Synthesis, in vitro metabolic studies, and antitumour activity of methyl analogues of ifosfamide

Article abstract of DOI:10.1002/1521-4184(200109)334:8/9<291::AID-ARDP291>3.0.CO;2-J

Synthesis of 2-chloro-1,1-dimethylethyl and 2-chloro-2,2-dimethylethyl analogues of ifosfamide was performed via aziridine intermediate. In vitro metabolic activation showed that both compounds are metabolised at a rate similar to the parent drug. However, their anticancer activity against L1210 leukaemia in mice was lower as compared with ifosfamide. The reduction of antitumour efficiency of examined analogues is probably caused by a lower ability to cross-link DNA by their final, active metabolites.

Full text of DOI:10.1002/1521-4184(200109)334:8/9<291::AID-ARDP291>3.0.CO;2-J

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 291–294
Konrad Misiuraa),
Methyl analogues of ifosfamide 291
Karina Kardackaa),
Synthesis, in vitro metabolic studies, and
antitumour activity of methyl analogues of
ifosfamide
Halina Kusnierczykb)
a) Department of Bioorganic Chemis-
try, Centre of Molecular and Macro-
molecular Studies, Polish Academy of
Sciences, Sienkiewicza 112, 90-363
Lodz, Poland
Synthesis of 2-chloro-1,1-dimethylethyl and 2-chloro-2,2-dimethylethyl analogues of
ifosfamide was performed via aziridine intermediate. In vitro metabolic activation showed
that both compounds are metabolised at a rate similar to the parent drug. However, their
anticancer activity against L1210 leukaemia in mice was lower as compared with
ifosfamide. The reduction of antitumour efficiency of examined analogues is probably
caused by a lower ability to cross-link DNA by their final, active metabolites.
b)
Department of Tumour Immunol-
ogy, Institute of Immunology and Ex-
perimental Therapy, Polish Academy
of Sciences, R. Weigla 12, 53-114
Wroclaw, Poland
Key Words: Anticancer drugs; Alkylating agents; Oxazaphosphorine; Ifosfamide
Received: May 21, 2001 [FP584]
Introduction
Ifosfamide [N,3-bis(2-chloroethyl)tetrahydro-2H-1,3,2-ox-
azaphosphorin-2-amine 2-oxide] (IF) belongs to the most
effective anticancer drugs currently used in clinical oncol-
ogy[1]. Therapeutic application of IF in high doses is limited
by several side effects; among them neurotoxicity[2] and
nephrotoxicity[3] are of the greatest concern. Methylene
Blue was used to diminish these side effects, albeit without
much improvement[4]. It was postulated[3] that neurotoxicity
and nephrotoxicity are connected with metabolic transfor-
mation of IF and the release of chloroacetaldehyde, as the
result of hydroxylations at C-1 atoms of 2-chloroethyl
groups. Suppressing hydroxylation at C-1 atoms by intro-
duction of deuterium atoms at these positions yielded
analogues with higher antitumour activity as compared with
unlabelled standards[5]. These results suggested that elimi-
nation of undesired hydroxylation by putting geminal methyl
groups at C-1 positions may give new ifosfamide analogues
exerting better therapeutic activity. To check this hypothe-
sis, a tetramethyl congener of IF, viz. compound 1, was
designed.
Scheme 1
The key intermediate 2 was obtained in 34% total yield.
However, its debenzylation or chlorination, both tried by
several methods, was unsuccessful. Most probably, a
strong steric influence of methyl groups caused the ob-
served lack of reactivity of 2 in those reactions. Such steric
inhibition may also effect hydroxylation at C-4 atom of
1,3,2-oxazaphosphorine ring, which is crucial for the acti-
vation of this class of drugs.
Results and Discussion
Synthesis of compound 1 was attempted by a variety of
routes (Scheme 1).
———
To avoid the possible steric inhibition during metabolic
activation, the synthesis of dimethyl compound, N-(2-
chloro-1,1-dimethylethyl)-3-(2-chloroethyl)tetrahydro-2H-
1,3,2-oxazaphosphorin-2-amine 2-oxide (3), was under-
taken (Scheme 2).
