Cancer chemotherapy: A SN-38 (7-Ethyl-10-hydroxycamptothecin) glucuronide prodrug for treatment by a PMT (Prodrug monoTherapy) strategy

Article abstract of DOI:10.1016/S0960-894X(02)01080-6

A glucuronide-based prodrug of SN-38 (7-ethyl-10-hydroxycamptothecin) has been synthesized for use in a Prodrug MonoTherapy Strategy (PMT). Since this prodrug is significantly less cytotoxic than SN-38 itself and efficiently releases the drug in vitro in the presence of β-D-glucuronidase, it can be considered as an appropriate candidate for cancer treatment by a PMT strategy.

Full text of DOI:10.1016/S0960-894X(02)01080-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 947–950
Cancer Chemotherapy: A SN-38 (7-Ethyl-10-hydroxycamptothecin)
Glucuronide Prodrug for Treatment by a PMT (Prodrug
MonoTherapy) Strategy
Stephane Angenault,a Sylvie Thirot,a Frederic Schmidt,a,* Claude Monneret,a
Bruno Pfeifferb and Pierre Renardb
aUMR176 CNRS/Institut Curie, 26, rue d’Ulm, 75248 Paris Cedex 05, France
b
´
Laboratoires Servier 1, rue Carle Hebert, 92415 Courbevoie Cedex, France
Received 11 September 2002; revised 12 November 2002; accepted 4 December 2002
Abstract—A glucuronide-based prodrug of SN-38 (7-ethyl-10-hydroxycamptothecin) has been synthesized for use in a Prodrug
MonoTherapy Strategy (PMT). Since this prodrug is significantly less cytotoxic than SN-38 itself and efficiently releases the drug in
vitro in the presence of b-d-glucuronidase, it can be considered as an appropriate candidate for cancer treatment by a PMT
strategy.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
20(S)-Camptothecin 1, an antitumor alkaloid first iso-
lated by Wall and co-workers in 1966,1 inhibits the
activity of topoisomerase I2,3 and displays antitumor
activity in various experimental tumor models.4 The
clinical tests of camptothecin as an anticancer agent
were aborted due to its non-mechanism related toxicity
and an extremely poor solubility profile. In an attempt
to circumvent these problems, researchers discovered
new derivatives of camptothecin. Among them, Irinote-
can (CPT-11 or Campto1) 2 and Topotecan (Hycam-
tin1) were the first derivatives used in several
indications including colon or ovarian cancer. More-
over, CPT-11 has also been shown to reduce angiogen-
esis.5 Newer analogues are also in clinical development
in order to find compounds with improved cytotoxicity
and to solve selectivity problems.
In fact, Irinotecan3,6À8 is a prodrug which is activated
by esterases9 to generate the more potent metabolite,
SN-38 3, which is in turn, extensively conjugated by
Scheme 1. Structures of compounds 1–3.
UDP-glucuronosyltransferase 1A1 into the inactive SN-
38 glucuronide (SN-38G) (Scheme 1).
In view of the narrow therapeutic index of CPT-11, we
decided to prepare prodrugs of SN-38 able to release the
active compound selectively at the tumor site. For this
targeting strategy, we planned to use human b-d-glu-
curonidase as the enzyme for the cleavage. In normal
*Corresponding author. Fax: +33-1-4234-6631; e-mail: frederic.
schmidt@curie.fr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01080-6

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S. Angenault et al. / Bioorg. Med. Chem. Lett. 13 (2003) 947–950
tissues, this glycosidase is localized in the lysosome
compartment of the cell and found at only low concen-
tration in the blood. In contrast, it has been shown by
enzyme histochemistry that it is specifically liberated
into the extracellular matrix of solid tumors, mainly in
necrotic areas. Based on these data, a new therapeutic
procedure called Prodrug MonoTherapy10À12 was pro-
posed. By applying this strategy to SN-38, we expected
to get enhanced solubility, sufficient hydrolytic and
proteolytic stability, and overall improvement in tumor
selectivity.
We report here the synthesis and in vitro biological tests
of a new prodrug of SN-38. Glucuronide prodrugs of 9-
aminocamptothecin were recently described.13 As
expected, these prodrugs were more soluble than 9-ami-
nocamptothecin itself, were well detoxified and then
released the drug by enzymatic hydrolysis.14 But, to our
knowledge, no prodrug of SN-38 able to liberate the
active compound in a selective manner has been described.
Following a proposal from Katzenellenbogen for pro-
drug design,15 self-immolative prodrugs have been pro-
posed16 for activation by tumor enzymes in targeting
strategies. Indeed, superior enzymatic hydrolysis17 of
such prodrugs is one of the advantages of self-immola-
tion.
Scheme 2. Synthesis of prodrug 7.
Chemistry
SN-38 was synthesized according to the publication of
Sawada et al.6 An ethylation of camptothecin, followed
by a N-oxidation and a photochemical rearrangement
afforded the drug 3 with a overall yield of 27%.
The glucuronic-spacer moiety 5 was obtained starting
from d-glucurono-3, 6-lactone and 2-amino-4-nitrophe-
nol, following our procedure.18,19
The carbamoyle chloride 5 was put into reaction with
SN-38 and an excess of DMAP and triethylamine to
give the coupling compound 6 in 83% yield. With pyri-
dine instead of DMAP, the yield dropped to 59%.
Finally, a two-step deprotection, with sodium methylate
in methanol for the cleavage of acetate groups, and with
sodium hydroxide for the methyl ester of the glucuronic
moiety followed by a neutralization gave the final pro-
drug 7 in its lactone form (Scheme 2).
This prodrug 7 was planned to liberate the active SN-38
following Scheme 3.
After enzymatic cleavage, the carbamate function of 7
reacts through an intramolecular cyclisation20 with
spontaneous liberation of the drug and formation of the
benzoxazolone 8.
Scheme 3. Liberation of SN-38 from prodrug 7.
compared to that of free SN-38 in the absence or in the
presence of b-d-glucuronidase (100 mg/mL). The mea-
surements were done using the microculture tetrazolium
assay (MTA)21 during 48-h periods.
Biological Results
First of all, the cytotoxicity of prodrug 7 was evaluated
against HT-29 (human colon cancer cells) cells and

