[1-3]-thiazine-fulleropyrrolo derivatives of C60 and C70 as HIV-inhibitor agents

Certain embodiments useful as HIV-inhibitor agents are directed to fullerene derivatives tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salts (Formula I) or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salts (Formula II), while further embodiments are directed to α, β, γ, and/or δ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt.

BACKGROUND

The present invention relates generally to the field of medicine. More particularly, it concerns fullerene compositions and treatments using the same for HIV infection.

Human immunodeficiency virus (HIV) infects and destroys the immune system of human body. HIV infects CD4+T cells, a lymphocyte that activates our immune system against numerous infections. As the number of CD4+T cells in the body decrease, cell mediated immunity decreases and a HIV infected person develops acquired immunodeficiency syndrome (AIDS). An infected person becomes more susceptible to infections and cancers, even to those which rarely occur to a healthy individual.

The number of HIV infected individuals is increasing continuously over the years on a global scale, but currently there is no cure or effective vaccine against HIV virus. Currently highly active anti-retroviral therapy (HAART) is often used to treat AIDS. HAART consists of a combination of antiretroviral agents. Although HAART slows progression of the disease and decreases risk of death, adverse effects are very common due to large dose and long term administration. Moreover drug-resistant viruses and genomic mutation in HIV makes the therapy less effective over time.

Fullerenes have been valuable compounds in different fields and their functionalization has expanded their applications from biology to materials science. Particular attention has been given to their biological applications towards the inhibition of the human immunodeficiency virus (HIV). It is well known that C60and C70are capable of inhibiting HIV protease, presumably thru interactions with the hydrophobic cavity of the protease. These interactions are believed to be possible because the diameter of the HIV-protease cavity can host the C60or C70derivatives. It is not well understood what kinds of interactions dominate, not only because they will depend on the type of derivative but also because it has not been possible to prove that the fullerene-protease interactions indeed occur inside the cavity. Several research groups have used modeling software to study the possible fullerene derivative interactions with the HIV-protease and results suggest that it is likely to occur inside the cavity. From the latter results researchers have designed potential fullerene derivative candidates that can effectively interact with the HIV protease cavity and inhibit HIV infectivity.

Most of the current anti-HIV drugs are not effective after mutations of the virus occur. As a result, HIV becomes resistant to those drugs and infection of healthy cells follows. Alternative drugs for effective treatment of HIV are necessary to prevent its transmission and infection of healthy cells, especially after mutation occurs.

SUMMARY

Embodiments of the invention relate to the design of a new class of fullerene derivatives as biologically active materials against HIV. The Acquired Immunodeficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV) has spread worldwide since its discovery. The effects of this contagious disease in the health and economy of both developed and under-developed countries are outrageous. Therefore, immeasurable efforts to discover and/or design and synthesize biologically active materials that can work as anti-HIV agents are being expended by scientists worldwide.

The two main anti-HIV agents for HIV growth are those that target the HIV reverse transcriptase and those that inhibit the HIV-protease. It has been shown in the literature that specific [60]-fullerene derivatives actively interact and inhibit the HIV-protease.

Described herein is the design, synthesis, and characterization of quaternary salts of [1,3]thiazine [60]- and [70]-fulleropyrrolo derivatives (Schemes 1 and 2,FIG. 11andFIG. 12, respectively) as a new class of anti-HIV agents aiming for alternative treatments for the disease.

The reaction conditions to obtain the [70]-fullerene derivatives are followed as for the synthesis of the [60]-fullerene derivatives. While for C60there is only one possible regioisomer for the mono-adduct derivatives, for C70it is possible to obtain a mixture of three mono-regioisomers (α, β, γ) but a fourth (δ) can possibly be obtain as well in significantly lower yields.

As for the disclosure, the quaternary ammonium salts of isolated pure mono- and bis-regioisomers of C60and C70and regioisomeric mixtures of mono-, bis- and tris-regioisomers of C60and C70are disclose as biologically active materials for HIV inhibition and other applications.

Certain embodiments are directed to C60fullerene derivatives tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula I,FIG. 13) or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula II,FIG. 14). Representative formulas are provided as Formula I and Formula II where n can be 1, 2, or 3.

Certain embodiments are directed to administration of one or both of tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula I) or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula II) to treat and reduce HIV infection. The fullerene derivative(s) can be administered alone or in combination with each other or other anti-cancer therapies.

