Discovery of indazole-pyridinone derivatives as a novel class of potent and selective MNK1/2 kinase inhibitors that protecting against endotoxin-induced septic shock

Article abstract of DOI:10.1016/j.ejmech.2020.113057

The mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNKs 1/2) and their downstream target eIF4E, play a role in oncogenic transformation, progression and metastasis. These results provided rationale for development of first MNKs inhibitors, currently in clinical trials for cancer treatment. Inhibitors of the MNKs/eIF4E pathway are also proposed as treatment strategy for inflammatory conditions. Here we present results of optimization of indazole-pyridinone derived MNK1/2 inhibitors among which compounds 24 and 26, selective and metabolically stable derivatives. Both compounds decreased levels of eIF4E Ser206 phosphorylation (pSer209-eIF4E) in MOLM16 cell line. When administered in mice compounds 24 and 26 significantly improved survival rates of animals in the endotoxin lethal dose challenge model, with concomitant reduction of proinflammatory cytokine levels – TNFα and IL-6 in serum. Identified MNK1/2 inhibitors represent a novel class of immunomodulatory compounds with a potential for the treatment of inflammatory diseases including sepsis.

Full text of DOI:10.1016/j.ejmech.2020.113057

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
Research paper
Discovery of indazole-pyridinone derivatives as a novel class of potent
and selective MNK1/2 kinase inhibitors that protecting against
endotoxin-induced septic shock
,
Agnieszka Dreas a *, Katarzyna Kucwaj-Brysz a, Karolina Pyziak a, Urszula Kulesza a,
Ewelina Wincza b, Charles-Henry Fabritius a, Kinga Michalik a, Ewelina Gabor-Worwa a,
Aniela Gołas a, Mariusz Milik a, Magdalena Masiejczyk a, Eliza Majewska a,
c
c
c
ꢀ
ꢀ
ꢀ
Kazimiera Pysniak , Urszula Wojcik-Trechcinska , Zuzanna Sandowska-Markiewicz ,
a
c
,
, **
Krzysztof Brzozka , Jerzy Ostrowski d, Tomasz Rzymski a, Michal Mikula c
ꢀ
a
ꢀ
Ryvu Therapeutics S.A., H. L. Sternbacha 2, 30-394, Krakow, Poland
b
ꢀ
ꢀ
Selvita S.A., Bobrzynskiego 14, 30-348, Krakow, Poland
c Department of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, 02-781, Warsaw, Poland
d Department of Gastroenterology, Hepatology and Clinical Oncology, Centre of Postgraduate Medical Education, 02-781, Warsaw, Poland
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 (MAPK)-interacting kinases 1 and 2 (MNKs 1/2) and their down-
stream target eIF4E, play a role in oncogenic transformation, progression and metastasis. These results
provided rationale for development of first MNKs inhibitors, currently in clinical trials for cancer
treatment. Inhibitors of the MNKs/eIF4E pathway are also proposed as treatment strategy for inflam-
matory conditions. Here we present results of optimization of indazole-pyridinone derived MNK1/2
inhibitors among which compounds 24 and 26, selective and metabolically stable derivatives. Both
compounds decreased levels of eIF4E Ser206 phosphorylation (pSer209-eIF4E) in MOLM16 cell line.
When administered in mice compounds 24 and 26 significantly improved survival rates of animals in the
endotoxin lethal dose challenge model, with concomitant reduction of proinflammatory cytokine levels
Received 11 September 2020
Received in revised form
25 November 2020
Accepted 26 November 2020
Available online xxx
Keywords:
MNK1/2 kinases
Kinase inhibitors
Sepsis
e TNF
compounds with a potential for the treatment of inflammatory diseases including sepsis.
© 2020 Elsevier Masson SAS. All rights reserved.
a and IL-6 in serum. Identified MNK1/2 inhibitors represent a novel class of immunomodulatory
Inflammatory diseases
1. Introduction
Established role in cancer and high druggability fueled develop-
ment of first inhibitors [7,8]. MNK1 and MNK2 share substantial
similarity in their coding sequences and protein structures [9].
MNK1 and MNK2 transcripts are alternatively spliced, giving rise to
two distinct isoforms, with MNK1a/MNK2a representing a full-
length protein and MNK1b/MNK2b, lacking the short MAPK bind-
ing C-terminal domain [10e12]. MNKs are phosphorylated at the C-
terminus [13] by either ERK or p38 mitogen-activated protein ki-
nases [14], hence the MNK1b/2b isoform poorly responds to acti-
vation and exhibits a low basal activity. In its turn, MNK1a
represents an inducible isoform characterized by low basal levels of
expression [15], while MNK2a is highly constitutively expressed
[12]. MNKs play important roles in controlling post-transcriptional
gene expression through Ser209 phosphorylation of the eukaryotic
translation initiation factor 4E (eIF4E) [12,15,16]. Study involving
double KO of MNK1/2 in mice indicates that MNK kinases are the
The mitogen-activated protein kinase (MAPK) interacting pro-
tein kinases 1 and 2 (MNK1 and MNK2) have been extensively
studied in the scope of their contribution to the development and
progression of solid tumors and hematological malignancies [1e6].
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; Boc,
tert-butyloxycarbonyl protecting group; DIPEA, diisopropylethylamine; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; LPS, Lipo-
polysaccharide; NEt3, trietylamine; rt, room temperature; SAR, structure-activity
relationship; TFA, trifluoroacetic acid; TLC, thin layer chromatography; WB, West-
ern blot; XPhos, 2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl.
* Corresponding author.
** Corresponding author.
E-mail addresses: agnieszka.dreas@ryvu.com (A. Dreas), michal.mikula@pib-nio.
pl (M. Mikula).
https://doi.org/10.1016/j.ejmech.2020.113057
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al., Discovery of indazole-pyridinone derivatives as a novel class of potent and
selective MNK1/2 kinase inhibitors that protecting against endotoxin-induced septic shock, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.113057

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
only proteins able to phosphorylate eIF4E [17]. This observation
marked eIF4E phosphorylation as a potential biomarker for MNK1/
2 inhibition studies. Recently, a number of studies suggested that
eIF4E preferentially enhances the translation of mRNA coding
tumor-associated proteins [18]. However, the control of 50Cap-
dependent translation may not be the sole function of MNK kinases.
Additionally, these kinases demonstrate an ability to influence
Characterization of the first hits for the MNK1/2 kinases.
mRNA stability of TNFa or IL6, therefore playing an important role
in modulating immune responses via mediation of cytokine pro-
duction [19,20]. This pose an interesting avenue in the research, as
TNFa is an important mediator of chronic and acute inflammation,
including septic shock [21,22]. Sepsis is one of major health prob-
lems affecting millions of individuals world-wide each year. It ranks
among the most common causes of death in hospitalized patients
and represents a major healthcare financial burden world-wide
[23]. Despite intense research efforts and clinical trials no specific
treatment has been developed for this syndrome so far [24]. Several
oncogenic and immune signaling pathways including PI3K/mTOR,
RAS, ERK/p38-MAPK and Toll-like receptors pathways converge at
the MNK kinases node to integrate signals into the initiation of
translation and regulation of vital downstream processes of tumor
cell biology and immune system [25]. As the MNKs are the only so
far established kinases that activate oncogenic eIF4E this gave the
rationale to develop nontoxic chemical inhibitors targeting these
kinases for cancer treatment with possible reposition to treat
autoimmune and inflammatory disorders, including sepsis. A large
number of studies utilizing transient MNKs knockdown or phar-
macological inhibition provided evidence on important MNK’s role
in mediating the production of multiple pro-inflammatory cyto-
kines [8,19,26,27]. These studies highlighted importance on MNKs
kinase node in mediating signals critical for pro-inflammatory re-
sponses, therefore the development of MNKs specific inhibitors
could be a novel therapeutic approach for the treatment of in-
flammatory diseases, including sepsis.
Although MNK1 and MNK2 kinases play critical roles in auto-
immune disorders and malignancies, expression of these proteins
is not essential for normal growth and homeostasis in mice
[17,20,28,29]. This distinction between normal and pathogenic
function makes the MNKs particularly interesting as potential drug
targets, with a broad therapeutic window. First MNK1/2 inhibitors
(e.g. CGP052088, CGP57380 and Cercosporamide) mainly served as
probes for target validation due to their low selectivity and potency.
Further development led to the discovery of more specific and
potent inhibitory agents [30e33], and eventually three MNKs in-
hibitors, namely ETC-206 [34], eFT508 [7] and BAY1143269 (full
structure is still undisclosed) [8] developed by Experimental
Therapeutics Centre, Effector Therapeutics and Bayer AG, respec-
tively, have entered clinical trials in oncology [1] (Fig. 1).
Hit1
Hit2
MNK1 IC50 [nM]
MNK2 IC50 [nM]
56
12
315
75
compounds 24 and 26, endowed with high in vitro target selectivity.
Oral treatment with the compound 26 decreased serum levels of
TNFa and IL-6, significantly extended survival and improved clin-
ical symptoms in a mouse model of endotoxin induced sepsis.
These data indicate that the compound 26 could be considered for
further development as an effective treatment of inflammatory
diseases.
2. Results and discussion
2.1. Hit identification
First hits targeting MNK1/2 kinases were identified by the
screening of the internal library of compounds using balanced re-
action conditions for both kinases at Km ATP concentration and
ADP-Glo™ bioluminescent detection assay. As shown in Table 1,
two hit compounds 1 and 2 were identified, sharing the same motif
(pyridinone) in its structure. Hit 1 containing disubstituted (at
positions C-3 e pyridinone and C-6 e bromine) aza-indazole core
showed IC50 of 56 and 12 nM for MNK1 and MNK2 kinases,
respectively. The second chemotype had the same pyridinone
moiety, however connected via the carbon atom at the C-6 position
of an indazole core. Hit 2 showed IC50 of 315 and 75 nM for MNK1
and MNK2 kinases, respectively.
Kinome selectivity of compounds 1 and 2 was tested at 1 mM
concentration by using KINOMEscan (DiscoverX) against 414 ki-
nases. As demonstrated in Fig. 2A, hit 1 turned out as a multi-target
compound, inhibiting significant number of kinases from panel. On
the contrary, hit 2 showed excellent selectivity profile in the same
panel with low selectivity score (S(35) ¼ 0.01). By using the <35%
threshold of control activity (% Ctrl), only 3 kinases (except of
Herein, we present the optimization of low molecular weight
dual MNK1/2 inhibitors, which resulted in identification of
Fig. 1. Structures of MNKs inhibitors in clinical trials.
2

