A single thiourea group is not enough to get stable thiourea lipoplexes

Article abstract of DOI:10.1016/j.bmc.2008.01.029

The preparation of a new family of lipothiourea is reported using an automatic synthetic workstation. In these compounds the headgroups were made from single thiourea derivatives. The physicochemical properties and the transfection efficiency of several members of the family were studied. It was found that in the presence of DMPC small lipoplexes could be prepared. In opposite to the previously described di- and tri-lipothioureas most of these liposomes are unstable overtime. In addition, even the stable ones show no transfecting efficiency. All these data demonstrate that at least two thiourea groups are necessary to produce stable lipoplexes, to condense DNA and to give efficient transfection.

Full text of DOI:10.1016/j.bmc.2008.01.029

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4003–4008
A single thiourea group is not enough
to get stable thiourea lipoplexes
Eihab Kabha, Claire Jacquement, Gae¨lle Pembouong, Nathalie Mignet,
Daniel Scherman and Jean Herscovici*
Inserm, U640, Paris F-75006, France
CNRS, UMR8151, Paris F-75006, France
Ecole Nationale Supe´rieure de Chimie de Paris, Unite´ de Pharmacologie Chimique et Ge´ne´tique, Paris F-75005, France
Universite´ Paris Descartes, Faculte´ de Pharmacie Paris, F-75270, France
Institut de Chimie Mole´culaire de Paris Centre, CNRS FR 2769, F-75005, France
Received 18 September 2007; revised 8 January 2008; accepted 16 January 2008
Available online 19 January 2008
Abstract—The preparation of a new family of lipothiourea is reported using an automatic synthetic workstation. In these com-
pounds the headgroups were made from single thiourea derivatives. The physicochemical properties and the transfection efficiency
of several members of the family were studied. It was found that in the presence of DMPC small lipoplexes could be prepared. In
opposite to the previously described di- and tri-lipothioureas most of these liposomes are unstable overtime. In addition, even the
stable ones show no transfecting efficiency. All these data demonstrate that at least two thiourea groups are necessary to produce
stable lipoplexes, to condense DNA and to give efficient transfection.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
solve this problem is the use of neutral derivatives.14
However, with the exception of glycocluster, neutral lip-
osomes has no or very low-transfecting efficiency.15
Anion receptors have been extensively studied using
charged species or neutral hydrogen bond donor.1,2
Among neutral receptors thioureas have attracted a spe-
cial interest because of the acidic nature of the NH pro-
tons that led to strong hydrogen bond.3 On the other
hand phosphate anions4 show the strongest affinity
among carboxylate and inorganic anions for thioureas.5
Withthesedatainmindweproposedtoreplacethecationic
head of the synthetic vector by a thiourea one (Fig. 1). Our
previous studies indicated that compounds bearing two to
four thiourea groups were able to condense DNA16,17 and
to deliver it to the cell.18 Recently we showed that T-shape
lipodithioureas transfect cells with the same efficiency as
cationic derivatives used asreferencewhenthe experiments
were performed in the presence of serum.
Chemical vectors6–8 are with viruses9,10 and physical
methods,11,12 the three main processes for gene transfer.
During the past year a great effort was made toward the
preparation of synthetic vector of nucleic acids. Due to
the polyanionic nature of nucleic acid and of plasmic
membrane most of them are derived from cationic lipid
or cationic polymer. The cationic nature of the DNA li-
pid or DNA polymer complexes is one of the main prob-
lems of their poor in vivo efficiency. These complexes
aggregate with blood protein, which in turn, leads to
their elimination.13 One of the solutions proposed to re-
These results raised the question of the number of thio-
urea group necessary for a stable interaction between
non cationic lipids and nucleic acids. In this report we de-
scribe the synthesis of a small library of lipomonothiourea
and the evaluation of their transfecting properties.
2. Results and discussion
2.1. Synthesis
Keywords: Liposomes; Thiourea; DNA condensation; Lipoplexes.
*
The synthesis was carried out in four steps (Scheme 1).
First, didecylamine was reacted with Boc-protected
Corresponding author. Tel.: +33 1 44 27 67 48; fax: +33 1 53 10 12
95; e-mail: jean-herscovici@enscp.fr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.029

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E. Kabha et al. / Bioorg. Med. Chem. 16 (2008) 4003–4008
O
R
S
S
O
HN
H
N
H
N
H
N
N
n
NH
S
n
N
H
S
N
H
N
H
N
N
HN
5
H
N
H
n
N
HN
O
S
O
X
NH
S
S
n
5
O
R
HN
O
O
N
H
N
O
H
O
Figure 1. Lipopolythioureas.
glycine in the presence of DCC and N-hydroxy-
succinimide to give 2 in 50–60% yield. After removal
of the tert-butoxy carbamate, using trifluoroacetic acid,
the resulting amine 3 was treated with carbon disulfide
in the presence of p-TSCl, DIPEA to give the lipoisoth-
iocyanate 4 in 89% yield. Finally, The scaffold 4 was
treated with different amines onto a Chemspeed ASW
2000 synthetic workstation and the lipomonothiourea li-
brary was obtained in 50–60% yield.
