A Multifunctional HBED-Type Chelator with Dual Conjugation Capabilities for Radiopharmaceutical Development

Abstract

Bifunctional HBED chelators are hexadentate complexing ligands (chelators) that tightly coordinate to trivalent gallium and, additionally, are able to bind to bioactive molecules. In nuclear medicine, HBED-based radiopharmaceuticals are used as powerful radiotracers for tumor imaging. Among variants of bifunctional HBED chelators, HBED-CC is the most well-known; it possesses two terminal carboxylic acid groups that are able to undergo bioconjugation by amide-bond formation. However, to permit bioconjugation through click coupling, we previously modified the structure of HBED-CC and introduced HBED-NN chelator bearing two azide functions. We have now combined the conjugation capabilities of HBED-CC and HBED-NN chelators in one molecule and have created HBED-NC, which possesses both azide and carboxylic acid groups. The advantage of HBED-NC is that it provides options for constructing either monomeric or heterodimeric radiolabeling precursors. This work describes the synthesis of HBED-NC by either of two pathways.

Key words

HBED chelators - click chemistry - azides - radiopharmaceuticals - radiotracers

• References and Notes

• 1 New address: K. Kopka, Helmholtz-Zentrum Dresden-Rossendorf e.V., Institute of Radiopharmaceutical Cancer Research, Bautzner Landstraße 400, 01328 Dresden, Germany.
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• 23 Compounds 1, 2, and 4 were prepared by the reported methods.^15,16 Characterization data for these compounds, general experimental methods, and all NMR spectra are provided in the Supporting Information.
• 24 2-{[(2-Aminoethyl)amino]methyl}-4-(2-azidoethyl)phenol (3) To a solution of compound 1 (1.0 g, 5.3 mmol, 1.0 equiv) in CH₂Cl₂ (5 mL) was added tert-butyl (2-aminoethyl)carbamate (1.0 g, 6.3 mmol, 1.2 equiv). The mixture was stirred for 1 h at r.t. then diluted with CH₂Cl₂ and washed with 0.5 M aq NaHSO₃. The organic phase was dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The resulting orange solid was dissolved in warm F₃CCH₂OH (40 mL) at 40–50 °C, then cooled in an ice bath. NaBH₄(0.47 g, 12.45 mmol, 2.5 equiv) was added in portions, and the mixture was allowed to warm to r.t. and stirred overnight under an inert atmosphere. The reaction was quenched with H₂O (100 mL) and the resulting mixture was extracted with CH₂Cl₂. The organic layer was dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was dissolved in a 4 M solution of HCl in 1,4-dioxane (10 mL) and the mixture was stirred for 5 min at r.t. Et₂O (40 mL) was added and the mixture was stirred for another 10 min. The precipitate was collected by filtration, washed with Et₂O, and dried under vacuum to give salt 3 as white solid; yield: 1.4 g (4.54 mmol, 87%). ¹H NMR (400.13 MHz, D₂O, 25 °C): δ = 7.29 (m, 2 H, CHCHCCH), 6.98 (d, ³ J _H–H = 8.3 Hz, 1 H, CHCHCCH), 4.33 (s, 2 H, ^BnCH ₂ ), 3.56 (t, ³ J _H–H = 6.7 Hz, 2 H, CH₂CH₂ N₃), 3.45 (m, 4 H, CH₂ CH₂ NH), 2.87 (t, ³ J _H–H = 6.7 Hz, 2 H, CH₂ CH₂N₃). ¹³C{¹H} NMR (100.61 MHz, D₂O, 25 °C): δ = 153.81 (COH), 132.06 (CHCHCCH), 130.92 (CHCHCCH), 116.88 (CCH₂N), 115.81 (CHCHCCH), 52.22 (CH₂N₃), 47.38 (^Bn CH₂), 43.44 (^BnCH₂NHCH₂), 35.39 (CH₂NH₂), 33.44 (CH₂CH₂N₃). HRMS (ESI⁺, MeOH): m/z [M + H]⁺ calcd for C₁₁H₁₈N₅O: 236.1506; found: 236.1539 (100%). (27) Methyl 3-(3-{[(2-{[5-(2-Azidoethyl)-2-hydroxybenzyl]amino}ethyl)amino]methyl}-4-hydroxyphenyl)propanoate (5) Method 1: To a mixture of compound 3 (0.34 g, 1.1 mmol, 1.1 equiv) and aldehyde 2 (0.21 g, 1.0 mmol, 1.9 equiv) in dry MeOH (6 mL) was added anhyd Et₃N (420 μL, 1.0 mmol, 3 equiv), and the mixture was stirred at r.t. for 30 min. The volatiles were then completely removed under reduced pressure, and