Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard

Article abstract of DOI:10.1016/S0960-894X(00)00353-X

A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared. Upon exposure to E. coli β-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00353-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1835±1837
Synthesis and Cytotoxic Activity of a Glucuronylated Prodrug
of Nornitrogen Mustard
Sebastien Papot,a Damien Combaud,a Klaus Bosslet,b,y Manfred Gerken,b,{
Jorg Czech b,{ and Jean-Pierre Gesson a,*
a
Á Â
Laboratoire de Synthese et Reactivite des Substances naturelles, Universite de Poitiers et CNRS,
40, Av du Recteur Pineau, F-86022 Poitiers, France
Â
Â
bHMR, PO Box 1140, D-35001 Marburg, Germany
Received 3 April 2000; revised 5 June 2000; accepted 20 June 2000
AbstractÐA new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug
HMR 1826, has been prepared. Upon exposure to E. coli b-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against
LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate
carbamic acid to the inactive chloroethyl oxazolidinone. # 2000 Elsevier Science Ltd. All rights reserved.
The use of non-toxic prodrugs, selectively activated at
the tumor site by an antibody±enzyme conjugate, has
been proposed by Bagshawe to improve cancer chemo-
therapy.1 Several enzymes have been used in this strategy
known as ADEPT (Antibody Directed Enzyme±Prodrug
Therapy),2 and in this respect b-glucuronidase (GUS),
®rst considered by Ro‚er3 and Bosslet,4 appears parti-
cularly interesting. Furthermore, Bosslet et al.5 have
shown that a stable homogeneous fusion protein can be
constructed from humanized GUS. Taking into account
earlier observations by Fishman6 and others7 indicating
that GUS accumulates in the necrotic area surrounding
cancer cells, Bosslet8 proposed that glucuronylated pro-
drugs may be used alone in the treatment of tumors with
size over 2±3 mm in diameter (PMT: Prodrug Mono-
Therapy). For example, a doxorubicin (DOX) prodrug,
HMR 1826, has demonstrated superior ecacy in the
treatment of various tumors in mice and monkeys, both
in ADEPT and PMT strategies.9
bioprecursor of the alleged active species,11 phosphor-
amide mustard 2 (together with acrolein). Farquhar12
has recently prepared the glucuronylated prodrug ester
3 which upon exposure to a carboxylate esterase and
GUS a€ords 2. However, 3 appears to be rather
unstable (t
5 h) in phosphate bu€er (pH 7.4; 37 ꢀC) and
2
1
furthermore the high hydrophilicity of 2 results in poor
penetration into cells. Since 2 is itself a precursor of the
nornitrogen mustard 4 and chloroethyl aziridine 5,11 it
seems worthwhile to prepare prodrugs of 4 (which
rapidly a€ords 5 under physiological conditions). Such a
prodrug, readily activated by b-lactamase, has been pre-
pared by Alexander using a carbamate linker function,
but stability and toxicity data have not been reported.13
However, cancer chemotherapy protocols frequently
employ several antitumor drugs. For example, DOX is
used in combination with cyclophosphamide in the treat-
ment of non-Hodgkin lymphomas, breast and genital
cancers. Thus, the design of a glucuronylated prodrug of
cyclophosphamide 1 may be of interest.10 1 is indeed a
*Corresponding author. Fax: +33-5-4945-3501; e-mail: jean-pierre.
gesson@univ-poitiers.fr
yPresent adress: Schering AG, D-13342 Berlin, Germany.
{Present adress: Aventis Pharma, D-65926 Franfurt, Germany.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00353-X

1836
S. Papot et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1835±1837
The preparation and evaluation of 9, a glucuronylated
prodrug incorporating the same spacer group as HMR
1826 are now reported. This material was obtained in
three steps from the known activated carbonate 6.4,9
First, condensation of the HCl salt of 4 with 6 (DMF,
pyridine, 45 ꢀC, 48 h) a€orded 7 (71%) which was then
deacylated to 8 (cat NaOMe, MeOH, 20 ꢀC, 24 h, 50%).
