A Flexible Synthesis of 68Ga-Labeled Carbonic Anhydrase IX (CAIX)-Targeted Molecules via CBT/1,2-Aminothiol Click Reaction

We herein describe a flexible synthesis of a small library of 68Ga-labeled CAIX-targeted molecules via an orthogonal 2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction. Three novel CBT-functionalized chelators (1–3) were successfully synthesized and labeled with the positron emitter gallium-68. Cross-ligation between the pre-labeled bifunctional chelators (BFCs) and the 1,2-aminothiol-acetazolamide derivatives (8 and 9) yielded six new 68Ga-labeled CAIX ligands with high radiochemical yields. The click reaction conditions were optimized to improve the reaction rate for applications with short half-life radionuclides. Overall, our methodology allows for a simple and efficient radiosynthetic route to produce a variety of 68Ga-labeled imaging agents for tumor hypoxia.


Introduction
Positron tomography scanners detect pairs of γ-rays originating from a radionuclide decaying by positron emission. The signal recorded by the scanner allows in vivo visualization and quantification of biological processes at the cellular or molecular level. Due to its high sensitivity and non-invasive properties, positron emission tomography (PET) plays an important role in cancer patient management, as a diagnostic and prognostic tool. 68 Ga is one of the most attractive positron emitters for PET imaging due to its physical properties, such as short half-life (t 1/2 = 67.7 min) and high positron abundance (β + : 89%), and its availability via a 68 Ge/ 68 Ga generator. The success of the [ 68 Ga]DOTATE (NETSPOT ® ) in the PET imaging of somatostatin receptor-positive neuroendocrine tumors (NETs), as well as recent the U.S. Food and Drug Administration (FDA) approval of this agent, demonstrate the growing interest in the utility of 68 Ga in PET imaging [1]. Moreover, its coordination chemistry and chemical tools are continuously being improved, facilitating the development of novel PET tracers based on this radiometal [2][3][4].
Tumor hypoxia represents a negative therapeutic indicator due to its multiple contributions to tumor invasiveness, radiotherapy and chemotherapy resistances [5,6]. Therefore, targeted imaging of hypoxic tumors would allow for the assessment of the response to antineoplastic treatments. Carbonic anhydrase IX (CAIX) is a transmembrane metalloenzyme that is strongly upregulated by hypoxia-inducible factor 1-α (HIF1-α) under tumor hypoxia [7]. The function of CAIX is to maintain intracellular acid-base homeostasis by catalyzing the interconversion of carbon dioxide (CO 2 ) and bicarbonate (HCO 3 − ); thus CAIX assists malignant cancer cell survival in the absence of oxygen [8,9]. Overexpression of CAIX has been reported in many types of malignancies, such as Molecules 2019, 24, 23; doi:10.3390/molecules24010023 www.mdpi.com/journal/molecules renal cell carcinoma, bladder, breast, lung and ovarian cancers. However, its expression in normal tissues is highly restricted [10][11][12][13][14]. Moreover, unlike the other members of this enzyme family, CAIX is abundantly present on the extracellular membrane of cancer cells. Therefore, CAIX represents a promising biomarker for tumor hypoxia detection. Over the last decade, several CAIX-targeted radioligands based on aromatic sulfonamide pharmacophores, such as acetazolamides (AAZs) and benzene-sulfonamides, have been developed [15][16][17]. By taking advantage of the high binding affinity of aromatic sulfonamide to CAIX, they have been labeled with a variety of radionuclides (i.e., 18 F, 64 Cu, 68 Ga, 99m Tc, 111 In) and evaluated in preclinical CAIX-positive tumor models. However, most of these probes suffered from low tumor uptake, weak selectivity and stability when evaluated in vivo. The development of this type of probe is still in its early stage and current probes have not been optimized yet [18,19]. Comparison of the target selectivity, physicochemical and pharmacodynamic properties of structurally diverse radiolabeled sulfonamides might provide useful information for probe optimization. Thus, we sought to develop an efficient synthetic method to generate a small library of CAIX-targeted compounds by applying a mild and universal two-step orthogonal labeling protocol. 68 Ga-labeling is usually performed in an aqueous buffer at low pH and high temperature [20,21]. However these harsh labeling conditions are sometimes not suitable for the direct labeling of fragile biomolecules. Our alternative radiochemical strategy is the two-step labeling approach where a bifunctional chelator (BFC) is radiolabeled and then conjugated to a biovector under biologically friendly conditions. Although sulfonamides are not particularly sensitive to the labeling conditions, we decided to opt for a two-step labeling approach in order to facilitate the implementation of a library-based synthesis of our new CAIX PET tracers ( Figure 1). The biovectors and the BFCs were prepared separately, to generate diverse compounds without the need to develop and optimize individually their syntheses. BFCs were functionalized with a 2-cyanobenzothiazole (CBT) clickable group, whereas the CAIX ligands were modified with the complementary 1,2-aminothiol functionality.
