Cu-Free 1,3-Dipolar Cycloaddition Click Reactions To Form Isoxazole Linkers in Chelating Ligands for <i>fac</i>-[M^I(CO)₃]⁺ Centers (M = Re, ^99mTc)

Isoxazole ring formation was examined as a potential Cu-free alternative click reaction to Cu^I-catalyzed alkyne/azide cycloaddition. The isoxazole reaction was explored at macroscopic and radiotracer concentrations with the <i>fac</i>-[M^I(CO)₃]⁺ (M = Re, ^99mTc) core for use as a noncoordinating linker strategy between covalently linked molecules. Two click assembly methods (<i>click, then chelate</i> and <i>chelate, then click</i>) were examined to determine the feasibility of isoxazole ring formation with either alkyne-functionalized tridentate chelates or their respective <i>fac</i>-[M^I(CO)₃]⁺ complexes with a model nitrile oxide generator. Macroscale experiments, alkyne-functionalized chelates, or Re complexes indicate facile formation of the isoxazole ring. ^99mTc experiments demonstrate efficient radiolabeling with <i>click, then chelate</i>; however, the <i>chelate, then click</i> approach led to faster product formation, but lower yields compared to the Re analogues

pH-Controlled Coordination Mode Rearrangements of “Clickable” Huisgen-Based Multidentate Ligands with [M^I(CO)₃]⁺ (M = Re, ^99mTc)

The viability of the Huisgen cycloaddition reaction for clickable radiopharmaceutical probes was explored with an alkyne-functionalized 2-[(pyridin-2-ylmethyl)­amino]­acetic acid (PMAA) ligand system, <b>3</b>, and <i>fac</i>-[M^I(OH₂)₃(CO)₃]⁺ (M = Re, ^99mTc). Two synthetic strategies, (1) <i>click, then chelate</i> and (2) <i>chelate, then click</i>, were investigated to determine the impact of assembly order on the reactivity of the system. In the c<i>lick, then chelate</i> approach, <i>fac</i>-[M^I(OH₂)₃(CO)₃]⁺ was reacted with the PMAA ligand “clicked” to the benzyl azide, <b>5</b>, to yield two unique coordination species, <i>fac</i>-[M^I(CO)₃(O,N_amine,N_py<b>-5</b>)], M = Re (<b>8</b>), ^99mTc (<b>8A</b>), and <i>fac</i>-[M^I(CO)₃(N_tri,N_amine,N_py<b>-5</b>)], M = Re (<b>9</b>), ^99mTc (<b>9A</b>), where coordination is through the triazole (N_tri), central amine (N_amine), pyridine (N_py), or carboxylate (O). Depending on the reaction pH, different ratios of complexes <b>8</b>(<b>A</b>) and <b>9</b>(<b>A</b>) were observed, but single species were obtained of (O,N_amine,N_py) coordination, <b>8</b>(<b>A</b>), in basic pHs (>9) and (N_tri,N_amine,N_py) coordination, <b>9</b>(<b>A</b>), in slightly acidic pHs (<4). In the <i>chelate, then click</i> approach, the (O,N_amine,N_py) coordination of [M^I(CO)₃]⁺ was preorganized in the alkyne-functionalized <i>fac</i>-[M^I(CO)₃(O,N_amine,N_py<b>-3</b>)], M = Re (<b>6</b>), ^99mTc (<b>6A</b>), followed by standard Cu^I-catalyzed Huisgen “<i>click</i>” conditions at pH ≈ 7.4, where the (O,N_amine,N_py) coordination mode remained unchanged upon formation of the triazole product in the clicked molecule. Despite the slow substitution kinetics of the low-spin d⁶ metal, the coordination modes (O,N_amine,N_py) and (N_tri,N_amine,N_py) were found to reversibly intraconvert between <b>8</b>(<b>A</b>) and <b>9</b>(<b>A</b>) based upon changes in pH that mirrored the (O,N_amine,N_py) coordination in basic pHs and (N_tri,N_amine,N_py) coordination in acidic pHs. Comparison of the Re and ^99mTc analogs also revealed faster intraconversion between the coordination modes for ^99mTc

pH-Controlled Coordination Mode Rearrangements of “Clickable” Huisgen-Based Multidentate Ligands with [M^I(CO)₃]⁺ (M = Re, ^99mTc)

The viability of the Huisgen cycloaddition reaction for clickable radiopharmaceutical probes was explored with an alkyne-functionalized 2-[(pyridin-2-ylmethyl)­amino]­acetic acid (PMAA) ligand system, <b>3</b>, and <i>fac</i>-[M^I(OH₂)₃(CO)₃]⁺ (M = Re, ^99mTc). Two synthetic strategies, (1) <i>click, then chelate</i> and (2) <i>chelate, then click</i>, were investigated to determine the impact of assembly order on the reactivity of the system. In the c<i>lick, then chelate</i> approach, <i>fac</i>-[M^I(OH₂)₃(CO)₃]⁺ was reacted with the PMAA ligand “clicked” to the benzyl azide, <b>5</b>, to yield two unique coordination species, <i>fac</i>-[M^I(CO)₃(O,N_amine,N_py<b>-5</b>)], M = Re (<b>8</b>), ^99mTc (<b>8A</b>), and <i>fac</i>-[M^I(CO)₃(N_tri,N_amine,N_py<b>-5</b>)], M = Re (<b>9</b>), ^99mTc (<b>9A</b>), where coordination is through the triazole (N_tri), central amine (N_amine), pyridine (N_py), or carboxylate (O). Depending on the reaction pH, different ratios of complexes <b>8</b>(<b>A</b>) and <b>9</b>(<b>A</b>) were observed, but single species were obtained of (O,N_amine,N_py) coordination, <b>8</b>(<b>A</b>), in basic pHs (>9) and (N_tri,N_amine,N_py) coordination, <b>9</b>(<b>A</b>), in slightly acidic pHs (<4). In the <i>chelate, then click</i> approach, the (O,N_amine,N_py) coordination of [M^I(CO)₃]⁺ was preorganized in the alkyne-functionalized <i>fac</i>-[M^I(CO)₃(O,N_amine,N_py<b>-3</b>)], M = Re (<b>6</b>), ^99mTc (<b>6A</b>), followed by standard Cu^I-catalyzed Huisgen “<i>click</i>” conditions at pH ≈ 7.4, where the (O,N_amine,N_py) coordination mode remained unchanged upon formation of the triazole product in the clicked molecule. Despite the slow substitution kinetics of the low-spin d⁶ metal, the coordination modes (O,N_amine,N_py) and (N_tri,N_amine,N_py) were found to reversibly intraconvert between <b>8</b>(<b>A</b>) and <b>9</b>(<b>A</b>) based upon changes in pH that mirrored the (O,N_amine,N_py) coordination in basic pHs and (N_tri,N_amine,N_py) coordination in acidic pHs. Comparison of the Re and ^99mTc analogs also revealed faster intraconversion between the coordination modes for ^99mTc

