Synthesis of side-chain-substituted ifosfamide analogs

Article abstract of DOI:10.1002/jhet.5570380517

Three analogs of the cytostatic drug ifosfamide incorporating 1-methyl-2-chloroethyl side chains were designed and prepared as an attempt to obtain drugs of lower toxicity.

Full text of DOI:10.1002/jhet.5570380517

Synthesis of Side-chain-substituted Ifosfamide Analogs
Angelo Paci, Dominique Guillaume* and Henri-Philippe Husson
1131
Sep-Oct 2001
†
Laboratoire de Chimie thérapeutique, UMR 8638 du CNRS,
Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris cedex 06, France
Received April 9, 2001
Three analogs of the cytostatic drug ifosfamide incorporating 1-methyl-2-chloroethyl side chains were
designed and prepared as an attempt to obtain drugs of lower toxicity.
J. Heterocyclic Chem., 38, 1131 (2001).
Ifosfamide (1a, 2-(2-chloroethylamino)-3-(2-chloro-
chloroacetaldehyde (4, Figure). This latter is suspected of
being responsible of the observed neurotoxicity, urotoxic-
ity and cardiotoxicity of the oxazaphosphorines [3].
Furthermore, it has also been observed that the deactiva-
tion route has a faster kinetic in the case of 1a compared to
1b, hence inducing higher blood levels of 4. The release of
4 in the blood stream is a serious drawback particularly in
the case of high-dose treatment and generally prevents the
use of ifosfamide that is however intrinsically a more
efficient alkylating drug than cyclophosphamide.
Though the reasons for this difference in term of side
chain metabolism are still unclear, we postulated that it
could be due to an easier access by the oxidative enzymatic
core to the 1-position of the 2-chloroethyl side chains of
1a. Indeed, the two chloroethyl chains of 1b substituting
the same nitrogen atom, the bulky bis(2-chloroethyl)amino
group is probably less accessible to the enzyme.
Consequently we reasoned that substitution of this
sensitive 1-position by a methyl group would induce some
hindrance that could reduce or even eliminate the
possibility of oxidation with minimum perturbation of the
alkylating properties. We did not envisage a substitution of
the 2-position or a 1,2-disubstitution since, in the
cyclophosphamide series, these kinds of derivatives have
been shown to be poorly therapeutically efficient [4].
Thus, we designed three compounds (16a-c) in which a
single or both 2-chloroethyl residues of 1a have been
replaced by a 1-methyl-2-chloroethyl chain. We herein
wish to report the synthesis of these three compounds.
Synthesis of 2-alkylamino-3-substitued-2H-1,3,2-
oxazaphosphorine 2-oxide can generally be easily
accomplished according two routes. Either a 2-chloro-3-
substituted-2H-1,3,2-oxazaphosphorine 2-oxide, prepared
ethyl)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) is a
structural isomer of cyclophosphamide (1b, 2-[bis(2-
chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine
2-oxide) differing only in the position of the alkylating
groups (Figure).
Both compounds are efficient antitumor agents
employed as a racemic mixture in the chemotherapy of
several tumors [1]. They also share a similar enzyme-
catalyzed activation process necessary for the therapeutic
activity [2]. This process begins by the formation of
4-hydroxylated derivatives (2) and ultimately ends with
the release of an isophosphoramide mustard (3) that is the
true alkylating species (Figure). Concomitantly, a compet-
itive deactivation pathway occurs by oxidation of the
chloroethyl groups and leads to the liberation of
by condensing an aminoalcohol with POCl , reacts with an
3
amine [5,6] or POCl is first stoichiometrically condensed
3
with an amine and the resulting species is cyclized using
an appropriate aminoalcohol [7].
The synthesis of 16a-c was performed following an
identical strategy that follows the first route (Scheme). In
both cases a suitable chloro-amino-alcohol was condensed
with POCl to afford the corresponding 2-chloro-3-
3
substituted-2H-1,3,2-oxazaphosphorine 2-oxide that was
subsequently condensed under basic conditions with the
selected chloroalkylamine hydrochloride to afford the
desired ifosfamide analog.
