New Taxol (paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Article abstract of DOI:10.1016/j.bmc.2006.03.002

Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 2′-OH of the drug by a carbonate function, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid residue. Both pr

Full text of DOI:10.1016/j.bmc.2006.03.002

Bioorganic & Medicinal Chemistry 14 (2006) 5012–5019
New Taxolꢀ (paclitaxel) prodrugs designed for ADEPT and
PMT strategies in cancer chemotherapy
*
´ ´
Abdessamad El Alaoui, Nabendu Saha, Frederic Schmidt, Claude Monneret and
Jean-Claude Florent
UMR 176 CNRS/Institut Curie, Centre de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France
Received 10 January 2006; revised 27 February 2006; accepted 3 March 2006
Available online 22 March 2006
Abstract—Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 20-OH of the drug by a carbonate func-
tion, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid res-
idue. Both prodrugs were well detoxified and easily cleaved in the presence of b-D-glucuronidase with fast removal of the spacer,
releasing paclitaxel. The arylamino spacer-containing prodrug, more stable than the corresponding nitro analogue, was selected
for further studies.
ꢁ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Paclitaxel (Taxolꢀ) and Docetaxel (Taxotereꢀ) are
potent anticancer agents that are currently used for the
treatment of patients with ovarian, breast, non-small cell
lung (NSCL) and prostate cancers. They possess a
unique mechanism of action by binding to tubulin and
promoting stable and non-functional microtubule for-
mation14,15 which finally leads to disrupted mitosis and
cell death. Recent findings have shown that paclitaxel
initiates apoptosis through multiple mechanisms.16,17
However, the clinical usefulness of these drugs is partic-
ularly hampered by their poor solubility, along with the
aforementioned general problems, that is, lack of
selectivity and development of multidrug resistance in
clinic.18,19 Therefore, it remains of interest to improve
their use, at least to gain in solubility and selectivity,
by preparing water-soluble prodrugs selectively
activable in tumour areas. Since endogenous extracellu-
lar b-D-glucuronidase20–23 is highly present in necrotic
tumours, glucuronide prodrugs fulfil these two require-
ments and can be used in prodrug monotherapy
(PMT). Moreover, in the case of non-necrotic areas of
tumours where the enzyme concentration is low, follow-
ing the antibody directed enzyme prodrug therapy
(ADEPT) strategy, introduction of exogenous b-D-
glucuronidase via a fusion protein consisting of an
anti-CEA antibody linked to a b-D-glucuronidase has
been already assessed.24 Therefore, some time ago, we
undertook and reported the synthesis of paclitaxel
prodrugs designed to be activated by b-glucuronidase
such as prodrugs 125 and 226 consisting of a b-glucuronide
Despite recent progress in cancer therapy, a major chal-
lenge remains to increase antitumour selectivity of cyto-
toxic drugs that are still currently used. Indeed, the lack
of selectivity of such drugs is a serious drawback typical-
ly associated with severe side-effects, insufficient drug
concentrations that would completely eradicate the tu-
mour and, for most of them, emergence of drug resis-
tance after prolonged treatment. Among different ways
to overcome these problems, one is to use non-cytotoxic
prodrugs designed to locally deliver the antitumour
agent by specific activation.1–3 According to this strate-
gy called ‘tumour activated prodrug’ (TAP),4–6 some
prodrugs have reached the preclinical or clinical trial
steps.7 This tumour-specific activation may occur by
hypoxic environment of solid tumours, by localisation
to tumour antigen, by tumour-associated proteases
(PSA, PSMA) or by enzyme selectively present in the tu-
mour. In the case of tumour-associated enzyme activa-
tion, the active enzyme may be present in the tumour,
like in PMT8,9 strategies, or targeted to the tumour site,
as in ADEPT, GDEPT, or VDEPT approaches.10–13,6
Keywords: Taxol; Paclitaxel; Prodrug; Glucuronide.
*
Corresponding author. Tel.: +33 1 42346664; fax: +33 1 42346631;
e-mail: frederic.schmidt@curie.fr
0968-0896/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.002

A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
5013
moiety, a self-immolative spacer and paclitaxel. In the
case of 1, relevant detoxification of the drug was ob-
served25 but along with an inefficient enzyme activation
of the prodrug, which was attributed to a steric hin-
drance of the glycosidic linkage, as ascertained by
molecular modelling. With still a suitable detoxification,
an improved enzymatic cleavage was obtained26 by
using an elongated spacer as in 2, but the synthesis of
such a prodrug became laborious for two main reasons:
removal, at the last step, of the benzyl ester as the pro-
tecting group of the acidic function of the glucuronic
acid gave moderate yields (54% and 24%, respectively)
but also led to partial reduction of the nitro function.26
2. Chemistry
Figure 2. Structures of prodrugs 3 and 4.
The priority was then to investigate the possibility of
replacing the benzyl ester of the glucuronic acid moiety
by an allyl ester. As, to our knowledge, this protecting
group had never been used in the case of paclitaxel glu-
curonides, the challenge was to find adequate conditions
to remove it in the presence of the very sensitive taxane
skeleton. Simultaneously, we expected to either keep or
reduce the nitro group in view of better understanding
the role of the nitro or amino-containing spacer with
respect to the prodrug stability and to the enzymatic
cleavage Figure 1.
The two planned molecules were 3 and 4 (Fig. 2).
Prodrug 3 was synthesized according to Scheme 1 start-
ing from the intermediate 5 already described by us.26
The first step consisted in replacing the methyl ester
group by an allyl ester. In order to avoid successive
saponification and reesterification, we tried a one-step
transesterification reaction in the presence of an excess
of allyl alcohol. A catalyst compatible with the silyl
ethers was found within the titanium IV derivatives.
