WO2015194954A1 - 6,7-dioxyalkyltetrahydroisoquinoline compounds - Google Patents

Abstract

The present invention relates to a 6,7-dioxyalkyltetrahydroisoquinoline compound, or a salt or solvate thereof according to formula I: (formula I), (I) wherein R represents hydrogen or a fluorinated alkyl group, and R₂ and R₃ independently represents hydrogen or an alkyl group.

Description

6,7-Dioxyalkyltetrahvdroisoquinoline Compounds

The present invention relates to 6,7-dioxyalkyltetrahydroisoquinoline compounds, their use as radiopharmaceuticals for P-glycoprotein imaging at the blood-brain barrier, and their synthesis process.

Background of the invention

P-glycoprotein (P-gp) is an efflux transporter protein on the luminal membrane of the brain endothelial cells, as disclosed in Clin. Pharmacol. Then, vol. 94, no. 1 , pp. 3-9, 2013. P-gp belongs to a family of transporters with similar function active at the blood- brain barrier (BBB),( amongst which the Breast Cancer Resistance Protein (BCRP) and Multidrug Resistance-associated Protein 1 (MRP1 ) are most important. This family of transporters is also known as the ATP-binding cassette (ABC) transporters, as their function is ATP dependent, as set out in Nat. Struct. Mol. Biol., vol. 1 1 , no. 10, pp. 918- 927, 2004.

They recognize small hydrophobic exogenous compounds which have diffused through the BBB and are effluxed back into the blood after recognition by the ABC transporters.

Consequently, P-gp not only protects the brain from harmful compounds, but can also reduce the uptake of drug that need to act in the brain G. M. Graff, Candace L; Pollack, "Drug transport at the BBB and the choroid plexuse," Curr. Drug Metab., vol. 5, pp. 95-108, 2004.

Moreover, P-gp function is altered in some neurological diseases. For instance, decreased P-gp function has been found in Alzheimer Disease (AD, see Brain, vol. 135, no. Pt 1 , pp. 181-9, Jan. 2012) and Parkinson Disease (PD, see J Neural Transm, vol. 1 15, pp. 1001-1009, 2008), while it has been hypothesized that P-gp function is increased in epilepsy, see Epilepsia, vol. 48, no. 9, pp. 1774-1784, 2007.

Positron emission tomography (PET) is a molecular imaging technique that is able to measure tissue concentrations of compounds labeled with positron emitting isotopes as a function of time in a non-invasive manner. To study P-gp functions with PET, radiolabeled substrates as well as radiolabeled inhibitors could be applied, as proposed in Curr. Top. Med. Chem., vol. 10, no. 17, pp. 1703-14, Jan. 2010.

The most widely used substrate radiopharmaceutical for P-gp imaging is [¹¹C]verapamil. It has been used either as racemic mixture or as (fl)-enantiomer [8] and has been utilized in clinical studies, see Clin. Pharmacol. Then, vol. 91 , no. 2, pp. 227- 33, Feb. 2012, and ibid., pp. 941-946, 2009. Since Verapamil is metabolized rapidly to numerous metabolites, interpretation of the measured activity in the tissue may not be conclusive. Also, it may result in a low signal to noise ratio, i.e., a modest increase of brain uptake after P-gp inhibition, which severely may limit its usefulness.

The P-gp inhibitors laniquidar, tariquidar and elacridar have been labeled with carbon-1 1 but prove to behave as substrates at tracer levels and are not specific for P- gp but are also transported by BCRP, see ACS Chem. Neurosci., vol. 2, no. 2, pp. 82- 89, Feb. 201 1 , and Drug Metab. Dispos., vol. 41 , no. 4, pp. 754-62, Apr. 2013.

Most of the labeled compounds which are currently in use as radiopharmaceuticals for P-gp are labeled with carbon-1 1.

A disadvantage of the use of carbon-1 1 is the fact that a cyclotron is required on site due to the short half-life of 20 min for carbon-1 1 , which makes a widespread use difficult. Furthermore, a longer half-life would enable prolonged imaging times and also more subjects could be dosed from radiopharmaceutical synthesis.

Fluorine-18 labeled radiopharmaceuticals have been proposed in variety of applications, such as [¹⁸F]fluoropaclitaxel has been used in the tumour imaging, as disclosed in Nucl. Med. Biol. 2007 Oct, 34(7), pp. 823-31 .

Tariquidar and elacridar have been labeled also with 1 -[¹⁸F] fluoroethyl as set out in Bioorg. Med. Chem., vol. 19, no. 2, pp. 861-70, Jan. 201 1 . However, the utility of 1 - [¹⁸F]fluoroelacridar was found very limited due to the low radiochemical yield and observed defluorination in vivo, as shown in Bioorg. Med. Chem., vol. 19, no. 7, pp. 2190-8, Apr. 201 1 .

Accordingly, there remains the need for radiopharmaceuticals to specifically track P-gp, which can be transported to imaging centres without on-site cyclotron, with good in vivo activity and a higher half-life than 1 1 -Carbon labeled compounds, and their use acquisition of prolonged imaging times. Yet further, this may also allow dosing more subjects from radiopharmaceutical synthesis.

Applicants have now identified and prepared a new class of F-labeled 6,7- dioxyalkyltetrahydroisoquinoline acting as P-gp substrates . When ¹⁸F-radiolabeled, these compounds also permit to specifically track P-gp presence. SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a 6,7-Dioxyalkyltetrahydroisoquinoline compound, or a salt or solvate thereof according to formula I:

(I) wherein Ri represents hydrogen or a fluorinated alkyl group, and R₂ and R₃ independently represents hydrogen or an optionally tritiated alkyl group.

In a second aspect the present invention also relates to a compound according to formula I as set out above, a solvate or salt thereof for use as an in vivo substrate and/or inhibitor for P-gp.

In a third aspect, the present invention also relates to a radiopharmaceutical formulation comprising the compound according to the invention, and its use in an in vivo diagnostic or imaging method.

In a fourth aspect, the present invention also relates to a method for the in vivo diagnosis or imaging of P-gp related disease or behaviour in a subject, preferably a human, comprising administration of a compound or a formulation according to the invention.

In a fifth aspect, the present invention also relates to a process for the preparation of a compound according to the invention, preferably a [¹⁸F]-fluorinated compound, the process, comprising:

(a) reacting a precursor compound according to formula (I) wherein Ri represents the group -R-L wherein R- represents an alkyl group, and L is a leaving group under conditions permitting a nucleophilic substitution upon reaction with [¹⁸F] fluoride, or

(b) reacting a precursor compound according to formula (I) wherein Ri is hydrogen, under alkylation conditions with a [¹⁸F] labeled alkyl reagent, and

(c) isolating the obtained compound from the reaction mixture. The present invention also relates to a process for the preparation of a compound according to the invention, preferably a [¹⁸F] -fluorinated compound, the process comprising reacting a precursor compound according to formula (I) of claim 1 wherein Ri is a leaving group with a compound -R-L wherein R- represents a fluorinated or [¹⁸F] fluorinated alkyl group , and L is a leaving group under conditions permitting a nucleophilic substitution, and isolating the compound from the reaction mixture.

