The Synthesis of a Bis(thiosemicarbazone) Macrocyclic Ligand and the Mn(II), Co(II), Zn(II) and 68Ga(III) Complexes

A 1,4,7,10-tetraazacyclododecane (cyclen) variant bearing two thiosemicarbazone pendant groups has been prepared. The ligand forms complexes with Mn2+, Co2+ and Zn2+. X-ray crystallography of the Mn2+, Co2+ and Zn2+ complexes showed that the ligand provides a six-coordinate environment for the metal ions. The Mn2+ and Zn2+ complexes exist in the solid state as racemic mixtures of the Δ(δ,δ,δ,δ)/Λ(λ,λ,λ,λ) and Δ(λ,λ,λ,λ)/Λ(δ,δ,δ,δ) diastereomers, and the Co2+ complex exists as the Δ(δ,δ,δ,δ)/Λ(λ,λ,λ,λ) and Δ(λ,λ,λ,δ)/Λ(δ,δ,δ,λ) diastereomers. Density functional theory calculations indicated that the relative energies of the diastereomers are within 10 kJ mol−1. Magnetic susceptibility of the complexes indicated that both the Mn2+ and Co2+ ions are high spin. The ligand was radiolabelled with gallium-68, in the interest of developing new positron emission tomography imaging agents, which produced a single species in high radiochemical purity (>95%) at 90 °C for 10 min.


Introduction
Thiosemicarbazone functional groups are versatile N,S donors that can coordinate metal ions as neutral or anionic ligands, with the resulting complexes displaying diverse coordination chemistry. In general, thiosemicarbazones are easy to synthesise by way of a condensation reaction between a thiosemicarbazide and an aldehyde or ketone. The structures can be modified in multiple ways, allowing for the generation of tri-, tetra-, pentaor even hexadentate ligands, as well as multinuclear complexes and coordination polymers [1][2][3][4][5][6][7][8][9]. Modifications to the thiosemicarbazone substituents can also result in dramatic changes to the structural, physical and biological properties of the metal complexes [10,11].
Hybrid ligands containing thiosemicarbazone groups and additional donor atoms can introduce modifiable properties to the complexes for a variety of biological applications [12][13][14]. N-heterocyclic thiosemicarbazones have been investigated for their pharmacological properties, which have shown that the metal complexes can display bioactivities which differ from those of either the ligand or the metal ion [15][16][17][18]. Lipophilic Mn 2+ , Zn 2+ and Ga 3+ complexes have demonstrated anti-tumour activity due to their ability to facilitate intracellular delivery of the free ligand upon metal dissociation or transmetallation to the Fe 3+ or Cu 2+ complexes [19][20][21][22][23][24]. Cobalt thiosemicarbazonato complexes have been isolated in the Co 2+ and Co 3+ oxidation states and have been investigated as redox-active prodrugs for hypoxia targeting and as anti-bacterial and anti-cancer agents [25][26][27]. Zinc ions have been found to play an important role in medicine, with compounds developed for treating neurodegeneration as well as anti-diabetic, anti-tumour, anti-bacterial, anti-microbial and anti-inflammatory agents [16,[28][29][30][31][32][33][34].

NMR Studies
The complex [ZnHL](BPh 4 ) was characterised by 1 H and 13 C{ 1 H} NMR spectroscopy. The presence of one equivalent of tetraphenylborate and a single hydrazinic nitrogen proton (δ = 10.01 ppm) indicated deprotonation of the ligand at the hydrazinic nitrogen of the coordinated pendant arm. The ligand resonances in the 1 H NMR spectrum for the thiosemicarbazonato coordinated pendant arm of [ZnHL](BPh 4 ) have shifted compared to the spectrum for H 2 L and the non-coordinated arm ( Figure 1). For example, the methyl group in the pendant arm at δ = 1.96 ppm in the 1 H NMR spectrum of H 2 L splits into two peaks at δ = 1.96 and 2.03 ppm for the complex. The two methyl groups attached to the macrocycle shifted from δ = 2.45 ppm for H 2 L to δ = 2.31 ppm for [ZnHL](BPh 4 ). A significant shift was observed for the terminal NH proton of the thiosemicarbazonato arm from δ = 8.22 ppm to 6.56 ppm. Although the signals due to the protons of the macrocycle of [ZnHL](BPh 4 ) (δ = 2.40-3.04 ppm) are somewhat broad, variable-temperature analysis of the 1 H NMR spectra indicated that, unlike H 2 L, this was not temperature dependent ( Figure S6).  Figure S6).