the residue was dissolved in warm F₃CCH₂OH (8 mL) at 40–50 °C. The resulting solution was cooled in an ice bath and NaBH₄ (0.19 g, 5 mmol, 5 equiv) was then added in portions. The mixture was then allowed to warm to r.t. and stirred overnight under an inert atmosphere. The reaction was quenched with H₂O (~70 mL), and the mixture was extracted with CH₂Cl₂. The extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure to give product 5 in quantitative yield. The NMR spectrum of this product showed no significant impurities, and it was therefore used without further purification in the next step. However, for spectroscopic characterization a portion was purified by reverse-phase HPLC to give a colorless solid. Method 2: The same procedure as described in Method 1 was applied to aldehyde 1 (0.19 g, 1.0 mmol, 1 equiv) and salt 4 (0.36 g, 1.1 mmol, 1.1 equiv). Compound 5 was again obtained in quantitative yield. ¹H NMR (400.13 MHz, CDCl₃, 25 °C): δ = 7.00 (m, 2 H, COHCHCH), 6.79 (m, 4 H, CCHC and COHCHCH), 3.97 (d, 4 H, ^BnCH ₂ ), 3.66 (s, 3 H, CH₃ ), 3.44 (t, ³ J _H–H = 7.2 Hz, 2 H, CH₂CH₂ N₃), 2.80 (m, 8 H, NH–C₂ H₄ –NH, CH₂ CH₂CO₂ and CH₂ CH₂N₃), 2.57 (t, ³ J _H–H = 8.0 Hz, 2 H, CH₂CH₂ CO₂). ¹³C{¹H} NMR (100.61 MHz, CDCl₃, 25 °C): δ = 173.62 (CO₂CH₃), 156.91 (COH–ArCO₂ or COH–ArN₃), 156.42 (COH–ArCO₂ or COH–ArN₃), 131.31 (C–C₂H₄–CO₂), 129.25 (CCHC–C₂H₄–CO₂), 128.99 (CCHC–C₂H₄–N₃), 128.73 (CHCHC–C₂H₄–CO₂), 128.50 (CHCHC–C₂H₄–N₃), 122.36 (CCHC–C₂H₄–CO₂), 122.19 (CCHC–C₂H₄–N₃), 116.76 (CHCHC–C₂H₄–CO₂ or CHCHC–C₂H₄–N₃), 116.56 (CHCHC–C₂H₄–CO₂ or CHCHC–C₂H₄–N₃), 52.82 (CH₂ CH₂N₃), 52.66 (d, ^Bn CH₂), 51.62 (CO₂ CH₃), 48.02 (d, NHCH₂ CH₂NH), 36.12 (CH₂ CH₂CO₂), 34.60 (CH₂CH₂N₃), 30.16 (CH₂CH₂CO₂). HRMS (ESI⁺, MeCN–MeOH): m/z [M + H]⁺ Calcd for C₂₂H₃₀N₅O₄: 428.2292; Found: 428.2294 (59%). 3-[3-({[3-[[5-(2-Azidoethyl)-2-hydroxybenzyl](2-tert-butoxy-2-oxoethyl)amino]-1-(tert-butoxycarbonyl)propyl]amino}methyl)-4-hydroxyphenyl]propanoic Acid (HBED-NC) A mixture of compound 5 (0.89 g, 2.03 mmol, 1 equiv), Na₂CO₃ (0.86 g, 8.13 mmol, 4 equiv), and tert-butyl 2-bromoacetate (630 μL, 4.23 mmol, 2.1 equiv) in anhyd MeCN (40 mL) was stirred under reflux overnight. The mixture was cooled to r.t., filtered, and concentrated under reduced pressure. The residue was dissolved in warm MeOH (30 mL, ~50 °C), and 4 M aq NaOH (10 mL) was slowly added. The resulting mixture was stirred for 2 h then the pH was adjusted to 4–5 with 1 M aq HCl. The crude product was extracted with EtOAc and the extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The product was isolated by reverse-phase HPLC as a colorless solid; yield: 0.07 g (0.11 mmol, 5.4%) ¹H NMR (400.13 MHz, CDCl₃, 25 °C): δ = 7.00 (m, 2 H, COHCHCH), 6.76 (m, 4 H, CCHC, COHCHCH and COHCHCH), 3.69 (s, 2 H, ^BnCH₂ –ArN₃), 3.65 (s, 2 H, ^BnCH₂ –ArCO₂Me), 3.40 (t, ³ J _H–H = 7.2 Hz, 2 H, CH₂CH₂ N₃), 3.14 (d, 4 H, NCH₂ -t-BuO₂), 2.82 (t, ³ J _H–H = 7.6 Hz, 2 H, CH₂ CH₂CO₂H), 2.75 (t, ³ J _H–H = 7.2 Hz, 2 H, CH₂ CH₂N₃), 2.65 [br s, 4 H, N(CH₂ )₂N], 2.59 (t, ³ J _H–H = 7.6 Hz, 2 H, CH₂CH₂ CO₂H), 1.44 (d, 18 H, CH₃ ). ¹³C{¹H} NMR (100.61 MHz, CDCl₃, 25 °C): δ = 176.16 (CO₂H), 170.22 (d, CO₂ t-Bu), 156.31 (COH–ArCO₂ or COH–ArN₃), 155.89 (COH–ArCO₂ or COH–ArN₃), 130.92 (C–C₂H₄–CO₂H), 129.69 (C–C₂H₄–N₃), 129.53 (CCHC–C₂H₄–CO₂H), 129.33 (CCHC–C₂H₄–N₃), 129.05 (CHCHC–C₂H₄–CO₂H), 128.66 (CHCHC–C₂H₄–N₃), 121.80 (CCHC–C₂H₄–CO₂H), 121.67 (CCHC–C₂H₄–N₃), 116.73 (CHCHC–C₂H₄–CO₂H), 116.58 (CHCHC–C₂H₄–N₃), 82.33 [(CO₂ CCH₃)–ArCO₂H], 82.24 [(CO₂ CCH₃)–ArN₃], 58.04 (^Bn C), 55.73 [(NCH₂CO₂)–ArCO₂H], 55.60 [(NCH₂CO₂)–ArN₃], 52.84 (CH₂ CH₂N₃), 50.35 (NCH₂ CH₂N), 36.13 (CH₂ CH₂CO₂H), 34.67 (CH₂CH₂N₃), 29.99 (CH₂CH₂CO₂H), 28.16 (CH₃). HRMS (ESI ⁺ , MeCN–MeOH): m/z [M + H]⁺ Calcd for C₃₃H₄₈N₅O₈ ⁺: 642.3497; Found: 642.3503 (100%).