Correspondence: Prof. K. Misiura, Department of Bioorganic
Chemistry, Centre of Molecular and Macromolecular Studies, Pol-
ish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
E-mail: kmisiura@bio.cbmm.lodz.pl
Fax: +48 42 681 54 83
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/0809/0291 $17.50 +.50/0

292 Methyl analogues of ifosfamide
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 291–294
Scheme 2
Reaction of 2-chlorotetrahydro-2H-1,3,2-oxazaphosphor-
ine 2-oxide (4) with 2-chloro-1,1-dimethylethylamine hydro-
chloride[6] in the presence of triethylamine did not provide
intermediate 5, probably due to instability of the latter
compound under reaction conditions. Therefore the di-
methylaziridinyl compound 6 was produced by reaction of
chlorophosphate 4 with 1,1-dimethylaziridine. Unexpect-
edly, the process of opening the 1,1-dimethylaziridynyl ring
in the intermediate 6 by hydrochloric acid[7] led not only to
the desired 1,1-dimethyl compound 5 but also to the 2,2-
dimethyl isomer 7 in a ratio of 23:77, respectively (31P NMR
assay). On using a solution of HCl in benzene in this
reaction compounds 5 and 7 were formed in a ratio of 58:42,
which was not affected by changing the solvent to meth-
ylene chloride or THF. Any attempt to separate these regioi-
somers by crystallisation or chromatographic methods
failed. Thus, a mixture of 5 and 7 was chloroacetylated[8] in
the presence of a 4-molar excess of triethylamine hydro-
chloride[9] to give compounds 8 and 9, which could have
been separated by column chromatography on silica gel.
Reduction of the carbonyl group in both regioisomers 8 and
9 by sodium borohydride in the presence of boron trifluoride
etherate[9] gave a 1,1-dimethyl IF analogue, compound 3,
and its 2,2-dimethyl isomer, compound 10, respectively.
To examine the effect of the presence of geminal methyl
groups on metabolic hydroxylation in compounds 3 and 10,
they were subjected to the in vitro microsomal activation by
procedure established for oxazaphosphorines[10]. IF was
used as a reference compound in these studies. The rates
of metabolism of the examined compounds were analysed
by HPLC[11] and are shown in Table 1.
Table 1. The amount of metabolised compound during in vitro
metabolic activation.
————————————————————————————–
Time [min]
IF [%]
3 [%]
10 [%]
————————————————————————————–
10
20
40
60
30
40
53
58
34
43
49
65
32
38
43
60
————————————————————————————–

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 291–294
Methyl analogues of ifosfamide 293
It was found that compounds 3 and 10 were metabolised
at similar rates to IF. Probably, the lack of hydroxylation of
3 at the exocyclic 2-chloroethyl chain is compensated by a
higher rate of hydroxylation of C-4 in the ring and/or in the
endocyclic chain.
Acknowledgement
This work was supported by State Committee for Scientific
Research (KBN), Grant No 4 P05F 023 15. The authors wish to
thank Prof. W. J. Stec for critical reading of manuscript.
Experimental
The influence of methyl modification of IF analogues on
their therapeutic efficiency was examined by evaluation of
the antitumour activity of 3 and 10 against L1210 leukaemia
in mice. The values of effective doses (ED50, providing
50% increase of lifespan over control) were found to be
equal to 90, 183, and 237 [mg/kg] for IF, 3, and 10,
respectively. Compound 3 was less active then IF and, as
expected, compound 10 was considerably less active then
the reference drug. Probably the occurrence of other unde-
sired effects overcomes the therapeutic benefit of the re-
lease of lower amounts of chloroacetaldehyde during
metabolism of 3.
Methylene chloride, benzene, tetrahydrofuran, and triethyl-
amine were dried by distillation over calcium hydride. 2-Chloro-
tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide was obtained
by the method of Jones[13]. 1,1-Dimethylaziridine was prepared
from 2-amino-2-methyl-1-propanol[14]. Microsomes were iso-
lated from rat liver[10]. Glucose-6-phosphate dehydrogenase
(Type V) was from Sigma.
