WO2009002203A1

WO2009002203A1 - Polyfunctional fullerene c60 aminoacid derivatives

Info

Publication number: WO2009002203A1
Application number: PCT/RU2007/000337
Authority: WO
Inventors: Alexandr Ivanovich Kotelnikov; Valentina Semenovna Romanova; Gennady Nikolaevich Bogdanov; Nina Petrovna Konovalova; Oleg Ivanovich Pisarenko; Raisa Alekseevna Kotelnikova; Irina Igorevna Faingold; Elena Sergeevna Frog; Yury Nikolaevich Bubnov; Sergei Mikhailovich Aldoshin; Mikhail Ivanovich Davydov
Original assignee: Institut Problem Khimicheskoi Fiziki Rossiiskoi Akademii Nauk (Ipkhf Ran); Federalnoe Gosudarstvennoe Uchrezhdenie Rossysky Kardiologichesky Nauchno-Proizvodstvenny Kompleks Federalnogo Agenstva Po Zdravookhraneniyu I Sotsialnomu Razvitiyu Roszdrava
Priority date: 2007-06-19
Filing date: 2007-06-19
Publication date: 2008-12-31
Also published as: CA2687557A1; WO2009002203A8

Abstract

The invention relates to novel polyfunctional fullerene C60 aminoacid derivatives of formula (1), wherein R=H, mono- or dihydroxyalkyl, aminoalkyl, haloidalkyl, mono- or dinitroxyalkyl or maleinimide mm; N-Z are the α,β,Ϝ, ω-aminoacid of general formula NH-CmH2m-COOM or (II) -N- COOM, wherein m=2-5 and M is a nitroxyalkyl group, an alkyl group or an alkali metal salt, or a biologically active dipeptide, to methods for the production thereof and to a method for binding fullerene derivatives with SH-containing proteins. The invention also relates to the use of nitroxyalkyl-(fullerenyl)aminoacids as nitrogen monoxide donors and to the use of said nitroxyalkyl-(fullerenyl)aminoacids in the form of quick-acting vasodilatators for antihypertensive therapy. A method for inhibiting a metastasis process and a method for enhancing antileukemik activity of cyclophosphamide are also disclosed.

Description

POLYFUNCTIONAL AMINO ACID DERIVATIVES

FULLERENA C ₆₀

DESCRIPTION The invention relates to new multifunctional amino acid derivatives of C ₆₀ fullerene having biological activity, as well as to methods for their preparation and a method for covalently linking fullerene derivatives to SH-containing proteins. In addition, the invention relates to the use of nitroxyalkyl-N- (fullerenyl) amino acids as donors of nitrogen monoxide, as well as the use of nitroxyalkyl-N- (fullerenyl) amino acids as quick-acting vasodilators for antihypertensive therapy. The invention also relates to a method for inhibiting the metastasis process and a method for enhancing the antileukemic activity of cyclophosphamide. It is known that the products of equimolar addition of amino acids and peptides to fullerene C ₆₀ / N- (monohydrogen-substituted) substituted amino acids or peptides / [Romapova VS, V. A. Tsuguarkip, Yu. I. Luakhovetsku, ZN RAMES M.E. VOGRIP // Russiap Scheme. BuIl., 1994, vol. 6, pp. 1090-1091.] Constitute a special class of organic compounds, which can be considered as potential antimetabolites belonging to a certain subclass of physiologically active substances. A wide range of biological activity of amino acid derivatives of fullerene (ACE) is due to the unique structure of the carbon spheroid, its ability to convert oxygen to a singlet state [Tokuuama H., Nakama S., Nakama E. // J. Am. Chem. Sos, 1993, vol. 115, pp. 7918], to exhibit membranotropic and antiradical properties [Kotelpikova R. A., Kotelpikova A. L, Vgdapov GN, Rampapova VS, Kuleshova EF, Rams ZN, Vograp M.E. // SUBSTITUTE SHEET (RULE 26) FEBS Letters. - 1996. 389. - P. 111-114], antiviral activity [Frog EC, Kotelnikova PA, Bogdanov G.H., Stolko BH, Fayngold II, Kushch AA, Fedorova HE, Medzhidova AA, Romanova V. S . // Technologies of living systems. - 2006. 3. 2. - P. 42-46] and the cytotoxic effect [Nakamuga E., Tukuuama H., Yamago S., Shigaki T., Sugiuga Y. // BuIl. Chem. Soc. Jarap. - 1996. 69. - P. 2143-2151]. Known prerequisites for the study of cardiovascular effects under the influence of fullerene derivatives [Syrensky A. V., Galagudza MM, Egorova E. I. Arterial hypertension, 2004, 10 (3), 15-20.]. For this reason, fullerene C _{60 is} considered in chemical pharmacology as a very promising carrier of functional groups with one or another type of biological activity. These include alkyl groups of a cytotoxic effect, anthracyclines, as well as donors of nitric monoxide.

