7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones designed by a “cut and glue” strategy are dual aurora a/vegf-r kinase inhibitors

Article abstract of DOI:10.3390/molecules26061611

Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.

Full text of DOI:10.3390/molecules26061611

molecules
Article
7-(2-Anilinopyrimidin-4-yl)-1-benzazepin-2-ones Designed by a
“Cut and Glue” Strategy Are Dual Aurora A/VEGF-R
Kinase Inhibitors
Mehmet Karatas 1,2, Apirat Chaikuad 3,4 , Bianca Berger 1, Michael H. G. Kubbutat 5, Frank Totzke 5,
Stefan Knapp 3,4 and Conrad Kunick 1,2,
*
1
Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
Beethovenstraße 55, 38106 Braunschweig, Germany
2
3
4
5
Zentrum für Pharmaverfahrenstechnik (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A,
38106 Braunschweig, Germany
Structural Genomics Consortium, BMLS, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany;
chaikuad@pharmchem.uni-frankfurt.de (A.C.); knapp@pharmchem.uni-frankfurt.de (S.K.)
Institut für Pharmazeutische Chemie, Johann Wolfgang-Goethe-Universität, Max-von-Laue-Straße 9,
60438 Frankfurt am Main, Germany
Reaction Biology Europe GmbH, 79108 Freiburg, Germany; M.Kubbutat@reactionbiology.de (M.H.G.K.);
F.Totzke@reactionbiology.de (F.T.)
*
Correspondence: c.kunick@tu-bs.de; Tel.: +49-531-391-2754
Abstract: Although overexpression and hyperactivity of protein kinases are causative for a wide
range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only
a limited number of these enzymes. To identify new chemotypes addressing alternative protein
kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and
reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and
proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of
two representatives of the new chemotype in complex with Aurora A showed the ligand orientation
in the ATP binding pocket and provided the basis for rational structural modifications. Congeners
with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two
members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer
Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range
in the majority of the cell lines.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Karatas, M.; Chaikuad, A.;
Berger, B.; Kubbutat, M.H.G.; Totzke,
F.; Knapp, S.; Kunick, C.
7-(2-Anilinopyrimidin-4-yl)-1-
benzazepin-2-ones Designed by a
“Cut and Glue” Strategy Are Dual
Aurora A/VEGF-R Kinase Inhibitors.
Molecules 2021, 26, 1611. https://doi.
org/10.3390/molecules26061611
Academic Editor: Brullo Chiara
Keywords: anilinopyrimidine; Aurora kinase; benzazepinone; molecular docking; protein kinase
inhibitor; sulfamide; X-ray structure analysis
Received: 12 February 2021
Accepted: 7 March 2021
Published: 14 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Hyperactivity of protein kinases is connected to various human tumors and frequently
contributes to the uncontrolled cell growth found in cancer diseases. Thus, more than
forty protein kinase inhibitors have been introduced within the recent two decades as
anticancer drugs, some of them with remarkable success [1–3]. However, these drugs
already address just a small fraction of the various cancer-related protein kinases, and
therefore the search for new chemotypes acting as inhibitors of these enzymes is still a major
research topic in medicinal chemistry [1]. Pyrimido[5,4-d][1]benzazepin-6-ones 1 are a
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
class of potent dual inhibitors of the polo-like kinase 1 (PLK1) and the vascular endothelial
growth factor receptor kinase 2 (VEGF-R2) with antiproliferative activity against cancer cell
lines [
interactions with the ATP binding pocket of protein kinases, namely hydrogen bond donor
and acceptor groups like the anilinopyrimidine and the benzazepinone elements [ ]. We
considered a rearrangement of these structural features with the goal to generate new
4]. Obviously, molecules such as 1 comprise structural features that are favorable for
2,5
Molecules 2021, 26, 1611. https://doi.org/10.3390/molecules26061611
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1611
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chemical matter with protein kinase inhibitory activity. Thus, we applied a “cut and
glue” strategy by dissecting the benzazepinone and the pyrimidine ring systems and
reconnecting them by a covalent bond between the respective 7- and the 4-position [6,7].
The so designed structure 2a contains no further substituents (R = H) and was therefore
considered as prototype within the series of similar compounds (Figure 1). Evaluation of
2a in a panel of 24 cancer-related protein kinases revealed that Aurora A kinase and the
receptor kinases VEGF-R2 and VEGF-R3 were inhibited with IC50 values below 10 µM.
Further structural modifications of this scaffold were directed to improve the potency
regarding Aurora A. Concomitant inhibition of the VEGF receptor kinases was considered
desirable, since various established multiple kinase inhibitors applied as anticancer drugs
also act on this target [
8], e.g., sorafenib (IC50 VEGF-R2 = 90 nM) [9], or cabozantinib (IC50
VEGF-R2 = 0.035 nM) [10].
Figure 1. “Cut and glue” strategy: the 1-benzazepin-2-one and the anilinopyrimidine parts of the
PLK-inhibitor scaffold were detached at the annulation site and reconnected at the 7- and the
1
4-positions of the ring systems, resulting in the title structures 2a–n. For residues R refer to Table 3.
The Aurora kinases A, B and C are serine/threonine kinases involved in the regula-
tion of the cell division process [11]. Although sharing structural similarity, they vary in
cellular and subcellular localization. Thus, Aurora B is expressed in virtually all cells, while
Aurora C is mainly expressed in germ cells. Aurora A is involved in the transition from
the G2 to the M phase of the cell cycle by promoting centrosome maturation and mitotic
spindle development. Aurora B and Aurora C are involved in the binding of chromosomes
to kinetochores and in the segregation of chromosomes. All Aurora kinases appear to
act as oncogenes by promoting cell proliferation and cell survival [12]. Dysfunction or
overexpression of Aurora kinases has been observed in various human tumors, such as
breast, lung, ovarian, prostate and colon cancer [13
number of Aurora kinase inhibitors have been developed as anticancer drugs, of which
several were evaluated in clinical trials [14 17]. The depicted structures PF-03814735 (
IC50 Aurora A = 0.8 nM), CYC116 ( , Ki Aurora A = 8 nM), MLN8054 ( , IC50 Aurora
A = 4 nM) and alisertib ( , also known as MLN8237, IC50 Aurora A = 1.2 nM) [14] dis-
,14]. Consequently, in recent years a
–
3,
4
5
6
play 2-anilinopyrimidine elements and in this respect share a certain degree of structural
similarity with members of the new compound class 2 (Figure 2). Alisertib is probably
the most advanced Aurora kinase inhibitor in clinical studies. However, in a randomized
phase III trial in patients with relapsed and refractory peripheral T-cell lymphoma (PTCL),
alisertib has not been significantly superior over the comparator drug regimens (prala-
trexate, romidepsin or gemcitabine, respectively) [18]. Recently it has been shown that
alisertib selectively kills HPV-positive cervical cancer cells, and for a targeted application
an intravaginal alisertib-loaded ring was constructed [19]. Based on this, a developing
research area on Aurora kinases as therapeutic targets and new inhibitor chemotypes are
of interest.

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Figure 2. Aurora kinase inhibitors displaying 2-anilinopyrimidine partial structures [14].
With the aim to generate structure–activity relationship information, a small collection
of congeners of prototype 2a was synthesized. Based on the results of crystal structure anal-
ysis and on corresponding docking experiments, we identified the aniline as a promising
area for molecular modification. Besides small customary substituents that were added for
decoration of the aniline ring, we also introduced sulfamoylaminoalkoxy groups, which
were envisaged to modify the structures without decreasing the biological activity and
without increasing the lipophilicity. Evaluation of representatives 2e and 2n of the new
protein kinase inhibitor class in the cancer cell line panel of the National Cancer Institute
(NCI) revealed considerable antiproliferative activity on tumor cell lines in the single digit
micromolar concentration range.
2. Results
2.1. Chemistry
The title compounds were prepared starting from α-tetralone (7), which was reacted
with hydrazoic acid to yield 1-benzazepine-2-one 8 by means of a Schmidt reaction [20].
Subsequent Friedel-Crafts acylation with acetyl chloride in carbon disulfide led to the
acetophenone derivative 9 [21]. The enaminone 10 was then prepared from 9 by prolonged
heating with dimethylformamide dimethyl acetal [22]. Eventually, heating 10 with appro-
priate N-arylguanidinium nitrates 11a–n under conventional [23] or microwave conditions
furnished the anilinopyrimidines 2a–n (Scheme 1).
For the attachment of larger substituents with sulfamide substructures, the phenols
2m and 2n were reacted with 1-bromo-3-chloropropane in the presence of cesium carbonate
by means of a Williamson synthesis. The resulting chloroalkane 12 was then reacted with a
suitable sulfamide in the presence of a base (potassium carbonate or sodium hydride, re-
spectively) to yield the sulfamoylaminoalkoxylated derivatives of series 14. The procedure
is outlined for the example 14d in Scheme 2.

Molecules 2021, 26, 1611
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◦
60 C, 30 min, 67% [20];
Scheme 1. Anilinopyrimidines 2a
–
n
. Reagents and conditions: (i) NaN3, H2SO4, AcOH, rt
→
◦
(ii) AcCl, AlCl3, CS2, reflux, 1 h, then rt, 3 h, 67% [21]; (iii) DMF-DMA, 110–115 C, 13 h, 59% [22]; (iv) propan-2-ol, NaOH,
reflux, 15–30 h, (v) propan-2-ol, NaOH, microwave irradiation: 150 W, 140 C, 30–90 min or (vi) DMF, NaOH, microwave
◦
◦
irradiation: 150 W, 175 C, 30–90 min. For substituents R refer to Table 3.
Scheme 2. Preparation of sulfamoylaminoalkoxy-substituted compounds 14a–g exemplified by the synthesis of 14d.
Reagents and conditions: (i) 1-bromo-3-chloropropane, Cs2CO3, ACN, reflux, 2 h, 25% and (ii) K2CO3, NaI, DMF, 80 ◦C,
22 h, 34%.
2.2. Molecular Docking
For a structure-based design of congeners of the prototype 2a, insight into the ori-
entation mode of the structures as ligands in the Aurora A protein was required. It was
assumed that the molecules would bind in the ATP binding pocket via their anilinopyrimi-
dine substructure towards the hinge region, similar to the binding mode of MLN8054 (5)
displayed in the corresponding cocrystal structure with Aurora-A (PDB: 2WTV) [24]. Dock-
ing experiments using GOLD [25] and the mentioned crystal structure however yielded
poses that appeared unlikely, since the anilinopyrimidine structure was not addressing the
hinge area (Figure 3A,C). Therefore, we applied a constraint for a hydrogen bond between
Ala213 and the NH-group of the aniline moiety of the ligands, as exemplified for congener
2m in Figure 3B,D.
Employing the constraint, the docking procedure indeed placed the prototype 2a
and its congeners in the ATP binding pocket similar to the orientation of MLN8054 (5) in
the original crystal structure (PDB: 2WTV) [24] as depicted for 2m in Figure 3B,D. Direct
interactions were predicted with three hydrogen bonds between the ligand 2m and Ala213
and Glu211 of the hinge region of Aurora A. As described below, the crystal structures of
congeners 2a and 2c later corroborated the general binding orientation predicted under
this constraint.

Molecules 2021, 26, 1611
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Figure 3. Two different binding modes of 2m (sand brown) in the ATP binding pocket of aurora kinase A (PDB: 2WTV,
aquamarine [24]) predicted by docking experiments. Hydrogen bonds are shown in red chain lines. Left ( ): Without
application of constraints; right ( ): constraints were defined between the nitrogen atom of the aniline partial structure of
2m and the carbonyl group of Ala213 (gk+3). ( ): The NH of the lactam ring interacted with Ala213 (gk+3) via a hydrogen
bond (D–A = 2.71 Å). In addition, a hydrogen bond (D–A = 2.84 Å) between the hydroxy group of 2m and Glu260 was
predicted. ( ): The hydrogen bond defined as constraint (D–A = 3.00 Å) was observed. Two additional hydrogen bonds
were formed between the hinge region (Ala213 and Glu211) and the pyrimidine ring of 2m, involving the C(6)H group as a
donor and ring nitrogen 1 as an acceptor. ( ): Surface of the ATP-binding pocket and the ligand without constraints. The
anilinopyrimidine partial structure fills the phosphate binding region. ( ): Surface representation of the binding pocket
A,C
B,D
A
B
C
D
with defined constraint. The hydroxy group was directed towards the solvent, while the benzazepine-2-one partial structure
filled the hydrophobic region and the phosphate binding region.
2.3. Crystal Structure Analyses
To confirm the binding mode of the members of the new Aurora A inhibitor class, we
determined the crystal structures of the kinase in complex with the representative inhibitors
2a and 2c (Table 1). The two cocrystallized inhibitors shared similar binding modes,
which were in agreement with the ones suggested by the docking analyses employing
the constraint between the aniline NH and Ala213 of the protein (Figure 4). While the
anilinopyrimidine elements of the inhibitors adopted flat configurations in the ATP adenine
binding pocket, the benzazepine-2-one partial structures filled the hydrophobic region.
Towards the hinge area, the pyrimidine ring formed a weak hydrogen bond from C(6)H
to Glu211. Another hydrogen bond was detected between the carbonyl group of Ala213
and the NH group of the aniline element. The third contact was established between the
pyrimidine nitrogen atom 1 and the NH group of Ala213. In the structure with 2c, an
additional interaction between the methoxy group of the ligand and Arg137 was observed.
The main result drawn from the docking studies and the crystal structure analyses was
the finding that the 3- and 4-positions of the aniline ring are oriented towards the entrance
of the ATP binding pocket. Therefore, these positions were assumed as suitable attachment
points for further substituents that would not interfere unfavorably with binding.

