Synthesis and antitumor activity of nonsymmetric phosphoric acid triamides

Full text of DOI:10.1023/A:1010351028826

Pharmaceutical Chemistry Journal
Vol. 34, No. 10, 2000
SYNTHESIS AND ANTITUMOR ACTIVITY OF NONSYMMETRIC
PHOSPHORIC ACID TRIAMIDES
L. Kosykhova,1 A. Palaima,1 and Z. Stumbryavichyute1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 10, pp. 12 – 14, October, 2000.
Original article submitted December 6, 1999.
Phosphonitrites attract the attention of researchers be-
cause of their broad spectrum of biological activity [1, 2]. In
particular, a rather thorough investigation was devoted to a
group of symmetric phosphoric and thiophosphoric acid
triamides possessing antitumor and antileukemic properties
[2 – 4].
of the synthesized compounds I – XII were confirmed by
1
data of elemental analyses and 31P and H NMR measure-
ments. The assignment of signals in the 1H NMR spectra was
based on their chemical shifts, multiplicity, and integral in-
tensity. The chemical shifts observed in the 31P NMR spectra
fall within the interval from 14.3 to 26.6 ppm, which is typi-
cal of compounds of this type.
The purpose of this study was to look for new agents
possessing antitumor activity in a series of nonsymmetric
phosphoric acid triamides (I – XII). The structures of these
compounds contain cytoactive ethyleneimine and/or
di(2-chloroethyl)amino groups and the residues of
aminocyclohexane, cis- or trans-4-aminocyclohexanecarbo-
xylic acid ethyl esters, or other aliphatic and heterocyclic
amines.Compounds I – XII were synthesized by sequential
phosphorylation of the initial amines by phosphoric acid
chloroanhydrides in the presence of hydrogen chloride ac-
ceptors. The initial reagents were N,N-dimethylamido-,
N,N-di(2-chloroethyl)amido-, and N-ethyleneimidophospho-
ric acid dichloroanhydrides (R1POCl2). Reactions of these
substances with a twofold excess of aminocyclohexane or
cis (trans )-4-aminocyclohexanecarboxylic acid ethyl ester
yielded monochloroanhydrides of the corresponding diami-
dophosphoric acids. These reactions were conduced in anhy-
drous diethyl ether in the temperature interval from – 8 to
0°C; the excess amine was necessary for binding hydrogen
chloride. In order to avoid unnecessary losses, the final
monoanhydrides were not isolated from reaction mixtures
and (upon separation of the initial amine hydrochlorides) im-
mediately brought into reaction with the following amine
(ethyleneimine, morpholine, monoethanolamine, or dime-
thylamine) in the same temperature interval. All the target
triamides appear as crystalline compounds which are diffi-
cult to purify.
O
R1
P
N
R
H
R2
I – XII
R
R
R2
I
H
H
N
N
O
II
N(CH2CH2Cl)2
H(CH3)2
N
N
N
N
N
N
III
IV
V
cis-COOH
trans-COOH
H
N(CH3)2
N(CH3)2
VI
VII
VIII
cis-COOH2H5
trans-COOC2H5
H
N(CH2CH2Cl)2
N(CH2CH2Cl)2
N(CH2CH2Cl)2
N
N
O
O
We have selected conditions ensuring suppression of the
reactions leading to the formation of symmetric triamides
and providing for a maximum yield and purity of the target
products I – XII. The proposed compositions and structures
IX
cis-COOC2H5
N(CH2CH2Cl)2
X
H
N(CH2CH2Cl)2
N(CH2CH2Cl)2
N(CH2CH2Cl)2
N(CH3)2
XI
XII
cis-COOC2H5
N(CH3)2
1
H
NHCH2CH2OH
Institute of Biochemistry, Vilnius, Lithuania.
525
0091-150X/00/3410-0525$25.00 © 2001 Plenum Publishing Corporation

526
L. Kosykhova et al.
TABLE 1. Acute Toxicity and Antitumor Activity of Compounds
I – XII
EXPERIMENTAL CHEMICAL PART
The 1H NMR spectra were measured at 35°C on a
Hitachi R-22 spectrometer at a working frequency of
90 MHz, using deuterochloroform as the solvent and HMDS
as the internal standard. The chemical shifts are presented in
the d-scale (ppm) relative to tetramethylsilane. The 31P NMR
spectra were recorded at 25°C on a Bruker HX-90-E spec-
trometer at a working frequency of 36.43 MHz, using chloro-
form as the solvent and a 85% H3PO4 solution as the internal
standard.
