Synthesis and biological activity of homologous piperidine-containing alkyl glycerolipids

Article abstract of DOI:10.1134/S1068162009050161

The paper provides the synthesis of new phosphorless glycerolipids with an ether bond that contain heterocyclic bases in the polar domain. The physico-chemical characteristics and biological activity of the prepared compounds were studied.

Full text of DOI:10.1134/S1068162009050161

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 5, pp. 641–644. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © S.G. Romanova, G.A. Serebrennikova, A.A. Shtil, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 5, pp. 709–713.
Synthesis and Biological Activity of Homologous
Piperidine-Containing Alkyl Glycerolipids
S. G. Romanovaa,1, G. A. Serebrennikovaa, and A. A. Shtilb
a Lomonosov Moscow State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 119571 Russia
b Blokhin Russian Oncological Research Center, Russian Academy of Medical Sciences, Moscow, Russia
Received January 27, 2009; in final form, February 05, 2009
Abstract—The paper provides the synthesis of new phosphorless glycerolipids with an ether bond that contain
heterocyclic bases in the polar domain. The physico-chemical characteristics and biological activity of the pre-
pared compounds were studied.
Key words: cationic glycerolipids, lipids with an ether bond, amines of heterocyclic series, antineoplastic activ-
ity, cancer chemotherapy
DOI: 10.1134/S1068162009050161
Y+ is ammonium or sulfonium aliphatic group contain-
ing the short alkyl substituent (C1−C3) or heterocyclic
head with the positively charged nitrogen or sulfur
atom (pyridinium, thiazolinium, etc.); and
INTRODUCTION
Works concerning the chemical synthesis and bio-
logical characteristics of new agents for cancer chemo-
therapy are now in active progress.2 The studies are
aimed at the optimization of properties of drugs used
now in medical practice [1], i.e., at increasing the selec-
tivity of antiproliferative anticancer agents and enhanc-
ing their therapeutic index, which is the ratio of the
maximum permissible dose to the minimum effective
dose. The design of new chemical compounds with a
broad spectrum of biological activity is a topical task of
bioorganic chemistry. Chemical modifications can
change the limits and even trends of drug therapeutics.
In the past few years, researchers’attention has been
drawn to alkyl glycerolipids, among which those with
antibacterial, anti-HIV-1, and anticancer action were
discovered [2–4]. Phosphorless glycerolipids were
marked as prospective anticancer agents [5].
Q– is counterion (Hal–, AcO–, TsO–, etc.)
The majority of lipids of this class contain at the C1
and/or C2 atoms of glycerol acyl or alkyl substituents
consisting of not less than 16 carbon atoms. These com-
pounds can be used as transfection agents for chemo-
therapy [6]. From this point of view, synthetic lipids
containing mono- and polyunsaturated long-chain sub-
stituents at C1/C2 atoms of the glycerol skeleton are not
so attractive. Depending on the stated problem, one or
another modification of the C2 substituent is carried
out. Compounds containing a short-chain substituent
(one to four carbon atoms) at the second position can be
potential anticancer agents for cancer chemotherapy
[6, 7].
XR'
The study of the biological activity of a modification
series of cationic glycerolipids containing aliphatic
bases in the hydrophobic part of the molecule showed
that the structure of their polar domain predominantly
influenced the cytotoxic properties of these compounds
(Table 1) [6, 8].
,
OR''
Z
Y+ Q–
where X is O, S, OCONH;
R' is the long-chain (C10–C20) alkyl, alkenyl, or acyl
substituent;
R'' is the long-chain (C10–C20) or short-chain (C1–C4)
alkyl substituent;
Z can be absent or be the spacer (C1–C8) of alkyl, acyl,
or amide type;
Aliphatic cationic glycerolipids containing the N,N-
dimethylaminoethanol residue demonstrated a high
cytotoxic activity towards K562 cells, while glycerolip-
ids bearing the N,N,N1,N1-tetramethylethylenediamine
residue in the polar domain at the same molar concen-
trations did not appear to be active for this cell type [8].
1
Corresponding author; phone: +7 (495) 936-89-03; e-mail: romfill@
Parallel with aliphatic glycerolipids derivatives, het-
erocycle-containing lipids are also biologically active.
In particular, the high cytotoxicity in vitro of pyridine-
and naphtaline-containing compounds is known [9]. At
mail.ru
2
Abbreviations: MePip, N-methylpiperidine; MTT, water soluble
vital dye, 3-(4,5-dimethyl-tiazole-2-yl)-2,5-diphenyl tetrazolium
bromide
641

