Prodrug Mono Therapy: Synthesis and biological evaluation of an etoposide glucuronide-prodrug

Article abstract of DOI:10.1016/S0968-0896(03)00108-1

A glucuronide-based prodrug of etoposide has been synthesized for a Prodrug Mono Therapy strategy. The aim is to selectively liberate the active compound by β-D-glucuronidase already present in necrotic tumours. Outside from these sites, this enzyme is kn

Full text of DOI:10.1016/S0968-0896(03)00108-1

Bioorganic & Medicinal Chemistry 11 (2003) 2277–2283
Prodrug Mono Therapy: Synthesis and Biological
Evaluation of an Etoposide Glucuronide-Prodrug
Frederic Schmidt* and Claude Monneret
UMR 176 CNRS/Institut Curie, Section Recherche, 26, rue d’Ulm, 75248 Paris Cedex 05, France
Received 3 December 2002; accepted 4 February 2003
Abstract—A glucuronide-based prodrug of etoposide has been synthesized for a Prodrug Mono Therapy strategy. The aim is to
selectively liberate the active compound by b-d-glucuronidase already present in necrotic tumours. Outside from these sites, this
enzyme is known to be localised inside the lysosomes. The three components of this prodrugare the glucuronic acid (substrate of
the enzyme), the spacer (for a faster cleavage), and the active etoposide. In vitro, the prodrug was shown to be less cytotoxic and
more water-soluble than etoposide itself. Finally, in the presence of the b-d-glucuronidase, cleavage of the prodrug with complete
release of the drughas been observed.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
active.6ꢀ8 It was unambiguously shown, by enzyme histo-
chemistry, that necrotic areas in human cancers are the
site in which lysosomal b-glucuronidase was liberated
extracellularly in high local concentration. According to
investigations, extracellular b-glucuronidase originates
from monocytes and granulocytes concentrated within
the necrotic areas. Based on these facts, a new ther-
apeutic procedure called ProdrugMono Therapy
(PMT) was proposed.6ꢀ8 Further investigations
showed, in the case of a human perfused lung, that a
doxorubicin prodrug(HMR1826) could release the free
doxorubicin due to the presence of enzymatically active
‘extralysosomal’ b-glucuronidase.9 The level of doxo-
rubicin after lungperfusion with HMR1826 was about
7-fold higher than after perfusion with doxorubicin
itself.
Lack of selectivity is a major limitation in cancer
chemotherapy. Most drugs are not selective enough to
be used in optimal doses without severe drawbacks. A
solution is to target the active drugs to the cancer cells.
Many trials usingfor instance antibody-drughave been
conducted but without real success1 until the recently
approved mylotarg.2 Indeed, a number of problems
have to be solved such as the stability of the conjugate
and its tumour penetration, internalisation and anti-
tumour activity.3
Prodrugs can be used in order to release the active drug
from an inactive precursor. In this particular case,
selectivity can be managed by an enzymatic cleavage.
The enzyme can be targeted in a first step at the tumour
site by a Mab-antigen recognition, like in an Antibody
Directed Enzyme ProdrugTherapy (ADEPT) strategy.
The second step will be the injection of the prodrug,
which results in the liberation of the drug.
All these results led us to consider this strategy as
available to increase the delivery of oncostatic drugs in
human tumours.10
4
Etoposide 1, or VP-16, is a semi-synthetic derivative of
podophyllotoxin,11,12 which exerts its antitumour
activity by stabilization of the ternary complex involv-
A more convenient strategy is the use of an already
present enzyme.5 The prerequisite is to identify an
enzyme specifically localized around the tumour cells.
Indeed, a lysosomal enzyme, b-glucuronidase, is liber-
ated extracellularly in necrotic tumours and remains
13ꢀ15
ingthe drug, DNA and Topoisomerase II. Estab-
lished indications of etoposide are testicular and small-
16ꢀ18
cell lungcancer,
as well as the use in paedia-
trics for the treatment of neuroblasma. Cancer leu-
kaemia, and Kaposi’s sarcoma represent other
clinical indications.
