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 xxx (xxxx) xxx

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://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

ꢀ

Kazimiera Pysniak , Urszula Wojcik-T rechcinska , 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

^cDepartment of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, 02-781, Warsaw, Poland

^dDepartment 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.

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.2020.1 13057

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 [1 7]. 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

[1 7,20,28,29]. 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).

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 K_mATP 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 IC₅₀of 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 IC₅₀of 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 Zn^2þbinding

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 180^ꢀ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 (LLE_MNK2

¼

4.8 and

LLE_MNK2¼ 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)*CH₂Cl₂to 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/NEt₃in anhydrous CH₂Cl₂[41]. Few selected pyridinone

derivatives with R₄¼ 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 Cl_int¼ 157

F were also metabolically unstable (Mouse Cl_intbetween 160 and

246 l/min/mg), however compounds 13 m-Cl, 16 m-CF₃and 22 m-

NHCOCH₃showed moderate improvement in stability (Cl_intbe-

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

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 3^rdposition 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 Cl_int< 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 -NH₂group.

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 Cl_int< 10 ml/

more bulky, electron-withdrawing groups, such as -CF_3,as a

possible alternative to the ﬂuorine or chlorine atoms on the aryl

group (compounds 15e17). The most potent compound 16 contains

a -CF₃group in the meta position. Interestingly, compound 17 with

the -CF₃in 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/IC₅₀values 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

2

H

F

H

Cl

H

CF₃

H

F

H

Cl

H

CF₃

H

Cl

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

H

CONH₂

H

CONH₂

H

NH₂

H

CONH₂>2000

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

Cl_int[ml/min/mg]

% remaining at 60 min

2

H

F

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

43

1073

1763

2.5. Selectivity proﬁling of compounds 24 & 26

8

9

>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]

Cl_int[ml/min/mg]

23

24

25

26

27

28

H

F

H

F

Cl

32

4

5

6

21

8

79

12

19

11

56

55

<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

93

>95

466

>500

>10

90

ND

172

275

2.6. Biochemical studies using recombinant MNK1 and MNK2

For better characterization of compounds 24 and 26, the inhi-

bition constant (K_i) 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. IC₅₀values 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, K_i14 nM was measured for

both compound 24 and 26. For MNK2, K_i11 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

other

CMGC

mutant

AGC

other

CMGC

lipid

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 T_max(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

T_max(h)

C_max(ng/mL)

T_1/2(h)

AUC_0-t(ng/h$mL)

AUC_0-∞(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 Na₂SO_4,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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%,

t_R¼ 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%, t_R¼ 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%, t_R¼ 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, 11_þ8.7, 106.5, 55.6;

m.p. >210 ^ꢀC decomp.; LC-MS (m/z) 330.1 (MþH) ; HPLC purity

(method C): 94%, t_R¼ 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%, t_R¼ 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%,

t_R¼ 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%,

t_R¼ 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%,

t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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

NaHCO₃and extracted with EtOAc (three times). The organic layers

were combined, dried over MgSO_4,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%, t_R¼ 7.1 min; HRMS

m/z [Mþ1]^þcalcd 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%, t_R¼ 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%, t_R¼ 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%, t_R¼ 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

IC₅₀experiments 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, 113_þ.6, 106.1,

51.9, 24.4; m.p. 148e153 ^ꢀC; LC-MS (m/z) 374.1 (MþH) ; HPLC

purity (method A): 100%, t_R¼ 11.9 min.

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 (K_i) 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. IC₅₀experiments for MNK2

iodo-1-(triphenylmethyl)pyrazolo[3,4-b]pyridine

(170

mg,

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. IC₅₀and 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%,

t_R¼ 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%, t_R¼ 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 T₀is 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.

!

C_buffer

% PPB ¼ 1 ꢃ

x 100%

C_plasma

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

V_d*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 IC₅₀value.