Unless stated otherwise, the progress of each reaction was
monitored by silica gel TLC. 1H and 31P NMR spectra were
recorded in CDCl3 on a Bruker AC-200. Mass spectrometric
analyses (chemical ionisation in iso-butane) were performed
using a Finnigan MAT 95 apparatus. HPLC analyses were
performed on LDC Analytical (UV detection at 200 nm) using
Econoshere C-8 (Alltech) column (flow 1 ml/min) and 40%
acetonitrile (Ultra gradient grade) as eluent.
The process of DNA alkylation is crucial for anticancer
activity of oxazaphosphorine drugs. The final, active meta-
bolite of IF, isophosphoramide mustard (iPAM), exerts
cross-linking activity toward double-stranded DNA via az-
iridynyl intermediates[12]. Most probably, final active meta-
bolites of 3 and 10, compounds 11 and 12, respectively,
undergo intermolecular cyclization to aziridinyl intermedi-
ates at much slower rates, as compared to that for iPAM
(Scheme 3).
1,1-Dimethylaziridinyltetrahydro-2H-1,3,2-oxazaphos-
phorine 2-oxide (6)
To
a
stirred solution of 2-chlorotetrahydro-2H-1,3,2-
oxazaphosphorine 2-oxide (4) (6.66 g, 30 mmole) in dry
methylene chloride (30 ml) was added a solution of 1,1-di-
methylaziridine (3.0 ml, 33 mmole) and dry triethylamine (5.0
ml, 36 mmole) in dry methylene chloride (20 ml). After 1 h the
reaction mixture was concentrated and the resulting crude
product (5.48 g) was purified by silica-gel chromatography in
chloroform containing 10–50% acetone. Compound 6 (2.75 g,
48% yield) was obtained as an oil: TLC (chloroform/acetone
1:1) Rf 0.47; 1H NMR δ: 1.40 (s, 3H), 1.41 (s, 3H), 1.48–1.57
(m, 2H), 2.17 (d, J = 14.2 Hz, 2H), 3.02 (br. s, 1H), 3.18–3.44
(m, 2H), 4.28–4.52 (m, 2H); 31P NMR δ 15.52; MS m/z 191.0
(M+H).
Mixture of N-(2-chloro-1,1-dimethylethyl)tetrahydro-2H-
1,3,2-oxazaphosphorin-2-amine 2-oxide (5) and N-(2-
chloro-2,2-dimethylethyl)tetrahydro-2H-1,3,2-oxazaphos
phorin-2-amine 2-oxide (7)
To a stirred solution of 1,1-dimethylaziridinyltetrahydro-2H-
1,3,2-oxazaphosphorine 2-oxide (6) (2.28 g, 12 mmole) in dry
benzene (48 ml) was added ca 1 M HCl/C6H6 dropwise until wet
pH paper showed a excess of acid. The reaction mixture was
concentrated, providing a mixture of 5 (58%, δP 8.28) and 7
(42%, δP 11.77) as a solid (2.64 g, 97% yield).
N-(2-chloro-1,1-dimethylethyl)-3-chloroacetyltetrahydro-
2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (8) and
N-(2-chloro-2,2-dimethylethyl)-3-chloroacetyltetrahydro-
2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (9)
Scheme 3
To a stirred solution of the mixture of N-(2-chloro-1,1-di-
methylethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-
oxide (5) and N-(2-chloro-2,2-dimethylethyl)tetrahydro-
2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (7) (2.27 g,
10 mmole total) and triethylamine hydrochloride (5.51 g, 40
mmole) in chloroform (stabilised with amylene) (100 ml) chlo-
roacetyl chloride (0.96 ml, 12 mmole) was added dropwise.
