Synthesis and properties of lipophilic derivatives of 5-fluorouracil

Article abstract of DOI:10.1134/S1068162013030138

A series of N¹-acyl derivatives of 5-fluorouracil (5-FU) bearing the residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and adamantane-1-carboxylic acids have been synthesized. The relative hydrolysis rates for the derivatives

Full text of DOI:10.1134/S1068162013030138

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 3, pp. 299–305. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © A.V. Semakov, A.A. Blinkov, G.P. Gaenko, A.G. Vostrova, J.G. Molotkovsky, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39, No. 3, pp. 338–
345.
Synthesis and Properties of Lipophilic Derivatives of 5ꢀFluorouracil
A. V. Semakov, A. A. Blinkov, G. P. Gaenko, A. G. Vostrova, and J. G. Molotkovsky1
Shemyakin—Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997 Russia
Received December 12, 2012; in final form, December 24, 2012
Abstract—A series of
toylaminobenzoic, ꢀoleoylaminobenzoic, and adamantaneꢀ1ꢀcarboxylic acids have been synthesized. The
relative hydrolysis rates for the derivatives under physiological conditions (pH 7.2 and 37 ) have been deterꢀ
mined, and it has been shown that the resistance of these compounds to hydrolysis increases as the steric
accessibility of the amide group at residue
1 of 5ꢀFU decreases. The derivatives easily incorporate into the
lipid bilayer; their liposomal preparations show a marked cytostatic activity on human breast lymphoma cells
(LD50 ~1 M) and are of interest as potential antitumor preparations. In addition, a fluorescent analogue of
N pꢀmyrisꢀ
1ꢀacyl derivatives of 5ꢀfluorouracil (5ꢀFU) bearing the residues of palmitic,
p
°C
N
µ
the above derivatives, 1ꢀ[8ꢀ(3ꢀperylenyl)octanoyl]ꢀ5ꢀfluorouracil, has been synthesized, which is intended
for studying the behavior of 5ꢀFU derivatives in cells and tissues by instrumental methods.
Keywords: 5ꢀfluorouracil, Nꢀacyl derivatives, cytostatic activity, lipophilic predrugs, liposomes, fluorescent anaꢀ
logue
DOI: 10.1134/S1068162013030138
1
INTRODUCTION
some of these derivatives showed that their therapeutic
index is much higher compared with the original
5ꢀFU; nevertheless, this effect still remains limited.
5ꢀFluorouracil is an antimetabolite antitumor
agent, which was first obtained in 1957 [1] and is also
currently used to advantage in clinical practice owing
to a wide spectrum of activity against various, primaꢀ
rily solid, tumors and the capacity to permeate
through the hematoencephalic barrier [2]. An addiꢀ
tional important point is the relatively low cost of
5ꢀFU. However, the substantial systemic toxicity of
5ꢀFU and the relatively rapid development of resisꢀ
tance of tumors to it [3, 4], problems common to some
degree to all ATAs, necessitate a search for more perꢀ
fect ways of its application, primarily those that
enhance the selectivity of the incorporation of the
drug into malignant cells and tissues compared with
normal, thereby increasing its therapeutic index.
The possibility of incorporating 5ꢀFU into the
aqueous interior of liposomes was studied; however,
the small waterꢀsoluble molecule was found to pass
through liposomal bilayers with a great velocity [10],
which cast some doubt upon the possibility of using
these preparations as ATAs. It was proposed to retain
5ꢀFU in the aqueous space of liposomes as a part of the
copper complex; this preparation showed a higher
effectiveness in (in vivo) experiments compared with
native 5ꢀFU [11]. A common disadvantage of these
approaches is the complicated methodology of incorꢀ
porating the waterꢀsoluble medicinal substances into
the aqueous interior of liposomes and the incompleteꢀ
ness of incorporation.
One of these ways is the design of derivatives that
are 5ꢀFU precursors, including those that increase the
lipophilicity of the 5ꢀFU molecule. Lipophilicity
facilitates the incorporation of drugs into tumor cells
(where 5ꢀFU is released), which differ from normal by
a higher fluidity and disorder of membranes [5].
Among the first derivatives was 1ꢀfuryl derivative,
which showed the efficacy of this approach [6]. At
present, hundreds of 5ꢀFU derivatives of different
kinds are known (see reviews [7–9]). Clinical trials of
LꢀFUs incorporated into nanosized carriers, priꢀ
marily liposomes, to be more precise, liposomal bilayꢀ
ers, have great advantages in this respect (see reviews
[12, 13]). These are the ease and completeness of
incorporation of LꢀFU into liposomes, low losses of
the drug in the blood flow, and the possibility of its
direct transmembrane transfer to tumor cells; the latꢀ
ter can change the intracellular traffic of the preparaꢀ
tion.
