
==== Front
Bioconjug Chem
Bioconjug Chem
bc
bcches
Bioconjugate Chemistry
1043-1802
1520-4812
American Chemical Society

39185789
10.1021/acs.bioconjchem.4c00313
Article
Cyclohexanediamine Triazole (CHDT) Functionalization Enables Labeling of Target Molecules with Al18F/68Ga/111In
https://orcid.org/0000-0002-2876-9925
Sihver Wiebke *†
https://orcid.org/0000-0002-0474-8492
Walther Martin †#
Ullrich Martin †#
Nitt-Weber Anne-Kathrin †
Böhme Jenny †
Reissig Falco †
Saager Magdalena †
https://orcid.org/0000-0002-7571-4732
Zarschler Kristof †
Neuber Christin †
Steinbach Jörg †
Kopka Klaus †‡
Pietzsch Hans-Jürgen †‡
https://orcid.org/0000-0001-7462-7111
Wodtke Robert †
https://orcid.org/0000-0002-1610-1493
Pietzsch Jens †‡
† Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstraße 400, 01328 Dresden, Germany
‡ Technische Universität Dresden, School of Science, Faculty of Chemistry and Food Chemistry, Mommsenstraße 4, 01069 Dresden, Germany
* E-mail: w.sihver@hzdr.de.
26 08 2024
18 09 2024
35 9 14021416
08 07 2024
15 07 2024
11 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by/4.0/ Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).

The Al18F-labeling approach offers a one-step access to radiofluorinated biomolecules by mimicking the labeling process for radiometals. Although these labeling conditions are considered to be mild compared to classic radiofluorinations, improvements of the chelating units have led to the discovery of (±)-H3RESCA, which allows Al18F-labeling already at ambient temperature. While the suitability of (±)-H3RESCA for functionalization and radiofluorination of proteins is well established, its use for small molecules or peptides is less explored. Herein, we advanced this acyclic pentadentate ligand by introducing an alkyne moiety for the late-stage functionalization of biomolecules via click chemistry. We show that in addition to Al18F-labeling, the cyclohexanediamine triazole (CHDT) moiety allows stable complexation of 68Ga and 111In. Three novel CHDT-functionalized PSMA inhibitors were synthesized and their Al18F-, 68Ga-, and 111In-labeled analogs were subjected to a detailed in vitro radiopharmacological characterization. Stability studies in vitro in human serum revealed among others a high kinetic inertness of all radiometal complexes. Furthermore, the Al18F-labeled PSMA ligands were characterized for their biodistribution in a LNCaP derived tumor xenograft mouse model by PET imaging. One radioligand, Al[18F]F-CHDT-PSMA-1, bearing a small azidoacetyl linker at the glutamate-urea-lysine motif, provided an in vivo performance comparable to that of [18F]PSMA-1007 but with even higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. Overall, our results highlight the suitability of the novel CHDT moiety for functionalization and radiolabeling of small molecules or peptides with Al18F, 68Ga, and 111In and the triazole ring seems to entail favorable pharmacokinetic properties for molecular imaging purposes.

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pmcIntroduction

Noninvasive imaging by positron emission tomography (PET) and single photon emission computed tomography (SPECT) has become an essential technique in nuclear medicine for the diagnosis of pathological conditions such as neurodegenerative diseases,1,2 cardiac diseases,3 inflammatory or infectious diseases,4 and cancer.5 For PET applications, a suitable vector molecule (small molecule, peptidomimetic, peptide, or protein) that targets the respective process of interest, e.g., a ligand that binds to a cell surface receptor, is equipped with a positron (β+) emitting radionuclide. Although a broad range of such radionuclides with suitable nuclear properties for PET imaging exists and their production is established,6 fluorine-18 is still one of the most frequently applied radionuclides. This originates from its sufficiently long half-life (109.8 min) that enables even multistep radiosyntheses and facilitates the transport of the final radiopharmaceutical to distant application sites within a satellite concept. Furthermore, the high percentage of β+ emission (97%) and the low positron energy (Emax = 0.635 MeV) favors the quality of the PET images.7,8

Driven by its almost ideal nuclear properties for PET imaging, the (radio)chemical toolbox for the introduction of fluorine-18 has expanded tremendously allowing the radiofluorination of basically all kinds of target molecules from small molecules to proteins.9 Generally, 18F-labeled radiotracers are accessed via direct labeling of their respective precursors10 or via indirect labeling using initially prepared 18F-labeled building blocks (prosthetic groups).8 In case of [18F]fluoride as 18F-species, direct labeling usually requires the workup of the crude aqueous solution via anion exchange and subsequent drying to provide reactive [18F]fluoride for efficient labeling reactions.11 Subsequent 18F-labeling is often performed in water-free organic solvents and at high temperatures. However, such conditions would usually not maintain the structural integrity of peptides and proteins. Furthermore, the structural complexity of these molecules bearing several functional groups lowers the labeling efficiency.12 Therefore, to enable the efficient direct labeling of such complex biomolecules, labeling strategies that rely on the formation of heteroatom- and metal–[18F]fluoride bond formation have been developed. In contrast to C–F bond formation, the introduction of [18F]fluoride is possible even under mild conditions and in the presence of water.12,13

Of these alternative 18F-labeling strategies, the Al18F-labeling approach appears particularly appealing.14,15 Based on the strong binding of fluoride to several metals, McBride et al.16 discovered the successful complexation of (Al[18F]F)2+ by common chelators including DTPA, NOTA, and NODA. In terms of complexation efficiency and complex stability, the cyclic pentadentate ligand NODA provided the best results due to the favorable N3O2 donor set, which leaves a sixth coordination site at the aluminum open for fluoride.16−19 Similar to labeling reactions with radiometals, the Al18F-labeling approach offers the opportunity for radiofluorination of target molecules via lyophilized kits.20 However, efficient Al18F-labeling usually entails temperatures of 100 °C, which is comparable to the conditions often applied for 68Ga- or 64Cu-labeling. Such high temperatures are tolerated by small peptides but certainly not by proteins.12

Aiming at Al18F-labeling at lower temperatures, Cleeren et al.21 developed acyclic pentadentate ligands with a N2O3 donor set. Of these new ligands, H3L3 (Figure 1) provided an acceptable stability of the Al18F-complex in vitro and bone uptake of Al[18F]F-L3 and a PSMA ligand conjugate was low at 60 min p.i. Later, the same group introduced a cyclohexane moiety in their ligand scaffold, resulting in the ligand called (±)-H3RESCA or rac-H3RESCA (REStrained Complexing Agent, Figure 1) which enabled Al18F-labeling at very mild conditions (pH 4.6, ambient temperature) in a short period of time (12 min).22 Moreover, the stability of the Al[18F]F-RESCA-complex, in particular with regards to defluorination, is sufficient for in vivo applications. The introduction into biomolecules is realized by an TFP or maleimide functionalized (±)-H3RESCA,23 which are commercially available. However, so far only few examples of biomolecules that were labeled by the Al18F-RESCA-method are reported, including human serum albumin, a nanobody against the Kupffer cell marker CIRg, affibodies against HER2, and a peptidic uPAR ligand.22,24−26

Figure 1 Structures of previously reported H3L3 and (±)-H3RESCA and the novel (±)-CHDA-tBu for generating CHDT-functionalized molecules.

