Radiosynthesis and Preclinical Investigation of 11C-Labelled 3-(4,5-Diphenyl-1,3-oxazol-2-yl)propanal Oxime ([11C]SZV 1287)

Article abstract of DOI:10.1002/cmdc.202000389

The radiosynthesis, as well as the in vivo and ex vivo biodistribution of the ¹¹C radiolabelled 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime (6, [¹¹C]SZV 1287) are reported. SZV 1287 is a novel semicarbazide-sensitive amine oxidase

Full text of DOI:10.1002/cmdc.202000389

Accepted Article
Title: Radiosynthesis and preclinical investigation of 11C labelled 3-
(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime ([11C]SZV 1287)
Authors: Viktória Forgács, Enikő Németh, Barbara Gyuricza, Adrienn
Kis, Judit P. Szabó, Pál Mikecz, Péter Mátyus, Zsuzsanna
Helyes, Ádám István Horváth, Tamás Kálai, György
Trencsényi, Anikó Fekete, and Dezső Szikra
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000389
Link to VoR: https://doi.org/10.1002/cmdc.202000389
01/2020

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
Radiosynthesis and preclinical investigation of 11C labelled 3-(4,5-
diphenyl-1,3-oxazol-2-yl)propanal oxime ([11C]SZV 1287)
Viktória Forgács[a,b], Enikő Németh[a]; Barbara Gyuricza[a,b], Adrienn Kis[a,c], Judit P. Szabó[a,c], Pál
Mikecz[a], Péter Mátyus[d], Zsuzsanna Helyes[e,f], Ádám István Horváth[e,f], Tamás Kálai[g,h], György
Trencsényi[a], Anikó Fekete*[a], and Dezső Szikra[a]
[a]
V. Forgács, E. Németh, B. Gyuricza, A. Kis, J.P. Szabó, Dr. P. Mikecz, Dr. G. Trencsényi, Dr. A. Fekete, Dr. D. Szikra
Division of Nuclear Medicine and Translational Imaging, Department of Medical Imaging, Faculty of Medicine
University of Debrecen
Nagyerdei krt. 98., H-4032 Debrecen (Hungary)
E-mail: fekete.aniko@science.unideb.hu
[b]
V. Forgács, B. Gyuricza
Doctoral School of Chemistry, Faculty of Science and Technology
University of Debrecen
Egyetem tér 1., H-4032 Debrecen (Hungary)
A. Kis, J.P. Szabó
Doctoral School of Clinical Medicine, Faculty of Medicine
University of Debrecen, Nagyerdei krt. 98., H-4032 Debrecen (Hungary)
Prof. Dr. P. Mátyus
[c]
[d]
Institute of Digital Health Sciences, Faculty of Health and Public Administration
Semmelweis University
Ferenc tér 15., H-1094 Budapest (Hungary)
Prof.Dr. Z. Helyes, Dr. Á.I. Horváth
Department of Pharmacology and Pharmacotherapy, Medical School
University of Pécs
Szigeti str. 12., H-7624 Pécs (Hungary)
Prof.Dr. Z. Helyes, Dr. Á.I. Horváth
[e]
[f]
Molecular Pharmacology Research Group & Centre for Neuroscience, Szentágothai Research Centre
University of Pécs
Szigeti str. 12., H-7624 Pécs (Hungary)
[g]
[h]
Prof.Dr. T. Kálai
Institute of Organic and Medicinal Chemistry, Medical School
University of Pécs
Szigeti str. 12., H-7624 Pécs (Hungary)
Prof.Dr. T. Kálai
MTA-PTE Chronic Pain Research Group
Szigeti str. 12., H-7624 Pécs (Hungary)
Supporting information for this article is given via a link at the end of the document.
Abstract: The radiosynthesis, as well as the in vivo and ex vivo
biodistribution of the 11C radiolabelled 3-(4,5-diphenyl-1,3-oxazol-2-
yl)propanal oxime (6, [11C]SZV 1287) are reported. SZV 1287 is a
novel SSAO inhibitor and a promising candidate to be a novel
analgesic for the treatment of neuropathic pain. Its radiolabelling was
developed via a four-step radiosynthesis which started from the
of primary amines. The first molecularly defined member of the
family was amine oxidase copper containing (AOC), originally
known as vascular adhesion protein-1, which has also essential
role in the transmigration of leukocytes from the bloodstream
into inflamed tissues.[1,2] Thus, increased SSAO activity was
observed in many inflammation-associated diseases, such as
inflammatory liver diseases, diabetes, congestive heart failure,
atherosclerosis, severe obesity and Alzheimer’s disease.[3–6]
Several studies described the anti-inflammatory properties[2,7–11]
and the analgesic actions of SSAO inhibition.[12,13]
3-(4,5-Diphenyl-1,3-oxazol-2-yl)propanal oxime (SZV 1287) is a
novel SSAO inhibitor. This compound is an oxime analogue of
the cyclooxygenase (COX) inhibitor (called oxaprozin) which
was developed and patented by Mátyus et al.[14,15] (Figure 1). In
addition, SZV 1287 has dual antagonistic effect on transient
receptor potential vanilloid 1 and ankyrin 1 (TRPV1 and TRPA1)
receptors which are expressed predominantly on capsaicin-
sensitive peptidergic sensory neurons mediating pain and
inflammation.[16]
reaction of Grignard reagent
2
with [11C]CO2 to produce
[11C]oxaprozin (3). In the next step this carboxylic acid 3 was directly
reduced to yield aldehyde 4, then it was converted to the oxime 6.
