HBED-NN: A Bifunctional Chelator for Constructing

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HBED-NN: A Bifunctional Chelator for Constructing Radiopharmaceuticals Ata Makarem, Karel D. Klika, German Litau, Yvonne Remde, and Klaus Kopka J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00832 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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The Journal of Organic Chemistry

HBED-NN: A Bifunctional Chelator for Constructing Radiopharmaceuticals Ata Makarem,*,† Karel D. Klika,‡ German Litau,† Yvonne Remde,† and Klaus Kopka†,§ †German

Cancer Research Center (DKFZ), Division of Radiopharmaceutical Chemistry, INF 223, D-69120 Heidelberg, Germany ‡German Cancer Research Center (DKFZ), Molecular Structure Analysis, INF 280, D-69120 Heidelberg, Germany §German Cancer Consortium (DKTK), Heidelberg, Germany ABSTRACT: Radiometal-based radiopharmaceuticals bearing bifunctional HBED chelators are powerful radiotracers for cancer diagnosis and therapy. Bifunctional HBED chelators make strong complexes with trivalent gallium and are able to bind to bioactive molecules through covalent bonds. However, thus far no bifunctional HBED chelator capable of direct conjugation via click chemistry has been reported. We hereby introduce HBED-NN as a structurally new bifunctional HBED chelator for direct click coupling. We also investigated the complex chemistry of [Ga-(HBED-NN)] for potential use in gallium-based radiopharmaceuticals.

Radiopharmaceuticals are important drugs for tumor diagnosis and cancer treatment.1–3 Among the various types of radiopharmaceuticals introduced to date, radiometal-based radiopharmaceuticals bearing the bifunctional complexing agent HBED4 have been drawing the attention of different research groups during the past years.5–15 The most important example in this category is [68Ga]Ga-PSMA-11 for use in noninvasive prostate cancer diagnosis.7 Bifunctional HBED chelators build strong complexes with trivalent gallium through N2O4 coordinating sites and are conjugated to bioactive molecules (e.g., antibodies).16,17 As a typical synthetic strategy, ester-protected variants of HBED are employed as chelator precursors for the synthesis of metal-based (radio)pharmaceuticals with the HBED framework (Scheme 1).18–22 Scheme 1. Typical Synthetic Pathway for HBED-Based Radiopharmaceuticals t-BuO2C

1) bioconjugation

CO2t-Bu N

HO2C

OH

R

CO2H N

2) t-Bu deprotection

N

N OH

R

HO

G

HO

G

G = modified bioactive molecule

HBED chelator (ester protected)

free HBED chelator O

3) radiolabeling

O

O N

N

O G

G O

O

chemoselective transformation.28,29 Furthermore, the corresponding aromatic 1,2,3-triazole is much stronger than ordinary covalent linkages such as amide, ester, thioether, etc. In recent years, new and useful bioactive molecules bearing alkyne groups with various structures and different targeting properties have been introduced,30–32 and potentially they could be used in combination with HBED chelator types for the development of radiopharmaceuticals. This is especially attractive for the synthesis of precursors for radiolabeling conjugated with macromolecules such as proteins.33 However, to the best of our knowledge, there are no reported examples of bifunctional HBED chelators which accomplish direct “click” coupling. Herein we present HBED-NN34 as a protected diazide variant of the bifunctional HBED chelator. The two terminal azide groups available on this structure successfully undergo CuAAC reactions. The presence of two functional groups also provides the possibility for unsymmetrical conjugation.15,19,22 The current work describes the synthesis, CuAAC reaction and complex chemistry of the HBED-NN chelator type for application in radiopharmaceutical chemistry. For this purpose, we have simulated the general synthetic route of HBED-based radiopharmaceuticals for the preparation of a hexadentate [Ga(HBED-NN)] complex (Scheme 2). The HBED-NN chelator entered into defined CuAAC reactions as well as complexation reactions under various conditions. The synthesis of esterprotected HBED-NN (1) was accomplished in 5 steps (Scheme 3). However, It is essential to understand the behavior of any structurally new chelator in both conjugation and coordination reactions before planning (radio)pharmaceutical syntheses with bioactive molecules.

HBED-based radiopharmaceutical

The CuAAC23 (“click”) reaction is an important tool for the conjugation of targeting molecules to bifunctional chelators.24– 27 In contrast to other commonly used methods24 (e.g., amide coupling), the CuAAC reaction is an irreversible and

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resting states and rate-determining steps.35–37 The CuAAC reaction for chelator 1 was optimized on phenyl acetylene and copper(II) acetate/sodium ascorbate in CH3CN/H2O was selected as the catalytic system. The reaction between phenyl acetylene and compound 1 was conducted both at room temperature as well as at an elevated temperature. At 80 °C in the presence of 0.05 equiv Cu(OAc)2 and 0.10 equiv sodium ascorbate, the reaction was relatively fast and after 15 min it was complete with an isolated yield of 59%. By contrast, at room temperature the reaction progress was very slow. To explore the chelator behavior in the presence of an excess amount of copper catalyst, the CuAAC reaction of phenyl acetylene with compound 1 was tested with 0.5 equiv of Cu(OAc)2 and 1.0 equiv of sodium ascorbate at room temperature, though using excess amounts of copper source in CuAAC reactions usually results in copper-containing byproducts.38,39 The reaction of 1 with a 10-fold excess of the catalytic mixture was finished after 15 min and, as expected, the amount of byproducts had increased whereby the isolated yield was only 47%. The CuAAC reaction between compound 1 and phenyl acetylene with acetic acid added was also checked since in some reported cases, catalytic amounts of acetic acid increased the reaction rate by affecting the catalytic cycle and without causing side reactions.36,40,41 However, no change in the reaction rate for compound 1 and phenyl acetylene was observed. CuAAC reactions of 1 with ethyl propiolate and propargyl alcohol were conducted at 80 °C with 0.05 equiv Cu(OAc)2 and 0.10 equiv sodium ascorbate and isolated yields were determined as 43% and 50%, respectively [Table S1 in the Supporting Information (SI)].

