Synthesis of New Guanidine-Containing Amphiphiles and Their Pyrene Analog for Liposomal Delivery Systems and Visualization in Target Cells

Article abstract of DOI:10.1134/S1070428019120030

New cationic amphiphiles with a guanidine head group have been synthesized starting from lipoamino acids with the goal of creating drug and genetic material delivery systems. Their analog containing a pyrene fragment has also been obtained as a potential agent for visualization of liposome transport in various cells.

Full text of DOI:10.1134/S1070428019120030

ISSN 1070-4280, Russian Journal of Organic Chemistry, 2019, Vol. 55, No. 12, pp. 1826–1831. © Pleiades Publishing, Ltd., 2019.
Russian Text © The Author(s), 2019, published in Zhurnal Organicheskoi Khimii, 2019, Vol. 55, No. 12, pp. 1827–1833.
Synthesis of New Guanidine-Containing Amphiphiles
and Their Pyrene Analog for Liposomal Delivery Systems
and Visualization in Target Cells
A. A. Losevaa, U. A. Budanovaa,* and Yu. L. Sebyakina
a Lomonosov Moscow Institute of Fine Chemical Technology,
MIREA—Russian Technological University, Moscow, 119454 Russia
*e-mail: c-221@yandex.ru
Received April 22, 2019; revised September 17, 2019; accepted September 20, 2019
Abstract—New cationic amphiphiles with a guanidine head group have been synthesized starting from
lipoamino acids with the goal of creating drug and genetic material delivery systems. Their analog containing
a pyrene fragment has also been obtained as a potential agent for visualization of liposome transport in
various cells.
Keywords: cationic amphiphiles, guanidines, diamines, pyrene, drug delivery systems.
DOI: 10.1134/S1070428019120030
Cationic biocompatible non-viral vectors are
widely used as delivery systems for drugs and
genetic material; among these, cationic liposomes
are considered safest [1]. The transfection efficiency
and toxicity of cationic lipids are largely determined
by their structure. Multivalent cationic amphiphiles
possess a higher liposome surface charge and are more
efficient in genetic material delivery to a cell nucleus
than their singly charged analogs [2]. The use of natural
polyamines such as spermidine and spermine enables
interaction with DNA phosphate groups to form strong
complexes with the transferred material [3]. Cationic
peptide vectors are also extensively studied [4]; they
are more advantageous than other non-viral systems
due to their low toxicity and the ability to tightly pack
and protect genetic material, as well as to recognize
target-specific receptors on the cell membrane and
deliver the package to the cell [5–8].
Scheme 1.
S
S
NBoc
Boc2O
HOCH2CH2NH2
Boc
Boc
Boc
OH
H2N
NH2
N
N
N
N
H
H
H
H
1
2
O
O
O
O
H
H
H
H2N
N
BocN
N
N
OC16H33
OC16H33
OC16H33
OC16H33
O
OC16H33
OC16H33
2, DCC
O
O
NH
HO
O
Boc
O
O
O
3
4
O
O
H
N
H
HN
N
O
OC16H33
OC16H33
CF3COOH
O
NH3
CF3COO–
O
5
1826

SYNTHESIS OF NEW GUANIDINE AMPHIPHILES
1827
Scheme 2.
O
O
Boc
N
H2N
H
OC16H33
O
OH
O
N
ClCH2COOH
Boc2O
N
O
HO
OC16H33
Boc
OH
NH2
OH
N
H
O
OC16H33
OC16H33
6
7
H
N
Boc
N
H
O
DCC, DMAP
O
O
O
6
+
7
H
Boc
N
N
O
N
O
OC16H33
H
OC16H33
8
H
N
H3N
2CF3COO–
O
CF3COOH
O
O
O
H
N
N
O
H3N
O
OC16H33
OC16H33
9
Boc
NH
H
N
Boc
N
N
O
1
H
Boc
NH
O
O
O
H
N
Boc
N
N
N
O
OC16H33
H
O
OC16H33
10
NH3
H
N
2CF3COO–
NH3
HN
N
O
H
CF3COOH
O
O
O
H
N
N
O
HN
N
O
OC16H33
H
OC16H33
11
Sen and Chaudhuri [9] described the properties of
guanidine-based cationic amphiphiles with hydro-
carbon fragments [9]. Taking into account the ability of
a guanidinium group to bind to biological counterions
and involve them in transport through plasma mem-
branes, the design of guanidinium-containing systems
for delivery of biologically active compounds to cells is
a challenging problem [10].
fluorophores such as pyrene derivatives. Liposomes
formed by amphiphiles containing a pyrenylmethanol
fragment as a hydrophobic moiety and mono-, di-,
or polyamine head groups showed a strong ability to
inhibit tumor cell proliferation and the possibility of
simultaneously visualizing their penetration into target
cells [11].