Whereas, by using the commercial titanium tetra-iso-
propoxide, a mixture of isopropyl and allyl ester was ob-
tained, in situ preparation of titanium tetra-allylate and
subsequent addition of the methyl ester gave exclusively
the expected ester 6.
At the same time, we envisioned improving the kinetics
of cleavage by attaching the spacer to the 20-OH of
paclitaxel by a carbonate instead of a carbamate. The
stability of a carbonate function in position 20 of the
paclitaxel depends on its structural environment.
In the literature, some 20-paclitaxel carbonates have
been reported to be hydrolysed in serum,27 whereas oth-
ers were stable enough to be used in TAP strategies. This
is the case of several prodrugs to be activated by
b-D-glucuronidase, plasmine or bioreduction in order
to release the free paclitaxel such as those described by
Scheeren et al.28–30
Selective deprotection of the TIPS group from the pri-
mary benzylic alcohol in the presence of the secondary
TBS ethers was achieved by using TBAF/AcOH in
DMF according to the literature.31
The benzylic alcohol of 7 was then activated with
DSC as a N-hydroxy succinimide ether in the presence
of a base. The obtained carbonate 8, too unstable to
be purified, was directly engaged in the next step by
addition of the required amino-alcohol to the crude
reaction mixture. Carbamate 9, obtained in good yield
(96%), was subsequently activated again with DSC as
a N-hydroxy succinimide carbonate. Carbonate 10
could be purified in 79% yield. Next step was the cou-
pling with paclitaxel which worked fine (yield: 93%)
by using stoichiometric DMAP in dichloromethane.
Under theses conditions, only the more reactive C-20
alcohol of the side chain of paclitaxel reacted. Depro-
tection of the TBS groups was conducted with HF/Pyr
complex.
The last step involved removal of the allyl ester group.
Our first trials with classical reagents such as
Pd(PPh3)4/pyrrolidine32 or Pd(PPh3)4/morpholine27
resulted in the C-20 carbonate cleavage of the paclitaxel,
probably due to the nucleophilicity of these heterocyclic
bases. However, the allyl ester was successfully removed
Figure 1. Structures of prodrugs 1 and 2.

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A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
Scheme 1. Synthesis of prodrugs. Reagents: (i) Ti(OAll)4/AllOH (69%); (ii) TBAF/AcOH/DMF (88%); (iii) DSC/iPr2NEt; (iv) MeNHCH2CH2OH
(96% two steps); (v) DSC/iPr2NEt/DMAP (79%); (vi) paclitaxel/DMAP/NEt3 (93%); (vii) HF/Pyr (75%); (viii) Pd(PPh3)4/NEt3/CH3CO2H (80%); (xi)
Pd(PPh34/NEt3/HCO2H (81%).
using the described reagent Pd(PPh3)4/formic acid/
triethylamine.33,34 During this reaction, the nitro group
of the spacer was reduced to an amino group leading to
4 in good yield (81%). In order to improve the yield of
the reaction, we tried to use the same reactant in acetic
acid instead of formic acid. Interestingly, by using
Pd(PPh3)4/acetic acid/triethylamine, deprotection of the
allyl ester occurred without reduction of the nitro group,
allowing 3 directly from 12 in 80% yield. To our knowl-
edge, it is the first time that such selectivity was observed
for this kind of reaction by replacing formic acid by ace-
tic acid in the reagent. An explanation could be that for-
mates act as reducing agents in the presence of the
palladium catalyst.
3.2. Cytotoxicity
The cytotoxicities of prodrugs 3 and 4 in the presence or
in the absence of b-D-glucuronidase were measured on
HT-29 (colon cancer) cell lines and compared to the
cytotoxicity of the free drug.
The IC50 of prodrugs 3, 4 and free paclitaxel were,
respectively, 28.9, 11.3 and 0.16 nM. In the presence of
b-D-glucuronidase, the cytotoxicities of prodrugs 3 and
4 were restored to that of the free paclitaxel. The ratios
of cytotoxicities between the prodrug and the free drug
were, respectively, 180 and 70 for 3 and 4. These values
are compatible with ADEPT or PMT strategies where a
relatively non-cytotoxic prodrug releases a cytotoxic
compound.
3. Biological results
3.1. Stability
3.3. Kinetics of drug release
The kinetics of transformation of the prodrugs 3 and 4
into the active drug were measured by HPLC and com-
pared to that of the previously obtained prodrug 2. The
planned enzymatic cleavage reaction was as depicted in
Scheme 2.
Stability of prodrugs 3 and 4 was measured by HPLC
in a phosphate buffer solution at pH 7.2 or in a 10%
FCS (foetal calf serum) in a phosphate buffer solution.
Prodrug 4 was stable over 24 h, with no visible cleav-
age of the carbonate bond in position 20 of the paclit-
axel. Prodrug 3, with its nitro group on the spacer,
was less stable in the same solutions. Monitoring
HPLC analysis with UV detection showed a 57%
decrease of the latter prodrug peak in a phosphate
buffer solution and a 76% decrease in the serum solu-
tion over 24 h. Since the nitro group causes the mole-
cule to be more sensitive, it appeared preferable to use
an amino group on the spacer, like in prodrug 4, for
further studies.
Escherichia coli b-D-glucuronidase was used as the
activating enzyme. For both prodrugs 3 and 4,
cleavage of the prodrug was observed with simulta-
neous appearance of Taxolꢀ and cyclized spacer
(Fig. 3).
In our comparison, we used the same concentrations of
prodrugs 2, 3 and 4 (190 lM) and of b-D-glucuronidase
(4.3 U/mL) and then measured the disappearance of the

A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
5015
faster enzymatic cleavage than the prodrug 2 with a car-
bamate function.
Between prodrugs 3 and 4, which differ only by a nitro
or an amino group on the spacer, the kinetics were al-
most identical.