Inspired by the general suitability of the Al18F-RESCA-method, we envisaged a complementary approach for the introduction of RESCA-like complexing agents into biomolecules. For this purpose, we sought to structurally modify (±)-H3RESCA by replacing the 2-(p-tolyl))acetic acid moiety by a propargyl moiety, which would transform into a 1,4-disubstituted-1,2,3-triazole ring upon coupling to azide-functionalized biomolecules via CuAAC (copper(I)-catalyzed azide–alkyne cycloaddition, Figure 1). Apart from this alternative functionalization strategy, we hypothesized that the triazole ring could participiate in coordination to metal ions (resulting in a N3O3 donor set), which could support thus the efficient and stable complexation of other metal ions in addition to Al3+. 1,2,3-Triazoles are known to participate as ligands in metal complexes including complexes of rhenium, technetium, copper, zinc, and platinum,27−30 but there are also occasional reports on coordination to aluminum31 and indium.32 To the best of our knowledge, studies on complexation of other metal ions, in particular radiometals for PET and SPECT imaging, by (±)-H3RESCA have not been reported so far. Furthermore, the 1,2,3-triazole ring entails an increased hydrophilicity compared to the benzyl ring in (±)-H3RESCA which might positively affect the pharmacokinetics of respective conjugates.33

Herein, we present the synthesis of the novel cyclohexanediamine alkyne (±)-CHDA-tBu (CycloHexaneDiamine Alkyne) in which the three acetic acid moieties are tBu-protected to allow an efficient CuAAC with azide-functionalized biomolecules. (±)-CHDA-tBu was then coupled to two simple model azides (5-azido pentanoic acid and benzyl azide) to generate the CHDT (CycloHexaneDiamine Triazole)-functionalized molecules CHDT-Pe and CHDT-Bn. Al18F-labeling but also labeling with radiometals such as 68Ga3+ and 64Cu2+ was tested and the Al18F-labeled compounds were subjected to ex vivo biodistribution studies after injection into healthy mice. Subsequently, (±)-CHDA-tBu was used to prepare three novel CHDT-functionalized PSMA ligands (CHDT-PSMA-1/2/3, Scheme 1). PSMA (prostate specific membrane antigen) is a type II transmembrane glycoprotein with cocatalytic metallopeptidase activity34 and emerged as attractive target for the radionuclide diagnosis and treatment of prostate cancer,35,36 owing to its high abundance in this type of cancer.37,38 Although numerous radiolabeled PSMA ligands were developed in the past and some compounds are already in clinical use, there is still an interest in novel radioligands, in particular 18F-labeled ligands.39CHDT-PSMA-1/2/3 were labeled with (Al18F)2+ but also 68Ga3+ and 111In3+ to demonstrate the potential of the CHDT moiety for complexing other metal ions in addition to Al3+. The respective nine radioligands were radiopharmacologically characterized in vitro with a focus on serum stability, saturation binding analyses and internalization behavior using monolayers and spheroids of PSMA-positive LNCaP cells. Moreover, the in vivo radiopharmacological characterization of Al18F-labeled CHDT-PSMA-1/2/3 was performed in LNCaP-tumor bearing mice in comparison to the well-known PSMA ligand [18F]PSMA-1007.

Scheme 1 Synthesis of (±)-CHDA-tBu (A) and of the CHDT-Functionalized Compounds (B)

Reagents and conditions: (a) benzaldehyde, NaBH4, CH3OH, 12 h, Ar; (b) tert-butyl bromoacetate, DIPEA, CH2Cl2, 12 h; (c) Pd/C, ammonium formate, 2.5 h, 65 °C; (d) propargyl bromide, triethyl amine, CH3CN, 12 h; (e) R′-N3 (benzyl azide/5-azidopentanoic acid/4–6), CuSO4, sodium ascorbate, tert-butanol/water, 24 h; (f) TFA/CH2Cl2 (9:1, v/v), 12 h.

Results and Discussion

Synthesis of (±)-CHDA-tBu and the CHDT-Functionalized Compounds

The synthesis of (±)-CHDA-tBu was accomplished in four steps starting with racemic trans-1,2-diaminocyclohexane in orientation to the procedure for the preparation of (±)-H3RESCA-TFP or (±)-H3RESCA-Mal.40,41 The first step comprised the monobenzylation by the use of benzaldehyde and sodium borohydride. This was followed by alkylation with tert-butyl bromoacetate under basic conditions (DIPEA). Subsequently, the benzyl group was removed by hydrogenolysis using Pd/C and ammonium formate. For the last step to (±)-CHDA-tBu, the propargyl group was introduced by reaction with propargyl bromide and triethyl amine. All steps proceeded in good to excellent yields and (±)-CHDA-tBu was obtained in an overall yield of 41%. The tert-butyl groups had to be kept for the introduction into biomolecules to enable an efficient coupling via CuAAC as the unprotected CHDT moiety is also able to complex Cu2+ (see below).

For model compounds CHDT-Pe and CHDT-Bn, 5-azidopentaoic acid and benzyl azide, respectively, were coupled via CuAAC. Subsequent removal of the tert-butyl groups by TFA treatment afforded both compounds in good yields of 46% and 81%, respectively, over these two steps. A similar procedure was followed for the synthesis of the PSMA ligands CHDT-PSMA-1/2/3, for which the respective tert-butyl protected and azide-functionalized precursor compounds (4–6) were synthesized (see Chemistry section in Supporting Information). CHDT-PSMA-1/2/3 were obtained in overall yields of 22–65% for the coupling and deprotection steps. Regarding the linker entities between the glutamyl-urea-lysine (KuE) PSMA binding motif and the CHDT moiety, CHDT-PSMA-1 was designed to bear the smallest possible azide linker (apart from the use of ε-azido-norleucine). In contrast, CHDT-PSMA-3 was designed in orientation to PSMA-617 (l-2NaI and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCH)) by substituting the DOTA chelator with the CHDT moiety and a hexanoyl linker. For CHDT-PSMA-2, d-1NaI was chosen instead of l-2NaI and AMCH was omitted. Benesova et al.42 previously demonstrated that l-2NaI is better tolerated than l-1NaI due to a more favorable orientation within the binding pocket of PSMA. Moreover, l-2NaI is superior to d-2NaI. However, d-1NaI was not tested, which prompted us to test this particular amino acid in the context of the present study.

Complexation of (Al[18F]F)2+, [68Ga]Ga3+, [64Cu]Cu2+, and [111In]In3+

Initially, CHDT-Pe and CHDT-Bn were used to test the complexation of (Al[18F]F)2+. The complexation under acidic conditions (pH 5.0) at precursor amounts of 50 μg (72 nmol) and a temperature between 25 and 40 °C proceeded with high radiochemical conversions (RCC) of >80% (Figure S1 in Supporting Information). Subsequently, we also envisaged complexation of [68Ga]Ga3+ and [64Cu]Cu2+ by the CHDT moiety. To our delight, complexation of these two radiometals was indeed possible (shown for CHDT-Pe in Figure S1) under similar conditions as applied for Al18F-labeling. This could indicate that the triazole ring participates in binding of these radiometal species as hypothesized by us. However, it is not excluded that (±)-H3RESCA is also able to complex other radiometal species but such data are not reported so far. Motivated by the initial radiolabeling experiments with CHDT-Pe and CHDT-Bn, radiolabeling of the novel CHDT-functionalized PSMA ligands, CHDT-PSMA-1/2/3, was performed. In addition to (Al[18F]F)2+, [68Ga]Ga3+, and [64Cu]Cu2+, we also tried the complexation of [111In]In3+. Complexation at 40 °C and a precursor amount of 10 μg (7.5, 8.3, and 10.5 nmol for CHDT-PSMA-1/2/3, respectively) resulted in high radiochemical conversions (>95%) for all radiometal species (Figure S2 in Supporting Information). Overall, we demonstrated that the transformation of the original (±)-H3RESCA ligand to the novel CHDT moiety still allows Al18F-labeling, but also labeling with [68Ga]Ga3+, [64Cu]Cu2+, and [111In]In3+.

While the 68Ga/64Cu/111In-labeled compounds were not further processed for their radiopharmacological characterization, residual unbound 18F-species were removed from the reaction mixture by the addition of hydroxyapatite. It is worth noting, that the treatment with hydroxyapatite should be conducted rather short (30 s) as it not only binds unbound 18F-species but leads also to decomplexation of the Al18F-complex, which ultimately lowers the radiochemical yield and apparent molar activity. Alternatively, unbound 18F-species could also be removed by solid-phase extraction using suitable cartridges. This processing would also improve radiochemical yield and molar activity values. However, herein we decided to follow the hydroxyapatite treatment to ensure [18F]fluoride (and Al18F species)-free radioligand formulations. For the radiolabeled CHDT-PSMA-1/2/3, radiochemical purities of >95% (Figure S3 in Supporting Information) and apparent molar activities of 10 (±1, Al18F), 24 (±3, 68Ga), and 24 (±5, 111In) MBq/nmol, respectively (mean ± SD values over all compounds), were achieved.

