Cobalamin derivatives used for diagnosis and treatment anomalous cell proliferation

FIELD: medicine.

SUBSTANCE: invention refers to cobalamin derivatives of the formula , where Rb means spacer chelating group of the formula , where n is 2, 3 or 4; Rc, Rd, Re and RR denote hydrogen, and X is cyano; or where Rd indicates spacer chelating group of the formula , where n is 3; Rb, Rc, Re, and RR denote hydrogen, and X is cyano, or where Rb indicates spacer chelating group of the formula , where n is equal to 2; Rc, Rd, Re and RR denote hydrogen, and X is cyano. These compounds are accumulated to a much lesser extent in blood and non-malignant organs such as kidneys and liver, compared with the rate of accumulation in neoplastic tissues, and moreover they are quickly removed from blood. The invention also refers to injectable pharmaceutical composition for diagnosis of tumors including cobalamin derivative carrying a radioactive metal atom. In addition, the invention refers to diagnosis of tumors in mammals, including (a) compliance vitamin B12-free diet for mammals predisposed to neoplastic disease, (b) subsequent administration of cobalamin derivative carrying a radioactive metal atom. The invention also refers to use of cobalamin derivative carrying a radioactive metal atom, in method of diagnosis of tumors in mammals and to obtain pharmaceutical composition designed for use in method of diagnosis of neoplastic diseases in mammals.

EFFECT: improvement of composition application.

13 cl, 2 tbl, 6 dwg, 35 ex

 

The scope of the invention

The present invention relates to methods of imaging and suppress unwanted rapidly proliferating cells in multicellular organisms.

Background of invention

Abnormal proliferation of cells, especially hyperproliferate, is the source of many diseases, of which the most serious is cancer. Only in the United States each year diagnosed with cancer registered in approximately 1.5 million people and death from cancer at 0.5 million people. Currently, despite considerable progress in the treatment of cancer, treatment methods are characterized by numerous disadvantages. The main problems associated with severe side effects of anticancer agents and the development of sustainable generation of cancer cells, as well as early localization, precise localization of the tumor and metastasis.

Hyperproliferative cells, as many types of cancer cells depend on the increased inflow of nutrients, growth factors, energy and vitamins. In this regard, there is an opportunity to deliver drugs in these unwanted cells using a path of transport of vitamins, which are essential for cell growth and in most cases, the supply of which is limited.

Cobalamin (Cbl), also known as vitamin B12, present is in the form of cyanocobalamin (CN-Cbl), hydroxocobalamin (OH-Cbl) or aquacobalamin (H2Oh-Cbl), is vital, and its concentration in the body is extremely small. Higher organisms, including humans, have to obtain the vitamin from food. The biosynthesis of cobalamin is only some prokaryotes, such as anaerobic bacteria. Cobalamin is essential for the normal function of the nervous system and is necessary for normal metabolism of carbohydrates, proteins and fats. Cobalamin is utilized in several vital intracellular metabolic pathways. As methylcobalamin (Me-Cbl) it is used as a cofactor for methodinstances. In the form of 5'-deoxyadenosylcobalamin (Ado-Cbl) it acts in conjunction with methylmalonyl-SOA-mutate when turning methylmalonyl-COA in succinyl-COA. The lack of cobalamin can lead to pernicious anemia. Cobalamin is also included in the recovery process of the conversion of ribonucleotides to deoxyribonucleotides in the formation of DNA.

In mammals, in most cases, the uptake by cells of cobalamin is regulated by serum transport proteins and cell membrane receptors. There are two types of cobalamin-binding proteins in plasma: non-protein transcobalamin II (TC) and glycosylated proteins transcobalamin I and III (TCI and TCIII), is also called R-binding proteins or haptocorrin. TCI and TCIII are characterized by immunoprotective reaction and may differ only in carbohydrate composition. TCI is the primary R-binding protein found in the bloodstream. For simplicity, the term TCI used to refer to both types of R-binding proteins TCI and TCIII. Both types of transport proteins (vectors) TCI and TCII circulate in the bloodstream of mammals in the form of partially saturated (Golubinci) or partially unsaturated (apalaci) cobalamin. In mammalian cells there is also a non-vector system poglosheniya of cobalamin, which in normal cells is sufficiently low efficiency (see Sennet S. and Rosenberg LE, Ann. Rev. Biochem. 50, 1053-86 (1981)).

Function TCII is the delivery of plasma cobalamin in all metabolically active cells by the mechanism of receptor-mediated endocytosis. It is known that accelerated cell proliferation in the occurrence and development of tumors (neoplasia) directly causes increased consumption associated with cobalamin TCII from the bloodstream by receptor-mediated endocytotic mechanism of absorption. In many studies found that increased regulation of a number of TCII receptors observed in malignant cell lines to meet the increasing metabolic demands in the production of thymidine and methionine, in the reactions metelyova the Oia in the DNA synthesis and energy supply of cells by the mechanism of mitochondrial metabolism.

The main TCII receptor is present in all types of tissues, while the second TCII receptor with greater specificity to the authorities, the so-called megalin, mainly expressed in the proximal tubules of the kidney and certain other types of absorptive epithelium. After endocytotic internalization TCII degraded in the lysosomes and free cobalamin is transported into the cytoplasm and in the nuclear membrane, where it turns into Me-Cbl and Ado-Cbl. These two forms are a function of the active coenzymes vitamin B12. The important role of TCII evidenced by the fact that hereditary congenital TCII deficiency causes megaloblastic anemia, severe neurological damage and death, if not to treat excess cobalamin.

Almost all cells are able to produce TCII. Many cells, such as hepatocytes, fibroblasts, nerve cells, enterocytes and macrophages synthesize increased amounts TCII. It is assumed that the primary source TCII is the endothelium of blood vessels. Approximately 20-30% of circulating cobalamin associated with TCII in the form of holo-TCII, which is metabolically effective manner that provides the internalization of cobalamin in all tissues (see E. Rothenberg, and others, in the book: Chemistry and Biochemistry of B12, ed R.Banerjee, New York, NY, cc.441-473 (1999)).

TCI is present in blood and plasma, and that the same in most exocrine secretions and other types of liquids. This protein is mainly produced in the tissues of the anterior intestine, gastric mucosa, salivary and lacrimal glands and secretory epithelium of the inner ear. TCI, unlike TCII, supplies not associated with cobalamin directly to absorption by cells, characterized by a prolonged half-life in the blood and thus at any given moment, holds more than 75% of circulating cobalamin (and corrina). Almost all TCI circulates in the form of holo-TCI. Its role is poorly understood. It is assumed that it functions as a bacteriostatic agent, which prevents the delivery of all types of cobalamines and korinov in microorganisms. This protein stabilizes also adenosylcobalamin and protects it from photolysis. In contrast to the TCI, the concentration of which in circulation is higher compared to TCII, level TCII can grow rapidly at the expense of synthesis de novo APO-TCII in response to incoming cobalamin. TCI builds up quite slowly and largely not stimulated in response to any trigger pulse (see the above book Alpers and D. Russell G., Chemistry and Biochemistry of B12, cc.411-441).

Up to the present time non-vector delivery of cobalamin in mammalian cells was not considered as an alternative way of absorption of cobalamin derivative of hyperproliferative cells. No calls from the opinion the fact that that physiologically important mechanisms of absorption of cobalamin benign cells pleocytosis include vectors TCII and TCI (as well as its own factor in the digestive tract). However, according to studies in vivo and in vitro that the free cobalamin is also able to penetrate through the plasma membrane without the participation of the vector proteins. Direct proof of the additional capacity of absorption of free cobalamin obtained in the study of children with congenital and full TCII deficiency, in whom parenteral free cobalamin was a marked remission of clinical and chemical characteristics of intracellular cobalamin deficiency (see Hall, S.E., and others, Blood, 53, 251-263 (1979)). In the study in vitro was observed absorption of free cobalamin HeLa cells and fibroblasts. In HeLa cells the uptake of free cobalamin is from 1% to 2% from acquisitions associated with TCII of cobalamin. In human fibroblasts accumulation of free cobalamin with an interval of 2 h is approximately 20% associated with TCII vitamin. System free absorption of vitamin a in human fibroblasts has been studied quite extensively, as described in the article in the Berliner N. and Rosenberg LE, Metabolism, 30, 230-236 (1981)). The absorption of free CN-[57Co]-Cbl investigated in the two-stage system: the initial absorption occurs with the high speed on the mechanism of saturation and specifically inhibited by excess unlabeled CN-Cbl and OH-Cbl, the absorption process is terminated after 30 minutes the Second stage of absorption is slower in linear dependence on time is not inhibited by an excess of its cobalamin and does not plateau even after 8 h, which shows the characteristic of non-specific process. The first stage of absorption is highly specific for the process of penetration through the membrane, which is mediated by protein and sensitive to sulfhydryl reagents, additionally, this process significantly inhibited by cycloheximide (see Sennet S. Rosenberg LE, Ann. Rev. Biochem. 50, 1053-86 (1981)). These properties are consistent with the presence of mediated protein rapid absorption of free cobalamin in mammals.