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.

The compounds according to the invention may advantageously be used as part of a pharmaceutical composition for use in the treatment and diagnostics in brain disorders., such as neurodegenerative diseases, including but not limited to Alzheimer and Parkinson disease.

The present invention relates to a 6,7-Dioxyalkyltetrahydroisoquinoline compound, or a salt or solvate thereof according to formula I wherein Ri represents a fluorinated alkyl group, and R₂ and R₃ independently represents hydrogen or an alkyl group.

Preferably, R-i represents a fluorinated alkyl group comprising from 1 to 4 carbon atoms. More preferably, Ri represents -CH₂F or -CH₂CH₂F. Again more preferably, Ri is a [¹⁸F]-fluorinated alkyl group. One of R₂ or R₃ preferably represents hydrogen, and the other of R₂ or R₃ represents an alkyl group.

Again more preferably, R₂ or R₃ each represents an alkyl group, preferably ethyl or , methyl, most preferably wherein both of R₂ and R₃ represent a methyl group.

The tetraline moiety is preferably bound to the dioxyalkyltetrahydroisoquinoline moiety via a saturated or unsatured propylene spacer moiety.

A particularly preferred compound is 5-(1 -(2-fluoroethoxy))-[3-(6,7-dimethoxy-3,4- dihydro-1 H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalene (Compound 14 in Figure 3), and the [¹⁸F] version thereof (([¹⁸F]14 in Figure 5, also depicted as compound 9-2 in Figure 9). Other preferred compounds are depicted in Figure 9. The radiolabeled compounds according to the invention can advantageously be used as diagnostic imaging agents for in vivo imaging of the activity of the P- glycoprotein (P-gp) function with positron emission tomography (PET) or single photon emission computed tomography (SPECT).

BRIEF DESCRIPTION OF THE FIGURES

These and further features can be gathered from the claims, description and drawings and the individual features, both alone and in the form of sub-combinations, can be realized in an embodiment of the invention and in other fields and can represent advantageous, independently protectable constructions for which protection is hereby claimed. Embodiments of the invention are described in greater detail hereinafter relative to the drawings, wherein:

Fig. 1 to 5 disclose various reaction schemes

Fig. 6 discloses cerebral kinetics of the ¹⁸F labelled tracers (MC210 = ¹⁸F labelled Compound 4 ([¹⁸F]4b]), MC224 = ¹⁸F labelled Compound 8 ([¹⁸F]8]), MC225 = ¹⁸F labelled Compound 14 ([¹⁸F]14])). Data are SUV-PET values for the entire brain, expressed as mean ± SEM (n = 3-6).

Fig. 7 discloses SUV-PET images of [18F]- compound 14 in control mouse (left), Mdr1 a/b(-/-) mouse (middle) and Mdr1 a/b(-/-)Bcrp1 (-/-) mouse (right), sagittal view. Vinci software was used to create the images.

Fig. 8 shows a biodistribution study of all the tracers performed after the scans. Data are SUV expressed as mean ± SEM (n = 3-6).

Fig. 9 discloses further compounds 9-1 to 9-8 according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

P-glycoprotein, abbreviated further herein as P-gp, is also known as permeability glycoprotein, multidrug resistance protein 1 (MDR1 ), ATP-binding cassette sub-family B member 1 (ABCB1 ), or cluster of differentiation 243 (CD243).

P-gp appears to operate by allowing substrates to enter either from an opening within the inner leaflet of a membrane wherein the protein sits, or from an opening at the cytoplasmic side of the protein. ATP then can bind at the cytoplasmic side of the protein. Following binding, ATP hydrolysis shifts the substrate into a position to be excreted from the cell. Release of the phosphate from the original ATP molecule then occurs concurrently with substrate excretion. ADP is released, and a new molecule of ATP binds to the secondary ATP-binding site. Hydrolysis and release of ADP and a phosphate molecule resets the protein, so that the process can start again.

P-gp is an important protein of the cell membrane that pumps many foreign substances out of cells, by acting an ATP-dependent efflux pump with broad substrate specificity. It exists in animals, fungi and bacteria and likely evolved as a defence mechanism against harmful substances. P-gp has been found to be extensively distributed and expressed in the intestinal epithelium where it pumps xenobiotics, e.g. toxins or drugs back into the intestinal lumen, in liver cells where it pumps them into bile ducts, in the cells of the proximal tubular of the kidney where it pumps them into urine- conducting ducts, and in the capillary endothelial cells comprising the blood-brain barrier and blood-testis barrier, where it pumps them back into the capillaries.

Some cancer cells were found to express large amounts of P-gp, which can render these cancers multi-drug resistant. Also the ability of P-gp to transport the many substrates accounts for the many roles of P-gp including regulating the distribution and bioavailability of drugs, whereby overexpression can reduce the absorption of drugs that are substrates for P-gp, resulting in a reduced bioavailability; whereas decreased P-gp expression, supratherapeutic plasma concentrations and drug toxicity can occur.

The BBB is a major impediment to the entry of many therapeutic drugs into the brain due to the barrier being composed of specialized endothelial cells that prevent various substances from entering the brain. Here P-gp plays a particular role in the transport of compounds out of the brain across the BBB.

The invention is also directed to a method for the in vivo diagnosis or imaging of P-gp activity in a subject, preferably a human, comprising administration of a radiolabeled compound according to the invention. Administration of the compound is preferably administrated in a radiopharmaceutical formulation comprising the compound or its salt or solvate and one or more pharmaceutically acceptable excipients in a form suitable for administration to humans. The radiopharmaceutical formulation is preferably an aqueous solution additionally comprising a pharmaceutically acceptable buffer, a pharmaceutically acceptable solubiliser such as, but not limited to, ethanol, tween or phospholipids, pharmaceutically acceptable stabilizer solutions and/or antioxidants such as, but not limited to, ascorbic acid, gentisic acid or p-aminobenzoic acid.

The invention advantageously also relates to a radiopharmaceutical formulation comprising a radiolabeled compound according to the invention and to a radiopharmaceutical formulation comprising the radiolabeled compound according to the invention for use as an in vivo diagnostic or imaging method, wherein the method is preferably positron emission tomography (PET) or single photon emission computed tomography (SPECT).

The radiolabeled compounds have a relatively short half time and are thus preferably prepared shortly before use in the above referred to in vivo diagnosis or imaging of P-gp related diseases. Suitably the compound is synthesised for the greater part to obtain a non-radiolabeled precursor compound. This non-radiolabeled precursor compound can by means of a relatively simple synthesis be reacted with a radiolabeled compound to obtain the radiolabeled compound of the present invention. The invention is also directed to any novel precursor described below and to the below processes to prepare the radiolabeled compounds.

