First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT

Article abstract of DOI:10.1016/j.bmc.2003.12.013

Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or

Full text of DOI:10.1016/j.bmc.2003.12.013

Bioorganic & Medicinal Chemistry 12 (2004) 969–977
First enzymatically activated Taxotere prodrugs designed for
ADEPT and PMT
Emmanuel Bouvier, Sylvie Thirot, Frederic Schmidt* and Claude Monneret
UMR176 CNRS/Institut Curie, Section Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France
Received 26 September 2003; accepted 12 December 2003
Abstract—Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs
of docetaxel (Taxotere1) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in
ADEPT or PMT. They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the
spacer substitution. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis
kinetics. As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological
evaluations.
# 2003Elsevier Ltd. All rights reserved.
1. Introduction
and evaluated;6À7 some of them displayed better solubi-
lities together with better toxicities than the parent drug.
A direct 7-O-glucosyl docetaxel analogue was reported
to be twice as water-soluble as paclitaxel but with simi-
lar activity.8 Amino acid conjugates linked to the
20-hydroxyl of a 30-cyclopropyl docetaxel analogue were
also prepared and tested in vivo.9 Some showed better
activity than docetaxel in ILS (Increase of Life Span) on
B16 melanoma-bearing mice.
Paclitaxel (Taxol1) 1,1 a diterpene considered as one of
the most promising antitumor drugs of the 1990s, and
its semisynthetic analogue docetaxel (Taxotere1) 2,2
have become indispensable drugs in clinics (Fig. 1). The
main chemical differences between paclitaxel 1 and
docetaxel 2 are the substitutions at the 30-nitrogen on
the side chain (BOC instead of benzoyl) and the 10-
position of the taxoid core (free alcohol instead of ace-
tate). Although being among the very first examples of a
new mechanism of action (promotion and stabilization
of microtubules) and showing high potency against
solid tumors, they bring about a number of undesirable
side effects, and moreover their poor water-solubility
hampers their clinical application. Namely, they have to
be co-injected with a detergent, which induces reactions
such as hypersensitivity.3,4
Since our aim was to selectively deliver docetaxel at the
tumor site for an increased efficacy of the treatment, we
considered converting the active compound into a
water-soluble prodrug. The specific delivery will be
based on an enzymatic (b-d-glucuronidase) hydrolysis
according to an ADEPT10 (Antibody Directed Enzyme
Prodrug Therapy) or a PMT11 (Prodrug Mono Ther-
apy) strategy. In an ADEPT strategy, the enzyme is first
targeted at the tumor site by Mab-antigen recognition.
In a second step, the enzyme acts as a catalyst to cleave
the injected prodrug locally and release the cytotoxic
drug. In a PMT strategy, the already present b-d-glu-
curonidase found in necrotic tumor areas is used
instead.12
To address these problems, numerous researches on
analogues, prodrugs5 and derivatives of paclitaxel have
been carried out; in contrast with docetaxel, very few
attempts have been undertaken to overcome these diffi-
culties and improve the drug properties. Previously,
some glycosyl derivatives of docetaxel at the 20, 7 or 10
positions, including a glycolate spacer, were prepared
In the present paper, we report the synthesis of two
docetaxel prodrugs 4 and 5 (Fig. 2) in which a glu-
curonic moiety is linked to a double spacer containing a
para-hydroxybenzyl alcohol connected to a diamine
tether via a carbamate linkage. This system was shown
Keywords: Taxotere; Docetaxel; Prodrug; ADEPT.
* Corresponding author. Tel.: +33-1-42346664; fax: +33-1-42346631;
e-mail: frederic.schmidt@curie.fr
0968-0896/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.013

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E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
to be efficient in releasing drugs in the presence of b-d-
glucuronidase.13,14 For instance a paclitaxel prodrug 3
(Fig. 2) was shown to be efficient.13 The spacer is linked
to the 20 position of docetaxel 2 via a carbamate. The 20
position was selected because it is known that its modi-
fication leads to a dramatic loss of cytotoxic activity,
and the carbamate linkage because it is relatively stable
in the presence of esterases. The drug release mechanism
from 4 or 5 is depicted on Scheme 1. On the other hand,
we decided to use two types of substituents on the aro-
matic part of the spacer, a nitro or an amino function in
the ortho position of the phenol involved in the glycosi-
dic linkage. Indeed most of the prodrugs based on a
para-hydroxybenzyl spacer reported in the ADEPT or
PMT literature contained a nitro substituent to decrease
the pKa of the phenol and consequently improve the
rate of the self-immolative decomposition of the spacer.
However we must remind that for paclitaxel prodrug 3,
we could not access to the corresponding nitro contain-
ing spacer prodrug since some intriguing problems rela-
ted to the reduction of the nitro group occurred during
a benzyl hydrogenolysis.13 Nevertheless, liberation of
paclitaxel from the amino containing spacer prodrug
was very efficient. Therefore it was of interest to prepare
both types in the case of docetaxel and to compare sta-
bility, cytotoxicity and kinetics of enzymatic hydrolysis.
So, at the outset of our project, we considered the
synthesis of prodrugs 4 and 5 as described in the retro-
synthetic pathway (Scheme 2). Whereas the preparation
of 4 was straightforward and used the same pathway as
Figure 1. Structures of paclitaxel 1 and docetaxel 2.
Figure 2. Structure of prodrugs.
Scheme 1. Release of docetaxel from prodrug.

