Fluorescent A_2A and A₃ adenosine receptor antagonists as flow cytometry probes

Abstract

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A_2A and A₃ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A_2AAR were K_i 144–316 nM for 10, 12, and 19, and at A₃AR affinity of K_i 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A_2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A₃AR characterization in whole cells by flow cytometry (K_d 11.8 nM), and its bitopic interaction mode with an A₃AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A_2AAR, ~ 50-fold vs. A₁AR and A_2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A₃AR.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11302-022-09873-3.

Introduction

Fluorescent ligands of the adenosine receptor (AR) family have provided insights into the orthosteric and allosteric ligand binding sites and receptor dimerization that conventional radioligands could not achieve [1–3]. The first high affinity fluorescent ligand of an adenosine receptor 1 (Fig. 1) was for the A_2AAR, based on the ability of the terminal carboxylate group of selective agonist CGS21680 to be extended through amides without loss of binding affinity [4]. Other agonist fluorescent ligands in the same structural class, such as 2, were used to detect A_2AAR heterodimerization with the D₂ dopamine receptor [5]. A fluorescent N⁶-substituted A₃AR agonist, 3, was used to characterize receptor-receptor interactions in A₃AR homodimers [6]. Antagonist fluorescent ligands have been reported for the A₁AR, A_2AAR, A_2BAR, and A₃AR [7–14]. These probes, such as triazolo[1,5-c]quinazolin-5-yl)amine MRS5449 5, facilitate drug screening using flow cytometry of whole cells and other techniques to discover novel antagonists [8]. The bitopic nature of these fluorescent-tethered ligands often causes deviation from the simple bimolecular interaction between ligand and target receptor, as was shown for various A_2AAR antagonists [13]. The binding of the fluorophore moiety can allosterically modulate the pharmacophore affinity at the orthosteric site, depending on precise distal modification of the functionalized chain. For example, xanthine derivative XAC-X-BY630 (CA200634) 4 and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative MRS7416 6 each displayed negative cooperativity in A_2AAR binding [13]. A covalently binding fluorescent antagonist derived from A_2AAR antagonist ZM241385 was recently reported [15].

Fig. 1.

Structures of six reported fluorescent agonists (1–3) and antagonists (4–6) for the ARs

Since AR ligands are in development for various metabolic, neurological, inflammatory, and malignant conditions [16–21], additional AR fluorescent ligands are needed to expand the range of tool compounds for drug discovery. Here we conjugated known fluorophores to two known AR antagonist chemotypes, i.e., 1,3-dipropylxanthines aryl-functionalized at the C8 position and triazolo[1,5-c]quinazolin-5-yl)amines functionalized through acylation at the N⁵ position [8, 22, 23]. These versatile scaffolds can be directed toward multiple AR subtypes based on their functionalization, and also dependent on the species being studied [24]. For example, 1,3-dialkyl-8-phenylxanthines, although originally reported as selective rat A₁AR antagonists [22], have been modified to achieve selectivity for the human (h) A_2AAR, A_2BAR, or A₃AR [24]. N-(2-Aminoethyl)-2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)phenoxy)acetamide (xanthine amine congener, XAC, 7, Scheme 1) was designed to serve as a functionalized congener for coupling to reporter groups and carrier moieties [22] and served as a precursor here. A triazolo[1,5-c]quinazolin-5-yl)amine CGS15943 8 (Scheme 2) binds to both A_2AAR and A₃AR and when acylated at the N⁵ position can be highly selective for the hA₃AR (Scheme 2) [24]. In this study, we focus on the hA_2AAR and hA₃AR, using a wide range of tethered fluorophores, including the AlexaFluor (AF), JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes [25, 26]. JF dyes, which are useful for super resolution microscopy and live cell imaging, do not photobleach as easily as other dyes, and some can be used for fluorescence detection of target protein binding without removing the supernatant [25]. The FNIR-Tag fluorophore was recently reported by Schnermann and coworkers as a tag for in vivo detection of NIR fluorescence [26].

Scheme 1.

Synthesis of XAC-fluorophore conjugates. Reagents and conditions: (a) functionalized fluorophore active ester (TFP or NHS), DMSO, DIPEA/TEA, rt in dark/wrapped in Al foil, 3–18 h, 40% to quantitative yields. Compound 9 was prepared as reported [13]

Scheme 2.

Synthesis of CGS15943-derived fluorescent conjugate 19 by two routes. Reagents and conditions: (a) 6-azidohexan-1-amine, DMF, sodium ascorbate, CuSO₄.5H₂O, rt, 18 h, 6%; (b) DMSO, triethylamine, Sulfo-Cy7-NHS ester 20, rt, 4 h, 24%. (c) 6-azidohexan-1-amine, DMF, rt, 4 h; (d) DMF, sodium ascorbate, CuSO₄.5H₂O, rt, 18 h, 5.3%. Compound 17 was prepared as reported [8]

Materials and methods

Chemistry

Materials and methods

AF-dyes were purchased from Thermo Fisher Scientific (New York, USA); CGS15943 and JF dyes were from Tocris (Bio-techne, Minneapolis, MN, USA). Cyanine dyes were purchased from Lumiprobe (Cockeysville, MD, USA). All other chemicals and solvents were from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous solvents were obtained directly from commercial sources. All reactions were carried out under argon atmosphere using anhydrous solvents. Room temperature or rt refers to 25 ± 2 °C. NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts are given in ppm (δ), calibrated to the residual solvent or TMS signals for hydrogen, carbon, and internally calibrated by solvent frequency for other nuclei (MestReNova 10.0.2). Exact mass measurements were performed on a proteomics optimized Q-TOF-2 (Micromass, Waters, Milford, MA, USA) mass spectrometer equipped with a standard electrospray ionization (ESI) and modular LockSpray TM interface. The RP-HPLC was performed using Luna 5 µm C18(2)100A, AXIA, 21.2 × 250 mm column (Phenomenex, Torrance, CA, USA). Purity was determined using C18-XDB, 5 µm, 4.6 × 250 mm column (Agilent, Santa Clara, CA, USA), and a 0 → 100% linear gradient of acetonitrile/10 mM triethylammonium acetate (TEAA) as mobile phase at flow rate of 1.0 mL/min. Purity of all the tested compounds was > 95% at 254 nm and/or the respective absorption wavelength in nm, unless noted otherwise.

