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DOI: 10.1055/a-2230-1003
Discovery of a Cannabinoid CB2 Receptor Fluorescent Probe Based on a Pyridin-2-yl-benzyl-imidazolidine-2,4-dione Scaffold
This work was supported by the Dutch Research Council (Nederlandse Organisatie voor Wetenschappelijk Onderzoek; NWO, Navistroke #15851).
Abstract
Cannabinoid receptor type 2 (CB2R) agonists have therapeutic potential for the treatment of (neuro)inflammatory diseases. Fluorescent probes enable the detection of CB2R in relevant cell types and serve as a chemical tool in cellular target engagement studies. Here, we report the structure-based design and synthesis of a new CB2R selective fluorescent probe. Based on the cryo-EM structure of LEI-102 in complex with the CB2R, we synthesized 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione analogues in which we introduced a variety of linkers and fluorophores. Molecular pharmacological characterization showed that compound 22, containing a Cy5-fluorophore with an alkyl-spacer, was the most potent probe with a pK i of 6.2 ± 0.6. It was selective over the cannabinoid CB1 receptor and behaved as an inverse agonist (pEC50 5.3 ± 0.1, E max –63% ± 6). Probe 22 may serve as a chemical tool in target and lead validation studies for the CB2R.
Supporting Information
- Supporting information for this article is available online at https://doi.org/10.1055/a-2230-1003.
- Supporting Information
Publication History
Received: 08 November 2023
Accepted after revision: 14 December 2023
Accepted Manuscript online:
14 December 2023
Article published online:
30 January 2024
© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by/4.0/)
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
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- 23 General Procedure: All reagents and solvents were purchased from commercial sources and were of analytical grade. Reagents and solvents were not further purified before use. All moisture-sensitive reactions were performed under inert atmosphere. Solvents were dried using 4Å molecular sieves prior to use when anhydrous conditions were required. Water used in reactions was always demineralized. Analytical thin-layer chromatography (TLC) was routinely performed to monitor the progression of a reaction and was conducted on silica gel 60 F254 plates. Reaction compounds on the TLC plates were visualized by UV irradiation (λ254) and/or spraying with potassium permanganate solution (K2CO3 (40 g), KMnO4 (6 g), and H2O (600 mL)), ninhydrin solution (ninhydrin (1.5 g), n-butanol (100 mL) and acetic acid (3.0 mL)) or molybdenum solution ((NH4)6MO7O24·4H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10 g/L) in sulfuric acid (10%)) followed by heating as appropriate. Purification by flash column chromatography was performed using silica gel 60 (40–63 μm, pore diameter of 60Å). Solutions were concentrated using a rotary evaporator.
- 24 Preparation of 29: A mixture of 28 (2.70 g, 10.0 mmol, 1 equiv), 25 (5.72 g, 20.1 mmol, 2 equiv), K2CO3 (8.32 g, 60.2 mmol, 6 equiv) and 18-crown-6 (0.53 g, 2.0 mmol, 0.2 equiv) in DMF (55 mL) was heated (50 °C) overnight. After cooling to r.t., the mixture was diluted with H2O (40 mL) and Et2O (40 mL). The layers were separated and the aqueous layer was extracted thrice with Et2O. The combined organic layer was washed five times with H2O and once with brine, dried (MgSO4), filtered, and the solvent evaporated under reduced pressure. The crude product was purified with flash column chromatography (SiO2, 20–50% EtOAc in pentane) to yield the product (2.67 g, 6.3 mmol, 62%) as a yellow solid. 1H NMR (850 MHz, CDCl3): δ = 7.48 (d, J = 8.4 Hz, 2 H), 7.18 (d, J = 8.1 Hz, 2 H), 4.71–4.67 (m, 2 H), 4.34 (d, J = 15.3 Hz, 1 H), 4.06 (hept, J = 6.5 Hz, 1 H), 3.72 (d, J = 17.1 Hz, 1 H), 3.66 (d, J = 17.2 Hz, 1 H), 3.51 (d, J = 7.9 Hz, 2 H), 1.37 (s, 9 H), 1.17 (d, J = 6.8 Hz, 3 H). 13C NMR (214 MHz, CDCl3): δ = 170.11, 157.03, 155.73, 134.58, 132.24, 129.93, 122.26, 79.32, 49.10, 46.16, 45.52, 44.97, 28.41, 18.63. LC-MS (ESI, 10-90): t R = 7.64 min; m/z = 425.53 [M + H]+.
