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combination with chemotherapeutic agent or radiotherapy.7,18,19
Small molecules inhibitors targeting CA-IX are either coumarin sui-
cidal inhibitors or sulfonamides moiety.13,20,21 Sulfonamide ligand
with high affinity to central metal ion of an enzyme can be utilized
for the targeted delivery of potent cytotoxic drugs into solid
tumors, which is otherwise showed ineffective biodistribution to
desired tumor site and accumulated in normal tissues.19,22 Devel-
opment of CA-IX inhibitors as an anticancer agent also enhanced
the development of PET imaging ligand for diagnostics and small
molecule-drug conjugates (SMDCs) for therapeutic purpose in both
solid and metastatic tumors. Acetazolamide, a prototype23 of sul-
fonamides, is a clinically approved pan-CA inhibitor with good pro-
file,21,24 and is also known to possess antitumor activity alone or in
combination.18,25 Suppression of tumor metastasis in lung carci-
noma was also acquired by acetazolamide.26 99mTc-labeled aceta-
zolamide was even used as a ligand for development of PET
imaging radiotracer for imaging purpose27 and SMDCs with aceta-
zolamide ligand for pharmacodelivery of cytotoxic drug at tumor
site.28 However, most of the known radiotracers for imaging CA-
IX as 99mTc-labeled acetazolamide ligand, 124I-cG250 or 124I- and
89Zr-labeled antiCA-IX monoclonal antibodies were performed
well predominantly for in vivo imaging of CA-IX in hypoxia inde-
pendent renal cell carcinoma SKRC-52,27,29 even though CA-IX is
also expressed in various hypoxic tumor models.30 Only few PET
tracer as 68Ga-NOTGA-(AEBSA)3 and 18F-AmBF3-(ABS)3 with tri-
meric sulfonamide moieties achieved the proposed role. Surpris-
ingly, the tracer with trimeric sulfonamide moieties showed
higher uptake with selective targeting to CA-IX compared to mono-
meric and dimeric isoforms.22 Reported studies raised a question
that CA-IX is really a universal target for imaging of all solid tumors
other than SKRC-52. One review was published performing valida-
tion of CA-IX target for hypoxic tumor imaging.31 The review pro-
posed that CA-IX is an unreliable target for hypoxic imaging due to
the facts that CA-IX expression is cancer type-dependent, not all
hypoxic tumors express CA-IX, expression of CA-IX is undetectable
in certain cancer types, and re-oxygenation of previously hypoxic
cancer cells induces expression of CA-IX. However, no in vivo
experimental study was carried out to support this conclusion
yet. Thus, further validation of CA-IX as a target for development
of clinically feasible hypoxia imaging PET tracer is required to be
performed. In the view of validation of CA-IX target for hypoxic
tumor imaging, we designed and synthesized a new [18F]-PET tra-
cer (1) based on acetazolamide as shown in Scheme 1. In vivo PET
imaging and biodistribution studies of [18F]-PET tracer (1) were
performed in CA-IX positive 4T1 and HT-29 skin xenograft Balb/c
mice tumor models. 4T1 and HT-29 cancer cells are known to
express CA-IX within hypoxic microenvironment. Imaging of PET
tracer was also performed in CA-IX expressed lung metastatic
tumors model formed by 4T1 breast cell lines.11 The in vivo PET
images and biodistribution profile provided insight about the
behavior of our novel PET tracer [18F]-acetazolamide (1) on tumor
model.
The precursor 6 for [18F]-labeling was synthesized to couple
with [18F]-PEG-alkyne (9) to form central triazole ring by copper
(I)-catalyzed click chemistry as shown in Scheme 1. The precursor
6 was synthesized starting with hydrolysis of commercially avail-
able acetazolamide (2) giving compound 3. Compound 3 was
reacted with succinic anhydride in DMF with heating to obtain car-
boxylic acid (4). Compound 4 was subjected to amide coupling
reaction with azide-PEG linker (2-(2-(2-azidoethoxy)ethoxy)
ethan-1-amine) (5) using BOP which finally provided the precursor
(6) for click reaction. This precursor was then used for synthesis of
radiolabeled [18F]-acetazolamide (1) through the click reaction
with [18F]-PEG-alkyne (9). Radiolabeling of the mesylated PEG-
alkyne linker (7) successfully provided [18F]-PEG-alkyne precursor
(9) in 32.5% radiochemical yield with >99% of radiochemical purity.
The click reaction of precursor (6) with [18F]-PEG-alkyne (9) was
performed to obtain final [18F]-PET tracer (1) in radiochemical
yield of 93.73% and radiochemical purity >99% after HPLC purifica-
tion (see Fig. S1 and Table S1, ESIy).
Scheme 1. Reaction conditions and reagents: A. (a) conc. HCl, EtOH, reflux, quant. yield; (b) succinic anhydride, DMF, 100 °C, 96%; (c) 2-(2-(2-azidoethoxy)ethoxy)ethan-1-
amine (5), BOP, iPrNEt2, DMF, rt, 53%; B. (d) TBAF, THF, 80 °C, 33%; (e) 18F/[2,2,2]-cryptand, CH3CN, 100 or 80 °C, 33%; C. (f) CuI, iPrNEt2, CH3CN, rt, 94%.