Preparation of a novel radiotracer targeting the EphB4 receptor via radiofluorination using spiro azetidinium salts as precursor

Article abstract of DOI:10.1002/jlcr.3526

The visualization of Eph receptors, which are overexpressed in various tumor entities, using selective small molecule Eph inhibitors by means of positron emission tomography is a promising approach for tumor imaging. N-(Pyrimidinyl)indazolamines represent a class of compounds, which are known to have high affinity especially for the EphB4 receptor. Radiofluorination of these compounds could provide a highly specific imaging agent and was investigated using a classical nucleophilic introduction of [¹⁸F]fluoride as well as a less common nucleophilic ring-opening reaction of azetidinium salts. In the past, radiofluorinations using azetidinium precursors were demonstrated to result in high radiochemical yields in short periods. For this purpose, an azetidinium precursor based on the N-(pyrimidinyl)indazolamine lead compound was developed, and radiofluorination was successfully accomplished. The respective [¹⁸F]radiotracer was quickly prepared with high radiochemical purity >97% and in a radiochemical yield of 34%.

Full text of DOI:10.1002/jlcr.3526

Short Note
Preparation of a novel radiotracer targeting the EphB4 receptor via
radiofluorination using spiro azetidinium salts as precursor
Jens Wiemer,a,† Jörg Steinbacha,b, Jens Pietzscha,b and Constantin Mamata,b,*
a) Institut für Radiopharmazeutische Krebsforschung, Helmholtz-Zentrum Dresden-
Rossendorf, Bautzner Landstraße 400, D-01328 Dresden, Germany
b) Fachbereich Chemie und Lebensmittelchemie, Technische Universität Dresden, D-01062
Dresden, Germany
* Correspondence to: Constantin Mamat, Institut für Radiopharmazeutische Krebsforschung,
Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstraße 400, D-01328 Dresden,
Germany; E-mail: c.mamat@hzdr.de
† Current address: Department of Nuclear Medicine, Ludwig-Maximilians-University of
Munich, Munich, Germany
Short title: Radiofluorinations using spiro precursors
Key words: EphB4 ligand / spiro salts / N-(pyrimidinyl)indazolamine
Abstract. The visualization of Eph receptors, which are overexpressed in various tumor
entities, using selective small molecule Eph inhibitors by means of PET is a promising
approach for tumor imaging. N-(Pyrimidinyl)indazolamines represent a class of compounds,
which are known to have high affinity especially for the EphB4 receptor. Radiofluorination
of these compounds could provide a highly specific imaging agent and was investigated using
a classical nucleophilic introduction of [18F]fluoride as well as a less common nucleophilic
ring opening reaction of azetidinium salts. In the past, radiofluorinations using azetidinium
precursors were demonstrated to result in high radiochemical yields in short periods of time.
For this purpose, an azetidinium precursor based on the N-(pyrimidinyl)indazolamine lead
compound was developed and radiofluorination was successfully accomplished. The
respective [18F]radiotracer was quickly prepared with high radiochemical purity >97% and in
a radiochemical yield of 34%.
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Introduction.
Aliphatic radiofluorinations commonly involve the nucleophilic introduction of [18F]fluoride
into alkyl compounds containing suitable leaving groups. To this end, there are two main,
well-established methods for the synthesis of the resulting tertiary [18F]fluoroethylamines and
[18F]fluoropropylamines as illustrated in Figure 1. The first method consists of preparing a
[18F]fluoropropyl or ethyl moiety (e.g. 1-bromo-3-[18F]fluoropropane or 2-[18F]fluoroethyl
tosylate)1 and subsequent introduction of this building block into secondary amine precursors
using a nucleophilic displacement reaction. This two-step reaction results in overall RCYs
ranging from 10 to 51%.2,3,4,5,6,7 In the second strategy, a tertiary amine with a propyl residue,
furnished with a good leaving group at the terminal carbon atom, is directly reacted with
[18F]fluoride. The use of tosylate or mesylate as leaving groups is preferred, leading to RCYs
ranging from 33–70%.2,8,9,10,11,12,13,14 A RCY of 88% was reported when using bromine as the
leaving group.15
A third approach, rarely used in the past, consists of the application of aziridinium (three
membered rings) and azetidinium (four membered rings) salts as precursors (Figure 1). The
reactions of nucleophiles with aziridinium ions caused by ring opening of substituted
heterocycles is reported to be facile16 especially for the introduction of fluorine-18.17,18,19
However, only little is known about ring opening reactions using azetidinium salts. It is
known that these reactions lead to unbranched propyl chains when the heterocycle is
unsubstituted20,21,22 with yields of 57–68%.
The introduction of fluorine-18 via azetidinium mesylates was first investigated by
18
Kiesewetter and Eckelman in 2004 with RCYs for the F-containing compounds ranging
from 56–75% using n.c.a [18F]fluoride.23 Furthermore, azetidinium salts were used to
successfully label molecules, which are part of pyrido[2,3-d]pyrimidine-scaffold, with
fluorine-18 in high RCYs (81-90%)24 As Pyrido[2,3-d]pyrimidines are known as cyclin-
dependent kinase (CDK) inhibitors. Finally, two radiofluorinated bioorthogonal building
blocks 1-(2-azidoethyl)-4-(2-[18F]fluoroethyl)piperazine ([18F]AFP) and 1-(but-3-yn-1-yl)-4-
(2-[18F]fluoroethyl)piperazine ([18F]BFP) were prepared (RCYs 29 and 31%, d.c.)25 for mild
radiolabeling via the Cu-catalyzed and strain-promoted click reaction as well as both variants
of the Staudinger Ligation in the case of [18F]AFP.
