ITPD20090110A1 - COMPLEX AZOTURO MONOCATIONIC DISSYMETRICS OF THE TECHNIQUE - Google Patents

Description

Description of the industrial invention entitled:

â € œMONOCATIONIC AZOTIDE DISSYMMETRIC COMPLEXES OF THE TECHNIQUEâ € Field of invention

The present invention relates to monocation asymmetric azide complexes of radioactive metals, in particular of technetium-99m (<99m> Tc), capable of accumulating in different target organs and / or tissues and in particular in the myocardium and in tumor tissues, and their use in nuclear medical diagnostics as radio tracers, in particular for cardiac and tumor imaging.

State of the art

Many of the radionuclides used in the preparation of radiopharmaceuticals are represented by transition metals. Among these, the <99m> Tc, thanks to its chemical-physical characteristics, is considered the radionuclide of choice for use in nuclear medicine. Technetium, in fact, decays through the emission of a monochromatic γ radiation with an energy of 141 keV. It also has a half-life of 6.02 hours, ie long enough to allow the radiochemist to complete the synthesis of the complex and the doctor to obtain useful images, and short enough for the radiation dose absorbed by the patient to be minimal. Furthermore, the <99m> Tc is readily available at low cost through portable generators of <99> Mo- <99m> Tc, which can be installed in any nuclear medicine center and can be easily incorporated into suitable molecules, thus giving origin to compounds that can be used as diagnostic agents. Among the first technetiated tracers developed and currently in clinical use, the <99m> Tc-Sestamibi or <99m> Tc-MIBI (Cardiolite <®>) and the <99m> Tc-tetrofosmin (Myoview <®>) described for the first time respectively in 1987 in the patent EP0233368 and in 1989 in the patent EP0337654. Both of these <99m> Tc complexes have found extensive use as radio-tracers in myocardial perfusion.

Furthermore, the <99m> Tc-Sestamibi was one of the first tracers to be used in the diagnosis of some types of tumor and validated as a substrate of MDR-Pgp (multidrug resistance proteins) and therefore as a tracer in the in vivo visualization through the use of tomographic techniques of MDR-Pgp expression. MDR-Pgp are particular trans-membrane glycoproteins, ATP dependent, produced by the MDR-1 gene. These proteins regulate the efflux of numerous cytotoxic agents (with different structural and functional characteristics) thus making the tumor cells resistant to the action of different drugs.

This use is possible thanks to the accumulation mechanism of the tracer in the cells within the mitochondrial structures, driven by a process of passive diffusion and supported by an electrical gradient generated by a high mitochondrial transmembrane potential, which determines an irreversible entrapment. Its accumulation is particularly pronounced, up to ten times greater, in sensitive tumor cells that have a higher mitochondrial density than that of normal cells, due to a greater difference in electrical gradient (60 mV). A reduction in the intracellular accumulation of this tracer can be related to various biological phenomena, some of which are associated with the over-expression of the trans-membrane glycoproteins MDR-Pgp. In these cells the accumulation of <99m> Tc-Sestamibi is inversely proportional to the expression levels of Pgp, as demonstrated by the increased uptake of the tracer after exposure of the same to modulators of the MDR-1 glycoprotein (verapamil, cyclosporine A , elacridar and valspodar). By blocking the function of MDR-Pgp, these latter substances induce an increase in the concentration of the tracer inside the cells (Agrawal M, Abraham J, Balis F M, et al. Increased <99m> Tcsestamibi accumulation in normal liver and drug-resistant tumors after the administration of the glycoprotein inhibitor, XR9576 Clin. Cancer Res., 2003; 9: 650â € “656; Sharma V. Radiopharmaceuticals for assessment of multidrug resistance P-glycoprotein-mediated drug transport activity. Bioconjug Chem.

2004; 15: 1464â € “14 74).

The recent discovery of a new and efficient method for the production of Tc-99m complexes containing the terminal multiple bond technetium-nitrogen [<99m> Tcâ ‰ ¡N] <2 +> (WO 89/086557; WO 92/00982; WO 93/01839) has opened the possibility to evaluate the biological activity of completely different classes of Tc-99m azide complexes and to propose them as specific perfusion or receptor radio-drugs. The growing interest in this new category of compounds is linked precisely to the presence of the group [<99m> Tcâ ‰ ¡N] <2+>, which gives the complexes, in which it is contained, a high stability and inertia to substitution, and resistance to redox processes. Consequently, several <99m> Tc (N) -complexes have recently been developed and evaluated as potential diagnostic agents. Examples are bi-substituted symmetrical neutral azide complexes of the type [Tc (N) (DTC) 2] described in WO 90/06137, in which DTC represents a ligand that can be of the dithiocarbamic type, such as for example: diethylidithiocarbamate, of n -propyl dithiocarbamate and N-ethyl, N-ethoxy dithiocarbamate. Other radioactive metal azide complexes proposed as cardiac perfusion agents are disclosed in EP 0949265 and WO02 / 09771. In particular, bi-substituted asymmetric monocation azide complexes of the type [Tc (N) (PNP) (DTC)] <+> are described, in which DTC can be a dithiocarbamic ligand containing linear carbon or ethereal chains and PNP a diphosphinoamine ligand. Among these the complex [<99m> Tc (N) DBODC (PNP5)] <+> [DBODC = bis (N-ethoxyethyl) -dithiocarbamate; PNP5 = bis (dimethoxypropylphosphinoethyl) -ethoxyethylamine], abbreviated to <99m> Tc (N) -DBODC (5) (described in patent application WO02 / 09771) has shown interesting biological properties and is currently undergoing a preliminary clinical study as potential cardiac perfusion agent.

Similarly to what observed for [<99m> Tc] -Sestamibi, <99m> Tc (N) DBODC (PNP5) is irreversibly localized in mitochondrial structures by virtue of a negative transmembrane electrical gradient, as demonstrated by subcellular distribution studies, conducted on isolated myocardium of rat. Furthermore, metabolism studies conducted in order to clarify the mechanism of rapid removal of this complex from non-target organs have shown that <99m> Tc (N) DBODC (PNP5) is eliminated in its native form, and that excretion is It is mediated by Pgp / MDR-Pgp transporters, aspects that allow us to glimpse the possibility of extending the diagnostic applicability of this complex and its derivatives from the cardiology to the oncology field. (C. Bolzati, M. Cavazza-Ceccato, S. Agostini, S. Tokunaga, D. Casara, G. Bandoli Subcellular Distribution and Metabolism Studies of the Potential Myocardial Imaging Agent [<99m> Tc (N) (DBODC) (PNP5 )]. J Nucl Med 2008; 49: 1336â € “1344).

Patent application WO2007 / 083395 describes: an intermediate for the synthesis of diphosphinoamine ligands, numerous diphosphinoamine ligands and some dithiocarbamate ligands, including pyrrolidinedithiocarbamate, piperidindithiocarbamate and 4-ethyl piperazindithiocarbamate complexes [ 99m> Tc (N) (PNP) (DTC)] <+>, in which the ligand DTC can be pyrrolidinedithiocarbamate, piperidindithiocarbamate and 4-ethylpiperazindithiocarbamate. The preparation of these complexes is not described in detail and consequently no tissue uptake test is given, although it is intended to be used as radio-tracers in cardiac imaging.

Summary

The present invention relates to a radiopharmaceutical usable in nuclear medical diagnostics represented by a technetium-99m monocation asymmetric azide complex capable of accumulating in the different organs and in particular in the myocardium and in tumor tissues and usable as a radiotracer.

For these radiopharmaceuticals the active principle is represented by a bi-substituted dissymmetric monocation azide complex, therefore consisting of a group [Tcâ ‰ ¡N] <2+> coordinated with two different polidentate ligands: a diphosphinoamine ligand and a dithiocarbamic ligand alicyclic.

Therefore in a first aspect the subject of the invention are asymmetric monocation azide complexes of technetium-99m represented by the general formula (I)

[Tc <V> (N) (PNP) (DTC-L)] <+> (I)

- Tc <V> (N) Ã ̈ [<99m> Tcâ ‰ ¡N] <2+>;

- PNP is a diphosphinoamine ligand;

- DTC-L is an alicyclic dithiocarbamic binder

and pharmaceutically acceptable salts thereof

in which:

- the PNP ligand is represented by the general formula (II)

R <2>

R1 R <1>

PCH2CH2NCH2CH2P

R <1> R <1> (II)

where is it:

- R <1> is an alkyl or ethereal group, the latter represented by the general formula (III)

- (CH2) mO (CH2) mâ € ™ CH3 (III)

where m is an integer in the range 1â ‰ ¤mâ ‰ ¤4 and mâ € ™ is an integer in the range 0â ‰ ¤mâ € ™ â ‰ ¤3;

- R <2> is a hydrogen atom, an alkyl group or a variously substituted alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, a cyclic ether or an ether group having the meaning of general formula (III) or an ester group of the type â € “(CH2) mâ € ™ â € ™ C (O) Râ € where mâ € ™ â € ™ is an integer within the interval 0â ‰ ¤mâ € ™ â € ™ â ‰ ¤3 and R <â € ™ â € ™> is a hydrogen atom, an alkyl group or a variously substituted alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, or an ethereal group having the meaning of the general formula (III); - the alicyclic dithiocarbamic binder DTC-L is represented by the general formula (IV)

S CN <n> X R <3>

S.

(IV)

where is it:

- n is an integer in the range 3â ‰ ¤nâ ‰ ¤8;

- X is a carbon atom or a heteroatom chosen from N, O, S;

- R <3> is a hydrogen atom, an alkyl group or a variously substituted linear or cyclic alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, or a linear acetal or ketal ether group or cyclic, a pharmacophore group or a biomolecule with the proviso that the DTC-L ligand is not pyrrolidinedithiocarbamate, piperidindithiocarbamate or 4-ethyl piperazindithiocarbamate.

Preferably the variously substituted alkyl groups represented by R <3> of the ligand DTC-L are represented by the following general formulas (V), (VI), (VII):

R <4>

R <5> (V)

where R <4> and R <5> are independently of each other a variously substituted alkyl or aryl group, also containing heteroatoms selected from N, O, S, variously spaced;

R <6>

(CH2) pY ((CH2) p'CH3) p "(VI)

where R <6> is a variously substituted alkyl or aryl group, also containing heteroatoms selected from N, O, S, variously spaced,

p is an integer in the range 0â ‰ ¤pâ ‰ ¤4; Y is a carbon atom or a heteroatom such as N, O, S, pâ € ™ is an integer within the interval 0â ‰ ¤pâ € ™ â ‰ ¤4, pâ € ™ â € ™ Ã ̈ an integer in the interval 1â ‰ ¤pâ € â ‰ ¤3;

(CH2) pY ((CH2) p'CH3) p "

<(CH2) pY ((CH2) p'CH3) p "> (VII)

p is an integer within the range 0â ‰ ¤pâ ‰ ¤4, Y is a carbon atom or a heteroatom chosen from N, O, S, pâ € ™ is an integer within the range 0â ‰ ¤pâ € ™ â ‰ ¤4, pâ € ™ â € ™ is an integer in the interval 1â ‰ ¤pâ € ™ â € ™ â ‰ ¤3.

When R <3> is a pharmacophore group, this is preferably chosen from the group consisting of 2 methoxyphenyl piperazine, derivatives of benzylguanidine, and of norepinephrine, while when it is a biomolecule, this is preferably chosen from the group consisting of bioactive peptides , fatty acids.

The azide complexes of technetium-99m of general formula (I) have been shown to possess biological activities suitable for their use as radio-tracers.

Therefore, a further object of the invention is the use of azide complexes of technetium-99m of general formula (I) as radio-tracers for diagnostic purposes, in particular for cardiac and tumor imaging.

