A new paclitaxel prodrug for use in ADEPT strategy

Article abstract of DOI:10.1039/b306236h

A new paclitaxel prodrug intended for use in Antibody-Directed Prodrug Therapy (ADEPT) or Prodrug Monotherapy (PMT) has been prepared. This prodrug was originally designed to be activated into the drug by human β-glucuronidase. In order to enhance the liberation rate of paclitaxel, an elongated spacer system including a nitro-aromatic derivative and a N,N′-methylethylenediamine was incorporated between the sugar moiety and the drug. Indeed, this new prodrug proved to be activated significantly faster than a former paclitaxel prodrug containing a conventional spacer.

Full text of DOI:10.1039/b306236h

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A new paclitaxel prodrug for use in ADEPT strategy
Emmanuel Bouvier, Sylvie Thirot, Frédéric Schmidt* and Claude Monneret
UMR 176 CNRS-Institut Curie, Section Recherche 26 rue d’Ulm, 75248 Paris Cedex 05,
France. E-mail: Frederic.Schmidt@curie.fr; Fax: ϩ33-1/42346631; Tel: ϩ33-1/42346664
Received 3rd June 2003, Accepted 28th July 2003
First published as an Advance Article on the web 29th August 2003
A new paclitaxel prodrug intended for use in Antibody-Directed Prodrug Therapy (ADEPT) or Prodrug
Monotherapy (PMT) has been prepared. This prodrug was originally designed to be activated into the drug by
human β-glucuronidase. In order to enhance the liberation rate of paclitaxel, an elongated spacer system including
a nitro-aromatic derivative and a N,NЈ-methylethylenediamine was incorporated between the sugar moiety and
the drug. Indeed, this new prodrug proved to be activated significantly faster than a former paclitaxel prodrug
containing a conventional spacer.
Introduction
Despite an increasing number of potent anticancer agents,
lack of selectivity is still a major drawback in clinical oncology.
The use of monoclonal antibodies (MAb) as well as tumor-
associated enzymes to efficiently target tumor cells represents a
promising way forward.1–4
In the Antibody-Directed Enzyme Prodrug Therapy
(ADEPT),5–15 an enzyme linked to a MAb directed at a tumor
The prodrugs of the second class were designed for
enhancing drug selectivity (Fig. 1).
site catalyses the activation of a prodrug into the corresponding
cytotoxic drug. The first step involves the targeting of the
desired enzyme to tumor cells by means of antibody–antigen
recognition in the form of an enzyme–MAb conjugate or a
fusion protein. Then, following tumor localisation and systemic
clearance, a non-active prodrug is administered, which, after
enzymatic cleavage, will release the active compound only at
tumor cells. A higher level of cytotoxic agent in tumor tissue
can be achieved via this concept, as shown for doxorubicin.16–17
The success of a rational approach strongly depends upon
several factors. These include the kind of cytotoxic agent, the
choice of the MAb and the enzyme. In order to reduce hetero-
and immunogenicity, Bosslet and co-workers were the first to
build a fusion protein made of human ß--glucuronidase
combined with a humanised anti-CarcinoEmbryonic Antigen
(CEA) MAb BW431.18–21 Furthermore, following histo-
chemical studies, ß--glucuronidase was found in the inter-
cellular medium of necrotic areas.22–23 This fact allows the
potentially useful Prodrug Mono Therapy (PMT) protocol,
which is the use of prodrugs alone without any externally added
enzyme.24
Fig. 1 General structure of prodrugs.
The first ADEPT-designed paclitaxel prodrug was reported
by Rodrigues et al.40 It required a ß-lactamase activation
followed by self-immolation of an aminobutyryl linker. How-
ever, it suffered from a poor release rate, since completion of the
self-immolation was reached after 16 h. During this time, the
intermediate can escape from the tumor site, which is incon-
sistent with selective targeting. It also has the potential draw-
back of immunogenicity already encountered with exogenous
enzymes.41
Scheeren and co-workers designed two kinds of paclitaxel
prodrugs. The first were devised for ADEPT, including a
spacer (aminobutyrate or p-aminophenylacetate) connected to
ß-glucuronic acid via a carbamate function, and linked to the
2Ј-hydroxy of paclitaxel by an ester bond.42 Although release
of paclitaxel occurred, problems of fast non-specific hydrolysis
were encountered in buffer solution (pH = 6.8), and this
may be reinforced in plasma, since it is well known that esters of
paclitaxel suffer from low chemical stability due to circulat-
ing enzymes.33 They also made prodrugs based on tumor-
associated protein plasmin activation (Prodrug MonoTherapy
strategy).43 For this purpose, a tripeptide was linked, via
Paclitaxel 1 (Taxol®)25 has been introduced several years
ago in cancer chemotherapy due to its remarkable antitumor
activity, especially against ovarian, breast and lung cancers,26
for which it has been approved by the FDA. It shows a unique
mechanism of action by promoting and stabilizing micro-
tubules from tubulin.26–28 The major drawbacks in the use of
paclitaxel are its poor selectivity resulting in a low therapeutic
index and side-effects such as myelosuppression, mucositis or
peripheral neuropathy.29–30 Moreover, due to its low water-solu-
bility, it is administered as a solution in Chremophor EL®, a
polyoxyethylated castor oil, which may be responsible for
hypersensitivity reactions.29–31 For all these reasons, paclitaxel is
an attractive candidate for ADEPT and/or PMT strategies, as a
prodrug could solve the problems of toxicity while enhancing
both selectivity and solubility.
an amide bond, to
a spacer (p-aminobenzyl, ethylene-
diamine or a combination of both) which in turn was con-
nected to the 2Ј-hydroxy of paclitaxel by a carbonate or a
carbamate function. Recently they designed the first bio-
reductive-activated prodrugs of paclitaxel with a carbonate
bond at the 2Ј-position.
To date, there are two classes of paclitaxel prodrugs; the first
included the addition of ionic groups that could be hydrolysed
by circulating enzymes such as esterases or phosphatases.32–39
They still lack selective release.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
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Scheme 1 Paclitaxel liberation.
For our part, we have previously synthesised the three-com-
ponent paclitaxel prodrug 2 for ADEPT by linking a glucuronyl
derivative of 2-N-methylamine-4-nitrophenol to the 2Ј-hydroxy
group through a carbamate linkage.45 The prodrug displayed a
relevant detoxification (about 700-fold) and a high stability in
buffer and plasma (half-life of 45.5 4 hours in the latter) with
no detected free drug. However, even in the presence of a rel-
ative high concentration of enzyme, the enzymatic hydrolysis
half-life was still 115 min. On the basis of the already known
structure of human ß--glucuronidase,46 molecular modelling
calculations indicated that this might be due to steric hindrance.
HMR 1826,49 was used as the starting material (Scheme 2). Due
to the sensitivity of the paclitaxel skeleton under both acidic or
basic conditions,50–51 the acetyl protecting groups had to be
replaced by tert-butyldimethylsilyl ethers and the methyl ester
by a benzyl ester; both types of protecting groups have been
shown to be compatible with our synthetic scheme.45 The
second expected difficulty was the sequential manipulation of
the two amine groups of ethylene diamine in order to link one
of them to paclitaxel and the other one to the moiety contain-
ing the glucuronate.
Our approach started with the reduction of aldehyde 6 to
alcohol 7 by sodium borohydride. The glucuronate 7 contained
three different kinds of hydroxy groups, one primary benzylic
alcohol, three secondary alcohols protected by acetate, and one
carboxylic acid protected as a methyl ester. Our aim was to
obtain a benzyl ester with silylated secondary hydroxy groups
and a free primary alcohol. Our first trial was based on the fact
that it should be possible to selectively deprotect a primary
silylated hydroxy group in the presence of secondary ones.
Treatment of compound 7 with sodium hydroxide in acetone
led to the fully deprotected glucuronide 8. Exhaustive silylation
using TBSOTf in pyridine, followed by esterification of the
carboxylic function with benzyl alcohol using DCC, gave
intermediate 9. However, the crucial selective desilylation of the
benzylic alcohol turned out to be very challenging. Despite
many trials (TBAF in THF, HF–pyridine in THF, adjustment
of reaction temperature and duration) such selectivity could
not be cleanly achieved in the correct yield.
Moreover, the poor yield observed in the preparation of 9,
led us to reconsider our synthetic scheme and use a stepwise
process for the deprotection/reprotection steps of the sugar
moiety,52 in order to differentiate the hydroxy on the benzylic
position prior to the protection of the sugar hydroxy groups.