Radioligands [³H]cyclopentyl-1,3-dipropylxanthine ([³H]DPCPX, 164 Ci/mmol), [³H]-2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine ([³H]CGS21680, 30.5 Ci/mmol), and [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide ([¹²⁵I]I-AB-MECA, 2170 Ci/mmol) for A₁, A_2A, and A₃ receptors, respectively, were purchased from PerkinElmer (Waltham, MA, USA). DMEM medium and 1 M Tris–HCl (pH 7.5) were purchased from Mediatech, Inc. (Herndon, VA, USA). Adenosine deaminase was from Worthington Biochemical Corp. (Lakewood, NJ, USA). AR ligands 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) and CGS21680 were from Tocris (Ellisville, MO). XAC was synthesized at NIDDK, National Institutes of Health (Bethesda, MD). All other materials were from Sigma-Aldrich (St. Louis, MO, USA) or other standard commercial sources and of analytical grade. 6-Amino-N-(5-((3-((2-(2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)phenoxy)acetamido)ethyl)amino)-3-oxopropyl)amino)-5-oxopentyl)hexanamide (XAC245, 9) was synthesized in the TFA salt form, as described in Gao et al. [13].

General procedure for the synthesis of XAC-fluorophore analogues (10–16)

A stock solution of XAC245.TFA (9, 6.0 mg, 7.26 µmol) and DIPEA (4.0 µL, 21.79 µmol) in anhydrous DMSO (596 µL) was prepared. To the TFP (2,3,5,6-tetrafluorophenyl)/NHS (N-hydroxysuccinimide)-ester of the fluorophore (1.0 eq.) was added XAC245.TFA-DIPEA-DMSO stock solution (1.5 eq.) under argon atmosphere at room temperature. The reaction vial was covered with Al foil and stirred at room temperature for 18 h. The product was purified by C18-RP-HPLC.

10, XAC245-AF488 triethylamine salt

Reacting AF488-5-TFP ester (1.0 mg, 1.13 µmol) with XAC245 stock solution (150 µL, 1.70 µmol) gave the product as an orange solid (0.88 mg, 59%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 05/95 → 45/55 in 40 min, flow rate = 5.0 mL/min, R_t = 39.14 min). ¹H NMR (400 MHz, D₂O) δ 8.20 (d, J = 1.9 Hz, 1H), 7.92 (dd, J = 7.9, 1.9 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 7.9 Hz, 1H), 6.98 (d, J = 9.3 Hz, 2H), 6.96–6.91 (m, 2H), 6.77 (d, J = 9.3 Hz, 2H), 4.53 (s, 2H), 3.98 (t, J = 7.5 Hz, 2H), 3.87 (t, J = 7.7 Hz, 2H), 3.47–3.33 (m, 5H), 3.29 (t, J = 6.8 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.32 (t, J = 6.8 Hz, 2H), 2.21 (t, J = 7.0 Hz, 2H), 2.05 (t, J = 7.2 Hz, 2H), 1.62 (dp, J = 15.8, 7.7 Hz, 8H), 1.51–1.33 (m, 7H), 0.89 (td, J = 7.4, 4.1 Hz, 6H). HRMS m/z [M-H]⁻ for C₅₆H₆₅O₁₇N₁₁S₂ calculated 1226.3923, found 1226.3918.

11, XAC245-AF647 bis-triethylamine salt

Reacting AF647-NHS ester (1.0 mg, 0.80 µmol) with XAC245 stock solution (130 µL, 1.60 µmol) gave the product as a blue-violet solid (1.80 mg, quantitative, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 20/80 → 35/65 in 40 min, flow rate = 5.0 mL/min, R_t = 26.12 min). ¹H NMR (400 MHz, D₂O) δ 8.04–7.86 (m, 2H), 7.86–7.72 (m, 5H), 7.31 (d, J = 8.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.42 (t, J = 12.4 Hz, 1H), 6.24 (d, J = 13.5 Hz, 1H), 6.13 (d, J = 13.5 Hz, 1H), 4.57 (s, 2H), 4.18 (s, 2H), 4.12 (t, J = 7.9 Hz, 2H), 3.97 (t, J = 7.6 Hz, 2H), 3.82 (t, J = 7.7 Hz, 2H), 3.45–3.28 (m, 6H), 3.06–2.89 (m, 9H), 2.36 (t, J = 6.8 Hz, 2H), 2.17 (dt, J = 19.4, 7.4 Hz, 2H), 2.11–2.04 (m, 3H), 2.01 (t, J = 7.2 Hz, 2H), 1.68 (q, J = 7.6 Hz, 2H), 1.65–1.56 (m, 9H), 1.56–1.49 (m, 1H), 1.40 (dp, J = 21.8, 7.3 Hz, 4H), 1.14 (dt, J = 17.1, 8.0 Hz, 3H), 0.89 (dt, J = 18.6, 7.4 Hz, 6H). HRMS m/z [M-H]⁻ for C₇₁H₉₇O₂₀N₁₁S₄ calculated 1550.5716, found 1550.5736.

12, XAC245-JF646

Reacting JF646-NHS ester (1.0 mg, 1.70 µmol) with XAC245 stock solution (210 µL, 2.5 µmol) gave the product as a light blue solid (1.0 mg, 50%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 50/50 → 100/00 in 40 min, flow rate = 5.0 mL/min, R_t = 32.32 min). ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (t, J = 5.6 Hz, 1H), 8.17 (t, J = 5.6 Hz, 1H), 8.11–7.97 (m, 3H), 7.93 (t, J = 5.5 Hz, 1H), 7.77 (t, J = 5.7 Hz, 1H), 7.70 (t, J = 5.6 Hz, 1H), 7.66 (s, 1H), 7.01 (d, J = 8.3 Hz, 2H), 6.71 (d, J = 2.7 Hz, 2H), 6.62 (d, J = 8.7 Hz, 2H), 6.32 (dd, J = 8.7, 2.7 Hz, 2H), 4.50 (s, 2H), 3.98 (t, J = 7.3 Hz, 2H), 3.83 (q, J = 7.0 Hz, 10H), 3.18 (ddt, J = 18.0, 11.3, 6.3 Hz, 7H), 2.97 (q, J = 6.5 Hz, 2H), 2.42 (q, J = 7.1 Hz, 2H), 2.29 (p, J = 7.3 Hz, 4H), 2.20 (t, J = 7.0 Hz, 2H), 2.01 (td, J = 7.4, 2.4 Hz, 4H), 1.72 (q, J = 7.4 Hz, 2H), 1.55 (q, J = 7.4 Hz, 2H), 1.43 (dq, J = 14.5, 7.2 Hz, 5H), 1.36–1.15 (m, 8H), 0.97–0.79 (m, 9H), 0.60 (s, 3H), 0.49 (s, 3H). HRMS m/z [M + H]⁺ for C₆₄H₇₉O₁₀N₁₁Si calculated 1190.5859, found 1190.5859.

