Synthesis of novel lipophilic tetraamines with cytotoxic activity

Article abstract of DOI:10.1016/j.mencom.2019.11.003

New lipophilic polyamines in which natural or synthetic tetraamines were linked via the terminal NH2 groups to diglycerides and/or to short-chained aliphatic substituents were synthesized, and their cytotoxic activity was tested. Relationship between the structure of the synthesized compounds and their cytotoxicity and hemolytic effect was evaluated.

Full text of DOI:10.1016/j.mencom.2019.11.003

Mendeleev
Communications
Mendeleev Commun., 2019, 29, 616–618
Synthesis of novel lipophilic tetraamines with cytotoxic activity
Ksenia A. Perevoshchikova,a Artemiy I. Nichugovskiy,a Alexandra K. Isagulieva,b Nina G. Morozova,a
Igor V. Ivanov,a Mikhail A. Maslov*a and Alexander A. Shtilb
a M. V. Lomonosov Institute of Fine Chemical Technologies, MIREA – Russian Technological University,
119571 Moscow, Russian Federation. E-mail: mamaslov@mail.ru
b N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2019.11.003
R
m
4
n
3
3
3
New lipophilic polyamines in which natural or synthetic
tetraamines were linked via the terminal NH2 groups to
diglycerides and/or to short-chained aliphatic substituents
were synthesized, and their cytotoxic activity was tested.
Relationship between the structure of the synthesized
compounds and their cytotoxicity and hemolytic effect was
evaluated.
Et
H
10
8
4
C18H37
O
4HCl
Et
3
EtO
H
H
H
H
H
Et
H
3
2
2
3
2
2
6
N
N
N
N
n
R
m
n
4
2
0
HCT116
K562
Natural polyamine (PA) spermine and its precursors (spermidine
and putrescine) are essential for cell growth in eukaryotes. These
PAs affect the conformational state of DNA, modulate the
chromatin structure, stabilize the lipid bilayer, and are crucial for
theregulationofionchannelsandgeneexpression.1,2 Intracellular
polyamine concentrations are highly regulated at the levels of
biosynthesis, uptake, and catabolism. Elevated intracellular
levels of PAs are known to be associated with cancer and other
diseases related to altered proliferation.3 Therefore, targeting the
metabolic pathways of these PAs has emerged as a promising
strategy for cancer treatment.4
A variety of symmetric or non-symmetric terminally
substituted natural PAs and their synthetic alkylated analogues
have been synthesized and investigated in different experimental
models.5,6 These compounds exhibited significant antitumour
effects modulated by the inhibition of PA biosynthesis or the
induction of the catabolic enzymes that affect PAs.7–9
Symmetrically substituted bis(alkylated)polyamine N1,N11-
diethylnorspermine (DENSpm) being one of the most
investigated PAs was evaluated in Phases I and II clinical trials,
however, its therapeutic efficacy was not confirmed.6
Non-phosphorous cationic ether glycerolipids (CELs)
represent a group of potential candidates for antitumour drug
therapy.10 These compounds are structurally close to Edelfosine
(1-O-octadecyl-2-O-methylglycero-3-phosphocholine), a phos-
phate-containing ether glycerolipid.11 In contrast to Edelfosine,
CELs are resistant to intercellular phospholipases and thus can
induce tumour cell apoptosis without exhibiting any hemolytic
effects.10 In our previous works, we have synthesized a series of
CELs which were modified with different polar heads,12 and
a strong correlation between their structure and biological
activities has been established.10,12,13 As a part of our ongoing
investigations, in this study we have designed and synthesized
a series of lipophilic polyamines (LPAs) in which the terminal
NH2 moieties of natural (spermine) and synthetic (norspermine
and triethylenetetramine) polyamines were linked to diglycerides
or short-chain aliphatic substituents, or both. Variations in the
chain length of the diglyceride substituents and the composition
of the PA backbone allowed us to get insight into the structure–
activity relationship for this series of LPAs.
Diverse modified PAs are often obtained by alkylation of
protected amines during chain elongation process14 or through
the formation of nitrogen-containing compounds and their
subsequent reduction to amines15–17, as well as by Michael
addition reaction.18 The preparation of alkylated PA conjugates
is most often carried out by alkylation of protected polyamines
with halogen derivatives.19–21
In this study, we employed the synthetic approach based on
the Fukuyama reaction27 between 2-nitrobenzenesulfonamides
obtained from regio-protected PAs and bromo derivatives of the
dialkylglyceroles. The removal of the 2-nitrophenylsulfonyl
groups gave desired secondary amines. This useful synthetic
approach led to monoalkylated LPAs as well as their
unsymmetrically substituted dialkylated analogues.
