Bioisosteric Replacement of Arylamide-Linked Spine Residues with N-Acylhydrazones and Selenophenes as a Design Strategy to Novel Dibenzosuberone Derivatives as Type i 1/2 p38α MAP Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00508

The recent disclosure of type I 1/2 inhibitors for p38α MAPK demonstrated how the stabilization of the R-spine can be used as a strategy to greatly increase the target residence time (TRT) of inhibitors. Herein, for the first time, we describe N-acylhydrazone and selenophene residues as spine motifs, yielding metabolically stable inhibitors with high potency on enzymatic, NanoBRET, and whole blood assays, improved metabolic stability, and prolonged TRT.

Full text of DOI:10.1021/acs.jmedchem.0c00508

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Article
Bioisosteric Replacement of Arylamide-linked Spine Residues by
N-Acylhydrazones and Selenophenes as Design Strategy to Novel
Dibenzosuberone Derivatives as Type I½ p38# MAP Kinase Inhibitors
Júlia Galvez Bulhões Pedreira, Philipp Nahidino, Mark Kudolo, Tatu Pantsar,
Benedict-Tilman Berger, Michael Forster, Stefan Knapp, Stefan A. Laufer, and Eliezer J. Barreiro
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00508 • Publication Date (Web): 28 May 2020
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Journal of Medicinal Chemistry
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Bioisosteric Replacement of Arylamide-linked Spine Residues by N-
Acylhydrazones and Selenophenes as Design Strategy to Novel
Dibenzosuberone Derivatives as Type I½ p38 MAP Kinase
Inhibitors
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Júlia G. B. Pedreiraa,b, Philipp Nahidinoc, Mark Kudoloc, Tatu Pantsar,c,d, Benedict-Tilman Berger,e,f,
Michael Forsterc, Stefan Knapp,e,f, Stefan Lauferc,g,h, Eliezer J. Barreiroa,b
aLaboratory of Evaluation and Synthesis of Bioactive Substances (LASSBio), Federal University of Rio de Janeiro (UFRJ),
21944-971, Rio de Janeiro, RJ, Brazil.
bGraduate Program of Chemistry (PGQu), Chemistry Institute, UFRJ, 21941-909, Rio de Janeiro, RJ, Brazil.
cDepartment of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls Universität,
Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
dSchool of Pharmacy, University of Eastern Finland, Yliopistonranta 1, 70210 Kuopio, Finland.
eInstitute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am
Main, Germany
fStructural Genomics Consortium (SGC), Buchman Institute for Life Sciences, Johann Wolfgang Goethe-University, Max-
von-Laue-Str. 15, D-60438 Frankfurt am Main, Germany
gCluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of
Tübingen, 72076 Tübingen, Germany
hTübingen Center for Academic Drug Discovery & Development (TüCAD2), 72076 Tübingen, Germany
ABSTRACT: The recent disclosure of type I½ inhibitors for p38 MAPK demonstrated how the stabilization of the R-spine can be
used as a strategy to greatly increase the target residence time (TRT) of inhibitors. Herein, we describe first time N-acylhydrazone
and selenophene residues as spine-motives, yielding metabolically stable inhibitors with high potency on enzymatic, NanoBRET and
whole blood assays, improved metabolic stability and prolonged TRT.
Skepinone-L (1, Figure 1A) was one of the first highly
selective p38 inhibitors developed. It is very potent,
displaying an IC50 of 5 nM and no relevant off-target activity
up to 1 µM within a panel of 402 kinases.8,9 It is ATP-
competitive, and classified as type I inhibitor. The crystal
structure of 1 in complex with p38 reveals an unique hinge-
INTRODUCTION
Almost 20 years ago, with the elucidation of the kinome,
protein kinases have become one of the most actively studied
pharmacological targets.1 The potential for modulating the
often increased activity of such proteins in the treatment of
various pathologies has become increasingly attractive as a
new therapeutic approach. 2,3 One of the most studied protein
kinases is the p38 Mitogen-activated protein kinase (MAPK).