Starting from compound 7, a selective deacetylation giving
ester 10 was achieved using NaOMe in dry MeOH. A fully
silylated derivative 11 was then obtained by reaction with
TBSOTf. The switch from the methyl ester to a benzyl ester was
attempted according to a described method by saponification
and reesterification,52 but was unsuccessful on 11 in our hands.
As selective desilylation of 9 proved to be also challenging, this
approach was consequently abandoned and the introduction of
different silyl groups was undertaken.
A selective silylation of the primary benzyl hydroxy could be
effected using TPSCl, and then the remaining secondary
hydroxy groups were protected using TBSOTf. The switch from
the methyl ester to a benzyl ester by saponification and reesteri-
fication52 was unsuccessful on 13 in our hands. A good altern-
ative turned out to be a transesterification procedure using
Ti(OEt)4 and benzyl alcohol,53 which had the advantage of
avoiding any free acid handling. So the fully protected
compound 14 was obtained from 6 in 44% overall yield.
The design for preparing a glucuronide-containing prodrug
of paclitaxel with better enzymatic kinetics led us to choose a
linker that would move the glucuronic moiety away from the
taxane core. We envisioned using two spacers linked together by
a carbamate function such as a para-hydroxybenzyl alcohol
linked to N, NЈ-dimethyl ethylenediamine; this double spacer
has proved efficient with a nitrogen mustard.47 Interestingly,
during the course of our work, a plasmin activated prodrug was
published by Scheeren et al.48 incorporating a double spacer.
The first spacer was an aniline derivative and the second N, NЈ-
dimethyl ethylenediamine derivative linked through carbamate
functionalities. This kind of spacer was shown to be effective
for the liberation of paclitaxel by plasmin.48
The 2Ј position of paclitaxel was selected for derivatisation as
it is known that its modification leads to a dramatic loss of
cytotoxic activity.32–33 Moreover this position is more reactive
than the hindered 7-hydroxy and the tertiary 1-hydroxy.35 Thus
the glucuronide-based paclitaxel prodrug 3, which was designed
for the liberation of the free drug as depicted in Scheme 1, was
synthesized and tested in vitro. This is the subject of the present
report.
Results and discussion
Synthesis
The 3-nitro-4-hydroxybenzaldehyde glucuronide 6, previously
reported during the synthesis of the doxorubicin prodrug
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
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Scheme 2 Prodrug synthesis (first part).
The next step was the introduction of the diamine tether.
Biological results
Using a reported procedure (1 eq. TBAF–AcOH),54 selective
desilylation of the benzyl hydroxy was cleanly performed to
afford 15. Next this hydroxy was activated as a p-nitro-
phenylchlorocarbonate, and 16 was condensed with the mono-
protected diamine 17 (Scheme 3).55 This afforded the benzyl
carbamate 18. It is noteworthy that an improved reaction yield
was achieved using a one-pot procedure by avoiding the iso-
lation of the sensitive activated carbonate. Dry 3 M HCl in
EtOAc removed the BOC protecting group giving the depro-
tected amine as a hydrochloride salt 19. No attempts were made
to isolate the free amine, which would obviously cyclise very
rapidly on the carbamate function with liberation of the start-
ing free benzylic alcohol. The chlorocarbamoylation of the
resulting amine by phosgene cleanly led to the activated carb-
amoyl chloride 20, which was ready for the coupling step with
paclitaxel. The amine liberated from its salt by triethylamine
reacted fast enough with phosgene to avoid the intermolecular
cyclisation. Compound 20 proved to be stable enough to be
purified by chromatography.
Remarkably, the coupling reaction between compound 20
and paclitaxel 1 was achieved regioselectively in good yield
(87%) by using a large excess of DMAP. The three silyl groups
of the resulting compound 21 were then easily removed with an
excess of the HF–pyridine complex in dry acetonitrile. Finally,
reductive cleavage of the benzyl ester needed a large excess of
cyclohexadiene with palladium-on-charcoal and gave the prod-
rug 3, which was isolated in moderate yield (24%). Moreover, it
must be noted that the nitro group was reduced to an amine
under these conditions. This was rather surprising compared
with the previous behaviour of such a nitro-containing spacer
used to obtain prodrug 2,45 since this procedure allowed us to
keep the nitro group intact.
Our aim was to obtain a prodrug more suitable than the for-
mer45 for the in vivo tests, especially concerning the enzymatic
cleavage. The spacer was modified and our choice was a cascade
of two linkers (Scheme 1), an aromatic part and a derivative of
diaminoethylene. Another difference to the former prodrug was
the presence of an amino group instead of a nitro group on the
aromatic spacer.
Prodrug 3 was tested in vitro for detoxification, stability and
kinetics of enzymatic cleavage.
A requirement for ADEPT strategy is that a relatively non-
cytotoxic prodrug releases a cytotoxic drug. Cytotoxicity meas-
urements were done on L1210 cell lines. IC50 of the prodrug
(1500 nM) was lower than that of the drug (9.8 nM) by a factor
of 153. This value is approximately of the same magnitude as
the values found for other stable paclitaxel prodrugs (Table 1).
The cytotoxicity ratio between the prodrug and the free drug
depends on the cell line, the stability of the prodrug, and its
structure. As a general rule, the more hydrophilic prodrugs are
less prone to crossing the cell membranes and reaching their
cellular targets. To date, the best detoxification for a paclitaxel
prodrug has been reached by plasmin activated prodrugs. These
compounds have two amine nitrogens and are used as their bis-
chlorhydrate. Usually, it can be considered that a factor of at
least 100 is needed for an ADEPT strategy; this is the case for
prodrug 3, even if the value is slightly lower than for prodrug 2
(722). One explanation could be the relative log P (octanol/
water partition coefficient) values of paclitaxel and both prod-
rugs 2 and 3. They were calculated using the ClogP module
from ChemDraw®.
Paclitaxel 1: 4.7308
Prodrug 2: 3.5745
Prodrug 3: 4.062
Prodrug 2 being more hydrophilic than 3 is also the less
cytotoxic prodrug.
Consistent with these figures is the observation that prodrug
3 is less water-soluble than the former; so, all measurements
were conducted with a concentration of 30 µg mLϪ1 (22.9 µmol)
instead of 250 µg mLϪ1 for prodrug 2.
There is still a precedent with prodrugs including a spacer
bearing ortho amino groups like NEt2, NHCOCH3, NHCOCF3
in the literature,56 but to date, not with NH2. It remains interest-
ing to know the behaviour of this latter prodrug, and thus, to
study its use as prodrug in an ADEPT or PMT strategy.
In summary, prodrug 3 was obtained in 16% overall yield
relative to paclitaxel.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
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from a peak corresponding to the prodrug 3, three peaks
appeared successively. The last one was identified by HPLC
comparison with the free paclitaxel. The first one corresponded
to the spacer 4. The second was an intermediate of the reaction,
since it first appeared then disappeared progressively. For
identification of this instable compound, we performed LC/MS
experiments. We could obtain the mass spectra of this com-
pound and characterise it as being the paclitaxel still linked to
the ethylenediamino derivative 23.
The half-life of intermediate 23 was measured by using an
excess of enzyme (50 µg mLϪ1, 240 units mLϪ1) in order to
cleave the glucuronic moiety almost instantaneously. The curve
corresponding to 23 gave a half-life of 13 minutes (Fig. 2).
Scheeren et al observed the same intermediate starting from a
different prodrug and measured a T1/2
of 47 minutes.48 The
cycl
difference could be explained by the concentration of starting
prodrug and the nature of buffer and enzyme in the solution.
Cleavage of prodrug 3 was followed by using an enzymatic
concentration of 2.5 µg mLϪ1 (12 units mLϪ1) (Fig. 3).
Fig. 2 Disappearance of intermediate 23. Prodrug 3 concentration:
30 µg mLϪ1 (22.9 µmol) Enzyme concentration: 240 units mLϪ1
.
Fig. 3 Enzymatic cleavage of prodrug 3. Prodrug 3 concentration:
30 µg mLϪ1 (22.9 µmol) Enzyme concentration: 12 units mLϪ1
.
Scheme 3 Prodrug synthesis (second part).
Two facts were very different from the data of the former
prodrug 2.45 The cleavage required a much lower β--glucuro-
nidase concentration, but an intermediate species was observed.
