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

Abstract

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.

Short abstract

New pyridylthiosemicarbazide ligands conjugated to the tumor targeting peptides octreotate and cyclic-RGD peptides were used to form {M=O}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 offering the possibility of using radionuclides of technetium and rhenium as imaging and 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 γ-ray 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 (^99mTc) and therapeutic (^186/188Re) 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_vβ₃ 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 [ReO₄]⁻ 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}

Figure 1.

(a) 6-Hydrazinonicotinic acid. (b) Metal complex (M = Tc or Re) with 6-hydrazinonicotinimide (HYNIC) acting as a monodentate ligand. It is necessary to complete the coordination sphere with coligands (L). (c) Metal complex (M = Tc or Re) with 6-hydrazinonicotinimide (HYNIC) acting as a bidentate ligand. (d) Pyridylphenylthiocarbazide (SHYNIC) ligand. (e) Re^V-oxo complex featuring two SHYNIC ligands.⁴⁴

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^VO}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 H₂L^1–4, and Their Ester Derivatives, H₂L^1–4(OMe) and {ReO}³⁺ Complexes

Synthesis of 6-hydrazinonictonic acid (HYNIC), 2, required treatment of 6-chloronicotinic acid (1) with aqueous hydrazine.⁴⁷ Ligands H₂L¹ to H₂L⁴ were prepared by reaction of either the ethyl, tert-butyl, phenyl, or nitrophenyl isothiocyanate with 6-hydrazinonictonic acid (1) in anhydrous N,N-dimethylacetamide (DMA) (Scheme 1).

Scheme 1. Synthesis of Ligands H₂L¹–H₂L⁴ and Their Methyl Ester Derivatives H₂L¹(OMe)–H₂L⁴(OMe).

The rhenium complexes of the methyl ester derivatives of H₂L^1–3 complexes, [ReO(HL^1–3(OMe))₂]⁺, can be prepared by reaction of the either trans-[ReOCl₃(PPh₃)₂] or [tBu₄N][ReOCl₄] with the two equivalents of ligand (Scheme 2). The IR spectra for the three complexes, [ReO(H₂L^1–3(OMe))₂]⁺, display medium intensity bands at ν̅ 960–963 cm^–1 characteristic of Re=O stretches.⁴⁸ Bands, which occur between ν̅ 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.

Scheme 2. Synthesis of Rhenium Complexes [ReO(HL^1-3)(OMe))₂]⁺.

Analysis of the complexes by ¹H 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(HL¹(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-d₆), 3.86 (CHCl₃-d), and 3.92 (DMSO-d₆) for complexes [ReO((HL^1,2,3)(OMe))₂]⁺ respectively. The [ReO(HL¹(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(HL¹(OMe))₂]CF₃CO₂ suitable for X-ray crystallographic analysis were obtained by evaporation of a solution of the compound that had been purified by semipreparative HPLC using an aqueous/CH₃CN 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,Strans 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.

Figure 2.

(a) ORTEP representation of [ReO(HL¹(OMe))₂]⁺ (50% probability ellipsoids). The trifluoroacetate counterion and hydrogen atoms (except those bound to nitrogen) are omitted. (b) Representation of hydrogen bonded centrosymmetric dimer with the two remaining H-bond donors capped by water molecules.

The Re–O1 bond distance (1.679(3) Å) is typical for five-coordinate, rhenium(V)-monooxo complexes and consistent with IR spectroscopy (Re=O, ν̅ 963 cm^–1).⁵²⁻⁵⁵ The N2–C6 and N6–C14 bond lengths (1.287(6) Å) are significantly shorter than the other bonds within the chelate ring suggesting significant sp² hybridized C=N bond character. The Re–N bond distances average 2.05 Å and are marginally shorter than typical Re–N bonds (ca. 2.15–2.18 Å) suggesting some degree of multiple bond character.^51,56 The Re–N1–N2 and Re–N5–N6 bond angles average 124°, suggesting an approximately sp² hybridized nitrogen. The Re–S bond distances average 2.29 Å and are similar to the Re–S bond distances in rhenium(V) complexes with amino thiolate ligands and Re–S bond distances in rhenium complexes with thiosemicarbazonato ligands.^51,57,58

Table 1. Selected Bond Lengths (Å) and Angles (deg) for the Rhenium Complex for [ReO(HL¹(OMe))₂]CF₃CO₂^a.

Bond Lengths
Re–O(1)	1.679(3)	Re–S(1)	2.2820(11)	Re–S(2)	2.2917(11)
N(1)–C(1)	1.350(6)	Re–N(1)	2.049(4)	Re–N(5)	2.050(4)
C(6)–N(3)	1.343(6)	N(1)–N(2)	1.422(5)	N(5)–N(6)	1.418(5)
C(14)–N(7)	1.350(6)	N(2)–C(6)	1.287(6)	N(6)–C(14)	1.287(6)
N(5)–C(9)	1.356(6)	C(6)–S(1)	1.784(5)	C(14)–S(2)	1.769(4)
Bond Angles
O(1)–Re–S(1)	115.44(11)	N(1)–Re–S(1)	80.74(11)
O(1)–Re–S(2)	116.75(11)	N(5)–Re–S(2)	80.23(11)
O(1)–Re–N(1)	101.73(15)	N(5)–Re–S(1)	89.71(11)
O(1)–Re–N(5)	102.61(15)	N(1)–Re–S(2)	87.98(11)
N(1)–Re–N(5)	155.64(15)	C(1)–N(1)–Re	125.0(3)
S(1)–Re–S(2)	127.80(4)	C(9)–N(5)–Re	126.2(3)
Torsion Angles
N(4)–C(1)–N(1)–Re	169.5(3)	N(6)–C(14)–N(7)–C(15)	13.8(7)
N(8)–C(9)–N(5)–Re	173.3(3)	N(2)–C(6)–N(3)–C(7)	5.1(7)
N(4)–C(1)–N(1)–N(2)	–10.5(6)	N(2)–C(6)–S(1)–Re	3.7(4)
N(8)–C(9)–N(5)–N(6)	–7.8(6)	N(6)–C(14)–S(2)–Re	4.1(4)
C(2)–C(1)–N(1)–N(2)	167.1(4)	N(3)–C(6)–S(1)–Re	–174.7(3)
C(10)–C(9)–N(5)–N(6)	168.4(4)	N(7)–C(14)–S(2)–Re	–175.1(3)

^aTrifluoroacetate counterion bond lengths and angles are not provided.

Table 2. Summary of Crystal Data and Structure Refinement for [ReO(HL¹(OMe))₂]CF₃CO₂^a.

data collection	compound details	data collection	compound details
empirical formula	C20H26N8O5ReS2.H2O.CF3CO2	V (Å3)	1551.05(13)
formula weight	839.84	Z	2
crystal size (mm3)	0.26 × 0.10 × 0.022	Dcalc. (Mg m–3)	1.797
crystal system	triclinic	μ (mm–1)	9.599
space group	P1̅	F(000)	828
T (K)	130.00(10)	reflections measured	10363
λ (Å)	1.54184	independent reflections	5568 [Rint = 0.0449]
a (Å)	10.9154(4)	final R indices [I > 2σ(I)]	R1 = 0.0329
b (Å)	11.1201(6)		wR(F2) = 0.0794
c (Å)	13.2651(7)	final R indices (all data)	R1 = 0.0398
α (deg)	86.355(4)		wR(F2) = 0.0831
β (deg)	74.965(4)	goodness-of fit on F2	1.027
γ (deg)	87.273(4)

^aCrystals were grown from a concentrated solution of the complex in methanol.

The potential of the substituted pyridylthiosemicarbazide (SHYNIC) ligands H₂L^1–4 to be modified with amino acids using standard solid phase peptide synthesis techniques was first accomplished by attaching l-lysine to H₂L¹ to give H₂L¹(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, H₂L¹, 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

Scheme 3. “On-Resin” Formation of H₂L¹(Lys) and the Corresponding Rhenium-oxo Complex, [ReO(HL¹(Lys))₂]⁺.

The rhenium complex, [ReO(HL¹(Lys))₂]⁺, was prepared by adding either trans-[ReOCl₃(PPh₃)₂] or [tBuN][ReOCl₄] 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-[ReOCl₃(PPh₃)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(HL¹(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(HL¹(Lys))₂]⁺ complex by electrospray ionization mass spectrometry (ESI-MS) reveals the expected peaks. Analysis by ¹H NMR shows the singlet attributed to the aromatic proton on the pyridine ring (pyH²) shifts upon coordination to the metal center from δ 8.48 in H₂L¹(Lys) to 8.58 ppm in [ReO(HL¹(Lys))₂]⁺. The four downfield ¹³C{¹H} NMR signals in [ReO(HL¹(Lys))2]⁺, 173.0 (CO₂H), 169.0 (N=CS), 165.4 (CONH), and 163.6 (pyC⁶), were assigned using HSQC and HMBC techniques.

