Improved Conjugation, 64-Cu Radiolabeling, in Vivo Stability, and

Article Cite This: J. Med. Chem. 2018, 61, 8774−8796

pubs.acs.org/jmc

Improved Conjugation, 64-Cu Radiolabeling, in Vivo Stability, and Imaging Using Nonprotected Bifunctional Macrocyclic Ligands: Bis(Phosphinate) Cyclam (BPC) Chelators Tomás ̌ David,†,‡ Veronika Hlinová,† Vojtěch Kubíček,† Ralf Bergmann,‡ Franziska Striese,‡ Nicole Berndt,§,∥ Dávid Szöllő si,⊥ Tibor Kovács,#,∇ Domokos Máthé,⊥ Michael Bachmann,‡,○,◆ Hans-Jürgen Pietzsch,‡ and Petr Hermann*,† Downloaded via KAOHSIUNG MEDICAL UNIV on October 14, 2018 at 10:35:05 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030, 128 40 Prague, Czech Republic Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany § Partner Site Dresden, German Cancer Consortium (DKTK), Fetscherstrasse 74, 01307 Dresden, Germany ∥ German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ⊥ Department of Biophysics and Radiation Biology, Semmelweis University, Tű zoltó utca 37−47, H-1094 Budapest, Hungary # Institute of Radiochemistry and Radioecology, University of Pannonia, Egyetem St. 10, H-8200 Veszprém, Hungary ∇ Social Organization for Radioecological Cleanliness, P.O. Box 158, H-8200 Veszprém, Hungary ○ Tumor Immunology, University Cancer Center (UCC), “Carl Gustav Carus” Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany ◆ National Center for Tumor Diseases (NCT), “Carl Gustav Carus” Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany ‡

S Supporting Information *

ABSTRACT: Bifunctional derivatives of bis(phosphinate)bearing cyclam (BPC) chelators bearing a carboxylate, amine, isothiocyanate, azide, or cyclooctyne in the BP side chain were synthesized. Conjugations required no protection of phosphinate or ring secondary amine groups. The ring amines were not reactive (proton protected) at pH < ∼8. For isothiocyanate coupling, oligopeptide N-terminal α-amines were more suitable than alkyl amines, e.g., Lys ω-amine (pKa ∼7.5−8.5 and ∼10−11, respectively) due to lower basicity. The Cu-64 labeling was efficient at room temperature (specific activity ∼100 GBq/μmol; 25 °C, pH 6.2, ∼100 ligand equiv, 10 min). A representative Cu-64-BPC was tested in vivo showing fast clearance and no nonspecific radioactivity deposition. The monoclonal anti-PSCA antibody 7F5 conjugates with thiocyanate BPC derivative or NODAGA were radiolabeled and studied in PC3-PSCA tumor bearing mice by PET. The radiolabeled BPC conjugate was accumulated in the prostate tumor with a low off-target uptake, unlike Cu-64-labeled NODAGA−antibody conjugate. The BPC chelators have a great potential for theranostic applications of the Cu-64/Cu-67 matched pair.

■

Similarly to other metal radioisotopes, 64/67Cu must be administered in the form of a thermodynamically stable and kinetically inert complex to avoid nonspecific deposition of the metal radioisotope in tissues. The most commonly used ligands for metal complexation in molecular imaging are derivatives of macrocyclic ligands, NOTA, DOTA, or TETA, with various functional groups (Chart 1). These imaging agents are commonly a conjugate of a bifunctional chelator and a targeting vector molecule

INTRODUCTION Theranostic applications of new radiopharmaceuticals are based on pairs of radionuclides for diagnostic imaging and therapeutic treatment. For the diagnostics, the β+-emitting radioisotopes are used and, for the therapy, the β−-emitting radioisotopes are utilized.1 One of these pairs is 64Cu for positron emission tomography (PET) and 67Cu for the therapy. The imaging radioisotope 64Cu stands out for its high availability, suitable properties (relatively long half-life τ1/2 = 12.7 h and soft positron radiation 19% β+, Eβ+max = 656 keV), and theranostic combination with the therapeutic radioisotope 67 Cu (τ1/2 = 61.8 h, 100% β−, Eβ− = 390−575 keV).2−5 © 2018 American Chemical Society

Received: June 11, 2018 Published: September 5, 2018 8774

DOI: 10.1021/acs.jmedchem.8b00932 J. Med. Chem. 2018, 61, 8774−8796

Journal of Medicinal Chemistry

Article

Chart 1. Structure of Ligands Discussed in the Texta

Most macrocyclic complexes intended for medicine have good thermodynamic stability and kinetic inertness. However, slow complex formation is often the main problem for their utilization in radiopharmaceuticals. Radiolabeling efficiency with copper radioisotopes has been improved, over the original TETA and DOTA derivatives, using NOTA/NODAGA derivatives13−19 and sarcophagin-based ligands, e.g., diamsar20−23 (Chart 1). Radiolabeling can be also enhanced by modifying the macrocycle pendant arms, and the methylphosphonic acid group has been particularly successful in cross-bridged cyclams derivatives (Chart 1). For example, CBTE2P shows good labeling with 64Cu, even at room temperature,24−29 although CB-TE2A requires prolonged heating at high temperature. Recently, cyclams with picolinate pendant arm(s) (e.g., TE1PA, Chart 1) have been suggested as ligands for efficient radiocopper incorporation.30−33 Among ligand skeletons, cyclam-based ligands are known to have the best thermodynamic selectivity for Cu(II). We have previously shown that Cu(II) selectivity is preserved in phosphonic acid cyclam derivatives and is associated with the good kinetic inertness of Cu(II) complexes of these ligands.34−36 On the basis of our experience with cyclam phosphonic acid derivatives 34 and on studies on simple geminal bis(phosphinates),37,38 we have recently designed cyclam-based ligands with one geminal bis(phosphinate) (te1bpin) or geminal phosphino-phosphonate (te1pp) pendant arm (Chart 1) for fast complexation of Cu(II) ions.39 The pendant arms effectively help to transfer Cu(II) ions from the bulk solution to cyclam units. Consequently, very efficient radiolabeling with 64Cu was achieved, even significantly outperforming the most commonly used ligands, DOTA and NOTA (Chart 1), in both radiochemical yield and specificity. Despite the structural similarity of geminal bis(phosphinates) to geminal bis(phosphonates), which are routinely used for bone targeting,40 neither bis(phosphinates)41 nor te1bpin, te1pp, or their complexes39 show any hydroxyapatite adsorption. Therefore, these ligands may be used in nuclear medicine as metal ion carriers for diagnosis or therapy in various tissues because no bone uptake is expected. To further capitalize on the excellent radiolabeling properties of bis(phosphinate)-cyclams, we decided to prepare their bifunctional derivatives. To introduce the bifunctional group onto te1bpin, the reactive group could be located at various positions of the ligand core: (i) Most commonly, it is attached to one of three secondary nitrogen atoms of the macrocycle. However, the attachment of additional alkyl groups to cyclam

a

The ligand diamsar is an example of the sarcophagin class of ligands, whereas CB-TE2A and CB-TE2P are examples of cross-bridged cyclam derivatives.

(oligopeptide, antibody or its fragments, or hormone, among others). To form the conjugates, carboxylates or amines are the most commonly used coupling groups in amide-forming reactions exploring a range of coupling agents, both in the laboratory6,7 and at industrial scale.8 The other reactions are couplings with isothiocyanate or bioorthogonal click reactions,9 as copper-catalyzed azide−alkyne cycloaddition (CuAAC), strain-promoted azide−alkyne cycloaddition (SPAAC), or inverse electron-demand Diels−Alder click reaction (iEDDA). Bifunctional ligands containing these click groups, including also those for copper radioisotopes, have been synthesized and developed for nuclear medicine applications.10−12 Scheme 1. Synthesis of Bis(phosphinic Acid) Precursorsa

(i) Hex-5-ynoic acid, Et3B, MeOH, dioxane, air, room temperature, 4 h; (ii) dibenzylamine, (CH2O)n, 6 M aq HCl/THF 1:1, 80 °C, overnight; (iii) trimethylsilylchloride, N,N-diisopropyl-ethylamine (DIPEA), room temperature, 1 h; (iv) (1) p-nitrobenzyl bromide, CH2Cl2, room temperature, 3 d, (2) EtOH; (v) hexamethyldisilazane (HMDS), 110 °C, overnight; (vi) (1) t-butyl acrylate, CH2Cl2, room temperature, overnight, (2) EtOH; (vii) trifluoroacetic acid, room temperature, overnight. a

8775



Article

Scheme 2. Synthesis of Bifunctional Cyclam Derivatives with a Bis(phosphinate) Pendant Arma

(i) cyclam, (CH2O)n, H-phosphinic acid, 6 M aq HCl, 60 °C (for L1-COOH and L2-COOH) or 80 °C (for 6 and 7), overnight; (ii) (NH4)H2PO2, 10% Pd/C, H2O, 60 °C, overnight; (iii) H2 (atmospheric pressure), 10% Pd/C, H2O, room temperature, 3 h; (iv) CSCl2, CCl4/ diluted aq HCl, room temperature, overnight; (v) (1) NaNO2, diluted aq HCl, 1 °C, 40 min, (2) NaN3, H2O, room temperature, 3 h. a

previously published protocol (see SI).54 Although the isolated yield of compound 1 was rather low (∼30%), the combination of inexpensive starting materials and an easy workup allowed the gram-scale production of the precursor. All other pendant arm intermediates (2, 3, and 5) were prepared from methylene-bis(phosphinic acid), which contains two identical P−H bonds of similar reactivity. Theoretically, the excess of methylene-bis(phosphinic acid) over reaction partners could be used to attain monosubstitution. Because methylenebis(phosphinic acid) is by far the most expensive reactant, the stoichiometry was chosen accordingly, just a minor excess of methylene-bis(phosphinic acid) or even less than an equimolar amount. Although inevitably leading to a statistical mixture of di-, mono-, and nonsubstituted methylene-bis(phosphinic acids), this approach was more cost-effective than using a large excess of the most valuable component. Mannich-type reactions between methylene-bis(phosphinic) acid, paraformaldehyde, and dibenzylamine yielded compound 2 (Scheme 1). Because of the poor solubility of dibenzylamine in acidic conditions,37 various solvent mixtures were used (SI, Table S1), including a mixture of aq HCl and THF, which led to compound 2, albeit with various side products, such as Phydroxymethylated and P-oxidized species and 4-chlorobutanol (resulting from THF opening by HCl). Nevertheless, the scale-up of this procedure combined with ion exchange workup easily generated compound 2 at sufficient purity for the subsequent synthesis of macrocyclic intermediate 6 (Scheme 1); the P-hydroxomethylated byproduct is inert in the synthesis of 6. Mixing aq HCl and MeOH led to a cleaner reaction mixture but with lower conversion. The reaction carried out in 50% aq AcOH led to a high conversion rate (∼80%) and a clean reaction mixture, and, after HPLC purification, pure 2 was obtained at 59% isolated yield. The structure of compound 2 was unambiguously confirmed by Xray diffraction analysis (SI, Table S2, Figures S2 and S3). The 4-nitrobenzyl derivative 3, the key intermediate in the synthesis

nitrogen atoms significantly reduces the kinetic inertness of the complexes.42,43 (ii) The reactive group on macrocycle carbon atoms (e.g., in BAT, Supporting Information (SI), Figure S1) are mostly available only via multistep synthesis.44−47 (iii) Substitution at the N−C−P carbon atom are, in general, rather low-yielding reactions. (iv) Substitution at the P−C−P methylene group is also available, and P-alkyl-substituted methylene-bis(phosphinates) are fully stable, unlike derivatives with both phosphorus atoms with P−H bonds.41,48 (v) Substitution at the terminal phosphorus atom of the bis(phosphinate) group (i.e., formal replacement of the P−H bond in te1bpin), has been overlooked until now. Synthetically, the P−H bond is relatively well accessible for replacement.49 Therefore, we explored mainly the last structural modification of the parent chelator,50−53 and we also studied a possible utilization of fully unprotected chelators in conjugation reactions. To our best knowledge, this is the first detailed study to use macrocycles with unprotected secondary amine groups in conjugation reactions. To define the usefulness of this class of ligands, we carried out a comparative radiolabeling study, and representative labeled chelator and an antibody conjugate, in comparison to a NODAGA-NCS conjugate (SI, Figure S1), were also tested in vivo.

■

RESULTS Synthesis of Bis(phosphinic Acid) Precursors. To introduce bifunctionality onto the bis(phosphinate) pendant arm, bis(phosphinic acid) precursors 1−5 (Scheme 1) were prepared in the first step of the synthetic sequence. Experimental and characterization details are given in the SI. The starting methylene-bis(phosphinic acid) is easily obtained from commercial CH2(PCl2)2. The derivative 1 with a 5-carboxypentyl group at the P,P-bridging methylene carbon atom was prepared by double radical addition of sodium hypophosphite to hex-5-ynoic acid according to a 8776



Article

observed in EtOH. Simple filtration of the catalyst and lyophilization generated L4-NH2 at 85% isolated yield. The aniline derivative L4-NH2 in the powdered zwitterionic form can be stored in a refrigerator for months without any degradation. The isothiocyanate ligand L5-NCS was prepared in a reaction of L4-NH2 with thiophosgene in a water−CCl4 mixture (Scheme 2), as described in literature for the analogous DOTA monophosphinic acid derivative.55 The conversion of aniline group into the corresponding arylisothiocyanate is a challenging reaction due to the presence of unprotected secondary amine groups. When the reaction was conducted in neutral or slightly acidic pH, multiple compounds were found in the reaction mixture. However, the reactivity of macrocycle amines was fully suppressed at pH ≤ 1 because they are fully protonated (i.e., proton protected, see below) and the desired product, L5-NCS, was prepared at ∼90% conversion. The extraction workup of the water−CCl4 mixture generated the product at ∼90% purity, which sufficed for further conjugation reactions. Nevertheless, L5-NCS was further purified by preparative HPLC (61% isolated yield). The azide-containing ligand L6-N3 was synthesized by diazotation of the aniline moiety of L4-NH2 using sodium nitrite followed by treatment of the reaction mixture with sodium azide (Scheme 2). The low-temperature diazotation (at ∼0 °C) proceeds quantitatively; however, a slight excess of L4-NH2 (in respect to NaNO2) and gradual addition of NaNO2 at room temperature were necessary to minimize the formation of byproducts (e.g., ring-N-nitroso derivative; details are discussed in SI, Figure S4). The azide ligand L6-N3 was purified by preparative HPLC at 63% isolated yield. Conjugation Reactions. Protected macrocycles are mostly used for conjugation reactions. As we had efficient procedure for synthesis of the unprotected chelators, we wanted to investigate their utilization in conjugation reactions. However, conjugation of the bare bis(phosphinate)-containing cyclam derivatives through the aforementioned groups is challenging due to the unavoidable presence of other potentially reactive groups in the molecules: the unprotected macrocycle secondary amine and the free phosphinate groups. To achieve the aim, the conjugation reactions were carried out with model substrates, i.e., amines, amino acids, or oligopeptides, to determine the optimal conditions under which more complex/ valuable biomolecules can be used or other reactive groups can be introduced. The reaction course was optimized to assess the simplest conditions with an easy workup, not necessarily for quantitative yield, and the shortest reaction time possible. The structures of all substrates used and their abbreviations are shown in SI, Figure S5. In addition, because of the presence of the highly polar and ionic cyclam-bis(phosphinate) zwitterionic fragment, all studied ligands, L1−L6 (Scheme 2), are well soluble in water but poorly soluble in nonaqueous solvents. Thus, the conjugation reactions were performed mostly in aqueous buffers (the organic cosolvents were exclusively added to dissolve the other substrate). Amide Coupling Reactions. In the amide coupling reactions, the carboxylate group is activated to react with an amine group and two strategies have been commonly used for the activation. In the first, the intermediate with the esteractivated carboxylate group was isolated and then used in the amide-forming reaction (preprepared ester = PPE method). In the second method, the active carboxylate ester was directly prepared in the reaction mixture containing the amine (in situ