2.2. Physico-chemical studies
Different attempts were performed to suspend the lipids
in aqueous media. They were formulated via an etha-
nolic injection method19 in the presence of DPPC as a
ratio 1/1 or as a lipidic film with DMPC as a ratio
DMPC/LMTU 0.65:0.45. Both ways allowed the forma-
tion of nanometer range particles with the characteris-
tics indicated below (Table 1). Compounds 5 and 6a
could also be formulated but the formulations were
poorly stable and we did not pursue our efforts on these
two compounds. The size of the particles formed was in
the same range 100–130 nm and zeta potential close to
zero. Aggregation of these formulations after few days
of storage at 4 ꢁC could be expected considering the
logP of these compounds (5 6.26, 6a 6.83).18
NH
1
i
O
H
N
O
N
O
2
ii
O
NH2
N
3
iii
iv
v
S
O
Compound 6b did not show a very low electrophoretic
mobility as expected, giving a zeta potential of
6.5 1.5 mV. Zeta potential of compound 6c was
2.5 2 at pH 7 and 35 0.5 mV at pH 5.5 relating to
the protonation of the imidazole ring.
C
N
N
O
4
H
H
N
N
LMTU/DNA interaction was evaluated by dynamic
light scattering experiments. A fixed amount of DNA
was added to an increasing amount of lipid to form
the complexes (Table 2). The lipid/DNA ratio is given
as the amount of thiourea function per phosphate. A
size increase occurred below a ratio of 1 TU/PO leading
to an aggregation, confirming the high interaction effi-
ciency that we previously observed between the thiourea
moiety and the phosphates.16 We cannot exclude that
the thiourea function not only reacts on DNA phos-
phate but also on the phosphate functions of phospho-
lipids. However, we have proven on the trithiourea
lipid that removing the colipid did not change the lipop-
N
S
5
O
H
N
R
N
S
6
OH
O
H2N
H2N
OH
6b (55 %)
NH2
N
N
O
6c (33%)
6a (57 %)
HO
OH
H2N
OH
NH2
NH2
6d (50%)
H2N
OH
6g (54%)
6e (54 %)
6f (100%)
O
H2N
Table 1. Characteristics of the particles LMTU/DMPC obtained via
the film hydration technique measured by dynamic light scattering
experiments
H2N
OH
6i (50%)
OH
6h (50%)
H2N
6j (60%)
H2N
Size (nm)
PDI
Zeta potential (mV)
OH
6k (60%)
6b
6c
99.3
130 (pH 5.5)
0.02
0.07
6.5 1.5
35 0.5
2.5 2 (pH 7)
Scheme 1. Preparation of the Lipomonothiourea. Reagents and
conditions: (i) N-hydroxysuccinimide, DCC, THF, 60%; (ii) trifluoro-
acetic acid, rt, 70% (iii) carbon disulfide, p-TSCl, DIPEA, CH2Cl2,
89%; (iv) amine, DMF, 50–60%.
PDI is the polydispersity index; zeta potential (mV) was measured in
20 M NaCl, pH 5.5.

E. Kabha et al. / Bioorg. Med. Chem. 16 (2008) 4003–4008
4005
Table 2. Size of the lipomonothiourea/DNA complexes in PBS as
measured by dynamic light-scattering
All chemicals were purchased from Sigma–Aldrich–Flu-
ka. Other solvents and products were used without fur-
ther purification. Reactions were monitored by thin-
layer chromatography using Merck precoated 60 F254
silica gel plates. Column chromatography was per-
formed over SDS (Peypin France) 35–70 m silica gel
according to the method of Still, Khan, and Mitra or
by using small column flash chromatography (SFC)
according to the following procedure. A plastic syringe
was filled with silica gel (product/silica gel 1:5) and con-
nected to a vacuum pump. The column was equilibrated
with heptane then the sample dissolved in minimum of
dichloromethane was added to the top. The column
was eluted by ten fractions of a heptane/ethyl acetate
mixture. The volume was equal to the silica gel volume.
For each fraction, the amount of ethyl acetate was in-
creased 10% (v/v) to 100% ethylacetate in heptane.
The 1H and 13C NMR spectra were recorded on a BRU-
KER Advance DRX-300 spectrometer at 300.13 MHz
TU/PO
Z-average nm (PDI)
6b/DMPC/DNA
6c/DMPC/DNA
1
5
461.9 (0.38)
8032 (0.11)
761.6 (0.77)
113.2 (0.27)
135.7 (0.09)
331.9 (0.312)
641.8 (0.7)
/
10
20
40
344.5 (0.4)
158.4 (0.4)
The polydispersity index is indicated in brackets.
olythiourea/DNA interaction.16 The system undergoes a
precipitation and then stabilizes at a ratio TU/PO = 20.
A similar pattern was evidenced for the two lipids.
