Rhenium and Technetium-oxo Complexes with Thioamide Derivatives of Pyridylhydrazine Bifunctional Chelators Conjugated to the Tumour Targeting Peptides Octreotate and Cyclic-RGDfK

This research aimed to develop new tumor targeted theranostic agents taking advantage of the similarities in coordination chemistry between technetium and rhenium. A γ-emitting radioactive isotope of technetium is commonly used in diagnostic imaging, and there are two β– emitting radioactive isotopes of rhenium that have the potential to be of use in radiotherapy. Variants of the 6-hydrazinonicotinamide (HYNIC) bifunctional ligands have been prepared by appending thioamide functional groups to 6-hydrazinonicotinamide to form pyridylthiosemicarbazide ligands (SHYNIC). The new bidentate ligands were conjugated to the tumor targeting peptides Tyr3-octreotate and cyclic-RGD. The new ligands and conjugates were used to prepare well-defined {M=O}3+ complexes (where M = 99mTc or natRe or 188Re) that feature two targeting peptides attached to the single metal ion. These new SHYNIC ligands are capable of forming well-defined rhenium and technetium complexes and offer the possibility of using the 99mTc imaging and 188/186Re therapeutic matched pairs.


■ INTRODUCTION
The technetium-99m isotope has excellent properties for detection with single photon emission computed tomography (SPECT) due to its low energy and nonparticulate gamma-ray emission (t 1/2 = 6.01 h, E max = 141 keV γ-ray emission, λ < 10 pm). Despite recent concerns over production related shortages of technetium-99m and the advent of positron emission tomography technetium-99m retains its importance to nuclear medicine to the extent that the isotope is used in over 80% of nuclear imaging procedures worldwide. 1 The heavier third row Group VII congener, rhenium, has an ionic radius similar to technetium due to the lanthanide contraction. Technetium and rhenium display similar coordination chemistry often resulting in essentially isostructural technetium and rhenium complexes. It is common for technetium and rhenium complexes to be essentially isostructural. There are two isotopes of rhenium that are of potential use in targeted radiotherapeutics, rhenium-186 (t 1/2 = 89.3 h, E max = 1.07 MeV β − particle emission, 137 keV γray emission) and rhenium-188 ((t 1/2 = 16.9 h, E max = 2.12 MeV β − particle emission, 155 keV γ-ray emission). The similar coordination chemistry of technetium and rhenium offers the possibility of using their radioisotopes as an imaging ( 99m Tc) and therapeutic ( 186/188 Re) matched pair using a single targeted ligand to form essentially isostructural complexes. 2−6 One approach to targeted imaging and therapy is to incorporate appropriate metal radionuclides into coordination complexes that are attached to biological targeting vectors such as tumor targeting peptides, antibodies or antibody fragments. Peptides that feature the −RGD-(arginine-glycine-aspartic acid) fibronectin fragment such as the cyclic-RGDfK pentapeptide (cRGDfK) bind to α v β 3 integrin receptors that are overexpressed in certain invasive tumors including osteosarcomas, glioblastoma, melanomas, and breast cancer, and can be used to selectively target tumor cells. 7−16 Metabolically stabilized somatostatin analogues such as octreotide and octreotate bind to somatostatin subtype 2 receptors (sstr2) that are overexpressed in many types of neuroendocrine tumors compared to relatively low levels of expression in other tissues and organs. 17−21 Tumor targeting technetium based imaging agents can be prepared using 6-hydrazinonicotinamide (HYNIC) derivatives conjugated to targeting molecules as ligands to form technetium(III) diazenido complexes. 22−31 The HYNIC ligand forms remarkably stable technetium complexes, and manipulation of the carboxylate functional group to attach a variety of targeting molecules is generally straightforward. HYNIC binds to technetium through the terminal hydrazine nitrogen but probably forms bidentate complexes through coordination to the pyridyl nitrogen. 