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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
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.
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The cannabinoid receptor type 2 (CB2R) is a class A G protein-coupled receptor (GPCR) with a role in inflammation and neurodegenerative diseases, making it an interesting target for drug discovery for multiple applications.[1] Confirming target engagement and understanding the biological cascades that lead to the therapeutic effect of a therapeutic agent are necessary for the successful translation of compounds into the clinic. To date, no CB2R-selective drugs have yet reached the market.[2] Fluorescent probes are ideal for detecting CB2R in relevant cell types, target engagement studies and to explore the roles of CB2R in healthy and pathological systems.[3] To this end, several fluorescent probes have been reported.[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] We have also contributed to this field and reported 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione LEI-121 (Figure [1]A) as a CB2R selective bifunctional probe that captured CB2R upon photoactivation.[15] An incorporated alkyne served as a ligation handle for the introduction of fluorescent reporter groups. LEI-121 enabled target engagement studies and visualization of endogenously expressed CB2R in HL-60 and primary human immune cells using flow cytometry.[15] However, LEI-121 is a two-step probe that requires copper-catalyzed azide-alkyne cycloaddition (click-reaction) with a fluorophore to visualize the receptor. For live imaging purposes, it would be beneficial to avoid the copper-mediated click reaction, which is toxic to cells, and to have a one-step fluorescent probe in which the fluorophore is incorporated into the structure of the ligand.
Previously, we have reported the three-dimensional structure of CB2R in complex with the 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione analogue LEI-102 using cryogenic electron microscopy (Figure [1]).[16] This provided an excellent opportunity for the structure-based design of novel one-step fluorescent CB2R probes. To validate this reasoning, we set out to design, synthesize, and characterize novel one-step fluorescent CB2R probes based on the 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione series.
Design
The cryo-EM structure of human CB2R in complex with LEI-102 was inspected to find the best exit vector on the scaffold for the introduction of a spacer that could be coupled to various fluorophores. The isobutyl and benzylic positions were positioned equally well to serve as an attachment point for spacer plus fluorophore. For synthetic reasons, the isobutyl position was chosen to introduce an optionally methyl substituted ethylamine (compounds 1–3) as attachment point for various alkyl (compounds 4–9) or polyethylene glycol (PEG) (compounds 10–18) spacers. The most promising scaffold-spacer compound (5) was coupled to four different fluorophores, i.e., BODIPY 493/503, BODIPY-TMR-X, DY480-XL, and Cy-5, resulting in compounds 19–22 (Scheme 1). BODIPY dyes have the advantages of high quantum yield, high molar extinction coefficients and brightness, as well as being relatively insensitive to the pH of their environment.[17] [18] Cy5 is frequently used and emits in the near-infrared range, which is not affected by biological auto-fluorescence, shows high stability, is moderately insensitive to solvent polarity, is highly water soluble, and has one of the highest molar extinction coefficients.[19–21] DY-480XL has a large Stokes shift, which increases the signal-to-noise by lowering the chance of self-quenching, self-absorption, and excitation source cross-talk.[22]
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Synthesis
The synthesis[23] of the CB2R-selective fluorescent probes based on LEI-102 19–22 started with the construction of the pyridinylbenzylimidazolidine-2,4-dione intermediates 1–18. Commercially available alaninol (d/l, Scheme [2]) was carbamate protected (23, 24), then subsequent iodination gave iodide derivatives 25 and 26, respectively. Meanwhile reductive amination of 4-bromobenzaldehyde and aminoacetamide led to compound 27. After cyclization, the formed imidazolidinedione 28 was alkylated with tert-butyl-(2-bromoethyl)carbamate, 25, or 26 which afforded compounds 29–31.[24] Next, oxidation of 6-bromo-3-fluoro-2-methylpyridine with m-CPBA followed by a Boekelheide rearrangement led to 32. After mesylation of the primary alcohol, the mesyl 33 was substituted with piperidine (34). Subsequently, borylation of 29–31 with pinacolboronic ester and conjugation to 34 via a Suzuki coupling gave 35–37,[25] and final acidolysis gained pharmacophores 1–3 (Scheme [2]).[26]
The various alkyl and ethyleneglycol based spacers (42–45, 51) were synthesized according to the generic synthesis depicted in Scheme [3]A/B. Alkyl spacers C8 and C12 were synthesized from 8-aminooctanoic acid and 12-aminododeanoic acid, respectively. The glycol spacers PEG2 and PEG3 were synthesized from 3-(2-(2-aminoethoxy)ethoxy)propanoic acid and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid, respectively. The amino acids were N-Boc protected (38–41)[27] and subsequently converted into the O-Su active esters 42–45.[28] The PEG4 glycol spacer required several additional steps (Scheme [3]B). Tosylation of tetraethylene glycol (46) was followed by azide substitution (47) of the sulfonate ester. This allowed a Michael addition to tert-butyl acrylate under TBAF conditions to give 48. Acidolysis of the ester to give 49 was followed by reduction of the azide and simultaneous N-Boc protection to give acid 50. The last step introduced the N-succinimide ester to give 51.