Reaction mixture was kept at 50 °C for 2.5 h and then was
washed with water (2 × 50 ml), dried (Na2SO4) and solvent
evaporated. Obtained mixture of 8 and 9 (2.09 g, 67%) was
separated by column chromatography on silica gel eluted with
a mixture ethyl acetate and n-hexane (2:1, v/v). Appropriate
In our previous studies on IF analogues possessing the
2-XCH2CH2NH moiety (X = Br, OSO2C6H4CH3,
OP(O)(C2H5) and other good leaving groups)[7] we proved
the importance of the rate of aziridinyl intermediate forma-
tion on anticancer activity of these group of compounds.
Thus it can be concluded that introduction of methyl sub-
stituents into 2-chloroethyl groups of ifosfamide leads to the
decrease of the antitumour activity of such analogues,
probably due to the lower ability of their final, active meta-
bolites to cross-link DNA.

294 Methyl analogues of ifosfamide
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 291–294
fractions were combined, concentrated, and crystallised from a
mixture of ethyl acetate and n-hexane (1:1, v/v). Compound 8
(1.05 g) was obtained as crystals: mp 139–140 °C; TLC (chlo-
roform/acetone 1:1) Rf 0.54; 1H NMR δ: 1.40 (s, 3H), 1.43 (s,
3H), 1.87–2.18 (m, 2H), 3.26–3.39 (m, 3H), 3.51, 3.68 (AB,
J = 11.0 Hz, 2H), 4.30–4.48 (m, 2H), 4.60, 4.77 (AB, J = 11.0
Hz, 2H); 31P NMR δ 0.54; MS m/z 303.0 (M+H, 2Cl); elemental
analysis for C9H17Cl2N2PO3: calc./found C 35.66/35.90, H
5.65/5.76, N 9.24/9.18, P 10.22/10.31. Compound 9 (0.89 g)
was also obtained as crystals: mp 123–124 °C; TLC (chloro-
form/acetone 1:1) Rf 0.46; 1H NMR δ: 1.60 (s, 6H), 2.00–2.18
(m, 2H), 3.08–3.42 (m, 4H), 3.75(br. s, 1H), 4.24–4.41 (m, 2H),
4.50, 4.65 (AB, J = 14.4 Hz, 2H); 31P NMR δ 3.82; MS m/z 303.0
(M+H, 2Cl). Anal. C, H, N, P.
sample was extracted with ethyl acetate (3 × 3 ml). Organic
solutions were combined, concentrated and redissolved in ace-
tonitrile (1 ml). Obtained samples were analysed on HPLC.
Antitumour activity
Antitumour effects of compounds 3, 10, and IF were evaluated
in vivo after single-dose treatment (day 1 only) of mice bearing
L1210 leukaemia. CDF1 mice were inoculated i.p. with 105
ascitic tumour cells suspended in 0.2 ml of PBS. The experi-
ments were conducted and activity of tested compounds evalu-
ated according to the NIH/NCI in vivo standard screening
protocols[15]. Effective doses providing 50% increase of
lifespan of treated animals over control (ED50) were estimated
graphically from least square-fitted dose-effect curves
N-(2-chloro-1,1-dimethylethyl)-3-(2-chloroethyl)tetra-
hydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (3) and
N-(2-chloro-2,2-dimethylethyl)-3-(2-chloroethyl)tetra-
hydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (10)
References
[1] L. R. Morgan, E. F. Harrison, G. E. Hawke, Semin. Oncol.
1991, 67, 2980–2983.
To a stirred solution of N-(2-chloro-1,1-dimethylethyl)-3-chlo-
roacetyltetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-ox-
ide (8) (0.61 g, 2 mmole) in dry THF (5 ml) sodium borohydride
(1.06 g, 2.8 mmole) was added in one portion and then boron
trifluoride etherate (0.45 ml, 3.6 mmole) was added dropwise.
After 1 h the reaction mixture was poured into water (10 ml) and
concentrated to ca 8 ml. The resulting suspension was ex-
tracted with chloroform (2 × 10 ml). Organic layers were com-
bined, dried (Na2SO4) and the solvent evaporated. Product 3
was crystallised from a mixture of ethyl ether and n-hexane (2:1,
v/v): 0.43 g (74% yield), mp. 75–76 °C; TLC (chloroform/ethanol
19:1) Rf 0.58; 1H NMR δ: 1.35 (s, 3H), 1.40 (s, 3H), 1.81–2.06
(m, 2H), 2.92 (br. s, 1H), 3.16–3.34 (m, 4H), 3.44–3.60 (m, 2H),
[2] L. D. Lewis, C. A. Meanwell, Lancet 1990, 335, 175–176.
