Synthesis and biological evaluation of novel 3′-difluorovinyl taxoids

Article abstract of DOI:10.1016/j.jfluchem.2012.07.007

A series of 3′-difluorovinyl taxoids with C10 modifications, as well as those with C2 and C10 modifications, were strategically designed to block the metabolism by cytochrome P-450 3A4 enzyme and synthesized. These novel difluorovinyl taxoids were evaluated for their cytotoxicity against drug-sensitive human breast (MCF7), multidrug-resistant (MDR) human ovarian (NCI/ADR), human colon (HT-29) and human pancreatic (PANC-1) cancer cell lines. 3′-Difluorovinyl taxoids exhibit several to 16 times better activity against MCF7, HT-29 and PANC-1 cell lines and up to three orders of magnitude higher potency against NCI/ADR cell line as compared to paclitaxel. Structure-activity relationship study shows the critical importance of the C2 modifications on the activity against MDR cancer cell line, while the C10 modifications have a rather minor effect on the potency with some exceptions. The effect of the C2 modifications on potency against MCF7 cell line increases in the following order: H < F < Cl < N₃. Among the twenty five 3′-difluorovinyl taxoids evaluated, eight taxoids exhibited less than 100 pM IC₅₀ values against MCF7 cell line. Difluorovinyl taxoids induced GTP-independent tubulin polymerization much faster than paclitaxel. Then, the resulting microtubules were stable to Ca²⁺-induced depolymerization, indicating strong stabilization of microtubules. Molecular modeling study indicated that a difluorovinyl taxoid binds to β-tubulin in a manner that is consistent with the REDOR-Taxol structure. The difluorovinyl group appears to mimic the isobutenyl group to some extent, but with very different electronic property, which may account for the unique activities of difluorovinyl taxoids.

Full text of DOI:10.1016/j.jfluchem.2012.07.007

Journal of Fluorine Chemistry 143 (2012) 177–188
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis and biological evaluation of novel 30-difluorovinyl taxoids
Larissa Kuznetsova a, Liang Sun a, Jin Chen a, Xianrui Zhao a, Joshua Seitz a, Manisha Das a, Yuan Li a,
Jean M. Veith c, Paula Pera c, Ralph J. Bernacki c, Shujun Xia d, Susan B. Horwitz d, Iwao Ojima a,b,
*
a Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, United States
b Institute of Chemical Biology & Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, United States
c Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, NY 14263, United States
d Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, United States
A R T I C L E I N F O
A B S T R A C T
A series of 30-difluorovinyl taxoids with C10 modifications, as well as those with C2 and C10
modifications, were strategically designed to block the metabolism by cytochrome P-450 3A4 enzyme
and synthesized. These novel difluorovinyl taxoids were evaluated for their cytotoxicity against drug-
sensitive human breast (MCF7), multidrug-resistant (MDR) human ovarian (NCI/ADR), human colon (HT-
29) and human pancreatic (PANC-1) cancer cell lines. 30-Difluorovinyl taxoids exhibit several to 16 times
better activity against MCF7, HT-29 and PANC-1 cell lines and up to three orders of magnitude higher
potency against NCI/ADR cell line as compared to paclitaxel. Structure–activity relationship study shows
the critical importance of the C2 modifications on the activity against MDR cancer cell line, while the C10
modifications have a rather minor effect on the potency with some exceptions. The effect of the C2
modifications on potency against MCF7 cell line increases in the following order: H < F < Cl < N3. Among
the twenty five 30-difluorovinyl taxoids evaluated, eight taxoids exhibited less than 100 pM IC50 values
against MCF7 cell line. Difluorovinyl taxoids induced GTP-independent tubulin polymerization much
faster than paclitaxel. Then, the resulting microtubules were stable to Ca2+-induced depolymerization,
indicating strong stabilization of microtubules. Molecular modeling study indicated that a difluorovinyl
Article history:
Received 15 April 2012
Received in revised form 14 July 2012
Accepted 16 July 2012
Available online 28 July 2012
Keywords:
Anticancer agent
Taxoid
Fluoro-taxoid
b-Lactam Synthon Method
Difluorovinyl
Baccatin
Structure–activity relationship
Microtubules
taxoid binds to
b-tubulin in a manner that is consistent with the REDOR-Taxol structure. The
difluorovinyl group appears to mimic the isobutenyl group to some extent, but with very different
electronic property, which may account for the unique activities of difluorovinyl taxoids.
ß 2012 Elsevier B.V. All rights reserved.
1. Introduction
lung cancer and Kaposi’s sarcoma [8,9]. These drugs have also been
approved for the treatment of prostate, neck and cervical cancer
The fact that a large number of fluorinated compounds have
been approved by the FDA for medical use clearly demonstrates the
importance of fluorine in medicinal chemistry [1–4]. In drug
design, fluorine is now recognized as the second ‘‘favorite
heteroatom’’, after nitrogen [5]. The replacement of a C–H or C–
O bond with a C–F bond in biologically active compounds often
introduces beneficial properties such as higher metabolic stability,
increased binding to target molecules, increased lipophilicity and
membrane permeability [6,7].
[8,9]. Paclitaxel and docetaxel bind to
b-tubulin, one half of the
a,b
-heterodimer subunit that comprises microtubules, stabilizing
the structure and preventing depolymerization. These drugs exert
their cytotoxic effects through the disruption of microtubule
dynamics, which causes the cell cycle arrest at the G2/M stage,
leading to apoptosis through signaling cascade [10,11].
Paclitaxel and docetaxel possess potent antitumor activity, but
chemotherapy with these drugs involves numerous undesirable
side effects as well as drug resistance. Resistance to paclitaxel is
mainly attributed to multi-drug resistance (MDR), which is caused
by the over-expression of ABC transporters, such as P-glycoprotein
(Pgp), an efflux pump responsible for the removal of cytotoxic
agents [12,13]. In addition to MDR, point mutations in the drug
Paclitaxel (Taxol1) and its semi-synthetic analog docetaxel
(Taxotere1) have been approved for the treatment of advanced
ovarian cancer, metastatic breast cancer, melanoma, non-small cell
binding pocket of b-tubulin as well as the altered expression of b-
tubulin isoforms contribute to drug resistance [14,15]. Therefore,
there is an obvious need to develop new taxane anticancer agents
with fewer side effects, superior pharmacological properties, and
improved efficacy against various classes of tumors, especially
* Corresponding author at: Department of Chemistry, State University of New
York at Stony Brook, Stony Brook, NY 11794-3400, United States.
Tel.: +1 631 632 1339; fax: +1 631 632 7942.
E-mail address: iojima@notes.cc.sunysb.edu (I. Ojima).
0022-1139/$ – see front matter ß 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2012.07.007

178
L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
against drug-resistant human cancer. Our extensive structure–
activity relationship (SAR) studies of taxoid anticancer agents have
led to the discovery and development of new generation taxoids
bearing non-aromatic substituents at the C30 position (isobutenyl
and isobutyl group in particular) and various acyl groups at the C10
position [16,17] as well as meta-substituted benzoyl groups at the
C2 position [17,18]. These new generation taxoids bearing an
isobutenyl or isobutyl group at C30 and modifications at C10 and/or
C2-benzoate exhibit 2–3 orders of magnitude higher potency than
paclitaxel and docetaxel against drug-resistant cancer cell lines
expressing MDR phenotype [16–18]. We have also developed 30-
trifluoromethyl and 30-difluoromethyl taxoids with C10 as well as
C10/C2 modifications in a similar manner [19–21]. This study has
shown that trifluoromethyl and difluoromethyl groups are viable
modifiers of the C30 position and a number of highly potent fluoro-
taxoids have been identified. Nevertheless, the isobutenyl group
appears to be the best substituent at C30 as far as the cytotoxicity is
concerned. However, our recent study on the metabolic stability of
30-isobutyl- and 30-isobutenyl-taxoids revealed that there was a
marked difference in metabolism between the new generation
taxoids (e.g. SB-T-1214, SB-T-1216 and SB-T-1103) and that of
docetaxel and paclitaxel [22]. The CYP 3A4 enzyme in the
cytochrome P450 family in humans was found to metabolize
these taxoids through hydroxylation primarily at the two allylic
methyl groups of the 30-isobutenyl group and the methyne moiety
of the 30-isobutyl group (Fig. 1).
2. Results and discussion
2.1. Synthesis of 30-difluorovinyl taxoids
A series of novel 30-difluorovinyl taxoids 6 were synthesized
from (3R,4S)-TIPSO-4-difluorovinyl-
b-lactam 4 and modified
baccatins 5 using the highly efficient Ojima–Holton coupling
protocol based on the ‘‘
[24–27].
b-Lactam Synthon Method’’ (Schemes 1–4)
Enantiopure (3R,4S)-4-difluorovinyl-
sized through a Wittig-type reaction of (3R,4R)-4-formyl-
b
-lactam 2 was synthe-
-lactam
b
1 (>99% ee) with phosphorus ylide, generated in situ by the
reaction of HMPT with CF2Br2, in the presence of zinc (Scheme 2).
Difluoromethylenation reaction of ketones and aldehydes using
HMPT/CF2Br2 combination has been studied with and without Zn
[28–30]. In the reaction of 4-formyl-
was found to be critical. Under optimized conditions (1/HMPT/
CF2Br2/Zn = 1/10/4/5) (see Section 3), difluorovinyl- -lactam 2
was obtained in 80–84% yield (Scheme 2). (3R,4R)-4-Formyl-
lactam 1 (>99% ee) was prepared from (3R,4R)-1-PMP-4-acetoxy-
4-(2-methylprop-2-enyl)azetidin-2-one obtained via enzymatic
optical resolution of the racemic
protocol (Scheme 1) [19]. Removal of the PMP group in the
presence of ceric ammonium nitrate (CAN), followed by the
introduction of t-Boc moiety afforded enantiopure (3R,4S)-3-
b-lactam 1, the presence of Zn
b
b
-
b-lactam, using our published
TIPSO-4-difluorovinyl-b-lactam 4 in high overall yield (Scheme 2).
This result makes a sharp contrast to the fact that the tert-butyl
group of the C30N-Boc moiety is the single predominant metabolic
site for docetaxel and the C30-phenyl and C6-methylene moieties
are the sites for paclitaxel [23]. Accordingly, we have addressed the
unique metabolic profiles of the new generation taxoids by
designing a series of 30-difluorovinyl taxoids in order to block the
oxidation by CYP 3A4, which should enhance the metabolic
stability and activity in vivo. The chemical synthesis, full
characterization, biological evaluations, SAR and molecular
modeling study of novel 30-difluorovinyl taxoids are described
herein.
Modifications of 10-deacetylbaccatin III (10-DAB) at C2 and C10
were performed, using our published protocol [17,21], with four
substituents (i.e., MeO, F, N3 and Cl) at the C2 benzoate and five acyl
groups at C10 to produce 20 different baccatins 5a-1 ꢀ 5e-4
(Scheme 3). In addition, five baccatins 5a–d (X = H) with only C10
modifications were prepared, using our published protocol [17].
The standard Ojima–Holton coupling conditions were used for
the ring-opening coupling of 4-difluorovinyl-b-lactam 4 with 25
baccatins 5 in the presence of LiHMDS in THF at ꢁ40 8C. Removal of
the silyl protecting groups afforded 30-difluorovinyl taxoids 6 in
moderate to excellent overall yield (Scheme 4).
O
O
O
O
O
OH
N
O
O
OH
O
O
OH
O
O
O
O
O
NH
O
NH
O
O
NH
O
O
O
O
O
O
O
OAc
O
OAc
O
OAc
O
O
HO
O
O
HO
HO
OH
OH
OH
SB-T-1214
SB-T-1216
SB-T-1103
CYP 3A4
CYP 3A4
CYP 3A4
O
O
O
O
HO
NH
N
O
OH
O
O
O
O
OH
OH
O
O
O
O
NH
O
O
O
O
NH
O
O
O
O
O
O
O
OAc
O
OAc
O
OAc
O
O
O
O
HO
HO
HO
HO
OH
OH
HO
OH
OH
OH
OH
Fig. 1. Primary sites of hydroxylation on the second-generation taxoids by the P450 family of enzymes.

