MXPA06007155A - Cobalamine derivatives useful for diagnosis and treatment of abnormal cellular proliferation - Google Patents

Abstract

The invention relates to cobalamin derivatives (a) having no binding affinity or low binding affinity to the transport protein transcobalamin II TCII) and (b) retaining activity as a vitamin B12 substitute, optionally carrying a therapeutic and/or diagnostic agent, such as a radioactive metal. These compounds have a much reduced accumulation rate in blood and benign organs, such as kidney and liver, compared to the accumulation rate in neoplastic tissues, and are more rapidly eliminated from blood. The invention further relates to a method of diagnosis and a method of treatment of a neoplastic disease or an infection by microorganisms in a mammal comprising (a) exposing the mammal to a period of a vitamin B12 - free diet, and (b) subsequently applying a cobalamin derivative of the invention carrying a diagnostic and/or therapeutic agent. By selecting cobalamin derivatives acting as vitamin B12 substitutes, the risk of the formation of resistant off -spring in neoplastic tissue is much reduced.

Description

GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, Published: ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), with international search report European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FL FR, GB, GR, HU, IE, IS, IT, LT, LU, MC, NL, PL , PT, RO, For two-letter codes and oiher abbreviations, referto the "GuidSE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, ance Notes on Codes and Abbrenñations" appearing at the beginGQ, GW, ML, MR, NE, SN, TD, TG). No ofeach regular issue of the PCT Gazette.

COBALAMINE DERIVATIVES USEFUL FOR DIAGNOSIS AND TREATMENT OF ABNORMAL CELLULAR PROLIFERATION Field of the Invention The invention relates to methods for imaging and destroying rapidly proliferating undesirable cells in multicellular organisms.

Background of the Invention Abnormal cell proliferation, notably hyperproliferation, is the source of numerous diseases, the most severe being cancer. In the United States alone, approximately 1.5 million people are diagnosed with cancer and 0.5 million die from this each year. The fight against cancer has seen some success but also some setbacks. Several side effects of anticancer drugs and the development of resistant offspring of cancer cells are major problems such as the anticipated and accurate localization of tumors and metastases. Hyperproliferative cells, such as many cancer cells, depend on an increased supply of nutrients, growth factors, energy and vitamins. Using the route of supply of a vitamin, which is essential for cell growth and often is specifically the supply, may possibly transport drugs to these unwanted cells. Cobalamin (Cbl), also known as vitamin B12 and present as cyano-cobalamin (CN-Cbl), hydroxy-cobalamin (HO-Cbl) or acuo-cobalamin (H20-Cbl), is essential for life and its concentration in the body is very low. Higher organisms including humans have to obtain the vitamin from their blood. The biosynthesis of cobalamin is limited to some prokaryotic organisms, such as anaerobic bacteria. Cobalamin is important for the proper function of the nervous system and is necessary for the proper metabolism of carbohydrates, proteins and fats. Cobalamin is used in essential intracellular metabolic pathways. As methyl-cobalamin (Me-Cbl), it functions as a co-factor for methionine synthase. As 5'-deoxyadenosyl-cobalamin (Ado-Cbl), it works with methylmalonyl-CoA-mutase in the rearrangement of methylmalonyl-CoA to succinyl-CoA. A deficiency of cobalamin can result in pernicious anemia. Cobalamin is also included in the reductive conversion of ribonucleotides to deoxyribonucleotides to generate DNA. In mammals, most of the cellular uptake of cobalamin is regulated by the serum transport proteins and cell membrane receptors. There are two types of cobalamin binding proteins in plasma: the non-glycosylated protein, transcobalamin II (TCII) and the glycosylated proteins, transcobalamin I and III (TCI and TCIII), also called R-binding proteins or haptocorrins. TCI and TCIII are immunologically cross-reactive and probably differ only in their carbohydrate composition. TCI is the first R-linker found in circulation. For reasons of simplicity, the term TCI will be used when referring to both TCI and TCIII r-linker proteins. Both types of transport proteins (TCI and TCII vectors circulate in the mammalian current either partially saturated (holo), or partially unsaturated (apo) with cobalamin.An vector-free uptake system for cobalamin with a rather low efficiency in Normal cells are also present in mammalian cells (see, Sennte, C and Rosenberg, LE, Ann.Rev. Biochem.5_0, 1053-86 (1981)) .TCII works in the distribution of cobalamin plasmid to all metabolically active cells by receptor-mediated endocytosis. It is also known that accelerated cell proliferation in neoplasia mainly leads to increased consumption of TCII loaded with cobalamin from the circulation by receptor-mediated endocytotic uptake. The promotion of expression in the number of TCII receptors has been demonstrated extensively in malignant cell lines to find the increased metabolic demand for thymidine and methionine production, mutilation reactions for DNA synthesis and cellular energy through mitochondrial metabolism. The general TCII receptor is present in all tissues while the second TCII receptor, more organ specific, called megalin, is expressed in excess in the proximal kidney tubules and several other absorbent epithelia. After endocytotic internalization, TCII degrades in lysosomes and free cobalamin is transported to the cytoplasm and within the cell membrane, where it is converted to Me-Cbl and Ado-Cbl. These two forms are operating as the active co-enzymes of vitamin B12. The essential role of TCII is well established by the observation that inborn deprivation, inherited from TCII leads to megaloblastic anemia, damaging neurological disorders and death if not treated with excess cobalamin. Almost all cells are capable of generating TCII. Many cells such as hepatocytes, fibroblasts, nerve cells, enterocytes and macrophages synthesize high amounts of TCII. It is assumed that the vascular endothelium is the main source of TCII. Approximately 20-30% of circulating cobalamin binds to TCII as a holo-TCII. This is the metabolically efficient form that ensures the internalization of cobalamin in all tissues (see Rothernberg, E. et al., Chemistry and Biochemistry of B12, ed R. Banerjee, New York, NY., 1999, pp. 441- 473). TCI is present in blood and plasma as well as in most exocrine secretions and other fluids. They are generated mainly in tissues from the front of the digestive tract, gastric mucosa, salivary and lacrimal glands and secretory epithelium of the inner ear. TCI, different from TCII, does not seem to distribute its cobalamin mainly for its cellular uptake, it has a prolonged half-life in the blood, and in this way it retains more than 75% of circulating cobalamin (and corrina) at any given time. Almost all TCI circulates as holo-TCI. Your paper is not completely understood. It has been proposed that it functions as a bacteriostatic agent by preventing the supply of all classes of cobalamines and corrinas to microorganisms. It can also stabilize adenosyl-cobalamin and protect it from photolysis. In contrast to TCI, which has a higher concentration than TCII in circulation, the level of TCII can be raised very rapidly by de novo synthesis of apo-TCII in response to incoming cobalamin. TCI is generated in a rather slow manner and can not be substantially stimulated in response to any activating impact (see, Alpers, D. and Russell, G., in: chemistry and Biochemistry of B12, supra, pp. 411- 441). Up to now, uptake without cobalamin vector into mammalian cells has not been considered as an alternative route to deliver cobalamin derivatives to hyperproliferative cells. It is indisputable that the physiologically important mechanisms for the uptake of cobalamin by benign mammalian cells require the TCII and TCI vectors (and intrinsic factor in the digestive tract). However, the in vivo and in vi tro data show that free cobalamin is also able to cross the plasma membrane without the implication of a vector protein. Direct evidence for an additional capacity to capture free cobalamin comes from the study of congenital and totally deficient children in TCII, in whom the parenteral administration of free cobalamin resulted in a remarkable remission of the clinical and chemical signs of intracellular cobalamin deficiency (see Hall, CE, et al., Blood, 5_3, 251-263 (1979)). The in vi tro studies showed the uptake of free cobalamin in HeLa cells and fibroblasts. In HeLa cells, the uptake of free cobalamin is between 1% and 2% of what is seen for cobalamin bound to TCII. With human fibroblasts, the accumulation of free cobalamin in an interval of two hours gives an account of approximately 20% of that indicated with vitamin bound to TCII. The system of uptake of free vitamin in human fibroblasts has been studied in some detail by Berliner and Rosenberg (Berliner, N. and Rosenberg, L.E., Metabolism. 3_0 '230-236 (1981)). The CN- [57Co] -Cbl free uptake has been established as a biphasic system. The initial uptake component is fast, saturable and specifically inhibited by CN-Cbl and OH-Cbl, unlabeled, in excess, and completed within 30 minutes. The second component of uptake is slower, linear with time and was not inhibited by cobalamin not marked in excess, and did not reach the plateau even after 8 hours, suggesting the characteristic attributes of a non-specific process. The initial mode of uptake has properties of a highly specific, protein-mediated membrane crossing; is sensitive to sulfhydryl reagents and markedly inhibited by cycloheximide (Sennet, C. and Rosenberg, L.E., Ann. Rev. Biochem. 5_0, 1053-86 (1981)). These properties are consistent with the presence of a facilitated uptake system mediated by free cobalamin protein in mammals. It is well established that many bacteria and all eukaryotic protists are auxotrophic for vitamin B12 and are capable of binding with greater affinity than mammalian intrinsic factor, TCI and TCII. Bacterial and protozoal B12 binding proteins are cell surface proteins that operate without vectors capable of binding to a wide variety of corrinas (including free cobalamin) with high avidity. Therefore, the detection of bacterial infections in the context of a complete body image, after the application of a radiolabelled cobalamin derivative, was not surprising (Collins, DA et al., Mayo Clin. Proc. 75, 568-580 (2000)). The development of hyperproliferative forms of mammalian cells can also lead to the development of multi-step carcinogenesis of more efficient forms of the cobalamin uptake system, without vector, already present. Approaches have been published and patented for using cobalamin as a carrier for a wide variety of biologically active agents, including isotopes of radioactive metals (see, Collins, D.A., U.S. Patent Application No. 2003/0144198). The results obtained in animals and humans, when radiolabeled cobalamin derivatives are used, showed tumor tissue labeling, but also a strong accumulation of radioactivity in healthy tissues, such as kidney and liver. Therefore, imaging and radiotherapy are far from optimal. The potential for major damage to some healthy parts of the body limits the applications described so far. There is an obvious need for compounds, compositions and methods for administration of cobalamin, therapeutic and diagnostic derivatives to rapidly proliferating cells in higher concentrations compared to normal cells. It is the object of the present invention to provide new methods for identifying, synthesizing, characterizing and applying cobalamin derivatives with higher specificity for cells with abnormally high proliferation, while preventing the development of resistant cell lineage.