The [11C]SZV 1287 was administered to male NMRI mice. The
animals were examined with dynamic PET/MR imaging for 90
minutes. Biodistribution studies were performed at 10, 30, 60 and
120 minutes post injection. The accumulation of the labelled
compound was observed in the brain of the animals. The main
excretion pathway was found to be through the liver and intestines.
These studies provide preliminary information for pharmacokinetic
characterization of the SZV 1287.
The recently described Metabolism-Activated Multitargeting
(MAMUT) concept[17] is based on the designed combination of
synergistic pharmacological effects of a parent drug and its
active metabolite(s). In order that a new type of broad-spectrum
anti-inflammatory profile could be achieved through combination
1. Introduction
Semicarbazide-sensitive amine oxidase (SSAO) is a copper
containing enzyme family which catalyses oxidative deamination
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
of two anti-inflammatory mechanisms of action, the MAMUT
concept was implemented for SZV-1287. In the case of SZV
1287 this concept means that it possesses a semicarbazide-
sensitive amine oxidase (SSAO) inhibitory effect, while its
metabolite, oxaprozin inhibits cyclooxygenase (COX) enzyme.
However, the unique pharmacodynamic profile of SZV 1287 is
due to the fact that it has a significant analgesic effect besides
its anti-inflammatory effect by antagonizing transient receptor
potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) cationic
channels on primary sensory neurons. Because of its unique
mechanism of action, it is considered to be a promising
analgesic drug candidate currently being developed for chronic
neuropathic pain, which is an unmet medical need.[12,13,18]
being performed. These PET imaging studies provide important
information for its distribution and help to understand its site of
action.
2. Results and Discussion
2.1 Radiosynthesis
11C isotope is an important positron emitter which is widely used
for the preparation of PET radiopharmaceuticals. The short half-
life (20.4 min) of this radionuclide makes its application
challenging since the radiolabelling have to be performed as fast
as possible. A five-step synthesis route was developed for the
preparation of 11C labelled 3-(4,5-diphenyl-1,3-oxazol-2-
yl)propanal oxime ([11C]SZV 1287) (Scheme 1).
Preclinical investigation of the pharmacokinetic properties of
drug candidates is a very important part of the drug development
process. Aldehyde oximes are generally metabolized fast in the
blood, thus our goal was to study the biodistribution of
radiolabelled SZV 1287 and its metabolites after intravenous
administration to provide information about its site of action.
Positron Emission Tomography (PET) is a functional imaging
technique which enables visualizing molecular events in living
organisms. Its major fields of clinical application are diagnosing
diseases, monitoring disease progression and detecting
treatment respond by using a suitable radiopharmaceutical. In
addition, PET is an effective tool for non-invasively monitoring
the biodistribution and clearance of radiolabelled bioactive
compounds and drugs. Due to its extremely high sensitivity and
selectivity, this technique is able to determine the concentration
of radiolabelled molecule at subpicomolar range, thus it has
significant advantages in the initial phases of drug development
process. PET imaging requires a radiotracer having a positron
emitter isotope built into the molecule at low concentration
(nanomolar range) and consequently it does not induce
pharmacological effect. The biodistribution studies using the
PET imaging could prove that the radiolabelled drug reaches the
tissue of interest and it does not accumulate in non-target sites
by causing potential toxicity. Therefore, this PET technique is
suitable for initial drug candidate screening in humans[19a,b] and
can contribute useful information to the conventional
pharmacokinetic (PK) studies. Labelling with the short-lived
O
Br
Ph
N
Ph
1
Mg, I2, MeI, Et2
O
40 oC, 1h
O
MgBr
Ph
N
Ph
BMPA ,
2
O
11CH2
OH
THF-Et2
1 min
O
O
O
4
11COOH
11CH
Ph
1. Et2O, -20 oC, 5min
2. 1 M HCl, 1 min
Ph
Ph
14N( p,a)11CO2
+
O
N
N
N
Ph
Ph
Ph
5
3
NH2OH.HCl
NaOAc*3 H2O, EtOH
10 min
O
11CH
Ph
N
OH
N
Ph
[11C]SZV 1287 ( 6)
Scheme 1. Radiosynthesis of [11C]SZV 1287.