This protein is activated upon cellular stress and it regulates
biosynthesis of various proinflammatory cytokines. Of the
four isoforms of p38 (, ,  and ), the p38 is the only
isoform that is ubiquitously expressed in all tissues and has
been associated with tumor development upon deregulation of
expression levels. Therefore, p38 is considered an attractive
target for cancer treatment.4 Eli Lilly’s compound,
LY2228820, is the most recent example to reach phase II
clinical trials for treatment of ovarian cancer.5 Despite major
efforts to target this protein, still no p38 MAPK inhibitors
have been approved by the FDA, which is most likely due to
off-target toxicity and lack of efficacy in vivo.6,7
interaction via
a
so called glycine flip with the
dibenzosuberone scaffold (PDB ID: 3QUE).8 The induction
of this glycine flip enables the great improvement of
selectivity compared to other p38 MAPK inhibitors. 10,11
Many efforts were made to subsequently develop optimized
inhibitors from 1, which led to the identification of
compounds 2 and 3 (Figure 1B-D). Both also displayed an
exceptional potency in enzymatic assays (IC50 < 3 nM),
together with an outstanding selectivity within a broad panel
of kinases.12–14 These compounds, although initially designed
as type II inhibitors, showed an atypical binding mode in the
p38 MAPK. The crystal structure (PDB ID: 5TCO) revealed
an edge-to-face interaction between the aromatic residues of
the eastern amide and Phe169 of the DFG motif (Figure 1C),
inducing a DFG-in conformation typically seen with type I
inhibitors. This interaction involves an important regulatory
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structural element, the regulatory spine (R-spine), which is to advance through the later development stages and maintain
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assembled upon kinase activation.15 This spine is formed by
Leu86, Leu75, Phe169 and His148. Thus, compounds 2 and 3
appeared to stabilize the R-spine and induce an active-like
conformation of the kinase. For their capacity to combine type
I and II interactions, these inhibitors were named as type I½.
efficacy in vivo.
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The combination of all the distinctive interaction
opportunities in the p38 MAPK is a promising and
interesting feature for the development of new inhibitors that
are eligible to proceed in the drug development process, since
it may provide improved selectivity and increased efficacy in
vitro and in vivo. In this work, we focus on the design of new
dibenzosuberone compounds by a bioisosteric replacement at
the R-spine interacting residue, with the aim to maintain the
excellent potency of the underlying compound class, together
with improved target residence time and metabolic stability.
In earlier studies we demonstrated that these interactions
and stabilization of the R-spine greatly influences the
inhibitor’s target residence time (TRT).14 While 1 has a
relatively short TRT of 88 s, inhibitor 2 has a slightly
prolonged TRT of 184s and finally 3 shows a substantial
increase to 746 s. The latter represents a difference of one
order of magnitude compared to 1, and this improvement is
directly related to the R-spine stabilization. The increase in
TRT observed from 2 to 3 was attributed to the significantly
higher electron density of the thiophene compared to phenyl
residue, which resulted in a stronger interaction with the
Phe169 residue. Optimization of the TRT evolved to a
promising design concept in medicinal chemistry over the last
years, since it is supposed to bring along several advantages
such as improved PK, increased in vivo efficacy, and reduced
adverse effects.16 Compounds 2 and 3, however, suffer from
metabolic instability. In vitro studies demonstrated that these
compounds undergo metabolic degradation, especially via
eastern amide hydrolysis, resulting in a loss of the R-spine
interacting residue. This is a challenge to overcome in order
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Structure Based Design. With the lead structures 2 and 3
as starting points, our initial design approach was to introduce
a new and metabolically stable functional group to replace the
amide, that could also be suitable for interactions with the R-
spine (Figure 1D). As the dibenzosuberone scaffold is crucial
to induce the glycine flip in the hinge, we kept it unchanged
as a main building block. We also opted to maintain the
morpholinoethyl-amide substitution to occupy HRII, since it
was known from previous SAR studies that it displays a well-
balanced profile acting as a solubilizing group.13
As an alternative for the eastern amide, we proposed the N-
acylhydrazone (NAH) group combined with the same set of
R-spine interacting residues as prototypes 2 and 3, resulting
Figure 1. (A) Chemical structure of Skepinone-L (1). (B) Chemical structure of 2. Type I ½ inhibitors contain an additional R-spine
interacting moiety. (C) Crystal structure of compound 2 in complex with p38 MAPK (PDB ID: 5TCO). Highlighted are the R-spine
(orange), DFG residues Asp168 and Gly170 (red) and dibenzosuberone interacting residues Met109 and Gly110 from the hinge region (blue),
notice the glycine-flip of Gly110. The type I½ R-spine interacting moiety displays an edge-to-face - interaction with Phe169. The amide
forms H-bonds to the side chain of Glu71 and to the backbone of Asp168 of the DFG motif (D) Schematic representation of the structural
design of the new N-acylhydrazone derivatives 4a-b and selenophene isosters 4c and 5.