Brief Description of the Invention The present invention is based on the observation that, in contrast to cobalamin itself, cobalamin derivatives with little or much reduced binding to the transport protein transcobalamin III (TCII), if properly applied, have a very low accumulation ratio in blood and benign organs, such as kidney and liver, compared to the accumulation in neoplastic tissues, and they are more rapidly eliminated from the blood. By selecting cobalamin derivatives that act as substitutes for vitamin B12, the risk of formation of resistant offspring in the neoplastic tissue is greatly reduced. The invention relates to cobalamin derivatives. (a) that they have no binding affinity or have low binding affinity to transcobalamin II, and (b) that they retain activity as a substitute for vitamin B12. In particular, the invention relates to cobalamin derivatives, (a) having less than 20%, preferably less than 5%, of binding affinity to transcobalamin II compared to the binding affinity of unmodified cobalamin in a binding test, and (b) retaining more than 2% of the activity as a substitute for vitamin B12 in a growth trial. Examples of compounds of the invention with little or no binding affinity for TCII are specific cobalamin derivatives that carry a therapeutic and / or diagnostic agent, such as a radioactive metal. The compounds of the invention are selected based on the results of a binding test with purified TCII and a growth assay using Lactobacillus delbrueckii as the test organism. The invention also relates to a method of diagnosing a neoplastic disease or an infection by microorganisms in a mammal, comprising: (a) exposing the animal suspected of being afflicted with a neoplastic disease or infection to a period of a vitamin B12-free diet, and (b) subsequently applying a cobalamin derivative of the invention having an agent of diagnosis. The invention also relates to a method of treating a mammal suffering from a neoplastic disease or an infection by microorganisms, comprising: (a) exposing the mammal in need of treatment to a period of a diet free of vitamin B12, and (b) subsequently applying a cobalamin derivative of the invention having a therapeutic agent. The invention also relates to the use of a cobalamin derivative according to the invention in a method of diagnosing a neoplastic disease or an infection by microorganisms or in a method of treating a mammal suffering from a neoplastic disease or an infection by microorganisms. The invention further relates to pharmaceutical compositions comprising cobalamin derivatives of the invention, in particular pharmaceutical compositions suitable for diagnostic applications and pharmaceutical compositions suitable for therapeutic applications, and to the use of these pharmaceutical compositions in a diagnostic method and in a method of therapeutic treatment, respectively. The invention also relates to intermediates for the preparation of compounds useful in a diagnostic or therapeutic treatment according to the invention, in particular to compounds substituted with chelators for binding radioactive metals, but having no metal or non-radioactive metal bound to the chelator The cobalamin derivatives according to the invention are of particularly high value for the diagnosis and / or treatment of aggressive neoplastic diseases, of rapid progress such as cancers and / or diagnosis and / or treatment of local infections by pathogenic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating the radioactively labeled cyanocobalamin-b-propyl-PAMA-OEt interaction of Example 11, a TCII-non-binder, with transport proteins in a gel shift assay. t = time, cpm = counts per minute. A) Gel filtration analysis of the radioactive labeled derivative on a Superdex ™ 75 column (Pico elutes at 1.5 kDa). B) Gel filtration analysis of the mixed derivative with TCI (peak displacement from 1.5 kDa to 44 kDa) C) Gel filtration analysis of the derivative mixed with TCII (peak elutes at 1.5 kDa indicating that cyanocobalamin-b-propyl-PAMA -OEt is essentially a non-binding TCII Figure 2 is a graph illustrating the interaction of radioactively labeled cyanocobalamin-b-butyl-PAPcet from Example 5, a TCII-linker, with transport proteins in a gel displacement assay t = time, cpm = counts per minute A) Gel filtration analysis of the radioactively labeled derivative on a Superdex ™ 75 column (peak elutes at 1.5 kDa). B) 7 Gel filtration analysis of the mixed derivative with TCI (peak displacement from 1.5 kDa to 44 kDa). C) Gel filtration analysis of the derivative mixed with TCII (peak displacement from 1.5 kDa to 60 kDa indicating that cyanocobalamin-b-butyl-PAPAcet does not bind to TCII). Figures 3, 4, 5 and 6; Bar graphs illustrating tissue distribution. y axis: percent of injected radioactivity or gram of tissue, x-axis: Organs 1) Blood, 2) Heart, 3) Spleen, 4) Kidney, 5) Stomach, 6) Intestine, 7) Liver, 8) Muscle, 9) Bone, 10) Tumor. Figure 3: Distribution in tissue after injection i. v. of radioactive cyanocobalamin (S7Co-CN-Cbl) in mice fed normal food. Figure 4: Distribution in tissue after injection i. v. of radioactive cyanocobalamin (S7Co-CN-Cbl) in mice fed food deficient in vitamin B12. Figure 5: Distribution in tissue after injection i. v. of radioactive cyanocobalamin-b-propyl-PAM-OEt (Example 11) in mice fed normal food. Figure 6-: Distribution in tissue after injection i.v. of radioactive cyanocobalamin-b-propyl-PAMA-OEt (Example 11) in mice fed food deficient in Vitamin B12.

Detailed Description of the Invention Cobalamin derivatives with low or very low binding affinity to the cobalamin (or transport protein) TCII vector protein, when applied to mammals exposed to vitamin B12 diet, exhibit a very low accumulation in blood and in crucial organs, such as kidney and liver, while maintaining high rates of uptake in hyperproliferative cells and thus, allowing more accurate diagnosis and therapy of neoplastic diseases and local infections by microorganisms. The compounds of the invention which have low binding affinity to TCII and- retain B12 activity are for example the compounds of the formula (I) wherein Rb, Rc, Rd and Re, independently of each other, are a chelator-separating group, an antiproliferative therapeutic agent or antibiotic, a sterically demanding organic group with 4 to 20 carbon atoms, or hydrogen; RR is a separator-chelator group or an antiproliferative or antibiotic therapeutic agent, each connected through a Z-linker, or hydrogen; with the proviso that at least three of the residues Rb, Rc, Rd, Re and RR are hydrogen and at least one of the residues Rb, Rc, Rd and Re is different from hydrogen; X is a monodentate ligand; and the central cobalt (Co) atom is optionally in the form of a radioactive isotope. In a particular embodiment, Re is hydrogen. A monodentate ligand X is for example cyano; halogen, such as fluoro, chloro, bromo or iodo, cyanate, isocyanate, thiocyanate or isothiocyanate; alkyl, linear or branched and comprising from 1 to 25 carbon atoms, preferably from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl or isobutyl, or also n-hexyl or n -decyl and optionally substituted by hydroxy, methoxy or amino, for example, hydroxymethyl, methoxymethyl, aminomethyl, hydroxyethyl or methoxyethyl; a nitrile R-CN; an isonitrile R-NC, a carboxylate R-COO "or a thiolate RS", wherein R is alkyl, linear or branched and 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-isocyanide, phenyl-isocyanide, acetate, propionate, benzoate, methylthiolate, ethylthiolate, n-hexylthiolate or thiophenolate; a phosphite (RO) 3P wherein the substituents R are identical or different and represent alkyl comprising from 1 to 6 carbon atoms or aryl, for example phenyl or optionally substituted naphthyl, such as trimethylphosphite, methyldiphenylphosphite, triphenylphosphite or tri-o-ring tolylphosphite; hydroxy or aqueous; or a 5'-deoxyadenosyl group or a related nucleoside. Preferably, X is cyano, methyl, hydroxy, aqueous or a 5'-deoxyadenosyl group. More preferred is cyano. A separator-chelator group as a substituent Rb, Rc, Rd, Re or RR is a chelator for metal atoms bonded to the NH or 0 function of cobalamin by a spacer, and optionally has a metal atom, in particular a radioactive metal atom. The compounds of the formula (I) in which the chelator-separator group does not have a metal atom are intermediates which are to be used in the preparation of the compounds useful in a method of diagnosis and / or therapeutic treatment in accordance with the invention. An antiproliferative therapeutic agent or an antibiotic as a substituent Rb, Rc, Rd, Re or RR is an antibiotic agent selected from aminoglycoside antibiotics, such as gentamicin, tetracyclines, antimetabolites, such as selenomethionine, macrolides, such as erythromycin and trimethoprim, or an antiproliferative agent selected from antimetabolites, such as 5-fluorouracil, alkylating agent, such as oxaliplatin, dacarbazine, cyclophosphamide or carboplatin, a cell cycle inhibitor, such as vinblastine or docetaxel, a DNA breaking agent (topoisomerase inhibitor, intercalator , strand breaking agent), such as doxorubicin, bleomycin or topotecan, a compound that interferes with the signal transduction pathway, such as a modifier of caspase activity, agonists or cell death receptor antagonist, and a modifier of nucleases, phosphatases and kinases, such as imatinib-mesylate, dexamethasone, phorbol-myristate acetate, cyclosporin A, corkine, or tamoxifen, either directly bound to the NH or O function of cobalamin or covalently bound by a separator. A spacer is an aliphatic chain of 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, for example 2 to 5 carbon atoms, wherein one or two carbon atoms can be replaced by nitrogen atoms and / or oxygen and the aliphatic chain can be replaced by hydroxy, oxo or amino. In particular, two adjacent carbon atoms can be replaced by an amide function -NH-CO or an ester function -O-CO-. The particular separators that connect the NH or O function of cobalamin with a chelator are ethylene, propylene, butylene or pentylene groups or these groups where a carbon is replaced by oxygen or nitrogen, or where a carbon atom is replaced by oxygen or nitrogen and the adjacent carbon atom is replaced by oxo. A chelator is a compound having two, three or more donor atoms selected from nitrogen, oxygen and sulfur at a distance such as to bind to a metal atom. Particular chelators are tridentate chelators that have three metal-binding sites that comprise N, O and / or S donor atoms at a distance from each other that allows the joining of metal atoms. The nitrogen atoms as donor atoms are for example part of an aliphatic amine, an aromatic amine or an aromatic heterocycle containing nitrogen. Oxygen atoms as donor atoms are, for example, alcohols, ethers, esters or carboxy functions. The sulfur atoms as donor atoms are, for example, thioethers or thiols. The donors can be connected, for example by aliphatic carbon chains or chains comprising amide bonds and / or ether functions, and can be derived from amino acids, polyethers, and the like. Preferred chelators are chelators of formula (II) to (IX). The carboxyl groups can be present as esters that are cleaved concomitantly with the complex formation with a metal atom to produce a coordinating carboxylate group. In these esterified chelators, the ester may be an alkyl ester wherein alkyl is linear or branched and comprises from 1 to 25 carbon atoms, optionally from one to five carbon atoms which is replaced by nitrogen or oxygen, or one or two carbon atoms replaced by sulfur or phosphorus, and which are optionally substituted by phenyl, pyridyl, hydroxy, halogen, cyano, oxo or amino. The ester can also be an aryl or heteroaryl ester, wherein the aryl or heteroaryl has from 3 to 10 carbon atoms and zero, one or two oxygen atoms, zero, one, two or three nitrogen atoms or zero or one sulfur atoms. These ester residues may be suitably substituted in order to render them cleavable under particular reaction conditions, for example as described for esters commonly used as protecting groups for carboxylic acids, see, Green, TW, and Wuts, PGM, Protective groups in organic synthesis, Wiley 1999. Esterified chelators, for example, esterified by methyl, ethyl or cyanoethyl, are also included in the definition of preferred chelators.