The following, non-limiting experiments illustrate the invention.

Material and methods

Chemicals and cell lines

All reagents and solvents were obtained from commercial suppliers (Merck, Sigma- Aldrich, Rathburn and B. Braun) and were used without further purification. (R)- norverapamil was purchased from ABX advanced biochemical compounds. Human liver microsomes were obtained from BD Gentest (20 mg/ml_). Cell culture reagents were purchased from Celbio s.r.l. CulturePlate 96/wells plates were bought from Perkin-Elmer Life Science. MDCK-MDR1 , MDCK-BCRP and MDCK-MRP1 cell lines were a gift from Professor P. Borst, NKI-AVL Institute, Amsterdam, Netherlands. Caco-2 cells were a gift from Dr. Aldo Cavallini and Dr. Caterina Messa from the Laboratory of Biochemistry, National Institute for Digestive Diseases, 'S. de Bellis', Bari, Italy.

Comparative examples 4b, 8, and Example 1 (compound 14) were identified as

P-gp substrates in the cell experiments. These compounds all comprise conformationally restricted non-basic moieties such as phenyloxazole (4b), biphenyl (8) or tetrahydronaphthalene (14) respectively, and are set out herein as representatives of novel classes of compounds. Furthermore, the compounds shared the same basic 6,7- dioxytetrahydroisoquinoline moiety as in tariquidar or elacridar.

The compounds were prepared, and then labeled with fluorine-18 and their brain uptake was investigated with PET imaging in P-gp expressing FVB mice as well as in the Mdr1 a/b^(~ Bcrp1^(~ knockout strain of Mice. In addition, P-gp selectivity of [¹⁸F] 14 was investigated in Mdr1a/b ^~ mice. The results of these three novel compounds was compared to those obtained with (fl)-[¹¹C]verapamil. Comparative Example 4b

Figure 1 shows the reaction scheme for the synthesis of Compound of compound 4 (A): SOCI₂, Et₃N, NH₄OH, CH₂CI₂; (B): 1 ,3-dichloroacetone; (C): Na₂C0₃, DMF.

Compounds 4a, b were synthesized as described in Figure 1 . Synthesis of compounds 2a-4a has been reported previously, and spectral data of these compounds were identical to those previously described in MedChem, vol. 4, no. 2, pp. 188-95, Feb. 2009. 4-Fluorobenzamide (2b) was prepared from the corresponding commercially available 4-fluorobenzoic acid (1 b, 1 mmol), which was treated with an excess of SOCI2 (7 mmol) in presence of Et₃N (0.1 mmol) under reflux for 1 h. The resulting acyl chloride was added to a mixture (45 mL) of NH₄OH (28 %), H₂0 and CH₂CI₂ (1 : 1 : 1 v/v/v). The mixture was stirred at room temperature for 4 h. The organic layer was separated from the aqueous layer and washed with 2 M NaOH (3 x 10 mL). The organic solution was dried over Na₂S0₄ and evaporated. The residue was purified by silica gel column chromatography (CH₂CI₂/MeOH 95:5 v/v). The yield of brown solid 2b was 30 %. GC- MS m/z: 139 (M⁺, 67), 123 (100), 95 (72).

4-(Chloromethyl)-2-(4-fluorophenyl)oxazole (3b) was prepared condensing amide 2b with 1 ,3-dichloroacetone (2 mmol) while heating at 200 ^°C for 5 h. After cooling to room temperature, water (10 mL) and CHCI₃ (10 mL) were added. The aqueous phase was extracted with CHCI3 (3 ^χ 50 mL), and the combined organic layers were dried over Na₂S0₄ and evaporated. The residue was purified on silica gel column chromatography (EtPt/AcOEt 8:2 v/v) yielding 30 % of 3b as a brown solid. GC-MS m/z: 213 (M⁺+2, 19), 21 1 (M⁺, 57), 176 (78). ¹H NMR (CDCI₃) δ: 4.32 (s, 2H, CH₂), 7.12-7.16 (m, 2H), 7.6 (s, 1 H), 8.03 (dd, 2H, J = 1 .9, 8.1 Hz).

3b (1 mmol) was alkylated with 6,7-dimethoxy-1 ,2,3,4-tetrahydroisoquinoline (1.2 mmol) in DMF (20 mL) using Na₂C0₃ as a base (2 mmol). The mixture was refluxed overnight. DMF was evaporated and the residue was partitioned between H₂0 (20 mL) and CHCI3 (20 mL). The organic phase was separated, the aqueous phase was extracted with CHCI3 (3 ^χ 50 mL) and the collected organic fractions were dried over Na₂S0₄ and evaporated. The residue was purified on silica gel column chromatography (CH₂CI₂/EtOAc 1 : 1 v/v) yielding 74 % 4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)- yl)methyl)-2-(4-fluorophenyl)oxazole (4b) as a brown oil. Hydrochloride salt was recrystallized from MeOH/Et₂0. Mp: 232-235 ^°C. ESI⁺/MS m/z: 369 [M+H]⁺, 391 [M+Na]⁺. ESr/MS/MS m/z: 352 (16), 230 (50), 176 (98), 121 (100). ¹ H NMR (CDCI3) δ: 2.94 (s, 4H, CH₂NCH₂), 3.75-3.79 (m, 4H, NCH₂CH₂), 3.82 (s, 3H, CH₃), 3.88 (s, 3H, CH₃), 6.50 (s, 1 H), 6.58 (s, 1 H), 7.1 1-7.17 (m, 2H), 7.67 (s, 1 H), 8.03 (dd, 2H, J = 5.5, 8.8 Hz). Purity of final compound was established by combustion analysis of the corresponding hydrochloride salt, confirming a purity >95 %. (C,N,H) C2i H2i FN203HCI(H₂0)o.5.

Synthesis of Comparative Example 2 (Compound 8).

Figure 2 shows the reaction scheme for the synthesis of Compound 8: (A): SOCI₂ and Et₃N; CH₂CI₂ and 1 .2 % NaOH; 6,7-dimethoxytetrahydroisoquinoline; (B) LiAIH₄, THF; (C) 2-fluoroethyl tosylate, NaH, DMF

Compound 8 and its precursor 7 were synthesized as reported in Figure 2.