*Correspondingauthor. Tel.: +33-1-4234-6664; fax: +331-4234-6631;
e-mail: frederic.schmidt@curie.fr
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00108-1

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F. Schmidt, C. Monneret / Bioorg. Med. Chem. 11 (2003) 2277–2283
In spite of this widespread clinical use, amonglimi-
tations is the very poor water-solubility of the drug.
Formulation with Tween1 80, polyethylene glycol
and ethanol results in acute mortality. To solve this
problem, in the 19900s, the group of Bristol-Myers
Squibb initiated a program to discover an appro-
priate prodrug. This led to the development of eto-
poside phosphate 2, BMY-404811.19ꢀ21 Etopophos is
rapidly converted to the parent drugin vivo and,
therefore, has been introduced in clinics with the same
profile as etoposide itself. On this account, although
esterification of the phenol function significantly
decreases, not only the activity but also the toxicity,
both are almost restored by enzymatic cleavage. This
indicates that there was no marked gain in selectivity
with this type of prodrug.
to obtain a water-soluble compound and a selective
liberation of the drug 1 at the necrotic tumours due
to the presence of active b-glucuronidase within these
areas.
Synthesis
In the prodrug 3, etoposide was connected to the b-d-
glucuronic moiety by a self-immolative spacer. The role
of the spacer is to facilitate the enzymatic cleavage of
the prodrugas it was shown that, in some cases, direct
connection gave a bad enzymatic hydrolysis turnover.
We chose a spacer already used in the laboratory for
other drugs, with success for the kinetics results.25ꢀ27
The mechanism of drugrelease was planned as in
Scheme 1.
For the other two types of prodrugs of etoposide
includinga carbamate or carbonate spacer at 4 0 recently
described,22,23 the release of the drugwas induced by a
retro-aldol/retro-Michael reaction catalysed by an
aldolase antibody 38C2 and by carboxylesterases,
respectively.
For the synthesis of prodrug 3, we could not use methyl
25,26
esters and acetate as protectingrgoups.
For the
removal of the ester groups from the b-d-glucuronic
moiety, the basic conditions are not compatible with the
etoposide structure. Even under slightly basic condi-
tions, podophyllotoxin derivatives are known to epi-
merise and give picropodophyllins which are
unfortunately inactive (Scheme 2).28,29
The main problems of the synthesis were the protecting
groups and the selectivity of fixation between the differ-
ent hydroxyl groups present in the etoposide structure.
Our first choice was silylated groups for both hydroxyl
and carboxyl groups of the glucuronic acid. The alco-
hols were transformed into tert-butyldimethylsilyl
ethers, and the acid into a trimethylsilylethanol ester.30,31
The last deprotection step was conducted with fluoride
anion. As we could not use the basic TBAF, we tried HF/
Pyr or HF/NEt3. Both reagents permit deprotection of
24
In this paper followingour patent, we report the
synthesis of a prodrugof etoposide 3. Our aim was
Scheme 1. Release of etoposide from prodrug 3.

F. Schmidt, C. Monneret / Bioorg. Med. Chem. 11 (2003) 2277–2283
2279
Biological Activity
Solubility
In water and under comparable conditions, prodrug 3 is
approximately 200-fold more soluble than the corre-
spondingdrug. Thus, while the solubility of etoposide is
about 0.1 mg/mL, the solubility of prodrug 3 is about
20 mg/mL.
Cytotoxicity
Scheme 2. Epimerisation of podophyllotoxin.
On L1210 cell line, prodrug 3 gave an IC50 value of 50.2
mM. After hydrolysis by b-glucuronidase (100 mg/mL),
increased cytotoxicity was obtained with an IC50 value
of 0.93 mM, which was very closely related to the IC50
value of the etoposide itself (0.834 mM). This means that
the prodrugwas detoxified by a factor of about 50.
the alcohols but we did not succeed in removingthe
silylated ester. For this deprotection, only TBAF was
described.30,31
Finally, we obtained the prodrug 3 following Scheme 3.
