HK1119428A

HK1119428A - Imaging 18f or 11c-labelled alkylthiophenyl guanidines

Info

Publication number: HK1119428A
Application number: HK08111298.8A
Authority: HK
Inventors: Edward George Robins; Erik Arstad
Original assignee: Ge Healthcare Limited
Priority date: 2005-06-23
Filing date: 2006-06-23
Publication date: 2009-03-06

Description

Imaging18F or11C-labelled alkylthiophenylguanidines

The present invention relates to the field of medical imaging, in particular Positron Emission Tomography (PET) technology, and provides compounds and methods for imaging Central Nervous System (CNS) receptors.

The N-methyl-D-aspartate (NMDA) receptor is a major subtype of glutamatergic receptors and is widely believed to play a key role in long-term depression (long term depression), long-term stimulation (long term potentiation) and growing neuronal plasticity (quantitative neuronal plasticity). NMDA-induced excitotoxicity has been found in a number of CNS disorders such as stroke, brain or spinal injury, epilepsy, alzheimer's disease and Huntington's disease, which results at least in part from overactivity or prolonged stimulation of NMDA receptors. Several compounds have been investigated as potential radioligands for studying NMDA receptor ion channel sites in vivo using PET. However, most of these compounds have the disadvantage of weak blood-brain barrier penetration or strong non-specific binding.

WO94/27591 describes certain substituted guanidine compounds and their use in therapy. WO2004/007440 describes radiolabeled guanidine derivatives and their use for imaging Central Nervous System (CNS) receptors, which have been shown to have to be purified by complicated High Performance Liquid Chromatography (HPLC) after synthesis and to give only low to moderate yields but require relatively long preparation times of about 45 minutes. There is therefore a need for improved labelling chemistry in terms of overall yield, preparation time and ease of purification. Further, in order to ensure longer scanning times and increase the availability of these tracers, there is a further need for radioligands for the NMDA receptor.

Accordingly, one aspect of the present invention provides a compound of formula (I):

or a salt or solvate thereof, wherein

R¹Is hydrogen or C_1-4An alkyl group;

R²and R⁴Are each independently selected from C_1-4Alkyl group, [ 2 ]¹¹C]-C_1-4Alkyl and [ alpha ], [ alpha¹⁸F]-C_1-4Fluoroalkyl with the proviso that R is²And R⁴At least one of which is [ alpha ], [¹¹C]-C_1-4Alkyl or [ alpha ], [ alpha¹⁸F]-C_1-4A fluoroalkyl group; and

R³is halogen.

R¹Preferably hydrogen or methyl, more preferably methyl.

R²Or R⁴One of them is preferred¹¹CH₃、-¹¹CH₂CH₃Or-¹¹CH₂CH₂CH₃、-CH₂ ¹⁸F、-CH₂CH₂ ¹⁸F or-CH₂CH₂CH₂ ¹⁸F, more preferably-¹¹CH₃or-CH₂ ¹⁸F; another R²Or R⁴The radical is preferably methyl.

R³Preferably in relation to the group-SR²In the para position of the phenyl ring, preferably, R³Is chlorine.

group-SR⁴Preferably in the meta position relative to the guanidine bridge.

Accordingly, in a preferred aspect of the present invention there is provided a compound of formula (Ia):

or a salt or solvate thereof, wherein R¹、R²、R³And R⁴As defined for the compounds of formula (I).

Most preferred compounds of formula (I) include:

n- (2-chloro-5-, [ solution ]¹⁸F]Fluoromethylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine;

n- (2-chloro-5- (2-)¹⁸F]Fluoroethylthio)) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine;

n- (2-chloro-5-methylthio) -phenyl-N' - (3-, [ solution ]¹⁸F]Fluoromethylthio) -phenyl-N' -methylguanidine;

n- (2-chloro-5-methylthio) -phenyl-N' - (3- (2-, [ solution of ] A¹⁸F]Fluoroethylthio)) -phenyl-N' -methylguanidine;

n- (2-chloro-5-, [ solution ]¹¹C]Methylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine;

n- (2-chloro-5-methylthio) -phenyl-N' - (3-, [ solution ]¹¹C]Methylthio) -phenyl-N' -methylguanidine;

n- (2-chloro-5-, [ solution ]¹¹C]Ethylthio) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine; and

n- (2-chloro-5-methylthio) -phenyl-N' - (3-, [ solution ]¹¹C]Ethylthio) -phenyl-N' -methylguanidine, or a salt or solvate of any of them.

Suitable salts according to the invention include physiologically acceptable acid addition salts, such as those derived from inorganic acids, for example hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric or sulfuric acid, and those derived from organic acids, such as tartaric, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, methanesulfonic and p-toluenesulfonic acids.

As indicated below, the compounds of formula (I) and (Ia) have utility as radioligands for the NMDA receptor. Thus, according to a further aspect of the invention there is provided a compound of formula (I) or (Ia) as defined above, or a salt or solvate thereof, for use in an in vivo diagnostic or imaging method such as PET. Suitably, a compound of formula (I) or (Ia) as defined above, or a salt or solvate thereof, may be used for NMDA receptor imaging in healthy human volunteers.

Suitably, the compounds of formula (I) or (Ia) or salts or solvates thereof are useful for in vivo NMDA receptor imaging and thus have utility in the diagnosis of MMDA-mediated disorders such as stroke, brain or spinal injury, epilepsy, alzheimer's disease or Huntington's disease. There is therefore further provided the use of a compound of formula (I) or (Ia), or a salt or solvate thereof, in the manufacture of a radiopharmaceutical for use in the in vivo diagnosis or imaging of NMDA-mediated diseases.

Alternatively, there is provided a method for in vivo diagnosis or imaging of NMDA-induced disease in a subject, preferably a human, comprising administering a compound of formula (I) or (Ia) or a salt or solvate thereof. The method is particularly preferred for in vivo diagnosis or imaging of stroke, brain or spinal injury, epilepsy, Alzheimer's disease, or Huntington's disease.

