Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers

Article abstract of DOI:10.1016/j.bmc.2015.06.040

Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [¹¹C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired ¹⁸F-radiotracer under the chosen conditions.

Full text of DOI:10.1016/j.bmc.2015.06.040

Bioorganic & Medicinal Chemistry 23 (2015) 6025–6035
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of indazolylpyrimidine derivatives as high-affine
EphB4 receptor ligands and potential PET radiotracers
Kristin Ebert a, , Jens Wiemer a, , Julio Caballero b, Martin Köckerling c, Jörg Steinbach a,d, Jens Pietzsch a,d
,
Constantin Mamat a,d,
⇑
a Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden, Germany
b Universidad de Talca, Centro de Bioinformática y Simulación Molecular, 2 Norte 685, Casilla 721, Talca, Chile
c Universität Rostock, Institut für Chemie, Anorganische Festkörperchemie, Albert-Einstein-Straße 3a, D-18057 Rostock, Germany
d TU Dresden, Fachbereich Chemie und Lebensmittelchemie, D-01062 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing
hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular
imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of
the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations
with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed
to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on
this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluo-
rine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to pre-
vent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient
radiolabeling strategy using [11C]methyl iodide was established which afforded the isotopically labeled
radiotracer in 30–35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammo-
nium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead
to the desired 18F-radiotracer under the chosen conditions.
Received 22 April 2015
Revised 5 June 2015
Accepted 11 June 2015
Available online 2 July 2015
Keywords:
In silico studies
Radiolabeling
Carbon-11
Fluorine-18
Spiro precursor
Eph receptor
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
EphB4 with its preferential ligand ephrinB2 is one of the most
important Eph receptors. Pro-tumorigenic effects of EphB4 activa-
Members of the Eph receptor tyrosine kinase family play an
essential role in the pathogenesis of cancer1 and, therefore, they
are promising candidates for noninvasive molecular imaging pur-
poses, for example, by positron emission tomography (PET).
These receptors belong to the largest family of receptor tyrosine
kinases and they are divided into two groups, class A and B,
depending on their structure and binding to the GPI-anchored class
A ephrins or transmembrane proteins (ephrins) of class B.2,3
Cellular survival, re-organization of the cytoskeleton, cell attach-
ment, and motility was regulated due to the cellular communica-
tion system provided by Eph receptors and ephrins.4 They are
involved in physiological processes like embryonic development
and angiogenesis5,6 as well as in pathophysiological processes like
tumorigenesis and tumorangiogenesis.7
tion are discussed for prostate8 and breast cancer, where EphB4
knockdown resulted in reduced tumor growth in mice.9
Inconsistent outcome of EphB4/ephrinB2 signaling is also
described for tumorigenesis,10 with anti-tumorigenic effects of
activated EphB4 in mouse melanoma and breast cancer cells, and
reduction or loss of EphB4 expression in human colorectal tumors
and invasive breast carcinoma.11–13
Several potent Eph kinase inhibitors were reported either based
on high molecular weight compounds like peptides,14–16 which
block the extracellular domain of the appropriate receptor, or on
small organic molecules,17 which bind to the intramolecular ATP
binding pocket. To date, only peptide-based radiotracers containing
Cu-64 or In-111 like I18,19 but also with F-18 like II,20,21which are
specific for Eph receptors, are known for multimodal imaging pur-
poses using PET or SPECT. Due to the favorable chemical and biolog-
ical properties, small organic inhibitors (e.g., IIIa or IV) published by
Bardelle et al. are the basis of our research (Scheme 1).22–25
Recently, the novel fluorine-18-containing radiotracer IIIb based
on the benzodioxolylpyrimidine structural motif was developed
⇑
Corresponding author. Tel.: +49 351 260 2805; fax: +49 351 260 2915.
E-mail address: c.mamat@hzdr.de (C. Mamat).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2015.06.040
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

6026
K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
O
F
(
N
NH
O
N
O
NH
)n
O
64Cu-DOTA-TNYLFSPNGPIARAW
NH
R
N
F
O
N
N
N
N
( )n
NH
N
N
I
O
S
O
HN
N
N
N
N
Cl
N
N
SNEWILPRLPQHL-triazolyl-[18F]FP
N
N
N
N
N
N
H
N
N
H
N
II
O
N
N
N
IV
N
N
H
N
IIIa: R = H
O
N
N
(
F
)n
R = CH2CH218F
IIIb:
O
H
O
1a
1b
: n = 1, : n = 2
2a
2b
: n = 1, : n = 2
3a
3b
: n = 1, : n = 2
Scheme 1. Structures of selected EphB4 radiotracers and lead structure used in this
paper.
Scheme 2. Proposed structures of fluorine-18 radiotracers and highlighted labeling
positions.
and analyzed via in vitro and in vivo studies.26 Unfortunately,
despite the high affinity of the original inhibitor IIIa to the EphB4
receptor (IC50 = 90 nM, recombinant EphB4), the tracer IIIb showed
a substantial uptake in tumor cells (A375) but 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 (clogP = 3.77) or a rather low affinity. To over-
come these problems, a novel lead structure was chosen based on
a indazolylpyrimidine core containing inhibitor IV with a much
higher affinity to the EphB4 receptor (IC50 = 1.3 nM, clogP = 3.42).24
In this regard, a convenient radiolabeling route to novel carbon-
11 and fluorine-18 radiotracers including a synthesis plan for the
insertion of protecting groups is pointed out. Prior to synthesis
and labeling, in silico docking studies are accomplished for finding
the best labeling position in case of the fluorine-18 introduction.
Afterwards, the corresponding precursors and reference com-
pounds for 11C/18F labeling are synthesized.
2. Results and discussion
2.1. In silico studies
Figure 1. Conformational comparison of the ligand IMPPD (two conformations)
from the crystal structure 2x9f (cyan) and compound IV (yellow).
Contrary to a radiolabeling with carbon-11 it is not possible to
apply an isosterical labeling procedure with fluorine-18 due to the
absence of fluorine-19 in the inhibitor lead molecule IV. To over-
come this fact, single hydrogen atoms or functional groups like
hydroxyl residues were normally replaced by fluorine-18/-19.27
In most of the cases, this replacement leads to a change of the
chemical and biological properties of the respective molecule. To
fix this problem, acceptable labeling positions are necessary to
determine using in silico docking experiments.
In general, three labeling positions were proven in the original
molecule for the introduction of fluorine-18/19 as pointed out in
Scheme 2. The simplest comprises the introduction of a fluoroalkyl
moiety on the free secondary nitrogen atom of original inhibitor IV
which would lead to structures 1a,b. The second variant comprises
a change of one of the morpholino groups of IV by a piperazine
residue in the first step. In the second step, a fluoroalkyl chain
was introduced into the piperazine moiety which gives 2a,b. The
last possibility comprehends change of the methyl group attached
to the secondary nitrogen atom of IV by a fluoroalkyl group (3a,b).
It was pointed out earlier, that both nitrogen atoms of the indazole
moiety form hydrogen donor and acceptor bonds to the side chain
‘S-shaped’ conformation. In this case, the indazole N01 NH and
N02 nitrogen form hydrogen donor and acceptor bonds to the side
chain carbonyl of Glu664 and the backbone NH of Asp758, respec-
tively (Fig. 1). RMSD values considering the common heavy atoms
between both conformations of IMPPD and docked conformation
obtained for IV were 0.483 and 0.434 Å. The low RMSD values
demonstrate that docking of IV reproduces IMPPD orientation.
On the basis of an X-ray structure analysis of the EphB4 recep-
tor including a similar inhibitor molecule (IMPPD), the secondary
nitrogen atom (highlighted in red in 1a,b) of the original inhibitor
IV has an important H-bond interaction with backbone of Met696
of the EphB4 receptor hinge region. According to this, it is expected
that compounds 1a,b should not have high interactions to the
active site of EphB4 when ethyl or propyl groups are placed on this
amine group. Therefore, compounds 1a,b are not supposed to func-
tion as good EphB4 inhibitors.
Compounds 3a and 3b seemed to be favorable candidates. They
have the same orientation with respect to the ligand from the
reported X-ray structure, the change of methyl by F-propyl group
should be tolerated since the pocket containing this group is large
and contains hydrophobic residues, but the occupancy of this
pocket by bigger hydrophobic groups can be unfavorable due to
the presence of some hydrophilic groups. The fluoropropyl group
of 3b is located close to side chains of Leu747, Lys647 (catalytic
lysine) and Val629, establishing hydrophobic interactions (Fig. 2).
Additionally, according to docking, 2a,b seemed to be good can-
didates, too. They have also the same orientation with respect to
the ligand from the reported X-ray structure and both the F-ethyl
and the F-propyl group are tolerated. The F-propyl group of 2b is
oriented towards the solvent. It is difficult to explain the potency
carboxylate of the conserved aC-helix glutamate (Glu664) and the
backbone NH of the DFG triad Asp758, respectively.24
First, we docked compound IV in available crystal structure that
contains a similar inhibitor molecule. The crystal structure of the
complex between EphB4 and the inhibitor N4-(1H-indazol-4-yl)-
N2-(3-(methylsulfonyl)phenyl)pyrimidin-2,4-diamine
(IMPPD)
(pdb: 2x9f) was selected; it contains two conformations of the
crystalized ligand. The structure shown in Figure 1 confirms
the predicted binding mode of IV in the ATP binding site, with
the pyrimidine N-1 and C-2 anilino N–H forming hydrogen
acceptor and donor bonds to the hinge region at Met696 and the
indazole heterocycle buried in the binding pocket, resulting in a

K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6027
Table 1
Docking energy scores for tested compounds
Compound
ICM scoring (kJ/mol)
IV
ꢀ435.65
ꢀ373.51
ꢀ396.93
ꢀ454.92
ꢀ429.99
ꢀ430.03
ꢀ440.55
1a
1b
2a
2b
3a
3b
O
N
part A
R1
part B
R2
N
N
N
N
N
N
N
H
O
R1 = H, Me, EOE, PMB
2 = H, Me, (CH2)3F
R
Figure 3. General composition of radiotracers, references and precursors.
modified procedure published by Slade et al.29 Additionally,
methylated compound 5c was prepared from 4 with 48% yield as
reference for the latter carbon-11-labeling. In case of the introduc-
tion of both the PMB and the methyl group, the respective 2H-re-
gioisomer was obtained supplementary.30 No 2H-regioisomer
was found when applying the EOE group.
Next, the nitro group of compounds 4 and 5a–c was reduced
with H2/Pd–C to obtain amines 6a–d. The resulting amino group
attached to position 4 of the indazole moiety is necessary for the
following introduction of the pyrimidine residue. Thus, 6a–d were
reacted with 2,4-dichloropyrimidine in absolute ethanol and DIPEA
to obtain 7a–d in yields from 17% to 57%. The last step afforded the
methylation of the secondary amine. Therefore, 7a–c were treated
with methyl iodide to give 8a–c in high yields of 88–91%. The reac-
tion path and the conditions were figured out in Scheme 3.
The molecular structure of EOE-protected compound 5b was
validated independently by a single-crystal X-ray analysis and
the resulting molecular structure of 5b is shown in Figure 4.31
Next, 3,5-di(morpholino)aniline 9 as second substructure (part
Figure 2. EphB4/compound 3b complex. (a) Conformation proposed by docking;
(b) interactions of the F-propyl group of the ligand (green spheres) with the
hydrophobic wall composed by Val629, Lys647, and Leu747 (gray dots).
when groups are oriented to the solvent using modeling, because
there are no interactions with protein residues. However, it is pos-
sible that 2a and 2b are potent inhibitors because the main inter-
actions with the kinase (in the hinge region) are established for
these compounds. Docking energy scores were similar for com-
pounds IV, 2a, 2b, 3a, and 3b (Table 1).