Detailed description of the invention

The present invention relates to radio-drugs for diagnostic use consisting of dissymmetric bi-substituted azide monocation complexes of <99m> Tc, in which the [Tcâ ‰ ¡N] <2+> group is coordinated with two different polydentate ligands: a ligand diphosphinoamine and an alicyclic dithiocarbamic ligand, able to reach a high and persistent cardiac localization and high heart / lung and heart / liver ratios and therefore can be used in the first instance in cardiac imaging. Furthermore, these compounds have been shown to be able to irreversibly localize in the mitochondrial structures of cancer cells, thus allowing their use as radio-tracers to be extended to the oncological field as well.

The monocation asymmetric azide complexes of Technetium-99m of general formula (I) object of the present invention, of which the corresponding spatial configuration is reported below, are characterized by the presence of a group [<99m> Tcâ ‰ ¡N] <2+ > coordinated with two different poly-toothed ligands: a diphosphinoamine ligand coordinated with the metal by means of the two pi-ï € acceptor phosphorus atoms; a mononegative bidentate ligand such as an alicyclic dithiocarbamate of formula IV.

R <1>

R <1> N

P S

Tc CN <n>

R <1> X R <3>

R <1> P S

No.

R <2>

In the present invention the diphosphinoamine ligand is preferably a compound of formula (II) as defined above, while the alicyclic dithiocarbamate ligand and some of its derivatives are according to the general formula (IV) with the meanings defined above.

Specific examples of PNP diphosphinoamine and DTC-L dithiocarbamic ligands according to the present invention, and preferred for the purposes thereof, are reported below:

- diphosphinoamine binders:

bis (dimethoxypropylphosphinoethyl) methoxyethylamine,

bis (dimethoxypropylphosphinoethyl) ethoxyethylamine,

bis (dimethoxypropylphosphinoethyl) methoxypropylamine,

bis (dimethoxypropylphosphinoethyl) ethoxypropylamine,

bis(dietossipropilfosfinoetil)metossietilamina, bis(dietossipropilfosfinoetil)etossietilamina, bis(dietossipropilfosfinoetil)metossipropilamina, bis(dietossipropilfosfinoetil)etossipropilamina, bis(dimetossietilfosfinoetil)etossietilamina, bis(dimetossietilfosfinoetil)metossietilamina, bis(dimetossietilfosfinoetil)metossipropilamina, bis(dimetossietilfosfinoetil)etossipropilamina, bis( dietossietilfosfinoetil)metossietilamina, bis(dietossietilfosfinoetil)etossietilamina, bis(dietossietilfosfinoetil)metossipropilamina, bis(dietossietilfosfinoetil)etossipropilamina, bis(dimetilfosfinoetil)metossietilamina, bis(dimetilfosfinoetil)etossietilamina, bis(dimetilfosfinoetil)metossipropilamina, bis(dimetilfosfinoetil)etossipropilamina, bis(dietilfosfinoetil) methoxyethylamine, bis (diethylphosphinoethyl) ethoxyethylamine, bis (diethylphosphinoethyl) methoxypropylamine, bis (diethylphosphinoethyl) ethoxypropylamine, bis (dipropylphosphinoethyl) methoxyethylamine, bis (dipropylphosphinoethyl) ethoxyethylamine, bis (dipropyl) etil)metossipropilamina, bis(dipropilfosfinoetil)etossipropilamina, bis(diisopropilfosfinoetil)metossietilamina, bis(diisopropilfosfinoetil)etossietilamina, bis(diisopropilfosfinoetil)metossipropilamina, bis(diisopropilfosfinoetil)etossipropilamina, bis(dimetilfosfinoetil)metilamina, bis(dimetilfosfinoetil)etilamina, bis(dimetilfosfinoetil) propylamine,

bis (diethylphosphinoethyl) methylamine,

bis (diethylphosphinoethyl) ethylamine, bis (diethylphosphinoethyl) propylamine, bis (dipropylphosphinoethyl) methylamine, bis (dipropylphosphinoethyl) ethylamine, bis (dipropylphosphinoethyl) propylamine, bis (diisopropylphosphinoethyl) methylamine, bis (dipropylphosphinoethyl) ethylamine, bis (dipropylphosphinoethyl) propylamine, bis (diisopropylphosphinoethyl) methyl

- alicyclic dithiocarbamate binders:

azepine dithiocarbamate,

azocane dithiocarbamate,

1-methyl- [1,4] diazepan dithiocarbamate,

3- (dimethylamino) pyrrolidine dithiocarbamate,

3- (diethylamino) pyrrolidine dithiocarbamate,

4-isopropylpiperidine dithiocarbamate,

4,4-dimethoxy piperidine dithiocarbamate,

4,4-diethoxyperidine dithiocarbamate,

4,4-diethoxyethylpiperidine dithiocarbamate,

4,4-diethoxypropylpiperidine dithiocarbamate,

4,4-bis- (2-ethoxy-ethoxy) -piperidine dithiocarbamate, 1-ethylomopiperazine dithiocarbamate,

morpholin dithiocarbamate,

thiomorpholin dithiocarbamate,

2,6-dimethylmorpholin dithiocarbamate,

4 morpholin piperidine dithiocarbamate,

4 N (2 methoxyphenyl) piperazine dithiocarbamate, 4,4-piperidindiol dithiocarbamate,

2,2,6,6-tetramethyl-4-piperidinone dithiocarbamate, 4-piperidinol dithiocarbamate,

2,2,6,6-tetramethyl-4-piperidinol dithiocarbamate, 1,4-dioxo-8-azaspiro [4,5] decan dithiocarbamate, (7,7,9,9-tetramethyl-1,4-dioxa-8 -azaspiro [4.5] dec-2-yl) methanol dithiocarbamate,

2-aza-5-oxabicyclo (2.2.1) -eptane dithiocarbamate,

4,7-epoxy-7-methyl-2,3,3a, 4,5,6,7,7a-octahydro-1h-isoindol dithiocarbamate. The most preferred of the PNP binders are:

bis (dimethoxypropylphosphinoethyl) methoxyethylamine, bis (dimethoxypropylphosphinoethyl) ethoxyethylamine, bis (diethoxypropylphosphinoethyl) methoxyethylamine, bis (diethoxyethylamine, bis (dimethoxyethylphosphinoethyl) ethoxyethinoethyl) ethoxyethinoethyl

while the most preferred among the DTC-L alicyclic ligands are:

morpholin dithiocarbamate,

4 N (2 methoxy phenyl) piperazine dithiocarbamate,

1,4-dioxo-8-azaspiro [4,5] decan dithiocarbamate,

thiomorpholin dithiocarbamate,

azepine dithiocarbamate,

azocane dithiocarbamate,

1-methyl- [1,4] diazepan dithiocarbamate.

Both binders are prepared according to preparation methods known to a person skilled in the art.

In particular, the PNP diphosphinoamine ligands that can be used for the azide complexes of technetium-99m according to the invention have been synthesized in accordance with what is described in the previously cited EP0949265 patent, while the dithiocarbamate ligands are prepared through a classical synthesis method which involves the treatment in a basic environment for soda and at low temperature of the appropriately selected cyclic amine with carbon disulfide (Pasqualini, R., Duatti, A., Bellande, E., Comazzi, V., Brucato, V., Hoffschir, D. , Fagret, D., and Comet, M. (1994) Bis (dithiocarbamate) nitride technetium-99m radiopharmaceuticals: a class of neutral myocardial imaging agents. J. Nucl. Med.

35, 334-341; Zhang, J., Wang, X., Li, C. Y., (2002) Synthesis and biodistribution of a new <99m> Tc nitrido complex for cerebral imaging. J. Nucl. Med. Biol. 29, 665-669).

According to a first method, the azide complexes of <99m> Tc can be prepared in a single step by adding the pertechnetate ion to a reaction mixture consisting of the selected PNP and DTC-L ligands, a reducing agent, a donor of N <groups. 3->, a stabilizer, a solubilizer and a pH regulator.

According to another method, the azide complexes of <99m> Tc can be prepared with a process comprising the following steps:

- preparation of a mixture of intermediate azides containing [<99m> Tc (N)] <2+> by reduction of the pertechnetate ion in the presence of a reducing agent, a donor of N <3-> groups, a stabilizer and a regulator of pH at room temperature;

- addition to the mixture containing the [<99m> Tc (N)] <2+> of the selected binders PNP and DTC-L at a temperature between RT and 100 ° C in the presence of a solubilizing agent.

The preparation method of the <99m> Tc complexes according to the invention based on the two phases indicated above can also provide that it is azide [<99m> Tc (N)] <2+> to be added to the reaction mixture substantially consisting of the binders PNP and DTC-L and the solubilizing agent or vice versa that the binders are added to the azide.

In detail, the first step allows the synthesis, after 30 min at room temperature, of a mixture of intermediate complex azide containing the core [<99m> Tc (N)] <2+>, obtained following the reduction of the pertechnetate ion carried out in the presence of a suitable reducing agent, such as for example tin chloride (SnCl2), and of a donor of N <3-> groups, such as for example succinyl dihydrazide (SDH) and ethylenediaminetetraacetic acid (EDTA). The presence of EDTA is necessary in order to avoid, after the addition of the dithiocarbamic ligand, the precipitation of the neutral complex Sn (DBODC) 2. The preparation is then completed by the simultaneous addition of the PNP and DTC ligands to the same reaction vial in such a way as to obtain, after 30 min at 80-100 ° C, the quantitative formation of the final product. Alternatively, the complexes can be prepared using a simplified synthesis route which sees the production of the final complex in high yield following the addition of the pertechnetate ion to a vial containing all the reagents. The azide complexes of technetium-99m according to the invention can also be produced extemporaneously ready for use using a kit equipped with the components necessary for the formation of the complexes themselves. Typically for this purpose it is possible to have a preparation of the complexes in two steps which involves the use in sequence of a first vial containing a donor of the azide group, a reducing agent, a stabilizer and a pH regulator and a second vial containing the two different binders, a diphosphinoamine compound and an alicyclic dithiocarbamic compound DTC-L, dissolved in a suitable solvent.

The pertechnetate eluted from a suitable generator is added to the first vial. A physiological solution is added to the second vial to dissolve the contents and an appropriate amount of the latter is added to the first vial. The final product is obtained after heating at 80-100 ° C.

Alternatively, all components can be prepared in a single vial, to which the pertechnetate (Na <99m> TcO4) eluted by a suitable generator is added. The final product is obtained after heating at 80-100 ° C.

The donor of the azide group is an essential component in the formation of the azide complex of <99m> Tc: dithiocarbazic acid and its derivatives, hydrazine and its derivatives are used for this purpose.

As reducing agent are used stannous chloride, hydrogen sulfite salts, borohydride salts, aliphatic and aromatic tertiary amines and their derivatives, and in particular stannous chloride, sodium hydrogen sulfite, sodium boron hydride, aliphatic tertiary phosphines and tris (m-sulfonate phenyl) phosphines.

As stabilizer, ethylenediamino tetraacetic acid (EDTA) or ethylenetriamino pentaacetic acid (DTPA) are preferable.

Phosphate buffers, carbonate buffers and sodium hydroxide are preferred as pH regulators.

Depending on the case, a solubility promoter or a surfactant can be added in order to facilitate the reaction kinetics and improve the solubility and bioavailability of the final complex.

The contents of the aforementioned vials can be kept in solution or in the form of lyophilisate, facilitating their conservation and use.

The dissymmetric azide complexes of <99m> Tc according to the invention and obtained following the coordination of these two different bidentate ligands PNP and DTC-L with the same metal center are characterized by a high stability in vitro and in vivo and they have proved capable of fulfilling the purposes of the present invention and possessing better biodistribution and subcellular uptake characteristics than known and clinically used <99m> Tc complexes.