Scheme 2. Preparation of Hexadentate [Ga-(HBED-NN)] Complex Using a HBED-NN Chelator Conjugated to Phenyl Acetylene as Vector Model t-BuO2C

CO2t-Bu N

N OH

HO2C 1) phenyl acetylene Cu(OAC)2/Na+ ascorbate N N3 H2O/CH3CN OH 80 oC, 1 h

2) r.t., 5 h TFA/CHCl3

HO N3

Ar =

Ar

HO Ar

isolated yield: 58%

1

HBED-NN chelator (protected)

CO2H N

N N N

2 free chelator

Ph

O nat

GaCl3 NaOAc DMF/H2O

O [Ga-(HBED-NN)]Na

N

G

(racemic mixture)

pH 4

O

90 oC, 4 min

G

O O

3

isolated yield: 81%

N

Ga

G=

(CH2)2 Ar

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O

proven geometry for one of the two enantiomers with the complexing oxygens of the carboxylate groups trans to each other and the phenolic oxygens cis (isomer II)

Thus, we next investigated the click chemistry of the HBEDNN chelator (1) using three small terminal alkynes, viz. phenyl acetylene, ethyl propiolate and propargyl alcohol, in a standard CuAAC reaction with compound 1 (Scheme 3). These alkynes were selected because they show different kinetics in CuAAC reactions due to their different acidities which influence catalyst

Scheme 3. Synthesis and CuAAC Reactions of Ester-Protected HBED-NN OH

OH

NaN3

N3

isolated yield: 96%

1b

isolated yield: 93%

N3 H N

N3

N H HO

Na2CO3 CH3CN reflux, 5 h

CF3CH2OH 0 oC to r.t. overnight inert gas

N HO

quantitative yield

isolated yield: 95%

1d t-BuO2C

N

N

N

Cu(OAc)2/ Na+ ascorbate H2O/CH3CN

overall yield: 65% HBED-NN (protected)

1

N N N

R

OH

R

HO

For investigating the complex chemistry of HBED-NN, it was necessary to use a representative vector model with a simple structure and uncomplicated spectral pattern in the 1H NMR spectrum, especially in the upfield region where protons from the chelate cage appear. Phenyl acetylene met these requirements and therefore its click product phenyl 1,2,3triazole 2 was used as the complexing agent. Compound 2 was synthesized in two steps: firstly the CuAAC reaction was performed on structure 1 followed by the removal of the t-butyl protecting groups using trifluoroacetic acid (Scheme 2). Compound 2 (1 equiv) was then reacted with natGa3+ (1.5 equiv)

CO2t-Bu N

N3

OH N3

NaBH4

N3

N3

CO2t-Bu N

BrCH2CO2t-Bu

isolated yield: 77%

1e

OH

1c

t-BuO2C OH

NH2

toluene r.t., 30 min

CH3CN reflux, overnight N3

1a

H2 N

CHO

N

DMF 45 C, 1.5 h inert gas o

Br

OH

MgCl2 , Et3N paraformaldehyde

N R

N

HO

yield: 43-59%

1aa : R = Ph, 1ab: R = CO2Et, 1ac: R = CH2OH

in DMF–H2O media (pH ~4) at both room temperature as well as at an elevated temperature. At 90 °C, chelation went very fast and was complete in 4 min, while at room temperature the reaction was comparatively slow. However, only the first 10 min of the reaction was perused because complexation of HBED derivatives with longer reaction times have limited use in [68Ga]Ga-radiolabeling (t½ = 68 min). The room temperature reaction was quenched after 10 min and the mixture analyzed by 1H NMR.


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The Journal of Organic Chemistry Previous reports of Ga-(HBED)-type complexes have simply assumed a structure or perpetuated a prior assumed structure,16,42,44 though some reports17,42–44 have recognized the possibility of three NMR distinguishable geometric isomers for a hexadentate Ga-(HBED)-type complex (Figure 1), viz. octahedral complexes with either carboxylate group oxygens cis to each other and the phenolic oxygens trans to each other (isomer I), the carboxylate group oxygens trans to each other and the phenolic oxygens cis to each other (isomer II), or a cis,cis arrangement (isomer III). All three isomers are chiral-atmetal constructs and thus any structure formed under symmetric conditions will be a racemic mixture of the Δ and Ʌ enantiomers, as has been shown.16,17,42 The formation of these geometric isomers is temperature and pH dependent, and at 90 °C the reaction results in the formation of a predominant isomer, ostensibly the thermodynamically favored one.42–44 It is worth noting that reports of multiple species resulting from a reaction may not only be mixtures of isomers, they may also include incompletely formed HBED complexes, e.g., with other ligands also in attendance such as water replacing one phenolic binding.43 The three geometric isomers may exhibit different pharmaceutical profiles, though oddly while the enantiomers have been shown to be distinct in this respect,42 a mixture of species actually appeared to be indifferent to the composition of the sample.44 (PhO)

(OAc)

O

N

N

Ga (OAc)O

(OAc)

O

N

N Ga

O (OAc) O(PhO)

isomer I PhO- ligands trans OAc- ligands cis

O

(PhO)

O(OAc)

O

N

N Ga

O(PhO)

isomer II PhO- ligands cis OAc- ligands trans

O

(OAc)

O O(PhO)

(PhO)

isomer III PhO- ligands cis OAc- ligands cis

Figure 1. Possible geometrical isomers of Ga-HBED complexes. The geometry of the predominant isomer is invariably an open question, but considering the X-ray-determined crystal structures of analogous HBED complexes43,45,46 with Ga3+, Ti4+ and Fe3+, the product obtained here is expected to attain isomer II (Scheme 2). Applying free HBED-NN chelator 2, the complexation of natGa3+ at 90 °C resulted in only one geometric isomer. Product purity was proven by HPLC and NMR and the isolated yield was 81%. The room temperature-conducted reaction retained reasonable selectivity whereby 1H NMR analysis of the crude reaction mixture revealed 49% of the favored isomer with only 6% of another complex – presumably another isomer or a mixed ligand complex – together with 45% of unreacted chelator 2. As a comparison, the synthesis of [natGa-PSMA-HBED-CC] complex at room temperature and pH ~4 provided 50% of the favored isomer and 50% of another species.44 Full NMR characterization of the synthesized [Ga-(HBEDNN)] complex (3) provided spectra that were fully consistent with the postulated geometry (Scheme 2). Accordingly, there are only three distinct methylene environments in the chelate cage, thereby isomer III is eliminated as all methylene groups are distinct in this structure. Two equivalent pairs of diastereotopic protons are each present for the methylene groups adjacent to the carboxylate group and the benzylic methylenes, thus two spin systems consisting of two doublets each with a relative integration of two protons for each signal