- 25 Preparation of 35: A stirred and degassed mixture of 29 (2.67 g, 6.3 mmol, 1.1 equiv), Pd(dppf)Cl2 (0.24 g, 0.3 mmol, 0.05 equiv), bis(pinacolato)diboron (2.40 g, 9.5 mmol, 1.5 equiv) and KOAc (2.70 g, 27.5 mmol, 4.4 equiv) in DMF (30 mL) was heated (75 °C) overnight. The reaction was diluted at r.t. with H2O and EtOAc. The layers were separated and the aqueous layer extracted thrice with EtOAc. The combined organic layer was washed with sat. NaHCO3 (aq), H2O, and brine, dried (MgSO4), filtered and the solvent evaporated under reduced pressure. The crude residue was used without further purification. The crude residue was co-evaporated thrice with chloroform and re-dissolved in degassed toluene/EtOH (30 mL, 4:1, v/v). To the stirred mixture was added 34 (1.64 g, 6.0 mmol, 1 equiv), K2CO3 (3.46 g, 25.0 mmol, 4 equiv) and Pd(PPh3)4 (0.50 g, 0.4 mmol, 0.06 equiv). After heating (75 °C) overnight, the reaction was diluted at r.t. with H2O and EtOAc. The layers were separated and the aqueous layer was extracted thrice with EtOAc. The combined organic layer was washed with H2O and brine, dried (MgsO4), filtered and the solvent evaporated under reduced pressure. The crude product was purified with flash column chromatography (SiO2, 0–4% MeOH in DCM) to yield a brown oil (1.40 g, 2.6 mmol, 42%). 1H NMR (400 MHz, CDCl3): δ = 7.94 (d, J = 8.3 Hz, 2 H), 7.60 (dd, J = 8.6, 3.5 Hz, 1 H), 7.41 (t, J = 8.8 Hz, 1 H), 7.35 (d, J = 7.9 Hz, 2 H), 4.82–4.68 (m, 2 H), 4.47 (d, J = 15.2 Hz, 1 H), 4.05 (p, J = 7.1 Hz, 1 H), 3.82 (d, J = 2.7 Hz, 2 H), 3.72 (d, J = 8.8 Hz, 2 H), 3.52 (d, J = 6.6 Hz, 2 H), 2.62–2.55 (m, 4 H), 1.59 (p, J = 5.7 Hz, 4 H), 1.38 (s, 11 H), 1.17 (d, J = 6.7 Hz, 3 H). 13C NMR (101 MHz, CDCl3): δ = 170.27, 157.94 (d, J = 258.1 Hz), 157.06, 155.71, 151.88 (d, J = 4.9 Hz), 145.63 (d, J = 15.1 Hz), 138.62, 135.95, 128.59, 127.61, 127.55, 123.70 (d, J = 20.4 Hz), 120.39 (d, J = 4.1 Hz), 79.26, 58.44 (d, J = 3.0 Hz), 54.36, 49.10, 46.49, 45.60, 44.80, 28.43, 25.98, 24.18, 18.62. LC-MS (ESI, 10-90): t R = 5.47 min; m/z = 540.20 [M + H]+.
