FIELD OF THE INVENTION

The present invention is in the field of isotopic labeling for functional and radiodiagnostic imaging and concerns the preparation of radiolabeled agents for use in such imaging. More specifically, the present invention concerns novel azidoalkyl derivatives of targeting molecules, i.e. molecules having an affinity to a specific target such as a ligand for a receptor, which may be utilized for the preparation of " ^m Tc labeled agents which serve for radioimaging.

The present invention further concerns the preparation of these azidoalkyl derivatives, radiolabelled agents prepared therefrom and the preparation of these agents and kits containing said derivatives.

LIST OF REFERENCES

1. Scatton B. et al, Neurology 32, 1039 (1982). 2. Lee T. et al. Nature, 273, 59 (1978)

3. Reisine T.D, et al. Life Sci. 21, 1123 (1977).

4. Goetz C.G. et al., Clin. Neuropharmacol. 5, 3 (1982)

5. Lee T. and Seeman P.S., "In Receptors for Neuro-transmitters and Peptide Hormones" (Eds. Pepeu G. Kuhar M.J. and Enna S.J.) p. 435 New York, Raven Press 1980.

6. Wagner H.N. Jr. et al., Science 221, 1264 (1983).

7. Wong D.F. et al. Science, 234, 1558 (1986).

8. Maziere B. et al. Life Sci. 35, 1349 (1984).

9. Arnett CD. et al. J. Nucl. Med. 27, 1878 (1987).

10. Arnett CD. et al. Life Sci. 36, 1359 (1985). 11. Satyamurthy N. et al, Nucl. Med. Biol. 13, 617 (1986).

12. Welch M.J. et al, Nucl. Med. Biol. 15, 83 (1986) and references cited therein.

13. Cholon S. et al, Nucl. Med. Biol.17, 389 (1990).

14. Ballinger J.R. et al, Appl. Radiat. Isot. 40, 547 (1988). 15. Baldas J, Int. J. Appl. Radiat. Isot. 26, 133 (1985).

16. Pusqualini, R, et al, Appl. Radiat. Isot. 43, 1329 (1992).

17. Janssen Pharmaceutica, Analytical Report (1990).

18. Kiesewetter D.O. et al, Appl. Rad. Isot. 37, 1181 (1986).

19. Hassner A. and Stern M, Angew. Chem. lnt, Ed. Engl. 25, 478 (1986).

20. Hassner A. et al, J. Org. Chem, 55, 2304 (1989).

The above references will be referred to in the following text by indicating in brackets their relevant number in the above list.

BACKGROUND OF THE INVENTION AND PRIOR ART

Imaging is a tool of major importance in medical diagnosis, evaluation of treatment efficacy and in basic medical research. Recently the advent of functional imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) turned functional brain imaging into a field of increasing importance in the area of medical imaging.

The in vivo imaging of D ₂ -dopamine receptors (to be referred to at times as "D ₂ receptors") is very important for basic research, diagnosis

and therapy of various neurological and psychiatric disorders such as Parkinson's disease (Scatton et al. 1982; Lee et al. 1980), Huntington's chorea (Reisine et al, 1977), tardive dyskinesia (Goetz et al, 1982) and schizophrenia (Lee et al, 1980). Spiperone, a neuroleptic molecule which has a high affinity for D ₂ receptors has been used for D, receptors imaging mostly by the PET technique. The spiperone derivatives prepared for use in PET imaging have been labeled with ⁿ C (Wagner et al, 1983; Wong et al, 1986), ⁷⁶ Br (Maziere et al, 1984) and ¹⁸ F (Arnett et al, 1987; Arnett et al, 1985; Satyamurthy et al, 1986; Welch et al, 1986) with the ¹⁸ F being the most useful due to its relatively longer half-life and its relative ease of production and superior emission characteristics. However the relative short half-life of these radioisotopes, their high prices and the very high costs involved in the establishment of a PET center greatly reduces the wide¬ spread use of D ₂ receptors imaging by this technique. In view of the fact that SPECT type imaging is a more accessible technology in that the equipment is less costly and in that the radioactive substances therefore are more easily manufactured, considerable efforts have been invested in developing suitable γ -emitting agents in general and such which are specific for D ₂ -receptor imaging in particular. Spiperone labeled with ¹²⁵ I has recently been prepared and animal studies have shown its specific uptake by the striatum which contains the most dense population of D ₂ receptors (Cholon et al, 1990). However, ¹²⁵ I labeling has a number of disadvantages which considerably limit its use, for one it is known that iodine-labeled materials are often unstable under physiological conditions; additionally, ¹²⁵ I has a short half-life; and furthermore the preparation of radioactive iodine is relatively expensive.