The advantages of azetidinium salts as precursor are a convenient preparation from the open-
chained compound, milder reaction conditions and a fast reaction. The strained four-
membered ring, two sites for the nucleophilic attack of the [18F]fluoride and the ammonium
residue as good leaving group enable a quick reaction time.
Our aim consisted of the development of a new radiolabeling procedure for a fluorine-18-
containing radiotracer using azetidinium salts based on inhibitor molecules for the EphB4
receptor, because EphB4 with its preferential ligand, ephrinB2, also from a
radiopharmacologist’s point of view is one of the most important Eph receptors.26 Pro-
tumorigenic effects of EphB4 activation have been described for prostate cancer27 and breast
cancer, whereas EphB4 knockdown resulted in reduced tumor growth in mice.28 Inconsistent
outcome of EphB4/ephrinB2 signalling is also described for tumorigenesis. Anti-tumorigenic
effects of activated EphB4 in mouse melanoma and breast cancer cells were observed, and
reduction or loss of EphB4 expression in human colorectal tumors and invasive breast
carcinoma.29,30,31
Several potent Eph kinase inhibitors were reported either based on high molecular weight
compounds like peptides,32,33 which block the extracellular domain of the appropriate
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receptor or based on small organic molecules34,35,36 which bind to the intramolecular ATP
binding pocket. To date, mainly peptide-based radiotracers containing Cu-64, In-111 or F-18
are known for multimodal imaging purposes using PET or SPECT which are specific for Eph
receptors.37,38,39
Due to their favorable chemical and biological properties, small organic inhibitors (e.g. I or
III) published by Bardelle et al. are the basis of our research.40,41,42,43 Recently, the novel
fluorine-18-containing radiotracer II based on the benzodioxolylpyrimidine structural motif
was developed and analyzed via in vitro and in vivo studies.44 Unfortunately, despite the high
affinity of the original inhibitor I to the EphB4 receptor (IC50 = 90 nM, recombinant EphB4),
the tracer II showed only a moderate uptake in tumor cells (A375) and no uptake in vivo in
the respective tumor (A375) bearing mice. This can partially be explained due to its
unfavorable (bio)chemical properties like a high lipophilicity (logP = 3.77). To overcome
these problems, a novel lead structure was chosen based on a N-(pyrimidinyl)indazolamine
core containing inhibitor III with an excellent affinity to the Eph receptor (IC50 = 1.3 nM)42
and practical physicochemical properties like a logP = 2.98.
For this purpose, we have choosen compound III as basis published by Bardelle et al. in
2010.42 Previous in silico studies were accomplished to find the best labeling positions
18
(structures IV, V, and VI in Figure 2).45 Based on these findings, we tried to prepare an F-
labeled compound V by replacing the methyl group of the original compound III, but without
success. According to the in silico studies, compound VI should have comparable affinity.
Thus, we aimed to prepare the radiotracer according this structure and to use azetidinium
salts as precursor.
Results and Discussion.
18
Synthesis. The original compound, the resulting F-tracer and its precursor consists of two
molecules parts as shown in Figure 3. The first part of the inhibitor (part A) was synthesized
as previously described.45 The second structure (part B) was synthesized from 4-(3-iodo-5-
nitrophenyl)morpholine (1) which was reacted with N-Boc-piperazine in a Buchwald-
Hartwig reaction with xantphos and Pd2dba3. The cross-coupled product 2 was obtained in
71% yield. Afterwards, the protecting group was removed quantitatively, leading to 3 which
was then reacted with either 1-fluoro-3-iodopropane leading to 4 (80% yield) or 3-
bromopropanol leading to 5 (69% yield). Next, the nitro groups in 2, 4, and 5 were reduced
yielding anilines 6-8 (95%, 78% and 100% yields, respectively). The complete pathway to
anilines 6-8 is shown in Scheme 1.
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Finally, both parts A and B were connected. Thus, anilines 7 and 8 were reacted with 9 to
give the EOE-protected reference compound 10 (62% yield) and the hydroxyl compound 11
(29% yield). The EOE group of 10 was cleaved under acidic conditions using 1 M HCl to
give the final reference compound 14.
To prepare the precursor, compound 11 was first treated with p-TsCl but no suitable product
was isolable. Then, compound 11 was treated with MsCl to give the open chained compound
12. After purification, 12 was dissolved in different solvents (dichloromethane, chloroform,
acetonitrile) and warmed to prepare the final azetidinium precursor 13. The progress of the
1
reaction was monitored by H NMR. For example, the signal for the mesyl group in 12 was
found at δ = 3.01 ppm whereas the same signal for the mesylate of azetidinium salt 13 was
found at δ = 2.76 ppm. The whole synthesis pathway is pointed out in Scheme 2.
Radiolabeling. Both precursors 12 and 13 were used for the radiolabeling. However under the
used labeling conditions, 12 converted into 13; no differences between 12 and 13 were found.
Previously published conditions were used: K[18F]F, K2CO3, K222, anhydrous acetonitrile to
give [18F]10 (tR = 30.7 min).45 For the removal of the EOE group, [18F]10 was treated with 2
M HCl to yield [18F]14 (tR = 11.9 min) (Scheme 3/Figure 3). After deprotection, a spot at Rf =
18
0.37 (solvent: dichloromethane/methanol = 9/1) belonging to the final F-tracer [18F]14 was
determined by comparison with the non-radioactive compound 13 using radio-TLC analyses
(Figure 4). Purification was done using semi-preparative HPLC yielding [18F]14 in 34%
RCY.
logP determination: The lipophilicities of the original inhibitor III and the fluorine-
containing compound 14 were determined according to the shake flask method reported
elsewhere.46 A logP value of 2.98 was determined for the original inhibitor III and a value of
2.55 for compound 14. Both compounds seemed to be more hydrophilic compared to the
previous prepared radiotracers. The results are summarized in Table 1.
