WO1996020928A1

WO1996020928A1 - Piperidine-based sigma receptor ligands

Info

Publication number: WO1996020928A1
Application number: PCT/AU1995/000885
Authority: WO
Inventors: Rikki Noel Waterhouse; Thomas Lee Collier; Joanne Christina O'brien
Original assignee: Australian Nuclear Science & Technology Organisation
Priority date: 1995-01-03
Filing date: 1995-12-29
Publication date: 1996-07-11
Also published as: AUPN037195A0

Abstract

Compounds of formula (I) or pharmaceutically acceptable salts therof wherein X = O, CO or CHOH; R' is fluoroalkyl of 2 to 5 carbon atoms and 1 to 11 fluorine atoms; mono- and di-substituted benzyl wherein the substituents are selected from NO2, CN, I, Br, F, OH, OCH3 or OCH2OCH3 the substituents being at the meta or para positions; iodoalkenyl groups of 3 to 6 carbon atoms; bromoalkenyl groups of 3 to 6 carbon atoms; alkyl groups of 1 to 5 carbon atoms; or hydroxyalky containing 2 to 5 carbon atoms and 1 to 3 hydroxyl groups; R' is phenyl optionally substituted with one or two groups at the meta or para positions selected from methyl, ethyl, I, Br, CN, NO2, OH, CF3, OR wherein R is phenyl, 4-cyanophenyl, 4-nitrophenyl, CF3, methyl or ethyl; iodoalkenyl of 3 to 6 carbon atoms; bromoalkenyl of 3 to 6 carbon atoms; fluoroalkyl of 1 to 5 carbon atoms and 1 to 11 fluorine atoms; or alkyl groups of 1 to 5 carbon atoms as piperidine based sigma-1 receptor ligands suitable for use in diagnosis and therapy of diseases such as psychosis and cancer.

Description

PIPERIDINE-BASED SIGMA RECEPTOR LIGANDS

Technical Field

The present invention relates to piperidine-based sigma-1 receptor ligands for use in diagnosis and therapy of selected diseases that might involve sigma receptors, such as psychosis and cancers. In particular, the present invention relates to piperidine-based sigma-1 receptor radioligands for imaging using positron emission tomography (PET) or single photon emission computerised tomography (SPECT).

Background Art

The sigma receptor is implicated to be involved in various neurological disorders such as schizophrenia and tardative diskynesia. Two sigma receptor subtypes are currently known. These are termed sigma-1 and sigma- 2. It has been suggested the sigma receptors may be a link between the nervous system, the immune system and endocrine system. These receptors are found in the organs of the immune and endocrine systems as well as in the heart, brain and lungs. Furthermore, it has been reported that many types of cancer cells exhibit a high density of sigma receptors, possibly allowing for a site to target for cancer detection.

A few radiolabeled sigma receptor ligands have been reported in the journals and in patent literature. These radioligands are deficient in that they are proven non-selective for the sigma receptor, or in that they do not cross the blood/brain barrier in sufficient amounts and do not have appropriate lipophilicity for low non-specific binding.

Gilligan et al has reported novel piperidine sigma receptor ligands as potential anti-psychotic drugs. None of these have been shown to be suitable radioligands for the evaluation of sigma receptor densities using computed tomography. The relative lipophilicity of these compounds has also not been determined.

To date, no proven selective sigma-1 receptor radioligand is commercially available for use in computed tomography. One of the major stumbling blocks in the evaluation of the biological role of the sigma receptors is the lack of a useful subtype specific sigma receptor radioligand for in vitro and in vivo use.

The present inventors have prepared a novel series of compounds which interacts selectively with the sigma-1 receptor and that have the appropriate lipophilicity for crossing the blood/brain barrier. Radiolabeled analogues have also been prepared from the appropriate precursor for in vivo evaluation of the sigma-1 receptors densities by positron emission tomography (PET) or single photon emission computerised tomography (SPECT).

Disclosure of Invention

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

or pharmaceutically acceptable salts thereof wherein

X = O, CO or CHOH;

R' is

- fluoroalkyl of 2 to 5 carbon atoms and 1 to 11 fluorine atoms;

- mono- and di-substituted benzyl wherein the substituents are selected from NO₂, CN, I, Br, F, OH, OCH₃ or OCH₂OCH₃ the substituents being at the meta or para positions;

- iodoalkenyl groups of 3 to 6 carbon atoms;

- bromoalkenyl groups of 3 to 6 carbon atoms;

- alkyl groups of l to 5 carbon atoms; or

- hydroxyalkyl containing 2 to 5 carbon atoms and 1 to 3 hydroxyl groups;

R'' is

phenyl optionally substituted with one or two groups at the meta or para positions selected from methyl, ethyl, I, Br, CN, NO₂, OH, CF₃, OR wherein R is phenyl, 4-cyanophenyl, 4-nitrophenyl, CF₃, methyl or ethyl;

- iodoalkenyl of 3 to 6 carbon atoms

- bromoalkenyl of 3 to 6 carbon atoms

- fluoroalkyl of 1 to 5 carbon atoms and 1 to 11 fluorine atoms or

- alkyl groups of 1 to 5 carbon atoms.

In a second aspect, the present invention provides radiolabeled derivatives of compounds of formula (I) wherein the compounds of formula (I) are radiolabeled with one or more isotopes selected from ¹⁸F, ¹¹C, ¹²³I , ¹²⁵I, ¹³¹I, ¹²⁴I and ⁷⁶Br.

Typically, the alkyl and alkenyl groups can be straight or branched chain.

The haloalkenyl groups can be present as their cis or trans geometric isomer.

Preferred compounds of the present invention are outlined in the following pages. The molecular structures, corresponding in vi tro evaluations and lipophilicity determinations are included for each compound.

Lipophilicity is indicated as a log P_7.5 value because the determinations are done at pH=7.5. In general, log P_7.5 values between 2.4 and 4.0 indicate that a compound will cross the blood/brain barrier and not be subject to a lot of non-specific binding that is found with compounds having log P value > 4.0. This is important mainly if the compound is to be used as a diagnostic imaging agent. Compounds having log P values > 4 can still be good therapeutic agents.

In a third aspect, the present invention provides a method of preparing compounds of formula (I) which comprises

(A) when R" is optionally substituted phenyl and

R' is fluoroalkyl, substituted benzyl, alkyl or hydroxy alkyl

treating an amine of formula (II)

with the appropriate fluoroalkyl halide, benzyl halide, alkylhalide or hydroxyalkyl halide in the presence of a base;

(B) when R" is optionally substituted phenyl and

R' is haloalkenyl

(i) treating an amine of formula (II) with an alkynyl halide in the presence of a base

(ii) treating the resulting alkyne with tributyltin hydride in the presence of a catalyst and (iii) treating the resulting vinyl stannanes with bromine or iodine;

(C) when R" is alkyl or haloalkyl and R' is substituted benzyl

treating the appropriate benzylamine alcohol with a metal hydride and reacting the resulting alkoxide with the appropriate alkylhalide or haloalkyl halide; or

(D) when R" is haloalkenyl and R' is substituted benzyl

(i) treating the appropriate benzylamine alcohol with a metal hydride and reacting the resulting metal alkoxide with an alkynyl halide

(ii) treating the resulting alkyne with tributyltin hydride in the presence of a catalyst and (iii) treating the resulting vinyl stannanes with bromine or iodine.

Preferably, the alkynyl halide is propargyl bromide and the reaction is conducted in anhydrous organic solvent such as dichloromethane. Preferably, the base is sodium or potassium carbonate. The reaction of the alkyne with tributyltin hydride is preferably conducted in a non-polar organic solvent such as toluene or benzene and where the catalyst is a radical initiator such as azo(bis)isobutyronitrile.

In a fourth aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula (I), a pharmaceutically acceptable salt thereof or a radiolabeled derivative thereof in a pharmaceutically acceptable carrier.

In another aspect, the present invention provides compounds of formula (III)

wherein X = O, CO or CHOH; n is an integer 1 to 5;

R' is

- fluoroalkyl of 2 to 5 carbon atoms and 1 to 11 fluorine atoms;

- mono- and di- substituted benzyl wherein the substituents are selected from NO₂, CN, I, Br, F, OH, OCH₃ or OCH₂OCH₃ the substituents being at the meta or para positions;

- iodoalkenyl groups of 3 to 6 carbon atoms;

- bromoalkenyl groups of 3 to 6 carbon atoms;

- alkyl groups of 1 to 5 carbon atoms; or

- hydroxyalkyl containing 2 to 5 carbon atoms and 1 to 3 hydroxyl groups.

