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Jan 15, 2015 - A series of Re and 99mTc complexes were synthesized with picolinic acid as ... In the last two decades, fac-[MI(CO)3]+ (M = Re, 99mTc) ...
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Influence of Functionalized Pyridine Ligands on the Radio/Chemical Behavior of [MI(CO)3]+ (M = Re and 99mTc) 2 + 1 Complexes Thomas R. Hayes,† Patrice A. Lyon,† Charles L. Barnes,‡ Steven Trabue,§ and Paul D. Benny*,† †

Department of Chemistry, Washington State University, Pullman, Washington 99164, United States Department of Chemistry, University of Missouri, Columbia, Missouri 65211, United States § USDA, National Soil Tilth Laboratory, Ames, Iowa 50011, United States ‡

S Supporting Information *

ABSTRACT: While a number of chelate strategies have been developed for the organometallic precursor fac-[MI(OH2)3(CO)3]+ (M = Re, 99mTc), a unique challenge has been to improve the overall function and performance of these complexes for in vivo and in vitro applications. Since its discovery, fac[MI(OH2)3(CO)3]+ has served as an essential scaffold for the development of new targeted 99mTc based radiopharmaceuticals due to its labile aquo ligands. However, the lipophilic nature of the fac-[MI(CO)3]+ core can influence the in vivo pharmacokinetics and biodistribution of the complexes. In an effort to understand and improve this behavior, monosubstituted pyridine ligands were used to assess the impact of donor nitrogen basicity on binding strength and stability of fac-[MI(CO)3]+ in a 2 + 1 labeling strategy. A series of Re and 99mTc complexes were synthesized with picolinic acid as a bidentate ligand and 4substituted pyridine ligands. These complexes were designed to probe the effect of pKa from the monodentate pyridine ligand both at the macro scale and radiochemical concentrations. Comparison of X-ray structural data and radiochemical solution experiments clearly indicate an increase in overall yield and stability as pyridine basicity increased.

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INTRODUCTION In the last two decades, fac-[MI(CO)3]+ (M = Re, 99mTc) based complexes have come to the forefront of development of new radiopharmaceutical probes for 99mTc diagnostic imaging1−4 and 188/186Re radiotherapy.5,6 A number of ligand strategies (mono-, bi, and tridentate) have been investigated with a variety of different ligand donor types. Aromatic amines (e.g., triazole, imidazole, and pyridine) have continued to be a major contributor in ligand design as monodentate or part of a multidentate ligand for the fac-[MI(CO)3]+ core.7 Recent trends in the [99mTcI(CO)3]+ radiopharmaceutical field have primarily been oriented toward the application of new biologically active molecules to a few common chelates (e.g., histidine, dipicolylamine (DPA), iminodiacetic acid) or organometallic ligands (e.g., cyclopentadienyl derivatives (Cp)).8 While Alberto has continued development of modified Cp rings to improve efficiency, research has declined with regards to the development of new chelate strategies with improved characteristics (e.g., hydrophilicity, binding strength, and single isomers).9 High in vivo stability is a major consideration in designing new chelates to avoid transchelation or decomplexation of the metal and transferring to blood proteins, which would alter the biodistribution profile from targeted to nonspecific uptake in organs involved in clearance pathways. While most fac-[MI(CO)3]+ complexes saturate the coordination sphere with a combination of three donors in a chelate, monodentate ligands can also be utilized. However, substitution of monodentate ligands can readily occur in weak ligands due © 2015 American Chemical Society