The compounds of formula (I) or (Ia) or salts thereof are preferably administered in the form of a radiopharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable excipient. In the present invention a "radiopharmaceutical formulation" is defined as a formulation comprising a compound of formula (I) or (Ia) or a salt thereof in a form suitable for administration to a human. Administration is preferably by injection of the formulation as an aqueous solution. Such formulations may optionally further contain other ingredients such as buffers, pharmaceutically acceptable solubilizers (e.g. cyclodextrins or surfactants such as Pluronic (Pluronic), Tween (Tween) or phospholipids), pharmaceutically acceptable stabilizers or antioxidants (e.g. ascorbic acid, gentisic acid or p-aminobenzoic acid).

The dosage of the compounds of formula (I), (Ia) or salts thereof will vary depending upon the particular compound administered, the weight of the patient, and other variables which will be apparent to those skilled in the art. In general, the dosage is from 0.1nmol/kg to 50nmol/kg, preferably from 1nmol/kg to 5 nmol/kg.

Compounds of formula (I), (Ia) or salts or solvates thereof may be prepared from the corresponding compounds of formula (II):

wherein R is²Or R⁴One of which is hydrogen or a thiol protecting group such as benzyl and the other is hydrogen, C_1-4Alkyl or thiol protecting groups such as benzyl; r¹Is hydrogen or C_1-4Alkyl radical, R³Is halogen; by (i) removing any thiol protecting group, and (ii) reacting with a suitable haloalkane [ alpha ], [ beta ]¹¹C]C_1-4alkyl-X or [ alpha ], [ alpha¹⁸F]-C_1-4fluoroalkyl-Y, wherein X and Y are independently halogen, preferably chlorine, iodine or bromine, or other suitable leaving groups such as aryl or alkyl sulfonates, e.g., tosylate, triflate or mesylate.

The reaction with the alkyl halide is preferably carried out in a suitable solvent such as N, N-Dimethylformamide (DMF), acetone, dichloromethane, chloroform, dimethyl sulfoxide, methanol, ethanol, propanol, isopropanol, tetrahydrofuran or acetonitrile and in the presence of a base, suitably an inorganic base such as potassium carbonate, potassium hydroxide or sodium hydride, or an organic base such as a trialkylamine such as triethylamine, diisopropylethylamine or dimethylaminopyridine.

The compounds of formula (II) are useful intermediates for the preparation of PET tracers of formula (I) and, thus, form a further aspect of the invention.

According to another aspect of the present invention there is provided a process for the preparation of a compound of formula (I):

wherein:

R¹is hydrogen or C_1-4An alkyl group;

R²and R⁴Each independently selected from C_1-4Alkyl group, [ 2 ]¹¹C]-C_1-4Alkyl and [ alpha ], [ alpha¹⁸F]-C_1-4Fluoroalkyl with the proviso that R is²And⁴at least one of which is [ alpha ]¹¹C]-C_1-4Alkyl and [ alpha ], [ alpha¹⁸F]-C_1-4A fluoroalkyl group; and

R³is halogen;

which comprises the reaction of a compound of formula (II):

wherein R is²Or R⁴One is hydrogen or a thiol protecting group such as benzyl and the other is hydrogen, C_1-4Alkyl or thiol protecting groups such as benzyl; r¹Is hydrogen or C_1-4Alkyl radical, R³Is halogen;

by (i) removing any thiol protecting group, and (ii) reacting with a suitable haloalkane [ alpha ], [ beta ]¹¹C]C_1-4alkyl-X or [ alpha ], [ alpha¹⁸F]-C_1-4fluoroalkyl-Y reaction, wherein X and Y are independently halogen, preferably chlorine, iodine or bromine, or other suitable leaving groups such as aryl or alkyl sulfonates, e.g., tosylate, triflate or mesylate; in a suitable solvent and in the presence of a base.

According to another aspect of the present invention there is provided a kit for the preparation of a radiopharmaceutical formulation comprising a compound of formula (II) as defined above. In using the kit, the compound of formula (II) may be converted to the corresponding compound of formula (I) using the methods described above.

A compound of formula (II) wherein R²Is hydrogen or a thiol protecting group, and may be selected from compounds of formula (III):

wherein R is³Is halogen, P¹Is a thiol protecting group as described below; prepared by reaction with a compound of formula (IV):

wherein R is¹Is hydrogen or C_1-4Alkyl radical, R⁴As defined for the desired compound of formula (II). The coupling of the compound of formula (III) with the compound of formula (IV) can be carried out without solvent or in the presence of a high-boiling aprotic solvent such as chlorobenzene, toluene or xylene. The reaction may be carried out at elevated temperature, for example at 50-200 ℃, suitably at about 160 ℃. After the reaction, the protecting group P¹Removal may be as follows.

Suitable methods for thiol group protection and deprotection can be found in Protecting Groups in organic Synthesis, Theodora W.Greene and Peter G.M.Wuts, John Wiley&Found in Sons inc. Suitable thiol protecting groups include aralkyl groups such as benzyl or p-methoxybenzyl, which may be preceded by a radiolabelling step, for example by treatment with an acid such as a lewis acid such as AlCl₃And (4) processing and removing.

The synthesis of compounds of formula (II) from compounds of formula (III) and formula (IV) is illustrated in scheme 1.

Scheme 1

The compounds of formula (III) and (IV) can be prepared from commercially available starting materials using the procedures described in scheme 1 and examples or similar procedures.

A compound of formula (II) wherein R⁴Is hydrogen or a thiol protecting group, and may be derived from a compound of formula (V):

wherein R is¹Is hydrogen or C_1-4Alkyl radical, P²Is a thiol protecting group as described above; prepared by reaction with a compound of formula (VI):

wherein R is³Is halogen, R²As defined for the desired compound of formula (II). The coupling of the compound of formula (V) with the compound of formula (VI) can be carried out by a method analogous to that described for the coupling of the compound of formula (III) with the compound of formula (IV). After the reaction, the protecting group P²Can be removed as described above.

