Synthesis and in vitro antileukemic activity of some new 1,3-(oxytetraethylenoxy)cyclotriphosphazene derivatives

Article abstract of DOI:10.1021/jm0490078

A new series of 1,3-(oxytetraethylenoxy)cyclotriphosphazene derivatives bearing 2-chloroethylamine or salicylaldehyde (2-hydroxybenzaldehyde) or its Schiff base (after condensation with 2-chloroethylamine) units and having also 2-naphthyl or anthraquinone

Full text of DOI:10.1021/jm0490078

806
J. Med. Chem. 2006, 49, 806-810
Synthesis and in Vitro Antileukemic Activity of Some New
1,3-(Oxytetraethylenoxy)cyclotriphosphazene Derivatives§
Mariola Siwy,† Danuta Se¸k,*,† Boz˘ena Kaczmarczyk,† Iwona Jaroszewicz,‡ Anna Nasulewicz,‡ Marzena Pelczyn˜ska,‡
Dmitry Nevozhay,‡ and Adam Opolski*,‡
Center of Polymer Chemistry, PAS, 34 M. Curie-Sklodowska Street, 41-800 Zabrze, Poland, and
Institute of Immunology and Experimental Therapy, PAS, 12 R. Weigl Street, 53-114 Wroclaw, Poland
ReceiVed December 6, 2004
A new series of 1,3-(oxytetraethylenoxy)cyclotriphosphazene derivatives bearing 2-chloroethylamine or
salicylaldehyde (2-hydroxybenzaldehyde) or its Schiff base (after condensation with 2-chloroethylamine)
units and having also 2-naphthyl or anthraquinone groups as cosubstituents has been synthesized. The in
vitro cytotoxic activity of these compounds against a panel of four cancer cell lines has been studied. Most
of the compounds exhibited antiproliferative activity in the range of the international criterion for synthetic
agents (4 µg/mL) against the MOLT4, L 1210, HL-60, and P388 cell lines chosen for testing.
Introduction
compounds is their high cumulative bone marrow toxicity and
other undesirable side effects. Therefore, a great deal of research
has been devoted to overcome these drawbacks and also to
improve solubility of aziridinyl cyclophosphazene. The latter
property was achieved by incorporation of a crown ether
structure into the aziridinyl cyclophosphazene which caused
good solubility of the compound in water (∼10%w/w) and also
a possibility of metal cation complexation.
In the search for anticancer drugs many compounds with
different structures have been tested. Even though some of them
exhibit therapeutical properties and are widely used, the hunt
for new substances especially those with improved efficiency
and no side effects is still an important research goal. Among
others, compounds with a cyclophosphazene core bearing
different substituents seem to be good candidates for anticancer
drugs. A great number of reports have described the antitumor
activity of (1-aziridynyl)cyclophosphazenes,1-9 mainly hexa-
(1-aziridynyl)cyclophosphazene MYKO 63.
The cytostatic activity of 1,3-(oxytetraethylenoxy)-1,3,5,5-
tetra(1-aziridynyl)cyclotriphosphazatriene has been tested in
vitro using five AIDS-related human lymphoma cell lines and
leukemia CCRF-CEM. The compound exhibited remarkable
cytotoxic activity.11 Further modification of this compound by
replacement of one aziridine group with an intercalating agent,
i.e., 2-naphthol or an anthraquinone derivative, has been carried
out.12
In previous works of our group a series of tetraaziridinylcy-
clophosphazenes bearing various spiro substituents whose
structures are shown below were tested and their cytotoxic
It was found that introduction of the 2-naphthol structure
decreased the in vitro antiproliferative activity of the compound
against human and mouse leukemic cells (MOLT 4, P 388, L
1220, HL-60) while the anthraquinone group enhanced cytotoxic
activity in comparison with the 1,3-(oxytetraethylenoxy)-1,3,5,5-
tetra(1-aziridinyl)cyclotriphosphazatriene without intercalating
agent. However, for the synthesis of the compounds described
above, it is necessary to use aziridine which in itself is a
carcinogen. Another group of compounds capable of DNA
alkylation are those with a 2-chloroethylamine moiety.10,13-15
To the authors’ best knowledge there are no previous reports
activity against L1210 cells was confirmed.8,9 The cytostatic
activity of aziridynyl groups is believed to result from the
formation of a carbocation capable of interacting with DNA
via alkylation.1,10 However, a severe disadvantage of these
§ In memory of Krystyna Brandt.
