Methylthiazolyl Tacn Ligands for Copper Complexation and Their

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Methylthiazolyl Tacn Ligands for Copper Complexation and Their Bifunctional Chelating Agent Derivatives for Bioconjugation and Copper-64 Radiolabeling: An Example with Bombesin Amaury Guillou,† Luís M. P. Lima,‡ David Esteban-Gómez,§ Nicolas Le Poul,† Mark D. Bartholomä,⊥ Carlos Platas-Iglesias,§ Rita Delgado,‡ Véronique Patinec,† and Raphaël Tripier*,† Downloaded via EASTERN KENTUCKY UNIV on January 29, 2019 at 08:25:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

UFR des Sciences et Techniques, UMR-CNRS 6521, Université de Bretagne Occidentale, 6 avenue Victor le Gorgeu, C.S. 93837, 29238 Brest Cedex 3, France ‡ Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal ⊥ Department of Nuclear Medicine, Saarland University−Medical Center, Kirrbergerstrasse, 66421 Homburg, Germany § Departamento de Química, Facultade de Ciencias & Centro de Investigaciones Científicas Avanzadas, Universidade da Coruña, 15071 A Coruña, Spain S Supporting Information *

ABSTRACT: We present here the synthesis of two new bifunctionalized azachelators, no2th-EtBzNCS and Hno2th1tha, as bioconjugable analogues of two previously described diand trimethylthiazolyl 1,4,7-triazacyclononane (tacn) ligands, no2th and no3th, for potential uses in copper-64 (64Cu) positron emission tomography imaging. The first one bears an isothiocyanate group on the remaining free nitrogen atom of the tacn framework, while the second one presents an additional carboxylic function on one of the three heterocyclic pendants. Their syntheses required regiospecific N-functionalization of the macrocycles. In order to investigate their suitability for in vivo applications, a complete study of their copper(II) chelation was performed. The acid−base properties of the ligands and their thermodynamic stability constants with copper(II) and zinc(II) cations were determined using potentiometric techniques. Structural studies were conducted in both solution and the solid state, consolidated by theoretical calculations. The kinetic inertness in an acidic medium of both copper(II) complexes was determined by spectrophotometry, while cyclic voltammetry experiments were performed to evaluate the stability at the copper(I) redox state. UV−vis, NMR (of the zinc complexes), electron paramagnetic resonance spectroscopy, and density functional theory studies showed excellent agreement between the solution structures of the complexes and their crystallographic data. These investigations unambiguously prove that these bifunctional derivatives display similar coordination properties as their no2th and no3th counterparts, opening the door to targeted bioapplications. The no2th-EtBzNCS and Hno2th1tha ligands were then conjugated to a bombesin antagonist peptide for targeting the gastrinreleasing peptide receptor (GRPr). To highlight the potential of the two chelators for radiopharmaceutical development, the 64 Cu-radiolabeling properties, in vitro stability, and binding affinity to GRPr of the corresponding bioconjugates were determined. Altogether, the results of this work warrant the further development of 64Cu-based radiopharmaceuticals comprising our novel bifunctional chelators.

■

INTRODUCTION

soft acid−base (HSAB) principle, and chelate and macrocyclic effects. The chemistry of tacn derivatives was developed rather late compared to tetraaza analogues such as cyclen and cyclam. However, considerable efforts from different research groups in macrocyclic organic chemistry during the last 15 years afforded

1,4,7-Triazacyclononane (tacn or [9]aneN3) belongs to the family of saturated azamacrocycles, very well-known for their coordination properties with metal cations, ranging from transition to heavy metals or lanthanides when the central cyclic polyamine is appropriately functionalized.1 Several applications of interest are exploiting their coordination properties, which can be fine-tuned using Pearson’s hard and © XXXX American Chemical Society

Received: November 23, 2018

A

DOI: 10.1021/acs.inorgchem.8b03280 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

bifunctional chelating agent (BCA) derivative of no2th containing an isothiocyanate group (no2th-EtBzNCS; Chart 1), suitable for coupling to lysine moieties of biovectors, was

various straightforward strategies for the preparation of tacn derivatives.2 As a result, tacn-based scaffolds are now more commonly used in applications where stable metal complexes are required, especially in medical applications such as imaging and therapy. For example, a lanthanide(III) complex of tacn Nfunctionalized with coordinating chromophore antennas presents excellent properties for optical imaging.3 Concerning applications in nuclear medicine, the gallium-68 (68Ga) chelate of the tacn triacetate derivative, designated as H3nota, is used in positron emission tomography (PET) imaging.4 However, the 68Ga β+ emitter possesses a rather short half-life (68 min) and does not present a β−-emitting counterpart for therapy applications. In contrast, the copper(II) complex of H3nota can be used with the copper-64 (64Cu)/copper-67 (67Cu) theranostic pair thanks to the six-coordination offered by the H3nota ligand. The results obtained with [64/67Cu(nota)]− radiopharmaceuticals were quite encouraging, especially compared with the H4dota analogue, which is now recognized in nuclear medicine as an inappropriate chelator for both copper(II) isotopes because of the low kinetic inertness of the corresponding complexes.5 On the other hand, H3nota was shown to be a promising 64Cu chelator (few examples with 67 Cu are known considering its currently limited availability) and used in various bifunctional forms through its conjugation with biovectors.6 However, it is now admitted that, even if no one could predict it, the influence of the metal complexes on the biological properties is not linked only to the coordination properties of the chelates. Indeed, depending on the biological targets, it is important to have an array of complexes of different size, shape, lipophilicity, and charge.7 In this context, the synthesis and coordination chemistry of new tacn derivatives bearing coordinative pendants other than acetate functions were recently investigated.8 Among them, we proved that methylimidazole and especially methylthiazolyl derivatives are efficient chelators for copper(II) and reported their potential use as part of radiopharmaceuticals.9,10 In this regard, no2th and no3th have both been synthesized, and their coordination with copper cations was evaluated. We found that the [Cu(no2th)]2+ complex presents a N5 coordination sphere and very good kinetic and thermodynamic properties. Electrochemical studies in water evidenced a reversible Cu2+ ⇄ Cu+ system, with the copper(I) complex being stable within the time scale of these experiments. On the other hand, no3th presents low solubility in an aqueous medium, thus limiting the usual physicochemical characterization in solution. Despite that, the results showed a very interesting inertness of the [64Cu(no3th)]2+ radiocomplex in human serum, and especially an important inertness in buffered solutions along a large pH range, compared to both H3nota and H4dota chelates. The in vivo studies without targeted biovectors were later performed, confirming these results.9,10 Considering these previous encouraging studies, we sought to incorporate the no2th and no3th platforms in radiopharmaceutical models. However, their structures lack the necessary supplementary function allowing their conjugation to a targeting biomolecule. The conjugation to the targeting vector must retain the coordination ability of the chelator. The dissymmetric structure of C-functionalized tacn derivatives11 makes their synthesis rather challenging, while the regiospecific N-functionalization of tacn is, on the other hand, much more straightforward. Thus, we decided to investigate the introduction of a coupling function via a nitrogen atom of the macrocycle or through one of the coordinating arms. A

Chart 1. Ligands Discussed in This Study

synthesized thanks to the free secondary amine of the ligand available. The preparation of bifunctional analogues of no3th is more challenging because the tri-N-functionalization of the tacn backbone does not leave a macrocyclic nitrogen atom available for further derivatization. Consequently, we envisaged the introduction of an additional acetate function on the α position of the nitrogen atom of one of the thiazolyl groups, which could also be linked to lysine residues upon activation (Hno2th1tha; Chart 1). In this paper, we present the synthesis of the two new bifunctional ligands, no2th-EtBzNCS and Hno2th1tha. For the latter, we have evaluated the impact of the additional acid function on its coordination chemistry by a comparison with the properties of no2th and no3th. We have tested the suitability of these chelators for standard peptide chemistry, studied their radiolabeling efficiency after conjugation, and performed preliminary biological evaluations. The two bifunctional methylthiazolyl derivatives have been conjugated to a small model peptide to perform the first in vitro studies of a tacn-thiazolyl-based radiopharmaceutical after radiolabeling with 64Cu. Optimization of the radiolabeling with 64Cu and preliminary biological investigations, including chelator and cation challenging studies and IC50 experiments, have been performed. The gastrin-releasing peptide receptor (GRPr) overexpressed in several tumor types, including prostate cancer (PCa) and breast cancer, has been selected for biological evaluations.12 A comparison with the reference H4dota ligand has been undertaken to evaluate the potential of this new family of tacn-based radiopharmaceuticals.