Com-
pound
LD50,
Tumor
strain
TD, mg/kg ´
TGI, %
LTI, %
mg/kg
5 days
I
260
W-256
SM-1
PLS
90
90
95.6*
85.1*
36.7*
92.2*
100.0*
38.3*
…
…
…
80
…
II
60
W-256
SM-1
PLS
15
…
20
…
10
…
LLC
20
–34.2*
…
The data of elemental analyses agreed with the results of
analytical calculations according to the empirical formulas.
The completion of reactions was monitored and the product pu-
rity was checked by TLC on Al2O3 plates (activity degree II)
eluted in a benzene – ether – methanol (4 : 2 : 1) solvent sys-
tem. The spots were developed by exposure to iodine vapor
and by treatment with the Vas’kovskii – Kostetskii reagent.
General method for the synthesis of phosphoric acid
triamides (I – XII). To a solution of 0.025 mole of
dichloroanhydrides R1POCl2 in 200 ml of anhydrous diethyl
ether, cooled to –8 – 0°C, was slowly added with stirring a
solution of 0.05 mole of aminocyclohexane or cis (trans )-4-
aminocyclohexanecarboxylic acid ethyl ester in 100-ml of
the same solvent. The mixture was stirred for 2 h at a temper-
ature in the above interval and allowed to stand overnight in
a refrigerator. The precipitated hydrochloride was separated
by filtration. To the monochloroanhydride solution, cooled to
– 8 – 0°C, was slowly added with stirring a solution of
0.05 mole morpholine in 50 ml of anhydrous diethyl ether, or
a solution of 0.05 mole monoethanolamine in a mixture of
50 ml of anhydrous chloroform and 20 ml of anhydrous
ether, or dimethylamine (to saturation). Upon completion of
the reaction (judged by TLC), the precipitated hydrochloride
was separated by filtration. The filtrate was evaporated in
vacuum and the residue was recrystallized.
III
307
W-256
SM-1
S-180
PLS
15
98.1*
98.3*
51.3*
37.9*
…
50
…
70
…
50
…
LLC
50
21.6
48.0*
20.2
–22.6
…
L-1210
LLC
100
500
400
130
80
…
IV
V
2060
890
…
L-1210
W-256
SM-1
S-180
W-256
SM-1
S-180
LLC
…
98.3*
0.6
…
150
70
35.3*
99.7*
97.3*
37.8
…
…
VI
730
…
90
…
70
…
40
–10.8
–9.3
35.4*
–4.0
…
L-1210
LLC
70
…
VII
447
650
100
100
100
90
…
L-1210
W-256
SM-1
S-180
L-1210
W-256
SM-1
S-180
W-256
SM-1
S-180
L-1210
W-256
SM-1
S-180
W-256
SM-1
W-256
S-180
L-1210
LLC
…
VIII
–7.9
–27.9
–41.2*
…
…
70
…
40
1.3
…
IX
X
640
230
300
200
200
80
20.0
–31.5
–11.7
–57.5
–22.0
8.1
…
Compound I. Yield, 2.01 g (30%); m.p., 71 – 72°C
(benzene); C12H24N3O2P; 1H NMR spectrum (d, ppm):
0.8 – 2.0 (m, 10H, C5H10-cyclohexyl), 1.94 (d, 4H,
aziridine); 2.38 (bm, 1H, NH), 2.98 (m, 2H,
CH–cyclohexyl), 3.09 (m, 4H, CH2NCH2), 3.54 (m, 4H,
CH2OCH2).
Compound II. Yield, 3.50 g (43%); m.p., 54 – 56°C
(ether); C12H24Cl2N3OP; 1H NMR spectrum (d, ppm):
0.8 – 2.0 (m, 10H, C5H10-cyclohexyl), 1.96 (d, 4H,
aziridine); 2.49 (bm, 1H, NH), 2.90 (m, 1H,
CH–cyclohexyl), 3.05 (m, 4H, CH2NCH2), 3.65 (m, 4H,
2CH2Cl).
