General
2-Chloro-4-fluorobenzonitrile was purchased from TCI Europe (Zwijndrecht, Belgium). All other chemicals and solvents were obtained from Acros Organics (Geel, Belgium), Sigma-Aldrich (Bornem, Belgium), or VWR International (Leuven, Belgium). For ascending thin layer chromatography (TLC), pre-coated aluminum backed plates (Silica gel 60 with fluorescent indicator UV 254 nm, 0.2 mm thickness; Macherey-Nagel, Düren, Germany) were used. Molecular mass measurements were performed on a time-of-flight mass spectrometer (LCT, Micromass, Manchester, UK) equipped with an orthogonal electrospray ionization (ESI) interface. Acquisition and processing of data was done using MassLynx software (version 3.5, Micromass). Mass data were rounded to the whole number. 1H nuclear magnetic resonance (NMR) spectra were acquired with a Bruker Avance II spectrometer (400 MHz, 5 mm probe, Fällanden, Switzerland). Chemical shifts are reported in parts per million relative to tetramethylsilane (δ = 0). Coupling constants are reported in hertz (Hz). Splitting patterns are defined by s (singlet), d (doublet), t (triplet), or m (multiplet). The clogD and PSA values were calculated using, respectively, Marvinsketch 5.7.0 software and the calculator from Daylight (http://www.daylight.com/meetings/emug00/Ertl/tpsa.html). HPLC analysis was performed on an L2130 LaChrom Elite pump (Hitachi, Tokyo, Japan) connected to a UV L2400 LaChrom Elite spectrometer (Hitachi). Radiolabeled compounds were analyzed by passage of the HPLC eluate (after passing through the UV detector) over a 3-inch NaI(Tl) scintillation detector connected to a single channel analyzer (GABI box; Raytest, Straubenhardt, Germany). Data were acquired and analyzed using a RaChel (Lablogic, Sheffield, UK) or GINA Star (Raytest) data acquisition system. Quantification of radioactivity during biodistribution and metabolite studies was performed using an automated gamma counter equipped with a 3-inch NaI(Tl) well crystal coupled to a multichannel analyzer, mounted in a sample changer [2480 Wizard (Mackie, 2008), Perkin Elmer, Massachusetts, USA]. The values are corrected for background radiation, physical decay and counter dead time.
Animals were housed in individually ventilated cages in a thermo-regulated (22°C), humidity-controlled facility under a 12 h light/12 h dark cycle, with free access to food and water. All animal experiments were performed in compliance with the principles set by the Belgian law relating to the conduct of animal experimentation, after approval from the university (KU Leuven) animal ethics committee.
Organic synthesis
Methazoic acid (1)
Caution:
Methazoic acid is a potential explosive, heat, and shock sensitive compound. To a solution of 10 M NaOH (3 mL) at 0°C was added dropwise MeNO2 (0.5 mL) and the resulting mixture was stirred for 15 min. The reaction mixture was allowed to warm to room temperature, stirred for 15 min and a second amount of MeNO2 (0.5 mL) was added. After stirring until a clear brown solution was formed (~30 min), ice-cold water (50 mL), and concentrated hydrochloric acid (~11.6 M HCl, 3 mL) were slowly added to the mixture. The resultant solution was extracted with diethyl ether (Et2O; 2 × 100 mL) and the organic layers were collected and dried over magnesium sulfate (MgSO4). The solvent was carefully removed under reduced pressure without heating to yield compound 1 (~1 g) as an orange-yellow viscous oil, which was used in the synthesis of 4, without any further purification.
3-nitro-6-hydroxyquinoline (4)
Step A:
To a solution of 2-nitro-5-hydroxybenzaldehyde (2; 1.20 g, 7.18 mmol, 1.0 eq) in ethanol (EtOH; 30 mL) was added Fe powder (1.48 g, 26.57 mmol, 3.7 eq), followed by acetic acid (2.4 mL) and H2O (0.8 mL). After stirring at room temperature for 16 h, the dark brown reaction mixture was diluted with EtOH (30 mL), filtered through a sintered funnel, and concentrated in vacuo. The crude mixture was extracted with dichloromethane (DCM; 2 × 200 mL), the combined organic layers were dried over MgSO4 and concentrated in vacuo. Compound 3 was obtained as a crude yellow powder, which was used in step B without any further purification.