For comparative in vitro and in vivo studies, [18F]PSMA-1007 (in-house prepared according to GMP and AMG) was used (RCP > 99%, Figure S3). Furthermore, as radioligand for the subsequent competition assay, [177Lu]Lu-PSMA-617 was prepared under standard conditions (pH 5.0, 90 °C, 20 min, RCY > 99%, Figure S3).

Distribution Coefficients (logD7.4) and Stability Studies

The distribution coefficients logD7.4 of the Al18F-, 68Ga-, and 111In-labeled PSMA conjugates were determined and are summarized in Table 1. Among the three PSMA conjugates, radiolabeled CHDT-PSMA-1 is the most hydrophilic compound, irrespective of the radionuclide, which appears reasonable due to the short azido alkyl linker and the absence of a naphthylalanine residue compared to the other two compounds.

Table 1 Summary of logD7.4 Values and Serum Stability of the Radiolabeled PSMA Ligandsa

 	 	Conjugates	
 	Radiolabel	CHDT-PSMA-1	CHDT-PSMA-2	CHDT-PSMA-3	
logD7.4	Al18F	–3.60 ± 0.14	–3.34 ± 0.09	–3.43 ± 0.13	
68Ga	–3.36 ± 0.04	–3.11 ± 0.06	–3.09 ± 0.10	
111In	–4.23 ± 0.08	–3.44 ± 0.07	–3.87 ± 0.07	
Percentage of intact radioligand after 3 h in human serum	Al18F	98.9 ± 0.1	99.1 ± 0.2	99.3 ± 0.1	
68Ga	96.6 ± 2.2#	97.6 ± 0.1	99.1 ± 0.4	
111In	99.5 ± 0.1	98.8 ± 0.4	99.2 ± 0.2	
*	99.6 ± 0.1	98.4 ± 0.4	99.1 ± 0.2	
**	99.3 ± 0.2	98.7 ± 0.7	99.4 ± 0.4	
a Data shown are mean values (±SEM) of three experiments (#two experiments). Each experiment was performed in triplicate. Percentage of intact 111In-labeled conjugates after 24 h (*) and after 48 h (**). Percentage of intact radioligand was assessed by radio-TLC using iTLC-SG strips as stationary phase and 2 M ammonium acetate/methanol (1:1, v/v) as eluent. The data of the individual experiments are listed in Table S1 in Supporting Information.

Stability studies of the Al18F-, 68Ga-, and 111In-labeled PSMA conjugates were conducted in human serum and the percentages of intact radioligand are summarized in Table 1. Analysis of serum samples was performed by radio-TLC and radio-HPLC to correctly assess disintegration of the radiolabeled molecules caused by released radiometal species and metabolization. All compounds showed an excellent stability over 3 h with values for residual intact radioligand >96%. Moreover, the 111In-labeled compounds appeared also to be stable even after a prolonged incubation period of 24 and 48 h (Table 1). These results illustrate that the novel CHDT moiety provides a high kinetic inertness of the respective Al18F-, 68Ga-, and 111In-complexes under physiological conditions in vitro.

Although [64Cu]Cu-complexation by the CHDT moiety is also possible, the kinetic inertness of the resulting complex appears rather low. This was exemplarily demonstrated for [64Cu]Cu-CHDT-PSMA-1 in a protein challenge experiments using human serum and analysis by radio-SDS-PAGE (Figure S4 in Supporting Information). There was a significant transchelation of [64Cu]Cu2+ to albumin (20%), while other copper chelators such as TETA, DOTA, NOTA, cyclam, or diamSar show only minimal 64Cu-transchelation under the same conditions.43 In contrast, for [68Ga]Ga-CHDT-PSMA-1 under similar conditions no transchelation of [68Ga]Ga3+ in human serum to transferrin, which has a high affinity for Ga3+,44 was observed (Figure S5 in Supporting Information), which highlights the higher kinetic inertness of the 68Ga-complex compared to the 64Cu-complex. In this context, Ga3+ and In3+ can be classified as hard acidic cations according to the Pearson’s hard–soft acid–base theory and favor hard donor atoms (e.g., anionic oxygen), while Cu2+ is rather a borderline acid and favors soft donor atoms (e.g., nitrogen).45 Consequently, it appears comprehensible that the acyclic CHDT moiety with N2O3 donor set (without triazole ring) is sufficient for stable complexation of Ga3+ and In3+. In contrast, although the 1,2,3-triazole was intended to provide an additional nitrogen donor for metal complexation, this coordination (if coordination occurs at all) is not sufficient for stably complexing Cu2+. It is worth noting that previously a DTPA analog bearing a trans-1,2-diaminocyclohexane backbone (CHX-A″) has been developed for complexation of 212Bi,46,47 but this chelator is also able to complex a series of other radiometals including 177Lu, 86Y, 68Ga, and 111In.45,48,49 Furthermore, an EDTA analog bearing a trans-1,2-diaminocyclohexane backbone (4-ICE) allows stable complexation of radiometals, such as 111In, 57Co, and 47Sc, but produced a less stable complexation of 67Cu as seen by a higher activity uptake in the liver of a respective antibody conjugate compared to conjugates bearing other chelators.50 In this context, it is known that a strong binding of copper ions is favored by macrocyclic ligands due to the so-called macrocyclic effect,51 which adds to the above-mentioned HSAB concept for explaining the low kinetic inertness of the [64Cu]Cu-CHDT complex.

Characterization of PSMA Binding

Motivated by the stability results for Al18F, 68Ga, and 111In-labeled CHDT-PSMA-1/2/3, we envisaged their further radiopharmacological characterization. First, the binding affinities of the novel compounds and three reference ligands (PSMA-1007, PSMA-617, and KuE (lysine-urea-glutamate, see Chemistry section in Supporting Information)) to PSMA were determined in a competition binding assay using LNCaP cell homogenates and [177Lu]Lu-PSMA-617 as radioligand (Figure 2 and Table 2). In this context, LNCaP cells are known for their high amount of PSMA,52 which was also herein confirmed by Western blotting (Figure S6 in Supporting Information). While PSMA-1007, PSMA-617, and CHDT-PSMA-3 show binding affinities by means of Ki values in the single-digit nanomolar range, CHDT-PSMA-1/2 exhibit significantly lower affinities (49 and 115 nM). The comparable binding affinity of CHDT-PSMA-3 and PSMA-617 appears reasonable due to the high structure similarity, in particular the l-2-NaI and AMCH moieties. Similarly, the lower binding affinity of CHDT-PSMA-2 can be rationalized due to the unfavorable orientation of the naphthyl moiety in 1-NaI within the PSMA binding site,42 which is obviously independent of the configuration (l or d) of the 1-naphthylalanine residue. CHDT-PSMA-1 exhibits an even lower binding affinity than CHDT-PSMA-2 indicating that the attachment of the CHDT moiety close to the KuE binding motif is detrimental to the binding to PSMA. In this context, although other PSMA ligands with a chelator moiety close to the KuE binding motif and a high binding affinity are known, e.g., PSMA-11 (6-aminohexanoyl linker between KuE and HBED-CC), the binding affinity seems to depend on the identity of the chelator moiety (Ki values of 12 and 37.6 nM for PSMA-11 and its DOTA analog, respectively).53 The lowest binding affinity was observed for the unmodified lysine-urea-motif (KuE), which agrees with the original report for this PSMA binding motif by Maresca et al.54

Figure 2 Competition binding of CHDT-PSMA-1/2/3 compared to known PSMA ligands using LNCaP cell homogenates [177Lu]Lu-PSMA-617 was used as radioligand (12 nM). Data shown are mean values (±SEM) of 2–8 separate experiments (see Experimental Section for details and Table S2 in the Supporting Information for the corresponding data), each performed in triplicate or quadruplicate.