Recent studies have revealed various aspects of the antitumor effect of exogenous NO donors [Konovalova NP // Technologies of living systems. 2004. 1. 3.- C.42-48]. They increase the effectiveness of known cytostatics, inhibit the development of metastases of experimental tumors and modulate the sensitivity of drug-resistant tumors to cytostatic therapy. Nitric oxide is a unique intracellular multifunctional regulator of metabolism, blood vessel homeostasis, blood pressure and organ perfusion. Most exogenous nitric oxide donors recommended for the treatment of myocardial ischemia and acute heart failure are widely used as vasodilators and platelet aggregation inhibitors.

A fairly extensive bibliography of patents on the medical chemistry of fullerenes does not contain information regarding new polyfunctional amino acid derivatives of fullerene (ACE) containing biologically active groups, peptides or proteins, methods for their preparation are also not disclosed.

The present invention is the creation of new multifunctional amino acid derivatives of fullerene (ACE),

SUBSTITUTE SHEET (RULE 26) possessing inhibitory activity against tumor metastases, as well as enhancing the antileukemic activity of cyclophosphamide, and which may be suitable as donors of nitric oxide or as vasodilators of rapid action for antihypertensive therapy. The multifunctional amino acid derivatives of fullerene C ₆₀ in accordance with the invention have the formula (1)

where R = H, mono - or dihydroxyalkyl, aminoalkyl, haloalkyl, mono - or dinitroxyalkyl, maleimide; NZ is a fragment of α, β, γ, ω-amino acids of the general formula -NH-

Coom

- NC _m H _2m —COOM or V where m = 2-5, and M represents a nitroxyalkyl group, an alkyl group or an alkali metal salt, or a dipeptide, wherein the alkyl groups contain 1-6 carbon atoms.

The inventors of the present invention first developed methods for producing ACE by substituting a mobile proton in monohydrofullerenyl amino acids, as well as by esterifying the carboxyl monohydrofullerenyl amino acid.

In accordance with this, when a monohydrofullerenyl amino acid derivative interacts with brommaleiminimide, a mobile proton is replaced with the formation of maleimide derivatives:

where NZ = a fragment of synthetic or natural α, β, γ, ω-amino acids (glycine, alanine, arginine, serine, γ-aminobutyric, ω-aminocaproic

SUBSTITUTE SHEET (RULE 26) acids, proline, etc.), its alkali metal salt or lower ester.

The proton mobility of monohydrofullerenyl amino acids is due to the electronegativity of the fullerene spheroid, which determines the high polarization of the C-H bond and the predominant direction of electrophilic substitution reactions along it:

n is 2-5; p = 0-2 where X = Cl-; Br-; J-.

When -N-Z represents a fragment of synthetic or natural α, β, γ,

ω-amino acids of the general formulas —NH — C _m H _2m —COOM, or

where m =

2-5, M represents a nitroxyalkyl group, an alkyl group where the alkyl groups include 1-6 carbon atoms, or an alkaline earth metal salt, and in the case of alkali metal salts of amino acids, the reaction of monohydrofullerenyl amino acids with nitroxylalkyl halides proceeds according to two reaction centers according to the equation:

where M = K, Na; m = 2-5, n = 2-5

It was noted that even with a slight increase in the values of n, m, and p, the already high hydrophobicity of the compounds sharply increases. Their complete loss of hydrophilicity negatively affects their membranotropy, and,

SUBSTITUTE SHEET (RULE 26) therefore, on the potential biological activity of synthesized NO donors.

Since the structure of ACE can be classified as a class of antimetabolites, we have proposed methods for the directed synthesis of fullerene derivatives covalently linked to biologically active peptides and proteins.