B) was synthesized from the respective nitro compound as pointed
23,32
out in the literature.
Crystals of 9 suitable for a single crystal
X-ray analysis were grown from a saturated ethyl acetate/petro-
leum ether solution and the molecular structure was expressed
in Figure 5.31
2.2. Preparation
Finally, two reactions paths were figured out for the connection
of substructures A (7a–d and 8a–c) with B (compound: 9). In the
first path, the connection reaction was done under acid catalysis.
Thus, compounds 7a–d and 8a were dissolved in anhydrous diox-
ane and stirred at 90 °C overnight. The desired products 10, 11, 14
and 16 were obtained in good yields (44–83%) except for 7b which
led to deprotected derivative 16. Hereupon, it was necessary that
both EOE protected substances 7b and 8b were reacted with 9 in
a Pd-catalyzed Buchwald–Hartwig amination. In addition, 7c and
8c were reacted as well using the same conditions and the result-
ing compounds 12–15 were obtained in similar yields (62–83%)
compared to the acid catalyzed reaction path A. All results were
pointed out in Scheme 4 and Table 2. The final step requires the
removal of the protecting groups. Unfortunately, all our attempts
to eliminate the PMB protecting group from 10 and 11 failed (see
later in detail). Based on these results, the EOE group is exposed
to be the better choice for this purpose and was applied instead
for this protection purpose. In this context, original inhibitor 17,
which serves as reference for the radiolabeling with [11C]methyl
The synthesis path for all reference compounds and precursors
for an isotopically labeled tracer with carbon-11 and a novel fluo-
rine-18 containing radiotracer based on previous in silico studies
was developed. In general, the inhibitor molecule IV as lead struc-
ture, the new radiotracers, the appropriate references and their
corresponding precursors consist of two substructures A and B
(Fig. 3) which were prepared separately.
The first substructure (part A) of the compounds was prepared
starting from indazole derivative 4.28 The development of a pro-
tecting group strategy was essential to avoid unwanted alkylation
reactions at the nitrogens of indazole 4 in case of the introduction
of carbon-11, which will be accomplished using [11C]CH3I, and fur-
ther for the preparation of fluorine-18 precursors and references
(see later). Thus, the p-methoxybenzyl (PMB) as well as the ethox-
yethyl (EOE) group were chosen. Compound 5a (1H-isomer) was
yielded with 48% when 4 was reacted with p-methoxybenzyl chlo-
ride whereas compound 5b was obtained in 80% yield when 4 was
treated with ethyl vinyl ether under acidic conditions after a

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K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
and filtration of the precipitated silver salts, the final spiro precur-
sor 23 was yielded after purification via column chromatography.
The full reaction sequence was pointed out in Scheme 5.
NH2
NO2
R1
R1
N
N
HN
N
N
d)
e)
N
N
f)
N
N
N
N
N
N
R1
R1
Cl
N
Cl
: R1 = PMB
6a: R1 = PMB
6b: R1 = EOE
6c: R1 = Me
6d: R1 = H
: R1 = PMB
7a
8a: R1 = PMB
8b: R1 = EOE
5a
2.3. Carbon-11 radiolabeling
a)
b)
7b: R1 = EOE
7c: R1 = Me
7d: R1 = H
4: R1 = H
5b: R1 = EOE
5c: R1 = Me
: R1 = Me
8c
c)
Carbon-11 enables the radiolabeling of bioactive molecules
without changing the chemical structure and the biochemical
properties due to the isotopic exchange. The most commonly
applied labeling method consists of the [11C]methylation using
Scheme 3. Synthesis path for substructure A. Reagents and conditions: (a) 4-
methoxybenzyl chloride, Cs2CO3, DMF, 110 °C, 2 h; (b) ethyl vinyl ether, p-TsOH, rt,
1 h; (c) MeI, NaH, 0 °C, 1.5 h; (d) Pd/C-H2, MeOH–CH2Cl2, rt, 1 d; (e) 2,4-
dichloropyrimidine, DIPEA, EtOH, 90 °C, 2 d; (f) MeI, Cs2CO3, DMF, rt, 4 h.
[
11C]CH3I or [11C]CH3OTf.33,34 The original inhibitor contains an
amine bound methyl group which is excellently suitable for the
isotopic labeling with carbon-11. In first experiments, the optimal
labeling conditions for the literature known compound 17 was
investigated. Problematic, three secondary amino functions are
present in this starting molecule feasible to be alkylated. Prior to
the radiolabeling, it was evaluated, which of the three amino func-
tions in precursor 16 are preferred to be methylated. For this pur-
pose, 16 was treated with 1 equiv of methyl iodide. In addition to
the desired original inhibitor 17 (61% yield) which serves as refer-
ence, the twice alkylated compound 15 was yielded (14%).
However, the threefold methylated product was not found even
when treated with a higher excess of MeI. Therefore, it was neces-
sary to develop a protecting strategy only for the indazole moiety
to avoid the introduction of the methyl group at this place and in
this case a twofold labeling of 16 with [11C]CH3I. As indicated
previously in Scheme 3, the p-methoxybenzyl group and the
ethoxyethyl group were applied for the protection of the indazole
ring. Both groups are stable under strong basic conditions. Thus, it
should be possible to remove these PGs either with mineral acids
(EOE),29 with DDQ (PMB)35 or with strong acids like TFA
(EOE/PMB).36 However, preliminary inquiries pointed out that it
was impossible to cleave the PMB group with DDQ as well as with
TFA under different conditions.
iodide, was obtained when 13 was deprotected with 1 M HCl at
ambient temperature or with p-TsOH at 100 °C for 24 h in a high
yield of 92%.
Two positions of the original inhibitor molecule IV were envis-
aged for the incorporation of fluorine-18/19. Optimally, the methyl
group of the original inhibitor IV was replaced by the 3-fluoro-
propyl moiety. Thus, compound 7b was treated with NaH for
30 min. Afterwards, 1-fluoro-3-iodopropane was added and 18a
(98%) was yielded after aqueous workup. Next, the p-TsOH cat-
alyzed reaction with aniline 9 was accomplished to obtain the
EOE protected fluorine-19 containing reference compound 19a.
After the workup, the EOE protecting group was removed immedi-
ately using 1 M HCl and reference compound 20 was achieved in
59% yield.
Further, the respective precursor was synthesized as follows. In
the presence of Cs2CO3 as base, compound 7b was treated with 3-
iodopropanol which was previously prepared from 3-bromo-
propanol and NaI in a Finkelstein exchange reaction. In the next
step, 18b was reacted with aniline 9 under Buchwald–Hartwig
conditions to give 19b. However, under these conditions, starting
material was still present. A complete conversion of 9 was not
observed. To fix this problem, the p-TsOH catalyzed reaction with
aniline 9 was accomplished to obtain the EOE protected hydroxy-
propyl compound 19b. Next, 19b was first tried to tosylate with
p-TsCl and Et3N as base to yield precursor 21a, however, no isolat-
able product was obtained. As an alternative, mesyl chloride was
used with DIPEA as mild base at ambient temperature; however,
the open-chained compound 21b was also not obtainable.
Instead the azoniaspiro compound 22 was isolated in 83% yield,
but no signal was found for the mesyl group in the 1H NMR analy-
sis. Thus, the solvent of the crude reaction mixture of 22 was
removed and the residue was immediately dissolved in acetoni-
trile. Silver mesylate was added and the mixture was stirred for
30 min in the dark. After changing the solvent to dichloromethane
Based on these results, the EOE protected compound 12
(tR = 8.6 min) was chosen as precursor for the introduction of
[
11C]CH3I. The labeling reaction proceeded in two steps. In a first
experiment, 12 was treated with 0.5 M NaOH followed by alkyla-
tion with [11C]CH3I leading to [11C]13 (tR = 11.1 min). In a next
experiment, subsequent deprotection of the EOE group with 1 M
HCl solution (200
11C]17 (tR = 5.8 min). Optimized conditions were found to be
approx. 1.2 mg of precursor 12 under basic conditions (40 L of
lL) for 2 min at 60 °C was executed to yield
[
l
0.5 M NaOH) for 2 min at 80 °C. Unknown by-products were found
at tR = 2.5–3.5 min and residual [11C]CH3I at tR = 9.25 min which
can be separated via semi-preparative HPLC purification. The
results are summarized in Scheme
6 and selected HPLC
Figure 4. ORTEP presentation of the molecular structure of 5b with the displace-
Figure 5. ORTEP presentation of the molecular structure of 9 with the displacement
ment ellipsoids drawn at the 50% probability level.
ellipsoids drawn at the 50% probability level.

K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6029
R3
O
R3
N
O
N
O
O
R2
R2
N
R1
N
N
N
N
N
R1
N
N
N
N
a) or b)
N
N
R1
N
N
HN
a) or b)
N
N
N
N
c)
+
N
O
N
N
N
N
O
N
N
H
N
NH2
Cl
7a-d, 8a-c
9
N
N
H
10- 16
Cl
N
O
Cl
N
7b
O
18a: R3 = F
18b
c)
: R1 = EOE, R3 = F
19a
R1
PMB
PMB
EOE
EOE
Me
R2
: R3 = OH
19b: R1 = EOE, R3 = OH
20: R1 = H , R3 = F
d)
O
7a
8a
7b
8b
7c
8c
7d
10
H
Me
H
11
12
13
14
15
16
N
N
N
NH
N
Me
H
Me
H
e)
R5
OR4
O
N
Me
N
N
N
H
H
O
N
O
17
N
N
N
N
O
N
N
N
N
N
Scheme 4. Synthesis path for precursors and reference compound 17. Reagents and
conditions: (a) p-TsOH, dioxane, 90 °C, overnight; (b) Cs2CO3, Xantphos, Pd2dba3,
dioxane, 100 °C, overnight; (c) HCl, EtOH, 1.5 h.
O
N
N
N
H
: R5 = Cl
22
O
f)
N
N
H
21a: R4 = Ts
21b: R4 = Ms
23: R5 = OMs
24: R5 = F
g)
O
Scheme 5. Synthesis path for precursor 23 and reference compound 20. Reagents
and conditions: (a) 1-fluoro-3-iodopropane, NaH, DMF, 80 °C, overnight; (b) 3-
bromopropanol, NaI, acetone, rt, overnight then Cs2CO3, DMF, 80 °C, overnight; (c)
9, p-TsOH, dioxane, 90 °C, overnight; (d) HCl, 1.5 h; (e) MsCl, DIPEA, DCM, 0 °C, 1 h;
(f) AgOMs, ACN, 1 h, rt, dark; (g) AgF, ACN, 1 h, rt, dark.
chromatograms were shown in Figure 6. In a typical experiment,
[
11C]17 was obtained after purification via radio-HPLC in a RCY
of 30–35% (d.c.) within a synthesis time of 20 min (after EOB) start-
ing from approx. 1–1.5 GBq [11C]CH3I.
