WO1995011234A1

WO1995011234A1 - Radiolabelled ligands for muscarinic receptors

Info

Publication number: WO1995011234A1
Application number: PCT/AU1994/000643
Authority: WO
Inventors: Michael Kassiou; Andrew G. Katsifis; Richard M. Lambrecht; Rodney Hicks
Original assignee: Australian Nuclear Science & Technology Organisation
Priority date: 1993-10-22
Filing date: 1994-10-21
Publication date: 1995-04-27

Abstract

Compounds of formula (I), radiolabelled compounds or pharmaceutically acceptable salts thereof where X- is a suitable counter ion; R¿1? is H; alkyl, alkenyl, alkynyl, phenyl or phenylalkyl each optionally substituted; R2 is one or more selected from hydrogen, halogen, radioactive isotopes of halogen, OR, CN, CF3, NO2, COOR, NRR', SR, COR, CONRR', SO3R, SO2NRR', SOR and SO2R where R and R' are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted phenyl or optionally substituted phenylalkyl; wherein the optional substituents are one or more selected from alkyl, alkenyl, alkynyl, phenyl, halogen, radioactive isotopes of halogen, OR, CN, CF3, NO2, COOR, NRR', SR, COR, CONRR', SO3R, SO2NRR', SOR and SO2R where R and R' are as defined above suitable for imaging muscarinic receptors of the heart.

Description

RADIOLABELLED LIGANDS FOR MUSCARINIC RECEPTORS

Technical Field The present invention relates to novel benzetimide derivatives and in particular to novel N-derivatives of Dexetimide suitable for PET studies and SPECT studies of muscarinic receptors.

Background Art

The presence of muscarinic acetylcholine receptors

( AChRs) in the mammalian system, predominantly in the heart, pancreas, intestinal smooth muscle and several structures of the brain, mediate the effects of acetylcholine and other cholinergic drugs. Changes in mAChR density occur in various physiological, pharmacological and clinical conditions. In the heart, mAChR mediate a slowing in the rate of contraction as well as a decrease in the force of contraction. Delfolge et al. (1990) have reported that altered muscarinic receptor distribution in the heart may be a cause of cardiac arrhythmia which lead to cardiac arrest. Varastet et al. (1992) have reported attempts to image myocardial mAChR involving the use of radiotracers such as [^1:LC]MQNB and [^1XC]MTRB with limited SPECT radiotracers available. Recently, atsumura et al. (1991) reported the use of [¹²³I] -4-iododexetimide, ( [¹²³I]IDEX) , a potent mAChR antagonist, for in vivo studies of myocardial mAChR which proved unsuccessful due to its low heart to lung ratio (H/L) and high non-specific binding.

The present inventors have prepared novel quaternary ammonium derivatives of benzetimide and in particular derivatives of dexetimide for PET studies and SPECT studies of myocardial mAChR.

Disclosure of the Invention In one aspect, the present invention provides compounds of formula (I)

radiolabelled compounds or pharmaceutically acceptable salts thereof where X- is a suitable counter ion;

R_x is H; alkyl, alkenyl, alkynyl or phenylalkyl each optionally substituted;

R₂ is one or more selected from hydrogen, halogen, radioactive isotopes of halogen, OR, CN, CF₃, N0₂, COOR, NRR', SR, COR, CONRR*, S0₃R, S0₂NRR', SOR and S0₂R where R and R' are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted phenyl or optionally substituted phenylalkyl; wherein the optional substituents are one or more selected from alkyl, alkenyl, alkynyl, phenyl, halogen, radioactive isotopes of halogen, OR, CN, CF₃, N0₂, COOR, NRR', SR, COR, CONRR', S0₃R, S0₂NRR', SOR and S0₂R where R and R' are as defined above.

Preferably, X^~ is a halide such as iodide but the skilled addressee would understand that the counter ion X^~ is dependent upon the reagent used to alkylate or arylate the nitrogen atom to provide the novel quaternary ammonium compounds.

The optionally substituted alkyl, phenyl or phenylalkyl groups may incorporate the isotopes C-ll, F- 18, Br-75, Br-76, Br-77 or 1-123. For example, methyl, ethyl, propyl, isopropyl, butyl, can be where one or more of the carbon atoms is C-ll. As a further example, fluoroethyl, fluoropropyl, fluorobutyl can be where fluoro represents F-19 or F-18. Where R_x is optionally substituted phenyl, the substituents can be F-18, 1-123, 1-124, 1-125, 1-131, Br-75, Br-76 or Br-77. Where R is halogen or has a halogen substituent, the halogen can be I, Br, F or Cl or F-18, 1-123, 1-124, I- 125, 1-131, Br-75 or Br-76.

For alkyl, alkenyl, alkynyl and phenylalkyl the carbon chain length is preferably ^- Cq .

The astrict (*) in the structural formula (I) represents a stereogenic (chiral) centre and it is to be understood that the present invention includes within its scope racemic mixtures of compounds of formula (I) as well as the individual isomers.

