Synthesis, radiolabelling and initial biological characterisation of 18F-labelled xanthine derivatives for PET imaging of Eph receptors

Article abstract of DOI:10.1039/d0ob00391c

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC₅₀ values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([¹⁸F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([¹⁸F]3) rather than the methyl group of the phenoxy moiety ([¹⁸F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [¹⁸F]2 and [¹⁸F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq μmol^-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [¹⁸F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [¹⁸F]2 did not accumulate at these cells. Since both tracers [¹⁸F]3 and [¹⁸F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.

Full text of DOI:10.1039/d0ob00391c

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Biomolecular Chemistry
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Synthesis, radiolabelling and initial biological
characterisation of 18F-labelled xanthine deriva-
Cite this: DOI: 10.1039/d0ob00391c
tives for PET imaging of Eph receptors†‡
Marc Pretze, §¶a,b Christin Neuber, §a Elisa Kinski,a Birgit Belter,a
a,b
Martin Köckerling, c Amedeo Caflisch, d Jörg Steinbach,
a,b
Jens Pietzsch a,b and Constantin Mamat
*
Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for mole-
cular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives
were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range
(1–40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase
domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase
inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-
binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at
the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl
group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis
route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert
fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield
with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol−1. In line with the docking
studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2
and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these
cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable
for in vivo molecular imaging of Eph receptors with PET.
Received 22nd February 2020,
Accepted 31st March 2020
DOI: 10.1039/d0ob00391c
rsc.li/obc
kinases and, at present, comprises 14 members divided into
two groups, A and B.1 Their corresponding, membrane-
Introduction
The Eph (erythropoietin-producing hepatoma cell line) recep- attached (GPI-anchored) ephrin
A and (transmembrane
tor family represents the largest subfamily of receptor tyrosine located) ephrin B ligands bind to the Eph receptors in a juxta-
crine manner which provides the condition for Eph receptor
forward and ephrin reverse signaling.2
aHelmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazeutische
Eph receptors and their ephrin ligands play a crucial role
Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden, Germany.
during embryogenesis by regulating spatial patterning, vascu-
E-mail: c.mamat@hzdr.de
bTechnische Universität Dresden, Fakultät Chemie und Lebensmittelchemie, D-01062
lar development, and axon guidance.3,4 Moreover, the Eph/
ephrin system is involved in tissue homeostasis, for instance
of bone, intestine, and the immune system.5,6 All these effects
are based on the regulation of cell–cell communication as well
as cell attachment, shape, and motility by Eph receptors and
their ephrin ligands.7
Beside their role during developmental processes it became
evident that the Eph/ephrin system plays an important role in
the pathogenesis of many diseases.5 In this regard, Eph recep-
tors are often dysregulated in cancer, whereby expression can
be both up and down regulated.8 Eph receptors can act both as
tumour promotor and tumour suppressor, depending, e.g., on
the cellular context, ligand stimulation, and the kind of result-
Dresden, Germany
cUniversität Rostock, Institut für Chemie – Anorganische Festkörperchemie, Albert-
Einstein-Straße 3a, D-18059 Rostock, Germany
dDepartment of Biochemistry, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland
†Dedicated to the memory of Prof. Rudolf Münze (Central Institute for Nuclear
Research Rossendorf, Germany), deceased in February 2020.
‡Electronic supplementary information (ESI) available: NMR spectra of com-
pounds. CCDC 1976810 and 1976126. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/d0ob00391c
§These authors contributed equally to this work.
¶Current
address:
Klinik
und
Poliklinik
für
Nuklearmedizin,
Universitätsklinikum Carl Gustav Carus der TU Dresden, Fetscherstraße 74,
D-01307 Dresden, Germany.
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ing signaling crosstalk.8,9 In particular, EphA2 and EphB4 results prompted us to search for an improved lead structure.
receptors are highly expressed in a number of tumour entities, In this regard, compound 1 (designated as compound 3 in ref.
including breast, prostate, lung, and ovarian cancer, and are 30) containing a xanthine skeleton published by Nevado in
often associated with poor prognosis8,10 as well as therapy 2009 and 2013 was chosen as lead compound.30,31 For lead
resistance.11,12 In brief, EphA2 receptor induces proliferation, compound 1, a high affinity in the low nanomolar range
migration, and invasion of tumour cells, resulting in higher (1–40 nm) was described for 10 Eph receptors including
tumourigenicity and enhanced tumour vascularisation.1,13 EphA2 and EphB4, the two receptors which are mainly
EphB4 and its cognate ligand ephrin B2, by contrast, affect involved in cancer progression and therapy resistance.
tumour growth mostly by regulation of tumour angiogenesis
In the present paper, a synthesis route to two novel fluo-
and vascular remodelling, which also may result in an rine-18-containing derivatives ([18F]2/3) of lead compound 1
unfavourable tumour microenvironment, e.g. hypoxia.14–16 In and the respective precursors for radiolabelling as well as a
line with this, EphB4 has been linked to tumour therapy resis- labelling procedure to insert fluorine-18 is presented. Prior to
tance, for instance when using the DNA damaging agent cis- synthesis and radiolabelling, in silico docking studies were
platin or the VEGF targeting antibody bevacizumab.17,18 accomplished to find the best position for labelling with fluo-
Therefore, they are emerging targets for the functional charac- rine-18. Based on these results first in vitro experiments were
terisation of tumours by non-invasive molecular imaging pur- performed to investigate both binding behavior to EphA2 and
poses like positron emission tomography (PET) or single EphB4 overexpressing human A375 melanoma cells and meta-
photon emission computed tomography (SPECT), and the deri- bolic stability.
vation of corresponding therapeutic applications.
In light of the involvement of the Eph/ephrin system in
several pathologies including cancer, a wide range of agents
targeting Eph receptors and their corresponding ephrins arose
in the last years. To date, several potent Eph kinase inhibitors
Results
In silico design
are reported in the literature either based on biomacro- As most of the pharmaceuticals in clinical use do not contain
molecules like peptides,19–21 which bind to the extracellular fluorine, an isosteric radiolabelling with fluorine-18 is not
ligand binding domain of the appropriate receptor, or based possible if these molecules are also interesting for molecular
on small organic molecules,22 which are able to block the imaging. Instead of this, single hydrogen atoms or functional
intracellular ATP-binding pocket. Selected compounds were groups like hydroxy groups are typically replaced by fluorine-
chosen to be radiolabelled with either technetium-99m 23 for 18.32 However, this replacement can lead to a change in the
SPECT or with copper-64 24 and fluorine-18 25 like peptide I 26 pharmacological behavior of the compounds. Thus, the search
as well as with carbon-11 like derivative IIIa 27 for PET of an acceptable labelling position is mandatory and can be
(Scheme 1).
performed by docking studies, particularly, if the (holo) struc-
In the past, we developed the fluorine-18-containing radio- ture of the target protein has been reported.
tracers IIb 28 based on the benzodioxolylpyrimidine structural
The chosen lead compound 1, containing a xanthine skel-
motif and IIIb 29 containing the indazolylpyrimidine core. eton, has been published by the group of Nevado.30 To investi-
Compound IIb showed a substantial uptake in human mela- gate local selectivity (inhibitory activity on a single branch of
noma cells (A375) in vitro but no uptake in the corresponding the kinome dendrogram), lead compound 1 was tested by an
melanoma xenograft model in vivo despite of the high affinity enzymatic assay with [γ-33P]ATP against a panel of 11 Eph
of the original compounds to the EphB4 receptor. These receptor kinases. Due to the high sequence identity of Eph
receptors (60–90%) and their small gatekeeper residue threo-
nine in the ATP-binding site, compound 1 showed IC50 values
in the nanomolar range for nearly all Eph receptors (IC50: 2.9
nM (EphA1), 2.3 nM (EphA2), 3.3 nM (EphA4), 3.0 nM
(EphA5), 4.5 nM (EphA8), 1.1 nM (EphB1), 1.2 nM (EphB2),
and 1.6 nM (EphB4)).30 In general, the size of the gatekeeper
residue of the hydrophobic pocket of the ATP-binding site
determines affinity as well as selectivity of RTK inhibitors.33
Both EphA2 and EphB4 belong to the subfamily of kinases
(∼20% of the 518 human kinases) with a small (threonine)
gatekeeper (Thr693).
Moreover, compound 1 was tested for its global selectivity
(inhibitory activity on the whole kinome) and was shown to
strongly inhibit six further kinases (out of 85 tested), namely
Src, Lck, Yes1, CSK, BTK, and HER-4, which all have a threo-
nine gatekeeper.30 Overall, lead compound 1 showed high
Scheme 1 Structures of 11C- and 18F-labelled radiotracers I–III based
on Eph inhibitors prepared by our group.
inhibition against 21 out of 143 tested kinases, about half of
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which were chosen because of their small gatekeeper residue, of the phenyl ring. In contrast, the fluorinated propyl residue
and 10 of them are Eph kinases. of compound 3 could be accommodated without steric con-
Explicit solvent molecular dynamics simulations of the flicts as it points towards the exposed region of the ATP-
complex of the kinase domain and a precursor of lead com- binding site (Fig. 1).
pound 1 (compound 3 in ref. 30), had revealed involvement of
carbonyl C6vO and amide group N1–H in hydrogen bonds
Chemistry
with the backbone polar groups of Met696 of EphB4.30 For the
development of the two novel 18F-based radiotracers [18F]2 and
N-Modified aminouracils were applied as basis for the prepa-
ration of the original lead compound 1, both 18F-radiotracers
[18F]3, we decided to choose two sites of the original inhibitor
[18F]2,3, the non-radioactive reference compounds 2,3 and
that are not directly involved in binding interactions.
