Synthesis and biological activity of a novel water-soluble methano[60]fullerene tetracarboxylic derivative

Article abstract of DOI:10.1016/j.mencom.2013.11.006

A reaction of 3,3',4,4'-tetrakis(methoxycarbonyl)benzophenone tosylhydrazone with fullerene C₆₀ was used to obtain a novel methano-[60]fullerene derivative bearing four carboxylic groups, whose sodium and potassium salts are water-soluble. The compound possesses antiviral and anticancer activity as well as pronounced antioxidant properties in combination with a low toxicity.

Full text of DOI:10.1016/j.mencom.2013.11.006

Mendeleev
Communications
Mendeleev Commun., 2013, 23, 323–325
Synthesis and biological activity of a novel water-soluble
methano[60]fullerene tetracarboxylic derivative
Alexey B. Kornev,*a Alexander S. Peregudov,b Vyacheslav M. Martynenko,a Galina V. Guseva,a
Tatiana E. Sashenkova,a Alexander Yu. Rybkin,a Irina I. Faingold,a Denis V. Mishchenko,a
Raisa A. Kotelnikova,a Nina P. Konovalova,a Jan Balzarinic and Pavel A. Troshina
a Institute of Problems of Chemical Physics, Russian Academy of Sciences, 142432 Chernogolovka,
Moscow Region, Russian Federation. Fax: +7 496 522 3507; e-mail: kornev@icp.ac.ru
b A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 119991 Moscow,
Russian Federation
c Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
DOI: 10.1016/j.mencom.2013.11.006
A reaction of 3,3',4,4'-tetrakis(methoxycarbonyl)benzophenone tosylhydrazone with fullerene C60 was used to obtain a novel methano-
[60]fullerene derivative bearing four carboxylic groups, whose sodium and potassium salts are water-soluble. The compound possesses
antiviral and anticancer activity as well as pronounced antioxidant properties in combination with a low toxicity.
O
O
Fullerenes and their functional derivatives possess antiviral, anti-
bacterial and anticancer activity along with efficient antioxidant
and radioprotector properties.1 Some new fullerene derivatives
are active against Mycobacterium tuberculosis2 and two viruses
of Herpesviridae family.3 They are also promising for drug4 and
gene delivery5 and in medical diagnostics (e.g., MRI contrast
agents).6 Unmodified fullerenes7 and some of their derivatives8
increase lifespan of laboratory animals and improve their cognitive
performance which makes the development of fullerene-based
pharmaceuticals against age-related disorders promising.
Low solubility of pristine fullerenes in aqueous media leading
to non-optimal pharmacokinetics and serious excretion difficulties
is a problem to be solved.9 Incorporation of ionic groups into a
molecule is one means to enhance solubility in water.10–14 Fullerenes
modified with protonated or quaternized amine groups often exhibit
high toxicity typical of polyamines.15 On the contrary, fullerene
derivatives bearing several carboxyl groups (typically, more than
three) show reasonable solubility in water and low toxicity.16–19
Previous syntheses of such compounds involved use of large
and sophisticated organic addends18 or multiple additions to the
fullerene cage,19 which can exert undesired influence on stereo-
electronic, photophysical and biological properties of a resulting
fullerene derivative.
R
R
R
R
i
O
O
O
O
O
O
3 R = CO2Me
2
R
R
R
R
R
R
R
R
iii
ii
N
NH
Ts
4 R = CO2Me
5 R = CO2Me
1 R = CO2H
iv
Scheme 1 Reagents and conditions: i, MeOH, SOCl2, D, 3 h; ii, TsNHNH2,
MeOH, D, 2 h; iii, C60, MeONa, Py, 1,2-DCB, 100°C, 3 h, then D, 10 h;
iv, MeONa, PhCl–MeOH–H2O, 70°C, 15 min, then H2O/HCl.
purification. Fullerene C60 (1.5 g, 2.13 mmol) was dissolved in 1,2-dichloro-
benzene (200 ml) in an argon atmosphere. Hydrazone 4 (2.0 g, 3.43 mmol),
MeONa (187 mg, 3.43 mmol) and pyridine (5 ml) were added. The mixture
was stirred at 100°C for 3 h, cooled, filtered, concentrated in vacuo to the
volume of 50 ml and subjected to the column chromatography on silica
(40–60 mm, 70 Å, Acros). Elution with pure toluene produced 210 mg of
unreacted C60. The product 5 was eluted with toluene–methanol (99:1 v/v)
mixture as a dark red-brown solution giving after concentration 860 mg
of a black solid. 1H NMR analysis of the obtained material revealed the
presence of ca. 20% of unisomerized fulleroid with an open-cage struc-
ture as an impurity. To complete the isomerization, product was dissolved
again in 50 ml of 1,2-dichlorobenzene in an argon atmosphere, heated at
reflux for 10 h and purified by column chromatography as above. The
methanofullerene derivative 5 was obtained as a dark solid in 42% yield
(840 mg) based on consumed C60. 1H NMR (CDCl3, 600 MHz) d: 3.96
(s, 6H), 3.98 (s, 6H), 7.87 (d, 2H), 8.35 (d, 2H), 8.49 (s, 2H). 13C NMR,
d: 52.93, 53.02, 55.36, 77.45, 129.64, 131.32, 132.34, 132.51, 133.82,
138.26, 141.08, 141.11, 142.11, 142.16, 143.03, 143.05, 143.07, 143.81,
144.51, 144.79, 144.80, 145.04, 145.25, 145.29, 146.50, 167.16, 167.73.