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in the corresponding inhibitors 4a and 4b (Figure 1D). This
bioisosteric replacement was proposed to be able to maintain
key interactions in the HRI, and also target the DFG motif for
the R-spine stabilization.
Biological evaluation. The N-acylhydrazone derivatives (4a
and 4b), along with the selenophene isosters (4c and 5), were
initially tested in an ADP-glo assay23 to determine inhibitory
activity for p38 MAPK (Table 1). In this initial screening, all
compounds displayed low nanomolar activity. Since the
measured IC50 values are ranging near the lower detection limit
of this assay system, it is difficult to derive proper structure-
activity relationships based on this data between these
substituents. Nonetheless, the inhibition profile primarily
demonstrates that introduction of the NAH moiety (compounds
4a-c) and the isosteric substitution in the aromatic system
maintains the potency for the enzyme inhibition, compared to
prototypes 2 and 3.
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Additionally, the amide replacement was supposed to be
advantageous for the metabolic profile, since NAHs have been
previously described as being more stable than amides, and do
17,18
not undergo cleavage as easily.
Finally, as a strategy to
explore specifically the R-spine interaction, we also designed
two additional compounds based on the isosteric substitution of
thiophene for selenophene (4c and 5). As the electronic density
of the selenophene ring is different from its O- or S-isomers, the
design of organo-selenium compounds has recently attracted
attention as a strategy to explore new type of interactions with
a target-protein.19–22 In the same way as the electronegativity
decreases within the group of chalcogenes (O >> S > Se), the
electronic density of the corresponding five-membered
heterocycles increases from furan to thiophene to selenophene.
In this regard, our hypothesis was that this is supposed to
positively affect the interaction with Phe169 and therefore
increase the TRT via an enhanced stabilization of the R-spine.
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Scheme 1: Synthetic route for preparation of aniline
intermediate 8 and 11a.
RESULTS AND DISCUSSION
aReagents and reaction conditions: (i) Boc2O, DMAPcat, TEA,
THF, r.t., 47%; (ii) H2, Pd/C, MeOH, r.t., 59%; (iii)
dimethoxypropane, TsOH, MeOH, r.t., 85%; (iv) H2, Pd/C, EtOAc,
r.t., 95%.
Chemistry. The synthesis of the new dibenzosuberone
derivatives started with the preparation of the aniline linkers, as
described in Scheme 1. Intermediate 8 was synthetized by bis
protection of the amino group in 2-fluoro-5-nitroaniline (6) with
di-tert-butyl dicarbonate (Boc2O) followed by reduction of the
nitro group in 7 with Pd/C under hydrogen atmosphere. For the
preparation of intermediate 11, 2-fluoro-5-nitrobenzaldehyde
(9) was acetal-protected in MeOH with dimethoxypropane,
followed by subsequent reduction of nitro group with hydrogen
on Pd/C.
After these promising results, the compounds were evaluated
in a cellular environment using a NanoBRET assay (Table 1).