Preferably, the compound of formula (I) is [⁷⁶Br] -N- methyl-4-bromodexetimide or [¹²³I] -N-methyl-4- iododexetimide.

In a second aspect, the present invention provides a method of preparation of compounds of formula (I) , radiolabelled compounds or pharmaceutically acceptable salts thereof which comprises:

A. (i) treating norbenzylbenzetimide with the appropriately substituted benzyl derivative as follows:

(ii) followed by alkylation or arylation as follows:

where X is a suitable leaving group; B. where R_λ is R₂PhCH₂- step A(i) as above with 2 molar equivalents of the benzyl derivative; or

C. where R₂ is a radioactive halogen **R₂

(i) step A(i) as above where R₂ is (CH₃)₃Si; (ii) treatment with Na**R₂ or NH₄**R₂;

(iii) followed by step A(ii) as above. The benzyl derivative is preferably benzylhalide, more preferably benzylbromide.

In a third aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula (I) , radiolabelled compounds and/or salts thereof in a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier is saline. In a fourth aspect, the present invention provides a method of imaging in a subject of muscarinic receptors of the heart which comprises administering to the subject a radiolabelled compound of formula (I) as defined above.

In a further aspect, the present invention provides a method of diagnosis in a subject of disorders related to muscarinic receptors of the heart which comprises administering to the subject a radiolabelled compound of formula (I) as defined abovee.

Typically, the disorders which can be diagnosed are ischemia, arrythmia and diabetes.

The present invention further provides radiolabelled compounds of formula (I) and/or pharmaceutically acceptable salts thereof as defined above as radiopharmaceuticals for imaging muscarinic receptors of the heart.

The present invention also provides the use of radiolabelled compounds of formula (I) and/or pharmaceutically acceptable salts thereof as defined above as radiopharmaceuticals in the manufacture of medicaments for imaging muscarinic receptors of the heart. Brief Description of Drawings

Figure 1 shows specificity and stereoselective of

[¹²³I]MIDEX binding at 10 minutes [demonstrated by coinjecting a cold load of levetimide (LEV 0.15 mg/kg) , dexetimide (DEX 0.15 mg/kg) and methyl-quinuclidinyl benzylate ( QNB 1 mg/kg) ] .

Figure 2 shows the uptake of [⁷⁶Br] -MBDEX when the peripheral muscarinic receptors were blocked by the coinjection of a load of cold methyl-quinuclidinyl benzylate (MQNB, 1 mg/kg) .

Modes for Carrying Out the Invention

Dexetimide and its derivatives were prepared according to literature procedures or modifications of these procedures. Racemic benzetimide was prepared by the procedure of Hermans et al. (1968). The resolution of benzetimide to dexetimide and levetimide was carried out according to the procedure of Wijngaarden (1969) as shown in Scheme 1. The preparation of halogenated dexetimides follows the procedures of Wilson (1989) and Dah-ren Hwang (1991) . Norbenzetimide is prepared from benzetimide by catalytic transfer hydrogenolysis. If benzetimide hydrochloride is used this is preferably converted to the free amine before catalytic hydrogenolysis. Benzetimide is commercially available or can be prepared according to the above procedure. Dexetimide is also commercially available.

The pharmaceutically acceptable salts are prepared by conventional techniques in the art known to the skilled addressee.

The pharmaceutical formulations or medicaments according to the present invention can be prepared by the skilled addressee according to techniques known in the art. The effective amount of the active compound required for imaging or therapy will vary both with the tissue to be imaged, the condition under treatment and the host undergoing imaging or treatment, and is ultimately at the discretion of the physician. Synthesis and Resolution of Benzetimide

(R)-Lθvetlmldθ (x=H) (S)-Dθxθti liiθ (x=H)

SCHEME 1 [⁷⁶Br] -4-bromodexetimide

The radiosynthesis of [⁷⁶Br] -4-bromodexetimide ( [⁷⁶Br] -BDEX) was carried out by bromination via electrophilic bromodesilylation with no carrier added [⁷⁶Br]NH₄. During the preparation of this radiopharmaceutical a number of reaction conditions and reagents were examined. Oxidising agents such as peracetic acid and dichloramine-T were evaluated. Peracetic acid reactions were carried out in acetic acid with radiochemical yields of 6%. Dichloramine-T reactions were conducted in both methanol and TFA solvent with radiochemical yields of 10% and 24% respectively. The chloramine-T reactions were most efficient but were also concentration and solvent dependent. The optimum labelling conditions were found to be the use of chloramine-T (10^"3M) in 0.IN HC1 at room temperature for 15 min followed by addition of a sodium metabisulfite solution. Under these conditions the radiochemical yield reaches 80%. The purification and isolation of the radiotracer from the reaction mixture was carried out by

HPLC on a μ-Bondapak C18 column (300 x 7.8mm) with a mixture of acetonitrile and ammonium acetate buffer