The efficacy of a drug in the liposomal form comꢀ
pared with its native form increases also as a result of
the soꢀcalled passive transport due to a higher permeꢀ
ability of defective capillary walls in tumors for lipoꢀ
somes [14]. A further increase in the selectivity of drug
delivery to tumors is afforded by the incorporation of
Abbreviations: ATA, antitumor agent; 5ꢀFU and LꢀFU, 5ꢀfluoꢀ
rouracil and its lipophilic derivative; DIPEA, diisopropylethylꢀ
amine; ePC, egg phosphatidylcholine; HBL, human breast lymꢀ
phoma; PAB,
p
ꢀaminobenzoyl.
Corresponding author: phone/fax: 8 (495) 330ꢀ6601; eꢀmail:
igmol@ibch.ru.
1
299

300
SEMAKOV et al.
Because we had a wide experience in synthesizing
O
O
and applying lipophilic derivatives of melphalan,
methotrexate, and other ATAs and their liposomal
preparations, which showed a high efficacy [23], we
set ourselves the task of obtaining similar derivatives of
5ꢀFU, which, on the one hand, should be capable of
transferring 5ꢀFU in the composition of liposomes
into tumors and releasing it there (not earlier) and, on
the other hand, be quite stable in operation and in
storage. In this case, we had to bear in mind that
F
F
1. Me SiNHSiMe /Me SiCl
3
HN3
3
3
HN
5
2. RCOCl
1
O
N
R
O
N
R
RCOCl/iꢀPr EtN
2
(5ꢀFU)
(LꢀFU, I–V)
chemically firm
Nꢀderivatives of 5ꢀFU possess no
(
(
I), R = COC15H31
antitumor activity (see, e. g., [24]); therefore, LꢀFU to
be designed had to be sufficiently (but not extremely)
labile to release the active principle.
II), R =
III), R =
IV), R =
CO
CO
CO
NHCOC13H27
NꢀAcyl LꢀFUs were chosen for this purpose
because of the ease of their synthesis and presumed
facility of incorporation into liposomes; the major
problem was to choose acids whose derivatives would
satisfy the above criteria and would not form addiꢀ
tional toxic compounds upon intracellular release of
5ꢀFU. In the present paper, the synthesis of a series of
(
(
NHCO(CH2)7CH=CH(CH2)7CH3
2'
3'
4'
1'
novel lipophilic Nꢀacyl LꢀFUs (I)–(IV), their stability
in aqueous medium, and their cytotoxic activity in
vitro are described. In addition, we describe here the
synthesis of the fluorescently labeled [8ꢀ(3ꢀperyleꢀ
2''
1''
(
V), R =
CO(CH2)7
3''
nyl)octanoyl analogue (V); we synthesized this derivaꢀ
tive considering the necessity of further study of the
intracellular traffic and metabolism of lipophilic Lꢀ
FU derivatives by instrumental methods.
Fig. 1. Scheme of synthesis of lipophilic 5ꢀFU derivatives.
1ꢀPalmitoylꢀ5ꢀFU (I) was chosen as a reference
standard. This substance is similar to 1ꢀstearoylꢀ5ꢀFU
whose properties are described in detail in [21]. When
choosing other derivatives for the study, we were
guided by few available data on their stability in aqueꢀ
ous medium. Although the authors of [22] present no
ATAs into liposomes loaded with ligands affine for the
surface components of tumor cell membranes, e. g.,
with antibodies to specific proteins (see reviews [15,
16]).
direct evidence on the stability of the оꢀtoluyl 5ꢀFU
derivative (to which the 3ꢀacyl structure is assigned) to
hydrolysis, the indirect evidence suggests that this
compound is more stable than the stearoyl analogue
[21]. We also proposed that this greater stability is due
to a lesser steric accessibility of the amide bond in the
toluyl derivative compared with the stearoyl derivative.