Found (%): C, 87.08; H, 1.78. Calc. for C81H18O8 (%): C, 86.94; H, 1.62.
Here we report the synthesis of a novel water-soluble fullerene
1 modified with a reasonably small solubilizing organic addend
having four carboxylic groups (Scheme 1).† At the first step the
†
3,3',4,4'-Tetrakis(methoxycarbonyl)benzophenone 3. Dianhydride 2
(35 g, 0.11 mol) was added to 150 ml of methanol and heated at reflux
for 3 h. After cooling, thionyl chloride (105 g, 0.88 mol) was added with
intense stirring. The resulting solution was stirred for 3 days at room
temperature and then concentrated at the rotary evaporator. The resulting
residue was dissolved in 200 ml of diethyl ether, washed with 5% aqueous
solution of K2CO3 (2×100 ml), dried over calcined MgSO4, filtered
and concentrated to give 36.7 g (78%) of compound 3 as a viscous liquid.
1H NMR (CDCl3, 600 MHz) d: 3.90 (s, 6H), 3.91 (s, 6H), 7.63 (d, 2H),
7.67 (d, 2H), 7.90 (s, 2H).
Compound 5. p-Toluenesulfonylhydrazine (7.0 g, 37 mmol) was added
to a solution of 3 (16.6 g, 37.0 mmol) dissolved in 100 ml of methanol.
The mixture was heated at reflux for 2 h and cooled. Methanol was removed
under reduced pressure leaving 21.8 g (quantitative) of tosylhydrazone
4 as a cream-coloured resin which was used at the next step without
© 2013 Mendeleev Communications. All rights reserved.
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Mendeleev Commun., 2013, 23, 323–325
commercially available 3,3',4,4'-benzophenonetetracarboxylic
dianhydride 2 was converted to tetramethyl ester 3 and then
tosylhydrazone 4. The Hummelen–Wudl reaction20 of compound
4 with fullerene C60 produced methanofullerene 5.
Our initial attempts to hydrolytically cleave the ester groups
in compound 5 under the typical acidic conditions20,21 were unsuc-
cessful. However, we have found that the ester groups in 5 can be
easily hydrolyzed under alkaline conditions using a small excess
(1.05 equiv. per one ester group) of methanolic sodium hydroxide
solution. The water-soluble target compound 1 was prepared in
four steps from easily available precursors with good overall yield
of 40% based on consumed C60.
Tetracarboxylic compound 1 in the form of free acid does
not show any noticeable solubility in pure water at neutral pH
(a behaviour characteristic of vast majority of polycarboxylic
fullerene derivatives). However, the corresponding tetrasodium
salt (designated below as 1-Na) and tetrapotassium salt (1-K)
of 1 exhibited high solubility in water (>100 mg cm–3) giving
transparent solutions stable over at least 6 months. High solubility
of 1-Na allowed us to investigate its biological activity in few
in vitro and in vivo systems.
Table 1 Antioxidant and antiradical activity of compound 1-Na compared
to butylated hydroxytoluene (BHT).
MDA
Standard Luminol chemi- Standard
Compound
Control
1-Na (10–5 m) 47
BHT (10–5 m) 59
concentration deviation luminescence
deviation
(%)
(% to control) (%)
(% to control)
100
12
7
100
82
8
7
5
7
81
determined to be 46% for the fullerene-treated mice in comparison
with the control group. Thus, compound 1-Na showed a weak,
but still evident anticancer action in the mouse tumor model.
The synthesized fullerene derivative 1 (in the form of sodium
salt) exhibited antiviral and anticancer activity. Although, the
revealed therapeutic effects are not strong enough to consider
practical implementation of this compound, its very low toxicity
makes the application of this compound as a biocompatible form
of fullerene C60 promising, e.g., for antioxidative, neuroprotective
or drug delivery applications.