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
MNK2) were identified as hits (AAK1 32% Ctrl, ERK8 20% Ctrl and
HASPIN 20% Ctrl). Those results prompted us to focus on further
exploration of hit 2.
change of the side chain of phenylalanine 227 in the 2HW7 com-
plex. This new position was not entirely consistent with the
conformation adopted by this residue in the native protein
complexes.
In order to create an alternative version of the docking grid for
MNK2, we used one of the apo structures of MNK2. At that time,
there were two publicly available structures in PDB, namely 2AC3
and 2AC5. The former is a wild type, apo MNK2 in DFG/D-out
conformation, the latter is a D228G mutant, apo MNK2 in DFG/D-
in conformation. One of difficulties was that the binding sites of
the kinase structures without ligand (apo) were too small to
contain the ATP competitive ligand. Additionally, Phe227 in a DFG/
D-out conformation was at the center positions of the ATP binding
site. Induced Fit Docking (IFD) procedure, from the Schrodinger
molecular modeling package removed small steric hindrances in
the binding site. Changes introduced in the local conformation of
the side chains and a flexible loop allowed docking of ligands to the
protein model.
Selected representative active compounds were docked using
the default IFD procedure from Protein Preparation Wizard [36].
The IFD-modified template on the basis of the MNK2 structure
2AC5 from PDB was selected given best docking score values and
consistency of the docking poses. This model of MNK2 structure
was used for SAR and evaluation rational ligand optimization. An
example of such pose and analysis for hit compound 2 is presented
in Fig. 3. In this pose, the main observed interaction is among atom
2-N from indazole moiety with Met162 residue in the hinge region
of MNK2. Moreover, an important hydrogen bond interaction is
created between a carbonyl group from pyridinone moiety and
Lys113 residue.
2.2. Molecular studies
The structural aspects of MNK1 and MNK2 kinases were already
broadly discussed in literature [1]. We may only emphasize here
that despite the fact that they share the typical, two-domain, pro-
tein kinase architecture, the structures of MNKs contain several
distinctive elements. The prominent characteristic is the replace-
ment, of well conserved “DFG” sequence motif with “DFD” in the
MNKs, which may influence both structural and functional aspects
of their activity. Additionally, they are distinguished by the pres-
ence of the atypical sequence inclusions, including a Zn2þ binding
motif containing four cysteines, near the C terminus.
In order to rationally improve protein/ligand interactions, hit
compounds were docked to known structures of MNK2 from Pro-
tein Data Bank (PDB) [35] using the Glide program from the
Schrodinger Suite [36]. In the first approach, we docked com-
pounds to the available active form structure of the MNK2 D228G
mutant with staurosporine (PDB code: 2HW7) [37], however re-
sults were inconclusive, because of generally low values of docking
scores and ambiguity of the protein/ligand interaction patterns.
Poses of the compounds docked to this template were sometimes
flipped by 180ꢀ in the MNK binding site, with no essential differ-
ence in the docking scores. One of the possible explanation was that
the presence of high molecular weight staurosporine in the binding
site caused substantial changes in the conformation of the binding
site, amino acid side chains and some backbone atoms of amino
acids from loops located in the vicinity of the ATP binding sites.
Additionally, the combined effect of the D228G mutation and steric
interactions of staurosporine ligand forced
a conformational
Fig. 2. Comparison of kinome selectivity panels for hit 1 (A) and 2 (B). Larger circle indicate higher affinity binding.
3

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
2.3. Chemistry
Although both hit compounds 1 & 2 were promising with
respect to
a
potential optimization (LLEMNK2
¼
4.8 and
LLEMNK2 ¼ 4.3 for hits 1 and 2, respectively), our medicinal chem-
istry effort focused on the expansion of hit 2. The first goal was to
improve the potency by keeping high selectivity and good physi-
cochemical/ADME properties. As shown in Fig. 4, the lead structure
compound I was divided into two structural regions for hit opti-
mization e the bicyclic core and the substitution on the pyridinone
R.
The synthesis of the different bicyclic systems 1 to 5 is shown in
Scheme 1. A Miyaura borylation reaction [38] of 1-benzyl-5-bromo-
1,2-dihydropyridin-2-one [39] resulted in the formation of the
boronic ester 29 (with additional boronic acid from 11% to 29%),
which was subsequently coupled with 5-bromo-3-iodo-1-(triphe-
nylmethyl)pyrazolo[3,4-b]pyridine [40] in Suzuki reaction to give
compound 30 (Route A). Removal of the trityl protecting group
from intermediate 30 in TFA conditions led to the hit 1 with
moderate yield. Hit 2 (Route B) was synthesized using 1-benzyl-5-
bromo-1,2-dihydropyridin-2-one and commercial 6-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole. Similarly, com-
pounds 3, 4 and 5 (Route C) have been obtained by coupling of
boronic ester/acid 29 with the corresponding 6-bromoindole, 6-
bromo-1-methyl-1H-indazole and 6-bromo-2-methyl-1H-inda-
zole, respectively, with moderate yield.
The series of derivatives modified on the pyridinone (R-part e
Fig. 4) have been synthesized in a similar way (Scheme 2). The
appropriate boronic esters 32a-c, prepared from N-substituted 5-
bromopyridin-2(1H)-ones 31a,b,d were reacted with 6-bromo-
1H-indazole in the presence of PdCl2(dppf)*CH2Cl2 to afford the
final compounds 6, 7 and 9. N-acylation of 5-bromo-pyridin-2(1H)-
one with 4-morpholine carbonyl chloride provided compound 31c
that was finally coupled with commercial 6-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-indazole to give desired derivative 8
with moderate yield.
Fig. 4. General structure of compound I.
three-step synthesis starting from 5-bromopyridin-2(1H)-one, us-
ing the same type of reactions as described above. First, a set of
different N-substituted 5-bromopyridin-2(1H)-one 33a-m was
prepared, as outlined on Scheme 3. Additionally, pyridinone 33m
was converted into the acetamide derivative 33n using acetyl
chloride/NEt3 in anhydrous CH2Cl2 [41]. Few selected pyridinone
derivatives with R4 ¼ H (33a,b,d,g,h) were transformed to the
corresponding boronic esters (34a-e).
Suzuki coupling reactions between key intermediates 33a-l, 33n
and 6-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole led to
final compounds 10e17, 20, 22 and intermediates 35, 36 and 37 as
described in Scheme 4. Obtained intermediates 35 and 36 were
converted into the final compounds 18 and 19 by basic hydrolysis of
cyano group. Moreover, compound 21 was achieved from com-
pound 37 by deprotection of Boc-group with a good yield.
The second group of compounds 23e28 was accomplished us-
ing the same type of coupling reaction between corresponding
boronic esters/acid 29, 34a-e and 6-bromo-1H-indazol-3-amine
with moderate to good yield (Scheme 5).
2.4. SAR study and ADME profiling
The first group of compounds 3e5 was synthesized to check the
importance of the nitrogen atoms of the indazole core (Table 2).
According to the docking studies, the nitrogen in position 2 creates
an interaction with the hinge region. The replacement of the
indazole moiety by an indole (compound 3) resulted in the loss of
biochemical potency as predicted. The same result was observed for
the compounds 4 and 5 when the nitrogen atom in position 1 or 2,
respectively, was methylated. Additional methyl group could
change geometry of the compound in the binding pocket and
impair essential interactions with the hinge region.
The series of derivatives bearing different substituents on
benzyl ring (10e28) (Schemes 4 and 5) have been achieved by a
In order to increase the potency of hit compound 2, we intro-
duced modifications of the benzyl part (R substitution of the pyr-
idinone (Fig. 4)). The incorporation of a longer, more flexible linker
than methylene group (compound 6) or the addition of a carbonyl
group (compound 7) resulted in inactive compounds (Table 3). The
same result was obtained for compound 8 with a morpholine
moiety. Finally, the complete removal of the aryl ring (compound 9)
resulted in the loss of potency as well, indicating the crucial role of
the benzyl group for the activity of hit compound 2. Further
modifications of hit compound 2 involved decoration of the benzyl
moiety.
Compounds 10e22 were synthesized with various substitutions
on the aryl group as shown in Table 4. First, the effect of halogen
atoms in different positions was examined. The fluorine atom
(compound 10) or chlorine atom (compound 12) in ortho (R2)
resulted in 2-fold activity improvement or the same level of ac-
tivity, respectively, compared to hit compound 2 against MNK2. In
case of MNK1 kinase, 7-fold activity improvement was observed for
compound 10 and 4-fold for compound 12, when compared to
compound 2. Substitutions in the meta position (R3) resulted also in
the minor 2-fold improvement for both compounds 11 and 13
against MNK2. Notably, for MNK1 kinase, the tendency was
Fig. 3. Docking pose of compound “hit 2” in MNK2 binding site, generated with use of
Glide program (Schrodinger). The used template MNK2 structure was obtained by
applying Induced Fit Docking procedure to the MNK2 structure 2AC5 from PDB, as it is
described in text.
4

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of bicyclic compounds 1e5. Reagents and conditions: (a) bis(pinacolato)diboron, KOAc, XPhos, PdCl2(dppf)*CH2Cl2, 1,4-dioxane, 100 ꢀC; (b) sat. aq. sol. NaHCO3,
PdCl2(dppf)*CH2Cl2, acetonitrile/toluene (2/1), 60 ꢀC, 50%; (c) TFA, CH2Cl2, rt, 64%; (d) Cs2CO3, PdCl2(dppf)*CH2Cl2, 1,4-dioxane/water (2/1), microwave, 125 ꢀC, 26e63%.
Scheme 2. Synthesis of compounds 6e9. Reagents and conditions: (a) corresponding bromo- and iododerivatives, 60% NaH, DMF, 0 ꢀC to rt, 12e95%; (b) 4-morpholine carbonyl
chloride, DIPEA, acetonitrile, rt, 98%; (c) bis(pinacolato)diboron, KOAc, XPhos, PdCl2(dppf)*CH2Cl2, 1,4-dioxane, 100 ꢀC; (d) Cs2CO3, PdCl2(dppf)*CH2Cl2, 1,4-dioxane/water (2/1),
microwave, 125 ꢀC, 32e53%.
opposite, with 4-fold increased activity of compound 11 with the
fluorine atom and 8-fold improvement for compound 13 with the
chlorine atom. The incorporation of two chlorine atoms in both
meta (R3) and para (R4) positions (compound 14) led to a minor
decrease in the activity against MNK2 kinase, however 5-fold in-
crease for MNK1 kinase. These results prompted us to introduce
5

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
showed the same trend in increased activity e meta > ortho > para.
Further exploration of the meta position with primary amine
(compound 21) and retro amide (compound 22) resulted in either
no improvement of potency or slightly, 2-fold, increase of the ac-
tivity against both MNK1 and MNK2 kinase, respectively.
ADME profiling of hit compound 2, revealed high microsomal
clearance Clint ¼ 157
F were also metabolically unstable (Mouse Clint between 160 and
246 l/min/mg), however compounds 13 m-Cl, 16 m-CF3 and 22 m-
NHCOCH3 showed moderate improvement in stability (Clint be-
tween 47 and 63 l/min/mg). As a potential strategy for further
ml/min/mg (Table 5). Compounds 10 o-F, 11 m-
m
m
improvement of clearance applied overall reduction of lipophilicity
in the current chemical series.
The introduction of a more polar group into the benzylic moiety
resulted in an increase of the metabolic stability but disappoint-
ingly in a decrease of the biochemical activity on both MNK1 and
MNK2. In order to improve the activity, a primary amine was
introduced in the 3rd position of the indazole moiety to compounds
10, 11, 13, 16, 22 with the most promising decoration on the benzyl
moiety (Table 6). This insertion resulted in compounds 23e28
showing a significant improvement of the metabolic stability in
Scheme 3. Synthesis of intermediates 33a-n and 34a-e. Reagents and conditions: (a)
corresponding benzyl bromide or chloride, 60% NaH, DMF, 0 ꢀC to rt, 24e96%; (b)
bis(pinacolato)diboron, KOAc, XPhos, PdCl2(dppf)*CH2Cl2, 1,4-dioxane, 100 ꢀC; (c)
acetyl chloride, NEt3, CH2Cl2, 0 ꢀC to rt, 85%.
mouse microsomes (for most compounds Mouse Clint < 10 ml/min/
mg). At the same time, introduction of the amine moiety in the
hinge region translated in an increase of the activity against MNK1/
2 kinases for all compounds (improvement in potency from 1.5 to 9-
fold for MNK2 and 3 to 4-fold for MNK1) compared to analogs
without -NH2 group.
Compounds with halogen substituents in benzylic moiety (24 o-
F and 26 m-Cl) were selected for further profiling given their high
activity against MNK1/2 and low microsomal clearance Clint < 10 ml/
more bulky, electron-withdrawing groups, such as -CF3, as a
possible alternative to the fluorine or chlorine atoms on the aryl
group (compounds 15e17). The most potent compound 16 contains
a -CF3 group in the meta position. Interestingly, compound 17 with
the -CF3 in para position turned out to be less active with a
micromolar activity against both kinases. Compounds 18e20 with a
polar amide group in ortho, meta and para position were less potent
compared to hit compound 2. Nevertheless, compounds 18e20
min/mg.
Notably, compounds 24 and 26 did not inhibit any tested
CYP450 isoforms (IC50 > 10 M), showed high plasma protein
binding and acceptable solubility (Table 7).
m
Scheme 4. Synthesis of compounds 10e22. Reagents and conditions: (a) Cs2CO3, PdCl2(dppf)*CH2Cl2, 1,4-dioxane/water (2/1), microwave, 125 ꢀC, 7e70%; (b) acetyl chloride, NEt3,
CH2Cl2, 0 ꢀC to rt, 85%; (c) K2CO3, 30% H2O2, MeOH/H2O (5/1), 0 ꢀC to rt, 46e72%; (d) 4 M HCl in 1,4-dioxane, rt, 89%.
6

