1. Introduction

Currently, more than 90% of infectious diseases in humans are caused by viruses. The most well-known include the influenza virus, human immunodeficiency virus (HIV), and Ebola virus, which have caused serious damage [1–5]. Although several anti-HIV and anti-Ebola drugs, such as saquinavir, ritonavir, T20, lopinavir, ribavirin, tenofovir, and remdesivir, have been developed, their efficacy is not satisfactory. Severe acute respiratory syndrome (SARS), which broke out in China in 2003, is a respiratory infection caused by a coronavirus. So far, there is no specific medicine for SARS. The novel coronavirus, SARS-CoV-2, now circulating worldwide, is more infectious than SARS or HIV. For patients infected with SARS-CoV-2, there are no specific antiviral drugs.

Fullerenes and their derivatives exert significant inhibitory effects against HIV [1], herpes simplex virus (HSV) [2], influenza virus [6], Ebola virus [7], cytomegalovirus (CMV) [8], and other viruses in vitro and in vivo (Figure 1). Fullerenes and their derivatives, as a class of new, broad-spectrum antiviral drugs, have attracted increasing attention as a potential treatment for SARS-CoV-2.

Figure 1. Possible interactions between fullerene molecules and coronavirus, in which fullerene molecules inhibit virus replication.

Fullerenes are insoluble in water and polar media, their use in biomedicine is extremely complicated. To increase biocompatibility, the cage structure of fullerenes needs to be modified with appropriate hydrophilic functional groups. The modified structure and properties of the carbon cage may facilitate new applications in different biological systems. Because the fullerene carbon cage has multiple modifiable reaction sites, many fullerene derivatives with well-defined structures have been synthesized using regioselective functional group derivatization strategies. Therefore, fullerenes serve as ideal scaffolds for different bioactive drugs. 

2. Classification, Molecular Structures and Antiviral Activity of Fullerene and their Derivatives

In the past, hundreds of fullerene derivatives have been synthesized and used to inhibit viruses in vitro. Most of these derivatives are water soluble. These fullerene de-rivatives can be classified as the following six types: (1) amino acid, peptide, and primary amine derivatives; (2) piperazine and pyrrolidine derivatives; (3) carboxyl derivatives; (4) hydroxyl derivatives; (5) glycofullerene derivatives; and (6) fullerene complexes. Numerous antiviral studies have been conducted to evaluate fullerene C₆₀ and its derivatives.

Figure 2. The six types of water-soluble fullerene derivatives used as virus inhibitors.

In 1993, Friedman et al. [1] reported a landmark study based on model building and experimental evidence. Theoretical calculations revealed that the hydrophobic cavity of HIVP accommodates C₆₀ molecules; the spherical C₆₀ molecules fit perfectly within the active site, facilitating strong interactions between HIVP and fullerene.  Sijbesma et al. [9] synthesized the water-soluble fullerene derivative bis (phenylenaminosuccinic acid)-C₆₀ (1) (Figure 2). In vitro studies revealed that 1 inhibited acute and chronic HIV-1 infection in human peripheral blood mononuclear (PBM) cells, with a half-effective concentration (EC₅₀) of 7.0 µM, while showing no cytotoxicity to uninfected PBM cells.

In 2016, Echegoyen and co-workers [3] reported a novel cationic N,N-dimethyl C₇₀ fullerene-pyrrolidine iodized salt derivative (2), with fullerene C₇₀ as the starting material (Figure 2), that inhibits more than 99% of HIV-1 infectivity with an EC₅₀ of 0.41 µM. An analysis of the life cycle of HIV-1 suggested that 2 inhibits viral maturation by influencing the processing of Gag and GAG-POL. Significantly, 2 does not inhibit protease activity in vitro, and strongly interact with immature HIV capsid proteins in a pull-down assay. Moreover, 2  blocks infection by viruses carrying either a mutant protease that is resistant to multiple protease inhibitors, or the mutant Gag protein, which is resistant to the mature inhibitor bevirimat. This finding differed from previous reports that fullerene derivatives affect HIV-1 protease activity in vitro. Echegoyen et al. [10] then proposed that fullerene-pyrrolidine derivatives act through a novel anti-HIV-1 mechanism, rather than interacting with other capsid proteins as previously reported. 

In 2007, Troshin and coworkers [11] proposed an effective method for the synthesis of water-soluble fullerene carboxylic acid derivatives. With C₆₀Cl₆ as the starting point, C₆₀(Ar)₅Cl, with ester groups linked to aryl groups, was obtained via the simple and efficient Friedel–Crafts arylation of C₆₀Cl₆  with methyl esters of phenylacetate at 100 °C. The fullerene carboxylic acid derivative (3) was prepared in almost a quantitative yield by removing the methyl group from the methyl ester under acidic conditions, as shown in Figure 2. Compound 3, with five carboxyl groups, is insoluble in water but soluble in DMSO. In order to improve the water solubility, potassium carbonate was added to neutralize the carboxylic acid group of 3 and form the corresponding ionic potassium salts, with a water solubility of up to 50–100 mg/mL at pH < 7.5. A virus-induced cytopathicity assay revealed that the fullerene carboxylic acid potassium derivative has pronounced anti-HIV-1 activity, with an IC₅₀ of 1.20 ± 0.44 μM and a low cytotoxicity (>52 μM).