are present for these protons. The methylenes in the – NCH2CH2N– bridge are diastereotopic and, as well, the chemically equivalent protons from each methylene pair are also magnetically inequivalent, thus a complex higher-order sub-spectrum results. Likewise, the protons of the equivalent phenyl substituents at the terminus of the scaffold also provide a complex higher-order sub-spectrum while the two isolated triazole protons are present as one singlet. The protons of the equivalent phenolic groups provide the expected pattern for a 1,2,4-substituted phenyl while the methylene protons adjacent to the triazole moiety appear higher-order due to the chiral-atmetal stereocenter. From models of isomer II (see SI), only one proton of the methylenes adjacent to the carboxylate group should have sizeable NOE’s with the protons of the –NCH2CH2N– bridge while both benzylic protons should have sizeable NOE’s with the protons of the –NCH2CH2N– bridge. For isomer I, the converse applies whereby only one of the benzylic protons should have sizeable NOE’s with the –NCH2CH2N– bridge protons while both protons of the carboxylate group methylenes should have sizeable NOE’s with these protons. From the NOESY spectrum, the observed NOE’s for these protons unequivocally implied isomer II. Depictions of these stereo relationships and the NOESY spectrum are presented in the SI. Additionally, the couplings between the protons on the  carbons to the coordinating nitrogen atom and the other two  carbons also provide an indication of the structural isomer adopted since the dihedral angles were either highly optimal (i.e., either very large or very small) – and therefore conducive to providing a correlation in the HMBC spectrum optimized for a long-range coupling of 8 Hz – or were otherwise not due to dihedral angles of intermediate size. Thus, these 3JH,C’s can be assigned as either expected to be observable or not observable in the HMBC spectrum acquired here. For example, for isomer II, while both methylene protons adjacent to the carboxylate group are suitably disposed in terms of dihedral angles to possess sizeable 3JH,C’s to the respective nearest carbon of the – NCH2CH2N– bridge, neither of the benzylic protons are so disposed. For isomer I, the converse applies whereby both benzylic protons are suitably disposed in terms of dihedral angles to possess sizeable 3JH,C’s to the respective nearest carbon of the –NCH2CH2N– bridge, while neither of the carboxylate group methylene protons are so disposed. In the HMBC spectrum, only the two methylene protons adjacent to the carboxylate group provided correlations to the – NCH2CH2N– bridge carbons, thereby providing further support for isomer II as the structure of the product. (See the SI for presentation of this example portion of the HMBC spectrum.) Overall, of the 12 considered relationships present in the molecule, 6 should yield correlations while 6 should not, and indeed the HMBC spectrum revealed observations fully in accordance with these expectations for isomer II. For isomer I, the converse applies, thus distinctly eliminating isomer I as the structure of the product. The experimental observations for the 12 possible correlations are presented in Table S2 in the SI. An interesting point raised by a referee was the possible occurrence of the self-disproportion of enantiomers (SDE),47 i.e., the spontaneous fractionation of nonracemic material into enantioenriched and -depleted fractions when any physicochemical process is applied to a nonracemic sample.48 Since only racemates were prepared herein, this is of no concern presently but would be of considerable interest if in future work



enantioselective preparation of the chelator was undertaken, either during sample preparation or after pharmacological administration.42,49 Pertinently, there is also the possibility of analogous SDE-type processes occurring between other stereochemically related species due to intermolecular association,48 e.g. between diastereomers or between geometric isomers, but such occurrences have never been enunciated. Although additional Ga3+ complexes have been observed here and elsewhere,42–44 whether they represent geometric isomers or incompletely formed HBED complexes43 is uncertain as their characterization was lacking. For readers unfamiliar with the SDE in all its manifestations, they are referred to the literature for detailed descriptions of the phenomenon.48 In conclusion, we have introduced HBED-NN as a diazide variant of the bifunctional HBED chelator, which coordinates trivalent gallium successfully and results in a hexadentate [Ga(HBED-NN)] complex as a single geometric isomer. NMR experiments unequivocally confirmed the geometry of this complex. This study shows that the HBED-NN chelator-type is potentially an excellent candidate for the development of new precursors for radiolabeling with the positron emitter gallium-68.

EXPERIMENTAL SECTION General Experimental Methods Chemicals and solvents used in this work were supplied by the division of Radiopharmaceutical Chemistry at the German Cancer Research Center or bought commercially. Reactions involving air-sensitive reagents were carried out under an atmosphere of nitrogen or argon using standard Schlenk techniques. The progress of chemical reactions was monitored by using HPLC and TLC techniques. For column chromatography, silica gel with grain size between 0.063 and 0.200 mm was employed as stationary phase. For thin layer chromatography commercially, available ready-to-use plates supplied by Macherey & Nagel (POLYGRAM© SIL G/UV254) were used as stationary phase. The following RP-HPLC system was used for analysis: Agilent (1100 series) reverse phase system, equipped with a multi-wavelength detector (MWD) and a Chromolith RP-18e analytical column. The following RP-HPLC system was used for purification: LaPrep P314 (VWR International, Radnor, USA) equipped with a DAD and a Nucleodur Sphinx RP 5 μm preparative column. IR spectra were recorded with a Bruker Lumos, Germanium ATR-crystal, and the following abbreviations were used to describe both the intensity and profile of the signals: w (weak), m (medium), s (strong), br (broad). NMR spectra were recorded with a Bruker Avance III (400 MHz) NMR spectrometer. Chemical shifts (δ) are reported in ppm relative to TMS and the spectra calibrated with respect to the solvent peaks.50 The assignment of signals in the NMR spectra was achieved by interpretation of two-dimensional NMR spectra (COSY, HMBC, HSQC and NOSEY). For processing, analysis and interpretation of NMR spectra, the program TopSpin 3.5 pl7 was used. Mass spectra were recorded by the Mass Spectrometry Service Facility of the Department of Chemistry at Ruprecht-Karls-Universität-Heidelberg and the Division of Radiopharmaceutical Chemistry at the German Cancer Research Center. The following instruments were employed: Bruker ApexQe hybrid 9.4 T FT-ICR (HR-ESI), Bruker MALDI-MS Daltonics Microflex and JEOL JMS-700 magnetic sector (EI). Elemental analyses were carried out by the Laboratory of Microanalysis of the Department of Chemistry at the Ruprecht-KarlsUniversität-Heidelberg on vario EL and vario MICRO cube instruments by Elementar Analysensysteme GmbH. The determination of melting points was done in Büchi (B-540) melting point apparatus. Synthesis of 4-(2-azidoethyl)phenol (1b): The compound was synthesized according to a literature procedure with some modification.51 A mixture of 4-(2-bromoethyl)phenol (1a)