Therefore, search for drug delivery agents among
synthetic cationic amphiphiles with guanidine-based
head groups and their fluorescent analogs is important
In order to visualize the delivery process mediated
by guanidinium-based carriers, it is reasonable to use
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LOSEVA et al..
1828
Scheme 3.
NBoc
NH
Boc
NH2
NH3
N
H
H
H
1
SOCl2, EtOH
H2N
OH
H3N
OEt
N
N
OEt
Boc
2Cl–
12
O
O
NBoc
O
13
NBoc
Boc
KOH
N
NH
H
H
H
N
N
OH
Boc
OH
NBoc
O
14
O
O
H
H
DCC, HOBt
Boc
N
Boc
N
+
N
OH
N
H
O
H
O
O
15
16
CF3COO–
H3N
O
CF3COOH
H
N
O
O
17
NBoc
Boc
N
NH
O
14
H
H
H
H
N
N
N
Boc
O
NBoc
O
18
NH
2CF3COO–
H
H3N
NH
O
CF3COOH
H
N
H3N
N
O
NH
O
19
for a deeper insight into their biodistribution processes
and intracellular localization, as well as for the design
of most efficient delivery systems.
The goal of the present work was to synthesize new
cationic amphiphiles 5 and 11 with polar guanidine
head groups, as well as a pyrene-containing guanidine
tripeptide (OrnGlyGly) 19, as subjects for further study
of their intracellular localization and functional activity
of polar head groups in target cells.
[12] to 2-aminoethanol in the presence of mercury
chloride as catalyst [13] gave protected guanidine 2.
L-Glutamic acid dihexadecyl ester was modified with
a succinic acid residue [14], and the resulting succin-
amic acid derivative 3 was esterified with alcohol 2
in the presence of N,N′-dicyclohexylcarbodiimide
(DCC) and N-hydroxysuccinimide (NHS) as carboxyl
group activators. The subsequent deprotection of 4
by treatment with trifluoroacetic acid afforded target
compound 5 in 60% yield. The structure of 5 was
confirmed by 1H NMR and mass spectra.
The synthesis of compound 5 is outlined in
Scheme 1. The addition of Boc-protected thiourea 1
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 55 No. 12 2019

SYNTHESIS OF NEW GUANIDINE AMPHIPHILES
1829
Scheme 2 illustrates the synthesis of amphiphile 11.
The hydrophobic moiety of 11 was built up from
L-glutamic acid dihexadecyl ester which was treated
with chloroacetic acid to obtain dicarboxylic acid 6.
Reaction of the latter with Boc-protected N-(2-amino-
ethyl)-2-aminoethanol 7 [15], followed by deprotection,
guanidination with thiourea 1, and final deprotection,
produced compound 11. The MALDI mass spectrum of
11 contained the molecular ion peak with m/z 969.794
[M]+. Molecule 11 features increased number of
cationic groups due to branching in the head group and
the presence of ethylenediamine fragments.
and trifluoroacetic acid (TFA, Biochem) were used
without further purification.
1
The H NMR spectra were recorded on a Bruker
WM-400 spectrometer (Germany) at 400 MHz using
chloroform-d as solvent and hexamethyldisiloxane as
internal standard. The IR spectra were measured on a
Bruker Equinox 55 spectrometer (Germany) with
Fourier transform. The mass spectra (MALDI) were
obtained with a Finnigan MAT Vision 2000 time-of-
flight mass spectrometer using 2,5-dihydroxybenzoic
acid as matrix. Elemental analysis was performed
using a Thermo Finnigan Flash EA 1112 Series CHNS
analyzer. Sorbfil plates (Krasnodar, Russia) were used
for thin-layer chromatography with the following
solvent systems as eluents: chloroform–methanol, 9:1
(A), 20:1 (B); toluene–ethyl acetate, 5:1 (C); toluene–
chloroform–butan-2-one–propan-2-ol, 10:6:3:1 (D).