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Table 1. Data collection and refinement statistics of the Aurora A complex with ligands 2a and 2c.
2a
7AYI
2c
7AYH
PDB Accession Code
Data collection
Swiss light source X06SA
44.43–2.86 (3.01–2.86)
P 6122
Data collection
beamline
Resolution a (Å)
space group
cell dimensions
a (Å)
Swiss light source X06SA
44.28–2.80 (2.95–2.80)
P 6122
82.6
82.6
83.4
83.4
b (Å)
170.1
168.2
c (Å)
α (deg)
β (deg)
90
90
90
90
γ (deg)
120
120
no. unique reflections a
Completeness a (%)
I/σI a
8526 (1186)
100.0 (100.0)
25.7 (2.1)
0.038 (0.975)
1.000 (0.797)
8.4 (8.6)
2014/25
25.2
9066 (1279)
99.9 (100.0)
19.3 (2.1)
0.046 (0.968)
0.999 (0.818)
8.5 (9.0)
2004/27
27.3
a
Rmerge
CC(1/2)
Redundancy a
no. atoms in refinement (P/L) b
Rfact (%)
Rfree (%)
29.7
121/115
0.011
0.96
94.35
0
31.3
133/112
0.011
1.14
95.12
0
Bf (P/L/O) b (Å2)
rmsd bond c (Å)
rmsd deviation angle c (deg)
Ramachandran favored
Ramachandran outlier
a Values in brackets show the statistics for the highest resolution shells. b P/L indicate protein and ligand molecule
c
respectively. rms indicates root-mean-square deviation.
Figure 4. Cocrystal structures of Aurora A in complex with 2a
triad of hydrogen bonds from the inhibitors to the amino acids Glu211 and Ala213 of the hinge area is observed. (
(
A
) and 2c
(
B
). (
A
): The characteristic donor–acceptor–donor
): In the
B
complex with 2c, an additional weak hydrogen bond between the methoxy group and Arg137 (D–A = 3.26 Å) is formed.
Hydrogen bonds are shown in red chain lines.
2.4. Protein Kinase Inhibition
The inhibitory activity of prototype 2a was determined in a panel of 24 tumor-relevant
protein kinases using a radiometric assay. Only the kinases Aurora A, VEGF-R2 and
VEGF-R3 were inhibited with single digit micromolar IC50 values (Table 2). Structural
modifications were then implemented, particularly directed to improve inhibition of Au-
rora A. The so-designed congeners with small substituents at the aniline ring 2b
–2n were
examined for inhibition of Aurora A and of VEGF-R2, VEGF-R3 and Aurora B for compari-

Molecules 2021, 26, 1611
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son (Table 3). For the group of analogues with sulfamoylaminoalkoxy substituents 14 the
results for inhibition of Aurora A are displayed in Table 4.
Table 2. Inhibition profile of 2a against a panel of cancer-related protein kinases a.
Protein Kinase
IC50 (µM)
Protein Kinase
IC50 (µM)
AKT1
ARK5
>100
>100
2.4
26
60
IGF1-R
INS-R
MET
>100
>100
>100
>100
44
>100
>100
>100
74
Aurora-A
Aurora-B
CDK2/CycA
CDK4/CycD1
CK2-alpha1
EGF-R
PAK4
PDGFR-beta
PDK1
PLK1
SAK
SRC
TIE2
VEGF-R2
VEGF-R3
19
>100
>100
>100
>100
>100
16
EPHB4
ERBB2
FAK
FLT3
19
2.8
5.9
a
As a measure of assay quality, the Z’-factor [26,27] was used. The Z’ factor did not drop below 0.4 and was above 0.6 in most cases,
indicating a very good to excellent assay quality.
Table 3. Kinase inhibitory activity of 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones 2a
–2n against
Aurora A, Aurora B, VEGF-R2 and VEGF-R3, represented by either IC50 value (
µM) or residual
activity at 10 µM (%) a.
Compound
R
Aurora A
Aurora B
VEGF-R2
VEGF-R3
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
4-CH3
4-OCH3
4-OC2H5
4-OH
4-Cl
4-Br
4-I
4-NO2
2-OH
2-Cl
2-Br
2.4 µM
2.4 µM
1.8 µM
1.5 µM
0.99 µM
9.4 µM
3.5 µM
3.5 µM
>100 µM
>100 µM
74%
26 µM
16 µM
18 µM
20 µM
3.5 µM
40 µM
18 µM
52 µM
>100 µM
>100 µM
87%
2.8 µM
1.8 µM
1.3 µM
2.0 µM
1.1 µM
11 µM
5.4 µM
17 µM
11 µM
8.4 µM
74%
5.9 µM
4.3 µM
2.5 µM
3.0 µM
1.5 µM
19 µM
4.1 µM
6.2 µM
35 µM
19 µM
72%
2j
2k
2l
73%
116%
78%
64%
2m
2n
3-OH
3-OH, 4-OCH3
1.7 µM
1.4 µM
6.7 µM
4.5 µM
0.78 µM
0.63 µM
0.54 µM
0.55 µM
a
As a measure of assay quality, the Z’-factor [26,27] was used. The Z’ factor did not drop below 0.4 and was
above 0.6 in most cases, indicating a very good to excellent assay quality.

Molecules 2021, 26, 1611
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Table 4. Aurora A kinase inhibition a and kinetic solubility b of 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones 2a
,m,n and
14a–g.
Compound
R1
R2
Aurora-A IC50 (µM)
Kinetic Solubility (µM)
2a
2m
2n
H
H
-OCH3
H
-OH
-OH
2.4
1.7
1.4
50.3 (48.5–52.0)
112 (109–114)
92.6 (92.4–92.8)
14a
H
H
1.8
25.3 c
14b
3.0
19.6 (18.8–20.4)
14c
14d
14e
H
H
4.5
1.5
1.1
25.3 (23.8–26.8)
25.2 (20.2–30.2)
21.4 (20.7–22.1)
OCH3
14f
OCH3
OCH3
2.5
25.0 (24.4–25.7)
34.2 (31.0–36.9)
14g
0.82
a
As a measure of assay quality, the Z’-factor [26
,
27] was used. The Z’ factor in the test run with compounds 14a
–
14g was 0.57, indicating
b
a very good assay quality. Inhibition data for 2a taken from Table 2 for comparison. Determined by nephelometry as a mean of two
measurements, range given in brackets. Singlicate measurement.
c
2.5. Kinetic Solubility
Solubility in aqueous media is of utmost importance for orally administered drugs, as
it significantly influences absorption from the gastrointestinal tract. Poorly soluble drugs
are hardly suitable as development candidates, since in the course of drug development
solubility often decreases because of molecule enlargement [28
active ingredients with moderate permeability and potency should display a solubility
of at least 60 g/mL [31], which corresponds to a concentration of 150 M at an average
–30]. As a rule of thumb,
µ
µ
molecular weight of 400. Since the unsubstituted prototype 2a showed a significantly lower
solubility then this benchmark value, the modification of the prototype either involved
the addition of only very small additional substituents to the aniline ring (compounds
2b
–2n) or of sulfamoylaminoalkyloxy elements (compounds 14a–g). Although increasing
molecular weight, the latter substituents contribute additional sp3-hybridized carbon

Molecules 2021, 26, 1611
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atoms [32] and the polar sulfamide group and therefore were expected to cause at least no
solubility impairment. Table 4 shows the kinetic solubility of the representatives of series
14, determined by nephelometry [33].
2.6. Antiproliferative Activity on Cancer Cell Lines
The new dual Aurora A/VEGF-R inhibitors 2e and 2n were selected by the National
Cancer Institute for screening in its broad cancer cell line panel [34–36]. The NCI in vitro
assay is performed by incubation of cells with test compounds for 48 h and subsequent
cell fixation with trichloroacetic acid. The pink dye sulforhodamin B is added, which
binds to the proteins of the fixed cells. After washing and solubilizing the bound stain, the
optical density is measured spectrophotometrically to determine the relative proliferation
of treated and untreated cells [37,38]. Results of the assays with 2e and 2n are shown in
Table 5.
Table 5. In vitro antiproliferative activity of 2e and 2n on cultured cancer cell lines in the cancer cell line screen of the NCI.
Data displayed as log10 GI50 [M] values a.
Cell Line
Cancer
2e
2n
Cell Line
Cancer
2e
2n
MDA-MB-435 b
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
NCI/ADR-RES b
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
786-0
CCRF-CEM
HL-60(TB)
K-562
RPMl-8226
SR
Leukemia
Leukemia
Leukemia
Leukemia
Leukemia
NSCL
NSCL
NSCL
NSCL
NSCL
NSCL
NSCL
NSCL
NSCL
Colon
Colon
Colon
Colon
Colon
Colon
Colon
CNS
CNS
CNS
CNS
Melanoma
Melanoma
Melanoma
−5.60
−5.86
−5.43
−5.49
−5.88
−5.35
−4.46
−4.96
−5.18
−4.53
>−4.00
−5.39
−5.50
−5.35
n.a.
−5.06
−5.44
−5.49
−5.29
−5.59
−5.32
−5.36
−5.38
−5.47
−5.22
−5.47
−5.66
−5.39
−5.41
−5.50
−5.37
−5.28
−5.50
−5.10
−4.36
−5.09
−6.67
−5.04
−4.93
−4.51
−5.33
−5.00
−4.58
−4.64
−5.42
−5.34
−4.88
−5.32
−5.35
−5.13
−5.35
−5.66
−4.99
−5.26
>−4.00
−5.36
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Renal
Renal
Renal
Renal
Renal
Renal
Renal
Renal
−5.44
−5.68
−5.24
−5.51
−5.38
−5.04
−5.60
>−4.00
−5.23
−5.25
−5.19
−5.39
−5.52
−5.36
−5.32
−5.59
−5.38
−5.73
−5.21
−5.42
−5.76
−5.31
−5.47
−5.37
>−4.00
−5.31
−5.29
−5.81
−5.28
−5.04
−5.04
−5.00
−4.83
−5.03
−5.52
−4.85
−5.00
−5.12
−5.06
−5.01
−5.28
−5.34
−4.43
−5.38
−5.25
−5.57
−5.24
−4.63
−5.52
−4.90
−5.30
−5.29
>−4.00
−5.67
−5.25
−5.39
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-539
SNB-75
U251
A498
ACHN
CAKl-1
RXF 393
SN12C
TK-10
U0-31
PC-3
Prostate
Prostate
Breast
Breast
Breast
Breast
Breast
DU-145
MCF7
MDA-MB-231/ATCC
HS 578T
LOX IMVI
MALME-3M
M14
BT-549
T-47D
a
GI50, Concentration for 50% growth inhibition relative to untreated cells; NSCL, non-small cell lung cancer; CNS, central nervous system
Although frequently designated as breast cancer cell lines, MDA-MB-435 is of melanoma origin [39] and NCI/ADR-RES is of ovarian
b
origin [40].
3. Discussion
Although significant progress in tumor therapy was achieved in the last two decades
with numerous protein kinase inhibitors, relatively few of the tumor-relevant protein
kinases have been exploited as targets in clinical therapy by selective inhibitors. New,
alternative basic structures are of interest for the development of appropriate drugs acting
on these previously unaddressed protein kinases [1,2]. We assumed that structural elements
of known protein kinase inhibitors are particularly well suited as binding motifs for
complementary regions in the ATP binding pocket of kinases and that new chemotypes

Molecules 2021, 26, 1611
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for protein kinase inhibitors could be identified by recombining such partial structures.
We have termed the corresponding procedure “cut and glue” method and applied it
to the already published compound class of dual PLK1/VEGF-R2 inhibitors 1. Formal
decomposition of this initial structure on the annulation side between the azepinone
ring and the pyrimidine ring and relinking between positions 7 and 4 of these cycles
resulted in the new basic structure 2 (Figure 1). Initially, this approach did not target any
specific structure of a particular protein kinase. Instead, the prototype 2a was screened
for inhibition of 24 tumor relevant protein kinases by assessment of IC50 values with a
radiometric assay (Table 2).
The results show that 2a inhibited three tumor relevant protein kinases in the single-
digit micromolar concentration range, namely Aurora A, VEGF-R2 and VEGF-R3, albeit
with IC50 values being at least three orders of magnitude higher than displayed by the
established inhibitors mentioned in the introduction. The inhibition of the VEGF receptor
kinases was not very surprising, since the initial structure
1
also inhibits these targets [
4].
In contrast, no inhibition of PLK1 was observed in 2a in contrast to
1
. Instead, an inhibition
of the Aurora A kinase by 2a was identified. For comparison, the 2-anilinopyrimidine
substructure is the common characteristic structural unit of well-known Aurora A kinase
inhibitors such as PF-03814735 (
3) [41], CYC116 (4) [42], MLN8054 (5) [43] and alisertib
(
6) [44]. A publicly accessible X-ray structure of a complex of the inhibitor MLN8054 and
Aurora A shows that the inhibitor in the ATP binding pocket is bound to the amino acids
of the hinge region via the 2-anilinopyrimidine [24]. Our docking analyses with structural
class
2 inhibitors could not reproduce this binding mode at first, but instead a binding type
was obtained in which the lactam structure of the seven-membered ring addressed the
hinge region as shown for 2m in Figure 3A,C. Despite a good filling of the binding pocket
by the inhibitor, we considered this orientation improbable because it only forms a single
hydrogen bridge to the hinge region.
Therefore, we crystallized Aurora A with the characteristic representatives 2a and
2c, whose structures were elucidated by X-ray analysis. These structures showed high
analogy to the binding of MLN8054 to the protein and met our expectation that the 2-
anilinopyrimidine structure is oriented towards the hinge region, where a triad of hydrogen
bonds to the amino acids Glu211 and Ala213 is formed (Figure 3). By setting a constraint
between Ala 213 and the aniline-NH in the docking experiments, corresponding poses
for newly designed analog structures were generated, as shown in Figure 3B,D. Based on
this orientation, substituents were attached to positions 3 and 4 of the aniline substructure
of 2a for optimization purposes. The results of the tests presented in Table 3 show that
small substituents in position 4 (halogen, hydroxy and alkoxy) only slightly change the
inhibitory activity on Aurora A and B as well as on VEGF-R2 and VEGF-R3. In contrast, a
nitro group in position 4 (2i) causes the complete loss of aurora inhibition and also leads to
a significant reduction in activity at the VEGF receptor kinases. Similarly, substituents in
the 2-position (2j–2l) are detrimental for kinase inhibition, presumably due to twisting of
the aniline ring from its original plane for steric reasons.
For further optimization, 2a was decorated with larger substituents in position 3 of
the aniline ring, which is facing the solvent according to the X-ray structure. Through this
modification we intended to generate additional contacts with the protein kinase near the
entrance area of the ATP binding pocket. Enlargement of drug molecules, however, often
results in increased lipophilicity and in decreased aqueous solubility. Poor solubility is
often observed in protein kinase inhibitors and represents a major problem in preclinical
drug development [45
,46]. An orally administered drug of medium potency and medium
permeability should have a solubility of at least 60
µg/mL [31], which corresponds to a
concentration of 150 µM at a relative molar mass of 400.
For the compounds presented here, the kinetic solubility (solubility of the fastest
precipitating form) was assessed. Since this property can be determined relatively quickly, it
is suitable for comparative considerations in a number of similar compounds. Displaying a
kinetic solubility of about 50 µM, even the unsubstituted basic structure 2a was significantly