Several regioselective click reactions, such as the Huisgen's cycloaddition, the Staudinger ligation, or the inverse electron demand Diels-Alder reaction (IEDDA) can be applied to the two-step radiosynthesis. In this study, we chose to apply the naturally occurring click reaction between 2-cyanobenzothiazole and 1,2-aminothiol because of its orthogonality, biocompatibility, fast kinetics, and metabolic stability of the reagents and product [22][23][24]. To the best of our knowledge, application of this chemistry to 68 Ga-labeling has not been reported due to the lack of amenable chemical reagents. Therefore, we describe herein the synthesis of three new CBT-functionalized macrocyclic chelators (1-3) that can be labeled with 68 Ga. Then we study their conjugation to acetazolamide derivatives (8 and 9) via the CBT/1,2-aminothiol click reaction. A small library of six novel 68 Ga-labeled CAIX-targeted imaging probes was obtained and their stability in phosphate buffered saline (PBS) and in a transchelation challenge assay were assessed.
Molecules 2018, 23 2 of 13 highly restricted [10][11][12][13][14]. Moreover, unlike the other members of this enzyme family, CAIX is abundantly present on the extracellular membrane of cancer cells. Therefore, CAIX represents a promising biomarker for tumor hypoxia detection. Over the last decade, several CAIX-targeted radioligands based on aromatic sulfonamide pharmacophores, such as acetazolamides (AAZs) and benzene-sulfonamides, have been developed [15][16][17]. By taking advantage of the high binding affinity of aromatic sulfonamide to CAIX, they have been labeled with a variety of radionuclides (i.e., 18 F, 64 Cu, 68 Ga, 99m Tc, 111 In) and evaluated in preclinical CAIX-positive tumor models. However, most of these probes suffered from low tumor uptake, weak selectivity and stability when evaluated in vivo. The development of this type of probe is still in its early stage and current probes have not been optimized yet [18,19]. Comparison of the target selectivity, physicochemical and pharmacodynamic properties of structurally diverse radiolabeled sulfonamides might provide useful information for probe optimization. Thus, we sought to develop an efficient synthetic method to generate a small library of CAIX-targeted compounds by applying a mild and universal two-step orthogonal labeling protocol. 68 Ga-labeling is usually performed in an aqueous buffer at low pH and high temperature [20,21]. However these harsh labeling conditions are sometimes not suitable for the direct labeling of fragile biomolecules. Our alternative radiochemical strategy is the two-step labeling approach where a bifunctional chelator (BFC) is radiolabeled and then conjugated to a biovector under biologically friendly conditions. Although sulfonamides are not particularly sensitive to the labeling conditions, we decided to opt for a two-step labeling approach in order to facilitate the implementation of a librarybased synthesis of our new CAIX PET tracers ( Figure 1). The biovectors and the BFCs were prepared separately, to generate diverse compounds without the need to develop and optimize individually their syntheses. BFCs were functionalized with a 2-cyanobenzothiazole (CBT) clickable group, whereas the CAIX ligands were modified with the complementary 1,2-aminothiol functionality.