We could evidence a lipid/DNA interaction by dynamic
light scattering while DNA was not retained on an aga-
rose gel when the previous complexes 6b/DMPC/DNA
and 6c/DMPC/DNA were loaded on a gel. DNA mi-
grated into the wells even when using a low electropho-
retic field. This suggests that even though one thiourea
function appears to be enough for the lipid to interact
with DNA, this association is probably weaker than
the one obtained with the lipids bearing two thiourea
functions for which DNA was partially retained in the
wells or for the previously reported lipopolythiourea,
which fully blocked DNA migration on the gel.18 Final-
ly, we evaluated the transfection capacity of the com-
plexes on B16 cells, using a luciferase encoding gene.
No transfection could be evidenced, probably due to
the lack of stability of the complexes in the biological
medium.
1
for H and 75.47 MHz for 13C. The NMR spectra were
processed using Xwinnmr (Bruker) or SwaN-MR. Mass
Spectrometry was carried out on a Shimazu 2010A LC–
MS on ESI mode. Dimyristoyl phosphatidylcholine
(DMPC) and Dipalmytoylphosphatidylcholine (DPPC)
were purchased from Aventi Polar Lipids (Alabaster
Alabama). logP calculation was performed using Mar-
vinSketch (Chemaxon Ltd Budapest Hungary). Particle
diameter was determined by dynamic light scattering on
a Zeta Sizer NanoSeries Malvern (Malvern Instruments,
France).
4.2. Synthetic procedures
4.2.1. tert-Butyl N-{[bis(decyl)carbamoyl]methyl} carba-
mate (2). Boc-glycine (5.2 g, 29.7 mmol), DCC (15.25 g,
74 mmol) and N-hydroxysuccinimide (3.42 g, 30 mmol)
were dissolved in dry THF (200 mL). The reaction mix-
ture was stirred for 3 h at room temperature, didecyl-
amine (7.43 g, 25 mmol) was then added and the
resulting mixture was stirred overnight at the same tem-
perature. The precipitated DCU was removed by filtra-
tion, and the filtrate was evaporated under reduced
pressure. The residual oil was dissolved in Et2O
(600 mL) and the solution was stirred overnight. An
additional amount of DCU was removed by filtration,
and the filtrate was washed with NaHCO3 (3·
200 mL), water, and brine. The organic layer was dried
(MgSO4) and evaporated under reduced pressure. Puri-
fication by chromatography (diethyl ether) yielded
5.85 g (51%) of tert-butyl N-{[bis(decyl)carbam-
oyl]methyl}carbamate (2) as yellow oil.
3. Conclusion
In conclusion we have developed a new family of lip-
othiourea using an automation strategy. Among these
compounds thioureas 5, 6a, 6b, and 6c were studied
for their physicochemical and transfecting properties.
As it could be expected from the properties of thio-
urea/phosphate complexes the described lipopolythiou-
reas were able to interact with DNA in the presence of
DMPC leading to small particles using a thiourea/phos-
phate ratio of the same magnitude that was previously
described.17,18 However, lipoplexes 5 and 6a were poorly
stable. In addition, the more stable lipothiourea 6b did
not possess any transfecting properties. All these data
demonstrated that at least two thiourea groups are nec-
essary to produce stable lipoplexes, to condense DNA
and to give efficient transfection.
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.29 (m, 28H, H-20–H-90), 1.3 (m, 4H, H-80), 1.48 (s, 9H,
Boc), 3.15 (m, 2H, H-10), 3.34 (m, 2H, H-10), 3.975 (d,
2H,J = 7 Hz, H-2), 5.06 (br s, 1H, NH). 13C NMR
(CDCl3) d (ppm) 14.4 (C-100), 23.1 (C-90), 27.4 (C-30),
28.5 (C-Boc), 42.4 (C-20), 32.3 (C-80), 46.4 (C-10), 47.4
(C-10). MS (ESI) MH+ = 355 (MH+-Boc).
4. Experimental
4.1. Material
All solvents were purchased from Carlo Erba-SDS (Pey-
pin France). Dichloromethane was distilled from P2O5,
and THF was distilled from Na in the presence of ben-
4.2.2. 2-Amino-N,N-bis(decyl)acetamide (3). Carbamate
2 (5.84 g, 12.86 mmol) was dissolved in trifluoroacetic
acid (55 mL) in a round bottomed flask equipped with
˚
zophenone. DMF was dried over 3 A molecular sieves.

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E. Kabha et al. / Bioorg. Med. Chem. 16 (2008) 4003–4008
a CaCl2 tube and the mixture was stirred for 1 h. TFA
was evaporated and the residue was dissolved in
CH2Cl2, washed successively with NaHCO3 (·3), water
and brine. The organic layer was dried (MgSO4), evap-
orated under a reduced pressure afford 3 g (65%) of 2-
amino-N,N-bis(decyl)acetamide 3 as yellow oil.