Radiotracers [18F]2 and [18F]3 are based on the replacement of
their respective precursors 14a,b. In the first step, unsymmetri-
cally alkylated urea derivatives 4a–c were prepared from the
a methyl group by a fluoropropyl group at the phenoxy moiety
and uracil residue of the lead compound 1, respectively
(Scheme 2). Advantageously, the fluoropropyl moiety is less
sterically hindered and has a comparable in vivo stability as
respective amines and sodium cyanate.36 Next, compounds
4a–c were reacted with ethyl 2-cyanoacetate and sodium etha-
nolate in a Traube purine synthesis37 to the N-substituted
uracil derivatives 5a–c. The preparation of the bromoxanthins
fluoroethyl, but it is much more stable compared to the fluoro-
was accomplished according to the literature.30 For this
methyl residue.34,35
purpose, all three uracil derivatives 5a–c were treated with
Starting from the crystal structure of compound 1 in the
NaNO2 under acidic conditions which resulted in the nitroso
derivatives 6a–c (yields: 79–83%). Next, the diamino com-
pounds 7a–c were prepared by the reduction of 6a–c with
sodium dithionite. Due to the instability of the free diamines,
compounds 7a–c were subsequently converted into the respect-
ive xanthines 8a–c by treatment with triethyl orthoformate and
complex with the kinase domain (PDB code 4GK2), manual
docking of the two derivatives was carried out with the
program WITNOTP, which is available from the homepage of
one of the authors (AC). The docking of compound 2 resulted
in steric clashes of the propyl chain with the N-terminal or
C-terminal domain of the kinase depending on the orientation
p-TsOH. The last step required the insertion of bromine.
Therefore, 8a–c were treated with elemental bromine for 3 h at
65 °C. Bromo derivatives 9a–c were obtained in high yields of
74–84%. The whole reaction sequence to the brominated
xanthines starting from the appropriate ureas is shown in
Scheme 3.
During the synthesis of the urea derivatives 4a–c and the
bromo xanthines 9a–c, it was possible to obtain single crystals
of 4b and 9a suitable for single crystal X-ray analyses. The
molecular structures of 4b and 9a are presented in Fig. 2 and
3, respectively.
The compound 4b crystallises in the monoclinic and 9a in
Scheme 2 Literature known lead compound 1 and proposed structures
of fluorine-containing-compounds 2 and 3 with highlighted labelling
positions.
the triclinic crystal system. The structures confirm the identi-
ties of the two compounds and the correctness of the reaction
pathways. All bond distances in both structures are within the
expected ranges. In crystals of 4b, the molecules are arranged
Fig. 1 Predicted binding mode of compound 3 in the ATP-binding site
of the EphA3 receptor. The position of the fluorine atom is emphasised
by a cyan sphere and hydrogen atoms are shown only for the propyl
chain.
Scheme 3 Reaction sequence to brominated xanthine derivatives 9a–c
starting from urea derivatives 4a–c.
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Fig. 2 Molecular structure of 4b in the crystal with atom labelling
scheme (ORTEP plot, 50% probability level).
Scheme 4 Reaction sequence to the fluorinated reference compounds
2,3 and their tosylated precursors 14a,b.
Fig. 3 Molecular structure of 9a in the crystal with atom labelling
scheme (ORTEP plot, 50% probability level).
The full reaction sequence to the final compounds is shown in
Scheme 4.
The last step to prepare the non-radioactive reference com-
pounds 2 and 3 involved the introduction of fluorine into 13a,
b. Attempts to use 14a,b for a nucleophilic introduction of flu-
oride were not successful. Alternatively, hydroxy compound
13a was treated with Deoxofluor in anhydrous THF under
ambient temperature for 1 h which led to reference compound
3 in a yield of 10%. The second reference compound was syn-
thesised from 13b with DAST which led to 2 in a yield of 80%.
Treatment of 13a,b with p-TsCl in N-methylmorpholine yielded
the desired precursors 14a (96%) and 14b (77%) for the later
radiolabelling procedure. The use of DMF as solvent should be
avoided due to the formation of unexpected by-products.39
such that hydrogen bonds are formed between the NH2 group
(around N1) and the O1 atoms of neighbouring molecules at
distances of 2.927(1), 2.948(1), and 3.033(1) Å. In crystals of 9a
the molecules are arranged, such that the xanthine units as
well as the phenyl rings of the benzyl group are arranged paral-
lel to same units of neighboring molecules, but outside of dis-
tances of π–π-interactions. Hydrogen bonds exist between the
H atoms of both NH groups (N1 and N3) to O1 and O2,
respectively, of neighboring molecules at distances of 2.6875
(9) and 2.817(1) Å.
The next reaction sequence to obtain the final compounds
started with the synthesis of 2-bromo-1-(5-hydroxy-2-methyl-
phenyl)ethanone (10),30 which was then connected to
xanthines 9a–c. The desired compounds 11a–c were obtained
Radiolabelling
in nearly quantitative yields of >99%. At this stage, the ben- The radiosyntheses of both radiotracers [18F]2 and [18F]3 were
zylgroup of 11a as well as the methyl group of 11b were inevita- achieved in a one-step procedure by classical aliphatic nucleo-
ble to be cleaved with BBr3 in dichloromethane to give 11d philic substitution of the tosylate group in precursors 14a,b
(yield: 99% starting from 11a and 84% starting from 11b). The with n.c.a. [18F]fluoride as shown in Scheme 5. Necessarily, the
final cyclisation reaction of 11c,d with alkoxyanilines 12a,b to tosyl group attached to the phenyl moiety of 14a,b has to be
the imidazopurinediones 13a–c was accomplished under cleaved simultaneously. Labelling conditions were optimised
Lewis acid catalysis (AlCl3), which is important for the success for [18F]2 with regard to various reaction parameters such as
of the reaction. Otherwise, only open-chained products were solvent, reaction temperature, synthesis time, and also in com-
obtained.38 The synthesis was executed either with 2-methox- bination with microwave (mw) conditions.
yaniline (12a) and 9c to yield 13a (62%) or with 2-(3-hydroxy-
The use of [18F]TBAF in t-butanol40 and acetonitrile under
propoxy)aniline (12b) and 11b to give 13b (83% yield). A standard heating and mw conditions (15 and 30 W) for 10 min
further optimisation consisted in the application of four showed no conversion. When using 70 W for 10 min, a new
equivalents of the respective aniline derivative (12a or 12b). In spot was detected on the TLC with a Rf value of 0.55 (solvent:
addition, the original inhibitor 13c was also obtained by the chloroform/MeOH = 9/1, silica gel), but not at the Rf value of
reaction of 11c with 2-methoxyaniline (12a) in a yield of 88%. the reference compound 2 (Rf = 0.37). The more lipophilic spot
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Table 1 Evaluation of the labelling conditions used for [18F]2
Entry Conditions Heating Time
18F]15
[
[
18F]2
1
2
3
4
[
[
[
18F]TBAF, CH3CNa
18F]TBAF, CH3CNa
18F]TBAF, CH3CN
rt
60 min
0
0
5
2
0
0
0
30 W
70 W
rt
10 min
60 min
10 min
K[18F]F, K222, K2CO3,
CH3CN
Trace
5
6
7
8
K[18F]F, K222, K2CO3,
CH3CN
30 W
50 W
50 W
100 W
10 min
5
2
K[18F]F, K222, K2CO3,
CH3CN
40 min <2
70 min
10 min Dec.
10
13
Dec.
K[18F]F, K222, K2CO3,
CH3CN
0
K[18F]F, K222, K2CO3,
CH3CN
a tert-BuOH was tested as well
Scheme 5 Radiolabelling procedure for the synthesis of tracers [18F]2
and [18F]3.
indicated the still tosyl group-containing tracer [18F]15a of
approx. 5%.
The use of K[18F]F/K222/K2CO3 mixture in acetonitrile at
room temperature yielded the spot at Rf = 0.55 as well and a
very weak spot at Rf = 0.37. Finally, the use of mw conditions
(10 min, 30 W) with the afore mentioned labelling conditions
was successful and delivered a spot for the desired product at
Rf = 0.37. An elongation of 40 min at 50 W delivered the tracer
[18F]2 in 10% RCY and tosylated [18F]15a in <2% RCY (Fig. 4).
Finally, after 70 min at 50 W only the desired product [18F]2
was detected in 13% RCY (Table 1 and Fig. 5). The unusual
long labelling time is related to the cleavage of the second
tosyl group of the former precursor molecule, which occurred
as well under these conditions, but very slowly in comparison
to the radiofluorination step. Thus, the by-products [18F]15a,b,
still containing the tosyl group bound to the phenol unit, were
Fig. 5 (Radio-)HPLC chromatograms of purified [18F]2 (γ-trace: red line,
tR = 13.7 min), reference compound 2 (UV-trace: bold black line, tR
=
13.7 min) and precursor 14a (UV-trace: black line, tR = 17.4 min).
first formed and then [18F]2/3 after an elongation of the reac-
tion time.
The purification of [18F]2 was executed using two
Chromafix® C18 cartridges. For this purpose, the reaction
mixture was diluted with deionised water and the resulting
solution was transferred to the cartridges. The first elution was
done with deionised water to remove remaining [18F]fluoride.
The still trapped [18F]2 was eluted with MeOH and then finally
purified using semipreparative HPLC to obtain 21–69 MBq
(1–6% d.c.) RCY with a Am = 10.3 5.1 GBq µmol−1. Radio-TLC
and radio-HPLC analyses showed no other radioactive by-pro-
ducts (Fig. 5). Radiotracer [18F]3 was prepared via a two step
procedure. First, the aliphatic tosylate group was converted
under standard radiofluorination conditions using K222
,
K2CO3 and [18F]F− at 100 °C for 30 min, whereas the aromatic
tosylate was not affected. Afterwards, aqueous NaOH was
added and the mixture was again heated at 100 °C for 15 min
to cleave the remaining aromatic tosyl group. Purification was
done via semi-preparative HPLC to obtain [18F]3 in RCYs
between 3–5%.
