Convenient synthesis of polycationic amphiphiles by the Fukuyama reaction

Article abstract of DOI:10.1016/j.mencom.2009.09.005

Cholesterol-containing spermine derivatives for gene delivery were synthesised via the Fukuyama reaction.

Full text of DOI:10.1016/j.mencom.2009.09.005

Mendeleev
Communications
Mendeleev Commun., 2009, 19, 250–252
Convenient synthesis of polycationic
amphiphiles by the Fukuyama reaction
Ivan A. Petukhov, Mikhail A. Maslov,* Nina G. Morozova and Galina A. Serebrennikova
M. V. Lomonosov Moscow State Academy of Fine Chemical Technology, 119571 Moscow, Russian Federation.
Fax: +7 495 936 8901; e-mail: mamaslov@mail.ru
DOI: 10.1016/j.mencom.2009.09.005
Cholesterol-containing spermine derivatives for gene delivery were synthesised via the Fukuyama reaction.
Natural polyamines such as putrescine, spermidine and spermine
play a significant role in biological systems by affecting DNA
replication and translation, membrane stability, and activity of
kinases and topoisomerases.1 The broad activity spectrum of poly-
amines gave impetus for creating various bioactive compounds
on their basis, including synthetic ionophores,2–4 antiviral5 and
antitumor6 agents, endotoxin antagonists7 and DNA condensing
agents.8,9
The ability of natural polyamines to bind DNA molecules
and pack it into small dense particles was used to create trans-
porting agents for delivery of therapeutic nucleic acids (DNA,
RNA and oligonucleotides) into cells in order to provide a
therapeutic effect.8–11 It was found that lipophilic derivatives
of polyamines condense DNA more efficiently than poly-
amines12 and the difference in the numbers of nitrogen atoms
and methylene units between them determines the capability of
polyamines to interact with DNA differently, causes DNA to
change conformation1,13 and affects the efficiency of nucleic
acid transfer.14
Lipopolyamine molecules can be considered as a combina-
tion of two structural units, viz., a hydrophobic substituent
and a polyamine linked by a certain type of chemical bond.
Cholesterol1,13,15,16 and other steroids,17 as well as long-chain
hydrocarbon substituents,18–22 are most commonly used as the
hydrophobic domain. The polyamine component can be of natural
or synthetic origin.1,13–21 The covalent bonding of structural
units is performed directly or via spacers of various length17–19
via a carbamoyl1,13,16 or amide19,20 bond. The binding type deter-
mines the stability of the cationic amphiphile and its toxicity,
which should be taken into account when developing carrier
systems. It is known that alkyl lipids possess high stability and
are hence more toxic than acyl lipids that are readily hydrolyzed
by endogenous esterases in a cell. The carbamoyl group provides
the most reasonable balance between amphiphile stability and
toxicity.10
lipopolyamines, in which cholesterol is bound to the polycation
via spacers of various lengths, a spacer group is first attached to
the polyamine matrix and only then the resulting fragment is
linked to the activated hydrophobic component.7,15,16,19 The
overall yield of the target compounds in such a synthesis varies
from 14 to 25%.7,16 A solid-phase method was proposed for
synthesising cholesterol-containing polycationic amphiphiles
using a 2-chlorotritylchloride resin, which allowed the yield of
lipopolyamines to be increased to 87%.23
Here we present a convenient method for synthesising poly-
cationic amphiphiles based on cell molecular components such
as cholesterol and spermine. We searched for new DNA delivery
agents possessing low cytotoxicity and high activity by varying
the length and type of the spacer between the spermine and
cholesterol. The synthetic approach is based on the Fukuyama
reaction between 2-nitrobenzenesulfonamides, which are
obtained from primary amines, and alkyl halides, followed by
removal of the 2-nitrobenzenesulfonyl group to give a secondary
amine.24,25 To implement this approach, at the first stage, we
obtained bromo derivatives of cholesterol 1a–c† (see Online
Supplementary Materials).
Lipopolyamines were synthesised from compound 2, which
was obtained from spermine by regioselective introduction/
removal of protective groups by a known procedure.19 N-Sul-
fonylation of compound 2 by treatment with 2-nitrobenzene-
sulfonyl chloride in triethylamine gave sulfonamide 3 in 88%
yield.‡ The key step of the synthesis involved condensation of
protected polyamine 3 with brominated derivatives of cholesterol
1a–c in the presence of cesium carbonate. In a typical proce-
dure, cesium carbonate (0.254 mmol) and the corresponding
bromide (1a–c) (0.306 mmol) were successively added to a
solution of compound 3 (0.254 mmol) in dry DMF (3 ml). The
†
1a: mp 112–114 °C. 1b: mp 92–94 °C. 1c: mp 106–108 °C.
