A new multi-charged C60 derivative: Synthesis and biological properties

Article abstract of DOI:10.1002/1099-0690(200209)2002:17<2928::AID-EJOC2928>3.0.CO;2-I

A new water-soluble multi-charged monoadduct fullero[60]pyrrolidine derivative with three ethylene glycol chains and three ammonium groups has been synthesized by means of two alternative synthetic pathways. Increasing the concentration of this C₆₀ derivative did not show a significant modification of concentration of superoxide anion radical O₂-, generated by the xanthine/xanthine oxidase system. Moreover, this C₆₀ derivative was ineffective for neuroprotection in quantitative assessment of a neuronal injury considered to occur via radicals. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200209)2002:17<2928::AID-EJOC2928>3.0.CO;2-I

FULL PAPER
A New Multi-Charged C60 Derivative: Synthesis and Biological Properties
Claudia Cusan,[a] Tatiana Da Ros,*[a] Giampiero Spalluto,[a] Sarah Foley,[b]
Jean-Marc Janot,[b] Patrick Seta,[b] Christian Larroque,[c] Maria Cristina Tomasini,[d]
Tiziana Antonelli,[d] Luca Ferraro,[d] and Maurizio Prato[a]
Keywords: Cycloaddition / Fullerenes / Neuroprotection / Bioorganic chemistry
A
new water-soluble multi-charged monoadduct fuller-
generated by the xanthine/xanthine oxidase system. More-
over, this C60 derivative was ineffective for neuroprotection
in quantitative assessment of a neuronal injury considered to
o[60]pyrrolidine derivative with three ethylene glycol chains
and three ammonium groups has been synthesized by means
of two alternative synthetic pathways. Increasing the concen- occur via radicals.
tration of this C60 derivative did not show a significant modi-
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
·−
fication of concentration of superoxide anion radical O2
,
Introduction
these species during oxidative stress is capable of inducing
biological damage.[11] The antioxidant properties of C60
could therefore be used to prevent free radical stress and all
the related diseases.
In this field, several C60 derivatives have been investigated
as neuroprotecting agents.[12,13] In particular, two C60-tris-
(malonic acid) derivatives C60[C(COOH)2]3, formed as two
different isomers, called C3 (1) and D3 (2), depending on
the addition pattern of the carbon cage, have proved to be
the most promising compounds for preventing neuronal
damage (Figure 1).[14,15]
The unusual spheroidal shape, the exclusive presence of
carbon atoms in their structures and the big π-electron
cloud make fullerenes good candidates for novel ap-
proaches to chemotherapy. Unmodified fullerenes, although
relatively soluble in some apolar solvents, are virtually in-
soluble in biologically relevant media.[1] Thus, the attach-
ment of hydrophilic groups to C60 is necessary to render
the fullerene soluble in aqueous solutions and hence more
amenable to biological experiments.[2] So far, fullerene de-
rivatives have shown interesting properties in different fields
such as apoptosis, neuroprotection, anti-HIV therapy, anti-
bacterial and DNA-photocleavage activities.[3Ϫ6] In particu-
lar, neuroprotection activity seems to be related to the
fullerene’s ability to take up many radicals on the carbon
cage (up to 34 methyl radicals on a single C60 molecule, as
demonstrated by Krusic[7]).
The formation of reactive oxygen species (ROS), includ-
ing superoxide radical anion (O2·Ϫ), or ROO· and ·OH rad-
icals, is considered to be one of the processes involved in
neuronal injury. One mechanism of production of these
ROS is the hyperstimulation of N-methyl--aspartate
(NMDA) glutamate receptors.[8Ϫ10] The overproduction of
Figure 1. Isomeric C60-tris(malonic acid) derivatives 1 (C3) and 2
(D3)
The presence of six carboxylic functions improves the
solubility in biological media and avoids the aggregation
problems commonly encountered in polar solvents. As a re-
sult of its better penetration into lipids, compound 1 has
been studied in more detail than other isomers and it has
been demonstrated to be a good radical scavenger against
superoxide anions and hydroxyl radicals at micromolar con-
centrations. In vivo studies have also shown that 1 is effect-
ive in neuroprotection by chronic administration.[15]
[a]
`
Dipartimento di Scienze Farmaceutiche, Universita di Trieste,
Piazzale Europa 1, 34127 Trieste, Italy
Fax: (internat.) ϩ 39-040/52-572
E-mail: daros@units.it
I. E. M. UMR 5635 CNRS,
[b]
[c]
[d]
1919 route de Mende, 34293, Montpellier, Cedex 5, France
INSERM U128
1919 route de Mende, 34293, Montpellier, Cedex 5, France
Dipartimento di Medicina Clinica e Sperimentale Ϫ Sezione di
`
Farmacologia, Universita di Ferrara,
Via Fossato di Mortara 17Ϫ19, 44100 Ferrara, Italy
2928
 WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0917Ϫ2928 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 2928Ϫ2934

A New Multi-Charged C60 Derivative
FULL PAPER
Starting from these observations, we have designed a new
Compounds 4 and 7 were the common key-products util-
water-soluble fullerene derivative 3, with three ethylene ized in the two different synthetic pathways.