Found that many bacteria and all eukaryotic protists are auxotrophic for vitamin B12 and associate it with a higher affinity compared to the native factor mammals, TCI and TCII. Linking B12 proteins from bacteria and protozoa are proteins that interact in non-vector mechanism with the cell surface and is able to link many of Corrino (including myself cobalamin) with high affinity. Therefore, there is a possibility of detection of bacterial infections in the visualization of the whole body after injection of radioactively labeled derivative was cobal the mine (see article D.A. Collins and others, Mayo Clin. Proc. 75, 568-580 (2000)). The development of hyperproliferative forms of mammalian cells can lead to the development during multistage carcinogenesis of more efficient forms of existing non-vector systems of the absorption of cobalamin.

Currently published and patented approaches to the use of cobalamin as a carrier for a wide range of biologically active agents, including radioactive isotopes of metals (see patent D.A. Collins, US Pat. Appl. No.2003/0144198). When tested on animals and patients using radioactively labelled derivatives of cobalamin was observed incorporation of label in the tumor tissue, but there are also significant accumulation of radioactivity in healthy tissues, such as kidneys and liver. Therefore, imaging and radiotherapy are not yet optimal. Currently the use of these published methods are limited to possible damage some of the healthy sections of the body.

Thus, there is a need for compounds, compositions and methods for the introduction of diagnostic and therapeutic derivative of cobalamin in quickly proliferous cells in higher concentrations compared with normal cells. The object of the present invention is the development of new methods for the identification, synthesis, is harakteristiki and the introduction of the derivative of cobalamin with high specificity in the cells, characterized by abnormally high proliferation, and exclude the development of sustainable generation of cells.

A brief description of the invention

The present invention is based on the fact that unlike the cobalamin derivative of cobalamin, which is not associated or linked to a much lesser extent with the transport protein, transcobalamin II (TCII), when appropriate way of introducing accumulate to a much lesser extent in the blood and benign organs such as the kidneys and liver, as compared with the rate of accumulation in neoplastic tissues and, in addition, are rapidly removed from the blood. When using cobalamin derivative acting as substitutes for vitamin B12, significantly reduces the risk of the formation of the sustainable generation of neoplastic tissues.

The invention relates to a derivative of cobalamin,

(a) does not have a binding affinity or low binding affinity towards transcobalamin II and

(b) preserving the activity of substitute vitamin B12.

Primarily, the invention relates to a derivative of cobalamin,

(a) with less than 20%, preferably less than 5%, of the binding affinity towards transcobalamin II compared to the binding activity of unmodified cobalamin, which determine Aut analysis of the binding, and

(b) preserving more than 2% of the activity of substitute vitamin B12, which is determined by the method of analysis of cell growth.

Examples of compounds according to the present invention, which has a low binding activity against TCII or not possessing such activity include specific derivative of cobalamin containing therapeutic and/or diagnostic agent, such as a radioactive metal. Compounds of the present invention is chosen according to the results determine the binding activity using purified TCII and analysis of cell growth using as the study organism bacteria Lactobacillus delbrueckii.

The present invention relates also to a method of diagnosing a neoplastic disease or infection of the microorganism in a mammal, including

(a) a diet that does not contain vitamin B12, for a mammal that is susceptible to neoplastic disease or infection, and

(b) the subsequent introduction of the derivative of cobalamin according to the invention, containing the diagnostic agent.

Similarly, the present invention relates to a method of treatment of a mammal suffering from a neoplastic disease or infection of the microorganism, including

(a) a diet that does not contain vitamin B12, to a mammal in need of treatment, and

b) the subsequent introduction of the derivative of cobalamin according to the invention, containing therapeutic agent.

The present invention relates also to the use of a derivative of cobalamin in the method for the diagnosis of neoplastic disease, or infection of the microorganism or in the treatment of a mammal suffering from a neoplastic disease or infection of the organism.

In addition, the present invention relates to pharmaceutical compositions containing cobalamin derivatives of the present invention, primarily to compositions suitable for diagnosis, and to pharmaceutical compositions suitable for treatment, as well as to the use of such pharmaceutical compositions in the method of diagnosis and treatment, respectively.

The present invention also relates to intermediate compounds used to obtain compounds suitable for the diagnosis or treatment of the present invention, first and foremost to compounds containing chelating substituents for binding of radioactive metals, but does not contain associated with chelate Deputy metal or non-radioactive metal.

Derivative of cobalamin in the present invention are primarily valuable for the diagnosis and/or treatment of aggressive, rapidly progressing neoplastic diseases, such as cancer, and/or diagnosis and/or treatment of local infections patoh is the R of microorganisms.

Brief description of drawings

Figure 1 presents graphs of the analysis of the interaction of radio-labelled cyanocobalamin-b-propyl-PAMA-OEt (example 11), not TCII binding transport proteins by gel-filtration on the shift of the molecular weight (y - axis the time on the x-axis is the number of pulses per minute (imp./min)),

(A) gel filtration of radioactively labeled derivative on a column of Superdex 75 (peak corresponds to the molecular weight of 1.5 kDa),

(B) gel filtration derivative in a mixture with TCI (the shift in molecular weight from 1.5 kDa and 44 kDa),

(C) gel filtration derivative in a mixture with TCII (peak corresponds to the molecular weight of 1.5 kDa, i.e. cyanocobalamin-b-propyl-PAMA-OEt virtually no contact with TCII).

Figure 2 presents graphs of the analysis of the interaction of radio-labelled cyanocobalamin-b-butyl-PAPAcet (example 5), TCII binding transport proteins by gel-filtration on the shift of the molecular weight (y - axis the time on the x-axis is the number of pulses per minute (imp./min)),

(A) gel filtration of radioactively labeled derivative on a column of Superdex 75 (peak corresponds to the molecular weight of 1.5 kDa),

(B) gel filtration derivative in a mixture with TCI (the shift in molecular weight from 1.5 kDa and 44 kDa),

(C) gel filtration derivative in a mixture with TCII (shift in molecular weight from 1.5 kDa to 60 kDa, that is, C is anakapalli-b-butyl-PAPAcet associated with TCII).

Figure 3, 4, 5 and 6 presents the distribution diagram in tissues

on the y - axis the percentage entered radioactivity per gram of tissue,

on the x - axis agencies: 1) blood, 2) heart 3) spleen, (4) kidney, and (5) stomach, 6) bowel, 7) liver, 8) muscle, 9) bone, 10) tumor.

Figure 3 presents the diagram of distribution after intravenous injection of radioactive cyanocobalamin (57Co-CN-Cbl) in tissues of mice treated with normal food.

4 shows the diagram of distribution after intravenous injection of radioactive cyanocobalamin (57Co-CN-Cbl) in tissues of mice treated with food without vitamin B12.

Figure 5 presents the diagram of distribution after intravenous injection of radioactive cyanocobalamin-β-propyl-PAMA-OEt (example 11) in tissues of mice treated with normal food.

Figure 6 presents the diagram of distribution after intravenous injection of radioactive cyanocobalamin-β-propyl-PAMA-OEt (example 11) in tissues of mice treated with food without vitamin B12.

Detailed description of embodiments of the present invention

With the introduction of mammals, observing not contain vitamin B12 diet, cobalamin derivative exhibiting extremely low binding affinity against cobalamin-vector protein (or transport protein) or not possessing such activity observed is a significant reduction in the accumulation in the blood and vital organs, such as the kidneys and liver, and is supported with a high level of absorption in hyperproliferative cells, which allows the use of such derivatives for a more accurate diagnosis and treatment of neoplastic diseases and local infections of microorganisms.

The compounds of the present invention, showing a low binding affinity against TCII and preserving the activity of vitamin B12 include, for example, the compounds of formula (I)

where Rb, Rc, Rdand Reindependently from each other mean a spacer elements chelate group, an antibiotic or an anti-proliferative therapeutic agent, an organic group with a certain configuration, containing from 4 to 20 carbon atoms, or hydrogen,

RRmeans spacer elements chelate group, an antibiotic or an anti-proliferative therapeutic agent, each of which is connected through a bridging group Z, or hydrogen,

provided that at least three of the remainder of Rb, Rc, Rd, Reand RRmean hydrogen and at least one of Rb, Rc, Rdand Redoes not mean hydrogen,

X means monodentate ligand, and

the Central atom of cobalt (Co) is not necessarily in the form of a radioactive isotope.

In a preferred embodiment, the image is etenia R emeans hydrogen.

Monodentate ligand X is, for example, cyano,

halogen, such as fluorine, chlorine, bromine or iodine, cyanate, isocyanate, thiocyanate or isothiocyanate,

alkyl straight or branched chain, containing from 1 to 25 carbon atoms, preferably from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl or isobutyl or n-hexyl or n-decyl, and optionally substituted groups: hydroxy, methoxy or amino, for example hydroxymethyl, methoxymethyl, aminomethyl, hydroxyethyl or methoxyethyl,

nitrile R-CN, isonitrile R-NC, carboxylate R-COO-or tiolet R-S-where R means alkyl straight or branched chain comprising from 1 to 15 carbon atoms, preferably from 1 to 6 carbon atoms, or aryl, for example phenyl or naphthyl, such as acetonitrile, propionitrile, benzonitrile, methyl isocyanate, phenylisocyanate, acetate, propionate, benzoate, mertiolate, editionat, n-exertional or thiophenolate,

postit (RO)3R, where the substituents R are identical or different and denote alkyl containing from 1 to 6 carbon atoms, or aryl, such as optionally substituted phenyl or naphthyl, such as trimethylphosphite, methyldiphenylphosphine, triphenylphosphine or tri-ortho-tolylphosphino,

hydroxy or the rest of AquaCity, or

the remainder of the 5'-deoxyadenosine or rodstvennoj the nucleoside.