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 5. Synthesis of compounds 23e28. Reagents and conditions: (a) Cs2CO3, PdCl2(dppf)*CH2Cl2, 1,4-dioxane/water (2/1), microwave, 125 ꢀC, 22e78%.
Table 2
SAR on the indazole core/IC50 values for derivatives.
Table 4
SAR of MNK2 and MNK1 kinase inhibitors.
Compound
Structure
MNK2 IC50 [nM]
75
MNK1 IC50 [nM]
315
2
Compound R2
R3
R4
MNK2 IC50 [nM] MNK1 IC50 [nM]
3
>2000
>2000
2
H
F
H
Cl
H
H
CF3
H
H
H
F
H
Cl
Cl
H
CF3
H
H
H
H
H
Cl
H
H
CF3
H
75
37
38
78
40
97
100
32
1112
340
106
315
47
74
80
37
62
133
40
1168
878
388
>2000
287
163
10
11
12
13
14
15
16
17
18
19
20
21
22
4
5
>2000
>2000
>2000
>2000
H
H
H
CONH2
H
H
H
H
CONH2
H
NH2
H
CONH2 >2000
H
H
84
43
NHCOCH3
Table 5
Metabolic stability of most potent compounds.
Table 3
SAR on the replacement of the benzyl group.
Compound
R2
R3
Mouse Microsomes
Clint [ml/min/mg]
% remaining at 60 min
2
H
F
H
H
H
H
H
H
F
Cl
6.5
1.2
5.6
39
32
157
246
160
52
63
47
Compound
R
MNK2 IC50 [nM]
75
MNK1 IC50 [nM]
315
10
11
13
16
22
2
CF3
NHCOCH3
6
7
>2000
>2000
43
1073
1763
2.5. Selectivity profiling of compounds 24 & 26
8
9
>2000
>2000
>2000
>2000
Both compounds e 24 and 26 were tested in a wide kinome
panel (>400 kinases) at 1 M concentration to determine their
target selectivity. As shown on Fig. 5A, the majority of kinases were
m
-CH3
7

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 6
Improvement of potency and metabolic stability.
Compound
R2
R3
MNK2 IC50 [nM]
MNK1 IC50 [nM]
Clint [ml/min/mg]
23
24
25
26
27
28
H
F
H
H
H
H
H
H
F
Cl
32
4
5
6
21
8
79
12
19
11
56
55
<10
<10
<10
<10
<10
14
CF3
NHCOCH3
Table 7
kinome selectivity. The off-targets were identified mostly from
CMGC (CLK, DYRK, ERK, HIPK) and AGC (GRK and SGK) kinase
family (Table 8).
ADME properties of compounds 24 and 26.
Experiment
Compound 24
Compound 26
CYP IC50
[
m
M]
3A4
1A2
2B6
2C19
2D6
mouse
rat
human
Soerensen buffer
water
>10
>10
>10
>10
>10
93
>95
>95
466
>500
>10
>10
>10
>10
>10
90
ND
ND
172
275
2.6. Biochemical studies using recombinant MNK1 and MNK2
For better characterization of compounds 24 and 26, the inhi-
bition constant (Ki) on MNK1 and MNK2 has been determined. The
compounds were tested in the presence of increasing ATP con-
centrations: 0.1 ꢁ Km, 0.3 ꢁ Km, 1 ꢁ Km, 3 ꢁ Km, 7 ꢁ Km of ATP for
each kinase. IC50 values for both compounds shifted over 3 times
between 0.3 and 7 ꢁ Km of ATP (data not shown), therefore
competitive inhibition model has been applied to evaluate the in-
hibition constant (Fig. 6). For MNK1, Ki 14 nM was measured for
both compound 24 and 26. For MNK2, Ki 11 nM and 5.5 nM were
measured for compound 24 and 26, respectively.
PPB
Kinetic solubility [
mM]
ND e not determined.
not affected by compound 24. Low selectivity score (S(35) ¼ 0.017)
indicate a small number of kinases that compound 24 interact with
(ERK7 25% Ctrl, PASK 24% Ctrl, STK 17A 6% Ctrl, TBK1 35% Ctrl and
TSF1 33% Ctrl). The second profiled compound 26 (Fig. 5B) dis-
played also very good selectivity (S(35) ¼ 0.051), confirming broad
2.7. Biological evaluation
Number of studies utilizing siRNA knockdown or small molecule
inhibitors provided evidence on MNK’s role in fine-tuning inflam-
matory and immune processes by influencing cytokines expression
levels (reviewed in Ref. [20]). The first synthetic small-molecule
MNK inhibitor, CGP57380, was shown to attenuate TNFa produc-
tion in a dose-dependent manner in mice macrophages [42]. Sub-
sequent studies using bone marrow-derived macrophages isolated
from a mouse model of Crohn’s disease and challenged with LPS
revealed inhibition of the TNFa, IL6, MCP-1 and induction of IL10
levels by CGP57380 [19]. The work by Fortin et al. revealed that the
CGP57380 attenuated production of several chemokines, including
IL8, MIP-1-alpha and MIP-1-beta upon LPS- and TNFa-treatment, at
Table 8
Activity of compound 26 towards selected off-target kinases.
Target
Kinase family
% Ctrl @1000 nM
BIKE
BMPR1B
CLK1
CLK4
DRAK1
ERK8
TK
TKL
1.2
19
27
10
1.5
5.7
22
23
24
30
24
34
6.2
4.4
30
25
CMGC
CMGC
other
CMGC
mutant
AGC
other
CMGC
lipid
atypical
atypical
atypical
AGC
FLT3
GRK4
HASPIN
HIPK4
PIP5K2B
RIOK1
RIOK2
RIOK3
SGK
Fig. 5. Selectivity e kinome panel for compounds 24 (A) and 26 (B). (Inhibition ¼ 100%
SGK3
AGC
-%Ctrl).
8

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 6. The results from ATP competition test for compounds 24 and 26.
the same time reversing the antiapoptotic effect of LPS and TNF
a
in
Both compounds 24 and 26 showed dose and time-dependent in-
hibition of Ser209 without significant effects on total eIF4E levels
(Fig. 7).
human neutrophils [43]. More recently, the efficacy of another
MNK kinase inhibitor, BAY1143269, was tested ex vivo on human
whole blood challenged with LPS, revealing its potency to repress
TNFa, IL6, MCP-1 and IL-1b pro-inflammatory cytokines production
2.7.2. In vivo studies
[8]. Collectively, these studies confirmed that pharmacological in-
hibition of MNK kinases can alter the cyto- and chemokines pro-
duction in different immune cells, raising the possibility that
selective targeting of MNK kinases may provide a potential novel
therapeutic approach for the treatment of systemic inflammation,
including sepsis.
Previous studies established MNK kinases as important regula-
tors of pro-inflammatory cytokine production and cytokine
signaling [20], therefore we tested if our compounds could have
protective effects in mouse model of endotoxin-induced septic
shock. In vivo mouse pharmacokinetics profile of both compounds
24 and 26 was determined after p.o. and i.v. dosing. The study
revealed a relatively short half-life of both compounds in plasma
and reached about 2 h (after p.o. administration) or 3 h (i.v.) for
compound 24 and 1.8 h (p.o.) or 2 h (i.v.) for compound 26.
Nevertheless, both compounds showed oral bioavailability (33%
and ~100% for compounds 24 and 26, respectively). At Tmax (0.25 h
for both compounds) after p.o. administration, maximum plasma
concentration for compound 26 was about 3-times higher than for
compound 24 (1050 ng/ml versus 335 ng/ml, respectively).
2.7.1. In vitro studies
MNK kinases are the only proteins able to directly phosphory-
late eIF4E at serine 209, what makes this modification a perfect
biomarker for MNK1/2 inhibition studies. Cellular activity of com-
pounds against MNK1/2 kinases could be conveniently tracked by
measuring inhibitory effects in MOLM16 cell line characterized by a
high basal levels of eIF4E Ser209 phosphorylation (pSer209-eIF4E).
Fig. 7. Compounds 24 and 26 inhibit eIF4e phosphorylation in MOLM 16 cell line. MOLM16 cells were treated with indicated concentrations of compounds for 2h or 24h. Cell lysates
were analyzed by immunoblotting with the eIF4E (C.sig.; 9741L), Phospho-eIF4E (Ser209) (C.sig.; 9742L) and b-Actin (Sigma; A2066) antibodies.
9

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 9
Pharmacokinetics parameters of compound 24 and 26 in mice (dose: p.o 5 mg/kg; i.v 2 mg/kg).
Compound
Tmax (h)
Cmax (ng/mL)
T1/2 (h)
AUC0-t (ng/h$mL)
AUC0-∞ (ng/h$mL)
F %
33
24 (p.o)
24 (i.v.)
26 (p.o)
26 (i.v.)
0.25
0.08
0.25
0.08
334.9
2.4
3.3
1.9
2.1
517.3
629.4
2149.2
833.9
674.2
677.3
2409.6
855.4
1671.9
1050.2
1493.1
~100
Difference in pharmacokinetics properties was also reflected in the
plasma exposure, being 4-times higher for compound 26 than for
compound 24 (Table 9). Based on these results, to assess efficacy of
the compounds 24 and 26, twice daily (BID) p.o. administration was
proposed. Both compounds were well tolerated by the animals in
long-term in vivo studies with BID p.o. dosing. Compound 26 was
safe for the tumor-bearing mice at the dose of 50 mg/kg BID:
general condition, body weight and blood clinical chemistry pa-
rameters were not affected during the 37-days treatment [3]. In the
case of compound 24, the dose of 100 mg/kg was administered p.o.
with BID schedule to tumor-bearing mice for 17 consecutive days,
and was well tolerated by the animals as assessed by the lack of
clinical observations or body weight loss (data not shown).
The treatment of mice with either compound 24 or 26 probes
before LPS lethal dose challenge followed by drugs administration
BID significantly improved survival rates of animals (Fig. 8). Com-
pound 24 and 26 treated groups exhibited significant recovery with
ameliorated clinical symptoms including lethargy, hunched
posture, and piloerection, compared with those in the LPS group
alone. Given the protective effect of compounds on mice survival
we next repeated experiment with 27 h endpoint following LPS
challenge and surveyed levels of cytokines in serum. We found that
compound 26, but not 24, significantly reduced level of proin-
was developed as dual inhibitors of MNK1/2 kinases. Identified hit
2 proved to be moderately active against MNK1/2 kinases but se-
lective in a kinome panel. Therefore, our goal was to improve po-
tency while keeping high selectivity. Indeed, primary SAR analysis
revealed that we managed to improved activity, however ADME
profiling revealed high microsomal clearance of compounds.
Decreasing overall lipophilicity by adding primary amine into the
hinge region increase biochemical activity (19-fold for MNK2 and
from 26-fold for MNK1 compared to hit 2) and decreased hepatic
clearance, without compromising overall favorable selectivity
profile. In vivo studies using murine model of the endotoxin-
induced sepsis indicated that selective MNK1/2 inhibitors are a
novel promising class of compounds with a strong immunomod-
ulatory activity and could be considered as candidate drugs for
treatment of inflammatory syndromes including sepsis.
4. Experimental procedures
4.1. General experimental protocol
All chemicals were purchased at the highest commercial quality
and used without further purification, unless otherwise stated.
Reactions were monitored by thin-layer chromatography (TLC)
carried out on Sigma Aldrich silica gel plates (60 F254) using UV
light as visualizing agent. Sigma Aldrich silica gel 63e200 mesh size
was used for column chromatography. 1H (400 MHz), 13C (101 MHz)
nuclear magnetic resonance spectra were recorded at ambient
temperature on Bruker Avance III HD and 1H (300 MHz) on Varian
flammatory TNF
abundances reduction was observed for other proteins including IL-
22 and IL-1 with compound 26 treatment (data not shown). To the
a and IL-6 (Fig. 9). A trend, but not significant, in
b
best of our knowledge this is the first study showing that phar-
macological inhibition of MNKs as mediators of pro-inflammatory
cytokine production exhibits highly protective effects and amelio-
rates clinical symptoms of the endotoxin induced sepsis.
Mercury-VX. Chemical shifts (d) are reported in parts per million
(ppm) and coupling constants (J) are given in hertz (Hz). The
following abbreviations were used to explain the multiplicities:
s ¼ singlet, d ¼ doublet, dd ¼ doublet of doublets, m ¼ multiplet.
UPLC-MS spectra were obtained by Shimadzu UFLC system equip-
3. Conclusions
In summary, a novel series of indazole-pyridinone derivatives
ped with Waters Acquity UPLC HSS C18 (50 mm ꢁ 2.1 mm x 1.8
mm)
Fig. 8. Compound 24 and 26 improve mice survival in endotoxin-induced septic shock. Mice were treated per os with compound 26 (25 mg/kg) or 24 (75 mg/kg), and 2 h later LPS
(20 mg/kg) was given i.p. to mice to assess effects of LPS on survival rate. Compounds 26 and 24 were given every 12 h following LPS injection during the experiment. The mice
survival rate was evaluated using KaplaneMeier curves (n ¼ 8).
10