Molecules 2021, 26, 1611
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less soluble than the target value. While the introduction of phenolic hydroxyl groups
increased the solubility (e.g., 2m, n, Table 4), phenols are prone to rapid metabolic turnover,
which is considered a disadvantage in drug design. We therefore introduced sulfamide
structures into the 3-position, which were bound to the aniline ring via a hydrocarbon
chain and an ether oxygen atom (compounds 14a–g). The sulfamide structure should
form additional contacts in the entrance area of the ATP binding pocket and limit the
lipophilicity of the substances by its polarity. The additional sp3-hybridized carbon atoms
of the linker chain should also have a positive effect on solubility [32]. Docking experiments
with the compounds 14a–14g designed in this way resulted in useful poses, which were
characterized by additional contacts in the form of hydrogen bonds between ligand and
protein. Figure 5 shows the result of a docking experiment with the sulfamide-substituted
compound 14b in the protein structure taken from PDB 7AYH. In this orientation, the
morpholinosulfamoylpropoxy chain protrudes from the ATP binding pocket into the
solvent as desired, but can still form a pair of hydrogen bonds to the guanidino group of
Arg220 in the entry region. In this pose, the inhibitor is also pushed into the binding pocket
so far that a hydrogen bridge is formed between the carbonyl carbon atom of the lactam
group and Lys162.
Figure 5. Docking result of 14b in the ATP binding pocket of Aurora A Kinase (PDB 7AYH). (A): Hydrogen bonds are
formed between Glu211 and the CH group of the pyrimidine ring (D–A = 2.59 Å), between the aniline NH group and
Ala213 (D–A = 3.17 Å) and between the pyrimidine nitrogen atom and Ala213 (D–A = 2.54 Å). There is also a hydrogen
bond between the carbonyl group of benzazepin-2-one and Lys162. The sulfonyl group interacts with the amino acids
Lys141 and Arg220. (B): Surface representation of the ATP-binding pocket and ligand 14b.
The results listed in Table 4 only partially met our expectations with the representatives
of the 14 series. Although the compounds exhibited inhibitory activity on the Aurora A
kinase in the same order of magnitude as the most potent Series
2 representatives, a
significant improvement over the unsubstituted basic structure 2a was not achieved. In
addition, a slight decrease in solubility was observed compared to the unsubstituted
structure 2a. In view of these results, the inhibition of VEGF receptor kinases was not
further investigated for the congener series 14.
Simultaneous inhibition of the Aurora A kinase and the VEGF-R2/3 receptor kinases
may represent an interesting dual activity for the treatment of cancer. The National Cancer
Institute selected the compounds 2e and 2n as representatives with particularly high in-
hibitory activity on the above kinases for testing in the IVCLS (in vitro cell line screening).
In this screening, compounds are tested for antiproliferative activity on about 60 in vitro
cultivated tumor cell lines [34–36,47]. Based on this very broad screening on cell lines from
nine tumor entities the general antiproliferative potency of compounds is determined. In
addition, the bioinformatic tool COMPARE allowed one to draw conclusions on the molec-
ular target of the compounds from the activity patterns on the different cell lines [47–51]
Similar molecular mechanisms of action of two compounds are assumed if a Pearson
correlation coefficient (PCC) of > 0.6 is calculated by COMPARE [52 53]. Table 5 shows the
.
,

Molecules 2021, 26, 1611
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GI50 values for test compounds 2e and 2n. For the majority of the cell lines tested, both
compounds caused growth inhibition in the single-digit micromolar concentration range.
Only the cell line MDA-MB-231/ATCC showed high resistance to both test compounds
with GI50 values >100 µM. The selectivity profile of the two compounds is not very pro-
nounced given the overall very similar proliferation inhibition values across all cell lines.
A COMPARE analysis determined a PCC = 0.61 for the patterns of test compounds 2e and
2n, which is consistent with an analogous mechanism of action. However, whether this
mechanism is actually based on the inhibition of the inhibited Aurora kinases and VEGF
receptor kinases cannot be deduced from these profiles.
The new compound class
used to discover new chemotypes for protein kinase inhibitors with unexpected selectivity
profiles. However, established Aurora A kinase inhibitors such as the compounds
presented in Figure 2 with IC50 values in the single-digit nanomolar range are far more
potent than the representatives of the new compound class . The results presented here
2 is an example of how the “cut and glue” technique is
3–6
2
indicate that a structural modification merely by substitution at the aniline ring of 2a is not
sufficient for a significant optimization. Instead, in future studies modifications should be
made to the benzazepinone element of
2 based on the structural information presented
here. Such further studies appear to be worthwhile in view of the strong antiproliferative
properties of 2e and 2n. In further studies, based on the “cut and glue” technique, the
benzazepinone and anilinopyrimidine scaffolds can also be linked via other positions.
Such alternative combinations open pathways to multiple new chemotypes that are also
expected to display biological activity.
4. Materials and Methods
4.1. General Information
The starting materials and reagents were purchased from Acros Organics (Geel, Bel-
gium), Alfa Aesar (Karlsruhe, Germany) and Sigma-Aldrich (Steinheim, Germany). All
purchased reagents and solvents were used without further purification. Silica gel 60 Å
was used for purification by column chromatography. Reaction monitoring was performed
using thin layer chromatography (TLC): Polygram SIL G/UV254, 0.2 mm silica gel 60,
40 mm × 80 mm (Macherey-Nagel, Düren, Germany), visualization by UV light (254 and
366 nm). The melting points (m.p.) were detected in open-glass capillaries on an electric
variable heater (Barnstead Electrothermal IA 9100, Southend-on-Sea, Essex, UK). The in-
frared spectra were recorded on a Thermo Nicolet FT-IR 200 spectrometer (Thermo Nicolet,
1
Madison, WI, USA) using KBr pellets. H-NMR, 13C-NMR and 13C-DEPT135 spectra were
recorded on Bruker Avance DRX-400, Bruker Avance III 400, Bruker Avance II 600 or Bruker
Avance III HD 500 spectrometers (Bruker Biospin, Rheinstetten, Germany) (at the NMR
laboratories of the Chemical Institutes of the Technische Universität Braunschweig) in
d6-DMSO with tetramethylsilane as an internal standard (δ = 0 ppm). Chemical shifts
are presented as parts per million (ppm) in relation to internal standard. Atmospheric
pressure chemical ionization (APCI) spectra were determined with an expressionL CMS
spectrometer (Advion, Ltd., Harlow, UK); the APCI source was coupled with ASAP (atmo-
spheric solids analysis probe) and the measurements were performed in the positive mode.
The elemental analyses were performed on a CE Instruments Flash EA® 1112 Elemental
Analyzer (Thermo Quest, San Jose, CA, USA). Purity of test compounds was determined by
high performance liquid chromatography (Merck Hitachi Elite LaChrom system; Hitachi
High Technologies Corporation, Tokyo, Japan) diode array detector: L-2450; pump: L-2130;
autosampler: L-2200; organizer box: L-2000; column: Merck LiChroCART 125-4, LiChro-
spher 100 RP-18 (5 µm) (Merck, Darmstadt, Germany); flow rate: 1.000 mL/min; detection
wavelength: 254 nm and 280 nm (isocratic); overall run time: 15 min (isocratic); AUC, 100%
method; ts = dead time related to DMSO; tms = retention time. For isocratic elution different
acetonitrile/water mixtures were used. Microwave-assisted syntheses were carried out in
a CEM focused microwaveTM synthesis system, type Discover; ChemDriverTM application
software program; reaction vessel: 10 mL with Teflon septum (closed system, CEM GmbH,

Molecules 2021, 26, 1611
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Kamp-Lintfort, Germany), equipped with a Jun-Air compressor (Blue Line Model, Jun-Air
International, Nørresundby, Denmark). Preparative HPLC: LaPrep P 110 HPLC pump;
LaPrep P 311 spectral photometer; LaPrep P 216 fraction collector; injection loop 5 mL;
software: EZChrom Elite, version 3.1.4.; column 125 mm LiChrospher 100 RP-18, 12 µM
(Merck, Darmstadt, Germany); detection wavelength 245 nm, flow rate 40 mL/min, run
time 20–60 min. For preperative HPLC, samples of 20–60 mg were dissolved in a mixture
(4 mL) of DMSO and the indicated eluent using a sandwich injection procedure between
two DMSO layers (0.5 mL each).
4.2. Syntheses
4.2.1. Syntheses of Intermediates
Synthesis procedures and analytical characterization of enaminone 10, N-arylguanidinium
nitrates 11a–n, and N-alkylated sulfamides (13 and its analogues) are presented in the
Supplementary Materials.
4.2.2. General Procedure A for the Synthesis of 7-(2-Anilinopyrimidin-4-yl)-1,3,4,5-tetrahy-
dro-2H-1-benzazepin-2-ones 2b,d,e,f,g,h,i,k,l
7-[3-(Dimethylamino)acryloyl]-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (10) (1 eqi-
valent), an appropriate N-arylguanidinium nitrate (1–1.2 equivalents) and sodium hydrox-
ide (1–2 equivalents) are dissolved in DMF (2–4 mL) and heated in a 10 mL microwave
◦
vessel at 175 C by using a microwave reactor (150 Watt, maximum pressure: 290 psi).
After cooling to room temperature, a precipitate is formed, which is collected by filtration,
washed with water and purified by recrystallization.
4.2.3. General Procedure B for the Synthesis of
7-(2-Anilinopyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-1-benzazepin-2-ones 2c,m,n
7-[3-(Dimethylamino)acryloyl]-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (10) (1 eqi-
valent), an appropriate N-arylguanidinium nitrate (1–1.2 equivalents) and sodium hy-
droxide (1–2 equivalents) are dissolved in propan-2-ol (2–4 mL) and heated at 140 ◦C in a
10 mL microwave vessel using a microwave reactor (150 Watt, maximum pressure: 290 psi).
After cooling to room temperature, a precipitate is formed, which is collected by filtration,
washed with water and purified by recrystallization.
4.2.4. General Procedure C for the Synthesis of 2-Methyl-1,2,5-thiadiazolidine-1,1-dioxide
Derivatives 14a,e
2-Methyl-1,2,5-thiadiazolidine-1,1-dioxide (13a) (1 equivalent) is dissolved in DMF
(9–30 mL). Sodium hydride (60% dispersion in mineral oil, 1.2–1.6 equivalents) is grad-
ually added to the solution at 0 ◦C. Subsequently the mixture is stirred for 1 h at room
temperature under N2-atmosphere. Then the appropriate haloalkane (0.03–1.3 equivalents)
is added in portions to the mixture at 0 ◦C. After stirring for 15–48 h at room temperature,
the mixture is quenched with water (10 mL) and extracted with ethyl acetate (3
×
30 mL).
The combined organic phases are washed with saturated sodium chloride solution (30 mL),
dried with sodium sulfate and evaporated to dryness. The residue is purified by column
chromatography and/or crystallization.
4.2.5. General Procedure D for the Synthesis of N-Alkylated Sulfamide Derivatives
14b,c,d,f,g
The appropriate haloalkane (1 equivalent) is dissolved in DMF (9 mL). Successively
potassium carbonate (4–5 equivalents), N-alkylated sulfamide 14b–d (4–5 equivalents), and
sodium iodide (0.6 equivalents) are added to the suspension and the mixture is heated for
◦
22–51 h at 80 C. After cooling to room temperature, water (9 mL) is added and the mixture
is extracted with ethyl acetate (3
water (2 50 mL), dried with sodium sulfate and evaporated to dryness. The residue is
purified by preparative HPLC.
×
20 mL). The combined organic phases are washed with
×