Synthesis of Peptide-Conjugated Ligands, H₂L^1–3(cRGDfK) and H₂L^1–3(Tyr³-Octreotate), and Corresponding Rhenium Complexes [ReO((HL^1–3)(cRGDfK))₂] and [ReO((HL^1–3)(Tyr³-Octreotate))₂]

The cyclic pentapeptide, cRGDfK, was prepared using standard solid phase peptide synthesis techniques with Fmoc (Fmoc = 9-fluorenylmethyloxycarbonyl) protected amino acids, using HATU/DIPEA coupling methodology on chlorotrityl resin. The Fmoc protecting groups were removed with 20% piperidine in DMF on the solid phase, followed by cleavage from the resin and cyclization using a small modification of published procedures.⁶⁴ The ligands, H₂L^1–3, were conjugated to cRGDfK using standard peptide coupling conditions (HATU, DIPEA) to give H₂L^1–3(cRGDfK) (Scheme 4). The new conjugates were purified by semipreparative RP-HPLC and characterized by electrospray mass spectrometry and analytical HPLC. The reaction for the ethyl-SHYNIC derivative, H₂L¹(cRGDfK), resulted in a higher isolated yield (80%) than H₂L²(cRGDfK) (24%) and H₂L³(cRGDfK) (55%). Analysis of aqueous solutions of H₂L^1–3(cRGDfK) by RP-HPLC revealed no degradation over a 24 h.

Scheme 4. Synthesis of H₂L^1-3(cRGDfK) and [ReO(HL^1-3(cRGDfK))₂]⁺.

The {ReO}³⁺ complexes of H₂L^1–3(cRGDfK) were prepared by adding [ReOCl₄]⁻ in DMF at ambient temperature followed by isolation and purification using semipreparative RP-HPLC (Scheme 4). Analysis of the complexes by ESI-MS revealed the 2+ molecular ion peaks at m/z 926.343, 954.372, and 974.371 for [ReO(HL¹(cRGDfK))₂]⁺, [ReO(HL²(cRGDfK))₂]⁺, and [ReO(HL³(cRGDfK))₂]⁺ respectively.

An intramolecular disulfide bridge between the second and seventh cysteine residues in Tyr³-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 H₂L^1–3 to Tyr³-octreotate was complicated by degradation of the pyridylthiocarbazide (SHYNIC) ligands (H₂L^1–3) in the presence of the two cysteine thiol containing residues in the linear peptide, leading to loss of H₂S identified in the ESI-MS by a loss of 34 atomic mass units. This loss of H₂S from the ligand was most prominent during reactions attempting intramolecular oxidation of thiol groups in the two cysteine residues. The loss of H₂S 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 H₂S from the ligands (Scheme 5).

Scheme 5. Suggested Mechanism for the Formation of a Carbodiimide from H₂L^1–3(Tyr³-Oct) Resulting in Loss of H₂S.

As conventional off-resin oxidative cyclization methodologies were inadequate for this synthesis, H₂L^1–3(Tyr³-Oct) was prepared entirely on solid support, where intramolecular oxidation/cyclization preceded bioconjugation of H₂L^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 (H₂L^1–3) is reacted at the deprotected d-phenylalanine N-terminus. Cleavage and deprotection of remaining protecting groups are achieved by treatment with trifluoracetic acid (Scheme 6).

Scheme 6. Reaction Scheme for the Formation of Octreotate-Derived Ligands H₂L^1-3(Tyr³-Oct) and Corresponding Complexes, [ReO(HL^1-3(Tyr₃-Oct))₂]⁺.

The rhenium complexes of H₂L^1–3(Tyr³-Oct) could be prepared on-resin or in-solution by treatment with [tBu₄N][ReOCl₄] in methanol (Scheme 6). The on-resin approach is potentially of interest in producing radioactive complexes in high specific activity as unreacted [ReO₄]⁻ 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(Tyr³-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).

Figure 3.

RP-HPLC chromatogram (UV absorbance, a.u., λ 254 nm) of Tyr³-octreotate rhenium complexes, [ReO(HL^1–3(Tyr³-Oct))₂]⁺. Inset: Positive ion ESI-MS data of major isotope peaks corresponding to each complex as the tripositive cation, [ReO(HL^1–3(Tyr³-Oct))₂]³⁺.

Preparation of [¹⁸⁸Re(HL¹ (Tyr³-Oct))₂]⁺

Preliminary radiolabeling of H₂L¹(Tyr³-Oct) with radioactive ¹⁸⁸Re was performed using generator-produced [¹⁸⁸ReO₄]⁻. A solution of [¹⁸⁸ReO₄]⁻, in an aqueous mixture of sodium chloride (0.9% w/v concentration) and sodium tartrate, was reduced with stannous chloride. This mixture was then reacted with H₂L¹(Tyr³-Oct) at 100 °C, leading to the formation of [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺ in ca. 67% radiochemical yield. The compound was characterized by analytical reversed phased HPLC (Figure 4), where [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺ (retention time = 11.6 min, detected using a NaI(Tl)) elutes with a similar retention time to the nonradioactive analogue [^natReO(HL¹(Tyr³-Oct))₂]⁺ (retention time = 11.3 min, detected at λ₂₂₀), where ^natRe refers to naturally abundant Re isotopes. The small difference in retention times is due to the different configurations of the radioactivity and UV detectors. This elution profile of [ReO(HL¹(Tyr³-Oct))₂]⁺ is distinct from that of the free ligand, H₂L¹(Tyr³-Oct) that elutes at 9.7 min under the same conditions. Unreacted ¹⁸⁸Re species, presumably [¹⁸⁸ReO₄]⁻, elute with the solvent front at 2.1 min (Figure 4).

Figure 4.

RP-HPLC chromatogram (UV absorbance, λ 224 nm and radiation detection) of [^188/natReO(HL¹(Tyr³-Oct))₂]⁺ and free ligand H₂L¹(Tyr³-Oct). The signal at 2.1 min in the chromatogram of [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺ corresponds to unreacted ¹⁸⁸Re species.

Preparation of ^99mTc-Labeled Complexes, [^99mTcO(HL^1–3(cRGDfK))₂]⁺ and [^99mTcO(HL^1–3(Tyr³-Oct))₂]⁺

The technetium-99m complexes [^99mTcO(HL^1–3(cRGDfK))₂]⁺ and [^99mTcO(HL^1–3(Tyr³-Oct))₂]⁺ were prepared in ca. 60–80% radiochemical yield using mild conditions and relatively simple procedures (Supporting Information, Figures S1 and S2). Preparation of [^99mTcO(HL^1–3(cRGDfK))₂]⁺ and [^99mTcO(HL^1–3(Tyr³-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 [^99mTcO₄]⁻ 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 ^99mTc 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 ^99mTc 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.

Table 3. RP-HPLC Retention Times (min) for Ligands and [MO(HL)₂]⁺ Complexes (M = Re, 99 mTc)·L¹ (ethyl), L² (t-butyl), and L³ (phenyl) Compounds^a.

compound	H2Lx	rhenium complex	technetium complex
L1-cRGDfK	10.0	11.4	11.5
L2-cRGDfK	9.3	10.3	10.9
L3-cRGDfK	10.9	12.0	12.7
L1-(Tyr3-Oct)	10.1	11.4	11.9
L2-(Tyr3-Oct)	10.9	11.7	12.1
L3-(Tyr3-Oct)	11.0	12.9	13.0

^aLinear gradient from 0 to 90% Buffer B to A. Buffer A: 0.1% TFA in Milli-Q water. Buffer B: 0.1% TFA in CH₃CN.

The stability of [^99mTcO(HL³(Tyr³-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, H₂L^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 ^99mTc 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 five-membered 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 Tyr³-octreotate, and these conjugates form complexes with rhenium to give well-defined single species, [ReO((HL^1–3)(cRGDfK))₂] and [ReO((HL^1–3)(Tyr³-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 “on-resin”, and the latter approach may prove useful in isolating radioactive ^188/186Re 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.^14,67−72 It is possible to prepare [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺ in ∼67% yield from generator produced [¹⁸⁸ReO₄]⁻, and improved yields should be possible by optimizing the reaction conditions. The analogous technetium complexes, [TcO((HL^1–3)(cRGDfK))₂] and [TcO((HL^1–3)(Tyr³-Octreotate))₂], were prepared directly from [^99mTcO₄]⁻ with tin chloride acting as a reducing agent. 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 (^99mTc) and therapeutic (^186/188Re) 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 (¹H NMR at 400 MHz and ¹³C{¹H} NMR at 101 MHz) or a Varian FT-NMR 500 spectrometer (¹H NMR at 500 MHz and ¹³C{¹H} NMR at 126 MHz) at 298 K. Chemical shifts were referenced to residual solvent peaks and are quoted in ppm relative to TMS.

Fmoc-l-amino acids, Fmoc-d-amino acids, Nvoc-Cl, HATU, DIC, Wang resin, 2-chlorotrityl, Fmoc-Lys(ivDde)–OH, and Fmoc-Cys(Acm)–OH were purchased from standard commercial sources.

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 in vacuo. A solution of TFA (97.5%) and Milli-Q water (2.5%) was added to the crude material to deprotect the peptide, followed by removal of the trifluoroacetic acid by sparging with a stream of N₂. The peptide was precipitated with diethyl ether, isolated by centrifugation (3 min, 3600 rpm) and dissolved in Milli-Q water (5 mL), and finally purified by semiprep RP-HPLC (Column 1); Gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 40% B to A, over 40 min (1.0% min^–1), UV detection at λ 220 nm with a flow rate of 5 mL min^–1.