S. Angenault et al. / Bioorg. Med. Chem. Lett. 13 (2003) 947–950
949
Conclusion
Table 1. Cytotoxicity of compounds 3 and 7
Compd
Cytotoxicity
IC50 (nM)
Cytotoxicity IC50 (nM)
in presence of b-d-glucuronidase
As the data of prodrug 7 (cytotoxicity, stability and
kinetics) are compatible for a PMT strategy, this com-
pound has been selected for in vivo tests. Other spacers
will be reported in a full paper.
3
7
27.3
1981
26
The prodrug 7 was 70 times less cytotoxic than the drug,
but in the presence of the enzyme the cytotoxicity of the
free drug was completely restored (Table 1).
Acknowledgements
We are grateful to Laboratoires Servier for financial
support and for a fellowship to S.A. A part of this
research was also funded by Grant 5544 from the
Association pour la Recherche sur le Cancer (ARC).
Another important point to determine was the stability
of the prodrug in solution. To test this, an in vitro sta-
bility study of 7 in a pH 7.2 phosphate buffer was
undertaken over a 2-day period and followed by
HPLC.22 During that time, except an equilibrium
between the lactone ring and the inactive carboxylate
form of camptothecin23 which is pH-dependent with
predominance of the former form in acidic medium and
of the open form in basic medium according to Scheme
4, the prodrug 7 displayed high stability.
References and Notes
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Scheme 4. Opening of the lactone.
Finally, the cleavage of the prodrug 7 by Escherichia
coli b-d-glucuronidase was measured by HPLC in a pH
7.2 phosphate buffer at 37 ꢀC (Fig. 1).24 In order to
avoid the multiplication of peaks due to the coexistence
of the open and closed forms, we chose basic (pH 9)
HPLC conditions. The peaks were identified by com-
parison with samples of the prodrug 7, the drug 3, and
the cyclised spacer 8. Unfortunately, as the prodrug 7
and drug 3 were mainly but not completely opened, we
needed to add both open and close forms for the inter-
pretation. After addition of the enzyme, the prodrug
peaks decreased whereas those corresponding to SN-38
and cyclised spacer increased. For a prodrug concen-
tration of 300 mM and an enzyme concentration of
10 units/mL, the half-life of 7 was 12.7 min.
13. Leu, Y.-L.; Roffler, S.; Chern, J.-W. J. Med. Chem. 1999,
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Straub, R.; Czech, J.; Gerken, M.; Bosslet, K. Anti-Cancer
Drug Des. 1999, 14, 93.
Figure 1. Kinetics of cleavage of prodrug 7.

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19. Schmidt, F.; Ungureanu, I.; Duval, R.; Pompon, A.;
Monneret, C. Eur. J. Org. Chem. 2001, 2129.
60% phosphate buffer (0.02 M, pH 3) and 40% acetonitrile
with UV detection at 265 nm.
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22. For the stability measurements, a solution of 250 mg/mL
of prodrug in 0.02 M phosphate buffer (pH 7.2) was incubated
at 37 ꢀC. Aliquots (40 mL) were taken at various times and
analyzed by HPLC after dilution with eluent (120 mL). HPLC
conditions: analysis was carried out on a reverse-phase column
(Spherisorb C18) using isocratic conditions (1 mL/min) of
23. Akimoto, K.; Kawai, A.; Ohya, K. Chem. Pharm. Bull.
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24. For the kinetic measurements, a solution of 300 mM of
prodrug in 0.02 M phosphate buffer (pH 7.2) was incubated at
37 ꢀC in the presence of 10 units/mL of b-d-glucuronidase (E.
coli). Aliquots (40 mL) were taken at various times and ana-
lyzed by HPLC after dilution with eluent (120 mL). HPLC
conditions: analysis was carried out on a reverse-phase column
(WATERS X-terra, 250/4.6 mm) using isocratic conditions
(1 mL/min) of 70% triethylammonium acetate buffer (0.1%,
pH 9) and 30% acetonitrile with UV detection at 265 nm
(extracted from PDA 3-D spectra).