to the absence of the chelate effect. This can occur either through a dissociative process where the ligand is lost followed by coordination of solvent (e.g. water) or another ligand or through a direct substitution by another competing ligand. Monodentate ligands also provide the unique capability to explore the electronic changes by the addition of functional groups on a ligand and directly measuring the role in complex stability without the influence of chelate effect obscuring results. In the past decade, pyridine based ligands have been utilized as a cornerstone for designing complexes with the fac[MI(CO)3]+ core. Several common examples of ligands containing pyridine moieties, e.g., 2,2′-bipyridine,10,11 picolinic acid (pic), 7 DPA,12 and asymmetric pyridine-based ligands,7,13−18 have been utilized with Re and Tc (Figure 1). The aromatic nature of pyridine offers an attractive platform to explore the effect of basicity on donor strength that could applied to any of these ligand systems. Inclusion of functional groups, either electron withdrawing (NO2, Cl, and CO2H) or donating (NR2 and OR), on pyridine can be used to tune the basicity of the nitrogen and impact the overall properties of the metal complex.19 In addition to the electronic nature of the functional group, the ring position (2, 3, or 4) of the substituent also contributes significantly to the basicity of the ligand. However, substitution at the 2 position leads to Received: October 15, 2014 Published: January 15, 2015 1528

DOI: 10.1021/ic502520x Inorg. Chem. 2015, 54, 1528−1534

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Inorganic Chemistry Table 1. pKa’s of Selected 4-Subsituted Pyridines

Figure 1. Examples of pyridine containing multidentate ligands.

of a series of 4-substituted pyridine ligands. A series of pyridines (pyridine (py), 4-hydroxypyridine (4-HP), 4-methoxypyridine (4-MP), 4-aminopyridine (4-AP), N,N-dimethylaminopyridine (DMAP), and 4-nitropyridine (4-NP)) were added to complex 1 as mondentate ligands to yield the corresponding 2 + 1 complex. Macroscale reactions were carried out with fac[ReI(CO)3]+ for standard chemical characterization and structural characterization through X-ray crystallography. The corresponding tracer level radioactive complexes were also prepared with fac-[99mTcI(CO)3]+ and used to assess complexation efficiency and stability to transchelation of the pyridine ligands. The experimental data of the macroscale Re and radiochemical Tc experiments illustrate clear trends with respect to pyridine basicity.

increased steric interactions with the metal center or adjacent ligands in the complex that would negatively influence coordination. Substitution at the 3 or 4 position relative to the nitrogen provides a wide range of pKa’s without increasing steric interactions with the metal or adjacent ligands.20 Recently, there have been several reports of functionalized mono-11,21 and tridentate22 pyridine based ligands for fac[MI(CO)3]+. In a recent publication by our group, DPA, a wellestablished chelate for the fac-[MI(CO)3]+ core was modified to include a 3-carboxylate functionality on both of its pyridine rings.22 This functionalization significantly changed the excretion pathway in vivo from a liver/kidney ratio of 2.3 with DPA to 0.6 with the modified chelate. While tumor uptake did not appear impacted by the pyridine functionalization, higher than expected intestinal clearance suggests the overall stability of the complex may have been impacted by the carboxylate functionalization of pyridine. While the in vitro testing of the carboxylate complexes indicated high stability, these results highlight the necessity for in vivo analysis for accurate assessment of the chelate’s overall behavior. Although these studies contained substituted pyridine compounds, limited information is known about the overall influence of the substitution of pyridine on the properties of the corresponding fac-[MI(CO)3]+ complexes. In this study, the effect of substitution at the 4-position of pyridine rings was explored using a 2 + 1 strategy with the fac[MI(CO)3]+ core. This strategy permits the independent assessment of the functionalized pyridines as monodentate ligands, while a bidentate ligand remains constant. Monodentate pyridines provide an avenue to examine the influence of ligand basicity while eliminating the influence of the chelate effect on stability and dissociation rates. The 4 position on the pyridine ligands was selected because it provides the greatest range of pKa values and minimizes steric interferences (Table 1). A neutral bidentate pic complex, fac-[M I (OH 2 )(CO)3(pic)], 1, was used as a platform for the complexation

