Pentadentate and Hexadentate Pyridinophane Ligands Support Reversible Cu(II)/Cu(I) Redox Couples

Two new ligands were synthesized with the goal of copper stabilization, N,N′-(2-methylpyridine)-2,11-diaza[3,3](2,6)pyridinophane (PicN4) and N-(methyl),N′-(2-methylpyridine)-2,11-diaza[3,3](2,6)pyridinophane (PicMeN4), by selective functionalization of HN4 and TsHN4. These two ligands, when reacted with various copper salts, generated both Cu(II) and Cu(I) complexes. These ligands and Cu complexes were characterized by various methods, such as NMR, UV-Vis, MS, and EA. Each compound was also examined electrochemically, and each revealed reversible Cu(II)/Cu(I) redox couples. Additionally, stability constants were determined via spectrophotometric titrations, and radiolabeling and cytotoxicity experiments were performed to assess the chelators relevance to their potential use in vivo as 64Cu PET imaging agents.

were integrated using an orientation matrix determined from the narrow frame scans.Apex II and SAINT software packages (Bruker Analytical X-Ray, Madison, WI, 2008) were used for data collection and data integration.Analysis of the integrated data did not show any decay.Final cell constants were determined by global refinement of xyz centroids of reflections from the complete data sets.Collected data were corrected for systematic errors using SADABS Bruker Analytical X-Ray, Madison, WI, 2008) based on the Laue symmetry using equivalent reflections.Crystal data and intensity data collection parameters are listed in Tables S4-S11.7] The structures were solved by direct methods and refined successfully.Full matrix least-squares refinements were carried out by minimizing Σw(Fo 2 −Fc 2 ) 2 .The non-hydrogen atoms were refined anisotropically to convergence.The hydrogen atoms were treated using appropriate riding model.
The crystallographic datasets have been deposited at CCDC under the record numbers 2049802, 2049803, 2049804, and 2049805.

Acidity and Stability Constant Determination
Spectrophotometric pH-titrations were performed for determination of the acidity constants for the ligands and the stability constants for their Cu(II) complexes.In general, Ligands (60 μM, 0.1 M KCl) or the Ligands + Cu(II) (60 μM, 0.1 M KCl), were titrated with small aliquots of 0.15 M KOH from low pH to high pH at room temperature until no more spectral changes (pH range 2-11).The starting low pH is achieved by addition of 0.5M HCl diluted in Chelex-treated water, until the desired starting pH is reached.About 50 UV−vis spectra were collected from the tra on experiments, the corresponding acidity and stability constants were then calculated using the HypSpec computer program (Protonic Software, UK), a commonly used method for studying metal complexes. 8e ligand-Cu(I) stability constant were calculated from half-wave potentials E f and the Cu II complex stability constants using the Nernst relationship. 9For a reversible redox couple, E f , were obtained by averaging the anodic and cathodic peak potentials.The value of the aqueous Cu(II/I) redox couple ( °), 0.230 V vs Ag/AgCl, was obtained from a control of Cu(OTf) 2 in the same aqueous conditions the complex CVs were measured in ( and acetonitrile (0.1% TFA) mobile phase with a gradient of 0-100% acetonitrile over 15 min with a flow rate of 1 mL/min.

LogD Determination of 64 Cu-Labeled Complexes
A 5 μL aliquot of 64 Cu-labeled complexes (0.37 MBq, 10 μCi) was added to a two-phase system consisting of an organic n-octanol and aqueous buffer PBS of pH 7.4 (500 μL/each).The mixture was vortexed at 1000 rpm for 1 h to allow distribution of the radiolabeled compound between the two phases and then given 30 min without agitation for the layers to separate.Aliquots (100 μL) were withdrawn from aqueous and n-octanol layers, and the amount of radioactivity was counted in an automated γ counter.The distribution coefficient was quantified using the following equation of log D oct = log([M] oct /[M] aq ).The experiment was conducted with five replicates, and the average of the different measurements was recorded as the final log D oct value for each compound.

Syntheses of Pic N4, PicMe N4, and their Cu complexes
Synthesis of Pic N4 The Pic N4 (51 mg, 0.12 mmol) and copper (II) triflate, Cu II (OTf) 2 , (43 mg, 0.12 mmol) were added to a 20 mL vial.MeCN (1 mL) was added to the solids and the color immediately changed from one white and one light green solids to a blue solution.The reaction was stirred overnight.The MeCN solution mixture was filtered through a 0.2 μm nylon syringe filter and set up for diethyl ether diffusion recrystallization at room temp over two days.When the large blue elongated crystals had formed, the pale yellow/orange solution was decanted away and the crystals rinsed with diethyl ether and pentane.The crystals were then dried under reduced pressure to remove excess solvent (78.2mg, 83.3%