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 9. Compound 26, but not 24 reduces levels of TNF
a
and IL-6 in serum upon endotoxin challenge. Mice were treated per os with either compound 26 (50 mg/kg) or 24 (75 mg/
kg), and 1 h later LPS (20 mg/kg) was given i.p. Additional drugs doses were administrated 12 h and 24 h following initial treatment and mice were scarified 4 h later with serum
collection for downstream analyses. Cytokines levels were measured with Mouse Cytokine Magnetic Panel on Luminex platform. *p < 0.05; Mann-Whitney U test.
column using flow rate of 0.5 mL/min. A gradient of water/aceto-
nitrile with 0.1% of formic acid was used as an eluent. LC-MS ana-
lyses were performed on Bruker Amazon SL. Analytical separation
was carried out on Waters Symmetry column C18 3.9 ꢁ 150mm
dried over Na2SO4, filtered off and concentrated. The crude product
was purified by column chromatography on silica gel (dichloro-
methane/methanol 9/1) to give a proper amide.
1-Benzyl-5-{5-bromo-1H-pyrazolo[3,4-b]pyridine-3-yl}pyridine-
2(1H)-one (1). Trifluoroacetic acid (2 mL) was added to a solution of
compound 30 (90 mg, 0.14 mmol) in dichloromethane (2 mL). The
reaction mixture was stirred at room temperature overnight and
then evaporated to dryness. The crude product was purified by
flash chromatography (dichloromethane/methanol 95/5) to afford
1 (35 mg, 64%) as a light yellow solid: 1H NMR (400 MHz, DMSO‑d6)
5 mm using flow rate of 1.0 ml/min (ROTT-C18 e method A) or flow
rate of 1.2 mL/min (BCM-30 e method C) with detection at 254 nm
or on Kinetex XB C18 4.6 ꢁ 50mm 2.6 m column using flow rate of
m
0.5 mL/min (Kinetex-BCM e method B). The mobile phase was
water (solvent A) and acetonitrile (solvent B) both containing 0.1%
formic acid. Purity of the tested compounds (ꢂ90%) was deter-
mined using these methods. HRMS analysis was performed on a Q
Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrom-
eter. Yields refer to chromatographically and spectroscopically pure
compounds unless otherwise stated.
d
13.96 (s,1H), 8.86 (d, J ¼ 2.1 Hz,1H), 8.63 (d, J ¼ 2.1 Hz,1H), 8.51 (d,
J ¼ 2.6 Hz, 1H), 8.12 (dd, J ¼ 9.5, 2.6 Hz, 1H), 7.36 (d, J ¼ 4.3 Hz, 4H),
7.32e7.25 (m, 1H), 6.60 (d, J ¼ 9.5 Hz, 1H), 5.30 (s, 2H); 13C NMR
(101 MHz, DMSO‑d6)
d 161.2, 151.5, 149.9, 139.5, 138.9, 137.8, 137.3,
132.8, 129.0, 128.0, 127.9, 121.0, 113.1, 112.4, 112.1, 52.0; m.p. >270 ꢀC
decomp.; LC-MS (m/z) 381.3 (MþH)þ; HPLC purity (method B):
100%, tR ¼ 6.5 min.
4.2. Synthesis
1-Benzyl-5-(1H-indazol-6-yl)-1,2-dihydropyridin-2(1H)-one (2).
Synthesized as described in the General Procedure A using 6-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole and 1-
benzyl-5-bromo-1,2-dihydropyridin-2(1H)-one in 63% yield as a
The synthesis of the final compounds 1e28 and their in-
termediates 30, 35e37 will be described below. The synthesis of the
remaining intermediates 29, 31a-d-34a-e can be found in the
Supporting Information.
beige amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
d 13.11 (s,
1H), 8.33 (d, J ¼ 2.7 Hz,1H), 8.07 (s,1H), 7.93 (dd, J ¼ 9.4, 2.7 Hz,1H),
4.2.1. General Procedure A for the synthesis of compounds 2e17, 20,
22e28 and 35e37. Suzuki coupling
7.81 (d, J ¼ 8.4 Hz, 1H), 7.64 (s, 1H), 7.47e7.24 (m, 6H), 6.56 (d,
J ¼ 9.4 Hz, 1H), 5.22 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 161.1,
To a solution of corresponding boronic ester/acid (2.0 eq.) in a
mixture of 1,4-dioxane/water (2/1) and caesium carbonate (2.5 eq.)
in a microwave vial was added suitable halide (1.0 eq.) and [1,10-
bis(diphenylphosphino)ferrocene] palladium(II) chloride complex
with dichloromethane (0.03 eq.). The reaction mixture was heated
at 125 ꢀC for 30 min under microwave irradiation. The reaction
mixture was filtered through Celite, washed with EtOAc and
concentrated in vacuo. The crude product was purified by flash
chromatography (dichloromethane/methanol 95/5) to afford
proper product.
140.9, 140.0, 137.9, 137.2, 134.4, 133.9, 129.0, 128.2, 128.0, 122.3,
121.5, 120.5, 119.3, 119.2, 106.7, 52.0; LC-MS (m/z) 302.1 (MþH)þ;
HPLC purity (method C): 100%, tR ¼ 7.8 min.
1-Benzyl-5-(1H-indol-6-yl)-1,2-dihydropyridin-2(1H)-one
(3).
Synthesized as described in the General Procedure A using boronic
ester/acid 29 and 6-bromoindole in 26% yield as an yellow solid: 1H
NMR (400 MHz, DMSO‑d6)
d
11.13 (s, 1H), 8.16 (d, J ¼ 2.6 Hz, 1H),
7.85 (dd, J ¼ 9.4, 2.7 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz, 1H), 7.51 (s, 1H),
7.41e7.32 (m, 5H), 7.32e7.25 (m, 1H), 7.22e7.16 (m, 1H), 6.54 (d,
J ¼ 9.4 Hz, 1H), 6.43 (s, 1H), 5.22 (s, 2H); 13C NMR (101 MHz,
DMSO‑d6)
d 161.0, 140.2, 138.0, 136.9, 136.0, 129.5, 129.0, 128.2,
4.2.2. General Procedure B for the synthesis of compounds 18 and
19. Hydrolysis of nitrile
127.9, 127.3, 126.5, 120.9, 120.4, 117.þ6, 108.6, 101.4, 51.8; m.p.
110e112 ꢀC; LC-MS (m/z) 300.8 (MþH) ; HPLC purity (method B):
99%, tR ¼ 7.0 min.
To a stirred solution of the corresponding nitrile (1.0 eq.) in a
mixture of methanol/water (5/1) potassium carbonate (6.0 eq.) was
added. After cooling to 0 ꢀC 30% aqueous solution of hydrogen
peroxide (23.0 eq.) was added dropwise and the reaction mixture
was allowed to reach room temperature and stirred overnight.
Methanol was evaporated and the reaction mixture was diluted
with EtOAc. The layers were separated and the aqueous layer was
further extracted with EtOAc. The organic layers were combined,
1-Benzyl-5-(1-methyl-1H-indazol-6-yl)-1,2-dihydropyridin-
2(1H)-one (4). Synthesized as described in the General Procedure A
using boronic ester/acid 29 and 6-bromo-1-methyl-1H-indazole in
43% yield as a brown oil: 1H NMR (400 MHz, DMSO‑d6)
d 8.37 (d,
J ¼ 2.7 Hz, 1H), 8.02 (d, J ¼ 10.7 Hz, 2H), 7.85 (s, 1H), 7.79 (d,
J ¼ 8.4 Hz, 1H), 7.40e7.33 (m, 5H), 7.33e7.24 (m, 1H), 6.59 (d,
11