Materials and Methods. All reagents and organic solvents of reagent grade or better were used as purchased from Aldrich, Acros, or Fluka without further purification. Rhenium starting material fac[ReI(OH2)3(CO)3](SO3CF3) was prepared by literature methods from Re2(CO)10 purchased from Strem.23,24 99mTc was obtained in the form of Na[99mTcO4] from Cardinal Health (Spokane, WA) and the fac-[99mTcI(OH2)3(CO)3]+ complex was prepared using a commercially available Isolink kit from Covidien. fac-[ReI(OH2)(CO)3(pic)], 1, was prepared as previously described.7 1H and 13C NMR spectra were recorded on a Varian 300 MHz instrument at 25 °C. FT-IR spectra were obtained on a Thermo Nicolet 6700 FT-IR with an ATR cell and analyzed with OMNIC 7.1 software. Mass spectra of the samples were obtained by electrospray ionization directly by infusing the samples into an Agilent 1100 Ion Trap LC/MS/MS and scanning from 50 to 1200 m/z with the drying gas at 12 mL min−1 at 350 °C and nebulizer pressure set at 50 psig. Separation and identification of compounds were conducted on a PerkinElmer Series 200 high pressure liquid chromatograph (HPLC) equipped with a UV/vis Series 200 detector and a Radiomatic 610TR detector. Utilizing a Varian Pursuit XRs 5 μm particle and 250 × 4.6 mm C-18 column, the compounds were separated with a reverse phase gradient system beginning with a 2 mM pH 7.4 phosphate buffer aqueous phase and shifting gradually to methanol. HPLC analysis was performed using 0−1.0 min (75% phosphate, 25% MeOH), 1.0−12.0 min (25% to 100% MeOH linear gradiant), 12−17 min (100% MeOH), and 17.0−25.0 min (75% phosphate, 25% MeOH) at a flow rate of 1.0 mL/min. fac-[ReI(CO)3(pic)(py)], 2. Complex 1 (40 mg, 0.10 mmol) was dissolved in absolute EtOH (5 mL) with stirring. Pyridine (15 μL, 0.18 mmol) was then added, and the solution was heated to 70 °C for 4 h. The solution was allowed to come to rt, H2O (3 mL) was added, and the EtOH was removed under reduced pressure. The resulting solution was cooled to 5 °C for 16 h, and the resulting precipitate was collected by vacuum filtration to give 2 as an off-white solid (12.3 mg, 30.6%). Crystals for X-ray diffraction analysis were obtained by slow evaporation of a methanolic solution. Anal. Calcd for C14H9N2O5Re: C 35.67, H 1.92, N 5.94; Found: C 35.08, H 1.94, N 5.83; 1H NMR (300 MHz, CDCl3) δ 8.88 (d, 1H, J = 4.9 Hz), 8.59 (d, 2H, J = 4.9 Hz), 8.14 (d, 1H, J = 7.7 Hz), 8.02 (t, 1H, J = 7.7 Hz), 7.80 (t, 1H, J = 8.7 Hz), 7.63 (t, 1H, J = 5.5 Hz), 7.34 (t, 2H, J = 7.7 Hz); 13C NMR

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DOI: 10.1021/ic502520x Inorg. Chem. 2015, 54, 1528−1534