30 (C-20–C-70), 32 (C-80), 46.9 (C-10), 46.9 (C-2), 47.7
(C-10). MS (ESI) MH+ = 429, MNa+ = 451, HPLC pur-
ity by ELSD 98%.
4.2.5. Automated thioureas synthesis. The reactions were
performed in an ASW2000 automated synthesis work-
station (Chemspeed Augst Switzerland). Amines
(0.08 mmol) were dissolved in dry DMF (1 mL), added
to solutions of N,N-bis(decyl)-2-isothiocyanatoaceta-
mide 4 (0.08 g, 0.2 mmol) in dry DMF (1 mL). The solu-
tions were stirred for 16 h at 70 ꢁC. All the products
were purified by SFC chromatography (Hep-
tane ! EtOAc ! MeOH) to yield the thioureas in 50–
60% yields as yellow oil.
1H NMR (CDCl3) d (ppm) 0.87 (t, 6H, J = 7 Hz, H-100),
1.26 (m, 4H, H-90), 1.27 (m, 28H, H-30–H-80), 1.49 (m,
2H, H-20), 1.5 (m, 2H, H-20), 3.14 (m, 2H, H-10), 3.32
(m, 2H, H-10), 4.09 (s, 2H, H-2).13C NMR (CDCl3) d
(ppm) 14.1 (C-100), 23.1 (C-90), 27–30.3 (C-20–C-70),
32.3 (C-80), 41.7 (C-2), 46.7 (C-10), 47.4 (C-10), 171.7
(C-1), MS (ESI) MH+ = 355.
4.2.3. N,N-Bis(decyl)-2-isothiocyanatoacetamide (4)
4.2.3.1. Procedure 1. A solution of DCC (0,757 g,
3,67 mmol) in dry CH2Cl2 (25 mL) was added dropwise
to a cold (ꢀ7 ꢁC/NH4Cl/ice) mixture of 2-amino-N,N-
bis(decyl)acetamide 3 (1.301 g, 3.67 mmol), and carbon
disulfide (1.50 mL, 24.96 mmol) in dry CH2Cl2
(25 mL). The solution was stirred for 30 min at 0 ꢁC
then for 25 h at room temperature. The solvent was
evaporated under reduced pressure. Purification by
SFC chromatography (Heptane/EtOAc) yielded 1,13 g
(89%) of N,N-bis(decyl)-2-isothiocyanatoacetamide (4)
as yellow oil.
4.2.6. N,N-Bis(decyl)-2-({[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyl]carbamothioyl}amino)acetamide (6a). 2,2-Di-
methyl-1,2-dioxalane-4-methanamine was added to a
solution of 4 to yield 0.060 g of N,N-bis(decyl)-2-({[(2,2-
dimethyl-1,3-dioxolan-4-yl)methyl]carbamothioyl} amino)-
acetamide 6a (57%).
1H NMR (CDCl3) d (ppm) 0.89 (t, 6H, J = 7 Hz, H-100),
1.28 (m, 32H, H-30–H-90), 1.35 (s, 3H, H-8), 1.41 (s, 3H,
H-9), 3.25 (m, 2H, H-10), 3.35 (m, 2H, H-10), 3.67 (br s,
2H, H-4), 3.67 (dd, 1H, J = 9 Hz, H-6), 4.04 (dd, 1H,
J = 9 Hz, H-6), 4.32 (m, 1H, H-5), 4.43 (s, 2H, H-2),
7.05 (br s, 1H, NH), 7.55 (br s, 1H, NH). 13C NMR
(CDCl3) d (ppm) 14.4 (C-100), 23.1 (C-90), 25.6 (C-9),
27.3 (C-8), 27.4 (C-30) 28 (C-70), 29.1 (C-60), 29.8 (C-40
and C-50), 30.0 (C-20), 32.3 1(C-80), 46.7 (C-10), 47.4
(C-10), 67 (C-6), 75 (C-5), 109 (C-7), 168 (C-1), 182.7
(C-3). MS (ESI) MH+ = 529, M+Na = 551; HPLC pur-
ity by ELSD 98%.
4.2.3.2. Procedure 2. p-Toluenesulfonyl chloride
(0,7 g, 3.67 mmol) in dry CH2Cl2 (25 mL) was added
dropwise to a cold (ꢀ7 ꢁC/NH4Cl/ice) solution of 2-ami-
no-N,N-bis(decyl)acetamide 3 (1.301 g, 3.67 mmol) and
carbon disulfide (1.50 ml, 24.96 mmol) in dry CH2Cl2
(25 mL). The solution was stirred for 30 min at 0 ꢁC
then for 25 h at room temperature. Purification by
SFC chromatography (Heptane/EtOAc) yield 1.13 g
(89%) of N,N-bis(decyl)-2-isothiocyanatoacetamide (4)
as yellow oil.
4.2.7. N,N-Bis(decyl)-2-{[(2,3-dihydroxypropyl)carbamo-
thioyl]amino}acetamide (6b). 3-Amino-1,2-propanediol
was added to a solution of 4 to yield 0.054 g of N,
N-bis(decyl)-2-{[(2,3-dihydroxypropyl) carbamothioyl]
amino}acetamide 6b (55%).