It was logical to try Nꢀaroyl LꢀFUs; however, aroyl resꢀ
idues had to be modified by increasing their lipophilicꢀ
ity, since the data reported in [22] indicate that the
In the choice of LꢀFU intended for the incorporaꢀ
tion into liposomes, its
Nꢀacyl derivatives, primarily
fatty acid derivatives, merit attention. In physicoꢀ
chemical parameters, their chains best fit nonpolar
residues of liposomal bilayers, promoting in such a way
the incorporation of LꢀFU into them. The methods of
N
ꢀacylation of 5ꢀFU [17, 18] and related heterocycles
[19] have been well elaborated; in this case, the acylaꢀ
tion of 5ꢀFU occurs as a rule at the N1ꢀposition of the
heterocycle. However, the study of the properties of
о
ꢀtoluyl 5ꢀFU derivative is poorly retained in the lipoꢀ
somal bilayer. Therefore, two other LꢀFUs, (II) and
III), were obtained by incorporating into 5ꢀFU the
PAB residue additionally lipophilized by the ꢀmyrisꢀ
toyl or the
ꢀoleoyl group, respectively. Finally, N1
some acylated heterocycles showed that aliphatic N1
ꢀ
(
N
acyl derivatives of 5ꢀFU have a low stability; in aqueꢀ
ous systems and under weakly acid conditions, they
N
ꢀ
(adamantaneꢀ1ꢀcarbonyl)ꢀ5ꢀFU (IV) as a 5ꢀFU
derivative with the most restricted access to the amide
group was synthesized (Fig. 1).
are easily hydrolyzed [20, 21]. Thus, the lifetime (t1/2
)
of N1ꢀdodecanoylꢀ5ꢀFU is ~5 min at pH 7.4 and 37
°С
[20]. As far as one can judge, 1ꢀstearoylꢀ5ꢀFU has
approximately the same stability; at pH > 8, the stabilꢀ
ity of this compound is substantially higher [21]. On
the other hand, as stated by Sun et al. [22], the 5ꢀFU
ꢀtoluyl derivative incorporated into liposomes is quite
stable.
To obtain a fluorescent probe simulating behavior
of other LꢀFUs, 5ꢀFU was acylated by 8ꢀ(3ꢀperyleꢀ
nyl)octanoic acid. This acid was chosen for the followꢀ
ing reasons. The 3ꢀperylenyl fluorophore is chemically
rather stable and highly sensitive; its spectral characꢀ
о
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SYNTHESIS AND PROPERTIES OF LIPOPHILIC DERIVATIVES
301
Firstꢀorder constants of the rate of hydrolysis of lipophilic
5ꢀFU derivatives in acetonitrile–20 mM TrisꢀHCl, pH
7.2, at 37
teristics (excitation at 400–450 nm, emission at 450–
500 nm) are convenient for confocal microscopy and
flowꢀcytofluorimetry studies. The fluorophoreꢀcarryꢀ
ing acid was chosen so that the total length of the fluꢀ
orescently labeled acyl was close to the length of palmꢀ
itoyl, one of the most widely occurring fatty acid resiꢀ
dues of cell membranes.
°
C
–1
Derivative
k, s
(
(
(
(
I
)
0.31
0.12
0.13
0.055
II
III
IV
)
8ꢀ(3ꢀPerylenyl)octanoic acid was synthesized
according to the scheme described for the homologous
8ꢀ(3ꢀperylenyl)butanoic acid [25]: perylene was acyꢀ
lated with an excess of suberic acid dichloride accordꢀ
ing to Friedel–Crafts to give 7ꢀ(3ꢀperylenoyl)hepꢀ
tanoic acid, which was then reduced according to
Wolff–Kishner/Huang–Minlon to give 8ꢀ(3ꢀperyleꢀ
nyl)octanoic acid. This acid was obtained in a similar
way by Hermetter and Oskolkova; the synthesis is
briefly described in a patent [26].
)
)
Experimental section). TLC of LꢀFUs (I)–(III) on
silica gel was performed only in nonaqueous systems.
At this step of the investigation, we restricted ourꢀ
selves to a comparison of the hydrolysis rates of (
IV) in a system containing TrisꢀHCl buffer, pH 7.2, at
37 , i. e., under conditions close to those under
which ATAs are in clinical use. Owing to a moderate
I)–
(
°С
The palmitoyl derivative (I) was synthesized by two
alternative methods: the acylation of 5ꢀFU with acid
chloride in the presence of a base [17, 18] or through a solubility of LꢀFUs in aqueous solutions, the hydrolyꢀ
trimethylsilyl 5ꢀFU derivative [19, 21]. The two methꢀ sis was carried out in a mixture acetonitrile–buffer
ods give close results; the fluorescently labeled anaꢀ 4 : 1; the course of hydrolysis was controlled densitoꢀ
logue (V) was obtained using the second method, and
the other derivatives (II)–(IV) were synthesized by the
first method, which is more convenient in experiment.
metrically after the separation of the starting LꢀFU
and 5ꢀFU by TLC (see the Experimental section).