Carnosine (β-alanyl histidine), which is a natural antioxidant of muscle and nerve tissues, was used as one of them. N- (monohydrofullerenyl) alanyl histidine, a direct analogue of carnosine containing the fullerene sphere, was synthesized.

The resulting compound is characterized by increased membranotropy and the ability to inhibit the process of peroxide oxidation of lipids of biological membranes.

Another example of the modifying effect of ACE on proteins and enzymes is the covalent attachment of the maleimide derivative of monohydrofullerenyl proline to the hemoglobin macromolecule that we performed:

This method is universal in nature and is suitable for the introduction of a SUBSTITUTE SHEET (RULE 26) the fullerene spheroid into any SN-containing proteins, ensuring its transmembrane transfer to the cell.

Recent studies in the field of molecular cardiology have established the important role of nitric oxide in the regulation of vascular homeostasis. The formation of NO during biotransformation of exogenous nitric oxide donors causes a decrease in coronary vascular tone, platelet aggregation and adhesion, which improves hemodynamic characteristics and increases blood flow.

All obtained nitrates based on the amino acid derivatives of fullerene C ₆₀ (NAPP) generate nitrogen monoxide during their biotransformation, as evidenced by their inhibitory effect on the catalytic activity of mitochondrial cytochrome C oxidase. HAPF-1 and HAPF-2, the structural formulas of which are presented below, exhibit moderate antitumor activity and pronounced antimetastatic activity.

It is known that among the most common therapeutic agents for the treatment of diseases of the cardiovascular system are drugs based on nitroglycerin. However, they are characterized by a number of disadvantages and side effects that limit the scope of their clinical use.

When studying the effect of nitroxyalkyl-N-fullerenylamino acid derivatives, in particular, HAPF-1 on the coronary, contractile and pumping functions of the isolated heart of Wistar rats, it was found that HAPF-1 is a fast-acting vasodilator that reduces blood pressure and heart rate and causes relaxation coronary vessels with less depressive effect on myocardial function in comparison with nitroglycerin (NG) SUBSTITUTE SHEET (RULE 26) [Pisarenko O. I., Serebryakova L. I., Studneva I. M., Tskitishvili O. V. // Bull. exp biol. honey. - 2006. 141 (3) - S. 267-269] This means that compounds of this class open the prospect of creating original vasodilators for antihypertensive therapy.

The following examples of the preparation of the claimed compounds and studies of their biological activity should not be understood as limiting the scope of the invention, but only as examples of a preferred embodiment of the invention.

Example 1 Synthesis of methyl ester of N-nitroxyethylfullerenyl proline

To a solution of 0.849 g (0.001M) of N-monohydrofullerenyl proline methyl ester in pyridine was added a solution of 1.25 g (0.01 M) of 2-chloro-nitroxyethane in 10 ml of pyridine and the mixture was kept for 6 hours at room temperature. Next, the reaction mixture was dialyzed for 20 hours.

The residue was dried in air, obtaining the target product with a close to quantitative yield. Absorption bands (1340 and 1540 cm ^"1 ) corresponding to the nitrate group appeared in the IR spectra of N- (nitroxy-fillelerenyl) proline methyl ester.

Example 2 Synthesis of N-nitroxyethyl-fullerenylproline nitroxyethyl ester

To a solution of 0.86 g (0.001 M) of the Na salt of N-monohydrofullerenyl proline in 50 ml of pyridine was added 2.5 g (0.02 M) of 2-chlorophenoxyethane in 20 ml of pyridine and kept at room temperature for 8 hours . Then the reaction mixture was dialyzed for 20 hours. The residue was dried to obtain the desired product in close quantitative yield.

SUBSTITUTE SHEET (RULE 26)

ONO ₂

In the IR spectra of the obtained N- (nitroxyfullerenyl) proline nitroxyethyl ester, absorption bands (1340 and 1540 cm ^"1 ) corresponding to the nitrate group appeared. Example 3

Synthesis of sodium salt of N- [monohydrofullerenyli-carnosine

To a solution of 100 mg of fullerene C ₆₀ (0.138 mmol) in 50 ml of toluene was added a 10-fold excess of sodium carnosine in 50 ml of water.