Table 2 Ki Values of CHDT-PSMA-1/2/3 Compared to Known PSMA Ligands Determined from Competition Binding Assaysa

 	Ki (nM)	
CHDT-PSMA-1	115 (13)	
CHDT-PSMA-2	49 (8)	
CHDT-PSMA-3	8.1 (1.9)	
PSMA-617	4.2 (0.4)	
PSMA-1007	1.7 (0.3)	
KuE	11,300 (2,900)	
a Data shown are mean values (±SEM) from 2 to 8 separate experiments (see Table S3 in the Supporting Information for the corresponding data), each performed in triplicate or quadruplicate using LNCaP cell homogenates.

In addition to assessing the binding affinities for nonlabeled CHDT-PSMA-1/2/3, we sought to characterize the saturation binding of the Al18F-, 68Ga-, and 111In-labeled compounds using LNCaP cells cultured in monolayers and as spheroids. The saturation binding curves for Al[18F]F-CHDT-PSMA-1/2/3 are depicted in Figure 3, while the graphs for [18F]PSMA-1007 and 68Ga- and 111In-labeled CHDT-PSMA-1/2/3 can be found in Figures S7–S9 in the Supporting Information. The binding parameters (Kd and Bmax) for Al[18F]F-CHDT-PSMA-1/2/3 and [18F]PSMA-1007 are summarized in Table 3 (for the 68Ga- and 111In-labeled compounds see Tables S4 and S5 in the Supporting Information). For the saturation binding assays, we recorded surface-bound radioligand (named “binding”), which refers to bound radioligand that can be removed by acid wash, and internalized or acid-resistant radioligand (named “internalization”), which refers to radioligand that remains cell-bound after acid wash. Consequently, two saturation binding plots were obtained and two sets of binding parameters (Kd/Bmax and Kd,int/Bmax,int) were derived.

Figure 3 Exemplary saturation binding curves for Al[18F]F-CHDT-PSMA-1/2/3 toward LNCaP cells extracellular saturation binding (“binding”) of Al[18F]F-CHDT-PSMA-1/2/3 toward intact LNCaP cells as monolayer (A1/B1/C1) and LNCaP spheroids (A3/B3/C3) and intracellular saturation binding (“internalization”) toward intact LNCaP cells (A2/B2/C2) and LNCaP spheroids (A4/B4/C4). Data for total (gray triangles), nonspecific (open triangles, in the presence of 800 μM KuE) and specific binding (Al[18F]F-CHDT-PSMA-1 in red, Al[18F]F-CHDT-PSMA-2 in green, and Al[18F]F-CHDT-PSMA-3 in blue triangles) are shown as mean values (±SD) of one representative experiment, which was performed in duplicate (nonspecific binding) or triplicate (total binding). The corresponding data for these graphs are listed in Table S6 in the Supporting Information.

Table 3 Parameters for Extracellular and Intracellular Saturation Binding of Al[18F]F-CHDT-PSMA-1/2/3 and [18F]PSMA-1007a

 	 	Binding	Internalization	
Conjugate	LNCaP	Kd (nM)	Bmax (pmol/mg)	Kd,int (nM)	Bmax,int (pmol/mg)	
CHDT-PSMA-1	M	26.4 ± 11.0	1.87 ± 0.57	18.4 ± 7.3	1.28 ± 0.96	
S	41.5 ± 26.4	1.49 ± 0.36	37.4 ± 10.7	0.49 ± 0.22	
CHDT-PSMA-2	M	12.9 ± 4.0	1.33 ± 0.10	14.4 ± 4.8	0.79 ± 0.16	
S	15.8 ± 4.4	1.07 ± 0.13	15.7 ± 0.8	0.33 ± 0.15	
CHDT-PSMA-3	M	19.7 ± 8.4	2.18 ± 0.97	14.4 ± 7.3	1.34 ± 0.32	
S	38.6 ± 25.8	0.90 ± 0.47	36.4 ± 14.6	0.86 ± 0.34	
PSMA-1007	M	5.4 ± 3.6	1.11 ± 0.06	10.1 ± 5.9	1.02 ± 0.21	
S	12.9 ± 1.2	0.86 ± 0.57	25.8 ± 18.6	0.67 ± 0.32	
a Data shown are mean values (±SEM) of 2–4 separate experiments (see Table S7 in the Supporting Information for the corresponding data). n.d. denotes not determined, M denotes monolayer, and S denotes spheroids.

Although the binding affinities of nonlabeled CHDT-PSMA-1/2 were almost 1 order of magnitude lower than that of CHDT-PSMA-3, the Kd values (cell surface binding) of the radiolabeled analogs, independent of the particular radiolabel (Al18F, 68Ga, 111In), were comparable (factor < 4). For example, the Kd values for the Al18F-labeled compounds were 26.4, 12.9, and 19.7 nM, respectively, using LNCaP cells as monolayer. This might indicate in case of CHDT-PSMA-1/2 that the CHDT moiety as complex with one of the above-mentioned radiometal species is better tolerated than the nonlabeled moiety. The most obvious difference after complexation is the overall charge of this moiety, with three negative charges due to the carboxyl groups being present in the nonlabeled state compared to an overall charge of −1 (Al18F) or ±0 (68Ga, 111In) after complexation. The longer linker entities between KuE binding motif and the CHDT moiety in CHDT-PSMA-3 compared to the other two analogs might separate the CHDT moiety too far away from the PSMA binding site to exert any interactions with PSMA. Consequently, the complexation state of CHDT is not as crucial for the binding affinity as for the other two compounds. While there is no clear trend for the Kd values in dependence on the particular radiolabel, the Bmax values were consistently higher by a factor of 2–3 for the Al18F-labeled compounds (1.33–2.18 pmol/mg) compared to the 68Ga- and 111In-labeled analogs (0.40–0.79 pmol/mg). In this context, [18F]PSMA-1007 was also characterized in saturation binding experiments, which revealed a lower Kd value (5.4 nM) but a comparable Bmax value (1.11 pmol/mg) compared to the Al18F-labeled CHDT-PSMA-1/2/3.

The results for the cell surface binding of the radioligands are largely conserved when analyzing the saturation binding data for the internalized fraction. This demonstrates that internalization is also concentration-dependent and that a constant fraction of around 50% of total-bound radioligand is internalized after 2 h (as Bmax and Bmax,int values are comparable). In contrast to the pronounced binding of all radioligands to LNCaP cells, binding to PSMA-negative PC3 cells was negligible (exemplarily shown for Al[18F]F-CHDT-PSMA-1 in Figure S10 in Supporting Information), which further confirms their PSMA-specificity.

The results obtained for saturation binding using LNCaP cells cultured in monolayers are also conserved when using LNCaP spheroids. However, the Kd values were consistently higher and Bmax values lower when comparing the data sets obtained for monolayer and spheroids. When the cells form a spheroid, the environment of the individual cells changes. An extracellular matrix is formed using adhesion molecules, and alterations regarding cell-to-cell communication and interaction, the orientation of cell structures, the ability of proliferation, aggregation, differentiation as well as the stiffness, oxygen, nutrient, and metabolic gradient occur.55−57 For LNCaP cells, abundance of PSMA was previously shown to be conserved in spheroids compared to monolayers.58 Therefore, regarding the somewhat reduced binding capacity toward spheroids observed herein originates most likely from a limited permeation potency of the radioligands into the spheroids and thus, the apparent amount of binding sites is reduced.

In addition to the saturation binding analyses of cell surface-bound and internalized radioligand, the time-dependent internalization of the radiolabeled CHDT-PSMA-1/2/3 was investigated (10 min, 1 h, and 2 h). Specific internalization of the Al18F-labeled CHDT-PSMA-1/2/3 and [18F]PSMA-1007 are shown in Figure 4 (the data for the 68Ga- and 111In-labeled compounds are shown in Figure S11 in the Supporting Information). Internalization increased for all radiolabeled compounds over time with the highest values among the novel PSMA ligands obtained for CHDT-PSMA-3, irrespective of the particular radiolabel. The internalization of Al[18F]F-CHDT-PSMA-3 was even comparable to that of [18F]PSMA-1007. In contrast, radiolabeled CHDT-PSMA-1/2 exhibited a lower internalization. In agreement with the aforementioned high Bmax values for the Al18F-labeled compounds, internalization was consistently higher for the Al18F-labeled CHDT-PSMA-1/2/3 compared to their 68Ga- and 111In-labeled counterparts.