L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
179
AcO
O
TIPSO
TIPSO
O
CHO
PMP
i, ii
85-90%
iii
N
N
N
72-78%
PMP
PMP
O
1
i)-KOH, THF, 0 °C; ii) TIPSCl, Et3N, DMAP, CH2Cl2; iii) O3, MeOH/CH2Cl2, -78 °C, Me2S
Scheme 1. Synthesis of (3R,4R)-1-PMP-3-TIPSO-4-formyl-b-lactam (1).
SB-T-121303 (30-isobutenyl in all cases), are also listed for
comparison [16,17]. Marked effect of C2-benzoate modification
at the meta position is clearly observed on the potency against
drug-sensitive and drug-resistant cell lines. The potency of these
difluorovinyl taxoids against NCI/ADR depends on the nature of
meta substituents of the C2-benzoate moiety, and the potency
increases in the following order: F < Cl ꢂ MeO < N3. Several
difluorovinlyl taxoids with 2,10-modifications exhibit impressive
potency, in that their IC50 values are in <100 pM range (78–92 pM)
against MCF7, and in sub-nanomolar range (0.34–0.50 nM) against
NCI/ADR, which is three orders of magnitude more potent than
paclitaxel (entries 12, 15, 21, 22 and 25). The resistance factor for
these difluorovinyl taxoids is 3.7–6.4, while that for paclitaxel is
250. Difluorovinyl taxoid 6b-2 (entry 17) exhibits extremely high
potency (IC50 71 pM) against MCF7 cell line, but the resistance
factor is 24. Difluorovinyl taxoids (6a–d) with unmodified C2-
benzoate moiety (entries 6–10) also exhibit substantially en-
hanced potency against MCF7 and NCI/ADR cell lines as compared
to paclitaxel. These difluorovinyl taxoids possess excellent potency
against HT-29 and PANC-1 cell lines as well. The potencies of
difluorovinyl taxoids with 2,10-modifications have not been
evaluated against HT-29 and PANC-1 cell lines yet, but it is
reasonable to assume that these novel taxoids would exhibit
exceptional potency against these cancer cell lines. It should also
be noted that difluorovinyl taxoid 6a (SB-T-12851) showed
exceptional potency (IC50 = 5.6 pM) against CFPAC-1 human
pancreatic cancer cell line, wherein paclitaxel (IC50 = 15.8 nM)
and difluorovinyl taxoid 6d (IC50 = 2.2 nM) indicated the existence
of a certain level drug resistance.
F
F
TIPSO
O
TIPSO
O
CHO
PMP
HMPT, CF2Br2
N
N
Zn, THF
80-84%
PMP
1
2
F
F
F
Boc2O
Et3N, DMAP
TIPSO
TIPSO
O
CAN
F
N
N
MeCN/H2O
92%
CH2Cl2
O
H
Boc
96%
4
3
Scheme 2. Synthesis of (3R,4S)-1-Boc-3-TIPSO-4-difluorovinyl-b-lactam (4).
2.2. Biological evaluation of 30-difluorovinyl taxoids
2.2.1. Cytotoxicity of 30-difluorovinyl taxoids
Cytotoxicity of the 30-difluorovinyl taxoids was evaluated in
vitro against drug-sensitive MCF7 human breast cancer cell line
and drug-resistant NCI/ADR human ovarian cancer cell line
expressing MDR phenotype. Selected difluorovinyl taxoids were
also assayed against HT-29 human colon cancer cell line and PANC-
1 human pancreatic cancer cell line. As Table 1 shows, all
difluorovinyl-taxoids are substantially more potent than paclitaxel
against all cancer cell lines examined. The potencies of three
representative new generation taxoids, SB-T-1213, SB-T-1214 and
TESO
O
OTES
X
CO2H
TESO
HO
O
OTES
i,ii
O
10-DAB
TESO
93%
iii
OAc
O
O
HO
X
O
TESO
85-90%
OAc
OH
X = MeO, F, N3, Cl
HO
O
RCO-O
O
OTES
OTES
iv, v
RCOCl
O
O
HO
HO
vi
71-95%
OAc
O
OAc
O
HO
O
HO
O
for 2 steps
67-98%
X
X
5
i)-TESCl, imidazole, DMF, r.t.; ii) sodium bis(2-methoxyethoxy)aluminumhydride,
THF, -10 ºC; iii) DIC, DMAP, CH2Cl2; iv) HF/pyridine, pyridine/MeCN; v) TESCl,
imidazole, DMF, RT, 2 h; vi) LiHMDS, THF.
Scheme 3. Synthesis of modified baccatin III (5).

180
L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
F
TIPSO
R1O
O
OTES
F
N
O
Boc
4
O
HO
i. LiHMDS, THF, -40o C
OAc
O
HO O
ii. HF/Py, MeCN/Py
0o C - r.t.
X
5
49-96%
RO
O
OH
O
Fig. 2. Tubulin polymerization with SB-T-12851 (6a), SB-T-12852 (6b), SB-T-12854
(6d), paclitaxel and GTP: microtubule protein 1 mg/mL, 37 8C, GTP 1 mM, drug
10
mM.
O
F
NH
O
O
O
2.2.2. Tubulin polymerization and microtubule stabilization
by 30-difluorovinyl taxoids
OAc
O
HO O
OH
F
The activities of three difluorovinyl taxoids, SB-T-12851 (6a),
SB-T-12853 (6c) and SB-T-12854 (6d), for tubulin polymerization
and microtubule stabilization were examined in comparison to
paclitaxel. In this spectrophotometric assay, changes in absorbance
values provide a direct measure of turbidity, indicating the degree
of tubulin polymerization. As Fig. 3 shows, these three difluor-
ovinyl taxoids induced GTP-independent tubulin polymerization
much faster than paclitaxel (Fig. 2). Thus, the turbidity of the
tubulin solution treated by difluorovinyl taxoids reaches a plateau
6
X
R = Ac, c-PrCO, EtCO, Me2NCO, MeOCO
X = H, MeO, F, N3, Cl
Scheme 4. Synthesis of 30-difluorovinyl-taxoids (6).
Table 1
In vitro cytotoxicity (IC50 nM)a of 30-difluorovinyl-taxoids.
Entry
Taxoid
X
R
MCF7b (breast)
NCI/ADRc (ovarian)
R/Sd
HT-29e (colon)
PANC-1f (pancreatic)
1
2
Paclitaxel
Docetaxel
SB-T-1213
SB-T-121303
SB-T-1214
6a
H
Ac
1.2
300
235
4.0
250
283
27
3.6
–
25.7
H
H
0.83
0.15
0.30
0.17
0.099
0.12
0.12
0.13
0.14
0.25
0.092
0.34
0.11
0.078
0.13
0.071
0.23
0.17
0.12
0.092
0.092
0.13
0.13
0.078
0.14
0.12
0.13
0.94
0.099
–
–
3
H
EtCO
0.31
–
4
MeO
H
EtCO
0.33
2.1
1.1
12
9.6
22.6
3.68
1.19
5.85
0.65
1.58
–
5
c-PrCO
Ac
0.36
0.41
0.85
0.34
0.46
–
6
H
0.95
6.03
1.2
7
6b
H
c-PrCO
EtCO
50
10
33
8
6c
H
9
6d
H
Me2NCO
MeOCO
Ac
4.27
1.29
1.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
6e
H
9.2
6a-1
MeO
MeO
MeO
MeO
MeO
F
6.0
5.2
1.7
8.7
6.4
–
–
6b-1
c-PrCO
Et-CO
Me2NCO
MeOCO
Ac
0.48
0.57
0.96
0.50
1.53
1.72
2.54
2.25
1.85
0.34
0.45
0.38
0.46
0.40
0.70
0.50
0.45
2.6
–
–
6c-1
–
–
6d-1
–
–
6e-1
–
–
6a-2
12
–
–
6b-2
F
c-PrCO
EtCO
24
11
13
15
–
–
6c-2
F
–
–
6d-2
F
Me2NCO
MeOCO
Ac
–
–
6e-2
F
–
–
6a-3
N3
N3
N3
N3
N3
Cl
3.7
–
–
6b-3
c-PrCO
EtCO
4.9
2.9
3.5
5.3
5.0
4.2
3.5
2.8
–
–
6c-3
–
–
6d-3
Me2NCO
MeOCO
Ac
–
–
6e-3
–
–
6a-4
–
–
6b-4
Cl
c-PrCO
EtCO
–
–
6c-4
Cl
–
–
6d-4
Cl
Me2NCO
MeOCO
–
–
6e-4
Cl
1.16
12
–
–
a
The concentration of compound that inhibits 50% of the growth of a cancer cell line after 72 h drug exposure.
Human breast carcinona.
b
c
d
e
f
Multidrug-resistant human ovarian carcinoma.
R/S =drug-resistance factor = IC50 (drug-resistant cell line)/IC50 (drug-sensitive cell line).
Human caucasian colon adenocarcinoma.
Human pancreatic carcinoma.

L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
181
quickly and does not change with time. The resulting microtubules
were stable to Ca2+-induced depolymerization, indicating strong
stabilization of microtubules, which is well known for paclitaxel.
This observation may suggest that the microtubules formed by
these novel difluorovinyl taxoids are different from those formed
by paclitaxel.
and shorter microtubules is related to the rapid polymerization of
tubulin observed with these difluorovinyl taxoids (see Fig. 2).
There is some morphological similarity between those micro-
tubules generated by the action of difluorovinyl taxoids and those
by second generation taxoids such as SB-T-1213 and SB-T-1214,
but the formation of thinner, shorter and straight microtubules
appear to be unique to difluorovinyl taxoids.
2.2.3. Electron microscopy analysis of microtubules treated with
selected difluorovinyl taxoids
2.3. Molecular modeling study on the bioactive structure
The microtubules formed by treatment of tubulin with the
three difluorovinyl taxoids were further analyzed by electron
microscopy to study their morphology and structure in compari-
son to those formed in the presence of GTP or paclitaxel. The
electron micrographs of microtubules formed by treatment with
SB-T-12851 (6a), SB-T-12852 (6b), SB-T-12854 (6d), paclitaxel and
GTP are shown in Fig. 3. Microtubules formed in the presence of
GTP and paclitaxel are long and thick (Fig. 3a and b), while those
formed by the difluorovinyl taxoids (Fig. 3c–e) appear to be much
thinner and shorter in length, which indicates substantial
difference in their properties as compared to those formed by
paclitaxel. It is strongly suggested that the formation of thinner
of 30-difluorovinyl taxoids in
b-tubulin
Recently, the ‘‘REDOR-Taxol’’ structure has been shown to be the
most plausible bioactive conformation of paclitaxel [31,32]. To
probe the binding conformations of 30-difluorovinyl taxoids, SB-T-
12853 (6c) was selected as representative and docked to the
paclitaxel binding pocket of the b-tubulin subunit using the REDOR-
Taxol coordinates. With the protein backbone fixed, the energy of
the system was minimized (InsightII 2000, CVFF), allowing the side
chains to find their lowest energy conformations. A structurally
closely related non-fluorinated second-generation taxoid, SB-T-
1213, was also docked into
b-tubulin following the same protocol
Fig. 3. Electromicrographs of microtubules: (a) GTP; (b) paclitaxel; (c) 6a; (d) 6b; (e) 6d.