The resulting heterogeneous system was stirred under heating at 7O ⁰ C in argon for 8 hours. Then the organic layer was separated, the solvent was distilled off, the residue was dissolved in 100 ml of water, and 100 ml of a saturated solution of sodium chloride was added to it. The resulting solution was dialyzed until sodium chloride was completely removed (reaction of the absence of chlorine ion in the washings). The resulting aqueous solution was evaporated, and 130 mg of N- [monohydrofullerenyl] -carnosine sodium salt was obtained (quantitative yield).

Upon hydrolysis of the obtained product in 6N hydrochloric acid, an equimolar amount (found 2.16 ± 0.2 μM and 2.16 ± 0.02 μM) of β-alanine and histidine corresponding to the calculated value (calculated 2.20 μM, is formed according to the HPLC analysis ) Example 4

Synthesis of N-G (N-maleimidyl) fullerenyl-L-proline methyl ester

To a solution of 0.849 g (0.001 mol) of N- (monohydrofueller) L-proline methyl ester in 50 ml of pyridine was added a solution of 1.76 g (0.01 mol) of bromomaleimide in 10 ml of pyridine and the mixture was stirred at room temperature for 8 hours . Then the reaction mass was dialyzed, the residue was dried to obtain N - [(N-maleimidyl) fullerenyl] -L-proline methyl ester in quantitative yield. The IR spectrum of the obtained product contains characteristic absorption bands at 1720 and 1355 cm ^"1 , confirming the presence of a maleimide

SUBSTITUTE SHEET (RULE 26) fragment.

Example 5 Covalent attachment of the maleimide derivative of Cp proline to protein macromolecules A special procedure was developed for the covalent attachment of the maleimide derivative of fullerenylproline to SH-groups of proteins. By way of example, such an attachment is made to the SH groups of human hemoglobin and albumin.

In the case of human hemoglobin, two SH-groups were modified, which are included in the structure of cysteine-93 in two β-subunits of the hemoglobin macromolecule. To a solution of met-hemoglobin in the state in 5XlO concentration ^"4 M in 0.1 M phosphate buffer at pH = 6,5 was added maleimide derivative of C-proline _used in threefold excess relative to the protein. The reaction was conducted for 30 minutes at 2O ⁰ C. an excess of water-soluble fullerene derivative maleimide was separated by column Serhadeh G-15, using as elyuenta phosphate buffer. The withdrawn from zero volume fraction accession maleimide derivative to hemoglobin was monitored spectrophotometrically. The concentration of hemoglobin opr fissioning of a characteristic absorption band at a wavelength of 407 nm (ε = 7,06xl0 ⁵ Nf ¹ CM ^"1), acceding maleimide concentration fullerene derivative - by absorbance at a wavelength of 315 nm. It was assumed that at this wavelength for maleimidyl-C ₆ o-proline ε = 2.10><10 ⁴ M ^-1 CM ^"1 and for hemoglobin it was ε = 9.81 ^χ lθ ⁴ M ^-1 CM ¹ . As a result of computer simulation of the total absorption spectrum of the product from the individual absorption spectra of hemoglobin and the maleimide derivative of fullerene, it was obtained that, as a result of the reaction, on average 1.6 molecules of the maleimide derivative of fullerene are attached to one hemoglobin macromolecule.

By a similar technique, the maleimide derivative of C ₆₀ proline was added to the reactive CH group of human albumin. To a solution of albumin at a concentration of 5XlO ^"4 M in OD M phosphate buffer at pH = 6.5 was added the maleimide derivative C ₆₀ -

SUBSTITUTE SHEET (RULE 26) proline in threefold excess in relation to protein. The reaction was carried out for 30 minutes at ^{2 °} C. The excess water-soluble maleimide derivative of fullerene was separated on a Serhadex G-25 column using phosphate buffer as an eluent. Column chromatography was performed twice to exclude the sorption of the fullerene derivative on albumin due to hydrophobic interactions. In the fraction with zero volume, the addition of the maleimide derivative to albumin was controlled by spectrophotometric method. The concentration of the joined maleimide derivative of the C ₆₀ proline spectrophotometrically, taking for albumin ε = 3.65><10 ⁴ M ¹ CM ^"1 at 279 nm and ε = l, 4><10 ³ M 'cm ^{' 1} at 310 nm, and maleimide derivative for o-C ₆ ppolina - ε = 3,30><April ¹⁰ M ¹ CM ^"1 at 279 nm and ε = 2,70 ^χ l0 ^4M ^'W ¹ at 310 nm. ε = 3.65 * 10 ⁴ M- ¹ CM ^"1 at 279 nm and ε = l, 4 ^χ lθ ³ M ^-1 CM ¹ at 310 nm. As a result of analysis of the absorption of the reaction mixture after double chromatography at two wavelengths, it was determined that the reaction to a single macromolecule albumin joins the average molecule 1.1 maleimide fullerene derivative. as a control was determined by amperometric titration that during attachment maleimide derivative C ₆₀ -ppolina to human albumin titrated number of free SH groups in albumin decreases from I, 05 ± 0.2 to 0, l ± 0.2.