HOOC yy, N "N NH, H (10 (lll) (IV) (V) HOOC NH HOOC N COOH N = (VI) (il) (VIII) (IX) The radioactive metals considered are radioisotopes such as 9 mTc, 99mTc, 188Re, 185Re, 1: L1In, 90Y, SCu, 67Cu and 177Lu, in particular 99mTc, 188Re '18SRe and llxIn. The radioactive isotopes of Co considered are, for example, 57Co and 60Co. A sterically demanding organic group having 4 to 20 carbon atoms is for example an alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group optionally substituted by hydroxy, alkoxy, oxo, amino, carboxy, carbamoyl or alkoxycarbonyl. Examples of aryl groups are phenyl, methylphenyl, dimethylphenyl, hydroxyphenyl or naphthyl. Examples of heteroaryl groups are pyridyl, pyrrolyl, imidazolyl or benzimidazolyl. In an alkyl chain, the carbon atoms can be replaced by nitrogen or oxygen atoms. For example, in an alkyl chain, a carbon atom can be replaced by a nitrogen (or oxygen) atom, and the neighboring carbon atom is replaced by oxo, which thus represents a carboxamide (or ester function, respectively ). Particular examples of a sterically demanding organic group are iso-butyl, tert-butyl, tert-pentyl, o-tolyl, o-methylbenzyl, or 2,6-dimethylbenzyl. A linker Z connecting RR with a spacer-chelator group or an antiproliferative therapeutic agent or antibiotic is a bond or a linker selected from the group of phosphates, phosphonates, carboxylic esters or alkylene of 1 to 10 carbon atoms and combinations thereof. This linker connects the spacer-chelator group or the therapeutic agent optionally comprising a spacer as defined hereinabove to the oxygen atom of cobalamin. Compounds that are derivatized in RR but where Rb, R °, Rd and Re are all hydrogen are recognized by CTs and are still enzymatically active, and therefore are excluded from the scope of the invention. The selection of a compound of the invention is based on the following criteria: (a) no or very low binding affinity, for example less than 20%, in particular less than 10%, preferably less than 5%, so more preferably less than 2% binding affinity, to TCII compared to the binding of cobalamin (unmodified); and (b) activity as a substitute for vitamin B12 in a growth test using a bacterium or mammalian cell line dependent on vitamin B12, eg, more than 2% activity, in particular more than 10% activity, preferably more than 20% activity compared to the activity of vitamin B12 of cobalamin (unmodified). To test the binding affinity of the cobalamin derivatives (Cbl) to TCII, an in vi tro test is carried out with partially purified TCII obtained from the blood of rabbits. Recombinant TCII produced with an E. coli expression system can also be used as a substrate. The cobalamin derivatives of the invention have to maintain their function as substitutes for vitamin B12. As a result, the risk of resistance development leads to cells with high proliferation rates to be very well reduced. In all likelihood, mutant cells that are no longer able to capture cobalamin derivatives with little or no TCII binding activity will have lost the advantage of their predecessor cells in achieving high proliferation rates through a mechanism of vitamin uptake B12 independent of TCII, highly efficient. To test the B12 activity of a cobalamin derivative, an assay is carried out using Lactobacillus delbrueckii, an internationally recommended test strain for cyanocobalamin (CN-Cbl). Complementing cyanocobalamin to a cyanocobalamin-free assay medium results in a growth response of the auxotrophic cyanocobalamin bacterial strain that can be measured by a quantitative solid diffusion plate assay. The test is used to determine to what degree (in%) the cobalamin derivative is capable of replacing the cyanocobalamin as a living support catalyst. The invention relates to a method of diagnosis and method of treatment of neoplastic diseases and local infections by microorganisms in a mammal, comprising (a) exposing the mammal to a period of vitamin B12-free diet, (b) subsequently applying a cobalamin derivative of the invention having a diagnostic or therapeutic agent, and using the cobalamin derivatives of the invention in this method. The positive effect of applying non-binding cyanocobalamin derivatives to TCII in their biodistribution in mammals exposed to a vitamin-free diet is illustrated in Table 1.

Table 1: Tissue distribution 24 hours after injection i. v. of labeled, radioactive derivatives in mice Example 5: Cyanocobalamin-b-butyl-PAPAcet Example 6: Cyanocobalamin-b-butyl-aminocarboxymethyl-His-OMe Example 8: Cyanocobalamin-c-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-propyl-PAMA-OEt Example 20: Cyanocobalamin-b-propyl-His-OMe Example 288: Cyanocobalamin-b-ethyl-Triamine Example 25: Cyanocobalamin-5 '- phosphocolamin-His-OMe The results of the biodistribution analysis collected in Table 1 indicate that non-TCII linkers, for example, the compounds of the invention as described in Examples 10, 11, 12, 18 and 22, have an accumulation comparatively high in tumor, five times more or more than in blood and at least half of the amount found in the critical organs kidney and liver. The compounds of Examples 5, 6, 8, 14, 20 and 25 do not fall under the definition of compounds of the invention since they bind to TCII, and are described herein only as reference compounds. The cobalamin derivatives according to the invention are of particularly high value for the diagnosis and / or treatment of rapidly progressive, aggressive neoplastic diseases, such as cancers. The compounds of the invention can be used for the treatment of highly proliferative cells of human origin comprised in malignancies such as melanoma, fibrosarcoma, ovarian cancer, pancreatic carcinoma, osteosarcoma and acute leukemia, to mention only a few examples and are capable of deriving the endocytosis mediated by TCII. The method of the invention allows a specific protection of the benign organs of the harmful uptake of cobalamin derivatives, mediated by TCII, which have a radioactive isotope and / or which have a non-radioactive cell-destroying agent. The compounds of the invention are not only useful in the formation of cancer images and cancer therapy, but also for the visualization and potential treatment of local infections by microorganisms depending on a high and direct uptake of cobalamins. The compounds of the invention having an antiproliferative agent are useful for transporting the agent in an inactive form to the hyperproliferative cells where they can exert their action after intracellular amidolysis. In a method of treating a neoplastic and / or infectious disease, a compound of the invention having a suitable therapeutic agent can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of combinations fixed, or administration of a compound of the invention and one or more other therapeutic agents that are stratified or given independently of each other, or the combined administration of fixed combinations and one or more other therapeutic agents. A compound of the invention may be administered, in addition to or additionally, especially for tumor therapy in combination with chemotherapy, immunotherapy, surgical intervention, or a combination thereof. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies. The invention further relates to pharmaceutical compositions comprising cobalamin derivatives of the invention, in particular pharmaceutical compositions suitable for diagnostic applications and pharmaceutical compositions suitable for therapeutic applications. Preferred are pharmaceutical compositions for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. The compositions comprise the active ingredient alone or together with a pharmaceutically acceptable carrier. The dose of the active ingredient depends on the disease to be treated and the species, its age, weight and individual condition, the pharmacokinetic data of the individual and the mode of administration.

