A flexible synthesis of 68Ga-labeled carbonic anhydrase IX (CAIX)-targeted molecules via CBT/1,2-aminothiol click reaction

Article abstract of DOI:10.3390/molecules24010023

We herein describe a flexible synthesis of a small library of⁶⁸Ga-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 new⁶⁸Ga-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 of⁶⁸Ga-labeled imaging agents for tumor hypoxia.

Full text of DOI:10.3390/molecules24010023

molecules
Article
A Flexible Synthesis of 68Ga-Labeled Carbonic
Anhydrase IX (CAIX)-Targeted Molecules via
CBT/1,2-Aminothiol Click Reaction
Kuo-Ting Chen 1, Kevin Nguyen 2, Christian Ieritano 2, Feng Gao 2 and Yann Seimbille 1,2,
*
1
Department of Radiology and Nuclear Medicine, Erasmus MC, University Medical Center Rotterdam,
Wytemaweg 80, 3015 CN Rotterdam, The Netherlands; k.chen@erasmusmc.nl
Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, BC V6T2A3, Canada;
knguyen7@ualberta.ca (K.N.); cieritano@edu.uwaterloo.ca (C.I.); rggaofeng@hotmail.com (F.G.)
Correspondence: y.seimbille@erasmusmc.nl; Tel.: +31-10-703-8961
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 November 2018; Accepted: 19 December 2018; Published: 21 December 2018
Abstract: 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 (
emitter gallium-68. Cross-ligation between the pre-labeled bifunctional chelators (BFCs) and the
1,2-aminothiol-acetazolamide derivatives ( and
) yielded six new 68Ga-labeled CAIX ligands with
1–3) were successfully synthesized and labeled with the positron
8
9
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.
Keywords: carbonic anhydrase IX; 68Ga-labeling; click reaction; compound library
1. 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. 68Ga is one of the most attractive positron emitters for PET
imaging due to its physical properties, such as short half-life (t1/2 = 67.7 min) and high positron
abundance (β
+: 89%), and its availability via a 68Ge/68Ga generator. The success of the [68Ga]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 68Ga 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 [2–4].
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
(CO2) and bicarbonate (HCO3−); 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

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renal cell carcinoma, bladder, breast, lung and ovarian cancers. However, its expression in normal
tissues is highly restricted [10 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 [15–17]
By taking advantage of the high binding affinity of aromatic sulfonamide to CAIX, they have been
labeled with a variety of radionuclides (i.e., 18F, 64Cu, 68Ga, 99mTc, 111In) 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. 68Ga-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 [22–24]. To the best of our knowledge, application
of this chemistry to 68Ga-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
8
–3
) that can be labeled with 68Ga. Then we study their conjugation to acetazolamide derivatives
and 9
) via the CBT/1,2-aminothiol click reaction. A small library of six novel 68Ga-labeled
CAIX-targeted imaging probes was obtained and their stability in phosphate buffered saline (PBS) and
in a transchelation challenge assay were assessed.
Figure 1. Design of a small library of carbonic anhydrase IX (CAIX) radioligands via a two-step
orthogonal labeling concept.

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2. Results and Discussions
The preparation of the CBT-bearing chelators
1–3
is illustrated in Scheme 1. For the synthesis of
NODA-pyCBT (
with 2,6-bis(bromomethyl)pyridine under basic conditions in the presence of cesium carbonate to
give the CBT-pyridinyl in 69% yield. N-alkylation of the bis-tert-butyl NODA chelator with
was performed under reflux to give the protected NODA-pyCBT ( ) in 88% yield. Removal of the
tert-butyl protecting groups under acidic conditions gave 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 and , the amino-CBT intermediate was prepared from , as previously
described [25]. Subsequently, 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.
1), commercially available 2-cyano-6-hydroxy-benzothiazole (4) was first O-alkylated
5
5
6
1
2
3
7
4
7
Scheme 1. Synthesis of the 2-cyanobenzothiazole (CBT)-bearing chelators (1–3). Reagents and
conditions: (a) 2,6-bis(bromomethyl)pyridine, Cs2CO3, THF, 50 ◦C, 16 h, 69%; (b) di-tert-butyl
2,2’-(1,4,7-triazonane-1,4-diyl)diacetate, K2CO3, KI, ACN, reflux, 16 h, 88%; (c) TFA, thioanisole,
DCM, rt, 18 h, 85%, (d) DOTA-NHS or NODAGA-NHS, triethylamine, DMF, rt, 16 h, 29% (for 2) or
83% (for 3).
With the three CBT-functionalized chelators (
optimization of the 68Ga-labeling conditions. We first evaluated the influence of pH on 68Ga-labeling
efficiency. The results showed that a nearly quantitative yield of [68Ga]-
was obtained at the
and ) gave excellent
1–3) in hand, we next turned our attention to the
1
optimal pH of 4.5 to 5.5 (Figure S1). Similarly, the other two CBT-precursors (
2
3
68Ga-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
temperatures. The RCYs were monitored by radio-high performance liquid chromatography (HPLC).
1–3
(~10 nmol) with 68GaCl3 in sodium acetate buffer (0.2 M, pH 5.5) for 15 min at different
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 68Ga-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 68Ga-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 68Ga 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 68Ga3+ at lower