Step B:
To a stirred solution of 3 in either tetrahydrofuran (THF) or EtOH (10 mL) was added a solution of compound 1 (~1 g) in a mixture of H2O (5 mL) and 5 M HCl (5 mL). The dark brown mixture was heated to 80oC and stirred for 2 h. The solvent was evaporated under reduced pressure, the crude material was extracted with DCM (3 × 100 mL) and the solution was again concentrated under reduced pressure and purified over silica column to give 4 as a pale brown solid. 1H NMR (CDCl3): δ 7.10– 7.17 (m, 1H, Ar), 7.41–7.45 (m, 1H, Ar), 8.11 (s, 1H, Ar), 8.67 (s, 1H, Ar), 9.24 (s, 1H, Ar). MS (ESI) m/z: 191.2 [(M+H)+, 100%].
3-nitro-6-methoxyquinoline (5a)
To a solution of compound 4 (260 mg, 1.37 mmol, 1 eq) in acetone (15 mL), K2CO3 (283 mg, 2.05 mmol, 1.5 eq) and MeI (5 eq) were added and the reaction mixture was stirred at 60 °C for 4 h. Completion of reaction was confirmed by TLC (EtOAc/heptane 1:1). Acetone was removed under reduced pressure and the crude residue was diluted with DCM (100 mL) and washed with H2O (3 × 25 mL). The organic layer was dried over MgSO4 and the solvent was removed under reduced pressure. Silica gel column purification yielded 5 (171 mg) as pale yellow crystalline needles. Yield: 61%. TLC (EtOAc/heptane 1:1): Rf = 0.55. 1H NMR (CDCl3): δ 3.94 (s, 3H, OMe), 7.19–7.23 (m, 1H, Ar), 7.50–7.55 (m, 1H, Ar), 8.05 (s, 1H, Ar), 8.86 (s, 1H, Ar), 9.44 (s, 1H, Ar). MS (ESI) m/z: 205.2 [(M+H)+, 100%].
6-(2-fluoroethoxy)-3-nitroquinoline (5b)
To a solution of compound 4 (260 mg, 1.37 mmol, 1 eq) in DMF (15 mL), K2CO3 (283 mg, 2.05 mmol, 1.5 eq) and FEtBr (5 eq) were added and the reaction mixture was stirred at 50°C for 2 h. Completion of reaction was confirmed by TLC (EtOAc/heptane 1:1). The crude residue was diluted with DCM (100 mL) and washed with H2O (3 × 25 mL). The organic layer was dried over MgSO4 and the solvent was removed under reduced pressure. Silica gel column purification yielded 5b (230 mg) as pale yellow crystalline needles. Yield: 67%. TLC (EtOAc/heptane 1:1): Rf = 0.60. 1H NMR (CDCl3): δ 4.36 (dt, 2H, J 2.4 & 19.4), 4.84 (dt, 2H, J 2.4 & 39.2), 7.22 (s, 1H, Ar), 7.59 (d, 1H, J 9.2, Ar), 8.10 (d, 1H, J 9.2, Ar), 8.87 (s, 1H, Ar), 9.47 (s, 1H, Ar). MS (ESI) m/z: 236.8 [(M+H)+, 100%].
3-amino-6-quinolinols (6a-c)
3-Aminoquinolin-6-ol (6a) To a solution of compound 4 (330 mg, 1.74 mmol, 1 eq) in EtOH (10 mL), Fe powder (485 mg, 8.68 mmol, 5 eq) was added and the mixture was stirred. After adding acetic acid (1 mL) and H2O (0.2 mL), the reaction mixture was heated to 60oC and stirred for 3 h. The Fe powder was removed by filtering the reaction mixture over a sintered funnel. The filtrate was collected, dried over MgSO4 and concentrated to yield 6a (100 mg) as a pale yellow powder. Yield: 36%. TLC (DCM/MeOH, 95:5): Rf = 0.21. MS (ESI) m/z: 161.0 [(M+H)+, 100%].
6-Methoxyquinolin-3-amine (6b) Compound 6b was synthesized starting from compound 5a in accordance with the procedure described for compound 6a. Yellow solid was obtained in 45% yield. TLC (DCM/MeOH 95:5): Rf = 0.25. MS (ESI) m/z: 174.7 [(M+H)+, 100%].