Fig. 4 Radiolabelling
of
[
18F]2:
radio-TLC
analysis
(eluent:
CHCl3 : MeOH 9 : 1), [18F]2 (Rf = 0.37) and [18F]15a (Rf = 0.55) after
40 min at 50 watt.
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Radiopharmacological investigations
To give a first impression of the later (radio-)pharmacological
behavior of the radiofluorinated tracers, log P(D) values were
calculated and compared with the experimentally determined
values for the fluorinated compounds 2 and 3 as well as the
lead compound 1 using ChemDraw and ACD labs (Table 2).
Experimental determination using the shake flask method
revealed suitable log P values of 2.2 and 2.9 for compound 2
and 3, respectively, which are comparable with the log P value
of the lead compound 1.
Cellular binding and uptake of [18F]2 and [18F]3 was accom-
plished using A375 melanoma cells with low Eph receptor
expression (A375) as well as transfected A375-EphA2 and A375-
EphB4 cells, which are characterised by a substantial increased
expression of EphA2 and EphB4, respectively (Fig. 6A).
Cellular binding and uptake of radiotracer [18F]2 was lower
than the uptake of [18F]3 resulting in less and more than
50% ID mg−1 (injected dose per mg protein), respectively
(Fig. 6B/C). With regard to [18F]2 there was no difference in cel-
lular tracer binding and uptake between non-transfected A375
cells and A375-EphA2 or A375-EphB4 cells (Fig. 6B). The
missing increase of tracer binding and uptake by time may
result from a lack in Eph receptor affinity and/or an active
tracer efflux due to activity of multi drug resistance (MDR) pro-
teins like P-glycoprotein (P-gp). With regard to the latter,
Lafleur et al. already recognised that compound 1 is a sub-
strate for P-gp transporters, since inhibitory activity of com-
pound 1 decreased by almost two orders of magnitude when
investigating IC50 in EphB4-overexpressing cells instead of by
using recombinant protein (130 nM in cellular assay vs. 5 nM
in enzymatic assay) as a result of efficient substrate efflux.31 In
contrast to [18F]2, cellular binding and uptake of [18F]3 was
remarkably increased in A375-EphA2 and A375-EphB4 cells in
comparison to A375 cells, amounting up to 110 and 90% ID
mg−1, respectively (Fig. 6C).
The specificity of radiotracer uptake was tested by perform-
ing blocking experiments with all three cell lines after pre-
incubation with lead compound 1 or the non-radioactive refer-
ence compound 2. Cellular binding and uptake of [18F]2 in
A375-EphA2 cells could be significantly blocked by pre-incu-
bation with compound 2 (100 µM) at all investigated time
points, whereas tracer accumulation in A375-EphB4 cells could
be blocked only after 120 min (Fig. 6D). Pre-incubation with
lead compound 1 (50 µM) significantly decreased tracer
accumulation in all three cell lines at 30, 60, and 120 min
(Fig. 6E). With regard to this, it has to be kept in mind that
lead compound 1 showed IC50 values in the low nanomolar
Fig. 6 Cellular binding and uptake of radiotracer [18F]2 and [18F]3 over
a period of 120 min at 37 °C in non-transfected as well as EphA2- and
EphB4-transfected A375 melanoma cells. (A) Representative Western
blots for EphA2 and EphB4 in (1) A375, (2) A375-EphB4, and (3) A375-
EphA2 cells. (B/C) Cellular binding and uptake of radiotracers [18F]2 and
[
18F]3 in A375, A375-EphA2, and A375-EphB4 cells after 5, 30, 60, and
120 min at 37 °C. (D/E) For blocking experiments, cellular binding and
uptake of radiotracers [18F]2 and [18F]3 was investigated in all three cell
lines after pre-incubation with compound 2 and lead compound 1,
respectively. Values represent mean
standard deviation (SD) from at
least two independent experiments each performed in quadruplicate.
range (1–40 nM) against almost all Eph receptors due to their
high sequence identity (60–90%) and their small gatekeeper
residue (threonine) of the hydrophobic pocket of their ATP-
binding site.13 Since we could demonstrate mRNA expression
of EphA1–4, EphA7, and EphB1-B4 in A375 cells in a previous
study,48 this might be the reason for the inhibition of
radiotracer binding and uptake even in non-transfected A375
cells.
First in vitro stability tests were accomplished with [18F]2
and [18F]3 in rat blood and plasma at 37 °C for up to 60 min
and analysed via radio-TLC and radio-HPLC. No radiodefluori-
nation or other degradation processes were observed for both
[18F]2 and [18F]3 within at least 60 min. Further in vivo metab-
olite analyses using rats showed a fast blood clearance of [18F]2
and [18F]3 at 60 min p.i. resulting in radioactivity concen-
trations insufficient for metabolite analysis by radio-TLC or
radio-HPLC. Additionally, a nearly complete degradation of
[18F]2 and [18F]3, respectively, to a more hydrophilic metabolite
was found in the urine. Exemplarily, PET imaging with [18F]2
confirmed a very fast blood clearance and elimination via
hepatobiliary and, of minor importance, via renal route even
after 5 min p.i. in the rat (Fig. 7).
Table 2 Calculated and determined log P(D) values for 2, 3, and the
lead 1
Compound
Lead 1
2
3
Calculated log P41
Calculated log D(pH 7.4)
Determined log P
2.86
3.04
—
3.16
3.48
2.2
3.16
3.44
2.9
42
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rate and metabolic stability in vivo to finally assess feasibility
of our newly developed radiotracer [18F]3 for in vivo molecular
imaging with PET. Such radiotracers may allow (i) for charac-
terisation of tumours and metastasis with regard to their mole-
cular fingerprint and the functional expression of (‘drugable’)
targets and (ii) for monitoring of (early) effects of (Eph) tar-
geted therapies. As an example, an on-going phase I clinical
trial (NCT03374943) is investigating the EphA3-targeting anti-
body KB004 in patients with recurrent glioblastoma.45,46 By
combining an initial PET imaging sequence using [89Zr]KB004
followed by the therapeutic application of KB004 promising
clinical responses in the absence of any dose-limiting toxicities
or drug-related side effects have been reported initially. With
regard to the approach chosen here, we generally point out
that a high degree of selectivity is not always desirable for
therapeutic purposes, and that inhibitors targeting multiple
kinases, for example, which are involved in tumor angio-
Fig. 7 Representative PET images (MIP, maximum intensity projection)
after i.v. injection of [18F]2 into a rat. To illustrate fast elimination of
[
60–300 s, and 300–600 s) up to 10 min p.i. Different organs are marked
as H-heart, V-vein, K-kidney, L-liver, I-intestine. Images are depicted at
same SUV scale.
18F]2 MIPs are delineated for different time frames (0–30 s, 30–60 s,
Discussion
Due to promiscuity of the Eph/ephrin system and missing genesis or cancer progression, may have an increased efficacy
selectivity of radiotracers targeting the hydrophobic pocket of and a better ability to circumvent resistance mechanisms. In
the ATP-binding site43,44 we consciously decided to test a quantitative molecular imaging it should be carefully con-
multi-Eph receptor tracer approach by choosing an imidazo- sidered whether, for example, multi-Eph targeting is desirable.
purinedione derivative developed by Nevado/Lafleur (lead com- This depends on the type of tumor under investigation and its
pound 1) as lead compound for radiolabelling with fluorine- molecular fingerprint, especially with regard to the up- or
18. Due to the high sequence identity of Eph receptors down-regulation of the individual Eph receptors. A very selec-
(60–90%) and their small gatekeeper residue (threonine) in the tive tracer approach can make an important contribution to
hydrophobic pocket of the ATP-binding site, lead compound 1 the identification of exactly this individual molecular finger-
showed IC50 values in low nanomolar range (1–40 nM) against print, which in turn provides important information on the
EphA1–5, EphA8, and EphB1–4. For EphA2 and EphB4, which response to therapy.
are mainly involved in cancer progression and therapy resis-
tance, IC50 values in an enzymatic assay using [γ-33P]ATP were
2.3 nM and 1.6 nM, respectively. Moreover, lead compound 1
was shown to strongly inhibit six further kinases (out of 85
Experimental
Materials and methods
tested), namely Src, Lck, Yes1, CSK, BTK, and HER-4, which all
have a threonine gatekeeper.30
All reagents were purchased from commercial suppliers and
For purposes of nuclear imaging, especially with PET, we were used without further purification. Compounds 4b–7b
developed two derivatives of lead compound 1, each bearing a were prepared according to van Muijlwijk-Koezen et al.44 and
[18F]fluoropropyl side chain instead of a methyl group at the compounds 4c–13c were prepared according to Lafleur et al.30
phenoxy ring ([18F]2) and uracile ([18F]3), respectively. Docking Analytical TLC was performed on pre-coated Silica Gel 60 F254
studies suggested a diminished binding potential to the plates (Merck) and results read under UV-light (λ = 254 nm).
