Synthesis of the Novel AT1 Receptor Tracer [18F]Fluoropyridine-Candesartan via Click Chemistry

Abstract

A novel 7-((4-(3-((2-[¹⁸F]fluoropyridin-3-yl)oxy)propyl)-1H-1,2,3-triazol-1-yl)methyl)-1H-benzo[d]imidazole derivative of the angiotensin II type-1 receptor (AT₁R) blocker candesartan, [¹⁸F]fluoropyridine-candesartan, was synthesized via the copper-catalyzed azide-alkyne cycloaddition click reaction between 2-[¹⁸F]fluoro-3-(pent-4-yn-1-yloxy)pyridine ([¹⁸F]FPyKYNE) and the tetrazole-protected azido-candesartan derivative, followed by acid deprotection. This three-step, two-pot, and two-step purification synthesis was done within 2 h. The use of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA) as a Cu(I) stabilizing agent increased the overall radiochemical yield by 4-fold (10 ± 2%, n = 13) compared to the reaction without THPTA (2.4 ± 0.2%, n = 3; decay-corrected from ¹⁸F produced at the end-of-beam). Complete separation of [¹⁸F]FPyKYNE from its nitro precursor and [¹⁸F]fluoropyridine-candesartan from the deprotected azido-candesartan allowed for high molar activities (>380 GBq/μmol) of the tracer. The use of 0.1% trifluoroacetic acid in water for reformulation and the addition of sodium ascorbate to the final formulation (1.6 ± 0.2 GBq/mL, n = 3) prevented tracer radiolysis with >97% radiochemical purity for a period of up to 10 h after the end-of-synthesis. A significant reduction in the uptake (86 ± 3%, n = 8) of the tracer was observed ex vivo in rats (at 20 min postinjection) in the AT₁R-rich kidney cortex following pretreatment with saturating doses of the AT₁R antagonist candesartan or losartan. This specific binding to AT₁R was confirmed in vitro in the rat renal cortex (autoradiography) by a reduction of 26 ± 5% (n = 12) with losartan coincubation (10 μM). These favorable binding properties support further studies to assess the potential of [¹⁸F]fluoropyridine-candesartan as a tracer for the positron emission tomography imaging of renal AT₁R.

Scheme 1. Synthesis of Precursors (2, 7, and 10) and Reference Compound (9)

Figure 1

Representative semipreparative HPLC chromatograms. (A) Silica Column: [¹⁸F]FPyKYNE ([¹⁸F]3) (t_R = 11 min) purification from 2 (t_R = 4 min, eluted with 70:30 dichloromethane (CH₂Cl₂)/isopropanol after switch , following [¹⁸F]3 collection). (B) C18 Column: [¹⁸F]fluoropyridine–candesartan ([¹⁸F]9) (t_R = 30 min) purification, [¹⁸F]3 (t_R = 17.5 min), 10 (t_R = 24 min), and 7 (t_R = 3.5 min, eluted with 100% EtOH after switch , following [¹⁸F]9 collection).

Scheme 2. Radiosynthesis of [¹⁸F]Fluoropyridine–Candesartan ([¹⁸F]9)

Figure 2

Representative analytical HPLC chromatograms. (A) [¹⁸F]Fluoropyridine–candesartan ([¹⁸F]9) formulation (t_R = 4.9 min). (B) Coinjection of the tracer [¹⁸F]9 (t_R = 5.13 min) with the nonradioactive standard 9 (t_R = 5.2 min).

Figure 3

Stability of [¹⁸F]fluoropyridine–candesartan at various radioactive concentration levels at EOS. Effect of using 0.1% TFA in water for reformulation (yellow ▲ 1.63 GBq/mL) and 10 mg/mL NaAsc/saline in the final formulation (blue ― 1.6 ± 0.2 GBq/mL, n = 3) compared to no radiolysis inhibitors (green ◇ 0.42 GBq/mL and red ■ 2.07 GBq/mL). (*) The plotted values correspond to mean ± SD (n = 3).

Figure 4

[¹⁸F]Fluoropyridine–candesartan relative uptake in the plasma, renal cortex, outer medulla, and heart by ex vivo biodistribution in rats at 20 min postinjection. Effect of AT₁R blocking with pre-injection (IV) of candesartan (10 mg/kg) or losartan (30 mg/kg) 20 min prior to tracer injection. Histogram represents mean ± SD (n = 3–4). (***) and (****) represent p values <0.001 and <0.0001, respectively.

Figure 5

[¹⁸F]Fluoropyridine–candesartan (0.74 and 1.3 nM) binding in the rat kidney cortex after 90 min incubation at room temperature in an autoradiography study. Effect of AT₁R blocking with 10 μM losartan coincubation. (A) Representative autoradiography images [(a,c), controls; (b,d), 10 μM losartan] with 0.74 and 1.3 nM [¹⁸F]9, respectively. (B) Histogram represents mean ± SD (n = 6 each group). (***) and (****) represent p values <0.001 and <0.0001, respectively.