- 26 Preparation of 1: To a solution of 35 (1.40 g, 2.6 mmol, 1 equiv) in ACN (5 mL) was added 4 M HCl in 1,4-dioxane (2.7 mL, 10.8 mmol, 4.1 equiv). After the reaction was heated (80 °C) for 2 h, the ACN was evaporated under reduced pressure. The mixture was basified with 1 M NaOH (aq) until pH 10. The aqueous layer was extracted with CHCl3/MeOH (7:1, v/v). NaCl was added for increased separation. The combined organic layer was dried (MgSO4), filtered, and the solvent evaporated under reduced pressure. The crude product was purified with flash column chromatography (SiO2, 0–4% MeOH in DCM with 2% Et3N (v/v)) to yield the product (0.98 g, 2.2 mmol, 86%) as an orange oil. 1H NMR (400 MHz, CDCl3): δ = 7.96 (d, J = 8.2 Hz, 2 H), 7.62 (dd, J = 8.6, 3.5 Hz, 1 H), 7.42 (t, J = 8.8 Hz, 1 H), 7.34 (d, J = 8.3 Hz, 2 H), 4.61 (s, 2 H), 3.83 (d, J = 2.7 Hz, 2 H), 3.78 (s, 2 H), 3.54–3.38 (m, 2 H), 3.32–3.19 (m, 1 H), 2.59 (s, 4 H), 1.60 (p, J = 5.6 Hz, 6 H), 1.46–1.36 (m, 2 H), 1.13 (d, J = 6.4 Hz, 3 H). 13C NMR (101 MHz, CDCl3): δ = 170.35, 157.98 (d, J = 258.1 Hz), 157.25, 151.84 (d, J = 4.9 Hz), 145.80 (d, J = 15.0 Hz), 138.79, 135.92, 128.66, 127.63, 123.73 (d, J = 20.4 Hz), 120.43 (d, J = 4.2 Hz), 58.55 (d, J = 3.2 Hz), 54.45, 49.15, 47.19, 46.61, 46.21, 26.06, 24.23, 22.10. LC-MS (ESI, 10-90): t R = 3.74 min; m/z = 440.33 [M + H]+. HRMS: m/z calcd for [C24H30FN5O2 + H]+: 440.24563; found: 440.24533.
- 27 Preparation of 39: To a cooled (0 °C) and stirred mixture of 12-aminododecanoic acid (1.50 g, 7.0 mmol, 1 equiv) and Et3N (1.9 mL, 13.9 mmol, 2 equiv) in acetone/H2O (14 mL, 1:1, v/v) was added dropwise Boc2O (1.67 g, 7.7 mmol, 1.1 equiv) in acetone (4 mL). After stirring at r.t. overnight, the acetone was evaporated under reduced pressure. The aqueous layer was acidified with 1 M HCl to pH 4 before being extracted thrice with EtOAc. The combined organic layer was washed with brine, dried (MgSO4), and filtered. The solvent was evaporated under reduced pressure to yield the product (2.09 g, 6.6 mmol, 95%) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 4.54 (s, 1 H), 3.10 (m, 2 H), 2.34 (t, J = 7.4 Hz, 2 H), 1.63 (p, J = 7.4 Hz, 2 H), 1.51–1.40 (m, 11 H), 1.38–1.21 (m, 14 H). 13C NMR (101 MHz, CDCl3): δ = 179.28, 156.27, 79.12, 40.64, 33.98, 30.02, 29.44, 29.42, 29.34, 29.24, 29.18, 29.02, 28.44, 26.78, 24.69. LC-MS (ESI, 10-90): t R = 8.44 min; m/z = 315.60 [M + H]+.
- 28 Preparation of 43: To a stirred solution of 39 (2.09 g, 6.6 mmol, 1.3 equiv) in DCM (35 mL) was added EDC·HCl (0.99 g, 5.2 mmol, 1 equiv) and N-hydroxysuccinimide (1.66 g, 14.4 mmol, 2.8 equiv). After stirring at r.t. for 3 days, the reaction was quenched with sat. NH4Cl (aq). The layers were separated and the aqueous layer was extracted once with DCM. The combined organic layer was dried (MgSO4), filtered, and the solvent evaporated under reduced pressure. The crude product was purified with flash column chromatography (SiO2, 10–40% EtOAc in pentane) to yield the product (0.88 g, 2.1 mmol, 40%) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 4.54 (s, 1 H), 3.12 (q, J = 6.8 Hz, 2 H), 2.86 (d, J = 4.3 Hz, 4 H), 2.62 (t, J = 7.5 Hz, 2 H), 1.76 (p, J = 7.4 Hz, 2 H), 1.50–1.42 (m, 11 H), 1.35–1.25 (m, 14 H). 13C NMR (101 MHz, CDCl3): δ = 169.34, 168.83, 165.27, 156.11, 79.12, 40.76, 31.07, 30.18, 29.61, 29.54, 29.41, 29.39, 29.17, 28.88, 28.56, 26.92, 25.72, 24.69. LC-MS (ESI, 10-90): t R = 8.87 min; m/z = 412.60 [M]+.
