K. Arimitsu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127400
Fig. 2. PET/CT images of mice after administration of the 18F-labeled STZ derivatives. (A) [18F]7a (17.2 MBq), (B) [18F]7b (13.4 MBq), (C) [18F]6a (28.0 MBq), (D)
[
18F]6b (33.7 MBq).
denitroso intermediates in the synthesis of [18F]7a and 7b, were also
examined. In an in-vitro study of fluorescently-labeled STZ deriva-
tives,35,36 it was found that denitroso derivatives were taken up by cells
through GLUT2 in addition to the nitroso products. Therefore, we also
expected the biodistribution of [18F]6a and [18F]6b as GLUT2 imaging
probes. PET data were acquired for 60 min soon after injection and
reconstructed into images with dynamic frames of 12 × 15 s, 7 × 60 s
and 5 × 600 s. Images at representative timepoints are shown in Fig. 2.
Images of mice injected with [18F]7a and [18F]7b showed high levels of
radioactivity accumulating in the liver and kidney within 5 min of
administration. We also observed intestinal accumulation after 20 min
and clearance through the liver. GLUT2 is known to be expressed pri-
marily in the liver, kidney, and intestine.9 In addition, we performed
Western blot analysis using ddY mice of same age as used for PET study,
and confirmed that GLUT2 is expressed in these tissues of ddY mice
(Fig. 3). The relatively high accumulations of [18F]7a and [18F]7b in
the liver and kidney were similar to previously reported results ob-
tained with carbon-14-labeled STZ.33,34 Surprisingly, [18F]6a and [18F]
6b accumulated only in the kidney and bladder soon after adminis-
STZ derivative was recognized by GLUT2 in-vitro,35,36 we thought the
distribution of [18F]6a and [18F]6b might be attributable to rapid renal
excretion. However, further examination is required. We evaluated
differences arising from the stereochemistry of the anomers and found
that the β anomers [18F]7b and [18F]6b accumulated in the kidney to a
greater extent than the α anomers [18F]7a and [18F]6a.
Fig. 3. The expression of GLUT2 in liver, pancreas, kidney, and small intestine.
(A and B) GLUT2 expression in total protein of liver (A) and pancreas (B).
GAPDH and β-actin were used as the loading control. (C and D) GLUT2 ex-
pression in membrane protein of kidney (C) and small intestine (D). Na+/K+
ATPase was used as the loading control.
-
2). Non-radioactive compounds 5a and 5b were obtained without any
byproducts after fluorinating 4a and 4b with tetrabutylammonium
fluoride. Hydrolysis of 5a and 5b and subsequent nitrosylation gave the
desired products 7a and 7b. As expected, the ureas 6a, 6b, 7a, and 7b
were relatively stable under various chromatographic purification
conditions. However, 7a and 7b were not stable under reversed-phase
high performance liquid chromatography (HPLC) conditions.
We synthesized radioactive [18F]7a and [18F]7b as illustrated in
Scheme 4. Nucleophilic substitution of 4a and 4b was performed using
potassium [18F]fluoride. Acetonitrile was a more suitable solvent for
this reaction than tetrahydrofuran and 1,4-dioxane. Ten minutes was
insufficient for the reaction to go to completion, so the reaction was
allowed to proceed for 20 min. The mixture was heated to 90 °C for 18F-
fluorination, and methanolysis was performed immediately after re-
moving the solvent to yield [18F]6a and [18F]6b. [18F]6a and [18F]6b
were purified and analyzed using a reversed-phase HPLC system
equipped with a radioisotope detector. Final nitrosylation proceeded
rapidly to afford [18F]7a and [18F]7b, which were then purified on a
reversed-phase Sep-Pak solid phase extraction column. The radio-
chemical purities were estimated via autoradiography using thin-layer
chromatography plates and found to be near 90% (84%–99%).
The ex-vivo biodistributions of [18F]7a and [18F]7b were also in-
vestigated (Table 1). Like the PET study, marked accumulation of both
compounds in the liver, kidney, and intestines was observed. Mean-
while, their clearances were relatively rapid. If [18F]7a,b are expected
to add a [18F]fluorohexyl group to the 7-position nitrogen atom in
guanine base by a mechanism similar to STZ alkylation,27,32 the
clearance of radioactivity from these tissue may be considered too
rapid. This may be attributed to the elimination of [18F]fluoride ions
caused by the formation of DNA cross-linking. Nitrosourea antitumor
agents such as nimustine, ranimustine, and carmustine that contain 2-
PET imaging was performed using normal mice to evaluate the
dynamic whole-body distributions of the 18F-labeled STZ derivatives.
Male ddY mice were anesthetized with isoflurane and injected in-
travenously with [18F]7a or [18F]7b. [18F]6a and [18F]6b, which were
4