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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Sitnikov et al.
to a solution of palmitic acid (50 mg, 0.19 mmol) in anhydrous
THF (2 mL) under an inert atmosphere. The reaction mixture
was stirred for 3 h at 25 °С. The mixture was dissolved in 1 mL of
THF under an inert atmosphere and added dropwise to sodium
phenolate of CAꢀ4, which was obtained by the addition of NaH
(4.2 mg, 0.18 mmol) as a 60% suspension in mineral oil to
a solution of CAꢀ4 (50 mg, 0.16 mmol) in anhydrous THF
(0.5 mL). The reaction mixture was stirred for 1 h at 25 °С and
then concentrated by evaporation. The residue was dissolved in
ethyl acetate (20 mL) and extracted three times with a 5% soluꢀ
tion of NaOH (10 mL). The organic layer was dried over anꢀ
hydrous Na2SO4. The product was isolated by column chromaꢀ
tography on silica gel (ethyl acetate—petroleum ether (1 : 4)
mixture as eluent). Product 1 was obtained as a white solid in
a yield of 71 mg (80%). Found (%): С, 73.29; H, 9.09. C34H50O6.
Calculated (%): С, 73.61; H, 9.08. 1H NMR, δ: 0.88 (t, 3 H, Me,
J = 8.0 Hz); 1.25 (m, 24 H, СH2); 1.71 (m, 2 H, С(О)СH2СH2);
2.52 (t, 2 H, С(О)СH2СH2, J = 8.0 Hz); 3.70 (s, 6 H, ОMe);
3.79, 3.83 (both s, 3 H each, ОMe); 6.43, 6.47 (both d, 1 H each,
HC=C, J = 12.0 Hz); 6.50 (s, 2 H, H(2´), H(6´)); 6.84 (d, 1 H,
H(5″), J = 8.0 Hz); 7.00 (d, 1 H, H(2″), J = 2.0 Hz); 7.11 (dd, 1 H,
H(6″), J = 2.0 Hz, J = 8.0 Hz). 13C NMR, δ: 14.1, 22.7, 25.0,
29.0, 29.2, 29.3, 29.5, 29.6, 29.5, 29.7, 31.9, 33.9, 55.8, 55.9,
60.9, 105.7, 111.9, 123.1, 127.6, 128.6, 129.4, 130.0, 132.4, 137.0,
139.5, 150.2, 152.9, 171.7.
5ꢀ[(Z)ꢀ2ꢀ(3,4,5ꢀTrimethoxyphenyl)ethenyl]ꢀ2ꢀmethoxyphenyl
oleate (2). Oxalyl chloride (80 μL, 0.87 mmol) was added to
a solution of oleic acid (0.21 g, 0.76 mmol) in anhydrous THF
(2 mL) under an inert atmosphere. The reaction mixture was
stirred for 3 h at 25 °С. The mixture was evaporated and dried.
The residue was dissolved in THF (2 mL) under an inert atmoꢀ
sphere, and added dropwise to sodium phenolate of CAꢀ4, which
was obtained by the addition of NaH (15 mg, 0.64 mmol) as
a 60% suspension in mineral oil to a solution of CAꢀ4 (0.18 g,
0.58 mmol) in anhydrous THF (1 mL). The reaction mixture
was stirred for 1 h at 25 °С and concentrated by evaporation. The
residue was dissolved in ethyl acetate (30 mL) and extracted
three times with a 5% solution of NaOH (10 mL). The organic
layer was dried over anhydrous Na2SO4. The product was isolatꢀ
ed by column chromatography on silica gel (ethyl acetate—petroꢀ
leum ether (1 : 4) as eluent). Compound 2 (230 mg, 67%) was
obtained as a colorless oil. Found (%): С, 74.29; H, 9.04.
C36H52O6. Calculated (%): С, 74.45; H, 9.02. 1H NMR, δ: 0.88
(m, 3 H, Me); 1.31 (m, 20 H, СH2); 1.72 (m, 2 H, С(О)СH2СH2);
2.03 (m, 4 H, СH2СH=СH); 2.52 (t, 2 H, С(О)СH2СH2,
J = 8.0 Hz); 3.70 (s, 6 H, ОMe); 3.79, 3.83 (both s, 3 H each,
ОMe); 5.35 (m, 2 H, СH=СH); 6.43, 6.47 (both d, 1 H each,
ArHC=C, J = 12.0 Hz); 6.50 (s, 2 H, H(2´), H(6´)); 6.84 (d, 1 H,
H(5″), J = 8.0 Hz); 6.99 (d, 1 H, H(2″), J = 2.0 Hz); 7.11
(dd, 1 H, H(6″), J = 2.0 Hz, J = 8.0 Hz). 13C NMR, δ: 14.1,
22.7, 25.0, 27.2, 29.0, 29.2, 29.3, 29.5, 29.7, 31.9, 33.9, 55.8,
55.9, 60.9, 105.9, 112.0, 123.2, 127.6, 128.6, 129.5, 129.7, 130.0,
132.4, 137.2, 139.5, 150.3, 153.0, 171.7.
