BENZAZEPIN-1-OL-DERIVED PET LIGANDS WITH HIGH IN VIVO NMDA SPECIFICITY

Abstract

The present invention is directed to benzazepin-1-ol-derived compounds for use in the diagnosis of NMDA receptor-associated diseases or disorders by positron emission tomography (PET). The invention also relates to a method for the diagnosis of NMDA-receptor-associated diseases or disorders by administering to a patient in need of such diagnosis a radioactively labelled compound of the invention in an amount effective for PET imaging of NMDA receptors, recording at least one PET scan, and diagnosing an NMDA-receptor-associated disease or disorder from an abnormal NMDA receptor expression pattern on the PET scan. NMDA-receptor-associated diseases or disorders that can be diagnosed with the radioactively labelled benzazepin-1-ol-derived compounds include but are not limited to neurodegenerative diseases or disorders, Alzheimer's disease, depressive disorders, Parkinson's disease, traumatic brain injury, stroke, migraine, alcohol withdrawal and chronic and neuropathic pain.

Claims

1. A method for the diagnosis of NMDA-receptor-associated diseases or disorders comprising the following steps: (a) administering to a patient in need of such diagnosis a radioactively labelled compound in an amount effective for PET imaging of NMDA receptors, (b) recording at least one PET scan, and (c) diagnosing the patient as having an NMDA-receptor-associated disease or disorder from an abnormal NMDA receptor expression pattern on the PET scan, wherein the radioactively labelled compound has formula (I) ##STR00010## wherein at least one atom of formula (I) is a radiolabeled atom, selected from the group of .sup.11C-, .sup.18F-, .sup.13N-, or .sup.15O-atom, suitable for positron emission tomography detection (PET); one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is independently selected from the group consisting of hydrogen, fluorine, —(C.sub.1-C.sub.4)alkyl, fluorinated —(C.sub.1-C.sub.4)alkyl, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2CH.sub.2—, —O(C.sub.1-C.sub.4)alkyl, fluorinated —O(C.sub.1-C.sub.4)alkyl, —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; R.sup.5 is selected from the group consisting of hydrogen, —(C.sub.1-C.sub.6)alkyl, fluorinated —(C.sub.1-C.sub.6)alkyl, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—; Y is selected from the group consisting of (C.sub.1-C.sub.6)alkyl, preferably C.sub.5-alkyl or C.sub.4-alkyl, (C.sub.1-C.sub.6)alkoxyalkyl, (C.sub.1-C.sub.6)polyethyleneglycoyl, (CH.sub.2).sub.2—O—(CH.sub.2).sub.2—R.sup.6, —(CH.sub.2).sub.3—O—R.sup.6, —(CH.sub.2).sub.4—O—R.sup.6, (C.sub.1-C.sub.6)heteroalkyl, —(CH.sub.2).sub.3—X—R.sup.6 and —(CH.sub.2).sub.4—X—R.sup.6, wherein X is sulfur or SO.sub.2, ##STR00011## —(CH.sub.2).sub.3—CO—R.sup.6, —(CH.sub.2).sub.2—CO—N(CH.sub.3)—CH.sub.2—R.sup.6, and —CO—(CH.sub.2).sub.3—R.sup.6, wherein R.sup.8 is one or more hydrogen or fluorine; R.sup.6 is selected from the group consisting of substituted or non-substituted (C.sub.5-C.sub.6)aryl, substituted or non-substituted (C.sub.5-C.sub.6)heteroaryl, substituted or non-substituted phenyl or pyridyl, and ##STR00012##  wherein Z is selected from the group consisting of hydrogen, fluorine and nitrile, wherein R.sup.7 is selected from the group consisting of hydrogen, fluorine, —(C.sub.1-C.sub.6)alkyl, fluorinated —(C.sub.1-C.sub.6)alkyl, —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 pharmaceutically acceptable salts or solvates thereof.

2. The method of diagnosis according to claim 1, wherein the at least one radiolabeled atom is a .sup.11C-atom.

3. The method of diagnosis according to claim 1, 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.

4. The method of diagnosis according to claim 1, wherein at least one of R.sup.1, R.sup.3 or R.sup.5 comprise a .sup.11C-atom.