(45:55) as the mobile phase and a flow rate of 2.5 ml/min while UV absorption was measured at 239nm. Radiochemical and chemical purities assessed by radio-TLC and HPLC were 98% with a specific activity > 11 GBq/μmol. [⁷⁶Br] -N-methyl-4-bromodexetimide

The radiosynthesis involves the preparation of [^7GBr] -4-bromodexetimide ( [⁷⁶Br] -BDEX) first by electrophilic bromodesilylation as described, followed by treatment with CH₃I. The reaction was carried out by dissolving [⁷⁶Br] -BDEX (50 MBq) in tributyl phosphate (lOOμL) followed by an addition of an excess of CH₃I (200μL) . Allowing the reaction mixture to stand at room temperature for 10 min resulted in the formation of the quaternary salt in a 34% radiochemical yield. When the temperature is increased to 90°C, radiochemical yields reached 95%. After evaporating excess methyl iodide and cooling the reaction mixture, isolation and purification of the radiopharmaceutical was carried out by HPLC on a μ-Bondapak C18 column (300 x 7.8 mm) while the UV absorption was measured at 239 nm. With acetonitrile and 0.IM ammonium acetate buffer (45.55, v:v) as mobile phase and a flow rate of 2.5 mL/min, the retention times of [⁷⁶Br] -MBDEX and [⁷⁶Br] -BDEX were 27 and 37 min respectively. Radiochemical and chemical purities assessed by radio-TLC and HPLC were 99% and the specific activity of [⁷⁶Br] -MBDEX was identical to that of [⁷⁶Br] - BDEX (>11 GBq/μmol)_,. [¹²³I] -N-methyl-4-iododexetimide ( [¹²³I]MIDEX)

The radiosynthesis of [¹²³I]MIDEX involves the initial preparation of [¹²³I] IDEX by electrophilic iododesilylation in trifluoroacetic acid in the presence of chloramine-T (similar to the procedure of Wilson et al., 1989) followed by treatment of [¹²³I] IDEX with excess methyl iodide (300 μl) the methylation reaction is carried out by dissolving [¹²³I] IDEX (10 mCi) in tributyl phosphate (50-100 μl) followed by addition of methyl iodide. The reaction mixture was tightly stoppered and heated at 90° for 10-15 min. After evaporating the excess methyl iodide and cooling the mixture, isolation and purification were carried out by preparative HPLC on a μ-Bondapak C-18 RP column (300 x 7.8 mm) with a mobile phase consisting of acetonitrile and 0.IM ammonium acetate buffer (45:55 v:v) and a flow rate of 2.5 ml/min. The retention times of [¹²³1] IDEX and [¹²³I]MIDEX were 38 and 26 min respectively. Radiochemical yields of 80-90 % were achieved with radiochemical and chemical purities greater than 97%. The specific activity of [¹²³I]MIDEX was similar to [¹²³I] IDEX (> 2000 mCi/μmol) . [¹²³I]N- methyl-4-iodolevetimide ( [¹²³I]MILEV) was also synthesized in radiochemical yields and specific activity similar to [^{1 3}I]MIDEX. UV absorption was measured at 239nm and radioactivity measured on a Berthold system. AFFINITY AND BIODISTRIBUTION STUDIES [⁷⁶Br] -BDEX - 9 -

The affinity and selectivity of [⁷⁶Br] -BDEX for muscarinic receptors was investigated in vitro and in vivo in rats and non human primates. In vitro, the binding parameters of this new radioligand were evaluated on homogenates of rat cerebral cortex membranes. Analysis of saturation data using a non linear least square regression method revealed a single population of binding sites (nH = 0.99 B_maχ=130±7pmol/g tissue) with an apparent dissociation constant of 1.9 ± 0.3nM. In vivo, the biodistribution of this new tracer was studied in rats. In the brain, 2h post injection, the -highest concentrations (about 5 x 10 ^"3 of the injected dose per gram) were found in the frontal cortex, the hippocampus and the striata while the cerebellar radioactive concentration was very similar to that of the plasma. Ex vivo autoradiographic studies have shown that this preferential accumulation was totally prevented by the co-administration of a cold load (0.1 mg/kg) of a muscarinic antagonist, scopolamine. A metabolite study performed on cortex, cerebellum and plasma samples showed that 1 hr post injection, 98, 79 and 20% of the radioactivity respectively was related to unchanged [⁷⁶Br] -BDEX. [¹²³I]MIDEX Rat biodistribution studies were performed and showed high heart uptake (2.4%ID/g) 10 minutes after injection with a heart to lung radioactivity concentration ratio (H/L) of 5.1. The H/L ratio decreased rapidly to 2.2 after 30 minutes and reached unity at 60 minutes. No uptake of [^{1 3}I]MIDEX was observed in the brain. The specificity and stereoselectivity of [¹²³I]MIDEX binding at 10 minutes was demonstrated by coinjecting a cold load of levetimide (LEV 0.15 mg/kg), dexetimide (DEX 0.15 mg/kg) and methyl- quinuclidinyl benzylate (MQNB 1 mg/kg) (fig. 1) . With DEX and MQNB the heart uptake was reduced to 0.20 and 0.13 %ID/g displacing 92% and 95% of the activity respectively while LEV maintained high heart uptake (2.2 %ID/g) . Interestingly, the kidney uptake was 21% ID/g and remained constant over a period of 30 minutes. Preliminary SPECT studies carried out on rabbit and dog suggest that [¹²³I]MIDEX has the potential of being developed as a SPECT radiotracer for the characterisation of myocardial muscarinic receptors.