EphB4 receptor for [18F]2. However, in vitro assay pointed out a 1H NMR, 13C NMR and 19F NMR spectra were recorded on a
high stability of [18F]2 in rat blood. In first cellular binding Varian Inova-400 or an Agilent 400-DD2 spectrometer with
and uptake experiments, we could not observe a time-depen- ProbeOne at 400, 101, and 376 MHz, respectively. Chemical
dent increase in cellular binding and uptake of [18F]2 in shifts are reported in ppm with tetramethylsilane (1H, 13C) and
EphA2- or EphB4-overexpressing A375 melanoma cells, which trichlorofluoromethane (19F) as internal standard, respectively.
is in line with the docking prediction, but might also be the MS spectra were obtained on a Micromass Quattro-LC spectro-
consequence of MDR protein, e.g., P-gp, mediated tracer efflux. meter using electron spray (ESI) as ionisation method. Melting
Therefore, we focused on compound [18F]3, bearing a [18F] points were recorded on a Cambridge Instruments Galen III
fluoropropyl side chain instead of a methyl group at the uracil apparatus and are uncorrected. Single-crystal X-ray diffraction
residue. Docking experiments predicted a conserved binding data of 4b (CCDC 1976810) and 9a (CCDC 1976126)‡ were col-
affinity for this compound. In line with this, we found an lected with a Bruker-Nonius Apex-X8 CCD-diffractometer using
increased cellular binding and uptake of [18F]3 to graphite-monochromated MoKα radiation (λ = 0.71073 Å). The
A375 melanoma cells overexpressing EphA2 or EphB4. diffraction measurements were done at −150 °C. The struc-
Moreover, blocking with compound 1 revealed specificity of tures were solved by Direct Methods and refined against F2 on
binding. Since this tracer was quite stable in rat blood in vitro all data by full-matrix least-squares using the SHELX suite of
over 60 min and, therefore, might be suitable for in vivo appli- programs.47 All non-hydrogen atoms were refined anisotropi-
cations, future studies now have to focus on blood clearance cally; all hydrogen atoms bound to C atoms were placed on cal-
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culated positions and refined using a riding model. Final R
6-Amino-1-(3-(benzyloxy)propyl)-5-nitrosopyrimidine-2,4
values converged at R1 = 0.035 (4b) and R1 = 0.030 (9a). (1H,3H)-dione (6a). Compound 5a (1.46 g, 5.30 mmol) was
Analytical HPLC was performed on a VWR/Hitachi Elite La suspended in a mixture of water (30 mL) and acetic acid
Chrome HPLC system, equipped with a reverse phase column (12 mL), NaNO2 (548 mg, 7.96 mmol) was added and the
(Nucleosil 100-5C18 Nautilus), a UV-diode array detector mixture was maintained at 50 °C for 1 h and at 4 °C overnight.
(254 nm) and a scintillation radiodetector (Raytest, Gabi Star) The resulting purple precipitate was filtered and washed with
at a flow rate of 1 mL min−1 (eluent: acetonitrile/water, 30 : 70 ice water. The resulting solid was dried at 80 °C for 3 h and
+ 0.1% TFA). The radioactive compound was identified using compound 6a was obtained as deep purple solid (1.13 g, 70%);
analytical radio-HPLC by comparison of the retention time tR mp 118–120 °C; Rf = 0.34 (ethyl acetate : EtOH = 6 : 1); 1H NMR
of the reference compound. Semi-preparative radio-HPLC was (400 MHz, DMSO-d6): δ 13.34 (s, 1H, NH2), 11.48 (s, 1H, NH),
performed on a Jasco HPLC system (Nucleosil Standard 9.06 (s, 1H, NH2), 7.35–7.25 (m, 5H, HAr), 4.42 (s, 2H, CH2Ph),
3
3
C18 100 Å 7μ, Macherey-Nagel, 250 × 16 mm, eluent: CH3CN/ 3.88 (t, J = 7.2 Hz, 2H, NCH2), 3.48 (t, J = 6.1 Hz, 2H, OCH2),
H2O + 0.1% TFA/0 → 1 min: 90% H2O, 1 → 18 min: 90% → 1.83–1.77 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6): δ
10% H2O, 18 → 25 min: 10% H2O, flow rate: 4 mL min−1). 160.3 (CvO), 151.1 (CNH2), 148.8 (CvO), 138.8 (CNvO),
Decay-corrected RCYs were quantified by integration of radio- 138.3 (C-i), 128.2 (C-m), 127.5 (C-o), 127.4 (C-p), 71.9 (CH2Ph),
active peaks on a radio-TLC using a radio-TLC scanner (Fuji, 67.1 (OCH2), 38.1 (NCH2), 26.5 (CH2); MS (ESI+): m/z 327 (M+ +
BAS2000). [18F]fluoride was produced by the 18O(p,n)18F Na, 50%), 305 (M+ + H, 100).
nuclear reaction utilizing the PET cyclotron Cyclone 18/9 (IBA,
Belgium) by irradiation of [18O]H2O.
5,6-Diamino-1-(3-(benzyloxy)propyl)pyrimidine-2,4(1H,3H)-
dione hydrochloride (7a). Sodium dithionite (1.14 g,
5.57 mmol) was added portionwise to a suspension of 6a
(1.13 g, 3.71 mmol) in water (20 mL) and the resulting solution
Synthetic procedures
1-(3-(Benzyloxy)propyl)urea (4a). Under Ar, 1-(3-(benzyloxy) was heated at 70 °C for 1 h (the color turned to light-green).
propan-1-amine (2.35 g, 14.22 mmol) was dissolved in water Next, the volume of the solution was reduced by half and then
(10 mL) and NaNCO (1.11 g, 17.07 mmol) was added in five cooled. The precipitate was filtered, washed with ice water and
portions within 10 min. Afterwards, the mixture was heated at the resulting solid was dried at 90 °C for 3 h and compound
100 °C for 30 min, cooled to rt and was allowed to stand over- 7a was obtained as light-green solid (1.12 g, 99%); mp
night. The precipitated product (scratch with glas rod) was fil- 261–262 °C (decomp.); Rf = 0.14 (ethyl acetate : MeOH = 2 : 1);
tered and washed with a small amount of cold water to give 4a 1H NMR (400 MHz, DMSO-d6): δ 10.53 (s, 1H, NH), 7.37–7.26
3
as colorless solid (1.10 g, 37%); mp 80–82 °C; Rf = 0.44 (ethyl (m, 5H, HAr), 6.09 (s, 2H, NH2), 4.45 (s, 2H, CH2Ph), 3.86 (t, J
1
3
acetate : MeOH = 6 : 1); H NMR (400 MHz, CDCl3): δ 7.40–7.27 = 7.2 Hz, 2H, NCH2), 3.47 (t, J = 6.3 Hz, 2H, OCH2), 2.83 (s,
(m, 5H, HAr), 4.99 (br s, 1H, NH), 4.61 (br s, 2H, NH2), 4.49 (s, 2H, NH2), 1.84–1.77 (m, 2H, CH2) ppm; 13C NMR (101 MHz,
2H, CH2Ph), 3.58 (t, 3J = 5.7 Hz, 2H, NCH2), 3.28 (t, 3J = 6.1 Hz, DMSO-d6): δ 159.6 (CvO), 149.3 (CNH2), 145.4 (CvO), 138.4
2H, OCH2), 1.83–1.77 (m, 2H, CH2) ppm; 13C NMR (101 MHz, (Cipso), 128.2 (Cmeta), 127.6 (Cortho), 127.4 (Cpara), 96.3
CDCl3): δ 158.8 (CvO), 138.1 (C-i), 128.5 (C-m), 127.8 (C-p), (NCCvO), 71.9 (CH2Ph), 67.1 (OCH2), 30.7 (NCH2), 28.0 (CH2).
127.7 (C-o), 73.1 (CH2Ph), 68.5 (OCH2), 38.8 (NCH2), 29.7 (CH2) MS (ESI+): m/z 313 (M+ + Na, 20%), 291 (M+ + H, 100). Due to
ppm; MS (ESI+): m/z 231 (M+ + Na, 100%), 209 (M+ + H, 35).
the instability of that compound, it should be immediately
6-Amino-1-(3-(benzyloxy)propyl)pyrimidine-2,4(1H,3H)-dione converted into its hydrochloride. Thus, the product (1.12 g)
(5a). Elemental sodium (1.86 g, 80.7 mmol) was dissolved in was treated with ice-cold HCl (37%, 15 mL) and stirred for 1 h
absolute EtOH (100 mL) and 4a (5.6 g, 53.8 mmol) and ethyl at rt. The HCl was removed under vacuum and the remaining
cyanoacetate (6.09 g, 53.8 mmol) were added to this solution solid dried under vacuum to yield the hydrochloride 7a as
and the resulting mixture was heated to 60 °C for 16 h. An hygroscopic pale red solid (1.27 g, 99%); mp 128–130 °C
organge precipitate appeared which disappeared after addition (decomp.); Rf = 0 (ethyl acetate : MeOH = 2 : 1); 1H NMR
+
of conc. HCl to pH ≈ 3 under formation of a colorless precipi- (400 MHz, DMSO-d6): δ 11.14 (s, 1H, NH), 9.31 (s, 3H, NH3 ),
tate. This was filtered and washed with EtOH (10 mL) and ethyl 7.64 (s, 2H, NH2), 7.37–7.26 (m, 5H, HAr), 4.44 (s, 2H, CH2Ph),
3
3
acetate (20 mL). The solvent was removed and the remaining 3.91 (t, J = 7.2 Hz, 2H, NCH2), 3.48 (t, J = 6.2 Hz, 2H, OCH2),
residue was purified via column chromatography (ethyl acetate 1.84–1.77 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6): δ
→ ethyl acetate : EtOH = 10 : 1) and 5a was obtained as bright 158.7 (CvO), 149.9 (CNH2), 149.5 (CvO), 138.4 (Cipso), 128.2
yellow solid (3.8 g, 51%); mp 171–173 °C; Rf = 0.30 (ethyl (Cmeta), 127.5 (Cortho), 127.4 (Cpara), 83.0 (NCCvO), 71.9
acetate : EtOH = 6 : 1); 1H NMR (400 MHz, DMSO-d6): δ 10.30 (s, (CH2Ph), 67.0 (OCH2), 39.5 (NCH2), 27.6 (CH2) ppm; MS
1H, NH), 7.37–7.26 (m, 5H, HAr), 6.73 (s, 2H, NH2), 4.53 (s, 1H, (ESI+): m/z 313 (M+ + Na − HCl, 20%), 291 (M+ − Cl, 100).