29,32 In all the crystallographically characterized technetium and rhenium complexes with one or more HYNIC-like ligands, such as 2-hydrazinopyridine, the pyridyl nitrogen is also coordinated to the metal center. 33 A variety of coligands such as tricine, nicotinic acid, EDDA, and phosphines (TPPTS, TPPDS, TPPMS = tri/bi/sodium triphenylphosphine tri/di/monosulfonate) are required to complete the coordination sphere and stabilize the metal oxidation state, and this leads to a high degree of uncertainty in the exact nature of the primary coordination sphere as well as challenges in ensuring structural homogeneity in the formulated product. Variation of the coligand can modify the in vivo metabolism and excretion. 34 A well-established "ternary ligand system" involves combining the HYNIC ligand with tetradentate tricine and monodentate trisodium 3,3′,3″phosphanetriyltris(benzenesulfonate) (TPPTS) coligands, but the possibility of forming multiple isomers adds complications. 33,35−39 Despite the superficial similarities in coordination chemistry between technetium and rhenium extrapolation of the HYNIC strategy to radioactive rhenium isotopes is challenging presumably due to their differences in kinetic lability and redox chemistry. 33 Some of the difficulty in isolating pure Re-HYNIC-peptide conjugates can be understood by considering the reaction of [ReO 4 ] − with 2-hydrazinopyridine (used as model for HYNIC). 33,40 This reaction results in relatively complex coordination chemistry due to the ability of the pyridylhydrazine derived ligands to coordinate as either monodentate or bidentate ligands and the existence of protic equilibria as well as the formation of complexes where two pyridylhydrazine derived units are coordinated to the rhenium ( Figure 1). 26,33,40−43 Modification of the terminal hydrazinic nitrogen of hydrazinopyridine to incorporate an additional thiourea functional group results in a ligand system that is capable of forming well-defined, very stable complexes with {Re V O} 3+ cores while retaining the bioconjugation possibilities well established for HYNIC. 44,45 A preliminary communication reported the structural characterization of a Re V -oxo complex featuring two pyridylphenylthiocarbazide (SHYNIC) ligands ( Figure 1). 44 In this manuscript we extend this concept by synthesizing a family of different substituted pyridylthiosemicarbazide ligands with carboxylate or ester functional groups that were used to tether octreotate and cyclic-RGD peptides to the ligands. The new ligands were used to prepare {MO} 3+ complexes (where M = Tc or Re) that feature two targeting peptides attached to the single metal ion. These modified HYNIC ligands are capable of forming well-defined rhenium and technetium complexes and offer the possibility of using the two radionuclides as imaging and therapeutic matched pairs. ■ RESULTS AND DISCUSSION Synthesis of H 2 L 1−4 , and Their Ester Derivatives, H 2 L 1−4 (OMe) and {ReO} 3+ Complexes. Synthesis of 6hydrazinonictonic acid (HYNIC), 2, required treatment of 6chloronicotinic acid (1) with aqueous hydrazine. 47 Ligands H 2 L 1 to H 2 L 4 were prepared by reaction of either the ethyl, tertbutyl, phenyl, or nitrophenyl isothiocyanate with 6-hydrazinonictonic acid (1) The rhenium complexes of the methyl ester derivatives of H 2 L 1−3 complexes, [ReO(HL 1−3 (OMe)) 2 ] + , can be prepared by reaction of the either trans-[ReOCl 3 (PPh 3 ) 2 ] or [tBu 4 N]-[ReOCl 4 ] with the two equivalents of ligand (Scheme 2). The IR spectra for the three complexes, [ReO(H 2 L 1−3 (OMe)) 2 ] + , display medium intensity bands at ν ̅ 960−963 cm −1 characteristic of ReO stretches. 48 Bands, which occur between ν ̅ 1553 and 1557 cm −1 due to carbonyl stretching of the ester functional group, shift approximately 150 cm −1 lower in energy when compared to the metal-free ligands.