Imaging of Cardiac Muscarinic Receptors with 1-123 N- Methyl-4-Iododexetimide [¹²³I]MIDEX

Muscarinic cholinergic receptors mediate vagal effects on the heart. With vagal reflexes being more sensitive than sympathetic reflexes to subendocardial ischemia, parasympathetic denervation may be important in the genesis of cardiac arrhythmias in ischemic heart disease. Cardiac muscarinic receptor ligands for PET have been characterized but single photon agents have not previously been available.

Serial imaging in anaesthetised dogs revealed rapid cardiac uptake and stable heart to lung activity ratios of >2.7:1 between 10 and 30 minutes making SPECT feasible. The L-isomer, 1-123 levetimide had no detectable myocardial uptake suggesting specific, stereo- selective receptor binding. Cold methyl-quinuclidinyl benzylate (MQNB) rapidly displaced myocardial MIDEX activity to background levels. SPECT revealed higher activity in the inferior than anterior wall consistent with the known regional variation of cardiac parasympathetic innervation. Thus, MIDEX shows promise as an imaging agent of cardiac muscarinic receptor distribution. [⁷⁶Br] -MBDEX

In vivo biodistribution studies in rat showed a high uptake in the heart (2% ID/g) that decreased with a half- life of "3-4 min; 5 min after injection, the heart to lung radioactivity concentration ratio (H/L) was 3.5. No uptake of [⁷⁶Br] -MBDEX was observed in the brain.

When the peripheral muscarinic receptors were blocked by the co-injection of a load of cold methyl-quinuclidinyl benzylate (MQNB, 1 mg/kg) , the heart uptake was reduced to 0.12% ID/g (H/L = 0.27) (fig. 2) . Radio-TLC analysis indicated that [⁷⁶Br] -MBDEX was rapidly metabolised in plasma (20% of unchanged tracer 10 min after injection) and showed that the radioactive metabolites were essentially polar by-products. In PET studies in dogs,

[⁷⁶Br] -MBDEX accumulated rapidly in the heart; the uptake remained in a plateau for 10 min and then decreased with a half-life of 20 min. Due to a rapid wash-out in the lungs, 3 min after injection, the heart to lung ratio reached a value of 8. The uptake of [⁷⁶Br] -MBDEX was displaced by MQNB (30μg/kg) that demonstrated the specificity and saturability of the binding of

[⁷⁶Br] -MBDEX to muscarinic receptors in the heart. From these preliminary animal studies it appears that [⁷⁶Br] -N- methyl-4-bromodexetimide has the potential of being developed as a useful PET radiotracer for the characterisation of heart muscarinic receptors.

Biodistribution studies in rats with [¹²³I]MIDEX and

[¹²³I] IDEX indicate significant uptake of both tracers in the heart (Table 1) . However, a significant reduction in lung and brain uptake is observed for [¹²³I]MIDEX (Table 2) .

TABLE 1. Biodistribution Studies of [1-123] -IDEX and [1-123] -MIDEX in Wistar Rats % injected dose/gram

TABLE 2. Biodistribution of 1-123

N-Methyl-4-iododexetimide in Wistar Rats % injected dose/gram

A heart uptake of (2.4% ID/g) 10 min after injection of [¹²³I]MIDEX with a heart to lung ratio (H/L) of 5.1 is observed. The H/L ratio decreased rapidly to 2.2 after 30 minutes and reached unity at 60 minutes. The specificity and stereoselectivity of [¹²³I]MIDEX binding at 10 minutes were demonstrated by coinjecting a cold load of levetimide (LEV 0.15 mg/kg), dexetimide (DEX 0.15 mg/kg) and methyl-quinuclidinyl benzylate (MQNB 1 mg/kg) . A significant reduction in heart uptake was observed with DEX and MQNB to 0.2 and 0.13 %ID/g (displacing 92% and 95% of the activity) respectively. With LEV, high heart uptake of [¹²³I]MIDEX was maintained (2.2%ID/g) . Biodistribution studies with [¹²³I] -N-methyl-4- Iodolevetimide ( [¹²³I]MILEV) , indicated no uptake in the organs of interest (Table 3) . The less active enantiomer

( [¹²³I]MILEV) can therefore be used to determine the amount of non-specific binding. The specific binding of

[¹²³I]MIDEX was estimated to be 94%. Both isomers have shown high activity in the kidneys. TABLE 3. Biodistribution of 1-123

N-Methyl-4-iodolevetimide in Wistar Rats % injected dose/gram

The above observations were confirmed by kinetic SPECT studies on rabbits and dogs. Good heart uptake was observed with no significant accumulation in the lungs.