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RESULTS AND DISCUSSION

Ligand and Complex Syntheses. The no2th ligand was synthesized and fully characterized in a previous work,9 while the coordination chemistry of no3th was not investigated in detail before.10 Syntheses are described in the Supporting Information. The no3th ligand 1 (synthesis A, Scheme 1) was obtained in 81% yield after purification by chromatography by the reaction of thiazolyl bromide (3.3 equiv) with tacn in acetonitrile. The synthesis of Hno2th1tha·nHCl (ligand 3, B


Article

Inorganic Chemistry Scheme 1. Schematic Synthesis of All Compounds Studied in This Work

performed by potentiometric titrations in aqueous solution at 25.0 ± 0.1 °C and 0.10 ± 0.01 M KNO3. The determined overall protonation constants βiH and the corresponding stepwise constants KiH of Hno2th1tha are presented in Table 1, while a species distribution diagram calculated from these data is provided in Figure S13. The Hno2th1tha ligand displays three protonation constants versus only two for no2th, which is related to the presence of the carboxylic acid function on the additional pendant arm. The first protonation constant is much lower than that found for no2th, which may be explained by the absence of a secondary amine in Hno2th1tha combined with the electron-withdrawing effect of the methylthiazolyl pendant arms. The second protonation constant, assigned to a macrocyclic amine just like the first one, is conversely higher than that for no2th. The third and last protonation constant should correspond to the single carboxylic acid function on one pendant arm because no protonation of the thiazolyl nitrogen atoms could be found for no2th. The complexation behavior of Hno2th1tha toward copper(II) and zinc(II) was studied by potentiometric titrations performed with each metal cation added at ca. 0.9 and 1.5 equiv of the ligand amount because of the possible formation of complex species with different metal-to-ligand (M/L) ratios. Because the corresponding copper(II) and zinc(II) complexes are extensively formed from very low pH values, the stability constants of the ML species with both cations had to be determined by competition titrations with ethylenediaminetetraacetic acid dipotassium salt (K2H2edta). These ML values were then kept fixed to calculate the remaining stability constants with each cation by a simultaneous analysis of the titrations performed using different M/L ratios. In this process,

synthesis B, Scheme 1) started from the previously described no2th ligand,9 which was consequently reacted with a slight excess (1.2 equiv) of 2-(chloromethyl)thiazolyl-4-carboxylic acid methyl ester, which was obtained based on an established procedure.13 Purification and subsequent acidic hydrolysis with 6 M HCl gave the expected ligand in 91% yield (Scheme 1). Finally, no2th-EtBzNCS (synthesis C, Scheme 1) was obtained by using a three-step procedure, which involved (i) the substitution reaction of no2th with 1-bromo-2-(4nitrophenyl)ethane in acetonitrile with an excess of K2CO3, yielding the tri-N-alkylated triazacyclononane derivative no2th-EtBzNO2 (4) in 50% yield, (ii) the reduction with SnCl2 using a 12 M HCl/methanol (HCl/MeOH; 3:1) mixture, which generated the corresponding reduced compound no2th-EtBzNH2 (5; 22%), and (iii) the reaction of the NH2 function with thiophosgene CSCl2 in a HCl/CHCl3 (2:3) mixture, to give no2th-EtBzNCS·nHCl (6) in 89% yield. Characterization data of intermediates and final ligands, including 1H and 13C NMR and high-resolution mass spectrometry (HRMS) spectra, are given in Figures S1−S12. Acid−Base Properties of the Ligand and Complexation Studies in Aqueous Solution. Useful insights into the chemical behavior of the ligands can be obtained by investigating the acid−base and metal complexation properties in aqueous solution. Unfortunately, no3th, no2th-EtBzNO2, and its reduced derivative no2th-EtBzNH2 were all found to be quite insoluble in an aqueous medium because of their lipophilic character. Additionally, no2th-EtBzNCS could not be studied because of the reactivity of the isothiocyanate function. Thus, only Hno2th1tha could be investigated in this regard and compared to the previously reported compound no2th.9 All acid−base and metal complexation studies were C


Article

Inorganic Chemistry Table 1. Protonation Constants (Overall, log βiH, and Stepwise, log KiH) of the Hno2th1tha Ligand and Its Overall Stability Constants (log β) for the Copper(II) and Zinc(II) Complexes in Aqueous Solution at 25.0 ± 0.1 °C and 0.10 ± 0.01 M KNO3a equilibrium reactionb

Hno2th1thac

no2thd

L + H ⇄ HL L + 2H+ ⇄ H2L L + 3H+ ⇄ H3L L + H+ ⇄ HL HL + H+ ⇄ H2L H2L + H+ ⇄ H3L Cu2+ + L ⇄ CuL Cu2+ + L + H+ ⇄ CuHL Cu2+ + L ⇄ CuLH−1 + H+ 3Cu2+ + 2L ⇄ Cu3L2 3Cu2+ + 2L ⇄ Cu3L2H−2 + 2H+ Zn2+ + L ⇄ ZnL Zn2+ + L + H+ ⇄ ZnHL Zn2+ + L ⇄ ZnLH−1 + H+

9.47(1) 12.65(2) 14.5(1) 9.47 3.18 1.8 19.8(1)e 22.76(1) 8.86(2) 46.53(4) 33.24(3) 16.0(1)e 18.56(2) 4.85(7)

11.03 13.48

+

11.03 2.45 20.77 23.60 11.59

16.28 18.30 5.75

a

Values for the no2th ligand are also given for comparison. bL denotes a ligand in general; the charges of ligand-containing species are omitted for clarity. cValues in parentheses are the standard deviations in the last significant figures. dReference 9. eValues determined from competition titrations with K2H2edta.

it was found that Hno2th1tha is able to form copper(II) complex species of 1:1 and 3:2 (M/L) ratios, while only complexes of a 1:1 (M/L) ratio were found for zinc(II). The overall stability constants (log β) obtained for all complexes are presented in Table 1, while species distribution diagrams calculated from these data are reported in Figure 1 for copper(II) and in Figure S14 for zinc(II). To compare the thermodynamic stability of the Hno2th1tha complex with its analogues, the pM values (pM = −log [Mn+]) that take into account the different basicities of the ligands were also calculated (Table 2). Both of the stability constants obtained and the pM values calculated at pH = 7.4 demonstrate the very high thermodynamic stability of the copper(II) complex of Hno2th1tha, which is comparable to those of the no2th9 and H3nota ligands.14,15 The pM values also show a high selectivity of Hno2th1tha for copper(II) over zinc(II), comparable to that seen for no2th and even higher than that for H3nota. Overall, the acid function added on one thiazolyl arm has a direct impact on the copper(II) coordination chemistry because it can lead, in the presence of excess metal cation, to the formation of trinuclear complexes. To avoid the presence of the latter species, the preparation of the copper(II) complex must be performed with a slight excess of ligand or should be followed by a final high-performance liquid chromatography (HPLC) purification. In any case, for further bioconjugation, the carboxylic acid will be transformed into an amide function that will reduce the formation of the trinuclear species. It is also noteworthy that for radiopharmaceutical applications the formation of a trinuclear species is not a concern because the (radio)metal concentration is very low. Synthesis of the Complexes and X-ray Structural Studies. Considering the potentiometric results, syntheses of the copper(II) and zinc(II) complexes were then only investigated with the aim of isolating the ML species. The [Cu(no3th)]2+ and [Zn(no3th)]2+ complexes were synthe-

Figure 1. Species distribution diagrams for the copper(II) complexes of Hno2th1tha at 1:1 (top) and 1.5:1 (bottom) Cu2+/Hno2th1tha ratios. CHno2th1tha = 1.0 mM. L denotes the ligand.

Table 2. Calculated pM Valuesa at pH = 7.4 for the Complexes of Hno2th1tha Compared to Those of no2th and H3nota ligand

Cu2+

Zn2+

Hno2th1tha no2thb H3nota

17.74 17.15 17.61c; 17.55d

13.89 12.65 14.28c; 16.54d

Calculated at CM = 1.0 × 10−5 and 100% excess of ligand. bReference 9. cReference 14. dReference 15.

a

sized by the addition of the ligand to 1.2 equiv of the metal perchlorate salts in refluxing acetonitrile during 12 h. These complexes were isolated, respectively, as blue (94%) and white (97%) crystals by slow evaporation of the solvent, as confirmed by the HRMS analysis (Figures S15 and S16). In both cases, the obtained crystals were good enough for X-ray diffraction studies. The structures of the two complexes are shown in Figures 2 and 3, while Tables S1 and S2 present respectively all of the crystallographic data and the bond distances of the coordination spheres of the metal ions. The crystal structure of [Cu(no3th)]2+ is very similar to that reported previously.10 The crystal structure of [Zn(no3th)]2+ is completely isostructural with its copper(II) analogue, with both complexes indeed crystallizing in the P21/c space group. In both complexes, the metal center is six-coordinate, with three nitrogen donors of the tacn backbone and three nitrogen donor atoms of the thiazolyl pendants coordinating to the D