X preferably denotes cyano, methyl, hydroxy, the residue of aquability or 5'-deoxyadenosine, most preferably cyano.

Spacer elements chelate group as substituents Rb, Rc, Rd, Reor RRmeans chelate group to metal atoms attached to NH - or-O-group of cobalamin through spacer elements group, and optionally contains a metal atom, especially a radioactive atom of metal.

The compounds of formula (I), in which the spacer elements chelate group does not contain a metal atom, are used as intermediate derivatives to obtain compounds intended for use in the methods of diagnosis and/or treatment according to the present invention.

If the substituent Rb, Rc, Rd, Reor RRuse an antibiotic or an anti-proliferative therapeutic agent, an antibiotic selected from the group comprising aminoglycoside antibiotics such as gentamicin, tetracycline, antimetabolites, such as Selenomethionine, macrolides such as erythromycin and trimethoprim, and antiproliferative agent selected from the group including antimetabolites such as 5-fluorouracil, an alkylating agent, such as oxaliplatin, dacarbazine, cyclophosphamide or carboplatin, an inhibitor of the cell cycle, such as vinblastine or docetaxel, degr deruosi DNA agent is a topoisomerase inhibitor, intercalator, splitting chain agent, such as doxorubicin, bleomycin or topotecan, connection, affecting the transmission signal, such as a modifier of the activity of a caspase, agonist or antagonist of the receptor cell death, and the modifier nucleases, fasthosts and kinases, such as of imatinib mesilate, dexamethasone, monistat-phorbol acetate, cyclosporine a, quercetin or tamoxifen, and these connections are attached to NH - or-O-group of cobalamin covalent bond either directly or through a spacer.

The spacer means aliphatic chain containing from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, for example from 2 to 5 carbon atoms, where one or two carbon atoms replaced by nitrogen atoms and/or oxygen, and aliphatic chain substituted by hydroxy groups, oxo, or amino. First of all, the two adjacent carbon atom is replaced by aminogroup-NH-CO - or ester group-O-CO-.

Examples of spacers connecting NH - or-O-group of cobalamin with a chelate group include ethylene, propylene, butylene or pentile, or group in which one carbon atom is replaced by an oxygen atom or nitrogen, or one carbon atom is replaced by an oxygen atom or nitrogen, or an adjacent carbon atom substituted with oxo.

Chelate compound contains two, three or more donor atoms selected from nitrogen, oxygen and sulfur, to the which are located at a sufficient distance to bind to the metal atom. First of all, chelate compounds include tridentate chelate compounds containing three binding site with metal, including donor atoms of nitrogen, oxygen and/or sulfur, and the distance between them is sufficient to bind to the metal atoms. The nitrogen atoms as donor atoms are part of, for example, aliphatic amine, aromatic amine or nitrogen-containing aromatic heterocycle. Oxygen atoms as donor atoms are part of, for example, alcohols, esters and ethers or carboxylic groups. Sulfur atoms as donor atoms are part of, for example, thioethers or thiols. Donors attached through, for example, aliphatic hydrocarbon chain or a chain consisting of amide linkages, and are derived from amino acids, polyesters, etc.

The preferred chelate compounds are chelate compounds of formulas (II)to(IX). Carboxyl groups can be present in the form of esters, which hatshepsuts simultaneously with the formation of a complex with a metal atom, with the formation of the coordinating carboxylate groups. Such esterified chelate compounds, that is, esters, may include alkyl esters in which the alkyl group is straight or branched and contains from 1 to 25 carbon atoms, and neoba is consequently from one to five carbon atoms are replaced by nitrogen atoms or oxygen, or one or two carbon atoms replaced by atoms of sulfur or phosphorus, and these atoms optionally substituted groups: phenyl, pyridyl, hydroxy, halogen, cyano, oxo, or amino. Ester also includes aryl or heteroaryl ether, in which the aryl or heteroaryl includes from 3 to 10 carbon atoms or zero, one, or two atoms of oxygen, zero, one, two or three nitrogen atom or zero or one sulfur atom. Such ester residues appropriately substituted to ensure the removal of ester groups in certain reaction conditions, for example, as described for ester groups in their use as protective groups for carboxyl groups, see the book Green T.W. and Wuts P.G.M., Protective groups in organic synthesis, Wiley (1999).

To the esterified chelate compounds are preferably compounds in the form of methyl, ethyl or cyanomethylene esters.

As radioactive metals using radioactive isotopes, such as94mTc99mTc188Re,186Re,111In90Y64Cu67Cu and177Lu, primarily99mTc188Re,186Re, and111In.

Radioactive isotopes With include, for example,57And60Co.

To organic groups with a specific conformation containing from 4 to 20 carbon atoms, include, for example, alkyl, cycloalkyl, the aryl arylalkyl, heteroaryl or heteroaromatic, optionally substituted groups: hydroxy, alkoxy, oxo, amino, carboxy, carbarnoyl or alkoxycarbonyl. Examples of aryl groups include phenyl, were, dimetilfenil, hydroxyphenyl or naphthyl. Examples of heteroaryl groups include pyridyl, pyrrolyl, imidazolyl or benzimidazolyl. In the alkyl chain carbon atoms are replaced by nitrogen atoms or oxygen. For example, in the alkyl chain by one carbon atom is replaced by an atom of nitrogen (or oxygen), and the adjacent carbon atom substituted by oxopropoxy education carboxamide (or ester groups, respectively). Typical examples of organic groups with a specific conformation include isobutyl, tert-butyl, tert-pentyl, ortho-tolyl, ortho-methylbenzyl or 2,6-dimethylbenzyl.

The linking group Z, R Rwith spacer elements chelate group or antibiotic or anti-proliferative agent, means linking or bridging group which is selected from the group comprising phosphates, phosphonates, esters of carboxylic acids or alkylene containing from 1 to 10 carbon atoms, or combinations thereof. Such a connecting group connecting the spacer elements chelating group or a therapeutic agent, optionally containing spacer elements group, as defined above, with oxygen atom in the structure of the cobalamin.

Compounds that are modified by the group RRbut in which Rb, Rc, Rdand Reall mean hydrogen, and which are bound to transport proteins vehicle and at the same time exhibit enzymatic activity, are excluded from the scope of the present invention.

The choice of compounds of the present invention is based on the following criteria:

(a) the absence of binding affinity or significantly reduced binding affinity in relation to TCII, for example less than 20%, especially less than 10%, preferably less than 5%, more preferably less than 2%, compared to the unmodified cobalamin, and

(b) activity as a substitute for vitamin B12 defined using the growth of bacteria or cell lines mammals that are dependent on vitamin B12, is, for example, more than 2% activity, first su is th more than 10%, preferably more than 20% activity compared with the activity of vitamin B12, which shows a non-modified cobalamin.

To determine the binding affinity of the derivative of cobalamin (Cbl) with TCII the in vitro assays performed using partially purified TCII, isolated from the blood of the rabbit. As the substrate can also be used recombinant TCII, which is produced in an expression system E. coli.

Derivative of cobalamin in the present invention should be proactive as a substitute of vitamin B12. In this regard, significantly reduced the risk of development of resistance, which leads to the formation of cells with a high proliferation rate. In all likelihood mutant cells, which does not absorb cobalamin derivative with a low binding activity against TCII or not possessing such activity, lose their ability of progenitor cells to maintain a high level of proliferation by a mechanism of highly efficient absorption of vitamin B12-independent TCII.

To determine the activity of the derivative of cobalamin in the vitamin B12 used the analysis in the presence of Lactobacillus delbrueckii, which is recommended worldwide as a standard strain for identification of cyanocobalamin (CN-Cbl). Adding cyanocobalamin in an environment that does not contain cyanocobalamin, in response, causes growth of the cyanocobalamin-auxotrophic bacterial strain, which can be determined by the method of quantitative solid-phase diffusion method on the plate. This method of analysis used to determine the ability of the derivative of cobalamin (in %) to replace cyanocobalamin as a vital catalyst.

The present invention relates also to method of diagnosis and treatment of neoplastic diseases and local infections of microorganisms in a mammal, including

(a) the diet of mammals that do not contain vitamin B12,

(b) subsequent maintenance derivative of cobalamin according to the present invention, containing diagnostic or therapeutic agent,

and cobalamin derivative according to the invention in the specified method.

The positive impact of the introduction of non-binding TCII derivative of cobalamin on their biodistribution in mammals after diet that does not contain the vitamin, described in table 1.

Table 1
The tissue distribution in mice 24 h after intravenous injection of radioactively labelled derivatives
Example5 681011121418202225
Mutual. with TCII+++---+-+-+
Blood2,302,402,200,100,090,041,200,062,100,250,18
Kidney14,1015,8016,501,360,391,0840,00the 10.4019,90 3,54116,00
Liver9,407,408,101,450,440,948,003,9021,063,9020,70
Tumorof 7.907,303,600,731,616,133,00to 6.809,202,903,16

Example 5: cyanocobalamin-b-butyl-PAPAcet,

example 6: cyanocobalamin-b-butylaminoethyl-His-OMe,

example 8: cyanocobalamin-with-butyl-PAPAcet,

example 10: cyanocobalamin-b-ethyl-PAMA-OEt,

example 11: cyanocobalamin-b-propyl-PAMA-OEt,

example 12: cyanocobalamin-b-butyl-PAMA-OEt,

example 14: cyanocobalamin-b-hexyl-PAMA-OEt,

example 18: cyanocobalamin-d-cut-FRAME-OEt,

example 20: cyanocobalamin-b-propyl-His-OMe,

example 22: cyanocobalamin-b-ethyldiamine,

example 25: cyanocobalamin-5'-postcolumn-His-OMe.