In fact, these complexes have demonstrated a pharmacokinetic profile in the different organs through uptake mechanisms of subcellular distribution and pharmacokinetics quite similar, if not superior, to those of other monocation complexes such as complexes with DTC = pyrrolidinedithiocarbamate, piperidindithiocarbamate or 4-ethyl -piperazindithiocarbamate and PNP = diphosphinoamines mentioned in patent application WO2007 / 083395, <99m> Tc (N) DBODC (PNP5) described in patent application WO02 / 09771, [<99m> Tc] -Sestamibi, and [<99m> Tc ] -Tetrofosmin.

Furthermore, the complexes according to the invention are also picked up by tumor cells with much higher accumulation percentages than those presented by the reference complexes, for which this uptake is known (i.e. [<99m> Tc] -Sestamibi, with this demonstrating that they can actually be used in the imaging of tumor tissues and in the evaluation of the expression and functionality of MDR-Pgp.

These aspects therefore support the possibility of extending the diagnostic applicability of these new complexes and other derivatives from the cardiological field to the oncological field in the visualization and monitoring of neoplastic forms characterized by a high mitochondrial density, as well as in the study of mediated drug resistance. and not from Pgps.

In fact, from a clinical point of view, the in vivo evaluation of the expression and functionality of MDR-Pgp allows to: select patients who can benefit from therapeutic treatments (eg with chemotherapy); distinguish whether the drug resistance that has been established is mediated by Pgp or not and then select patients who can benefit from treatment with Pgp inhibitors. Furthermore, functional measures that define the state of MDR-Pgp through the application of non-invasive techniques such as SPECT represent a greater advantage over conventional in vitro assessment techniques that require biopsy samples.

In fact, the results of the biodistribution studies show that the incorporation of alicyclic dithiocarbamate ligands, as defined by the meanings of the general formula (I), in the structure of the molecular fragment [Tc (N) (PNP)] <2+> allows to increase cardiac accumulation in a short time from the administration of this new class of complexes, making them particularly suitable for the development of diagnostic agents with superior characteristics compared to those currently on the market, as well as this incorporation is particularly significant in the uptake in tumor cells of these complex. Their preferential uptake at the subcellular level in the mitochondria also makes them particularly useful for diagnostic purposes in the oncology sector, where they can be used in the evaluation of any drug resistance mediated by MDR-Pgp and therefore to select patients who can benefit from treatment with inhibitors. of the Pgp.

Therefore, for the purposes of the invention, the azide complexes according to the invention can be formulated in radiopharmaceuticals to be used for diagnostic purposes, combining in an aseptic environment the appropriate reagents (such as donors of the azide group, reducing agents, stabilizers, chelators) with additives acceptable for pharmaceutical use, such as for example: stabilizers such as ascorbic acid and paraaminobenzoates; pH correctors, such as carbonate and phosphate buffers; solubilizers, such as cyclodextrins and meglumins; and excipients such as D-mannitol. Furthermore, these radiopharmaceuticals can be prepared in the form of kits to be reconstituted at the time of use by adding a fresh solution of sodium pertechnetate (Na <99m> TcO4) eluted by a suitable generator to the selected binders.

The complexes of technetium-99m of general formula (I) object of the invention can be used as radiopharmaceuticals that can be administered by conventional parenteral routes, such as intravenous at the dosage which can be determined by the levels of radioactivity necessary for the acquisition of the image by means of appropriate instrumentation and parameters attributable to the characteristics of the patient such as: age, body weight, sex, pathology according to what is known to a person skilled in the art. When a radiopharmaceutical containing <99m> Tc is administered for human use its dosage is between 37 and 1850 MBq, preferably 185-740 MBq expressing the radioactivity of <99m> Tc.

The present invention is illustrated below through some detailed examples, but not limited to specific examples, and by biodistribution and subcellular uptake studies in human tumor cell lines.

Experimental part

Chemical Reagents, the analytical methods used and their abbreviations are listed below. The complexes with DTC-L = pyrrolidinedithiocarbamate, piperidindithiocarbamate, 4-N-ethyl piperazine dithiocarbamate were prepared for the purpose of comparison with the azide complex of general formula (I) object of the invention.

Amino diphosphin compounds (PNPx, x = 3, 5, 7, 10)

PNP3 abbreviated as (3) = bis (dimethoxypropylphosphinoethyl) methoxyethylamine;

PNP5 abbreviated as (5) = bis (dimethoxypropylphosphinoethyl) ethoxyethylamine;

PNP7 abbreviated as (7) = bis (dimethoxyethylphosphinoethyl) ethoxyethylamine;

PNP10 abbreviated as (10) = bis (dimethoxyethylphosphinoethyl) methoxyethylamine have been synthesized following the procedure cited in patent application WO2007 / 083395.

Dithiocarbamic binders (DTC-Ln, n = 1-5, 17, 21-24):

DTC-L1 = pyrrolidinedithiocarbamate;

DTC-L2 = piperidindithiocarbamate;

DTC-L3 = morpholin dithiocarbamate;

DTC-L4 = 4 N ethyl piperazine dithiocarbamate;

DTC-L5 = 4 N (2methoxy phenyl) piperazine dithiocarbamate;

DTC-L17 = 1,4-dioxo-8-azaspiro [4,5] decan dithiocarbamate.

DTC-L21 = thiomorpholin dithiocarbamate,

DTC-L22 = azepine dithiocarbamate,

DTC-L23 = azocane dithiocarbamate,

DTC-L24 = 1-methyl- [1,4] diazepan dithiocarbamate.

In detail, in a two-necked flask immersed in a suitably cooled water bath, 1.6 mmoles of an aqueous solution of NaOH are slowly added to 1.6 moles of cyclic amine suspended in 10 mL of distilled water. of carbon disulfide (100 µL, 4 x 25µL) drop by drop.

The reaction mixture is then left to stir at room temperature for 1 h. The formation of a yellow-orange solution is observed. The solvent is removed and the resulting crude solid residue is recrystallized from ethanol and ether. White crystals of the corresponding dithiocarbamate are obtained.

DTC-L1 is commercially sourced and was purchased by Sigma-Aldrich. DTC-L2 piperidindithiocarbamate. Yield = 41%. Anal. calculated for C6H10NNaS2 (PM 183.27): C 39.32, H 5.50, N 7.64, S 34.99 Found: C 40.97, H 6.01, N 7.56, S 35.24. IR (KBr cm <-1>) 2930 (-CH2); 2852 (-CH2); 1449 (CH2); 1409 (C-N); 1218 (C = S). <1> H NMR (D2O) Î ́: 4.19-4.15 (t, 4H, J = 5.3 Hz -CH2CH2CH2N-), 1.72-1.65 (m, 4H, -CH2CH2CH2N-), 1.58- 1.64 (m, 2H, -CH2CH2CH2N-). <13> C NMR (D2O) Î ́: 202.2 (C = S), 52.6 (CH2CH2CH2N-), 25.2 (-CH2CH2CH2N-), 21.9 (CH2CH2CH2N-).

DTC-L3 morpholin dithiocarbamate. Yield = 48%. Anal. Calculated for C5H8NNaOS2 (PM 185.25): C 32.42, H 4.35, N 7.56, O 8.64, S 34.62 Found: C 31.97, H 4.58, N 7.06, O 8.94, S 35.24. IR (KBr cm <-1>) 2930 (-CH2); 1418 (C-N); 1219 (C = S). <1> H NMR (D2O) Î ́: 4.24 (m, 4H, -OCH2CH2N-), 3.62 (m, 4H, -OCH2CH2N-). <13> C NMR (D2O) Î ́ : 212.0 (C = S), 68.91 (-OCH2CH2N-), 54.15 (-OCH2CH2N-).

DTC-L4 4-N-ethyl piperazine dithiocarbamate. Yield = 60%. Anal. calculated for C7H13N2NaS2 (PM 212.04): C 39.60, H 6.17, N 13.19, S 30.21. Found: C 40.97, H 7.58, N 13.86, S 30.54. IR (KBr cm <-1>) 2969 (-CH3); 2818 (-CH2); 1415 (C-N); 1227 (C = S). <1> H NMR (D2O) Î ́: 4.32-4.16 (m, b, 4H, CH2CH2N (CS2)), 2.52 (m, b, 4H (CH3CH2) NCH2CH2N-), 2.47- 2.39 (q, b, 2H, J = 7.3 Hz CH3CH2N <), 1.05-0.98 (t, 3H, CH3CH2N <). <13> C NMR (D2O) Î ́: 202.10 (C = S), 50, 91 ((CH3CH2) NCH2CH2), 50.75 (CH3CH2N <), 49.81 (-CH2CH2N (CS2)), 10.00 (CH3CH2N <).

DTC-L5 4-N- (2methoxy phenyl) piperazine dithiocarbamate. Yield = 76%. Anal. Calculated for C12H15N2NaOS2 (PM 290.05): C 49.63, H 5.21, N 9.65, O 5.51, S 22.08. Found: C 50.97, H 5.99, N 9.89, O 5.94, S 23.24. IR (KBr cm <-1>) 1464 (F, C-N); 1236 (F, C = S). <1> H NMR (D2O) Î ́: 7.01-6.96 (m, 4H, Ar), 4.37 (m, 4H, CH2CH2N (CS2)), 3.74 (s, 3H, OCH3 ), 2.95 (m, 4H, CH2CH2N-Ar). <13> C NMR (D2O) Î ́: 214.10 (C = S), 155.21, 143.42 (2C, -Ar) 125.35-115.53 (4C, -Ar ), 62.03 (2C, CH2CH2N (CS2)), 57.95 (- OCH3), 53.21 (-CH2CH2N-Ar).

DTC-L17 1,4-dioxo-8-azaspiro [4,5] decan dithiocarbamate. Yield = 69%.

<1> H-NMR (D2O): Î ́ (ppm): 4.40 (m, 4H, -CH2NCH2-); 4.07 (s, 4H, -OCH2CH2O-);

1.84 (m, 4H, -CH2NCH2-). <13> C-NMR (D2O): Î ́ (ppm): 208.66 (s, S2CN); 107.50 (s, CO2); 64.47 (s, -OC-CO-); 49,413 (s, -C-N-C-); 34.09 (s, -C-C-C-).

DTC-L21 thiomorpholin dithiocarbamate. Yield 60%.

<1> H-NMR, (D2O): Î ́ (ppm) = 4.6 (m, 4H, -CH2NCH2-); 2.7 (m, 4H, -CH2SCH2-). <13> C-NMR, (D2O): Î ́ (ppm) = 209.09 (s, (S2) CN); 54.13 (s, H2C-N-CH2); 27.07 (s, -CH2-S-CH2-).

DTC-L22 azepine dithiocarbamate. Yield 62%.

<1> H-NMR, (D2O): Î ́ (ppm) = 4.15 (t, 4H, -CH2-N-CH2-); 1.8 (m, 4H, -N-CH2CH2-CH2CH2-CH2CH2-N-); 1.55 (m, 4H -N-CH2CH2-CH2CH2-CH2CH2-N-). <13> C-NMR, (D2O): Î ́ (ppm) = 206.68 (s, (S2) CN); 55.75 (s, H2C-N-CH2); 26.81 (s, -N-CH2CH2-CH2CH2-CH2CH2-N-); 26,359 (s, -N-CH2CH2-CH2CH2-CH2CH2-N-).

DTC-L23 azocane dithiocarbamate. Yield 63%.

<1> H-NMR, (D2O): Î ́ (ppm) = 4.10 (t, 4H, -CH2-N-CH2-); 1.85 (m, 4H, -CH2-CH2-N-CH2CH2-); 1.5 (m, 6H). <13> C-NMR, (D2O): Î ́ (ppm) = 207.08 (s, (S2) CN); 56.34 (s, H2C-N-CH2); 26.82 (s, -N-CH2CH2CH2CH2-); 26.25 (s, 2C -N-CH2CH2CH2CH2-); 25.09 (s, 2C, -N-CH2CH2CH2CH2-).

DTC-L24 1-methyl- [1,4] diazepan dithiocarbamate. Yield 54%.