Stability
The hydroxyl protectingrgoups were kept, but the
acid was protected as a benzyl ester. The inter-
mediate 526 was deprotected and then reprotected as
TBDMS ethers for the hydroxyl groups and as a
benzyl ester for the carboxylic acid. The amine
function of 7 was activated with phosgene and then
coupled to etoposide 1 at the phenolic hydroxyl
group. Under the controlled conditions used (over-
stœchiometric DMAP and one equivalent carbamoyle
chloride), the alcohol groups of the etoposide glucose
moiety did not react and only the 40-OH phenol group
was coupled. Deprotection of the glucuronide moiety
was then achieved to convert 9 into prodrug 3. The
TBDMS groups were removed with HF/Pyridine giving
10 and the benzyl ester with cyclohexadiene over palla-
dium.32,33 These conditions enabled to keep the aro-
The stability of 3 was followed by HPLC measurements
in buffer solution at pH 7.2 for 24 h. We could not detect
any evolution. So, in the absence of b-d-glucuronidase,
prodrug 3 is stable in vitro under these conditions.
Kinetics of drug release
Prodrug 3 (500 mg/mL, 0.521 mM) was incubated with
Escherichiacoli b-d-glucuronidase (20 mg/mL, 26.2
units/mL). Aliquot samples were analysed by HPLC at
different times (Fig. 1).
Examination of the curves indicated that the prodrug
was rapidly hydrolysed, the only products detected
beingthe etoposide 1 and the cyclised spacer 4. No
intermediate containingthe spacer still attached to the
etoposide was seen. This is consistent with a fast enzy-
matic cleavage (half-life of 3 is <25 min) and a fast
cleavage of the spacer.
matic
nitro
function,
contrary
to
classical
hydrogenation conditions which are known to reduce
the nitro function very well. The overall yield starting
from etoposide was 15%.
Scheme 3. Synthesis of prodrug 3.

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F. Schmidt, C. Monneret / Bioorg. Med. Chem. 11 (2003) 2277–2283
Electrospray 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. Chromato-
graphies were conducted over silica gel [Merck 60
(230–400 mesh)].
In vitro cytotoxicity
Cytotoxicity was tested against L1210 (mouse leukemic
cell line) cells usingthe MTA assay.
L1210 cells were cultivated in RPMI 1640 medium
(Gibco) supplemented with 10% fetal calf serum, 2
mM l-glutamine, 100 units/mL penicillin, 100 g/mL
streptomycin, and 10 mM HEPES buffer (pH=7.4).
Cytotoxicity was measured by the microculture tetra-
zolium assay (MTA). Cells were exposed to graded
concentrations of drug(nine serial dilutions in tripli-
cate) for 48 h. Results are expressed as IC50, the con-
centration which reduced by 50% the optical density of
treated cells with respect to the optical density of
untreated controls.
Figure 1. Enzymatic cleavage of prodrug 3.
It is also assumed that the liberated compound is indeed
etoposide and not picroetoposide, which was synthe-
sised followinga described procedure.
nation by comparison of the retention times was in
agreement with the fact that, during all the synthesis,
the trans-fused lactone was not epimerised into cis-fused
lactone.
34
HPLC exami-
For the cell cycle analysis, L1210 cells (5Â105 cells/
mL) were incubated for 21 h with various concentra-
tions of drugs. Cells were then fixed by 70% ethanol
(v/v), washed, and incubated in PBS containing100
mg/mL RNAse and 50 mg/mL propidium iodide for 30
min at 20 ꢁC. For each sample, 10,000 cells were ana-
lysed on a XLMCL flow cytometer (Beckman Coulter,
France).