Article

Inorganic Chemistry

prismatic rather than distorted octahedral.17 In the case of [Zn(no3th)]2+, we obtained S(Oh) = 7.87 and S(TP) = 2.41, revealing again a distorted trigonal-prismatic coordination. The trigonal-prismatic polyhedron leads to trans angles that differ considerably from the ideal values of 180° expected for an octahedral coordination (∼145−154°; Table S2). The Cu−N distances fall within the range 2.05−2.26 Å, with three of the bonds (2.21−2.26 Å) being considerably longer than the remaining three (2.05−2.07 Å). Similar trends in bond distances were observed previously for copper(II) complexes with tacn derivatives containing pyridyl or aniline pendant arms.18 The distortion of the coordination polyhedron in [Cu(no3th)]2+ can be attributed to the Jahn−Teller effect because the corresponding Zn−N distances fall within a narrower range (2.12−2.25 Å). The three pendant arms of the ligand are twisted to the same side with respect to the pseudo-C3-symmetry axis of the complex, generating two possible helical structures generally denoted as Δ or Λ. Furthermore, the tacn unit forms three chelate rings upon coordination to the metal ion, resulting in (δδδ) or (λλλ) conformations. Inspection of the crystal data reveals the presence of two enantiomeric forms of the complexes in the crystal lattice that correspond to the centrosymmetrically related Δ(δδδ)/Λ(λλλ) enantiomeric pair. A similar situation was observed previously for a zinc(II) complex with a tacn-based ligand containing aniline pendant arms.19 Considering the higher solubility of the methylthiazolylcarboxylate derivative in an aqueous medium, another synthetic procedure was employed to obtain copper(II) and zinc(II) complexes of Hno2th1tha, which present a higher solubility in water. Additionally, syntheses in aqueous media match better the conditions typically used for radiolabeling. Both complexes were synthesized by the reaction of the ligand with 1 equiv of the corresponding metal perchlorate salts in water at pH ∼ 6 (the pH was controlled along the reactions). The complexes were isolated with >92% yield as blue-green and white solids, respectively, after evaporation and washing with MeOH. All complexes were purified by HPLC to avoid any trace of trinuclear species (see the HRMS analysis; Figures S17 and S18). Attempts for crystallizing the copper(II) and zinc(II) complexes of Hno2th1tha, characterized in a potentiometric study, were carried out by dissolving the corresponding solids in water at acidic and neutral pH (6−7). The [Cu(no2th1tha)]+ complex leads, only after slow evaporation of a solution of the complex under acidic conditions (1 M HCl), to the formation of single crystals suitable for X-ray diffraction determination. The study reveals an endo copper(II) complex in an “opened” form resulting from the protonation of one nitrogen atom of a methylthiazolyl arm (Figure 4). The copper(II) center has a N4Cl coordination sphere. The additional Cu−N4 distance of 2.819 Å is too long to be considered as a bond distance. One of the thiazolyl arms is protonated at this pH, so the N6 atom is not engaged in coordination to the copper(II) cation. Thus, the copper center is five-coordinated by the three amine nitrogen atoms of the macrocycle, a nitrogen atom of one of the methylthiazolyl pendant arms and an additional chloride ligand. The metal coordination environment can be described as distorted square-pyramidal, where the basal plane of the pyramid is defined by N1, N2, N5, and Cl1 and N3 is occupying the apical position. The bond distance involving the apical donor atom [Cu1−N3 = 2.356(3) Å] is longer than those to donor

Figure 2. View of the X-ray structure of the [Cu(no3th)]2+ complex cation. Representation with 70% thermal ellipsoid probability. Hydrogen atoms and counterions are omitted for clarity.

Figure 3. View of the X-ray structure of the [Zn(no3th)]2+ complex cation. Representation with 70% thermal ellipsoid probability. Hydrogen atoms and counterions are omitted for clarity.

metal (copper or zinc) center. The unit cell contains two perchlorate counterions that balance the positive charge of the complex cations. The coordination environment around the metal centers in the [Cu(no3th)]2+ and [Zn(no3th)]2+ complexes is closer to trigonal-prismatic than to octahedral. The upper triangular face of the trigonal prisms is defined by the three nitrogen atoms of the pendant arms, while the lower tripod is delineated by the nitrogen atoms of the tacn unit. These two planes are virtually parallel, intersecting at 0.9 and 0.7° for the complexes of Cu2+ and Zn2+, respectively. The mean twist angles of these triangular faces are 23.6 and 20.4°, revealing an important distortion of the coordination polyhedron from trigonalprismatic (ideal value 0°) to octahedral (ideal value 60°) coordination. This was confirmed by performing shape measures with the aid of the SHAPE program.16 The shape measure S(P) is a quantitative measure of the agreement between a given polyhedron and reference polyhedra, with S(P) = 0 corresponding to a structure fully coincident with the reference polyhedron. This analysis provides for [Cu(no3th)]2+ S(P) values of 6.78 and 3.11 for the octahedral and trigonal-prismatic reference polyhedra. Shape measures with S(Oh) > 4.42 and S(TP) < 4.42 characterize polyhedra better described as distorted trigonalE


Article

Inorganic Chemistry

The copper center in [Cu(no2th-EtBzNO2)](ClO4)2 is fivecoordinate, and the coordination environment is comparable to that of the complex of the nonbifunctional analogue [Cu(no2th)](ClO4)2. The asymmetric unit is composed of two complex cations [Cu(no2th-EtBzNO2)]2+ and four perchlorate units as counteranions. The copper center is also five-coordinate with a distorted square-pyramidal geometry with three nitrogen atoms from the tacn ring and two nitrogen atoms from the thiazolyl arms as the donor atoms. The introduction of the bifunctional arm induces a slight elongation of the Cu−N3 bond compared to the parent complex (Δ = 0.038 Å; Table S4). Spectroscopic Studies. The paramagnetic nature of the [Cu(no3th)]2+ and [Cu(no2th1tha)]+ complexes does not allow its characterization using NMR, so the structural investigation in solution by NMR was performed with the diamagnetic zinc(II) analogues (Figures S20−S23). The 1H and 13C NMR spectra of [Zn(no3th)]2+ were recorded in deuterated acetonitrile (CD3CN) or dimethyl sulfoxide (DMSO)-d6 because of poor solubility in aqueous media. A comparison of the 1H NMR spectra of the ligand and complex reveals significant chemical shifts induced by coordination to the metal cation, in particular for the proton nuclei of the thiazolyl units and the methylene CH2 protons of the pendant arms. The 13C NMR spectrum displays five signals, which points to an effective C3v symmetry of the complex in solution. The 1H nuclei of the tacn unit give two signals that correspond to the protons pointing in different directions with respect to the ideal C3 axis of the complex. Overall, the NMR spectra are consistent with fast interconversion at room temperature between the Δ(δδδ) and Λ(λλλ) enantiomers, which requires both inversion of the tacn unit [(δδδ) ↔ (λλλ)] and rotation of the pendant arms [Δ ↔ Λ]. The 1H NMR spectrum of [Zn(no2th1tha)]+ recorded in a D2O solution reveals important chemical shifts induced by coordination to the metal ion, as well as an increased complexity of the region where aliphatic signals are observed. The 13C NMR spectrum presents 13 signals, which is consistent with an effective Cs symmetry of the complex in solution, as evidenced previously for [Zn(no2th)]3+.9 The absorption spectra in the visible region of the [Cu(no3th)]2+, [Cu(no2th1tha)]+, and [Cu(no2th-EtBzNO2)]2+ complexes were acquired in N,N-dimethylformamide (DMF)/water (1:1) solutions because of the low solubility of the complexes in water (Figure S24). All complexes display one d−d transition band in the 600−700 nm range (Table 3). The structures of the three complexes were also investigated by electron paramagnetic resonance (EPR) spectroscopy, with spectra acquired in frozen samples (130 K) also in DMF/water (1:1) solutions (Figure 6). The EPR spectra were all simulated (Figure S25) using a single paramagnetic species because no additional species with different structures were observed. The spectra of the complexes consist of three of the four lines expected for gz, while the superhyperfine interactions due to the presence of the nitrogen atoms are not observed. The values of g and hyperfine coupling constants A obtained by simulation of the experimental spectra (Table 3) indicate three different principal values of g, with gz > (gx + gy)/2 and the lowest g ≥ 2.03, which is characteristic of mononuclear copper(II) complexes in a rhombic symmetry with elongation of the axial bonds and a dx2−y2 ground state. These data, together with λmax (ε) of the visible absorption bands, are

Figure 4. View of the X-ray structure of [Cu(H2no2th1tha)Cl]Cl2. Hydrogen atoms and counterions are removed for clarity.

atoms of the basal plane (1.97−2.27 Å; Table S3), as usually observed for copper(II) complexes with square-pyramidal coordination.20 The Cu1−Cl1 distance is typical of a chloride ligand occupying a basal position in square-pyramidal copper(II) complexes.21 This structure shows that even under strong acidic conditions the copper(II) ion remains lodged in the cavity of the macrocyclic ligand in a protonated state. The fact that the complex is still present in an endo form under acidic conditions confirms the formation of a highly stable complex. The [Cu(no2th-EtBzNO2)]2+ complex was also synthesized using a slight excess of copper(II) perchlorate in a solution of the ligand and in refluxing acetonitrile during 12 h. The complex was isolated as a blue crystalline solid after recrystallization in MeOH and several washings with the same solvent in order to remove excess copper(II) perchlorate. Single crystals suitable for X-ray diffraction were obtained by slow evaporation from a water/acetonitrile (9:1) solution. A representative view of the [Cu(no2th-EtBzNO2)]2+ complex cation is given in Figure 5. The HRMS analysis is provided in Figure S19.