In another aspect, the present invention provides compounds of formula (IV)

wherein X= O, CO, or CHOH; n is an integer 1 to 5;

R'' is

- phenyl optionally substituted with one or two groups at the meta or para positions selected from methyl, ethyl, I, Br, CN, NO₂, OH, CF₃, OR wherein R is phenyl, 4-cyanophenyl, 4-nitrophenyl, CF₃, methyl or ethyl;

- iodoalkenyl of 3 to 6 carbon atoms

- bromoalkenyl of 3 to 6 carbon atoms

- fluoroalkyl of 1 to 5 carbon atoms and 1 to 11 fluorine atoms or

- alkyl groups of 1 to 5 carbon atoms.

In another aspect, the present invention provides a method of imaging sigma-1 receptors in a subject comprising administering to the subject a radiolabeled derivative of a compound of formula (I) followed by imaging using positron emission tomography (PET) or single photon emission computerised tomography (SPECT).

In yet another aspect, the present invention provides a method of diagnosis of presence of sigma-1 receptors in a subject which comprises administering to the subject a radiolabeled derivative of a compound of formula (I).

In another aspect, the present invention provides the use of radiolabeled compounds of formula (I) in the manufacture of a medicament for the diagnosis or imaging of sigma-1 receptor sites in a subject.

Brief Description of Drawings

Figure l shows the effect of selected compounds on the uptake of 1-123 TPCNE in various brain regions of rats. Adult male AAW rats (n=3)

Figure 2 shows the effect of selected compounds on the uptake of 1-123 TPCNE in various organs. Adult male AAW rats (n=3). Modes for Carrying Out the Invention

Chemistry (general) for non-radioactive compounds

R" = phenyl optionally substituted

The amines indicated by Formula 1 are precursors to many of the final compounds of the invention and they can be synthesised according to Scheme I.

The first step in the synthesis of the amine precursors is the reduction of the ester group of ethyl isonipecotate, 1, to an alcohol to give 4-hydroxymethyl piperidine, 2. This reduction was accomplished by refluxing 1 in anhydrous tetrahydrofuran in the presence of 4 molar equivalents of lithium aluminium hydride. The amine 2 is then protected with a Boc group by reacting 2 with di-tert-butyl dicarbonate in dichloromethane as a solvent. The resulting product 3, is then reacted with one molar equivalent of methane sulfonyl chloride in anhydrous dichloromethane in the presence of triethylamine to provide the mesylate 4. Compound 4 was then reacted with the sodium salt of the appropriate phenol derivative in an anhydrous solvent such as tetrahydrofuran or N,N-dimethylformamide. Good results were obtained using dimethylformamide as the solvent and using 4 equivalents of the appropriate phenolic sodium salt. The Boc group of the resulting product 5, is easily removed by stirring 5 at room temperature in a mixture of 60:40 v/v dichloromethane/trifluoroacetic acid to provide the corresponding amine.

Compounds of formula (I) having the following formula 2

when R"=phenyl optionally substituted and R'= fluoroalkyl groups, mono- and di-substituted benzyl groups, alkyl groups or hydroxyalkyl groups can be prepared according to Scheme Ila.

Alkylation of the amines takes place in an anhydrous solvent such as dichloromethane or N,N,-dimethylformamide by reacting the amine with the appropriate alkyl halide, fluoroalkyl halide, benzyl halide or hydroxyalkyl halide in the presence of a base such as potassium carbonate or sodium carbonate.

If R'' = phenyl optionally substituted and R' = cis or trans iodoalkenyl or cis or trans bromoalkenyl groups then these can be prepared according to Scheme IIb .

The appropriate amine, eg 6, is reacted with an alkynyl halide, such as propargyl bromide, in an anhydrous organic solvent such as dichloromethane in the presence of a base, usually sodium or potassium carbonate. The alkyne of the type of 7 is then reacted with tributyltin hydride in a non-polar organic solvent such as toluene or benzene. The reaction takes place at reflux in the presence of a radical initiator such as azo(bis)isobutyronitrile in an inert atmosphere. The subsequent vinyl stannanes can have the cis or trans configuration, although the major product is the trans isomer. The vinyl stannanes can be reacted with either iodine or bromine in an organic solvent, such as dichloromethane, in the absence of light to provide the corresponding vinyl iodides or vinyl bromides, respectively, such as 9.

If R'' in Formula 2 is iodoalkenyl groups, bromoalkenyl groups, fluoroalkyl groups or alkyl groups and R' is mono- and di-substituted benzyl groups, then these can be prepared according to Scheme III.

The appropriate alcohol, eg 2 is reacted with the appropriately substituted benzyl halide in an anhydrous organic solvent such as dichloromethane or N,N-dimethylformamide in the presence of a base, preferably potassium or sodium carbonate. The products of this reaction have the formulas indicated by structure 10. If one wishes to prepare an alkyl or fluoroalkyl derivative of 10, then 10 can be reacted with a metal hydride, preferably sodium hydride, in an anhydrous organic solvent such at tetrahydrofuran. The resulting sodium alkoxide is then reacted with an appropriate alkyl or fluoroalkyl halide to provide the desired product. If one wishes to synthesise the vinyl halide derivatives such as those indicated by 13 , the metal alkoxide salt of 10 is reacted with an alkynyl halide, such as propargyl bromide. This reaction produces compounds of the type as indicated in structure 11. The alkyne of the type of 7 is then reacted with tributyltin hydride in a non-polar

organic solvent such as toluene or benzene. The reaction takes place at reflux in the presence of a radical initiator such as azo(bis)isobutyronitrile in an inert atmosphere. The subsequent vinyl stannanes can have the cis or trans configuration, although the major product is the trans isomer. The vinyl stannanes can be reacted with either iodine or bromine in an organic solvent, such as dichloromethane, in the absence of light to provide the corresponding vinyl iodides or vinyl bromides, respectively, such as 13.

Compounds of formula (I) having structure 3

where R' = - fluoroalkyl groups, mono- and di-substituted benzyl groups, alkyl groups of 1 to 5 carbon atoms, hydroxyalkyl groups and X = I, Br, OH, OR (R is phenyl, 4-cyanophenyl, 4-nitrophenyl, methyl or ethyl) can be prepared according to Scheme IV.

Commercially available 4-picoline is reacted in an anhydrous organic solvent, such as tetrahydrofuran, with a mild base, preferably trimethylsilylamide, to provide the corresponding sodium salt, 4. Compound 4 is then reacted with the appropriately substituted ethyl benzoate in an anhydrous solvent to provide compounds having the structures indicated by 15. The benzyl salt of 15 is synthesised by reacting 15 with a benzyl halide, such as benzyl bromide, in an organic solvent such as dichloromethane. Hydrogenation of the pyridine ring of 16 is accomplished with hydrogen gas using platinum dioxide as a catalyst and ethanol as the solvent. The reaction takes place well at room temperature and 25 lbs

of pressure.

The next step in the synthesis is the removal of the benzyl group. This can be accomplished in two steps. First, 17 is treated with methyl chloroformate in benzene and the resulting mixture is heated at reflux for 12 hours. Secondly, the resulting carbamate, 18, is hydrolyzed using either acid or base hydrolysis, preferably 2 N HCl in methanol 1:1 v/v at reflux for 24 hours. The resulting amine can then be alkylated using the appropriate fluoroalkylhalide, alkyl halide, benzyl halide or hydroxyalkyl halide to give the corresponding alkylated amines such as compound 20.

The corresponding vinyl halides can be synthesised by using Scheme V. The appropriate amine, eg 19 is reacted with the appropriate alkynyl halide, preferably propargyl bromide, in an organic solvent such as dichloromethane or N,N-dimethylformamide in the presence of a base, such as potassium carbonate. This reaction produces compounds of the type as indicated by structure 21. The alkyne of the type of 21 is then reacted with tributyltin hydride in a non-polar organic solvent such as toluene or benzene. The reaction takes place at reflux in the presence of a radical initiator such as azo(bis)isobutyronitrile in an inert atmosphere. The subsequent vinyl stannanes can have the cis or trans configuration, although the major product is the trans isomer. The vinyl stannanes can be reacted with either iodine or bromine in an organic solvent, such as dichloromethane, in the absence of light to provide the corresponding vinyl iodides or vinyl bromides, respectively, such as 23 .

If X = CN, then a slightly different approach is be taken as the cyano group is not so stable to the hydrogenation step. If a cyano group is required, then the corresponding bromo precursor of 19 is refluxed with copper (II) cyanide in an anhydrous organic solvent (Uemura et al). This reaction provides the corresponding cyano derivatives and subsequent reactions can be carried out according to Schemes IV and V.