3
CH), 4.44 (s, 2H, CH2Ph), 3.82 (t, J = 7.3 Hz, 2H, NCH2), 3.46
3-(3-(Benzyloxy)propyl)-1H-purine-2,6(3H,7H)-dione
(8a).
(t, J = 6.3 Hz, 2H, OCH2), 1.83–1.77 (m, 2H, CH2); 13C NMR Compound 7a (2.0 g, 6.12 mmol) was dissolved in anhydrous
(101 MHz, DMSO-d6): δ 162.2 (CvO), 155.6 (CNH2), 151.1 DMF (30 mL) under an argon atmosphere. Triethyl orthoformi-
(CvO), 138.3 (C-i), 128.1 (C-m), 127.4 (C-o), 127.3 (C-p), 75.4 ate (2.29 mL, 13.77 mmol) and p-toluenesulfonic acid (mono-
(CH), 71.8 (CH2Ph), 67.0 (OCH2), 38.2 (NCH2), 27.7 (CH2) ppm; hydrate, 150 mg, 0.79 mmol) were added and the mixture was
3
MS (ESI+): m/z 298 (M+ + Na, 40%), 276 (M+ + H, 30).
stirred at 70 °C for 3.5 h. After cooling to rt, ice water (10 mL)
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was added and the mixture was allowed to crystallise at 0 °C, saturated bicarbonate solution (20 mL) was slowly added
approx. 4 °C overnight. Next, the solid was filtered and washed and the solution extracted with ethyl acetate (3 × 25 mL). The
with cold water. After drying at 90 °C, compound 8a was combined organic layers were dried over MgSO4, the solvent
obtained as yellowish solid (76%, 1.4 g); mp 241–242 °C; Rf = was removed and the crude product was recrystalised with a
1
0.51 (ethyl acetate : MeOH = 4 : 1); H NMR (400 MHz, DMSO- small amount of dichloromethane to obtain 9b as pale-yellow
1
d6): δ 13.47 (s, 1H, NHCH), 11.07 (s, 1H, NHCvO), 8.01 (s, 1H, solid (750 mg, 84%); H NMR (400 MHz, DMSO-d6): δ 11.17 (s,
3
3
3
CH), 7.35–7.25 (m, 5H, HAr), 4.41 (s, 2H, CH2Ph), 4.03 (t, J = 1H, NHCvO), 3.92 (t, J = 7.3 Hz, 2H, NCH2), 3.35 (t, J = 6.1
3
7.0 Hz, 2H, NCH2), 3.49 (t, J = 6.1 Hz, 2H, OCH2), 1.97–1.90 Hz, 2H, OCH2), 3.19 (s, 3H, CH3), 180–1.89 (m, 2H, CH2);
(m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6): δ 154.7 13C-NMR (101 MHz, DMSO-d6):
δ 153.7 (CvO), 150.5
(CvO), 150.8 (CvO), 149.3 (CH), 140.5 (NCN), 138.5 (C-i), (NCCvO), 149.3 (CvO), 124.3 (CBr), 109.6 (NCN), 69.5 (OCH2),
128.2 (C-m), 127.4 (C-o), 127.3 (C-p), 106.9 (NCCvO), 71.9 57.9 (CH3), 27.7 (CH2) ppm; MS (ESI+): m/z 327 (M+ + Na, 81Br,
(CH2Ph), 67.4 (OCH2), 39.8 (NCH2), 27.9 (CH2) ppm; MS 97%), 325 (M+ + Na, 79Br, 100), 305 (M+ + H, 81Br, 40), 303 (M+
(ESI+): m/z 323 (M+ + Na, 40%), 301 (M+ + H, 100).
3-(3-(Methyloxy)propyl)-1H-purine-2,6(3H,7H)-dione
+ H, 79Br, 42).
(8b).
3-(3-(Benzyloxy)propyl)-8-bromo-7-(2-(5-hydroxy-2-methyl-
Compound 7b (2.56 g, 10.2 mmol) was dissolved in anhydrous phenyl)-2-oxoethyl)-1H-purine-2,6(3H,7H)-dione
(11a).
DMF (30 mL) under an argon atmosphere. Triethyl orthoformi- Compound 9a (930 mg, 2.45 mmol) and DIPEA (633 mg,
ate (3.66 mL, 22.0 mmol) and p-toluenesulfonic acid (monohy- 4.90 mmol) were dissolved in anhydrous DMF (5 mL) and
drate, 251 mg, 1.32 mmol) were added and the mixture was 2-bromo-1-(5-hydroxy-2-methylphenyl)ethanone (10, 843 mg,
stirred at 70 °C for 3.5 h. After cooling to rt, ice water (15 mL) 3.68 mmol) dissolved in DMF (5 mL) was added dropwise and
was added and the mixture was allowed to crystallise at the resulting solution was stirred at 50 °C overnight.
approx. 4 °C overnight. Next, the solid was filtered and washed Afterwards, the solvent was removed and the crude product
with cold water and acetone. After drying, compound 8b was was purified using column chromatography (chloroform →
1
obtained as yellowish solid (55%, 1.25 g); mp 221 °C; H NMR chloroform : MeOH = 25 : 1) to obtain 11a as solid (1.3 g, 99%);
1
(400 MHz, DMSO-d6): δ 13.38 (s, 1H, NHCH), 11.03 (s, 1H, mp 172–173 °C; Rf = 0.18 (ethyl acetate); H NMR (400 MHz,
NHCvO), 7.98 (s, 1H, CH), 3.98 (t, 3J = 7.2 Hz, 2H, NCH2), 3.36 DMSO-d6): δ 11.32 (s, 1H, NH), 9.74 (s, 1H, OH), 7.37–7.27 (m,
3
3
3
(t, J = 6.2 Hz, 2H, OCH2), 3.20 (s, 3H, CH3), 1.83–1.91 (m, 2H, 6H, HAr), 7.18 (d, J3,4 = 8.3 Hz, 1H, H-3), 6.96 (dd, J3,4 = 8.3
CH2) ppm; 13C NMR (101 MHz, DMSO-d6): δ 154.7 (CvO), Hz, 4J4,6 = 2.5 Hz, 1H, H-4), 5.70 (s, 2H, CH2CvO), 4.43 (s, 2H,
150.8 (CvO), 149.3 (CH), 140.6 (NCN), 107.2 (NCCvO), 69.5 CH2Ph), 4.02 (t, 3J = 6.9 Hz, 2H, NCH2), 3.51 (t, 3J = 6.0 Hz, 2H,
(OCH2), 57.8 (CH3), 27.8 (CH2) ppm; MS (ESI+): m/z 247 (M+ + OCH2), 2.29 (s, 3H, CH3), 1.99–1.91 (m, 2H, CH2) ppm; 13C
Na, 40%), 225 (M+ + H, 100).
NMR (101 MHz, DMSO-d6): δ 194.1 (CH2C̲vO), 155.4 (C-5),
3-(3-(Benzyloxy)propyl)-8-bromo-1H-purine-2,6(3H,7H)-dione 154.1 (CvO), 150.2 (CvO), 148.7 (NCN), 138.4 (C-i), 134.6
(9a). Elemental bromine (144 μL, 2.83 mmol) was added drop- (C-1), 133.0 (C-3), 129.2 (C-2), 128.2 (C-m), 128.0 (CBr), 127.5
wise to a solution of 8a (710 mg, 2.36 mmol) and NaOAc (C-o), 127.3 (C-p), 119.7 (C-4), 115.5 (C-6), 109.0 (NC
̲
(321 mg, 4.72 mmol) in glacial acetic acid (25 mL) and the 71.9 (CH2Ph), 67.3 (OCH2), 54.3 (C
̲
resulting mixture was stirred at 65 °C for 3 h. After cooling to (CH2), 19.7 (CH3) ppm; MS (ESI+): m/z 551 (M+ + Na, 81Br,
0 °C, saturated bicarbonate solution (20 mL) was slowly added 90%), 549 (M+ + Na, 79Br, 100), 527 (M+ + H, 81Br, 83), 529 (M+
and the solution extracted with ethyl acetate (3 × 25 mL). The + H, 79Br, 80).
combined organic layers were dried over MgSO4, the solvent
was removed and the crude product was recrystalised with a phenyl)-2-oxoethyl)-1H-purine-2,6(3H,7H)-dione
3-(3-(Methyloxy)propyl)-8-bromo-7-(2-(5-hydroxy-2-methyl-
(11b).
small amount of dichloromethane to obtain 9a as pale-yellow Compound 9b (758 mg, 2.50 mmol) and DIPEA (485 mg,
solid (750 mg, 84%); mp 214–216 °C; Rf = 0.64 (ethyl 3.75 mmol) were dissolved in anhydrous DMF (5 mL) and
1
acetate : MeOH = 4 : 1); H NMR (400 MHz, DMSO-d6): δ 11.16 2-bromo-1-(5-hydroxy-2-methylphenyl)ethanone (10, 630 mg,
(s, 1H, NHCvO), 7.35–7.26 (m, 5H, HAr), 4.41 (s, 2H, CH2Ph), 2.75 mmol) dissolved in DMF (5 mL) was added dropwise and
3
3
3.97 (t, J = 7.0 Hz, 2H, NCH2), 3.48 (t, J = 6.0 Hz, 2H, OCH2), the resulting solution was stirred at 50 °C overnight.