For prodrug 3 (30 µg mLϪ1), the half-life of the prodrug was
10 minutes for an enzyme concentration of 12 units mLϪ1. For
comparison, we measured the kinetics of cleavage of prodrug 2
with the same concentration of prodrug. In the latter case, an
Stability of prodrug 3 was measured by HPLC (UV detec-
tion) in a phosphate buffer at 37 ЊC (pH 7.2). The prodrug
proved to be stable during a 24-hour run, and no paclitaxel was
liberated from the prodrug during this time.
Enzymatic cleavage was studied using E. coli β--glucuron-
idase at 37 ЊC, at pH 7.2 in 0.02 M phosphate buffer. Starting
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
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Table 1 Comparison of cytotoxicities for paclitaxel prodrugs
References
Activation enzyme
Cytotoxicity ratioa
Cell lines
ClogP (from ChemDraw®)
40
42
β-lactamase
β--glucuronidase
10
95
1.1
135
SK-BR-3
OVCAR-3
2.9236
3.3781
2.7830
3.2735
43
Plasmin
1100–20000 (average 8000)
MCF-7
4.9453–7.1763 (calculations on
neutral
species, not on the chlorhydrates)
EVSA-T
WIDR
IGROV
M19
A498
H226
H226
MCF7
EVSA-ϩT
WIDR
IGROV
M19
44
48
Bioreduction
Plasmin
1–462
4.8784–7.5460
A498
MCF-7
1.6–213
6.7667–7.0700 (calculations on
neutral
species, not on the chlorhydrates)
EVSA-T
WIDR
IGROV
M19
A498
H226
LoVo
45 (compound 2)
β--glucuronidase
722
3.5745
a Measured as IC50(prodrug)/IC50(Free paclitaxel).
enzyme concentration of 240 units mLϪ1 was needed to obtain
a half-life of 5.5 hours. At this enzyme concentration, cleavage
of prodrug 3 was almost instantaneous. The difference can be
explained by steric interactions. In prodrug 2, the glucuronate
and the paclitaxel are in ortho positions on the aromatic spacer,
giving a bad steric interaction for the approach of the enzyme
towards the glycosidic bond. In prodrug 3, the glucuronate and
paclitaxel are in para positions, so the glycosidic bond is much
more accessible and the kinetics are much faster.
These results showed also that good kinetic results can be
observed with an amino aromatic spacer instead of a nitro
aromatic one. It was known that spacers with substituted amino
groups like NEt2, NHCOCH3, NHCOCF3 were compatible,56
but to our knowledge it is the first time that it has also been
proved for a NH2 spacer with a prodrug suitable for an ADEPT
strategy.
were recorded on a Nermag R 10-10C spectrometer. Electro-
spray ionisation mass spectra (ESI-MS) were acquired on an
API3000 LC-MS-MS spectrometer, and HR-MS were recorded
on a LCTOF (Micromass) Spectrometer. Chromatography was
conducted over silica gel (Merck 60 (230–400 Mesh)).
For the NMR description, the following numeration
was chosen: “a” for aromatic “g” for glucuronic acid, “e” for
ethylenediamine, and “p” for paclitaxel (see Fig. 4).
If there was any ambiguity in 1D-NMR experiments, 1H and
13C assignments were determined by 2-D-NMR experiments
(COSY, HMQC).
Methyl
{[2-nitro-4-(hydroxymethyl)phenyl]-2,3,4-tri-O-
acetyl-ꢀ-D-glucopyranosid}uronate 749. Aldehyde 6 (1.45 g, 3
mmol) in CHCl3 (20 mL) and iPrOH (5 mL) was degassed
under argon. After addition of 2.25 g of silica, the medium was
cooled to 0 ЊC and NaBH4 (230 mg, 12 eq.) was added portion-
wise. After 1 h at 0 ЊC, the solution was allowed to reach room
temperature. The solution was filtered on Celite 545 and the
pad washed with CH2Cl2 (20 mL). The filtrate was concentrated
under vacuum, and the compound was recrystallized from
ethanol, affording 1.19 g (82%) of the reduction compound as a
microcrystalline powder; mp 175 ЊC (from EtOH); (Found: C,
49.75; H, 4.81; N, 2.96. C20H23NO13 requires C, 49.49; H: 4.78;
N, 2.89%); δH (DMSO-d6): 7.80 (1H, d, J 1.5 Hz, a3), 7.62 (1H,
dd, J 8.3 and 1.3 Hz, a5), 7.38 (1H, d, J 8.7 Hz, a6), 5.71 (1H, d,
J 7.8 Hz, g1), 5.45 (2H, m, OH, g3), 5.11 (2H, m, g2, g4), 4.72
(1H, d, J 9.9 Hz, g5), 4.51 (2H, s, CH2OH); 3.64 (3H, s, CH3O);
2.01 (9H, s, CH3C(O)); δC (DMSO-d6): 169.5, 169.3 and 168.8
(MeC(O)), 167.0 (CO2Me), 146.9, 140.2 and 138.6 (Cqaromatics),
132.0 (a5), 122.3 (a3), 117.7 (a6), 98.1 (g1), 71.0 (g5), 70.8 (g3),
70.0 (g2), 68.7 (g4), 61.3 (CH2OH), 52.7 (CH3O), 20.6
(CH3C(O)); m/z [CI ϩ NH3] 503 (M ϩ NH4)ϩ.
Conclusion
In conclusion, we have prepared a new paclitaxel prodrug,
which contains two self-immolative spacers. Such a prodrug is
endowed with acceptable reduced cytotoxic effect and activated
by β-glucuronidase to release paclitaxel under conditions com-
patible with in vivo experiments. It is also noteworthy that for
this class of glucuronic acid-containing prodrugs this work
represents the first example of a spacer bearing an amino group
in the ortho position of the phenol involved in the glycosidic
linkage.
Experimental
General
Melting points (mp) were measured using an Electrothermal
digital melting point apparatus and are uncorrected. 1H-NMR
(300 MHz) and 13C-NMR (75 MHz) spectra were recorded on
Bruker AC 300 spectrometer – chemical shifts δ in ppm and J in
Hz. Chemical ionisation CI-MS (NH3) or FAB mass spectra,
Benzyl {[2-nitro-4-(O-tert-butyldimethylsilylmethyl)phenyl]-
2,3,4-tri-(O-tert-butyldimethylsilyl)-ß-D-glucopyranosid}uronate
9. Benzyl alcohol 7 (1.6 g, 3.3 mmol) was dissolved in 40 mL of
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
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Fig. 4 NMR attributions for prodrug 3.
acetone at 0 ЊC. A molar solution (25 mL) of aqueous sodium
hydroxide was added dropwise and stirring was continued
for 5 min. The medium was then neutralized with 1 M HCl,
evaporated and the compound was purified on a silica gel
column (eluent CH3CN–H2O, 7 : 3). The recuperated com-
pound was dissolved in distilled pyridine (15 mL) in the
presence of DMAP (5.6 mg). After cooling to 0 ЊC, 15 mL of
TBSOTf were added dropwise to the medium under argon,
and the reaction was left for 18 h at room temperature. The
reaction mixture was then concentrated, the residue taken
up in toluene (120 mL) and the insoluble pyridinium triflate
salt filtered. The filtrate was washed with 20 mL of water,
decanted and dried over MgSO4. Finally the organic layer was
concentrated under reduced pressure. The residue was then
solubilized in anhydrous CH2Cl2 (10 mL) under argon, in
the presence of 200 mg of DMAP. The mixture was cooled to
0 ЊC, benzyl alcohol (0.4 mL, 1 eq.) was added, then DCC
(770 mg, 1 eq.) as a solution in 3 mL of anhydrous CH2Cl2,
and stirring was maintained overnight. The solution was con-
centrated, taken up with cyclohexane, the insoluble urea was
filtered, and the compound was purified twice by chromato-
graphy (CH2Cl2–CyH, 5 : 1 then Tol–MeOH, 1 : 0 97.5 :
2.5), finally leading to 230 mg (7%) of a yellow oil. δH (CDCl3):
7.73 (1H, d, J 2.1 Hz, a3), 7.38 (1H, dd, J 8.7 and 2.1 Hz,
a5), 7.30 (5H, m, Bn), 7.08 (1H, d, J 8.7 Hz, a6), 5.52 (1H,
d, J 5.7 Hz, g1), 5.11 (2H, s, Bn), 4.65 (2H, s, CH2OSi), 4.44
(1H, d, J 1.8 Hz, g3), 4.37 (1H, m, g4), 4.03 (1H, d, J 5.6 Hz,
g2), 3.82 (1H, d, J 3.7 Hz, g5), 0.90, 0.87, 0.83 and 0.80
(9H, s, (CH3)3C), 0.14 (3H, s, CH3Si), 0.11 (3H, s, CH3Si), 0.03
(3H, s, CH3Si), Ϫ0.02 (3H, s, CH3Si), 0.07 (6H, s, CH3Si);
δC (CDCl3): 168.6 (CO2), 149.1 and 140.2 (Cqaromatics), 135.3
(2 CqaromaticsC), 131.4 (a5), 128.6, 128.4 and 128.4 (CHaromatics),
123.1 (a3), 116.9 (a6), 99.7 (g1), 78.8 (g3), 77.0 (g5), 75.2 (g2),
72.3 (g4), 67.0 (CH2Ph), 63.6 (CH2OTBS), 26.0, 25.9 and 25.8
((CH3)3C), 18.5, 18.1 and 18.0 (Me3CSi), Ϫ4.4, Ϫ4.5, Ϫ4.6,
Ϫ4.7, Ϫ4.9, Ϫ5.0 and Ϫ5.2 (CH3Si); m/z [CI ϩ NH3] 909
(M ϩ NH4)ϩ.
