WO2014098593A1

WO2014098593A1 - Verapamil like compounds

Info

Publication number: WO2014098593A1
Application number: PCT/NL2013/050925
Authority: WO
Inventors: Renske RAAPHORST; Gert LUURTSEMA; Albert Dirk Windhorst; Adriaan LAMMERTSMA
Original assignee: Stichting Voor De Technische Wetenschappen; Stichting Vu-Vumc; Rijksuniversiteit Groningen; Academisch Ziekenhuis Groningen
Priority date: 2012-12-21
Filing date: 2013-12-19
Publication date: 2014-06-26
Also published as: NL2010036C2; WO2014098593A9

Abstract

The invention is directed to a compound, a salt or a solvate thereof according to (I) wherein R₁ is an optionally branched fluoroalkyl group or [¹⁸F]fluoroalkyl group, R₂, R₃, R₄ and R₅ are eacn independently a methyl, [³H]methyl, fluoroalkyl or [¹⁸F]fluoroalkyl group, and R₆ is a (R)-cyano group or a (S)-cyano group; or wherein R₁ is a methyl group, and R₃ is a R₃ is a fluoroalkyl group or a [¹⁸F]fluoroalkyl group, R₂, R₄ and R₅ are each independently a methyl, [³H]methyl, fluoroalkyl or [¹⁸F]fluoroalkyl group, and R₆ is a (R)-cyano group or a (S)-cyano group; or wherein if R₁ is hydrogen, at least one of R₂, R₃, R₄ is an optionally branched fluoroalkyl group or [¹⁸F]fluoroalkyl group.

Description

VERAPAMIL LIKE COMPOU DS

The present invention relates to verapamiMike compounds or verapamil analogues, their use as a P-gSycoprotein inhibitor or substrate, a radiopharmaceutical formulation comprising specific verapamil like compounds, compound, a radiopharmaceutical formulation comprising specific verapamiMike compounds for use in vivo diagnostic or in vivo imaging method, a method for the in vivo diagnosis or In vivo imaging of a P- glycoprotein related disease in a subject, and a method to prepare the novel verapamiMike compounds.

Positron emission tomography (PET) is a nuclear medicine imaging technique that produces images of functional processes of the body. Radiotracers are used in PET as diagnostic tools and to image tissue concentration of molecules of interest,

P-giycoprotem, further referred to as P-gp herein, is an ABC efflux transporter mainly expressed in the btood brain barrier,a s disclosed in Mature 1984; 309:626-628, and Science 1983, 221:1285-1288. it transports xenobiotic and structurally diverse compounds out of the cell. Overexpression of P-gp leads to decreased uptake of pharmaceuticals aimed at the brain. Furthermore, this protein is involved in diseases like epilepsy, Alzheimer's disease and Parkinson's disease. Imaging of this particular protein may therefore give insights into its role in the healthy and diseased state of a person.

A known tracer for P-gp is (iicjverapamti, as disclosed in J. Nucl. Med. 1996; 37;

1571-1575. Originally utilized as a calcium channel blocker drug, it was recognized as to be a

P-gp substrate as well. fUcjVerapamii is labeled with carbon-ll and is employed clinically as a diagnostic tool, see for instnance Eur. j. Clin. Pharmacol. 2001; 56:827-829. its (H) and (S) enantiomers have been found to exhibit different in vivo pharamcokinetics, see for instance J. Label. C and Radiopharm. 2002, 45:1199-1207.

A disadvantage of the P-gp [^Cjverapamil compound is that it has a very short half- life of only 20 minutes. This short half-life limits the application of this compound. The object of this invention is to provide an alternative for [^Cjverapamii.

This is attained by the following compound: A compound, a salt or a solvate thereof according to (i), wherein R is an optionally branched fluoroaikyi group or [¹⁸F]fiuoroaikyl group, ^R4 ^ar»d % ^{are eacr}» independently a methyl, f³H]methyi , fluoroaikyi or 18

[ F]fluoroalkyl group, and R is a ( ?J-cyano group or a (S)-cyano group; or wherein is hydrogen or a methyl group, R2, R3, R4 and R5 are each independently a methyl,

[³H]methyl , fluoroalkyi or [¹⁸F]fluoroalkyl group, and R5 is a (R)-cyano group or a (S)-cyano group, wherein at least one of R2, R3, R4 and R5 is a fluoroalkyi or [¹⁸F]fluoroalkyl group.

