Compounds and their synthesis

Abstract

The present invention relates to sulfonium salts of formula (I): (I), their preparation, and utility as precursors for preparing functionalized organic compounds, wherein R.sub.1 and R.sub.2 are the same or different and each is independently selected from an optionally substituted aryl group, an optionally substituted alkynyl group, an optionally substituted alkenyl group, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aralkyl group, an optionally substituted arylalkenyl group, an optionally substituted heteroaryl group, an optionally substituted heterocyclyl group, an optionally substituted amine, an optionally substituted alkoxy group, an optionally substituted thioether group, an optionally substituted phosphine group, an optionally substituted boron species, an optionally substituted carbene, an organometallic moiety, and a halide, or R.sub.1 and R.sub.2 are joined together to form an optionally substituted sulfur-containing ring; W is a bond, an optionally substituted alkynylene group, an optionally substituted alkenylene group, and optionally substituted alkylene group, an optionally substituted heterocyclyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group; R.sub.3 is a moiety comprising at least one basic group, provided that when R.sub.3 does not contain any carbon atoms, W is not a bond; X is an anionic species; and n is an integer selected from 1 to 5. ##STR00001##

Claims

1. A sulfonium salt according to formula (I): ##STR00042## wherein R.sub.1 and R.sub.2 are joined together to form, together with the sulfur atom, an optionally substituted dibenzothiophene ring; W is an optionally substituted aryl group or an optionally substituted heteroaryl group; R.sub.3 is a moiety comprising at least one basic group, wherein the basic group of R.sub.3 is a Brnsted base and/or Lewis base and is selected from a primary amine, secondary amine, tertiary amine, amidine, guanidine, enamine, hydrazine, hydrazone, hydroxylamine, imine, an N-containing-heterocyclyl group, and an N-containing-heteroaryl group; X is an anionic species; and n is an integer selected from 1 to 5, wherein WR.sub.3 is a group capable of binding to a biological target and/or is a biologically active agent, wherein the aryl group of the optionally substituted aryl group of W is a C.sub.6-C.sub.16 aryl group, and wherein the heteroaryl group of the optionally substituted heteroaryl group of W is a 5-6 membered monocyclic or 8-16 membered fused bicyclic or tricyclic heteroaryl group.

2. A sulfonium salt according to claim 1, wherein W is a C.sub.6-C.sub.14 aryl group.

3. A sulfonium salt according to claim 1, wherein WR.sub.3 is a fragment of a known pharmacologically active agent.

4. A sulfonium salt according to claim 1, wherein X is selected from halide, triflate, mesylate, tosylate, tetrafluoroborate, and hexafluoroantimonate.

5. A method of preparing a sulfonium salt according to claim 1, the method comprising i) treating a thioether according to formula (II) ##STR00043## with an acidic compound so as to form an acid-base adduct by virtue of the basic group of R.sub.3; ii) treating the adduct with a compound according to the formula [(R.sub.2).sub.2I]m.sup.+Z.sup.m or formula [(R.sub.1)(R.sub.2)I]m.sup.+Z.sup.m, optionally in the presence of a catalyst, or where R.sub.1 and R.sub.2 are joined together to form an optionally substituted sulfur-containing ring in formula (I), treating an adduct of formula (II) formed in step (i) in which R.sub.1 contains at least one unsaturated bond, with an acid or electrophilic species so as to cause formation of the optionally substituted sulfur-containing ring; and iii) recovering the product sulfonium salt, wherein R.sub.1, R.sub.2, W, and R.sub.3 are as defined in claim 1, Z is an anionic species, and m is an integer selected from 1 to 5.

6. A method according to claim 5, wherein the acidic compound is a protic acid.

7. A method according to claim 6, wherein the acidic compound is selected from sulfuric acid, fluorosulfuric acid, nitric acid, phosphoric acid, fluoroantimonic acid, fluoroboric acid, hexafluorophosphoric acid, chromic acid, boric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, acetic acid, citric acid, formic acid, hydrogen chloride, hydrogen bromide, and hydrogen iodide.