The hydrolysis of all LꢀFUs tested is described by
the firstꢀorder kinetics, which is indicated by the expoꢀ
nential time dependence of substrate decrease (data
not shown); the rate constants are given in the table.
The structures of (I)–(V) were confirmed by the data
of 1H NMR, mass, UV, and fluorescence spectra (see
the Experimental section).
All LꢀFUs (I)–(V) possess no marked solubility in
The mechanism of hydrolytic decomposition of
Nꢀ
water but incorporate easily into ePC liposomes in the
molar ratio 1 : 9 (see the Experimental section); the gel
filtration of liposomal preparations through Sephadex
LHꢀ20 showed an almost 100% incorporation (data
not shown). It should be noted that none of the fracꢀ
tions was found to contain substantial amounts of
5ꢀFU upon gel filtration (as determined by TLC),
although liposomes were prepared by dispersion in
aqueous buffer with pH 7.2; presumably, the incorpoꢀ
ration into the liposome bilayer strongly shields the
amide bond of the acyl residue, thereby preventing
hydrolysis.
acyl (aroyl) 5ꢀFU derivatives has not been studied; the
information on this subject is scanty, and it is only
known that the stability of 1ꢀstearoylꢀ5ꢀFU is minimal
at pH ~ 4 [21]. However, it is clear that the resistance
to hydrolytic cleavage of the aboveꢀdescribed LꢀFUs
(
I
)–(IV) increases in the following order: palmitoylꢀ5ꢀ
FU < ꢀmyristoylꢀPABꢀ5ꢀFU = ꢀoleoylꢀPABꢀ5ꢀ
N
N
FU < adamantoylꢀ5ꢀFU, which fully agrees with the
increase in the steric restriction of access to the amide
bond of the acyl group. At the same time, the close
cytotoxic activity of LꢀFUs (I)–(IV) (Fig. 2) strongly
suggests that, in tumor cells, they all undergo hydrolysis
with the release of the active starting compound, 5ꢀFU.
As was mentioned above, the stability of the prepaꢀ
rations in aqueous media is one of their most imporꢀ
tant characteristics, although it remains still unclear
how great must be their optimal stability to achieve the
maximum antitumor activity and therapeutic index
and in what media. The significance of the minimum
stability of LꢀFUs for convenient manipulation during
isolation, purification, preparation of compounds,
etc. is evident from the fact that the most unstable of
The cytostatic activity of LꢀFUꢀloaded liposomes
in HBL cells was estimated by the standard trypan blue
test. The IC50 value was determined by extrapolating
the data on cell survival depending on the concentraꢀ
tion of liposome preparations added to the medium
(Fig. 2). The IC50 values for all preparations tested were
close to ~1 µM and exceeded the IC50 value for LꢀFU
seven to ten times. The lower cytostatic activity of the
liposomal forms of the ATA is a natural phenomenon
and was described many times (see, e. g., [27]). It
should be noted that, in in vivo experiments, the
reverse situation can occur (and is often observed),
namely, a higher antitumor activity of liposome prepꢀ
our LꢀFUs, palmitoylꢀ5ꢀFU (I), is completely
decomposed on purification by chromatography on
silica gel (the stability is higher in reverseꢀphase chroꢀ
matography in aprotic solvents). The most stable
derivative adamantoylꢀ5ꢀFU (IV) withstands flash
chromatography on silica gel; LꢀFUs of intermediate
stability (II
) and (III) endure rapid filtration through arations carrying lipophilic prodrugs of ATAs as comꢀ
silica gel and reverseꢀphase chromatography (see the pared with these agents per se (see, e. g., [28]).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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302
SEMAKOV et al.
the presence of calcined Na2CO3 (3 h at 25
sive SOCl2 was removed by evaporation with dry toluꢀ
ene. 8ꢀ(3ꢀPerylenyl)octanoic acid, mp 220–223
(decomp.), was obtained as described in [25]; its H
NMR spectrum and behavior in TLC correspond to
those described in [26].