glycol chains and the same number of ammonium groups
In synthetic route 1 (Scheme 2), 7 was allowed to react
for increasing water solubility, as a potential neuroprotect- with an excess of tert-butyl bromoacetate to obtain 8,
ing agent.
which, after simultaneous deprotection of amino and carb-
oxylic functions under catalytic hydrogenation conditions,
gave amino acid 9. The cycloaddition step was performed
in the presence of C60, paraformaldehyde and 9 in refluxing
toluene (Scheme 2).[18Ϫ20]
The rational design of this compound was aimed at ob-
taining a mono-functionalized water-soluble C60 derivative
with increased potential free radical loading capacity, since
a higher number of addends on the fullerene moiety should
decrease its radical scavenging properties.[7]
Results and Discussion
Synthesis
Two different synthetic approaches have been utilized to
reach the goal, as depicted below, in which a solubilizing
appendage was introduced on a suitably functionalized
fulleropyrrolidine or was already present in the amino acid
used in the cycloaddition step.
In both cases, the synthetic pathway started from the
monoprotected 2,2Ј-(ethylenedioxy)diethylamine (4),[16]
which was allowed to react with benzyl bromoacetate to
give amino ester 5.[17] Protection of the amino group with
benzyl chloroformate led to 6, which was deprotected with
trifluoroacetic acid to yield the corresponding salt 7
(Scheme 1).
Scheme 2. i: K2CO3, tert-butyl bromoacetate, THF; ii: H2, Pd/C,
methanol; iii: C60, paraformaldehyde, toluene, ∆; iv: TFA, CH2Cl2;
v: EDC·HCl, HOBt, 4, dioxane; vi: TFA, CH2Cl2
Fulleropyrrolidine 10 was obtained in 20% yield, while
subsequent deprotection with trifluoroacetic acid afforded
the bis(acid) 11. The latter was coupled with 4 in the pres-
ence of EDC·HCl and HOBt to give 12, which, after depro-
tection, afforded the desired final compound 3. However,
purification of derivative 12 was very difficult and the yield
very low (5%).
To circumvent this problem, we focused our attention on
synthetic pathway 2, in which the final ramification (amino
acid 15) was performed before the cycloaddition. Pathway 2
starts with the reaction of key-product 4 with bromoacetyl
bromide in the presence of TEA, leading to compound 13
(Scheme 3). This compound was allowed to react with 7,
giving amino ester 14, which was treated with hydrogen in
the presence of Pd/C (10%), leading to amino acid 15. The
cycloaddition step was performed in the presence of C60,
paraformaldehyde and 15 in refluxing toluene affording the
Scheme 1. i: ZCl, DCM; ii: TFA, DCM
Eur. J. Org. Chem. 2002, 2928Ϫ2934
2929

T. Da Ros et al.
FULL PAPER
desired product 12 with higher yields (14%) and avoiding
Derivative 13 was used as alkylating agent with N-
the purification problems found in synthetic pathway 1. De- (benzyloxycarbonyl)-2,2Ј-bis(ethylenedioxydiethylene)-
protection of 12 with trifluoroacetic acid afforded the de- diamine (17)[21] to obtain 18, which gave 19 after hydro-
sired compound 3 in quantitative yield.
genolysis. Total deprotection of 19 by HCl/methanol
afforded the desired triamino compound 16 as its hydro-
chloride salt.
Reactivity Towards Radicals: Chemical Assays for
Superoxide Anion
As easily observed by UV/Vis spectrophotometry,[22] so-
lutions of 3 show aggregation at concentration values of
approximately 10Ϫ5  not only in water but also in mixtures
of water/methanol (75:25) and water/DMF (75:25) where
the solubility reaches values of 0.34 and 0.46 m, respect-
ively. This was deduced from the deviation from the
LambertϪBeer law. In fact, the optical density changes
non-linearly with the fullerene concentration increase, lead-
ing to saturation above 10Ϫ5 .
The reactivity of the fullerene derivative 3 towards O2
·Ϫ
,
thought to be one of the oxy radicals involved in neuronal
degeneration,[8Ϫ10] was studied by spectrophotometric as-
says employing the xanthine/xanthine oxidase system for
the generation of superoxide radicals and ferricytochrome c
reduction for the measurement of superoxide concentration.
Conversion of xanthine to uric acid by xanthine oxidase in
the presence of oxygen is accompanied by the production
of O2·Ϫ. The capture of this species in situ in a buffer solu-
tion is possible using ferricytochrome c (Fe3ϩ) resulting in
the formation of ferrocytochrome c (Fe2ϩ) which absorbs
optically at 550 nm.[23] The absorbance change (increase in
absorbance ∆ε(550) ϭ 2.1 ϫ 104 Ϫ1 cmϪ1) due to the pro-
duction of ferrocytochrome by an electron-transfer reaction
between superoxide ions and ferricytochrome c is a measure
of the reaction. In all experiments the optical density in-
creased rapidly during the first 10 min and then leveled off
between 20 and 30 min. The change in absorbance at
550 nm during the first 5 min of the reaction was used to
calculate the reduction of the ferricytochrome c and was
found to be linear within the 95% confidence limit. The
presence of 3 did not cause a significant modification of
the concentration of O2·Ϫ, within the experimental errors
observed for the determination of the reduced cytochrome
c (Table 1).