Conclusion. In this short note, we have demonstrated the successful radiolabeling of a
radiotracer based on the N-(pyrimidinyl)indazolamine core structure suitable for the
visualization of the EphB4 receptor. [18F]14 was prepared via a nucleophilic ring opening
reaction of the appropriate azetidinium salt precursor 13 in a RCY of 34% after cleavage of
the EOE protecting group. The precursor 13 was formed from the respective open-chained
mesyl compound 12. Further analyses were accomplished showing an improved lipophilicity
with a logP of 2.55 compared to the recently published tracer II. Biological and
radiobiological studies are in progress and will be reported separately.
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Experimental
Materials and Methods
All reagents were purchased from commercial suppliers and used without further purification.
Compounds 1 and 9 were prepared according to the literature. Analytical TLC was performed
on pre-coated Silica Gel 60 F254 plates (Merck) and the results visualized under UV-light ( =
254 nm). 1H NMR, 13C NMR and 19F NMR spectra were recorded on a Varian Inova-400 or
Agilent DD2-400 (OneNMR Probe) spectrometer at 400, 101, and 376 MHz, respectively.
Chemical shifts are reported in ppm with tetramethylsilane (1H, 13C) and
trichlorofluoromethane (19F) as internal standard, respectively. MS spectra were obtained on
a Xevo TQ-S (Waters) by electron spray (ESI) as the ionization method. Melting points were
determined using a Cambridge Instruments Galen III apparatus and are uncorrected.
Analytical HPLC was performed on a VWR/Hitachi Elite La Chrome HPLC system,
equipped with a reverse phase column (Nucleosil 100-5C18 Nautilus), a UV-diode array
detector (254 nm) and a scintillation radiodetector (Raytest, Gabi Star) at a flow rate of 1
mL/min (eluent: acetonitrile/water, 20:80 + 0.1% TFA, isocratic). The radioactive compound
was identified with analytical radio-HPLC by comparison of the retention time of the
reference compound. Decay-corrected RCYs were quantified by integration of radioactive
peaks on a radio-TLC using a radio-TLC scanner (Fuji, BAS2000). [18F]Fluoride was
produced by the 18O(p,n)18F nuclear reaction utilizing the PET cyclotron Cyclone 18/9 (IBA,
Belgium); [18O]H2O was irradiated.
tert-Butyl 4-(3-morpholino-5-nitrophenyl)piperazine-1-carboxylate 2
Compound 1 (600 mg, 1.80 mmol) was dissolved in anhydrous toluene (15 mL) in a Schlenk tube under argon
atmosphere. 1-Boc-piperazine (672 mg, 3.60 mmol) and Cs2CO3 (2.93 g, 8.99 mmol) were added. Pd2dba3 and
xantphos were added in catalytic amounts. The mixture was stirred at 90 °C for 24 h. After cooling to rt, the
reaction was quenched with H2O (15 mL) and extracted with EtOAc (3 x 15 mL). The organic layer was dried
over Na2SO4 and concentrated under reduced pressure and the crude product was purified by flash column
chromatography (PE–EtOAc = 2:1  1:1) to yield compound 2 (634 mg, 90%) as an orange solid. Mp. 202°C;
Rf = 0.66 (PE–EtOAc = 1:1); 1H NMR: (400 MHz, CDCl3): δ = 1.49 (9H, s, CH3), 3.18–3.24 (8H, m, CH2N),
3.59 (4H, t, 3J = 5.1, CH2N), 3.87 (4H, t, 3J = 4.9, CH2O), 6.64 (1H, t, 4J = 2.1, 2-H), 7.26 (2H, d, 4J = 2.1 Hz,
4/6-H); 13C NMR (101 MHz, CDCl3): δ = 28.6 (CH3), 49.1 (CH2N), 66.8 (CH2O), 80.3 (Cq), 102.2, 102.7 (C-
4/6), 108.6 (C-2), 150.3 (C-1), 152.7, 152.8 (C-3/5), 154.8 (C=O); MS (ESI+): m/z 415 (49) [M++Na], 393 (100)
[M++H]; elemental analysis calcd. for C19H28N4O5 (392.45): C, 58.15, H, 7.19, N, 14.28; found: C, 58.44, H,
7.50, N, 14.42%.
4-(3-Nitro-5-(piperazin-1-yl)phenyl)morpholine 3
Compound 2 (1.85 g, 4.71 mmol) was dissolved in anhydrous dichloromethane (40 mL), trifluoroacetic acid (4
mL) was added dropwise and the mixture was stirred overnight at rt. After the removal of the solvent, 50 mL of
a 10% K2CO3 solution was added and the aqueous layer extracted with dichloromethane (3 x 15 mL). The
organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure to yield compound 3
(1.36 g, 99%) as an orange solid without further purification. Mp 144 °C; Rf = 0.17 (CHCl3–MeOH = 9:1); 1H
NMR: (400 MHz, CDCl3): δ = 1.68 (1H, br. s, NH), 3.04 (4H, t, 3J = 4.9, CH2N), 3.21 (8H, t, 3J = 4.9, CH2N),
3.87 (4H, t, 3J = 4.9, CH2O), 6.65 (1H, t, 4J = 2.1, 6-H), 7.23 (1H, t, 4J = 2.0, HAr), 7.27 (1H, t, 4J = 2.0, HAr);
13C NMR (101 MHz, CDCl3): δ = 46.1, 49.2, 50.1 (3 x CH2N), 66.8 (CH2O), 101.7, 102.4 (C-4/6), 108.1 (C-2),
150.3 (C-1), 152.7, 153.1 (C-3/5); MS (ESI+): m/z 293 (100) [M++H]; elemental calcd. for C14H20N4O3 (292.33):
C, 57.52, H, 6.90, N, 19.17; found: C, 57.36, H, 6.94, N, 19.04%.
