Synthesis and cytotoxicity of novel phosphorusless analogues of edelfosine

Article abstract of DOI:10.1134/S1068162008060150

Modified series of phosphorusless edelfosine analogues bearing the polar heads of aliphatic bases, N,N-dimethylethanolamine and N,N,N1,N ¹-tetramethylethylenediamine, were synthesized, with the length of the spacer varying from three to four methylene units. The cytotoxic characteristics of the compounds synthesized were studied.

Full text of DOI:10.1134/S1068162008060150

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 6, pp. 743–746. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © S.G. Romanova, A.A. Shtil’, G.A. Serebrennikova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 6, pp. 827–830.
Synthesis and Cytotoxicity of Novel Phosphorusless
Analogues of Edelfosine
S. G. Romanovaa, 1, A. A. Shtil’b, and G. A. Serebrennikovaa
a Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571 Russia
b Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478 Russia
Received October 2, 2007; in final form, October 29, 2007
Abstract—Modified series of phosphorusless edelfosine analogues bearing the polar heads of aliphatic bases,
N,N-dimethylethanolamine and N,N,N1,N1-tetramethylethylenediamine, were synthesized, with the length of
the spacer varying from three to four methylene units. The cytotoxic characteristics of the compounds synthe-
sized were studied.
Key words: antineoplastic activity, edelfosine, edelfosine analogues, ether lipids
DOI: 10.1134/S1068162008060150
INTRODUCTION
The practical use of ether glycerolipids requires the
solution of problems of the chemical synthesis of this
class of compounds. Preparation of Edelfosine and its
analogues is a multistage process, which requires long-
term purification at every step. This decreases the yield
of the target product and increases its cost. Further-
more, these syntheses deal with toxic phosphorus-con-
taining reagents.
In the last few years the structure–activity special-
ties of the representatives of a new lipid class, cation
nonphosphorus ether glycerolipids, are under intensive
study2. Glycerophospholipid Edelfosine (1-é-octade-
cyl-2-é-methyl-rac-glycero-3-phosphocholine) pos-
sessing antitumor activity is their prototype.
The main features of the antiproliferative action of
antitumor lipids, the possibility of inhibition of cell
proliferation and the selectivity of antiproliferative
action, were previously established; the attempt to cor-
relate the results of the study afore-mentioned with the
literal data on the parent phosphorus-containing com-
pounds was also undertaken [3, 4]. The most pro-
nounced antitumor activity is observed in for the series
CH
O
C H
18 37
2
H C O HC
O
P
3
+
CH
O
O CH CH N (CH )
2 2 3 3
2
–
O
Edelfosine (Et-18-OMe)
Nowadays Edelfosine is used for antitumor therapy. of ether glycerolipids. The biological tests [5[a1]] of
The advantage of Edelfosine and the related com- the nonphosphorus glycerolipids synthesized allowed
pounds over many of antitumor drugs is in the absence us to amplify the main requirements to the structure of
of mutagenic effect of these compounds [1, 2].
alkyl glycerolipids reported in literature [6[a2]].
1
XR1
OR2
ZY+ Q–
X = O, S, OCONH; R = alkyl, alkenyl, acyl (C –C ), alkyls with aromatic and heterocyclic moieties;
10 20
2
R = long-chain (C –C ) or short-chain (C –C ) alkyl and acyl substituents; Z = spacer moieties of vari-
10 20
1
4
+
ous types, can be absent; Y = ammonium, sulfonium, or heterocyclic moieties with positively charged
–
–
–
–
nitrogen and sulfur atoms; Q = counterions Hal , AcO or TosO .
It turned out that in most cases the long-chain amine bond should be present at C1 of the glycerol
backbone. The short-chain (ë1–ë4) alkoxy group is
required at C2 of the glycerol backbone. The spacer
region between the polar domain and the glycerol back-
bone can be absent or to be a connective moiety of
alkyl, acyl, or amide type (ë1−ë8). As a rule, the cation
hydrocarbon residue attached by the ether, thioether, or
1
Corresponding author; phone: +7 (495) 936-8903; e-mail: rom-
fill@mail.ru.