The results of the analysis bioresidues what I in table 1, indicate that not satybaldina with TCII compounds, for example compounds of the present invention, described in examples 10, 11, 12, 18 and 22, substantially accumulate in tumors, 5 times or more compared with blood and at least more than half of the amount specified in the vital organs, the kidneys and liver. The compounds obtained in examples 5, 6, 8, 14, 20 and 25 do not apply to compounds according to the invention, since they are associated with TCII and described only for comparison.

Derivative of cobalamin according to the present invention, first of all, are valuable for the diagnosis and/or treatment of aggressive, rapidly progressing tumor diseases, such as cancer. Compounds according to the invention can be used for treatment of cells with a high degree of proliferation in the human body, involved in the development of malignant tumors, such as melanoma, fibrosarcoma, ovarian carcinoma, carcinoma of the pancreas, osteocarcinoma and acute leukemia, as these compounds are not sensitive to indirect TCII endocytosis. The method according to the invention allows a selective manner to protect benign organs from indirect TCII destructive absorption of cobalamin derivative containing a radioactive isotope and/or non-radioactive Agay is t, destroying cells.

Compounds of the present invention can be used not only for visualization of tumors and cancer treatment, but also for visualization and possible treatment of local infections of microorganisms that depend on direct absorption cobalamine in a high degree.

Compounds of the present invention, containing an antiproliferative agent, can be used to transport agent in an inactive form in hyperproliferative cells, where it exerts its action after intracellular amidolysis.

The method of treatment of cancer and/or infectious diseases is the introduction of the compounds according to the invention, containing a suitable therapeutic agent, alone or in combination with one or more other therapeutic agents, and their estimated combinational treatment with the use of fixed combinations or the connection according to the invention and one or more other therapeutic agents are administered sequentially or administered independently of each other or jointly impose a fixed combination with one or more other therapeutic agents. The connection according to the invention it is possible to introduce, in addition, or in addition, specifically for the treatment of tumors in combination with chemotherapy, immunotherapy, surgical intervention, or use of combines the th following methods of treatment. Equally possible long-term therapy along with adjuvant therapy in conjunction with other treatment strategies.

In addition, the present invention relates to pharmaceutical compositions comprising the derivatives of cobalamin according to the invention, primarily to pharmaceutical compositions suitable for diagnosis, and to pharmaceutical compositions intended for the treatment.

A preferred pharmaceutical composition for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. The compositions comprise the active ingredient alone or in a mixture with a therapeutically priemlemim carrier. The dose of active ingredient depends on the disease that needs to be cured, and from the subject, its age, weight and individual condition, the specific pharmacokinetic data and on a way of introduction.

Ways to get

Compounds according to the invention receive standard methods known in the field of chemistry.

Preferably cyanocobalamin, i.e. the compound of formula (I), where Rb, Rc, Rd, Reand RRmean hydrogen, and X is cyano, hydrolyzing under controlled conditions, for example, diluted inorganic acid, and allocate the resulting mixture of monocyclic, in which one of carbamoyl groups CONH2 converted into COOH. Bis-acid get in the same way.

Then cyanocobalamin-b-, C-, d - or f-acid, i.e. the compound of formula (I), where CONHRb, CONHRc, CONHRdor CONHRereplaced by COOH, respectively, and X is cyano, communicates with the appropriate amine Rb-NH2, Rc-NH2, Rd-NH2and Re-NH2accordingly, under standard conditions with the formation of amide, for example, as described for the synthesis of peptides. Functional groups in the residues Rb, Rc, Rdand Rewho can react the formation of amide linkages, preferably protect, and then after the formation of the amide bond of the protective group is removed by a standard method. To obtain compounds in which the spacer includes an amide group, use the reaction of cyanocobalamin-b-, C-, d - or e-acid with diamine H2N(CH2)nNH2in standard conditions of formation of amide linkages, and then condense the received H2N(CH2)n-cyanocobalamin with the corresponding carboxylic acid under standard amide formation conditions of communication and get a connection with the substituents Rb, Rc, Rdand Rerespectively.

To obtain compounds in which Rcdoes not mean hydrogen, the preferred method includes education is about-lactone with a subsequent recovery of the disclosure lactoovo cycle reactions described in the article by Brown and others, Inorg. Chem. 3038 (1995).

To obtain compounds in which RRdoes not mean hydrogen, cyanocobalamin (or a derivative of cobalamin, in which Rb, Rc, Rdor Rethat does not mean the hydrogen interacts with the connection RR-L, where L means suitable activating leaving group, with formation of ester bonds, for example halogen, the residue is mixed anhydride or other activating residue for the formation of esters of carboxylic, phosphoric or phosphonic acids, which are commonly used in the synthesis of peptides and nucleic acids.

The following examples are provided only to illustrate the present invention and do not limit its scope.

Examples

In this work, we used reagents Merck, Dietikon (CH), Aldrich or Fluka, Buchs (CH) brand HC without additional purification.

THIEF - hexaphosphate 1-benzothiazolylthio(dimethylamino)phosphonium

DCC - dicyclohexylcarbodiimide

DIPEA - diisopropylethylamine

EDC - 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Fmoc - (N-fluoren-9 ylethoxy)carbonyl

HOSu - N-hydroxysuccinimide

MES - 2-(4-morpholinyl)econsultancy acid

CT room temperature

TBTU - tetrafluoroborate benzotriazol-1-yl-N-tetramethylurea

TEAR - phosphate triethylamine

Theos - 2-trimethylsilylethynyl

TFU - triperoxonane the acid

The analysis method GHUR spent in the system Merck-Hitachi L-7000, with a radiometric detector EG&G Berthold LB 508, using column Waters Xterra RP8 (particle size 5 μm, 1×100 mm), flow rate 1 ml/min, detection at 250 and 360 nm. Solventandmean aqueous buffer solutions. Sodium acetate buffer solutionandreceived under stirring to 2.9 ml of acetic acid and 4,55 ml of 2 M sodium hydroxide in 900 ml of water and 100 ml of methanol. Tris-buffer solutionandreceived by dissolving Tris(hydroxymethyl)aminomethane (605 mg) in water, adding 2 M HCl to a pH of 8.2, bring the volume to 1000 ml, and add acetonitrile (10 ml). Solventbalways means methanol.

Preparative GHUR spent in the system Varian Prostar, equipped with two pumps Prostar 215 and detector Prostar 320 UV/., using column Waters Xterra Prep RP8 (particle size 5 µm, 3×100 mm and 30×100 mm). The flow rate of 4 ml/min for column 3×100 mm and 30 ml/min for column 30×100 mm

Spectra UV/view. filmed on the spectrometer Varian Cary 50, IR spectra were taken on the spectrometer Bio-Rad FTS-45 samples in the form of KBr pellets. Mass spectra (MS(ESI)ionization electrospray) filmed spectrometer Merck Hitachi M-8000. In the analysis of compounds of rhenium determined mass for isotope187Re. NMR spectra were taken on a spectrometer Bruker DRX 500 MHz. Chemical shifts were determined relative to the residual proton solution is a dye, which was used as standard.

A small number of derivative of cobalamin (several mg) was absoluely in aqueous solution connection with the use of cartridge Chromafix RP18ce, then the cartridge is thoroughly washed with water. The desalted product was then suirable with methanol, the solvent was removed in vacuum and the product was dried in high vacuum. Large number (>50 mg) was absoluely by phenol extraction, as described in Meth. Enzymol. 18(3), p.43, 1971.

Ethyl ester of (N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid (propyl-PAMA-Oet) was obtained as described for petranella article Schibli and others (Nucl. Med. Biol. 30, 465, 2003). The connection collisional in alkaline conditions. Then received the BOC-protected intermediate compound and kept. Protective BOC-group was removed immediately before further modification under stirring in dilute aqueous HCl. Ethyl - and hexylphosphonic received a similar method.

Carbonate Re[N-3-aminopropyl-N-pyridine-2-ylmethylamino]acetic acid (CO)3) was obtained in the interaction of (N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid (with remote protective group) with (Net4)2[Re(OH)2)3(CO)3].

Triptorelin methyl ester 1-carboxymethyl-N-Fmoc-histidine were obtained as described in article Pak and others (Chem. Eur. J., 9, 2053-2061, 2003). The counterion is replaced with chlorine the ID when mixing the compounds in 0.05 M HCl and subsequent evaporation in vacuum at room temperature.

Methyl ester of 3-aminopropyl-N-theos-histidine were obtained as described in article Staveren and other (Organic & Molecular Chemistry 2, 2593, 2004).

4-Nitrophenyloctyl ester of 3-(N-2-cyanometallates)-N-pyridin-2-ylmethylamino)propionic acid was obtained as described by Kunze (Dissertation, University of Zurich, 2004).