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4.2.6. Detailed Chemical Synthesis Procedures and Characterization
7-(2-Anilinopyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2a): 7-[3-(Dimethy-
lamino)acryloyl]-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (10, 77 mg, 0.33 mmol), N-
arylguanidinium nitrate (11a, 65 mg, 0.33 mmol) and sodium hydroxide (13 mg, 0.33 mmol)
in 4 mL DMF were heated at reflux for 16.5 h. After cooling to room temperature, the
mixture was poured on water (50 mL). A precipitate was formed, which was collected by
filtration, washed with water and purified by crystallization from ethanol to yield colorless
◦
crystals (36 mg, 36%). m.p.: 249 C; IR (KBr): max 3247 (NH), 3183 (NH), 1668 cm−1 (C=O);
ῦ
1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.13–2.23 (m, 4H, CH2-CH2), 2.78–2.81 (m, 2H,
CH2), 6.96 („t”, J = 7.3 Hz, 1H, CH2), 7.12 (d, J = 5.3 Hz, 1H, ArH), 7.32 (t, J = 7.6 Hz, 2H,
ArH), 7.38 (d, J = 5.3 Hz, 1H, pyrimidine-H), 7.84 (m, 2H, ArH), 8.04 (dd, J = 8.2, 2.0 Hz,
1H, ArH), 8.09 (d, J = 1.7 Hz, 1H, ArH), 8.52 (d, J = 5.2 Hz, 1H, pyrimidine-H), 9.63 (s, 1H,
NH), 9.77 (s, 1H, NH); 13C-NMR (d6-DMSO, 101 MHz):
δ (ppm) = 27.8, 30.3, 33.1 (CH2),
107.5, 118.8 (2C), 121.3, 121.6, 125.9, 128.4 (2C), 128.5, 158.8 (CH), 132.6, 133.7, 140.6, 141.5,
160.1, 163.1, 173.3 (C); C20H18N4O (330.38): calc. C 72.71, H 5.49 N 16.96, found C 72.41, H
5.53, N 16.65; APCI-MS: m/z (%) 331 [M+H]+ (100); HPLC (isocr.): 99.1% at 254 nm, 99.8%
at 280 nm, tms = 2.63 min, tm (DMSO) = 1.02 min (ACN/H2O 50:50).
7-[2-(4-Toluidino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2b): Synthesis ac-
cording to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-2H-
benzo[b]azepin-2-one (10, 258 mg, 1.00 mmol) and N-(4-methylphenyl)guanidinium nitrate
(
11b, 233 mg, 1.10 mmol), reaction tim◦e 2.5 h. Recrystallization from ethyl acetate gave a
−1
ῦ
yellow powder (66 mg, 19%). m.p.: 223 C; IR (KBr): max 3282 (NH), 3195 (NH), 1668 cm
(C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 2.15–2.25 (m, 4H, CH2-CH2), 2.27 (s, 3H,
CH3), 2.77–2.81 (m, 2H, CH2), 7.10–7.13 (m, 3H, ArH), 7.34 (d, J = 5.3 Hz, 1H, pyrimidine-H),
7.70–7.72 (m, 2H, ArH), 8.03 (dd, J = 8.1, 2.0 Hz, 1H, ArH), 8.07 (d, J = 2.0 Hz, 1H, ArH), 8.49
(d, J = 5.1 Hz, 1H, pyrimidine-H), 9.50 (s, 1H, NH), 9.74 (s, 1H, NH); 13C-NMR (d6-DMSO,
126 MHz):
δ (ppm) = 20.3 (CH3), 27.7, 30.2, 33.1 (CH2), 107.2, 118.8 (2C), 121.6, 125.8, 128.3,
128.8 (2C), 158.7 (CH), 130.0, 132.6, 133.7, 138.0, 141.4, 160.1, 163.0, 173.2 (C); C21H20N4O
(344.41): calc. C 73.23, H 5.85 N 16.27, found C 73.18, H 5.85, N 15.69; APCI-MS: m/z (%) 345
[M+H]+ (100); HPLC (isocr.): 98.9% at 254 nm, 99.5% at 280 nm, tms = 2.86 min, tm (DMSO)
= 1.03 min (ACN/H2O 60:40).
7-[2-(4-Methoxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2c): Synthe-
sis according to General Procedure B with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 103 mg, 0.4 mmol) and N-(4-methoxyphenyl)guanidinium
nitrate (11c, 110 mg, 0.480 mmol), reaction time 30 min. Recrystallization from ethanol
◦
ῦ
70% gave ochre-colored crystals (82 mg, 57%). m.p.: 195 C; IR (KBr):
3276 (NH),
max
3189 (NH), 1670 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.15–2.22 (m, 4H,
CH2-CH2), 2.77–2.81 (m, 2H, CH2), 3.74 (s, 3H, OCH3), 6.90–6.93 (m, 2H, ArH), 7.10 (d,
J = 8.3 Hz, 1H, ArH), 7.31 (d, J = 5.3 Hz, 1H, pyrimidine-H), 7.69–7.73 (m, 2H, ArH), 8.02
(dd, J = 8.3 Hz, J = 2.0 Hz, 1H, ArH), 8.06 (d, J = 2.0 Hz, 1H, ArH), 8.47 (d, J = 5.3 Hz,
1H, pyrimidine-H), 9.43 (s, 1H, NH), 9.75 (s, 1H, NH); 13C-NMR (d6-DMSO, 126 MHz):
δ
(ppm) = 55.1 (CH3), 27.7, 30.2, 33.1 (CH2), 106.2, 113.7 (2C), 120.5 (2C), 121.6, 125.8, 128.3,
158.7 (CH), 132.6, 133.6, 133.7, 141.4, 154.7, 160.2, 163.0, 173.2 (C); C21H20N4O2 (360.41): calc.
C 69.98, H 5.59 N 15.55, found C 69.78, H 5.66, N 15.05; APCI-MS m/z (%) 361.2 [M+H]+
(100), 375.2 [M+15]+ (30); HPLC (isocr.): 99.7% at 254 nm, 99.8% at 280 nm, tms = 2.62 min,
tm (DMSO) = 1.03 min (ACN/H2O 60:40).
7-[2-(4-Ethoxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2d): Synthe-
sis according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 266 mg, 1.00 mmol) and N-(4-ethoxyphenyl)guanidinium
nitrate (11d, 258 mg, 1.10 mmol), reaction time1 h. Recrystallization from ethanol gave vio-
◦
let crystals (170 mg, 45%). m.p.: 205 C; IR (KBr): max 3278 (NH), 3181 (NH), 1671 cm−1
ῦ
1
(C=O); H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 1.32 (t, J = 7.1 Hz, 3H, CH3), 2.10–
2.24 (m, 4H, CH2-CH2), 2.77–2.81 (m, 2H, CH2), 3.99 (q, J = 7.1 Hz, 2H, CH2 (OCH2CH3)),
6.88–6.92 (m, 2H, ArH), 7.10 (d, J = 8.3 Hz, 1H, ArH), 7.31 (d, J = 5.3 Hz, 1H, pyrimidine-

Molecules 2021, 26, 1611
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H), 7.67–7.72 (m, 2H, ArH), 8.02 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 8.06 (d, J = 2.0 Hz, 1H,
ArH), 8.46 (d, J = 5.3 Hz, 1H, pyrimidine-H), 9.42 (s, 1H, NH), 9.75 (s, 1H, NH); 13C-NMR
(d6-DMSO, 101 MHz):
δ (ppm) = 14.7 (CH3), 27.8, 30.2, 33.1, 63.1 (CH2), 106.9, 114.3 (2C),
120.6 (2C), 121.6, 125.8, 128.3, 158.9 (CH), 132.7, 133.6, 133.7, 141.4, 153.4, 160.2, 163.0, 173.2
(C); C22H22N4O2 (374.44): calc. C 70.57, H 5.92 N 14.96, found C 70.44, H 5.99, N 14.68;
APCI-MS m/z (%) 375.2 [M+H]+ (100); HPLC (isocr.): 99.3% at 254 nm, 99.6% at 280 nm, tms
= 3.71 min, tm (DMSO) = 1.02 min (ACN/H2O 50:50).
7-[2-(4-Hydroxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2e): Synthe-
sis according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 103 mg, 0.40 mmol) and N-(4-hydroxyphenyl)guanidinium
nitrate (11e, 103 mg, 0.480 mmol), reaction time 1 h. Recrystallization from ethanol 70%
◦
ῦ
gave ochre-colored crystals (60 mg, 43%). m.p.: 291 C; IR (KBr)
3251 (NH), 3196
max
1
(NH), 1653 cm−1 (C=O); H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 2.15–2.22 (m, 4H,
CH2-CH2), 2.76–2.80 (m, 2H, CH2), 6.71–6.75 (m, 2H, ArH), 7.09 (d, J = 8.3 Hz, 1H, ArH),
7.27 (d, J = 5.3 Hz, 1H, pyrimidine-H), 7.53–7.57 (m, 2H, ArH), 8.00 (dd, J = 8.2, 2.1 Hz, 1H,
ArH), 8.05 (d, J = 1.9 Hz, 1H, ArH), 8.44 (d, J = 5.2 Hz, 1H, pyrimidine-H), 9.03 (s, 1H, OH),
9.28 (s, 1H, NH), 9.75 (s, 1H, NH); 13C-NMR (d6-DMSO, 101 MHz):
δ (ppm) = 27.8, 30.3,
33.1 (CH2), 106.7, 114.9 (2C), 121.1 (2C), 121.6, 125.8, 128.3, 158.7 (CH), 132.1, 132.8, 133.7,
141.4, 152.2, 160.4, 162.9, 173.3 (C); C20H18N4O2 (346.38): calc. C 69.35, H 5.24 N 16.17,
found C 69.54, H 5.26, N 15.86; APCI-MS m/z (%) 347.2 [M+H]+ (100); HPLC (isocr.): 97.9%
at 254 nm, 98.9% at 280 nm, tms = 2.23 min, tm (DMSO) = 1.02 min (ACN/H2O 35:65).
7-[2-(4-Chloroanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2f): Synthesis
according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 103 mg, 0.400 mmol) and N-(4-chlorophenyl)guanidinium
nitrate (11f, 112 mg, 0.480 mmol), reaction time 1 h. Recrystallization from ethanol gave
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a beige-green powder (70 mg, 48%). m.p.: 268 C; IR (KBr): max 3266 (NH), 3188 (NH),
1672 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.16–2.23 (m, 4H, CH2-CH2),
2.75–2.83 (m, 2H, CH2), 7.12 (d, J = 8.3 Hz, 1H, ArH), 7.35–7.39 (m, 2H, ArH), 7.42 (d,
J = 5.3 Hz, 1H, pyrimidine-H), 7.86–7.90 (m, 2H, ArH), 8.04 (dd, J = 8.3, 2.0 Hz, 1H, ArH),
8.08 (d, J = 2.0 Hz, 1H, ArH), 8.54 (d, J = 5.2 Hz, 1H, pyrimidine-H), 9.77 (s, 1H, NH), 9.80
(s, 1H, NH); 13C-NMR (d6-DMSO, 126 MHz):
δ (ppm) = 27.7, 30.2, 33.1 (CH2), 107.9, 120.1,
121.6 (2C), 125.9, 128.3 (2C), 128.4, 158.7 (CH), 124.6, 132.4, 133.7, 139.6, 141.6, 159.8, 163.1,
173.2 (C); C20H17ClN4O (364.83): calc. C 65.84, H 4.70, N 15.36, found C 65.89, H 4.68,
N 14.96; APCI-MS m/z (%) 365.2 [M+H]+ (100); HPLC (isocr.): 97.1% at 254 nm, 97.9% at
280 nm, tms = 2.47 min, tm (DMSO) = 1.03 min (ACN/H2O 60:40).
7-[2-(4-Bromoanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2g): Synthesis
according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-2H-
benzo[b]azepin-2-one (10, 258 mg, 1.00 mmol) and N-(4-bromophenyl)guanidinium nitrate
(
11g, 291 mg, 1.05 mmol), reaction time 45◦min. Recrystallization from ethanol gave ochre-
ῦ
colored crystals (185 mg, 45%). m.p.: 239 C; IR (KBr): max 3258 (NH), 3183 (NH), 1672
cm−1 (C=O); H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 2.15–2.22 (m, 4H, CH2-CH2),
1
2.78–2.86 (m, 2H, CH2), 7.11 (d, J = 8.3 Hz, 1H, ArH), 7.42 (d, J = 5.3 Hz, 1H, pyrimidine-H),
7.47–7.51 (m, 2H, ArH), 8.04 (dd, J = 8.2, 2.1 Hz, 1H, ArH), 8.08 (d, J = 1.9 Hz, 1H, ArH), 8.54
(d, J = 5.3 Hz, 1H, pyrimidine-H), 9.76 (s, 1H, NH), 9.78 (s, 1H, NH); 13C-NMR (d6-DMSO,
101 MHz):
δ (ppm) = 27.8, 30.2, 33.1 (CH2), 107.9, 120.6 (2C), 121.7, 126.0, 128.4, 131.2
(2C), 158.8 (CH), 112.6, 132.4, 133.8, 140.1, 141.6, 159.8, 163.2, 173.3 (C); C20H17BrN4O
(409.28): calc. C 58.69, H 4.19, N 13.69, found C 58.65, H 4.29, N 13.28; APCI-MS m/z
(%) 409.1 [M+H]+ (100); HPLC (isocr.): 97.4% at 254 nm, 97.6% at 280 nm, tms = 5.66 min,
tm (DMSO) = 1.03 min (ACN/H2O 65:35).
7-[2-(4-Iodoanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2h): Synthe-
sis according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 103 mg, 0.400 mmol) and N-(4-iodophenyl)guanidinium
nitrate (11h, 156 mg, 0.480 mmol), reaction time 1.5 h. Recrystallization from ethanol
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gave violet crystals (122 mg, 67%). m.p.: 275 C; IR (KBr):
3253 (NH), 3177 (NH),
max