E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
971
for 3, we envisioned that 5 could be obtained from the
common precursor 9 by means of an hydrogenation, or
through troc deprotection conditions (Zn and AcOH)
that would also reduce the nitro into an amino group.15
without reduction of the nitro group on the spacer
moiety, yielded 4 (34% from 2, Scheme 3). This result
was quite surprising at first sight, bearing in mind the
failure of this reaction using paclitaxel as the drug.13
Molecular modelling (HyperChem, field MM+) of the
compounds may support this fact as we have observed
that the two faces of the benzyl ester of prodrug 3 are
hindered by p-stacking with the 2-benzoate and inter-
actions with the side chain of paclitaxel on the other
side. This steric hindrance in the approach of the benzyl
group explains the need for harsher conditions in order
to cleave this protecting group by hydrogenolysis,
whereas the nitro group of the spacer is very accessible.
So we could not succeed in cleaving the benzyl ester
without reducing the nitro to an amino group. In com-
parison, the ester in 13 is relatively free on one face, the
other being involved in p-stacking interactions with
the phenyl ring of the spacer. This fact could account
2. Chemistry
First of all, docetaxel 2 and its 7,10-ditroc protected
derivative 12 were prepared starting from oxazoline and
7,10-ditroc-DAB (10-deacetyl-baccatin III) using pub-
lished procedures.16,17 For 4, a regioselective carba-
moylation at the 20 hydroxyl of 2 with the previously
described intermediate 1013 cleanly led to the fully pro-
tected prodrug 9; as expected the less reactive hydroxyl
groups of docetaxel did not react. Then desilylation of 9
with HF-Pyridine, followed by selective hydrogenolysis
of 13 with cyclohexadiene and palladium on charcoal,
Scheme 2. Retrosynthetic scheme of compounds 4 and 5.
Scheme 3. Synthesis of prodrug 4.

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E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
for its easy removal in our conditions without reduction
of the nitro group.
ester with concomitant reduction of the nitro group to
yield 5 quantitatively.
Alternatively, the ditroc derivative 12 was used instead
of 9 to achieve the synthesis of prodrug 5 (Scheme 4).
Coupling of 12 with 10 led to 14 (70% yield), which was
first deprotected with Zn/AcOH with simultaneous
reduction of the nitro group to an amine giving 15. The
next desilylation step proved to be very challenging and
compound 16 was obtained in a rather modest yield
(20%). Indeed, since the HF/Pyridine reagent was not
strong enough to deprotect all the silyl groups, we
instead used the Et3N/3HF reagent, which had been
efficient to remove silyl groups from nucleosides and
nucleotides.18 In fact, the best way to afford 5 appeared
to be the hydrogenation of compound 13 which quanti-
tatively led to the deprotected amino prodrug (Scheme
4). So, the troc protecting groups were removed prior to
coupling with the carbamoyle chloride 10, and then
hydrogenolysis with H2 and Pd/C cleaved the benzyl
For HPLC comparison, spacers 6 and 7 were prepared
as described in Scheme 5.
3. Biological results
The key issues of these studies were to quantify the
differences between the two docetaxel prodrugs (with a
nitro- or an amino-substituted spacer) as well as
between the previously obtained paclitaxel prodrug and
the new docetaxel ones containing the same kind of
spacer.
3.1. Stability
First of all, stabilities of the prodrugs 4 and 5 were
measured by HPLC (UV detection) in a phosphate
Scheme 4. Synthesis of prodrug 5.
Scheme 5. Synthesis of spacers 6 and 7.

E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
973
Figure 3. Enzymatic cleavage of prodrug 4.
Figure 4. Enzymatic cleavage of prodrug 5.
buffer at 37 ꢀC (pH 7.2). Both prodrugs proved to be
stable during a 24 h-run, and no docetaxel was liberated
from the prodrugs during this time.
3.2. Cytotoxicity
Cytotoxities of the prodrug were also measured. For an
ADEPT or a PMT strategy, the prodrug must be less
cytotoxic than the corresponding drug.
The IC50 measured for L1210 cell lines were:
Prodrug 4: 4.86 mM Docetaxel 2: Spacer 6: 75.3 mM
14.4 nM
Prodrug 5: 2.69 mM
Spacer 7: 45.8 mM
Figure 5. Comparison of the disappearance of the three prodrugs.
It is noteworthy that the spacers are much less cytotoxic
than the drug and therefore cannot interfere. The ratios
of IC50 between prodrug and drug are 337 for 4 and 187
for 5, which correspond to a good detoxification. These
values are compatible with an ADEPT or a PMT strategy.
3.3. Kinetics of drug release
The in vitro kinetics studies are very important because
they first validate the approach if the prodrug is cleaved
and then are used to select the best prodrugs for in vivo
experiments. Prodrugs 4 and 5 were incubated with
Escherichiacoli b-d-glucuronidase at 37ꢀC and the solu-
tions were analysed by HPLC (UV detection). The
enzymatic hydrolyses of both prodrugs were very similar,
showing the appearance of two peaks characterized as
docetaxel and spacer 6 for 4 and spacer 7 for 5 by
comparison with the synthesized samples (Figs 3and 4 ).
The peak corresponding to the prodrug decreased until its
complete disappearance and both spacer and docetaxel
reached a plateau. The observed half-lives of prodrug
disappearance (Fig. 5) were approximatically 10 min for
both prodrugs, which was almost the same as the one
previously reported 313 for the same concentration of
prodrug (29.6 mmol), and of enzyme (12 units/mL).
Figure 6. Structure of intermediate 18.
The striking difference between 3 and 4 or 5 was that,
for the two latter examples, no intermediate compound
such as docetaxel linked to the diamine tether could be
detected, whereas for 3, compound 18 (Fig. 6) was
detected and characterized by LC-MS.13 This again
stressed the divergent reactivities between paclitaxel and
docetaxel already observed in the hydrogenolysis step
(see above). It seems that this ethylenediamine-docetaxel
intermediate cyclised too fast to be detected and only
docetaxel was seen.
4. Conclusion
The absence of major differences between the kinetics
for 4 and 5 could be explained by the fact that the
immolative step involving two different substituted
phenols is not rate-determining.