As concentration of the enzyme in necrotic area is
dependent on the nature and localisation of the tumour,
it is difficult to predict whether the concentration nee-
ded for the cleavage of etoposide prodrug will be
reached in vivo. Moreover, it must be taken into con-
sideration that human b-d-glucuronidase is less efficient
than E. coli b-d-glucuronidase at physiological pH.35
Nevertheless, it has been established that doxorubicin
was liberated efficiently from the prodrugHMR1826 in
a human perfused lung.9
HPLC conditions
Good separation in a short delay was obtained with a
reversed-phase phenyl analytical column (Spherisorb
250Â4.6) usingisocratic conditions (1 mL/min) of 60%
phosphate buffer (0.02 M, pH 3) and 40% acetonitrile
with UV detection at 254 nm. Usingthese conditions,
the retention times of 1, 3, 4 were respectively 4.9, 3.4,
and 5.8 min.
Conclusions
The prodrug 3 as described has all the in vitro require-
ments for use in a PMT strategy in cancer chemo-
therapy. Less cytotoxic than the free drug, this prodrug
is stable at pH 7.2 and releases etoposide by the action
of b-d-glucuronidase. In vivo experiments are planned
to evaluate more precisely the enzymatic cleavage inside
necrotic tumours.
Stability of compounds in a buffer solution
A solution of 500 mL of prodrugin 0.02 M phosphate
buffer (pH 7.2) was incubated for various times at 37 ꢁC.
Aliquots (100 mL) were taken at various times and ana-
lysed by HPLC after dilution with eluent (300 mL).
Experimental
Meltingpoints (mp) were taken on a Koffler Bench and
are uncorrected. Optical rotations were obtained on a
Perkin-Elmer 241 polarimeter (589 nm). Specific rota-
tions ([a]D) are reported in deg/dm, and the concen-
tration (c) is given in g/100 mL in the specific solvent.
Infrared spectra were recorded on a Perkin-Elmer 1600
Enzymatic cleavage by E. coli ꢀ-D-glucuronidase
Hydrolysis was investigated by incubating a solution of
500 mg/mL (0.521 mM) of prodrug 3 and 20 mg/mL
(26.2 units/mL) of E. coli b-d-glucuronidase in 0.02 M
phosphate buffer (pH 7.2) at 37 ꢁC. Aliquots (100 mL)
were taken at various times and analysed by HPLC
after dilution with 300 mL of eluent.
1
FTIR spectrometer (n in cmꢀ1). H NMR (300 MHz)
and 13C NMR (75 MHz) spectra were recorded on Bru-
ker AC 300 spectrometer — chemical shifts d in ppm
and J in Hz. Chemical ionisation (CI-MS; NH3, positive
ion mode) or FAB (positive ion mode) mass spectra,
were recorded on a NermagR 10-10C spectrometer.
For the NMR description, the followingnumeration
was chosen: ‘a’ for aromatic, ‘e’ for Etoposide, ‘g’ for
glucose, ‘G’ for glucuronic acid.

F. Schmidt, C. Monneret / Bioorg. Med. Chem. 11 (2003) 2277–2283
2281
purified by two successive chromatographies, the first
with CH2Cl2, and the second with CH2Cl2/cyclohexane:
5/1. Compound 7 was obtained as a yellow resin (1.83 g,
44% from 6). C38H64NO9Si3; [a]D ꢀ2.3 (c 0.98 in
CHCl3); nmax/cmꢀ1(CDCl3) 1762 (C¼O ester), 1623
(aromatics), 1530, 1343 (NO2); dH (CDCl3) 7.53 (1H,
dd, Jo=9, Jm=3, a5), 7.35 (1H, d, Jm=3, a3), 7.33–7.26
(5H, Ph), 6.83 (1H, d, Jo=9, a6), 5.62 (1H, d, J=6,
G1), 5.11 (s, 2H, CH2Ph), 4.59 (1H, q, J=5, NH), 4.52
(1H, G3), 4.38 (1H, G4), 4.02 (1H, t, J=6, G2), 3.87
(1H, d, J=3.5, G5), 2.86 (3H, d, J=5, N–CH3), 0.91
(18H, Si–C–CH3), 0.86 (9H, Si–C–CH3), 0.15 (3H, Si–
CH3), 0.14 (3H, Si–CH3), 0.12 (6H, Si–CH3), 0.08 (3H,
Si–CH3), ꢀ0.01 (3H, Si–CH3); dC (CDCl3) 168.4 (G6),
148.8 (a1), 143.7 (a2), 140.5 (a4), 135.1 (Ph quaternary),
128.5–128.4–128.3 (Ph tertiary), 112.5 (a5), 112.0 (a6),
104.1 (a3), 98.9 (G1), 78.9 (G3), 77.2 (G5), 75.7 (G2),
72.1 (G4), 67.0 (CH2Ph), 29.4 (NMe), 25.7 (Si–C–CH3),
18.0–17.9 (Si–C–CH3)ꢀ4.5–4.6–4.7–5 (Si–CH3); m/z
(CI) 777 [M+H]+.