* Corresponding authors. D.S.: tel.:+4832 2716077, +4832 2732214,
fax: +4832 2712969. e-mail: danuta.sek@cchp-pan.zabrze.pl. A.O.: tel.
+4871 3371172 ext.371, fax: +4871 3371172 ext. 366. e-mail: opolski@
iitd.pan.wroc.pl.
† Center of Polymer Chemistry.
‡ Institute of Immunology and Experimental Therapy.
10.1021/jm0490078 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/20/2005

1,3-(Oxytetraethylenoxy)cyclotriphosphazene DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 807
Figure 1. Structures of the compounds 1-11.
concerning the synthesis and cytostatic activity of cyclophos-
phazatriene substituted with an oxytetraethylenoxy group (crown
ether) and a 2-chloroethylamine group, as are presented in this
work.
2-Chloroethylamine was bonded directly to the cyclophos-
phazatriene ring or was introduced in the form of a Schiff base.
In the latter case 2-chloroethylamine was condensed with a
previously substituted cyclophosphazene ring with 2-hydroxy-
benzaldehyde (salicylaldehyde). Syntheses of the compounds
having additional intercalating groups (2-naphthol, anthraquino-
ne) were also carried out.
expected to enhance cytotoxic activity of the compounds in
comparison to the ones having only alkylating agent.
The compounds synthesized were tested for their antiprolif-
erative activity in vitro against the cells of four human and
mouse leukemic lines. The results are presented in Table 1. All
the compounds bearing alkylating and intercalating groups,
except 2, revealed antiproliferative activity against the cells
tested. However, the compounds 5 and 6 did not exhibit activity
in the range of the international activity criterion for synthetic
agents (4 µg/mL).19
In the compounds 2 and 6, the 2-chloroethylamine moiety is
directly bonded to phosphorus atoms. This might account for
its low DNA alkylating activity. On the other hand, the presence
of the anthraquinone structure in 6 induced antiproliferative
activity similar to that of the compound 5, having unsubstituted
chlorine atoms. In contrast to 6, a compound with aziridinyl
substituents instead of 2-chloroethylamine groups exhibited
antiproliferative activity against the same leukemic cells.
However, the tetraaziridinyl compound (without intercalator)
was also inactive similar to 2.12
Results and Discussion
In this work, two series of derivatives of 1,3-(oxytetraethyl-
enoxy)-1,3,5,5-tetrachlorocyclotriphosphazatriene (1, precursor)
have been synthesized (Figure 1) and their structures confirmed
by elemental analysis, mass spectroscopy, FTIR, 1H NMR, and
31P NMR. In the first group, all chlorine atoms of the precursor
1 were substituted with 2-chloroethylamine being directly
connected with phosphorus atoms of the cyclotriphosphazatriene
ring as in 2 or 2-chloroethylamine was bonded via an oxyphe-
nylene unit and was in the form of aldimine as in 4. In the
second group intercalating anthraquinone or 2-naphthol struc-
tures were introduced additionally (6, 8). Their presence was
A sufficient cytostatic activity was exhibited by compounds
4 and 8 in which the alkylating agent is in the form of a Schiff
base. The presence of an intercalating agent in 8 did not
influence its activity significantly. Similarly, introduction of the

808 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Siwy et al.
Table 1. In Vitro Antiproliferative Activity of the Compounds (Figure
1)
Experimental Section
Synthesis of the Investigated Compounds 1-11. Structures
are presented in Figure 1.