The synthesis of compounds of formula (II) from compounds of formula (V) and formula (VI) is illustrated in scheme 2.

Scheme 2

The compounds of formula (V) and (VI) can be prepared from commercially available starting materials using the procedures described in scheme 2 and examples or similar procedures.

The invention will now be illustrated in detail by way of examples, in which the following abbreviations are used:

HPLC: high performance liquid chromatography

UV: ultraviolet light

TLC: thin layer chromatography

EtOAC: ethyl acetate

IR: infrared ray

min(s): minute (min)

Example 1: synthesis of N- (2-chloro-5-fluoromethylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine Example 1(i) Synthesis of 3-amino-4-chlorobenzenethiol

To a cold solution (0 ℃) of tin (II) chloride (11.260g, 59.40mmol) in 10ml of concentrated hydrochloric acid was slowly added 4-chloro-3-nitro-benzenesulfonyl chloride (1.690g, 6.60mmol) in portions. The resulting suspension was kept cool and stirred for 15 minutes, then the mixture was heated to reflux for 1 hour. After cooling to room temperature, the mixture was diluted with water (100ml) and NaHCO₃Slowly neutralize. The aqueous phase was extracted with chloroform (4X 50ml), the organic phase was separated, Na₂SO₄And (5) drying. The solvent was removed in vacuo to give a bright yellow solid. Silica gel column chromatography using 1: 1 chloroform/hexane as the mobile phase afforded 3-amino-4-chlorobenzenethiol (0.632g, 60%) as a white solid.

¹H NMRδ(CDCl₃)7.08(d, 1H, | J | ═ 8.5Hz, aryl H), 6.67(d, 1H, | J | ═ 2.0Hz, aryl H), 6.59(dd, 1H, | J | ═ 8.5 and 2.0Hz, aryl H), 4.03(br s, 2H, NH₂)，3.37(s，1H，SH)。

EXAMPLE 1(ii) Synthesis of (5-benzylthio-2-chloro) -aniline and (5-benzylthio-2-chloro) -aniline hydrochloride

To a cooled (0 ℃ C.) solution of 3-amino-4-chlorobenzenethiol (0.630g, 3.92mmol) in dry Tetrahydrofuran (THF) (15ml) was added n-butyllithium (1.6M in hexane, 2.45ml, 3.92mmol) and the reaction mixture was stirred rapidly. To this mixture was slowly added benzyl bromide (0.47ml, 3.92mmol) and the reaction mixture was stirred rapidly and warmed to room temperature over about 1 hour. The solvent was removed under reduced pressure to give a crude product, which was purified by silica gel column chromatography using 1: 1 dichloromethane/hexane as the mobile phase. (5-benzylsulfanyl-2-chloro) -aniline (0.805g, 82%) was isolated as a bright white solid.

To a cooled solution (0.805g, 3.21mmol) of (5-benzylsulfanyl-2-chloro) -aniline (0.805 g) in anhydrous diethyl ether (10ml) at 0 ℃ was slowly added anhydrous hydrochloric acid (1M, 5.0ml, 5mmol) in diethyl ether. The resulting precipitate was isolated by filtration, washed with diethyl ether (2X 5ml) and dried in vacuo. As a white solid, isolated in almost quantitative yield(5-benzylsulfanyl-2-chloro) -aniline hydrochloride (0.865g, 94%). Example 1(iii) Synthesis of methyl- (3-methylthio-phenyl) cyanamide

To a cooled solution (0 ℃) of 3-methylthio-aniline (1.850g, 13.30mmol) in anhydrous diethyl ether (10ml) was added a solution (10ml) of cyanogen bromide (0.704g, 6.65mmol) in diethyl ether. Note that: cyanogen bromide is highly toxic. The resulting solution was stirred at room temperature overnight. The resulting mixture was filtered to remove the precipitate, the diethyl ether filtrate was washed with 1M HCl (20ml) and brine (20ml), then the solvent was removed in vacuo to give an oily yellow residue. The crude product was purified by column chromatography on silica gel using 95: 5 dichloromethane/ethyl acetate to give (3-methylsulfanyl-phenyl) cyanamide (0.570g, 52%) as a nearly colorless oil, which crystallized upon standing.

A flame-dried Schlenk flask was charged with sodium hydride (60% in mineral oil, 0.14g, 3.5mmol), 3-methylsulfanyl-phenylaminonitrile (0.493g, 3.00mmol) and anhydrous THF (5ml) under a nitrogen atmosphere. The mixture was stirred rapidly and heated to 70 ℃ for about 0.5 hour. Upon cooling to room temperature, methyl iodide (0.37ml, 6.00mmol) was added dropwise and the mixture was stirred at room temperature overnight. The resulting clear, colorless solution was concentrated in vacuo, then water (30mL) and diethyl ether (40mL) were added. Separating the organic phase from Na₂SO₄Drying and removal of diethyl ether in vacuo afforded a crude residue. Purification by silica gel column chromatography using dichloromethane as the mobile phase gave the title compound as a pale yellow oil (0.388g, 73%).

¹H NMRδ(CDCl₃)7.26(m, 1H, aryl H), 6.96(m, 2H, aryl H), 6.81(m, 1H, aryl H), 3.31(s, 3H, NCH)₃)，2.48(s，3H，SCH₃)。

Example 1(iv) Synthesis of N- (5-benzylsulfanyl-2-chloro) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine hydrochloride Become into

To a 10ml flame-dried round bottom flask equipped with a magnetic stir bar were added methyl- (3-methylthiophenyl) cyanamide (0.185g, 1.04mmol) and (5-benzylthio-2-chloro) -aniline hydrochloride (0.297g, 1.04 mmol). The flask was vented and refilled with nitrogen three times, then the flask was sealed under nitrogen and heated to 160 ℃ for 3 hours. Upon cooling to room temperature, the pale orange residue was dissolved in a minimum volume of dichloromethane (0.5-1ml) and purified by column chromatography on silica gel using a 0-10% methanol gradient in dichloromethane. Removal of the solvent under high vacuum gave the title compound as a glassy white solid (0.357g, 74%).