Example 6

The antimetastatic activity of HAPF-1 and HAPF-2 and their combinations with cyclophosphamide (CF) was studied on two models of metastatic solid tumors: LL carcinoma and B-16 melanoma in BDFi mice. In the experimental and control groups of mice, the average number of lung metastases was determined and the percentage inhibition of metastases was calculated: IIM = ((No-N _к ) / (N _к )) ^χ l00. Statistical processing of the results of the study was carried out according to Student. The results were considered reliable at p <0.05. The results obtained are presented in Table 1. (Experimental evaluation of antitumor drugs in the USSR and

USA "edited by Z. P. Sof'ina, A. B. Syrkin (USSR), A. Goldin, A. Klein (USA) Moscow," Medicine ", 1980, pp. 76-86.) SUBSTITUTE SHEET (RULE 26) Table 1

Antimetastatic activity of HAPF-1 and HAPF-2

Data on the antitumor activity of NAPF, which was determined in accordance with the methodology disclosed in ((Experimental evaluation of antitumor drugs in the USSR and the USA) edited by Z. P.

Sof'ina, A.B. Syrkina (USSR), A. Goldin, A. Klein (USA) Moscow,

"Medicine", 1980, p. 73) are shown in FIG. 1 and 2.

In FIG. 1 shows an increase in antileukemic activity (increase in life expectancy (ILS,%) of BDF-I mice with P-388 leukemia) cyclophosphamide (SOMg / kg; 1; 6 days) when combined with HAPP-1 (30 mg / kg; 1 -6 days) and HAPF-2 (ZOMg / kg; 1-6 days).

Figure 2 shows the results of an increase in the chemosensitizing effect of HAPF-1 and HAPF-2 (% of cured BDF-I mice with P-388 leukemia) when combined with cyclophosphamide (single doses and administration modes as in Fig. 1).

Example 7

In ipviv experiments, Wistar rats were intravenously injected with HAPF-1 or NG (nitroglycerin) at equimolar doses (2.6 x 10 ^"5 mM / kg). Hemodynamics was characterized by blood pressure (BP) and heart rate (HR). normalized values are given in Table 2.

SUBSTITUTE SHEET (RULE 26) table 2

The effect of NO donors on normalized values of hemodynamic parameters in rats under ip vivo conditions

The effect of intravenous administration of NO donors on the duration of a decrease in blood pressure in rats was studied under ip vivo conditions (Fig. 3).

The nitroxy derivative of fullerene in a wide range of concentrations weakly affects the duration of a decrease in blood pressure and significantly (two to three times) reduces this indicator compared to nitroglycerin.

The data were obtained in a series of 6 experiments and the results are presented in FIG. 3.

Example 8

The generally accepted methodology [Pisarenko O. I., Shulzhenko V. S, Studneva I. M., Timoshin A. A. // Cardiology. - 2004. 44 (4) - S. 65-70] studied the effect of HAPF-1 on the functions of the left ventricle of the isolated heart of rats and coronary vessels at a concentration of HAPF-1 3.5 x 10 ^"5 M. The normalized pressure values obtained (D) developed by the left ventricle, the intensity of contractions (IS) and coronary flow (CP) are shown in Table 3.

Table 3

Changes in the functional characteristics of the left ventricle and the intensity of the coronary flow under the influence of NO donors

The significance of differences is confirmed by Student's criterion at P <0.05.

SUBSTITUTE SHEET (RULE 26) LITERATURE

1. Romapova VS, V. A. Tsuguarkip, Yu. I. Luhovetsku, ZN Ropes M.E. VOGRIP. Addition of amino acids and dipeptides to fullerene C ₆₀ gjviпg risе to mopoadduсts. - Russiap Chem. BuIl., 1994, vol. 6, pp. 1090-1091. 2. Tokuuama H., Nakamuga S., Nakamuga E. Rhotoipduced bioshemisal activitof fullerene sarbohulis asid. - J. Am. Chem. Sos, 1993, vol.l 15, pp. 7918.