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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
point of view, similar 68Ga-labeling efficiencies of
and suggest that the replacement of a carboxylate
with a pyridine ring does not alter the chelation of NOTA-type chelators to 68Ga3+. Furthermore, due
to the intrinsic ultraviolet (UV) absorption of the pyridine ring, is more easily monitored than
3. Interestingly, from a structural
1
2
1
2
during the compound preparation. Thus, the NODA-pyridine analog could be a viable alternative to
NOTA for 68Ga labeling.
A
B
Figure 2.
(
A
) Comparison of RCYs for precursors 1–3 B) Comparison of RCYs
at 37, 65 and 90 ◦C. (
◦
when varying amounts of precursor is used. All the reactions were performed at 90 C by incubation
in sodium acetate buffer (0.2 M, pH 5.5) for 15 min.
Assessment of the stability of the complexes ([68Ga]-
1–3) in both PBS buffer and through
a transchelation challenge study was performed. 68Ga-labeled CBTs were incubated in PBS (0.2 M,
pH 7.4) in the absence or presence of EDTA (34 mM) at 37 ◦C for 2 h, and subsequently analyzed by
radio-HPLC. All the labeled compounds were stable in the neutral PBS buffer over a period of 2 h
(Figure S2). More than 98% of [68Ga]-
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
1,2-aminothiol functionalized compounds.
Preparation of two acetazolamide derivatives (
PEG2) and a 1,2-aminothiol moiety were carried out by solid phase synthesis. In general, synthesis
of and 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 , whereas was prepared according
8 and 9) containing a linker (Asp-Arg-Asp or
8
9
8
9
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 68Ga-CAIX probes ([68Ga]-L14-L16).

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Scheme 2. Preparation of 8 and 9 by solid-phase synthesis. Reagents and conditions: (a) Fmoc-SPPS;
(b) 13, CuI, TBTA, sodium ascorbate, DMF, rt, 16 h; (c) TFA/TIPS/H2O, rt, 3 h, 32% (for
(for 9).
8) or 37%
The conjugates [68Ga]-L14-L16 were obtained by CBT/1,2-aminothiol click ligation of the
68Ga-labeled BFCs ([68Ga]-
3) and the two acetozalamides ( and ) (Scheme 3). The efficiency
of click reaction between [68Ga]- to form [68Ga]-L14b was first tested at pH 7.4. At different
and
1–
8
9
1
9
time points, the reaction was quenched by addition of 10% acetic acid and analyzed by radio-HPLC to
monitor the progress of the reaction. As illustrated in Figure 3, the formation of [68Ga]-L14b could
already be observed at 10 min (RCY of 44%), demonstrating the fast kinetics of the click reaction.
Identity of the clicked product ([68Ga]-L14b) was confirmed by HPLC comparison of a non-radioactive
standard (Figure S3). Longer reaction time resulted in improved radiochemical yields. Surprisingly,
the conjugation of
(Figure S4). RCYs of 54 and 78% were obtained when [68Ga]-
(Table 1, entry 1 and 2), as compared to 81 and 99% for the reaction between [68Ga]-
8
to [68Ga]-
1
was not as efficient as the click reaction between
was treated with for 30 and 60 min
and . Similar
9 1
and [68Ga]-
1
8
1
9
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 68Ga-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 [68Ga]- at pH 9.0 in PBS. To our delight, the RCY of [68Ga]-L14a was significantly
1 and 8
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
25 nmol resulted in an excellent RCY (99%) after 20 min incubation time (Table 1, entry 6). Then, we
applied the above slightly basic conditions to the ligation of the two other 68Ga-CBTs ([68Ga]-
and
[68Ga]- ). [68Ga]-L14b, [68Ga]-L15a and [68Ga]-L15b were successfully obtained
) with AAZs ( and
8 from 10 to
2
3
8
9
with high RCYs (95–99%) within 20 min. These radiochemical products were amenable for direct
utilization in preclinical tests without further purification. Lower RCYs of 88 and 82% were found
for [68Ga]-L16a and [68Ga]-L16b, respectively. It might be explained by a weaker complexation of
68Ga3+ in the DOTA chelator (the stability constants (log KML) of Ga-DOTA and Ga-NOTA are 26 and
31, respectively), and therefore a small amount of free 68Ga was released from the [68Ga]-
during the conjugation reaction [28 29]. It confirms that BFCs and are preferred over BFCs
68Ga-labeling, since the 68Ga complexation in
and is more efficient and stable than in (Figure 2B
could be very valuable for labeling with other
3
complex
for
,
1
2
3
1
2
3
and Figure S5). Nevertheless, our DOTA-based BFC
radiometals, such as 90Y and 177Lu.
3