Figure 5. View of the X-ray structure of the [Cu(no2th-EtBzNO2)]2+ complex cation. Representation with 70% thermal ellipsoid probability. Hydrogen atoms, counterions and disorder were removed for clarity. F


Article

Inorganic Chemistry

Table 3. UV−Vis and EPRa Parameters Obtained for the Copper(II) Complexes of the Studied Ligands in DMF/Water Solutions ligand no3th Hno2th1tha

no2th-EtBzNO2 no2thf

exp calcd exp calcdd calcde exp exp calcd

λmax (ε)b

gx

gy

gz

Axc

Ayc

Azc

695 (115)

2.041 2.049 2.040 2.060 2.038 2.039 2.037 2.033

2.108 2.089 2.073 2.067 2.070 2.071 2.075 2.079

2.246 2.193 2.229 2.187 2.162 2.212 2.213 2.165

8.5 2.9 1.5 18.4 11.8 1.2 7.3 18.8

25.2 57.9 11.5 33.6 40.0 25.4 46.4 52.8

153.1 166.2 175.8 175.3 180.0 181.5 175.7 173.5

640 (136)

600 (157) 620 (160)

From simulation of the experimental spectra. bλmax in nm; ε in M−1 cm−1. cValues of Ai × 104 cm−1. dCalculated for the six-coordinated structure obtained with DFT. eObtained for the five-coordinated structure. fFrom ref 9 in aqueous solution. a

The results of these calculations are shown in Table 3, together with the corresponding experimental data. The g and A tensors calculated for [Cu(no2th)]2+ are in good agreement with the experiment, confirming that this complex adopts in solution a structure that is very similar to that observed in the solid state. Thus, we conclude that the [Cu(no2th-EtBzNO2)]2+ complex adopts a similar structure. The [Cu(no3th)]2+ complex is characterized by higher gy and gz values than the [Cu(no2th)]2+ analogue as well as a lower value of Az. This trend is well reproduced by DFT calculations, confirming that the [Cu(no3th)]2+ complex retains in solution the distorted trigonal-prismatic coordination observed in the solid state. This trigonal-prismatic coordination in [Cu(no3th)]2+ is also accompanied by a red shift of the d−d absorption bands in the electronic spectra (Table 3). The EPR parameters measured for the [Cu(no2th1tha)]+ complex are very similar to those of [Cu(no2th)]2+, with a slightly higher value of gz. Thus, the measured EPR parameters suggest that the methylthiazolyl-4-carboxylate arm is not involved in coordination to the copper(II) ions. To gain further insight into the structure of the [Cu(no2th1tha)]+ complex, we performed DFT calculations, which yielded the optimized geometries of the five- and six-coordinated forms of the complex (Figure 7). The coordination environment calculated for the five-coordinated form is comparable to that calculated for [Cu(no2th)]2+. For the six-coordinated complex, the calculated Cu−N distance involving the methylthiazolyl-4-carboxylate group (2.389 Å) is considerably longer than the corresponding distances to the nitrogen atoms of the methylthiazolyl arms (2.065 and 2.034 Å). The corresponding distances calculated for [Cu(no3th)]2+ are 2.338, 2.059, and 2.049 Å. Thus, the steric hindrance brought by the introduction of the carboxylate group results in a significant elongation of the corresponding Cu−N bond. The relative Gibbs free energies obtained with DFT favor the sixcoordinated species by only 0.07 kcal mol−1, which suggests that both forms are likely present in solution. The EPR parameters calculated for the five-coordinated form of [Cu(no2th1tha)]+ are virtually identical with those calculated for [Cu(no2th)]2+, while the six-coordinated complex presents a slightly higher gz value. The experimental gz value obtained for [Cu(no2th1tha)]+ is slightly higher than that of [Cu(no2th)]2+, which suggests that the six-coordinated form of the complex is present in solution, at least to some extent. The A tensor is calculated as the sum of the isotropic Fermi contact (FC) term, the spin-dipolar (SD) term, and the spin− orbit coupling (SOC) contribution.23 The breakdown of the Az values calculated for [Cu(no2th)]2+ and [Cu(no3th)]2+

Figure 6. Experimental EPR spectra for the copper(II) complexes of the no3th, Hno2th1tha, and no2th-EtBzNO2 ligands obtained in frozen DMF/water (1:1) solutions.

consistent with distorted square-pyramidal or distorted octahedral (trigonal-prismatic) geometries. Density functional theory (DFT) calculations were carried out to gain further information on the structure of this family of complexes in solution (see the computational details below). Geometry optimization at the TPSSh/TZVP level was followed by calculation of the EPR parameters (g and A tensors) by using both the TPSSh and TPSS0 functionals. The latter is a variation of the hybrid TPSSh functional (10% exchange) with an increased amount of exact exchange (25%). The TPSS0 functional was found to provide more accurate g tensors of copper(II) complexes than TPSSh, while TPSSh gives A tensors with better agreement with the experiment.9,22 G


Article

Inorganic Chemistry

The redox properties of the three complexes [Cu(no3th)](ClO 4 ) 2 , [Cu(no2th1tha)](ClO 4 ), and [Cu(no2thEtBzNO2)](ClO4)2 were studied in a 0.1 M LiClO4 aqueous solution at pH = 6.8 on a glassy carbon electrode under argon (Figure 8). The corresponding voltammograms of the three complexes, recorded under identical conditions, exhibit quasireversible redox behavior (Figure 8A1,B1,C1), consistent with a stable copper(I) species at the CV time scale (0.02 V s−1 < v < 2 V s−1). As shown in Table 4, half-wave potential (E1/2) values range between −0.40 and −0.27 V versus Ag/AgCl. They are substantially more positive than that previously reported for the [Cu(no2th)](ClO4)2 complex (E1/2 = −0.47 V vs Ag/AgCl).9 According to these values, the different ligands can be ranked with regard to their ability to stabilize copper(II) over copper(I) as no2th > no3th > no2thEtBzNO2 > Hno2th1tha, with the no2th chelator forming the most redox-stable copper(II) complex in this series. This trend is in full agreement with variation of the computed Cu−N bond lengths for this family of complexes (Table S12). Notably, all half-wave potential values (vs NHE) found for these complexes are more positive than the standard potential of bioreducers such as NADH (E0 = −0.32 V vs NHE). This implies that all of these complexes are likely being reduced to copper(I) in a biological medium. In order to evaluate the stability of the resulting copper(I) complexes out of the CV time scale, exhaustive electrolyses of the three copper(II) complexes (no3th > no2th-EtBzNO2 > Hno2th1tha) were performed under an inert atmosphere. In all cases, the CV obtained after electrolysis showed a quasi-reversible redox behavior (Figure 8A2,B2,C2) at the same E1/2 value as that found before electrolysis. Moreover, no copper(0) deposit was observed on the working electrode after electrolysis. This behavior is different from that obtained with the no2th copper complex, which slowly evolves at the copper(I) redox state.9 Here, the copper(II) complexes do not dissociate upon monoelectronic reduction, hence suggesting a pronounced kinetic stability of the corresponding copper(I) species. Dissociation Studies: Kinetic Stability of the Copper(II) Complexes. The kinetics of the acid-assisted dissociation of the [Cu(no2th1tha)]+ complex was studied under pseudofirst-order conditions in an aqueous solution at 25.0 °C. The dissociation of the complex was monitored by following the changes in the d−d absorption band of the complex and comparing it to the already studied no2th and no3th analogues. Even if highly acidic media are not found in vivo, these experiments provide important information on the behavior of the complex in highly competitive media, while also allowing a comparison of the copper(II) complexes of different ligands with regard to their inertness. In 1 and 5 M HCl media at 25 °C, no changes in the absorption spectrum were observed over time, indicating that the complex does not undergo dissociation under these conditions. In 5 M HCl at 90 °C (Figure S26), a gradual dissociation is observed with a halflife time higher than 100 h, which is comparable to that of [Cu(no3th)]2+ (Table 5). This behavior can be linked to the structure presented in Figure 4, which shows the complex in its protonated form. Bioconjugation, 64Cu Radiolabeling, and Initial Biological Studies. To further explore the potential of the two new bifunctional chelators for envisaged radiopharmaceutical applications, we decided to evaluate Hno2th1tha and no2thEtBzNCS in combination with a GRPr targeting peptide. The GRPr is overexpressed in several tumor types including

Figure 7. Structures of the [Cu(no2th1tha)]+ complex obtained with DFT calculations (TPSSh/TZVP). Bond distances are given in angstroms.

(Table S5) shows that the contributions of the sum of the FC and SD terms are virtually identical. The lower Az value of [Cu(no3th)]2+ is related to a more important contribution of the SOC term. Similarly, the g tensor contains contributions from the relativistic mass correction, the diamagnetic spin− orbit term, and the paramagnetic spin−orbit term. The higher gz value calculated for [Cu(no3th)]2+ is the result of a larger paramagnetic spin−orbit contribution (Table S6). Thus, the gz values increase and the Az values decrease as a result of more important spin−orbit contributions, which correlates with the simultaneous red shift of the d−d absorption bands in the electronic spectra. Optimized Cartesian coordinates obtained with DFT for [Cu(no3th)]2+ and [Cu(no2th1tha)]+ are also given in Tables S7 and S11. The distances of the metal coordination environments obtained with DFT calculations (and compared to X-ray when possible) are given in Table S12. Electrochemical Properties. Release of 64Cu from a radiolabeled bioconjugate can be observed in a biological medium after the reduction of copper(II) to copper(I) by endogenous enzymes and subsequent demetalation due to the instability of corresponding copper(I) complexes. However, the release of 64Cu from a radiopharmaceutical should be avoided because this leads to the accumulation of radioactivity in nontarget organs and, thus, to lower signal-to-noise ratios. Consequently, the redox properties of the copper(II) complexes of no3th, Hno2th1tha, and no2th-EtBzNO2 were investigated by cyclic voltammetry (CV) in water to obtain detailed information about the redox potential of these three compounds for further use in radiopharmaceutical development. H


Article

Inorganic Chemistry

Figure 8. CVs (0.1 V s−1 scan rate) before (A1, B1, and C1) and after (A2, B2, and C2) electrolysis of the complexes at a glassy carbon electrode in water/0.1 M LiClO4 under an argon atmosphere: (A) 1 mM [Cu(no3th)](ClO4)2, pH = 6.8; (B) 5 mM [Cu(no2th1tha)](ClO4), pH = 6.8; (C) 2 mM [Cu(no2th-EtBzNO2)](ClO4)2, pH = 6.8.

