3.1. Synthetic Procedures and Characterization

2,6-Pyridinedicarboxylic acid, 4-chloro-2,6-dimethyl ester (1). Chelidamic acid (15 g, 27.3 mmol, 1 eq) was dissolved in SOCl₂ (120 mL) in a three-necked round-bottom flask and anhydrous DMF (1 mL) was added under N₂ flow. The suspension was refluxed overnight. Upon completion the SOCl₂ was removed by distillation under reduced pressure and the solid was suspended in MeOH (120 mL) at 0 °C. The resulting mixture was refluxed overnight. After cooling down, the product was crystallized and collected by filtration through a Buchner funnel, and washed with cold MeOH. Pure product 1 was obtained as a white solid (13 g, yield = 83%). ¹H-NMR (400 MHz, CDCl₃) δ (ppm) = 8.28 (s, 2H, Py), 4.02 (s, 6H, Me).

2,6-Dimethyl 4-[2-ethoxy-1-(ethoxycarbonyl)-2-oxoethyl]-2,6-pyridinedicarboxylate (2). 2,6-Pyridinedicarboxylic acid, 4-chloro-2,6-dimethyl ester 1 (5 g, 21.8 mmol, 1 eq) was dissolved in dry DMSO (100 mL) under N₂ flow. Diethyl malonate (6.7 mL, 43.2 mmol, 2 eq) and Cs₂CO₃ (14.2 g, 43.6 mmol, 2 eq) were added and the resulting solution was stirred overnight at 100 °C. Then, the mixture was cooled down, diluted with water and acidified with HCl 1M until pH < 7. The aqueous solution was extracted with DCM and the organic phases washed with H₂O and brine until neutral pH. After drying over anhydrous Na₂SO₄, filtration and evaporation of the solvent, product 2 was obtained as a solid (6.8 g, yield = 88%) which was used in the next step without further purification. ¹H-NMR (400 MHz, CDCl₃) δ (ppm) = 8.64 (s, 2H), 4.22–4.15 (q, ³J = 7.05 Hz, 4H), 4.01 (s, 1H), 4.00 (s, 6H), 1.29–1.24 (t, ³J = 7.03 Hz, 6H). ¹³C-NMR (150 MHz, CDCl₃) δ (ppm) = 166.7, 163.9, 148.3, 137.15, 67.1, 61.5, 53.4, 14.0.

2,6-Pyridinedicarboxylic acid, 4-(2-methoxy-2-oxoethyl)-2,6-dimethyl ester (3). Compound 2 (6.8 g, 19.1 mmol, 1 eq) was suspended in 100 mL of MeOH, NaOH 1M (100 mL) was added and the resulting mixture was refluxed for 3 h. After cooling down and acidification with H₂SO₄ until pH = 1 (decarboxylation and re-esterification steps), the solution was stirred overnight at room temperature. Then, the solvent was evaporated and the solid was suspended in DCM and filtered through a Buchner funnel. The crude was purified by LC chromatography (80 g SiO₂ column, solid state sampling, Hex/EtOAc gradient elution), affording pure product 3 as a white solid (3 g, yield = 60%). ¹H-NMR (400 MHz, CDCl₃) δ (ppm) = 8.21 (s, 2H, Py), 3.99 (s, 6H, Me), 3.77 (s, 2H, -CH₂), 3.71 (s, 3H, Me). ¹³C-NMR (150 MHz, CDCl₃) δ (ppm) = 169.5, 164.9, 148.5, 145.8, 128.9, 53.2, 52.6, 40.1.

General procedure for the synthesis of 4 and 5. In a typical reaction, compound 3 (0.5 g, 1.9 mmol, 1 eq) was dissolved in 15 mL of DMSO dry under N₂ flow. An appropriate amount of salicylaldehyde (170 μL, 1 eq, for 4) or iodo-salicylaldehyde (232 mg, 1 eq, for 5) and piperidine (1 eq) were added, and the mixture was stirred at 90 °C until complete conversion (HPLC). Solution was cooled down, the product precipitated and then it was centrifugated and washed with HCl 1M and finally dried under reduced pressure. Pure products were obtained in excellent yields.

Compound 4. Yellow solid, yield = 80%. ¹H-NMR (400 MHz, d₆-DMSO) δ (ppm) = 8.72 (s, 1H, H-4), 8.66 (s, 2H, Py), 7.88–7.86 (d, ³J = 7.67 Hz, 1H, H-8), 7.73–7.69 (t, ³J = 7.34 Hz, 1H, H-7), 7.50–7.48 (d, ³J = 8.5 Hz, 1H, H-5), 7.46–7.41 (t, ³J = 7.66 Hz, 1H, H-6), 3.95 (s, 6H, -Me). ¹³C-NMR (150 MHz, d₆-DMSO) δ (ppm) = 265.0, 159.5, 153.9, 148.4, 145.5, 144.3, 133.6, 129.9, 127.1, 125.4, 123.1, 119.5, 116.5, 53.3.

Compound 5. Yellow solid, yield = 84%. ¹H-NMR (400 MHz, d₆-DMSO) δ (ppm) = 8.64 (s, 3H, H-4 + Py), 8.24–8.23 (d, ³J = 2.07 Hz, 1H, H-5), 8.00–7.96 (dd, ³J = 1.93 Hz, 8.56 Hz, 1H, H-7), 7.34–7.31 (d, ³J = 8.59 Hz, 1H, H-8), 3.95 (s, 6H, Me). ¹³C-NMR (150 MHz, d₆-DMSO) δ (ppm) = 164.9, 159.0, 153.6, 148.5, 145.2, 142.8, 141.5, 137.8, 127.1, 123.9, 121.7, 118.9, 88.9, 53.3.