Linear protected Tyr³-octreotate peptide, dPhe-Cys(Trt)-Tyr(tBu)-dTrp(tBoc)-Lys(tBoc)-Thr(tBu)-Cys(Trt)-Thr(tBu)–OH,was synthesized using standard automated Fmoc SPPS procedures on a 2-chorotrityl chloride or Wang resin unless otherwise specified.⁷³

Analytical reversed phase high performance liquid chromatography (RP-HPLC) was undertaken using an Agilent 1100 Series HPLC system at a flow rate of 1 mL min^–1 with either Column 1: A Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5.0 μm) or Column 2: A Phenomenex Aeris Peptide XB-C18 column (250 mm × 4.6 mm, 3.6 μm). Solvent gradients for analytical analysis were either using System A: Gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 100% B over 25 min and UV detection at λ 214, 254, and 280 nm, System B: Gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 60% B over 30 min and UV detection at λ 214, 220, 254, 280, and 350 nm or System E: Gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 20 to 80% B, over 30 min and UV detection at λ 220, 254, and 350 nm with a flow rate of 1 mL min^–1.

Semipreparative reversed phase high performance liquid chromatography (semiprep RP-HPLC) was performed using an Agilent 1200 series preparative HPLC unit with variable wavelength detector. An automated Agilent 1200 fraction collector collected 0.5–4 mL fractions. Peak separation was achieved using either Column 3: Kinetex C18 100 Å, AXIA column (150 mm × 21.2 mm, 5 μm), Column 4: Phenomenex Synergi Hydro-RP 80 Å (50 mm × 21.2 mm, 4 μm), Column 5: Varian Pursuit XRs C18 100 Å (150 × 21.2 mm, 5 μm) or Column 6: SGE ProteCol C18 120 Å (250 mm × 10 mm, 5 μm). Gradient elution, flow rate, and wavelength detection are compound specific and are detailed under the Experimental Section of a particular compound. Each fraction collected above 400 mAU was analyzed using ESI-MS and analytical HPLC.

Analytical HPLC traces of radiolabeled ¹⁸⁸Re 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 H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 100% B over 20 min and UV detection at λ 220 nm.

Analytical HPLC traces of radiolabeled ^99mTc 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 H₂O) and Buffer B (0.1% TFA in CH₃CN) 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(HL¹(OMe))₂]TFA on an Oxford Diffraction SuperNova 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 ų 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).

Ligand Synthesis

Note: The designation of H₂L^1–4refers to the structure with a carboxylic acid-substituted, pyridylhydrazine with the different thiocarbahydrazide functional groups (Et, tBu, and Ph respectively). Further derivatization of the carboxylate functional group is represented by placing the substituted group at the carboxylic carbon in replacement of the OH group (e.g., H₂L^1–3(OMe), denotes the substitution of a methoxy at the carbonyl carbon to give a methyl ester.

6-[(2-Ethylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, H₂L¹

To a suspension of 6-hydrazino-3-pyridinecarboxylic acid²² (0.45 g, 2.9 mmol) in anhydrous ethanol (5 mL) was added ethyl isothiocyanate (0.51 mL, 5.8 mmol) under an atmosphere of nitrogen. The suspension was heated to reflux for 16 h then cooled to RT, and the precipitate that formed was collected by filtration, washed with cold ethanol (20 mL), and diethyl ether (20 mL) to give H₂L¹ as a colorless powder (0.67 g, 95%). ¹H NMR [DMSO-d₆, 500 MHz]: δ (ppm) 9.39 (1H, s, NH), 9.01 (1H, s, NH), 8.63 (1H, d, J = 1.7 Hz, pyH²), 8.19 (1H, t, J = 4.8 Hz, NH), 8.04 (1H, dd, J = 8.8, 2.2 Hz, pyH⁴), 6.53 (1H, d, J = 8.8 Hz, pyH⁵), 3.45 (2H, dt, J = 13.3, 6.8 Hz, CH₂), 1.03 (3H, t, J = 7.1 Hz, CH₃); ¹³C{¹H} NMR [DMSO-d₆, 125.7 MHz]: δ (ppm) 181.5 (C, NCS), 166.5 (C, CO₂H), 162.0 (C, pyC⁶), 150.4 (C, pyC²), 138.8 (C, pyC⁴), 117.8 (C, pyC³), 105.6 (C, pyC⁵), 38.4 (C, CH₂), 14.6 (C, CH₃); IR: ν̅_max (cm^–1) 2981 (s/sh, N–H), 1605 (s/sh, C=O), 1539 (s/sh), 1319 (m/sh), 1282 (s/sh), 1245 (s/sh), 782 (s/sh); ESI-MS (⁺): m/z calc’d for C₉H₁₃N₄O₂S 241.0759, found 241.0817 {[M + H]⁺, 100%}; RP-HPLC (Column 1, System A): R_T (min) 7.2.

6-[2-(tert-Butylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, H₂L²

To a suspension of 6-hydrazino-3-pyridinecarboxylic acid (0.45 g, 2.9 mmol) in anhydrous DMA (5 mL) was added tert-butyl isothiocyanate (0.56 mL, 4.4 mmol) under an atmosphere of nitrogen. The suspension was heated to 85 °C and after 30 min became a yellow mixture, which was heated at 85 °C for a further 3 h. The mixture was concentrated by evaporation under reduced pressure to a volume of approximately 1 mL and cold diethyl ether (15 mL) was added. The suspension was stirred vigorously overnight at RT. The precipitate was collected via filtration and washed with copious amounts of cold diethyl ether to afford an off-white solid (0.68 g, 86%). ¹H NMR [MeOH-d₄, 500 MHz]: δ (ppm) 8.73 (1H, d, J = 2.2 Hz, pyH²), 8.18 (1H, dd, J = 8.7, 2.2 Hz, pyH⁴), 6.76 (1H, dd, J = 8.7, 0.7 Hz, pyH⁵), 1.51 (9H, s); ¹³C{¹H} NMR [MeOH-d₄, 125.7 MHz]: δ (ppm) 181.0 (C, NCS), 166.1 (C, CO₂H), 162.0 (C, pyC⁶), 150.4 (C, pyC²), 138.8 (C, pyC⁴), 117.8 (C, pyC³), 105.6 (C, pyC⁵), 54.7 (C, -C(CH₃)₃), 29.0 (3C, -C(CH₃)₃); IR: ν̅_max (cm^–1) 1604 (s/sh, C=O), 1533 (s/sh), 1280 (s/sh), 1252 (s/sh), 1133 (m/sh), 1002 (m/sh), 779 (s/sh); HRMS (ESI⁺): m/z calc’d for C₁₁H₁₇N₄O₂S 269.1072, found 269.1110 {[M + H]⁺, 100%}; RP-HPLC (Column 1, System A): R_T (min) 10.6.

6-[2-(Phenylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, H₂L³

To a suspension of 6-hydrazino-3-pyridinecarboxylic acid (0.45 g, 2.9 mmol) in anhydrous dimethylacetamide (DMA) (5 mL) was added phenyl isothiocyanate (0.59 mL, 4.4 mmol) under an atmosphere of nitrogen. The suspension was heated to 65 °C and after 5 min became a yellow solution, which was heated at 65 °C for a further 2 h. The mixture was concentrated by evaporation under reduced pressure to a volume of approximately 1 mL, and cold diethyl ether (15 mL) was added A precipitate was collected by filtration and washed with copious amounts of cold diethyl ether to afford an off-white powder (0.81 g, 97%). ¹H NMR [DMSO-d₆, 400 MHz]: δ (ppm) 9.88 (1H, br s, NH), 9.84 (1H, br s, NH), 9.22 (1H, br s, NH), 8.67 (1H, d, J = 1.9 Hz, pyH²), 8.07 (1H, d, J = 8.4 Hz, pyH⁴), 7.47 (2H, d, J = 7.2 Hz, ArH), 7.30 (2H, t, J = 7.2 Hz, ArH), 7.13 (1H, t, J = 7.2 Hz, ArH), 6.66 (1H, d, J = 8.8 Hz, pyH⁵); ¹³C{¹H} NMR [MeOH-d₄, 125.7 MHz]: δ (ppm) 181.3 (C, NCS), 166.4 (C, CO₂H), 161.7 (C, pyC⁶), 150.3 (C, pyC²), 139.2 (C, pyC⁴), 138.7 (C, ArC¹), 127.9 (2C, ArC^3,5), 125.6 (C, ArC⁴), 124.9 (2C, ArC^2,6), 117.8 (C, pyC³), 106.1 (C, pyC⁵); IR: ν̅_max (cm^–1) 1607 (s/sh, C=O), 1596 (s/sh), 1537 (s/sh), 1280 (s/sh), 1254 (s/sh), 1140 (m/sh), 1019 (m/sh), 782 (m/sh); HRMS (ESI⁺): m/z calc’d for C₁₃H₁₃N₄O₂S 289.0759, found 289.0749 {[M + H]⁺, 100%}; RP-HPLC (Column 1, System A): R_T (min) 9.9.