The invention preferably relates to a compound according to general formula (I) wherein R^ is hydrogen, an optionally branched [¹⁸F]fluoroalkyl group, R2, R3, R4 and R5 are each independently a methyl, [³H]methyl , fluoroalkyi or [¹⁸F]fluoroalkyl group, and Rg is a {R)- cyano group or a (S)-cyano group. It further preferably relates to a compound according to gen eral formula (I) wherein R^ is a methyl group and R3 is a [l⁸F]fluoroalkyl group.

The compound according to this invention can be referred to as a verapamil like compound. The fluoroalkyi analogs of verapamil according to the above may be provided in an unlabeled and radiolabeled state. The radiolabeled compounds may advantageously be used as a radiopharmaceutical for use as a tracer for P-gp with a longer half-life than the existing tracers. The fluorine-18 labeled P-gp tracer is preferred, since the half-life of fluorine-18 is 110 minutes compared to 20 min for carbon-11. This is more practical and allows transport of the radiolabeled compound to hospitals which do not have their own cyclotron capacities. The tritium labeled compounds according to the present invention may advantageously be used for in vitro studies to investigate the interaction with P-gp at low concentration levels, better mimicking the in vivo situation than with unlabeled compounds.

In a first aspect, the subject invention relates to a first group of preferred verapamil like compounds are compounds wherein R^ is a fluoroalkyi group or more preferably a

[l⁸F]fluoroalkyl group. Preferably the alkyl group has 1 to 3 carbon atoms of which methyl or ethyl are especially suited. Preferably R2, R3, R4 and R5 are a methyl group. If R^ is a fluo roalkyi group R2, R4 and R5 may be all a methyl group and R3 is a [^HJmethyl group. In a second aspect, the subject invention also relates to a second group of compounds, wherein is a methyl group, and R3 is a [¹⁸F]fluoroalkyl group. Preferably R2, R4 and R5 are a methyl groups. Suitably the alkyl group has 1 to 3 carbon atoms, suitably methyl or ethyl.

Brief Description of the Figures

Figures 1 to 13 depict the molecular structures of exemplary compounds according to the invention, wherein Figure 13 shows ( ?)-0-[¹⁸F]fluoroethylverapamil.

Figure 14 shows the resulting ex vivo biodistribution of (R)-N- [¹⁸F]fluoroethylverapamil in male wister rats.

Figure 15 shows the ex vivo biodistribution of (R)-N-[¹⁸F]fluoroethylverapamil at 15 minutes in male wistar rats, after treatment with 15 mg/kg tariquidar (1^st set of blocks), compared to the ex vivo biodistribution of (R)-N-[^lsF]fluoroethylverapamil at 15 minutes in non-treated male wistar rats (second set of blocks). For comparison the result of the same experiments for ex vivo biodistribution of (R)-[^uC]verapamil is depicted in the third and fourth set of blocks, respectively.

Figure 16 shows the metabolite analysis of (R)-N-[¹⁸F]fluoroethylverapamil in male wistar rats.

Figure 17 discloses the ex vivo biodistribution, corrected for metabolism, of (R)-N- [¹⁸F]fluoroethylverapamil at 15 minutes in male wistar rats, after treatment with 15 mg/kg tariquidar, compared to the ex vivo biodistribution of (R)-N-[¹⁸F]fluoroethylverapamil at 15 minutes in non-treated male wistar rats.

Figure 18 shows two base line PET scans of 450g Wistar rats, without (left), and with blocking by 15mg Tariquidar (right).

Figure 19 shows time activity curves of the brain uptake of (fl)-N- [¹⁸F]fluoroethylverapamil.

Detailed Description

The non-radiolabeled compounds according to this first group may advantageously be used as reference compounds. Examples of such preferred compounds are illustrated in the attached Figures as compounds 1: (5)- and (/?)-N-Fluoroethylverapamil, wherein R^ is a fluoroethyl group and R2, R3, R4 and R5 are each a methyl group; compounds 2: (S)~ and (/?)- N-Fluoromethylverapamil, wherein is a fluoromethyl group and R2, R3, R4 and R5 are each a methyl group.