5. The method of diagnosis according to claim 1, wherein one of R.sup.1, R.sup.3 is —O[.sup.11C]H.sub.3 or R.sup.5 is —[.sup.1C]H.sub.3.

6. The method of diagnosis according to claim 1, wherein one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently 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 the other of R.sup.1 to R.sup.4 are hydrogen or fluorine.

7. The method of diagnosis according to claim 1, 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

8. The method of diagnosis according to claim 1, wherein R.sup.3 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.1, R.sup.2 and R.sup.4 are hydrogen or fluorine.

9. The method of diagnosis according to claim 1, wherein R.sup.5 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—.

10. The method of diagnosis according to claim 1, wherein Y is selected from the group consisting of —(CH.sub.2).sub.i—R.sup.6 and —(CH.sub.2).sub.e—O—(CH.sub.2).sub.e—R.sup.6, wherein i is an integer from 2 to 6, and e is an integer from 1 to 3.

11. The method of diagnosis according to claim 1, wherein R.sup.6 is selected from the group consisting of phenyl, ##STR00013## wherein Z is hydrogen or nitrile and wherein R.sup.7 is selected from the group consisting of fluorine, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—.

12. The method of diagnosis according to claim 1, wherein the compound is R-configured at carbon 1.

13. The method of diagnosis according to claim 1, wherein the compound is S-configured at carbon 1.

14. The method of diagnosis according to claim 1, 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.5 is hydrogen, Y is —(CH.sub.2).sub.4—R.sup.6, and R.sup.6 is substituted or un-substituted phenyl.

15. The method of diagnosis according to claim 1, wherein R.sup.3 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.1, R.sup.2 and R.sup.4 are hydrogen or fluorine, R.sup.5 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—R.sup.6 or —(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—R.sup.6, and R.sup.6 is selected from the group consisting of phenyl, ##STR00014##  wherein Z is hydrogen or nitrile and wherein R.sup.7 is selected from the group consisting of fluorine, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—.

16. The method of diagnosis according to claim 1, wherein R.sup.3 is selected from the group consisting of fluorine, —CH.sub.3, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2— and FCH.sub.2CH.sub.2CH.sub.2— and R.sup.1, R.sup.2 and R.sup.4 are hydrogen, R.sup.5 is hydrogen, Y is —(CH.sub.2).sub.4—R.sup.6, and R.sup.6 is substituted or un-substituted phenyl.

17. The method of diagnosis according to claim 1, wherein the compound is selected from the group consisting of ##STR00015## wherein R.sup.9 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.2—, and FCH.sub.2CH.sub.2CH.sub.2—; R.sup.10 is selected from the group consisting of hydrogen, CH.sub.3, —CH.sub.2F,—CD.sub.2F, FCH.sub.2CH.sub.2—, FCH.sub.2CH.sub.2—, and FCH.sub.2CH.sub.2CH.sub.2—; R.sup.11 is selected from the group consisting of fluorine, —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 Z is selected from the group consisting of hydrogen and nitrile.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIGS. 1A and 1B. FIG. 1A shows [.sup.11C]NB1 binding to rat brain slices by in vitro autoradiography. Baseline and blocking conditions as indicated. The two slices were incubated and exposed simultaneously. FIG. 1B shows the analytical HPLC-profile of the enantiomeric separation of racemic NB1.

[0082] FIGS. 2A, 2B and 2C. FIG. 2A shows PET scans of rat brain with [.sup.11C]WMS1405, averaged from 0 to 60 min p.i. FIG. 2B shows time-activity curves (TAC) of whole brain of the scans shown in FIG. 2A. FIG. 2C shows racemate baseline and blockade as in FIGS. 2A and 2B, and TAC of a displacement experiment with 1 mg/kg eliprodil 20 min after scan start/tracer injection.