Upon treatment with 1 equiv of Ba(OH)2 (MeOH, 20 ꢀC,
2 h) followed by acidi®cation with Amberlyst IRC-50, 9
was isolated as a homogeneous (HPLC, 1H and 13C
NMR) compound in 36% yield.14
and 37 ꢀC (no decomposition of 9 was observed without
GUS under these conditions after 24 h). Fast hydrolysis
of 9 was observed by HPLC17 (Fig. 1) with formation of
4-hydroxymethyl 2-nitrophenol, which is the known
metabolite of the HMR 1826 spacer group, together
with oxazolidinone 11. No trace of 4 or 5 was observed.
Hence, the observed lower cytotoxicity of 9 in the pre-
sence of GUS does not result from failure of glycosidic
cleavage and spacer decomposition. The intermediate
carbamic acid 10, in equilibrium under these conditions
with the corresponding carboxylate, is suciently stable
to cyclize to the inactive carbamate 11. Such a process is
known to occur in humans since 11 is detected in the
urine of patients treated with cyclophosphamide as a
result of carboxylation of 4 in the blood stream.11c
Similarly, decomposition of 9 with bovine liver GUS at
pH 516,17 led to the same results without detection of 4
in HPLC. Even at this more acidic pH, cyclization of 10
to 11 was the only observed process.
Cytotoxicity tests15 were carried out at 37 ꢀC against
LoVo colon cancer cells with 9 alone, 9 and an excess of
GUS (Escherichia coli) and with the HCl salt of 4. The
cytotoxicity of 9 was very low (IC50 ꢁ 400 mM) compared
to 4 (IC50=15 mM) as expected from introduction of a
carbamate group. Indeed, the cytotoxicity measured for
9 in the presence of GUS was found to be reduced
compared to 4 (IC50=95 mM).
In conclusion, a non-toxic glucuronylated prodrug of
nornitrogen mustard, incorporating the HMR 1826
spacer group, has been shown to generate, after enzymatic
To explain this result, 9 was incubated with GUS (E.
coli, as above)16 in 0.02 M phosphate bu€er at pH 7.2

S. Papot et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1835±1837
1837
4. Jacquesy, J.-C.; Gesson, J.-P.; Monneret, C.; Mondon, M.;
Renoux, B.; Florent, J.-C.; Koch, M.; Tillequin, F.; Sedlacek,
H. H.; Gerken, M.; Kolar, C.; Gaudel, G.; Bosslet, K.; Czech,
J.; Ho€mann, D.; Seeman, G. Eur. Pat. Appl. EP 511,917,
1992, Chem. Abstr. 1994, 120, 271070s.
5. Bosslet, K.; Czech, J.; Lorenz, P.; Sedlacek, H. H.; Schuer-
mann, M.; Seeman, G. Br. J. Cancer 1992, 65, 234.
6. (a) Fishman, W. H.; Anlyan, A. J. J. Biol. Chem. 1947, 169,
449. (b) Fishman, W. H.; Baker, J. R.; Borges, P. R. F. Cancer
1959, 12, 240.
7. Sperker, B.; Backman, J. T.; Kroemer, H. K. Clin. Phar-
macol. 1997, 33, 18.
8. Bosslet, K.; Czech, J.; Ho€mann, D. Tumor Targeting
1995, 1, 45.
9. Florent, J.-C.; Gaudel, G.; Mitaku, S.; Monneret, C.; Ges-
son, J.-P.; Jacquesy, J.-C.; Mondon, M.; Renoux, B.; Andria-
nomenjanahary, S.; Michel, S.; Koch, M.; Tillequin, F.;
Gerken, M.; Czech, J.; Straub, R.; Bosslet, K. J. Med. Chem.
1998, 41, 3572.
10. For a study of a similar prodrug of a phenol mustard see:
Lougestay-Madec, R.; Florent, J.-C.; Monneret, C.; Nematti, F.;
Poupon, M.-F. Anti-Cancer Drug Design 1998, 13, 995.
