Syntheses and biological activity of C-3'-difluoromethyl-taxoids

Article abstract of DOI:10.1016/S0968-0896(00)00093-6

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly deve

Full text of DOI:10.1016/S0968-0896(00)00093-6

Bioorganic & Medicinal Chemistry 8 (2000) 1619±1628
Syntheses and Biological Activity of C-30-Di¯uoromethyl-Taxoids
Iwao Ojima,a,* Songnian Lin,a John C. Slater,a Tao Wang,a Paula Pera,b
Ralph J. Bernacki,b Cristiano Ferlinic and Giovanni Scambiac
aDepartment of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-340, USA
bDepartment of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Elm and Carlton Streets,
Bu€alo, NY, 14263, USA
cDepartment of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo A. Gernelli, 8-00168, Rome, Italy
Received 1 February 2000; accepted 6 March 2000
AbstractÐA series of new taxoids bearing di¯uoromethyl group at the C-30 position and modi®cations at the C-10 and C-14 posi-
tions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly
developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40±70 times
better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis
analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative
cancer cells. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Over the past several years, Taxol1 (paclitaxel)1 and its
semisynthetic analogue, Taxotere1 (docetaxel),2 have
been at the forefront of the development of potent
anticancer agents.3 12 These drugs possess the same
unique and e€ective mechanism of action13,14 and exhi-
bit strong activity against several cancers that are not
e€ectively treated with other chemotherapeutic drugs.
Paclitaxel received FDA approval for the treatment of
advanced ovarian cancer (December, 1992) and breast
cancer (April, 1994), while docetaxel was approved for
the treatment of breast cancer (May, 1996). Clinical
trials for other cancers, as well as combination thera-
pies, are also currently in progress worldwide.7,15 While
these two drugs have made signi®cant progress in the
treatment of various forms of cancer, reports have also
indicated that their use results in an assortment of
undesirable side-e€ects.16 In addition to these side-
e€ects, the onset of multi-drug resistance (MDR)17
clearly illustrates the necessity for the development of
the ``second-generation taxoids'' that exhibit fewer
undesirable side-e€ects and improved activity against
drug-resistant tumors.
In the course of our structure±activity relationship
(SAR) study of taxoids, we set out to incorporate
¯uorine into taxoids to study the e€ects of ¯uorine-
incorporation on the cytotoxicity of the resulting ana-
logues,12,18 blocking of known metabolic pathways
of taxoids,19 21 and the use of ¯uorine(s) as probe(s)
for the conformational analysis of these antitumor
agents.22 24 The synthesis and biological activities of
other ¯uorinated taxoids have also been reported by
Bristol-Myers Squibb laboratories.25 29
As a part of our continuing SAR study of taxoids,11,12
we have become interested in developing new taxoids
bearing a di¯uoromethyl group at C-30 position (1a±e
and 2a±f, Fig. 1). We have also studied the ability of a
new CF2H-taxoid, which possesses the strongest cyto-
toxicity in this series against drug-resistant cancer cells,
in inducing apoptosis in drug-sensitive and drug-resis-
tant cancer cells, since the aim of chemotherapy is the
induction of apoptosis in cancer cells. We describe here
the synthesis and biological activities of a series of C-30-
dephenyl-30-di¯uoromethyldocetaxel analogues derived
*Corresponding author. Tel.: +1-631-632-7890; fax: +1-631-632-
7942; e-mail: iojima@notes.cc.sunysb.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00093-6

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I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
Figure 1. 30-CF2-taxoids 1 and 2.
from 10-deacetylbaccatin III (DAB) and 14b-hydroxy-
10-deacetylbaccatin III (14b-OH-DAB).
with extremely high enantiomeric purity (>98% ee) was
prepared in excellent yield through a highly ecient
chiral enolate-imine cyclocondensation reaction devel-
oped in these laboratories.30,34 37 Ozonolysis of the 4-
alkenyl-b-lactam at low temperature a€orded the corre-
sponding 4-formyl-b-lactam 6 in quantitative yield.
Treatment of 4-formyl-b-lactam 6 with 3.0 equiv of
dialkylaminosulfur tri¯uorides (DAST)38 in dichloro-
methane for 5 h at room temperature a€orded 1-PMP-
3-OTIPS-4-CF2H-b-lactam 7 in good yield. There was
no detectable epimerization of the b-lactam in the
¯uorination step (Scheme 1). Subsequent treatment of
b-lactam 7 with ceric ammonium nitrate (CAN) remov-
ing the N-PMP group, followed by t-Boc protection of
the resulting N-H-b-lactam, a€orded 1-t-Boc-3-OTIPS-
4-CF2H-b-lactam 8 in good yield (Scheme 1).
Results and Discussion
Synthesis
A series of novel C-30-di¯uoromethyl-taxoids was syn-
thesized from DAB and 14b-OH-DAB using the coup-
ling protocols developed in these laboratories.9,11,12,30 7-
TES-baccatin III and its 10-modi®ed analogues 3a±e
(TES=triethylsilyl) were prepared in two steps from
DAB, employing previously published methods.31,32
The C-7 hydroxyl group of DAB was protected as a
triethylsilyl ether, followed by modi®cation of the C-10
hydroxyl group with chlorotriethylsilane, acyl chlorides,
or N,N-dimethylcarbamoyl chloride using LiHMDS as
the base to a€ord baccatins 3a±e in high to excellent
yields (Fig. 2).33 7-TES-10-modi®ed-baccatin-1,14-car-
bonates 4a±f were also prepared in good to excellent
yields from 14b-OH-DAB,34 through (a) protection of
the C-7 hydroxyl group of 14b-OH-DAB as a triethyl-
silyl ether, (b) protection of 1,14-cis-diol as cyclic 1,14-
carbonate with triphosgene and pyridine, and (c) mod-
i®cation of C-10 hydroxyl group with chlorotriethyl-
silane, acyl chlorides, or N,N-dimethylcarbamoyl
chloride (Fig. 2).
Novel taxoids bearing a di¯uoromethyl group at the
C-30 position and modi®cation at the C-10 position
were synthesized by applying the ring-opening coupling
protocol developed in these laboratories.9,11,12,30,36,37,39
The coupling of 4-CF2H-b-lactam 8 with 7-TES-10-
modi®ed-baccatins 3 or 7-TES-10-modi®ed-baccatin-
1,14-carbonates 4 was carried out under the standard
conditions of our protocol. Thus, treatment of a mix-
ture of baccatin (3 or 4) and b-lactam 8 in THF with
LiHMDS or NaHMDS at 40 ꢀC for 30 min a€orded
the corresponding coupling products, which were
treated with HF/pyridine to give desired 30-CF2H-tax-
oids, 1 and 2, in good to excellent yields (Scheme 2 and
Table 1).