182
L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
Fig. 4. (a) Proposed binding conformation of SB-T-12853 (6c) in tubulin; (b) overlay of SB-T-12853 (cyan) and SB-T-1213 (magenta) in tubulin.
for comparison. The resulting computer-generated binding struc-
tures of these taxoids and their overlay are shown in Fig. 4.
As Fig. 4 shows, both SB-T-12853 and SB-T-1213 form a very
stable H-bond with His229, consistent with the REDOR-Taxol
structure. Their overlay (Fig. 4b) shows almost complete overlap in
the baccatin moieties of SB-T-12853 and SB-T-1213, with small but
appreciable difference in the side chain positions. The result may
indicate that difluorovinyl group mimics isobutenyl group, but
there is a difference in size and electronic nature between two
groups. The difluorovinyl group is between vinyl and isobutenyl
groups in size and two fluorine atoms may mimic electronically
two hydroxyl groups rather than two methyl groups.
In our recent study on the metabolic stability of difluorovinyl
taxoids 6a–d against P-450 family enzymes, almost no appreciable
metabolites were detected, which suggests that not only the
metabolism at C30 is effectively blocked, but also oxidative
metabolism on other parts of the taxoid molecule, including the
C30N-t-Boc and C6 methylene moieties, is suppressed. The results
will be published elsewhere. Thus, our strategic incorporation of a
difluorovinyl group in place of an isobutenyl group at C30 has been
proven to be successful to block the observed metabolism of the
isobutenyl moiety in second-generation taxoids (see Fig. 1).
Accordingly, the difluorovinyl group is a very unique structural
component in medicinal chemistry and may serve as a versatile
modifier of pharmacological properties in a manner similar to the
trifluoromethyl group.
used as the internal standard for 1H and 13C NMR spectra, while CFCl3
as the standard for 19F NMR spectra. Melting points were measured
on a Thomas Hoover Capillary melting point apparatus. Optical
rotations were measured on a Perkin-Elmer Model 241 polarimeter.
TLC was performed on Merck DC-alufolien with Kieselgel 60F-254
and column chromatography was carried on silica gel 60 (Merck;
230–400 mesh ASTM). Chiral HPLC analysis for the determination of
enantiomeric excess was carried out with a Waters HPLC assembly.
HPLC assembly consisted of a Waters M45 solvent delivery system, A
Waters Model680 gradient controller, and a WaterM440 detector (at
254 nm), equipped with a Spectra Physic Model SP4270 integrator
and uses a DAICEL-CHIRACEL OD chiral column (25 ꢃ 0.46 cm i.d.),
employing hexane/2-propanol (99.5/0.5, v/v) as the solvent system
with a flow rate of 1.0 ml/min. HPLC analysis for determination of
isomeric ratio was carried out with the same Water HPLC assembly
using 5
m Spherical Silica column employing hexane/2-propanol/
dichloromethane (15/1/1, v/v/v) as the solvent system with a flow
rate 1.0 ml/min, or hexane/2-propanol/dichloromethane (10/1/1, v/
v/v) as the solvent system with a flow rate 1.4 ml/min. High
resolution mass spectra were obtained from the Mass Spectrometry
Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL.
3.2. Materials
The chemicals were purchased from Aldrich and Sigma and
purified before use by standard methods. Tetrahydrofuran
was freshly distilled under nitrogen from sodium metal
and benzophenone. Dichloromethane was also distilled immedi-
ately prior to use under nitrogen from calcium hydride. 7-
Triethylsilylbaccatin III (5a) was prepared by the literature
method [33]. 7-Triethylsilyl-10-deacetyl-10-acylbaccatins (5b–e)
were prepared by the previously reported method [16]. 2-
Debenzoyl-2-(3-methoxybenzoyl)-7-triethylsilylbaccatin III (5a-1),
3. Experimental
3.1. General method
1H, 13C and 19F NMR spectra were recorded on Varian 300, 400
or 500 MHz NMR spectrometer in CDCl3. Tetramethylsilane was

L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
183
2-debenzoyl-2-(3-fluorobenzoyl)-7-triethylsilylbaccatin III (5a-2),
2-debenzoyl-2-(3-azidobenzoyl)-7-triethylsilylbaccatin III (5a-3), 2-
debenzoyl-2-(3-chlorobenzoyl)-7-triethylsilylbaccatin III (5a-4),
2-debenzoyl-2-(3-methoxybenzoyl)-7-triethylsilyl-10-deacetyl-10-
acylbaccatins (5bꢀe-1), 2-debenzoyl-2-(3-fluorobenzoyl)-7-triethyl-
silyl-10-deacetyl-10-acylbaccatins (5bꢀe-2), 2-debenzoyl-2-(3-azi-
dobenzoyl)-7-triethylsilyl-10-deacetyl-10-acylbaccatins (5bꢀe-3),
and 2-debenzoyl-2-(3-chlorobenzoyl)-7-triethylsilyl-10-deacetyl-
10-acylbaccatins (5bꢀe-4) were prepared by the previously reported
methods [21].
brine, dried over anhydrous MgSO4, and concentrated under
reduced pressure. Crude material was purified by flash chroma-
tography on silica to give 1-t-Boc-4-difluorovinyl-
colorless oil (599 mg, 96% yield): [
20 +24.17 (c 14.4, CHCl3); 1H
NMR (CDCl3, 300 MHz): 1.04–1.17 (m, 21 H), 1.49 (s, 9 H), 4.49
b-lactam 4 as
a
]
D
d
(ddd, J = 1.6 Hz, 13.8 Hz, 23.7 Hz, 1 H), 4.75 (m, 1 H), 5.04 (d,
J = 5.7 Hz, 1 H), 6.59 (bs, 1 H); 13C NMR (CDCl3, 100 MHz): 12.0,
17.8, 28.2, 53.6, 74.5, 77.2, 83.9, 147.9, 158.5, 165.3; 19F NMR
(282 MHz, CDCl3):
d
ꢁ81.20 (d, J = 31.0 Hz, 1 F), ꢁ85.83 (dd,
J = 5.6 Hz, 29.3 Hz, 1F). HRMS (FAB+, m/z): Calcd. for C19H33F2NO4-
SiꢄNa+, 428.2039; Found, 428.2050.
3.3. Synthesis of (+)-(3R,4S)-1-tert-butoxycarbonyl-3-
triisopropylsiloxy-4-(2,2-difluoro-ethenyl)azetidin-2-one (4)
3.4. Synthesis of 30-difluorovinyl taxoids
3.3.1. (3R,4S)-1-(4-Methoxyphenyl)-3-triisopropylsiloxy-4-(2,2-
difluorovinyl)azetidin-2-one (2)
A typical procedure is described for the synthesis of 10-acetyl-
30-dephenyl-30-(2,2-difluoroethenyl)docetaxel (6a). All other 30-
difluorovinyl taxoids were synthesized in the same manner.
Hexamethylphosphorous
triamide
(HMPT)
(4.58 mL,
24.6 mmol) was added to a solution of dibromodifluoromethane
(1.51 mL, 9.84 mmol) in THF (75 mL) under nitrogen at 0 8C, which
immediately yielded white precipitate. To this mixture was added
Difluoromethyl-b-lactam 4 (190 mg, 0.468 mmol) and 5a
(234 mg, 0.335 mmol) were dissolved in dry THF (35 mL). The
mixture was cooled to ꢁ40 8C, and LiHMDS (1 M THF solution,
0.67 mL) was added. The reaction mixture was stirred for 2 h,
and quenched with aqueous saturated ammonium chloride. The
aqueous layer was extracted with ethyl acetate, and the
combined organic phases were washed with brine and dried
over anhydrous MgSO4. The crude product was purified on silica
gel column (hexanes/ethyl acetate = 6/1) to give 7-TES-10-Ac-30-
dephenyl-20-TIPSO-30-(2,2-difluoroethenyl)docetaxel (356 mg,
96%).
a suspension of 4-formyl-b-lactam 1 (927 mg, 2.46 mmol) and Zn
(802 mg, 12.3 mmol) in THF (100 mL) at 0 8C. The ice bath was
removed and the reaction mixture was refluxed for 40 min. Ether
was added to the reaction mixture. After cooling to room
temperature, the reaction mixture was filtered through Celite
and concentrated. The reddish residue was dissolved in ethyl
acetate (50 mL), washed with water (20 mL ꢃ 3), brine
(20 mL ꢃ 3), dried over anhydrous MgSO4, and concentrated in
vacuo to afford a crude product as an yellow-orange oil. The crude
product was purified by flash chromatography on silica gel
To
a
solution of the 7-TES-20-TIPS-taxoid (356 mg) thus
obtained in 14 mL of a 1:1 mixture of pyridine and acetonitrile
cooled to 0 8C was added 3.5 mL of HF/pyridine (7/3). The reaction
mixture was allowed to warm to room temperature and stirred for
24 h. The reaction was then quenched with 10 mL of aqueous
saturated sodium bicarbonate and extracted with ethyl acetate.
The combined organic layers were washed with copper sulfate and
brine, dried over anhydrous MgSO4 and concentrated. The residue
was purified by chromatography on silica gel using hexanes/ethyl
acetate (1/1) as eluent to afford 6a (SB-T-12851) (263 mg, 96%
yield) as a white solid.
(hexane/EtOAc = 3/1) to afford 1-PMP-4-difluorovinyl-
b-lactam
2 (851 mg, 84% yield) as a white solid: 1H NMR (CDCl3, 300 MHz)
d
1.08–1.15 (m, 21 H), 3.79 (s, 3 H), 4.54 (ddd, J = 1.5 Hz, 6.3 Hz,
16.5 Hz, 1 H), 4.83 (m, 1 H), 5.14 (d, J = 5.1 Hz, 1 H), 6.87 (d,
J = 9.0 Hz,2H), 7.32 (d, J = 9.0 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz)
12.1, 17.9, 54.1, 55.8, 75.8, 76.9, 77.4, 114.8, 118.6, 130.9, 156.7,
164.9; 19F NMR (282 MHz, CDCl3)
d
ꢁ80.80 (d, J = 32.7 Hz, 1F),
ꢁ86.34 (dd, J = 2.8 Hz, 28.2 Hz, 1F). HRMS (FAB+, m/z): Calcd. for
C
21H31F2NO3SiꢄH+, 412.2114; Found, 412.2127.
3.3.2. (3R,4S)-3-Triisopropylsilanyloxy-4-(2,2-
difluoroethenyl)azetidin-2-one (3)
3.4.1. 10-Acetyl-30-dephenyl-30-(2,2-difluoroethenyl)docetaxel (6a)
20
Yield 92% for 2 steps; mp 155–160 8C; [
a
]
D
ꢁ74.83 (c 2.86,
To a solution of 1-PMP-4-difluorovinyl-
b
-lactam 2 (688 mg,
CHCl3); 1H NMR (CDCl3, 400 MHz)
d 1.15 (s, 3H), 1.25 (s, 3 H), 1.30
1.67 mmol) in acetonitrile (50 mL) and H2O (10 mL) at ꢁ10 8C was
added dropwise a solution of ceric ammonium nitrate (3.74 g,
6.69 mmol) in water (40 mL) with stirring. The reaction mixture
was stirred for 2 h and diluted with water (60 mL) and aqueous
saturated Na2SO3 (60 mL). The aqueous layer was extracted with
ethyl acetate, and the combined organic layer was washed with
water, dried over MgSO4 and concentrated in vacuo. The crude
product was purified on a silica gel column (hexanes/EtOAc = 3/1)
(s, 9H), 1.68 (s, 3 H), 1.75 (bs, 1 H), 1.88 (m, 4 H), 2.24 (s, 3 H), 2.33
(m, 2 H), 2.39 (s, 3H), 2.49 (d, J = 3.6 Hz, 1H), 2.55 (m, 1 H), 3.52 (d,
J = 5.6 Hz, 1 H), 3.81 (d, J = 7.2 Hz, 1 H), 4.17 (d, J = 8.4 Hz, 1 H), 4.28
(d, J = 2.8 Hz, 1 H), 4.31 (d, J = 8.4 Hz, 1 H), 4.44 (m, 1 H), 4.58 (ddd,
J = 1.2 Hz, 9.6 Hz, 24.8 Hz, 1 H), 4.87 (t, J = 8.8 Hz, 1 H), 4.96 (m, 2
H), 5.66 (d, J = 7.2 Hz, 1 H), 6.24 (t, J = 8.8 Hz, 1 H), 6.30 (s, 1 H), 7.49
(t, J = 7.6 Hz, 2 H), 7.61 (t, J = 7.2 Hz, 1 H), 8.11 (d, J = 7.6 Hz, 2H); 13
NMR (CDCl3, 75.5 MHz) 9.8, 14.4, 15.1, 21.1, 22.1, 22.5, 26.9, 28.3,
C
d
to give NH free 4-difluorovinyl-
white solid: 1H NMR (CDCl3, 400 MHz)
4.54 (m, 2 H), 5.04 (dd, J = 1.6 Hz, 2.4 Hz, 1 H), 6.59 (bs, 1 H); 13C
NMR (CDCl3, 100 MHz) 12.1, 17.8, 50.4, 77.1, 79.3, 157.6, 169.4; 19
b
-lactam 3 (469 mg, 92% yield) as a
35.7, 43.5, 45.7, 48.2, 58.8, 72.4, 72.9, 75.8, 76.7, 79.3, 80.7, 81.3,
84.6, 128.9, 129.3, 130.4, 133.4, 133.9, 142.4, 155.1, 156.7, 158.0,
d
1.03–1.18 (m, 21 H), 4.44–
167.3, 170.5, 171.5, 172.7, 203.9; 19F NMR, (CDCl3, 282 MHz)
d
F
ꢁ84.29 (dd, J = 25.7 Hz, 36.4 Hz, 1 F), ꢁ86.22 (d, J = 34.7 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C41H51F2NO15ꢄH+, 836.3300; Found,
836.3278.
NMR (282 MHz, CDCl3)
d
ꢁ82.33 (d, J = 34.7 Hz, 1F), ꢁ87.50 (dd,
J = 9.3 Hz, 25.7 Hz, 1 F).
3.3.3. (+)-(3R,4S)-1-(tert-Butoxycarbonyl)-3-triisopropylsiloxy-4-
3.4.2. 30-Dephenyl-30-(2,2-difluoroethenyl)-10-
(2,2-difluoroethenyl)azetidin-2-one (4)
cyclopropanecarbonyldocetaxel (6b)
20
To
a
solution of 4-difluorovinyl-
b-lactam
3
(469 mg,
Yield 88% for 2 steps; white solid; mp 171–177 8C; [a]
D
1.54 mmol), triethylamine (0.75 mL, 5.38 mmol), and DMAP
(43 mg, 0.35 mmol) in CH2Cl2 (9 mL) was added Boc2O (398 mg,
1.77 mmol) at room temperature. The reaction mixture was stirred
for 18 h and quenched with water. The reaction mixture was
diluted with ethyl acetate and the organic layer was washed with
ꢁ73.71 (c 5.44, CHCl3); 1H NMR (CDCl3, 400 MHz)
d 0.98 (m, 2 H),
1.13 (m, 2 H), 1.15 (s, 3 H), 1.26 (s, 3 H), 1.30 (s, 9 H), 1.66 (s, 3 H),
1.78 (m, 2 H), 1.87 (m, 4 H), 2.31 (m, 2 H), 2.38 (s, 3 H), 2.53 (m, 1 H),
2.59 (d, J = 3.2 Hz, 1 H), 3.57 (bs, 1 H), 3.80 (d, J = 6.8 Hz, 1 H), 4.17
(d, J = 8.4 Hz, 1 H), 4.28 (m, 2 H), 4.40 (m, 1 H), 4.58 (ddd, J = 1.6 Hz,