°С); excesꢀ
100
1
°С
1
80
60
40
20
0
5
6
3
Column chromatography was carried out using silꢀ
ica gel Kieselgel 60 and LiChrosorb RPꢀ18 (for
reverseꢀphase chromatography) (Merck). For TLC,
plates with the fluorescent indicator Kieselgel 60 F254
and without indicator Kieselgel 60 (Merck), as well as
plates with reverse phase RP18 F254s were used. Detecꢀ
tion in TLC was with phosphomolybdic acid (A), by
UV irradiation (B) (5ꢀFU and its derivatives quench
the fluorescence of the indicator), and with an acidiꢀ
fied KMnO4 solution (C) (5ꢀFU and LꢀFU appear as
yellow spots against the roseꢀcolored background).
Evaporation in vacuo was carried out at 8–15 mmHg
2
4
0.1
1
10
100
Concentration
,
µ
M
Fig. 2. Effect on the growth of HBL cells in vitro of (
control (empty) ePC liposomes, ( ) free 5ꢀFU, ( ) 1ꢀ
palmitoylꢀ5ꢀfluorouracil (I), ( ) 1ꢀ( ꢀmyristoylꢀPABꢀ5ꢀ
fluorouracil (II), 1ꢀ( ꢀoleoylꢀPABꢀ5ꢀfluorouracil
III), and ( ) 1ꢀadamantoylꢀ5ꢀfluorouracil (IV), incorpoꢀ
1)
2
3
4
p
and a temperature of <40°С.
(
5
)
p
(
6
Mass spectra were recorded on a MALDI TOF
mass spectrometer UltraFlex (Bruker Daltonics, Gerꢀ
many): N2 laser, 337 nm, matrix 2.5ꢀdihydroxybenꢀ
zoic acid, and on an ESIꢀTOF mass spectrometer
МХꢀ5311 (IAP RAS, St. Petersburg) with nanospray
rated into liposomes containing LꢀFU–ePC 1 : 9
(mol/mol); incubation for 48 h. On the abscissa, the conꢀ
centration of 5ꢀFU (for curves 2–6); curve
for the ePC liposome concentrations corresponding to
ePC concentrations for curves 2–6
1 was plotted
.
1
ionization and registration of positive ions; H NMR
spectra (
δ, ppm, J, Hz) were recorded on a Bruker
In conclusion, the results of this study suggest that
some ꢀacyl derivatives of 5ꢀFU having a sufficient
stability in aqueous media may be of substantial interꢀ
est as promising ATAs, considering the wide spectrum
of activity of the initial agent.
W
М
ꢀ700 device (United States). Electronic spectra of
N
substances were measured on an SFꢀ256UVI specꢀ
trometer (LOMO Fotonika, St. Petersburg) in ethaꢀ
nol; fluorescence spectra (in ethanol) were recorded
on a Hitachi Fꢀ4000 spectrofluorimeter (Japan); the
slit width at excitation and emission was 3 nm.
1ꢀPalmitoylꢀ5ꢀfluorouracil (I).
ride (215 L, 0.58 mmol) was added dropwise to a susꢀ
pension of 5ꢀFU (50 mg, 0.385 mmol) in a mixture of
anhydrous dioxane (2 mL) and DIPEA (137 L,
0.58 mmol) in an atmosphere of argon, and the mixꢀ
ture was stirred for 1 h at 40–50 . The course of the
reaction was controlled by TLC in benzene—ethyl
acetate 7 : 1, on silica gel F254; detection: A, B, and C.
The mixture was evaporated in vacuo, and the residue
A. Palmitoyl chloꢀ
EXPERIMENTAL
µ
The chemicals used:
pꢀaminobenzoic acid,
DIPEA, hexamethyldisilazane, trimethylchlorosiꢀ
lane, 5ꢀfluorouracil (5ꢀFU) (Sigma, United States);
oleic and adamantaneꢀ1ꢀcarboxylic acids, perylene,
pyridine (Fluka, Germany); silica gel 60 and DCꢀAluꢀ
folien Kieselgelꢀ60 plates for TLC (Merck, Gerꢀ
many); and 1ꢀpalmitoylꢀ2ꢀoleoylphosphatidylcholine
(POPC) (Avanti, United States). Other reagents were
from Reakhim (Russia). DIPEA was distilled over
ninhydrin, then over powdery KOH; methanol was
distilled over magnesium methylate, and chloroform
and methylene chloride were distilled over phosphorus
pentoxide. Other solvents were used after conventional
purification. PBS, рН 7.2, was prepared from a conꢀ
centrate (Sigma, United States).