8. A method according to claim 5, wherein the catalyst is a transition metal coordination complex.

9. A method according to claim 8, wherein the catalyst is a copper (II) complex.

10. A method of preparing a compound according to formula (III): ##STR00044## the method comprising i) treating a sulfonium salt according to claim 1 with a species capable of generating a nucleophile Y, optionally in the presence of a base and/or chelating agent; and ii) recovering the compound according to formula (III), wherein R.sub.3 and W are as defined in claim 1.

11. A method according to claim 10, wherein the base is selected from sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, and caesium carbonate.

12. A method according to claim 10, wherein the chelating agent is a cryptand or a crown ether.

13. A method according to claim 12, wherein the chelating agent is selected from 21-cryptand, 211-cryptand, 221-cryptand, 222-cryptand, 222B-cryptand, 12-crown-4, 15-crown-5, 18-crown-6, dibenzo-18-crown-6, and diaza-18-crown-6.

14. A method according to claim 10, wherein the nucleophile is selected from a hydrocarbon species capable of forming a carbon anion, an amine, an amide, an alkoxy group, a phenolate, a thiolate, a thiophenolate, a cyanate, a thiocyanate, and a halide.

15. A method according to claim 14, wherein the nucleophile is a halide.

16. A method according to claim 10, wherein the sulfonium salt is linked to a surface of a solid phase adsorbent.

17. A method according to claim 16, wherein the solid phase adsorbent is a reversed phase adsorbent.

18. A method according to claim 16, wherein the solid phase adsorbent is presented in conjunction with a column or cartridge.

19. A halogenated compound obtainable by the method of claim 10.

20. A solid phase adsorbent comprising a sulfonium salt according to claim 1.

21. A solid phase adsorbent according to claim 20, wherein the solid phase adsorbent is presented in conjunction with a column or cartridge.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Figures containing multiple results have been ordered in accordance with the ordering presented in the respective legend.

(2) FIG. 1. Analytical results of the reaction of precursor P4 to fluorinated compound 4. HPLC UV detection (254 nm) showed that after 5 minutes precursor P4 (expected retention time 10 min) had been consumed almost quantitatively and the two expected products, the desired fluorinated compound 4 as well as the by-product diphenyl sulphide, had been formed.

(3) FIG. 2. Identification of the radiolabelled product of the reaction of Example 8. HPLC chromatogram showing UV (254 nm; left peak) and radioactivity (right peak) detection.

(4) FIG. 3. Analytical results (HPLC chromatograms showing radioactivity detection) of the reaction of precursors P5 and P9 to fluorinated compound [.sup.18F]5.

(5) FIG. 4. Analytical results of reaction 1 of Example 10. HPLC chromatogram (top panel: radioactivity detection; bottom panel: UV detection at 254 nm) of the quenched crude reaction mixture after heating (110 C.) indicating formation of the desired radiolabelled product in very good yield but also revealing side product formation and decomposition of precursors.

(6) FIG. 5. Analytical results of reaction 3 of Example 10. HPLC chromatogram (top panel: radioactivity detection; bottom panel: UV detection at 254 nm) of the quenched crude reaction mixture after UV irradiation (254 nm) indicating formation of the desired radiolabelled product in moderate yield but also revealing light-induced decomposition of precursors.

(7) FIG. 6. Analytical results of reaction 4 of Example 10. HPLC chromatogram (top panel: radioactivity detection; bottom panel: UV detection at 254 nm) of the quenched crude reaction mixture after stirring at room temperature indicating formation of the desired radiolabelled product in good yield and without formation of side products/decomposition of precursors.

(8) FIG. 7. Biodistribution results 5, 30 and 60 minutes after administration of an .sup.18F-labelled prodrug. The synthesis of the compound involved labelling of sulfonium salt P3, followed by reaction of the obtained radiolabelled intermediate [.sup.18F]3 with phenylmagnesium bromide in a Grignard reaction.

(9) FIG. 8. Biodistribution results 5 and 30 minutes after administration of an .sup.18F-labelled prodrug. The synthesis of the compound involved labelling of sulfonium salt P3, followed by reaction of the obtained radiolabelled intermediate [.sup.18F]3 with phenylmagnesium bromide in a Grignard reaction and subsequent acylation of the alcohol with acetyl chloride.