We have reported the synthesis of the first docetaxel
prodrugs to date aimed at enzyme activation in ADEPT
or PMT strategies. These prodrugs differ only by the
substitution of the aromatic ring. Indeed, a nitro group

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E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
on the aromatic ring of the spacer could be preserved
during the hydrogenolysis step, a reaction that could
not have been achieved during deprotection of the
already reported paclitaxel prodrug 3.13 Therefore
nitro- and amino-spacer containing prodrugs of doc-
etaxel were obtained and compared. Their in vivo eval-
uation showed a relevant detoxification and good
kinetics of enzymatic hydrolysis.
5.3. Stability of compounds in a buffer solution
A solution of 29.6 mmol of prodrug in 0.02 M phosphate
buffer (pH 7.2) was incubated at 37 ꢀC. Aliquots (50 mL)
were taken at various times and analysed by HPLC
after dilution with eluent (150 mL).
5.4. Enzymatic cleavage by E. coli ꢀ-D-glucuronidase
The double self-immolative spacer used was found to be
as effective with docetaxel as with paclitaxel, with only a
slight difference for the spacer release between docetaxel
prodrugs containing the nitro and the amino spacer.
These are encouraging results in the field and will be
further investigated.
A solution of 29.6 mmol of prodrug in 0.02 M phosphate
buffer (pH 7.2) was incubated at 37 ꢀC in the presence of
12 units/mL of b-d-glucuronidase (E. coli). Aliquots (50
mL) were taken at various times and analysed by HPLC
after dilution with eluent (150 mL).
5.4.1. 20-O-[N,N0-Dimethyl-N0-benzyl-[4(À2,3,4-tri-O-tert-
butyldimethylsilyl-ꢀ-D-glucopyranosyl uronate-3-nitro-
benzyloxycarbonyl]ethylenediamin]docetaxel (9). 200 mg
(0.248 mmol) of docetaxel 2 and a large excess of
DMAP (300 mg, 2.45 mmol) were dissolved in 20 mL of
freshly distilled CH2Cl2 and placed under argon atmos-
phere. Then 284 mg (1.1 equiv) of the carbamoyl chloride
1013 were added, followed by dropwise addition of 0.2
mL of TEA. After 6 h, the mixture was diluted with 20
mL of CH2Cl2, washed with water and brine, and the
compound was purified by column chromatography
(eluent CH2Cl2–MeOH 97.5/2.5!95/5) to give the cou-
pling product 9 267 mg (73%) as a gum. 1H NMR
(CDCl3) d 8.15 (m, 2H, Ho Bz2), 7.85 (m, 1H, Ha), 7.48
(m, 4H, Hm Bz, Hp Bz, Hb), 7.30 (m, 10H, Haro), 7.13(d,
J=8.7 Hz, 1H, Hc), 6.99 (d, J=9.5 Hz, 1H, NH), 6.32
5. Experimental
Melting points (mp) were measured using an Electro-
thermal digital melting point apparatus and are uncor-
1
rected. H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were recorded on a Bruker AC 300 spectro-
meter-chemical shifts d in ppm and J in Hz. Chemical
ionisation (CI-MS; NH3, positive ion mode) or FAB
(positive ion mode, either MB (‘magic bullet’), or NBA
(3-nitrobenzyl alcohol) as matrix) mass spectra were
recorded on a Nermag R 10-10C spectrometer. Electro-
spray ionisation mass spectra (ESI-MS) were acquired
with a quadripole instrument with a mass of charge
(m/z) range of 2000. The Nermag R 10-10 mass
spectrometer used was equipped with an analytical
atmospheric pressure electrospray source. The chroma-
tographies were conducted over silica gel [Merck 60
(230–400 Mesh)].
0
(m, 1H, H13), 5.65 (m, 1H, H3 ), 5.58 (d, J=5.6 Hz, 1H,
00
H1 ), 5.48–5.24 (m, 3H, H2, 3 , 10), 5.13(m, 2H, CH 2 Bn),
5.09 (m, 2H, CH2O), 4.98 (m, 1H, H5), 4.50 (m, 1H,
00
H3 ), 4.40 (s, 1H, H4 ), 4.29–4.06 (m, 3H, H7, H20), 4.17
00
00
(d, J=3.6 Hz, 1H, H2 ), 4.00 (m, 1H, H2 ), 3.89 (d,
0
00
J=2.2 Hz, 1H, H5 ), 3.62–3.02 (m, 4H, CH2N), 2.92–
For the NMR descriptions, the numbers correspond to
the baccatin core, the 0 to the taxoid side chain and the 00
to the glucuronic- spacer moiety.
2.81 (m, 6H, CH3N), 2.52 (m, 4H, H6a, CH3 Ac4), 2.43–
2.15 (m, 2H, H14), 1.96 (s, 3H, H18), 1.84 (m, 1H, H6b),
1.73(m, 3H, H 19), 1.28 (m, 9H, Me BOC), 1.21 (s, 3H,
H16), 1.10 (s, 3H, H17), 0.89, 0.87, 0.83(s, 9H)
(CH3)3CSiMe2), 0.16, 0.11, 0.11, 010, 0.05, 0.01 (s, 3H)
(CH3Si); 13C NMR (CDCl3) d 211.5 (C9), 169.8, 168.9,
5.1. In vitro cytotoxicity
Cytotoxicity was tested against L1210 (mouse leukemic
cell line) cells using the MTA assay.