¹H NMRδ(d₆-DMSO)9.70(br s, 1H, NH), 8.01(br s, 1H, NH), 7.39-7.08(m, 11H, aryl H), 7.09(m, 1H, aryl H), 4.24(s, 2H, CH)₂)，3.39(s，3H，N-CH₃)，2.45(s，3H，S-CH₃)。

Example 1 (v): synthesis of N- (2-chloro-5-mercapto) -phenyl-N '(3-methylthio) phenyl-N' -methyl guanidine hydrochloride

A flame dried Schlenk flask was charged with aluminum chloride (0.293g, 2.20mmol) and anhydrous toluene (5ml) under a nitrogen atmosphere. To the resulting stirred suspension was added a solution of N- (5-benzylsulfanyl-2-chloro) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine hydrochloride (0.250g, 0.54mmol) in toluene and the reaction mixture was stirred rapidly at room temperature for 2 hours. The resulting mixture was diluted with methanol (5mL) to give a colorless, homogeneous solution. The solvent was removed in vacuo to give a colourless residue which was dissolved in dichloromethane (3ml), filtered and the filtrate purified using silica gel column chromatography using a 0-10% methanol gradient in dichloromethane. The title compound was isolated as a glassy white solid (0.130g, 64%).

By the reaction of a compound in K₂CO₃The hydrochloride salt was heated in acetone in the presence and then separated using silica gel column chromatography using a 0-10% methanol gradient in dichloromethane to produce a sample of the free base.

¹H NMRδ(CDCl₃)7.29(br s, 1H, aryl H), 7.28(m, 1H, aryl H), 7.13(m, 2H, aryl H), 7.11(d m, 1H, aryl H), 7.06(dd, 1H, aryl H), 7.01(d m, 1H, aryl H), 3.48(br s, 1H, SH), 3.36(s, 3H, NCH)₃)，2.49(s，3H，SCH₃)。

Example 1(vi) Synthesis of N- (2-chloro-5-fluoromethylthio) -phenyl-N '- (3-methylthio) phenyl-N' -methylguanidine

A flame dried Schlenk flask was charged with N- (2-chloro-5-mercapto) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine hydrochloride (0.037g, 0.10mmol), triethylamine (0.020g, 0.20mmol) and anhydrous dichloromethane (2-3ml) under a nitrogen atmosphere and cooled to 0 ℃ in an ice bath. Fluorobromomethane gas was bubbled through the dark reaction mixture for 30 seconds, then the reaction was allowed to slowly warm to room temperature. After 2 hours, the resulting pale yellow solution was concentrated in vacuo to give a crude residue which was redissolved in dichloromethane (1ml) and purified using silica gel column chromatography with a 0-10% gradient of methanol in dichloromethane. Removal of the solvent under high vacuum gave the title compound as a pale yellow oil (0.024g, 68%).

¹H NMRδ(CDCl₃)7.32(m, 2H, aryl H), 7.20(m, 1H, aryl H), 7.17(m, 1H, aryl H), 7.16-7.06(m, 2H, aryl H), 7, 04(m, 1H, aryl H), 5.71(d, 2H, | J | ═ 52.8Hz, CH₂F)，3.41(s，3H，N-CH₃)，2.50(s，3H，S-CH₃)。

Example 2: n- (2-chloro-5-, [ solution ] ¹⁸ F]Fluoromethylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine

The title compound is prepared using a method analogous to the method in example 1(vi), but using [ 2 ]¹⁸F]Fluorobromomethane as the haloalkylating agent, anhydrous acetonitrile as the solvent and cesium carbonate as the base. The product is identified by N- (2-chloro-5-)¹⁸F]Confirmation of HPLC coelution of fluoromethylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine with the reference sample prepared in example 1 (vi).

HPLC method

For the HPLC analytical conditions tested, the most efficient chromatographic separation between the precursor N- (2-chloro-5-mercapto) phenyl-N ' -3 ' - (methylthio) -phenyl-N ' -methylguanidine and the control standard N- (2-chloro-5-fluoromethylthio) -phenyl-N ' - (3-methylthio) -phenyl-N ' -methylguanidine was found to be as follows: 5 μ -Luna C-18(2) column (250X 4.6mm), MP55/45 acetonitrile/0.01M (NH)₄)₂HPO₄Flow rate 1ml/min, UV 254 nm. The retention time of the N- (2-chloro-5-mercapto) phenyl-N ' -3 ' - (methylthio) -phenyl-N ' -methylguanidine precursor was 20.0 minutes, while the retention time of the N- (2-chloro-5-fluoromethylthio) -phenyl-N ' - (3-methylthio) -phenyl-N ' -methylguanidine was 9.70 minutes.

Example 3: process for preparation of N- (2-chloro-5- (2-fluoro-ethylthio) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine Synthesis of

A flame-dried Schlenk flask equipped with a reflux condenser was charged with N- (2-chloro-5-mercapto) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine hydrochloride (0.030g, 0.08mmol), potassium carbonate (0.022g, 0.16mmol) and anhydrous acetone (2 ml). To the mixture was added a solution of 2-fluoroethyl tosylate (0.017g, 0.080mmol) in acetone (1ml), and the reaction was heated to reflux under a nitrogen atmosphere for 3 days. After cooling to room temperature the solvent was removed in vacuo and the residue was redissolved in dichloromethane (1 ml). Purification using silica gel column chromatography using 0-10% gradient methanol in dichloromethane afforded the title compound as a pale yellow oil (0.021g, 68%).