Synthesis of compounds 5-7 was previously reported and spectral data of these compounds were identical to those previously described [18]. 8 was synthesized by reaction of 2-fluoroethyl tosylate with 7 as described below. 2-Biphenyl-4-yl-2- fluoroethoxy-6,7-dimethoxy-1 ,2,3,4-tetrahydro-isoquinoline (8) was obtained in a yield of 45 %. (C,N,H) C26H28FNO3HCI. ESr/MS m/z; 444 [M+Na]⁺, ESI⁺/MS/MS m/ 441 (100). ¹H NMR (CDCI3) δ: 2.83-2.76 (m, 4H), 3.57 (s, 2H), 3.71 (s, 2H), 3.80 (s, 3H, CH₃), 3.83 (s, 3H, CH₃), 4.21 (t, 1 H, J = 1 .2 Hz), 4.30 (t, 1 H, J =1 Hz), 4.70 (t, 1 H, J = 1 .6 Hz), 4.86 (t, 1 H, J = 1 Hz), 6.45 (s, 1 H), 6.60 (s, 1 H), 7.01 (d, 2H, J = 8 Hz), 7.42-7.55 (m, 6H).

Synthesis of 2-fluoroethyl tosylate

Synthesis of 2-fluoroethyl tosylate was performed by making a solution of 2- fluoroethanol (2 mmol) and p-toluenesulfonyl chloride (1.1 mmol) in 5 M NaOH (1 .6 mmol), which was stirred at room temperature for 24 hours. The reaction mixture was diluted with CH₂CI₂ and the organic phase was washed with 10 % NaOH. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The crude product was purified by chromatography on a silica gel column with CH₂CI₂ to give a colorless oil that was stored in the freezer (yield 92 %). ESI⁺/MS m/z: 241 [M+Na]⁺, ESI⁺/MS/MS m/z 241 (74), 97 (100). ¹ H NMR (CDCI₃) δ: 2.45 (s, 3H, CH₃), 4.21 (t, 1 H, J = 4 Hz, CH₂), 4.30 (t, 1 H, J = 4 Hz, CH₂), 4.49 (t, 1 H, J = 4 Hz, CH₂), 4.64 (t, 1 H, J = 4 Hz, CH₂), 7.34 (d, 2H, J = 8 Hz), 7.80 (d, 2H, J = 8 Hz).

General procedure for fluoroethylation

A suspension of NaH (60 %, 1 mmol) in DMF dry (3 ml_) was stirred at room temperature for 10 minutes. A solution of phenol precursor (1 mmol 7 or 13) in DMF (1 mL) was added and the solution was stirred for 1 hour. A solution of 2-fluoroethyl tosylate was added (2 mmol) in DMF (1 mL) and the reaction mixture was stirred for 4 hours. Water was added until effervescence ceased. The solvent was evaporated and the residue was partitioned between H₂0 (20 mL) and CHC (20 mL). The organic phase was separated, the aqueous phase was extracted with CHCI3 (3 ^χ 50 mL) and the collected organic fractions were dried over Na₂S0₄ and evaporated. The residue was purified on silica gel column chromatography (CHCI₃/MeOH 19: 1 v/v) and recrystallized from MeOH/Et₂0. Synthesis of Example 1 Compound 14

Figure 3 illustrates the synthesis of Compound 14: (A): CH₃S0₂CI, Et₃N, CH₂CI₂, T = - 10 °C; (B): cyclopropylMgBr, 3 N HCI, THF; (C): 6,7-dimethoxy-1 , 2,3,4- tetrahydroisoquinoline, Na₂C0₃, DMF; (D): H₂, Pd/C (5 %); (E): 2-fluoroethyl tosylate, NaH, DMF

Compounds 14 and its precursor 13 were synthesized as reported in Figure 3. Synthesis of compounds 9-13 was previously reported and spectral data of these compounds were identical to those previously described in J Med Chem, vol. 52, no. 14, pp. 4524-4532, Jul. 2009.

14 was synthesized by reaction of 2-fluoroethyl tosylate with 13 by following the general procedure reported above. 5-(1 -(2-fluoroethoxy))-[3-(6,7-Dimethoxy-3,4- dihydro-1 H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen (14) was obtained in a yield of 38 %. (C,N, H) C₂₆H₃5FN0₃HCI. ESI⁺/MS m/z: 450 [M+Na]⁺, ESI⁺/MS/MS m/ 420 (47), 388 (100). ¹ H NMR (CDCI₃) δ: 1 .63-1 .85 (m, 9H), 2.60-2.84 (m, 8H), 3.55 (s, 2H), 3.83 (s, 3H, CH₃), 3.84 (s, 3H, CH₃), AA A (t, 1 H, J =3 Hz), 4.24 (t, 1 H, J =3 Hz), 4.66 (t, 1 H, J =3 Hz), 4.83 (t, 1 H, J =3 Hz), 6.52 (s, 1 H), 6.58-6.63 (m, 2H), 6.84-7.10 (m, 2H).

Synthesis of 2-bromoethyl tosylate

A volume of 3 mL of DCM was added to toluenesulfonyl chloride (5.0 mmol) in round bottom flask and mixture was brought to 0°C. Triethylamine (5.0 mmol) was added and 2-bromoethanol (4.0 mmol) was added dropwise. Mixture was stirred for 1 h at 0°C. It was brought to room temperature, washed with water (10 mL), brine (10 mL) and dried over Na₂S0₄. Crude product was concentrated under reduced pressure into brown liquid. Product was purified by silica gel chromatography (EtOAc/hexane 1 :9 v/v). Product was isolated as a colorless liquid (yield 49 %). ¹ H NMR (500 MHz, CDCI₃) δ: 7.84 (d, 2H, J = 5 Hz), 7.39 (d, 2H, J = 10 Hz), 4.31 (t, 2H, J = 5 Hz), 3.50 (t, 2H, J = 1 Hz), 2.48 (s, 3H).

Radiochemistrv

Fluorine-18 tracers

Production, trapping and drying of the fluoride was done in the same way for all the fluorine-18 tracers. Figure 4 shows the reaction scheme for the radiosynthesis of [¹⁸F]-labeled compound 4.

A QMA anion exchange cartridge (Waters Sep-Pak light Accell Plus QMA) was eluted with 5 mL of 1.4 % Na₂C0₃ solution and washed with water until pH was neutral. The QMA was dried using argon or nitrogen gas flow. [¹⁸F]fluoride was produced by irradiation of [¹⁸0]water with the Scanditronix MC-17 cyclotron via the ¹⁸0(p,n)¹⁸F nuclear reaction. The Zymark robotic system was used for the labeling. The aqueous [¹⁸F]fluoride was passed through the QMA to recover the ¹⁸0-enriched water. The [¹⁸F]fluoride was eluted from the cartridge with 3-5 mg of K₂C0₃ in 1 mL of water and collected to a vial with 15 mg of Kryptofix 2.2.2. 1 mL of dry acetonitrile was added to the vial and solvents were evaporated at 130 °C. Three subsequent additions of 0.5 mL acetonitrile were added and evaporated. In the [¹⁸F] -4 radiosynthesis (scheme 4), 2 mg of precursor 4a in 0.5 mL of DMF was added to the dried [¹⁸F]/KF/Kryptofix complex and reacted for 30 minutes at 160 °C. After reaction the vial was cooled for 2 minutes and 0.5 mL of HPLC eluent (0.05 M NaOAc/MeOH/THF 55:27: 18 (v/v/v)) was added. The product was purified by RP-HPLC (column Symmetryshield RPS 5 pm 7.8 χ 300 mm, flow 2 mL/min, 254 nm, R_t([¹⁸F] COMPOUND 4) = 15 min). Tracer was collected into a bottle containing 80 mL of sterile water. After mixing with helium, the solution was transferred to an Oasis HLB 1 cc (30 mg) extraction cartridge. [¹⁸F] COMPOUND 4 was trapped to the cartridge which was washed twice with 8 mL of water and the product was eluted with 1 mL of ethanol through a Millipore Millex LG filter (0.2 pm). A volume of 4 mL of 0.9 % NaCI was also added to reduce the ethanol concentration to 20 %. Quality control (QC) was performed in HPLC using Xterra C18 5 um 4,6 χ 250 mm column and 0.05 M NaH₂P0₄/MeOH/THF 55:27: 18 (v/v/v) as eluent with a flow of 1 mL/min.