Figure 4 Internalization behavior of the Al18F-labeled CHDT-PSMA-1/2/3 (A–C) and [18F]PSMA-1007 (D) Time-depending internalization using LNCaP cells as monolayer with specific internalization and specific internalization in the presence of MβCD (3 mM). Data shown are mean values (±SEM) of 3 or 7 separate experiments (see Table S8 in the Supporting Information for the corresponding data), each performed in triplicate or quadruplicate. Same color coding as in Figure 3.

Regarding the mechanism of ligand induced PSMA internalization, it is known that this process occurs mainly via clathrin-coated pits.59−61 Previously, Matthias et al.62 demonstrated for fluorophore-labeled PSMA inhibitors and by using stimulated emission depletion (STED) nanoscopy that PSMA internalization upon inhibitor binding proceeds also via clathrin-dependent endocytosis. Herein, we also characterized the time-dependent internalization of the radiolabeled compounds in the presence of the methylated cyclic oligosaccharide methyl-β-cyclodextrin (MβCD),63,64 a known endocytosis inhibitor. Treatment with MβCD was associated with a significantly reduction in internalization for all PSMA radioligands examined herein, which indicates that the novel radioligands induce upon binding PSMA internalization via endocytosis.

Ex Vivo Biodistribution and PET Imaging

Due to the original use of (±)-H3RESCA for complexation of (Al[18F]F)2+, we sought to subject the Al18F-labeled compounds developed herein bearing the novel CHDT moiety to a detailed in vivo radiopharmacological characterization. First, the biodistribution of the two radiolabeled model compounds, Al[18F]F-CHDT-Pe and Al[18F]F-CHDT-Bn, was studied at 60 and 240 min p.i. in healthy SKH1 mice to primarily assess the activity uptake in bone tissue and thus to get information about the in vivo stability of the Al[18F]F-CHDT complex (Tables S9 and S10 in the Supporting Information). While Al[18F]F-CHDT-Pe was mainly excreted into the urine (79% ID in urine at 60 min p.i.), the benzyl analog was predominantly excreted via the hepatobiliary route (51% ID in intestine at 60 min p.i.). This different in vivo behavior is reasonable considering the structures of both compounds with the aliphatic carboxylic acid moiety likely mediating the renal excretion pathway of Al[18F]F-CHDT-Pe. Activity uptake in bone tissue was assessed on the basis of the femur. Values of around 0.4%ID/g were measured, which did not increase up to 240 min p.i. This suggests a sufficient stability of Al[18F]F-CHDT complex in vivo regarding the release of [18F]F– or related species that would accumulate permanently in the bone tissue.

We furthermore investigated the biodistribution and suitability for targeting tumor-associated PSMA of Al18F-labeled CHDT-PSMA-1/2/3 by PET imaging up to 120 min p.i. For this purpose, the PSMA-positive LNCaP tumor xenograft was used. Representative PET images at selected time points p.i. are shown in Figure 5, time-activity curves and total tissue uptakes based on PET images are illustrated in Figure 6. For comparison, [18F]PSMA-1007 was also examined in this tumor model. For all radiofluorinated PSMA ligands, the LNCaP tumor was clearly visible and PSMA-specific uptake was confirmed by coadministration of 2-(phosphonomethyl)pentanedioic acid) (2-PMPA), which significantly reduced the tumor uptake (Figure 6C). Total tumor uptake was comparable for Al[18F]F-CHDT-PSMA-1/3 and [18F]PSMA-1007, while uptake of Al[18F]F-CHDT-PSMA-2 was significantly lower (Figure 6A and C). Considering rather comparable binding affinities of all novel Al18F-labeled PSMA ligands (Table 3), the inferior tumor targeting capability of Al[18F]F-CHDT-PSMA-2 might primarily be a consequence of its altered pharmacokinetic behavior. [18F]PSMA-1007 and Al[18F]F-CHDT-PSMA-1/3 were mainly renally excreted, while Al[18F]F-CHDT-PSMA-2 exhibited a higher uptake in the organs associated with hepatobiliary excretion compared to kidneys and bladder (Figure 6B and D). Apparently, the presence of the naphthylalanine residue shifts the excretion route to hepatobiliary excretion, which is partly compensated for Al[18F]F-CHDT-PSMA-3 by the AMCH moiety. Although total uptake in bone tissue was low for all compounds (Figure 6C), the average SUVmean values at 120 min p.i. appeared to be slightly higher for the CHDT-functionalized PSMA ligands (0.30–0.51) compared to [18F]PSMA-1007 (0.21, Figure 6A). This is in agreement with the results from ex vivo biodistribution studies of the two radiofluorinated model compounds and further supports our view that the Al[18F]F-CHDT complex is sufficiently stable in vivo.

Figure 5 PET images of LNCaP xenograft mice after injection of Al18F-labeled CHDT-PSMA-1/2/3 compared to [18F]PSMA-1007 (A–D) Maximum intensity projections at specific time points; (b) bone, (g) gall bladder, (h) heart, (i) intestine, (k) kidneys, (l) liver, (p) parotid glands, (t) tumor, (u) urinary bladder; (SUV) standardized uptake value. Compounds names for the Al18F-labeled PSMA ligands were abbreviated.

Figure 6 Pharmacokinetics of Al18F-labeled CHDT-PSMA-1/2/3 compared to [18F]PSMA-1007 data shown are from quantitative analysis of PET images. (A) time courses of activity concentrations in specific tissues (see Tables S11–S14 for the time-dependent SUVmean data); (B) time courses of excreted and/or retained activity fractions in renal (kidney + urinary bladder) and hepatobiliary (gall bladder + liver + intestine) organs; (C–D) areas under time-activity curves of specific tissues and excretory organs, (AUC) area under curve 0–120 min after radioligand injection, inhibition of PSMA-specific uptake with 2-PMPA administered simultaneously with the radioligand at a dose of 50 mg/kg; (SUV) standardized uptake value; mean values (an =4, bn =3, ± SD), statistical significance of differences compared to [18F]PSMA-1007: * p < 0.05; † p < 0.01; # p < 0.001. Compounds names for the Al18F-labeled PSMA ligands were abbreviated.

A more detailed view on the biodistribution and tumor uptake of Al[18F]F-CHDT-PSMA-1 reveals that its in vivo performance is at least comparable to that of [18F]PSMA-1007. In fact, the tumor and kidney uptakes were similar but blood clearance was faster, which resulted in significantly higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. (Table 4). The good in vivo performance of Al[18F]F-CHDT-PSMA-1 is also striking considering the results of previously developed PSMA ligands by Cleeren et al. bearing acyclic pentadentate chelating units, i.e., Glu-urea-Lys(Ahx)L3 and PSMA-RESCA1 (Figure 7).21,65 A PSMA-specific tumor uptake was also seen for both Al18F-labeled ligands, but Al[18F]F-PSMA-RESCA1 underlay a significant hepatobiliary excretion, which is less favorable for imaging purposes. Consequently, our objective of making the chelating unit more hydrophilic by the introduced triazole ring seems to be successful, although we should mention that the Ahx linker present in PSMA-RESCA1 might also add a certain hydrophobicity. In contrast, Al[18F]F-Glu-urea-Lys(Ahx)L3 showed a more pronounced uptake in bone tissue. Overall, our data for Al[18F]F-CHDT-PSMA-1 indicate that this radioligand is an interesting candidate for clinical translation.

