CA2687557A1 - Polyfunctional fullerene c60 amino acid derivatives - Google Patents

Polyfunctional fullerene c60 amino acid derivatives Download PDF

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CA2687557A1
CA2687557A1 CA002687557A CA2687557A CA2687557A1 CA 2687557 A1 CA2687557 A1 CA 2687557A1 CA 002687557 A CA002687557 A CA 002687557A CA 2687557 A CA2687557 A CA 2687557A CA 2687557 A1 CA2687557 A1 CA 2687557A1
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amino acid
fullerene
nitroxyalkyl
fullerenyl
derivative
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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
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Federalnoe Gosudarstvennoe Uchrezhdenie Rossysky Kardiologichesky Nauchno-Proizvodstvenny Kompleks Federalnogo Agenstva Pozdravookhraneniyu I Sotsialnomu Razvitiyu Roszdrava
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    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P9/12—Antihypertensives
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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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 .alpha.,.beta.,.gamma., .omega.-aminoacid of general formula NH-CmH2 m-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

SPECIFICATION
The invention relates to novel polyfunctional fullerene C60 amino acid derivatives that have biological activity and also to a method for the production thereof and to a method for covalent binding fullerene derivatives to SH-containing proteins. Furthermore, the invention relates to the use of nitroxyalkyl-N-(fullerenyl)amino acids as nitrogen monoxide donors and also to the use of nitroxyalkyl-N-(fullerenyl)amino acids as quick-acting vasodilators for antihypertensive therapy. The invention also relates to a method for inhibiting a metastasis process and a method for enhancing antileukemic activity of cyclophosphamide.
It is known that the products of an equimolar attachment of amino acids and peptides to fullerene C60 /N-(monohydrofullerenyl)substituted amino acids or peptides/
[Romanova V.S., V.A. Tsyryapkin, Yu.I. Lyakhovetsky, Z.N. Parnes M.E. Vol'pin // Russian Chem.
Bull., 1994, vol. 6, pp. 1090-1091] compose a special class of organic compounds, which may be considered as potential antimetabolites relating to a certain subclass of physiologically active substances.
The broad spectrum of the biological activity of fullerene amino acid derivatives (FAD) is due to the unique structure of the carbonic spheroid, the capability thereof to convert oxygen to a singlet state [Tokuyama H., Nakamura S., Nakamura E. // J. Am. Chem. Soc., 1993, vol. 115, pp. 7918], manifest membranotropic and antiradical properties [Kotelnikova R.A., Kotelnikov A.I., Bogdanov G.N., Romanova V.S., Kuleshova E.P., Parnes Z.N., Vol'pin M.E.
// FEBS
Letters. - 1996. 389. - P. 111-114], antiviral activity [Frog E.S., Kotelnikova R.A., Bogdanov G.N., Shtolko V.N., Finegold I.I., Kush A.A., Fedorova N.E., Mejidova A.A., Romanova V.S. //
Technology of live systems. - 2006. 3. 2. - pp. 42-46] and cytotoxic action [Nakamura E., Tokuyama H., Yamago S., Shiraki T., Sugiura Y. // Bull. Chem. Soc. Japan. -1996. 69. - pp.
2143-2151]. Prerequisites to the study of cardiovascular effects due to the effect of fullerene derivatives are known [Sirensky A.V., Galagudza M.M., Egorova E.I. Arterial hypertension, 2004, 10 (3), 15-20]. It is for this reason in particular that fullerene C60 is given consideration in chemical pharmacology as a very promising carrier of functional groups that have one or another kind of biological activity. These include alkylating aggregations of cytotoxic action, anthracyclines, and also donors of nitrogen monoxide.
Different aspects of antitumor action of exogenic NO donors have been found by researchers in recent years [Konovalova N.P. // Technology of living systems. -2004. 1.3 - pp.
42-48]. They increase the effectiveness of the action of known cytostatics, slow the development of metastasis of experimental tumors and modulate the sensitivity of drug-resistant tumors to cytostatic therapy.
Nitrogen oxide is a unique intracellular polyfunctional regulator of metabolism,
2 homeostasis of blood vessels, arterial pressure and perfusion of the organs.
The majority of nitrogen oxide exogenic donors that are recommended for treatment of myocardial ischemia and acute cardiac insufficiency are widely used as vasodilatators and inhibitors of aggregation of thrombocytes.
A sufficiently broad bibliography of patents concerning the medicinal chemistry of fullerenes does not contain information in respect to novel polyfunctional amino acid derivatives of fullerene (ADF) containing biologically active groups, peptides or proteins. Methods for the preparation thereof are not disclosed either.
The object of the instant invention is to create novel polyfunctional amino acid derivatives of fullerene (ADF) that have inhibiting activity in respect to tumor deposits, and also enhance the antileukemic activity of cyclophosphamide, and which may be suitable as nitrogen monoxide donors or as quick action vasodilators for antihypertension therapy.
The polyfunctional fullerene C60 amino acid derivatives in accordance with the invention have the formula (1) R
N-Z
where R = H, mono- or dihydroxyalkyl, aminoalkyl, haloidalkyl, mono- or dinitroxyalkyl, maleinimide;