Preparation of [( Pic N4)Cu I ]OTf: 2·OTf
The Pic N4 (0.24 g, 5.8 mmol) and tetrakisacetonitrile copper (I) triflate, [(MeCN) 4 Cu I ]OTf, (0.22 g, 5.9 mmol) were added to a 20 mL vial.MeCN (3 mL) was added to the solids and the color immediately changed from two white solids to a deep orange/brown solution.The reaction was stirred for one hour.
The solution was concentrated by vacuum to roughly 1 mL and the solution was split to make two sets of recrystallizations: 1) 0.5 mL of concentrated MeCN solution and 3 mL of dry diethyl ether layered on top and 2) 0.5 mL of concentrated MeCN solution and 3 mL of dry toluene layered on top.These recrystallizations were placed in the -35 °C freezer for four days.The large, dark-orange crystals had formed on the bottom of the vial in each recrystallization.These were rinsed with ether and harvested (200.(1.12 g, 8.67 mmol) and a catalytic amount of tetrabutylammonium bromide (0.020 g) was added and the solution stirred at reflux for two days.To work up, ice was added to the warm reaction solution, the solution basified with a small amount of NaOH to reach a pH of 13, and the aqueous layer was extracted with DCM (3 x 500 mL).The organic layer was dried with potassium carbonate, and the solvent removed by rotary evaporation.The resulting tan solid was dried well under vacuum.(2.16 g, 89%).
Using an addition funnel, 2-pyridine carboxaldehyde (435 μL, 4.57 mmol) dissolved in 80 mL of DCM was slowly dripped in.The reaction vial was left to stir under a nitrogen atmosphere at room temperature overnight.To the reaction solution, saturated sodium bicarbonate solution (250 mL) was added as the solution stirred vigorously.The solution was then filtered using a coarse frit funnel to remove the molecular sieves and the vessel rinsed well with excess DCM.The organic layer was then separated, dried with anhydrous potassium carbonate, and then the solvent removed under reduced pressure rotary evaporation The white/off-white powder was then dried on vacuum line for at least five hours and stored in a desiccator (

Synthesis of PicH N4
Under N 2 , TsPic N4 (2.98 g, 6.14 mmol) was dissolved in 90% sulfuric acid (65 mL).This mixture was refluxed at 100 °C for 2.5 hours.After cooling, the solution was diluted with DI water (50 mL).In an ice bath, NaOH solid was added to the solution to basify to bring the pH to 14.The resulting solution was extracted with DCM (3 x 600 mL).The combined organic layers were dried over anhydrous potassium carbonate, and filtered.The filtrate was concentrated to dryness to give a white solid (

Synthesis of PicMe N4
Under N 2 , PicH N4 (1.77 g, 5.34 mmol) was dissolved in concentrated formic acid (150 mL, 3.98 mol) and 40% formaldeyde solution (15 mL, 163 mmol).This mixture was refluxed at 110 °C for 24 hours.After 24 hours, the reaction mixture was removed from the heat and 15 mL of 1 M HCl solution was stirred in.The solution was stirred for an additional 30 minutes.The solution was then concentrated to dryness via rotary evaporation and the residue was basified using KOH.The basic aqueous fraction was extracted with DCM and organic layers were combined and dried with potassium carbonate.The organic layer was then concentrated to dryness to give a white residue.This residue was purified by hot heptane extraction ( The PicMe N4 (54.4 mg, 1.54 mmoles) and Cu II (OTf) 2 , (55.1 mg, 1.53 mmoles) were added to a 20 mL vial.
MeCN (1 mL) was added to the solids and the color immediately changed from white solids to a green solution which faded to blue.The reaction was stirred for 4 hours.The solution mixture was then concentrated down to a blue-green solution.This solution was then layered with toluene and stored in the -35 °C freezer overnight.The solution was then decanted to leave a blue-green residue which was rinsed with pentane to yield a blue-green powder which was dried under reduced pressure (50.3 mg, 47%

3 Figure S32 .
Figure S32.Projection view with 30% probability ellipsoids: H atoms and disorder components omitted for clarity.
1,  96485.3329 •  ,  8.3145  •  •  ,  298.15  (Thermo Fisher Scientific) was added to each well and incubated for 60 min at 37 °C.Fluorescence intensity was measured at 590 nm (excitation wavelength = 560 nm) on a SpectraMax M2e plate reader.Radiolabeling 64 Cu was produced by a (p,n) reaction on enriched 64 Ni on a TR-19 biomedical cyclotron (Cyclotron Corporation, Berkeley, CA) at Mallinckrodt Institute of Radiology, S5 Washington University School of Medicine, and purified with an automated system using standard procedures.Radiolabeling studies were performed at the Experimental Molecular Imaging Laboratory (EMIL) in the Beckman Institute for Advanced Science and Engineering at UIUC.A stock solution of 64 CuCl 2 was diluted with a 10-fold excess of 0.1 M ammonium acetate (NH 4 OAc) buffer (pH 5.5).Labeling of PicN4 and PicMeN4 with 64 Cu was achieved by adding 20 μL of 1 mM compounds to 7.
The PicMe N4 (0.298 g, 0.84 mmol) and [(MeCN)4Cu I ]OTf (0.295 g, 0.78 mmol) were added to a 20 mL vial.MeCN (3 mL) was added to the solids and the color immediately changed from two white solids to a deep orange/brown solution.The reaction was stirred for one hour in the dark.The solution was concentrated by vacuum to roughly 1 mL and the MeCN solution was recrystallized by diethyl ether diffusion at -35 °C freezer.The large, dark-orange crystals had formed on the bottom of the vial in each recrystallization.