Methods of Production The compounds of the invention are prepared by standard methods known in the art. Preferably, the cyanocobalamin, ie the compound of the formula (I), wherein Rb, Rc, Rd, Re and RR are hydrogen and X is cyano, is hydrolysed under controlled conditions, eg, dilute mineral acid, and the mixture obtained from mono-acids, wherein one of the carbamoyl groups, CONH2 is converted to COOH, are separated. The bis-acids can be obtained in a similar manner. The cyanocobalamin-b, c, do e-acid, ie the compound of the formula (I), wherein CONHRb, CONHRc, CONHRd or CONHRe is replaced by COOH, respectively, and X is cyano, then it can be reacted with a corresponding amine Rb-NH2, RC-NH2, Rd-NH2 and Re-NH2, respectively, under normal conditions for amide formation, for example, as is known in the chemistry of peptides. The functional groups in the residues Rb, Rc, Rd and Re that interfere with the formation of amides are preferably in the protected form, and are deprotected by normal methods after the formation of amides. For the preparation of compounds wherein the separator comprises an amide function, it is also possible to react cyanocobalamin-b, c, e-acid with a diamine H2N (CH2) nNH2 under the normal conditions of amide formation, and to condense additionally the cyanocobalamin worked with H2N (CH2) n obtained with a corresponding carboxylic acid again under normal conditions of amide formation to generate the substituent Rb, Rc, Rd and Re, respectively.

For the preparation of compounds, wherein Rc is different from hydrogen, the preferred method is the formation of the c-lactone followed by a reductive reaction of lactone ring opening according to Brown et al. , Inorg. Chem. 1995, 3038. For the preparation of the compounds, wherein RR is different from hydrogen, cyanocobalamin (or a cyanocobalamin derivative in which Rb, Rc, Rd or Re is different from hydrogen) is reacted with RR-L , wherein L is a suitable leaving group of activation to form an ester linkage, for example, halogen, the residue of a mixed anhydride or other of the usual activation residues for formation of carboxylic ester, phosphate or phosphonate usual in the synthesis of peptides and nucleic acids. The following examples serve to illustrate the invention without limiting the invention in its scope.

Examples The reagent grade chemicals were from Merck, Dietikon (CH), Aldrich of Fluka, Buchs (CH) and were used without further purification. BOP = 1-benzotriazolyloxy tris (dimethylamino) phosphonium hexafluorophosphate DCC = dicyclohexylcarbodiimide DIPEA = diisopropylethylamine EDC = l-ethyl-3- (3-dimethylaminopropyl) carbodiimide Fmoc = (9H-fluoren-9-ylmethoxy) carbonyl HOsu = N-hydroxysuccinimide MES = 2- (4-morpholinyl) ethanesulfonic acid RT = room temperature TBTU = benzotriazol-1-yl-N-tetramethyluronium tetrafluoroborate TEAP = triethylammonium phosphate Teoc = 2-trimethylsilyl-ethoxycarbonyl TFA = trifluoroacetic acid HPLC analyzes were performed on a system Merck-Hitachi L-7000 equipped with a radioelectric detector EG &G Berthold LB 508, using a Waters XTerra RP8 column (particle size 5 μm, 1 x 100 mm) and a flow rate of 1 mL / min. The chromatograms were recorded at 250 and 360 nm. The solvent a were predominantly aqueous buffers. The sodium acetate buffer was prepared by mixing 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 was prepared by dissolving tris (hydroxymethyl) -aminomethane (605 mg) in water, adding 2 M HCl to reach a pH of 8.2, adjusting the volume to 1000 ml, and adding acetonitrile (10 ml). Solvent b was always methanol. The preparative HPLC separations were carried out in a Varian Prostar variant system equipped with two Prostar 215 pumps and a Prostar 320 UV / Vis detector, using Waters XTerra Prep columns. RP8 (particle size 5 μm, 3 x 100 mm and 30 x 100 mm). The flow rates were 4 ml / min for the 3 x 100 mm column and 30 ml / min for the 30 x 100 mm column. The UV / Vis spectra were recorded on a Varian Cary 50 spectrometer, the IR spectra were recorded on a Bio-Rad FTS-45 spectrometer with the samples on compressed KBr pills. The ionization mass spectra were recorded by electrospray (ESI-MS) on a Merck Hitachi M-8000 spectrometer. In rhenium compounds, isotope 187RE values are reported. The NMR spectra are recorded on a Bruker DRX 500 MHz spectrometer. The chemical shifts are reported relative to the residual solvent protons as a reference. The cobalamin derivatives (mg amounts) were scrubbed by applying an aqueous solution of the compound to a Chromafix RP18ce cartridge, followed by complete rinsing with water. The desalted product was then evaluated with methanol, the solvent was removed in vacuo and the product dried under high vacuum. Larger amounts (above 50 mg) were scrubbed by extraction with phenol as described in Meth. Enzymol. 1971, 18 (3), p. 43. (N-3-aminopropyl-N-pyridin-2-ylmethyl-amino) acetic acid ethyl ester (propyl-PAMA-OEt) was prepared as described for the pentyl analogue by Schibli et al., (Nucí. Med. Biol. 2003, 30, 465). The compound is prone to cyclization under basic conditions. Therefore, the Boc-protected intermediate was stored and Boc was removed just before further functionalization by shaking in dilute aqueous HCl. The ethyl and hexyl derivatives were prepared in an analogous manner. RE ([N-3-aminopropyl-N-pyridin-2-ylmethyl-amino] acetic acid) (C0) 3 was prepared by reacting the acid (N-3-aminopropyl-N-pyridin-2-ylmethyl-amino) acetic completely deprotected with y (Net4) 2 [Re (0H2) 3 (CO) 3]. Methyl 1-carboxymethyl-N-Fmoc-histidinate trifluoroacetate was prepared as described by Pak et al. , (Chem. Eur. J. 2003, 9, 2053-2061). The counterion was exchanged to chloride by stirring the compound in HCl 0. 05 M, followed by evaporation in vacuo at room temperature. Methyl 3-aminopropyl-N-Teoc-histidinate was prepared as described by Staveren et al. (Organic &Molecular Chemistry 2004, 2, 2593). 3- (N-2-Cyanoethoxycarbonylmethyl) -N-pyridin-2-ylmethyl-amino) -propionic acid 4-nitro-phenyl ester was prepared as described by Kunze (Dissertation, University of Zurich, 2004).

Example 1: Cyanocobalamin monocarboxylic acids (b, d e) Vitamin B12 (1.88 g, 1.39 mmol) is hydrolyzed in 0.1 M HCl (190 ml) as described by Pathare et al. (Bioconjugate Chem. 1996, 217). The purification is modified as follows: The Dowex column allows, after desalination by extractions with phenol, the isolation of three fractions, one that exclusively contains d-acid, a second one that exclusively contains b-acid and d-acid, and a third that exclusively contains b-acid and e-acid. The mixture of b-acid and d-acid is separated by preparative HPLC (column: Waters XTerra Prep. RP8, 5 μm, 30 x 100 mm, gradient a / b 0.5% min "1 starting from a of 100% acetate) The mixture of b-acid and e-acid is separated in the same system but using the Tris buffer as solvent a.Cyanocobalamin-b-acid is isolated in a yield of 280.6 mg (14.9%), cyanocobalamin-d-acid in a yield of 131.5 mg (7.0%), and cyanocobalamin-e-acid in a yield of 94.26 mg (5.0%).

Example 2: Cyanocobalamin-b- (2-aminoethyl) amide [cyanocobalamin-b-ethylamine] Cyanocobalamin-b- (2-aminoethyl) amide was prepared as described by Pathare et al. (Bioconjugate Chem. 1996, 217) for synthesis of the dodecane analogue Ethylenediamine (132 mg, 0.147 ml, 2.2 mmol) was dissolved in a mixture of DMF / H20 (10 ml, l / lv / v). The pH was adjusted to 5 by addition of 1 M HCl. To the solution were added cyanocobalamin-b-acid (60.0 mg, 44.4 μmol) and KCN (57 mg, 0.87 mmol), followed by adjustment of pH 5.5. Then, EDC (84.2 mg, 0.43 mmol) and HOSu (50.6 mg, 0.44 mmol) were added. The mixture was stirred at room temperature for 3 days, and extra portions of EDC and HOSu were added at 24 hour intervals. For the treatment, the mixture was evaporated to dryness in vacuo, followed by preparative HPLC purification (acetate system, gradient: 0.5% min "1 starting from buffer to 100%) to give 34 mg (55%) of cyanocobalamin- b- (2-aminoethyl) amide, MS (MeOH; ESI-pos.): m / z = 1398.8 [M + H] +, 1420.1 [M + Na) +, 669.4 [M + H] 2+, 711.1 [ M + H + Na] 2+.

Example 3: Cyanocobalamin-b- (4-aminobutyl) amide [cyanocobalamin-b-butylamine] was prepared as described above for the synthesis of the ethyl analogue. MS (MeOH, ESI-pos.): M / z = .1427.1 [M + 1] +, 714.5 [M + 3] 2+.