Examples of suitable radiolabeled compounds according to this First Group are compound 5: (5)- and (fiJ-N-t^Fjfluoroethylverapamil, wherein R^ is a [^FJfluoroethyl group and R2, R3, R4 and R5 are each a methyl group; compound 6, (S)- and ( ?)-N-

[^^F]fluoromethylverapamil, wherein R^ is a [l^F]fluoromethyl group and R2, R3, R4 and R5 are each a methyl group.

The radiolabeled compounds are preferably prepared starting from a suitable precursor. These precursors are actually the more important compounds as they are the compounds which are stored and used to prepare the radiolabeled compounds hours before their actual use. These compounds should have a molecular structure which enables one to easily, preferably by means of one synthesis step, prepare the desired radiolabeled compound. Applicants found that compounds 9 and 10, wherein R^ is a fluoromethyl or fluoroethyl group and R2, R4 and R5 are a methyl group and R3 is hydrogen are suited precursors for preparing compounds 7 and 8 respectively by means of a methylation.

Preferred precursor compounds are compounds wherein ^ is hydrogen and wherein group R^ is substituted by the desired fluoroalkyl group by means of an alkylation.

Compounds 11, wherein R^ is hydrogen, R2, R4 and R5 are a methyl group and R3 is hydrogen are suited intermediate compounds to prepare compounds 9 and 10, which compounds 9 and 10 are in turn suitable precursors to prepare compounds 7 and 8 respectively.

For a second group of compounds R^ is a methyl group. Preferably R2, R4 and R5 are a methyl groups and R3 is a fluoroalkyl group or a more preferably a [^FJfluoroalkyl group. Suitably the alkyl group has 1 to 3 carbon atoms, suitably methyl or ethyl. Examples are compound 3, (S)- and (/?)-0-fluoroethylverapamil, wherein R^, 2, R4 and R5 are each a methyl group and R3 is a fluoroethyl group; compound 4, (5)- and (R)-0- fluoromethylverapamil, wherein Rj_, R2, R4 and R5 are each a methyl group and R3 is a fluoromethyl group. Examples of radiolabeled compounds are compound 12, (5)- and {R)-0-

[!^FJfluoroethylverapamil, wherein Rj_, R2, R4 and R5 are each a methyl group and R3 is a - [l^F]fluoroethyl group and compound 13, (S)- and ( ?)-0-[l^F]fluoromethylverapamil, wherein R^, R2, R4 and R5 are each a methyl group and R3 is a -[^Fjfluoromethyl group.

The above radiolabeled compounds are preferably prepared from a precursor compound wherein R3 is hydrogen or a leaving protective group that preferably increases the nucleophilicity of the oxygen atom, such as preferably alkyltosylate.

The - optionally actived- hydroxyl group may be substituted by the desired fluoroalkyl group by means of a!kylation. An example of a suitable precursor compound is compound 14, (S)- and ( ?)-desmethylverapamil, wherein R^, R2, R4 and R5 are each a methyl group and R3 is hydrogen.

The radio labelled compounds and non-radio labelled compounds according to the present invention may be purified according to those methods known to the person skilled in the art, for example by means of HPLC purification or Solid Phase Extraction (SPE). The HPLC purification is preferable carried out on a preparative HPLC column packed with reverse phase material such as, but not limited to, C18, C18-EPS or C8, a mobile phase consisting of a mixture of methanol, ethanol or acetonitrile mixed with water or water containing buffer like, but not limited to, ammonium dihydrogen phosphate or an acid like phosphoric acid or trifluoracetic acid . The Solid Phase Extraction (SPE) is preferably performed on a Sep-Pak or similar device, such as, but not limited to, a C18, a tC18, a Silica or a n Oasis Sep-Pak. The compound is preferably eluted from the Sep-Pak with a solvent suitable for injection in vivo, like ethanol.

The above treated compounds may be formulated to a desired formulation for their intended use. For example the collected HPLC fraction from the preparative HPLC, containing a com pound according to the invention may be diluted with water or water containing such as, but not limited to, sodium hydroxide or hydrogen chloride. The diluted fraction as prepared is preferably trapped on a bonded silica sample preparation devices for solid-phase extraction for sample preparation, e.g. Sep-Pak or similar, but not limited to, such as preferably a C18, tC18, Silica or an Oasis Sep-Pak (Sep-Pak is a registered trademark of Water Corporation), and the compound is subsequently preferably eluted from the Sep- Pak with a solvent suitable for injection in vivo, such as ethanol. The obtained eluate is preferable diluted with pharmaceutically acceptable buffers such as, but not limited to 0.9% sodi um chloride, sodium dihydrogenphosphate 7.09 mM in 0.9 % sodium chloride or citrate buffer, pharmaceutically acceptable solubilisers such as, but not limited to, ethanol, tween or phospholipids and/or with pharmaceutically acceptable stabilizers or antioxidants such as, but not limited to, ascorbic acid, gentisic acid or p-aminobenzoic acid.

