Design and synthesis of novel fluorescently labeled analogs of vemurafenib targeting MKK4

Article abstract of DOI:10.1016/j.ejmech.2020.112901

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

Full text of DOI:10.1016/j.ejmech.2020.112901

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of novel fluorescently labeled analogs of
vemurafenib targeting MKK4
,
Theresa Kircher a, Tatu Pantsar a b, Andreas Oder c, Jens Peter von Kries c,
Michael Juchum a, Bent Pfaffenrot a, Philip Kloevekorn a, Wolfgang Albrecht d,
,
, e, *
Roland Selig a d, Stefan Laufer a
a Institute of Pharmaceutical Chemistry, Eberhard Karls University of Tuebingen, Morgenstelle 8, 72076, Tuebingen, Germany
b School of Pharmacy, University of Eastern Finland, Yliopistonranta 1C, 70210 Kuopio, Finland
c
€
Leibniz-Forschungsinstitut Fuer Molekulare Pharmakologie, FMP, Robert-Rossle-Straße 10, 13125, Berlin, Germany
d HepaRegeniX GmbH, Eisenbahnstraße 63, 72072, Tuebingen, Germany
e Tuebingen Center for Academic Drug Discovery, Morgenstelle 8, 72076, Tuebingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under
investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small
molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver
diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large
compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor
vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent com-
pound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-
TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suit-
able tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of
MKK4.
Received 21 July 2020
Received in revised form
18 September 2020
Accepted 29 September 2020
Available online xxx
Keywords:
Mitogen-activated protein kinase kinase 4
Vemurafenib
Fluorescence polarization assay
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
(Fig. 1) was shown to have strong off-target activity on MKK4 [3].
One method to find new lead structures for inhibiting kinases is
The Mitogen-Activated Protein Kinase Kinase 4 (MKK4), a
member of the Mitogen-activated protein kinase family, plays an
important role in the regulation of cell signaling caused by cellular
stress or inflammatory cytokines. In 2013, Wüstefeld et al.
demonstrated that silencing of MKK4 in mice via shRNA is sub-
stantially involved in liver regeneration and stimulates hepatocyte
proliferation [1]. As a result, by this genetic silencing of MKK4,
MKK7 and apoptosis signal resulting kinase 1 (ASK1) become
upregulated. This leads to a higher phosphorylation of c-Jun N-
terminal kinase (JNK1) and finally to enhanced activation of the
transcription factors activating transcription factor (ATF2) and ETS-
like transcription factor (ELK1), which are responsible for cell dif-
ferentiation and increased hepatocyte proliferation [2]. At the same
time, FDA-approved fibrosarcoma B (BRAF) inhibitor vemurafenib 1
a fluorescence polarization (FP)-based assay [4]. FP provides fast
and detailed information about inhibitor-protein interaction and
can be adopted for high-throughput screenings of compound li-
braries using competition assays [5]. The method can distinguish
between bound and unbound compounds by analyzing polarized
light emission after excitation [6]. Earlier, 1 has successfully been
tagged with the fluorophore BODIPY to enable in vivo cell imaging
[7].
Finding new small molecule inhibitors of MKK4 may represent a
starting point for the treatment of acute and chronic liver diseases.
To this end, a MKK4-targeting fluorescent could be used as a tracer
for fluorescence-based competition assay systems against com-
pound libraries. Here, we generated a series of compounds based
on 1, with varying linkers combined with a fluorophore. These ef-
forts resulted in a MKK4-specific probe that maintains high binding
affinity and meets the requirements of a tracer compound for high-
throughput fluorescence polarization assays.
* Corresponding author. Auf der Morgenstelle 8, 72076, Tuebingen, Germany.
E-mail address: stefan.laufer@uni-tuebingen.de (S. Laufer).
https://doi.org/10.1016/j.ejmech.2020.112901
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: T. Kircher, T. Pantsar, A. Oder et al., Design and synthesis of novel fluorescently labeled analogs of vemurafenib
targeting MKK4, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112901

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
2. Ligand design
In combination with the docking results, the structural insights
suggested that it might be beneficial to position the bulky fluo-
rophore 5-TAMRA in the adjacent cleft outside the binding pocket
of MKK4 (Fig. 2C). The positively charged lysine residue, K118,
would be suitable for an ionic interaction with the negatively
charged carboxylate group in the fluorophore (Figs. 2C and 1),
fixating the ligand molecule. An optimal position for this group
would be ensured by the connecting linker system with up to six
carbons.
