Modification of the Antitumor Effect of Doxorubicin by Complexesof a-Fetoprotein
475
1
TABLE 1. Yields and H NMR Spectra of the Synthesized Compounds
Phosphorus content, %
1
Compound Yield, %
37
H NMR spectrum (in CD
3
SOSD
3
): d, ppm
f
R *
Found
6.29
Calcd.
6.26
I
0.9 – 1.0 (t, 3H, w-CH ); 2.0 – 2.1 (t, 2H, CH CO); 2.2 – 2.4 (m, 4H, 2CH CH=CH);
0.09
3
2
2
2
1
.7 – 2.9 (bs, 10H, 5HC=CHCH
2
CH=CH); 3.9 – 4.0 (bs, 2H, CH
2
OP); 4.3 – 4.4 (m,
H, NHCHCO); 5.2 – 5.4 (bs, 12H, 6HC=CH); 8.2 – 8.4 (m, 3H, NH, 2POH)
CHOP); 2.0 – 2.1 (t, 2H); 2.2 – 2.4 (m, 4H);
.7 – 2.9 (bs, 10H); 4.1 – 4.3 (m, 2H, 2CH); 5.2 – 5.4 (bs, 12H); 8.2 – 8.4 (m, 3H)
0.9 – 1.0 (t, 3H); 2.0 – 2.1 (t, 2H); 2.2 – 2.4 (m, 4H); 2.7 – 2.9 (bs, 10H); 2.9 – 3.1 (m,
H); 4.3 – 4.4 (m, 1H); 5.2 – 5.4 (bs, 12H); 7.0 – 7.2 (q, 4Harom); 8.2 – 8.4 (m, 3H)
0.9 – 1.0 (t, 3H); 2.0 – 2.1 (t, 2H); 2.2 – 2.4 (m, 4H); 2.7 – 2.9 (bs, 10H); 3.4 – 3.5 (bs,
H, CH NH); 3.9 – 4.0 (bs, 2H, CH OP); 5.2 – 5.4 (bs, 12H); 8.2 – 8.4 (m, 3H)
0.9 – 1.0 (t, 3H); 1.3 (s, 8H, 4CH ); 2.0 – 2.4 (m, 6H, 2CH CH=CH, CH CO);
CH=CH); 3.9 – 4.0 (bs, 2H); 4.3 – 4.4 (m, 1H);
.2 – 5.4 (bs, 8H); 8.2 – 8.4 (m, 3H)
II
43
26
44
35
6.12
5.38
6.94
6.57
6.09
5.43
6.87
6.58
0.9 – 1.0 (t, 3H); 1.2 – 1.4 (d, 3H, CH
2
3
0.09
0.10
0.12
0.08
III
IV
V
2
2
2
2
2
2
2
2
5
.7 – 2.9 (bs, 6H, 3HC=CHCH
2
VI
40
23
41
6.33
5.73
7.18
6.39
5.67
7.25
0.9 – 1.0 (t, 3H); 1.2 – 1.4 (m, 11H); 2.0 – 2.4 (m, 6H); 2.7 – 2.9 (bs, 6H); 4.1 – 4.3
m, 2H); 5.2 – 5.4 (bs, 8H); 8.2 – 8.4 (m, 3H)
0.9 – 1.0 (t, 3H); 1.3 (s, 8H); 2.0 – 2.4 (m, 6H); 2.7 – 2.9 (bs, 6H); 2.9 – 3.1 (m, 2H);
.3 – 4.4 (m, 1H); 5.2 – 5.4 (bs, 8H); 7.0 – 7.2 (q, 4H); 8.2 – 8.4 (m, 3H)
0.9 – 1.0 (t, 3H); 1.3 (s, 8H); 2.0 – 2.4 (m, 6H); 2.7 – 2.9 (bs, 6H); 3.4 – 3.5 (bs, 2H);
.9 – 4.0 (bs, 2H); 5.2 – 5.4 (bs, 8H); 8.2 – 8.4 (m, 3H)
0.08
0.09
0.12
(
VII
VIII
4
3
*
TLC conditions: silica gel, chloroform – methanol – 28% aqueous ammonia (9 : 7 : 2, v/v).
therapeutic effect of doxorubicin by the synthesized com-
pounds.
EXPERIMENTAL CHEMICAL PART
The results obtained in the first series of experiments
showed (Table 2) that administration of free doxorubicin
The experiments were performed with doxorubicin from
Pharmacia AB (Sweden). AFP was isolated from
retroplacental serum of parturient women as described in
(
3 mg/kg, i.v.) to the test mice with the model tumor pro-
[10]. Elemental analysis for phosphorus was performed us-
duced an antitumor effect characterized by a 30% inhibition
of the growth of Ehrlich ascitic carcinoma cells. It was estab-
lished that the complex of doxorubicin with compound IV
ing a method described in [11].
The binding constant (K ) of [3H]-arachidonic acid was
a
determined by graphic analysis using a Scatchard program.
and a-oncofetal protein (DR + IV + AFP) is a more effective
inhibitor of tumor cell growth as compared to the antibiotic
introduced separately. At the same time, ligand II in an anal-
ogous mixture exhibited cytotoxicity at the same level as that
of free doxorubicin, while the complexes with compounds
I and III were inactive.
The values of K and the concentrations of ligands I – VIII
a
producing a 50% inhibition of the arachidonic acid binding
were used to determine the competitive inhibition constants
as described in [12].
N-Docosahexanoyl-O-phospho-L-serine (I). Docosa-
hexaenoic acid (164 mg, 0.5 mmole) and triethylamine
In the second series of experiments performed against
the background of more intense growth of Ehrlich ascitic
carcinoma in mice, doxorubicin administered in the same
dose by the standard scheme reliably inhibited the tumor
growth with respect to the number of viable cells but did not
influenced ascites production. The DR + VIII + AFP com-
plex showed a certain tendency for increasing the antitumor
action of doxorubicin. The administration of ligands V – VII
in the complex composition did not significantly modify the
antitumor activity of the antibiotic.
(
52 mg, 0.51 mmole) were dissolved in 3 ml of a THF – ace-
tonitrile (1 : 2) mixture and the solution was cooled to
18°C. To this solution was added n-butylchloroformate
70 mg, 0.51 mmole) and the reaction mixture was allowed
–
(
to stand at –18°C for 20 min. Then the precipitate of
triethylamine hydrochloride was separated and the remaining
mixed anhydride solution was added to a solution of L-serine
methylate hydrochloride (1 mmole) in 1 ml of methanol
(also cooled to –18°C). The reaction mixture was kept for
2
0 min at –18°C and stirred for 2 h at room temperature.
Thus, the results of our experiments show that N-acyl de-
rivatives of aminoethanol phosphate IV and VIII in com-
plexes with AFP increase the antitumor effect of
doxorubicin. Other compounds containing structural ele-
ments representing hydroxyamino acid residues do not sig-
nificantly modify the antitumor activity of this antibiotic.
Then 10 ml of 0.5 M HCl cooled to 5°C was added and the
mixture was extracted with 20 ml of diethyl ether. The ether
extract was washed with 10 ml of water, dried over anhy-
drous Na SO , and evaporated. The residue was dissolved in
chloroform (2 ml) and applied onto a chromatographic col-
umn filled with alumina (alkaline, activity degree II accord-
2
4