"Tc is the most widely used radioisotope in today's nuclear medicine. This is due to the fact that the energy of its γ-irradiation is suitable for medical purposes, it is readily available and it has a low price.

In most cases, the approach to ^99m Tc complexation has generally been to bind "" c to a known chelate and thereafter modify the chelate to enable its binding to the molecule responsible for localization in the body. This approach invariably causes large changes in the physical and biological characteristics of the ^99m Tc labeled molecule.

It was found several years ago that azide anion may form inorganic complexes with ^99m Tc (Baldas, 1985).

A " ^m Tc spiperone complex which has been prepared recently and which structure has not been determined in detail was found to be only moderately stable in aqueous solutions and to have a very low brain uptake (Ballinger et al, 1988).

In spite of the long-felt need for an efficient and inexpensive agent for use in functional imaging of D ₂ receptors and the like, no such agents are available to date.

OBJECTS OF THE INVENTION

It is therefore the object of the present invention to provide novel compounds useful for the preparation of " ^m Tc labeled agents which may be used for functional and diagnostic radioimaging. It is a further object of the present invention to provide a kit which may be used by a diagnostic laboratory for the preparation of ^99m Tc labeled agents for use in radioimaging.

It is a still further object, according to said preferred embodiment to provide such a kit which may be used for the preparation of ^99m Tc labelled agents for use in D ₂ receptors radioimaging.

SUMMARY OF THE INVENTION

The invention is based on a new approach of direct labeling of a targeting molecule with ^99m Tc. In accordance with the invention novel compounds useful for the preparation of ^99m Tc complexes which may be utilised in radioimaging are provided.

The complexes prepared according to the present invention do not use bulky chelates to bind the ^99m Tc to the targeting molecules and accordingly there are only minimal changes in the structural and binding characteristics of said targeting molecule.

The present invention thus provides a compound of the formula I:

T -R -Y

wherein

T is a targeting group derived from a molecule T which has an affinity to a specific body tissue said targeting group essentially retaining the specific affinity of said molecule; R is a branched or straight alkane chain having 1 to 6 carbon atoms; and Y is azide (-N ₃ ).

T may be derived from a large number of T molecules each one having an affinity to a specific tissue. Such molecules may be ligands of receptors, various agents which have an affinity to certain types of cells or tissue, agents which avoid certain cells or tissue and the like. Examples of such T molecules are the following: (i) Ligands of dopamine receptors such as azidoalkylspiperone derivatives, represented by the following formula II

or various benzamides having the following formula III

(ii) specific ligands of brain and heart muscarinic receptors such as quinalidinyl benzylate (QNB) derivatives of the general formula IV

(iii) synthetic ionophorcs which may be utilized in brain imaging such as those having the general formula V

R,

\

C - [ CH ₂ O (CH ₂ ) _n CONHCHR (CH ₂ ) _m CO (NOHR;) ] ₂ V

R /

In the above formulae II-V, groups R _a to R. independently represent the group RH, wherein R is as defined for formula I, and m and n are integers which are independently 0-6. R in the above Formulae I-V has preferably 1 to 4 carbon atoms, with 2 carbon atoms being the most preferred.

In accordance with a preferred embodiment T is spiperone, which is a ligand of the D ₂ receptor and which is represented by the following formula VI

The present invention also provides a process for preparing a compound of the formula I comprising the steps:

(a) providing a T' molecule, in a reactive form if required;

(b) reacting the molecule of step (a) with a di-p-tosylate having the general formula VII

wherein R is as defined above for formula I; and

(c) reacting the product of step (b) with a compound capable of donating an azide anion, e.g. amberlite azide. The reaction product of step (c) in the above process may suitably be separated from the reaction mixture by chromatography. In accordance with the present invention, there is also provided a method for the preparation of a radioisotopic agent useful for in vivo imaging comprising:

(a) preparing an aqueous solution of the compound of formula I;

(b) preparing a reaction mixture by adding an acid and a solution of pertechnetate of ^99m Tc (a salt solution of ^99m TcO ₄ ^~ ); ,

(c) providing appropriate reaction conditions whereby a complex of ^99m Tc with the compound of formula I is formed.