N-Z represents a fragment of a,(3,y,co-amino acid of the general formula -NH-CmH2m-COOM or COOM
-N

where m = 2-5, and M represents a nitroxyalkyl group, an alkyl group or an alkali metal salt, or dipeptide, wherein the alkyl groups contain 1-6 carbon atoms.
The authors of the instant invention were the first to develop methods for the preparation of ADF by replacing a movable proton in monohydrofullerenyl-amino acids and also by esterifying the carboxide of monohydroful lerenyl -amino acid.
In accordance with the aforesaid, upon the interaction of a derivative of monohydrofullerenyl amino acids with brommaleinimide, replacement of the movable proton takes place with the formation of maleinimide derivatives:
3 .91 N H O N

+ Br-N
I N-Z N-Z
O
O

where N-Z = a fragment of synthetic or natural a, y, co-amino acids (glycine, alanine, arginine, serine, y-aminobutyric, (o-aminocapronic acids, proline and others), a salt of the alkali metal thereof or ether.
The mobility of the proton of the monohydrofullerenyl-amino acids is due to the electronegative spheroid of the fullerene, which determines high polarization of the C-H bond and the preferred directivity thereon of the electrophilic replacement reaction :

0 .~ ,,, H CnH2n-p - (ONO2)p+l ~ / ~
~ + X - CnH2n-p - (ONO2)p+l -~ I -\ /
`~ ~ N-Z N-Z
n=2-5; p=0-2 where X = Cl-; Br-; J-.

In the case where -N-Z represents a fragment of synthetic or natural a, (3, y, co-amino acids of the general formula -NH-CmH2ri-COOM or COOM
-N

where m = 2-5, M represents a nitroxyalkyl group, an alkyl group, wherein the alkyl groups contain 1-6 carbon atoms, or a salt of an alkali-earth metal, wherein in the case of salts of alkali metals of amino acids, the reaction of monohydrofullerenyl-amino acids with nitroxylalkyl halogenides takes place along two reaction centers according to the equation:

H
+ X - CnH2n_p - (ONO2)p+i --' N - CmH2m - COOM
4 CnH2n-p - (ONO2)p+-~ -N - CmH2m - COO - CH2n-p - (ON02)p+1 H

where M = K, Na; m=2-5, n=2-5.
It is noted that even in the case of a small increase of the meanings of n, m and p, the relatively high, without that increase, hydrophobic nature of the compounds sharply increases. A
complete loss thereby of the hydrophilicity has a negative effect on the membrane activity thereof and consequently on the potential biological activity of synthesized NO donors.
Since in accordance with its structure ADP may be related to the class of antimetabolites, we proposed methods for the directed synthesis of fullerene derivatives that are covalently linked to biologically active peptides and proteins.
Camosine (R-alanylhistidine), which is a natural antioxidant of muscular and nerve tissues, was used as one of them. N-(monohydrofullerenyl) alanylhistidine, a direct analog of carnosine, comprising a fullerene sphere, was synthesized.

II H
NH2-CH2-CH2-C-N-CH-COONa ( - I + CH2 -=T
~i N~

-CH2-C-NH-CH-COONa OAH

CH2 The obtained compound is distinguished by increased membrane activity and the capability of inhibiting the process of peroxide oxidation of biological membranes.
Another example of the modifying action of the ADP on proteins and enzymes may be our implemented covalent attachment of the maleinimide derivative of the monohydrofullerenyl proline to the hemoglobin macromolecule:

N ~ N I -S-HeM
O + HeM - SH ----- O

N N I
COONa COONa This method is universal and is suitable for introducing a fullerene spheroid into any SH-containing proteins, providing for the transmembrane transfer thereof into a cell.
Studies carried out in recent years in the field of molecular cardiology have made it possible to establish the important role of nitrogen oxide in the control of vascular homeostasis.
The formation of NO upon the biotransformation of exogenic donors of nitrogen oxide causes a reduction of the tonus of coronary vessels, aggregation and adhesive capability of thrombocytes, which promotes enhancement of the hemodynamic characteristics and increase of the blood flow.
All of the obtained nitrates that are based on fullerene C60 amino acid derivatives (NFAD) in the process of their biotransformation generate nitrogen monoxide, which is shown by their inhibiting action against the catalytic activity of mitochondrial cytochrome-C oxidase.
NFAD-1 and NFAD-2, the structural formulas of which are presented below, exhibit moderate antitumor action and expressed antimetastatic activity.