184
L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
9.6 Hz, 24.8 Hz, 1 H), 4.87 (t, J = 8.8 Hz, 1 H), 4.97 (m, 2 H), 5.66 (d,
J = 7.2 Hz, 1 H), 6.24 (t, J = 8.0 Hz, 1 H), 6.29 (s, 1 H), 7.49 (t,
3.4.6. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
methoxybenzoyl)-10-acetyldocetaxel (6a-1)
J = 7.6 Hz, 2 H), 7.60 (t, J = 7.6 Hz, 1 H), 8.11 (d, J = 7.2 Hz, 2 H); 13
C
Yield 76% for 2 steps; white solid; mp 8C; [
a
]
D
ꢁ74.42 (c 2.15,
20
NMR (CDCl3, 75.5 MHz)
d
9.4, 9.6, 9.8, 13.2, 22.5, 27.0, 28.3, 35.7,
CHCl3); 1H NMR (CDCl3, 400 MHz)
d 1.15 (s, 3 H), 1.26 (m, 3 H), 1.30
43.5, 45.9, 48.2, 58.8, 72.4, 72.9, 73.3, 75.3, 75.6, 76.6, 79.3, 80.7,
81.3, 84.7, 128.9, 129.3, 130.4, 133.4, 133.9, 142.4, 155.1, 156.7,
(s, 9 H), 1.68 (s, 3 H), 1.76 (s, 1 H), 1.88 (m, 4 H), 2.24 (s, 3 H), 2.33
(m, 2 H), 2.38 (s, 3 H), 2.49 (d, J = 3.5 Hz, 1 H), 2.56 (m, 1 H), 3.47 (bs,
1 H), 3.81 (d, J = 7.5 Hz, 1 H), 3.90 (s, 3 H), 4.15 (d, J = 8.0 Hz, 1 H),
4.27 (m, 1 H), 4.36 (d, J = 8.0 Hz, 1 H), 4.41 (dd, J = 6.0 Hz, 10.5 Hz, 1
H), 4.58 (ddd, J = 1.0 Hz, 9.5 Hz, 24.5 Hz, 1 H), 4.87–4.93 (m, 2 H),
4.97 (dd, J = 2.0 Hz, 9.0 Hz, 1 H), 5.67 (d, J = 7.5 Hz, 1H), 6.24 (t,
J = 8.5 Hz, 1 H), 6.30 (s, 1 H), 7.15 (dd, J = 2.0 Hz, 8.0 Hz, 1 H), 7.40 (t,
J = 7.5 Hz, 1 H), 7.62 (s, 1 H), 7.72 (d, J = 8.0 Hz, 1 H); 13C NMR
167.3, 170.5, 172.6, 175.3, 204.0; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.32 (dd, J = 25.4 Hz, 36.4 Hz, 1 F), ꢁ86.30 (d, J = 36.7 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C43H53F2NO15ꢄH+, 862.3456; Found,
862.3445.
3.4.3. 30-Dephenyl-30-(2,2-difluoroethenyl)-10-propanoyldocetaxel
(6c)
(CDCl3, 100 MHz) d 9.5, 14.8, 20.8, 21.8, 22.3, 26.7, 28.1, 35.5, 43.2,
20
Yield 64% for 2 steps; white solid; mp 175–181 8C; [
a
]
D
45.6, 55.3, 58.5, 72.1, 72.7, 73.1, 75.1, 75.5, 76.4, 77.2, 79.0, 80.5,
81.2, 84.4, 114.0, 120.7, 122.7, 129.7, 130.3, 133.1, 142.1, 154.8,
159.7, 166.9, 170.2, 171.2, 203.5; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.55 (dd, J = 33.8 Hz, 36.4 Hz, 1 F), ꢁ86.24 (d, J = 36.4 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C42H53F2NO16ꢄH+, 866.3405; Found,
866.3439.
ꢁ82.83 (c 5.01, CHCl3); 1H NMR (CDCl3, 400 MHz)
d 1.14 (s, 3 H),
1.24 (m, 6 H), 1.30 (s, 9 H), 1.67 (s, 3 H), 1.78 (m, 1 H), 1.87 (m, 4 H),
2.31 (m, 2 H), 2.38 (s, 3H), 2.53 (m, 4 H), 3.55 (bs, 1 H), 3.81 (d,
J = 6.8 Hz, 1 H), 4.17 (d, J = 8.4 Hz, 1 H), 4.29 (m, 2 H), 4.39 (m, 1 H),
4.56 (ddd, J = 1.6 Hz, 9.6 Hz, 24.8 Hz, 1 H), 4.86 (t, J = 8.8 Hz, 1 H),
4.96 (m, 2 H), 5.66 (d, J = 7.2 Hz, 1 H), 6.25 (t, J = 8.4 Hz, 1 H), 6.30 (s,
1 H), 7.49 (t, J = 7.6 Hz, 2 H), 7.60 (t, J = 7.2 Hz, 1 H), 8.11 (d,
3.4.7. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
J = 7.2 Hz, 2 H); 13C NMR (CDCl3, 75.5 MHz)
d
9.2, 9.8, 15.1, 22.1,
methoxybenzoyl)-10-cyclopropanecarbonyldocetaxel (6b-1)
20
22.5, 26.9, 27.8, 28.4, 35.7, 43.5, 45.9, 48.2, 58.8, 72.2, 72.4, 72.9,
73.3, 75.3, 75.6, 76.6, 77.4, 79.3, 80.7, 81.3, 84.6, 128.9, 129.3,
130.4, 133.5, 133.9, 142.2, 155.1, 156.7, 167.3, 170.5, 172.6, 174.8,
Yield 96% for 2 steps; white solid; [
a
]
D
ꢁ77.03 (c 5.79, CHCl3);
1H NMR (CDCl3, 500 MHz)
d
0.99 (m, 2 H), 1.10 (m, 2 H), 1.15 (s, 3
H), 1.25 (s, 3 H), 1.28 (s, 9 H), 1.66 (s, 3 H), 1.74–1.81 (m, 2 H), 1.83–
1.89 (m, 1 H), 1.87 (s, 3 H), 2.31 (m, 2 H), 2.37 (s, 3 H), 2.53 (m, 1 H),
2.59 (d, J = 3.5 Hz, 1 H), 3.55 (bs, 1 H), 3.79 (d, J = 7.5 Hz, 1 H), 3.88
(s, 3 H), 4.16 (d, J = 8.5 Hz, 1 H), 4.26 (d, J = 2.5 Hz, 1 H), 4.34 (d,
J = 8.5 Hz, 1 H), 4.40 (m, 1 H), 4.57 (dd, J = 10.0 Hz, 24.5 Hz, 1 H),
4.86 (t, J = 8.5 Hz, 1 H), 4.96 (m, 2 H), 5.65 (d, J = 7.0 Hz, 1 H), 6.23 (t,
J = 9.0 Hz, 1 H), 6.28 (s, 1 H), 7.13 (dd, J = 2.0 Hz, 8.0 Hz, 1 H), 7.38 (t,
J = 7.5 Hz, 1 H), 7.63 (s, 1 H), 7.79 (d, J = 7.5 Hz, 1 H); 13C NMR
203.9; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.31 (dd, J = 23.7 Hz,
34.7 Hz, 1 F), ꢁ86.23 (d, J = 36.4 Hz, 1 F); HRMS (FAB+, m/z): Calcd.
for C42H53F2NO15ꢄH+, 850.3456; Found 850.3450.
3.4.4. 30-Dephenyl-30-(2,2-difluoroethenyl)-10-
dimethylcarbamoyldocetaxel (6d)
20
Yield 84% for 2 steps; white solid; mp 166–170 8C; [a]
D
ꢁ70.48 (c 6.3, CHCl3); 1H NMR (CDCl3, 400 MHz)
d
1.15 (s, 3 H),
(CDCl3, 75.5 MHz) d 9.2, 9.4, 9.5, 13.0, 14.8, 22.0, 22.3, 22.4 26.7,
1.25 (s, 3 H), 1.30 (s, 9 H), 1.67 (s, 3 H), 1.84 (m, 1 H), 1.89 (m, 4 H),
2.31 (m, 2 H), 2.38 (s, 3 H), 2.53 (m, 1 H), 2.96 (s, 3 H), 3.05 (s, 3 H),
3.24 (bs, 1 H), 3.64 (d, J = 5.6 Hz, 1 H), 3.80 (d, J = 6.8 Hz, 1 H), 4.17
(d, J = 8.4 Hz, 1 H), 4.29 (m, 2 H), 4.44 (m, 1 H), 4.57 (dd, J = 10.0 Hz,
25.2 Hz, 1 H), 4.86 (t, J = 8.8 Hz, 1 H), 4.97 (d, J = 9.2 Hz, 1 H), 5.02 (d,
J = 9.6 Hz, 1 H), 5.66 (d, J = 7.2 Hz, 1 H), 6.24 (m, 2 H), 7.49 (t,
28.1, 35.5, 43.2, 45.6, 55.3, 58.5, 72.1, 72.6, 73.1, 75.1, 75.3, 76.4,
77.2, 79.0, 80.4, 81.0, 84.4, 114.0, 120.6, 122.7, 129.7, 130.3, 133.2,
142.1, 154.8, 156.4, 159.7, 166.9, 170.2, 172.4, 175.1, 203.8; 19F
NMR, (CDCl3, 282 MHz)
d
1
ꢁ84.62 (dd, J = 25.7 Hz, 36.7 Hz, 1 F),
ꢁ86.29 (d, J = 36.4 Hz,
F); HRMS (FAB+, m/z): Calcd. for
C
44H55F2NO16ꢄH+, 892.3562; Found, 892.3599.
J = 7.6 Hz, 2H), 7.59 (t, J = 7.2 Hz, 1 H), 8.10 (d, J = 7.6 Hz, 2 H); 13
NMR (CDCl3, 75.5 MHz) 9.6, 15.1, 22.1, 22.5, 27.1, 28.3, 35.6, 36.3,
C
d
3.4.8. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
36.8, 43.5, 45.8, 48.2, 58.7, 72.6, 72.9, 73.3, 75.4, 76.3, 76.7, 77.4,
79.4, 80.6, 81.3, 84.6, 128.9, 129.4, 130.4, 133.7, 133.9, 142.7, 155.1,
156.3, 156.5, 167.3, 170.4, 171.3, 203.9; 19F NMR, (CDCl3, 282 MHz)
methoxybenzoyl)-10-propanoyldocetaxel (6c-1)
20
Yield 58% for 2 steps; white solid; [
a
]
D
ꢁ79.78 (c 3.66, CHCl3);
1H NMR (CDCl3, 500 MHz)
d
1.15 (s, 3 H), 1.22–1.25 (m, 6 H), 1.30
d
ꢁ84.31 (dd, J = 25.7 Hz, 37.2 Hz, 1 F), –86.23 (d, J = 36.4 Hz, 1 F);
(s, 9 H), 1.67 (s, 3 H), 1.71 (s, 1 H), 1.79 (s, 1 H), 1.86–1.91 (m, 4 H),
2.32 (m, 2 H), 2.38 (s, 3 H), 2.38–2.59 (m, 3 H), 3.52 (bs, 1 H), 3.82 (d,
J = 7.0 Hz, 1 H), 3.89 (s, 3 H), 4.18 (d, J = 8.5 Hz, 1 H), 4.27 (bs, 1 H),
4.35 (d, J = 8.5 Hz, 1 H), 4.40 (m, 2 H), 4.58 (ddd, J = 1.5 Hz, 10.0 Hz,
25.0 Hz, 1 H), 4.87 (t, J = 9.0 Hz, 1 H), 4.96 (m, 2 H), 5.66 (d,
J = 6.5 Hz, 1 H), 6.24 (t, J = 9.0 Hz, 1 H), 6.31 (s, 1 H), 7.15 (dd,
J = 2.5 Hz, 8.5 Hz, 1 H), 7.39 (t, J = 7.5 Hz, 1 H), 7.65 (s, 1 H), 7.71 (d,
HRMS (FAB+, m/z): Calcd. for C42H54F2N2O15ꢄH+, 865.3565; Found
865.3562.
3.4.5. 30-Dephenyl-30-(2,2-difluoroethenyl)-10-methoxycarbonyl-
docetaxel (6e)
20
Yield 90% for 2 steps; white solid; mp 144–148 8C; [a]
D
ꢁ77.06 (c 6.8, CHCl3); 1H NMR (CDCl3, 400 MHz)
d
1.15 (s, 3 H),
J = 8.0 Hz, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d 9.0, 9.5, 14.8, 21.9,
1.24 (s, 3 H), 1.29 (s, 9 H), 1.68 (s, 3 H), 1.78 (m, 1 H), 1.88 (m, 1 H),
1.91 (s, 3 H), 2.31 (m, 2 H), 2.39 (s, 3 H), 2.53 (m, 2 H), 3.55 (d,
J = 5.6 Hz, 1 H), 3.78 (d, J = 7.2 Hz, 1 H), 3.86 (s, 3 H), 4.17 (d,
J = 8.4 Hz, 1 H), 4.29 (m, 2 H), 4.38 (m, 1 H), 4.57 (ddd, J = 1.6 Hz,
9.6 Hz, 24.8 Hz, 1 H), 4.86 (t, J = 8.8 Hz, 1 H), 4.96 (m, 2 H), 5.66 (d,
J = 6.8 Hz, 1 H), 6.11 (s, 1 H), 6.23 (t, J = 8.0 Hz, 1 H), 7.49 (t,
22.3, 26.7, 27.5, 28.1, 35.4, 43.2, 45.6, 55.3, 58.3, 72.1, 72.6, 73.1,
75.1, 75.3, 76.4, 77.2, 79.0, 81.1, 84.4, 114.0, 122.7, 129.7, 130.3,
133.2, 141.9, 154.8, 159.7, 166.9, 170.2, 174.6, 203.7; 19F NMR,
(CDCl3, 282 MHz)
d
ꢁ84.58 (dd, J = 25.7 Hz, 36.7 Hz, 1 F), ꢁ86.26 (d,
J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C43H55F2NO16ꢄH+,
880.3562; Found 880.3578.
J = 7.6 Hz, 2 H), 7.60 (t, J = 7.2 Hz, 1 H), 8.10 (d, J = 7.2 Hz, 2 H); 13
NMR (CDCl3, 75.5 MHz) 9.7, 15.2, 22.1, 22.5, 26.8, 28.3, 35.8, 43.4,
C
d
3.4.9. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
45.9, 48.2, 55.8, 58.8, 72.1, 72.3, 72.8, 73.3, 75.2, 76.7, 77.4, 78.4,
79.2, 80.7, 81.2, 84.6, 128.9, 129.3, 130.4, 133.0, 133.9, 143.2, 155.1,
155.9, 156.5, 167.3, 170.6, 172.3, 204.1; 19F NMR, (CDCl3, 282 MHz)
methoxybenzoyl)-10-dimethylcarbamoyldocetaxel (6d-1)
Yield 74% for 2 steps; white solid; 1H NMR (CDCl3, 500 MHz)
d
1.15 (s, 3H), 1.25 (s, 3 H), 1.29 (s, 9 H), 1.67 (s, 3 H), 1.76 (bs, 1 H),
1.88 (m, 1 H), 1.89 (s, 3 H), 2.31 (m, 2 H), 2.38 (s, 3 H), 2.53 (m, 1 H),
2.96 (s, 3 H), 3.04 (s, 3H), 3.52 (bs, 1 H), 3.81 (d, J = 7.0 Hz, 1 H), 3.90
(s, 3 H), 4.17 (d, J = 8.0 Hz, 1 H), 4.27 (s, 1 H), 4.35 (d, J = 8.0 Hz, 1 H),
d
ꢁ84.30 (dd, J = 23.7 Hz, 34.7 Hz, 1 F), ꢁ86.22 (dd, J = 34.7 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C41H51F2NO16ꢄH+, 852.3249; Found
852.3227.