13, XAC245-JF549

Reacting JF549-NHS ester (1.15 mg, 2.10 µmol) with XAC245 stock solution (260 µL, 3.15 µmol) gave the product as a dark pink solid (0.83 mg, 40%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 05/95 → 95/05 in 40 min, flow rate = 5.0 mL/min, R_t = 30.10 min). ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.15 (d, J = 9.9 Hz, 2H), 8.07–7.89 (m, 3H), 7.78 (s, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 6.98 (s, 2H), 6.56 (s, 1H), 6.50 (d, J = 8.6 Hz, 1H), 6.21 (d, J = 2.2 Hz, 2H), 6.19–6.10 (m, 2H), 4.48 (s, 2H), 3.97 (s, 2H), 3.85 (t, J = 7.3 Hz, 9H), 3.67 (d, J = 11.0 Hz, 1H), 3.27–3.06 (m, 40H), 2.96 (d, J = 6.6 Hz, 2H), 2.42 (q, J = 7.1 Hz, 2H), 2.37–2.24 (m, 3H), 2.19 (t, J = 7.0 Hz, 2H), 2.00 (d, J = 6.5 Hz, 4H), 1.72 (d, J = 7.8 Hz, 2H), 1.54 (d, J = 7.1 Hz, 1H), 1.43 (s, 5H), 1.36–1.11 (m, 6H), 1.00–0.77 (m, 10H). HRMS m/z [M + H]⁺ for C₆₂ H₇₃N₁₁O₁₁ calculated 1148.5569, found 1148.5585.

14, XAC245-Sulfo-Cy7

Reacting Sulfo-Cy7-NHS ester (1.06 mg, 1.26 µmol) with XAC245 stock solution (156 µL, 1.90 µmol) gave the product as a blue solid (1.61 mg, 91%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 05/95 → 100/00 in 40 min, flow rate = 5.0 mL/min, R_t = 27.36 min). ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (s, 1H), 8.11–8.04 (m, 2H), 7.97 (d, J = 5.7 Hz, 1H), 7.83–7.66 (m, 4H), 7.62 (ddd, J = 9.7, 5.5, 2.7 Hz, 1H), 7.28 (dd, J = 13.1, 8.3 Hz, 1H), 7.09 (d, J = 8.9 Hz, 2H), 6.53 (s, 1H), 6.14 (dd, J = 14.2, 6.8 Hz, 2H), 4.54 (s, 2H), 4.10 (s, 2H), 4.01 (t, J = 7.3 Hz, 2H), 3.92–3.81 (m, 2H), 3.61 (s, 3H), 3.25–3.07 (m, 5H), 2.96 (q, J = 6.4 Hz, 4H), 2.20 (t, J = 7.1 Hz, 2H), 2.10–1.95 (m, 6H), 1.88–1.70 (m, 3H), 1.65 (d, J = 3.2 Hz, 10H), 1.56 (dq, J = 14.0, 7.2 Hz, 2H), 1.48–1.38 (m, 2H), 1.25–1.10 (m, 2H), 1.04 (q, J = 7.8, 7.3 Hz, 6H), 0.88 (dt, J = 11.3, 7.4 Hz, 6H). HRMS m/z [M + H]⁺ for C₇₂H₉₅O₁₄N₁₁S₂ calculated 1402.6580, found 1402.6597.

15, XAC245-Sulfo-Cy7.5

Reacting Sulfo-Cy7.5-NHS ester (1.4 mg, 1.20 µmol) with XAC245 stock solution (150 µL, 1.80 µmol) gave the product as a blue solid (1.52 mg, 69%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 05/95 → 45/55 in 40 min, flow rate = 5.0 mL/min, R_t = 38.58 min). ¹H NMR (400 MHz, D₂O) δ 8.72 (dd, J = 14.2, 9.1 Hz, 2H), 8.56 (d, J = 11.4 Hz, 2H), 8.31 (d, J = 8.4 Hz, 1H), 7.48 (dt, J = 30.3, 14.5 Hz, 3H), 7.34 (d, J = 8.2 Hz, 2H), 7.06 (s, 1H), 6.57 (d, J = 8.3 Hz, 2H), 5.79 (dd, J = 28.7, 13.8 Hz, 2H), 4.34 (s, 2H), 3.92–3.65 (m, 6H), 3.53–3.28 (m, 9H), 3.03 (t, J = 6.8 Hz, 2H), 2.73 (d, J = 12.4 Hz, 2H), 2.40 (t, J = 6.8 Hz, 2H), 2.27–2.04 (m, 8H), 1.96 (t, J = 7.5 Hz, 2H), 1.74 (s, 12H), 1.46 (s, 8H), 1.39–1.31 (m, 1H), 1.12 (s, 1H), 0.94 (d, J = 8.5 Hz, 1H), 0.80 (dt, J = 15.2, 7.2 Hz, 8H). HRMS m/z [M + H]⁺ for C₈₀H₉₇O₂₀N₁₁S₄ calculated 1660.5872, found 1660.5852.

16, XAC245-FNIR-Tag

Reacting FNIR-Tag-NHS ester (0.5 mg, 0.43 µmol) with XAC245 stock solution (55 µL, 0.65 µmol) gave the product as a green solid (0.53 mg, 70%, purified by RP-HPLC, linear gradient of CH₃CN-10 mM TEAA in H₂O (v/v) 05/95 → 65/35 in 40 min, flow rate = 5.0 mL/min, R_t = 36.35 min). ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (s, 1H), 7.95 (t, J = 13.0 Hz, 5H), 7.80 (s, 2H), 7.74 (t, J = 5.6 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.28 (d, J = 14.1 Hz, 2H), 4.46 (s, 2H), 4.36 (d, J = 6.7 Hz, 3H), 3.99 (d, J = 25.1 Hz, 3H), 3.80 (dt, J = 10.4, 6.3 Hz, 5H), 3.65 (s, 1H), 3.51 (dd, J = 5.9, 3.5 Hz, 4H), 3.22 (s, 4H), 3.18 (s, 3H), 3.10–3.02 (m, 1H), 2.97 (d, J = 6.2 Hz, 1H), 2.58 (s, 3H), 2.42 (q, J = 7.1 Hz, 14H), 2.21 (q, J = 7.1 Hz, 3H), 2.05–1.96 (m, 5H), 1.86 (s, 13H), 1.68 (s, 10H), 1.53 (d, J = 7.1 Hz, 1H), 1.43 (s, 4H), 1.34–1.16 (m, 5H), 0.93 (t, J = 7.1 Hz, 20H), 0.86 (dd, J = 14.1, 7.2 Hz, 6H). HRMS m/z [M + H]⁺ for C₈₈H₁₂₈O₂₁N₁₂S₂ calculated 1753.8837, found 1753.8864.