2
Abbreviations: DMAE, N,N-dimethylethanolamine; TMEDA,
1
1
N,N,N ,N -tetramethylethylenediamine.
743

744
ROMANOVA et al.
head is of ammonium or sulfonium type with short (ë1– ammonium iodide (IVa), rac-N-{4-[(2-methoxy-3-octa-
ë3) substituents or a hetero atom involved in heterocy-
clic systems (e.g., pyridinium or thiasolinium cation
heads). A halogen ion can serve as a counterion.
We have developed an approach to the synthesis of
novel nonphosphorus ether glycerolipids suited to the
requirements afore-mentioned. The glycerolipids under
study designated as (III‡), (IIIb), (IVa), and (IVb) were
synthesized and characterized for the first time.
decyloxy)propyl]oxycarbonylbutyl}-N,N-dimethyl-N-(2-
hydroxyethyl)ammonium iodide (IIIb), and rac-N-{4-
[(2-methoxy-3-octadecyloxy)propyl]oxycarbonylbutyl}-
N,N-dimethyl-N-(2-dimethylaminoethyl)ammonium
iodide (IVb).
The choice of the amine type is dictated by the high
antitumor activity of glycerolipids containing the resi-
due of N,N-dimethylethanolamine as a polar head (the
so-called reverse choline type cationic head) [6]. Fur-
thermore, in order to clarify the influence of the
hydroxyl group on the cytotoxicity, N,N,N1,N1-tetrame-
thylethylenediamine containing the dimethylamino
moiety instead of the hydroxyl group was chosen as a
polar head. The possibility of creation of dicationic
compounds that widens the area of search for antitumor
agents is another specialty of these lipids. The key com-
pounds in the synthesis of lipids (IIIa), (IIIb), (IVa),
and (IVb), rac-1-octadecyl-2-methyl-3-(4-bromobu-
tanoyl)glycerol (IIa) and rac-1-octadecyl-2-methyl-3-
(5-bromopentanoyl)glycerol (IIb), were obtained by
acylation of the starting diglyceride (I) with 4-bro-
mobutyric and 5-bromovaleric acid chlorides, respec-
tively.
RESULTS AND DISCUSSION
To study the structure–function characteristics of
nonphosphorus analogues of Edelfosine and to deter-
mine their biological activity, new modification series of
cationic ether glycerolipids differing in the spacer length
represented by the residues of valeric or butyric acids and
in the type of the polar head formed by DMAE or
TMEDA residues (see scheme) have been synthesized.
The influence of an individual structure-forming compo-
nent on the activity was estimated for N,N-{4-[(2-meth-
oxy-3-octadecyloxy)propyl]oxycarbonylpropyl}-N,N-
dimethyl-N-(2-hydroxyethyl)ammonium iodide (IIIa),
rac-N-{4-[(2-methoxy-3-octadecyloxy)propyl]oxycar-
bonylpropyl}-N,N-dimethyl-N-(2-dimethylaminoethyl)
CH2 OC18H37
CH OMe
CH2 OH
(I)
CH2 OC18H37
CH OMe
CH2 OC18H37
A–B
C–D
CH OMe
CH2 OCO(CH2)nBr
CH2 OCO(CH2)nY
(IIa): n = 3;
(IIb): n = 4
(IIIa): n = 3; (IIIb): n = 4; Y = DMAE
(IVa): n = 3; (IVb): n = 4; Y = TMEDA
Scheme. Synthesis of phosphorusless cationic glycerolipids of alkyl type.
A: (IIa), Br(CH ) COCl, Py/CHCl , 20°C, 0.5 h; B: (IIb), Br(CH ) COCl, Py/CHCl , 20°C, 0.5 h;
2 3
3
2 4
3
C: (IIIa) Ë (IIIb): DMAE, NaI/DMSO, 55°C, 4.5 h; D: (IVa) Ë (IVb): TMEDA, NaI/DMSO, 50°C, 5 h.