The activity in the heart was markedly reduced when MQNB was administered to the animals 15 minutes after

[¹²³I]MIDEX administration. In a separate experiment, no uptake of [¹²³I]MILEV was observed clearly illustrating stereoselective binding to the mAChR.

The specificity and stereoselectivity of [¹²³I]MIDEX for myocardial mAChR has been demonstrated. The high heart to lung ratio and the availability of [¹²³I]MILEV for the estimation of non-specific binding suggests that in the absence of significant lung uptake [¹²³I]MIDEX has the potential of being developed as a useful SPECT radiotracer for imaging studies of myocardial mAChR. EXPERIMENTAL Example 1

(S) - (+) -3-phenyl-3- (4-piperidinyl) -2, 6-piperidinedione 3. (Ref 8) Briefly S (+) -Dexetimide hydrochloride (2.0 g) was converted to the free amine by treatment of the hydrochloride salt with IN NaOH 8 ml in methanol (20 ml) . The methanol was removed under reduced pressure and the residue extracted with dichloromethane (3X50 ml) . The organic phase was dried (Na₂S0₄) and the solvent evaporated to give a white solid 1.5 g. The residue was dissolved in 60 ml of methanol, to which 3 ml of 88% formic acid and 1.5 g of palladium black were added. The mixture was then stirred at room temperature until the disappearance of the starting material, monitoring by TLC silica gel, MeOH/CH₂Cl₂/NH₄OH=l/9/0.5, R_f = 0.9; about 3 hr. Pd/C was removed by filtration. The filtrate was concentrated under vacuum to a small volume and a cold 2N NaOH solution was carefully added to the residue until it was basic. The mixture was extracted with CH₂C1₂ and the organic layer dried over sodium sulphate, filtered and concentrated under vacuum to give a white solid 0.8g. If necessary the product can be further purified by flash chromatography using the above solvent system. Example 2 (a)

(S) -3-Phenyl-3- [1- [ (4-iodophenyl)methyl] -4-piperidinyl] - 2,6-piperidinedione. (4-Iodo-dexetimide) 4 (Ref 4) A mixture of 3 (250 mg, 0.92 mmol) 4-iodobenzyl bromide (273 g 0.92 mmol) and potassium carbonate (0.19 g, 1.8 mmol) in ethanol 20 ml was heated to reflux for 3-4 hours and then cooled. The mixture was filtered to remove inorganics and the solvent evaporated to leave a pale yellow solid. This solid was treated with boiling ether (3X20 ml) and filtered. The filtrate was washed with water, dried over anhydrous sodium sulfate and filtered. Hydrogen chloride gas was passed through the filtrate and the resultant white precipitate collected by vacuum filtration. Dissolution in methanol (20 ml) filtration and evaporation of the methanol gave a white solid as the hydrochloride salt (340 mg, 76%) . Example 2 (b) The above compound can be prepared in a more convenient manner and in higher yields by heating to reflux a mixture of 3 (O.lOg, 0.36 mmol) and 4-iodobenzyl bromide

(0.107 g, 0.36 mmol) in the presence of potassium carbonate (O.lOg, 0.72 mmol) in dry toluene (30 ml) for 6-12 h under nitrogen. After cooling, the toluene was evaporated and the residue triturated with water. The aqueous mixture was extracted into chloroform (3X30ml) dried (Na₂S0₄) and evaporated to give a white solid

(0.155g, 89%) of the neutral compound. Trace amounts of the starting amine which may be present in the reaction mixture can be removed by column chromatography dichloromethane:methanol (95:5) . Example 3 (a) [1- [ (4-bromophenyl)methyl] -4-piperidinyl] -2, 6- piperidinedione. (4-bromodexetimide) 5

A mixture of 3 (0.250g, 0.92 mmol) 4-bromobenzyl bromide

(230 mg 0.92 mg) and potassium carbonate (0.19 g, 1.8 mmol) in ethanol 20 ml was heated to reflux for 3-4 hours and then cooled. The mixture was filtered to remove inorganics and the solvent evaporated to leave a pale yellow solid. This solid was treated with boiling ether

(3X20 ml) and filtered. The filtrate was washed with water, dried over anhydrous sodium sulfate and filtered.