We selected the 5-Carboxytetramethylrhodamine single isomer
(5-TAMRA) as a fluorophore in our synthetic strategy. Rhodamine
derivatives possess high quantum yields, which can have a positive
effect on the sensitivity of the fluorescent screen. As a significant
number of compounds from screening libraries demonstrates a
Designing an appropriate ligand system with retaining high
binding affinity is an iterative process and has strong influence on
the fluorescence polarization assay results. At first, we investigated
the X-ray structure of 1 bound to BRAF (PDB ID: 4rzv, 3og7) [8]. The
azaindole core of 1 forms two hydrogen bonds to the hinge-region.
The para-chloro substituent of the phenyl protrudes out of the ATP
binding pocket towards the solvent in a solvent-accessible interface
(Fig. 2A). On the other side of the molecule, the sulfonamide interacts
with the DFG motif of BRAF forming a hydrogen bond between the
NH of D592 and the sulfonamide nitrogen, whereas the terminal
aliphatic propyl group occupies a lipophilic back pocket [9].
Three MKK4 structures have been published to date (PDB IDs:
3vut [10]; 3aln; 3alo [11]). Two of these structures are in complex
with adenylyl-imidodiphosphate and one is an apo structure, none
is in complex with inhibitors. In these structures, the lipophilic back
pocket is not observed. Thus, we were first unable to observe a
similar binding mode for 1 in MKK4 based on these structures.
However, our molecular dynamics (MD) simulations [12], revealed
the opening of this lipophilic back pocket. Docking of 1 to this MD-
derived MKK4 structure resulted in a similar binding mode as
observed with BRAFe1 (Fig. 2B). This binding orientation provided
us with a rational starting point for the ligand design of fluorescent
derivatives for MKK4.
Fig. 2. Binding mode of 1 to BRAF and MKK4 as a starting point for fluorescent ligand
design. (A) Crystal structure of 1 in complex with BRAF (PDB ID: 4rzv). (B) Docking
pose of 1 in MD derived MKK4 structure. Similar binding mode of 1 is observed with
MKK4 as with BRAF. The fluorophore occupies a cleft between the N- and C-lobe in
MKK4. (C) Docking pose of fluorescent compound 45 in MKK4. In AeC: residues that
show polar interactions (dashed yellow lines) with the ligand are labeled in the figures.
(For interpretation of the references to color in this figure legend, the reader is referred
to the Web version of this article.)
Fig. 1. Overview of the fluorophore attachment strategy. Structure of 1; scaffold with
X ¼ CH or N and R ¼ propyl or benzyl (highlighted in blue) attached by a linker with
n ¼ 1e6 (yellow) to the fluorophore 5-TAMRA (pink). (For interpretation of the ref-
erences to color in this figure legend, the reader is referred to the Web version of this
article.)
2

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
strong fluorescent emission similar to FITC (exc. 491 nm, em.
516 nm) at the used screening concentration of 10 mM, we used 5-
TAMRA (exc. 546 nm, em. 516 nm) for labelling which shows a shift
to the red fluorescence like rhodamine. Thereby we prevent inter-
ference in FP-readout by most fluorescent compounds.
In order to obtain the maximum polarization effect, the local
mobility should be kept as small as possible for the fluoroprobe
bound to the target protein. Therefore, it is mandatory to minimize
unfavorable repulsive interactions between the fluorophore and
the protein. A screening of differing linkers is necessary to identify
the best possible compromise between affinity and fluorophore
mobility. Thereby the aromatic moiety of the former para-chloro
residue serves as connection between linker and the scaffold of 1
which could be introduced via CeC-coupling. Using pyridinyl- and
pyrimidinyl-moiety instead of phenyl could help to reduce the
rotational movement, because this part of the molecule seems to be
close to the binding cleft.
The deprotection in TFA/toluene and amide coupling with 5-
TAMRA (single isomer) afforded the fluorescent compounds 8
and 9. The low solubility of the compounds in organic solvents and
the following column chromatography resulted in low yields.
To substitute the propyl residue with benzyl, the fluorescent
compounds 10 and 11 (Table 1) were prepared starting from (3-
amino-2,6-difluorophenyl)(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-
yl)methanone (detailed reaction path can be found in SI) [14].
In Scheme 1 the synthetic route to the linkage systems is
described. For the retro amide linkage with n ¼ 1 commercially
available 4-bromoaniline or 4-bromopyridine-2-amine were
coupled using uronium salts (HATU, HBTU) or CDI to yield 12 and
13. Retro amides have been used, as the corresponding amide with
n ¼ 1 is chemically not stable. For amide linkages with n ¼ 2e6
containing phenyl, pyridinyl and pyrimidinyl moieties the carbox-
ylic acid was activated with oxalyl chloride and connected to the
Boc protected diamines in moderate to good yields.