Molecules 2021, 26, 1611
16 of 26
1665 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.15–2.22 (m, 4H, CH2-CH2),
2.78–2.82 (m, 2H, CH2), 7.11 (d, J = 8.3 Hz, 1H, ArH), 7.41 (d, J = 5.3 Hz, 1H, pyrimidine-H),
7.63–7.65 (m, 2H, ArH), 8.04 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, ArH), 8.08 (d, J = 1.8 Hz, 1H,
ArH), 8.54 (d, J = 5.3 Hz, 1H, pyrimidine-H), 9.77 and 9.78 (s, 1H, NH and s, 1H, NH;
overlapping); 13C-NMR(d6-DMSO, 126 MHz):
δ (ppm) = 27.7, 30.2, 33.1 (CH2), 107.8, 120.9
(2C), 121.6, 125.9, 128.4, 137.0 (2C), 158.7 (CH), 83.9, 132.3, 133.7, 140.5, 141.6, 159.8, 163.1,
173.2 (C); C20H17IN4O (456.28): calc. C 52.65, H 3.76, N 12.28, found C 52.20, H 3.72, N
11.96; APCI-MS m/z (%) 457.1 [M+H]+ (100); HPLC (isocr.): 95.1% at 254 nm, 96.8% at
280 nm, tms = 2.07 min, tm (DMSO) = 1.03 min (ACN/H2O 65:35).
7-[2-(4-Nitroanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2i): Synthe-
sis according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 232 mg, 0.900 mmol) and N-(4-nitrophenyl)guanidinium
nitrate (11i, 243 mg, 1.00 mmol), reaction time 1.5 h. Purification by decocting in 200 mL
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ethanol gave a brown powder (145 mg, 43%). m.p.: 333 C; IR (KBr):
3271 (NH),
max
3194 (NH), 1671 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.17–2.24 (m, 4H,
CH2-CH2), 2.80–2.84 (m, 2H, CH2), 7.14 (d, J = 8.3 Hz, 1H, ArH), 7.57 (d, J = 5.3 Hz, 1H,
pyrimidine-H), 8.07–8.13 (m, 4H, ArH), 8.23–8.27 (m, 2H, ArH), 8.65 (d, J = 5.3 Hz, 1H,
pyrimidine-H), 9.79 (s, 1H, NH), 10.46 (s, 1H, NH); 13C-NMR (d6-DMSO, 126 MHz):
δ
(ppm) = 27.7, 30.2, 33.1 (CH2), 109.4, 117.6 (2C), 121.7, 125.0 (2C), 126.1, 128.5, 158.9 (CH),
132.0, 133.8, 140.2, 141.8, 147.2, 159.3, 163.4, 173.2 (C); C20H17N5O3 (375.38): calc. C 63.99,
H 4.56, N 18.66, found C 63.64, H 4.58, N 18.44; APCI-MS m/z (%) 376.2 [M+H]+ (100);
HPLC (isocr.): 99.3% at 254 nm, 99.6% at 280 nm, tms = 2.97 min, tm (DMSO) = 1.02 min
(ACN/H2O 50:50).
7-[2-(2-Hydroxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2j): A mix-
ture of 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (10, 194 mg,
0.750 mmol), N-(2-hydroxyphenyl)guanidinium nitrate (11j, 171 mg, 0.800 mmol) and
sodium hydroxide (32 mg, 0.80 mmol) was heated in 4 mL propan-2-ol to reflux for 23 h.
After cooling to room temperature, a precipitate was formed, which was collected by
filtration, washed with water and purified via column chromatography (ethyl acetate:
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ethanol) to yield a yellow powder (52 mg, 19%). m.p.: 249 C; IR (KBr): max 3256 (NH),
1
3188 (NH), 1666 cm−1 (C=O); H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 2.12–2.24 (m,
4H, CH2-CH2), 2.78–2.81 (m, 2H, CH2), 6.81–6.91 (m, 3H, ArH), 7.11 (d, J = 8.3 Hz, 1H,
ArH), 7.40 (d, J = 5.3 Hz, 1H, pyrimidine-H), 8.04 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 8.08 (d,
J = 2.0 Hz, 1H, ArH), 8.13 (dd, J = 8.1, 2.3 Hz, 1H, ArH), 8.19 (s, 1H, OH), 8.52 (d, J = 5.3 Hz,
1H, pyrimidine-H), 9.77 (s, 1H, NH), 10.01 (s, 1H, NH); 13C-NMR (d6-DMSO, 101 MHz):
δ
(ppm) = 27.8, 30.2, 33.1 (CH2),107.5, 115.1, 119.1, 120.3, 121.6, 122.7, 125.9, 128.4, 158.8
(CH),127.9, 132.3, 133.8, 141.6, 146.9, 160.0, 163.3, 173.3 (C); C20H18N4O2 (364.38): calc. C
69.35, H 5.24, N 16.17, found C 69.74, H 5.30, N 15.61; HPLC (isocr.): 99.6% at 254 nm, 99.6%
at 280 nm, tms = 4.54 min, tm (DMSO) = 1.03 min (ACN/H2O 30:70).
7-[2-(2-Chloroanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2k): Synthe-
sis according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 232 mg, 0.900 mmol) and N-(2-chlorophenyl)guanidinium
nitrate (11k, 233 mg, 1.00 mmol), reaction time 1.3 h. Recrystallization from ethyl acetate
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ῦ
gave a colorless powder (105 mg, 32%). m.p.: 214 C; IR (KBr): max 3400 (NH), 3175 (NH),
1672 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.14–2.23 (m, 4H, CH2-CH2),
2.76–2.79 (m, 2H, CH2), 7.08 (d, J = 8.3 Hz, 1H, ArH), 7.15 („dt“, J = 7.8, 1.5 Hz, 1H, ArH),
7.34–7.41 (m, 2H, ArH), 7.52 (dd, J = 8.1, 1.5 Hz, 1H, ArH), 7.96–8.00 (m, 2H, ArH), 8.04 (d,
J = 2.0 Hz, 1H, ArH), 8.48 (d, J = 5.3 Hz, 1H, pyrimidine-H), 8.73 (s, 1H, NH), 9.74 (s, 1H,
NH); 13C-NMR (d6-DMSO, 101 MHz):
δ (ppm) = 27.7, 30.2, 33.1 (CH2), 108.0, 121.6, 124.8,
125.1, 125.9, 127.3, 128.4, 129.3, 158.8 (CH), 126.4, 132.3, 133.7, 136.5, 141.6, 160.2, 163.2,
173.2 (C); C20H17ClN4O (364.83): calc. C 65.84, H 4.70, N 15.36, found C 65.69, H 4.74, N
14.95; APCI-MS m/z (%) 365.2 [M+H]+ (100), 329.3 [M-35] +(26); HPLC (isocr.): 99.6% at
254 nm, 99.7% at 280 nm, tms = 3.09 min, tm (DMSO) = 1.03 min (ACN/H2O 60:40).

Molecules 2021, 26, 1611
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7-[2-(2-Bromoanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2l): Synthesis
according to General Procedure A with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 181 mg, 0.700 mmol) and N-(2-bromophenyl)guanidinium
nitrate (11l, 213 mg, 0.770 mmol), reaction time 55 min. Recrystallization from ethyl acetate
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gave brown crystals (109 mg, 38%). m.p.: 216 C; IR (KBr):
1660 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
(ppm) = 2.14–2.23 (m, 4H, CH2-CH2),
2.76–2.78 (m, 2H, CH2), 7.07–7.11 (m, 2H, ArH), 7.39–7.44 (m, 2H, ArH), 7.68 (dd, J = 8.1
3413 (NH), 3175 (NH),
max
δ
,
1.5 Hz, 1H, ArH), 7.93–7.96 and 7.96–7.99 (dd, J = 8.1, 1.5 Hz, 1H, ArH and dd, J = 8.3, 2.1 Hz,
1H, ArH; overlapping), 8.03 (d, J = 2.0 Hz, 1H, ArH), 8.48 (d, J = 5.1 Hz, 1H, pyrimidine-H),
8.65 (s, 1H, NH), 9.74 (s, 1H, NH); 13C-NMR (d6-DMSO, 126 MHz):
δ (ppm) = 27.7, 30.2,
33.1 (CH2), 107.9, 121.5, 125.4, 125.6, 125.8, 127.9, 128.3, 132.3, 158.9 (CH), 117.7, 132.5, 133.6,
137.8, 141.5, 160.2, 163.1, 173.2 (C); C20H17BrN4O (409.28): calc. C 58.69, H 4.19, N 13.69,
found C 58.63, H 4.22, N 13.35; APCI-MS m/z (%) 410.2 [M+H]+ (100); HPLC (isocr.): 99.3%
at 254 nm, 99.9% at 280 nm, tms = 3.49 min, tm (DMSO) = 1.03 min (ACN/H2O 60:40).
7-[2-(3-Hydroxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2m): Syn-
thesis according to General Procedure B with 7-[3-(dimethylamino)acryloyl]-1,3,4,5-tetrahydro-
2H-benzo[b]azepin-2-one (10, 129 mg, 0.500 mmol) and N-(3-hydroxyphenyl)guanidinium
nitrate (11m, 107 mg, 0.500 mmol), reaction time 30 min. Recrystallization from ethanol
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gave beige-colored crystals (66 mg, 38%). m.p.: 243 C; IR (KBr):
3270 (NH), 3191
max
1
(NH), 1650 cm−1 (C=O); H-NMR (d6-DMSO, 400 MHz):
δ (ppm) = 2.12–2.26 (m, 4H,
CH2-CH2), 2.76–2.83 (m, 2H, CH2), 6.38 (ddd, J = 8.1, 2.3, 0.8 Hz, 1H, ArH), 7.06 and 7.12
(t, J = 8.1 Hz, 1H, ArH and d, J = 8.3 Hz, 1H, ArH; overlapping), 7.20–7.24 (m, 1H, ArH),
7.37 (d, J = 5.3 Hz, 1H, pyrimidine-H), 7.41 (t, J = 2.3 Hz, 1H, ArH), 8.05 (dd, J = 8.1, 2.0 Hz,
1H, ArH), 8.09 (d, J = 1.8 Hz, 1H, ArH), 8.52 (d, J = 5.3 Hz, 1H, pyrimidine-H), 9.26 (s, 1H,
OH), 9.76 (s, 1H, NH); 13C-NMR (d6-DMSO, 101 MHz):
δ (ppm) = 27.7, 30.2, 33.1 (CH2),
106.0, 107.4, 108.5, 109.8, 121.6, 125.9, 128.4, 129.0, 158.7 (CH), 132.5, 133.7, 141.4, 141.6,
157.4, 160.1, 163.0, 173.2 (C); C20H18N4O2 (346.38): calc. C 69.35, H 5.24, N 16.17, found
C 68.99, H 5.16, N 15.83; APCI-MS m/z (%) 347.2 [M+H]+ (100); HPLC (isocr.): 99.8% at
254 nm, 99.9% at 280 nm, tms = 2.61 min, tm (DMSO) = 1.03 min (ACN/H2O 40:60).
7-[2-(3-Hydroxy-4-methoxyanilino)pyrimidin-4-yl]-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
(
2n): Synthesis according to General Procedure B with 7-[3-(dimethylamino)acryloyl]-
1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (10, 258 mg, 1.00 mmol) and N-(3-hydroxy-4-
methoxyphenyl)guanidinium nitrate (11o, 244 mg, 1.00 mmol) reaction time 1 h. Recrystal-
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lization from ethanol gave brown crystals (136 mg, 36%). m.p.: 269 C; IR (KBr): max 3544
(NH), 3280 (NH), 1670 cm−1 (C=O); 1H-NMR (d6-DMSO, 400 MHz):
δ
(ppm) = 2.12–2.26
(m, 4H, CH2-CH2), 2.75–2.83 (m, 2H, CH2), 3.74 (s, 3H, OCH3), 6.87 (d, J = 8.8 Hz, 1H,
ArH), 7.10 (d, J = 8.3 Hz, 1H, ArH), 7.18 (dd, J = 8.6, 2.5 Hz, 1H, ArH), 7.31 (d, J = 5.3 Hz,
1H, pyrimidine-H), 7.37 (d, J = 2.5 Hz, 1H, ArH), 8.04 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 8.07 (d,
J = 2.0 Hz, 1H, ArH), 8.47 (d, J = 5.3 Hz, 1H, pyrimidine-H), 8.89 (s, 1H, OH), 9.33 (s, 1H,
NH), 9.76 (s, 1H, NH); 13C-NMR (d6-DMSO, 101 MHz):
δ (ppm) = 56.1 (CH3), 27.8, 30.3,
33.1 (CH2), 106.9, 107.9, 109.9, 112.8, 121.6, 125.9, 128.4, 158.7 (CH), 132.7, 133.7, 134.3, 142.4,
142.7, 146.4, 160.2, 163.0, 173.3 (C); C21H20N4O3 (376.41): calc. C 67.01, H 5.36, N 14.88,
found C 66.68, H 5.41, N 14.69; APCI-MS m/z (%) 377.2 [M+H]+ (100); HPLC (isocr.): 99.9%
at 254 nm, 100.0% at 280 nm, tms = 2.37 min, tm (DMSO) = 1.02 min (ACN/H2O 50:50).
7-{2-[(3-(3-Chloropropoxy)phenyl)amino]pyrimidin-4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-
one (12): 7-{2-[(3-Hydroxyphenyl)amino)pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-
2-one (2m) (199 mg, 0.574 mmol) and cesium carbonate (375 mg, 1.15 mmol) were sus-
pended in ACN (30 mL) and heated for 30 min at reflux. 1-Bromo-3-chloropropane (289 µL,
2.89 mmol) was added dropwise to the hot solution and refluxed for 2 h. After cooling to
room temperature the suspension was evaporated and the crude product was purified by
column chromatography (ethyl acetate/dichloromethane/triethylamine 3:1:0.1) to obtain a
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yellow solid (60 mg, 25%). m.p.: 183–185 C; IR (KBr):
1H-NMR (d6-DMSO, 600 MHz):
(ppm) = 2.13–2.25 (m, 6H, 3
2H, CH2), 3.82 (t, J = 6.5 Hz, 2H, CH2), 4.11 (t, J = 6.0 Hz, 2H, CH2), 6.56 (ddd, J = 8.1, 2.5,
3435 (NH), 1671 (C=O) cm−1
;
max
δ
×
CH2), 2.79 (t, J = 7.2 Hz,