Several regioselective click reactions, such as the Huisgen's cycloaddition, the Staudinger ligation, or the inverse electron demand Diels-Alder reaction (IEDDA) can be applied to the two-step radiosynthesis. In this study, we chose to apply the naturally occurring click reaction between 2cyanobenzothiazole and 1,2-aminothiol because of its orthogonality, biocompatibility, fast kinetics, and metabolic stability of the reagents and product [22][23][24]. To the best of our knowledge, application of this chemistry to 68 Ga-labeling has not been reported due to the lack of amenable chemical reagents. Therefore, we describe herein the synthesis of three new CBT-functionalized macrocyclic chelators (1-3) that can be labeled with 68 Ga. Then we study their conjugation to acetazolamide derivatives (8 and 9) via the CBT/1,2-aminothiol click reaction. A small library of six novel 68 Ga-labeled CAIXtargeted imaging probes was obtained and their stability in phosphate buffered saline (PBS) and in a transchelation challenge assay were assessed.

Results and Discussions
The preparation of the CBT-bearing chelators 1-3 is illustrated in Scheme 1. For the synthesis of NODA-pyCBT (1), commercially available 2-cyano-6-hydroxy-benzothiazole (4) was first O-alkylated with 2,6-bis(bromomethyl)pyridine under basic conditions in the presence of cesium carbonate to give the CBT-pyridinyl 5 in 69% yield. N-alkylation of the bis-tert-butyl NODA chelator with 5 was performed under reflux to give the protected NODA-pyCBT (6) in 88% yield. Removal of the tert-butyl protecting groups under acidic conditions gave 1 in 85% yield. Notably, thioanisole was used as a cation scavenger to prevent the degradation of the cyano group during the deprotection. For the preparation of 2 and 3, the amino-CBT intermediate 7 was prepared from 4, as previously described [25]. Subsequently, 7 was conjugated to DOTA-NHS or NODAGA-NHS under mild basic conditions to give NODAGA-CBT (2) or DOTA-CBT (3) in 29 and 83% yield, respectively.

Results and Discussions
The preparation of the CBT-bearing chelators 1-3 is illustrated in Scheme 1. For the synthesis of NODA-pyCBT (1), commercially available 2-cyano-6-hydroxy-benzothiazole (4) was first Oalkylated with 2,6-bis(bromomethyl)pyridine under basic conditions in the presence of cesium carbonate to give the CBT-pyridinyl 5 in 69% yield. N-alkylation of the bis-tert-butyl NODA chelator with 5 was performed under reflux to give the protected NODA-pyCBT (6) in 88% yield. Removal of the tert-butyl protecting groups under acidic conditions gave 1 in 85% yield. Notably, thioanisole was used as a cation scavenger to prevent the degradation of the cyano group during the deprotection. For the preparation of 2 and 3, the amino-CBT intermediate 7 was prepared from 4, as previously described [25]. Subsequently, 7 was conjugated to DOTA-NHS or NODAGA-NHS under mild basic conditions to give NODAGA-CBT (2) or DOTA-CBT (3) in 29 and 83% yield, respectively. With the three CBT-functionalized chelators (1-3) in hand, we next turned our attention to the optimization of the 68 Ga-labeling conditions. We first evaluated the influence of pH on 68 Ga-labeling efficiency. The results showed that a nearly quantitative yield of [ 68 Ga]-1 was obtained at the optimal pH of 4.5 to 5.5 ( Figure S1). Similarly, the other two CBT-precursors (2 and 3) gave excellent 68 Gacomplexation (>90%) under identical pH conditions. Next, the effect of reaction temperature on radiochemical yields (RCYs) was evaluated. We performed the reaction by incubating the precursors 1-3 (~10 nmol) with 68 GaCl3 in sodium acetate buffer (0.2 M, pH 5.5) for 15 min at different temperatures. The RCYs were monitored by radio-high performance