[0083] FIGS. 3A and 3B show PET scans of rat brain with [.sup.18F]WMS1410. FIG. 3A. A(−) stereoisomer, FIG. 3B. B(+) stereoisomer. Baseline blockade (1 mg/kg eliprodil co-injection) and displacement (1 mg/kg eliprodil 20 min after scan start/tracer injection) scans.

[0084] FIGS. 4A and 4B show [.sup.11C]WMS1405 indirectly images modulation of NMDA receptors by the signal receptor. FIG. 4A. In vitro displacement of [.sup.3H](+)pentazocine, a signal receptor ligand with Kd 1.6 nM (Lever et al., Synapse 2006, 59, 350-358.), by WMS1405, from its binding to rat brain membranes. One out of three independent experiments. Triplicate incubations with standard deviations. Solid line, fit IC50 function. Upper broken line, binding of 2.9 nM [.sup.3H](+)pentazocine alone. Lower broken line, blocking with 0.1 nM haloperidol. Ki of WMS1405 for signal receptor was 1-10 μM, indicating no significant binding to signal receptor. FIG. 4B In vivo, pentazocine and haloperidol reduced [.sup.11C]WMS1405 binding to similar levels as under blocking conditions with eliprodil, despite the absence of competition at the signal receptor between WMS1405 and pentazocine in vitro. Baseline and blockade (circles) as in FIGS. 2B and 2C. Squares, co-injection of 2.5 mg/kg (+)-pentazocine. Haloperidol (0.13 mg/kg) co-injection with the tracer had a similar effect as (+)-pentazocine (data not shown).

EXAMPLES

Example 1—Chemistry

[0085] The general methodology for the synthesis of benzazepin-1-ols is known in the art, e.g. from Tewes et al., Chem Med Chem 2010, 5, 687-695. A representative synthetic path as used for producing the present PET ligands is shown in scheme 1 below. The synthetic route of scheme 1 can be adapted by commonly known methods to deliver derivatives of benzazepin-1-ols and substantially all of the PET ligands of 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.

##STR00007##

Example 2—C-11 Radiolabeling

[0086] No-carrier-added (n.c.a.) [.sup.11C]CO.sub.2 was produced via the .sup.14N(p,α).sup.11C nuclear reaction by bombardment of nitrogen gas fortified with 0.5% oxygen using a Cyclone 18/9 cyclotron (18-MeV, IBA, Louvain-la-neuve, Belgium). [.sup.11C]Iodomethane ([.sup.11C]CH.sub.3I) was generated in a two-step reaction sequence involving the catalytic reduction of [.sup.11C]CO.sub.2 to [.sup.11C]methane over a supported nickel catalyst and subsequent gas phase iodination [Larsen et al, Appl Radiat Isot, 48, 153-157, 1997]. [.sup.11C]CH.sub.3I was bubbled into the reaction vial containing precursor NB1 (1 mg) and cesium carbonate (5 mg) in DMF (0.5 mL). After the transfer was complete, the closed reaction vial was heated at 90° C. for 3 min. The reaction mixture was diluted with water (1.5 mL), and the crude product was purified using semi-preparative HPLC column (reversed-phase Sunfire C18 column Waters, Ireland, 5 μm, 10×150 mm, product peak at 12.8 min). The collected product was diluted with water (10 mL), trapped on a C18 cartridge (Waters, Ireland), preconditioned with 5 mL EtOH and 10 mL water), washed with water (5 mL) to remove traces of HPLC eluent. The product was eluted with 0.8 ml ethanol into a sterile pyrogen-free penicillin vial which contains 9.2 mL water for injection. For quality control, an aliquot of the formulated solution was injected into an analytical HPLC system. The identity of the .sup.11C-labeled product was confirmed by comparison with the retention time of its nonradioactive reference compound NB1 (5.9 min). The specific radioactivity was determined by matching the area under UV absorbance peak at 230 nm, which co-eluted with the radiolabeled product, to a standard calibration curve calculated using known concentrations of the non-radioactive reference compound NB1. .sup.11C-radiolabeling was achieved by reacting the phenolic salt of NB1 with [.sup.11C]CH.sub.3I in DMF. Typically, specific activities ranged between 290±90 GBq/μmol with a total activity of 7.4±1.9 GBq (ca. 60-72% radiochemical yield, decay corrected) at the end of synthesis (n=17) and a total synthesis time from end of bombardment was 35-40 min. The final product was of >99% radiochemical purity as confirmed by HPLC analysis. The identity of the tracer was confirmed by co-injection with non-radioactive Me-NB1.