Radiochemistry:

Radioiodination and radiobromination of vinylstannanes:

The radioiodinations and radiobrominations can be carried out using the procedure outlined in Scheme VI. Briefly, the vinylstannanes or arylstannanes can be synthesised according to literature methods and then radiolabeled using either Na¹²³I, Na¹²⁵I, Na¹²⁴I, Na¹³¹I or Na Br or other isotopes of iodine or bromine in the presence of an oxidizing agent, such as chloramine-T.

All radioiodinations and radiobrominations are carried out using standard literature methods (Moerlein et al,

Seevers et al, Leusind et al (1967), Leusind et al

(1968), however, the stannane precursors are novel. Several of these compounds could also be radiolabelled with isotopes of astatine, such as astatine-211 for purposes of radiotherapy for cancer.

Radiofluorinations:

1⁸F-fluoroalkylations:

The formation of the ¹⁸F-fluoroalkyl derivatives can be synthesised according to published methods (Chi et al). Briefly, the appropriate bromohydroxyalkane is reacted with trifluoromethanesulfonyl chloride in anhydrous dichlormethane in the presence of 2,6-lutidine. The corresponding triflate is then reacted with Na¹⁸F to form a bromo [¹⁸F]fluoroalkane which is then used to alkylate amines of the type 6 or 19 to form the desired ¹⁸F-labeled sigma receptor radioligands. Alternatively, the corresponding alkylmesylates or alkyltosylates can be prepared and reacted with ¹⁸F-fluoride in the presence of a solubilising agent in an organic solvent, preferably dimethylformamide or acetonitrile.

The [¹⁸F]fluorobenzyl compounds can also be synthesised using procedures outlined in the literature (Mach et al). These radiofluorination reactions are demonstrated in Schemes VII and VIII.

Radiolabeling with carbon-11 (¹¹C):

Several of the compounds in this invention can be radiolabeled with C using known literature methods

(Dannals et al and Langstrom et al! Substrates for the radiolabeling include [¹¹C]methyl iodide, [¹¹C]benzyl bromide and [¹¹C]ethyl iodide. Each of these radiolabeled alkylating agents can be prepared and then reacted with the appropriate amine precursor, such as 6 or 19, or with the alkali metal salts of compound 10 in Scheme III. Examples of these radiolabeling reactions are provided in Scheme IX.

The pharmaceutically acceptable salts of compounds of formula (I) are prepared by conventional techniques in the art known to the skilled addressee.

The pharmaceutical formulations 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. Radiolabeled compounds of formula (I) are useful ligands for in vivo evaluation of the sigma-1 receptor densities using positron emission tomography (PET) or single photon emission computerised tomography (SPECT). Such selective radioligands can be used in the detection of various forms of cancer using PET or SPECT including melanoma and cancers of the lung, ovary, breast, brain and colon. These radioligands might also be useful to evaluate the status of the cancer during the course of treatment. The same radioligands might also be used for the diagnosis of schizophrenia and, possibly, other neurological disorders.

The radiolabeled analogues of the compounds of formula (I) can be readily synthesised from the appropriate precursor for use as PET or SPECT sigma receptor radioligands. The radioligands of the present invention have appropriate lipophilicity for crossing the blood/brain barrier. Some of the compounds of formula

(I) can be labeled with more than one radio isotope without changing the original structure of the compound. As several of the compounds have high affinity for the sigma receptor site (Ki<10 nM), these compounds may also be useful as therapeutic agents for disease that involve the sigma receptor such as schizophrenia and depression.

The advantages of the present invention can be summarised as follows:

the compounds bind selectively to sigma-1 receptors in vi tro; the radiolabeled analogues can be easily synthesised;

the compounds exhibit good in vivo stability;

the compounds readily cross the blood/brain barrier; option for radiolabeling using PET isotopes (¹¹C,

¹⁸F, ⁷⁶Br, ¹²⁴I) or SPECT isotopes (¹²³I, ¹³¹I, ¹²⁵I).

Specific embodiments of the present invention are illustrated by the following preparative examples. It will be understood, however, that the invention is not confined to the specific limitations set forth in the individual examples.

EXPERIMENTAL SECTION:

Analytical data were obtained for the compounds described below using the following general procedures. Proton and Carbon NMR spectra were recorded on a Joel 400

MHz FT-NMR spectrometer; chemical shifts were recorded in ppm (δ) from an internal tetramethylsilane standard in deuterochloroform, deuteroethanol or deuterodimethylsulfoxide and coupling constants (J) are reported in Hz. Mass spectra were obtained by fast atom bombardment methods (FAB). Melting points were recorded on a

GallenKramp melting point apparatus and are uncorrected.

Elemental analysis was performed by Atlantic Microlabs

Inc. in Norcross Georgia, USA.

Reagents were purchased from commercial sources and were used without further purification. Chromatography was performed on silica gel using the solvent systems as indicated below. For mixed solvent systems, the ratios are given with respect to volumes.

The intermediate amines leading to compounds of

Formula (I) are exemplified in Tables I, II, IV, VI and

VIII. Compounds of Formula (I) are exemplified in Tables

III, V, VII, IX and X. All reactions were performed under a blanket of nitrogen gas unless otherwise specified.

EXAMPLE 1

Synthesis of 1- ( trans- Iodopropen- 2' -yl ) - 4 - (4' - cyano¬phenoxymethyl ) piperidine A: 1-(tert-Butoxycarbonyl)-4-hydroxymethylpiperidine

A 6.50 gram portion (56.4 mmol) of 4-hydroxymethylpiperidine was dissolved in 80 ml ethanol-free dichloromethane and to this was added di- tert-butyl dicarbonate (13.97g, 62.1 mmol). The resulting mixture was stirred at room temperature for 30 minutes and then the solvent was removed by vacuum. The product, 1-(tert-butoxycarbonyl)-4-hydroxymethylpiperidine, was isolated from the resulting oil by column chromatography (ethyl actetate/ethanol, 17:3) to provide 11.8 grams (54.4 mmol, 96%) of a white, crystalline solid: mp 73°C; ¹H-NMR: δ 1.03-1.17 (m, 2H), 1.42 (s, 9H), 1.55-1.73 (m, 3H), 2.56-2.73 (m, 2H), 3.34 (t, 1H, J = 5.40), 3.41 (t, 2H, J = 5.40), 4.00-4.14 (m, 2H); MS m/z 216; Anal, calcd for C₁₁H₂₁NO3: C, 61.37, H, 9.8, N, 6.51; Found C, 61.46, H, 9.82, N, 6.56;

B: 1-(tert-Butoxycarbonyl)-4-(methanesulfonyloxymethyl)piperidine

1-(tert-Butoxycarbonyl)-4-hydroxymethylpiperidine (3.60 g, 16.6 mmol) was dissolved in 50 ml of ethanol-free dichloromethane and to this solution was added anhydrous triethylamine (8.10 ml, 58.0 mmol) followed by methanesulfonyl chloride (1.54 ml, 19.6 mmol). This mixture was stirred at 0 C for 60 minutes. The resulting dark brown solution containing yellow precipitate was poured into a separatory funnel and 100 ml of 0.1M potassium carbonate solution was added. The product was extracted in to dichloromethane (3 X 100ml) and the organic extracts were combined, dried over magnesium sulfate, and the solvent removed in vacuo to give a pale yellow oil. The product was purified by column chromatography (hexanes/ethyl acetate 2.5:1.0) to provide a clear, colorless oil (4.51g, 15.3 mmol, 92.9%) which solidified upon standing to a white, crystalline solid: mp 76-77°C; ¹H-NMR: δ 1.15-1.28 (m, 2H), 1.46 (s, 9H), 1.82 (d, 2H, J = 11.40), 1.90-1.97 (m, 1H), 2.67-2.77 (m, 2H), 3.02 (s, 3H), 4.07 (d, 2H, J = 6.55), 4.12-4.25 (m, 2H); Ea (calc.) C:68.33, H: 7.65, N: 8.85 (found) C: 68 .21, H: 7 . 61, N: 8 .77 ; MS m/z 294

C : 1-(tert-Butoxycarbonyl)-4-(4'-cyanophenoxymethyl)piperidine

A 3.55 g portion (29.8 mmol) of 4-cyanophenol was dissolved in 30 ml anhydrous N, N-dimethyl formamide and to this was added sodium hydride (894 mg, 22.4 mmol) in several portions over a 10 minute period. This light-brown mixture was stirred at room temperature for an additional 10 minutes and then 1-(tert-butoxycarbonyl)-4- (methanesulfonyloxymethyl)piperidine (2.20g, 7.45 mmol) was added. The resulting solution was heated to 60°C for 16 hours and then cooled to room temperature. The dark-brown solution was next poured into a separatory funnel along with 200 ml of 1N sodium hydroxide solution. The product was extracted into dichloromethane (3 X 100 ml) and the organic layers were combined, dried over magnesium sulfate, and the solvent removed in vacuo to give a dark-brown oil. The product was purified by column chromatography (hexanes/ethyl acetate 2.5:1) to provide a white solid (2.30 g, 7.22 mmol, 97V): 1H-NMR: 2.10-3.12 (m, 2H), 1.47 (s, 9H), 1.80-1.90 (m, 2H), 1.94-2.00 (m, 1H), 2.68-2.80 (m, 2H), 3.80 (d, 2H, J = 6.61), 3.85 (d, 2H, J = 5.71), 6.93 (d, 2H, J = 9.00), 7.58 (d, 2H, J = 9.0); MS: 317.