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Inorganic Chemistry (75 MHz) δ 197.5, 196.2, 193.8, 172.8, 152.2, 151.3, 150.8, 140.2, 139.1, 128.7, 127.8, 126.3; IR (neat powder, cm−1) 2023, 1916, 1881, 1864, 1664; MS (m/z): [M + Na]+ 495.0. fac-[ReI(CO)3(pic)(4-HP)], 3. Complex 1 (40 mg, 0.10 mmol) was dissolved in methanol (5 mL) with stirring. 4-Hydroxypyridine (13.9 mg, 0.15 mmol) was then added, and the solution was heated to 70 °C for 4 h. The solution was allowed to come to rt, concentrated to half volume, and allowed to slowly evaporate to yield 3 (17.2 mg, 36.2%). Crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of a methanolic solution. Anal. Calcd for C14H9N2O6Re: C 34.50, H 1.86, N 5.75; Found: C 34.22, H 2.01, N 5.73; 1H NMR (300 MHz, CD3OD) δ 9.02 (d, 1H, J = 5.5 Hz), 8.23−8.17 (m, 3H), 8.07 (d, 1H, J = 7.1 Hz), 7.81 (t, 1H, J = 5.2 Hz), 6.76 (d, 2H, J = 7.1 Hz); 13C NMR (75 MHz, DMSO-d6) δ 197.9, 197.6, 195.6, 172.2, 166.9, 153.4, 153.0, 149.7, 141.9, 130.1, 127.4, 114.5; IR (neat powder, cm−1) 2020, 1891, 1588; MS (m/z): [M-H]− 486.8. fac-[ReI(CO)3(pic)(4-MP)], 4. Complex 1 (40 mg, 0.10 mmol) was dissolved in absolute EtOH (5 mL) with stirring. 4-Methoxypyridine (20 μL, 0.20 mmol) was then added, and the solution was heated to 70 °C for 4 h. The solution was allowed to come to rt, H2O (3 mL) was added, and the EtOH was partially removed under reduced pressure. The resulting mixture was heated to dissolve the solids and allowed to slowly cool. The resulting solution was cooled to 5 °C for 16 h, and the resulting precipitate was collected by vacuum filtration and dried in vacuo to give 4 as a white powder (36.3 mg, 74.3%). Crystals for X-ray diffraction analysis were obtained by slow diffusion of cyclohexane into an EtOAc solution. Anal. Calcd for C15H11N2O6Re: C 35.93, H 2.21, N 5.59; Found: C 35.94, H 2.16, N 5.54; 1H NMR (300 MHz, CDCl3) δ 8.85 (d, 1H, J = 5.2 Hz), 8.34 (d, 2H, J = 7.4 Hz), 8.13 (d, 1H, J = 7.7 Hz), 8.01 (t, 1H, J = 7.7 Hz), 7.61 (t, 1H, J = 5.2 Hz), 6.77 (d, 1H, J = 7.6 Hz), 3.84 (s, 3H); 13C NMR (75 MHz) δ 197.7, 196.4, 194.0, 172.8, 167.3, 153.2, 151.2, 150.8, 140.1, 128.5, 127.8, 112.2, 56.2; IR (neat powder, cm−1) 2029, 1920, 1887, 1656; MS (m/z): [M + Na]+ 525.0. fac-[ReI(CO)3(pic)(4-AP)], 5. Complex 1 (40 mg, 0.10 mmol) was dissolved in absolute EtOH (5 mL) with stirring. 4-Aminopyridine (11 mg, 0.11 mmol) was then added, and the solution was heated to 70 °C for 4 h. The solution was allowed to come to rt, H2O (3 mL) was added, and the EtOH was partially removed under reduced pressure. The resulting mixture was heated to dissolve the solids and allowed to slowly cool. The resulting solution was cooled to 5 °C for 16 h, and the resulting precipitate was collected by vacuum filtration and dried in vacuo to give 5 as a white powder (41.7 mg, 88%). Crystals for X-ray diffraction analysis were obtained by slow evaporation of a mixed methanol−water solution. Anal. Calcd for C14H10N3O5Re: C 34.57, H 2.07, N 8.64; Found: C 34.82, H 2.21, N 8.75; 1H NMR (300 MHz, CD3OD) δ 8.99 (d, 1H, J = 5.2 Hz), 8.18 (t, 1H, J = 7.7 Hz), 8.06 (d, 1H, J = 8.0 Hz), 7.84−7.76 (m, 3H), 6.41 (d, 2H, J = 6.9 Hz); 13C NMR (75 MHz, DMSO-d6) δ 196.9, 196.7, 194.3, 174.0, 156.6, 152.1, 150.9, 149.5, 140.6, 129.2, 127.1, 109.7; IR (neat powder, cm−1) 2016, 1877, 1640; MS (m/z): [M + Na]+ 510.0. fac-[ReI(CO)3(pic)(DMAP)], 6. Complex 1 (40 mg, 0.10 mmol) was dissolved in absolute EtOH (5 mL) with stirring. DMAP (13.1 mg, 0.11 mmol) was then added, and the solution was heated to 70 °C for 3.5 h. The solution was allowed to come to rt, H2O (3 mL) was added, and the EtOH was partially removed under reduced pressure. The resulting mixture was cooled to 5 °C for 16 h, and the resulting precipitate was collected by vacuum filtration and dried in vacuo to give 6 as a white powder (31.7 mg, 63.2%). Crystals for X-ray diffraction analysis were obtained by slow evaporation of a mixed methanol−water solution. Anal. Calcd for C16H14N3O5Re: C 37.35, H 2.74, N 8.17; Found: C 37.58, H 2.73, N 8.34; 1H NMR (300 MHz, CDCl3) δ 8.84 (d, 1H, J = 5.2 Hz), 8.13 (d, 1H, J = 7.7 Hz), 8.02−7.96 (m, 3H), 7.57 (t, 1H, J = 6.9 Hz), 6.33 (d, 2H, J = 7.4 Hz), 2.98 (s, 6H); 13C NMR (75 MHz) δ 198.1, 196.9, 194.6, 172.9, 154.6, 151.1, 150.9, 150.8, 139.9, 129.3, 127.6, 107.9, 39.4; IR (neat powder, cm−1) 2016, 1863, 1669; MS (m/z): [M + Na]+ 538.0. 99m Tc Labeling Studies. A solution of pic in MeOH (200 μL, 10 μM) was diluted with MeOH (800 μL) and 50 mM pH 8 phosphate buffer (800 μL). The solutions were sparged for 5 min under N2 prior