Methyl 6-Chloropyridine-3-carboxylate, 3

A suspension of 6-chloronicotinic acid (5.0 g, 32 mmol) in methanol (100 mL) was cooled to 0 °C, and H₂SO₄ (0.5 mL) was added dropwise. The mixture was heated at 60 °C for 8 h. The crude reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate and washed with sat. NaHCO₃. The organic layer was dried over MgSO₄, filtered, and evaporated to dryness in vacuo. The pale yellow solid was recrystallized from methanol to form clear, plate-like crystals (5.0 g, 91%). ¹H NMR [CHCl₃-d, 400 MHz]: δ (ppm) 9.00 (1H, dd, J = 2.4, 0.7 Hz, pyH²), 8.25 (1H, dd, J = 8.3, 2.4 Hz, pyH⁴), 7.42 (1H, dd, J = 8.3, 0.8 Hz, pyH⁵), 3.96 (3H, s, CH₃); ¹³C{¹H} NMR [CHCl₃-d, 125.7 MHz]: δ (ppm) 165.0 (C, CO₂Me), 155.8 (C, pyC⁶), 151.3 (C, pyC²), 139.8 (C, pyC⁴), 125.2 (C, pyC³), 124.4 (C, pyC⁵), 52.8 (C, OCH₃); ESI-MS (⁺): m/z calc’d for C₇H₇ClNO₂ 172.0165, found 171.9965 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 8.7.

Methyl 6-Hydrazinyl-3-pyridinecarboxylate, 4

Methyl 6-chloropyridine-3-carboxylate, 3, (0.10 g, 0.58 mmol), and hydrazine hydrate (50–60% N₂H₄, d = 1.029 g cm^–3, 0.10 mL, approximately 3 equiv) were added to a 5 mL microwave vial, which was evacuated and purged with N₂. Degassed methanol (5 mL) was added to the vial, and the suspension was subjected to microwave irradiation for 15 min at 105 °C. The mixture was filtered and concentrated in vacuo. The residue obtained was dissolved in ethyl acetate and washed with sat. NaHCO₃ and sat. NaCl. The organic layer was collected and dried over Na₂SO₄, filtered, and concentrated to afford a chalky, yellow powder (0.08 g, 81%). ¹H NMR [CHCl₃-d, 600 MHz]: δ (ppm) 8.95 (1H, d, J = 2.4 Hz, pyH²), 8.21 (1H, dd, J = 8.3, 2.4 Hz, pyH⁴), 7.40 (1H, d, J = 8.3 Hz, pyH⁵), 3.92 (3H, s, OCH₃); ¹³C{¹H} NMR [CHCl₃-d, 151 MHz]: δ (ppm) 165.0 (C, CO₂Me), 155.7 (C, pyC⁶), 151.1 (C, pyC²), 139.7 (C, pyC⁴), 125.1 (C, pyC³), 124.3 (C, pyC⁵), 52.7 (C, OCH₃); HRMS (ESI⁺): m/z calc’d for C₇H₁₀N₃O₂ 168.0773, found 168.0777 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 7.6.

Methyl 6-[(2-Ethylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylate, H₂L¹(OMe)

Crude methyl 6-hydrazinyl-3-pyridinecarboxylate, 4, (approximately 0.06 g, 0.67 mmol), was added to anhydrous ethanol (8 mL) and heated until entirely dissolved. Ethyl isothiocyanate (60 μL, 0.67 mmol) was added to the solution and stirred at reflux for 3 h. The solution was cooled to RT. A stream of N₂ was blown over the solution, and the resulting precipitate was collected by filtration and washed with copious amounts of diethyl ether to afford a colorless crystalline solid (0.05 g, 51%). ¹H NMR [DMSO-d₆, 400 MHz]: δ (ppm) 9.41 (1H, s, -NHNHCS), 9.10 (1H, s, -NHNHCS), 8.66 (1H, d, J = 1.8 Hz, pyH²), 8.21 (1H, br t, J = 6.5 Hz, -NHCH₂CH₃), 8.06 (1H, dd, J = 8.8, 2.1 Hz, pyH⁴), 6.55 (1H, d, J = 8.8 Hz, pyH⁵), 3.80 (3H, s, OCH₃), 3.45 (2H, quin., J = 6.6 Hz, CH₂CH₃), 1.03 (3H, t, J = 7.1 Hz, CH₂CH₃); ¹³C{¹H} NMR [DMSO-d₆, 151 MHz]: δ (ppm) 181.3 (C, NCS), 165.4 (C, CO₂CH₃), 162.2 (C, pyC⁶), 150.2 (C, pyC²), 138.5 (C, pyC⁴), 116.7 (C, pyC³), 105.9 (C, pyC⁵), 51.9 (C, CO₂CH₃), 38.4 (C, CH₂), 14.6 (C, CH₃); IR: ν̅_max (cm^–1) 3143 (m/br, N–H), 2977 (m/br), 1710 (s/sh, C=O), 1533 (m/br), 1295 (s/sh), 1254 (s/sh), 1240 (m/sh), 1119 (w/sh), 781 (m/sh); HRMS (ESI⁺): m/z calc’d for C₁₀H₁₅N₄O₂ 255.0916, found 255.0938 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 9.2.

Methyl 6-[(2-tert-Butyllcarbamothioyl)hydrazinyl]-3-pyridinecarboxylate, H₂L²(OMe)

The compound 6-[2-(tert-butylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, (H₂L²) (0.2 g, 0.74 mmol), was added to deoxygenated and dried CH₃OH (20 mL). Sulfuric acid (∼0.5 mL) was added dropwise, and the reaction was heated to 50–60 °C for 3 h. Addition of aqueous NaOH (0.1 M) caused a precipitate to form (pH 7–8). The precipitate was collected by filtration as a gray powder (0.13 g, 63%). ¹H NMR [CHCl₃-d, 500 MHz]: δ (ppm) 8.74 (1H, d, J = 1.7 Hz, pyH²), 8.16 (1H, dd, J = 8.7, 2.2 Hz, pyH⁴), 6.76 (1H, dd, J = 8.7, 0.3 Hz, pyH⁵), 3.86 (3H, s, CO₂CH₃), 1.47 (9H, s, (CH₃)₃); ¹³C{¹H} NMR [CH₃OH-d₄, 125.7 MHz]: δ (ppm) 180.9 (C, NCS), 165.6 (C, CO₂CH₃), 161.4 (C, pyC⁶), 150.6 (C, pyC²), 140.1 (C, pyC⁴), 119.8 (C, pyC³), 106.1 (C, pyC⁵), 53.8 (C, -OCH₃), 52.1 (C, -C(CH₃)₃), 28.7 (3C, -C(CH₃)₃); IR: ν̅_max (cm^–1) 1721 (s/sh, C=O), 1523 (m/sh), 1285 (s/sh), 1250 (s/sh), 1133 (m/sh), 775 (s/sh); ESI-MS (⁺): m/z calc’d for C₁₂H₁₉N₄O₂S 283.1229, found 283.0450 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 12.9.

Methyl 6-[(2-Phenylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylate, H₂L³(OMe)

The same procedure and reagents were used as for the synthesis of H₂L¹(OMe), except using phenyl isothiocyanate (45 μL, 0.38 mmol) and methyl 6-hydrazinyl-3-pyridinecarboxylate (63 mg, 0.38 mmol) to afford a white, microcrystalline solid (0.08 g, 71%). ¹H NMR [CH₃OH-d₄, 400 MHz]: δ (ppm) 8.77 (1H, s, pyH²), 8.19 (1H, d, J = 8.8 Hz, pyH⁴), 7.51 (2H, dd, J = 8.1 Hz, ArH^2,6), 7.35 (2H, t, J = 7.6 Hz, ArH^3,5), 7.21 (1H, t, ArH⁴), 6.85 (1H, d, J = 8.8 Hz, pyH⁵), 3.89 (3H, s, CO₂CH₃); ¹³C{¹H} NMR [DMSO-d₆, 125.7 MHz]: δ (ppm) NCS resonance not detected, 165.0 (C, CO₂Me), 161.6 (C, pyC⁶), 150.4 (C, pyC²), 138.8 (C, ArC¹), 137.7 (C, pyC⁴), 128.0 (2C, ArC^3,5), 127.0 (C, ArC⁴), 124.6 (2C, ArC^2,6), 116.6 (C, pyC³), 105.3 (C, pyC⁵), 51.8 (C, CO₂CH₃); IR: ν̅_max (cm^–1) 3262 (m/sh, N–H), 1707 (s/sh, C=O), 1618 (m/sh), 1537 (s/sh), 1274 (s/sh), 1133 (m/sh), 742 (m/sh); ESI-MS (⁺): m/z calc’d for C₁₄H₁₅N₄O₂S 303.0916, found 303.1274 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 13.1.