(3R,4S)-1-PMP-3-TIPSO-4-(2-methylprop-1-enyl)-b-lactam
(5, PMP=p-methoxyphenyl, TIPSO=triisopropylsiloxy)
Figure 2. C-10-modi®ed baccatins 3 and 4.

I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
1621
Scheme 1. Synthesis of optically pure 4-CF2H-b-lactam 8.
Scheme 2. Coupling of baccatins 3 and 4 with b-lactam 8.
Table 1. Synthesis of 30-CF2-taxoids
As Table 2 shows, all of the 30-CF2H-taxoid-1,14-car-
bonates (2) exhibit activity comparable to those of
paclitaxel and docetaxel against drug-sensitive cell line
LCC6-WT, and 3±7 times better activity than those of
paclitaxel against drug-resistant cancer cell line LCC6-
MDR. Most 30-CF2H taxoids (1) in this series exhibit 3±
6 times better activity against drug-sensitive cell line
LCC6-WT, and 40±70 times better activity against
drug-resistant cancer cell line LCC6-MDR than that of
paclitaxel. All these new taxoids show 2±3 orders of
magnitude higher potency than that of doxorubicin
against both drug-sensitive and drug-resistant cell lines.
Baccatin b-Lactam
R
Taxoid Yield (%, 2 steps)
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
8
8
8
8
8
8
8
8
8
8
8
H
Ac
Me2N-CO
Et-CO
Me3CCH2-CO
H
Ac
Me2N-CO
Et-CO
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
71
54
51
62
57
59
86
79
60
81
73
Me3CCH2-CO
c-Pr-CO
There is no correlation between the activities against the
drug-sensitive and drug-resistant cell lines for both
series of taxoids. For example, taxoid 1e (R=tert-butyl-
acetyl), the least active against the drug-sensitive cell
line LCC6-WT, exhibits strongest cytotoxicity against
the drug-resistant cell line LCC6-MDR. In the same
manner, taxoid 2f (R=cyclopropanecarbonyl), that is
the least active against LCC6-WT, shows the highest
potency against LCC6-MDR in this series.
Cytotoxicity assay
Cytotoxicities of these new 30-CF2H-C-10-modi®ed-tax-
oids, 1 and 2, were evaluated in vitro against a human
breast cancer cell line, LCC6-WT, and the corre-
sponding drug-resistant cell line, LCC6-MDR.40 The
results are shown in Table 2. The assay results for
paclitaxel, docetaxel, and doxorubicin are also shown for
comparison.
It should be noted that the C-10 modi®cation sub-
stantially improves the cytotoxicity of taxoids (1a versus

1622
I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
Table 2. In vitro cytotoxicity (IC50, nM)a of 30-CF2H-taxoids 1 and 2
Table 3. DNA fragmentation rate (%)a of MDA-MB 231,b MCF-7
ADRr,c CEM,d and CEM VBLre cells after treatment with taxoids
Taxoid
R
LCC6-WTb
LCC6-MDRc
Cancer cell
C (nM) SB-T-12843 Paclitaxel Docetaxel Control
(1e)
Paclitaxel
Docetaxel
Doxorubicin
Ð
Ð
Ð
H
Ac
3.1
1.0
346
120
2900
43.5
8.79
8.20
5.69
5.04
>100d
64
180
0.96
0.50
0.48
1.0
1.34
3.2
1.9
2.8
MDA-MB 231
0
0.01
0.1
0.5
2
Ð
2.8
4.6
6.2
39.3
Ð
5.5
2.8
4.2
3.3
Ð
3.5
5
13
52
1.55
Ð
Ð
Ð
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
2f
Me3N-CO
Et-CO
Me2CCH2-CO
H
Ð
MCF-7 ADRr
0
20
50
200
500
Ð
Ð
7.1
5.6
8.1
7.8
Ð
7.5
5
9.4
7.7
4.1
Ð
Ð
Ð
Ð
14.4
52.8
76.2
89.5
Ac
Me2N-CO
Et-CO
Me3CCH2-CO
c-Pr-CO
53
63
133
49
3.2
3.2
5.6
CEM
0
10
50
100
500
1000
5000
10000
Ð
Ð
Ð
9.8
13.2
12
13.4
14
8.1
Ð
Ð
Ð
Ð
Ð
Ð
Ð
22.7
19.3
33.7
30.8
46.9
52.2
25.1
13.6
14.4
16.6
14.9
15.8
13.9
14.8
aThe concentration of compound which inhibits 50% of the growth of
the human tumor cell line after 72 h drug exposure.
bLCC6-WT: human breast carcinoma.
cLCC6-MDR: MDR1 transduced human breast carcinoma.
d20% inhibition at 100 nM.
15
15
CEM VBLr
0
10
50
100
500
1000
5000
10000
Ð
Ð
9.8
13.2
12
13.4
14
Ð
1.55
Ð
Ð
Ð
Ð
Ð
Ð
Ð
22.7
19.3
33.7
30.8
46.9
52.2
25.1
13.6
14.4
16.6
14.9
15.8
13.9
14.8
1b±e; 2a versus 2b±d, f). However, in contrast to the C-
30-alkyl and alkenyl series of taxoids previously repor-
ted,33,34 the e€ect of the size of the C-10 substitution on
cytotoxicity is very small except for the case of 2e.
15
15
Apoptosis assay
aData obtained after 72 h of cell exposure to the taxoids.
bMDA-MB 231: human breast cancer cell line.
Besides blocking cell replication by inhibiting the depoly-
merization of microtubules, paclitaxel, like vincristine,
was also found to induce apoptosis.41 This appears to
suggest that perturbation of the microtubule assembly
could trigger programmed cell death. Since the 30-
CF2H-taxoids exhibit exceptional potency in the cyto-
toxicity assay, it would be of great interest to study the
activity of these taxoids in inducing apoptosis. Thus, we
selected compound SB-T-12843 (1e) to perform the
apoptosis analysis against four cell lines, i.e., leukemia
cells, CEM and CEM VBLr; breast cancer cells, MDA-
MB 231 and MCF-7 ADRr. The CEM VBLr and
MCF-7 ADRr cell lines overexpress P-gp protein.