This similarity between amino and nitro spacers was pre-
viously observed with docetaxel prodrugs.35 An explana-
tion could be that the glycosidic enzymatic cleavage is
rate-determining and that the following step, namely
self-immolation of both spacers, is fast (Scheme 2).
4. Conclusion
In the light of these experiments it appears that, from
the two new prodrugs obtained, prodrug 4 can be select-
ed for further in vivo tests for its better stability and lib-
eration kinetics of the active compound in the presence
of the activating enzyme.
The spacer of prodrug 4 could also be used in the syn-
thesis of other prodrugs, such as prodrugs of antitumour
compounds possessing a free hydroxy group, like etopo-
side or camptothecin derivatives like SN-38.
Scheme 2. Hydrolysis of prodrugs 3 and 4.
During this synthesis, we have shown a difference in reac-
tivity between two reagents (Pd(PPh3)4/formic acid/
triethylamine and Pd(PPh3)4/acetic acid/triethylamine)
for the removal of an allyl ester-protecting group. This
can be applied to other syntheses in which the starting
material contains a sensitive and reducible group.
5. Experimental
Melting points (mp) were measured using an electrother-
mal digital melting point apparatus and are uncorrected.
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra
were recorded on a Bruker AC 300 spectrometer—
chemical shifts d in parts per million and J in hertz.
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.
Electrospray ionisation mass spectra (ESI-MS) were ac-
quired using 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. Chromato-
graphies were conducted over silica gel (Merck 60
(230–400 Mesh)).
Figure 3. Cleavage of prodrug 4 by b-D-glucuronidase.
starting prodrug (Fig. 4). The results clearly showed the
faster cleavage of prodrugs 3 and 4 (Fig. 4). So, the
prodrugs 3 and 4 bearing a carbonate function exhibited
For the NMR descriptions, the numbers are as in
Figure 5.
5.1. Allyl {[2-nitro-4-(O-tert-butyldiphenylsilylmethyl)phen-
yl]-2,3,4-tri-(O-tert-butyldimethylsilyl)-b-D-glucopyrano-
sid}uronate (6)
To a solution of Ti-isopropoxide (0.094 mL, 0.32 mmol)
in toluene (10 mL) under argon, allyl alcohol (0.058 mL,
Figure 4. Comparison of cleavage between prodrugs 2, 3 and 4.

5016
A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
0
00
H1 ), 5.32–5.19 (dt, 2H, J = 10.4 Hz and J = 1.2 Hz,
CH2@CH), 4.68 (s, 2H, CH2Oarom), 4.60 (d, 2H,
00
J = 5.8 Hz, CH@CH2O), 4.51 (d, 1H, J = 1.3 Hz, H3 ),
00
4.40 (d, 1H, J = 3.2 Hz, H4 ), 4.04 (d, 1H, J = 5.7 Hz,
00
00
H2 ), 3.85 (d, 1H, J = 3.4 Hz, H5 ), 0.92, 0.87 and 0.82
(s, 27H, (CH3)3CSi), 0.15, 0.13, 0.12, 0.06 and 0.00 (s,
18H, (CH3)2Si); 13C NMR (CDCl3) d 170.0 (CO2 allyl),
149.8 (Ce), 141.5 (Cf), 137.3 (Cb), 132.9 and 132.8 (Cc,
CH2@CH), 124.7 (Ca), 119.2 (CH2@CH), 117.2 (Cd),
Figure 5. NMR attributions.
0.85 mmol) was added. The reaction mixture was re-
fluxed and isopropanol was removed. Then, after 1 h,
fresh allyl alcohol (0.85 mmol) was added and the same
procedure was repeated three times. Tetrasilyl com-
pound 5 in 10 mL toluene was added to the reaction ves-
sel and reflux was maintained for four additional hours.
Water was added to the reaction mixture, and the solu-
tion was filtered through Celite and extracted with dieth-
yl ether (6· 20 mL). After drying over MgSO4 and
evaporation, the crude residue was purified by chroma-
tography (toluene). A pale yellow liquid (0.07 g, 69.0%)
was isolated. On multigram scale, the yield was less
00
00
00
00
100.3 ðC1 Þ, 80.1 ðC3 Þ, 78.4 ðC5 Þ, 77.2 ðC2 Þ, 73.5
00
ðC4 Þ, 67.1 (CH–CH2O), 63.5 (CH2arom), 26.4. 26.3
and 26.1 ((CH3)3CSi), 18.8 (2C) ((CH3)3CSi), ꢁ4.2,
ꢁ4.3, ꢁ4.4, ꢁ4.6 and ꢁ4.7 ((CH3)2Si); m/z [CI+NH3]
745.5 (M+NH4)+.