The cationic head was introduced to the lipid mole- K562 (leukemia), HCT-116 (colon cancer), and MCF-
cule by quaternization of the corresponding tertiary 7 (breast cancer) cultured cell lines.
amine with bromides (IIa) and (IIb) in the presence
In seemed to be appropriate to include cationic lipid
of NaI.
(V) previously synthesized in our laboratory [6], which
contained DMEA in the polar domain and the ethyl
It is known that quaternization proceeds rather
substituent instead of the methyl moiety in C2 of the
slowly (20–70 h) at heating in the presence of the amino
glycerol backbone, to the test program.
component [6]. In our case, the reaction time varied
from 3 to 5 h. The substitution of the more reactive
iodide for bromide in the Finkelstein reaction allowed
us to decrease the reaction time and to mollify the tem-
perature conditions of the process in order to avoid the
resinification of t he reaction products, and, thus, to
increase the yield of the target compounds.
Lipids (IIa), (IIIb), and (V) appeared to be the most
toxic for K562 cells (IC50 4.0 0.8; 3.9 1.0, and
3.6 1.2 µM, respectively). In the case of Edelfosine,
the value of IC50 is 4.7 0.9 µM. Compounds (IVa) and
(IVb) appeared to display almost no cytotoxicity
towards these cell lines.
Thus, estimation of cytotoxicity of the new com-
pounds showed that the length of the spacer region has
no essential effect on the activity of the cationic glycer-
olipids with an aliphatic base as a polar domain. The
structure of the polar domain has the marked influence
Bromides (IIa) and (IIb) were put into interaction
with DMAE and TMEDA to give lipids (IIIA), (IIIb),
(IVa), and (IVb) differing in the length of the spacer
group and the structure of the cationic head.
The study of the cytotoxic activity of the com- on the cytotoxicity of glycerolipids: lipids with the
pounds obtained was carried out using MTT test [7] on reversed choline cationic head are toxic for K562 cells,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

SYNTHESIS AND CYTOTOXICITY OF NOVEL PHOSPHORUSLESS
745
whereas the compounds with TMEDA cationic head 1ç NMR: 0.84 (3 H, t, J 6.8, ((ëç2)15ëç3); 1.20 (30 H,
have no cytotoxic effect on the cell lines under study.
br. s, (ëç2)15ëç3); 1.45–1.58 (2 H, m, éëç2ëç2),
1.75–1.89 (2 H, q, ëç2ëç2Br); 2.36 (2 H, t, J 7.0,
éëéëç2), 3.33–3.46 ëç2éëç2ëç2; ëçéëç3;
ëç2Br); 3.58 (3 H, s, éëç3), 4.08 (1 H, dd, J = 5.8 and
10.6, ëççaéëé), 4.23 (1 H, dd, J = 3.9 and 10.6,
ëççbéëé).
EXPERIMENTAL
Solvents ands reagents used in this study were dis-
tilled. The compounds synthesized and the monitoring
of the reactions were carried out by TLC on Silica gel
60 (Merck, Germany) precoated plates using (A) chlo-
roform and (B) 4 : 1 chloroform–methanol as develop-
ing systems. Spots were visualized by treating with
10% phosphomolybdic acid and subsequent heating.
Column chromatography was carried out on Silica
gel 60 (0.040–0.063 µm, Merck, Germany) in (1) chlo-
roform and (2) 6 : 1 chloroform–methanol. Melting
points were taken on a Bohetius (Germany) instrument.