642
ROMANOVA et al.
the same time, compounds bearing aromatic cationic
heads were shown to be liable to induce acute hemoly-
sis. The hemolytic activity of compounds significantly
impedes the transition to the in vivo level because it is
necessary to choose the necessary concentration of the
used compounds and to find the approaches to mini-
mize it.
This work presents the synthesis of piperidine-con-
taining phosphorless cationic glycerolipids, new agents
of the examined class. The studied glycerolipids (IIIa),
(IIIb), (IVa), and (IVb) (scheme) were prepared and
analyzed for the first time.
I–
OC H
18 37
B
OMe
+
OCO(CH ) N
2 n
OC18H37
OMe
OH
OC H
18 37
(IIIa,b)
A
CH
3
OMe
OC H
18 37
OCO(CH ) Br
I–
2 n
OMe
OCO(CH ) N
(I)
(IIa,b)
a: n = 3; b: n = 4
C
+
OH
2 n
CH
(IVa,b)
3
Reactants and synthesis conditions: A, Br(CH ) COCl, Py/CHCl , –5°C, 20 min;
2 n
3
B, MePip, MeCOEt/NaI, 60°C, 4 h; C, 4-hydroxy-MePip, MeCOEt/NaI, 60°C, 4.5 h.
Synthesis of heterocyclic homologous pairs of glycerolipids.
RESULTS AND DISCUSSION
The intended products (scheme) were synthesized in
a yield of ~70% under conditions of the quaternization
of the corresponding tertiary bases, i.e., N-methylpipe-
ridine and 4-hydroxy-N-methylpiperidine. The reaction
was carried out in dry methylethyl ketone in the pres-
ence of sodium iodide.
The synthesized lipids contained N-methylpiperi-
dine and 4-hydroxy-N-methylpiperidine in the polar
domain, which were separated from the glycerol resi-
due by three and four methylene units. These products
were submitted to the investigation protocol for reveal-
ing their cytotoxic activity towards cancer cells of the
K562 and U937 lines (human leukemia cells) and the
HCT116 line (large intestine adenocarcinoma cells).
This study was performed using the standard method of
the MTT test [10]. The IC50 values of the synthesized
compounds for cancer cells of the K562 and U937 lines
are presented in Table 2.
Key compounds in the synthesis of lipids (III‡),
(IIIb), (IVa), and (IVb), i.e., rac-1-octadecyl-2-
methyl-3(4-bromobutanoyl)glycerol (IIa) and rac-1-
octadecyl-2-methyl-3(5-bromopentanoyl)glycerol
(IIb), were synthesized through the acylation of parent
diglycerol (I) with chloroanhydrides of 4-bromobutiric
and 5-bromovaleric acids, respectively.
Table 1. Cytotoxic activity of aliphatic derivatives of phos-
phorless glycerolipids with an ether bond towards the human
leukemia cell line K562* [8]
OC18H37
I–
OR
OCO(CH2)nX
n
R
ï
IC50, ÏÍå
The results correlate with the cytotoxic activity val-
ues for previously synthesized pyridine-containing
alkyl glycerolipids (Table 3) [11].
2
3
4
4
3
4
CH3 +N(CH3)2CH2CH2OH
4.5 1.0
3.9 1.2
3.6 1.0
4.2 0.8
>60
It was previously shown when studying the
hemolytic activity of pyridine-containing compounds
that the maximum permissible values for hemolysis
were significantly exceeded [11]. At the same time, the
hemolytic activity of the newly prepared piperidine-
containing compounds was not revealed in the studied
concentration range. The high cytotoxicity of com-
pounds (III‡), (IIIb), (IVa), and (IVb) towards a num-
ber of cancer cells, as well as the absence of their
CH3
CH3
C2H5
CH3 +N(CH3)2CH2CH2N(CH3)2
CH3
>60
Note: *The time of incubation of cells with lipids was 72 h.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