5.3. N-Methyl-N-allyl-[4-(2,3,4-tri-O-tert-butyldimethylsi-
lyl-b-D-glucopyranosyl)uronate-3-nitrobenzyloxycarbonyl]-
2-amino-ethanol (9)
To a solution of silyl compound 7 (0.05 g; 0.069 mmol)
in dry CH2Cl2 (2.0 mL) under argon, N,N-disuccimidyl
carbonate (0.026 g, 0.1 mmol) and (iPr)2NEt (0.03 mL,
0.17 mmol) were added. The reaction mixture was stir-
red for 2 h at room temperature, and N-methyl-ethanol
(0.006 mL, 0.084 mmol) was added. The reaction mix-
ture was then stirred at room temperature for 1 h. After
evaporation, the residue was purified by chromatogra-
phy (CH2Cl2/MeOH: 99:1 then 95:5). A pale yellow li-
reproducible ranging from 55% to 70%; Rf = 0.674
20
D
(cyclohexane/EtOAc 7:3); ½aꢀ ꢁ12.0 (c 1.13, CHCl3);
1H NMR (300 MHz, CDCl3)
d
7.74 (d, 1H,
J = 1.9 Hz, Ha), 7.68–7.64 (m, 4H, Ho/2Si), 7.42 (m,
7H, Hm/p/2Si, Hc), 7.18 (d, 1H, J = 8.1 Hz, Hd), 5.93–
5.80 (m, 1H, CH2@CH–CH2), 5.58 (d, 1H, J = 5.6 Hz,
0
00
H1 ), 5.33–5.19 (dt, 2H, J = 10.4 Hz and J = 1.2 Hz,
CH2@CH), 4.70 (s, 2H, CH2Oarom), 4.61 (d, 2H,
quid (54.7 mg, 96.0%) was isolated; Rf = 0.264
20
D
00
J = 5.6 Hz, CH@CH2O), 4.51 (d, 1H, J = 1.6 Hz, H3 ),
(CH2Cl2/MeOH 9.75:0.25); ½aꢀ ꢁ13.8 (c 1.84, CHCl3);
1H NMR (300 MHz, CDCl3)
d
7.84 (d, 1H,
00
00
4.41 (m, 1H, H4 ), 4.06 (d, 1H, J = 5.5 Hz, H2 ), 3.85
00
(d, 1H, J = 3.4 Hz, H5 ), 1.10 (s, 9H, (CH3)3CSi/2),
J = 2.1 Hz, Ha), 7.53 (dd, 1H, J = 2.1 Hz and
J = 8.6 Hz, Hc), 7.17 (d, 1H, J = 8.6 Hz, Hd), 5.92–
0.93. 0.88 and 0.87 (s, 27H, (CH3)3CSi), 0.16, 0.14,
0.13, 0.07 and 0.02 (s, 18H, (CH3)2Si); 13C NMR
(CDCl3) d 170.3 (CO2 allyl), 150.8 (Ce), 141.9 (Cf),
137.3 (CHo/m/2Si), 136.6 (Cb), 134.8 (Cc), 133.3 and
133.2 (Cqarom/2Si, CH2@CH), 131.7 (CHp/2Si),
129.6 (CHo/m/2Si), 124.9 (Ca), 120.7 (CH2@CH), 118.5
5.78 (m, 1H, CH2@CH–CH2), 5.59 (d, 1H, J = 5.6 Hz,
0
00
H1 ), 5.32–5.19 (dt, 2H, J = 10.3 Hz and J = 1.4 Hz,
CH2@CH), 5.09 (s, 2H, CH2Oarom), 4.60 (d, 2H,
00
J @ 5.8 Hz, CH@CH2O), 4.49 (d, 1H, J = 1.4 Hz, H3 ),
00
4.40 (s, 1H, H4 ), 4.05 (d, 1H, J = 5.6 Hz, H2 ), 3.85
00
00
00
00
00
(Cd), 101.3 ðC1 Þ, 80.5 ðC3 Þ, 79.2 ðC5 Þ, 77.0 ðC2 Þ,
00
(d, 1H, J = 3.5 Hz, H5 ), 3.79 (s, 2H, CH2OH), 3.46 (s,
00
74.1 ðC4 Þ, 67.8 (CH–CH2O), 66.0 (CH2arom), 28.6
2H, CH2N), 2.99 (s, 3H, NCH3), 0.95, 0.92, 0.86, 0.84
and 0.83 (s, 27H, (CH3)3CSi), 0.18–0.01 (s, 18H,
(CH3)2Si); 13C NMR (CDCl3) d 168.5 (CO2 allyl),
157.3 (OCONCH3), 150.0 (Ce), 140.2 (Cf), 133.8 (Cb),
131.7 (Cc), 130.8 and 130.6 (CH2@CH), 125.4 (Ca),
((CH3)3CSi/2), 27.6 and 27.5 ((CH3)3CSi), 21.0
((CH3)3CSi/2), 19.8 and 19.7 ((CH3)3CSi), ꢁ2.6, ꢁ2.7,
ꢁ2.8, ꢁ2.9, ꢁ3.1 and ꢁ3.2 ((CH3)2Si); m/z [CI+NH3]
983.5 (M+NH4)+.
00
00
119.1 (CH2@CH), 117.2 (Cd), 99.4 ðC1 Þ, 78.9 ðC3 Þ,
5.2. Allyl {(2-nitro-4-hydroxymethylphenyl)-2,3,4-tri-(O-
tert-butyldimethylsilyl)-b-D-glucopyranosid}uronate (7)
00
00
00
76.9 ðC5 Þ, 75.4 ðC2 Þ, 72.4 ðC4 Þ, 68.2 (CH–CH2O),
65.8 (CH2arom), 61.2 and 60.7 (CH2OH), 52.1 and 51.1
(CH2N), 35.9 and 35.5 (NCH3), 26.8, 26.3 and 24.9
((CH3)3CSi), 18.3, 18.2 and 18.1 ((CH3)3CSi), ꢁ4.4
(2C) and ꢁ4.7 ((CH3)2Si); m/z [CI+NH3] 846.2
(M+NH4)+.