Spots were visualized by heating in open flame (spirit
lamp); compounds with carbon–carbon multiple bonds
were detected by treatment with a 10% solution of
potassium permanganate, and those with free amino
groups, with a 5% ninhidrin solution, followed by
heating to 50–80°C. Column chromatography was
performed on silica gel (0.060–0.200 mm, 60 Å; Acros
Organics, Belgium).
Pyrene-containing fluorophore 19 was synthesized
by a block method (Scheme 3). Bis-guanidine L-orni-
thine derivative 14 was obtained by reaction of
L-ornithine ethyl ester dihydrochloride (12) with
Boc-protected thiourea 1 and subsequent alkaline
hydrolysis of the ester group. The IR spectrum of 14
showed absorption bands typical of a free carboxy
group. Intermediate block 17 was synthesized by ad-
dition of pyren-1-ylmethanol to commercially avail-
able Boc-Gly-Gly (15) in the presence of DCC
and 1-hydroxybenzotriazole (HOBt), followed by
1
deprotection. The H NMR spectrum of 17 contain no
signal assignable to tert-butoxycarbonyl group, but
signals of the CH2 groups of the Gly-Gly fragment
[δ 3.45 and 3.90 ppm, s (2H each)] and pyrene residue
(δ 7.75–8.50 ppm) were present. The amidation of
carboxylic acid 14 with amino acid 17 in the presence
of DCC, NHS, and 4-dimethylaminopyridine (DMAP)
and removal of the Boc protecting groups from 18
afforded conjugate 19. The structures of 14, 17, and 19
were confirmed by 1H NMR, IR, and mass spectra. The
MALDI mass spectrum of 19 displayed the molecular
ion peak with m/z 546.285 [M]+.
N,N″-Bis(tert-butoxycarbonyl)-N′-(2-hydroxy-
ethyl)guanidine (2). Thiourea 1, 0.50 g (1.81 mmol),
was added to a solution of 0.11 g (1.81 mmol) of
2-aminoethanol in THF, and the mixture was stirred for
10 h at room temperature. The product was purified by
recrystallization from methanol. Yield 0.05 g (86%),
1
Rf 0.90 (D). H NMR spectrum, δ, ppm: 1.42 s (18H,
t-Bu), 3.54–3.62 m (2H, CH2), 3.78 t (2H, CH2),
4.36 t (1H, OH), 5.40–5.48 m (1H, CH), 8.70 d (1H,
NH), 11.41 d (1H, NH).
Thus, we have proposed synthetic approaches to
new guanidine-based cationic amphiphiles and their
pyrene-containing analog starting from lipoamino acids
and diamines. The synthesized compounds are planned
to be used as drug and genetic material delivery agents,
as well as for visualization of intracellular localization
of cationic liposomes and lipoplexes derived therefrom.
N′-{2-[(4-{[1,5-Bis(hexadecyloxy)-1,5-dioxo-
pentan-2-yl]amino}-4-oxobutanoyl)oxy]ethyl}gua-
nidinium trifluoroacetate (5). Compound 3 [14],
0.07 g (0.10 mmol), was dissolved in methylene
chloride, 30 mg (0.15 mmol) of DCC and 16 mg
(0.11 mmol) of NHS were added, and the mixture was
stirred for 1 h at 0°C. The precipitate was filtered off,
0.03 g (0.20 mmol) of compound 2 was added to the
filtrate, and the mixture was stirred for 24 h at room
temperature. The product was isolated by preparative
thin-layer chromatography on silica gel using solvent
system C as eluent and was deprotected by treatment
with trifluoroacetic acid in methylene chloride for
3 h. The solvent and volatile compounds were removed
under reduced pressure. Yield 14.00 mg (60%), amor-
EXPERIMENTAL
Commercially available L-ornithine (Acros
Organics), 2-chloroacetic acid (Sigma Aldrich),
L-aspartic acid (L-Asp, Sigma Aldrich), thiourea
(Sigma Aldrich), 2-aminoethanol (Acros Organics),
di-tert-butyl dicarbonate (Boc2O, Sigma Aldrich),
N,N′-dicyclohexylcarbodiimide (DCC, Sigma Aldrich),
1
phous material, Rf 0.20 (B). H NMR spectrum, δ,
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 55 No. 12 2019

LOSEVA et al..