to addition of fac-[99mTc(OH2)3(CO)3]+ solution (200 μL). The resulting solution was then heated for 30 min at 95 °C. HPLC analysis of this solution showed >98% radiochemical purity of fac[99mTcI(OH2)(CO)3(pic)]. This solution was used without further purification. Solutions of substituted pyridines in MeOH (100 μL, 0.1 M) and 50 mM phosphate buffer (400 μL, pH 8 for complexes 2a, 4a−7a and pH 7 for complex 3a) were added to a sealed vial. The solutions were sparged for 5 min with N2. fac-[99mTcI(OH2)(CO)3(pic)] solution (100 μL) was then added, and the solutions were heated at 90 °C for 30 min. Samples were then analyzed by radio-HPLC to determine percent conversion. 99m Tc Stability Studies. Solutions of complexes 2a−6a were purified by radio-HPLC. Purified solutions were then mixed 1:1 with either 2 mM histidine or 2 mM cysteine in 10 mM pH 7.4 phosphate buffer to give a final concentration of 1 mM amino acid in N2 sparged sealable vials. The solutions were then placed in a 37 °C water bath and samples were analyzed at 1 h by radio-HPLC. Percent stability was determined by comparison of the peak area of the purified complex to all other 99mTc species in solution. X-ray Crystal Structure Determination. For all structures intensity data were obtained on a Bruker APEX II CCD Area Detector system using the ω scan technique with Mo Kα radiation from a graphite monochromator. Data were collected at −173 °C. Intensities were corrected for Lorentz and polarization effects. Equivalent reflections were merged, and absorption corrections were made using the multiscan method.25 Space group, lattice parameters and other relevant information are given in the Supporting Information. The structures were solved by direct methods with fullmatrix least-squares refinement, using the SHELX package26−28 with the aid of the program X-SEED.29 All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atoms were placed at calculated positions and included in the refinement using a riding model, with fixed isotropic U.