General procedure for the synthesis of HL₁ and HL₂. In a typical reaction, diester 4 (100 mg, 0.29 mmol, 1 eq) or 5 (100 mg, 0.21 mmol, 1 eq) was dissolved in a 3:1 mixture of MeOH/H₂O. Solid NaOH (15 eq) was added and the mixture was refluxed for 2 h. Upon completion (HPLC), MeOH was evaporated and the solution was acidified with HCl 1M, causing the precipitation of the product. After centrifugation, the solid was washed with water and dried under reduced pressure. Pure products were obtained in good yields.

Compound HL₁. Yellow solid, yield = 90%. ¹H-NMR (400 MHz, triethylammonium salt in D₂O) δ (ppm) = 8.27 (s, 1H, H-4), 8.20 (s, 2H, Py), 7.70 (d, ³J = 7.57 Hz, ⁴J = 1.06 Hz, 1H, H-5), 7.61 (t, ³J = 7.75 Hz, ⁴J = 1.50 Hz, 1H, H-7), 7.38 (d, ³J = 7.49 Hz, 1H, H-8), 7.35(t, ³J = 7.57 Hz, 1H, H-6). ¹³C-NMR (150 MHz, triethylammonium salt in D₂O) δ (ppm) = 177.6, 172.9, 165.7, 153.5, 148.7, 134.6, 130.7, 127.9, 125.3, 125.1, 121.2, 119.7, 114.5. HRMS [M − H]⁻ m/z = 310.03571, calculated for C₁₆H₉NO₆ m/z = 311.04244. HPLC purity at 254 nm (triethylammonium salt) 98.5%, t_R = 5.69 min.

Compound HL₂. Yellow solid, yield = 60%. ¹H-NMR (400 MHz, d₆-DMSO) δ (ppm) = 8.64 (s, 1H, H-4), 8.61 (s, 2H, Py), 8.25–8.24 (d, ³J = 2.03 Hz, 1H, H-5), 7.99–7.96 (dd, ³J = 2.27 Hz, 8.80 Hz, 1H, H-7), 7.33–7.31 (d, ³J = 8.84 Hz, 1H, H-8). ¹³C-NMR (150 MHz, d₆-DMSO) δ (ppm) = 165.9, 159.1, 153.6, 149.1, 145.1, 142.6, 137.8, 126.7, 124.2, 121.8, 118.9, 88.9. HRMS [M − H]⁻ m/z = 435.93236, calculated for C₁₆H₈INO₆ m/z = 436.94018. HPLC purity at 254 nm (triethylammonium salt) 98%, t_R = 6.54 min.

Complex Na₃[Eu(L₁)₃]. Compound HL₁ (20 mg, 0.06 mmol, 1 eq) was dissolved in deionized water (30 mL) and NaOH 1M was added (130 mL, 2 eq), monitoring solution pH by litmus paper to be between 7 and 8. EuCl₃·6H₂O was added (5.5 mg, 0.02 mmol, 0.33 eq), the pH regulated to 7 and the mixture was stirred for 1h. The solution was then filtrated on a cellulose acetate filter (0.45 µm) and then concentrated to a volume of 1 mL and the precipitation of the complex was observed. After centrifugation, the solid was washed with water and dried under reduced pressure. The complex Na₃[Eu(L₁)₃] was obtained as pure product in good yield (15 mg, yield = 61%). ¹H-NMR (400 MHz, D₂O) δ (ppm) = 7.41 (t, ³J = 7.85 Hz, 3H, H-7), 7.29 (d, ³J = 6.62 Hz, 3H, H-8), 7.19 (t, ³J = 7.11 Hz, 3H, H-6), 7.14 (d, ³J = 8.50 Hz, 3H, H-5), 6.96 (s, 3H, H-4), 4.33 (s, 6H, Py). HRMS [M − Na]⁻ m/z = 1125.98462, calculated for C₄₈H₂₁EuN₃Na₂O₁₈⁻ m/z = 1125.98289. HPLC purity at 254 nm 96%, t_R = 5.80 min. Elemental analysis calculated (%) for C₄₈H₂₁EuN₃Na₃O₁₈ × 0.15 NaCl × 0.5 H₂O: C 50.19, H 1.84, N 3.66; found C 49.65, H 2.15, N 3.35.

Complex Na₃[Eu(L₂)₃]. Compound HL₂ (20 mg, 0.05 mmol, 3 eq) was dissolved in deionized water (20 mL) and NaOH 1M was added (92 mL, 2 eq), monitoring solution pH by litmus paper to be between 7 and 8. EuCl₃·6H₂O was added (5.6 mg, 0.02 mmol, 1 eq), the pH regulated to 7 and the mixture was stirred for 1h. The solution was then filtrated on a cellulose acetate filter (0.45 µm) and then concentrated to a volume of 1 mL and the precipitation of the complex was observed. After centrifugation, the solid was washed with water and dried under reduced pressure. The complex Na₃[Eu(L₂)₃] was obtained as pure product in good yield (12.5 mg, yield = 56%). ¹H-NMR (400 MHz, d₆-DMSO) δ (ppm) = 7.62 (s, 6H, Py), 7.60 (s, 3H, H-4), 6.93 (bs, 3H, H-8), 6.90 (bs, 3H, H-7), 6.89 (bs, 3H, H-5). HRMS [M − Na]⁻ m/z = 1503.67371, calculated for C₄₈H₁₈EuI₃N₃Na₂O₁₈⁻ m/z = 1503.67281. HPLC purity at 254 nm 95%, t_R = 6.70 min. Elemental analysis calculated (%) for C₄₈H₁₈EuI₃N₃Na₃O₁₈ × 0.45 NaCl × 0.3 H₂O: C 37.77, H 1.19, N 2.75; found C 37.22, H 1.43, N 2.46.