Example 4: Cyanocobalamin-b-ethyl-PAPAcet Cyanocobalamin-b-ethylamine (Example 2, 24 mg, 17.2 μmol) was dissolved in a mixture of DMF / DMSO (5 ml, 4/1 v / v). To this mixture was added 3- [N-2-cyanoethoxy-carbonylmethyl-N-pyridin-2-ylmethyl-amino] -propionic acid 4-nitrophenyl ester (14 mg, 34.1 μmol) and DIPEA (5 μl, 29 μmol). . After stirring at room temperature for 24 hours, the mixture was evaporated to dryness in vacuo. Purification by preparative HPLC (acetate system, gradient: 0.5% min "1 starting from buffer to 100%) gave 20 mg (70%) of cyanocobalamin-b-ethyl-PAPAcet as a red solid MS (MeOH, ESI -pos.): m / z = 1672.1 [M + H] +, 836.9 [M + H] 2+ Example 5: Cyanocobalamin-b-butyl-PAPAcet Cyanocobalamin-b-butylamine (Example 3, 5.5 mg, 3.9 μmol) and 4-nitrophenyl ester of 3- [N-2-cyanoethoxy-carbonylmethyl-N-pyridin-2-acid were dissolved. -ylmethyl-amino] propionic (2.5 mg, 6.1 μmol) in a mixture of dry DMSO (0.5 ml) and DMF (0.5 ml). DIPEA (5 μl, 29 μmol) was added to reach a pH between 8 and 9, and the mixture was stirred at room temperature. After 5 hours, HPLC analysis confirmed complete production of the product. The solvent was partially evaporated in vacuo to allow the product to precipitate on the addition of ethyl ether. The suspension was centrifuged and decanted three times to give a fine powder. Purification by preparative HPLC (acetate system, gradient: 0.5% min "1 starting from buffer to 100%) gives the pure product in yield of 2.7 mg (41%) ESI-MS: m / z = 850.1 [M + 2] 2 UV / Vis:? nm (emol l "1 cm" 1) = 279.1 (17300, 361.0 (31200), 519.9 (8700), 552.0 (9700).

Example 6: Cyanocobalamin-b-butyl-aminocarboxymethyl-His-OMe A solution of cyanocobalamin-b-butylamine (49.6 mg, 34.8 μmol) in dry DMSO (2 ml) was added 1-carboxymethyl-N-Fmoc-histinate hydrochloride. methyl (35.5 μmol) and BOP (46.2 mg, 104. 4 μmol). DIPEA (12 μL, 70.0 μmol) was added, and the solution was stirred at room temperature for 16 hours. HPLC analysis confirmed complete conversion of the starting material of cobalamin into the intermediate compound protected with Fmoc. The intermediate compound was precipitated by adding diethyl ether, and the suspension was centrifuged and decanted three times to give a fine powder. The intermediate compound was dissolved in DMF (5 ml), and piperidine (225 μl) was added. After stirring at room temperature for 1.5 hours, the product was precipitated upon addition of diethyl ether, and the suspension was centrifuged and decanted three times to give a fine powder. Purification by preparative HPLC (acetate system, gradient: 1% min "1 starting from buffer to 100%) gave the pure product in a yield of 17.1 mg (32.1%). UV / Vis:? Nm (emol l" 1 cm "1) = 279.1 (19200), 361.0 (24700), 521.0 (9600), 551.1 (10700).

Example 7 Cyanocobalamin-c- (4-aminobutyl) -amide [cyanocobalamin-c-butyl [amine] Cyanocobalamin-c-acid was prepared as described by Brown et al., (Inorg, Chem. 1995, 3038). 1,4-Diaminobutane (0.059 ml, 0.59 mmol) was dissolved in a mixture of DMF / H20 (10 ml, l / l v / v). The pH was adjusted to 5.2 by addition of 1 M HCl. To the solution was added cyanocobalamin-c-acid (16.0 mg, 11.8 μmol), KCN (15.3 mg, 0.236 mmol), EDC (9.0 mg, 47.2 μmol) and HOSu. (5.4 mg, 47.2 μmol). The mixture was stirred at room temperature for 4 days, and extra portions of EDC and HOSu were added. After another day, additional portions of EDC and HOSu were added again. After a total of 6 days, HPLC analysis confirmed complete conversion of the cobalamin derivative. For workup, the mixture was evaporated to dryness in vacuo, followed by preparative HPLC purification (RP C18 column), 1 mM HCl as a buffer, gradient: from 20% methanol to 50% methanol in 30 minutes) to give 9.8 mg (58%) of cyanocobalamin-c-butylamine. MS (MeOH, ESI-pos.): M / z = 1427.7 [M + 2] +, 713.5 [M + 1] 2+ Example 8: Cyanocobalamin-c-butyl-PAPAcet Cyanocobalamin-c-butylamine was reacted (7.0 mg, 4.9 μmol) and 3- [N ~ 2-cyanoethoxycarbonylmethyl-N-pyridin-2-ylmethyl-amino] propionic acid 4-nitrophenyl ester (3.8 mg, 9.2 μmol) and purified as described in the synthesis of cyanocobalamin -b-butyl-PAPAcet (Example 5) to give the crude product in a yield of 3.8 mg (78%). ESI-MS: m / z = 1701.0 [M + l] +, 850.1 [M + l] 2+ UV / Vis:? Nm (emol l "1 cm" 1) = 278.1 (14500), 362.1 (25400), 550.0 (7900).

Example 9: Cyanocobalamin-b-butyl-PAPA-Re (CO) 3 Cyanocobalamin-b-butylamine (Example 3, 24.6 mg, 17.2 μmol) and Re (CO) 3 (3- [N-carboxymethyl-N- acid] were dissolved. pyridin-2-ylmethyl-amino] propionic) (9.1 mg, 17.2 μmol) were dissolved in DMSO. BOP (22.9 mg, 51.7 μmol) and DIPEA (2.94 μl, 17.2 μmol) were added, and the mixture was stirred at room temperature. DIPEA and BOP were added daily for 4 days. HPLC analysis confirmed formation of two products. They were precipitated in the addition of ethyl ether. The suspension was centrifuged and decanted three times to give a fine powder. The purification for preparative HPLC (acetate system, gradient: 0.5% min "1 starting from buffer to 100%) allowed the isolation of the main product peak in a yield of 2.3 mg (7.0%) ESI-MS: m / z = 1917.5 [M + 2] +, 959.9 [M + 4] + Example 10: Cyanocobalamin-b-ethyl-PAMA-OEt Cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (0.8 ml). Subsequently DMF (2 ml) and Net3 (0.1 ml) were added. In a different flask, 5 equivalents of (N-2-aminoethyl-N-pyridin-2-ylmethylamino) acetic acid ethyl ester hydrochloride were dissolved. (ethyl-PAMA-OEt) (prepared by cleavage of the Boc-protected derivative by stirring in a mixture of absolute EtOH / 2M HCl (7.5 ml 4/1 v / v) overnight and subsequent removal of the volatiles in vacuo in DMF / Net3 mixture (4.5 ml, 8/1 v / v) The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol) After stirring at room temperature for 45 minutes, the solvent was added to the mixture. The residue was purified by preparative HPLC (acetate system, gradient: 1.0% min "1 starting from buffer to 100%) to give 12 mg (51%) of cyanocobalamin-b-ethyl-PAMA-OEt as red solid MS (MeOH, ESI-pos.): m / z = 1575.8 [M + H] +, 788.7 [M + H] 2+, 799.3 [M + H + Na] 2.

Example 11: Cyanocobalamin-b-propyl-PAMA-OEt A solution of freshly prepared (N-3-aminopropyl-N-pyridin-2-ylmethyl-amino) acetic acid ethyl ester (361 μmol) in water (1 ml) added to cyanocobalamin-b-acid (65.0 mg, 48.1 μmol). EDC (46.1 mg, 240 μmol) was added and the pH adjusted to 5.5 with 0.1 M NaOH. After stirring at room temperature for 15 hours, HPLC analysis (sodium acetate buffer) showed approximately 50% formation of the product. EDC (46.1 mg, 240 μmol) was added again, but prolonged stirring at room temperature does not lead to further formation of the product. The solvent was removed i13 vacuo, and the residue was purified by preparative HPLC (gradient a / b 0.5% min "1 starting from buffer to 100% acetate.) The main fraction was collected, the solvent was removed in vacuo, and the product was sloped to give cyanocobalamin-b-propyl-PAMA-OEt in a yield of 25.8 mg (16.2 μmol, 33.3%) ESI-MS: m / z = 806.5 [M + l + Na] 2, 795.6 [M +2] +. UV / Vis:? Nm (emol l "1 cm" 1) = 278.0 (8500), 361.1 (26500), 549.1 (8000).

Example 12: Cyanocobalamin-b-butyl-P7AMA-OET Cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (0.8 ml). Subsequently DMF (2 ml) and Net3 (0.1 ml) were added. In a separate flask, about 5 equivalents of (N-4-aminobutyl-N-pyridin-2-ylmethyl-a) n-acetic acid ethyl ester hydrochloride (butyl-PAMA-OEt) (prepared by cleavage of the protected derivative) were dissolved. with Boc by acceptance in a mixture of absolute EtOH / 2M HCl (7.5 ml 4/1 v / v) overnight and subsequent removal of the volatiles in vacuo) in a mixture of DMF / Net3 (4.5 ml; v / v). The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 1.0% min "1 starting from buffer to 100%) to give 15 mg (63%) of cyanocobalamin-b-butyl-PAMA-OEt as a red solid.