2.4. Fluorine-18 radiolabeling
O
N
O
N
The introduction of radiofluorine should be realized by the
nucleophilic displacement with [18F]fluoride. For this purpose,
the protic hydrogen of the indazole moiety in the original inhibitor
was blocked with the EOE protecting group. The other secondary
amine should not influence the radiolabeling as it was shown by
us recently.26 Furthermore, we could demonstrate that precursors
which contain an azoniaspirononane moiety are highly suitable in
case of the nucleophilic introduction of [18F]Fꢀ in excellent radio-
chemical yields and in case of the latter purification.37,38
*
HN
N
N
R
N
N
N
N
a)
O
N
N
N
N
H
N
12
N
N
H
O
O
11C]13: R = EOE
=
11C
*
[
b)
[
11C]17: R = H
Scheme 6. Radiolabeling of 12 with [11C]CH3I followed by deprotection with HCl to
yield [11C]17. Reagents and conditions: (a) [11C]MeI, NaOH, DMF, 80 °C, 2 min; (b)
HCl, DMF, 80 °C, 2 min.
In the first step, EOE-precursor 23 was treated with [18F]fluoride
in acetonitrile at 100 °C for 30 min. HPLC analyses showed a signal
at tR = 3.0 min (c-trace) which did not belong to the non-radioac-
tive EOE reference compound 19a (tR = 13.5 min, UV trace). After
cleavage of the EOE group with 2 M HCl a signal of an unknown
species at tR = 7 min appeared which did not belong to 19F-refer-
ence 20. This result was also obtained after changing the solvent
(DMF, DMSO), rising the temperature (100–150 °C) and elongating
the reaction time (up to 2 h). This could indicate, that the
[
18F]fluorine is not covalently bound to the rest of the molecule.
In addition, free [18F]fluoride was still found as shown by radio-
TLC analyses. It is more likely, that compound [18F]24 appeared
instead of [18F]20 having non-covalently bound [18F]fluorine as
Figure 6. (Radio-)HPLC chromatograms of 12 (UV trace), [11C]13 (
c
trace), and
18F]fluoride. A possible explanation for this indication could be
[
11C]17 (
c
trace).
[
that the formed spiro ammonium moiety of precursor 20 between
the two aromatic rings is highly stable and the positive charge is
delocalized. In this case, a nucleophilic attack of the neighboring
carbon atom of the four-membered ring is not possible.
In order to prove and verify this, 22 was treated with AgF in ace-
tonitrile to change chloride to fluoride (see Scheme 5). The 19F
NMR of resulting derivative 24 (yield: 27%) showed a singlet at
18F
O
N
MsO
N
O
N
N
N
N
N
N
N
N
O
O
N
N
N
N
H
N
N
H
O
23
[
18F]19a
Table 2
O
Scope of the connection of different substructures A with 9
Entry
Educt
R1
R2
Path
Product
Yield (%)
O
N
[
18F]F
1
2
3
4
5
6
7
7a
8a
7b
8b
7c
8c
7d
PMB
PMB
EOE
EOE
Me
H
Me
H
Me
H
Me
H
A
A
(A) B
B
(A) B
B
10
11
(16) 12
13
14
78
44
(21) 73
62
(73) 83
80
N
N
N
O
N
N
N
H
N
F]
18
24
[
O
Me
H
15
16
A
79
Scheme 7. Possible results from [18F]radiolabeling of precursor 23.

6030
K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
ꢀ120 ppm (compared to ꢀ220.0 ppm of 20) and no coupling to
protons was observed. This was furthermore confirmed by the
analysis of the 1H and 13C NMR spectra. This finding evidences, that
the fluorine is not covalently bound in 24. In fact, the spiro ammo-
nium cation with the appropriate fluoride as anion was formed.
Additionally, only a peak at m/z = 585 which belongs to the spiro
compound was found in MS analyses of 24 (compared to
m/z = 605 for 19a or m/z = 533 for 20, both with covalently bound
fluorine). Based on these results, compound 24 was also analyzed
by HPLC. A comparison of the tR-values clearly pointed out that
24 does not belong to the above described unknown peak in the
scanner (Fuji, BAS2000). [18F]Fluoride and [11C]CH4 were obtained
utilizing the PET cyclotron Cyclone 18/9 (IBA, Belgium).
[
[
18F]Fluoride was produced by the 18O(p,n)18F nuclear reaction,
18O]H2O was irradiated.
[
11C]CH4 was produced by the
14N(p,
a)
11C nuclear reaction by irradiation of N2 gas containing
10% H2 using an aluminum target. Radiosyntheses were performed
in an automated nucleophilic synthesizer TracerLabFxC (GE) that
was modified in terms of direct trapping of [11C]CH4.
4.2. Synthesis
c
[
-trace, too. Thus, at this stage it is not possible to prepare
Compounds 4, 5a, 5c, 6c, 6d, 7d, 9, 15 and 16 were prepared
according to the literature. Additional details and procedures can
be found in the Supporting information.
18F]20 as [18F]radiotracer using this method (Scheme 7).
3. Conclusion
4.2.1. 1-(1-Ethoxyethyl)-4-nitro-1H-indazole (5b)
Ethyl vinyl ether (3.978 g, 35.17 mmol) was added to a solution
of 4-nitro-1H-indazole (4) (3 g, 18.39 mmol) and p-toluene sul-
fonic acid (350 mg, 1.839 mmol) in 50 mL anhydrous dichloro-
methane. After stirring at rt for 1 h, the reaction mixture was
quenched with water (20 mL) and saturated bicarbonate solution
(20 mL). After stirring for 30 min, the aqueous layer was extracted
with dichloromethane (3 ꢁ 25 mL), the combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (PE–EtOAc = 20:1) to yield compound 5b (3.4 g, 80%) as yellow
solid. Mp 58 °C; Rf = 0.54 (PE–EtOAc = 1:1); dH (400 MHz, CDCl3)
1.14 (3H, t, 3J = 7.1, CH3CH2), 1.81 (3H, 3J = 6.1, CH3CH), 3.28 (1H,
dq, 2J = 9.2, 3J = 7.1, CH2), 3.49 (1H, dq, 2J = 9.2, 3J = 7.1, CH2), 5.97
(1H, q, 3J = 6.1, CH), 7.51 (1H, dd, 3J = 7.8, 3J = 8.4, 6-H), 8.12 (1H,
d, 3J = 8.4, 5-H), 8.17 (1H, d, 3J = 7.8, 7-H), 8.52 (1H, s, 3-H); dC
(101 MHz, CDCl3) 14.9, 21.2 (2ꢁ CH3), 64.2 (CH2), 88.1 (CH),
117.9 (C-7), 118.2 (C-3a), 118.6 (C-5), 125.6 (C-6), 132.8 (C-3),
139.9 (C-7a), 140.8 (C-4); MS (ESIꢀ): m/z 235 (100, M+); elemental
analysis calcd for C11H13N3O3 (235.24): C, 56.16, H, 5.57, N, 17.86;
found: C, 56.44, H, 5.72, N, 16.87.
A successful synthesis of precursors as well as reference com-
pounds for 11C-/18F-radiolabeling based on a highly affine EphB4
receptor inhibitor is demonstrated. As a prerequisite, docking stud-
ies were accomplished pointing out the preferred position in the
original molecule for the introduction of a 3-[18F]fluoropropyl moi-
ety which was not mandatory for the isotopic labeling with car-
bon-11. For radiolabeling purposes,
a convenient protecting
group strategy was established based on the application of the
ethoxyethyl (EOE) group. Radiolabeling with carbon-11 was suc-
cessfully accomplished leading to the isotopically labeled radio-
tracer whereas the radiofluorination with [18F]fluoride did not
lead to the desired radiotracer under the chosen conditions.
Thus, our ongoing research is focused on an alternative preparation
of the [18F]radiotracer based on this lead structure and on the
(radio)biological evaluation of the [11C]radiotracer.
4. Experimental
4.1. General
All reagents were purchased from commercial suppliers and
were used without further purification. Analytical TLC was per-
formed on pre-coated Silica Gel 60 F254 plates (Merck) and results
read under UV-light (k = 254 nm). 1H NMR, 13C NMR as well as 19F
NMR spectra were recorded on a Varian Inova-400 or on an Agilent
DD2-400 (OneNMR Probe) spectrometer at 400, 101, and 376 MHz,
respectively. Chemical shifts are reported in ppm with tetra-
methylsilane (1H, 13C) and trichlorofluoromethane (19F) as internal
standard, respectively. Mass spectrometric (MS) data were
obtained on a Quattro/LC mass spectrometer (MICROMASS) or on
a Xevo TQ-S (Waters) by electron spray ionization (ESI). Melting
points were recorded on a Cambridge Instruments Galen III appa-
ratus and are uncorrected. Diffraction data of 5b and 9 were col-
lected with a Bruker-Nonius Apex-X8 CCD-diffractometer using
4.2.2. 4-Amino-1-(4-methoxybenzyl)-1H-indazole (6a)
Compound 5a (875 mg, 3.08 mmol) was dissolved in metha-
nol/dichloromethane (30 mL, v:v = 1:1), treated with Pd/C
(500 mg) and stirred at rt and 1 bar H2 for 24 h. Subsequently,
the catalyst was filtered and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (PE–EtOAc = 1:1 ? 1:2) to yield compound 6a (697 mg, 89%)
as
a brown solid. Mp 99 °C; Rf = 0.31 (PE–EtOAc = 1:1). dH
(400 MHz, CDCl3) 3.75 (3H, s, CH3), 5.47 (2H, s, CH2), 6.31 (1H, d,
3
3J5,6 = 7.5, 5-H), 6.77 (1H, d, J7,6 = 8.3, 7-H), 6.82 (2H, dd, 3J = 8.6,
3
3
H-o), 7.12 (1H, dd, J5,6 = 7.5, J7,6 = 8.3, 6-H), 7.16 (2H, dd,
3J = 8.6, H-m), 7.97 (1H, s, 3-H); dC (101 MHz, CDCl3): d = 52.7
(CH3), 55.4 (CH2), 99.6 (C-7), 103.8 (C-5), 114.3 (C-m), 115.2 (C-
3a), 128.1 (C-6), 128.8 (C-o), 129.3 (C-i), 130.5 (C-3), 140.5 (C-
7a), 159.3 (C-p); MS (ESI+): m/z 254 (100, M++H); elemental analy-
sis calcd for C15H15N3O (253.30): C, 71.13, H, 5.97, N, 16.59; found:
C, 71.43, H, 6.06, N, 16.52.
graphite-monochromated Mo–K radiation (k = 0.71073 Å). The
a
diffraction measurement was done at ꢀ100 °C and the structures
were solved by Direct Methods using Shelxs-97 and refined against
F2 on all data by full-matrix least-squares with Shelxl-97.39 All
non-hydrogen atoms were refined anisotropically; all hydrogen
atoms bonded to C atoms were placed on calculated positions
and refined using a riding model. 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,
30:70 + 0.1% TFA). 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
4.2.3. 1-(1-Ethoxyethyl)-1H-indazol-4-amine (6b)
Compound 5b (3.4 g, 14.45 mmol) was dissolved in metha-
nol/dichloromethane (30 mL, v:v = 1:1) and Pd/C (500 mg) was
added. This mixture was treated with H2 (approx. 1 bar) at rt for
24 h. Subsequently, the catalyst was filtered and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (PE–EtOAc = 2:1) to yield compound 6b (1.22 g,
41%) as a brown oil. Rf = 0.58 (PE–EtOAc = 2:1); dH (400 MHz,
CDCl3) 1.12 (3H, t, 3J = 7.0, CH3CH2), 1.78 (3H, d, 3J = 6.2, CH3CH),
3.25 (1H, dq, 2J = 9.3, 3J = 7.0, CH2), 3.43 (1H, dq, 2J = 9.3, 3J = 7.0,
CH2), 5.84 (1H, q, 3J = 6.2, CH), 6.36 (1H, d, 3J = 7.4 Hz, 5-H), 7.06

K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6031
(1H, d, 3J = 8.4, 7-H), 7.17 (1H, dd, 3J = 8.4, 3J = 7.4, 6-H), 7.94 (1H, s,
3-H); dC (101 MHz, CDCl3) 14.9, 21.2 (2ꢁ CH3), 64.2 (CH2), 88.1
(CH), 117.9 (C-7), 118.2 (C-3a), 118.6 (C-5), 125.6 (C-6), 132.8 (C-
3), 139.9 (C-7a), 140.8 (C-4); MS (ESI+): m/z 228 (20, M++Na), 206
(100, M++H); elemental analysis calcd for C11H15N3O (205.26): C,
64.37, H, 7.37, N, 20.47; found: C, 63.92, H, 7.20, N, 19.35.