The cyclotron produced [11C]CO2 is a common starting material
for the radiosynthesis of different organic molecules such as
carboxylic acids, aldehydes, and alcohols. The 11C introduction
was designed via carboxylation of a suitable Grignard reagent
(organo-magnesium halide) with [11C]carbon dioxide. First, the
synthesis of the Grignard reagent 2 was carried out from 2-(2-
bromomethyl)-4,5-diphenyl-1,3-oxazole (1) in argon atmosphere.
In the next step Grignard reagent 2 was reacted with [11C]CO2
which was produced via 14N(p,α)11C nuclear reaction by high-
energy proton bombardment of nitrogen gas in cyclotron. The
[11C]CO2 was trapped on 4Å molecular sieves. After heating, the
released [11C]CO2 was reacted with the freshly prepared
Grignard reagent at -20 °C. Then the reaction mixture was
positron emitting 11C radionuclide (t = 20.4 min) does not
½
change the chemical structure as well as biochemical features of
the molecule and results in a suitable radiotracer for PET
imaging.
O
N
N
OH
heated to room temperature and stirred for
5 minutes.
Subsequently, hydrochloric acid (1 M) was added to the mixture
to quench the reaction. Overall trapping efficiency varied
between 33% and 63%. The reaction mixture was extracted with
diethyl ether, then the organic phase containing the 11C labelled
propionic acid derivative 3 was transferred into another reactor
and concentrated. Based on radio-HPLC chromatogram the
residue contained approximately 60-70% [11C]3-(4,5-diphenyl-
1,3-oxazol-2-yl)propionic acid (3).
For the synthesis of aldehydes the most frequently used method
is the reduction of different carboxylic acid derivatives (e.g.
esters, acyl chlorides and nitriles) with mild reducing agents.
However, there are only few reducing agents and methods for
SZV-1287
Figure 1. Chemical structure of SZV 1287.
In this work we report the radiosynthesis of the 11C-labelled SZV
1287 and the evaluation of this PET probe distribution by in vivo
dynamic PET/MR imaging and ex vivo biodistribution in mice at
10, 30, 60 and 120 minutes post injection.
The detailed PK analysis and toxicity studies of the analgesic
candidate SZV 1287 for the preclinical dossier are currently
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
the direct reduction of carboxylic acids into aldehydes. Since we
intended to reduce the number of the synthetic steps as much
as possible, therefore the previously described bis(N-
methylpiperazinyl)aluminum hydride reagent (BMPA complex)
by Hubert et al.[20] was applied for the conversion of carboxylic
acid 3 into aldehyde 4. The radio-HPLC chromatogram showed
the crude product contained the 11C labelled aldehyde derivative
4 and 10-20% alcohol byproduct 5. It can be explained by the
partial overreduction of the carboxylic acid 3.
Finally, the mixture of aldehyde 4 and alcohol 5 was reacted with
hydroxyl ammonium chloride and sodium acetate in ethanol. The
reaction mixture contained 33-78% [11C]3-(4,5-diphenyl-1,3-
oxazol-2-yl)propanal oxime ([11C]SZV 1287, 6). Although the
formation of the oxime 6 from the aldehyde 4 was quantitative,
the yield was limited by the ratio of the aldehyde 4 and alcohol 5
formed in the reduction. First we intended to use the same type
reverse phase columns for analytical and semipreparative HPLC
methods too, but the application of the reverse phase column for
the purification of 11C labelled SZV1287 did not provide good
separation. Therefore, we modified our initial concept and used
normal phase silica column in the case of semipreparative HPLC
purification. Thus the isolation of the desired 11C labelled oxime
6 was performed on a normal phase silica column (WATERS
NovaPak Silica) by using ethyl acetate and dichloromethane
30:70 as an eluent (see Figure 2). The [11C]SZV 1287 (6) was
collected between 9 and 11 minutes. Two peaks can be seen in
this region of the radio-HPLC chromatogram because 11C
labelled oxime 6 is a mixture of E/Z stereoisomers and these
stereoisomers were separated from each other on the normal
phase silica column (see Figure 2), while both of them were co-
eluated on the reverse phase column, Lichrospher 100 RP18
(see Figure 3 and Figure 4).
