The invention relates to a novel ¹⁸ F-labelled compound useful in studying anomalies in the dopaminergic neurotransmission of brain in neurological and psychiatric diseases.

Tropane derivatives labelled with radioisotopes have been previously used as selective tracers for dopamine transporter, e.g. in the diagnosis or study of the Parkinson's disease, misuse of some narcotic substances, and schizophrenia. 2β-carbomethoxy-3β-(4-fluorophenyl)- tropane labelled with C isotope or [ ^j-β-CFT or [ ⁿ C]WIN 35,428 or 8-(methyl- ⁿ C)-3-(4-fluorophenyl)-8- azabicyclo[3.2.l]octane-2-carboxylic acid methyl ester, [lR-(exo, exo)], has been used in PET studies in humans (1,2), (PET = .Positron Emission Tomography). 2β-carbo- ethoxy-3B-(4-iodophenyl)tropane labelled with I isotope or [ I]-β-CIT or [ I]RTI-55 or 8-(methyl)-3-(4-iodo- phenyl- 123I)-8-azabicyclo[3.2.l]octane-2-carboxylic acid methyl ester, [lR-(exo, exo)], and 2β-carbomethoxy-3β-(4- iodophenyl)tropane labelled with ^U C isotope or [ ^π C]-β-CIT or [ ⁿ C]RTI-55 or 8-(methyl- ^π C)-3-(4-iodophenyl)-8-aza- bicyclo[3.2.l]octane-2-carboxylic acid methyl ester, [1R- (exo, exo)] are other examples of tropane derivatives labelled with radioisotopes which have been employed in human imaging (3,4).

The drawback with the above ^u C-labelled tropane derivatives' is the short half life of ^U C (20.4 minutes). The biological properties of iodosubstituted compounds do not correspond to those of fluorosubstituted compounds.

Iodoisotope I 12 ₃ decays through so-called single gamma emission and consequently this isotope cannot be used in

PET studies.

Radioactive fluorine isotope ( 18F) has a longer half life (T ₁₂ = 109.8 min) than the ^l C isotope. Hence it is better suited to a radioactive label since kinetics of the above tropane derivatives in the brain is relatively slow. ⁱ⁸ F isotope (like ⁿ C isotope) decays through positron emission (β+) creating a directed beam which in turn makes PET imaging possible.

The prerequisite in the radioactive labelling of substances used in in vivo diagnostics is high specific radioactivity (specific radioactivity = the amount of radionuclide per mass of the substance) of the tracer. The molecules in question are generally very toxic so that they cannot be used in large quantities. For example, about 20 nmol (5.5 μg) of 2β-carbomethoxy-3β-(4-fluoro¬ phenyl)tropane labelled with ^U C isotope was injected to the patients and this quantity of the substance was not observed to have any adverse effects (1). Furthermore, the radiochemical synthesis of the tracer must be rapid owing to the half life of ¹⁸ F isotope.

Previously it has not been possible to use ¹⁸ F isotope for the electrophilic labelling of tropane derivatives because it was not possible to produce sufficient amounts of electrophilic ¹⁸ F isotope with a specific radioactivity higher than about 10 mCi/μmol.

Quite recently, a new method has been developed for the production of ¹⁸ F-labelled fluorine gas ([ ¹⁸ F]F ₂ ) with high specific radioactivity (5). This F-labelled gas may be used directly in the labelling synthesis, or acetylhypo- fluorite ( [ ¹⁸ F]CH ₃ COOF) may be prepared therefrom for subsequent use in the labelling synthesis (5,6). The

principal steps of the method are the following:

1) Water enriched with oxygen-18 isotope is irradiated with protons to produce 18F isotope as a solvatized ion in water.

2) The solvatized F ion is dried and activated with a cryptand. The negatively charged F ion produced is an efficient nucleophilic fluorination reagent.