Methyl {[2-nitro-4-(O-tert-butyldimethylsilylmethyl)phenyl]-
2,3,4-tri-(O-tert-butyldimethylsilyl)-ß-D-glucopyranosid}uronate
11. To a solution of the deacetylated compound 10 (470 mg, 1.3
mmol) in 10 mL DMF were added 875 mg (5.8 mmol) of
TBSCl, 792 mg (11.6 mmol) of imidazole and 79 mg (0.65
mmol) of DMAP. The solution was heated at 80 ЊC for 48 h,
and allowed to return to room temperature. 5 mL of anhydrous
pyridine and 5 g (18.9 mmol) of TBSOTf were added dropwise
to the solution. After stirring at room temperature for one night
and evaporation, the residue was taken up in toluene and the
insoluble pyridinium triflate was removed by filtration. After
washing with water, brine, and drying over MgSO4, the com-
pound was purified on silica gel (eluent CH2Cl2). An oil was
obtained, which crystallized; after recrystallization from etha-
nol, 900 mg (85%) of the methyl ester were collected as white
crystals; mp 90–91 ЊC (from EtOH) (Found; C, 55.62; H, 9.01;
N, 1.74; Si, 13.85. C38H73NO10Si4 requires C, 55.91; H, 9.01; N,
1.72; Si, 13.76); δH (CDCl3): 7.79 (1H, d, J 2.1 Hz, a3), 7.47 (1H,
dd, J 8.7 and 2.1 Hz, a5), 7.12 (1H, d, J 8.7 Hz, a6), 5.58 (1H, d,
J 5.9 Hz, g1), 4.70 (2H, s, CH2O), 4.51 (1H, d, J 1.4 Hz, g3),
4.39 (1H, d, J 6.0 Hz, g4), 4.04 (1H, d, J 5.9 Hz, g2), 3.84 (1H,
d, J 3.8 Hz, g5), 3.72 (3H, s, CH3O), 0.94 (18H, s, CH3)3CSi),
0.87 and 0.83 (9H, s, (CH3)3CSi), 0.17–0.09 (18H, m, CH3Si),
0.07 and 0.00 (3H, s, CH3Si); δC (CDCl3): 169.6 (CO2), 149.1,
140.0 and 135.3 (Cqaromatics), 131.5 (a5), 123.2 (a3), 116.4
(a6), 99.4 (g1), 79.0 (g3), 77.0 (g2), 75.4 (g5), 72.5 (g4), 63.6
(CH2O), 52.5 (CH3O), 26.0, 25.9 (2C) and 25.8 ((CH3)3C), 18.5,
18.1 (2C) and 18.0 (Me3CSi), Ϫ4.4, Ϫ4.5, Ϫ4.6, Ϫ4.6, Ϫ4.7–
5.0, Ϫ5.1 and Ϫ5.2 (2C) (CH3Si); m/z [CI ϩ NH3] 833
(M ϩ NH4)ϩ.
Methyl [2-nitro-4-(O-tert-butyldiphenylsilylmethyl)phenyl-ß-
D-glucopyranosid]uronate 12. To a solution of 400 mg (1.11
mmol) of methyl [2-nitro-4-(hydroxymethyl)phenyl-β--gluco-
pyranosid]uronate 10 and 173 mg (2.3 eq.) of imidazole in 10
mL of anhydrous DMF, 0.49 mL (1.7 eq.) of TBDPSCl were
added dropwise; after 3 h, an extra quantity of 0.1 mL was
added. After stirring for 5 h at room temperature, 30 mL of
water were added to the medium, which was then extracted with
4 × 20 mL of EtOAc. After drying over MgSO4 and concen-
tration under high vacuum, the compound was purified by
chromatography on a silica gel column (eluent CH2Cl2–MeOH,
97.5 : 2.5), giving 568 mg (85%) of the monosilylated com-
pound as a white foam; mp 60–61 ЊC; δH (CDCl3): 7.68 (1H, d,
J 1.6 Hz, a3), 7.61 (4H, dd, J 7.5 and 1.4 Hz, Ho SiPh2), 7.42
Methyl
[2-nitro-4-(hydroxymethyl)phenyl-ꢀ-D-glucopyran-
osid]uronate 10. To a solution of the triacetylated benzyl
alcohol 7 (2 g, 4.12 mmol) in 40 mL of methanol cooled to
Ϫ15 ЊC, 84 mg (0.5 eq.) of NaOMe were added. The mixture
was allowed to reach room temperature overnight. The medium
was then neutralized with Dowex 50X8–200 resin and the
compound was purified by chromatography on a silica gel
column (eluent CH2Cl2–MeOH, 9 : 1). After recrystallization
from ethanol, the deacetylated compound was obtained as a
white powdery solid (958 mg, 65%); mp 175 ЊC; (Found:
C, 47.05; H, 4.81; N, 3.94. C20H23NO13 requires C, 46.80; H:
4.77; N, 3.90%); δH (CD3OD): 7.80 (1H, d, J 2.0 Hz,
a3), 7.56 (1H, dd, J 8.7 and 2.1 Hz, a5), 7.36 (1H, d, J 8.7 Hz,
a6), 5.16 (1H, d, J 7.2 Hz, g1), 4.61 (2H, s, CH2OH), 4.08 (1H,
d, J 9.7 Hz, g5), 3.76 (3H, s, CH3O), 3.64 (1H, t, J 9.4 Hz, g4),
3.56–3.46 (2H, m, g2, g3); δC (CD3OD): 171.1 (CO2), 150.3,
142.7 and 138.5 (Cqaromatics), 133.5, 124.6 and 119.3 (CHaromatics),
103.0 (g1), 77.5 (g2 or g3), 77.2 (g5), 74.8 (g2 or g3), 73.1
(g4), 64.0 (CH2OH), 58.7 (CH3O); m/z [CI ϩ NH3] 377
(M ϩ NH4)ϩ.