CHz-CH2ONOz CH2-CH20N02 VNH-(CH2)5-C00CH2CH20N02 ~/~~ ~ \ ~ N NFAD-2 It is known that preparations based on nitroglycerine relate to the most widely-spread therapeutic agents for the treatment of cardiovascular diseases. However, they are characterized by a number of drawbacks and side effects, which limit the scale of their clinical use.
It was found upon a study of the effect of nitroxyalkyl-N-fullerenyl amino acid derivatives, in particular NFAD-1, on the coronary, contractile and pumping function of an isolated heart of rats of the Wistar strain of rats that NFAD-1 is a rapid-action vasodilatator that reduces arterial pressure and the frequency of heart contractions and causes weakening of the coronary vessels with a less, as compared with nitroglycerine (NG), depressive effect on the function of the myocardium [Pisarenko 0.1., Serebikova L.I., Studneva I.M., Ckitishvili O.V. //
Bull. of exper. biological medicine - 2006. 141(3) - pp. 267-269]. This means that compounds of this class open the way for the creation of original vasodilatators for antihypertension therapy.
The following examples of the preparation of the claimed compounds and studies of the biological activity thereof should not be understood as limiting the scope of the instant invention, and are only presented as examples of a preferable embodiment of the invention.

Example 1 Synthesis of methyl ester of N-nitroxy ethylfullerenyl proline A solution of 1.25 g (0.01 M) of 2-chloro-nitroxyethane in 10 ml of pyridine was added to a solution of 0.849 g(0.001 M) of N-monohydrofullerenyl proline methyl ester in pyridine and the mixture was held at room temperature for 6 hours. Then the reaction mixture was subjected to dialysis in the course of 20 hours.
:~, ,~ H CH2CH2ONO2 + C1CH2CH2ONO2 -~ ~ -N N

The residue was dried in air, obtaining the desired product close to the theoretical yield.
Absorption bands (1340 and 1540 cm'1), corresponding to the nitrate group, appeared in the infrared spectra of the N-(nitroxyfullerenyl)proline methyl ester.
Example 2 Synthesis of nitroxy ethyl ester of N-nitroxy ethylfullerenyl 12roline A solution of 2.5 g (0.02 M) of 2-chloro-nitroxyethane in 20 ml of pyridine was added to a solution of 0.86 g(0.001 M) of a Na-salt of N-monohydrofullerenyl proline in 50 ml of pyridine and the mixture was held at room temperature for 8 hours. Then the reaction mixture was subjected to dialysis in the course of 20 hours. The residue was dried in air, obtaining the desired product close to the theoretical yield.
,, ~ H CH2CH2ONO2 ~ ~ i ~
I - + 2 C1CH2CH2ONO2 ~ ~ -~ i ~ N~ N

