Discovery of a Cannabinoid CB₂ Receptor Fluorescent Probe Based on a Pyridin-2-yl-benzyl-imidazolidine-2,4-dione Scaffold

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.

Key words

cannabinoid CB2 receptor - fluorescent probes - LEI-102

Publication History

Received: 08 November 2023

Accepted after revision: 14 December 2023

Accepted Manuscript online:
14 December 2023

Article published online:
30 January 2024

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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.