L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
185
4.45 (dd, J = 6.0 Hz, 10.5 Hz, 1 H), 4.58 (ddd, J = 1.5 Hz, 10.0 Hz,
25.0 Hz, 1 H), 4.87 (t, J = 8.5 Hz, 1 H), 4.97 (m, 2 H), 5.65 (d,
J = 7.0 Hz, 1 H), 6.25 (m, 2 H), 7.14 (dd, J = 1.5 Hz, 7.5 Hz, 1 H), 7.39
(t, J = 8.0 Hz, 1 H), 7.65 (s, 1 H), 7.71 (d, J = 7.5 Hz, 1 H); 13C NMR
131.3, 133.0, 142.2, 154.8, 160.9, 164.2, 165.8, 170.2, 175.1, 203.7;
19F NMR, (CDCl3, 282 MHz)
ꢁ86.20 (d, J = 34.7 Hz, 1 F), ꢁ111.72 (dd, J = 9.3 Hz, 14.6 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C43H52F3NO15ꢄH+, 880.3362; Found,
880.3346.
d
ꢁ84.22 (dd, J = 23.7 Hz, 36.7 Hz, 1 F),
(CDCl3, 75.5 MHz) d 9.3, 14.9, 22.3, 26.9, 28.1, 35.4, 36.0, 36.6, 43.2,
45.5, 55.3, 58.5, 72.4, 72.7, 73.1, 75.2, 76.1, 76.4, 79.2, 80.4, 81.2,
84.6, 114.0. 120.7, 122.7, 129.7, 130.3, 133.5, 133.7, 142.4, 154.8,
3.4.13. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
156.1, 159.7, 166.9, 170.1, 205.6; 19F NMR, (CDCl3, 282 MHz)
d
fluorobenzoyl)-10-propanoyldocetaxel (6c-2)
20
ꢁ84.60 (dd, J = 23.9 Hz, 34.9 Hz, 1 F), ꢁ86.30 (d, J = 36.4 Hz, 1 F);
HRMS (FAB+, m/z): Calcd. for C43H56F2N2O16ꢄH+, 895.3671; Found
895.3676.
Yield 71% for 2 steps; white solid; mp 161–165 8C; [a]
D
ꢁ71.57(c 8.3, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 1.14 (s, 3 H), 1.24
(m, 6 H,), 1.31 (s, 9 H), 1.67 (s, 3 H), 1.72 (bs, 1 H), 1.88 (m, 4 H), 2.32
(m, 2 H), 2.39 (s, 3 H), 2.53 (m, 3 H), 3.51 (bs, 1 H), 3.82 (d, J = 7.0 Hz,
1 H), 4.16 (d, J = 8.5 Hz, 1 H), 4.28 (d, J = 1.5 Hz, 1 H), 4.30 (d,
J = 8.5 Hz, 1 H), 4.42 (dd, J = 6.5 Hz, 10.5 Hz, 1 H), 4.58 (ddd,
J = 1.5 Hz, 9.0 Hz, 24.5 Hz, 1 H), 4.87 (m, 1 H), 4.96 (m, 2 H), 5.64 (d,
J = 6.5 Hz, 1 H), 6.23 (t, J = 8.0 Hz, 1 H), 6.31 (s, 1 H), 7.31 (dt,
J = 2.0 Hz, 7.0 Hz, 1 H), 7.48 (ddd, J = 5.5 Hz, 7.5 Hz, 13.0 Hz, 1 H),
7.80 (d, J = 8.5 Hz, 1 H), 7.91 (d, J = 7.5 Hz, 1 H); 13C NMR (CDCl3,
3.4.10. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
methoxybenzoyl)-10-methoxycarbonyldocetaxel (6e-1)
20
Yield 89% for 2 steps; white solid; [
a
]
D
ꢁ68.98 (c 4.61, CHCl3);
1H NMR (CDCl3, 500 MHz)
d
1.15 (s, 3 H), 1.24 (m, 3 H), 1.29 (s, 9 H),
1.69 (s, 3 H), 1.78 (bs, 1 H), 1.88 (m, 1 H), 1.91 (s, 3 H), 2.32 (m, 2 H),
2.38 (s, 3 H), 2.49 (d, J = 4.5 Hz, 1 H), 2.56 (m, 1 H), 3.53 (bs, 1 H),
3.78 (d, J = 6.5 Hz, 1 H), 3.87 (s, 3 H), 3.89 (s, 3 H), 4.18 (d, J = 8.5 Hz,
1 H), 4.27 (d, J = 3.0 Hz, 1 H), 4.36 (d, J = 8.5 Hz, 1 H), 4.41 (m, 1 H),
4.59 (dd, J = 9.5 Hz, 24.5 Hz, 1 H), 4.86 (t, J = 9.0 Hz, 1 H), 4.95 (m, 2
H), 5.66 (d, J = 7.5 Hz, 1 H), 6.12 (s, 1 H), 6.24 (t, J = 8.5 Hz, 1 H), 7.15
(d, J = 2.0 Hz, 7.5 Hz, 1 H), 7.39 (t, J = 8.0 Hz, 1 H), 7.64 (s, 1 H), 7.71
75.5 MHz)
d 8.9, 9.5, 14.8, 21.9, 22.2, 26.7, 27.5, 28.1, 35.5, 43.2,
45.6, 58.5, 72.1, 72.5, 73.1, 75.3, 75.4, 76.3, 79.1, 80.9, 84.4, 116.9,
120.8, 125.9, 130.4, 131.3, 133.0, 142.1, 154.6, 160.9, 164.2, 165.8,
170.2, 174.6, 203.6; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.24 (dd,
J = 23.9 Hz, 34.9 Hz, 1 F), ꢁ86.22 (d, J = 36.4 Hz, 1 F); ꢁ111.72 (dd,
(d, J = 7.0 Hz, 1 H); 13C NMR (CDCl3, 100 MHz)
d
9.5, 14.9, 21.8, 22.3,
J = 9.3 Hz, 14.6 Hz,
1
F); HRMS (FAB+, m/z): Calcd. for
26.6, 28.1, 35.3, 43.1, 45.6, 55.6, 55.6, 58.5, 72.0, 72.6, 73.1, 75.0,
77.2, 78.2, 79.0, 80.5, 81.1, 84.4, 114.0, 120.7, 129.7, 130.3, 132.8,
142.9, 154.8, 155.7, 159.7, 166.9, 170.3, 203.9; 19F NMR, (CDCl3,
C42H52F3NO15ꢄH+, 868.3362; Found 868.3352.
3.4.14. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
282 MHz)
d
ꢁ84.60 (dd, J = 25.7 Hz, 36.7 Hz, 1 F), ꢁ86.27 (d,
fluorobenzoyl)-10-dimethyl-carbamoyldocetaxel (6d-2)
J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C42H53F2NO17ꢄH+,
Yield 71% for 2 steps; white solid; [
a
]
D
ꢁ85.33 (c 1.5, CHCl3);
20
882.3354; Found 882.3353.
1H NMR (CDCl3, 500 MHz)
d 1.16 (s, 3 H), 1.26 (s, 3 H), 1.31 (s, 9 H),
1.60 (bs, 1 H), 1.67 (s, 3 H), 1.84 (m, 1 H), 1.89 (m, 1 H), 1.91 (s, 3 H),
2.33 (m, 2 H), 2.40 (s, 3 H), 2.55 (m, 1 H), 2.97 (s, 3 H), 3.05 (s, 3 H),
3.40 (bs, 1 H), 3.82 (d, J = 7.0 Hz, 1 H), 4.17 (d, J = 7.5 Hz, 1 H), 4.29
(s, 1 H), 4.31 (d, J = 7.5 Hz, 1 H), 4.45 (dd, J = 6.0 H, 10.5 Hz, 1 H),
4.58 (ddd, J = 1.5 Hz, 9.0 Hz, 24.5 Hz, 1 H), 4.87 (m, 2 H), 4.99 (d,
J = 8.0 Hz, 1 H), 5.64 (d, J = 7.0 Hz, 1 H), 6.26 (m, 2 H), 7.32 (dt,
J = 2.0 Hz, 8.0 Hz, 1 H), 7.49 (ddd, J = 6.0 Hz, 8.5 Hz, 13.5 Hz, 1 H),
7.81 (d, J = 9.5 Hz, 1 H), 7.92 (d, J = 8.0 Hz, 1 H); 13C NMR (CDCl3,
3.4.11. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
fluorobenzoyl)-10-acetyldocetaxel (6a-2)
20
Yield 72% for 2 steps; white solid; mp 165–168 8C; [a]
D
ꢁ73.29 (c 7.0, CHCl3); 1H NMR (CDCl3, 400 MHz)
d 1.15 (s, 3H), 1.26
(s, 3 H), 1.31 (s, 9 H), 1.68 (m, 4 H), 1.89 (m, 4 H), 2.24 (s, 3 H), 2.33
(m, 2 H), 2.39 (s, 3 H), 2.47 (bs, 1 H), 2.56 (m, 1 H), 3.46 (bs, 1 H),
3.82 (d, J = 7.0 Hz, 1 H), 4.16 (d, J = 8.5 Hz, 1 H), 4.28 (s, 1 H), 4.31 (d,
J = 8.5 Hz, 1 H), 4.42 (dd, J = 6.5 Hz, 11.0 Hz, 1 H), 4.58 (ddd,
J = 1.0 Hz, 9.0 Hz, 26.0 Hz, 1 H), 4.90 (m, 2H), 4.97 (dd, J = 2.0 Hz,
9.0 Hz, 1 H), 5.64 (d, J = 7.0 Hz, 1 H), 6.23 (t, J = 8.0 Hz, 1 H), 6.30 (s, 1
H), 7.31 (dt, J = 2.0 Hz, 8.0 Hz, 1 H), 7.49 (ddd, J = 6.0 Hz, 8.5 Hz,
13.5 Hz, 1 H), 7.80 (d, J = 9.0 Hz, 1 H), 7.91 (d, J = 7.5 Hz, 1 H); 13C
75.5 MHz)
d 9.3, 14.7, 22.3, 26.9, 28.1, 35.4, 36.0, 36.6, 43.2, 45.5,
58.5, 72.4, 72.7, 73.1, 75.6, 76.1, 76.4, 77.2, 79.3, 80.4, 81.1, 84.6,
110.7, 120.8, 125.9, 130.4, 131.3, 133.9, 142.7, 155.1, 157.4, 161.2,
164.1, 166.1, 170.2, 172.3, 205.5; 19F NMR, (CDCl3, 282 MHz)
d–
84.13 (dd, J = 25.4 Hz, 36.4 Hz, 1 F), ꢁ86.13 (d, J = 36.6 Hz, 1 F),
NMR (CDCl3, 75.5 MHz) d 9.5, 14.8, 20.8, 21.8, 22.2, 26.7, 28.1, 35.5,
ꢁ111.73 (dd, J = 9.3 Hz, 14.6 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for
43.2, 45.6, 58.5, 72.1, 72.5, 73.1, 75.5, 76.3, 79.1, 80.4, 80.9, 84.4,
117.0, 120.8, 125.9, 130.4, 131.2, 132.9, 142.2, 154.9, 156.7, 160.9,
164.2, 165.9, 170.2, 171.2, 203.5; 19F NMR, (CDCl3, 282 MHz)
C
42H53F3N2O15ꢄH+, 883.3471; Found 883.3433.
d
3.4.15. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
ꢁ84.23 (dd, J = 25.7 Hz, 34.7 Hz, 1 F), ꢁ86.21 (d, J = 36.7 Hz, 1 F),
fluorobenzoyl)-10-methoxycarbonyldocetaxel (6e-2)
ꢁ111.7 (dd, J = 9.3, 14.6 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for
Yield 72% for 2 steps; white solid; mp 157–160 8C; [a]
D
20
C
41H50F3NO15ꢄH+, 854.3205; Found, 854.3207.
ꢁ70.31 (c 6.5, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 1.15 (s, 3 H),
1.25 (s, 3 H), 1.29 (s, 9 H), 1.65 (bs, 1 H), 1.69 (s, 3 H), 1.88 (m, 1 H),
1.92 (s, 3 H), 2.31 (m, 2 H), 2.39 (s, 3 H), 2.46 (bs, 1 H), 2.57 (m, 1 H),
3.48 (bs, 1 H), 3.79 (d, J = 7.5 Hz, 1 H), 3.87 (s, 3 H), 4.16 (d,
J = 8.5 Hz, 1 H), 4.28 (s, 1 H), 4.31 (d, J = 8.5 Hz, 1 H), 4.39 (dd,
J = 7.0 Hz, 10.5 Hz, 1 H), 4.58 (dd, J = 9.0 Hz, 24.5 Hz, 1 H), 4.87–
4.94 (m, 2H), 4.97 (d, J = 8.0 Hz, 1 H), 5.65 (d, J = 7.0 Hz, 1 H), 6.12 (s,
1 H), 6.23 (t, J = 9.0 Hz, 1 H), 7.31 (dt, J = 2.5 Hz, 8.5 Hz, 1 H), 7.49
(ddd, J = 5.0 Hz, 7.5 Hz, 13.5 Hz, 1 H), 7.79 (d, J = 8.5 Hz, 1 H), 7.91
3.4.12. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
fluorobenzoyl)-10-cyclopropane-carbonyldocetaxel (6b-2)
20
Yield 78% for 2 steps; white solid; mp 157–161 8C; [a]
D
ꢁ77.04(c 7.1, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 0.99 (m, 2 H),
1.12 (m, 2 H), 1.15 (s, 3 H), 1.26 (s, 3 H), 1.30 (s, 9 H), 1.66 (s, 3 H),
1.77 (m, 2 H), 1.86 (m, 1 H), 1.87 (s, 3 H), 2.33 (m, 2 H), 2.39 (s, 3 H),
2.53 (m, 1 H), 2.61 (bs, 1 H), 3.55 (bs, 1 H), 3.80 (d, J = 7.0 Hz, 1 H),
4.17 (d, J = 8.0 Hz, 1 H), 4.28 (s, 1 H), 4.29 (d, J = 8.0 Hz, 1 H), 4.40
(dd, J = 7.0 Hz, 10.5 Hz, 1 H), 4.58 (dd, J = 8.5 Hz, 23.5 Hz, 1 H), 4.86
(m, 1 H), 4.97 (m, 2 H), 5.63 (d, J = 7.0 Hz, 1 H), 6.24 (t, J = 9.5 Hz, 1
H), 6.29 (s, 1 H), 7.49 (dt, J = 2.5 Hz, 8.5 Hz, 1 H), 7.60 (ddd,
J = 6.0 Hz, 8.0 Hz, 13.5 Hz, 1 H), 7.78 (d, J = 9.5 Hz, 1 H), 7.90 (d,
(d, J = 8.0 Hz, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d 9.4, 14.9, 21.8,
22.2, 26.6, 28.1, 35.3, 35.6, 43.1, 45.6, 47.9, 55.6, 58.5, 72.1, 72.5,
73.1, 75.4, 76.3, 78.2, 79.2, 80.5, 81.0, 84.6, 117.1, 120.8, 126.0,
130.4, 131.3, 132.7, 143.1, 154.9, 155.7, 160.9, 164.2, 165.9, 170.3,
203.8; 19F NMR, (CDCl3, 282 MHz)
36.4 Hz, 1 F), ꢁ86.17 (d, J = 36.7 Hz, 1 F), ꢁ111.71 (dd, J = 9.3 Hz,
14.6 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C41H50F3NO16ꢄH+,
870.3154; Found 870.3146.
d
ꢁ84.15 (dd, J = 25.4 Hz,
J = 7.5 Hz, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d
9.2, 9.5, 13.0, 14.7,
21.9, 22.2, 26.7, 28.1, 35.5, 43.2, 45.6, 48.0, 58.5, 72.1, 72.5, 73.1,
75.3, 75.5, 76.3, 79.1, 80.4, 80.9, 84.4, 117.0, 120.8, 125.9, 130.4,