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RESULTS AND DISCUSSION Pyridine-based ligands are commonly used in the complexation of the fac-[M I(CO) 3 ] + core. Examination of pyridine substitution could determine potential targets for modification of existing ligands such as DPA and 2-((pyridin-2-ylmethyl)amino)acetate. Pic was used in this study as a bidentate ligand because it yields a neutral bidentate complex with the fac[MI(CO)3]+ core, eliminating the need for a counterion. A series of substituted pyridines were chosen, primarily by their pKa range (Table 1) to determine the effect of the electron donating capability of the pyridine on bond strength. Determining the effect of substitution in monodentate pyridine systems can be used to estimate the effect a substitution may have in a bi- or tridentate system. Understanding these effects will assist in the development of novel pyridine based ligands with tunable solubility or additional positions for biomolecule conjugation. Synthesis and Characterization of fac-[ReI(CO)3]+ Complexes. Preparation of the fac-[ReI(OH2)(CO)3(pic)], 1, was performed as previously described.7 To investigate the overall effect of pyridine substitution on the stability of the complexes a series of 4-subsituted monodentate ligands were used and compared to results with pyridine. Para substitution of the pyridine ring was chosen to minimize steric effects on the complexation while the different substituents were chosen to give a wide range of pyridine pK a ’s (Table 1). fac[ReI(CO)3(pic)(py)], 2, fac-[ReI(CO)3(pic)(4-MP)], 4, fac[ReI(CO)3(pic)(4-AP)], 5, and fac-[ReI(CO)3(pic)(DMAP)], 6, were synthesized by dissolving 1 in EtOH and reacting it with the corresponding pyridine for 3.5−4 h at 70 °C to give the complex in 30.6%, 74.3%, 88.0%, and 63.2% yield, 1530

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Inorganic Chemistry

Complexes were characterized by 1H NMR, 13C NMR, IR, MS, and elemental analysis. 1H NMR for complexes 2 and 4−6 showed splitting patterns and chemical shifts similar to both the monodentate pyridines and pic complex 1 with slight upfield shifts in the spectra. Complex 3 had larger downfield shifts than those observed in the other complexes. 13C NMR for complexes 2−6 showed similar chemical shifts for the picolinic acid ligand as 1.7 The peaks corresponding to the coordinated monodentate ligands were similar to the free ligands with slight downfield shifts in most cases. One exception, the 4-HP 2 + 1 complex 3, had a significant upfield shift of the 4 position carbon (166.9) compared to the free ligand (181.3). The coordinated 4-HP is similar to the chemical shift observed in 4MP suggesting the complexed 4-HP is no longer able to tautomerize to its keto 4-pyridinone form. IR confirmed the presence of the fac-[ReI(CO)3]+ core in each of the complexes. MS was performed on complex 3 in negative ion mode and yielded a molecular ion peak [m/z] of 486.8 corresponding to [M-H]− likely due to loss of the phenolic proton. All other complexes were analyzed in positive ion mode and gave corresponding peaks for their respective [M + Na]+ ion with an m/z of 495.0 (2), 525.0 (4), 510.0 (5), and 538.0 (6).

respectively, after precipitation with water (Scheme 1). Synthesis of fac-[ReI(CO)3(pic)(4-HP)], 3, was prepared by Scheme 1. Synthesis of 2 + 1 Complexes with Re and

99m

Tc

reacting complex 1 with 4-HP in MeOH and crystallized by slow evaporation to give a 36.2% isolated yield. The Re complex of 4-NP was not synthesized due to the low solution stability of the ligand.30

Figure 2. Crystal structure of 2−6 with thermal ellipsoids at 30% probability. Hydrogens and included solvent have been omitted for clarity. 1531

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Inorganic Chemistry Table 2. Selected Bond Lengths around the Re Center in Complexes 2−6 complex

Re(1)−N(1)

Re(1)−(O1)

Re(1)−N(2)

Re(1)−C(7)

Re(1)−C(8)

Re(1)−C(9)

2 3 4 5 6

2.180(2) 2.169(2) 2.162(2) 2.177(2) 2.177(2)

2.1276(17) 2.158(2) 2.1426(18) 2.144(19) 2.138(18)

2.207(2) 2.202(2) 2.203(2) 2.192(2) 2.191(2)

1.908(3) 1.901(3) 1.906(3) 1.905(3) 1.906(3)

1.917(3) 1.922(3) 1.916(3) 1.916(3) 1.924(3)

1.920(3) 1.926(3) 1.924(3) 1.932(3) 1.915(3)

