Binding affinities of the CB1R antagonists / agonists

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Chapter 3 Experimental

General Information

All reactions were carried out in flame- or oven-dried glassware under a dry nitrogen atmosphere. All reagents were purchased as reagent grade and used without further purification. Dimethyl formamide was degassed and dried using an LC Technical SP-1 solvent purification system. Ethanol was distilled over Mg(OEt)2. Ethyl acetate, methanol, and petroleum ether were distilled prior to use. All other solvents were used as received unless stated otherwise. RP-HPLC was performed with an Agilent 1100 using a Jupiter C18 300 Å, 5 µm, 2.0 mm x 250 mm column at a flow rate of 0.2 mLmin-1 with a DAD Detector operating at 262, 280 and 320 nm. A suitably adjusted gradient of 5% B to 100% B was used, where solvent A was 0.1% HCOOH in H2O and B was 20 % A in MeCN. Flash chromatography was carried out using 0.063-0.1 mm silica gel with the desired solvent. Thin layer chromatography (TLC) was performed using 0.2 mm Kieselgel F254 (Merck) silica plates and compounds were visualised using UV irradiation at 254 or 365 nm and/or staining with a solution of potassium permanganate and potassium carbonate in aqueous sodium hydroxide. Preparative TLC was performed using 500 µm, 20 x 20 cm UniplateTM (Analtech) silica gel TLC plates and compounds were visualised using UV irradiation at 254 or 365 nm. Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. Infrared spectra were obtained using a Perkin-Elmer Spectrum 100 FTIR spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in wavenumbers (cm-1). NMR spectra were recorded as indicated on either a Bruker Avance 400 spectrometer operating at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei, a Bruker DRX-400 spectrometer operating at 400 MHz for 1H nuclei, 100 MHz for 13C nuclei, a Bruker Avance AVIII-HD 500 spectrometer operating at 500 MHz for 1H nuclei, 125 MHz for 13C nuclei or a Bruker Avance 600 spectrometer operating at 600 MHz for 1H nuclei, 150 MHz for 13C nuclei. 1H and 13C chemical shifts are reported in parts per million (ppm) relative to CDCl3 (1H and 13C) or (CD3)2SO (1H and 13C). High resolution mass spectra were recorded on a Bruker micrOTOF-Q II mass spectrometer with ESI ionisation source. Ultraviolet-visible spectra were run as H2O solutions on a Shimadzu UV-2101PC scanning spectrophotometer.

General procedure for the mono-Boc-protection of diamines

GP 1A:
To an ice-cooled solution of the respective diamine (44.0 mmol, 5.0 equiv.) in chloroform (30 mL) was added Boc2O (1.92 g, 8.80 mmol, 1 equiv.) in chloroform (15 mL) over 1h under vigorous stirring. The solution was allowed to warm to r.t. and stirred for additional 14 h. The precipitate was filtered, the solvent was removed in vacuo and the residue dissolved in ethyl acetate (100 mL). The organic layer was washed with brine (2 x 20 mL), the aqueous layer was extracted with ethyl acetate (1 x 15 mL), the combined organic layers were dried with MgSO4 filtered and the solvent removed in vacuo to afford the respective mono-Boc-protected diamine.
GP 1B:
To a solution of the respective diamine (27.5 mmol, 3.0 equiv.) in methylene chloride (50 mL) and DIPEA (1.56 mL, 9.16 mmol, 1.0 equiv.) was added Boc2O (2.0 g, 9.16 mmol, 1 equiv.) dissolved in methylene chloride (20 mL) over a period of 1 h under vigorous stirring. The mixture was stirred for an additional hour, the solvent was removed in vacuo and the crude product was purified over silica to afford the respective mono-Boc-protected diamine.

General procedure for amide formation via acid-chloride NK25:

GP 2:
To an ice-cooled solution of a mono-Boc-protected amine (2.17 mmol, 1.2 equiv.) in methylene chloride (20 mL) and NEt3 (1.25 mL, 9.0 mmol, 5.0 equiv.) was slowly added acid chloride NK25 (720 mg, 1.8 mmol, 1.0 equiv.). The solution was stirred at r.t. for another 30 minutes after which it was diluted with EtOAc (30 mL) and washed with 1 N aq. HCl (10 mL) and brine (10 mL), dried with MgSO4, filtered and the solvent removed in vacuo. Purification over silica afforded the desired Boc-protected amide.