For the NMR attribution, some publications were very
helpful.36ꢀ39
2-Methylamino-4-nitrophenyl-ꢀ-D-glucopyranosiduronic
acid (6). To a solution of 526 (2 g, 4.13 mmol) in 50 mL
acetone at 0 ꢁC, a 1 N NaOH aqueous solution (50 mL)
Benzyl [2-(N-chloroformyl-N-methylamino)-4-nitrophe-
nyl-2,3,4-tri-O-(tert-butyldimethylsilyl)-ꢀ-D-glucopyrano-
sid]uronate (8). To a solution of 7 (350 mg, 0.45 mmol)
in 20 mL CH2Cl2 at 0 ꢁC, a solution of phosgene (700
mL, 1.35 mmol) in toluene was added. Then triethyla-
mine (1.13 mL, 8.16 mmol) was added dropwise. After
30 min at 0 ꢁC, the reaction was quenched with 10 mL
water. The organic phase was separated and washed
with 10 mL brine, dried over magnesium sulphate and
evaporated. The residue was purified by chromato-
graphy (EtOAc/cyclohexane: 1/13) to afford a colour-
less viscous oil (352 mg, 93%). C39H63N2O10ClSi3; [a]D
ꢀ5 (c 1 in CHCl3); nmax/cmꢀ1 (CDCl3), 1734 (C¼O
carbamoyle chloride), 1594 (aromatics), 1528, 1349
(NO2); dH (CDCl3) 8.24 (1H, dd, Jo=9, Jm=3, a5), 8.14
(1H, d, Jm=3, a3), 7.34–7.28 (5H, Ph), 7.22 (1H, d,
Jo=9, a6), 5.73 (1H, d, J=5.5, G1), 5.07–5.05 (2H, 2 s,
CH2Ph), 4.43 (1H, G3), 4.40 (1H, G4), 3.96 (1H, G2),
3.88 (1H, d, J=3, G5), 3.25 (3H, s, N–CH3), 0.95–0.86
(27H, Si–C–CH3), 0.08 (12H, Si–CH3), 0.07 (3H, Si–
CH3), 0.02 (3H, Si–CH3); dC (CDCl3) 168.0 (G6), 157.5
(NCOCl), 148.6 (a1), 142.1 (a2), 134.7 (a4), 133.1 (Ph
quaternary), 128.3–128.1–125.7 (Ph tertiary), 125.5 (a5),
115.9 (a6), 99.3 (a3), 99.2 (G1), 78.6 (G3), 76.4 (G5),
75.5 (G2), 71.6 (G4), 66.9 (CH2Ph), 45.5–44.2 (NMe),
25.6–25.5 (Si–C–CH3), 17.7–13.5–12.6 (Si–C–CH3)
ꢀ4.6–4.8–4.9–5.2 (Si–CH3); m/z (CI) 856 [M+NH4]+.
Benzyl [4-nitrophenyl-2-[(etoposide-40-O-carbonyl)methyl-
amino]-2,3,4-tri-O-(tert-butyldimethylsilyl)-ꢀ-D-glucopyr-
anosid]uronate (9). DMAP (124.5 mg, 1.035 mmol) was
added to a solution of 8 (0.51 g, 0.609 mmol) and eto-
poside (287 mg, 0.487 mmol) in CH2Cl2 (107 mL).