6-(2-Fluoroethoxy)quinolin-3-amine (6c) Compound 6c was also synthesized starting from compound 5b as described for compound 6a. Colorless powder was obtained in 60% yield (175 mg). RF 0.25 (9.5:0.5 DCM:MeOH). 1H NMR (MeOD): δ 4.55 (dt, 2H, J 3.5 & 28.8), 5.01 (dt, 2H, J 3.4 & 47.8), 7.35 (d, 1H, J 9.2, Ar), 7.25 (s, 1H, Ar), 7.51 (s, 1H, Ar), 7.98 (d, 1H, J 9.2, Ar) 8.54 (s, 1H, Ar). MS (ESI) m/z: 206.9 [(M+H)+, 100%].
2-chloro-4-fluoro-n′-hydroxybenzamidine (8)
To a solution of 2-chloro-4-fluoro-benzonitrile (7; 1.0 g, 6.45 mmol, 1.0 eq) in MeOH was added NH2OH·HCl (0.58 g, 8.36 mmol, 1.3 eq), Na2CO3 (1.0 g, 9.64 mmol, 1.5 eq) and H2O (4 mL), and the reaction mixture was stirred for 4 h at 60°C. The solvent was evaporated under reduced pressure and the crude residue was extracted with DCM (2 × 500 mL). The combined organic layers were dried over MgSO4 and the solvent was removed under reduced pressure. The crude compound 8 (1.18 g, 97%) was used as such in the next step without any further purification. RF 0.41 (1:1 hexane:EtOH). MS (ESI) m/z: 189.0 [(M+H)+, 100%].
3-(3-(2-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid (9)
A solution of compound 8 (1.0 g, 5.32 mmol, 1.0 eq) and succinic anhydride (0.53 g, 5.32 mmol, 1.0 eq) in toluene (15 mL) was stirred for 3 h at 110°C. Toluene was evaporated under reduced pressure, the crude residue was extracted with DCM (3 × 250 mL), the combined organic extracts were dried over MgSO4 and concentrated under reduced pressure. The crude residue was purified using silica gel column chromatography (EtOAc/heptane gradient 10–30% EtOAc) and compound 9 was obtained as a white solid (425 mg). Yield: 28%. RF 0.25 (1:1 hexane:EtOH). 1H NMR (DMSO-d6): δ 2.56 (t, 2H, J 6.7, CH2), 2.94 (t, 2H, J 6.9, CH2), 7.11–7.18 (m, 1H, Ar), 7.39–7.44 (m, 1H, Ar), 7.65–7.71 (m, 1H, Ar). MS (ESI) m/z: 269.29 [(MH¬), 100%].
Methyl-3-(3-(2-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5-yl)propanoate (10)
Compound 9 (100 mg, 0.370 mmol, 1 eq) was dissolved in dry DCM (4 mL) followed by the addition of DCC (229 mg, 1.11 mmol, 3 eq), DMAP (90 mg, 0.740 mmol, 2 eq), and dry MeOH (0.5 mL). The reaction mixture was stirred for 16 h at room temperature. The reaction mixture was concentrated under reduced pressure, DCM (50 mL) was added and the solution was washed with H2O (2 × 20 mL). After drying the organic layer over MgSO4, the solvent was removed and the crude product purified through silica gel column chromatography (EtOAc/heptane gradient 0–10% EtOAc) yielded pure compound 10. Colorless oil (63% yield, 66 mg). RF 0.55 (1:1 hexane:EtOH). 1H NMR (CDCl3): δ 2.54 (t, 2H, J 7.3, CH2), 2.89 (t, 2H, J 7.3, CH2), 3.33 (s, 3H, CH3), 6.66–6.72 (m, 1H, Ar), 6.85–6.89 (m, 1H, Ar), 7.50–7.55 (m, 1H, Ar). MS (ESI) m/z: 285.1 [(MH+), 100%].
3-(3-(2-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5-yl)propanal (11)
To a solution of compound 10 (100 mg, 0.352 mmol, 1 eq) in dry DCM (5 mL) cooled to −80°C was added drop-wise over 5 min DIBAL-H 1.5 M in toluene (0.107 mL, 0.528 mmol, 1.5 eq) and the reaction mixture was stirred at the same temperature for 1 h. The reaction was quenched with a few drops of H2O, DCM (50 mL) was added and the mixture was washed with H2O (2 × 20 mL). The organic layer was dried over MgSO4 and the solvent was removed under reduced pressure. The crude residue was purified through silica gel flash chromatography using a mixture of heptane/EtOAc (2:1) to yield the desired aldehyde 11 (40 mg). Colorless powder (40 mg, 45%). RF 0.40 (1:1 hexane:EtOH). 1H NMR (CDCl3): δ 2.72 (t, 2H, J 6.8, CH2), 2.87 (t, 2H, J 6.9, CH2), 6.67–6.70 (m, 1H, Ar), 6.85–6.89 (m, 1H, Ar), 7.54–7.56 (m, 1H, Ar), 9.49 (s. 1H, CHO). MS (ESI) m/z: 255.1 [(MH+), 100%].