The compounds of Table I may be prepared by the method outlined in Example IC using the appropriately substituted phenol and the appropriate anhydrous polar solvent.

D: 4-(4'-Cyanophenoxymethyl)piperidine

1- (tert-Butoxycarbonyl)-4- (4'-cyanophenoxymethyl)piperidine (950 mg, 2.98 mmol) was stirred together with 15 ml of a mixture of dichloromethane and trifluoroacetic acid (3:2) for 30 minutes at room temperature. Next, the solution was basified by the addition of 200 ml of 1M potassium carbonate followed by 50 ml 0.1N potassium hydroxide solution. The product was extracted into dichloromethane (2 X 75 ml) and the organic extracts were combined, dried over magnesium sulfate, and the solvent removed in vacuo to provide the product (612 mg, 2.83 mmol, 95%) as an off-white solid: mp 109-110°C; ¹Η-NMR: δ 2.10-3.12 (m, 2H), 1.47 (s, 9H), 1.80-1.90 (m, 2H), 1.94-2.00 (m, 1H), 2.68-2.80 (m, 2H), 3.80 (d, 2H, J = 6.61), 3.85 (d, 2H, J = 5.71), 6.93 (d, 2H, J = 9.00), 7.58 (d, 2H, J = 9.0); MS m/z 317; Anal, calcd for C₁₈H₂₄N₂O₃: C, 68.33; H, 7.65; N, 8.85. Found: C, 68.21; H, 7.61; N, 8.77.

The compounds of Table II may be prepared by the method outlined in Example ID using the conditions as described.

E: 1-Propargyl-4-(4'-cyanophenoxymethyl)piperidine

To 20 ml of ethanol-free dichloromethane was added 4-(4'-cyanophenoxymethyl)piperidine (500 mg, 2.31 mmol), potassium carbonate (1.29 g, 9.24 mmol) and propargyl bromide (0.22 ml, 2.8 mmol). The resulting solutions was stirred at room temperature for 2 hours and a yellow precipitate formed over time. The reaction mixture was then diluted with 100 ml of distilled water and the product was extracted into dichloromethane (2 X 50 ml). The organic extracts were combined, dried over magnesium sulfate, and the solvent removed in vacuo at room temperature to provide the crude product as a dark-yellow oil. The product was purified by column chromatography (ethyl acetate) to give a clear, colorless oil (290 mg,

1.14 mmol, 49%) that solidified upon standing: mp 93°C; ¹H

NMR: δ 1.21-1.35 (m, 2H), 1.55-1.66 (m, 1H), 1.85-1.91

(m, 2H), 2.22-2.34 (m, 3H), 2.97 (d, 2H, J = 11.54), 3.33

(d, 2H, 0.01), 3.85 (d, 2H, J = 5.97), 6.93 (d, 2H, J = 9.00), 7.58 (d, 2H, J = 9.00); MS m/z 254; Anal, calcd for C₁₆H₁₈N₂O: C, 75.56; H, 7.14; N, 11.01. Found: C, 75.46; H, 7.15; N, 10.99.

The compounds of Table III may be prepared by the method described in Example IE by using the appropriate alkylating agent and organic solvent.

F: 1-(trans-(tri-n-Butyltin)propen-2'-yl)-4-(4'-cyanophenoxymethyl)piperidine

1 - Propargyl-4-(4'-cyanophenoxymethyl)piperidine ( 130 mg, 0.51 mmol) was dissolved in 10 ml anhydrous toluene and to this solution was added of tri-n-butyltin hydride (0.18 ml, 0.66 mmol) and a catalytic amount (20 mg) of azobisisobutyronitrile. This solution was heated at reflux for 16 hours and then cooled to room temperature. The solvent was removed in vacuo and the product was purified by column chromatography (toluene/ethyl acetate 17:3) to provide a clear colorless oil (219 mg, 0.40 mmol, 82%): ¹H-NMR: δ 0.85-0.98 (m, 9H), 1.25-1.39 (m, 14H), 1.51-1.76 (m, 6H), 1.81-1.90 (m, 2H), 2.02-2.12 (m,

1H), 3.03-3.20 (m, 4H), 3.83 (d, 2H, J = 6.01), 5.95-6.20

(m, 2H, J_ab = 19.5), 6.92 (d, 2H, J = 8.90), 7.57 (d, 2H,

J = 8.90); MS m/z 545; Anal, calcd for C₂₈H₄₆N₂OSn: C,

61.66; H, 8.50; N, 5.14. Found: C, 61.89; H, 8.56; N, 5.07.

The compounds in Table IV can be prepared by the method described in Example IF by using the appropriate piperidine alkyne substrate.

G: 1-(trans-Iodopropen-2-yl)-4-(4'-cyanophenoxymethyl)piperidine

To 10 ml of dichloromethane was added 1-(trans-(tri-n-butyltin)propen-2'-yl)-4-(4'-cyanophenoxymethyl )piperidine (200 mg, .37 mmol) and to this was added in the absence of light and over a period of 10 minutes a solution of iodine (140 mg, 0.55 mmol) in dichloromethane

(5 ml). This solution was stirred at room temperature for another 15 minutes and then the excess iodine was destroyed by the addition of 5 ml 0.1 M sodium hydrogen sulfite solution. The reaction mixture was then diluted with 50 ml distilled water and the product was extracted into dichloromethane (2 X 25 ml). The organic extracts were combined, dried over magnesium sulfate, and the solvent removed in vacuo to provide a crude yellow oil. The product was purified by column chromatography (ethyl acetate/ethanol 9:1) to give a clear, colorless oil (120 mg, 0.31 mmol, 86%): ¹H-NMR: 1.32-1.47 (m, 2H), 1.80-1.89

(m, 3H), 1.96-2.07 (m, 2H), 2.92-3.04 (m, 4H), 3.84 (d,

2H, J = 6.0), 6.24-6.44 (m, 1H, Jab = 14.2), 6.57-6.65 (m, 1H, J_ab = 14.2), 6.93 (d, 2H, J = 9.0), 7.56 (d, 2H, J = 9.0); FAB HRMS 383.0620 (M+H)⁺; calcd for C₁₆H₁₉N₂OI: 383.0622.

The compounds detailed in Table V can be prepared by the method described in Example IG by choosing the appropriate stannane precursor and the appropriate halogenating agent.

Example 250

Preparation of 1-(4'-Cyanobenzyl)-4-trans-iodopropen-2'-yloxymethyl-piperidine

H: 1-(4'-Cyanobenzyl)-4-hydroxymethylpiperidine

4-Hydroxymethylpiperidine (1.20 g, 10.4 mmol) was dissolved in 30 ml ethanol-free dichloromethane and to this was added potassium carbonate (5.76 g, 41.7 mmol) and 4-cyanobenzyl bromide (2.27 g, 11.5 mmol). The resulting mixture was heated with stirring at 40 °C for 2 hours. Next, the reaction mixture was cooled to room temperature and diluted with 100 ml distilled water. The product was extracted into dichloromethane (3 x 50 ml) and the organic extracts were combined, dried over sodium sulfate, and the solvent removed in vacuo to provide a light yellow oil. The product was purified by column chromatography (ethyl acetate/ethanol 9:2) to provide a clear, colorless oil that solidified upon standing to a white solid (1.40 g, 6.1 mmol, 58%): 12.4-1.33 (m, 2H), 1.50-1.65 (m, 4H), 1.95-2.05 (m, 2H), 2.86 (s, 2H, J = 11.44), 3.47-3.54 (m, 4H), 7.45 (d, 2H, J = 8.09), 7.61 (d, 2H, J = 8.09).

The compounds described in Table VI can be prepared by the methods used in Example 250 H by choosing the appropriately substituted halobenzyl reagent and an appropriate organic solvent.