Cellular evaluation is an important step to verify compound
permeability and whether they maintain the activity observed at
the isolated protein in a much more dynamic system where
cellular concentrations of the competitive substrate ATP are
present. Overall, a good agreement between the enzymatic and
the cellular data was proven with low nanomolar IC50 values,
demonstrating a good cell permeability and cellular target
engagement.
Both aniline derivatives 8 and 11 were coupled via a
Buchwald-Hartwig amination with key intermediate 12 (see
Supplementary)14 using standard cross-coupling conditions, to
afford either 13 or 14 (Scheme 2). For the preparation of the
NAHs, intermediate 13 was submitted to a one-pot deprotection
and condensation sequence with the corresponding hydrazides
28, 29 or 32 (SI) to afford the desired final compounds 4a-c.
Subsequently, we assessed the activity in a human whole-
blood TNF- assay (Table 1). This test system represents a
reliable model for more complex biological environments,
where factors such as solubility, cell-cell-interaction and others,
influence the compounds activity and efficacy.24 Within the
NAH series, the TNF- assay revealed a significant loss of
potency from phenyl (4a), to the thiophene (4b), and to the
selenophene (4c) substituent.
To prepare the amide 5, intermediate 14 was first deprotected
with TFA to yield the aniline 15 followed by standard amide
coupling via the acid chloride derived by the activation of the
carboxylic acid 31 (SI).
Scheme 2. Preparation of the dibenzosuberone derivatives 4a-c and 5a
aReagents and conditions: (i) 8 or 11, K2CO3, BrettPhos Pd G3, 1,4-dioxane, 90˚C, 87 – 93%; (ii) (1) H2SO4, EtOH, r.t., (2) ArN2H4, EtOH,
room temperature 75 – 95%; (iii) TFA, DCM, r.t., 54%; (iv) (1) 31, oxalyl chloride, DMF, THF, (2) acid chloride, Et3N, THF, 0˚C, 46%.
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Table 1: Biological data of the Dibenzosuberone derivatives.
3
4
5
6
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9
TNF [nM]c
47  13
R
ADP-Glo [nM]a
NanoBRET [nM]b
TRT [s]d
4a
4b
4c
5
19.7
6.3  1.1
830  146
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29.7
16
15.5  1.2
30.4  1.2
4.9  1.2
4.3  2.0
N.D.
276  66
490  168
101  35
38  1.1
43  0.8
1341  222
1013  146
1596  22e
184  37e
746f
24.6
4
2
3
12
aresults from an activity-based ADP-Glo assay (Promega); bresults from a cellular reporter-displacement NanoBRET
assay (Promega); cresults from a human whole blood TNF-α release assay; dresults from a time-resolved FRET assay; emeasurement from
duplicate; fsingle measurement; N.D. = not determined
This may be attributed to solubility issues or differences in
plasma protein binding, given that thiophene and selenophene
are more lipophilic when compared to phenyl. Nevertheless, the
NAH phenyl derivative 4a displays a good activity, with an IC50
value of 47 nM, which is comparable to its corresponding amide
analogue 2 with an IC50 value of 38 nM (determined from
previous studies).13 For the selenophene-substituted amide 5,
we also observed a 2-fold loss of potency when compared to
amide analogues 2 or 3, but again this is most probably
correlated with the higher lipophilicity as already discussed for
the NAHs.