Figure 5 illustrates the reaction scheme for the radiosynthesis of [¹⁸F] COMPOUND 8 and [¹⁸F] Compound 14

In the synthesis of [¹⁸F]COMPOUND 8 ([¹⁸F]8) and [¹⁸F]COMPOUND 14 ([¹⁸F]14) (scheme 5) 15 μί of 2-bromoethyl tosylate (15) in 1 ml of 1 ,2-dichlorobenzene was added to the dried fluoride complex. Distillation of the formed [¹⁸F]bromoethyl fluoride (16) at 90 degrees was started immediately with a helium gas flow to the second vial in room temperature containing 2 mg of precursor (7 or 13) and 3 mg of NaH in 0.5 mL of DMF. After 15 min of distillation, vial 2 was reacted for 5 min at 80 degrees in the synthesis of [¹⁸F]COMPOUND 14 and 10 min in the synthesis of [¹⁸F]COMPOUND 8. After reaction, 0.5 mL of HPLC eluent was added (0.1 M NaOAc/MeCN 5.5:4.5 (v/v)) and the product was purified by RP-HPLC (column Symmetryshield RPS 5 pm 7.8 χ 300 mm, flow 3 mL/min, 254 nm, R_t([¹⁸F]COMPOUND 14, [¹⁸F]COMPOUND 8) = 10 min). Tracer was collected into a bottle containing 80 mL of sterile water. After mixing with helium, the solution was transferred to an Oasis HLB 1 cc (30 mg) extraction cartridge. The product was trapped to the cartridge which was washed twice with 8 mL of water and the product was eluted with 1 mL of ethanol through a Millipore Millex LG filter (0.2 pm). A volume of 4 mL of 0.9 % NaCI was also added to reduce the ethanol concentration to 20 %. Alltech Alltima C18 5 urn 4.6 χ 250 mm was used as a column in the QC with either the same eluent as in prep. HPLC ([¹⁸F]COMPOUND 8) or MeCN/water 1 : 1 + 0.1 % TFA ([¹⁸F]COMPOUND 14) (1 mL/min).

Comparative Example 3: (f?H¹¹ C1verapamil

The synthesis of (fl)-[^{1 1}C]verapamil was performed as described with some modifications in Appl. Radiat. Isot. , vol. 57, no. 4, pp. 505-7, Oct. 2002.

[¹¹ C]CH₄, produced directly in the target using ¹⁴N(p,a)¹¹ C nuclear reaction, was trapped in the liquid nitrogen. [¹¹ C]methane was first converted into [¹¹ C]methyl iodide and further into [1 1 C]methyl triflate by passage through a column containing silver- triflate impregnated graphitized carbon. [¹¹ C]Methyl triflate was bubbled into (R)- norverapamil (0.5 mg) solution in 0.5 mL of acetonitrile. The reaction mixture was heated for 5 min at 120 °C. Water (0.5 mL) and MeCN (0.5 mL) were added, solution was injected into HPLC (column Symmetryshield RPS 5 pm 7.8 χ 300 mm) and purified with 25 mM NaH₂PO₄ pH 7.0/MeCN/MeOH (41 :37:22 v/v/v) as mobile phase (flow rate 5 mL/min, UV detection at 210 nm). The eluted fraction containing [¹¹ C]verapamil was collected into 80 mL of sterile water. Organic solvents were removed by passing the mixture through an Oasis HLB 1 cc (30 mg) extraction cartridge, following rinsing of the cartridge (twice) with 8 mL of saline solution (0.9 % NaCI). (fl)-[¹¹ C]-Verapamil was eluted by passing 0.8 mL of EtOH through the cartridge and formulated with 4 mL of saline solution. Quality was controlled on HPLC using Alltima C18 5 pm 4,6 x 250 mm column and 0.1 M NaH₂PO₄ (pH 3)/MeCN (65:35 v/v) as eluent (flow rate 1 .5 mL/min, UV detection at 210 nm). Distribution coefficient Log D

n-Octanol (0.5 ml_) and phosphate buffered saline (PBS, pH = 7.2, 0.5 ml_) were pipetted in 1 : 1 ratio into Eppendorf tubes. The tracer solution (~1 MBq, 100 μΙ_) was added, tubes were vortexed for 1 minute and centrifuged for 5 minutes at 6000 rpm. Samples (100 μΙ_) of octanol and PBS layer were counted on a γ-counter (LKBG- Compugamma CS 1282, Wallac). The distribution coefficient Log D was calculated as log(A₀ctanoi/ApBs)- Measurements were done in triplicate. Animals

All animal studies were in compliance with the local ethical guidelines and protocols were approved by the Institutional Animal Care and Use Committee of the University of Groningen.

Male FVB wild type mice (28.2 ± 1 .8 g), Mdr1 a/b^(~ Bcrp1^(~ constitutive knockout mice (28.6 ± 1 .1 g) and Mdr1a/b^(~ constitutive knockout mice (27.9 ± 2.3 g) developed from the FVB line were purchased from Taconic. After arrival, animals were acclimatized at least 7 days in the Central Animal Facility of the University Medical Center Groningen. Mice had access to food and water ad libitum and were kept under a 12 h light-dark cycle. During experiments, mice were anesthetized with 2 % isoflurane in medical air and warmed with a heating pad.