(5 g, 24.86 mmol, 1 equiv) and NaN3 (8.08 g, 124.35 mmol, 5 equiv) in dry DMF (80 mL) was stirred at 45 °C under an inert gas atmosphere for 1.5 h. The reaction mixture was diluted with water (500 mL) and extracted with CH2Cl2. The organic layer was dried over Na2SO4, filtered and the solvent removed under reduced pressure. The residue was dissolved in diethyl ether (100 mL) and washed with HCl solution (2.5% × 1) and brine (× 3). The organic layer was dried over Na2SO4, filtered and evaporated to give azide 1b (3.88 g, 23.78 mmol, 95.6%) as a yellow oil. Spectroscopic data is identical to previously reported results.52 1H NMR (400 MHz, CDCl3, 25 °C):  = 7.09 (d, 3JH–H = 8.6 Hz, 2H, m-CH), 6.78 (d, 3JH–H = 8.6 Hz, 2H, ortho-CH), 4.69 (s, 1H, OH), 3.46 (t, 3JH–H = 7.2 Hz, 2H, CH2N3), 2.82 (t, 3JH–H = 7.2 Hz, 2H, CH2C) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 154.5 (COH), 130.4 (CCH2), 130.1 (meta-CH), 115.6 (ortho-CH), 52.8 (CH2N3), 34.6 (CH2C) ppm. Synthesis of 5-(2-azidoethyl)-2-hydroxybenzaldehyde (1c): This new compound was synthesized using standard orthoformylation.53 To a mixture of compound 1b (3.88 g, 23.78 mmol, 1equiv), paraformaldehyde (5.71 g, 190.14 mmol, 8 equiv) and anhydrous MgCl2 (4.53 g, 47.58 mmol, 2 equiv) in absolute acetonitrile (100 mL), was added dry triethylamine (13.26 mL, 95.12 mmol, 4 equiv). The reaction mixture was stirred overnight under reflux conditions and then cooled to room temperature. The mixture was diluted with water (500 mL) and HCl (5%, 100 mL) and then the product was extracted with toluene. The organic layer was washed several times with brine, dried over Na2SO4 and filtered. The solvent was removed under reduced pressure to give compound 1c (4.25 g, 22.21 mmol, 93.4%) as a yellow oil on high purity. For structural characterization, this product was further purified by preparative HPLC. IR (reflection):  = 3308 (w, br), 2858 (m), 2090 (s), 1652 (s) cm−1. 1H NMR (400 MHz, CDCl3, 25 °C):  = 10.92 (s, 1H, OH), 9.89 (s, 1H, CHO), 7.40 (m, 2H, CHCCH), 6.97 (d, 3JH–H = 8 Hz, 1H, CHCOH), 3.52 (t, 3JH–H = 7.0 Hz, 2H, CH2N3), 2.88 (t, 3JH–H = 7.0 Hz, 2H, CH2C) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 196.5 (CHO), 160.7 (COH), 137.6 (CHCHCOH), 133.7 (CCHC), 129.7 (CHCCH), 120.7 (CCHO), 118.2 (CHCOH), 52.5 (CH2N3), 34.4 (CH2C) ppm. MS (EI+): m/z [2M]2+ Calcd for C18H18N6O4: 191.1; Found: 191.1 (11%), [M−(CH2N3)]+ Calcd for C8H7O2: 135.0; Found: 135.0 (100%). Elemental Analysis (analysis no. 42014): Anal. Calcd for C9H9N3O2 C, 56.54; H, 4.75; N, 21.98. Found: C, 56.75; H, 4.61; N, 22.38. Synthesis of 2,2'-{[ethane-1,2-diylbis(azaneylylidene)]bis (methaneylylidene)}bis[4-(2-azidoethyl)phenol] (1d): Ethylene diamine (0.37 mL, 0.33 g, 5.50 mmol, 1 equiv) was added to a solution of compound 1c (2.00 g, 10.46 mmol, 1.9 equiv) in toluene (10 mL). The reaction mixture was stirred at room temperature for 30 min and then n-hexane (~40 mL) was added to the mixture. The precipitate was filtered and washed with n-hexane to yield imine 1d (2.01 g, 4.95 mmol, 94.6%) as an orange solid. Considerable impurities were not detected by NMR spectra and thus the compound was used in the next step without further purification. However, for spectroscopic characterization, the product was washed with a dilute solution of sodium hydrogen sulfite and water, while for elemental analysis it was purified by preparative HPLC. MP 114-116 °C. IR (ATR):  = 2926 (w), 2124 (s), 1638 (s), 1279 (m) cm−1. 1H NMR (400 MHz, CDCl3, 25 °C MHz):  = 8.33 (s, 2H, CHN), 7.15 (dd, 3JH–H = 8.5 Hz, 4JH–H = 2.3 Hz, 2H, CHCHCOH), 7.07 (d, 4JH–H = 2.3, 2H, CCHC), 6.90 (d, 4JH–H = 8.5 Hz, 2H, CHCHCOH), 3.94 (s, 4H, CH2Nimine), 3.45 (t, 3JH–H = 7.1 Hz, 4H, CH2N3), 2.81 (t, 3JH–H = 7.1 Hz, 4H, CH2CH2N3) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 166.4 (CHN), 160.0 (COH), 132.9 (CHCHCOH), 131.7 (CCHC), 128.2 (CHCCH), 118.7 (CCHN), 117.4 (CHCHCOH), 59.9 (CH2Nimine), 52.7 (CH2N3), 34.49 (CH2CH2N3) ppm. MS (MALDI): m/z [M+H]+ Calcd for C20H23N8O2: 407.2; Found: 407.2 (100%). Elemental Analysis (analysis no. 42993): Anal. Calcd for C20H22N8O2: C, 59.10; H, 5.46; N, 27.57. Found: C, 59.14; H, 5.59; N, 27.66.