X-Ray Crystal Structures of [MnHL](BPh4), [CoHL](BPh4) and [ZnHL](BPh4)
The coordination compounds [MnHL](BPh4), [CoHL](BPh4)·(C3H6O) and [ZnHL](BPh4)·1.67(C3H6O) crystallised in the monoclinic P21/c space group. Each asymmetric unit contains two discrete diastereomeric complexes, [M1HL] + and [M2HL] + , that are present as racemic mixtures of enantiomers in the unit cell. A single pendant thiosemicarbazonato arm is coordinated to the M 2+ centre through the sulfur (S1) and azomethinic (Nazo) nitrogen donor atoms (N5), along with the four macrocyclic (Nmac) nitrogen atoms (N1, N2, N3 and N4), forming six five-membered chelate rings. The Mn 2+ , Co 2+ and Zn 2+ structures are consistent with the loss of one proton from the ligand. The C12-S1 bond lengths for the Mn 2+ (1.745(2) Å), Co 2+ Figure 2. A list of metal to donor bond lengths is given in Table 2. The donor atoms N1, N2, N3, N4, N5 and S1 are analogous to the donor atoms N11, N12, N13, N14, N15 and S3 of the Mn2, Co2 and Zn2 complexes.   shown in Figure 2. A list of metal to donor bond lengths is given in Table 2. The donor atoms N1, N2, N3, N4, N5 and S1 are analogous to the donor atoms N11, N12, N13, N14, N15 and S3 of the Mn2, Co2 and Zn2 complexes.  The bonds between the metal ions and N3/N13 are significantly longer than the other bonds between the metal and nitrogen donor atoms of the macrocycle. This is presumably a result of the steric effects of the uncoordinated thiosemicarbazone pendant arm. The metal to sulfur donor bond lengths are typical of Mn 2+ and Zn 2+ thiosemicarbazonato complexes [11,20]. The Mn1, Mn2, Zn1, Zn2 and Co1 ions sit above the plane defined by the four nitrogen atoms of the macrocycle by 1.1 Å, while the Co2 ion sits above the plane by 1.0 Å. The bonds between the metal ions and N3/N13 are significantly longer than the other bonds between the metal and nitrogen donor atoms of the macrocycle. This is presumably a result of the steric effects of the uncoordinated thiosemicarbazone pendant arm. The metal to sulfur donor bond lengths are typical of Mn 2+ and Zn 2+ thiosemicarbazonato complexes [11,20]. The Mn1, Mn2, Zn1, Zn2 and Co1 ions sit above the plane defined by the four nitrogen atoms of the macrocycle by 1.1 Å, while the Co2 ion sits above the plane by 1.0 Å.

Magnetic Susceptibility
The distorted coordination geometries of the [MnHL](BPh 4 ) and [CoHL](BPh 4 ) complexes suggested that the metal ions were high-spin 3d 5 and 3d 7 , respectively. To confirm this hypothesis, the magnetic susceptibility of both the Mn 2+ and Co 2+ complexes were measured. The The difference from the spin-only value is due to the mixing of angular momentum from the excited state and spin-orbit coupling [81]. The Co 2+ value indicates a spin quartet ground state, obtained from six-coordinate octahedral or trigonal prismatic geometry [82].

Density Functional Theory Calculations
Complexes incorporating the cyclen scaffold with pendant groups are known to have multiple stereoisomeric forms that result from the combination of the two chiral elements [83]. The diastereomers can have distinctly different coordination geometries and properties [77,84]. Furthermore, formation of a predominant diastereoisomer can result when there is a free-energy difference between the diastereomers [85,86]. Density functional theory (DFT) calculations were used to investigate the energetics of each species identified from the X-ray crystallography. The calculations were performed using the Becke, 3-parameter, Lee-Yang-Parr (B3LYP) functional for all complexes investigated. The standard Ahlrichs valence triple-ξ including polarization functions (TZVP) basis set was used for the high-spin Co 2+ complexes and the DGDZVP basis set for the Zn 2+ complexes. These combinations of functional and basis sets were chosen because they have shown good agreement with experimental values for similar combinations of ligands and metals [87,88]. For comparison with the XRD data, values for selected bond lengths of the optimised structures are shown in  Figures S16-S23. A bond length difference of~0.074 Å for Zn-S and~0.078 Å for Zn-N mac and Zn-N azo was observed between the DFT optimised and XRD experimental values that was attributed to solvent effects. DFT analysis of the [ZnHL] + complexes in the presence of water indicated four energy minima corresponding to the following diastereomeric pairs: ∆(δ,δ,δ,δ) and Λ(λ,λ,λ,λ) for [Zn1HL] + , and ∆(λ,λ,λ,λ) and Λ(δ,δ,δ,δ) for [Zn2HL] + . According to these calculations, the minimum energy conformation corresponds to the Λ(λ,λ,λ,λ) isomer, with the relative energies of the ∆(δ,δ,δ,δ), ∆(λ,λ,λ,λ) and Λ(δ,δ,δ,δ) isomers being 0.34, 1.27 and 2.53 kJ mol −1 , respectively. The optimisation of the Co 2+ structures indicated that the minimum energy conformation corresponds to the ∆(δ,δ,δ,δ) isomer, with the relative energies of the Λ(λ,λ,λ,λ), ∆(λ,λ,λ,δ) and Λ(δ,δ,δ,λ) isomers being 4.17, 8.14 and 8.14 kJ mol −1 , respectively.