To a solution of allyltetrasilyl 6 (0.1 g, 0.1 mmol) in
DMF (5.4 mL) and acetic acid (6.2 lL, 0.1 mmol) under
argon, TBAF (1 M in THF, 0.103 mL, 0.15 mmol) was
added and stirred at room temperature for 2 h. The
solution was extracted with ether (6· 20 mL). The ethe-
real layer was washed with saturated NaHCO3 (3·
10 mL), then with water. The organic layer was dried
over MgSO4 and evaporated. The crude residue was
purified by chromatography (MeOH/CH2Cl2 2:8). A
5.4. 1-{N-Methyl-N-allyl-[4-(2,3,4-tri-O-tert-butyldime-
thylsilyl-b-D-glucopyranosyl)uronate-3-nitrobenzyloxy-
carbonyl]-2-amino-ethoxycarbonyl}-pyrrolidine-2,5-dione
(10)
white solid (66 mg, 88.0%) was isolated; Rf = 0.261
To a solution of silyl compound 9 (311 mg, 0.375 mmol)
in dry dichloromethane, N,N-disuccimidylcarbonate
(480 mg, 2.25 mmol, three times 2 equiv every 2 h, added
by small portions), DMAP (91.6 mg, 0.75 mmol,
2 equiv) and (iPr)2NEt (163.3 lL, 0.973 mmol,
2.5 equiv) were added. The reaction mixture was stirred
20
D
(cyclohexane/EtOAc 7:3); mp 136 ꢂC; ½aꢀ ꢁ19.2 (c
1
1.65, CHCl3); H NMR (300 MHz, CDCl3) d 7.81 (d,
1H, J = 1.9 Hz, Ha), 7.52 (dd, 1H, J = 2.0 Hz and
J = 8.6 Hz, Hc), 7.16 (d, 1H, J = 8.6 Hz, Hd), 5.92–
5.79 (m, 1H, CH2@CH–CH2), 5.58 (d, 1H, J = 5.7 Hz,

A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
5017
overnight at room temperature. After evaporation, the
residue was purified by chromatography on a silica gel
column (CH2Cl2/EtOAc 1:1) affording 288 mg (79%)
137.1, 133.5, 132.8, 131.9 and 129.2 (Cqarom and C11),
131.4, 130.2, 129.0, 128.7, 128.6, 128.4 and 127.1
(CHarom, Cc, CH2@CH), 126.6 (Ca), 118.8 (CH2@CH),
00
of compound 10 as an oil; Rf = 0.465 (cyclohexane/
116.8 (Cd), 99.3 ðC1 Þ, 84.4 (C5), 81.0 (C4), 78.9 (C1),
20
D
EtOAc 1:1); ½aꢀ ꢁ14.7 (c 1.02, CHCl3); 1H NMR
78.7 ðC3 Þ, 76.6, 76.4 and 75.5 (C10, C2 , C2 , C7, C4
00
0
00
00
00
(300 MHz, CDCl3) d 7.84 (d, 1H, J = 1.6 Hz, Ha), 7.53
(d, 1H, J = 8.1 Hz, Hc), 7.17 (d, 1H, J = 8.1 Hz, Hd),
5.83 (m, 1H, CH2@CH–CH2), 5.58 (d, 1H, J = 5.3 Hz,
and C5 Þ, 75.1 (2C) (C20, C2), 72.1 (C13), 66.5 (CH2O-
CO), 65.9 (CH–CH2O), 65.6 (CH2arom), 58.4 (C8), 52.7
0
ðC3 Þ, 47.3 and 46.2 (CH2N), 45.5 (C3), 43.1 (C15), 35.6
00
H1 ), 5.22 (m, 2H, CH2@CH), 5.12 (d, 2H, J = 4.5 Hz,
(2C) (NCH3 and C6), 26.7 (C16), 25.8 and 25.7
((CH3)3CSi), 22.6 (C14), 22.1 (CH3Ac4), 25.8 and 25.7
((CH3)3CSi), 20.9 (C17), 20.8 (CH3Ac10), 17.9
((CH3)3CSi), 14.7 (C18), 9.6 (C19), ꢁ4.5, ꢁ4.6, ꢁ4.7,
ꢁ5.0 and ꢁ7.0 ((CH3)2Si); m/z [FAB+NaI] 1730.2
(M+Na)+, 1731.4 (M+Na+1)+ (Found M+Na+,
1730.7131, C86H117N3O27Si3Na requires 1730.7080).
CH2Oarom), 4.58 (d, 2H, J = 5.6 Hz, CH@CH2O), 4.49
00
00
(s, 2H, H3 and H4 ), 4.39 (s, 2H, CH2OCO), 4.03 (d,
00
00
1H, J = 5.3 Hz, H2 ), 3.84 (d, 1H, J = 3.3 Hz, H5 ),
3.61 (m, 2H, CH2N), 3.01 (s, 3H, NCH3), 2.83 (s, 4H,
CH2–CH2), 0.92, 0.86 and 0.82 (s, 27H, (CH3)3CSi),
0.15, 0.13, 0.11, 0.05 and ꢁ0.00 (s, 18H, (CH3)2Si);
13C NMR (CDCl3) d 168.6, 168.5 and 168.3 (CO esters,
CO anhydride), 155.9 and 155.6 (OCO and
OCONCH3), 151.5 (Ce), 149.7 (Cf), 140.0 (Cb), 133.8
(Cc), 131.4 (CH2@CH), 125.5 (Ca), 118.9 (CH2@CH),
5.6. 20-O-{[N-Methyl-N-allyl-4-(b-D-glucopyranosiduro-
nate)-3-nitrobenzyloxycarbonyl]-2- amino-ethyl}-oxycar-
bonyl-paclitaxel (12)
00
00
00
116.6 (Cd), 99.2 ðC1 Þ, 78.7 ðC3 Þ, 76.7 ðC5 Þ, 75.1
00
00
ðC2 Þ, 72.1 (CH2OCO), 69.1 ðC4 Þ, 66.0, 65.8 and 65.6
(CH2arom and CH–CH2O), 48.3 (CH2N), 35.9 (NCH3),
25.8, 25.7 and 25.4 ((CH3)3CSi) and (CH2–CH2anhyd),
17.9 (3C) ((CH3)3CSi), ꢁ4.5, ꢁ4.6, ꢁ4.7, ꢁ5.0 and
ꢁ5.1 ((CH3)2Si); m/z [CI+NH3] 987.5 (M+NH4)+.