(1H, dd, J 8.8 and 1.7 Hz, a5), 7.35 (6H, m, Hm, SiPh2), 7.26
p
(1H, d, J 8.7 Hz, a6), 5.17 (1H, s, OH), 5.07 (1H, d, J 6.8 Hz,
g1), 4.83 (1H, s, OH), 4.67 (1H, s, OH), 4.64 (2H, s, CH2O),
4.12 (1H, d, J 9.5 Hz, g5), 3.89 (1H, m, H4), 3.79 (2H, m, g2,
g3), 3.76 (3H, s, CH3O), 1.05 (9H, s, (CH3)3CSi); δC (CDCl3):
168.9 (CO2), 148.9, 140.1 and 136.1 (Cqaromatics), 135.1 (Co m
/
SiPh2), 132.8 (Cqaromatics SiPh2), 131.7 (a5), 129.9 (Cp
SiPh2), 127.8 (Co m SiPh2), 122.7 (a3), 118.1 (a6), 101.9
/
(g1), 75.2 (g2 or g3), 75.0 (g5), 72.7 (g2 or g3), 71.0 (g4), 64.0
(CH2), 52.7 (CH3O), 26.7 ((CH3)3C), 19.2 (Me3CSi); m/z [CI ϩ
NH3] 615 (M ϩ NH4ϩ) (Found: M ϩ NH4ϩ, 615.2369,
C30H39O10N2Si requires 615.2374); m/z [FAB ϩ NaI]: 620
(M ϩ Na)ϩ.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
3348

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Methyl {[2-nitro-4-(O-tert-butyldiphenylsilylmethyl)phenyl]-
2,3,4-tri-(O-tert-butyldimethylsilyl)-ß-D-glucopyranosid}uronate
13. Methyl [2-nitro-4-(O-tert-butyldiphenylsilylmethyl)phenyl-
ß--glucopyranosid] uronate 12 (568 mg, 0.95 mmol), pyridine
(6.25 mL) and DMAP (187.5 mg, 1.53 mmol) were dissolved in
50 mL of freshly distilled CH2Cl2, placed under argon and
cooled in an ice-bath. Then, TBSOTf (5.5 mL, 23.9 mmol) was
cautiously added dropwise. The reaction medium was allowed
to reach room temperature overnight. After evaporation, the
residue was taken up in toluene, the insoluble salt was filtered,
and the organic layer was washed with brine and dried over
MgSO4. After chromatography (eluent CH2Cl2), 775 mg (84%)
of a yellowish oil were obtained. δH (CDCl3): 7.77 (1H, d, J 2.1
Hz, a3), 7.69 (4H, m, Ho SiPh2), 7.48 (1H, dd, J 8.8 and 2.0 Hz,
concentration, the compound was purified on a silica gel col-
umn (eluent CH2Cl2–MeOH, 98 : 2), giving 198 mg (85%) of the
benzyl alcohol as a white solid; mp 111–112 ЊC; δH (CDCl3):
7.77 (1H, d, J 2.0 Hz, a3), 7.44 (1H, dd, J 8.7 and 2.1 Hz, a5),
7.31 (5H, m, Bn), 7.12 (1H, d, J 8.7 Hz, a6), 5.56 (1H, d, J 5.7
Hz, g1), 5.14 (2H, s, Bn), 4.66 (2H, s, CH2OH), 4.52 (1H, d,
J 1.5 Hz, g3), 4.41 (1H, m, g4), 4.05 (1H, d, J 5.6 Hz, g2), 3.85
(1H, d, J 3.6 Hz, g5), 2.03 (1H, se, OH), 0.91, 0.88 and 0.83 (9H,
s, (CH3)3CSiMe2), 0.16, 0.11, 0.06 and 0.00 (3H, s, CH3Si), 0.12
(6H, s, CH3Si); δC (CDCl3): 168.6 (CO2), 149.4, 140.1, 135.3 and
134.8 (Cqaromatics), 132.3 (a5), 128.6 and 128.4 (Co, Bn), 128.4
m
(Cp Bn), 123.8 (a3), 116.9 (a6), 99.6 (g1), 78.9 (g3), 76.8 (g5),
75.2 (g2), 72.2 (g4), 67.1 (CH2, Bn), 63.7 (CH2OH), 25.9 (2C)
and 25.8 ((CH3)3CSiMe2), 18.1 and 18.0 ((CH3)3CSiMe2), Ϫ4.4,
Ϫ4.5, Ϫ4.6, Ϫ4.7, Ϫ5.0 and Ϫ5.0 (CH3Si); m/z [CI ϩ NH3] 795
(M ϩ NH4)ϩ (Found: M ϩ NH4ϩ, 795.4096, C38H67O10N2Si3
requires 795.4104).
a5), 7.42 (6H, m, Hm, SiPh2), 7.15 (1H, d, J 8.7 Hz, a6), 5.61
p
(1H, d, J 5.8 Hz, g1), 4.73 (2H, s, CH2O), 4.53 (1H, d, J 1.6 Hz,
g3), 4.43 (1H, dd, J 3.6 and 1.6 Hz, g4), 4.10 (1H, d, J 5.8 Hz,
g2), 3.88 (1H, d, J 3.7 Hz, g5), 3.74 (3H, s, CH3O), 1.11 (9H, s,
(CH3)3CSiPh2), 0.96, 0.90 and 0.86 (9H, s, (CH3)3CSiMe2), 0.19
(6H, s, CH3Si), 0.17, 0.16, 0.10 and 0.04 (3H, s, CH3Si);
δC (CDCl3): 169.6 (CO2), 149.1, 140.1 and 134.9 (Cqaromatics),
Benzyl
[(2-nitro-4-(4-nitrophenoxycarbonyloxymethyl)-
phenoxy)-2,3-tert-butyldimethylsilyl)-ꢀ-D-glucopyranosid]-
uronate 16. Compound 15 (145 mg, 0.187 mmol) with one drop
of anhydrous pyridine was dissolved in 5 mL of anhydrous
CH2Cl2 and placed under argon. Then, 85 mg (2.3 eq.) of
4-nitrophenyl chloroformate were poured onto the mixture in
one portion. After leaving overnight at room temperature,
water was added and the organic layer was carefully washed
with a saturated NaHCO3 solution, and with H2O, then dried
(MgSO4). After chromatography on silica gel (eluent CH2Cl2),
95 mg (54%) of the expected activated carbonate were obtained
as a yellow oil. δH (CDCl3): 8.27 (2H, d, J 9.2 Hz, Har/o-NO2), 7.90
(1H, d, J 2.0 Hz, a3), 7.53 (1H, dd, J 8.7 and 2.1 Hz, a5), 7.38
(2H, d, J 9.2 Hz, Har/m-NO2), 7.31 (5H, m, Bn), 7.19 (1H, d, J 8.7
Hz, a6), 5.60 (1H, d, J 5.6 Hz, g1), 5.23 (2H, s, Bn), 5.13 (2H, s,
CH2O), 4.52 (1H, d, J 1.2 Hz, g3), 4.41 (1H, m, g4), 4.06
(1H, d, J 5.5 Hz, g2), 3.86 (1H, d, J 3.6 Hz, g5), 0.90, 0.88 and
0.83 (s, 9H, (CH3)3CSiMe2), 0.16, 0.11, 0.06 and 0.01 (3H, s,
CH3Si), 0.12 (6H, s, CH3Si); δC (CDCl3): 168.4 (CO2), 155.4,
152.4, 150.6, 145.5, 140.1 and 135.1 (Cqaromatics and CO3), 134.3
(a5), 128.5 and 128.4 (Co, m, p Bn), 127.8 (Cqaromatics), 126.1 (a3),
125.4 (Car/o-NO2), 121.8 (Har/m-NO2), 115.7 (a6), 99.5 (g1), 78.9 (g3),
76.6 (g5), 75.1 (g2), 72.1 (g4), 69.2 (CH2 Bn), 67.1 (CH2O), 25.8
(2C) and 25.8 ((CH3)3CSiMe2), 18.0 (2C) and 18.0 ((CH3)3-
CSiMe2), Ϫ4.5, Ϫ4.6, Ϫ4.6, Ϫ4.8 and Ϫ5.0 (2C) (CH3Si); m/z
[CI ϩ NH3] 960 (M ϩ NH4)ϩ.
135.6 (Co m SiPh2), 133.1 (Cqaromatics SiPh2), 131.5 (a5), 130.0
/
(Cp SiPh2), 127.9 (Co m SiPh2), 123.3 (a3), 116.5 (a6), 99.5 (g1),
/
78.9 (g3), 77.0 (g5), 75.4 (g2), 72.5 (g4), 64.3 (CH2), 52.4
(CH3O), 26.9 ((CH3)3CSiPh2), 25.9 and 25.8 (CH3)3CSiMe2),
19.3 (Me3CSiPh2), 18.1, 18.0 and 18.0 ((CH3)3CSiMe2), Ϫ4.4,
Ϫ4.5, Ϫ4.6, Ϫ4.7, Ϫ4.9 and Ϫ5.0 (CH3Si); m/z [CI ϩ NH3] 957
(M ϩ NH4)ϩ (Found: M ϩ NH4ϩ, 957.4977, C40H81O10N2Si4
requires 957.4968).
Benzyl {[2-nitro-4-(O-tert-butyldiphenylsilylmethyl)phenyl]-
2,3,4-tri-(O-tert-butyldimethylsilyl)-ß-D-glucopyranosid}uronate
14.