<1> H-NMR, (D2O): Î ́ (ppm) = 2.05 (m, 2H, -N-CH2CH2CH2-N (CH3) -); 2.45 (s, 3H, -CH3); 2.75 (m, 2H, (S2) CN-CH2CH2-N (CH3) -); 2.9 (m, 2H, (S2) CN-CH2CH2-N (CH3) -); 4.2 (m, 2H, -N-CH2CH2CH2-N (CH3) -); 4.35 (m, 2H, -N-CH2CH2CH2-N (CH3) -). <13> C-NMR, (D2O): Î ́ (ppm) = 209.16 (s, S2CN-); 45.00 (s, -CH3); 25.73 (s, -N-CH2CH2CH2-N (CH3) -); 52.91 (s, -N-CH2CH2CH2-N (CH3) -); 53.68 (s, (S2) CN-CH2CH2-N (CH3) -); 55.89 (s, (S2) CN-CH2CH2-N (CH3) -); 56.20 (s, (S2) CN-CH2CH2-N (CH3) -).

Preparation of Dissymmetric Complexes [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> (x = 3,5,7,10) (n = 1-5, 17, 21-24 ).

Dissymmetric complexes of the type [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> are obtained in high yield (90-98%) through the synthetic procedures described below; in all cases, the pH, measured at the end of the reaction, is in the range 6.5-7.5. The radiochemical yields (RCY) of the products obtained through the different synthetic procedures

they are between 90-98%.

Method A: In a vial containing 5.0 mg of succinyl dihydrazide (SDH), 5 mg of

tetraacetic ethylenediamine acid (EDTA), 0.10 mg SnCl2 suspended in 0.10 ml of saline solution and 1.0 ml of ethyl alcohol (EtOH) 0.250 ml of [Na

<99m> TcO4] (50.0 MBqâˆ'3.0 GBq). The mixture is incubated at room temperature for 15 min. Subsequently, 1.0 mg of the diphosphinoamine binder PNPx dissolved 0.10 ml in EtOH are added to the solution containing the mixture of [<99m> Tcâ ‰ ¡N] mix <2+> intermediates. The solution is kept at room temperature for 30 min. Finally, 2 mg of DTC-Ln dithiocarbamic binder dissolved in 0.20 ml of saline solution are added to the reaction mixture. In all cases, the radiochemical purity of the final complex evaluated, after 30 min at 80 ° C, by TLC and HPLC chromatography is always greater than 90%.

Method B: to the mixture of [<99m> Tcâ ‰ ¡N] mix <2+> prepared as indicated above, 2 mg of DTC-Ln dissolved in 0.20 ml of saline solution are added. The solution is incubated at room temperature for 30 min. The reaction mixture is finally treated with 1.0 mg of a PNPx binder dissolved in 0.10 ml of EtOH and heated at 80 ° C for 30 min. The products obtained have the same chromatographic profile as those prepared with method A. In this case, the RCY of the final complex is between 85-90%.

Method C: 1.0 mg of PNPx dissolved in 0.10 ml of EtOH and 2 mg of DTC-Ln dissolved in 0.20 ml of saline solution. RCY, determined after 30 min at 80 ° C by TLC and HPLC, is always greater than 95%.

Method D: In a vial containing 5.0 mg of succinyl dihydrazide (SDH), 5 mg of ethylenediamine tetraacetic acid (EDTA), 0.10 mg SnCl2 suspended in 0.10 ml of saline, 1.0 mg of the aminodiphosphine binder PNPx dissolved in 0.10 ml in EtOH and 1.0 ml of EtOH 0.250 ml of Na [<99m> TcO4] (50.0 MBqâˆ'3.0 GBq) are added. The mixture is incubated at room temperature for 15 min. Subsequently, 2 mg of DTC-Ln binder dissolved in 0.20 ml of saline solution are added to the solution containing the mixture of [<99m> Tc (N) (PNPx)] <2+> intermediates. In all cases, the radiochemical purity of the final complex evaluated, after 30 min at 80 ° C, by TLC and HPLC chromatography is always greater than 90%. Method E: In a vial containing 5.0 mg of succinyl dihydrazide (SDH), 5 mg of ethylenediamine tetraacetic acid (EDTA), 0.10 mg SnCl2 suspended in 0.10 ml of saline, and 1.0 ml of EtOH 0.250 ml of Na [<99m > TcO4] (50.0 MBqâˆ'3.0 GBq), 1.0 mg of the aminodiphosphinic binder PNPx dissolved 0.10 ml in EtOH and 2 mg of DTC-Ln binder dissolved in 0.20 ml of saline solution. In all cases, the radiochemical purity of the final complex evaluated, after 30 min at 80 ° C, by TLC and HPLC chromatography is always greater than 90%.

Method F: In a vial containing 5.0 mg of succinyl dihydrazide (SDH), 5 mg of ethylenediamine tetraacetic acid (EDTA) and 0.10 mg SnCl2 suspended in 0.10 ml of saline, 1-2 ml of Na [<99m> TcO4] are added (50.0 MBqâˆ'3.0 GBq), 1.0 mg of the PNPx aminodiphosphinic binder dissolved 0.5 ml of a saline solution containing 2 mg / ml of γ-cyclodextrin and 2 mg of DTC-Ln binder dissolved in 0.20 ml of saline. In all cases, the radiochemical purity of the final complex evaluated, after 30 min at 80 ° C, by TLC and HPLC chromatography is always greater than 90%.

Chromatographic analysis:

Dissymmetric complexes of the type [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> prepared with the above-mentioned ligands and as described below were analyzed by TLC (Thin-layer chromatography) ) and HPLC (high performance liquid chromatography).

TLC analyzes were carried out using C18 F254S (Merck, Milan, Italy) as the fixed phase and a salt / methyl alcohol (MeOH) / tetrahydrofuran (THF) / acetic acid (HAc) (g) (2/8 / 1/1) or alternatively SiO2F254 plates (Merck, Milan, Italy) and a mixture of ethyl alcohol (EtOH) / CHCl3 / toluene (Tol) / NH4Ac 0.5 M (5/3/3/1). The activity on the plates is detected and measured using a Cyclone <ï £ ̈> Instrument scanner equipped with phosphorus imaging screens and OptiQuant image analysis software (Packard, Meridian, CT).

HPLC analyzes were conducted using a Beckman System Gold instrument equipped with a Model 126 pump system, 210A injection valve, Module 166 UV detector, and Model B-FC-3200 Bioscan radioactive detector. 100 µL loop. Analyzes are conducted using a Beckman ODS reversed phase column (5 µm, 4.6 × 45 mm), and a Beckman ODS reverse phase column (5 µm, 4.6 x 250 mm).

Complex Purification [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> (x = 3,5,7,10) (n = 1-5, 17, 21-24)

Before starting the in vitro and in vivo studies in order to eliminate the influence of any impurities, the complexes are purified as follows:

Method A. A SEP PAK C-18 (Waters) cartridge is conditioned with ethyl alcohol (EtOH) and deionized H2O. The reaction mixture containing the radium complex [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> is diluted with deionized water and loaded on the SEP PAK C-18, (approximately 90% of the initial activity is retained by the resin). The column is then washed in succession with H2O and EtOH 35%.

The complex (for 90% of the charged activity) is eluted with 1 ml of a 9/1 mix of EtOH / saline solution. After purification, the radiochemical purity (RCP) of the complexes is> 95%.

Method B. A cartridge containing a SEP PAK CM (Waters) cation exchange resin is conditioned with deionized H2O. The reaction mixture containing the radium complex [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> is diluted with deionized water and loaded on the SEP PAK CM, (60-90% of the € ™ initial activity is retained by the resin). The column is then washed in succession with H2O and EtOH. The complex (for 90% of the charged activity) is eluted with a mixture of EtOH / saline solution (9/1). After purification, the CPR of the radio complexes is> 95%.

Method C: It involves the serial use of method A and B described above.

Logkâ € ™ 0 measurements.

The evaluation of the lipophilic characteristics of the radio-complexes was performed through the HPLC chromatographic determination of the corresponding Log Kâ € ™ 0 value.

Logk'0 (the value of Log Kâ € ™ at 0% organic solvent) is extrapolated from Logk ', where k' is the capacity factor that measures the partition of the compound between a non-polar stationary phase and a mobile phase polar. Logk 'values are determined using an HPLC, equipped with Beckman ODS C18 pre-column, (5 µm, 4.6 x 45 mm) and Beckman ODS C18 column, (5 µm, 4.6 x 250 mm). Mobile phase different compositions of a mixture of phosphate buffer H2PO4 <-> / HPO4 <2-> 0.02 M, pH 7.4 and methyl alcohol (MeOH). Flow 1 mL / min. For each compound it is necessary to measure at least three retention time values obtained at three different concentrations of methanol in the mobile phase.

Logk'0Ã is extrapolated from the linear part of the curve

Log k '= a bC

where C is the concentration of the organic solvent and Log k 'is the Log [(tr-t0) / t0] [tr retention time of the compounds (min) and t0 dead time corresponding to the retention time of the Na pertechnetate <99m> TcO4 (2.77 min.)].

The results are reported in Table 1.

For comparison the value of Logk'0 of the known complexes [<99m> TcN (DTCL1) (3)] <+>, [<99m> TcN (DTCL2) (3)] <+>, [<99m> TcN (DTCL2) (5)] <+>, [<99m> TcN (DTCL2) (10)] <+>, [<99m> TcN (DTCL4) (3)] <+>, <99m> Tc (N) - DBODC ( 3/5) (gold standard of the series, described in patent application WO02 / 09771) and complexes known and already used in the clinic [<99m> Tc] -MIBI are obtained under the same conditions.

Table 1

TLC TLC HPLC%

<99m> Tc-Complexes RFRFRT% RCY Log k0â € ™ bound to C18 SiO2 (min) proteins [<99m> TcN (DTCL3) (3)] <+> 0.65 0.41 7.89 95 2.29

[<99m> TcN (DTCL5) (3)] <+> 0.41 0.57 17.33 95 3.47

<0.30 95 2.40 0.20>

[<99m> TcN (DTCL17) (3)] <+> 0.20 11.40

± 0.03 <95 2.92 2.67>

[<99m> TcN (DTCL17) (5)] <+> 12.45

± 0.13 <0.53 71 2.11 1.67>

[<99m> TcN (DTCL17) (7)] <+> 0.46 12.66

± 0.12

[<99m> TcN (DTCL17) (10)] <+> 0.46 0.53 10.88 72 2.11

[<99m> TcN (DTCL21) (5)] <+> 0.50 0.50 94.7

[<99m> TcN (DTCL22) (5)] <+> 0.40 0.60 88.6

[<99m> TcN (DTCL23) (5)] <+> 0.30 0.5 88.6

[<99m> TcN (DTCL24) (5)] <+> 0.20 0.1 95.6

[<99m> TcN (DTCL1) (3)] <+> 11.24 95 2.51

[<99m> TcN (DTCL2) (3)] <+> 0.52 <0.54>

11.73 <95 3.09 2.13>

± 0.03 [<99m> TcN (DTCL2) (5)] <+> 14.82 92 3.20 1.64

[<99m> TcN (DTCL2) (10)] <+> 0.44 0.62 11.87 75 2.25

[<99m> TcN (DTCL4) (3)] <+> 0.31 0.44 10.93 94 2.71

[<99m> TcN (DBODC) (3)] <+> 0.43 16.06; 95 3.35 <0.53> ± 0.04 3 3.68 1.10 [<99m> TcN (DBODC) (5)] <+> 9

18.13

± 0.09 [<99m> Tc (MIBI)] <+> 9.15 3.22

Stability Studies

The in vitro stability of radio complexes (defined as variation of radiochemical purity, RCP, of the same as a function of time) is determined through transchelation studies against cysteine or glutathione (A) and serum and hepatic biotransformation studies (B).