To reveal the performance of 1-Na as a potential neuropro-
tective agent, the inhibition of the lipid peroxidation and radical
scavenging activity were studied in ex vivo models using a well-
known antioxidant BHT (3,5-di-tert-butyl-4-hydroxytoluene)
as a reference compound. Malondialdehyde (MDA) production
characterizing lipid peroxidation level was measured spectro-
photometrically in the rat brain homogenates after the reaction
with thiobarbituric acid.23 Radical scavenging activity of 1-Na
was determined using the luminol chemiluminescence assay
applied to the rat brain homogenates after initiation of the lipid
peroxidation with tert-butyl hydroperoxide.24,25 The obtained
results are shown in Table 1. Compound 1-Na at a concentration
of 10–5 mol dm–3 suppressed MDA production down to 47% of its
control value in comparison with 59% for BHT applied at the
same concentration. Thus, 1-Na showed somewhat higher
antioxidant activity compared to the reference antioxidant.
However, the chemiluminescence of luminol was quenched only
by 18% which implies that 1-Na does not perform as an efficient
scavenger of active oxygen species formed during the lipid
peroxidation. It is even possible that 1-Na catalyzes peroxide
dismutation like some other fullerene derivatives reported
previously.8 Detailed study of this aspect is now underway.
In conclusion, we have synthesized a novel highly water-
soluble C60 derivative which exhibited promising biological pro-
perties such as low toxicity in combination with noticeable anti-
viral, antitumor and antioxidant activity.
First of all, we studied a cytotoxicity of compound 1-Na in
a range of eukaryotic cell lines (murine leukemia cells L1210/0,
human T-lymphocyte cells Molt4/C8 and CEM/0, human cervix
carcinoma cells HeLa and human osteosarcoma cells Ost/TK–).
At the highest investigated dose of 100 mg cm–3 (86 mm) this
derivative did not reveal any sign of cytostatic action.
Antiviral activity of 1-Na was investigated against HIV-1 and
HIV-2 in an in vitro assay according to procedure described
previously.21 The compound showed comparable activity against
both virus strains and was characterized by mean effective inhibitory
concentrations IC50 of 20 10 (HIV-1) and 29 1 mM (HIV-2).
The results suggest that 1-Na is not a very promising antiviral
agent since its activity is ca. 10 times lower compared to many
other fullerene derivatives.17,21,22 However, the observed low
cytostatic activity of this compound implies that its biological
activity might be investigated using various in vivo models.
The acute toxicity of 1-Na was evaluated after intraperitoneal
(i.p.) injection in mice (male BDF1 hybrids, weight 20–25 g)
with determination of main toxicological parameters: maximum
tolerable dose LD0, median lethal dose LD50 and absolute lethal
dose LD100. The values were found to be 400, 500 and 700 mg
kg–1, respectively, characterizing this compound as a very low-
toxic substance.
The anticancer activity of 1-Na was assessed on murine
leukemia P388 tumor-bearing mice. For this purpose, leukemia
P388 (106 cells) was i.p. inoculated into male BDF1 hybrid mice.
After that, compound 1-Na was given at the dose of 167 mg kg–1
(1/3 LD50) as i.p. injections on the 1st, 3rd, 4th and 6th days after
tumor transplantation. The increase of average lifespan (ILS) was
This work was supported by the Russian Foundation for
Basic Research (grant nos. 12-03-31719-mol_a and 12-03-33031-
mol_a_ved), the Russian President Science Foundation (grant no.
MK-6177.2013.3), Russian Ministry for Science and Education
(contract no. 14.512.11.0127) and the KU Leuven (GOA 10/014).
61,61-Bis(3,4-dicarboxyphenyl)methano[1,9](C60-Ih)[5,6]fullerene 1.
Compound 5 (300 mg, 0.27 mmol) was dissolved in 150 ml of chloro-
benzene under heating at 70°C. The addition of the solution of 122 mg
of MeONa (1.13 mmol, 1.05 equiv.) in 3 ml of MeOH–H2O (9:1 v/v)
to the solution of 5 induced immediate precipitation of some insoluble
product and decolouration of the solution. The mixture was heated at
70°C for further 15 min and cooled. The precipiated solid was collected
by centrifugation, dissolved in 10 ml of deionized water and quenched
with 1 ml of 5% HCl. The precipitated tetraacid 1 was collected by
centrifugation, washed several times with water and dried in vacuo. The
yield of 1 obtained as a dark brown powder was 275 mg (96%). 1H NMR
(DMSO-d6, 600 MHz) d: 7.86 (d, 2H), 8.75 (d, 2H), 8.80 (s, 2H).
13C NMR (DMSO-d6, 150 MHz) d: 56.34, 65.40, 128.68, 129.37, 132.85,
133.93, 134.32, 137.46, 140.45, 141.36, 142.08, 142.17, 142.85, 142.95,
143.81, 144.24, 144.54, 144.63, 144.77, 145.13, 145.15, 146.50, 148.56,
168.58, 168.94. ESI-MS, m/z: 1061.0 [M–H]–.
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Received: 30th July 2013; Com. 13/4174
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