1H NMR spectra (δ, ppm; J, Hz) were registered on a
Bruker MSL-200 (200 MHz) spectrometer in CDCl3
using tetramethylsilane as the internal standard.
rac-N-{4-[(2-Methoxy-3-octadecyloxy)propyl]
oxycarbonylbutyl}-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium iodide (IIIb). A mixture of rac-1-octade-
cyl-2-methyl-3-(5-penthanoyl)glycerol
(0.379
g,
0.73 mmol), DMSO (1.6 ml), NaI (0.33 g, 2.2 mmol),
and DMEA (0.09 ml, 0.08 g, 0.87 mmol) was kept for
4.5 h at 55°ë at occasional stirring. The mixture was
cooled, diluted with chloroform (20 ml), and washed
with 1% HCl (2 × 25 ml) to pH < 7 and water (5 ×
30 ml). The aqueous layer was extracted with chloro-
form (15 ml). The organic layer was dried with Na2SO4
and concentrated in vacuo. The residue was chromato-
graphed on silica using system (2); yield 0.300 g
rac-1-Octadecyl-2-methylglycerol
(I)
was
1
obtained according to the standard procedure [6, 8]; 1H
NMR: 0.83 (3 H, t, J6.8, ((ëç2)15ëç3); 1.24 (30 H, br. s,
(ëç2)15ëç3); 1.49–1.56 (2 H, m, éëç2ëç2), 3.31 (1 H,
m, ëçéëç3), 3.36 (3 H, Ò, éëç3), 3.48–3.52 (4 H, m,
ëç2éëç2; ëç2éç). Found, %: C 73.64; H 12.90;
C22H46O3. Calc., %: C 73.68; H 12.93.
(82.7%); Rf = 0.56 (B); H NMR: 0.83 (3 H, t, J 6.8,
((ëç2)15ëç3); 1.24 (30 H, br. s, (ëç2)15ëç3); 1.49–1.56
(2 H, m, éëç2ëç2), 1.78–1.84 (4 H, m,
(ëç2)2ëç2N+); 2.44 (2 H, t, J 6.9, éëéëç2); 3.38 (6 H,
s, N+(ëç2)3), 3.43–3.47 (4 H, m, ëç2éëç2ëç2); 3.58–
3.69 (3 H, m, N+ëç2ëç2éç, ëçéëç3), 3.75–3.81
(2 H, m, (ëç2)3ëç2N+), 4.09 (1 H, dd, J = 5.9 and 11.8,
ëççaéëé), 4.13–4.22 (2 H, m, N+ëç2éç), 4.26
(1 H, dd, J = 4.2 and 11.8, ëççbéëé). Found, %:
C 53.75; H 8.76; N 2.26; C31H64O5NI. Calc., %:
C 53.71; H 8.73; N 2.21.
5-Bromovaleric and 4-bromobutyric acid chlo-
rides were obtained by treating the corresponding acids
with the excess (10 equiv.) of thionyl chloride in anhy-
drous chloroform (24 h at 20°C). The solvent and the
excess of thionyl chloride were removed in vacuo. The
resulting acid chlorides were put into reaction without
additional purification.
rac-N-{4-[(2-Methoxy-3-octadecyloxy)propyl]
oxycarbonylpropyl}-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium iodide (IIIa) was obtained as described
for (IIIb) starting from a solution of rac-1-octadecyl-2-
rac-1-Octadecyl-2-methyl-3-(5-bromopenthanoyl)
glycerol (IIb). Pyridine (0.5 ml) and then a solution of
5-bromopenthanoyl chloride (0.22 ml, 1.2 mmol) in
anhydrous chloroform (1.5 ml) were added dropwise at
stirring at 0°ë to a solution of rac-1-octadecyl-2-meth-
ylglycerol (I) (0.290 g, 2 mmol) in anhydrous chloro-
form. The mixture was stirred for 30 min at 20°ë,
diluted with chloroform (20 ml), washed with 1% HCl
(3 × 25 ml) and water (2 × 25 ml), and dried with
Na2SO4. The residue was chromatographed in system
(1[a3]) to give 0.379 g (91.2%) of the target product;
Rf 0.66 (Ä). 1ç NMR: 0.86 (3 H, t, J 6.8, ((ëç2)15ëç3);
1.23 (30 H, br. s, (ëç2)15ëç3); 1.47–1.59 (2 H, m,
éëç2ëç2), 1.71–1.91 (4 H, m, ((ëç2)2ëç2ëç2Br);
2.35 (2 H, t, J 7.0, éëéëç2), 3.35–3.46 (5 H, m,
ëç2éëç2ëç2; ëçéëç3; ëç2Br); 3.60 (3 H, s, éëç3),
4.07 (1 H, dd, J 5.8, 10.6, ëççaéëé); 4.25 (1 H, dd, J
3.9, 10.6, ëççbéëé).