SYNTHESIS AND BIOLOGICAL ACTIVITY OF HOMOLOGOUS
643
Table 2. The biological activity of piperidine-contaning ho- Table 3. Cytotoxic activity of pyridine-containing alkyl
mologous pairs of alkyl glycerolipids*
glycerolipids towards the human leukemia cell line K562
and large intestine adenocarcinoma cells line HCT116* [11]
Lipid
(IIIa)
(IIIb)
(IVa)
(IVb)
OC18H37
I–
IC50, µM (K562)
IC50, µM (U937)
4.5 0.9 4.8 0.6 4.0 0.5 4.1 0.8
22.1 0.6 20.4 0.3 16.2 0.4 15.8 0.3
OR
+
Z
N
Note: *The time of incubation of cells with lipids was 72 h.
IC50, µM
R
Z
Lipid
hemolytic activity, allow one to consider these com-
pounds as new potential anticancer agents among phos-
for K562
for HCT116
phorless heterocyclic derivatives of cationic alkyl glyc- CH3
OCO(CH2)3
12.6
12.5
12.6
12.8
4.6
2.4
2.3
2.6
2.8
7.3
4.7
1.2
39.0
a
b
c
d
e
f
erolipids.
CH3
C2H5
C2H5
CH3
C2H5
H
OCO(CH2)4
OCO(CH2)3
OCO(CH2)4
EXPERIMENTAL
–
–
–
–
We used distilled solvents and reagents of Russian
(Khimmed, Ecros) and foreign (Merck, Germany)
manufacture. Chromatographic isolation and purifica-
tion of compounds were carried out on Kieselgel 60
(40–63 µm; Merck, Germany) silica gel.
3.1
–
g
h
C18H37
36.2
Note; *The time of incubation of cells with lipids was 72 h.
1H NMR spectra (δ, ppm; SSIC, Hz) for compounds
in deuterochloroform solutions were registered on a
Bruker MSL-200 (200 MHz) pulsed Fourier spectrom-
eter. Tetramethylsilane was used as the internal stan-
dard.
rac-1-Octadecyl-2-methyl-3-(4-bromobutanoyl)gly-
cerol (IIa) and rac-1-Octadecyl-2-methyl-3-(5-bro-
mopentanoyl)glycerol (IIb) were prepared as previ-
ously described in [6, 12].
Mass spectra were recorded on a Finnigan MAT
900XL-TRAP (San Jose, United States) spectrometer
by the ESI method.
rac-N-{4-[2-methoxy-3-octadecyloxy)propyl]oxy-
pentanoyl}-N-methylpiperidinium iodide (IIIa).
1-Methylpiperidine (0.18 ml, 1.58 mmol) was added to
the solution of compound (IIa) (0.103 g, 0.198 mmol)
in anhydrous methylethyl ketone (2.5 ml). The reaction
mixture was stirred for four hours at 60°ë. The solvent
was removed in a vacuum and the residue was chro-
matographed in a system of chloroform–methanol,
15 : 1. The yield of (IIIa) was 0.073 g (68.1%).Rf 0.47
(chloroform–methanol, 4 : 1). 1H NMR, δ: 0.84 (3 H, t,
J 6.8, ((ëç2)15ëç3); 1.24 (30 H, br.s, (ëç2)15ëç3); 1.49–
1.56 (2 H, m, éëç2ëç2); 1.70–1.82 (2 H, m,
ëç2ëç2N+; 4 ç CH2-β; 4 ç CH2-γ); 2.46 (2 H, t, J 7.0,
éëéëç2); 3.34–3.36 (3 H, s, ëçéëç3); 3.28–3.42
(8 H, s, N+ëç3; CH2OCH2CH2; CHOCH3); 3.62–3.74
(2 H, m, CH2CH2N+; 4 ç CH2-α); 4.08–4.11 (1 H, dd, J
5.8, 10.6, ëççaéëé); 4.14–4.21 (1 H, dd, J 3.9, 10.6,
ëççbéëé).
The reactions were monitored by TLC on PTSKh-
AF-V-UF Sorbfil plates (Russia) for compounds (I),
(IIa), and (IIb) and on Kieselgel 60 plates (Merck, Ger-
many) for the rest of the compounds. The compounds
were revealed by sprinkling the plates with 10% phos-
phor-molybdic acid followed by baking.
The optic absorption of the probes in the solutions
when studying their hemolytic activity was measured
on a KFK-3 photocolorimeter.
The K562 and U937 cell lines (human leukemia
cells) and HCT116 (large intestine adenocarcinoma
cells) were cultivated under the previously described
conditions [8, 11].
Chemical Synthesis
rac-N-{5-[(2-methoxy-3-octadecyloxy)propyl]oxy-
butanoyl}-N-methylpiperidinium iodide (IIIb) was
synthesized analogously to derivative (IIIa) starting
from (IIb) (0.124 g, 0.238 mmol) and 1-methylpiperi-
rac-1-Octadecyl-2-methylglycerol (I) was pre-
pared from glycerol according to the standard method
[12].
Chloroanhydrides of 5-bromovaleric and 4-bro- dine (0.2 ml, 1.67 mmol) in a yield of 0.091 g (70%).
mobutiric acids were synthesized by the treatment of Rf 0.45 (chloroform–methanol, 4 : 1). 1H NMR, δ: 0.86
the corresponding acids with an excess (10 equiv) of (3 H, t, J 6.8, ((ëç2)15ëç3); 1.19 (30 H, br. s,
thionyl chloride in dry chloroform (24 h at 20°ë). The (ëç2)15ëç3); 1.52–1.57 (2 H, m, éëç2ëç2); 1.70–
solvent and the excess of thionyl chloride were 2.00 (2 H, m, (ëç2)ëç2N+; 4ç CH2-β; 4ç CH2-γ);
removed in a vacuum. The resulting chloroanhydrides 2.41 (2 H, t, J 7.0, éëéëç2); 3.34–-3.36 (3 H, s,
were used in the following synthesis without additional ëçéëç3); 3.31–3.40 (8 H, s, N+ëç3; CH2OCH2CH2;
purification.
CHOCH3); 3.68–3.76 (2 H, m, CH2CH2N+; 4ç CH2-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