Molecules 2021, 26, 1611
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1.0 Hz, 1H, ArH), 7.11 (d, J = 8.3 Hz, 1H, ArH), 7.20 (t, J = 8.1 Hz, 1H, ArH), 7.32 (ddd,
J = 8.3, 2.0, 0.94 Hz, 1H, ArH), 7.40 (d, J = 5.3 Hz, 1H, ArH), 7.72 (t, J = 2.3 Hz, 1H, ArH),
8.05 (dd, J = 8.2, 2.2 Hz, 1H, ArH), 8.10 (d, J = 2.1 Hz, 1H, ArH), 8.54 (d, J = 5.3 Hz, 1H, ArH),
9.66 (s, 1H, NH), 9.78 (s, 1H, NH); 13C-NMR (d6-DMSO, 151 MHz): δ (ppm) = 27.7, 30.2,
31.7, 33.1, 42.0, 63.9 (CH2), 105.0, 107.0, 107.6, 111.4, 121.6, 125.8, 128.3, 129.2, 158.6 (CH),
132.5, 133.7, 141.5, 141.8, 158.8, 160.0, 163.0, 173.2 (C); C23H23ClN4O2 (422.91); APCI-MS:
m/z (%) = 423 [M+H]+ (100).
7-{2-[(3-(3-Chloropropoxy)-4-methoxyphenyl)amino]pyrimidin-4-yl}-1,3,4,5-tetrahydro-2H-
benzo[b]azepin-2-one: 7-{2-[(3-Hydroxy-4-methoxyphenyl)amino)pyrimidin-4-yl)-1,3,4,5-
tetrahydro-2H-benzo[b]azepin-2-one (2n) (100 mg, 0.266 mmol) and cesium carbonate (173
mg, 0.531 mmol) were suspended in ACN (30 mL) and heated for 30 min at reflux. 1-
Bromo-3-chloropropane (32 µL, 0.32 mmol) was added dropwise to the hot solution and
heated for 4 h to reflux. After cooling to room temperature, the suspension was evaporated
and the crude product was purified by colu◦mn chromatography (ethyl acetate) to obtain
ῦ
a yellow solid (27 mg, 22%). m.p.: 180–182 C; IR (KBr): max 3419 and 3287 (NH), 1670
(C=O) cm−1; 1H-NMR (d6-DMSO, 600 MHz):
δ
(ppm) = 2.12–2.25 (m, 6H, 3
×
CH2), 2.78 (t,
J = 7.0 Hz, 2H, CH2), 3.74 (s, 3H, CH3), 3.82 (t, J = 6.4 Hz, 2H, CH2), 4.10 (t, J = 6.0 Hz, 2H,
CH2), 6.94 (d, J = 8.8 Hz, 1H, ArH), 7.10 (d, J = 8.2 Hz, 1H, ArH), 7.30 (dd, J = 8.8, 2.4 Hz,
1H, ArH), 7.33 (d, J = 5.3 Hz, 1H, ArH), 7.66 (s, 1H, ArH), 8.04 (dd, J = 8.3, 2.1 Hz, 1H,
ArH), 8.06 (d, J = 2.1 Hz, 1H, ArH), 8.49 (d, J = 5.1 Hz, 1H, ArH), 9.45 (s, 1H, NH), 9.77
(s, 1H, NH); 13C-NMR (d6-DMSO, 151 MHz):
δ (ppm) = 56.0 (CH3), 27.7, 30.2, 31.8, 33.1,
42.0, 65.0 (CH2), 106.0, 107.1, 111.3, 112.7, 121.6, 125.8, 128.2, 158.8 (CH), 132.7, 133.7, 134.3,
141.4, 143.9, 147.5, 160.1, 162.9, 173.2 (C); C24H25ClN4O3 (452.94); APCI-MS: m/z (%) = 453
[M+H]+ (100).
7-{2-[(3-(3-Bromopropoxy)phenyl)amino]pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-
2-one: 1,3-Dibromopropane (590 µL, 5.79 mmol) was added dropwise to a suspension
of 7-{2-[(3-hydroxyphenyl)amino)pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-
one (2m; 201 mg, 0.580 mmol) and anhydrous potassium carbonate (160 mg, 1.16 mmol) in
dry acetone (7 mL). Subsequently, the solution was refluxed for 24 h. After cooling, ethyl ac-
etate (15 mL) was added and the solution was washed with water (2
layer was dried with sodium sulfate and evaporated to dryness. Purification by column
chromatography (ethyl acetate/triethylamine 50:1) yielded a yellow solid (135 mg, 50%).
×
20 mL). The organic
◦
m.p.: 182–185 C; IR (KBr): max 3283 and 3192 cm−1 (NH), 1667 cm−1 (C=O); 1H-NMR
ῦ
(500 MHz, DMSO-d6):
δ
(ppm) = 2.12–2.24 (m, 4H, 2
×
CH2), 2.27 (quint., J = 6.4 Hz, 2H,
CH2), 2.79 (t, J = 7.0 Hz, 2H, CH2), 3.69 (t, J = 6.5 Hz, 2H, CH2), 4.10 (t, J = 6.0 Hz, 2H, CH2),
6.56 (ddd, J = 8.0, 2.5, 0.9 Hz, 1H, ArH), 7.11 (d, J = 8.3 Hz, 1H, ArH), 7.20 (t, J = 8.2 Hz, 1H,
ArH), 7.30–7.37 (m, 1H, ArH), 7.40 (d, J = 5.2 Hz, 1H, ArH), 7.72 (t, J = 2.3 Hz, 1H, ArH),
8.05 (dd, J = 8.3, 2.1 Hz, 1H, ArH), 8.10 (d, J = 2.1 Hz, 1H, ArH), 8.54 (d, J = 5.3 Hz, 1H, ArH),
9.66 (s, 1H, NH), 9.78 (s, 1H, NH); 13C-NMR (126 MHz, d6-DMSO): δ (ppm) = 27.7, 30.2,
31.3, 31.9, 33.1, 65.0 (CH2), 105.0, 107.0, 107.6, 111.4, 121.6, 125.8, 128.3, 129.2, 158.8 (CH),
132.5, 133.7, 141.5, 141.8, 158.6, 160.0, 163.0, 173.2 (C); C23H23BrN4O2 (467.37); APCI-MS:
m/z (%) = 467 [M+H]+ (10).
7-{2-[(3-(3-(5-Methyl-1,1-dioxido-1,2,5-thiadiazolidin-2-yl)propoxy)phenyl)amino]pyrimidin-4-yl}-
1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (14a): Synthesis according to General Procedure C with
2-methyl-1,2,5-thiadiazolidine-1,1-dioxide (200 mg, 1.47 mmol), sodium hydride (60% disper-
sion in mineral oil, 72 mg, 1.8 mmol) and 7-{2-[(4-(3-bromopropoxy)phenyl)amino]pyrimidin-
4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (130 mg, 0.278 mmol) in DMF (9 mL). After
a reaction time of 48 h, the product was purified by column chromatography with ethy-
lacetate/dichloromethane (5:1) and obtained as a colorless solid (18 mg, 12%). m.p.: 183–
◦
185 C; IR (KBr): max 3266 (NH), 1660 (C=O), 1577 cm−1; 1H-NMR (d6-DMSO, 600 MHz):
δ (ppm) = 1.97–2.05 (m, 2H, CH2), 2.13–2.20 (m, 2H, CH2), 2.20–2.25 (m, 2H, CH2), 2.60 (s, 3H,
CH3), 2.79 (t, J = 7.0 Hz, 2H, CH2), 3.12 (t, J = 7.1 Hz, 2H, CH2), 3.22–3.28 (m, 2H, CH2), 3.29–3.33
(m, 2H, CH2), 4.05 (t, J = 6.1 Hz, 2H, CH2), 6.55 (dd, J = 8.1, 2.5 Hz, 1H, ArH), 7.11 (d, J =
8.3 Hz, 1H, ArH), 7.20 (t, J = 8.1 Hz, 1H, ArH), 7.34 (dd, J = 8.2, 2.0 Hz, 1H, ArH), 7.40 (d, J =
ῦ

Molecules 2021, 26, 1611
19 of 26
5.2 Hz, 1H, ArH), 7.67 (t, J = 2.3 Hz, 1H, ArH), 8.04 (dd, J = 8.2, 2.1 Hz, 1H, ArH), 8.10 (d, J
= 2.1 Hz, 1H, ArH), 8.54 (d, J = 5.2 Hz, 1H, ArH), 9.65 (s, 1H, NH), 9.77 (s, 1H, NH); 13C-NMR
(d6-DMSO, 151 MHz):
δ (ppm) = 33.4 (CH3), 27.1, 27.7, 30.2, 33.1, 44.4, 45.4, 47.0, 64.5 (CH2),
105.1, 107.0, 107.6, 111.3, 121.6, 125.9, 128.3, 129.2, 158.7 (CH), 132.5, 133.7, 141.5, 141.8, 158.8,
160.0, 163.0, 173.2 (C); C26H30N6O4S (522.62); HRMS (EI): m/z [M]+• calc. 522.20438, found
522.20448; APCI-MS: m/z (%) = 523 [M+H]+ (14); HPLC (isocr.): 99.8% at 254 nm, 99.8% at
280 nm, tms = 8.29 min, tm (DMSO) = 1.21 min (ACN/H2O 50:50); HPLC (gradient): 98.8% at
254 nm, tms = 10.3 min, tm = 1.17 min; λmax 212, 281 nm.
N-{3-[3-((4-(2-Oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7-yl)pyrimidin-2-yl)amino)phenoxy]-
propyl}morpholine-4-sulfonamide (14b): According to General Procedure D, potassium carbon-
ate (196 mg, 1.42 mmol), morpholine-4-sulfonamide (236 mg, 1.42 mmol) and sodium iodide
(26 mg, 0.17 mmol) were added to a solution of 7-{2-[(3-(3-chloropropoxy)phenyl)amino]pyri-
midin-4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (12) (120 mg, 0.284 mmol) in DMF
(9 mL). The resulting yellow suspension was heated at 80 ◦C for 51 h. The crude product
was purified by preparative HPLC (ACN/H2O 40:60) to yield a colorless solid (22 mg,
◦
◦
ῦ
14%); m.p.: 221 C (discoloration starting at 102 C); IR (KBr): max 3429 (NH), 1667 (C=O),
1578 cm−1; 1H-NMR (d6-DMSO, 600 MHz):
(ppm) = 1.92 (quint., J = 6.8 Hz, 2H, CH2),
2.14–2.25 (m, 4H, 2 CH2), 2.79 (t, J = 7.0 Hz, 2H, CH2), 2.99 (t, J = 4.8 Hz, 4H, 2 CH2),
3.10 (q, J = 7.0 Hz, 2H, CH2), 3.59 (t, J = 4.8 Hz, 4H, 2 CH2), 4.03 (t, J = 6.1 Hz, 2H, CH2),
δ
×
×
×
6.54 (dd, J = 8.1, 2.5 Hz, 1H, ArH), 7.11 (d, J = 8.3 Hz, 1H, ArH), 7.20 (t, J = 8.1 Hz, 1H, ArH),
7.33–7.36 (m, 1H, ArH), 7.38–7.43 (m, 2H, ArH und NH), 7.68 (t, J = 2.3 Hz, 1H, ArH), 8.04
(dd, J = 8.3, 2.1 Hz, 1H, ArH), 8.11 (d, J = 2.3 Hz, 1H, ArH), 8.54 (d, J = 5.3 Hz, 1H, ArH), 9.65
(s, 1H, NH), 9.77 (s, 1H, NH); 13C-NMR (d6-DMSO, 151 MHz):
δ (ppm) = δ (ppm) = 27.7,
29.1, 30.2, 33.1, 39.4 (overlapping with DMSO peak), 45.7 (2 C), 64.3, 65.4 (2 C) (CH2), 105.1,
107.0, 107.6, 111.2, 121.6, 125.9, 128.3, 129.1, 158.7 (CH), 132.5, 133.7, 141.5, 141.8, 158.8,
160.0, 163.0, 173.2 (C); C27H32N6O5S (552.65); HRMS (ESI): m/z (C27H33N6O5S, monocation)
calc. 553.22277, found 553.22303; MS (ESI, positive): m/z (%) = 553 [M]+ (100), 466 [M+–86]
(19), 381 [M+ –171] (25); HPLC (isocr.): 99.4% at 254 nm, 99.2% at 280 nm, tms = 6.70 min, tm
(DMSO) = 1.20 min (ACN/H2O 40:60); HPLC (gradient): 97.5% at 254 nm, tms = 17.5 min
tm = 1.40 min; λmax 216, 278 nm.
,
N-{3-[3-((4-(2-Oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7-yl)pyrimidin-2-yl)amino)phenoxy]
propyl}-pyrrolidine-1-sulfonamide (14c): According to General Procedure D, potassium carbon-
ate (131 mg, 0.948 mmol), pyrrolidine-4-sulfonamide (179 mg, 1.19 mmol) and sodium io-
dide (23 mg, 0.15 mmol) were added to a solution of 7-{2-[(3-(3-chloropropoxy)phenyl)amino]
pyrimidin-4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (12) (100 mg, 0.237 mmol) in
◦
DMF (9 mL). The resulting yellow suspension was heated at 80 C for 22 h. The crude
product was purified by preparative HPLC (ACN/H◦2O 40:60) to yield a colorless solid
◦
ῦ
(20 mg, 16%); m.p.: 225 C (discoloration starting at 100 C); IR (KBr): max 3434 (NH), 1667
(C=O), 1578 cm−1; 1H-NMR (d6-DMSO, 600 MHz):
δ
(ppm) = 1.73–1.82 (m, 4H, 2 × CH2),
1.91 (quint., J = 6.9 Hz, 2H, CH2), 2.13–2.25 (m, 4H, 2 × CH2), 2.79 (t, J = 7.2 Hz, 2H, CH2),
3.09 (q, J = 7.0 Hz, 2H, CH2), 3.11–3.15 (m, 4H, 2 × CH2), 4.03 (t, J = 6.1 Hz, 2H, CH2),
6.54 (ddd, J = 8.1, 2.5, 1.0 Hz, 1H, ArH), 7.11 (d, J = 8.5 Hz, 1H, ArH), 7.15 (t, J = 5.9 Hz,
1H, NH), 7.19 (t, J = 8.2 Hz, 1H, ArH), 7.32 (ddd, J = 8.3, 2.1, 0.9 Hz, 1H, ArH), 7.40 (d, J
=
5.3 Hz, 1H, ArH), 7.68 (t, J = 2.4 Hz, 1H, ArH), 8.04 (dd, J = 8.3, 2.1 Hz, 1H, ArH), 8.11
(d, J = 2.3 Hz, 1H, ArH), 8.54 (d, J = 5.1 Hz, 1H, ArH), 9.65 (s, 1H, NH), 9.76 (s, 1H, NH);
13C-NMR (d6-DMSO, 151 MHz):
(ppm) = 24.9 (2C), 27.7, 29.0, 30.2, 33.1, 39.4 (overlapping
δ
with DMSO peak), 47.5 (2C), 64.4 (CH2), 105.0, 107.0, 107.6, 111.2, 121.6, 125.9, 128.3, 129.1,
158.8 (CH), 132.5, 133.7, 141.5, 141.8, 158.8, 160.0, 163.0, 173.2 (C); C27H32N6O4S (536.65);
HRMS (ESI): m/z (C27H33N6O4S, monocation) calc. 537.22785, found 537.22812; MS (ESI,
positive): m/z (%) = 537 [M+H]+ (100), 381 [M+
−
155] (15), 466 [M+
−
70] (11); HPLC (isocr.):
99.6% at 254 nm, 99.9% at 280 nm, tms = 10.3 min, tm (DMSO) = 1.20 min (ACN/H2O 40:60);
HPLC (gradient): 99.6% at 254 nm, tms = 10.6 min, tm = 1.23 min; λmax 213, 280 nm.
N-{3-[3-((4-(2-Oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7-yl)pyrimidin-2-yl)amino)phenoxy]
propyl}-azetidine-1-sulfonamide (14d): According to General Procedure D, potassium carbon-