11. (a) Shulman-Roskes, E. M.; Noe, D. A.; Gamcsik, M. P.;
Marlow, A. L.; Hilton, J.; Hausheer, F. H.; Colvin, O. M.;
Ludeman, S. M. J. Med. Chem. 1998, 41, 515. (b) le Roux, C.;
Modro, A. M.; Modro, T. A. J. Org. Chem. 1995, 60, 3832. (c)
Chan, K. K.; Hong, P. S.; Tutsch, K.; Trump, D. L. Cancer Res.
1994, 54, 6421.
Figure 1. (a) HPLC analysis of an assay after incubation of 9 with
GUS for 45 min. Peak noted as X corresponds to 3-nitro-4-hydroxy
benzyl alcohol; (b) HPLC analysis of a mixture of 11 and 4 (for con-
ditions see ref 17).
12. Ghosh, A. K.; Farquhar, D. Tetrahedron Lett. 1997, 38,
8795.
13. Eaton, M. A. W.; Pratt, A. J.; Alexander, R. P. Eur. Pat.
Appl. EP 392,745 1990.
cleavage, oxazolidinone 11 as a result of a side reaction
of the intermediate carbamic acid. Although cyclization
may be very fast in this case, it may thus be worthwhile
to study the rate of decarboxylation of such prodrug
intermediates, depending on amine substrates and con-
ditions, to avoid di€usion of the hydrophilic carbamic
acid from the tumor area.
14. Selected data for 9: mp 155 ꢀC, [a]D 570 (c 0.07, H2O);
d1H (CD3OD) 3.50±3.80 (m, 11H), 3.95 (d, 1H, J=9.3 Hz),
5.16 (s, 2H), 5.19 (broad s, 1H), 7.45 (d, 1H, J=8.7 Hz), 7.62
(dd, 1H, J=8.7 and 2.1 Hz), 7.87 (d, 1H, J=2.1 Hz); d13C
(CD3OD) 42.4, 43.0, 50.8, 51.1, 67.3, 73.0, 74.6, 76.9, 77.6,
102.6, 119.1, 126.1, 132.9, 135.0, 142.2, 151.1, 157.6, 170.9; SM
(FAB+): 535 (MN+a), 371, 273, 242, 214; exact mass (FAB HR)
M+Na calcd 535.0498 g mol 1, exp. 535.0509 g mol
.
1
Acknowledgements
15. Cytotoxicity tests were carried out as described in ref 9,
except for the use of LoVo cells.
Financial support from HMR and the `Ligue Nationale
Contre le Cancer, Comite de Charente-Maritime' is
gratefully acknowledged.
16. b-Glucuronidase from E. coli and from bovine liver were
purchased from Sigma (respectively, Cat G-7896, type X-A and
Cat G-0501, type B-10). Assay with b-glucuronidase from E. coli:
90 units of enzyme and 2 mmol of prodrug were incubated in 0.02
M phophate bu€er pH 7 at 37 ꢀC. Assay with b-glucuronidase
from bovine liver: 100 units of enzyme and 2 mmol of prodrug
were incubated in a 0.02 M phophate bu€er pH 5 at 37 ꢀC.
17. HPLC conditions: A C18 reverse phase column (Lichro-
sorb, 5 mm, 250Â4.6 mm) was used, the mobile phase was
MeCN±0.02 M phosphate bu€er, pH 7.0 or 5.0 (70:30) with a
¯ow rate of 0.8 mL min 1. A UV detector WatersTM 486 was
set at 254 nm. An HPLC Refractive Index detector Model
8120 from ICS.N and detection system was driven by PICx
version PIC3 0-4-0 from Normasoft. Retention times for pro-
drug, 3-nitro-4-hydrobenzyl alcohol, oxazolidinone and nor-
nitrogen mustard (and/or chloroethylaziridine), respectively,
are 3.2 , 4 , 4.3 and 5.5 min.
References and Notes
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C. In Studies in Natural Product Chemistry; Atta-ur-Rahman,
Ed.; Amsterdam, 1998; Vol. 21. (c) Syrigos, K. N.; Epenetos,
A. A. Anticancer Res. 1999, 19, 605.
3. Ro‚er, S. R.; Wang, S. M.; Chern, J. W.; Yeh, M. Y.;
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Cancer Res. 1992, 52, 4484.