1,3-(Oxytetraethylenoxy)-1,3,5,5-tetrachlorocyclotriphosph-
azatriene 1. This compound was prepared as reported previ-
ously.16,17
1,3-(Oxytetraethylenoxy)-1,3,5,5-tetra(2-chloroethylamine)-
cyclotriphosphazatriene 2. 2-Chloroethylamine hydrochloride
(2.06 g, 17.6 mmol) and triethylamine (TEA) (4.857 g, 48.0 mmol)
in dry benzene (100 mL) were stirred for 1 h at room temperature
under an argon atmosphere. Subsequently, 1 (0.938 g, 2.0 mmol)
in dry benzene (20 mL) was added. The mixture was stirred for 29
h in boiling solvent and then cooled and filtered. The filtrate was
evaporated under reduced pressure. The crude product was column-
chromatographed on silica gel using hexane:THF (tetrahydrofuran)
(1:1 and next 2:3 and 1:2, v/v) as eluent. The oily substance (yield
48.5%) was crystallized from a mixture of hexane:CHCl3 (1:3, v/v)
to provide 2: mol mass (g/mol) found/calcd M ) 642.1/641, mp
84.70 °C. Anal. (C16H36Cl4N7O5P3) C, H, N.
1,3-(Oxytetraethylenoxy)-1,3,5,5-tetra(phenyloxy-2-carboxal-
dehyde)cyclotriphosphazatriene 3. 1 (0.938 g, 2.0 mmol), 2-hy-
droxybenzaldehyde (1.495 g, 12 mmol), and sodium hydride (60%)
(0.48 g, 12 mmol) in THF (80 mL) were stirred for 27 h at room
temperature under an argon atmosphere. The mixture was filtered
and the filtrate evaporated under reduced pressure. The crude
product was column-chromatographed on silica gel using hexane:
THF (3:2 next 1:1 v/v) as eluent. The oily substance (yield 49.5%)
was crystallized from mixture of hexane:CHCl3 (1:3, v/v) to provide
3: mol mass (g/mol) found/calcd M ) 812.6/811.48, mp 100.8
°C. Anal. (C36H36N3O13P3) C, H, N.
a ID50: the dose of compound that inhibits cell proliferation by 50%.
2-naphthol structure, instead of anthraquinone, in compound 11
did not cause any change in activity. It is very interesting that
compounds 3, 7, and 10, having aldehyde groups in their
substituents, also exhibited high antiproliferative activity that
was little enhanced by the anthraquinone moiety in the
compound 7. In fact, the activity of the compounds having
aldehyde and Schiff base groups differ very little.
1,3-(Oxytetraethylenoxy)-1,3,5,5-tetra(phenyloxy-2-{2-chloro-
ethylimino})cyclotriphosphazatriene 4. 2-Chloroethylamine hy-
drochloride (0.445 g, 3.8 mmol) and triethylamine (1.154 g, 11.4
mmol) in dry benzene (20 mL) were stirred for 1 h at room
temperature in an argon atmosphere. Subsequently, 3 (0.772 g, 0.95
mmol) in dry benzene (20 mL) was added. The mixture was stirred
for 4 h in boiling solvent. The mixture was then filtered and benzene
evaporated under reduced pressure. The yield of the oily substance
4 was 51.2%, mol mass (g/mol) found/calcd M ) 1058.0/1057.48.
Anal. (C44H52Cl4N7O9P3) C, H, N.
For comparison, activity of compounds 3, 4, 7, and 8 against
normal cells (BALB/ 3T3) was also examined. As can be seen
in Table 1, the antiproliferative activity of the compounds is
much higher against the cancer cells than against the normal
cells.
The following results summarize our investigations dealing
with antiproliferative activity in vitro against cells of human
and mouse leucemic lines (MOLT4, P388, L1210, HL-60) of
the new compounds:
1-{2-[1,3-(Oxytetraethylenoxy)-3,5,5-tri(2-chloroethylamine)-
cyclotriphosphazatrien-1-yl]aminoethylamino}anthraquinone 6.