Methyl
{[2-nitro-4-(O-tert-butyldiphenylsilylmethyl)-
phenyl]-2,3,4-tri-(O-tert-butyldimethylsilyl)-ß--glucopyrano-
sid} 13 uronate (775 mg, 0.827 mmol), 2 mL (19.2 mmol) of
BnOH, 0.2 mL (0.8 mmol) of Ti(OEt)4 (pract. 85%) and 5 g of 4
Å molecular sieves were reacted at reflux for 48 h in 30 mL of
anhydrous toluene. The mixture was then diluted with 50 mL
of water and extracted with Et2O (5 × 30 mL), then succes-
sively washed with water and brine, dried over MgSO4 and con-
centrated under high vacuum. After chromatography (eluent
CH2Cl2), 602 mg (72%) of a colourless oil were obtained. δH
(CDCl3): 7.71 (1H, d, J 2.3 Hz, a3), 7.66 (5H, m, Ho SiPh2 and
a5), 7.42 (6H, m, Hm, p SiPh2), 7.31 (5H, m, Bn), 7.13 (1H, d, J
8.7 Hz, a6), 5.56 (1H, d, J 5.6 Hz, g1), 5.15 (2H, s, Bn), 4.69
(2H, s, CH2O), 4.52 (1H, d, J 1.8 Hz, g3), 4.42 (1H, m, g4), 4.07
(1H, d, J 5.5 Hz, g2), 3.86 (1H, d, J 3.6 Hz, g5), 1.10 (9H, s,
(CH3)3CSiPh2), 0.91, 0.88 and 0.85 (9H, s, (CH3)3CSiMe2), 0.17,
0.12, 0.07 and 0.03 (3H, s, CH3Si), 0.13 (6H, s, CH3Si); δC
(CDCl3): 168.6 (CO2), 149.1, 140.2, 135.4 and 134.9 (Cqaromatics),
N,NЈ-Dimethyl-N-benzyl-[4-(2,3,4-tri-O-tert-butyldimethyl-
silyl-ß-D-glucopyranosyl)uronate-3-nitrobenzyloxycarbonyl]-NЈ-
(tert-butoxycarbonyl) ethylenediamine 18. Method 1: to 81 mg
(0.085 mmol) of the activated carbonate 16 and 170 mg (11 eq.)
of the mono-BOC amine 1755 in solution in 5 mL of dry
CH2Cl2 were added 3 drops of pyridine. The mixture was stirred
for 5 h at room temperature, diluted with CH2Cl2 (20 mL) and
water (10 mL), then extracted with 3 × 30 mL of a saturated
NaHCO3 solution and dried (Na2SO4). After purification by
chromatography (eluent CH2Cl2–MeOH, 98.5 : 1.5), 38 mg
(45%) of the diamino compound were obtained.
Method 2: benzyl alcohol 15 (600 mg, 0.771 mmol) and 4
drops of Et3N were dissolved in 30 mL of anhydrous CH2Cl2
and placed under argon. Then, 250 mg (1.265 mmol) of
4-nitrophenyl chloroformate in solution in anhydrous CH2Cl
(5 mL) were added dropwise. After stirring for 2.5 h at room
temperature, 1.5 g (8.25 mmol) of the amine 17 was added to
the medium and stirring was continued overnight. The same
treatment as in method 2 enabled us to obtain 488 mg (64%) of
the desired coupling compound as a pale yellow syrup.
135.6 (Co m SiPh2), 133.1 (Cqaromatics SiPh2), 131.4 (a5), 130.0
/
(Cp SiPh2), 128.6 and 128.4 (Co, Bn), 128.4 (Cp Bn), 127.9
m
(Co m SiPh2), 123.2 (a3), 117.0 (a6), 99.7 (g1), 78.8 (g3), 77.0
/
(g5), 75.2 (g2), 72.4 (g4), 67.1 (CH2 Bn), 64.3 (CH2OSi), 26.9
((CH3)3CSiPh2), 25.9 and 25.8 ((CH3)3CSiMe2), 19.4 (Me3CS-
iPh2), 18.1, 18.1 and 18.0 ((CH3)3CSiMe2), Ϫ4.4, Ϫ4.5,
Ϫ4.6, Ϫ4.7, Ϫ4.9 and Ϫ4.9 (CH3Si); m/z [CI ϩ NH3] 1033
(M ϩ NH4)ϩ (Found: M ϩ NH4ϩ,1033.5288, C54H85O10N2Si4
requires 1033.5281).
Benzyl
[(2-nitro-4-hydroxymethylphenyl)-2,3,4-tri-(O-tert-
butyldimethylsilyl)-ß-D-glucopyranosid]uronate 15. Compound
14 (276 mg, 0.298 mmol) and acetic acid (17 µL, 1 eq.) were
dissolved in 15 mL of anhydrous DMF and placed under
argon. Then, 298 µL (1 eq.) of TBAF (1 M in THF) were added
dropwise. The reaction was followed by TLC (it lasted notably
longer when larger quantities were used). The reaction was then
quenched with 15 g of ice and the medium was extracted with
ether (6 × 20 mL). After washing with a saturated aqueous
NaHCO3 solution then with water, drying (MgSO4) and
δH (CDCl3): 7.81 (1H, s, a3), 7.44 (1H, m, a5), 7.31 (5H, m,
Bn), 7.14 (1H, d, J 8.7 Hz, a6), 5.57 (1H, d, J 5.7 Hz, H1), 5.13
(2H, s, Bn), 5.06 (2H, s, CH2O), 4.51 (1H, s, g3), 4.40 (1H, m,
g4), 4.05 (1H, d, J 5.5 Hz, g2), 3.85 (1H, d, J 3.2 Hz, g5), 3.38
(4H, m, e1, e2), 2.95 (3H, s, CH3N), 2.87–2.80 (3H, m, CH3N),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 4 3 – 3 3 5 2
3349

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1.43 (9H, s, (CH3)3CO), 0.90, 0.87 and 0.83 (9H, s, (CH3)3-
CSiMe2), 0.16, 0.06 and 0.01 (3H, s, CH3Si), 0.11 (9H, s,
CH3Si); δC (CDCl3): 168.5 (CO2), 155.8 and 149.9 (NCO2),
140.1, 135.3 (2C) and 130.5 (Cqaromatics), 133.5 (a5), 128.6 and
dissolved in 10 mL of freshly distilled CH2Cl2 over P2O5. Under
argon, 32 mg (0.037 mmol) of paclitaxel, then, dropwise, 0.2
mL of Et3N were added. After 6 h, the solution was diluted
with CH2Cl2 (20 mL), washed with water and brine, and dried
over Na2SO4. Purification on a thin silica gel column (60, 0.015–
0.040 mm, Merck) (eluent CH2Cl2–MeOH, 98 : 2), yielded 57
mg (87%) of the coupling compound as a white foam; mp 183
ЊC; δH (CDCl3): 8.53 (1H, d, J 9.7 Hz, p3Ј-NH), 8.15 (2H, d,
J 7.2 Hz, p2-Bz (o)), 7.78 (3H, m, p2-Bz (m) and a3), 7.65–7.15
(17H, m, Haromatics), 7.10 (1H, m, a6), 6.30 (2H, m, p10, p13),
6.12 (1H, m, p3Ј), 5.66 (1H, m, p1), 5.57 (1H, d, J 6.3 Hz, g1),
5.48 (1H, d, J 2.7 Hz, p2Ј), 5.13 (2H, m, CH2, Bn), 4.98 (1H, m,
p5), 4.64 (2H, ABq, J 12.6 Hz, CH2O), 4.50 (1H, m, g3), 4.47
(1H, m, p7), 4.40 (1H, s, g4), 4.17 (2H, ABq, J 8.6 Hz, p20),
4.05 (1H, d, J 5.4 Hz, g2), 3.84 (2H, m, p3, g5), 3.65–3.02 (4H,
m, e1, e2), 2.95–2.89 (6H, m, CH3N), 2.58 (s, p4-OAc), 2.52
(1H, m, p6α), 2.42 (2H, d, J 2.3 Hz, p14), 2.21 (3H, s, p10-OAc),
2.09 (3H, m, 3H, p18), 2.00 (1H, m, p6β), 1.68 (3H, s, p19), 1.22
(3H, s, p16), 1.11 (3H, s, p17), 0.90, 0.87 and 0.83 (9H, s,
(CH3)3CSiMe2), 0.16, 0.06, and 0.00 (3H, s, CH3Si), 0.12 (9H, s,
CH3Si); δC (CDCl3): 204.1 (p9), 171.4, 170.1, 169.0, 168.4, 168.3
and 167.1 (MeC (O)C4, MeC(O)C10, PhCO2, p3Ј-PhC(O)Ј, p1Ј
and CO2Bn), 155.9 and 149.9 (NCO2), 154.9, 143.3, 140.1
(p12), 137.6, 135.2, 134.6, 132.5, 130.0 and 129.3 (7 Cqaromatics
and p11), 133.6, 133.3, 131.4, 130.3, 129.0, 128.8 (2C), 128.6,
128.4, 128.1, 127.9, 127.1, 126.7 and 125.0 (14 CHaromatics), 116.8
(a6), 99.4 (g1), 84.5 (p5), 81.0 (p4), 79.2 (p1); 78.9 (g3), 76.7,
75.7 (2C) and 75.2 (2C) (p2, p10, p2Ј, g2, g5), 72.2 (p7, g4), 71.4
(p13), 76.5 (p20), 67.1 (CH2, Bn), 65.5 (CH2O), 58.5 (p8), 52.8
(p3Ј), 47.4 and 46.1 (e1, e2), 45.6 (p3), 43.2 (p15), 35.7 (CH3N),
35.6 (p6), 26.8 (p16), 25.9 (2C) and 25.8 ((CH3)3CSiMe2), 22.8
(p14 and p4-OAc), 22.4 (p17), 20.9 (p10-OAc), 18.0 (2C)
and 18.0 ((CH3)3CSiMe2), 14.9 (p18), 9.7 (p19), Ϫ4.5, Ϫ4.6,
Ϫ4.6, Ϫ4.8, Ϫ5.0 and Ϫ5.0 (CH3Si); m/z [FAB ϩ NaI] 1794
(M ϩ Na)ϩ (Found: M ϩ Naϩ, 1793.7540, C91H122O26N4Si3Na
requires 1793.7553).