3) The 18F ion is reacted with CH ₃ I to yield a relatively inert [ ¹⁸ F]CH ₃ F gas labelled with ¹⁸ F isotope.

4) The obtained [ F]CH ₃ F gas is chromatographically purified and transferred to a reaction chamber with the carrier gas mixture (noble gas / fluorine gas). The gas mixture is subjected to a high voltage. During the electric discharge gaseous F ₂ is produced wherein part of the F-atoms are ¹⁸ F isotopes. This ¹⁸ F-labelled F ₂ gas is highly reactive but unlike the fluorine gas obtained by the previous methods it also has a very high specific radioactivity. Classical methods produce fluorine gas with a specific radioactivity of 3-10 mCi/μmol whereas this new method produces fluorine gas with a specific radioactivity of over 100 mCi/μmol, preferably about 1500 mCi/μmol.

The high specific radioactivity 18F-fluorine gas prepared by the above method has been used for fluorine labelling of L-dopa (7) .

The characteristic features of the invention appear in claims 1, 3, 6, and 8.

Consequently the invention relates, among other things, to a novel chemical compound which has the structural formula

(I)

and which is useful in the diagnostics of dopaminergic diseases like the Parkinson's disease and schizophrenia.

The invention also relates to mixtures of the compound (I) with the corresponding non-radioactive compound (II),

The invention further relates to a method for producing the compound (I) and its mixtures with the compound (II)

Formula (I) covers all stereoisomers of the compound. Literature knows the following stereoisomers of the corresponding non-radioactive compound (II): a) 3-(4-

fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2- carboxylic acid methyl ester, [lR-(exo, exo)]-, (reference 8), b) 3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]- octane-2-carboxylic acid methyl ester, [lR-(2-exo,3- endo)]-, (reference 9), c) 3-(4-fluorophenyl)-8-methyl-8- azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester, [lS-(2-endo,3-exo) ]-, (reference 8), d) 3-(4-fluoro¬ phenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester, [IS-(exo, exo)]-, (reference 10), and e) 3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1Joctane- 2-carboxylic acid methyl ester, [lR-(2-endo, 3-exo)]-, (reference 8). Consequently, the formula (I) includes particularly the stereoisomers [lR-(exo, exo)]-, [lR-(2- exo,3-endo)]-, [lS-(2-endo,3-exo)]-, [IS-(exo, exo)]-, and [lR-(2-endo, 3-exo)]- of. the compound 3-(4-fluorophenyl- 18F)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester.

However, the compound (I) is advantageously 3-(4-fluoro- phenyl-18F)-8-methyl-8-azabicyclo[3.2.1]octane-2-carbox- ylic acid methyl ester, [lR-(exo, exo)]- or [ ¹⁸ F]-2β- carbomethoxy-3fi-(4-fluorophenyl)tropane. The compound (II) is a substance (code WIN 35428) patented by Sterling Drug, Inc. in 1972 and it is known as a very selective tracer of dopamine transporter.

A mixture comprising the compounds according to the formulas (I) and (II) must comprise the compound (I) in such an amount that the specific radioactivity of the mixture at the time of injection is at least 25 mCi/μmol. In practice, however, the specific radioactivity at the time of injection is preferably at least about 100 mCi/μmol. Very good imaging results are obtained if the corresponding value is at least about 250 mCi/μmol.

A compound according to the formula (I) and its mixture

with a compound of the formula (II) is prepared so that a compound of the formula (III)

wherein M is Sn, Hg or Si , and R _{l f} R ₂ , and R ₃ , which may be the same or different groups , are Ci-Cβ-alkyl or phenyl groups , are reacted either

- with ¹⁸ F-labelled fluorine gas or [ ¹⁸ F]F ₂ , or

- with ¹⁸ F-labelled acetylhypof luorite or [ ¹⁸ F] CH ₃ COOF .