Table 4 Tumor-to-Organ Contrast in PET Imaging with Al18F-Labeled CHDT-PSMA-1/2/3 Compared to [18F]PSMA-1007a

Radioligand	Tu/Blood	Tu/Kidney	Tu/Liver	Tu/Muscle	Tu/Bone	
[18F]PSMA-1007i	14 ± 3.0	0.5 ± 0.2	16 ± 6.8	20 ± 6.9	18 ± 7.9	
Al[18F]F-CHDT-PSMA-1ii	72 ± 44*	0.7 ± 0.4	15 ± 3.9	124 ± 65*	6.2 ± 0.6	
Al[18F]F-CHDT-PSMA-2ii	32 ± 16	0.7 ± 0.3	22 ± 17	72 ± 32	2.3 ± 0.7*	
Al[18F]F-CHDT-PSMA-3ii	43 ± 17	1.6 ± 0.5*	22 ± 14	116 ± 59	15 ± 11	
a SUV mean ratios 105–120 min after radioligand injection; (Tu) tumor; mean values (in = 4, iin = 3, ± SD); statistical significance of differences compared to [18F]PSMA-1007: * p < 0.05.

Figure 7 Structures of PSMA ligands Glu-urea-Lys(Ahx)L321,65 and PSMA-RESCA165

Conclusion

Previously reported acyclic pentadentate chelating units including (±)-H3RESCA turned out to facilitate efficient Al18F-labeling at low temperatures with sufficient complex stabilities for in vivo applications. Based on (±)-H3RESCA, our newly developed alkyne-functionalized building block allows the straightforward functionalization of biomolecules via click chemistry and simultaneously forms the actual complexing unit with a triazole ring (CHDT) upon conjugation. Apart from its conserved suitability to complex (Al[18F]F)2+, we showed that the CHDT moiety also enables an efficient complexation of [68Ga]Ga3+ and [111In]In3+ and that the respective complexes exhibit a high kinetic inertness at least under physiological conditions in vitro. Furthermore, the preclinical characterization of the Al18F-labeled CHDT-functionalized PSMA ligands revealed that the triazole ring might also positively affect the in vivo behavior of small molecule or peptide-derived conjugates. In particular, the in vivo performance of Al[18F]F-CHDT-PSMA-1 is even comparable to that of [18F]PSMA-1007 rendering this novel PSMA ligand a promising candidate for clinical translation. Moreover, the activity uptake in bone tissue was really low and highlights the high stability of the Al[18F]F-CHDT-complex in vivo. For the [68Ga]Ga- and [111In]In-CHDT-complexes, the kinetic inertness in vivo needs to be further investigated. Overall, our results highlight that the excellent complexing properties of (±)-H3RESCA for (Al[18F]F)2+ can be further modified by chemical modifications to enable the efficient complexation of other radiometals and to improve the pharmacokinetics of respective biomolecule conjugates, which might pave the way for broadening the radiopharmaceutical use of this chelator type.

Experimental Section

General

For the syntheses of the PSMA conjugates and model compounds all chemicals were obtained by commercial suppliers and used without further purification. Solvents were obtained by Fisher Scientific and anhydrous solvents were supplied by Sigma-Aldrich. PSMA-617 was purchased from ABX (Germany). KuE was prepared as previously described.66,67 Nuclear magnetic resonance spectra were recorded on an Agilent Technologies 400 MR spectrometer consisting of 400/54 premium compact magnet, 400 MR console and 400 MHz OneNMRProbe PT probe head (400 MHz for 1H, 101 MHz for 13C and 376 MHz for 19F). Spectra were processed by using the program MestreNova (version 14.2.1-27684). NMR chemical shifts were referenced to the residual solvent resonances relative to tetramethylsilane (TMS; 1H and 13C) and trichlorofluoromethane (CFCl3; 19F). Mass spectra (ESI) were obtained on a Waters Xevo TQ-S mass spectrometer (driven by the Mass Lynx software) or an Advion ExpressIon CMS spectrometer. The following molar masses of the final compounds were used for calculations (all molar masses were calculated including two TFA molecules): CHDT-Pe (697.53 g/mol), CHDT-Bn (687.54 g/mol), CHDT-PSMA-1 (956.75 g/mol), CHDT-PSMA-2 (1210.10 g/mol, CHDT-PSMA-3 (1335.27 g/mol).

Chromatography

Thin-layer chromatography (TLC) was performed on Merck silica gel F-254 aluminum plates with visualization under UV (254 nm). Preparative column chromatography was carried out on the Flash Chromatography “Isolera Four” from Biotage using appropriate “Sfär” columns (SNAP HC-Sfär; 5, 10, or 25 g, depending on amount of crude product and solvent mixtures). Analytical RP-HPLC was performed on a VWR Hitachi system using an analytical Zorbax 300SB-C18 column, 100 × 4.6 mm (Agilent Technologies) and CH3CN/water (0.1% TFA) as mobile phase and a flow rate of 1 mL/min. Purification by RP-HPLC was performed on a semipreparative HPLC system (AlphaCrom, Rheinfelden, Switzerland) equipped with 2 × pumps (Varian, PrepStar 218 Solvent Delivery Module) and a UV/vis detector (Varian, ProStar 325, wavelength of detection 254 nm). A Microsorb C18 60-8 column (Viarian Dynamax 250 × 21.4 mm) was used as stationary phase and a binary gradient system of 0.1% CF3COOH/water (solvent A) and 0.1%CF3COOH/CH3CN at a flow rate of 10 mL/min served as the eluent. For UPLC-DAD-MS, a system from Waters (ACQUITY UPLC I class system including an ACQUITY UPLC PDA e λ detector coupled to a Xevo TQ-S mass spectrometer) was used. An ACQUITY UPLC BEH C18 column (1.7 μm, 130 Å, 100 × 2.1 mm, equipped with a ACQUITY UPLC BEH C18 VanGuard Precolumn, 1.7 μm, 130 Å, 5 × 2.1 mm) was used as stationary phase. A binary gradient system of 0.1% CH3COOH/water (solvent A) and 0.1% CH3COOH in CH3CN/CH3OH (1:1, v/v, solvent B) at a flow rate of 0.4 mL/min served as the eluent.

Analytical radio-HPLC was performed using a system from Knauer (Knauer Smartline Gabi Advanced Scientific Instruments, Smartline Pump 1000, UV-detector 2500, Manager 5000, Software: EZChrom Elite, Client/Server Version 3.2.7) with a Jupiter C-18 column (5 μm, 300 Å, 4.6 × 250 mm, Phenomenex, USA) as stationary phase. For the 18F-, 68Ga-, and 111In-labeled CHDT-PSMA-1/2/3, the following binary gradient system of water with 0.1% acetic acid (solvent A) and acetonitrile with 0.1% acetic acid (solvent B) was used: 0 to 3 min 95% A, 3 to 20 min 95% A to 5% A, 20 to 25 min 5% A, 25 to 28 min 5% A to 95% A, 28 to 37 min 95% A, flow rate 1 mL/min. For [177Lu]Lu-PSMA-617, the following binary gradient system of water with 0.1% TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B) was used: 0 to 4 min 95% A, 4 to 15 min 95% A to 5% A, 15 to 20 min 5% A, 20 to 25 min 5% A to 95% A, 25 to 30 min 95% A, flow rate 1 mL/min.68 For radio-TLC, iTLC-SG strips (Agilent Technologies, USA) were used. An aliquot (0.3 μL, 1 μL for stability studies in human serum) of the respective radiolabeled compound was applied to the strip. The eluent was 2 M ammonium acetate/methanol (1:1, v/v). At the end, the stripes were exposed to a phosphor image plate (Fuji) and the scanned images (BAS 3000, Fuji, Raytest, Germany) were evaluated by the image analyzer program AIDA (Version 5.1 SP 4, Raytest, Germany).