¹H NMRδ(CDCl₃)7.30(m, 2H, aryl H), 7.19(br m, 1H, aryl H), 7.13(br m, 3H, aryl H), 6.94(m, 1H, aryl H), 4.53(dt, 2H, | J | ═ 6.6 and 47.0Hz, CH₂F)，3.41(s，3H，N-CH₃) 3.12(dt, 2H, | J | ═ 6.6 and 20.5Hz), 2.51(S, 3H, S-CH)₃)。

Example 4N- (2-chloro-5- (2-, [ 2 ]) ¹⁸ F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine

The title compound is prepared using a method analogous to that of example 3, but using 2-, [ toluene sulfonic acid ]¹⁸F]Fluoroethyl ester as the haloalkylating agent, a 1: 2 mixture of anhydrous acetonitrile/ethanol as the solvent and cesium carbonate as the base, and the product is identified by N- (2-chloro-5-, [ 2- ]¹⁸F]HPLC co-elution confirmation of fluoroethylthio) -phenyl-N '- (3-methylthio) phenyl-N' -methylguanidine with the reference sample prepared in example 3.

HPLC method

For the HPLC analytical conditions tested, the most efficient chromatographic separation between the precursor N- (2-chloro-5-mercapto) phenyl-N ' -3 ' - (methylthio) -phenyl-N ' -methylguanidine and the control standard N- (2-chloro-5- (2-fluoro-ethylthio)) -phenyl-N ' - (3-methylthio) -phenyl-N ' -methylguanidine was found to be as follows: 5 μ -Luna C-18(2) column (250X 4.6mm), MP55/45 acetonitrile/0.01M (NH)₄)₂HPO₄Flow rate 1ml/min, UV 254 nm. N- (2-chloro-5-mercapto) benzeneThe retention time of the base-N ' -3 ' - (methylthio) -phenyl-N ' -methylguanidine precursor was 20.0 minutes, while the retention time of N- (2-chloro-5-fluoroethylthio) -phenyl-N ' - (3-methylthio) -phenyl-N ' -methylguanidine was 9.40 minutes.

Example 5N- (2-chloro-5- (2-, [ 2 ]) ¹¹ C]Ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine Become into

The title compound is prepared using a method analogous to that of example 3, but using 2-, [ 2 ], [¹¹C]Iodoethane is used as the haloalkylating agent.

Example 6: synthesis of N- (2-chloro-5-methylthio) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine

To a round bottom flask equipped with a magnetic stirrer was added sodium methoxide (1.4mg, 26.6. mu. mol), N- (2-chloro-5-mercapto) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidinium hydrochloride (5.0mg, 13.3. mu. mol) and anhydrous methanol (1 ml). The reaction mixture was stirred rapidly for 5 minutes under nitrogen, then the mixture was further treated with methyl iodide (1.8. mu.l, 30. mu. mol). After stirring at room temperature for 15 minutes, the solvent was removed in vacuo and the residue was analyzed by HPLC.

Example 7: n- (2-chloro-5-, [ solution ] ¹¹ C]Synthesis of methylthio-phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine

The title compound is prepared using a method analogous to that of example 6, but using [ 2 ]¹¹C]Methyl iodide is used as a methylating agent.

Example 8: n- (2-chloro-5-methylthio) -phenyl-N' - (3-methylthio) methaneSynthesis of phenyl-N' -methylguanidine Example 8 (i): synthesis of 2-chloro-5- (methylthio) aniline hydrochloride

To a stirred solution of 2-chloro-5- (methylthio) benzoic acid (5g, 24.67mmol) in t-butanol (20mL) was added triethylamine (5.25mL, 37.8 mmol). After stirring slightly, diphenylphosphoryl azide (6mL, 27.60mmol) was added dropwise. The reaction mixture was slowly heated to reflux for 6 hours and then cooled to room temperature. The solvent was removed under reduced pressure and the crude reaction mixture was dissolved in tetrahydrofuran (12.5mL) and then 12.5mL of trifluoroacetic acid (1: 1) was added. The reaction mixture was heated to reflux for 6 hours, cooled to room temperature and the solvent evaporated. The reaction mixture was treated with NaOH (25%) to pH 12 while cooling in an ice-water bath. The product was extracted repeatedly into ethyl acetate (4X 25mL) and the organic layer was washed with water (10 mL). The combined extracts were passed over MgSO₄Dried and concentrated in vacuo to give a yellow oil. The product is purified by column chromatography (SiO)₂Gradient hexane/ethyl acetate) and the collected sample was dissolved in ether and treated with HCl/ether (10mL, 1M) to give white crystals. The title compound was a white solid (3.73g, 87% yield); mp: 180 ℃ and 181 ℃; TLC: Hexane/Ethyl acetate (9: 1) R_f＝0.51；MS(Cl)m/e 174(M+1，C₇H₈ClNS) and M/e 191(M + NH)₃)，¹H NMR(CDCl₃)δ(ppm)7.2-6.7(m，3H，Ar-H)，2.5(s，3H，S-CH₃)。

Example 8(ii) Synthesis of 3- (benzylthio) aniline

To a stirred solution of sodium hydroxide (2.1g, 52.5mmol) in water (4ml) cooled in an ice bath was added dropwise a solution of 3-aminothiophenol (4.8g, 38.4mmol) in ethanol (20ml) followed by benzyl chloride (5g, 39.5mmol) in ethanol (5 ml). After the addition, the reaction mixture was stirred at room temperature for 4 hours to become a brown solution with a white precipitate. After the precipitate was filtered off, the filtrate was concentrated, and the residue was dissolved in methylene chloride (40 ml). The dichloromethane solution was washed three times with aqueous sodium hydroxide (0.5M, 3X 40ml) and once with water (40 ml). MgSO (MgSO)₄After drying, filtration and methylene chloride vacuumConcentration gave a thick yellow oily crude product. Further by flash chromatography (SiO)₂hexane/CH₂Cl₂0-100%) to give 3- (benzylthio) aniline (6.77g, 82%) as a pale yellow oil which solidified to a white solid after standing at room temperature. Thin layer chromatography: methylene chloride, R_f＝0.37；¹H-NMR(CDCl₃)δ(ppm)6.6-7.4(m，9H，Ar-H)，4.15(s，2H，S-CH2)。

Example 8(iii) Synthesis of 3- (benzylthio) phenylaminonitrile.