H4dota for radiometal complexation (Figure 9).26 We thus decided to use the peptide Ahx-[D-Phe6,Statine13]bombesin(6−14) (Ahx-JMV594) as a model compound for the evaluation of our bifunctional chelators (Figure 9), in which the Pip spacer as in RM2 was replaced by 6-aminohexanoic acid. First, we were interested if Hno2th1tha and no2thEtBzNCS can be employed in standard peptide chemistry. For preparation of the GRPr targeting bioconjugates, both ligands were consequently reacted with the terminal NH2

prostate and breast cancer and is therefore a key target for noninvasive diagnosis by PET.12 Many bombesin-like radiotracers targeting GRPr have been developed in recent years.24 More recently, it was shown that GRPr antagonists are superior to agonists because of their increased tumor uptake and retention compared to agonists.25 A prominent example in this respect is the radiotracer [68Ga]Ga(RM2), which is based on the peptide antagonist [D-Phe6,Statine13]bombesin(6−14) (JMV594) and additionally comprises a 4-amino-1-carboxymethylpiperidine (Pip) spacer and the bifunctional chelator I


Article

Inorganic Chemistry Table 4. Electrochemical Data (E/V vs Ag/AgCl) at v = 0.1 V s−1 in a 0.1 M LiClO4 Aqueous Solution at pH = 6.8 for the Studied Copper(II) Complexes [Cu(no2th)]2+ a [Cu(no3th)]2+ [Cu(no2th1tha)]+ [Cu(no2th-EtBzNO2]2+

Epcb

Epac

E1/2

ΔEpd

−0.51 −0.45 −0.32 −0.37

−0.44 −0.36 −0.23 −0.25

−0.47 −0.40 −0.27 −0.32

0.07 0.09 0.09 0.12

in purities of >98% and 93%, respectively (see the HPLC and HRMS spectra in Figures S27−S34). Because the most important prerequisite for novel chelating agents is their capability to complex the radiometal of interest, the labeling properties of no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 with 64Cu were assessed in detail at different pH values and temperatures (Figure 10). The

a

Data from ref 9. bEpc: cathodic peak potential; cEpa: anodic peak potential; dΔEp = Epa − Epc.

Table 5. Acid-Assisted Dissociation of the Complexes Copper(II) Studied by UV−Vis λmax (nm) 1 M HCl, rt 5 M HCl, rt 5 M HCl, 95 °C

[Cu(no3th)]2+10

[Cu(no2th1tha)]+

[Cu(no2th)]+9

650 inert inert 55 h

635 inert inert ≥100 h

620 inert 95%) at room temperature (r.t.) within 15 min at pH = 5.5. An increase in the pH to 7.2 and 8.2, respectively, resulted in slightly lower radiochemical conversions (RCCs) of ∼60− 87%. Interestingly, the RCC was lower for the five-coordinated no2th-EtBzNCS than for the six-coordinated Hno2th1tha at pHs > 7 and r.t. In contrast, both bioconjugates labeled quantitatively (≥98%) at elevated temperatures (95 °C) at pH = 7.4 and 8.2. Because quantitative labeling was achieved at pH = 5.5, both conjugates were consequently labeled at pH = 5.5 for further experiments providing molar activities of Am = ∼20 MBq nmol−1. For the subsequent in vitro tests, radiolabeling was performed at 45 °C to ensure quantitative RCC, which was confirmed by HPLC showing radiochemical purities of >95% (Figures S35 and S36). Next, we studied the stability/inertness of 64Cu-labeled no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 by ligand-challenge and copper-exchange experiments (Table 6). No significant transchelation was noted for both radiolabeled conjugates after incubation in an 75000-fold molar excess of diethylenetriamine pentaacetate (H5dtpa). While for the potentially hexadentate no2th1tha in [64Cu]Cu-no2th1tha-Ahx-JMV594 the corresponding radiometal complex remained essentially intact with 99.8% for up to 24 h, the five-coordinated complex showed marginal transchelation with ∼97% of intact [64Cu]Cu-no2th-EtBzNCS-Ahx-JMV594. To assess further the kinetic stability of both 64Cu-labeled compounds, copper-exchange experiments were performed with slight modifications to the literature procedure.10,27 At physiological pH, no exchange of 64Cu was observed in the presence of a 40-fold molar excess of natCu2+ after 24 h of incubation time, confirming a high kinetic stability of both radiometalated complexes. Finally, the distribution coefficients log Doct/PBS were determined for 64Cu-labeled no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 (Table 6). Both radiolabeled bioconjugates were more lipophilic than 64Cu-labeled RM2, which can be attributed to the differences in the spacer region and the corresponding metal complexes. In [64Cu]Cu(RM2),

Figure 9. Various bioconjugates synthesized and/or discussed in this study.

group of resin-bound and side-chain-protected peptide AhxJMV594. In the case of no2th-EtBzNCS, the NCS group was reacted with the N-terminus of the peptide by simple mixing in the presence of N,N-diisopropylethylamine (DIPEA) to give the final conjugate no2th-EtBzNCS-Ahx-JMV594(7) in the usual yield (3%) after deprotection and cleavage using trifluoroacetic acid (TFA)/water (H2O)/triisopropylsilane (TIS). For conjugation of Hno2th1tha, the carboxylic acid functionality was transformed into an active ester using the coupling reagent 1-[bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU). Coupling proceeded smoothly, and the final conjugate no2th1tha-Ahx-JMV594(8) was also obtained in standard yield (5%) after deprotection. Subsequent purification by semipreparative reversed-phase HPLC (RP-HPLC) provided both conjugates no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 and their natCu-metalated derivatives J


Article

Inorganic Chemistry Table 6. Various Data from the Bioconjugates and Their Radiolabeled Isotopes no2th-EtBzNCS-AhxJMV594(7) molecular formula mass calcd mass found HPLCUV−vis tR [min] HPLCradio tR [min]a H5dtpa challengeb copper exchange log Doct/PBS IC50 [nM]

no2th1tha-AhxJMV594(8)

C84H119N21O12S3 1710.8581 1710.8581 24.06

C80H115N21O13S3 1672.8061 1672.8067 22.6

24.1 ± 0.3

4.5 ± 0.1

[nat/64Cu]-7 2+

[CuC84H119N21O12S3] 886.3897 886.3824 21.40 23.07 97.2 ± 3.45 99 ± 0.10 −0.83 ± 0.00 46.5 ± 0.3

[nat/64Cu]-8

RM2

[CuC80H115N21O13S3]2+ 867.3637 867.3624 20.0 23.03 99.8 ± 0.10 99 ± 0.10 −1.64 ± 0.03 3.0 ± 0.4

−2.27 ± 0.02 12.5 ± 0.2

a

UV−vis and radioactivity detector in series. b% intact.

the [64Cu]Cu(dota) complex is negatively charged, resulting in an ionic character at the N-terminus in combination with the positive charge of the Pip spacer. In contrast, both bioconjugates no2th-EtBzNCS-Ahx-JMV594 and no2th1thaAhx-JMV594 are equipped with a neutral and potentially more lipophilic Ahx spacer and carry a 2-fold positive charge at the N-terminus through the corresponding copper complexes because both ligands do not possess any charge-compensating groups like H4dota. It is worth noting that a stark difference in the lipophilicity was seen between the two novel conjugates no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594. Substitution of the methylthiazolyl acid arm in Hno2th1tha with an ethylbenzyl arm as for no2th-EtBzNCS significantly increased the lipophilicity of the entire bioconjugate, most likely because of the lipophilic nature of the benzyl moiety. Competitive Cell Binding Studies. To further demonstrate the potential application of no2th-EtBzNCS and Hno2th1tha for radiopharmaceutical applications, we performed competitive cell binding experiments on the GRPrexpressing PCa cell line PC-3 using [125I]I-[Tyr4]-bombesin as the radioligand. The metal-free as well as natCu complexes of no2th-EtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 inhibited the binding of [125I]I-[Tyr4]-bombesin in a dosedependent manner. RM2 was used as a control and tested simultaneously in each experiment. The corresponding IC50 values are given in Table 6, while the corresponding curves are given in Figures S37 and S38. Interestingly, the nature of the metal chelator as well as the geometry of the corresponding copper complex impacted the affinity for GRPr. For example, no2th-EtBzNCS-Ahx-JMV594 exhibited a 2-fold lower affinity than RM2. Complexation of Cu2+ by no2th-EtBzNCS-AhxJMV594 resulted in a decrease in the affinity with an approximately 4-fold higher IC50 value than RM2. Interestingly, the metal-free conjugate no2th1tha-Ahx-JMV594 had a ∼3-fold higher affinity than RM2. In contrast to the results obtained for metal-free and natCu complexes of no2thEtBzNCS-Ahx-JMV594, metal coordination in the case of no2th1tha-Ahx-JMV594 did not significantly alter the affinity for GRPr with an IC50 value comparable to that of the metalfree conjugate. The inhibitory constants of our novel conjugates are comparable to IC50 values of bombesin antagonists reported in the literature.28 An inhibitory constant of 7.7 ± 3.3 nM has been reported for RM2, which is slightly lower than the IC50 value of 12.5 ± 0.2 nM determined in this work.25 However, care should be taken when comparing biological data from different experiments because of differences in the experimental setup in the competitive binding assays. For example, we performed a solution-based assay in

