Synthesis and antitumour activity of stereoisomers of 4-hydroperoxy derivatives of ifosfamide and its bromo analogue

Article abstract of DOI:10.1016/S0014-827X(02)01202-8

Racemic mixtures and laevorotatory enantiomers of cis- and trans-4-hydroperoxyifosfamide and 4-hydroperoxybromofosfamide possess high antitumour activity both in vitro and in vivo. However, no major differences in biological activity were observed among t

Full text of DOI:10.1016/S0014-827X(02)01202-8

Il Farmaco 57 (2002) 315–319
www.elsevier.com/locate/farmac
Synthesis and antitumour activity of stereoisomers of
4
-hydroperoxy derivatives of ifosfamide and its bromo analogue
a,
a
b
b
Konrad Misiura *, Daria Szymanowicz , Halina Kusnierczyk , Joanna Wietrzyk ,
b
Adam Opolski
a
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112,
9
0-363 Lodz, Poland
Department of Tumour Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12,
3-114 Wroclaw, Poland
b
5
Received 15 November 2001; accepted 19 January 2002
Abstract
Racemic mixtures and laevorotatory enantiomers of cis- and trans-4-hydroperoxyifosfamide and 4-hydroperoxybromofos-
famide possess high antitumour activity both in vitro and in vivo. However, no major differences in biological activity were
´
observed among these stereoisomers. © 2002 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
Keywords: Ifosfamide; Bromofosfamide; 4-Hydroperoxyifosfamide; Antitumour activity
1
. Introduction
are connected with metabolic transformation of IF and
a release of chloroacetaldehyde following the hydroxy-
lations at C-1 atoms of 2-chloroethyl groups. IF is a
pro-drug activated in vivo via cytochrome P-450 medi-
ated oxidation of C-4 atom of 1,3,2-oxazaphosphorine
ring [11]. It is conceivable that the use of pre-activated,
Ifosfamide (IF) like its structural isomer cyclophos-
phamide is an anticancer alkylating agent widely used
for the treatment of variety of human tumours [1]. IF is
a chiral molecule and both of its enantiomers have been
synthesized [2]. We have found that laevorotatory enan-
tiomer of ifosfamide [(−)-IF] is more active than the
parent racemic drug against several experimental tu-
mours in mice [3]. Within the process of search for new
and more active congeners of IF, bromo analogues
have been prepared [4]. Also, among stereoisomers of
bromo analogues of IF stereo-differentiation in
antitumour activity [5] and pharmacokinetics [6] was
observed. Based on these results (S)-(−)-3-(2-
bromoethyl) - N - (2 - chloroethyl)tetrahydro - 2H - 1,3,2-
oxazphosphorine-2-amine 2-oxide [7] [compound CBM-
4
-hydroperoxy derivatives of IF and (−)-BF could
diminish these side effects.
Racemic cis- and trans-4-hydroperoxyifosfamide
were obtained previously and their antitumour activity
proved [12,13]. In this study we examined the influence
of the configuration, on phosphorus atom in 4-hy-
droperoxy derivatives, on their antitumour activity. Ob-
tained results shed some additional light on the
problem of stereo-differentiation of biological activity
of IF and BF enantiomers.
1
1, (−)-BF] became subject to the Phase I clinical trials
2
. Experimental
in Poland. Therapeutic application of high-dose IF [8,9]
or (−)-BF [10] is limited by several side effects; among
them neurotoxicity and nephrotoxicity are of the great-
est concern. It was postulated [8] that these side effects
2.1. Chemistry
2.1.1. General synthetic procedure
Racemate or laevorotatory enantiomer of IF or BF
(
20 mmol) was dissolved in a mixture of acetone (80
*
Corresponding author.
E-mail address: kmisiura@bio.cbmm.lodz.pl (K. Misiura).
ml), water (40 ml), and 30% hydrogen peroxide (12 ml).
´
0
014-827X/02/$ - see front matter © 2002 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01202-8