In certain aspects an tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In certain aspects an tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In a further aspect an α isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The α isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In a further aspect β isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The β isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In an another aspect γ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The γ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In an another aspect δ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt is administered to treat and reduce HIV infection. The δ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt can be administered alone or in combination with one or more additional anti-HIV therapies.

In an another aspect a mixture of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt isomers is administered to treat and reduce HIV infection. The mixture of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt isomers can be administered alone or in combination with other anti-HIV therapy. In certain aspects the mixture can comprise 5, 10, 20, 30, 30, 40, 50, 60, 70, 80, or 90%, including all values and ranges there between, of an α isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt with respect to the total amount of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt. In a further aspect the mixture can comprise 5, 10, 20, 30, 30, 40, 50, 60, 70, 80, or 90%, including all values and ranges there between, of a β isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt with respect to the total amount of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt. In still a further aspect the mixture can comprise 5, 10, 20, 30, 30, 40, 50, 60, 70, 80, or 90%, including all values and ranges there between, of a γ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt with respect to the total amount of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt. In still a further aspect the mixture can comprise 5, 10, 20, 30, 30, 40, 50, 60, 70, 80, or 90%, including all values and ranges there between, of a δ isomer of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt with respect to the total amount of tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt.

In certain aspects one or more tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt isomer(s) are administered to a subject in need of an anti-HIV treatment. In certain aspects the isomers are administered within 1, 5, 10, 20, 30, or 60 minutes or hours of each other. In a further aspect the isomers are administered concurrently. In certain aspects the isomers are formulated in the same composition. In a further aspects the HIV patient has a second viral infection. The second viral infection can be, but is not limited to hepatitis B virus (HBV), hepatitis C virus (HCV), or cytomegalovirus (CMV).

Certain aspects include methods for treating an HIV co-infection in an HIV patient comprising administering to a patient an effective amount of a one or more isomers of tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt. The patient can be co-infected with hepatitis B virus, hepatitis C virus, cytomegalovirus, or other virus.

The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).

“Subject”, “individual,” “host” and “patient” are used interchangeably herein, to refer to any animal, e.g., mammal, human or non-human. Generally, the subject is a mammalian subject. Exemplary subjects include, but are not necessarily limited to, humans, non-human primates, cattle, sheep, goats, pigs, dogs, cats, and horses, with humans being of particular interest.

Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention.

As used herein, the term “IC50” refers to an inhibitory dose that results in 50% of the maximum response obtained.

The term half maximal effective concentration (EC50) refers to the concentration of a drug that presents a response halfway between the baseline and maximum after some specified exposure time.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

DESCRIPTION

The inventors have designed and synthesized new fullerene derivatives that can effectively inhibit an HIV protease. As a result, a new class of fullerene derivatives, [1,3]thiazinefulleropyrrolo and their pyrrolidinium salts, were prepared (FIG. 1andFIG. 2). The pyrrolidinium salts were tested for their activity against the HIV protease. Studies demonstrate that the [60]-derivatives exhibit potent inhibition of late stage HIV. The infectivity was determined to be less than 2% using DMSO as the 100% infectivity control (FIG. 3). These results suggest that this new class of fullerene derivatives can be used for the effective treatment of HIV patients.

Certain aspects described herein are directed to treatment for the human immunodeficiency virus (HIV) by administering C60or C70fullerene derivatives. HIV is a pathogenic retrovirus and the causative agent of acquired immune deficiency syndrome (AIDS) and related disorders (Barre-Sinossi et al., 1983, Science220:868-870; Gallo et al., 1984, Science224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinossi et al., 1983, Science220:868-870; Gallo et al., 1984, Science224:500-503) and HIV-2 (Clavel et al., 1986, Science223:343-346; Guyader et al., 1987, Nature326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4+ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and untimely death.

HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus 1988, Science240:1427-1439). The HIV viral particle consists of a viral core, made up of proteins designated p24 and p18. The viral core contains the viral RNA genome and those enzymes required for replicative events. Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kD precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains noncovalently associated with gp41, possibly in a trimeric or multimeric form (Hammerwskj old and Rekosh, 1989, Biochem. Biophys. Acta989:269-280).