Scheme 3. i: Bromoacetyl bromide, TEA, CH2Cl2; ii: 7, TEA,
CH2Cl2; iii: H2, Pd/C, methanol; iv: C60, paraformaldehyde, tolu-
ene, ∆
To better evaluate the effect of fullerene derivative 3 as a
neuroprotecting agent, the branched chain without C60 (16)
was synthesized for use as a control in the biological as-
says (Scheme 4).
Table 1. Kinetics of cytochrome c reduction by superoxide anion
(O2·Ϫ); all values are the average of three experiments
[Compound 3] [µ]
µ reduced cytochrome c minϪ1
0.0
2.26
5.64
11.3
505 Ϯ 50
410 Ϯ 41
405 Ϯ 40
489 Ϯ 48
Scheme 4. i: 13, TEA, CH2Cl2; ii: H2, Pd/C, methanol; iii: HCl,
methanol
2930
Eur. J. Org. Chem. 2002, 2928Ϫ2934

A New Multi-Charged C60 Derivative
FULL PAPER
Biological Evaluation
is ineffective. A possible explanation of this undesirable tox-
icity can be ascribed to the lipophilic character of 3, which,
coupled with its hydrophilic part, confers surfactant proper-
ties favoring its interaction with cell membranes with their
possible disruption and subsequent cell death.
We analyzed the fullerene derivative 3 in a model of neuro-
protection in cerebral cortical cells. In fact, for evaluating
the protection against glutamate-induced neuronal death,
compound 3 and its analogue 16 were tested in cortical cell
culture treated with 100 µ glutamate. As shown in Fig-
ure 2, 24 h after exposure to glutamate a metabolic impair-
ment was observed, as indicated by the decrease (53 Ϯ 4%,
n ϭ 25, of the control cells) in the cell viability based on
the MTT assay.
Conclusion
A new water-soluble multi-charged monoadduct fullero-
[60]pyrrolidine derivative with three ethylene glycol chains
and three ammonium groups has been synthesized by me-
ans of two alternative synthetic pathways. In spectrophoto-
metric assays using the reduction of ferricytochrome c, this
new C60 derivative did not show a significant reaction with
O2·Ϫ, the oxy radical considered to be one of the species
involved in neuronal degeneration.[8Ϫ10] In a neuroprotec-
tion model, in cerebral cortical cell death induced by glu-
tamate, the compound was not only found to be ineffective,
but also showed a significant concentration-dependent tox-
icity. Nevertheless, the results presented here give useful in-
formation about the chemical requirements necessary for a
C60 derivative to have neuroprotective activity. This seems
to be correlated more with the number of substitutions than
with their nature. An increased number of polar groups on
C60 probably prevents its interaction with the cell mem-
brane, and consequently its disruption. Further work in this
field should therefore focus on multiple addition products
to guarantee a better solubility and a lower degree of ag-
gregation than monoadducts.
Experimental Section
Synthesis
General Remarks: 1H and 13C NMR spectra were recorded at
200 MHz and 50 MHz, respectively, in CDCl3, unless otherwise
noted. Chemical shifts are given in parts per million (δ) relative to
tetramethylsilane. To record ES-MS spectra, the compounds were
dissolved in THF/methanol (4:1), unless otherwise noted. FT-IR
spectra were recorded using NaCl cells (oils) or KBr powder
(DRIFT system). Yields are reported as absolute values without
taking into account C60 recovery (30Ϫ40% of the initial fullerene
was routinely recovered). C60 was purchased from Bucky-USA
(99.5%); all other reagents and solvents were used as purchased
from Fluka, Aldrich, J. T. Baker and Cambridge Isotope Laborat-
ories. Silica gel NM Kieselgel 60 (70Ϫ230 mesh ASTM) was ob-
tained from MachereyϪNagel and Merck. The syntheses of 4,[16]
5[17] and 17[21] were performed as described in the literature.
Figure 2. Cell viability assessed with MTT assay 24 h after 100 µ
exposure (10 min) in cortical cell culture; effect of 3 and 16 in the
presence (panel A) and in the absence (panel B) of glutamate; the
results are expressed as a percentage of control cells not exposed
to glutamate; the values represent the mean Ϯ SEM; significantly
different from Glu and 3 (1 µ) ϩ Glu: ** P Ͻ 0.01; significantly
different from 3 (10 µ) ϩ Glu: oo P Ͻ 0.01 (panel A); significantly
different from 3 (1 µ): •• P Ͻ 0.01; significantly different from 3
(10 µ): ᭞᭞ P Ͻ 0.01 (panel B); according to one-way ANOVA
with Newman-Keuls test for multiple comparisons
Synthesis of 6: ZCl (7.5 mL, 44.8 mmol) in 40 mL of CH2Cl2 was
added to a solution of 5 (8.1 g, 20.3 mmol) in dichloromethane
(60 mL). The mixture was stirred over a period of 2 h at room temp.