18, CGS15943-3T6-amine

To a suspension of compound 17 [8] (20 mg, 0.053 mmol) in DMF (1.0 mL) was added sequentially a freshly prepared solution of sodium ascorbate (1 M, 132 µL, 0.132 mmol), 6-azidohexane-1-amine [27] (15 mg, 0.106 mmol) and CuSO₄.5H₂O (1 M, 27 µL, 0.027 mmol) and the mixture stirred at room temperature for 18 h. The solvent was evaporated under high vacuum, and the residue was purified by silica-gel column chromatography to afford the 18 as a minor product (1.5 mg, 6%; R_f = 0.3, TLC eluent = 10% MeOH in CH₂Cl₂ + 1% Et₃N). ¹H NMR (400 MHz, DMSO) δ 8.16 (dd, J = 8.0, 2.6 Hz, 1H), 7.97–7.78 (m, 2H), 7.75–7.63 (m, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 3.8 Hz, 1H), 6.71 (s, 1H), 4.33–4.17 (m, 2H), 2.69 (t, J = 8.7 Hz, 2H), 2.63–2.51 (m, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.82–1.65 (m, 2H), 1.44–1.11 (m, 6H). HRMS m/z [M + H]⁺ for C₂₅H₂₈O₂N₉Cl calculated 522.2133, found 522.2134.

19, CGS15943-3T6-Sulfo-Cy7

First route

A mixture of compound 18 (1.5 mg, 2.87 µmol) and triethylamine (2.0 µL, 14.0 µmol) in anhydrous DMSO (150 µL) was added to Sulfo-Cy7-NHS ester 20 (1.0 mg, 1.20 µmol) under insert atmosphere and stirred at room temperature for 4 h. The product was purified immediately by RP-HPLC to afford compound 19 as a dark-blue solid (0.34 mg, 24%; purified using Agilent C18-XDB 4.6 × 250 mm column and linear gradient of CH₃CN/10 mM aq. TEAA (v/v) 20/80 → 55/45 in 20 min at 1.00 mL/min, R_t = 17.34 min). ¹H NMR (400 MHz, DMSO) δ 8.39 (s, 1H), 8.00 (s, 1H), 7.91 (s, 2H), 7.75 (s, 3H), 7.61–7.33 (m, 4H), 7.25–7.34 (m, 3H), 6.77 (s, 1H), 6.52 (s, 1H), 6.11–6.19 (m, 2H), 4.28 (t, J = 7.4 Hz, 2H), 4.09 (m, 2H), 3.61 (s, 3H), 2.94 (m, 6H), 2.67–2.77 (m, 4H), 1.91–2.02 (m, 4H), 1.78 (m, 2H), 1.65 (s, 8H), 1.52 (m, 2H), 1.13–1.31 (m, 6H), 1.12 (t, J = 7.1 Hz, 4H). HRMS m/z [M-H]⁻ for C₆₂H₇₀O₉N₁₁S₂Cl calculated 1210.4410, found 1210.4409. Purity—94.69% at 750 nm; 88.20% at 254 nm.

Second route

To a solution of compound 20 (100 mM, 13 µL, 0.013 mmol) in DMF was added 6-azidohexan-1-amine (100 mM, 15 µL, 0.015 mmol) and the mixture stirred at room temperature for 4 h. The reaction mixture was evaporated under a stream of nitrogen gas. Freshly prepared solution of sodium ascorbate (50 mM, 158 µL, 0.079 mmol), CuSO₄∙5H₂O (50 mM, 30 µL, 0.015 mmol), and compound 17 (100 mM, 32 µL, 0.032 mmol) were added and the mixture stirred at room temperature for 18 h. The product was purified by RP-HPLC, using the same conditions as above, to afford compound 19 as a dark-blue solid (1.96 mg, 5.3%).

To avoid freeze–thaw cycles, compound 19 was dissolved in EtOH and divided into small aliquots that were dried and stored at − 80 °C for use in binding assays. Radioligand binding was performed as described in Gao et al. [13].

Flow cytometry

Saturation assay: determination of dissociation constant

Flow cytometry was run using a CytoFLEX B4-RO-VO System (Beckman Coulter, Brea, CA, USA). The instrument includes 13 band pass filters which can be repositioned as needed, and it is available with different configurations to provide application flexibility. Violet side scatter resolution: (VSSC) < 200 nm blue laser wavelength: 488 nm; power: 50 mw; beam spot size: 5 × 80 μm; red laser wavelength: 638 nm; power: 50 mw; beam spot size: 5 × 80 μm; violet laser wavelength: 405 nm; power: 80 mw; beam spot size: 5 × 80 μm; flow cell dimensions: 420 μm × 180 μm internal diameter; signal processing: fully digital system with a 7-decade data display; carryover: single tube format < 1.0%; plate loader format < 0.5%.

HEK293 cells expressing either human A₃AR or human A_2AAR were plated in black 96-well plates overnight in complete DMEM media (DMEM, FBS (10%), penicillin/steptomycin (100 U/mL)). Media was then aspirated, and fresh complete media containing a range of concentrations of fluorescent ligand was added to appropriate cells. Non-specific binding was prepared through the addition of XAC (14 µM final concentration) at each concentration of fluorescent ligand. Cells were incubated for 1 h at 37 °C. Media was then aspirated, and cells were detached with non-enzymatic Cellstripper and resuspended in 200 µL PBS (without Ca²⁺, Mg²⁺). Cell fluorescence was measured on a CytoFLEX B4-RO-VO system. For the detection of 19 and 12, a 638-nm laser was employed and a 780/60 bandpass filter and a 660/20 bandpass filter were used, respectively. The data were analyzed using CellQuest Software. Specific binding was calculated by subtracting non-specific binding from total binding.