PET procedure

Mice were injected with a fluorine-18 tracer (5.3 ± 1 .9 MBq, 0.1 mL, 64 ± 22 ng of [¹⁸F]COMPOUND 4, 7.0 ± 7.0 ng of [¹⁸F]COMPOUND 8, 0.3 ± 0.01 ng of [¹⁸F]COMPOUND 14) or (fl)-[¹¹C]verapamil (7.3 ± 3.2 MBq, 0.2 mL) in the penile vein under isoflurane anesthesia. [¹⁸F]COMPOUND 4 was injected on the table and animals were transferred into the microPET camera (microPET Focus 220, Siemens Medical Solutions USA, Inc.) causing a few minutes delay between injection and start of the 60 minute dynamic emission scan. All the other tracers were injected directly on the camera following the start of the 30 minute dynamic scan. A transmission scan of 515 s with a ⁵⁷Co point source was performed for correction of attenuation and scatter by tissue after an emission scan. Mice were terminated by cervical dislocation. Several organs and tissues were excised, weighed and radioactivity was measured with a γ- counter. Blood sample and half of the brain were taken for the metabolite analysis. Radioactivity accumulation in the organs was expressed as standardized uptake value (SUV), using the following formula: [tissue activity concentration (MBq/g)]/[injected dose (MBq)/body weight (g)].

Metabolite analysis

In vitro metabolism was examined in human liver microsomes. 4 mM solution (14 μΙ_) of COMPOUND 4, COMPOUND 14, COMPOUND 8 or (fl)-verapamil solution in DMSO was pipetted into test tubes to have final concentration of 20 μΜ. Verapamil was used as a reference compound, because its metabolic pathways are known [21 ]. PBS (2576 μΙ_) and human liver microsomes (70 μΙ_, final protein concentration 0.5 mg/ml_) were added, and tubes were preincubated at 37 °C for 5 minutes. NADPH solution (20 mM, 140 μΙ_) was freshly made in PBS, and was added to tubes in final concentration of 1 mM to start the reaction. The first sample (400 μΙ_) was taken immediately and proteins were precipitated by addition of acetonitrile + 0.1 % formic acid (2 V). Other samples were taken at time points 15, 30, 45, 60, 90 and 120 min. Three control tubes were also incubated for 120 min: one without microsomes, one without NADPH and one without test compound. In these tubes the volume of a missing compound was replaced by PBS. Sample tubes were vortexed and centrifuged for 6 min at 12000 rpm. Supernatants were analyzed by UPLC/MS/MS (Waters Xevo QTof MS, column ACQUITY UPLC BEH C18 1 .7 μηπ, eluent H₂O/CH₃CN + 0.1 % HCOOH, flow 0.6 mL/min, ESI positive mode). Metabolynx XS software (Waters) was used for the analysis of metabolites. Found mass peaks were integrated and the area was converted to percentage considering the parent area at T = 0 min as 100 %. Percentage of the parent compound was expressed as function of time. Results from 2-3 experiments were fitted using second order polynomial fitting in GraphPad Prism software (data not shown).

In vivo the metabolites were analyzed in the plasma and brain. Terminal arterial whole blood samples (0.5 mL) taken after the scans were centrifuged for 5 minutes at 6000 rpm to obtain plasma. Plasma samples were precipitated by 2 volume addition of acetonitrile, centrifuged and 2.5-10 μΙ_ samples of supernatant were applied on a thin layer chromatography (TLC) plate (F-254 silica gel plates, Sigma-Aldrich). Metabolism in the brain was investigated by dissecting half of the brain and homogenizing it with 0.9 ml of acetonitrile. Brain homogenate was centrifuged and supernatant was applied on TLC. Plates were eluted with ethylacetate and methanol, ratio 95:5 for [¹⁸F]COMPOUND 4 and [¹⁸F]COMPOUND 14 and ratio 99: 1 for [¹⁸F]COMPOUND 8 (R_f[¹⁸F]COMPOUND 4 = 0.57, R_f[¹⁸F]COMPOUND 14 = 0.41 , R_f[¹⁸F]COMPOUND 8 = 0.69). Radioactivity of the eluted plates was analyzed by phosphor storage imaging. Exposed screens were scanned with a Cyclone phosphor storage system (PerkinElmer Life and Analytical Science). Percentage of the intact tracer was calculated by region of interest (ROI) analysis using OptiQuant software (PerkinElmer Life and Analytical Science).

PET data analysis

A 60 minute scan was separated into 21 frames: 6 ^χ 10, 4 ^χ 30, 2 ^χ 60, 1 ^χ 120, 1 ^χ 180, 4 ^χ 300 and 3 ^χ 600 s of which 30 min scan consequently had the first 18 frames. Emission sinograms were normalized and corrected for attenuation and radioactive decay. The sinograms were iteratively reconstructed (two-dimensional ordered subsets expectation maximization (OSEM2D) with Fourier rebinning, 4 iterations and 16 subsets). The final data sets consisted of 95 slices with a slice thickness of 0.8 mm and an in-plane image matrix of 128 x 128 pixels. The voxel size was 0.9 mm x 0.9 mm x 0.8 mm. The Inveon Research Workplace software (Siemens Medical Solutions USA, Inc.) was used for the data analysis. All the frames were summed, and a PET image was co-registered with an MRI template. Whole brain ROI based on the MRI template was generated. Radioactivity concentration was converted to SUV values and plotted as a time-activity curve (TAC). Statistical analysis

Differences between control and knockout animals were calculated for statistical significance using two-sided unpaired Student's f test. A P value of less than 0.05 was considered statistically significant. Results: Cell experiments

Three different cell experiments were performed with the non-radioactive MC- compounds to examine the substrate potential: apparent permeability experiment in Caco-2 cells, Calcein-AM experiment to determine selectively the potency (EC50) towards each transporter and ATP depletion assay. Experimental setup is described in detail elsewhere [16] [22]. A compound is defined as a substrate when it is transported and it activates ATPase. The ratio of drug transport through Caco-2 monolayers in the basolateral-apical and apical-basolateral directions (P_apP BA/AB) was higher than 2 for all the compounds (9.5, 7.9 and 6.1 respectively for COMPOUND 4, COMPOUND 8 and COMPOUND 14) as presented in table 1 . EC50 values were determined in the Calcein-AM experiment in Madin-Darby Canine Kidney cells overexpressing selectively each transporter.

All compounds showed high affinity towards P-gp, most active being COMPOUND 14 (EC₅₀ = 0.35 μΜ).

Only COMPOUND 4 had some affinity to Bcrp (15 μΜ), but none of the compounds were found to have affinity to Mrpl All the compounds activated ATPase. Based on the results from all the experiments, compounds were classified as substrates.