Clickable, Hydrophilic Ligand for <i>fac-</i>[M^I(CO)₃]⁺ (M = Re/^99mTc) Applied in an <i>S</i>‑Functionalized α‑MSH Peptide

The copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (<b>1</b> and <b>3</b>) for <i>fac</i>-[M^I(CO)₃]⁺ (M = Re/^99mTc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (<b>3</b>) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of <i>fac</i>-[^99mTc^I(CO)₃]⁺ complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The <i>fac</i>-[Re^I(CO)₃(<b>3</b>)] complex (<b>4</b>) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between <b>3</b> and <i>fac</i>-[^99mTc^I(OH₂)₃(CO)₃]⁺. The corresponding ^99mTc complex (<b>4a</b>) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log <i>P</i> analysis compared to an analogous complex with nonfunctionalized pyridine rings (<b>2a</b>). An α-MSH peptide functionalized with an azide was labeled with <i>fac</i>-[M^I(CO)₃]⁺ using both <i>click, then chelate</i> (CuAAC reaction with <b>1</b> or <b>3</b> followed by metal complexation) and <i>chelate, then click</i> (metal complexation of <b>1</b> and <b>3</b> followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on <i>fac</i>-[M^I(CO)₃]⁺ complexation within a peptide framework. The peptides from the <i>click, then chelate</i> strategy had different HPLC <i>t</i>_R’s and in vitro stabilities compared to those from the <i>chelate, then click</i> strategy, suggesting nonspecific coordination of <i>fac</i>-[M^I(CO)₃]⁺ using this synthetic route. The <i>fac</i>-[M^I(CO)₃]⁺-complexed peptides from the <i>chelate, then click</i> strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC₅₀ analyses, and led to moderately high uptake in B16F10 melanoma cells. Log <i>P</i> analysis of the ^99mTc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (<b>10a′</b>) compared to the peptide with the unmodified DPA chelate (<b>9a′</b>). In vivo biodistribution analysis of <b>9a′</b> and <b>10a′</b> showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for <b>10a′</b> compared to predominantly hepatic accumulation for <b>9a′</b>. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted <i>fac</i>-[M^I(CO)₃]⁺ complexes in future studies

Clickable, Hydrophilic Ligand for <i>fac-</i>[M^I(CO)₃]⁺ (M = Re/^99mTc) Applied in an <i>S</i>‑Functionalized α‑MSH Peptide

The copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (<b>1</b> and <b>3</b>) for <i>fac</i>-[M^I(CO)₃]⁺ (M = Re/^99mTc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (<b>3</b>) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of <i>fac</i>-[^99mTc^I(CO)₃]⁺ complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The <i>fac</i>-[Re^I(CO)₃(<b>3</b>)] complex (<b>4</b>) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between <b>3</b> and <i>fac</i>-[^99mTc^I(OH₂)₃(CO)₃]⁺. The corresponding ^99mTc complex (<b>4a</b>) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log <i>P</i> analysis compared to an analogous complex with nonfunctionalized pyridine rings (<b>2a</b>). An α-MSH peptide functionalized with an azide was labeled with <i>fac</i>-[M^I(CO)₃]⁺ using both <i>click, then chelate</i> (CuAAC reaction with <b>1</b> or <b>3</b> followed by metal complexation) and <i>chelate, then click</i> (metal complexation of <b>1</b> and <b>3</b> followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on <i>fac</i>-[M^I(CO)₃]⁺ complexation within a peptide framework. The peptides from the <i>click, then chelate</i> strategy had different HPLC <i>t</i>_R’s and in vitro stabilities compared to those from the <i>chelate, then click</i> strategy, suggesting nonspecific coordination of <i>fac</i>-[M^I(CO)₃]⁺ using this synthetic route. The <i>fac</i>-[M^I(CO)₃]⁺-complexed peptides from the <i>chelate, then click</i> strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC₅₀ analyses, and led to moderately high uptake in B16F10 melanoma cells. Log <i>P</i> analysis of the ^99mTc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (<b>10a′</b>) compared to the peptide with the unmodified DPA chelate (<b>9a′</b>). In vivo biodistribution analysis of <b>9a′</b> and <b>10a′</b> showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for <b>10a′</b> compared to predominantly hepatic accumulation for <b>9a′</b>. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted <i>fac</i>-[M^I(CO)₃]⁺ complexes in future studies