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. 2011 Oct 13;54(19):6874-87.
doi: 10.1021/jm2008562. Epub 2011 Sep 16.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

Jillian G Baker  1 , Luke A Adams, Karolina Salchow, Shailesh N Mistry, Richard J Middleton, Stephen J Hill, Barrie Kellam
Affiliations
Free PMC article

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

Jillian G Baker et al. J Med Chem. .
Free PMC article

Abstract

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

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Figures

Figure 1
Figure 1
Previously reported fluorescent β-AR ligands.
Scheme 1
Scheme 1. Synthetic Route to BODIPY 630/650-X-Labeled Propranolol Derivatives
Reagents and conditions: (i) for 10a, (2S)-glycidyl-3-nitrobenzenesulfonate, NaH, DMF, 56%; for 10b, epichlorohydrin, K2CO3, 2-butanone, Δ. (ii) 11a–c, DMF/H2O (9:1), 85 °C, 16 h, 35–70%. (iii) Pd/C, H2, MeOH, rt, 3 h, quantitative. (iv) BODIPY-X-630/650-OSu, DMF, rt, 4 h. (v) Benzyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (15), DMF/H2O (9:1), 85 °C, 16 h, 35–67%.
Scheme 2
Scheme 2. Synthetic Route to BODIPY 630/650-X-Labeled Alprenolol Derivatives
Reagents and conditions: (i) for 20a, (2S)-glycidyl-3-nitrobenzenesulfonate, NaH, DMF, 56%; for 20b, epichlorohydrin, K2CO3, 2-butanone, Δ, 54%. (ii) tert-Butyl 2-aminoethylcarbamate, DMF/H2O (9:1), 85 °C, 16 h, 35–70%. (iii) 2 M HCl in dioxane, 89–94%. (iv) BODIPY-X-630/650-OSu, DMF, rt, 4 h, 68–98%. (v) tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, DMF/H2O (9:1), 85 °C, 16 h, 77%.
Scheme 3
Scheme 3. Synthetic Route to BODIPY 630/650-X-Labeled Pindolol Derivatives
Reagents and conditions: (i) epichlorohydrin, Cs2CO3, microwave (MW) 120 °C, 30 min, 97%. (ii) 11a, DIPEA, iPrOH/MeCN/H2O (7:2:1), MW 90 °C, 60 min, 48%. (iii) Pd/C, H2, MeOH, rt, 3 h, 88%. (iv) BODIPY-X-630/650-OSu, DMF, rt, 4 h. (v) Benzyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (15), iPrOH/MeCN/H2O (7:2:1), MW 90 °C, 60 min, 40%.
Figure 2
Figure 2
Fluorescent ligand displacement of 3H-CGP 12177 specific binding at the human β-AR in whole cells. Inhibition of 3H-CGP 12177 specific binding to whole cells by compounds 18a, 18b, 14c, and 14f in (a) β1 cells, (b) β2 cells, and (c) β3 cells. Nonspecific binding was determined by (a) 10 μM CGP 20712A and (b and c) 10 μM ICI 118551.(1) The concentrations of 3H-CGP 12177 present in each case are (a and b) 1.3 nM and (c) 18 nM. Data points are mean ± SE of triplicate determinations. These single experiments are representative of (a and b) six and (c) four separate experiments.
Figure 3
Figure 3
Fluorescent ligand 26a displacement of 3H-CGP 12177 specific binding at the human β-adrenoceptors in whole cells. Inhibition of 3H-CGP 12177 specific binding to whole cells by compound 26a in (a) β1 and β2 cells and (b) β3 cells. Nonspecific binding was determined by (a) 10 μM CGP 20712A (β1) and (b) 10 μM ICI 118551 (β2 and β3). The concentrations of 3H-CGP 12177 present in each case are (a) 1.7 nM and (b) 6.4 nM. Data points are mean ± SE of triplicate determinations. These single experiments are representative of (a) six and (b) five separate experiments.
Figure 4
Figure 4
Fluorescent ligand 18a is a competitive antagonist at all three human β-AR as measured via a whole cell reporter gene assay. CRE-SPAP production in the absence and presence of 10, 100, and 1000 nM compound 18a in (a) β1 cells following stimulation by cimaterol, (b) β1 cells following stimulation by CGP 12177, (c) β2 cells following stimulation by salbutamol, and (d) β3 cells following stimulation by fenoterol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline, and that in response to 10, 100, or 1000 nM compound 18a alone. Data points are mean ± SE of triplicate determinations. These single experiments are representative of (a) five, (b) seven, (c) 10, and (d) four separate experiments. The Schild slope for part (a) is 1.08.
Figure 5
Figure 5
Partial agonism effects of compound 26a are competitively antagonized at all three human β-AR. CRE-SPAP production in the absence and presence of 10, 100, and 1000 nM compound 26a in (a) β1 cells following stimulation by cimaterol, (b) β1 cells following stimulation by CGP 12177, (c) β2 cells following stimulation by salbutamol, and (d) β3 cells following stimulation by fenoterol. Bars represent basal CRE-SPAP production, that in response to 10 μM isoprenaline, and that in response to 10, 100, or 1000 nM compound 26a alone. Data points are mean ± SE of triplicate determinations. These single experiments are representative of (a) five, (b) eight, (c) four, and (d) five separate experiments.
Figure 6
Figure 6
Confocal visualization of the effect of ICI 118551 or CGP 20712A on the binding of the BODIPY-630/650 derivative of propranolol 18a to CHO K1 cells expressing the β2-AR. Antagonists were incubated with cells for 30 min prior to addition of 3nM 18a for 10 min. Imaging of cells was undertaken in the continued presence of fluorescent ligand. The lower right-hand panel shows the binding of 18a to control CHO-K1 cells.
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