liquid chromatography (HPLC). As illustrated in Figure 2A, precursors 1-3 were efficiently labeled at 90 °C (88-97% RCYs). However, lowering the temperature was dramatically detrimental to the 68 Ga-coordination, as RCYs below 25% were observed at 37 and 65 °C for all BFCs. These findings suggest that high temperature is required to provide high yields of 68 Ga-labeled CBT-derivatives 1-3. To investigate the effect of precursor amount on RCYs, reactions were performed under the optimal pH and temperature conditions defined above. All three precursors could be efficiently labeled with 68 Ga at a chelator amount equal or superior to 1.5 nmol ( Figure 2B). We noticed that 1 and 2 are more prone to complex with 68 Ga 3+ at lower concentrations (~0.2 nmol) than the DOTA chelator 3, which means a higher molar activity could be obtained by using NODA-pyCBT or NODAGA-CBT than precursor 3. Interestingly, from a With the three CBT-functionalized chelators (1-3) in hand, we next turned our attention to the optimization of the 68 Ga-labeling conditions. We first evaluated the influence of pH on 68 Ga-labeling efficiency. The results showed that a nearly quantitative yield of [ 68 Ga]-1 was obtained at the optimal pH of 4.5 to 5.5 ( Figure S1). Similarly, the other two CBT-precursors (2 and 3) gave excellent 68 Ga-complexation (>90%) under identical pH conditions. Next, the effect of reaction temperature on radiochemical yields (RCYs) was evaluated. We performed the reaction by incubating the precursors 1-3 (~10 nmol) with 68 GaCl 3 in sodium acetate buffer (0.2 M, pH 5.5) for 15 min at different temperatures. The RCYs were monitored by radio-high performance liquid chromatography (HPLC). As illustrated in Figure 2A, precursors 1-3 were efficiently labeled at 90 • C (88-97% RCYs). However, lowering the temperature was dramatically detrimental to the 68 Ga-coordination, as RCYs below 25% were observed at 37 and 65 • C for all BFCs. These findings suggest that high temperature is required to provide high yields of 68 Ga-labeled CBT-derivatives 1-3. To investigate the effect of precursor amount on RCYs, reactions were performed under the optimal pH and temperature conditions defined above. All three precursors could be efficiently labeled with 68 Ga at a chelator amount equal or superior to 1.5 nmol ( Figure 2B). We noticed that 1 and 2 are more prone to complex with 68 Ga 3+ at lower concentrations (~0.2 nmol) than the DOTA chelator 3, which means a higher molar activity could be obtained by using NODA-pyCBT or NODAGA-CBT than precursor 3. Interestingly, from a structural point of view, similar 68 Ga-labeling efficiencies of 1 and 2 suggest that the replacement of a carboxylate with a pyridine ring does not alter the chelation of NOTA-type chelators to 68 Ga 3+ . Furthermore, due to the intrinsic ultraviolet (UV) absorption of the pyridine ring, 1 is more easily monitored than 2 during the compound preparation. Thus, the NODA-pyridine analog could be a viable alternative to NOTA for 68 Ga labeling.
Molecules 2018, 23 4 of 13 structural point of view, similar 68 Ga-labeling efficiencies of 1 and 2 suggest that the replacement of a carboxylate with a pyridine ring does not alter the chelation of NOTA-type chelators to 68 Ga 3+ . Furthermore, due to the intrinsic ultraviolet (UV) absorption of the pyridine ring, 1 is more easily monitored than 2 during the compound preparation. Thus, the NODA-pyridine analog could be a viable alternative to NOTA for 68 Ga labeling.  Figure  S2). More than 98% of [ 68 Ga]-1-3 remained intact, even when challenged by a large excess of EDTA. The high stability of the radiocomplexes warrants their utility in further conjugations with 1,2aminothiol functionalized compounds.
