Synthesis and properties of a fluorescent-labeled triglyceride derivative of the antitumor drug merphalan

Article abstract of DOI:10.1023/B:RUBI.0000015776.60923.96

A new fluorescent probe, a 3-perylenoyl derivative of the lipophilized antitumor drug merphalan (sarcolysine), was synthesized. The probe is suitable for studying intracellular traffic and metabolism of merphalan and its derivatives. The perylenoyl fluore

Full text of DOI:10.1023/B:RUBI.0000015776.60923.96

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 1, 2004, pp. 71–74. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 1, 2004, pp. 80–83.
Original Russian Text Copyright © 2004 by Boldyrev, Grechishnikova, Pavlova, Molotkovsky.
Synthesis and Properties of a Fluorescent-Labeled Triglyceride
Derivative of the Antitumor Drug Merphalan
1
I. A. Boldyrev, I. V. Grechishnikova, Yu. B. Pavlova, and Jul. G. Molotkovsky
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia
Received September 13, 2002; in final form, December 16, 2002
Abstract—A new fluorescent probe, a 3-perylenoyl derivative of the lipophilized antitumor drug merphalan
(sarcolysine), was synthesized. The probe is suitable for studying intracellular traffic and metabolism of mer-
phalan and its derivatives. The perylenoyl fluorescence is partially quenched by the merphalan chromophore,
which broadens the probe potentialities.
Key words: fluorescent probe, merphalan, 3-perylenoyl, synthesis
1
INTRODUCTION
RESULTS AND DISCUSSION
Liposomal preparations for the delivery of antican-
cer drugs are now actively studied. In recent years, they
have also found a practical use, since, in the majority of
cases, their cytotoxic efficiency increases and their sys-
temic toxicity decreases (see, e.g., review [1]). When
designing new formulations of anticancer drugs for
such delivery, we have previously reported the synthe-
sis of rac-1,2-dioleoyl-3-sarcolysylglycerol (I) [2], a
lipophilic derivative of merphalan, (4-[bis(2-chloro-
ethyl)amino]-DL-phenylalanine. This compound mani-
fested a substantial cytotoxic activity in tumor cell cul-
tures in vitro [3] and an appreciable antitumor activity
in vivo [4, 5].
1-Octadecenylglycerol (selachyl alcohol) (II) was
used as a starting compound in the synthesis. The
hydrophobic chain in this compound is linked to the
glycerol residue with ether instead of ester bond, which
a little differentiates it from triglyceride (I) in structure.
However, ether bond is very close to ester bond in its
polarity and is more stable to the hydrolysis by intra-
cellular catabolic enzymes. Due to this, our probe may
live longer in the studied system. The acylation of
selachyl alcohol with 3-(3-perylenoyl)propionic acid
by carbodiimide method predominantly proceeded at
position 3 of glycerol residue; (III) was isolated by col-
umn chromatography. Its acylation with N-Boc-mer-
phalan by carbodiimide method resulted in (IV), which
was deprotected to the desired probe (V) by treatment
with trifluoroacetic acid. In addition to the synthetic
scheme and chromatographic behavior, the structures
The knowledge of pathways and mechanisms of
drug penetration into cells and tissues is of great impor-
tance for drug design. At present, fluorescence meth-
ods, in particular, the confocal fluorescence microscopy
(CFM), are widely used for these studies (see, e.g., [6]).
A prerequisite for using this method must be either an
intrinsic fluorescence of a studied compound [7] or a
fluorophore label in its molecule. Bearing in mind
studying pharmacodynamics of (I) and similar com-
pounds using CMF and other fluorescence techniques,
we synthesized its fluorescent analogue, rac-1-(Z-9-
octadecenyl)-2-sarcolysyl-3-[3-(3-perylenoyl)propionyl]-
glycerol (V) (scheme). This bears a fluorescent peryle-
noyl residue, which proved to be a highly efficient
probe in fluorescent microscopy and flow cytometry
[8].
O
CH2O
O
CHO
NH2
Cl
CH2O
N
O
Cl
1
Corresponding author; phone/fax: +7 (095) 330-6601; e-mail:
jgmol@ibch.ru
(I)
1068-1620/04/3001-0071 © 2004 MAIK “Nauka /Interperiodica”