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
J ¼ 9.5 Hz, 1H), 5.22 (s, 2H), 4.08 (s, 3H); 13C NMR (101 MHz,
(d, J ¼ 3.4 Hz), 124.4 (d, J ¼ 14.6 Hz), 122.4, 121.6, 120.4, 119.2, 119.1,
115.8 (d, J ¼ 21.0 Hz), 106.7, 46.8; m.p. 173e175 ꢀC; LC-MS (m/z)
320.0 (MþH)þ; HPLC purity (method C): 99%, tR ¼ 8.2 min.
1-[(3-Fluorophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
DMSO‑d6) d 161.2, 140.7, 140.0, 137.8, 137.3, 134.3, 132.7, 129.0, 128.1,
128.0, 122.8, 121.7, 120.4, 119.1, 119.0, 106.3, 52.1, 35.8; LC-MS (m/z)
316.3 (MþH)þ; HPLC purity (method C): 99%, tR ¼ 9.4 min.
1-Benzyl-5-(2-methyl-2H-indazol-6-yl)-1,2-dihydropyridin-
dihydropyridin-2(1H)-one (11). Synthesized as described in the
2(1H)-one (5). Synthesized as described in the General Procedure A
using boronic ester/acid 29 and 6-bromo-2-methyl-2H-indazole in
43% yield as a brown amorphous solid: 1H NMR (400 MHz,
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33b in 21% yield
as a beige amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.14 (s,
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
d
DMSO‑d6)
d
8.37 (d, J ¼ 2.7 Hz, 1H), 8.05e7.98 (m, 2H), 7.84 (d,
1H), 8.37 (d, J ¼ 2.7 Hz, 1H), 8.07 (d, J ¼ 1.2 Hz, 1H), 7.95 (dd, J ¼ 9.5,
2.7 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.66 (s, 1H), 7.41 (td, J ¼ 8.1,
6.1 Hz,1H), 7.35 (dd, J ¼ 8.5,1.5 Hz,1H), 7.26e7.20 (m, 2H), 7.17e7.10
(m, 1H), 6.57 (d, J ¼ 9.5 Hz, 1H), 5.22 (s, 2H); LC-MS (m/z) 320.0
(MþH)þ; HPLC purity (method C): 99%, tR ¼ 8.3 min.
J ¼ 1.3 Hz, 1H), 7.81e7.76 (m, 1H), 7.42e7.31 (m, 5H), 7.34e7.24 (m,
1H), 6.59 (d, J ¼ 9.5 Hz, 1H), 5.22 (s, 2H), 4.08 (s, 3H); 13C NMR
(101 MHz, DMSO‑d6)
d 161.2, 140.7, 140.0, 137.8, 137.3, 134.3, 132.7,
129.0, 128.1, 128.0, 122.8, 121.7, 120.4, 119.1, 119.0, 106.3, 52.1, 35.8;
LC-MS (m/z) 316.3 (MþH)þ; HPLC purity (method B): 97%,
tR ¼ 6.4 min.
1-[(2-Chlorophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
dihydropyridin-2(1H)-one (12). Synthesized as described in the
5-(1H-Indazol-6-yl)-1-(2-phenylethyl)-1,2-dihydropyridin-2(1H)-
one (6). Synthesized as described in the General Procedure A using
boronic ester/acid 32a and 6-bromo-1H-indazole in 42% yield as a
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33c in 65% yield
as a white amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.12
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
d
beige amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
d
13.11 (s,
(s, 1H), 8.27 (d, J ¼ 2.6 Hz, 1H), 8.10e8.04 (m, 1H), 8.01 (dd, J ¼ 9.5,
2.7 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.66 (s, 1H), 7.56e7.49 (m, 1H),
7.39e7.28 (m, 3H), 7.00e6.91 (m, 1H), 6.61 (d, J ¼ 9.5 Hz, 1H), 5.49
1H), 8.06 (s, 1H), 7.94 (d, J ¼ 2.7 Hz,1H), 7.86 (dd, J ¼ 9.3, 2.7 Hz, 1H),
7.77 (d, J ¼ 8.4 Hz, 1H), 7.51 (s, 1H), 7.38e7.22 (m, 5H), 7.20 (dd,
J ¼ 8.5, 1.3 Hz, 1H), 6.52 (d, J ¼ 9.4 Hz, 1H), 4.27e4.18 (m, 2H),
3.06e2.98 (m, 2H); LC-MS (m/z) 316.3 (MþH)þ; HPLC purity
(method C): 95%, tR ¼ 8.3 min.
(s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 161.1, 141.0, 140.4, 137.6,
134.8, 134.3, 133.9, 132.4, 129.9, 129.5, 128.4, 127.9, 122.4, 121.6,
120.5, 119.2, 106.7, 50.3; LC-MS (m/z) 336.3 (MþH)þ; HPLC purity
(method B): 99%, tR ¼ 6.3 min.
5-(1H-Indazol-6-yl)-1-(2-oxo-2-phenylethyl)-1,2-dihydropyridin-
2(1H)-one (7). Synthesized as described in the General Procedure A
using boronic ester/acid 32b and 6-bromo-1H-indazole in 32% yield
1-[(3-Chlorophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
dihydropyridin-2(1H)-one (13). Synthesized as described in the
as a light brown solid: 1H NMR (400 MHz, DMSO‑d6)
d
13.13 (s, 1H),
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33d in 7% yield
as a brown amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.12
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
8.21 (d, J ¼ 2.7 Hz, 1H), 8.13e8.06 (m, 3H), 8.00 (dd, J ¼ 9.5, 2.7 Hz,
1H), 7.82 (d, J ¼ 8.5 Hz, 1H), 7.77e7.72 (m, 1H), 7.68e7.59 (m, 3H),
d
7.33 (dd, J ¼ 8.5, 1.5 Hz, 1H), 6.58 (d, J ¼ 9.5 Hz, 1H), 5.61 (s, 2H); 13
C
(s, 1H), 8.38 (d, J ¼ 2.6 Hz, 1H), 8.07 (s, 1H), 7.94 (dd, J ¼ 9.5, 2.7 Hz,
1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.66 (s, 1H), 7.47 (s, 1H), 7.41e7.32 (m,
4H), 6.57 (d, J ¼ 9.5 Hz, 1H), 5.21 (s, 2H); 13C NMR (101 MHz,
NMR (101 MHz, DMSO‑d6)
d 193.3, 161.1, 141.0, 140.3, 138.2, 135.1,
134.5, 133.9, 129.4, 128.4, 122.3, 121.6, 119.9, 119.2, 11þ8.7, 106.5, 55.6;
m.p. >210 ꢀC decomp.; LC-MS (m/z) 330.1 (MþH) ; HPLC purity
(method C): 94%, tR ¼ 7.2 min.
DMSO‑d6) d 161.1,140.9,140.3,137.2, 134.3,133.9, 133.5, 130.9,128.2,
128.0, 127.0, 122.4, 121.5, 120.5, 119.4, 119.3, 106.7, 51.7, 40.0; LC-MS
(m/z) 336.2 (MþH)þ; HPLC purity (method C): 98%, tR ¼ 9.8 min.
1-[(3,4-Dichlorophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
5-(1H-Indazol-6-yl)-1-(morpholine-4-carbonyl)-1,2-
dihydropyridin-2(1H)-one (8). Synthesized as described in the
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 31c in 53% yield
as a brown amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.24
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
dihydropyridin-2(1H)-one (14). Synthesized as described in the
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33e in 60% yield
as a brown amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.12
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
d
(s, 1H), 8.71 (d, J ¼ 2.6 Hz, 1H), 8.28 (dd, J ¼ 8.4, 2.6 Hz, 1H), 8.14 (s,
1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.83 (s, 1H), 7.47 (dd, J ¼ 8.4, 1.3 Hz, 1H),
7.31 (d, J ¼ 8.4 Hz, 1H), 3.71e3.65 (m, 4H), 3.63 (s, 2H), 3.46 (s, 2H);
LC-MS (m/z) 325.1 (MþH)þ; HPLC purity (method C): 90%,
tR ¼ 6.3 min.
d
(s, 1H), 8.39 (d, J ¼ 2.6 Hz, 1H), 8.07 (s, 1H), 7.94 (dd, J ¼ 9.5, 2.7 Hz,
1H), 7.81 (d, J ¼ 8.5 Hz, 1H), 7.70 (d, J ¼ 2.0 Hz, 1H), 7.66 (s, 1H), 7.63
(d, J ¼ 8.3 Hz, 1H), 7.40 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.35 (dd, J ¼ 8.4,
1.5 Hz, 1H), 6.57 (d, J ¼ 9.5 Hz, 1H), 5.19 (s, 2H); 13C NMR (101 MHz,
5-(1H-Indazol-6-yl)-1-methyl-1,2-dihydropyridin-2(1H)-one (9).
Synthesized as described in the General Procedure A using boronic
ester/acid 32c and 6-bromo-1H-indazole in 53% yield as a brown
DMSO‑d6) d 161.1, 140.9, 140.4, 138.9, 137.2, 134.3, 133.9, 131.5, 131.2,
130.7, 130.6, 128.8, 122.4, 121.5, 120.5, 119.5, 119.3, 106.8, 51.3; LC-
MS (m/z) 371.4 (MþH)þ; HPLC purity (method C): 97%,
tR ¼ 11.1 min.
amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
d 13.11 (s, 1H), 8.19
(d, J ¼ 2.7 Hz, 1H), 8.07 (s, 1H), 7.89 (dd, J ¼ 9.4, 2.7 Hz, 1H), 7.80 (d,
5-(1H-Indazol-6-yl)-1-{[2-(trifluoromethyl)phenyl]methyl}-1,2-
dihydropyridin-2(1H)-one (15). Synthesized as described in the
J ¼ 8.4 Hz, 1H), 7.65 (s, 1H), 7.34 (dd, J ¼ 8.5, 1.6 Hz, 1H), 6.51 (d,
J ¼ 9.4 Hz, 1H), 3.54 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d
161.6,
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33f in 48% yield
as a beige solid: 1H NMR (400 MHz, DMSO‑d6)
13.12 (s, 1H), 8.30
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
141.0, 139.7, 138.2, 134.6,133.9, 122.3,121.5, 119.6,119.3, 118.6, 106.5,
37.5; LC-MS (m/z) 226.0 (MþH)þ; HPLC purity (method C): 96%,
tR ¼ 3.3 min.
d
(d, J ¼ 2.7 Hz,1H), 8.09e8.02 (m, 2H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.69 (s,
1H), 7.64 (t, J ¼ 7.6 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz, 1H), 7.36 (dd, J ¼ 8.5,
1.6 Hz, 1H), 6.93 (d, J ¼ 7.8 Hz, 1H), 6.63 (d, J ¼ 9.5 Hz, 1H), 5.44 (s,
1-[(2-Fluorophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
dihydropyridin-2(1H)-one (10). Synthesized as described in the
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33a in 30% yield
as a beige solid: 1H NMR (400 MHz, DMSO‑d6)
13.12 (s, 1H), 8.27
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
2H); 13C NMR (101 MHz, DMSO- d6)
d 161.1, 141.0, 140.6, 137.8, 136.1,
134.3, 133.9, 133.5, 128.1, 127.0, 126.6 (q, J ¼ 5.2 Hz), 126.2 (q,
J ¼ 30.4 Hz), 124.9 (q, J ¼ 273.9 Hz), 122.4, 121.5, 120.6, 119.4, 119.3,
106.7, 49.3; m.p. >205 ꢀC decomp.; LC-MS (m/z) 370.0 (MþH)þ;
HPLC purity (method C): 98%, tR ¼ 10.5 min.
d
(d, J ¼ 2.7 Hz, 1H), 8.07 (t, J ¼ 1.3 Hz, 1H), 7.96 (dd, J ¼ 9.5, 2.7 Hz,
1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.69e7.63 (m, 1H), 7.40e7.31 (m, 2H),
7.27e7.15 (m, 3H), 6.56 (d, J ¼ 9.5 Hz, 1H), 5.27 (s, 2H); 13C NMR
5-(1H-Indazol-6-yl)-1-{[3-(trifluoromethyl)phenyl]methyl}-1,2-
dihydropyridin-2(1H)-one (16). Synthesized as described in the
(101 MHz, DMSO‑d6)
137.5, 134.4, 133.90, 130.1 (d, J ¼ 4.4 Hz), 130.0 (d, J ¼ 3.1 Hz), 125.0
d
161.1, 160.6 (d, J ¼ 245.2 Hz), 141.0, 140.3,
General
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
12