General procedure for Boc deprotection:

GP 3:
The Boc-protected amide (1.2 mmol, 1.0 equiv.) was dissolved in a 1:1 mixture of DCM/TFA (10 mL). Upon completion of the deprotection the solvent was removed in vacuo, the residue dissolved in EtOAc (20 mL) and washed with a sat. aqueous NaHCO3 solution (10 mL), followed by drying with MgSO4 and filtering. The solvent was removed in vacuo and the crude product purified via flash chromatography.

General procedure for the preparation of isothiocyantes:

GP 4:
The free amine (0.5 mmol, 1.0 equiv) was dissolved in abs. EtOH (2 mL) followed by subsequent addition of CS2 (300 µL, 5.0 mmol, 10.0 equiv.) and NEt3 (70 µL, 0.5 mmol, 1.0 equiv) at rt. The solution was stirred for 15 minutes after which time Boc2O(s) (98 mg, 0.45 mmol, 0.9 equiv.) and DMAP (3 mg, 0.03 mmol, 0.05 equiv.) were added. Upon completion of the isothiocyanate formation the solvent was removed in vacuo and the crude product was purified via flash chromatography to afford the desired isothiocyanate.

General procedure for the synthesis of bromoacetamides:

GP 5:
The respective free amine (0.5 mmol, 1.0 equiv.) was dissolved in methylene chloride (3 mL) and NEt3 (140 µL, 1.0 mmol, 2.0 equiv.). The solution was cooled to 0 °C after which bromoacetyl bromide (53 µL, 0.6 mmol, 1.2 equiv.) was added. Upon completion of the reaction the solvent was removed in vacuo and the crude product was purified via flash chromatography to afford the desired bromoacetamide.

General procedure for the synthesis of maleimides:

GP 6A:
The respective free amine (0.5 mmol, 1.0 equiv.) was dissolved in methylene chloride (3 mL) and maleic anhydride (0.55 mmol, 1.1 equiv.) was added. After 50 minutes the solvent was removed in vacuo and the free acid was purified over silica (Step 1). It was then dissolved in acetic anhydride (2 mL) and refluxed for 30 minutes. After cooling to room temperature the solution was carefully quenched with NH4Cl(s) and stirred at r.t. for another 10 minutes after which time it was diluted with EtOAc (10 mL) and washed with water (10 mL) and brine (10 mL), dried with MgSO4, filtered and the solvent removed in vacuo. Purification over silica afforded the desired maleimide (Step 2).

GP 6B:
The respective free amine (0.5 mmol, 1.0 equiv) was dissolved in methylene chloride (3 mL) and maleic anhydride (0.55 mmol, 1.1 eqiuv.) was added. After 50 minutes the solvent was removed in vacuo and the residue was then dissolved in acetic anhydride (2 mL) and refluxed for 30 minutes. After cooling to room temperature the solution was carefully quenched with NH4Cl(s) and stirred at r.t. for another 10 minutes after which time it was diluted with EtOAc (10 mL) and washed with water (10 mL) and brine (10 mL), dried with MgSO4, filtered and the solvent removed in vacuo. Purification over silica afforded the desired maleimide.

1,6 Dimethoxynaphthalene NK227

To a suspension of 1.6-dihydroxynaphtalene (7.0 g, 43.7 mmol, 1.0 equiv.) and K2CO3 (22.3 g, 161.7 mmol, 3.7 equiv.) in acetone (200 mL) was added dropwise Me2SO4 (14.5 mL, 152.6 mmol, 3.5 equiv.) under a nitrogen atmosphere. The reaction mixture was then refluxed for 5 h and subsequently cooled to rt and quenched with a 10% aq. ammonia solution (50 mL) and stirred for another 30 min. The insoluble salts were removed by filtration and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (hexanes/DCM 2:1) to give NK227 as a white solid (7.2 g, 88%).
1H NMR (300 MHz, CDCl3):                        δ = 8.20 (d, J = 9.00 Hz, 1H), 7.42 – 7.32 (m, 2H), 7.19 – 7.11 (m, 2H), 6.71 (dd, J = 6.40, 2.20 Hz, 1 H), 4.00 (s, 3 H), 3.93 ppm (s, 3 H).
Spectroscopic data is consistent with that reported in the literature.82