It is known (Pasqualini et al, 1992) that phosphines or diphospines, particularly such with the general formula: P(R ₅ ) ₃ or wherein

R ₅ is independently in each case alkyl, aryl, arylalkyl, heteroaryl, and hεteroarylalkyl, optionally substituted by a functional group such as SO ₃ , CN, OH, SH and NH ₃ ; X is CH,; and n is 1, 2 or 3;

can be used as oxygen acceptors in organometallic reactions such as the one described above with TcO ₄ ^" . Using such an oxygen acceptor is expected to increase the yield of the final spiperone- ^99m Tc complex and accordingly it is preferred in accordance with the invention to add such an an oxygen acceptor to the reaction mixture in step (b) above.

The solution obtained in method step (c) may be used for imaging as is or alternatively and preferably, the obtained complex is separated from the solution prior to its use. In some specific cases it might be necessary to add a base to neutralise the resulting hydronium salt of said target molecule in the complex prior to its use.

In the case of azidoethylspiperone (represented hereinbelow in short as sp-(CH ₂ ) ₂ N ₃ ) the direct covalent binding of ^Wm Tc to the azide moiety of azidoethylspiperone may be carried out according to the following reaction scheme:

SP-(CH,) ₂ -N ₃ + H _A X _B + TcO ^" ₄ 100°C SP-(CH ₂ ) ₂ -N = Tc(X), wherein (i) X is an inorganic anion, such as I ^" , Br ^~ , Cl ^" ; A = 1; B = 1; and C =

4; or

(ii) X is an organic anion of the structure R^OO ^" wherein R ¹ is hydrogen or a - branched or straight chain alky] optionally substituted ^' by halogen atoms, e.g. 1 to 3, or by hydroxyl, e.g. 1 to 3, or an organic anion of the structure R ² (COOO ^~ ) ₂ wherein R ² is a -C ₆ branched or straight chain alkylene optionally substituted by halogen atoms, e.g. 1 to 3, or hydroxyl, e.g. 1 to 3. If X is a monocarboxylic acid anion c = 4, a = 1 and b = 1. If X is a dicarboxylic acid anion c = 2, a = 2 and b = 1.

In principle, any acid capable of reducing the technetium from the heptavalent state to the lower valency needed for preparing the complex may be used in accordance with the present invention. The acids may be

inorganic or organic acid, hydrochloric acid and succinic acid being presently preferred.

Examples of specific pertechnetates of ^99m Tc that can be used in accordance with the above method of the present invention are Na ^99m Tc0 ₄ , NH ₄ ^99m TcO ₄ or K" ^m TcO ₄ .

Because ^99m Tc has a half life time of about 6 hrs it is preferred that the actual preparation of the complex be carried out only shortly before the intended use of the agent.

The present invention further provides an agent useful for radioimaging being a complexation product of the compounds of formula I and of "" c.

The invention further provides a kit which may be utilized by a diagnostic laboratory for the preparation of the above complexation product comprising at least one container holding a compound of formula I optionally containing one or more additional containers holding a suitable acid solution.

EXAMPLES:

The present invention will now be illustrated with reference to azidoethylspiperone and its synthesis. It is understood that the invention is not limited thereto.

Spiperone

Spiperone hydrochloride (500 mg) was dissolved by heating in distilled water (105 ml). After cooling IN NaOH (1.23 ml) was added. The solution was filtered and then dried under high vacuum resulting in 376 mg of spiperone (82% yield). The product was identified by TLC, m.p. and microanalysis. The m.p. was found to be 208-210°C (lit m.p. 208.6- 210°C (Janssen Pharmaceutica, 1990)). Micronalysis:

P-tosylatoethylspiperone

To a solution of spiperone (342 mg) in hot benzene (40 ml) tetrabutyl ammonium hydrogen sulfate (78 mg) and 50% w/v NaOH (1.22 ml) were added. After 10 minutes reflux ethylene glycol di-p-tosylate in benzene (718 mg/10 ml) was added in one portion. The solution was refluxed vigorously with stirring during 20 min. Water was then added and the reaction mixture was extracted four times with methylene chloride.