3: molecular sieves 4 Å (1.0 g), triethylamine (0.239 ml, 1.716 mmol)
‡
and 2-nitrobenzenesulfonyl chloride (0.228 g, 1.02 mmol) were added to
a cooled (0 °C) solution of compound 2 (0.431 g, 0.858 mmol) in dry
CH2Cl2 (5 ml). The reaction mixture was stirred for 1 h at 24 °C. The
molecular sieves were filtered off and washed with CH2Cl2; the solvent
was removed in vacuo. The residue was chromatographed on a column
with silica gel using a CHCl3–MeOH mixture (50:1) as the eluent.
Compound 3 was obtained (0.507 g, 88%). 1H NMR (Bruker DPX-300,
300 MHz, CDCl3, internal standard SiMe4) d: 1.27 [br. s, 31H, 3CMe3
and NCH2(CH2)2CH2N], 1.52–1.71 (m, 4H, 2NCH2CH2CH2N), 2.92–3.11
(m, 8H, 4NCH2), 3.11–3.30 (m, 4H, 2NHCH2), 7.59–7.71 (m, 2H),
7.71–8.89 (m, 1H) and 8.00–8.11 (m, 1H, C6H4). 13C NMR, d: 25.96,
28.54, 28.59, 28.93, 37.91, 41.03, 43.78, 44.23, 46.82, 47.05, 79.16, 79.74,
80.02, 125.25, 130.93, 132.68, 133.51, 148.29, 156.17. MS (MALDI-
TOF Bruker Ultraflex spectrometer), m/z: 710.341 [M + Na]+; calc. for
C31H53N5NaO10S: 710.3411 [M + Na]+.
Despite considerable interest in the lipophilic derivatives of
polyamines, syntheses of these compounds are often labor-
intensive and involve multiple stages. For example, to obtain
X
(
)
n
Br
O
1a X = CO, n = 3
1b X = NHCO, n = 3
1c X = NHCO, n = 5
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© 2009 Mendeleev Communications. All rights reserved.

Mendeleev Commun., 2009, 19, 250–252
Boc
Boc
Boc
Boc
NO2
N
N
NH
N
N
NH
NH
i
ii
O
S
O
NH2
X
(
)
n
N
O
Boc
Boc
O S O
N
NHBoc
NO2
2
3
N
Boc
Boc
4a–c
a X = CO, n = 3
b X = NHCO, n = 3
c X = NHCO, n = 5
iii
iv
X
X
4 HCl
(
)
( )
n
HN
O
HN
O
n
H
N
NHBoc
N
NH2
N
N
H
Boc
Boc
5a–c
6a–c
Scheme 1 Reagents and conditions: i, 2-NO2C6H4SO2Cl, TEA, 24 °C; ii, bromides 1a–c, Cs2CO3, DMF, 60 °C; iii, PhSH, K2CO3, DMF, 24 °C; iv, 4 N HCl,
dioxane, 24 °C.
reaction mixture was stirred for 1 h at 60 °C, and the precipitate
was filtered off using Celite® 545. Removal of the solvents
in vacuo followed by chromatographic isolation gave com-
pounds 4a–c in 86–94% yields. The resulting compounds were
characterized by mass-spectrometric data, which were shown
structures of the resulting compounds were confirmed by NMR
spectroscopic and mass-spectrometric data.¶
Thus, using the Fukuyama reaction, we synthesised poly-
cationic amphiphiles based on spermine and cholesterol, which
differ in the length of the spacer group (4 or 6 methylene links)
and bonding type (ester or carbamoyl bond).
1
to contain the expected molecular ion signals, and by H and
13C NMR spectroscopic data, in which aggregate signals of the
polyamine and hydrophobic components were observed.§
Unblocking of amino groups in compounds 4a–c started in
early removal of 2-nitrobenzenesulfonyl protecting group on
treatment with thiophenol in the presence of potassium carbonate
to give secondary amines 5a–c in high yields. The final removal
of tert-butoxycarbonyl groups was achieved by treatment of com-
pounds 5a–c with 4 N HCl in dioxane for 2 h. After recrystallisa-
tion from a chloroform–ethanol mixture, lipophilic polyamines
6a–c were obtained in 77, 78 and 99% yields, respectively. The
This work was supported by the Russian Foundation for
Basic Research (grant no. 07-03-00632a) and a Federal goal-
oriented program (contract no. 02.512.11.2200).