Example 1

Monocarboxylic acid cyanocobalamin (b, d and f)

Vitamin B12 (1.88 g, of 1.39 mmole) hydrolyzed in HCl (0.1 M, 190 ml), as described in article Pathare and others (Bioconjugate Chem. 217, 1996). Purification was performed by a modified method: after desalting by phenol extraction was performed chromatography on a column of Dowex and received three fractions. The first fraction contained mainly d-acid, the second fraction contained mainly b-acid and d-acid, and the third fraction contained mainly b-acid and acid. A mixture of b-acids and d-acid was shared by preparative GHUR (column Waters Xterra Prep RP8, 5 μm, 30×100 mm, gradienta/b0,5% min-1as the initial buffer solution used 100% acetate buffer solutionand). A mixture of b-acids and e-acids were separated in a similar system, but as solventandused a Tris-buffer solution. To receive cyanocobalamin-b-acid (output 280,6 mg, 14.9 per cent), cyanocobalamin-d-acid (exit 131, 5mm mg, 7.0%) and cyanocobalamin-s-acid (output 94,26 mg, 5.0 percent).

Example 2

Cyanocobalamin-b-(2-amino-ethyl)AMI the(cyanocobalamin-b-ethylamine)

Cyanocobalamin-b-(2-amino-ethyl)amide were obtained as described in article Pathare and others (Bioconjugate Chem. 217, 1996) for the synthesis of dodecylpropane. The Ethylenediamine (132 mg, 0,147 ml, 2.2 mmole) was dissolved in a mixture of DMF/N2About (10 ml, 1:1 vol./vol.). The solution was acidified by adding 1 M HCl to pH 5, the resulting solution was added cyanocobalamin-b-acid (60,0 mg, 44.4 mmol) and KCN (57 mg, of 0.87 mmole) and the pH was brought to 5.5. Then was added EDC (84,2 mg, 0.43 mmole) and HOSu (50,6 mg of 0.44 mmole). The mixture was stirred at RT for 3 days and an additional portion of the EDC and HOSu was added with an interval of 24 h the Mixture was evaporated to dryness in vacuo and was purified preparative GHUR (sodium acetate buffer solution, gradient: 0,5% min-1as the initial buffer solution used 100% acetate buffer solutionand), received 34 mg (yield 55%) of cyanocobalamin-b-(2-amino-ethyl)amide. MS (Meon, ESI-put.): m/z 1398,8 [M+H]+, 1420,1 [M+Na]+, 699,4 [M+H]2+, 711,1 [M+H+Na]2+.

Example 3

Cyanocobalamin-b-(4-aminobutyl)amide[cyanocobalamin-b-butylamine]

Specified in the title compound was obtained as described above for the synthesis of atenalol. MS (Meon, ESI-put.): m/z 1427,1 [M+1]+, 714,5 [M+3]2+.

Example 4

Cyanocobalamin-b-ethyl-Raritet

Cyanocobalamin-b-ethylamine (example 2, 24 mg, 17.2 mmol) was dissolved in a mixture of DMF/DMSO (5 ml, 4:1, vol/vol.). To the mixture was added 4-nitro niloy ester of 3-[N-2-cyanometallates-N-pyridine-2-ylmethylamino]propionic acid (14 mg, 34.1 per μmol) and DIPEA (5 μl, 29 mcmole). The mixture was stirred at RT for 24 h, evaporated to dryness in vacuo and was purified preparative GHUR (sodium acetate buffer solution, gradient: 0,5% min-1as the initial buffer solution used 100% acetate buffer solutionand), received 20 mg (yield 70%) of cyanocobalamin-b-ethyl-Raritet in a solid red color. MS (Meon, ESI-put.): m/z 1672,1 [M+H]+, 836,9 [M+H]2+.

Example 5

Cyanocobalamin-b-butyl-Raritet

Cyanocobalamin-b-butylamine (example 3, and 5.5 mg, 3.9 mmol) and 4-nitrophenyloctyl ester of 3-[N-2-cyanometallates-N-pyridine-2-ylmethylamino]propionic acid (2.5 mg, 6.1 mmol) was dissolved in dry DMSO (0.5 ml) and DMF (0.5 ml), was added DIPEA (5 μl, 29 mcmole) to a pH of 8 to 9 and the mixture was stirred at room temperature. After 5 h according to the analysis GHWR the reaction is fully completed. The solvent was partially evaporated under vacuum, adding ethyl ether, the product precipitated. The suspension was centrifuged three times decantation, when it received a fine powder. After purification preparative GHUR (sodium acetate buffer solution, gradient: 0,5% min-1as the initial buffer solution used 100% acetate buffer solutionand) was obtained pure product (yield 2.7 mg, 41%). MS (ESI): m/z 850,1 [M+2]2+. In the/.: λ/nm (ε/mol l -1cm-1)=279,1 (17300), 361,0 (31200), 519,9 (8700), 552,0 (9700).

Example 6

Cyanocobalamin-b-butylaminoethyl-His-OMe

A solution of cyanocobalamin-b-butylamine (49,6 mg, 34.8 mmol) in dry DMSO (2 ml) was added to the hydrochloride of the methyl ester of 1-carboxymethyl-N-Fmoc-histidine (35.5 mmol) and the THIEF (46.2 mg, 104,4 of µmol)was then added DIPEA (12 μl, 70.0 mmol) and the solution was stirred at RT for 16 h According to the analysis GHUR there was complete conversion of the source of cobalamin in the intermediate Fmoc-protected connection that adding diethyl ether to precipitate. The resulting suspension was centrifuged three times decantation, when it received a fine powder. The intermediate compound was dissolved in DMF (5 ml) was added piperidine (225 µl). After stirring at RT for 1.5 h product adding diethyl ether to precipitate. The resulting suspension was centrifuged three times decantation, when it received a fine powder. After purification preparative GHUR (sodium acetate buffer solution, gradient: 1% min-1as the initial buffer solution used 100% acetate buffer solutionand) was obtained pure product (yield of 17.1 mg, 32.1 per cent). UV/.: λ/nm (ε/mol l-1cm-1)=279,1 (19200), 361,0 (24700), 521,0 (9600), 551,1 (10700).

Example 7

Cyanocobalamin-s-(4-aminobutyl)amide(yanocobalamin-with-butylamine)

Cyanocobalamin-with-acid was obtained as described in the article by Brown and others (Inorg. Chem. 3038, 1995). 1,4-Diaminobutane (0,059 ml of 0.59 mmole) was dissolved in a mixture of DMF/N2About (10 ml, 1:1 vol./vol.). The solution was acidified by adding 1 M HCl to a pH of 5.2, the resulting solution was added cyanocobalamin-with-acid (16.0 mg, 11.8 mmol), KCN (15.3 mg, 0,236 mmole), EDC (9.0 mg, 47.2 mmol) and HOSu (5.4 mg, 47.2 mmol). The mixture was stirred at RT for 4 days. and added an additional portion of the EDC and HOSu. After 1 day. again added an additional portion of the EDC and HOSu. After 6 days according to the analysis GHUR there was complete conversion of cobalamin-derived. The mixture was evaporated to dryness in vacuo and was purified preparative GHUR (column RP C18, as a buffer solutionandused 1 mm HCl, gradient: from 20% methanol 50% methanol for 30 min), when it got to 9.8 mg (yield 58%) cyanocobalamin-with-butylamine. MS (Meon, ESI-put.): m/z 1427,7 [M+2]+, 713,5 [M+1]2+.

Example 8

Cyanocobalamin-with-butyl-Raritet

Cyanocobalamin-with-butylamine (7,0 mg, 4.9 mmol) and 4-nitrophenyloctyl ester of 3-[N-2-cyanometallates-N-pyridine-2-ylmethylamino]propionic acid (3.8 mg, 9.2 μmol) was injected into the reaction and was purified as described for the synthesis of cyanocobalamin-b-butyl-Raritet (example 5), it was obtained a pure product (yield 3.8 mg, 78%). MS (ESI): m/z 1701,0 [M+1]+, 850,1 [M+1]2+. UV/.: λ/nm (ε/mol l-1cm-1)=278,1 (14500), 362,1 (25400), 550,0 (7900).

Example 9

Cyanocobalamin-b-butyl-RAR-Re(CO)3

Cyanocobalamin-b-butylamine (example 3, to 24.6 mg, 17.2 mmol) and Re(CO)3(3-[N-carboxymethyl-N-pyridine-2-ylmethylamino]propionic acid (9.1 mg, 17.2 mmol) was dissolved in DMSO, was added to the THIEF (22.9 mg, 51.7 mmol) and DIPEA (2,94 μl, 17.2 mmol), the mixture was stirred at room temperature. DIPEA and the THIEF was added daily for 4 days. According to the analysis GHUR was observed the formation of two products that adding ethyl ether to precipitate. The resulting suspension was centrifuged three times decantation, when it received a fine powder. After purification preparative GHUR (sodium acetate buffer solution, gradient: 1% min-1as the initial buffer solution used 100% acetate buffer solutionand) received a fraction of the main product (the output of 2.3 mg, 7.0 percent). MS (ESI): m/z 1917,5 [M+2]+, 959,9 [M+4]4+.