of the bifunctional ligands L4-NH2, L5-NCS, and L6-N3, was prepared by the Arbuzov reaction with phosphite [(Me3SiO)2P]2CH2 prepared in situ by silylation of methylene-bis(phosphinic acid) using trimethylsilyl chloride in the presence of a base (Scheme 1). Its reaction with slightly more than one equivalent of 4-nitrobenzyl bromide yielded 3 at an unexpectedly high conversion rate (∼85%). Apparently, the reactivity of the other silylphosphite group decreased when attaching the first 4-nitrobenzyl moiety, surprisingly resulting in the low abundance of the bis(4-nitrobenzyl)-substituted derivative in the reaction mixture. Consequently, a high isolated yield (69%) was obtained after the ion exchange workup. The carboxylic acid intermediate 5 was also prepared by a two-step process similar to a previously reported procedure.53 In the first step, hexamethyldisilazane (HMDS) was used to convert methylene-bis(phosphinic) acid to its silylphosphite derivative at high temperature, followed by its reaction with t-butyl acrylate to yield 4. Surprisingly, no reaction with t-butyl acrylate was observed when silylation was performed using trimethylsilyl chloride and N,N-diisopropylethylamine in dichloromethane. The scale-up of the HMDS procedure and ion exchange workup led to a mixture of monoand disubstituted derivatives, which are hardly separable without HPLC. Therefore, the t-butyl esters were deprotected using TFA, and the mixture of the acids (the disubstituted acid is an inert impurity in the following reaction steps) was directly used to synthesize the bifunctional ligand L2-COOH (Scheme 2). To prepare pure intermediate 5, the t-butyl esters forms were separated by preparative HPLC, yielding pure compounds 4 and ultimately 5 (after TFA deprotection) was isolated at 22% yield in a two-step process. Generally, only the remains of the starting methylenebis(phosphinic acid) have to be removed as the disubstituted products are not reactive in the next step and can be easily removed. Synthesis of Bifunctional Ligands. The bis(phosphinic acid) intermediates 1−3 and 5 were treated with paraformaldehyde and with an excess of cyclam (Scheme 2). Similarly to the synthesis of te1bpin,39 the excess of cyclam prevents the formation of multiply substituted cyclam derivatives, thereby enabling us to easily recover cyclam during the ion-exchange resin workup. The reactions yielded the carboxylate ligands L1COOH (58% yield) and L2-COOH (21% yield over three steps from the methylene-bis(phosphinic acid)) as well as the macrocyclic intermediates 6 (74% yield) and 7 (71% yield). Benzyl groups in compound 6 were removed by transfer hydrogenation in 10% Pd/C catalyst using ammonium formate or ammonium hypophosphite. Both reagents enabled the quantitative removal of benzyl groups; however, some decomposition of the substrate and/or the product was also observed (an additional P−H signal was observed in the 31P NMR spectrum of the reaction mixture). Moreover, in the case of ammonium formate, N-formylated byproducts were also formed (characteristic signals at 7.8−8.2 ppm were observed in the 1H NMR spectrum of the reaction mixture). Under the best conditions (30% catalyst loading, 60 °C, overnight, ammonium hypophosphite as the hydrogen source), the amine ligand L3-NH2 was isolated at 66% yield after ion-exchange workup. The nitro group of 7 was quantitatively converted to the primary amine group of L4-NH2 by hydrogenation at 10% Pd/ C in water by continuously bubbling hydrogen gas into the reaction mixture (Scheme 2). Surprisingly, no reaction was 8777



Article

Scheme 3. Amide Coupling Reactionsa

a

(i) aq buffer−MeCN 1:1, pH 5.1−7.4; (ii) EDC·HCl, NHS, aq buffer−MeCN 3:1, pH 5.1−7.4. Optimized conditions: L3-VGG, MES−NaOH (pH 6.8), room temperature, 48 h, ∼75% conversion, ∼45% yield (PPE method); L4-BTN, MES−NaOH (pH 6.2), room temperature, 48 h, ∼85% conversion, ∼65% yield; L4-DBCO, MES−NaOH (pH 6.2), room temperature, 48 h, ∼75% conversion, ∼60% yield.

adding an excess of activating reagents increased the abundance of side products (formation of N-acylisourea and aminolysis of NHS; for details, see below). Therefore, their initial amounts were reduced to 1.0 equiv of EDC and 0.5 equiv of NHS. The coupling reaction was faster at higher pH due to the higher abundance of the deprotonated side arm amino group of L3-NH2 (expected pKa ∼8−9).37 However, the higher pH also led to the faster hydrolysis of the NHS ester, thereby decreasing the activation efficiency. Thus, the highest conversion to L3-VGG (Scheme 3) obtained when using the ISA method was ∼50% at pH 6.8 after 2 d and with ∼50% of unreacted HO-VGG-Cbz. A similar pH-dependent process was also observed when using the PPE method. Here, the tripeptide was initially identified as an NHS active ester and, thus, the reaction proceeds at significantly higher conversion rates than those of the ISA method. The L3-VGG conjugate was produced at ∼75% conversion under optimized conditions (pH 6.8, macrocycle−tripeptide molar ratio 1:1, 2 days) and at 45% isolated yield. L3-NH2 binding to the tripeptide through the P−CH2−NH−C(O) fragment was unambiguously confirmed by 2D NMR (SI, Figure S6). Because the PPE method is more efficient, it was used for L4-NH2 (Scheme 2) coupling. The coupling was tested with NHS-biotin and NHS-β-CDBCO to introduce a common biological vector (biotin in L4-BTN) or another functional group (dibenzocyclooctyne group in L4-DBCO), respectively (Scheme 3, SI, Table S5). The later approach exemplifies a one-step conversion of a bifunctional ligand (bearing amino group) to another, which is suitable for strain-promoted azide−alkyne cycloaddition (SPAAC). The weakly basic aromatic amine of L4-NH2 (expected pKa ∼ 5, similar to aniline) led to higher yields at pH 5.1 and 6.2 than those

activation = ISA method). In the present study, the EDC− NHS pair (EDC = N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide used as the condensation reagent, NHS = Nhydroxy-succinimide as the activating alcohol), commonly used in amide coupling reactions performed in aqueous media, was chosen because the same carboxylate NHS ester is the active reagent in both strategies. To assess the effect of the free ring amine and pendant phosphinate groups, N-protected tripeptide HO-Val-Gly-GlyCbz (HO-VGG-Cbz) and the macrocycle 7 (Scheme 2) containing no amino group in the pendant arm were used as model compounds (Scheme 3). The tripeptide was activated in situ (called below ISA method) in the presence of the macrocycle 7 (1.5 equiv of each EDC and NHS were used) or the isolated solid tripeptide NHS-ester (NHS-VGG-Cbz) was reacted with the macrocycle 7 (called below PPE method). Both amide coupling reactions were performed at the pH range 5.1−7.4, with equimolar amounts of the tripeptide and of the macrocycle 7 (SI, Table S3). In all cases, no amide coupling product was detected. In the ISA strategy at pH 5.1, the NHSVGG-Cbz active ester could even be quantitatively produced in the presence of nonaltered macrocycle 7. The activation slowed and lost efficiency with the increase in pH due to hydrolysis of both the NHS ester and EDC itself. In the PPE approach, NHS-VGG-Cbz slowly hydrolyzed to HO-VGGCbz and, as expected, this decomposition accelerated when increasing the pH. In addition, the free phosphinic acid/ phosphinate groups are also inert under these conditions because no changes were observed in the groups. The amide coupling reactions of the same peptide (HOVGG-Cbz) with L3-NH2 (Scheme 2) were tested in the pH range 5.1−7.4 (Scheme 3, SI, Table S4). In the ISA method, 8778



Article

Scheme 4. Conjugations of Ligands with Carboxylic Acid in the Pendant Arm, L1-COOH and L2-COOHa

a

(i) EDC·HCl, NHS, aq buffer−MeCN 3:1, pH 5.1−7.4; (ii) TBTU or COMU, DIPEA, dry DMSO. Optimized conditions: L1-PEA, MES− NaOH (pH 5.1), room temperature, 24 h, ∼75% conversion, ∼60% yield; L1-Phe, MES−NaOH (pH 6.2), room temperature, 24 h, ∼75% conversion, ∼50% yield (ISA); L2-Phe, TBTU, room temperature, 180 min, ∼80% conversion, ∼45% yield.

obtained when using L3-NH2 with a more basic aliphatic amino group (e.g., conversion rates ∼70% for L4-BTN vs ∼5% for L3-VGG at pH 5.1 after 24 h). The pH was further lowered, and the reaction rate at pH 4.7 dramatically decreased due to protonation of the aniline group of L4-NH2 (data not shown). At pH 6.8 and 7.4, the differences between the coupling efficiency of L3-NH2 and L4-NH2 were small. The maximal conversion rates were observed at pH 6.2 after 2 d (∼75% for both L4-BTN and L4-DBCO with equimolar amounts of reagents). These conversion rates may not be satisfactory for valuable substrates, yet almost quantitative conversion (≥95%) to L4-DBCO (SI, Table S6) was achieved with a 3-fold excess of L4-NH2 at pH 5.1 or 6.2 after 1 day when adding an excess of ligand and lengthening the reaction time. The coupling reactions of the ligand L1-COOH were performed (Scheme 4) with two amine substrates differing in the basicity of the primary amino group: (2-phenyl)ethylamine (PEA, pKa = 9.8)56 and t-butyl ester of L-phenylalanine (HPhe-tBu; predicted57 pKa = 7.1 ± 0.3). These two substrates are models for coupling to rather basic amino group in a peptide side chain (e.g., ε-amino group of lysine) and in a terminal α-amino group of (an)a (oligo)peptide chain, respectively. Using the preprepared active ester of the ligand (PPE method) was unsuccessful. The reaction between the ligand and EDC/NHS led to mixtures with several byproducts, which were difficult to purify. In situ activation (ISA method) was more successful, but using an excess of EDC and NHS generated undesired byproducts resulting from the EDCassisted aminolysis of NHS (details in SI, Table S7), as only recently described in literature. 58 Thus, the reaction stoichiometry was optimized for the L1-COOH conjugation with PEA, yielding L1-PEA (Scheme 4, SI, Table S7). The highest conversion was achieved with 0.5 equiv of NHS. The higher amount of NHS increased the abundance of byproducts, whereas the lower amount of NHS decreased the

reaction rate. The best conditions were then applied for amide coupling of both amine substrates (SI, Table S7). In this particular reaction, the basicity of the amine substrates was rather unimportant and, under the best conditions, ∼75% conversion rates were attained. The conversion rates at pH 5.1 and 6.2 were higher than those attained at pH 6.8 and 7.4. At higher solution pH, the coupling became less efficient due to the EDC-assisted aminolysis of NHS, yielding β-alanine derivatives (PEA-βAlan and Phe-βAlan; for more details, see SI, Table S8). The isolated yields obtained under optimized conditions were 60% and 50% for L1-PEA and L1-Phe (Scheme 4), respectively. Surprisingly, L2-COOH and H-Phe-tBu conjugation under the conditions (ISA method) used for L1-COOH failed to yield any product. Thus, a different type of amide conjugation reaction was tested, which has been successfully used for amide coupling to other ligands with free phosphinic and carboxylic acid groups, e.g., the TRAP ligand family (SI, Figure S1),50 using nonaqueous coupling with TBTU or COMU as coupling agents (Scheme 4). Unfortunately, the very low solubility of the studied bifunctional ligands in polar aprotic solvents considerably hampered testing nonaqueous coupling conditions. Nevertheless, the reactions can be performed in a suspension of the ligand in dry DMSO (SI, Table S9). The ligand, L2-COOH, was fully dissolved after adding three equivalents of TBTU. At this point, the conversion rate was still too low (∼15%). This indicates that the coupling agent was likely hydrolyzed by traces of water (L2-COOH could be isolated only as a hydrate). The addition of another portion of TBTU (another 3 equiv) caused a sharp and fast increase in conversion rate (∼80% after 1 h), whereas the third portion of TBTU (another 3 equiv) decreased the yield of L2-Phe (Scheme 4) and caused the formation of additional byproducts. When the coupling was repeated with COMU, a lower coupling efficiency was achieved (∼60%), even when 8779



Article

Scheme 5. Conjugation Reactions of L5-NCSa

a

(i) aq buffer−MeCN 3:1, pH 6.2−10.1; (ii) aq buffer without MeCN, pH 7.4−10.1. Optimized conditions: L5-GGY, MOPS−NaOH (pH 8.0), room temperature, 24 h, ∼90% conversion, ∼65% yield; L5-NAP, H3BO3−LiOH (pH 10.1), room temperature, 24 h, ∼30% conversion, ∼15% yield (∼45% H-Acp-βAla-NAP recovery).