316
K. Misiura et al. / Il Farmaco 57 (2002) 315–319
Obtained solution was bubbled with ozone (rate 0.5
g/h) at 0 °C. After 4 h reaction mixture was concen-
trated up to ca. 50 ml and obtained suspension was
extracted with chloroform (3×50 ml). Organic layers
activity was determined according to In vivo Cancer
Models (NIH Publication No. 84-2635) by comparing
the mean survival time of the treated group with that of
the control group, and expressed as percentage of in-
creased life span over control (ILS%). Doses providing
50% ILS (ED ) were estimated graphically from the
were combined, dried with MgSO , and solvent evapo-
4
rated. Resulting mixture was separated on silica gel
50
using chloroform/acetone 1:1 as an eluent providing
least-square-fitted dose–effect curves. Similarly, doses
curative for 50% of treated animals were estimated
from curves where the effect was expressed as the
percentage of long-term survivors (in 2-month observa-
tion period).
4
-keto derivatives (R 0.52), trans (R 0.45), and cis
f
f
isomers (R 0.34) of the appropriate 4-hydroperoxy
f
compounds.
2.1.2. HPLC analysis conditions
Reverse-phase C8 column (Alltech Econoshere, 5 mm,
50 mm); eluent, isocratic 25% acetonitrile; flow 1.5
2
3. Results
ml/min; u 200 nm.
Stereoisomers of 4-hydroperoxyifosfamide (4-HOO-
IF) and 4-hydroperoxy-bromofosfamide (4-HOO-BF)
were obtained by the ozonolysis of racemic IF, (−)-IF,
racemic BF, and (−)-BF performed in the presence of
hydrogen peroxide (Scheme 1).
3
1
2
.1.3. P NMR analysis conditions
Spectra of 100 mM racemic 4-ketoifosfamide and 50
mM Pr(tfc) showed a presence of two signals of 8.7
3
and 7.2 ppm with 1:1 ratio. Spectra of (−)-4-ketoifos-
famide obtained from a mixture of isomers coming
from (−)-cis- and (−)-trans-4-HOO-IF, performed as
above, revealed the presence of only one signal of 8.7
ppm.
From each substrate both cis- and trans-isomers of
4-hydroperoxy compound together with 4-keto side-
product, were formed in nearly equimolar ratio. Ob-
tained products were separated by careful silica gel
1
column chromatography and were characterized by H-
31
2
.2. Cytotoxic acti6ity in 6itro
and P NMR, and CI-MS. Their purity was examined
31
by TLC, HPLC, and P NMR. Yields of products and
selected physicochemical data are presented in Table 1.
Stability of 4-hydroperoxy compounds (conc. 10
mM) in 0.1 M phosphate buffer, pH 7.4 and 0.1 M
Tris–HCl buffer, pH 7.5 was measured using HPLC
method. It was found that all tested compounds un-
dergo fast cisltrans isomerization. For each com-
pound this isomerization was completed within 15 min
at room temperature. By prolonging incubation time it
was also found that 4-HOO-IF and 4-HOO-BF are
chemically unstable in these buffers and collapse with
spontaneous release of most probably 4-hydroxy
derivatives (HPLC analysis, TR 2.77, 3.12 min), which
decompose further to several products. The above men-
tioned isomerization was earlier observed [13] for
racemic cis- and trans-4-hydroperoxyifosfamide dis-
solved in chloroform in the presence of p-toluenesul-
fonic acid. It was postulated [13,14] that this process
proceeded with epimerization at phosphorus atom and
retention of configuration at C-4 atom. However, by
using optically active form of 4-HOO-IF we proved
that cisltrans isomerization did not change the
configuration at P-atom. (−)-cis-4-HOO-IF (conc. 50
mM) was epimerized in 0.1 M Tris–HCl buffer with
pH 7.5 within 1 h and, without isolation, reduced to
The following established in vitro human cancer cell
lines were employed: SW707 (rectal adenocarcinoma),
MCF-7 (breast carcinoma), KB (cervical carcinoma),
and HCV29T (bladder cancer). The cytotoxicity assay
was performed after 72-h exposure of the cultured cells
to varying concentrations (from 0.1 to 100 mg/ml) of
the tested agents. The cells attached to the plastic were
fixed by gently layering cold 50% trichloroacetic acid
on top of the culture medium in each well. The plates
were incubated at 4 °C for 1 h and then washed five
times with tap water. The background optical density
was measured in the wells filled with culture medium,
without the cells. The cellular material fixed with TCA
was stained with 0.4% sulphorhodamine B dissolved in
1
% acetic acid for 30 min. Unbound dye was removed
by rinsing (4×) with 1% acetic acid. The protein-
bound dye was extracted with 10 mM unbuffered Tris
base for the determination of optical density (at 540
nm) in a computer-interfaced, 96-well microtitre plate
reader Multiskan RC photometer. Each compound in
given concentration was tested in triplicates in each
experiment, which was repeated 3–5 times.
2
.3. Antitumour acti6ity against L1210 leukaemia
4
-ketoifosfamide by adding 0.1 M iron(II) sulphate.
Leukaemia cells were implanted i.p. in CD2F1 mice
Obtained 4-ketoifosfamide was isolated by extraction
with ethyl acetate and its enantiomeric purity was
examined by P NMR spectroscopy performed in the
5
at 10 cells/mouse. Each experimental group was com-
posed of six mice. Tested compounds were administered
i.p. 24 h after tumour implantation (day 1). Antitumour
31
presence of a chiral shift reagent Pr(tfc) . It was found
3