MDA-MB 231 cells were preferred over the MDR-
negative MCF-7 wt, because this cell line does not
overexpress P-gp protein under the selective pressure of
xenobiotics. The data for paclitaxel and docetaxel were
also obtained for comparison.
cMCF-7 ADRr: human breast adriamycin (ADR)-resistant cancer cell
line.
dCEM VBLr: human leukemia CEM vinblastine (VBL)-resistant cell
line.
eCEM: human leukemia CEM cell line.
showing the exceptional activity of SB-T-12843 (1e)
against P-gp overexpressing cells. The same phenomena
are observed in breast cancer cells where SB-T-12843
starting from 50 nM induces a dose-dependent increase
in the DNA fragmentation of MCF-7 ADRr cells. SB-
T-12843 (1e) exhibits a similar potency to that of doc-
etaxel and a higher potency than that of paclitaxel in
MDA-MB231 cells.
These ®ndings indicate that SB-T-12843 (1e) can induce
apoptosis in the P-gp negative cells at a level similar to
that of docetaxel and higher than that of paclitaxel. On
the other hand, in the P-gp positive cells SB-T-12843
(1e) induces a remarkably higher level of apoptosis at
lower concentrations as compared to those of paclitaxel
and docetaxel. Further studies are currently in progress in
these laboratories to ascertain if the enhanced apoptotic
ability of the taxoids against P-gp positive MDR cancer
cells is closely linked to their ability to induce the dis-
ruption of microtubules and the cell division cycle arrest.
We scored the occurrence of apoptosis by evaluating
DNA fragmentation by ¯ow cytometry, a marker of
apoptosis in a number of experimental models,42
including cells tested for taxoids.43 Table 3 shows the
quantitative data of DNA fragmentation rate obtained
from analysis of SB-T-12843 (1e) against the four cell
lines assayed. The rates for paclitaxel and docetaxel in
the same assay are also shown for comparison purposes.
Figure 3 summarizes the DNA fragmentation data for
SB-T-12843 (1e), paclitaxel, and docetaxel by normal-
izing the results as percentage of the control. As Figure
3 illustrates, the three drugs exhibit similar ability in
inducing apoptosis in CEM cells, while only SB-T-
12843 induces apoptosis in CEM VBLr cells. This ®nd-
ing is consistent with the cytotoxicity assay results
Conclusions
Novel taxoids bearing a di¯uoromethyl group at the
C-30 position and modi®cations at the C-10 and C-14

I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
1623
Figure 3. Apoptosis assay of paclitaxel, docetaxel, and SB-T-12843 (1e) on MDA-MB 231, MCF-7 ADRr, CEM and CEM VBLr cells.
positions have been synthesized and their biological
activities examined. The newly developed taxoids exhi-
bit higher potency against drug-sensitive cell line LCC6-
WT, and 40±70 times higher potency against drug-
resistant cancer cell line LCC6-MDR as compared to
that of paclitaxel. One of these novel CF2-taxoids, SB-
T-12843 (1e), is found to possess exceptional activity in
inducing apoptosis in both MDR-bearing and MDR-
negative cancer cells.
California, Riverside Mass Spectrometry Facility, Riv-
erside, CA; or Mass Spectrometry Laboratory, Uni-
versity of Illinois at Urbana-Champaign, Urbana, IL.
Materials. The chemicals were purchased from Aldrich
Co. and Sigma, and puri®ed before use by standard
methods. Tetrahydrofuran was freshly distilled under
sodium metal and benzophenone. Dichloromethane was
also distilled immediately prior to use under nitrogen
from calcium hydride. 7-TES-10-modi®ed baccatins 3a±
e and 7-TES-10-modi®ed-baccatin-1,14-carbonates 4a±f
were prepared using our published procedures.33,34
(3R,4S)-1-PMP-3-TIPSO-4-(2-methylprop-1-enyl)-b-
lactam (5) was prepared through a highly ecient chiral
ester enolate-imine cyclocondensation reaction pre-
viously reported from our laboratories.30,34,36,37
Experimental
Chemical synthesis
1
General methods. H and 13C NMR spectra were mea-
sured on a Bruker AC-250 NMR spectrometer or a
Varian 300 NMR spectrometer using tetramethylsilane
as the internal standard. Melting points were measured
on a Thomas Hoover Capillary melting point apparatus
and are uncorrected. Optical rotations were measured
on a Perkin±Elmer Model 241 polarimeter. IR spectra
were recorded on a Perkin±Elmer Model 1600 FT-IR
spectrophotometer. TLC was performed on Merck DC-
alufolien with Kieselgel 60F-254 and column chroma-
tography was carried out on silica gel 60 (Merck; 230±
400 mesh ASTM). Elemental analyses were performed
at M-H-W Laboratory, Phoenix, AZ. High resolution
mass spectra were obtained from the University of
(3R,4S)-1-(4-Methoxylphenyl)-3-triisopropylsiloxy-4-for-
mylazetidin-2-one (6). Nitrogen gas was bubbled into a
solution of 4-(2-methyl-2-propenyl)-b-lactam 5 (500 mg,
1.239 mmol) in MeOH:CH2Cl2 (1:2, 27 mL) at 78 ꢀC
for 3 min. Then, O3 gas was bubbled into the solution
till the color of the solution turned blue (2 min), and N2
was bubbled into the solution for another 3 min. Dime-
thyl sul®de (0.458 mL, 6.194 mmol) was added to the
solution and the reaction mixture was allowed to warm
to room temperature. The reaction mixture was stirred
for 3 h at room temperature. The solvents were removed
in vacuo and the residue was further puri®ed on a neu-

1624
I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
tral alumina column using hexane:ethyl acetate (4:1 fol-
lowed by 2:1) as the eluant to a€ord 6 as a white solid
(467.5 mg, quantity): mp 78±80 ꢀC; [a]d20 +158.0 (c 0.50,
added, at 0 ꢀC, 315 mg (1.44 mmol) of di-tert-butyl
dicarbonate in 2 mL of methylene chloride. The reaction
mixture was stirred at room temperature for 3 h, quen-
ched with saturated aqueous NH4Cl, and extracted with
ethyl acetate. The combined organic layers were washed
with saturated aqueous NH4Cl and brine, dried over
MgSO4, ®ltered, and concentrated in vacuo. The crude
material was puri®ed by column chromatography on
silica gel (hexane:EtOAc=5:1) to a€ord product 8 as a
1
CHCl3); H NMR (250 MHz, CDCl3) d 1.06 (m, 21H),
3.74 (s, 3H), 4.43 (dd, J=5.1, 3.8 Hz, 1H), 5.25 (d,
J=5.4 Hz, 1H), 6.81 (d, J=8.8 Hz, 1H), 7.22 (d,
J=8.8 Hz, 1H), 9.72 (d, J=3.8 Hz, 1H); 13C NMR
(62.9 MHz, CDCl3) d 11.6, 17.4, 55.3, 64.2, 78.6, 114.4,
117.8, 130.7, 156.7, 164.2, 199.6. Anal. calcd for C20H31
NO4Si: C, 63.63; H, 8.28; N, 3.71. Found: C, 63.80; H,
8.05; N, 3.72.