The invention is also directed to the salts and solvates of the compounds described above. Suitable salts according to the invention, include physiologically acceptable acid addition salts such as those derived from mineral acids, but not limited to, hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric or sulphuric acids or those derived from organic acids such as, but not limited to, tartaric, fumaric, malonic, citric, benzoic, trifluoroacetic, lactic, glycolic, gluconic, methanesulphonic or p-toluenesulphonic acids.

Applicants found that the compounds according to the invention may be

advantageously be used as a P-glycoprotein inhibitor or substrate. More specifically the compounds may be used as part of a pharmaceutical formulation for use in diagnostics of/studying diseases like, but not limited to, epilepsy, Alzheimer's disease and Parkinson's disease.

The invention is especially directed to a radiopharmaceutical formulation

comprising the above described radiolabeled compounds. More especially the invention is directed to a radiopharmaceutical formulation comprising the above described radiolabeled

[!^F] compounds for use in an in-vivo diagnostic or an in-vivo imaging method. The diagnostic or imaging method of the P-glycoprotein will provide insight into diseases where this protein plays a role, like for example epilepsy, Alzheimer's disease and Parkinson's disease. The imaging method may be positron emission tomography (PET).

The invention is thus also directed to a method for the in vivo diagnosis or in vivo imaging of a P-glycoprotein related disease in a subject, preferably a human, comprising administration of a radiolabeled [^F]compound according to the invention or a formulation comprising such a a radiolabeled

In a preferred embodiment, the norverapamil analogue of (I) may be employed, wherein R]=hydrogen. Suitable precursors or compounds include are those depicted in the following structural formulae la,b to 5 a,b, namely (R)-0-[18F]fluoronorverapamil and (S)-O- [18F]fluoronorverapamil (la,b); (R)-O-fluoronorverapamil and (S)-O-fluoronorverapamil (2a,b) that may serve as "cold", i.e. not radiolabeled references for la, lb, 3a or 3b, respectively, namely (R)-0-[18F]fluoronorverapamil and (S)-0-[18F]fluoronorverapamil; Tritium labeled (R)-O-fluoronorverapamil and (S)-O-fluoronorverapamil (3a_;b); Precursors of tritium labeled (R)-O-fluoronorverapamil and (S)-O-fluoronorverapamil (4a, b); and precursors of (R)-0-[18F]fluoronorverapamil and (S)-0-[18F]fluoronorverapamil (5a, b).

In a solution of 5-((3,4-dimethoxyphenethyl)amino)-2-(3,4-dimethoxyphenyl)-2- isopropylpentanenitrile (100 mg, 0.227 mmol) (J Med Chem 1999, 42, 1687-1697) in 2 ml DMF, l-bromo-2-fluoroethane (0.068 ml, 0.908 mmol) and potassium carbonate (125 mg, 0.908 mmol) were added and stirred at 65°C overnight. Consumption of the starting amine was observed on TLC (MeOH:DCM 5:95, v/v) and the reaction mixture was diluted with 20 ml ethyl acetate and the mixture was washed with water (3 x 20 ml), brine (20 ml) and dried over sodium sulfate. The volatiles were removed by rotary evaporation and the remainder was purified by flash column chromatography (MeOH:DCM = 1:99, v/v) to afford 5-((3,4- dimethoxyphenethyl)(2-fluoroethyl)amino)-2-(3,4-dimethoxyphenyl)-2- isopropylpentanenitrile. ¹H-NM R (250 M Hz, CDCI₃) δ: 6.76 (m, 6H), 4.53 (m, 1H), 4.34 (m,

1H), 3.86 (m, 12H), 2.77 (m, 1H), 2.66 (m, 4H), 2.52 (t, 2H, J= 6.8), 2.06 (m, 2H), 1.82 (m, 1H), 1.49 (m, 1H), 1.17 (d, 3H, J= 6.63), 0.78 (d, 3H, J= 6.63) ¹³C-NM R (62.9 M Hz, CDCI₃) δ:

148.908, 148.718, 148.171, 147.208, 132.849, 130.593, 121.468, 120.466, 118.596, 111.967, 111.084, 110.950, 109.478, 83.983, 81.313, 77.507, 77.000, 76.490, 56.386, 37.914, 35.414, 33.015, 23.400, 18.910, 18.558

Example 2

To a solution of 5-bromo-2-(3,4-dimethoxyphenyl)-2-isopropylpentanenitrile (60 mg, 0.176 mmol) in 1 ml DMF 4-(2-aminoethyl)-2-methoxyphenol (44.2 mg, 0.265 mmol) and potassium carbonate (36.6 mg, 0.265 mmol) were added. The reaction was stirred overnight at room temperature. The mixture was diluted with diethyl ether and washed with water, brine and dried over Na₂S0₄. The volatiles were removed by rotary evaporation and the crude remainder was purified by flash column chromatography (MeOH:DCM= 5:95, v/v) to afford 2-(3,4-dimethoxyphenyl)-5-((4-hydroxy-3-methoxyphenethyl)amino)-2- isopropylpentanenitrile. ¹H-NMR (250 MHz, CDCI₃) δ: 6.83 (m, 4H), 6.65 (m, 2H), 3.86 (m, 9H), 2.80 (b, 4H), 2.66 (t, 2H, J= 6.3), 2.08 (m, 3H), 1.89 (dt, 1H, J= 4.5 12.4), 1.59 (m, 1H), 1.17 (d, 3H, J= 6.63), 0.78 (d, 3H, J= 6.63) ¹³C-NMR (125.78 M Hz, CDCI₃) δ: 148.955, 148.226, 146.505, 144.065, 131.235, 130.408, 121.321, 121.132, 118.608, 114.384, 111.195, 111.018, 109.457, 55.964, 55.849, 55.811, 50.772, 49.080, 37.852, 35.507, 25.570, 18.928, 18.554 Example 3

To a solution of 2-(3,4-dimethoxyphenyl)-5-((4-hydroxy-3-methoxyphenethyl)amino)-2- isopropylpentanenitrile (16 mg, 0.038 mmol) in 1 ml acetonitrile l-bromo-2-fluoroethane (8.38 μΙ, 0.113 mmol) and DIPEA (9.83 μΙ, 0.056 mmol) were added. The reaction was brought to 70°C and was stirred overnight. TLC analysis (MeOH :DCM=5:95) still showed remaining starting material and extra l-bromo-2-fluoroethane (10 μΙ, 0.134 mmol) was added and the reaction was stirred at 70°C for additional 6 hours. The volume was reduced by rotary evaporation. The crude product was dissolved in ethyl acetate, washed with sat. NaHC0₃ and dried over Na₂S0₄. The volatiles were removed by rotary evaporation and the remaining crude product was purified by gradient flash column chromatography

(MeOH:DCM=2:98 to 5:95, v/v) to afford 2-(3,4-dimethoxyphenyl)-5-((2-fluoroethyl)(4- hydroxy-3-methoxyphenethyl)amino)-2-isopropylpentanenitrile. ¹H-NMR (250 MHz, CDCI₃) δ: 6.83 (m, 4H), 6.63 (m, 2H), 5.47 (b, 1H), 4.53 (m, 1H), 4.34 (m, 1H), 3.87 (m, 9H), 2.77 (q, 1H, J= 4.8), 2.63 (m, 4H), 2.51 (t, 2H, J= 6.8), 2.06 (m, 2H), 1.81 (dt, 1H, J= 4.5 12.4), 1.50 (m, 2H), 1.17 (d, 3H, J= 6.63), 0.78 (d, 3H, J= 6.63)

Example 4

[ F]2-bromofluoroethane (Bioorganic & Medicinal Chemistry 2009, 17, 7441-7448) was added to a suspension of 5-((3,4-dimethoxyphenethyl)amino)-2-(3,4-dimethoxyphenyl)-2- isopropylpentanenitrile (5 mg) and potassium carbonate (5 mg) in 1 ml DMF. The obtained mixture was brought to 100 °C for 30 minutes and cooled to 20 °C. The reaction was quenched with 1 ml acetonitrile and purified by HPLC, using a Kromasil 100-10-C18 250*22 mm column (Kromasil is a registered trademark of EKA Chemicals AB ) concentrated on a tC- IS Sep-Pak and formulated in a phosphate buffer/saline mixture, with a yield of 10-30%, as corrected for decay, a purity of > 95%, and a specific activity > 20 GBq/μΐΎΐοΙ.