Triethylamine (0.14 mL, 1.035 mmol) was added drop-
wise, and the mixture was stirred for 16 h at room tem-
perature. After evaporation, the residue was purified by
chromatography (CH2Cl2/CH3CN: 8/2). A white solid
(0.39 g, 57%) was isolated. C68H94N2O23Si3; mp 171 ꢁC;
[a]D +1.3 (c 0.99 in CHCl3); nmax/cmꢀ1 (CDCl3) 1770
(CO ester), 1729 (CO carbamate), 1601 (aromatics),
1525, 1348 (NO2); dH (CDCl3) 8.67 (d, Jm=2, 1H, a3),
8.10 (dd, Jo=9, Jm=2, 1H, a5), 7.30 (5H, Ph 7.04 (d,
ꢁ
was added dropwise. After stirringfor 5 min at 0 C, the
mixture was neutralized with 1 N HCl at pH 4, evapo-
rated and purified by column chromatography
(CH3CN/H2O: 80/20). The solid was heated in boiling
methanol and filtered to eliminate silica. After evapora-
tion, a bright orange solid (100%) was obtained.
C13H16N2O9; mp 172 ꢁC; [a]D ꢀ53 (c 0.96 in MeOH);
n
max/cmꢀ1 (KBr) 3400 (O–H), 1588 (aromatics), 1530,
1343 (NO2); dH (DMSO) 7.46 (1H, dd, Jm=3, Jo=9,
a5), 7.19 (1H, d, Jm=3, a6), 7.12 (1H, d, Jo=9, a3), 6.04
(1H, q, J=5, N-H), 5.68 (1H, br, s, OH), 5.13 (1H, br, s,
OH), 4.85 (1H, d, J=7, G1), 3.57–3.17 (4H, G2, G3,
G4, G5), 2.80 (3H, d, J=5, N–CH3); dC (DMSO) 172.4
(G6), 149.8 (a1), 143.0 (a2), 140.5 (a4), 113.3 (a5), 111.6
(a6), 102.3 (a3), 101.4 (G1), 75.6, 74.1, 73.0, 72.1 (G2,
G3, G4, G5), 29.4 (NMe); m/z (ESꢀ) 343 [MꢀH]ꢀ.
2-Methylamino-4-nitrophenyl-2,3,4-tri-O-(tert-butyldime-
thylsilyl)-ꢀ-D-glucopyranosiduronic acid. DMAP (0.1 g)
was added to a solution of 6 (1.87 g, 5.43 mmol) in 20
mL pyridine. The mixture was cooled to 0 ꢁC and TBS
triflate (12 mL, 52.3 mmol) were added dropwise. After
48 h at room temperature, the mixture was evaporated
and the residue was taken in toluene (200 mL). The
insoluble pyridinium triflate was filtered, and the filtrate
evaporated. The product, obtained as a yellow resin
(3.68 g), was used crude in the next step. Any attempt of
purification by chromatography resulted in loss of the
compound.
Benzyl [2-methylamino-4-nitrophenyl-2,3,4-tri-O-(tert-bu-
tyldimethylsilyl)-ꢀ-D-glucopyranosid]uronate (7). DMAP
(0.3 g, 2.45 mmol) was added to the preceding crude
compound (3.64 g, 5.31 mmol) in 20 mL CH2Cl2. After
coolingto 0 ꢁC, benzyl alcohol (0.5 mL, 4.9 mmol) and
DCC (1.095 g, 5.31 mmol) were successively added.
