BENZAZEPIN-L,7-DIOL-DERIVED RADIOLABELED LIGANDS WITH HIGH IN VIVO NMDA SPECIFICITY

Abstract

The present invention is directed to benzazepin-1,7-diol-derived compounds (I) for use in the diagnosis of NMDA (N-methyl-D-aspartate) receptor-associated diseases or disorders by positron emission tomography (PET), single-photon emission computed tomography (SPECT), liquid based-scintillation- and/or autoradiography-based assays. The invention also relates to a method for the diagnosis of NMDA receptor-associated diseases or disorders by administering to a patient or a sample of a patient in need of such diagnosis a compound of the invention in an amount effective for PET imaging, SPECT imaging, liquid based-scintillation- and/or autoradiography-based assays of NMDA receptors, recording at least one PET or SPECT scan, liquid based-scintillation or autoradiography result, and diagnosing an NMDA receptor-associated disease or disorder from an abnormal NMDA receptor expression pattern on the PET or SPECT scan, in the liquid based-scintillation or autoradiography result. The present invention also provides a method for evaluating a putative NMDA-receptor antagonist in a liquid scintigraphy detection assay or an autoradiography assay using the compounds of the present invention.

##STR00001##

Claims

1.-19. (canceled)

20. A method for the diagnosis of NMDA receptor-associated diseases or disorders, the method comprising: (a) administering to a patient or a sample of a patient in need of such diagnosis a compound of formula (I) in an amount effective for PET imaging, SPECT imaging, liquid based-scintillation- and/or autoradiography-based assays of NMDA receptors, (b) recording at least one PET scan, SPECT scan, liquid based-scintillation or autoradiography result, optionally by ex vivo analysis, and (c) diagnosing an NMDA-receptor-associated disease or disorder from an abnormal NMDA receptor expression pattern on the PET scan, the SPECT scan or the autoradiography result, or an abnormal NMDA receptor expression pattern in the liquid-based scintillation result, wherein the chemical structure of formula (I) is: ##STR00010## at least one atom of formula (I) is a radiolabeled atom suitable for detection in a method selected from the group consisting of positron emission tomography (PET), single-photon emission computed tomography (SPECT), liquid based-scintillation counting assays, and autoradiography; one of R.sup.1, R.sup.2 and R.sup.3 is independently selected from the group consisting of hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine, —OH, —CN, —NO.sub.2, —NH.sub.2, —SH.sub.2, —(C.sub.1-C.sub.4)alkyl, fluorinated —(C.sub.1-C.sub.4)alkyl, and fluorinated —O(C.sub.1-C.sub.4)alkyl, and the other of R.sup.1 to R.sup.3 are hydrogen or fluorine; wherein T is .sup.3H; R.sup.4 is selected from the group consisting of hydrogen, —(C.sub.1-C.sub.4)alkyl, fluorinated —(C.sub.1-C.sub.4)alkyl, chlorinated —(C.sub.1-C.sub.4)alkyl, brominated —(C.sub.1-C.sub.4)alkyl, deuterated —(C.sub.1-C.sub.4)alkyl, and tritiated —(C.sub.1-C.sub.4)alkyl; Y is selected from the group consisting of (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkyl substituted with at least one deuterium, tritium, .sup.18F, .sup.19F, chlorine, bromine, iodine, OH, or a combination thereof, (C.sub.1-C.sub.6)alkoxyalkyl, (C.sub.1-C.sub.6)polyethyleneglycoyl, and (C.sub.1-C.sub.6)heteroalkyl; R.sup.5 is selected from the group consisting of substituted or non-substituted (C.sub.5-C.sub.6)aryl, and substituted or non-substituted (C.sub.5-C.sub.6)heteroaryl; and YR.sup.5 is selected from the group consisting of: ##STR00011## wherein R.sup.7 is one or more hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine or (C.sub.1-C.sub.7)alkyl substituted with at least one deuterium, tritium, fluorine, chlorine, bromine, iodine or OH; and pharmaceutically acceptable salts or solvates thereof.