The compound according to formula (III) is preferably 3- (4-trimethylstannylphenyl)-8-methyl-8-azabicyclo[3.2.1]- octane-2-carboxylic acid methyl ester, [lR-(exo, exo)]- or 2β-carbomethoxy-3β-(4-trimethylstannylphenyl)tropane, which is a commercially available substance. An example of another substance according to formula (III) known in literature is a compound where M is Sn and R R ₂ , and R ₃ are butyl groups (11).

In order to give the end product, which is used as the tracer, a sufficiently high specific radioactivity it is important that the reagent ([ ¹⁸ F]F ₂ or [ ¹⁸ F]CH ₃ COOF) has a certain minimum specific radioactivity in the beginning of

the synthesis of the tracer. Specific radioactivity diminishes as a function of time due to radioactive decay.

If radioactive fluorine gas i.e. [ 18F]F ₂ is used, the fluorine gas should comprise 18F isotope in such an amount that the specific radioactivity of the gas is at least about 100 mCi/μmol, however, preferably at least about 1000 mCi/μmol. If on the other hand radioactive acetylhypofluorite or [ F]CH ₃ C00F is used, the reagent

18 must comprise the compound CH ₃ C00 F in such an amount that the specific radioactivity of the mixture is at least about 50 mCi/μmol, however, preferably at least about 500 mCi/μmol.

The invention will be described in more detail in the following non-limiting examples. The following materials and methods were used in the tests described as examples:

The reagents and solvents were of analytical grade and were commercially available. Kryptofix 222 (4, 7, 13, 16, 21, 24 - Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) , potassium carbonate (K ₂ C0 ₃ ), glacial acetic acid (CH ₃ C00H) and ammonium acetate (CH ₃ C00NH ₄ ) were from Merck; methyl iodide (CH ₃ I) and acetonitrile from Aldrich, and Freon 11 (CFC1 ₃ ) from Fluka. The labelling precursor or 2β-carbo- methoxy-3β-(4-trimethylstannylphenyl)tropane was purchased from Research Biochemicals Inc. in vials each containing 50 μg of the substance. In all tests, the labelling reac¬ tion was made in the same one ml vials in which the labelling precursor was supplied.

Example 1

Preparation of the labelling reagents

Preparation of the radionuclide:

[ ¹⁸ F]-fluoride was prepared by irradiating 400 μl ¹⁸ 0-enriched water (94 %, Isotec, Inc.) in an open silver chamber with protons (10-12 μA, 16 MeV, 60-90 min) using the Abo Akademi 103 cm isochronous cyclotron. The solvatized [ ¹⁸ F]F ^" was transferred through silicon tubes into a reaction flask in the radiocheraistry laboratory.

Preparation of the labelling reagents ([ ¹⁸ F]F ₂ and [ ¹⁸ F]- CH ₃ COOF) :

Details of the preparation process appear in the earlier publications (5, 6, 7). The [ ¹⁸ F]Kryptofix/K ⁺ F ^" complex was allowed to react with methylene iodide to yield [ ¹⁸ F]CH ₃ F. This was isolated with a Haysep Q (80/100 mesh, Ohio Valley Specialty Chemical) gas chromatography column (inner diameter 0.8 cm, length 30 cm) using neon (99.995 %, Aga Special gases) as the sweep gas. The [ ¹⁸ F]CH ₃ F was collected at liquid nitrogen temperature, after which it together with the neon/fluorine gas was passed into the reaction chamber (5) at a pressure of 3.7 bar. The mixture was subjected to a voltage of 20 kV (current 300 μA) for 10 seconds through the metal electrodes in the chamber thereby yielding [ F]F ₂ as the reaction product. Either [ ¹⁸ F]F ₂ -gas or acetylhypofluorite [ ¹⁸ F]CH ₃ COOF prepared further therefrom may be used as the fluorination reagent for the labelling of the precursor.