Molecules 2021, 26, 1611
20 of 26
ate (157 mg, 0.1.14 mmol), azetidine-1-sulfonamide (193 mg, 1.42 mmol) and sodium iodide
(26 mg, 0.17 mmol) were added to a solution of 7-{2-[(3-(3-Chloropropoxy)phenyl)amino]pyrimidin-
4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (12) (121 mg, 0.285 mmol) in DMF (9 mL). The
resulting yellow suspension was heated at 80 ◦C for 22 h. The crude product was purified
by preparative HPLC (ACN/H2O◦40:60) to yield a colorless solid (50 mg, 34%); m.−p1.:
◦
ῦ
248 C (discoloration starting at 115 C); IR (KBr): max 3280 (NH), 1668 (C=O), 1579 cm
;
1H-NMR (d6-DMSO, 500 MHz):
δ (ppm) = 1.92 (quint., J = 6.6 Hz, 2H, CH2), 2.01–2.11 (m,
2H, CH2), 2.12–2.26 (m, 4H, 2
CH2), 3.68 (t, J = 7.7 Hz, 4H, 2
×
CH2), 2.79 (t, J = 7.0 Hz, 2H, CH2), 3.12 (q, J = 7.0 Hz, 2H,
CH2), 4.03 (t, J = 6.1 Hz, 2H, CH2), 6.55 (ddd, J = 8.2, 2.4,
×
0.9 Hz, 1H, ArH), 7.11 (d, J = 8.2 Hz, 1H, ArH), 7.20 (t, J = 8.2 Hz, 1H, ArH), 7.24 (t, J =
6.0 Hz, 1H, ArH), 7.30–7.36 (m, 1H, NH), 7.39 (d, J = 5.2 Hz, 1H, ArH), 7.68 (t, J = 2.1 Hz,
1H, ArH), 8.04 (dd, J = 8.2, 2.1 Hz, 1H, ArH), 8.11 (d, J = 2.1 Hz, 1H, ArH), 8.54 (d, J = 5.2
Hz, 1H, ArH), 9.64 (s, 1H, NH), 9.76 (s, 1H, NH); 13C-NMR (d6-DMSO, 126 MHz):
δ (ppm)
= 14.4, 27.7, 29.2, 30.2, 33.1, 39.4 (overlapping with DMSO peak), 49.7 (2C), 64.4 (CH2),
105.0, 107.0, 107.6, 111.2, 121.6, 125.9, 128.3, 129.1, 158.8 (CH), 132.5, 133.7, 141.5, 141.8,
158.8, 160.0, 163.0, 173.2 (C) (the signal at 158.8 ppm corresponds to one quaternary and one
tertiary carbon atom); C26H30N6O4S (522.62); HRMS (EI): m/z [M]+• calc. 522.20438,found
522.20251; MS (ESI, positive): m/z (%) = 523 [M]+ (100), 466 [M+–56] (18), 381 [M+–141] (5);
HPLC (isocr.): 96.9% at 254 nm, 99.8% at 280 nm, tms = 7.68 min, tm (DMSO) = 1.20 min
(ACN/H2O 40:60); HPLC (gradient): 99.1% at 254 nm, tms = 10.2 min, tm = 1.27 min;
λ
max
212, 281 nm.
7-(2-((4-Methoxy-3-(3-(5-methyl-1,1-dioxido-1,2,5-thiadiazolidin-2-yl)propoxy)phenyl)amino)
pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (14e): According to General Pro-
cedure C, with 2-methyl-1,2,5-thiadiazolidine-1,1-dioxide (142 mg, 1.04 mmol), sodium
hydride (60% dispersion in mineral oil, 50 mg, 1.3 mmol) and 7-{2-[(3-(3-chloropropoxy)-4-
methoxyphenyl)amino]pyrimidin-4-yl}-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (69 mg,
0.15 mmol) in DMF (9 mL). After a reaction time of 48 h, the product was purified by col-
umn chromatography with ethylacetate/ethanol (1:0
→
0.75:0.25
→
0.5:0.5
→
0:1). After
recrystallization with ethanol the product was obtained as a yellow solid (20 mg, 24%);
◦
ῦ
m.p.: 198–201 C; IR (KBr):
600 MHz): (ppm) = 2.02 (quint., J = 6.4 Hz, 2H, CH2), 2.10–2.26 (m, 4H, 2
3264 (NH), 1670 (C=O), 1578 cm−1; 1H-NMR (d6-DMSO,
max
δ
×
CH2), 2.59
(s, 3H, CH3), 2.78 (t, J = 7.0 Hz, 2H, CH2), 3.12 (t, J = 7.1 Hz, 2H, CH2), 3.22–3.27 (m, 2H,
CH2), 3.28–3.33 (m, 2H, CH2), 3.74 (s, 3H, OCH3), 4.04 (t, J = 6.1 Hz, 2H, CH2), 6.93 (d, J =
8.9 Hz, 1H, ArH), 7.10 (d, J = 8.3 Hz, 1H, ArH), 7.31 (d, J = 2.4 Hz, 1H, ArH), 7.32–7.34 (m,
1H, ArH), 7.61 (d, J = 2.4 Hz, 1H, ArH), 8.03 (dd, J = 8.2, 2.1 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H,
ArH), 8.48 (d, J = 5.2 Hz, 1H, ArH), 9.44 (s, 1H, NH), 9.76 (s, 1H, NH); 13C-NMR (d6-DMSO,
151 MHz):
δ (ppm) = 33.4, 56.0 (CH3), 27.2, 27.7, 30.2, 33.1, 44.5, 45.3, 47.0, 65.6 (CH2), 105.9,
107.0, 111.1, 112.7, 121.6, 125.8, 128.2, 158.8 (CH), 132.7, 133.7, 134.3, 141.4, 143.9, 147.7,
160.1, 162.9, 173.2 (C); C27H32N6O5S (552.65); APCI-MS: m/z (%) = 553 [M+H]+ (18); HRMS
(EI): m/z [M]+• calc. 552.21494, found 552.21475; HPLC (isocr.): 98.2% at 254 nm, 97.3% at
280 nm, tms = 5.35 min, tm (DMSO) = 1.19 min (ACN/H2O 40:60); HPLC (gradient): 95.6%
at 254 nm, tms = 9.81 min, tm = 1.16 min; λmax 298 nm.
N-{3-[2-Methoxy-5-((4-(2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7-yl)pyrimidin-2-yl)amino)
phenoxy]-propyl}morpholine-4-sulfonamide (14f): According to General Procedure D, potas-
sium carbonate (229 mg, 1.66 mmol), morpholine-4-sulfonamide (275 mg, 1.66 mmol) and
sodium iodide (30 mg, 0.20 mmol) were added to a solution of 7-(2-((3-(3-chloropropoxy)-4-
methoxyphenyl)amino)pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (150 mg,
0.331 mmol) in DMF (9 mL). The resulting yellow suspension was heated at 80 ◦C for 23 h.
The crude product was purified by preparative HPLC (ACN/H2O 40:60) to yield a colorless
◦
◦
ῦ
solid (12 mg, 6%). m.p.: 176 C (discoloration starting at 100 C); IR (KBr):
(NH), 1672 (C=O), 1578 cm−1; 1H-NMR (d6-DMSO, 600 MHz):
(ppm) = 1.93 (quint., J
= 6.4 Hz, 2H, CH2), 2.12–2.25 (m, 4H, 2 CH2), 2.78 (t, J = 7.1 Hz, 2H, CH2), 2.97–3.02
(m, 4H, 2 CH2), 3.11 (q, J = 7.0 Hz, 2H, CH2), 3.57–3.61 (m, 4H, 2 CH2), 3.74 (s, 3H,
OCH3), 4.04 (t, J = 6.0 Hz, 2H, CH2), 6.92 (d, J = 8.8 Hz, 1H, ArH), 7.10 (d, J = 8.3 Hz, 1H,
3433
max
δ
×
×
×