(10) FIG. 9. Biodistribution results 5 and 30 minutes after administration of compound [.sup.18F]2 after direct labelling of sulfonium salt P2 with fluoride-18.

(11) FIG. 10. Biodistribution results 5, 30 and 60 minutes after administration of compound [.sup.18F]5 after direct labelling of sulfonium salt P5 with fluoride-18.

(12) FIG. 11. Biodistribution results 5, 15, 30, 45, 60, 90 and 120 minutes after administration of an .sup.18F-labelled compound corresponding to compound [.sup.18F]6.

(13) FIG. 12. Biodistribution results 5, 60 and 120 minutes after administration of an .sup.18F-labelled carboxylate compound corresponding to a compound derived from P5.

(14) FIG. 13. Brain metabolite analysis 60 minutes after administration of compound [.sup.14]6.

(15) FIG. 14. Biodistribution results 30, 60, 90 and 120 minutes after administration of the compound [.sup.18F]6, with P-glycoprotein activity inhibited by concomitant administration of the P-glycoprotein inhibitor tariquidar (+TQD) indicating that the metabolite as shown in FIG. 13 is transported by the efflux pump.

(16) FIG. 15. Blood and brain uptake of compound [.sup.18F]6 over time, with and without inhibition of P-glycoprotein activity by concomitant administration of TQD. Results exhibit a high brain uptake as well as a good brain to blood ratio suitable for nuclear imaging. The tracer is consistently washed out of the brain by P-glycoprotein. Inhibition of the efflux pump results in a plateau between 60 and 90 minutes after injection, indicating the window for quantitative imaging of the efflux pump. The upper line (from the left most point of the line) relates to brain uptake+TQD, the second from top line relates to brain uptake, the second from bottom line relates to blood uptake+TQD, and the bottom line relates to blood uptake.

EXAMPLES

Example 1General Procedure for the Preparation of Sulfonium Salts

(17) The respective diphenyl sulphide was dissolved in chlorobenzene (1.0 ml/100 mg) and treated with trifluoromethanesulfonic acid (1 equivalent per amine). Under inert atmosphere, diphenyliodoniumtriflate (1 equivalent) and copper (II) benzoate (0.05 equivalents) were added and the mixture was heated at 125 C. for 1 h. During this time, a brown oil separated from the colourless solution. After cooling, diethyl ether was repeatedly added (310 ml) to the brown oil and subsequently decanted to remove chloro- and iodobenzenes. The crude mixture was purified by flash column chromatography on silica. The resulting oil was taken up in methylene chloride and washed with sodium hydroxide solution (2 N) and a saturated solution of sodium triflate. The organic layer was dried over magnesium sulphate, filtered and concentrated under reduced pressure to give the desired precursor as oil. Compounds were characterised by NMR and high resolution mass spectrometry.

Example 2Preparation and Characterisation of Compound P3 ((4-(1-(4-Acetoxybutyl)piperidine-4-carbonyl)phenyl)diphenylsulfonium trifluoromethanesulfonate)

(18) Under inert atmosphere, 4-(4-(4-(phenylthio)benzoyl)piperidin-1-yl)butyl acetate (100 mg, 0.24 mmol) was dissolved in chlorobenzene (1 ml). Trifluoromethanesulfonic acid (0.02 ml, 0.24 mmol), diphenyliodonium triflate (100 mg, 0.24 mmol) and copper(II) benzoate (4 mg, 0.012 mmol) were added and the mixture was heated at 125 C. for 1 h. After cooling, diethyl ether (310 ml) was repeatedly added to the brown oil and subsequently decanted to remove chloro- and iodobenzenes. The brown oil was purified by column chromatography (methylene chloride:methanol=10:0.fwdarw.9:1). The resulting oil was taken up in methylene chloride and washed with sodium hydroxide solution (2 N) and a saturated solution of sodium triflate. The organic layer was dried over magnesium sulphate, filtered and concentrated under reduced pressure to give a colourless oil (90 mg, 60%).