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
dioxaborolan-2-yl)-1H-indazole and pyridinone 33g in 66% yield as
d
13.10 (s, 1H), 8.19 (d, J ¼ 2.7 Hz, 1H), 8.07 (s, 1H), 7.91 (dd, J ¼ 9.4,
a red amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
d
13.12 (s,1H),
2.7 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.63 (s, 1H), 7.32 (dd, J ¼ 8.5,
1.5 Hz, 1H), 6.98 (t, J ¼ 7.7 Hz, 1H), 6.59e6.43 (m, 4H), 5.09 (s, 2H),
8.43 (d, J ¼ 2.7 Hz, 1H), 8.08 (s, 1H), 7.95 (dd, J ¼ 9.5, 2.7 Hz, 1H),
7.84e7.78 (m, 2H), 7.72e7.65 (m, 3H), 7.60 (t, J ¼ 7.7 Hz, 1H), 7.35
(dd, J ¼ 8.5,1.5 Hz,1H), 6.58 (d, J ¼ 9.5 Hz,1H), 5.30 (s, 2H); 13C NMR
5.07 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 161.1, 149.4, 141.0,
139.8, 138.5, 137.2, 134.5, 133.9, 129.5, 122.3, 121.5, 120.4, 119.3,
118.9, 115.4, 113.6, 113.2, 106.6, 51.9; LC-MS (m/z) 317.2 (MþH)þ;
HPLC purity (method C): 99%, tR ¼ 11.8 min.
(101 MHz, DMSO‑d6)
d 161.2, 140.9, 140.3, 139.2, 137.3, 134.3, 133.9,
132.4, 130.1, 129.6 (q, J ¼ 31.6 Hz), 125.1 (q, J ¼ 3.8 Hz), 124.8 (q,
J ¼ 3.9 Hz), 124.6 (q, J ¼ 272.4 Hz), 122.4, 121.5, 120.5, 119.4, 119.3,
106.8, 51.9; LC-MS (m/z) 370.1 (MþH)þ; HPLC purity (method C):
98%, tR ¼ 10.6 min.
N-(3-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]
methyl}phenyl)acetamide (22). Synthesized as described in the
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33n in 62% yield
as a light yellow solid: 1H NMR (400 MHz, DMSO‑d6)
13.13 (s, 1H),
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
5-(1H-Indazol-6-yl)-1-{[4-(trifluoromethyl)phenyl]methyl}-1,2-
dihydropyridin-2(1H)-one (17). Synthesized as described in the
d
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33h in 40% yield
as a beige amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.12 (s,
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
9.96 (s, 1H), 8.30 (d, J ¼ 2.7 Hz, 1H), 8.07 (s, 1H), 7.94 (dd, J ¼ 9.5,
2.7 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.65 (s, 1H), 7.60e7.54 (m, 1H),
7.48 (t, J ¼ 1.8 Hz, 1H), 7.34 (dd, J ¼ 8.4, 1.5 Hz, 1H), 7.27 (t, J ¼ 7.9 Hz,
1H), 7.04 (d, J ¼ 7.6 Hz, 1H), 6.56 (d, J ¼ 9.5 Hz, 1H), 5.19 (s, 2H), 2.01
(s, 3H); m.p. 132e135 ꢀC; LC-MS (m/z) 359.3 (MþH)þ; HPLC purity
(method A): 99%, tR ¼ 14.6 min.
d
1H), 8.39 (d, J ¼ 2.6 Hz,1H), 8.07 (s,1H), 7.96 (dd, J ¼ 9.5, 2.6 Hz,1H),
7.81 (d, J ¼ 8.4 Hz, 1H), 7.73 (d, J ¼ 8.1 Hz, 2H), 7.67 (s, 1H), 7.58 (d,
J ¼ 8.0 Hz, 2H), 7.35 (dd, J ¼ 8.5, 1.4 Hz, 1H), 6.58 (d, J ¼ 9.5 Hz, 1H),
5.31 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
161.1, 142.5, 140.9,
5-(3-Amino-1H-indazol-6-yl)-1-benzyl-1,2-dihydropyridin-
2(1H)-one (23). Synthesized as described in the General Procedure
A using boronic ester/acid 29 and 3-amino-6-bromo-1H-indazole in
140.3, 137.4, 134.3, 133.9, 128.9, 125.9 (q, J ¼ 4.1 Hz), 122.4, 121.5,
120.5, 119.4, 119.3, 106.7, 51.9; LC-MS (m/z) 370.1 (MþH)þ; HPLC
purity (method C): 94%, tR ¼ 10.8 min.
43% yield as a white solid: 1H NMR (400 MHz, DMSO‑d6)
d 11.43 (s,
2-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]methyl}
benzamide (18). Synthesized as described in the General Procedure
B using nitrile 35 in 46% yield as a brown amorphous solid: 1H NMR
1H), 8.27 (d, J ¼ 2.7 Hz, 1H), 7.88 (dd, J ¼ 9.5, 2.7 Hz, 1H), 7.72 (d,
J ¼ 8.4 Hz, 1H), 7.40e7.32 (m, 4H), 7.35e7.24 (m, 2H), 7.11 (dd,
J ¼ 8.3, 1.5 Hz, 1H), 6.54 (d, J ¼ 9.4 Hz, 1H), 5.36 (s, 2H), 5.21 (s, 2H);
(400 MHz, DMSO‑d6)
d
13.10 (s, 1H), 8.33 (d, J ¼ 2.7 Hz, 1H), 8.09 (s,
13C NMR (101 MHz, DMSO‑d6)
d 161.2, 158.9, 158.5, 147.6, 143.0,
1H), 8.09e8.04 (m, 1H), 7.96 (dd, J ¼ 9.4, 2.7 Hz, 1H), 7.80 (dd,
J ¼ 8.5, 0.8 Hz, 1H), 7.65 (s, 1H), 7.59e7.54 (m, 2H), 7.41 (td, J ¼ 7.5,
1.6 Hz, 1H), 7.40e7.29 (m, 2H), 7.11 (dd, J ¼ 7.6, 1.3 Hz, 1H), 6.57 (d,
139.8, 137.8, 137.6, 137.3, 129.0, 128.2, 128.0, 122.1, 120.5, 118.7, 118.þ2,
112.4, 106.7, 52.0; m.p. 210e213 ꢀC; LC-MS (m/z) 317.2 (MþH) ;
HPLC purity (method C): 100%, tR ¼ 4.2 min.
J ¼ 9.4 Hz, 1H), 5.41 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
170.9,
5-(3-Amino-1H-indazol-6-yl)-1-[(2-fluorophenyl)methyl]-1,2-
dihydropyridin-2(1H)-one (24). Synthesized as described in the
General Procedure A using boronic ester/acid 34a and 3-amino-6-
161.4,140.1,137.8,136.0,135.9, 134.4,130.6,128.2,128.1,127.6,122.3,
121.5, 120.4, 119.2, 119.1, 106.7, 50.1; LC-MS (m/z) 345.2 (MþH)þ;
HPLC purity (method C): 96%, tR ¼ 4.8 min.
bromo-1H-indazole in 57% yield as
(400 MHz, DMSO‑d6)
J ¼ 9.5, 2.7 Hz, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.39e7.32 (m, 2H),
7.27e7.20 (m, 1H), 7.20e7.16 (m, 2H), 7.11 (dd, J ¼ 8.4, 1.5 Hz, 1H),
6.54 (d, J ¼ 9.5 Hz, 1H), 5.37 (s, 2H), 5.26 (s, 2H); 13C NMR (101 MHz,
a
beige solid: 1H NMR
11.45 (s,1H), 8.22 (d, J ¼ 2.7 Hz,1H), 7.92 (dd,
4-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]methyl}
benzamide (19). Synthesized as described in the General Procedure
B using nitrile 36 in 72% yield as a white amorphous solid: 1H NMR
d
(400 MHz, DMSO‑d6)
d
13.17 (s, 1H), 8.34 (d, J ¼ 2.7 Hz, 1H), 8.07 (s,
1H), 7.97e7.91 (m, 2H), 7.88e7.77 (m, 3H), 7.66 (s, 1H), 7.43 (d,
J ¼ 8.0 Hz, 2H), 7.37e7.31 (m, 2H), 6.57 (d, J ¼ 9.4 Hz, 1H), 5.26 (s,
DMSO‑d6)
d
161.1, 160.6 (d, J ¼ 245.1 Hz), 149.6, 142.5, 140.3, 137.3,
134.4, 130.0 (d, J ¼ 8.4 Hz), 130.0 (d, J ¼ 4.0 Hz), 125.0 (d, J ¼ 3.3 Hz),
124.4 (d, J ¼ 14.6 Hz), 121.4, 120.3, 119.4, 116.3, 115.8 (d, J ¼ 21.0 Hz),
113.6, 106.1, 46.8; m.p. 209e212 ꢀC; LC-MS (m/z) 335.3 (MþH)þ;
HPLC purity (method C): 99%, tR ¼ 4.7 min.
2H); 13C NMR (101 MHz, DMSO‑d6)
d 168.0, 161.1, 141.0, 140.9, 140.2,
137.3, 134.3, 134.0, 133.8, 128.2, 127.9, 122.3, 121.5, 120.5, 119.3,
106.8, 51.9; LC-MS (m/z) 345.0 (MþH)þ; HPLC purity (method A):
99%, tR ¼ 13.4 min.
5-(3-Amino-1H-indazol-6-yl)-1-[(3-fluorophenyl)methyl]-1,2-
dihydropyridin-2(1H)-one (25). Synthesized as described in the
General Procedure A using boronic ester/acid 34b and 3-amino-6-
bromo-1H-indazole in 72% yield as a brown solid: 1H NMR
3-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]methyl}
benzamide (20). Synthesized as described in the General Procedure
A using 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-inda-
zole and pyridinone 33k in 70% yield as a brown solid: 1H NMR
(400 MHz, DMSO‑d6)
d
11.45 (s, 1H), 8.31 (d, J ¼ 2.6 Hz,1H), 7.90 (dd,
(400 MHz, DMSO‑d6)
d
13.11 (s, 1H), 8.35 (d, J ¼ 2.7 Hz, 1H), 8.07 (s,
J ¼ 9.5, 2.7 Hz, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.40 (td, J ¼ 8.2, 6.3 Hz,
1H), 7.35 (dd, J ¼ 1.5, 0.8 Hz, 1H), 7.26e7.18 (m, 2H), 7.17e7.09 (m,
2H), 6.55 (d, J ¼ 9.4 Hz, 1H), 5.37 (s, 2H), 5.21 (s, 2H); 13C NMR
1H), 7.97 (s, 1H), 7.94 (dd, J ¼ 9.5, 2.7 Hz, 1H), 7.89 (s, 1H), 7.80 (t,
J ¼ 8.4 Hz, 2H), 7.66 (s, 1H), 7.52 (dt, J ¼ 7.8, 1.4 Hz, 1H), 7.43 (t,
J ¼ 7.6 Hz, 1H), 7.38e7.32 (m, 2H), 6.57 (d, J ¼ 9.5 Hz, 1H), 5.26 (s,
(101 MHz, DMSO‑d6)
d
162.6 (d, J ¼ 243.7 Hz), 161.1, 149.6, 142.5,
2H); 13C NMR (101 MHz, DMSO‑d6)
d
168.1, 161.1, 141.0, 140.1, 138.0,
140.7 (d, J ¼ 7.4 Hz), 140.2, 137.0, 134.4, 131.0 (d, J ¼ 8.3 Hz), 124.3 (d,
J ¼ 2.7 Hz), 121.3, 120.4, 119.7, 116.3, 115.1 (d, J ¼ 21.9 Hz), 114.8 (d,
J ¼ 20.9 Hz), 113.6, 106.1, 51.6; m.p. 82e85 ꢀC; LC-MS (m/z) 335.2
(MþH)þ; HPLC purity (method C): 99%, tR ¼ 4.6 min.
137.3,135.1,134.4,133.9,131.0,128.9,127.6,126.9,122.3,121.5,120.5,
119.3, 119.2, 106.7, 52.0; m.p. > 220 ꢀC decomp.; LC-MS (m/z) 345.1
(MþH)þ; HPLC purity (method C): 95%, tR ¼ 4.4 min.
1-[(3-Aminophenyl)methyl]-5-(1H-indazol-6-yl)-1,2-
5-(3-amino-1H-indazol-6-yl)-1-[(3-chlorophenyl)methyl]-1,2-
dihydropyridin-2(1H)-one (26). Synthesized as described in the
General Procedure A using boronic ester/acid 34c and 3-amino-6-
dihydropyridin-2(1H)-one (21). To a Boc-protected compound 37
(86 mg, 0.20 mmol) 4 M HCl in 1,4-dioxane was added (2 mL). After
stirring at room temperature for 3 h, the reaction mixture was
diluted with water, neutralized with saturated aqueous solution of
NaHCO3 and extracted with EtOAc (three times). The organic layers
were combined, dried over MgSO4, filtered off and concentrated.
The crude product was purified by column chromatography on
silica gel (dichloromethane/methanol 9/1) to give 21 as a white
amorphous solid (41 mg, 89%): 1H NMR (400 MHz, DMSO‑d6)
bromo-1H-indazole in 78% yield as
(400 MHz, DMSO‑d6)
a
grey solid: 1H NMR
11.45 (s,1H), 8.32 (d, J ¼ 2.5 Hz,1H), 7.90 (dd,
d
J ¼ 9.5, 2.7 Hz, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.47 (br s, 1H), 7.42e7.33
(m, 4H), 7.13 (dd, J ¼ 8.4, 1.5 Hz, 1H), 6.55 (d, J ¼ 9.5 Hz, 1H), 5.36 (s,
2H), 5.20 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 161.1, 149.6, 142.5,
140.3, 140.3, 137.0, 134.4, 133.5, 130.9, 128.2, 128.0, 127.0, 121.3,
120.4, 119.7, 116.3, 113.6, 106.1, 51.6; m.p. 175e177 ꢀC; LC-MS (m/z)
13