Table 3. HPLC Retention Times for fac-[MI(CO)3] and 99m Tc Yields

Crystallography. Single crystals of 2−6 were obtained and analyzed by X-ray diffraction analysis. Complete experimental parameters and tables of bond angles and distances for each of these compounds can be found in the Supporting Information (SI, Tables S1−S10). In all of the structures, the fac[ReI(CO)3]+ core was arranged in a distorted octahedral orientation of ligands (Figure 2). Single crystals of each of the monodentate complexes were obtained and found to pack in the monoclinic P21/c (2 and 4), triclinic P-1 (3 and 5), or monoclinic C2/c (6) spacegroups. Selected bond lengths around Re(1) are listed in Table 2. The length of the Re(1)−C, Re(1)−O(1), Re(1)−N(1) bonds were analogous in all of the compounds. Comparison of the Re(1)−N(2) bond length, however, shows a distinct trend with increasing pKa causing a decrease in bond length (Figure 3).

complex 1a 2(a) 3(a) 4(a) 5(a) 6(a) 7(a)

R H OH OMe NH2 N(Me)2 NO2

Re r.t. (min) 11.6 13.1 8.5 13.9 13.3 14.2

99m

Tc r.t. (min) 11.8 13.5 8.7 14.2 13.6 14.5 13.2

99m

Tc % yield >98% 45% 20% 71% 98% 94% 7%

assess the changes in efficiency of complexation and stability due to changes in substitution and pyridine pKa. fac[99mTcI(OH2)(CO)3(pic)], 1a, was synthesized by reacting fac-[99mTcI(OH2)3(CO)3]+ solution with pic in a pH 8 phosphate buffer at 90 °C. This reaction solution was used directly for all further studies. Coordination studies were run with 10 mM concentrations of monodentate ligand in pH 7 (3a) or 8 (2a, 4a-7a) phosphate buffer at 90 °C for 30 min. pH 7 phosphate buffer for 3a was used due to potential deprotonation of the hydroxyl group which could change the electron density of the pyridine nitrogen. A higher pH was used to avoid loss of complexation yield due to pyridine protonation.11 Yield calculations were made by comparing peak areas of complex 1a with the [2 + 1] product (Table 3). Reactions were run in triplicate and the resulting yields were plotted against the pKa of the pyridinium species (Figure 4).

Figure 3. Relationship between pyridine basicity and Re(1)−N(2) bond length. Plot of pKa’s of pyridines for complexes 2 and 4−6 (square) and 3 (diamond). Linear regression analysis was performed both with (dashed line, R2 = 0.6905) and without (solid line, R2 = 0.9929) the complex 3 Re(1)−N(2) bond distance.

DMAP has the shortest Re(1)−N(2) bond length at 2.191(2) Å, while pyridine has a 2.207(2) Å length. 4-HP complex 3 with a monodentate pKa of 3.25 does not follow this trend due to its shorter bond length of 2.202(2) Å, which is shorter than observed for pyridine (pKa 5.23). A possible explanation for this lies in the packing exhibited in this crystal structure. There is hydrogen bonding evident between the hydroxyl O(3) and the O(2) carbonyl of a neighboring molecule. With a bond length of 2.597 Å, it is possible that this causes a significant degree of deprotonation of the hydroxyl, which would result in a partial negative charge on O(3) and increased electron density in the ring. Incorporation of the data point for the 4-HP complex gave a significant decrease in R2 value of 0.6905, however, elimination of the point results in an excellent correlation (R2 = 0.9929) between pKa and bond length. 99m Tc Labeling and Stability. Re analogs 2−6 were used for the identification of their corresponding fac-[99mTcI(CO)3]+ complexes 2a−6a by HPLC (Table 3, SI, Figures S1−S5). Synthesis of the fac-[99mTcI(CO)3]+ analogs was performed to

Figure 4. Radiochemical yield in relationship to pyridine pKa for the synthesis of complexes 2a−7a. Data is presented as % yield ± standard deviation (n = 3).