186
L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
3.4.16. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
(dd, J = 6.5 Hz, 10.5 Hz, 1 H), 4.57 (ddd, J = 1.0 Hz, 9.0 Hz, 24.0 Hz,
1 H), 4.85 (t, J = 8.0 Hz, 1 H), 4.97 (m, 2 H), 5.65 (d, J = 7.0 Hz, 1 H),
6.23 (t, J = 9.5 Hz, 1 H), 6.25 (s, 1 H), 7.23 (dd, J = 1.5 Hz, 8.5 Hz, 1
H), 7.47 (t, J = 8.0 Hz, 1 H), 7.80 (s, 1 H), 7.89 (d, J = 8.0 Hz, 1 H); 13
C
azidobenzoyl)-10-acetyl-docetaxel (6a-3)
20
Yield 49% for 2 steps; white solid; [
a
]
D
ꢁ70.59 (c 3.23, CHCl3);
1H NMR (CDCl3, 400 MHz)
d
1.15 (s, 3 H), 1.26 (s, 3 H), 1.30 (s, 9 H),
1.67 (s, 3 H), 1.69 (bs, 1 H), 1.88 (m, 4 H), 2.24 (s, 3 H), 2.32 (m, 2 H),
2.39 (s, 3 H), 2.49 (bs, 1 H), 2.56 (m, 1 H), 3.48 (bs, 1 H), 3.82 (d,
J = 7.0 Hz, 1 H), 4.16 (d, J = 8.5 Hz, 1 H), 4.26 (s, 1 H), 4.33 (d,
J = 8.5 Hz, 1 H), 4.42 (dd, J = 6.5 Hz, 10.5 Hz, 1 H), 4.57 (ddd,
J = 1.5 Hz, 9.5 Hz, 25.0 Hz, 1 H), 4.85 (t, J = 8.5 Hz, 1 H), 4.93 (d,
J = 9.5 Hz, 1 H), 4.98 (d, J = 7.5, 1 H), 5.66 (d, J = 7.5 Hz, 1 H), 6.22 (t,
J = 9.0 Hz, 1 H), 6.29 (s, 1 H), 7.23 (dd, J = 1.5 Hz, 8.0 Hz, 1 H), 7.48 (t,
J = 8.0 Hz, 1 H), 7.81 (s, 1 H), 7.89 (d, J = 8.0 Hz, 1 H); 13C NMR
NMR (CDCl3, 75.5 MHz) d 9.3, 14.9, 22.3, 26.9, 28.1, 35.4, 36.0,
36.6, 43.2, 58.5, 72.4, 72.6, 73.1, 75.6, 76.1, 76.3, 79.2, 80.1, 84.7,
120.7, 120.2, 124.3, 126.7, 130.2, 130.9, 133.4, 140.9, 142.6, 154.9,
156.1, 166.1, 170.3, 205.5; 19F NMR, (CDCl3, 282 MHz)
(dd, J = 25.7 Hz, 34.7 Hz, 1 F), ꢁ86.17 (d, J = 36.7 Hz, 1 F); HRMS
(FAB+, m/z): Calcd. for C42H53F2N5O15ꢄH+, 906.3579; Found
906.3588.
d
ꢁ84.06
(CDCl3, 100 MHz)
d
9.5, 14.9, 20.8, 21.9, 22.4, 26.7, 28.1, 35.5, 43.2,
3.4.20. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
45.6, 58.5, 72.1, 72.6, 73.1, 75.4, 75.5, 76.4, 79.1, 80.4, 81.0, 84.4,
120.1, 124.4, 126.7, 130.2, 133.0, 140.9, 142.2, 154.8, 166.1, 170.3,
azidobenzoyl)-10-methoxycarbonyldocetaxel (6e-3)
20
Yield 77% for 2 steps; white solid; [
a
]
ꢁ66.67 (c 3.9, CHCl3);
D
171.2, 203.6; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ83.99 (dd, J = 25.7 Hz,
1H NMR (CDCl3, 500 MHz)
d 1.15 (s, 3 H), 1.24 (s, 3 H), 1.29 (s, 9 H),
36.7 Hz, 1 F), ꢁ86.12 (d, J = 35.0 Hz, 1 F); HRMS (FAB+, m/z): Calcd.
1.69 (s, 3 H), 1.71 (bs, 1 H), 1.89 (m, 1 H), 1.92 (s, 3 H), 2.32 (m, 2 H),
2.39 (s, 3 H), 2.49 (bs, 1 H), 2.56 (m, 1 H), 3.51 (bs, 1 H), 3.79 (d,
J = 7.0 Hz, 1 H), 3.87 (s, 3 H), 4.17 (d, J = 8.5 Hz, 1 H), 4.26 (s, 1 H),
4.33 (d, J = 8.5 Hz, 1 H), 4.40 (dd, J = 7.0 Hz, 11.0 Hz, 1 H), 4.57 (ddd,
J = 1.0 Hz, 9.0 Hz, 24.5 Hz, 1 H), 4.85 (t, J = 8.5 Hz, 1 H), 4.96 (m, 2
H), 5.66 (d, J = 7.5 Hz, 1 H), 6.12 (s, 1 H), 6.22 (t, J = 9.0 Hz, 1 H), 7.23
(dd, J = 1.5 Hz, 6.5 Hz, 1 H), 7.48 (t, J = 7.5 Hz, 1 H), 7.81 (s, 1 H), 7.89
(d, J = 8.0 Hz, 1 H); 13C NMR (CDCl3, 100 MHz)
d 9.5, 14.9, 21.8, 22.4,
26.6, 28.1, 35.4, 43.1, 45.6, 55.6, 55.6, 58.5, 72.0, 72.5, 73.1, 75.4,
76.3, 78.2, 79.1, 80.4, 81.0, 84.3, 120.1, 124.4, 126.7, 130.2, 130.8,
132.7, 140.9, 143.0, 154.7, 155.7, 166.1, 170.4, 203.8; 19F NMR,
for C41H50F2N4O15ꢄH+, 877.3314; Found, 877.3351.
3.4.17. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
azidobenzoyl)-10-cyclopropanecarbonyldocetaxel (6b-3)
20
Yield 78% for 2 steps; white solid; [
a
]
D
ꢁ67.39 (c 5.09, CHCl3);
1H NMR (CDCl3, 500 MHz)
d
1.02 (m, 2 H), 1.15 (m, 2 H), 1.17 (s, 3
H), 1.28 (m, 3 H), 1.32 (s, 9 H), 1.68 (s, 3 H), 1.76 (bs, 1 H), 1.79 (m, 1
H), 1.86–1.91 (m, 1 H), 1.90 (s, 3 H), 2.34 (m, 2 H), 2.41 (s, 3 H), 2.56
(m, 1 H), 2.62 (bs, 1H), 3.54 (d, J = 5.5 Hz, 1 H), 3.83 (d, J = 7.5 Hz, 1
H), 4.18 (d, J = 8.5 Hz, 1 H), 4.28 (d, J = 3.0 Hz, 1 H), 4.35 (d,
J = 8.5 Hz, 1 H), 4.43 (dd, J = 6.5 Hz, 10.0 Hz, 1 H), 4.58 (ddd,
J = 1.5 Hz, 9.0 Hz, 24.5 Hz, 1 H), 4.87 (t, J = 8.5 Hz, 1 H), 4.98 (m, 2
H), 5.67 (d, J = 7.0 Hz, 1 H), 6.24 (t, J = 8.0 Hz, 1 H), 6.31 (s, 1 H), 7.25
(dd, J = 1.5 Hz, 8.0 Hz, 1 H), 7.49 (t, J = 8.0 Hz, 1 H), 7.82 (s, 1 H), 7.90
(CDCl3, 282 MHz)
d
ꢁ84.01 (dd, J = 25.7 Hz, 36.7 Hz, 1 F,), ꢁ86.13
(d, J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C41H50F2N4O16ꢄH+,
893.3263; Found 893.3269.
(d, J = 8.0 Hz, 1 H); 13C NMR (CDCl3, 100 MHz)
d
9.2, 9.4, 9.5, 13.0,
3.4.21. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
14.9, 22.0, 22.3, 26.7, 28.1, 35.5, 43.2, 45.6, 58.5, 72.1, 72.6, 73.1,
75.3, 75.5, 76.4, 79.1, 80.4, 81.0, 84.5, 120.1, 124.4, 126.7, 130.2,