- 29 General preparation of Key Intermediates KI (4–18): A mixture of 1, 2 or 3 (1 equiv), Et3N (6 equiv) and O-Su ester (42, 43, 44, 45 or 51, 1 equiv) in DCM (0.3 M) was stirred at r.t. for 1–3 h. The reaction mixture was diluted with H2O and DCM. The layers were separated and the aqueous layer was extracted thrice with DCM. The combined organic layer was washed with H2O and brine, dried (MgSO4), filtered, and the solvent evaporated under reduced pressure. The crude product was purified using preparative HPLC and freeze-dried twice.
- 30 Preparation of 5: Compound 5 was synthesized according to general preparation KI29 using 1 (92.0 mg, 21.0 μmol, 1 equiv) and 43 (87.0 mg, 21.0 μmol, 1 equiv). The product was obtained as a white solid (75.2 mg, 0.10 mmol, 49%). 1H NMR (400 MHz, CDCl3): δ = 7.97 (d, J = 8.0 Hz, 2 H), 7.63 (dd, J = 8.6, 3.5 Hz, 1 H), 7.43 (t, J = 8.8 Hz, 1 H), 7.34 (d, J = 8.0 Hz, 2 H), 6.00 (d, J = 8.1 Hz, 1 H), 4.62 (s, 2 H), 4.54 (s, 1 H), 4.39–4.24 (m, 1 H), 3.86 (d, J = 2.5 Hz, 2 H), 3.76 (q, J = 17.3 Hz, 2 H), 3.64–3.51 (m, 2 H), 3.09 (q, J = 6.7 Hz, 2 H), 2.62 (s, 4 H), 2.11 (t, J = 7.6 Hz, 2 H), 1.66–1.54 (m, 6 H), 1.44 (s, 13 H), 1.34–1.23 (m, 14 H), 1.19 (d, J = 6.7 Hz, 3 H). 13C NMR (101 MHz, CDCl3): δ = 173.25, 170.39, 158.01 (d, J = 258.3 Hz), 157.26, 151.82, 138.76, 135.80, 128.55, 127.65, 123.75 (d, J = 20.2 Hz), 120.46, 77.48, 77.16, 76.84, 58.34, 54.31, 49.22, 46.60, 45.07, 44.23, 40.77, 37.03, 30.19, 29.64, 29.58, 29.47, 29.41, 28.57, 26.93, 25.95, 25.70, 24.19, 18.45. LC-MS (ESI, 10-90): t R = 7.10 min; m/z = 737.33 [M + H]+. HRMS: m/z calcd for [C41H61FN6O5 + H]+: 737.47602; found: 737.47562.
- 31 Preparation of 52: A mixture of 5 (435 mg, 0.6 mmol, 1 equiv) and TFA (5 mL, 64.9 mmol, 110 equiv) in DCM (10 mL) was stirred at r.t. for 2 h. The volatile compounds were evaporated under reduced pressure. The crude was re-dissolved in DCM and 1 M (aq) NaOH was added until pH 10. The layers were separated and the aqueous layer extracted thrice with chloroform. The combined organic layer was dried (MgSO4), filtered and the solvent evaporated under reduced pressure to yield the product (376 mg, 0.59 mmol, quant.) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ = 7.94 (d, J = 8.3 Hz, 2 H), 7.60 (dd, J = 8.6, 3.5 Hz, 1 H), 7.41 (t, J = 8.8 Hz, 1 H), 7.33 (d, J = 8.3 Hz, 2 H), 6.09 (d, J = 8.1 Hz, 1 H), 4.60 (s, 2 H), 4.35–4.25 (m, 1 H), 3.81 (d, J = 2.6 Hz, 2 H), 3.75 (q, J = 17.5 Hz, 2 H), 3.62–3.49 (m, 2 H), 2.74 (t, J = 7.5 Hz, 2 H), 2.58 (s, 4 H), 2.10 (t, J = 7.5 Hz, 2 H), 1.59 (p, J = 5.6 Hz, 4 H), 1.56–1.46 (m, 2 H), 1.44–1.37 (m, 2 H), 1.29–1.18 (m, 16 H), 1.17 (d, J = 6.7 Hz, 3 H). 13C NMR (126 MHz, CDCl3): δ = 173.48, 170.46, 162.32, 162.05, 157.93 (d, J = 258.3 Hz), 157.21, 156.90, 151.95 (d, J = 5.0 Hz), 145.52 (d, J = 15.1 Hz), 138.63, 135.82, 128.52, 127.58, 123.78 (d, J = 20.2 Hz), 120.56 (d, J = 5.0 Hz), 58.29 (d, J = 2.5 Hz), 54.37, 53.54, 50.57, 49.19, 46.49, 44.78, 44.18, 40.98, 36.86, 30.40, 29.40, 29.36, 29.27, 29.24, 29.18, 28.92, 28.82, 26.63, 25.88, 25.60, 24.13, 18.27. LC-MS (ESI, 10-90): t R = 4.84 min; m/z = 637.53 [M + H]+.