N-(3-(3-(2-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5-yl)propyl)-6-methoxyquinolin-3-amine (MA2)
Compound 11 (87.0 mg, 0.343 mmol, 1 eq) and amine 6b (60.0 mg, 0.343 mmol, 1 eq) were dissolved in dichloroethane (DCE; 3 mL), followed by the addition of Ti(O-iPr)4 (1.68 mL, 5.142 mmol, 15 eq) at room temperature. After stirring for 1 h, the reaction mixture was added dropwise to a stirred solution of NaBH4 (77.8 mg, 2.057 mmol, 6 eq) in dry MeOH (6 mL) at 0oC. After the addition was complete the mixture was brought to room temperature and stirred for 2 h. The reaction was quenched with a few drops of 1 M HCl and solvents were evaporated under reduced pressure. The residue was dissolved in DCM (100 mL), the solution was washed with H2O (2 × 15 mL) and the organic layer was dried and concentrated by vacuum evaporation. The crude product was purified over silica gel chromatography using mixtures of DCM/MeOH as eluent. Pale yellow oil (70 mg), yield: 49%. TLC (hexane/EtOH 1:1): Rf = 0.21. 1H NMR (CDCl3): δ 2.25–2.30 (m, 2H, H3b), 3.10–3.16 (m, 2H, H3c), 3.40 (t, 2H, J 6.7, H3a), 3.87 (s, 3H, OMe), 6.86–6.88 (m, 1H, Ar), 6.94–6.97 (m, 1H, Ar), 7.02–7.11 (m, 2H, Ar), 7.21–7.27 (m, 2H, Ar), 7.78–7.83 (m, 1H, Ar), 7.85–7.91 (m, 1H, Ar), 8.25 (br s, 1H, NH). MS (ESI) m/z: 412 [(M+H)+, 100%].
3-(3-(3-(2-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5-yl)propylamino)quinolin-6-ol (MA1)
MA1 was synthesized starting from compound 6a in accordance with the procedure described for MA2. Pale yellow solid (90 mg), yield: 57%. TLC (hexane/EtOH 1:1): Rf = 0.20. MS (ESI) m/z: 398 [(M+H)+, 100%]. 1H NMR (CDCl3): δ 2.50–2.56 (m, 2H, H3b), 3.40–3.46 (m, 2H, H3c), 3.60–3.65 (m, 2H, H3a), 7.16–7.26 (m, 4H, Ar), 7.42–7.48 (m, 1H, Ar), 7.65–7.70 (m, 1H, Ar), 8.09–8.13 (m, 1H, Ar), 8.24–8.28 (m, 1H, Ar), 8.45 (s, 1H, NH). HRMS (ESI) Calcd. for C20H17ClFN4O2 [M+H]+: 399.1018. Found: 399.1058.
6-(2-fluoroethoxy)-n-(3-(3-(2-chloro-4-fluorophenyl) -1,2,4-oxadiazol-5-yl)propyl)quinolin-3-amine (MA3)
Yellow crystalline solid (55 mg, 51%). RF 0.25 (1:1 hexane:EtOH). 1H NMR (CDCl3): δ 2.20–2.30 (m, 2H), 3.11 (t, 2H, J 7.2), 3.37 (t, 2H, J 6.8), 4.25 (dt, 2H, J 4.2 & 27.7), 4.77 (dt, 2H, J 4.1 & 47.4), 6.86 (d, 1H, J 2.7, Ar), 6.92 (d, 1H, J 2.6, Ar), 7.03–7.11 (m, 2H, Ar), 7.23–7.28 (m, 1H, Ar), 7.78–7.89 (m, 2H, Ar), 8.25 (d, 1H, J 2.7, Ar). HRMS (ESI) Calcd. for C22H20ClF2N4O2 (MH+): 445.1237. Found: 445.1276.