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Scheme 3. Synthesis of [68Ga]-14 to [68Ga◦]-16 via CBT/1,2-aminothiol click reaction. Reagents and
conditions: (a) TCEP·HCl, PBS, pH 9.0, 37 C, 20 min.
Figure 3. Radio-high performance liquid chromatography (HPLC) monitoring of the progress of click
1 and 9 1
reaction between [68Ga]- . The retention time of [68Ga]- and [68Ga]-L14b are 10.0 and 10.3 min,
respectively (HPLC system A).
Table 1. Evaluation of the click reaction between [68Ga]-1 and 8 under varying conditions.
Entry
8 (nmol)
Buffer pH
Time (min)
RCY (%) a
1
2
3
4
5
6
10
10
10
10
10
25
7.4
7.4
7.4
9.0
9.0
9.0
30
60
180
30
60
20
54
78
98
85
99
99
a
Radiochemical yield (RCY) of [68Ga]-L14a was determined by radio-HPLC.

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Stability studies were performed by incubation of our 68Ga-labeled CAIX-targeted probes
([68Ga]-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
[68Ga]-L14-L16 were not subject to radiolytic degradation over this time period (Table 2). Lipophilicity
of the 68Ga-labeled ligands was determined by measurement of the LogD7.4. 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 PEG2 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 D7.4 values were found to be affected when using different 68Ga-labeling chelators.
For instance, [68Ga]-L14a and [68Ga]-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.
Table 2. Determination of LogD7.4 and stability of [68Ga]-L14-L16.
Compound
68Ga]-L14a
68Ga]-L14b
68Ga]-L15a
68Ga]-L15b
68Ga]-L16a
68Ga]-L16b
Stability (%) a
Log D7.4
>99
>99
>99
>99
>99
>99
−1.95 ± 0.03
−2.03 ± 0.10
−2.68 ± 0.02
−2.71 ± 0.05
−3.20 ± 0.06
−3.29 ± 0.14
[
[
[
[
[
[
The stability tests were performed in phosphate buffered saline (PBS) at 37 ◦C for 2 h. Results are expressed as
a
percentage (%) of intact ligand after incubation.
3. Materials and Methods
3.1. 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. 68Ga was obtained from a 68Ga/68Ge
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, D2O, CDCl3 or CD3OD 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).
3.2. 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:

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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.
3.3. HPLC Conditions for Purification
The HPLC purifications were performed on a Phenomenex semi preparative RP-C18 column
(Aqua®, 5
µ
m, 10
×
250 mm) at a flow rate of 3.0 mL/min. A gradient of solvent A and B (t = 0–5 min,
90% A; t = 5–30 min, 90 to 0% A) was applied.
3.4. Chemistry and Radiolabeling
6-((6-(Bromomethyl)pyridin-2-yl)methoxy)benzo[d]thiazole-2-carbonitrile (5). To a solution of 2,6-bis
(bromomethyl)pyridine (460 mg, 1.74 mmol) and 2-cyano-6-hydroxybenzothiazole (200 mg, 1.14 mmol)
in THF (50 mL) was added Cs2CO3 (450 mg, 1.38 mmol). The reaction was heated at 50 ◦C for 16 h
then cooled to rt and filtered. The filtrate was concentrated and purified by flash chromatography
1
(hexanes/EtOAc, 6:4) to afford
5
as a white solid (284 mg, 69%). H NMR (400 MHz, CDCl3):
δ
8.10 (d,
1H, J = 9.2 Hz), 7.76 (t, 1H, J = 7.6 Hz), 7.40–7.47. (m, 3H), 7.34 (dd, 1H, J = 2.4, 9.2 Hz), 5.30 (s, 2H), 4.58
(s, 2H). ESI-MS: m/z 360.0 [M + H]+.
Di-tert-butyl 2,2’-(7-((6-(((2-cyanobenzo[d]thiazol-6-yl)oxy)methyl)pyridin-2-yl)met-hyl)-1,4,7-triazonane-
1,4-diyl)diacetate (
0.14 mmol), K2CO3 (76 mg, 0.52 mmol) and KI (0.3 mg, 1.81
dropwise a solution of (50 mg, 0.14 mmol) in acetonitrile (3 mL). The reaction was allowed to stir at
6
). To a mixture of di-tert-butyl 2,2’-(1,4,7-triazonane-1,4-diyl)diacetate (49 mg,
µmol) in acetonitrile (2 mL) was added
5
rt for 1 h, then refluxed for 16 h. The reaction was cooled to rt, filtered and concentrated to give
a yellow oil (77 mg, 88%). The product was directly used without further purification. H NMR (400
6
as
1
MHz, CDCl3):
δ 8.07 (d, 1H, J = 9.2 Hz), 7.68 (t, 1H, J = 7.6 Hz), 7.51 (br, 1H), 7.42 (br, 1H), 7.33 (m, 2H),
5.26 (m, 2H), 3.86 (s, 2H), 3.30 (s, 4H), 2.87 (m, 12H), 1.43 (s, 18H). ESI-MS: m/z 637.3 [M + H]+, 659.3
[M + Na]+.
2,2’-(7-((6-(((2-Cyanobenzo[d]thiazol-6-yl)oxy)methyl)pyridin-2-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic
Acid (NODA-pyCBT,
1). TFA (1 mL) was added dropwise to a solution of crude 6 (24 mg, 37.67 µmol),
thioanisole (20%) in 1 mL DCM at 0 ◦C and allowed to stir at rt for 18 h. The reaction mixture was
concentrated under reduced pressure and purified by semi preparative HPLC to yield
1
as a yellow
1
solid (17 mg, 85%). H NMR (400 MHz, CD3OD):
δ
8.12 (d, 1H, J = 9.2 Hz), 7.95 (t, 1H, J = 7.6 Hz), 7.80
(m, 1H), 7.68 (m, 1H), 7.60 (m, 1H), 7.44 (m, 1H), 5.40 (s, 2H), 4.47 (s, 2H), 3.55 (m, 4H), 3.24 (s, 6H),
2.94 (s, 6H). High-resolution mass spectrometry (HRMS) (ESI): m/z calcd. for [C25H28N6O5S + H]+
525.1920, found: 525.1917. The compound purity was determined by UV-HPLC (254 nm), showing
greater than 95% (Figure S7).
2,2’-(7-(1-Carboxy-4-((2-((2-cyanobenzo[d]thiazol-6-yl)oxy)ethyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)
diacetic Acid (NODAGA-CBT, 2). To a solution of 7 (8 mg, 0.02 mmol) in DMF (1 mL) was added 33 µL
of triethylamine (0.24 mmol). Subsequently, a solution of NODAGA-NHS ester (15 mg, 0.02 mmol) in
0.15 mL of DMF was added to the reaction mixture. The vial was placed under a positive pressure of
argon and the solution was stirred for 16 h at rt. Solvent was removed under vacuum and the residue
was dissolved in H2O/ACN (1:1) and purified by semi-preparative HPLC to yield
2 as a pale-yellow
solid upon lyophilization (4 mg, 29%). 1H NMR (400 MHz, CD3OD):
δ
8.07 (d, 1H, J = 9.2 Hz), 7.68
(m, 1H), 7.33 (dd, 1H, J = 2.4, 9.2 Hz), 4.33–4.36 (m, 2H), 3.68–3.88 (m, 6H), 3.43 (m, 1 H), 2.89–3.25 (m,
12H), 2.36–2.48 (m, 2H), 1.91–2.08 (m, 2H). ESI-MS: m/z 595.3 [M + H3O]+. The compound purity was
determined by UV-HPLC (254 nm), showing greater than 95% (Figure S7).