Figure. Schematic representation of the oxidative activation and deactiva-
tion pathways of 1.

1132
A. Paci, D. Guillaume and H.-P. Husson
Vol. 38
Reagents, conditions and yields: i: SOCl , 75%; ii: DIC, Ph-Me, reflux, 74%; iii: NaH, DMF, Br(CH ) OAc, 120 °C, 80%; iv: N H , 95%; v: HCl gas,
2
2 3
2 4
70%; vi: POCl , CH Cl , Et N; vii: NH (CH ) Cl, HCl, Et N, reflux, 60%; viii: Et N, reflux, 35% (16c) 52% (16b).
3
2
2
3
2
2 2
3
3
Preparation of the 2-Chloro-3-substituted-2H-1,3,2-oxaza-
phosphorine 2-oxide Moities 5 and 6.
Synthesis of 6 whose N-3 atom is substituted with a
1-methyl-2-chloroethyl side chain was performed by
applying a strictly similar protocole to oxazolidinone 11
that was prepared in 74% yield by refluxing racemic
2-aminopropanol and N-N'-carbonyldiimidazole (DIC) in
toluene [8]. Compound 6 and its synthetic intermediates
were obtained in yields almost identical to those obtained
for the preparation of 5.
We first decided to prepare 5 whose N-3 atom is substi-
tuted with a chloroethyl side chain. Reaction of 2-oxazo-
lidinone 7 with 3-bromopropylacetate in anhydrous DMF
at 120 °C and in the presence of NaH afforded N-substi-
tuted 2-oxazolidinone 8 in 80% yield. Deacetylation of this
latter, performed in almost quantitative yield by use of
aqueous hydrazine was followed by the opening of the
oxazolidinone (HCl gas) furnishing 10 in 70% yield.
Cyclization of 10 into 5 (65% yield) was accomplished in
Preparation of Diastereomers 16.
Although compounds 5 and 6 are relatively stable, we
observed that derivatives 16 were obtained in much better
yields when the synthetic procedure was carried out
refluxing dichloromethane by use of POCl in the pres-
3
ence of 3 equivalents of Et N (Scheme).
3

Sep-Oct 2001
Synthesis of Side-chain-substituted Ifosfamide Analogs
1133
Compound 8 was obtained as an oil; yield 82%; IR (neat) 2930,
rapidly and without isolation of the 2-chloro-oxazaphos-
phorine 2-oxide 5 and 6. Consequently, and as reported in
the phosphotriamide series [9], we currently synthesized
compounds 16 using a one pot procedure.
Synthesis of 16a was realized in 60% yield by directly
adding to a refluxing solution of 6 in 2-chloroethylamine
-1
1
1732, 1430, 1244 cm ; H-NMR (CDCl ): δ 1.95 (m, 2H), 2.12
3
(s, 3H), 3.46 (t, J= 7, 2H), 3.71 (m, 2H), 4.18 (t, J=6, 2H), 4.40
13
(m, 2H) ; C-NMR (CDCl ): δ 20.8, 27.3, 42.1, 45.6, 62.9, 63.6,
3
+
160.8, 172.6 ; MS m/z (rel. int., %) 205 (C H NO +NH
100), 188 (C H NO +H , 23).
8 13
Compound 12 was obtined as an oil; yield 80%; IR (neat)
,
8 13
4
4
+
4
-1
1
and 2 equivalents of Et N.
2930, 1738, 1732 cm ; H-NMR: δ 1.23 (d, J= 6, 3H), 1.94
3
Synthesis of 16b,c required the preparation of 1-methyl-
2-chloroethylamine hydrochoride 15. This was achieved in
75% yield by treatment of (±)-2-aminopropanol with
(m, 2H), 2.01 (s, 3H), 3.14 (m, 1H), 3.45 (m, 1H), 3.82 (m, 2H),
4.05 (t, J=6, 2H), 4.35 (t, J= 6.5, 1H); C-NMR: δ 17.9, 20.8,
13
26.3, 38.6, 51.0, 61.6, 68.7, 157.9, 170.8 ; MS m/z (rel. int., %)
+
+
219 (C H NO +NH , 100), 202 (C H NO +H , 46).