1H NMR (CDCl3) d (ppm) 0.86 (t, 6H, J = 7 Hz, H-100),
1.25 (m, 32H, H-30–H-90), 1.54 (m, 4H, H-20), 3.06 (m,
2H, H-10), 3.33 (m, 2H, H-10), 4.25 (s, 2H, H-2).13C
NMR (CDCl3) d (ppm) 14.18 (C-100), 22.75 (C-90),
26.92–29.61 (C-20–C-70), 31.95 (C-80), 46.99 (C-2),
46.47 (C-10), 47.47 (C-10), 139.55 (C-3), 164.01 (C-1).
MS (ESI) MH+ = 397, HPLC purity 98% by ELSD.
1H NMR (CDCl3) d (ppm) 0.89 (t, 6H, J = 7 Hz, H-100),
1.3 (m, 4H, H90), 1.18–1.46 (m, 20H, H-30–H-70), 1.324
(m, 4, H-80), 1.52 (m, 2H, H-20), 1.62 (m, 2H, H-20), 3.27
(m, 2H, H-10), 3.31 (m, 2H, H-10), 3.60 (m, 4H, H-4 and
H-6), 3.9 (m, 1H, H-5), 4.47 (s, 2H, H-2), 7.56 (br s, 1H,
NH), 7.78 (br s, 1H, NH). 13C NMR (CDCl3) d (ppm)
14.4 (C-100), 23.1 (C-90), 27–30 (C-20–C-70), 32.3 (C-
80), 46.9 (C-4), 46.9 (C-10), 47.9 (C-10), 64 (C-6), 71.5
(C-5), 169.4 (C-1), 182.4 (C-3). MS (ESI) MH+ = 488,
HPLC purity by ELSD 98%.
4.2.4. N,N-Bis(decyl)-2-[(methylcarbamothioyl)amino]-
acetamide (5). Isothiocyanate (0.082 g, 1.12 mmol) was
added to a solution of 2-amino-N,N-bis(decyl)acetamide
3 (0.199 g, 0.56 mmol), triethylamine (0.227 g, 2.24
mmol) in dry CH2Cl2 (5.6 mL). The reaction solution
was stirred 6 h at room temperature. Purification by chro-
matography (Heptane/EtOAc) to yield 0.15 g (62%) of
N,N-bis(decyl)-2-[(methylcarbamothioyl)amino]acetamide
5 as yellow oil.
4.2.8. N,N-Bis(decyl)-2-({[3-(1H-imidazol-1-yl)propyl]-
carbamothioyl}amino)acetamide (6c). 1-(3-Aminopro-
pyl)imidazole was added to a solution of 4 to yield
N,N-bis(decyl)-2-({[3-(1H-imidazol-1-yl)propyl]carbamo-
thioyl}amino)acetamide 6c 0.033 g (33%).
1H NMR (CDCl3) d (ppm) 0.87 (t, 6H, J = 7 Hz, H-100),
1.26 (m, 4H, H90), 1.32 (m, 4H, H-80), 1.38–1.17 (m,H,
20H, H-30–H-70), 1.5 (m, 2H, H-20), 1.59 (m, 2H, H-
20), 2.99 (s, 3H, H-4), 3.23 (m, 2H, H-10), 3.23 (m, 2H,
H-10), 7.18 (br s, 1H, NH), 7.53 (br s, 1H, NH). 13C
NMR (CDCl3) d (ppm) 14.4 (C-100), 22.8 (C-90), 27–
1H NMR (CDCl3) d (ppm) 0.90 (t, 6H, J = 7 Hz, H-100),
1.30 (m, 4H, H90), 1.21–145 (m, 20H, H-30–H-70), 1.324
(m, 4H, H-80), 1.52 (m, 2H, H-20), 1.63 (m, 2H, H-20),
2.14 (m, 2H, H-5), 3.26 (m, 2H, H-10), 3.33 (m, 2H,
H-10), 3.51 (m, 2H, H-4), 4.09 (m, 2H, H-6), 4.44 (s,

E. Kabha et al. / Bioorg. Med. Chem. 16 (2008) 4003–4008
4007
2H, H-2), 7.02 (s, 1H, H-8), 7.11 (s, 1H, H-7), 7.53 (br s,
2H, NH), 7.85 (br s, 1H, H-9). 13C NMR (CDCl3) d
(ppm) 14.4 (C-100), 23.1 (C-90), 27–30 (C-20–C-70), 32.3
(C-80), 30.8 (C-5), 41.4 (C-40), 45.2 (C-60), 46.7 (C-10),
46.7 (C-2), 47.7 (C-10), 120 (C-8), 129 (C-7), 138 (C-9).