1830
ppm: 0.90 t (6H, CH3), 1.30 s [52H, (CH2)13], 1.54–
1.70 m (4H, β-CH2), 1.98–2.40 m (2H, β-CH2, Glu),
2.50 t (2H, δ-CH2, Glu), 2.55–2.65 m (4H, CH2, Suc),
2.80 t (2H, NHCH2CH2), 3.44–3.56 m (4H, OCH2),
3.92–4.18 m (4H, α-CH2), 4.48–4.52 m (1H, α-H, Glu),
6.44 s (3H, NH). Found, %: C 67.58; H 10.79; N 7.21.
C44H84N4O7. Calculated, %: C 67.65; H 10.84; N 7.17.
as eluent. The product was dissolved in 20 mL of
methanol, and potassium hydroxide was added at
room temperature until pH 12. The mixture was stirred
for 3 h at room temperature, KU-9 (H+) exchanger
was added, and the mixture was stirred for 3 h more
and filtered. Yield of 14 0.210 g (78%), Rf 0.25 (A).
IR spectrum (film), ν, cm–1: 3353 (OH), 3103 (NH),
2929 (CH3), 1740 (C=O), 1251 (C–O–C), 1171 (C–N).
1H NMR spectrum, δ, ppm: 1.50 s (36H, t-Bu), 1.62–
1.90 m (4H, β,γ-CH2), 3.26–3.56 m (2H, δ-CH2), 3.80 s
(1H, CH), 7.80 q (2H, NH).
9-[1,5-Bis(hexadecyloxy)-1,5-dioxopentan-2-yl]-
7,11-dioxo-6,12-dioxa-3,9,15-triazaheptadecane-
1,17-diaminium bis(trifluoroacetate) (9). Diacid 6,
0.50 g (0.70 mmol), was dissolved in methylene
chloride, 0.18 g (0.90 mmol) of DCC and 12 mg
(0.10 mmol) of DMAP were added, and the mixture was
stirred for 1 h at 0°C. The precipitate was filtered off,
0.85 g (2.81 mmol) of 7 was added to the filtrate, and
the mixture was stirred for 24 h at room temperature.
The product was isolated by column chromatography
and was deprotected by treatment with trifluoroacetic
acid in methylene chloride for 3 h. The solvent was
removed under reduced pressure. Yield 0.15 g (31%),
2-Oxo-2-{[2-oxo-2-(pyren-1-ylmethoxy)ethyl]-
amino}ethan-1-aminium trifluoroacetate (17).
Compound 15, 0.15 g (0.60 mmol), was dissolved in
anhydrous methylene chloride, 0.23 g (1.14 mmol) of
DCC and 0.135 g (1.14 mmol) of HOBt in DMF were
added, and the mixture was stirred for 2 h at 0°C. The
precipitate of dicyclohexylurea was filtered off, 0.14 g
(0.60 mmol) of pyren-1-ylmethanol was added to the
filtrate, and the mixture was stirred for 24 h at room
temperature. The product was purified by column
chromatography using solvent system D as eluent.
Intermediate 16 thus obtained was dissolved in 50 mL
of chloroform, a solution of 0.20 g (1.68 mmol) of
50% trifluoroacetic acid in chloroform was added drop-
wise, and the mixture was stirred for 1 h on a magnetic
stirrer until a white solid separated. The solvent and
excess trifluoroacetic acid were removed under reduced
1
Rf 0.15 (C). H NMR spectrum, δ, ppm: 0.81 t (6H,
CH3), 1.21 s [52H, (CH2)13], 1.47–1.64 m (4H, β-CH2),
1.68 s (4H, NCH2CO), 1.79–2.01 m (2H, β-CH2, Glu),
1.96 t (4H, NH2), 2.03–2.40 m (2H, δ-CH2, Glu),
2.46 t (1H, CH, Glu), 3.20–3.44 m (2H, α-CH2),
3.56–3.74 m (4H, CH2NH), 4.02 t (4H, CH2O), 4.20–
4.30 m (8H, NHCH2CH2NH3), 4.31 t (4H, CH2NH),
4.84–5.36 m (2H, NH).