In 2013, Eropkin et al. [12] synthesized fullerenol 4 (Figure 2). In vitro studies revealed that fullerenols containing 12~14 hydroxyl groups are insoluble in water and have no biological activity when introduced into cell culture as suspensions. The fullerenols with 18~24 and 30~38 hydroxyl groups show broad spectrum antiviral activity in vitro against the human influenza viruses H1N1 and H3N2, avian influenza virus A (H5N1), adenovirus, human HSV, and respiratory syncytial virus. C₆₀(OH)_18–24 demonstrates better antiviral activity than C₆₀(OH)_30–38. Moreover, the three water-soluble fullerenols exhibit no toxicity in vitro to human and animal cells.

In 2013, Martin and coworkers [13] designed and synthesized a class of hexakis-adduct [14] glycofullerene derivatives (5), containing 36 mannoses (Figure 2), and used them to inhibit cell infection by pseudotyped Ebola virus particles. This was the first time that glycofullerene derivatives were demonstrated to effectively inhibit cell infection. In the pseudotyped Ebola infection model, the antiviral activity of 5 was in the low nanomolar range, with an IC₅₀ of 0.3 μM. The glycofullerene with 12 galactosyl had no inhibitory effect on virus infection, indicating that the inhibitory effect is dependent on mannose. Interestingly, only an increase in the valence of glycofullerene resulted in a loss of the antiviral effect. This phenomenon is related to the spatial crowding of sugars at the surface of glycofullerene. Martin et al. speculated that the high binding affinity occurs not only because of the extensive spatial presentation of multivalent ligands, but also the frequent interactions between the ligands and corresponding receptors. They demonstrated that a rational design of compounds with the same valency but longer spacers can significantly increase the antiviral activity, likely due to more efficient interactions with receptors. Therefore, the selection of suitable scaffolds (such as spherical fullerenes) to achieve multivalence, as well as the accessibility and flexibility of ligands, are key factors for improving antiviral activity.

In 2012, Yang and coworkers [15] prepared a fullerene [60] liposome complex 6 (Figure 2) and studied its anti-H1N1 activity in vivo. The fullerene liposome complex 6 significantly reduces the average lung virus yields and lung index; prolongs the mean time to death; and decreases the mortality of mice infected with H1N1. In addition, 6 has good water solubility and low toxicity, and its anti-influenza activity in vivo is much higher than that of rimantadine. Therefore, 6 is a promising clinical candidate drug against influenza infection.

3. Conclusions and Prospects

Numerous water-soluble fullerene derivatives or fullerene complexes have shown broad-spectrum antiviral potential, mainly because fullerenes have three advantages. First, pristine fullerenes are hydrophobic, which is conducive to the formation of strong hydrophobic interactions with the active site surfaces of viruses. Second, hydrophilic groups with various functions (such as amino, carboxyl, amino acid, hydroxyl, pyrrolidine, and sugar groups) can be used to selectively modify the unique spherical skeleton of fullerenes via organic reactions. Third, fullerenes and their derivatives exhibit no or low cytotoxicity at relatively high concentrations. Although fullerenes are promising prospective antiviral drugs, antiviral research on fullerenes requires improvement. Most fullerene derivatives exhibit good antiviral effects in vitro, but the antiviral mechanism has not been thoroughly studied. Additionally, most of the studies on fullerenes have only focused on virus inhibition in vitro; there have been few antiviral studies in vivo, and relevant clinical studies involving fullerenes have not been conducted. Viruses constantly threaten human health. Fullerenes have become an important molecular platform for the development of antiviral drugs. Research on fullerenes as antiviral drugs urgently needs the joint efforts of scientists working in synthesis, molecular design, biology, and medicine. Some fullerene derivatives display inhibitory activity against multiple types of viruses. Therefore, fullerene derivatives have the potential to become a class of broad-spectrum antiviral drugs effective against SARS-CoV-2, which remains a global threat. 