Molecules 2021, 26, 1611
21 of 26
ArH), 7.29 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 7.33 (d, J = 5.3 Hz, 1H, ArH), 7.35 (t, J = 5.7 Hz,
1H, NH), 7.64 (s, 1H, ArH), 8.03 (dd, J = 8.3, 2.3 Hz, 1H, ArH), 8.08 (d, J = 2.3 Hz, 1H, ArH),
8.48 (d, J = 5.1 Hz, 1H, ArH), 9.45 (s, 1H, NH), 9.75 (s, 1H, NH); 13C-NMR (d6-DMSO,
151 MHz):
δ (ppm) = 55.9 (CH3), 27.7, 29.1, 30.2, 33.1, 39.4 (overlapping with DMSO peak),
45.7 (2C), 65.4 (2C), 65.5 (CH2), 105.6, 107.0, 111.0, 112.6, 121.6, 125.8, 128.2, 158.8 (CH),
132.7, 133.7, 134.3, 141.4, 143.8, 147.6, 160.1, 162.9, 173.2 (C); C28H34N6O6S (582.68); HRMS
(ESI): m/z (C28H35N6O6S, monocation) calc. 583.23333, found 583.23362; MS (ESI, positive):
m/z (%) = 583 [M]+ (100), 381 [M+–201] (37), 102 [M+–480] (74); HPLC (isocr.): 94.8% at
254 nm, 95.1% at 280 nm, tms = 5.43 min, tm (DMSO) = 1.20 min (ACN/H2O 40:60); HPLC
(gradient): 95.9% at 254 nm, tms = 9.77 min, tm = 1.27 min; λmax 289 nm.
N-{3-[2-Methoxy-5-((4-(2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7-yl)pyrimidin-2-yl)amino)
phenoxy]-propyl}azetidine-1-sulfonamide (14g): According to General Procedure D, potas-
sium carbonate (95 mg, 0.69 mmol), azetidine-1-sulfonamide (117 mg, 0.859 mmol) and
sodium iodide (16 mg, 0.11 mmol) were added to a solution of 7-(2-((3-(3-chloropropoxy)-4-
methoxyphenyl)amino)pyrimidin-4-yl)-1,3,4,5-tetrahydro-2H-benzo[b]azepin-2-one (78 mg,
0.17 mmol) in DMF (9 mL). The resulting yellow suspension was heated at 80 ◦C for 48 h.
The crude product was purified by preparative HPLC (ACN/H2O 40:60) to yield a colorless
◦
3279 (NH), 1669 (C=O), 1578 cm−1
;
ῦ
solid (12 mg, 6%); m.p.: 167–170 C; IR (KBr):
1H-NMR (d6-DMSO, 600 MHz):
(ppm) = 1.93 (quint., J = 6.6 Hz, 2H, CH2), 2.02–2.10 (m,
2H, CH2), 2.13–2.26 (m, 4H, 2 CH2), 2.78 (t, J = 7.0 Hz, 2H, CH2), 3.13 (q, J = 7.0 Hz, 2H,
CH2), 3.68 (t, J = 7.4 Hz, 4H, 2 CH2), 3.74 (s, 3H, OCH3), 4.04 (t, J = 6.1 Hz, 2H, CH2),
max
δ
×
×
6.92 (d, J = 8.9 Hz, 1H, ArH), 7.10 (d, J = 8.3 Hz, 1H, ArH), 7.19 (t, J = 5.8 Hz, 1H, NH),
7.29 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 7.33 (d, J = 5.3 Hz, 1H, ArH), 7.65 (s, 1H, ArH), 8.03
(dd, J = 8.3, 2.3 Hz, 1H, ArH), 8.08 (d, J = 2.3 Hz, 1H, ArH), 8.48 (d, J = 5.3 Hz, 1H, ArH),
9.45 (s, 1H, NH), 9.75 (s, 1H, NH); 13C-NMR (d6-DMSO, 151 MHz):
δ (ppm) = 55.9 (CH3),
14.4, 27.7, 29.2, 30.2, 33.1, 39.4 (overlapping with DMSO peak), 49.7 (2C), 65.5 (CH2), 105.6,
107.0, 111.0, 112.6, 121.6, 125.8, 128.2, 158.8 (CH), 132.7, 133.7, 134.3, 141.4, 143.8, 147.7,
160.1, 162.9, 173.2 (C); C27H32N6O5S (552.65); HRMS (EI): m/z [M]+• calc. 552.21494, found
552.21431; APCI-MS: m/z (%) = 553 [M+H]+ (7); HPLC (isocr.): 97.9% at 254 nm, 98.9% at
280 nm, tms = 6.20 min, tm (DMSO) = 1.29 min (ACN/H2O 40:60); HPLC (gradient): 96.6%
at 254 nm, tms = 9.98 min, tm = 1.17 min; λmax 289 nm.
4.3. Molecular Docking
Docking studies were performed using GOLD [25]. The protein and the ligands were
prepared using MOE (Molecular Operating Environment, 2018.01, Chemical Computing
group, Montreal, QC, Canada). Energy minimization was performed using the QuickPrep
function in MOE and structures were saved as mol2 files. Docking runs were performed
using the wizard of GOLD in the HERMES interface (version 1.6.2). Missing hydrogen
atoms were added, and the ligand and all water molecules were removed from the protein
structure. The binding site was defined as a zone of 10 Å around the cocrystallized inhibitor.
For scoring, the chemscore kinase function was applied. Docking accuracy was set to 200%,
10 docking runs for each ligand were performed, the function “generate diverse solutions”
was activated, and the option “allow early termination” was turned off. The results of
the docking experiments were analyzed and visualized using UCSF Chimera (version
1.11.2) [54].
4.4. Crystallization of Aurora A Complexes, Data Collection and Structure Determination
Crystallizations of the Aurora A complexes were carried out as described previ-
ously [55]. In brief, the recombinant kinase dom◦ain of Aurora A was mixed with the
compounds, and crystallization was performed at 4 C using the conditions containing 18%
PEG 3350 and 5% tacsimate pH 7 (for 2a) or 24% PEG 3350 and 0.2 M sodium malonate
pH 7. Diffraction data collected at SLS beamline X06SA were processed using XDS [56
and scaled with AIMLESS [57]. Molecular replacements were performed using Phaser [58
]
]
and the coordinates of Aurora A (PDB 5ONE) [55]. Model rebuilding was performed

Molecules 2021, 26, 1611
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in COOT [59], alternated with refinement in REFMAC [60]. The final structures were
validated for geometric correctness with MolProbity [61], and were deposited under PDB
accession codes 7AYI and 7AYH.
4.5. Protein Kinase Inhibition Assays
The screening for protein kinase inhibition was performed with the radiometric 33Panqinase
activity assay at Reaction Biology Europe GmbH (Freiburg) using a robotic system (Beckmann
Coulter/SagianRoboter) and 96-well plates (FlashPlates, Perkin/Elmer/NEN, Boston, MA,
USA). In brief, the transfer of 33P-phosphate into the substrate was measured with a
microplate scintillation counter (Microbeta Trilux, Wallace). Protein kinases were ex-
pressed in Sf9 cells as recombinant glutathione S-transferase (GST) or polyhistidine (His)-
tagged proteins in baculovirus. Isolation and purification of expressed kinases were
performed with affinity chromatography by using GSH agarose (Sigma) or Ni-NTA
agarose (Qiagen). The standard solution for each enzyme contained: 60 mM HEPES-
NaOH (pH 7.5), 3 mM MgCl2, 3
PEG20000 and 1 M [
-33P]-ATP (approximately 5
ical order the reaction mixture was prepared: 20
lution in water, 5 L of the compound in 10% DMSO and 20
mixture (50:50). Each kinase contained different substrates in different concentrations:
125 ng/50 µL Poly(Glu, Tyr)4:1 for 20 ng/50 L IGF1-R, 25 ng/50 L IGF1-R, 20 ng/50 µL
L EGF-R, 10 ng/50 L EPHB4, 20 ng/50
L SRC, 100 ng/50 L FAK, 200 ng/50 L FAK,
LTIE2; 250 ng/50 L Poly(Glu,
L tetra (LRRWSLG) for 50 ng/50 L Aurora-A,
L PDK1; 250 ng/50 L tetra (LRRWSLG) for 50 ng/50
Aurora-B, 200 ng/50 µL Aurora-B; 125 ng/50 L Histon H1 for 100 ng/50 L CDK2/CycA;
250 ng/50 µL Rb-CTF for 50 ng/50 µL CDK/CycD1; 500 ng/50 µL Rb-CTF for 50 ng/50 µL
CDK4/CycD1; 125 ng/50 µL Poly(ala, Glu, Lys, Tyr)6:2:5:1 for 25 ng/50 L INS-R, 50 ng/50 µL
L FLT3, 20 ng/50 µL
L Casein for 200 ng/50 L CK2-
L PLK1, 200 ng/50 µL
L AKT1; 125 ng/50 L p53-CTM
LCK2-alpha1; 250 ng/50 L MEK-KM for
µ
M sodium orthovanadate, 1.2 mM DTT, 50
105 cpm per well). In chronolog-
L standard solution, 5 L ATP so-
L of enzyme/substrate
µg/mL
µ
γ
×
µ
µ
µ
µ
µ
µ
EGF-R, 25 ng/50 µL EGF-R, 40 ng/50
EPHB4, 200 ng/50 µL ERBB2, 10 ng/50
µ
µ
µL
µ
µ
µ
50 ng/50 µL VEGF-R2, 100 ng/50
Tyr)4:1 for 200 ng/50 L TIE2; 500 ng/50
25 ng/50 µL PAK4, 25 ng/50
µ
L VEGF-R3, 200 ng/50
µ
µ
µ
µ
µ
µ
µ
µL
µ
µ
µ
PDGFR-beta, 100 ng/50
MET, 50 ng/50 L MET, 100 ng/50
alpha1, 200 ng/50
CK2-alpha1; 1000 ng/50
for 150 ng/50 L CK2-alpha1, 200 ng/50
µ
L PDGFR-beta, 100 ng/50
µ
L FLT3, 150 ng/50
µ
µ
µL MET; 250 ng/50
µ
µ
µ
L PLK1; 1000 ng/50
µL Casein for 200 ng/50 µ
µLGSK3 (14–27) for 100 ng/50
µ
µ
µ
µ
µ
20 ng/50
µ
L B-RAF-VE; autophosphorylation at ARK5, COT and SAK. After incubation
◦
(80 min, 30 C) the reaction was stopped by adding 50
µ
L 2% (v/v) phosphoric acid to
L 0.9% (v/v) saturated
sodium chloride or water. The measured radioactivity, conditioned by the incorporation of
-33P]-phosphate, is proportional to kinase activity. Furthermore, low control and high
the reaction mixture. The microplate was washed twice with 200
µ
[
γ
control were measured to determine the residual activity (%). As low controls, mixtures
with substrate, but without enzyme were used to determine the unspecific binding of
[γ
-33P]-phosphate to the well plate. Mixtures with enzyme, but without inhibitor were
used as high controls to determine maximum enzyme activity. The IC50-values of the
distinct compounds were calculated with Quattro Workflow V1.1.0.8 (Quattro Research
GmbH, München, Germany). Dose response curves were generated by measurement of the
residual activity of ten different inhibitor concentrations (0.1 mM, 30.0 µM, 10 µM, 3.0 µM,
1.0
µM, 0.3
µM, 0.1
µM, 30.0 nM, 10.0 nM and 3.0 nM). The residual activity was calculated
using Equation (1):
(cpm compound − low control)
residual activity (%)= 100×
(1)
(high control − low control)
4.6. Kinetic Solubility
The kinetic solubility of test compounds was determined by nephelometry. Dilution
series of test compounds in DMSO with eight to ten different concentrations were prepared.
The dilutions were pipetted (2 µL) into the cavities of a 96 well plate (CorningTM UV-

Molecules 2021, 26, 1611
23 of 26
transparent flat-bottom microtiter plate; Corning Incorporated, Kennebunk, ME, USA). A
phosphate buffer solution pH 7.4 (198 µL) was added, so that the DMSO concentration in
the measured solutions was 1%. Three wells were filled with DMSO and phosphate buffer
pH 7.4 (2:198) and measured as blank values. The 96 well plate prepared in this way was
measured with the NEPHELOstar® Plus nephelomete◦r (BMG Labtech GmbH, Ortenberg,
Germany, software: Omega 5.11) at a temperature of 25 C. Previously, the plate was shaken
at 500 rpm for 10 s and the laser intensity was set to 80%. The beam focus was set to 2.50 mm.
If the test substance is completely soluble, only a scattering close to the blank value is
detected. As soon as a critical concentration is reached in which the kinetic solubility of
the test compound is exceeded, solid particles precipitate. Undissolved particles in the
sample scatter the laser beam. The intensity of the scattered light is determined, which is
proportional to the number of particles in the solution. The blank values were determined
and subtracted from the measured values of the test compounds. By measuring different
concentrations of the test substances, different intensities of the scattered light, indicated as
RNU (relative nephelometric units), were determined. Kick off curves were obtained by
plotting concentrations against the RNU values. The point of intersection of the horizontal
and of the ascending part of the kick-off curve indicated the kinetic solubility concentration.
5. Conclusions
The new chemotype 2 was created from the dual PLK1/VEGF-R2 inhibitor 1 by the
“cut and glue” method, i.e., formal cutting the structure and joining at different molecular
locations. The syntheses of the new structures were performed by standard methods,
a key step being the reaction of an enaminone with aromatic guanidium nitrates. The
prototype 2a turned out to be an inhibitor of the protein kinases Aurora A, VEGF-R2
and VEGF-R3. Based on the results of X-ray structure analyses and docking experiments,
2a was chemically modified by attachment of substituents to the aniline ring. The most
potent of the resulting compounds exhibited inhibition of the Aurora A kinase in the
single-digit micromolar concentration range. The additional attachment of sulfamide
structures via propoxy linkers led to analogues with retained protein kinase inhibitory
activity, but reduced the solubility. Two representatives of the new substance class showed
very interesting antiproliferative activity in the NCI in vitro cell line screening, justifying a
further structural optimization of the new chemotype. A further optimization based on the
X-ray structures presented here should focus on modifications of the molecular regions
that potentially form additional interactions in the center of the binding pocket.
Supplementary Materials: The following are available online: Synthesis of 10, syntheses of N-
arylguanidinium nitrates, syntheses of N-alkylated sulfamides, Figure S1: IR spectrum of 14a
,
Figure S2: APCI-MS spectrum of 14a, Figure S3: 1H-NMR spectrum of 14a, Figure S4: 13C-NMR
spectrum of 14a, Figure S5: HPLC chromatogram of 14a–gradient method, Figure S6: HPLC chro-
matogram of 14a–isocratic method.
Author Contributions: Conceptualization, C.K.; formal analysis, M.K., A.C., B.B. and F.T.; inves-
tigation, M.K., A.C., B.B. and F.T.; data curation, M.K., A.C., B.B. and F.T.; writing—original draft
preparation, M.K., A.C. and C.K.; writing—review and editing, M.H.G.K. and S.K.; visualization,
M.K.; supervision, C.K.; project administration, M.H.G.K. and C.K.; funding acquisition, S.K. and
C.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the European Commission, Contract No LSHB-CT-2004-503467
(to B.B. and C.K.). We acknowledge support by the German Research Foundation and the Open Access
Publication Funds of Technische Universität Braunschweig. S.K. and A.C. acknowledge support by
the SGC, a registered charity (no: 1097737) that receives funds from; AbbVie, Bayer AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome
Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond,
Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant 875510), Janssen, Merck KGaA
(aka EMD in Canada and US), Merck & Co (aka MSD outside Canada and US), Pfizer, São Paulo
Research Foundation-FAPESP, Takeda and Wellcome. S.K. is grateful for support from the German
translational Cancer Network DKTK and the Frankfurt Cancer Institute (FCI).

Molecules 2021, 26, 1611
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Data Availability Statement: Structure data of 2a and 2c in complex with Aurora A kinase were de-
posited at the protein data bank (http://www.rcsb.org) under code numbers 7AYI and 7AYH, respectively.
Acknowledgments: The authors thank staff at Swiss Light Source for their assistance during crystal-
lographic data collection.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are not available from the authors.
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