4-(3-(4-(3-Fluoropropyl)piperazin-1-yl)-5-nitrophenyl)morpholine 4
Compound 3 (500 mg, 2.57 mmol), 1-fluoro-3-iodopropane (482 mg, 2.57 mmol) and Et3N (346 mg, 3.42
mmol) were dissolved in anhydrous THF (20 mL) and the reaction mixture was stirred at 60 °C overnight. The
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reaction was quenched with H2O (30 mL), extracted with dichloromethane (3 x 20 mL), the organic layer was
dried over Na2SO4 and the solvent removed. Purification was performed by flash chromatography (CHCl3–
MeOH = 20:1) to yield compound 4 as an orange solid (479 mg, 80%). Mp 102 °C; Rf = 0.82 (CHCl3–MeOH =
10:1); 1H NMR: (400 MHz, CDCl3): δ = 1.85–2.00 (2H, m, CH2CH2F), 2.55 (2H, t, 3J = 7.4, CH2N), 2.62 (4H, t,
3J = 4.8, CH2N), 3.21 (4H, t, 3J = 5.0, CH2N), 3.25 (4H, t, 3J = 5.0, CH2N), 3.86 (4H, t, 3J = 5.0, CH2O), 4.54
(2H, dt, 2JH,F = 47.2, 3J = 5.9, CH2F), 6.65 (1H, t, 4J = 2.1, 2-H), 7.23 (1H, t, 4J = 1.9, HAr), 7.27 (1H, t, 4J = 2.0,
HAr); 13C NMR (101 MHz, CDCl3): δ = 28.1 (d, 2JC,F = 19.4, CH2CH2F), 48.9, 49.2, 53.1 (3 x CH2N), 54.3 (d,
4JC,F = 5.3, CH2N), 66.8 (CH2O), 82.5 (d, 1JC,F = 165.1, CH2CH2F), 101.7, 102.4 (C-6/4), 108.0 (C-2), 150.3 (C-
5), 152.6, 152.7 (C-1/3); 19F NMR (376 MHz, CDCl3): δ = –220.6; MS (ESI+): m/z 352 (100) [M++H];
elemental calcd. for C17H25FN4O3 (352.40): C, 57.94, H, 7.15, N, 15.90; found: C, 57.76, H, 7.08, N, 15.35.
3-(4-(3-Morpholino-5-nitrophenyl)piperazin-1-yl)propan-1-ol 5
Compound 3 (500 mg, 1.71 mmol), 3-bromopropan-1-ol (380 mg, 2.73 mmol) and Et3N (0.38 mL, 2.73 mmol)
were dissolved in anhydrous THF (15 mL), NaI (50 mg) was added and the reaction mixture was stirred at 60
°C overnight. The mixture was treated with H2O (20 mL), extracted with dichloromethane (3 x 20 mL), the
organic layer was dried over Na2SO4 and the solvent removed. Purification was performed by flash
chromatography (CHCl3–MeOH = 10:1) to yield compound 5 as an orange solid (530 mg, 88 %). Mp 128 °C, Rf
= 0.53 (CHCl3–MeOH = 9:1); 1H NMR: (400 MHz, CDCl3): δ = 1.78 (2H, qi, 3J = 5.6, CH2CH2OH), 2.65–2.72
(6H, m, 3 x CH2N), 3.21 (4H, t, 3J = 4.8, CH2N), 3.26 (4H, t, 3J = 5.0, CH2N), 3.83 (2H, t, 3J = 5.3, CH2OH),
3.86 (4H, t, 3J = 4.8, CH2O), 6.63 (1H, t, 4J = 2.1, 2-H), 7.23 (1H, t, 4J = 2.0, HAr), 7.26 (1H, t, 4J = 2.0, HAr). 13C
NMR (101 MHz, CDCl3): δ = 27.3 (CH2CH2O), 48.9, 49.1, 53.2, 58.7 (4 x CH2N), 64.5, 66.8 (2 x CH2O),
101.9, 102.6 (C-6/4), 108.1 (C-2), 150.3 (C-5), 152.5, 152.7 (C-1/3); MS (ESI+): m/z 351 (100, M++H);
elemental calcd. for C17H26N4O4 (350.41): C, 58.27, H, 7.48, N, 15.99; found C, 58.06, H, 7.51, N, 16.13.
3-(-4-tert-Butoxycarbonyl)piperazino-5-morpholinoaniline 6
Compound 2 (930 mg, 2.37 mmol) was dissolved in CHCl3/MeOH (20 mL, v:v = 1:1) and Pd/C (500 mg) was
added. The resulting mixture was treated with H2 (approx. 1 bar) at rt for 24 h. Afterwards, the catalyst was
filtered and washed with CHCl3 (10 mL). The solvent of the combined filtrates was removed under reduced
pressure to yield compound 6 (819 mg, 95%) as colorless solid. Mp 173 °C; Rf = 0.06 (PE–EtOAc = 1:1); 1H
NMR: (400 MHz, CDCl3): δ = 1.47 (9H, s, CH3), 3.07–3.12 (8H, m, CH2N), 3.54 (4H, t, 3J = 5.2, CH2N), 3.82
(4H, t, 3J = 4.9, CH2O), 5.84 (2H, d, 4J = 1.7, 2/6-H), 5.93 (1H, t, 4J = 1.7, 4-H); 13C NMR (101 MHz, CDCl3): δ
= 28.6 (CH), 49.7, 49.8 (2 x CH2N), 67.1 (CH2O), 80.0 (Cq), 96.0, 96.2 (C-2/4), 96.6 (C-6), 148.1 (C-3), 153.5,
153.6 (C-1/5), 154.9 (C=O); MS (ESI+): m/z 363 (100, M++H); elemental calcd. for C19H30N4O3 (362.47): C,
62.96, H 8.34, N, 15.46; found: C, 63.07, H, 8.40, N, 15.38%.