To a stirred solution of 3- (benzylthio) aniline (4.6g, 21.4mmol) in anhydrous diethyl ether (25ml) at 0-4 ℃ was slowly added a solution of cyanogen bromide (1.42g, 13.4mmol) in anhydrous diethyl ether (10 ml). After the addition, the reaction mixture was stirred at room temperature for 12 hours to become a brown solution with a white precipitate. The precipitate was removed by filtration and the filtrate was washed with aqueous HCl (1M, 3X 40ml) and then brine (40 ml). The ether solution was passed over MgSO₄Drying, filtration and concentration in vacuo gave the crude product as a yellow oil. It is further processed by flash chromatography (SiO)₂，CH₂Cl₂EtOAc, 0-20%) to afford 3- (benzylthio) phenylaminonitrile (2.82g, 55% yield) as a white solid. TLC: dichloromethane/ethyl acetate (93: 7), R_f＝0.64；¹H-NMR(CDCl₃)δ(ppm)7.2-6.7(m，9H，Ar-H)，4.12(s，2H，S-CH₂).IR(KBr)：3178cm^-1(secondary N-H), 3023-3085cm^-1(C-H fragrance extension), 2227cm^-1(CN)。

Example 8(iv) Synthesis of 3- (benzylthio) phenyl-N-methylaminonitrile

To a solution of 3- (benzylthio) phenylaminonitrile (0.80g, 3.33mmol) in acetonitrile (8mL) was added diisopropylethylamine (0.65g, 5.0mmol) followed by methyl iodide (0.94g, 6.66 mmol). The reaction mixture was refluxed at 80-85 ℃ for 3 hours. After removal of the solvent the residue was dissolved in dichloromethane (40ml) and the organic solution was washed with water (40 ml). MgSO (MgSO)₄After drying, filtration and concentration of the dichloromethane solution in vacuo gave a yellow colorCrude product as an oil. Column chromatography (SiO)₂hexane/CH₂Cl₂50% -100%) to give 3- (benzylthio) phenyl-N-methylaminonitrile (0.67, 80% yield) as a pale yellow oil. CH (CH)₂Cl₂ R_f＝0.45；¹H-NMR(CDCl₃)δ(ppm)7.3-6.8(m，9H，Ar-H)，4.06(s，S-CH₂，2H)，3.57(s，3H，N-CH₃)。

EXAMPLE 8(v) Synthesis of N- (2-chloro-5-methylsulfanyl) -phenyl-N ' -3 ' - (methylsulfanyl) -phenyl-N ' -methylguanidine

To a 25ml dry flask equipped with a water condenser were added 3- (benzylthio) phenyl-N-methylaminonitrile (0.65g, 2.56mmol), 2-chloro-5- (methylthio) aniline hydrochloride (0.54g, 2.56mmol) and 1ml chlorobenzene. The flask was then purged with nitrogen and then heated at 150 ℃ for 3 hours with stirring. The reaction mixture was cooled to room temperature. After removal of chlorobenzene in vacuo, a thick glassy oily crude product was obtained. Flash chromatography (SiO)₂，CH₂Cl₂MeOH, 0-20%) to give guanidine hydrochloride as a solid (0.9g, 85% yield): thin layer chromatography: CH (CH)₂Cl₂/MeOH(9∶1)，R_f＝0.34；¹H NMR(CDCI₃)δ(ppm)6.8-7.3(m，12H，Ar-H)，4.06(s，S-CH₂，2H)，3.57(s，3H，N-CH₃)，2.35(s，3H，S-CH₃)。

Example 8(vi) Synthesis of N- (2-chloro-5-methylthio) -phenyl-N ' -3 ' -mercaptophenyl-N ' -methylguanidine

To a 25ml dry flask, under nitrogen, was added aluminum trichloride (125mg, 0.94mmol) followed by dropwise addition of a solution of N- (2-chloro-5-methylthio) -phenyl-N ' -3 ' - (benzylthio) -phenyl-N ' -methylguanidine (100mg, 0.23mmol) in toluene (2 ml). The mixture was stirred at room temperature under nitrogen overnight. The reaction was quenched by the use of acetic acid (0.5ml) and then concentrated in vacuo to give a crude product as a viscous oil. Flash chromatography (SiO)₂，CH₂Cl₂MeOH, 0-20%) to afford the title product as a glassy solid (70mg, 96% yield): TLC: CH (CH)₂Cl₂/MeOH(9∶1)，R_f＝0.14；¹H NMR(CDCl₃)δ(ppm)6.8-7.3(m，7H，Ar-H)，3.35(s，3H，N-CH₃)，2.4(s，3H，S-CH₃)。

Example 8(vii) Synthesis of N- (2-chloro-5-methylsulfanyl) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine HPLC method

For the HPLC analytical conditions tested, the most efficient chromatographic separation between the precursor N- (2-chloro-5-methylsulfanyl) -phenyl-N-3 '-mercaptophenyl-N' -methylguanidine, N- (2-chloro-5-mercapto) -phenyl-N '-3' - (methylthio) -phenyl-N '-methylguanidine and the control standard N- (2-chloro-5-methylthio) -phenyl-N' -3 '- (methylthio) -phenyl-N' -methylguanidine was found to be as follows: mu-Bondapak C-18 column (300X 7.8mm), MP 60/40 acetonitrile/0.05M (NH)₄)₂HPO₄Flow rate 2ml/min, UV 254 nm. The retention times for the 3 ' -demethylthio precursor and the 5-demethylthio precursor were 6.65 and 6.01 minutes, respectively, while the retention time for N- (2-chloro-5-methylthio) -phenyl-N ' - (3-methylthio) -phenyl-N ' -methylguanidine was 11.81 minutes.