H₂L¹(Lys)

Dichloromethane (10 mL) was added to 2-chlorotrityl resin (1.00 mmol g^–1 loading) (3.1 g, 3.1 mmol) swelling the resin. A mixture of N_α-tBoc-N_ε-Fmoc-l-Lys (1.7 g, 3.6 mmol) and DIPEA (2.1 mL, 12 mmol) in dichloromethane (15 mL) was added to the resin, which was then shaken at ambient temperature for 2 h. The resin was filtered and the filtrate was discarded. The resulting resin was washed with dichloromethane (3 mL), DMF (3 mL), dichloromethane (3 mL), and finally diethyl ether (3 mL). After several hours of suction drying the resin was weighed and loading determined (0.77 mmol g^–1). To the dried resin-bound tBoc-Lys(Fmoc)–OH (0.51 g, 0.39 mmol) was added a DMF/piperidine (80:20 v/v) solution (20 mL), which was manually stirred. After 20 min, the supernatant was removed by filtration through a sintered frit, and the remaining resin was washed with DMF (3 mL). A positive TNBSA assay (2,4,6-trinitrobenzenesulfonic acid in methanol (5% w/v)) was used to indicate deprotection of the Fmoc protected epsilon (ε) amine. A mixture of HATU (285 mg, 0.75 mmol), H₂L¹ (176 mg, 0.75 mmol), and DIPEA (261 μL, 1.5 mmol) in DMF (4 mL) was added to the resin and reacted for 4 h at ambient temperature. The liquid was drained and the resin was washed successively with dichloromethane (3 mL), then DMF (3 mL), and again with dichloromethane (3 mL). A TFA/H₂O/dichloromethane (18:2:80 v/v) solution was added to half the resin (approximately 0.19 mmol), and the mixture shaken for 1 h and then filtered. The filtrate was concentrated in vacuo and diethyl ether (40 mL) was added to form a suspension, which was centrifuged (3 min, 3600 rpm) and the supernatant was discarded. The crude material was dissolved in H₂O/CH₃CN (90:10 v/v), filtered (Millipore 0.45 μm porosity syringe filter), and then purified by semipreparative RP-HPLC (Column 5). The HPLC system involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 20 to 40% B to A, over 20 min (1.0% min^–1) then 40–60% over 40 min (0.5% min^–1) and UV detection at 220 nm with a flow rate of 5 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford an off-white, fluffy compound (10 mg, 14%). ESI-MS (⁺): m/z calc’d for C₁₅H₂₅N₆O₃S 369.1709, found 369.1798 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 6.00, (Column 2, System B) 24.0.

H₂L²(N_αOAc-Lys-OMe)

A solution of HATU (0.23 g, 0.56 mmol) and DIPEA (0.29 mL, 1.1 mmol) in DMSO (1 mL) was added to H₂L² (0.15 g, 0.56 mmol) and shaken for 2 min. N_α-Acetyl-l-lysine methyl ester hydrochloride (0.10 g, 0.42 mmol) in DMSO (1 mL) was added and shaken for 2 h. Diethyl ether (40 mL) was added and the mixture was centrifuged. The supernatant was discarded and the remaining yellow oil was purified by semipreparative RP-HPLC (Column 5). The HPLC system involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 10 to 60% B to A, over 50 min (1.0% min^–1) and UV detection at λ 220 and 254 nm with a flow rate of 5 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a colorless fluffy solid (0.19 g, 27%). ¹H NMR [MeOH-d₄, 600 MHz]: δ (ppm) 8.48 (1H, d, J = 2.1 Hz, pyH²), 8.29 (1H, dd, J = 9.1, 2.2 Hz, pyH⁴), 7.05 (1H, d, J = 9.1 Hz, pyH⁵), 4.38 (1H, dd, J = 8.9, 5.2 Hz, αCH), 3.70 (3H, s, CO₂CH₃), 3.38 (2H, t, J = 7.2 Hz, εCH₂), 1.97 (3H, s, COCH₃), 1.87–1.84 (1H, m, βCH₂), 1.75–1.69 (1H, m, βCH₂), 1.65–1.61 (2H, m, δCH₂), 1.53 (9H, s, (CH₃)₃), 1.49–1.41 (2H, m, γCH₂); ¹³C{¹H} NMR [MeOH-d₄, 150.9 MHz]: δ (ppm) 184.0 (C, NCS), 174.2 (C, -COCH₃), 173.4 (C, -CO₂CH₃), 165.9 (C, CONH), 159.1 (C, pyC⁶), 149.4 (C, pyC²), 141.4 (C, pyC⁴), 123.7 (C, pyC³), 110.6 (C, pyC⁵), 54.9 (C, -C(CH₃)₃), 53.7 (C, αCH₂), 52.7 (C, −CO₂CH₃), 40.7 (C, εCH₂), 32.1 (C, βCH₂), 29.8 (C, δCH₂), 28.9 (3C, -C(CH₃)₃), 24.2 (C, −COCH₃), 22.3 (C, γCH₂); HRMS (ESI⁺): m/z calc’d for C₂₀H₃₃N₆O₄S 453.2284, found 453.2288 {[M + H]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 12.5.

H₂L¹(cRGDfK)

To a solution of 6-[(2-ethylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, (H₂L¹) (16 mg, 66 μmol) in DMF (0.5 mL) was added HATU (25 mg, 66 μmol) and DIPEA (23 μL, 0.13 mmol). A solution of cRGDfK (10 mg, 17 μmol) in DMF (0.3 mL) was added to the initial mixture and then shaken for 2 h at ambient temperature. Diethyl ether (40 mL) was added and the subsequent suspension centrifuged (3 min, 3600 rpm). The supernatant was discarded and the process repeated. The remaining solid was dissolved in a H₂O/CH₃CN (90:10 v/v) solution, filtered (Millipore 0.45 μm porosity syringe filter), and then purified by semipreparative RP-HPLC (Column 4). The HPLC system involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 50% B to A, over 65 min (1.3% min^–1) and UV detection at λ 220, 254, 275, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a colorless solid (10 mg, 80%). ESI-MS (⁺) m/z calc’d for [C₃₆H₅₂N₁₃O₈S]⁺ 826.378, found 826.376, calc’d for [C₃₆H₅₃N₁₃O₈S]²⁺ 413.693, found 413.692; RP-HPLC (Column 2, System A): R_T (min) 11.3.

H₂L²(cRGDfK)

The same procedure was used as for H₂L¹(cRGDfK), except H₂L¹ was replaced with 6-[(2-tert-butylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, (H₂L²) (5.3 mg, 19 μmol), and other reagent quantities were adapted accordingly; HATU (6.5 mg, 17 μmol), DIPEA (45 μL, 0.13 mmol), and cRGDfK (10 mg, 17 μmol). Semipreparative RP-HPLC purification (Column 4) involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 40% B to A, over 80 min (0.5% min^–1) and UV detection at λ 220, 254, 275, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a light yellow solid (3.4 mg, 24%). ESI-MS (⁺) m/z calc’d for [C₃₈H₅₆N₁₃O₈S]⁺ 854.4096, found 854.4097, calc’d for [C₃₈H₅₇N₁₃O₈S]²⁺ 427.7087, found 427.7083; RP-HPLC (Column 2, System A): R_T (min) 12.9.

H₂L³(cRGDfK)

The same procedure was used as for H₂L¹(cRGDfK), except H₂L¹ was replaced with 6-[(2-phenylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid, (H₂L³) (5.2 mg, 19 μmol) and other reagent quantities were adapted accordingly; HATU (6.8 mg, 18 μmol), DIPEA (45 μL, 0.13 mmol), and cRGDfK (10 mg, 17 μmol). Semipreparative RP-HPLC purification (Column 4) involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 40% B to A, over 80 min (0.5% min^–1) and UV detection at λ 214, 220, and 254 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a colorless solid (8.6 mg, 55%). ESI-MS (⁺) m/z calc’d for [C₄₀H₅₂N₁₃O₈S]⁺ 874.3783, found 874.3380, calc’d for [C₄₀H₅₃N₁₃O₈S]²⁺ 437.6930, found 437.6992; RP-HPLC (Column 2, System A): R_T (min) 14.2.