Diglycerides were obtained from Bn-O-protected rac-
glycidol25 via the oxirane ring opening with long-chained
aliphatic alcohols (C10–C18) in the presence of sodium hydride
and subsequent O-alkylation with bromoethane.26 Bromo
diglyceride derivatives 1a–e were synthesized from the
correspondingdiglyceridesontreatmentwithtetrabromomethane
and triphenylphosphine in 82–98% yields (see Online
Supplementary Materials).
Regioselectively protected PAs 2a–c (Scheme 1) were
prepared by the conversion of primary amines into the
corresponding amides on treatment with 2-nitrophenylsulfonyl
chloride in a four-step procedure as described previously.22,23 An
effective differentiation of the amino groups of spermine,
norspermine, or triethylenetetramine was achieved by the
trifluoroacetylation of the primary amino groups24 followed by
N-Boc protection of the secondary ones.
The consequent condensation of the PA derivatives 2a–c with
equimolar amounts of compounds 1a–e was carried out at 85 °C
for 25 h in the presence of cesium carbonate (see Scheme 1).27
This reaction afforded both monoalkylation products 3a–g
(17–33% yields) and symmetric dialkylated derivatives 4a–g
(10–15% yields).
© 2019 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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Mendeleev Commun., 2019, 29, 616–618
OR
OEt
Br
Boc Boc
RO
i
RO
OR
H
N
H
N
N
m
N
EtO
Boc Boc
EtO
Boc Boc
OEt
H
Ns
Ns
n
n
N
N
N
m
N
N
N
N
m
N
n
n
n
n
1a–e
2a–c
Ns
Ns
Ns
Ns
a n = 3, m = 4
b n = m = 3
c n = m = 2
a R = C18H37
b R = C16H33
c R = C14H29
d R = C12H25
e R = C10H21
3a–g
4a–g
Ns = 2-nitrophenylsulfonyl
RO
RO
4HCl
iii, iv
ii
EtO
EtO
Boc Boc
3a–g
Et
H
H
H
H
N
N
N
N
n
N
N
N
N
n
Et
n
m
m
n
Ns
Ns
RO
4HCl
iii, iv
6a–g
5a–g
3a,b,f,g
EtO
H
H
H
N
N
N
NH2
n
3–6: a R = C18H37, n = 3, m = 4
b R = C16H33, n = 3, m = 4
c R = C14H29, n = 3, m = 4
n
m
7a–d
d R = C12H25, n = 3, m = 4
e R = C10H21, n = 3, m = 4
a R = C18H37, n = 3, m = 4
b R = C16H33, n = 3, m = 4
c R = C18H37, n = m = 3
d R = C18H37, n = m = 2
f
R = C18H37, n = m = 3
g R = C18H37, n = m = 2
Scheme 1 Reagents and conditions: i, Cs2CO3, DMF, 85 °C; ii, EtBr, Cs2CO3, DMF, 85 °C; iii, PhSH, K2CO3, 24 °C; iv, 4 n HCl·dioxane, 24 °C.
To obtain unsymmetrically disubstituted LPAs 5a–g, the
(a)
intermediates 3a–g were treated with ethyl bromide under the
Fukuyama conditions (yields 66–91%). The 2-nitrophenyl-
sulfonyl protecting groups in compounds 5a–g were removed
using thiophenol in the presence of potassium carbonate. The
removal of the N-Boc protective group using HCl in dioxane led
to the final LPAs 6a–g. The monosubstituted LPAs 7a–d were
synthesized in a similar manner as described above for the
compounds 6a–g. All LPAs obtained, including their
intermediates, were characterized by 1H, 13C NMR spectroscopy
and mass spectrometry (see Online Supplementary Materials).
The newly synthesized LPAs 6a–g and 7a–d were tested
against the human chronic myelogenous leukemia (K562), colon
(HCT116) and breast (MCF7) adenocarcinoma cells using the
MTT-assay.28 LPAs induced tumour cell death in the micromolar
concentration range, and their potencies (IC50 values) were 4–5-
fold higher than those of the reference cationic ether glycerolipids
(CL, rac-N-methyl-N'-{4-[(2-ethoxy-3-octadecyloxy)prop-1-
yloxycarbonyl]butyl}imidazolium iodide), Edelfosine and
DENSpm (Figure 1, Table S1 and Figure S2 of the Online
Supplementary Materials).