CL_int

¼

T₁₌₂

where, V_de volume of distribution equals:

1

V_d¼

ꢀ

ꢁ

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 IC₅₀inhibition 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

CYP2B6

Tranylcypromine

50

Tranylcypromiine

10

EFC

2.5

CEC

25

10

30

CYP2C19

CYP2D6

CYP3A4

Quinidine

0.05

AMMC

1.5

15

5

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

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 1 1 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

26

334.35

350.81

335.16

351.15

198.04

226.06

198.06

226.06

33.056

25.522

34.169

26.008

1.97

2.08

16

A. Dreas, K. Kucwaj-Brysz, K. Pyziak et al.

European Journal of Medicinal Chemistry xxx (xxxx) xxx

[19] R.M. Rowlett, C.A. Chrestensen, M. Nyce, M.G. Harp, J.W. Pelo, F. Cominelli, et

al., MNK kinases regulate multiple TLR pathways and innate proin ﬂ ammatory

cytokines in macrophages, Am. J. Physiol. Gastrointest. Liver Physiol. 294

(2008) G452eG459.

Development, Poland, INNOTECH Program (INNOTECH-K1/HI1/16/

157483/NCBR/12) and the Polish National Science Center, Poland

[2014/14/M/NZ5/00462 to MMikula].

[20] S. Joshi, L.C. Platanias, Mnk kinases in cytokine signaling and regulation of

cytokine responses, Biomol. Concepts 3 (2012) 127e139.

[21] S. Lv, M. Han, R. Yi, S. Kwon, C. Dai, R. Wang, Anti-TNF-a therapy for patients

Declaration of competing interest

with sepsis: a systematic meta-analysis, Int. J. Clin. Pract. 68 (2014) 520e528.

[22] C.E. Spooner, N.P. Markowitz, L.D. Saravolatz, The role of tumor necrosis factor

in sepsis, Clin. Immunol. Immunopathol. 62 (1992) S11eS17.

[23] R. Plevin, R. Callcut, Update in sepsis guidelines: what is really new? Trauma

Surg. Acute Care Open 2 (2017), e000088.

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

[24] S. Jain, Sepsis: an update on current practices in diagnosis and management,

Am. J. Med. Sci. 356 (2018) 277e286.

[25] H.K. de Jong, T. van der Poll, W.J. Wiersinga, The systemic pro-in ﬂ ammatory

response in sepsis, J. Innate Immun. 2 (2010) 422e430.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 13057.

[26] B.G. Chousterman, F.K. Swirski, G.F. Weber, Cytokine storm and sepsis disease

pathogenesis, Semin. Immunopathol. 39 (2017) 517e528.

[27] S.M. Opal, T. van der Poll, Endothelial barrier dysfunction in septic shock,

J. Intern. Med. 277 (2015) 277e293.

[28] P. Seidel, Q. Sun, L. Costa, D. Lardinois, M. Tamm, M. Roth, The MNK-1/eIF4E

pathway as a new therapeutic pathway to target in ﬂ ammation and remod-

elling in asthma, Cell. Signal. 28 (2016) 1555e1562.

References

ꢀ

[1] A. Dreas, M. Mikulski, M. Milik, C.-H. Fabritius, K. Brzozka, T. Rzymski,

[29] J. Hou, F. Lam, C. Proud, S. Wang, Targeting Mnks for cancer therapy, Onco-

target 3 (2012) 118e131.

[30] X.-B. Huang, C.-M. Yang, Q.-M. Han, X.-J. Ye, W. Lei, W.-B. Qian, MNK1 in-

hibitor CGP57380 overcomes mTOR inhibitor-induced activation of eIF4E: the

mechanism of synergic killing of human T-ALL cells, Acta Pharmacol. Sin. 39

(2018) 1894e1901.

[31] Q. Wen, W. Wang, J. Luo, S. Chu, L. Chen, L. Xu, et al., CGP57380 enhances

ef ﬁ cacy of RAD001 in non-small cell lung cancer through abrogating mTOR

inhibition-induced phosphorylation of eIF4E and activating mitochondrial

apoptotic pathway, Oncotarget 7 (2016) 27787e27801.

[32] J.K. Altman, A. Szilard, B.W. Konicek, P.W. Iversen, B. Kroczynska, H. Glaser, et

al., Inhibition of Mnk kinase activity by cercosporamide and suppressive ef-

fects on acute myeloid leukemia precursors, Blood 121 (2013) 3675e3681.

[33] U. Knauf, C. Tschopp, H. Gram, Negative regulation of protein translation by

mitogen-activated protein kinase-interacting kinases 1 and 2, Mol. Cell Biol.

21 (2001) 5500e5511.

Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1

and MNK2) as targets for cancer therapy: recent progress in the development

of MNK inhibitors, Curr. Med. Chem. 24 (2017) 3025e3053.

[2] S. Joshi, L.C. Platanias, Mnk kinase pathway: cellular functions and biological

outcomes, World J. Biol. Chem. 5 (2014) 321e333.

[3] Y. Zhan, J. Guo, W. Yang, C. Goncalves, T. Rzymski, A. Dreas, et al., MNK1/2

inhibition limits oncogenicity and metastasis of KIT-mutant melanoma, J. Clin.

Invest. 127 (2017) 4179e4192.

[4] E.M. Kosciuczuk, D. Saleiro, L.C. Platanias, Dual targeting of eIF4E by blocking

MNK and mTOR pathways in leukemia, Cytokine 89 (2017) 116e121.

[5] N. Siddiqui, N. Sonenberg, Signalling to eIF4E in cancer, Biochem. Soc. Trans.

43 (2015) 763e772.

[6] X. Yuan, H. Wu, H. Bu, P. Zheng, J. Zhou, H. Zhang, Design, synthesis and

biological evaluation of pyridone-aminal derivatives as MNK1/2 inhibitors,

Bioorg. Med. Chem. 27 (2019) 1211e1225.

[7] S.H. Reich, P.A. Sprengeler, G.G. Chiang, J.R. Appleman, J. Chen, J. Clarine, et al.,

Structure-based design of pyridone-aminal eFT508 targeting dysregulated

translation by selective mitogen-activated protein kinase interacting kinases 1

and 2 (MNK1/2) inhibition, J. Med. Chem. 61 (2018) 3516e3540.

[34] H. Yang, L.R. Chennamaneni, M.W.T. Ho, S.H. Ang, E.S.W. Tan, D.A. Jeyaraj, et

al., Optimization of selective mitogen-activated protein kinase interacting

kinases 1 and 2 inhibitors for the treatment of blast crisis leukemia, J. Med.

Chem. 61 (2018) 4348e4369.

€

[8] S. Santag, F. Siegel, A.M. Wengner, C. Lange, U. Bomer, K. Eis, et al., BAY

[35] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, et al.,

The protein Data Bank, Nucleic Acids Res. 28 (2000) 235e242.

1143269, a novel MNK1 inhibitor, targets oncogenic protein expression and

shows potent anti-tumor activity, Canc. Lett. 390 (2017) 21e29.

[9] A.J. Waskiewicz, A. Flynn, C.G. Proud, J.A. Cooper, Mitogen-activated protein

kinases activate the serine/threonine kinases Mnk1 and Mnk2, EMBO J. 16

(1997) 1909e1920.

[10] A. O ’Loghlen, V.M. Gonzalez, D. Pineiro, M.I. Perez-Morgado, M. Salinas,

M.E. Martín, Identi ﬁ cation and molecular characterization of Mnk1b, a splice

variant of human MAP kinase-interacting kinase Mnk1, Exp. Cell Res. 299

(2004) 343e355.

€

[36] Schrodinger. Schrodinger Release 2018-1, LLC, New York, 2016.

€

[37] R. Jauch, M.-K. Cho, S. Jakel, C. Netter, K. Schreiter, B. Aicher, et al., Mitogen-

activated protein kinases interacting kinases are autoinhibited by a reprog-

rammed activation segment, EMBO J. 25 (2006) 4020e4032.

[38] J.Y. Ramphal, B.O. Buckman, K. Emayan, J.B. Nicholas, S.D. Seiwert, Anti-

ﬁbrotic pyridinones [Internet]. 2015 [cited 2019 Jun 7]. Available from:

https://patents.google.com/patent/WO2015153683A1/en.

ꢀ

~

ꢀ

[39] R. Benarous, F. Chevreuil, B. Ledoussal, S. Chasset, F.L. Strat, Inhibitors of viral

replication, their process of preparation and their therapeutical uses

[Internet]. 2014 [cited 2019 Jun 7]. Available from: https://patents.google.

com/patent/WO2014057103A1/en?oq¼WO2014057103A1.

[11] K. Slentz-Kesler, J.T. Moore, M. Lombard, J. Zhang, R. Hollingsworth,

M.P. Weiner, Identi ﬁ cation of the human Mnk2 gene (MKNK2) through pro-

tein interaction with estrogen receptor beta, Genomics 69 (2000) 63e71.