H₂L¹(Tyr³-Oct)

Linear protected Tyr³-octreotate peptide (dPhe-Cys(Acm)-Tyr(tBu)-dTrp(tBoc)-Lys(tBoc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OH) was prepared manually by standard Fmoc SPPS on Wang resin preloaded with H-Thr(tBu)–OH (100 mg, 0.5 mmol g^–1). Amino acid coupling: Coupling of each amino acid was achieved by addition to the resin of a premade solution of Fmoc-amino acid (2 mmol, 4 equiv), HATU (1.9 mmol, 3.8 equiv), and DIPEA (4 mmol, 8 equiv) in DMF (5 mL). The resin mixture was shaken in a reaction vessel equipped with a sintered bottom for 1 h then filtered and washed with DMF (3 mL), dichloromethane (3 mL), and again with DMF (3 mL). Successful coupling was confirmed by a negative TNBSA test. Fmoc deprotection: Cleavage of the Fmoc groups was achieved after each successive coupling reaction. A DMF/piperidine (80:20 v/v) solution was added to the protected resin, which was contained in a reaction vessel with a sintered frit. The mixture was stirred for 5 min, and then the resin was filtered and washed with cleavage solution, then DMF (2 mL), dichloromethane (2 mL) and again DMF (3 mL). Complete deprotection was confirmed with a positive TNBSA test. Acm-group deprotection and on-resin cyclization: After the final Fmoc deprotection of resin-bound Fmoc-dPhe-OH, thallium(III) trifluoroacetate (Tl(CF₃CO₂)₃) (0.54 g, 1.0 mmol, 2 equiv) in DMF (4 mL) was added to the resin, and the mixture was stirred for 2 h. The liquid was decanted from the resin and the resin was washed with DMF (15 mL), dichloromethane (3 mL), and diethyl ether (2 mL). SHYNIC coupling: Half the resin-bound peptide (approximately 0.25 mmol) was used for the following reaction. H₂L¹ (0.18 g, 0.75 mmol, 3 equiv) was dissolved in DMF (3 mL) and HATU (0.29 g, 0.75 mmol, 3 equiv) added to preactivate the ligand. DIPEA (0.26 mL, 1.5 mmol, 6 equiv) was added and the mixture shaken for 2 min until full dissolution had occurred. The yellow solution was added to the dry resin in a reaction vessel equipped with a sintered frit and shaken for 2 h. The reaction solution was drained and the resin was washed with DMF (15 mL), dichloromethane (3 mL) and again DMF (3 mL). Resin and acid-sensitive protecting group cleavage: The petide-loaded resin was treated with a TFA/TIPS/H₂O (96:1.5:2.5 v/v/v) solution (20 mL) and shaken for 2 h. The mixture was filtered and sparged with a steady stream of N₂ to reduce the volume by 90%. Cold diethyl ether (40 mL) was added to precipitate the peptide that was isolated by centrifugation (3 min, 3600 rpm). The crude material was dissolved in CH₃CN/H₂O (50:50 v/v) and lyophilized. The solid was dissolved in CH₃CN/H₂O (40:60 v/v) and filtered (Millipore 0.45 μm porosity syringe filter). The compound was purified by semipreparative RP-HPLC (Column 5) with a linear gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 20 to 80% B to A, over 120 min (0.5% min^–1) and UV detection at λ 214, 220, 254, 280, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a pale yellow solid (56 mg, 18%). HRMS (⁺) m/z calc’d for [C₅₈H₇₅N₁₄O₁₃S₃]⁺ 1271.4800, found 1271.4760, calc’d for [C₅₈H₇₅N₁₄O₁₃S₃]²⁺ 636.2439, found 636.2416; RP-HPLC (Column 2, System A): R_T (min) 13.0.

H₂L²(Tyr³-Oct)

The same procedure was used as for H₂L¹(Tyr³-Oct), except H₂L¹ was replaced with H₂L² (0.20 g, 0.75 mmol, 3 equiv), and all other reagents were used according to their reported equivalencies. The precipitate after lyophilization was dissolved in CH₃CN/H₂O (8 mL, 50:50 v/v) and filtered (Millipore 0.45 μm porosity syringe filter). The compound was purified by semipreparative RP-HPLC (Column 5) with an isocratic step gradient system of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN). The elution method involved 28% B for 10 min then 32% B for 40 min (desired peak at 34 min) and UV detection at λ 214, 220, 254, 280, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a fluffy, colorless compound (18 mg, 5.5%). HRMS (⁺) m/z calc’d for [C₆₀H₇₉N₁₄O₁₃S₃]⁺ 1299.5113, found 1299.5110, calc’d for [C₆₀H₈₀N₁₄O₁₃S₃]²⁺ 650.2596, found 650.2599; RP-HPLC (Column 2, System A): R_T (min) 14.4.

H₂L³(Tyr³-Oct)

The same procedure was used as for H₂L¹(Tyr³-Oct), except H₂L¹ was replaced with H₂L³ (0.22 g, 0.75 mmol, 3 equiv). The precipitate after lyophilization was dissolved in CH₃CN/H₂O (10 mL, 50:50 v/v) and filtered (Millipore 0.45 μm porosity syringe filter). The compound was purified by semipreparative RP-HPLC (Column 5) with a linear gradient system of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 20% to 50% B to A over 60 min (0.5% min^–1) (or isocratic elution at 29% Buffer B) and UV detection at λ 254 nm with a flow rate of 7 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a fluffy white solid (14 mg, 4.2%). HRMS (⁺) m/z calc’d for [C₆₂H₇₅N₁₄O₁₃S₃]⁺ 1319.4800, found 1319.4849, calc’d for [C₆₂H₇₅N₁₄O₁₃S₃]²⁺ 650.2596, found 636.2425; RP-HPLC (Column 2, System A): R_T (min) 14.1.

Synthesis of Rhenium Complexes

Note: Unless specified Re represents ‘rhenium with natural isotope abundance’.

[ReO(HL¹(OMe))₂]Cl

To H₂L¹(OMe) (90 mg, 0.35 mmol) and (tBu₄N)[ReOCl₄] (103 mg, 0.18 mmol) was added anhydrous MeOH (13 mL). The mixture immediately turned deep red and was stirred at room temperature for 4 h. The reaction mixture was filtered, diethyl ether (ca. 15 mL) was added, and the resulting precipitate was collected by filtration to give [ReO(HL¹(OMe))₂]Cl as a dark-red, microcrystalline solid (0.11 g, 84%). ¹H NMR [DMSO-d₆, 400 MHz]: δ (ppm) 8.63 (2H, s, pyH²), 8.26 (2H, dd, J = 9.3, 1.8 Hz, pyH⁵), 7.87 (2H, d, J = 9.4 Hz, pyH⁴), 7.43 (2H, br m, NH), 3.89 (6H, s, OCH₃), 3.47 (4H, qd, J = 13.6, 7.0 Hz, CH₂CH₃), 1.16 (6H, t, J = 7.5 Hz, CH₂CH₃); ¹³C{¹H} NMR [CH₃CN-d₃, 150.9 MHz]: δ (ppm) 173.7 (2C, CO₂CH₃), 165.1 (2C, N=CS), 162.5 (2C, pyC⁶), 140.5 (2C, pyC⁵), 139.9 (2C, pyC²), 123.9 (2C, pyC⁴), 122.0 (2C, pyC³), 52.3 (2C, CO₂CH₃), 42.6 (2C, CH₂CH₃), 14.5 (2C, CH₂CH₃); IR: ν̅_max (cm^–1) 1553 (s/sh, C=O), 1287 (s/sh), 963 (m/sh, Re=O), 764 (m/sh); HRMS (ESI⁺): m/z calc’d for ReC₂₀H₂₆N₈O₅S₂ 709.0916, found 709.0898 {[M]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 14.8.

Histidine and Cysteine Challenge Experiments

A 50-fold excess of histidine or cysteine was added to a solution of [ReO(HL¹(OMe))₂]⁺ (1 mg/L) in PBS Buffer (10 mM, pH 7.4, 4 mL) and the mixture was heated to 37 °C. At 2, 4, and 24 h after initiation of the experiment, 10 μL aliquots of the reaction mixture were diluted with 90 μL of Milli-Q water and analyzed using analytical HPLC methods. The HPLC traces showed little or no decomposition (<5%) of the rhenium complex at all the time-points.

[ReO(HL²(OMe))₂]BPh₄

To a stirred suspension of trans-[ReOCl₃(PPh₃)₂] (0.15 g, 0.17 mmol) in MeOH (10 mL) was added H₂L²(OMe) (0.10 g, 0.35 mmol) and 2 drops of Et₃N. The mixture was heated at reflux for 16 h, then filtered, and diethyl ether was added to the filtrate. The resulting precipitate was collected by filtration, washed with cold diethyl ether, and then dissolved in MeOH. Addition of excess NaBPh₄ resulted in the precipitation of a red-brown precipitate that was collected, and washed with hot hexane (10 mL) to afford [ReO(HL²(OMe))₂]BPh₄ (72 mg, 39%). ¹H NMR [CHCl₃-d, 600 MHz]: δ (ppm) 8.10 (2H, d, J = 9.5 Hz, pyH²), 7.75 (2H, d, J = 9.4 Hz, pyH⁵), 7.62 (8H, m, BArH), 7.33 (2H, m, pyH⁴), 6.93 (8H, t, J = 7.1 Hz, BArH), 6.72 (4H, t, J = 7.1 Hz, BArH), 3.86 (6H, s, CO₂CH₃), 1.39 (18H, s, (CH₃)₃); ¹³C{¹H} NMR [CHCl₃-d, 150.9 MHz]: δ (ppm) 164.5, 142.5, 135.8–135.8 (8C, BArC), 133.9–133.8, 128.9, 128.6, 128.5, 127.5, 126.2–126.1 (8C, BArC), 122.0 (4C, BArC), 54.9 (2C, -OCH₃), 52.6 (2C, -C(CH₃)₃), 29.0 (6C, -C(CH₃)₃); IR: ν̅_max (cm^–1) 1555 (s/sh, C=O), 1284 (s/sh), 960 (m/sh, Re=O), 756 (m/sh); HRMS (ESI⁺): m/z calc’d for ReC₂₄H₃₄N₈O₅S₂ 765.1651, found 765.1559 {[M]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 15.0.