1
colorless oil (452 mg, 88%): H NMR (CDCl3) d 1.06±
1.21 (m, 21 H), 1.48 (s, 9H), 4.20±4.31 (m, 1H), 5.11 (d,
J=5.8 Hz, 1H), 5.99 (ddd, J=54.2, 54.2, 4.8 Hz, 1H);
13C NMR (CDCl3) d 11.74, 17.44, 17.50, 27.83, 57.32
(m), 76.02, 84.18, 147.51, 164.73; 19F NMR (CDCl3) d
122.9 (ddd, J=297.9, 53.8, 8.9 Hz, 1F), 127.4 (ddd,
J=297.9, 55.0, 8.5 Hz, 1F). Anal. calcd for C18H33
F2NO4Si: C, 54.94; H, 8.45; N, 3.56. Found: C, 55.02;
H, 8.33; N, 3.68.
(3R,4S)-1-(4-Methoxyphenyl)-3-triisopropylsiloxy-4-di-
¯uoromethylazetidin-2-one (7). To a solution of 800 mg
(2.12 mmol) of 4-formyl-b-lactam 6 in 13 mL of methyl-
ene chloride was added dropwise 0.84 mL (6.37 mmol)
of diethylaminosulfur tri¯uoride at room temperature.
The reaction was monitored by TLC, quenched after 5 h
with water, and extracted with ethyl acetate. The com-
bined organic layers were washed with water, dried over
MgSO4, ®ltered, and concentrated in vacuo. The crude
material was puri®ed by column chromatography on
silica gel (hexane:EtOAc=6:1) to a€ord 665 mg (78%)
of the product 7 as a light yellow oil: [a]2d0 +55.9 (c 0.34,
General procedure for synthesis of 30-dephenyl-30-di-
¯uoro-taxoids 1 and 2. A general procedure is described
for the synthesis of 30-dephenyl-30-di¯uoromethyl-
docetaxel 1a: To a solution of 7,10-di-TES baccatin III
3a (54 mg, 0.07 mmol) and 4-di¯uoromethyl-b-lactam 8
(52 mg, 0.13 mmol) in 2 mL dry THF was added 1.0 M
NaHMDS in THF (0.11 mL, 0.11 mmol) dropwise at
40 ꢀC, and the solution was stirred at 40 ꢀC for
30 min. The reaction was quenched with aqueous satu-
rated ammonium chloride solution (5 mL), and the
aqueous layer was extracted with dichloromethane
(25 mLÂ3). The combined extracts were then dried over
anhydrous magnesium sulfate and concentrated in
vacuo. The residue was puri®ed on a silica gel column
using hexane:ethyl acetate (6:1) as the eluant to a€ord
the coupling product 20-triisopropylsilyl-30-di¯uoro-
methyl-7,10-di-triethylsilyldocetaxel as a white solid
(66 mg, 82.5%): 1H NMR (CDCl3) d 0.52±0.73 (m
12H), 0.92±1.21 (m, 45H), 1.30 (s, 9H), 1.61±1.65 (m,
4H), 1.82±1.89 (m, 4 H), 2.20±2.37 (m, 4H), 2.43±2.58
(m, 1H), 3.83 (d, J=6.9 Hz, 1H), 4.15 (d, J=8.3 Hz,
1H), 4.25±4.48 (m, 3H), 4.83 (br s, 1 H), 4.89 (d,
J=8.6 Hz, 1H), 5.00 (d, J=10.2 Hz, 1H), 5.15 (s, 1H),
5.65 (d, J=6.9 Hz, 1H), 5.77 (ddd, J=56.1, 55.8, 6.3 Hz,
1H), 6.10 (t, J=8.8 Hz, 1H), 7.48 (t, J=7.4 Hz, 2H),
7.57 (t, J=7.4 Hz, 1H), 8.11 (d, J=7.4 Hz, 2H).
1
CHCl3); H NMR (CDCl3) d 1.12±1.26 (m, 21H), 3.78
(s, 3H), 4.31±4.40 (m, 1H), 5.17 (d, J=5.1 Hz, 1H), 6.07
(ddd, J=55.7, 53.5, 5.3 Hz, 1H), 6.87 (d, J=8.8 Hz,
2H), 7.45 (d, J=8.8 Hz, 2H); 13C NMR (CDCl3) d
11.89, 17.65, 55.39, 58.41(m), 75.75, 75.84, 114.21,
118.93, 130.65, 156.73, 165.10; 19F NMR (CDCl3) d
119.3 (ddd, J=298.7, 53.5, 7.1 Hz, 1F), 123.7 (ddd,
J=298.7, 56.2, 7.1 Hz, 1F). Anal. calcd for C20H31
NO3SiF2: C, 60.12; H, 7.82; N, 3.51. Found: C, 60.81;
H, 7.84; N, 3.60.
(3R,4S)-1-tert-Butoxycarbonyl-3-triisopropylsiloxy-4-di-
¯uoromethylazetidin-2-one (8). To a solution of 638 mg
(1.6 mmol) of 4-di¯uoromethyl-b-lactam 7 in 40 mL of
CH3CN at 10 ꢀC was added 3.5 g (6.4 mmol) of
ceric(IV) ammonium nitrate (CAN) dropwise over a
period of 1 h at 10 ꢀC. The reaction mixture was stir-
red at 5 ꢀC for an additional 2 h and quenched with
the addition of 75 mL of water. The reaction mixture
was extracted with ethyl acetate and the combined
organic layers were washed with 5% NaHSO3, 10%
Na2CO3, 5% NaHSO3, and brine. The organic layer
was then dried over MgSO4, ®ltered, and concentrated
in vacuo. Puri®cation of the crude product by column
chromatography on silica gel (hexane:EtOAc=4:1 to 2:1)
a€orded 397 mg (85%) of the N-H-b-lactam as a light
yellow oil: [a]d20 +33.3 (c 0.18, CHCl3); 1H NMR
(CDCl3) d 1.09±1.21 (m, 21H), 3.79±3.91 (m, 1H), 5.07
(br s, 1H), 5.86 (ddd, J=54.2, 54.1, 6.5 Hz, 1H), 6.79 (br
s, 1H); 13C NMR (CDCl3) d 11.74, 17.44, 55.24 (m),
77.85, 77.97, 169.02; 19F NMR (CDCl3) d 121.3 (ddd,
J=299.4, 53.7, 7.3 Hz, 1F), 122.6 (ddd, J=299.4,
54.2, 7.0 Hz, 1F). Anal. calcd for C13H25F2NO2Si: C,
53.21; H, 8.59; N, 4.77. Found: C, 53.08; H, 8.72; N,
4.87.