To a solution of N, N-dimethylformamide or anhydrous ethanol (250. mu.l) containing N- (2-chloro-5-methylthio) -phenyl-N '-3-mercaptophenyl-N' -methylguanidine (0.5-0.8mg), potassium butoxide (0.5-1.0mg, 2-4 equivalents relative to the precursor) was added iodomethane (0.3-0.6mg, 1-2 equivalents relative to the precursor). The resulting mixture was stirred at room temperature for 5 minutes, then added by adding 100. mu.l HPLC mobile phase (0.05M (NH)₄)₂HPO₄) And (4) quenching. An aliquot of the reaction mixture was injected into an HPLC column for analysis. Based on the results of HPLC analysis, it was shown that the yield of the title product was higher than 75% on all test trials using N, N-dimethylformamide or ethanol as solvent, that the chemical consistency was good, and that the separation between the title product and its desmethylthio precursor was sufficiently efficient for semi-preparative separation in radiochemistry (hot chemistry).

Example 9N- (2-chloro-5-methylthio) -phenyl-N' - (3-, [ solution ] ¹¹ C]Synthesis of methylthio-phenyl-N' -methylguanidine

Reduction by lithium aluminum hydride [ 2 ]¹¹C]CO₂And then iodinated with hydroiodic acid and distilled to prepare the product of [ 2 ], [¹¹C]Methyl iodide was added to a vial containing a solution of N- (2-chloro-5-methylsulfanyl) -phenyl-N '-3-mercaptophenyl-N' -methylguanidine (0.5mg), potassium butoxide (0.8mg) in N, N-dimethylformamide (300. mu.l). The labelling chemistry was carried out at room temperature for 5 min and the reaction mixture was purified by addition of 100. mu.l HPLC mobile phase (0.05M (NH)₄)₂HPO₄) And (6) quenching. An aliquot was taken from the reaction mixture and injected into the radioactive HPLC system. The analysis was performed under the same chromatographic conditions as in example 8. The radioactive peak, which eluted with a retention time of 11.81 minutes, was confirmed as the title product by co-elution under the same analytical conditions with a non-radioactive control standard. Based on [ 2 ]¹¹C]Methyl iodide, the decay-corrected radiochemical yield of the title product was found to be greater than 90%. After the cyclotron bombardment is finished, the secondary membrane¹¹C]CO₂Initially, the total time of the radiosynthesis was within 20 minutes.

Biological examples

N- (2-chloro-5- (2-) ¹⁸ F]Biological fraction of fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine And (5) distributing data.

Materials and methods

N- (2-chloro-5- (2-)¹⁸F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine prepared according to example 4 (synthetic formulation of 3% ethanol in 0.9% w/v saline solution), having a radiochemical purity of-99%, and a specific activity of 4-16GBq/nmol upon injection^-1. The biodistribution and blood data were from 11 adult male Sprague-Dawley rats (body weight range 269-329 g; mean. + -. S.E.: 300. + -.18 g). N- (2-chloro-5- (2-)¹⁸F]Fluorine-ethyleneThio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine was injected directly into the tail vein of each isoflurane (isoflurane) anesthetized rat. Each animal was then allowed to recover from anesthesia. At the indicated time post-injection, rats were sacrificed by head and neck dislocation under anesthesia and brain and body tissues were rapidly sampled.

Biodistribution

Data were obtained using two sets. Rats were given an average of-86 MBq (85.3 MBq on the first day of the experiment and 87.3MBq on the second day of the experiment), 0.20ml in volume (-3% alcohol synthetic formulation), by direct intravenous injection via the tail vein. Co-injected N- (2-chloro-5- (2-, [ 2- ])¹⁸F]The mass of the fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine is 0.7-2.9nmol.kg^-1. Details of the method, as well as sample preparation and calculation, can be found in Hume et al, nuclear.med.biol. (1991) 18: 339-. Data were normalized for radioactivity injected and body weight, resulting in:

'uptake units (uptake units)' (cpm.g.)^-1Wet heavy tissue) (cpm.g. injected)^-1Body weight)^-1

Results

The radioactivity concentration data are additionally collated in table 1 (peripheral tissues) and table 2 (brain). Since no metabolite study was conducted, it was reflected in the presence of the parent N- (2-chloro-5- (2-), [ 2 ], ]¹⁸F]The proportion of total radioactivity of the label associated with fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine is unknown. Note that: blood and plasma samples were collected post mortem from the ventricles.

Distribution in vivo

The data are summarized in table 1. For the sampled tissues, skeletal muscle, skin and testis showed low initial content of 0.4 uptake units, which was maintained during the experiment. Bone showed an initial high uptake of 1.4, which decreased to-0.8 after 90 minutes of the experiment, indicating no sign of defluorination. High initial uptake (-30 uptake units) was observed in the lungs, which rapidly decreased to 2 uptake units at 90 minutes. Similar curves were observed in the kidney and heart. A slower rate of radioactivity loss was observed in the liver, spleen and intestine.

TABLE 1

Intravenous injection of N- (2-chloro-5- (2-)¹⁸F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine radioactivity distribution in rat peripheral organs and body fluids as a function of time. No blood volume (bloodvolume) correction was performed. Data are in 'uptake units'. The organization code is as follows: 1 bone, 2 skeletal muscle, 3 skin, 4 urine, 5 fat, 6 testes, 7 small intestine, 8 small intestine content, 9 large intestine, 10 large intestine content, 11 spleen, 12 liver, 13 kidney, 14 stomach, 15 lung, 16 heart (ventricle). Plasma data corresponding to the time of the sample are also shown for the control (17).