time (∼60% after 3 d). At higher pH (9.3 and 10.1), the hydrolysis of the isothiocyanate group became the dominant reaction. The distant aliphatic amino group of the H-Acp-βAlaNAP is much more basic than the amino group of H-GGYOH, and, thus, its conjugation with L5-NCS to yield L5-NAP (Scheme 5) at pH 7.4 and 8.0 was ineffective; the isothiocyanate group was only gradually hydrolyzed in the reaction mixture. At higher pH (9.3 a 10.1) and with an excess of ligand, the desired L5-NAP conjugate was produced, albeit in a low quantity (∼25−30%; SI, Table S10). Although the purification of the product from the reaction mixture was possible (Figure 1), this conjugation approach is clearly unsuitable for valuable substrates. Strain-Promoted Click Reactions. Alkyne−azide click chemistry is a popular coupling strategy due to its compatibility with a broad range of functional groups and the relatively good synthetic availability of alkyne- and azidemodified substrates. The reaction is commonly catalyzed by Cu(I) ion or its complexes. However, such a strategy may not be used with cyclam-based ligands because Cu(I) ions are easily oxidized and Cu(II) ions are rapidly and irreversibly complexed by the cyclam moiety. An alternative is the strainpromoted alkyne−azide cycloaddition (SPAAC), which can be performed without metal catalysis and has been successfully applied in radiochemistry.10−12 Therefore, the SPAAC reactions between an azide group and a dibenzocyclooctyne (DBCO) moiety were also investigated here. The coupling reactions were tested with ligands bearing either an azide group (L6-N3) or a DBCO group (L4-DBCO, prepared by amide coupling from L4-NH2). The L6-N3 was conjugated with δ-CDBCO to yield L6-CDBCO (Scheme 6), and it can be considered a model reaction for conjugations to DBCOlabeled biomolecules. The L4-DBCO was conjugated with 3azido-7-hydroxy-coumarin (HAC),61 which was chosen as a representative of a fluorescent moiety, producing a fluorescent ligand after conjugation, L4-FC (Scheme 6). The resulting conjugate may be used for bimodal PET−optical imaging. The results are summarized in SI, Table S11. Both reactions (at 10 mM) were significantly faster than aqueous amide coupling reactions described above, e.g., L6-CDBCO was quantitatively

using a large excess of COMU (9 equiv). This was likely caused by the higher sensitivity to hydrolysis of this coupling agent than that of TBTU.7 Thus, using TBTU as the coupling agent, the L2-Phe conjugate was isolated at 45% yield. Such nonaqueous approach (TBTU activation, initial suspension of L1-COOH in DMSO, DIPEA) also afforded L1-Phe (38% isolated yield), showing that organic solvents with poorly soluble unprotected ligands can also be used for conjugation (Scheme 4) even though the aqueous ISA method produces cleaner reaction mixtures that are easier to purify by HPLC. Isothiocyanate Coupling Reactions. The coupling reaction is selective for amines, and its main problem is concurrent hydrolysis of the −NCS group which is progressively faster at higher pH. Therefore, pH stability of the L5-NCS was tested. At pH ∼ 6 and at room temperature, its complete hydrolysis lasted approximately three months, and the main product of this slow hydrolysis was the symmetrical thiourea dimer, L2-TU (SI, Figure S7). At pH ∼ 8−9, the hydrolysis was completed within 1−2 days and multiple products were formed. The products can be likely ascribed to intermolecular reactions between the isothiocyanate group and the secondary cyclam amines. To investigate the potential of L5-NCS (Scheme 2) as an unprotected bifunctional ligand, its coupling reactions were tested with two unprotected oligopeptides differing in the basicity of the free amino group (Scheme 5): N-terminal αamino group in H-Gly-Gly-Tyr-OH (H-GGY-OH, predicted59 pKa ∼ 8.0) and distant aliphatic amino group in H-Acp-βAlaNle-Asp-His-DPhe-Arg-Trp-Gly-NH 2 (i.e., H-Acp-βAlaNAP,60 estimated59 pKa ∼ 10.3−10.7). The results (SI, Table S10) indicate that the basicity of the amine-containing substrate has a dominant effect on the conjugation efficiency. At pH 7.4 and 8.0, the conjugation of the low-basicity amine group in H-GGY-OH with L5-NCS was faster than the hydrolysis of the ligand isothiocyanate group, and, thus, the reactions afforded almost quantitatively L5-GGY (Scheme 5) after 24 h (>50% conversion in the first 3 h). At lower pH (6.2), the conjugation was significantly slower (∼25% of L5GGY after 24 h), but the sufficient stability of L5-NCS at low pH allowed achieving a reasonable conversion rate after a long 8780



Article

insolubility of DBCO and coumarine derivatives in water. The coupling reactions quantitatively yielded L6-CDBCO and L4FC from an equimolar mixture of the corresponding reactants. No interference from the free ring amine or pendant phosphinate groups was observed. The conjugates are well soluble in water. 64 Cu Radiolabeling. The effect of the derivatization of the bis(phosphinate) pendant arm on radiolabeling properties was assessed. The high labeling efficiency of the parent bis(phosphinate) ligands, te1bpin and te1pp, with [64Cu]CuCl2 was previously described39 and was now compared with an extended set of the most important established ligands, i.e., with cyclam, DOTA, NOTA, CB-TE2A, and diamsar (Chart 1). The relatively low molar excess of the ligands over the molar amount of the copper radioisotope was used to highlight differences among the chelators and show specific activity that can be achieved. Under these conditions, the cyclam bis(phosphinate) derivatives were apparently the most efficient ligands in the radiolabeling reactions (Figure 2A). To test possible changes in the labeling efficiency of the parent bifunctional ligands and their conjugates, the labeling of te1bpin, of selected functionalized ligands (L2-COOH and 7; Scheme 2) and of their conjugates (L1-PEA, L4-BTN, L4DBCO, and L5-GGY; Schemes 3−5) was directly compared under previously described conditions (i.e., at pH 6.2, 25 °C and with ∼100 equiv of ligand excess over no-carrier-added (NCA) [64Cu]CuCl2; 10 min labeling time (Figure 2B).39 Under the conditions used (only ∼100-fold molar excess of the ligands over a molar amount of [64Cu]CuCl2), ligand labeling not only was fast and efficient but also led to the high specific activities of the prepared radiopharmaceuticals (up to ∼100 GBq/μmol), which is a highly desirable property considering their possible applications (see below). In Vivo Characterization of L6-N3 Labeled with 64Cu. The 64Cu-labeled prototypic chelator L6-N3 (Scheme 2), [64Cu-(L6-N3)], was evaluated in vivo in male Wistar rats. Dynamic PET showed fast distribution of the [64Cu-(L6-N3)] in the rat organism (Figure 3 and SI, Figures S42−S44). The low molecular weight of the labeled complex enabled fast renal elimination. However, a remarkable amount of the complex was also eliminated through the liver into the feces. The resulting half-life in the blood was 19.2 min, and that also determined the clearance in the heart, kidneys, and muscle, with half-lives 18.4, 17.0, and 20.6 min, respectively. A slower

Figure 1. 1H NMR spectra cut of L5-NCS (A), H-Acp-βAla-NAP oligopeptide (B), and their L5-NAP conjugate (C). Integral values were rounded to integers for clarity. Color coding of the peptide moiety is the same as in Scheme 5. Full-range image as well as similar 1 H NMR traces of all other conjugates are shown in the SI (Figures S32−S41).

formed within 30 min. As expected, lower reactant concentrations led to a longer reaction time, e.g., 2 h for completion with L4-FC at 2 mM. Both SPAAC conjugates were obtained as a mixture of regioisomers as indicated by the structure of the DBCO moiety (partial isomer separation was achieved for L6-CDBCO, SI, Figure S8). The reactivity of the cycloalkyne and azide groups is not significantly affected by the pH because they cannot be protonated. Thus, the SPAAC reactions were performed in a nonbuffered mixture(s) of water and organic solvent(s) due to Scheme 6. Conjugates Pepared Uing SPAAC Click Reactionsa

a (i) Nonbuffered water−MeCN−DMSO 2:1:1, room temperature, 30 min, ≥99% conversion, ∼85% yield (sum of two fractions, each with ∼80% regioisomeric purity); (ii) nonbuffered water−MeCN 4:1, room temperature, 120 min, ≥99% conversion, ∼75% yield (as 1:1 mixture of regioisomers).

8781



Article

NODAGA per the antibody molecule (SI, Figure S49), and purity was confirmed by size exclusion chromatography (SI, Figure S50). The L5-7F5 conjugate was reacted with NCA 64 Cu (pH 5.5, 37 °C, 20 min), giving radiolabeled conjugate with immunoreactivity of 76%. The biodistribution in mouse subcutaneous xenografted PC3-PSCA tumor cells was followed by PET. The 64Cu-(L5-7F5) was clearly accumulated in the tumor with increasing concentration over 2 days (Figure 5). At day 2, a metastasis at a large kidney vessel was also visible (Figures 6 and 7). The very low 64Cu-activity in the liver at day 2 demonstrates the high in vivo stability of the 64Cu-L5 complex and confirms negligible release of 64Cu from the labeled antibody in vivo. At the same time, the 64Cu-labeled antibody was still in the circulation as the heart, large vessels in the trunk, and the liver were slightly visible. It also confirmed the high specificity and quality of the conjugated and radiolabeled anti-PSCA 7F5 antibody. In contrast to these data, the biodistribution and kinetics of the [64Cu(NODAGA7F5)] showed much lower tumor and significantly higher liver accumulation. To attach the chelators to the antibody, a 50fold excess of the chelators over antibody was used, and all other conjugation and radiolabeling conditions were identical. The reactions with both bifunctional chelators were performed in the same time. The conjugation of L5-NCS was less efficient, resulting to ∼3 L5 moieties per the antibody molecule; however, it is optimal for the application. On the other hand, the conjugation of NODAGA-NCS was more effective, giving ∼6 NODAGA chelators per the antibody molecule; in spite of more chelators per antibody molecule, it resulted to the lower molar activity of the conjugate and to a lower tumor and higher liver accumulations. The Figure 8 shows that the absolute tumor uptake of [64Cu(L5-)] and, similarly, the final tumor-to-blood ratio for [64Cu(L5-7F5)] was clearly higher than that for [64Cu(NODAGA-7F5)]. The tumor uptake of [64Cu(L5-7F5)] continues to increase over all time in contrast to that of [64Cu(NODAGA-7F5)]. This leads to the significantly better visibility of the tumor in the PET images (Figure 6).

Figure 2. Comparison of the NCA [64Cu]Cu2+ radiolabeling efficiency of te1bpin and te1pp with other common ligands (A) and that of te1bpin (as a control, blue) with the bifunctional ligands (L2-COOH and L4-NH2, yellow) and their conjugates (L1-PEA, L4BTN, L4-DBCO, and L5-GGY, green) (B) under identical conditions (0.5 M MES−NaOH buffer, pH 6.2, ∼100 equiv of the ligands over molar amount of 64Cu, 25 °C, 10 min labeling). The data shown are a mean of three experiments, each conducted with 9−11 MBq [64Cu]CuCl2). The results denoted with asterisks were adopted from our previous study.39

■

DISCUSSION AND CONCLUSIONS To keep convenient radiolabeling efficiency of the parent ligands, te1bpin and te1pp,39 reactive functional groups for conjugation were introduced on bis(phosphinic acid) pendant arm, i.e., far away from the metal binding macrocyclic unit. The chelators were prepared directly from unprotected cyclam and appropriately substituted bis(phosphinic acids), and, thus, the number of necessary reaction steps to get final bifunctional ligands were reduced. The monosubstituted cyclam derivatives prepared in this way contain secondary amine and free phosphinic acid groups. However, the presence of both groups in unprotected form brings a potential cross-reactivity problem in conjugation reactions. The unprotected macrocyclic ligands with the secondary amine groups have only been rarely used in conjugation reactions thus far; only the picolinate-containing macrocycle TE1PA (Chart 1) was recently used for antibody coupling.63 Beside utilization of fully protected ring amines, the macrocycle secondary amines can be converted to tertiary ones by an alkylation. This modification leads to less stable complexes as it was shown by comparison of behavior of, e.g., copper(II) complexes with TETA and TE2A (Chart 1) derivatives64−67 or with cyclam derivatives with an increasing number of Npropionate pendant arms.68 Therefore, the expected problem

clearance with a half-time of 28.6 min was observed only in the liver. The highest remaining activity was measured in the kidneys with a SUV of 1.9. The elimination into the bladder followed one-phase kinetics, with a half-time of 17.0 min, which was also comparable to the blood clearance. After 2 h, 66.5%ID was excreted into urine. The PET images showed that the kidney cortex did not retain the [64Cu-(L6-N3)]. This observation was in agreement with the ex vivo biodistribution data after 1 h (Figure 4, and SI, Tables S12 and S13). Metabolite analyses of the [64Cu-(L6-N3)] were performed using radio-HPLC and radio-TLC. The data confirm the very high metabolic stability of the [64Cu-(L6-N3)] in arterial blood plasma samples because it was found unchanged in feces and urine (SI, Figures S45−S48). In Vivo Evaluation of 64Cu-Labeled L5-NCS and NODAGA-NCS Conjugates with 7F5 Antibody. The isothiocyanate derivatives, L5-NCS (Scheme 2) and NODAGA-NCS (SI, Figure S1), were conjugated (pH 7.9, 24 h) to bivalent anti-PSCA 7F5 antibody of the IgG type which targets prostate stem cell antigen (PSCA). The antibody was developed by the M. Bachmann group earlier.62 Mass spectrometry showed an average of 2.9 of L5 or 6.0 of 8782



Article

Figure 3. Distribution of [64Cu-(L6-N3)] in a rat measured by PET at 1, 5, 20, and 90 min after a single intravenous injection. The maximum intensity projection is scaled to the maximum (A) or to individual color to visualize the organs with lower activity concentration (B). Organ abbreviations: H = heart, VC = vena cava, K = kidneys, I = intestine, B = bladder.

also determined by the basicity of amine group in the substrate. The N-terminal α-amino group of peptides (pKa ∼ 7.5−8.5) can be efficiently coupled with L5-NCS at high conversion rates at pH range 7−7.5, where the proton protection is still efficient. However, the coupling with more basic alkyl amines (pKa ∼ 10−11) was less efficacious and various byproducts were observed. The optimal pH range was 7−7.5 as the reaction with −NCS group requires higher pH than that the amide coupling to proceed with reasonable rate. The observed dependence of the reaction yield/conversion on the substrate basicity has general importance as isothiocyanate-containing compounds are among the most commonly used bifunctional ligands. An excessively high pH (pH ≥ 9) leads to hydrolysis of the isothiocyanate group. The amines (commonly aniline derivatives with low amine group basicity) formed by (aryl)isothiocyanates hydrolysis easily react with another isothiocyanate molecule, yielding a symmetrical thiourea dimer,55 and similar reactivity was observed for L5-NCS. Our findings suggest that isothiocyanate conjugation to N-terminal α-amino groups of (oligo)peptides (pKa ∼ 8) should be more favorable than commonly used conjugations to the ε-amino group of Lys residues or to aliphatic amine groups (pKa > 10). Efficacy of the radiolabeling may be explained by the complexation mechanism reported in our previous study.39 The bis(phosphinic acid) group serves as an anchor catching the metal radioisotope and bringing it close to its final destination inside the macrocycle. Modification of the bis(phosphinic acid) group did not alter radiolabeling unlike radiolabeling of chelators, where the conjugation takes place

with secondary ring amine groups should be solved in a different way. We turned our attention to special properties of the macrocyclic amine groups. The macrocycle amine groups are highly basic (values of their first two protonation constants, log K1 and log K2, are each higher than 10), and all four nitrogen atoms are sharing these two protons due to intramolecular hydrogen bonds.34,39 Therefore, all secondary amine groups should be effectively protonated (i.e., proton protected) in neutral or slightly acidic solutions and they should be progressively less available for coupling reactions (e.g., amide formation) with decreasing solution pH. The other groups, bis(phosphinic acids), are rather strong acids and, under tested conditions, they are fully deprotonated37−39 and present as non-nucleophilic phosphinate groups. Therefore, the phosphinate groups should not affect the activation of carboxylic groups in the ligands. These assumptions have been already confirmed as phosphinic acid analogues of amino acids69−71 or, more recently, macrocycle derivatives50,72,73 in a free zwitterionic form have been used in coupling reactions. We utilized the proton protection in the amide and thiourea forming reactions. The carboxylic group in L1-COOH/L2COOH were activated with the water-compatible EDC/NHS couple or with TBTU in DMSO suspension. At formal pH < 7, no coupling to the ring amine groups was detected and excellent regioselective coupling of substrates to the side chains of the pendant arm was observed. The optimal pH range for the reaction was 6.2−6.8. In isothiocyanate coupling, the proton protection has to be coupled with an appropriate choice of substrate. We observed that the coupling efficiency is 8783



Article

Figure 4. Biodistribution of [64Cu-(L6-N3)] in rats at 5 and 60 min after a single intravenous injection (values in (A) %ID and in (B) SUV, mean ± SEM, n = 4). Abbreviations: w.c. = with content, Hard. gl. = Harderian glands, WAT = white adipose tissue.