COONa COOCH2CH2ONO2 Absorption bands (1340 and 1540 cm"1), corresponding to the nitrate group, appeared in the infrared spectra of the N-(nitroxyfullerenyl)proline methyl ester.
Example 3 Synthesis of a sodium salt of N-[monohydrofullerenyll-carnosine A ten-time excess of sodium salt of carnosine in 50 ml of water was added to a solution of 100 mg of fullerene C60 (0.138 mmol) in 50 ml of toluene. The obtained heterogenic system was stirred while being heated at 70 C in argon for 8 hours. Then the organic layer was separated, the solvent distilled off, the residue dissolved in 100 ml of water and 100 ml of a saturated solution of sodium chloride were added thereto. The obtained solution was subjected to dialysis until there was a complete removal of sodium chloride (a reaction for the absence of a chlorine ion in the wash water). The obtained aqueous solution was boiled off and 130 mg of sodium salt of N-[monohydrofullerenyl]-carnosine obtained (theoretical yield).
Upon hydrolysis of the obtained product in 6N of hydrochloric acid, an equimolar amount (2.16 0.2 M and 2.16 0.02 M are found) of (3-alanine and histidine is formed, corresponding to the calculated value (2.20 M is calculated), in accordance with HPLC-analysis.
Example 4 Synthesis of N-[(N-maleinimidyl fullerenyl]-L-proline methyl ester A solution of 1.76 g (0.01 mol) of brommaleinimide in 10 ml of pyridine was added to a solution of 0.849 g(0.001 mol) of N-(monohydrofullerenyl)-L-proline methyl ester in 50 ml of pyridine and the mixture stirred at room temperature for 8 hours. Then the reaction mass was subjected to dialysis, the residue dried, obtaining an N-[(N-maleinimidyl)fullerenyl]-L-proline methyl ester with a theoretical yield. In the infrared spectrum of the obtained product there are typical absorption bands at 1720 and 1355 cm"1, which confirm the presence in the molecule of a maleinimide fragment.
Example 5 Covalent attachment of a maleinimide derivative of C60- roline to macromolecules of proteins A special method was developed for the covalent attachment of a maleinimide derivative of fullerenyl proline to SH-groups of proteins. As an example, such an attachment to SH-groups of human hemoglobin and albumin was carried out.
In the case of human hemoglobin, the modification of two SH-groups, which are included in the structure of cystein-93 in two R-subunits of the hemoglobin macromolecule, was carried out. A maleinimide derivative of C60-proline in a three-time excess in respect to the protein was added to the solution of hemoglobin in a met-state in a concentration of 5x 104 M in a 0.1 M
phosphate buffer with pH=6.5. The reaction was carried out for 30 min at 20 C.
The excess of the water-soluble maleinimide derivative of fullerene was separated on a Sephadex G-15 column, using a phosphate buffer as the eluent. In the fraction exiting with a zero volume, the attachment of the maleinimide derivative to the hemoglobin was controlled by the spectrophotometric method. The concentration of the hemoglobin was determined in accordance with the characteristic absorption band at a wavelength of 407 nm (s=7.06x105 M"lcm"'), the concentration of the attaching maleinimide derivative of fullerene - according to absorption at a wavelength of 315 nm. Wherein it was taken that at that wavelength for the maleinimidyl-C6o-proline s=2.10x 104 M"lcm 1 and for hemoglobin - s=9.81 x 104 M" lcm 1. As a result of computer modeling of the sum spectrum of absorption of the product from individual spectra of absorption of hemoglobin and the maleinimide derivative of fullerene, it seems that as a result of the reaction, on the average 1.6 molecules of a maleinimide derivative of fullerene attach to one macromolecule of hemoglobin.
The attachment of the maleinimide derivative of C60-proline to the reaction-capable group of human albumin was carried out by a similar method. A maleinimide derivative of C60-proline with a three-time excess in respect to the protein was added to a solution of albumin in a concentration of 5x10-4 M in a 0.1 M phosphate buffer at pH=6.5. The reaction was carried out for 30 min at 20 C. The excess of the water-soluble maleinimide derivative of fullerene was separated on a Sephadex G-25 column, using a phosphate buffer as the eluent.
Chromatography on the column was carried out twice in order to eliminate the sorption of the fullerene derivative on the albumin due to hydrophobic interaction. In the fraction exiting with a zero volume, the attachment of the maleinimide derivative to the albumin was controlled by the spectrophotometric method. The concentration of the attaching maleinimide derivative of C60-proline spectrophotometrically, taking s=3.65x104 M"lcm I at 279 nm and s=1.4x103 M-lem"I at 310 nm for albumin, and for the maleinimide derivative of C60-proline -=3.30x104 M"Icm-1 at 279 nm and s=2.70x104 M"lcm"1 at 310 nm, E=3.65x104 M"lcm"1 at 279 nm and E=1.4x103 M-icm-i at 310 nm. As a result of analysis of absorption of the reaction mixture after two-time chromatography at two wavelengths, it was determined that as a result of the reaction, on the average 1.1 molecules of the maleinimide derivative of fullerene attach to one macromolecule of albumin. As control, it was determined by the method of amperometric titration that in the process of attaching the maleinimide derivative of C60-proline to human albumin, the number of free titrated SH-groups in the albumin decreases from 1.05 0.2 to 0.1 0.2.