130.8, 133.1, 140.9, 142.3, 154.8, 166.1, 170.3, 175.1, 203.7; 19F
chlorobenzoyl)-10-acetyl-docetaxel (6a-4)
20
Yield 57% for 2 steps; white solid; mp 170–175 8C; [a]
D
ꢁ72.93 (c 4.1, CHCl3); 1H NMR (CDCl3, 400 MHz)
d 1.14 (s, 3 H),
NMR, (CDCl3, 282 MHz)
d
1
–84.02 (dd, J = 25.7 Hz, 34.9 Hz, 1 F),
1.25 (s, 3 H), 1.31 (s, 9 H), 1.67 (s, 3 H), 1.73 (s, 1 H), 1.89 (m, 4 H),
2.24 (s, 3 H), 2.33 (m, 2 H), 2.39 (s, 3 H), 2.51 (bs, 1 H), 2.55 (m, 1 H),
3.51 (bs, 1 H), 3.81 (d, J = 7.5 Hz, 1 H), 4.15 (d, J = 8.5 Hz, 1 H), 4.29
(m, 2 H), 4.41 (dd, J = 6.5 Hz, 11.0 Hz, 1 H), 4.58 (ddd, J = 1.0 Hz,
10.0 Hz, 24.5 Hz, 1 H), 4.86 (m, 1 H), 4.93 (d, J = 9.0 Hz, 1 H), 4.98 (d,
J = 7.5, 1 H), 5.62 (d, J = 7.5 Hz, 1 H), 6.21 (t, J = 9.5 Hz, 1 H), 6.30 (s, 1
H), 7.45 (t, J = 7.5 Hz, 1 H), 7.58 (dd, J = 1.5 Hz, 8.5 Hz, 1 H), 8.00 (d,
ꢁ86.14 (d, J = 34.9 Hz,
F); HRMS (FAB+, m/z): Calcd. for
C
43H52F2N4O15ꢄH+, 903.3470; Found, 903.3469.
3.4.18. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
azidobenzoyl)-10-propanoyldocetaxel (6c-3)
20
Yield 93%; white solid; [
(CDCl3, 500 MHz)
a
]
D
ꢁ67.77 (c 3.32, CHCl3); 1H NMR
d
1.14 (s, 3 H), 1.22–1.25 (m, 6 H), 1.30 (s, 9 H),
J = 7.5 Hz, 1 H), 8.12 (s, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d 9.5, 14.8,
1.67 (s, 3 H), 1.71 (s, 1 H), 1.85–1.91 (m, 4 H), 2.32 (m, 2 H), 2.39 (s,
3 H), 2.47–2.59 (m, 3 H), 3.48 (d, J = 4.5 Hz, 1 H), 3.82 (d, J = 7.5 Hz, 1
H), 4.16 (d, J = 8.5 Hz, 1 H), 4.26 (bs, 1H), 4.33 (d, J = 8.5 Hz, 1 H),
4.43 (dd, J = 7.0 Hz, 11.0 Hz, 1 H), 4.56 (ddd, J = 1.5 Hz, 9.0 Hz,
24.5 Hz, 1 H), 4.86 (t, J = 8.0 Hz, 1 H), 4.92 (d, J = 9.5 Hz, 1 H), 4.98 (d,
J = 8.0 Hz, 1 H), 5.66 (d, J = 7.5 Hz, 1 H), 6.22 (t, J = 8.0 Hz, 1 H), 6.31
(s, 1 H), 7.23 (dd, J = 1.5 Hz, 7.5 Hz, 1 H), 7.48 (t, J = 7.5 Hz, 1 H), 7.81
20.8, 21.8, 22.1, 26.7, 28.1, 35.4, 43.1, 45.6, 58.5, 72.1, 72.5, 73.1,
75.5, 76.3, 79.2, 80.4, 80.9, 84.3, 128.2, 130.0, 130.3, 130.9, 132.9,
134.8, 142.2, 154.8, 165.7, 170.2, 171.2, 203.5; 19F NMR, (CDCl3,
282 MHz)
d
ꢁ84.15 (dd, J = 25.7 Hz, 36.7 Hz, 1 F), ꢁ86.12 (dd,
J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C41H50ClF2NO15ꢄH+,
870.2910; Found, 870.2891.
(s, 1 H), 7.89 (d, J = 8.0 Hz, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d
9.0,
3.4.22. 30-Dephenyl-30-(2,2-difluorovinyl)-2-debenzoyl-2-(3-
9.5, 14.9, 21.7, 22.4, 26.7, 27.5, 28.1, 35.5, 43.2, 45.6, 58.5, 72.1,
72.6, 73.1, 75.1, 75.3, 75.4, 76.4, 79.1, 81.0, 84.5, 120.1, 124.4,
126.7, 130.2, 130.8, 133.2, 140.9, 154.8, 159.7, 166.1, 170.3, 174.6,
chlorobenzoyl)-10-cyclopropanecarbonyldocetaxel (6b-4)
20
Yield 73% for 2 steps; white solid; mp 161–166 8C; [a]
D
ꢁ78.97(c 5.8, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 1.02 (m, 2 H),
203.7; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ83.99 (dd, J = 25.7 Hz,
1.15 (m, 2 H), 1.16 (s 3 H), 1.28 (m, 3 H), 1.32 (s, 9 H), 1.68 (s, 3 H),
1.70 (bs, 1 H), 1.80 (m, 1 H,), 1.86–1.89 (m, 4 H), 2.33 (m, 2 H), 2.41
(s, 3 H), 2.56–2.59 (m, 2H), 3.51 (bs, 1 H), 3.82 (d, J = 7.0 Hz, 1 H),
4.16 (d, J = 8.5 Hz, 1 H), 4.29 (s, 1 H), 4.31 (d, J = 8.5 Hz, 1 H), 4.41
(dd, J = 7.0 Hz, 10.5 Hz, 1 H), 4.59 (dd, J = 9.0 Hz, 25.0 Hz, 1 H), 4.89
(m, 1 H), 4.98 (m, 2 H), 5.63 (d, J = 7.0 Hz, 1 H), 6.24 (t, J = 9.0 Hz, 1
H), 6.31 (s, 1 H), 7.46 (t, J = 8.5 Hz, 1 H), 7.60 (dd, J = 1.0 Hz, 7.0 Hz, 1
H), 8.01 (d, J = 7.5 Hz, 1 H), 8.13 (s, 1 H); 13C NMR (CDCl3, 100 MHz)
34.7 Hz, 1 F), ꢁ86.12 (d, J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd.
for C42H52F2N4O15ꢄH+, 891.3470; Found 891.3473.
3.4.19. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
azidobenzoyl)-10-dimethylcarbamoyldocetaxel (6d-3)
20
Yield 79% for 2 steps; white solid; [
a
]
D
ꢁ75.39 (c 4.51, CHCl3);
1H NMR (CDCl3, 500 MHz)
d
1.15 (s, 3 H), 1.25 (s, 3 H), 1.30 (s, 9 H),
1.66 (s, 3 H), 1.75 (bs, 1 H), 1.87 (m, 1 H), 1.90 (s, 3 H), 2.31 (m, 2 H),
2.39 (s, 3 H), 2.54 (ddd, J = 7.0 Hz, 10.0 Hz, 15.5 Hz, 1 H), 2.96 (s, 3
H), 3.04 (s, 3 H), 3.23 (bs, 1 H), 3.56 (bs, 1 H), 3.81 (d, J = 7.0 Hz, 1 H),
4.16 (d, J = 8.5 Hz, 1 H), 4.27 (s, 1 H), 4.32 (d, J = 8.5 Hz, 1 H), 4.45
d 9.2, 9.4, 13.0, 14.9, 21.9, 22.2, 26.7, 28.1, 35.4, 43.2, 45.6, 48.0,
58.5, 72.1, 72.6, 73.1, 75.3, 75.5, 76.3, 77.2, 79.2, 80.4, 81.0, 84.4,
128.3, 130.0, 130.3, 130.9, 133.0, 133.7, 134.8, 142.3, 154.8, 165.7,
170.2, 175.1, 203.7; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.15 (dd,