After 12 h at room temperature, the mixture was eva-
porated and taken in cyclohexane (250 mL). The inso-
luble urea was filtered. The filtrate was evaporated and

2282
F. Schmidt, C. Monneret / Bioorg. Med. Chem. 11 (2003) 2277–2283
Jm=2, 1H, a6), 6.84 (s, 1H, e5), 6.55 (s, 1H, e8), 6.38–
6.22 (2H, e20, e60), 6.03 (d, J=1, 1H, e10A), 6.02 (s,
J=1, 1H, e10B), 5.77 (d, J=6, 1H, G1), 5.16 (AB,
J=12, 1H, CH2(A)Ph), 5.09 (AB, J=12, 1H,
CH2(B)Ph), 4.92 (d, J=3, 1H, g4), 4.77 (q, J=5, 1H,
g7), 4.68 (d, J=8, 1H, g1), 4.59 (d, J=5.5, 1H, e1), 4.52
(br, 1H, G3), 4.41 (br, 2H, e11A, G4), 4.22–4.15 (2H,
g6equ, e11B), 4.05 (d, J=6, 1H, G2), 3.91 (d, J=3.5,
1H, G5), 3.82–3.68 (7H, g3, OCH3), 3.59 (t, J=10, 1H,
g6ax), 3.44 (t, J=8, 1H, g2), 3.36 (1H, g5), 3.27 (5H, e2,
e4,N–CH3), 2.86 (m, 1H, e3), 1.39 (d, J=5, 3H, g8),
0.96 (9H, Si–C–CH3), 0.91 (9H, Si–C–CH3), 0.89 (9H,
Si–C–CH3), 0.21– (ꢀ0.02) (18H, Si–CH3); dC (CDCl3)
174.9 (e9), 168.4 (G6), 156.2, 153.5, 153.2, 152.1, 148.9,
147.4, 142.0, 137.6, 135.2, 132.7, 132.5 (C quaternary,
carbamate) 128.6, 128.5, 128.3 (Ph), 126.6 (a3), 123.6
(a5), 114.3 (a6), 110.9 (e8), 109.1 (e5), 106.8 (e20, e60),
102.1 (e10), 101.7 (g1), 99.9 (g7), 98.1 (G1), 79.8 (g5),
79.0 (G3), 77.0 (G5), 76.8 (G2), 74.6 (g2), 73.9 (g4), 73.1
(g3), 72.4 (G4), 68.1 (g6), 67.9 (e11), 67.0 (CH2Ph), 66.5
(e4), 56.0 (O-CH3), 44.0 (e1), 41.3 (e2, e3), 37.5, 37.0 (N-
CH3), 26.6, 25.9, 25.8, 25.7 (Si–C–CH3), 20.3 (g8), 18.1,
18.0, 17.9 (Si–C–CH3,), ꢀ4.2, ꢀ4.5, ꢀ4.6, ꢀ4.7, ꢀ4.9,
ꢀ5.7 (Si–CH3); m/z (FAB+) 1413 [M+Na]+.
(63.6 mg, 0.06 mmol) in ethanol (1.8 mL). The mixture
was stirred at 45 ꢁC for 15 h. After filtration over Celite
and evaporation, the crude product was purified by
chromatography (CH3CN/H2O: 90/10). The compound
3 was isolated as a beige powder (17 mg, 29%).
C43H46N2O23; mp 186 ꢁC; [a]D +6.5 (c 0.85 in MeOH);
n
max/cmꢀ1 (KBr) 3426 (O–H), 1770 (CO ester), 1717
(CO carbamate), 1603 (aromatics), 1505, 1378 (NO2);
dH (DMSO) 8.40 (1H, a3), 8.17 (d, J=9, 1H, a5), 7.46
(d, J=9, 1H, a6), 7.02 (s, 1H, e5), 6.55 (s, 1H, e8), 6.28
(2H, e20, e60), 6.02 (s, 2H, e10), 5.29 (2H, OH), 5.18 (1H,
G1), 4.95 (1H, OH), 4.72 (q, J=5, 1H, g7), 4.58 (2H, g1,
e1), 4.27 (1H, e11A), 4.08 (1H, e11B), 3.66 (s, 6H,
OCH3), 3.62–3.09 (14H, NCH3, e4, g2, g3, g4, g5, g6,
G2, G3, G4, G5), 3.07 (1H, e2), 2.91 (1H, e3), 1.24 (d,
J=5, 3H, g8); dC (DMSO) 175.2 (e9), 172.2 (G6), 158.2,
153.2, 151.8, 148.45, 147.0, 141.6, 139.1, 132.7, 129.6,
128.0 (C quaternary, carbamate), 126.0 (a3), 124.6 (a5),
116.9 (a6), 110.6 (e5), 110.4 (e8), 108.0 (e20, e60), 102.2
(g1), 102.0 (e10), 101.9 (G1), 99.9 (g7), 80.8 (g4), 75.0
(e2), 74.4–74.0 (G4, g2), 73.4 (G2), 72.5 (G3), 68.4 (e11),
68.0 (G5), 66.4 (g3, g5, g6, e4), 56.5 (OCH3), 43.9 (e1),
41.0 (e3), 37.8 (N–CH3), 21.0 (g8); m/z (ES+) 981
[M+Na]+, 997 [M+K]+.