21. The method of claim 1, wherein the radiolabeled atom is selected from the group consisting of .sup.3H-atom, .sup.11C-atom, .sup.14C-atom, .sup.18F-atom, .sup.13N-atom, .sup.15O-atom, .sup.123I-atom, .sup.124I-atom, .sup.125I-atom, and .sup.131I-atom.

22. The method of claim 1, wherein the radiolabeled atom is a positron emitting radiometal.

23. The method of claim 22, wherein the positron emitting radiometal is .sup.64Cu or .sup.68Ga.

24. The method of claim 21, wherein the radiolabeled atom is a .sup.11C-atom, .sup.18F-atom, .sup.123I-atom, .sup.124I-atom, .sup.125I-atom, or .sup.131I-atom.

25. The method of claim 20, wherein at least one of R.sup.4 or R.sup.5 comprises a .sup.11C-atom, .sup.18F-atom, .sup.123I-atom, .sup.124I-atom, .sup.125I-atom, or .sup.131I-atom.

26. The method of claim 25, wherein R.sup.4 is —OCH.sub.2CH.sub.2—.sup.18F, —OCH.sub.2CH.sub.2CH.sub.2—.sup.18F, OCH.sub.2CH.sub.2—.sup.123I, or —OCH.sub.2CH.sub.2CH.sub.2—.sup.123I, R.sup.6 is —.sup.18F or —.sup.123I, or a combination thereof.

27. The method of claim 20, wherein one of R.sup.1, R.sup.2 and R.sup.3 is independently selected from the group consisting of —CH.sub.2F, —CD.sub.2F, —CT.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2CH.sub.2—, OCH.sub.2F, —OCD.sub.2F, FCH.sub.2CH.sub.2O—, and FCH.sub.2CH.sub.2CH.sub.2O—.

28. The method of claim 20, wherein R.sup.4 is selected from the group consisting of —CH.sub.2F, —CD.sub.2F, —CT.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2CH.sub.2—, FCD.sub.2CD.sub.2-, FCD.sub.2CD.sub.2CD.sub.2-, FCT.sub.2CT.sub.2-, FCT.sub.2CT.sub.2CT.sub.2-, —CH.sub.2Cl, —CD.sub.2Cl, —CT.sub.2I, ClCH.sub.2CH.sub.2—, ClCH.sub.2CH.sub.2CH.sub.2—, ClCD.sub.2CD.sub.2-, ClCD.sub.2CD.sub.2CD.sub.2-, ClCT.sub.2CT.sub.2-, ClCT.sub.2CT.sub.2CT.sub.2-, —CH.sub.2I, —CD.sub.2I, —CT.sub.2I, ICH.sub.2CH.sub.2—, ICH.sub.2CH.sub.2CH.sub.2—, ICD.sub.2CD.sub.2-, ICD.sub.2CD.sub.2CD.sub.2-, ICT.sub.2CT.sub.2-, and ICT.sub.2CT.sub.2CT.sub.2-.

29. The method of claim 20 or 28, wherein R.sup.1 to R.sup.3 are hydrogen, fluorine, chlorine, bromine or iodine.

30. The method of claim 20, wherein Y is selected from the group consisting of C.sub.5-alkyl, C.sub.4-alkyl, CH.sub.2—CH.sub.2—FCH—CH.sub.2N—, CH.sub.2—FCH—CH.sub.2CH.sub.2N—, CH.sub.2CH.sub.2—ICH—CH.sub.2N—, CH.sub.2—ICH—CH.sub.2CH.sub.2N—, CH.sub.2CH.sub.2—CD.sub.2-CH.sub.2N—, CH.sub.2—CD.sub.2-CD.sub.2CH.sub.2N—, CH.sub.2CH.sub.2—CT.sub.2-CH.sub.2N—, CH.sub.2—CT.sub.2-CT.sub.2CH.sub.2N—, or CH.sub.2—CHOH—CH.sub.2—CH.sub.2N—, —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—R.sup.5, —(CH.sub.2).sub.3—O—R.sup.5, —(CH.sub.2).sub.4—O—R.sup.5, —(CH.sub.2).sub.3—X—R.sup.5 and —(CH.sub.2).sub.4—X—R.sup.5, wherein X is sulfur or SO.sub.2, ##STR00012## —(CH.sub.2).sub.3—CO—R.sup.5, —(CH.sub.2).sub.2—CO—N(CH.sub.3)—CH.sub.2—R.sup.5, and —CO—(CH.sub.2).sub.3—R.sup.5.

