Note: Descriptions are shown in the official language in which they were submitted.
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POLYFUNCTIONAL FULLERENE C60 AMINO ACID DERIVATIVES
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,
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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:
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0
.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
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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.
0
II H
NH2-CH2-CH2-C-N-CH-COONa
( - I + CH2 -=T
~i
N~
0
11
NH-CH2
-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:
0 0
N ~ N I -S-HeM
O + HeM - SH ----- O
N N I
COONa COONa
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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
COOCH3
NFAD-1
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.
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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
COOCH3 COOCH3
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
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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
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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.)
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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
CA 02687557 2009-11-17
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.
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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.
