Synthesis and Radiosynthesis of Prospective 2-Nitroimidazole Hypoxia­ PET Tracers via Thiazolidine Ligation with 5-Fluorodeoxyribose (FDR)

Abstract

The first prospective fluorinated PET tracers for imaging hypoxia obtained via thiazolidine-ligation are reported. Three 1,2-thiol-amine linkers were combined with four different 2-nitroimidazole spacers via amide or urea bond formation. The resulting compounds were submitted to thiazolidine-ring-forming ligation reaction with the fluorinated carbohydrate l-5-fluoro-5-deoxy-ribose (FDR), affording the desired candidate PET tracers in variable yields. The same ligation reactions performed on l-ribose – a by-product of [¹⁸F]FDR radiosynthesis – under conditions mimicking a radiochemical production showed that the fluorinated adducts can be efficiently purified and isolated by HPLC. Finally, one of the prospective hypoxia tracers was successfully produced in radiolabelled form in 29.2% radiochemical yield from [¹⁸F]FDR.

Key words

hypoxia - radiofluorination - thiazolidines - nitroimidazole - bio-orthogonal ligation

• References and Notes

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• 22 Synthesis of 4c: Hydrazine monohydrate (124 μL, 2.52 mmol) was added to a solution of 13 (340 mg, 0.84 mmol) in EtOH (5 mL) and the reaction mixture was heated at reflux for 3 h. After cooling to 0 °C the resulting white precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved with Et₂O (5 mL) and the resulting white precipitate was filtered, then the filtrate was concentrated under reduced pressure to afford 4c (448 mg, 98.4%) as a yellow oil. ¹H NMR (CDCl₃, 400 MHz): δ = 4.16 (br, 1 H), 3.00 (dd, J = 11.6, 6.1 Hz, 1 H), 2.65–2.54 (m, 2 H), 2.47 (d, J = 11.6 Hz, 1 H), 1.73–1.49 (m, 4 H), 1.60 (s, 6 H), 1.44–1.41 (m, 2 H), 1.33 (s, 9 H). ¹³C NMR (CDCl₃, 100 MHz): δ = 152.3, 79.8, 69.4, 64.1, 41.8, 31.3, 30.9, 30.2, 29.6 (2C), 28.4 (3C). MS (ESI): m/z calcd for C₁₃H₂₆N₂O₂S: 275.1 [M+H]⁺, 297.2 [M+Na]⁺ 303.2 [M+K]⁺; found: 275.2 [M+H]⁺, 297.2 [M+Na]⁺, 303.1 [M+K]⁺
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• 27 A shorter synthesis of 14d has been reported, see: Joyard Y., Azzouz R., Bischoff L., Papamicaël C., Labar D., Bol A., Bol V., Vera P., Grégoire V., Levacher V., Bohn P.; Bioorg. Med. Chem.; 2013, 21: 3680; and references therein. However, imidazolyl carbinol 24 was already being used in our labs for a related project, therefore it was used as an intermediate for 14d
• 28 Synthesis of 25a: DIPEA (156 μL, 0.92 mmol) and HATU (350 mg, 0.92 mmol) were added to a solution of 4a (200 mg, 0.77 mmol) in anhydrous CH₂Cl₂ (10 mL) at 0 °C and the mixture was allowed to react at r.t. for 1 h. Then the amino derivative 14a (260 mg, 1.53 mmol) dissolved in CH₂Cl₂ (2 mL) was added to the mixture. After 16 h under stirring, the mixture was washed with a 0.5 M aq. NaOH solution (3 × 6 mL) and then with a 0.1 M aq. HCl solution (3 × 6 mL), dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (Hex/EtOAc, from 8:2 to 7:3) to afford 25a (229 mg, 72.3%) as a yellow oil. ¹H NMR (CDCl₃, 400 MHz, mixture of rotamers): δ = 7.32 (s, 1 H), 7.08 (s, 1 H), 6.55 (br, 1 H), 4.72 (br, 1 H), 3.42–3.11 (m, 4 H), 1.82 (s, 3 H), 1.73 (s, 3 H), 1.42 (s, 9 H). ¹³C NMR (CDCl₃, 100 MHz): δ = 171.7, 153.3, 144.7, 128.4, 127.0, 81.8, 71.4, 67.5, 47.4, 36.0, 31.0, 29.3, 28.9, 28.4 (3C). MS (ESI): m/z calcd for C₁₇H₂₇N₅O₅S: 436.2 [M+Na]⁺, 452.0 [M+K]⁺; found: 436.1 [M+Na]⁺, 452.0 [M+K]⁺