31. The method of claim 30, wherein R.sup.5 is selected from the group consisting of substituted or non-substituted phenyl or pyridyl.

32. The method of claim 20, wherein R.sup.5 is selected from the group consisting of substituted or non-substituted phenyl or pyridyl.

33. The method of claim 20, wherein one of R.sup.1, R.sup.2 and R.sup.3 is independently selected from the group consisting of —H, -D, -T, —CH.sub.3, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2CH.sub.2—, —OCH.sub.3, —OCH.sub.2F, —OCD.sub.2F, FCH.sub.2CH.sub.2O— and FCH.sub.2CH.sub.2CH.sub.2O—, and the other of R.sup.1 to R.sup.4 are hydrogen or fluorine.

34. The method of claim 20, wherein R.sup.1 is selected from the group consisting of —CH.sub.3, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2CH.sub.2—, —OCH.sub.3, —OCH.sub.2F, —OCD.sub.2F, FCH.sub.2CH.sub.2O— and FCH.sub.2CH.sub.2CH.sub.2O—, and R.sup.2, R.sup.3 and R.sup.4 are hydrogen or fluorine.

35. The method of claim 20, wherein R.sup.4 is selected from the group consisting of hydrogen, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—.

36. The method of claim 20, wherein Y is selected from the group consisting of —(CH.sub.2).sub.i—R.sup.5 and —(CH.sub.2).sub.e—O—(CH.sub.2).sub.f—R.sup.5, wherein i is an integer from 2 to 6, and e and f are independently selected from 1, 2 or 3.

37. The method of claim 20, wherein R.sup.5 is selected from the group consisting of phenyl, ##STR00013## wherein Z is selected from the group consisting of hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine, cyano and nitrile, and wherein R.sup.6 is selected from the group consisting of hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine, cyano, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—.

38. The method of claim 37, wherein R.sup.6 is selected from the group consisting of hydrogen, tritium, fluorine and iodine.

39. The method of claim 20, wherein R.sup.6 is selected from the group consisting of hydrogen, tritium, fluorine and iodine.

40. The method of claim 20, wherein the compound is R-configured at carbon 1.

41. The method of claim 20, wherein the compound is S-configured at carbon 1.

42. The method of claim 20, wherein R.sup.1 is selected from the group consisting of —OCH.sub.3, —OCH.sub.2F, —OCD.sub.2F, FCH.sub.2CH.sub.2O— and FCH.sub.2CH.sub.2CH.sub.2O— and R.sup.2, R.sup.3 and R.sup.4 are hydrogen, R.sup.4 is hydrogen, Y is —(CH.sub.2).sub.4—, and R.sup.5 is substituted or unsubstituted phenyl.

43. The method of claim 20, wherein R.sup.4 is selected from the group consisting of hydrogen, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—, Y is —(CH.sub.2).sub.4— or —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—, and R.sup.5 is selected from the group consisting of phenyl, ##STR00014## wherein Z is hydrogen, tritium or nitrile and wherein R.sup.6 is selected from the group consisting of fluorine, iodine, —CH.sub.2F, —CD.sub.2F, FCH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—.

44. The method of claim 20, wherein R.sup.4 is hydrogen, Y is (CH.sub.2).sub.4, and R.sup.5 is substituted or un-substituted phenyl.