Analysis of the complexes by 1 H NMR data reveals that the two coordinated ligands are magnetically equivalent, with three resonances at δ 8. 63, 8.25, and 7.86 ppm corresponding to the six pyridinyl CH protons for [ReO(HL 1 (OMe)) 2 ] + , and similar resonances for the phenyl and tert-butyl derivatives. The pyridine proton which is closest to the rhenium ion shifts from δ 6.55 ppm in free ligand to δ 7.86 ppm in the complex. The methyl ester functional group gives rise to singlets at δ 3.89 (DMSO-d 6 ), 3.86 (CHCl 3 -d), and 3.92 (DMSO-d 6 ) for complexes [ReO((HL 1,2,3 )(OMe)) 2 ] + respectively. The [ReO-(HL 1 (OMe)) 2 ] + complex was stable to cysteine and histidine challenge experiments with very little decomposition evident (<5%), as detected by analysis by HPLC and UV/vis spectroscopy, when incubated at 37°C for 24 h in the presence of a 100-fold excess of cysteine and histidine.
Red crystals of [ReO(HL 1 (OMe)) 2 ]CF 3 CO 2 suitable for Xray crystallographic analysis were obtained by evaporation of a solution of the compound that had been purified by semipreparative HPLC using an aqueous/CH 3 CN mobile phase with 0.1% trifluoracetic acid (Figure 2a). The compound crystallizes in the triclinic space group, P1̅ , and the rhenium ion is in a distorted square pyramidal environment with the oxo group in the apical position relative to the pseudo basal plane of two five-membered chelate rings. Each thiocarbahydrazide functional group is doubly deprotonated and serves as a dianionic ligand fragment and a N,N/S,S trans configuration about the Re-oxo. The selective formation of the N,N/S,S trans geometric isomer presumably reflects a strong "trans effect", although steric requirements may also play some role. 49−51 Protonation of the pyridyl nitrogen atom in each ligand results in each ligand having a single negative charge and resulting an overall monocationic complex. The two pyridinium protons and the hydrogen atoms of the ethylamino functional group are involved in hydrogen bonds interactions leading to a hydrogen bonded centrosymmetric dimer (Figure 2b) with the two remaining H-bond donors capped by water molecules.
The Re−O1 bond distance (1.679(3) Å) is typical for fivecoordinate, rhenium(V)-monooxo complexes and consistent with IR spectroscopy (ReO, ν ̅ 963 cm −1 ). 52 51,57,58 The potential of the substituted pyridylthiosemicarbazide (SHYNIC) ligands H 2 L 1−4 to be modified with amino acids using standard solid phase peptide synthesis techniques was first accomplished by attaching L-lysine to H 2 L 1 to give H 2 L 1 (Lys). The doubly N-protected lysine derivative, N α -tBoc-N ε -Fmoc-L-Lys, was immobilized on chlorotrityl resin, and the N ε -Fmoc group was removed by treatment with piperidine. The ligand, H 2 L 1 , was added to the resin in a mixture of DMF followed by the coupling agent HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate) in the presence of N,N-diisopropylethylamine (DIPEA). The product was cleaved from the resin using trifluoroacetic acid that also resulted in the deprotection of the N α -t-butoxycarbonyl group (Scheme 3). This lysine amino acid conjugate provides an amino acid with an appended chelator, which with appropriate protecting groups, could be incorporated into biological targeting molecules with total site specificity via solid-phase peptide synthesis. 59−63 The rhenium complex, [ReO(HL 1 (Lys)) 2 ] + , was prepared by adding either trans-[ReOCl 3 (PPh 3 ) 2 ] or [tBuN][ReOCl 4 ] suspended in DMF to the reaction mixture, while the ligand remained immobilized on the resin. Performing the complexation while the ligand remained immobilized on the resin and with the amino group still protected ensured that the functional groups of the lysine did not complicate the coordination chemistry. When green trans-[ReOCl 3 (PPh 3 ) 2 ] is used as the starting material the green colored suspension gradually changes to colorless, and the resin beads turn dark red indicative of the formation of [ReO(HL 1 (Lys)) 2 ] + . After being stirred at room temperature, the resin was washed with dimethylformamide and dichloromethane and cleaved off the resin with a 10% trifluoroacetic acid/dichloromethane mixture (Scheme 