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The Journal of Organic Chemistry Synthesis of 2,2'-{[ethane-1,2diylbis(azanediyl)]bis(methylene)}bis[4-(2-azidoethyl)phenol] (1e): Compound 1d (1.91 g, 4.69 mmol, 1 equiv) was dissolved in warm 2,2,2-trifluoroethanol (40 mL, ~50 °C) and then cooled in an ice-bath. To this solution was added NaBH4 (0.71 g, 18.78 mmol, 4 equiv) in portions and then the reaction mixture was allowed to warm up to room temperature. After being stirred overnight under an inert gas atmosphere, the reaction was quenched with water (100 mL), dried over Na2SO4, filtered and evaporated to provide product 1e as a colorless to pale yellow solid in quantitative yield. NMR showed no considerable impurities and thus this compound was used in the next step without further purification. However, for structural characterization product 1e was further purified by preparative HPLC. MP 99-102 °C. IR (ATR):  = 3287 (m), 2833 (w), 1614 (m), 1458 (s) cm−1. 1H NMR (400 MHz, CDCl3, 25 °C):  = 7.02 (dd, 3JH–H = 8.3 Hz, 4JH–H = 2.1 Hz, 2H, CHCHCOH), 6.83 (d, 4JH–H = 2.1 Hz, 2H, CCHC), 6.78 (d, 3JH–H = 8.3 Hz, 2H, CHCHCOH), 3.98 (s, 4H, BnCH2), 3,44 (t, 3JH–H = 7.2 Hz, 4H, CH2N3), 2.84 (s, 4H, CH2NH), 2.78 (t, 3JH–H = 7.2 Hz, 4H, CH2CH2N3) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 156.9 (COH), 129.3 (CHCHCOH), 129.0 (CCHC), 128.7 (CHCCH), 122.35 (BnCH2C), 116.8 (C(OH)CHCH), 52.9 (CH2N3), 52.7 (BnCH2), 48.0 (CH2NH), 34.70 (CH2CH2N3) ppm. MS (MALDI): m/z [M+H]+ Calcd for C20H27N8O2: 407.2; Found: 407.2 (100 %). HRMS (ESI+): m/z [MH+(H2N-C2H4-NH2)+2(PhOH-C2H4-N3)]+ Calcd for C38H53N16O4: 797.4430; Found: 797.4417 (99%). Synthesis of di-tert-butyl 2,2'-(ethane-1,2-diylbis{[5-(2-azidoethyl)2-hydroxybenzyl]azanediyl}) diacetate [HBED-NN-di(t-Bu ester)] (1) A mixture of compound 1f (0.56 g, 1.36 mmol, 1 equiv), anhydrous Na2CO3 (0.58 g, 5.46 mmol, 4 equiv) and tert-butyl 2-bromoacetate (0.56 g, 2.86 mmol, 2.1 equiv) was suspended in anhydrous acetonitrile (25 mL) and stirred under reflux conditions for 5 h, and then the reaction mixture was cooled to room temperature and filtered. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (Rf = 0.4, hexane–EtOAc, 2:1). The obtained product was recovered in ethanol to give HBEDNN-di(t-Bu ester) (1) as a colorless solid (0.76 g, 1.05 mmol, 77%). MP 93-95 °C. IR (ATR): 2975 (w), 2091 (s), 1733 (s), 1500 (m), 1152 (s) cm−1. 1H NMR (400 MHz, CDCl3, 25 °C):  = 9.62 (s, 2H, COH), 7.02 (dd, 3JH–H = 8.2 Hz, 4JH–H = 2.2 Hz, 2H, CHCHCOH), 6.79 (d, 3JH– 4 H = 8.2 Hz, 2H, CHCHCOH), 6.76 (d, JH–H = 2.2 Hz, 2H, CCHC), 3.71 (s, 4H, BnCH2), 3.42 (t, 3JH–H = 7.3 Hz, 4H, CH2N3), 3.16 (s, 4H, CH2CO2), 2.77 (t, 3JH–H = 7.3 Hz, 4H, CH2CH2N3), 2.68 (s, 4H, N-C2H4N), 1.46 (s, 18H, CH3) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 170.1 (CO2), 156.4 (COH), 129.7 (CCHC), 129.6 (CHCHCOH), 128.6 (CHCCH), 121.8 (BnCH2C), 116.8 (CHCHCOH), 82.2 (CCH3), 58.10 (BnCH2), 55.6 (CH2CO2), 52.9 (CH2N3), 50.4 (N-C2H4-N), 34.7 (CH2CH2N3), 28.2 (CH3) ppm. HRMS (ESI+): m/z [M+H]+ Calcd for C32H47N8O6: 639.3613; Found: 639.3634 (18%), [M+Na]+, Calcd for C32H46N8NaO6: 661.3433; Found: 661.3451 (36%). Elemental Analysis (analysis no. 41992): Anal. Calcd for C32H46N8O6: C, 60.17; H, 7.26; N, 17.54. Found: C, 60.17; H, 7.29; N, 17.28. Syntheses and characterization of substrates 1aa, 1ab and 1ac: These compounds were synthesized according to the standard CuAAC reaction.54 To a mixture of HBED-NN (0.1 g, 0.16 mmol, 1 equiv) and a terminal alkyne (0.39 mmol, 2.5 equiv) in CH3CN/H2O (0.5 mL: 0.3 mL) were added 78 µL of an aqueous Cu(OAc)2 solution (0.1 M; 7.8 µmol, 0.05 equiv) and 156 µL of an aqueous sodium ascorbate solution (0.1 M; 15.6 µmol, 0.1 equiv). The reaction mixture stirred at 80 °C and reaction progress was followed by HPLC and TLC. After being stirred for 1 h, the reaction mixture was cooled to room temperature and the product isolated by preparative HPLC. Note: Product 1aa was additionally filtered through a short column packed with silica gel (eluent: CHCl3). Isolated yields for 1aa, 1ab and 1ac were 59% (0.08 g, 0.09 mmol), 43% (0.06 g, 0.07 mmol) and 50% (0.06 g, 0.08 mmol), respectively. Each product is a colorless solid. Synthesis of compound