optimal number of ∼3 chelators per antibody. The conjugate was labeled under mild conditions fully compatible with large proteins, resulting in a high molar activity for excellent tumor imaging in the PC-3-PSCA mouse model. The study clearly showed that there is no nonspecific off-target uptake of 64Cu. Thus, no significant transchelation/demetalation took place in vivo as another form of 64Cu would be localized in the liver at the delayed time point. Therefore, the L5-SCN seems to be an excellent bifunctional chelator for conjugations and 64Curadiolabeling of sensitive peptides and proteins under mild conditions. The improved in vivo behavior of the 64Cu-(L57F5) may be caused by the smaller number of chelators per antibody (∼3) compared to that of NODAGA-7F5 (∼6). These results point to lower influence of the BPC chelator on the binding to the tumor and to the higher stability of the BPC copper(II) complex against transchelation and, therefore, the lower accumulation of the released 64Cu in the liver.78 In conclusion, a new family of bifunctional ligands derived from te1bpin, bis(phosphorus acid) cyclams = BPC, were synthesized and studied as potential 64Cu2+ chelators for applications in nuclear medicine. Their anchor groups (−CO2H, −NH2, −NCS, −N3, cyclooctyne) were purposely attached to the distant position on the bis(phosphinate) fragment, and, thus, the promising 64Cu radiolabeling properties of the te1bpin unit remained intact. The coupling reactions of bifunctional BPC with various model substrates, including amino acids, oligopeptides, or a fluorescent dye, were studied to assess their conjugation potential and, above all, to simplify the procedure for possible applications. The

on a group involved in metal ion complexation.74 The apparent molar activity up to ∼100 GBq/μmol was attained as approximately only 100-fold molar excess of the ligands over a molar amount of [64Cu]CuCl2 was required for complete labeling. Behavior in vivo was tested on two systems. More detailed study was carried out on simple labeled bifunctional ligand, [64Cu-(L6-N3)]. With the complex, no noticeable uptake or retention of the labeled complex was observed except for the excretory organs. Furthermore, the results also indirectly confirmed that no decomplexation occurred in vivo. The compounds are stable in the blood of rats and show no affinity to protein components of the rat plasma. In an attempt to improve the tumor-targeting efficacy of antibodies, new formats with modified, multivalent properties have been generated.75 These formats imitated the structure of native IgG or other protein scaffolds creating mostly monospecific, bivalent antibodies. Recently, novel multispecific, di-, tri-, or tetravalent, antibodies have been developed to maximize tumor targeting capabilities through enhanced biodistribution and functional affinity. The increasing application of these new antibody derived molecules require simple, low-temperature, fast, and specific radiolabeling for in vivo applications. As a model of these new formats, we chose the highly specific high affinity IgG type monoclonal antibody 7F5 which is directed to the prostate stem cell antigen (PSCA). PSCA is overexpressed prostate tumor tissues.76,77 Biodistribution PET studies were performed with PSCA positive tumor bearing mouse. The conjugation gave an 8784



Article

Figure 5. Orthogonal sections of PET studies of [64Cu(L5-7F5)] (A) and [64Cu(NODAGA-7F5)] (B) conjugates after a single intravenous injection in PC3-PSCA tumor bearing mice at different time points. At 90 min pi (i.e., at day 0), at day 1 and at day 2, the duration of each experiment was 1 h. Abbreviations: T = tumor.

vivo by metabolite analysis, biodistribution studies, and PET scans. The data clearly showed the very high metabolic stability of the 64Cu chelate unit because no decomplexation was detected. Except for the excretory organs, no prominent uptake and retention was observed. Conjugation of a representative chelator to PSCA specific mAb and mild 64Cu labeling led to a high specific molar activity and excellent tumor imaging. The PET biodistribution studies confirmed the high in vivo stability of the copper complex. The direct comparison with the radiolabeled analogous NODAGA-7F5 conjugate supports these findings. Thus, the bis(phosphinate)-bearing cyclam-based ligands, BPCs, are highly promising chelators for 64Cu or other copper radioisotopes. Upon addition of other functional group for conjugation to targeting units, such as peptides, oligonucleotides, or the new antibody formats, they represent a new tool for theranostic nuclear medicine applications.

unprotected phosphinate groups have no effect on the coupling reactions. For the first time, ligands with unprotected secondary ring amine groups were directly used in conjugation reactions. At pH < 8.0−8.5, the cyclam moiety is diprotonated, and, therefore, the ring secondary amines are effectively proton protected and unreactive. Proton protection of ring amine groups is a new concept which can be widely used in conjugation chemistry for very basic macrocyclic ligands. Amine group basicity in the substrate plays an important role in thiocyanate coupling. The reaction is more effective with amines with pKa ≤ ∼8.5, e.g., with an α-amine group in amino acids or aromatic amines. The importance of the basicity of the amine group in substrates was investigated in detail for the first time. Derivatives bearing azide or strained cyclooctyne were easily used in copper-free click coupling reactions, and the reactions were quantitative, fast, and free of byproducts. The NCA [64Cu]CuCl2 radiolabeling efficiency of the BPC ligands and of their conjugates was similar to that of the parent ligand, te1bpin, showing that the suggested change of the ligands on distant atoms has no effect on their coordination with Cu2+ ions. All studied compounds show very high radiolabeling efficiency, leading to a significantly higher specific activity than that described for other commonly used macrocyclic chelators. It confirms again50,79,80 that using properly designed phosphinic acid pendant arm(s) is a good strategy to achieve conjugation flexibility (due to the distant bifunctional site) without compromising the radiolabeling efficiency or the high specific activity of radiopharmaceuticals. A prototypic representative, [64Cu-(L6-N3)], was evaluated in

■

EXPERIMENTAL SECTION

Material and Methods. The commercially available (Fluka, Aldrich, Chematech, Strem) chemicals had synthetic purity and were used as received. Various substrates for conjugation reactions were obtained from Click Chemistry Tools (USA). The te1bpin, NOTA, and DOTA were available from our previous works.39 The 3-azido-7hydroxy-coumarin61 and CB-TE2A81 were synthesized according to the published procedures. Ligand diamsar was kindly donated by (the late) L. Spiccia group (Melbourne). Compound 1 was prepared by a modification of the published procedure,54 and details are given in the SI. Paraformaldehyde was filtered from an aged aqueous formaldehyde solution and was dried over P2O5 in vacuum desiccators. 8785



Article

Figure 6. PET/MRI of [64Cu(L5-7F5)] conjugate in a PC3-PSCA tumor bearing mouse at day 2 after injection: all orthogonal sections were positioned through the tumor (A), all orthogonal sections were positioned through the metastasis (B). Abbreviations: T = tumor, M = metastasis. Methylene-bis(phosphinic acid) was obtained by careful hydrolysis of commercial CH2(PCl2)2 according to the published procedure.37,82 Full structure of the substrates (and their abbreviations) used in conjugation reactions are given in SI (Figure S5). All chiral amino acids were L-isomers unless specified otherwise. The following buffers were used for conjugation reactions: MES−NaOH (pH 5.1, 6.2 and 6.8), MOPS−NaOH (pH 7.4 and 8.0), and H3BO3−LiOH (pH 9.3 and 10.1); MES = 2-(N-morpholino)ethanesulfonic acid, MOPS = 3(N-morpholino)propanesulfonic acid. The 1D/2D NMR experiments were run on Bruker Avance III with cold probe (600 MHz; 1H and 13 C{1H}), Varian S300 (300 MHz; 31P and 31P{1H}), or on Bruker Avance III HD (400 MHz; 31P and 31P{1H}) spectrometers (chemical shift in ppm, coupling constants in Hz, mc means macrocycle). For clarity reasons, characterization 13C and 31P NMR integrals were rounded to one decimal point. The 31P NMR spectra were referenced to external 85% aq H3PO4 (δP = 0.0 ppm), and the 1H and 13C NMR spectra to external or internal t-BuOH (δH = 1.25 ppm, δC = 30.29 ppm) or 1,4-dioxane (δH = 3.75 ppm). The signals arising from counterions (AcOH and TFA) are not included in the list of characteristic signals. Characterization 31P, 1H, and 13C{1H} NMR spectra of all synthesized compounds are presented in SI (Figures S9−S31; for clarity reasons, 1H and 31P NMR integrals were rounded to one decimal digit and integers, respectively). The qNMR technique (calibrated with maleic acid as the external standard, δH = 6.42 ppm) was used for determination of analytical concentration of stock solutions of substrates for conjugations and stock solutions of ligands for radiolabeling. The pH/pD was measured with combined glass electrode calibrated with standard buffers (pD = pH + 0.4). The ESIMS spectra were recorded on Bruker Esquire 3000 spectrometer with ion-trap detection in negative or positive modes. Silica gel 60 F254 on aluminum foils (Merck) were used for TLC. Elemental analyses are

presented in the format: calculated (found). HPLC was performed on analytical column (C8, ReproSil Gold, 5 μm, 120 Å, 150 mm × 4.6 mm, Dr. Maisch GmbH, Germany) and preparative column (C8(2), Luna, 10 μm, 100 Å, 250 mm × 21.2 mm, Phenomenex). Four HPLC gradient methods were used (Table 1). Presented HPLC traces are shown with respect to the dead volume of the columns (∼2 and ∼4 min for the analytical and preparative ones, respectively). Because the reactants and products have, generally, different molar absorption coefficients, the HPLC conversion was mostly estimated as fraction of reactant remaining in the solution after the reaction compared to initial reactant amount. Because of the submillimolar scale of the conjugation reactions, their conversions and isolated yields were rounded to 5%. Amount of TFA present in the samples of conjugates purified by preparative HPLC was not taken into account in the calculation of the yields, and, thus, the yields might be slightly overestimated. All final compounds had at least 95% purity according to multinuclear NMR and HPLC. Syntheses. Syntheses and characterization data of the starting simple bis(phosphinic acids) 1−5 and the NHS-VGG-Cbz tripeptide are given in the SI. Synthesis of 6-[(1,4,8,11-Tetraazacyclotetradec-1-yl)-methylenephosphinico]-6-(hydroxyphosphinico)-hexanoic Acid (L1-COOH). In a 20 mL glass vial, cyclam (1.50 g, 7.50 mmol, 3.8 equiv) and 1 (1.24 g, 5.08 mmol, 2.6 equiv) were dissolved in aq HCl (6 M, 10 mL). Paraformaldehyde (59.2 mg, 1.97 mmol, 1.0 equiv) was added in one portion, and the vial was quickly closed with a stopper. The resulting suspension was stirred at 70 °C overnight. After cooling to room temperature, the reaction mixture was evaporated to dryness and further coevaporated with water. The residue was purified on strong cation exchange resin (Dowex 50, ∼100 mL, H+-form, water → 10% aq pyridine). The pyridine fraction was evaporated to dryness 8786



Article

Table 1. HPLC Gradients Used Throughout the Work (Solvent A = H2O, Solvent B = 0.1% TFA in H2O; Solvent C = MeCN) method M1 M2 M3 M4

A−B−C 55:40:5 → 20:40:40 A−B−C 85:10:5 → 0:10:90 → 0:10:90 A−B−C 75:10:15 → 35:10:55 A−B−C 65:10:25 → 50:10:40

analytical (min)

preparative (min)

30 5+5

70 11.4 + 11.4

10 10

22.8 22.8

2.51−2.79 (mc, N−CH2−P, m, 18H); 7.05 (PH, d, 1H, 1JHP = 525). 13 C{1H} δ 23.8 (CH2, t, 3JCP = 3); 25.6 (mc, s); 26.9 (mc, s); 27.2 (CH2, s); 30.8 (CH2, dd, 2JCP = 8, 2JCP = 4); 38.3 (CH2−CO, s); 45.0 (P−CH−P, dd, 1JCP = 79, 1JCP = 73); 45.8 (mc, s); 46.1 (mc, s); 46.4 (mc, s); 46.7 (mc, s); 47.3 (mc, s); 48.2 (mc, s); 53.3 (N−CH2−P, d, 1 JCP = 103); 54.1 (mc, d, 3JCP = 7); 54.7 (mc, d, 3JCP = 6); 184.6 (CO, s). 31P δ 25.7 (PH, dm, 1P, 1JHP = 525); 36.3 (P−CH2−N, m, 1P). ESI-MS: (−) 454.7 [M − H+]−; (+) 457.0 [M + H+]+. ESI-HRMS: (+) 457.2336 [M + H+]+ (calcd for [C17H39N4O6P2]+ = 457.2340). TLC (i-PrOH−conc aq ammonia−water 7:3:3): Rf ∼0.5. HPLC (M2): Rf ∼2.5 min. EA (C17H38N4O6P2·2H2O, MR = 492.5): C 41.5 (41.3); H 8.6 (8.5); N 11.4 (11.4). Synthesis of 3-[(1,4,8,11-Tetraazacyclotetradec-1-yl)-(methylenephosphinico-methylenephosphinico)]-propanoic Acid (L 2 COOH). Cyclam (1.19 g, 5.95 mmol, ∼5.5 equiv), the crude 5 (∼75% purity, with ∼25% of the disubstituted byproduct, 340 mg, ∼1.1 mmol, 1.0 equiv) and paraformaldehyde (58 mg, 1.93 mmol, ∼1.8 equiv) were dissolved in aq HCl (6 M, 10 mL). The flask was quickly closed with a stopper, and the resulting mixture was stirred at 60 °C overnight. The reaction mixture was evaporated to dryness, and the residue was coevaporated several times with water. The residue was dissolved in water and purified on strong anion exchange resin (Amberlite IRA 402, ∼100 mL, OH-form, water → 10% aq AcOH). The excess of cyclam was eluted off with water. The acetate fraction containing product was evaporated to dryness and further coevaporated several times with water. The residue was purified on strong cation exchange resin (Dowex 50, ∼50 mL, H+-form, water → 10% aq pyridine). The pyridine fraction was evaporated to dryness and the residue coevaporated several times with water. The residue was redissolved in water (50 mL) and lyophilized. Product was obtained in the zwitterionic form as white hygroscopic powder. Conversion: (31P NMR) ≥95% (16 h). Yield: 181 mg (21%, 3 steps, based on methylene-bis(phosphinic acid), ≥99% purity). NMR (D2O + LiOD, pD ≥12): 1H δ 1.71 (mc, p, 2H, 3JHH = 6); 1.76 (mc, p, 2H, 3 JHH = 6); 1.91 (P−CH2−C, m, 2H); 2.05 (P−CH2−P, t, 2H, 2JHP = 16); 2.37 (CH2−CO, m, 2H); 2.65 (mc, t, 2H, 3JHH = 5); 2.69−2.79 (mc, N−CH2−P, m, 16H). 13C{1H} δ 25.6 (mc, s); 27.2 (mc, s); 29.7 (P−CH2−C, d, 1JCP = 96); 31.2 (CH2−CO, s); 33.5 (P−CH2−P, t,

Figure 7. PET/MRI of [64Cu(L5-7F5)] conjugate in a PC3-PSCA tumor bearing mouse at day 2 after injection: maximum intensity projection (MIP) of PET (A) and of PET/MRI overlay (B). Abbreviations: T = tumor, M = metastasis. and coevaporated several times with water. The crude product was purified by column chromatography (SiO2, ∼100 g, i-PrOH−conc aq ammonia−water 7:3:3, Rf ∼ 0.5). Fractions containing product were combined and evaporated to dryness. The residue was redissolved in water (50 mL), and the solution was treated with charcoal and filtered through a syringe microfilter (0.22 μm). The filtrate was purified on strong cation exchange resin (Dowex 50, ∼50 mL, H+-form; water → 10% aq pyridine) to remove ammonia. The pyridine fraction containing product was evaporated to dryness and further coevaporated several times with water. The residue was redissolved in water (100 mL), and the resulting solution was lyophilized. Product was obtained in a zwitterionic form as colorless solidified oil. Conversion (31P NMR): ∼90% (16 h). Yield: 545 mg (56%, 1 step, based on paraformaldehyde, ∼95% purity). NMR (D2O + CsOD, pD ≥12): 1H δ 1.38 (CH2, m, 1H); 1.50 (CH2, m, 3H); 1.64 (mc, p, 2H, 3 JHH = 5); 1.66 (P−CH−P, t, 1H, 2JHP = 15); 1.67 (CH2, bm, 2H); 1.69 (mc, p, 2H, 3JHH = 5); 2.12 (CH2−COOH, t, 2H, 3JHH = 7);