MS (ESI) MH+ = 522, HPLC purity by ELSD 98%.
oxy)ethanol was added to a solution of 4 to yield N,
N-bis(decyl)-2-({[2-(2-hydroxyethoxy)ethyl]carbamothi-
oyl}amino)acetamide 6g 0.054 g (54%).
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.30 (m, 4H, H90), 1.21–145 (m, 20 H, H-30–H-70), 1.34
(m, 4H, H-80), 1.53 (m, 2H, H-20), 1.60 (m, 2H, H-20),
3.23 (m, 2H, H-10), 3.356 (m, 2H, H-10), 3.36 (m, 2H,
H-4), 3.61 (m, 4H, H-5 and H-6), 3.73 (m, 2H, H-7),
4.14 (s, 1H, OH), 4.43 (s, 2H, H-2), 6.82 (s, 1H, NH),
7.72 (s, 1H, NH). 13C NMR (CDCl3) d (ppm) 14.4 (C-
100), 22.9 (C-90), 26.8–30 (C-20–C-70), 32.3 (C-80), 46.7
(C-4 and C-10), 47.5 (C-10), 62.1 (C-7), 74.2 (C-5, C-6).
MS (ESI) MH+ = 503, M+Na = 525, HPLC purity by
ELSD 95%.
4.2.9. N,N-Bis(decyl)-2-{[(3-hydroxypropyl)carbamothioyl]-
amino}acetamide (6d). 3-Amino-L-propanol was added
to a solution of 4 to yield N,N-bis(decyl)-2-{[(3-
hydroxypropyl)carbamothioyl]amino}acetamide 6d
0.048 g (50%).
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.305 (m, 4H, H90), 1.24–145 (m, 20H, H-30–H-70), 1.364
(m, 4H, H-80), 1.55 (m, 2H, H-20), 1.64 (m, 2H, H-20),
1.75 (m, 2H, H-5), 3.26 (m, 2H, H-10), 3.33 (m, 2H,
H-10), 3.36(m, 2H, H-4), 3.64 (m, 2H, H-4), 3.72 (m,
2H, H-6), 4.5 (s, 2H, H-2), 6.90 (s, 1H, NH), 7.45 (s,
1H, NH).13C NMR (CDCl3) d (ppm) 14.4 (C-100),
22.8 (C-90), 26.8–30 (C-20–C-70), 32.3 (C-80), 32.3 (C-
5), 46.9 (C-10), 46.9 (C-4), 46.9 (C-2), 47.7 (C-10), 59.3
(C-6), 169 (C-1), 182 (C-3). MS (ESI) MH+ = 473,
M+Na = 495, HPLC purity by ELSD 98%.
4.2.13.
N,N-Bis(decyl)-2-({[2-(4-hydroxyphenyl)ethyl]-
carbamothioyl}amino)acetamide (6h). Tyramin was
added to a solution of 4 to yield N,N-bis(decyl)-2-({[2-
(4-hydroxyphenyl) ethyl]carbamothioyl}amino)acetam-
ide 6h 0.054 g (50%).
1H NMR (CDCl3) d (ppm) 0.92 (t, 6H, J = 7 Hz, H-100),
1.32 (m, 4H, H-90), 1.20–1.45 (m, 20H, H-30–H-70), 1.32
(m, 4H, H-80), 1.48 (m, 2H, H-20), 1.59 (m, 2H, H-20),
2.85 (t, 2H, J = 6 Hz, H-5), 3.23 (m, 4H, H-10), 3.7 (br
s, 2H, H-4), 4.41 (s, 2H, H-2), 6.31 (br s, 1H, OH),
6.67 (s, 1H, NH), 6.79 and 7.02 (AA‘XX’ system), 7.22
4.2.10. N,N-Bis(decyl)-2-{[3-(2-hydroxypropyl)carbamo-
thioyl]}acetamide (6e). 1-Amino-2-propanol was added
to a solution of 4 to yield N,N-bis(decyl)-2-[3-(2-
hydroxypropyl)carbamothioyl]acetamide 6e 0.051 g
(54%).
13
(s, 1H, NH). C NMR (CDCl3) d (ppm) 14.4 (C-100),
23.1 (C-90), 27–30 (C-20–C-70), 32 (C-80), 34.2 (C-5),
45.7 (C-4), 46.7 (C-2 and C-10), 116 and 130 (AA‘XX’).
MS (ESI) MH+ = 535, MNa+ = 557. HPLC purity by
ELSD 98%.
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.2 (d, 3H, H-6), 1.305 (m, 4H, H90), 1.26–145 (m, 20H,
H-30–H-70), 1.283 (m, 4H, H-80), 1.53 (m, 2H, H-20),
1.65 (m, 2H, H-20), 3.25 (m, 2H, H-10), 3.23 (m, 2H,
H-4), 3.35 (m, 2H, H-10), 3.76 (br s, 1H, OH), 3.96 (m,
1H, H-5), 4.38 (dd, 1H, H-2), 4.65 (dd, 1H, H-2),
7.132 (br s, 1H, NH), 7.904 (br s, 1H, NH). 13C NMR
(CDCl3) d (ppm) 14.4 (C-100), 21.3 (C-6), 23.1 (C-90),
27–30 (C-20–C-70), 32.3 (C-80), 46.9 (C-10), 46.9 (C-4),
47.9 (C-10), 67 (C-5). MS (ESI) MH+ = 473,
M+Na = 495. HPLC purity by ELSD 98%.