Competitive binding assay

For competitive binding experiments, chemically transfected HEK293 cells expressing either human A₃AR or human A_2AAR were plated in black 96-well plates overnight in complete DMEM media (DMEM, FBS (10%), penicillin/streptomycin (100 U/mL)). The media was then aspirated and increasing concentrations of ligand in complete DMEM media were incubated with 12 (250 nM) for hA_2AAR or 19 (100 nM) for hA₃AR expressing cells at 37 °C for 1 h. XAC (14 uM) was used to determine non-specific binding. Media was then aspirated, and cells were detached with non-enzymatic Cellstripper and resuspended in PBS (without Ca²⁺, Mg²⁺). Cell fluorescence was measured on a Beckman Coulter CytoFLEX B4-RO-VO system. For the detection of 19 and 12, a 638-nm laser was employed and a 780/60 bandpass filter and a 660/20 bandpass filter were used, respectively. The data were analyzed using CellQuest Software. Specific binding was calculated by subtracting non-specific binding from total binding. The inhibition constants (K_i), dissociation constants (K_d), and kinetic constants were calculated using Prism, version 9.0.0 (GraphPad, San Diego, CA).

Determination of off-rate

Chemically transfected HEK293 cells expressing human A₃AR were plated in black 96-well plates overnight in complete DMEM media (DMEM, FBS (10%), penicillin/streptomycin (100 U/mL)). Media was then aspirated, and fresh complete media containing a saturating concentration of 19 (400 nM) was added to each well. Cells were then incubated for 1 h at 37 °C. Media was then aspirated and PBS (without Ca²⁺, Mg²⁺) containing a saturating concentration of XAC (100 µM) was added to each well. Upon addition of XAC, data collection immediately began and continued for various timepoints using a Beckman Coulter CytoFLEX B4-RO-VO system with a 638-nm red laser and a 780/60 bandpass filter. Dissociation of 19 was followed by the change in mean fluorescence intensity (MFI) vs time. Data were analyzed using CellQuest Software.

The laser wavelengths of the longpass/bandpass filters used for the flow cytometry experiment were not optimal for compound 19. The λ_max of the Sulfo-Cy7 fluorophore of 19 is 750 nm. Therefore, the 638-nm laser used to excite 19 resulted in low fluorescence emission consistent with non-optimal excitation. However, the emission signal resulting from the 638-nm excitation was sufficient to obtain a signal.

Confocal microscopy

HEK293 cells transiently expressing human A_2AAR were plated in 8-well Ibibi µ-slides and grown overnight in complete DMEM media (DMEM, FBS (10%), containing penicillin/streptomycin (100 U/mL)). The media was removed, and total binding was determined by treatment of cells with 500 nM 12 in complete DMEM media. Non-specific binding was determined by cotreatment of cells with 500 nM 12 and 50 µM XAC. Cells were incubated with treatments for 1 h. Media was removed and DPBS (w/Ca²⁺ & Mg²⁺) was added.

Cell fluorescent imaging

A Zeiss LSM 700 confocal scanhead (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) mounted on a Zeiss AxioObserver.Z1 microscope, running ZEN 2.3 software, was used to simultaneously collect brightfield with fluorescence overlay images. The objective lens used was the Zeiss Plan-Apo 63 × /1.4 Oil DIC. The 639-nm laser was used for illumination/excitation, at 28.4% power. Emission was filtered by a 640-nm longpass filter. Imaging was set at a scan field of 203.12 µm × 203.12 µm with a pinhole of 61 µm, which corresponds to a confocal sectioning thickness of 1.0 µ.

Molecular modeling

The model of hA₃AR was retrieved from a previous work [28], where it was obtained through homology modeling using an antagonist-bound inactive structure of hA₁AR (PDB ID: 5UEN [29]) as a template, minimization of non-conserved residues and induced fit docking of the high affinity A₃AR antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide (MRS1220).

The structure was prepared with the Protein Preparation Wizard tool [30] (Schrödinger suite, Maestro 2021–2 [31], New York, NY, USA). The predicted tautomeric state of histidines was HIE (hydrogen at Nε nitrogen) for H79, H95, H124, H158 and H304, and HID (hydrogen at Nδ nitrogen) for H272.

Compound 19 was docked at the receptor orthosteric binding site using Glide-XP [32–34], employing a grid centered on the centroid of F168 and N250, and with inner and outer box dimensions of 10 Å and 46 Å, respectively. The conformational search was restricted constraining the scaffold of compound 9 to the reference pose of MRS1220, using the maximum common substructure option and an RMSD tolerance of 2 Å. A maximum of 20 poses was set as output, and a post-docking minimization phase was included. One pose was generated.

Results

Chemistry

To prepare various fluorescent compounds for this study, derivatives of XAC 7 and CGS15943 8 with a linker containing a primary amine group were prepared and coupled with respective chromophores (Schemes 1 and 2). XAC245-amine TFA salt 9 was synthesized by successive addition of N-Boc-amino acids as reported earlier [13]. The pharmacologically active XAC-fluorophore conjugates 10–16 were synthesized by the reaction of corresponding amines with activated fluorophore esters under basic conditions (Scheme 1). Cyanine dyes appear in compound 11 and NIR probes 14–16. The two JF dye conjugates containing azetidine moieties are 12 (JF649) and 13 (JF549). Compound 16 contains a net neutral heptamethine cyanine with a stable C4′-O-alkyl linker as reported [26]. This fluorophore has a λ_max of 765 nm, with emission at 788 nm. Sulfo-Cy7 conjugate 19 has a λ_max of ~ 750 nm, with emission at ~ 773 nm. The spectral characteristics of each fluorophore are given in Supporting Information.

For the synthesis of an 8-derived fluorescent compound 19, a click reaction between alkyne 17 [8] and an azide [27] gave the product 18 in low yields (Scheme 2). The reaction had unreacted starting material 17 and the major by-product 8, both of which were recovered. An amide bond formation by the amino group in compound 18 with activated carbonyl of Sulfo-Cy7-NHS ester 20 gave the desired product CGS15943-3T6-Sulfo-Cy7 19 in acceptable yields. A second, more efficient route involved the coupling of the fluorophore as an active ester 20 to 6-azidohexan-1-amine, followed by click reaction of the resulting azide 21 to the alkyne-functionalized pharmacophore 17. The overall yield of the second route (compound 19 compared to the amount of compound 17) was ~ tenfold higher than the first route.