45. The method of claim 20, wherein the compound is selected from the group consisting of ##STR00015## wherein R.sup.7 is selected from the group consisting of fluorine, chlorine, bromine, iodine, deuterium, tritium, —CH.sub.2F, —CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—; and X and Z are independently selected from the group consisting of hydrogen, deuterium, tritium, fluorine, iodine and nitrile.

46. The method of claim 20, wherein the compound is selected from the group consisting of ##STR00016## wherein X and Z are independently selected from the group consisting of hydrogen, deuterium, tritium, fluorine, iodine and nitrile; and the fluorine is .sup.19F if X and/or Z are tritium and the fluorine is .sup.18F if X and/or Z are not.

47. The method of claim 20, wherein the NMDA-receptor-associated disease or disorder is selected from the group consisting of neurodegenerative diseases or disorders, Alzheimer's disease, depressive disorders, Parkinson's disease, traumatic brain injury, stroke, migraine, alcohol withdrawal and chronic and neuropathic pain.

48. A method for evaluating a putative NMDA-receptor antagonist, the method comprising: (a) providing a compound of formula (I), (b) performing a step selected from the group consisting of: (i) measuring competitive binding affinity of a putative NMDA-receptor antagonist and the compound of step (a) in a liquid scintigraphy detection-based assay; and (ii) performing an in vitro or ex vivo autoradiography assay, wherein the putative NMDA-receptor antagonist is used for blocking or displacing the compound of step (a); (c) determining whether the putative NMDA-receptor antagonist is an NMDA-receptor antagonist based on the displacement of the compound of step (a) from the NMDA-receptor by the putative NMDA-receptor antagonist, wherein the compound comprises a tritium or .sup.14C-atom; the chemical structure of formula (I) is: ##STR00017## at least one atom of formula (I) is a radiolabeled atom suitable for detection in a method selected from the group consisting of positron emission tomography (PET), single-photon emission computed tomography (SPECT), liquid based-scintillation counting assays, and autoradiography; one of R.sup.1, R.sup.2 and R.sup.3 is independently selected from the group consisting of hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine, —OH, —CN, —NO.sub.2, —NH.sub.2, —SH.sub.2, —(C.sub.1-C.sub.4)alkyl, fluorinated —(C.sub.1-C.sub.4)alkyl, and fluorinated —O(C.sub.1-C.sub.4)alkyl, and the other of R.sup.1 to R.sup.3 are hydrogen or fluorine; wherein T is .sup.3H; R.sup.4 is selected from the group consisting of hydrogen, —(C.sub.1-C.sub.4)alkyl, fluorinated —(C.sub.1-C.sub.4)alkyl, chlorinated —(C.sub.1-C.sub.4)alkyl, brominated —(C.sub.1-C.sub.4)alkyl, deuterated —(C.sub.1-C.sub.4)alkyl, and tritiated —(C.sub.1-C.sub.4)alkyl; Y is selected from the group consisting of (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkyl substituted with at least one deuterium, tritium, .sup.18F, .sup.19F, chlorine, bromine, iodine, OH, or a combination thereof, (C.sub.1-C.sub.6)alkoxyalkyl, (C.sub.1-C.sub.6)polyethyleneglycoyl, and (C.sub.1-C.sub.6)heteroalkyl; R.sup.5 is selected from the group consisting of substituted or non-substituted (C.sub.5-C.sub.6)aryl, and substituted or non-substituted (C.sub.5-C.sub.6)heteroaryl; and YR.sup.5 is selected from the group consisting of: ##STR00018## wherein R.sup.7 is one or more hydrogen, deuterium, tritium, fluorine, chlorine, bromine, iodine or (C.sub.1-C.sub.7)alkyl substituted with at least one deuterium, tritium, fluorine, chlorine, bromine, iodine or OH; and pharmaceutically acceptable salts or solvates thereof.