Figure 3. Analytical radio-HPLC chromatogram of the formulated [11C]SZV
1287 using reverse phase column (Lichrospher 100 RP18). UV (254 nm);
lower: RA.
The radiochemical yield from trapped [11C]CO2 was 1.8 ± 0.1%
(end of synthesis, decay corrected) and the molar activity of
formulated [11C]SZV 1287 was 4.3-6.5 GBq/µmol. To improve
the radiochemical yield many reaction conditions were tested in
the case of each step, but these experiments did not led to
better results. Therefore, we increased the starting activity to
meet the requirements of the animal experiments. The low
radiochemical yield was caused by the four moderate yield steps
and the partial overreduction of the carboxylic acid 3. In addition,
high activity loss was observed during transfer and in the course
of the extraction steps and preparative HPLC purification (strong
absorption to the column).
2.2. Chromatographic separation of SZV 1287 and related
compounds
For the optimisation of the radiosynthesis we have developed a
radio-HPLC method. Among the tested stationary phases, the
non-endcapped Lichrospher 100 RP18 gave the best separation
utilizing the differences in the polarity and hydrogen bond
donor/acceptor ability of the functional groups of the analytes.
The aim of the chromatographic development was to separate
all compounds of interest from each other, namely 3-(4,5-
diphenyl-1,3-oxazole-2-yl)propanal oxime (SZV 1287, 6), the
precursor used for the radiolabelling reaction, i.e. (2-(2-
bromoethyl)-4,5-diphenyl-1,3-oxazole (1)), the intermediates of
the radiosynthesis (3-(4,5-diphenyl-1,3-oxazole-2-yl)propionic
acid (oxaprozin, 3), 3-(4,5-diphenyl-1,3-oxazole-2-yl)propanal
(4)) and the possibly formed side product (2-ethyl)-4,5-diphenyl-
1,3-oxazole (7)). The synthesis and the characterization of
compound 1 are described in Supplementary Material and the
other compounds were synthesized according to a previously
described method.[14]
Figure 2. Preparative HPLC chromatogram of the final reaction mixture using
normal phase silica column (WATERS NovaPak Silica).
The [11C]SZV 1287 (6) was prepared under the optimized
reaction conditions nine times starting from [11C]CO2 from 10
minutes irradiation with 44 µA beam current. The whole process
took about 70 minutes and the radioactivity of the product varied
between 8 and 70 MBq. Radiochemical purity was higher than
90% in every case. The analytical radio-HPLC chromatogram of
the formulated [11C]SZV 1287 (6) is shown in Figure 3. where
the radiochemical purity was 91.4%.
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
After quantitative SUV analysis of the dynamic PET images
(Figure 6) we found that the radiotracer accumulation in the liver
is constantly increasing in the first 4-9 minutes post injection
(SUVmean: 5-6) and then decreasing with time. In parallel the
radioactivity of the intestines increased from 30 to 90 min post
injection (SUVmean: approx. 3.5 at 90 min). Another route of
elimination from the body was observed through the urinary
system.
Figure 4. UV chromatogram (254 nm) of reference compounds using reverse
phase column (Lichrospher 100 RP18).
The HPLC chromatogram of these reference compounds is
shown in Figure 4. The compounds were baseline-separated
with symmetrical peaks and without tailing.
2.3 In vivo imaging in mice
2.3.1. In vivo biodistribution of [11C]SZV 1287
For the determination of the biodistribution of the [11C]SZV 1287
molecule, dynamic PET scans were performed using healthy
control animals. Representative decay-corrected PET/MRI
images are shown in Figure 5. By the qualitative analysis of the
PET images it was clearly visualized that [11C]SZV 1287 was
excreted through the liver and the intestines, and also the
kidneys with the urine. Notable accumulation was also found in
the Harderian-glands and moderate uptake was found in the
heart and brain (Figure 5).
Figure 6. Representative mean time-activity curve (TAC) of [11C]SZV 1287 in
selected organs and tissues of a healthy control NMRI mouse.
The elimination was faster via the kidneys than the liver.
Radioactivity appeared in the urine 25 minutes after the injection
and increased exponentially (Figure 6). No significant
accumulation was observed in other organs in the chest or in the
abdomen.
Figures 6. and 7. show that the [11C]SZV 1287 is able to cross
the blood-brain-barrier. Significant accumulation was observed
in the Harderian glands behind the eyes and in the brain in the
first minutes post injection, thereafter the radiotracer
accumulation decreased rapidly (Figure 6). It was earlier
reported that high accumulation of different radiotracers (e.g.:
18FDG, 11C-PIB, 18F-fallypride) was observed in the Harderian
glands of rodents during PET investigation,[21] which makes the
evaluation of the PET images of the brain difficult. The cause of
this uptake is not explained yet.