HIV is targeted to CD4+ T lymphocytes because the CD4 surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature312: 767-768, Maddon et al., 1986, Cell47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4+ receptor molecules, while gp41 anchors the envelope glycoprotein complex in the viral membrane (McDougal et al., 1986, Science231:382-385; Maddon et al., 1986, Cell47:333-348) and thus explains HIV's tropism for CD4+ cells.

Fullerenes are carbon compounds composed entirely of carbon, in the form of a hollow sphere, ellipsoid, tube, or other shapes. Cylindrical fullerenes are called carbon nanotubes and spherical fullerenes are sometimes referred to as “buckyballs.” C70fullerene or [70]fullerene is a cage like molecule composed of 70 carbon atoms joined together by single and double bonds to form a hollow sphere with 25 hexogonal and 12 pentagonal rings. The rings of the [70]fullerene can be chemically modified. In certain aspects [70]fullerenes can be modified with pyrrolidines.

In certain embodiments described herein fullerenes and derivatives thereof can have antiviral activity and can be used for treatment of viral infections, such as HIV-infection. Fullerenes derivatives can be designed to bind or interact with and inhibit HIV protease, which is essential for HIV lifecycle. Inhibiting HIV protease inhibits HIV replication and infection.

Methods for making the fullerene derivative described herein comprise, but are not limited to mixing C60or C70fullerene, DL-Homocysteine, and paraformaldehyde in chlorobenzene. The resulting mixture was dissolved with sonication and refluxed overnight under N2gas atmosphere. After this time, the solvent was removed under reduced pressure and the mixture chromatographed using silica gel. Carbon disulfide (CS2) was used as the mobile phase to elute the unreacted C70, the polarity was then increased using CS2:CHCl3(1:1) to elute the desire mono-adducts (F1: gamma-1; F2: beta-1; F3: gamma-2, beta-2-, alpha-1).

Various chemical definitions related to such compounds are provided as follows.

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e. unbranched) or branched carbon chain, which may be fully saturated, mono- or polyunsaturated.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. In certain embodiments, the heteroatoms are selected from the group consisting of S and N. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive.

The terms “cycloalkyl” and “heterocyclyl,” by themselves or in combination with other terms, means cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocyclyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Pyrrolidine, also known as tetrahydropyrrole, is a saturated heterocycle with the molecular formula (CH2)4NH, a cyclic secondary amine. A thiazine moiety is a 6 membered heterocylic moiety containing a ring of four carbon, one nitrogen and one sulfur atom.

The term “aryl” means a polyunsaturated, aromatic, hydrocarbon substituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3 rings that are fused together or linked covalently). One example of a polycyclic compound is the fullerenes. A fullerene is a molecule of carbon in the form of a hollow sphere, ellipsoid, tube, and many other shapes. Spherical fullerenes are also called buckyballs. Cylindrical ones are called carbon nanotubes or buckytubes. Fullerenes are similar in structure to graphite, which is composed of stacked graphene sheets of linked hexagonal rings; but they may also contain pentagonal (or sometimes heptagonal) rings. The discovery of fullerenes greatly expanded the number of known carbon allotropes, which until recently were limited to graphite, diamond, and amorphous carbon such as soot and charcoal.

A particular fullerene is the C70fullerene. The C70fullerene consists of 70 carbon atoms. It is a cage-like fused-ring structure made of 25 hexagons and 12 pentagons, with a carbon atom at the vertices of each polygon and a bond along each polygon edge.

In certain aspects a thiazine moiety is used as a substituent to a pyrrolidine group on a C60or C70fullerene as described herein.

An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs. Unless otherwise specified, the compounds described herein are meant to encompass their isomers as well.

A “stereoisomer” is an isomer in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers that are not enantiomers.

A regioisomer is where a functional group or other substituent changes position on a parent structure. For example, a hydroxyl group can occupy three different positions on an n-pentane chain forming three different compounds or a pyrrolidine can occupy different positions on a fullerene.