1aa in the presence of 0.5 equiv of Cu(OAc)2 and 1.0 equiv of sodium ascorbate at room temperature gave 47% ( 0.06 g, 0.07mmol) as isolated yield. In this case, Cu(OAC)2 and sodium ascorbate were added directly and the reaction mixture stirred for 30 min. Characterization data for di-tert-Butyl 2,2'-[ethane-1,2-diylbis({2hydroxy-5-[2-(4-phenyl-1H-1,2,3-triazol-1yl)ethyl]benzyl}azanediyl)]diacetate (1aa): IR(ATR): 2929 (w), 1726 (m), 1152 (s), 765 (m). 1H NMR (400 MHz, CDCl3, 25 °C):  = 9.57 (s, br, 2H, COH), 7.76 (dd, 4H, 3JH–H = 7.8 Hz, 4JH–H = 1.4 Hz, Phsubstituent, ortho-H), 7.52 (s, 2H, CHN), 7.38 (t, 4H, 3JH–H = 7.6 Hz, Ph-substituent, meta-H), 7.30 (m, 2H, Ph-substituent, para-H), 6.97 (dd, 2H, 3JH–H = 8.2 Hz, 4JH–H = 2.2 Hz, CHCHCOH), 6.76 (d, 2H, 3JH–H = 8.1 Hz, CHCHCOH), 6.52 (d, 2H, 4JH–H = 2.2 Hz, CCHC), 4.56 (t, 4H, 3JH–H = 7.2 Hz, CCH2CH2N), 3.53 (s, 4H, BnCH2), 3.12 (t, 4H, 3JH– H = 7.2 Hz, CCH2CH2N), 3.08 (s, 4H, CH2CO2), 2.45 (s, 4H, N-C2H4N), 1.46 (s, 18H, CH3) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 170.4 (CO2), 156.6 (COH), 147.5 (NCCH), 130.8 (Ph-substituent, ipso-C), 129.9 (BnCH2C), 129.5 (CCHC), 129.0 (CHCHCO), 128.2 (Ph-substituent, meta-C), 127.6 (Ph-substituent, para-C), 125.8 (Phsubstituent, ortho-C), 122.1 (CCH2CH2N), 120.25 (CHN), 116.9 (CHCHCO), 82.3 (CCH3), 57.8 (BnC), 55.7 (CH2CO2), 52.2 (CCH2CH2N), 49.8 (N-C2H4-N), 36.1 (CCH2CH2N), 28.2 (CH3) ppm. HRMS (ESI+): m/z [M+H]+ Calcd for C48H59N8O6: 843.4552; Found: 843.4556 (100%). Characterization data for diethyl 1,1'-{[({2,2,13,13-tetramethyl-4,11dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl}bis(methylene))bis(4hydroxy-3,1-phenylene)]bis(ethane-2,1-diyl)}bis(1H-1,2,3-triazole-4carboxylate) (1ab): IR(ATR): 2980 (w), 1722 (s), 1153 (s), 775 (m). 1H NMR (400 MHz, CDCl , 25 °C):  = 9.63 (s, br, 2H, COH), 7.83 (s, 3 2H, CHN), 6.92 (dd, 4H, 3JH–H = 8.3 Hz, 4JH–H = 2.2 Hz, CHCHCOH), 6.76 (d, 3JH–H = 8.3 Hz, 2H, CHCHCOH), 6.55 (d, 4JH–H = 2.2 Hz, 2H, CCHC), 4.58 (t, 4H, 3JH–H = 7.2 Hz, CCH2CH2N), 4.39 (q, 4H, 3JH–H = 7.2 Hz, CH3CH2), 3.62 (s, 4H, BnCH2), 3.16 (s, 4H, CH2CO2), 3.11 (t, 4H, 3JH–H = 7.0 Hz, CCH2CH2N), 2.60 (s, 4H, N-C2H4-N), 1.46 (s, 18H, CCH3), 1.39 (t, 6H, 3JH–H = 7.2 Hz, CH3CH2) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 170.4 (CO2t-Bu), 160.9 (CO2Et), 156.7 (COH), 140.1 (NCCH), 129.8 (CCHC), 129.3 (CHCHCO), 127.9 (CHN), 127.0 (CHCCH), 122.2 (BnCH2C), 117.1 (CHCHCO), 82.4 (CCH3), 61.4 (CH2CH3), 57.9 (BnC), 55.7 (CH2CO2), 52.4 (CCH2CH2N), 50.0 (N-C2H4-N), 35.9 (CCH2CH2N), 28.2 (CCH3), 14.5 (CH2CH3) ppm. HRMS (ESI+): m/z [M+H]+ Calcd for C42H59N8O10: 835.4349; Found: 835.4356 (100%). Elemental Analysis (analysis no. 42221): Anal. Calcd for C42H58N8O10: C, 60.42; H, 7.00; N, 13.42. Found: C, 60.20; H, 6.85; N, 13.03. Characterization data for di-tert-Butyl 2,2'-{ethane-1,2-diylbis[(2hydroxy-5-{2-[4-(hydroxymethyl)-1H-1,2,3-triazol-1yl]ethyl}benzyl)azanediyl]}diacetate (1ac): IR(ATR): 3323 (w), 2931 (w), 1725 (m), 1154 (s). 1H NMR (400 MHz, CDCl3, 25 °C):  = 7.33 (s, 2H, CHN), 6.95 (dd, 4H, 3JH–H = 8.2 Hz, 4JH–H = 2.2 Hz, CHCHCOH), 6.76 (d, 3JH–H = 8.2 Hz, 2H, CHCHCOH), 6.49 (d, 4JH–H = 2.1 Hz, 2H, CCHC), 4.72 (s, 4H, CH2OH), 4.50 (t, 4H, 3JH–H = 7.3 Hz, CCH2CH2N), 3.58 (s, 4H, BnCH2), 3.18 (s, 4H, CH2CO2), 3.10 (t, 4H, 3JH–H = 7.3 Hz, CCH2CH2N), 2.60 (s, 4H, N-C2H4-N), 1.46 (s, 18H, CCH3) ppm. 13C{1H} NMR (100 MHz, CDCl3, 25 °C):  = 170.51 (CO2t-Bu), 156.5 (COH), 147.6 (NCCH), 129.9 (CCHC), 129.4 (CHCHCO), 127.6 (CHCCH), 122.2 (CHN or BnCH2C), 122.1 (CHN or BnCH2C), 117.0 (CHCHCO), 82.5 (CCH3), 57.8 (BnC), 56.7 (CH2OH), 56.0 (CH2CO2), 52.1 (CCH2CH2N), 50.2 (N-C2H4-N), 36.1 (CCH2CH2N), 28.2 (CCH3) ppm. HRMS (ESI+): m/z [M+H]+ Calcd for C38H55N8O8: 751.4137; Found: 751.4142 (100%). Synthesis of 2,2'-[ethane-1,2-diylbis({2-hydroxy-5-[2-(4-phenyl1H-1,2,3-triazol-1-yl)ethyl]benzyl}azanediyl)]diacetic acid (2): Compound 1 (0.60 g, 0.94 mmol, 1 equiv) and phenyl acetylene (0.24 g, 2.35 mmol, 2.5 equiv) were dissolved in CH3CN (2 mL). To this mixture were added 470 µL of an aqueous Cu(OAc)2 solution (0.1 M; 47 µmol, 0.05 equiv) and 940 µL of an aqueous sodium ascorbate solution (0.1 M; 94 µmol, 0.1 equiv). After being stirred at 80 °C for 1 h, the reaction mixture was cooled to room temperature and filtered through a thick layer of silica gel (eluent: CHCl3). The solvent was