which the PC-3 cells were suspended directly in the media in filter plates, whereas in some reports, cells were allowed to adhere to the surface of the well plate or competitive binding was performed directly on cryosections of tumors. Nonetheless, the IC50 values of the metal-free and copper-labeled conjugates compare well to the inhibitory constants reported for similar bombesin antagonists.29 A single positive charge at the N-terminus of GRPr antagonists has been proposed to result in improved GRPr affinity.30 To test this hypothesis, we replaced the Pip spacer as in RM2, being protonated at physiological pH and creating a positive charge, with a neutrally charged Ahx spacer. Here, the positive charges are created either by protonation of the ligand at physiological pH for the metal-free conjugates no2thEtBzNCS-Ahx-JMV594 and no2th1tha-Ahx-JMV594 (see potentiometric data) or by complexation of the 2-fold positively charged copper(II) cation. With regard to these Nterminal modifications, we obtained mixed results. Compared to RM2, a lower affinity for GRPr was noted for both the metal-free and corresponding copper(II) complexes of no2thEtBzNCS-Ahx-JMV594. In contrast, a ∼3-fold higher affinity than that of RM2 was found for no2th1tha-Ahx-JMV594 and its corresponding copper(II) complex. Noteworthy is the influence of metal complexation on the binding affinity. While metal complexation for no2th-EtBzNCS-Ahx-JMV594 resulted in a decrease in the affinity, no significant differences were observed for no2th1tha-Ahx-JMV594 and the corresponding copper complex. Besides the (complex) charge, additional factors obviously have an impact on the GRPr affinity such as the lipophilicity, shape, and geometry of the metal complex, and further experiments are warranted to elucidate the effect of the metal binding moiety on the GRPr affinity in more detail.

■

CONCLUSIONS We recently investigated the synthesis and coordination chemistry of tacn derivatives bearing two or three methylthiazolyl pendants with copper(II), namely, no2th and no3th, reporting their promising use as 64Cu radiopharmaceuticals compared to both H3nota and H4dota chelates. In this paper, we carried out a complete study of their complexation properties, the corresponding bifunctional analogues were developed, and their potential for applications in PET imaging or therapy were explored. In this respect, the bifunctional derivatives no2th-EtBzNCS and Hno2th1tha were prepared by direct N-functionalization of the previously described no2th, showing the versatility of the synthetic route. We unambiguously proved, in both the solid state and solution, K


Article

Inorganic Chemistry

General Procedure for the Complex Syntheses in Aqueous Solution. The ligand (0.1 mmol) was dissolved in ultrapure water (10 mL). The pH was raised to 6 with 1 M KOH, and 0.9 equiv of the metal(II) perchlorate salt was added. The pH was raised again to 6, and the mixture was refluxed for 12 h. The solvent was removed, and the residue was taken up in MeOH. The complex, which remained insoluble in MeOH, was washed twice with MeOH, dissolved in water, and dried under reduced pressure. [Cu(no2th1tha)](ClO4): green powder (54 mg, 95%). ESIHRMS. Calcd for [CuC22H30N6S2 + H]2+: m/z 263.5204. Found: m/z 263.5207. Monocrystals of [Cu(H2no2th1tha)Cl]Cl2 descripted in Figure 4 were obtained by slow evaporation of the acidic solution. Crystals were then filtered, washed with cold water, and directly used for X-ray diffraction. [Zn(no2th1tha)](ClO4): white powder (52 mg, 92%). ESIHRMS. Calcd for [ZnC19H24N6O2S3 + H]2+: m/z 264.0202. Found: m/z 264.0199. Potentiometric Studies. The potentiometric setup was described previously.32 Titrations were performed in aqueous solutions at 0.10 ± 0.01 M KNO3 and 25.0 ± 0.1 °C. The titrants were a KOH solution prepared at ca. 0.1 M from a commercial ampule and standardized by application of the Gran method33 and also a standard HNO3 solution at 0.1 M used on back-titrations as prepared from a commercial ampule. A standard solution of K2H2edta was prepared at 0.050 M from a commercial ampule. Ligand (commonly designated as L) solutions were prepared at ca. 2.0 × 10−3 M in water acidified with HNO3 to pH ≈ 2. The Cu2+ and Zn2+ solutions were prepared at ca. 0.05 M from analytical-grade nitrate salts and standardized by complexometric titrations34 with the standard K2H2edta solution. Sample solutions for titration contained approximately 0.03−0.05 mmol of ligand in a 30 mL starting volume, and in complexation titrations, metal cations were added at ca. 0.9 and 1.5 equiv of the ligand amount. The electromotive force during titrations was measured after calibration of the electrode by titration of a standard HNO3 solution at 2.0 × 10−3 M. The [H+] of the solutions was determined by measurement of the electromotive force of the cell, E = E°′ + Q log [H+] + Ej, and the term pH is defined as −log [H+]. E°′ and Q were determined from the acidic pH region of the calibration titrations. The liquid-junction potential, Ej, was found to be negligible under the experimental conditions used. The value of Kw = [H+][OH−] was found to be equal to 10−13.78 by titrating a solution of known hydrogen-ion concentration at the same ionic strength in the alkaline pH region, considering E°′ and Q to be valid for the entire pH range. The protonation constants of H4edta and the thermodynamic stability constants of its copper(II) and zinc(II) complexes used in competition titration refinements were taken from the literature.35 Each titration consisted of 120−150 equilibrium points in the range of pH = 2.5−11.0, and at least two replicate titrations were performed for each particular system. Back-titrations were always performed at the end of each direct complexation titration in order to determine if equilibrium was attained throughout the entire pH range. Competition titrations using H4edta were performed for Hno2th1tha with copper(II) and zinc(II) to determine the stability constants of the ML complex species with both cations, at a 1:1:1 (M/L/edta) ratio using 0.03−0.05 mmol of ligand. The refinements of the full set of stability constants for the copper(II) and zinc(II) complexes were done using direct titrations with both metal ratios simultaneously, while inserting the stability constant for the ML species previously determined by competition as a fixed value in the speciation model. The potentiometric data were refined with the HYPERQUAD software,36 while species distribution diagrams and pM values were calculated using the HySS software.37 The overall equilibrium constants βiH and βMmHhLl are defined by βiH = [HhL]/[H]h[L] and βMmHhLl = [MmHhLl]/[M]m[H]h[L]l. Differences, in the log units, between the values of protonated (or hydrolyzed) and nonprotonated constants provide the stepwise (log K) reaction constants (being KMmHhLl = [MmHhLl]/[MmHh−1Ll][H]). The errors quoted are the

that the two BCAs conserve their affinity for copper ions as well as their selectivity over zinc(II). Both ligands were then conjugated to a bombesin antagonist peptide targeting the GRPr, and corresponding bioconjugates were successfully radiolabeled with 64Cu under mild conditions. The introduction of additional functional groups for bioconjugation did not alter the complex stability, as demonstrated by the stability tests in vitro. In a cell-based assay for determination of the binding affinity to GRPr, we obtained mixed results for our novel conjugates. While the metal-free and corresponding copper(II) complexes of the Hno2th1tha derivative exhibited a higher affinity than the current gold-standard RM2, the corresponding no2th-EtBzNCS conjugate showed a lower affinity. Obviously, apart from the charge at the N-terminus of GRPr antagonists, additional factors such as the lipophilicity and geometry of the corresponding radiometal chelate impact the GRPr affinity. Our results clearly demonstrate the potential of the described ligands for radiopharmaceutical applications, especially in terms of complex stability/inertness, bioconjugation, and radiolabeling properties, and warrant further development of 64Cu-labeled radiopharmaceuticals based on our novel chelators. A second interesting point is the possible behavior of the methylthiazolyl acid derivative to form polynuclear complexes in excess copper(II) metal ion in solution. Knowing the importance of such complexes in coordination chemistry, this point will also constitute one of our investigation objectives in further works.

