Anti-HIV properties of cationic fullerene derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.05.069

A series of regioisomeric bis-fulleropyrrolidines bearing two ammonium groups have been synthesized and their activities against HIV-1 and HIV-2 have been evaluated. Two trans isomers have been endowed with interesting antiviral properties, confirming the importance of the relative positions of the substituent on the C₆₀ cage. In addition, reduced amphiphilicity of molecules to other compounds previously reported decreases their cytotoxicity in CEM cell cultures. None of the compounds showed any inhibitory activity against a variety of DNA and RNA viruses other than HIV.

Full text of DOI:10.1016/j.bmcl.2005.05.069

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3615–3618
Anti-HIV properties of cationic fullerene derivatives
Silvia Marchesan,a Tatiana Da Ros,a,* Giampiero Spalluto,a
Jan Balzarinib and Maurizio Pratoa
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
bRega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 28 February 2005; accepted 11 May 2005
Abstract—A series of regioisomeric bis-fulleropyrrolidines bearing two ammonium groups have been synthesized and their activities
against HIV-1 and HIV-2 have been evaluated. Two trans isomers have been endowed with interesting antiviral properties, confirm-
ing the importance of the relative positions of the substituent on the C60 cage. In addition, reduced amphiphilicity of molecules to
other compounds previously reported decreases their cytotoxicity in CEM cell cultures. None of the compounds showed any inhib-
itory activity against a variety of DNA and RNA viruses other than HIV.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
This study allowed the identification of some structural
requirements necessary for bearing anti-HIV-1 proper-
ties. In fact, from this evaluation, it has been possible to
underline the fact that specific relative positions of the
two substituents (trans-2) and positive charges close to
the carbon cage are prerequisite for antiviral activity. In
contrast, bulky polar chains on a C60 sphere induce cyto-
toxicity and seem to reduce potency, suggesting a signifi-
cant steric control. The toxic action could be attributed to
the amphiphilic character of this class of compounds, as
recently reported.19,20
There is ample evidence to suggest that fullerene and its
derivatives possess properties which hint at their use in
biomedicine, such as DNA photocleavage,1,2 neuropro-
tection,3,4 and inhibition of apoptosis.5,6 Moreover,
Wudl and co-workers demonstrated that the HIV prote-
ase (HIV-P) could be complexed and inhibited by C60,
which perfectly fits into the catalytic site.7,8 However,
lack of solubility of fullerenes in polar solvents and
biological fluids restricts their use in pharmacological
studies.9 For this reason in the last decade many efforts
have been made to overcome this problem, obtaining
promising results.10,11
Taking into account these preliminary observations, we
synthesized, as potential anti-HIV agents, a series of C60
derivatives (3–7, Fig. 2) bearing two ammonium salts
close to the carbon cage, with the aim of defining an opti-
mal combination of substituents on the C60, which could
lead to good potency and low cytotoxicity. The trans-2
and trans-4, and the mixture of isomers have already been
used by other laboratories to perform biological studies
on growthinhibition of Escherichia coli (mixtureof regioi-
somers)21 and the respiratory chain inhibition (trans-2
and trans-4).22
Exploration of anti-HIV properties of fullerene
derivatives has been performed by different research
groups,12–16 and up to now, the best result has been that
of dendrofullerene 1 (Fig. 1), prepared by Brettreich and
Hirsch,10 which has an EC50 of 0.22 lM in human lym-
17
phocytes acutely infected by HIV-1LAI
.
Recently, our group has reported a series of di-substituted
water-soluble C60 derivatives capable of inhibiting HIV-1
infection in cells at low micromolar concentrations.18
Derivative 2, the trans-2 isomer, proved to be the most
interesting compound of this series in terms of activity.
2. Results and discussion
Derivatives 3–7 have been prepared by the [3+2] dipolar
cycloaddition of azomethine ylides to C60, as briefly
summarized in Scheme 1.23
Keywords: Fullerene; Fulleropyrrolidine; Bis-adducts; Anti-HIV activity.
*
Corresponding author. Tel.: +39 040 5583110; fax: +39 040 52572;
e-mail: daros@units.it
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.069