and then washed with basic, acidic and neutral water. The organic
phase was dried with CaCl2 and the solvents were removed by evap-
oration, giving the pure product 6 as a colorless oil (10.8 g,
20.3 mmol, yield 99%). FT-IR: ν˜ ϭ 3367, 2936, 1715, 1707, 1508,
1460, 1365, 1245, 1178, 999, 914, 741 cmϪ1. 1H NMR: δ ϭ 1.41 (s,
9 H), 3.24 (m, 2 H), 3.39Ϫ3.49 (m, 6 H), 3.52Ϫ3.64 (m, 4 H), 4.14
Pretreatment with 3 (1Ϫ100 µ) surprisingly enhanced
the glutamate-induced cell death, while 16 (10Ϫ100 µ) did
not effect the injury induced by the excitatory amino acid
(Figure 2, panel A). For better analyzing these results, the
effect of both derivatives (3, 16) alone on cell viability was
tested (Figure 2, panel B). It is clearly evident that while
compound 3 (1, 10, 30, 100 µ) behaves as a toxic agent
since it induces a concentration-dependent decrease of the
cell viability, derivative 16 at 30 and 100 µ concentration and 4.19 (s, 2 H), 4.98 (br. s, 1 H), 5.06 and 5.07 (s, 2 H), 5.15 (s,
Eur. J. Org. Chem. 2002, 2928Ϫ2934 2931

T. Da Ros et al.
FULL PAPER
2 H), 7.39Ϫ7.24 (m, 10 H) ppm. 13C NMR: δ ϭ 28.3, 40.2, 47.8, yielding 11 as a brown powder (35.0 mg, 34.6 µmol, yield 97%).
48.4, 50.1, 66.6, 67.3, 67.4, 70.0, 70.1, 70.2, 79.0, 127.6, 127.7,
FT-IR: ν˜ ϭ 3568, 2929, 1641, 1119, 520 cmϪ1. 1H NMR (DMSO):
127.8, 127.9, 128.1, 128.2, 128.2, 128.3, 128.4, 135.3, 136.3, 155.7, δ ϭ 2.96 (m, 2 H), 3.46 (s, 4 H), 3.37Ϫ3.54 (m, 2 H), 3.62 (t, J ϭ
169.6 ppm. ES-MS: m/z ϭ 531 [MHϩ].
4.5 Hz, 2 H), 3.76 (m, 4 H), 4.03 (t, J ϭ 6.0 Hz, 2 H), 4.59 (s, 4 H)
ppm. 13C NMR (DMSO): δ ϭ 53.7, 54.3, 59.2, 69.3, 69.9, 70.5,
70.6, 136.1, 140.0, 141.8, 141.9, 142.1, 142.5, 143.0, 144.4, 145.1,
145.3, 145.9, 146.0, 146.1, 147.2, 155.0, 176.0 ppm. ES-MS: m/z ϭ
1112 [MHϩ]. UV/Vis (CH2Cl2): λmax ϭ 306, 430, 461 (sh), 691 nm.
C72H22N2O6 (1011): calcd. C 85.54, H 2.19, N 2.77; found C 83.60,
H 2.23, N 2.65.
Synthesis of 7: A solution of 6 (12.5 g, 23.6 mmol) in CH2Cl2
(60 mL) and TFA (60 mL) was stirred at room temp. for 24 h then
the solvent and the acid were eliminated by evaporation and the
pure product 7 was isolated as a colorless oil (12.9 g, 23.6 mmol,
yield 99%). FT-IR: ν˜ ϭ 2945, 1710, 1690, 1463, 1360, 1197, 997,
1
803, 702, 601, 445 cmϪ1. H NMR: δ ϭ 3.03 (m, 2 H), 3.42Ϫ3.61
(m, 10 H), 4.06 (br. s, 2 H), 5.11 (m, 4 H), 7.38Ϫ7.13 (m, 10 H), Synthesis of 12: A solution containing 11 (15.0 mg, 14.8 µmol),
7.58 (br. s, 3 H) ppm. 13C NMR: δ ϭ 39.7, 48.1, 48.6, 49.9, 50.5, EDC·HCl (7.1 mg, 37.0 µmol), HOBt (3.5 mg, 37.0 µmol) and 4
66.6, 67.1, 67.7, 69.4, 70.1, 70.2, 127.7, 128.1, 128.2, 128.5, 128.5,
128.6, 129.1, 135.3, 136.3, 156.4, 169.8 ppm. EI-MS: m/z ϭ 431
(20) [Mϩ], 91 (100).
(9.2 mg, 37.0 µmol) in dioxane (10 mL) was stirred at 50 °C for 1 h
then the mixture was cooled to room temp. and the product was
purified by chromatography (eluent: toluene/methanol, 4:1). Com-
pound 12 was precipitated by addition of diethyl ether to a CH2Cl2
solution yielding a brown powder (1.1 mg, 0.7 µmol, yield 5%). ES-
MS: m/z ϭ 1472 [MHϩ].