To a solution of coupling product thus obtained
(66.0 mg, 0.057 mmol) in 4 mL of pyridine:acetonitrile
(1:1) was added dropwise HF:pyridine (70:30, 0.6 mL)
at 0 ꢀC, and the mixture was stirred at room tempera-
ture for 12 h. The reaction was quenched with aqueous
saturated sodium carbonate solution (5.0 mL). The
mixture was then diluted with ethyl acetate (70 mL),
washed with aqueous saturated copper sulfate solution
(10 mLÂ3) and water (10 mL), dried over anhydrous
magnesium sulfate and concentrated in vacuo. The
crude product was puri®ed on a silica gel column using
hexane:ethyl acetate (1:1) as the eluant to a€ord new
taxoid 1a as a white solid (38.0 mg, 86% yield): mp
20
158.0±160.0 ꢀC; [a]d 88.5 (c 0.26, CHCl3); H NMR
(CDCl3) d 1.11 (s, 3H), 1.22 (s, 3H), 1.32 (s, 9H), 1.72±
1.77 (m, 5H), 1.88 (s, 3H), 2.17±2.33 (m, 1H), 2.38 (s,
3H), 2.51±2.62 (m, 1H), 3.87 (d, J=6.9 Hz, 1H), 4.00 (br
1
To a solution of N-H-b-lactam thus obtained (384 mg,
1.31 mmol), 25 mg of DMAP, and 0.37 mL (2.62 mmol)
of triethylamine in 7 mL of dry methylene chloride was

I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
1625
s, 1H), 4.14±4.49 (m, 5H), 4.64 (br s, 1H), 4.94 (d,
J=8.7 Hz, 1H), 5.19 (d, J=10.1 Hz, 1H), 5.27 (s, 1H),
5.65 (d, J=6.9 Hz, 1H), 5.66±6.10 (m, 2H), 6.22 (t,
J=8.5 Hz, 1H), 7.49 (t, J=7.4 Hz, 2H), 7.60 (t,
J=7.4 Hz, 1H), 8.09 (d, J=7.4 Hz, 2H); 13C NMR
(CDCl3) d 9.64, 14.46, 20.75, 22.44, 26.30, 28.02, 35.45,
36.86, 43.16, 46.56, 54.58 (m), 57.75, 68.53, 71.94, 72.87,
74.56, 74.86, 78.62, 80.78, 81.05, 84.21, 114.13, 118.03,
128.77, 129.08, 130.22, 133.71, 136.04, 138.31, 155.15,
167.13, 170.34, 172.64; 19F NMR (CDCl3) d 128.6
(ddd, J=287, 56.1, 8.3 Hz, 1F), 126.1 (ddd, J=287,
55.5, 11.3 Hz, 1F). Anal. calcd for C38H49F2NO14: C,
58.38; H, 6.32. Found: C, 58.19; H, 6.51.
1H), 4.94 (d, J=9.4 Hz, 1H), 5.11 (d, J=10.0 Hz, 1H),
5.63±6.10 (m, 2H), 6.20±6.29 (m, 2H), 7.49 (t, J=
7.4 Hz, 2H), 7.60 (t, J=7.4 Hz, 1H), 8.11 (d, J=7.4 Hz,
2H); 13C NMR (CDCl3) d 9.56, 13.71, 14.80, 21.93,
22.20, 22.50, 26.72, 26.88, 28.00, 33.90, 35.41, 35.56,
43.24, 45.68, 54.58 (m), 58.59, 68.43, 72.18, 72.91, 75.00,
75.27, 79.03, 80.93, 81.09, 84.46, 114.11, 128.75, 129.04,
130.22, 133.38, 133.70, 141.73, 155.06, 167.16, 170.36,
172.35, 174.01, 203.51; 19F NMR (CDCl3) d 126.2 (ddd,
J=286.9, 55.5, 11.4 Hz, 1F), 128.6 (ddd, J=286.9,
56.0, 9.4 Hz, 1F). Anal. calcd for C41H53F2NO15: C,
58.77; H, 6.38. Found: C, 58.51; H, 6.45.
30-Dephenyl-30-di¯uoromethyl-10-(3,3-dimethylbutanoyl)-
30-Dephenyl-30-di¯uoromethyl-10-acetyldocetaxel (1b).
White solid; 54% yield (two steps); mp 158.0±160.0 ꢀC;
[a]2d0 113 (c 0.16, CHCl3); 1H NMR (CDCl3) d 1.15 (s,
3H), 1.25 (s, 3H), 1.31 (s, 9H), 1.68 (s, 3H), 1.89 (br s, 4
H), 2.24 (br s, 4H), 2.40 (s, 3H), 2.49±2.61 (m, 1H), 3.41
(br s, 1H), 3.81 (d, J=6.9 Hz, 1H), 4.17 (d, J=8.2 Hz,
1H), 4.31 (d, J=8.2 Hz, 1H), 4.36±4.47 (m, 2H), 4.63 (s,
1H), 4.95 (d, J=9.2 Hz, 1H), 5.04 (d, J=10.1 Hz, 1H),
5.64±6.10 (m, 2H), 6.20±6.30 (m, 2H), 7.50 (t, J=
7.4 Hz, 2H), 7.61 (t, J=7.4 Hz, 1H), 8.12 (d, J=7.4 Hz,
2H); 13C NMR (CDCl3) d 9.56, 14.83, 20.84, 21.90,
22.50, 26.72, 35.39, 35.59, 43.25, 45.65, 54.56 (m), 58.61,
68.42, 72.17, 72.94, 74.98, 75.51, 79.04, 81.07, 84.43,
114.09, 128.76, 129.06, 130.22, 133.26, 133.71, 141.67,
155.05, 167.18, 170.34, 171.25, 172.38, 203.61; 19F
NMR (CDCl3) d 126.24 (ddd, J=287.2, 55.5, 11.7 Hz,
1F), 128.56 (ddd, J=287.2, 56.2, 8.6 Hz, 1F). Anal.