evaporated under reduced pressure and the residue was dissolved in CHCl3/trifluoroacetic acid (1 mL: 4 mL). This mixture stirred for 5 h at room temperature. Then, the volatiles were removed under reduced pressure and the crude product purified by preparative HPLC to give compound 2 as a colorless solid (0.40 g, 0.54 mmol, 58%). Note: Compound 2 could also be synthesized directly from product 1aa. IR (ATR): 3036 (w), 1613 (m), 1195 (s), 765 (s). 1H NMR (400 MHz, MeOD, 25 °C):  = 8.13 (s, 2H, CHN), 7.74 (d, 4H, 3JH–H = 7.8 Hz, Phsubstituent, ortho-H), 7.40 (t, 4H, 3JH–H = 7.8 Hz, Ph-substituent, metaH), 7.32 (t, 2H, 3JH–H = 7.8 Hz, Ph-substituent, para-H), 7.03 (dd, 2H, 3J 4 H–H = 8.3 Hz, JH–H = 1.44 Hz, CHCHCO), 6.97 (s, br, 2H, CCHC), 3 6.78 (d, 2H, JH–H = 8.3 Hz, CHCHCO), 4.63 (t, 4H, 3JH–H = 6.7 Hz CCH2CH2N), 3.99 (s, 4H, BnCH2), 3.57 (s, 4H, CH2CO2), 3.15 (t, 4H, 3J 13C{1H} H–H = 6.7 Hz, CCH2CH2N), 3.05 (s, 4H, N-C2H4-N) ppm. NMR (100 MHz, MeOD, 25 °C):  = 171.8 (CO2), 158.6 (COH), 148.6 (NCCH), 133.8 (CCHC), 132.3 (CHCHCO), 131.6 (Ph-substituent, ipso-C), 130.0 (Ph-substituent, meta-C), 129.4 (Ph-substituent, paraC), 126.6 (Ph-substituent, ortho-C), 122.4 (CHN), 120.2 (CCH2CH2N and BnCH2C), 116.9 (CHCHCO), 54.2 (BnC), 54.0 (CH2CO2), 52.9 (CCH2CH2N), 50.9 (N-C2H4-N), 36.5 (CCH2CH2N) ppm. HRMS (ESI−): m/z [M−H]− Calcd for C40H41N8O6: 729.3155; Found: 729.3152 (100%). Synthesis of sodium gallium(III){2,2'-[ethane-1,2-diylbis({2-oxido5-[2-(4-phenyl-1H-1,2,3-triazol-1yl)ethyl]benzyl}azanediyl)]diacetate} (3): Stock solution S1: In a 1 mL flask, 100 µL of an aqueous GaCl3 solution (1 M) was mixed with 550 µL of an aqueous solution of NaOAc (1 M). The pH of the Stock solution was ~4. To a solution of compound 2 (20 mg, 27 µmol, 1.0 equiv) in DMF (2 mL) was added 275 µL of fresh S1 (containing 1.6 equiv Ga3+ and 8.6 equiv NaOAc) at room temperature. The reaction mixture was stirred at 90 °C for 15 min (full conversion reached in 4 min as determined by TLC). After the elapsed time, the reaction mixture was cooled to ~50 °C and the solvent removed under reduced pressure. The residue was washed with water and then with THF, or alternatively, a dilute NaHCO3 solution, and dried in vacuo to give complex 3 (isomer II) as a colorless solid (18 mg, 22 µmol, 81%). The room temperature gallium complexation was run under the same reaction conditions, with halfscale in quantities. The reaction was quenched after 10 min by diluting with water (~50 mL). Then, the aqueous mixture was immediately centrifuged and the residue was washed several times with water. 1H NMR analysis of the crude reaction mixture revealed 49% of isomer II, and 45% of the unreacted chelator (2) together with 6% of another complex species. IR (ATR): 2919 (w), 1652 (s), 1498 (m), 1367 (m), 769 (m) cm−1. 1H NMR (400 MHz, d6-DMSO, 25 °C):  = 8.43 (s, 2H, CHN), 7.80 (d, 4H, 3JH–H = 7.5 Hz, Ph-substituent, ortho-H), 7.44 (t, 4H, 3JH–H = 7.5 Hz, Ph-substituent, meta-H), 7.32 (t, 2H, 3JH–H = 7.5 Hz, Ph-substituent, para-H), 6.86 (dd, 2H, 3JH–H = 8.3 Hz, 4JH–H = 2.0 Hz, CHCHCO), 6.66 (d, 4JH–H = 2.0 Hz, 2H, CCHC), 6.46 (dd, 3JH–H = 8.3 Hz, 2H, CHCHCO), 4.53 (m, 4H, CCH2CH2N), 4.18 (d, 2H, 2JH–H = 12.9 Hz, BnC[Ha,Hb]), 3.52 (d, 2H, 2JH–H = 12.9 Hz, BnC[Ha,Hb]), 3.20 (d, 2H, 2JH–H = 17.2 Hz, C[Ha,Hb]CO2), 3.00 (t, 4H, 3JH–H = 7.3 Hz CCH2CH2N), 2.93 (d, 2H, 2JH–H = 17.2 Hz, C[Ha,Hb]CO2), 2.81 (m, 4H, N-C2H4-N) ppm. 13C{1H} NMR (100 MHz, d6-DMSO, 25 °C):  = 171.2 (CO2), 162.4 (PhOC), 146.0 (NCCH), 130.9 (Ph-substituent, ipsoC), 130.4 (CCHC), 129.3 (CHCHCO), 128.9 (Ph-substituent, meta-C), 127.7 (Ph-substituent, para-C), 125.1 (Ph-substituent, ortho-C), 122.6 (CCH2CH2N), 121.3 (CHN), 120.3 (BnCH2C), 120.1 (CHCHCO), 60.9 (BnC), 55.9 (CH2CO2), 55.3 (N-C2H4-N), 51.3 (CCH2CH2N), 35.2 (CCH2CH2N) ppm. HRMS (ESI−): m/z [M]− Calcd for C40H38GaN8O6: 795.2176; Found: 795.2181 (100%).