Radiolabelling with 68 Ga 3+
Preliminary radiolabelling studies were performed with 68 Ga 3+ eluted from a 68 Ge/ 68 Ga generator. The eluate was buffered with 1 M sodium acetate to pH 3.5 or 6 and reacted with H 2 L (0.5, 5, 50 and 500 µM) at ambient temperature, 40 and 90 • C for 10 min. The 68 min half-life of 68 Ga 3+ necessitates relatively short reaction times. The reaction mixtures were analysed by radio-HPLC, which showed a single product with a retention time (R T ) of 5.3 min while there was negligible retention of the [ 68 Ga]Ga 3+ ion on the C18 column (R T = 1.5 min). The synthesis of the non-radioactive Ga 3+ complex was attempted but isolation was unsuccessful. The radiolabelling reaction with DOTA was performed previously under the same conditions and used as a comparison [49]. The percentage of 68 Ga 3+ incorporation is dependent on ligand concentration, pH and temperature. 68 Ga 3+ incorporation was investigated at both pH 3.5 and pH 6 at 50 µM with an activity to ligand ratio of 0.2 MBq nmol −1 (Figure 5). A radiochemical yield (RCY) > 95% was achieved at pH 6 at both 40 and 90 • C but required a ligand concentration of 500 µM (Table 5). At pH 3.5, a RCY > 95% was achieved with a ligand concentration of 50 µM at 90 • C.

Radiolabelling with 68 Ga 3+
Preliminary radiolabelling studies were performed with 68 Ga 3+ eluted from 68 Ge/ 68 Ga generator. The eluate was buffered with 1 M sodium acetate to pH 3.5 or 6 an reacted with H2L (0.5, 5, 50 and 500 µM) at ambient temperature, 40 and 90 °C for min. The 68 min half-life of 68 Ga 3+ necessitates relatively short reaction times. The rea tion mixtures were analysed by radio-HPLC, which showed a single product with a r tention time (RT) of 5.3 min while there was negligible retention of the [ 68 Ga]Ga 3+ ion o the C18 column (RT = 1.5 min). The synthesis of the non-radioactive Ga 3+ complex was a tempted but isolation was unsuccessful. The radiolabelling reaction with DOTA w performed previously under the same conditions and used as a comparison [49]. Th percentage of 68 Ga 3+ incorporation is dependent on ligand concentration, pH and tem perature. 68 Ga 3+ incorporation was investigated at both pH 3.5 and pH 6 at 50 µM wi an activity to ligand ratio of 0.2 MBq nmol −1 (Figure 5). A radiochemical yield (RCY) 95% was achieved at pH 6 at both 40 and 90 °C but required a ligand concentration 500 µM (Table 5). At pH 3.5, a RCY > 95% was achieved with a ligand concentration 50 µM at 90 °C.  Under similar conditions, DOTA required concentrations of 50 µM or above to reach yields > 95%, similar to H 2 L. At pH 6, a RCY > 95% for DOTA was achieved with a concentration of 500 µM at 25 • C or 50 µM at 90 • C [49]. The RCY for DOTA with a concentration of 50 µM at 25 • C is~87% whereas H 2 L achieved only 2% RCY under the same conditions. These results demonstrate that H 2 L radiolabelling with 68 Ga 3+ is temperature dependent at both pH values studied, and that it does not possess the radiolabelling efficiency properties to be a potential alternative chelator for 68 Ga 3+ radiolabelling. This is perhaps unsurprising given the combination of soft base sulfur donor atoms and the hard acid Ga 3+ [89]. A ligand design incorporating oxygen donor-containing semicarbazone pendant arms may be better suited for use in 68 Ga 3+ radiopharmaceuticals [90].