1
4c: H NMR, d: 0.61 (s, 3H, 18-Me), 0.79 (d, 3H, 27-Me, J 6.5 Hz),
0.80 (d, 3H, 26-Me, J 6.5 Hz), 0.84 (d, 3H, 21-Me, J 6.5 Hz), 0.94 (s, 3H,
19-Me), 0.90–2.00 [m, 39H, Chol, NHCH2(CH2)2CH2N, 2NCH2CH2CH2N,
CH2(CH2)4CH2], 1.37 (br. s, 18H) and 1.39 (br. s, 9H, 3CMe3), 2.11–2.35
(m, 2H, 4-CH2), 2.92–3.13 (m, 10H, CH2NHCOO, 4NCH2), 3.13–3.33
[m, 6H, CH2NHCOOChol, CH2N(CO2)CH2], 4.40 (tt, 1H, 3-H, J 4.3 Hz,
J 11.3 Hz), 4.50–4.85 (m, 1H, NHCOOChol), 4.96–5.25 (m, 1H, NHBoc),
5.26–5.35 (m, 1H, 6-H), 7.51–7.59 (m, 1H), 7.59–7.70 (m, 2H) and
7.87–8.00 (m, 1H, C6H4). 13C NMR, d: 12.00, 18.87, 19.48, 21.20, 22.70,
23.00, 23.98, 24.43, 25.91, 26.31, 26.37, 27.60, 28.09, 28.14, 28.36,
28.60, 30.30, 32.05, 35.93, 36.34, 36.72, 37.16, 37.81, 38.74, 39.66,
39.90, 40.88, 42.47, 44.13, 44.67, 45.28, 46.82, 47.48, 50.19, 56.30,
56.85, 74.36, 79.65, 122.57, 124.29, 130.87, 131.73, 133.57, 133.68,
140.03, 148.20, 155.59, 156.18, 156.33. MS, m/z: 1221.124 [M + Na]+,
calc. for C65H110N6NaO12S: 1221.7800 [M + Na]+.
§
1
4a: H NMR, d: 0.61 (s, 3H, 18-Me), 0.79 (d, 3H, 27-Me, J 6.5 Hz),
0.80 (d, 3H, 26-Me, J 6.5 Hz), 0.83 (d, 3H, 21-Me, J 6.5 Hz), 0.94
(s, 3H, 19-Me), 0.90–2.00 [m, 38H, Chol protons, 2CH2(CH2)2CH2,
2NCH2CH2CH2N], 1.36 (br. s, 18H) and 1.39 (br. s, 9H, 3CMe3), 2.11–
2.33 (m, 4H, 4-CH2, CH2COO), 2.95–3.12 (m, 8H, 4NCH2), 3.12–3.30
[m, 6H, CH2NH, CH2N(CO2)CH2], 4.51 (tt, 1H, 3-H, J 4.2 Hz, J 11.2 Hz),
5.01–5.26 (m, 1H, NHCO), 5.26–5.34 (m, 1H, 6-H), 7.50–7.59 (m, 1H),
7.59–7.70 (m, 2H) and 7.87–8.00 (m, 1H, C6H4). 13C NMR, d: 11.94,
18.80, 19.39, 21.11, 22.03, 22.64, 22.89, 23.90, 24.36, 25.61, 25.79, 27.87,
28.07, 28.29, 28.53, 31.94, 31.98, 34.00, 35.86, 36.26, 36.68, 37.07,
37.67, 38.22, 39.60, 39.82, 42.40, 43.94, 44.54, 45.19, 46.77, 47.07, 50.12,
56.23, 56.78, 74.02, 79.58, 122.73, 124.24, 130.80, 131.73, 133.50, 133.58,
139.71, 148.13, 155.50, 156.10, 172.51. MS, m/z: 1178.741 [M + Na]+,
calc. for C63H105N5NaO12S: 1178.7378 [M + Na]+.
¶
Compounds 6a–c decompose above 300 °C.
6a: 1H NMR (D2O) d: 0.61 (s, 3H, 18-Me), 0.77 (d, 3H, 27-Me, J 6.5 Hz),
0.79 (d, 3H, 26-Me, J 6.5 Hz), 0.84 (d, 3H, 21-Me, J 6.5 Hz), 0.94 (s,
3H, 19-Me), 0.90–2.00 [m, 42H, Chol protons, NHCH2(CH2)2CH2N,
2NCH2CH2CH2N, CH2(CH2)2CH2], 2.11–2.32 (m, 2H, 4-CH2), 2.90–
3.15 (m, 16H, CH2COO, 7CH2N), 4.20–4.40 (m, 1H, 3-H), 5.20–5.35
(m, 1H, 6-H). MS (CI, 1100 LC/MS Agilent Technologies spectrometer),
m/z: 671.5 [M – 4HCl + H]+, calc. for C42H78N4O2: 670.6125 [M – 4HCl]+.