3-(4-(3-Fluoropropyl)piperazin-1-yl)-5-morpholinoaniline 7
Compound 4 (479 mg, 1.49 mmol) was dissolved in MeOH (50 mL) and Pd/C (150 mg) was added. The
resulting mixture was treated with H2 (approx. 1 bar) at rt for 24 h. Afterwards, the catalyst was filtered and
washed with CHCl3 (10 mL). The solvent of the combined filtrates was removed under reduced pressure to yield
1
compound 7 (438 mg, 100%) as brown syrup. Rf = 0.37 (CHCl3–MeOH = 10:1); H NMR: (400 MHz, CDCl3):
δ = 1.83–1.99 (2H, m, CH2CH2F), 2.52 (2H, t, 3J = 7.4, CH2N), 2.58 (4H, t, 3J = 5.0, CH2N), 3.10 (4H, t, 3J =
4.8, CH2N), 3.15 (4H, t, 3J = 5.1, CH2N), 3.82 (4H, t, 3J = 4.8, CH2O), 4.52 (2H, dt, 2JH,F = 47.1, 3J = 6.0,
CH2F), 5.81 (1H, t, 4J = 1.9, HAr), 5.85 (1H, t, 4J = 1.9, HAr), 5.93 (1H, t, 4J = 1.9, HAr); 13C NMR (101 MHz,
CDCl3): δ = 28.0 (d, 2JC,F = 19.7, CH2CH2F), 49.4, 49.8, 53.4 (3 x CH2N), 54.4 (d, 4JC,F = 5.5, CH2N), 67.1
(CH2O), 82.6 (d, 1JC,F = 164.4, CH2CH2F), 95.6, 95.8, 96.2 (3 x Ar-CH), 148.0, 153.4, 153.5 (3 x Ar-Cq); 19F
NMR (376 MHz, CDCl3): δ = –220.4; MS (ESI+): m/z 345 (9, M++Na), 323 (46, M++H); elemental calcd. for
C17H27FN4O (322.42): C, 63.33, H, 8.44, N, 17.38; found: C, 63.03, H, 8.41, N, 17.44.
3-(4-(3-Amino-5-morpholinophenyl)piperazin-1-yl)propan-1-ol 8
Compound 5 (1.10 g, 3.14 mmol) was dissolved in CHCl3 (20 mL) and Pd/C (200 mg) was added. The resulting
mixture was treated with H2 (approx. 1 bar) at rt for 24 h. Afterwards, the catalyst was filtered and washed with
CHCl3 (10 mL). The solvent of the combined filtrates was removed under reduced pressure to yield compound 8
(887 mg, 88%) as light brown syrup. Rf = 0.22 (CHCl3–MeOH = 10:1); 1H NMR: (400 MHz, CDCl3): δ = 1.80
(2H, qi, 3J = 5.2, CH2CH2OH), 2.70–2.80 (6H, m, 3 x CH2N), 3.10 (4H, t, 3J = 4.8, CH2N), 3.21 (4H, t, 3J = 4.8,
CH2N), 3.79–3.86 (6H, m, CH2O/CH2OH), 5.84 (2H, d, 4J = 2.0, 2-/5-H), 5.92 (1H, t, 4J = 2.0, 4-H). 13C NMR
(101 MHz, CDCl3): δ = 27.2 (CH2CH2O), 49.5, 49.8, 53.5, 58.9 (4 x CH2N), 64.5, 67.1 (2 x CH2O), 95.9, 96.0,
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96.4 (3 x Ar-CH), 148.1, 153.3, 153.5 (3 x Ar-Cq). MS (ESI+): m/z 321 (100, M++H); elemental calcd. for
C17H28N4O2 (320.43): C, 63.72, H, 8.81, N, 17.48; found: C, 63.88, H, 8.66, N, 17.28.
N2-(3-(4-(3-Fluoropropyl)piperazin-1-yl)-5-morpholinophenyl)-N4-(1-(1-ethoxyethyl)-1H-indazol-4-yl)-N4-
methylpyrimidin-2,4-diamine 10
Compound 7 (358 mg, 1.11 mmol) and compound 9 (405 mg, 1.22 mmol) were dissolved in anhydrous dioxane
(10 mL), p-TsOH·H2O was added in catalytic amounts and the resulting mixture was stirred at 90°C for 24 h.
After cooling to rt, saturated hydrogencarbonate solution was added (30 mL), the aqueous layer was extracted
with EtOAc (3 x 20 mL), the combined organic layers were dried over Na2SO4 and the solvent was removed.