Description

FIGURES

[0108] FIG. 1 shows a representative radiosynthesis of .sup.18F-labeled demethylated benzazepine-1,7-diol derivatives.

[0109] FIG. 2 shows a representative radiosynthesis of [.sup.3H]Me-NB1, [.sup.3H]OF-Me-NB1, [.sup.3H]PF-NB1 and [.sup.3H]MF-Me-NB1.

[0110] FIG. 3 shows a representative radiosynthesis of [.sup.3H]OF-NB1 FIG. 4 shows a general methodology for the synthesis of benzazepin-1-ols.

[0111] FIG. 5 shows an autoradiography comparison between [.sup.18F]OF-NB1 and [.sup.18F]OF-Me-NB1 in rat and mouse brain.

[0112] FIG. 6 Shows an autoradiography comparison between [.sup.18F]PF-NB1 and [.sup.18F]PF-Me-NB1 in rat and mouse brain

[0113] FIG. 7 shows the selective and specific binding of [.sup.18F]OF-NB1 in vitro to GluN2B in rat and mouse brain.

[0114] FIG. 8 shows an ex vivo metabolite study of (R)-[.sup.18F]OF-NB1.

[0115] FIG. 9 shows a comparison of time-activity curves (TACS) between [.sup.18F]OF-NB1 and [.sup.18F]OFMe-NB1 in rat brain

[0116] FIG. 10 shows a comparison of time-activity curves (TACS) between [.sup.18F]PF-NB1 and [.sup.18F]PFMe-NB1 in rat brain.

[0117] FIG. 11 shows time-activity curves (TACS) of (R)-[.sup.18F]OF-NB1 using different doses of the experimental GluN2B antagonist CP-101,606 in rat brain.

EXAMPLES

Example 1—Chemistry

[0118] The general methodology for the synthesis of benzazepin-1-ols is known in the art, e.g. from Tewes et al., ChemMedChem 2010, 5, 687-695. A representative synthetic path as used for producing the labeled compounds for use in the present invention is shown in FIG. 4. The synthetic route of FIG. 4 can be adapted by commonly known methods to deliver derivatives of benzazepin-1-ols and substantially all of the compounds for use in the present invention. The skilled person will routinely adapt the synthetic route to be suitable for the synthesis of any PET ligand of the present invention.