Putative binding mode of N-acylhydrazones. To ascertain
the putative binding mode of the NAH-derived compounds,
which contain substantially longer type I½ R-spine interacting
moieties compared to previously reported inhibitors, we utilized
in silico methods (see details in SI). Perhaps unsurprisingly,
docking approaches with Glide and Induced fit docking (IFD)25,
which allows a limited movement of the protein, resulted only
in reasonable poses for derivatives with smaller residues than
the here shown aromatic rings (Figure S2). To evaluate if this
type of binding mode could be realistic with compounds that
are carrying considerably larger substituents, we conducted
extensive molecular dynamics (MD) simulations with 4a and
4b (aggregate of 21.6 s) starting from a similar binding
conformation as observed in the aforementioned docking
studies. Indeed, in all of the simulations with these bulkier R-
Figure 2: Putative binding mode of NAHs. (A) Representative
spine interacting NAHs we observed stable binding (Figure. 2;
snapshots from MD simulations of 4a (cyan) aligned with crystal
Figures. S3-9; Supplementary movies). The interaction to
structure of 2 (blue) (PDB ID: 5TCO). The main differences
Met109 of the hinge appears extremely stable throughout the
simulations (Figures S7 and S9). R-spine interacting moieties
displayed sufficiently stable interactions and contacts to Phe169
and Leu75. The iminic nitrogen atom in the NAH displays water
mediated interactions to Asp168 (or Lys53). In comparison to
the crystal
compared to compound 2 are zoomed in (B). The fluorophenyl
moiety orientation of 4a is shifted deeper into the protein (single
black arrow). Moreover, type I ½ moiety of 4a is oriented deeper
into the pocket and capable of adapting different conformations in
the R-spine interacting region (black arrows).
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structure of the corresponding shorter phenylamide
fragmentation during the analysis. Nevertheless, there was no
evidence of oxidation in the selenophene moiety.
1
2
3
4
containing compound 2, the NAHs display a shift in the binding
orientation of the middle fluorophenyl group deeper into the
protein in a way that the type I½ residue is able to accommodate
proper binding and interactions with the R-spine residues (Fig.
S3; Fig. S8; Supplementary movies).
5
Determination of Target Residence Time (TRT). The set
of biological assays confirmed that the conducted structural
modifications did not compromise the potency of the
dibenzosuberone derivatives. Additionally, MD simulations
suggested that the NAH moiety could fit into the active pocket
and possibly uphold the key interactions with the protein. In
order to better correlate the data and to investigate the impact
of the different aromatic residues in the overall biological
profile of our compounds, we determined the TRT on the p38α
MAPK using a TR-FRET-based method (Table 1).
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Figure 3. In vitro metabolism of selected compounds. Graphical
representation of analyte decrease over 180 minutes in incubation
with HLM.
Interestingly, the NAH moiety appears to provide a
significant increase in the TRT. Comparison between specific
aromatic residues shows, that there was an increase of almost
5-fold between 2 and 4a, and almost 2-fold between 3 and 4b.
However, the same is not observed for the selenophene pair 4c
and 5, as the NAH derivative displays slightly decreased TRT,
which is possibly correlated to conformational constraints of
this fragment due to intramolecular chalcogen interactions (see
Supplementary).26–28 Nonetheless, MD simulations suggest that
NAH compounds reach deeper into the hydrophobic area within
the R-spine region and display additional water mediated
interactions from the imine nitrogen atom. These additional
interactions may play a decisive role in the increased TRT.
CONCLUSION
We were able to synthetize new N-acylhydrazone derivatives
of the dibenzosuberone p38 MAPK inhibitor series, with low
nanomolar activity and good metabolic stability. From a series
of assays of increasing levels of biological complexity, we
identified compound 4a, that displayed good biological profile,
with metabolic stability in microsomal studies, combined with
an increased TRT compared to its prototype 2.
From the set of inhibitors, we demonstrated that isosteric
replacement of sulfur to selenium does not affect the potency of
inhibition at the enzyme. Moreover, based on TRT
measurements we observed a more than 2-fold increase of the
selenophene isoster 5 compared to its thiophene analogue 3,
suggesting that this new residue is able to further stabilize the
spine by a  interaction with the Phe169. This work
demonstrates that the isosteric replacement is a valid strategy to
explore a new chemical space in drug discovery that could
improve target interaction. We believe that the bioisosteric
approach described in this study could ultimately be an useful
tool for Medicinal Chemistry research.