72
BOLDYREV et al.
the molar extinction coefficient at the absorption maxi-
CH2O
mum [11] in the same sample, is 1.00 for (III), 0.59 for
(IV), and 0.58 for (V) (in ethanol). In other words, the
quantum yield of the perylenoyl fluorophore in both
(IV) and (V) is almost two times lower than the normal
value due to the quenching by the merphalan residue.
1, 3-(3-Perylenoyl)propionic acid/DCC
2, N-Boc-merphalan/DCC
3, CF3COOH
CHOH
CH2OH
(II)
1
Obviously, the separation of these residues upon the
cleavage of one of the ester bonds in the probe (V), for
example, under the action of esterases will be accompa-
nied by an increase in the fluorescence intensity. Such
a methodological approach, when fluorophore and
quencher are combined in the same molecule and their
enzymatic separation enables studying the enzyme
activity, is attracting more and more attention (see, e.g.,
[12]). In our case, this property of probe (V) widens its
potentialities in studying the behavior of merphalan
derivatives in various systems.
CH2O
CHOH
O
CH2O
O
(III)
2
Preliminary experiments showed that the probe (V)
can be efficiently included into the liposomes prepared
from egg yolk phosphatidylcholine and distributes in
them rather uniformly at near 1% concentration. This is
an important property for successful CFM experimen-
tation with cells, which will be the subject of our sub-
sequent communications.
CH2O
CHO
NHX
Cl
N
O
Cl
O
EXPERIMENTAL
CH2O
DCC, 4-dimethylaminopyridine (DMAP), triethyl-
amine, and TFA were purchased from FLUKA (United
States); rac-1-(Z-9-octadecenyl)glycerol (selachyl
alcohol) from Supelco (United States); other reagents
and solvents from Reakhim (Russia). Anhydrous chlo-
roform was obtained by distillation over phosphorus
pentoxide. Other solvents of domestic production were
used after usual purification procedures. Column chro-
matography was performed on Kieselgel 60 (Merck,
Germany) and alumina (Reakhim, Russia). Precoated
plates Kieselgel 60 F254 and Kieselgel 60 (Merk, Ger-
many) and reversed-phase Nano-Sil C18-100 plates
(Macherey-Nagel, Germany) were used for TLC. The
spots were detected by (A) spraying with phosphomo-
lybdic acid, (B) under UV irradiation, and (C) by treat-
ment with Cl2–benzidine. 3-(3-Perylenoyl)propionic
acid [12] and N-Boc-sarcolysine [2] were obtained as
described previously. Selachyl alcohol (a preparation
stored for twenty years) was purified by column chro-
matography on alumina using 9 : 9 : 1 CH2Cl2–ethylac-
etate–methanol system for elution.
O
3
(IV)
(V)
X = Boc X = H
Scheme
of (III)–(V) were above all confirmed by 1H NMR and
mass spectra. Their fluorescence spectra were charac-
teristic of perylenoyl compounds [9] and demonstrated
λex 450–452 nm and λem 526–527 nm in ethanol (see
the Experimental section).
The fluorescent perylenoyl group in the probe (V) as
well as in the synthon (IV) is sufficiently close to the p-
bis(2-chloroethyl)aminophenyl group of merphalan,
which could quench the fluorophore. We have not
found in literature any data concerning the application
of the merphalan residue as a fluorescence quencher.
However, when planning the synthesis, we have pre-
sumed such a capability of this residue, which followed
from the basic principles of this phenomenon, which
Mass spectra were taken on a MALDI time-of-flight
Vision 2000 mass spectrometer (Thermobioanalysis,
UK) using 2,5-dihydroxybenzoic acid as a matrix and
N2 laser (3 ns pulse, maximal pulse energy of 250 µJ)
are now well-known [10]. It turned out that the fluores- or an ESI time-of-flight Finnigan MAT 900S instru-
cence of (IV) and (V) is indeed partially quenched in ment (injection in chloroform–methanol 1 : 1). UV
comparison with the emission of (III). Relative quan- spectra were registered on a LKB Ultrospec II spectro-
tum yield, represented as a ratio of the fluorescence photometer (Sweden). Fluorescence spectra were taken
intensity at the emission maximum (in relative units) to on a Hitachi F-4000 spectrofluorimeter in 10 × 10 mm
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS AND PROPERTIES OF A FLUORESCENT-LABELED TRIGLYCERIDE
73
1
quartz cuvettes at 20°ë. 1H NMR spectra (δ, ppm rela- (ε 12600); H NMR (δ, ppm): 0.89 (3H, t, J 6.7 Hz,
tive to TMS) were measured on a Bruker WM 500 spec- ëç3), 1.20–1.38 (24H, m, H2–H7 and H12–H17), 2.01