Example 2—F-18 Radiolabeling

[0119] [.sup.18F]fluoride was produced and trapped on an anion exchange cartridge (Waters SepPak Accell QMA cartridge carbonate, no pre-conditioning) and then eluted with a solution of Kryptofix 222 (6.3 mg/mL), K.sub.2C.sub.2O.sub.4 (1 mg/mL) and K.sub.2CO.sub.3 (0.1 mg/mL) in MeCN/H.sub.2O (4:1, 0.9 mL) followed by azeotropic drying with MeCN (3×1 mL) (Preshlock et al., ChemComm 2016). The reactivial was purged with air (20 mL) and the residue was re-dissolved in a solution of 6-8 mg boronic ester precursor 2a, 2b, 2c (FIG. 1) and 14 mg Cu(OTf).sub.2(py).sub.4 in 0.3 mL dry dimethylacetamide (DMA). The resulting solution was stirred at 120° C. for 20 minutes and subsequently diluted with 1.5 mL MeCN/H.sub.2O (1:1). Upon addition of 0.4 mL aq. NaOH (10 M), the mixture was stirred at 95° C. for 15 min. The product was purified by semi-preparative HPLC (Agilent Eclipse XBD-C18 column, 250×9.4 mm, 5 m, 0.1% H.sub.3PO.sub.4 in H.sub.2O (solvent A), MeCN (solvent B); 0.0-5.0 min, 20% B; 5.1-20.0 min, 20-35% B; 20.1-25.0 min, 35% B, 25.1-30.0 min, 35-60% B, 30.1-33.0 min, 60-90% B, 33.1-36.0 min, 90% B, 36.1-38.0 min, 90-20% B, 38.1-42.0 min, 20% B, flow rate 4 mL/min, UV detection at 230 nm). Collected fractions were diluted with 35 mL water and the product was trapped on a C18 light cartridge (Waters, pre-conditioned with 5 mL EtOH and 5 mL water). The cartridge was washed with 5 mL of water, followed by product elution with 0.5 mL EtOH. The product was formulated with 5% EtOH in saline to afford either [.sup.18F]OF-Me-NB1 3a, [.sup.18F]PF-Me-NB1 3b or [.sup.18F]MF-Me-NB1 3c (FIG. 1). Alternatively, the ethanol was evaporated at 90° C. followed by azeotropic drying with MeCN (3×0.8 mL). The residue was cooled down in an ice bath before re-dissolving it in 0.4 mL DCM. The vial was kept in the ice bath as 0.4 ml of BBr.sub.3 (1 M in DCM) was added simultaneously, followed by stirring at rt for 15 min. The DCM was evaporated to dryness and subsequently re-dissolved in aq. 0.1% H.sub.3PO.sub.4/MeCN (5:1, 3 mL). Afterwards, the product was purified by semi-preparative HPLC (Agilent Eclipse XBD-C18 column, 250×9.4 mm, 5 m, 0.1% H.sub.3PO.sub.4 in H.sub.2O (solvent A), MeCN (solvent B); 0.0-5.0 min, 20% B; 5.1-20.0 min, 20-35% B; 20.1-25.0 min, 35% B, 25.1-30.0 min, 35-60% B, 30.1-33.0 min, 60-90% B, 33.1-36.0 min, 90% B, 36.1-38.0 min, 90-20% B, 38.1-42.0 min, 20% B, flow rate 4 mL/min, UV detection at 230 nm). Collected fractions were diluted with 35 mL water and the product was trapped on a C18 light cartridge (Waters, pre-conditioned with 5 mL EtOH and 5 mL water). The cartridge was washed with 5 mL of water, followed by product elution with 0.5 mL EtOH. The product was formulated with 5% EtOH to afford either [.sup.18F]OF-NB1 4a, [.sup.18F]PF-NB1 4b or [.sup.18F]MF-NB1 4c (FIG. 1). Quality control was performed using an analytical HPLC system (Waters Atlantis T3 column, 150×4.6 mm, 3 m, 0.1% TFA in H.sub.2O (solvent A), MeCN (solvent B); 0.0-10.0 min, 40-60% B; 10.1-11.0 min, 60-40% B; 11.1-15.0 min, 40% B, flow rate 1 mL/min, UV detection at 230 nm). Identities of the radioligands were confirmed by co-injection with the corresponding nonradioactive reference compounds and molar activities were calculated based on established UV calibration curves of the reference compounds. The radiochemical conversion (RCC) ranged between 9-25% and radiochemical purities >95%. Molar activities ranged from 61-168 GBq/mol at the end of synthesis.