Benzyl [4-nitrophenyl-2-[(etoposide-40-O-carbonyl)methyl-
amino]-ꢀ-D-glucopyranosid]uronate (10). To a solution
of 9 (223.2 mg, 0.16 mmol) in pyridine (2.65 mL) at
0 ꢁC, HF/pyridine (2.65 mL, 70%) was added dropwise.
The mixture was stirred for 4 h at 0 ꢁC, then 10 h at
room temperature. After evaporation, the residue was
taken in 200 mL CH2Cl2, and washed with water; the
aqueous phase was extracted with CH2Cl2. The organic
phases were dried over magnesium sulphate, and the
compound was purified by chromatography (CH3CN).
The obtained product 10 was a beige solid (150 mg,
89%). C50H52N2O23; mp 170 ꢁC; [a]D ꢀ9.2 (c 1.1 in
CHCl3); nmax/cmꢀ1(CDCl3) 3406 (O–H), 1752 (CO
ester), 1713 (CO carbamate), 1602 (aromatics), 1525,
1346 (NO2); dH (CDCl3) 8.20 (br, 2H, a3, a5), 7.39 (br,
5H, Ph), 7.12 (d, J=9, 1H, a6), 6.95+5.62 (2H, e20, e60),
6.65 (s, 1H, e5), 6.57 (s, 1H, e8), 6.07 (s, 2H, e10), 5.34
(AB, J=12, 1H, CH2(A)Ph), 5.27 (AB, J=12, 1H,
CH2(B)Ph), 5.13 (d, J=6, 1H, G1), 5.03 (d, J=2, 1H,
G5), 4.70 (2H, g7, e1), 4.46 (2H, e11), 4.38 (1H, g1),
4.23 (dd, J=10, J=4, 1H, g4), 4.15 (d, J=10, 1H, G4),
3.91 (br, 1H, G3), 3.68 (G2), 3.66–3.52 (4H, e2, e4, g3,
g6), 3.51 (s, 9H, OCH3, NCH3), 3.42 (1H, g2), 3.33 (1H,
g5), 2.99 (1H, e3), 1.40 (d, J=5, 1H, g8); dC (CDCl3)
176.4 (e9), 167.5 (G6), 156.0, 152.9, 150.8, 148.0, 146.0,
141.5, 137.4, 134.0, 131.7, 131.3, 126.4, 125.5 (C qua-
ternary, carbamate), 127.8, 127.7, 127.4 (Ph tertiary),
123.3, 122.1 (a3, a5), 113.5 (a6), 110.5 (e8), 109.0 (e5),
107.8 (e20, e60), 100.8 (e10, G1), 98.8 (g7), 96.4 (g1), 78.8
(g4), 73.9 (G4, g2), 72.9 (G2), 72.5 (e4), 71.2 (G3), 69.7
(G5), 67.4 (g3, g6), 67.0–66.7 (e11, CH2Ph), 65.0 (g5),
55.3 (O-CH3), 43.2 (e1), 38.8–38.1 (e2, e3), 36.6 (N–
CH3), 19.4 (g8); m/z (FAB+) 1071 [M+Na]+.
Acknowledgements
This work was supported by grant 5544 from the ARC
(Association pour la Recherche sur le Cancer). The
technical assistance of Ludovic Maillard, Estelle Chen-
eau, Sandrine Lacombe for the syntheses is also
acknowledged. We are grateful to Laboratoires Servier
for performingthe cytotoxic measurements.
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