■

EXPERIMENTAL SECTION

Materials and Methods. Reagents were purchased from Acros Organics and from Aldrich Chemical Co. 1,4,7-Triazacyclononane (tacn) was purchased from CheMatech (Dijon, France). RM2 was obtained from ABX (Radeberg, Germany). [64Cu]CuCl2 was purchased from the Department of Preclinical Imaging and Radiopharmacy of the Eberhard-Karls-University (Tübingen, Germany). [64Cu]CuCl2 was produced via the 64Ni(p,n)64Cu route.31 2(Bromomethyl)thiazole and 2-(chloromethyl)-1,3-thiazole-4-carboxylic acid methyl ester were obtained by following published procedures.9,13,19 Acetonitrile, tetrahydrofuran, and water solvents were distilled before use. HRMS analyses were performed at ICOA, Orléans, France. NMR spectra were recorded at the “Services communs” of the University of Brest. 1H and 13C NMR spectra were recorded with Bruker Avance 500 (500 MHz), Bruker Avance 400 (400 MHz), or Bruker AMX-3 300 (300 MHz) spectrometers. Coupling constants are given in hertz. Synthesis. Ligand syntheses are given in the Supporting Information, while the syntheses and characterizations of complexes are described here. Caution! Although no problems arose during our experiments, perchlorate salts and their metal complexes are potentially explosive and should be handled with great care and in small quantity. General Procedure for the Complex Syntheses in Acetonitrile. The ligand (0.1 mmol) was dissolved in acetonitrile (10 mL), and 1.2 equiv of the metal(II) perchlorate salts, dissolved in acetonitrile (1 mL), was added. The mixture was stirred for 12 h under reflux. The solvent was removed, and the residue was taken up in MeOH. The desired complex remained insoluble in MeOH, while excess metal(II) perchlorate salt was soluble. The compound was filtered off, washed with MeOH (3 × 3 mL), then dissolved in acetonitrile, and dried under reduced pressure. [Cu(no3th)](ClO4)2: green powder (55 mg, 96%). ESI-HRMS. Calcd for [CuC18H24N6S3]2+: m/z 241.5255. Found: m/z 241.5260. [Zn(no3th)](ClO4)2: white powder (53 mg, 95%). ESI-HRMS. Calcd for [ZnC18H24N6S3]2+: m/z 242.0253. Found: m/z 242.0256. [Cu(no2th-EtBzNO2)](ClO4)2: blue powder (48 mg, 95%). ESIHRMS. Calcd for [CuC22H28N6O2S2]2+: m/z 473.1774. Found: m/z 473.1781. L


Article

Inorganic Chemistry standard deviations calculated by the fitting program for the experimental data in each system. Spectroscopic Studies. For NMR experiments with zinc(II) complexes, the solution of the zinc(II) complex with the no3th ligand was prepared at ca. 1.0 mM (final concentration) by mixing at 40 °C for 15 min with 1.0 equiv of Zn(ClO4)2·6H2O and no3th in CD3CN. A solution of the zinc(II) complex of the ligand Hno2th1tha was prepared at ca. 1.0 mM (final concentration) by adjusting the pD to 6.8 and mixing at 40 °C for 15 min with 1.0 equiv of Zn(ClO4)2· 6H2O and Hno2th1tha in D2O. Solutions of the copper(II) complexes of the three ligands were prepared at ca. 1.0 mM concentration by dissolution of the previously isolated complexes in DMF/water, and these solutions were used for both the visible and EPR studies. The visible spectra were acquired at 25 °C on a PerkinElmer Lambda 650 spectrophotometer. The EPR spectra of the frozen sample solutions were acquired at 130 K on a Bruker EMX 300 spectrometer operating in the X band and equipped with a continuous-flow cryostat for liquid nitrogen, at a microwave power of 2.0 mW and a frequency of 9.5 GHz. All spectra were simulated using a single paramagnetic species because no additional species with different structures were distinguishable. Simulations of the experimental spectra were performed with the SpinCount software.38 Electrochemistry. The electrochemical studies were performed in a glovebox (Jacomex; O2 < 1 ppm and H2O < 1 ppm) with a homedesigned three-electrode cell (WE, glassy carbon; RE, Ag/AgCl/NaCl (3 M); CE, graphite rod). The potential of the cell was controlled by using an Autolab PGSTAT 302 (Ecochemie) potentiostat monitored using a computer. The glassy carbon electrode was carefully polished before each experiment with a 1 μm alumina aqueous suspension and ultrasonically rinsed in water and then with acetone. Exhaustive electrolysis was performed with a graphite rod working electrode. The ultrapure (18 MΩ) deoxygenated water was used as received and kept under argon in the glovebox after degassing. Lithium perchlorate (Sigma-Aldrich, 99.99%) was used as a supporting electrolyte at 0.1 M concentration without purification. X-ray Diffraction Determinations. X-ray diffraction data were collected by François Michaud (University of Brest) with a X-Calibur2 CCD four-circle diffractometer (Oxford Diffraction), including a four-circle goniometer (KM4) and a two-dimensional CCD detector (SAPPHIRE 2). Three-dimensional X-ray diffraction data were collected on an X-Calibur-2 CCD four-circle diffractometer (Oxford Diffraction). Data reduction, including interframe scaling, Lorentzian, polarization, empirical absorption, and detector sensitivity corrections, was carried out using programs that are part of the CrysAlis software39 (Oxford Diffraction). Complex scattering factors were taken from the program SHELX9740 running under the WinGX program system.41 The structure was solved by direct methods with SIR-9742 and refined by full-matrix least squares on F2. All hydrogen atoms were included in calculated positions and refined in riding mode. Crystal data and details of the data collection and refinement are summarized in Table S1. Peptide Synthesis and Bioconjugation. The bombesin peptide Ahx-[D-Phe6,Statine13]bombesin(6−14) (Ahx-JMV594; Ahx = 6aminohexanoic acid) was prepared by standard fluorenylmethyloxycarbonyl chloride (Fmoc) chemistry using Rink amide resin on a CS Bio CS336X peptide synthesizer according to the manufacturer’s protocol. A 4-fold excess of coupling reagent HATU and corresponding Fmoc-protected amino acids (Iris Biotech GmbH, Marktredwitz, Germany) was used in each coupling step. Purification of the peptide conjugates was performed by semipreparative RPHPLC using a Knauer Smartline 1000 HPLC system in combination with a Macherey Nagel VP 250/21 Nucleosil 120-5 C18 column. The solvent system was A = H2O (0.1% TFA) and B = acetonitrile (0.1% TFA). The semipreparative HPLC gradient was 0−40 min 5−60% B at a flow rate of 12 mL min−1. Analytical HPLC was performed on an Agilent 1260 Infinity System equipped with an Agilent 1200 DAD UV detector (UV detection at 220 nm) and a Raytest Ramona radiation detector (Raytest GmbH, Straubenhardt, Germany) in series. A Phenomenex Jupiter Proteo (250 × 4.60 mm) column was used for