Example 3—Autoradiography

[0120] Rodent brain tissue was embedded in Tissue-Tek® (O.C.T.™ Tissue-Tek®, Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands). Horizontal rat and mouse brain sections of 10 m thickness were prepared on a cryostat (Cryo-Star HM 560 MV; Microm, Thermo Scientific, Wilmington, Del., USA). The tissue sections were mounted to SuperFrost Plus slides (Menzel, Braunschweig, Germany) and stored at −20° C. until further use. Prior to the autoradiography experiments, brain slices were initially thawed for 15 min on ice and subsequently preconditioned for 10 min at 0° C. in a buffer (pH 7.4) containing 30 mM HEPES, 0.56 mM MgCl.sub.2, 110 mM NaCl, 3.3 mM CaCl.sub.2, 5 mM KCl and 0.1% fatty acid free bovine serum albumin (BSA). Upon drying, the tissue sections were incubated with 1 mL of the respective radioligand (3 nM) for 15 minutes at 21° C. in a humidified chamber. For σ1R-blockade, 1 μM solution of either SA4503, fluspidine or (+)pentazocine was added to the radiotracer solution. For GluN2B-blockade experiments, 1 μM solution of either CERC-301, EVT 101 or CP101,606 was added to the radiotracer solution. The brain slices were washed for 5 min with a buffer (pH 7.4) containing 30 mM HEPES, 0.56 mM MgCl.sub.2, 110 mM NaCl, 3.3 mM CaCl.sub.2, 5 mM KCl and 0.1% fatty acid free bovine serum albumin (BSA) and further washed twice 3 min in a second buffer with the same ionic composition but without BSA. Tissue sections were dipped twice in distilled water, subsequently dried and exposed to a phosphor imager plate (Fuji, Dielsdorf, Switzerland) for 30 minutes. The films were scanned in a BAS5000 reader (Fuji) and images were generated using AIDA 4.50.010 software (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). Typical autoradiographies of methylated and demethylated NB1 are shown in FIGS. 5, 6 and 7. The distribution of the tracers conforms to the expression distribution of the GluN2B over the brain. Additionally, to establish specificity, GluN2B blockers (Ro 25-6981, CP101.606 (Sigma-Aldrich, Buchs, Switzerland)) were used. As a negative control for establishing selectivity, GluN2A blocker NVP-AAM077 (Sigma-Aldrich, Buchs, Switzerland) and the endogenous ligand glutamate were used.

Example 4—PET Scans of Rat Brain and Time-Activity Curves and Receptor Occupancy

[0121] Wistar rats were anesthetized with isoflurane and scanned for a period of 90 min in a PET/CT scanner (Super Argus, Sedecal, Madrid, Spain) upon tail-vein injection of 15-38 MBq, 0.6-1.7 nmol/kg (rats) of methylated and demethylated radiolabelled ligands. For anatomical orientation, PET scans were followed by computed tomography. Dose-response and receptor occupancy in Wistar rats were conducted by tail-vein injection of different doses (3, 10 and 15 mg/kg) of CP101,606 (GluN2B-antagonist, Sigma-Aldrich, Buchs, Switzerland) shortly before tracer administration The obtained data was reconstructed in user-defined time frames with a voxel size of 0.3875×0.3875×0.775 mm.sup.3 as previously described by our group (Haider et al., Eur. J. Med. Chem. 2018). Time-activity curves (TACs) were deducted by PMOD v3.7 (PMOD Technologies, Zurich, Switzerland) with predefined regions of interest. The results are given as standardized uptake values (SUVs), indicating the decay-corrected radioactivity per cm.sup.3 divided by the injected dose per gram body weight. Receptor occupancy evaluations were carried out as previously reported (Haider et al., Eur. J. Med. Chem. 2018). Baseline TACs for the methylated and demethylated benzazepines are depicted in FIGS. 9 and 10. TACs resulting from a typical receptor occupancy study using (R)[.sup.18F]-OF-NB1 is shown in FIG. 11.