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2,2’,2”-(10-(2-((2-((2-Cyanobenzo[d]thiazol-6-yl)oxy)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic Acid (DOTA-CBT, ). was prepared according to the method described above for
Semi-preparative HPLC purification provided 3 as a white solid upon lyophilization (12 mg, 83%).
3
3
2.
1H NMR (400 MHz, CD3OD):
δ 7.97–8.02 (m, 1H), 7.59–7.61 (m, 1H), 7.27–7.33 (m, 1H), 4.33 (t, 2H,
J = 4.8 Hz), 3.69–4.1 (m, 10 H), 3.58 (t, 2H, J = 4.8 Hz), 3.2–3.44 (m, 14H). ESI-MS: m/z 624.4 [M + H3O]+.
The compound purity was determined by UV-HPLC (254 nm), showing greater than 95% (Figure S7).
N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)hex-5-ynamide (13). The 5-hexynoic acid (100 mg, ◦0.89 mmol,
3.1 equiv.) was treated with an ex◦cess of thionyl chloride (2 mL, 27.56 mmol) at 0 C, and the
reaction mixture was heated to 70 C and stirred for 1 h. 5-Hexynoyl chloride was obtained by
evaporation of the excess of thionyl chloride and dried under vacuum. A solution of 5-amino-1,3,4-
thiadiazole-2-sulfonamide (50 mg, 0.28 mmol, 1.0 equiv.) in anhydrous DMF (2 mL) was slowly added
◦
into 5-hexynoyl chloride at 0 C under N2. The reaction mixture was warmed to rt and stirred for 48 h.
The reaction mixture was then concentrated and purified by flash chromatography (with a gradient of
EtOAc/Hexanes = 1:4 to 1:1 to 100% EtOAc; silica gel) to give the product as an off-white solid (42 mg,
1
55%). H NMR (400 MHz, DMSO-d6):
δ 13.02 (s, 1H), 8.31 (s, 2H), 2.82 (t, 1H, J = 2.4 Hz), 2.63 (t, 2H,
J = 7.2 Hz), 2.21–2.25 (m, 2H), 1.78–1.81 (m, 2H). ESI-MS: m/z 297.1 [M + Na]+.
K(C)-DRD-AAZ (
(Chemglass®, Vineland, NJ, USA). All the reactions were performed at rt with agitation at 150 rpm.
The washing steps were performed with DMF (5 mL 2) and DCM (5 mL 2). The capping was
carried out by using Ac2O (94.5 L, 1.00 mmol) in DMF (5 mL). Before the first amino acid coupling,
8). Compound 8 was prepared by solid-phase synthesis in a 10 mL reaction vessel
×
×
µ
Rink amide MBHA resin (100 mg, 0.65 mmol/g) was preswollen with DMF (5 mL) for 1 h, and
then treated with 20% piperidine in DMF (5 mL) for 1 h to remove the Fmoc-protecting group on
resin. A mixture of Fmoc-Lys[N-Boc-Cys(Trt)]-OH (159 mg, 0.19 mmol), HBTU (76 mg, 0.32 mmol),
Oxymapure (46 mg, 0.32 mmol), and DIPEA (83 µL, 0.65 mmol) in DMF (5 mL) was added to the resin