3). Analysis of [ReO(HL 1 (Lys)) 2 ] + complex by electrospray ionization mass spectrometry (ESI-MS) reveals the expected peaks. Analysis by 1 H NMR shows the singlet attributed to the aromatic proton on the pyridine ring (pyH 2 ) shifts upon coordination to the metal center from δ 8.48 in H 2 L 1 (Lys) to 8.58 ppm in [ReO(HL 1 (Lys)) 2 ] + . The four downfield 13     An intramolecular disulfide bridge between the second and seventh cysteine residues in Tyr 3 -octreotate improves the metabolic stability of the peptide, and this disulfide is often introduced by oxidation of the linear octapeptide with 2,2′dithiodipyridine. Unfortunately the bioconjugation of ligands H 2 L 1−3 to Tyr 3 -octreotate was complicated by degradation of the pyridylthiocarbazide (SHYNIC) ligands (H 2 L 1−3 ) in the presence of the two cysteine thiol containing residues in the linear peptide, leading to loss of H 2 S identified in the ESI-MS by a loss of 34 atomic mass units. This loss of H 2 S from the ligand was most prominent during reactions attempting intramolecular oxidation of thiol groups in the two cysteine residues. The loss of H 2 S results in the formation of a carbodiimide form of the SHYNIC ligands. The formation of carbodiimides from thioureas is well-known. 65 This degradation and loss of sulfur were not observed for RGD-based conjugates, suggesting that in the case of the octreotate conjugates, thiocarbazide-thiol-disulfide interchange/scrambling promotes the loss of H 2 S from the ligands (Scheme 5).
As conventional off-resin oxidative cyclization methodologies were inadequate for this synthesis, H 2 L 1−3 (Tyr 3 -Oct) was prepared entirely on solid support, where intramolecular oxidation/cyclization preceded bioconjugation of H 2 L 1−3 . The eight-residue peptide was synthesized by sequential addition of the amino acid residues via solid-phase peptide synthesis, using acetamidomethyl (Acm) protected cysteine residues followed by in situ Acm removal and simultaneous disulfide bond formation using thallium(III) trifluoroacetate. 66 Following cyclization, the preactivated SHYNIC derivative (H 2 L 1−3 ) is 13.2651 (7) final R indices (all data) a Crystals were grown from a concentrated solution of the complex in methanol.  4 ] in methanol (Scheme 6). The on-resin approach is potentially of interest in producing radioactive complexes in high specific activity as unreacted [ReO 4 ] − and other impurities such as colloidal rhenium could be readily removed by filtration of the resin. The pure complex can be cleaved from the resin with 50% trifluoracetic acid and is stable to this relatively high concentration of acid. Analysis by HPLC and ESI-MS confirmed the identity of the complexes, with the [ReO-(HL 1−3 (Tyr 3 -Oct)) 2 ] + complexes showing signals in the ESI-MS that could be attributed to the 3+ molecular ion with expected rhenium isotope peak patterns ( Figure 3).

Article
Preparation of [ 99m TcO(HL 1−3 (cRGDfK)) 2 ] + and [ 99m TcO-(HL 1−3 (Tyr 3 -Oct)) 2 ] + involved adding an excess of the appropriate ligand dissolved in aqueous sodium chloride (0.5 mg mL −1 , 0.9% NaCl, pH 7.4) to a mixture of [ 99m TcO 4 ] − that has been reduced by tin(II) chloride in 0.1 M HCl in the presence of tartrate (pH 1−4) at room temperature. The radiolabeled 99m Tc complexes were characterized by analysis by HPLC equipped with a radioactivity detector, and the elution profiles were compared to the analogous nonradioactive rhenium compounds (detected by UV absorbance). The close correlation between the retention times of the rhenium and technetium complexes strongly suggests that the complexes are isostructural ( Table 3). The small difference in the retention times between the traces for 99m Tc and Re complexes is, in part, due to the detector configurations but could also reflect the difference in polarity between the oxorhenium(V) and oxotechnetium(V) cores.
The stability of [ 99m TcO(HL 3 (Tyr 3 -Oct)) 2 ] + was assessed by incubation in human plasma at 37°C. The complex was stable for at least 2 h with only small amounts of degradation products (<5%) evident that, based on their retention times in analytical HPLC, are most likely due to degradation of the peptide.