In the preparation of radioactive acetylhypofluorite, the

[ 18F]F ₂ was conducted to the acetic acid/ammonium acetate solution thereby yielding radioactive acetylhypofluorite [ ¹⁸ F]CH ₃ COOF.

Example 2

3-(4-fluorophenyl-18F)-8-methyl-8-azabicyclo[3.2.1]octane -

2-carboxylic acid methyl ester, [lR-(exo, exo)]- or [ ¹⁸ F]- 2β-carbomethoxy-3β-(4-fluorophenyl)tropane

a) preparation using the [ 18F]F ₂ -reagent

[ F]F ₂ was passed directly into a vial containing 50 μg of 2β-carbomethoxy-3β-(4-trimethylstannylphenyl)tropane dissolved in 0.6 ml of Freon 11. The gas was bubbled until the solvent had evaporated. The solid residue was dissolved in one ml of the mobile phase of the high pressure liquid chromatograph and injected onto the liquid chromatograph column for purification. The purified end product was collected from the column outlet. The end product was chromatographically identified by comparing it with a reference standard (WIN 35,428, Research Biochemicals Int.). The specific radioactivity of the obtained product was about 400 mCi/μmol.

b) preparation using the [ ¹⁸ F]CH ₃ COOF-reagent

The [ 18F]acetylhypofluorite in acetic acid was passed into a vial containing 50 μg of 2β-carbomethoxy-3β-(4-tri- methylstannylphenyl)tropane. The desired end product was purified and identified as above. The specific radioactivity of the obtained product was about 400 mCi/μmol.

Animal experiments

Accumulation of [ F]CFT and metabolism in a living tissue were studied in test animals. Spraque-Dawley rats slightly anaesthetized with ether were injected i.v. with [ ¹⁸ F]CFT (about 200-400 μCi, specific radioactivity about 350 mCi/μmol). The rats were killed at different instants of time and the brains were cut into 16 μm slices with a

cryomicrotome. Distribution of the radioactivity in the slices were measured with a Molecular imager instrument (Bio Rad, USA), see Figs. 1 and 2. From the figures radioactivity of various brain regions was analyzed, e.g. striatum (basal ganglia) which is known to occupy a lot of dopamine reuptake sites, and cerebellum (little brain) which has a small number of such sites. By means of the different time points it was possible to determine the accumulation and the striatum to cerebellum ratio as a function of time, see Fig. 3. Specificity of the labelled CFT for dopamine transporter was tested by administering to some rats inactive medicament (GBR 12935 (RBI)) which has a strong affinity for dopamine transporter, 15 g/kg half an hour before the [ F]CFT injection. Fig. 2 shows that the inactive medicament has already saturated the reuptake sites of dopamine and no specific [ ¹⁸ F]CFT accumulation can be seen. Other rats were in a corresponding way given medicament which had strong affinity for serotonin transporter (citalopram). Similar displacement is not observed though citalopram affects the [ ¹⁸ F]CFT metabolism in the rat liver.

On the basis of the animal tests [ F]CFT is a promising tracer of dopamine transporter.

It is obvious to a specialist in the field that various embodiments of the invention may vary within the limits of the enclosed claims.

References:

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2. Frost JJ et al. , Ann Neurol 34:423-431 (1993)

3. Kuikka JT et al., Eur J Nucl Med 20:783-786 (1993) 4. Farde L et al. , Synapse 16:93-103 (1994)

5. Bergman J et al., J Nucl Med (suppl) 34 (1993) 69P

6. Solin 0 et al., J Label Comp Radiopharm 32 (1993) 137- 138

7. Bergman J et al., J Label Comp Radiopharm 35 (1994), Symposiun Abstract, 476-477

8. Clarke R et al., J Med Chem (1973), 16(1), 1260-7

9. Madras B & Meltzer K, Int. Pat. Publ. WO 94/04146

10. Milius R et al. , J Med Chem (1991) 34(5), 1728-31

11. Neu eyer JL et al., J med Chem 1991, 34, 3144-3146