl-N-[2-(1,1,1-trifluoro-2-methyl)pro-
pyloxycarbonyl]-30-dephenyl-30-(4-fluorophenyl)docetaxel
a mixture of acetonitrile : 5 mM ammonium acetate (55 : 45. v/v). The
flow rate was 0.6 mL/min and the injection volume was 20 mL.
(3c). Yield 52.5%; mp 166e168 ꢀC; [
a
]20D ꢁ23.8ꢀ (c 0.53 MeOH);
1H NMR (300 MHz, CD3COCD3):
d 1.15 (s, 3H), 1.20 (s, 3H), 1.58 (s,
4.5. Pharmacokinetics
6H), 1.72 (s, 3H), 1.90 (s, 3H), 2.29 (m, 2H), 2.40 (s, 3H), 1.84 and 2.46
(2m, 2H), 3.78 (s, 1H), 3.93 (d, J ¼ 7.2 Hz, 1H), 4.17 (s, 2H), 4.23 (m,
1H), 4.30 (m, 1H), 4.35 (s, 1H), 4.67 (m, 1H), 4.96 (m, 1H), 4.99 (d,
J ¼ 7.8 Hz, 1H), 5.23 (m, 1H), 5.24 (s, 1H), 5.68 (d, J ¼ 7.2 Hz, 1H), 6.10
(t, J ¼ 7.8 Hz, 1H), 7.18 (t, J ¼ 8.7 Hz, 2H), 7.19 (m, 1H), 7.54 (m, 2H),
7.56 (t, J ¼ 7.5 Hz, 2H), 7.67 (t, J ¼ 7.5 Hz, 1H), 8.10 (d, J ¼ 7.5 Hz, 2H);
The test compounds were dissolved in the mixture of cremo-
phor:etbanol (1:1), and then diluted further with sterile 0.154 M
NaCl such that i.v. dose of 5 mg/kg test compounds could be
delivered in a volume of 10 ml/kg. SpragueeDawley male rats
(200e220 g) with five rats in each group received food and water
ad libitum except on the evening prior to dosing, when all food was
removed and withheld until 2 h after dosing. Serial specimens were
collected via the retrobulbar vein at 5, 15, 30, 60, 120, 180, 240, 360,
480, 600, and 1440 min after dosing. Blood was collected by cardiac
puncture into heparinized syringes and stored on ice until centri-
fuged at 3000 rpm for 10 min to obtain plasma. Plasma and dosing
solutions were stored frozen at ꢁ20 ꢀC until analysis. The concen-
trations of the test compounds in the plasma were detected by LC-
MS-MS, and pharmacokinetic parameters were calculated from the
mean plasma concentration by non-compartmental analysis.
13C NMR (100 MHz, CDCl3):
d 9.4, 13.5, 18.9, 20.4, 22.1, 22.5, 26.1,
36.0, 36.7, 43.3, 46.6, 57.7, 71.6, 74.0, 74.3, 75.0, 76.0, 80.9, 84.1, 115.1,
128.5, 129.3, 129.9, 130.4, 133.1, 136.8, 165.8, 170.0, 172.5, 210.5;
ESIMS m/z 902.3 [M þ Na]þ; HRMS (MALDI) m/z calcd for
C43H49NO14F4Naþ: 902.2970 [M þ Na]þ, found 902.29814.
4.3. In vitro cytotoxicity
Potential cytotoxicity was evaluated against five human cancer
cell lines, i.e., breast carcinoma (MDA-MB-468), ovarian carcinoma
(HO-8910), non-small cell lung carcinoma (A549), and hepatoma
carcinoma (BEL-7402). The compounds were pre-dissolved in
DMSO and diluted with cell culture medium to at least five required
concentrations. The content of DMSO in the final concentrations
did not exceed 0.1%. At this content, DMSO was found to be
nontoxic to the cells tested. All cells were cultured in RPMI 1640
supplemented with 10% fetal bovine serum, at 37 ꢀC in a humidified
atmosphere of 5% CO2, The cells were seed at a density of
0.4e1.0 ꢂ 104 cells per well in 96-well microplates. After 24 h, the
cells were treated with 10 times diluted concentration test
compound. The cells were exposed to drugs for 72 h. Cell growth
was assayed using sulforhodamine B (SRB). The optical density (OD)
was read 520 nm. All cytotoxicity tests were performed three times
in quadruplicate. The IC50 values were calculated from curves
constructed by plotting cell survival (%) versus compound
Acknowledgments
Financial support by the National Natural Science Foundation of
China (No: 20772017), National Drug Innovative Program (No:
2009ZX09301-011), and the Shanghai Municipal Committee of
Science and Technology (No: 07DZ19713) are acknowledged. We
would like to thank Prof. B. Yang for the experiment of in vitro
cytotoxicity, and Prof. D.-F. Zhong for the experiments of metabolic
stability and pharmacokinetics.
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0.1M potassium phosphate buffer (pH 7.4) stock solutions of test
compound usually in DMSO or water) were prepared. Stock solution
of compounds was diluted into buffer to give a concentration of
5
m
mol/L with 0.5 mg/mL pooled human liver microsome (Lot 46262,
BD Gentest, US) and 1 mM NADPH in 200 L incubation solution. All
samples were incubated at 37 ꢀC and incubationwas ended byadding
200 L acetonitrile at 0, 5, 10, 15, 30, 60 min. Then the extraction
m
m
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v/v) was added and the proteins were removed bycentrifugation. The
supernatant was analyzed for the amount of remaining parent
compound by HPLC-MS. The HPLC/ITMS experiment was carried out
on an Agilent 6330LC/MSD Trap XCT ultra (Agilent Technologies,
Waldbronn, Germany). The mass spectrometer was equipped with
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100e1000. The Agilent 1200 high-performance liquid chromatog-
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