Purification was performed by flash chromatography (CHCl3–MeOH = 25:1) to yield compound 10 as a
brownish solid (428 mg, 62%). Rf = 0.63 (CHCl3–MeOH = 4:1); 1H NMR: (400 MHz, CDCl3): δ = 7.82 (d, 5J3,5
= 0.7 Hz, 1H, H-3), 7.80 (d, 3J6‘,5‘ = 6.0 Hz, 1H, H-6'), 7.68 (d, 3J7,6 = 8.6 Hz, 1H, H-7), 7.42 (dd, 3J6,5 = 7.4, 3J6,7
= 8.6 Hz, 1H, H-6), 7.07 (dd, 3J5,6 = 7.4 Hz, 5J5,3 = 0.7 Hz, 1H, H-5), 6.97 (s, 1H, NH), 6.90 - 6.88 (m, 2H, H-
2''/6''), 6.19 (t, 4J4‘‘,2‘‘ = 4J4‘‘,6‘‘ = 2.0 Hz, 1H, H-4'' ), 5.91 (q, 3J = 6.1 Hz, 1H, CH), 5.71 (d, 3J5‘,6‘ = 6.0 Hz, 1H,
H-5'), 4.53 (dt, 3J = 5.9 Hz; 2JH,F = 47.3 Hz, 2H, CH2F ), 3.85 (t, 3J = 4.9 Hz, 4H, CH2N), 3.63 (s, 3H, CH3),
3.49 (dq, 2J = 9.3 Hz, 3J = 7.1 Hz, 1H, CH2CH3), 3.32 (dq, 2J = 9.3 Hz, 3J = 7.1 Hz, 1H, CH2CH3), 3.23 (t, 3J =
4.9 Hz, 4H, CH2N), 3.17 (t, 3J = 4.9 Hz, 4H, CH2N), 2.61 (t, 3J = 4.9 Hz, 4H, CH2N), 2.53 (t, 3J = 7.4 Hz, 2H,
CH2N), 1.99 - 1.86 (m, 2H, CH2CH2CH2), 1.82 (d, 3J = 6.1 Hz, 3H, CHCH3), 1.17 (t, 3J = 7.1 Hz, 3H, CH2CH3).
13C NMR (101 MHz, CDCl3): δ = 163.0 (C-4‘), 159.8 (C-2‘), 155.8 (C-6‘),152.9, 152.6 (C-3’’/-5’’), 140.8 (C-
7a) 140.1 (C-4), 136.8 (C-1‘‘), 131.2 (C-3), 127.4 (C-6), 122.1 (C-3a), 119.4 (C-5), 110.6 (C-5’), 100.8 (C-7),
100.3 (C-4’’), 99.8, 97.3 (C-2’’/-6’’), 87.6 (CH), 82.4 (d, 1JC,F = 164.5 Hz, CH2F), 67.1 (CH2O), 64.1 (CH2),
54.5 (d, 3JC,F = 5.4 Hz, CH2N), 53.3, 49.9, 49.4 (3 x CH2N), 38.6 (CH3), 27.7 (d, 2JC,F = 18.8 Hz, CH2CH2F)
21.1, 15.0 (2 x CH3). 19F NMR (376 MHz, CDCl3): δ = – 220.2. MS (ESI+): m/z (%) = 618 (30) [M++H], 546
(100) [M++H–EOE].
N2-(3-(4-(3-Hydroxypropyl)piperazin-1-yl)-5-morpholinophenyl)-N4-(1-(1-ethoxyethyl)-1H-indazol-4-yl)-N4-
methylpyrimidin-2,4-diamine 11
Compound 8 (159 mg, 0.50 mmol) and compound 9 (181 mg, 0.55 mmol) were dissolved in anhydrous dioxane
(7 mL), p-TsOH·H2O was added in catalytic amounts and the resulting mixture was stirred at 90°C for 24 h.
After cooling to rt, saturated hydrogencarbonate solution was added (20 mL), the aqueous layer was extracted
with EtOAc (3 x 15 mL), the combined organic layers were dried over Na2SO4 and the solvent was removed.
Purification was performed by flash chromatography (CHCl3–MeOH = 20:110:1) to yield compound 11 as a
brownish oil (90 mg, 29%). Rf = 0.52 (CHCl3–MeOH = 4:1); 1H NMR (400 MHz, CDCl3): δ = = 7.81 (d, 5J3,5
0.7 Hz, 1H, H-3), 7.79 (d, 3J6‘,5‘ = 6.0 Hz, 1H, H-6'), 7.68 (d, 3J7,6 = 8.6 Hz, 1H, H-7), 7.41 ("t", 3J6,5 = 7.4 Hz,
=
3J6,7 = 8.6 Hz, 1H, H-6), 7.20 (s, 1H, NH), 7.07 (dd, 3J5,6 = 7.4 Hz, 5J5,3 = 0.7 Hz, 1H, H-5), 6.91 (t, 3J = 2.0 Hz,
1H, HAr), 6.88 (t, 3J = 2.0 Hz, 1H, HAr), 6.17 (t, 4J4‘‘,2‘‘ = 4J4‘‘,6‘‘ = 2.0 Hz, 1H, H-4'' ), 5.91 (q, 3J = 6.1 Hz, 1H,
CH), 5.71 (d, 3J5‘,6‘ = 6.0 Hz, 1H, H-5'), 3.85 - 3.80 (m, 6H, CH2O, CH2OH), 3.62 (s, 3H, CH3), 3.49 (dq, 2J =
9.3 Hz, 3J = 7.1 Hz, 1H, CH2CH3), 3.31 (dq, 2J = 9.3 Hz, 3J = 7.1 Hz, 1H, CH2CH3), 3.22 (t, 3J = 5.0 Hz, 4H,
CH2N), 3.16 (t, 3J = 4.8 Hz, 4H, CH2N), 2.69–2.66 (m, 6H, CH2N), 1.82 (d, 3J = 6.1 Hz, 3H, CHCH3), 1.78 -
1.73 (m, 2H, CH2CH2CH2), 1.17 (t, 3J = 7.1 Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3): δ = 162.9 (C-4‘),
159.5 (C-2‘), 155.1 (C-6‘),152.9, 152.7 (C-3’’/-5’’), 150.5 (C-1’’), 141.6 (C-7a) 140.1 (C-4), 131.6 (C-3), 127.4
(C-6), 122.3 (C-3a), 119.4 (C-5), 110.0 (C-5’), 100.4 (C-7), 100.0 (C-4’’), 99.3, 97.5 (C-2’’/-6’’), 87.4 (CH),
67.1, 64.5 (2 x CH2O), 64.0 (CH2), 58.9, 53.5, 50.0, 49.7 (4 x CH2N), 38.3 (CH3), 27.2 (CH2CH2CH2), 21.1,
15.0 (2 x CH3). MS (ESI+): m/z (%) = 616 (40) [M++H], 544 (100) [M+–EOE].