The synthesis of this compound required two steps (a, b). Step a:
1 (0.235 g, 0.5 mmol), 1-(2-aminoethylamino)anthraquinone (pre-
pared as described in the literature18) (0.266 g, 1.0 mmol), and
NaOH (0.2 g, 5 mmol) were dissolved in a mixture of hexane:
THF (1:1, v/v; 60 mL) and heated at 40 °C for 5 h. The mixture
was filtered and the filtrate evaporated under reduced pressure. The
crude product was column-chromatographed on silica gel using
hexane:THF (3:1, v:v) as eluent to provide 1-{2-[1,3-(oxytetraeth-
ylenoxy)-3,5,5-trichlorocyclotriphosphazatrien-1-yl]aminoethylamino}-
anthraquinone 5 as a red powder. Yield 58.2%, mol mass (g/mol)
found/calcd M ) 699.2/698.7, mp 149.4 °C. Anal. (C24H29Cl3N5O7P3)
C, H, N: calcd, 10.0; found, 9.58.
Step b: 2-Chloroethylamine hydrochloride (0.476 g, 4.07 mmol)
and triethylamine (1.123 g, 11.1 mmol) in dry benzene (60 mL)
were stirred for 1 h at room temperature in an argon atmosphere.
Subsequently, 5 (0.432 g, 0.618 mmol) in dry benzene (60 mL)
was added. The mixture was then stirred for 22 h in boiling solvent.
After cooling, the mixture was filtered and the solvent evaporated
under reduced pressure. The crude product was purified column-
chromatographically on silica gel using hexane:THF (2:1, v/v) as
eluent. Compound 6 possessed a greasy nature. Yield 18.7%, mol
mass (g/mol) found/calcd M ) 827.4/827.63 g/mol, mp 84.33 °C.
Anal. (C30H44Cl3N8O7P3) C, H, N.
(a) 1,3-(Oxytetraethylenoxy)-1,3,5,5-tetrachlorocyclotriphos-
phazatriene 1 and its mono- aminoanthraquinone-substituted
derivative 5 do not exhibit antiproliferative activity in the range
of the international criterion for synthetic agents.
(b) Compound 2 in which four chlorine atoms in compound
1 are substituted with a 2-chloroethylamine moiety also is
inactive in relation to the cell lines tested. Replacement of one
2-chloroethylamine residue in 2 by an aminoanthraquinone
moiety enhances antiproliferative activity but it is still lower
than the value of the international criterion.
(c) Substitution of four chlorine atoms in 1 with 2-oxyben-
zaldehyde groups (3) enhances the cytotoxic activity, being
sufficient to meet the international criterion. Slightly better
activity resulted from replacement of one 2-oxybenzaldehyde
group in 3 by an intercalating agent, i.e., aminoanthraquinone
(7) or 2-naphthol (10) residue.
(d) Cytotoxic activity of compound 4, a condensation product
of aldehyde groups in 3 with 2-chloroethylamine, is comparible
with compound 3. The presence of an intercalating agent does
little to enhance antiproliferative activity of compounds 8 and
11 in comparison with compound 4. These compounds also
exhibit activity in the range of the international criterion.
1-{2-[1,3-(Oxytetraethylenoxy)-3,5,5-tri(phenyloxy-2-carbox-
aldehyde)cyclotriphosphazatrien-1-yl]-aminoethylamino}-
anthraquinone 7. 5 (0.699 g, 1 mmol), 2-hydroxybenzaldehyde
(0.56 g, 4.5 mmol), and sodium hydride (60%) (0.18 g, 4.5 mmol)
in THF (60 mL) were stirred for 29 h at room temperature in an
Therefore, new compounds 3, 4, 7, 8, 10, and 11 may be
considered as the agents with highest potential antileukemic
activity and appear to be good candidates for more advanced
screening.

1,3-(Oxytetraethylenoxy)cyclotriphosphazene DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 809
argon atmosphere. The mixture was then filtered and the filtrate
evaporated under reduced pressure. The crude product was chro-
matographed on a silica gel column using hexane:THF (3:1, v:v)
as eluent to provide 7 as a red oil. Yield 44.1%, mol mass (g/mol)
found/calcd M ) 956.0/955.52. Anal. (C45H44N5O13P3) C, H, N.