Radiolabeling

[18F]fluoride (no-carrier-added) was produced on a cyclotron (30 MeV TR-Flex-Cyclotron, Advanced Cyclotron Systems Inc., Canada) by irradiation of [18O]H2O via the 18O(p,n)18F nuclear reaction. An aliquot of the aqueous [18F]fluoride solution (600 MBq) was withdrawn and sodium acetate buffer (0.1 M, pH 4.0) was added to reach a volume of 300 μL. For formation of (Al[18F]F)2+, 10 μL of AlCl3 (2 mM, in 0.1 M sodium acetate, pH 4) was added and the mixture was incubated for 10 min at 22 °C. Subsequently, 10 μL of the respective CHDT-functionalized compound (stock of 1 μg/μL) were added and the mixture was incubated for 20 min at 40 °C (300 rpm, Thermomixer Comfort, Eppendorf, Germany). For processing after successful labeling, a few crumbs of hydroxyapatite were added to the mixture. After 30 s, the suspension was shortly centrifuged (10 s at 10000g, Eppendorf 5415R), and the supernatant was transferred to a clean vial. [18F]PSMA-1007 was prepared according to a protocol from Cardinale et al.69

68Ga was eluted as [68Ga]GaCl3 from a 68Ge/68Ga generator of iThemba Laboratories (South Africa) in 0.6 M HCl. To 300 MBq of [68Ga]GaCl3, 1 M MES buffer (pH 6.0) was added until a pH of 5 to 6 was reached. Subsequently, 10 μL of the respective CHDT-functionalized compound (stock of 1 μg/μL) were added and the mixture was incubated for 20 min at 40 °C.

111In was purchased as [111In]InCl3 from Curium (United Kingdom). To 50 MBq of [111In]InCl3, 390 μL MES buffer (0.1 M, pH 5.5) and 10 μL of the respective CHDT-functionalized compound (stock of 1 μg/μL) were added and the mixture was incubated for 20 min at 40 °C.

[177Lu]LuCl3 was purchased from Isotope Technologies Munich AG (Germany). To 60 MBq of [177Lu]LuCl3, 90 μL MES buffer (0.1 M, pH 5.0) and 10 μL of PSMA-617 (stock of 1 μg/μL) were added and the mixture was incubated for 20 min at 90 °C.

[64Cu]CuCl2 was produced on the 30 MeV TR-Flex-Cyclotron (Advanced Cyclotron Systems, Inc., ACSI, Canada) by 64Ni(p,n)64Cu nuclear reaction as reported previously.70,71 To 12 MBq of [64Cu]CuCl3, 0.1 M MES buffer (pH 5.5) was added until a pH of 5 to 6 was reached. Subsequently, 10 μL of the respective CHDT-functionalized compound (stock of 1 μg/μL) were added and the mixture was incubated for 30 min at 50 °C.

n-Octanol/PBS Distribution Coefficient (logD7.4 Value)

The log D7.4 values were determined as follows: 500 μL of n-octanol and 450 μL of PBS (pH 7.4) were premixed prior to the addition of 50 μL of the respective radiolabeled compound. The mixture was vigorously stirred for 30 min at 22 °C, and thereafter centrifuged for 5 min at 7,500g at 22 °C. Aliquots of the n-octanol and the aqueous phases were transferred to measuring tubes, and the activity counts in both phases were measured in a gamma counter (Hidex Deutschland Vertrieb GmbH, Germany). The logarithm of the quotient of the activity counts from the n-octanol and the aqueous phase was calculated, which is equal to the log D7.4 value.

Serum Stability Assay

To investigate the serum stability of the radiolabeled CHDTA-PSMA-1/2/3, human serum (frozen, from male AB clotted whole blood; Sigma-Aldrich/Merck) was centrifuged for 5 min at 20,000g at 4 °C after thawing. The supernatant was sterile filtrated (filter pore 0.2 μm). Four parts serum and one part of radioligand were incubated at 37 °C with shaking. Aliquots were taken after 3 h and for the 111In-labeled compounds also after 24 and 48 h, and residual intact radiolabeled compound was determined by radio-TLC and/or radio-HPLC. For analysis by radio-HPLC, the aliquots were mixed with Supersol (mixture for protein precipitation that consists of 20% ethanol, 0.5% Triton X-100, 5 mM EDTA and 0.1% saponin)72 and kept on ice for 5 min. After centrifugation for 5 min at 20,000g at 4 °C, the supernatant was used for radio-HPLC analysis.

Biological In Vitro Assays

Binding assays were conducted using the high PSMA synthesizing human prostate carcinoma cell line LNCaP (ATCC CRL-1740) and the PSMA negative cell line PC3 (ATCC CRL-1435). The cells were grown as monolayer at 37 °C in a humidified atmosphere comprising 5% CO2 and 95% air in RPMI medium including 10% FCS (Biochrom AG). After washing the confluent cells twice with PBS and detaching them with trypsin/EDTA (0.05%/0.02%), the cells were suspended in RPMI and counted (Casy TT, Omni Life Science, Germany).

For the formation of spheroids, the LNCaP cells were incubated overnight in a normal culture bottle with biocompatible nanoparticles (1 × 10–6 cells with 80 μL of a suspension consisting of Au, Fe2O3 and poly-l-lysine called Nanoshuttle, Greiner Bio-One GmbH, Germany). After harvesting and cell counting, 10,000 cells/well of a 24-well plate or 7,000 cells/well of a 96-well plate (cell-repellent plates) were sown. The well plates were placed on matching plates with button magnets (Greiner Bio-One GmbH, Germany). The magnetized LNCaP cells formed suitable spheroids that were used after 5 d.

In order to assess the PSMA synthesis of LNCaP and PC3 cells, Western blotting was carried out. Cell lysates were prepared with RIPA buffer and 10 μg (protein level determined by Bio-Rad protein assay) was separated by SDS-PAGE (12% separating gel). This was followed by blotting onto a polyvinylidene fluoride membrane and blocking with 5% bovine serum albumin (BSA) in phosphate-buffered saline +0.1% Tween 20 (PBST) for 1 h, followed by an 1 h-incubation with appropriate primary antibodies (rabbit anti-PSMA 1:5,000; rabbit anti-β-actin 1:5,000, Cell Signaling Technology, USA) diluted with 5% BSA in PBST. After washing three times in PBST (à 5 min), the blots were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (antirabbit-HRP 1:20,000, Cell signaling Technology Inc., Danvers, USA) for 1 h in 5% BSA-TBST. The visualization of the HRPenzyme activity was accomplished with Western blotting luminol reagent according to manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX, USA). Enhanced chemiluminescence was detected with a blot scanner (C-DiGit Blot Scanner, LI-COR Bioscience GmbH, Germany).

Competition Binding Assay

The binding affinity of the nonlabeled CHDT-PSMA-1/2/3, PSMA-1007, PSMA-617, and KuE was assessed in competition binding assays using LNCaP cell homogenates (∼1 × 106 cells/mL using Potter-Elvehjem) and the radioligand [177Lu]Lu-PSMA-617 (n = 5 for CHDT-PSMA-1; n = 4 for CHDT-PSMA-2/3; n = 2 for PSMA-1007; n = 8 for PSMA-617; n = 6 for KuE). Homogenized cells were incubated with 40 μL of [177Lu]Lu-PSMA-617 (final concentration 12 nM) in the presence of increasing concentrations of the compounds at 37 °C for 60 min: CHDT-PSMA-1/2 (1.91 × 10–9 to 1.25 × 10–4 M), CHDT-PSMA-3 (3.89 × 10–10 to 2.55 × 10–5 M), PSMA-617 (9.15 × 10–11 to 1.20 × 10–5 M), and KuE (2.29 × 10–8 to 1.50 × 10–3 M). The assays were performed in a final volume of 200 μL. The incubation was stopped by washing the homogenate with cold PBS four times using a filter (Whatman GF/C, 90 min presoaked in 0.3% polyethylenimine) in a cell harvester (Brandel, USA). The radioactivity bound to the filter was measured in a gamma counter. Filter binding was determined for adjacent samples without cell homogenate. The experiments were performed two to four times in triplicates or quadruplicates. Inhibitory constants (Ki) were derived from nonlinear regression according to the model of “One site – Fit Ki” as implemented in GraphPad Prism 10. The Kd value of [177Lu]Lu-PSMA-617 was determined in saturation binding assays using LNCaP cell homogenates to be 15.2 (±2.1) nM.

Saturation and Internalization Assays

Saturation binding assays using cell homogenates of LNCaP cells (∼1 × 106 cells/mL) were performed for [68Ga]Ga-CHDT-PSMA-1/2 (0.2 to 50 nM) and using cell homogenates of PC3 cells (∼2 × 106 cells/mL) for Al[18F]F-CHDT-PSMA-1 (0.3 to 40 nM). To obtain the nonspecific binding adjacent samples received KuE (0.8 mM). The further procedure was carried out as described for the ’Competition Assay’ (see above).