Besides the azaindole (1H-pyrrolo[2,3-b]pyridine) core of 1, we
Scheme 3 describes the synthesis of fluorescent
derivatives 37e47 containing scaffold B (Fig. 3) The synthesis of 3-
chloro- -carboline was prepared according to literature [15] The
a-carboline
used the
a-carboline (9H-pyrido[2,3-b]indole) scaffold B (Fig. 3)
which turned out to have a promising binding affinity to MKK4. The
pyrimido[4,5-b]indole scaffolds were preliminarily designed by
Bayer in 2003 as MKK4 and MKK7 inhibitors [13]. Due to the ge-
ometry of azaindole, we altered the structure to 9H-pyrido[2,3-b]
a
resulting chloro-substituted carboline was acylated (compound 23)
and further converted to the borylated species (compound 24) in a
Miyaura borylation reaction. The bromo derivatives of Boc-
protected linkages were coupled in a Suzuki coupling under mi-
crowave irradiation to obtain 25e36. After deprotection with TFA/
toluene the amines were linked in a HATU-mediated amide
coupling to 5-TAMRA. Low yields were isolated in some cases due
to difficult column chromatography.
indoles (a-carboline) to connect the linkers at the 3-position and
the keto-bridge at position 6 of the three-ring system. Based on our
docking results, compound 45 with this scaffold occupies the cleft
nicely and forms the desired ionic interaction to K118 (Fig. 2C).
Hypothesizing that the sulfonamide part of the molecule pro-
trudes into a lipophilic backpocket of MKK4 (see Fig. 2), the more
lipophilic residue benzyl was reported to further improve binding
affinity to the target and was therefore used additionally to the
propyl residue [13].
4. Results
The binding affinities of all compounds were characterized us-
ing a commercial binding assay (KINOMEscan by DiscoverX). The
binding affinity is described by POC (percentage of control) where
low numbers indicate high binding affinities (see Experimental).
Almost all of the 15 synthesized fluorescently labeled derivatives
show high affinity to MKK4 (Table 1), some even higher than 1
3. Chemistry
The synthetic route to fluorescent azaindole derivatives 8 and 9
(Scheme 2) starts with 5-bromo-7-azaindole as commercially
available starting material which was acylated with 2,6-difluoro-3-
(propylsulfonamido)benzoic acid under Friedel-Crafts conditions to
yield 2 described in literature [14]. For the glycine-based linkage
system, the resulting bromo-derivative was directly connected to
(tert-butoxycarbonyl)glycine under Suzuki conditions to 3 and then
converted into the Boc-protected linkage mediated by HATU
yielding 4. Due to low conversion, the synthetic strategy was
changed for all other linkage systems by protecting the azaindole
nitrogen of 2 with 2,6-dichlorobenzoyl chloride to afford 5. Com-
pound 5 was then borylated with B2pin2 using Miyaura conditions
to 6 and subsequently connected to the prepared linkage 18
(Scheme 1) to afford the corresponding Boc-protected precursor 7.
(POCMKK4 ¼ 14). Also, the scaffold change from azaindole to
a-car-
boline is tolerated for almost all compounds (37e47). Only two de-
rivatives (9,41) have low binding affinity to the target compared to 1.
Changing the sulfonamide residue of
1 from propyl (8,
POCMKK4 ¼ 11) to benzyl (10, POCMKK4 ¼ 6.6) slightly increases the
affinity to the target. This confirms the hypothesis of an accessible
lipophilic back pocket, just as in BRAF.
In order to identify the optimal linker length, we prepared
linkage systems containing one to six carbon atoms between the
fluorophore and the template. All lengths were tolerated, but for
the azaindole and carboline scaffold the binding mode appeared to
be different. Good binding affinity with a POCMKK4 of Good binding
affinity with a POC11 of 11 were obtained for the azaindole scaf-
fold, the shortest linker system (compound 33) with one carbon
and a phenylamide moiety. The carboline derivative 45 were
obtained for the azaindole scaffold, the shortest linker system
(compound 33) with one carbon and a phenylamide moiety. The
carboline derivative 45 (Fig. 4) was found to be the most promising,
with a linker length of four carbons and a picolinamide moiety,
showing a binding affinity of POCMKK4 ¼ 1.3. Presumably, the
additional ring system of scaffold B has an influence on the posi-
tioning of the former para-chloro residue.
Changing the phenyl moiety to pyridinyl, or pyrimidyl has
nearly no impact on the binding affinity.
Comparing Boc protected compound 25 with the corresponding
5-TAMRA attached 37 (Table 2) binding affinity slightly increases.