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organo-met.#### Spectra and more details about the complex geometry are available in the SI.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] Notes

New address for German Litau: Faculty of Biology, Heidelberg University, INF 234, 69120 Heidelberg, Germany. The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Mrs. Jana Schmidt (technical assistant) from the German Cancer Research Center and Mrs. Petra Krämer from the IR spectroscopy service of the Organic Chemistry Institute, Heidelberg University. This work was partly funded by a grant of German Cancer Aid (Deutsche Krebshilfe); project number: 70112043.

REFERENCES (1) Velikyan, I. Molecular Imaging and Radiotherapy: Theranostics for Personalized Patient Management. Theranostics 2012, 2 (5), 424–426. https://doi.org/10.7150/thno.4428. (2) Ambrosini, V.; Fani, M.; Fanti, S.; Forrer, F.; Maecke, H. R. Radiopeptide Imaging and Therapy in Europe. J. Nucl. Med. 2011, 52, 42S–55S. https://doi.org/10.2967/jnumed.110.085753. (3) Chen, K.; Conti, P. S. Target-Specific Delivery of Peptide-Based Probes for PET Imaging. Adv. Drug Deliv. Rev. 2010, 62 (11), 1005– 1022. https://doi.org/10.1016/j.addr.2010.09.004. (4) HBED full name: N,N’-bis(2-hydroxybenzyl)ethylenediamineN,N’-diacetic acid. (5) Eder, M.; Knackmuss, S.; Le Gall, F.; Reusch, U.; Rybin, V.; Little, M.; Haberkorn, U.; Mier, W.; Eisenhut, M. 68Ga-Labelled Recombinant Antibody Variants for Immuno-PET Imaging of Solid Tumours. Eur. J. Nucl. Med. Mol. Imaging 2010, 37 (7), 1397–1407. https://doi.org/10.1007/s00259-010-1392-6. (6) Eder, M.; Schäfer, M.; Bauder-Wüst, U.; Hull, W. E.; Wängler, C.; Mier, W.; Haberkorn, U.; Eisenhut, M. 68Ga-Complex Lipophilicity and the Targeting Property of a Urea-Based PSMA Inhibitor for PET Imaging. Bioconjug. Chem. 2012, 23 (4), 688–697. https://doi.org/10.1021/bc200279b. (7) Afshar-Oromieh, A.; Zechmann, C. M.; Malcher, A.; Eder, M.; Eisenhut, M.; Linhart, H. G.; Holland-Letz, T.; Hadaschik, B. A.; Giesel, F. L.; Debus, J. J.; et al. Comparison of PET Imaging with a 68Ga-Labelled PSMA Ligand and 18F-Choline-Based PET/CT for the Diagnosis of Recurrent Prostate Cancer. Eur. J. Nucl. Med. Mol. Imaging 2014, 41 (1), 11–20. https://doi.org/10.1007/s00259-0132525-5. (8) Verburg, F. A.; Krohn, T.; Heinzel, A.; Mottaghy, F. M.; Behrendt, F. F. First Evidence of PSMA Expression in Differentiated Thyroid Cancer Using [68Ga] PSMA-HBED-CC PET/CT. Eur. J. Nucl. Med. Mol. Imaging 2015, 42 (10), 1622–1623. https://doi.org/10.1007/s00259-015-3065-y. (9) Rauscher, I.; Maurer, T.; Beer, A. J.; Graner, F.-P.; Haller, B.; Weirich, G.; Doherty, A.; Gschwend, J. E.; Schwaiger, M.; Eiber, M. Value of 68Ga-PSMA HBED-CC PET for the Assessment of Lymph Node Metastases in Prostate Cancer Patients with Biochemical Recurrence: Comparison with Histopathology After Salvage Lymphadenectomy. J. Nucl. Med. 2016, 57 (11), 1713–1719. https://doi.org/10.2967/jnumed.116.173492. (10) Haberkorn, U.; Eder, M.; Kopka, K.; Babich, J. W.; Eisenhut, M. New Strategies in Prostate Cancer: Prostate-Specific Membrane Antigen (PSMA) Ligands for Diagnosis and Therapy. Clin. Cancer Res. 2016, 22 (1), 9–15. https://doi.org/10.1158/1078-0432.CCR-150820. (11) Pyka, T.; Weirich, G.; Einspieler, I.; Maurer, T.; Theisen, J.; Hatzichristodoulou, G.; Schwamborn, K.; Schwaiger, M.; Eiber, M.


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The Journal of Organic Chemistry 68Ga-PSMA-HBED-CC

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The Journal of Organic Chemistry


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HBED-NN: A Bifunctional Chelator for Constructing

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