EtOH = 2:6:1); dH (400 MHz, CDCl3) 4.11 (3H, s, CH3), 6.62 (1H, d,
3J = 5.9, H-50), 7.19 (1H, d, 3J = 7.5, 5-H), 7.31 (1H, d, 3J = 8.6, 7-H),
7.41 (1H, dd, 3J = 8.6, 3J = 7.5, 6-H), 7.91 (1H, d, 4J = 0.7, 3-H), 8.14
3
(1H, d, J = 5.9, 60-H); dC (101 MHz, CDCl3) 35.7 (CH3), 102.7 (C-7),
107.0 (C-50), 114.1 (C-5), 118.9 (C-3a), 126.8 (C-6), 129.6 (C-3),
129.9 (C-4) 141.1 (C-7a), 158.1 (C-20), 160.7 (C-60), 162.4 (C-40);
MS (ESI+): m/z 262 (20, M++H, 37Cl), 260 (58, M++H, 35Cl); elemental
analysis calcd for C12H10ClN5 (259.69): C, 55.50, H, 3.88, N, 26.97;
found: C, 54.97, H, 4.00, N, 26.63.
4.2.4. N-(2-Chloropyrimidin-4-yl)-1-(4-methoxybenzyl)-1H-ind-
azol-4-amine (7a)
2,4-Dichloropyrimidine (156 mg, 1.045 mmol) and DIPEA
(142 mg, 1.095 mmol) were added to 6a (252 mg, 0.995 mmol) dis-
solved in absolute ethanol (5 mL). This mixture was allowed to stir
under reflux for 2 d. After cooling to rt, water (5 mL) was added,
the aqueous layer was extracted with EtOAc (3 ꢁ 10 mL). The com-
bined organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (PE–EtOAc = 2:1 ? 1:1) to yield compound 7a
(207 mg, 57%) as light red solid. Mp 67 °C; Rf = 0.45 (PE–
EtOAc = 1:2); dH (400 MHz, CDCl3) 3.77 (3H, s, CH3), 5.55 (2H, s,
4.2.7. N-(2-Chloropyrimidin-4-yl)-1-(4-methoxybenzyl)-N-met-
hyl-1H-indazol-4-amine (8a)
Cs2CO3 (179 mg, 0.548 mmol) and MeI (29 mg, 0.205 mmol)
were added to 7a (50 mg, 0.137 mmol) dissolved in anhydrous
DMF (3 mL) at 0 °C. Then, the solution was stirred at rt for 4 h.
Afterwards, water (15 mL) was added and the aqueous layer was
extracted with EtOAc (3 ꢁ 15 mL). The combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (PE–EtOAc = 1:1) to yield compound 8a (50 mg, 96%) as pale
yellow syrup. Rf = 0.55 (PE–EtOAc–EtOH = 2:6:1); dH (400 MHz,
CDCl3) 3.59 (1H, s, NCH3), 3.77 (1H, s, OCH3), 5.55 (2H, s, CH2),
3
CH2), 6.65 (1H, d, J = 5.9, 500-H), 6.84 (2H, dd, 3J = 8.7, H-m), 7.16
(1H, d, 3J = 7.4, 5-H), 7.19 (2H, dd, 3J = 8.7, H-o), 7.29 (1H, d,
3J = 8.4, 7-H) 7.35 (1H, dd, 3J = 7.4, 3J = 8.4, 6-H), 7.96 (1H, s, 3-H),
3
3
8.15 (1H, d, J = 5.9, 600-H); dC (101 MHz, CDCl3) 53.2 (CH3), 55.5
6.06 (1H, d, J = 6.0, H-500), 6.85 (2H, d, 3J = 8.5, H-o), 7.01 (1H, t,
(CH2), 103.0 (C-500), 107.8 (C-7), 114.4 (C-m), 120.0 (C-3a), 127.2
(C-6), 128.5 (C-100), 129.0 (C-o), 129.9 (C-4), 130.7 (C-3), 140.9 (C-
7a), 158.3 (C-600), 158.4 (C-p), 159.5 (C-200), 162.6 (C-400); MS
(ESIꢀ): m/z 366 (40, M+ꢀH, 37Cl), 364 (100, M+ꢀH, 35Cl); elemental
analysis calcd for C19H16N5OCl (365.82): C, 62.38, H, 4.41, N, 19.14;
found: C, 62.62, H, 4.52, N, 18.73.
3J = 4.8, 3J = 3.4, 6-H), 7.23 (2H, d, 3J = 8.5, H-m), 7.39–7.40 (2H,
3
m, 5/7-H), 7.80 (1H, s, 3-H), 7.84 (1H, d, J = 6.0, 600-H); dC
(101 MHz, CDCl3) 38.2, 53.2 (2ꢁ CH3), 55.4 (CH2), 103.9 (C-7),
109.6 (C-500), 114.3 (C-o), 118.8 (C-5), 121.1 (C-3a), 127.4 (C-6),
128.4 (C-10), 129.0 (C-m), 131.0 (C-3), 136.2 (C-4), 141.1 (C-7a),
156.4 (C-600), 159.5 (C-40), 160.8 (C-200), 163.5 (C-400); MS (ESI+):
m/z 382 (28, M++H, 37Cl), 380 (70, M++H, 35Cl); elemental analysis
calcd for C19H16N5OCl (365.82): C, 62.38, H, 4.41, N, 19.14; found:
C, 62.62, H, 4.52, N, 18.73.
4.2.5. N-(2-Chloropyrimidin-4-yl)-1-(1-ethoxyethyl)-1H-indazol-
4-amine (7b)
2,4-Dichloropyrimidine (1.51 g, 10.14 mmol) and DIPEA (1.06 g,
8.19 mmol) were added to 6b (1.6 g, 7.80 mmol) dissolved in abso-
lute ethanol (25 mL). This mixture was allowed to stir under reflux
for 2 d. After cooling to rt, water (20 mL) and 10% NaHSO4 solution
(2 mL) was added, the aqueous layer was extracted with EtOAc
(3 ꢁ 20 mL). The combined organic layers were dried over MgSO4
and the solvent was removed under reduced pressure.
Purification was done by column chromatography (PE–
EtOAc = 2:1 ? 1:1) to yield compound 7b (1.3 g, 52%) as colorless
solid. Mp 160 °C; Rf = 0.39 (PE–EtOAc = 1:2); dH (400 MHz, CDCl3)
1.15 (3H, t, 3J = 7.1, CH3CH2), 1.81 (3H, d, 3J = 6.1, CH3CH), 3.28
(1H, dq, 2J = 9.2, 3J = 7.1, CH2), 3.48 (1H, dq, 2J = 9.2, 3J = 7.1, CH2),
4.2.8. N-(2-Chloropyrimidin-4-yl)-1-(1-ethoxyethyl)-N-methyl-
1H-indazol-4-amine (8b)
Cs2CO3 (615 mg, 1.887 mmol) and MeI (134 mg, 0.944 mmol)
were added to 7b (200 mg, 0.629 mmol) dissolved in anhydrous
DMF (5 mL) at 0 °C. Then, the solution was stirred at rt for 4 h.
Afterwards, water (15 mL) was added and the aqueous layer was
extracted with EtOAc (3 ꢁ 15 mL). The combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (PE–EtOAc = 1:1) to yield compound 8b (183 mg, 88%) as col-
orless solid. Mp 105 °C. Rf = 0.26 (PE–EtOAc = 2:1). dH (400 MHz,
CDCl3) 1.14 (3H, t, 3J = 7.1, CH3CH2), 1.78 (3H, d, 3J = 6.1, CH3CH),
3.29 (1H, dq, 2J = 9.3, 3J = 7.1, CH2), 3.47 (1H, dq, 2J = 9.3, 3J = 7.1,
CH2), 3.58 (3H, s, CH3), 5.90 (1H, q, 3J = 6.1, CH), 6.07 (1H, d,
3J = 6.0, 50-H), 7.04 (1H, d, 3J = 7.4, 5-H), 7.42 (1H, dd, 3J = 7.4,
3J = 8.5, 6-H), 7.72 (1H, d, 3J = 8.5, 7-H), 7.76 (1H, s, 3-H), 7.84
3
5.91 (1H, q, 3J = 6.1, CH), 6.65 (1H, d, J = 5.9, 50-H), 7.20 (1H, d,
3J = 7.4, 5-H), 7.27 (1H, s, NH), 7.40 (1H, dd, 3J = 7.4, 3J = 8.5, 6-H),
7.62 (1H, d, 3J = 8.5, 7-H), 7.93 (1H, s, 3-H), 8.17 (1H, d, 3J = 5.9,
60-H); dC (101 MHz, CDCl3) 15.0, 21.1 (2ꢁ CH3), 64.1 (CH2), 87.5
(CH), 102.9 (C-7), 109.1 (C-50), 114.9 (C-5), 120.4 (C-3a), 127.3
(C-6), 130.0 (C-3), 130.7 (C-4), 140.0 (C-7a), 158.7 (C-20), 161.1
(C-60), 162.6 (C-40). MS (ESI+): m/z 342 (10, M++Na, 37Cl), 340 (20,
M++Na, 35Cl), 320 (32, M++H, 37Cl), 318 (100, M++H, 35Cl); elemen-
tal analysis calcd for C15H16N5OCl (317.77): C, 56.69, H, 5.08, N,
22.04; found: C, 57.12, H, 5.25, N, 21.53.
3
(1H, d, J = 6.0, 60-H); dC (101 MHz, CDCl3) 14.9, 21.1, 38.2 (3ꢁ
CH3), 64.1 (CH2), 87.5 (CH), 103.8 (C-7), 110.8 (C-50), 119.2 (C-5),
121.7 (C-3a), 127.4 (C-6), 131.0 (C-3), 136.2 (C-4), 140.1 (C-7a),
156.4 (C-60), 160.8 (C-20), 163.5 (C-40); MS (ESI+): m/z 334 (35,
M++H, 37Cl), 332 (100, M++H, 35Cl),; elemental analysis calcd for
C16H18N5OCl (331.80): C, 57.92, H, 5.47, N, 21.11; found: C,
4.2.6. 4-Amino-N-(2-chloropyrimidin-4-yl)-1-methyl-1H-indaz-
ole (7c)
58.28, H, 5.54, N, 20.78.
2,4-Dichloropyrimidine (1.34 g, 9.02 mmol) and DIPEA (1.22 g,
9.45 mmol) were added to 6c (1.26 g, 8.59 mmol) dissolved in
absolute ethanol (25 mL). This mixture was allowed to stir under
reflux for 2 d. After cooling to rt, water (20 mL) was added, the
aqueous layer was extracted with EtOAc (3 ꢁ 25 mL). The com-
bined organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (PE–EtOAc = 2:1 ? 1:1) to yield compound 7c
(1.054 g, 47%) as light red solid. Mp 169 °C; Rf = 0.44 (PE–EtOAc–
4.2.9. N-(2-Chloropyrimidin-4-yl)-N,1-dimethyl-1H-indazol-4-
amine (8c)
Cs2CO3 (655 mg, 2.01 mmol) and MeI (142 mg, 1.00 mmol)
were added to 7c (174 mg, 0.67 mmol) dissolved in anhydrous
DMF (5 mL) at 0 °C. Then, the solution was stirred at rt for 4 h.