(TO). Transchelation studies with L-cysteine and glutathione of the radio-complexes are carried out by incubating, at 37 ° C for 24 h, 100 µl of the solution of the purified radio-compound (vide supra) with a mixture of 0.2 M phosphate buffer, pH 7.4 ( 250 µl), H2O (100 µl) and 50 µl of L-cysteine solubilized in H2O at different concentrations (10 mM, 1 mM). As a control, an equal volume of water is added to replace the L-cysteine solution.

Transchelation studies against glutathione are conducted by applying a similar protocol using 50 µl of aqueous solution of glutathione (10 mM). In both cases, product CPR is evaluated by chromatography (TLC and HPLC) at 15 minutes, 1, 2 and 24 hours.

(B). Biotransformation studies of the radio-complexes are performed by incubating, at 37 ° C for 24 h, 100 µl of the purified complex solution (vide supra) with 900 µl of the following media: a) physiological solution; b) 0.2 M phosphate buffer, pH 7.4; c) rat liver homogenate; d) rat serum; e) human serum.

At established times (15 minutes, 1, 2, 3 and 24 h) aliquots (50 µL) are taken and diluted with phosphate buffer (950 µL, 0.02 M, pH = 7.4). Preparations a), b), d) and e) (100 µL), suitably diluted with 900 µL of phosphate buffer (0.02 M, pH 7.4), are analyzed directly by HPLC. Elution conditions: Waters Symmetry 300 C4 pre-column (5 µm, 3.9 x 20 mm) and a Waters Symmetry 300 C4 column (5 µm, 4.6 x 150 mm), eluted with solvent A = H2O (with 0.1% v / v acid trifluoroacetic acid, pH 3) and solvent B = MeCN (with 0.1% v / v of trifluoroacetic acid), with a flow of 1 ml / min. Method: 0-2 min, B = 15% v / v; 2-20 min, B = 65% v / v; 20-22 min, B = 15% v / v; 22-25 min, B = 15% v / v).

Sample c) must be purified, before HPLC analysis, by means of OASIS HLB extraction cartridges. It is loaded onto a column, previously washed with 1 mL of methyl alcohol (MeOH) and conditioned with 1 mL of H2O, washed with 3 mL of MeOH at 5% v / v and eluted in a single fraction with 1 mL of mixture of EtOH / aqueous solution of NaCl 0.9% 90/10 v / v. Every single fraction, including the filter, is counted in the gamma counter. 90% of the initial activity is collected in the elution fraction which is analyzed by TLC and HPLC (100 µL). The stability of the complexes is assessed by TLC, both directly on the incubated samples and on the purified fraction, without substantial changes in yield in terms of RCP. The complexes studied were the complexes reported in tab. 1 from [<99m> TcN (DTCL3) (3)] <+> to [<99m> TcN (DTCL24) (5)] <+> and proved to be stable.

Biodistribution Studies.

Biodistribution studies were performed on the purified products and suitably diluted with a physiological solution, in order to obtain an EtOH concentration lower than 5% v / v suitable for in vivo injection.

Female Sprague-Dawley rats weighing 180-200 g were anesthetized with a mixture of Zoletil (40 mg / kg) and Xylazine (2 mg / kg) administered intraperitoneally.

0.1 ml (containing 10 µCi) of the solution containing the labeled and purified complex was injected through the jugular vein.

Groups of animals (n = 3) were sacrificed at different times. Immediately after the sacrifice, a sample of blood and urine and the organs of interest were taken. The washed and weighed tissue samples were measured in a gamma counter.

As a reference of the activity actually injected, the activity of four solutions prepared at the moment of injection is counted. The values, expressed as a percentage of injected activity per gram of tissue (% dose / g), are obtained by applying the following formula, which takes into account the dose actually injected:

(cpm organ) 1% dose / g = â € ¢ â € ¢ 100 (cpmactivity actually injected) (g of the sample)

The results obtained for the new heterocomplexes according to the invention in comparison with known Tc complexes [<99m> TcN (DTCL1) (3)] <+>, [<99m> TcN (DTCL2) (3)] <+> , [<99m> TcN (DTCL2) (5)] <+>, [<99m> TcN (DTCL2) (10)] <+>, [<99m> TcN (DTCL4) (3)] <+>, < 99m> Tc (N) -DBODC (3/5) and of the technician tracers currently in clinical use <99m> Tc-Sestamibi (Cardiolite <®>) and <99m> Tc-tetrofosmin (Myoview <®>) are shown in the Tables 2-5.

Table 2

<99m> Tc- complex organs 2 min 10 min 30 min 60 min 120 min

Blood 0.22 0.06 0.02 0.01 0.00 ± 0.01 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Heart 1.89 2.38 2.33 2.34 2.40 ± 0.41 ± 0.04 ± 0.05 ± 0.40 ± 0.08 Lungs 1.06 0.87 0.72 0.61 0.10 ± 0.18 ± 0.05 ± 0.02 ± 0.02 ± 0.06 Liver 4.89 4.51 1.13 0.66 0.35 ± 3.29 ± 0.08 ± 0.07 ± 0.03 ± 0.07

[<99m> Tc (N) (DTC- Spleen 0.53 0.50 0.28 0.03 0.14 L3) (3)] <+> ± 0.13 ± 0.03 ± 0.04 ± 0.01 ± 0.01

Kidneys 4.85 5.34 3.89 3.18 2.50 ± 0.84 ± 0.23 ± 0.08 ± 0.26 ± 0.23 Intestine 5.58 2.60 12.35 2.79 15.97

± 2.26 ± 0.38 ± 2.12 ± 0.13 ± 11.12 Muscle 0.32 0.26 0.31 0.59 0.42 ± 0.10 ± 0.02 ± 0.01 ± 0.18 ± 0.04 Urine 79.30 42.42 39.84 15.71

± 13.64 ± 14.12 ± 4.15 Blood 0.35 0.07 0.05 0.03 0.03

± 0.00 ± 0.01 ± 0.01 ± 0.00 ± 0.01 Heart 1.74 2.09 2.43 3.07 2.49

± 0.06 ± 0.05 ± 0.08 ± 0.08 ± 0.10 Lungs 1.37 0.86 0.90 1.01 1.17

± 0.43 ± 0.09 ± 0.09 ± 0.11 ± 0.31 Liver 5.21 2.13 1.09 1.62 1.43

± 1.14 ± 0.08 ± 0.10 ± 0.49 ± 0.11

[<99m> Tc (N) (DTC- Spleen 1.48 1.20 1.13 2.77 2.52 L5) (3)] <+> ± 0.07 ± 0.03 ± 0.10 ± 0.07 ± 0.62

Kidneys 12.60 10.81 11.71 11.27 10.98

± 2.50 ± 1.36 ± 0.25 ± 0.60 ± 0.36 Intestine 10.43 16.74 11.34 15.27 41.30

± 0.79 ± 1.61 ± 0.64 ± 0.10 ± 6.67 Muscle 0.15 0.21 0.17 0.37 0.37

± 0.01 ± 0.01 ± 0.02 ± 0.14 ± 0.11 Urine 14.51 23.49 18.33 12.46

± 0.79 ± 5.64 ± 2.29

Table 3

<99m> Tc- Organs 2 min 10 min 30 min 60 min 120 min complex

[<99m> Tc (N) (DTC- Blood 0.24 0.05 0.02 0.01 0.01 L17) (3)] <+> ± 0.02 ± 0.01 ± 0.00 ± 0.00 ± 0.00

Heart 3.78 3.73 4.00 3.62 3.58

± 0.31 ± 0.30 ± 0.21 ± 0.10 ± 0.16 Lungs 1.93 1.10 1.22 0.97 0.97

± 0.07 ± 0.10 ± 0.15 ± 0.07 ± 0.01 Liver 1.85 0.43 0.24 0.13 0.08

± 0.02 ± 0.07 ± 0.06 ± 0.01 ± 0.00 Spleen 1.65 1.33 0.86 0.49 0.31

± 0.07 ± 0.14 ± 0.09 ± 0.03 ± 0.04 Kidney 12.21 6.61 5.51 5.17 3.46

± 0.73 ± 0.88 ± 0.26 ± 0.88 ± 0.17 Intestine 2.44 4.89 6.29 8.28 5.44

± 0.09 ± 1.23 ± 0.18 ± 0.66 ± 1.70 Muscle 0.44 0.63 0.60 0.41 0.40

± 0.02 ± 0.01 ± 0.03 ± 0.01 ± 0.02 Urine 8.71 67.97 47.41 35.10 26.2

± 1.13 ± 2.80 ± 7.65 ± 7.61 ± 5.12

Blood 0.36 0.02 0.01

± 0.18 ± 0.00 ± 0.00 Heart 5.21 4.50 3.79

± 0.61 ± 0.39 ± 0.60 Lungs 1.12 1.17 0.85

± 0.55 ± 0.05 ± 0.08 Liver 1.16 0.30 0.15

± 0.43 ± 0.01 ± 0.02 Spleen 1.70 0.94 0.59

<99m> ± 0.06 ± 0.04 ± 0.05

[Tc (N) (DTC-Kidneys 12.03 8.43 5.47 L17) (5)] <+>

± 0.73 ± 1.10 ± 0.44 Intestine 5.27 16.59 14.45

± 1.09 ± 0.11 ± 1.78 Muscle 0.35 0.39 0.37

± 0.22 ± 0.07 ± 0.04 Urine 9.50 36.67 29.90

± 3.84 ± 2.42 ± 7.40

Blood 0.36 0.08 0.04

± 0.03 ± 0.00 ± 0.01 Heart 3.91 4.76 4.58

± 0.41 ± 0.71 ± 0.08 Lungs 1.48 1.23 1.10

± 0.14 ± 0.14 ± 0.15 Liver 1.89 0.31 0.17

± 0.03 ± 0.08 ± 0.03

[<99m> Tc (N) (DTC- Spleen 1.71 0.89 0.52 L17) (7)] <+> ± 0.05 ± 0.03 ± 0.05

Kidneys 14.91 11.67 7.21

± 0.60 ± 0.03 ± 0.40 Intestine 12.13 13.95 9.51

± 4.09 ± 4.12 ± 0.81 Muscle 0.30 0.43 0.45

± 0.04 ± 0.10 ± 0.21 Urine 52.01 35.93

± 10.17 ± 11.79

Blood 0.69 0.17 0.07

± 0.20 ± 0.01 ± 0.01 Heart 3.26 3.20 2.89

± 0.19 ± 0.22 ± 0.26

[<99m> Tc (N) (DTC- Lungs 1.85 1.12 0.95 L17) (10)] <+> ± 0.09 ± 0.05 ± 0.05

Liver 2.41 1.18 1.09

± 0.47 ± 0.16 ± 0.15 Spleen 1.75 0.91 0.50

± 0.06 ± 0.04 ± 0.05 Kidneys 13.09 5.30 4.11

± 2.05 ± 0.03 ± 0.30 Intestine 8.46 12.76 14.27

± 0.60 ± 0.49 ± 0.59 Muscle 0.61 0.31 0.26

± 0.29 ± 0.01 ± 0.03

Urine 43.73

± 1.31

Table 4

<99m> Tc-complex organs 2 min 10 min 30 min 60 min 120 min Blood 0.20 0.08 0.03 0.01 0.01

± 0.02 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Heart 3.36 3.27 3.53 3.02 4.00

± 0.15 ± 0.37 ± 0.59 ± 0.32 ± 0.51 Lungs 1.62 0.82 0.99 1.06 0.91

± 0.36 ± 0.09 ± 0.26 ± 0.10 ± 0.00 Liver 1.81 0.75 0.18 0.14 0.10

± 0.06 ± 0.18 ± 0.02 ± 0.03 ± 0.02 [<99m> Tc (N) (DTC- Spleen 1.33 1.32 0.71 0.41 0.25 L1) (3)] <+> ± 0.08 ± 0.22 ± 0.15 ± 0.06 ± 0.02