0
168.3, 167.0 (MeC(O)C4, fCO2, C1 , CO2Bn), 155.9,
154.9, 149.9 (NCO2), 140.1, 139.3, 138.0, 135.4, 135.1,
129.3(Cq aro, C11, C12); 133.9, 133.5, 130.2, 128.7, 128.5,
128.3, 127.7, 126.3 (CHaro), 125.5 (Ca), 116.8 (Cc), 99.3
L1210 cells were cultivated in RPMI 1640 medium
(Gibco) supplemented with 10% fetal calf serum, 2 mM
l-glutamine, 100 units/mL penicillin, 100 mg/mL strep-
tomycin, and 10 mM HEPES buffer (pH=7.4). Cyto-
toxicity was measured by the microculture tetrazolium
assay (MTA). Cells were exposed to graded concentra-
tions of drug (nine serial dilutions in triplicate) for 48 h.
Results are expressed as IC50, the concentration which
reduced by 50% the optical density of treated cells with
respect to the optical density of untreated controls.
00
(C1 ), 84.4 (C5), 81.0 (C4), 79.5 (Me3C), 78.9 (C1), 78.8
00
(C3 ), 76.6 (C20), 77.8, 75.8, 75.5 (C2/10, C2 and C5 ),
00
00
0
00
75.2 (C3 ), 74.5 (C2/10), 72.2 (C4 ), 71.7 (C13), 71.5 (C7),
67.0 (CH2 Bn), 65.7 (CH2O), 57.5 (C8), 54.2 (C3), 50.6,
0
47.4, 47.0, 46.2 (CH2N), 46.5 (C2 ), 43.1 (C15), 36.8 (C6),
35.6, 35.5 (CH3N), 28.2, 28.2 (Me BOC), 26.3(C 16),
25.8 (2C), 25.7 ((CH3)3CSiMe2), 22.7 (CH3 Ac4), 22.6
(C14), 21.0 (C17), 18.0 (2C), 17.9 ((CH3)3CSiMe2), 14.3
(C18), 10.0 (C19), À4.5, À4.6, À4.7, À4.8, À5.0, À5.1
(CH3Si); m/z (FAB+ MB+NaI) 1747.8 M+Na+,
1748.8 M+Na+H+; HRMS: (C87H124O26N4Si3Na) m/
z calcd: 1747.7709, found: 1747.7657.
5.2. HPLC conditions
Analysis was carried out on a reverse-phase column
(Lichrospher RP18e, 250Â4 mm, 5 mm) using isocratic
conditions (1 mL/min) of 53% phosphate buffer
(0.02 M, pH 3) and 47% acetonitrile with UV detection
at 226 nm (extracted from PDA 3-D spectra).
5.4.2.
20-O-[N,N0-Dimethyl-N-benzyl-4-(ꢀ-D-glucopyr-
anosiduronate)-3-nitrobenzyloxycarbonylethylenediamin]-
docetaxel (13). 267 mg (0.155 mmol) of the silylated

E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
975
00
(Ca), 117.5 (Cc), 102.3(C ), 82.2, 80.8, 79.2 (C1,
compound 9 were dissolved in 10 mL of anhydrous
CH3CN with 0.5 mL of anhydrous pyridine. The reac-
tion mixture was placed under argon and cooled to 0 ꢀC
in an ice bath. 1.5 mL of 70% HF–pyridine complex
were then added dropwise, and the mixture allowed to
reach rt overnight. The solution was cooled again and
quenched with 50 mL of satd NaHCO3. After extraction
with 2Â50 mL of EtOAc and drying over Na2SO4, the
residue was purified on a silica gel column (eluent
CH2Cl2–MeOH 95/5!9/1) to afford 185 mg (87%) of
the desilylated compound 13 as a sticky foam, together
with 10 mg of the starting material and 50 mg of a
monosilylated compound. H NMR (CDCl3) d 8.07 (s,
2H, Ho Bz2), 7.79 (m, 1H, Ha), 7.56 (m, 1H, Hp Bz), 7.48
(m, 3H, Hm Bz, and Hb), 7.32 (m, 11H, Haro), 6.63(m,
0
1H, NH), 6.19 (m, 1H, H13), 5.60 (s, 1H, H3 ), 5.50–4.94
00
(m, 5H, H2, 3, 5, 10, 1 ), 5.17 (m, 4H, CH2 Bn, CH2O),
,
Me3C), 77.5, 77.2, 76.6, 76.5, 75.6, 74.5, 73.4, 73.1
1
4
0
(C2,7,10,20,3 ,2 ,3 ,4 ,5 ), 66.9 (CH2O), 58.8 (C8), 56.4 (C3),
00 00 00 00
0
49.6, 49.3, 49.0 (CH2N), 47.7 (C2 ), 44.4 (C15), 37.5 (C6),
36.6 (CH3N), 28.8 (Me BOC), 28.0 (C16), 23.3
(CH3Ac4), 21.9 (C17), 20.5 (C14), 14.8 (C18), 10.5 (C19);
m/z (ESI-) 1291.5 MÀHÀ; HRMS: (C62H76O26N4) m/z
calcd: 1291.4670, found: 1291.4608.