methyl-3-(4-bromobutanoyl)glycerol
(0.365
g,
0.72 mmol) in DMSO (1.5 m), NaI (0.32 g, 2.16
mmol), and DMEA (0.09 ml, 0.08 g, 0.86 mmol); yield
0.382 g (62.9%); Rf = 0.58 (B); 1H NMR: 0.85 (3 H, t,
J 6.8, ((ëç2)15ëç3); 1.26 (30 H, br. s, (ëç2)15ëç3);
1.51–1.55 (2 H, m, éëç2ëç2), 1.81 (2 H, m,
(ëç2ëç2N+); 2.41 (2 H, t, J 6.9, éëéëç2); 3.36 (6 H, s,
N+(ëç2)3), 3.43–3.47 (4 H, m, ëç2éëç2ëç2); 3.56–
3.62 (3 H, m, N+ëç2ëç2éç, ëçéëç3), 3.78–3.82
(2 H, m, (ëç2ëç2N+), 4.11 (1 H, dd, J = 5.9 and 11.8,
ëççaéëé), 4.12–4.23 (2 H, m, N+ëç2éç), 4.26
(1 H, dd, J = 4.2 and 11.8, ëççbéëé). Found, %:
C 55.91; H 9.75; N 2.17; C30H62O5NI. Calc., %:
C 55.97; H 9.70; N 2.24.
rac-N-{4-[(2-Methoxy-3-octadecyloxy)propyl]
oxycarbonylbutyl}-N,N-dimethyl-N-(2-dimethy-
rac-1-Octadecyl-2-methyl-3-(4-bromobutanoyl) laminoethyl)ammonium iodide (IVb) was prepared
glycerol (IIa) was prepared by the procedure described as described for (IIIb) starting from a solution of rac-
for (IIb) starting from rac-1-octadecyl-2-methylglyc- 1-O-octadecyl-2-O-methyl-3-O-(5-bromopenthanoyl)
erol (I) (0.285 g, 0.796 mmol) in anhydrous chloroform glycerol (0.230 g, 0.44 mmol) in DMSO (0.6 ml), NaI
(0.5 ml), pyridine (0.5 ml), and a solution of 4-bro- (0.20 g, 1.32 mmol), and TMEDA (0.08 ml, 0.06 g,
1
mobutyric acid chloride (0.12 ml, 0.20 g, 1.2 mmol) in 0.53 mmol); yield 0.142 g (80.1%); Rf = 0.56 (B); H
chloroform (1.5 ml); yield 0.365 g (90.6%); Rf 0.64 (Ä). NMR: 0.89 (3 H, t, J 6.8, ((ëç2)15ëç3); 1.22 (30 H, br.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

746
ROMANOVA et al.
s, (ëç2)15ëç3); 1.52–1.56 (2 H, m, éëç2ëç2), 2.24
(4 H, m, ((ëç2)2ëç2N+); 2.29 (6 H, s, N(ëç3)2),
2.36–2.38 (2 H, m, éëOëç2); 2.73 (2 H, m,
(ëç2ëç2N+)); 3.21 (6 H, s, N+(CH3)2), 3.32 (3 ç, s,
CH2OCH3), 3.91 (1 H, dd, J = 5.9 and 11.8,
ëççaéëé), 4.15–4.25 (6 H, m, CH2OCH2,
CH2N+(ëç2)3CH2), 4.28 (1 H, dd, J = 4.2 and 11.8,
ëççbéëé). Found, %: C 59.44; H 10.39; N 2.01;
C33H69O4N2I. Calc., %: C 59.37; H 10.34; N 2.10.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research (project no. 04–3-32452).
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