Organization time minute (n ═ number of data points)

Brain distribution

The data are summarized in table 2. All tissues had a relatively high initial uptake of 4 uptake units 2 minutes after intravenous injection of the radioligand. Then the activity gradually decreased, reaching-0.4 uptake units 90 minutes after radioligand injection. Small signals were obtained in hippocampus (hippopmus) and cortex relative to cerebellum, increasing from-0.8 to 1.3 over the first 40 minutes, and decreasing to 1 over 90 minutes.

N- (2-chloro-5- (2-)¹⁸F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine peripheral clearance (periherral clearance) is via the kidney to the urine and via the intestine. Intravenous injection of rat brain N- (2-chloro-5- (2-, [ solution of ] 2- ]¹⁸F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine at the earliest sample time(2min) showed high uptake of radioactivity. Small diversity is difficult to detect due to physiological 'blockade' or receptor quiescence (restingstate). Since the cerebellum showed the lowest retention after-60 min, a small signal was seen in the hippocampus when the data were expressed relative to cerebellum radioactivity (known as the region of high NMDA receptor density; Bowery et al (1988) Br. J. Pharmacol.93: 944. 954).

TABLE 2

N- (2-chloro-5- (2-)¹⁸F]Fluoro-ethylthio)) -phenyl-N '- (3-methylthio) -phenyl-N' -methylguanidine the radioactivity distribution in the brain tissue of rats after intravenous injection was measured as a function of time. Data are in 'uptake units'. Asterisks indicate the average number from 2 or 3 rats per time point. Where n is 3, the mean ± SD value is shown. All other values were from 1 rat per time point. The organization numbers are as follows: 1 olfactory tubercles (olfactorytubercles), 2 entorhinal cortex (entorhinal cortex), 3 hypothalamus (hypothalamus), 4 thalamus (thalamus), 5 prefrontal cortex (prefrontal cortex), 6 striatum (striata), 7 somatosensory cortex (somasensory cortex), 8 hippocampus, 9 occipital cortex (occipital cortex), 10 hypothalamus (interfereioluli), 11 superior colliculi (superior colliculi), 12 pons and medulla (pons with canaliculus) and 13 cerebellum. Likewise, the plasma data (17) are displayed for comparison with the blood data (18).

Organization time minute (n ═ number of data points)

Claims

1. A compound of formula (I);

or a salt or solvate thereof, wherein:

R¹is hydrogen or C_1-4An alkyl group;

R²and R⁴Are each independently selected from C_1-4Alkyl group, [ 2 ]¹¹C]-C_1-4Alkyl radicals and[¹⁸F]-C_1-4fluoroalkyl with the proviso that R is²And R⁴At least one of which is [ alpha ], [¹¹C]-C_1-4Alkyl or [ alpha ], [ alpha¹⁸F]-C_1-4A fluoroalkyl group; and R³Is halogen.

2. A compound according to claim 1 of formula (Ia):

or a salt or solvate thereof, wherein R¹、R²、R³And R⁴As defined in claim 1.

3. A compound according to claim 1 or 2 selected from:

n- (2-chloro-5-methylthio) -phenyl-N' - (3-, [ solution ]¹¹C]Ethylthio) -phenyl-N' -methylguanidine; or a salt or solvate of any of them.

4. A compound according to any one of claims 1 to 3 for use in an in vivo diagnostic or imaging method such as PET.

5. Use of a compound according to any one of claims 1-3 for the preparation of a radiopharmaceutical for the in vivo diagnosis or imaging of NMDA-mediated diseases.

6. A radiopharmaceutical formulation comprising a compound according to any one of claims 1 to 3 together with a pharmaceutically acceptable excipient.

7. A method for in vivo diagnosis or imaging of NMDA-mediated diseases in a subject, preferably a human, comprising administering a compound according to any one of claims 1-3.

8. A compound of formula (II):

or a salt thereof, wherein R²Or R⁴One of which is hydrogen or a thiol protecting group such as benzyl and the other is hydrogen, C_1-4Alkyl or thiol protecting groups such as benzyl; r¹Is hydrogen or C_1-4Alkyl radical, R³Is halogen.

9. A compound of formula (II) according to claim 8, selected from:

n- (5-benzylsulfanyl-2-chloro) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine;

n- (2-chloro-5-mercapto) -phenyl-N '- (3-methylsulfanyl) -phenyl-N' -methylguanidine;

n- (2-chloro-5-methylsulfanyl) -phenyl-N ' -3 ' - (benzylsulfanyl) -phenyl-N ' -methylguanidine;

n- (2-chloro-5-methylsulfanyl) -phenyl-N ' -3 ' -mercaptophenyl-N ' -methylguanidine;

or a salt thereof.

10. A process for the preparation of a compound of formula (I) or a salt or solvate thereof:

wherein:

R¹is hydrogen or C_1-4An alkyl group;

R³is halogen;

which comprises the reaction of a compound of formula (II):

wherein R is²Or R⁴One of which is hydrogen or a thiol protecting group such as benzyl and the other is hydrogen, C_1-4Alkyl or thiol protecting groups such as benzyl; r¹Is hydrogen or C_1-4Alkyl radical, R³Is halogen;

the reaction is carried out by (i) removing any thiol protecting group, and (ii) reacting with a suitable alkyl halide¹¹C]C_1-4alkyl-X or [ alpha ], [ alpha¹⁸F]-C_1-4fluoroalkyl-Y, wherein X and Y are independently halogen, preferably chlorine, iodine or bromine, or other suitable leaving groups such as aryl or alkyl sulfonates, e.g., tosylate, triflate or mesylate; in a suitable solvent and in the presence of a base.

11. A kit for the preparation of a radiopharmaceutical formulation comprising a compound of formula (II) as defined in claim 8 or 9.