10
8
6
6a
7a
4
2
0
(b)
10
8
6f
6
7c
**
4
2
The effect of norspermine-containing compound 6f on
HTC116, MFC7 and K562 cells was higher than the effect of
0
(c)
****
10
8
25
***
6a
**
***
***
***
20
15
10
5
6f
6g
CL
6
***
***
6g
7d
****
4
*
2
n.d.
n.d.
0
0
Figure 1 The impact of the polyamine structure on the cytotoxicity against
tumour cells. The cytotoxicity data against the myelogenous leukaemia
(K562), colon (HCT116) and breast adenocarcinoma (MCF-7) cells are
presented as mean value SEM for n = 3 independent experiments. *P < 0.1,
***P < 0.001, ****P < 0.0001 vs. CL; paired t-test.
Figure 2 Differences between the cytotoxic activity of unsymmetrically
dialkylated (6) and monoalkylated (7) lipophilic polyamine derivatives (a) 6a
and 7a, (b) 6f and 7c, (c) 6g and 7d, against the tumour cells. The cytotoxicity
data against K562, HTC116 and MCF-7 cells are presented as mean values
SEM for n = 3 experiments. **P < 0.01, ****P < 0.0001; paired t-test.
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Mendeleev Commun., 2019, 29, 616–618
3 E. W. Gerner and F. L. Meyskens, Jr., Nat. Rev. Cancer, 2004, 4, 781.
spermine- or triethyltetramine-based LPAs. Moreover, a four-
fold increase in the cytotoxicity against the MCF7 cells was
observed for compound 6f in comparison with natural spermine
analogue 6a (see Figure 1). For all the cell lines tested, the
cytotoxicity of the spermine-based compounds 6a–e was hardly
influenced by the length of the O-alkyl chain at C1 position of
the glycerol backbone (see Online Supplementary Materials,
Figure S1). The cytotoxicity of these compounds against the
HCT116 cells (IC50 6.20–2.97 µmol dm–3) was comparable with
the effect against the MCF7 (IC50 5.80–1.82 µmol dm–3) cell
lines, whereas it was lower against the K562 cells (IC50 2.25–
1.30 µmol dm–3). However, LPA 6a with octadecyl residue was
less active than other analogues against HCT116 and MCF7
cells but more effective against K562 cells.
Interestingly, the unsymmetrically dialkylated PA derivatives
6f–g were more effective against the HTC116 and K562 cells
than their monoalkylated analogues 7c–d with free terminal NH2
group (Figure 2). Accordingly, no significant difference in the
cytotoxic activity of spermine-based PAs 6a and 7a was observed
against all tested cell lines.
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The evaluation of the ability to disrupt human erythrocytes
revealed that novel LPAs did not mediate any distinguish hemolytic
effect, similarly to the reference compounds CL25 and DENSpm,
whereas Edelfosine stronger induced the erythrocytes damage
(Table S2, see Online Supplementary Materials). Moreover, the
hemolytic activity decreased with the decrease in the length of the
C1-positioned O-alkyl chain of compounds 6b–e. Norspermine-
based LPAs 6f and 7c had less effect than their spermine analogues
6b and 7b. The presence of ethyl group at the terminal NH2 group
did not affect the hemolytic activity in the case of spermine LPAs
but decreased that for norspermin LPAs.
In summary, new lipophilic tetraamine derivatives have been
designed and synthesized using the Fukuyama reaction.
The results of the MTT-assay allow one to consider these
compounds as potential antitumour agents. Using different
combinations of substituents, we have demonstrated that
unsymmetrically dialkylated lipophilic polyamines with
norspermine backbones are the promising candidates for the
further biological investigations. The synthesis and the biological
screening of optically active LPAs are currently underway.
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This study was supported in part by the Ministry of Education
and Science of the Russian Federation (base part of state
assignment 4.9671.2017/BP) and by the Russian Foundation for
Basic Research (project no. 18-33-20192).
25 E. V. Shmendel, K. A. Perevoshchikova, D. K. Shishova, T. S. Kubasova,
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Online Supplementary Materials
Supplementary data associated with this article (general
synthetic procedures, characterization of compounds and details
for cytotoxicity and hemolytic activity assays) can be found in
the online version at doi: 10.1016/j.mencom.2019.11.003.
28 T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
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Received: 13th May 2019; Com. 19/5921
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