L. Kuznetsova et al. / Journal of Fluorine Chemistry 143 (2012) 177–188
187
J = 25.7 Hz, 34.7 Hz, 1 F), ꢁ86.16 (d, J = 36.7 Hz, 1 F); HRMS (FAB+,
m/z): Calcd. for C43H52ClF2NO15ꢄH+, 896.3066; Found, 896.3036.
containing 3 M glycerol. Tubulin stored in liquid nitrogen was
centrifuged just before use and protein concentration adjusted to
1 mg/mL. The concentration of tubulin in MTP is 85% and that is
taken into consideration when the ratios of tubulin to drug are
presented in Fig. 1. Microtubule assembly was carried out with
3.4.23. 30-Dephenyl-30-(2,2-difluorovinyl)-2-debenzoyl-2-(3-
chlorobenzoyl)-10-propanoyldocetaxel (6c-4)
20
Yield 72% for 2 steps; white solid; mp 154–158 8C; [
a
]
10
m
M
SB-T-12851, SB-T-12852 and SB-T-12854. Paclitaxel
D
ꢁ76.73(c 4.9, CHCl3); 1H NMR (CDCl3, 500 MHz):
d
1.14 (s, 3 H),
(10
mM) was also used for comparison purpose. Calcium chloride
1.22–1.25 (m, 6 H), 1.31 (s, 9 H), 1.67 (s, 3 H), 1.71 (s, 1 H), 1.86–
1.91 (m, 4 H), 2.32 (m, 2 H), 2.39 (s, 3 H), 2.40–2.59 (m, 3 H), 3.49
(bs, 1 H), 3.82 (d, J = 7.0 Hz, 1 H), 4.15 (d, J = 8.5 Hz, 1 H), 4.29 (m, 2
H), 4.40 (dd, J = 6.0 Hz, 10.5 Hz, 1 H), 4.58 (ddd, J = 1.5 Hz, 9.0 Hz,
24.5 Hz, 1 H), 4.87 (m, 1 H), 4.92 (d, J = 9.0 Hz, 1 H), 4.98 (dd,
J = 1.5 Hz, 9.5 Hz, 1 H), 5.62 (d, J = 7.5 Hz, 1 H), 6.22 (t, J = 9.5 Hz, 1
H), 6.31 (s, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 7.56 (dd, J = 1.0 Hz, 8.0 Hz, 1
H), 8.00 (d, J = 7.5 Hz, 1 H), 8.12 (s, 1 H); 13C NMR (CDCl3, 75.5 MHz)
(6 mM) was added to the assembly reaction after 50 min to follow
the calcium-induced microtubule depolymerization.
3.4.27. Electron microscopy
Aliquots (50
mL) were taken from in vitro polymerization assays
at the end of the reaction and placed onto 300-mesh carbon-
coated, formavar-treated copper grids. Samples were then stained
with 20
mL of 2% uranyl acetate and viewed with a JEOL model
d
8.9, 9.5, 14.9, 21.8, 22.2, 26.7, 27.5, 28.1, 35.5, 43.2, 45.7, 58.5,
100CX electron microscope.
72.2, 72.6, 73.1, 75.3, 75.5, 76.3, 79.2, 80.4, 81.0, 84.4, 128.3, 130.1,
130.3, 130.9, 133.1, 133.7, 134.8, 142.1, 154.8, 165.7, 170.2, 174.6,
3.4.28. Computational methods
203.6; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.14 (dd, J = 23.7 Hz,
The structures of SB-T-12853 and SB-T-1213 in the 1JFF
coordinate [36] were produced by directly changing the groups
of REDOR-Taxol in the 1JFF complexes using the Builder module in
the InsightII 2000 program (CVFF). Then, these structures were
energy-minimized in 5000 steps or until the maximum derivative
was found to be <0.001 kcal/A by means of the conjugate gradients
method using the CVFF force field and a distance-dependent
dielectric. The backbone of the protein was fixed throughout the
energy minimization. After the energy minimization, the snap-
shots were overlaid by superimposing the backbones of the
proteins.
34.7 Hz, 1 F), ꢁ86.13 (d, J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd.
for C42H52ClF2NO15ꢄH+, 884.3066; Found 884.3057.
3.4.24. 30-Dephenyl-30-(2,2-difluoroethenyl)-2-debenzoyl-2-(3-
chlorobenzoyl)-10-dimethylcarbamoyldocetaxel (6d-4)
20
Yield 91% for 2 steps; white solid; mp 170–173 8C; [a]
D
ꢁ88.09 (c 2.1, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 1.17 (s, 3 H),
1.27 (s, 3 H), 1.33 (s, 9 H), 1.62 (bs, 1 H), 1.69 (s, 3 H), 1.84–1.90 (m,
2 H), 1.93 (s, 3H), 2.34 (m, 2H,), 2.42 (s, 3 H), 2.56 (m, 1 H), 2.98 (s, 3
H), 3.06 (s, 3 H), 3.25 (d, J = 2.5 Hz, 1 H), 3.52 (d, J = 5.5 Hz, 1 H), 3.83
(d, J = 7.0 Hz, 1 H), 4.18 (d, J = 8.5 Hz, 1 H), 4.29 (s, 1 H), 4.32 (d,
J = 8.5 Hz, 1 H), 4.46 (dd, J = 6.5 Hz, 11.0 Hz, 1 H), 4.58 (ddd,
J = 1.5 Hz, 10.0 Hz, 24.5 Hz, 1 H), 4.93 (m, 2 H), 5.01 (d, J = 10.0 Hz, 1
H), 5.63 (d, J = 7.5 Hz, 1 H), 6.25 (t, J = 9.0 Hz, 1 H), 6.27 (s, 1 H), 7.47
(t, J = 7.5 Hz 1 H), 7.62 (dd, J = 1.0 Hz, 9.5 Hz, 1 H), 8.03 (d, J = 8.0 Hz,
Acknowledgments
This work has been supported by grants from the National
Institutes of Health (GM42798 and CA103314 to I.O.; CA083185
and CA077263 to S.B.H.; CA 73872 to R.J.B.). Generous support
from Indena SpA, is gratefully acknowledged.
1 H), 8.15 (s, 1 H); 13C NMR (CDCl3, 75.5 MHz)
d 9.3, 14.9, 22.2, 26.9,
28.1, 35.4, 36.0, 36.6, 43.2, 45.5, 58.5, 72.4, 72.6, 73.1, 75.6, 76.1,
76.3, 79.3, 80.4, 81.1, 84.6, 128.3, 130.1, 130.4, 130.9, 133.3, 133.7,
134.8, 142.6, 154.8, 156.1, 165.7, 171.1, 205.5; 19F NMR, (CDCl3,
References
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d
ꢁ84.16 (dd, J = 23.9 Hz, 34.9 Hz, 1 F), ꢁ86.17 (d,
J = 36.7 Hz, 1 F); HRMS (FAB+, m/z): Calcd. for C42H53ClF2N2O15ꢄH+,
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chlorobenzoyl)-10-methoxycarbonyldocetaxel (6e-4)
20
Yield 70% for 2 steps; white solid; mp 161–165 8C; [a]
D
ꢁ72.09 (c 4.3, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 1.16 (s, 3H), 1.26
(m, 3 H), 1.32 (s, 9 H), 1.63 (bs, 1 H), 1.71 (s, 3 H), 1.91 (m, 1 H), 1.94
(s, 3 H), 2.33 (m, 2 H), 2.42 (s, 3 H), 2.46 (bs, 1 H), 2.59 (m, 1 H), 3.46
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J = 8.5 Hz, 1 H), 5.64 (d, J = 7.5 Hz, 1 H), 6.13 (s, 1 H), 6.23 (t,
J = 8.5 Hz, 1 H), 7.49 (t, J = 8.0 Hz, 1 H), 7.60 (d, J = 7.5 Hz, 1 H), 8.01
(d, J = 8.0 Hz, 1 H), 8.14 (s, 1 H); 13C NMR (CDCl3, 75.5 MHz)
14.9, 21.7, 22.2, 26.6, 28.1, 35.3, 35.6, 43.1, 45.6, 55.6, 58.5, 72.1,
72.5, 73.2, 75.4, 76.3, 78.2, 79.2, 80.5, 80.9, 84.4, 128.3, 130.1,
130.3, 130.9, 132.6, 133.7, 134.9, 143.1, 154.9, 155.7, 165.7, 170.2,
d 9.4,
203.8; 19F NMR, (CDCl3, 282 MHz)
d
ꢁ84.16 (dd, J = 23.7 Hz,
34.7 Hz, 1 F), ꢁ86.17 (dd, J = 34.9 Hz, 1 F); HRMS (FAB+, m/z): Calcd.
for C41H50ClF2NO16ꢄH+, 886.2859; Found 886.2845.
3.4.26. Tubulin polymerization assay
Assembly and disassembly of calf brain microtubule protein
(MTP) was monitored spectrophotometrically (Beckman Coulter
DU 640, Fullerton, CA) by recording changes in turbidity at 350 nm
at 37 8C [34,35]. MTP was diluted to 1 mg/mL in MES buffer

188
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