Example 5

2-bromo [ F]fluoroethane was synthesized starting from 2-bromoethyltosylate in DMF, which was added to a dried ¹⁸F/K₂.2.2/I 2C03 solution. This mixture was heated for 10 min at 90 °C, after which the product was distilled at 90 °C through a heated (200°C) AgOTf column into a cooled (0 °C) vessel containing 1.5 mg (3.5 μιηοΙ) of nor-verapamil and 5.0 mg (3.6 μιηοΙ) of K₂C0₃. This reaction mixture was heated to 120 °C for 15 min during stirring and purified by HPLC, using a Luna lOu C18(2) 100A, 250 x 10.00 mm column (Luna is a registered trademark of Phenomenex, Inc.) , 72:28 ACN/5 mM K₃P0₄), resulting in (R)-N- [¹⁸F]fluoroethylverapamil in 5% radiochemical yield decay-corrected. The radiochemical purity was >99% detected with HPLC ( Kromasil C18 lOu, 250 x 4.6 mm, 40:60 ACN/H20 + 0.1% TFA, as obtained from Grace Corp.) and no observable UV peaks were detected. Example 6

Biodistribution Study: The biodistribution of (/?)-/V-[¹⁸F]fluoroethylverapamil was studied in healthy Wistar rats. The animals were injected with +/- 50 M Bq of the tracer and sacrificed after 5, 15, 30 and 60 min (Λ/=3 or 4). The indicated organs were collected, weighted and counted for activity. No increased bone uptake was observed, indicating that no

defluorination had occurred. The tracer had a low, but observable, uptake in the brain which is stable over time, as illustrated by the results depicted in Figure 14.

Example 7 Blocking study: The behavior of ( ?)-/V-[¹⁸F]fluoroethylverapamil with respect to P-gp was tested in a blocking study, with the use of the well-known P-gp inhibitor tariquidar. The inhibitor was injected i.v. in healthy Wistar rats at a 15 mg/kg dose during a 15 minute injection period. 5 minutes after the tariquidar injection, 50 MBq of the (R)-N- [¹⁸F]fluoroethylverapamil was injected. The animals were sacrificed 15 minutes after the (/?)- A/-[¹⁸F]fluoroethylverapamil injection and the indicated organs were collected, weighted and counted for activity.

The data shows a 2-fold increased brain uptake of the tracer in the P-gp blocked brain 15 minutes after tracer injection. This is a significant increase, however, a lower increase compared to [ C]verapamil. The brain uptake of [ C] verapamil increases 10-fold in the presence of tariquidar, as illustrated in Figure 15.

Example 8

Metabolite studies: The metabolism of (/?)-/V-[¹⁸F]fluoroethylverapamil was assessed in blood plasma and brain of healthy male Wistar rats. The rats were injected with 35-50 MBq of (/?)-/V-[¹⁸F]fIuoroethylverapamil and were sacrificed at 5, 15 and 60 minutes after injection (from left to right in Figure 16).

Homogenized brain and blood plasma were passed over a SPE cartridge to separate the polar from the non-polar fractions. The activity of all fractions was measured and only the non-polar fractions were analyzed with HPLC.

( ?)-/V-[¹⁸F]fluoroethylverapamil was found to rapidly metabolize, 46%, 21% and 4% of the parent tracer is detected after 5, 15 and 60 minutes, respectively, as illustrated in Figure 16, which discloses the metabolite analysis of (R)-N-[¹⁸F]fluoroethylverapamil in male wistar rats. Without wishing to be bound to any particular theory, it is believed that the high fraction of polar metabolites may be explained by the cleavage of [¹⁸F]fluoroethyl group. Although only 21% of the parent compound remained after 15 minutes, still an increased tracer uptake in the brain is observed. Since the polar metabolites are very unlikely to be P-gp substrates, it may hence be assumed that this uptake is only initiated by the parent ( ?)- /V-[¹⁸F]fluoroethylverapamil.