Table 1 . Results of the cell experiments with non-radioactive compounds

Compound P_app P-gp Bcrp Mrp1 ATPase

BA/AB EC₅O (MM) EC₅O (MM) EC₅O (MM) activation COMPOUND 9.5 21 15 M OO Yes

4

COMPOUND 7.9 0.54 >100 >100 Yes

8

COMPOUND 6.1 0.35 >100 >100 Yes

14

Radiolabeling

[¹⁸F]COMPOUND 4 was synthesized via nucleophilic aromatic substitution reaction in 90 minutes with a total radiochemical yield of 3.2 ± 2.6 %. The radiochemical purity was >96 % and the specific activity 29 ± 13 GBq/pmol. Radiochemical yield (decay corrected) is calculated for the formulated product from the end of bombardment. The measured Log D was 2.6. [¹⁸F]COMPOUND 8 and [¹⁸F]COMPOUND 14 were synthesized in a two pot method in 70-80 minutes. They were produced with a radiochemical yield of 1 1 .9 ± 4.7 %. Specific activity for both tracers was >100 GBq/pmol. Radiochemical purity was >98 % for [¹⁸F]COMPOUND 8 and >95 % for [¹⁸F]COMPOUND 14. Log D value was measured as 2.9 for [¹⁸F]COMPOUND 8 and 3.0 for [¹⁸F]COMPOUND 14. (fl)-[¹¹C]verapamil was produced in 39 min with a radiochemical yield of 8.2 ± 4.9 % and specific activity of 34.1 ± 16.5 GBq/pmol. Radiochemical yield is calculated for the formulated product from the trapped activity of [¹¹C]CH₄. PET data and biodistribution

Figure 6 shows the cerebral kinetics of the tracers. Data are SUV-PET values for the entire brain, expressed as mean ± SEM (n = 3-6).

Figure 7 shows SUV-PET images of [¹⁸F]COMPOUND 14 in control mouse (left), Mdr1 a/b^{( />} mouse (middle) and Mdr1 a/b ^~ Bcrp1 ^~ mouse (right), sagittal view. Vinci software was used to create the images.

In the [¹⁸F]COMPOUND 4 scan the peak of activity in the brain was missed, due to the delay between tracer injection and start of the scan. On average, the first 7.7 minutes were not recorded. However, it is clear from the time-activity curves (figure 1 ) that there is no difference in the brain uptake between knockout and control animals. In the biodistribution study (figure 3), the uptake in all the organs and tissues was also similar between the strains. The cerebral kinetics of [¹⁸F]COMPOUND 8 were not significantly different between the strains either. In addition, the shape of the time-activity curve was different than expected. There is no initial peak of uptake, but instead the curves are slowly rising until reaching a plateau after 10 min. In the periphery [¹⁸F]COMPOUND 8 had nonspecific uptake in all the collected organs and tissues. In the biodistribution data no significant difference between the strains was found. Notable is the uptake in plasma, which could refer to binding to plasma proteins.

[¹⁸F]COMPOUND 14 had almost the same maximum uptake in the Mdr1 a/b^(~ Bcrp1^(~ mice as [¹¹C]verapamil, but the excretion from the brain was slower. Uptake in the control mice was higher than for [¹¹C]verapamil. The area under curve (AUC) 0-30 min of [¹¹C]verapamil in Mdr1 a/b ^~ Bcrp1 ^~ mice was 3.7 fold bigger than in the control mice. With [¹⁸F]COMPOUND 14 the difference was twofold. Maximum knockout/control ratio with [¹⁸F]COMPOUND 14 was reached in 15-20 minutes when for [¹¹C]verapamil it was 5 minutes. There was no statistically significant difference between the [¹⁸F]COMPOUND 14 uptake in Mdr1 a/b^h Bcrp1^h and Mdr1 a/b^(~ knockout mice (figure 7). The area under the curve of Mdr1a/b ^~ mice constitutes to about 86 % of the AUC of Mdrla/b^Bcrpl^mice. In the biodistribution study the uptake of [¹¹C]verapamil was significantly different between the strains in testes and brain at time point 45 min. [¹⁸F]COMPOUND 14 was excreted to the urine slower than [¹¹C]verapamil, most of the uptake was in the liver and pancreas. Statistical differences between control animals and Mdr1 a/b ^~ Bcrp1 ^~ knockouts were found in the spleen, small intestine, testes and brain. Between Mdr1a/b ^~ knockouts and controls the difference was found in whole blood, plasma, heart, liver, spleen, small intestine, urine and brain. In contrast to the brain, the radioactivity uptake in many P-gp containing peripheral organs was actually higher in control animals than in the knockouts.

Figure 8 shows a biodistribution study of all the tracers performed after the scans. Data are SUV expressed as mean ± SEM (n = 3-6).

In addition to TACs, we calculated brain-to-plasma radioactivity ratios from the biodistribution data. Therefore, the values represent only one time point after the scan (Table 2). Brain-to-plasma radioactivity ratios of [¹⁸F]COMPOUND 14 suggest that radioligand is P-gp selective and is not transported by Bcrp. The difference in brain uptake between control and P-gp knockout was 4.1 -fold for [¹⁸F]COMPOUND 14 and 7.1 -fold for [¹¹C]-verapamil. Furthermore, baseline brain uptake of [¹⁸F] COMPOUND 14 was approximately 4-fold higher than for [¹¹C]verapamil.

Table 2 Metabolism

Phase I metabolism of the non-radioactive compounds was investigated in the human liver microsomes, to study the possible metabolites and metabolism rate. Control tubes without microsomes and without NADPH were included to see if the compound metabolized/degraded without them and if the microsomes were working adequately. Tubes without the test compound were analyzed to detect impurities in the chemicals or in the LC-MS system. In the experiment performed in human liver microsomes, 60% of the intact COMPOUND 4 was still present after 75 min. Decomposition of the compound caused a formation of 6,7-dimethoxy-1 ,2,3,4-tetrahydroisoquinoline, which was identified by MS as a primary metabolite. In vivo plasma samples were analyzed by radio-TLC. Majority of the metabolites were hydrophilic (>85 %, R_f = 0). After 75 min, 43 ± 8 % of the parent tracer was still intact. Brain metabolism data is not available. COMPOUND 8 was stable in the microsome study and no metabolites were found. In the light of in vivo data, this could be due to the protein binding. Only the supernatant was analyzed, but the amount of the compound in the precipitate is not known. At 45 min, 78 ± 10 % of the parent [¹⁸F]COMPOUND 8 was intact in the plasma and 69 ± 10 % in the brain, based on the radio-TLC. All the observed metabolites were hydrophilic (R_f = 0). Demethylation and defluoroethylation was observed for COMPOUND 14 in microsome incubations. After 45 min, 80 % of the parent was still intact. In vivo at the same time point the number was 69 ± 1 1 % in the plasma and 96 ± 0.8 % in the brain. All the found metabolites were hydrophilic in the nature (R_f =0-0.3). In vivo [¹¹C]verapamil metabolite analysis was not possible due to the low activity. However, metabolism of (fl)-[¹¹C]verapamil is known in rats, see Nucl. Med. Biol., vol. 32, no. 1 , pp. 87-93, Jan. 2005.

At 30 min post-injection, 47 % of the parent tracer was intact in plasma and at 60 min 28 %. In the brain at 30 min 68 % of the intact tracer was found and at 60 min 47 %. Typical metabolites are O- and A/-demethylated compounds as well as A/-dealkylated one. These were found also in the microsome experiment where about 60 % of the parent compound was intact at 45 min.