Figure 7. Representative PET/MRI images of a healthy control mouse after
intravenous injection of
cerebellum; red arrow: cerebrum.
[
11C]SZV 1287 (90 min summ.). White arrows:
Figure 5. Representative axial (A,D), coronal (B,E) and sagittal (C,F) PET/MRI
images of a healthy control mouse after intravenous injection of [11C]SZV 1287
(90 min summ.). White arrows: Harderian glands; grey arrows: liver; red arrow:
stomach; blue arrows: kidney; orange arrow: urine; yellow arrow: heart; black
arrow: brain.
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
The detectable radioactivity using in vivo PET imaging reached
its maximum at the first minute post injection (SUVmean
approx.: 3) in mouse brain, then decreased with time (Figure 6).
The highest uptake of [11C]SZV 1287 was found in the
cerebellum (SUVmean: 3.18-4.79) 1 minute post injection.
1287 and its metabolite (oxaprozin, 3) could be discovered in the
blood, while unidentified polar metabolites could be found in the
urine.
SZV 1287 rapidly enters the brain, and it is quickly metabolized,
but its irreversible SSAO inhibitory effect in the central and
peripheral nervous systems can explain its potent analgesic
effects. These preclinical studies reveal valuable details about
the in vivo behavior of this novel analgesic drug candidate.
2.3.2 Ex vivo biodistribution studies
The ex vivo biodistribution of [11C]SZV 1287 in healthy control
animals was assessed by gamma-counter measurements.
Higher accumulation of [11C]SZV 1287 was found in the liver and
in the small intestine than in other organs at each investigated
time point (see Supplementary material Figure 1). These results
suggested that [11C]SZV 1287 was excreted mainly through the
liver and intestines due to its lipophilic property. Furthermore,
high activity was found in the kidney, which implies that some
unidentified polar metabolites was rapidly washed out through
the urinary system. [11C]SZV 1287 concentration of all organs
decreased from 10 to 120 minutes (see Supplementary material
Figure 1). Similar results were found by Malfatti et al.[22], where
the biodistribution and pharmacokinetics of a new oxime
(RS194B) were investigated. They also found higher liver and
kidney accumulation and rapid renal clearance in guinea pigs
after intravenous administration of the radiolabelled oxime,
furthermore, this observation was dose-dependent.
4. Experimental Section
4. Materials and methods
4.1 General
All reagents and solvents were obtained from Sigma-Aldrich and VWR,
and used without further purification. For the HPLC system HPLC-MS
grade acetonitrile, methanol (Fisher Solutions) and deionized water (Milli-
Q, 18.2 MΩcm−1) were used. 3-(4,5-diphenyl-1,3-oxazole-2-yl)propanal
oxime (SZV 1287, 6), 2-(2-bromoethyl)-4,5-diphenyl-1,3-oxazole (1), 3-
(4,5-diphenyl-1,3-oxazole-2-yl)propionic acid (3), 3-(4,5-diphenyl-1,3-
oxazole-2-yl)propanal (4) and 2-ethyl)-4,5-diphenyl-1,3-oxazole (7) were
provided by Institute of Organic and Medicinal Chemistry, University of
Pécs and synthesized as described previously.[14] The synthesis and the
characterization of compound 1 are described in Supplementary Material.
4.2 Chemistry
2.3.4. Determination of metabolites
4.2.1 Cyclotron production of [11C]CO2
The investigation of metabolization of [11C]SZV 1287 was
attempted by radio-HPLC separation of the blood- and urine
samples taken during biodistribution experiments. Radio-HPLC
analysis of the samples is described in the Supplementary
Material. The low injected activity did not allow repeated
measurements. The sample with the highest activity was
examined in every case.
[
11C]CO2 was produced in a GE PETtrace cyclotron by 14N(p, α)11C
nuclear reaction using 16.5 MeV proton bombardment of nitrogen gas
containing 1 % oxygen. The beam current (44 µA) was kept constant,
and the produced activity was adjusted by changing the irradiation time
between 1 and 10 minutes. The obtained activity of [11C]CO2 was
approximately 3.4 GBq/minute.
Unfortunately the administered low activity did not make
possible the correct identification of metabolites arising from the
short half-life of the 11C isotope and the low activity of the HPLC
samples. According to the obtained radio-HPLC chromatograms
the [11C]SZV 1287 and its metabolite, the carboxylic acid 3
(oxaprozin) were identified in the blood (see Supplementary
material Figure 2), whereas unidentified polar metabolites were
detected in the urine (see Supplementary material Figure 3).