(19) .sup.1H NMR (DMSO-d.sub.6, 600 MHz) (ppm): 8.26 (d, 2H, J=8.64 Hz, ph-2,6H), 7.92 (d, 2H, J=8.64 Hz, ph-3,5H), 7.93-7.86 (m, 6H, ph-2,4,6H), 7.80 (t, 4H, J=7.86 Hz, ph-3,5H), 4.00 (t, 2H, J=6.48 Hz, but-4H.sub.2), 3.46 (br s, 1H, pip-4H), 2.98 (br s, 2H, pip-2,6H.sup.eq), 2.43 (br s, 2H, but-1H.sub.2), 2.20 (br s, 2H, pip-2,6H.sup.ax), 2.00 (s, 3H, COCH.sub.3), 1.81 (d, 2H, J=12.24 Hz, pip-3,5H.sup.eq), 1.60-1.50 (m, 6H, but-2,3H.sub.2, .sup.13C NMR (DMSO-d.sub.6, 150 MHz) : 201.67 (CO.sup.ketone), 170.47 (CO.sup.acetyl), 139.56 (ph-4C), 134.60 (ph-3,5C), 131.62 (ph-2-6C), 130.48 (ph-2,6C), 129.88 (ph-1C), 124.88 (ph-1C), 120.69 (q, .sup.1J.sub.C,F=320.55 Hz, CF.sub.3), 63.67 (but-4C), 57.10 (but-1C), 52.14 (pip-2,6C), 42.84 (pip-4C), 27.68 (but-3C), 26.02 (pip-3,5C), 22.33 (but-2C), 20.78 (COCH.sub.3). .sup.19F NMR (DMSO-d.sub.6, 282 MHz): 78.20 (CF.sub.3). TOF MS ES+: 488.2274 (100%, calc 488.2259).

Example 3Sulfonium Salt Precursors

(20) Each sulfonium salt was isolated as a triflate salt.

(21) ##STR00009## ##STR00010## ##STR00011## ##STR00012##

Example 4Preparation of Sulfonium Salt Precursors Bearing Aliphatic Moieties

(22) A thioether bearing an alkenyl residue in the ortho position (with R.sup.1 and R.sup.2 being alkyl or aryl groups) can be treated with an electrophile (e.g. Br.sub.2 or ICl) to cyclise to a benzothiophenium ion. If residue R.sub.3 is, for example, an aliphatic structure that also exhibits a basic moiety, sulfonium salts for the functionalisation of aliphatic residues can be prepared.

(23) ##STR00013##

Example 5General Method for Functionalisation of Sulfonium Salts

(24) Under inert conditions, the respective sulfonium salt precursor was added to a species capable of generating a nucleophile Y (as defined above) with or without addition of a base and/or chelator in a suitable solvent (or solvent mixture). The mixture was stirred for times between 1 and 15 minutes at temperatures up to 150 C., usually up to 60 C. Work-up, purification and isolation were performed according to the physicochemical properties of the reactants used and according to the scale of the reaction (preparative/analytical).

Example 6Preparation of ethyl 4-(4-(4-fluorobenzoyl)piperidin-1-yl)butanoate (4) from (4-(1-(4-ethoxy-4-oxobutyl)piperidine-4-carbonyl)phenyl)diphenylsulfonium trifluoromethanesulfonate (P4) on an Analytical Scale

(25) To a solution of potassium fluoride (0.09 mg) in water (28 l) was added a solution of Kryptofix-222 and potassium bicarbonate (30 mM each) in acetonitrile (0.5 ml) containing 15% water. The resulting solution was azeotropically dried at 100 C. and under a stream of nitrogen. Acetonitrile (0.5 ml) was added, and the distillation was continued, this procedure being repeated. The reaction vial was subsequently closed and, after cooling to ambient temperature, (4-(1-(4-ethoxy-4-oxobutyl)piperidine-4-carbonyl)phenyl)diphenylsulfonium trifluoromethanesulfonate (P4; 1 mg, 1 equivalent related to fluoride) dissolved in dimethyl sulfoxide (0.5 ml) was added. The mixture was stirred for 5 minutes at room temperature. It was subsequently quenched with water (4.5 ml) and analysed by HPLC using a Chromolith Performance RP18-e column (1004.6 mm) at room temperature. The mobile phase consisted of water and methanol (each containing 0.5% TFA). Elution started with an isocratic solvent mixture containing 10% methanol that, after 5 minutes was increased to 90% in 17 minutes (FIG. 1).