Afterwards, water (15 mL) was added and the aqueous layer was
extracted with EtOAc (3 ꢁ 15 mL). The combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column

6032
K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
chromatography (PE–EtOAc = 1:1) to yield compound 8c (166 mg,
91%) as pale yellow solid. Mp 142 °C; Rf = 0.55 (PE–EtOAc–
EtOH = 2:6:1); dH (400 MHz, CDCl3) 3.58 (3H, s, NCH3), 4.11 (3H,
elemental analysis calcd for C34H38N8O3 (606.72): C, 67.31, H,
6.31, N, 18.47; found: C, 67.24, H, 6.36, N, 18.23.
s, CH3), 6.03 (1H, d, J = 6.1, 50-H), 7.02 (1H, dd, 3J = 6.5, 3J = 7.9,
4.2.12. N2-(3,5-Dimorpholinophenyl)-N4-(1-(1-ethoxyethyl)-1H-
indazol-4-yl)pyrimidin-2,4-diamine (12)
3
6-H), 7.41 (1H, d, 3J = 6.5, 5-H), 7.44 (1H, d, 3J = 7.9, 7-H), 7.74
3
(1H, s, 3-H), 7.82 (1H, d, J = 6.1, 60-H); dC (101 MHz, CDCl3) 36.0,
Compound 7b (220 mg, 0.69 mmol) and compound 9 (182 mg,
0.69 mmol) were dissolved in anhydrous dioxane (5 mL), Cs2CO3
(615 mg, 1.89 mmol) was added. Xantphos and Pd2(dba)3 were
added in catalytic amounts and the mixture was stirred at 100 °C
for 12 h. The reaction was quenched with water (15 mL) and half-
saturated bicarbonate solution (4 mL) was added. The aqueous
layer was extracted with EtOAc (3 ꢁ 15 mL). The combined organic
layers were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (CHCl3–MeOH = 98:2) to yield compound 12 (276 mg, 73%) as
pale brown solid. Mp 110 °C; Rf = 0.49 (CHCl3–MeOH = 9:1); dH
(400 MHz, CDCl3) 1.14 (3H, t, 3J = 7.1, CH3CH2), 1.80 (3H, d,
3J = 6.1, CH3CH), 3.13 (8H, t, 3J = 4.8, CH2N), 3.27 (1H, dq, 2J = 9.3,
3J = 7.1, CH2), 3.46 (1H, dq, 2J = 9.3, 3J = 7.1, CH2), 3.82 (8H, t,
38.1 (2ꢁ CH3), 103.8 (C-7), 109.1 (C-50), 118.7 (C-5), 120.7 (C-3a),
127.3 (C-6), 130.5 (C-3), 136.1 (C-4), 141.7 (C-7a), 156.4 (C-60),
160.8 (C-20), 163.5 (C-40); MS (ESI+) m/z 276 (35, M++H, 37Cl), 274
(100, M++H, 35Cl); elemental analysis calcd for C13H12N5Cl
(273.72): C, 57.04, H, 4.42, N, 25.59; found: C, 57.51, H, 4.51, N,
25.30.
4.2.10. N2-(3,5-Dimorpholinophenyl)-N4-(1-(4-methoxybenzyl)-
1H-indazol-4-yl)pyrimidin-2,4-diamine (10)
Compound 7a (189 mg, 0.516 mmol) and compound 9 (150 mg,
0.568 mmol) were dissolved in anhydrous dioxane (5 mL) and p-
TsOH in catalytic amounts was added. Afterwards, the reaction
mixture was stirred at 90 °C for 1 d. The reaction was quenched
with water (15 mL) and half-saturated bicarbonate solution
(4 mL). The aqueous layer was extracted with EtOAc (3 ꢁ 15 mL).
The combined organic layers were dried over MgSO4 and the sol-
vent was removed under reduced pressure. Purification was done
by column chromatography (CHCl3–MeOH = 98:2) to yield com-
pound 10 (239 mg, 78%) as light brown solid. Mp 122 °C;
Rf = 0.69 (CHCl3–MeOH = 95:5); dH (400 MHz, CDCl3) 3.07 (8H, t,
3J = 4.7, CH2N), 3.74 (3H, s, CH3), 3.77 (8H, t, 3J = 4.7, CH2O), 5.49
4
3J = 4.8, CH2O), 5.89 (1H, q, 3J = 6.1, CH), 6.16 (1H, t, J = 2.0, 400-4),
3
4
6.25 (1H, d, J = 5.9, 500-H) 6.78 (1H, d, J = 2.0, 200/600-H), 7.06 (1H,
s, NH), 7.17 (1H, s, NH), 7.33–7.38 (2H, m, 5/6-H), 7.48–7.50 (1H,
m, H-7), 7.97 (1H, s, 3-H), 8.08 (1H, d, 3J = 5.9, 500-H); dC (101 MHz,
CDCl3) 15.1, 21.1 (2ꢁ CH3), 49.9 (CH2N), 64.0 (CH2), 67.2 (CH2O),
87.2 (CH), 97.4 (C-50), 98.8 (C-7), 100.4 (C-200/500), 107.1 (C-5),
113.5 (C-400), 119.7 (C-3a), 127.4 (C-6), 131.0 (C-3), 131.7 (C-4),
139.9 (C-7a), 141.3 (C-100), 153.0 (C-300/500), 157.6 (C-60), 160.3 (C-
20), 161.3 (C-40); MS (ESI+): m/z 545 (100, M++H); elemental analysis
calcd for C29H36N8O3 (544.65): C, 63.95, H, 6.66, N, 20.57; found: C,
64.01, H, 6.65, N, 20.55.
4
3
(2H, s, CH2), 6.13 (1H, t, J = 1.9, 4000-H), 6.21 (1H, d, J = 5.8, 5-00H),
6.75 (2H, d, J = 1.9, 2000/6000-H), 6.81 (2H, d, 3J = 8.6, H-o), 7.13 (1H,
4
d, 3J = 8.3, 5-H), 7.15 (2H, d, 3J = 8.6, H-m), 7.27 (1H, dd,
3J = 7.6 Hz, 3J = 8.3, 6-H), 7.33 (1H, d, 3J = 7.6, 7-H), 7.53 (1H, s,
4.2.13. N2-(3,5-Dimorpholinophenyl)-N4-(1-(1-ethoxyethyl)-1H-
indazol-4-yl)-N4-methylpyrimidin-2,4-diamine (13)
3
NH), 7.55 (1H, s, NH), 7.96 (1H, s, 3-H), 8.04 (1H, d, J = 5.8, 600-
H); dC (101 MHz, CDCl3) 50.3 (CH2N), 53.4 (CH3), 55.9 (CH2Ph),
67.6 (CH2O), 97.7 (C-500), 99.3 (C-7), 100.8 (C-2000/6000), 106.4 (C-5),
113.4 (C-4000), 114.8 (C-m), 119.4 (C-3a), 127.8 (C-6), 129.2 (C-i),
129.3 (C-o), 131.2 (C-3), 131.9 (C-4), 141.2 (C-7a), 141.6 (C-1000),
153.4 (C-3000/5000), 157.5 (C-600), 159.8 (C-p), 160.3 (C-200), 161.6 (C-
400); MS (ESI+): m/z 593 (100, M++H); elemental analysis calcd for
Compound 8b (133 mg, 0.40 mmol) and compound 9 (106 mg,
0.40 mmol) were dissolved in anhydrous dioxane (5 mL), Cs2CO3
(392 mg, 1.20 mmol) was added. Xantphos and Pd2(dba)3 were
added in catalytic amounts and the mixture was stirred at 90 °C
for 24 h. The reaction was quenched with water (15 mL) and
half-saturated bicarbonate solution (4 mL) was added. The aque-
ous layer was extracted with EtOAc (3 ꢁ 15 mL). The combined
organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (CHCl3–MeOH = 98:2 ? 95:5) to yield compound
13 (140 mg, 62%) as brown solid. Mp 82 °C; Rf = 0.57 (CHCl3–
MeOH = 9:1); dH (400 MHz, CDCl3) 1.17 (3H, t, 3J = 7.1, CH3CH2),
1.81 (3H, d, 3J = 6.1, CH3CH), 3.16 (8H, t, 3J = 4.7, CH2N), 3.31 (1H,
dq, 2J = 9.3, 3J = 7.1, CH2), 3.49 (1H, dq, 2J = 9.3, 3J = 7.1, CH2), 3.62
C33H36N8O3 (592.69): C, 66.87, H, 6.12, N, 18.91; found: C, 66.51,
H, 6.14, N, 18.99.
4.2.11. N2-(3,5-Dimorpholinophenyl)-N4-(1-(4-methoxybenzyl)-
1H-indazol-4-yl)-N4-methylpyrimidin-2,4-diamine (11)
Compound 8a (137 mg, 0.36 mmol) and compound 9 (104 mg,
0.40 mmol) were dissolved in anhydrous dioxane (5 mL) and p-
TsOH in catalytic amounts was added. Afterwards, the reaction
mixture was stirred at 100 °C for 1 d. The reaction was quenched
with water (15 mL) and half-saturated bicarbonate solution
(4 mL). The aqueous layer was extracted with EtOAc (3 ꢁ 15 mL).
The combined organic layers were dried over MgSO4 and the sol-
vent was removed under reduced pressure. Purification was done
by column chromatography (CHCl3–MeOH = 98:2) to yield com-
pound 11 (239 mg, 78%) as light red solid. Mp 111 °C; Rf = 0.25
(EtOAc); dH (400 MHz, CDCl3) 3.16 (8H, t, 3J = 4.7, CH2N), 3.61
(3H, s, CH3), 3.77 (3H, s, CH3), 3.84 (8H, t, 3J = 4.7, CH2O), 5.55
3
(3H, s, CH3), 3.84 (8H, t, 3J = 4.7, CH2O), 5.71 (1H, d, J = 6.0, 50-H),
4
5.91 (1H, q, 3J = 6.1, CH), 6.17 (1H, s, J = 1.8, 400-H), 6.90 (2H, d,
4J = 1.8, 200/600-H), 7.07 (1H, d, 3J = 7.4, 5-H), 7.29 (1H, s, NH), 7.41
(1H, dd, 3J = 7.4, 3J = 8.4, 6-H), 7.68 (1H, d, 3J = 8.4, 7-H), 7.81 (1H,
3
s, 3-H), 7.82 (1H, d, J = 6.0, 60-H); dC (101 MHz, CDCl3) 15.0, 21.1,
38.2 (3ꢁ CH3), 50.0 (CH2N), 64.0 (CH2), 67.1 (CH2O), 87.4 (CH),
97.5 (C-50), 98.6 (C-7), 99.8 (C-200/500), 109.9 (C-5), 119.3 (C-3a),
122.3 (C-400), 127.3 (C-6), 131.6 (C-3), 137.6 (C-4), 140.1 (C-7a),
141.9 (C-100), 152.9 (C-300/500), 155.8 (C-60), 159.9 (C-20), 162.9 (C-
40); MS (ESI+): m/z 559 (100, M++H); elemental analysis calcd for
3
(2H, s, CH2), 5.70 (1H, d, J = 6.0, 500-H), 6.17 (1H, s, 4000-H), 6.85
4
(2H, dd, 3J = 8.7, H-m), 6.90 (2H, d, J = 2.0, 2000/6000-H), 7.03 (1H,
C30H38N8O3 (558.67): C, 64.50, H, 6.86, N, 20.06; found: C, 64.45,
dd, 3J = 6.7, 3J = 7.8, 6-H), 7.17 (1H, s, NH), 7.22 (2H, dd, 3J = 8.7,
H, 6.92, N, 20.01.