Example 13: Cyanocobalamin-b-butyl-PAMA-OH The 9H-fluoren-9-ylmethyl ester of bromoacetic acid was prepared from bromoacetyl bromide and 9H-fluorenylmethanol in dry THF at 0 ° C. It was prepared ([(4-tert-butoxy-carbonylamino-butyl) -pyridin-2-ylmethyl-amino] -acetic acid 9H-fluoren-9-ylmethyl ester from Boc-NH- (CH2) NH2, pyridine- 2-aldehyde and 9H-fluoren-il-methyl ester of bromoacetic acid according to the procedure used by Schibli et al., (Nucí.Med. Biol. 2003, 30, 465) for the synthesis of Boc-pentyl-PAMA-OMe. dissolved cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) in DMSO (0.8 ml), DMF was subsequently added (2 ml) and Net3 (0.1 ml). In a different flask, approximately 5 equivalents of [(4-amino-butyl) -pyridin-2-ylmethyl-amino] -acetic acid 9-fluoren-9-ylmethyl ester (butyl-PAMA-OFm) (prepared by cleavage of the Boc protected derivative by stirring in a mixture of trifluoroacetic acid / CH2Cl2 (4 ml 1/2 v / v) for 1 hour and subsequent removal of the volatiles in vacuo) in a mixture of DMF / Net3 (4.5 ml; / 1 v / v). The two solutions were shaken, followed by addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 1.5% min "1 starting from buffer to 100%) to give 15 mg of cyanocobalamin-b-butyl-PAMA-OFm as a red solid. Butyl-PAMA-OFm (15 mg) in 3 ml of a mixture of HNEt2 / DMF (2/1, v / v) and stirred at room temperature for 2 hours.The solvent was removed in vacuo, and the residue was purified by preparative HPLC (acetate system, burning: 1.0% min "1 starting from buffer to 100%) to give 9 mg of cyanocobalamin-butyl-PAMA-OH as a red solid.

Example 14: Cyanocobalamin-b-hexyl-PAMA-OEt Cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (0.8 ml). Subsequently DMF (2 ml) and Net3 (0.1 ml) were added. In a separate flask, about 5 equivalents of (N-6-aminohexyl-N-pyridin-2-ylmethyl-amino) acetic acid ethyl ester hydrochloride (hexyl-PAMA-OEt) (prepared by cleavage of the protected derivative with Boc by stirring in a mixture of absolute EtOH / 2M HCl (7.5 ml 4/1 v / v) overnight and subsequent removal of the volatiles in vacuo) in a mixture of DMF / Net3 (4.5 ml, 8/1 v / v). The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 1.0% min "1 starting from buffer to 100%) to give 10 mg (41%) of cyanocobalamin-b-butyl-PAMA-OEt as a red solid. MS (MeOH, ESI-pos.): M / z = 816.9 [M + 2H] +, 1632 [M + H] + Example 15: Cyanocobalamin-b-ethyl-PAMA-Re (CO) 3_ Cyanocobalamin-b-ethyl-PAMA-OEt was dissolved (Example 10, 11 mg, 7.0 μmol) in 4 ml in a 2 M NaHCO 3 solution. a solution of (Net4) 2 [ReBr3 (CO) 3] (14.2 μmol) in 1.5 ml of MeOH. The mixture was heated at 85 ° C for 1 hour. After allowing the mixture to reach room temperature, it was purified by preparative HPLC (acetate system, gradient: 2.0% per min, starting from buffer a). Yield 11 mg (86%).

Example 16: Cyanocobalamin-b-propyl-PAMA-Re (CO) 3 Cyanocobalamin-b-acid (26.7 mg, 19.8 μmol), Re ([N-3-aminopropyl-N-pyridin-2-ylmethyl-amino] was dissolved. acetic acid) (C0) 3 (29.2 mg, 60 μmol), EDC (11.5 mg, 60 μmol) and HOSu (6.9 mg, 60 μmol) in a mixture of water (5 ml) and DMSO (0.5 ml), and the pH is adjusted to 5.5 with dilute HCl and NaOH. After 5 hours of stirring at room temperature, HPLC analysis (acetate buffer) shows about 33-% product formation. EDC and HOSu are added again. The mixture is stirred at room temperature for 3 days, with addition of EDC and HOSu at 24 hour intervals. The water is removed in vacuo, and the product is precipitated by adding diethyl ether. The suspension in oil is centrifuged and decanted. Washing with diethyl ether is repeated twice until a fine precipitate is formed. The crude product is stirred under high vacuum, purified by preparative HPLC (gradient a / b 1% min "1 starting from buffer to 100% acetate) and desalinated to give cyanocobalamin-b-propyl-PAMA-Re (CO 3 in a yield of 9.1 mg (23%).

ESI-MS: m / z = 1831.7 [M + 1] +, 916-1 [M + 1] 2+ UV / Vis:? Nm (emol l "1 cm" 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 μmol) was dissolved in DMSO (0.8 ml). Subsequently DMF (2 ml) and Net3 (0.1 ml) were added. In a different flask approximately 5 equivalents of [Re ([N-3-aminopropyl-N-pyridin-2-ylmethyl-amino] acetic acid) (CO) 3] CF3COOH (prepared by Boc cleavage of the protected complex in CH2C12) was dissolved. and TFA (2/1 v / v) for 1 hour at 0 ° C, followed by removal of the volatiles at room temperature in vacuo) in a mixture of DMF / Net3 (4.5 ml, 8/1 v / v). The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 2.0% min "1 starting from buffer to 100%) to give 11 mg (40%) of cyanocobalamin-b-hexyl-PAMA-Re (CO) 3. MS (MeOH, ESI-pos.): M / z = 936.5 [M + 2H] 2+, 948.3 [M + H + Na] 2+, 1873.8 [M + H] +.

Example 18: Cyanocobalamin-d-propyl-PAMA-OEt. Cyanocobalamin-d-acid (9.3 mg, 6.9 μmol) was reacted with (N-3-aminopropyl-N-pyridin-2-ylmethyl-amino) -acetic acid ethyl ester. (7 μmol) and EDC (6.6 mg, 34 μmol) as described for the synthesis of cyanocobalamin-b-propyl-PAMA-OEt (Example 11). The product was isolated in a yield of 3.6 mg (33%). ESI-MS: m / z = 1612 [M + Na] +, 1590 [M + 1] +, 806 [M + 1 + Na] 2+, 795. 1 [m + 2] 2+. UV / Vis:? Nm (emol l "1 cm" 1) = 279.0 (13400), 361.1 (23300), 549. 1 (7200).

Example 19: Cyanocobalamin-d-propyl-PAMA-Re (CO) 3 Cyanocobalamin-d-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (1.5 ml). Subsequently DMF (2 ml) and Net3 (0.1 ml) were added. In a separate flask, 5 equivalents of Re ([N-3-aminopropyl-N-pyridin-2-ylmethyl-amino] acetic acid) (C0) 3 were dissolved in a mixture of DMF / Net3 (4.5 ml; v / v). The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 2.0% min "1 starting from buffer to 100%) to give 20 mg (73%) of cyanocobalamin-d-propyl-PAMA-Re (CO) 3.

Example 20: Cyanocobalamin-b-propyl-His-OMe Cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (0.8 ml). Subsequently, DMF was added (2 ml) and Net3 (1 ml). In a different flask, about 4 equivalents of methyl 3-aminopropyl-N-Teoc-histidinate were dissolved in DMF. The mixtures were added together, and TBTU (32.1 mg, 0.1 mmol) was added. The mixture was stirred for 45 minutes, and was subsequently evaporated to dryness in vacuo. Purification by preparative HPLC (acetate system: gradient: 2.0% per min, starting from buffer a) gave 16 mg of a red solid (67%). MS (MeOH, ESI-pos.): M / z = 1710.4 [M + H] +, 855.0 [M + 2H] 2+, 866.7 [M + Na + H] 2+. One sample was dissolved 19 mg of this compound Teoc protected in a mixture of TFA / CH2C12 (4/1 v / v) at 0 ° C. After stirring for 4 hours at this temperature, the analytical HPLC showed complete conversion of the starting material. The solvent was removed in vacuo at room temperature. To the residue was added dry Et20, followed by removal of the solvent in vacuo. This step was performed three times in total, in order to remove any trace of TFA. Purification by preparative HPLC (acetate system, gradient: 0.5% per min, starting from buffer to 100%) yielded 11 mg of the title compound.

MS (MeOH, ESI-pos.): M / z = 1565.2 [M + H] +, 1587.2 [M + Na] +, 783.4 [M + 2] 2+, 794.1 [M + Na + H] 2+.