and agitated for 2 h. After a washing/capping sequence, the Fmoc-l-Lys(N-Boc-l-Cys(Trt))-resin was
obtained. Subsequent couplings with Fmoc-l-Asp(tBu)-OH (80 mg, 0.19 mmol), Fmoc-L-Arg(Pbf)-OH
(126 mg, 0.19 mmol), Fmoc-L-Asp(tBu)-OH (80 mg, 0.19 mmol) and 5-azidopentanoic acid (28 mg,
0.19 mmol) were accomplished according to the same experimental protocol to give the azido-peptidic
resin. The coupling reactions were performed in DMF with HBTU (5.0 equiv.), OxymaPure (5.0 equiv.)
and DIPEA (10.0 equiv.) for 2 h. Fmoc deprotection was achieved by treatment of the resin with
piperidine (20%) in DMF for 0.5 h. The amide formation and Fmoc deprotection were monitored by
Kaiser test. Double couplings or deprotection were performed when the reaction was not completed.
The click reaction was carried out by mixing the azido-peptidic resin, 13 (53 mg, 0.19 mmol), CuI (4 mg,
0.02 mmol), TBTA (10 mg, 0.02 mmol) and sodium ascorbate (34 mg, 0.20 mmol) in DMF (5 mL) for
16 h. Cleavage from the resin and deprotection of the AAZ analog were conducted by treatment of
the resin with TFA/TIPS/H2O (3 mL, v/v/v = 95/2.5/2.5) at rt for 3 h. The cleavage cocktail was
concentrated, washed with ice-cold ether (12 mL
×
3) and the residue purified by semi-preparative
7.84 (s, 1H), 4.67–4.73 (m,
1
HPLC to give
8
as a white solid (22 mg, 32%). H NMR (400 MHz, D2O):
δ
2H), 4.29–4.36 (m, 3H), 4.23–4.27 (m, 1 H), 4.17 (t, 1H, J = 6.0 Hz), 3.17–3.35 (m, 4H), 3.06 (dd, 1H,
J = 4.0, 5.6 Hz), 2.88–3.03 (m, 3H), 2.79–2.88 (m, 4H), 2.66–2.70 (m, 2H), 2.31 (t, 2H, J = 7.6 Hz), 2.12–2.18
(m, 2H), 1.69–1.91 (m, 6H), 1.51–1.66 (m, 6H), 1.33–1.45 (m, 2H). ESI-MS: m/z 1034.37 [M + H]+. The
compound purity was determined by UV-HPLC (254 nm), showing greater than 95% (Figure S7).
K(C)-PEG-AAZ (9). The preparation of 9 was similar to the preparation of 8. The synthesis was started
by loading Fmoc-Lys[N-Boc-Cys(Trt)]-OH onto Rink amide MBHA resin (100 mg, 0.65 mmol/g),
followed by subsequent conjugation with Fmoc-AEEAc-OH (75 mg, 0.19 mmol), 5-azidopentanoic acid
(28 mg, 0.19 mmol) and 13 (53 mg, 0.19 mmol). After cleavage and deprotection, the crude product
1
was washed with cold-ether and purified by HPLC to give
(400 MHz, D2O):
9
as white solid (19 mg, 37%). H NMR
δ
7.68 (s, 1H), 4.12–4.18 (m, 4 H), 3.96 (s, 2H), 3.53 (dd, 4H, J = 4.4, 5.2 Hz), 3.46 (t,