K. Misiura et al. / Il Farmaco 57 (2002) 315–319
317
Scheme 1.
Table 1
Yields of products and selected physicochemical data of 4-hydroperoxy derivatives
Comp.
Yield (%)
[a]D
RT (RP-HPLC) (min)
l (31P NMR) (ppm)
rac. cis-4-HOOꢀIF
rac. trans-4-HOOꢀIF
8
16
9
10
9
21
6
12
4.81
5.44
4.81
5.44
5.32
5.98
5.32
5.98
8.82
8.93
8.82
8.93
8.66
8.82
8.66
8.82
(
(
−)-cis-4-HOOꢀIF
−)-trans-4-HOOꢀIF
−39.1
−38.6
rac. cis-4-HOOꢀBF
rac. trans-4-HOOꢀBF
(
(
−)-cis-4-HOOꢀBF
−)-trans-4-HOOꢀBF
−38.9
−36.2
Table 2
Cytotoxic activities of 4-hydroperoxy derivatives
that obtained (−)-4-ketoifosfamide possessed 100%
enantiomeric purity. Earlier, we have proved [15] by
chemical correlation that (−)-4-ketoifosfamide has the
Sample
Cell line/ID50 a (mg/ml)
KB SW707
same S configuration as (−)-(S)-IF. The same analysis
p
of stereochemistry at P-atom was performed for a mix-
ture of isomers obtained from (−)-trans-4-HOO-IF and
no epimerization at phosphorus was found. These results
of stereochemical analysis of cisltrans isomerization of
HCV29T MCF-7
rac. trans-4-HOO-IF 32.691.2
rac. cis-4-HOO-IF 37.991.1
−)-trans-4-HOO-IF 33.791.0
4.191.4 3.291.1 4.491.4
3.391.0 2.991.3 3.591.0
3.391.1 3.091.2 3.291.0
3.491.1 3.391.1 4.591.4
6.491.6 5.591.2 17.691.1
(
4-HOO-IF is consistent with the kinetic data obtained by
(−)-cis-4-HOO-IF
39.091.0
Borch for cisltrans isomerization of 4-hydroperoxy-
cyclophosphamide [16], for which elimination–addition
of hydrogen peroxide mechanism was proposed.
Cytotoxic activity of obtained 4-hydroperoxy com-
pounds was analysed on several human tumour cell lines
rac. trans-4-HOO-BF 32.391.4
rac. cis-4-HOO-BF
40.491.0 27.191.2 12.391.9 30.091.1
−)-trans-4-HOO-BF 38.091.1 26.091.0 26.891.1 33.291.1
(
(
−)-cis-4-HOO-BF
49.891.0 31.391.2 22.991.5 33.391.1
a
The dose of compound that inhibits proliferation rate of the
(
Table 2).
tumour cells by 50% as compared to the control untreated cells.

318
K. Misiura et al. / Il Farmaco 57 (2002) 315–319
Fig. 1. Dose-dependent increase in life span (A) and percentage of long term survivors (B) in L 1210 leukaemia-bearing mice following i.p.
administration of stereoisomers of 4-hydroperoxy derivatives of ifosfamide.
It was found that chloro analogues [IF and (−)-IF
derivatives] are more active than bromo congeners.
However, the differences in cytotoxicity between cis-
and trans-isomers as well as between racemic and opti-
cally pure compounds were negligible.
ties in N-dichloroethylation metabolic pathways of IF
[17] and cyclophosphamide [18] were also recently
observed.
4-Hydroperoxy derivatives of IF and (−)-IF were
Acknowledgements
also active against L1210 leukaemia in mice (Fig. 1).
All examined compounds have similar therapeutic
efficacy in this model. Effective doses (ED ) were in a
The authors wish to thank Professor W.J. Stec for
the critical reading of the manuscript. This project was
financially assisted by the State Committee for Scien-
tific Research, Grant No 4 P05F 023 15.
50
range of 14–22 mg/kg, while curative doses (CD )
50
were 45–82 mg/kg, and they were 3–5 times lower
when compared with the values obtained for IF.
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