Example 21: Cyanocobalamin-b-propyl-His-Re (CO) 3 Cyanocobalamin-b-acid (20.0 mg, 14.8 μmol) was dissolved in DMSO (0.8 ml). • Subsequently, DMF (2 ml) and NEt3 (0.1 ml) were added. In a different flask, approximately 5 equivalents of [Re (methyl-3-aminopropyl-N-Teoc-histidinate) (CO) -CF3COOH (prepared by Boc cleavage of the protected complex in CH2C12 and TFA (2/1 v / v) for 1 hour at 0 ° C, followed by removal of the volatiles at room temperature in vacuo) in a mixture of DMF / Net3 (4.5 ml, 8/1 v / v). The two solutions were mixed, followed by the addition of TBTU (32.1 mg, 0.1 mmol). After stirring at room temperature for 45 minutes, the solvent was removed in vacuo. The residue was purified by preparative HPLC (acetate system, gradient: 2.0% min "1 starting from buffer to 100%) to give 7 mg (73%) of cyanocobalamin-b-propyl-His-Re (C0) 3. MS (MeOH, ESI-pos.): 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-ethyl-triamine Triethylene tetramine (55.4 μl, 369 μmol) was dissolved in a mixture of DMF (2.5 ml) and water (2.5 ml). KCN (9.6 mg, 147 μmol) was added and the pH adjusted to 6 by the addition of aqueous HCl. Cyanocobalamin-b-acid (10.0 mg, 7.4 μmol), EDC (5.7 mg, 29 μmol) and HOSu (3.4 mg, 29 μmol) were added. The same amounts of EDC and HOSu were added after 6 hours, 24 hours, 48 hours and 120 hours. Analysis by HPLC (acetate buffer) exhibited slow product formation, reaching a conversion of 75% after 48 hours which did not exceed with prolonged agitation. After stirring for 144 hours, the solvent was removed in vacuo, and the product was purified by preparative HPLC using 0.1% aqueous TFA as a buffer and methanol as solvent b, with a gradient of 1% min "1 starting from buffer to 80%) The product was isolated as cyanocobalamin-b-ethyl-Triamine x 3TFA in a yield of 7.5 mg (55%) ESI-MS: m / z = 743.1 [M + 2] 2+. UV / Vis : nm (emol l "1 cm" 1) = 278.0 (13000), 316.0 (23100), 519.0 (6500), 549.0 (7200).

Example 23: Cyanocobalamin-b-ethyl-Triamine-Re (CO) 3 Cyanocobalamin-b-ethyl-Triamine (5 mg, 2. 7 μmol) and (Et 4 N) 2 [ReBr 3 (CO) 3] (2.2 mg, 2.9 μmol) in phosphate buffer, pH 7.4 (0.1 M, 0.33 ml) at 50 ° C. After 1 hour, the HPLC analysis showed complete conversion of the starting materials into a product. After 4 hours, the reaction mixture was scrubbed to give a product which is, according to the HPLC analysis, a mixture of two stereoisomers in an appropriate ratio of 2/1. The same pattern of two stereoisomers was found in the labeling of cyanocobalamin-b-ethyl-triamine with 99mTC. ESI-MS: m / z = 1755.9 [M + l] +, 878.5 [M + 2] 2+ Example 24: Cyanocobalamin-5'-phosphocholamine A solution of cyanocobalamin (30 mg, 22.14 μmol), DCC (457 mg, 2214 mmol) and N-Fmoc-phosphocolamine (78.9 mg, 217.2 μmol) in dry DMF, (2 ml) and dry pyridine (1 ml) was stirred under N2 atmosphere at room temperature for 24 hours. After the addition of 2 ml of water the precipitated dicyclohexylurea was completely filtered, and the water and pyridine were evaporated at 60 ° C under reduced pressure. The residual solution was diluted to a volume of 8 ml with a 5% piperidine solution of DMF and stirred at room temperature for 2.5 hours. The product was precipitated with diethyl ether, centrifuged and washed several times. The product was precipitated with diethyl ether, centrifuged and washed several times. The crude product was purified by preparative HPLC (gradient: 100%? 20% a, 0%? 80% MeOH in 30 min, a = 0.1% AcOH, 10% acetonitrile in water, 10 ml / min flow, column: M &; N VP 250/21 Nucleosil 100-7 C18). Yield: 82% as a red solid. 31 P NMR (500, CD30D) d 0.00 (s, 1 P), 0.53 (s, 1 P) MS (ESI + MeOH): m / z = 1478 [M + 1] +, 762 [M + 2 + 2 Na] 2+ Example 25: Cyanocobalamin-5'-phosphocolamin-His-OMe Cyanocobalamin-5'-phosphocolamin (50 mg, 33.8 μmol) and methyl-1- (carboxymethyl) -N-Fmoc-histidinate hydrochloride (25 mg, 50.7 μmol) were dissolved. ) in dry DMSO (4 ml) and the pH was adjusted to 6-7 with 24 μl of DIPEA. BOP (45 mg, 101.5 μmol) was added to the solution as a solid and stirred at room temperature. After 1 hour, the pH of the reaction mixture became acidic and adjusted to neutral again. After 5 hours, there was no detectable starting material by analytical HPLC. After precipitation with diethyl ester, the product was subjected to deprotection in a 1: 1 mixture of DMF and piperidine (10 ml) for 1.5 hours. After reprecipitation and purification with preparative HPLC as described for cyanocobalamin-5'-phosphocolamine (Example 24), the product was obtained in 46% yield. NMR 31P (500, D20) d-2.16, -0.37; MS (ESI +, MeOH): m / z 1690 (M + 1) +, 845.6 (M + 2) +.

Example 26: Cyanocobalamin-5'-phosphocolamine-His-Re (CO) 3 The same procedure was used as described for cyanocobalamin-5'-phosphocolamine-His-OMe (Example 25), using the Re (CO) 3 complex of 1- (carboxymethyl) histidinate instead of methyl-1- (carboxymethyl) -N-Fmoc-histidinate hydrochloride. The yield to the crude product was 37%. 31 P NMR (500, D20, 333K) d 0.97, 2.23 MS (ESI +, MeOH): m / z = [M + 1] +, 1945, 929 (fragment). IR (KBr, cm "1): 3400, 2128, 2020, 1901, 1902, 1664, 1499, 1399, 1219, 1073.

Table 2: Structure of cobalamin derivatives of formula (I), X = CN, of the examples Example 27: General labeling procedure Solutions of the precursor [99mTc (OH2) 3 (CO) 3] + of [99p, Tc04] "were prepared using a boranocarbonate kit as described by Alberto et al., (J. Am. Chem. Seo., 123, 3135-3136) A 10 ml glass vial with a rubber stopper was dispensed with N2, 20 μl of a solution of cyanocobalamin derivative (0.01 M in water), 20 μl of MES buffer were added. (1.0 M) and 200 μl of a solution [99mTc (OH2) 3 (CO) 3] + and the reaction mixture was maintained at 75 ° C for 1 to 2 hrs. HPLC analysis with? -detection was performed to build complete conversion of 99mTc species under these conditions, the ester protecting groups in the chelators were cleaved to give the carboxylate complexes. For in vivo studies and for binding studies to transport vectors, very high specific activity was demanded, therefore, 100 μl of labeled solution was injected into an analytical HPLC system to separate the hot from the cold vitamin derivative. The fraction of the product eluted with the highest gamma activity (approximately 300 μl) was diluted with normal saline at a concentration of 10 μCi per animal before injection i. v. The separation conditions were: acetate buffer, column XTerra RP8, gradient: 0% methanol (0 min), 30% methanol (15 min), 100% methanol (25 min) for byd derivatives, and the system TEAP as described by Schibli et al., (Bioconjugate Chem. 2000, 343-351) for the other compounds.

Example 28: Preparation of transcobalamin II (TCII) from rabbit whole blood TCII is purified by chromatography defined on a cyanocobalamin-agarose matrix (Sigma). The gel (5 ml) was first washed with 200 ml of 50 mM Tris / 1 M NaCl, pH 8.0, then with 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 / 1M NaCl 200 ml of centrifuged whole blood was applied twice (first time 5000 rpm, 15 min, second time, 20,000 rpm, 20 min at 4 ° C) to the defined column, and the column was washed sequentially as before. The bound TCII is eluted with 20 ml of 4.0 M guanidine-HCl / 50 mM Tris, pH 8.0, and in a second step with guanidine 7.5 M HCl / 50 mM Tris, pH 8.0. Most of the bound TCII is already eluted with 4 M guanidine-HCl. The waves are extensively dialyzed against H20 for 2 days at 4 ° C. Typical yields are 5-30 nmol / 1 which translates to 7.5-10 μg of TCII (MW: 50 kDa) per rabbit.

Example 29: Preparation of transcobalamin II bacteria (recombinant TCII) The transcobalamin cDNA is expressed in strain FAI13 of E. coli, a K12 derivative with double annihilation in trxB and gor genes, where the cytoplasm forms an oxidizing environment and allows the formation of disulfide. The transcobalamin II protein contains a PreScission protease site followed by the N-terminal histidine tag. The protein is isolated from the soluble fractions of the E. coli extracts using nickel chelation sepharose. The cyanocobalamin of transcobalamin II bound to the chelation column is removed with 8 M urea, and subsequently the transcobalamin II is released by imidazole. In some of the preparations, the His tag is removed by a specific protease.

Example 30: Preparation of transcobalamin I (TCI, haptocorrin) As a source of transcobalamin I, saliva is used from vegetarian human subjects.The saliva is centrifuged at 20,000 rpm, 20 min at 4 ° C, mixed 1: 1 with PBS and sterile filtered. The binding capacity of transcobalamin I is usually 10 ng / ml.

Example 31: Interaction of cyanocobalamin derivatives with TCI and TCII transport proteins) (Figure 1 and Figure 2) The interaction of radiolabelled cyanocobalamin derivatives (57Co, 99Tc, 188Re, l? AIn) is measured by gel shift assays. The radiolabelled cyanocobalamin (0.05 ng to 1 ng) is allowed to react with an excess of transport proteins for 15 minutes at room temperature. This mixture is applied to a gel filtration column (Superdex 75, Amersham Biosciences) in the run buffer PBS and 0.1% Tween 20. The biologically active cyanocobalamin, which binds to transport proteins, moves from a molecular weight of about 1.4 kDa to 40-70 kDa, depending on the transport protein. Titration of the binding capacity of the transport proteins is made with 51Co-cyanocobalamin (ICN Biomedicals GmbH, Germany: 10 μCi / 50 ng).