Example 7General Fluorination Procedure Using 18F

(26) Fluoride-18 in water (50-150 MBq) was trapped on a Sep-Pak Accell Plus QMA Plus Light Cartridge (Waters) and released with a solution of Kryptofix-222 and potassium bicarbonate (30 mM each) in acetonitrile (0.5 ml) containing 15% water. The resulting solution was azeotropically dried at 100 C. and under a stream of nitrogen. Acetonitrile (0.5 ml) was added, and the distillation was continued, this procedure being repeated. The reaction vial was subsequently closed and, after cooling to ambient temperature, the sulfonium precursor dissolved in dimethyl sulfoxide (0.5 ml) was added. The mixture was stirred for 15 minutes at 110 C. After cooling, it was quenched with water (1.5 ml) and purified by HPLC using a Chromolith SemiPrep RP18-e column (10010 mm) at room temperature. The mobile phase consisted of water and methanol (each containing 0.5% TFA). Gradient elution starting with 10% methanol content that was increased to 90% allowed for isolation of the respective radioactive product (method slightly modified for each product). The identity of the radiolabelled product was confirmed by HPLC co-injection of the non-radiolabelled analogue.

Example 8Preparation and Characterisation of [18F](4-Fluorophenyl)(piperidin-4-yl)methanone ([18F]1)

(27) Fluoride-18 in water (115.0 MBq) was trapped on a Sep-Pak AccellPlus QMA Plus Light Cartridge (Waters) and released with a solution of Kryptofix-222 and potassium bicarbonate (30 mM each) in acetonitrile (0.5 ml) containing 15% water. The resulting solution was azeotropically dried at 100 C. and under a stream of nitrogen. Acetonitrile (0.5 ml) was added, and the distillation was continued, this procedure being repeated. The reaction vial was subsequently closed and, after cooling to ambient temperature, diphenyl(4-(piperidine-4-carbonyl)phenyl)sulfonium trifluoromethanesulfonate (P1) dissolved in dimethyl sulfoxide (0.5 ml) was added. The mixture was stirred for 15 minutes at 110 C. After cooling, it was quenched with water (1.5 ml) and purified by HPLC using a Chromolith SemiPrep RP18-e column (10010 mm) at room temperature. The mobile phase consisted of water and methanol (each containing 0.5% TFA). At a flow rate of 5 ml/minute, isocratic elution (10% methanol content for 5 minutes) was followed by gradient elution starting with 10% methanol that was increased to 90% in 15 minutes. [.sup.18F](4-Fluorophenyl)(piperidin-4-yl)methanone ([.sup.18F]1) was isolated. In this specific example, the analytical radiochemical yield (RCY) was 31%, and the decay-corrected isolated RCY was 21%. The identity of the radiolabelled product was confirmed by HPLC co-injection of the non-radiolabelled analogue (compound 1) using a Chromolith Performance RP18-e column (1004.6 mm) at room temperature. The mobile phase consisted of water and methanol (each containing 0.5% TFA). At a flow rate of 3 ml/minute, the organic content was increased from 1% to 40% in 15 minutes. Compounds showed a retention time of 5.1 minutes (FIG. 2). The radiochemical purity of the isolated labelled product was 99%.