General Procedures
Reagents were purchased from standard commercial sources unless otherwise stated and used without further purification. Cyclen was purchased from Strem Chemicals (Newburyport, MA, USA). Nuclear magnetic resonance (NMR) data were collected on a Bruker AVANCE III 600 ( 1 H at 600.27 MHz, 13 C{ 1 H} at 150.95 MHz) (Bruker, Billerica, MA, USA). Spectra were processed using MestReNova 10.0 software. DMSO-d 6 was obtained from Cambridge Isotope Laboratories Inc. Chemical shifts (δ) are reported in parts per million (ppm) with respect to TMS and are referenced to residual solvent peaks. Coupling constants (J) are reported in Hz. Unless specified, all NMR spectra were recorded at 25 • C. Low-resolution mass spectrometry (LR-MS) was carried out using an Agilent 1260 Infinity liquid chromatograph system coupled with a 6120 series quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) in MeOH using ESI. High-resolution mass spectrometry (HR-MS) was carried out with an Agilent 6540 UHD Accurate Mass Q-TOF LCMS (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was fitted with the Agilent Jet Stream Source using ESI. Positive detection is shown by the charge on the ion, e.g., [M + H] + for a positive protonated ion. All calculated values were determined using the PerkinElmer software ChemDraw ® Professional 19.0 to four decimal places. HPLC traces of both radiolabelled and non-radiolabelled complexes were acquired using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) with a Phenomenex Luna C18(2) column (4.6 mm × 150 mm, 5 µm), a 1 mL/min flow rate gradient elution of 0.05% TFA in 5% MeCN in H 2 O to 100% MeCN over 15 min at 25 • C with UV spectroscopic detection at 254 nm and 280 nm. Data were processed and analysed using Laura radio chromatography software (Lablogic, Brandon, FL, USA). Magnetic susceptibility was measured at room temperature by calibrating a Johnson Matthey MSB balance with [Ni(en) 3 ]S 2 O 3 at 295 K and diamagnetic corrections of the paramagnetic susceptibilities were calculated using standard Pascal's constants [91,92].

Radiolabelling with 68 Ga
68 Ga was eluted from an Eckert and Ziegler 68 Ge/ 68 Ga generator system (Eckert and Ziegler, Berlin, Germany). Aqueous HCl solution (0.1 M, 5 mL) was passed through the generator and the eluate was collected in five 1 mL fractions. Aqueous NaOAc (1 M) was added to the fourth fraction (1 mL, containing~77.6 MBq 68 Ga) to increase the pH to either pH 3.5 or pH 6. Aliquots from the pH adjusted fraction were used for radiolabelling reactions. H 2 L was dissolved in DMSO (1 mg/mL) and diluted with ultrapure water. 68 Ga (15 µL,~1.16 MBq in pH adjusted solution) was added to chelator solutions (105 µL) to provide solutions with chelator concentrations ranging from 0.5-500 µM and the final reaction solution was incubated at 25 • C, 40 • C or 90 • C for 10 min. The reaction solutions were analysed via radio-HPLC (5-20 µL injection).

Single-Crystal X-ray Diffraction Procedure
Low-temperature (123 K or 173 K) X-ray intensity data were collected using a Rigaku XtaLAB Synergy diffractometer (Rigaku Oxford Diffraction, Chalgrove, Oxford, United Kingdom) fitted with a Hypix6000HE hybrid photon counting detector and MoKα (λ = 0.71073 Å) or CuKα (λ = 1.54184 Å) radiation. Data were processed, including a multiscan absorption correction, using the proprietary diffractometer software package CrysAlisPro v1.171.39.46 [93]. The structure was solved by conventional methods and refined on F 2 using full matrix least squares using the SHELX 2018/3 software suite [94]. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms attached to carbon were placed in calculated positions and were refined using a riding model. The positions of the acidic hydrogen atoms attached to nitrogen were initially located in the difference Fourier map but were included in calculated positions and refined using a riding model. In the [CoHL](BPh 4 ) and [ZnHL](BPh 4 ) complexes, solvent molecules were successfully modelled. However, after refinement of the primary molecule [MnHL](BPh 4 ), residual electron density was assumed to be isolated solvent molecules located in the crystal lattice. These were accounted for using PLATON/SQUEEZE [95]. CSD reference numbers 2072659-2072661.

DFT Calculations
Density functional theory (DFT) calculations were performed using the Gaussian 16 program package with the Becke, 3-parameter, Lee-Yang-Parr (B3LYP) functional and the DGDZVP basis set for the Zn 2+ complexes [96][97][98][99][100]. The B3LYP functional and the standard Ahlrichs valence triple-ξ including polarization functions (TZVP) basis set were used for the high-spin Co 2+ complexes [101,102]. The geometries of the various complexes were fully optimised without imposing any symmetry constraint. No imaginary frequencies were found at the optimised molecular geometries, which indicate that they are real minima of the potential energy surface. The complexes were optimised in aqueous solution by using the polarizable continuum model with the integral equation formalism variant (IEFPCM), which creates a solvent cavity via a set of overlapping spheres [103]. The calculated relative energies of the complexes include nonpotential energy contributions. Calculation results were visualized and interpreted using GaussView version 6.1.1 [104].

Supplementary Materials:
The following are available online: 1 H and 13 C{ 1 H} NMR spectra, ESI-MS spectra, HPLC spectra and computational methods.