Example 32: Marking of cyanocobalamin derivatives with 188 Re-tricarbonyl The preparation of 188 Re-tricarbonyl and the labeling of the cyanocobalamin derivatives is carried out in a reaction in the kettle. 7.5 mg of BH3NH3 are mixed with 20 mg of sodium ascorbate, 100 μl of cyanocobalamin derivatives (10-3 M), 900 μl of a generator eluate [188 Re0] (0.9% saline solution, 40 mCi at 270 mCi) , 20 mg of H3P04 (85%) and gasified with carbon monoxide (CO) for 20 minutes. The mixture is heated for half an hour to 2 hours at 60 ° C and for a half to 2 hours at 90 ° C. The labeled cyanocobalamin is separated from the unlabeled one on an inverted phase HPLC column (Waters Xterra RP8) in phosphate buffer with a linear gradient of methanol. The active fraction is diluted with normal saline before injection i. v. at a concentration of 10 μCi per animal for imaging purposes and up to 2 mCi for therapeutic treatments.

Example 33: Sensitivity of spheroids from tumor cells to ionizing radiation In radiobiology, the similarity of radiation response between spheroids and mice that have tumor xenograft makes the spheroids a good alternative model to in vivo irradiation studies. The multicellular tumor spheroids are cultured in rotating flasks with continuous agitation at 37 ° C at an average diameter of 400 μm. The spheroids are harvested, washed in fresh media and then incubated for 1 hour with labeled or cold cyanocobalamin derivatives 188Re in 24-well flat bottom tissue culture plates. The dose range is 1 μCi at 20 μCi per well. Cytotoxicity is assessed for fluorescence viability markers, by measurements of the diameter of the complete spheroids and by a clonogenic assay of spheroids dispersed on semi-solid agar.

Example 34: Biodistribution of radiolabeled cyanocobalamin derivatives in mice (Figures 3, 4, 5, 6) For biodistribution studies with 57Co-cyanocobalamin, 0.2 μCi / in 1 ng of radiolabeled cyanocobalamin are mixed with 180 μl of normal saline and injected iv in balb / c mice that have tumor (Syngeneic mouse B16-F10 melanoma). After a specified time (5 minutes to 24 hours), the animals are sacrificed, the organs are weighed and counted in a gamma counter. For biodistribution studies with 99mTc-labeled cyanocobalamin, 10 μCi / 0.5 ng of the labeled cyanocobalamin are mixed with normal saline and used as before. For biodistribution with cyanocobalamin 1: L1In-labeled, 2 μCi / ng of radiolabeled cyanocobalamin are mixed with normal saline and used as before. To study the effect of deficient vitamin B12 feed, the biodistribution of labeled cyanocobalamin is compared in mice fed normal food with biodistribution in mice fed with vitamin B12 deficient food for a period of 2 weeks.

Example 35: Studies of 188Re-labeled Cyanocobalamin Derivative Therapy in Mice Having Tumor For therapy studies, the syngeneic melanoma tumor is grown in balb / ca mice approximately 200 mg in size (measured by calibrator. Increased (0.1 to 2 mCi) of radiolabelled cyanocobalamin constructs and cold constructs.Tumor volume is assessed by measurement with a calibrator.When the tumor reaches a size of 800 mg, animals are sacrificed.In a series of experiments, Animals are treated with a fractionated regimen: the radiolabeled cyanocobalamin is given 3 times a week.The animals are observed for 60 days for re-growth of the tumors.

Claims (25)

1. CLAIMS 1. Cobalamin derivative, characterized in that (a) it has no binding affinity or has low binding affinity to transcobalamin II, and (b) retains activity as a substitute for vitamin B12.
2. 2. A cobalamin derivative according to claim 1, characterized in that (a) it has less than 20% binding affinity to transcobalamin II compared to the binding affinity of unmodified cobalamin in a binding test, and (b) retains more than 2% of the activity as a substitute for vitamin B12 in a growth trial.
3. 3. A cobalamin derivative according to claim 1, characterized in that (a) it has less than 10% binding affinity to transcobalamin II, compared to the binding affinity of unmodified cobalamin in a binding test, and (b) ) retains more than 10% of activity as a substitute for vitamin B12 in a growth trial.
4. 4. Cobalamin derivative according to claim 1, characterized in that (a) it has less than 5% binding affinity to transcobalamin II compared to the binding affinity of unmodified cobalamin in a binding test, and (b) ) retains more than 10% of activity as a substitute for vitamin B12 in a growth trial.
5. 5. Cobalamin derivative according to any of claims 1 to 4, characterized in that it has a therapeutic and / or diagnostic agent.
6. 6. Cobalamin derivative according to any of claims 1 to 5, characterized in that it has a radioactive metal.
7. 7. Cobalamin derivative according to any of claims 1 to 6, of the formula (I) characterized in that Rb, Rc, Rd and Re, independently of each other, are a chelator-separating group, an antiproliferative therapeutic agent or antibiotic, a sterically demanding organic group with 4 to 20 carbon atoms, or hydrogen; RR is a separator-chelator group or an antiproliferative or antibiotic therapeutic agent, each connected through a Z-linker, or hydrogen; with the proviso that at least three of the substitutes Rb, Rc, Rd, Re and RR are hydrogen and at least one of the residues Rb, Rc, Rd and Re is different from hydrogen; X is a monodentate ligand; and the central cobalt (Co) atom is optionally in the form of a radioactive isotope.
8. 8. Derivative of cobalamin according to claim 7, characterized in that Re is hydrogen.
9. 9. A cobalamin derivative according to claim 7 or 8, characterized in that the separator-chelator group comprises a separator, which is an aliphatic chain of 2 to 10 carbon atoms, wherein one or two carbon atoms can be replaced by nitrogen and / or oxygen atoms and the aliphatic chain may be substituted by hydroxy, oxo or amino, and a chelator, which is a compound having two, three or more donor atoms selected from nitrogen, oxygen and sulfur at a distance such as to bind to a metal atom, and optionally a metal atom.
10. 10. Cobalamin derivative according to claim 9, characterized in that the chelator is selected from the chelators of formula (II) to (IX) HOOC "no HN NH, H di) (III) HOOC NH HOOC COOH N = 0 (VI) (VII) (VIII) (IX) 5 wherein the carboxyl groups may be present as esters.
11. 11. The cobalamin derivative according to any of claims 6 to 10, characterized in that the radioactive metal is 94mTc, 99mTc, 188Re, 186Re, 1: L1In, or 9Y? ß4Cu ¡ßyCu Q 177LU_
12. 12 Cobalamin derivative according to any of claims 7 to 11, characterized in that X is cyano, methyl, hydroxy, aqueous or a 5'-deoxyadenosyl group.
13. 13. Derivative of cobalamin according to claim 12, characterized in that X is cyano.
14. 14. A transcobalamin derivative according to any of claims 7 to 12, characterized in that the central cobalt atom is a 57Co or 60Co radioisotope.
15. 15. A cobalamin derivative according to claim 10, characterized in that R is a chelator-separating group optionally having a metal atom, the separator is an aliphatic chain of 2 to 4 carbon atoms, and the chelator is of the formula ( II), wherein the COOH group is optionally in the form of an ester; Rc, Rd, Re and RR are hydrogen; and X is cyano.
16. 16. Derivative of cobalamin according to claim 15, characterized in that Rb is a chelator-separating group optionally having a metal atom, the separator is an aliphatic chain of 4 carbon atoms, and the chelator is of the formula (II) , wherein the COOH group is in the form of an ethyl ester; Rc, Rd, Re and RR are hydrogen; and X is cyano.
17. 17. A cobalamin derivative according to claim 10, characterized in that Rd is a spacer-chelator group optionally having a metal atom, the spacer is an aliphatic chain of 3 carbon atoms, and the chelator is of the formula (II) , wherein the COOH group is optionally in the form of an ester; Rb, Rc, Re and RR are hydrogen; and X is cyano.
18. 18. Cobalamin derivative according to claim 10, characterized in that Rb is a chelator-separating group optionally having a metal atom, the separator is an aliphatic chain of 2 carbon atoms, and the chelator is of the formula (III); Rc, Rd, Re and RR are hydrogen; and X is cyano.
19. 19. Pharmaceutical composition, characterized in that it comprises a cobalamin derivative according to any of claims 1 to 18.
20. 20. Method of diagnosis of a neoplastic disease or an infection by microorganisms in an animal, characterized in that it comprises: (a) putting the mammal suspected of being afflicted by a neoplastic disease or an infection to a period of a vitamin B12-free diet, and (b) subsequently applying a cobalamin derivative according to any of claims 1 to 18 having a diagnostic agent.
21. 21. Method of treatment of a mammal suffering from a neoplastic disease or infection by microorganisms, characterized in that it comprises: (a) exposing the mammal in need of treatment to a period of vitamin B12-free diet, and (b) subsequently applying a cobalamin derivative according to any of claims 1 to 18, which has a therapeutic agent.
22. 22. Use of a cobalamin derivative according to any of claims 1 to 18, in a method of diagnosing a neoplastic disease or an infection by microorganisms or in a method of treating a mammal suffering from a neoplastic disease or an infection by microorganisms.
23. 23. Use according to claim 22, in the formation of cancer images.
24. 24. Use of a cobalamin derivative according to any of claims 1 to 18 for the manufacture of a pharmaceutical composition for use in a method of diagnosis of a neoplastic disease or an infection by microorganisms or in a method of treating an animal that suffers from a neoplastic disease or infection by microorganisms.
25. 25. Use according to claim 24, of a cobalamin derivative for the preparation of a pharmaceutical composition for the formation of cancer images.