Thus, the incorporation of the bulky fluorophore 5-TAMRA has no
negative effect on the binding affinity with the chosen linkers.
Fig. 3. Structures of azaindole (A) and a-carboline (B) scaffolds used as hinge-binding
motives.
3

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of linkage systems 12e22.
aReaction conditions: (a) HATU or HBTU or CDI, Et3N or DIPEA, DMF or THF, RT, 17 h, 36e59%; (b) (i) (COCl)2, DCM, DMF, RT, 1 h; (ii) corresponding amine, DMF, 0 ꢀC, 3 h, 43% e
quant.
Scheme 2. Synthesis of compound 8 and 9 with 5-TAMRA attached to derivatives with azaindole scaffold A.
aReaction conditions: (a) AlCl3, 2,6-difluoro-3-(propylsulfonamido)benzoic acid, (COCl)2, DCM, 50 ꢀC, 85%; (b) 2,6-dichlorobenzoyl chloride, Et3N, DMAP, THF, 0 ꢀC, 17 h, 68%; (c)
B2pin2, KOAc, Pd(PPh3)2Cl2, 1,4-dioxane, 80 ꢀC, 2 h, 68%; (d) K2CO3, Pd(PPh3)4, 1,4-dioxane/H2O (1:1, v/v), 55 ꢀC, 72 h, 49%; (e) 5-TAMRA, HATU, DIPEA, DMF, rt, 17 h, 30e73%; (f) 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, K2CO3, Pd(PPh3)4, MW (50 W) 1,4-dioxane/H2O (1:1, v/v), 110 ꢀC, 40 min, 52%; (g) (tert-butoxycarbonyl) glycine, DIPEA, HATU,
DMF, rt, 17 h, 84%.
Surprisingly, the selectivity profile (Fig. 5) of compound 45 reveals
no binding to the off-target BRAF (POCBRAF ¼ 92) which is the target
for 1 that was used as a starting point for the ligand design. The su-
perimposition of the docking pose of 45 bound to MKK4 and the
crystalstructure of BRAFin complex with1 demonstrates that BRAFis
missing the cleft outside the binding pocket, which allows the
beneficial positioning of the 5-TAMRA moiety. The weak interaction
between 45 and BRAF can be explained by severe steric clashes,
especially related to BRAF residues D479, K473, W531 and E533.
Further investigations at lower assay concentrations on MKK4
(Table 3) showed that 45 reveals sustained high binding affinity.
To investigate whether compound 45 can be used to find new
small molecules in a fluorescence polarization assay, we validated
the interaction of known inhibitors 53e55 of MKK4 (see SI) in the
presence of 45 (Fig. 6) [14]. These compounds differ in their binding
affinity towards MKK4. The positive control 53 has a high binding
affinity to MKK4 with a POC of 0.25 at a concentration of 100 nM
and showed an EC50-value of 31 nM. The reference compound 54
has a weaker binding affinity to MKK4 (POCMKK4 ¼ 11) than 53 and
shows a decreased EC50-value of 164 nM. 55 was used as negative
control (POCMKK4 ¼ 47) and resulted in an EC50-value of 628 nM.
Using MKK4 at 64 nM and compound 45 at 20 nM in a competition
binding experiment, all known inhibitors showed EC50-values
fitting their binding properties and could be properly classified.
Hence, compound 45 is a promising candidate which can be
implemented for fluorescence polarization high-throughput-
4

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 3. Synthesis of compound 37e47 with 5-TAMRA attached to derivatives with a-carboline scaffold B.
aReaction conditions: (a) AlCl3, 2,6-difluoro-3-(propylsulfonamido)benzoic acid, (COCl)2, DCM, 50 ꢀC, 17 h, 69%; (b) B2pin2, KOAc, XPhos Pd G3, MW (50 W), 1,4-dioxane, 110 ꢀC,
40 min, 89%; (c) appropriate linkages 12e22, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O (1:1, v/v), MW (50 W), 110 ꢀC, 40 min, 24e65%; (d) (i) toluene/TFA (10:1, v/v), (ii) 5-TAMRA, HATU,
DIPEA, DMF, rt, 17 h, 20e65%.
screening to find new small molecule MKK4 inhibitors from large
compound libraries.
Fit docking [20e22] was conducted using default parameters with
extra precision (XP) accuracy.
The figures were generated with PyMOL (The PyMOL Molecular
€
5. Conclusion
Graphics System, Version 2.0 Schrodinger, LLC.).
Biological assays: All compounds were investigated by a com-
mercial binding assay by DiscoverX (Kinomescan™) at a screening
concentration of 100 nM, which uses an immobilised ligand
competing with the measured compound for the kinase. For
identification of compound 45 as a probe for HTS, a fluorescence
polarization assay with MKK4 and known inhibitors of the kinase
was established [23].