To 115 mg (0.067 mmol) of the coupling compound 11,
dissolved in 1.5 mL of anhydrous pyridine at 0 ꢂC, was
added, dropwise, at 0 ꢂC, 1 mL of a 70% HF–pyridine
complex. After 2 h at 0 ꢂC, the mixture was allowed to
reach room temperature and stirring was continued for
26 h. The reaction mixture was cooled to 0 ꢂC and
15 mL of saturated NaHCO3 was added. The medium
was then extracted with EtOAc (4· 10 mL), washed with
water, dried over MgSO4, concentrated and chromato-
graphed on a silica gel (CH3CN/toluene 1:1 then 8:2).
5.5. 20-O-{N-Methyl-N-allyl-[4-(2,3,4-tri-O-tert-butyl-
dimethylsilyl-b-D-glucopyranosyl)uronate-3-nitrobenzyl-
oxycarbonyl]-2-amino-ethyl}-oxycarbonyl-paclitaxel (11)
Carbonate 10 (200 mg, 0.206 mmol) and 50.3 mg
(0.412 mmol, 2 equiv) of DMAP were dissolved in
20 mL of freshly distilled dichloromethane. Under ar-
gon, 123 mg (0.144 mmol) of Taxol and then 50 lL
(0.412 mmol, 2 equiv) of triethylamine were added.
After 24 h, the solution was diluted with CH2Cl2
(25 mL), washed with water, brine, dried over MgSO4
and concentrated. After purification on a silica gel col-
umn (CH2Cl2/EtOAc 1:1), 230 mg (93%) of the coupling
A white solid (69 mg, 75%) was isolated; Rf = 0.245
20
D
(CH2Cl2/MeOH 95:5); mp 116 ꢂC; ½aꢀ ꢁ20.2 (c 1.0,
1
CHCl3); H NMR (300 MHz, CDCl3) d 8.15 (d, 2H,
J = 7.4 Hz, HoBz2), 7.72 (m, 3H, HmBz and Ha), 7.53–
7.13 (m, 13H, Harom), 6.31 (m, 2H, H10 and H13), 6.03
0
(d, 1H, J = 1.9 Hz, H3 ), 5.91 (m, 1H, CH2@CH–CH2),
5.69 (d, 1H, J = 6.8 Hz, H2), 5.47 (d, 1H, J = 1.9 Hz,
0
H2 ), 5.31 (m, 2H, CH2@CH), 4.98 (m, 4H, H5, CH2Oar-
00
and H ), 4.70 (d, 2H, J = 5.2 Hz, CH@CH2O), 4.50
om
1
00
(m, 1H, H3 ), 4.32–4.21 (m, 4H, H4 , CH2OCO and H7),
00
compound was isolated as a white solid; Rf = 0.340
20
D
00
00
00
(CH2Cl2/MeOH 97:3); mp 128–129 ꢂC; ½aꢀ ꢁ38.4ꢂ (c
4.18–3.61 (m, 7H, H20, H3, H5 , H2 , 2OH ), 3.51 (m,
2H, CH2N), 3.13 (m, 1H, OH0), 2.91 (d, 3H,
J = 3.4 Hz, NCH3), 2.56 (m, 1H, H6a), 2.48 (s, 3H,
CH3Ac4), 2.35 (s, 3H, CH3Ac10), 1.99 (s, 2H, H14),
1.91 (s, 3H, H18), 1.68 (s, 3H, H19), 1.44 (m, 1H, H6b),
1.21 (s, 3H, H16), 1.13 (s, 3H, H17); 13C NMR (CDCl3)
d 203.8 (C9), 171.2, 170.0, 168.0, 167.9, 167.2 and 166.9
1
0.85, CHCl3); H NMR (300 MHz, CDCl3) d 8.15 (d,
2H, J = 7.3 Hz, HoBz2), 7.75 (m, 3H, HmBz and Ha),
7.59 (d, 1H, J = 7.2 Hz, Hc), 7.53–7.25 (m, 11H, Harom),
7.14 (m, 1H, Hd), 6.29 (s, 2H, H10 and H13), 6.01 (m,
0
1H, H3 ), 5.82 (m, 1H, CH2@CH–CH2), 5.69 (d, 1H,
00
J = 7.02 Hz, H2), 5.59 (d, 1H, J = 5.6 Hz, H1 ), 5.43
0
0
0
(d, 1H, J = 1.9 Hz, H2 ), 5.25 (m, 2H, CH2@CH), 4.99
(d, 3H, J = 9.1 Hz, H5 and CH2Oarom), 4.59 (d, 2H,
(MeCO(C4), MeCO(C10), /CO2, /CON3 , C1 , CO2 al-
lyl), 155.8, 155.7 and 154.0 (OCO and OCONCH3),
149.7 (Cf), 142.4 (C12), 140.3, 136.6, 133.6, 132.8,
132.0 and 129.2 (Cqarom and C11), 131.1, 130.2, 129.0,
128.7, 128.6, 128.5, 128.2, 127.2, 127.0, 126.6, 125.2,
124.7 and 124.5 (CHarom, Ca, Cc, CH2@CH), 119.0
00
J = 5.5 Hz, CH@CH2O), 4.49 (s, 1H, H3 ), 4.40 (s, 1H,
00
H4 ), 4.32 (d, 2H, J = 8.2 Hz, CH2OCO), 4.21 (m, 1H,
H7), 4.12 (2H, ABq, J = 7.1 Hz, H20), 4.04 (d, 1H,
00
00
J = 5.3 Hz, H2 ), 3.85 (m, 2H, H3 and H5 ), 3.51 (m,