Example 9Compounds Functionalised with Fluorine-18

(28) TABLE-US-00001 Product Ana- (standard reagents and conditions unless lytical Isolated specified otherwise: RCY RCY Precursor (each isolated as a triflate salt) [.sup.18F]F.sup., K.sub.222, KHCO.sub.3, DMSO, 15 min) [%] [%] Compound P1 Compound [.sup.18F]1 110 C.: 31 110 C.: 21 Compound P2 Compound [.sup.18F]2 110 C.: 33 110 C.: 21 Compound P3 Compound [.sup.18F]3 110 C.: 61 50 C.: 36 110 C.: 39 50 C.: 19 0 Compound P5 Compound [.sup.18F]5 110 C.: 21 110 C.: 10 Compound P6 Compound [.sup.18F]6 110 C.: <10 n.d. Compound P9 Compound [.sup.18F]5 110 C.: 31 110 C.: 15 Compound P10 Compound [.sup.18F]10 110 C.: <10 n.d. Compound P11 Compound [.sup.18F]11 110 C.: 84 50 C.: 80 25 C.: 68 110 C.: 54 50C.: 51 25 C.: 41 0 Compound P13 Compound [.sup.18F]13 110 C.: 68 50 C.: 61 25 C.: 31 110 C.: 54 50 C.: 40 25 C.: 22 Compound P14 Compound [.sup.18F]14 50 C.: 37 50 C.: 24 Compound P15 Compound [.sup.18F]15 110 C.: 73 50 C.: 34 n = 1 n.d. Compound P16 Compound [.sup.18F]16 150 C., 15 min: 40 150 C., 15 min: 17 Compound P17 Compound [.sup.18F]16 150 C.: 40 150 C.: 13 0 Compound P18 Compound [.sup.18F]16 110 C.: 53 110 C.: 16
These results show that sulfonium salts bearing amines, alcohols and esters functionalities were successfully labelled with fluoride-18 in moderate to good yields. Activation of the para-substituted aryl ring (compounds [.sup.18F]1-[.sup.18F]3) generally led to higher yields as compared to those compounds that were not activated (compound [.sup.18F]5). However, alkene compound [.sup.18F]5 could be labelled in 10% isolated radiochemical yield (RCY), which is enough to allow tracer preparation for imaging. Interestingly, compounds exhibiting hydrogen bond donors like the secondary amine in compound [.sup.18F]1 or the primary alcohol in compound [.sup.18F]2 could also be labelled without protecting groups. In addition, a comparison of sulfonium salts P5 and P9 showed that salts containing deactivated aryl substituents, i.e. the anisole groups in P9, exhibited excellent regioselectivity, with no detected formation of [.sup.18F]4-fluoroanisole products (FIG. 3). A corresponding result was obtained for P14 and P15. On the other hand, a small amount of [.sup.18F]4-fluorobenzene was isolated in the case of P5. Conformational restraints in P17 also led to good regioselectivity. Each experiment was performed in triplicate (unless stated otherwise, i.e. n=1 means that the experiment was performed once) and the radiochemical yield (RCY) determined as an average. Transformations P16-P18 to [.sup.18F]16 are for reference only.

Example 10Fluorination at Room Temperature

(29) Triarylsulfonium salts can undergo photolytic cleavage after irradiation with short UV light following a heterolytic pathway. In order to evaluate this mechanism as a potential fluorination/labelling method, the reaction of precursor compound P4 to tracer compound [.sup.18F]4 was further investigated. Reactions at room temperature with and without UV irradiation (254 nm) were performed and compared to that performed as described above.

(30) TABLE-US-00002 Analytical RCY Reaction Conditions [%] 1 110 C., electric lighting 47.7 2 Room temperature, electric lighting 18.8 3 Room temperature, UV (254 nm) 11.6 4 Room temperature, under light exclusion 17.5

(31) Conventional reaction conditions (heating at 110 C.) yielded almost 50% of radioactive product. When the reaction was performed at room temperature ca. 20% conversion was observed. The yield dropped to about 10% under UV irradiation, whereas reactions under light exclusion do not show differences to reactions performed without shielding from electric lighting. Importantly, only the reaction performed at ambient temperature did not show any side products, whilst energy input (heat or UV light) caused the formation of side products. UV chromatograms also showed significant differences; heating clearly led to decomposition of the sulfonium precursor and induced generation of side products. UV-irradiated reactions proceed much cleaner. However, side products most probably resulting from the photolytic cleavage of the precursor can be identified. Neither decomposition nor formation of side products was observed after performing reactions at room temperature, which significantly facilitates purification steps (FIGS. 4-6).