1
pressure. Yield 0.13 g (48%), Rf 0.50 (A). H NMR
N′1,N′2-{9-[1,5-Bis(hexadecyloxy)-1,5-dioxo-
pentan-2-yl]-7,11-dioxo-6,12-dioxa-3,9,15-triaza-
heptadecane-1,17-diyl}diguanidium bis(trifluoro-
acetate) (11). Compound 9, 0.10 g (0.11 mmol),
was dissolved in THF, 0.063 g (0.22 mmol) of 1,
0.074 g (0.27 mmol) of HgCl2, and a catalytic amount
of triethylamine were added, and the mixture was
stirred for 10 h at room temperature. The product was
isolated by recrystallization from methanol. Protecting
groups were removed by treatment with trifluoroacetic
acid in methylene chloride for 3 h. Yield 0.14 g (56%),
amorphous powder, Rf 0.70 (A). Mass spectrum:
m/z 969.784 [M]+. Found, %: C 63.19; H 10.79;
N 12.87. C44H84N4O7. Calculated, %: C 63.12; H 10.70;
N 12.99. M 969.793.
spectrum, δ, ppm: 3.45 s (2H, CH2), 3.90 s (2H, CH2),
4.91 s (2H, CH2O), 5.22–5.58 m (2H, NH2), 7.75–
8.50 m (9H, Harom).
N′1,N′2-((4S)-5-Oxo-5-{[2-oxo-2-(pyren-1-ylme-
thoxy)ethyl]amino}pentane-1,4-diyl)diguanidinium
bis(trifluoroacetate) (19). Compound 14, 0.21 g
(0.34 mmol), was dissolved in THF, 0.14 g
(0.68 mmol) of DCC, 78 mg (0.68 mmol) of NHS,
and 83 mg (0.68 mmol) of DMAP were added, and
the mixture was stirred for 24 h at 0°C. The precipitate
of dicyclohexylurea was filtered off, a solution of
0.13 g (0.34 mmol) of 17 in 100 mL of THF was added
to the filtrate, and the mixture was stirred for 48 h at
25°C. Intermediate product 18 was isolated by column
chromatography using solvent system D as eluent.
It was dissolved in 50 mL of chloroform, a solution
of 0.20 g (1.68 mmol) of 30% trifluoroacetic acid in
chloroform was added dropwise, and the mixture was
stirred for 1 h on a magnetic stirrer until a white solid
separated. The solvent was removed under reduced
pressure. Yield 0.05 g (55%), white powder, Rf 0.51
(2S)-2,5-Bis{[N,N′-bis(tert-butoxycarbonyl)car-
bamimidoyl]amino}pentanoic acid (14). A solution
of 1.00 g (3.60 mmol) of 1 in 3 mL of DMF was added
at room temperature to a solution of 0.3 g (1.78 mmol)
of ester 12 in 3 mL of DMF. The solvent was removed
under reduced pressure, and compound 13 was isolated
by column chromatography using solvent system D
1
(A). H NMR spectrum, δ, ppm: 1.70–2.00 m (4H,
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SYNTHESIS OF NEW GUANIDINE AMPHIPHILES
1831
kov, A.M., Kovalenko, E.I., Markvicheva, E.A., and
Khaitov, M.R., Colloids Surf., B, 2018, vol. 167, p. 328.
https://doi.org/10.1016/j.colsurfb.2018.04.003
β-CH2, γ-CH2, Orn), 3.37–3.43 m (2H, δ-CH2, Orn),
3.42–3.48 m (2H, CH2), 3.50–3.70 m (2H, CH2), 4.30 s
(1H, CH), 5.48 s (2H, CH2O), 7.75–8.50 m (9H, Harom).
Mass spectrum: m/z 546.285 [M]+. Found, %: C 61.59;
H 6.31; N 20.77. C28H34N8O4. Calculated, %: C 61.52;
H 6.27; N 20.50. M 546.270.
7. Koloskova, O.O., Nikonova, A.A., Budanova, U.A.,
Shilovskiy, I.P., Kofiadi, I.A., Ivanov, A.V.,
Smirnova, O.A., Zverev, V.V., Sebyakin, Yu.L.,
Andreev, S.M., and Khaitov, M.R., Eur. J. Pharm.
Biopharm., 2016, vol. 102, p. 159.
FUNDING
https://doi.org/10.1016/j.ejpb.2016.03.014
This study was performed under financial support by the
Russian Foundation for Basic Research (project no. 17-04-
01141-a).
8. Zhang, X.-X., La Manna, C.M., Kohman, R.E.,
McIntosh, T.J., Han, X., and Grinstaff, M.W., Soft
Matter, 2013, vol. 9, p. 4472.
https://doi.org/10.1039/C3SM27633C
CONFLICT OF INTERESTS
The authors declare the absence of conflict of interests.
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RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 55 No. 12 2019