Synthesis of 8: A solution of 7 (3.0 g, 5.5 mmol), K2CO3 (1.9 g,
13.8 mmol) and tert-butyl bromoacetate (2.0 mL, 13.8 mmol) in
THF (50 mL) was stirred at room temp. over a period of 48 h. The
solvent was then removed by evaporation and the crude material
was purified by chromatography (eluent: CH2Cl2/ethyl acetate, 9:1)
Synthesis of 13: Bromoacetyl bromide (1.2 mL, 13.9 mmol) was ad-
ded dropwise to a cold solution of 4 (3.4 g, 13.9 mmol) and TEA
(2.2 mL, 15.9 mmol) in CH2Cl2 (20 mL). After 1 h, the mixture was
to give 8 as a colorless oil (2.2 g, 3.4 mmol, yield 61%). FT-IR: ν˜ ϭ
1
2931, 1720, 1710, 1460, 1145, 744, 701 cmϪ1. H NMR: δ ϭ 1.41 washed with brine and the collected organic phases were dried with
(s, 18 H), 2.89 (t, J ϭ 5.9 Hz, 2 H), 3.44 (s, 4 H), 3.45 (m, 4 H),
CaCl2. The product was purified by chromatography (eluent: petro-
3.54 (m, 6 H), 4.13 and 4.17 (s, 2 H), 5.06 and 5.07 (s, 2 H), 5.14 leum ether/ethyl acetate, 7:3, then ethyl acetate) and crystallized
(s, 2 H), 7.27 (m, 5 H), 7.32 (m, 5 H) ppm. 13C NMR: δ ϭ 28.2, from diethyl ether yielding 13 as a white solid (4.5 g, 12.0 mmol,
50.3, 53.3, 56.4, 56.4, 56.7, 66.7, 67.4, 70.2, 70.4, 70.4, 70.5, 80.8,
127.7, 127.9, 128.2, 128.4, 128.5, 135.5, 136.4, 155.9, 156.2, 169.7, 1641, 1539, 1250, 1100, 657 cmϪ1
170.7 ppm. EI-MS: m/z ϭ 658 (5) [Mϩ], 601 (3), 557 (50), 528 (10), 3.26 (m, 2 H), 3.51 (m, 6 H), 3.58 (m, 4 H), 3.84 (s, 2 H), 4.98 (br.
yield 86%). M.p. 57Ϫ58 °C. DRIFT: ν˜ ϭ 3296, 3077, 2875, 1711,
.
1H NMR: δ ϭ 1.43 (s, 9 H),
501 (100), 393 (30), 91 (100).
s, 1 H), 6.93 (br. s, 1 H) ppm. 13C NMR: δ ϭ 28.1, 39.8, 40.0,
69.0, 69.8, 69.9, 78.8, 155.6, 165.6 ppm. ES-MS: m/z ϭ 370 [MHϩ].
C13H25BrN2O5 (369.3): calcd. C 42.29, H 6.82, N 7.59; found C
42.00, H 6.72, N 7.51.
Synthesis of 9: A solution of 8 (1.0 g, 1.5 mmol) in methanol
(12 mL) was stirred under hydrogen pressure with 10% Pd/C
(0.05 g) as catalyst. After 24 h, the crude material was purified by
filtration through Celite and the solvent evaporated to yield 9 as a
colorless oil (0.6 g, 1.5 mmol, yield 99%). FT-IR: ν˜ ϭ 3440, 2926,
Synthesis of 14: Compound 13 (1.9 g, 5.2 mmol) was added in seven
portions (one per day) to a solution of 7 (200.0 mg, 0.4 mmol) and
1727, 1646, 1368, 1150, 847 cmϪ1 1H NMR (D2O): δ ϭ 1.28 (s, 151.2 µL (1.1 mmol) of TEA in CH2Cl2 (50 mL). After one week,
.
18 H), 2.74 (t, J ϭ 5.4 Hz, 2 H), 3.11 (t, J ϭ 5.1 Hz, 2 H), 3.31 (s, the mixture was purified by chromatography (eluent: ethyl acetate,
4 H), 3.45 (m, 2 H), 3.51 (m, 6 H), 3.62 (t, J ϭ 4.0 Hz, 2 H) ppm. then ethyl acetate/2-propanol, 4:1), yielding 14 as a colorless oil
13C NMR (D2O): δ ϭ 26.9, 46.4, 48.6, 52.5, 56.0, 65.0, 68.2, 69.0, (172.0 g, 0.2 mmol, 46% yield). DRIFT: ν˜ ϭ 3398, 2923, 1702,
69.1, 69.2, 82.9, 171.6 ppm. EI-MS: m/z ϭ 434 (10) [Mϩ], 333 (30),
1687, 1454, 1284, 1132, 843 cmϪ1 1H NMR: δ ϭ 1.41 (s, 18 H),
.
116 (60), 70 (100).