Materials: All reagents and (anhydrous) solvents are commer-
cially available and were used without further purification.
NMR: 1H and 13C NMR spectra were obtained with Bruker
Avance 200, Bruker Avance 400 or Bruker Avance 600. The spectra
were obtained in the indicated solvent and calibrated against the
In this study we report on the design of a fluorescent compound
addressing the molecular target MKK4 with high affinity. Installing
the bulky fluorophore 5-TAMRA and an appropriate linker at the
para-chloro side of 1 has no negative effect on binding and confirms
the assumption about the orientation of 1 bound to MKK4 based on
docking experiments. For targeting MKK4 with a fluorescent tool
compound, the linker system of 45 comprising four carbons ap-
pears the most promising. Moreover, changing the propyl residue
of 1 to more lipophilic residues increases the binding affinity. Also,
the replacement of the azaindole- to an
tolerated.
a-carboline-core is well-
Compound 45 was designed based on the information of the off-
target activity of 1 towards MKK4 and represents a potent fluo-
rescent ligand which can be used for a high-throughput screen to
find new chemical entities as promising MKK4 inhibitors. Addi-
tionally, the results obtained herein provide insight into the
possible binding mode of 1 to MKK4.
residual proton peak of the deuterated solvent. Chemical shifts (
are reported in parts per million.
Mass Spectrometry: Mass spectra were obtained by TLC-MS
(ESI) and from the Mass Spectrometry Department (HRMS), Insti-
d)
€
tute of Organic Chemistry, Eberhard Karls Universitat Tübingen.
TLC: Analyses were performed on fluorescent silica gel 60 F254
plates (Merck) and visualized under UV illumination at 254 and
366 nm.
6. Experimental
Column Chromatography. Column chromatography was per-
Molecular modelling: All the molecular modelling was con-
formed on Davisil LC60A 20e45
mm silica from Grace Davison and
€
ducted with Maestro (Schrodinger Release 2019-3: Maestro,
Geduran Si60 63e200 m silica from Merck for the precolumn
m
€
Schrodinger, LLC, New York, NY, 2019) with OPLS3 and OPLS3e force
using an Interchim PuriFlash 430 automated flash chromatography
system.
fields [16,17].
For the docking we used the Desmond [18] MD simulation-
derived structure of MKK4 (derived from PDB ID: 3alo) which
was prepared with Protein Preparation Wizard using default set-
tings [19]. Prior to the docking, the ligands were prepared with
HPLC: The purity of all compounds is, unless otherwise stated,
>95% and was determined via reverse-phase high-performance
liquid chromatography on Hewlett-Packard HP 1090 series II LC
equipped with a UV diode array detector (DAD, detection at 230
and 254 nm). The chromatographic separation was performed on a
€
LigPrep (Schrodinger, LLC, New York, NY, 2019). Finally, the Induced
5

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
7. Synthesis
Binding affinities of the synthesized fluorescent derivates on MKK4.
The representative preparation of compound 45 is given. All
other compounds were synthesized following similar procedures.
Detailed descriptions and analytics can be found in the Supporting
Information.
7.1. N-(3-(3-chloro-9H-pyrido[2,3-b]indole-6-carbonyl)-2,4-
difluorophenyl)propane-1-sulfonamide (23)
3-Chloro-9H-pyrido[2,3-b]indole (1.03 g, 5.08 mmol, 1 eq) and
AlCl3 (3.39 g, 25.41 mmol, 5 eq) were dissolved in DCM (50 mL), and
stirred at room temperature for 60 min. Meanwhile, 2,6-difluoro-3-
(propylsulfonamido)benzoic acid (2.13 g, 7.62 mmol, 1.5 eq) was
Compound
Scaffolda
R1
Linker
MKK4b
[POC]
c
n
R2
X, Y
suspended in DCM (20 mL) and oxalyl chloride (654