calcd for C40H51F2NO15: C, 58.32; H, 6.24. Found: C,
58.07; H, 6.01.
docetaxel (1e). White solid; 57% yield (two steps); mp
ꢀ
141.0±143.0 C; [a]d20 86.3 (c 0.51, CHCl3); H NMR
(CDCl3) d 1.10 (s, 9H), 1.14 (s, 3H), 1.23 (s, 3H), 1.30 (s,
9H), 1.67±1.73 (m, 4H), 1.87 (br s, 4H), 2.19±2.46 (m,
6H), 2.49±2.68 (m, 1H), 3.46 (br s, 1H), 3.81 (d,
J=6.9 Hz, 1H), 4.17 (d, J=8.3 Hz, 1H), 4.30 (d,
J=8.3 Hz, 1H), 4.38±4.52 (m, 2 H), 4.63 (br s, 1H), 4.95
(d, J=9.3 Hz, 1H), 5.06 (d, J=10.0 Hz, 1H), 5.64±6.10
(m, 2H), 6.20±6.27 (m, 2H), 7.49 (t, J=7.4 Hz, 2H), 7.60
(t, J=7.4 Hz, 1H), 8.11 (d, J=7.4 Hz, 2H); 13C NMR
(CDCl3) d 9.58, 144.20, 14.80, 22.00, 22.51, 26.78, 28.00,
29.64, 31.03, 35.48, 43.22, 45.64, 47.93, 54.58 (m), 58.62,
68.53, 72.24, 72.93, 75.02, 75.18, 76.51, 79.07, 80.94,
81.18, 84.49, 114.11, 128.76, 129.05, 130.22, 133.57,
133.70, 141.69, 155.05, 167.16, 170.35, 172.34, 172.47,
203.52; 19F NMR (CDCl3) d 126.2 (ddd, J= 287.2,
55.5, 11.4 Hz, 1F), 128.5 (ddd, J=287.2, 56.3, 8.5 Hz,
1F). Anal. calcd for C44H59F2NO15: C, 60.06; H, 6.76.
Found: C, 60.12; H, 6.99.
1
30 -Dephenyl-30 -di¯uoromethyl-10-(N,N-dimethyl-carba-
moyl)docetaxel (1c). White solid; 51% yield (two steps);
mp 168.0±170.0 ꢀC; [a]d20 52.0 (c 0.25, CHCl3); 1H
NMR (CDCl3) d 1.15 (s, 3H), 1.24 (s, 3H), 1.30 (s, 9H),
1.66 (s, 3H), 1.74±1.82 (m, 1H), 1.90 (br s, 4H), 2.21±
2.37 (m, 1H), 2.39 (s, 3H), 2.44±2.58 (m, 1H), 2.95 (s,
3H), 3.04 (s, 3H), 3.58 (br s, 1H), 3.80 (d, J=6.9 Hz,
1H), 4.16 (d, J=8.3 Hz, 1H), 4.30 (d, J=8.3 Hz, 1H),
4.38±4.49 (m, 2H), 4.63 (br s, 1H), 4.96 (d, J=8.6 Hz, 1
H), 5.13 (d, J=10.1 Hz, 1H), 5.63±6.10 (m, 2H), 6.18±
6.29 (m, 2H), 7.49 (t, J=7.4 Hz, 2H), 7.59 (t, J=7.4 Hz,
1H), 8.11 (d, J=7.4 Hz, 2H); 13C NMR (CDCl3) d 9.38,
14.90, 22.32, 22.51, 26.70, 28.00, 35.40, 36.06, 36.66,
43.25, 45.63, 54.56 (m), 58.51, 68.45, 68.64, 72.46, 72.90,
75.11, 76.12, 79.13, 80.85, 81.10, 84.67, 110.24, 114.14,
116.04, 128.75, 129.06, 130.24, 133.54, 133.68, 142.26,
155.09, 156.12, 167.15, 170.29, 172.34, 205.59; 19F
NMR (CDCl3) d 126.2 (ddd, J=286.7, 55.7, 10.6 Hz,
1F), 128.2 (ddd, J=286.7, 56.0, 9.2 Hz, 1F). Anal.
calcd for C41H54F2NO15: C, 57.75; H, 6.38. Found: C,
57.53; H, 6.38.
30 -Dephenyl-30 -di¯uoromethyldocetaxel-1,14-carbonate
(2a). White solid; 59% yield (two steps); mp 184±
187 ꢀC; [a]d20 25.0 (c 0.32, CHCl3); 1H NMR (CD3OD)
d 1.25 (s, 3H), 1.27 (s, 3H), 1.37 (s, 9H), 1.43 (m, 1H),
1.73 (s, 3H), 1.79 (m, 1H), 1.95 (s, 3H), 2.44 (m, 1H),
2.52 (s, 3H), 3.84 (d, J=7.5 Hz, 1H), 4.24 (m, 3H), 4.52
(m, 1H), 4.71 (br s, 1H), 4.91 (d, J=6.8 Hz, 1H), 5.00
(d, J=9.0 Hz, 1H), 5.29 (s, 1H), 5.69±6.17 (dt, J=56.4,
6.5 Hz, 1H), 6.07 (d, J=7.5 Hz, 1H), 6.41 (d, J=6.5 Hz,
1H), 6.80 (d, J=10.0 Hz, 1H), 7.55 (t, J=7.4 Hz, 2H),
7.67 (t, J=7.1 Hz, 1H), 8.10 (d, J=7.7 Hz, 2H); 13C
NMR (CD3OD) d 10.5, 14.6, 22.5, 23.0, 26.2, 28.7, 37.3,
43.0, 49.4, 59.3, 70.2, 70.8, 72.3, 75.3, 75.6, 77.0, 81.3,
81.8, 85.7, 87.4, 89.7, 103.2, 116.4, 129.6, 130.2, 131.2,
135.4, 136.8, 138.2, 165.8, 166.4, 167.4, 169.0, 172.4,
209.3. Anal. calcd for C39H47O16N1F2: C, 56.86; H,
5.75; N, 1.70. Found: C, 57.09; H, 5.73; N, 1.68.