Example 10

Cyanocobalamin-b-ethyl-PAMA-OEt

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. hydrochloride ethyl ester (N-2-amino-ethyl-N-pyridine-2-ylmethylamino)acetic acid (ethyl-PAMA-OEt) (which was obtained by cleavage of the BOC-protected derivative with stirring in a mixture of absolute EtO/2 M HCl (7.5 ml, 4:1 vol./about.) during the nights, and removing the volatile components under vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 1% min-1as the initial buffer solution used 100% acetate buffer solutionand), received 12 mg (yield 51%) of cyanocobalamin-b-ethyl-PAMA-OEt in a solid red color. MS (Meon, ESI-put.): m/z 1575,8 [M+H]+, 788,7 [M+H]2+, 799,3 [M+H+Na]2+.

Example 11

Cyanocobalamin-b-propyl-PAMA-OEt

In cyanocobalamin-b-acid (65,0 mg, 48.1 mmol) was added a freshly prepared solution of ethyl ester (N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid (361 mmol) in water (1 ml), then was added EDC (46,1 mg, 240 mcmole) and podslushivaet 0.1 M NaOH to pH 5.5. After stirring at RT for 15 h according to the analysis GHUR (sodium acetate buffer solution) was observed the formation of the product is approximately 50%. Then added another portion of the EDC

(46,1 mg, 240 mcmole), but upon further stirring at room temperature for more product education was not observed. The solvent was removed in vacuum and the residue was purified preparative GHUR (gradient:and/b05% min -1as the initial buffer solution used 100% acetate buffer solutionand). The main fraction was collected, the solvent was removed in vacuum and the product was absoluely, got cyanocobalamin-b-propyl-PAMA-OEt (yield of 25.8 mg, 16.2 mmol, 33,3%). MS (ESI): m/z 806,5 [M+1+Na]2+, 795,6 [M+2]2+. UV/.: λ/nm (ε/mol l-1cm-1)=278,0 (8500), 361,1 (26500), 549,1 (8000).

Example 12

Cyanocobalamin-b-butyl-PAMA-OEt

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. hydrochloride ethyl ester (N-4-aminobutyl-N-pyridine-2-ylmethylamino)acetic acid (butyl-PAMA-OEt) (which was obtained by cleavage of the BOC-protected derivative with stirring in a mixture of absolute EtOH/2 M HCl (7.5 ml, 4:1 vol./about.) during the nights, and removing the volatile components under vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 1% min-1as the initial buffer solution used 100% acetate buffer solutionand), received 15 mg (yield 63%) of cyanocobalamin-b-butyl-PAMA-OEt in a solid red t the ETA.

Example 13

Cyanocobalamin-b-butyl-FRAME-HE

N-Fluoren-9-ymetray ether bromoxynil acid was obtained from bromoacetamide and N-fluorenylmethyl in dry THF at 0°C. BOC-Butyl-PAMA-OFm (N-fluoren-9-ymetray ether [(4-tert-butoxycarbonylamino)pyridine-2-ylmethylamino]acetic acid) was obtained from Boc-NH-(CH2)4NH2, pyridine-2-aldehyde and N-fluoren-9-Eletropaulo ether bromoxynil acid according to the method described in the article Schibli and others (Nucl. Med. Biol. 30, 465, 2003) for the synthesis of BOC-pentyl-FRAME-OMe.

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. N-fluoren-9-Eletropaulo ether [(4-aminobutyl)pyridine-2-ylmethylamino] acetic acid (butyl-PAMA-OFm) (which was obtained by cleavage of the BOC-protected derivative with stirring in a mixture triperoxonane acid/CH2Cl2(4 ml, 1:2 vol./about.) within 1 h, and removing volatile components under vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 1,5% min-1as the initial buffer solution used 100% acetate BU is Erny solution and), received 15 mg of cyanocobalamin-b-butyl-PAMA-OFm in a solid red color.

Cyanocobalamin-b-butyl-PAMA-OFm (15 mg) was dissolved in 3 ml of a mixture of HNEt2/DMF (2:1 vol./about.) and stirred at RT for 2 h the Solvent was removed in vacuo, the residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 1,0% min-1as the initial buffer solution used 100% acetate buffer solutionand), to receive 9 mg of cyanocobalamin-b-butyl-FRAME IT in a solid red color.

Example 14

Cyanocobalamin-b-hexyl-PAMA-OEt

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. hydrochloride ethyl ester (N-6-aminohexyl-N-pyridine-2-ylmethylamino)acetic acid (hexyl-PAMA-OEt) (which was obtained by cleavage of the BOC-protected derivative with stirring in a mixture of absolute EtOH/2 M HCl (7.5 ml 4:1 vol./about.) during the nights, and removing the volatile components under vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 1,0% min-1as for the material of the buffer solution used 100% acetate buffer solution and), while received 10 mg (yield 41%) of cyanocobalamin-b-hexyl-PAMA-OEt in a solid red color. MS (Meon, ESI-put.): m/z 816,9 [M+2H]+, 1632 [M+H]+.

Example 15

Cyanocobalamin-b-ethyl-FRAME-Re(CO)3

Cyanocobalamin-b-ethyl-PAMA-OEt (example 10, 11 mg, 7.0 μmol) was dissolved in 4 ml of 2 M solution of NaHCO3that solution was added (NEt4)2[ReBr3(CO)3] (11 mg, 14.2 mmol) in 1.5 ml Meon. The mixture was heated at 85°C for 1 h, cooled to CT and was purified preparative GHUR (sodium acetate buffer solution, the gradient is: 2.0% min-1as the initial buffer solution used buffer solutionandoutput 11 mg, 86%.

Example 16

Cyanocobalamin-b-propyl-FRAME-Re(CO)3

Cyanocobalamin-b-acid (26,7 mg, 19.8 μmol), sodium carbonate Re[N-3-aminopropyl-N-pyridine-2-ylmethylamino]acetic acid(CO)3(29,2 mg, 60 mcmole), EDC (11,5 mg, 60 mcmole) and HOSu (6.9 mg, 60 mcmole) was dissolved in a mixture of water (5 ml) and DMSO (0.5 ml), the pH was brought diluted HCl and NaOH to 5.5. After stirring at RT for 5 h according to the analysis GHUR (acetate buffer solution) was observed the formation of the product is approximately 33%. The mixture was again added EDC and HOSu. The mixture was stirred at room temperature for 3 days, with an additional portion of the EDC and HOSu was added with an interval of 24 hours the Water was removed in vacuum, the product was besieged added the eat diethyl ether. The oil suspension was centrifuged and decantation, the precipitate was twice washed with diethyl ether before the formation of fine sediment. The crude product was dried in high vacuo, was purified preparative GHUR (gradient:and/b, 1% min, as the initial buffer solution used 100% acetate buffer solutionand) and the product was absoluely, got cyanocobalamin-b-propyl-FRAME-Re(CO)3(release 9.1 mg, 23%). MS (ESI): m/z 1831,7 [M+1]+, of 916.1 [M+1]2+. UV/.: λ/nm (ε/mol l-1cm-1)=278,0, 361,1, 519,9, 551,1.

Example 17

Cyanocobalamin-b-hexyl-PAMA-Re(CO)3

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. carbonate Re([N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid(CO)3·CF3COOH (which was obtained by cleavage of the BOC-protective groups of the complex in a mixture of CH2Cl2and TFU (2:1 vol./about.) within 1 h at 0°C and removing volatile components at RT in vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, the gradient is: 2.0% min-1as the beginning of Lenogo buffer solution used 100% acetate buffer solution and), received 11 mg (yield 40%) of cyanocobalamin-b-hexyl-FRAME-Re(CO)3. MS (Meon, ESI-put.): m/z 936,5 [M+2H]2+, 948,3 [M+H+Na]2+, 1873,8 [M+H]+.

Example 18

Cyanocobalamin-d-cut-FRAME-OEt

Cyanocobalamin-d-acid (9.3 mg, 6.9 μmol) was injected into the reaction with ethyl ether (N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid (7 mcmole) and EDC (6.6 mg, 34 μmol)as described for the synthesis of cyanocobalamin-b-propyl-PAMA-OEt (example 11), when this has been specified in the header of the product (output 3.6 mg, 33%). MS (ESI): m/z 1612 [M+Na]+, 1590 [M+1]+, 806 [M+1+Na]2+, 795,1 [M+2]2+. UV/.: λ/nm (ε/mol l-1cm-1)=279,0 (13400), 361,1 (23300), 549,1 (7200).

Example 19

Cyanocobalamin-d-cut-FRAME-Re(CO)3

Cyanocobalamin-d-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (1.5 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. carbonate Re([N-3-aminopropyl-N-pyridine-2-ylmethylamino)acetic acid(CO)3was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, the gradient is: 2.0% min-1as the initial buffer solution used 100% acetate buffer solutionand), while the floor is Ali 20 mg (yield 73%) cyanocobalamin-d-cut-FRAME-Re(CO) 3.

Example 20

Cyanocobalamin-b-propyl-His-OMe

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(1 ml). In another bowl, approximately 4 EQ. methyl ether, 3-aminopropyl-N-Teoc-histidine was dissolved in DMF. Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred for 45 min, then was evaporated to dryness in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, the gradient is: 2.0% min-1as the initial buffer solution used buffer solutionand), received 16 mg solid red (yield 67%). MS (Meon, ESI-put.): m/z 1710,4 [M+H]+, 855,0 [M+2H]+, 866,7 [M+Na+H]2+.