mL, 1.5 mmol,
8
9
A
A
A
A
B
B
B
B
B
B
B
Propyl
Propyl
Benzyl
Benzyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
1
4
1
4
1
2
3
4
5
6
1
1
2
2
2
1
2
2
2
2
2
1
-
-
-
-
11.0
57.0
6.6
13.0
13.0
19.0
13.0
4.3
1.5 eq) and DMF (500 L) were added dropwise. After the gas
m
evolution stopped, both mixtures were combined and stirred at
50 ꢀC until no starting material was left. The reaction was stopped
by adding MeOH and water at 0 ꢀC, the aqueous phase was then
extracted with ethyl acetate. The organic phase was concentrated
and the forming precipitate (1.53 g, 3.3 mmol, 65^%) was collected
and dried under vacuum conditions. 1H NMR (200 MHz, DMSO)
10
11
37
38
39
40
41
42
43
X, Y ¼ CH
X, Y ¼ CH
X, Y ¼ CH
X, Y ¼ CH
X, Y ¼ CH
X, Y ¼ CH
X ¼ N
Y ¼ CH
X ¼ N
Y ¼ CH
X ¼ N
Y ¼ CH
X ¼ N
Y ¼ CH
X, Y ¼ N
-
32.0
6.6
8.4
d
12.63 (s, 1H), 9.85 (s, 1H), 8.88 (d, J ¼ 1.8 Hz, 1H), 8.80 (s, 1H), 8.48
(d, J ¼ 2.0 Hz, 1H), 8.06 (d, J ¼ 8.6 Hz, 1H), 7.75e7.60 (m, 2H), 7.34 (t,
J ¼ 8.7 Hz, 1H), 3.21e3.11 (m, 2H), 1.85e1.61 (m, 2H), 0.93 (t,
44
45
46
B
B
B
Propyl
Propyl
Propyl
3
4
6
2
2
2
2
4.5
1.3
4.8
J ¼ 7.3 Hz, 3H) ppm. 13C NMR (50 MHz, DMSO)
d 187.1, 156.22 (dd,
J ¼ 246.8, 6.8 Hz), 152.38 (dd, J ¼ 250.0, 8.4 Hz), 151., 145.4, 143.8,
129.4, 129.2, 128.6, 128.3, 128.47 (d, J ¼ 7.5 Hz), 125.8, 123.1, 122.14
(dd, J ¼ 13.4, 3.7 Hz), 119.9, 117.52 (dd, J ¼ 23.9, 22.1 Hz), 116.8,
112.60 (dd, J ¼ 21.5, 2.5 Hz), 112.4, 53.5, 16.9, 12.6 ppm. ESI-MS (m/z)
462.1 [MꢂH]-.
47
1
B
A
Propyl
Propyl
4
-
9.3
14.0
a
Scaffold: (A) azaindole, (B) a-carboline.
Screening concentration 100 nM.
R2: 1 ¼ Retroamide, 2 ¼ Amide.
b
c
7.2. N-(2,4-difluoro-3-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-9H-pyrido[2,3-b]indole-6-carbonyl)phenyl)
propane-1-sulfonamide (24)
N-(3-(3-chloro-9H-pyrido[2,3-b]indole-6-carbonyl)-2,4-
difluorophenyl)propane-1-sulfonamide (514 mg, 1.1 mmol, 1 eq),
4,4,40,40,5,5,50,50-octamethyl-2,20-bi(1,3,2-dioxaborolane) (295 mg,
1.16 mmol, 1.1 eq) and KOAc (217 mg, 2.2 mmol, 2 eq) were sus-
pended in 1,4-dioxane/H2O (1:1, v/v, 4 mL) and purged with argon
for 15 min. Subsequently XPhos Pd G3 (29 mg, 33 mmol, 3 mol %)
was added and stirred under microwave irradiation (50 W, 115 ꢀC)
for 40 min. After TLC analysis revealed full consumption of the
starting material, ethyl acetate was added, and the organic layer
was washed with water and saturated sodium chloride solution.
The organic layer was dried over sodium sulfate and the solvent
was removed under vacuum conditions. After column chromatog-
raphy (DCM/EE, v/v, 0e50 %), the pure product could be obtained as
an off-white solid (550 mg, 990
DMSO) 12.54 (s, 1H), 9.78 (s, 1H), 8.96 (s, 1H), 8.87 (s, 1H), 8.71 (s,
m
mol, 89 %). 1H NMR (200 MHz,
d
Fig. 4. Structure of compound 45 (fluorophore highlighted in pink). (For interpretation
of the references to color in this figure legend, the reader is referred to the Web version
of this article.)
1H), 8.09 (d, J ¼ 9.0 Hz, 1H), 7.72e7.57 (m, 1H), 7.33 (t, J ¼ 8.7 Hz,
1H), 3.24e3.08 (m, 1H), 1.74 (dd, J ¼ 15.1, 7.5 Hz, 1H), 1.33 (s, 3H),
0.93 (t, J ¼ 7.3 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO)
d 187.6,
Phenomenex Luna 5u C8 column (150 mm ꢁ 4.6 mm, 5
oven temperature. The injection volume was 5 L, and the gradient
m
m) at 35 ꢀC
158.0, 157.9, 157.9, 156.69 (dd, J ¼ 246.7, 6.7 Hz), 155.5, 155.4, 155.4,
154.8, 154.1, 154.0, 153.4, 152.79 (dd, J ¼ 249.2, 8.0 Hz), 151.6, 151.5,
143.4, 136.5, 129.65 (d, J ¼ 9.3 Hz), 129.2, 127.8, 126.6, 122.40 (dd,
J ¼ 13.2, 3.2 Hz), 121.1, 118.00 (t, J ¼ 22.9 Hz), 115.7, 112.92 (dd,
J ¼ 22.7, 3.8 Hz), 112.6, 84.3, 53.9, 25.1, 17.1, 13.0 ppm. ESI-MS (m/z)
554.1 [MꢂH]-.