Preparation of two acetazolamide derivatives (8 and 9) containing a linker (Asp-Arg-Asp or PEG2) and a 1,2-aminothiol moiety were carried out by solid phase synthesis. In general, synthesis of 8 and 9 was initiated by immobilizing a SPPS compatible Fmoc-dipeptide onto a Rink amide MBHA resin (Scheme 2) [25]. Subsequent conjugations with Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH and 5-azidopentanoic acid were required for 8, whereas 9 was prepared according to a similar protocol by using Fmoc-NH-AEEAc-OH instead of the three Fmoc-protected amino acids. The acetazolamide intermediate 13 was then incorporated to the azido-resins to complete the chemical sequence through a Cu(I)-catalyzed Huisgen's cycloaddition. Cleavage and global deprotection were performed by the treatment of the acetazolamide-resins with a solution of TFA/TIPS/H2O (v/v/v = 95/2.5/2.5) to give 8 and 9 in an overall yield of 32% and 37%, respectively, after HPLC purification based on initial resin loading. The pure acetazolamides 8 and 9 were then applied to the preparation of the 68 Ga-CAIX probes ([ 68 Ga]-L14-L16). Preparation of two acetazolamide derivatives (8 and 9) containing a linker (Asp-Arg-Asp or PEG 2 ) and a 1,2-aminothiol moiety were carried out by solid phase synthesis. In general, synthesis of 8 and 9 was initiated by immobilizing a SPPS compatible Fmoc-dipeptide onto a Rink amide MBHA resin (Scheme 2) [25]. Subsequent conjugations with Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH and 5-azidopentanoic acid were required for 8, whereas 9 was prepared according to a similar protocol by using Fmoc-NH-AEEAc-OH instead of the three Fmoc-protected amino acids.   Figure S3). Longer reaction time resulted in improved radiochemical yields. Surprisingly, the conjugation of 8 to [ 68 Ga]-1 was not as efficient as the click reaction between 9 and [ 68 Ga]-1 ( Figure S4). RCYs of 54 and 78% were obtained when [ 68 Ga]-1 was treated with 8 for 30 and 60 min ( Table 1, entry 1 and 2), as compared to 81 and 99% for the reaction between [ 68 Ga]-1 and 9. Similar observations have previously been reported for other 1,2-aminothiol substrates, where the click reaction rate was presumably affected by the differences of chemical structures, configurations and electronic distribution [26,27]. Although a high RCY (98%) could be reached by extending the reaction time to 180 min ( Table 1, entry 3), it was not practical for 68 Ga-labeling considering its short half-life. We previously demonstrated that the ligation between 2-cyano-6-hydroxybenzothiazole and L-cysteine in PBS at pH 9.0 is 4-times more efficient than at pH 7.4 [27]. Thus, we evaluated the click reaction between [ 68 Ga]-1 and 8 at pH 9.0 in PBS. To our delight, the RCY of [ 68 Ga]-L14a was significantly improved to 85% after 30 min reaction time (as opposed to 54% at pH 7.4), and a nearly quantitative yield was observed after 60 min (Table 1, entries 4 and 5). We also checked if a higher concentration of 1,2-aminothiol-AAZ would accelerate the reaction. An increase in the amount of 8 from 10 to 25 nmol resulted in an excellent RCY (99%) after 20 min incubation time (Table 1, [28,29]. It confirms that BFCs 1 and 2 are preferred over BFCs 3 for 68 Ga-labeling, since the 68 Ga complexation in 1 and 2 is more efficient and stable than in 3 ( Figure 2B and Figure S5). Nevertheless, our DOTA-based BFC 3 could be very valuable for labeling with other radiometals, such as 90 Y and 177 Lu.        Stability studies were performed by incubation of our 68 Ga-labeled CAIX-targeted probes ([ 68 Ga]-L14-L16) in PBS at 37 • C for 2 h. All our compounds showed excellent stability, with more than 99% of intact radioligand after 2 h incubation. It indicates that our radiolabeled compounds [ 68 Ga]-L14-L16 were not subject to radiolytic degradation over this time period ( Table 2). Lipophilicity of the 68 Ga-labeled ligands was determined by measurement of the LogD 7.4 . All our radiolabeled compounds were hydrophilic and water-soluble, and therefore they are more prone to be cleared via the kidney [30]. In general, replacing the PEG 2 linker by a peptide inker (Asp-Arg-Asp) had little effect on the molecular lipophilicity, suggesting that this charged peptide linker can be used for a comparison of the overall charge effects with its corresponding PEG surrogate on in vivo pharmacokinetics. In contrary, the Log D 7.4 values were found to be affected when using different 68 Ga-labeling chelators. For instance, [ 68 Ga]-L14a and [ 68 Ga]-L14b exhibited significantly higher hydrophobicity than the other two series compounds. Those compounds with various physicochemical properties could be used for a comparison of their in vivo effects to guide the direction of probe optimization.