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S. Marchesan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3615–3618
Figure 1. Structures of compounds 1 and 2.
Table 1. EC50 and CC50 assay results for compounds 3–7
Compound
EC50, lMa
HIV-2
0.21 ( 0.07) 0.2 to 1.0
CC50, lMa CEM cells
HIV-1
3
4
5
6
7
2.93 ( 1.20)
0.35 ( 0.07) 0.70 ( 0.42) 9.04 ( 0.18)
1.08 ( 0.57) 2.50 ( 1.90) 12.5 ( 7.54)
>25 >25
2.50 ( 0.71) >10
>125
28.7 ( 1.27)
a Values are means of three experiments; standard deviation is given in
parentheses.
HIV-2(ROD), as previously described.26,27 The cytostat-
ic activity was also evaluated in CEM cell cultures (Ta-
ble 1).
As shown in Table 1, compounds 3 and 4 showed inter-
esting anti-HIV properties in the high nanomolar range,
confirming the observations previously reported18
regarding the preferred position of the substituent on
the carbon cage of C60. In fact, the trans series (3–5)
has proved to be more potent than the corresponding
isomer cis-3 (7) (about 2- to 10-fold), while 6 (equatori-
al) turned out to be totally inactive.
Figure 2. Structures of compounds 3–7.
The reaction of C60 with formaldehyde and N-methyl-
glycine in refluxing toluene afforded a mixture of mono-
adduct and bis-adducts. After the monoadduct was
isolated, the isomeric bis-adducts 8–12 were purified
by flash chromatography and then by preparative
HPLC with a purity >99%.24,25 Compounds 3–7 were
obtained by methylation of derivatives 8–12 by methyl
iodide and their purity was checked by ES-MS.
Analyzing the trans series, it seemed that compounds 3
and 4 showed the best biological profiles (0.21 and
0.35 lM, respectively), while a shift in the pyrrolidine
ring to the trans-4 position led to a 5-fold decrease of
potency against HIV-1. Furthermore, the peculiar activ-
ity versus HIV-2 should be noticed. In fact, while com-
pound 2 was almost inactive against this subtype
(>4 lM), compounds 3 and 4 showed an interesting
activity against both virus subtypes.
The fullerene derivatives were administered to lympho-
cyte CEM cell cultures infected with HIV-1(IIIB) or
Scheme 1. Synthesis of compounds 3–7.

S. Marchesan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3615–3618
3617
A different analysis on toxicity should be performed, con-
sidering the different profiles between substituent position
and toxicity with respect to the anti-HIV properties. In
fact, considering both anti-HIV and cytotoxicity proper-
ties, the best compounds of this series were the trans-3 iso-
mer, which presents a CC50/EC50 ratio of 26, which was
higher than reference compound 2 (CC50/EC50 = 12).
molecular modeling studies, and to Dott. Fabio Hollan
(CSPA, University of Trieste) for his continuous sup-
port with mass spectrometry. This work was carried
out with partial support from the EU (Project No.
QLRT-2000-0291 and RTN network ÔWONDER-
FULLÕ), MIUR (cofin prot. 2004035502), and the Uni-
versity of Trieste.
Preliminary computational studies were performed on
compounds 2 and 3 utilizing MOE. The dipolar mo-
ments were 8.8 D for 2 and 3.2 for 3. The relative hydro-
phobic surface, expressed as (hydrophobic surface/total
surface) · 100, has also been calculated and in the case
of 2 it is 76%, while for 3 it is 92%, demonstrating a sig-
nificant reduction in the amphiphilic character of the
N,N-dimethylfulleropyrrolidine trans-2 with respect to
2. Therefore, differences in toxicity between the two con-
sidered compounds could be attributed to differences in
amphipathicity, a reason these compounds could be
considered useful probes for biological studies. These re-
sults are in agreement with our recent study on toxicity
and on the hemolytic effect of C60 derivatives, from
which the toxic effect also seems to be related to the
amphiphilic character of the molecules.19
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This study confirms and increases further our knowledge
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Acknowledgments
We are grateful to Mrs. Ann Absillis for her technical
assistance, to Dr. Paolo Braiuca for his help in the

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