Example 5—In Vitro Binding Affinities

[0122] The conduction of the binding competition assay for the GluN2B is already known from the current state of the art (Szermerski et al, ChemMedChem, 2018) as well as the assay for 61 receptor (σ1R) (Chu et al, Current Protocols in Pharmacology, 2015). Portions of rat membrane homogenates of ˜1 mg protein/mL (for GluN2B subunit IC.sub.50 determination) and ˜2 mg protein/mL (for σ1R IC.sub.50 determination) were used in the assay (determined by the method of Bradford). The radioligands used were [.sup.3H]ifenprodil (Perkin Elmer) for the GluN2B binding competition assay and [.sup.3H](+)-pentazocine (Perkin Elmer) for the σ1R binding competition assay. For the tested ligands, a dilution series of 8 different concentrations were prepared ranging from 30 pM up to 30 nM. For the GluN2B subunit binding assay, [.sup.3H]ifenprodil was incubated together with the substrate and 1 mg/ml total protein in HEPES buffer (30 mM, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl.sub.2), 1.2 mM MgCl2, pH 7.4) in a total volume of 200 μl at 25° C. for 60 min under mechanical shaking (110 Rpm). The σ1R binding competition binding assay was performed similarly with modifications in the total protein concentration, incubation time and temperature of 2 mg protein/mL, 37° C. and 150 min, respectively. Termination of the incubation was completed by dilution with 3 ml HEPES buffer followed by filtration through glass microfiber filters (Whatman GF/C 25 mm) pre-incubated in 0.05% polyethylenimine solution. To measure the activity, scintillation fluid (10 ml/scintillation vial, Ultima Gold, Perkin Elmer) was utilized and the activity was measured by a Packard 2200CA TRI-CARB liquid scintillation analyzer. The resulting IC.sub.50 values were transformed into Ki values using the equation of Cheng and Prusoff (Cheng Y, Prusoff WH, 1973, Biochem Pharmacol. 22 (23)). The Ki values for methylated (OF-Me-NB1 and PF-Me-NB1) and demethylated (OF-NB1 and PF-NB1) are depicted in Table 1 below.

TABLE-US-00001 TABLE 1 GluN2B subunit and σ1R Ki values of the benzoadepine-1-ol derivatives GluN2B Affinity σ1R Affinity Selectivity Compounds in nM in nM (σ1R/GluN2B) OF-Me-NB1 37 32 0.9 PF-Me-NB1 56 12 0.2 OF-NB1 10.36 ± 4.75 (n = 3) 410 39.6 PF-NB1 10.44 ± 3.90 (n = 3) 544 52.1

Example 6—In Vitro Binding Affinities

[0123] Wistar rats were injected with 242-704 MBq (14.8-27.5 nmol/kg) of (R)-[.sup.18F]OF-Me-NB1 and (R)-[.sup.18F]OF-NB1. Samples of the brain extracts at predefined times (15, 30 and 60 min) were obtained and analyzed by radio-UPLC as previously reported (Haider et al., Eur. J. Med. Chem. 2018). This class of compounds was found to be metabolically stable in the brain as no radiometabolites were detected up to 60 min in the brain. A typical metabolite study is shown in FIG. 8 for (R)-[.sup.18F]OF-NB1.

Example 7—Tritium Labeling

[0124] For methylated ligands, a typical tritium-labeling approach is presented in FIG. 2. To a stirred solution of (R)-NB1 (0.5 mg, 1.6 μmol) in dry DMF (0.2 mL) was added cesium carbonate (2.5 mg, 7.7 μmol) and the resulting suspension was stirred for 5 min at ambient temperature. A solution of [.sup.3H]iodomethane (15 MBq, ARC, St Lous, US, molar activity 1.4 GBq/mol) in DMF (0.1 mL) was added dropwise and the reaction mixture was stirred for 1 h at 90° C. The mixture was diluted with aqueous TFA buffer (0.1%, 0.7 mL) and subsequently injected into a semiproparative HPLC system (Merck Hitachi) equipped with L-6200A pump system, a D-6000 interface, an L-4250 UV-VIS detector and a semipreparative column (Waters Sunfire Prep C18 column (150×10.0 mm, 5 m). A gradient system of 0.1% aqueous TFA (solvent A) and MeCN (solvent B) was used for the purification as follows: 0-5 min 20% B, 5-40 min 20-95% B, 40-41 min 95-20% B. The product was collected, diluted with 4 mL of water and passed through a Waters Sep Pak C18 light cartridge (preconditioned with 5 mL ethanol and 5 mL water). The cartridge was washed with 5 mL of water and the product eluted with 1 mL of ethanol. Quality control was performed with the same HPLC system and gradient. The synthesis was successfully performed under carrier-added and non-carrier added conditions and the final product was stored at −20° C. Another example of introducing tritium into the demethylated-NB1 ligands would be through aromatic tritiation as shown in FIG. 3 (McCarthy et al, J. Label. Compd. Radiopharm, 1997).