128.4 (Co,
Bn), 125.1 (a3), 117.0 (a6), 99.4 (g1), 79.7
m,
p
(Me3CO), 78.8 (g3), 76.8 (g5), 75.2 (g2), 72.2 (g4), 67.1 (CH2,
Bn), 65.7 and 65.4 (CH2O), 46.8 (m, 2C, e1, e2), 35.5, 35.0 and
34.6 (2C, CH3N), 28.5 ((CH3)3CO), 25.9 and 25.8 (2C)
((CH3)3CSiMe2), 18.1 (2C) and 18.0 ((CH3)3CSiMe2), Ϫ4.4,
Ϫ4.5, Ϫ4.6, Ϫ4.7, Ϫ4.9 and Ϫ5.0 (CH3Si); m/z [CI ϩ NH3]
1009 (M ϩ NH4)ϩ.
N,NЈ-Dimethyl-N-benzyl-[4-(2,3,4-tri-O-tert-butyldimethyl-
silyl-ß-D-glucopyranosyl)uronate-3-nitrobenzyloxycarbonyl]-
ethylenediamine hydrochlorate 19. BOC derivative 18 (270 mg,
0.27 mmol) was suspended in a freshly prepared 3 M HCl solu-
tion in EtOAc (20 mL) previously cooled to 0 ЊC. Stirring was
maintained at this temperature until complete BOC dis-
appearance (TLC monitoring). The reaction medium was then
concentrated and the crude salt obtained was used directly for
the next step. δH (CD3OD): 7.91 (1H, s, a3), 7.64 (1H, m, a5),
7.36 (5H, m, Bn), 7.27 (1H, d, J 8.2 Hz, a6), 5.65 (1H, d, J 6.0
Hz, g1), 5.16 (4H, s, Bn and CH2O), 4.71 (1H, s, H3), 4.46 (1H,
d, J 3.5 Hz, g4), 4.01 (1H, d, J 6.0 Hz, g2), 3.91 (1H, d, J 3.5 Hz,
g5), 3.63 (2H, m, CH2N), 3.21 (2H, m, CH2N), 3.00 (3H, s,
CH3N), 2.72 (3H, s, CH3N), 0.94, 0.90 and 0.85 (9H, s,
(CH3)3CSiMe2), 0.16, 0.15, 0.08 and 0.00 (3H, s, CH3Si), 0.14
(6H, s, CH3Si); δC (CD3OD): 170.0 (CO2), 150.5 (NCO2), 141.5,
136.6 (2C) and 129.6 (Cqaromatics), 132.2 (a5), 129.5 and 129.4
(Co,
Bn), 129.2 (a3), 117.4 (a6), 100.4 (g1), 80.3 (g3), 78.2
m,
p
(g5), 77.1 (g2), 73.5 (g4), 68.3 (CH2, Bn), 67.1 (CH2O), 49.4 (m,
2C, CH2N), 34.0 (2C, CH3N), 26.4, 26.3 and 26.2 ((CH3)3-
CSiMe2), 18.9 ((CH3)3CSiMe2), Ϫ4.2, Ϫ4.2, Ϫ4.5, Ϫ4.6, Ϫ4.6
and Ϫ4.8 (CH3Si); m/z [FAB ϩ] 892 (M – HCl ϩ H)ϩ.
N,NЈ-Dimethyl-N-benzyl-[4-(2,3,4-tri-O-tert-butyldimethyl-
silyl-ß-D-glucopyranosyl)uronate-3-nitrobenzyloxycarbonyl]-
ethylenediamine carbamoyl chloride 20. Salt 19 (250 mg, 0.027
mmol) and 0.1 mL (0.2 mmol) of phosgene in solution in 20%
toluene were suspended in 50 mL of anhydrous CH2Cl2, placed
under an argon atmosphere and cooled to 0 ЊC. Then, 3 drops
of Et3N were added and the temperature was maintained at
0 ЊC for 2.5 h. The reaction mixture was then concentrated and
purified on a silica gel column (eluent EtOAc); 230 mg (90%)
of carbamoyl chloride were obtained as a pale yellow oil.
δH (CDCl3): 7.82 (1H, s, a3), 7.47 (1H, m, a5), 7.31 (5H, m, Bn),
7.15 (1H, d, J 8.6 Hz, a6), 5.58 (1H, d, J 5.5 Hz, g1), 5.13 (2H, s,
Bn), 5.07 (2H, s, CH2O), 4.51 (1H, s, g3), 4.40 (1H, m, g4), 4.05
(1H, d, J 6.0 Hz, g2), 3.85 (1H, d, J 3.6 Hz, g5), 3.75–3.35 (4H,
m, e1, e2), 3.13–2.96 (6H, m, CH3N), 0.90, 0.87 and 0.83 (9H, s,
(CH3)3CSiMe2), 0.16, 0.12, 0.11, 0.07, 0.06 and 0.01 (3H, s,
CH3Si); δC (CDCl3): 168.1 (CO2), 156.0, 155.8, 155.4 and 155.3
(NCO2), 149.9, 149.7, 149.5 and 149.5 (NCOCl), 148.5, 139.8
and 135.0 (Cqaromatics), 133.9, 133.4 and 133.2 (a5), 130.4, 130.3,
130.1 and 130.0 (Cqaromatics), 130.0 (Co m Bn), 128.3 (Co m and
Bn), 125.3, 125.2, 124.8 and 124.8 (a3), 116.6 (a6), 99.1 (g1),
78.6 (g3), 76.6 (g5), 75.0 (g2), 71.9 (g4), 66.3 (CH2, Bn), 65.7,
65.5, 65.4 and 65.3 (CH2O), 50.4, 50.1, 48.9 and 48.2 (e1, e2),
46.8, 46.4, 46.0 and 45.7 (CH2N), 38.9, 38.6, 37.0 and 36.8
(CH3N), 35.2, 35.0, 34.9 and 34.4 (CH3N), 25.6 (2C) and 25.5
((CH3)3CSiMe2), 17.8 (2C) and 17.7 ((CH3)3CSiMe2), Ϫ4.7,
Ϫ4.8, Ϫ4.9, Ϫ5.0, Ϫ5.2 and Ϫ5.3 (CH3Si); m/z [CI ϩ NH3] 971
(100%), 973 (61.4) (M ϩ NH4)ϩ (Found: M ϩ NH4ϩ, 971.4451
(100%), 973.4450 (63.9),C44H76O12N4ClSi3 requires 971.4456,
973.4427).