6b: 1H NMR (D2O) d: 0.61 (s, 3H, 18-Me), 0.77 (d, 3H, 27-Me, J 6.5 Hz),
0.79 (d, 3H, 26-Me, J 6.5 Hz), 0.84 (d, 3H, 21-Me, J 6.5 Hz), 0.94
(s, 3H, 19-Me), 0.90–2.00 [m, 42H, Chol protons, NHCH2(CH2)2CH2N,
2NCH2CH2CH2N, CH2(CH2)2CH2], 2.11–2.32 (m, 2H, 4-CH2), 2.90–3.15
(m, 16H, 8CH2N), 4.15–4.40 (m, 1H, 3-H), 5.18–5.38 (m, 1H, 6-H). MS,
m/z: 686.6 [H – 4HCl + H]+, calc. for C42H79N5O2: 685.6234 [M – 4HCl]+.
6c: 1H NMR (D2O) d: 0.61 (s, 3H, 18-Me), 0.77 (d, 3H, 27-Me, J 6.5 Hz),
0.79 (d, 3H, 26-Me, J 6.5 Hz), 0.84 (d, 3H, 21-Me, J 6.5 Hz), 0.94 (s,
3H, 19-Me), 0.90–2.05 [m, 46H, Chol protons, NHCH2(CH2)2CH2N,
2NCH2CH2CH2N, CH2(CH2)4CH2], 2.13–2.33 (m, 2H, 4-CH2), 2.91–3.15
(m, 16H, 8CH2N), 4.15–4.40 (m, 1H, 3-H), 5.18–5.38 (m, 1H, 6-H). MS,
m/z: 714.6 [M – 4HCl + H]+, calc. for C44H83N5O2: 713.6547 [M – 4HCl]+.
1
4b: H NMR, d: 0.61 (s, 3H, 18-Me), 0.79 (d, 3H, 27-Me, J 6.5 Hz),
0.80 (d, 3H, 26-Me, J 6.5 Hz), 0.84 (d, 3H, 21-Me, J 6.5 Hz), 0.94 (s, 3H,
19-Me), 0.90–2.00 [m, 38H, Chol, 2CH2(CH2)2CH2, 2NCH2CH2CH2N],
1.36 (br. s, 18H) and 1.39 (br. s, 9H, 3CMe3), 2.11–2.33 (m, 2H,
4-CH2), 2.91–3.12 (m, 10H, CH2NHBoc, 4NCH2), 3.12–3.30 [m,
6H, CH2NHCOOChol, CH2N(CO2)CH2], 4.40 (tt, 1H, 3-H, J 4.2 Hz,
J 11.3 Hz), 4.55–4.96 (m, 1H, NHCOOChol), 4.96–5.25 (m, 1H, NHBoc),
5.25–5.33 (m, 1H, 6-H), 7.50–7.59 (m, 1H), 7.59–7.70 (m, 2H) and
7.87–8.00 (m, 1H, C6H4). 13C NMR, d: 11.97, 18.84, 19.44, 21.17, 22.67,
22.92, 23.95, 24.40, 25.61, 26.01, 27.20, 27.57, 28.11, 28.30, 28.58,
32.01, 35.90, 36.31, 36.54, 36.69, 37.13, 37.80, 38.70, 39.63, 39.87, 40.36,
42.44, 44.16, 44.70, 45.57, 46.70, 50.15, 56.27, 56.82, 74.36, 79.66, 122.56,
124.27, 130.83, 131.77, 133.48, 133.61, 139.98, 148.18, 155.56, 156.16,
156.35. MS, m/z: 1193.762 [M + Na]+, calc. for C63H106N6NaO12S:
1193.7487 [M + Na]+.
– 251 –

Mendeleev Commun., 2009, 19, 250–252
13 A. J. Geall, M. A. W. Eaton, T. Baker, C. Catterall and I. S. Blagbrough,
Online Supplementary Materials
FEBS Lett., 1999, 459, 337.
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.mencom.2009.09.005.
14 M. Keller, M. R. Jorgensen, E. Perouzel and A. D. Miller, Biochemistry,
2003, 42, 6067.
15 I. S. Blagbrough, A. J. Geall and A. P. Neal, Biochem. Soc. Trans., 2003,
31, 397.
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Received: 17th March 2009; Com. 09/3298
– 252 –