[12] G.C. Scheper, J.L. Parra, M. Wilson, B. Van Kollenburg, A.C.O. Vertegaal, Z.-

G. Han, et al., The N and C termini of the splice variants of the human

mitogen-activated protein kinase-interacting kinase Mnk2 determine activity

and localization, Mol. Cell Biol. 23 (2003) 5692e5705.

[40] D. Demong, T.J. Greshock, R.K. Chang, X. Dai, H. Liu, J.A. Mccauley, et al.,

Compounds inhibiting leucine-rich repeat kinase enzyme activity [Internet].

2015 [cited 2019 Jun 7]. Available from: https://patents.google.com/patent/

WO2015026683A1/en?oq ¼ WO2015026683A1.

ꢀ

[13] J.L. Parra, M. Buxade, C.G. Proud, Features of the catalytic domains and C

[41] H. Zhang, P. Chen, G. Liu, Palladium-catalyzed oxidative arylalkylation of

unactivated alkenes: dual CeH bond cleavage of anilines and acetonitrile,

Synlett 23 (2012) 2749e2752.

termini of the MAPK signal-integrating kinases Mnk1 and Mnk2 determine

their differing activities and regulatory properties, J. Biol. Chem. 280 (2005)

37623e37633.

[42] K. Andersson, R. Sundler, Posttranscriptional regulation of TNFalpha expres-

sion via eukaryotic initiation factor 4E (eIF4E) phosphorylation in mouse

macrophages, Cytokine 33 (2006) 52e57.

[14] R. Fukunaga, T. Hunter, MNK1, a new MAP kinase-activated protein kinase,

isolated by a novel expression screening method for identifying protein ki-

nase substrates, EMBO J. 16 (1997) 1921e1933.

[43] C.F. Fortin, T.Z. Mayer, A. Cloutier, P.P. McDonald, Translational control of

human neutrophil responses by MNK1, J. Leukoc. Biol. 94 (2013) 693e703.

[44] M.E. Starr, J. Ueda, H. Takahashi, H. Weiler, C.T. Esmon, B.M. Evers, et al., Age-

dependent vulnerability to endotoxemia is associated with reduction of

anticoagulant factors activated protein C and thrombomodulin, Blood 115

(2010) 4886e4893.

[45] B. Shrum, R.V. Anantha, S.X. Xu, M. Donnelly, S.M. Haeryfar, J.K. McCormick, et

al., A robust scoring system to evaluate sepsis severity in an animal model,

BMC Res. Notes 7 (2014) 233.

[15] A.J. Waskiewicz, J.C. Johnson, B. Penn, M. Mahalingam, S.R. Kimball,

J.A. Cooper, Phosphorylation of the cap-binding protein eukaryotic translation

initiation factor 4E by protein kinase Mnk1 in vivo, Mol. Cell Biol. 19 (1999)

1871e1880.

[16] J.-L. Parra-Palau, G.C. Scheper, M.L. Wilson, C.G. Proud, Features in the N and C

termini of the MAPK-interacting kinase Mnk1 mediate its nucleocytoplasmic

shuttling, J. Biol. Chem. 278 (2003) 44197e44204.

[17] T. Ueda, R. Watanabe-Fukunaga, H. Fukuyama, S. Nagata, R. Fukunaga, Mnk2

and Mnk1 are essential for constitutive and inducible phosphorylation of

eukaryotic initiation factor 4E but not for cell growth or development, Mol.

Cell Biol. 24 (2004) 6539e6549.

[18] Y. Mamane, E. Petroulakis, L. Rong, K. Yoshida, L.W. Ler, N. Sonenberg, eIF4E –

from translation to transformation, Oncogene 23 (2004) 3172e3179.

[46] D.B. Feldman, J.C. Seely, Necropsy Guide: Rodents and the Rabbit, CRC Press,

Boca Raton, Fla, 1988. Available from: https://www.taylorfrancis.com/books/

9781482216073.

17

Article Doi

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

DOI: 10.1016/j.ejmech.2020.113057

Source and publish data:

Authors:

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

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

Products guided by the article

R&D Labs maybe for 1492148-75-0

Relevant to this article

Hot Product