1.95–1.90 (m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6): δ Afterwards, the solvent was removed and the crude product
153.6 (CvO), 150.5 (CvO), 149.3 (CCO), 138.4 (C-i), 128.2 was purified using column chromatography (chloroform →
(C-m), 127.5 (C-o), 127.3 (C-p), 124.3 (CBr), 109.6 (NCN), 80.0 chloroform : MeOH = 20 : 1) to obtain 11b as colorless solid
(NCCvO), 79.3 (NCN), 71.9 (CH2Ph), 67.3 (OCH2), 38.6 (1.1 g, 97%); mp 213 °C; Rf = 0.38 (chloroform : MeOH = 9 : 1);
(NCH2), 27.8 (CH2) ppm; MS (ESI+): m/z 403 (M+ + Na, 81Br, 1H NMR (400 MHz, DMSO-d6): δ 11.34 (s, 1H, NH), 9.75 (s, 1H,
97%), 401 (M+ + Na, 79Br, 100), 381 (M+ + H, 81Br, 53), 379 (M+ OH), 7.35 (d, 3J4,6 = 2.3 Hz, 1H, H-6), 7.18 (d, 3J3,4 = 8.4 Hz, 1H,
3
4
+ H, 79Br, 55).
H-3), 6.96 (dd, J3,4 = 8.4 Hz, J4,6 = 2.3 Hz, 1H, H-4), 5.71 (s,
3
3
3-(3-(Methyloxy)propyl)-8-bromo-1H-purine-2,6(3H,7H)-dione 2H, CH2CvO), 3.97 (t, J = 7.1 Hz, 2H, NCH2), 3.39 (t, J = 6.0
(9b). Elemental bromine (360 μL, 7.08 mmol) was added drop- Hz, 2H, OCH2), 3.22 (s, 3H, CH3), 2.28 (s, 3H, CH3), 1.86–1.92
wise to a solution of 8b (1.25 g, 5.57 mmol) and NaOAc (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6): δ 194.2
(750 mg, 9.14 mmol) in glacial acetic acid (25 mL) and the (CH2C
resulting mixture was stirred at 65 °C for 3 h. After cooling to (NCN), 134.6 (C-1), 133.0 (C-3), 129.2 (C-2), 128.0 (CBr), 119.7
̲vO), 155.4 (C-5), 154.1 (CvO), 150.2 (CvO), 148.7
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(C-4), 115.5 (C-6), 109.0 (NC
̲
7-(5-Hydroxy-2-methylphenyl)-8-(2-(3-hydroxypropoxy)phenyl)-
54.3 (C
(ESI+): m/z 575 (M + Na, 81Br, 85%), 573 (M+ + Na, 79Br, 87), Compound 11c (500 mg, 1.27 mmol), 2-(3-hydroxy(propoxy))
553 (M+ + H, 81Br, 23), 551 (M+ + H, 79Br, 25).
aniline (12b, 1.81 g, 10.82 mmol) and AlCl3 (anhydrous,
̲
(13b).
8-Bromo-7-(2-(5-hydroxy-2-methylphenyl)-2-oxoethyl)-3-(3-hydro- 393 mg, 2.95 mmol) were dissolved in absolute EtOH (20 mL)
xypropyl)-1H-purine-2,6(3H,7H)-dione (11d). Starting from 11a: and the mixture was heated at 175 °C for 3 d in a high pressure
BBr3 (1 M in CH2Cl2, 7.4 mL, 7.36 mmol) was slowly added to reactor (quarz glass tube). Afterwards, the solvent was removed
a suspension of 11a (1.38 g, 2.64 mmol) in anhydrous CH2Cl2 and the crude product was purified by column chromato-
(20 mL) at 0 °C and the solution was stirred for 1 h at 0 °C. graphy (CHCl3 : EtOH 15 : 1). Afterwards, the resulting solid
Afterwards, water was added for quenching and the formed was washed with ethyl acetate to obtain 13b as a greyish solid
precipitate was filtered, washed with CH2Cl2 (2 × 10 mL) and (326 mg, 56%); Rf = 0.45 (CHCl3 : MeOH = 5 : 1); 1H NMR
water (2 × 10 mL) and dried in vacuo to obtain 11d as colorless (400 MHz, DMSO-d6): δ 0.97 (s, 1H, 1-NH), 9.25 (s, 1H, PhOH),
3
solid (1.15 g, 99%). Starting from 11b: BBr3 (1 M in CH2Cl2, 7.74 (s, 1H, CH), 7.39 (dt, J = 8.0 Hz, 4J = 1.7 Hz, 1H, H-4′),
3
4
3
7.0 mL, 9.96 mmol) was slowly added to a suspension of 11b 7.32 (dd, J = 7.7 Hz, J = 1.7 Hz, 1H, H-6′), 7.14 (dd, J = 8.5
(1.0 g, 2.22 mmol) in anhydrous CH2Cl2 (20 mL) at 0 °C and Hz, 4J = 1.2 Hz, 1H, H-3′), 6.95–6.99 (m, 2H, H-3, H-5′), 6.63
3
4
4
the solution was stirred for 1 h at 0 °C. Next, water was added (dd, J3,4 = 8.3 Hz, J4,6 = 2.6 Hz, 1H, H-4), 6.59 (d, J4,6 = 2.6
3
for quenching and the formed precipitate was filtered, washed Hz, 1H, H-6), 4.41 (t, J = 5.1 Hz, 1H, OH), 3.91–4.04 (m, 2H,
with CH2Cl2 (2 × 10 mL) and water (2 × 10 mL) and dried in OCH2), 3.28–3.34 (m, 5H, CH2OH, NCH3), 2.10 (s, 3H, PhCH3),
vacuo to obtain 9d as colorless solid (813 mg, 84%); mp 1.71–1.61 (m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6): δ
229–230 °C; Rf = 0.50 (ethyl acetate : MeOH = 8 : 1); 1H NMR 154.6 (Cq), 154.2 (CvO), 153.4 (Cq), 153.0 (Cq), 151.1 (CvO),
(400 MHz, DMSO-d6): δ 11.33 (s, 1H, NH), 9.73 (s, 1H, HOPh), 147.6 (NCN), 131.8 (C-1), 130.9 (C-3), 130.8 (C-4′), 129.7 (C-6′),
7.35 (d, 4J4,6 = 2.5 Hz, 1H, H-6), 7.18 (d, 3J3,4 = 8.3 Hz, 1H, H-3), 127.8 (Cq), 127.6 (Cq), 122.4 (Cq), 120.4 (Cq), 117.9 (C-6), 116.3
3
4
6.96 (dd, J3,4 = 8.3 Hz, J4,6 = 2.5 Hz, 1H, H-4), 5.71 (s, 2H, (C-4), 113.3 (C-3′), 106.4 (CHAr), 99.1 (NC̲CvO), 65.3 (OCH2),
3
3
CH2CvO), 3.97 (t, J = 7.5 Hz, 2H, NCH2), 3.51 (t, J = 6.4 Hz, 57.1 (CH2OH), 31.8 (CH2), 28.8, 18.8 (2 × CH3); MS (ESI+): m/z
2H, OCH2), 2.29 (s, 3H, CH3) 1.85–1.78 (m, 2H, CH2) ppm; 13C 484 (M+ + Na, 100%), 462 (M + H, 60).
NMR (101 MHz, DMSO-d6): δ 194.6 (CH2CvO), 155.4 (C-5),
154.1 (CvO), 150.2 (CvO), 148.7 (NCN), 134.5 (C-1), 133.0 2,3,4,8-tetrahydro-1H-imidazo[2,1-f]purin-7-yl)phenyl
4-Methyl-3-(1-methyl-2,4-dioxo-8-(2-(3-(tosyloxy)propoxy)phenyl)-
4-methyl-
(C-3), 129.2 (C-2), 128.0 (CBr), 119.7 (C-4), 115.5 (C-6), 109.0 benzenesulfonate (14a). Compound 13b (250 mg, 0.54 mmol)
(NCCvO), 58.5 (OCH2), 54.3 (CH2CvO), 38.7 (NCH2), 31.0 was dissolved in anhydrous N-methyl morpholine (5 mL), tosyl
(CH2), 19.8 (CH3) ppm; MS (ESI+): m/z 461 (M + Na, 81Br, 25%), chloride (437 mg, 2.29 mmol) was added and the mixture was
459 (M+ + Na, 79Br, 23), 439 (M+ + H, 81Br, 100), 437 (M + H, allowed to stirr at rt overnight. Water was added to the reaction
79Br, 97).