Also, in agreement with our hypothesis, substitution in the
amide for the selenophene isoster (5) revealed a great increase
of TRT compared to its prototypes 2 and 3. This is most
probably due to an ability of the selenophene ring to make a
stronger  interaction with the Phe169 residue
In vitro metabolism. Considering the good overall biological
profile obtained for derivative 4a, combined with a prolonged
TRT, we selected this compound to be evaluated for its
metabolic stability. Additionally, we included compound 5 to
obtain a more detailed insight into the biotransformation profile
of this isoster. There are only sparse descriptions of metabolic
stability of seleno-aromatic compounds, but from comparison
to thiophene, it may be expected to proceed via oxidation of the
ring, which is known to have great implications on the toxicity
of thiophene-containing bioactive compounds.29 Selenium,
however, has a lower redox potential, so that oxidation reactions
in a biological system could differ substantially when compared
from sulfur.30,31 To investigate the metabolic stability of the
chosen compounds, the experiments were performed with
human liver microsomes (HLM).
EXPERIMENTAL SECTION
Chemistry. All starting materials, reagents and (anhydrous)
solvents were commercially available and were used without further
purification. 1H and 13C NMR spectra were obtained with Bruker
Avance 200 or Bruker Avance 400 spectrometer. The spectra were
calibrated against the residual proton peak of the used deuterated
solvent. Chemical shifts () are reported in parts per million. Mass
spectra were obtained by Advion DC-MS (ESI) and from the MASS
Spectrometry Department (FAB-MS; ESI-HRMS), Institute of Organic
Chemistry, Eberhard-Karls-Universität Tübingen. HPLC analysis was
carried out on an Agilent 1100 Series LC with Phenomenex Luna C8
column (150 x 4.6 mm, 5 µm) and detection was performed with a UV
DAD at 254 nm and 230 nm wavelength. Elution was carried out with
the following gradient: 0.01 M KH2PO4, pH 2.30 (solvent A), MeOH
(solvent B), 40 % B to 85 % B in 8 min, 85 % B for 5 min, 85 % to
40 % B in 1 min, 40 % B for 2 min, stop time 16 min, flow 1.5 ml/min.
Thin-layer-chromatography (TLC) analyses were performed on
fluorescent silica gel 60 F254 plates (Merck) and visualized under UV
illumination at 254 and 366 nm. Column chromatography was
performed on Davisil LC60A 20−45 μm silica from Grace Davison and
Geduran Si60 63−200 μm silica from Merck for the precolumn using
an Interchim PuriFlash 430 automated flash chromatography system.
All synthesized final compounds had purity of at least 95% as
determined by HPLC at two different wavelengths (230 nm / 254 nm).
After 180 minutes of incubation, there was no significant
formation of metabolites from the NAH derivative 4a (Figure
3). This corroborates with the hypothesis that the NAH
functional group is more stable than an amide and appears not
to undergo hydrolysis in this model of the first phase of
metabolism. For compound 5 we observed a more prominent
degradation with only 43% of the parent compound remaining
after the full incubation time. The major metabolite could be
identified with m/z of 489. This equals the mass of the amide
hydrolysis metabolite, as previously observed for compounds 2
and 3. Other species formed from incubation could not be
identified as common metabolites, and might originate from
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The representative preparation of compound 4a is given here. All
other final compounds were synthesized following similar procedures
and detailed descriptions and analytic can be found in the SI.
ASSOCIATED CONTENT
Supporting Information
1
2
3
4
5
6
7
8
9
2-(Dimethoxymethyl)-1-fluoro-4-nitrobenzene (10) A volume of 545
µL of 2,2-dimethoxypropane (4.44 mmol, 1.5 eq.) and a catalytic
amount of p-toluenesulfonic acid (0.05 g, 0.29 mmol, 0.1 eq.) were
added to a solution of the aldehyde (9) (0.5 g, 2.95 mmol, 1 eq.) in
absolute methanol (10 mL) and stirred overnight at room temperature.
The reaction mixture was then poured into 30 mL of saturated K2CO3
aqueous solution and extracted using dichloromethane. The organic
extracts were dried using Na2SO4, filtrated and concentrated under
reduced pressure. The crude product was purified on flash
chromatography (0-30% EtOAc/PE) to obtain 10 as a transparent oil.