2H, CH2N), 2.91 (s, 3H, NCH3), 2.55 (m, 1H, H6a),
2.46 (s, 3H, CH3Ac4), 2.40 (m, 1H, H14), 2.22 (s, 3H,
CH3Ac10), 2.20 (m, 1H, H14), 1.93 (s, 3H, H18), 1.84
(m, 1H, H6b), 1.67 (s, 3H, H19), 1.24 (s, 3H, H16), 1.13
(s, 3H, H17), 0.92, 0.86 and 0.83 (s, 27H, (CH3)3CSi),
0.15, 0.13, 0.11, 0.06, 0.03 and ꢁ0.00 (s, 18H, (CH3)2Si);
13C NMR (CDCl3) d 203.7 (C9), 171.1, 169.8, 168.2,
167.8, 167.2 and 166.8 (MeCO(C4), MeCO(C10), /
00
(CH2@CH), 118.7 (Cd), 102.4 ðC1 Þ, 84.4 (C5), 81.0
0
00
(C4), 79.0 (C1), 75.6, 75.0 and 74.8 (C10, C2, C2 , C2 ,
00
C7 and C4 ), 72.8, 72.0 (2C) and 70.9 (C13, C20, C3
00
00
and C5 ), 66.3 and 65.4 (CH2OCO, CH–CH2O,
0
CH2arom), 58.3 (C8), 52.7 ðC3 Þ, 47.5 and 46.3 (CH2N),
45.7 (C3), 43.1 (C15), 35.8 and 35.5 (NCH3), 33.8 (C6),
26.7 (C16), 24.3 (C17), 22.6 (C14), 22.1 (CH3Ac4), 20.8
(CH3Ac10), 14.7 (C18), 9.7 (C19); m/z [FAB+NaI]
1388.6 (M+Na)+ (Found M+Na+ 1388.4526,
C68H75N3O27Na requires 1388.4486).
0
0
CO2., /CON3 , C1 , CO2 allyl), 155.9, 155.8 and 144.9
(OCO and OCONCH3), 149.7 (Cf), 142.5 (C12), 140.1,

5018
A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
5.7. 20-O-{[N-Methyl-N-4-(b-D-glucopyranosiduronate)-
3-nitrobenzyloxycarbonyl]-2-amino-ethyl}-oxycarbonyl-
paclitaxel (3)
00
H5), 4.97 (s, 1H, H1 ), 4.68 (m, 3H, CH2Oarom and
00
H7), 4.32–4.23 (m, 5H, H20, CH2OCO and H5 ), 3.80
00
00
00
(m, 1H, H3), 3.53 (m, 5H, CH2N, H2 , H3 , H4 ), 2.89
(s, 3H, NCH3), 2.45 (m, 4H, H6a and CH3Ac4), 2.14
(m, 2H, H14), 1.93–1.80 (m, 7H, CH3Ac10, H18 and
H6b), 1.64 (s, 3H, H19), 1.12 (s, 6H, H16 et H17); RMN
13C (MeOD) d 203.7 (C9), 170.2, 169.9, 168.8 and
Allyl ester 12 (30 mg, 0.022 mmol) was dissolved in 1 mL
of THF, and then was added a mixture of Et3N (9.2 lL,
0.066 mmol)–AcOH (0.88 lL, 0.044 mmol) in 23 lL
THF. After bubbling of argon gas for 10 min, a few crys-
tals of palladium tetrakistriphenylphosphine were added.
After 30 min understirringat room temperature(no start-
ing material was detected on TLC), the volatiles were re-
moved in vacuo, and the residue was purified on silica gel
(CH3CN/H2O 95:5). Lyophilisation afforded 23.2 mg
0
166.2 (2C) (MeCO(C4), MeCO(C10), /CO2, /CON3 ,
0
C1 , CO2), 156.4 (2C), 153.9 and 149.4 (OCO and
OCONCH3), 149.4 (Cf), 140.8, 140.3 and 136.6 (Cqar-
om), 133.9, 133.5, 133.2, 131.5, 131.2, 129.9, 129.8,
128.7, 128.4, 127.1, 126.6 and 124.1 (CHarom), 117.7
00
and 117.5 (Cd, Cc, Ca), 101.0 ðC1 Þ, 84.4 (C5), 80.8
(80%) of prodrug 3 as a white solid; Rf = 0.197
(C4), 77.5 (C1), 77.2, 76.0 (2C) and 75.3 (C20, C10, C2,
20
D
0
00
00
00
C2 , C3 , C5 ), 74.8, 71.8 and 70.8 (C13, C7, C2 and
(CH3CN/H2O 9:1); mp 180 ꢂC; ½aꢀ ꢁ56 (c 0.9, MeOH);
1
0
00
C4 ), 68.6, 65.8 and 65.4 (CH2OCO, CH2arom), 57.7
H NMR (300 MHz, MeOD) d 9.12 (m, 1H, NH3 ), 8.13
0
(d, 2H, J = 6.9 Hz, HoBz2), 7.77–7.24 (m, 16H, Harom,
Ha, Hc, Hd), 6.45 (s, 1H, H10), 6.08 (m, 1H, H13), 5.83
0
(m, 1H, H3 ), 5.65 (d, 1H, J = 6.8 Hz, H2), 5.45 (m, 1H,
(C8), 53.7 ðC3 Þ, 47.0 and 46.7 (C3, CH2N), 43.1 (C15),
36.1 (C6), 35.0 (NCH3), 25.5 (C16), 22.8, 21.8 and 20.9
(C17, C14), (CH3Ac4), 19.4 (CH3Ac10), 13.6 (C18), 9.0
(C19); m/z [MALDI] 1296.54 (M+H)+ (Found M+Na+,
1318.4459, C65H73N3O25Na requires 1318.4431).