µ
°
С
was washed with heptane at 50
benzene, and dried in vacuo. The yield of derivative
), which was completely identical to the preparation
obtained by the method , was 120 mg (85.3%).
°С, crystallized from
(
I
B
B. A mixture of 5ꢀFU (200 mg, 1.54 mmol), hexaꢀ
methyldisilazane (5.4 mL, 26 mmol), and trimethylꢀ
chlorosilane (1.54 mL, 12 mmol) was stirred for 4 h at
110–120
Thionyl chloride was distilled over linseed oil, and was removed in vacuo, acetonitrile (10 mL) and palmꢀ
palmitoyl chloride was obtained by the boiling of itoyl chloride (467 L; 423 mg, 1.54 mmol) were
palmitic acid with an excess of thionyl chloride in benꢀ added to the residue (colorless liquid), and the mixture
zene followed by distillation, bp 110 С/1 mmHg. In a was stirred for 4 h at 80 . The course of the reaction
similar way, myristoyl chloride was obtained from was controlled by TLC in benzene–ethyl acetate 7 : 1,
myristic acid, bp 136–141 С/2 mmHg, and adamanꢀ on a plate with the fluorescent indicator; detection: A,
taneꢀ1ꢀcarbonyl chloride, from adamantaneꢀ1ꢀcarꢀ B, and C. The precipitate formed after cooling to
boxylic acid, bp ~130 . Oleoyl chloride was obtained room temperature was aspirated after 6 h, washed with
by the reaction of thionyl chloride with oleic acid in cold benzene and crystallized again from benzene.
°С (bath), the excess of silylating reagents
µ
°
°С
°
°C
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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SYNTHESIS AND PROPERTIES OF LIPOPHILIC DERIVATIVES
303
Derivative (
470 mg (83%); mp 99–101
cated system; UV, λmax, nm (
ESIꢀMS, : 391.229 [
+ Na]+ C20H33FN2O3Na,
391.238), 130.56 [
– acyl + H]+; 1H NMR: 0.89
(3 H, t, СН3), 1.26 (24 H, br m (СH2)12), 1.72 (2
m, СH2СH2СО), 3.12 (2 H, m, СH2СО), 8.30 (1 H,
d, F–H 5, CF=CH), 8.34 (1 H, br s, NH).
I
) was obtained as a colorless powder; yield UV, λmax, nm (
С; f 0.6 in the above indiꢀ : 401.305 [
, М–1 cm–1): 272 (9000);
ε
M
, М–1 cm–1): 274 (3.3
+ Н]+
×
104), ESIꢀMS,
°
R
m/
z
.
ε
p
ꢀOleoylaminobenzoic acid was converted to acid
m/
z
M
chloride (dense oil, quantitative yield) as described
above, which was then used without additional purifiꢀ
cation.
M
M
H,
J
A solution of pꢀmyristoylꢀPAB chloride (0.31 g,
0.75 mmol) in dry dioxane (2 mL) was added under
stirring in an atmosphere of argon to a suspension of
5ꢀFU (106 mg, 0.81 mmol) in dry dioxane (2 mL) and
DIPEA (0.26 mL, 1.4 mmol), and the mixture was
1ꢀ( ꢀMyristoylaminobenzoyl)ꢀ5ꢀfluorouracil (II).
–Myristoylaminobenzoic acid was obtained by the
acylation of ꢀaminobenzoic acid by an equivalent
amount of myristoyl chloride in pyridine (3 h at 10
and 1 h at 25 ). After conventional treatment and
p
p
p
°С
stirred at 60
~2 h). The course of the reaction was controlled as
described for analogue (II). The mixture was allowed
to stand for 18 h at 25 and then evaporated twice
with benzene. The solution of the residue in benzene
(50 mL) was washed with water ( 30 mL, separation
°С until the complete dissolution of 5ꢀFU
°
С
(
crystallization from the chloroform–acetone mixture,
the yield was 80%. TLC: system benzene–ethyl aceꢀ
tate–AcOH 75 : 15 : 2; Rf 0.39 (detection: A); mp
°
С
224–226
°
С
; UV, λmax, nm (
ε
, М–1 cm–1): 273 (3.4
×
104). Literature data [29]: mp 220–222
°С.