Affinity determination

The AR affinity of the fluorescent derivatives was initially determined using standard radioligand binding methods [10, 13] in membranes of HEK293 cells overexpressing each receptor: A₁, A_2A, and A₃ (Table 1). The highest affinity was observed for the fluorescent conjugates 10, 12, and 19 at the A_2AAR (K_i 144–316 nM). The A₁AR affinity was generally low (K_i > 1 µM) and only percent inhibition is indicated. The A₃AR and A_2AAR affinities were consistently higher than at the A₁AR with the highest A₃AR affinity (K_i 21.6 nM) observed for 19. Compounds 10 and 13–15 were inactive at the A₃AR. Thus, compound 12 was highly selective for the A_2AAR compared to the A₁AR, but only 3.4-fold selective compared to A₃AR. Compounds 13–15 were > two-fold selective for the A_2AAR compared to the A₃AR. Binding affinity was measured for two compounds at the hA_2BAR, using [³H]8-cyclopentyl-1,3-dipropylxanthine ([³H]DPCPX) as radioligand in membranes of transfected HEK293 cells [28]. At a concentration of 1 µM, compounds 12 and 19 inhibited binding by only 57.1% and 38.5%, respectively. Thus, these two fluorescent ligands were selective for their respective target receptors compared to the A_2BAR. 

Table 1.

Fluorescent ligand binding affinities^a at three AR subtypes

No	Compound	hA1, Ki (nM) or % inhib. at 1 µM	hA2A Ki (nM) or % inhib. at 1 µM	hA3 Ki (nM) or % inhib. at 1 µM
XAC 7 and its conjugates
7	XACb	8.3 ± 1.1	32.1 ± 5.9	38.3 ± 6.7
9	XAC-245b	35%	198 ± 33	220 ± 36
10	XAC245-AF488	19%	316 ± 24	40 ± 3%
11	XAC245-AF647	18%	37 ± 21%	41 ± 5%
12	XAC245-JF646c	53%	144 ± 24	495 ± 89
13	XAC245-JF549	29%	882 ± 215	36.9 ± 4.3%
14	XAC245-Sulfo-Cy7 (750 nm)d	20%	1290 ± 470	49.7 ± 11.8%
15	XAC245-Sulfo-Cy7.5 (778 nm)d	27%	1120 ± 310	30.8 ± 3.5%
16	XAC245-FNIR-Tag (765 nm)d	27%	956 ± 232	232 ± 53
CGS15943 8 and its conjugates
8	CGS15943e	3.5	1.2	35
5	MRS5449e	87 ± 24	73 ± 8	6.4 ± 2.5
17	Alkyne deriv.e	170 ± 40	51 ± 10	33.5 ± 10.1
19	CGS15943-3T6-Sulfo-Cy7c	53%	244	21.6 ± 4.0

^aDetermined with membranes of HEK293 cell expressing each receptor and using the following radioligands (final concentration in incubation medium), unless noted: A₁, [³H]DPCPX (0.7 nM); A_2A, [³H]CGS21680 (8.1 nM); A₃, [.¹²⁵I]I-AB-MECA (0.15 nM). Results are expressed as mean ± SEM from at least 3 independent experiments

^bA_2A and A₃ AR affinities reported in Gao et al. [13]. A₁AR affinity of XAC (using [³H]R-PIA) is from Kecskés et al. [46]

^c12, MRS7774; 19, MRS7535

^dExcitation wavelength

^eAR affinities reported in Kozma et al. [8]

Application to flow cytometry and fluorescence microscopy

The objective was to determine the suitability of these fluorescent conjugates for use in binding assays by flow cytometry and in fluorescence microscopy. The conjugate with the highest A₃AR affinity was compound 19 , with a radioligand binding K_i value of 21.6 nM. The fluorophore of 19 was a NIR dye with a maximum excitation wavelength of 750 nm, which could be sufficiently exited with a red laser (638 nm) to obtain significant emission due to the high quantum yield (0.88) of the dye. A saturation experiment (Fig. 2) using human embryonic kidney (HEK) 293 cells overexpressing the A₃AR indicated an acceptably low level of non-specific binding (determined using 14 µM XAC). Non-specific binding was linear with respect to the fluorescent ligand concentration (up to 200 nM). The specific binding of 19 was saturable up to a concentration of 200 nM. A K_d value of 11.8 ± 0.7 nM was determined by nonlinear regression analysis using a one-site specific binding model (Eq. 1: y = B_max*x/(K_d + x). In non-AR-expressing control HEK293 cells, specific binding of 19 was absent. 

Fig. 2.

Representative saturation binding assay with 19 using FCM in HEK293 cells expressing hA₃AR (A, B) or non-transfected HEK293 cells (C, D), N = 2 for all experiments. Fluorescence intensity was measured using a Beckman Coulter CytoFLEX B4-RO-VO System with a 638-nm laser and a 780/60 bandpass filter. (A) Total binding of HEK293 cells expressing hA₃AR was determined by treating cells with a range of concentrations of 19, and non-specific binding was determined in the presence of XAC (14 µM). (B) Specific binding of 19 at HEK293 cells expressing hA₃AR, calculated as total binding MFI minus non-specific binding MFI. The dissociation constant (K_d) was calculated to be 11.8 ± 0.7 nM. Results reported as mean ± SEM. Note that the Y-axis in (B) has a different scale compared to other plots. (C) Total and nonspecific binding of 19 with non-transfected HEK293 cells. (D) Lack of specific binding of 19 with non-transfected HEK293 cells

Kinetic experiments showed the off-rate to be 0.0118 nM⁻¹ min⁻¹ (Fig. 3), while the on-rate calculated from binding saturation K_d and k_off was 0.334 min⁻¹ (N = 1).

Fig. 3.

Kinetic determination of k_off for 19 at hA₃AR in HEK293 cells expressing hA₃AR (N = 2). Cells were treated with a saturating concentration of 19 (400 nM) in complete DMEM media and incubated for 1 h at 37 °C. Media was removed, and PBS (without Ca²⁺, Mg²⁺) containing XAC (100 µM) was added to the cells. MFI was measured over time. Data were fit to a monophasic decay curve. Apparent k_off was measured to be 0.334 min⁻¹ (N = 1). Results reported as mean ± SEM

Fluorescent binding inhibition (Fig. 4) by known A₃AR agonist IB-MECA and AR antagonist XAC was performed using a fixed concentration of 19 (100 nM) resulting in sigmoidal inhibition curves with Hill coefficients ~ 1. This high concentration (~ 10X the K_d of 19) was needed to obtain an adequate signal. The K_i values of 71 and 171 nM for IB-MECA and XAC, respectively, were of the same rank order, but roughly an order of magnitude higher than radioligand binding K_i values obtained for the same ligands at the hA₃AR. Their reported K_i values using an A₃AR agonist radioligand were 1.8 and 13.8, respectively [8].