[ReO(HL³(OMe))₂]Cl

The same procedure was used as for [ReO(HL¹(OMe))₂]⁺, except H₂L¹(OMe) was replaced with H₂L³(OMe) (0.10 g, 0.33 mmol). All other reagents quantities were used accordingly; (tBu₄N)[ReOCl₄] (95 mg, 0.16 mmol) and MeOH (20 mL). The reaction afforded a red microcrystalline solid (69 mg, 51%). ¹H NMR [DMSO-d₆, 400 MHz]: δ (ppm) 9.84 (2H, s, NH), 8.79 (2H, s, pyH²), 8.42 (2H, dd, J = 9.1, 1.7 Hz, pyH⁵), 8.03 (2H, d, J = 9.2 Hz, pyH⁴), 7.73 (4H, d, J = 7.8 Hz, ArH^2,6), 7.32 (4H, t, J = 7.9 Hz, ArH^3,5), 6.95 (2H, t, J = 7.3 Hz, ArH⁴), 3.92 (6H, s, CO₂CH₃); ¹³C{¹H} NMR [DMSO-d₆, 150.9 MHz]: δ (ppm) CO₂Me signal not detected, 164.2 (2C), 145.5 (2C), 142.2 (2C, pyC⁵), 139.5 (2C, pyC²), 130.0 (2C, ArC^3,5), 129.3 (4C, ArC⁴), 128.9 (2C, ArC¹), 122.3 (2C, pyC⁴), 121.4 (2C, pyC³), 117.6 (4C, ArC^2,6), 52.6 (2C, CO₂CH₃)₃); IR: ν̅_max (cm^–1) 1557 (s/sh, C=O), 1282 (s/sh), 960 (m/sh, Re=O), 752 (m/sh); HRMS (ESI⁺): m/z calc’d for ReC₂₈H₂₆N₈O₅S₂, 805.1014, found 805.1004 {[M]⁺, 100%}; RP-HPLC (Column 2, System A): R_T (min) 15.4.

[ReO(HL²(N_α-Ac-Lys-OMe))₂]⁺

To H₂L²(N_α-Ac-Lys-OMe) (15 mg, 33 μmol) in MeOH (1 mL) was added a solution of (tBu₄N)[ReOCl₄] (0.9 mg, 15 μmol) in MeOH (0.3 mL). The mixture was stirred at ambient temperature for 2 h, then diluted with Milli-Q water and purified by semipreparative RP-HPLC (Column 4). The HPLC system involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 10 to 20% B to A, over 10 min (1.0% min^–1), then 20 to 40% B to A over 40 min (0.5% min^–1) and UV detection at λ 214, 220, 254, and 280 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a red solid (5 mg, 27%, assuming [ReO(HL²(N_α-Ac-Lys-OMe))₂]CF₃CO₂). ¹H NMR [MeOH-d₄, 600 MHz]: δ (ppm) 8.68 (2H, s, pyH²), 8.36 (2H, d, J = 9.6 Hz, pyH⁵), 8.03 (2H, d, J = 9.5 Hz, pyH⁴), 4.43–4.40 (2H, m, αCH), 3.72 (6H, s, CO₂CH₃), 3.43 (4H, q, J = 6.6 Hz, εCH₂), 1.99 (6H, s, COCH₃), 1.92–1.86 (4H, m, βCH₂), 1.78–1.72 (4H, m, δCH₂), 1.71–1.66 (4H, m, γCH₂), 1.50 (18H, s, (CH₃)₃); ¹³C{¹H} NMR [MeOH-d₄, 150.9 MHz]: δ (ppm) 174.2 (2C, COCH₃), 173.4 (2C, CO₂CH₃), 167.2 (2C, N = CS), 165.1 (2C, CONH), 163.3 (2C, pyC⁶), 140.7 (2C, d, pyC⁵), 139.1 (2C, d, pyC²), 124.5 (2C, pyC⁴), 122.6 (2C, pyC³), 55.3 (2C, -C(CH₃)₃), 53.7 (C, αCH₂), 52.7 (C, −CO₂CH₃), 40.8 (2C, εCH₂), 32.1 (2C, βCH₂), 29.9 (2C, δCH₂), 28.9 (6C, -C(CH₃)₃), 24.3 (2C, COCH₃), 22.3 (2C, γCH₂); HRMS (ESI⁺) m/z calc’d for [ReC₄₀H₆₂N₁₂O₉S₂]⁺ 1105.3762, found 1105.4079, calc’d for [ReC₄₀H₆₂N₁₂O₉S]²⁺ 553.1920, found 553.2094; RP-HPLC (Column 2, System A): R_T (min) 16.9.

[ReO(HL¹(Lys))₂]⁺

To resin-bound H₂L¹(Lys) (approximately 0.19 mmol) was added trans-[ReOCl₃(PPh₃)₂] (78 mg, 0.09 mmol) suspended in DMF (5 mL). The slurry was stirred for 2 h. The green suspension turned colorless and the resin turned dark red. The red resin was isolated by filtration and washed with DMF (3 mL), dichloromethane (3 mL), and again with DMF (3 mL). The rhenium complex was cleaved from the resin by treatment with TFA/dichloromethane (50:50 v/v) solution (30 mL) for 3 h. The mixture was sparged with N₂ to reduce the volume to 10% of the original volume. Cold diethyl ether (40 mL) was added, the mixture was centrifuged (3 min, 3600 rpm), and the supernatant was discarded. This process was repeated twice. The precipitate was dissolved in CH₃CN (1.0 mL) and then Milli-Q water (2.0 mL) was added. The complex was purified by semipreparative RP-HPLC purification (Column 5) involved a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 10 to 70% B to A, over 120 min (0.5% min^–1) and UV detection at λ 214, 220, and 254 nm, with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a red, fluffy solid (22 mg, 23%, assuming [ReO(HL¹(Lys))₂]CF₃CO₂). ¹H NMR [MeOH-d₄, 600 MHz]: δ (ppm) 8.58 (2H, s, pyH²), 8.34 (2H, dd, J = 9.5, 1.8 Hz, pyH⁵), 7.97 (2H, d, J = 9.5 Hz, pyH⁴), 3.80 (2H, t, J = 6.1 Hz, αCH), 3.60 (4H, dtd, J = 20.2, 13.2, 7.0 Hz, −CH₂CH₃), 3.46 (4H, dd, J = 8.9, 5.1 Hz, εCH₂), 1.99–1.92 (4H, m, βCH₂), 1.72 (4H, dt, J = 14.5, 7.3 Hz, δCH₂), 1.60–1.50 (4H, m, γCH₂), 1.27 (6H, t, −CH₂CH₃); ¹³C{¹H} NMR [MeOH-d₄, 150.9 MHz]: δ (ppm) 172.8 (2C, CO₂H), 169.0 (2C, N=CS), 165.4 (2C, CONH), 163.6 (2C, pyC⁶), 140.5 (2C, pyC⁵), 138.9 (2C, pyC²), 123.9 (2C, pyC⁴), 122.5 (2C, pyC³), 54.9 (2C, αCH), 42.5 (2C, CH₂CH₃), 40.6 (2C, εCH₂), 31.5 (2C, βCH₂), 29.9 (2C, δCH₂), 23.4 (2C, γCH₂), 14.9 (2C, CH₂CH₃); ESI-MS (⁺) m/z calc’d for [ReC₃₀H₄₆N₁₂O₇S₂]⁺ 937.2611, found 937.2574, calc’d for [ReC₃₀H₄₇N₁₂O₇S₂]²⁺ 469.1345, found 469.1355, calc’d for [ReC₃₀H₄₈N₁₂O₇S₂]³⁺ 313.0256, found 313.0939; RP-HPLC (Column 2, System A): R_T (min) 10.1.

[ReO(HL¹(cRGDfK))₂]⁺

A solution of H₂L¹(cRGDfK) (5 mg, 6.1 μmol) in MeOH (250 μL) was added to a mixture of [tBu₄N][ReOCl₄] (1.8 mg, 3.0 μmol) in MeOH (250 μL). The mixture was shaken for 2.5 h, then diluted with Milli-Q water (5 mL), filtered (Millipore 0.45 μm porosity syringe filter), and purified by semiprep RP-HPLC (Column 6). The HPLC system involved isocratic elution of 77% Buffer A (0.1% TFA in H₂O) and 23% Buffer B (0.1% TFA in CH₃CN) (0.5% min^–1) and UV detection at λ 214, 220, 254, 280, and 350 nm with a flow rate of 5 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a white solid (3.1 mg, 53%, assuming [ReO(HL¹(cRGDfK))₂]CF₃CO₂). HRMS (ESI⁺) m/z calc’d for [ReC₇₂H₁₀₁N₂₆O₁₇S₂]²⁺ 926.3413, 926.3433, calc’d for [ReC₇₂H₁₀₂N₂₆O₁₇S₂]³⁺ 617.8968, found 617.9939; RP-HPLC (Column 2, System A): R_T (min) 11.0.