Kidneys 11.68 8.38 5.62 4.45 3.62

± 1.21 ± 0.47 ± 1.07 ± 0.27 ± 0.34 Intestine 4.77 11.11 10.76 13.81 11.76

± 0.25 ± 0.21 ± 4.71 ± 1.14 ± 4.46 Muscle 0.35 0.42 0.53 0.44 0.43

± 0.02 ± 0.04 ± 0.02 ± 0.04 ± 0.01 Urine 50.94 43.59 22.93 25.22

± 0.83 ± 1.66 ± 1.05

[<99m> Tc (N) (DTC- Blood 0.21 0.04 0.02 0.01 0.00 L2) (3)] <+> ± 0.02 ± 0.01 ± 0.00 ± 0.00 ± 0.00 Heart 3.49 4.16 3.40 3.67 3.67

± 0.36 ± 0.32 ± 0.13 ± 0.55 ± 0.30 Lungs 1.06 0.87 0.97 1.04 0.79

± 0.08 ± 0.12 ± 0.15 ± 0.35 ± 0.05 Liver 2.39 0.54 0.23 0.18 0.13

± 0.32 ± 0.09 ± 0.12 ± 0.05 ± 0.05 Spleen 3.20 2.85 1.12 1.08 0.56

± 0.14 ± 0.30 ± 0.15 ± 0.23 ± 0.05 Kidney 12.81 11.74 6.19 5.81 4.07

± 0.29 ± 0.15 ± 0.66 ± 0.28 ± 0.11 Intestine 1.99 4.48 10.56 12.36 13.20

± 0.11 ± 0.21 ± 0.80 ± 1.10 ± 0.71 Muscle 0.19 0.21 0.28 0.29 0.25

± 0.02 ± 0.01 ± 0.00 ± 0.01 ± 0.00 Urine 46.84 22.04 9.82 9.29

± 0.58 ± 1.19 ± 0.73

Blood 0.45 0.36 0.34 0.28 0.27

± 0.03 ± 0.06 ± 0.06 ± 0.01 ± 0.02 Heart 3.45 3.49 3.67 3.10 3.64

± 0.25 ± 0.45 ± 0.11 ± 0.39 ± 0.13 Lungs 1.70 1.40 1.33 1.04 1.18

± 0.04 ± 0.09 ± 0.04 ± 0.04 ± 0.02 [<99m> Tc (N) (DTC- Liver 2.28 0.76 0.33 0.26 0.17 L2) (5)] <+> ± 0.33 ± 0.04 ± 0.03 ± 0.02 ± 0.04

Spleen 3.01 2.04 1.34 0.76 0.43

± 0.19 ± 0.06 ± 0.05 ± 0.01 ± 0.13 Kidney 15.99 10.51 7.56 5.02 3.97

± 0.39 ± 0.79 ± 0.17 ± 0.83 ± 0.41 Intestine 4.46 8.97 6.20 11.35 7.12

± 0.89 ± 1.44 ± 2.26 ± 1.10 ± 2.45 Muscle 0.53 0.53 0.64 0.54 0.51

± 0.03 ± 0.07 ± 0.00 ± 0.07 ± 0.10 Urine 27.89 53.83 31.72 25.6

± 5.00 ± 3.40 ± 6.28 ± 6.37

Blood 0.64 0.37 0.13 0.04 0.02

± 0.13 ± 0.06 ± 0.01 ± 0.01 ± 0.01 Heart 3.98 3.79 3.58 3.63 3.73

± 0.34 ± 0.44 ± 0.32 ± 0.46 ± 0.26 Lungs 1.72 1.48 1.30 1.40 1.15

± 0.06 ± 0.09 ± 0.14 ± 0.27 ± 0.27 Liver 3.21 2.76 0.98 0.99 0.69

± 0.32 ± 0.04 ± 0.01 ± 0.17 ± 0.17 [<99m> Tc (N) (DTC-Spleen - - - - -L2) (10)] <+>

Kidneys 15.25 10.51 7.82 5.54 3.57

± 2.77 ± 0.59 ± 0.30 ± 0.77 ± 0.40 Intestine 11.83 8.97 10.83 13.74 8.12

± 1.56 ± 1.44 ± 1.75 ± 4.88 ± 1.45 Muscle 0.31 0.34 0.37 0.23 0.16

± 0.10 ± 0.02 ± 0.05 ± 0.02 ± 0.04 Urine 27.89 24.00 36.54 39.54

± 5.00 ± 0.05 ± 3.90 ± 5.90

Blood 0.16 0.03 0.01 0.02 0.01

± 0.04 ± 0.02 ± 0.00 ± 0.00 ± 0.00 Heart 3.15 2.93 3.06 3.25 2.79

± 0.31 ± 0.53 ± 0.26 ± 0.46 ± 0.59 Lungs 1.16 1.06 077 1.01 1.03

± 0.03 ± 0.13 ± 0.03 ± 0.04 ± 0.19 [99mTc (N) (DTC- Liver 0.79 0.64 0.24 0.21 0.13 L4) (3)] + ± 0.02 ± 0.05 ± 0.05 ± 0.06 ± 0.03 Spleen 0.79 0.59 0.43 0.43 0.29

± 0.02 ± 0.01 ± 0.05 ± 0.04 ± 0.03 Kidney 7.54 5.41 5.25 4.85 3.79

± 1.00 ± 0.07 ± 0.34 ± 0.17 ± 0.36 Intestine 2.01 7.66 5.07 8.68 27.86

± 0.19 ± 5.30 ± 2.52 ± 1.63 ± 14.59 Muscle 0.19 0.32 0.16 0.35 0.50

± 0.03 ± 0.06 ± 0.02 ± 0.05 ± 0.09 Urine 46.84 22.04 9.82 9.29

± 0.58 ± 1.19 ± 0.73

Table 5

<99m> Tc-complex organs 2 min 10 min 30 min 60 min 120 min Blood 0.13 0.03 0.02 0.02 0.01

± 0.004 ± 0.02 ± 0.00 ± 0.01 ± 0.00 Heart 2.85 2.82 3.01 2.98 2.82

<99m> ± 0.09 ± 0.05 ± 0.30 ± 0.04 ± 0.04 Tc (N) DBODC (3)

Lungs 1.18 0.63 0.55 0.59 0.34

± 0.06 ± 0.04 ± 0.02 ± 0.01 ± 0.02 Liver 1.53 0.43 0.18 0.10 0.06

± 0.29 ± 0.12 ± 0.02 ± 0.01 ± 0.01

Blood 0.14 0.03 0.02 0.02 0.01

± 0.05 ± 0.01 ± 0.00 ± 0.01 ± 0.00

<99m> Heart 2.95 2.84 3.02 2.91 2.75 Tc (N) DBODC (5)

± 0.09 ± 0.05 ± 0.30 ± 0.04 ± 0.04 Lungs 0.88 0.64 0.57 0.39 0.29

± 0.06 ± 0.05 ± 0.02 ± 0.01 ± 0.02 Liver 2.53 1.43 0.64 0.20 0.11

± 0.25 ± 0.12 ± 0.02 ± 0.01 ± 0.01 Blood 0.13 0.015

± 0.04 ± 0.00

<99m> Tc-Sestamibi Heart 3.09 3.29

± 0.017 ± 0.39 Lungs 1.71 0.45

± 0.09 ± 0.02 Liver 3.03 1.28

± 0.29 ± 0.15

Blood 0.14 0.03

± 0.02 ± 0.01

<99m> Tc-Tetrofosmin Heart 2.69 2.73

± 0.21 ± 0.14 Lungs 1.22 0.4

± 0.07 ± 0.01 Liver 1.96 0.49

± 0.39 ± 0.03

Tables 6 and 7 show respectively the variation in cardiac accumulation (% ID / g), the heart / lung and heart / liver ratios of the azide complexes object of the invention as a function of time in comparison with the previously mentioned known complexes. [<99m> TcN (DTCL1) (3)] <+>, [<99m> TcN (DTCL2) (3)] <+>, [<99m> TcN (DTCL2) (5)] <+>, [< 99m> TcN (DTCL2) (10)] <+>, [<99m> TcN (DTCL4) (3)] <+>, <99m> Tc (N) -DBODC (3/5), <99m> Tc- Sestamibi and <99m> Tc-Tetrofosmin.

Table 6

<99m> Tc-complexes 2 min 10 min 30 min 60 min 120 min [<99m> Tc (N) (DTC- 1.89 2.38 2.33 2.34 2.40 L3) (3)] <+> ± 0.41 ± 0.04 ± 0.05 ± 0.40 ± 0.08

[<99m> Tc (N) (DTC- 1.74 2.09 2.43 3.07 2.49 L5) (3)] <+> ± 0.06 ± 0.05 ± 0.08 ± 0.08 ± 0.10

[<99m> Tc (N) (DTC- 3.78 3.73 4.00 3.62 3.58 L17) (3)] <+> ± 0.31 ± 0.30 ± 0.21 ± 0.10 ± 0.16

[<99m> Tc (N) (DTC- 5.21 4.50 3.79

L17) (5)] <+> ± 0.61 ± 0.39 ± 0.60

[<99m> Tc (N) (DTC- 3.36 3.27 3.53 3.02 4.00 L1) (3)] <+> ± 0.15 ± 0.37 ± 0.59 ± 0.32 ± 0.51 [<99m> Tc (N) (DTC- 3.49 4.16 3.40 3.67 3.67 L2) (3)] <+> ± 0.36 ± 0.32 ± 0.13 ± 0.55 ± 0.30 [<99m> Tc (N) (DTC- 3.45 3.49 3.67 3.10 3.64 L2) (5)] <+> ± 0.25 ± 0.45 ± 0.11 ± 0.39 ± 0.13 [<99m> Tc (N) (DTC- 3.95 3.79 3.58 3.63 3.73 L2) (10)] <+> ± 0.34 ± 0.44 ± 0.32 ± 0.46 ± 0.26 [<99m> Tc (N) (DTC - 3.15 2.93 3.06 3.25 2.79 L4) (3)] <+> ± 0.31 ± 0.53 ± 0.26 ± 0.46 ± 0.59 <99m> Tc (N) DBODC (3) 2.85 2.82 3.01 2.98 2.82 ± 0.09 ± 0.05 ± 0.30 ± 0.04 ± 0.04 <99m> Tc (N) DBODC (5) 2.95 2.84 3.02 2.91 2.75 ± 0.09 ± 0.05 ± 0.30 ± 0.04 ± 0.04 <99m> Tc-Sestamibi 3.09 3.29

± 0.17 ± 0.39

<99m> Tc-Tetrofosmin 2.69 2.73

± 0.21 ± 0.14

Table 7

<99m> Tc-complex Reports 2 min 10 min 30 min 60 min 120 min [<99m> Tc (N) (DTC- Heart / Lung 2.07 3.99 3.57 2.85 4.40 <L1) (3)] +> Heart / Liver 1.86 4.36 19.61 21.57 40.00