Figure 8. PET kinetics in tumor (A) and blood (B) of [64Cu(L5-7F5)] (blue) and [64Cu(NODAGA-7F5)] (red) conjugates in PC-3-PSCA tumor bearing mice after a single intravenous injection. Duration of each measurement was 1 h. The tumor-to-blood ratios are shown in C. 8787



Article

1

The residue was dissolved in water (100 mL), and the solution was filtered through a syringe microfilter (0.22 μm). The filtrate was lyophilized. Product was obtained in a form of nonstoichiometric hydrochloride as off-white powder. Conversion: (HPLC, M2) ≥95% (16 h). Yield: 412 mg (66%, 1 steps, based on 6·AcOH·1.5H2O, ∼95% purity). NMR (D2O, pD ∼2): 1H δ 2.05 (mc, p, 2H, 3JHH = 6); 2.23 (mc, p, 2H, 3JHH = 6); 2.36 (P−CH2−P, t, 2H, 2JHP = 16); 2.89 (mc, bm, 2H); 3.00 (P−CH2−N, d, 2H, 2JHP = 6); 3.08−3.19 (mc, P− CH2−N, bm, 4H); 3.26−3.42 (mc, bm, 6H); 3.44−3.61 (mc, bm, 6H). 13C{1H} δ 22.5 (mc, s); 22.9 (mc, s); 34.3 (P−CH2−P, dd, 1JCP = 84, 1JCP = 80); 39.7 (P−CH2−N, d, 1JCP = 98); 42.1 (mc, s); 43.4 (mc, s); 44.1 (mc, s); 44.9 (mc, s); 45.0 (mc, s); 45.9 (mc, s); 53.8 (P− CH2−N, d, 1JCP = 109); 54.5 (mc, bs); 55.8 (mc, d, 3JCP = 8). 31P{1H} δ 23.9 (CH2−P−CH2, d, 1P, 2JPP = 6); 30.9 (CH2−P−CH2, bm, 1P). ESI-MS: (−) 383.9 [M − H+]−; (+) 385.7 [M + H+]+; 407.9 [M + Na+]+. TLC (EtOH−conc aq ammonia 1:1): Rf ∼0.6. HPLC (M1 or M2): no retention. EA (C13H33N5O4P2·3.5HCl·2.5H2O, MR = 558.0): C 28.0 (28.3); H 7.5 (7.6); N 12.6 (12.6). Synthesis of P-({Hydroxy[(4-nitrophenyl)methyl]phosphinyl}methyl)-P-(1,4,8,11-tetraazacyclotetradec-1-yl-methyl)-phosphinic Acid (7). Paraformaldehyde (169 mg, 5.63 mmol, 1.2 equiv) was added to suspension of cyclam (3.38 g, 16.9 mmol, 3.6 equiv) and 3 (1.31 g, 4.69 mmol, 1.0 equiv) in aq HCl (6 M; 60 mL), and the flask was quickly closed with a stopper. The resulting mixture was stirred at 60 °C overnight. The reaction mixture was evaporated to dryness, and the residue was coevaporated several times with water. The residue was dissolved in water and purified on strong cation exchange resin (Dowex 50, ∼200 mL, H+-form, water → 10% aq pyridine). The pyridine fraction was evaporated to dryness, and the residue was coevaporated several times with water. The residue was purified on strong anion exchange resin (Dowex 1, ∼100 mL, OH-form, water → 10% aq AcOH). The acid fraction was evaporated to dryness, and the residue was once coevaporated with water. The residue was once more purified on strong cation exchange resin (Dowex 50, ∼75 mL, H+-form, water → 10% aq pyridine). The pyridine fraction was evaporated to dryness, and the residue was coevaporated several times with water. The solid residue was dried under vacuum at 80 °C for 24 h. Product was obtained in a zwitterionic form as a fine bright-yellow powder. Conversion: (31P NMR) ≥95% (16 h). Yield: 1.67 g (71%, 1 steps, based on 3, ≥99% purity). NMR (D2O + LiOD, pD ≥12): 1H δ 1.70 (mc, p, 2H, 3JHH = 5); 1.76 (mc, p, 2H, 3JHH = 6); 2.01 (P− CH2−P, t, 2H, 2JHP = 16); 2.55−2.83 (mc, N−CH2−P, bm, 18H); 3.32 (CH2−C−CH, m, 2H); 7.56 (CH−C−CH2, dd, 2H, 3JHH = 9, 4 JHP = 2); 8.21 (CH−C−N, d, 2H, 3JHH = 9). 13C{1H} δ 25.4 (mc, s); 27.0 (mc, s); 33.2 (P−CH2−P, t, 1JCP = 79); 40.8 (P−CH2−C, d, 1JCP = 86); 46.0 (mc, s); 46.2 (mc, s); 46.7 (mc, s); 46.8 (mc, s); 47.9 (mc, s); 48.9 (mc, s); 54.5 (mc, d, 3JCP = 8); 54.8 (mc, s, 3JCP = 5); 55.6 (P−CH2−N, d, 1JCP = 110); 124.3 (CH−C−N, d, 4JCP = 3); 131.3 (CH−C−CH2, d, 3JCP = 5); 144.6 (CH−C−CH2, d, 2JCP = 9); 146.6 (CH−C−N, s, 5JCP = 3). 31P{1H} δ 31.6 (CH2−P−CH2, d, 1P, 2JPP = 9); 32.9 (CH2−P−CH2, d, 1P, 2JPP = 9). ESI-MS: (−) 489.7 [M − H+]−; (+) 491.9 [M + H+]+; 513.9 [M + Na+]+; 529.8 [M + K+]+. TLC (EtOH−conc aq ammonia 1:1): Rf ∼0.7. HPLC (M2): Rf ∼5.7 min. EA (C19H35N5O6P2·0.5H2O, MR = 500.5): C 45.6 (45.2); H 7.3 (7.6); N 14.0 (13.8). Synthesis of P-({[(4-Aminophenyl)methyl]hydroxyphosphinyl}methyl)-P-(1,4,8,11-tetraazacyclotetradec-1-yl-methyl)-phosphinic Acid (L4-NH2). In a 100 mL flask, a solution of 7·0.5H2O (512 mg, 1.02 mmol, 1.0 equiv) in water (150 mL) was gently flushed with argon and Pd/C catalyst (10%; 253 mg) was added. Gaseous hydrogen was continuously bubbled through the reaction mixture at room temperature for 3 h. The reaction mixture was filtered through a syringe microfilter (0.22 μm). The filtrate was directly lyophilized. Product was obtained in a zwitterionic form as fine white foam. Conversion: (HPLC, M2) ≥95% (16 h). Yield: 445 mg (85%, 1 steps, based on 7·0.5H2O, ≥ 95% purity). NMR (D2O + LiOD, pD ≥12): 1 H δ 1.72 (mc, p, 2H, 3JHH = 5); 1.79 (mc, p, 2H, 3JHH = 6); 1.96 (P− CH2−P, t, 2H, 2JHP = 15); 2.62−2.88 (mc, N−CH2−P, bm, 18H); 3.02 (CH2−C−CH, d, 2H, 2JHP = 17); 6.82 (CH−C−N, d, 2H, 3JHH = 8); 7.18 (CH−C− CH2, dd, 2H, 3JHH = 8, 4JHP = 2). 13C{1H} δ 25.0

JCP = 77); 46.2 (mc, s); 46.3 (mc, s); 46.8 (mc, s); 46.9 (mc, s); 47.6 (mc, s); 48.8 (mc, s); 54.4 (mc, bs); 54.8 (mc, s); 55.2 (N−CH2−P, d, 1 JCP = 109); 183.2 (CO, d, 3JCP = 19). 31P{1H} δ 33.4 (P−CH2−N, d, 1P, 2JPP = 10); 36.6 (P−CH2−CH2, d, 1P, 2JPP = 10). ESI-MS: (−) 402.8 [M − H3O+]−; 426.7 [M − H+]−; (+) 428.9 [M + H+]+; 450.8 [M + Na+]+; 466.8 [M + K+]+. ESI-HRMS: (−) 427.1875 [M − H+]− (calcd for [C15H33N4O6P2]− = 427.1880). TLC (i-PrOH−conc aq ammonia−water 7:3:3): Rf ∼0.5. HPLC (M1 or M2): no retention. EA (C15H34N4O6P2·4H2O, MR = 500.5): C 36.0 (36.0); H 8.5 (8.8); N 11.2 (11.2). Synthesis of P-[({[Bis(phenylmethyl)amino]methyl}hydroxyphosphinyl)methyl]-P-(1,4,8,11-tetraazacyclotetradec-1-ylmethyl)-phosphinic Acid (6). Cyclam (3.26 g, 16.3 mmol, ∼5.4 equiv), the crude 2 (∼75% purity, with ∼25% of hydroxymethylated byproduct, 1.44 g, ∼3.0 mmol, 1.0 equiv) and paraformaldehyde (160 mg, 5.33 mmol, ∼1.8 equiv) were dissolved in aq HCl (6 M, 60 mL). The flask was quickly closed with a stopper, and the resulting mixture was stirred at 80 °C overnight. The reaction mixture was evaporated to dryness, and the residue was coevaporated several times with water. The residue was dissolved in water and purified on strong cation exchange resin (Dowex 50, ∼200 mL, H+-form, water → 5% aq ammonia). The aqueous fraction was discarded, and the ammonia fraction containing crude product was evaporated to dryness. The residue was purified on strong anion exchange resin (Dowex 1, ∼100 mL, OH−-form, water → 20% aq AcOH). The aqueous fraction was discarded and the acetate fraction was evaporated to dryness and the residue was coevaporated several times with water. The resulting paleyellow oil was dissolved in water (40 mL), treated with charcoal, and filtered on fine glass frit. The filtrate was evaporated to dryness, and the residue was several times coevaporated with water and dried under vacuum at 80 °C for 3 d. Product was obtained in the form of nonstoichiometric acetate salt as pale-yellow powder. Conversion: (31P NMR) ≥95% (16 h). Yield: 1.46 g (18%, 2 steps, based on methylene-bis(phosphinic acid), ≥95% purity). ESI-MS: (−) 563.8 [M − H+]−; (+) 566.0 [M + H+]+; 588.0 [M + Na+]+; 604.0 [M + K + ] + . TLC (EtOH−conc aq ammonia 2:1): R f ∼0.8. EA (C27H45N5O4P2·AcOH·1.5H2O, MR = 657.2): C 53.4 (53.4); H 8.0 (7.7); N 10.7 (10.6). Portion of the product (130 mg) was dissolved in water (5 mL). The resulting solution was purified by preparative HPLC (M2). The fractions with product were combined and transferred to a 250 mL round-bottom flask using additional water, and the solution was lyophilized to give a product (≥99% purity as determined by NMR) in the form of nonstoichiometric trifluoroacetate salt as fine white foam (117 mg). NMR (D2O, pD ∼2): 1H δ 2.02 (mc, p, 2H, 3JHH = 6); 2.06 (P−CH2−P, dd, 2H, 2JHP = 17, 2JHP = 15); 2.12 (mc, p, 2H, 3JHH = 6); 2.80 (mc, t, 2H, 3JHH = 6); 2.85 (N− CH2−P, d, 2H, 2JHP = 6); 3.02 (mc, t, 2H, 3JHH = 5); 3.20 (mc, bm, 2H); 3.24−3.35 (mc, N−CH2−P, bm, 8H); 3.39 (mc, bm, 2H); 3.50 (mc, t, 2H, 3JHH = 7); 4.56 (CH2−Ph, s, 4H); 7.43−7.75 (Ph, m, 10H). 13C{1H} δ 22.6 (mc, s); 23.4 (mc, s); 34.8 (P−CH2−P, dd, 1JCP = 85, 1JCP = 78); 42.6 (mc, s); 44.4 (mc, s); 45.1 (mc, s); 45.6 (mc, s); 45.9 (mc, bs); 46.7 (mc, bs); 51.1 (P−CH2−N, d, 1JCP = 91); 53.1 (P−CH2−N, d, 1JCP = 111); 54.2 (mc, d, 3JCP = 4); 55.3 (mc, d, 3JCP = 11); 58.7 (CH2−Ph, d, 3JCP = 4); 129.1 (Ph, s); 129.2 (Ph, s); 130.1 (Ph, s); 131.5 (Ph, s). 31P{1H} δ 20.9 (P−CH2−N, d, 1P, 2JPP = 7); 31.3 (P−CH2−N, d, 1P, 2JPP = 7). HPLC (M2): Rf ∼6.0 min. Synthesis of P-{[(Aminomethyl)hydroxyphosphinyl]methyl}-P(1,4,8,11-tetraazacyclotetradec-1-yl-methyl)-phosphinic Acid (L3NH2). In a 100 mL flask, a solution of 6·AcOH·1.5H2O (721 mg, 1.10 mmol, 1.0 equiv) in water (100 mL) was gently flushed with argon. Ammonium hypophosphite (920 mg, 11.1 mmol, 10 equiv) and Pd/C catalyst (10%, 310 mg) were added. The reaction mixture was stirred at 60 °C overnight. The catalyst was filtered off through a syringe microfilter (0.22 μm). The filtrate was evaporated to dryness, and the residue was purified on strong cation exchange resin (Dowex 50, ∼100 mL, H+-form, water → 5% aq ammonia). The ammonia fraction containing product was evaporated to dryness, and the residue was purified on strong anion exchange resin (Dowex 1, ∼50 mL, OH−form, water → 3% aq HCl). The HCl fraction was evaporated to dryness, and the residue was coevaporated several times with water. 8788