30 -Dephenyl-30 -di¯uoromethyl-10-acetyldocetaxel-1,14-
carbonate (2b). White solid; 86% yield (two steps); mp
188±189 ꢀC; [a]d20 40.0 (c 0.15, CHCl3); 1H NMR
(CDCl3) d 1.27 (s, 3H), 1.32 (s, 12H), 1.71 (s, 3H), 1.85
(m, 1H), 1.89 (s, 3H), 2.24 (s, 3H), 2.49 (s, 3H), 2.54 (m,
1H), 3.71 (d, J=7.3 Hz, 1H), 4.21 (d, J=8.4 Hz, 1H),
4.28 (d, J=8.4 Hz, 1H), 4.39 (dd, J=10.5, 6.8 Hz, 1H),
4.50 (m, 1H), 4.72 (s, 1H), 4.84 (d, J=6.6 Hz, 1H), 4.91
(d, J=8.9 Hz, 1H), 5.27 (d, J=10.0 Hz, 1H), 5.90 (td,
J=56.1, 5.5 Hz, 1H), 6.09 (d, J=7.5 Hz, 1H), 6.27 (s,
1H), 6.44 (d, J=6.2 Hz, 1H), 7.49 (t, J=7.3 Hz, 2H),
30-Dephenyl-30-di¯uoromethyl-10-propanoyldocetaxel (1d).
White solid; 62% yield (two steps); mp 134.0±136.0 ꢀC;
1
[a]2d0 108 (c 0.26, CHCl3); H NMR (CDCl3) d 0.94 (t,
J=7.2 Hz, 3H), 1.14 (s, 3H), 1.21±1.52 (m, 14H), 1.69
(br s, 4H), 1.90 (br s, 4H), 2.21±2.59 (m, 7H), 3.51 (br s,
1H), 3.81 (d, J=6.8 Hz, 1H), 4.16 (d, J=8.3 Hz, 1H),
4.30 (d, J=8.3 Hz, 1H), 4.39±4.47 (m, 2H), 4.62 (br s,

1626
I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
7.60 (t, J=7.2 Hz, 1H), 8.08 (d, J=7.4 Hz, 2H); 13C
NMR (CDCl3) d 9.7, 14.8, 20.7, 22.5, 23.0, 25.8, 28.0,
35.5, 41.8, 45.2, 54.3 (m), 58.7, 68.9, 69.2, 71.7, 74.7,
75.0, 79.5, 80.5, 81.2, 84.2, 88.1, 110.3, 114.2, 118.1,
127.8, 129.0, 130.0, 133.7, 134.1, 139.0, 151.8, 155.3,
164.7, 170.9, 171.4, 202.0. Anal. calcd for C41H49
F2NO17: C, 56.88; H, 5.70; N, 1.62. Found: C, 56.66; H,
5.88; N, 1.74.
156.9, 164.8, 166.6, 170.9, 171.1, 171.5, 201.6. Anal.
calcd for C45H57F2NO17: C, 58.63; H, 6.23; N, 1.52.
Found: C, 58.48; H, 6.18; N, 1.51.
30-Dephenyl-30-di¯uoromethyl-10-cyclopropanylcarbonyl-
docetaxel-1,14-carbonate (2f). White solid; 73% yield
(two steps); mp 186±188 ꢀC; [a]d20 40.4 (c 0.52, CHCl3);
1H NMR (CDCl3) d 0.85 (m, 1H), 1.03±1.44 (m, 21H),
1.70 (s, 3H), 1.79 (m, 1H), 1.89 (s, 3H), 2.44 (m, 4H),
3.72 (d, J=7.5 Hz, 1H), 4.22 (dd, J=17.2, 8.5 Hz, 2H),
4.39 (m, 1H), 4.50 (m, 1H), 4.71 (br s, 1H), 4.84 (d,
J=6.6 Hz, 1H), 4.90 (d, J=8.6 Hz, 1H), 5.17 (d,
J=10.0 Hz, 1H), 5.67±6.07 (dt, J=56.4, 6.5 Hz, 1H),
6.08 (d, J=7.6 Hz, 1H), 6.25 (s, 1H), 6.44 (d, J=6.5 Hz,
1H), 7.49 (t, J=7.5 Hz, 2H), 7.58 (t, J=7.1 Hz, 1H),
8.04 (d, J=7.4 Hz, 2H); 13C NMR (CDCl3) d 9.4, 9.7,
12.9, 14.9, 22.5, 23.2, 26.0, 28.1, 35.4, 41.8, 45.1, 58.7,
68.9, 69.3, 71.8, 74.5, 75.3, 76.0, 79.5, 84.3, 87.3, 88.1,
127.9, 129.0, 130.0, 134.1, 139.1, 152.0, 155.3, 164.5,
170.8, 171.5, 174.9, 202.2. Anal. calcd for C43H51
O17N1F2: C, 57.91; H, 5.76; N, 1.57. Found: C, 57.70;
H, 5.89; N, 1.63.
30 -Dephenyl-30 -di¯uoromethyl-10-(N,N-dimethylcarba-
moyl)docetaxel-1,14-carbonate (2c). White solid; 79%
yield (two steps); mp 174±176 ꢀC; [a]d20 45.0 (c 0.27,
1
CHCl3); H NMR (CDCl3) d 1.30 (m, 15H), 1.70 (s,
3H), 1.83 (m, 1H), 1.91 (s, 3H), 2.48 (s, 3H), 2.53 (m,
1H), 2.96 (s, 3H), 3.04 (s, 3H), 3.71 (d, J=7.4 Hz, 1H),
4.21 (d, J=8.4 Hz, 1H), 4.28 (d, J=8.4 Hz, 1H), 4.44
(m, 2H), 4.70 (s, 1H), 4.84 (d, J=6.6 Hz, 1H), 4.92 (d,
J=8.5 Hz, 1H), 5.44 (d, J=10.0 Hz, 1H), 5.90 (td,
J=56.3, 5.1 Hz, 1H), 6.08 (d, J=7.5 Hz, 1H), 6.22 (s,
1H), 6.46 (d, J=5.9 Hz, 1H), 7.49 (t, J=7.3 Hz, 2H),
7.60 (t, J=7.2 Hz, 1H), 8.08 (d, J=7.4 Hz, 2H); 13C
NMR (CDCl3) d 9.5, 14.9, 22.5, 23.4, 25.9, 28.0, 35.3,
36.1, 36.7, 41.8, 45.2, 54.3 (m), 58.6, 68.9, 69.3, 72.0,
74.9, 75.4, 75.9, 79.7, 80.6, 81.0, 84.5, 87.4, 88.3, 110.4,
114.3, 118.2, 127.9, 129.0, 130.0, 133.9, 134.1, 139.6,
152.0, 155.3, 155.8, 164.7, 170.9, 171.4, 203.9. Anal.
calcd for C42H52F2NO17: C, 56.37; H, 5.86; N, 3.13.