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
350.9 (MþH)þ; HPLC purity (method C): 100%, tR ¼ 7.1 min; HRMS
m/z [Mþ1]þ calcd for C19H15ClN4O 351.1007, found 351.1007.
5-(3-Amino-1H-indazol-6-yl)-1-{[3-(trifluoromethyl)phenyl]
(MþH)þ; HPLC purity (method B): 98%, tR ¼ 5.5 min.
tert-Butyl N-(3-{[5-(1H-indazol-6-yl)-2-oxo-1,2-dihydropyridin-
1-yl]methyl}phenyl)carbamate (37). Synthesized as described in the
methyl}-1,2-dihydropyridin-2(1H)-one
(27).
Synthesized
as
General
dioxaborolan-2-yl)-1H-indazole and pyridinone 33l in 23% yield
as a white amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
13.11
Procedure
A
using
6-(4,4,5,5-tetramethyl-1,3,2-
described in the General Procedure A using boronic ester/acid 34d
and 3-amino-6-bromo-1H-indazole in 22% yield as a brown
d
amorphous solid: 1H NMR (400 MHz, DMSO‑d6)
d
11.4 (s, 1H), 8.53
(s, 1H), 9.34 (s, 1H), 8.26 (d, J ¼ 2.7 Hz, 1H), 8.07 (s, 1H), 7.93 (dd,
J ¼ 9.5, 2.7 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.65 (s, 1H), 7.46 (s, 1H),
7.40e7.32 (m, 2H), 7.22 (t, J ¼ 7.9 Hz, 1H), 6.94 (dt, J ¼ 7.6, 1.3 Hz,
1H), 6.56 (d, J ¼ 9.4 Hz, 1H), 5.17 (s, 2H), 1.45 (s, 9H); 13C NMR
(d, J ¼ 2.7 Hz, 1H), 8.03 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd, J ¼ 9.5, 2.7 Hz,
1H), 7.79 (s, 1H), 7.68 (t, J ¼ 6.9 Hz, 2H), 7.61 (d, J ¼ 7.7 Hz, 1H), 7.58
(s, 1H), 7.43 (dd, J ¼ 8.6, 1.5 Hz, 1H), 6.58 (d, J ¼ 9.5 Hz, 1H), 5.43 (s,
2H), 5.30 (s, 2H); LC-MS (m/z) 385.1 (MþH)þ; HPLC purity (method
C): 91%, tR ¼ 7.0 min.
(101 MHz, DMSO‑d6)
d 161.1, 153.2, 141.0, 140.2, 140.0, 138.4, 137.3,
134.5, 133.9, 129.2, 122.3, 121.7, 121.5, 120.4, 119.3, 119.1, 117.8, 106.7,
79.5, 51.9, 28.6; LC-MS (m/z) 416.9 (MþH)þ; HPLC purity (method
B): 100%, tR ¼ 6.7 min.
N-(3-{[5-(3-Amino-1H-indazol-6-yl)-2-oxo-1,2-dihydropyridin-
1-yl]methyl}phenyl)acetamide (28). Synthesized as described in the
General Procedure A using boronic ester/acid 34e and 3-amino-6-
bromo-1H-indazole in 67% yield as
(400 MHz, DMSO‑d6)
a
grey solid: 1H NMR
4.3. Biological section
d
11.44 (s, 1H), 9.95 (s, 1H), 8.23 (d, J ¼ 2.7 Hz,
1H), 7.89 (dd, J ¼ 9.5, 2.7 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz, 1H), 7.57 (d,
J ¼ 7.9 Hz, 1H), 7.47 (d, J ¼ 2.0 Hz, 1H), 7.34 (s, 1H), 7.26 (t, J ¼ 7.9 Hz,
1H), 7.12 (dd, J ¼ 8.4, 1.5 Hz, 1H), 7.03 (d, J ¼ 7.6 Hz, 1H), 6.54 (d,
J ¼ 9.4 Hz, 1H), 5.36 (s, 2H), 5.18 (s, 2H), 2.01 (s, 3H); 13C NMR
4.3.1. In vitro kinase assay
IC50 experiments for MNK1 [Carna Bioscience, #02e145] were
performed on white, 96-wells plate with 20
250 M substrate peptide GRSRSRSRSR [Lipopharm]. Kinase reac-
tion was run for 2 h at room temperature in assay buffer: 60 mM
HEPES, 3 mM MgCl2, 3 mM MnCl2, 50 g/ml PEG20, 3 M sodium
mM ATP [Promega] and
m
(101 MHz, DMSO‑d6)
d 168.8, 161.1, 149.6, 142.5, 140.0, 138.4, 137.0,
134.5, 129.3, 122.7, 121.3, 120.4, 119.4, 118.6, 118.4, 116.3, 113þ.6, 106.1,
51.9, 24.4; m.p. 148e153 ꢀC; LC-MS (m/z) 374.1 (MþH) ; HPLC
purity (method A): 100%, tR ¼ 11.9 min.
m
m
ortovanadate, 1 mM DTT, pH 7.5. ADP-Glo Kinase Assay kit [Prom-
ega, #V9103] was used to detect ADP produced in the kinase re-
action. For inhibition constant (Ki) experiments the compounds
were tested in MNK1 reaction with 5 different ATP concentrations:
1-Benzyl-5-[5-bromo-1-(triphenylmethyl)pyrazolo[3,4-b]pyridin-
3-yl]pyridine-2(1H)-one (30). The boronic ester/acid 29 (100 mg,
0.44 mmol, 1.0 eq.) was dissolved in mixture of acetonitrile/toluene
(2/1). Saturated aqueous solution of NaHCO3 (1.5 mL), 5-bromo-3-
2
m
M, 6
[Carna Bioscience, #02e146] were performed on white, 96-wells
plate with 100 M ATP [Promega] and 160 M substrate peptide
mM, 20 mM, 60 mM, 140 mM. IC50 experiments for MNK2
iodo-1-(triphenylmethyl)pyrazolo[3,4-b]pyridine
(170
mg,
m
m
0.31 mmol, 1.4 eq.) and [1,10-bis(diphenylphosphino)ferrocene]
palladium(II) chloride complex with dichloromethane (7 mg, 0.03
eq.) were added and reaction mixture was flushed with argon for
10 min. Then, the reaction mixture was heated at 60 ꢀC for 7 h, after
cooling to room temperature, filtered through Celite, washed with
EtOAc and concentrated in vacuo. The crude product was purified
by flash column chromatography (dichloromethane/methanol 99/
1) to afford 30 (96 mg, 50%) as a beige amorphous solid: 1H NMR
GRSRSRSRSR [Lipopharm]. Kinase reaction was run for 2 h at room
temperature in assay buffer was 50 mM MOPS, 5 mM MgCl2,
0.4 mM EDTA, 1 mM DTT, pH 7.5. ADP-Glo Kinase Assay kit
[Promega, #V9103] was used to detect ADP produced in the kinase
reaction. For inhibition constant experiments the compounds were
tested in MNK2 reaction with 5 different ATP concentrations:
10 mM, 30 mM, 100 mM, 300 mM, 700 mM. IC50 and inhibition con-
stant values were determined by fitting variable slope (four pa-
rameters) model or competitive inhibition model, respectively, in
GraphPad Prism 7.04 Software.
(400 MHz, DMSO‑d6)
1H), 8.37 (d, J ¼ 2.2 Hz, 1H), 7.84 (dd, J ¼ 9.5, 2.6 Hz, 1H), 7.35 (d,
J ¼ 3.7 Hz, 4H), 7.31e7.21 (m, 16H), 6.58 (d, J ¼ 9.5 Hz, 1H), 5.28 (s,
d
8.86 (d, J ¼ 2.1 Hz, 1H), 8.49 (d, J ¼ 2.6 Hz,
2H); 13C NMR (101 MHz, DMSO‑d6)
d
161.2, 151.0, 148.8, 142.7, 138.7,
4.3.2. Cell culture
138.3,137.8,137.6,133.0,129.9,129.0,128.0,127.9,127.9,127.4,121.2,
115.7, 113.4, 111.6, 78.5, 52.0; UPLC (m/z) 625.0 (MþH)þ; 97%,
tR ¼ 4.9 min.
MOLM-16 [DSMZ; ACC 555] cell line was cultured in RPMI 1640
containing 20% FBS, 1% pyruvate and 1% penicillin/streptomycin at
37 ꢀC in a humidified incubator with 5% CO2. All media and Feta
Bovine Serum (FBS) were purchased from Gibco.
2-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]methyl}
benzonitrile (35). Synthesized as described in the General Procedure
A using 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-inda-
zole and pyridinone 33i in 62% yield as a beige amorphous solid: 1H
4.3.3. Western blot analysis
Compounds 24 and 26 were synthesized internally according to
procedures described above. Drugs were dissolved in DMSO, and
10 mM aliquots were stored at -80 ꢀC. The following commercially
available antibodies were purchased from Cell Signaling Technol-
ogy: phospho-eIF4e [Ser209, catalog 9741L], eIF4e [catalog 9742L].
b-Actin [catalog A2066] antibody was purchased from Sigma. Cells
were treated with either compound 24 or 26 at the indicated times,
and pellets were harvested to obtain protein extracts. Cell pellets
were washed with cold PBS and lysed in ice-cold RIPA buffer with
phosphatase inhibitors [Thermo Scientific, 89900]. After sonication,
cell lysates were centrifuged at 16 900 g for 10 min. The superna-
tants were collected, and protein concentrations were determined
using Bradford reagent [Sigma, B6916]. Equal amounts of protein in
each well was resolved on MiniProtean Gels [Biorad, 456e8036]
and transferred using Trans-Blot Turbo PVDF Transfer Packs [Bio-
rad, 1704157]. Membranes were blocked for 40 min at room tem-
perature in 5% fat-free milk, incubated with primary antibodies
NMR (400 MHz, DMSO‑d6)
d
13.14 (s, 1H), 8.38 (d, J ¼ 2.5 Hz, 1H),
8.08 (s, 1H), 8.01 (dd, J ¼ 9.5, 2.7 Hz, 1H), 7.90 (d, J ¼ 7.6 Hz, 1H), 7.82
(d, J ¼ 8.4 Hz, 1H), 7.75e7.65 (m, 2H), 7.50 (t, J ¼ 8.0 Hz, 1H), 7.37
(dd, J ¼ 8.5, 1.3 Hz, 1H), 7.22 (d, J ¼ 7.6 Hz, 1H), 6.58 (d, J ¼ 9.5 Hz,
1H), 5.40 (s, 2H); LC-MS (m/z) 326.7 (MþH)þ; HPLC purity (method
B): 99%, tR ¼ 5.5 min.
4-{[5-(1H-Indazol-6-yl)-2-oxo-1,2-dihydropyridin-1-yl]methyl}
benzonitrile (36). Synthesized as described in the General Procedure
A using 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-inda-
zole and pyridinone 33j in 61% yield as a beige amorphous solid: 1H
NMR (400 MHz, DMSO‑d6)
d
13.12 (s, 1H), 8.38 (d, J ¼ 2.6 Hz, 1H),
8.07 (s, 1H), 7.96 (dd, J ¼ 9.5, 2.6 Hz, 1H), 7.89e7.78 (m, 3H), 7.67 (s,
1H), 7.54 (d, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 9.4 Hz, 1H), 6.58 (d,
J ¼ 9.5 Hz, 1H), 5.29 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 161.1,
143.4, 140.9, 140.4, 137.4, 134.3, 133.9, 132.9, 129.0, 122.4, 121.5,
120.5, 119.4, 119.3, 119.1, 110.7, 106.8, 52.1; LC-MS (m/z) 326.7
14