5.4.4. 20-O-[N,N0-Dimethyl-N0-benzyl-[4(À2,3,4-tri-O-tert-
butyldimethylsilyl-ꢀ-D-glucopyranosyl uronate-3-nitro-
benzyloxycarbonyl]ethylenediamin]-7,10-ditroc-docetaxel
(14). To 100 mg (97 mmol) of ditroc-docetaxel 12 and
174 mg of DMAP (1.42 mmol) in 15 mL of CH2Cl2 were
added 131 mg (1.3 eq.) of the carbamoyl chloride 1013
dissolved into 1.5 mL of CH2Cl2. After 2 h, the mixture
was diluted with CH2Cl2, washed with water and brine,
and dried over Na2SO4. After chromatography (eluent
CH2Cl2–MeOH 98/2), 187 mg (93%) of the coupling
1
00
4.46 (s, 1H, H7), 4.26 (s, 1H, H3 ), 4.14 (s, 1H, H4 ), 4.26
00
0
and 4.14 (s, 1H, H20), 3.87 (m, 2H, H2 , 5 ), 3.76 (m, 4H,
00
1
00
H2 , OH
0
00
00
), 3.33–3.00 (m, 4H, CH2N), 2.91–2.76
2 , 3 , 4
product 14 were isolated as a gum. H NMR (CDCl3) d
(m, 6H, CH3N), 2.67 (m, 1H, H6a), 2.60–2.20 (m, 2H,
H14), 2.44 (s 3H, CH3 Ac4), 1.88 (s, 3H, H18), 1.86 (m,
1H, H6b), 1.28 (s, 9H, Me BOC), 1.14 (s, 3H, H16), 1.05 (s,
3H, H17); 13C NMR (CDCl3) d 211.5 (C9), 170.1, 169.0,
168.4, 167.0 (MeC(O)C4, fCO2, C1 , CO2Bn), 155.7, 155.2,
149.8 (NCO2), 140.2, 139.4, 137.8, 135.4, 135.1, 131.9,
129.4 (Cqaro, C11, C12), 133.6, 130.2, 128.8, 128.6, 128.4,
8.11 (m, 2H, Ho Bz2), 7.86 (m, 1H, Ha), 7.60 (m, 1H, Hp
Bz), 7.52 (m, 3H, Hm Bz, Hb), 7.31 (m, 10H, Haro), 7.13
(d, J=8.7 Hz, 1Hc), 7.04 (d, J=10.6 Hz, 1H, NH), 6.34
(m, 1H, H13), 6.26 (s, 1H, H10), 5.70 (s, 1H, H2), 5.58
0
00
0
(m, 2H, H5, 1 ), 5.36 (m, 2H, CH2O), 5.23(m, 1H, H 3 ),
0
5.13(s, 2H, CH 2 Bn), 5.00 (m, 2H, H7, 2 ), 4.89 (m, 1H),
4.76 (m, 2H, CH2CCl3), 4.59 (m, 1H) (CH2CCl3), 4.50
00
128.1, 126.5 (CHaro), 124.9 (Ca), 118.4 (Cc), 101.7 (C1 ),
00
(s, 1H, H3 ), 4.40 (s, 1H, H4 ), 4.33 and 4.18 (m, 1H,
00
00
H20), 4.05 (d, J=5.6 Hz, 1H, H2 ), 3.94 (m, 1H, H3),
84.4 (C5), 81.0 (C1), 80.3(Me C), 78.9 (C4), 76.5 (C20),
3
00 00 00 00
75.2, 75.0, 74.5, 72.7, 71.7, 70.8 (C2,7,10,13,2 ,3 ,4 ,5 ), 67.4
00
3.84 (d, J=3.6 Hz, 1H, H5 ), 3.75–3.00 (m, 4H, CH2N),
0
(CH2 Bn), 65.7 (CH2O), 57.5 (C8), 54.5 (C3), 46.5 (C2 ),
47.7, 46.3(CH N), 43.1 (C15), 36.6 (C6), 35.4 (CH3N),
2
34.5 (C14), 28.3, 28.2 (Me BOC), 26.4 (C16), 22.6
(CH3Ac4), 21.0 (C17), 14.3(C 18), 10.1 (C19); m/z (FAB+
MB+NaI) 1405.8 M+Na+; HRMS: (C69H82O26N4Na)
m/z calcd: 1405.5115, found: 1405.5145.
2.88 (m, 6H, CH3N), 2.67 (m, 1H, H6a), 2.62 (s, 3H,
CH3Ac4), 2.43(m, 2H, H 14), 2.09 (s, 3H, H18), 2.05 (m,
1H, H6b), 1.85 (s, 3H, H19), 1.34 (s, 3H, H16), 1.27 (s,
9H, Me BOC), 1.18 (s, 3H, H17), 0.90 (m, 9H), 0.87,
0.83(s, 9H) ((C H3)3CSi), 0.16, 0.05, 0.00 (s, 3H), 0.11 (s,
9H) (CH3Si); 13C NMR (CDCl3) d 201.1 (C9), 170.1,
0
169.1, 168.4, 167.0 (MeC(O)C4, fCO2, C1 , CO2Bn),
5.4.3. 20-O-[N,N0-Dimethyl-N-4-(ꢀ-D-glucopyranosiduro-
nate)-3-nitrobenzyloxycarbonylethylenediamin]docetaxel
4. 64 mg (46 mmol) of 13 in solution in 4 mL of EtOH in
the presence of 10% Pd/C (60 mg) was heated at 40–
50 ꢀC, then 50 mL of cyclohexa 1,4-diene (10 equiv) were
added to the mixture. After 2 h, 50 mL of diene were
added again and the reaction was continued overnight
at rt. A third equivalent of diene was added and, after
an additional 24 h stirring, the mixture was filtered on
Celite 545. The crude product was then purified by
chromatography on silica gel (eluent CH3CN–H2O 9/1)
to give 43mg (53%) of the solid nitro prodrug 4. Mp
156.0, 154.8, 153.3, 153.1, 150.0 (NCO2), 143.8, 140.2,
138.0, 135.2, 129.1 (Cqaro, C11, C12), 133.9 (Cb), 131.4,
130.3, 128.8, 128.6, 128.4, 127.8, 126.4, 125.6 (CHaro),
00
125.1 (Ca), 116.9 (Cc), 99.4 (C1 ), 94.2 (CCl3), 83.8 (C5),
00 00
), 75.8
3 /4
80.7 (C4), 80.3(Me C), 79.6 (C10), 79.3(C
3
0
(CH2O), 75.2 (C3 ), 74.4 (C2), 72.2 (C3 /4 ), 71.4 (C4), 67.1
00 00
0
00
(CH2 Bn), 65.8 (C2 ), 76.8 (CH2CCl3), 76.5 (C5 ), 75.2
00
(C20), 74.4 (C2 ), 56.1 (C3), 50.9 (C8), 46.9 (m, CH2N),
43.2 (C15), 35.6 (m, CH3N), 33.3 (C6), 28.3, 28.2 (Me
BOC), 26.3(C 16), 25.9 (2C), 25.8 ((CH3)3CSi), 22.7 (CH3
Ac4), 22.6 (C14), 21.3(C 17), 18.0 (2C), 17.9 ((CH3)3CSi),
14.7 (C18), 10.8 (C19), À4.5, À4.6, À4.6, À4.7À4.9, À5.0
(CH3Si); m/z (FAB+ NBA+NaI) calcd: 2097.8 M
(35Cl537Cl)+Na+ (88%), 2098.8 M (35Cl537Cl)+Na+H+
(88%), 2099.8 M (35Cl437Cl2)+Na+ (100%); HRMS:
(C139H126O30N435Cl6Si3Na) m/z calcd: 2095.5794, found:
2095.5823(23%), (C 139H126O30N435Cl537ClSi3Na) m/z
calcd: 2097.5764, found: 2097.5806 (77%), (C139H126O30-
N435Cl437Cl2Si3Na) m/z calcd: 2099.5837, found:
2099.5734 (100%), (C139H126O30N435Cl337Cl3Si3Na) m/z
calcd: 2101.5702; found: 2101.5823(67%).