Example 11Trapping of Fluoride-18 by Sulfonium Salts on Solid Phase Extraction Cartridges

(32) (A)Fluoride Trapping on a Strata C-18 SPE Column

(33) Precursor compound 4 (2 mg dissolved in 2 ml water containing 5% methanol) was loaded on the preconditioned (5 ml methanol, followed by 10 ml water) SPE column. Fluoride-18 from the target water (59.11 MBq in 1 ml water) was trapped leading to 58.65 MBq of radioactivity on the column. It was subsequently washed with water (2 ml.fwdarw.55.48 MBq), tetrahydrofuran (0.5 ml.fwdarw.48.97 MBq) and dried under a stream of nitrogen for five minutes (.fwdarw.p 40.84 MBq).

(34) (B)Fluoride Trapping on a Sep-Pak Light C-18 SPE Cartridge

(35) Triphenylsulfoniumtriflate (1 mg dissolved in 1 ml water containing 10% methanol) was loaded on the preconditioned (5 ml methanol, followed by 10 ml water) SPE cartridge. Fluoride-18 from the target water (20 MBq in 1 ml water) was trapped leading to 15.6 MBq of radioactivity on the cartridge. It was subsequently washed with water (1 ml.fwdarw.14.96 MBq), acetonitrile (0.5 ml.fwdarw.14.25 MBq) and dried under a stream of nitrogen for ten minutes (.fwdarw.12.49 MBq).

Example 12Application of the Reported Chemistry to the Production of Radiotracers Investigated In Vivo

(36) A series of radiolabelled tracers was prepared using the reported chemistry and investigated in vivo (Balb/C or FVB mice) by biodistribution studies and/or metabolite analysis. Examples in FIGS. 7-10 highlight the versatility of the reported chemistry that allows for tracers with a variety of backbone structures and side chains. By deliberately modifying chemical moieties the tracer can be fine-tuned towards up-take in tissues of interest, e.g. brain, heart or lungs. FIGS. 11-15 show the results of the in vivo investigation of a potential pro-drug tracer that was developed to monitor the efflux pump P-glycoprotein at the blood-brain barrier.

(37) Mice:

(38) Female Balb/C mice and FVB mice were obtained from Charles River UK. When used, they were eight to eleven weeks old and weighing approximately 20 g. All biological work was carried out by licensed investigators in accordance with the UK Home office's Animals (Scientific procedures) Act 1986.

(39) Biodistribution Studies:

(40) The respective radiotracer (0.5-1 MBq formulated in a saline solution containing 5% ethanol) was administered intravenously into the tail vein. At designated time points between five and 120 minutes after injection, mice were anesthetized with isoflurane (5% mixed with medical air at a flow of 2 ml/min) and sacrificed by cardiac puncture. The organs of interest (blood, colon, stomach, spleen, kidneys, liver, heart, lungs, tail, femur, skull, and brain) were sampled, weighed, and the radioactivity content was measured by automated gamma counting (Cobra Multi Gamma Model 5010-Packard, UK). Results were normalised to the radioactivity found in 1% of the injected dose per gram bodyweight. All experiments were performed in duplicates or triplicates and analysed using Microsoft Office Excel 2007.

(41) Metabolite Analysis:

(42) Brains were homogenised in saline (0.5 ml) and ethanol (0.5 ml). Samples were deproteinated by adding ethanol (0.5 ml) and subsequent centrifugation (3 min, 13,000 rpm). The resulting supernatant was separated from the pellet, diluted with saline (0.5 ml) and analysed by radio-HPLC, using a Luna 3 m C8(2) 100 , LC column (304.6 mm). The mobile phase consisted of water and methanol containing 0.02% ammonium hydroxide and was used for gradient elution (from 30% of methanol to 90% in 15 min, then 5 minutes at 90%). The flow rate was 1 ml/min and the UV absorbance detector was set at 254 nm. All experiments were performed in triplicates.