2.35 (br. s, 2 H), 2.75 (t, J ϭ 5.1 Hz, 2 H), 3.26 (m, 4 H), 3.54 (s,
4 H), 3.36Ϫ3.48 (m, 30 H), 3.55 (s, 4 H), 4.10 and 4.16 (s, 2 H),
5.05 and 5.07 (s, 2 H), 5.14 (s, 2 H), 7.27 (m, 5 H), 7.33 (m, 5 H)
ppm. 13C NMR: δ ϭ 28.1, 38.3, 39.9, 47.5, 48.1, 49.7, 49.8, 53.2,
58.8, 66.3, 66.4, 67.0, 67.1, 69.0, 69.3, 69.7, 78.6, 127.2, 127.4,
127.5, 127.6, 127.8, 128.0, 128.1, 128.1, 134.9, 135.0, 135.8, 135.9,
155.4, 155.5, 155.7, 169.2, 170.4 ppm. ES-MS: m/z ϭ 1006 [MHϩ],
1029 [M ϩ Naϩ].
Synthesis of 10: A mixture of C60 (502.0 mg, 0.7 mmol), paraform-
aldehyde (104.5 mg, 3.5 mmol) and 9 (502.0 mg, 1.2 mmol) in tolu-
ene (500 mL) was heated at reflux for 2 h. After cooling the solu-
tion to room temp., the product was purified on a chromatography
column (eluent: toluene, then toluene/ethyl acetate, 9:1), and then
the pure compound 10 was precipitated by addition of methanol
to a CH2Cl2 solution giving a brown solid (160.5 mg, 0.2 mmol,
yield 20%). DRIFT: ν˜ ϭ 2908, 2805, 1735, 1461, 1360, 1146, 759, Synthesis of 15: A solution of 14 (1.1 g, 1.1 mmol) in methanol
1
518, 441 cmϪ1. H NMR: δ ϭ 1.45 (s, 18 H), 2.96 (t, J ϭ 5.8 Hz,
(20 mL) was stirred under hydrogen pressure, with 10% Pd/C
2 H), 3.35 (t, J ϭ 5.5 Hz, 2 H), 3.49 (s, 4 H), 3.69 (t, J ϭ 6.1 Hz, (0.02 g) as catalyst. After 24 h, the crude material was purified by
2 H), 3.79 (m, 4 H), 4.03 (t, J ϭ 5.5 Hz, 2 H), 4.49 (s, 4 H) ppm.
13C NMR: δ ϭ 28.2, 53.4, 54.2, 56.7, 58.4, 70.4, 70.5, 70.5, 70.6,
filtration through Celite and the solvent evaporated, giving the pure
product 15 as a colorless oil (0.9 g, 1.1 mmol, yield 99%). FT-IR:
70.8, 80.8, 136.1, 140.0, 141.8, 142.0, 142.1, 142.5, 143.0, 144.4, ν˜ ϭ 3335, 2870, 1700, 1530, 1365, 1250, 1115, 730 cmϪ1. 1H NMR
145.2, 145.3, 145.6, 145.9, 146.0, 146.1, 147.2, 155.0, 170.7 ppm. (D2O): δ ϭ 1.24 (s, 18 H), 2.63 (t, J ϭ 4.9 Hz, 4 H), 3.06 (t, J ϭ
ES-MS: m/z ϭ 1123 [MHϩ]. UV/Vis (CH2Cl2): λmax ϭ 325, 430
nm. C80H38N2O6 (1122): calcd. C 85.55, H 3.41, N 2.49; found C
85.50, H 3.32, N 2.48.
5.1 Hz, 4 H), 3.14 (s, 4 H), 3.25 (br. t, J ϭ 4.9 Hz, 2 H), 3.31Ϫ3.42
(m, 20 H), 3.48 (s, 4 H), 3.89 (m, 8 H) ppm. 13C NMR: δ ϭ 28.2,
38.8, 40.0, 58.5, 58.5, 58.6, 65.8, 69.2, 69.8, 70.0, 78.8, 155.7, 169.3,
170.5 ppm. ES-MS: m/z ϭ 783 [MHϩ].
Synthesis of 11: Trifluoroacetic acid (5 mL) was added to a solution
of 10 (40.0 mg, 35.6 µmol) in CH2Cl2 (5 mL) and the reaction mix-
ture was stirred for 24 h at room temp. The solvent was evaporated
and the salt was washed with toluene and dried under vacuum,
Synthesis of 12: A mixture of C60 (52.0 mg, 72.0 µmol), paraformal-
dehyde (10.8 mg, 0.3 mmol) and 15 (283.0 mg, 0.4 mmol) in toluene
(50 mL) was heated at reflux for 1 h. After cooling the solution to
2932
Eur. J. Org. Chem. 2002, 2928Ϫ2934

A New Multi-Charged C60 Derivative
FULL PAPER
room temp., the product was purified by chromatography (eluent:
toluene, then toluene/methanol, 97:3) and then compound 12
(14.0 mg, 9.5 µmol, yield 14%) was precipitated as a brown solid
by addition of diethyl ether to a CH2Cl2 solution. DRIFT: ν˜ ϭ
3424, 2918, 1703, 1680, 1456, 1371, 1092, 452 cmϪ1. 1H NMR: δ ϭ
1.43 (s, 18 H), 2.82 (t, J ϭ 4.8 Hz, 2 H), 3.24Ϫ3.39 (m, 12 H),
3.46Ϫ3.57 (m, 16 H), 3.61 (s, 4 H), 3.78 (m, 4 H), 4.05 (t, J ϭ
5.7 Hz, 2 H), 4.50 (s, 4 H), 5.21 (br. t, J ϭ 5.7 Hz, 2 H), 7.54 (br.
s, 2 H) ppm. 13C NMR: δ ϭ 28.4, 39.0, 40.2, 40.4, 54.1, 55.1, 59.4,
68.4, 69.8, 70.2, 70.2, 70.7, 79.2, 141.8, 140.1, 136.7, 142.0, 142.1,
142.5, 143.0, 144.4, 145.2, 145.3, 145.5, 145.9, 146.0, 146.1, 147.2,
154.8, 155.9, 170.7 ppm. ES-MS: m/z ϭ 1472 [MHϩ]. UV/Vis
(CH2Cl2): λmax ϭ 314, 457 (sh), 544 (sh), 702 nm. C94H66N6O12
(1472): calcd. C 76.72, H 4.52, N 5.71; found C 74.90, H 4.56,
N 5.46.
Biology
General: Cytochrome c, xanthine and xanthine oxidase (Sigma)
were all used as received. All solvents used were of spectroscopic
grade and were used without further purification.
Spectrophotometric Assay for Superoxide Anion (O2·Ϫ): Superoxide
radicals were generated by employing the xanthine/xanthine oxid-
ase/cytochrome c system. The reaction was initiated by the addition
of xanthine oxidase (7.5 ϫ 10Ϫ3 units) to the incubation mixture
and the reaction was followed in terms of the reduction of
cytochrome c and the corresponding increase in the absorbance at
550 nm. The reduction of ferricytochrome c into ferrocytochrome
c was determined using the molar absorption coefficients of 9
mmolϪ1 cmϪ1 and 27.7 mmolϪ1 cmϪ1 for the oxidized and reduced
forms, respectively. Cytochrome c was fully oxidized as demon-
strated by the absence of the absorption band at 550 nm. All assays
were performed in triplicate at 25 °C. The incubation mixture con-
sisted of 10 µ cytochrome c, 0.05 m xanthine and 0.1 m EDTA
in a buffer solution of KH2PO4/K2HPO4 (50 m, pH ϭ 7.4), along
with the indicated concentration of fullerene derivative. A total vol-
ume of 3 mL was used for all experiments.
Synthesis of 3: Trifluoroacetic acid (2 mL) was added to a solution
of 12 (14.0 mg, 9.5 µmol) in CH2Cl2 (2 mL), and the reaction mix-
ture was stirred for 3 h at room temp. The solvent was then evapor-
ated and the salt washed with toluene and dried under vacuum,
yielding 3 as a brown powder (14.3 mg, 9.5 µmol, yield 99%).
DRIFT: ν˜ ϭ 3505, 2921, 1682, 1432, 1129, 832, 520, 414 cmϪ1. 1H
NMR (CD3OD): δ ϭ 2.86 (br. s, 2 H), 3.12 (br. t, 4 H), 3.20 (m, 4
H), 3.34Ϫ3.37 (m, 8 H), 3.51Ϫ3.71 (m, 20 H), 3.85 (m, 2 H), 4.06
(br. s, 2 H), 4.61 (br. s, 2 H) ppm. 13C NMR (CD3OD): δ ϭ 38.3,
38.8, 42.7, 57.8, 63.0, 66.0, 67.9, 68.7, 69.4, 69.5, 70.2, 135.8, 139.5,
141.3, 141.6, 142.0, 142.6, 144.0, 144.6, 144.8, 145.0, 145.6, 146.7,
154.4 ppm. ES-MS: m/z ϭ 1272 [Mϩ], 635 [M/2]ϩ. UV/Vis (H2O):
λmax ϭ 217, 271, 337 nm. C92H54F12N6O16 (1727): calcd. C 63.97,
H 3.15, N 4.86; found C 63.70, H 3.27, N 4.96.
Evaluation of Neuroprotection in Cortical Cell Culture: Cerebral
cortical cells were prepared from 1 d old SpragueϪDawley.[24] After
resuspension in the plating medium, the cells were counted and
then plated on poly--lysine-coated (5 µg/mL) 24-well plates
(NUNC) at a density of 5 ϫ 105 cells/plate. The culture medium
consisted of Eagle’s Basal Medium supplemented with inactivated
fetal calf serum, 25 m KCl, 2 m glutamine and 100 µg/mL genta-
mycin. Cultures were grown at 37 °C in a humidified atmosphere,
5% CO2/95% air. Cytosine arabinoside (10 µ) was added within
24 h of plating to prevent glial cell replication. The cultures were
used in experiments after 8 d in vitro. The culture medium was
removed and 100 µ glutamate was added in 1 mL of Mg2ϩ-free
KrebsϪRinger bicarbonate buffer at 37 °C in 5% CO2/95% air.