160 ꢀC (dec.) H NMR (CDCl3) d 8.12 (d, J=6.4 Hz,
1
2H, Ho Bz), 7.83(m, 1H, H ), 7.63–7.39 (m, 9H, Hp, m
Bz, Haro, Hb), 7.24 (1H, Hc), 6.09 (m, 1H, H13), 5.63(d,
a
0
J=6.4 Hz, 1H, H3 ), 5.46 (br s, 1H, NH), 5.29 (s, 2H,
00
H3 , 10), 5.16–5.00 (m, 5H, H2, 5, 1 , CH2O), 4.19 (s, 3H,
0
H7, H20), 3.82 (m, 2H, H2 , 5 ), 3.75–3.50 (m, 8H, H2 , 3 ,
00
00
00
00
, CH2N), 3.02–2.78 (m, 6H, CH3N), 2.41 (m, 4H, H6a,
4
CH3 Ac4), 2.60–2.20 (m, 2H, H14), 1.93(s, 3H, H 18),
1.82 (m, 1H, H6b), 1.68 (s, 3H, H19), 1.38 (s, 9H, Me
BOC), 1.14 (s, 3H, H16), 1.12 (s, 3H, H17); 13C NMR
(CDCl3) d 211.4 (C9), 171.5, 171.0, 167.7 (MeC(O)C4,
5.4.5. 20-O-[N,N0-Dimethyl-N0-benzyl-[4(À2,3,4-tri-O-tert-
butyldimethylsilyl-ꢀ-D-glucopyranosyl uronate-3-amino-
benzyloxycarbonyl]ethylenediamin]docetaxel (15). Com-
pound 14 (187 mg, 0.09 mmol) was dissolved in 20 mL
0
fCO2, C1 ), 157.9, 157.1, 151.2 (NCO2), 141.7, 140.0,
139.8, 139.0, 137.7, 130.0 (Cqaro, C11, C12), 134.7 (Cb),
132.7, 131.4, 131.2, 129.8, 129.6, 128.2 (CHaro), 123.1

976
E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
of EtOAc under vigorous stirring in the presence of a
large excess of zinc (ca. 250 mg) and 2 mL of AcOH.
After heating at 45 ꢀC for 4 h, the reaction medium was
filtered, then quenched with water and extracted with
2Â30 mL of EtOAc. The organic layers were washed
with water and brine, and dried over Na2SO4. Flash
chromatography (eluent:EtOAc) gave 85 mg (56%) of
hydrogen atmosphere (1 bar). After filtration on Celite
545 and concentration of the filtrate, the compound was
purified by chromatography (eluent CH3CN–H2O 9/1)
to give 11 mg (67%) of the prodrug 5.
Method 2: 35 mg (25 mmol) of the nitrobenzyl 13 were
suspended in 5 mL of pure EtOH with 10% Pd/C (60
mg), then stirred for a further 4 h under hydrogen
atmosphere (1 bar). After filtration on Celite 545 and
concentration of the filtrate, the compound was purified
by chromatography (eluent CH3CN–H2O 9/1), giving
the solid prodrug 5 in quantitative yield (32 mg).