Fig. 4.

Competitive binding of 19 with a non-selective antagonist, XAC, and an A₃AR-selective agonist, IB-MECA, in HEK293 cells expressing hA₃AR determined using FCM. Fluorescence intensity was measured using a Beckman Coulter CytoFLEX B4-RO-VO System with a 638-nm laser and a 780/60 bandpass filter. A K_i of 171 ± 29 nM (N = 2) was calculated for XAC and 71 ± 17 nM (N = 2) for IB-MECA based on a K_d of 11.8 nM for 19 at hA_2AAR

A_2AAR flow cytometry (Fig. 5 and Fig. 6) and microscopic cell imaging data (Fig. 7) were determined using the conjugate 12 showing the highest A_2AAR affinity. A FCM binding saturation experiment demonstrated a K_d value of 222 nM at the hA_2AAR by Scatchard analysis, which is close to its binding K_i value in Table 1. Competition for A_2AAR binding by non-selective AR antagonist, XAC, and A_2AAR-selective agonist, CGS21680, was determined using FCM. The sigmoidal inhibition curves indicated K_i values of 53 and 783, respectively, compared to K_i values of 38.3 and 16.3 nM using an agonist radioligand [13]. Thus, the affinity determined for antagonist XAC was similar to previous values, but not the affinity of agonist CGS21680, using this antagonist fluorescent ligand. By comparison, the inhibition of the hA_2AAR binding in whole cells using fluorescent antagonist 6 by agonists was complex, possibly due to multiple affinity states of this GPCR for agonists [10]. The cell images (Fig. 7B, C) show specific binding on the outer cell membrane, which disappeared when 50 µM XAC was present to block the fluorescent binding (Fig. 7E, F). Some cells showed no binding, which is reasonable considering that a transient transfection of the HEK293 cells was used.

Fig. 5.

Representative saturation binding assay with 12 using FCM in HEK293 cells expressing hA_2AAR. Fluorescence intensity was measured using a Beckman Coulter CytoFLEX B4-RO-VO System with a 638-nm laser and a 660/20 bandpass filter. (A) Total binding of HEK293 cells expressing hA_2AAR was determined by treating cells with a range of concentrations of 12 and non-specific binding determined in the presence of XAC (14 µM). (B) Specific binding of 12 at HEK293 cells expressing hA_2AAR, calculated as total binding MFI minus non-specific binding MFI. The dissociation constant (K_d) was determined to be 222 ± 64 nM (N = 3)

Fig. 6.

Competitive binding of 12 with antagonist, XAC and A_2AAR-selective agonist, CGS21680 in HEK293 cells expressing hA_2AAR. Fluorescence intensity was measured using a Beckman Coulter CytoFLEX B4-RO-VO System with a 638-nm laser and a 660/20 bandpass filter. A K_i of 53 ± 6 nM (N = 2) was calculated for XAC and 783 ± 52 nM (N = 2) for CGS21680 based on a K_d of 222 nM for 12 at hA_2AAR

Fig. 7.

Confocal microscopy images of HEK293 cells transfected with hA_2AAR (transient). (A) and (D) are bright field images, (B) and (E) are bright field images with fluorescent overlay, and (C) and (F) are fluorescent images. The cells in images (A), (B), and (C) were treated with 12 (500 nM) for 1 h at 37 °C to measure the total binding of 12. The cells in images (D), (E), and (F) were treated with both 12 (500 nM) and XAC (50 µM) to measure the non-specific binding of 12

Computational studies

The hA₃AR interaction with antagonists was previously modeled using computational docking based on X-ray crystallographic structures of an antagonist-bound hA_2AAR (PDB ID: 4EIY) [35, 36]. Here, a hypothetical binding mode was predicted for compound 19 at hA₃AR orthosteric binding site using molecular docking based on a structurally similar template (Fig. 8). The structure of hA₃AR was obtained through homology modeling, using as template an antagonist-bound hA₁AR structure in the inactive state (PDB ID: 5UEN [29]), as recently reported [28]. The model was previously optimized by induced fit docking of the known hA₃AR antagonist MRS1220 (K_i ≈ 0.7 nM), which shares with compound 19 the 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amido scaffold [28]. The maximum common substructure between compound 19 and MRS1220 was used to constrain the docking search of compound 19. The predicted pose of the ligand interacts with F168 (EL2) through a π-π stacking interaction, with N250 (TM6) through a bidentate H-bond engaging N3 and the amido group at position 5. The carbonyl oxygen of the 5-amido group can be stabilized by a H-bond with Q167 on EL2, and further interactions of extracellular residues may involve the Sulfo-Cy7 group: in particular, in the predicted poses, the two sulfonic groups are interacting through a H-bond with the amido group of V259 on EL3 and through an ionic H-bond with the side chain of R173 in EL2. Since the linker connecting the fluorophore to the triazoloquinazoline scaffold is flexible and its interactions are solvent exposed, we expect them just to be transient and not to contribute extensively to the ligand stabilization. The flexibility and lack of stable interactions of a fluorophore-conjugated moiety is pointed out in a recently determined X-ray structure of a stabilized hA_2AAR bound to a BODIPY-conjugated pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine high affinity antagonist, which was the first X-ray structure of a GPCR bound to a fluorophore-conjugated ligand [37]. The chain linking the fluorophore to the N7-position of the pyrazolotriazolopyrimidine scaffold folds back upon the ELs of the receptor, pointing out of the orthosteric pocket, but the fluorophore could not be unambiguously solved because of the high flexibility of the linker. Instead, the triazolopyrimidine scaffold assumes the typical pattern of interactions at the hA_2AAR involving the bidentate hydrogen bond with N253 (TM6) and π-π interaction with F168(EL2), resembling the predicted binding mode of the triazolopyrimidine substructure of compound 19 at hA₃AR binding site.

Fig. 8.