3
H-o), 7.33–7.36 (2H, m, 5/6-H), 7.80 (1H, d, J = 6.0, 600-H), 7.84
4.2.14. N2-(3,5-Dimorpholinophenyl)-N4-(methyl-1H-indazol-4-
yl)-pyrimidin-2,4-diamine (14)
Compound 7c (71 mg, 0.27 mmol) and compound 9 (80 mg,
0.27 mmol) were dissolved in anhydrous toluene (5 mL) and
Cs2CO3 (352 mg, 1.08 mmol) was added. Xantphos and Pd2(dba)3
were added in catalytic amounts and the mixture was stirred at
(1H, s, 3-H); dC (101 MHz, CDCl3) 38.2 (CH3), 50.0 (CH2N), 53.1
(CH3), 55.4 (CH2Ph), 67.2 (CH2O), 97.6 (C-500), 98.6 (C-7), 99.8 (C-
2000/6000), 108.7 (C-5), 114.3 (C-30/50), 119.0 (C-3a), 121.7 (C-4000),
127.3 (C-6),128.7 (C-10), 129.0 (C-20/60), 131.6 (C-3), 137.6 (C-4),
141.1 (C-7a), 141.9 (C-1000), 153.0 (C-3000/5000), 156.0 (C-600), 159.5
(C-40), 159.9 (C-200), 162.9 (C-400); MS (ESI+): m/z 607 (90, M++H);

K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6033
90 °C for 24 h. The reaction was quenched with water (15 mL) and
the aqueous layer was extracted with EtOAc (3 ꢁ 15 mL). The com-
bined organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (EtOAc ? EtOAc–EtOH = 10:1) to yield compound
14 (109 mg, 83%) as light brown solid. Mp 113 °C; Rf = 0.35 (EtOAc–
EtOH = 3:1); dH (400 MHz, CDCl3) 3.11 (8H, t, 3J = 4.8, CH2N), 3.81
5), 127.5 (C-6), 130.9 (C-3), 134.8 (C-4), 140.2 (C-7a), 156.7 (C-
20), 160.8 (C-60), 163.4 (C-40); dF (376 MHz, CDCl3) –220.7; MS
(ESI+): m/z 380 (34, M++H, 37Cl), 378 (100, M++H, 35Cl); elemental
analysis calcd for C18H21ClFN5O (377.84): C, 57.22, H, 5.60, N,
18.54; found: C, 57.25, H, 5.74, N 18.28.
4.2.17. N-(2-Chloropyrimidin-4-yl)-1-(1-ethoxyethyl)-N-(3-hyd-
roxypropyl)-1H-indazol-4-amine (18b)
4
(8H, t, 3J = 4.8, CH2O), 4.08 (1H, s, CH3), 6.16 (1H, t, J = 2.0, 400-
3
4
H), 6.23 (1H, d, J = 5.8, 50-H), 6.77 (2H, d, J = 2.0, 200/600-H), 7.05
3-Bromopropanol (262 mg, 1.89 mmol) and NaI (300 mg,
2.00 mmol) were dissolved in acetone (5 mL) and the mixture
was stirred at rt overnight. Afterwards, the solvent was changed
against anhydrous DMF (5 mL), 7b (364 mg, 1.15 mmol) and
Cs2CO3 (1.2 g, 3.68 mmol) were added and the resulting mixture
was stirred at 80 °C overnight. Afterwards, water was added, the
mixture was extracted with EtOAc (3 ꢁ 20 mL), the combined
organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. Purification was done by column
chromatography (PE–EtOAc = 1:1 ? 1:2) to yield compound 18b
(350 mg, 51%) as yellowish oil. Rf = 0.63 (CHCl3–EtOH = 95:5); dH
(400 MHz, CDCl3) 1.17 (3H, t, 3J = 7.0, CH3), 1.70–1.83 (5H, m,
CH3/CH2CH2O), 3.31 (1H, dq, 2J = 9.2, 3J = 7.2, CH2), 3.49 (1H, dq,
2J = 9.2, 3J = 7.2, CH2), 3.72 (2H, br s, CH2N), 3.89 (1H, br s, OH),
4.06–4.37 (2H, m, CH2O), 5.88–5.95 (2H, m, CH, HPyr), 7.02 (1H,
d, 3J = 7.4, 5-H), 7.45 (1H, dt, 3J = 7.4, 3J = 8.5, 6-H), 7.74 (1H, s, 3-
H), 7.78 (1H, d, 3J = 8.5, 7-H), 7.85 (1H, d, 3J = 6.0, HPyr); dC
(101 MHz, CDCl3) 15.0, 21.1, (2ꢁ CH3), 31.0 (CH2CH2O), 46.6
(CH2N), 58.4 (CH2O), 64.2 (CH2), 87.8 (CH), 103.9 (C-7), 111.4 (C-
50), 120.5 (C-5), 122.3 (C-3a), 127.6 (C-6), 130.8 (C-3), 134.2 (C-
4), 140.2 (C-7a), 156.9 (C-60), 160.6 (C-20), 163.9 (C-40); MS (ESI+):
m/z 398 (100, M++Na, 35Cl), 285 (42, 285 M+ꢀEOE–OH); elemental
analysis calcd for C18H22ClN5O2 (375.85): C, 57.52, H, 5.90, N,
18.63; found: C, 57.55, H, 5.81, N, 18.74.
(1H, s, NH), 7.11 (1H, s, NH) 7.19 (1H, ‘t’, 3J = 4.8, 6-H), 7.34–7.38
3
(2H, m, 5/7-H), 7.94 (1H, s, 3-H), 8.07 (1H, d, J = 5.8, 60-H); dC
(101 MHz, CDCl3) 35.9 (CH3), 49.8 (CH2N), 67.1 (CH2O), 97.5 (C-
200/600), 98.8 (C-50), 100.4 (C-7), 112.9 (C-5), 118.7 (C-3a), 127.3
(C-6), 130.4 (C-3), 131.6 (C-4), 141.3 (C-7a), 152.9 (C-500/300),
157.5 (C-100), 160.2 (C-60), 161.3 (C-20), 171.1 (C-40); MS (ESI+):
m/z 487 (100, M++H); elemental analysis calcd for C26H30N8O2
(486.57): C, 64.18, H, 6.21, N, 23.03; found: C, 64.28, H, 6.13, N,
23.16.
4.2.15. N2-(3,5-Dimorpholinophenyl)-N4-methyl-N4-(1-methyl-
1H-indazol-4-yl)pyrimidin-2,4-diamine (15)
Compound 8c (92 mg, 0.34 mmol) and compound 9 (89 mg,
0.34 mmol) were dissolved in anhydrous dioxane (5 mL) and
Cs2CO3 (328 mg, 1.01 mmol) was added. Xantphos and Pd2(dba)3
were added in catalytic amounts and the mixture was stirred at
90 °C for 24 h. The reaction was quenched with water (15 mL)
and the aqueous layer was extracted with EtOAc (3 ꢁ 15 mL). The
combined organic layers were dried over MgSO4 and the solvent
was removed under reduced pressure. Purification was done by
column chromatography (CHCl3–MeOH = 98:2) to yield compound
15 (135 mg, 80%) as brown solid. Mp 78 °C; Rf = 0.69 (acetone–
EtOAc = 2:1); dH (400 MHz, CDCl3) 3.16 (8H, t, 3J = 4.8, CH2N),
3.61 (3H, s, CH3), 3.83 (8H, t, 3J = 4.8, CH2O), 4.11 (3H, s, CH3),
3
4
5.69 (1H, d, J = 6.0, 50-H), 6.13 (1H, t, J = 2.0, 400-H), 6.90 (2H, d,
4.2.18. N2-(3,5-Dimorpholinophenyl)-N4-(1-(1-ethoxyethyl)-1H-
indazol-4-yl)-N4-(3-fluoropropyl)pyrimidin-2,4-diamine (19a)
and N2-(3,5-dimorpholinophenyl)-N4-(3-fluoropropyl)-N4-(1H-
indazol-4-yl)pyrimidin-2,4-diamine (20)
4J = 2.0, 200/600-H), 7.05 (1H, d, 3J = 7.1, 5-H), 7.36–7.45 (2H, m,
3
6/7-H), 7.80 (1H, s, 3-H), 7.80 (1H, d, J = 6.0, 60-H); dC (101 MHz,
CDCl3) 6.0, 38.2 (2ꢁ CH3), 50.0 (CH2N), 67.1 (CH2O), 97.4 (C-50),
98.6 (C-7), 99.8 (C-200/600), 108.3 (C-5), 118.8 (C-400), 121.2 (C-3a),
127.2 (C-6), 131.1 (C-3), 137.5 (C-4), 141.6 (C-7a), 141.9 (C-100),
152.9 (C-300/500), 155.7 (C-60), 159.9 (C-20), 162.9 (C-40); MS (ESI+):
m/z 501 (100, M++H); elemental analysis calcd for C27H32N8O2
(500.60): C, 64.78, H, 6.44, N, 22.38; found: C, 64.91, H, 6.38, N,
22.50.
Compounds 18a (327 mg, 0.87 mmol) and
9
(207 mg,
0.79 mmol) were dissolved in anhydrous dioxane (10 mL), p-
TsOH was added in catalytic amounts and the mixture was stirred
at 90 °C for 48 h. After cooling to rt, saturated bicarbonate solution
(15 mL) was added and the aqueous layer was extracted with
EtOAc (3 ꢁ 15 mL). The combined organic layers were dried over
MgSO4 and the solvent was removed under reduced pressure to
yield the crude compound 19a. Rf = 0.48 (CHCl3–EtOH = 95:5); dH
(400 MHz, CDCl3) 1.17 (3H, t, 3J = 7.3, CH3), 1.81 (3H, d, 3J = 6.0,
CH3), 2.00–2.15 (2H, m, CH2), 3.18 (8H, t, 3J = 4.5, CH2N), 3.32
(1H, dq, 2J = 9.2, 3J = 7.0, CH2), 3.49 (1H, dq, 2J = 9.2, 3J = 7.0, CH2),
3.86 (8H, t, 3J = 4.7, CH2O), 4.23 (2H, t, 3J = 7.2, CH2N), 4.50 (2H,
4.2.16. N-(2-Chloropyrimidin-4-yl)-1-(1-ethoxyethyl)-N-(3-fluo-
ropropyl)-1H-indazol-4-amine (18a)
Compound 7b (400 mg, 1.26 mmol) was dissolved in anhydrous
DMF (5 mL), NaH (60% in mineral oil, 127 mg, 3.18 mmol) was
added and the mixture was maintained at rt for 30 min.