mixture, the precipitate was filtered off and purified via
7-(5-Hydroxy-2-methylphenyl)-1-(3-hydroxypropyl)-8-(2-meth- column chromatography (petroleum ether : ethyl acetate 1 : 5)
oxyphenyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
(13a). to obtain 14a as a pale yellow solid (400 mg, 96%); Rf = 0.79
Compound 11d (1.15 g, 2.64 mmol), 2-methoxyaniline (12a, (CHCl3 : MeOH = 10 : 1); 1H NMR (400 MHz, DMSO-d6): δ 11.01
5.8 mL, 52.8 mmol) and AlCl3 (anhydrous, 393 mg, (s, 1H, NH), 7.82 (s, 1H, CH), 7.62 (d, 3J = 8.3 Hz, 2H, Ts-
3
2.95 mmol) were dissolved in absolute EtOH (40 mL) and the
mixture was heated at 175 °C for 3 d in a high pressure reactor
H
H
ortho), 7.51 (d, J = 8.5 Hz, 2H, Ts-Hortho), 7.45–7.35 (m, 4, Ts-
3
3
meta), 7.39 (d, J = 8.0 Hz, 2H, PhOTs-Hmeta), 7.32 (d, J = 8.1
(quarz glass tube). Afterwards, the solvent was removed and Hz, 2H, Hmeta), 7.14 (dd, 3J = 7.9 Hz, 4J = 1.5 Hz, 1H, H-3′),
the crude product was purified by column chromatography 7.16–7.08 (m, 2H, H-3′,5′), 6.97–6.99 (m, 2H, H-3, H-6), 6.77
3
4
(ethyl acetate → ethyl acetate : MeOH 20 : 1) to obtain 13a as a (dd, J3,4 = 8.3 Hz, J4,6 = 2.5 Hz, 1H, H-4), 3.99–3.86 (m, 4H,
greyish solid (730 mg, 62%); mp 282–283 °C; Rf = 0.32 (ethyl OCH2 + NCH2), 3.29 (s, 3H, NCH3), 2.41 (s, 3H, MeTs), 2.36 (s,
acetate : MeOH = 10 : 1); 1H NMR (400 MHz, DMSO-d6): δ 10.97 3H, MeTs), 2.11 (s, 3H, CH3), 1.91–1.68 (m, 2H, CH2) ppm; 13C
(s, 1H, NH), 9.73 (s, 1H, HOPh), 7.74 (s, 1H, CH), 7.42–7.37 (m, NMR (101 MHz, DMSO-d6): δ 153.7, 153.5, 152.9, 151.1, 147.5,
3
2H, H-4′, H-6′), 7.12 (d, J3,4 = 8.3 Hz, H-3), 7.00–6.97 (m, 2H, 146.4, 145.8, 144.8, 137.3, 132.4 (2 × CvO + 8 × CAr), 131.5,
H-3′,5′), 6.62 (dd, 3J3,4 = 8.3 Hz, 4J4,6 = 2.5 Hz, 1H, H-4), 6.55 (d, 131.0 (2 × CHAr), 130.9 (CHAr), 130.1, 130.0 (2 × Ts-m), 129.8
3
3
4J4,6 = 2.5 Hz, 1H, H-6), 4.45 (t, J = 5.4 Hz, 1H, OH), 3.92 (t, J (CAr), 129.6 (CHAr), 128.6 (CAr), 128.2, 127.2 (2 × Ts-o), 113.5
3
= 7.1 Hz, 2H, NCH2), 3.62 (s, 3H, OCH3), 3.41 (dt, JH,H = 6.1 (C-6), 107.2 (CH), 99.2 (NCCvO), 67.6, 63.9 (CH2), 28.8
3
Hz, JH,OH = 5.4 Hz, 2H, HOCH2), 2.11 (s, 3H, CH3), 1.80–1.74 (NCH3), 28.0 (CH2), 21.1, 21.0 (2 × TsCH3), 19.0 (CH3) ppm; MS
(m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6): δ 154.7 (ESI+): m/z 770 (M+ + H, 90%), 598 (M − OTs, 15).
(C-2′), 154.5 (CvO), 153.4 (C-5), 152.4 (Cq), 150.8 (CvO), 147.3
3-(8-(2-Methoxyphenyl)-2,4-dioxo-1-(3-(tosyloxy)propyl)-2,3,4,8-
(NCN), 131.8 (CvCPh), 130.9 (C-3), 130.8 (C-4′), 129.5 (C-5′), tetrahydro-1H-imidazo[2,1-f]purin-7-yl)-4-methylphenyl 4-methyl-
127.8 (C-1), 127.7 (C-1′), 122.3 (C-2), 120.7 (C-6′), 117.9 (C-4), benzenesulfonate (14b). Compound 13a (200 mg, 0.44 mmol)
116.3 (C-3′), 112.8 (C-6), 106.3 (CH), 99.1 (NC̲CvO), 58.4 was dissolved in anhydrous N-methyl morpholine (5 mL), tosyl
(OCH2), 55.6 (OCH3), 38.7 (NCH2), 30.9 (CH2), 18.7 (CH3) ppm; chloride (350 mg, 1.84 mmol) was added and the mixture was
MS (ESI+): m/z 484 (M+ + Na, 100%), 462 (M + H, 45).
allowed to stirr at rt overnight. Water was added to the reaction
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mixture, the precipitate was filtered off and purified via and the aqueous solution extracted with ethyl acetate (3 ×
column chromatography (petroleum ether : ethyl acetate 1 : 5) 20 mL). The crude product was purified by column chromato-
to obtain 14b as a pale yellow solid (260 mg, 77%); mp graphy (CH2Cl2 : EtOH = 30 : 1) to give 1b as a greyish solid
1
89–90 °C; Rf
=
0.71 (CHCl3 : MeOH
=
10 : 1); 1H NMR (80 mg, 80%); mp 308 °C; Rf = 0.60 (CHCl3 : EtOH = 5 : 1); H
(400 MHz, DMSO-d6): δ 10.95 (s, 1H, NH), 7.79 (s, 1H, CH), NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H, NH), 9.27 (s, 1H,
7.64 (d, 3J = 8.3 Hz, 2H, Hortho), 7.48 (d, 3J = 8.4 Hz, 2H, PhOTs- HOPh), 7.76 (s, 1H, CH), 7.40 (dt, J = 7.9 Hz, J = 1.6 Hz, 1H,
3
4
3
4
3
H
H
ortho), 7.46–7.43 (m, 1H, H-6′), 7.39 (d, 3J = 8.0 Hz, 2H, PhOTs- H-4′), 7.30 (dd, J = 7.8 Hz, J = 1.6 Hz, 1H, H-6′), 7.19 (dd, J =
3
4
meta), 7.32 (d, J = 8.1 Hz, 2H, Hmeta), 7.31–7.29 (m, 1H, H-4′), 8.5 Hz, J = 1.1 Hz, 1H, H-3′), 6.96–7.00 (m, 2H, H-3, H-5′), 6.64
7.16–7.10 (m, 2H, H-3′,5′), 6.98 (dt, 3J3,4 = 8.0 Hz, 5J3,6 = 0.9 Hz, (dd, 3J3,4 = 8.3 Hz, 4J4,6 = 2.6 Hz, 1H, H-4), 6.59 (d, 4J4,6 = 2.6 Hz,
3
3
H-3), 6.88 (d, J4,6 = 2.6 Hz, 1H, H-6), 6.73 (dd, J3,4 = 8.0 Hz, 1H, H-6), 4.39–4.28 (m, 2H, CH2F), 3.98–4.09 (m, 2H, OCH2),
4J4,6 = 2.6 Hz, 1H, H-4), 4.01 (t, J = 6.3 Hz, 1H, TsOCH2), 3.83 3.32 (s, 3H, NCH3), 2.09 (s, 3H, PhCH3), 2.00–1.81 (m, 2H, CH2)
3
(t, 3J = 6.5 Hz, 2H, NCH2), 3.54 (s, 3H, OCH3), 2.36 (s, 3H, ppm; 13C NMR (101 MHz, DMSO-d6): δ 154.6 (Cq), 153.9 (CvO),
PhOTs-CH3), 2.35 (s, 3H, TsCH3), 2.18 (s, 3H, CH3), 1.93–1.90 153.4 (Cq), 152.8 (Cq), 151.0 (CvO), 147.5 (NCN), 131.7 (C-1),
(m, 2H, CH2) ppm; 13C NMR (101 MHz, CD3CN): δ 156.0 (C-2′), 130.9 (C-3), 130.8 (C-4′), 129.7 (C-6′), 127.8 (Cq), 127.6 (Cq), 122.5
154.2 (CvO), 154.0 (C-5), 151.9 (C(N)3), 148.9 (CvO), 147.9 (Cq), 120.7 (Cq), 117.9 (C-6), 116.3 (C-4), 113.4 (C-3′), 106.5
(NCN), 147.3 (PhTsOpara), 146.3 (TsOpara), 138.9 (C-1), 132.6 (CHHetar), 99.1 (NCC̲
vO), 80.4 (1JC,F = 162.4 Hz, CH2F), 64.0
(CvCPh), 132.3 (PhTsOipso
,
TsOipso), 131.6 (C-3), 131.0 (3JC,F = 5.5 Hz, OCH2), 29.5 (d, 2JC,F = 19.5 Hz, CH2), 28.8, 18.7 (2
(PhTsOmeta), 130.9 (TsOmeta), 130.5 (C-5′), 130.0 (C-2), 129.3 × CH3) ppm; 19F NMR (376 MHz, DMSO-d6): δ −220.7 ppm; MS
(PhTsOortho), 129.2 (C-4′), 128.6 (TsOortho), 126.6 (C-5′), 125.9 (ESI+): m/z 486 (M+ + Na, 100%), 464 (M+ + H, 30).
(C-1′), 124.0 (C-4), 123.2 (C-6′), 121.8 (C-3′), 113.7 (C-6), 107.9
8-(2-(3-[18F]Fluoropropoxy)phenyl)-7-(5-hydroxy-2-methyl-
(CH), 100.6 (NCCvO), 69.6 (OCH2), 56.4 (OCH3), 40.3 (NCH2), phenyl)-1-methyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
28.2 (NCH2CH2), 21.7 (TsOCH3), 19.8 (CH3) ppm; MS (ESI+): ([18F]2). An anion-exchange cartridge (Waters, Sep-Pak Light
m/z 770 (M+ + H, 90%), 598 (M − OTs, 15).