The [11C]CO2 from the cyclotron was trapped quantitatively on 4Å
molecular sieves (Alltech, 80-100 mesh) at room temperature. The
molecular sieves was heated to 350 °C and the released [11C]CO2 was
transferred with helium sweep gas (10 ml/min) to the reactor for
entrapment.
4.2.2 Radiosynthesis of [11C]SZV 1287 (6)
Radiolabelling was performed on a Scansys TracerMaker system in a
Von Gahlen hotcell. The Grignard reagent was prepared by suspending
magnesium turnings (5 mg, 0.20 mmol) in anhydrous diethyl ether (0.5
mL, freshly distilled from sodium) in the presence of catalytic amount of
iodine and methyl-iodide under argon atmosphere. Then 2-(2-bromo-
ethyl)-4,5-diphenyl-1,3-oxazol (1) (10 mg, 0.03 mmol) in anhydrous
diethyl ether (0.5 mL) was added. The mixture was heated at 40 °C for 1
hour. The [11C]CO2 from the cyclotron was trapped quantitatively on 4Å
molecular sieves (Alltech, 80-100 mesh) at room temperature. The
molecular sieves was heated to 350 °C and the released [11C]CO2 was
transferred with helium sweep gas (10 ml/min) to the reactor for
entrapment. The [11C]CO2 was bubbled into the Grignard reagent
solution at -20 °C with a low (10 ml/min) helium flow. When the activity in
the reactor reached a plateau it was heated to room temperature and left
to react for 5 min with constant helium bubbling. The reaction was
stopped with the addition of 1 mL of 1 M hydrochloric acid. [11C]Carbon
dioxide waste was collected on an Ascarite column connected after the
reactor.
3. Conclusion
We have successfully developed and accomplished
a
radiolabelling procedure for the preparation of 11C labelled 3-
(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime ([11C]SZV 1287, 6)
in four active synthesis steps. The radiosynthesis process took
about 70 minutes and resulted in 8-70 MBq labelled product with
>90% radiochemical purity. The [11C]SZV 1287 was
administered to NMRI mice to investigate its in vivo properties
with non-invasive dynamic PET/MR imaging and ex vivo
biodistribution studies. These experiments showed that the
labelled compound was accumulated moderately in the brain
and excreted through mainly the liver and the intestines.
The metabolic stability of [11C]SZV 1287 was examined with
radio-HPLC technique, but the administered low activity did not
allow precise identification of metabolites. However the [11C]SZV
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
For in vivo PET imaging studies NMRI mice (n=3) were injected with
5.44±1.28 MBq of [11C]SZV 1287 via the lateral tail vein. Animals were
anaesthetized by 3% isoflurane (Forane) with a dedicated small animal
anesthesia device (Tec3 Isoflurane Vaporizer) and dynamic (90 min)
PET scans were performed (mouse whole body) using the preclinical
nanoScan PET/MRI system (Mediso Ltd., Hungary). Reconstructed
images were analyzed using the InterView™ FUSION (Mediso Ltd.,
Hungary) image analysis software. Radiotracer uptake was expressed in
terms of standardized uptake values (SUVs). SUV was calculated as
follows: SUV = [VOI activity (Bq/mL)]/[injected activity (Bq)/animal weight
(g)], assuming a density of 1 g/mL.
Diethyl ether (2 mL) was added two times to the reaction mixture, using a
syringe pump and mixed thoroughly with bubbling helium. The organic
phase, containing 11C labelled propionic acid derivative (3) was
separated and transfered into another reactor through a needle using
helium pressure. During the transfer, the ether phase was passed
through a drying cartridge, filled with dried magnesium sulphate and 4Å
molecular sieves. The collected ethereal solution was concentrated in the
second reactor with 100 ml/min He flow at 70 °C.
After cooling, the residue was dissolved in
a solution of BMPA
complex[20] in the mixture of tetrahydrofurane and diethyl ether 1:1 (0.5
mL). After one minute reaction time saturated NaCl (1 mL) was added to
quench the reduction process. The reaction mixture was extracted with
diethyl ether (2x1 mL) manually. The ethereal solution was concentrated
to dryness to give the crude product, containing [11C]3-(4,5-diphenyl-1,3-
4.3.3 Ex vivo biodistribution studies
For ex vivo biodistribution studies NMRI mice (n=12) were injected
intravenously with 5.62±1.21 MBq of [11C]SZV 1287. After 10, 30, 60 and
120 min incubation time mice were euthanized with 5% Forane. Tissue
samples were taken from selected organs and the weight and the
radioactivities of the samples were measured with calibrated analytical
lab balance and gamma counter. The uptake was expressed as decay
corrected %ID/g tissue.
oxazol-2-yl)propanal (4).