Yield: 545 mg (2.53 mmol, 85%); 1H NMR (400 MHz, CDCl3): δ 8.51
(dd, 1H), 8.25 (m, 1H), 7.21 (t, 1H), 5.63 (s, 1H), 3.39 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ (ppm): 162.5, 144.3, 127.4, 126.2, 124.9,
116.7, 97.5, 53.8.
Additional experimental details for preparation of the compounds;
1H NMR, 13C NMR, HR-MS, HPLC molecular modelling, MD
simulations, and biological assays. The supplementary movies and
full-length raw trajectories are freely available at
https://doi.org/10.5281/zenodo.3696749.
strings (CSV) file available.
Molecular
formula
AUTHOR INFORMATION
Corresponding Author
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Dr. Stefan Laufer: Phone + 49 7071 2972459 Email:
stefan.laufer@uni-tuebingen.de
Dr. Eliezer J. Barreiro: Phone +55 21 25626644 E-mail addresses:
ejbarreiro@ccsdecania.ufrj.br, ejbarreiro@gmail.com Both
corresponding authors contributed equally (shared)
3-(Dimethoxymethyl)-4-fluoroaniline (11) Intermediate 10 (0.54 g,
2.53 mmol) was dissolved in EtOAc (10 mL) and 10 wt % of palladium
on activated carbon was added. The reaction was stirred at room
temperature for 4 hours under hydrogen atmosphere. Afterwards, the
reaction mixture was filtered over Celite® and the solvent removed
under pressure to afford 11 as a brown oil. Yield 465 mg (2.51 mmol,
Funding Sources
The authors would like to thank CAPES/INCT-INOFAR (Project
No 88887.3182253/2019-00), DAAD (Project. No. 5749963) and
the Baden-Württembergisches Brasilien-Zentrum from the
University of Tübingen for funding of this project. S.L. and iFIT
are funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) under Germany's Excellence
Strategy - EXC 2180 – 390900677. TüCAD2 is funded by the
Federal Ministry of Education and Research (BMBF) and the
Baden-Württemberg Ministry of Science as part of the Excellence
Strategy of the German Federal and State Governments. T.P.
acknowledges the Orion Research Foundation sr for financial
support and funding from the European Union’s Horizon 2020
research and innovation programme under the Marie Sklodowska-
Curie grant agreement No 839230. SK and B-TB are grateful for
support by the German cancer network DKTK, the Frankfurt
Cancer Centre (FCI) as well as the SGC, a registered charity that
receives funds from AbbVie, Bayer Pharma AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman Institute
for Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA), Janssen, Merck KGaA Darmstadt Germany, MSD,
Novartis Pharma AG, Ontario Ministry of Economic
1
98%); H NMR (400 MHz, CDCl3): δ 6.87–6.82 (m, 2H), 6.62–6.58
(m, 1H), 5.53 (s, 1H), 3.56 (s, 2H), 3.38 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ (ppm): 168.4, 142.5, 125.0, 116.5, 115.9, 114.0, 98.8, 53.8.
8-((3-(Dimethoxymethyl)-4-fluorophenyl)amino)-N-(2-
morpholinoethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-
3-carboxamide (13) The dibenzosuberone intermediate (12, 0.5 g, 1.25
mmol, 1 eq.), aniline 11 (278 mg, 1.50 mmol, 1.2 eq.) and K2CO3 (519
mg, 3.76 mmol, 3 eq.), were suspended in dry dioxane (10 mL) in a
Schlenck flask. The mixture was degassed and backfilled with argon
three times. BrettPhos Pd G3 precatalyst (45 mg, 0.05 mmol, 0.04 eq)
was added and the degassing procedure was repeated. The reaction was
heated to 90˚C and stirred for 4h. After cooling, the reaction was
quenched with a saturated solution of NH4Cl and then extracted with
DCM. The organic layer was dried over Na2SO4 and evaporated in a
rotary evaporator. The crude product was purified using flash
chromatography (0 - 8% MeOH/DCM) to obtain 13 as a yellow solid.