[0087] The radiolabeling procedure was carried out identically for both enantiomers of the precursor NB1 and the obtained results for specific activity, total activity, synthesis time and radiochemical purity were in the same range when compared to the racemic mixture.

##STR00008##

Example 3—F-18 Radiolabeling

[0088] [.sup.18F]fluoride was produced by bombardment of enriched .sup.18O-water using a Cyclone 18/9 cyclotron (18-MeV; IBA, Belgium). The aqueous .sup.18F-fluoride was delivered from cyclotron to the hot cell and trapped on an anion exchange cartridge (Waters, Ireland, SepPak Accell QMA cartridge carbonate). After elution with a tetrabutylammonium hydroxide solution (0.18 M in MeOH, 1 mL) into a reaction vessel the solvents were evaporated at 90° C. under reduced pressure with a gentle inflow of nitrogen gas. Azeotropic drying was carried out three times using 1 mL of acetonitrile each.

[0089] A solution of 5 mg ethylene ditosylate in 0.5 mL acetonitrile was added and the reaction mixture was stirred at 100° C. for 7 min. After dilution with water (2.5 mL) the crude product was purified by semi-preparative HPLC column (Sunfire, Waters, Ireland, C18 column, 5 μm, 10×150 mm). [.sup.18F]fluoroethyl tosylate was diluted with 40 mL of water and trapped on a C18 light cartridge (Waters, Ireland, preconditioned with 5 mL EtOH and 10 mL water). The cartridge was washed with water (2 mL) and the [.sup.18F]fluoroethyl tosylate was eluted with 0.5 mL of N,N-dimethylformamide into a reaction vessel previously loaded with 1 mg of precursor 3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol (NB1, which was synthesized according to the published synthetic route described by Tewes et. al, Chem Med Chem. 2010, 5, 687-95) and 5 mg of cesium carbonate in 0.2 mL of N,N-dimethylformamide. The reaction mixture was stirred for 120° C. for 10 min, diluted with water (2.3 mL) and purified by using the same semi-preparative HPLC column (product peak at 11.3 min). The collected product fraction of [.sup.18F]fluoroethyl-NB1 was diluted with water (15 mL) and trapped on a C18 light cartridge (Waters, Ireland, preconditioned with 5 mL EtOH and 10 mL water). After washing the cartridge with water (5 mL) the product was eluted with ethanol (0.5 mL) through a sterile filter (0.2 μm). The volume of ethanol was decreased to 0.1 mL under reduced pressure and the resulting solution diluted with 1.9 mL of water to give a final ethanol concentration of 5%. An aliquot of the final formulation was analyzed by using a reversed-phase column (Atlantis T3, Waters, Ireland, C18, particle size 3 μm, 4.6×150 mm) and the identity of the tracer was confirmed by comparison with the retention time of the non-radioactive reference compound (6.3 min). The specific activity was calculated by comparison of the UV (absorbance at 230 nm) peak area under the curve with a calibration curve of the reference compound. The total activity was 684±53 MBq (enantiomer A) and 621±364 MBq (enantiomer B) at the end of synthesis (n=2). The decay corrected radiochemical yield was 1.5±0.4% (enantiomer A) and 0.9±0.2% (enantiomer B) with a total synthesis time of 120-145 min. Radiochemical purity was >99% in all cases as confirmed by HPLC analysis and the identity of the tracer was confirmed by comparison with the retention time of the cold reference.

[0090] The radiolabeling procedure was carried out identically for both enantiomers of the precursor NB1.