1-{2-[1,3-(Oxytetraethylenoxy)-3,5,5-tri(phenyloxy-2-{2-chloro-
ethylimino})cyclotriphosphazatrien-1-yl]aminoethylamino}an-
thraquinone 8. 2-Chloroethylamine hydrochloride (0.121 g, 1.04
mmol) and triethylamine (0.316 g, 3.1 mmol) in dry benzene (20
mL) were stirred at room temperature in an argon atmosphere. After
1 h, 7 (0.33 g, 0.345 mmol) in dry benzene (20 mL) was added
and the stirring continued for an additional 4 h in boiling solvent.
The mixture was filtered, and the filtrate was evaporated under
reduced pressure. The yield of 8 (obtained as a red oil) was
95%, mol mass (g/mol) found/calcd M ) 1140.3/1139.99.Anal.
(C57H56N8O10P3) C: calcd, 53.68; found, 53.28; H; N: calcd, 9.82;
found, 9.36.
1,3-(Oxytetraethylenoxy)-1-(2-naphthyloxy)-3,5,5-trichloro-
cyclotriphosphazatriene 9. 1 (0.938 g, 2 mmol), 2-naphthol (0.294
g, 2 mmol), and sodium hydride (60%) (0.08 g, 2 mmol) in THF
(120 mL) were stirred for 2 h at room temperature. The mixture
was filtered and the filtrate evaporated under reduced pressure and
finally washed with hot water. The yield of 9 was 99%, mol mass
[g/mol] M ) 576.5 g/mol. Anal. (C18H23Cl3N3O6P3) C, H, N.
1,3-(Oxytetraethylenoxy)-1-(2-naphthyloxy)-3,5,5-tri(phenyl-
oxy-2-carboxaldehyde)cyclotriphosphazatriene 10. 9 (1.15 g, 1.99
mmol), 2-hydroxybenzaldehyde (1.116 g, 8.96 mmol), and sodium
hydride (60%, 0,358 g, 8.96 mmol) in THF (140 mL) were stirred
at room temperature for 24 h. The mixture was filtered and the
filtrate evaporated under reduced pressure. The crude product was
chromatographed on a silica gel column using hexane:THF (2:1,
v/v) as eluent. The yield of 10 (as a red oil) was 50.8%, mol mass
(g/mol) found/calcd M ) 834.1/833.36. Anal. (C39H38N3O12P3) C,
H, N.
in RPMI medium supplemented with glutamine, antibiotics, and
10% fetal bovine serum.
Antiproliferative Assays. Cells were plated in 96-well plates
(Sarstedt, Costar) at a density of 104 cells per well in 100 µL of
culture medium. After 24 h, testing agents were added to the cells
in concentrations ranging from 100 to 0.1 µg/mL. The incubation
continued for an additional 72 h.
The MTT technique was applied for the cytotoxicity screening.
Twenty microliters of MTT solution (MTT: 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (Sigma); stock solution: 5
mg/mL) was added to each well and incubated for 4 h. The
mitochondria of viable cells convert the pale yellow MTT to a navy-
blue colored formazan. After the incubation time was completed,
80 µL of the lysing mixture was added to each well (lysing
mixture: 225 mL of dimethylformamide, 67.5 g of sodium dodecyl
sulfate (Sigma), and 275 mL of distilled water). After 24 h, when
the formazan crystals had dissolved, the optical densities of the
samples were read on an Multiskan RC photometer (Labsystems)
at 570 nm.
Acknowledgment. This work was supported by Ministry
of Science and Information Technology, grant No. 4T09A 127
22.
Supporting Information Available: Spectroscopic date (FTIR,
1H NMR, 31P NMR) and elemental analysis of the compounds
synthesized and investigated are available free of change via the
Internet at http://pubs.acs.org.
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1,3-(Oxytetraethylenoxy)-1-(2-naphthyloxy)-3,5,5-tri(phenyl-
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(1.184 g, 11.7 mmol) in dry benzene (35 mL) were stirred for 1 h
at room temperature under an argon atmosphere. After 1 h, 10 (1.1
g, 1.3 mmol) in dry benzene (35 mL) was added and the mixture
stirred for a further 4 h in boiling solvent. Subsequently, the solvent
(benzene) was evaporated under reduced pressure. The yield of 11
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P
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Collection by courtesy of Professor Spik and Dr. Mazurier
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JM0490078