Article

JPP = 9). ESI-MS: (−) 485.8 [M − H+]−; (+) 488.0 [M + H+]+; 509.9 [M + Na+]+; 531.9 [M + K+]+. ESI-HRMS: (+) 488.2301 [M + H+]+ (calcd for [C19H36N7O4P2]+ = 488.2299). TLC (EtOH−conc aq ammonia 2:1): Rf ∼0.7. EA (C19H35N7O4P2·TFA·2H2O, MR = 637.5): C 39.6 (39.7); H 6.3 (6.1); N 15.4 (15.3). HPLC (M2): Rf ∼5.9 min. General Procedure for Synthesis and Purification of Conjugates. The corresponding reagents (mostly used as solutions) were mixed in a glass vial (4 mL) and briefly shaken on vortex. The resulting solution (having specified concentration of components) was stirred for a given time at room temperature. Conversion of the reaction was monitored by HPLC. Reaction mixture was then filtered through syringe microfilter (0.22 μm) and purified on preparative HPLC. The fractions with product were transferred to a 100 mL round-bottom flask using additional H2O, and the solution was lyophilized. Product (purity determined by NMR) was obtained as fine lightweight foam. Synthesis of L1-PEA Conjugate. The 2-phenyl-ethylamine (PEA, 200 mM solution in water−MeCN 1:1, 200 μL, 40.0 μmol, 1.0 equiv) and NHS (100 mM solution in MeCN, 200 μL, 20.0 μmol, 0.5 equiv) were added to a solution of L1-COOH·2H2O (19.8 mg, 40.2 μmol, 1.0 equiv) in MES−NaOH buffer (1 M, pH = 5.1, 1.00 mL, 1.00 mmol, 25 equiv) in water (1.60 mL) and MeCN (870 μL). Then, EDC·HCl (7.7 mg, 40.2 μmol, 1.0 equiv) was added, and the resulting solution (containing ∼11.5 mM PEA and ∼11.5 mM L1-COOH in a mixture of aqueous buffer−MeCN 3:1) was stirred 24 h. Mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼6.1 min. Conversion: (M2) ∼45% (3 h); ∼75% (24 h). Yield: 12.9 mg (∼60%, 1 step, based on PEA, ≥95% purity). NMR (D2O + CsOD, pD ∼3). 1 H δ 1.34−2.29 (CH−(CH2)4−CO, mc, bm, 13H); 2.43−3.81 (N− CH2−P, Ph−CH2−CH2, mc, bm, 22H); 7.03 (PH, bd, 1H, 1JHP = 532); 7.31 (Ph, m, 3H); 7.39 (Ph, m, 2H). 13C{1H} δ 22.7 (CH− CH2, bs); 23.2 (mc, s); 24.1 (mc, bs); 26.1 (CH2, s); 29.2 (CH2, bs); 35.2 (CH2−Ph, bs); 36.2 (CH2−CO, s); 41.1 (CH2−NH−CO, s); 43.5 (mc, s); 43.8 (P−CH−P, bm); 44.9 (mc, bs); 45.8 (mc, s); 46.4 (mc, s); 46.6 (mc, bs); 47.4 (mc, bs); 52.3 (P−CH2−N, bm); 55.1 (mc, s); 56.1 (mc, d, 3JCP = 12); 127.2 (Ph, s); 129.3 (Ph, s); 129.6 (Ph, s); 140.0 (Ph, s); 177.4 (CO, s). 31P δ 26.0 (PH, dm, 1P, 1JPH = 532); 34.3 (P−CH2−N, m, 1P). ESI-MS: (−) 558.0 [M − H+]−; (+) 560.2 [M + H+]+; 582.2 [M + Na+]+; 598.1 [M + K+]+. ESI-HRMS: (+) 560.3126 [M + H+]+ (theor [C25H48N5O5P2]+ = 560.3126). TLC (i-PrOH−conc. aq ammonia−water 7:3:3): Rf ∼0.7. Synthesis of L1-Phe Conjugate. MES−NaOH buffer (1 M, pH = 6.2, 1045 μL, 1.05 mmol, 25 equiv), water (1.70 mL), and MeCN (860 μL) were added to a mixture of L1-COOH·2H2O (20.7 mg, 42.0 μmol, 1.0 equiv), H-Phe-tBu·HCl (10.8 mg, 41.9 μmol, 1.0 equiv), and NHS (400 mM solution in MeCN, 52 μL, 21.0 μmol, 0.5 equiv). Then, EDC·HCl (8.1 mg, 42.3 μmol, 1.0 equiv) was added and the resulting solution (containing ∼11.5 mM H-Phe-tBu and ∼11.5 mM L1-COOH in aqueous buffer−MeCN 3:1) was stirred for 24 h. The mixture was then purified on preparative HPLC (first using M2 and the fraction with product was repurified using M4). HPLC (M2): Rf ∼ 6.6 min; (M4): Rf ∼6.5 min. Conversion: (M2): ∼55% (3 h), ∼75% (24 h). Yield: 13.5 mg (∼50%, 1 step, based on H-Phe-tBu·HCl, ≥ 95% purity). NMR (D2O + CsOD, pD ∼2): 1H δ 1.43 (CH3, s, 9H); 1.45 (CH2, p, 2H, 3JHH = 8); 1.55 (CH2, p, 2H, 3JHH = 8); 1.74 (CH2, bm, 2H); 1.83 (P−CH−P, bt, 1H, 2JHP = 16); 2.05 (mc, p, 2H, 3JHH = 5); 2.17 (mc, p, 2H, 3JHH = 6); 2.26 (CH2−CO, t, 2H, 3JHH = 7); 2.60−3.73 (N−CH2−P, Ph−CH2, mc, bm, 20H); 4.56 (NH−CH− CO, t, 1H, 3JHH = 8); 7.05 (PH, dd, 2H, 1JHP = 533, 3JHH = 4); 7.32 (Ph, d, 2H, 3JHH = 7); 7.35 (Ph, t, 1H, 3JHH = 7); 7.41 (Ph, t, 2H, 3JHH = 7). 13C{1H} δ 22.8 (CH−CH2, s); 22.9 (mc, s); 23.4 (mc, s); 26.0 (CH2, s); 27.8 (CH3, s); 29.2 (CH2, t, 2JCP = 6); 35.7 (CH2−CO, m); 37.3 (CH2−Ph, s); 43.0 (mc, s); 43.5 (P−CH−P, bm); 44.2 (mc, s); 45.1 (mc, s); 45.5 (mc, s); 46.2 (mc, s); 46.4 (mc, bs); 52.5 (P−CH2− N, bm); 55.1 (mc, s); 55.7 (NH−CH−CO, s); 56.1 (mc, d, 3JCP = 11); 84.6 (C−CH3, s); 127.8 (Ph, s); 129.4 (Ph, s); 129.9 (Ph, s); 137.2 (Ph, s); 173.2 (CO−O, m); 177.4 (CO−CH2, m). 31P δ 26.0 (PH, dm, 1P, 1JPH = 533); 34.3 (P−CH2−N, m, 1P). ESI-MS: (−) 658.0 [M − H+]−; (+) 660.2 [M + H+]+; 682.1 [M + Na+]+. ESIHRMS: (+) 660.3652 [M + H+]+ (theor [C30H56N5O7P2]+ =

(mc, s); 26.7 (mc, s); 32.7 (P−CH2−P, t, 1JCP = 78); 39.4 (P−CH2− C, d, 1JCP = 91); 45.7 (mc, s); 46.2 (mc, s); 46.7 (mc, s); 46.8 (mc, s); 48.7 (mc, s); 49.4 (mc, s); 54.8 (mc, m); 54.9 (mc, m); 55.0 (P− CH2−N, d, 1JCP = 109); 117.3 (CH−C−N, d, 4JCP = 3); 126.4 (CH− C−CH2, d, 2JCP = 8); 131.4 (CH−C−CH2, d, 3JCP = 5); 144.7 (CH− C−N, d, 5JCP = 3). 31P{1H} δ 33.7 (CH2−P−CH2, d, 1P, 2JPP = 9); 34.6 (CH2−P−CH2, d, 1P, 2JPP = 9). ESI-MS: (−) 459.8 [M − H+]−; (+) 461.9 [M + H+]+; 483.9 [M + Na+]+; 499.9 [M + K+]+. ESIHRMS: (+) 462.2398 [M + H+]+ (calcd for [C19H38N5O4P2]+ = 462.2394). TLC (EtOH−conc aq ammonia 1:1): Rf ∼0.6. HPLC (M1 or M2): no retention. EA (C19H37N5O4P2·3H2O, MR = 515.5): C 44.3 (44.6); H 8.4 (8.8); N 13.6 (13.9). Synthesis of P-({Hydroxy[(4-isothiocyanatophenyl)methyl]phosphinyl}methyl)-P-(1,4,8,11-tetraazacyclotetradec-1-yl-methyl)-phosphinic acid (L5-NCS). In a 20 mL glass vial, L4-NH2·3H2O (40.0 mg, 77.6 μmol, 1.0 equiv) was dissolved in water (3 mL). Then, aq HCl (1.120 M, 138 μL, 154 μmol, 2.0 equiv) was added followed by addition of freshly prepared solution of CSCl2 (12 μL, 157 μmol, 2.0 equiv) in CCl4 (3 mL). The resulting mixture was vigorously stirred at room temperature for 1 h. Then, the organic layer was separated and discarded, and the aqueous layer was filtered through a syringe microfilter (0.22 μm). The filtrate was then successively purified by preparative HPLC (M2). The fractions containing product were transferred to a 100 mL round-bottom flask using additional water, and the solution was lyophilized. Product was obtained in the form of a trifluoroacetate salt as fine white foam. Conversion: (HPLC, M2) ≥95% (1 h). Yield: 35.8 mg (61%, 1 steps, based on L4-NH2· 3H2O, ≥99% purity). NMR (D2O, pD ∼2). 1H δ 2.04 (mc, p, 2H, 3 JHH = 6); 2.13 (mc, p, 2H, 3JHH = 7); 2.16 (P−CH2−P, dd, 2H, 2JHP = 17, 2JHP = 14); 2.61−3.73 (mc, N−CH2−P, P−CH2−C, bm, 20H); 7.32 (CH, m, 4H). 13C{1H} δ 23.1 (mc, s); 23.6 (mc, s); 32.4 (P− CH2−P, t, 1JCP = 80); 40.3 (P−CH2−C, dd, 1JCP = 90, 3JCP = 3); 43.1 (mc, s); 44.5 (mc, s); 45.5 (mc, s); 45.6 (mc, s); 46.4 (mc, s); 46.5 (mc, s); 53.5 (P−CH2−N, d, 1JCP = 107); 55.0 (mc, s); 55.9 (mc, d, 3JCP = 11); 126.5 (CH−C−N, d, 4JCP = 3); 129.6 (CH−C−N, d, 5JCP = 3); 131.6 (CH−C−CH2, d, 3JCP = 5); 134.3 (NCS, s); 134.7 (CH−C− CH2, d, 2JCP = 8). 31P{1H} δ 30.2 (CH2−P−CH2, bm, 1P); 35.6 (CH2−P−CH2, d, 1P, 2JPP = 10). ESI-MS: (−) 501.8 [M − H+]−; (+) 504.0 [M + H+]+. ESI-HRMS: (+) 504.1959 [M + H+]+ (calcd for [C20H36N5O4P2S]+ = 504.1958). EA (C20H35N5O4P2S·2TFA·1.5H2O, MR = 758.6): C 38.0 (38.3); H 5.3 (4.9); N 9.2 (8.9); S 4.2 (4.1). HPLC (M2): Rf ∼6.3 min. Synthesis of P-({[(4-Azidophenyl)methyl]hydroxyphosphinyl}methyl)-P-(1,4,8,11-tetraazacyclotetradec-1-yl-methyl)-phosphinic acid (L6-N3). In a 4 mL brown glass vial, L4-NH2·3H2O (25.1 mg, 48.7 μmol, 1.3 equiv) was dissolved in aq HCl (243.8 mM, 625 μL, 152 μmol, 4.0 equiv) and the resulting solution was cooled in an ice bath (1 °C). Freshly prepared aq NaNO2 (293 mM, 130 μL, 38.1 μmol, 1.0 equiv) was added in 13 portions over 40 min (10 μL every 3 min) while the bath temperature was kept at 1 °C. After the last addition, freshly prepared aq NaN3 (586 mM, 129 μL, 72.6 μmol, 2.0 equiv) was added in one portion and nitrogen gas bubbles started to evolve immediately. The vial was removed from ice bath and stirred at room temperature for 3 h. The reaction mixture was purified on preparative HPLC (M2). The fractions containing product were transferred to a 100 mL round-bottom flask using additional water, and the solution was lyophilized. Product was obtained in the form of a nonstoichiometric trifluoroacetate salt as fine white foam. Conversion: (HPLC, M2) ∼95% (3 h). Yield: 15.4 mg (63%, 1 steps, based on NaNO2, ≥99% purity). NMR (D2O, pD ∼3): 1H δ 2.07 (mc, p, 2H, 3JHH = 6); 2.13 (mc, p, 2H, 3JHH = 7); 2.20 (P− CH2−P, t, 2H, 2JHP = 16); 2.80−3.57 (mc, N−CH2−P, P−CH2−C, bm, 20H); 7.11 (CH−C−N, d, 2H, 3JHH = 8); 7.34 (CH−C−CH2, dd, 2H, 3JHH = 8, 4JHP = 2). 13C{1H} δ 22.6 (mc, s); 22.7 (mc, s); 32.3 (P−CH2−P, t, 1JCP = 80); 39.3 (P−CH2−C, d, 1JCP = 91); 42.4 (mc, s); 43.6 (mc, s); 44.3 (mc, s); 44.9 (mc, s); 45.4 (mc, s); 46.0 (mc, s); 53.7 (P−CH2−N, d, 1JCP = 105); 54.6 (mc, s); 55.6 (mc, d, 3JCP = 8); 119.9 (CH−C−N, d, 4JCP = 3); 130.9 (CH−C−CH2, d, 2JCP = 8); 131.8 (CH−C−CH2, d, 3JCP = 5); 139.0 (CH−C−N, d, 5JCP = 3). 31 1 P{ H} δ 29.6 (CH2−P−CH2, bm, 1P); 37.1 (CH2−P−CH2, d, 1P,