Afterwards, 1-fluoro-3-iodopropane (497 mg, 2.64 mmol) was
added and the mixture was stirred at 80 °C overnight. After cooling
to rt, water (15 mL) was added and the aqueous layer was
extracted with EtOAc (3 ꢁ 15 mL). The combined organic layers
were dried over MgSO4 and the solvent was removed under
reduced pressure. Purification was done by column chromatogra-
phy (PE–EtOAc = 1:1) to yield compound 18a (468 mg, 98%) as
light red oil. Rf = 0.54 (PE–EtOAc = 1:2); dH (400 MHz, CDCl3) 1.18
(3H, t, 3J = 7.1, CH3), 1.80 (3H, d, 3J = 6.1, CH3), 2.04–2.19 (2H, m,
CH2CH2F), 3.31 (1H, dq, 2J = 9.3, 3J = 7.1, CH2), 3.49 (1H, dq,
2J = 9.3, 3J = 7.1, CH2), 4.20 (2H, br s, CH2N), 4.54 (2H, dt,
2JH,F = 47.1, 3J = 5.8, CH2F), 5.88–5.96 (2H, m, CH, HPyr), 7.05 (1H,
d, 3J = 7.1, 5-H), 7.45 (1H, t, 3J = 7.9, 6-H), 7.74 (1H, s, 3-H), 7.76
(1H, d, 3J = 8.5, 7-H), 7.85 (1H, d, 3J = 6.1, HPyr); dC (101 MHz,
2
dt, JH,F = 47.2, 3J = 5.8, CH2F), 5.56 (1H, d, 3J = 6.0, HPyr), 5.92 (1H,
q, 3J = 6.0, CH), 6.17 (1H, t, 3J = 1.6, HAr), 6.83 (2H, d, 3J = 1.6, HAr),
7.06 (1H, d, 3J = 7.5, 5-H), 7.43 (1H, dd, 3J = 6.9, 3J = 8.2, 6-H), 7.71
(1H, 3J = 8.2, 7-H), 7.77 (1H, d, 4J = 0.8 Hz, 3-H), 7.80(1H,
3J = 6.0 Hz, HPyr); dF (376 MHz, CDCl3) ꢀ225.2; MS (ESI+): m/z 627
(72, M++Na); 605 (100, M++H); 432 (76, M+ꢀ2 ꢁ Morph).
Subsequently, the crude compound 19a (approx. 300 mg) was trea-
ted with 1 M HCl (6 mL) for 1.5 h at rt. Afterwards, saturated bicar-
bonate solution was added, the mixture was extracted with EtOAc
(4 ꢁ 15 mL), the combined organic layers were dried over MgSO4
and the solvent was removed under reduced pressure.
Purification was done by column chromatography (DCM–
MeOH = 19:1) to yield compound 20 (381 mg, 27% after two steps)
as yellowish solid. Mp 196 °C; Rf = 0.30 (DCM–EtOH = 19:1); dH
(400 MHz, CDCl3) 1.98–2.12 (2H, m, CH2), 3.17 (8H, t, 3J = 5.0,
CH2N), 3.85 (8H, t, 3J = 4.6, CH2O), 4.25 (2H, t, 3J = 7.2, CH2N),
2
CDCl3) 15.0, 21.1 (2ꢁ CH3), 29.1 (d, JC,F = 19.8 Hz, CH2CH2F), 47.1
3
1
(d, JC,F = 5.4 Hz, CH2N), 64.2 (CH2O), 82.0 (d, JC,F = 165.7 Hz,
2
CH2F), 87.7 (CH), 103.9 (C-7), 111.2 (C-50), 120.4 (C-3a), 122.4 (C-
4.50 (2H, dt, JH,F = 47.2, 3J = 5.7, CH2F), 5.56 (1H, d, 3J = 6.1, HPyr),

6034
K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6.18 (1H, t, 3J = 1.9, HAr), 6.83 (2H, d, 3J = 1.9, HAr), 7.07 (1H, d,
3J = 6.9, 5-H), 7.22 (1H, br s, NH), 7.43–7.53 (2H, m,6-H, 7-H),
7.78 (1H, d, 3J = 6.1, HPyr), 7.87 (1H, s, 3-H); dC (101 MHz, CDCl3)
(C-5), 127.6 (C-6), 130.1 (C-3), 133.3 (C-4), 140.1 (C-7a), 140.3
(C-100), 152.6 (C-300/-500),153.3 (C-60), 154.7 (C-20), 157.0 (C-40); MS
(ESI+): m/z 585 (90, M+ꢀOMs), 513 (100, M+ꢀOMsꢀEOE); MS
(ESIꢀ): m/z 95 (100, OMsꢀ); elemental analysis calcd for
2
3
29.5 (d, JC,F = 19.3 Hz, CH2CH2F), 46.5 (d, JC,F = 5.2 Hz, CH2N),
1
50.0 (CH2N), 67.1 (CH2O), 82.0 (d, JC,F = 165.4 Hz, CH2F), 97.8,
C33H44N8O6S (680.82): C, 58.22, H, 6.51, N, 16.46; found: C,
98.9, 100.5, 109.6, 120.5, 121.4, 127.9, 133.1, 135.7, 141.4, 141.8,
153.0, 155.2, 159.5, 162.8; dF (376 MHz, CDCl3) ꢀ220.0; MS
(ESI+): m/z 533 (100, M++H); elemental analysis calcd for
58.19, H, 6.45, N, 16.38.
4.2.21. 1-(2-((3,5-Dimorpholinophenyl)amino)pyrimidin-4-yl)-
1-(1-(1-ethoxyethyl)-1H-indazol-4-yl)azetidin-1-ium fluoride
(24)
C28H33FN8O2 (532.61): C, 63.14, H, 6.25, N, 21.04; found: C,
63.25, H, 6.21, N, 20.99.
Compound 19b (538 mg, 0.89 mmol) was dissolved in DCM
(20 mL), DIPEA (5 mL) and MsCl (307 mg, 2.68 mmol) were added
at 0 °C. After stirring at 0 °C for 30 min, the reaction was quenched
with saturated bicarbonate solution (15 mL), the aqueous layer
extracted with DCM (3 ꢁ 10 mL), the combined organic layers
dried over Na2SO4 and the solvent removed to obtain the crude
22. Subsequently, the crude was dissolved in ACN (5 mL), AgF
(40 mg, 0.20 mmol) was added and the mixture was stirred in
the dark at rt for 1 h. The solvent was changed by DCM (5 mL),
the solid filtered and washed with DCM (2 mL). Next, the solvent
was removed and the crude product was purified by column chro-
matography to yield 24 (146 mg, 27%) as syrup/solid. Rf = 0.20
(DCM–MeOH = 10:1); dH (400 MHz, CDCl3) 1.17 (3H, t, 3J = 7.0 Hz,
CH3), 1.80 (3H, d, 3J = 6.1 Hz, CH3), 2.39–2.52 (2H, m, Spiro-CH2),
3.11 (8H, t, 3J = 4.6 Hz, CH2N), 3.27–3.36 (1H, m, CH2), 3.50 (1H,
dq, 2J = 9.3 Hz, 3J = 7.1 Hz, CH2), 3.77 (8H, t, 3J = 4.6 Hz, CH2O),
3.87 (2H, m, Spiro-NCH2), 4.46 (2H, m, Spiro-CH2), 5.17 (1H, br s,
4.2.19. 3-((2-(3,5-Dimorpholinophenylamino)pyrimidin-4-yl)-
(1-(1-ethoxyethyl)-1H-indazol-4-yl)amino)propan-1-ol (19b)
Compounds
9
(309 mg, 0.82 mmol) and 18b (201 mg,
0.69 mmol) were dissolved in anhydrous dioxane (6 mL), p-TsOH
was added in catalytic amounts and the mixture was stirred at
90 °C overnight. After cooling to rt, saturated bicarbonate solution
(15 mL) was added and the aqueous layer was extracted with
EtOAc (3 ꢁ 15 mL). The combined organic layers were dried over
MgSO4 and the solvent was removed under reduced pressure.
Purification was done by column chromatography (PE–
EtOAc = 2:1 ? 1:1) to yield compound 19b (320 mg, 77%) as yel-
lowish oil. Rf = 0.52 (DCM–MeOH = 4:1); dH (400 MHz, CDCl3)
1.17 (3H, t, 3J = 7.0 Hz, CH3), 1.69–1.77 (2H, m, CH2), 1.81 (3H, d,
3J = 6.1, CH3), 3.16 (8H, t, 3J = 4.8, CH2N), 3.31 (1H, dq, 2J = 9.2,
3J = 7.0, CH2), 3.49 (1H, dq, 2J = 9.2, 3J = 7.0, CH2), 3.66 (2H, t,
3J = 5.5, CH2N), 3.84 (8H, t, 3J = 4.8, CH2O), 4.25 (2H, br s, CH2OH),
3
5.51 (1H, d, J = 5.9, 50-H), 5.91 (1H, q, 3J = 6.1, CH), 6.20 (1H, t,
H
Ar), 5.93 (1H, q, 3J = 6.1 Hz, CH), 6.14 (1H, br s, Hpyr), 6.38 (2H,
br s,
Ar), 7.14 (3J = 7.4 Hz, 1H, d, H-5), 7.48 (3J = 8.6 Hz,
3J = 1.7, HAr), 6.77 (2H, d, 3J = 1.5, HAr), 7.03 (1H, d, 3J = 7.4, 5-H),
7.09 (1H, s, NH), 7.42 (1H, dd, 3J = 7.4, 3J = 8.4, 6-H), 7.70–7.81
(3H, m, 3-H/60-H/7-H); dC (101 MHz, CDCl3) 15.0, 21.1 (2ꢁ CH3),
31.2 (CH2), 46.0 (CH2N), 50.0 (CH2N), 58.6 (CH2OH), 64.1 (OCH2),
67.1 (OCH2), 87.5 (CH), 97.6, 99.3, 101.1, 110.6, 120.9, 122.9,
127.4, 131.3, 135.3, 140.1, 141.1, 153.0, 156.2, 159.1, 163.2; MS
(ESI+): m/z 603 (5, M++H), 531 (100, M+ꢀEOE); elemental analysis
calcd for C32H42N8O4 (602.73): C, 63.77, H, 7.02, N, 18.59; found: C,
63.85, H, 7.00, N, 18.63.
H
3J = 7.5 Hz, 1H, dd, H-6), 7.58 (1H, br s, HPyr), 7.83 (3J = 8.6 Hz, 1H,
d, H-7), 7.88 (1H, s, H-3); dC (101 MHz, CDCl3) 14.9 (CH3), 19.4
(CH2), 21.1 (CH3), 41.3, 42.5, 54.1, 64.2 (4ꢁ CH2N), 66.9 (CH2O),
87.6 (CH), 112.5, 119.8, 120.9, 127.7, 130.0, 133.5, 139.9, 153.0,
154.9, 157.6; dF (376 MHz, CDCl3) ꢀ120.0; MS (ESI+): m/z 585
(73, M+ꢀF), 513 (100, M+ꢀFꢀEOE); elemental analysis calcd for
C32H41FN8O3 (604.72): C, 63.56, H, 6.83, N, 18.53; found: C,
63.42, H, 6.89, N, 18.61.
4.2.20. 1-(2-((3,5-Dimorpholinophenyl)amino)pyrimidin-4-yl)-
1-(1-(1-ethoxyethyl)-1H-indazol-4-yl)azetidin-1-ium methane-
sulfonate (23)
4.3. Radiosynthesis
4.3.1. N2-(3,5-Dimorpholinophenyl)-N4-(1H-indazol-4-yl)-N4-[11C]-
methylpyrimidin-2,4-diamine ([11C]17)
Compound 19b (538 mg, 0.89 mmol) was dissolved in DCM
(20 mL), DIPEA (5 mL) and MsCl (307 mg, 2.68 mmol) were added
at 0 °C. After stirring at 0 °C for 1 h, the reaction was quenched
with saturated bicarbonate solution (15 mL), the aqueous layer
extracted with DCM (3 ꢁ 10 mL), the combined organic layers
dried over Na2SO4 and the solvent removed to obtain the crude
22. Subsequently, the crude was dissolved in ACN (5 mL), AgOMs
(40 mg, 0.20 mmol) was added and the mixture stirred in the dark
at rt for 1 h. The solvent was changed to DCM (5 mL), the solid was
filtered and washed with DCM (2 mL). Next, the solvent was
removed and the crude 23 purified by column chromatography
(DCM–MeOH = 100:1 ? 10:1) to yield 23 (195 mg, 33%) as syrup.