Hydrogen chloride gas was passed through the filtrate and the resultant white precipitate collected by vacuum filtration. Dissolution in methanol (20 ml) filtration and evaporation of the methanol gave a white solid as the hydrochloride salt (0.33 g, 82%) . Example 3 (b) The above compound can be easily prepared in higher yields by heating to reflux a mixture of 3 (0.250g, 0.92 mmol) and 4-bromobenzyl bromide (0.230g, 0.92 mmol) in the presence of potassium carbonate (0.19 g, 1.8 mmol) in dry toluene (30 ml) for 6-12 h under nitrogen. After cooling, the toluene was evaporated and the residue triturated with water. The aqueous mixture was extracted into chloroform (3X30ml) dried (Na₂S0₄) and evaporated to give a white solid (0.344 g, 85%) of the neutral compound. Trace amounts of the starting amine which may be present in the reaction mixture can be removed by column chromatography dichloromethane:methanol (95:5) . Example 4 (S) -3-Phenyl-3- [1- [ (4- iodophenyl ) methyl] - 4 - N- methylpiperidinyl] -2, 6-piperidinedione iodide 6. 4-Iododexetimide 4 (0.70g, 1.4 mmol) was dissolved in dry benzene (10 ml) and treated with one equivalent of methyl iodide (0.203 g, 0.09 ml, 1.4 mmol) . The solution was tightly stoppered and left to stand in the dark overnight. The resultant white precipitate was filtered, washed with benzene and then dried under high vacuum to give the N-methyl-4-iododexetimide as a white solid

(0.80g, 90%) . ¹H NMR spectrum (CDC1₃) δ; 1.2 - 2.2 m, (9H) ; 3.1 s, CH₃; (3H) ; 3.25 - 4.12 m, (4H) ; 5.1 d, (1H) ; 5.2 d, (1H) ; 7.2 d, (2H) , (Ar) ; 7.3 - 7.45, m, (5H) , (Ar) ; 7.7 d, (2H) , (Ar) ; 8.1, s, (1H) . Example 5 (S) -3-Phenyl-3- [1- [ (4 -bromophenyl ) methyl] - 4 -N- methylpiperidinyl] -2, 6-piperidinedione iodide 7.

4-bromodexetimide 5 (0.692 g, 1.57 mmol) was dissolved in dry benzene (10 ml) and treated with one equivalent of methyl iodide (0.22g, 0.1 ml) . The solution was tightly stoppered and left to stand in the dark overnight. The resultant white precipitate was filtered, washed with benzene and then dried under high vacuum to give the N- methyl-4-bromodexetimide as a white solid (0.81g, 85%)

¹H NMR spectrum (CDC1₃) δ; 1.2 - 2.7 m, (9H) ; 3.12 s, CH₃,

(3H) ; 3.34 - 4.1 m, (4H) ; 5.1, d, (1H) ; 5.2 d, (1H) ; 7.2 d, (2H) ; 7.25 - 7.45 , (5H) , (Ar) ; 7.7 d, (2H) , (Ar) ; 8.2 s, (1H) . Example 6

(S) -3-Phenyl-3- [1- (phenylmethyl) -4-N-methylpiperidinyl] - 2, 6-piperidinedione iodide 8. Dexetimide 3 (0.10 g, 0.27 mmol) was dissolved in dry benzene (10 ml) and treated with one equivalent of methyl iodide (40 mg, 0.27 mmol) . The solution was tightly stoppered and left to stand in the dark overnight. The resultant white precipitate was filtered, washed with benzene and then dried under high vacuum to give the N- methyldexetimide as a white solid. ^XH NMR spectrum (CDCI₃) δ; 1.2 - 2.8 m, (9H) ; 3.13 s, CH₃, (3H) ; 3.3 - 4.16 m (4H) ; 5.05 d, (1H) , 5.14 d, (1H) ; 7.2 - 7.7 m, (10H) , (Ar) ; 8.2 s, (1H) . Mass spectrum: Fab (+ve) , (glycerol) . 377 (M+) , 285, 188. Example 7 (S) -3-Phenyl-3- [1- [ (4- iodophenyl) methyl] -4-N- [ (4- iodophenyl)methyl]piperidinyl] -2, 6-piperidinedione iodide 9.

A mixture of the amine 3 (110 mg, 0.4 mmol) 4-iodobenzyl bromide (290 mg, 0.97 mmol) and K₂C0₃ (0.12 g, 0.94 mmol) in toluene (15 ml) was stirred at reflux for 12 hours. The mixture was cooled and the toluene removed under reduced pressure. The residue was triturated with water extracted into chloroform (3x20 ml) . The organic phase was dried (Na₂S0₄) filtered, and evaporated to dryness to give a white solid. Further purification was carried out with column chromatography using dichloromethane:methanol (70:30) . Example 8