Docking pose of compound 19 (C atoms in green) at the hA₃AR (gray) orthosteric binding site. Residues within 3 Å of the ligand are shown as sticks

Discussion

Since the first instance of fluorescence observed for a chemical compound, i.e., quinine in 1845, much of the innovation of new specialized fluorophores has been driven by the proven utility of fluorescent compounds as biomolecular labels, cellular stains, and mechanistic probes [25, 38–40]. Part of the impetus to develop high affinity fluorescent ligands for ARs also arose from the cost and regulatory restrictions associated with the use of radioligands. Widespread availability of robust fluorescent AR ligands would make this research more economical and accessible to researchers worldwide.

Currently, rhodamine dyes are one of the most useful classes of fluorescent small molecules due to their brightness, the ease by which a large library of spectrally diverse dyes can be made through structural modification, and their characteristic chemical transformation from the non-fluorescent lactone form to the fluorescent zwitterionic form upon binding to their molecular target [25, 40]. Upon fluorophore binding, the lactone-zwitterion equilibrium is shifted toward the fluorescent zwitterion form, such that bound ligand fluoresces while non-bound ligand remains colorless [40]. This property of modified rhodamine dyes potentially eliminates the need for washing excess ligand when conducting cell imaging and binding studies [40]. One such rhodamine fluorophore is JF646, which can be easily coupled via the NHS ester to a wide variety of agonists and antagonists for live cell imaging and pharmacological characterization of different cellular receptors [40].

Fluorescent A_2AAR antagonist 12, which contains the JF646 fluorophore, was effective as a high-affinity flow cytometry probe for the A_2AAR (K_d 222 ± 64 nM). The success of 12 in flow cytometric competitive binding assays with canonical antagonist XAC suggested that 12 is a suitable A_2AAR fluorescent ligand for antagonist drug screening. Competitive binding of 12 with agonist CGS21680 did follow monophasic, sigmoidal inhibition. Thus, agonist screening using 12 appears to be more complex than antagonist screening. The potential of 12 as an A_2AAR dye for fluorescent confocal microscopy was also confirmed, with 12 efficiently labeling the hA_2AAR in a specific manner. The selective activation of JF646 fluorescent activity only upon binding of the ligand to its molecular target, allowed for wash free imaging of hA_2AAR in live cells.

Fluorescent A₃AR antagonist 19 (K_d 11.8 ± 0.7 nM), containing the NIR Sulfo-Cy7, displayed high affinity and modest selectivity (11-fold vs. A_2AAR, roughly 50-fold vs. A₁AR and A_2BAR) and a good specific-to-nonspecific binding ratio. While 19 was demonstrated to be an effective flow cytometric probe for the A₃AR, the high excitation wavelength required to obtain a strong fluorescent emission signal presents a challenge since many flow cytometers are not equipped with far-red lasers. The greatest potential of 19 could be its application for in vivo imaging as a diagnostic NIR agent for imaging and/or therapy targeting the A₃AR [41, 42], although extensive pharmacokinetic characterization and additional control experiments would be required for in vivo studies. Near infrared light is particularly well suited for in vivo imaging because of the low levels of biomolecular absorption and general cell autofluorescence in this wavelength range [43]. NIR fluorophore-tagged GPCR ligands have been used for live-cell imaging experiments, for example, the human orexin type 2 receptor [44]. A Cy5.5 fluorophore conjugated to a peripherally selective, high affinity agonist of long half-life was used to image the oxytocin receptor in living mice [45].

Conclusions

We have introduced novel fluorescent conjugates of known AR pharmacophore functionalized congeners in two chemical series, 8-aryl-1,3-dialkylxanthines and triazolo[1,5-c]quinazolin-5-yl)amine. Among the conjugates synthesized and characterized are a JF probe 12, which binds and can be imaged at the A_2AAR in whole cells, and an A₃AR NIR antagonist probe 19, containing a Sulfo-Cy7 NIR dye, of even higher affinity, which can be used as a screening tool in flow cytometry for drug discovery. We also predicted a bitopic interaction mode of 19 with an A₃AR homology model.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AN

Acetonitrile

AR

Adenosine receptor

BY

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)

CGS15943

9-Chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine

CGS21680

2-[p-(2-Carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine

DIPEA

Diisopropylethylamine

DMEM

Dulbecco’s modified Eagle medium

DMF

N,N-Dimethylformamide

DPCPX

Cyclopentyl-1,3-dipropylxanthine

EL

Extracellular loop

IE

Interaction energy

FBS

Fetal bovine serum

GPCR

G protein-coupled receptor

HEK

Human embryonic kidney

I-AB-MECA

N⁶-(4-Amino-3-iodobenzyl)adenosine-5′-N-methyluronamide

MD

Molecular dynamics

MFI

Mean fluorescence intensity

NHS

N-Hydroxysuccinimide

NIR

Near infrared

RMSD

Root mean squared deviation

TEAA

Triethylammonium acetate

TFA

Trifluoroacetic acid

TFP

2,3,5,6-Tetrafluorophenyl

TM

Transmembrane helical domain

XAC

N-(2-aminoethyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)phenoxy]acetamide

ZM241385

4-[2-[7-Amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethyl]phenol

Kiran S. Toti,

was a post-doctoral fellow in Dr Jacobson’s research group at NIH, USA, from 2014-2020. He holds a PhD degree from Ghent University, Belgium, for the dissertation titled ‘Synthesis and Biological Evaluation of 4-Hydroxymethyl Deleted, Transposed and Modified Nucleosides’ under the guidance of Prof. Serge Van Calenbergh. Kiran has 10+ years medicinal chemistry research experience working on GPCR, kinase and polymerase small molecule modulators involving nucleosi(t)ides and heterocycles. At present, he is an Assoc. Scientist in the group of Prof. Dennis Liotta at the Chemistry Department, Emory University.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases, Intramural Research Program (grant no. ZIADK031117).

Data availability

The original data from this study will be made available upon reasonable request.

Declarations

Conflict of interest

Kiran S. Toti declares that he has no conflict of interest. Ryan G. Campbell declares that she has no conflict of interest. Hobin Lee declares that she has no conflict of interest. Veronica Salmaso declares that she has no conflict of interest. R. Rama Suresh declares that he has no conflict of interest. Zhan-Guo Gao declares that he has no conflict of interest. Kenneth A. Jacobson declares that he has no conflict of interest.

Ethical approval

Not applicable; no animal studies are included.

Consent to participate

Not applicable; no patient studies are included.