Sample specified theos-protected compound (19 mg) was dissolved in a mixture of TFU/CH2Cl2(4:1, vol/about.) at 0°C, stirred at this temperature for 4 h, while according to the analysis GHUR there was complete conversion of the starting material. The solvent was removed in vacuum at RT, the residue was added Et2O, and then the solvent was removed in vacuum. The specified stage was repeated three times to remove traces of TFU. The residue was purified preparative GHUR (sodium acetate buffer solution, gradient: 0,5% min-1as the initial buffer solution used 100% buffer solutionand ), received 11 mg specified in the connection header. MS (Meon, ESI-put.): m/z 1565,2 [M+H]+, 1587,2 [M+Na]+, 783,4 [M+2H]2+, 794,1 [M+Na+H]2+.

Example 21

Cyanocobalamin-b-propyl-His-Re(CO)3

Cyanocobalamin-b-acid (20.0 mg, 14.8 mmol) was dissolved in DMSO (0.8 ml), then was added DMF (2 ml) and NEt3(0.1 ml). In another bowl, about 5 EQ. carbonate Re (methyl ester of 3-aminopropyl-N-theos-histidine)(CO)C]·CF3COOH (which was obtained by cleavage of the BOC-group secure complex in CH2Cl2and TFU (2:1 vol./about.) within 1 h at 0°C and removing volatile components at RT in vacuum) was dissolved in a mixture of DMF/NEt3(4,5 ml, 8:1.vol.). Both solutions were mixed, was added TBTU (32.1 mg, 0.1 mmole), was stirred at RT for 45 min, the solvent was removed in vacuum. The residue was purified preparative GHUR (sodium acetate buffer solution, the gradient is: 2.0% min-1as the initial buffer solution used 100% buffer solutionand), received 7 mg (yield 73%) of cyanocobalamin-b-propyl-His-Re(CO)3. MS (Meon, ESI-put.): m/z 911.6 [M+2H]2+, 923,2 [M+H+Na]2+, 933,9 [M+2Na]2+, 1822,1 [M+H]+, 1845,6 [M+Na]+.

Example 22

Cyanocobalamin-b-ethyldiamine

Triethylenetetramine (55,4 μl, 369 mcmole) was dissolved in a mixture of DMF (2.5 ml) and water (2.5 ml)was added KCN (9.6 mg, 47 mcmole) and acidified solution by adding aqueous HCl to pH 6. In the resulting solution was added cyanocobalamin-b-acid (10.0 mg, 7.4 μmol), EDC (5.7 mg, 29 mcmole) and HOSu (3.4 mg, 29 mcmole). A similar number of EDC and HOSu was added after 6, 24, 48 and 120 hours According to the analysis GHUR (sodium acetate buffer solution) has been a slow formation of the product, i.e. after 48 h received 75%, but upon further stirring for educational product was not observed. After stirring for 144 h the solvent was removed in vacuum and the product was purified preparative GHUR, eluent: aqueous solution of 0.1% TFU as a buffer solutionandand methanol as solventb(gradient: 1% min-1as the initial buffer solution used 80% buffer solutionand), received 7.5 mg (yield 55%) as trifenatate cyanocobalamin-b-ethyl-analogue (STFU). MS (ESI): m/z 743,1 [M+2]2+. UV/.: λ/nm (ε/mol l-1cm-1)=278,0 (13000), 316,0 (23100), 519,0 (6500), 549,0 (7200).

Example 23

Cyanocobalamin-b-ethyldiamine-Re(CO)3

Cyanocobalamin-b-ethyldiamine (5 mg, 2.7 mmol) and (Et4N)2[ReBr3(CO)3] (2.2 mg, 2.9 mmol) was stirred in phosphate buffer solution, pH 7.4 (0.1 M, 0.33 ml) at 50°C. After 1 h according to the analysis GHUR there was complete conversion of starting materials into the product. After 4 h the reaction mixture was absoluely, when it received the product, which is the data the analysis GHUR contained a mixture of two stereoisomers in a ratio of about 2:1. A similar composition of stereoisomers is set up by using labeled cyanocobalamin-b-ethyldiamine (99mTc). MS (ESI): m/z 1755,9 [M+1], 878,5 [M+2]2+.

Example 24

Cyanocobalamin-5'-postcolumn

The solution of cyanocobalamin (30 mg, 22,14 of mmol), DCC (457 mg, 2,214 mmole) and N-Fmoc-phosphocholine (78,9 mg, 217.2 mmol) in dry DMF (2 ml) and dry pyridine (1 ml) was stirred in an atmosphere of N2at room temperature for 24 hours After adding 2 ml of water precipitated in the sediment dicyclohexylphosphino was filtered, and the water and the pyridine was evaporated at 60°C under reduced pressure. The resulting solution was diluted to a volume of 8 ml solution of piperidine in DMF (5%) and was stirred at room temperature for 2.5 hours the Product was planted in diethyl ether, centrifuged and washed several times. The crude product was purified preparative GHUR (gradient: 100%→20%and, 0%→80% Meon for 30 min,a=0,1% Asón, 10% acetonitrile in water, flow rate 10 ml/min, column M&N VP 250/21 Nucleosil 100-7 C18). When it received the product in a solid red (82%). MS (ESI+, Meon): m/z 1478 [M+1]+, 762 [M+2+2Na]2+.

31P-NMR (500 CD3OD): δ 0,00 (s, 1P), of 0.53 (s, 1P).

Example 25

Cyanocobalamin-5'-postcolumn-His-OMe

Cyanocobalamin-5'-phosphocholine (50 mg, 33.8 mmol) and hydrochloride methyl ester 1-(carboxymethyl)-N-Fmoc-histidine (25 mg, 50.7 mkm is La) was dissolved in dry DMSO (4 ml) and the pH is brought to 6-7 by the addition of 24 μl of DIPEA. To the solution was added a THIEF (45 mg, 101.5 mmol) as a solid and stirred at RT. After 1 h was formed acid solution, which was neutralized. After 5 h according to the analytical GHUR there was complete consumption of starting material. After planting residue in diethyl ether, the crude product was removed protective group in a mixture of DMF and piperidine (10 ml, 1:1) for 1.5 hours After re-planting and cleaning preparative GHUR as described for cyanocobalamin-5'-phosphocholine (example 24)was obtained the desired product (yield 46%). MS (ESI+, Meon): m/z 1690 (M+1)+, 845,6 (M+2)+.

31P-NMR (500, D2O): δ -2,16, -0,37.

Example 26

Cyanocobalamin-5'-postcolumn-His-Re(CO)3

The specified connection was obtained in the same way as described for cyanocobalamin-5'-postcolumn-His-OMe (example 25), but replacing methyl ester hydrochloride 1-(carboxymethyl)-N-Fmoc-histidine complex Re(CO)3and 1-(carboxymethyl)histidine. To receive the purified product (37%). MS (ESI+, Meon): m/z 1945, 929 [M+1]+.

31P-NMR (500, D2O, 333 K): δ 0,97, 2,23.

IR (KBr, cm-1): 3400, 2128, 2020, 1901, 1902, 1664, 1499, 1399, 1219, 1073.

Table 2
The structure of the cobalamin derivative of the formula (I), where X represents CN obtained in examples
ExampleR=HR≠HSpacerChelate groupSpacer elements chelate group
4Rc=H, Rd=H, RR=HRb:ethyl-NHCOCH2CH2(n=2)PAPAcet
5Rc=H, Rd=H, RR=HRb:butyl-NHCOCH2CH2(n=4)PAPAcet
6Rc=H, Rd=H, RR=HRb:butyl-NHCOCH2(n=4)His-OMe
8Rc=H, Rd=H, RR=HRc:butyl-NHCOCH2CH2(n=4)PAPAcet
10Rc=H d=H, RR=HRb:ethyl (n=2)PAMA-OEt
11Rc=H, Rd=H, RR=HRb:propyl (n=3)PAMA-OEt
12Rc=H, Rd=H, RR=HRb:butyl (n=4)PAMA-OEt
13Rc=H, Rd=H, RR=HRb:butyl (n=4)PAMA-OH
14Rc=H, Rd=H, RR=HRb:hexyl (n=6)PAMA-OEt
18Rc=H, Rd=H, RR=HRd:propyl (n=3)PAMA-Oet
20Rc=H, Rd=H, rR=hRb:propyl (n=3)/td> His-OMe
22Rc=H, Rd=H, rR=hRb:ethyl (n=2)Triamine
25Rc=H, Rd=H, RR=HRR:phosphate-ethyl-NHCOCH2His-OMe

Example 27

A General method of introducing labels

The synthesis of the precursor of [99mTc(OH)2)3(CO)3]+received from [99mTcO4]-using a set of breakability reagents, as described in article Alberto and others, J. Am. Chem. Soc. 123, 3135-3136. Glass vial of 10 ml with a rubber tube was purged with nitrogen, then added 20 μl of a solution derived cyanocobalamin (0.01 M in water), 20 μl of MES buffer solution (1.0 M) and 200 μl of a solution of [99mTc(HE2)3(CO)3]+the reaction mixture was stirred at 75°C for 1 to 2 hours completing the transformation of atoms99mTc controlled method GHUR using γ-detector. In these conditions was observed useplaceholdernotice protective groups chelating agents and the formation of carboxylate complexes.