m
of the used method was the following (flow, 1.5 mL/min), with
0.01 M KH2PO4, pH 2.3 (solvent A), methanol (solvent B): from 40%
B to 85% B in 8 min, 85% B for 5 min, from 85% to 40% B in 1 min, 40%
B for 2 min, stop time 16 min.
6

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 2
Table 3
Binding affinities of 45 at different screening concentrations.
Binding affinities of Boc-protected carboline 25 and the corresponding 5-TAMRA
derivative 37.
Compound
MKK4 [POC]
100 nM
1.3
30 nM
4.1
10 nM
18.0
45
Compound
MKK4b [POC]
R
25
37
Boc
5-TAMRA
9.2
13.0
b
Screening concentration 100 nM.
Fig. 6. MKK4 Fluorescence polarization assay. Compound 45 in competition with
MKK4 inhibitors 53 (red), 54 (blue) and 55 (green) showed EC50-values of 31, 164 and
628 nM respectively. (For interpretation of the references to color in this figure legend,
the reader is referred to the Web version of this article.)
under microwave irradiation (50 W, 110 ꢀC) for 45 min. After TLC
analysis revealed full consumption of the starting material, ethyl
acetate was added, and the organic layer was washed with water
and saturated sodium chloride solution. The organic layer was
dried over sodium sulfate and the solvent was removed under
vacuum conditions. After column chromatography (DCM/EE, v/v,
0e75 %) the pure product could be obtained as a colorless solid
(199 mg, 271 m d 12.57 (s, 1H),
mol, 75 %). 1H NMR (200 MHz, DMSO)
9.83 (s, 1H), 9.14 (d, J ¼ 2.1 Hz, 1H), 8.90 (d, J ¼ 2.0 Hz, 1H), 8.85 (s,
1H), 8.53 (t, J ¼ 5.6 Hz, 1H), 8.10e7.87 (m, 5H), 7.76e7.58 (m, 2H),
7.35 (t, J ¼ 8.7 Hz,1H), 6.81 (t, J ¼ 5.4 Hz,1H), 3.28 (d, J ¼ 5.6 Hz, 2H),
3.22e3.11 (m, 2H), 2.95 (d, J ¼ 6.0 Hz, H), 1.85e1.62 (m, 2H), 1.47 (s,
4H), 1.37 (s, 9H), 0.92 (t, J ¼ 7.4 Hz, 3H) ppm. 13C NMR (50 MHz,
DMSO)
d 187.0, 165.7, 155.6, 152.5, 146.1, 143.5, 140.5, 133.2, 128.4,
127.9, 127.8, 126.5, 120.8, 115.7, 77.4, 53.5, 39.5, 28.3, 16.8, 12.6 ppm.
Fig. 5. Compound 45 is selective for MKK4 with low affinity towards BRAF. Super-
imposition of docking pose of 45 (purple) in MKK4 (hidden) and the crystal structure
of BRAF (PDB ID: 4rzv) (see Fig. 2A and C). The nearby amino acids of BRAF within 4 Å
of 45 are highlighted in red in the figures. The yellow arrows indicate the major ste-
rically clashes between BRAF and 45. The molecular surface of BRAF is shown in red
surface. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
ESI-MS (m/z) 742.8 [MþNa]þ.