Example 6 The antimetastatic activity of NFAD-1 and NFAD-2 and combinations thereof with cyclophosphamide (CP) was studied on two models of metastasizing solid tumors:
carcinoma LL
and melanoma B-16 in mice of the BDF, strain. The average number of metastasis in the lungs were determined in the experimental and control groups of mice and the index of inhibition of metastasis was calculated in percentage: IIM= ((N -Nk)/(Nk))x100. Statistical processing of the results of the study was carried out according to Student. The results were considered to be reliable with p < 0.05. The obtained results are presented in Table 1.

("Experimental assessment of antitumor preparations in the USSR and USA"
edited by Z.P. Sofina, A.B. Sirkin (USSR), A. Goldin, A. Kline (USA), Moscow, "Meditsina," 1980, pp.
76-86.) Table 1 Antimetastatic activity of NFAD-1 and NFAD-2 No. Preparations IIM, %
(unit doses, mg/kg) Carcinoma LL Melanoma B- 16 1 CF (30) 21 -NFAD-1 (50) 20 -CF+NFAD-1 (30+50) 48 -2 CF (30) - 31 NFAD-2 (83) - 10 CF+NFAD-2 (30+83) - 38 Data in respect to the antitumor activity of NFAD, which were determined in accordance with the method disclosed in the "Experimental assessment of antitumor preparations in the USSR and USA" edited by Z.P. Sofina, A.B. Sirkin (USSR), A. Goldin, A. Kline (USA), Moscow, "Meditsina," 1980, p. 73, are presented in Figs. 1 and 2.
Fig. 1 shows enhancement of antileukemic activity [increase of longevity (ILS, %) of mice of the BDF-1 strain with leukemia P-388] of cyclophosphamide (30 mg/kg; 1-6 days) upon the combination thereof with NFAD-1 (30 mg/kg; 1-6 days) and NFAD-2 (30 mg/kg;
1-6 days).
Fig. 2 shows the results in respect to an increase of the chemosensitizing effect of NFAD-1 and NFAD-2 (% of cured mice of the BDF-1 line with leukemia P-388) in the case of the combination thereof with cyclophosphamide (unit doses and regimens of administration - as in Fig. 1).

Example 7 In experiments in vivo NFAD-1 or NG (nitroglycerine) was administered intravenously in equimolar doses (2.6 x 10-5 mM/kg) to rats of the Wistar strain. The circulatory dynamics were characterized by meanings of the arterial pressure (AP) and the heart rate (HR). The obtained normalized meanings are presented in Table 2.

Table 2 Effect of NO donors on normalized values of chemodynamic indexes in rats under in vivo conditions NO donor AD HR
Nitroxyethyl-N-fullerenylproline (NFAD-1) 0.17 0.03 0.22 0.02 Nitroglycerine (NG) 0.22 0.04 0.20 0.03 A study has been made of the effect of the intravenous administration of NO
donors on the length of the period during which there is a reduction of the arterial pressure in rats under in vivo conditions (Fig. 3). A nitroxy derivative of fullerene in a wide range of concentrations has a weak effect on the length of the period of reduced arterial pressure and significantly (by two-three times) reduces this index as compared with nitroglycerine.
The data were obtained in a series of 6 experiments and the results are presented on Fig.
3.
Example 8 The effect of NFAD-1 on the function of the left ventricle of an isolated heart of a rat and the function of coronary vessels at a concentration of NFAD-1 of 3.5 x 10-5 M
was studied with the use of a generally accepted method [Pisarenko 0.1., Shulzhenko V.S., Studneva I.M., Timoshin A.A. // Cardiology. - 2004. 44(4) - pp. 65-70]. The obtained normalized meanings of the pressure (P), developed by the left ventricle, the intensity of contraction (IC) and the coronary flow (CF) are presented in Table 3.
Table 3 Changes of the functional characteristics of the left ventricle and intensity of the coronary flow under the effect of NO donors NO donor P IC CF
NFAD-1 0.64 0.48 0.90 Nitroprussid 0.49 0.38 0.80 The reliability of the differences is confirmed by the Student criterion with P<0.05.