Yield 600 mg (1.09 mmol, 87%); 1H NMR (400 MHz, CDCl3) δ 8.13
(d, 1H), 7.93 (dd, 1H), 7.69 (dd, 1H), 7.31-7.26 (m, 3H), 7.06-7.04 (m,
1H), 6.93 (t, 1H), 6.88 (bs, 1H), 6.78-6.74 (m, 1H), 5.53 (s, 1H), 5.43
(s, 1H), 3.74 (t, 4H), 3.56 (q, 2H), 3.36 (s, 6H), 3.21-3.09 (m, 4H), 2.62
(bs, 2H), 2.60 (bs, 4H). 13C NMR (100 MHz, CDCl3) δ 196.4, 166.6,
154.1, 144.4, 141.3, 140.5, 140.4, 140.2, 139.6, 133.5, 133.3, 131.3,
129.5, 127.5, 127.4, 125.3, 125.1, 119.0, 117.0, 116.4, 98.8, 67.0, 57.0,
36.3, 33.6, 28.8.
Development and Innovation, Pfizer, São Paulo Research
Foundation-FAPESP, Takeda, and the Wellcome Trust.
ACKNOWLEDGMENT
8-((3-((2-Benzoylhydrazineylidene)methyl)-4-fluorophenyl)amino)-
N-(2-morpholinoethyl)-5-oxo-10,11-dihydro-5H-
We thank Jens Strobach and Katharina Bauer for biological testing.
The authors wish to acknowledge CSC – IT Center for Science,
Finland, for computational resources.
dibenzo[a,d][7]annulene-3-carboxamide (4a). The acetal intermediate
(13, 0.1 g, 0.18 mmol) was solubilized in EtOH (5 mL) and a 250 µL
of H2SO4 was added. The reaction mixture was stirred at room
temperature for 30 min, and then the corresponding hydrazide was
added (25 mg, 1 eq.). Stirring was maintained at room temperature for
4 h. The solvent was then removed under pressure and the product was
precipitated by addition of water in an ice bath. The precipitate was
filtrated under vacuum and crude product was purified using flash
chromatography (0 - 5% MeOH/DCM) to afford the NAH compound
4a at 89% yield as a yellow solid (0.1 g). 1H NMR (400 MHz, CDCl3)
δ 10.79 (s, 1H), 8.50 (s, 1H), 8.32 (s, 1H), 8.03 (d, 1H), 7.96 (d, 2H),
7.88 (d, 1H), 7.59 (bs, 1H), 7.54–7.50 (m, 1H), 7.42 (t, 2H), 7.29 (s,
1H), 7.17-7.15 (m, 2H), 6.90 (t, 1H), 6.81 (d, 1H), 6.73 (s, 1H), 6.63 (s,
1H), 3.75-3.73 (t, 4H), 3.61-3.59 (q, 2H), 2.99-2.96 (m, 4H), 2.69-2.67
(m, 2H), 2.66-2.57 (bs, 4H).13C NMR (100 MHz, CDCl3) δ: 191.3,
167.2, 165.1, 156.3, 148.5, 145.7, 145.3, 141.4, 140.2, 139.9, 139.2,
137.2, 134.3, 133.0, 132.8, 132.5, 131.2, 129.3, 129.0, 128.8, 127.8,
124.3, 119.4, 116.6, 114.9, 113.0, 66.8, 57.3, 53.5, 36.4, 35.93, 34.7.
HPLC: 6.127 min (96,05 %). ESI-HRMS [M+H]+ calculated:
620.26676, found: 620,266687.
ABBREVIATIONS
ATP, adenosine triphosphate; R-spine, regulatory spine; TRT,
target residence time; NAH, N-acylhydrazone; MAP, mitogen
activated protein; TNF, tumor necrosis factor; HLM, human liver
microsome; MD, molecular dynamics; HR, hydrophobic region.
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