After 10 min, glutamate was quickly removed. The cells were re-
turned to the incubator in their culture medium for 24 h, when
injury was assessed. To determine the degree of neuroprotection,
the tested compounds (3, 16) were added to the cultures 20 min
before glutamate and maintained in contact with the cells during
and 30 min after glutamate exposure. At the end of the treatment
with the tested compounds, the cells were washed three times and
then returned to their culture medium. Quantitative assessment of
neuronal injury was obtained by a colorimetric assay for cell sur-
vival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-
ium bromide (MTT) as previously described by Mosmann.[25] In
live cells mitochondrial enzymes have the capacity to transform
MTT tetrazolium salt into MTT formazone. Briefly, MTT tetrazol-
ium salt was added to the cultures and incubated for 4 h at 37 °C.
The precipitated dye was dissolved in 2-propanol with 1  HCl
and quantified colorimetrically (absorbance at 570 nm). The results
were expressed as the percentage of vital neurons compared with
the cells not exposed to glutamate.
Synthesis of 18: Compound 13 (2.7 g, 7.2 mmol) was added in seven
portions (one per day) to a solution of 17 (100.0 mg, 0.3 mmol)
and TEA (151 µL, 1.1 mmol) in CH2Cl2 (50 mL). After one week,
the mixture was purified by chromatography (eluent: ethyl acetate,
then ethyl acetate/2-propanol, 24:1), yielding 18 as a colorless oil
(101.0 mg, 0.1 mmol, yield 34%). DRIFT: ν˜ ϭ 3333, 2869, 1713,
1531, 1455, 1390, 1366, 1246, 1115, 1045, 863, 779, 738, 699 cmϪ1
.
1H NMR: δ ϭ 1.40 (s, 18 H), 2.74 (t, J ϭ 11.8 Hz, 2 H), 3.12Ϫ3.66
(m, 38 H), 5.06 (s, 2 H), 5.19 (br. s, 2 H), 5.89 (br. s, 1 H), 7.27Ϫ7.35
(m, 5 H), 7.55 (br. s, 2 H) ppm. 13C NMR: δ ϭ 28.5, 39.1, 40.4,
40.9, 55.1, 59.5, 66.7, 69.1, 69.9, 70.2, 79.3, 128.2, 128.2, 128.5,
136.6, 156.0, 156.6, 170.9 ppm. ES-MS: m/z ϭ 449 [(M ϩ K)/2]ϩ,
858 [M]ϩ, 880 [M ϩ Na]ϩ, 898 [M ϩ K]ϩ.
Synthesis of 19: A solution of 18 (0.1 g, 0.1 mmol) in methanol
(10 mL) was stirred under hydrogen pressure, with 10% Pd/C
(0.05 g) as catalyst. After 24 h, the crude material was purified by
filtration through Celite, the solvent was evaporated to give 19 as
a colorless oil (84.0 mg, 0.1 mmol, yield 99%). FT-IR: ν˜ ϭ 3329,
1
2871, 1703, 1529, 1360, 1274, 1113, 733 cmϪ1. H NMR: δ ϭ 1.22
(s, 18 H), 2.64Ϫ2.76 (t, J ϭ 10.5 Hz, 2 H), 2.95Ϫ3.10 (m, 6 H),
3.18Ϫ3.30 (m, 8 H), 3.32Ϫ3.60 (m, 24 H) ppm. 13C NMR: δ ϭ
27.2, 38.3, 38.6, 39.1, 57.8, 65.9, 67.8, 68.3, 68.9, 69.1, 80.5, 157.6,
172.0 ppm. ES-MS: m/z ϭ 724 [M]ϩ, 747 [M ϩ Na]ϩ.
Synthesis of 16: HCl gas was bubbled through a cold solution of
19 (80.0 mg, 0.1 mmol) in methanol (15 mL) for 15 min, then the
solvent was removed by evaporation affording the pure product 16
as a colorless oil (95.3 mg, 0.1 mmol, yield 99%). FT-IR: ν˜ ϭ 3370,
Acknowledgments
1
2034, 1678, 1281, 1113, 1024 cmϪ1. H NMR: δ ϭ 2.98Ϫ3.08 (br.
m, 6 H), 3.24Ϫ3.35 (br. m, 6 H), 3.43Ϫ3.69 (m, 24 H), 3.87Ϫ3.95 This work was supported by the University of Trieste (Fondi 60%),
(br. s, 4 H) ppm. 13C NMR: δ ϭ 38.6, 55.9, 65.9, 68.1, 69.0, 69.9,
by MURST (PRIN 2001, prot. no. 2001063932), by CNR (Agenzia
165.8 ppm. EI-MS: m/z ϭ 439 (25), 482 (100), 525(75) [M]ϩ, 561 2000) and by the European Community (TMR program
(25) [M ϩ Cl]ϩ, 597 (25) [M ϩ 2 Cl]ϩ.
BIOFULLERENES, contract no. ERB FMRX-CT98-0192).
Eur. J. Org. Chem. 2002, 2928Ϫ2934
2933

T. Da Ros et al.
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