9 15 9 13
4
4
4
SOCl [10]. Condensation of 15 with 5 or 6, in the
2
conditions depicted for the preparation of 16a led to the
isolation of 16b (52%) and 16c (35%), respectively.
Synthesis of 3-(2-Oxo-1,3-oxazolidin-3-yl)-propan-1-ol (9) and
3-(4-Methyl-2-oxo-1,3-oxazolidin-3-yl)-propan-1-ol (13).
To a solution of 8 or 12 (346 and 372 mg respectively,
1.85 mmol) in 5 mL of methanol was added at room temperature
1.1 equivalent of hydrazine hydrate. The solution was heated at
50 °C for 4 hours and 0.2 equivalent of hydrazine hydrate were
re-added. The solution was allowed to return to room temperature
and stirred for 2 hours. The solution was concentrated under
reduced pressure and the residue flash chromatographed using
dichloromethane/methanol (98/1) as an eluent. Compounds 9 and
13 were obtained in 95% yield and used for the next step without
further characterization.
Conclusion.
We have been able to prepare three ifosfamide analogs
designed to prevent the enzymatic oxidation of the side
chains and hence the release of chloroacetaldehyde, a
highly toxic metabolite. Although, chirality of the phos-
phorus atom is not a critical factor for the therapeutic use,
chirality on the side chains has still to be evaluated. In case
of importance, our synthetic strategy can easily be applied,
on a large scale, in asymmetric series using commercially
available optically pure 2-aminopropanol. Biological data
and importance of the side-chain chirality will be the
subject of a forthcoming communication.
Synthesis of 3-(2-Chloroethylamino)-propan-1-ol (10) and 3-(2-
Chloro-1-methylethylamino)-propan-1-ol (14).
A solution of oxazolidinone 9 or 13 (1 and 1.1 g respectively,
6.89 mmol) in 30 mL of THF was placed under a stream of HCl
at room temperature for 30 minutes. The solution was refluxed
for 4 hours then stirred at room temperature overnight. The
solution was concentrated under reduced pressure and the residue
flash chromatographed using dichloromethane/methanol (9/2) as
an eluent.
EXPERIMENTAL
IR spectra were recorded on a Perkin Elmer FTIR-1600 spec-
trometer. NMR spectra were obtained with a Bruker AC-300
instrument using CD OD as a solvent unless otherwise indicated.
3
Compound 10 was obtained as a powder; yield 73%; IR (neat)
1
13
H and C chemical shifts are reported from residual non-
-1
1
3380, 1634, 1435 cm ; H-NMR: δ 2.02 (m, 2H), 3.25 (t, J=7,
2H), 3.51 (t, J=7, 2H), 3.73 (t, J=7, 2H), 4.02 (t, J=7, 2H);
31
deuteriated solvent traces. P-NMR spectra were calibrated
using phosphoric acid as external reference. Coupling constants
(J) are given in Hz. Chromatographic separations were carried
out on a silicagel column (Merck; 230-400 mesh). TLC were
developped on Merck 60F
after spraying a 5% ethanolic solution of phosphomolybdic acid.
Ionization of the samples for mass analysis was performed by
electronic impact or chemical ionization using NH as vector.
Some synthetic intermediates are potentially carcinogenic and
consequently should be handled with care.
13
C-NMR 29.5, 40.4, 47.7, 50.3, 60.8; MS m/z (rel. int., %) 138
+
+
(C H NOCl+H , 100), 140 (C H NOCl+H , 31).