Flash chromatographic separation of the crude product (with methylene chloride -methanol 95:5) afforded 340 mg (60% yield) of the desired product as a light foam. The product (p-tosylatoethylspiperone)

was identified by Η and ¹³ C NMR (Kiesewetter et al, 1986).

Azidoethylspiperone

P-tosylatoethylspiperone (261 mg) was dissolved in acetonitrile (20.5 ml) and amberlite azide (10.2 gr), prepared according to Hassner (Hassner et al, 1986; Hassner et al, 1989) was added to the solution. The slurry was shaken in a water bath at 50°C overnight.

Filtration and evaporation afforded 175 mg crude product which was purified by flash chromatography (methylene chloride - methanol 95:5). 120 mg of the oily product (56% yield) were obtained.

Azidoethylspiperone having the formula

was obtained and identified by -H. and ¹³ C NMR and mass spectrometry.

Spectrometric analysis:

NMR data of spiperone azide: Η NMR (CDC1 ₃ )(<5): 7.15(2H)(J _HF =9HZ); 8.04(2H)(J _HF =5.5HZ); 3.04(2H);

2.01(2H); 2.56(2H); 2.87(4H); 1.72(2H); 2.63(2H); 4.77(2H); 3.59(4H);

6.94(2H); 7.28(2H); 6.91(1H).

Η NMR(C ₆ D ₆ )( 5): 6.69(2H)(J _HF =8.5Hz); 7.70(2H)(J _HF =5.5Hz); 2.54(2H);

1.88(2H); 2.33(2H); 3.05(2H); 2.70(2H); 1.58(2H); 2.70(2H); 4.15(2H); 2.71(2H); 2.90(2H); 6.83(2H); 7.18(2H); 6.83(1H).

133.7; 198.5; 36.9; 21.9; 57.5; 49.5; 29.4; 60.7; 174.8; 64.6; 40.9; 49.5;

142.9; 116.5; 129.2; 119.7.

Mass spectrum of azidoethylspiperone: 465.3(100%)(M ⁺ +1); 437.2(45 %)(M ⁺ -28;M ⁺ -N ₂ ); 396.1(5%)(M ⁺ -69;M ⁺ - H ₃ N ₃ ); 339.1(6%)(M ⁺ -126; (M ⁺ -C ₄ H ₆ N ₄ O).

Complexation of azidoethylspiperone with ^99,n TcO ₄ ^"

The direct covalent binding of ^99m Tc to the azidoethyl moiety of the azidoethylspiperone was carried out as follows:

Azidoethylspiperone (2 mg) was dissolved in saline (1 ml) by heating and stirring for 15 min at 100°C. 2.6 ml 10 N HCl were added while continuing heating during 4 min. 1 ml saline solution of " ^m TcO ^" was added and the reaction mixture was heated further during 30 min.

After cooling, the reaction mixture was separated by one and two dimensional chromatography on silica TLC plates. One dimensional TLC was performed in acetonitrile. Two dimensional chromatography was performed in MeOH-Benzene-CH ₂ Cl ₂ - DDW (2:1:1:0.05). The plates were cut into strips and counted. The labeled spiperone complex had a yield of 30-50%.

Complexation of rhenium with azidoethylphenyl

Rhenium (Re) is a compound of group Vllb of the periodic table of chemical elements, to which group technetium also belongs, and accordingly the chemical properties of these two compounds are very similar. The preparation of a complex with Re is thus very similar to the preparation of a complex with " ^m Tc.

34 mg of phenylethylazide having the following formula:

was dissolved in 42 ml ethanol and heated to 70°C for 15 min. Then 110 ml of a 10 N solution of HCl was added and the resulting solution was maintained at the same temperature for an additional 4 min. 52.9 mg of KReO ₄ in 42 ml saline were then added and the reaction mixture was maintained at 70°C for one and a half hours.

The reaction was then neutralized with sodium bicarbonate at pH7. The complex having the suggested formula

was then extracted with CH,C1 ₂ (4 repeats) and washed with saline at pH7. The extraction product was separated on preparative silica T.L.C. plates using CH ₂ C1 ₂ as the eluting solvent. Fraction 3 (R)=0.4) was isolated and identified by mass spectroscopy as having the mass corresponding to the above Re complex.