Hydrolytic pathway of glufosfamide, a new phosphorylated anticancer agent

Article abstract of DOI:10.1080/10426500212229

³¹P NMR was used to demonstrate that glufosfamide is hydrolyzed into isophosphoramide mustard (IPM) with a half-life of about 25 h at 37°C and pH 7.4 in buffer as well as in human plasma. Through a cascade of reactions, IPM gives rise to three

Full text of DOI:10.1080/10426500212229

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Phosphorus, Sulfur, and Silicon and the Related
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Hydrolytic Pathway of Glufosfamide, a New
Phosphorylated Anticancer Agent
a
a
a
Marie-Caroline Aoust de Rouvèze , Veronique Gilard , Robert Martino , Myriam Malet-
a
b
Martino & Ulf Niemeyer
a
Biomedical NMR Group, IMRCP Laboratory , Université Paul Sabatier , 118, route de
Narbonne, Toulouse, 31062, France
b
ASTA Medica AG , Franckfurt am Main, Germany
Published online: 27 Oct 2010.
To cite this article: Marie-Caroline Aoust de Rouvèze , Veronique Gilard , Robert Martino , Myriam Malet-Martino & Ulf
Niemeyer (2002) Hydrolytic Pathway of Glufosfamide, a New Phosphorylated Anticancer Agent, Phosphorus, Sulfur, and Silicon
and the Related Elements, 177:6-7, 1735-1738, DOI: 10.1080/10426500212229
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Phosphorus, Sulfur and Silicon, 2002, Vol. 177:1735–1738
Copyright �C 2002 Taylor & Francis
1
042-6507/02 $12.00 + .00
DOI: 10.1080/10426500290093333
HYDROLYTIC PATHWAY OF GLUFOSFAMIDE, A
NEW PHOSPHORYLATED ANTICANCER AGENT
a
a
Marie-Caroline Aoust de Rouv e` ze, Veronique Gilard,
a
a
b
Robert Martino, Myriam Malet-Martino, and Ulf Niemeyer
Biomedical NMR Group, IMRCP Laboratory, Universit e´ Paul
a
Sabatier, 118, route de Narbonne, 31062 Toulouse, France and
ASTA Medica AG, Franckfurt am Main, Germanyb
31P NMR was used to demonstrate that glufosfamide is hydrolyzed
into isophosphoramide mustard (IPM) with a half-life of about 25 h at
�
3
7 C and pH 7.4 in buffer as well as in human plasma. Through a cas-
cade of reactions, IPM gives rise to three final products, dihydroxyIPM,
phosphate ion, and phosphorylethanolamine, which is by far the major
compound.
Keywords: Glufosfamide; hydrolysis; isophosphoramide mustard;
3
1
P NMR
INTRODUCTION
The anticancer drug glufosfamide (�-D-glucosylisophosphoramide mus-
tard, glc-IPM) entered a clinical phase II trial in 1999. In this drug,
IPM is coupled to the C1 of D-glucose in a �-glycosidic bond. Inside
the cells, glc-IPM is mainly cleaved by glucosidases into D-glucose and
1
IPM, but nonenzymatic hydrolysis could also contribute to its decay.
In this study, we investigated the time course of glc-IPM hydrolysis in
buffered solutions at pH 7.4 and in human urine and plasma. Since the
first stage of hydrolysis gives IPM, the chemical stability and the fate
of IPM was also determined in the same conditions.
RESULTS AND DISCUSSION
31P NMR monitoring of the glc-IPM hydrolysis in cacodylate buffer
at pH 7.4 shows that this compound (� = 18.29 ppm) gives IPM
Address correspondence to R. Martino. E-mail: martino@chimie.ups-tlse.fn
1
735

1
736
M.-C. A. de Rouv e` ze et al.
(
� = 13.60 ppm) as the first intermediate, which is transformed into
the monoaziridinyl derivative of IPM (monoAzIPM) (� = 17.90 ppm)
Figure 1 and Scheme 1). This is in accordance with the fact that IPM
(
gives also monoAzIPM at the same pH (data not shown). The rates of
degradation of glc-IPM were � 12–25-fold slower than those of IPM,
depending on the temperature and medium (Table I). Up to now, the
chemical stability of glc-IPM in any aqueous medium has not been re-
�
ported. This study shows that its half-life (t1/2) at 37 C was � 1 day
at pH 7.4 in buffer and plasma, slightly shorter (� 0.8 day) at pH 6 in
urine, and slightly longer (� 1.2 day) at pH 8 in deproteinized plasma.
The t1/2 of IPM (Table I) were in agreement with the literature data
�
(
350 min at pH 7.0 and 25 C and in the range 49–84 min at pH 7.0–7.4
�
2,3
at 37 C).
SCHEME 1 Hydrolytic pathway of glc-IPM at pH 7.4.a
All the compounds are represented in neutral form. Structures were char-
a
1
13
31
acterized from one- and two-dimensional H, C, and P NMR experiments.
b
�
�
are given at pH 7.4 in cacodylate buffer and 37 C. They are expressed in
ppm relative to 85% H PO as external standard.
3
4

Hydrolytic Pathway of Glufosfamide
1737
3
1
FIGURE 1
P NMR time course of the main hydrolytic products of glc-IPM
�
at pH 7.4 (cacodylate buffer) and 37 C.
On the basis of the formation in time and the temporal patterns of
the degradation compounds of glc-IPM at pH 7.4, its hydrolytic pathway
can be summarized as illustrated in Scheme 1. IPM, monoAzIPM, and
monohydroxyIPM (monoOHIPM) are intermediates. DihydroxyIPM
(
diOHIPM), phosphorylethanolamine (PEA), and phosphate ion (Pi) are
the final products, PEA being the major one (Figure 1).
Identical hydrolytic profiles are observed for glc-IPM and IPM in
urine and plasma; however, in plasma, additional 31P signals were
TABLE I 31P NMR-Derived Kinetic Data for Hydrolysis of glc-IPM
and IPM
Glc-IPM
IPM
Conditions
T
(n)a
t1/2a
(n)a
t1/2a
�
Cacodylate buffer
25 C
(3)
(4)
(5)
(4)
(1)
127 � 5 h
25 � 3 h
20 � 3 h
24.5 � 1 h
30 h
(3)
(5)
(6)
(5)
373 � 33 min
65 � 12 min
98 � 14 min
107 � 16 min
�
7
.4
37 C
b
�
Urine pH 6.0
37 C
Plasma pH 7.4b
37 C
�
�
Deproteinized plasma
37 C
b
pH 8.3
a(n): number of experiments; t1/2: half-life.
b
Initial pH can increase or decrease by 1–1.5 pH units, depending on the
temperature, concentration of the solution, and duration of the experiments.

1
738
M.-C. A. de Rouv e` ze et al.
detected in the resonance area of IPM at 13.93 ppm (broad), 14.57 ppm,
and 13.25 ppm. These � suggest that these compounds correspond very
probably to adducts of a nucleophile present in plasma with IPM via its
aziridinyl derivatives.
In conclusion, this study demonstrated that glc-IPM is nonenzyma-
tically hydrolyzed into IPM and determined the successive steps of its
hydrolysis.
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