[<99m> Tc (N) (DTC- Heart / Lung 3.29 4.78 3.50 3.53 4.64

<L2) (3)] +> Heart / Liver 1.46 7.70 14.78 20.39 28.23

[<99m> Tc (N) (DTC- Heart / Lung 2.02 2.49 2.75 2.98 3.08

<L2) (5)] +> Heart / Liver 1.51 4.59 11.12 11.92 21.41

[<99m> Tc (N) (DTC- Heart / Lung 1.78 2.74 3.24 3.93 23.40

<L3) (3)] +> Heart / Liver 0.39 0.53 2.060 3.54 6.86

[<99m> Tc (N) (DTC- Heart / Lung 2.71 2.79 3.97 3.21 2.70

<L4) (3)] +> Heart / Liver 1.08 4.57 12.75 15.47 21.46

[<99m> Tc (N) (DTC- Heart / Lung 1.27 2.43 2.70 3.04 2.98

<L5) (3)] +> Heart / Liver 0.33 0.98 2.23 1.89 2.44

[<99m> Tc (N) (DTC- Heart / Lung 1.96 3.39 3.28 3.73 3.69

<L17) (3)] +> Heart / Liver 2.04 8.87 16.67 27.85 44.75

[<99m> Tc (N) (DTC- Heart / Lung 4.67 3.84 4.48

L17) (5)] <+> Heart / Liver 4.49 15.06 25.10

[<99m> Tc (N) (DTC- Heart / Lung 2.64 3.88 4.16

L17) (7)] <+> Heart / Liver 2.07 15.55 27.54

[<99m> Tc (N) (DTC- Heart / Lung 1.77 2.86 3.04

L17) (10)] <+> Heart / Liver 1.35 2.70 2.65

<99m> Tc (N) DBODC Heart / Lung 3.24 4.48 5.47 5.88 8.29

<(3)> Heart / Liver 1.86 6.56 16.72 29.42 47.00 <99m> Tc (N) DBODC Heart / Lung 3.35 4.43 5.29 7.46 9.48

<(5)> Heart / Liver 1.16 1.98 4.71 14.55 25.00

<99m> Tc-Sestamibi Heart / Lung 1.81 7.31

Heart / Liver 1.02 2.57

<99m> Tc-Tetrofosmin Heart / Lung 2.20 6.66

Heart / Liver 1.37 5.57

As can be seen from the results obtained, the complexes according to the invention of the general formula [Tc <v> (N) (PNP) (DTC-L)] <+> (DTC-L = alicyclic dithiocarbamates; PNP = aminodipphosphines) ( I), even when compared with the known complexes, in which DTC-L = pyrrolidinedithiocarbamate, piperidindithiocarbamate and 4-ethyl piperazindithiocarbamate, revealed interesting biological properties characterized by: a high and persistent cardiac uptake; rapid blood clearance; a low and transient hepatic and pulmonary uptake. The rapid and quantitative elimination of activity from these organs, called non-target organs, allows to obtain high heart / liver and heart / lung ratios for these complexes, especially when compared to those of <99m> Tc (N) -DBODC (5) and of the technician tracers currently in clinical use <99m> Tc-Sestamibi (Cardiolite <®>) and <99m> Tc-Tetrofosmin (Myoview <®>), allowing to obtain images characterized by a high diagnostic quality. This favorable pharmacokinetic profile is highlighted shortly after administration, in 2 min p.i.

In vitro studies of subcellular uptake in human carcinoma cell lines

Cell lines

2008 (human ovarian cancer) and MCF7 (human breast cancer).

Amino diphosphinic binders:

PNP3 abbrev. (3) = bis (dimethoxypropylphosphinoethyl) methoxyethylamine

PNP5 abrev. (5); bis (dimethoxypropylphosphinoethyl) ethoxyethylamine

PNP7 abrev. (7); bis (dimethoxyethylphosphinoethyl) ethoxyethylamine

PNP10 abrev. (10); bis (dimethoxyethylphosphinoethyl) methoxyethylamine.

Dithiocarbamic binders

DTC-L17; 1,4-dioxo-8-azaspiro [4,5] decan dithiocarbamate.

Dissymmetric Complexes

[<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> (n = 17) (X = 3,5,7,10).

The uptake of <99m> Tc-complexes according to the invention in tumor cells was evaluated in vitro using the following cell lines: 2008 human ovarian cancer (provided by Prof. G. Marverti Dept. of Biomedical Sciences, University of Modena , Italy) and MCF-7 human breast cancer (ATCC, Rockville, MD). The <99m> Tc-Sestamibi and <99m> Tc (N) DBODC (5) complexes are used as a reference comparison.

The cell lines are maintained in logarithmic growth phase at 37 ° C in an atmosphere of CO25%, using the medium RPMI-1640 (Euroclone, Celbio, Milan Italy), containing fetal bovine serum (FBS 10%), HEPES buffer (25 mM ), L-glutamine (4 mM), penicillin (50 units / mL), streptomycin (50 mg / mL).

For uptake studies, cells are detached from culture plates with trypsin (0.05%) - EDTA (0.53 mM), washed and resuspended with RPMI-1640 to achieve a concentration of 5 x 10 <6> cells / mL.

The experiments are conducted by incubating the cells in previously sterilized glass tubes. Before each experiment the cells are preincubated for 30 min at 4 ° and 37 ° C.

In a test tube containing 350 µl of a suitably selected cell suspension, 6.5 µCi / 5 µL saline of the purified radiocomplex are added. Each sample is incubated for 120 min. at 4 ° or 37 ° C. At defined time intervals (5, 15, 30, 60, 90 and 120 min), from each sample an aliquot (8 µL) is taken and stratified on cold FBS (350 µL) contained in a 500 µL Eppendorf microtube. µl (n = 3). After centrifugation (14000 g x 2 min), the microtubes are frozen in an ethanol and dry ice bath.

The underside of the microtubes containing the cell pellet is cut and placed in a tube for counting. The remaining part of the microtube with the supernatant is, on the other hand, placed in a second counting tube. The activity of both fractions is determined using a gamma counter (Packard). The percentage of activity associated with the cells is calculated by applying the following formula:

% uptake = [cpm (pellet)] / [cpm (pellet) cpm (supernatant)] x 100.

All experiments were carried out in duplicate and repeated 4 times.

The amount of supernatant present in the pellet is determined to be <1%.

Cellular vitality

Cell viability assessed before and during the tryptan blue experiments was> 90%.

Results

The data obtained from the study of cell uptake kinetics, evaluated at 4 ° and 37 ° C, of the complexes [<99m> Tc <V> (N) (PNPx) (DTC-Ln)] <+> compared with that obtained for the reference compounds <99m> Tc (N) DBODC (5) and <99m> Tc-Sestamibi are reported in tables 8 and 9.

The experiments carried out at 37 ° C allow to evaluate the net cellular accumulation correlated to the ATP-dependent processes, while those carried out at 4 ° C allow to determine non-specific absorption of the complexes solely correlated to the lipophilic characteristics of the product (non-specific uptake) .

Tab. 8 uptake in the 2008 cell line

<99m> Tc-accomplished 5 min 15 min 30 min 60 min 90 min [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (5)] <+ > 2.10 ± 0.83 1.7610.50 3.0711.48 2.9111.12 2.2510.83 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 6.79 ± 0.72 8.6112.54 9.9712.91 11.1112.28 13.1813.19 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (3)] <+> 1 .8410.59 1.6010.82 1 .7710.49 1.8111.00 1 .3410.95 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 9.5212.48 12.4313.13 12.9312 .23 15.1012.92 16.2813.53 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (7)] <+> 1 .4010.74 1 .3510.48 1.0010 .41 1.4510.51 2.0510.86 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 8.9811.50 13.7512.19 15.3312.52 17.2213. 23 16.9213.16 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (10)] <+> 1.4710.91 1.5510.91 2.0511.31 2.5910. 74 3.5010.83 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 8.7111.81 13.5512.36 15.6311.94 17.8412.15 19.8412.68

(a) (a) (a) (a) (a) <99m> Tc (N) (DBODC- (5)

1 .7810.49 1 .8010.47 2.2110.77 3.1210.73 3.2711.37

(a) T = 4 ° C

(b) (b) (b) (b) (b) (b) T = 37 ° C

9.1911.21 8.4810.67 10.1311.63 11.1511.46 11.1011.80

(a) (a) (a) (a) (a) Sestamibi 2.2110.81 2.2610.53 2.0210.0.70 1.6010.45 2.2610.94 (a) T = 4 ° C (b) (b) (b) (b ) (b) (b) T = 37 ° C 10.25 ± 2.39 10.33 ± 2.40 10.77 ± 2.75 10.69 ± 1.77 10.9711.51

Tab. 9 uptake in the MCF7 cell line

Tc-complexes 5 min 15 min 30 min 60 min 90 min [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (5)] <+> 1. 6810.34 1.8510.64 2.8211.03 2.7311.02 2.2710.87 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 5.0210.71 5.6410.97 6.2310.87 5.7710.93 6.3010.60 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (3)] <+> 1 .7410.60 2.3910.59 2.2710.93 2.0010.30 2.1610.23 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 4.6110.71 6.4911.07 6.8610.58 6.9211 .17 6.5910.67 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (7)] <+> 1 .9710.32 2.3610.45 2.6810.54 2.2910 .49 2.5910.70 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 9.4511.61 15.9312.39 19.6114.29 21.5814.47 21.7714. 67 [<SSfm> Tc (N) (DTC- (a) (a) (a) (a) (a) L17) (10)] <+> 1 .8210.53 2.1410.57 2.3010.58 2.1210.68 2.9610. 36 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 6.9711.09 13.0611.50 15.9112.35 18.6613.63 19.1314.75 <uum> Tc (N) (DBODCJ5) (a) (a) (a) (a) (a) (a) T = 4 ° C 1.8510.61 2.5610.62 2.3710.55 2.5910.63 2.6110.51 (b) T = 37 ° C (b) (b) (b) (b) (b)

5.2010.68 5.9011.22 5.9710.97 6.4711 .45 6.6911.09

(a) (a) (a) (a) (a) Sestamibi 1.B210.50 2.0310.42 2.1810.56 2.3110.63 2.3610.92 (a) T = 4 ° C (b) (b) (b) (b) (b) (b) T = 37 ° C 7.14 ± 0.81 8.2611.05 8.6011.37 8.4311 .34 7.4111.49 The data reveal in both cell lines a rapid and high uptake of the complexes [<99m> Tc < V> (N) (PNPx) (DTC-Ln)] <+> higher than that observed for the two reference products.

The diagnostic radiopharmaceuticals containing <99m> Tc according to the invention administered at dosages for human use comprised between 37 and 1850 MBq, preferably between 185-740 MBq, expressing the radioactivity of <99m> Tc, are devoid of acute toxicity.

Kit for the preparation of radiopharmaceuticals for diagnostic imaging One and two pass formulation: the vials are prepared as follows, frozen in dry ice and freeze-dried.

Two-pass formulation One-pass formulation

Run 1 Run 2 Run 1 Run 2 Vial SDH 5 mg 5 mg Vial SDH 5 mg 5 mg 1 1

EDTA 5 mg 5 mg EDTA 5 mg 5 mg SnCl2x 2H2O 0.1 0.1 SnCl2x 2H2O 0.1 0.1 mg mg mg mg Buffer 1 mL 1 mL Buffer 1 mL 1 mL Phosphate Phosphate

Vial PNP5 1.5 3.5 PNP5 1.5 3.5 2 mg mg mg mg DTC-L17 2 mg 0.5 DTC-L17 2 mg 0.5 mg mg γ-cyclodestin 3.5 2 mg γ -cyclodestin 3.5 2 mg mg mg

From the lyophilisate, the radiopharmaceutical containing the azide complex of <99m> Tc can be obtained as follows: 1 or 2 mL of [Na <99m> TcO4] eluted from <99> Mo- <99m> Tc generator are added to vial 1 which, suitably stirred, is placed to incubate at room temperature for 15 min. At the end of the incubation, 1.5 mL of physiological solution is instead added to vial 2 to dissolve its contents and 1 mL of the latter is taken and subsequently added to vial 1. After shaking, the resulting solution is heated at 100 ° C for 15 min to obtain the final product.

Alternatively, all components can be prepared in a single vial to which 1 or 2 mL of [Na <99m> TcO4] eluted from <99> Mo- <99m> Tc generator are added. The resulting solution is then heated to 100 ° C for 20 min.

No effect on the reaction yield is determined by the variation of the quantity of the different reactants.