644
ROMANOVA et al.
α); 4.02–4.09 (1 H, dd, J 5.8, 10.6, ëççaéëé); 4.14– with the given dose to that in the control wells (the latter
values were taken as 100%).
4.21 (1 H, dd, J 3.9, 10.6, ëççbéëé).
Hemolytic activity. The control and experimental
probes were incubated for 5 min at 37°C. Each experi-
mental probe contained 0.154 M NaCl (pH 7.2, 5 ml),
the studied compound (20 µl in DMSO), and whole
blood (20 µl). The control probes did not contain the
studied compounds. After the incubation completion,
the probes were centrifuged for 10 min at 600 g. The
optical absorption of the supernatants (D1) was mea-
sured at 540 nm (λmax for hemoglobin) in a cuvette with
a 1-cm optical way. Probes were subjected to total
hemolysis by the addition of 20% aqueous Triton
X-100 (0.1 ml) under thorough stirring. The optical
absorption was measured at the same wave length (D2).
The extent of hemolysis was calculated as the ratio of
the number of dead cells to the total amount of hemo-
lyzed cells and expressed in percents (D1/D2 × 100).
4 rac-N-{4-[(2-methoxy-3-octadecyloxy)propyl]oxy-
pentanoyl}-N-methyl-4-hydroxypiperidinium bro-
mide (IVa). 4-Hydroxymethylpiperidine (0.31 ml,
2.763 mmol) was added to the solution of compound
(IIa) (0.144 g, 0.276 mmol) in anhydrous methylethyl
ketone (2.0 ml). The reaction mixture was stirred for
3.5 h at 60°ë. The solvent was removed in a vacuum
and the residue was chromatographed in a system of
chloroform–methanol, 30 : 1. The yield of (IIIa) was
72.1%. Rf 0.37 (chloroform–methanol, 5 : 1). 1H NMR,
δ: 0.84 (3 H, t, J 6.8, ((ëç2)15ëç3); 1.20 (30 H, br. s,
(ëç2)15ëç3); 1.48–1.54 (2 H, m, éëç2ëç2); 1.70–1.82
(2 H, m,(ëç2)ëç2N+); 2.08–2.26 (4 H, m, CH2-β); 2.46
(2 H, t, J 7.0, éëéëç2); 3.24 (3 H, s, N+ëç3); 3.38–
3.54 (10 H, m, CH2OCH2CH2; CHOCH3; s. ëçéëç3);
3.56–3.78 (2 H, m, CH2CH2N+; 4 ç CH2-α); 4.04–4.12
(1 H, dd, J 5.8, 10.6, ëççaéëé); 4.16–4.26 (1 H, dd,
J 3.9, 10.6, ëççbéëé).
ACNOWLEDGMENTS
The work is supported by grants of Russian Founda-
tion for Basic Research (project no. 07-03-00632-a).
rac-N-{5-[(2-methoxy-3-octadecyloxy)propyl]oxyb-
utanoyl}-N-methyl-4-hydroxypiperidinium bromide
(IVb) was synthesized analogously to derivative (IVa)
starting from (IIb) (0.105 g, 0.211 mmol) and 4-
hydroxymethylpiperidine (0.124 ml, 1.06 mmol) in a
yield of 67.9%. Rf 0.35(chloroform–methanol, 5 : 1).
1H NMR, δ: 0.86 (3 H, t, J 6.8, ((ëç2)15ëç3); 1.22 (30
H, br. s, (ëç2)15ëç3); 1.46–1.56 (2 H, m, éëç2ëç2);
1.72–1.80 (2 H, m, (ëç2)ëç2N+); 2.02–2.24(4 H, m,
CH2-β); 2.48 (2 H, t, J 7.0, éëéëç2); 3.22 (3 H, s,
N+ëç3); 3.38–3.54 (10 H, m, CH2OCH2CH2; CHOCH3;
s. ëçéëç3); 3.54–3.82 (2 H, m, CH2CH2N+; 4 ç CH2-
α); 4.08–4.12 (1 H, dd, J 5.8, 10.6, ëççaéëé); 4.14–
4.20 (1 H, dd, J 3.9, 10.6, ëççbéëé).
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009