Functional and binding experiments
The competition binding experiments and functional assays were performed by Roche Healthcare.
Cell culture
CHO-K1 beta-arrestin cells (DiscoveRx, Fremont, CA) expressing hCB2, mouse CB2 (mCB2), and human CB1 (hCB1) were cultured in F-12 Nutrient Mixture (HAM) supplemented with 10% fetal bovine serum (FBS), 300 μg/mL hygromycin and 800 μg/mL geneticin (G418). Cells were incubated in a humidified atmosphere at 37°C with 5% CO2.
cAMP assay
cAMP was measured using cAMP-Nano-TRF detection kit (Roche Diagnostics, Penzberg, Germany). Cells were seeded 17–24 h prior to the experiment 3 × 104 cells per well in a black 96-well plate with flat clear bottom (Corning, Wiesbaden, Germany) in growth medium and incubated in 5% CO2 at 37°C in a humidified incubator. The growth medium was exchanged with Krebs Ringer bicarbonate buffer with 1 mmol/L 3-isobutyl-1-methylxanthine (IBMX), 0.1% fatty acid-free bovine serum albumin (BSA) and incubated at 30°C for 60 min. Agonist was added to a final assay volume of 100 μL and the mixture was incubated for 30 min at 30°C. The assay was stopped by the addition of 50 μL 3x lysis reagent and shaken for 2 h at room temperature. The time-resolved energy transfer was measured using an LF502 Nanoscan FLT (IOM, Berlin, Germany), equipped with a laser as excitation source. cAMP content was determined from the function of a standard curve spanning from 10 to 0.13 nmol/L cAMP.
Radioligand binding assay
Stably transfected cells were treated for 24 h with 5 mM butyrate in growth medium before harvesting, followed by homogenization in 15 mmol/L Hepes, 0.3 mmol/L EDTA, 1 mmol/L EGTA, 2 mmol/L MgCl2, complete EDTA-free protease inhibitor (Roche Applied Science, Rotkreuz, Switzerland), pH 7.4 using a glass potter and centrifugation at 47,800 g at 4°C for 30 min. The pellet was then rehomogenized twice in the same buffer and centrifuged (47,800 g, 4°C, 30 min). The final pellet was then resuspended in 75 mmol/L Tris, 0.3 mmol/L EDTA, 1 mmol/L EGTA, 12.5 mmol/L MgCl2, 250 mmol/L sucrose, pH 7.4 at a protein concentration of 1 to 3 mg/mL, aliquoted, frozen on dry ice and stored at −80°C. Saturation binding was performed with 0.05 to 2.6 nM [3H]CP55940 (Perkin Elmer) and 1.0 μg of membrane protein. CP55940 (10 μM) was used to define non-specific binding. More than 95% of the total binding signal was specific. Assay buffer consisted of 50 mmol/L Tris-HCl, 5 mmol/L MgCl2, 2.5 mmol/L EGTA, and 0.1% fatty acid-free BSA, pH 7.4. Assays were initiated by addition of membranes in a final volume of 250 μL/well. Mixtures were incubated for 3 h at room temperature and then vacuum filtered and rinsed with wash buffer (50 mmol/L Tris-HCl, 5 mmol/L MgCl2, 2.5 mmol/L EGTA, and 0.5% fatty acid-free BSA, pH 7.4) on a Filtermate cell harvester through Packard GF/B filters presoaked in 0.3% polyethylenimine.
For competition binding, membrane preparations were incubated with 0.3 nM [3H]CP55940 in the presence or absence of increasing concentrations of ligands for 60 min at 30°C in a final volume of 0.2 mL of 50 mmol/L Tris-HCl, 5 mmol/L MgCl2, 2.5 mmol/L EGTA, 0.1% fatty acid-free BSA, and 1% DMSO, pH 7.4, buffer, gently shaking. Non-specific binding was defined with 10 μM CP55940. All binding reactions were terminated by vacuum filtration onto 0.5% polyethylenimine presoaked GF/B filter plates (Packard) followed by seven brief washes with 2 mL of ice-cold binding buffer containing 0.5% fatty acid-free BSA. Plates were dried at 50°C for 1 h and liquid scintillation counting was used to determine bound radiolabel. IC50 values and Hill slopes were determined by a four parameter logistic model using ActivityBase (ID Business Solution, Guilford, UK). pKi values were determined by the generalized Cheng-Prusoff equation (Yung-Chi and Prusoff, 1973).