The three pharmacophores (1–3) were conjugated under basic conditions to the five spacers (42–45, 51) to yield 15 CB2R probe precursors 4–18 (Scheme [3]C).[29] The most potent of theses series, namely compound 5,[30] was N-Boc deprotected to give 52 [31] and thereafter conjugated to the four fluorophores (Scheme [3]D), affording the BODIPY 493/503 (19), BODIPY TMR-X (20), DY480-XL (21) and Cy5 (22)[32] LEI-102 based probes.
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Molecular Pharmacology
Compounds 1–22 were tested at 1 μM in a [3H]CP-55,940 radioligand displacement assay to determine their affinity for the CB2R and CB1R. Compounds with less than 50% displacement were considered inactive. The results are shown in Table [1].
a Binding affinities were determined either as displacement (%) or pK i using a [3H]CP-55,940 radioligand displacement assay on CHO cells stably over-expressing either hCB2R_bgal or hCB1R_bgal. The displacement percentages represent the percentage of radioligand displaced from the receptor at 1 μM compound. Total binding at vehicle concentration was set at 0%, while non-specific binding was set at 100%. Potency values (pEC50) were obtained for compounds with displacement ≥35% using a [35S]GTPγS assay on CHO cells stably over-expressing hCB2R_bgal. The maximum effect (E max in %) was normalized to reference full agonist CP-55,940. All values are presented as the mean ± SEM of at least two independent experiments performed in triplicate. n.d. not determined.
Compound 5 was the only scaffold that was active on hCB2R (63% ± 5 at 1 μM), but not hCB1R (pK i <5), therefore the four fluorophores were only conjugated to this scaffold. Subsequently, the binding affinity (pK i), potency (pEC50) and efficacy (E max) of compounds 19–22 was determined in a functional [35S]GTPγS assay (Table [1] and Figure [2]). Compound 22 displayed a pK i (CB2R) of 6.2 ± 0.6 and was inactive on CB1R. The other fluorescent probes were inactive at CB2R. In contrast to LEI-102 and the parent compound 1, compound 22 was an inverse agonist with a pEC50 of 5.3 ± 0.1 and E max of –63% ± 6. Previously, we have noted that very small structural changes in a compound may change the functional behavior of the CB2R.[15] It is not clear from a structural point of view what is the cause of this switch in functionality. We speculate that the alkyl linker may prevent the conformational change needed by one or more of the seven transmembrane helices in the binding site to activate the receptor.
A structure-based approach in combination with rational design was used to develop a one-step fluorescent probe for the cannabinoid CB2 receptor based on a pyridin-2-yl-benzyl-imidazolidine-2,4-dione scaffold. Ultimately probe 22 was synthesized with an alkyl spacer and Cy5 fluorescent dye. Probe 22 demonstrated reasonable CB2R affinity (pK i 6.2 ± 0.6), was selective over CB1R, and behaved as an inverse agonist (EC50 5.3 ± 0.1, E max –63% ± 6). It is envisioned that probe 22 can be used to visualize CB2R in cells without the need for copper-assisted click reactions.
Author Contributions
Design: A.P.A.J. (Antonius P.A. Janssen), A.H. (Asad Halimi), L.V.d.P. (Laura V. de Paus); Conducting Experiments, A.H., L.V.d.P.; Writing-Original Draft, L.V.d.P.; Writing Review & Editing, M.v.d.S., L.H.H. (Laura H. Heitman), R.J.B.H.N.v.d.B (Richard J. B. H. N. van den Berg).
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Conflict of Interest
The authors declare no conflict of interest.
Supporting Information
- Supporting information for this article is available online at https://doi.org/10.1055/a-2230-1003.
- Supporting Information
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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.
Corresponding Authors
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/)
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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.