If the observed increase in brain uptake after the tariquidar treatment is corrected for metabolites, a 4-fold increase can be calculated. Example 9

PET Imaging studies: The PET imaging study confirmed an increased uptake of (R)-N- [¹⁸F]fluoroethylverapamil in the brain in combination with the inhibitor tariquidar (15 mg/kg). On day 1, a baseline scan was performed on big healthy Wistar rats (N=3) with (R)- N-[¹⁸F]fluoroethylverapamil followed by a scan with Na¹⁸F to locate the bones. The second day the animals were injected with 15 mg/kg tariquidar and after 30 minutes {R)-N- [¹⁸F]fluoroethylverapamil was administered. The time-activity curve (Figure 17) shows a 4- fold increased brain uptake of the tracer in the blocked animals in the first 10 minutes of the scan. However, still after 60 minutes, when according to metabolite studies only 3% of the tracer is still intact, the brain uptake is still 2-fold higher compared to the baseline scans, as illustrated in Figure 18.

Figure 19 depicts the time-activity curve, which shows a 4-fold increased brain uptake of the tracer in the blocked animals in the first 10 minutes of the scan. However, still after 60 minutes, when according to metabolite studies only 3% of the tracer is still intact, the brain uptake is still 2-fold higher compared to the baseline scans.

The results of the in vivo studies of ( ?)-/V-[¹⁸F]fluoroethylverapamil show that a 4-fold increase in the brain uptake when blocked with tariquidar could eb achieved, which indicates the possibility to utilize this tracer as a diagnostic tool to investigate the P-gp function in combination with P-gp blockers. While this particular tracer was found to be rapidly metabolized, other verapamil-like compounds were designed and synthezised with one of the methoxy groups on the aromatic rings replaced by fluoroalkyl groups, e.g. a fluoroethyl group, which showed a slower metabolisation. The present invention also preferably relates to the synthesis of the precursor for this new tracer, (R)-0- [¹⁸F]fluoroethylverapamil, and to the compound and its enatiomer.

Claims

Compo nd, a salt or a solvate thereof according to (I)

wherein is an optionally branched fluoroalkyi group or [ F]fluoroalkyl group,

R2, R3, R4 and R5 are each independently a methyl, [³H]methyl , fluoroalkyi or [¹⁸F]fluoroalkyl group, and

Rg is a (fl -cyano group or a (S)-cyano group; or

wherein R^ is hydrogen or a methyl group,

R2, R3, R4 and R5 are each independently a methyl, [³H] methyl , fluoroalkyi or [¹⁸F]fluoroalkyl group, and

R5 is a (R)-cyano group or a (S)-cyano group,

wherein at least one of R2, R3, R4 and R5 is a fluoroalkyi or [¹⁸F]fluoroalkyl group.

Compound according to claim 1, wherein the fluoroalkyi group or

[¹⁸F]fluoroalkyl group of R^ comprises 1 to 3 carbon atoms.

Compound according to claim 2, wherein the alkyl group is methyl or ethyl.

Compound according to any one of claims 2-3, wherein R^ is a

[¹⁸F]fluoroalkyl group.

Compound according to any one of claims 2-3, wherein R2, R3, R4 and R5 are a methyl group or wherein R2, R4 and R5 are a methyl group and R3 is hydrogen. 6. Compound according to any one of claims 2-3, wherein R2, R4 and R5 are a methyl group and R3 is a [³H] methyl group.

7. Compound according to claim 6, wherein is a methyl group, R2, R4 and R5 are a methyl groups and R3 is a fluoroalkyl group or a [¹⁸F]fluoroalkyl group preferably comprising from 1 to 3 carbon atoms.

8. Compound according to claim 7, wherein the alkyl group is methyl or ethyl.

9. Compound according to any one of claims 7 or 8, wherein R3 is a

[¹⁸F]fluoroalkyl group.

10. The ( ?)- or (S)- Enantiomer of a compound according to any one of the

proceeding claims, or an enantiomerically en riched composition comprising the ( ?)- and the (S)- Entantiomer.

10. Compound according to any one of claims 1 to 10 for use as a P-glycoprotein inhibitor or substrate.

11. A radiopharmaceutical formulation comprising the compound according to claim 4, 6, 9 and/or 10.

12. A radiopharmaceutical formulation comprising the compound according to claim 4, 6, 9 and/or 10 for use in vivo diagnostic or in vivo imaging method.

13. A method for the in vivo diagnosis or in vivo imaging of a P-glycoprotein related disease in a subject, preferably a human, comprising administration of a compound according to any one of the claims 4. 6, 9 and/or 10 or a formulation according to claim 12.