Accell Plus QMA) was activated by rinsing with 5 mL of a 1 M
1-(3-Fluoropropyl)-7-(5-hydroxy-2-methylphenyl)-8-(2-meth- NaHCO3 solution and 10 mL of deionised H2O. It was charged
oxyphenyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
(3). with [18F]fluoride (2–3 GBq) and eluted with 1.5 mL of a solu-
Compound 13a (150 mg, 0.33 mmol) was dissolved in a tion of Kryptofix 2.2.2 (10 mg mL−1) and K2CO3 (13 mM) in
mixtrue of N-methyl morpholine (5 mL) and DCM (5 mL). The 7 mL CH3CN and 43 mL H2O. The solvents were evaporated
reaction mixture was cooled to −78 °C and DAST (200 µL, azeotropically by subsequent addition of three portions of
1.51 mmol) was added stepwise. After complete addition the 1 mL each of anhydrous CH3CN under a stream of nitrogen at
reaction mixture was stirred at rt overnight. Afterwards, ice was 110 °C. Precursor 14a (approx. 3 mg) was dissolved in 500 μL
added, and the aqueous phase extracted with CHCl3 (3 × of anhydrous CH3CN and the mixture was added to the [18F]
20 mL). The combined organic layers were washed with satu- fluoride-containing sealed vial. The resulting solution was
rated hydrogen carbonate solution (20 mL) and water (20 mL). heated at 100 °C for 30 min. Afterwards, aqueous NaOH
The crude product was purified by column chromatography (40 µL, 5 M) was added and the mixture heated at 100 °C for
(CHCl3 → CHCl3 : MeOH 30 : 1 → 20 : 1 → 10 : 1) to obtain 3 as 15 min. After cooling to rt, 120 µL of water were added and the
pale yellow solid (30 mg, 10%); mp 194–195 °C; Rf = 0.37 solution purified by semi-preparative radio-HPLC (peak collec-
(CHCl3 : MeOH = 9 : 1); 1H NMR (400 MHz, DMSO-d6): δ 9.23 tion tR: 24–28 min, gradient: 60% water, 40% CH3CN + 0.1%
(s, 1H, HOPh), 7.74 (s, 1H, CH), 7.41–7.37 (m, 2H, H-4′,6′), 7.12 TFA). To remove the solvent from the product, the solvent of
3
4
(dd, J3,4 = 8.0 Hz, J3,6 = 1.7 Hz, 1H, H-3), 7.02–6.97 (m, 2H, the collected product fraction was removed under reduced
H-3′,5′), 6.61 (dd, 3J3,4 = 8.0 Hz, 4J4,6 = 2.7 Hz, 1H, H-4), 6.54 (d, pressure. Analytical radio-HPLC: tR = 12.0 min; RCY: 57–129
4J4,6 = 2.7 Hz, 1H, H-6), 4.48 (dt, 3JH,F = 47.3 Hz, 3JH,H = 5.8 Hz, MBq (3–5%, d.c.); RCP: >98%; Am = 11.6 3.4 GBq µmol−1
2H, FCH2), 3.96 (t, J = 6.2 Hz, 2H, NCH2), 3.64 (s, 3H, OCH3),
.
3
1-(3-[18F]Fluoropropyl)-7-(5-hydroxy-2-methylphenyl)-8-(2-
2.11 (s, 3H, CH3), 1.94–1.93 (m, 2H, CH2) ppm; 13C NMR methoxyphenyl)-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
(101 MHz, DMSO-d6): δ 154.7 (Cq), 154.5 (CvO), 153.5 (Cq), ([18F]3). An anion-exchange cartridge (Waters, Sep-Pak Light
152.3 (Cq), 150.8 (CvO), 147.3 (NCN), 131.8 (C-1), 130.8 (C-3), Accell Plus QMA) was activated by rinsing with 5 mL of a 1 M
130.7 (C-4′), 129.5 (C-6′), 127.9 (Cq), 127.7 (Cq), 122.3 (Cq), NaHCO3 solution and 10 mL of deionised H2O. It was charged
120.7 (Cq), 117.9 (C-6), 116.3 (C-4), 112.8 (C-3′), 106.3 (CHHetar), with [18F]fluoride (∼2 GBq) and eluted with 1.5 mL of a solu-
99.2 (NC̲
CvO), 79.5 (1JC,F = 163.1 Hz, CH2F), 63.8 (3JC,F = 5.8 tion of Kryptofix 2.2.2 (10 mg mL−1) and K2CO3 (13 mM) in
2
Hz, OCH2), 29.5 (d, JC,F = 19.5 Hz, CH2), 30.8, 18.7 (2 × CH3) 7 mL CH3CN and 43 mL H2O. The solvents were evaporated
ppm; 19F NMR (376 MHz, DMSO-d6): δ −218.3 ppm; MS (ESI+): azeotropically by subsequent addition of three portions of
m/z 464 (M+ + H, 10%).
1 mL each of anhydrous CH3CN under a stream of nitrogen at
8-(2-(3-Fluoropropoxy)phenyl)-7-(5-hydroxy-2-methylphenyl)- 110 °C. Precursor 14b (approx. 3 mg) was dissolved in 300 μL
1-methyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione
(2). of anhydrous CH3CN and the mixture was added to the [18F]
Compound 13b (100 mg, 0.22 mmol) was suspended in anhy- fluoride-containing sealed vial. The resulting solution was
drous THF (5 mL) and deoxofluor (300 µL, 1.6 mmol) was heated under microwave conditions at 50 watt for 70 min.
added. After stirring at rt for 1.5 h, water (25 mL) was added After cooling to rt, 3 mL of water were added and the solution
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transferred to 2 connected Chromafix C18 ec-cartridges. The target selected human Eph receptors. Docking studies were
cartridges were washed with 20 mL of water and the tracer done to find suitable positions on the original inhibitor for a
eluted with 0.9 mL of MeOH. Further purification was done radiolabelling with fluorine-18. Two positions were figured out
via semi-preparative radio-HPLC (peak collection tR: and the respective non-radioactive references and precursors
9–11 min). To remove the solvent from the product, the solvent prepared followed by a straightforward radiolabelling pro-
of the collected product fraction was removed under reduced cedure with [18F]fluoride with radiochemical yields between
pressure. Analytical radio-HPLC: tR = 15.2 min; RCY: 21–69 3–15% and radiochemical purity >95% for [18F]2/3.
MBq (1–6%, d.c.); RCP: >98%; Am = 10.3 5.1 GBq µmol−1
.
First radiobiological studies were done in vitro on human
A375 melanoma cells showing a low uptake for [18F]2 (approx.
40% ID mg−1) and a substantial uptake for [18F]3 (60–110% ID
mg−1), but nearly no difference between the transfected A375-
EphA2 and A375-EphB4 cells compared to the non-transfected
A375 cells.
In vivo investigations showed a fast blood clearance and a
metabolisation of the radiotracers to a more lipophilic com-
pound in the urine after 60 min p.i., but no radiodefluorination.
Biological characterisation
Generation of EphA2- and EphB4-transgenic A375 cells.
Human A375 melanoma cells overexpressing EphA2 or EphB4
were prepared as described previously by us.28,48 In brief, A375
cells were stably transfected with a plasmid encoding for both
the reporter green fluorescent protein (GFP) and the EphA2
(A375-EphA2) or EphB4 receptor (A375-EphB4). GFP expression
was used to select transfected cells by fluorescence associated
cell sorting (FACS) and to routinely control stable expression of
EphA2 and EphB4.
Conflicts of interest
Verification of EphA2- and EphB4-overexpression. Enhanced
EphA2- and EphB4-expression in A375-EphA2 and A375-EphB4
cells, respectively, was verified by Western blot as described
previously.28,48
There are no conflicts to declare.
Acknowledgements
Cellular binding and uptake studies and blocking experi-
ments. Cellular binding and uptake of the radiotracers [18F]2
and [18F]3 were investigated in A375, A375-EphA2, and A375-
EphB4 cells for up to 120 min as previously described by us.28
For blocking experiments, cellular binding and uptake of
[18F]2 and [18F]3 was investigated after pre-incubation with
compound 2 for 30 min and lead compound 1 for 10 min,
respectively. Cellular binding and uptake was investigated in at
least two independent experiments, each performed in
quadruplicate.
The authors are grateful to Jaques Pliquett, Waldemar Herzog,
Stephan Preusche, and Tilow Krauss for their excellent techni-
cal and laboratory assistance. We are very thankful to Andrea
Suhr, Catharina Heinig, and Regina Herrlich for the in vitro
and in vivo experiments. This work is part of a research initiat-
ive within the Helmholtz-Portfoliothema “Technologie und
Medizin – Multimodale Bildgebung zur Aufklärung des In vivo-
Verhaltens von polymeren Biomaterialien”.
In vitro stability test. The in vitro stability was investigated
using rat blood or plasma. Thus, purified [18F]2 or [18F]3 was
diluted in 200 μL E153 infusion solution and then incubated
with 3 mL of rat blood or plasma for 60 min at 37 °C. Samples
were taken after time points of 1, 30 and 60 min, and analysed
by radio-TLC (silica gel, eluent: chloroform : methanol 9 : 1).
Animal experiments. For in vivo stability tests, radiotracers
[18F]2 and [18F]3 were injected i.v. into male Wistar rats (body
weight approx. 236 g, injected dose approx. 10 MBq) under
desflurane anesthesia (10% desflurane in 30% oxygen/air).
Using a catheter, blood samples from femoral artery were
taken at 1, 3, 5, 10, 20, 30, and 60 min p.i. The resulting loss of
volume was compensated by i.v. injection of E153. All animal
experiments were carried out according to the guidelines of
the German Regulations for Animal Welfare. The protocols
were approved by the local Ethical Committee for Animal
Experiments (AZ 24-9168.21-4/2004-1 and 24-9168.11-4/2012-1).
Notes and references
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Conclusions
Two novel fluorine-18-containing radiotracers based on the 11 X. Yang, Y. Yang, S. Tang, H. Tang, G. Yang, Q. Xu and
xanthine lead structure were prepared and characterised to
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