- 32 Preparation of Cy5 Probe 22: To a stirred and cooled (0 °C) mixture of cyanine-5-carboxylic acid (23.4 g, 48.4 μmol, 1.1 equiv) in DMF (7 mL) was added HOBt (8.09 mg, 52.8 μmol, 1.2 equiv), 52 (26.94 mg, 42.4 μmol, 1 equiv), DIPEA (18.4 μL, 105.6 μmol, 2.5 equiv) and EDC. HCl (9.69 mg, 50.5 μmol, 1.2 equiv). After stirring overnight at r.t., H2O (7 mL) and EtOAc (7 mL) was added. The layers were separated and the aqueous layer extracted thrice with EtOAc. The combined organic layer was washed with sat (aq) NaHCO3, five times with H2O, and once with brine, dried (MgSO4), filtered, and the solvent evaporated under reduced pressure. The crude product was purified with preparative HPLC and freeze-dried twice to yield the product (50.4 mg, 45.8 μmol, quant.) as a blue solid. 1H NMR (400 MHz, CDCl3): δ = 8.02–7.88 (m, 3 H), 7.67 (t, J = 5.7 Hz, 1 H), 7.61 (dd, J = 8.6, 3.5 Hz, 1 H), 7.41 (t, J = 8.8 Hz, 1 H), 7.38–7.29 (m, 6 H), 7.20 (dt, J = 11.9, 7.4 Hz, 2 H), 7.08 (dd, J = 17.4, 7.9 Hz, 2 H), 6.91 (t, J = 12.5 Hz, 1 H), 6.56 (d, J = 13.7 Hz, 1 H), 6.27 (d, J = 13.5 Hz, 1 H), 6.12 (d, J = 8.0 Hz, 1 H), 4.60 (s, 2 H), 4.35–4.23 (m, 1 H), 4.07 (t, J = 7.7 Hz, 2 H), 3.81 (d, J = 2.6 Hz, 2 H), 3.73 (q, J = 17.4 Hz, 2 H), 3.64–3.50 (m, 4 H), 3.19 (q, J = 6.4 Hz, 2 H), 2.57 (s, 4 H), 2.35 (t, J = 7.2 Hz, 2 H), 2.09 (t, J = 7.8 Hz, 2 H), 1.88–1.73 (m, 2 H), 1.70 (s, 6 H), 1.69 (s, 6 H), 1.61–1.48 (m, 10 H), 1.39 (q, J = 6.2 Hz, 2 H), 1.29–1.19 (m, 18 H), 1.16 (d, J = 6.7 Hz, 3 H). 13C NMR (101 MHz, CDCl3): δ = 173.51, 173.48, 173.25, 172.57, 170.39, 157.93 (d, J = 258.0 Hz), 157.22, 154.02, 152.97, 151.77 (d, J = 4.9 Hz), 145.79 (d, J = 15.0 Hz), 142.94, 142.03, 141.33, 140.76, 138.70, 135.81, 128.88, 128.73, 128.53, 127.59, 126.90, 125.49, 124.94, 123.69 (d, J = 20.4 Hz), 122.28, 122.20, 120.38 (d, J = 4.2 Hz), 111.03, 110.22, 104.93, 103.65, 58.55 (d, J = 3.1 Hz), 54.36, 49.50, 49.20, 49.05, 46.53, 45.01, 44.71, 44.16, 39.67, 36.96, 36.21, 29.80, 29.70, 29.65, 29.62, 29.54, 29.45, 29.40, 29.35, 28.16, 27.23, 27.10, 26.51, 26.06, 25.67, 25.34, 24.22, 18.39. LC-MS (ESI, 10-90): t R = 7.36 min; m/z = 1101.73 [M]+. HRMS: m/z calcd for [C68H90FN8O4 + H]+: 1101.70636; found: 1101.70696.