N2-(3-(4-(3-Fluoropropyl)piperazin-1-yl)-5-morpholinophenyl)-N4-(1H-indazol-4-yl)-N4-methylpyrimidin-2,4-
diamine 14
Compound 10 (384 mg, 0.62 mmol) was treated with HCl (12 mL, 1 M) and stirred at rt for 1.5 h. Afterwards, a
saturated hydrogencarbonate solution was added for neutralization, the aqueous layer was extracted with
chloroform (3 x 20 mL), the combined organic layers were dried over Na2SO4 and the solvent was removed.
Purification was performed by flash chromatography (CHCl3–MeOH = 30:1) to yield compound 14 as a
1
brownish oil (288 mg, 85%). Rf = 0.35 (CHCl3–MeOH = 9:1); H NMR: (400 MHz, CDCl3): δ = 10.56 (s, 1H,
NH), 7.91 (d, 5J3,5 = 0.8 Hz, 1H, H-3), 7.81 (d, 3J6’,5’ = 6.0 Hz, 1H, H-6'), 7.46 - 7.41 (m, 2H, H-6/-7), 7.10 (s,
1H, NH), 7.08 (dd, 3J5,6 = 6.5 Hz, 5J5,3 = 0.8Hz, 1H, H-5 ), 6.90 - 6.88 (m, 2H, H-2''/-6''), 6.19 (t, 4J4’’,2’’ = 4J4’’,6’’
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= 2.1 Hz, 1H, H-4'' ), 5.71 (d, 3J5’,6’ = 6.0 Hz, 1H, H-5'), 4.53 (dt, 3J = 6.0 Hz; 2JH,F = 47.3 Hz, 2H, CH2F), 3.85
(t, 3J = 4.8 Hz, 4H, CH2O), 3.63 (s, 3H, CH3), 3.22 (t, 3J = 4.8 Hz, 4H, CH2N), 3.16 (t, 3J = 4.8 Hz, 4H, CH2N),
2.61 (t, 3J = 4.8 Hz, 4H, CH2N), 2.54 (t, 3J = 7.4 Hz, 2H, CH2N), 1.99 - 1.86 (m, 2H, CH2). 13C NMR (101
MHz, CDCl3): δ = 163.0 (C-4‘), 159.9 (C-2‘), 155.7 (C-6‘),152.9, 152.9 (C-3’’/-5’’), 141.8 (C-7a) 141.7 (C-4),
137.5 (C-1‘‘), 133.2 (C-3), 127.8 (C-6), 120.7 (C-
1
3a), 119.3 (C-5), 109.1 (C-5’), 100.2 (C-7), 99.7 (C-4’’), 99.1, 97.4 (C-2’’/-6’’), 82.7 (d, JC,F = 164.5 Hz,
CH2F), 67.2 (CH2O), 54.5 (d, 3JC,F = 5.5 Hz, CH2N), 53.4, 50.0, 49.7 (3 x CH2N), 38.3 (CH3), 28.0 (d, 2JC,F
=
19.8 Hz, CH2CH2F). 19F NMR (376 MHz, CDCl3): δ = – 220.0 ppm. MS (ESI+): m/z (%) = 546 (100) [M++H].
N2-(3-(4-(3-Methylsulfonyloxypropyl)piperazin-1-yl)-5-morpholinophenyl)-N4-(1-(1-
ethoxyethyl)-1H-indazol-4-yl)-N4-methylpyrimidin-2,4-diamin 12
Compound 11 (65 mg, 0.11 mmmol) was dissolved in anhydrous dichloromethane (10 mL), MsCl (18 mg, 0.16
mmol) and DIPEA (27 mg, 0.21 mmol) were added and the resulting mixture was stirred for 1 h at rt.