5 12
5 11
Compound 14 was obtained as a powder; yield 70%; IR (neat)
plates and revealed by heating
254
-1 1
3400, 1634, 1432 cm ; H-NMR: δ 1.53 (d, J= 6.5, 3H), 2.02 (m,
2H), 3.25 (m, 2H), 3.75 (m, 3H), 3.84 (m, 2H), 4.06 (t, J=7, 2H);
13
C-NMR: δ 14.3, 29.5, 44.9, 45.9, 55.8, 60.5; MS m/z (rel. int.,
3
+
+
%) 152 (C H NOCl+H , 100), 154 (C H NO +H , 39).
6 14 8 13
4
Synthesis of 2-(2-Chloroethylamino)-3-(1-methyl-2-chloroethyl)-
tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide (16a), 2-(1-
Methyl-2-chloroethylamino)-3-(2-chloroethyl)tetrahydro-2H-
1,3,2-oxazaphosphorine 2-oxide (16b), and 2-(1-Methyl-2-
chloroethylamino)-3-(1-methyl-2-chloroethyl)tetrahydro-2H-
1,3,2-oxazaphosphorine 2-oxide (16c).
Synthesis of 3-(2-Oxo-1,3-oxazolidin-3-yl)-propylacetate (8) and
3-(4-Methyl-2-oxo-1,3-oxazolidin-3-yl)-propylacetate (12).
A solution of oxazolidinone (7 or 11 [6]) (190 and 220 mg
respectively, 2.2 mmol) in 4 mL of DMF was cooled at 0 °C.
Sodium hydride (1.25 equivalent) was carefully added and the
solution allowed to warm up to room temperature then heated to
50 °C for 1 hour. Then 3-bromopropylacetate (1.5 equivalent)
was added and the solution was refluxed for 2 hours. The organic
solution was cooled at room temperature then slowly poured in
1.5 mL of a saturated aqueous ammonium chloride. The crude
mixture was concentrated under reduced pressure and
compounds 8 or 12 were flash chromatographed using
dichloromethane/methanol (19:1) as an eluent.
To a solution of 10 or 14 (226 and 244 mg respectively,
1.3 mmol) in dichloromethane (100 mL), Et N (1 equivalent)
3
was added. Then, phosphorus oxychloride (1 equivalent) was
slowly and carefully added and the solution was cooled in case of
excessive heating. Triethylamine (2 equivalents) was further
added. The solution was stirred for 2 hours then the hydrochlo-
ride salt of the suitable chloroethylamine was added together
with two more equivalents of Et N. The solution was refluxed
3
for 6 hours. The solvent was then evaporated and the crude

1134
A. Paci, D. Guillaume and H.-P. Husson
Vol. 38
product was chromatographed using dichloromethane/methanol
(98:2) as an eluent. Compounds 16a, 16b and 16c were obtained
in pure form in 60%, 52%, and 35% yield, respectively.
Anal. Calcd for C H N O PCl •4H O: C, 29.93; H, 7.53;
9 19 2 2
2
2
N, 7.76. Found: C, 30.11; H, 7.39; N, 7.91.
Compound 16a was obtained as a thick oil; IR (neat) 3216,
REFERENCES AND NOTES
-1
1
1455, 1265 cm ; H-NMR (mixture of diastereomers): δ 1.32
(6H), 1.95 (m, 4H), 3.15-3.31 (m, 8H), 3.55 (m, 4H), 3.65
(m, 4H), 3.76 (m, 1H), 3.92 (m, 1H), 4.21 (m, 2H), 4.35 (m, 2H);
[†*] Permanent address: Laboratoire de Chimie Thérapeutique, 1
rue des Louvels, 80037 Amiens cedex 1, France. dominique.guil-
laume@sa.u-picardie.fr.
[1] D. L. Hill, A Review of Cycloposphamide, C. C. Thomas:
Springfield, Il, 1975; H. Burket and H. C. Voigt, Proceedings of the
International Holoxan Symposium, Düsseldorf, March 21-23, 1977.
[2] N. E. Sladek, Pharmacol. Ther., 37, 301 (1988); G. P. Kaijser,
J. H. Beijnen, A. Bult and W. J. M. Underberg, Anticancer Res., 14, 517
(1994).