Pharmaceutical compositions of the invention are useful in the treatment or prevention of viral infections in humans and contain as an active agent one or more isoform of tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula I) or tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine salt derivatives (Formula II). In certain aspects the pharmaceutical compositions contain at least one other antiviral agent, such as reverse transcriptase inhibitors, protease inhibitor, inhibitors of mRNA processing, inhibitors of protein glycosylation and inhibitors of viral fusion. Such agents include but are not limited to nucleoside analogs or chain terminators (e.g., dideoxynucleosides). Additional suitable therapeutic agents which may be used in combinational therapy include, but are not limited to 2-deoxy-D-glucose (2-dGlc), deoxynojirimycin, acycloguanosine, ribavirin (virazole), rifampicin (rifadin), adamantidine, rifabutine, ganciclover, (DHPG), fluoroiodoaracytosine, idoxurine, trifluorothymidine, adenine arabinoside (ara-A), ara-AMP, bromovinyldeoxyuridine, bromovinylarauracil (BV-araU by Bristol-Meyers Squibb (1-beta-D-arabinofuranoside-E-5-[2-bromovinyl]uracil)) rimantadine, arildone, diarylamidine, (S)-(p-nitrobenzyl-)6-thioinosine and phosphonoformate.

The compounds and compositions described herein may be administered using techniques well known to those in the art. Preferably, agents are formulated and administered systemically. Techniques for formulation and administration may be found in “Remington's Pharmaceutical Sciences”, 18th ed., 1990, Mack Publishing Co., Easton, Pa. Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. In certain aspects administration is intravenous. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.

A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).

Pharmaceutical compositions containing the compounds described herein can be administered to a human patient, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat a viral infection, in particular HIV infection. Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The antiviral activity exhibited by the compounds described herein may be measured, for example, by easily performed in vitro assays.

For the compounds of the present invention, alone or as part of a pharmaceutical composition, such doses are between about 0.001 mg/kg and 1 mg/kg body weight, preferably between about 1 and 100 μg/kg body weight, most preferably between 1 and 10 μg/kg body weight.

Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

EXAMPLES

Synthesis of Mono-, Bis- and Tris-[1,3]Thiazine[60]Fulleropyrrolidine

To a 250 mL round bottom flask were added C60(100 mg, 0.14 mmol), DL-Homocysteine (16 mg, 0.12 mmol) and paraformaldehyde (36 mg, 1.2 mmol) followed by 100 mL of chlorobenzene. The resulting mixture was dissolved with sonication and refluxed overnight under N2gas atmosphere. After this time, the solvent was removed under reduced pressure and the mixture chromatographed using silica gel. Carbon disulfide (CS2) was used as the mobile phase to elute the unreacted C60, the polarity was then increased using chloroform (CS2:CHCl310%, 30%, 50%, 75%) to elute the desire mono-, bis- and tris-adducts.

Synthesis of Mono- and Bis-[1,3]Thiazine[70]Fulleropyrrolidine

To a 250 mL round bottom flask were added C70(100 mg, 0.12 mmol), DL-Homocysteine (14 mg, 0.10 mmol) and paraformaldehyde (30 mg, 1.0 mmol) followed by 100 mL of chlorobenzene. The resulting mixture was dissolved with sonication and refluxed overnight under N2gas atmosphere. After this time, the solvent was removed under reduced pressure and the mixture chromatographed using silica gel. Carbon disulfide (CS2) was used as the mobile phase to elute the unreacted C70, the polarity was then increased using CS2:CHCl3(1:1) to elute the desire mono-adducts (F1: gamma-1; F2: beta-1; F3: gamma-2, beta-2-, alpha-1).

Synthesis of the Quaternary Ammonium Fullerene Salts

Synthesis of tetrahydro-1H—C60-fulleropyrrolidinium[1,2-c][1,3]thiazine iodide B. tetrahydro-1H—C60-fulleropyrrolo[1,2-c][1,3]thiazine A (0.01 mmol) was dissolved in methyl iodide (5 mL) and stirred for 36 h at room temperature to give a black precipitate. The solvent was dried and the precipitate was washed with CS2, CHCl3, toluene, MeOH and acetone in this order; to afford a brown powder, [1,3]thiazine[60]fulleropyrrolidinium iodide B. The yields were 65% for n=1, 74% for n=2 and 75% for n=3.

Synthesis of tetrahydro-1H—C70-fulleropyrrolidinium[1,2-c][1,3]thiazine iodide D. tetrahydro-1H—C70-fulleropyrrolo[1,2-c][1,3]thiazine C (0.01 mmol) was dissolved in methyl iodide (5 mL) and stirred for 36 h at room temperature to give a black precipitate. The solvent was dried and the precipitate was washed with CS2, CHCl3, toluene, MeOH and acetone in this order; to afford a black powder, [1,3]thiazine[70]fulleropyrrolidinium iodide D. The yields were 79% for n=1 and 71% for n=2.