2

8789



Article

660.3650). Alternative synthesis (larger scale): L1-COOH·2H2O (95.7 mg, 194 μmol, 1.3 equiv) and H-Phe-tBu·HCl (40.0 mg, 15.5 μmol, 1.0 equiv) were suspended in dry DMSO (2.5 mL). Then DIPEA (108 μL, 620 μmol, 4.0 equiv) and TBTU (150 mg, 467 μmol, 3.0 equiv) were added, and the mixture was stirred for 1 h (after which nearly clear solution was obtained). The mixture was then filtered through a syringe microfilter (0.22 μm), and the filtrate was purified on preparative HPLC (M2). Conversion: (M2): ∼80% (1 h). Yield: 61.3 mg (38%, 1 step, based on H-Phe-tBu·HCl, ≥95% purity). EA (C30H55N5O7P2·3TFA·1.5H2O, MR = 1028.8): C 42.0 (42.3); H 6.0 (5.6); N 6.8 (6.8). Synthesis of L2-Phe Conjugate. L2-COOH·4H2O (30.8 mg, 61.5 μmol, 1.5 equiv) and H-Phe-tBu·HCl (10.6 mg, 41.1 μmol, 1.0 equiv) were suspended in dry DMSO (1.50 mL). Then DIPEA (36.0 μL, 207 μmol, 5.0 equiv) and TBTU (39.8 mg, 124 μmol, 3.0 equiv) were added, and the mixture was stirred for 30 min. Then, another portion of DIPEA (36.0 μL, 207 μmol, 5.0 equiv) and TBTU (39.8 mg, 124 μmol, 3.0 equiv) was added and the mixture was stirred for further 60 min (after which majority of the initially insoluble L2-COOH was dissolved). The mixture was then purified by preparative HPLC (first using M2 and the fraction with product was repurified using M3). HPLC: (M2) Rf ∼6.4 min; (M3) Rf ∼6.5 min. Conversion: (M2) ∼15% (30 min); ∼80% (90 min). Yield: 11.6 mg (∼45%, 1 step, based on H-Phe-tBu·HCl, ≥99% purity). NMR (D2O, pD ∼ 2): 1H δ 1.43 (CH3, s, 9H); 1.88 (CH2−CO, m, 2H); 2.05 (mc, p, 2H, 3JHH = 6); 2.15 (mc, p, 2H, 3JHH = 7); 2.16 (P−CH2−P, t, 2H, 2JHP = 16); 2.46 (P−CH2−CH2, m, 2H); 2.56−3.85 (N−CH2−P, Ph−CH2, mc, bm, 20H); 4.56 (NH−CH−CO, dd, 1H, 3JHH = 8, 3JHH = 7); 7.34 (Ph, d, 2H, 3JHH = 8); 7.36 (Ph, t, 1H, 3JHH = 7); 7.43 (Ph, t, 2H, 3JHH = 7). 13C{1H} δ 23.1 (mc, s); 23.6 (mc, s); 27.7 (CH3, s); 28.3 (P− CH2−CH2, dd, 1JCP = 97, 3JCP = 3); 29.1 (P−CH2−CH2, d, 2JCP = 3); 32.6 (P−CH2−P, t, 1JCP = 79); 37.3 (CH2−Ph, s); 43.2 (mc, s); 44.5 (mc, s); 45.4 (mc, bs); 45.7 (mc, s); 46.4 (mc, s); 46.5 (mc, s); 53.4 (P−CH2−N, d, 1JCP = 108); 55.2 (mc, s); 55.8 (NH−CH−CO, s); 56.1 (mc, d, 3JCP = 12); 84.6 (C−CH3, s); 127.8 (Ph, s); 129.3 (Ph, s); 129.9 (Ph, s); 137.1 (Ph, s); 173.1 (CO−O, m); 175.9 (CO−CH2, d, 3JCP = 17). 31P δ 31.1 (P−CH−N2, m, 1P); 36.6 (P−CH2−CH2, d, 1P, 2JPP = 10). ESI-MS: (−) 630.0 [M − H+]−; (+) 632.3 [M + H+]+; 654.2 [M + Na+]+; 670.2 [M + K+]+. ESI-HRMS: (+) 632.3338 [M + H+]+ (calcd for [C28H52N5O7P2]+ = 632.3337). Synthesis of L3-VGG Conjugate. Freshly prepared solution of NHS-VGG-Cbz peptide (100 mM in MeCN−water 5:1, 159 μL, 15.9 μmol, 1.0 equiv) was added to L3-NH2·3.5HCl·2.5H2O (8.9 mg, 15.9 μmol, 1.0 equiv) dissolved in a mixture of aqueous MES−NaOH buffer (1.0 M, pH = 6.8, 398 μL, 398 μmol, 25 equiv), water (265 μL), and MeCN (555 μL). The reaction mixture (containing ∼11.5 mM NHS-VGG-Cbz and ∼11.5 mM L3-NH2 in aqueous buffer− MeCN 1:1) was stirred for 48 h. The mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼6.2 min. Conversion: (M2): ∼35% (3 h); ∼70% (24 h); ∼75% (48 h). Yield: 6.7 mg (∼60%, 1 step, based on NHS-VGG-Cbz, ≥99% purity). NMR (D2O, pD ∼3): 1H δ 0.95 (CH3, m, 6H); 1.87−2.25 (mc, P−CH2−P, CH3− CH−CH3, bm, 7H); 2.35−3.77 (mc, N−CH2−P, P−CH2−C, bm, 20H); 3.91 (CH2, m, 2H); 4.00 (CH2, m, 2H); 4.16 (CH−CO, d, 1H, 3JHH = 7); 5.17 (Ph−CH2−O, s, 2H); 7.37−7.49 (Ph, m, 5H). 13 C{1H} δ 18.2 (CH3, s); 19.2 (CH3, s); 23.2 (mc, s); 24.1 (mc, bs); 30.7 (CH3−CH−CH3, s); 32.2 (P−CH2−P, t, 1JCP = 80); 41.5 (P− CH2−N, dd, 1JCP = 105, 3JCP = 105); 42.9 (CH2, s); 43.5 (mc, s); 44.4 (CH2, s); 44.9 (mc, s); 45.9 (mc, s); 46.4 (mc, bs); 46.6 (mc, s); 47.3 (mc, bs); 53.4 (P−CH2−N, d, 1JCP = 108); 55.3 (mc, s); 56.1 (mc, d, 3 JCP = 12); 60.4 (CH−CO, s); 68.0 (Ph−CH2−O, s); 128.5 (Ph, s); 129.1 (Ph, s); 129.5 (Ph, s); 136.9 (Ph, s); 159.3 (CO−O, s); 172.0 (CO, s); 173.6 (CO, s); 173.7 (CO, d, 3JCP = 5). 31P{1H} δ 29.4 (N− CH2−P, d, 1P, 2JPP = 10); 31.2 (P−CH2−C, m, 1P). ESI-MS: (−) 731.0 [M − H+]−; (+) 733.2 [M + H+]+; 755.2 [M + Na+]+; 771.2 [M + K+]+. ESI-HRMS: (+) 733.3565 [M + H+]+ (calcd for [C30H55N8O9P2]+ = 733.3562). Synthesis of L4-BTN Conjugate. Freshly prepared solution of NHS-biotin (100 mM in MeCN−water 2:1, 146 μL, 14.6 μmol, 1.0 equiv) was added to L4-NH2·3H2O (7.5 mg, 14.5 μmol, 1.0 equiv)

dissolved in a mixture of MES−NaOH buffer (1.0 M, pH = 6.2, 365 μL, 365 μmol, 25 equiv), water (220 μL), and MeCN (535 μL). The resulting solution (containing ∼11.5 mM NHS-biotin and ∼11.5 mM L4-NH2 in aqueous buffer−MeCN 1:1) was stirred for 48 h. The mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼5.7 min. Conversion: (M2): ∼25% (3 h); ∼75% (24 h); ∼80% (48 h). Yield: 6.6 mg (∼65%, 1 step, based on NHS-biotin, ≥95% purity). NMR (D2O + CsOD, pD ∼6): 1H δ 1.50 (CH2, m, 2H); 1.64 (CH2, m, 1H); 1.75 (CH2, m, 1H); 1.77 (CH2, m, 2H); 1.86 (mc, bm, 1H); 1.90 (mc, bm, 2H); 2.00 (P−CH2−P, t, 2H, 2JHP = 16); 2.10 (mc, bm, 1H); 2.23 (N−CH2−P, bm, 1H); 2.45 (CH2−CO, t, 2H, 3 JHH = 7); 2.47 (mc, bm, 1H); 2.69 (mc, bm, 1H); 2.72 (mc, bm, 1H); 2.79 (CH2−S, d, 1H, 2JHH = 13); 2.84 (mc, bm, 1H); 2.89 (mc, bm, 1H); 2.92 (mc, bm, 1H); 2.98 (mc, bm, 1H); 3.00 (CH2−S, dd, 1H, 2 JHH = 13, 2JHH = 5); 3.01 (mc, bm, 1H); 3.03 (mc, bm, 1H); 3.04 (P−CH2−C, bm, 1H); 3.08−3.33 (mc, N−CH2−P, P−CH2−C, bm, 8H); 3.37 (CH−S, dt, 1H, 3JHH = 10, 3JHH = 5); 3.40 (mc, bm, 1H); 4.44 (NH−CH, dd, 1H, 3JHH = 8, 3JHH = 5); 4.61 (NH−CH, dd, 1H, 3 JHH = 8, 3JHH = 5); 7.34 (CH−C−CH2, dd, 2H, 3JHH = 8; 4JHP = 2); 7.40 (CH−C−N, d, 2H, 3JHH = 8). 13C{1H} 23.2 (mc, s); 25.6 (mc, s); 25.8 (CH2, s); 28.3 (CH2, s); 28.5 (CH2, s); 32.5 (P−CH2−P, dd, 1 JCP = 81, 1JCP = 78); 36.8 (CH2−CO, s); 40.1 (P−CH2−C, dd, 1JCP = 90, 3JCP = 2); 40.4 (CH2−S, s); 44.2 (mc, s); 45.9 (mc, s); 46.0 (mc, s); 46.3 (mc, s); 50.1 (mc, s); 50.2 (mc, s); 52.9 (N−CH2−P, d, 1JCP = 108); 54.8 (mc, s); 55.5 (mc, d, 1JCP = 14); 56.0 (CH−S); 60.9 (CH− NH, s); 62.8 (CH−NH, s); 122.9 (CH−C−N, d, 4JCP = 3); 131.0 (CH−C−CH2, d, 3JCP = 5); 132.7 (CH−C−CH2, d, 2JCP = 8); 135.6 (CH−C−N, d, 5JCP = 4); 166.1 (NH−CO−NH, s); 176.4 (CH2− CO, s). 31P{1H} δ 31.2 (CH2−P−CH2, d, 1P, 2JPP = 8); 33.0 (CH2− P−CH2, d, 1P, 2JPP = 8). ESI-MS: (−) 686.0 [M − H+]−; (+) 688.2 [M + H+]+; 710.2 [M + Na+]+; 726.2 [M + K+]+. ESI-HRMS: (+) 688.3172 [M + H+]+ (calcd for [C29H52N7O6P2S]+ = 688.3170). Synthesis of L4-DBCO Conjugate. A freshly prepared solution of NHS-β-CDBCO (100 mM in MeCN−water 2:1, 144 μL, 14.4 μmol, 1.0 equiv) was added to a mixture of L4-NH2·3H2O (22.3 mg, 43.3 μmol, 3.0 equiv), MES−NaOH buffer (1.0 M, pH = 5.1, 360 μL, 360 μmol, 25 equiv), water (220 μL), and MeCN (535 μL). The resulting solution (containing ∼11.5 mM NHS-β-CDBCO and ∼34.5 mM L4NH2 in a mixture of aq buffer−MeCN 1:1) was stirred for 24 h. The mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼6.7 min. Conversion: (HPLC, M2) ∼50% (3 h); ≥95% (24 h). Yield: 8.9 mg (∼80%, 1 step, based on NHS-β-CDBCO, ≥95% purity). NMR (D2O + CsOD, pD ∼6): 1H δ 1.86 (mc, bm, 1H); 1.91 (mc, bm, 2H); 2.01 (P−CH2−P, t, 2H, 3JHH = 15); 2.12 (mc, bm, 1H); 2.21 (CH2−CO, bm, 1H); 2.28 (N−CH2−P, bm, 1H); 2.36 (CH2−CO, bm, 2H); 2.46 (mc, bm, 1H); 2.60 (CH2−CO, bm, 1H); 2.69 (mc, bm, 1H); 2.73 (mc, bm, 1H); 2.84 (mc, bm, 1H); 2.86−3.08 (mc, P−CH2−C, bm, 6H); 3.08−3.35 (mc, P−CH2−C, N−CH2−P, bm, 8H); 3.40 (mc, bm, 1H); 3.73 (CH2−N−CO, bm, 1H); 5.05 (CH2−N−CO, bm, 1H); 7.12 (CH, bm, 1H); 7.14 (CH, bm, 2H); 7.27 (CH, bm, 2H); 7.31 (CH, bm, 1H); 7.34−7.50 (CH, bm, 4H); 7.53 (CH, bm, 1H); 7.62 (CH, bm, 1H). 13C{1H} δ 23.2 (mc, s); 25.7 (mc, s); 30.9 (CH2−CO, s); 32.4 (CH2−CO, s); 32.5 (P−CH2−P, t, 1 JCP = 79); 40.2 (P−CH2−C, d, 1JCP = 90); 44.2 (mc, s); 46.0 (mc, s); 46.1 (mc, s); 46.4 (mc, s); 50.1 (mc, s); 50.2 (mc, s); 54.0 (P−CH2− N, d, 1JCP = 107); 54.8 (mc, s); 55.5 (mc, d, 3JCP = 15); 56.2 (CH2− N−CO, s); 108.4 (arom, s); 115.2 (arom, s); 122.3 (CH−C−N, d, 4 JCP = 2); 122.4 (arom, s); 123.1 (arom, s); 126.5 (CH, s); 127.7 (CH, s); 128.7 (CH, s); 129.0 (CH, s); 129.5 (CH, s); 129.8 (CH, s); 129.9 (CH, s); 130.8 (CH−C−CH2, d, 3JCP = 3); 132.2 (C−CH2−P, d, 2JCP = 7); 132.5 (CH, s); 135.7 (C−NH−CO, d, 5JCP = 4); 148.4 (arom, s); 151.3 (arom, s); 173.3 (CO, s); 174.9 (CO, s). 31P{1H} δ 31.8 (CH2−P−CH2, d, 1P, 2JPP = 8); 33.6 (CH2−P−CH2, d, 1P, 2JPP = 8). ESI-MS: (−) 749.2 [M − H+]−. TLC (i-PrOH−AcOH−H2O 10:1:10): Rf = ∼0.2. ESI-HRMS: (+) 749.3342 [M + H+]+ (calcd for [C38H51N6O6P2]+ = 749.3340). Synthesis of L4-FC Conjugate. A solution of 3-azido-7-hydroxycoumarin (HAC, 32.9 mM in CD3OD, 90 μL, 2.96 μmol, 1.05 equiv) was added to a solution of L4-DBCO conjugate in D2O (3.40 mM, 8790