This plot showed a linear correlation between the pKa and the resulting radiochemical yield. The lower yield of the DMAP in relation to the 4-amino substituted pyridine is likely due to the pH of the reaction; DMAP, having a higher pKa, was protonated to a greater degree than the 4-AP (SI, Figure S6). It is likely that a employing a higher pKa buffer may have resulted in quantitative conversion to the 4-AP and DMAP complexes. Unlike the other pyridine complexes, synthesis of 1532

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Inorganic Chemistry 7a gave several product peaks which indicated the possibility of breakdown of the ligand before or after complexation with the 4-nitropyridine. Complexes 2a−6a were subjected to amino acid challenge studies with cysteine and histidine to determine their stability to transchelation (Table 4). Stability was not performed on

ligands in Tc and Re complexes. While the impact of basicity of pyridine was demonstrated in the 2 + 1 approach, this trend can readily be extended in the design of symmetric and asymmetric tridentate ligands containing pyridine for improved stability of 99m Tc and 186/188Re radiopharmaceuticals using the fac[MI(CO)3]+ core.

Table 4. Stability of fac-[99mTcI(CO)3]+ Complexes 2a−6a under Amino Acid Challenge (1 mM Cysteine or Histidine, 10 mM pH 7.4 Phosphate Buffer, 37 °C) Conditions

S Supporting Information *

complex

histidine

cysteine

2a 3a 4a 5a 6a

6% 55% 43% 65% 82%

3% 41% 16% 24% 68%

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ASSOCIATED CONTENT

Complete X-ray structural information for 2−6 available as a CIF file, speciation diagrams and additional characterization data of selected complexes as a PDF. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

CCDC numbers for complexes 2−6 are 1018869−1018873.

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AUTHOR INFORMATION

Corresponding Author

complex 7a due to its low yield. The amino acid challenges showed a higher stability to histidine than cysteine in all cases. Interestingly, all of the substituted ligands tested were more stable than pyridine complex 2a. After 1 h, only 6.3% of 2a remained when challenged with histidine and 2.9% with cysteine, showing the poor stability of pyridine as a monodentate ligand. When isolating 2a, formation of 1a was immediately observed indicating loss of the pyridine ligand under buffer conditions. This suggests that pyridine has a fast off rate and loss of the py ligand followed by coordination of histidine or cysteine may play a significant role in complex decomposition. In complexes 3a−6a the dissociation of the substituted pyridine ligand was not observed upon isolation, suggesting a slower off rate for the monodentate ligand. DMAP (6a) showed the highest stability of all of the complexes with 82.3% and 68.0% remaining with histidine and cysteine, respectively. This result was expected as the higher pKa of DMAP should create a stronger metal ligand bond and reduce the incidence of dissociation. Unexpectedly, the 4-HP complex 3a was also significantly more stable than pyridine and 4methoxypyridine, with 55.3% and 41.1% of 3a remaining with histidine and cysteine, respectively. This is likely due to the deprotonation of the hydroxyl group on the pyridine ring after coordination. The increased electron density likely yielded a stronger ligand than was indicated by its pKa and labeling efficiency.

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This worked was funded in part by the DOE, Radiochemistry and Radiochemistry Instrumentation Program (#DE-FG02-08ER64672) and Washington State University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Isolink kits were graciously provided by Dr. Mary Dyszlewski at Covidien. REFERENCES

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CONCLUSIONS Using a 2 + 1 labeling strategy, a range of pyridine substitutions were tested with the fac-[MI(CO)3]+ core to examine their effects on complexation. Changing substitutents on pyridine rings has a direct effect on the bond strength and stability of the resulting complexes. The magnitude of pKa was found to provide a good predicator of binding strength and complexation efficiency for the fac-[MI(CO)3]+ core. As pKa of the pyridine ligands increased, higher labeling efficiency and overall stability of the complexes was observed. Comparison of the Xray structural data from Re complexes and 99mTc studies, both delineate the importance of increasing the basicity of the pyridine donor by electron donating functional groups. Groups (e.g., −OMe and −NR2) provided a significant increase in yields and stability compared to unsubstituted pyridine. Careful selection of the pyridine substituent is critical to balance favorable pharmacokinetics properties (e.g., lipophilicity and charge) and coordination stability of pyridine functionalized 1533

DOI: 10.1021/ic502520x Inorg. Chem. 2015, 54, 1528−1534

Article

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DOI: 10.1021/ic502520x Inorg. Chem. 2015, 54, 1528−1534