After cooling the residue was dissolved in 96% (0.5 mL) ethanol and
mixed with a solution of 20 mg (0.29 mmol) hydroxyl ammonium chloride
and 20 mg (0.15 mmol) sodium acetate in 200 µL water. After 10 minutes
the [11C]3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime ([11C]SZV 1287,
6) was isolated with preparative HPLC. Purification of the labelled oxime
6 was performed on a WATERS NovaPak Silica 6 µm, 7.8 x 300 mm
column with ethyl acetate and dichloromethane 30:70, 4 mL/min flow with
UV (254 nm) and radioactivity detection. The product was eluted
between 9 and 11 minutes. After collection and evaporation to dryness it
was reconstituted with 5% ethanol in saline.
4.3.4. Investigation of metabolites
Samples were taken during biodistribution experiments and were
extracted with ethyl acetate and a 10 µL aliquot was injected onto the
column (see Supplementary material Figure
2 and Figure 3).
Chromatography system: Waters Acquity UPLC I-class liquid
chromatograph with a Berthold LB513 radioactivity detector, equipped
with a plastic scintillation cell.
4.2.3 Radio-HPLC method
Standard samples were prepared as follows: 1 mg material (1, 3, 4, 6
and 7) were dissolved in a 1.5 mL of a mixture 0.1 % phosphoric acid
and acetonitrile (60:40, v/v). The sample was obtained by mixing these
standard solutions. This aliquot was filtered with of 0.45 µm PTFE
Acknowledgements
syringe filter.
Authors wish to thank the Translational Imaging Centre of
Scanomed Ltd. (Debrecen, Hungary) for help provided in
PET/MRI imaging. This work was supported by Hungarian Grant
GINOP 2.2.1-15-2016-00020.
HPLC measurements were performed on a Waters Acquity UPLC I-Class
System equipped with a Binary Solvent Manager, a Sample Manager
(Flow-Through-Needle with 100 µL loop),
a Column Manager, a
Photodiode Array Detector. photomultiplier tube (Hamamatsu
A
Photonics), equipped with a plastic scintillator was used as radioactivity
detector. Data were evaluated by Empower 3 chromatography software.
The following four reversed-phase columns were tested for the
separation of the reference compounds: Kinetex XB-C18 (50 mm x 4.6
mm, 2.6 µm, 100 Å, Phenomenex), Nucleosil C18 (150 mm x 4.6 mm, 5
µm, 100 Å, Macherey-Nagel), Symmetry C18 (150 mm x 4.6 mm, 3.5 µm,
100 Å, Waters), Lichrospher 100 RP18 (250 mm x 4.6 mm, 5 µm, 100 Å,
Merck). The best separation was achieved with the Lichrospher 100
RP18 column, with the following gradient: A: 0.1 % phosphoric acid, B:
acetonitrile, from 40% B to 100% B in 8 minutes, followed by an isocratic
Keywords: carbon-11• radiochemistry • imaging agents • in vivo
biodistribution • positron emission tomography
[1]
[2]
A. M. O’Rourke, et al. J. Pharmacol Exp Ther. 2008, 342, 867–875.
(a) M. Salmi, et al. 2001, 14, 265–276.; (b) M. Salmi, S. Jalkanen,
Antioxid Redox Signal. 2019, 30, 314-332.
[3]
[4]
J. O’Sullivan, et al. Neurotoxicology. 2004, 25, 303–315.
F. Boomsma, F.H. Derkx, A. H. van der Meiracker, et al. Clin Sci (Lond).
1995, 88, 675–679.
hold for
1 minute. The separation took 13 minutes. The UV
chromatograms were integrated at λ=254 nm. Measurements were
carried out with a flow rate of 0.8 mL/min and injection volume 5-20 µL.
[5]
[6]
[7]
[8]
F. Boomsma, et al. Cardiovasc Res. 1997, 33, 387–391.
R. Kurkijarvi, et al. Gastroenterology. 2000, 119, 1096–1103.
C.S. Bonder, et al. Immunity. 2005, 23, 153–163.
S. Tokha, M. Lauknnen, S. Jalkanen, M. Salmi, FASEB J. 2001, 15,
373–382.
4.3 Biology
[9]
P.F. Lalor, et al. Vascular Adhesion Protein-1 mediates adhesion and
transmigration of lymphocytes on human hepatic endothelial cells. J
Immunol. 2002, 169, 983–992.