Molecules 2019, 24, 23
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2H, J = 5.2 Hz), 3.23 (dd, 2H, J = 4.8, 5.6 Hz), 3.01–3.18 (m, 4 H), 2.67 (d, 2H, J = 7.2 Hz), 2.51 (d, 2H,
J = 7.2 Hz), 2.10 (d, 2H, J = 7.2 Hz), 1.94–1.99 (m, 2H), 1.63–1.68 (m, 3H), 1.55–1.58 (m, 1H), 1.38–1.40
(m, 4H), 1.19–1.27 (m, 2H). ESI-MS: m/z 815.27 [M + Na]+. The compound purity was determined by
UV-HPLC (254 nm), showing greater than 95% (Figure S7).
Radiolabeling of NODA-pyCBT (
aqueous solution containing 0.05 M HCl and 3.0 M NaCl. 68GaCl3 (200
a solution of 1 (9.5 nmol) in DMSO (1 L) and sodium acetate buffer (0.2 M, pH 5.5, 700
1
). 68GaCl3 was eluated from the 68Ge/68Ga-Generator as an
L, 115 MBq) was added to
L). The
µ
µ
µ
mixture was incubated at 90 ◦C with shaking at 700 rpm for 15 min. The reaction conversion was
monitored by radio-TLC and a solution of NH4OAc/MeOH (1.0 M, v/v = 1:1) was used as mobile
phase. The reaction mixture was cooled for 5 min, and then passed through a C-18 light cartridge.
The cartridge was washed with water (10 mL) to remove unchelated-68Ga and followed by washed
with ethanol (1 mL) to give [68Ga]-
1 1 was obtained with a radiochemical yield of 85% (decay
. [68Ga]-
corrected) and a molar activity of 8.9 MBq/nmol after purification. The radiochemical purity of
[68Ga]-
1
was determined by radio-HPLC, showing greater than 99%. The retention time of [68Ga]-
1
was 10.0 min (HPLC system A).
Radiolabeling of NODAGA-CBT (
2
). [68Ga]-
2
(8.6 nmol) was labeled with 68GaCl3 (84 MBq) under
similar conditions as previously described for [68Ga]- . [68Ga]-
1 2 was obtained with a radiochemical
yield of 94% (d. c.) and a molar activity of 6.9 MBq/nmol after purification. The retention time of
[68Ga]-2 was 11.1 min (HPLC system A) and its radiochemical purity was greater than 95%.
Radiolabeling of DOTA-CBT (
the similar conditions of preparing [68Ga]-
of 85% (d. c.) with molar activity of 7.1 MBq/nmol after purification. The radiochemical purity of
3
). [68Ga]-
3
(8.2 nmol) was labeled by 68GaCl3 (98 MBq) by using
3
. The [68Ga]-
3 was obtained in the radiochemical yield
[68Ga]-
3
was determined by radio-HPLC, showing greater than 99%. The retention time of [68Ga]-
3
was 10.2 min (HPLC system A).
Conjugation of 68Ga-labeled bifunctional chelators (BFCs) ([68Ga]-
1
–3
) and acetazolamides (AAZs)
(
8
–
9
). Compound
pH 9.0, 400
L) were mixed in a 1.5 mL centrifuge tube at rt. To the mixture, 68Ga-labeled precursors
(2 nmol, 10–20 MBq) in 100
L of EtOH, was added, and the reaction was incubated at 37 ◦C with
8
(26
µ
g, 25 nmol) or
9
(20
µ
g, 25 nmol), TCEP
·
HCl (8 µg, 28 nmol) in PBS (0.2 M,
µ
µ
agitation (700 rpm) for 20 min. The radiochemical purity (RCP) and retention time (tR) of products
[68Ga]-L14a-L16b were analyzed by radio-HPLC (Table 3 and Figure S5).
Table 3. Conjugation of 68Ga-labeled bifunctional chelators (BFCs) and acetazolamides (AAZs) through
the click reaction.
68Ga-Labeled BFCs
AAZs
Products
RCP (%)
tR (min)/ HPLC System
68Ga]-1
68Ga]-1
68Ga]-2
68Ga]-2
68Ga]-3
68Ga]-3
[
68Ga]-L14a
8
9
8
9
8
9
98
99
96
95
88
82
9.4 / A
10.3 / A
9.9 / A
10.8 / A
9.5 / A
[
[
[
[
[
[
[
68Ga]-L14b
[
[
68Ga]-L15a
68Ga]-L15b
68Ga]-L16a
68Ga]-L16b
[
[
18.5 / B *
* The retention time of [68Ga]-3 is 16.7 min in HPLC system B.
3.5. Determination of LogD7.4
Distribution coefficients (LogD7.4 values) were determined by a shake-flask method. Sample
containing the radioligand in 5
and 600 L n-octanol. The vial was vortexed vigorously and then centrifuged for 10 min for phase
separation. Samples of the octanol (200 L) and aqueous (200 L) phases were taken and counted
by -counter. LogD7.4 value was calculated by using the equation: LogD7.4 = log [(counts in octanol
phase)/(counts in aqueous phase)]. All the experiments were performed in triplicates.
µL PBS (pH 7.4) was added to a vial containing 595 µL PBS (pH 7.4)
µ
µ
µ
γ

Molecules 2019, 24, 23
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3.6. 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 [68Ga]-L16b; HPLC system A
for other compounds).
4. Conclusions
We have developed a two-step orthogonal labeling method to prepare a small library of
68Ga-labeled CAIX ligands via a CBT/1,2-aminothiol click reaction. Three novel CBT-functionalized
chelators (1–3
) were synthesized and efficiently labeled with 68Ga 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 68Ga3+, but it enhances its hydrophobicity. Six new 68Ga-labeled CAIX-targeted molecules
were successfully prepared under optimal conditions by cross-ligation between our three 68Ga-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.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Conceptualization, K.-T.C. and Y.S.; investigation, K.-T.C., K.N., C.I. and F.G.;
writing—original draft preparation, K.-T.C.; writing—review and editing, Y.S, K.-T.C., K.N., C.I. and F.G.
Funding: We gratefully acknowledge the Leenaards Foundation (grant # 3699), NSERC, and the Department of
Radiology and Nuclear Medicine at Erasmus MC for financial support.
Acknowledgments: We thank the NMR facilities in the Chemistry department at the University of British
Columbia for providing support and resources. We would like to thank the Radiochemistry team in the
Department of Radiology and Nuclear Medicine at Erasmus MC for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).