Article

prepared solution of δ-CDBCO (3.0 mg, 9.00 μmol, 1.05 equiv) in a mixture of dry DMSO−dry MeCN (1:1; 440 μL). The reaction mixture (containing ∼10.0 mM L6-N3 and ∼10.5 mM δ-CDBCO) was then stirred for 30 min. The mixture was then purified on preparative HPLC (M1). HPLC (M1): Rf ∼19.8 min and ∼20.2 min for each regioisomer, respectively (anal.); ∼46 min and ∼47 min for each regioisomer, respectively (prep). Conversion: (M1) ∼90% (10 min); ≥99% (30 min). Yield: 2.9 mg and 3.0 mg (≥99% purity each fraction, ∼80% regioisomeric purity of each fraction). Combined yield: ∼85% (1 step, based on L6-N3). NMR (D2O, pD ∼2, first fraction, only signals from major regioisomer are presented): 1H δ 1.13 (CH2, m, 1H); 1.19 (CH2, m, 1H); 1.28 (CH2, m, 1H); 1.36 (CH2, m, 1H); 2.04 (mc, p, 2H, 3JHH = 6); 2.11 (mc, p, 2H, 3JHH = 7); 2.15 (P−CH2−P, t, 2H, 2JHP = 16); 2.20 (CH2, bm, 2H); 2.38 (CH2, dt, 1H, 2JHH = 15, 3JHH = 8); 2.55 (CH2, dt, 1H, 2JHH = 15, 3JHH = 7); 2.63−3.67 (mc, N−CH2−P, P−CH2−C, bm, 20H); 5.05 (CH2−N− CO, d, 1H, 2JHH = 18); 5.66 (CH2−N−CO, d, 1H, 2JHH = 18); 6.88 (CH, d, 1H, 3JHH = 8); 7.20 (CH, t, 1H, 3JHH = 8); 7.27 (CH, d, 1H, 3 JHH = 8); 7.38 (CH, m, 1H); 7.41 (CH, m, 1H); 7.44 (CH−C−CH2, d, 2H, 3JHH = 8); 7.49 (CH−C−N, m, 2H); 7.50 (CH, m, 1H); 7.53 (CH, m, 1H); 7.55 (CH, m, 1H). 13C{1H} δ 22.9 (mc, s); 23.2 (mc, s); 24.2 (CH2, s); 24.5 (CH2, s); 32.3 (P−CH2−P, t, 1JCP = 80); 33.2 (CH2, s); 34.0 (CH2, s); 40.2 (P−CH2−P, d, 1JCP = 89); 42.8 (mc, s); 44.1 (mc, s); 44.9 (mc, s); 45.3 (mc, s); 46.0 (mc, s); 46.3 (mc, s); 53.5 (N−CH2−P, d, 1JCP = 106); 54.8 (mc, s); 55.8 (CH2−N−CO, s); 55.9 (mc, d, 3JCP = 11); 126.0 (CH−C−N, m); 126.1 (arom, s); 127.0 (arom, s); 128.2 (CH, s); 128.3 (CH, s); 129.1 (CH, s); 129.5 (CH, s); 130.1 (CH, s); 130.2 (CH, s); 131.4 (CH−C−CH2, d, 3JCP = 5); 132.8 (CH, s); 133.0 (CH, s); 133.5 (arom, s); 133.8 (arom, s); 134.3 (CH−C−N, d, 5JCP = 3); 137.4 (CH−C− CH2, d, 2JCP = 8); 141.7 (arom, s); 145.1 (arom, s); 177.0 (CO−N, s); 178.8 (CO−O, s). 31 1 P{ H} δ 30.9 (CH2−P−CH2, bm, 1P); 34.5 (CH2−P−CH2, d, 1P, 2 JPP = 10). ESI-MS: (−) 818.8 [M − H+]−; (+) 821.1 [M + H+]+; 843.0 [M + Na+]+; 859.0 [M + K+]+. Radiochemistry. Comparative Radiochemical Labeling Experiments. For any ligand, three different batches of no-carrier-added (NCA) [64Cu]CuCl2 were utilized. Comparative ligand [64Cu]CuCl2 labeling experiments (CB-TE2A, cyclam, diamsar, NOTA, DOTA, te1bpin, and te1pp; Chart 1) were carried out under identical conditions as those published in our previous study.39 The identical protocol was used to label selected bifunctional ligands (L2-COOH and L4-NH2), conjugates (L1-PEA, L4-BTN, L4-DBCO, and L5GGY), and te1bpin (used as a reference compound). As labeling efficiency and reproducibility of te1bpin labeling has been well established in the previous study,39 the ligand was used as the control ligand in each labeling experiment to ensure mutual comparability of the results carried out with various 64Cu batches and the ligands. Stock solutions of the ligands (∼1−2 mM) were prepared by dissolution of the solid ligands in D 2O. Precise analytical concentration of the ligands in solution was determined by quantitative NMR (qNMR) technique with calibrated external maleic acid standard solution. These samples solutions were precisely diluted by deionized water to get the final ligand stock solutions (100 μM) for the labeling experiments. Retention factors in the TLC analysis were Rf ∼0.8−0.9 (free Cu) or Rf 0 (complexes of the studied ligands and conjugates). In Vitro/in Vivo Characterization of [64Cu-(L6-N3)]. Radiolabeling Material and Methods. All solvents were purchased from commercial sources (Sigma−Aldrich, Fluka, VWR, Fisher Scientific) and used without further purification. A Direct-Q3 UV water purification system from Millipore (Merck KGaA) was applied to produce ultrapure water with a resistivity of 18.2 MΩ/cm. The NCA [64Cu]Cu2+ was produced at the Helmholtz-Zentrum DresdenRossendorf on the Cyclone 18/9 cyclotron (IBA, Louvain-la-Neuve, Belgium) by 64Ni(p,n)64Cu nuclear reaction as reported previously.83 For the 64Cu labeling, aq [64Cu]CuCl2 (200 MBq in 0.01 M aq HCl, 120 μL, 0.3 M aq NH4OAc, pH 5.0) was added to 9.2 ± 6.7 nmol of L6-N3 and tempered at 80 °C for 10 min, resulting in a labeling yield and a radiochemical purity (decay-corrected) 94 ± 4.6% as determined by analytical radio high-performance liquid chromatog-

pD ∼6, 820 μL, 2.78 μmol, 1.0 equiv). Then, water−MeCN mixture (1:1, 492 μL) was added to a slightly opalescent yellow mixture to produce slightly yellow solution (containing ∼2.0 mM L4-DBCO and ∼2.1 mM HAC) which was stirred for 2 h. Mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼6.5 min. Conversion: (M2) ∼80% (30 min); ≥99% (2 h). Yield: ∼2.1 mg (∼75%, 1 step, based on L4-DBCO, ≥95% purity as equimolar mixture of two regioisomers. NMR (D2O + CsOD, pD ∼6): 1H δ 1.48−3.60 (mc, P− CH2−P, P−CH2−C, P−CH2−N, CH2−CO, bm, 30H); 4.67 (CH2− N−CO, d, 0.5H, 2JHH = 17); 5.16 (CH2−N−CO, d, 0.5H, 2JHH = 18); 5.77 (CH2−N−CO, d, 0.5H, 2JHH = 18); 6.18 (CH2−N−CO, d, 0.5H, 2JHH = 17); 6.62−8.68 (CH−C−CH2, CH−C−NH−CO, 8 × CH, 4 × CH, bm, 16 H). 31P{1H} δ 31.9 (CH2−P−CH2, m, 1P); 33.5 (CH2−P−CH2, d, 1P, bm). ESI-MS: (−) 949.1 [M − H+]−; (+) 951.0 [M + H+]+. TLC (i-PrOH−AcOH−water 10:1:10): Rf = ∼0.2. Synthesis of L5-GGY Conjugate. A freshly prepared solution of L5NCS (19.9 mM in D2O, 500 μL, 9.93 μmol, 1.0 equiv) was added to a mixture of H-GGY-OH peptide (10.0 mM in MeCN−water 1:1, 1000 μL, 10.0 μmol, 1.0 equiv), MOPS−NaOH buffer (1.0 M, pH = 8.0, 250 μL, 250 μmol, 25 equiv), and water (250 μL). The resulting solution (containing ∼5.0 mM H-GGY-OH and ∼5.0 mM L5-NCS in a mixture of aq buffer−MeCN 3:1) was stirred for 24 h. The mixture was then purified on preparative HPLC (M2). HPLC (M2): Rf ∼5.9 min. Conversion: (M2) ∼70% (3 h); ∼90% (24 h). Yield: 5.3 mg (∼65%, 1 step, based on H-GGY-OH, ≥95% purity). NMR (D2O, pD ∼3): 1H δ 2.01 (mc, bm, 2H); 2.06 (mc, p, 2H, 3JHH = 6); 2.12 (P− CH2−P, t, 2H, 2JHP = 15); 2.44−3.80 (mc, N−CH2−P, P−CH2−C, CH2, m, 22H); 3.90 (CH2, m, 2H); 4.21 (CH2, s, 2H); 4.64 (CH−N, dd, 1H, 3JHH = 9, 3JHH = 5); 6.85 (CH−C−OH, d, 2H, 3JHH = 8); 7.16 (CH−CH−CH2, d, 2H, 3JHH = 8); 7.32 (CH−C−NH, d, 2H, 3 JHH = 8); 7.38 (CH−C−CH2, d, 2H, 3JHH = 8). 13C{1H} δ 23.0 (mc, s); 23.8 (mc, bs); 32.4 (P−CH2−P, t, 1JCP = 80); 36.5 (CH2, s); 40.1 (P−CH2−C, d, 1JCP = 89); 42.9 (CH2, s); 43.3 (mc, s); 44.8 (mc, s); 45.6 (mc, s); 46.0 (mc, s); 46.4 (mc, s); 47.0 (mc, s); 48.2 (CH2, s); 53.5 (P−CH2−N, d, 1JCP = 107); 54.9 (mc, s); 55.1 (CH−N, s); 55.8 (mc, d, 3JCP = 11); 116.1 (CH−C−OH, s); 126.8 (CH−C−NH, s); 129.2 (C−CH2, s); 131.3 (CH−C−CH2, s); 131.5 (CH−C−CH2, d, 4 JCP = 5); 134.3 (C−CH2−P, d, 2JCP = 8); 135.8 (C−NH, s); 155.1 (C−OH, s); 171.9 (CO, s); 173.1 (CO, s); 175.7 (CO, s); 182.3 (CS, s). 31P{1H} δ 31.2 (N−CH2−P, m, 1P); 34.2 (P−CH2−C, d, 1P, 2JPP = 8). ESI-MS: (−) 796.8 [M − H+]−; (+) 799.0 [M + H+]+. ESIHRMS: (+) 799.3132 [M + H+]+ (calcd for [C33H53N8O9P2S1]+ = 799.3126). Synthesis of L5-NAP Conjugate. L5-NCS·1.5TFA·H2O (19.5 mg, 28.2 μmol, 2.6 equiv) and H-Acp-βAla-NAP peptide (12.0 mg, 10.8 μmol, 1.0 equiv) were dissolved in aq H3BO3−LiOH buffer (1.0 M, pH = 10.1, 2.70 mL, 2.70 mmol, 250 equiv), and the reaction mixture was stirred for 24 h. The mixture was then purified on preparative HPLC (M1). HPLC (M1): Rf ∼21. Conversion: (M1) ∼30% (3 h); ∼30% (24 h). Yield: 2.4 mg (∼15%; 1 step, based on H-Acp-βAlaNAP, ≥95% purity). Peptide recovery: 5.6 mg (∼45%). NMR: 1H (D2O, pD ∼4) δ 0.84 (CH3, t, 3H, 3JHH = 7); 1.03 (CH2, bm, 2H); 1.27 (CH2, bm, 4H); 1.36 (CH2, bm, 2H); 1.45−1.78 (CH2-peptide, bm, 8H); 1.72−2.15 (mc, P−CH2−P, bm, 6H); 2.19 (CH2, t, 2H, 3 JHH = 7); 2.49 (CH2, m, 2H); 2.55−3.64 (mc, N−CH2−P, N−CH2− C, CH2-peptide, bm, 36H); 3.79 (CH2, m, 2H); 4.12 (α-CH, dd, 1H, 3 JHH = 9, 3JHH = 6); 4.16 (α−CH, dd, 1H, 3JHH = 8, 3JHH = 6); 4.52 (α-CH, t, 1H, 3JHH = 8); 4.55 (α-CH, t, 1H, 3JHH = 7); 4.59 (α-CH, dd, 1H, 3JHH = 8, 3JHH = 6); 4.68 (α-CH, t, 1H, 3JHH = 8); 7.06 (CH, s, 1H); 7.14 (CH, t, 1H, 3JHH = 7); 7.17 (CH, bd, 2H, 3JHH = 8); 7.21 (CH, t, 1H, 3JHH = 7); 7.23 (CH, d, 2H, 3JHH = 8); 7.25 (CH, s, 1H); 7.31 (CH, t, 1H, 3JHH = 8); 7.32 (CH, bm, 2H); 7.36 (CH, t, 1H, 3JHH = 8); 7.46 (CH, d, 1H, 3JHH = 7); 7.63 (CH, d, 1H, 3JHH = 7); 8.53 (CH, s, 1H). 31P{1H} (D2O + CsOD, pD ∼7) δ 32.5 (CH2−P−CH2, m, 1P); 35.4 (CH2−P−CH2, m, 1P). ESI-MS: (−) 1614.5 [M − H+]−; (+) 1616.4 [M + H+]+. MALDI-HRMS: 1615.7735 [M] (calcd for [C73H111N21O15P2S1] = 1615.7764). Synthesis of L6-CDBCO Conjugate. A solution of L6-N3 (20.4 mM in D2O, pD ∼3, 420 μL, 8.57 μmol, 1.00 equiv) was added to a freshly 8791



Article

raphy (radio-HPLC) and radio thin-layer chromatography (radioTLC). The specific activity was 10.6 ± 6.6 GBq/μmol in five syntheses. The analytical radio-HPLC was performed on a Series 1200 device (Agilent Technologies, Santa Clara, CA, USA) equipped with a Ramona β/γ-ray detector (Raytest, Straubenhardt, Germany): eluent A, 0.1% (v/v) TFA in H2O; eluent B, 0.1% (v/v) TFA in MeCN. HPLC system: Zorbax SB-C18, 300 Å, 4 μm, 250 mm × 9.4 mm (Agilent); gradient elution using 95% eluent A for 5 min, 95% eluent A to 95% eluent B in 10 min, 95% eluent B for 5 min and 95% eluent B to 95% eluent A in 5 min, 3 mL/min, 50 °C, recovery of activity (decay-corrected) was >95%. The radio-TLC was carried out on aluminum cellulose plates with H2O with 0.1% TFA and MeCN with 0.1% TFA (90:10 v/v). The developed chromatograms were analyzed by autoradiography using imaging plates and the BAS 5000 (Fuji, Japan). Animal Experiments. All animal procedures were conducted in accordance with the ARRIVE guidelines, the guidelines set by the European Communities Council Directive (86/609 EEC). The local Ethical Committee for Animal Experiments approved the animal facilities and the protocol according to institutional guidelines at the HZDR (reference numbers 24D-9168.11-4/2007-2 and 24-9168.214/2004-1) or at the Semmelweis University approved by the Animal Care and Use Committee of the Semmelweis University (XIV-I-001/ 29-7/2012). Male Wistar rats (Harlan Winkelmann GmbH, Borchen, Germany, between 7 and 9 weeks of age) or nude male mice (Janvier Laboratories, France, 4−6 weeks old) were housed in an Animal Biosafety Level 1 (ABSL-1) acclimatized facility with a temperature of 22 ± 2 °C and humidity of 55 ± 5%. Animals were kept under a 12 h light cycle in temperature-controlled airflow cabinets (27 ± 1 °C) and had free access to standard pellet feed and water. In Vivo Biodistribution. The present study includes data obtained from a total of eight animals. Four rats (body weight 116 ± 8 g) for each time point were injected intravenously into a tail vein or subcutaneously with approximately 0.377 ± 0.004 MBq [64Cu-(L6N3)] corresponding to 3.28 ± 0.23 MBq/kg body weight in 0.5 mL of electrolyte solution E-153 (Serumwerk Bernburg, Germany). Animals were euthanized at 5 and 60 min postinjection. Blood and the major organs were collected, weighed, and counted in a cross-calibrated γcounter (Isomed 1000, Isomed GmbH, Dresden) and WIZARD automatic gamma counter (PerkinElmer, Germany). The radioactivity of the tissue samples was decay-corrected and calibrated by comparing the counts in tissue with the counts in aliquots of the injected radiotracer that had been measured in the γ-counter at the same time. The activity in the selected organs was expressed as percent-injected dose per organ (% ID), and the activity concentration in tissues and organs as standardized uptake value (SUV). Values are quoted as mean ± standard deviation (SD) for each group of four animals. Rat PET Biodistributions. The procedures are described in detail elsewhere.60 Rats were anesthetized using 9 ± 1% desflurane in 30% oxygen and placed on a heat mat. The animals were kept warm under anesthesia until the end of the scan with a total duration of 2 h. The anesthetized animals were localized in a prone position in the axial direction of the scanner. A needle catheter was installed in a lateral tail vein for injection using a syringe pump (0.5 mL/min). The PET studies of two rats were performed with the dedicated small animal PET NanoPET/CT (Mediso, Budapest, Hungary) or microPET P4 (Siemens Healthcare, Erlangen, Germany). Transmission correction was performed with a computer tomogram or transmission scan using a Co-57 point source that was performed before tracer application. Data were acquired over 120 min. Within 5 s of the start of data acquisition, approximately 30 MBq [64Cu-(L6-N3)] in saline was injected. PET images were iteratively reconstructed by a 3-D orderedsubset expectation maximization algorithm (3D OSEM/MAP) with transmission correction and with voxel size of 0.050 cm × 0.050 cm × 0.050 cm. No additional corrections were made of partial-volume effects and recovery. Three-dimensional regions of interest (ROI) were determined for subsequent data analysis. The standardized uptake values (SUV, g/mL) and standardized uptake ratios (SUR, as ratio of the SUVs of the tissue of interest and the blood SUV, derived

from a region over the caudal arteria abdominalis and vena cava) were used to quantify the activity uptake and kinetics. Statistical Analysis. Statistical analyses were carried out with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). The data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used for statistical evaluation. Means were compared using Student’s t test. A P value of

Improved Conjugation, 64-Cu Radiolabeling, in Vivo Stability, and

Recommend Documents