5-methoxy-3,4-dihydronaphthalen-2(1H)-one NK228

To a warm solution (40 °C) of NK227 (7.0 g, 37.2 mmol, 1.0 equiv.) in EtOH (120 mL) was carefully added small pieces of sodium (7.3 g, 320.0 mmol, 8.5 equiv.) over a period of 1 h. After the last addition the reaction mixture was refluxed for 2.5 h and subsequently cooled to rt. It was quenched with conc. HCl to pH < 2 and afterwards refluxed for 1 hour and then diluted with water and extracted with methylene chloride (3 x 100 mL). The combined organic layers were dried with MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 5:1) to give tetralone NK228 as a brown oil (4.32 g, 66%).
1H NMR (300 MHz, CDCl3):  δ = 7.14 (t, J = 8.1 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 3.84 (s, 3H), 3.54 (s, 2H), 3.07 (t, J = 6.7 Hz, 2H), 2.50 ppm (t, J = 6.7 Hz, 2H).
13C NMR (75 MHz, CDCl3): δ = 210.7, 156.4, 135.0, 128.2, 127.6, 126.9, 126.8, 125.0,                    108.5, 55.4, 44.6, 37.9, 21.0 ppm.82, 183 Spectroscopic data is consistent with that reported in the literature.82

5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine NK229

To a solution of tetralone NK228 (4.0 g, 22.7 mmol, 1.0 equiv.) in methylene chloride (75 mL) was added 1-propylamine (2.05 mL, 25.0 mmol, 1.1 equiv.) and AcOH (2.6 mL, 45.4 mmol, 2.0 equiv.) The mixture was stirred for 30 min at rt under a nitrogen atmosphere. Then NaBH3CN was added (4.28 g, 68.1 mmol, 3.0 equiv.) and the resulting suspension was stirred for 14h under a nitrogen atmosphere. The reaction mixture was concentrated in vacuo and the residue was dissolved in a mixture of EtOAc (70 mL) and aq. sat. NaHCO3 (70 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried with MgSO4, filtered and concentrated in vacuo. The crude oil was purified by flash chromatography (EtOAc/ULTRA 9:1) to give amine NK229 as a yellow oil (3.73 g, 75%).
1H NMR (300 MHz, CDCl3):δ = 7.10 (t, J = 8.1 Hz, 1 H), 6.68 (d, J = 8.1 Hz, 1 H), 6.71 (d, = 8.1 Hz, 1 H), 3.79 (s, 3 H), 3.43-3.27 (m, 1 H), 3.26-3.12 (m, 1 H), 3.11-2.93 (m, 4 H), 2.67-2.49 (m, 1 H), 2.43-2.28 (m, 1 H), 1.96-1.74 (m, 3 H), 1.02 ppm (t, J = 7.4 Hz, 3 H).
13C NMR (75 MHz, CDCl3):  δ = 157.1, 132.9, 127.0, 123.4, 121.2, 107.9, 55.3, 55.2, 47.1,          31.9, 25.3, 21.9, 19.5, 11.1 ppm.82

2-(4-nitrophenyl)acetyl chloride NK230

To a solution of 4-nitrophenylacetic acid (3.85 g, 21.2 mmol, 1.25 equiv.) in chloroform (40 mL) was added (COCl)2 (3.6 mL, 42.4 mmol, 2.5 equiv.) and DMF (0.1 mL) and the mixture stirred for 30 min. The reaction mixture was concentrated in vacuo and re-dissolved in toluene (3 x 15 mL). The brown solid NK230 was used as obtained in the next step.

5-methoxy-N-(4-nitrophenethyl)-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine NK231

To an ice-cooled solution of amine NK229 (3.73 g, 17.0 mmol, 1.0 equiv.) in methylene chloride (25 mL) and 1N aq. NaOH (20 mL) was added dropwise a solution of acid chloride NK230 (4.2 g, 21.2 mmol, 1.25 equiv.) in methylene chloride (20 mL). The solution was then warmed to rt and stirred for another 30 min. The reaction mixture was then concentrated in vacuo and the precipitate was dissolved in EtOAc (60 mL) and washed with brine (3 x 30 mL) and NaHCO3 (30 mL). The organic phase was dried with MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 3:2) to give amide NK231 as a yellow foam (6.0 g, 93%).
1H NMR (300 MHz, CDCl3):δ = 8.24 – 8.13 (m, 2H), 7.50 – 7.37 (m, 2H), 7.18 – 7.04 (m, 1H), 6.74 – 6.62 (m, 2H), 3.87 – 3.77 (m, 5H), 3.26 – 3.12 (m, 2H), 3.08 – 2.93 (m, 2H), 2.90 – 2.37 (m, 3H), 1.96 – 1.74 (m, 2H), 1.74 – 1.56 (m, 2H), 0.99 – 0.86 ppm (m, 3H).