References

1. Friedman, H.; DeCamp, D.L.; Sijbesma, R.P.; Srdanov, G.; Wudl, F.; Kenyon, G.L. Inhibition of the HIV-1 protease by fullerene derivatives: Model building studies and experimental verification. J. Am. Chem. Soc. 1993, 115, 6506–6509. https://doi.org/10.1021/ja00068a005.
2. Mashino, ; Shimotohno, K.; Ikegami, N.; Nishikawa, D.; Okuda, K.; Takahashi, K.; Nakamura, S.; Mochizuki, M. Human immunodeficiency virus-reverse transcriptase inhibition and hepatitis C virus RNA-dependent RNA polymerase inhibition activities of fullerene derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 1107–1109. https://doi.org/10.1016/j.bmcl.2004.12.030.
3. Castro, ; Martinez, Z.S.; Seong, C.-S.; Cabrera-Espinoza, A.; Ruiz, M.; Hernandez Garcia, A.; Valdez, F.; Llano, M.; Echegoyen, L. Characterization of New Cationic N,N-Dimethyl [70]fulleropyrrolidinium Iodide Derivatives as Potent HIV-1 Maturation Inhibitors. J. Med. Chem. 2016, 59, 10963–10973. https://doi.org/10.1021/acs.jmedchem.6b00994.
4. Yasuno, ; Ohe, T.; Takahashi, K.; Nakamura, S.; Mashino, T. The human immunodeficiency virus-reverse transcriptase inhibition activity of novel pyridine/pyridinium-type fullerene derivatives. Bioorg. Med. Chem. Lett. 2015, 25, 3226–3229. https://doi.org/10.1016/j.bmcl.2015.05.086.
5. Kornev, B.; Khakina, E.A.; Troyanov, S.I.; Kushch, A.A.; Peregudov, A.; Vasilchenko, A.; Deryabin, D.G.; Martynenko, V.M.; Troshin, P.A. Facile preparation of amine and amino acid adducts of [60]fullerene using chlorofullerene C60Cl6 as a precursor. Chem. Commun. 2012, 48, 5461–5463. https://doi.org/10.1039/C2CC00071G.
6. Tollas, ; Bereczki, I.; Borbás, A.; Batta, G.; Vanderlinden, E.; Naesens, L.; Herczegh, P. Synthesis of a cluster-forming sialylthio-d-galactose fullerene conjugate and evaluation of its interaction with influenza virus hemagglutinin and neuraminidase. Bioorg. Med. Chem. Lett. 2014, 24, 2420–2423. https://doi.org/10.1016/j.bmcl.2014.04.032.
7. Muñoz, ; Sigwalt, D.; Illescas, B.M.; Luczkowiak, J.; Rodríguez-Pérez, L.; Nierengarten, I.; Holler, M.; Remy, J.-S.; Buffet, K.; Vincent, S.P.; et al. Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection. Nat. Chem. 2016, 8, 50–57. https://doi.org/10.1038/nchem.2387.
8. Fedorova, E.; Klimova, R.R.; Tulenev, Y.A.; Chichev, E.V.; Kornev, A.B.; Troshin, P.A.; Kushch, A.A. Carboxylic Fullerene C60 Derivatives: Efficient Microbicides Against Herpes Simplex Virus and Cytomegalovirus Infections In Vitro. Mendeleev Commun. 2012, 22, 254–256. https://doi.org/10.1016/j.mencom.2012.09.009.
9. Sijbesma, ; Srdanov, G.; Wudl, F.; Castoro, J.A.; Wilkins, C.; Friedman, S.H.; DeCamp, D.L.; Kenyon, G.L. Synthesis of a fullerene derivative for the inhibition of HIV enzymes. J. Am. Chem. Soc. 1993, 115, 6510–6512. https://doi.org/10.1021/ja00068a006.
10. Martinez, S.; Castro, E.; Seong, C.-S.; Cerón, M.R.; Echegoyen, L.; Llano, M. Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity. Antimicrob. Agents Chemother. 2016, 60, 5731–5741. https://doi.org/10.1128/AAC.00341-16.
11. Troshina, A.; Troshin, P.A.; Peregudov, A.S.; Kozlovskiy, V.I.; Balzarini, J.; Lyubovskaya, R.N. Chlorofullerene C60Cl6: A precursor for straightforward preparation of highly water-soluble polycarboxylic fullerene derivatives active against HIV. Org. Biomol. Chem. 2007, 5, 2783–2791. https://doi.org/10.1039/B705331B.
12. Eropkin, Y.; Melenevskaya, E.Y.; Nasonova, K.V.; Bryazzhikova, T.S.; Eropkina, E.M.; Danilenko, D.M.; Kiselev, O.I. Synthesis and Biological Activity of Fullerenols with Various Contents of Hydroxyl Groups. Pharm. Chem. J. 2013, 47, 87–91. https://doi.org/10.1007/s11094-013-0901-x.
13. Luczkowiak, ; Muñoz, A.; Sánchez-Navarro, M.; Ribeiro-Viana, R.; Ginieis, A.; Illescas, B.M.; Martín, N.; Delgado, R.; Rojo, J. Glycofullerenes Inhibit Viral Infection. Biomacromolecules 2013, 14, 431–437. https://doi.org/10.1021/bm3016658.
14. Hirsch, ; Vostrowsky, O. C₆₀ Hexakisadducts with an Octahedral Addition Pattern—A New Structure Motif in Organic Chemistry. Eur. J. Org. Chem. 2001, 2001, 829–848. https://doi.org/10.1002/1099-0690(200103)2001:5<829::AID-EJOC829>3.0.CO;2-V.
15. Du, X. The antiviral effect of fullerene-liposome complex against influenza virus (H1N1) in vivo. Sci. Res. Essays 2012, 7, 705–711.

This entry is adapted from the peer-reviewed paper 10.3390/nano12152547