Afterwards, water (10 mL) was added and the aqueous layer was extracted with dichloromethane (3 x 5 mL),
the combined organic layers were dried over Na2SO4 and the solvent was removed at temperatures below 30°C
1
to yield compound 12 as a light brownish oil (64 mg, 87%). Rf = 0.55 (CHCl3–MeOH = 10:1); H NMR: (400
MHz, CDCl3): δ = 7.81 (d, 5J3,5 = 0.8 Hz, 1H, H-3), 7.79 (d, 3J6‘,5‘ = 6.0 Hz, 1H, H-6'), 7.68 (d, 3J7,6 = 8.5 Hz,
1H, H-7), 7.41 ("t", 3J6,7 = 8.5 Hz, 3J6,5 = 7.4 Hz, 1H, H-6), 7.12 (s, 1H, NH), 7.07 (dd, 3J5,6 = 7.4 Hz, 5J5,3 = 0.8
Hz, 1H, H-5), 6.89 (m, 2H, Har), 6.18 (t, 4J4’’,2’’ = 4J4’’,6’’ = 2.0 Hz, 1H, H-4'' ), 5.92 (q, 3J = 6.1 Hz, 1H, CH),
5.71 (d, 3J5‘,6‘ = 6.0 Hz, 1H, H-5'), 4.32 (t, 3J = 7.0 Hz, 2H, CH2N), 3.85 - 3.83 (m, 4H, CH2O), 3.62 (s, 3H,
CH3), 3.49 (dq, 2J = 9.3 Hz, 3J = 7.0 Hz, 1H, CH2CH3), 3.31 (dq, 2J = 9.3 Hz, 3J = 7.0 Hz, 1H, CH2CH3), 3.21 (t,
3J = 5.0 Hz, 4H, CH2N), 3.17 (t, 3J = 5.0 Hz, 4H, CH2N), 3.01 (s, 3H, CH3S), 2.59 (t, 3J = 5.0 Hz, 4H, CH2N),
2.51 (t, 3J = 7.0 Hz, 2H, CH2N), 1.99 - 1.93 (m, 2H, CH2), 1.81 (d, 3J = 6.1 Hz, 3H, CHCH3), 1.17 (t, 3J = 7.0
Hz, 3H, CH2CH3). 13C NMR (101 MHz, CDCl3): δ = 162.9 (C-4‘), 159.8 (C-2‘), 155.7 (C-6‘),152.9, 152.8 (C-
3’’/-5’’), 150.5 (C-1’’), 141.7 (C-7a) 140.1 (C-4), 131.6 (C-3), 127.4 (C-6), 122.3 (C-3a), 119.4 (C-5), 109.9 (C-
5’), 100.2 (C-7), 99.6 (C-4’’), 99.0, 97.5 (C-2’’/-6’’), 87.4 (CH), 68.5, 67.1 (2 x CH2O), 64.0 (CH2), 54.1, 53.4,
50.0, 49.7 (4 x CH2N), 38.2, 37.4 (2 x CH3), 26.6 (CH2), 21.1, 15.0 (2 x CH3). MS (ESI+): m/z (%) = 636 (100)
[M++H–EOE].
N2-(3-(4-(3-[18F]Fluoropropyl)piperazin-1-yl)-5-morpholinophenyl)-N4-(1H-indazol-4-yl)-N4-methylpyrimidin-
2,4-diamine [18F]14
An anion-exchange cartridge (Waters, Sep-Pak Light Accell Plus QMA) was activated by rinsing with 10 mL of
a 1M NaHCO3 solution and 10 mL of deionized H2O. [18F]Fluoride was trapped on the cartridge, eluted with a
Kryptofix K 2.2.2./K2CO3 solution (1.5 mL) into the reaction vessel and dried via azeotropic distillation with
anhydrous acetonitrile (3 x 2 mL) under a stream of nitrogen at 100°C for approx. 30 min. Precursor 12 or 13
(~3 mg) was dissolved in 500 µL of anhydrous acetonitrile, and the mixture was added to the [18F]fluoride-
containing sealed vial. The resulting solution was heated at 100°C for 30 min. Afterwards, HCl (100 µL / 2 M)
was added and the mixture was heated at 100°C for further 5 min. Next, the mixture was treated with 15 mL
deionized H2O and then passed through a C18 cartridge (LiChrolut RP-18/Merck). After elution with ethanol,
samples for analytical radio-TLC and radio-HPLC were taken. Analytical radio-TLC: Rf = 0.37
(dichloromethane/methanol, 9/1). Analytical radio-HPLC: tR = 11.9 min (eluent: acetonitrile/water 20/80 + 0.1%
TFA). Purification was done via semi-preparative radio-HPLC.
Acknowledgements. The authors thank the Fonds der Chemischen Industrie (FCI, Germany)
for financial support. The authors also thank the Helmholtz Association for funding a part of
this work through Helm olt - ortfolio opic ec nologie und edi in ultimodale
ildge ung ur uf l rung des n vivo-Ver altens von polymeren iomaterialien”. The
excellent technical assistance of Waldemar Herzog is gratefully acknowledged.
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Table 1. Comparison of calculated vs. determined logP values.
compound
calcd. logP47
3.30
calcd. logP48
2.77
expt. logP
n.d.
I
3.77
3.11
n.d.
II
3.42
3.87
3.40
3.34
2.98
2.55
III
14
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Figure 1. General strategies for the insertion of a [18F]fluoroethyl or [18F]fluoropropyl moiety.
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Figure 2. Overview of original inhibitors I and III as basis for the development of possible radiotracers IV – VI
and recently prepared [18F]tracer II.
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Figure 3. Radio-HPLC chromatogram of [18F]10 (EOE protected) and the final tracer [18F]14 (deprotected) after
purification.
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Figure 4. Radio-TLC chromatogram of the final tracer [18F]14 from the reaction mixture after deprotection.
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Scheme 1. Synthesis pathway for substructure B. Reagents and conditions: a) N-Boc-piperazine, Cs2CO3,
xantphos, Pd2dba3, 90°C, 2 d; b) TFA, DCM, rt, overnight; c) 1-fluoro-3-iodopropane, Et3N, THF, 60°C,
overnight; d) 3-bromopropanol, NaI, Et3N, THF, 60°C, overnight; e) Pd/C-H2, MeOH-CH2Cl2, rt, 24 h.
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Scheme 2. Synthesis pathway for precursor 13 and reference compound 14. Reagents and conditions: a)
p-TsOH, dioxane, 90°C, overnight; b) HCl, EtOH, 1.5 h; c) MsCl, DCM, rt, 60 min; d) acetonitrile, 60°C, 3 h.
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Scheme 3. General radiolabeling procedure for [18F]14 from precursors 12 and 13. Reagents and conditions: a)
K[18F]F, K2CO3, K222, acetonitrile, 100°C, 30 min.
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