[3] M. P. Goren, R. K. Wright, C. B. Pratt and F. E. Pell, Lancet II,
1219 (1986); J. Pohl, J. Stekar and P. Hilgard, Arzneim. Forsch., 39, 704
(1989); C. Joqueviel, M. Malet-Martino and R. Martino, Cell. Mol. Biol.,
43, 773 (1997).
[4] H. Arnold, F. Bourseaux and N. Brock, Arzneim.-Forsch., 11,
143 (1961).
[5] K. Misuiura, A. Okruszek, K. Pankiewcz, W. J. Stec, Z.
Czownicki and B. Utracka, J. Med. Chem., 26, 674 (1983).
[6] B. Kutscher, U. Niemeyer, J. Engel, A. Kleeman, P. Hilgard, J.
Pohl and G. Scheffler, Arzneim.-Forsch., 45, 323 (1995).
[7] S. M. Ludeman, D. L. Bartlett and G. Zon, J. Org. Chem., 44,
1163 (1979).
[8] J. R. Piper, C. R. Stringfellow Jr, R. D. Elliott and T. P.
Johnston, J. Med. Chem., 14, 345 (1971).
[9] D. Guillaume and G. R. Marshall, Synthetic Commun., 28,
2903 (1998).
31
P-NMR (mixture of diastereomers): δ 13.6, 14.4 ; MS m/z (rel.
+
int., %) 292 (C H N O PCl +NH , 100), 294 (C H
8 17 2 2
2
4
8 17-
+
+
N O PCl +NH , 38), 275 (C H N O PCl +H , 42), 277
2 2 8 17 2 2
2
4
2
+
(C H N O PCl +H , 12).
8 17 2 2
2
Anal. Calcd for C H N O PCl •3H O: C, 29.19; H, 7.04;
8 17 2 2
2
2
N, 8.51. Found: C, 29.23; H, 7.14; N, 8.67.
Compound 16b was obtained as a thick oil; IR (neat) 3210,
-1
1
1455, 1265 cm ; H-NMR (mixture of diastereomers): δ 1.32
(6H), 1.95 (m, 2H), 2.15 (m, 2H), 3.10-3.35 (m, 6H), 3.40
(m, 2H), 3.55-3.75 (m, 10H), 4.25 (m, 2H), 4.35 (m, 2H);
31
P-NMR (mixture of diastereomers): δ 13.6, 14.4 ; MS m/z (rel.
+
int., %) 292 (C H N O PCl +NH , 100), 294 (C H N O
8 17 2 2
2
4
8 17 2 2-
+
+
PCl +NH , 40), 296 (C H N O PCl +NH , 4), 275 (C
2
4
8 17 2 2
2
4
2
8-
+
+
H
N O PCl +H , 23), 277 (C H N O PCl +H , 12), 279
17 2 2 8 17 2 2
2
(C H N O PCl , 8).
8 17 2 2
2
Anal. Calcd for C H N O PCl •2H O: C, 30.88; H, 6.80;
N, 9.00. Found: C, 30.61; H, 6.97; N, 9.01.
8 17 2 2
2
2
Compound 16c was obtained as a thick oil; IR (neat) 3216,
-1
1
1455, 1265 cm ; H-NMR: complex mixture of diastereomers;
31
P-NMR (mixture of diastereomers) 10.6, 11.4, 13.6 ; MS m/z
+
(rel. int., %) 306 (C H N O PCl +NH , 100), 308 (C H
9 19 2 2
2
4
9 19-
+
+
N O PCl +NH , 52), 310 (C H N O PCl +NH , 8), 289
2 2 9 19 2 2
2
4
2
4
2
+
+
(C H N O PCl +H , 23), 291 (C H N O PCl +H , 9),
[10] K. Kashiwabara, I. Kinoshita, T. Ito and J. Fujita, Bull. Chem.
Soc. Jpn., 54, 725 (1981).
9 19 2 2 9 19 2 2
2
293 (C H N O PCl , 2).
9 19 2 2
2