I: 1-(4'-Cyanobenzyl)-4-propargyloxymethylpiperidine

To 10 ml of anhydrous tetrahydrofuran at 0°C was added 1-(4'-cyanobenzyl)-4-hydroxymethylpiperidine (1.70 g, 7.4 mmol) followed by sodium hydride (384 mg, 9.6 mmol). The resulting mixture was stirred for 10 minutes and then propargyl bromide (.69 ml, 8.9 mmol) was added in one portion via syringe. Stirring continued for a subsequent 60 minutes and then the reaction was allowed to warm to room temperature. Next, 100 ml of distilled water was added to the reaction mixture and the product was then extracted into dichloromethane (3 × 50 ml). The organic layers were combined, dried over magnesium sulfate and the solvent removed in vacuo to yield a yellow oil. The product was purified by column chromatography to give a clear, colorless oil that solidified upon standing to a white, crystalline solid (1.47 g, 5.5 mmol, 74%): 1H-NMR: 1.22-1.37 (m, 2H), 1.50-1.70 (m, 3H), 1.90-1.98 (m, 2H), 2.40 (t, 1H, J = 2.41), 2.89 (d, 2H, J = 11.60), 3.30 (d, 2H, J = 6.55), 3.49 (s, 2H), 4.15 (s, 2H), 7.45 (d, 2H, 8.10), 7.60 (d, 2H, 8.10).

The compounds in Table VII can be prepared by the method outlined in EXAMPLE 250 I by choosing the appropriate alcohol and haloalkyl or haloalkyne derivative.

J: 1-(4'-Cyanobenzyl)-4-trans-tri-n-butyltinpropen-2'¬yloxymethylpiperidine

1-(4'-Cyanobenzyl)-4-propargyloxymethylpiperidin e

(1.40 mg, 4.89 mmol) was dissolved in 10 ml anhydrous toluene and to this solution was added tri-n-butyltin hydride (1.76 ml, 6.36 mmol) and a catalytic amount (20 mg) of azobisisobutyronitrile. This solution was heated at reflux for 16 hours and then cooled to room temperature. The solvent was removed in vacuo and the product was purified by column chromatography

(toluene/ethyl acetate 17:3) to provide a clear colorless oil (2.00 g, 3.46 mmol, 71%) : ¹H-NMR: 0.82-0.90 (m, 9H), 1.22-1.30 (m, 12H), 1.45-1.50 (m, 6H), 1.62-1.72 (m, 3H), 1.91-2.02 (m, 2H), 2.90 (d, 2H, J = 11.0), 3.25 (d, 2H, J = 6.40), 3.48 (s, 2H), 3.98 (d, 2H, J = 4.0), 5.98-6.25 (m, 2H), 7.45 (d, 2H, 8.10), 7.60 (d, 2H, 8.10).

The compounds in table VIII can be prepared by the method described in EXAMPLE 250 J by choosing the appropriate alkyne substrate.

K: 1-(4'-Cyanobenzyl)-4-trans-iodopropen-2'-yloxymethylpiperidine

To 10 ml of dichloromethane was added 1-(4'-cyanobenzyl)-4-trans-tri-n-butyltinpropen-2'-yloxymethylpiperidine (350 mg, .63 mmol) and to this was added in the absence of light and over a period of 10 minutes a solution of iodine (239 mg, 0.94 mmol) in dichlormethane (5 ml). This solution was stirred at room temperature for another 15 minutes and then the excess iodine was destroyed by the addition of 5 ml 0.1 M sodium hydrogen sulfite solution. The reaction mixture was then diluted with 50 ml distilled water and the product was extracted into dichloromethane (2 X 25 ml). The organic extracts were combined, dried over magnesium sulfate, and the solvent removed in vacuo to provide a crude yellow oil. The product was purified by column chromatography (ethyl acetate/ethanol 9:1) to give a clear, colorless oil (190 mg, 0.46 mmol, 73%): ¹H-NMR: 1.26-1.37 (m, 2H), 1.57- 1.75 (m, 3H), 1.96-2.08 (m, 2H), 2.84 (d, 2H, 11.44), 3.25 (d, 2H, 6.41), 3.53 (s, 2H), 3.89 (m, 2H), 6.33-6.48 (m, 1H), 6.57-6.66 (m, 1H), 7.45 (d, 2H, 8.10), 7.60 (d, 2H, 8.10); anal, calcd for C₁₇H₂₁N₂OI: C, 51.53; H, 5.34; N, 7.07; I, 32.02. Found: C, 51.37; H, 5.38; N, 7.01; I, 31.96.

The compounds in Table IX can be prepared by the method used in Example 250 K by choosing the appropriate vinyltin compound and halogenating agent.

Example 410

Preparation of 1-(2'-fluoroethyl)-4-(2'-(4"-bromo-phenyl)-2'-oxoethyl)piperidine

L: 1-(4'-Bromophenyl)-2-(4-pyridyl)ethanone

A solution of trimethylsilyl sodium amide (7.50 g, 40.0 mmol) in 100 ml anhydrous tetrahydrofuran was cooled to 0°C and to this was added dropwise over 30 minutes a solution of 4-picoline (3.95 g, 40.0 mmol) in anhydrous tetrahydrofuran. The resulting dark brown solution was stirred at 0°C for another 10 minutes after the addition was completed and then ethyl-4-bromobenzoate (3.33 ml, 20.00 mmol) in 20 ml anhydrous tetrahydrofuran was added dropwise over a 15 minute period. The mixture was then allowed to warm to room temperature and stirring continued for a subsequent 30 minutes. The reaction mixture was then diluted with 200 ml distilled water and the product was extracted into dichloromethane (3 × 70 ml). The organic extracts were combined, dried over magnesium sulfate and the solvent removed in vacuo to provide a yellow oil. The product was purified by column chromatography (ethyl acetate) to provide a white solid (3.96 g, 14.3 mmol, 71%): ¹H-NMR: 4.26 (s, 2H), 7.23 (d, 2H, J = 5.5), 7.63 (d, 2H, J = 7.0), 7.85 (d, 2H, J = 7.0) 8.58 (d, 2H, J = 5.5).

M: 1-Benzyl-4-(2'-(4"-bromophenyl)-2'-oxoethyl)-piperidine A 2.20 g (7.96 mmol) portion of 1-(4'-bromophenyl)-2-(4-pyridyl)ethanone was dissolved in 30 ml anhydrous acetonitrile and to this was added in one portion benzyl bromide (1.93 ml, 15.93 mmol). This mixture was heated to 45°C and stirred for 6 hours. The resulting yellow solution was cooled to 0°C and diluted with anhydrous petroleum ether (bp 60-80 ºC, 150 ml). Filtration of the precipitate provided the desired pyridinium salt as a yellow solid (2.43 g, 5.43 mmol, 68%). The product was not purified further but was used directly in subsequent reactions.

Platinum dioxide (.25g) was suspended in ethanol

(100ml) and to this was added the crude pyridinium salt

(500 mg, 1.3 mmol). This mixture was then degassed in vacuo and purged with hydrogen gas. The mixture was then stirred under hydrogen gas at 20 psi for 75 minutes.

After this time, the theoretical amount of hydrogen had been consumed and the suspension was filtered through

Celite. The solvent was removed in vacuo to provide a yellow oil. The product was purified by column chromatography to provide a light yellow oil (.450 mg, 93%): ¹H-NMR: 1.22-1.40 (m, 2H), 1.53-1.80 (m, 5H), 1.91-2.04 (m, 2H), 2.61 (t, 2H, J = 8.0), 2.91 (d, 2H, J = 11.0), 3.54 (s, 2H), 7.25-7.37 (m, 5H); MS: 372.

N: 1-Carbomethoxy-4-(2'-(4"-bromophenyl)-2'-oxoethyl)piperidine

1-Benzyl-4- (2 ' - (4" -bromophenyl) -2 ' -oxoethyl)piperidine (500 mg, 1.34 mmol) and methyl chloroformate (200 μL, 2.6 mmol) were reacted together in benzene (50 ml) at reflux for a total of 12 hours. The reaction mixture was then cooled to room temperature and the solvent was removed in vacuo. The product was isolated by column chromatography (ethyl acetate) to provide the product as a light yellow oil (320 mg, 0.94 mmol, 70%): ¹H-NMR: 1.15-1.42 (m, 2H), 1.72-1.80 (d, 2H, J = 13), 2.11-2.23 (m, 1H), 2.75-2.88 (m, 2H), 3.68 (s, 3H), 4.0-4.25, (m, 2H) 7.6 (d, 2H, J = 8.5), 7.8 (d, 2H, J = 8.5); MS: 340.