For saturation assays in well plates, monolayers of LNCaP (sowing 5 × 104 cells/well in 48-well plates 2 days before the assay), as well as LNCaP spheroids were incubated with radiolabeled CHDT-PSMA-1/2/3 and PSMA-1007 (0.2 nM to 100 nM) with adjacent samples receiving KuE (0.8 mM) at 37 °C for 2 h. The number of experiments can be derived from the data in the Tables S4, S5, and S7 in the Supporting Information. After removing the incubation medium and washing of cells with ice-cold PBS (containing Mg2+ and Ca2+) the surface-bound activity was stripped with 4 °C cold acid-wash buffer (0.2 M glycine, 0.15 M NaCl, pH 3.0) for 10 min. The acid wash buffer was transferred from the wells to measuring tubes, as was the PBS buffer after washing once. Cytosolic activity (internalized radioligand) was determined after treatment with cell lysis buffer (1% SDS in 0.1 M NaOH) at 37 °C for 30 min. Cell surface and cytosolic activity were measured separately in a gamma counter. Time-depending internalization assays were performed only with cells in monolayer. The incubation (radioligand concentration 13 nM) for these assays was terminated after 10 min, 1 and 3 h, with 111In and 177Lu additionally after 24 and 48 h at 37 °C, respectively. During the internalization assays adjacent samples received KuE (0.8 mM) for PSMA blocking, methyl-β-cyclodextrin (MβCD; 3 mM) or KuE together with MβCD, 1 h before radioligand application. The number of experiments can be derived from the data of Figure S11 and associated table and from Table S8 in the Supporting Information.

Dissociations constants (Kd and Kd,int), as well as maximal binding capacities (Bmax and Bmax,int), were derived from specific binding data by nonlinear regression according to the model of “One site-specific binding” as implemented in GraphPad Prism 10. Protein content was determined using the bicinchoninic acid protein assay kit (Pierce, Thermo Scientific) to calculate the binding capacities in pmol/mg and for the time-depending internalization the internalized percent of administrated dose per μg protein (% AD/μg protein).

Small Animal PET/CT Imaging

Tumor Xenograft Model

All animal experiments were performed in accordance with the German Animal Welfare regulations and were approved by the local ethics committee for animal experiments (DD24.1-51-31/449/49, Oct 26, 2018). A PSMA-positive prostate cancer xenograft model was generated by subcutaneous injection of human LNCaP cells into the right shoulder of 8–12 week-old male nude mice (Rj:NMRI-Foxn1nu/nu, Janvier Laboratories, Le Genest-Saint-Isle, France). Imaging studies were performed when subcutaneous tumors reached a diameter of at least 6 mm. General anesthesia of the animals was induced and maintained by inhalation of 10% (v/v) desflurane in 30/70% (v/v) oxygen/air. Animals were continuously warmed at 37 °C during anesthesia.

Positron Emission Tomography

Positron emission tomography (PET) was performed in vivo using the nanoScan PET/CT scanner (Mediso Medical Imaging Systems, Budapest, Hungary), especially suited for small animals. Each animal received 8–12 MBq of the reference compound [18F]PSMA-1007 or the radioligands Al[18F]F-CHDT-PSMA-1/2/3 in phosphate-buffered saline by intravenous injection via a tail vein catheter. At the time of injection, molar activities and radiochemical purities of the compounds were as follows: [18F]PSMA-1007 (15 MBq/nmol, >99%); Al[18F]F-CHDT-PSMA-1 (10 MBq/nmol, 99%); Al[18F]F-CHDT-PSMA-2 (6.3 MBq/nmol, 98%); Al[18F]F-CHDT-PSMA-3 (4.5 MBq/nmol, >99%). Specificity of target binding was assessed by blocking PSMA binding sites with 2-phosphonomethyl pentanedioic acid (2-PMPA), administered simultaneously with the radioligand at a dose of 50 mg/kg. With injection of the radioligand, emission of the annihilation photons was recorded continuously in coincidence mode of 1:5 for 120 min. A corresponding CT image was acquired with each PET scan and used for attenuation correction and anatomical referencing. From three-dimensional list mode data, events within the energy window of 400–600 keV were extracted and sorted into 36 time frames (f1–f36) with a sequence of 15 × 10 s, 5 × 30 s, 5 × 60 s, 4 × 300 s, 3 × 600 s, and 4 × 900 s. For each time frame, a PET image was reconstructed with the three-dimensional (3D) Tera-Tomo algorithm, with a voxel size of 0.4 mm, applying corrections for random events, scattering, attenuation, and decay.

Quantitative Analysis of PET Images

PET images were analyzed using ROVER (ABX, Radeberg, Germany) and displayed as maximum intensity projections with identical intensity scaling. Three-dimensional regions of interest (ROIs) were defined within an image series of selected time frames using fixed intensity thresholds for delineation of organs: bones at knee joint position (0–120 min, 55%), cardiac blood volume (0–1.7 min, 70%), gall bladder (0–120 min, 39%), intestine (0–120 min, 5%), kidneys (0–2.5 min, 15%), liver (0–2.5 min, 55%), muscle (0–120 min, 0%), parotid glands (0–120 min, 39%), tumor (0–120 min, 39%), and urinary bladder (0–120 min, 5%). Activity concentrations in tissue were determined as standardized uptake value (SUV = [MBq detected activity/mL tissue]/[MBq injected activity/g body weight]) and expressed as SUVmean (ROI-averaged). Time-activity curves were generated and analyzed using Prism (GraphPad Software, San Diego CA, USA). Data points of the time-activity curves were generated by extracting the maximum value at <1.7 min followed by averaging the subsequent values in subgroups with summarized midframe times of 2.1, 5.5, 11, 20, 38, 55, 82, and 113 min. Areas under curves (AUC) were calculated using the trapezoid rule. Statistical significance of mean differences was tested by analysis of variance (ANOVA) using the Šidák test.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.4c00313.Exemplary radio-TLC and radio-HPLC chromatograms, radio-SDS-PAGE radioluminograms for the serum stability assays of [64Cu]Cu-CHDT-PSMA-1 and [68Ga]Ga-CHDT-PSMA-1, Western Blot for PSMA-synthesis in LNCaP and PC3 cells, exemplary saturation binding curves of [18F]PSMA-1007, [68Ga]Ga-CHDT-PSMA-1/2/3, [111In]In-CHDT-PSMA-1/2/3 toward LNCaP cells and of Al[18F]F-CHDT-PSMA-1 toward PC3 cells, internalization data for [68Ga]Ga-CHDT-PSMA-1/2/3, [111In]In-CHDT-PSMA-1/2/3 using LNCaP cells, individual data and mean values for the determination of logD7.4, as well as the competition binding, saturation binding, and internalization assays shown in the main article and the Supporting Information, ex vivo biodistribution data of Al[18F]F-CHDT-Pe and Al[18F]F-CHDT-Bn, SUV-time data for all PET experiments, and synthetic procedures and analytical data (chemistry) of all compounds (NMR, ESI-MS, HPLC) (PDF)

Supplementary Material

bc4c00313_si_001.pdf

Author Contributions

# M.W. and M.U. contributed equally to this work.

The authors declare no competing financial interest.

Acknowledgments

The excellent technical assistance of Christian Jentschel (radiolabeling and analytics), Utta Herzog (cell culture), Andrea Suhr (radiolabeling, analytics, and assays), as well as Linda Belke and Bianca Kreisl (organic synthesis), is greatly acknowledged. The authors thank the head and staff of the animal research facility, Dr. Birgit Belter, Katrin Baumgart, and Helge Gläser. The authors further thank the staff of the Production of Radiopharmaceuticals Department for the production of [18F]fluoride and [64Cu]CuCl2.

Abbreviations Used

AMCH trans-4-(aminomethyl)cyclohexanecarboxylic acid

CHDA cyclohexanediamine alkyne

CHDT cyclohexanediamine triazole

PSMA prostate specific membrane antigen

RESCA restrained complexing agent

SUV standardized uptake value
==== Refs
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