LITERATURE
1. Romanova V.S., V.A. Tsyryapkin, Yu.I. Lyakhovetsky, Z.N. Parnes, M.E.
Vol'pin.
Addition of amino acids and dipeptides to fullerene C60 giving rise to monoadducts. - Russian Chem. Bull., 1994, vol. 6, pp. 1090-1091.
2. Tokuyama H., Nakamura S., Nakamura E. Photoinduced biochemical activity of fullerene carboxylic acid. - J. Am. Chem. Soc., 1993, vol. 115, p. 7918.
3. Kotelnikova R.A., Kotelnikov A.I., Bogdanov G.N., Romanova V.S., Kuleshova E.F., Parnes Z.N., Vol'pin M.E. Membranotropic properties of the water soluble amino acid and peptide derivatives of fullerene C60. // FEBS Letters. - 1996. 389. - pp. 111-114.
4. Frog E.S., Kotelnikova R.A., Bogdanov G.N., Shtolko V.N., Fingold I.I., Kush A.A., Fedorova N.E., Mejilova A.A., Romanova V.S. Effect of amino acid derivatives of fullerene C60 on the development of cytometaloviral infection. // Technology of live systems. - 2006. 3. 2. -pp. 42-46.
5. Nakamura E., Tokuyama H., Yamago S., Shiraki T., Suguira Y. Biological activity of water-soluble fullerenes. Structural dependence of DNA cleavage, cytotoxicity, and enzyme inhibitory activities including HIV-protease inhibition. // Bull. Chem. Soc.
Japan. - 1996. 69. -pp. 2143-2151.
6. Sirensky A.V., Galagudza M.M., Egorova E.I. Arterial hypertension, 2004, 10(3), 15-20.
7. Konovalova N.P. Nitrogen monoxide donors in experimental chemotherapy of tumors Technology of live systems. - 2004. 1. 3. - pp. 42-48.
8. Pisarenko 0.1., Serebryakova L.I., Studneva I.M., Ckitishvili O.V. // Bull.
of experim.
Biol. Med. - 2006. 141 (3) - pp. 267-269.
9. Pisarenko 0.1., Shulzhenko V.S., Studneva I.M., Timoshin A.A. //
Cardiology. - 2004.
434 (4) - pp. 65-70.
10. "Experimental assessment of antitumor preparations in the USSR and USA"
edited by Z.P. Sofina, A.B. Sirkin (USSR), A. Goldin, A. Kline (USA), Moscow, "Meditsina," 1980.

Claims (9)

1. Polyfunctional fullerene C60 amino acid derivatives of formula (1) where R = H, mono- or dihydroxyalkyl, aminoalkyl, haloidalkyl, mono- or dinitroxyalkyl, maleinimide;
N-Z represents a fragment of .alpha.,.beta.,.gamma.,.omega.-amino acid of the general formula -NH-C m H2m-COOM or where m = 2-5, and M represents a nitroxyalkyl group, an alkyl group or an alkali metal salt, or dipeptide.
2. A method for preparing a compound according to claim 1, comprising treatment of a monohydrofullerenyl amino acid alkyl ester with a excess of halogen derivative nitroxyalkyl in a pyridine solution at room temperature for 6-10 hours followed by dialysis of the reaction mixture.
3. A method for preparing compounds according to claim 1, comprising esterification of a carboxy group of fullerenyl amino acid by treatment of alkaline metal salt of fullerenyl amino acid with a excess of halogen derivative nitroxyalkyl in a pyridine solution at room temperature for 6-10 hours followed by dialysis of the reaction mixture.
4. A method for covalent binding fullerene derivatives to SH-containing proteins, characterized in that a corresponding protein is subjected to interaction with the maleinimide amino acid derivative of fullerene C60 according to claim 1.
5. Use of nitroxyalkyl-N-(fullerenyl)amino acid according to claim 1 as a nitrogen monoxide donor.
6. Use of nitroxyalkyl-N-(fullerenyl) amino acid according to claim 1 as rapid action vasodilatators for antihypertension therapy.
7. Use of nitroxyalkyl-N-(fullerenyl) amino acid according to claim 1 as chemosensibilizers in cytostatic chemotherapy of tumors.
8. A method of inhibiting the metastasis process, comprising administering a polyfunctional fullerene C60 derivative according to claim 1, in combination with cytostatics.
9. A method for enhancing antileukemic activity of cyclophosphamide, characterized in that cyclophosphamide is administered together with the polyfunctional fullerene C60 derivative according to claim 1.
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US9246104B2 (en) 2013-09-25 2016-01-26 Showa Denko K.K. Fullerene derivative, method of manufacturing fullerene derivative and solar cell

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JP4293735B2 (en) * 2001-01-19 2009-07-08 三栄源エフ・エフ・アイ株式会社 Fullerene derivative and composition comprising the same
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US9246104B2 (en) 2013-09-25 2016-01-26 Showa Denko K.K. Fullerene derivative, method of manufacturing fullerene derivative and solar cell
WO2015138819A1 (en) * 2014-03-14 2015-09-17 Livepet, Llc. Cellular enhancements through lipofullerene and peptide group combinations

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