• 29 Synthesis of 25f: A solution of 4c (152 mg, 0.56 mmol) in CH₂Cl₂ (2 mL) was added dropwise to a solution of CDI (90 mg, 0.56 mmol) in anhydrous CH₂Cl₂ (3 mL), at 0 °C under N₂ atmosphere, then the mixture was allowed to react at r.t. for 1 h. After 16 h under stirring, the mixture was added via syringe to a solution of 14a (226 mg, 1.33 mmol) in CH₂Cl₂ (3 mL) under N₂ atmosphere. After 16 h under stirring the mixture was concentrated under recued pressure. Purification by FC on silica gel (Hex/EtOAc, from 3:7 to 7:3) gave 25f (148 mg, 56.7%) as a yellow oil. ¹H NMR (CDCl₃, 400 MHz, 2 rotamers): δ = 7.35 (br, 1 H), 7.02 (br, 1 H), 5.55 (br, 2 H), 4.40 (t, J = 6.9 Hz, 2 H), 4.19 (br, 1 H), 3.24–2.94 (m, 5 H), 2.49 (d, J = 11.8 Hz, 1 H), 2.05–1.86 (m, 2 H), 1.81–1.66 (m, 2 H), 1.63 (s, 3 H), 1.61 (s, 3 H), 1.48–1.26 (m, 11 H). ¹³C NMR (CDCl₃, 100 MHz, 2 rotamers): δ = 159.0, 152.7, 144.6, 128.2, 127.1, 80.3, 69.4, 64.0, 47.8, 39.8, 36.6, 31.6, 31.4, 30.6, 30.1, 29.6, 28.4 (3C), 27.3. MS (ESI): m/z calcd for C₂₀H₃₄N₆O₅S: 471.2 [M+H]⁺, 493.2 [M+Na]⁺; found: 471.2 [M+H]⁺, 493.2 [M+Na]⁺
• 30 Synthesis of 3a: Compound 25a was dissolved in a TFA/H₂O/MeOH 3:2:1 mixture and heated to 65 °C for 2 h. Solvents were then concentrated under reduced pressure at 60 °C, then the residue was dissolved in ethanol and passed through a SiliaBond ^® carbonate pad to give the crude 3a as trifluoroacetate salt (55.7 mg, 74.8%). The compound was used in the next reaction without any further purification. ¹H NMR (CD₃OD, 400 MHz, in mixture with the dimer): δ = 7.62–7.54 (m, 1 H), 7.20–7.15 (m, 1 H), 4.60–4.42 (m, 2 H), 4.31–4.19 (m, 1 H), 3.52–3.33 (m, 2 H), 3.24–3.09 (m, 2 H), 2.19–2.00 (m, 2 H). ¹³C NMR (CD₃OD, 100 MHz, in mixture with the dimer): δ = 167.4, 144.6, 127.4, 127.2, 51.7, 47.4, 37.8, 36.3, 29.7. MS (ESI): m/z calcd for C₉H₁₅N₅O₃S: 274.1 [M+H]⁺, 296.1 [M+Na]⁺; found: 274.0 [M+H]⁺, 296.0 [M+Na]⁺
• 31 Synthesis of 1a: [¹⁹F]FDR ([¹⁹F]2) (5 mg, 0.033 mmol) was added to a solution of 3a (32.0 mg, 0.083 mmol) and DTT (12.8 mg, 0.083 mmol) in a 1 M sodium acetate buffer solution (pH 4.5), then the mixture was allowed to react at 30 °C for 20 min. Purification by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm; mobile phase: A (H₂O + 0.05% TFA), B (ACN + 0.05% TFA); gradient: from 5% B to 6% B in 15 min; flow: 5 mL min⁻¹; t_R : 12.5 min) gave 1a as trifluoroacetate salt (10.6 mg, 61.3 %). NMR analyses were performed after treatment of 1a with SiliaBond ^® carbonate (10% w/w) in EtOH, under gentle stirring for 1 h in order to freebase trifluoroacetate salt. ¹H NMR (CD₃OD, 400 MHz, – four diastereoisomers – two major isomer in ~3:2 ratio were identified): δ = 7.55 (d, J = 1.2 Hz, 1 H), 7.16 (d, J = 1.2 Hz, 1 H), 4.89–4.83 (m, 1 H), 4.62–4.42 (m, 4 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.09–4.04 (m, 1 H), 3.91 (dd, J = 7.4, 4.6 Hz, 1 H), 3.66 (dd, J = 7.4, 5.8 Hz, 1 H), 3.39–3.23 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H); δ (second isomer) = 7.57 (d, J = 1.2 Hz, 1 H), 7.17 (d, J = 1.2 Hz, 1 H), 4.92 (d, J = 2.6 Hz, 1 H), 4.67 (dd, J = 9.8, 3.0 Hz, 1 H), 4.63–4.42 (m, 3 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.04–3.95 (m, 1 H), 3.85–3.75 (m, 2 H), 3.39–3.14 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H). ¹³C NMR (CD₃OD, 100 MHz, – four diastereoisomers – two major isomers in ~3:2 ratio were identified): δ (first isomer) = 172.5, 144.7, 127.2, 127.1, 84.1 (d, J _CF = 167 Hz), 73.6 (d, J _CF = 7 Hz), 72.3, 72.1, 71.8 (d, J _CF = 18 Hz), 71.4, 65.8, 35.8, 34.9, 30.0; δ (second isomer) = 172.4, 144.7, 127.2, 127.1, 84.3 (d, J _CF = 167 Hz), 73.6 (d, J _CF = 7 Hz), 72.3, 72.0, 71.9, 71.9 (d, J _CF = 18 Hz), 70.2, 66.2, 36.6, 34.9. 29.9. ¹⁹F NMR (376 MHz, CD₃OD): δ (first isomer) = –233.0 (dt, J = 48.0, 22.7 Hz); δ (second isomer) = –233.6 (dt, J ₁ = 48.0 Hz, J ₂ = 22.3 Hz); MS (ESI): m/z calcd for: C₁₄H₂₂FN₅O₆S: 408.1 [M+H]⁺, 430.1 [M+Na]⁺; found: 408.0 [M+H]⁺, 430.0 [M+Na]⁺
• 32 Optimised radiosynthesis of [¹⁸F]1a: A solution of sodium acetate buffer (6 M, pH 4.5) was added to a solution of 3a (2.5 mg, 6.4 μmol), DTT (1 mg, 6.4 μmol) and [¹⁸F]FDR ([¹⁸F]2) (2.5–10 MBq) in 0.5–1.0 mL of H₂O to form a 70% v/v sodium acetate buffer solution (final concentration 4.2 M). After ~20 min the mixture was purified by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm) to give [¹⁸F]1a in 29% RCY (decay corrected)