(S) -3-Phenyl-3- [1- [ [4- (trimethylsilyl)phenyl]methyl] -4- piperidinyl] -2, 6-piperidinedione 10. A mixture of the amine 3 (150 mg, 0.55 mmol) and Na₂C0₃ (75 mg, 0.70 mmol) in ethanol (10 ml) was stirred at r e f l u x w h i l e a s o l u t i o n o f 4 - (trimethylsilyl)benzylbromide (0.137 g, 0.56 mmol) in ethanol (10 ml) was added dropwise over 1 hour. After stirring for a further 30 minutes the mixture was cooled and the ethanol removed under reduced pressure. The residue was taken up in Chloroform (20 ml) and washed with water (20 ml) and aqueous HC1 (1 M, 20 ml) . The organic phase was dried (Na₂S0₄) filtered, and evaporated to dryness to give a white solid. The material was triturated with petroleum ether and then dissolved in boiling THF (10 ml) . Upon cooling hexane was added to precipitate a white solid which was collected, washed with cold THF and dried to give the silyl-dexetimide (0.109g, 46%) . Example 9

(S) -3 -Phenyl -3 - [1- [ (4- [¹²³I] iodophenyl) methyl] -4- piperidinyl] -2-6-piperidinedione 11.

A freshly prepared solution of 10 (0.5-1.0 mg) in trifluoroacetic acid (TFA) (100 μl) was added to chloramine-T (1.5 mg) . The resultant solution was added to a vial containing aqueous sodium [¹²³I] iodide (5-120 μl, 0.5-100 mCi) . After 15 min the reaction mixture was neutralized with a mixture of concentrated aqueous ammonia (70 μl) in HPLC buffer (acetonitrile: 0.IM ammonium acetate 45:55) . The solution was then injected onto a semipreparative HPLC column. (Waters C18 Novapak, 250x7.8 mm) using a flowrate of 2.5 ml/min. The desired fraction (t_R 38 min) was collected and evaporated to dryness and used directly in the next step. Example 10 (S) -3-Phenyl-3- [l- [ (4- [¹²³I] iodophenyl ) methyl] -4-N- methylpiperidinyl] -2, 6-piperidinedione iodide. (N-Methyl- 4- [¹²³I] Iododexetimide) 12.

The above 4- [¹²³I] Iododexetimide 11 (0.5-50 mCi) was dissolved in tri-n-butylphosphate (50-100 μl) . This was then treated with methyl iodide (100-300 μl) tightly stoppered and heated to 80-90° for 15 min. The reaction mixture was cooled diluted with buffer 1 ml and the entire contents injected onto the HPLC column. (Conditions as above) . The N-methyl 4- [¹²³I] iodo¬ dexetimide, t_R 18-20 minutes, was collected, evaporated to dryness and dissolved into 0.9% saline. Adjustment of pH (6-8) and sterile filtration through a 0.22 μ filter gave the required product suitable for biological evaluation. The (R)-(-)- enantiomers were prepared in a similar fashion using (R) - (-) -3-Phenyl-3- (4-piperidinyl) -2, 6- piperidinedione as the starting material. Example 11 (S) -3 -Phenyl -3 - [1- [ (4- [⁷⁶Br] bromophenyl ) methyl] -4- piperidinyl] -2-6-piperidinedione (4- [⁷⁶Br]bromodexetimide 13.

A freshly prepared solution of 10 (0.5-1.0 mg) in trifluoroacetic acid (TFA) (100 μl) was added to chloramine-T (1.5 mg) . The resultant solution was added to a vial containing aqueous ammonium [⁷⁶Br]bromide (5-120 μl, 0.5-100 mCi) . After 15 min the reaction mixture was neutralized with a mixture of concentrated aqueous ammonia (70 μl) in HPLC buffer (acetonitrile: 0.IM ammonium acetate 45:55) . The solution was then injected onto a semipreparative HPLC column. (Waters C18 Novapak, 250x7.8 mm) using a flowrate of 2.5 ml/min. The desired fraction (t_R 38 min) was collected and evaporated to dryness and used directly in the next step. Example 12

(S) -3-Phenyl-3- [1- [ (4- [⁷⁶Br] bromophenyl ) methyl] -4-N- methylpiperidinyl] -2, 6-piperidinedione iodide. (N-Methyl- 4- [⁷⁶Br]bromodexetimide) 14. The above 4- [⁷⁶Br]bromodexetimide 11 (0.5-50 mCi) was dissolved in tri-n-butylphosphate (50-100 μl) . This was then treated with methyl iodide (100-300 μl) tightly stoppered and heated to 80-90° for 15 min. The reaction mixture was cooled diluted with buffer 1 ml and the entire contents injected onto the HPLC column. (Conditions as above) . The N-methyl 4-

[⁷⁶Br]bromodexetimide, t_R 18-20 minutes, was collected, evaporated to dryness and dissolved into 0.9% saline.

Adjustment of pH (6-8) and sterile filtration through a

0.22 μ filter gave the required product suitable for biological evaluation.

The (R)- (-)- enantiomers were prepared in a similar fashion using (R) -(-) -3-Phenyl-3- (4-piperidinyl) -2, 6- piperidinedione as the starting material . References. 1. Delfolge, J. , Janier, M. , Syrota, A., Crouzel, C, Vallois, J.M., Cayla, J., Lancon, J-M. , Mazier, B.M. Noninvasive Quantification of Muscarinic Receptors In Vivo with PET in the Dog Heart. Circulation 1990, 82 : 4 , 1494 .