O: 4 -(2'-(4"-Bromophenyl)-2'-oxoethyl)piperidine

1-Benzyl-4- (2 ' - (4" -bromophenyl) -2 ' -oxoethyl)piperidine was dissolved in a mixture of methanol (100 ml) and 1 N hydrochloric acid (50 ml) and the resulting solution was refluxed for 12 hours and then cooled to room temperature. The reaction mixture was basified with 1N sodium hydroxide solution and then the product was extracted into dichloromethane (3 × 100 ml). The organic extracts were combined, dried over sodium sulfate, and the solvent removed in vacuo to provide a yellow oil. This oil was not purified further but was used directly in subsequent reactions.

P: 1-(2'-Fluoroethyl)-4-(2'-(4"-bromophenyl)-2'- oxoethyl)piperidine

4-(2'-(4"-Bromophenyl)-2'-oxoethyl)piperidine (200 mg, 0.71 mmol) was dissolved in dichloromethane (20 ml) and to this was added anhydrous potassium carbonate (392 mg) and bromofluoroethane (60 mL, 0.78 mmol). This mixture was stirred at 35°C for 16 hours and then cooled to room temperature. Next, the mixture was diluted with distilled water and the product was extracted into dichloromethane (2 × 25 ml). The organic layers were combined, dried, and the solvent evaporated to provide the crude product as a yellow solid. The product was purified by column chromatography to give a yellow solid (155 mg, 0.48 mmol, 67%): ¹H-NMR: 1.34-1.53 (m, 2H), 1.74-1.83 (d, 2H, J = 12.8), 1.91-2.23 (m, 3H), 2.72 (dt, 2H, JHF = 27.08, JHH = 7.03), 2.85 (d, 2H, J = 7.0), 2.97 (d, 2H, J = 12.0), 4.58 (dt, 2H, JHF = 48.0, JHH = 7.03), 7.61 (d, 2H, J = 8.70), 7.81 (d, 2H, J = 8.70).

The compounds in Table X can be prepared by the method described in EXAMPLE 410 by choosing the appropriate amine precursors and alkylating agents.

Radiochemistry:

Radioiodinations:

Synt hes is of 1 - ( [¹²³I]-Iodopropen-2-ylmethoxy)piperidine

A solution of sodium [¹²³I]iodide (1.52 mCi) in sodium hydroxide solution (45 μl) was placed into a 10 ml wheaton vial and to this was added a solution of methanol and 0.017 M phosphate buffer (15:85, 200 μl),

N-chloramine-T (2 mg), and a solution of 1- (tributyltinpropen-2-ylmethoxy)piperidine in ethanol (2 mg stannane, 100 μl solution). The reaction mixture was allowed to stand at room temperature for 2 minutes and then 20 ml of 1N sodium metabisulfite solution was added. The reaction mixture was then diluted with 1 ml distilled water and the product was extracted into chloromethane (1 ml). The organic layer was passed through 100 mg sodium carbonate and then the solvent was removed under a stream of nitrogen gas. The product was purified by high pressure liquid chromatography to provide 1.24 mCi (80%) of the desired radiotracer. Radiochemical purity of the radiotracer was >99%. The specific activity of the product was determined to be > 2,100 μCi/mmol using the classical HPLC methods (reversed phase semi-preparative OBD column, mobile phase: ethanol/water (85:15), flow rate = 2.0 ml/min). The organic solvent was removed in vacuo and the product was dissolved in 10.0 ml sterile saline. The resulting preparation was passed through a .22 μM filter into an evacuated sterile vial. Preparations of this sort were suitable for use in the studies described in the in vivo experimental section.

All of the iodine containing compounds described in this patent application can be radiolabeled using this method if the appropriate stannane precursor is used. Alternatively, trialkylsilyl precursors may also be used and some of the compounds might also be prepared by halogen exchange methods.

Radiofluorinations:

Fluorination reactions, following basic method of Hatano et al. The preparation of the radiofluorinated compounds can take place by the method shown below or by simple nucleophilic 18F- displacement reactions using the corresponding alkyltosylate or alkylmesylate precursors.

In a 1 ml Reacti-Vial was added 18 mg of Kryptofix (K₂₂₂ ^®) and 16.9 mg of potassium carbonate (anhydrous). To this was added 100 μl of deionized water and 0.5 ml of anhydrous acetonitrile. The vessel was ultrasounded to effect dissolution of the materials. Add ^~200 μl of F-18 fluoride solution (^~5 mCi), seal flask and shake to mix. Remove cap to vial and place in aluminium block at 65-70°C under a stream of nitrogen gas. Evaporate 5 times with 0.5 ml aliquots of acetonitrile. Allow reaction mixture to cool to RT and add 0.4 ml of acetonitrile, followed by 6 μl of 1-bromo-2-trifluoromethansulfonylethane (bromoethyltriflate) 1 and the reaction vessel was sealed. The reaction was allowed to proceed at room temperature for 8 minutes. 9.2 mg of the amino compound 3 was dissolved in 0.4 ml of acetonitrile and this solution was added to reaction vial the vial was sealed. The reaction vial was heated at 90-95 ºC for 90 minutes. After heating for 90 minutes the reaction vial was cooled in ice and the vial opened and the solvent removed from the vial under a stream of nitrogen. 0.8 ml of 5% ethanol/ethyl acetate was added and the vial was shaken. 0.4 ml was injected on the HPLC (4.6 mm X 250 mm silica gel, 2.0 ml/min, 100% acetonitrile). The reaction mixture was spiked with authentic compound and reinjected. The radiation peak co-eluted with the authentic compound.

Specific activity based on starting with a maximum of 5 mCi of F-18 fluoride in ^~0.5 ml of target water. Detection limit of system 0.04 nmol. Normally 0.2 - 0.4 nmol found in sample. Therefore specific activity is about ^~70 mCi/μmol.

In Vivo Studies:

General Biodistribution Studies:

It is the aim of these studies to evaluate the effectiveness of selected radiolabeled piperidine derivatives as in vivo imaging agents for sigma receptors in the brain. This can be accomplished by determining the biodistribution of the radiolabeled agents in the brain and other organs at selected timepoints after iv administration. The information gained from animal biodistribution studies is essential in the evaluation of a radiopharmaceuticals for human use.

Method:

1. All rats (Australian Albino Wistar, AAW) were allowed food and water ad lib throughout the experiment except during the injection of the radiolabeled piperidine derivatives.

2. The amount of radioactivity injected into each rat was approximately 10 mCi in saline in a maximum volume of 100 μl . The injections took place in the tail vein of the rats.

3. During injections of the radiolabeled piperidine derivatives, no anaesthesia was used and the rats were restrained by hand.

4. At the appropriate time points, the rats were sacrificed by cervical fracture under the influence of CO₂. Various organs (eg. brain, liver, spleen, heart, blood intestines, lung, thyroid, kidneys) were evaluated for the presence of the radiolabeled piperidine agents used in these studies.

Blocking studies:

It is the aim of these experiments to evaluate in vivo the selectivity of radiolabeled sigma piperidine derivatives for the sigma receptor in order to help determine their effectiveness as imaging agents for sigma receptors in the brain and in tumours. This is accomplished by pre-administration of various well characterised drug in order to pre-block neuroreceptors in vivo before administration of the radiotracer. Observing the changes in the biodistribution of the radiotracer due to the pre-administration of a drug, helps one to deduce which receptors the radiotracer interacts with in vivo.

1. All rats were allowed food and water ad lib throughout the experiment except during the injection of the radiolabeled piperidine derivatives.

2. The amount of radioactivity injected into each rat was about 10 μCi in a maximum of 100 μl sterile saline (0.37 MBq - 0.93 MBq).

3. Each rat received two injections via the tail vein. These injections included the radiolabeled piperidine derivative in 100 μL sterile saline and the competing drug in 100 μL phosphate buffer solution at physiological pH (dose = 1mg/Kg).

4. During injections of the radiolabeled piperidine derivatives or competing agents, no anaesthesia was used and the rats were restrained by perspex.

5. All rats were sacrificed by an overdose of CO₂ or by cervical fracture under the influence of CO₂. Various organs (eg. brain, liver, spleen, heart, blood intestines, lung, thyroid, kidneys) were evaluated for the presence of the radiolabeled piperidine agent used in the study.

Studies in Mice with B16 Melanoma Tumours:

The aim of these experiments is to evaluate novel ^radiolabeled piperidine sigma ligands as potential imaging agents for malignant melanoma. Specifically, the localisation of radiolabeled piperidine sigma ligands in B16 melanoma tumour-bearing nude mice was determined.