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
overnight, washed 4 times in PBST, then incubated with HRP-
conjugated secondary antibodies for 1 h at room temperature. Af-
ter four PBST washes, blots were developed using ECL reagent
[Biorad, 170e5061] on ChemiDoc (Biorad MP Imaging System).
Quantitative image analysis was performed using ImageLab soft-
ware (Biorad).
concentration of filtrate was determined spectrophotometrically by
measurement of UV-VIS absorption spectrum. Concentrations were
calculated based on equations resulted from the calibration curves
(in duplicates, 8 calibration points, dilution factor x2, 500
3.91 M, including blank sample).
mM /
m
4.4.3. Protein plasma binding assay
The assay of plasma protein binding was performed using Rapid
Equilibrium Dialysis (RED) system of Thermo Scientific. Com-
pounds for PPB testing were prepared as 10 mM stock solutions in
DMSO. Plasma (human, mouse and rat) was spiked with investi-
gated compound (in triplicates) at chosen initial concentration
4.3.4. Cytokine signaling pathways analysis
The level of cytokines in mouse serum samples were measured
in duplicates using Cytokine & Chemokine 26-Plex Mouse Pro-
cartaPlex™ Panel [Thermo Fisher; EPX260-26088-901,] on Luminex
platform according to the manufacturer’s recommendations.
(5 mM for compound 24, 1 mM for compound 26) and transferred
into plasma compartment of RED device. Dialysis was carried out
against PBS buffer, pH 7.4, for 4 h at 37 ꢀC, while agitating
(300 rpm). Two reference compounds were used as control com-
pounds: Warfarin and Metoprolol. After the incubation, samples
were collected from both compartments. Plasma samples were
spiked with aliquot volume of buffer, while buffer samples were
spiked with aliquot volume of plasma to ensure a homogeneous
matrix. 8-point calibration curves (dilution factor x2, including
blank sample) were prepared in plasma spiked with buffer. Proteins
were precipitated with the addition of cold acetonitrile, next
samples were centrifuged at 2000 rpm for 15 min at 4 ꢀC. Com-
pound concentration was determined in both compartments using
LC-MS technique. HPLC-MS consisted of a HPLC of the Dionex, Ul-
timate 3000 and mass spectrometer of Bruker Daltonics, amaZon SL
(ESI-IT). Samples were analyzed in Single Ion Monitoring scan
mode.
4.4. In vitro ADMET assays
4.4.1. Metabolic stability assay
Metabolic stability assay was performed using mice microsomal
fraction (phase I of metabolism) obtained from XenoTech. Com-
pounds for metabolic stability testing were prepared as 10 mM
stock solutions in DMSO. Compounds were incubated in triplicates
at 1 mM initial concentration with microsomal fraction in presence
of NRS (NADPH-regenerating system, 1.3 mM). Final microsomal
incubation (0.3 mg/ml) contained following components in phos-
phate buffer pH 7.4. Control incubations were conducted without
cofactors. Reference compound e Verapamil was used as a positive
control. Incubations were carried out for 1 h on 96 well plates using
heated (þ37 ꢀC) orbital shaker (350 rpm). After the incubation,
reaction was quenched by addition of cold acetonitrile. Plate con-
taining samples was centrifuged at 2000 rpm for 15 min at 4 ꢀC.
Supernatants were analyzed using LC-MS technique. HPLC-MS
consisted of a HPLC of the Dionex, Ultimate 3000 and mass spec-
trometer of Bruker Daltonics, amaZon SL (ESI-IT). Samples were
analyzed in Single Ion Monitoring scan mode.
Percent loss of compound was calculated by normalization of
peak area against time 0, where peak area at T0 is 100%. Elimination
rate constant (h) was determined from the plot of ln (percent loss of
compound) as a time function and equal minor value of linear curve
slope. Half-life time was calculated from the following equation:
Based on concentration results from both compartments PPB
was calculated.
!
Cbuffer
% PPB ¼ 1 ꢃ
x 100%
Cplasma
4.4.4. CYP inhibition assay
CYP inhibition was performed with the use of recombinant
enzymes CYP1A2, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 obtained
from BD Biosciences. Compounds for CYP inhibition testing were
prepared as 10 mM stock solutions in DMSO. Isoform-specific
substrates were incubated at 37 ꢀC individually with P450 en-
zymes (Table 10) and a range of test compound concentrations (1.1,
0:693
k
T1=2
¼
Calculated half-life time was used for intrinsic clearance
calculation:
3.3 and 10 mM) in duplicates. Each isoform was tested separately
Vd*0:693
with one reference compound, known positive control inhibitor.
During preincubation, the 96-well black plates were scanned with a
fluorescence plate reader in order to eliminate false results origi-
nating from autofluorescence of the test compounds. At the end of
the incubation, product formation was monitored with fluores-
cence detection. A decrease in the formation of the metabolite
compared to “no inhibition” control samples was used to calculate
an IC50 value.
CLint
¼
T1=2
where, Vd e volume of distribution equals:
1
Vd ¼
ꢀ
ꢁ
mg
ml
protein concentration
4.4.5. In vivo PK
4.4.2. Kinetic solubility assay
All animals were handled in strict accordance with good animal
practice, and maintained according to the standards of pathogen-
free conditions. Experiment was approved by the 1st Local Ethical
Committee for Animal Research in Krakow, Poland [approval 197/
2014]. The pharmacokinetic profile of compound 24 and 26 was
assessed in 5-week-old female CD-1 mice (outbred colony, Charles
River Laboratories) (3 animals per time point). Compounds were
freshly dissolved in DMSO and then diluted in Captisol (Ligand) for
The kinetic solubility assay is performed to determine the sol-
ubility of compounds in the in vitro assays conditions. Assay in-
vestigates solubility based on precipitation process. Compounds for
kinetic solubility testing were prepared as 10 mM stock solutions in
DMSO. Investigated compounds were diluted (in triplicates) with
€
buffer of interest (water or Sorensen’s buffer pH 7.4) to final con-
centration 500 mM. Mixture was shaken on filter plate at 500 rpm
for 1.5 h at RT. After incubation time filter plate was placed inside
vacuum manifold and filtrated. Samples were collected and
administration with a volume of 10
oral (p.o.; 5 mg/kg) or i.v. (2 mg/kg) route. Animals were sacrificed
ml per 1g of body weight via the
15

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 10
Experimental conditions summary - cytochrome P450 IC50 inhibition determination assay.
P450 isoform
Positive control highest concentration (
m
M)
Substrate concentration (
m
M)
Enzyme concentration (nM)
Incubation length (min)
15
CYP1A2
Furafylline
5
CEC
5
5
CYP2B6
Tranylcypromine
50
Tranylcypromiine
10
EFC
2.5
CEC
25
10
10
30
30
CYP2C19
CYP2D6
CYP3A4
Quinidine
0.05
AMMC
1.5
15
5
30
30
Ketoconazole
0.05
BFC
50
at 8 time points (5, 15, and 30 min and 1, 2, 4, 7, and 24 h) and blood
samples harvested. Plasma samples were collected and stored at
-80 ꢀC for further analysis.
5. Endotoxin survival studies
16 weeks old C57BL/6 male mice, were housed in the Depart-
ment of Genetics, at the Maria Sklodowska-Curie National Research
Institute of Oncology. Experimental protocol was approved by the
2nd Local Ethical Committee for Animal Research in Warsaw,
Poland [approval WAW2/47/2017]. All animals will be checked for
any ongoing inflammation (rash, fever, pain, swelling) prior to the
experiment. The procedure was conducted by an experienced
veterinarian and animals are monitored following 2 h post-LPS
every 30 min and then every 6 h. Mice were injected intraperito-
neally with lethal dose of endotoxin solution in saline (lipopoly-
saccharide [LPS]; L9143-02; Sigma), 20 mg/kg [44], or with saline
alone and monitored every 2 h post injection for 6 h followed by
every 6 h for remaining days. Compounds 24 and 26 were recon-
stituted in DMSO 3% þ DMA 10% þ Captisol 20% formulation and
administrated per os, at the final concentration of 25 mg/kg and
75 mg/kg (survival experiment) or 50 mg/kg and 75 mg/kg, for
compound 26 and 24, respectively, an hour prior to LPS injection
and then with BID regimen every 12 h. The control group was
administrated with the DMSO/DMA/Captisol formulation. To pre-
vent dehydration animals were given subcutaneous fluids (sterile
normal saline) and had constant access to water. Mice were
euthanized by isoflurane overdose and spinal cord dislocation
when they became moribund according to the Murine Sepsis Score
[45]. Necropsy and organs examination was performed on each
mouse according to previous recommendations [46]. Tissues were
flash frozen in liquid nitrogen while blood collected for serum for
downstream analyses.
4.4.5.1. Sample analysis. Investigated compounds 24 and 26 were
dissolved in DMSO in order to obtain 10 mM stock solutions. So-
lutions were used for calibration curve preparation (16 calibration
points, dilution factor x2, 10 000 ng/ml / 0.2 ng/ml, including
blank sample). A solution of compounds 24 and 26 in acetonitrile
and serial dilutions were prepared. The calibration curve was pre-
pared in blank plasma. 5
blank plasma. 200 l of cold acetonitrile was added to each well.
Next, plasma samples with the compounds were prepared in the
same manner as samples. 50 l of each plasma sample was
dispensed into appropriate wells of 96-deep well plate. 200 l of
ml of each solution was added to 45 ml of
m
m
m
cold acetonitrile was added to each well. Plate was centrifuged
(2000ꢁg at 4 ꢀC for 20 min). 170
ml of supernatant was transferred
into a 96-well plate. The plate was covered with a silicone plate
mat. Samples were analyzed using LC-MS technique. Concentra-
tions of compounds in plasma samples were calculated using a
calibration curve prepared in blank mice plasma.
4.4.5.2. Conditions of the liquid chromatography-mass spectrometry.
Dionex Ultimate 3000 RS HPLC of the Thermo Scientific and
Thermo Scientific TSQ Quantiva model was used. The conditions
were: Column HPLC Ascentis Express C18 (5 cm ꢁ 2.1 mm, 2.7
mm),
the injection volume was 1 l. An acetonitrile gradient was used
m
with 0.1% formic acid with 0.1% of formic acid in water at a constant
flowrate of 0.6 ml/min. The programming was the following:
0e0.1 min 5% of ACN, 0.1e2.0 increase of 5e98%, 2.0e3.0 remaining
at 98% of ACN, 3.0e3.2 min decrease of 98e5% and 3.2e4.0 min 5%
of ACN. Mass spectrometry was conducted in tandem with the
triple stage quadrupole analyzer in SRM mode, equipped with an
heated-electrospray ionization source (H-ESI) in positive mode. The
conditions of the main parameters were: spray voltage of 3.5 kV,
sheath gas flow (Arb) of 52, auxiliary gas flow (Arb) of 16, sweep gas
flow (Arb) of 2, drying gas temperature of 420C, ion transfer tube
temperature of 356C and positive polarity. Table 11 shows the
values of precursor ions (m/z), retention time (RT) and collision
energy (V) optimized for the identification of each compound.
6. Statistical analysis
Statistical analysis was performed using the GraphPad Prism 6
software. The survival rates were tested with the log-rank Mantel-
Cox test while the serum cytokines levels with Mann-Whitney U
test. The significance of mean comparison is annotated as follows:
ns, non-significant (p ꢂ 0.05), *p < 0.05. Results were considered
significant when p-value < 0.05.
Funding sources
This work was supported by National Center for Research and
Table 11
Mass spectrometer SRM mode parameters.
Compound
Molecular weight (g/mol)
Precursor m/z (Th)
Product m/z (Th)
Collision energy (V)
RT (min)
24
24
26
26
334.35
334.35
350.81
350.81
335.16
335.16
351.15
351.15
198.04
226.06
198.06
226.06
33.056
25.522
34.169
26.008
1.97
1.97
2.08
2.08
16

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
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