analytical HPLC. The gradient was 0−1 min 5% B, 1−25 min 50% B at a flow rate of 1 mL min−1. no2th-EtBzNCS-Ahx-JMV594. Resin loaded with 0.2 mmol g−1 peptide (18.11 mg, 0.014 mmol) was put in 3 mL of dry DMF. Then, Hno2th-EtBzNCS (28.56 mg, 0.059 mmol) and DIPEA (10.5 μL, 0.059 mmol) were added, and the mixture was shaken for 2 h at rt. The resin was subsequently washed with DMF (3 × 2 mL) and CH2Cl2 (3 × 2 mL). After deprotection and cleavage with TFA/ H2O/TIS (95:2.5:2.5, v/v), the crude peptide conjugates were precipitated in ice-cold diethyl ether, centrifuged, and finally purified by semipreparative RP-HPLC. Yield: 1.03 mg, 3%. RP-HPLC (analytical, 220 nm): tR = 24.06 min. ESI-HRMS. Calcd for [C84H121N21O12S3 + H]+: m/z 1710.8581. Found: m/z 1710.8581. no2th1tha-Ahx-JMV594. Resin loaded with 0.2 mmol g−1 peptide (83 mg, 0.024 mmol) was introduced to 3 mL of dry DMF. Then no2th1tha (46 mg, 0.099 mmol), HATU (37 mg, 0.099 mmol), and DIPEA (17 μL, 0.099 mmol) were added, and the mixture was shaken for 2 h at rt. The resin was washed with DMF (3 × 2 mL) and CH2Cl2 (3 × 2 mL). After deprotection and cleavage with TFA/ H2O/TIS (95:2.5:2.5, v/v), the crude peptide conjugates were precipitated in ice-cold diethyl ether, centrifuged, and finally purified by semipreparative RP-HPLC. Yield: 1.71 mg, 5%. RP-HPLC (analytical, 220 nm): tR = 22.6 min. ESI-HRMS. Calcd for [C80H115N21O13S3 + H]+: m/z 1672.8061. Found: m/z 1672.8087. Preparation of the natCu Metallopeptides. The natCu metallopeptides were synthesized using a 1.5-fold excess of natCuCl2·2H2O (0.1 mM in ammonium acetate buffer). For each compound, the bioconjugates 7 and 8 (500 μg, 0.29 μmol) in 250 μL of H2O were mixed with 250 μL of a natCu stock solution containing 1.5 equiv of the corresponding metal salt (CuCl2·2H2O) and heated at 45 °C for 15 min. After cooling to rt, the complexes were purified using C18 Sep Pak cartridges, which were preconditioned with ethanol (EtOH) and H2O (5 mL each). After loading, the cartridge was washed with H2O (2 mL), and the product was eluted using EtOH/H2O (50:50, v/v; 2 mL). After evaporation of EtOH at ambient temperature, the samples were lyophilized to give the final metalated bioconjugates. nat Cu-no2th-EtBzNCS-Ahx-JMV594 (natCu-7). Yield: 488 μg, 0.27 μmol, 95%. RP-HPLC (analytical, 220 nm): tR = 21.40 min. ESIHRMS. Calcd for [CuC84H121N21O12S3]2+: m/z 886.3897. Found: m/ z 886.3824. nat Cu-no2th1tha-Ahx-JMV594 (natCu-8). Yield: 463 μg, 0.26 μmol, 92%. RP-HPLC (analytical, 220 nm): tR = 20.0 min. ESIHRMS. Calcd for [CuC80H115N21O13S3]2+: m/z 867.3637. Found: m/ z 867.3624. 64 Cu-Labeling Experiments. Radiolabeling was performed in 0.1 M buffered media [pH = 5.5 NaOAc, pH = 7.4 HEPES, and pH = 8.2 HEPES, where HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. Stock solutions of 7 and 8 were prepared in H2O (1 nmol μL−1). For labeling experiments, 5 μL of bioconjugate (1:100 dilution of a stock solution in the corresponding labeling buffer) was mixed with 119 μL of the corresponding buffer. To this solution was added 1 μL of [64Cu]CuCl2 in 0.1 M HCl (64Cu stock diluted to an activity concentration of ∼1−2 MBq μL−1) to give a final volume of 125 μL and a chelator concentration of 0.4 μM. After brief mixing, samples were either incubated at rt or 95 °C for 15 min. Analysis was initially performed by analytical RP-HPLC. The RCC was also checked by thin-layer chromatography (TLC) using silica gel microfiber strips (Agilent, iTLC-SG glass microfiber) and a dtpa solution (50 mM, pH = 7.4). No differences between the HPLC and TLC analyzed samples were observed so that determination was continued by TLC. While 64 Cu-7 and 64Cu-8 remained at the origin (Rf = 0), uncomplexed 64Cu (in the form of the corresponding dtpa complex) moved with the solvent front (Rf = 1). The TLC strips were analyzed by phosphorimaging, and the RCC was calculated. Each experiment was performed in triplicate. The identity of the 64Cu complexes was confirmed by the UV trace of the corresponding nonradioactive Cu2+ complexes. For H5dtpa-challenge and copper-exchange experiments, ∼20 MBq [64Cu]CuCl2 was added to 1 nmol of 7 and 8, respectively, in 0.1 M NaOAc pH = 5.5 and heated at 45 °C for 15 min. M


Article

Inorganic Chemistry H5dtpa Stability Measurements. Experiments were performed according to a previously described protocol.10 For each experiment, 1−2 MBq (1 μL) of [64Cu]Cu-7 or [64Cu]Cu-8 was mixed with 75 × 103-fold excess of H5dtpa as a competitive ligand. The samples were incubated for 24 h at rt. The dissociation of the radiopeptide was monitored by radio-TLC. Experiments were performed in triplicate. nat Cu-Exchange Experiments. Experimental conditions were based on a previously described procedure.10 Copper exchange was measured at pH = 7.4 in 0.1 M HEPES buffer. A nonradioactive Cu2+ solution was prepared as a stock solution of CuCl2·2H2O. For each experiment, 1−2 MBq (1 μL) of [64Cu]Cu-7 or [64Cu]Cu-8 was mixed with a 40-fold excess of natCu as the competitive cation. The mixture was incubated for 24 h at rt. The exchange rate between natCu and 64Cu was monitored by radio-TLC. Experiments were performed in triplicate. Lipophilicity (log Doct/PBS) Measurements. For log Doct/PBS measurements, 1−2 MBq of [64Cu]7, [64Cu]8, or [64Cu]RM2 (20 μL) was added to a presaturated mixture of pH = 7.4 phosphatebuffered saline (PBS; 480 μL) and octanol (500 μL). Samples were shaken for 30 min at rt and centrifuged at 21100g for 5 min, and 100 μL of each phase was counted for 1 min on a calibrated PerkinElmer 2480 Automatic Wizard Gamma Counter (Waltham, MA) by using a dynamic energy window of 400−600 keV for 64Cu (511 keV emission). Experiments were performed in triplicate. Determination of the Binding Affinity. The binding affinity profiles (IC50 values) of the metal-free peptides and the corresponding metallopeptides were determined using a cell-based competitive assay with 125I[Tyr4]-bombesin as the radioligand. RM2 was used as the reference. Experiments were performed using Merck multiscreen filter plates and punch kit. Briefly, each compound in different concentrations (1000−0 nM) was incubated in the presence of 0.15 nM 125I[Tyr4]-bombesin (PerkinElmer, Waltham, MA) at 37 °C for 1.5 h in 96-well plates seeded with 2 × 105 PC3 cells well−1. The total well volume was 150 μL. After incubation, the supernatant was removed by vacuum, and the wells were washed two times with 200 μL of cold PBS. Using the punch kit, the cell-associated radioactivity was transferred into counting tubes, and the radioactivity was measured using a PerkinElmer 2480 Automatic Wizard Gamma Counter. Data were fitted using nonlinear regression (GraphPad Prism). Experiments were performed at least in two independent experiments in triplicate. DFT Studies. Geometry optimizations of the [Cu(no2th)]2+, [Cu(no3th)]2+, and [Cu(no2th1tha)]+ complexes were performed with the Gaussian 09 package (revision D.01)43 using DFT calculations within the hybrid meta-GGA approximation with the TPSSh exchange-correlation function.44 The standard Ahlrichs’ valence triple-ξ including polarization functions (TZVP) basis set was used in these calculations.45 Solvent effects (water) were considered by using the integral equation formalism variant of the polarizable continuum model (IEFPCM).46 Frequency calculations were carried out to confirm the nature of the optimized geometries as local energy minima. Gibbs free energies were obtained at T = 298.15 K within the harmonic approximation. The default values for the integration grid (75 radial shells and 302 angular points) and the selfconsistent-field energy convergence criteria (10−8) were used in all calculations. Calculations of the g and A tensors were carried out using the ORCA program package (version 4.0.2).47,48 In these calculations, we used both the TPSSh and TPSS0 functionals.49 The TPSS0 functional is a 25% exchange version of TPSSh (10% exchange) that provides improved energetics.50 The center of the electronic charge was taken as the origin for the calculation of the g tensor, which is a gaugedependent property. The spin−orbit contributions to the hyperfine coupling constants and g values were computed with the spin−orbit mean-field approach (SOMF), employing the one-center approximation to the exchange term [SOMF(1X)].51 The basis set used for the EPR parameter calculations comprised Ahlrichs’ def2 TZVPP for ligand atoms and aug-cc-pVTZ-J for Cu.52 The aug-cc-pVTZ-J basis set was developed specifically for calculation of the EPR parameters, includes an improved description of the core region with four tight s-

type, one tight p-type, and one tight d-type functions, and presents a (25s17p10d3f2g)/[17s10p7d3f2g] contraction scheme. The RIJCOSX approximation53 was used to speed up calculations of the EPR parameter auxiliary basis sets constructed automatically by ORCA with the autoaux function.54 The convergence tolerances and integration accuracies of the calculations were increased from the defaults using the available TightSCF and Grid5 options (grid 7 for copper). Solvent effects were considered with the universal solvation model based on the solute electron density and on a continuum model (SMD).55

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b03280. All analytical characterizations of each compound by NMR and HRMS, HPLC chromatograms, UV−vis and EPR simulated spectra, DFT calculation details, optimized Cartesian coordinates obtained with DFT, and IC50 curves (PDF) Accession Codes

CCDC 1876232−1876235 contain 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 [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Luís M. P. Lima: 0000-0002-9729-330X David Esteban-Gómez: 0000-0001-6270-1660 Nicolas Le Poul: 0000-0002-5915-3760 Carlos Platas-Iglesias: 0000-0002-6989-9654 Rita Delgado: 0000-0002-0814-4960 Raphaël Tripier: 0000-0001-9364-788X Author Contributions

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.T. and V.P. acknowledge the Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique, and the “Service Commun” of NMR facilities of the University of Brest. They also thank the University of Brest and University of Bretagne-Loire for financial support given to A.G. in Freiburg. R.D. and L.M.P.L. acknowledge Fundaçaõ para a Ciência e a Tecnologia (FCT) for financial support under Project LISBOA-01-0145-FEDER007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020Programa Operacional Competitividade e Internacionalizaçaõ and by national funds through FCT, and L.M.P.L. also thanks the FCT for a postdoctoral fellowship (SFRH/BPD/73361/2010). C.P.-I. and D.E.-G. thank Centro de Supercomputación de Galicia for providing access to their supercomputing facilities. N


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Inorganic Chemistry

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M.D.B. thanks the University of Brest for the “Invited Professor” fellowship obtained in July 2018 and Prof. Matthias Eder for covering the costs of 64Cu.

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