2Ј-O-[N,NЈ-Dimethyl-N-benzyl-4-(ß-D-glucopyranosiduron-
ate)-3-nitrobenzyloxycarbonylethylenediamine]paclitaxel 22. To
150 mg (0.085 mmol) of the protected prodrug 21 and 0.3 mL
of anhydrous pyridine dissolved in anhydrous CH3CN (5 mL)
under argon was dropwise added, at 0 ЊC, 0.8 mL of a 70%
HF–pyridine complex. After 3 h at 0 ЊC, the mixture was
allowed to reach room temperature and stirring was continued
overnight. Hydrolysis at 0 ЊC was performed with saturated
NaHCO3 (125 mL), then the medium was extracted with
EtOAc (3 × 50 mL), dried over Na2SO4, concentrated and
chromatographed on silica gel (eluent CH2Cl2–MeOH, 95 : 5),
affording 90 mg (76%) of the desilylated compound as a white
foam; mp 154 ЊC; δH (CDCl3): 8.43 (1H, d, J 9.5 Hz, p3Ј-NH),
8.13 (2H, p2-Bz (o)), 7.78 (3H, m, p2-Bz (m) and a3), 7.65–7.24
(18H, m, Haromatics), 6.28 (2H, m, p10 and p13), 6.12 (1H, dd,
J 9.3 and 3.0 Hz, p3Ј), 5.66 (1H, m, p2), 5.46 (1H, d, J 2.9 Hz,
p2Ј), 5.23 (2H, CH2, Bn), 4.94 (1H, m, p5), 4.88 (1H, d, J 7.2
Hz, g1), 4.64 (2H, ABq, J 13 Hz, CH2O), 4.50 (1H, m, g3), 4.47
(1H, m, p7), 4.10 (2H, ABq, J 8.4 Hz, p20), 3.95 (1H, g5), 3.88
(1H, m, g4), 3.81 (1H, m, p3), 3.80–3.05 (8H, m, g3, e1, e2,
OH(g2, g3, g4)), 2.93–2.88 (6H, m, CH3N), 2.55 (3H, s,
p4-OAc), 2.45 (1H, m, p6α), 2.39 (2H, m, p14), 2.21 (3H, s, p10-
OAc), 1.98 (3H, s, p18), 1.88 (1H, m, p6β), 1.67 (3H, s, p19),
1.18 (3H, s, p16), 1.11 (3H, s, p17); δC (CDCl3): 204.0 (p9),
171.4, 170.1, 169.0, 168.4, 168.2 and 167.1 (p4-OAc, P10-OAc,
PhCO2, PhC(O)N3Ј, p1Ј and CO2Bn), 155.9 and 149.9 (NCO2),
154.9, 143.2, 140.3 (p12), 137.5, 134.9, 134.5, 132.6 132.1 and
129.3 (7 Cqaromatics and p11), 133.7, 131.5, 130.4, 128.8 (2C),
128.7 (2C), 128.7, 128.3, 128.1, 128.0, 127.1, 126.7 and 124.7
(14 CHaromatics), 119.1 (a6), 102.7 (g1), 84.5 (p5), 81.0 (p4), 79.3
(p1), 75.7 (2C), 75.2, 75.0 and 74.8 (p2, p10, p2Ј, g2 or g3, g5),
76.8 (p20), 72.8, 72.1, 71.4 and 70.9 (p7, p13, g2 or g3, g4), 67.7
(CH2 Bn), 65.4 (CH2O), 58.5 (p8), 53.0 (p3Ј), 47.4 and 46.1 (e1,
/
/
2Ј-O-{N,NЈ-Dimethyl-N-benzyl-[4-(2,3,4-tri-O-tert-butyl-
dimethylsilyl-ß-D-glucopyranosyl)uronate-3-nitrobenzyloxycarb-
onyl] ethylenediamine}paclitaxel 21. Carbamoyl chloride 20
(80 mg, 0.083 mmol) and 116 mg (ca. 10 eq.) of DMAP were
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e2), 45.7 (p3), 43.2 (p15), 35.7 (CH3N), 35.6 (p6), 26.8 (p16),
22.8 (p17), 22.6 (p14), 22.3 (p4-OAc), 20.9 (p10-OAc), 15.0
(p18), 9.7 (p19); m/z [FAB ϩ NaI] 1451 (M ϩ Na)ϩ. (Found:
M ϩ Naϩ, 1451.4979, C73H80O26N4Na requires 1451.4959).
taken at various times and analyzed by HPLC after dilution
with eluent (300 µL).
HPLC conditions: analysis was carried out on a reverse-
phase column (Lichrospher RP18e, 250 × 4 mm, 5 µm) using
isocratic conditions (1 mL/min) of 50% phosphate buffer (0.02
M, pH 3) and 50% acetonitrile with UV detection at 226 nm
(extracted from PDA 3D spectra).
2Ј-O-[N,NЈ-Dimethyl-N-4-(ß-D-glucopyranosiduronate)-3-
aminobenzyloxycarbonylethylenediamine]paclitaxel 3. To a sus-
pension of 90 mg (0.063 mmol) benzyl ester 22 and 10% Pd/C
(100 mg) in 10 mL abs. ethanol, 0.3 mL (50 eq.) cyclohexa-1,4-
diene were added and the mixture heated at 45 ЊC for 24 hours.
After filtration over Celite 545, the product was purified by
chromatography on silica gel (eluent CH3CN–H2O, 9 : 1).
Lyophilisation afforded 20 mg (24%) of prodrug 3 as a white
foam; mp 190 ЊC; δH (CD3OD): 9.12 (1H, m, p3Ј-NH), 8.13
(2H, p2-Bz (o)), 7.81 (2H, m, p2-Bz (m)), 7.67–7.28 (11H, m,
Haromatics), 7.05 (1H, m, a5), 6.67 (1H, m, a3), 6.50 (1H, m, a6),
6.46 (1H, m, p10), 6.15 (1H, m, p13), 6.05 (1H, m, p3Ј), 5.64
(1H, m, p2), 5.40 (1H, m, p2Ј), 4.99 (1H, m, p5), 4.76 (1H, m,
g1), 4.76 (2H, m, CH2O), 4.35 (1H, m, p7), 4.19 (4H, m, p20,
CH2O), 3.83 (2H, m, p3, g5), 3.60–3.34 (7H, m, g2, g3, g4, e1,
e2), 2.99–2.81 (6H, m, CH3N), 2.49–2.39 (4H, s, p4-OAc, p6α),
2.19 (2H, m, p14), 2.16 (3H, s, p10-OAc), 1.97 (3H, s, p18), 1.80
(1H, m, p6β), 1.66 (3H, s, p19), 1.12 (6H, s, p16, p17);
δC (CD3OD): 205.3 (p9), 171.6, 171.3, 171.0, 170.9, 167.7
(p4-OAc, p10-OAc, PhCO2, PhC(O)N3Ј, p1Ј), 158.0 and 146.6
(NCO2), 156.7, 142.5, 138.3 (Cqaromatics), 135.7, 134.8, 133.8,
131.4, 131.1, 130.5, 130.0, 129.5, 129.1, 128.7, 128.4, 128.1, and
124.4 (CHaromatics), 118.6 (a5, a6), 117.4 (a3), 104.6 (g1), 85.9
(p5), 82.2 (p4), 78.0, 77.4, 76.9 (2C) and 76.3 (2C) (p2, p10, p20,
p2Ј, g3, g5), 74.6, 73.3, 72.7 and 72.2 (p7, p13, g2, g4), 69.7
(CH2O), 59.2 (p8), 54.8 (p3Ј), 47.5 (p3), 47.1 and 46.9 (e1, e2),
44.5 (p15), 37.5 (p6), 37.1–35.2 (CH3N), 27.2 (p16), 23.3, 22.5,
22.4 (p17, p14, p4-OAc), 20.8 (p10-OAc), 14.7 (p18), 10.5 (p19);
m/z [ESϩ] 1309 (M ϩ H)ϩ. (Found: M ϩ Hϩ, 1309.4926,
C66H77O24N4 requires 1309.4928).
Kinetic measurements
A solution of 30 µg mLϪ1 of prodrug in 0.02 M phosphate
buffer (pH 7.2) was incubated at 37 ЊC in the presence of 12
units mLϪ1 of β--glucuronidase (E. coli). Aliquots (100 µL)
were taken at various times and analyzed by HPLC after
dilution with eluent (300 µL).
LC/MS measurements
LC/MS spectra were recorded on an API3000 LC-MS-MS
spectrometer with electrospray ionisation. The HPLC separ-
ation was carried out on a reverse-phase column (Nucleosil
C18, 150 × 2.1 mm, 3 µm) using isocratic conditions (0.2 mL
minϪ1) of 50% ammonium formate buffer (5 mM, pH 3.5) and
50% acetonitrile.
Acknowledgements
We are grateful to the Association pour la Recherche sur le
cancer (ARC) for Grant Number 5544 and a partial fellow-
ship to E. B. Cytotoxic measurements were performed at the
Laboratoires Servier. We thank Jean-Marc Clavel for effecting
the LC/MS measurements.
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