7.4. 5-((4-(5-(6-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-9H-
pyrido[2,3-b]indol-3-yl)picolinamido)butyl)carbamoyl)-2-(6-
(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate
(45)
Tert-butyl
zoyl)-9H-pyrido[2,3-b]indol-3-yl)picolinamido)butyl)carbamate
(109 mg, 134 mol, 2.5 eq) was dissolved in toluene/TFA (10:1, v/v)
(4-(5-(6-(2,6-difluoro-3-(propylsulfonamido)ben-
7.3. Tert-butyl (4-(4-(6-(2,6-difluoro-3-(propylsulfonamido)
benzoyl)-9H-pyrido[2,3-b]indol-3-yl)benzamido)butyl)carbamate
(34)
m
and stirred for 17 h. Then the solvent was evaporated under
reduced pressure and the residue was dissolved again in DMF
(0.5 mL). The mixture was added to a solution of 5-TAMRA (23 mg,
N-(2,4-difluoro-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-9H-pyrido[2,3-b]indole-6-carbonyl)phenyl)propane-1-
53
427
m
m
mol, 1 eq), HATU (20 mg, 53
mol, 8 eq) in DMF (1 mL). After 17 h at room temperature the
mmol, 1 eq) and DIPEA (75 mL,
sulfonamide (200 mg, 360
bromobenzamido)butyl)carbamate (147 mg, 396
K2CO3 (100 mg, 720 mol, 2 eq) were suspended in 1,4-dioxane/
H2O (1:1, v/v, 3.5 mL) and purged with argon for 15 min. Subse-
quently Pd(PPh3)4 (21 mg, 18 mol, 5 mol %) was added and stirred
m
mol,
1
eq), tert-butyl (4-(4-
mmol, 1.1 eq) and
solvent was removed under reduced pressure. The crude product
was purified using column chromatography (DCM/MeOH/TFA, v/v/
v, 90:9.5:0.5) to obtain the pure product as a purple solid (27 mg,
m
m
25 m d 12.60
mol, 47 %). mp 195 - 197 ꢀC, 1H NMR (400 MHz, DMSO)
7

T. Kircher, T. Pantsar, A. Oder et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
signaling, Elife 2 (2013) 1e25.
[4] F. Ansideri, A. Lange, A. El-Gokha, F.M. Boeckler, P. Koch, Fluorescence
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J ¼ 2.4 Hz, 1H), 8.87 (dd, J ¼ 11.2, 5.0 Hz, 1H), 8.84 (s, 1H), 8.45 (s,
1H), 8.41 (dd, J ¼ 8.2, 2.2 Hz,1H), 8.23 (dd, J ¼ 8.1,1.5 Hz,1H), 8.16 (d,
J ¼ 8.2 Hz, 1H), 8.03 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.69 (d, J ¼ 8.8 Hz, 1H),
7.64 (dt, J ¼ 9.1, 4.6 Hz, 1H), 7.32 (t, J ¼ 7.9 Hz, 1H), 6.54e6.45 (m,
6H), 3.42e3.35 (m, 4H), 3.17e3.08 (m, 2H), 2.93 (s, 12H), 1.72 (td,
J ¼ 15.0, 7.5 Hz, 2H), 1.63 (s, 4H), 0.91 (t, J ¼ 7.4 Hz, 3H) ppm. HPLC
tR ¼ 6.633 min, purity: 96 % (254.4 nm), 100 % (430.4 nm). IR (ATP)
[cmꢂ1] 1597, 1490, 1347, 1184, 1139. ESI-HRMS m/z [MþH]þ calcu-
lated: 1033.35131, measured: 1033.35.050.
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Author contributions
[9] G. Bollag, P. Hirth, J. Tsai, J. Zhang, P.N. Ibrahim, H. Cho, W. Spevak, C. Zhang,
Y. Zhang, G. Habets, E.A. Burton, B. Wong, G. Tsang, B.L. West, B. Powell,
R. Shellooe, A. Marimuthu, H. Nguyen, K.Y.J. Zhang, D.R. Artis, J. Schlessinger,
F. Su, B. Higgins, R. Iyer, K. D’Andrea, A. Koehler, M. Stumm, P.S. Lin, R.J. Lee,
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a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma, Na-
ture 467 (2010) 596e599.
All authors contributed to writing the manuscript. All authors
have approved the final version of the manuscript.
Notes
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The authors declare no competing financial interest.
Declaration of competing interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: SL is stock owner <3% of Heparegenix GmbH. However
this relationship has not influenced any work related to this
manuscript.
[13] T. Lowinger, M. Shimazaki, H. Sato, K. Tanaka, K. Marx, M. Yamamoto,
K. Urbahns, F. Gantner, Hiromi Qkigami, Kasake Nakashima,
Keisuke Takeshita, K. Bacon, H. Komura, N. Yoshida, WO 03/037898 A1, 2003.
Acknowledgements
€
[14] W. Albrecht, S. Laufer, R. Selig, P. Klovekorn, B. Praefke WO 2018/134254 Al,
2018.
We would like to thank the CSC e IT Center for Science, Finland
for computational resources. T.P. acknowledges European Union’s
Horizon 2020 research and innovation programme (Marie
Sklodowska-Curie grant agreement No 839230) and the Orion
Research Foundation sr for financial support.
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We would like to thank Kristine Schmidt for the language
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2020.112901.
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