4.2.14. N,N-Bis(decyl)-2-{[(6-hydroxyhexyl)carbamothioyl]-
amino}acetamide (6i). Amino-1-hexanol was added to
a solution of 4 to yield N,N-bis(decyl)-2-{[(6-hydrox-
yhexyl)-carbamothioyl]amino}acetamide 6i 0.0563 g
(50%).
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.30 (m, 4H, H-90), 1.21–1.37 (m, 26 H, H-30–H-70 and
H-5–H-7), 1.32 (m, 4H, H-80), 1.42 (m, 4H, H-20), 1.62
(m, 2H, H-8), 3.23 (m, 2H, H-4), 3.355 (m, 4H, H-10),
3.68 (t, 2H, H-9), 4.41 (s, 2H, H-2), 6.34 (s, 1H, NH),
7.06 (s, 1H, NH). 13C NMR (CDCl3) d (ppm) 14.5 (C-
100), 22.9 (C-90), 26.8–30 (C-20–C-70 and C-5–C-7),
32.3 (C-80), 32.9 (C-8), 46.6 (C-4), 47.2 (C-10 and C-2),
63 (C-9). MS (ESI) MH+ = 515, MNa+ = 537, HPLC
purity by ELSD 98%.
4.2.11. N,N-Bis(decyl)-2-{[(1-hydroxybutan-2-yl)carbamo-
thioyl]amino}acetamide (6f). 2-Amino-1-butanol was
added to a solution of 4 to yield N,N-bis(decyl)-2-{[(1-
hydroxybutan-2-yl)carbamothioyl]amino}acetamide 6f
0.067 g (100%).
1H NMR (CDCl3) d (ppm) 0.89 (t, 6H, J = 7 Hz, H-100),
1.263 (m, 4H, H-80), 1.305 (m, 4H, H-90), 0.97 (t, 3H, H-
7), 1.57 (m, 2H, H-6), 1.21–144 (m, 20H, H-30–H-70),
1.57 (m, 2H, H-20), 1.65 (m, 2H, H-20), 3.25 (m, 2H,
H-10), 3.173 (m, 1H, H-4), 3.356 (m, 2H, H-10), 3.57
(dd, 1H, H-5), 3.76 (dd, 1H, H-5), 4.12 (s, 2H, H-2),
7.74 (br s, 1H, NH), 7.78 (br s, 1H, NH). 13C NMR
(CDCl3) d (ppm) 10.9 (C-7), 14.4 (C-100), 22.8 (C-90),
24.8 (C-6), 27–30 (C-20–C-70), 32.3 (C-80), 46.7 (C-4),
47 (C-10), 47.8 (C-10), 64.7 (C-5). MS (ESI)
MH+ = 487, M+Na = 509, HPLC purity by ELSD 98%.
4.2.15. N,N-Bis(decyl)-2-{[(2-phenylethyl)carbamothioyl]
amino}acetamide (6j). 2-Phenyl-ethylamine was added to
a solution of 4 to yield N,N-bis(decyl)-2-{[(2-phenyl-
ethyl)carbamothioyl]amino}acetamide 6j 0.062 g (60%).
1H NMR (CDCl3) d (ppm) 0.921 (t, 6H, J = 7 Hz, H-
100), 1.32 (m, 4H, H-90), 1.20–145 (m, 20H, H-30–H-
70), 1.324 (m, 4H, H-80), 1.48 (m, 2H, H-20), 1.59 (m,
2H, H-20), 2.93 (t, 2H, J = 6 Hz, H-5), 3.30 (m, 4H, H-
10), 3.70 (br s, 2H, H-4), 4.42 (s, 2H, H-2), 6.52 (s, 1H,
NH), 7.23–7.33 (m, 5H, Ph).MS(ESI) MH+ = 518,
HPLC purity by ELSD 90%.
4.2.12. N,N-Bis(decyl)-2-({[2-(2-hydroxyethoxy)ethyl]-
carbamothioyl}amino)acetamide (6g). 2-(2-Aminoeth-

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E. Kabha et al. / Bioorg. Med. Chem. 16 (2008) 4003–4008
4.2.16. N,N-Bis(decyl)-2-{[(2-hydroxyethyl)carbamothioyl]-
amino}acetamide (6k). Ethanalamine was added to a
solution of 4 to yield 0.055 g N,N-bis(decyl)-2-{[(2-
hydroxyethyl)carbamothioyl]-amino}acetamide 6k (60%).
References and notes
1. Hisaki, I.; Sasaki, S.; Hirose, K.; Tobe, Y. Eur. J. Org.
Chem. 2007, 607–615, and references cited therein.
2. Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40,
486–516.
3. Fan, E.; Van Arman, S. A.; Kincaid, S.; Hamilton, A. D.
J. Am. Chem. Soc. 1993, 115, 369–370; Scheerder, B. J.;
Fochi, M.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Org.
Chem. 1994, 59, 7815–7820; Raposo, C.; Crego, M.;
1H NMR (CDCl3) d (ppm) 0.91 (t, 6H, J = 7 Hz, H-100),
1.20–145 (m, 28H, H-30–H-90), 1.56 (m, 2H, H-20), 1.66
(m, 2H, H-20), 3.35 (m, 4H, H-10), 3.73 (br s, 2H, H-4),
4.57 (s, 2H, H-2), 7.34 (s, 1H, NH), 7.95 (s, 1H, NH).
MS (ESI) MH+ = 459, M+Na = 481, HPLC purity by
ELSD 95%.
´
Almaraz, M.; Mussons, M. L.; Martın, M.; Weinrich, V.;
Caballero, M. C.; Moran, J. R. Chem. Lett. 1996, 478, 87–
´
110; Buhlmann, P.; Nishizawa, S.; Xiao, K. P.; Umezawa,
¨
Y. Tetrahedron 1997, 53, 1647–1654; Jagessar, R. C.;
Shang, M.; Scheidt, W. R.; Burns, D. H. J. Am. Chem.
Soc. 1998, 120, 11684–11692; Sasaki, S.; Citterio, D.;
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2309–2313.
4.3. Physico-chemical procedures
4.3.1. Preparation of the liposomes by the film/hydration
method. All compounds were formulated the same way.
Example is given for compound 6b. Compound 6b
(0.85 mg, 1.7 lmol) and DMPC (1.9 mg, 2.8 lmol) were
dissolved in 600 lL of CHCl3. After solvent removal un-
der vacuum, the film obtained was hydrated with 400 lL
of H2O at room temperature to give a multilamellar
liposome. Liposomes were successively heated at 37 ꢁC
and extruded to form a homogeneous population of
liposomes.
4. Xiao, X. P. Tetrahedron 1997, 53, 1647–1654.
5. Nishizawa, S.; Buhlmann, P.; Iwao, M.; Umezawa, Y.
¨
Tetrahedron Lett. 1995, 36, 6483–6486.
6. Rolland, A. Adv. Drug Deliv. Rev. 2005, 57, 669–673.
7. Bhattacharya, S.; Bajaj, A. Curr. Opin. Chem. Biol. 2005,
9, 647–655.
8. Gao, X.; Kim, K. S.; Liu, D. AAPS J. 2007, 23, E92–
E104.
9. Kootstra, N. A.; Verma, I. M. Ann. Rev. Pharmacol.
Toxicol. 2003, 43, 413–439.
4.3.2. Preparation of the complexes DMPC/LMTU/
DNA. Plasmid DNA (pVax2Luc18), 50 lL, 0.01 g/L in
sucrose 15%, was added to various amounts of
DMPC/LMTU liposomes at room temperature. The
mixture was vortexed during 10 s and left at room tem-
perature for 1 h before size measurements using water
viscosity and refractive index. TU/PO indicates the ratio
in nmol of thiourea function versus nmol of DNA
phosphates
10. Verma, I. M.; Weitzman, M. D. Ann. Rev. Biochem. 2005,
74, 711–738.
11. Al-Dosari, M. S.; Knapp, J. E.; Liu, D. Adv. Genet. 2005,
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12. Trollet, C.; Bloquel, C.; Scherman, D.; Bigey, P. Curr.
Gene Ther. 2006, 6, 561–578.
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´ ´
14. Arigon, J.; Prata, C. A.; Grinstaff, M. W.; Barthelemy, P.
Bioconjug. Chem. 2005, 16, 864–872.
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Horiuchi, S.; Sando, S.; Niidome, T. J. Am. Chem. Soc.
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4.3.3. Gel retardation experiments. Samples were de-
scribed as above (20 lL) and 5 lL of bromophenol blue
was added. The mixture was loaded on a 0.8% agarose
gel in TBE buffer (1 M Tris, 0.9 M boric acid, 0.01
EDTA) at 80 V/cm. DNA was revealed with ethidium
bromide and visualized under UV light.
18. Leblond, J.; Mignet, N.; Largeau, C.; Spanedda, M. V.;
Seguin, J.; Scherman, D.; Herscovici, J. Bioconjug. Chem.
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Acknowledgments
19. Thompson, B.; Mignet, N.; Hofland, H.; Lamons, D.;
Seguin, J.; Nicolazzi, C.; de la Figuera, N.; Meng, X. Y.;
Scherman, D.; Bessodes, M. Bioconjug. Chem. 2005, 16,
608–614.
This work was financially supported by INSERM,
´
CNRS, MNERT, the Region Ile de France (SESAME,
CPER). E.K. thanks the French Government for a Cha-
teaubriand Post-doctoral fellowship.