[3] R. Skinner, I. M. Sharkey, A. O. Pearson, A. W. Graft, J. Clin.
Oncol. 1993, 11, 173–190.
[4] A. Kupfer, C. Aeschlimann, B. Wermuth, T. Cerny, Lancet
1994, 343, 763–764.
[5] K. Misiura, R. W. Kinas, H. Kusnierczyk, unpublished results.
[&] A. S. Gudkova, G. M. Ostapchuk, I. V. Petrosian, O. A.
Reutov, Dokl. Akad. Nauk SSSR Ser. Khim. 1970, 194,
1086–1089.
[7] K. Misiura, R. W. Kinas, W. J. Stec, H. Kusnierczyk, C.
4.30–4.48 (m, 2H), 3.68–3.79 (m, 2H), 4.20–4.41 (m, 2H); 31
P
Radzikowski, A. Sonoda, J. Med. Chem. 1988, 31, 226–230.
NMR δ 8.67; MS m/z 289.1 (M+H, 2Cl); Anal. C, H, N, P.
[8] K. Pankiewicz, R. Kinas, W. J. Stec, A. B. Foster, M. Jarman,
J. M. S. Van Maanen, J. Am. Chem. Soc. 1979, 101, 7712–
7718.
Staring
from
N-(2-chloro-2,2-dimethylethyl)-3-chloroace-
tyltetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide (9)
and using the same procedure product 10 was obtained in 85%
yield: TLC (chloroform/ethanol 19:1) Rf 0.56; 1H NMR δ: 1.56
(s, 6H), 1.89–2.04 (m, 2H), 2.17 (br. s, 1H), 3.10 (d, J = 8.6 Hz,
2H), 3.16–3.30 (m, 4H), 3.36–3.52 (m, 2H), 3.58–3.65(m, 2H),
4.10–4.42 (m, 2H); 31P NMR δ 12.30; MS m/z 289.1 (M+H, 2Cl);
Anal. C, H, N, P. Attempts to crystallise 10 failed.
[9] K. Misiura, R. W. Kinas, H. Kunierczyk, C. Radzikowski, W.
J. Stec, Anti-Cancer Drugs 2001, 12, 453–458.
[10] T. A. Connors, P. J. Cox, P. B. Farmer, A. B. Foster, M.
Jarman, Biochem. Pharmacol. 1974, 23, 115–129.
[11] L. A. Burton, C. A. James, J. Chromatography 1988, 431,
450–454.
Microsomal activation
[12] J. H. Boal, M. Williamson, V. L. Boyd , S. M. Ludeman, W.
Solutions of 3, or 10, or IF (0.4 mM, 10 ml) in 0.1 M Tris-HCl pH
7.4 containing: 5 mM MgCl2, 6 mM glucose-6-phosphate, 0.3
mM NADP, glucose-6-phosphate dehydrogenase (0.8 U/ml)
and active or inactive microsomes equivalent to 1 g of rat liver
were incubated in oxygen atmosphere at 37 °C for 10, 20, 40,
and 60 min. After each period of time a sample was removed
(2 ml) and chilled in ice. After addition of a 0.4 mM solution of
cyclophosphamide in 0.1 M Tris-HCl pH 7.4 (0.2 ml) each
Egan, J. Med. Chem. 1989, 32, 1768–1773.
[13] R. N. Hunston, M. Jehangir, A. S. Jones, R. T. Walker,
Tetrahedron 1980, 36, 2337–2340.
[14] T. L. Cairns, J. Am. Chem. Soc. 1941, 63, 871–872.
[15] Experimental Therapeutics Program: In vivo cancer models
1976-1982, NIH publication No. 84-2635, Bethesda, MD.