4.3.1 Animals
NMRI mice (male, 12 weeks old; 20-22 g; n=15) (Charles River
Laboratories) were housed under sterile conditions in IVC cages at a
temperature of 26±2 °C, with 50±10% humidity and artificial lighting with
a circadian cycle of 12 h. Semi-synthetic diet (VRF1; Akronom Ltd.,
Budapest, Hungary) and drinking water were available ad libitum to all
the animals. Laboratory animals were kept and treated in compliance
with all applicable sections of the Hungarian Laws and regulations of the
European Union. Ethical license number: 6/2018/DEMÁB.
[10] C.M. Stolen, et al. Absence of the endothelial oxidase AOC3 leads to
abnormal leukocyte traffic in vivo. Immunity. 2005, 22, 105–115.
[11] T. Tábi, E. Szökő, A. Mérey, V. Tóth, P. Mátyus, K. Gyires, Study on
SSAO enzyme activity and anti-inflammatory effect of SSAO inhibitors
in animal model of inflammation. J Neural Transm (Vienna). 2013, 120,
963-967.
[12] Á. Horváth, A. Menghis, B. Botz, É. Borbély, et al. Analgesic and Anti-
Inflammatory Effects of the Novel Semicarbazide Sensitive Amine-
Oxidase Inhibitor SzV-1287 in Chronic Arthritis Models of the Mouse
Scientific Reports 2017, 7, 39863.
4.3.2 In vivo PET/MRI imaging
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
[13] Á. Horváth, V. Tékus, N. Bencze, N. Szentes, et al. Analgesic effects of
the novel semicarbazide-sensitive amine oxidase inhibitor SZV 1287 in
mouse pain models with neuropathic mechanisms: Involvement of
transient receptor potential vanilloid
1 and ankyrin 1 receptors
Pharmacol Res. 2018, 131, 231-243.
[14] P. Mátyus, et al. Compounds for inhibiting semicarbazide-sensitive
amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1) and uses
thereof for treatment and prevention of diseases. PCT international
application WO/2010/029379 (2010).
[15] Z. Helyes, P. Mátyus, V. Tékus, B. Scheich, Semicarbazide-sensitive
amine-oxidase inhibitors, as analgesics in traumatic neuropathy and
neurogenic inflammation. Hungarian and USA PCT P1400205 (2014).
[16] M. Payrits, É. Sághy, P. Mátyus, A. Czompa, et al. A novel 3-(4,5-
diphenyl-1,3-oxazol-2-yl)propanal oxime compound is
a
potent
Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and V1)
receptor antagonist, Neuroscience. 2016, 324, 151–162.
[17] P. Mátyus and C.L.L. Chai, Metabolism-Activated Multitargeting
(MAMUT): An Innovative Multitargeting Approach to Drug Design and
Development, Chem Med Chem. 2016, 11, 1197–1198
[18] B. Botz, K. Bölcskei, Z. Helyes, Challenges to Develop Novel Anti-
Inflammatory and Analgesic Drugs WIREs Nanomed. Nanobiotechnol.
2017, 9, e1427.
[19] (a) M. Bergstrom, A. Grahnen, B. Langstrom B. Positron emission
tomography microdosing: a new concept with application in tracer and
early clinical drug development. Eur J Clin Pharmacol. 2003; 59, 357-
66; (b) P.M. Matthews, E.A. Rabiner, J. Passchier, R.N. Gunn, Positron
emission tomography molecular imaging for drug development. Br J
Clin Pharmacol. 2011; 73, 175-186.
[20] T.D. Hubert, D.P. Eyman, D.F. Wiemer, A Convenient Synthesis of
Bis(N-methylpiperaziny1)aluminum Hydride:
A
Reagent for the
Reduction of Carboxylic Acids to Aldehydes. J Org Chem. 1984, 49,
2278-2279.
[21] M. Kim, S-K. Woo, J.W. Yu, Y.J. Lee, et al. Effect of Harderian
adenectomy on the statistical analyses of mouse brain imaging using
positron emission tomography. J Vet Sci. 2014, 15, 157–161.
[22] M.A. Malfatti, H.A. Enright, N.A. Be, et al. The biodistribution and
pharmacokinetics of the oxime acetylcholinesterase reactivator
RS194B in guinea pigs. Chem Biol Interact. 2017; 277, 159-167.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000389
ChemMedChem
FULL PAPER
Entry for the Table of Contents
A radiosynthesis has been developed for the preparation of [11C]SZV 1287. This radiolabelled compound was evaluated by in vivo
dynamic PET/MR imaging and ex vivo biodistribution studies. The [11C]SZV 1287 was accumulated in the brain of NMRI mice and
excreted through the liver. These studies provide preliminary details for the development of analgesic drug candidate SZV 1287.
8
This article is protected by copyright. All rights reserved.