N-(4-aminophenethyl)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine NK239

To an ice-cooled solution of BH3·SMe2 (4.6 mL, 48.6 mmol, 3.1 equiv.) in THF (70 mL) was slowly added a solution of nitroamide NK231 (6.0 g, 15.7 mmol, 1.0 equiv) in THF (20 mL). The reaction mixture was stirred for 15 min at 0 ºC and then refluxed for 4h. After cooling to rt the reaction mixture was carefully quenched with 6 N aq. HCl. The resulting solution was dissolved in water and basified with 4 N aq. NaOH to pH > 12. The aqueous solution was extracted with methylene chloride (3 x 40 mL). The combined organic layers were dried with MgSO4, filtered and concentrated in vacuo. The resulting brown oil was dissolved in EtOH (250 mL) and warmed to 50 °C. Hydrazine (10 mL, 205 mmol, 13.0 equiv) and a catalytic amount of Raney-nickel were added and stirred for 3h. After cooling to rt the mixture was filtered over kieselgur and concentrated in vacuo. The resulting yellow oil was purified by flash chromatography (hexanes/EtOAc/NEt3 50:50:1) to give NK239 as a yellow oil (3.6 g, 68% over 2 steps).
1H NMR (300 MHz, CDCl3): δ = 8.24 – 8.13 (m, 2H), 7.50 – 7.37 (m, 2H), 7.18 – 7.04 (m, 1H), 6.74 – 6.62 (m, 2H), 3.87 – 3.77 (m, 5H), 3.26 – 3.12 (m, 1H), 3.11 – 2.93 (m, 4H), 2.67 – 2.49 (m, 1H), 2.43 – 2.28 (m, 1H), 1.96 – 1.74 (m, 3H), 1.02 ppm (t, J = 7.4 Hz, 3 H).
13C NMR (75 MHz, CDCl3):  δ = 157.1, 144.3, 138.1 130.9, 129.5, 126.1, 125.3, 121.7,121.6, 115.2, 106.8, 56.8, 55.2, 53.3, 52.7, 35.1, 32.3, 25.8, 23.8, 22.2, 11.1 ppm.

6-((4-aminophenethyl)(propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol NK242

To a cooled solution (-78 °C) of NK239 (3.6 g, 10.7 mmol, 1.0 equiv.) in methylene chloride (70 mL) was slowly added a 1M BBr3 solution (21.4 mL, 21.4 mmol, 2.0 equiv.). The reaction mixture was stirred for 3h at -78 °C and then slowly allowed to warm to rt and stirred for additional 12h. The brown precipitate was dissolved in hot brine solution (150 mL) and the aqueous layer was washed with EtOAc (2 x 50 mL). The aqueous layer was then basified to pH > 9 with 1N aq. NaOH and extracted with methylene chloride (5 x 60 mL). The combined organic layers were dried with MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 1:1.5 + 1% NEt3) to give NK242 as a brown solid (2.5 g, 73%).
1H NMR (300 MHz, CDCl3):  δ = 7.05 – 6.95 (m, 3H), 6.73 – 6.54 (m, 4H), 3.07 – 2.48 (m, 10H), 2.16 – 2.04 (m, 1H), 1.70 – 1.45 (m, 3H), 0.90 ppm (t, J = 7.4 Hz, 3H).
13C NMR (75 MHz, CDCl3): δ = 153.4, 144.3, 138.5 130.9, 129.5, 126.4, 122.9, 121.7, 115.3, 111.9, 56.7, 53.2, 52.7, 34.9, 32.8, 25.7, 23.6, 22.1, 11.9 ppm.

Table of Contents
Chapter 1 Introduction 
1.1 G protein-coupled receptors
1.2 Distribution and structure of the CB1 receptor
1.3 Cannabinoids and the cannabinoid receptors
1.4 Dopamine receptors and their distribution
1.5 Coexpression and a hypothetical CB1-D2 receptor heterome
1.6 Covalent binding ligands .
1.7 Reactive electrophiles for covalent GPCR ligands
1.8 GPCR dimers
Chapter 2 Discussion 
2.1 Synthesis of CB1 inverse agonist ligands based on SR171416A
2.2 Synthesis of Rimonabant derivatives with short (≤ 6C) C3 substituents
2.3 Synthesis of CB1R-agonists
2.4 Biotinylated CB1 receptor ligands
2.5 Binding affinities of the CB1R antagonists / agonists
2.6 CB1 Inverse agonists ligands with long linkers and functional substituents.
2.7 Synthesis of the D2R agonist (±)-PPHT-NH2
2.8 Synthesis of CB1-D2 receptor heterodivalent ligands
2.9 Cholesterol containing ligands
2.10 Covalent Ligand Series for the Cannabinoid CB1 Receptor
Chapter 3 Experimental
GET THE COMPLETE PROJECT
Novel Divalent and Irreversibly- Binding Ligands for the CB1 Cannabinoid GPCR

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