For testing in vivo and to determine the binding with transport vectors requires extremely high specific activity. In this regard, 100 µl of tracer solution was injected into the system GHUR to separate hot (labeled) derivative of cobalamin from the cold (unlabeled) is derived. Before intravenous injection of the animal faction with the highest γ-radioactivity (approximately 300 μl) was diluted with saline to a concentration of 10 µci per animal. When chromatography used the following separation conditions: acetate buffer solution, column XTerra RP8, gradient: 0% methanol (0 min), 30% methanol (15 min), 100% methanol (25 min) for elution of b - and d-derived and the system TEAR, as described in article Schibli and others, Bioconjugate Chem. 343-351 (2000).

Example 38

The selection of transcobalamin II (TCII) from whole blood of the rabbit

TCII was purified by affinity chromatography using cyanocobalamin-agarose (Sigma). The agarose (5 ml) was washed first with 200 ml of 50 mm Tris/1 M NaCl, pH 8.0, and then 200 ml of 0.1 M glycine/0.1 M glucose/1 M NaCl, pH 10, and again with 200 ml of 50 mm Tris/1 M NaCl. 200 ml double-centrifuged whole blood (first at 5000 rpm for 15 min, then at 20,000 rpm for 20 min at 4°C) were applied to the affinity column and the column was washed sequentially as described above. Associated TCII was suirable 20 ml of 4.0 M guanidine hydrochloride/50 mm three is, pH 8.0, then 7.5 M guanidine hydrochloride/50 mm Tris, pH 8.0. The main number TCII eluted in 4 M guanidine hydrochloride. The samples were dialyzed against excess water for 2 days at 4°C. the Typical yield is 5 to 30 nmol/l or 7.5-10 mcg TS (MM 50 kDa) from one rabbit.

Example 29

Getting transcobalamin II of bacteria (recombinant TCII)

cDNA of transcobalamin II expressed in E. coli, strain FA113, modification K12 dual absence of trxB gene and gor gene, in which the cytoplasm contains oxidants and are formed disulfide bonds. Protein, transcobalamin II, contains the site of cleavage by PreScission protease and T-terminal his-tag fragment. Protein was isolated from the soluble fraction of E. coli extracts using Nickel-chelate chromatography on sepharose. Cyanocobalamin was separated from transcobalamin II associated with chelate column, with elution of 8 M urea, and then suirable transcobalamin II imidazole. In some preparations of his-tag fragment was tsalala specific protease.

Example 30

Getting transcobalamin I (TCI, haptocorrin)

As a source of transcobalamin I used the saliva vegetarians volunteers. The saliva was centrifuged at 20,000 rpm for 20 min at 4°C, mixed with FSB 1:1 and sterilized by filtration. Binding activity transcobalamin typically 10 ng/ml.

Example 31

The interaction of derivatives of cyanocobalamin to transport proteins TCI and TCII (figure 1 and figure 2)

The interaction of radioactively labelled derivatives of cyanocobalamin (57With,99mTc188Re,111In) was determined by the method of the shift of the peaks on the column for gel filtration. Radioactively labeled cyanocobalamin (from 0.05 to 1 ng) were incubated in the presence of an excess of transport proteins for 15 min at room temperature. The resulting mixture was applied to a column for gel filtration (superdex 75, firms Amersham Biosciencies) and washed work buffer solution FSB, containing 0.1% tween-20. Molecular weight biologically active cyanocobalamin, bind to transport proteins, changes (observed shift of the peak with approximately 1.4 MM up to 40-70 kDa kDa depending on transport protein). Titration of the binding activity of transport proteins was performed using57Co-cyanocobalamin (ICN Biomedicals GmbH, Germany; 10 µci/50 ng).

Example 32

The introduction of the label in the derivatives of cyanocobalamin using188Re-tricarbonyl

Getting188Re-tricarbonyl and labeled derivatives of cyanocobalamin were carried out in one stage in a single vessel. 7.5 mg BH3NH3was mixed with 20 mg of sodium ascorbate, 100 μl derived cyanocobalamin (10-3M), 900 μl of the eluate from the source 188ReO4]-(0.9% saline solution, from 40 to 270 MCI MCI), 20 mg of H3PO4(85%) and the mixture missed monoxide (CO) for 20 minutes the Mixture was heated for 30 min to 2 h at 90°C. Labeled cyanocobalamin was separated from unlabeled column for reversed-phase GHUR (Waters Xterra RP8) in phosphate buffer solution in a linear gradient of methanol. The active fraction was diluted with normal saline before intravenous injection to a concentration of 10 µci per animal for visualization and up to 2 MCI for treatment.

Example 33

The sensitivity of the spherical tumor cells to ionizing radiation

Adopted in radiobiology alternative model in vivo in the study of radiation are spherical cells, since these cells and xenografts inoculated into the tissue of the mouse, showing a similar response to radiation. Multicellular spherical tumor cells were grown in rotating flasks under continuous stirring at 37°C to an average diameter of 400 μm. Spherical cells were collected, washed with fresh medium and then incubated for 1 h in the presence or cold188Re-labeled derivative of cyanocobalamin in 24-hole culture tablets with a flat bottom. The range of radioactivity ranged from 1 to 20 µci µci in the hole. Cytotoxicity was assessed by the application of fluorescent markers of cell viability according to the diameter of the spherical cells and the method count of clonogenic analysis of dispersed spherical cells in semi-solid agar.

Example 34

The biodistribution of radioactively labelled derivatives of cyanocobalamin in mouse tissues (figure 3, 4, 5, 6)

To study bearsdley used57Co-cyanocobalamin, which was mixed in the amount of 0.2 µci/1 ng with 180 µl of normal saline and injected intravenously to balb/c mice inoculated with tumor (melanoma B16-F10 in syngeneic mice). After a certain period of time (from 5 min to 24 h), animals were scored, organs were weighed and radioactivity was determined on the counter γ-radiation. To study bearsdley used also99mTc-labeled cyanocobalamin, which was mixed in an amount of 10 µci/0.5 ng with normal saline and analyzed as described above. To study bearsdley used also111In-labeled cyanocobalamin, which was mixed in the amount of 2 µci/5 ng with normal saline and analyzed as described above. To study the effect does not contain vitamin B12 feed the biodistribution of labeled cyanocobalamin compared with mice that received normal food and biodistribution in mice that received food that does not contain vitamin B12, within 2 weeks.

Example 35

Treatment of mice with transplanted tumor using88Re-labeled derivatives of cyanocobalamin

For trials in the treatment of the drives of the working systems is available from syngeneic balb/c mice were grown to approximately 200 mg (measured with a caliper). Then, the animals were injected intravenously in increasing doses (from 0.1 to 2 MCI) compositions radioactively labelled cyanocobalamin and cold cyanocobalamin. Tumor size was measured with a caliper. If the size of the tumor reached 800 mg, animal scored. In a series of experiments, animals were treated according to the fractional rate: labeled cyanocobalamin were injected three times with an interval of 1 week. Animals were examined within 60 days and recorded the resumption of growth of tumors.

1. Derivative of cobalamin formula (I)

where Rbmeans spacer elements chelating group of the formula
,
where n is 2, 3 or 4;
Rc, Rd, Reand RRmean hydrogen; and X is cyano; or
where Rdmeans spacer elements chelating group of the formula
,
where n is 3;
Rb, Rc, Reand RRmean hydrogen; and X is cyano; or
where Rbmeans spacer elements chelating group of the formula
,
where n is 2;
Rc, Rd, Reand RRmean hydrogen; and X is cyano.

2. Derivative of cobalamin according to claim 1 of formula (I), where
Rbmeans spacer elements chelating group of the formula
,
where n is 2, 3 or 4;
Rc, Rd, Reand RRmeans vador is d; and X is cyano.

3. Derivative of cobalamin of claim 2, where n is equal to 4.

4. Derivative of cobalamin of claim 2, where n is equal to 3.

5. Derivative of cobalamin of claim 2, where n is equal to 2.

6. Derivative of cobalamin according to any one of claims 1 to 5 in combination with a radioactive metal selected from the group including57With,99mTc188Re and111In Telefonnaya function of spacer-chelator obtained.

7. Derivative of cobalamin of claim 6, skomplikowanie through reaction with [99mTc(HE2)3(CO)3]+or [188ReO4]-.

8. Injectable pharmaceutical composition for the diagnosis of neoplastic diseases, including derivative of cobalamin carrying radioactive metal atom according to claim 6 or 7.

9. A method for the diagnosis of neoplastic diseases in mammals, including
(a) compliance does not contain vitamin B12 diet mammal predisposed to neoplastic disease,
(b) the subsequent introduction of the derivative of cobalamin on any of PP or 7 carrying radioactive metal atom.

10. The use of a derivative of cobalamin according to claim 6 or 7, carrying radioactive metal atom, in the method for the diagnosis of neoplastic diseases of a mammal.

11. The use of claim 10 for imaging cancer.

12. The use of a derivative of cobalamin according to claim 6 or 7, carrying radioactive metal atom, to receive pharmaceutical composition, designed for use in the method for the diagnosis of neoplastic diseases of a mammal.

13. Use para.12 derivative of cobalamin carrying radioactive metal atom, to obtain a pharmaceutical composition intended for imaging cancer.



 

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