General Information
All chemicals were obtained from commercial suppliers and used without further purification. NODAGA-NHS ester and DOTA-NHS ester were obtained from CheMatech (Dijon, France). All solvents were anhydrous grade unless indicated otherwise. 68 Ga was obtained from a 68 Ga/ 68 Ge generator (IGG-100; Eckert and Ziegler Europe, Berlin, Germany). Reactions were magnetically stirred and monitored by thin-layer chromatography on Merck aluminum-backed pre-coated plates (Silica gel 60 F254) (Quebec, QC, Canada), and visualized with ultraviolet light or by staining with 10% phosphomolybdic acid in neat ethanol. Flash chromatography was performed on silica gel of 40-63 µm particle size. Concentration refers to rotary evaporation. Reverse-phase high-performance liquid chromatography (HPLC) was carried out on a Waters ® 2659 series system (Etten-Leur, The Netherlands) equipped with a diode array detector and a radio-detector. Nuclear magnetic resonance (NMR) spectra were recorded in DMSO-d6, D 2 O, CDCl 3 or CD 3 OD on diluted solutions on a Bruker AVANCE 400 (Leiderdorp, Leiden, The Netherlands) at ambient temperature. Chemical shifts are given as δ values in ppm and coupling constants J are given in Hz. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublets) and br (broad signal). Low-resolution electrospray ionization (ESI) mass spectra were recorded on a TSQ Quantum Ultra™ triple quadrupole mass spectrometer from Thermo Fisher Scientific ® (Bleiswijk, Lansingerland, The Netherlands). Fmoc-based solid-phase peptide synthesis (SPPS) was conducted on an C.S. Bio CS136 automated peptide synthesizer (Menlo Park, CA, USA).

High-Performance Liquid Chromatography (HPLC) Conditions for Analysis
The analyses of reaction were performed by HPLC on an analytical RP-C18 column (5 µm, 4.6 × 250 mm, Phenomenex Aqua ® , Torrance, CA, USA) at a flow rate of 1 mL/min. The UV signal was recorded at wavelength of 254 nm. The following solvents and eluting gradients were used: solvent A = 0.1% trifluoroacetic acid (TFA) in water (v/v); solvent B = 0.1% TFA in acetonitrile (v/v). HPLC system A, a gradient of solvent A and B: t = 0-20 min, 95 to 5% A; t =20-23 min, 5% A; HPLC system B, a gradient of solvent A and B: t = 0-25 min, 95 to 55% A; t = 25-27 min, 55 to 0% A; t = 27-30 min, 0% A were applied.

Stability and Challenge Studies
For the stability and challenge tests, the radiolabeled samples (~2 MBq) were mixed with 300 µL of PBS (0.1 M, pH 7.4) or a PBS/EDTA solution (34 mM EDTA in PBS), respectively. After 2 h incubation at 37 • C, the samples were analyzed by radio-HPLC (HPLC system B for [ 68 Ga]-L16b; HPLC system A for other compounds).

Conclusions
We have developed a two-step orthogonal labeling method to prepare a small library of 68 Ga-labeled CAIX ligands via a CBT/1,2-aminothiol click reaction. Three novel CBT-functionalized chelators (1-3) were synthesized and efficiently labeled with 68 Ga while retaining their clickable functionality. The physicochemical properties, such as lipophilicity, solubility and overall charges, of a NOTA-type chelator could be manipulated by modification or replacement of one of the carboxylate arms on NOTA. Replacement of a carboxylate with a pyridine ring has no detrimental effect on the chelation of 68 Ga 3+ , but it enhances its hydrophobicity. Six new 68 Ga-labeled CAIX-targeted molecules were successfully prepared under optimal conditions by cross-ligation between our three 68 Ga-labeled chelators and two acetazolamide derivates. The products exhibited high radiochemical purity and in vitro stability, allowing direct application for further biological evaluations. Our chemistry and materials provide a high degree of versatility to develop new target-specific imaging or therapeutic probes, but can also be considered for pretargeting applications. An in vitro receptor binding affinity assay, a cell internalization assay, an in vivo biodistribution and µPET studies are currently underway in our laboratory to explore the effects of the molecular composition on the physicochemical properties of our CAIX radioligands.