0
H2 ), 5.15 (d, 1H, J = 5.7 Hz, H5), 4.98 (s, 1H, H1 ), 4.36
00
(m, 3H, CH2Oarom and H7), 4.18 (s, 2H, H20), 3.99 (d,
00
1H, J = 8.9 Hz, H5 ), 3.82 (d, 1H, J = 6.8 Hz, H3), 3.72
00
(m, 2H, CH2OCO), 3.61–3.39 (m, 5H, CH2N, H2 ,
5.9. In vitro cytotoxicity
00
00
H3 ,H4 ), 2.90 (s, 3H, NCH3), 2.47–2.40 (m, 4H, H6a
and CH3Ac4), 2.25 (m, 2H, H14), 2.02 (s, 3H, CH3Ac10),
1.94 (m, 3H, H18), 1.86 (m, 1H, H6b), 1.64 (s, 3H, H19),
1.13 (s, 6H, H16 and H17); 13C NMR (MeOD) d 203.7
(C9), 170.2, 169.9, 168.8 (2C) and 166.2 (MeCO(C4), Me-
HT 29 (human colorectal adenocarcinoma) cells were
grown in RPMI 1640 (+)(L)-glutamine medium supple-
mented with 10% (v/v) foetal calf serum, 100 UI/mL
penicillin, 100 lg/mL streptomycin and 1.5 lg/mL
fungizone and kept under 5% CO2 at 37 ꢂC.
0
0
CO(C10), /CO2, /CON3 , C1 , CO2), 156.4 (2C), 153.9
and 149.7 (OCO and OCONCH3), 149.6 (Cf), 140.8
(2C), 136.6 (Cqarom), 133.9, 133.5, 133.2, 132.3, 131.7,
131.5, 131.0, 129.9, 129.8, 128.7, 128.4, 128.3, 128.1 and
127.1 (CHarom), 124.1 and 117.4 (Cd, Cc, Ca), 100.9
Ninety-six-well plates were seeded with 800 HT 29 cells
per well in 200 lL medium.
00
ðC1 Þ, 84.4 (C5), 80.8 (C4), 77.5 (C1), 77.1, 76.2, 76.0 and
Twenty-four hours later, chemicals dissolved in DMSO
were added for 72 h at a final concentration ranging be-
tween 0.5 nM and 10 lM in a fixed volume of DMSO
(1% final concentration). Controls received an equal vol-
ume of DMSO. The number of viable cells was measured
at 490 nm with the MTS reagent (Promega, Madison, WI)
and IC50 was calculated as the concentration of com-
pound eliciting a 50% inhibition of cell proliferation.
0
75.3 (C20, C10, C2, C2 , C3 , C5 ), 74.8, 73.0 (2C), 71.8
00
and 70.8 (C13, C7, C2 and C4 ), 65.4 and 65.4 (CH2OCO,
00
00
00
0
CH2arom), 57.7 (C8), 53.7 ðC3 Þ, 47.0 and 46.7 (C3, CH2N),
43.1 (C15), 36.0 (C6), 35.0 (NCH3), 25.5 and 25.0 (C16),
21.8 (2C) and 20.9 (C17, C14), (CH3Ac4), 19.4 (CH3Ac10),
13.5 (C18), 9.0 (C19); m/z [ESIꢁ] 1324.24 (M+H)+ (Found
M+Na+,
1348.4121,
C65H71N3O27Na
requires
1348.4173).
5.10. HPLC conditions
5.8. 20-O-{[N-Methyl-N-4-(b-D-glucopyranosiduronate)-
3-aminobenzyloxycarbonyl]-2-amino-ethyl}-oxycarbonyl-
paclitaxel (4)
Analysis was carried out on a reverse-phase column
(Lichrospher RP18e, 125 · 4 mm, 5 lm) 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).
Allyl ester 12 (40 mg, 0.029 mmol) was dissolved in
1.5 mL THF, followed by addition of Et3N (12.3 lL,
0.087 mmol)–HCOOH (0.88 lL, 0.058 mmol) mixture
in 25 lL THF. After bubbling of argon gas for
10 min, a few crystals of palladium tetrakistriphenyl-
phosphine were added. After 30 min under stirring at
room temperature (no starting material was detected
on TLC), the volatiles were removed in vacuo, and the
residue was purified on silica gel (eluent CH3CN/H2O
95:5). Lyophilisation afforded 31 mg (81%) of prodrug
5.11. Stability of compounds in a buffer solution
A solution of 190 lM of prodrug in 0.02 M phosphate
buffer (pH 7.2) or in 10% FCS (fetal calf serum) in phos-
phate buffer was incubated at 37 ꢂC. Aliquots (50 lL)
were taken at various times and analysed by HPLC after
dilution with eluent (150 lL).
4 as a white solid; Rf = 0.157 (CH3CN/H2O 9:1); mp
20
178–179 ꢂC; ½aꢀ ꢁ65 (c 1.25, MeOH); 1H NMR
5.12. Enzymatic cleavage by Escherichia coli
b-D-glucuronidase
D
0
(300 MHz, MeOD) d 9.20 (m, 1H, NH3 ), 8.12 (d, 2H,
J = 5.9 Hz, HoBz2), 7.97–7.25 (m, 16H, Harom), 6.45 (s,
0
1H, H10), 6.08 (m, 1H, H13), 5.85 (m, 1H, H3 ), 5.64
0
(d, 1H, J = 5.1 Hz, H2), 5.42–5.16 (m, 2H, H2 and
A solution of 190 lM of prodrug in 0.02 M phosphate
buffer (pH 7.2) was incubated at 37 ꢂC in the presence

A. E. Alaoui et al. / Bioorg. Med. Chem. 14 (2006) 5012–5019
5019
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Acknowledgments
We thank Sylvie THIROT for the HPLC measurements
and Thierry CRESTEIL (Institut de Chimie des Sub-
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