1
the amino compound 15 as an oil. H NMR (CDCl3) d
8.11 (m, 2H, Ho Bz2), 7.52 (m, 1H, Hp Bz2), 7.48 (m, 2H,
Hm Bz), 7.31 (m, 11H, Haro, NH), 6.82–6.70 (m, 3H, Ha,
b, c), 6.30 (m, 2H, H13), 5.67 (m, 2H, H2, 10), 5.47 (m,
00
1H, H1 ), 5.24 (m, 1H, H3 ), 5.12 (s, 2H, CH2 Bn), 4.98
0
00
(m, 1H, H5), 4.80 (m, 2H, CH2O), 4.44 (m, 1H, H3 ),
0
4.29–4.13(m, 5H, H 7, 20, 2 , 4 ), 3.96 (m, 2H, H3 , 2), 3.84
00
(m, 1H, H5 ), 3.60–3.00 (m, 4H, CH2N), 2.88–2.80 (m,
00
00
The obtained quantities of 5 were gathered together and
1
lyophilised. Mp 202 ꢀC (dec.) H NMR (CDCl3) d 8.12
6H, CH3N), 2.58 (m, 1H, H6a), 2.54 (s, 3H, CH3Ac4),
2.44 (m, 2H, H14), 1.97 (s, 3H, H18), 1.85 (m, 1H, H6b),
1.74 (s, 3H, H19), 1.31 (s, 3H, H16), 1.25 (s, 9H, Me
BOC), 1.11 (s, 3H, H17), 0.88 (s, 18H), 0.82 (s, 9H)
((CH3)3CSi), 0.15, 0.11 (s, 3H), 0.09 and 0.05 (s, 6H)
(CH3Si); 13C NMR (CDCl3) d 211.6 (C9), 171.2, 169.9,
(m, 2H, Ho Bz2), 7.64 (m, 1H, Hp Bz2), 7.57 (m, 2H, Hm
Bz), 7.38 (m, 4H, Haro), 7.10 (m, 1H, Haro), 6.99–6.64
(m, 3H, Ha, b, c), 6.09 (m, 2H, H13), 5.64 (m, 1H, H2),
0
5.44 (m, 2H, H10, 3 ), 5.12 (m, 1H, H5), 5.01 (m, 2H,
00
CH2O), 4.65 (m, 1H, H1 ), 4.19 (m, 3H, H7, 20), 3.88 (m,
0
00
00
00
00
2H, H3), 3.72 (m, 1H, H2 ), 3.50 (m, 4H, H2 , 3 , 4 , 5 ),
3.39–3.01 (m, 4H, CH2N), 2.86 (m, 6H, CH3N), 2.44
(m, 1H, H6a), 2.40 (s, 3H, CH3 Ac4), 2.18 (m, 1H, H14a),
2.05 (m, 1H, H14b), 1.93(s, 3H, H 18), 1.86 (m, 1H, H6b),
1.68 (s, 3H, H19), 1.39 (s, 9H, Me BOC), 1.15 (s, 3H,
H16), 1.12 (s, 3H, H17); 13C NMR (CDCl3) d 211.3(C 9),
0
168.8, 167.1 (MeC(O)C4, fCO2, C1 and CO2Bn), 156.4,
155.9, 155.2, 154.9 (NCO2), 144.5, 139.4, 138.5, 138.2,
135.3, 131.5, 129.4 (Cqaro, C11, C12), 133.5, 130.2, 128.7,
128.5, 128.3, 128.3, 127.6, 126.4 (CHaro), 118.4, 116.0,
00
115.9 (Ca, b, c), 100.3(C ), 84.4 (C5), 81.0 (C4), 80.0
1
00
(C1), 79.2 (C3 ), 76.6 (C20), 76.5, 75.4 (C2 , ), 76.1
00
00
0
171.5, 170.3, 167.8 (MeC(O)C4, fCO2, C1 ), 157.8 (m,
4
0
(C10), 74.9 (C2), 74.5 (C3 ), 72.5 (C7, 2 ), 71.7 (C13), 67.9
0
NCO2), 146.8, 145.0, 139.9, 139.6, 137.7, 135.0 (Cqaro,
C11, C12); 134.5, 131.4, 131.2, 129.9, 129.7, 129.3, 128.3,
00
(CH2Bn), 67.4 (C5 ), 66.9 (CH2O), 57.6 (C8), 47.7, 46.4
00
127.9 (CHaro), 118.8, 118.2, 113.9 (Ca, b, c), 104.6 (C1 ),
86.0 (C5), 82.2 (C4), 80.8 (C1), 79.2 (C20), 77.6, 77.5,
(CH2N), 43.2 (C15), 46.5 (C3), 43.1 (C15), 35.5 (CH3N),
29.7 (C6), 28.2 (Me BOC), 25.9 (2C), 25.8 ((CH3)3CSi),
25.6 (C14), 22.7 (CH3 Ac4), 21.0 (C17), 18.0, 18.0, 17.9
((CH3)3CSi), 14.3(C 18), 11.5 (C19), À4.1, À4.6, À4.6,
À4.7, À4.8, À5.0 (CH3Si); m/z (FAB+ NBA+NaI)
1718.5 M+Na+H+, 1717.5 M+Na+.
00
00
00
00
74.7, 73.4 (C2 , 3 , 4 , 5 ), 76.5 (C2), 75.6 (C10), 73.0 (C13),
0
0
72.6 (C7), 68. 9 (C2 ), 68.4 (CH2O), 58.8 (C8), 56.4 (C3 ),
47.9 (C3), 44.5 (C15), 46.5 (C3), 43.1 (C15), 39.7 (m,
CH2N), 37.5 (C6), 35.3 (CH3N), 28.7 (Me BOC), 23.3
(CH3 Ac4), 22.1 (C14), 21.7 (C17), 14.8 (C18), 10.5 (C19);
HRMS (ESI+): (C139H126O30N4) m/z calcd: 1263.5096;
found: 1263.5084.
5.4.6.
20-O-[N,N0-Dimethyl-N-benzyl-4-(ꢀ-D-glucopyr-
anosiduronate)-3-aminobenzyloxycarbonylethylenediamin]-
docetaxel (16). To 110 mg (0.80 mmol) of the amine 15
dissolved in 5 mL of anhydrous CH3CN and 0.5 mL of
pyridine, cooled to 0 ꢀC, 1 mL of the HF–Pyridine
complex was added dropwise. The mixture was allowed
to reach rt and stirring was pursued for 48 h. The reac-
tion was not completed, as compounds of intermediary
polarity which showed partial desilylation, were isolated
and characterized by mass spectrography. The mixture
of compounds was taken up, dissolved in 5 mL of THF
dried over 4 A molecular sieves, then 0.1 mL (1.5 equiv)
of NEt3/3HF were added. After 4 h, NEt3 was added
and the medium was concentrated and purified by
chromatography (eluent CH2Cl2–MeOH 95/5!9/1)
affording 22 mg (20%) of the desilylated compound 16
as an oil. As the problems encountered during the
reaction and the purification of the final product did
not allow full characterization of 16, only the spec-
troscopic mass analysis is indicated; m/z (FAB+ MB)
1353.9 M+H+.
5.4.8. 2-Nitro-4-hydroxymethylphenol (6). A solution of
4-hydroxy-3-nitro-benzaldehyde (200 mg, 1.19 mmol) in
methanol (20 mL) was cooled to 0 ꢀC; then NaBH4 (100
mg) was added. After 4 h at rt, the mixture was quen-
ched with 50 mL water and the solvent was evaporated.