Initiation of tumour growth:

Tumour growth was initiated in nude mice by a subcutaneous injection of B16 mouse melanoma cells

(approximately 1 X 10⁶ cells in 0.1 ml media). The biodistribution studies proceeded once the size of the tumors reached approximately 1cm in diameter. No anaesthesia was used during the injection of the B16 melanoma cells into the mice.

Biodistribution studies:

Each mouse received one injection of the appropriate radiotracer via the tail vein. The total amount of radioactivity injected was between 10 μCi and 20 μCi

(0.37 MBq - 0.74 MBq) contained in 100 μL of sterile saline. All injections were made without the use of anaesthesia and the mice were restrained by hand during the injections. At predetermined time points between 1hour and 48 hours, the mice were sacrificed by cervical fracture under the influence of CO₂ and the tumours and selected organs were assayed for radioactivity to determine the biodistribution of the radiotracer.

Other Characterisation: Log P evaluations and In Vitro

Binding Assays:

In Vi tro Binding assays:

The in vi tro binding assays were performed by an US company called NOVASCREEN^® in Baltimore, Maryland. All sigma compounds were tested for interaction with sigma, dopamine D1, D2, muscarinic M1, M2, M3, Alphal, Alpha2, Serotonin, PCP and NMDA receptors in vi tro at NovaScreen. Briefly, competitive binding assays were performed in either 250 or 500 μl volumes containing, by volume, 80% receptor preparations, 10% radioligand and 10% of test compound/cold ligand (non-specific binding determinant/ 4%DMSO (total binding determinant). All compounds were solubilized in neat DMSO which was diluted to a final concentration of 0.4% in the assay. Assays were terminated by rapid vacuum filtration (centrifugation for VIP assay) over Whatman glass fiber filters followed by rapid washing with cold buffer. Radioactivity was determined by either liquid scintillation or gamma spectrometry. Enzyme activity assays were similarly performed. Data was reduced by a software program proprietary to NOVASCREEN^®. Specific assay conditions, ie tissue preparation, buffers, incubation times, temperatures, and filter treatments are according to literature procedures and the list of references is available from NOVASCREEN^®.

Log P Calculations:

The lipophilicity of the sigma receptor ligands was examined by determination of the log P_7.5 value using a

HPLC method previously described (Brent et al, 1983).

Briefly, samples were analysed using a C18 column

(Goldpak Exsil 10mm, 4.6 × 250mm) and a mobile phase of

MeOH and phosphate buffer (85:15 v/v, pH = 7.5) at 1.0 ml/min. The lipophilicity of 2 was determined by comparison of the retention time of the compound to that of standards having known log P values. The standards used in our study were catechol, aniline, benzene, bromobenzene, ethyl benzene, trimethylbenzene and hexachlorobenzene dissolved in an appropriate solvent. Relative retention times, RRT (to catechol), were calculated, and a calibration curve of log P vs. log RRT was generated. The calibration equations were polynomial with r² of 0.994 or greater. All sample injections were done in triplicate and the results averaged to provide the final values. Using this method, the log P_7.5 value for 2 was 3.36, indicating that this ligand should readily cross the blood brain barrier but should not exhibit a large degree of non-specific binding as normally results with compounds having high lipophilicities.

Results of In Vivo Studies:

General Biodistribution Studies: In vivo experiments have been performed to evaluate the biodistribution of three of the proposed sigma receptor radioligands, 1-([¹²³I]-Iodopropen-2-yl)-4- (4'cyanophenoxymethoxy)piperidine (¹²³I-TPCNE), and 1-cyanobenzyl-4-(trans -[¹²³I]iodopropen-2'-ylmethoxy) piperidine (¹²³I-CNBN) and 1-(3-[¹⁸F]fluoropropyl)-4-(4-cyanophenoxymethyl)piperidine (¹⁸F-FPPCNE). For each study, three to five rats were used for each of the time points specified..

The results of the general biodistribution studies indicate that all radioligands crossed the blood/brain barrier and remained in the brain for the duration of the study (up to 24 hours for ¹²³I-TPCNE). Highest brain uptake was exhibited by ¹²³I-TPCNE (2% ID from 20 minutes to 4 hours) and [^1SF]FPPCNE (2.5% ID from 5 minutes to 2 hours). The retention of the radioligands in the brain, along with the small amount of activity found in the thyroid gland (for the 123I-labeled ligands) and the bone

(for 18F-FPPCNE) over the course of the study, indicates that all three of the radioligands are relatively stable to in vivo dehalogenation. Retention of radioactivity was also observed in other organs known to contain sigma receptors, including the lungs, heart, spleen and kidneys. There was significant uptake of all radioligands in the lung, however, this is not surprising as it is known that sigma receptors are present in lung tissues.

The distribution of ¹²³I-TPCNE in adult male AAW Wristar Rats is presented in Tables XI and XII. The data is presented as %ID and as %ID/gram. Similarly, the distribution of ¹²³I-CNBN is presented in Tables XIII and XIV and the distribution of ¹⁸F-FPPCNE is presented in Tables XV and XVI.

The distribution of the radioligands was also determined in various regions of the brain at several time points. The brain regions examined include the medulla pons (MP), cerebellum (CB), midbrain (MB), hippocampus (HP), striatum (ST), frontal cortex (FC), and posterior cortex (PC). For all radioligands examined, the highest density of radioactivity was found in the (posterior and frontal) cortex. This is in contrast to literature reports in which other sigma receptor radioligands were most concentrated in the cerebellum or hippocampus. The discrepancies may be due to the use of non-selective ligands in other previously reported studies. Also, uptake in the cortex is significantly better that several known radioligands reported in the literature (eg. 1.55% injected dose per gram at 4 hours for ¹²³1-TPCNE versus 0.6% injected dose per gram at 1 hour for [¹⁸F]-a-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol. The piperidine-based radioligands described in the invention may selectively bind the sigma-1 receptor in vivo, and this could explain their different distribution in the brain regions.

The regional brain distribution of ¹²³I-TPCNE is provided in Table XVII. The data obtained for ¹⁸F-FPPCN is shown within Tables XV and XVI. Similarly, the regional brain distribution of ¹²³I-CNBN is indicated within Tables XIII and XIV.

Results of Blocking Studies:

Regional brain distribution for all three compounds differed from previously reported sigma radioligands in that the highest density was found in the posterior cortex > frontal cortex > cerebellum and other brain regions. Most of this uptake was inhibited in vivo by pre-administration of known sigma ligands such as haloperidol (sigma-1, sigma-2, D₂), (+)-pentazocine (sigma-1 > sigma-2, phencyclidine PCP) and Dup 734

(sigma, serotinin 5HT₂) while the uptake was not prevented by pretreatment with ritanserin (serotonin

5HT₂ 5HT_1C), S-(-)-eticlopride (dopamine D₂), or atropine

(muscarinic M₁, M₂, M₃). The same effects were obvserved in other organs that exhibited uptake and retention of the radiotracers, specifically the lung, heart, spleen and lung. The pre-administration of (+) pentazocine at doses of 10 mg/kg prevented uptake of the radiolabeled sigma ligands in the brain. 1,3-Di(2-tolyl)guanidine (DTG, sigma-1 and sigma-2) prevented displacement of the radiotracer in the organs in the periphery. However, DTG does not cross the blood/brain barrier well enough to prevent uptake of the sigma radioligands in the brain. The results of these blocking studies indicate that the sigma radioligands described in our invention selectively label sigma receptors in vivo. However, it must be stated that due to the lack of a well-established sigma-1 and sigma-2 ligands for use in blocking studies, it is currently difficult to prove the in vivo selectivity of these ligands for the sigma-1 receptor.

Examples of results of the blocking studies to evaluate ¹²³I-TPCNE in the brain regions and in the periphery are provided in Figures I and II, respectively. Biodistribution studies in mice with B16 melanomas:

In recent years it has been shown that various types of human cancer cells express high densities of sigma receptors. It has therefore been postulated the radiolabeled compounds with affinity for sigma receptors may be useful for the in vivo detection of cancer and, in particular, malignant melanoma. Three of the radioligands described in this invention, 1-([¹²³I]- Iodopropen-2-yl) -4- (4'cyanophenoxymethoxy)piperi dine (¹²³I-TPCNE), 1- (4'-cyanobenzyl)-4- (trans-¹²³I-iodopropen- 2' -ylmethoxy )piperidine (¹²³I-CNBN) and 1- (hydroxyethyl)-4-(4-iodophenoxymethyl)piperidine (¹²³I-HEPIE) have been evaluated in nude mice with B16 melanoma. At 24 hours post-injection, the tumour uptake of radioactivity was moderate to high, ranging from 3.93 +/- 1.03 ID/g for ¹²³I-CNBN to 16.42 +/- 2.35% ID/g for ¹²³I-HEPIE (n = 5). The results of these distribution studies are shown in Tables XVIII-XXIII.