…
…
60
…
120
120
60
…
…
8.0
…
XI
960
–4.2
–30.7
–58.8
–16.3
81.0*
67*
100
60
…
…
XII
1410
8.4
300
300
2
…
…
TPP
…
Compound III. Yield, 2.72 g (36%); isolated by pre-
parative TLC on a plate with nonbonded Al2O3 in the form of
2
64*
…
1
…
98**
23**
a light-yellow oil, Rf = 0.64; C13H26N3O2P; H NMR spec-
…
trum (d, ppm): 1.17 (t, 3H, CH3CH2), 1.3 – 2.0 (m, 8H,
C4H8-cyclohexyl), 2.00 (d, 4H, aziridine); 2.38 (m, 1H,
CHCO), 2.58 (bm, 1H, NH), 2.70 (d, 6H, CH3NCH3), 3.22
(m, 1H, CHN), 4.03 (q, 2H, CH2O).
*
Difference from control reliable for p < 0.05.
Data from [9]; TPP = thiophosphamide (reference).
**

Synthesis and Antitumor Activity of Nonsymmetric Phosphoric Acid Triamides
527
Compound IV. Yield, 3.20 g (42%); m.p., 104 – 106°C
(ether); C13H26N3O3P; 1H NMR spectrum (d, ppm):
0.50 – 2.10 (m, 9H, C5H9-cyclohexyl), 1.17 (t, 3H,
CH3CH2), 1.97 (d, 4H, aziridine); 2.24 (bm, 1H, NH), 2.66
(d, 6H, CH3NCH3), 3.00 (m, 1H, CHN), 4.03 (q, 2H, CH2O).
Yields and physicochemical characteristics of com-
pounds I – XII were reported elsewhere [5, 6].
inhibited the growth of sarcoma M-1. Moderate antitumor ef-
fect was observed for compounds I – III on the Pliss
lymphosarcoma model and for compounds II, V, and VI on
the sarcoma 180 model. At the same time all these com-
pounds weakly affected the growth of other tumors and the
average lifetime. In comparison with the most active
triamides, the reference drug thiophosphamide exhibited a
lower antitumor activity with respect to Walker’s carcinosar-
coma (W-256), but more actively inhibited the growth of sar-
coma 180 and lymphoid leukosis (L-1200).
EXPERIMENTAL BIOLOGICAL PART
Thus, the results of our experiments showed that the
antitumor activity of nonsymmetric phosphoric acid triami-
des is determined for the most part by the nature of amide re-
sidues attached at the phosphorus atom. The antitumor effect
can be varied within a broad range by changing this residue,
the maximum antitumor activity being inherent in non-
symmetric triamides containing the ethyleneimine residue.
The acute toxicity of the synthesized compounds was
studied upon single intraperitoneal injections with 1% starch
jelly to male ICR mice weighing 20 – 22 g [7]. The toxicity
parameters were estimated according to the Litch-
field – Wilcoxon method. The data were statistically pro-
cessed in terms of the Student t-criterion [8].
The antitumor activity was studied on rats (90 – 110 g)
inoculated with strains of Walker’sascites carcinosarcoma
(W-256), sarcoma M-1 (SM-1), and Pliss lymphosarcoma
(PLS) and on C57BP and DBA12 mice inoculated with sar-
coma 180 (S-180), epidermoid Lewis lung carcinoma (LLC),
and lymphoid leukemia (L-1210). The strains of experimen-
tal tumor models were obtained from the Blokhin Oncologic
Research Center (Russian Academy of Sciences, Moscow).
The tumors were transplanted by conventional methods [9].
The compounds to be tested were intraperitoneally injected
at a single daily therapeutic dose (TD) over a period of 5
days. The antitumor activity was evaluated either by the per-
centage tumor growth inhibition (TGI) determined on the
seventh day after termination of treatment or by the percent-
age lifetime increase (LTI) in the test groups relative to un-
treated control. The animals were kept under standard condi-
tions recommended in the literature [9 – 12]. The acute tox-
icity and antitumor activity of the test compounds were
compared to the analogous data for thiophosphamide (TPP).
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RESULTS AND DISCUSSION
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activity as compared to that of the reference drug thiophos-
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sarcoma (W-256), and compounds I – III, VI, XII strongly
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