2. Varastet, M. , Brouillet, E., Chavoix, C, Prenant, C, Crouzel, C, Stulzaft, 0., Bottlaender, M. , Cayla, J., Maziere, B, Maziere, M. In vivo visualization of central muscarinic receptors using [11-C]quinuclidinyl benzilate and PET in baboons. Eur. <J. of Pharmacology. 1992, 213: 275-84.

3. Matsumura, K. , Uno, Y., Schefel, U. , Wilson, A.A. Dannals, R.F., Wagner, Jr. H.N. , J. Nuclear Med. 1991, 32: 76-80.

4. Wilson, A.A. Dannals, R.F., Ravert, H.T. , Frost, J.J., Wagner, Jr. H.N. J. Med. Chem. 1989, 32: 1057- 1602.

5. Maziere, Berger, G., Godot, J-M. , Prenant, C, Sastre, J. , Comar, D. [C-ll]Methiodide Quiniclidinyl

Benzilate a muscarinic antagonist for in-vivo studies of myocardial muscarinic receptors. J^". of Radioanalytical Chemistry. 1983. 76: 2, 305-309.

6. Hermans, Bert., Van Daele, Paul., Van De Westeringh, Cornells., Van De Eycken, Cyriel. , Boey, Jozef.,

Dockx, Josef., Jansen, Paul A.J. J. Org. Chem. 1968, 11: 797-800.

7. Van Wijngaarden, I Life Sciences . 1969, 8: 1, 517- 523. 8. Hwang, Da -Ren. , Dence, Carmen S., McKinnon, Zoe, Ann., Mathias, Carla, J., Welch, Michael, J., Nucl . Med. Biol . 1991, 18:2, 247-252.

Claims

1. Compounds of formula (I) ,

radiolabelled compounds or pharmaceutically acceptable salts thereof where X^~ is a suitable counter ion;

R*ι_ is H; alkyl, alkenyl, alkynyl, phenyl or phenylalkyl each optionally substituted;

R₂ is one or more selected from hydrogen, halogen, radioactive isotopes of halogen, OR, CN, CF₃, N0₂,

COOR, NRR', SR, COR, CONRR', S0₃R, S0₂NRR', SOR and

S0₂R where R and R' are independently selected from

H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted phenyl or optionally substituted phenylalkyl; wherein the optional substituents are one or more selected from alkyl, alkenyl, alkynyl, phenyl, halogen, radioactive isotopes of halogen, OR, CN,

CF₃, N0₂, COOR, NRR¹, SR, COR, CONRR', S0₃R, S0₂NRR',

SOR and S0₂R where R and R¹ are as defined above.

A compound of formula (I) according to claim 1 which is [⁷⁶Br] -N-methyl-4-bromodexetimide or [¹²³I] -N- methyl-4-iododexetimide.

A method of preparation of compounds of formula (I) , radiolabelled compounds or pharmaceutically acceptable salts thereof as defined in claim 1 or 2 which comprises:

(ii) followed by alkylation or arylation as follows:

where X is a suitable leaving group; B. where R_x is R₂PhCH₂- step A(i) as above with 2 molar equivalents of the benzyl derivative; or C. where R₂ is a radioactive halogen **R₂

(i) step A(i) as above where R₂ is (CH₃)₃Si; (ii) treatment with Na**R₂ or NH₄**R₂; (iii) followed by step A(ii) as above.

4. A pharmaceutical formulation comprising a compound of formula (I) , radiolabelled compounds and/or salts thereof as defined in claim 1 in a pharmaceutically acceptable carrier.

5. A pharmaceutical formulation according to claim 4 wherein the compound of formula (I) is [⁷⁶Br] -N- methyl-4-bromodexetimide or [¹²³I] -N-methyl-4- iododexetimide.

6. A pharmaceutical formulation according to claim 4 or 5 wherein the pharmaceutically acceptable carrier is saline. 7. A method of imaging in a subject of muscarinic receptors of the heart which comprises administering to the subject a radiolabelled compound of formula (I) as defined in claim 1 or 2. 8. A method of diagnosis in a subject of disorders related to muscarinic receptors of the heart which comprises administering to the subject a radiolabelled compound of formula (I) as defined in claim 1 or 2 and imaging muscarinic receptors. 9. Radiolabelled compounds of formula (I) and/or pharmaceutically acceptable salts thereof as defined in claim 1 or 2 as radiopharmaceuticals for imaging muscarinic receptors of the heart.

10. Use of radiolabelled compounds of formula (I) and/or pharmaceutically acceptable salts thereof as defined in claim 1 or 2 as radio pharmaceuticals in the manufacture of medicaments for imaging muscarinic receptors of the heart.