During these studies, there was no significant loss of the radioactivity from the tumour for up to 24 hours. For ¹²³I-TPCNE, positive tumour/tissue ratios (ID/g) were optimum at 24 hours for most organs, including the brain (2.3), muscle (6.2), skin (1.3), heart (4.7), blood (38.0) and lung (2.2). However, the best overall results were obtained using ¹²³I-HEPIE, for which tumour /tissue ratios at 24 hours were positive for most organs including the brain (5.2), muscle (22.8), skin (4.0), GIT (7.1), blood (54.7) and lung (5.3). These results indicate that these sigma receptor ligands might be useful for imaging malignant melanoma. Furthermore, due to its relatively high tumour uptake and lower uptake in non-target tissues, ¹²³I-HEPIE may have potential as a radiotherapeutic agent for treatment of melanoma.

In Vitro Sigma-1 versus Sigma-2 Selectivy

Selected compounds of the invention were examined in sigma-1 and sigma-2 receptor binding assays performed by an independent company (NovaScreen, Hanover, MD USA). The results of these studies are provided below. As is indicated, many compounds of the invention are selective for the sigma-1 receptor in vi tro. In general, compounds having Ki values > 10 nM are not effective as receptor imaging agents for computed tomography due to relatively fast dissociation from the site of interest. However, all of the compounds shown below have Ki values < 10 nM for the sigma-1 receptor and Ki values > 10 nM for the sigma-2 receptor. These compounds should therefore be useful for the in vivo evaluation of sigma-1 receptors using computed tomography.

In Vitro Binding Affinities of Selected Compounds for Sigma-1 and Sigma-2 Receptors

REFERENCES

1. Kentaro Hatano, Tatsuo Ido, Kiichi Ishiwata, Jun Hatazawa, Masatoshi Itoh, Koichiro Kawashima and Ren Iwata, Appl. Radiat. Isot. Vol 41 (6), pp 551-555 (1990). 2. Gilligan, P.J.; Cain, G.A.; Christos, T.E.; Cook, L.; Drummond, S.; Johnson, A.L.; Kergaye, A.A.; McElroy, J.F.; Rohrbach, K.W.; Schmidt, W.K.; Tam, S.W. "Novel Piperidine Sigma Receptor Ligands as Potential Antipsychotic Drugs" J. Med . Chem. 1992, 35,4344-4361. 3. Uemura, S.; Ikeda, Y. and Ichikawa, K. "Reaction of Arylthallium (III) compounds with Copper (II) and (I) Cyanides. Synthesis of Aryl Cyanide" Tetrahedron 1972, 28, 3025-3030.

4. Chi, Dae Yoon; Kilbourn, Michael R.; Katzenellenbogen, John A. and Welch, Michael J. "A Rapid and Efficient Method for the Fluoroalkylation of Amines and Amides. Development of a Method Suitable for Incorporation of the Short-Lived Positron Emitting Radionuclide Fluorine-181 " J. Org. Chem . 1987, 52, pp 658-664.

5. Moerlein, S.M. and Coenen, H. H. J. Med . Soc . Perk . Trans 1985, pp1941.

6. Seevers, R. and Counsell, R. Chem . Rev. 1982, pp 575-582.

7. Leusind, A. J.; Budding, H. A. and Drenth, W. J. J. Organometal . Chem . 1967, 9, pp285.

8. Leusink, A. J. and Budding, H. A. "J. Organometal . Chem . 1968, 11, pp533.

9. Mach, Robert H.; Elder, Todd S.; Morton, Thomas E.; Nowak, Peggy A.; Evora, Paul H.; Scripko, James G.;

Luedtke, Robert R.; Unsworth, Christopher D.; Filtz, Theresa; Rao, Anand V.; Molinoff, Perry B. and Ehrenkaufer, Richard L. E. "The Use of [18F]4-Fluorobenzyl Iodide (FBI) in PET Radiotracer Synthesis: Model Alkylation Series and Its Application in the Design of Dopamine D1 and D2 Receptor Based Imaging Agents" Nucl . Med. Biol . 1993, 20, pp 777-794.

10. Itzhak, Y.; Stein, I.; Zhang, S-H.; Kassim, C. O.; Cristante, D. "Sigma Ligands to C57 BL/6 Mouse Brain Membranes: Effects of Monoamine Oxidase Inhibitors and

Sub-Cellular Studies Suggests the Existence of Sigma-receptor Sub-types" J. Phrmacol . Exp . Ther. 1991, 257,141-148.

11. Dannals, R. F. and Langstrom, B. J. Nucl . Med. 1985, pp126.

12. Langstrom, B. and Lundqvist, H. "The preparation of 11C-methyl iodide and its use in the synthesis of 11C-Methyl-L-methionine" Int . J. Appl . Radiat. Isot . 1976, 27 pp 357-363.

13. Brent D. A., Sabatka J. J., Minick, D. J. and Henry D. W. - J. Med. Chem. 26:1014 (1983).

Claims

1. Compounds of formula (I)

or pharmaceutically acceptable salts thereof wherein X = O, CO or CHOH;

R' is

- fluoroalkyl of 2 to 5 carbon atoms and 1 to 11 fluorine atoms;

- iodoalkenyl groups of 3 to 6 carbon atoms ;

- bromoalkenyl groups of 3 to 6 carbon atoms ;

- alkyl groups of 1 to 5 carbon atoms; or

- hydroxyalkyl containing 2 to 5 carbon atoms and 1 to 3 hydroxyl groups;

R'' is

- iodoalkenyl of 3 to 6 carbon atoms

- bromoalkenyl of 3 to 6 carbon atoms

- fluoroalkyl of 1 to 5 carbon atoms and 1 to 11 fluorine atoms or

- alkyl groups of 1 to 5 carbon atoms.

2. Radiolabeled derivatives of compounds of formula (I) as defined in claim 1 wherein the compounds of formula

(I) are radiolabeled with one or more isotopes selected from ¹⁸F, ¹¹C, ¹²³I, ¹²⁵I, ¹³¹I, ¹²⁴I and ⁷⁶Br.

3. A method of preparing compounds of formula (I) which comprises

(A) when R" is optionally substituted phenyl and

R' is fluoroalkyl, substituted benzyl, alkyl or hydroxy alkyl treating an amine of formula (II)

(B) when R" is optionally substituted phenyl and

R' is haloalkenyl

(ii) treating the resulting alkyne with tributyltin hydride in the presence of a catalyst and

(iii) treating the resulting vinyl stannanes with bromine or iodine;

(C) when R" is alkyl or haloalkyl and R' is substituted benzyl

(D) when R" is haloalkenyl and R' is substituted benzyl (i) treating the appropriate benzylamine alcohol with a metal hydride and reacting the resulting metal alkoxide with an alkynyl halide

(iii) treating the resulting vinyl stannanes with bromine or iodine.

4. A pharmaceutical formulation comprising a compound of formula (I), as defined in claim 1, a pharmaceutically acceptable salt thereof or a radiolabeled derivative thereof in a pharmaceutically acceptable carrier.

5. Compounds of formula (III)

wherein X = O, CO or CHOH; n is an integer 1 to 5;

R' is

- fluoroalkyl of 2 to 5 carbon atoms and l to 11 fluorine atoms;

- iodoalkenyl groups of 3 to 6 carbon atoms;

- bromoalkenyl groups of 3 to 6 carbon atoms;

- alkyl groups of 1 to 5 carbon atoms; or

- hydroxyalkyl containing 2 to 5 carbon atoms and 1 to 3 hydroxyl groups.

6. Compounds of formula (IV)

wherein X = O, CO or CHOH; n is an integer 1 to 5;

R'' is

- iodoalkenyl of 3 to 6 carbon atoms - bromoalkenyl of 3 to 6 carbon atoms

- fluoroalkyl of 1 to 5 carbon atoms and 1 to 11 fluorine atoms or

- alkyl groups of 1 to 5 carbon atoms.

7. A method of imaging sigma-1 receptors in a subject comprising administering to the subject a radiolabeled derivative of a compound of formula (I) followed by imaging using positron emission tomography (PET) or single photon emission computerised tomography (SPECT).

8. A method of diagnosis of presence of sigma-1 receptors in a subject which comprises administering to the subject a radiolabeled derivative of a compound of formula (I).

9. Use of radiolabeled compounds of formula (I) in the manufacture of a medicament for the diagnosis or imaging of sigma-1 receptor sites in a subject.