3. Kotelpikova R. A., Kotelpikov AI, Vgdapov GN, Rampapova VS, Kuleshov EF, Rams ZN, Vogrep M.E. Меmbrapotgoris роrеtеs оf thе wаther sоluble аmіpо аsid аpd reрtid derivative оf fullerene C ₆₀ . // FEBS Letters. - 1996. 389. - P. 111-114.

4. Frog EC, Kotelnikova PA, Bogdanov G.N., Shtolko V. H., Fayngold II, Kushch AA, Fedorova HE, Medzhidova AA, Romanova VS The influence of amino acid derivatives of fullerene C ₆₀ on the development of cytomegalovirus infection . // Technologies of living systems. - 2006. 3. 2. - S. 42-46.

5. Nakamura E., Tokuuama H., Yamago S., Shigaki T., Sugiura Y. Biological assistance of a full-class. Structural dependence of DNA is cleavage, complete, upgrade, testability, testability, and NGV-testability. // BuIl. Chem. Soc. Jarap. - 1996. 69. - P. 2143-2151. 6. Syrensky A. V., Galagudza M. M., Egorova E. I. Arterial hypertension, 2004, 10 (3), 15-20.

7. Konovalova N.P. Nitrogen Monoxide Donors in Experimental Tumor Chemotherapy // Living Systems Technologies. 2004. 1. 3.- C. 42-48.

8. Pisarenko O. I., Serebryakova L. I., Studneva I. M., Tskitishvili O. V. // Bull. exp biol. honey. - 2006.141 (3) - S. 267 -269.

9. Pisarenko O. I., Shulzhenko V. S, Studneva I. M., Timoshin A. A. // Cardiology. - 2004.44 (4) - S. 65 -70.

10. ((Experimental evaluation of anticancer drugs in the USSR and the USA) edited by Z. P. Sof'ina, A. B. Syrkin (USSR), A. Goldin, A. Klein (USA) Moscow, "Medicine", 1980,

SUBSTITUTE SHEET (RULE 26)

Claims

CLAIM

1. Multifunctional amino acid derivatives of fullerene C _{60 of the} formula (1)

where R = H, mono- or dihydroxyalkyl, aminoalkyl, haloalkyl, mono- or dinitroxyalkyl, maleimide;

N-Z is a fragment of α, β, γ, ω-amino acids of the general formula -NH-

C _m H _2m -COOM or

where m = 2-5, and M represents a nitroxyalkyl group, an alkyl group or an alkali metal salt, or a dipeptide.

2. A method for producing a compound according to claim 1, comprising treating the monohydrofullerenyl amino acid alkyl ester with an excess of a halogen derivative of nitroxyalkyl in a pyridine solution at room temperature for 6-10 hours, followed by dialysis of the reaction mixture.

3. The method of producing compounds according to claim 1, comprising the esterification of carboxyl fullerenyl amino acid by treating the alkali metal salt of fullerenyl amino acid with an excess of a halogenated nitroxyalkyl in a pyridine solution at room temperature for 6-10 hours, followed by dialysis of the reaction mixture.

4. A method for covalently linking fullerene derivatives to SH-containing proteins, the method being that the corresponding protein is reacted with the maleimide amino acid derivative of fullerene C ₆₀ according to claim 1.

5. The use of nitroxyalkyl-N- (fullerenyl) amino acids according to claim 1 as a donor of nitric monoxide.

6. The use of nitroxyalkyl-N- (fullerenyl) amino acids according to claim 1 as quick-acting vasodilators for antihypertensive therapy.

SUBSTITUTE SHEET (RULE 26)

7. The use of nitroxyalkyl-N- (fullerenyl) amino acids according to claim 1 as chemosensitizers in cytostatic chemotherapy of tumors.

8. A method of inhibiting a metastasis process, comprising administering a multifunctional C ₆ o fullerene derivative according to claim 1, in combination with cytostatics.

9. A method of enhancing the antileukemic activity of cyclophosphamide, which consists in the fact that cyclophosphamide is administered together with polyfunctional derivatives of fullerene C ₆₀ according to claim 1.

SUBSTITUTE SHEET (RULE 26)