Results and Conclusions

A novel PET-tracer, Compound 14, intended for imaging P-glycoprotein function was synthesized and evaluated in control and knockout mice, and compared to comparative Examples 1 and 2 (Compounds 4 and 8, respectively).

All of the tracers were labeled with fluorine-18 to increase their applicability.

Compounds were characterized in vitro as substrates and were compared in vivo to the best known P-gp substrate [¹¹C]verapamil, Comparative Example 3.

[¹⁸F]COMPOUND 14 had higher uptake in the transporter knockout mice than in the control mice. [¹⁸F]COMPOUND 14 was evaluated both in Mdr1 a/b^(~ Bcrp1^(~ and Mdr1a/b^(~ mice. Uptake in the Mdr1a/b^(~ knockout was lower than in the Mdr1a/b^(~ Bcrp1^(~ knockout, indicating that Bcrp is involved in the transport. As for [¹¹C]verapamil, it has been found to be transported only by P-gp and not by Bcrp or Mrp1 in the BBB in nanomolar concentrations, see Nucl. Med. Biol., pp. 1-6, Jul. 2013. Uptake in control mice was higher for [¹⁸F]COMPOUND 14 than for [¹¹C]verapamil, which could be advantageous in the cases where P-gp is overexpressed, such as drug-resistant epilepsy, requiring that tracer doesn't bind unspecifically.

Upregulation of P-gp would probably not be detectable with substrates that have a very low baseline uptake, as PET signal would only be further decreased compared to the baseline.

[¹⁸F]COMPOUND 4 and [¹⁸F]COMPOUND 8, on the other hand, failed to show substrate behaviour in vivo. Neither of them had any difference in the brain uptake between Mdr1 a/b^(~ Bcrp1^(~ mice and control.

In addition, [¹⁸F]COMPOUND 8 had high uptake in plasma which could refer to protein binding.

Low radiochemical yield of [¹⁸F]COMPOUND 4 (3.2 %) would have also limited its use. In order to improve the yield, several different reaction temperatures and times were tried. Microwave and microfluidic synthesis modules were also attempted to conduct the labeling, but the radiochemical yield remained low.

In vitro COMPOUND 8 had EC₅₀ value in the same range as COMPOUND 14. The affinity of COMPOUND 4 was 6-fold lower than for COMPOUND 14, which could explain the poor in vivo results. One explanation to the conflicting results between in vitro characterization and in vivo evaluation could be, that the cell experiments were performed with micromolar concentrations of the compounds while imaging studies are performed in nanomolar concentrations.

Metabolically COMPOUND 14 was more stable than verapamil. In vivo metabolites were analyzed in plasma and brain and in vitro metabolism was investigated in liver microsomes. Although liver microsomes were from a human source and in vivo experiments were performed in mice, metabolism rate in microsomes was quite predictable. More than 96 % of the parent tracer [¹⁸F]COMPOUND 14 was still intact in the brain after 45 min, whereas [¹¹C]verapamil produces much more brain entering metabolites, as found earlier in rats in Nucl. Med. Biol., vol. 32, no. 1 , pp. 87- 93, Jan. 2005.

The in vivo stability of a tracer is of importance, because PET measures only total radioactivity and cannot distinguish radioactive metabolites from the parent tracer.

In summary, [¹⁸F]COMPOUND 14 was found to be a suitable representative compound for a novel class compound of PET tracers, as it shows high affinity to the P- gp in vivo, and thus represents a novel ¹⁸F-substrate tracer for imaging functional changes of P-glycoprotein in the BBB.

Claims

Claims

1 . A dioxyalkyltetrahydroisoquinoline compound, or a salt or solvate thereof according to formula I:

wherein Ri represents hydrogen or a fluorinated alkyl group, and R₂ and R₃ independently represent hydrogen or an optionally tritiated alkyl group.

2. A compound according to claim 1 , wherein Ri represents a fluorinated alky group comprising from 1 to 4 carbon atoms.

3. A compound according to claim 2, wherein Ri represents -CH₂F or -CH₂CH₂F.

4. A compound according to anyone of claims 1 to 3, wherein Ri is a [¹⁸F]-fluorinated alkyl group.

5. A compound according to anyone of the previous claims, wherein R₂ or R₃ represents hydrogen, and the other of R₂ or R₃ represents an alkyl group.

6. A compound according to anyone of the previous claims, wherein R₂ or R₃ each represents an alkyl group, preferably ethyl or methyl, most preferably wherein both of R₂ and R₃ represent a methyl group.

7. A compound according to anyone of the previous claims, wherein the tetraline moiety is bound to the dioxyalkyltetrahydroisoquinoline moiety via a saturated or unsatured propylene spacer moiety.

8. 5-(1 -(2-fluoroethoxy))-[3-(6,7-Dimethoxy-3,4-dihydro-1 H-isoquinolin-2-yl)-propyl]- 5,6,7,8-tetrahydronaphthalene (14).

9. 5-(1 -(2-[¹⁸F]fluoroethoxy))-[3-(6,7-Dimethoxy-3,4-dihydro-1 H-isoquinolin-2-yl)- propyl]-5,6,7,8-tetrahydronaphthalene ([¹⁸F]14).

10.5-(1 -Hydroxy-[3-(6,7-Dimethoxy-3,4-dihydro-1 H-isoquinolin-2-yl)-propyl]-5,6,7,8- tetrahydronaphthalene (13).

1 1 . A compound according to any one of the claims 1 to 9, a solvate or salt thereof for use as an in vivo substrate and/or inhibitor for P-gp, and/or for the in vivo diagnosis or imaging of P-gp related disease or behaviour in a subject, preferably a human.

12. A radiopharmaceutical formulation comprising the compound according to any one of claims 4 to 9.

13. A radiopharmaceutical formulation according to claim 12 for use in an in vivo

diagnostic or imaging method.

14. A method for the in vivo diagnosis or imaging of P-gp related disease or behaviour in a subject, preferably a human, comprising administration of a compound according to claim 4 to 1 1 or a formulation according to claim 12 or 13.

15. A process for the preparation of a compound according to any one of claims 4 to 9, comprising:

(a) reacting a precursor compound according to formula (I) wherein Ri

represents the group -R-L wherein R- represents an alkyl group, and L is a leaving group under conditions permitting a nucleophilic substitution upon reaction with [¹⁸F] fluoride, or

(b) reacting a precursor compound according to formula (I) wherein Ri is

hydrogen, under alkylation conditions with a [¹⁸F] labeled alkyl reagent, and

(c) isolating the obtained compound from the reaction mixture.

16. Use of a compound according to claim 4 to 9 or a formulation according to claim 12 or 13 for the in vivo diagnosis or imaging of P-gp related disease or behaviour in a subject, preferably a human, comprising administration of a compound according to claim 4 to 1 1 or a formulation according to claim 12 or 13.