After extraction with 3Â50 mL EtOAc and drying over
Na2SO4, the residue was purified by chromatography
(eluent CH2Cl2–MeOH 97/3), 141 mg (70%) 6 were
isolated as yellow needles. Mp 142–144 ꢀC 1H NMR
(CDCl3) d 10.56 (sl, 1H, OH), 8.11 (d, 1H, J=2.2 Hz,
H3), 7.60 (dd, 1H, J=8.6 and 2.2 Hz, H5), 7.16 (1H, d,
J=8.6 Hz, H6), 4.69 (s, 2H, CH2); 13C NMR (CDCl3):
154.5(Cq–O), 136.3 (C5), 135.6 and 133.2 (Cq), 123.0
(C3), 120.2 (C6), 63.6 (CH2); m/z [CI+NH3] 170
[M+H]+; HRMS (C7H10NO2) m/z calcd: 170.0453,
found: 170.0459.
5.4.9. 2-Amino-4-hydroxymethylphenol (7). Method 1: A
solution of 4-hydroxy-3-nitro-benzaldehyde 17 (205 mg,
1.23mmol) in methanol (20 mL) was hydrogenated
during 2 h in the presence of 10% Pd/C (206 mg). After
filtration over Celite 545 and chromatography (eluent
CH2Cl2–MeOH/95-5), 60 mg (25%) 7 were isolated as a
brown powder.
5.4.7. 20-O-[N,N0-Dimethyl-N-4-(ꢀ-D-glucopyranosiduro-
nate)-3-aminobenzyloxycarbonylethylenediamin]docetaxel
(5). Method 1: 18 mg (13 mmol) of the benzyl compound
16 were dissolved in 5 mL of pure ethanol in the pre-
sence of 60 mg of Pd/C 10%, then stirred for 24 h under

E. Bouvier et al. / Bioorg. Med. Chem. 12 (2004) 969–977
977
Method 2: A solution of nitrophenol 6 (141 mg, 0.833
mmol) in methanol (20 mL) was hydrogenated during 4
h in the presence of 10% Pd/C (96 mg). After filtration
over Celite 545 and chromatography (eluent CH2Cl2–
MeOH/95-5), 47 mg (40%) 7 were isolated.
4. Kingston, D. G. I. Trends Biotechnol. 1994, 12, 222.
5. Rodrigues, M.; Carter, P.; Wirth, C.; Mullins, S.; Lee, A.;
Blackburn, B. Chem. Biol. 1995, 2, 223.
6. Mandai, T.; Okumoto, H.; Hara, K.-j.; Mikuni, K.; Hara,
K.-z., Hiroki, H. Taxoid derivative and method of
producing thereof. Patent EP0882732 A 19981209 1998,
Japan.
7. Mandai, T.; Okumoto, H.; Oshitari, T.; Nakanishi, K.;
Mikuni, K.; Hara, K.-j.; Hara, K.-z.; Iwatani, W.;
Amano, T.; Nakamura, K.-I.; Tsuchyia, Y. Heterocycles
2001, 54 (2), 561.
8. Nikolakakis, A.; Haidara, K.; Sauriol, F.; Mamer, O.;
Zamir, L. O. Bioorg. Med. Chem. 2003, 11 (7), 1551.
9. Yamaguchi, T.; Harada, N.; Ozaki, K.; Arakawa, H.;
Oda, K.; Nakanishi, N.; Tsujihara, K.; Hashiyama, T.
Bioorg. Med. Chem. Lett. 1999, 9 (12), 1639.
10. Jung, M. Mini Rev. Med. Chem. 2001, 1 (4), 399.
11. de Groot, F. M. H.; Damen, E. W. P.; Scheeren, H. W.
Curr. Med. Chem. 2001, 8 (9), 1093.
Mp 142–144 ꢀC 1H NMR (CD3OD) d 6.75 (d, 1H,
J=1.7 Hz, H3), 6.66 (dd, 1H, J=8.0 Hz, H6), 6.58 (d,
1H, J 8.0 Hz and J=1.7 Hz, Hs), 4.42 (s, 2H, CH2); 13
C
NMR (CD3OD) d 145.6 (Cquaternary-O), 135.6 and 133.8
(Cquaternary), 119.2 (C5), 116.5 (C3), 115.1 (C6), 61.3
(CH2); m/z [CI+NH3] 140 [M+H]+; HRMS
(C7H10O2N) m/z calcd: 140.0712, found: 140.0710.
Acknowledgements
This work was partially supported by a grant 5544 to
E.B. from the ARC (Association pour la Recherche sur
le Cancer). We are grateful to Laboratoires Servier for
performing the cytotoxic measurements.
12. Bosslet, K.; Czech, J.; Hoffmann, D. Tumor Targeting
1995, 1, 45.
13. Bouvier, E.; Thirot, S.; Schmidt, F.; Monneret, C. Org.
Biomol. Chem. 2003, 1, 3343.
14. Lougerstay-Madec, R.; Florent, J.-C.; Monneret, C.;
Nemati, F.; Poupon, M.-F. Anti-Cancer Drug Des. 1998,
13 (8), 995.
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