[ReO(HL²(cRGDfK))₂]⁺

The same procedure was used as for [ReO(HL¹(cRGDfK))₂]⁺, except H₂L¹(cRGDfK) was replaced with H₂L²(cRGDfK) (8.0 mg, 9.4 μmol) in MeOH (250 μL); (tBu₄N)[ReOCl₄] (2.7 mg, 4.7 μmol) in MeOH (500 μL). The compound was purified by semipreparative RP-HPLC purification (Column 4) involved gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 20% over 20 min (1% min^–1), then 20 to 60% B to A over 40 min (0.5% min^–1) and UV detection at 214, 220, 254, 280, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated and lyophilized to afford a white solid (5.1 mg, ∼54%, assuming [ReO(HL²(cRGDfK))₂]CF₃CO₂). HRMS (ESI⁺) m/z calc’d for [ReC₇₆H₁₀₉N₂₆O₁₇S₂]²⁺ 954.3732, found 954.3715, calc’d for [ReC₇₆H₁₁₀N₂₆O₁₇S₂]³⁺ 636.5847, found 636.5837; RP-HPLC (Column 2, System A): R_T (min) 12.5.

[ReO(HL³(cRGDfK))₂]⁺

The same procedure was used as for [ReO(HL¹(cRGDfK))₂]⁺, except H₂L¹(cRGDfK) was replaced with H₂L³(cRGDfK) (4.0 mg, 4.6 μmol) in MeOH (200 μL), and other reagent quantities were adapted accordingly, [tBu₄N][ReOCl₄] (1.3 mg, 2.3 μmol) in MeOH (200 μL). The HPLC system (Column 6) involved a linear gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 0 to 20% over 20 min (1% min^–1), then 20 to 60% B to A over 40 min (0.5% min^–1) and UV detection at 214, 220, 254, 280, and 350 nm with a flow rate of 8 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a white solid (2.0 mg, ∼42%, assuming [ReO(HL³(cRGDfK))₂]CF₃CO₂). HRMS (ESI⁺) m/z calc’d for [ReC₈₀H₁₀₁N₂₆O₁₇S₂]²⁺ 974.3419, found 974.3711, calc’d for [ReC₈₀H₁₀₂N₂₆O₁₇S₂]³⁺ 649.8972, found 650.0023; RP-HPLC (Column 2, System A): R_T (min) 12.9.

[ReO(HL¹(Tyr³-Oct))₂]⁺

A solution of H₂L¹(Tyr³-Oct) (9.0 mg, 7.1 μmol) in MeOH (50 μL) was added to a mixture of (tBu₄N)[ReOCl₄] (2.1 mg, 3.5 μmol) and MeOH (100 μL). The reaction mixture was shaken vigorously for 2 h. Milli-Q water was added (4 mL) and the reaction was filtered (Millipore 0.45 μm porosity syringe filter). The compound was purified by semipreparative RP-HPLC purification (Column 6) with a gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 10 to 80% B to A, over 70 min (1.0% min^–1) and UV detection at λ 214, 220, and 254 nm, with a flow rate of 5 mL min^–1. Fractions containing the desired compound were identified by HPLC-MS, consolidated, and lyophilized to afford a red, fluffy material (6.8 mg, ∼ 68%, assuming [ReO(HL¹(Tyr³-Oct))₂]CF₃CO₂). ESI-MS (⁺) m/z calc’d for [ReC₁₁₆H₁₄₈N₂₈O₂₇S₆]²⁺ 1371.9470, found 1372.0024, calc’d for [ReC₁₁₆H₁₄₉N₂₈O₂₇S₆]³⁺ 914.9673, found 915.0145; RP-HPLC (Column 2, System A): R_T (min) 15.4.

[ReO(HL²(Tyr³-Oct))₂]⁺

The same procedure was used as for [ReO(HL¹(Tyr³-Oct))₂]⁺, except H₂L¹(Tyr³-Oct) was replaced with H₂L²(Tyr³-Oct) (8.0 mg, 6.2 μmol) in MeOH (200 μL) and other reagent quantities were adapted accordingly: (tBu₄N)[ReOCl₄] (1.8 mg, 3.1 μmol) in MeOH (100 μL). The compound was purified by semipreparative RP-HPLC (Column 6) with isocratic elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) at 38% Buffer B, over 60 min (1.0% min^–1) and UV detection at λ 214, 220, and 254 nm, with a flow rate of 4 mL min^–1. Fractions containing the desired compound were consolidated and lyophilized to afford a red, fluffy material (5.5 mg, ∼61%, assuming [ReO(HL²(Tyr³-Oct))₂]CF₃CO₂). ESI-MS (⁺) m/z calc’d for [ReC₁₂₀H₁₅₄N₂₈O₂₇S₆]²⁺ 1399.9784, found 1399.4720, calc’d for [ReC₁₂₀H₁₅₆N₂₈O₂₇S₆]³⁺ 933.6548, found 933.6460; RP-HPLC (Column 2, System A): R_T (min) 16.2.

[ReO(HL³(Tyr³-Oct))₂]⁺

Preloaded, resin-bound (Wang) ligand, H₂L³(Tyr³-Oct) (approximately 6.0 μmol), was swollen in CH₂Cl₂ and drained twice. Anhydrous DMF (2 × 10 mL) was added to the resin the mixture was stirred and drained. DMF (1 mL) was added to the washed resin and (tBu₄N)[ReOCl₄] (2.1 mg, 2.9 μmol) in DMF (50 μL) was added to the resin. The resin mixture was reacted for 2 h at RT. The solution was drained and the resin was washed with copious amounts of DMF (5 × 10 mL), CH₂Cl₂ (3 × 10 mL), and diethyl ether (10 mL). The crude material was cleaved off the resin by addition of TFA/CH₂Cl₂/TIPS/H₂O (50:46:1:3 v/v) (20 mL). The mixture was shaken for 1.5 h and filtered and the filtrate sparged with a stream of N₂ until the red mixture had reduced to approximately 0.5 mL. Diethyl ether (2 × 10 mL) was added to the crude material, and the resulting precipitate was collected via centrifugation (3 min, 3600 rpm). The compound was purified by semipreparative RP-HPLC purification (Column 6) by gradient elution of Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in CH₃CN) from 10 to 80% B to A, over 70 min (1.0% min^–1) and UV detection at λ 214, 220, and 254 nm, with a flow rate of 4 mL min^–1. Fractions containing the desired compound were consolidated and lyophilized to afford a red, fluffy material (5.0 mg, ∼58%, assuming [ReO(HL³(Tyr³-Oct))₂]CF₃CO₂). ESI-MS (⁺) m/z calc’d for [ReC₁₂₄H₁₄₈N₂₈O₂₇S₆]²⁺ 1419.9470, found 1419.9281, calc’d for [ReC₁₂₄H₁₄₉N₂₈O₂₇S₆]³⁺ 946.9673, found 946.1960; RP-HPLC (Column 2, System A): R_T (min) 16.2.

Synthesis of [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺

Rhenium-188 was produced from an ITG ¹⁸⁸W/¹⁸⁸Re generator (ITG Isotope Technologies, Garching, Germany). Generator-produced ¹⁸⁸ReO₄^– (86 MBq) in aqueous sodium chloride solution (200 μL, 0.9% w/v) was added to a sealed, N₂-purged vial containing sodium tartrate (0.2 mg) and stannous chloride (0.1 mg) in water (400 μL), followed by heating at 80 °C for 30 min. An aliquot of this solution (50 μL) was added to a separate sealed, N₂-purged vial containing H₂L¹(Tyr³-Oct) (100 μg) dissolved in water (100 μL). The reaction was left for 30 min at ambient temperature, after which an aliquot was removed for HPLC analysis, revealing that there was no reaction between ¹⁸⁸Re and peptide. The reaction vial was then heated at 100 °C for 1 h, after which an aliquot was analyzed by HPLC (column 1, system F). Radiochemical yield: 67%, retention time of [¹⁸⁸ReO(HL¹(Tyr³-Oct))₂]⁺ = 11.6 min, compared to retention time of [^natReO(HL¹(Tyr³-Oct))₂]⁺ = 11.3 min and H₂L¹(Tyr³-Oct) = 9.7 min.

Synthesis of Technetium-99m Complexes

Sodium pertechnetate, Na[^99mTcO₄] (1000 MBq), was eluted from a Gentech ⁹⁹ Mo/^99mTc sterile generator (Austin Health: Nuclear Medicine and Centre for PET, Australia, via ANSTO Health) as a 1.0 mL saline solution (0.9% v/v). A solution of SnCl₂ in 0.1 M HCl (0.5 mg mL^–1) was prepared and purged with nitrogen. Disodium tartrate dihydrate was dissolved in water (1 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 [^99mTcO₄]⁻ (0.1 mL in 0.9% saline, 108 MBq). The conjugated peptide, H₂L^1–4(cRGDfK) or H₂L^1–3(Tyr³-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 NaHCO₃ (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, [^99mTc(HL³(Tyr³-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).

Supporting Information Available

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

• Representative RP-HPLC chromatograms (PDF)

Accession Codes

CCDC 1543360 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Australian Research Council FT130100204 (P.S.D.). People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA Grant Agreement Number 299009 (MTM). The Centre of Excellence in Medical Engineering Centre funded by the Wellcome Trust and EPSRC (WT088641/Z/09/Z), the KCL and UCL Comprehensive Cancer Imaging Centre funded by CRUK and EPSRC in association with the MRC and DoH (England), and the NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London.

The authors declare no competing financial interest.