Rf = 0.20 (DCM–MeOH = 10:1); dH (400 MHz, CDCl3) 1.15 (3H, t,
3J = 7.0 Hz, CH3), 1.78 (3H, d, 3J = 6.0 Hz, CH3), 2.48–2.58 (2H, m,
Spiro-CH2), 2.67 (3H, s, Ms), 3.12 (8H, t, 3J = 4.9 Hz, CH2N), 3.29
(1H, dq, 2J = 9.3 Hz, 3J = 7.0 Hz, CH2), 3.48 (1H, dq, 2J = 9.3 Hz,
3J = 7.0 Hz, CH2), 3.77 (8H, t, 3J = 4.9 Hz, CH2O), 3.92 (2H, m,
Spiro-NCH2), 4.75 (2H, t, 3J = 5.8 Hz, Spiro-CH2), 5.66 (1H, d,
3J = 6.0 Hz, Hpyr), 5.92 (1H, q, 3J = 6.0 Hz, CH), 6.26 (1H, s, HAr),
6.76 (2H, s, HAr), 7.18 (3J = 7.4 Hz, 1H, d, H-5), 7.48 (3J = 6.1 Hz,
3J = 7.4 Hz, 1H, t, H-6), 7.77 (3J = 6.0 Hz, 1H, d, HPyr), 7.85
(3J = 6.1 Hz, 1H, d, H-7), 7.94 (1H, s, H-3), 10.3 (1H, s, NH); dC
(101 MHz, CDCl3) 14.9 (CH3), 20.2 (CH2), 21.1 (CH3), 39.6 (Ms),
46.2, 49.2, 49.5, 64.3 (4ꢁ CH2N), 67.0 (CH2O), 87.9 (CH), 96.9 (C-
200/-600), 101.4 (C-400), 104.6 (C-7), 112.8 (C-50), 120.8 (C-3a), 120.9
Labeling precursor 12 (approx. 1.2 mg) dissolved in DMF
(250 lL) and 0.5 M NaOH (40 lL) was placed into the reacting ves-
sel of the synthesizer unit. [11C]CH3I was transferred in a stream of
Helium into the vessel at ꢀ20 °C. After completion, the reactor was
sealed and heated at 80 °C for 3 min. Then, the reactor was cooled
to 60 °C and 1 M HCl (200 lL) was added and the mixture was
maintained for 2 min at 60 °C. Afterwards, the mixture was trans-
ferred onto a semi-preparative HPLC column. The product eluting
between 5 and 7 min was separated and diluted with 30 mL of
water, passed over a PR-18 cartridge and eluted with 0.75 mL of
EtOH and diluted with 6.5 mL of E153 solution. In a typical exper-
iment approx. 200 MBq of compound [11C]17 could be obtained
within 20 min after EOB starting from 1 to 1.5 GBq [11C]CH4 (30–
35% d.c. yield based on [11C]CH4).
4.4. Molecular modeling
Docking was performed using Glide module.40 Protein coordi-
nates were extracted from the X-ray crystal structure of the
EphB4 kinase domain in complex with a potent inhibitor (code
2x9f in PDB). A grid box of 20 Å ꢁ 20 Å ꢁ 20 Å was centered on
the center of mass of the inhibitor in this crystal structure covering
the ATP-binding site of the enzyme. The module LigPrep (version
2.5, Schrödinger, LLC, NY) was used to assign ionization states,

K. Ebert et al. / Bioorg. Med. Chem. 23 (2015) 6025–6035
6035
16. Ma, B.; Kolb, S.; Diprima, M.; Karna, M.; Tosato, G.; Yang, Q.; Huang, Q.;
Nussinov, R. Growth Factors 2014, 32, 236.
17. Noberini, R.; Lamberto, I.; Pasquale, E. B. Semin. Cell Develop. Biol. 2012, 23, 51.
18. Xiong, C.; Huang, M.; Zhang, R.; Song, S.; Lu, W.; Flores, L., II; Gelovani, J.; Li, C. J.
Nucl. Med. 2011, 52, 241.
19. Zhang, R.; Xiong, C.; Huang, M.; Zhou, M.; Huang, Q.; Wen, X.; Liang, D.; Li, C.
Biomaterials 2011, 32, 5872.
20. Pretze, M.; Kuchar, M.; Bergmann, R.; Steinbach, J.; Pietzsch, J.; Mamat, C.
ChemMedChem 2013, 8, 935.
stereochemistries, and ring conformations of the sketched ligands.
Docking parameters were used as in previous works,41 in extrapre-
cision (XP) mode during the search. The more energetically favor-
able conformation was selected as the best pose.
Acknowledgments
21. Pretze, M.; Mosch, B.; Bergmann, R.; Steinbach, J.; Pietzsch, J.; Mamat, C. J.
Label. Compd. Radiopharm. 2014, 57, 660.
22. Bardelle, C.; Cross, D.; Davenport, S.; Kettle, J. G.; Ko, E. J.; Leach, A. G.;
Mortlock, A.; Read, J.; Roberts, N. J.; Robins, P.; Williams, E. J. Bioorg. Med. Chem.
Lett. 2008, 18, 2776.
23. Bardelle, C.; Coleman, T.; Cross, D.; Davenport, S.; Kettle, J. G.; Ko, E. J.; Leach, A.
G.; Mortlock, A.; Read, J.; Roberts, N. J.; Robins, P.; Williams, E. J. Bioorg. Med.
Chem. Lett. 2008, 18, 5717.
Waldemar Herzog is gratefully acknowledged for his technical
support. The authors thank the Helmholtz Association for partly
supporting this work through Helmholtz-Portfolio Topic
‘Technologie und Medizin—Multimodale Bildgebung zur
Aufklärung des In-vivo-Verhaltens von polymeren Biomaterialien’.
24. Bardelle, C.; Barlaam, B.; Brooks, N.; Coleman, T.; Cross, D.; Ducray, R.; Green, I.;
Lambert-van der Brempt, C.; Olivier, A.; Read, J. Bioorg. Med. Chem. Lett. 2010,
20, 6242.
Supplementary data
25. Barlaam, B.; Ducray, R.; Lambert-van der Brempt, C.; Plé, P.; Bardelle, C.;
Brooks, N.; Coleman, T.; Cross, D.; Kettle, J. G.; Read, J. Bioorg. Med. Chem. Lett.
2011, 21, 2207.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.040.
26. Mamat, C.; Mosch, B.; Neuber, C.; Köckerling, M.; Bergmann, R.; Pietzsch, J.
ChemMedChem 2012, 7, 1991.
27. Pretze, M.; Pietzsch, D.; Mamat, C. Molecules 2013, 18, 8618.
28. Miloudi, A.; El Abed, D.; Boyer, G.; Galy, J.-P. Heterocycles 2006, 68, 2595.
29. Slade, D. J.; Pelz, N. F.; Bodnar, W.; Lampe, J. W.; Watson, P. S. J. Org. Chem.
2009, 74, 6331.
30. Ebert, K.; Köckerling, M.; Mamat, C. Crystals 2012, 2, 176.
31. CCDC-1034376 (5b) and CCDC-1029700 (9) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
32. Belfield, A. J.; Brown, G. R.; Foubister, A. J.; Ratcliffe, P. D. Tetrahedron 1999, 55,
13285.
33. Pretze, M.; Große-Gehling, P.; Mamat, C. Molecules 2011, 16, 1129.
34. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaud, M. Curr. Med. Chem. 2008, 15,
235.
35. Rahim, M. A.; Matsumura, S.; Toshima, K. Tetrahedron Lett. 2005, 46, 7307.
36. Williams, A. L.; Dandepally, S. R.; Kotturi, S. V. Mol. Diversity 2010, 14, 697.
37. Grosse-Gehling, P.; Wuest, F. R.; Peppel, T.; Köckerling, M.; Mamat, C.
Radiochim. Acta 2011, 99, 365.
38. Pretze, M.; Mamat, C. J. Fluorine Chem. 2013, 150, 25.
39. a) Sheldrick, G. M. SHELXS-97 and SHELXL-97, Programs for the Solution and
Refinement of Crystal Structures; University of Göttingen, 1997; b) Sheldrick, G.
M. Acta Crystallogr. 2008, A64, 112.
References and notes
1. Xi, H.-Q.; Wu, X.-S.; Wei, B.; Chen, L. J. Cell Mol. Med. 2012, 16, 2894.
2. Arvaniti, D.; Davy, A. Genes Dev. 2008, 22, 416.
3. Wimmer-Kleikamp, S. H.; Lackmann, M. IUBMB Life 2005, 57, 421.
4. Pasquale, E. B. Nat. Rev. Cancer 2010, 10, 165.
5. Coulthard, M. G.; Duffy, S.; Down, M.; Evans, B.; Power, M.; Smith, F.; Stylianou,
C.; Kleikamp, S.; Oates, A.; Lackmann, M.; Burns, G. F.; Boyd, A. W. Int. J. Dev.
Biol. 2002, 46, 375.
6. Cheng, N.; Brantley, D. M.; Chen, J. Cytokine Growth Factor Rev. 2002, 13, 75.
7. Mosch, B.; Reissenweber, B.; Neuber, C.; Pietzsch, J. J. Oncol. 2010. http://
dx.doi.org/10.1155/2010/135285.
8. Wang, B. Sci. Signal. 2011, 4, pe28.
9. Kumar, S. R.; Singh, J.; Xia, G.; Krasnoperov, V.; Hassanieh, L.; Ley, E. J.; Scehnet,
J.; Kumar, N. G.; Hawes, D.; Press, M. F.; Weaver, F. A.; Gill, P. S. Am. J. Pathol.
2006, 169, 279.
10. Ferguson, B. D.; Tretiakova, M. S.; Lingen, M. W.; Gill, P. S.; Salgia, R. Growth
Factors 2014, 32, 202.
11. Berclaz, G.; Flutsch, B.; Altermatt, H. J.; Rohrbach, V.; Djonov, V.; Ziemiecki, A.;
Dreher, E.; Andres, A.-C. Oncol. Rep. 2002, 9, 985.
12. Davalos, V.; Dopeso, H.; Castaño, J.; Wilson, A. J.; Vilardell, F.; Romero-
Gimenez, J.; Espín, E.; Armengol, M.; Capella, G.; Mariadason, J. M.; Aaltonen, L.
A., ; Schwartz, S., Jr.; Arango, D. Cancer Res. 2006, 66, 8943.
13. Huang, X.; Yamada, Y.; Kidoya, H.; Naito, H.; Nagahama, Y.; Kong, L.; Katoh, S.
Y.; Li, W. L.; Ueno, M.; Takakura, N. Cancer Res. 2007, 67, 9800.
14. Koolpe, M.; Dail, M.; Pasquale, E. B. J. Biol. Chem. 2002, 277, 46974.
15. Chrencik, J. E.; Brooun, A.; Recht, M. I.; Nicola, G.; Davis, L. K.; Abagyan, R.;
Widmer, H.; Pasquale, E. B.; Kuhn, P. J. Biol. Chem. 2007, 282, 36505.
40. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
41. Muñoz, C.; Adasme, F.; Alzate-Morales, J. H.; Vergara-Jaque, A.; Kniess, T.;
Caballero, J. J. Mol. Graphics Modell. 2012, 32, 39.