trometer (Germany) in CDCl3. Solvents were removed
on a rotary evaporator in a vacuum at bath temperature
below 40°ë.
(4H, m, CH2CH=), 2.87 (2H, t, J 7.3 Hz, ëç2ëéé),
2.97, 3.04 (2H, 2 m, CH2Ar), 3.42, 3.49 (6H, 2 m,
ëç2ëéAr, ëç2éëç2ëç2), 3.52, 3.62 (10H, 2 m,
Nëç2ëç2Cl, Oëç2ëç2), 4.24, 4.43 (2 H, 2 m,
ëç2OOë), 4.56 (1 H, s, NH), 4.98 (1 H, m, CHNH),
5.25 (1H, m, CHéç), 5.34 (2H, s, ëç=ëç), 6.58,
rac-1-(Z-9-Octadecenyl)-3-[3-(3-perylenoyl)pro-
pionyl]glycerol (III) and its 1,2-isomer. A 50% solu-
tion of DCC in CCl4 (100 µl) was added upon stirring
to a solution of selachyl alcohol (II) (40 mg, 7.04 (4 H, 2 m, phenylene), 7.54 (2H, m), 7.59 (1H, q),
0.12 mmol), 3-(3-perylenoyl)propionic acid (30 mg,
0.085 mmol), and DMAP (40 mg, 0.33 mmol) in anhy-
drous chloroform (2 ml). The reaction mixture was kept
for a day and treated with an additional DCC solution
(50 µl; 75 mg, 0.36 mmol in total). Then the reaction
mixture was diluted with CH2Cl2 (20 ml) and stirred
with 1 N H2SO4 (1 ml) for 30 min at cooling with ice.
The organic extract was twice chromatographed on a
silica gel column (~1 × 13 cm) using gradient elution
7.74 (1H, d), 7.79 (1H, d), 7.98 (1H, q), 8.21 (1H, q),
8.27 (3H, asymmmetric t), 8.59 (1H, t) (perylenyl); flu-
orescence: λex 450 nm in ethanol and 452 nm in chloro-
form, at λem 515 nm; λem 526 nm in ethanol and 506 nm
in chloroform at λex 450 nm; relative quantum yield
0.59.
rac-1-(Z-9-Octadecenyl)-2-merphalanyl-3-[3-(3-
perylenoyl)propionyl]glycerol (V). A solution of the
amide (IV) (10 mg, 9.4 µmol) in CH2Cl2 (0.5 ml) and
TFA (2 ml) was kept for 1 h at 35°C in argon atmo-
sphere, and the reaction mixture was twice coevapo-
rated with toluene. The residue was purified on a
LiChroprep RP-18 (1 g) column eluted with a 95 : 4.5 :
0.5 methanol–CH2Cl2–TFA mixture to yield 7.8 mg
(86%) of the amorphous red product (V); Rf 0.55 on
Nano-Sil C18-100 (89 : 10 : 1 methanol–CH2Cl2–TFA)
and 0.45 on Kieselgel 60 without indicator (94 : 5 : 1
CHCl3–isopropanol–CH3COOH), detection with A, B,
and ninhydrin; orange amorphous substance; MALDI
MS, m/z: 965 [2M + H]+; fluorescence: λex 450 nm in
ethanol and 452 nm in chloroform at λem 515 nm;
λem 527 nm in ethanol and 507 nm in chloroform at
λex 450 nm; relative quantum yield 0.58.
with ethyl acetate (5
15%) in pentane–CH2Cl2 (1 : 1)
at TLC monitoring (45 : 45 : 10 pentane–CH2Cl2–ethy-
lacetate, detection A, B, and C), to get fluorescent (III);
yield 26 mg (45%); Rf 0.18; and, probably, 1,2-isomer
of (III); yield 17 mg (30%); Rf 0.15 with the same flu-
orescence.
(III): amorphous orange substance; ESI-MS, m/z:
699.5 [M + Na]+; UV [ethanol): λmax 446.5 nm (ε
11500); 1H NMR (δ, ppm): 0.89 (3 H, t, J 6.8 Hz, 3ç,
ëç3), 1.3–1.4 (24 H, m, H2–H7, H12–H17), 2.01 (4 H,
m, H8, H11), 2.90 (2 H, t, J 6.4 Hz, 2ç, ëç2ëéé),
3.44, 3.52 (6 H, 2 m, ëç2ëéAr, ëç2éëç2), 4.05
(1H, s, CHéç), 5.35 (2 H, m, ëç=ëç); 7.54 (2 H, q),
7.61 (1 H, m), 7.74 (1 H, d), 7.79 (1 H, d), 7.99 (1 H,
d), 8.22 (1 H, d), 8.27 (3 H, asymmetric t), 8.59 (1 H, d)
(perylenyl); fluorescence: λex 451 nm in ethanol and
459 nm in chloroform, λem 515 nm, λem 527 nm in eth-
anol and 511 nm in chloroform at λex 450 nm, relative
quantum yield 1.00.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, projects nos. 00-04-48416 and
02-04-48287.
1,2-Isomer: ESI-MS, m/z: 700.5 [M + Na]+; fluores-
cence spectrum is the same as that of (III).
rac-1-(Z-9-Octadecenoyl)-2-(N-Boc-merphalanyl)-
3-[3-(3-perylenoyl)propionyl]glycerol (IV). A 50%
solution of DCC in CCl4 (0.29 mmol, 60 µl) was added
to a stirred solution of (III) (10 mg, 15 µmol), N-Boc-
merphalan (10 mg, 25 µmol), and DMAP (20 mg,
0.16 mmol) in anhydrous chloroform (0.7 ml). After a
day, the reaction mixture was treated with DCC as
described above and chromatographed on a silica gel
column (0.5 × 7.5 cm) eluted with a 10 : 10 : 1 pentane–
CH2Cl2–ethyl acetate system at TLC monitoring of the
eluate (see above). The product (IV) was additionally
filtered in CH2Cl2 through an alumina column (0.5 ×
4 cm) for removal of dicyclohexylurea traces; yield
11 mg (70%); a red amorphous solid; Rf 0.45 (45 : 45 :
10 pentane–CH2Cl2–ethyl acetate, detection A, B, and
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