2
×
of phases by centrifugation), dried over Na2SO4, and
evaporated. The filtration of the residue through silica
gel (1 g) in benzene–acetate 2 : 1 and then through
LiChrosorb RPꢀ18 (1 g) in a mixture of acetonitrile
DMF (5 L) was added to a suspension of
µ
p
ꢀmyrisꢀ
toylaminobenzoic acid (3 g) in a mixture of SOCl2
(10 mL) and benzene (30 mL), and the mixture was
stirred for 2 h and allowed to stand for 3 days. Then the
solution was filtered and dried at 20–30 Pa; the resultꢀ
ing pꢀmyristoylꢀPAB chloride was used without addiꢀ
tional purification. The completeness of the formation
of acid chloride was controlled by converting a sample
of the latter to methyl ether (TLC: Rf 0.56 in the above
indicated system) after the treatment with the
СН3ОН–pyridine mixture.
with 5% chloroform yielded 345 mg (87%) of 1ꢀpꢀoleꢀ
oylꢀPABꢀ5ꢀFU (III) as a chromatographically indiꢀ
vidual colorless powder; Rf ~ 0.5 in benzene–ethyl
acetate 2 : 1; mp 112–115
cm–1): 284 (3.6 104), 311 (3.5
: 536.301 [
+ Na]+; 1Н NMR (CD3OD–
CDCl3): 0.88 (3 H, t, 7.0, CH3), 1.22 (18 H, m,
(CH2)9), 1.33 (2 H, m, С 2СН3), 1.58 (2 H, m,
2СН2СО), 2.38 (2 H, t, 7.4, СН2СО), 4.37 (1 H,
5.5, =СН), 5.69 (q, 5.5, =СН), 7.65–7.77 (4 H, m,
Ar), 7.86 (1 H, d, F–H 7, CF=СН), 8.45 (1 H, s, NH).
°C
; UV, λmax, nm (
ε
, М–1
×
×
104); MALDIꢀMS,
m/z
M
J
Н
J
A solution of pꢀmyristoylꢀPAB chloride (155 mg,
0.42 mmol) in dry dioxane (2 mL) was added to a susꢀ
pension of 5ꢀFU (50 mg, 0.385 mmol) in dry dioxane
СН
q,
J
J
J
(1 mL) and DIPEA (134
ring in an atmosphere of argon, and the mixture was
stirred at 40 until the complete dissolution of 5ꢀFU
~1 h). The course of the reaction was controlled by
µL, 0.77 mmol) under stirꢀ
1ꢀAdamantoylꢀ5ꢀfluorouracil (IV). 5ꢀFU (100 mg,
0.77 mmol) was acylated with 1ꢀadamantoyl chloride
(139 mg, 0.7 mmol) in dioxane (1 mL) and DIPEA
(0.3 mL) as described for analogue (III). The resulting
mixture was purified by flash chromatography on silica
gel (3 g), the column was washed with chloroform, and
the product was eluted with chloroform–ethyl aceꢀ
°С
(
TLC on RPꢀ18 F254s plates in the system MeOH–
water 9 : 1; Rf of 5ꢀFU was ~0.9, and of product (II),
0.34. The mixture was evaporated in vacuo, and the
residue (amorphous mass) was crystallized at –15–
tate–
Rf 0.26 (control by TLC in the same system). The
yield of product IV) 61 mg (30%); mp >166
(decomposition); UV, λmax, nm (
, М–1 cm–1): 274
(4400); MALDIꢀMS, : 315.135 [
+ Na]+,
292.113 [
]+, 273.052 [ F]+; 1Н NMR (CD3OD–
tꢀBuOH 70 : 10 : 1 to give a substance with
20
20
°
С
from CH2Cl2 and then from toluene at –15–
after which it was dried at 20–30 Pa. Yield of
°С
(
°C
product
nm (
, М–1 cm–1): 278 (3.1
MALDIꢀMS, : 482.253 [
(CD3ODꢀCDCl3): 0.85 (3 H, t,
(18 H, m, (CH2)9), 1.34 (2 H, m,
H, m, 2СН2СО), 2.36 (2 H, t,
.57–7.61 (4 H, m, Ar), 7.73 (1 H, d, JF–H
CF=СН).
(
II) 51 mg (30%); mp 145–147
°
C
; UV, λmax
104);
+ Na]+; Н NMR
7.1, CH3), 1.22
2СН3), 1.57 (2
7.5, СН2СО),
,
ε
×
104), 311 (3.1
×
ε
1
m
M ⎯
/
z
M
m/z
M
M
J
CDCl3): 1.76 (6 H, m, 4'ꢀH), 1.94 (3 H, m, 3'ꢀH), 2.07
(6 H, m, 2'ꢀH), 7.38 (1H, br s, CF=СН).