Found: C, 56.31; H, 5.91; N, 3.04.
Cytotoxicity assay
Materials and methods. Human tumor cell lines
MDA435/LCC6-WT and MDR1 were provided by
Dr. R. Clarke, Lombardi Cancer Center, Georgetown
University School of Medicine. Cell lines were propa-
gated as monolayers in RPMI-1640 containing 5%
FCS, 5% NuSerum IV, 20 mM HEPES, 2 mM l-gluta-
mine at 37 ꢀC in a 5% CO2 humidi®ed atmosphere. The
doubling times for the cell lines range between 20 and
24 h.
30 -Dephenyl-30 -di¯uoromethyl-10-propanoyldocetaxel-
1,14-carbonate (2d). White solid; 60% yield (two steps);
mp 177±180 ꢀC; [a]d20 62.5 (c 0.24, CHCl3); H NMR
1
(CDCl3) d 1.20±1.40 (m, 20H), 1.71 (s, 3H), 1.89 (s, 3H),
2.39 (m, 7H), 3.73 (m, 2H), 3.85 (s, 3H), 4.22 (dd,
J=17.2, 8.5 Hz, 2H), 4.39 (m, 1H), 4.50 (m, 1H), 4.71
(br s, 1H), 4.84 (d, J=6.6 Hz, 1H), 4.90 (d, J=8.6 Hz,
1H), 5.15 (d, J=9.9 Hz, 1H), 5.67±6.07 (dt, J=56.4,
6.5 Hz, 1H), 6.08 (d, J=7.6 Hz, 1H), 6.27 (s, 1H), 6.44
(d, J=6.5 Hz, 1H), 7.49 (t, J=7.5 Hz, 2H), 7.58 (t,
J=7.1 Hz, 1H), 8.04 (d, J=7.4 Hz, 2H); 13C NMR
(CDCl3) d 9.0, 9.7, 14.9, 22.5, 23.1, 25.9, 27.5, 28.1, 35.5,
41.8, 45.2, 58.7, 68.8, 69.3, 71.8, 74.5, 75.2, 76.0, 79.5,
80.6, 81.4, 84.3, 87.3, 88.1, 127.9, 129.0, 130.0, 134.1,
138.9, 151.7, 155.3, 164.7, 170.8, 171.5, 174.3, 202.1.
Anal. calcd for C42H51O17N1F2: C, 57.33; H, 5.84; N,
1.59. Found: C, 56.97; H, 5.87; N, 1.61.
Growth inhibition assay. In 96 well microtiter plates
assessment of cell growth inhibition was determined
according to the methods of Skehan et al.40 Brie¯y, cells
were plated at 2000 cells/well in 96 well plates and
incubated at 37 ꢀC for 15±18 h prior to drug addition to
allow cell attachment. Compounds were dissolved in
100% DMSO and further diluted in RPMI-1640 con-
taining 10 mM HEPES. Each cell line was treated with
10 concentrations of compound (5 log range). After a
72 h incubation, 100 mL of ice-cold 50% TCA was
added to each well and incubated for 1 h at 4 ꢀC. Plates
were then washed 5 times with tap water to remove
TCA, low-molecular-weight metabolites and serum
proteins. Then, 50 mL of 0.4% sulforhodamine B (SRB)
was added to each well. Following a 5 min incubation at
room temperature, plates were rinsed 5 times with 0.1%
acetic acid and air dried. Bound dye was dissolved with
10 mM Tris base (pH 10.5) for 5 min on a gyratory
shaker. Optical density was measured at 570 nm.
30-Dephenyl-30-di¯uoromethyl-10-(3,3-dimethylbutanoyl)-
docetaxel-1,14-carbonate (2e). White solid; 81% yield
(two steps); mp 165±167 ꢀC; [a]d20 51.4 (c 0.12, CHCl3);
1H NMR (CDCl3) d 0.98 (s, 9H), 1.20 (s, 6H), 1.25 (s,
9H), 1.61 (s, 3H), 1.75 (m, 1H), 1.81 (s, 3H), 2.27 (s,
3H), 2.40 (s, 2H), 2.45 (m, 1H), 3.63 (d, J=7.5 Hz, 1H),
4.11 (d, J=8.5 Hz, 1H), 4.19 (d, J=8.4 Hz, 1H), 4.25
(dd, J=10.9, 6.7 Hz, 1H), 4.39 (m, 1H), 4.61 (s, 1H),
4.76 (d, J=6.7 Hz, 1H), 4.85 (d, J=8.9 Hz, 1H), 5.79
(td, J=56.0, 5.8 Hz, 1H), 5.99 (d, J=7.3 Hz, 1H), 6.23
(s, 1H), 6.31 (d, J=6.1 Hz, 1H), 7.40 (t, J=7.4 Hz, 2H),
7.52 (t, J=7.3 Hz, 1H), 7.94 (d, J=7.5 Hz, 2H); 13C
NMR (CDCl3) d 9.5, 14.3, 22.0, 22.6, 25.5, 27.8, 29.3,
30.8, 35.5, 41.5, 45.5, 47.6, 54.3 (m), 58.3, 68.5, 69.2,
70.9, 74.2, 75.8, 79.6, 80.4, 80.6, 84.3, 88.1, 110.4, 114.3,
118.2, 127.6, 128.8, 129.7, 133.7, 134.0, 138.6, 151.9,
Data analysis. Data were ®t with the Sigmoid-Emax
concentration±e€ect model44 with nonlinear regression,
weighted by the reciprocal of the square of the predicted
response. The ®tting software was developed at RPCI
with MicroSoft FORTRAN, and uses Marquardt45
algorithm as adapted by Nash46 for the nonlinear
regression. The concentration of drug which resulted in
50% growth inhibition (IC50) was calculated.

I. Ojima et al. / Bioorg. Med. Chem. 8 (2000) 1619±1628
1627
Apoptosis assay
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Stony Brook that is supported by a grant from National
Science Foundation (CHE 9413510).

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