animal a week earlier than for other animals because of
the intense growth of its tumor. Evidently, in this case,
injections should be started earlier and carried out more
frequently than once a week.
Thus, SiaLeXꢀliposomes with prodrug 2 in the dose
used by the CAꢀ4 residue (22 mg kg–1) showed the effiꢀ
cient antitumor effect in the regime of monotherapy.
A single injection of СА4Р to mice with transplanted breast
cancer cells МСаꢀ4 in a dose accepted in the clinic pracꢀ
tice (81 mg kg–1) did not inhibit the tumor growth.11 Notiꢀ
ceable inhibition was achieved only if the preparation was
injected after irradiation (by ~30% on the first 10 days),
and the curve of tumor growth almost coincided with the
curves obtained after irradiation without using any prepaꢀ
ration or after four injections of CA4Р at an interval of
a day. The strong inhibition of breast cancer growth (by
~80% during 15—20 days) was achieved due to the introꢀ
duction of CA4Р (15 mg kg–1) in immunoliposomes
Stealth® carrying mAb to Eꢀselectin and only after the
irradiation of the tumor, which considerably increases seꢀ
lectin expression.11 The effect of immunoliposomes on
tumor growth without irradiation almost did not differ
from the effect of CA4Р after irradiation, and only the
dose of the preparation introduced in liposomes was
5.4 times smaller.11 Note that in the cited work11 the tests
were carried out on the model of rapidly growing breast
cancer (during 10 days after the beginning of the treatꢀ
ment the tumors in the control group increased in volume
from 1 to 4 cm3). It is known27 that rapidly growing agꢀ
gressive tumors are more sensitive to chemotherapy. The
spontaneous model of slowly growing breast cancer, which
better reflects the corresponding human disease, was used
in our work.
Thus, we obtained the new liposome construction bearꢀ
ing the antimitotic agent combretastatin Aꢀ4 as the oleoyl
derivative and the carbohydrate ligand of selectins SiaLeX.
The results of primary testing in vivo showed a good antiꢀ
tumor effect exemplified by therapy of breast cancer. The
use of CAꢀ4 as an antivascular antitumor remedy for sysꢀ
temic injection can be improved due to the inclusion of its
lipophilic prodrug into liposomes equipped with the moꢀ
lecular address to the angiogenic endothelium of tumors.
Experimental
1
Н and 13С NMR spectra were recorded on a Bruker ARX
400 spectrometer (400 and 101 MHz, respectively) in CDCl3.
Chemical shifts are presented in the δ scale relative to Ме4Si.
Elemental analysis was carried out on a Perkin—Elmer Series II
CHN/O Analysis 2400 instrument. Commercially available reꢀ
agents (Aldrich, Alfa Aesar) were used as received. Prior to use
solvents were purified by standard methods. Petroleum ether
with b.p. 40—70 °С was used. Combretastatin Aꢀ4 was syntheꢀ
sized by the known procedure.14
Preparation of dispersions of drugꢀloaded liposomes. Phosphaꢀ
tidylcholine from egg yolk and PI from S. cerevisiae (Reakhim,
Russia) were used. SiaLeXꢀconjugate 3 was synthesized from
biscarboxymethyl ether of PEG (average weight 600 Da),
racꢀ1,2ꢀdioleylꢀ3ꢀ(3ꢀaminopropionyl)glycerol, and 3ꢀaminoproꢀ
pyl glycoside SiaLeX as described recently.15 Buffers were preꢀ
pared with a 1 mМ solution of EDTA: PBS, pH 7.06 is a physioꢀ
5ꢀ[(Z)ꢀ2ꢀ(3,4,5ꢀTrimethoxyphenyl)ethenyl]ꢀ2ꢀmethoxypheꢀ
nyl palmitate (1). Oxalyl chloride (49 μL, 0.57 mmol) was added
logical solution on a phosphate buffer (КН2PO4, 0.2 g L–1
;