С
Н
J
СН
7
7,
1ꢀ[8ꢀ(3ꢀPerylenyl)octanoylꢀ5ꢀfluorouracil
(V).
Freshly calcined sodium carbonate (16 mg, 0.15 mmol)
1ꢀ( ꢀOleoylaminobenzoyl)ꢀ5ꢀfluorouracil (III) was was added to a solution of 8ꢀ(3ꢀperylenyl)octanoic
obtained by the aboveꢀdescribed method from ꢀamiꢀ acid (30 mg, 76 mol) in dry methylene chloride
nobenzoic acid (1.08 g, 7.86 mmol) and oleoyl chloꢀ (1 mL), then thionyl chloride (45 L, 0.61 mmol) was
p
p
µ
µ
ride (2.37 mL, 8.14 mmol). The product was chroꢀ added under stirring in an atmosphere of argon, and
matographed on silica gel (45 g) by the elution with the mixture was agitated for 4 h. The mixture was
CHCl3–ethyl acetate–AcOH 95 : 5 : 1. The yield of
oleoylaminobenzoic acid was 2.43 g (76.9%); f 0.37 rated, concentrated by evaporation in dry benzene in
in the same system (detection: A); mp 201–204 vacuo, and dried for 1 h at 30 Pa. The acid chloride of
pꢀ
extracted with dry ether, and the extract was evapoꢀ
R
°С;
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 3
2013

304
SEMAKOV et al.
8ꢀ(3ꢀperylenyl)octanoic acid was used in the subseꢀ size of 100 nm (Nucleopore, United States) using a
quent reaction without additional purification.
5ꢀFU (5 mg, 36 mol) was boiled and stirred for 4 h
in an atmosphere of argon with hexamethyldisilazane
(128 L, 0.61 mmol) and trimethylchlorosilane
(45 L, 0.34 mmol) as described for the synthesis of
). After cooling, the excess of silylating agents was
removed in vacuo. The residue was diluted with dry
acetonitrile (250 L), a half of the aboveꢀdescribed
miniextruder (Avanti Polar Lipids, United States).
The content of 5ꢀFU in liposomal preparations was 10
mM.
µ
Cytotoxic activity of LꢀFU. HBL cells in the logaꢀ
rithmic growth phase were incubated for 48 h in a 24ꢀwell
plate in RPMI 1640 culture medium supplemented with
µ
µ
(
I
7% fetal serum containing 5 ×
103 cells/well with a lipoꢀ
somal preparation containing 10 mol % LꢀFU with
respect to lipid, with 5ꢀFU (addition in the form of a
10 mM aqueous solution; the final 5ꢀFU concentraꢀ
µ
acid chloride of 8ꢀ(3ꢀperylenyl)octanoic acid in the
form of a solution in dry acetonitrile was added, and
tion from 0.01 to 10 µM), or an aliquot of empty lipoꢀ
the mixture was stirred for 4 h at 80
the reaction was controlled by TLC (Kieselgel 60 F254
°С. The course of
somes of the same lipid concentration (control). The
number of viable cells was determined by the standard
trypan blue test; the survival (%) was estimated by the
equation (number of live cells in the experiment/numꢀ
)
in benzene–ethyl acetate 7 : 1; detection: A and B.
The reaction mixture was evaporated twice with benꢀ
zene, and product (
with toluene from the chloroform solution at –10
V) was separated by reprecipitation
ber of live cells in the control) × 100.
°С
and flash chromatography on reverse phase RPꢀ18
(Merck) in acetonitrile–methylene chloride. Yield:
8 mg (40%); the product was obtained as a dark red
amorphous substance having no clearly defined meltꢀ
ACKNOWLEDGMENTS
This work was supported by the Russian Foundaꢀ
tion for Basic Research (project no. 09ꢀ04ꢀ00313).
The authors would like to thank I.A. Boldyrev (IBCH
RAS) for help in data processing.
ing point (decomposition >150
aboveꢀindicated system, detection: A and B; UV, λmax
nm (
, М–1 cm–1): 260 (6.1 104), 423 (4.2 104),
447 (5.9 : 507.212 [
+ H]+;
°C); Rf 0.7 in the
,
ε
×
×
M
×
104); MALDIꢀMS,
m/z
1Н NMR: 1.27 (6 H, br m, (СН2)3СН2СН2СОО), 1.63
(2 H, m, СН2СН2СОО), 1.70 (2 H, m, ArСН2СН2),
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2013