Claims (19)

Claims 1. Unsymmetrical monocation azide complexes of technetium-99m represented by the general formula (I) [Tc <V> (N) (PNP) (DTC-L)] <+> (I) - Tc <V> (N) Ã ̈ [<99m> Tcâ ‰ ¡N] <2+>; - PNP is a diphosphinoamine ligand; - DTC-L is an alicyclic dithiocarbamic binder and pharmaceutically acceptable salts thereof in which - the PNP ligand is represented by the general formula (II) R <2> R1 R <1> PCH2CH2NCH2CH2P R <1> R <1> (II) where is it: - R <1> is an aromatic, alkyl or ethereal group; the latter represented by the general formula (III) - (CH2) mO (CH2) mâ € ™ CH3 (III) where m is an integer in the range 1â ‰ ¤mâ ‰ ¤4 and mâ € ™ is an integer in the range 0â ‰ ¤mâ € ™ â ‰ ¤3, - R <2> is represented by a hydrogen atom, an alkyl group or a variously substituted alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, a cyclic ether or an ether group having the meaning of the general formula (III) or an ester group of the type â € “(CH2) mâ € ™ â € ™ C (O) Râ € where mâ € ™ â € ™ is an integer within the interval 0â ‰ ¤mâ € ™ â € ™ â ‰ ¤3 and R <â € ™ â € ™> is represented by a hydrogen atom, an alkyl group or a variously substituted alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, or an ethereal group having the meaning of the general formula (III); - the alicyclic dithiocarbamic binder DTC-L is represented by the general formula (IV) S. CN <n> X R <3> S (IV) where is it: - n is an integer in the range 3â ‰ ¤nâ ‰ ¤8; - X is a carbon atom or a heteroatom chosen from N, O, S; - R <3> is a hydrogen atom, an alkyl group or a variously substituted linear or cyclic alkyl group, an aryl group, a variously substituted aryl group, an amino group, an amino acid, or a linear acetal or ketal ether group or cyclic, a pharmacophore group or a biomolecule, with the proviso that the DTC-L ligand is not pyrrolidinedithiocarbamate, piperidindithiocarbamate or 4-ethyl piperazindithiocarbamate. 2. Unsymmetrical monocation azide complexes of technetium-99m according to claim 1, wherein the variously substituted alkyl groups R <3> of the ligand DTC-L are represented by the general formulas (V), (VI), (VII): R <4> R <5> (V) where is it: R <4> and R <5> are independently of each other a variously substituted alkyl or aryl group, also containing heteroatoms selected from N, O, S, variously spaced; R <6> (CH2) pY ((CH2) p'CH3) p "(VI) where is it: R <6> is a variously substituted alkyl or aryl group, also containing heteroatoms selected from N, O, S, variously spaced, p is an integer in the range 0â ‰ ¤pâ ‰ ¤4; Y is a carbon atom or a heteroatom chosen from N, O, S, pâ € ™ is an integer within the interval 0â ‰ ¤pâ € ™ â ‰ ¤4, nâ € is an integer including in the interval 1â ‰ ¤nâ € â ‰ ¤3; (CH2) pY ((CH2) p'CH3) p " <(CH2) pY ((CH2) p'CH3) p "> (VII) p is an integer within the range 0â ‰ ¤pâ ‰ ¤4, X is a carbon atom or a heteroatom chosen from N, O, S, pâ € ™ is an integer within the range 0â ‰ ¤pâ € ™ â ‰ ¤4, nâ € is an integer in the range 1â ‰ ¤pâ € â ‰ ¤3. 3. Technetium-99m dissymmetric monocation azide complexes according to claim 1, wherein the pharmacophore group R <3> is selected from the group consisting of 2 methoxyphenyl piperazine, benzyl guanidine or norepinephrine derivatives. 4. Technetium-99m dissymmetric monocation azide complexes according to claim 1, wherein the biomolecule R <3> is selected from the group consisting of bioactive peptides, fatty acids. 5. Unsymmetrical monocation azide complexes of technetium-99m according to claim 1, wherein the PNP diphosphinoamine ligand is selected from the group consisting of bis (dimethoxypropylphosphinoethyl) methoxyethylamine, bis (dimethoxypropylphosphinoethyl) ethoxyethylamine, bis (dimethoxypropylphosphinoethyl) methoxypropylamine, bis (dimethoxypropylphosphinoethyl) ethoxypropylamine, bis (diethoxypropylphosphinoethyl) methoxyethylamine, bis (diethoxypropylphosphinoethyl) ethoxyethylamine, bis (diethoxypropylphosphinoethyl) methoxypropylamine, bis (diethoxypropylphosphinoethyl) ethoxypropylamine, bis (dimethoxyethylphosphinoethyl) ethoxyethylamine, bis (dimethoxyethylphosphinoethyl) methoxyethylamine, bis (dimethoxyethylphosphinoethyl) methoxypropylamine, bis (dimethoxyethylphosphinoethyl) ethoxypropylamine, bis (diethoxyethylphosphinoethyl) methoxyethylamine, bis (diethoxyethylphosphinoethyl) ethoxyethylamine, bis (diethoxyethylphosphinoethyl) methoxypropylamine, bis (diethoxyethylphosphinoethyl) ethoxypropylamine, bis (dimethylphosphinoethyl) methoxyethylamine, bis (dimethylphosphinoethyl) ethoxyethylphosphinoethylamine, bis (diethoxyphinoethylamine), bis (dimethylphosphinoethylamine) ethoxyphino ethylamine (diethoxyphinoethylamine) (diethoxyphino ethylamine) (diethoxyphino ethylamine) (diethoxyphino ethylamine) (diethoxyphino ethyl) dietilfosfinoetil)etossietilamina, bis(dietilfosfinoetil)metossipropilamina bis(dietillfosfinoetil)etossipropilamina, bis(dipropilfosfinoetil)metossietilamina, bis(dipropilfosfinoetil)etossietilamina, bis(dipropilfosfinoetil)metossipropilamina, bis(dipropilfosfinoetil)etossipropilamina, bis(diisopropilfosfinoetil)metossietilamina, bis(diisopropilfosfinoetil)etossietilamina , bis (diisopropylphosphinoethyl) methoxypropylamine, bis (diisopropylphosphinoethyl) ethoxypropylamine, bis (dimethylphosphinoethyl) methylamine, bis (dimethylphosphinoethyl) ethylamine, bis (dimethylphosphinoethyl) propylamine, bis (diethylphosphinoethyl) methylamine, bis (dimethylphosphinoethyl) ethylamine, bis (dimethylphosphinoethyl) ilphosphinoethyl) propylamine, bis (dipropylphosphinoethyl) methylamine, bis (dipropylphosphinoethyl) ethylamine, bis (dipropylphosphinoethyl) propylamine, bis (diisopropylphosphinoethyl) methylamine, bis (diisopropylphosphinoethyl) ethylamine, bis (diisopropylphosphinoethyl) propylamine. 6. Technetium-99m monocation dissymmetrical azide complexes according to claim 1, wherein the PNP diphosphinoamine ligand is selected from the group consisting of bis (dimethoxypropylphosphinoethyl) methoxyethylamine, bis (dimethoxypropylphosphinoethyl) ethoxyethylamine (dimethoxyphinoethyl) ethoxyethylamine (dimethoxyphinoethyl) ) -methoxyethylamine, bis (diethoxypropylphosphinoethyl) -methoxyethylamine, bis (diethoxypropylphosphinoethyl) -ethoxyethylamine. 7. Technetium-99m monocation dissymmetric azide complexes according to claim 1, wherein the dithiocarbamic ligand DTC-L is selected in the group consisting of azepine dithiocarbamate, azocane dithiocarbamate, 1-methyl- [1,4] diazepan dithiocarbamate, 3- (dimethylamino) pyrrolidine dithiocarbamate, 3- (diethylamino) pyrrolidine dithiocarbamate, 4-isopropylpiperidine dithiocarbamate, 4,4-dimethoxy piperidine dithiocarbamate, 4,4-diethoxyperidine dithioxycarbamate, 4,4-diethoxy diethylpyloxycarbamate, 4,4-diethoxy-diethylphyldine , 4-bis- (2-ethoxy-ethoxy) -piperidine dithiocarbamate, 1-ethylomopiperazine dithiocarbamate, morpholin dithiocarbamate, thiomorpholin dithiocarbamate, 2,6-dimethylmorpholin dithiocarbamate, 4 morpholine piperidine dithiocarbamate, 4-methyl-piperidine dithiocarbamate, 4-methyl methyl methyl methyl methiocarbamate, 4-methylcarbamate 4bammeryl dithiocarbamate , 4-piperidinediol dithiocarbamate, 2,2,6,6-tetramethyl-4-piperidinone dithiocarbamate, 4-piperidinol dithiocarbamate, 2,2,6,6-tetramethyl-4-piperidinol dithiocarbamate, 1 , 4-dioxo-8-azaspiro [4,5] decan dithiocarbamate, (7,7,9,9-tetramethyl-1,4-dioxa-8-azaspiro [4,5] dec-2-yl) methanol dithiocarbamate, 2-aza-5-oxabicyclo (2,2,1) -heptane dithiocarbamate, 4,7-epoxy-7-methyl-2,3,3a, 4,5,6,7,7a-octahydro-1h-isoindol dithiocarbamate . 8. Unsymmetrical monocation azide complexes of technetium-99m according to claim 1, wherein the ligand DTC-L is selected from the group consisting of morpholin dithiocarbamate, 4,4-N- (2methoxy phenyl) piperazine dithiocarbamate, 1,4-dioxo -8-azaspiro [4,5] decan dithiocarbamate, thiomorpholin dithiocarbamate, azepine dithiocarbamate, azocane dithiocarbamate, 1-methyl- [1,4] diazepan dithiocarbamate. 9. Technetium-99m dissymmetric monocation azide complexes according to claims 1-8, for use in a diagnostic method of visualization practiced on the human or animal body. 10. Technetium-99m dissymmetrical monocation azide complexes according to claim 9, for use in a radiodiagnostic method of cardiac visualization. 11. Technetium-99m dissymmetric monocation azide complexes according to claim 9, for use in a radiodiagnostic method of visualizing MDR-Pgp in tumor tissues. 12. Use of dissymmetric monocation azide complexes of technetium-99m of formula (I) as defined in claims 1-8 for the preparation of a radiodiagnostic agent for radiodiagnostic visualization practiced on the human or animal body. 13. Use of dissymmetric monocation azide complexes of technetium-99m of formula (I) according to claim 12, wherein the visualization is cardiac. 14. Use of dissymmetric monocation azide complexes of technetium-99m of formula (I) according to claim 12, wherein the visualization is of the MDR-Pgp in tumor tissues. 15. Kit suitable for the preparation of a radiopharmaceutical consisting of the dissymmetric monocation azide complexes of technetium-99m of formula (I) as defined in claims 1-8, comprising a bottle containing a donor of azide groups and a reducing agent and a bottle containing alicyclic dithiocarbamen (DTC-L) and diphosphinamine (PNP) ligands. 16. Kit suitable for the preparation of a radiopharmaceutical consisting of the dissymmetrical monocation azide complexes of technetium-99m of formula (I) as defined in claims 1-8, comprising a bottle containing a donor of azide groups, a reducing agent and the dithiocarbamen ligands (DTC-L) and diphosphinoamine (PNP). 17. Kit suitable for the preparation of a radiopharmaceutical according to claims 15-16, in which the contents of the bottles are lyophilized. 18. Kit suitable for the preparation of a radiopharmaceutical according to claims 15-16, in which the donor of azide groups is selected between dithiocarbazic acid and its derivatives and hydrazine and its derivatives. 19. Kit suitable for the preparation of a radiopharmaceutical according to claims 15-16, wherein the reducing agent is selected from stannous chloride, sodium hydrogen sulphite, sodium boron hydride, aliphatic tertiary phosphines and tris (msulfonate phenyl) phosphines.