##STR00009##

Example 4—[.SUP.11.C]NB1 Accumulates in Brain and is Displaced by GluN1/GluN2B NTD Modulators In Vitro

[0091] For in vitro autoradiography, horizontal rat brain slices (20 μm) of a male Wistar rat (492 g) were incubated for 30 min at room temperature with 5.4 nM [.sup.11C]NB1 alone or in combination with 10 μM either eliprodil or AAM077 in HEPES buffer (30 mM HEPES, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl.sub.2, 1.2 mM MgCl.sub.2, pH 7.4) containing 0.1% bovine serum albumin (HEPES/BSA). After incubation, the slices were washed with HEPES/BSA for 8 min and twice with HEPES buffer for 3 min followed by two 5-second rinses with water. The dried slices were exposed for 20 min to a phosphorimager plate, read in a phosphorimager BAS5000 (Fuji, Tokyo, Japan) and analyzed with the software AIDA v. 4.5 (Raytest Isotopenmessgerate GmbH, Germany).

[0092] In vitro autoradiography with rat brain slices revealed high binding in most brain regions which was blocked by Glu2B specific ligand eliprodil but not AAM077 (Glu2A specific ligand, Novartis Pharmaceuticals, Switzerland). Typical autoradiographs are shown in FIG. 1A.

Example 5—PET Scans of Rat Brain with [.SUP.11.C]WMS1405, Averaged from 0 to 60 Min p.i

[0093] Wistar rats (331-353 g) were under isoflurane anaesthesia (2.5-5% in oxygen/air 1/1) for all procedures and temperature and respiration were controlled with warm air and by adjusting the isoflurane dosage. A dynamic PET scan was started with a calibrated VISTA eXplore/Super Argus PET/CT scanner (Sedecal, Madrid, Sprain; axial field of view 4.8 cm) and at time zero, 32-52 MBq (0.44-0.78 nmol/kg, 250-300 μL) [.sup.11C]NB1 were injected via a tail vein. For blocking experiments by PET, injected doses of [.sup.11C]NB1 were 20-67 MBq, the dose of eliprodil was 1 mg/kg. Images and decay-corrected time-activity curves (TAC) were generated in PMOD v3.6 (PMOD Technologies Ltd., Zurich, Switzerland) with predefined regions of interest (ROI) implemented in PMOD and as shown in FIGS. 2A, 2B, and 2C. [.sup.11C]WMS1405 accumulates in NMDA-rich brain regions and is blocked and displaced, respectively, by the NMDA N2B-selective compound eliprodil. Stereoisomer B(+) accumulates more than the racemate, stereoisomer A(−) accumulates less than the racemate, both accumulate more than when the racemate is blocked by eliprodil.

Example 6—Time-Activity Curves of PET Scans of Rat Brain with Enantiomeric Pure [.SUP.18.F]WMS1410 Under Baseline and Blockade Conditions (FIGS. 3A and 3B)

[0094] Similar PET experimental procedure as mentioned above for [.sup.11C]WMS1405 was applied for imaging [.sup.18F]WMS1410 with Wister rat. [.sup.18F]WMS1410 accumulates in NMDA-rich brain regions and is blocked by eliprodil. Both stereoisomers accumulate and are blocked and displaced, respectively, by eliprodil.

Example 7—[.SUP.11.C]WMS1405 Indirectly Images Modulation of NMDA Receptors by the Signal Receptor (FIGS. 4A and 4B)

[0095] In vitro binding study with [.sup.3H](+)pentazocine, a ligand for signal receptor. WMS1405 does not compete for the binding of (+)pentazocine to signal, excluding WMS1405 as a signal receptor ligand (Tewes B, Chem Med Chem. 2010, 5, 687-95).

[0096] In vivo, pentazocine and haloperidol, a ligand for several neuroreceptors including signal receptor, reduce [.sup.11C]WMS1405 binding, albeit WMS1405 did not compete with pentazocine binding in vitro. This is strong evidence that PET with NR2B ligands such as WMS1405 or WMS1410 allows to indirectly image sigma receptor (in particular signal receptor) activity. It is known that signal receptor interacts with NMDA NR1A and modulates NMDA receptor function (Balasuriya D, J Neurosci. 2013, 33(46), 18219-24).