■ CONCLUDING REMARKS
The new pyridylthiocarbazide ligands (SHYNIC, H 2 L 1−3 ) described here offer a useful alternative to the standard HYNIC system. While HYNIC has proved a very successful and versatile bifunctional ligand for 99m Tc coligands are required to complete the coordination sphere of the metal ion and extrapolation to radioactive rhenium isotopes has been challenging. 3,63 This family of bidentate ligands form stable complexes with the {ReO} 3+ core with two ligands coordinated to a single metal ion. A rhenium complex with a methyl ester functional group has been characterized by X-ray crystallography and features the rhenium ion in a distorted square pyramidal environment with the oxo group in the apical position relative to the pseudo basal plane of two fivemembered chelate rings with a N,N/S,S trans configuration about the Re-oxo core. The basic ligands have been decorated with the tumor targeting peptides cyclic-RGD and Tyr 3octreotate, and these conjugates form complexes with rhenium to give well-defined single species, [ReO((HL 1−3 )(cRGDfK)) 2 ] and [ReO((HL 1−3 )(Tyr 3 -octreotate)) 2 ], without having to add coligands resulting in the formation of a single structural and geometrical isomer. It is possible to form the rhenium complexes using either standard solution chemistry or "onresin", and the latter approach may prove useful in isolating radioactive 188/186 Re analogues in high specific activity. These complexes feature two targeting peptides separated by 14 chemical bonds, and there is evidence that molecules containing more than one targeting peptide, sometimes referred to as bivalent, can display enhanced receptor binding due to simultaneous binding to more than one receptor on the surface on any given cell. 14 Comparison of HPLC profiles suggests the rhenium and technetium complexes are isostructural. The complexes described in this manuscript have two ligands coordinated to a single metal ion, whereas conventional HYNIC systems involve one HYNIC ligand binding to one metal ion. It is likely the two different systems will exhibit quite different biodistribution in vivo. These new systems warrant further investigation as potential theranostic agents employing an imaging ( 99m Tc) and therapeutic ( 186/188 Re) matched pair for a single targeted agent.
General Experimental. All reagents were purchased from standard commercial sources. Nuclear magnetic resonance (NMR) spectra were acquired on either an Agilent 400-MR ( 1 H NMR at 400 MHz and 13 C{ 1 H} NMR at 101 MHz) or a Varian FT-NMR 500 spectrometer ( 1 H NMR at 500 MHz and 13 C{ 1 H} NMR at 126 MHz) at 298 K. Chemical shifts were referenced to residual solvent peaks and are quoted in ppm relative to TMS.
Linear protected RGDfK peptide (Arg(Pbf)-Gly(tBoc)-Asp(OtBu)-dPhe-Lys(tBoc)) was synthesized manually using standard Fmoc solid phase peptide synthesis (SPPS) procedures on the 2-chlorotrityl chloride resin. The linear pentapeptide was cleaved from the resin (with retention of protecting groups) using 1% TFA in dichloromethane and shaking for 40 min. The mixture was filtered and the filtrate was reduced in volume to afford crude linear product. Cyclization involved reacting the crude material in a mixture of dichloromethane (1 mg mL −1 ), HATU (0.9 equiv), and DIPEA (6 equiv) at RT for 2 h, then evaporation to dryness
Analytical HPLC traces of radiolabeled 188 Re compounds were acquired using an Agilent 1200 LC system with in-line UV and gamma detection (Flow-Count, LabLogic). Peak separation was achieved using an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm), with column 1 and system F: Gradient elution of Buffer A (0.1% TFA in H 2 O) and Buffer B (0.1% TFA in CH 3 CN) from 0 to 100% B over 20 min and UV detection at λ 220 nm.
Analytical HPLC traces of radiolabeled 99m Tc compounds were acquired using a Shimadzu 10 AVP UV−visible spectrophotometer (Shimadzu, Kyoto, Japan) and a sodium iodide scintillation detector with two LC-10ATVP solvent delivery systems for solvents A and B. Peak separation was achieved using Column 7: Nacalai Tesque Cosomosil 5C18-AR Waters column (4.6 × 150 mm, 5 μm) (Kyoto, Japan) at a flow rate of 1 mL min −1 . Gradient elution followed System C: Gradient elution of Buffer A (0.1% TFA in H 2 O) and Buffer B (0.1% TFA in CH 3 CN) from 0 to 100% B over 20 min and UV detection at λ 254 nm.
X-ray structure determination and refinement was obtained for [ReO(HL 1 (OMe)) 2 ]TFA on an Oxford Diffraction Super-Nova CCD diffractometer using Cu−Kα radiation, and the temperature during data collection was maintained at 130.0(1) using an Oxford Cryosystems cooling device. The structure was solved by direct methods using SHELXT and refined using least-squares methods using SHELXL. 74,75 Thermal ellipsoid plots were generated using ORTEP-3 integrated within the WINGX suite of programs. 76 The trifluoroacetate counterion, although recognizable from the difference electron density maps, was badly disordered and could not be modeled satisfactorily. Application of the Squeeze procedure gave a void volume of 272 Å 3 containing 127 electrons, consistent with the presence of two trifluoroacetate anions per unit cell. 77 The charge on the complex is unambiguously (+1) given the presence of the two pyridinium protons which are involved in intramolecular hydrogen bonds and the ethylamino protons which are also involved in hydrogen bonds. The crystallographic data has been deposited in the Cambridge Structural Database (CCDC 1543360).

Inorganic Chemistry
Article mg mL −1 ) in a separate evacuated vial, and a 0.5 mL aliquot was taken from both solutions and mixed together. To the solution was added [ 99m TcO 4 ] − (0.1 mL in 0.9% saline, 108 MBq). The conjugated peptide, H 2 L 1−4 (cRGDfK) or H 2 L 1−3 (Tyr 3 -Oct), was dissolved in degassed Milli-Q water (1 mg mL −1 ), and 100 μL of this mixture was added to the technetium solution. The sample was neutralized with NaHCO 3 (pH 6.5, approximately 55 μL) then filtered or allowed to react without neutralizing at ambient temperature for 30 to 120 min. The samples were filtered (Supelco, Iso-disc Filter, 4 mm x 0.45 μm). Radiochemical yields were evaluated by reverse-phase high-performance liquid chromatography (Column 7, System C).
Stability Studies in Human Serum. Human blood samples were centrifuged with a Heraeus Labofuge 6000 centrifuge at 3000g for 10 min (Heraeus, Hanau, Germany). Radioactivity readings for serum stability studies were taken with a Capintec CRC-35R dose calibrator (Capintec, New Jersey, USA) and were measured in MBq. Centrifugation of radioactive compounds was undertaken using an Eppendorf 5415 D centrifuge (Eppendorf, Hamburg, Germany). Partition coefficient data were collected with a PerkinElmer, Wizard 1470 (PerkinElmer, Massachusetts, USA) automatic γ counter, which measured the radioactive decay of each sample in counts per minute (cpm).
For serum stability studies, blood from a healthy male (20 mL) was centrifuged (10 min, 3000 rpm) to separate blood plasma and red blood cells. The plasma was transferred to a separate vial and the red blood cells were discarded. An aliquot of plasma (0.6 mL) was added to labeled compound, [ 99m Tc(HL 3 (Tyr 3 -Oct)) 2 ] + (0.15 mL), the radioactivity was monitored and the mixture was then incubated at 37°C. Aliquots (0.1 mL) of the mixture were removed from heating at 10 min and after 2 h. Acetonitrile (0.1 mL) was added to the serum/tracer mix to precipitate serum proteins. The suspension was shaken for 5 min and then centrifuged (5 min, 13 200 rpm). The radioactivity of the supernatant and pellet was recorded. The supernatant (20 μL) was analyzed by analytical RP-HPLC (Column 7, System C) for UV and radioactivity analysis and the pellet were washed with acetonitrile (3 × 0.1 mL), and radioactivity levels were again recorded (radioactivity levels of the pellet were negligible).

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01247.